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Web of Science Databases=SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, IC, CCR-EXPANDED. Searched on 2009-03-16 by K. Hruska Topic=((paratuberculosis OR Johne's OR Johnes) AND (wild OR rabbit OR deer OR mouse OR boar OR elephant OR hippo* OR dog OR fox OR wolf OR badger OR possum OR hedgehog OR mole OR hare)) Timespan=All Years. Results: 299

1 Sleeman, J.M., Manning, E.J.B., Rohm, J.H. , Sims, J.P., Sanchez, S., Gerhold, R.W., Keel, M.K. (2009) Johne's Disease in a Free-Ranging White-tailed Deer from Virginia and Subsequent Surveillance for Mycobacterium avium subspecies paratuberculosis

Journal of Wildlife Diseases, 45, 201-206 Johne's disease (paratuberculosis) was diagnosed in a 2-yr-old, male, free-ranging White-tailed deer (Odocoileus virginianus) from Fauquier County, Virginia, USA, based oil histopathology and culture for Mycobacterium avium subspecies paratuberculosis. Clinical and pathologic findings included emaciation; loss of body fat; chronic diarrhea; severe, chronic, diffuse granulomatous colitis with intrahistiocytic acid-fast bacilli; moderate, chronic granulomatous lymphadenitis with intrahistiocytic acid-fast bacilli; as well as moderate chronic, multifocal, lymphoplasmacytic hepatitis. These findings are consistent with previous reports of Johne's disease in cervids. Subsequent targeted surveillance of 10 emaciated deer with diarrhea, as well as sampling of 72 asymptomatic deer for M. avium subsp. paratuberculosis using culture of multiple tissue types, as well as serology using an enzyme-linked immnunosorbent assay (ELISA) optimized for cervid antibody detection, did not reveal any additional eases of infection ill this geographic region. To date, this appears to be an isolated case of Johne's disease in a free-ranging white-tailed deer, and infection with the causative agent for Johne's disease appears to be all infrequent occurrence ill deer from this region. The origin of inflection was most likely domestic ruminants. This is the first report of clinical Johne's disease in a free-ranging white-tailed deer outside of the Florida Keys, USA. Stressors, such as high deer population density and low selenium levels, may have contributed to the development of clinical disease in this case and warrant further investigation Diarrhea/emaciation/INFECTION/Johne's disease/ Mycobacterium avium subspecies paratuberculosis/Odocoileus virginianus/ODOCOILEUS-VIRGINIANUS/paratuberculosis/white-tailed deer

2 Fernandez, J.G., Fernandez-De-Mera, I., Reyes, L.E., Ferreras, M.C., Perez, V., Gortazar, C., Fernandez, M. , Garcia-Marin, J.F. (2009) Comparison of three immunological diagnostic tests for the detection of avian tuberculosis in naturally infected red deer (Cervus elaphus) Journal of Veterinary Diagnostic Investigation, 21, 102-107 Cases Of tuberculosis due to Mycobacterium avium subsp. avium in 52 adult red deer (Cervus elaphus) from a farm were studied using different diagnostic techniques. Immunological probes consisted of the comparative cervical tuberculin (CCT) skin test, the interferon-gamma (IFN-gamma) assay, and 2 enzyme-linked immunosorbent assays (ELISAs) employing either avian purified protein derivatives or protoplasmatic antigen (PPA-3) as antigens. Three of the animals were euthanized due to severe weakness, loss of weight, and emaciation. Macroscopically, the 3 animals showed tuberculous lesions located mainly in lymph nodes of the digestive system and small intestine but also in other organs and lymph nodes. Polymerase chain reaction was carried out on samples from the 3 deer using primers to detect IS901, IS900, and IS6110, specific for Mycobacterium avium subsp. avium, Mycobacterium avium subsp. paratuberculosis, and Mycobacterium tuberculosis complex, including Mycobacterium bovis, respectively. The last 2 agents cause pathologies very similar to avian tuberculosis in deer. The 3 deer were strongly positive by both ELISAs, slightly positive by the IFN-gamma test, and 1 of 2 was positive by the CCT test. As with domestic ruminants, ELISA could detect deer in an advanced stage of infection, with large numbers of mycobacteria ASSAY/Avian tuberculosis/AVIUM/BCG VACCINATION/CERVIDS/deer/DISEASES/emaciation/enzyme-linked immunosorbent assay/GAMMA/INFECTION/interferon-gamma/Mycobacterium avium/MYCOBACTERIUM-BOVIS/ODOCOILEUS-VIRGINIANUS/paratuberculosis/WILDLIFE

3 Pogranichniy, R.M., Raizman, E., Thacker, H.L., Stevenson, G.W. (2008) Prevalence and characterization of bovine viral diarrhea virus in the white-tailed deer population in Indiana Journal of Veterinary Diagnostic Investigation, 20, 71-74 Bovine viral diarrhea (BVD) is one of the economically important diseases of cattle. For many years, different types of vaccines have been commercially available, yet this disease is hard to control in high-density population areas. Detection and isolation of bovine viral diarrhea virus (BVDV) from any potential

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reservoir is vital, especially when considering virus eradication from a herd or locale. One potential source is wild ruminants. Ear notches and lymph nodes were collected from the wild population of white-tailed deer (Odocoileus virginianus) during deer hunting season in Indiana and tested for BVDV with a commercial BVD antigen capture enzyme-linked immunosorbent assay. Two samples out of 745 collected samples were positive, and subsequently cp and ncp BVDV was isolated from 1 ear notch and I lymph node. These isolates were genotyped as type la and 16 based on sequence analysis of the 5' untranslated region (U-I-R). The results of the present study indicate that the prevalence of BVDV in the white-tailed deer population of Indiana is about 0.3%. Wild ruminants infected with BVDV should be taken into consideration during an eradication program of BVDV from the livestock population ASSAY/AVIUM SUBSPECIES PARATUBERCULOSIS/Bovine viral diarrhea/BVDV/CALVES/CATTLE/deer/Diarrhea/DISEASES/enzyme-linked immunosorbent assay/INFECTION/NEOSPORA-CANINUM/Odocoileus virginianus/ODOCOILEUS-VIRGINIANUS/PESTIVIRUSES/RUMINANTS/UNITED-STATES/viral infection/white-tailed deer

4 Lopez-Olvera, J.R., Vidal, D., Vicente, J., Perez, M., Lujan, L., Gortazar, C. (2009) Serological survey of selected infectious diseases in mouflon (Ovis aries musimon) from south-central Spain

European Journal of Wildlife Research, 55, 75-79 Serum samples from 101 mouflons (Ovis aries musimon) collected from July 2002 to January 2006 were tested for antibodies against Anaplasma spp., Brucella spp., bovine viral diarrhea virus, Chlamydophila abortus, Coxiella burnetii, Mycobacterium avium ssp. paratuberculosis, and Maedi-Visna virus. Mouflon came either from extensive farms or high ungulate density fenced hunting estates. Antibodies were detected against Anaplasma spp. (22.2%), C. burnetii (4.0%), M. avium ssp. paratuberculosis (1.0%) and C. abortus (1.0%). According to our results, mouflons could participate as a wild reservoir in the epidemiology of Anaplasma spp. infection and maybe Q fever, but they do not seem to contribute in the epidemiology of the rest of the studied infectious diseases in south-central Spain Anaplasma spp./ANTIBODIES/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BORDER DISEASE/Bovine viral diarrhea/CHAMOIS RUPICAPRA-PYRENAICA/Coxiella burnetii/DEER CERVUS-ELAPHUS/Diarrhea/DISEASES/Epidemiology/INFECTION/Mycobacterium avium/paratuberculosis/PYRENEAN CHAMOIS/RED DEER/SHEEP/Ungulate/VIRUS-INFECTION /WILD RUMINANTS/WILDLIFE

5 Whittington, R.J., Windsor, P.A. (2009) In utero infection of cattle with Mycobacterium avium subsp paratuberculosis: A. critical review and meta-analysis

Veterinary Journal, 179, 60-69 Mycobacterium avium subsp. paratuberculosis (Mptb) causes Johne's disease in ruminants. Disease control programmes aim to break the faecal-oral cow-calf transmission cycle through hygienic calf rearing and removal of affected cows from the herd, but these programmes do not take account of the potential for congenital infection. The aims of this study were to critically review research on in Utero infection, determine the prevalence of fetal infection in cattle through meta-analysis and estimate the incidence of calves infected via the in utero route. About 9% (95% confidence limits 6-14%) of fetuses from subclinically infected cows and 39% (20-60%) from clinically affected cows were infected with Mptb (P < 0.001). These are underestimates for methodological reasons. The estimated incidence of calf infection derived via the in utero route depends on within-herd prevalence and the ratio of sub-clinical to clinical cases among infected cows. Assuming 80:20 for the latter, estimates of incidence were in the range 0.44-1.2 infected calves per 100 cows per annum in herds with within-herd prevalence of 5%, and 3.5-9.3 calves in herds with 40% prevalence. These estimates were not markedly sensitive to the value chosen for the proportion of clinical cases. In utero transmission of Mptb could retard the success of disease control programmes if the opportunities for post natal transmission via colostrum/milk and environmental contamination were able to be controlled. The consequences of fetal infection for the calves so infected are discussed in the context of diagnosis and vaccination together with recommendations for future research. (c) 2007 Elsevier Ltd. All rights reserved AMERICAN WILD RUMINANTS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE PARATUBERCULOSIS/CALVES/CATTLE/Congenital infection/CROHNS-DISEASE/CULTURE METHODS/DAIRY-CATTLE/HERD PREVALENCE/INFECTION/Intrauterine/Johne's disease/JOHNES-DISEASE/Meta-analysis/Mycobacterium avium/PARA-TUBERCULOSIS/paratuberculosis/POLYMERASE-CHAIN-REACTION/ Review/RUMINANTS/SHEEP /Transmission

6 Sevilla, I., Li, L.L., Amonsin, A., Garrido, J.M., Geijo, M.V., Kapur, V., Juste, R.A. (2008) Comparative analysis of Mycobacterium avium subsp paratuberculosis isolates from cattle,

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sheep and goats by short sequence repeat and pulsed-field gel electrophoresis typing

Bmc Microbiology, 8, Background: Mycobacterium avium subsp. paratuberculosis (Map) causes the chronic enteritis called paratuberculosis mainly in cattle, sheep and goats. Evidences that point out an association between Map and Crohn's Disease in humans are increasing. Strain differentiation among Map isolates has proved to be difficult and has limited the study of the molecular epidemiology of paratuberculosis. In order to asses the usefulness of the PCR based short sequence repeat (SSR) analysis of locus 1 and locus 8 in the epidemiological tracing of paratuberculosis strains we here compare for the first time the results of SSR and SnaBI-SpeI pulsed-field gel electrophoresis (PFGE) typing methods in a set of 268 Map isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar). Results: A total of nineteen different multi-locus SSR (SSR1_SSR8) types were identified amongst the 268 isolates compared to the 37 multiplex profiles differentiated by the SnaBI-SpeI PFGE. SSR type 7_4 was the predominant genotype (51.2% of all isolates and 54.3% of cattle isolates), but combined with PFGE results the abundance of the most prevalent genotype (7_4&{2-1}) dropped down to 37.7%. SSR types 7_3 and 14_3 were significantly spread amongst isolates recovered from small ruminants. The comparison of SSR1_SSR8 and SnaBI-SpeI PFGE typing of these isolates has shown that both methods perform at similar discriminatory level. These were 0.691 and 0.693, respectively for SSR and PFGE as indicated Simpson's Index of Diversity, and 0.82 when calculated for combined SSR and PFGE genotypes. Overall, SSR1_SSR8 analysis seemed to detect higher levels of within-farm strain diversity and seemed to give higher year-related information. Combination of both typing methods revealed 20 multi-type farms out of the 33 bovine farms studied with more than one isolate. Conclusion: The particular SSR and PFGE typing approaches described here are in general agreement but they showed some discrepancies that might reflect differing evolutionary processes of Map strains. Both methods are able to reciprocally complement their results and neither should be replaced with the other if sufficient material and time is available. Overall, the results of our comparative analyses suggest that, based on current methodologies available, a combined approach that includes SSR and PFGE seems to provide the highest level of discrimination for Map strain typing with meaningful epidemiological information AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/CROHNS-DISEASE/deer/Epidemiology/FRAGMENT-LENGTH-POLYMORPHISM/GENETIC DIVERSITY/HYBRIDIZATION/IDENTIFICATION/IS1311/IS900/MOLECULAR EPIDEMIOLOGY/Mycobacterium avium/paratuberculosis/RFLP/RUMINANTS/SHEEP/STRAINS

7 Davidson, R.S., Marion, G., White, P.C.L., Hutchings, M.R. (2009) Use of host population reduction to control wildlife infection: rabbits and paratuberculosis

Epidemiology and Infection, 137, 131-138 Reduction in wildlife Populations is a common method for the control of livestock infections which have wildlife hosts, but its Success is dependent on the characteristics of the infection itself, as well as on the spatial and social structure of the wildlife host. Paratuberculosis (Mycobacterium (avium subsp. paratuberculosis; Map) is a widespread and difficult infection to control in livestock populations and also has possible links to Crohn's disease in humans. Rabbits have recently been identified as a key wildlife species in terms of paratuberculosis persistence in the environment and risk to the wider host community, including cattle. Here we use a spatially explicit stochastic Simulation model of Map dynamics in rabbit populations to quantify the effects of rabbit Population control on infection persistence. The model parameters were estimated from empirical Studies of rabbit Population dynamics and rabbit-to-rabbit routes of Map transmission. Three rabbit control strategies were compared: single unrepeated Population reductions based on removing individual animals; single unrepeated population reductions based on removal of entire social groups; and repeated annual population reductions based on removing individual animals. Unrealistically high rabbit culls (>95% population reduction) are needed if infection is to be eradicated from local rabbit Populations with a single one-off population reduction event, either of individuals or social groups. Repeated annual culls are more effective at reducing the prevalence of infection in rabbit Populations and eradicating infection. However, annual Population reductions of >40% Eire required over extended periods of time (many years). Thus, using an approach which is both highly conservative and parsimonious with respect to estimating lower bounds on the time to eradicate the infection, we find that Map is extremely persistent in rabbit Populations and requires significant and prolonged effort to achieve control AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BADGERS/BOVINE TUBERCULOSIS/CATTLE/DISEASE/IMPACT/INFECTION /MYCOBACTERIUM-BOVIS/ORGANIZATION/ORYCTOLAGUS-CUNICULUS/paratuberculosis/Transmission/WILDLIFE

8 Lyashchenko, K.P., Greenwald, R., Esfandiari, J., Chambers, M.A., Vicente, J. , Gortazar, C., Santos, N., Correia-Neves, M., Buddle, B.M., Jackson, R., O'Brien, D.J., Schmitt, S., Palmer, M.V., Delahay, R.J., Waters, W.R. (2008) Animal-side serologic assay for rapid detection of Mycobacterium bovis infection in multiple species of free-ranging wildlife

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Veterinary Microbiology, 132, 283-292 Numerous species of mammals are susceptible to Mycobacterium bovis, the causative agent of bovine tuberculosis (TB). Several wildlife hosts have emerged as reservoirs of M. bovis infection for domestic livestock in different countries. In the present study, blood samples were collected from Eurasian badgers (n = 1532), white-tailed deer (n = 463), brushtail possums (it = 129), and wild boar (it = 177) for evaluation of antibody responses to M. bovis infection by a lateral-flow rapid test (RT) and multiantigen print immunoassay (MAPIA). Magnitude of the antibody responses and antigen recognition patterns varied among the animals as determined by MAPIA; however, MPB83 was the most commonly recognized antigen for each host studied. Other seroreactive antigens included ESAT-6, CFP10, and MPB70. The agreement of the RT with culture results varied from 74% for possums to 81% for badgers to 90% for wild boar to 97% for white-tailed deer. Small numbers of wild boar and deer exposed to M. avium infection or paratuberculosis, respectively, did not cross-react in the RT, supporting the high specificity of the assay. In deer, whole blood samples reacted similarly to corresponding serum specimens (97% concordance), demonstrating the potential for field application. As previously demonstrated for badgers and deer, antibody responses to M. bovis infection in wild boar were positively associated with advanced disease. Together, these findings suggest that a rapid TB assay such as the RT may provide a useful screening tool for certain wildlife species that may be implicated in the maintenance and transmission of M. bovis infection to domestic livestock. (C) 2008 Elsevier B.V. All rights reserved ANTIBODIES/ANTIBODY-RESPONSES/ASSAY/AVIUM/BADGERS/BADGERS MELES-MELES/BOVINE TUBERCULOSIS/CATTLE/deer/DIAGNOSIS/DISEASE/DOMESTIC-ANIMALS/INFECTION/MICHIGAN/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/ODOCOILEUS-VIRGINIANUS/paratuberculosis/Serology/STAT-PAK ASSAY/Transmission/Tuberculosis/TUBERCULOSIS COMPLEX/white-tailed deer/WILDLIFE

9 Jaimes, N.G. , Flores, M.A.S., Cruz, O.A.H., Lopez, D.C., Ruiz, C.C.G., Reynoso, B.A., Aparicio, E.D., Gutierrez, V.R.T., Ordaz, A.C. (2008) Detection of Mycobacterium avium subspecies paratuberculosis by nested-PCR of ovine fecal samples

Veterinaria Mexico, 39, 377-386 Paratuberculosis is a chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (Map), which affects wild and domestic ruminants. Map is shed in feces from infected animals. Transmission of the infection takes place by oral ingestion of the bacterium from contaminated food and water with feces. With the objective to establish a paratuberculosis diagnosis in ovine by nested-PCR from fecal samples, 204 fecal and serum ovine samples were studied. Feces were evaluated by nested-PCR and bacterial culture, serum samples were analyzed by agar gel immunodiffusion (AGID). Nested-PCR yielded a 210 bp amplification product that corresponds to Map-IS900, in 61 out of 204 samples. From these, 43 were from AGID positive animals and 18 from negative animals. Seventeen Map strains were isolated by bacterial culture and AGID detected 91 positive animals. Nested-PCR allowed to detect, sooner, greater number of animals shedding bacillus, even when they had resulted negative to the serological test. This result is considered important because generally these animals, while remaining in the farm, constitute the main source of infection for the herd. Nested-PCR should be considered as an alternative, when a prompt result is required to know the health status of the herd with respect to paratuberculosis AVIUM/ AVIUM SUBSPECIES PARATUBERCULOSIS/BACTERIAL CULTURE/BOVINE FECES/CATTLE/CULTURE/DIAGNOSIS/DNA/INFECTION/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/NESTED-PCR/OVINE/paratuberculosis/POLYMERASE-CHAIN-REACTION/ RUMINANTS/SEROLOGICAL STUDY/SHEEP/STRAINS/TESTS/Transmission

10 Kopecna, M. , Parmova, I., Dvorska-Bartosova, L., Moravkova, M., Babak, V., Pavlik, I. (2008) Distribution and transmission of Mycobacterium avium subspecies paratuberculosis in farmed red deer (Cervus elaphus) studied by faecal culture, serology and IS900 RFLP examinations

Veterinarni Medicina, 53, 510-523 The objectives of this study were the determination of Mycobacterium avium subsp. paratuberculosis (MAP) distribution in organs of farmed red deer (Cervus elaphus) and the investigation of its vertical and horizontal spread among animals, using serology, cultivation and the standardized IS900 RFLP method. During the three year of study, the production of antibodies for MAP increased from 0 in the first year to 7.7% (positive) and 0 to 88.5% (dubious) in the third year of the study. The first performed global culture examination of faecal samples from 28 animals was negative for MAP. In the three subsequent examinations of animals, the following positivity was found: 5.9%, 34.6%, and 36.8%, respectively. In the last year of the study, clinical signs such as diarrhoea were observed in four animals. The animals with clinical symptoms and those that were found to be infected with MAP by serology or faecal culture were

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euthanized. MAP was isolated from the intestinal tract and pulmonary lymph (tracheobronchial or mediastinal lymph nodes) nodes of all studied animals. Apart of this MAP was also isolated from reproductive organs, such as the mammary gland, milk, uterus, amniotic fluid and testicles. Application of the IS900 RFLP method revealed that the prevailing MAP isolates were of RFLP type B-C1; this profile was found in all types of tissue samples as well as in faeces, milk and amniotic fluid. In five animals a mixed infection of two profiles B-C1 and B-C5 or B-C1 and B-C16 was detected ANTIBODIES/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BOVINE/CATTLE FARMS/Crohn's disease/CULTURE/deer/ ecology/Epidemiology/food safety/FRAGMENT-LENGTH-POLYMORPHISM/INFECTED COWS/INFECTION/IS900/Johne's disease/JOHNES-DISEASE/MILK/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/ODOCOILEUS-VIRGINIANUS/PARA-TUBERCULOSIS/paratuberculosis/RED DEER/ RFLP/Serology/Transmission/white-tailed deer/WILD RUMINANTS

11 Gronesova, P., Ficova, M., Mizakova, A., Kabat, P., Trnka, A., Betakova, T. (2008) Prevalence of avian influenza viruses, Borrelia garinii, Mycobacterium avium, and Mycobacterium avium subsp paratuberculosis in waterfowl and terrestrial birds in Slovakia, 2006

Avian Pathology, 37, 537-543 The prevalence of Borrelia, Mycobacteria and avian influenza virus (AIV) infections, together with the distribution of different AIV subtypes, was studied in migratory waterfowl and terrestrial birds trapped in three localities in Slovakia during 2006. Samples obtained from waterfowl captured in the Senianske Ponds area of Eastern Slovakia showed the highest diversity of AIV isolates. A total of 13 different subtypes were detected in 19 samples from this location (H1N2, H2N2, H3N2, H6N6, H7N6, H9N2, H9N5, H9N6, H10N5, H10N6, H12N6, H13N6, and H16N6). H3N5 virus was detected in 50% of passerines testing positive for AIV in the Parizske Wetlands, with H7N2, H9N2, H9N5, H12N1, and H13N2 infections also recorded at this locality. H9N5 virus predominated in passerines captured at Trnava Ponds, with isolates H1N6, H6N5, H7N2, H7N6, H10N3, and H10N6 also detected at this location. There were five cases where different AIV infections were detected in oropharyngeal and cloacal samples originating from the same bird (H13N6 and H1N2; H10N5 and H12N6; H9N5 and H6N5; H10N6 and H7N6; and H9N2 and H3N5 in the oropharynx and cloaca, respectively). Between 21% and 52% of captured birds tested positive for Borrelia burgdorferi sensu lato, with the proportion infected depending on bird species and locality. Samples were characterized by polymerase chain reaction-restriction fragment length polymorphism analysis and identified as Borrelia garinii species (either B/B' or R/R' pattern). Mycobacteria were detected in 42% and 26% of waders captured at Senianske Ponds and marsh-dwelling passerines captured in the Parizske Wetlands, respectively Interestingly, forest-dwelling passerine species caught in the Trnava Ponds region were tested negative for Mycobacteria A VIRUSES/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BURGDORFERI SENSU-LATO/CONFORMATION POLYMORPHISM ANALYSIS/FRAGMENT-LENGTH-POLYMORPHISM/INFECTION/INTERGENIC SPACER AMPLICONS/IXODES-RICINUS TICKS/LYME-DISEASE/MIGRATORY BIRDS/Mycobacterium avium/paratuberculosis/POLYMERASE-CHAIN-REACTION/WILD BIRDS

12 Kopecna, M. , Trcka, I., Lamka, J., Moravkova, M., Koubek, P., Heroldova, M., Mrlik, V., Kralova, A., Pavlik, I. (2008) The wildlife hosts of Mycobacterium avium subsp paratuberculosis in the Czech Republic during the years 2002-2007

Veterinarni Medicina, 53, 420-426 The objective of this study was to determine the wildlife hosts of Mycobacterium avium subsp. paratuberculosis (MAP) in the Czech Republic. A total of 8 796 wildlife animals were examined by culture of faecal or tissue samples during the years 2002-2007. MAP was isolated from 12 (0.5%) out of 2 296 red deer (Cervus elaphus), two (0.2%) out of 835 roe deer (Capreolus capreolus), 78 (5.7%) out of 1 381 fallow deer (Dama dama), 28 (3.2%) out of 866 mouflons (Ovis musimon), four (2.5%) out of 162 chamois (Rupicapra rupicapra) and from one (0.1%) out of 805 wild boar (Sus scrofa). MAP was not cultured from 82 badgers (Meles meles), 55 martens (Martes foina), one pine marten (Martes martes), 25 brown hares (Lepus europaeus), five rabbits (Oryctolagus cuniculus), nine European polecats (Mustela putorius), two steppe polecats (Mustela eversmannii), two American minks (Mustela vison), four raccoon dogs (Nyctereutes procyonoides) and four Eurasian otters (Lutra lutra). MAP was isolated from three (2.0%) out of 149 small terrestrial mammals: one (5.9%) out of 17 brown rats (Rattus norvegicus), one (1.7%) out of 59 common voles (Microtus arvalis) and one (2.6%) out of 39 lesser white-toothed shrews (Crocidura suaveolens). Culture examinations of 34 house mice (Mus musculus) and 2 113 pigeons (Columba livia f. domestica) were negative. All 123 in vitro growing MAP isolates from wild ruminants were of IS900 RFLP type B-C1. One mouflon infected with a MAP strain which did not grow on the tested media was after IS1311-PRA-PCR assessed as being infected with a "sheep" strain. The RFLP type of the MAP isolate from the wild boar was of the RFLP type A-C10. Although the detection of

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MAP in wildlife in the Czech Republic was not very high, their role as a potential risk factor for cattle should be considered AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BADGERS/ BOAR SUS-SCROFA/CATTLE/CULTURE/deer/Epidemiology/FALLOW DEER/FRAGMENT-LENGTH-POLYMORPHISM /INFECTIONS/IS900/IS900PCR/Johne's disease/JOHNES DISEASE/Mycobacterium avium/non-ruminant species/ORYCTOLAGUS-CUNICULUS/PARA-TUBERCULOSIS/paratuberculosis/PASSIVE VECTORS/RED DEER/RFLP/RUMINANTS/SYRPHID FLIES/WILD RUMINANTS/WILDLIFE

13 Moravkova, M., Trcka, I., Lamka, J., Pavlik, I. (2008) A mixed infection of Mycobacterium avium subsp paratuberculosis and M-a. hominissuis in one red deer (Cervus elaphus) studied by IS900 BstEII and IS1245 PvuII RFLP analyses: a case report

Veterinarni Medicina, 53, 445-451 A mixed infection with Mycobacterium avium subsp. paratuberculosis (MAP) and Mycobacterium avium subsp. hominissuis (MAH) in one naturally infected red deer stag from a game park is described. The animal was euthanized because of symptoms of poor condition, weight loss and chronic diarrhoea. In spite of that, pathological lesions were observed only in the mesenteric lymph nodes, which were five to ten times enlarged with confluent caseous granulomas of 1 to 10 mm in size. Mycobacteria were isolated from all studied samples: a mixed infection of MAP and MAH was confirmed by multiplex PCR for the detection of IS900, IS901, IS1245 and dnaJ. MAP of the identical IS900 BstEII RFLP type C1 was isolated from all tissue samples and faeces. MAH isolates were detected in six examined tissue samples, including three mesenteric lymph nodes with caseous granulomas. Only minor differences in the band numbers and position of four different IS1245 PvuII RFLP patterns of MAH isolates were found. It follows from these results that red deer may potentially be infected with MAH, when a MAP infection is under way AMPLIFICATION/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/deer/DIFFERENTIATION/Epidemiology/FARMED DEER/food safety/FRAGMENT-LENGTH-POLYMORPHISM/game park/INFECTION/IS900/Johne's disease/MOLECULAR EPIDEMIOLOGY/mycobacteriosis/Mycobacterium avium/paratuberculosis/RED DEER/ RESTRICTION/RFLP/STANDARDIZATION/STRAINS/WILD RUMINANTS/zoonosis

14 Roupie, V., Leroy, B., Rosseels, V., Piersoel, V., Noel-Georis, I., Romano, M., Govaerts, M., Letesson, J.J., Wattiez, R., Huygen, K. (2008) Immunogenicity and protective efficacy of DNA vaccines encoding MAP0586c and MAP4308c of Mycobacterium avium subsp paratuberculosis secretome

Vaccine, 26, 4783-4794 Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis of the small intestine in domestic and wild ruminants, causes Substantial losses to livestock industry. Control of this disease is seriously hampered by the lack of adequate diagnostic tools, vaccines and therapies. In this study, we have evaluated the vaccine potential of two MAP proteins, i.e. MAP0586c and MAP4308c, previously identified by postgenomic and immunoproteomic analysis of MAP secretome as novel serodiagnostic antigens. Immunizations of BALB/c and C57BL/6 mice with plasmid DNA encoding MAP0586c and MAP4308c induced strong Th1 type immune responses to both antigens, whereas antibody responses were only induced upon immunization with DNA encoding MAP4308c. Homologous boosting of DNA vaccinated mice with recombinant protein resulted in strong antibody responses against both proteins. Using synthetic overlapping peptides, immunodominant H-2(d) and H-2(b) restricted Th1 T cell epitopes were identified. Finally, MAP infected mice generated strong MAP0586c-specific T cell responses and MAP0586c DNA vaccination could protect BALB/c but not C57BL/6 mice against MAP challenge mice to the same extent as the Mycobacterium bovis BCG vaccine, indicating that this putative transglycosylase is an interesting vaccine candidate that warrants further investigation. (C) 2008 Elsevier Ltd. All rights reserved ANTIBODIES/ANTIBODY-RESPONSES/ANTIGEN 85A/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE PARATUBERCULOSIS/CROHNS-DISEASE/DISEASE/DNA/DNA vaccine/GAMMA-INTERFERON/IMMUNE-RESPONSES/INFECTED MICE/JOHNES-DISEASE/MAP0586c/MAP4308c/Mycobacterium avium/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/paratuberculosis/PHOSPHATE-TRANSPORT RECEPTOR/PLASMID DNA/RUMINANTS/secretome/Tuberculosis/WILD RUMINANTS

15 Florou, M., Leontides, L., Kostoulas, P., Billinis, C., Sofia, M., Kyriazakis, I., Lykotrafitis, F. (2008) Isolation of Mycobacterium avium subspecies paratuberculosis from non-ruminant wildlife living in the sheds and on the pastures of Greek sheep and goats

Epidemiology and Infection, 136, 644-652

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This study aimed to: (1) investigate whether non-ruminant wildlife interfacing with dairy sheep and goats of four Greek flocks endemically infected with Mycobaclerium avium subspecies paratuberculosis (MAP) harboured MAP and (2) genetically compare the strains isolated from the wildlife to those isolated from the small ruminants of these flocks. We cultured and screened, by polymerase chain reaction (PCR), pooled-tissue samples from 327 wild animals of 11 species for the MAP-specific IS900 insertion sequence. We also cultured faecal samples from 100 sheep or goats from each of the four flocks. MAP was detected in samples from 11 sheep, 12 goats. two mice, two rats, a hare and a fox. Only one rat had histopathological findings. Genetic typing categorized 21 isolates as cattle-type strains and two. from a house Mouse and a goat respectively, as sheep-type strains this is the first report of a rodent harbouring a sheep-type strain. The MAP types that were most frequently isolated amongst the sheep and goats of each flock were also the ones isolated from sympatric rodents; those isolated from the fox and hare also belonged to the predominant ruminant strains AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/CATTLE/deer/DNA HYBRIDIZATION/IDENTIFICATION/INFECTION/IS900/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/PARA-TUBERCULOSIS/paratuberculosis/POLYMERASE-CHAIN-REACTION/RABBITS ORYCTOLAGUS-CUNICULUS/RESTRICTION-ENDONUCLEASE ANALYSIS/RUMINANTS/SCOTLAND/SHEEP/STRAINS/WILDLIFE

16 Pedersen, K., Manning, E.J.B., Corn, J.L. (2008) Distribution of Mycobacterium avium subspecies paratuberculosis in the Lower Florida Keys

Journal of Wildlife Diseases, 44, 578-584 Johne's disease, a fatal and contagious gastrointestinal infection caused by Mycobacterium avium subsp. paratuberculosis (Map), was first diagnosed in an endangered Florida Key deer (Odocoileus virginianus clavium) in 1996 and later in six additional key deer deaths from 1998 to 2004. We investigated the geographic distribution of Map in the Lower Florida Keys from february 2005 through May 2006 via collection of blood and fecal pellets from 51 live-captured deer, collection of 550 fecal samples from the ground, and by necropsies of 90 carcasses. Tissue and fecal samples also were submitted from 30 raccoons (Procyon lotor), three fetal cats (Felis catus), an opossum (Didelphis virginiana), and a Lower Keys marsh rabbit (Sylvilagus palustris hefneri). Mycobacterium avium subsp. paratuberculosis was identified in 23 key deer fecal samples collected from the ground, tissue samples from two clinically ill Key deer, and from the mesenteric lymph node of a raccoon. The results of this study indicate that Map persists in the Key deer populaiton and environment at a low prevalence, but its distribution currently is limited to a relatively small geographic area within the range of Key deer AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/CLAVIUM/deer/DEER ODOCOILEUS-VIRGINIANUS/DISEASE/Florida Keys/INFECTION/Johne's disease/Key deer/MAMMALS/Mycobacterium avium/Mycobacterium avium subsp paratuberculosis/Mycobacterium avium subspecies paratuberculosis/Odocoileus virginianus/Odocoileus virginianus clavium/ODOCOILEUS-VIRGINIANUS/PARA-TUBERCULOSIS/paratuberculosis/Procyon lotor/RABBITS/rocoon/SCOTLAND/white-tailed deer

17 Robinson, M., O'Brien, R., Mackintosh, C. , Griffin, F. (2008) Differential immune responses of red deer (Cervus elaphus) following experimental challenge with Mycobacterium avium subsp paratuberculosis

Clinical and Vaccine Immunology, 15, 963-969 Immune responses of red deer (Cervus elaphus) that presented with different levels of paucibacillary pathology were profiled to detail immune changes during the progression of Johne's disease. Immune responses were monitored using an immunoglobulin G1 (IgG1) antibody enzyme- linked immunosorbent assay (ELISA), a gamma interferon (IFN-gamma) ELISA, and flow cytometry. Animals in the study were divided into outcome groups postmortem according to disease severity. All animals mounted IgG1 antibody and IFN-gamma responses to both the vaccination and experimental challenges. The Mycobacterium avium subsp. paratuberculosis- specific IgG1 antibody responses in the challenged group showed marked differences between infected and severely diseased animals. Slightly higher IFN-gamma responses were seen in infected animals compared with severely diseased animals. No significant changes were seen in the phenotype of lymphocyte populations investigated. Vaccination with killed M. avium subsp. paratuberculosis in mineral oil adjuvant reduced the level of severe disease; however, it obscured immunological differences between the infected and severely diseased groups. This suggests protection is not exclusively mediated via the presence of a type 1 response and, furthermore, the presence of a type 2 response is compatible with protection. These profiles provide information on the different immune processes in Johne's disease progression ANTIBODIES/ANTIBODY-RESPONSES/ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BLOOD MONONUCLEAR-CELLS/BOVINE-

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PARATUBERCULOSIS/CATTLE/CYTOKINE GENE-EXPRESSION/deer/DIAGNOSIS/DISEASE/EXPERIMENTAL-INFECTION MODEL/GAMMA/GAMMA-INTERFERON/IMMUNE-RESPONSES/interferon-gamma/Johne's disease/JOHNES-DISEASE/Mycobacterium avium/Mycobacterium avium subsp paratuberculosis/paratuberculosis/PATHOLOGY/RED DEER/SHEEP

18 Miller, L.A., Gionfriddo, J.P., Fagerstone, K.A., Rhyan, J.C., Killian, G.J. (2008) The single-shot GnRH immunocontraceptive vaccine (GonaCon (TM)) in white-tailed deer: Comparison of several GnRH preparations

American Journal of Reproductive Immunology, 60, 214-223 Problem An effective, single-injection, multi-year, GnRH contraceptive agent is needed to control reproduction in overabundant white-tailed deer populations. Method of study Two GnRH conjugates, GonaCon (TM) (GnRH-KLH) and GonaCon-B (TM) (GnRH-blue protein), were prepared in emulsion form as one-injection and two-injection immunocontraceptive vaccine formulations. In addition, the GnRH-KLH protein conjugate was lyophilized and suspended in AdjuVac (TM) adjuvant to produce a fifth vaccine formulation. Each formulation was administered to a group of five captive adult female white-tailed deer. Reproductive performance of treated female deer was monitored for 5 years to determine the comparative efficacy of the various treatments. Results The longevity of the contraceptive response (2-5 years) was strongly influenced by the design of the conjugate antigen, the adjuvant used, and the delivery form of the vaccine. Conclusion One-injection and two-injection formulations of GonaCon (TM) and GonaCon-B (TM) produced multi-year contraception in adult female white-tailed deer. GonaCon-B (TM) provided a longer lasting contraceptive effect ACTIVE IMMUNIZATION/ANTIFERTILITY VACCINE/ANTIGEN/AVIUM SUBSP PARATUBERCULOSIS/CELL/contraception /deer/gonadotropin-releasing hormone/GONADOTROPIN-RELEASING-HORMONE/infertility/MAMMALS/Odocoileus virginianus/overabundance/white-tailed deer/WILDLIFE

19 Moravkova, M., Hlozek, P., Beran, V., Pavlik, I., Preziuso, S., Cuteri, V., Bartos, M. (2008) Strategy for the detection and differentiation of Mycobacterium avium species in isolates and heavily infected tissues

Research in Veterinary Science, 85, 257-264 The members of Mycobacterium avium species, comprising M. avium subsp. paratuberculosis, M. a. hominissuis, M. a. avium, M. a. silvaticum, are currently the most prevalent opportunistic pathogenic mycobacteria causing mycobacterial infection in animals and humans. The ability to distinguish between these subspecies is of relevance for proper diagnosis and control programmes of the diseases. The aim of this study was to design a fast and specific PCR strategy for the detection and differentiation of M. avium subspecies from the solid plate cultures for use in routine veterinary diagnosis. We have developed a multiplex PCR based on IS900, IS901, IS1245 and the dnaJ gene. This method allows the detection of M. a. paratuberculosis, M. a. hominissuis and M. a. avium/M. a. silvaticum in one PCR reaction and theoretically enables mixed infections of M. a. paratuberculosis and M. a. avium or M. a. paratuberculosis and M. a. hominissuis to be revealed. The sensitivity of this multiplex PCR is 10(3) CFU for each bacterial strain in one PCR reaction, which also enabled the use of this test directly for DNA isolated from the tissue of the heavily infected sheep. (C) 2007 Elsevier Ltd. All rights reserved Avian tuberculosis/AVIUM/COMPLEX/CULTURE/DEER CERVUS-ELAPHUS/DIAGNOSIS/DIFFERENTIATION/DISEASE/DISEASES/DNA/IDENTIFICATION /INFECTION/INFECTIONS/insertion sequences/INTRACELLULARE/ IS900/IS901/Johne's disease/mycobacteriosis/Mycobacterium avium/paratuberculosis/PASTEURIZED COWS MILK/PCR/RESTRICTION ENDONUCLEASE ANALYSIS/SHEEP/STRAINS/SUBSP-PARATUBERCULOSIS

20 Woodbury, M.R., Chirino-Trejo, M., Mihajlovic, B. (2008) Diagnostic detection methods for Mycobacterium avium subsp paratuberculosis in white-tailed deer

Canadian Veterinary Journal-Revue Veterinaire Canadienne, 49, 683-688 This study compares the results and suitability of serological testing, microscopic examination, deoxyribonucleic acid (DNA) detection, and bacterial culture for detecting Mycobacterium avium subsp. paratuberculosis (Map) infection in asymptomatic farmed white-tailed deer (WTD) (Odocoileus virginianus). Deer were classified as infected if culture slants from their feces, lymph nodes, or ileum were positive, or if a polymerase chain reaction (PCR) assay detected Map DNA in any of its tissues. Deer identified as positive by agar gel immunodiffusion (AGID) testing or enzyme-linked immunosorbent assay (ELISA) but not by bacterial culture, Ziehl-Neelsen staining, or PCR assay were classified as suspect. Culture of tissues classified 10/16 (62.5%), histopathologic examination 1/16 (6.3%), tissue

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smears 4/16 (25%), culture slant (CS)-PCR on feces 12/15 (80%), CS-PCR on tissue 13/16 (81.3%), and direct PCR on uncultured tissues 5/16 (31.3%) deer as infected. The ELISA classified 2/15 (13.3%) deer as positive and therefore suspect. The AGID test was negative for all deer. Fifteen of 16 deer were positive by 1 or more tests; only 1 deer was negative on all 11 assays. The CS-PCR gave superior results oil antemortem fecal testing as well as postmortem tissue testing and can be recommended for improving the detection of Map in WTD at every stage of infection ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIAL CULTURE/CERVUS-ELAPHUS/CULTURE/deer/ DNA/enzyme-linked immunosorbent assay/FARMED RED DEER/FECES/INFECTION/JOHNES-DISEASE/Mycobacterium avium/Mycobacterium avium subsp paratuberculosis/NORTH-AMERICAN BISON/Odocoileus virginianus/ODOCOILEUS-VIRGINIANUS/paratuberculosis/PCR/POLYMERASE-CHAIN-REACTION/RUMINANT PARA-TUBERCULOSIS/SENSITIVITY/SPECIFICITY/SUBSP-PARATUBERCULOSIS/TESTS/white-tailed deer

21 Reyes-Garcia, R., Perez-de-la-Lastra, J., Vicente, J., Ruiz-Fons, F., Garrido, J.M., Gortazar, C. (2008) Large-scale ELISA testing of Spanish red deer for paratuberculosis Veterinary Immunology and Immunopathology, 124, 75-81 A role of wildlife species as paratuberculosis reservoirs is strongly suspected based on field and molecular epidemiologic evidence. This paper presents the first large-scale data on enzyme-linked immunosorbent assay (ELISA) against Mycobacterium avium subspecies paratuberculosis (MAP) antibodies in red deer from Spain, and tests the effect of host and environmental risk factors on antibody levels. A total of 257 out of 852 serum samples tested positive, yielding a total seroprevalence of 30.16% (95% CI 27.08-33.24). Sampling locality, presence of cattle and increasing age explained the variation in the individual ELISA optical density (OD) results. Data presented in this study strongly suggest that Spanish red deer are exposed to MAP. While contact with cattle was statistically significant, some wild populations showed the highest positivity to the ELISA. The results support the need of a careful study of MAP prevalence based on culture and molecular tools in order to clarify if deer play a significant role as paratuberculosis reservoirs for livestock, and if deer paratuberculosis is affecting hunting harvest, trophy quality, or wild animal welfare in Spain. (c) 2008 Elsevier B.V. All rights reserved ANTIBODIES/ASSAY/AVIUM/AVIUM SUBSP-PARATUBERCULOSIS/AVIUM SUBSPECIES PARATUBERCULOSIS/CATTLE/Cervus elaphus /CERVUS-ELAPHUS/CULTURE/deer/enzyme-linked immunosorbent assay/Epidemiology/Johne's disease/JOHNES-DISEASE/LINKED IMMUNOSORBENT-ASSAY/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/ODOCOILEUS-VIRGINIANUS/paratuberculosis/RED DEER/scrology/SEROLOGIC SURVEY/SOUTH-CENTRAL SPAIN/TESTS/TUBERCULOSIS-LIKE LESIONS/WILD BOAR/WILDLIFE

22 Beckler, D.R., Elwasila, S., Ghobrial, G., Valentine, J.F., Naser, S.A. (2008) Correlation between rpoB gene mutation in Mycobacterium avium subspecies paratuberculosis and clinical rifabutin and rifampicin resistance for treatment of Crohn's disease

World Journal of Gastroenterology, 14, 2723-2730 AIM: To investigate overlapping regions of the rpoB gene previously involved with rifamycin resistance in M. tuberculosis and seek correlation between rpoB mutations in clinical MAP strains with susceptibility to RIF and RFB. METHODS: We designed a molecular-based PCR method for the evaluation of rifabutin (RFB) and rifampicin (RIF) resistance based on probable determinant regions within the rpoB gene of MAP, including the 81 bp variable site located between nucleotides 1363 and 1443. The minimum inhibitory concentration (MIC) for RIF was also determined against 11 MAP isolates in attempt to seek correlation with rpoB sequences. RESULTS: We determined that MAP strain 18 had an MIC of > 30 mg/L and <= 5 mg/L for RIF and RFB respectively, and a significant and novel rpoB mutation C1367T, compared to an MIC of <= 1.0 mg/L for both drugs in the wild type MAR The 30-fold increase in the MIC was a direct result of the rpoB mutation C1367T, which caused an amino acid change Thr456 to Ile456 in the drug's binding site. In addition, MAP strain 185 contained five silent rpoB mutations and exhibited an MIC comparable to the wild-type. Moreover, our in vitro selected mutation in MAP strain UCF5 resulted in the generation of a new resistant strain (UCF5-RIF16r) that possessed T1442C rpoB mutation and an MIC > 30 mg/L and > 10 mg/L for RIF and RFB respectively. Sequencing of the entire rpoB gene in MAP strains UCF4, 18, and UCF5-RIF16r revealed an rpoB mutation A2284C further downstream of the 81 bp variable region in UCF4, accounting for observed slight increase in MIC. In addition, no other significant mutations were found in strains 18 and UCF-RIF16r. CONCLUSION: The data clearly illustrates that clinical and in vitro-selected MAP mutants with rpoB mutations result in resistance to RIF and RFB, and that a single amino acid change in the beta subunit may have a significant impact on RIF resistance. Unconventional drug susceptibility testing such as our molecular approach will be beneficial for evaluation of antibiotic effectiveness. This molecular approach may also serve as a model for other drugs used for treatment of MAP infections. (C) 2008 WJG. All rights reserved

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ANTIBIOTICS/ AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/CLARITHROMYCIN/Crohn's disease/DISEASE/ESCHERICHIA-COLI/HELICOBACTER-PYLORI/IDENTIFICATION/IMPACT/IN-VITRO ACTIVITY/INFECTION/INFECTIONS/minimum inhibitory concentration/MOLECULAR CHARACTERIZATION/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/Mycobacterium paratuberculosis/paratuberculosis/PCR/Rifabutin/rifampicin/rpoB/STRAINS/THERAPY/TISSUE/Tuberculosis

23 Horiuchi, N., Watarai, M., Kobayashi, Y., Omata, Y., Furuoka, H. (2008) Proliferative enteropathy involving Lawsonia intracellularis infection in rabbits (Oryctlagus cuniculus)

Journal of Veterinary Medical Science, 70, 389-392 Five rabbits suffering from diarrhea were diagnosed with proliferative enteropathy (PE). Histopathology revealed a thickened mucosa consisting of hyperplastic intestinal epithelium and infiltration of inflammatory cells mainly consisted of macrophages. In the affected epithelial cytoplasm, numerous curved bacillus-like Organisms were observed in the Warthin-Starry silver stain and electron microscopy observation. In polymerase chain reactions, Lawsonia intracellularis-specific DNA fragment were amplified from affected ileal tissue extracted DNA in each case and present 5 cases were confirmed to be L. intracellularis infection. Serum collected from the affected rabbit was immunohistochemically reactive with L. intracellularis in tissue sections from pigs with porcine proliferative enteropathy, as well as with tissue sections from the five affected rabbits. Thus, serum obtained from the affected rabbit may be applicable to immunohistochemical detection for L. intracellularis infection in other species BACTERIUM/CELL/Diarrhea /DNA/ENTERITIS/INFECTION/Lawsonia intracellularis/ORGANISM/paratuberculosis/POLYMERASE-CHAIN-REACTION/proliferative enteropathy/rabbit/RABBITS/TISSUE

24 Balseiro, A., Marin, J.F.G., Solano, P., Garrido, J.M., Prieto, J.M. (2008) Histopathological classification of lesions observed in natural cases of paratuberculosis in free-ranging fallow deer (Dama dama)

Journal of Comparative Pathology, 138, 180-188 Ninety-five adult fallow deer, legally hunted in the Regional Hunting Reserve of El Sueve (Northern Spain), were subjected to a post-mortem examination for paratuberculosis, samples being taken from the proximal and distal jejunum, proximal and distal ileum, ileocaccal valve and associated lymph nodes. The lesions were divided into four categories. Focal lesions (n = 19 cases) consisted of small granulomas., mainly in the jejunal and ileal lymph nodes. Multifocal lesions (n = 4) consisted of well-demarcated granulomas in the intestinal lymphoid tissue and also in the intestinal lamina propria. Diffuse multibacillary lesions (n = 2) were characterized by a severe granulomatous enteritis and lymphadenitis. Macrophages and numerous Langhans giant cells containing many mycobacteria were present, resulting in macroscopical changes in the normal gut morphology. These changes were found from the proximal jejunum to the ileocaecal valve, but lesions were always particularly severe in the distal jejunum. In diffuse intermediate (multibacillary-lymphocytic) lesions (n = 3) the infiltrate consisted of lymphocytes, macrophages and Langhans giant cells, with small numbers of mycobacteria. Mycobacterium avium subspecies paratuberculosis was identified by a polymerase chain reaction technique. The widespread Occurrence of paratuberculosis in fallow deer in this Reserve represents a potential source of infection for other susceptible species. (c) 2008 Elsevier Ltd. All rights reserved AVIUM/AVIUM SUBSP PARATUBERCULOSIS/AVIUM SUBSPECIES PARATUBERCULOSIS/ bacterial infection/BACTERIAL ISOLATION/CELL/Dama dama/deer/ENTERITIS/EXPERIMENTAL-INFECTION/FALLOW DEER/IMMUNE-RESPONSES /INFECTION/Johne's disease/JOHNES DISEASE/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/NATURALLY ACQUIRED PARATUBERCULOSIS/PARA-TUBERCULOSIS/paratuberculosis/paratuberculosis lesions/PATHOLOGICAL FINDINGS/POLYMERASE-CHAIN-REACTION/SHEEP/TISSUE

25 Begg, D.J., Whittington, R.J. (2008) Experimental animal infection models for Johne's disease, an infectious enteropathy caused by Mycobacterium avium subsp paratuberculosis

Veterinary Journal, 176, 129 -145 A critical literature review of experimental infection models for Johne's disease in farm and laboratory animals was conducted. A total of 73 references were admitted. They were published between 1938 and 2006 and covered species as diverse as cattle, sheep, goats, deer, mice, pigs and others. The factors that appeared to influence the outcome of experimental infections with Mycobacterium avium subsp. para tuberculosis (Mptb) were the species, breed and age of subject used for the infection, the route of

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infection, and the strain, dose and number of doses of Mptb used to inoculate the subjects. Natural paratuberculosis infection passes through stages, generally over a period measured in years. However, the endpoints chosen by researchers using experimental infections have been determined by the need for immunological, microbiological, pathological or clinical outcomes, and these were the likely factors determining the duration of the trials. Studies have been lacking in the use of a defined type strain of Mptb in pure culture prepared from an archived seed stock of Mptb that can be used at the same passage level in a later trial. Replication of experimental groups has been very uncommon, temporal replication equally rare, as have sufficiently long time scales so as to be able to observe a full range of immunological and pathological changes at different stages of the disease process. While it may be difficult to develop a satisfactory experimental infection model, there is room for improvement in the way experiments have been designed and carried out to date. Choice of animal species/breed of host and strain of Mptb used in an experimental model should be based on the purpose of the study (for example, vaccine efficacy trial, diagnostic test evaluation, pathogenesis study) and local needs. The strain of Mptb used should be typed using IS900 RFLP analysis, IS1311 sequence analysis and other genotypic methods, and preferably be from an archived low passage pure culture with viable bacteria enumerated using a sensitive method rather than from an uncharacterised and unrepeatable tissue homogenate. It is generally agreed that the faecal-oral route is the most important natural route of exposure and the oral route is therefore the preferred route of experimental inoculation to achieve Johne's disease that closely resembles natural infection. (C) 2007 Elsevier Ltd. All rights reserved AMERICAN WILD RUMINANTS/animal model/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIUM/CATTLE/CULTURE/deer/DEER CERVUS-ELAPHUS/DISEASE/experimental infection/EXPERIMENTAL ORAL INFECTION/EXPERIMENTAL-INFECTION/FECAL CULTURE/IMMUNE-RESPONSES/INFECTION/INFECTIONS/INTRAVENOUS INOCULATION/IS1311/IS900/Johne's disease/LABORATORY ANIMALS/Mycobacterium avium/Mycobacterium avium subsp/ Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/OVINE PARATUBERCULOSIS/PARA-TUBERCULOSIS/paratuberculosis/REGULATORY T-CELLS/Review/RFLP/SHEEP/SUBSP-PARATUBERCULOSIS /SWISS WHITE MICE/TISSUE/Tuberculosis

26 Zanetti, S. , Bua, A., Molicotti, P., Delogu, G., Mura, A., Ortu, S., Sechi, L.A. (2008) Identification of mycobacterial infections in wild boars in Northern Sardinia, Italy Acta Veterinaria Hungarica, 56, 145-152 During a six-month period a region of Northern Sardinia was monitored to check the presence of mycobacterial infections in wild boars. Forty-eight serum and 229 biopsy samples were collected from different animals and examined by both traditional diagnostic techniques (culture, bacterioscopic and molecular tests) and enzyme-linked immunosorbent assay (ELISA). The latter was used to determine the antibody response against both methylated and nonmethylated Heparin-Binding Haemagglutinin (HBHA) protein. Nine mycobacterial strains were isolated: three M. avium ssp. paratuberculosis (Map), three M. avium, one M. interjectum and two M scrofulaceum strains. By PCR, only one animal was positive for M bovis, whereas 10 animals were positive for Map. Out of the 48 sera tested, 19 showed a good humoral response to methylated HBHA and 17 to nonmethylated HBHA. Our data provide new information on the prevalence of mycobacterial infection among wild boars in Northern Sardinia and suggest that a more effective program should be developed to monitor mycobacterial infections in the wild animal population ANTIBODIES/ASSAY/AVIUM/BOVIS/CULTURE/DISEASE/enzyme-linked immunosorbent assay/HBHA/HEPARIN-BINDING HEMAGGLUTININ/humoral response/IDENTIFICATION/INFECTION/INFECTIONS/Mycobacterium bovis/paratuberculosis/PCR/STRAINS/TESTS/Tuberculosis/TUBERCULOSIS COMPLEX/WILD BOAR/wild boars

27 Mackintosh, C.G., Labes, R.E., Thompson, B.R. , Clark, R.G., de Lisle, G.W., Johnstone, P.D., Griffin, J.F.T. (2008) Efficacy, immune responses and side-effects of vaccines against Johne's disease in young red deer (Cervus elaphus) experimentally challenged with Mycobacterium avium subsp paratuberculosis New Zealand Veterinary Journal, 56, 1-9 AIMS: To test the efficacy of a commercially available and an experimental vaccine against Johne's disease in young red deer (Cervus elaphus), using experimental challenge with live virulent Mycobacterium avium subsp paratuberculosis (M. ptb), measure injection-site reactions, and assess the effects of vaccination and challenge on results of subsequent skin tests and ancillary blood tests for bovine tuberculosis (Tb). METHODS: Ninety 6-8-week-old red deer fawns were randomly allocated to three equal groups of 30, and received either a 1-ml S/C injection of either a commercially available whole-cell killed vaccine with a mineral-oil adjuvant (COM), or a live attenuated M. ptb experimental vaccine with a lipid adjuvant (EXP), or were unvaccinated controls. Ten weeks later (Week 10), all 90

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fawns received an oral challenge with similar to 10(8) cfu of a bovine strain of M. ptb daily for 4 days. The fawns were regularly weighed and monitored for clinical signs of Johne's disease, and regularly blood-sampled and tested for antibodies to M. ptb, using the Paralisa test, an IgG(1) ELISA, and for antibodies to Mycobacterium bovis, using a similar test. A mid-cervical tuberculin skin test (MCT) was administered at Week 23, and comparative cervical skin tests (CCTs) were administered at Weeks 37 and 57. All animals were electively killed at Week 59, injection sites inspected, gastrointestinal tracts examined for gross lesions, and samples taken for culture and histopathology. RESULTS: There were no clinical cases of Johne's disease but, at slaughter, more gross lesions in intestinal lymph nodes were observed in Control (20%) than COM animals (0%; p<0.05). This latter group also had less severe histopathological lesions in samples of intestines and lymph nodes compared with the Control group (p<0.05), but not deer in the EXP group. Over 89% of deer in all three groups were shown by culture to be infected with M. ptb, while only 21-33% of faecal samples were culture-positive. Time to positive culture was longer for COM vs EXP and Control groups (p<0.01), reflecting fewer M. ptb organisms in samples from the ileocaecal valve (ICV) in that group. Almost all (>= 90%) deer reacted to the MCT at Week 23, and there were no significant differences between groups. One or two deer in each group were classified as Tb reactors to the CCT at Week 37, and none were classified as Tb reactors to the CCT at Week 57. At the time of challenge, over 50% of deer in the COM group were classified as positive (9/28) or suspicious (7/28) for M. ptb antibodies in the Paralisa test, one animal in the EXP group was classified as suspicious, and all the Controls were negative. From Week 23 to the end of the trial, 25/28 (89%) deer in the COM group were Paralisa-positive or -suspicious. The proportion of animals in the EXP and Control groups that were Paralisa-positive peaked at Week 39 (60% and 55%, respectively). The majority of deer in the COM group had significant levels of antibody to M. bovis 10 weeks after vaccination, while the proportion of M. bovis-antibody positive Control deer rose gradually throughout the trial, reaching 23/30 (77%) at slaughter. Injection-site lesions in COM deer ranged from 10-38 mm in diameter 4 weeks after vaccination, and then resolved. Minimal injection-site lesions were observed in EXP deer. At slaughter, 14 months after vaccination, 19/28 deer in the COM group had 5-15-mm nodules that were easily trimmed from the carcass. CONCLUSIONS: The experimental challenge with M. ptb produced subclinical Johne's disease in the majority of deer, but did not cause any clinical disease. The number and severity of gross and microscopic lesions was significantly reduced in the COM compared with Control and EXP groups; vaccination of the EXP group did not appear to give significant protection. Deer vaccinated with the commercial vaccine are likely to give a false-positive reaction to the MCT but should have an avian reaction to the CCT, if it is carried out >12 months after vaccination. Most of the deer vaccinated with the commercial vaccine produced significant levels of antibodies against both M. ptb and M. bovis, which interfered with ancillary Tb tests. If this vaccine or similar oil-based vaccines are used on deer farms in the future, it may be advisable to only vaccinate animals destined for slaughter, that would not need to be Tb-tested, but would be `works-monitored' for evidence of Tb instead ANTIBODIES/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/BOVINE TUBERCULOSIS/BOVIS/Cervus elaphus/CERVUS-ELAPHUS/CULTURE/deer/DIAGNOSIS/DISEASE/FARMED DEER/IMMUNE-RESPONSES/INFECTION/Johne's disease/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium bovis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-BOVIS/NEW-ZEALAND/ORGANISM/OVINE PARATUBERCULOSIS/paratuberculosis/RED DEER/SHEEP/SUBSP-PARATUBERCULOSIS/TESTS/Tuberculosis/VACCINATION

28 Roupie, V., Rosseels, V., Piersoel, V., Zinniel, D.K., Barletta, R.G., Huygen, K. (2008) Genetic resistance of mice to Mycobacterium paratuberculosis is influenced by Slc11a1 at the early but not at the late stage of infection

Infection and Immunity, 76, 2099-2105 We have recently described the development of a luminescent Mycobacterium paratuberculosis strain of bovine origin expressing the luxAB genes of Vibrio harveyi. With this luminescent isolate, fastidious and costly enumeration of CFU by plating them on agar can be replaced by easy and rapid luminometry. Here, we have reevaluated the effect of Slc11a1 (formerly Nramp1) polymorphism on susceptibility to M. paratuberculosis, using this luminometric method. A series of inbred mouse strains were infected intravenously with luminescent M. paratuberculosis S-23 and monitored for bacterial replication in spleen, liver, and lungs for 12 weeks. The results indicate that, as for Mycobacterium avium subsp. avium, innate resistance to infection is genetically controlled by Slc11a1. In BALB/c, congenic BALB.B10-H2(b) (BALB/c background; H-2(b)), C57BL/6, and beige C57BL/6(bg/bg) mice (all Slc11a1(s)), bacterial numbers in spleen and liver remained unchanged during the first 4 weeks of infection, whereas in DBA/2 and congenic BALB/c.DBA/2 (C.D2) mice (both Slc11a1(r)) and in (C57BL/6 x DBA/2)F-1 mice (Slc11a1(s/r)), the bacterial numbers had decreased more than 10-fold at 4 weeks postinfection in both male and female mice. At later time points, additional differences in bacterial replication were observed between the susceptible mouse strains, particularly in the liver. Whereas bacterial numbers in the liver gradually decreased more than 100-fold in C57BL/6 mice between week 4

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and week 12, bacterial numbers were stable in livers from BALB/c and beige C57BL/6bg/bg mice during this period. Mycobacterium-specific gamma interferon responses developed earlier and to a higher magnitude in C57BL/6 mice than in BALB/c mice and were lowest in resistant C.D2 mice AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BCG GENE/BEIGE MICE/BOVINE/CROHNS-DISEASE/FORMERLY NRAMP1/GAMMA/GAMMA-INTERFERON/INFECTION/INFLAMMATORY-BOWEL-DISEASE/INTRACELLULAR INFECTIONS/JOHNES-DISEASE/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/NATURAL-RESISTANCE/NRAMP1 GENE/paratuberculosis/STRAINS

29 Robino, P., Nebbia, P., Tramuta, C., Martinet, M., Ferroglio, E., De Meneghi, D. (2008) Identification of Mycobacterium avium subsp paratuberculosis in wild cervids (Cervus elaphus hippelaphus and Capreolus capreolus) from Northwestern Italy

European Journal of Wildlife Research, 54, 357-360 Seventy-seven red deer (Cervus elaphus hippelaphus), 40 roe deer (Capreolus capreolus) from the Northwestern (NW) Alps (Turin Province, NW Italy) and 29 roe deer from the NW Apennines (Alessandria province, NW Italy) were examined for the presence of Mycobacterium avium subsp. paratuberculosis (MAP) by culture, IS900 nested polymerase chain reaction (PCR) and IS1311 PCR restriction endonuclease analysis for strain characterisation. MAP identification (nested PCR and/or culture) allowed us to detect 32.9% MAP-infected red deer and 22.5% infected roe deer in the NW Alps and 41.4% MAP infected roe deer in the NW Apennines. On the basis of the polymorphism present in the IS1311 sequence, all MAP isolates were characterised as cattle strains. Our results show that MAP circulates widely among populations of wild cervids in NW Italy AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/CERVIDS/Cervus elaphus/CERVUS-ELAPHUS/CULTURE/deer/IDENTIFICATION/IS1311/IS900/Italy/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/NESTED-PCR/paratuberculosis/PCR/POLYMERASE-CHAIN-REACTION/RED DEER/red deer (Cervus elaphus hippelaphus)/RESTRICTION/RESTRICTION ENDONUCLEASE ANALYSIS/roe deer (Capreolus capreolus)/STRAINS/SUBSP-PARATUBERCULOSIS

30 Thompson, B.R., Clark, R.G., Mackintosh, C.G. (2007) Intra-uterine transmission of Mycobacterium avium subsp paratuberculosis in subclinically affected red deer (Cervus elaphus)

New Zealand Veterinary Journal, 55, 308-313 AIM: To determine the rate of transmission of Mycobacterium avium subsp paratuberculosis (M. ptb) from hind to fetus in utero, and the risk of transmission from dam to fawn via infected colostrum and milk in subclinically affected red deer hinds. METHODS: Hinds were sourced from farms in Otago or Southland and selected for the study if they were positive to the immunoglobulin G1 (IgG1) modified enzyme-linked immunosorbent assay (ELISA) (Paralisa) and exhibited no clinical signs of Johne's disease. The hinds (n=35) were sent to a deer slaughter premises (DSP; n=31) or were killed on-farm (n=4). All post-mortem samples were collected from the fetus first and then from the dam, taking care to avoid cross contamination between samples. Fresh samples (n= 185) were collected for culture, and tissue samples (n= 72) were collected from 24 hinds and their fetuses for histopathological examination. RESULTS: A total of 24/35 hinds selected were suitable for inclusion in the study. Eighteen of these pregnant hinds were culture-positive for M. ptb, and 14 of these had culture-positive fetuses, representing a transmission rate of 78% (95% confidence interval (CI) = 0.58-0.98) from dam to fetus. Of the 16 mammary glands sampled, 11 (69%) were culture-positive for M. ptb while 12/15 (80%) mammary lymph nodes sampled were also culture-positive. CONCLUSIONS: This study demonstrated a high rate of transmission of M. ptb from dam to fetus in red deer, and a potential risk of transmission to fawns suckling from mothers that are subclinically affected with Johne's disease ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/Cervus elaphus/CERVUS-ELAPHUS/CULTURE/deer/DISEASE/enzyme-linked immunosorbent assay/FARMED DEER/FETUSES/INFECTED COWS/intra-uterine transmission/Intrauterine/Johne's disease/JOHNES-DISEASE/mammary gland/MILK/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/NEW-ZEALAND/paratuberculosis/RED DEER/SHEEP/SUBSP-PARATUBERCULOSIS/TISSUE/Transmission

31 Bannantine, J.P., Waters, W.R., Stabel, J.R., Palmer, M.V., Li, L.L., Kapur, V., Paustian, M.L. (2008) Development and use of a partial Mycobacterium avium subspecies paratuberculosis protein array Proteomics, 8, 463-474

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As an initial step toward systematically characterizing all antigenic proteins produced by a significant veterinary pathogen, 43 recombinant Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) expression clones were constructed, cataloged, and stored. NC filters were spotted with purified proteins from each clone along with a whole cell lysate of M. paratuberculosis. Spots on the resulting dot array consisted of hypothetical proteins (13), metabolic proteins (3), cell envelope proteins (7), known antigens (4), and unique proteins with no similarity in public sequence databases (16). Dot blot arrays were used to profile antibody responses in a rabbit and mouse exposed to M. paratuberculosis as well as in cattle showing clinical signs of Johne's disease. The M. paratuberculosis heat shock protein DnaK, encoded by ORF MAP3840 and a membrane protein (MAP2121c), were identified as the most strongly immunoreactive in both the mouse and rabbit hosts, respectively. MAP3155c, which encodes a hypothetical protein, was most strongly immunoreactive in sera from Johne's disease cattle. This study has enabled direct comparisons of antibody reactivity for an entire panel of over 40 proteins and has laid the foundation for future high throughput production and arraying of M. paratuberculosis surface proteins for immune profiling experiments in cattle ANTIBODIES/ANTIBODY-RESPONSES/ANTIGEN/antigens/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/CATTLE /CELL/deer/DIAGNOSIS/diagnostics/DISEASE/IDENTIFICATION/IMMUNE-RESPONSE/INFECTION/Johne's disease/JOHNES-DISEASE/LINKED-IMMUNOSORBENT-ASSAY/MAJOR ANTIGENS/MONOCLONAL-ANTIBODIES/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/rabbit

32 Wolf, K.N., DePerno, C.S., Jenks, J.A., Stoskopf, M.K., Kennedy-Stoskopf, S., Swanson, C.C., Brinkman, T.J., Osborn, R.G., Tardiff, J.A. (2008) Selenium status and antibodies to selected pathogens in white-tailed deer (Odocoileus virginianus) in southern Minnesota

Journal of Wildlife Diseases, 44, 181-187 To determine exposure to a variety of infectious diseases potentially important for native ungulates, livestock, and humans, serum samples from 114 (94 adults, 20 fawns) female white-tailed deer (Odocoileus virginianus) were collected during January 2000-03 from multiple locations in southeast (SE) and southwest (SW) Minnesota. Antibody prevalence was determined for the following pathogens: Mycobacterium avium subsp. paratuberculosis, Leptospira interrogans (six serovars), Anaplasma marginale, Borrelia burgdorferi, Brucella abortus, epizootic hemorrhagic disease virus, and bovine viral diarrhea virus (BVDV) types 1 and 2. Samples collected in 2001 were screened for antibodies against Anaplasma phagocytophilum, and whole blood was submitted for polymerase chain reaction (PCR) testing for A. phagocytophilumn and B. burgdorferi. In addition, serum selenium concentrations were evaluated for samples collected during 2001-03. Antibody prevalence and selenium concentration were compared by age-class and geographic region. Antibodies to all of the infectious agents except A. marginale and B. abortus were detected; when detected, antibody prevalence was highest in adults. Deer collected from SE Minnesota had a higher antibody prevalence to B. burgdorferi! than SW deer. Blood culture and PCR results for A. phagocytophilum and B. burgdorferi! were negative. Antibodies against BVDV (combined 2 types I and 2) were more prevalent (chi(2) =3.617, P <= 0.029) in deer collected in SW (41%) than in SE (25%) Minnesota. No statistically significant differences in serum selenium concentrations were detected when data were analyzed by age-class or by geographic location ANTIBODIES/AVIUM/BOVINE/Bovine viral diarrhea/BVDV/CULTURE/deer/Diarrhea/DISEASE/ DISEASES/INFECTION/infectious disease/LEPTOSPIRA/Minnesota/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/Odocoileus virginianus/ODOCOILEUS-VIRGINIANUS/paratuberculosis/PCR/POLYMERASE-CHAIN-REACTION/PREVALENCE/selenium/Serology/Ungulate/VIRAL DIARRHEA VIRUS/white-tailed deer

33 Bannantine, J.P., Paustian, M.L., Waters, W.R., Stabel, J.R., Palmer, M.V., Li, L.L., Kapur, V. (2008) Profiling bovine antibody responses to Mycobacterium avium subsp paratuberculosis infection by using protein arrays

Infection and Immunity, 76, 739-749 With the genome sequence of Mycobacterium avium subsp. paratuberculosis determined, technologies are now being developed for construction of protein arrays to detect the presence of antibodies against H. avium subsp. paratuberculosis in host serum. The power of this approach is that it enables a direct comparison of M. avium subsp. paratuberculosis proteins to each other in relation to their immunostimulatory capabilities. In this study, 93 recombinant proteins, produced in Escherichia coli, were arrayed and spotted onto nitrocellulose. These proteins include unknown hypothetical proteins and cell surface proteins as well as proteins encoded by large sequence polymorphisms present uniquely in M. avium subsp. paratuberculosis. Also included were previously reported or known M. avium subsp. paratuberculosis antigens to serve as a frame of reference. Sera from healthy control cattle (n = 3) and

15

cattle infected with either M. avium subsp. avium and Mycobacterium bovis were exposed to the array to identify nonspecific or cross-reactive epitopes. These data demonstrated a degree of cross-reactivity with the M. avium subsp. avium proteins that was higher than the degree of cross-reactivity with the more distantly related M. bovis proteins. Finally, sera from naturally infected cattle (n = 3) as well as cattle experimentally infected with M. avium subsp. paratuberculosis (n = 3) were used to probe the array to identify antigens in the context of Johne's disease. Three membrane proteins were the most strongly detected in all serum samples, and they included an invasion protein, an ABC peptide transport permease, and a putative GTPase protein. This powerful combination of genomic information, molecular tools, and immunological assays has enabled the identification of previously unknown antigens of M. avium subsp. paratuberculosis ANTIBODIES/ANTIBODY-RESPONSES/ANTIGEN/ANTIGEN DISCOVERY/antigens/ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/BOVIS/CATTLE/CELL/CELLULAR IMMUNE-RESPONSES/COMPLETE GENOME SEQUENCE/deer/DIAGNOSIS/DISEASE/ESCHERICHIA-COLI/EXPORTED PROTEIN/IDENTIFICATION/INFECTION/Johne's disease/JOHNES-DISEASE/LINKED-IMMUNOSORBENT-ASSAY/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium bovis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-BOVIS/paratuberculosis/SHEEP/SUBSP-PARATUBERCULOSIS/Tuberculosis

34 Ghalmi, F., China, B., Losson, B. (2007) Diagnosis and epidemiological surveillance of Neospora caninum

Annales de Medecine Veterinaire, 151, 123-149 Neospora caninum is a pathogenic parasite responsible for diseases mainly in cattle and dog but other animals could be infected ( small ruminants, equids, wild ruminants). In cow, the most frequent clinical manifestation is abortion. The main diagnosis methods are : serology detection of the parasite in infected tissues by immunohistochemisty and the observation of lesions in infected tissues. The polymerase chain reaction can be applied on different tissues types. Statistical methods allow the determination of the more powerful method. These serological techniques were used to determine the seroprevalence in animals. Studies indicated that the seroprevalence is higher in farm dogs compared to urban dogs or in aborting cows compared to non-aborting cows. The isolation of the parasite is usually performed from the brain of aborted bovine foetus. It is clear that the improvement of diagnosis methods will allow a better understanding of the parasite transmission ways and a better control of this recently described pathogen AVIUM SUBSPECIES PARATUBERCULOSIS/BOVINE/BOVINE ABORTED FETUSES/BUFFALOS BUBALUS-BUBALIS/CATTLE/DIAGNOSIS/DISEASE/DISEASES/FLUORESCENT-ANTIBODY TEST/FOXES VULPES-VULPES/LINKED-IMMUNOSORBENT-ASSAY/NEOSPORA-CANINUM/POLYMERASE-CHAIN-REACTION/RUMINANTS/Serology/TISSUE/TOXOPLASMA-GONDII ANTIBODIES/Transmission/VIRAL-DIARRHEA VIRUS/WILD RUMINANTS/WOLVES CHRYSOCYON-BRACHYURUS

35 Spraker, T.R. (2007) Diseases of importance of domestic ruminants and free-ranging North American cervids

Proceedings of the Fortieth Annual Conference American Association of Bovine Practitioners, 162-167 Numerous diseases of importance in domestic ruminants and free-ranging wildlife can be transmitted between each other. This section will be limited to diseases that are known to affect both wild and domestic animals. In most cases, the disease can be transmitted in both directions and have important impacts in both domestic and free-ranging animals. In some cases the disease is primarily in domestic animals and spills over into wildlife, and in others the disease is primarily in wildlife and spills over into domestic animals. Diseases covered in this presentation include tuberculosis, brucellosis, anthrax, paratuberculosis, malignant catarrhal fever, bluetongue/epizootic hemorrhage disease, elaeophoriasis and bovine virus diarrhea BOVINE/CERVIDS/Diarrhea/ DISEASE/DISEASES/DOMESTIC-ANIMALS/IMPACT/paratuberculosis/RUMINANTS/Tuberculosis/WILDLIFE

36 onso-Hearn, M., Patel, D., Danelishvili, L. , Meunier-Goddik, L., Bermudez, L.E. (2008) The Mycobacterium avium subsp paratuberculosis MAP3464 gene encodes an oxidoreductase involved in invasion of bovine epithelial cells through the activation of host cell Cdc42

Infection and Immunity, 76, 170-178 Mycobacterium avium subsp.paratuberculosis infection of cattle takes place through the intestinal mucosa. To identify M. avium subsp. paratuberculosis genes associated with the invasion of bovine

16

epithelial cells in vitro, we screened a library of transposon mutants. Several mutants of M. avium subsp. paratuberculosis were identified which invaded Madin-Darby bovine kidney (MDBK) epithelial cells less efficiently than wild-type (wt) M. avium subsp. paratuberculosis. The Delta Ox mutant had the transposon located in the MAP3464 gene, a putative oxidoreductase gene whose expression is upregulated upon bacterial contact with MDBK cells. Complete restoration of invasion comparable to that for the wt bacterium was achieved by introducing a copy of the complete oxidoreductase operon into the Delta Ox mutant. Immunoprecipitation and Western blot analysis indicated that wt M. avium subsp. paratuberculosis activates Cdc42 and RhoA pathways of internalization 15 and 60 min after infection of the host cell, respectively. The Delta Ox mutant, however, failed to activate the Cdc42 pathway. To determine whether an M. avium subsp. paratuberculosis protein delivered to the host cell mediates the entry of the wt bacterium by activation of the Cdc42 pathway, affinity precipitation of active Cdc42 from MDBK-infected cells followed by mass spectrometry was carried out. We identified a 17-amino-acid bacterial peptide associated with the Cdc42 of cells infected with wt M. avium subsp. paratuberculosis but not with the Delta Ox mutant. The sequence of the peptide matches MAP3985c, a hypothetical protein, possibly functioning as a putative Cdc42 effector. These findings reveal a novel signaling pathway activated during M. avium subsp. paratuberculosis entry that links the product of MAP3464 gene to activation of Cdc42 in the host cell ABILITY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIUM/BOVINE/CATTLE/CELL/DSBA/EXPRESSION/FAMILY/IDENTIFICATION/INFECTION/JOHNES-DISEASE/MEMBRANE/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis /PROTEINS/SUBSP-PARATUBERCULOSIS/VIBRIO-CHOLERAE/VIRULENCE

37 Palmer, M.V., Stabel, J.R., Waters, W.R., Bannantine, J.P., Miller, J.M. (2007) Experimental infection of white-tailed deer (Odocoileus virginianus) with Mycobacterium avium subsp paratuberculosis

Journal of Wildlife Diseases, 43, 597-608 Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of paratuberculosis or Johne's disease, a chronic enteric disease of domestic ruminants as well as some nondomestic ruminants. Paratuberculosis is characterized by a protracted subclinical phase followed by clinical signs such as diarrhea, weight loss, and hypoproteinemia. Fecal shedding of Map is characteristic of both the subclinical and clinical phases, and it is important in disease transmission. Lesions of paratuberculosis are characterized by chronic granulomatous enteritis and mesenteric lymphadenitis. Animal models of paratuberculosis that simulate all aspects of the disease are rare. Oral inoculation of 9-day-old white-tailed deer (Odocoileus virginianus) on 3 June 2002 with 1.87x 10(10) colony-forming units of Map strain K10 resulted in clinical disease (soft to diarrheic feces) as early as 146 days after inoculation; lesions consistent with paratuberculosis were observed in animals at the termination of the study. Intermittent fecal shedding of Map was seen between 28 and 595 days (4 March 2004) after inoculation. These findings suggest that experimental oral inoculation of white-tailed deer fawns may mimic all aspects of subclinical and clinical paratuberculosis AMERICAN WILD RUMINANTS/animal model/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CALVES/CATTLE/CULTURE/deer/Diarrhea/DISEASE/DOMESTIC SHEEP/ENTERITIS/experimental infection/EXPERIMENTAL-INFECTION/FECES/IMMUNE-RESPONSES/INFECTION/Jobne's disease/Johne's disease/JOHNES-DISEASE/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium avium subsp./MYCOBACTERIUM-AVIUM/Odocoileus virginianus/ODOCOILEUS-VIRGINIANUS/ORAL INOCULATION/PARA-TUBERCULOSIS/paratuberculosis/PATHOLOGY/RUMINANTS/SUBSP-PARATUBERCULOSIS/Transmission /white-tailed deer

38 Uzoigwe, J.C., Khaitsa, M.L., Gibbs, P.S. (2007) Epidemiological evidence for Mycobacterium avium subspecies paratuberculosis as a cause of Crohn's disease

Epidemiology and Infection, 135, 1057-1068 Mycobacterium avium subspecies paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants including cattle, sheep, goats, and farmed deer. Recently, this bacterium has received an increasingly wide interest because of a rapidly growing body of scientific evidence which suggests that human infection with this microorganism may be causing some, and possibly all, cases of Crohn's disease. Recent studies have shown that a high percentage of people with Crohn's disease are infected with M. avium subsp. paratuberculosis; whether the association of this bacterium and Crohn's disease is causal or coincidental is not known. Crohn's disease is a gastrointestinal disease in humans with similar histopathological findings to those observed in the paucibacillary form of Johne's disease in cattle. The search for risk factors in Crohn's disease has been frustrating. However, epidemiologists have gathered enough information that points to an association between M. avium subsp.

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paratuberculosis and Crohn's disease. This paper reviews epidemiological models of disease causation, the major philosophical doctrines about causation, the established epidemiological criteria for causation, and the currently known epidemiological evidence of M. avium subsp. paratuberculosis as a possible cause of Crohn's disease AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BACTERIUM/BIOPSY SPECIMENS/CATTLE/Crohn's disease/DAIRY-CATTLE/deer/DISEASE/ENTERITIS/ENVIRONMENTAL RISK-FACTORS/FARMED DEER/IN-SITU HYBRIDIZATION/INFECTION/INFLAMMATORY-BOWEL-DISEASE/Johne's disease/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PASTEURIZED COWS MILK/POLYMERASE CHAIN-REACTION/Review/RUMINANT PARA-TUBERCULOSIS/RUMINANTS/SHEEP/SUBCLINICAL JOHNES-DISEASE/ULCERATIVE-COLITIS

39 van Kooten, H.C.J., Mackintosh, C.G., Koets, A.P. (2006) Intra-uterine transmission of paratuberculosis (Johne's disease) in farmed red deer

New Zealand Veterinary Journal, 54, 16-20 Aim: To determine whether intra-uterine transmission of paratuberculosis (Johne's disease) occurs in farmed red deer (Cervus elaphus) in New Zealand. Methods: On four different farms, nine late-stage pregnant hinds with Johne's disease were slaughtered and samples were taken from them and their 10 fetuses. Samples of the hepatic, ileocaecal and mesenteric lymph nodes and the posterior ileum were collected from the hinds. The lung, liver, spleen, jejunum and ileum from the fetuses were sampled, as were the placentomes. Blood samples were tested using the 'Paralisa' test, a modified immunoglobulin G(1) (IgG(1)) enzyme-linked immuno-sorbent assay (ELISA). Tissue samples were cultured using the BACTEC system, and fixed samples were sectioned and histological slides examined. Results: All nine hinds and 9/10 fetuses (one hind had twins) were culture-positive for Mycobacterium avium subsp paratuberculosis (M. ptb). Six hinds had gross lesions of Johne's disease, while all hinds had characteristic histopathological lesions affecting the ileum, ileocaecal valve and associated lymph nodes. The only histopathological change observed in the fetuses was some mild inflammation in the lungs of one individual. Acid fast organisms (AFOs) were seen in histological sections of the lymph nodes and ileum of six hinds, and none were seen in tissues from the fetuses. These six hinds were Paralisa-positive, whereas the remaining hinds and fetuses were serologically-negative. Conclusions: These results confirm that there is a high risk of transmission of M. ptb from clinically affected hinds to their fetuses during pregnancy. Clinical Relevance: Johne's disease is an increasingly important disease responsible for deaths in young red deer. Recognising the influence of intra-uterine transmission on the spread of this disease may be an important step towards improved control of Johne's disease ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/Cervus elaphus/CERVUS-ELAPHUS/CULTURE/deer/DISEASE/FARMED RED DEER/FETUSES/intra-uterine transmission/Intrauterine/Johne's disease/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/NEW-ZEALAND/ORGANISM/paratuberculosis/ RED DEER/SUBSP-PARATUBERCULOSIS/TISSUE/Transmission

40 de Lisle, G.W., Cannon, M.C., Yates, G.F., Collins, D.M. (2006) Use of a polymerase chain reaction to subtype Mycobacterium avium subspecies paratuberculosis, an increasingly important pathogen from farmed deer in New Zealand

New Zealand Veterinary Journal, 54, 195-197 Aims: To review the number of microbiologically-confirmed cases of Johne's disease in farmed deer since 2000, and determine the prevalence of the bovine and ovine subtypes of Mycobacterium avium subsp paratuberculosis (M. paratuberculosis), using a highly specific polymerase chain reaction (PCR) test on samples from infected herds. Methods: The number of cases of M. paratuberculosis in farmed deer identified by culture or IS900 PCR was documented. A highly specific PCR test was applied to subtype M. paratuberculosis from BACTEC 12B cultures selected on the basis of one culture per deer herd, to give a wide coverage of herds in New Zealand. Results: From January 2001 to October 2005, M. paratuberculosis was isolated from 1,141 farmed deer, and has now been identified by microbiological testing in over 600 deer herds in New Zealand. The bovine subtype of M. paratuberculosis was shown by a highly specific PCR test to be present in 91/95 herds examined; the ovine subtype was found in the remaining four herds. Conclusions: Since 2000, there has been a substantial increase in both the number of microbiologically-confirmed cases of Johne's disease in farmed deer and the number of infected herds. Johne's disease is now widespread and common in deer herds throughout New Zealand. Whilst the bovine subtype of M. paratuberculosis predominates in deer herds in New Zealand in which Johne's disease has been confirmed, the occasional finding of the ovine subtype highlights the need to consider both sheep and cattle as potential sources of infection for farmed deer

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AVIUM/AVIUM SUBSP PARATUBERCULOSIS/AVIUM SUBSPECIES PARATUBERCULOSIS/BOVINE/CATTLE/CULTURE/deer/DISEASE/FARMED DEER/IDENTIFICATION/INFECTION /IS900/Johne's disease/ Mycobacterium avium/Mycobacterium avium subsp /Mycobacterium avium subsp paratuberculosis/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/NEW-ZEALAND/OVINE/paratuberculosis/PCR/POLYMERASE-CHAIN-REACTION/PREVALENCE/RESTRICTION ENDONUCLEASE ANALYSIS/Review/SHEEP/STRAINS/SUBSP-PARATUBERCULOSIS

41 Mackintosh, C.G., Labes, R.E., Clark, R.G., de Lisle, G.D., Griffin, J.F.T. (2007) Experimental infections in young red deer (Cervus elaphus) with a bovine and an ovine strain of Mycobacterium avium subsp paratuberculosis

New Zealand Veterinary Journal, 55, 23-29 AIMS: To compare the virulence of a 'bovine' and an 'ovine' strain of Mycobacterium avium subsp paratuberculosis ( M. ptb) in red deer (Cervus elaphus) after experimental inoculation orally, and to examine the relationship between the dose of the bovine strain given and immunological, clinical and histopathological outcomes in young red deer. METHODS: Newly-weaned 4-month-old male red deer (n=81) were randomly assigned to one of five groups. Three groups (n=16) received high ( 109 colony forming units (cfu); HB), medium ( 107 cfu; MB) or low ( 103 cfu; LB) oral doses of a bovine strain of M. ptb, one group (n=16) received medium ( 107 cfu; MO) doses of an ovine strain of M. ptb, and a Control group (n=17) was not dosed. The HB and Control groups were grazed together, the MB and LB groups were grazed together, and the MO group was grazed alone, in separate small paddocks on a quarantined area of the farm for 45 weeks. Liveweight, clinical signs and immunoglobulin G1 (IgG1) antibody levels were monitored for up to 45 weeks. Deer affected with Johne's disease were euthanised when they showed obvious clinical signs. Unaffected deer were slaughtered at the end of the trial ( Week 45), and all deer were necropsied. Faeces and tissue samples were cultured for M. ptb, and fixed tissues were examined for histopathology. RESULTS: Between 21 and 38 weeks post-challenge ( pc), 5/16 animals in the HB group developed early signs of Johne's disease and were euthanised. The remaining deer in the five groups were all apparently healthy and reached good liveweights (similar to 100 kg average), and were euthanised and examined 45 weeks pc. Three deer ( two HB and one MB) had small caseous lesions in their jejunal lymph nodes (JJLNs) and one HB animal had a small caseous lesion in a retropharyngeal lymph node. The remaining animals had no grossly-visible lesions. Mycobacterium avium subsp paratuberculosis was cultured from samples from 100% of the HB and MB animals, 50% of the LB group, 69% of the MO group and all Control animals. Thus all Control deer were infected by natural transmission from the HB group but none developed signs of clinical disease. Examination of histological sections of jejunum, ileocaecal valve (ICV) and associated lymph nodes showed a gradation of severity of lesions that was positively correlated (p < 0.001) with dose of the bovine strain administered; mean lesion severity scores were 4.8, 2.9 and 0.9 for HB, MB and LB groups, and 2.2 and 0.9 for the Control and MO groups, respectively. IgG1 antibody levels at the time of euthanasia were also correlated with lesion severity scores at slaughter (p < 0.001). CONCLUSIONS: The ovine strain of M. ptb used in this study was less virulent for red deer than the bovine strain. The correlation between dose of the bovine strain and the severity of lesions suggests that clinical Johne's disease in yearling red deer likely results from high oral challenge with a bovine strain whilst they are young. The minimum oral infective dose may be close to 10(3) cfu for this bovine strain ANTIBODIES/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/bovine and ovine strains/CATTLE/Cervus elaphus/CERVUS-ELAPHUS/deer/DIAGNOSIS/DISEASE/dose response/experimental infection/EXPERIMENTAL-INFECTION/FARMED DEER/FECAL CULTURE/INFECTION /INFECTIONS/Johne's disease/JOHNES-DISEASE/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/NEW-ZEALAND/ORAL INOCULATION/OVINE/paratuberculosis/RED DEER/ Serology/SHEEP/SUBSP-PARATUBERCULOSIS/TISSUE/Transmission/Tuberculosis/VIRULENCE

42 Mainar-Jaime, R.C., Woodbury, M.R., Chirino-Trejo, M. (2007) Survey on 'lumpy jaw' on deer farms in western Canada: Prevalence and distribution, and identification of associated factors

New Zealand Veterinary Journal, 55, 30-39 AIM: To investigate the prevalence and geographical distribution of 'lumpy jaw' (LJ) in a population of white-tailed deer (WTD; Odocoileus virginianus) and mule deer ( MD; Odocoileus hemionus) farms from the western Canadian provinces of Saskatchewan and Alberta, and to identify factors associated with its occurrence. METHODS: A cross-sectional study, in which the target population was all farmers of WTD and MD registered in Saskatchewan and Alberta, was conducted between July 2004 and January 2005. A questionnaire was mailed to all farmers requesting information about the presence of LJ and

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othernecrobacillosisrelated syndromes (footrot and fawn death syndrome), and various farm characteristics, during 2002, 2003 and 2004. Herd and within-herd incidences of disease were estimated. Global and local spatial analyses were performed to identify possible clusters of occurrence of LJ in the region. Logistic regression analysis was used to identify factors associated with the occurrence of LJ. RESULTS: A total of 139/268 (52%) deer farmers responded to the survey. Over the entire study period, 108/139 (78%) of farmers reported having cases of LJ in their herds, and in any given year the incidence amongst herds was about 40%. The presence of footrot was not associated with the presence of LJ. The proportion of fawns dying suddenly in 2004 was higher on farms affected by LJ than in those considered LJ-free (median of 11.1% and 0%, respectively; p < 0.001). Two areas in Saskatchewan were identified as having a higher herd prevalence of LJ (clusters) than all other areas. Density of animals, moving and handling animals, lack of basic hygiene measures, and bottle-feeding of fawns increased the odds of a herd being affected by LJ. CONCLUSIONS AND CLINICAL RELEVANCE: LJ should be considered a common disease in farmed deer in western Canada. The observed relationship between the occurrence of LJ and acute mortality of fawns emphasises the potential of this infection to result in significant economic loss. Intensive management of deer, characterised by high densities and frequent moving and handling of animals, may contribute significantly to the occurrence of LJ. Observed geographical clusters may reflect areas where management of deer was more intensive or the trading of deer more common bovine and ovine strains/deer/DISEASE/ dose response/FARMED DEER/FUSOBACTERIUM-NECROPHORUM/HERD PREVALENCE/ IDENTIFICATION/INFECTION/Johne's disease/MORTALITY/Mycobacterium paratuberculosis/NECROBACILLOSIS/Odocoileus virginianus/ODOCOILEUS-VIRGINIANUS/PREVALENCE/RED DEER/Serology/STRESS/white-tailed deer

43 Bohm, M., White, P.C.L., Chambers, J., Smith, L., Hutchings, M.R. (2007) Wild deer as a source of infection for livestock and humans in the UK

Veterinary Journal, 174, 260-276 Wild deer can feature in the epidemiology of a wide range of livestock and human diseases in the United Kingdom by representing a source of disease via various transmission routes. This review highlights current and possible future infections of deer in the UK which may have an impact on livestock and/or human health. Increases in deer abundance as well as range expansion are likely to exacerbate the potential for disease persistence due to the formation of multi-species deer assemblages, which may act as disease reservoirs. Climatic changes are likely to have a direct impact on the presence and abundance of various pathogens and their vectors, so that with a warming climate exotic diseases may play a role in future UK livestock and wildlife disease management. This paper highlights the need for a monitoring strategy for wildlife diseases, in particular infections in wild deer, in the UK. (c) 2006 Elsevier Ltd. All rights reserved AVIUM SUBSP PARATUBERCULOSIS/ climate change/deer/DISEASE/DISEASES/Epidemiology/IMPACT/INFECTION/INFECTIONS/infectious disease/MALIGNANT CATARRHAL FEVER/monitoring/MOUTH-DISEASE VIRUS/ODOCOILEUS-VIRGINIANUS-CLAVIUM/REINDEER RANGIFER-TARANDUS/Review/TICK-BORNE ENCEPHALITIS/TOXOPLASMA-GONDII ANTIBODIES/Transmission/WEST-NILE-VIRUS/white-tailed deer/WILDLIFE/YELLOWSTONE-NATIONAL-PARK

44 Palmer, M.V., Thacker, T.C., Waters, W.R. (2007) Vaccination of white-tailed deer (Odocoileus virginianus) with Mycobacterium bovis bacillus Calmette Guerin

Vaccine, 25, 6589 -6597 Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock. In Michigan, USA tuberculous white-tailed deer transmit M. bovis to cattle. One approach in dealing with this wildlife reservoir is to vaccinate deer in order to interrupt the cycle of deer to deer and deer to cattle transmission. Thirty-one white-tailed deer were assigned to one of three groups; 2 SC doses of 107 CFU of M. bovis BCG (n = 11); 1 SC dose of 107 CFU of M. bovis BCG (n = 10); or unvaccinated deer (n = 10). After vaccination, deer were inoculated intratonsilarly with 300 CFU of virulent M. bovis. Gross lesion severity scores of the medial retropharyngeal lymph node were significantly reduced in deer receiving 2 doses of BCG compared to unvaccinated deer. Vaccinated deer had fewer lymph node granulomas than unvaccinated deer, and most notably, fewer late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid-fast bacilli. BCG was isolated from 7/21 vaccinated deer as long as 249 days after vaccination. In one case BCG was transmitted from a vaccinated deer to an unvaccinated deer. In white-tailed deer BCG provides measurable protection against challenge with virulent M. bovis. However, persistence of vaccine within tissues as well as shedding of BCG from vaccinates remain areas for further investigation. Published by Elsevier Ltd

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AVIUM SUBSP PARATUBERCULOSIS/BCG/BCG VACCINATION/BOVIS/BRUSHTAIL POSSUMS/CATTLE/CROSS-REACTIVITY/deer/ENVIRONMENTAL MYCOBACTERIA/IMMUNE-RESPONSES/interferon-gamma/MICHIGAN/Mycobacterium bovis /MYCOBACTERIUM-BOVIS/NEONATAL CALVES/Odocoileus virginianus/ODOCOILEUS-VIRGINIANUS/ORAL VACCINATION/PULMONARY TUBERCULOSIS/TISSUE/Transmission/Tuberculosis/VACCINATION /white-tailed deer/WILDLIFE

45 Anderson, J.L., Meece, J.K., Koziczkowski, J.J., Clark, D.L., Radcliff, R.P. , Nolden, C.A., Samuel, M.D., Ellingson, J.L.E. (2007) Mycobacterium avium subsp paratuberculosis in scavenging mammals in Wisconsin

Journal of Wildlife Diseases, 43, 302-308 The presence of Mycobacterium avium subsp. paratuberculosis (MAP) in non-ruminant wildlife has raised questions regarding the role of these species in Johne's disease transmission. In this study we tested 472 tissues from 212 animals of six different species of scavenging mammals. All animals were taken from within a 210-square-mile area in Dane and Iowa counties of south central Wisconsin from September to May in 2003-04 and tested for the presence of MAP. We detected MAP-specific DNA in 81 of 212 (38%) scavenging mammals, in 98 of the 472 (21%) tissues; viable MAP was cultured from one coyote's ileum and lymph node tissue. Despite the low numbers of viable MAP isolated in this study, our data adds to the increasing evidence demonstrating the potential for transmission and infection of MAP in nonruminant species and provides possible evidence of interspecies transmission. The apparently high exposure of nonruminant wildlife provides potential evidence of a spillover of MAP to wildlife species and raises the question of spillback to domestic and wild ruminants. These results demonstrate the importance of understanding the role of wildlife species in developing management strategies for Johne's disease in domestic livestock AVIUM/AVIUM SUBSP PARATUBERCULOSIS/DISEASE/DNA/INFECTION/Johne's disease/JOHNES-DISEASE/MAMMALS/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/paratuberculosis/RUMINANTS/scavenging mammals/SCOTLAND/SHEEP/SUBSP-PARATUBERCULOSIS/TISSUE/Transmission/WILD RUMINANTS/WILDLIFE/Wisconsin

46 Hines, M.E. , Stabel, J.R., Sweeney, R.W., Griffin, F., Talaat, A.M., Bakker, D., Benedictus, G., Davis, W.C., de Lisle, G.W., Gardner, I.A., Juste, R.A., Kapur, V., Koets, A., McNair, J., Pruitt, G., Whitlock, R.H. (2007) Experimental challenge models for Johne's disease: A review and proposed international guidelines

Veterinary Microbiology, 122, 197-222 An international committee of Johne's disease (JD) researchers was convened to develop guidelines for JD challenge studies in multiple animal species. The intent was to develop and propose international standard guidelines for models based on animal species that would gain acceptance worldwide. Parameters essential for the development of long-term and short-term infection models were outlined and harmonized to provide a "best.fit" JD challenge model for cattle, goats, sheep, cervids, and mice. These models will be useful to study host-pathogen interactions, host immunity at the local and systemic level, and for evaluating vaccine candidates and therapeutics. The consensus guidelines herein list by animal species strains of Mycobacterium avium subsp. paratuberculosis used, challenge dose, dose frequency, age of challenge, route of challenge, preparation of inoculum, experimental animal selection, quality control, minimal experimental endpoints and other parameters. (c) 2007 Elsevier B.V. All rights reserved AMERICAN WILD RUMINANTS/animal models/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/CERVIDS/challenge/DEER CERVUS-ELAPHUS/DISEASE/experimental infection/EXPERIMENTAL ORAL INFECTION/EXPERIMENTAL PARA-TUBERCULOSIS/EXPERIMENTAL-INFECTION MODEL/INFECTION/Johne's disease/LINKED-IMMUNOSORBENT-ASSAY/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/Review/SHEEP/SMALL-INTESTINAL MUCOSA/STRAINS/SWISS WHITE MICE

47 Cirone, K.M., Morsella, C.G., Colombo, D. , Paolicchi, F.A. (2006) Viability of Mycobacterium avium subsp paratuberculosis in elaborated goat and bovine milk cheese maturity

Acta Bioquimica Clinica Latinoamericana, 40, 507-513 Mycobacterium avium subsp. paratuberculosis (Map) is the etiologic agent of Paratuberculosis or Johne's disease. It is a chronic enteritis affecting ruminants, domestic and wild species. Goats and cows

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infected with Map can excret mycobacteria in their milk. This fact represents a potential risk when people consume raw milk or non-pasteurized cheese. The objective of this work was to determine the viability of Map in cheese elaborated with experimentally infected caprine and bovine milk with different thermal treatments (65 T or 37 T). During maturity, Map was isolated in cheese with only treatment of milk with 37 T, keeping its viability until days 60 and 45 days in caprine and bovine cheese respectively. In caprine cheese the highest pH value was found between days 30 and 45 of maturity in agreement with the biggest number of Map's isolates, The maturity decreases the Map viability in elaborated cheese without thermal treatment, and the treatment with 65 degrees C of temperature plus cheese maturity process would be beneficial for the mycobacteria elimination in these products ARGENTINA/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/DISEASE/ENTERITIS/HEAT/INACTIVATION/Johne's disease/maturity/MILK/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PASTEURIZED COWS MILK/PCR/RUMINANTS/SUBSP-PARATUBERCULOSIS/TEMPERATURES/viability in cheese

48 Cho, D., Shin, S.J., Talaat, A.M., Collins, M.T. (2007) Cloning, expression, purification and serodiagnostic evaluation of fourteen Mycobacterium paratuberculosis proteins

Protein Expression and Purification, 53, 411-420 Fourteen proteins of potential diagnostic value for bovine paratuberculosis were identified in the culture filtrate of Mycobacterium paratuberculosis JTC303 by immunoblot and mass spectrometry. The goals of the present study were to express these 14 ORFs in Escherichia coli and evaluate their antigenicity. All 14 proteins were expressed in E. coli BL21(DE3) after transformation with the pET-22b(+) vector. Yields of insoluble proteins were higher than those of the soluble proteins. Polyclonal rabbit antibodies directed against culture filtrate of JTC303 strain confirmed that five of the expressed and purified proteins are culture filtrate components: ModD, Antigen 85C, PepA, MAP1693c, and MAP2168c. Evaluation of ModD as an ELISA solid-phase antigen on a set of bovine sera from well-characterized paratuberculosis cases and infection-free controls revealed that there was strong serum antibody reactivity to rModD in many infected cattle. However, the overall rModD ELISA sensitivity and specificity for bovine paratuberculosis was not greater than those of ELISAs using crude antigens such as cellular extract or culture filtrate for plate coating, as judged by area under the curve (AUC) of Receiver-operating curve (ROC) analysis. However, an ELISA using natural ModD as the solid-phase antigen had a higher sensitivity and AUC than did rModD suggesting diminution of antigenicity in rModD. Taken together, our results showed that the natural forms of the identified proteins may be useful for diagnosis of bovine paratuberculosis. (C) 2007 Elsevier Inc. All rights reserved 45/47-KILODALTON ANTIGEN COMPLEX/ANTIBODIES /ANTIGEN/antigens/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/BOVINE PARATUBERCULOSIS/BOVINE-PARATUBERCULOSIS/CATTLE/CULTURE/CULTURE FILTRATE/DIAGNOSIS/ELISA/ESCHERICHIA-COLI/EXPRESSION/FAP/FIBRONECTIN-ATTACHMENT PROTEIN /Johne's disease/JOHNES-DISEASE/LINKED-IMMUNOSORBENT-ASSAY/MAP/ModD/Mycobacterium avium subspecies paratuberculosis/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/PROTEINS/rabbit/RECOMBINANT PROTEINS/SENSITIVITY/serodiagnosis/SPECIFICITY/Tuberculosis

49 Wu, C.W., Livesey, M., Schmoller, S.K., Manning, E.J.B., Steinberg, H., Davis, W.C., Hamilton, M.J., Talaat, A.M. (2007) Invasion and persistence of Mycobacterium avium subsp paratuberculosis during early stages of Johne's disease in calves

Infection and Immunity, 75, 2110-2119 Infection with Mycobacterium avium subsp. paratuberculosis causes Johne's disease in cattle and is a serious problem for the dairy industry worldwide. Development of models to mimic aspects of Johne's disease remains an elusive goal because of the chronic nature of the disease. In this report, we describe a surgical approach employed to characterize the very early stages of infection of calves with M. avium subsp. paratuberculosis. To our surprise, strains of M. avium subsp. paratuberculosis were able to traverse the intestinal tissues within 1 h of infection in order to colonize distant organs, such as the liver and lymph nodes. Both the ileum and the mesenteric lymph nodes were persistently infected for months following intestinal deposition of M. avium subsp. paratuberculosis despite a lack of fecal shedding of mycobacteria. During the first 9 months of infection, humoral immune responses were not detected. Nonetheless, using flow cytometric analysis, we detected a significant change in the cells participating in the inflammatory responses of infected calves compared to cells in a control animal. Additionally, the levels of cytokines detected in both the ileum and the lymph nodes indicated that there were TH1-type-associated cellular responses but not TH2-type-associated humoral responses. Finally, surgical inoculation of a wild-type strain and a mutant M. avium subsp. paratuberculosis strain (with an

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inactivated gcpE gene) demonstrated the ability of the model which we developed to differentiate between the wild-type strain and a mutant strain of M. avium subsp. paratuberculosis deficient in tissue colonization and invasion. Overall, novel insights into the early stages of Johne's disease were obtained, and a practical model of mycobacterial invasiveness was developed. A similar approach can be used for other enteric bacteria ABILITY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIUM/BLOOD MONONUCLEAR-CELLS/CALVES/CATTLE/CELL/DAIRY HERDS/ DISEASE/EXPERIMENTAL-INFECTION/GENE-EXPRESSION/humoral response/IMMUNE-RESPONSE/IMMUNE-RESPONSES/INFECTION/Johne's disease/MODEL/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PATHOGENESIS/REAL-TIME/STRAINS/SUBCLINICAL PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/TISSUE

50 Dubey, J.P. , Schares, G., Ortega-Mora, L.M. (2007) Epidemiology and control of neosporosis and Neospora caninum

Clinical Microbiology Reviews, 20, 323-+ Neospora caninum is a protozoan parasite of animals. Until 1988, it was misidentified as Toxoplasma gondii. Since its first recognition in dogs in 1984 and the description of the new genus and species Neospora caninum in 1988, neosporosis has emerged as a serious disease of cattle and dogs worldwide. Abortions and neonatal mortality are a major problem in livestock operations, and neosporosis is a major cause of abortion in cattle. Although antibodies to N. caninum have been reported, the parasite has not been detected in human tissues. Thus, the zoonotic potential is uncertain. This review is focused mainly on the epidemioloy and control of neosporosis in cattle, but worldwide seroprevalences of N. caninum in animals and humans are tabulated. The role of wildlife in the life cycle of N. caninum and strategies for the control of neosporosis in cattle are discussed ANTIBODIES/AVIUM SUBSPECIES PARATUBERCULOSIS/BOVINE-LEUKEMIA-VIRUS/BUFFALOS BUBALUS-BUBALIS/CATTLE/DISEASE/Epidemiology/FOXES VULPES-VULPES/IN-VITRO ISOLATION/MORTALITY/NATURALLY INFECTED-DOG/NEOSPORA-CANINUM/POINT-SOURCE EXPOSURE/Review/TISSUE/TOXOPLASMA-GONDII ANTIBODIES/VIRAL-DIARRHEA VIRUS/white-tailed deer/WILDLIFE

51 Skoric, M., Shitaye, E.J., Halouzka, R., Fictum, P., Trcka, I., Heroldova, M., Tkadlec, E., Pavlik, I. (2007) Tuberculous and tuberculoid lesions in free living small terrestrial mammals and the risk of infection to humans and animals: a review

Veterinarni Medicina, 52, 144-161 The present study describes pathogenesis and morphology of tuberculous and tuberculoid lesions in small terrestrial mammals, above all, in rodents. The most serious infectious agents that cause tuberculous and tuberculoid lesions in these animals are also cited. Besides others, the diseases caused by pathogenic mycobacteria that are members of Mycobacterium tuberculosis and M. avium complexes, M. lepraemurium, tularaemia, brucellosis and salmonellosis are included in the present study ACID-FAST BACILLUS/Avian tuberculosis/AVIUM/BOAR SUS-SCROFA/BOVINE TUBERCULOSIS/CENTRAL-EUROPEAN COUNTRIES/COMPLEX/DISEASE/DISEASES/GOLDEN-HAMSTER/GRANULOMA-FORMATION/INFECTION/MAMMALS/MICROTI LLAMA-TYPE/mycobacteriosis/MYCOBACTERIUM-AVIUM COMPLEX/paratuberculosis/PATHOGENESIS/PULMONARY TUBERCULOSIS/Review/terrestrial mammals/TUBERCLE-BACILLI/Tuberculosis/VOLE BACILLUS/zoonoses

52 Leroy, B., Roupie, V., Noel-Georis, I., Rosseels, V., Walravens, K., Govaerts, M., Huygen, K., Wattiez, R. (2007) Antigen discovery: A postgenomic approach to paratuberculosis diagnosis Proteomics, 7, 1164-1176 Paratuberculosis is a chronic enteritis caused in domestic and wild ruminant species by Mycobacterium avium subsp. paratuberculosis (MAP) that is responsible for major economic losses to the agricultural industry. To date, no satisfactory therapeutic, vaccine, or diagnostic tools are available, globally impairing all control programs. In this study, we have undertaken a large-scale postgenomic analysis of MAP proteins, to identify specific antigens that colad potentially improve the diagnosis of paratuberculosis. Two complementary approaches; were implemented, the first one consisting in the systematic proteomic identification of proteins present in MAP culture filtrates (CFs), followed by the selection of MAP-specific proteins by BLAST query on available mycobacterial genomes. The resulting database represents the first established secretome of MAP and a useful source of potentially specific antigens. The second approach consisted in the immunoproteornic analysis of both MAP extracts and CFs, using sera from MAP-infected and uninfected cattle. Combining results obtained with both

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approaches resulted in the identification of 25 candidate diagnostic antigens. Five of these were tested in an ELISA assay for their diagnostic potential, on a limited panel of field sera, and the combination of three of them competed in performance with available commercial assays, reaching a test sensitivity of 94.74% and specificity of 97.92% ANTIGEN/ANTIGEN DISCOVERY/antigens/ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE-PARATUBERCULOSIS/BRONCHOALVEOLAR LAVAGE FLUID/CATTLE/CROHNS-DISEASE/CULTURE /CULTURE FILTRATE/DIAGNOSIS/diagnostic antigens/ELISA/ENTERITIS/HEAT-SHOCK-PROTEIN/IDENTIFICATION/immunoproteomics/JOHNES-DISEASE/LINKED-IMMUNOSORBENT-ASSAY/MAP/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/ PROTEINS/secretome/SENSITIVITY/SPECIFICITY

53 Sevilla, I. , Garrido, J.M., Geijo, M., Juste, R.A. (2007) Pulsed-field gel electrophoresis profile homogeneity of Mycobacterium avium subsp paratuberculosis isolates from cattle and heterogeneity of those from sheep and goats Bmc Microbiology, 7, Background: Mycobacterium avium subsp. paratuberculosis (Map) causes paratuberculosis in animals and is suspected of causing Crohn's Disease in humans. Characterization of strains led to classify paratuberculosis isolates in two main types, cattle type strains, found affecting all host species, and sheep type strains, reported affecting mainly sheep. In order to get a better understanding of the epidemiology of paratuberculosis a large set of Map isolates obtained from different species over the last 25 years have been characterized. Five-hundred and twenty isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar) and origins had been cultured and typed by IS1311 restriction-endonuclease-analysis. Two-hundred and sixty-nine isolates were further characterized by pulsed-field gel electrophoresis (PFGE) using SnaBI and SpeI endonucleases. Differences in strain isolation upon various media conditions were also studied. Results: All bovines, 4 and 26% of Spanish sheep and goats, respectively, and the deer and wild boar studied, carried IS1311-Cattle type strains. IS1311-Sheep type encompassed 96% and 74% of Spanish sheep and goats, and all three Portuguese sheep. Thirty-seven distinct multiplex PFGE profiles were found, giving 32 novel profiles. Profiles 2-1 and 1-1 accounted for the 85% of cattle isolates. Ten distinct profiles were detected in Spanish sheep, none of them with an incidence higher than 25%. Profile 16-11 (43%) and another three profiles were identified in Spanish caprine cultures. The hierarchical analysis, clustered all profiles found in cattle, "wild" hosts and some small ruminants within the same group. The other group included 11 profiles only found in Spanish sheep and goats, including Spanish pigmented profiles. Differences in growth requirements associated with isolate genotype were observed. Conclusion: Cattle in Spain are infected with cattle type strains, while sheep and goats are mainly infected with sheep type strains. Although 7H9 broth based culture media seem to broadly cover the growth requirements of most Map strains, the use of various solid media is recommended to reduce any recovery biases. High genetic homogeneity of isolates from cattle, and heterogeneity of those from sheep and goats have been detected AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/CATTLE/Crohn's disease/CULTURE/deer/DIAGNOSIS/DISEASE/DNA HYBRIDIZATION/Epidemiology/FRAGMENT-LENGTH-POLYMORPHISM/GENETIC DIVERSITY/IDENTIFICATION/INSERTION ELEMENT IS900/IS1311/MAP/MOLECULAR EPIDEMIOLOGY/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/RESTRICTION ENDONUCLEASE ANALYSIS/RESTRICTION-ENDONUCLEASE ANALYSIS/RUMINANTS/SHEEP/STRAINS/SUBSP-PARATUBERCULOSIS/WILD BOAR/WILD RUMINANTS

54 Manning, E.J.B., Cushing, H.F., Hietala, S., Wolf, C.B. (2007) Impact of Corynebacterium pseudotuberculosis infection on serologic surveillance for Johne's disease in goats Journal of Veterinary Diagnostic Investigation, 19, 187-190 False-positive results on serologic assays for Mycobacterium avium subsp. paratuberculosis (MAP) are believed to occur due to cross-reacting antibody produced by Corynebacterium pseudotuberculosis (C. pstb) infection in goats. This issue of compromised specificity was evaluated by testing 771 adult goats from 10 Midwestern goat herds in 2004. Assays for MAP infection included radiometric fecal culture and 2 enzyme-linked immunosorbent assays (ELISAs); ELISA-positive samples were tested by agar gel immunodiffusion (AGID). A synergistic hemolysin inhibition assay (SHI) was used to detect C. pstb antibody. Four infection status categories were evaluated. Category 1 goats (free of both MAP and C. pstb infection) tested negative on all MAP fecal cultures and SHI tests. Five of 181 goats were positive in both ELISAs, and 2 more were positive in ELISA-1 only. For Category 2 (MAP infected; no C. pstb infection), all animals were SHI negative. Six goats were fecal culture positive and strongly positive in both ELISAs; 2 more goats were positive only in ELISA-1. For Category 3 (C. pstb infected or vaccinated; no history of MAP infection), all fecal cultures were negative and 91% were SHI test-positive.

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In this population, only 2 goats were positive in both MAP ELISAs, while 84 additional goats were test-positive only on ELISA-1. In the absence of C. pstb infection, both ELISAs performed comparably, but when C. pstb infection was present the performance of ELISA-1 was significantly perturbed. Use of the ELISA-2 for goats is an effective and efficient method for Johne's disease surveillance in any goat herd ANTIBODIES/ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/caprine/caseous lymphadenitis/CULTURE/deer/DIAGNOSIS/DISEASE/ELISA/enzyme-linked immunosorbent assay/false positive /FECAL CULTURE/goat/IMPACT/INFECTION/Johne's disease/LINKED-IMMUNOSORBENT-ASSAY/MAP/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS INFECTION/paratuberculosis/SHEEP/SPECIFICITY/TESTS

55 Judge, J., Davidson, R.S., Marion, G., White, P.C.L., Hutchings, M.R. (2007) Persistence of Mycobacterium avium subspecies paratuberculosis in rabbits: the interplay between horizontal and vertical transmission

Journal of Applied Ecology, 44, 302-311 Paratuberculosis (Mycobacterium avium subspecies paratuberculosis; Map) is a widespread and difficult disease to control in livestock populations and also has possible links to Crohn's disease in humans. Rabbits (Oryctolagus cuniculus) have been identified recently as the key wildlife species in terms of paratuberculosis transmission to the wider host community. Here, we test the hypothesis that Map can persist in rabbit populations for extended periods of time in the absence of any external source of infection. A spatially explicit stochastic simulation model of a generic host-disease interaction was developed to quantify the interplay between vertical and horizontal routes of transmission needed for the persistence of Map in rabbit populations and to test the hypothesis. The model was parameterized based on empirical studies on rabbit population dynamics and on rabbit-to-rabbit routes of Map transmission. Predictions from the model suggest that any disease with susceptible-infected (SI) dynamics without disease-induced mortality can persist within a rabbit population in the absence of vertical transmission, providing the horizontal transmission coefficient, beta, is greater than approximately 0.012. The inclusion of any vertical transmission reduces the value of beta that is necessary for infection to persist. Paratuberculosis persists in rabbit populations at all values of the horizontal and vertical transmission parameters in the range estimated from the field data and in many cases at all values within 95% confidence intervals around this range. The persistence of Map infection in rabbit populations in the absence of external sources of infection suggests that they may act as a reservoir of infection for sympatric livestock. Synthesis and applications. Our findings, in combination with the ubiquitous distribution of rabbits in the United Kingdom and elsewhere, suggests that if Map becomes established in rabbit populations they are likely to provide widespread and persistent environmental distributions of infection and thus disease risk to livestock and potentially humans. Where local rabbit populations are infected they should be included in any future paratuberculosis control strategies. Because eradication of rabbits is often not a realistic option, control strategies should include reducing interspecific transmission risk from rabbits to livestock via the faecal-oral route AUSTRALIA/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BOVINE TUBERCULOSIS/Crohn's disease/DISEASE/epidemiological models/GENETIC-STRUCTURE/INFECTION/infection dynamics/Johne's disease/JOHNES-DISEASE/MAP/MODEL/MORTALITY/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/NEW-SOUTH-WALES/NEW-ZEALAND/ORYCTOLAGUS-CUNICULUS/paratuberculosis/POPULATION ECOLOGY/rabbit/RABBITS/Transmission/WILD RABBIT/ WILDLIFE/wildlife reservoir

56 Johansen, T.B., Olsen, I., Jensen, M.R., Dahle, U.R., Holstad, G., Djonne, B. (2007) New probes used for ISI2450 and ISI311 restriction fragment length polymorphism of Mycobacterium avium subsp avium and Mycobacterium avium subsp hominissuis isolates of human and animal origin in Norway Bmc Microbiology, 7, Background: Mycobacterium avium is an environmental mycobacterium that can be divided into the subspecies avium, hominissuis, paratuberculosis and silvaticum. Some M. avium subspecies are opportunistic pathogens for animals and humans. They are ubiquitous in nature and can be isolated from natural sources of water, soil, plants and bedding material. Isolates of M. avium originating from humans (n = 37), pigs (n = 51) and wild birds (n = 10) in Norway were examined by IS1245 and IS1311 RFLP using new and specific probes and for the presence of IS901 and ISMpa1 by PCR. Analysis and generation of a dendrogram were performed with the software BioNumerics. Results: IS1311 RFLP provided clear results that were easy to interpret, while IS1245 RFLP generated more complex patterns with a higher discriminatory power. The combination of the two methods gave additional discrimination between isolates. All avian isolates except one were M. avium subsp. avium with two copies of IS1311 and one copy of IS1245, while the isolates of human and porcine origin belonged to M. avium subsp. hominissuis. The isolates from human patients were distributed randomly among the clusters of porcine isolates. There were few identical isolates. However, one isolate from a human patient was identical to a porcine isolate. Regional differences were detected among the porcine

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isolates, while there was no clustering of human isolates according to type of clinical symptoms or geographical location of the patient's home addresses. Conclusion: The results demonstrate that a wide range of M. avium subsp. hominissuis are present in pigs and humans in Norway, and that some of these isolates are very similar. It remains to be determined whether humans are infected from pigs or if they are infected from common environmental sources AVIUM/COMPLEX/ENVIRONMENTAL MYCOBACTERIA/Epidemiology/FRAGMENT-LENGTH-POLYMORPHISM/IDENTIFICATION/INFECTION/IS1245/IS1311/IS901/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/NONTUBERCULOUS MYCOBACTERIA/paratuberculosis/PCR/RESTRICTION/RFLP/STRAINS/WATER/WILD BIRDS

57 Sharma, S., Mallick, G.P., Verma, R., Ray, S.K. (2007) Polymerase chain reaction (PCR) amplification of IS6110 sequences to detect Mycobacterium tuberculosis complex from formalin-fixed paraffin-embedded tissues of deer (Axis axis)

Veterinary Research Communications, 31, 17-21 AMPLIFICATION/AVIUM SUBSP PARATUBERCULOSIS/BOVIS/CATTLE/COMPLEX/deer/ORGANISMS/PCR/POLYMERASE-CHAIN-REACTION/TISSUE/Tuberculosis/TUBERCULOSIS COMPLEX

58 Crawford, G.C., Ziccardi, M.H., Gonzales, B.J., Woods, L.M., Fischer, J.K., Manning, E.J.B., Mazet, J.A.K. (2006) Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subsp avium infections in a tule elk (Cervus elaphus nannodes) herd

Journal of Wildlife Diseases, 42, 715-723 Between 2 August and 22 September 2000, 37 hunter-killed tule elk (Cervus elaphus nannodes) were evaluated at the Grizzly Island Wildlife Area, California, USA, for evidence of paratuberculosis. Elk were examined post-mortem, and tissue and fecal samples were submitted for radiometric mycobacterial culture. Acid-fast isolates were identified by a multiplex polymerase chain reaction (PCR) that discriminates among members of the Mycobacterium avium complex (MAC). Histopathologic evaluations were completed, and animals were tested for antibodies using a Johne's enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion. In addition, 104 fecal samples from tule elk remaining in the herd were collected from the ground and submitted for radiometric mycobacterial culture. No gross lesions were detected in any of the hunter-killed animals. Mycobacterium avium subsp. paratuberculosis (MAP) was cultured once from ileocecal tissue of one adult elk and was determined to be a strain (A18) found commonly in infected cattle. One or more isolates of Mycobacterium avium subsp. avium (MAA) were isolated from tissues of five additional adult elk. Gastrointestinal tract and lymph node tissues from 17 of the 37 elk (46%) examined had histopathologic lesions commonly seen with mycobacterial infection; however, acid-fast bacteria were not observed. All MAC infections were detected from adult elk (P = 0.023). In adult elk, a statistically significant association was found between MAA infection and ELISA sample-to-positive ratio (S/P)>= 0.25 (P=0.021); four of five MAA culture-positive elk tested positive by ELISA. Sensitivity and specificity of ELISA S/P >= 0.25 for detection of MAA in adult elk were 50% and 93%, respectively. No significant associations were found between MAC infection and sex or histopathologic lesions. Bacteriologic culture confirmed infection with MAP and MAA in this asymptomatic tule elk herd. The Johne's ELISA was useful in signaling mycobacterial infection on a population basis but could not discriminate between MAA and MAP antibodies. The multiplex PCR was useful in discriminating among the closely related species belonging to MAC ANTIBODIES/ASSAY/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BACTERIUM/CATTLE/Ceruits elaphus nannodes/Cervus elaphus/CERVUS-ELAPHUS/COMPLEX/CULTURE/DEER ODOCOILEUS-VIRGINIANUS/DIAGNOSIS/ELISA/enzyme-linked immunosorbent assay/Epidemiology/Grizzly Island Wildlife Area/INFECTION/INFECTIONS/IS900/Johne's disease/MAP/Mycobacterium avium/Mycobacterium avium ss.avium/mycobacterium avium ss.paratuberculosis/Mycobacterium avium subsp/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/paratuberculosis/PATHOGENESIS/PCR/POLYMERASE-CHAIN-REACTION/SENSITIVITY /SEROLOGICAL TESTS/SPECIFICITY/TISSUE/tule elk/white-tailed deer/WILD ANIMALS/WILDLIFE

59 Glawischnig, W., Steineck, T., Spergser, J. (2006) Infections caused by Mycobacterium avium subspecies Avium, Hominissuis, and Paratuberculosis in free-ranging red deer (Cervus elaphus hippelaphus) in Austria, 2001-2004

Journal of Wildlife Diseases, 42, 724-731 Between 2001 and 2004, 14 Austrian free-ranging red deer (Cervus elaphus hippelaphus) infected by Mycobacterium avium species were observed. Eight of the cases were from different geographical

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regions, and six originated from the same bunting area. The affected animals had signs of diarrhea, severe weight loss, and emaciation. On post-mortem examination, lymphadenitis associated with grossly enlarged mesenteric lymph nodes as well as multiple caseous or purulent nodular lesions in the thickened wall of the intestines were present in all animals. In 10 cases M. avium subsp. avium and in four cases M. a. hominissuis were isolated. In three red deer, a mixed infection with M. a. hominissuis and M. a. paratuberculosis was evident. Typing of M. a. avium and M. a. hominissuis isolates was performed by polymerase chain reaction (PCR) detection of insertion sequence IS901 and the virulence-associated macrophage-induced gene (mig), inverted repeat (IR) typing (IS1245/IS1311), and random amplified polymorph DNA (RAPD) analysis. While all M. a. avium and M. a. hominissuis contained the inig gene, IS901 was detected only in M. a. avium. The prevalence of IS901-positive isolates correlated well with the geographic location of affected animals. The IS901-containing isolates were shown to be genotypically closely related, as they exhibit similar patterns in IR-typing and in RAPID analysis. In contrast, IS901-negative isolates (M. a. hominissuis) displayed distinct profiles in both molecular systems AVIUM/Cervus elaphus/Cervus elaphus hippelaphus/CERVUS-ELAPHUS/deer/Diarrhea/DNA/DNA fingerprinting/emaciation/FARMED DEER/FRAGMENT-LENGTH-POLYMORPHISM/IDENTIFICATION/INFECTION/INFECTIONS/IS901/mycobacteriosis /Mycobacterium avium/Mycobacterium avium subsp avium/Mycobacterium avium subsp hominissuis/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/NEW-ZEALAND/paratuberculosis/PATHOLOGY/PCR/POLYMERASE-CHAIN-REACTION/PREVALENCE/RED DEER/STRAINS/Tuberculosis/WILD RUMINANTS

60 Harrenstien, L.A., Finnegan, M.V., Woodford, N.L., Mansfield, K.G., Waters, W.R., Bannantine, J.P., Paustian, M.L., Garner, M.M., Bakke, A.C., Peloquin, C.A., Phillips, T.M. (2006) Mycobacterium avium in pygmy rabbits (Brachylagus idahoensis): 28 cases

Journal of Zoo and Wildlife Medicine, 37, 498-512 The Columbia basin subpopulation of pygmy rabbit Brachylagus idahoensis was listed as endangered by the United States Fish and Wildlife Service in November 2001, and no pygmy rabbits have been seen in the wild since spring 2002. Captive propagation efforts have attempted to increase population size in preparation for reintroduction of animals into central Washington. Disseminated mycobacteriosis due to Mycobacterium avium has been the most common cause of death of adult captive pygmy rabbits. Between June 2002 and September 2004, mycobacteriosis was diagnosed in 28 captive adult pygmy rabbits (representing 29% of the captive population), in contrast to 18 adult pygmy rabbits dying of all other causes in the same time period. Antemortem and postmortem medical records were evaluated retrospectively to describe the clinical course of mycobacteriosis in pygmy rabbits, physical examination findings, and diagnostic test results in the diagnosis of mycobacteriosis in pygmy rabbits. Various treatment protocols, possible risk factors for mortality. and recommendations for prevention of mycobacteriosis were evaluated also. Compromised cell-mediated immunity appears to be the best explanation at this time for the observed high morbidity and mortality from mycobacterial infections in pygmy rabbits AVIUM/Brachylagus idahoensis/cell-mediated immunity/COMPLEX INFECTION/DENDROLAGUS-MATSCHIEI/DIAGNOSIS/EASTERN WASHINGTON/FASTING CONDITIONS/IMMUNOAFFINITY CAPILLARY-ELECTROPHORESIS/INFECTION/INFECTIONS/MORTALITY/mycobacteriosis/Mycobacterium avium/Mycobacterium avium complex/MYCOBACTERIUM-AVIUM/OSTEOMYELITIS/paratuberculosis/PHARMACOKINETICS/POLYMERASE-CHAIN-REACTION/pygmy rabbit/rabbit/RABBITS/UNITED-STATES/WILDLIFE

61 Kopecna, M. , Ondrus, S., Literak, I., Klimes, J., Horvathova, A., Moravkova, M., Bartos, M., Trcka, I., Pavlik, I. (2006) Detection of Mycobacterium avium subsp paratuberculosis in two brown bears in the central European carpathians

Journal of Wildlife Diseases, 42, 691-695 The incidence of mycobacterial infections was monitored in brown bears (Ursus arctos) in the National Park Low Tatras in the central European Carpathians in Slovakia. Tissue samples of 20 brown bears were examined microscopically and by culture for the presence of mycobacteria. Acid-fast rods were detected by Ziehl-Neelsen staining in a smear from the kidney of one brown bear, although the culture was negative for mycobacteria. Mycobacterium avium subsp. paratuberculosis, the causative agent of paratuberculosis in ruminants, was isolated from the intestinal mucosa of another two brown bears. The isolates were identified by polymerase chain reaction for the specific insertion sequence IS900. Using standardized IS900 restriction fragment length polymorphism (RFLP) analysis, the M. a. paratuberculosis isolates were classified as RFLP type B-Cl, which also were detected in the infected cattle in surrounding area. This study describes the first isolation of M. a. paratuberculosis from a brown

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bear. Our results confirm that animal species other than ruminants can become infected with M. a. paratuberculosis and can act as potential vectors and/or reservoirs of the infection AVIUM /AVIUM SUBSP PARATUBERCULOSIS/BITE /BOAR SUS-SCROFA/carnivores/CATTLE/CULTURE/Epidemiology/FRAGMENT-LENGTH-POLYMORPHISM/INFECTION/INFECTIONS/IS900/Johne's disease/Mycobacterium avium /Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/POLYMERASE-CHAIN-REACTION/RABBITS/RESTRICTION/RFLP/RUMINANTS/SCOTLAND/SUBSP-PARATUBERCULOSIS/TISSUE/WILD RUMINANTS/WILDLIFE

62 de Juan, L. , Alvarez, J., Romero, B., Bezos, J., Castellanos, E., Aranaz, A., Mateos, A., Dominguez, L. (2006) Comparison of four different culture media for isolation and growth of type II and type I/III Mycobacterium avium subsp paratuberculosis strains isolated from cattle and goats

Applied and Environmental Microbiology, 72, 5927-5932 Culture is considered the definitive technique for Johne's disease diagnosis, and it is essential for later applications of certain molecular typing techniques. In this study, we have tested four solid media (Herrold's egg yolk medium [HEYM] with sodium pyruvate and mycobactin [HEYMm-SP], HEYM with mycobactin and without sodium pyruvate [HEYMm], Middlebrook 7H11 with mycobactin [Mm], and Lowenstein-jensen with mycobactin [LJm]) for isolation of Mycobacterium avium subsp. paratuberculosis strains in 319 tissue samples from cattle herds and goat flocks. We have shown that each of the two main groups of M. avium subsp. paratuberculosis (type II and type I/III) has different requirements for growth in the culture media studied. The recommended solid media for isolation of type I/III strains are LJm and Mm, since the combination of both media allowed the recovery of all these strains. The most widespread culture medium, HEYM, is not suitable for the isolation of this group of M. avium subsp. paratuberculosis strains. Regarding the type II strains, HEYMm-SP was the medium where more strains were isolated, but the other three media are also needed in order to recover all type II strains. The incubation period is also related to the strain type. In conclusion, because the type of strain cannot be known in advance of culture, coupled with the fact that cattle and goats can be infected with both groups of strains, we recommend the use of the four solid media and the prolongation of the incubation period to more than 6 months to detect paratuberculous herds/flocks and to determine the true prevalence of the infection AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE PARATUBERCULOSIS/CATTLE/CROHNS-DISEASE/CULTURE/DIAGNOSIS/DISEASE/FECAL CULTURE/FRAGMENT-LENGTH-POLYMORPHISM/goat/INFECTION/Johne's disease/JOHNES-DISEASE/MOLECULAR EPIDEMIOLOGY/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/paratuberculosis/POLYMERASE CHAIN-REACTION/PREVALENCE/RESTRICTION ENDONUCLEASE ANALYSIS/STRAINS/SUBSP-PARATUBERCULOSIS/TISSUE/WILD RABBITS

63 Taylor, K.H., Taylor, J.F., White, S.N., Womack, J.E. (2006) Identification of genetic variation and putative regulatory regions in bovine CARD15 Mammalian Genome, 17, 892-901 Mutations in caspase recruitment domain 15 (CARD15) are associated with susceptibility to Crohn's disease and Blau Syndrome. We performed comparative analyses of the bovine, murine, and human CARD15 transcripts to elucidate functionality of bovine CARD15 and examine its potential role in bovine disease resistance. Comparative analyses of intronic sequence across seven divergent species were performed to identify putative regulatory element binding motifs. High levels of interspecies conservation in sequence, genomic structure, and protein domains were detected indicating common functionality for CARD15 in cattle, human, and mouse. We identified species-specific regulatory elements in the 5' and 3' untranslated regions, suggesting that modes of regulation may have diverged across species. Thirty-one conserved putative regulatory element binding motifs were identified in the CARD15 intronic sequence of seven species. To assess the extent of genetic diversity within bovine CARD15, 41 individuals from two subspecies were sequenced and screened for polymorphisms. Thirty-six single nucleotide polymorphisms (SNPs) were identified. Finally, 20 subspecies-specific haplotypes were predicted with 7 and 13 unique haplotypes explaining the diversity within B. taurus taurus and B. taurus indicus animals, respectively. Strong evidence for a simple causal relationship between these SNP loci and their haplotypes with Johne's disease was not detected BOVINE/CATTLE/Crohn's disease/CROHNS-DISEASE/DISEASE/EXPRESSION/GENETIC DIVERSITY/IDENTIFICATION/Johne's disease/KAPPA-B/MOUSE/MUTATION/NOD2/SUSCEPTIBILITY GENE

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64 Metzger-Boddien, C., Khaschabi, D., Schonbauer, M., Boddien, S., Schlederer, T., Kehle, J. (2006) Automated high-throughput immunomagnetic separation-PCR for detection of Mycobacterium avium subsp paratuberculosis in bovine milk International Journal of Food Microbiology, 110, 201-208 Two monoclonal antibody-mediated immunomagnetic separation PCR kits (AnDiaTec ParaTub-S (R) IMS-PCR-ELISA and ParaTub-SL (R) IMS-real time PCR) were developed and evaluated for the automated high-throughput detection of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) in bulk milk of naturally infected dairy herds and made commercially available. M. paratuberculosis are first isolated from milk by high-throughput immumomagnetic bead separation using a completely automated magnetic particle pipetting robot within 45 min and released subsequently for analysis directly into PCR amplification mixtures for real time PCR or for PCR-ELISA. The threshold detection level and specificity of the tests were evaluated first with different M. paratuberculosis pure cultures and artificially contaminated (spiked) bulk milk samples. Both experiments proved a good detection limit, specificity and reliability of the tests that consistently detected 20 or less M. paratuberculosis organisms from cattle, deer and mufflon in 1 ml milk. Experiments with more than 200 bulk milk samples that were tested in parallel with the PCR methods and with the cultural method in a second evaluation study demonstrated that both PCR tests are superior to culture and sufficiently sensitive to detect single shedders in pooled milk samples. The experiments proved that the newly developed tests are sensitive, specific and fast, and thus for the first time allow the standardized large-scale routine M. Paratuberculosis screening of bulk milk samples at acceptable costs. (c) 2006 Elsevier B.V. All rights reserved AMPLIFICATION/automated/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/CATTLE/COWS MILK/CROHNS-DISEASE/CULTURE/DAIRY HERDS/deer/DIAGNOSIS/HERD/HIGH-TEMPERATURE/high-throughput/IMS-PCR/INHIBITION/M.paratuberculosis/MILK/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/ORGANISM/ORGANISMS/paratuberculosis/PASTEURIZATION/PCR/PCR-ELISA/raw milk/real time PCR/REAL-TIME/SPECIFICITY/SUBSP-PARATUBERCULOSIS/TESTS

65 Eda, S., Bannantine, J.P., Waters, W.R., Mori, Y., Whitlock, R.H., Scott, M.C., Speer, C.A. (2006) A highly sensitive and subspecies-specific surface antigen enzyme-linked immunosorbent assay for diagnosis of Johne's disease

Clinical and Vaccine Immunology, 13, 837-844 Johne's disease (JD), or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Control of JD could be accomplished by diagnosis and good animal husbandry, but this is currently not feasible because commercially available diagnostic tests have low sensitivity levels and are incapable of diagnosing prepatent infections. In this study, a highly sensitive and subspecies-specific enzyme-linked immunosorbent assay was developed for the diagnosis of JD by using antigens extracted from the surface of M. avium subsp. paratuberculosis. Nine different chemicals and various intervals of agitation by vortex were evaluated for their ability to extract the surface antigens. Various quantities of surface antigens per well in a 96-well microtiter plate were also tested. The greatest differences in distinguishing between JD-positive and JD-negative serum samples by ethanol vortex enzyme-linked immunosorbent assay (EVELISA) were obtained with surface antigens dislodged from 50 mu g/well of bacilli treated with 80% ethanol followed by a 30-second interval of agitation by vortex. The diagnostic specificity and sensitivity of the EVELISA were 97.4% and 100%, respectively. EVELISA plates that had been vacuum-sealed and then tested 7 weeks later (the longest interval tested) had diagnostic specificity and sensitivity rates of 96.9 and 100%, respectively. In a comparative study involving serum samples from 64 fecal culture-positive cattle, the EVELISA identified 96.6% of the low-level fecal shedders and 100% of the midlevel and high-level shedders, whereas the Biocor ELISA detected 13.7% of the low-level shedders, 25% of the mid-level shedders, and 96.2% of the high-level shedders. Thus, the EVELISA was substantially superior to the Biocor ELISA, especially in detecting low-level and midlevel shedders. The EVELISA may form the basis for a highly sensitive and subspecies-specific test for the diagnosis of JD ABILITY/ANTIGEN/antigens/ASSAY/AVIUM/BOVINE PARATUBERCULOSIS/CATTLE/DAIRY-CATTLE/DIAGNOSIS/DISEASE/DISEASES/ELISA/enzyme-linked immunosorbent assay/FECAL CULTURE/ INFECTION/INFECTIONS/Johne's disease/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/paratuberculosis/PARATUBERCULOSIS INFECTION/RESPONSES/RUMINANTS/SENSITIVITY/SEROLOGICAL TESTS/SPECIFICITY/TESTS/WILD RUMINANTS

66 Shin, S.J., Wu, C.W., Steinberg, H., Talaat, A.M. (2006) Identification of novel virulence determinants in Mycobacterium paratuberculosis by screening a library of insertional mutants

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Infection and Immunity, 74, 3825-3833 Johne's disease, caused by Mycobacterium paratuberculosis infection, is a worldwide problem for the dairy industry and has a possible involvement in Crohn's disease in humans. To identify virulence determinants of this economically important pathogen, a library of 5,060 transposon mutants was constructed using Tn5367 insertion mutagenesis, followed by large-scale sequencing to identify disrupted genes. In this report, 1,150 mutants were analyzed and 970 unique insertion sites were identified. Sequence analysis of the disrupted genes indicated that the insertion of Tn5367 was more prevalent in genomic regions with G+C content (50.5 to 60.5%) lower than the average G+C content (69.3%) of the rest of the genome. Phenotypic screening of the library identified disruptions of genes involved in iron, tryptophan, or mycolic acid metabolic pathways that displayed unique growth characteristics. Bioinformatic analysis of disrupted genes identified a list of potential virulence determinants for further testing with animals. Mouse infection studies showed a significant decrease in tissue colonization by mutants with a disruption in the gcpE, pst4, kdpC, papA2, impA, umaA1 or fabG2_2 gene. Attenuation phenotypes were tissue specific (e.g., for the umaA1 mutant) as well as time specific (e.g., for the impA mutant), suggesting that those genes may be involved in different virulence mechanisms. The identified potential virulence determinants represent novel functional classes that could be necessary for mycobacterial survival during infection and could provide suitable targets for vaccine and drug development against Johne's and Crohn's diseases AVIUM SUBSP PARATUBERCULOSIS/CELLS/Crohn's disease/DISEASE/DISEASES/ESCHERICHIA-COLI/IDENTIFICATION/INFECTION/INTESTINAL PATHOPHYSIOLOGIC CHANGES/ISOPRENOID BIOSYNTHESIS/Johne's disease/JOHNES-DISEASE/MICE/MOUSE/Mycobacterium paratuberculosis/ MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/PARATUBERCULOSIS INFECTION/PREVALENCE/SMEGMATIS/TISSUE/Tuberculosis/VIRULENCE

67 Pickup, R.W., Rhodes, G., Bull, T.J., Arnott, S., Sidi-Boumedine, K., Hurley, M., Hermon-Taylor, J. (2006) Mycobacterium avium subsp paratuberculosis in lake catchments, in river water abstracted for domestic use, and in effluent from domestic sewage treatment works: Diverse opportunities for environmental cycling and human exposure

Applied and Environmental Microbiology, 72, 4067-4077 Mycobacterium avium subsp. paratuberculosis from infected animals enters surface waters and rivers in runoff from contaminated pastures. We studied the River Tywi in South Wales, United Kingdom, whose catchment comprises 1,100 km(2) containing more than a million dairy and beef cattle and more than 1.3 million sheep. The River Tywi is abstracted for the domestic water supply. Between August 2002 and April 2003, 48 of 70 (68.8%) twice-weekly river water samples tested positive by IS900 PCR. In river water, the organisms were associated with a suspended solid which was depleted by the water treatment process. Disposal of contaminated slurry back onto the land established a cycle of environmental persistence. A concentrate from 100 liters of finished water tested negative, but 1 of 54 domestic cold water tanks tested positive, indicating the potential for these pathogens to access domestic outlets. In the separate English Lake District region, with hills up to 980 m, tests for M. avium subsp. paratuberculosis in the high hill lakes and sediments were usually negative, but streams and sediments became positive lower down the catchment. Sediments from 9 of 10 major lakes receiving inflow from these catchments were positive, with sediment cores indicating deposition over at least 40 to 50 years. Two of 12 monthly 1-liter samples of effluent and a single 100-liter sample from the Ambleside sewage treatment works were positive for M. avium subsp. paratuberculosis. Since Lake Ambleside discharges into Lake Windermere, which is available for domestic supply, there is a potential for these organisms to cycle within human populations AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIAL PATHOGENS/BIOPSY SPECIMENS/CATTLE/CROHNS-DISEASE/IN-SITU HYBRIDIZATION/INFLAMMATORY-BOWEL-DISEASE/IS900/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/ORGANISM/ORGANISMS/paratuberculosis/PASTEURIZED COWS MILK/PCR/RISK-FACTORS/SHEEP/SUBSP-PARATUBERCULOSIS/TESTS/TUBERCULOSIS JOHNES DISEASE/UNITED-KINGDOM/WATER/WILD RABBITS

68 Eda, S., Elliott, B., Scott, M.C., Waters, W.R., Bannantine, J.P., Whitlock, R.H., Speer, C.A. (2005) New method of serological testing for Mycobacterium avium subsp paratuberculosis (Johne's disease) by flow cytometry

Foodborne Pathogens and Disease, 2, 250-262 Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Attempts to control JD have proven inordinately difficult due to low levels of

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sensitivity by currently available diagnostic tests, which are also incapable of detecting prepatent MAP infections. In the present work, we describe the use of a flow cytometry method (FCM) for serological diagnosis of subclinical and clinical JD in cattle. The FCM was capable of distinguishing MAP-infected from MAP-non-infected cattle as well as MAP from M. scrofulaceum and M. avium subsp. avium. Results of the FCM were compared to that of a commercially available ELISA using 82 serum samples from JD-positive and JD-negative dairy and beef cattle farms that were separated into the following groups: (1) sera from a JD-free farm; (2) sera from JD-positive farms that had tested negative by ELISA; and (3) sera from JD-positive farms that tested JD-positive by ELISA. The FCM found that groups 1-3 were 6.6%, 73.3%, and 97.3% positive for MAP infections, respectively. By using 30 fecal culture-negative samples from a JD-free farm and 21 fecal culture-positive samples from JD-positive farms, diagnostic sensitivity and specificity of the FCM were calculated to be 95.2% and 96.7%, respectively. A retrospective study of 10 JD-positive cows showed that the FCM detected MAP infections 6-44 months earlier than the fecal culture test. Further, the FCM specifically detected MAP infections in serum samples as early as 170 days after experimental inoculation of calves with MAP and did not react with calves inoculated with other mycobacteria. Production of IgG against MAP was detected by FCM in all the calves inoculated with MAP 240 days after inoculation, whereas positive anti-MAP IgG production was not detected in control calves or calves experimentally infected with M. avium subsp. avium or M. bovis. The FCM assay is rapid and is completed in less than 4 h. Moreover, the FCM is objective, technically easy and can be automated for handling large numbers of samples. This novel assay might form the basis of a highly sensitive and subspecies-specific test for the diagnosis of JD antigens/ASSAY/automated/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE PARATUBERCULOSIS/BOVIS/CALVES/CATTLE/CATTLE FARMS/CROHNS-DISEASE/CULTURE/DAIRY-CATTLE/DIAGNOSIS/DISEASE/DISEASES/ELISA/FECAL CULTURE/INFECTION/INFECTIONS/Johne's disease/LINKED-IMMUNOSORBENT-ASSAY/MAP/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/RUMINANTS/SENSITIVITY/SPECIFICITY/SUBSP-PARATUBERCULOSIS/TESTS/Tuberculosis/WILD RUMINANTS

69 Waters, W.R., Palmer, M.V., Thacker, T.C. , Payeur, J.B., Harris, N.B., Minion, F.C., Greenwald, R., Esfandiari, J., Andersen, P., McNair, J., Pollock, J.M., Lyashchenko, K.P. (2006) Immune responses to defined antigens of Mycobacterium bovis in cattle experimentally infected with Mycobacterium kansasii

Clinical and Vaccine Immunology, 13, 611-619 Cross-reactive responses elicited by exposure to nontuberculous mycobacteria often confound the interpretation of antemortem tests for Mycobacterium bovis infection of cattle. The use of specific proteins (e.g., ESAT-6, CFP-10, and MPB83), however, generally enhances the specificity of bovine tuberculosis tests. While genes for these proteins are absent from many nontuberculous mycobacteria, they are present in M. kansasii. Instillation of M. kansasii into the tonsillar crypts of calves elicited delayed-type hypersensitivity and in vitro gamma interferon and nitrite concentration responses of leukocytes to M. avium and M. bovis purified protein derivatives (PPDs). While the responses of M. katisasii-inoculated calves to M. avium and M. bovis PPDs were approximately equivalent, the responses of M. bovis-inoculated calves to M. bovis PPD exceeded their respective responses to M. avium PPD. The gamma interferon and nitrite responses of M. kansasii-inoculated calves to recombinant ESAT-6-CFP-10 (rESAT-6-CFP-10) exceeded corresponding responses of noninoculated calves as early as 15 and 30 days after inoculation, respectively, and persisted throughout the study. The gamma interferon and nitrite responses of M. bovis-inoculated calves to rESAT-6-CFP-10 exceeded the corresponding responses of M. kansasii-inoculated calves beginning 30 days after inoculation. By using a lipoarabinomannan-based enzyme-linked immunosorbent assay, specific serum antibodies were detected as early as 50 days after challenge with M. kansasii. By a multiantigen print immunoassay and immunoblotting, serum antibodies to MPB83, but not ESAT-6 or CFP-10, were detected in M. katisasii-inoculated calves; however, responses to MPB83 were notably weaker than those elicited by M. bovis infection. These findings indicate that M. kansasii infection of calves elicits specific responses that may confound the interpretation of bovine tuberculosis tests ANTIBODIES/ANTIGEN/antigens/ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/BOVINE TUBERCULOSIS/BOVIS/ CALVES/CATTLE/CFP-10/challenge/DIAGNOSIS/enzyme-linked immunosorbent assay/ESAT-6/GAMMA/GAMMA-INTERFERON/IMMUNE-RESPONSE/IMMUNE-RESPONSES/INFECTION/INTERFERON-GAMMA ASSAY/MURINE MACROPHAGES/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/NITRIC-OXIDE SYNTHASE/NONTUBERCULOUS MYCOBACTERIA/ODOCOILEUS-VIRGINIANUS/PROTEINS/PULMONARY TUBERCULOSIS/RESPONSES/SPECIFICITY/TESTS/Tuberculosis/white-tailed deer

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70 Thacker, T.C., Palmer, M.V., Waters, W.R. (2006) Correlation of cytokine gene expression with pathology in white-tailed deer (Odocoileus virginianus) infected with Mycobacterium bovis Clinical and Vaccine Immunology, 13, 640-647 Mycobacterium bovis-infected white-tailed deer (WTD) in northeast Michigan are a reservoir of mycobacteria that pose a threat to both domestic animals and humans. Relatively little work has been done to characterize the immune response of WTD to M. bovis infection; however, an understanding of the immune response to infection and pathogenesis may be critical to the development of an effective vaccine. Immunological responses to infection were characterized by monitoring cytokine gene expression in M. bovis-infected and uninfected deer. Peripheral blood leukocytes (PBL) from infected WTD expressed more gamma interferon (IFN-gamma), interleukin-12p40 JL-12p40), granulocyte-monocyte colony-stimulating factor, and IL-4 mRNA than did PBL from uninfected deer, however, differences were not detected in expression of IL-10 and transforming growth factor-P mRNA. Infected animals could be divided into two groups based on pathology. Lesions were confined primarily to the lymph nodes of the head in animals with less severe pathology. Animals with more severe pathology had lesions in the lung and associated lymph nodes as well as the lymph nodes of the head. More robust IFN-gamma mRNA expression correlated with pathology early in infection. These findings indicate that IFN-gamma expression likely plays a role in both protection and pathogenesis AVIUM SUBSP-PARATUBERCULOSIS/BOVIS/CATTLE/CELLS/deer/DOMESTIC-ANIMALS/Epidemiology/EXPRESSION /GAMMA/GAMMA-INTERFERON/GENE-EXPRESSION/HUMORAL IMMUNE-RESPONSES /IMMUNE-RESPONSE/INFECTION/MICHIGAN/monitoring/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/Odocoileus virginianus/ODOCOILEUS-VIRGINIANUS/PATHOGENESIS/PATHOLOGY/PREVALENCE/RECOGNITION/RESPONSES/Tuberculosis/white-tailed deer

71 Waters, W.R., Palmer, M.V., Thacker, T.C. , Bannantine, J.P., Vordermeier, H.M., Hewinson, R.G., Greenwald, R., Esfandiari, J., McNair, J., Pollock, J.M., Andersen, P., Lyashchenko, K.P. (2006) Early antibody responses to experimental Mycobacterium bovis infection of cattle

Clinical and Vaccine Immunology, 13, 648-654 Bovine tuberculosis persists as a costly zoonotic disease in numerous countries despite extensive eradication and control efforts. Sequential serum samples obtained from Mycobacterium bovis-infected cattle were evaluated for seroreactivity to mycobacterial antigens. Animals received M. bovis by aerosol, intratonsil, intranasal, or intratracheal inoculation. Assays included the multiantigen print immunoassay for determination of antigen recognition patterns, immunoblot analysis for sensitive kinetic studies, and the VetTB STAT-PAK test, a novel, rapid test based on lateral-flow technology. Responses to MPB83 were detected for all M. bovis-infected animals regardless of the route or strain of M. bovis used for inoculation. Other less commonly recognized antigens included ESAT-6, CFP-10, and MPB70. Responses to MPB83 were detectable as early as 4 weeks after inoculation, were boosted upon injection of purified protein derivatives for skin testing, and persisted throughout the course of each of the four challenge studies. MPB83-specific immunoglobulin M (IgM) was detected prior to MPB83-specific IgG detection; however, early IgM responses rapidly waned, suggesting a benefit of tests that detect both IgM- and IgG-specific antibodies. The VetTB STAT-PAK test detected responses in sera from 60% (15/25) of the animals by 7 weeks after challenge and detected responses in 96% (24/25) of the animals by 18 weeks. These findings demonstrate the potential for new-generation antibody-based tests for the early detection of M. bovis infection in cattle ANTIBODIES/ANTIBODY-RESPONSES/ANTIGEN/antigens/ASSAY/BOVINE/BOVINE TUBERCULOSIS/BOVIS/CATTLE/CFP-10/challenge/DIAGNOSIS/DISEASE/ESAT-6/GAMMA-INTERFERON/INFECTION/MICHIGAN/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/RECOGNITION/RESPONSES/SUBSP PARATUBERCULOSIS INFECTION/TESTS/Tuberculosis/white-tailed deer

72 Hasonova, L., Pavlik, I. (2006) Economic impact of paratuberculosis in dairy cattle herds: a review

Veterinarni Medicina, 51, 193-211 Paratuberculosis (PTB) is a disease which causes considerable economic losses to producers of livestock, particularly dairy cows. Nowadays PTB is one of the most prevailing and costly infectious diseases of dairy cattle. The purpose of the present study was to review economic losses, which may be caused by Mycobacterium avium subsp. paratuberculosis (MAP) above all in herds of dairy cattle. The most important losses caused by the presence of clinically ill animals have been thoroughly described: loss of milk production and poor body condition followed by death or culling. In contrast, losses arising from a subclinical disease have not been well documented and contradictory results have been

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published to date. The calculation of losses caused by PTB depends to a certain degree on the production system in a herd, efficiency level, herd management system and other factors. Direct economic losses are above all caused by decreased milk production concurrent to increased incidence of mastitis, changes in milk parameters and increased somatic cell counts, reproductive dysfunctions, poor feed conversion, shortened production age and increased predisposition to other diseases etc. Indirect economic losses are caused by premature culling of animals and their unrealized future income, expenses for nonactive production, herd replacement, diagnostic testing, "unnecessary" veterinary care and establishing disease control programmes. Genetic value of animals and their progeny is lost. Last but not least, the reputation of the farm where MAP infected animals are kept is lost for a long-time, which is also important Avian tuberculosis/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE PARATUBERCULOSIS/CATTLE/CELL/Crohn's disease/dairy cattle/DAIRY-CATTLE/DISEASE/DISEASES/economic losses/HERD/IMPACT/infectious disease/IS900/Johne's disease/JOHNES-DISEASE/LINKED-IMMUNOSORBENT-ASSAY/MAP/MILK/ MILK-PRODUCTION/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/PARA-TUBERCULOSIS/paratuberculosis/Review/SUBCLINICAL PARATUBERCULOSIS/WILD RUMINANTS

73 Trcka, I., Lamka, J., Suchy, R., Kopecna, M., Beran, V., Moravkova, M., Horvathova, A., Bartos, M., Parmova, I., Pavlik, I. (2006) Mycobacterial infections in European wild boar (Sus scrofa) in the Czech Republic during the years 2002 to 2005

Veterinarni Medicina, 51, 320-332 A total of 842 wild boar of differing ages, originating from 29 (37.7%) of the 77 districts in the Czech Republic, were examined during the hunting seasons from 2002 to 2005. Of them, 274 (32.5%) of the animals were wild specimens and 568 (67.5%) from game parks. Out: of 786 animals, the following were included in the study: 668 piglets, 61 juveniles, 32 adult males and 25 adult females. A total of 2 704 samples from various tissues and faeces were examined: 309 separately collected faecal samples from 309 (36.7%) animals, 2 332 samples from various tissues and 63 faecal samples from 533 (63.3%) animals. Mycobacteria were isolated from 75 (8.9%) animals from 11 of the districts. Neither a causative agent of bovine tuberculosis, nor any other members of Mycobacterium tuberculosis complex were isolated from any of the animals. From one (0.1%) animal, M. avium subsp. paratuberculosis of IS900 RFLP type A-C10 was isolated from intestinal lymph nodes, which was also isolated within the same district during other studies of cattle and free living ruminants. The causative agent of avian tuberculosis, M. a. avium (IS901+ and IS1245+), was isolated from 7 (0.8%) animals; among them tuberculous lesions were detected in intestinal lymph nodes, with gross tuberculous lesions visible on two animals. The causative agent of avian mycobacteriosis M. a. hominissuis (IS901- and IS1245+) was detected in lymph nodes without gross lesions in one (0.1%) animal. From 45 (5.5%) animals without lesions, atypical mycobacteria of the following nine species were isolated from pulmonary lymph nodes, small and large intestine, intestinal mucosa, and faeces: M. fortuitum, M. chelonae, M. scrofulaceum, M. triviale, M. terrae, M. phlei, M. abscessus, M. flavescens, and M. smegmatis. Due to a high density of wild boar and their large migration radius, they can be viewed as a potential source for mycobacterial infections as well as other infectious agents ANIMALS/Avian tuberculosis/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BOVINE/BOVINE TUBERCULOSIS/CATTLE/COMPLEX/DRIVE HUNTS/Epidemiology/FAMILY GROUPS/FRAGMENT-LENGTH-POLYMORPHISM/game park/INFECTION/INFECTIONS/IS900/ IS901/LOWER SAXONY/LYMPH-NODES/mycobacteriosis/paratuberculosis/PASSIVE VECTORS/RFLP/RUMINANTS/SMEGMATIS/TISSUE/Tuberculosis/TUBERCULOSIS COMPLEX/WILD ANIMALS/WILD BOAR/zoonosis

74 Fischer, O.A., Matlova, L., Dvorska, L., Svastova, P., Bartos, M., Weston, R.T., Pavlik, I. (2006) Various stages in the life cycle of syrphid flies (Eristalis tenax; Diptera : Syrphidae) as potential mechanical vectors of pathogens causing mycobacterial infections in pig herds

Folia Microbiologica, 51, 147-153 We defined the role of the syrphid fly Eristalis tenax in the survival and transmission of mycobacteria in pigs. The conditionally pathogenic mycobacterial (CPM) species Mycobacterium chelonae was isolated from 10 % of liquid dung samples, and both M chelonae and another CPM species M. fortuitum were isolated from 7 (78 %) of the examined E. tenax larvae collected from the same location. Mycobacteriosis of the lymph nodes of pigs from 3 infected farms was caused by M avium subsp. avium, M avium subsp. hominissuis, and M fortuitum. M avium subsp. avium and M avium subsp. hominissuis of identical genotype and serotypes and M fortuitum were isolated from 7 (1.9 %) larvae, 2 (7.4 %) puparia, and one (1.6 %) imago. The count of colony forming units isolated from larval skin covering (pouch) was higher (p <= 0.01) than that isolated from the internal organs of larvae. These results showed the

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potential for E. tenax larvae to spread mycobacteria throughout pig herds and the surrounding environment ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/ENVIRONMENT/HERD/INFECTION/INFECTIONS/INSERTION ELEMENT/IS1245/LYMPH-NODES/mycobacteriosis/PASSIVE VECTORS/SYRPHID FLIES/Transmission/TUBERCULOUS LESIONS/WILD RUMINANTS

75 O'Brien, R. , Mackintosh, C.G., Bakker, D., Kopecna, M., Pavlik, I., Griffin, J.F.T. (2006) Immunological and molecular characterization of susceptibility in relationship to bacterial strain differences in Mycobacterium avium subsp paratuberculosis infection in the red deer (Cervus elaphus) Infection and Immunity, 74, 3530-3537 Johne's disease (JD) infection, caused by Mycobacterium avium subsp. paratuberculosis, represents a major disease problem in farmed ruminants. Although JD has been well characterized in cattle and sheep, little is known of the infection dynamics or immunological response in deer. In this study, typing of M. avium subsp. paratuberculosis isolates from intestinal lymphatic tissues from 74 JD-infected animals showed that clinical isolates of M. avium subsp. paratuberculosis from New Zealand farmed red deer were exclusively of the bovine strain genotype. The susceptibility of deer to M. avium subsp. paratuberculosis was further investigated by experimental oral-route infection studies using defined isolates of virulent bovine and ovine M. avium subsp. paratuberculosis strains. Oral inoculation with high (10(9) CFU/animal) or medium (10(7) CFU/animal) doses of the bovine strain of M. avium subsp. paratuberculosis established 100% infection rates, compared to 69% infection following inoculation with a medium dose of the ovine strain. The high susceptibility of deer to the bovine strain of M. avium subsp. paratuberculosis was confirmed by a 50% infection rate following experimental inoculation with a low dose of bacteria (10(3) CFU/animal). This study is the first to report experimental M. avium subsp. paratuberculosis infection in red deer, and it outlines the strong infectivity of bovine-strain M. avium subsp. paratuberculosis isolates for cervines ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIUM/BOVINE/CATTLE/Cervus elaphus/CERVUS-ELAPHUS/deer/DISEASE/FARMED DEER/FARMED RED DEER/FRAGMENT-LENGTH-POLYMORPHISM/IMMUNE-RESPONSES/INFECTION/infection dynamics/IS900/Johne's disease/JOHNES-DISEASE/MOLECULAR CHARACTERIZATION/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/NEW-ZEALAND/ORAL INOCULATION/OVINE/paratuberculosis/PARATUBERCULOSIS INFECTION/PROTECTIVE EFFICACY/RED DEER/RUMINANTS/SHEEP/STRAINS/SUBSP PARATUBERCULOSIS INFECTION/SUBSP-PARATUBERCULOSIS/TISSUE/Tuberculosis/WILD RUMINANTS

76 Rosseels, V., Roupie, V., Zinniel, D., Barletta, R.G., Huygen, K. (2006) Development of luminescent Mycobacterium avium subsp paratuberculosis for rapid screening of vaccine candidates in mice

Infection and Immunity, 74, 3684-3686 Mycobacterium avium subsp.paratuberculosis is a slowly growing mycobacterial species, requiring 6 to 8 weeks of culture before colonies can be counted visually. Here, we describe the development of luminescent M. avium subsp. paratuberculosis expressing luxAB genes of Vibrio harveyi and its use for vaccine testing in an experimental mouse model, replacing fastidious CFU counting by rapid luminometry AG85/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/CULTURE/INFECTION/MICE/MODEL/MOUSE/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/SUBSP-PARATUBERCULOSIS/Tuberculosis

77 Spergser, J., Fuchs, K., Deutz, A. (2006) Molecular characterisation of M-avium subsp paratuberculosis isolated from cattle and wild animal species from Styria

Wiener Tierarztliche Monatsschrift, 93, 47-52 CATTLE/DNA/Epidemiology/FRAGMENT-LENGTH-POLYMORPHISM/IDENTIFICATION/IS1311/IS900/JOHNES-DISEASE/M.paratuberculosis/paratuberculosis/PFGE/POLYMERASE-CHAIN-REACTION/RAPD/RED DEER/RUMINANTS/Styria/SUBSP-PARATUBERCULOSIS/WILDLIFE

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78 Weiss, D.J. , Evanson, O.A., Souza, C.D. (2006) Mucosal immune response in cattle with subclinical Johne's disease

Veterinary Pathology, 43, 127-135 Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease, a chronic granulomatous enteritis of wild and domestic ruminants. During a long subclinical period, the organism persists in the intestine despite Systemic cellular and humoral immune responses. To explore the mucosal immune response in Johne's disease, we isolated mononuclear leukocytes from the ileum of cows naturally infected with M. avium subsp. paratuberculosis and from cows that were not infected. We evaluated the immunophenotype of these cells and the proliferative responses after the addition of M avium subsp. paratuberculosis sonicate or B-cell or T-cell mitogens. Although the percentage of T cells was increased in infected cows, these cells consisted mostly of memory (CD2(+)CD62L(-)) and regulatory (CD4(+)CD25(+)) T cells. Further evidence of immune hyporesponsiveness included a decrease in the percentage of T cells with an activated phenotype and a decrease in cells expressing major histocorripatibility factor class II (MHC class II). Unlike the spleen, ileal lymphocytes from infected cows failed to proliferate in response to M. avium subsp. paratuberculosis sonicate. Additionally, Heal lymphocytes from infected cows proliferated poorly in response to concanavalin A and pokeweed mitogen, Suggesting generalized T cell and B cell hyporesponsiveness. These results indicate that a state of tolerance may exist in the intestine of COWS subclinically infected with M. avium subsp. paratuberculosis organisms in subclinically infected cows. This effect may be induced, at least in part, by proliferation of regulatory T cells that nonspecifically suppress mucosal immune responsiveness AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE MACROPHAGES/bovine monocytes/CATTLE/CELL/CELLS/CYTOKINE GENE-EXPRESSION/DISEASE/ENTERITIS/GROWTH-FACTOR-BETA/ ILEAL TISSUES/IMMUNE-RESPONSE/IMMUNE-RESPONSES/INFECTED CATTLE/INFECTED COWS/INTERLEUKIN-10/Johne's disease/mucosal immunology/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/ORGANISM/ORGANISMS/paratuberculosis/PEYERS-PATCHES/regulatory T cells/RESPONSES/RUMINANTS/T-CELL RESPONSES

79 Bartos, M., Hlozek, P., Svastova, P., Dvorska, L., Bull, T., Matlova, L., Parmova, I., Kuhn, I., Stubbs, J., Moravkova, M., Kintr, J., Beran, V., Melicharek, I., Ocepek, M., Pavlik, I. (2006) Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900, IS901, IS1245 and IS901-flanking region PCR with internal standards

Journal of Microbiological Methods, 64, 333-345 From Mycobacterium avium species Mycobacterium avium subsp. paratuberculosis (n = 961), Mycobacterium a. avium (n = 677), Mycobacterium a. silvaticum (n = 5), and Mycobacterium a. hominissuis (n = 1566) were examined, and from Mycobacterium tuberculosis complex M. tuberculosis (n = 2), Mycobacterium bovis (n = 13), M. bovis BCG (n = 4), and Mycobacterium caprae (n = 10) were examined. From other mycobacterial species Mycobacterium intracellulare (n = 60) and atypical mycobacteria (n = 256) including Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium scrofulaceum, Mycobacterium gastri and other species of conditionally pathogenic mycobacteria were analysed. The internal standard molecules corresponding to insertion sequences IS900, IS901, IS1245, and flanking region (FR300) of IS901 were produced by PCR of alfalfa genome segment and inserted into plasmid vector. The resulting recombinant plasmid molecules were used as internal standards in coamplification with a total of 4729 mycobacterial collection strains and field isolates between 1996 and 2003. The size differences between amplicons obtained from IS900 (258 bp), IS901 (1108 bp), IS1245 (427 bp), and FR300 (300 bp) and from corresponding internal standard molecules ISIS900 (591 bp), ISIS901 (1 336 bp), ISIS1245 (583 bp), and IS901 flanking region of 300 bp ISFR300 (488 bp), respectively, allowed easy discrimination. The internal amplicons were visible by naked aye on agarose gel when 10(1), 10(3), 10(2), and 10(2) molecules for ISIS900, ISIS901, ISIS 1245, and ISFR300 were used in the PCR, respectively, when no bacterial DNA was added to the reaction. The system was tested to define the amount of internal standards that could be used in the PCR without affecting the amplification of the specific segment. Non-specific amplifications were observed in M. fortuitum with IS1245 PCR and mixed infections with M. a. avium and M. a. hominissuis from pigs and cattle were found. PCR results of typing were compared with serotyping and Accu-Probes analyses in selected field isolates. (C) 2005 Elsevier B.V. All rights reserved AMPLIFICATION/Avian tuberculosis/AVIUM/BCG/BOVINE TUBERCULOSIS/BOVIS/CATTLE/COMPLEX/Crohn's disease/DNA/DNA PROBES/FRAGMENT-LENGTH-POLYMORPHISM/human tuberculosis/IDENTIFICATION/INFECTION/INFECTIONS/INSERTION ELEMENT/insertion sequences/INTRACELLULARE/IS1245/IS900/IS901/Johne's disease/mycobacteriosis/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium bovis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-BOVIS/paratuberculosis/PCR/STRAINS/SUBSP PARATUBERCULOSIS/Tuberculosis/TUBERCULOSIS COMPLEX/WILD RUMINANTS

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80 Basagoudanavar, S.H., Goswami, P.P., Tiwari, V. (2006) Cellular immune responses to 35 kDa recombinant antigen of Mycobacterium avium paratuberculosis Veterinary Research Communications, 30, 357-367 Mycobacterium avium paratuberculosis is the causative agent of Johne disease, a chronic ulcerative intestinal condition in ruminant animals. Owing to the predominance of cellular response in subclinical forms of the infection, identification of M. a. paratuberculosis antigens eliciting host cell-mediated immune (CMI) reaction is crucial for early control of the disease. A 35 kDa protein of M. a. paratuberculosis was studied for its ability to elicit CMI responses using a mouse model. Lymphoproliferation and IFN-gamma response were used to measure the CMI response. Recombinant 35 kDa protein (P35) stimulated proliferation of mouse mononuclear splenocytes sensitized with M. a. paratuberculosis. The P35 elicited increased nitrite production from mononuclear splenocytes from M. a. paratuberculosis-sensitized mice. In addition, RT-PCR-based semiquantitative IFN-gamma measurement showed that stimulation with P35 is associated with significant expression of IFN-gamma mRNA in M. a. paratuberculosis-sensitized mouse splenocytes. The results indicate that the 35 kDa protein of M. a. paratuberculosis is associated with CMI response in the host 35 kDa protein/ABILITY/ANIMALS/ANTIGEN/antigens/AVIUM/BLOOD MONONUCLEAR-CELLS/CD4(+) T-CELLS/cellular immune response/COLORIMETRIC ASSAY/CROHNS-DISEASE/DISEASE/EXPRESSION/FRANCISELLA-TULARENSIS/GAMMA-INTERFERON/IDENTIFICATION/IMMUNE-RESPONSE/IMMUNE-RESPONSES/INFECTION/JOHNES-DISEASE/LYMPHOCYTE-PROLIFERATION/MICE/MODEL/MOUSE/Mycobacterium avium/Mycobacterium avium paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PROTEIN/RESPONSES/SUBSP PARATUBERCULOSIS

81 Palmer, M.V., Waters, W.R. (2006) Advances in bovine tuberculosis diagnosis and pathogenesis: What policy makers need to know Veterinary Microbiology, 112, 181-190 The mainstay of tuberculosis diagnosis in cattle and deer has been the tuberculin skin test. Recent advances have allowed the incorporation of blood based assays to the diagnostic arsenal for both cattle and deer. Use of defined and specific antigens has allowed for improved specificity of cell mediated assays in both cattle and deer and advances in antibody tests for tuberculosis have potential for use in free-ranging and captive cervid populations. Combined use of blood-based assays with skin testing will require further understanding of the effect of skin testing on the accuracy of blood based assays. Models of experimental infection of cattle have allowed for increased understanding of natural disease pathogenesis. Differences likely exist; however, between cattle and deer in both disease distribution and primary route of inoculation in naturally infected animals. Published by Elsevier B.V ANIMALS/ANTIBODIES/ANTIGEN/antigens/ASSAY/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/ BOVINE TUBERCULOSIS/CATTLE/CELL/Cervidae/CROSS-REACTIVITY/deer/DIAGNOSIS/DISEASE/experimental infection/EXPERIMENTAL-INFECTION/EXPERIMENTALLY INFECTED CATTLE/GAMMA-INTERFERON ASSAY/IMMUNE-RESPONSES/INFECTION/MODEL/mycobacteria/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/ODOCOILEUS-VIRGINIANUS/PATHOGENESIS/PATHOLOGY/RESPIRATORY TRACTS/SPECIFICITY/TEST-REACTOR CATTLE/TESTS/Tuberculosis/white-tailed deer

82 Chamberlin, W.M., Naser, S.A. (2006) Integrating theories of the etiology of Crohn's disease - On the etiology of Crohn's disease: Questioning the Hypotheses

Medical Science Monitor, 12, RA27-RA33 The most prominent theory describes the Crohn's Syndrome as a dysregulated, inappropriate immune response to otherwise innocuous bowel flora in a genetically Susceptible host. The autoimmune theory assumes that a specific infections agent does not exist. Data from mouse models, impairment of the mucosal epithelial barrier and the influence of gut flora are used to support the autoimmune theory. Critics claim that the dysregulated immune responses are not the primary disorder but secondary to all underlying infection. Two Other theories are also consistent with the same data. The immunodeficiency theory hypothesizes that defects in innate immunity leading to compensatory immune processes underlie the Crohn's phenotype and suggests that therapy should stimulate immunity rather than suppress it. The mycobacterial theory proposes that Mycobacterium avium subspecies paratuberculosis is One Of the causes of the Crohn's Disease syndrome. Mycobacterial molecules dysregulate immune signaling pathways as part of the organisms' evolved survival strategy. If MAP were to initiate the dysregulated immune response then the necessity to hypothesize that commensal gut flora provide the antigenic stimulus would be moot. Commensal bacteria would be relegated to a secondary role of modifying the immune response rather than occupying the central role of providing the initiating antigens. Critics claim

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that MAP is merely a secondary invader. The three theories differ primarily by emphasizing different aspects of the same overall process ANTIGEN/antigens/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/AVIUM SUBSPECIES PARATUBERCULOSIS/BACTERIUM/BIOPSY SPECIMENS/Crohn's disease/CULTURE/DISEASE/DNA/etiology/IMMUNE-RESPONSE/IMMUNE-RESPONSES/INFECTION/INFECTIONS/LASER CAPTURE MICRODISSECTION/MAP/MODEL/ MOUSE/mycobacteria/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/NOD2/ORGANISM/ORGANISMS/paratuberculosis/POLYMERASE CHAIN-REACTION/RESPONSES/THERAPY/TISSUES/Tuberculosis

83 Sechi, L.A. , Mara, L., Cappai, P., Frothingam, R., Ortu, S., Leoni, A., Ahmed, N., Zanetti, S. (2006) Immunization with DNA vaccines encoding different mycobacterial antigens elicits a Th1 type immune response in lambs and protects against Mycobacterium avium subspecies paratuberculosis infection Vaccine, 24, 229-235 Paratuberculosis, or Johne's disease, is a disease of domestic and wild ruminants that culminate with a chronic enteritis caused by Mycobacterium avium subsp. paratuberculosis. The aim of this work was to evaluate the type of immune response, Th1 or Th2, induced by DNA vaccinations in lambs of Sarda breed. Twenty-five lambs, serum negative for M. paratuberculosis, were selected at birth from equally serum negative mothers. The lambs were inoculated at 5 months of age with three different mycobacterial antigens cloned into a mammalian expression vector as fusion protein with the enhanced green fluorescent protein (pEGFP-N1). The animals were divided in five groups containing each five lambs. Each group was vaccinated as following (A: physiological solution; B: Gudair (TM), C: p-85A-Mav; D: p-85A-BCG; E: p-Hsp65). Immune response was evaluated by measuring the expression of INF-gamma (Th1 type response) and IL-10 (Th2 type response) by real-time PCR. Gene expression was estimated by comparing the results with that of P-actin. INF-gamma expression level was increased in lambs vaccinated with plasmids codifying mycobacterial antigens, in particular with p-Hsp65, in comparison with the controls suggesting stimulation of a Th1 immune response similar to that supported by natural infection of M. paratuberculosis. Moreover, animals were infected orally with live M. paratuberculosis. Three months after vaccination and again INF-gamma\ and IL-10 expression was evaluated in order to verify in vivo the protection level of the vaccines. Plasmids p-85A-BCG and p-Hsp65 seem to elicit a stronger protective immune response against M. paratuberculosis by evaluating the expression level of INF-gamma and evaluating the presence of M. paratuberculosis and animal cell organ damage post-mortem. (c) 2005 Elsevier Ltd. All rights reserved ANIMALS/ANTIGEN/antigens/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/CATTLE/CELL/CROHNS-DISEASE/DISEASE/DNA/DNA vaccine/DNA vaccines/ENTERITIS/EXPRESSION/GENE-EXPRESSION/IMMUNE-RESPONSE/INFECTION/interferon-gamma/Johne's disease/JOHNES-DISEASE/MESSENGER-RNA/mycobacteria/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PARATUBERCULOSIS INFECTION/PCR/PERIPHERAL-BLOOD/PROTEIN /real time PCR/REAL-TIME/RUMINANTS/SHEEP/T-CELLS/Th1 immune response/Tuberculosis/VACCINATION/WILD RUMINANTS

84 Judge, J., Kyriazakis, I., Greig, A., Davidson, R.S., Hutchings, M.R. (2006) Routes of intraspecies transmission of Mycobacterium avium subsp Paratuberculosis in rabbits (Oryctolagus cuniculus): a field study

Applied and Environmental Microbiology, 72, 398-403 Rabbits have been increasingly linked to the persistence of paratuberculosis (Johne's disease) in domestic ruminants in the United Kingdom. The aims of this study were to determine the routes of intraspecies transmission of Mycobacterium avium subspecies paratuberculosis (MAP) in rabbits and to estimate the probability of transmission via each route, in order to gain understanding of the dynamics of MAP in this host. Rabbits were sampled from two sites where MAP had previously been isolated from the livestock and rabbit populations. No pathology was noted in any animals, but the overall prevalence of MAP in rabbits was high at both sites studied, 39.7% and 23.0%, respectively. MAP was isolated from the testes, uterus, placenta, fetuses, and milk. This is the first time that the bacterium has been isolated from any of these tissues in a nonruminant wildlife species. These results suggest that transmission may occur vertically, pseudovertically, and horizontally. Vertical, i.e., transplacental, and/or pseudo-vertical, i.e., through the ingestion of contaminated milk and/or feces, transmission occurred in 14% of offspring entering the population at 1 month of age. As infection via these routes is only possible from infected adult females, this equates to a probability of infection via this route of 0.326. Probability of infection via horizontal transmission (including interspecies transmission) occurred at up to 0.037 per month. The

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presence of these routes of transmission within natural rabbit populations will contribute to the maintenance of MAP infections within such populations and, therefore, the environment ANIMALS/AUSTRALIA/ AVIUM/AVIUM SUBSP PARATUBERCULOSIS/AVIUM SUBSPECIES PARATUBERCULOSIS/BACTERIUM/BOVINE PARATUBERCULOSIS/DISEASE/ENVIRONMENT/FECAL CULTURE/FECES/FETUSES/INFECTED COWS/INFECTION/INFECTIONS/Johne's disease/JOHNES-DISEASE/MAP/MILK/mycobacteria/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/ORYCTOLAGUS-CUNICULUS/paratuberculosis/PATHOLOGY/POPULATIONS/PREVALENCE/rabbit/RABBITS/RUMINANTS/SCOTLAND/SENSITIVITY/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/TISSUE/TISSUES/Transmission/UNITED-KINGDOM/WILD RABBITS/WILDLIFE

85 Griffin, J.F.T., Spittle, E., Rodgers, C.R., Liggett, S., Cooper, M., Bakker, D., Bannantine, J.P. (2005) Immunoglobulin G1 enzyme-linked immunosorbent assay for diagnosis of Johne's disease in red deer (Cervus elaphus)

Clinical and Diagnostic Laboratory Immunology, 12, 1401-1409 This study was designed to develop a customized enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Johne's disease (JD) in farmed deer. Two antigens were selected on the basis of their superior diagnostic readouts: denatured purified protein derivative (PPDj) and undenatured protoplasmic antigen (PpAg). ELISA development was based on the antigen reactivity of the immunoglobulin G1 (IgG1) isotype, which is a highly specific marker for mycobacterial disease seroreactivity in deer. Sensitivity estimates and test parameters were established using 102 Mycobacterium paratuberculosis-infected animals from more than 10 deer herds, and specificity estimates were determined using 508 uninfected animals from 5 known disease-free herds. A receiver-operated characteristic analysis determined that at a cut point of 50 ELISA units, there was a specificity of 99.5% and sensitivities of 84.0% with PPDj antigen, 88.0% with PpAg, and 91.0% when the antigens were used serially in a composite test. Estimated sensitivity was further improved using recombinant protein antigens unique for M. paratuberculosis, which identified infected animals that were unreactive to PPDj or PpAg. While 80% of animals that were seropositive in the IgG1 ELISA had detectable histopathology, the assay could also detect animals with subclinical disease. The test was significantly less sensitive (75%) for animals that were culture positive for M. paratuberculosis but with no detectable pathology than for those with pathological evidence of JD (> 90%). When the IgG1 ELISA was used annually over a 4-year period in a deer herd with high levels of clinical JD, it eliminated clinical disease, increased production levels, and reduced JD-related mortality ANIMALS/ANTIBODY-RESPONSES/ANTIGEN/antigens/ASSAY/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/Cervus elaphus/CERVUS-ELAPHUS/CULTURE/deer/DIAGNOSIS/DISEASE/ELISA/enzyme-linked immunosorbent assay/FARMED DEER/HERD/INFECTION/Johne's disease/MORTALITY/mycobacteria/MYCOBACTERIUM-BOVIS/NEW-ZEALAND/paratuberculosis/PATHOLOGY/PROTEIN/RED DEER/SENSITIVITY/serodiagnosis/SEROLOGIC SURVEY/SPECIFICITY/Tuberculosis

86 Corn, J.L., Manning, E.J.B., Sreevatsan, S., Fischer, J.R. (2005) Isolation of Mycobacterium avium subsp paratuberculosis from free-ranging birds and mammals on livestock premises

Applied and Environmental Microbiology, 71, 6963-6967 Surveys for Mycobacterium avium subsp. paratuberculosis infection in free-ranging mammals and birds were conducted on nine dairy and beef cattle farms in Wisconsin and Georgia. Specimens were collected from 774 animals representing 25 mammalian and 22 avian species. Specimens of ileum, liver, intestinal lymph nodes, and feces were harvested from the larger mammals; a liver specimen and the gastrointestinal tract were harvested from birds and small mammals. Cultures were performed by using radiometric culture and acid-fast isolates were identified by 16S/IS900/IS1311 PCR and mycobactin dependency characteristics. M. avium subsp. paratuberculosis was cultured from tissues and feces from 39 samples from 30 animals representing nine mammalian and three avian species. The prevalence of infected wild animals by premises ranged from 2.7 to 8.3% in Wisconsin and from 0 to 6.0% in Georgia. Shedding was documented in seven (0.9%) animals: three raccoons, two armadillos, one opossum, and one feral cat. The use of two highly polymorphic short sequence repeat loci for analysis of 29 of the 39 strains identified 10 alleles. One allelic pattern broadly shared in domestic ruminants ("7,5") appeared in approximately one-third of the wildlife M. avium subsp. paratuberculosis isolates studied. Given the few cases of shedding by free-ranging animals compared to the volume of contaminated manure produced by infected domestic ruminant livestock, contamination of the farm environment by infected wildlife was

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negligible. Wildlife may, however, have epidemiological significance for farms where M. avium subsp. paratuberculosis recently has been eliminated or on farms free of M. avium subsp. paratuberculosis but located in the geographic vicinity of farms with infected livestock ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/CATTLE FARMS/CULTURE/deer/ENVIRONMENT/Epidemiology/FARMS/FECES/INFECTION/LYMPH-NODES/MAMMALS/mycobacteria/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PARATUBERCULOSIS INFECTION/PCR/PREVALENCE/RUMINANTS/SCOTLAND/STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/TISSUE/TISSUES/WILD ANIMALS/WILD RABBITS/WILDLIFE/Wisconsin

87 Whan, L., Ball, H.J., Grant, I.R., Rowe, M.T. (2005) Occurrence of Mycobacterium avium subsp paratuberculosis in untreated water in Northern Ireland

Applied and Environmental Microbiology, 71, 7107-7112 Mycobacterium avium subsp. paratuberculosis is the known cause of Johne's disease of both domestic and wild ruminants and has been implicated as a possible cause of Crohn's disease in humans. The organism is shed in the feces of infected animals and can survive for protracted periods in the environment and hence could be present in catchment areas receiving agricultural runoff. A limited survey was undertaken in Northern Ireland to test for M. avium subsp. paratuberculosis in untreated water entering nine water treatment works (WTWs) over a 1-year period. Three detection methods were employed, viz., immunomagnetic separation-PCR and culture on Herrold's egg yolk medium (HEYM) and BACTEC 12B medium, the latter both supplemented with mycobactins. Of the 192 untreated water samples tested, 15 (8%) tested M. avium subsp. paratuberculosis positive by one or more of the three detection methods. M. avium subsp. paratuberculosis was successfully isolated from eight untreated water samples, three by BACTEC culture and five by culture on HEYM. Although the highest incidence of M. avium subsp. paratuberculosis was found in spring, overall, there was no statistically significant difference between the seasons. No significant correlation was found between numbers of coliforms or fecal coliforms and the presence of M. avium subsp. paratuberculosis. In general, a higher incidence of M. avium subsp. paratuberculosis was found in untreated water entering those WTWs that had a high mean water pH value over the sampling period. This work indicates the need to determine the efficacy of water treatment processes to either kill or remove M. avium subsp. paratuberculosis from untreated water and the possible risks posed by contact with recreational water sources AIDS PATIENTS/ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/Crohn's disease/CULTURE/DECONTAMINATION/DISEASE/ENVIRONMENT/FECES/INFECTION/INTRACELLULARE/Johne's disease/MILK/mycobacteria/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/ORGANISM/paratuberculosis/RUMINANTS/SCOTLAND/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/WATER/WILD RUMINANTS

88 Dupont, C., Thompson, K., Heuer, C., Gicquel, B., Murray, A. (2005) Identification and characterization of an immunogenic 22 kDa exported protein of Mycobactefium avium subspecies paratuberculosis

Journal of Medical Microbiology, 54, 1083-1092 An exported 22 kDa putative lipoprotein was identified in an alkaline phosphatase gene fusion library of Mycobacterium avium subsp. paratuberculosis and expressed in Mycobacterium smegmatis. The full nucleic acid sequence of the gene encoding P22 was determined and the ORF was cloned into a mycobacterial expression vector, enabling full-length P22 to be produced as a C-terminal polyhistidine-tagged protein in M. smegmatis. N-terminal sequencing of the recombinant protein confirmed cleavage of a signal sequence. Native P22 was detected in culture supernatants and cell sonicates of M. avium subsp. paratuberculosis strain 31 6F using rabbit antibody raised to recombinant P22. Investigation of the presence of similar genes in other mycobacterial species revealed that the gene was present in Mycobacterium avium subsp. avium and similar genes existed in Mycobacterium intracellulare and Mycobacterium scrofulaceum. Database searches showed that P22 belonged to the LppX/LprAFG family of mycobacterial lipoproteins also found in Mycobacterium leprae and in members of the Mycobacterium tuberculosis complex. P22 shared less than 75% identity to these proteins. Recombinant P22 was able to elicit interferon-gamma secretion in blood from eight of a group of nine sheep vaccinated with a live attenuated strain of M. avium subsp. paratuberculosis (strain 31 6F) compared to none from a group of five unvaccinated sheep. Antibody to P22 was detected by Western blot analysis in 10 out of 11 vaccinated sheep, in two out of two clinically affected cows and in 11 out of 13 subclinically infected cows ANTIBODIES/ANTIGEN/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BACTERIAL

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LIPOPROTEINS/BOVIS/CELL/COMPLEX/CULTURE/EXPORTED PROTEIN/EXPRESSION/FAMILY/GENE/IDENTIFICATION/INFECTED COWS/INFECTION/interferon-gamma/INTRACELLULARE/JOHNES-DISEASE/LYMPHOCYTE-TRANSFORMATION/mycobacteria/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/paratuberculosis/PROTEIN/PROTEINS/ rabbit/SEROLOGICAL TESTS/SHEEP/SMEGMATIS/Tuberculosis/TUBERCULOSIS COMPLEX/TUBERCULOSIS DNA VACCINE/WHOLE-BLOOD

89 Palmer, M.V., Stoffregen, W.C., Carpenter, J.G., Stabel, J.R. (2005) Isolation of Mycobacterium avium subsp paratuberculosis (Map) from feral cats on a dairy farm with map-infected cattle

Journal of Wildlife Diseases, 41, 629-635 Paratuberculosis is an economically important disease of dairy cattle caused by Mycobacterium avium subsp. paratuberculosis (Map). The role of nonruminant, nondomestic animals in the epidemiology of paratuberculosis in cattle is unclear. To examine nonruminant, nondomestic animals for the presence of Map, 25 feral cats, nine mice (species unknown), eight rabbits (Sylvilagus floridanus), six raccoons (Procyon lotor), and three opossums (Didelphis virginiana) were collected from a mid-western dairy with known Map-infected cattle. Mycobacterium avium, subsp. paratuberculosis was isolated from the mesenteric lymph node from seven of 25 (28%) feral cats. Ileum was culture-positive for three of these seven cats, and an isolation of Map was also made from the ileum of one of nine (11%) mice. Tissue samples from other species were negative as determined by Map culture; microscopic lesions consistent with paratuberculosis were not seen in any animal. Restriction fragment polymorphism analysis of isolates from cats and dairy cattle suggest interspecies transmission. The means by which interspecies transmission occurred may be through ingestion of Map-contaminated feces or waste milk or through ingestion of Map-infected prey. Shedding of Map from infected cats was not evaluated. The epidemiologic role of Map-infected feral cats on dairy farms requires further investigation ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BISON/CATTLE/CULTURE/dairy cattle/DAIRY-CATTLE/deer/DISEASE/Epidemiology/FARMS/FECES/feral cats/Johne's disease/MAP/MICE/MILK/mycobacteria/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/ORAL INOCULATION/PARA-TUBERCULOSIS/paratuberculosis /PATHOLOGY/Procyon lotor/rabbit/RABBITS/RESTRICTION/SCOTLAND/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/TISSUE/Transmission/WILDLIFE

90 Judge, J., Kyriazakis, I., Greig, A., Allcroft, D.J., Hutchings, M.R. (2005) Clustering of Mycobacterium avium subsp paratuberculosis in rabbits and the environment: How hot is a hot spot?

Applied and Environmental Microbiology, 71, 6033-6038 Clustering of pathogens in the environment leads to hot spots of diseases at local, regional, national, and international levels. Scotland contains regional hot spots of Johne's disease (caused by Mycobacterium avium subsp. paratuberculosis) in rabbits, and there is increasing evidence of a link between paratuberculosis infections in rabbits and cattle. The spatial and temporal dynamics of paratuberculosis in rabbits within a hot spot region were studied with the overall aim of determining environmental patterns of infection and thus the risk of interspecies transmission to livestock. The specific aims were to determine if prevalence of paratuberculosis in rabbits varies temporally between seasons and whether the heterogeneous spatial environmental distribution of M. avium subsp. paratuberculosis on a large scale (i.e., regional hot spots) is replicated at finer resolutions within a hot spot. The overall prevalence of M. avium subsp. paratuberculosis in rabbits was 39.7%; the temporal distribution of infection in rabbits followed a cyclical pattern, with a peak in spring of 55.4% and a low in summer of 19.4%. Spatially, M. avium subsp. paratuberculosis-infected rabbits and, thus, the risk of interspecies transmission were highly clustered in the environment. However, this is mostly due to the clustered distribution of rabbits. The patterns of M. avium subsp. paratuberculosis infection in rabbits are discussed in relation to the host's socioecology and risk to livestock AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE TUBERCULOSIS/CATTLE/CROHNS-DISEASE/DISEASE/DISEASES/ENVIRONMENT/Epidemiology/GREAT-BRITAIN/INFECTION/INFECTIONS/Johne's disease/mycobacteria/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/ORYCTOLAGUS-CUNICULUS/paratuberculosis/PARATUBERCULOSIS INFECTION/POTENTIAL ROLE/PREVALENCE/rabbit/RABBITS/SCOTLAND/SEASONAL-CHANGES/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/Transmission/WILD RABBITS

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91 Koo, H.C., Park, Y.H., Ahn, J., Waters, W.R., Palmer, M.V., Hamilton, M.J., Barrington, G., Mosaad, A.A., Park, K.T., Jung, W.K., Hwang, I.Y., Cho, S.N., Shin, S.J., Davis, W.C. (2005) Use of rMPB70 protein and ESAT-6 peptide as antigens for comparison of the enzyme-linked immunosorbent, immunochromatographic, and latex bead agglutination assays for serodiagnosis of bovine tuberculosis

Journal of Clinical Microbiology, 43, 4498-4506 Current assays used to detect Mycobacterium bovis infection lack accuracy, especially for recently infected animals, or are impractical for rapid field diagnostic applications. To overcome these limitations with serological assays, a synthetic peptide derived from early secretory antigenic target 6 (ESAT6-p) and a recombinant major secreted immunogenic protein (rMPB70) of H. bovis were used in an enzyme-linked immunosorbent assay (EIA), an immunochromatographic assay (ICGA), and a latex bead agglutination assay (LBAA). Sera from noninfected, M. bovis-infected, or M. avium subsp. paratuberculosis-infected (by natural and experimental routes) animals were evaluated. Receiver operating characteristic analysis comparing optical density values from the EIA with results of bacterial culture or skin test, the reference test, established suitable cutoff values for assessing sensitivity and specificity. The EIA and LBAA, respectively, had sensitivities of 98.6 and 94.8%, specificities of 98.5 and 92.6%, and kappa values of 0.97 and 0.88 with ESAT6-p. The EIA, ICGA, and LBAA, respectively, had sensitivities of 96.8, 83.0, and 86.7%, specificities of 90.1, 99.4, and 97.8%, and kappa values of 0.87, 0.85, and 0.83 with rMPB70. Examination of serial samples of sera collected from experimentally M. bovis-infected cattle and deer revealed that ESAT6-p-specific responses developed early after infection whereas responses to rMPB70 developed later in the course of disease. The advantage of the LBAA and ICGA as initial tests for multiple species is a rapid reaction obtained in 2 to 3 h by LBAA or 20 min by ICGA without species-specific secondary antibodies under field conditions, thus allowing immediate segregation of suspect animals for further testing before culling ANIMALS/ANTIBODIES/ANTIBODY-RESPONSES/ANTIGEN/antigens/ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIAL CULTURE/BOVINE /BOVINE TUBERCULOSIS/BOVIS/CATTLE/CULTURE/CULTURE FILTRATE/deer/DISEASE/enzyme-linked immunosorbent assay/ESAT-6 /GAMMA-INTERFERON ASSAY/IMMUNE-RESPONSES/INFECTION/mycobacteria/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/PROTEIN/RESPONSES/SENSITIVITY/serodiagnosis/SKIN-TEST/SPECIFICITY/T-CELL RESPONSES/TEST-REACTOR CATTLE/TESTS/Tuberculosis/VIRULENT MYCOBACTERIUM-BOVIS/white-tailed deer

92 Godfroid, J., Delcorps, C., Irenge, L.M., Walravens, K., Marche, S., Gala, J.L. (2005) Definitive differentiation between single and mixed mycobacterial infections in red deer (Cervus elaphus) by a combination of duplex amplification of p34 and f57 sequences and Hpy188I enzymatic restriction of duplex amplicons

Journal of Clinical Microbiology, 43, 4640-4648 Severe emaciation and mortalities suggestive of mycobacterial infections were recently reported for both adult and young wild red deer (Cervus elaphus) in the southeastern part of Belgium. In deer, tuberculous lesions are not pathognomonic of Mycobacterium bovis infection due to gross and microscopic similarities with lesions caused by Mycobacterium avium subsp. paratuberculosis or M. avium subsp. avium. The aim of this study was to improve molecular methods for the species-specific identification of M. bovis, M. avium subsp. avium, and M. avium subsp. paratuberculosis in mycobacterial infections of deer. DNA banding patterns were assessed prior to and after Hpy188I restriction of f57-upstream (us)-p34 duplex amplicons. The duplex f57-us-p34 PCR differentiated M. bovis from M. avium subsp. paratuberculosis and M. avium subsp. avium infections, whereas the restriction step differentiated single M. avium subsp. paratuberculosis or M. avium subsp. avium infections from mixed M. avium subsp. paratuberculosis/M. avium subsp. avium infections. The endonuclease Hpy188I cleaves DNA between nucleotides N and G in the unique TCNGA sequence. This restriction site was found at position 168 upstream of the us-p34 initiation codon in all M. avium subsp. avium strains tested, regardless of their origin and the results of IS901 PCR. In contrast, the restriction site was abrogated in all M. avium subsp. paratuberculosis strains tested, independent of their origin, Mycobactin J dependency, and IS900 PCR results. Consequently, a two-step strategy, i.e., duplex us-p34-f57 PCR and Hpy188I restriction, allowed us to exclude M. bovis infection and to identify single (M. avium subsp. paratuberculosis or M. avium subsp. avium) or mixed (M. avium subsp. paratuberculosis/M. avium subsp. avium) infections in wild red deer in Belgium. Accordingly, we propose to integrate, in a functional molecular definition of M. avium subsp. paratuberculosis, the absence of the Hpy188I restriction site from the us-p34 amplicon AMPLIFICATION/AVIUM/AVIUM SUBSP-PARATUBERCULOSIS/BOVIS/Cervus elaphus/CERVUS-ELAPHUS/DAIRY-CATTLE/DECONTAMINATION/deer/DIAGNOSIS/DIFFERENTIATION/DNA/emaciation/FARMED DEER/IDENTIFICATION/INFECTION/INFECTIONS/IS900/IS901/JOHNES-DISEASE/MORTALITY/mycobacteria/Mycobacterium avium/Mycobacterium avium

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subsp/Mycobacterium bovis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-BOVIS/ODOCOILEUS-VIRGINIANUS/paratuberculosis/PCR/POLYMERASE-CHAIN-REACTION/RED DEER/RESTRICTION/STRAINS/TUBERCULOUS LESIONS/white-tailed deer

93 Maue, A.C., Waters, W.R., Davis, W.C., Palmer, M.V., Minion, F.C., Estes, D.M. (2005) Analysis of immune responses directed toward a recombinant early secretory antigenic target six-kilodalton protein-culture filtrate protein 10 fusion protein in Mycobacterium bovis-infected cattle Infection and Immunity, 73, 6659-6667 Cell-mediated immune responses are critical for protective immunity to mycobacterial infections. Recent progress in defining mycobacterial antigens has determined that region of difference 1 (RD1) gene products induce strong T-cell responses, particularly the early secretory antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP10). However, comprehensive analysis of the immune response towards these antigens is incompletely characterized. To evaluate recall responses to ESAT-6 and CFP10, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated in vitro with a recombinant ESAT-6 (rESAT-6)-CFP10 fusion protein and compared to responses induced by M. bovis-derived purified protein derivative. Following antigenic stimulation, activation marker expression was evaluated. Significant proliferative responses (P < 0.05) were evident in CD4(+), CD8(+), immunoglobulin M-positive, and CD172a(+) cell fractions after 6 days of culture. Expression of CD25 and CD26 was increased (P < 0.05) on CD4(+), CD8(+), and gamma delta T-cell-receptor-positive cells. CD4(+) and CD8(+) cells also exhibited significant changes (P < 0.05) in expression of CD45 isoforms. Using a flow cytometry-based proliferation assay, it was determined that CD45R expression is downregulated (P < 0.05) and that CD45RO expression is upregulated (P < 0.05) on proliferating (i.e., activated) CD4(+) cells. Collectively, data indicate that recall immune responses directed toward the rESAT-6-CFP10 fusion protein or purified protein derivative are comparable and that recall to mycobacterial antigens correlates with a CD45RO(+) phenotype ANTIGEN/antigens/ASSAY/AVIUM SUBSP PARATUBERCULOSIS/BCG/CALMETTE-GUERIN/CATTLE/CELL/CELLS/CULTURE/CULTURE FILTRATE/ESAT-6/EXPRESSION/GAMMA/GENE/IMMUNE-RESPONSE/IMMUNE-RESPONSES/INFECTION/INFECTIONS/interferon-gamma/MEMORY/MICE DEFICIENT/mycobacteria/PERIPHERAL-BLOOD/PROTEIN/RESPONSES/T-CELL ANTIGENS/T-CELL RESPONSES/TUBERCULOSIS INFECTION/white-tailed deer

94 Huntley, J.F., Stabel, J.R., Paustian, M.L., Reinhardt, T.A., Bannantine, J.P. (2005) Expression library immunization confers protection against Mycobacterium avium subsp paratuberculosis infection

Infection and Immunity, 73, 6877-6884 Currently, paratuberculosis vaccines are comprised of crude whole-cell preparations of Mycobacterium avium subsp. paratuberculosis. Although effective in reducing clinical disease and fecal shedding, these vaccines have severe disadvantages as well, including seroconversion of vaccinated animals and granulomatous lesions at the site of vaccination. DNA vaccines can offer an alternative approach that may be safer and elicit more protective responses. In an effort to identify protective M. avium subsp. paratuberculosis sequences, a genomic DNA expression library was generated and subdivided into pools of clones (similar to 1,500 clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging them with live, virulent M. avium subsp. paratuberculosis. Four clone pools resulted in a significant reduction in the amount of M. avium subsp. paratuberculosis recovered from mouse tissues compared to mice immunized with other clone pools and nonvaccinated, infected control mice. One of the protective clone pools was further partitioned into 10 clone arrays of 108 clones each, and four clone arrays provided significant protection from both spleen and mesenteric lymph node colonization by M. avium subsp. paratuberculosis. The nucleotide sequence of each clone present in the protective pools was determined, and coding region functions were predicted by computer analysis. Comparison of the protective clone array sequences implicated 26 antigens that may be responsible for protection in mice. This study is the first study to demonstrate protection against M. avium subsp. paratuberculosis infection with expression library immunization ANIMALS/ANTIGEN/antigens/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/DAIRY HERDS/DISEASE/DNA/DNA vaccine/DNA vaccines/EXPRESSION/GENE/IMMUNE-RESPONSE/IMMUNOGENICITY/INFECTION/MICE/MOUSE/mycobacteria/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PARATUBERCULOSIS INFECTION/PE-PGRS PROTEINS/RESPONSES/SUBSP PARATUBERCULOSIS/SUBSP PARATUBERCULOSIS INFECTION/SUBSP-PARATUBERCULOSIS/TISSUE/TISSUES/TUBERCULOSIS JOHNES DISEASE/VACCINATION

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95 Conraths, F.J., Kohler, H., Werner, O., Beer, M., Depner, K.R., Geue, L., Kaden, G., Staubach, C., Potzsch, C., Schares, G., Mettenleiter, T.C. (2005) The "Friedrich-Loeffler-Institut": Past, present and future of research in infectious diseases of animals

Berliner und Munchener Tierarztliche Wochenschrift, 118, 354-364 The Friedrich-Loeffler-Institut, founded in 1910 by Friedrich Loeffler, the discoverer of the first animal virus, foot-and-mouth disease virus, is the oldest virological research facility in the world. Beyond viruses, its area of competence has significantly expanded since its foundation and now also covers bacterial, parasitic and prion diseases of livestock, poultry and aquatic animals. Presently located at four sites within Germany (Insel Riems, Jena, Tubingen, Wusterhausen) the tasks of the institute as delineated in the Animal Disease Act encompass research on infectious animal diseases including zoonoses, import/export examinations, epidemiological studies in case of outbreaks of notifiable animal diseases, acting as reference laboratory for notifiable animal diseases and nationwide quality management of diagnosis of notifiable animal diseases. It is obliged to publish and maintain up-to-date diagnostic regimes for notifiable animal diseases, and it publishes a yearly report on animal health in Germany With the increasing importance of infectious diseases of animals, in particular those potentially harmful to man (zoonoses), the Friedrich-Loeffler-Institut will be moving into new facilities including laboratories and animal facilities up to the highest biosafety level at its main site Insel Riems on the occasion of its 100th anniversary Animal diseases/ANIMALS /AVIAN INFLUENZA/BOVINE NEOSPOROSIS/classical swine fever/CLASSICAL SWINE-FEVER/CROHNS-DISEASE/DAIRY-CATTLE/DIAGNOSIS/DISEASE/DISEASES/infectious disease/MYCOBACTERIUM-PARATUBERCULOSIS/NEOSPORA-CANINUM INFECTION/neosporosis/ORAL IMMUNIZATION/paratuberculosis/VERTICAL TRANSMISSION/WILD BOAR/zoonoses

96 Bulte, M., Schonenbrucher, H., Abdulmawjood, A. (2005) From farm to fork - Mycobacterium avium ssp paratuberculosis (MAP) as zoonotic agent?

Berliner und Munchener Tierarztliche Wochenschrift, 118, 377-385 Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of the paratuberculosis (Para Tb) in ruminants. In addition, this pathogen has been suspected to be implicated in the pathogenesis of Morbus Crohn disease (MC), causing chronic inflammatory intestine changes of humans. The participation of MAP in this illness is discussed intensively and has very contradictory opinions. On the one hand several times succeeded in proving MAP DNA in changed human tissues as well as, in recent time, the bacteria has been isolated from patient's blood. On the other hand there are many publications which support the opposite opinion. In critical evaluation of already available data, therefore the hypothesis can be formulated that MAP could possibly take part in the MC of humans. The reliable verification of this hypothesis will only be possible, if the diagnostic procedures can be refined upon the substantial deficit concerning the sensitiviiy and/or specificity of the diagnostic methods. In addition, till now there is lack of optimized statistically case control studies. The conceivable transmission of the bacteria to humans by the direct animal contact has been considered as possible vector, furthermore, MAP has been detected in pasteurised milk and other food of animal origin. The prevalence data, usually estimated by ELISA for milk cattle stock show over 80% prevalence in many counties of the Federal Republic of Germany with an individual case prevalence ranging between 1% and 17% in different stocks. Comparable data are present also from other countries as well as for small ruminants. MAP has been concerned as a global problem, moreover the high spreading rate of MAP in wild animal populations as well as the considerable ability of the bacteria to survive in different stages of the infectious- and contamination-cycle, which might hardly be broken through. Thus it requires intensive research efforts for the development of the methodical diagnostic process as basis for valid epidemiological investigations of animals, humans and food ABILITY/ANIMALS/AVIUM/BACTERIUM/BIOPSY SPECIMENS/CATTLE/CROHNS-DISEASE/detection methods/DISEASE/DNA/ECONOMIC-LOSSES/ELISA/food/GENETIC-VARIATION/IMMUNOMAGNETIC PCR/INFLAMMATORY-BOWEL-DISEASE/JOHNES-DISEASE/MAP/MAP-DNA/MILK/Morbus Crohn (MC)/mycobacteria/Mycobacterium avium/Mycobacterium avium ssp.paratuberculosis (MAP)/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/paratuberculosis/PATHOGENESIS/POLYMERASE CHAIN-REACTION/POPULATIONS/PREVALENCE/RUMINANTS/SPECIFICITY/SUBSP PARATUBERCULOSIS/TISSUE/TISSUES/Transmission

97 Hruska, K., Bartos, M., Kralik, P., Pavlik, I. (2005) Mycobacterium avium subsp paratuberculosis in powdered infant milk: paratuberculosis in cattle - the public health problem to be solved

Veterinarni Medicina, 50, 327-335

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Fifty one products of dried milk baby food purchased from 10 producers from seven countries available on the Czech market have been tested. IS900, the specific fragments for Mycobacterium avium subsp. paratuberculosis ( MAP) have been detected using PCR in 25 samples (49.0 %) and fragment f57 by real time PCR in 18 samples (35.3%). These results correspond to the epidemiological situation in Europe and are not unexpected. Paratuberculosis in cattle was almost unknown in the Czech Republic until 1990. An increase in the number of cows with paratuberculosis found in slaughterhouses and the incidence of Crohn's disease in the last decade is evident. The possible risk of MAP dead cells or bacterial structures in food is discussed in respect to autoimmune Crohn's disease. The national programmes of paratuberculosis control and certification of paratuberculosis- free herds should be strongly supported to decrease the risk for children and other people under higher risk. Producers should use MAP free milk for baby food production on a voluntary basis AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BULK-TANK MILK/CATTLE/CELL/CELLS/Crohn's disease/CROHNS-DISEASE/DISEASE/f57/food/HEAT-SHOCK PROTEIN/HERD/INFECTION/INFLAMMATORY-BOWEL-DISEASE/IS900/Johne's disease/JOHNES-DISEASE/MAP/MILK/mycobacteria/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/paratuberculosis control/PASTEURIZED COWS MILK/PCR/real time PCR/REAL-TIME/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/ULCERATIVE-COLITIS/UNITED-KINGDOM/WILD RUMINANTS

98 Waters, W.R., Palmer, M.V., Bannantine, J.P., Greenwald, R., Esfandiari, J., Andersen, P., McNair, J., Pollock, J.M., Lyashchenko, K.P. (2005) Antibody responses in reindeer (Rangifer tarandus) infected with Mycobactetium bovis Clinical and Diagnostic Laboratory Immunology, 12, 727-735 Despite having a very low incidence of disease, reindeer (Rangifer tarandus) are subject to tuberculosis (TB) testing requirements for interstate shipment and herd accreditation in the United States. Improved TB tests are desperately needed, as many reindeer are falsely classified as reactors by current testing procedures. Sera collected sequentially from 11 (experimentally) Mycobacterium bovis-infected reindeer and 4 noninfected reindeer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for antibody specific to M. bovis antigens. Specific antibody was detected as early as 4 weeks after challenge with M. bovis. By MAPIA, sera were tested with 12 native and recombinant antigens, which were used to coat nitrocellulose. All M. bovis-infected reindeer developed responses to MPB83 and a fusion protein, Acrl/MPB83, and 9/11 had responses to MPB70. Other antigens less commonly recognized included MPB59, ESAT-6, and CFPIO. Administration of purified protein derivatives for skin testing boosted serum antibody responses, as detected by each of the assays. Of the noninfected reindeer, 2/4 had responses that were detectable immediately following skin testing, which correlated with pathological findings (i.e., presence of granullomatous lesions yet the absence of acid-fast bacteria). The levels of specific antibody produced by infected reindeer appeared to be associated with disease progression but not with cell-mediated immunity. These findings indicate that M. bovis infection of reindeer elicits an antibody response to multiple antigens that can be boosted by skin testing. Serological tests using carefully selected specific antigens have potential for early detection of infections in reindeer ANTIBODIES/ANTIBODY-RESPONSES/ANTIGEN/ANTIGEN RECOGNITION/antigens/ASSAY/BACTERIUM/BOVIS/cell-mediated immunity/challenge/CROSS-REACTIVITY/DISEASE/ELISA/enzyme-linked immunosorbent assay/ESAT-6/HERD/IMMUNE-RESPONSES/INFECTION/INFECTIONS/LINKED-IMMUNOSORBENT-ASSAY/mycobacteria/ODOCOILEUS-VIRGINIANUS/PATHOLOGICAL FINDINGS/PROTEIN/PROTEIN MPB70/RESPONSES/SEROLOGICAL TESTS/SERUM ANTIBODIES/SUBSP PARATUBERCULOSIS INFECTION/TESTS/Tuberculosis/UNITED-STATES/white-tailed deer

99 Holstad, B.G., Sigurdardottir, O.G., Storset, A.K., Tharaldsen, J., Nyberg, O., Schonheit, J., Djonne, B. (2005) Description of the infection status in a Norwegian cattle herd naturally infected by Mycobacterium avium subsp paratuberculosis Acta Veterinaria Scandinavica, 46, 45-56 The Norwegian surveillance and control programme for paratuberculosis revealed 8 seroreactors in a single dairy cattle herd that had no clinical signs of Mybacterium avium subsp. paratuberculosis (M. a. paratuberculosis) infection. Paratuberculosis had been a clinical problem in goats several years previously in this herd. All 45 cattle were culled and a thorough investigation of the infection status was conducted by the use of interferon-gamma (IFN-gamma) immunoassay, measurement of antibodies, and pathological and bacteriological examination. In the IFN-gamma immunoassay, 9 animals gave positive results, and 13 were weakly positive, while 19 animals were negative. In the serological test, 10 animals showed positive reactions, and 5 were doubtful, while 30 animals gave negative reactions. There

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appeared to be a weak trend toward younger animals having raised IFN-gamma and older animals having raised serological tests. Histopathological lesions compatible with paratuberculosis were diagnosed in 4 animals aged between 4 and 9 years. Three of these animals had positive serological reaction and one animal gave also positive results in the IFN-gamma immunoassay. Infection was confirmed by isolation of M. a. paratuberculosis from 2 of these 4 animals. One single bacterial isolate examined by restriction fragment length polymorphism (RFLP) had the same profile, B-Cl, as a strain that had been isolated from a goat at the same farm several years previously. Despite many animals being positive in one or both of the immunological tests, indicative of a heavily infected herd, none of the animals showed clinical signs and only one cow was shown to be shedding bacteria. A cross-reaction with other mycobacteria might have caused some of the immunoreactions in these animals. It is also possible that the Norwegian red cattle breed is resistant to clinical infection with M. a. paratuberculosis ANIMALS/ANTIBODIES/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIUM/BOVINE JOHNES-DISEASE/CATTLE/dairy cattle/DAIRY HERDS/DAIRY-CATTLE/DIAGNOSIS/DOMESTIC SHEEP/ELISA/Epidemiology/FECAL CULTURE/FRAGMENT-LENGTH-POLYMORPHISM/goat/HERD/IFN-gamma/INFECTION/interferon-gamma/mycobacteria/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/RESTRICTION/RFLP/SEROLOGICAL TESTS/SUBCLINICAL PARATUBERCULOSIS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/TESTS/WILD RUMINANTS

100 Mackintosh, C.G., Labes, R.E., Griffin, J.F.T. (2005) The effect of Johne's vaccination on tuberculin testing in farmed red deer (Cervus elaphus)

New Zealand Veterinary Journal, 53, 216-222 AIM: To assess the degree of interference with bovine tuberculin testing in farmed red deer that vaccination of young deer with an oil-adjuvanted vs aqueous formulation of live attenuated Mycobacterium paratuberculosis Strain 316F vaccines would be likely to cause, and to compare immunological responses between vaccine formulations. METHODS: Five-month-old red deer (n=45) were randomly allocated to three treatment groups of 15 animals, which received either no vaccine, a single 2-ml dose of an oil-adjuvanted formulation or two 2-ml doses, 6 weeks apart, of an aqueous formulation of live attenuated M. paratuberculosis Strain 316F vaccine injected subcutaneously (S/C) in the neck (Control, Oil-adjuvant Ptb, and Aqueous Ptb groups, respectively). Injection-site reactions were described and measured on Weeks 3, 6 and 9. Animals were weighed and lymphocyte transformation tests (LTT) and antibody enzyme-linked immunosorbent assays (ELISA) using avian, bovine and Johnin tuberculin were conducted on blood samples collected at Weeks 0, 6, 12, 15, 24, 27, 36 and 39. A bovine mid-cervical skin test (MCT) was applied at Week 12, and comparative cervical skin tests (CCTs) at Weeks 24 and 36. At Week 42, the animals were slaughtered at a commercial deer slaughter premises and subjected to rigorous meat inspection. RESULTS: Two animals were eliminated at the start of the trial due to a positive cross-reaction with bovine tuberculin in the initial ITT. Almost all animals reacted to the MCT at Week 12, with mean skin thicknesses of 3.9, 2.9 and 1.0 mm for the Oil-adjuvant Ptb, Aqueous Ptb and Control groups, respectively. When the CCT was conducted at Week 24, 2/15 Oil-adjuvant Ptb, 2/14 Aqueous Ptb and 1/14 Control animals were classified as CCT-positive to bovine tuberculin. By Week 36, all animals were CCT-negative. The Oil-adjuvant Ptb vaccination resulted in high persistent levels of antibody that reacted with bovine tuberculin, compared with negligible levels in the Aqueous Ptb group. Overall, a single dose of the Oil-adjuvant Ptb vaccine in deer stimulated a vigorous, cross-reactive immune response, evidenced by high LTT, skin-test and antibody reactions to bovine tuberculin, with both cell-mediated and Immoral characteristics. By comparison, two doses of the Aqueous Ptb vaccine produced less cross-reactivity and a bias towards a cell-mediated response. The Oil-adjuvant Ptb vaccine resulted in moderate injection-site lesions that were quite persistent, whereas the Aqueous Ptb vaccine resulted in smaller nodules that regressed more quickly. CONCLUSIONS: Vaccination of farmed deer with an oil-adjuvanted Johne's vaccine has the potential to cause significant interference with routine tuberculin skin testing. The cross-reactivity should decline with time and the CCT should be able to clear MCT-positives, but there is a risk of false-positives to the blood test for tuberculosis (BTB), due to high persistent levels of antibody. The CCT could be used as a primary skin test in vaccinated deer on some farms. The Aqueous Ptb caused fewer problems with skin testing and produced significantly less bovine antibody than the Oil-adjuvant Ptb, but stimulated persistent cell-mediated immune responses that may provide some protection against Johne's disease ANIMALS/ANTIBODIES/ASSAY/blood testing/BOVINE/bovine tuberculosis control/BOVIS/Cervus elaphus/CERVUS-ELAPHUS/CROSS-REACTIVITY/deer/DIAGNOSIS/DISEASE/ELISA/enzyme-linked immunosorbent assay/FARMED DEER/FARMED RED DEER/FARMS/IMMUNE-RESPONSE/IMMUNE-RESPONSES/Johne disease/Johne's disease/LYMPHOCYTE-TRANSFORMATION/mycobacteria/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/neoparasec/NEW-ZEALAND/paratuberculosis /RED DEER/RESPONSES/skin testing/SKIN-TEST/TESTS/Tuberculosis/VACCINATION

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101 Deutz, A., Spergser, J., Wagner, P., Rosengarten, R., Kofer, J. (2005) Mycobacterium avium subsp paratuberculosis in wild animal species and cattle in Styria/Austria

Berliner und Munchener Tierarztliche Wochenschrift, 118, 314-320 Infections with Mycobacterium avium ssp. parotuberculosis (M. paratuberculosis) are increasingly recognised worldwide. In addition to an increased prevalence of paratuberculosis in Austrian cattle herds, recent years have also shown a rise in infections with M. paratuberculosis in wild red and roe deer, chamois and mouflon. During the period from June 2002 to September 2004, mesenteric lymph nodes were taken from a total of 483 wild animals hunted or found dead and from 338 deceased cattle. Samples were analysed using PCR and cultivation methods. In the case of pathomorphological changes or anamnestic indications, investigations also included an analysis of organ samples (e.g. liver, lung) or foetuses. The tests revealed that 129 wild animal samples (red deer, roe deer, chamois, mouflon, fallow deer, ibex, foxes, mountain hare, yellow-necked field mouse, and capercaillie) contained M.paratuberculosis. The major symptoms in the wild animals affected were weight loss and significantly enlarged mesenteric lymph nodes. Evidence of diarrhoea was only observed in about 15 % of the positive cases. The study for the first time provided evidence of intrauterine transmission of M. paratuberculosis in red deer (3 cases) and chamois (1 case) and succeded in the isolation of the pathogen from the liver, lung and subcutaneous granulomas of wild animals. Of the total of 338 mesenteric lymphnodes of cattle from 303 herds, 80 samples from 77 herds tested positive for paratuberculosis. Twentytwo wild animal and 3 cattle isolates have so far been molecularly typed using IS900-RFLP and RAPD analyses in order to prove epidemiological relationships between occurrences in cattle and wild animals. The increase of paratuberculosis in wild animal species is assumed to have been caused by the purchase of animals, a strong increase in suckler cow farming (cow-calf herds) with a concentration of pathogens in the environment and by inadequate feed hygiene for wild animals ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/CULTURE/deer/DEER CERVUS-ELAPHUS/ DNA/ENVIRONMENT/FALLOW DEER/FRAGMENT-LENGTH-POLYMORPHISM/HERD/INFECTION/INFECTIONS/Intrauterine/IS900/JOHNES-DISEASE/LYMPH-NODES/M.avium subsp paratuberculosis/M.paratuberculosis/MOLECULAR CHARACTERIZATION/MOUSE/mycobacteria/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PCR/POLYMERASE-CHAIN-REACTION/PREVALENCE/RAPD/RED DEER/RUMINANT PARA-TUBERCULOSIS/SHEEP/Styria/ SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS /TESTS/Transmission/WILD ANIMALS

102 Miltner, E., Daroogheh, K., Mehta, P.K., Cirillo, S.L.G., Cirillo, J.D., Bermudez, L.E. (2005) Identification of Mycobacterium avium genes that affect invasion of the intestinal epithelium

Infection and Immunity, 73, 4214-4221 Invasion of intestinal mucosa of the host by Mycobacterium avium is a critical step in pathogenesis and likely involves several different bacterial proteins, lipids, glycoproteins, and/or glycolipids. Through the screening of an M. avium genomic library in Mycobacterium smegmatis, we have identified a number of M. avium genes that are associated with increased invasion of mucosal epithelial cells. In order to further investigate these genes, we cloned six of them into a plasmid downstream of a strong mycobacterial promoter (L5 mycobacterial phage promoter), resulting in constitutive expression. Bacteria were then evaluated for increased expression and examined for invasion of HT-29 intestinal epithelial cells. The genes identified encode proteins that are similar to (i) M. tuberculosis coenzyme A carboxylase, (ii) M. tuberculosis membrane proteins of unknown function, (iii) M. tuberculosis FadE20, (iv) a Mycobacterium paratuberculosis surface protein, and (v) M. tuberculosis cyclopropane fatty acyl-phopholipid synthase. The constitutive expression of these genes confers to M. avium the ability to invade HT-29 intestinal epithelial cells with a severalfold increase in efficiency compared to both the wild-type H. avium and M. avium containing the vector alone. Using the murine intestinal ligated loop model, it was observed that the constitutive expression of M. avium proteins has a modest impact on the ability to enter the intestinal mucosa when compared with the wild-type control, suggesting that under in vivo conditions these genes are expressed at higher levels. Evaluation of the expression of these invasion-related genes indicated that under conditions similar to the intestinal lumen environment, the genes identified are upregulated. These data suggest that invasion of the intestinal mucosa is an event that requires the participation of several bacterial factors and the expression of the genes that encode them is less observed under standard laboratory growth conditions ABILITY/AVIUM/BACTERIUM/CELL/CELLS/COMPLEX/ENTRY/ENVIRONMENT/EXPRESSION/GENE/IDENTIFICATION/IMPACT/INFECTION/MEMBRANE/MODEL/mycobacteria/Mycobacterium avium/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/PATHOGENESIS/PROTEIN/PROTEINS/SALMONELLA/SMEGMATIS/Tuberculosis/TYPHIMURIUM

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103 Siguroardottir, O.G., Bakke-McKellep, A.M., Djonne, B., Evensen, O. (2005) Mycobacterium avium subsp paratuberculosis enters the small intestinal mucosa of goat kids in areas with and without Peyer's patches as demonstrated with the everted sleeve method Comparative Immunology Microbiology and Infectious Diseases, 28, 223-230 The main lesions of paratuberculosis in ruminants are in the small intestine. Previous studies have shown that the bacterium enters the small intestine through M cells found in the follicle-associated epithelium lining the domes of the Peyer's patches. The everted sleeve method, devised for the in vitro study of intestinal absorption, was used in this study to investigate the uptake of Mycobacterium avium subsp. paratuberculosis in goat intestine. Everted small intestinal sleeves of goat kids, prepared from areas with and without Peyer's patches, were incubated for 60 min in 3 H-labeled bacterial solution. The results of this study imply that the bacteria can enter the intestinal mucosa of the jejunum, both in areas with and without Peyer's patches. These findings indicate, therefore, that M. avium subsp. paratuberculosis bacteria not only enter through M cells but also through enterocytes. (c) 2005 Published by Elsevier Ltd AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIUM/CELL/CELLS/EPITHELIAL-CELLS/everted sleeves/FIBRONECTIN ATTACHMENT PROTEIN/follicle-associated epithelium/goat/goats/INFECTION/intestinal uptake/INVASION/LESIONS/M cells/MOUSE/mycobacteria/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/Peyer's patches/RUMINANTS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS

104 Biet, F., Boschiroli, M.L., Thorel, M.F., Guilloteau, L.A. (2005) Zoonotic aspects of Mycobacterium bovis and Mycobacterium avium-intracellulare complex (MAC)

Veterinary Research, 36, 411-436 Pathogens that are transmitted between the environment, wildlife, livestock and humans represent major challenges for the protection of human and domestic animal health, the economic sustainability of agriculture, and the conservation of wildlife. Among such pathogens, the genus Mycobacterium is well represented by M. bovis, the etiological agent of bovine tuberculosis, M. avium ssp. paratuberculosis ( Map) the etiological agent of Johne disease, M. avium ssp. avium (Maa) and in a few common cases by other emergent environmental mycobacteria. Epidemiologic surveys performed in Europe, North America and New Zealand have demonstrated the existence and importance of environmental and wildlife reservoirs of mycobacterial infections that limit the attempts of disease control programmes. The aim of this review is to examine the zoonotic aspects of mycobacteria transmitted from the environment and wildlife. This work is focused on the species of two main groups of mycobacteria classified as important pathogens for humans and animals: first, M. bovis, the causative agent of bovine tuberculosis, which belongs to the M. tuberculosis complex and has a broad host range including wildlife, captive wildlife, domestic livestock, non-human primates and humans; the second group examined, is the M. avium-intracellulare complex ( MAC) which includes M. avium ssp. avium causing major health problems in AIDS patients and M. avium ssp. paratuberculosis the etiological agent of Johne disease in cattle and identified in patients with Crohn disease. MAC agents, in addition to a broad host range, are environmental mycobacteria found in numerous biotopes including the soil, water, aerosols, protozoa, deep litter and fresh tropical vegetation. This review examines the possible reservoirs of these pathogens in the environment and in wildlife, their role as sources of infection in humans and animals and their health impact on humans. The possibilities of control and management programmes for these mycobacterial infections are examined with regards to the importance of their natural reservoirs AIDS PATIENTS/AMERICAN WILD RUMINANTS/ANIMALS/Avian tuberculosis/AVIUM /BOVINE/BOVINE TUBERCULOSIS/BOVIS/CATTLE/challenge/COMPLEX/CROHNS-DISEASE/DISEASE/ENVIRONMENT/ENVIRONMENTAL MYCOBACTERIA/IMPACT/INFECTION/INFECTIONS/Johne disease/MAP/mycobacteria/Mycobacterium/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/NEW-ZEALAND/NONTUBERCULOUS MYCOBACTERIA/paratuberculosis/POTABLE WATER/Review/RUMINANT PARA-TUBERCULOSIS/SUBSP PARATUBERCULOSIS/Tuberculosis/TUBERCULOSIS COMPLEX/TUBERCULOSIS JOHNES DISEASE/WATER/white-tailed deer/WILDLIFE/wildlife reservoir/zoonosis

105 McGuire, K., Glass, E.J. (2005) The expanding role of microarrays in the investigation of macrophage responses to pathogens

Veterinary Immunology and Immunopathology, 105, 259-275 In the last few years, microarray technology has emerged as the method of choice for large-scale gene expression studies. It provides an efficient and rapid method to investigate the entire transcriptome of a

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cell. No research field has benefited more from microarray technology than the study of the exquisite interplay between pathogens and hosts. Numerous microarray studies have now been published in this field, which have provided insights into the mechanisms of host defence and the tactics employed by pathogens to circumvent these protection strategies. These studies have led to a more comprehensive understanding of the host immune response and identified new avenues of research for potential control strategies against pathogens. In the past, research has concentrated on human and mouse microarrays to investigate host-pathogen interactions, regardless of the host species. This trend is changing with the ever-expanding sequence resources now available for many pathogen and host species, including livestock animals. The use of species-specific microarrays has furthered our understanding of host-pathogen interactions for particular organisms and aided in the annotation of unknown genes. Macrophages play a central role in the host's innate and adaptive immune responses to pathogens. These cells are in the first line of defence and interact with a wide range of pathogens; many of which have evolved strategies to circumvent the macrophage defence mechanisms and survive within these cells. In this report, we review the wealth of studies using microarray technology to investigate the response of macrophages to pathogens. These studies illustrate how microarray technology has expanded our understanding of the dialogue between macrophage and pathogen and provide examples of the benefits and pitfalls of using this technique. Furthermore, we discuss the resources available to use microarray analysis to study the immune response of a non-human, non-rodent species, the cow. (c) 2005 Elsevier B.V. All rights reserved ANIMALS/AVIUM SUBSP PARATUBERCULOSIS/BLOOD MONONUCLEAR-CELLS/BOVINE/CELL/CELLS/COMPLETE GENOME SEQUENCE/DNA-MICROARRAY/EQUALIZED CDNA LIBRARY/EXPRESSION/GENE/gene expression/GENE-EXPRESSION/GENE-EXPRESSION PROFILES/host-pathogen interactions/IMMUNE-RESPONSE/IMMUNE-RESPONSES/macrophage/MESSENGER-RNA EXPRESSION/microarray/MOUSE/MYCOBACTERIUM-TUBERCULOSIS INFECTION/ORGANISM/ORGANISMS/RESPONSES/Review/THEILERIA-ANNULATA/TUMOR-NECROSIS-FACTOR

106 Ellingson, J.L.E., Stabel, J.R., Radcliff, R.P., Whitlock, R.H., Miller, J.M. (2005) Detection of Mycobacterium avium subspecies paratuberculosis in free-ranging bison (Bison bison) by PCR

Molecular and Cellular Probes, 19, 219-225 Bacterial culture is the 'gold standard' for detecting Mycobacterium avium subspecies paratuberculosis (MAP) infection, but is time consuming. laborious, and recovery of organism varies with species of animal tested. PCR has been used for detection of MAP DNA in feces and tissues. We used PCR to detect MAP DNA isolated from tissues from 25 free-ranging North American bison (Bison bison), each with clinical signs compatible with Johne's disease. We report the performance of PCR to detect MAP DNA in both frozen and paraffin-embedded ileum, jejunum. and ileocecal lymph node samples collected at the time of slaughter. Specific oligonucleotide primers for PCR amplification were derived from 16S rRNA sequence M. avium subspecies (MAs) and insertion elements IS1245 (MAs avium), IS901 (MAs avium), IS900 (MAP). and hspX (MAP). Genomic DNA samples were prepared three different ways; crude DNA from frozen tissues, crude DNA from paraffin-embedded tissues. and purified DNA from paraffin-embedded tissues. An animal was considered infected if MAP DNA was detected in at least two separate tissues using the IS900 primer set. Using these criteria, 25 of 25 bison tested were positive for MAP. The data indicate that these free-ranging bison have been infected by MAP. (c) 2005 Elsevier Ltd. All rights reserved AMERICAN WILD RUMINANTS/AMPLIFICATION/AVIUM /AVIUM SUBSPECIES PARATUBERCULOSIS/BACTERIAL CULTURE/BISON/CULTURE/DISEASE/DNA/DOMESTIC SHEEP/EXPERIMENTAL-INFECTION/FECES/FRAGMENT-LENGTH-POLYMORPHISM/IDENTIFICATION/INFECTION/INSERTION ELEMENT/IS1245/IS900/IS901/Johne's disease/JOHNES DISEASE/MAP/MAP-DNA/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/ORGANISM/paraluberculosis : Johne's disease/paratuberculosis/PCR/POLYMERASE-CHAIN-REACTION/RUMINANT PARA-TUBERCULOSIS/TISSUE/TISSUES

107 Judge, J., Greig, A., Kyriazakis, I., Hutchings, M.R. (2005) Ingestion of faeces by grazing herbivores - risk of inter-species disease transmission

Agriculture Ecosystems & Environment, 107, 267-274 Grazing herbivores are in contact with faeces and the faecal-oral route is a common mode of transmission of parasite species that represent the most pervasive challenge to host fecundity and survival. Two grazing experiments were carried out to quantify the rate of faeces ingestion by grazing herbivores using the example of rabbits and the risk they pose to ruminants of paratuberculosis. Ten sheep and 10 cattle were each presented with three replicates of nine sward treatments, created through three sward heights and three densities of rabbit faeces and allowed to graze for short periods. Both

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sheep and cattle ingested rabbit faecal pellets, the latter showing no behavioural avoidance of rabbit faeces while grazing. Sward characteristics affected the probability and rate of ingestion of faeces and thus parasites. Level of faecal contamination affected the proportion of pellets ingested by sheep, and the probability of cattle ingesting one or more faecal pellets. Sward height affected the proportion of pellets ingested by cattle but not sheep, demonstrating there is the potential to reduce the risks of parasitism by managing grazing systems. (c) 2004 Elsevier B.V. All rights reserved CATTLE/challenge/DISEASE/disease transmission/faecal-oral route/FECAL AVOIDANCE/INFECTION/livestock disease/NEMATODES/paratuberculosis/POPULATION/rabbit/RABBITS/RABBITS ORYCTOLAGUS-CUNICULUS/RUMINANTS/SCOTLAND/SHEEP/Transmission/Tuberculosis/WILD RABBITS

108 Li, Y.J., Miltner, E., Wu, M., Petrofsky, M., Bermudez, L.E. (2005) A Mycobacterium avium PPE gene is associated with the ability of the bacterium to grow in macrophages and virulence in mice

Cellular Microbiology, 7, 539-548 PPE and PE gene families, which encode numerous proteins of unknown function, account for 10% of Mycobacterium tuberculosis genome. Mycobacterium avium genome has similar PPE and PE gene families. Using a temperature-sensitive phage phAE94 transposon mutagenesis system, a M. avium transposon library was created in the strain MAC109. Screening of individual mutants in human U937 macrophages for the ability to replicate intracellularly, we identified several attenuated clones. One of them, the 2D6 mutant, has a transposon interrupting a PPE gene (52% homologous to Rv 1787 in M. tuberculosis) was identified. The mutant and the wild-type strain had comparable ability to enter macrophages. Challenge of mice with the 2D6 mutant resulted in approximately 1 log and 2 log fewer bacteria in the spleen, at 1 and 3 weeks after infection, compared with the wild-type bacterium. The 2D6 mutant grows like the wild-type bacterium in vitro. Vacuoles containing the 2D6 mutant acidified to pH 4.8; whereas, vacuoles containing wild-type bacterium were only slightly acidic. It was also observed that, in contrast to the wild-type bacterium, the 2D6 mutant did not prevent phagosome-lysosome fusion, and it is only expressed within macrophage but not in 7H9 broth. These results revealed a role for this PPE gene in the growth of M. avium in macrophages and in virulence in mice ABILITY/ACIDIFICATION/AVIUM/BACTERIUM/challenge/EXPRESSION/FAMILY/GENE/INFECTION/INTRACELLULAR SURVIVAL/macrophage/MATURATION/MICE/MURINE MACROPHAGES/mycobacteria/Mycobacterium/Mycobacterium avium/MYCOBACTERIUM-AVIUM/paratuberculosis/PHAGOSOMAL PH/PROTEIN/PROTEINS/TRANSPOSON MUTAGENESIS/Tuberculosis/TUMOR-NECROSIS-FACTOR/VIRULENCE

109 Ayele, W.Y., Svastova, P., Roubal, P., Bartos, M., Pavlik, I. (2005) Mycobacterium avium subspecies paratuberculosis cultured from locally and commercially pasteurized cow's milk in the Czech Republic Applied and Environmental Microbiology, 71, 1210-1214 Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7 degrees C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp. paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp. paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk. M. avium subsp. paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from I of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp. paratuberculosis. These findings confirm that M. avium subsp. paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp. paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/CONDITIONS SIMULATING PASTEURIZATION/CROHNS-DISEASE/CULTURE/DAIRY HERDS/DIAGNOSIS/HEAT INACTIVATION/HERD/HIGH-TEMPERATURE/INFECTED COWS/INFECTION/IS900/JOHNES-

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DISEASE/MILK/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/paratuberculosis/PARATUBERCULOSIS INFECTION/PASTEURIZATION/PCR/raw milk/RAW-MILK/UNITED-KINGDOM /UNITED-STATES/WILD RUMINANTS

110 Alvarez, J., de Juan, L., Briones, V., Romero, B., Aranaz, A., Fernandez-Garayzabal, J.F., Mateos, A. (2005) Mycobacterium avium subspecies paratuberculosis in fallow deer and wild boar in Spain Veterinary Record, 156, 212-213 AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/deer/Epidemiology/FALLOW DEER/FRAGMENT-LENGTH-POLYMORPHISM/JOHNES-DISEASE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/SCOTLAND/WILD BOAR

111 Vaughan, J.A., Lenghaus, C., Stewart, D.J. , Tizard, M.L., Michalski, W.P. (2005) Development of a Johne's disease infection model in laboratory rabbits following oral administration of Mycobacterium avium subspecies paratuberculosis

Veterinary Microbiology , 105, 207-213 To assess the rabbit as a model for the study of Johne's disease pathogenesis, a breeding group of adult and juvenile New Zealand white rabbits were orally challenged with three doses of the Mycobacterium avium subspecies paratuberculosis wildtype bovine strain, CLIJ623. on three occasions. Faecal culture, post-mortem tissue bacteriological culture and histopathology were used to monitor the disease progression in the rabbits for more than 2 years. Of 4 adult and 16 juvenile orally dosed rabbits M. paratuberculosis organisms were recovered bacteriologically from two and three animals, respectively, using the BACTECT(TM) radiometric culture system. Tissue sites from which the bacteria were recovered included the mesenteric lymph nodes. ileocaecal valve. vermiform appendix, caecum, proximal colon and jejunum. Body weight loss, reduced abdominal fat and mild lesions were observed at necropsy in four infected rabbits. Diarrhoea and persistent faecal shedding of bacteria were not observed. Faecal culture did not yield any cultivable mycobacterial organisms on solid media. (C) 2004 Elsevier B.V. All rights reserved ANIMALS/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/bacteriology/BACTERIUM/BOVINE/CROHNS-DISEASE/CULTURE/DISEASE/HAMSTERS/IDENTIFICATION/INFECTION /Johne's disease/LESIONS/LYMPH-NODES/MICE/MODEL/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/NEW-ZEALAND/ORGANISM/ORGANISMS/paratuberculosis/PATHOGENESIS/rabbit/rabbit infection model/RABBITS/TISSUE

112 hackova-Kopecna, M., Bartos, M., Straka, M., Ludvik, V., Svastova, P., Alvarez, J., Lamka, J., Trcka, I., Treml, F., Parmova, I., Pavlik, I. (2005) Paratuberculosis and avian tuberculosis infections in one red deer farm studied by IS900 and IS901 RFLP analysis

Veterinary Microbiology, 105, 261-268 As the attempt to eradicate paratuberculosis in one red deer (Cervus elaphus) farm failed, all 167 red deer of different age groups were slaughtered and examined by culture for mycobacteria, and the farm a was closed down. Spleen and hepatic lymph nodes. mediastinal lymph node, ileocecal lymph node, and ileum were collected from each animal and examined (a total of 835 organs). Neither tuberculosis lesions nor pathognomic signs of paratuberculosis were detected. Among all microscopically negative for mycobacteria organs, Mycobacterium avium subsp. paratuberculosis alone was isolated from 165 organs, M. a. avium alone from 41 organs, and both pathogens from four organs. M. a. paratuberculosis alone was detected in 71 red deer, M. a. avium alone in 13 red deer and both pathogens in 18 red deer. Using standardised RFLP methods, three IS900 RFLP types B-C1, B-C16, and B-C32 were identified among 40 M. a. paratuberculosis isolates and four IS901 RFLP types N-B1, N-B3, N-B4, and P-B3 among 17 M. a. avium isolates. (C) 2004 Elsevier B.V. All rights reserved Avian tuberculosis/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/Cervus elaphus/CERVUS-ELAPHUS/CULTURE/deer/DISEASES/Epidemiology/FRAGMENT-LENGTH-POLYMORPHISM/INFECTION/INFECTIONS/INSERTION ELEMENT/IS900/IS901/Johne's disease/LESIONS/LYMPH-NODES/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/paratuberculosis/RED DEER/RFLP/risk assessment /small terrestrial vertebrates/STRAINS/Tuberculosis/TUBERCULOSIS INFECTION/WILD RUMINANTS/zoonoses

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113 Tuboly, S. , Kovacs, A., Lami, E., Nagy, G. (2005) Connections between Chron-disease in humans and Johne-disease (paratuberculosis) in cattle

Magyar Allatorvosok Lapja, 127, 106-112 On the basis of literature data, the authors present the common aetiology, pathogenesis, clinical signs, pathological and pathohistological changes, diagnosis and its difficulties and limits of treatment and prevention of human Chron-disease and bovine Johne-disease. Serological screening for paratuberculosis in cattle started in Hungary in the 80's, which showed cc. 50% positivity. Less than 10% of the 20 000 blood samples [cattle, sheep, goat, wild or zoo ruminants (deer, moufflon, antelope)] examined between 2000 and 2004 were positive. M. para tuberculosis was cultured from 13% of 2600 faeces samples originating form positive herds. Clinical form of Chron disease involves cc. 6000 persons in Hungary with great differences in the different parts of the country (Table 1.). The authors consider necessary to improve diagnostic examinations aiming at the detection of paratuberculosis and on the basis of it, to eradicate the disease BOVINE/CATTLE/COWS/CROHNS-DISEASE/deer/DIAGNOSIS/DISEASE/goat/HERD/INACTIVATION/INFLAMMATORY BOWEL-DISEASE/Johne disease/MILK/MYCOBACTERIUM-PARATUBERCULOSIS/PARA-TUBERCULOSIS/paratuberculosis/PATHOGENESIS/RUMINANTS/SHEEP/Tuberculosis

114 Raizman, E.A., Wells, S.J., Jordan, P.A., DelGiudice, G.D., Bey, R.R. (2005) Mycobacterium avium subsp paratuberculosis from free-ranging deer and rabbits surrounding Minnesota dairy herds

Canadian Journal of Veterinary Research-Revue Canadienne de Recherche Veterinaire, 69, 32-38 The objectives of this study were to estimate the prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) among deer and rabbits surrounding infected and noninfected Minnesota dairy farms using fecal culture, and to describe the frequency that farm management practices were used that could potentially lead to transmission of infection between these species. Fecal samples from cows and the cow environment were collected from 108 Minnesota dairy herds, and fecal pellets from free-ranging white-tailed deer and eastern cottontail rabbits were collected from locations surrounding 114 farms; all samples were tested using bacterial culture. In addition, a questionnaire was administered to 114 herd owners. Sixty-two percent of the dairy herds had at least 1 positive fecal pool or environmental sample. A total of 218 rabbit samples were collected from 90% of the herds, and 309 deer samples were collected from 47% of the herds. On 2 (4%) of the farms sampled, 1 deer fecal sample was MAP positive. Both farms had samples from the cow fecal pool and cow environment that were positive by culture. On 2 (2%) other farms, 1 rabbit fecal sample was positive by culture to MAP, with one of these farms having positive cow fecal pools and cow environmental samples. Pasture was used on 79% of the study farms as a grazing area for cattle, mainly for dry cows (75%) and bred or prebred heifers (87%). Of the 114 farms, 88 (77%) provided access to drylot for their cattle, mainly for milking cows (77/88; 88%) and bred heifers (87%). Of all study farms, 90 (79%) used some solid manure broadcasting on their crop fields. Of all 114 farms, the estimated probability of daily physical contact between cattle manure and deer or rabbits was 20% and 25%, respectively. Possible contact between cattle manure and deer or rabbits was estimated to occur primarily from March through December. The frequency of pasture or drylot use and manure spreading on crop fields may be important risk factors for transmission of MAP among dairy cattle, deer, and rabbits. Although the MAP prevalence among rabbits and deer is low, their role as MAP reservoirs should be considered AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIAL CULTURE/CATTLE/CATTLE HERDS/CERVUS-ELAPHUS/COWS/CULTURE/dairy cattle/DAIRY HERDS/DAIRY-CATTLE/deer/ENVIRONMENT/FARMS/FECAL CULTURE/HERD/INFECTION/INOCULATION/JOHNES-DISEASE/MAP/Minnesota/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PREVALENCE/rabbit/RABBITS/RISK-FACTORS/RUMINANT PARA-TUBERCULOSIS/SCOTLAND/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/Transmission/tule elk/white-tailed deer/WILD RUMINANTS

115 Denis, M., Wedlock, D.N., Buddle, B.M. (2005) Vaccination of brushtail possums, Trichosurus vulpecula, with Bacille Calmette-Guerin induces T lymphocytes that reduce Mycobacterium bovis replication in alveolar macrophages via a contact-dependent/nitric oxide-independent mechanism

Immunology and Cell Biology, 83, 57-66 The permissiveness of alveolar macrophages from brushtail possums for the replication of Mycobacterium bovis was examined. Mycobacterium bovis replication was indirectly measured by assessing bacterial metabolism via the incorporation of [3-H]-uracil by bacilli released from lysed

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macrophages previously infected with mycobacteria. Alveolar macrophages allowed substantial replication of virulent M. bovis, in contrast to Bacille Calmette-Guerin (BCG) Pasteur, which replicated poorly. The addition of crude lymphokines enhanced the metabolic activity of phagocytosed M. bovis in possum macrophages. Possum lymphokines enhanced the ability of possum macrophages to generate reactive oxygen intermediates, measured by the reduction of nitroblue tetrazolium, which is indicative of an activation process. Similarly, the addition of recombinant possum TNF-alpha enhanced the permissiveness of alveolar macrophages for M. bovis. In contrast to mouse peritoneal macrophages, possum alveolar macrophages did not release significant levels of nitric oxide (NO) after stimulation with M. bovis and/or lymphokines. However, the uptake of virulent M. bovis by possum macrophages was associated with an enhanced ability of cells to release TNF-alpha, whereas very low levels of TNF-alpha were released after infection with BCG. The addition of a selective inhibitor of inducible NO synthase had no impact on the replication of M. bovis or BCG in possum macrophages in the presence or absence of lymphokines. Co-culturing infected possum alveolar macrophages with autologous blood mononuclear cells from BCG-vaccinated possums led to a significant decrease in the metabolic activity of intracellular M. bovis. This effect was contact dependent and NO independent and was mediated by a population of CD3(+) cells. In addition, adding scavengers of reactive oxygen intermediates did not abrogate this phenomenon ABILITY/ALPHA TNF-ALPHA/AVIUM SUBSP PARATUBERCULOSIS/BCG/BOVIS/BRUSHTAIL POSSUMS/CALMETTE-GUERIN/CELL/CELLS/cytokine/EXPERIMENTAL-INFECTION/GAMMA-INTERFERON/GROWTH/HOST-DEFENSE/HUMAN-MONOCYTES/IFN-gamma/IMPACT/INFECTION/macrophage/MOUSE/mycobacteria/Mycobacterium/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/NITRIC-OXIDE/POPULATION/possum/PULMONARY TUBERCULOSIS/T cell/VACCINATION

116 Onwuamaegbu, M., Belcher, R., Soare, C. (2005) Cell wall-deficient bacteria as a cause of infections: a review of the clinical significance

Journal of International Medical Research, 33, 1-20 Cell wall-deficient bacteria (CWDB) are pleomorphic bacterial forms. These atypical organisms may occur naturally or they can be induced in the laboratory. Their presence has been known about for over a century, but a definite link to clinical disease outcomes has not been demonstrated. A number of case reports and laboratory studies suggest some disease associations, however. Considerable controversy surrounds the true relevance of CWDB to disease; there is a widespread belief that they may represent a response by the walled organism to adverse extracellular conditions like antibiotic pressure. This review looks at studies published between 1934 and 2003, which were identified by Dialog DataStar using the key words 'cell wall deficient bacteria and clinical significance and infections' and by further scanning the reference list at the end of the papers retrieved. We conclude that the evidence for the clinical significance of CWDB in disease is not compelling atypical bacteria/AVIUM SUBSP PARATUBERCULOSIS/BACTERIUM/CELL/cell wall-deficient bacteria/CHLAMYDIA-PNEUMONIAE/clinical significance/CROHNS-DISEASE PATIENTS/DISEASE/INFECTION/INFECTIONS/L-form/MARROW-TRANSPLANT RECIPIENTS/MYCOBACTERIUM-AVIUM/MYOCARDIAL-INFARCTION/ORGANISM/ORGANISMS/RANDOMIZED CONTROLLED-TRIAL/Review/RHEUMATIC FEVER/STAPHYLOCOCCUS-AUREUS/STREPTOCOCCAL-L FORMS/wild-type bacteria

117 Hattel, A.L., Shaw, D.P., Love, B.C., Wagner, D.C., Drake, T.R., Brooks, J.W. (2004) A retrospective study of mortality in Pennsylvania captive white-tailed deer (Odocoileus virginianus): 2000-2003

Journal of Veterinary Diagnostic Investigation, 16, 515-521 The postmortem records of 160 white-tailed deer (Odocoileus virginianus) submitted for necropsy examination from 59 separate Pennsylvania captive deer farms over a 3.5-year period were reviewed to determine the primary cause of death of each animal. The most common causes of death were bronchopneumonia (39 cases), enterocolitis (30 cases), malnutrition (13 cases), and trauma (11 cases). Other causes of mortality included severe gastrointestinal parasitism (6 cases), cellulitis with septicemia (5 cases), degenerative myopathy (4 cases), ruminal acidosis (4 cases), and nephritis (4 cases). The cause of death was undetermined in 13 of the 160 animals. Arcanobacterium pyogenes (19 cases), Fusobacterium necrophorum (10 cases), Escherichia coli (7 cases), and Mannheimia haemolytica (4 cases) were the most commonly isolated bacteria from the pneumonic lungs. Bacterial agents associated with enterocolitis included Clostridium perfringens (15 cases), E. coli (12 cases), and Mycobacterium avium subsp. paratuberculosis (2 cases). The majority (52.2%) of the death loss in white-tailed deer of known ages occurred in animals I year of age or less, with 46.2% of the bronchopneumonia cases and 50.0% of the enterocolitis cases occurring during this time period. Cases of degenerative myopathy, myocardial degeneration, hepatic necrosis, meningoencephalitis, peritonitis, and urolithiasis considered severe enough to be the primary cause of death appeared early in life,

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affecting deer 6 months of age or less in all cases. In conclusion, bronchopneumonia, enterocolitis, malnutrition, and trauma were considered the most common causes of death in confined white-tailed deer in this study ANIMALS/AVIUM/BACTERIUM/deer/DISEASE/ ESCHERICHIA-COLI/FARMS/FUSOBACTERIUM-NECROPHORUM/ILLINOIS/Minnesota/MORTALITY/mycobacteria /Mycobacterium/Mycobacterium avium /Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/Odocoileus virginianus/ODOCOILEUS-VIRGINIANUS/paratuberculosis/SURVIVAL/white-tailed deer

118 Robino, P. , Nebbia, P., Meneguz, P.G., De Meneghi, D. (2003) Survey on paratuberculosis in roe deer (Capreolus capreolus) and small ruminants in North-Western Italy Proceedings of the Seventh International Colloquium on Paratuberculosis, 472-476 The presence of paratuberculosis in roe deer (Capreolus capreolus) and in small ruminants grazing, on the same pasture lands, was investigated in a study area located in NW Apennines (Alessandria province). Serum samples (n = 94) and mesenteric lymph nodes (n = 47) were collected from 94 roe deer culled in Game Hunting Reserves (GHRs) within the study area. Sera from 109 small ruminants (56 goats, 53 sheep), bred on 7 farms located within or bordering the above GHRs, were also collected. Antibodies against Mycobacterium avium subsp. paratuberculosis (Map) were detected (ELISA test) in 13 roe deer (13.8%), 7 goats (12.5%) and 1 sheep (1.8%) sera. Sero-positive small ruminants were from 3 flocks: 4 of the 6 positive goats, from a flock of 19 with high sero-prevalence, showed specific clinical signs. Difference in sero-prevalence amongst small ruminant flocks, categorised by presence or absence of clinical signs, was statistically significant (p = 0.004, Fisher's exact text), while the difference in sero-prevalence between domestic and wild ungulates was not significant. Acid-fast organisms (Ziehl-Neelsen staining: ZN) were detected in 23 lymph node samples (49%), and Map DNA (Nested PCR, IS900) was found in 17 samples (36.2%). Agreement between results of ZN and Nested PCR was evaluated by Kappa coefficient (k = 0.40). Moreover, bacteriological tests isolated Map in 5 out of the 15 roe deer samples so far tested, and in one sick sero-positive goat slaughtered for examination. Although clinical and gross pathological signs of paratuberculosis have not been observed in the roe deer from the area,, our results suggest that Map is widely present within the roe deer population studied. Small ruminant sero-reactors seem to be clustered at flock/farm level, while sero-positive roe deer are present and distributed throughout the whole area. Epidemiological patterns of paratuberculosis infection in sympatric roe deer and small ruminant populations are being investigated ANTIBODIES/AVIUM/bacteriological culture/CERVUS-ELAPHUS/deer/DNA/ELISA/FARMED RED DEER/FARMS/goat/goats/INFECTION/IS900/Italy/JOHNES-DISEASE/LYMPH-NODES/MAP/MAP-DNA/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/Nested PCR/NESTED-PCR/ORGANISM/ORGANISMS/paratuberculosis/PARATUBERCULOSIS INFECTION/PCR/POPULATION/POPULATIONS/roe deer/RUMINANTS/SHEEP/sheep and goats/sympatric population/TESTS/Ungulate/Ziehl-Neelsen

119 Greig, A., Beard, P.M., Daniels, M.J., Henderson, D., Hutchings, M.R., Stevenson, K. (2003) The potential role of wildlife in the epidemiology of paratuberculosis in domestic animals Proceedings of the Seventh International Colloquium on Paratuberculosis, 358-360 Carcases of mammals and birds were collected on four farms where clinical cases of paratuberculosis were regularly encountered in farmed livestock and rabbits were known to be infected. Carcases were subjected to gross examination, before tissues (primarily intestine and associated lymph nodes) were collected for microbiology and histopathology. Isolates of Mycobacterium avium subsp. paratuberculosis (Map) were confirmed by demonstrating IS900 by PCR. Map was isolated from foxes (23/27), stoats (17/37), weasels (2/4), rats (3/35), wood mouse (3/88), hare (1/6), crow(36/60), rook (3/53) and jackdaw (1/38) and in the cases of foxes (12/26), stoats (1/13), weasels (2/4) and crows (1/60) was accompanied by lesions consistent with paratuberculosis. Significant numbers of bank voles, house mouse, feral and wood pigeons and house sparrow were sampled with negative results on culture ANIMALS/AVIUM/CULTURE/domestic animal/DOMESTIC-ANIMALS/Epidemiology/FARMS/IS900/LESIONS/LYMPH-NODES/MAMMALS/MAP/MOUSE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/paratuberculosis/PCR/POTENTIAL ROLE/rabbit/RABBITS/role/TISSUE/TISSUES/WILDLIFE

120 Bartos, M. , Svastova, P., Yayo, A.W., Machackova, M., Pavlik, I. (2003) RFLP analysis of Mycobacterium avium subsp paratuberculosis for the study of molecular epidemiology

Proceedings of the Seventh International Colloquium on Paratuberculosis, 23-24

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A total of 1750 strains of Mycobacterium avium subsp. paratuberculosis isolated from cattle, sheep, goats, wild ruminants: mouflon (Ovis musimon), red deer (Cervus elaphus) etc., non-vertebrates, small vertebrates: cat (Felis catus), rat (Rattus norvegicus), rabbit (Oryctolagus cuniculus) etc., external environment, milk from cattle (Bos taurus), mouflon, red deer, fallow deer (Dama dama) and other animals and Crohn's patients were examined by the use of standardised Restriction Fragment Length Polymorphism (RFLP) method with two restriction endonucleases (RE) PstI and BstEII, and standard IS900 probe prepared by PCR a non-radioactive labelled. For more detailed differentiation of PstI RFLP types three different hybridisation probes were developed. As RE PstI digests inside of the element IS900, one probe was designed so that it covered both part of the digested fragments (total probe) and two half-probe hybridised to different halves of IS900. A combination of hybridisation by these three probes enabled us to identify new RFLP subtypes inside the original types. We have analysed the similarity of certain BstEII RFLP types and noticed that some RFLP profiles might have been a mixture of several different strains. Such isolates were sub-cultivated and progeny from single bacterial colony was analysed ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/Cervus elaphus/CERVUS-ELAPHUS/Dama dama/deer/DIFFERENTIATION/DNA/ENVIRONMENT/Epidemiology/FALLOW DEER/FRAGMENT-LENGTH-POLYMORPHISM/goat/goats/half-probe/IS900/Johne's diseases/MILK/MOLECULAR EPIDEMIOLOGY/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/ORYCTOLAGUS-CUNICULUS/paratuberculosis/PCR/rabbit/RED DEER/RESTRICTION/RFLP/RUMINANTS/SHEEP/standardisation/STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/WILD RUMINANTS

121 Bannantine, J.P., Huntley, J.F.J., Miltner, E., Stabel, J., Bermudez, L.E. (2003) Antibodies specific to the Mycobacterium avium subsp paratuberculosis major membrane protein inhibit invasion of bovine epithelial cells Proceedings of the Seventh International Colloquium on Paratuberculosis, 49-54 Mycobacterium avium subsp. paratuberculosis (Map) initiates infection by crossing the intestinal epithelial cells of ruminant animals via a mechanism that remains to be fully elucidated. In this study, we observed that the Map major membrane protein (MMP), plays a role in invasion of bovine epithelial cells. The gene encoding MMP was cloned and expressed as a fusion protein with the maltose binding protein (MBP/MMP) in E. coli. Rabbit antisera were raised against a Map whole cell sonicate and MMP-specific antibodies were purified from rabbit sera by affinity chromatography. Immunoelectron microscopy of Map bacilli labeled with MMP-specific antibodies shows that the protein is localized to the surface of these bacteria. Both anti-MMP antibodies and MBP/MMP protein inhibited Map invasion of cultured Madin-Darby bovine kidney cells by 30%. Similar invasion experiments with Map incubated in low oxygen tension, a condition simulating the intestine, decreased invasion by 60%. Collectively, these data show that the 35-kDa MMP protein is a surface exposed protein that plays a role in invasion of epithelial cells. From these studies, we suggest that the major membrane protein is a virulence factor of Map that may be important in the initiation of infection in vivo 35-KILODALTON PROTEIN/ANIMALS/ANTIBODIES /AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIUM/BOVINE/ CELL/CELLS/E.coli expression/ENTRY/EPITHELIAL-CELLS/GENE/IDENTIFICATION/INFECTION/INTESTINAL-MUCOSA/INVASION/Johne's disease/LEPRAE/MAP/MEMBRANE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PEYERS-PATCHES/PROTEIN/rabbit/role/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/VIRULENCE

122 Huntley, J.F.J., Stabel, J.R., Bannantine, J.P. (2003) Expression library immunization of mice identified five genomic DNA clone pools that are protective against colonization with Mycobacterium avium subsp paratuberculosis

Proceedings of the Seventh International Colloquium on Paratuberculosis, 72-77 Johne's disease is a chronic granulomatous infection of cattle caused by Mycobacterium avium subsp. paratuberculosis (Map). Clinical disease is characterized by weight loss, diarrhea, decreased milk production, and ultimately death. To date, there is no effective treatment for paratuberculosis and current vaccines do not prevent infection, but rather delay the onset of clinical disease. Furthermore, currently available vaccines interfere with bovine tuberculosis skin testing and paratuberculosis diagnostic tests. In an effort to identify protective Map genomic sequences, an expression library of Map was generated and subdivided into 78 pools of clones. Each clone pool, which contained an average of 1500 clones, was evaluated by PCR with a set of seventeen known Map sequences. C57BL/6J mice (6 week old, female, 20 grams) were immunized with 2 mug of DNA in the abdominal area with each clone pool via gene gun delivery. The mice were boosted three weeks after initial immunization and inoculated in the tail vein with

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live, virulent Map (strain 19698, 10(8) bacteria per mouse) two weeks following the boost. Protective effects of each clone pool were evaluated based on culture of viable bacteria from liver, spleen, mesenteric lymph node, and ileum obtained at necropsy three months post-challenge. Five of twenty-six clone pools examined thus far have demonstrated at least a hundred-fold reduction in Map colonization in mice when compared to other clone pools and nonvaccinated, infected control mice AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIUM/BOVINE/BOVINE TUBERCULOSIS/CATTLE/CULTURE/Diarrhea/DISEASE/DNA/DNA vaccine/EXPRESSION/expression vector/ GENE/INFECTION/IS900/Johne's disease/MAP/MICE/MILK/MILK-PRODUCTION/MOUSE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/nucleic acid immunization/paratuberculosis/PCR/PROTEIN/skin testing/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/TESTS/Tuberculosis

123 Stahel, J.R., Palmer, M.P., Whitlock, R.H. (2003) Immune responses after oral inoculation of weanling bison or beef calves with a bison or cattle strain of Mycobacterium avium subsp paratuberculosis

Proceedings of the Seventh International Colloquium on Paratuberculosis, 90 -94 Paratuberculosis is endemic in domestic and wild ruminants worldwide. We designed the following study to compare host immune responses and pathologic changes in beef calves and bison calves after challenge with either a cattle (Bos taurus) or bison (Bison bison) strain of Mycobacterium avium subsp. paratuberculosis (Map). In the first part of the study, 6 bison and 6 beef calves were orally inoculated over a 2-week period with a cattle isolate of Map. In the second part, 6 bison and 6 beef calves were similarly inoculated with a bison strain of Map. Throughout each of the studies, blood and fecal samples were taken monthly for a 6-month infection period. Tissue samples were obtained at necropsy for culture and histopathologic analyses. Results from this study demonstrated that bison calves were more susceptible to tissue colonization than beef calves, regardless of bacterial strain. Although lesions were minimal they were most apparent in the jejunum and distal ileum. Interferon-gamma responses were noted in some calves by one month post-inoculation and were sustained longer in beef calves after challenge with the bison isolate. Antibody was not detected in either beef or bison calves during the 6-month infection period. These results indicate that the host response to strains of Map may differ between ruminant species ANTIBODIES/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BISON/CALVES/CATTLE/challenge/cross-species transmision/CULTURE/deer/DIAGNOSTIC-TESTS/experimental infection/HERD/IMMUNE-RESPONSE/IMMUNE-RESPONSES/INFECTION/INOCULATION/interferon-gamma/ JOHNES-DISEASE/LESIONS/MAP/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/ORAL INOCULATION/paratuberculosis/RESPONSES/RUMINANTS/STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/susceptibility/TISSUE/Tuberculosis/WILD RUMINANTS

124 Verna, A., Morsella, C., Casaro, A., Paolicchi, F. (2003) Serologic and pathologic characterization of infection for Mycobacterium avium subsp paratuberculosis in Red Deer from Argentina

Proceedings of the Seventh International Colloquium on Paratuberculosis, 95-98 Deer farming is now widespread throughout Argentina, having paratuberculosis been identified in red deer (Cervus elaphus) herds in Buenos Aires state. This study examined 332 animals with different ages (8 to 84 months), selected at random and divided into seven groups, 50% females and 50% males each. Other seven seropositive animals were selected for having been shown paratuberculosis clinical signs. Blood for indirect ELISAs (ELISA 1, and ELISA 2) and AGID was collected. Feces and tissues of Mycobacterium avium subsp. paratuberculosis (Map) for isolation were cultured on Herrold's with and without mycobactin. ELISA 2 test results showed (using deer anti-IgG) significant higher percentage of animals reactor than animals with AGID. While there were no significant differences found in percentages when ELISA 1 (using bovine anti-IgG) test was compared with AGID test. On the other hand ELISA 1 detected a higher percentage in 12 and 84 months age strata, whereas ELISA 2 and AGID test detected a high percentage in 17 and 84 months animals. The three tests analysis proved the highest seropositive incidence in animals occurred within the 84 month old males group. Both ELISAs absorbance values were highly correlated (r = 0.865). ELISA 1 test had 83.3% sensitivity while the AGID test turned out having 33% sensitivity. Mesenteric lymph nodes of animals with paratuberculosis had moderated to severe infiltration with macrophages (MO) as well as Langhans' giant cells (LC). Lesions observed were similar to IIIb and IIIc types established in small ruminants. All ZN were positive to MO and LC lymph nodes while intestine sections were positive to immunohistochemistry, showing the highest specificity to Map inside MO and LC

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agar gel immunodiffusion (AGID)/ANIMALS/ARGENTINA/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/CELL/CELLS/Cervus elaphus/CERVUS-ELAPHUS/deer/ELISA/enzyme linked immunosorbent (ELISA)/FECES/HERD/immunohistochemical/INFECTION/JOHNES-DISEASE/LESIONS/LYMPH-NODES/macrophage/MAP/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/RED DEER/RUMINANTS/SENSITIVITY/SPECIFICITY/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/TESTS/TISSUE /TISSUES

125 Schroen, C., Gwozdz, J., Roche, M., Bradley, T., McDonald, W., Condron, R. (2003) Evaluation of diagnostic tests for the diagnosis of Johne's disease in deer Proceedings of the Seventh International Colloquium on Paratuberculosis, 154-161 Two tests for the diagnosis of Johne's disease (JD) in live deer; pooled faecal culture (PFC) and a modified ELISA, were evaluated by comparison with individual faecal culture (IFC), culture of tissues (TC) and histopathology. Specimens were collected from 1222 deer from farms on which JD had been diagnosed and from 483 deer from properties with no evidence of the disease. In addition, samples of tissues were collected for culture and histopathological examination from 390 animals selected on the basis of faecal culture and ELISA results. The most sensitive method for confirming deer to be infected with Mycobacterium avium subsp. paratuberculosis (Map) was slaughter and culture of tissues and next sensitive was histopathological examination of affected lymph nodes and intestinal tissues. Individual faecal culture was positive for 47% of infected deer relative tissue culture. Pooled faecal culture was effective at detecting infected animals excreting greater than 200 (CI 30-621) cfu/g of Map in their faeces. The absorbed ELISA modified for deer was positive for 37.5-40 % of infected deer tested positive by IFC or TC. While both PFC and ELISA are suitable as whole herd tests to detect infected herds neither test is capable of detecting a large proportion of individual infected deer in a single test ANIMALS/AVIUM/CULTURE/deer/DIAGNOSIS/DIAGNOSTIC-TESTS/DISEASE/ELISA/FARMS/FECAL CULTURE/HERD/histopathological examination/Johne's disease/LYMPH-NODES/MAP/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/paratuberculosis/SEROLOGICAL TESTS/TESTS/TISSUE/tissue culture/TISSUES

126 Machackova, M., Lamka, J., Yayo, A.W., Parmova, I., Svastova, P., Amemori, T., Pavlik, I. (2003) Infection of ruminants by uncultivable strains of Mycobacterium avium subsp. paratuberculosis in the Czech Republic Proceedings of the Seventh International Colloquium on Paratuberculosis, 191-196 During the survey of paratuberculosis (1992-2001) in cattle, sheep, goats and wild ruminants: red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama), and mouflon (Ovis musimon), tissue samples of the small intestine and corresponding lymph nodes of 4212 animals were examined. From total number of examined animals, Ziehl-Neelsen microscopy of organs was positive in 308 ruminants. Subsequently culture results of these animals were positive in 271 cases (88.0%). In 249 heads of cattle (Bos taurus), microscopy positive, Mycobacterium avium subsp. para tuberculosis (Map) was isolated from 226 (90.4%) animals within 1.5 to 3 months of incubation: "relatively fast-growing" strains. On the contrary, from the 59 other ruminant species 25 (42.3%) were found positive for Map within 2 and 3 months of incubation and 20 (33.8%) within 4 and 8 months of incubation: "relatively slow-growing" strains. Tissue culture of Ziebl-Neelsen positive tissue samples from gastrointestinal tract from 14 (23.7%) animals was negative in seven (50.0%) mouflons, three (21.0%) fallow deer, one (7.3%) red deer and three (21.7%) sheep (Ovis aries). Any relationship between occurrence of uncultivable Map strains and age and sex of ruminants was not revealed. From a total of 30 "relatively slow-growing" strains isolated from sheep, goats and mouflons, 13 were examined by standardised Restriction Fragment Length Polymorphism (RFLP) analysis. Four RFLP types were identified as cattle strains (n - 12): AC 10, B-C 1, B-C2, and E-C1 and one RFLP type was found in sheep strain (n = 1): C-S1. No relationship was observed among RFLP types of above mentioned 13 "relatively slow-growing" strains and 604 "relatively fast-growing" strains in our database. In our study of incubation period, also another 382 ruminants were involved. These ruminants were positive in culture of gastrointestinal tract but ZiehlNeelsen microscopy was negative in all samples ANIMALS/AVIUM/CATTLE/CERVIDS/Cervus elaphus/CERVUS-ELAPHUS/CULTURE/Dama dama/deer/DNA HYBRIDIZATION/FALLOW DEER/FRAGMENT-LENGTH-POLYMORPHISM/goat/goats/IDENTIFICATION/incubation period/INFECTION/Johne's diseases/JOHNES DISEASE/LYMPH-NODES/MAP/MOLECULAR CHARACTERIZATION/mouflons/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/paratuberculosis/RED DEER/RESTRICTION/RESTRICTION-ENDONUCLEASE ANALYSIS/RFLP/roe

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deer/RUMINANTS/SHEEP/STRAINS/TISSUE/tissue culture/Tuberculosis/tule elk/uncultivable strains/WILD RUMINANTS/ Ziehl-Neelsen

127 Vaughan, J.A., Stewart, D.J., Stiles, P.L. , Lenghaus, C., Tizard, M., Michalski, W.P., Prowse, S.J. (2003) Bacteriology of Johne's disease in cattle, sheep, goats and rabbits

Proceedings of the Seventh International Colloquium on Paratuberculosis, 197-200 Bacteriologic detection of Mycobacterium avium subsp. paratuberculosis (Map) was used to confirm the shedding patterns, infection status and final disease profile of cattle, sheep, goats and rabbits in 3 infection experiments. Cattle, sheep and goats were orally dosed (4 doses at weekly intervals) with up to 2 x 10(10) cfu of a wild-type bovine Map isolate or with 15 - 20 g of bovine intestinal mucosal tissue from a naturally Johne's Disease (JD) infected cow. Faecal culture was performed pre-challenge and for 4 years post-challenge. Similarly a further group of cattle, sheep and goats were orally dosed with up to 2 x 10(10) cfu of a wild-type ovine Map isolate or with 15 - 20 g of ovine intestinal mucosal tissue from a naturally JD infected sheep. Faecal culture was carried out pre-challenge and for 31 months post-challenge. Adult and juvenile rabbits were given oral infective doses of 10(8) cfu of the wild-type bovine Map isolate on 3 occasions with faecal and autopsy tissue culture being used to monitor disease progression for more than 2 years. The BACTEC 460 radiometric culture system was used together with conventional culture for the recovery of Map from faecal specimens and post mortem tissues. The Growth Index (GI) was determined weekly using the BACTEC 460 and samples taken from vials initially with a GI > 100 and transferred to 4 varieties of Herrold's egg yolk agar along with modified Middlebrook 7H10 slopes. Ziehl-Neelsen stained smears, Mycobactin J dependency and PCR were used as confirmatory tests. Constant, intermittent and non-shedders were detected and both high and low Map colony numbers observed. Map was cultured from a variety of post mortem tissues from animals manifesting different stages of JD infection. Radiometric bacteriologic culture increased the recovery of bacterial colonies for both the bovine and ovine strains of Map ANIMALS/AVIUM/BACTEC-RCM/bacteriology/BOVINE/bovine and ovine strains/CATTLE/CULTURE/diagnosis-bacteria/DISEASE/goat/goats/GROWTH/INFECTION/Johne's disease/MAP/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/OVINE/paratuberculosis/PCR/rabbit/RABBITS/SHEEP/sheep and goats/STRAINS/TESTS/TISSUE/tissue culture/TISSUES/Ziehl-Neelsen

128 Yayo, A.W. , Fischer, O., Svastova, P., Alexa, M., Machackova, M., Pavlik, I. (2003) Dairy and beef cattle paratuberculosis survey in intensive and extensive farming conditions Proceedings of the Seventh International Colloquium on Paratuberculosis, 340-344 The study was carried out in a beef herd with 137 animals kept on pasture, and in a dairy herd with 96 animals confined in a stable. The objectives of this study were to determine which risk factors influenced the likelihood of the herds becoming infected, to compare the extent to which animals are infected in the two separate management practices and to design a suitable control programme. Following high rate of positive results from testing by serology and faecal culture, all animals were slaughtered. Tissue culture of 52.5 % animals in the beef herd and 54.7 % animals in the dairy herd were culture positive for Mycobacterium avium subsp. paratuberculosis (Map). Environmental samples (n = 776) such as wall scrapings, water, feed-leftovers, larvae and emerging adults of dipterous flies from barn and pasture; grass, soil, mud and pond water from the pasture were cultured. Map was isolated from 7.1 % of pasture samples and 4.3 % of samples from hams. The prevalence of the disease in both herds was similar. Infected cows were compared with their calves for evidence of prenatal or neonatal transmission of Map. Most of the beef calves were born from infected cows, whereas most infected dairy calves were born from healthy cows. Map was isolated in slaughtered calves between the age of two to six months. Two different Restriction Fragment Length Polymorphism (RFLP) types of Map strains were observed, RFLP type B-C1 in the beef herd and RFLP type B-C9 in the dairy herd. Because of the infection of both herds and contamination of premises and the environment a radical control programme was adopted ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CALVES/CATTLE/cattle breeds/COWS/CULTURE/DISEASE/ENVIRONMENT/FRAGMENT-LENGTH-POLYMORPHISM/HERD/INFECTED COWS/INFECTION/IS900/Johne's diseases/MAP/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/pasture/PREVALENCE/RABBITS/RESTRICTION/RFLP/ RISK-FACTORS/Serology/STRAINS/TISSUE/tissue culture/Transmission/WATER/WILD RUMINANTS

129 O'Brien, R., Chinn, N., Rodgers, C., Liggett, S., Spittle, E., Crosbie, P., Mackintosh, C., Beatson, N., Griffin, F. (2003) Johne's disease: an emerging problem in farmed deer

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Proceedings of the Seventh International Colloquium on Paratuberculosis, 353-357 The incidence of Mycobacterium avium subsp. paratuberculosis (Map) has been increasing steadily in New Zealand deer herds throughout the past decade. This has been oberved through the increased numbers of cases of Johne's disease (Jd) confirmed by microbiological culture and the identification of Map through the use of molecular techniques such as IS900 Polymerase Chain Reaction (PCR) (1). In recent years, our laboratory (DRL) has noted an ever increasing number of tuberculin reactor deer presenting with immunological profiles that are typical of neither M. bovis or M. avium and it appears likely that a proportion of such reactions are caused by infection with Map. This is perhaps ironic as the diagnosis of Map is rarely the primary goal; most specimens collected by meat inspectors from deer slaughter plants are submitted for M. bovis diagnosis. Up to one third of pathological specimens submitted for culture to date had histopathology considered as typical of tuberculosis (Tb). Moreover, more than 600 animals, from 300 deer herds (< 10 % of the New Zealand national herd), have been diagnosed with Jd over the past 15 years (1). Because there has been no policy to eradicate this disease, it is likely that the majority of these herds have remained infected and continue to contribute to the cumulative total of infected herds nationwide. At this time it is not possible to estimate the true prevalence of Jd in New Zealand deer herds. The fact that Jd is a non-notifiable disease in New Zealand makes it likely that the true prevalence of Jd is being underestimated. There is little incentive for individual farmers to confirm the presence of Jd within their herd and the absence of well refined methods to diagnose Jd accurately provides little assurance that, should the disease be diagnosed, it may be controlled within, or eradicated from, a deer herd. The objectives of this paper are twofold; to attempt to demystify some of the concerns about Jd diagnosis so that farmers may more easily commit to a programme for the control of Jd in New Zealand deer herds, and to highlight some of the research activites currently underway at our laboratory which, it is hoped, may go some way towards improving control of Jd in deer ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVIS/CULTURE/deer/DIAGNOSIS/DISEASE/FARMED DEER/HERD/IDENTIFICATION/immunodiagnosis/INFECTION/IS1311/IS900/Johne's disease/MAP/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/NEW-ZEALAND/paratuberculosis/PCR/POLYMERASE-CHAIN-REACTION/POLYMORPHISMS/PREVALENCE/SEQUENCE/Tuberculosis

130 de Lisle, G., Yates, G.F., Cavaignac, S.M., Collins, D.M., Paterson, B.M., Montgomery, R.H. (2003) Mycobacterium avium subsp paratuberculosis in feral ferrets - a potential reservoir of Johne's disease

Proceedings of the Seventh International Colloquium on Paratuberculosis, 361-362 Ferrets (Mustela putorius furo) were released in New Zealand in the 19(th) century for the control of rabbits. Currently they inhabit large portions of the North and South Islands, especially those areas with moderate to high numbers of rabbits. Mycobacterium bovis (M. bovis) was first isolated from feral ferrets in 1982 and they have been extensively studied to determine their role in the maintenance and spread of bovine tuberculosis. Recently, eight ferrets from the North and South Islands were identified with lesions in mesenteric lymph nodes and livers that contained acid-fast staining bacteria. The histological picture of these cases was not typical of that seen in ferrets infected with M. bovis and mycobacteria were not isolated from them using non-mycobactin supplemented media. However, IS900 was detected in these lesions by PCR. Subsequently, Mycobacterium avium subsp. paratuberculosis (Map) was isolated from these animals using Bactec 12B vials supplemented with mycobactin, egg yolk and antibiotics. Characterisation of the isolates by Southern blotting using a DNA probe from IS900 showed that six of the isolates were the "ovine" type and the remainder the "bovine" subtype of Map. Sources of infection for ferrets in New Zealand include, cattle, sheep, farmed deer and possibly rabbits. While rabbits are known to carry Map in some countries, in New Zealand they have not yet been examined for this organism. A wildlife reservoir of infection has major implications for the control of paratuberculosis ANIMALS/ANTIBIOTICS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIUM/BOVINE/BOVINE TUBERCULOSIS/BOVIS/CATTLE/CULTURE/deer/DISEASE/DNA/Epidemiology/ FARMED DEER/ferret/INFECTION/IS900/Johne's disease/LESIONS/LYMPH-NODES/MAP/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium bovis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-BOVIS/NEW-ZEALAND/ORGANISM/paratuberculosis/PCR/POLYMERASE CHAIN-REACTION/rabbit/RABBITS/role/SHEEP/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/Tuberculosis/WILDLIFE/wildlife reservoir

131 Hutchings, M.R., Daniels, M.J., Henderson, D., Greig, A. (2003) Potential wildlife to ruminant transmission routes for Mycobacterium avium subsp

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paratuberculosis

Proceedings of the Seventh International Colloquium on Paratuberculosis, 363-367 The recent isolation of Micobacterium avium subsp. paratuberculosis (Map) from nonruminant wildlife species opens up the possibility of transmission from wildlife to domestic ruminants via the faecal oral route. Here we determine the level of contact between cattle and rabbit faeces in the grazing environment (Experiment 1) and between cattle and rodent faeces via farm-stored concentrate feed (Experiment 2). The rates of deposition of rabbit faeces on grazing pastures and rodent faeces in feed stored on four farms with a history of paratuberculosis in cattle and wildlife were estimated by random stratified quadrat sampling. Experiment 1: Remote behaviour-monitoring systems in conjunction with stratified surveys of sward height were used to quantify the grazing behaviour of 57 cattle in relation to rabbit faeces-contaminated pasture. Prior to grazing each field, 4 pasture treatments were created by contaminating 40 plots (0.5 x 0.5 m(2)) with 0, 10, 50 or 250 rabbit faecal-pellets. Experiment 2: Ten cattle were presented individually with 3 repeats of 5 feed treatments: 3 levels of contamination of concentrate feed (none, 20 and 80 faecal-pellets / 400 g feed) x two rodent species (rat and mouse). The mean number of faecal-pellets deposited by adult rabbits on pasture was 7,357 2,571 faeces/ha/day. Grazing cattle did not avoid rabbit faeces and 54/57 cattle were recorded grazing seven 250-faecal-pellet plots. The mean number of faecal-pellets deposited by rodents in stored feed was 79.9 (95 % CI: 37.5-165.9) faeces / m(2) / month. Cattle ingested 40 % and 91 % of rat and mouse faecal-pellets respectively respectively during Experiment 2. Given that rabbit faeces contains up to 4 x 10(6) cfu Map/g and the experimental doses needed to produce disease in domestic ruminants range from 10(3) to 10(9) organisms, the ingestion of a few wildlife faecal-pellets may constitute an infective dose for cattle AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE TUBERCULOSIS/CATTLE/DISEASE/ENVIRONMENT/faeces/FARMS/FECES/grazing behaviour/intake/MAP/MOUSE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/ORGANISM/ORGANISMS/paratuberculosis/pasture/PELLETS/rabbit/RABBITS/rodents/RUMINANTS/SCOTLAND/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/Transmission/WILDLIFE

132 Kennedy, D.J., Hood, R., Allworth, M.B. (2003) Directions for the future control of Johne's disease caused by cattle types of Mycobacterium avium subsp paratuberculosis in Australia

Proceedings of the Seventh International Colloquium on Paratuberculosis, 429-434 The restricted distribution of Johne's disease in Australia resulted in States implementing regulatory controls for many years to try to stop the spread of infection to free areas and herds. This has contributed to the virtual absence of paratuberculosis in northern and western Australia. National zoning for Johne's disease, introduced in 1999, formally recognised the differential distribution of infection and should strengthen regional control. At the same time zoning permits more appropriate controls in infected areas and cattle enterprises than are required in protected and free zones. A new national approach to the control of cattle (C) types of Mycobacterium avium subsp. paratuberculosis (Map) is being developed by Animal Health Australia in conjunction with industry and government stakeholders. Within the infected areas of south-eastern Australia the dairy industry is focussing more on reducing contamination of farm and product and improving management of replacement heifers. It appears that the beef industry in Australia has very little disease but some infected herds have distributed the infection with sales of replacement cattle. Different risk-based strategies are being developed in both industries to allow trading of live cattle while still aiming to reduce the spread of paratuberculosis to non-infected herds and regions. The occurrence of C types of Map in alpaca and goats in Australia has diminished but outbreaks in deer herds in the past 2 years are presenting new challenges AUSTRALIA/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/challenge/control/deer/DISEASE/goat/goats/HERD/INFECTION/Johne's disease/MAP/mycobacteria/ Mycobacterium/Mycobacterium avium/ Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/risk/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS

133 Tobin, F., Wood, I., Doyle, C. (2003) The Victorian Ovine Johne's disease eradication program - Impact on sheep producers Proceedings of the Seventh International Colloquium on Paratuberculosis, 450-454 In December 1996 the Department of Natural Resources and Environment commenced an eradication program of Ovine Johne's Disease (OJD) in the state of Victoria, Australia. The program was based on compulsory slaughter and destocking for a minimum of 2 summers of all sheep, deer, goats and alpaca on properties where OJD was identified. The Victorian OJD Action Group - a group of farmers affected by this program - presented a paper to the 6th International Colloquium, held in Melbourne Australia in

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1998, highlighting the effect of an eradication program on sheep owners. This paper will update our 1998 paper, with specific reference to the circumstances, which ultimately forced the Victorian Government and the States major farmer representative body to terminate eradication in favour of a voluntary control and management program. Our paper will also reflect on the status of an ever-changing National OJD program, its prospects of success and its relationship to the OJD program in Victoria. It seems some natural justice has prevailed in Victoria, where common sense has achieved a partial gain at the expense of closed-door bureaucracy AUSTRALIA/control /deer/DISEASE/ENVIRONMENT/eradication program/goat/goats/IMPACT/Johne's disease/OVINE/Ovine Johne's disease/SHEEP/victoria

134 Koo, H.C., Park, Y.H., Ahn, J., Waters, W.R., Hamilton, M.J., Barrington, G., Mosaad, A.A., Palmer, M.V., Shin, S., Davis, W.C. (2004) New latex bead agglutination assay for differential diagnosis of cattle infected with Mycobacterium bovis and Myobacterium avium subsp para tuberculosis

Clinical and Diagnostic Laboratory Immunology, 11, 1070-1074 Extensive studies have shown that the current assays used to identify cattle infected with Mycobacterium bovis or Mycobacterium avium subsp. paratuberculosis are not sufficiently sensitive and specific to detect all infected animals, especially animals recently infected with the pathogens. In the present report we show that these limitations might be overcome with a latex bead agglutination assay (LBAA). With the specific immunodominant epitope (ESAT6-p) of M. bovis, we developed an LBAA and enzyme immunoassay (EIA) for that purpose and compared them with the "gold standard" culture method and skin test for their efficacy in detecting bovine tuberculosis. When sera from control healthy cows (n = 10), M. avium subsp. paratuberculosis-positive cattle (naturally infected, n = 16; experimentally infected, n = 8), and M. bovis-positive cattle (naturally infected, n = 49;experimentally infected, n = 20) were applied to an EIA and an LBAA developed with ESAT6-p, the two tests showed similar sensitivity (97.1% by EIA, 95.7% by LBAA), high specificity (94.2% by EIA, 100% by LBAA), and a positive correlation (kappa value, 0.85; correlation rate, 93.2%; correlation coefficient, 0.64). Receiver operating characteristic analysis of EIA results and comparison with the culture method determined a suitable cutoff value at 0.469, with an area under the curve of 0.991 (95% confidence interval, 0.977 to 1.0). As LBAA didn't show any positive reactions with sera from uninfected control cows or M. avium subsp. paratuberculosis-infected cattle, which were confirmed to be free of M. bovis by culture or PCR, LBAA using the ESAT6-p can be a rapid and useful M. bovis diagnostic assay. The data suggest that rapid, sensitive, and specific assays can be developed with peptides containing inummodominant epitopes present in proteins uniquely expressed in M. bovis or M. avium subsp. paratuberculosis for differential diagnosis of cattle infected with M. bovis or M. avium subsp. paratuberculosis ANIMALS/antigens/ASSAY/AVIUM/BCG/BOVINE/BOVINE TUBERCULOSIS/BOVIS/CATTLE/control/COWS/CULTURE/DIAGNOSIS/ESAT-6 PROTEIN/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium bovis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-BOVIS/PARA-TUBERCULOSIS/paratuberculosis/PCR/PROTEIN/PROTEINS/SENSITIVITY/SKIN-TEST/SPECIFICITY/TESTS/Tuberculosis/white-tailed deer

135 Ayele, W.Y., Bartos, M., Svastova, P., Pavlik, I. (2004) Distribution of Mycobacterium avium subsp paratuberculosis in organs of naturally infected bull-calves and breeding bulls

Veterinary Microbiology, 103, 209-217 Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, has particular importance in cattle due to the resulting chronic diarrhoea, weight loss, decreased production, infertility and eventual death. While faecal oral route of infection is generally recognised, reports about semen-derived infection are rare. The objective of this work was to assess whether M.a. paratuberculosis may disseminate from the gastrointestinal tract to reproductive organs, and compare this event between naturally infected bull-calves and breeding bulls. Ten bull-calves, aged 6-28 weeks and four breeding bulls were tested by serology, faecal and tissue culture, IS900 PCR and RFLP. In seven bull-calves M.a. paratuberculosis was isolated predominantly from mesenteric lymph nodes (75%); isolates from mucosa of the intestine constituted 25%. In three breeding bulls, M.a. paratuberculosis was isolated both from intestinal mucosa and mesenteric lymph nodes. Head and mediastinal lymph nodes, liver, spleen and semen of bull no. 1 (Holstein-Friesian); testes and epididymis of bull no. 2 (Piemonte); testes, epididymides and seminal vesicle of bull No. 3 (Hereford); and seminal vesicle of bull No. 4 (Simmental) tested positive by culture. Hot-start PCR revealed M. a. paratuberculosis in semen, seminal vesicle and intestinal tissue where culture isolation was difficult. Isolates from bull-calves and breeding bulls were of RFLP types B-C9 and B-C1, respectively. Bull-calves born in infected herd can be sources of infection when later used for natural mating or artificial insemination. Sub-clinically infected bulls release M.a. paratuberculosis into semen, consequently infecting the uterine environment of cows. (C) 2004 Elsevier B.V. All rights

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reserved artificial insemination/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE PARATUBERCULOSIS/CATTLE/COWS/CULTURE/ENVIRONMENT/FECAL CULTURE/FRAGMENT-LENGTH-POLYMORPHISM/germ cells/HERD/INFECTION/infertility/INTESTINAL-MUCOSA/IS900/Johne's disease/JOHNES-DISEASE/LYMPH-NODES/mycobacteria /Mycobacterium/Mycobacterium avium /Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/paratuberculosis/PCR /reproduction/RFLP/RISK-FACTORS/SEMEN/SEQUENCE/Serology/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/TISSUE/tissue culture/WILD RUMINANTS

136 Waters, W.R., Palmer, M.V., Bannantine, J.P., Whipple, D.L., Greenwald, R., Esfandiari, J., Andersen, P., McNair, J., Pollock, J.M., Lyashchenko, K.P. (2004) Antigen recognition by serum antibodies in white-tailed deer (Odocoileus virginianus) experimentally infected with Mycobacterium bovis Clinical and Diagnostic Laboratory Immunology, 11, 849-855 White-tailed deer (Odocoileus virginianus) have emerged as reservoirs of bovine tuberculosis in northern America. For tuberculosis surveillance of deer, antibody-based assays are particularly attractive because deer are handled only once and immediate processing of the sample is not required. Sera collected sequentially from 25 Mycobacterium bovis-infected and 7 noninfected deer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for immunoglobulin specific to M. bovis antigens. Various routes of experimental M. bovis infection, such as intratonsillar inoculation (n = 11), aerosol (n = 6), and exposure to infected deer (in contact, n = 8), were studied. Upon infection, specific bands of reactivity at similar to24 to 26 kDa, similar to33 kDa, similar to42 kDa, and similar to75 kDa to M. bovis whole-cell sonicate were detected by immunoblot. Lipoarabinomannan-specific immunoglobulin was detected as early as 36 days postchallenge, and responses were detected for 94% of intratonsillarly and "in-contact"- infected deer. In MAPIA, sera were tested with 12 native and recombinant antigens coated on nitrocellulose. All in-contact-infected (8 of 8) and 10 of 11 intratonsillarly infected deer produced antibody reactive with one or more of the recombinant/native antigens. Responses were boosted by injection of tuberculin for intradermal tuberculin skin testing. Additionally, three of six deer receiving a very low dose of M. bovis via aerosol exposure produced antibody specific to one or more recombinant proteins. M. bovis was isolated from one of three nonresponding aerosol-challenged deer. Of the 12 antigens tested, the most immunodominant protein was MPB83; however, a highly sensitive serodiagnostic test will likely require use of multiple antigens ANTIBODIES/ANTIGEN/ANTIGEN RECOGNITION/antigens /ARABINOMANNAN/ASSAY/BOVINE/BOVINE TUBERCULOSIS/BOVIS/CATTLE/deer/DIAGNOSIS/ELISA/enzyme-linked immunosorbent assay/IMMUNE-RESPONSES/INFECTION/INOCULATION/LINKED-IMMUNOSORBENT-ASSAY/LIPOARABINOMANNAN/mycobacteria/Mycobacterium/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/Odocoileus virginianus/ODOCOILEUS-VIRGINIANUS/PROTEIN/PROTEINS/RECOGNITION/RECOMBINANT PROTEINS/RESPONSES/serodiagnosis/SERUM ANTIBODIES/skin testing/SUBSP PARATUBERCULOSIS INFECTION/Tuberculosis/white-tailed deer

137 Fischer, O.A., Matlova, L., Dvorska, L., Svastova, P., Peral, D.L., Weston, R.T., Bartos, M., Pavlik, I. (2004) Beetles as possible vectors of infections caused by Mycobacterium avium species

Veterinary Microbiology, 102, 247-255 Mycobacteria were not isolated from any of 229 beetle imagoes of 29 species originating from 14 distinct localities in the Czech and Slovak Republics: 186 imagoes (34 samples) and 43 imagoes (12 samples) from the wild and herds with paratuberculosis infected ruminants, respectively. From 75 environmental samples taken from barns with infected ruminants, Mycobacterium avium subsp. paratuberculosis was isolated from five scrapings of the floors in barns and a feed processing room. From bran and peat taken from pig farms, M. a. hominissuis was diagnosed in 13% of 72 samples and in 69% of 70 samples, respectively. M. a. avium was isolated from 2 (2.9%) and atypical mycobacteria from 12 (17.1%) peat samples. In the respective experiments, larvae of Tenebrio molitor Linnaeus and Zophobas atratus Fabricius were infected in vitro with isolates of M. a. paratuberculosis of IS900 RFLP type B-C1 and M. a. avium of IS901 RFLP type F-C3. T molitor larvae were also infected with M. a. hominissuis by naturally contaminated bran and peat. M. a. paratuberculosis and M. a. avium were diagnosed in larvae of both species on days 1 to 3 post infection (p.i.). M. a. hominissuis was isolated from T molitor larvae fed by bran on days 4 to 9 p.i. and from imagoes on day 35 p.i. and from larvae fed by peat on days 4 to 14 p.i. RFLP types of all the isolates identified before infection and after isolation from larvae were

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identical. Thus, beetles could mechanically transmit mycobacteria, this hazard should be considered for both the implementation of control measures and feeding captive animals with larvae. (C) 2004 Elsevier B.V. All rights reserved ANIMALS/ARTHROPODS/Avian tuberculosis/AVIUM/CATTLE/COLEOPTERA/control/ENVIRONMENT/FARMS/HERD/INFECTION/INFECTIONS/IS1245/IS900/IS901/Johne's disease/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium complex/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/paratuberculosis/PASSIVE VECTORS/RFLP/RUMINANTS/SEROTYPES/STANDARDIZATION/SUBSP PARATUBERCULOSIS/Transmission/zoonoses

138 Hruska, K. (2004) Research on paratuberculosis: Analysis of publications 1994-2004

Veterinarni Medicina, 49, 271-282 The Web of Knowledge Results Analysis of papers published on paratuberculosis in 1994-2004 demonstrated the increasing interest in Mycobacterium avium subsp. para tuberculosis (MAP). In the analyzed period 1032 papers published by 2 519 authors affiliated with 738 institutions were indexed in the Web of Science database. The papers were published in 238 journals, 25 of which contained more than 55% of articles. The Top 50 authors, Top 50 institutions and Top 50 most frequently cited papers are listed in this review. The contribution of OIE Reference Laboratory for Paratuberculosis established in the Veterinary Research Institute, Brno, is assessed according to the number of publications (29), number of authors participating (79), number of institutions collaborating (41 from 17 countries) and their positions in the Top 50 lists. It is evident that the number of papers on Mycobacterium avium subsp. paratuberculosis, a species causing para tuberculosis in ruminants and possibly having a role in the development of Crohn's disease in at least some humans is significantly increasing AVIUM/AVIUM SUBSP-PARATUBERCULOSIS/BOVINE TUBERCULOSIS/Crohn's disease/CROHNS-DISEASE/DISEASE/FRAGMENT-LENGTH-POLYMORPHISM/MAP/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/NUCLEOTIDE-SEQUENCE/PARA-TUBERCULOSIS/paratuberculosis/PASSIVE VECTORS/PERIPHERAL-BLOOD/POLYMERASE CHAIN-REACTION/Review/role/RUMINANTS/Tuberculosis/Web of knowledge/Web of science/WILD RUMINANTS

139 Machackova, M., Svastova, P., Lamka, J., Parmova, I., Liska, V., Smolik, J., Fischer, O.A., Pavlik, I. (2004) Paratuberculosis in farmed and free-living wild ruminants in the Czech Republic (1999-2001)

Veterinary Microbiology, 101, 225-234 Due to the occurrence of the infection of Mycobacterium avium subspecies paratuberculosis among domestic ruminants and the rapid development of farmed deer industry and the market of cloven-hoofed game we have carried surveys of paratuberculosis, beginning in 1997, in the most common four species of wild ruminants in the Czech Republic [Pavlik et aL, Vet. Microbiol. 77 (2000) 231-251]. From 1999 the prevalence of paratuberculosis has been slightly reduced in all three types of husbandry of wild ruminants. Nevertheless paratuberculosis has been diagnosed in wild ruminants in three districts, in four game parks and in five farms. M. a. paratuberculosis was isolated from 128 (5.3%) out of 2, 403 wild ruminants of four animal species: 106 red deer, 2 roe deer, 4 fallow deer and 16 mouflons. In red deer farms, the highest number of clinical paratuberculosis cases was in yearling deer. RFLP type B-Cl of M. a. paratuberculosis predominated during the second period (1999-2001) in all types of husbandry with no relationship to wild ruminant species. New "cattle" RFLP types B-C5 and B-C16 of M. a. paratuberculosis were described in infected farmed red deer and one "intermediate" RFLP type R-I4 in fallow deer from one game park. The survival of M. a. paratuberculosis was found to be 4 months during winter in the pasture after destocking of all cattle infected with paratuberculosis. We found that non-vertebrates, wild ruminants or non-ruminant wildlife can be vectors and potentially become a risk factor in the spread of M. a. paratuberculosis infection. (C) 2004 Elsevier B.V. All rights reserved AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/CATTLE/deer/DEER CERVUS-ELAPHUS/DNA HYBRIDIZATION/FALLOW DEER/FARMED DEER/FARMED RED DEER/FARMS/FRAGMENT-LENGTH-POLYMORPHISM/game park/INFECTION/Johne's diseases/JOHNES-DISEASE/MOLECULAR EPIDEMIOLOGY/mouflons/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/PARA-TUBERCULOSIS/paratuberculosis/PARATUBERCULOSIS INFECTION/pasture/PREVALENCE/RED DEER/reservoir/RESTRICTION-ENDONUCLEASE ANALYSIS/RFLP/risk/risk assessment/roe deer/RUMINANTS/STRAINS/SURVIVAL/vector/WILD RUMINANTS/WILDLIFE

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140 Mackintosh, C.G., de Lisle, G.W., Collins, D.M., Griffin, J.F.T. (2004) Mycobacterial diseases of deer

New Zealand Veterinary Journal, 52, 163-174 The most significant mycobacterial diseases of free-living, captive and farmed deer are bovine tuberculosis, caused by Mycobacterium bovis, Johne's disease (paratuberculosis), caused by Mycobacterium avium subsp paratuberculosis (basonym M. paratuberculosis), and avian tuberculosis, caused principally by M. avium subsp avium. The first case of M. bovis infection in farmed deer was identified in New Zealand in 1978. In 1983, a voluntary scheme was introduced in New Zealand to control tuberculosis in farmed deer, followed by a compulsory tuberculosis control scheme in 1990. The primary control measure is the slaughter of infected animals, detected by skin testing and blood testing, together with movement control and vector control. The number of infected deer herds peaked in the mid 1990s at over 160 herds, but by 30 June 2002 this had been reduced to 79 (1.45%), and to 67 (1.23%) by June 2003. Deer-to-deer transmission occurs, but the majority of herd breakdowns are believed to be from infected vectors. Factors likely to affect the susceptibility of deer include age, environment, population density, exposure and genetics. Avian tuberculosis occasionally causes clinical disease in wild, captive and farmed deer in New Zealand and overseas. Mycobacterium intracellulare, and subspecies of M. avium other than M. paratuberculosis, are widespread throughout New Zealand and are thought to be largely responsible for the high level of sensitisation to avian purified protein derivative (PPD), which is used for comparison purposes in tuberculosis skin testing of deer in this country. Infections with these organisms are usually subclinical in farmed deer, although M. avium subsp avium commonly causes lesions in retropharyngeal, mesenteric and ileocaecal lymph nodes. These lesions cause problems because of their gross and microscopic similarity to those due to M. bovis infection. Birds and domestic animals are most likely to become infected via environmental contamination of food, water, bedding litter or soil, while carnivores or scavengers may also become infected by ingesting infected carcasses. Johne's disease has been reported in deer in the wild and in zoos, especially in North America, the United Kingdom (UK) and Europe. Since first being confirmed in farmed deer in New Zealand in 1979, the incidence of Johne's disease has increased steadily. To date, M. paratuberculosis has been identified in >600 farmed deer on 300 properties. The majority of cases have been identified from suspected tuberculous lesions submitted from deer slaughter plants. Clinically, Johne's disease in deer is similar to the disease in sheep and cattle, with typical signs of loss of weight and condition, and diarrhoea. However, outbreaks of Johne's disease frequently occur in young red deer, 8-15 months of age, whereas the clinical disease in sheep and cattle is sporadic and usually affects adults 3-5 years of age. The disease is characterised by a chronic granulomatous enteritis and lymphadenitis, especially affecting the jejunum and ileum and the mesenteric lymph nodes. Deer affected subclinically may have lesions in these lymph nodes at slaughter, which are grossly indistinguishable from those due to bovine tuberculosis. Because of the antigenic similarity between M. intracellulare and all the subspecies of M. avium, including M. paratuberculosis, the diagnostic tests for Johne's disease lack sensitivity and specificity, making control difficult ANIMALS/Avian tuberculosis/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/blood testing/BOVINE/BOVINE TUBERCULOSIS/BOVIS/carnivores/CATTLE/control/DAMA-DAMA/deer/DETECTING TUBERCULOSIS/DIAGNOSIS/DIAGNOSTIC-TESTS/DISEASE/DISEASES/domestic animal/DOMESTIC-ANIMALS/ELK CERVUS-ELAPHUS/ENTERITIS/ENVIRONMENT/Epidemiology/FALLOW DEER/FARMED DEER/FARMED RED DEER/food/HERD/INFECTION/INFECTIONS/INTRACELLULARE/Johne's disease/JOHNES DISEASE/JOHNES-DISEASE/LESIONS/LYMPH-NODES/M.avium subsp avium/mycobacteria/mycobacterial diseases/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium bovis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-BOVIS/NEW-ZEALAND/ORGANISM/ORGANISMS/paratuberculosis/POPULATION/prevention/PROTEIN/RED DEER/SENSITIVITY/SHEEP/skin testing /SPECIFICITY/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/susceptibility/TESTS/Transmission/Tuberculosis/TUBERCULOUS LESIONS/UNITED-KINGDOM/vector/WATER/white-tailed deer/zoonosis

141 Miyamoto, Y., Mukai, T., Takeshita, F., Nakata, N., Maeda, Y., Kai, M., Makino, M. (2004) Aggregation of mycobacteria caused by disruption of fibronectin-attachment protein-encoding gene

Fems Microbiology Letters, 236, 227-234 The fibronectin-attachment protein (FAP) is conserved among several species of mycobacteria. Although this protein is associated with attachment and internalization of bacteria to host cells via fibronectin, the physiological role of the protein still remains unclear. To investigate this point, we generated FAP gene disruptant in Mycobacterium smegmatis. The gene disruption, verified by Southern blot and PCR analysis, induced changes on the bacteria, which are associated with strong aggregation and alteration of cell surface properties. Increased hydrophobicity and Congo red accumulation was observed in the FAP gene disruptant. In addition, the complementation experiment demonstrated that the corresponding

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gene restored wild type morphology in the disruptant. These results indicate that the FAP affects the cell surface properties, and its deletion lead to enhanced aggregation of the M. smegmatis. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved AVIUM SUBSP PARATUBERCULOSIS/BACTERIUM/BINDING MOTIF/CELL/CELL-SURFACE/CELLS/ENVELOPE/EPITHELIAL-CELLS/FAP/FIBRONECTIN ATTACHMENT PROTEIN/FIBRONECTIN-ATTACHMENT PROTEIN/GENE/gene disruption/IDENTIFICATION/mycobacteria/Mycobacterium/Mycobacterium smegmatis/PCR/PERMEABILITY/PHAGOCYTOSIS/PROTEIN/role/SMEGMATIS/Tuberculosis

142 Waters, W.R., Nonnecke, B.J., Palmer, M.V., Robbe-Austermann, S., Bannantine, J.P., Stabel, J.R., Whipple, D.L., Payeur, J.B., Estes, D.M., Pitzer, J.E., Minion, F.C. (2004) Use of recombinant ESAT-6 : CFP-10 fusion protein for differentiation of infections of cattle by Mycobacterium bovis and by M. avium subsp avium and M. avium subsp paratuberculosis

Clinical and Diagnostic Laboratory Immunology, 11 , 729-735 Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (LCFP-10) are dominant gamma interferon (IFN-gamma)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental non-tuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-gamma and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-gamma responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis ANIMALS/ANTIGEN/antigens/ANTIGENS ESAT-6/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVIS/CALVES/CATTLE/CFP-10/CROSS-REACTIVITY/CULTURE/CULTURE FILTRATE/DIAGNOSIS/DIFFERENTIATION/ESAT-6/experimental infection/EXPERIMENTAL-INFECTION/GAMMA/ GAMMA-INTERFERON/HERD/IFN-gamma/IMMUNE-RESPONSES/INFECTION/INFECTIONS/INTERFERON/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium bovis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-BOVIS/NITRIC-OXIDE/NONTUBERCULOUS MYCOBACTERIA/paratuberculosis/PEPTIDES/PROTEIN/PROTEINS/RESPONSES/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/Tuberculosis/TUMOR-NECROSIS-FACTOR/white-tailed deer

143 Davidson, W.R., Manning, E.J.B., Nettles, V.F. (2004) Culture and serologic survey for Mycobacterium avium subsp paratuberculosis infection among Southeastern white-tailed deer (Odocoileus virginianus) Journal of Wildlife Diseases, 40, 301-306 From July 1998 through October 2002, radiometric culture (ileocecal lymph node, mesenteric lymph node, and feces) and serologic testing by enzyme-linked immunosorbent assay (ELISA) were used to survey white-tailed deer (Odocoileus virginianus) from the southeastern United States for infection by Mycobacterium avium subsp. paratuberculosis (Mptb) the causative agent of paratuberculosis (Johne's disease). Mycobacterium avium subsp. paratuberculosis was isolated from the ileocecal lymph node of one of 313 deer (0.3%) originating from 63 populations in Alabama, Arkansas, Florida, Georgia, Kentucky, Louisiana, Maryland, Mississippi, North Carolina, South Carolina, Tennessee, and West Virginia (USA). Six deer (2%) all from different populations, had ELISA results above a 0.25 sample-to-positive cutoff value, but none of the ELISA reactors originated from the population from which the single Mptb isolation was made. These six deer were seronegative when tested by agar gel immunodiffusion (AGID). Collectively, these data indicate that white-tailed deer currently do not constitute a broad regional reservoir for Mptb; however, further study is warranted to clarify the significance, if any, of

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infected deer to the epizootiology of paratuberculosis on a local scale. Adaptation and validation of an ELISA or another serologic assay for use with deer and other wildlife would markedly enhance Mpth surveillance among wild populations and would be a powerful tool for gaining information on the role of wild species in epidemiology of paratuberculosis AMERICAN WILD RUMINANTS/ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CULTURE/deer/DIAGNOSIS/DISEASE/ELISA/enzyme-linked immunosorbent assay/Epidemiology/FECES/HERD/INFECTION/Johne's disease/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium avium.subsp paratuberculosis/MYCOBACTERIUM-AVIUM/Odocoileus virginianus/ODOCOILEUS-VIRGINIANUS/PARA-TUBERCULOSIS/paratuberculosis/PARATUBERCULOSIS INFECTION/POPULATION/POPULATIONS/radiometric culture/reservoir/risk/role/SCOTLAND/SEROLOGIC SURVEY/SHEEP/SUBSP PARATUBERCULOSIS/SUBSP PARATUBERCULOSIS INFECTION/SUBSP-PARATUBERCULOSIS/survey/tule elk/United States/UNITED-STATES/white-tailed deer/WILDLIFE

144 Secott, T.E., Lin, T.L., Wu, C.C. (2004) Mycobactetium avium subsp paratuberculosis fibronectin attachment protein facilitates M-cell targeting and invasion through a fibronectin bridge with host integrins Infection and Immunity, 72, 3724-3732 Efficient attachment and ingestion of Mycobacterium avium subsp. paratuberculosis by cultured epithelial cells requires the expression of a fibronectin (FN) attachment protein homologue (FAP-P) which mediates FN binding by M. avium subsp. paratuberculosis. Invasion of Peyer's patches by M. avium subsp. paratuberculosis occurs through M cells, which, unlike other intestinal epithelial cells, express integrins on their luminal faces. We sought to determine if the interaction between FAP-P of M. avium subsp. paratuberculosis and soluble FN enabled targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo via these surface integrins. Wild-type and antisense FAP-P mutant M. avium subsp. paratuberculosis strains were injected alone or coinjected with blocking peptides or antibodies into murine gut loops, and immunofluorescence microscopy was performed to assess targeting and invasion of M cells by M. avium subsp. paratuberculosis. Nonopsonized M. avium subsp. paratuberculosis preferentially invaded M cells in murine gut loops. M-cell invasion was enhanced 2.6-fold when M. avium subsp. paratuberculosis was pretreated with FN. Invasion of M cells by the antisense FAP-P mutant of M. avium subsp. paratuberculosis was reduced by 77 to 90% relative to that observed for the control strains. Peptides corresponding to the RGD and synergy site integrin recognition regions of FN blocked M. avium subsp. paratuberculosis invasion of M cells by 75 and 45%, respectively, whereas the connecting segment 1 peptide was noninhibitory. Antibodies against the alpha5, alphaV, beta1, and beta3 integrin subunits inhibited M-cell invasion by 52 to 73%. The results indicate that targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo is mediated primarily by the formation of an FN bridge formed between FAP-P of M. avium subsp. paratuberculosis and integrins on M cells ADHESION /ANTIBODIES/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACILLUS-CALMETTE-GUERIN/BINDING PROTEIN/CELL/CELLS/control/EPITHELIAL-CELLS/EXPRESSION/FIBRONECTIN ATTACHMENT PROTEIN/HUMAN RESPIRATORY MUCOSA/INTESTINAL-MUCOSA/INVASION/M cells/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/paratuberculosis/PEPTIDES/Peyer's patches/PROTEIN/RECOGNITION/STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/SYNERGY SITE/Tuberculosis/YERSINIA-PSEUDOTUBERCULOSIS

145 Dinnella, C., Monteleone, E., Farenga, M.F., Hourigan, J.A. (2004) The use of enzymes for thermal process monitoring: modification of milk alkaline phosphatase heat resistance by means of an immobilization technique

Food Control, 15, 427-433 Milk alkaline phosphatase (AP) was immobilized by adsorption on to a synthetic polymer that is reversibly soluble-insoluble depending on the medium pH value. Immobilization experiments were conducted using different carrier concentrations. The relationship between carrier concentration and amount of added AP was expressed in terms of Molar Ratio (MR) which ranged from 0.6 to 16.0 mol of carrier available for each mole of enzyme. The effect of MR value on the activity and on the heat stability of the immobilized enzyme was investigated. The amount of active adsorbed enzyme depended on MR value and ranged from 13.3 +/- 1.4 to 8.0 +/- 0.3 Enzymatic Units (EU) for MR value of 2.0 and 16.0, respectively. An average D-63 degreesC value of 130.0 +/- 2.5 s was calculated for the enzyme free form and values equal to 148.0 +/- 4.4, 122.7 +/- 5.2, 154.3 +/- 7.7, 113.3 +/- 6.1 and 77.0 +/- 2.9 s were found for adsorbed enzyme samples with MR of 0.6, 2.0, 3.8, 9.8 and 16.0, respectively. Over the temperature from 63 to 71 degreesC the following z values were computed: 6.5 degreesC for the free

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enzyme, 11.4 and 4.6 degreesC for the adsorbed form with MR of 0.6 and 3.8 respectively. (C) 2003 Elsevier Ltd. All rights reserved BOVINE-MILK/D and z values modulation/enzymatic indicators/EUDRAGIT S-100/food safety/HEAT/HYDROLYSIS/INACTIVATION/INDICATORS/LIPASE/MILK/monitoring/MYCOBACTERIUM-PARATUBERCULOSIS/PASTEURIZATION/SALMONELLA/TIME-TEMPERATURE INTEGRATORS

146 Fredriksen, B., Donne, B., Sigurdardottir, O., Tharaldsen, J., Nyberg, O., Jarp, J. (2004) Factors affecting the herd level of antibodies against Mycobacterium avium subspecies paratuberculosis in dairy cattle

Veterinary Record, 154, 522-526 A case-control study was made of Norwegian dairy herds with high and low herd levels of antibodies against Mycobacterium avium subspecies paratuberculosis. A high proportion of the herds had a considerable number of seropositive cows, and environmental and management factors were examined for possible associations with the high serological levels of antibodies. The most important appeared to be: geographical location, red deer (Cervus elaphus) gaining access to the pastures for cattle, the observation of wild birds in the feed storage, and herds sharing common pasture with other herds of cattle. However, diagnostic tests showed that none of the animals in the case herds was infected with M a paratuberculosis ANIMALS/ANTIBODIES/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BOVINE PARATUBERCULOSIS /CATTLE/Cervus elaphus/ CERVUS-ELAPHUS/COWS/dairy cattle/DAIRY HERDS/DAIRY-CATTLE/deer/DEER CERVUS-ELAPHUS/DIAGNOSTIC-TESTS/EXPERIMENTAL-INFECTION/HERD/JOHNES-DISEASE/LINKED-IMMUNOSORBENT-ASSAY/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/pasture/RED DEER/SCOTLAND/TESTS/Tuberculosis/WILD BIRDS

147 Motiwala, A.S., Amonsin, A., Strother, M., Manning, E.J.B., Kapur, V., Sreevatsan, S. (2004) Molecular epidemiology of Mycobacterium avium subsp paratuberculosis isolates recovered from wild animal species

Journal of Clinical Microbiology, 42, 1703-1712 Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpson's diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACILLUS-ANTHRACIS/COMPLEX/CROHNS-DISEASE/CULTURE/DIFFERENTIATION/Epidemiology/FRAGMENT-LENGTH-POLYMORPHISM/INSERTION ELEMENT/INTRACELLULARE/IS1245/IS1311/IS900/JOHNES-DISEASE/MOLECULAR EPIDEMIOLOGY/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/NUMBER TANDEM REPEAT/paratuberculosis/PCR/POLYMERASE-CHAIN-REACTION/radiometric culture/RESTRICTION/SEQUENCE/SPECIFICITY/STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/Transmission/tule elk/United States/UNITED-STATES

148 Tryland, M., Olsen, I., Vikoren, T., Handeland, K., Arnemo, J.M., Tharaldsen, J., Djonne, B., Josefsen, T.D., Reitan, L.J. (2004) Serologic survey for antibodies against Mycobacterium avium subsp Paratuberculosis in free-

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ranging cervids from Norway

Journal of Wildlife Diseases, 40, 32-41 Affinity between protein-G and immunoglobulins from red deer (Cervus elaphus), moose (Alces alces), roe deer (Capreolus capreolus), and reindeer (Rangifer tarandus tarandus) was tested in a competition binding assay. Sera from red deer, reindeer, and moose inhibited the assay less than sera from cattle (less affinity), whereas sera from roe deer showed a slightly higher affinity to protein-G than did sera from cattle. The conclusion was made that protein-G could be used instead of anti-species antibodies for these cervid species, where the aim of the screening was to look for exposure or lack of exposure to mycobacteria in the tested populations. Serologic screening of 1,373 free-ranging cervids for antibodies against Mycobacterium avium subsp. paratuberculosis was conducted. All sera were tested by a protein-G-based antigen-absorbed enzyme-linked immunosorbent assay (ELISA). Seropositive moose (10/537; 1.9%), red deer (14/371; 3.8%), roe deer (6/49; 12.2%), and semidomesticated reindeer (11/325; 3.4%) were found, whereas wild reindeer (n=91) were seronegative. In addition, the red deer sera were tested with a commercial ELISA, by which two animals tested positive and nine were suspicious of having M. avium subsp. paratuberculosis antibodies. Tissue samples and feces from 10 moose originating from a population with a clustering of seropositive animals were investigated by histology and bacteriology with negative results. Paratuberculosis has never been diagnosed in free-ranging or farmed cervid species in Norway. Thus, further studies are indicated to prove that the present findings reflect an infection with M. avium subsp. paratuberculosis ANIMALS/ANTIBODIES/ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/bacteriology/CATTLE/CERVIDS/Cervus elaphus/CERVUS-ELAPHUS/deer/DEER CERVUS-ELAPHUS/ELISA/enzyme-linked immunosorbent assay/FECES/G-BINDING/IMMUNOGLOBULIN/INFECTION/JOHNES-DISEASE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/paratuberculosis/POPULATION/POPULATIONS/protein-G/RED DEER/roe deer/SCOTLAND/SEROLOGIC SURVEY/Serology/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/survey/TISSUE/WILD RABBITS/WILDLIFE

149 Deem, S.L. , Noss, A.J., Villarroel, R., Uhart, M.M., Karesh, W.B. (2004) Disease survey of free-ranging grey brocket deer (Mazama gouazoubira) in the Gran Chaco, Bolivia

Journal of Wildlife Diseases, 40, 92-98 Samples from 17 free-ranging hunter-killed grey brocket deer (Mazaina gouazoubira) in the Gran Chaco, Bolivia, were collected during June-August 1999. All 17 deer appeared to be in good condition at the time of death. Gross necropsies were performed, serum was collected for serologic evaluation of selected infectious disease agents, and feces and ectoparasites were collected for evaluation of internal and external parasites. Serologic tests were positive for antibodies against bovine respiratory syncytial virus and four Leptospira interrogans serovars, with questionable results for epizootic hemorrhagic disease virus serotypes 1 and 2. No antibodies were detected to Anaplasma marginate, Babesia bigemina, Babesia bovis, Babesia odocoilei, bluetongue virus (serotypes 2, 10, 11, 13, and 17), bovine viral diarrhea virus, Brucella abortus, foot-and-mouth disease virus, infectious bovine rhinotracbeitis virus, Mycobacterium avium subsp. paratuberculosis, and parainfluenza-3 virus. Sixty-four percent (7/11) of the deer had endoparasites. Amblyomma spp. ticks were found on seven deer, flies of the family Hippoboseidae on six deer, and lice on six deer ANTIBODIES/ANTIBODY/AVIUM/Bolivia/BOVINE/Bovine viral diarrhea/BOVIS/deer/Diarrhea/DISEASE/FAMILY/FECES/Gran Chaco/grey brocket deer/infectious disease/LEPTOSPIRA/Mazama gouazoubira/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/parasites/paratuberculosis/Serology/SEROTYPES/survey/TESTS/VIRAL DIARRHEA VIRUS/VIRUS/white-tailed deer

150 Basagoudanavar, S.H., Goswami, P.P., Tiwari, V., Pandey, A.K., Singh, N. (2004) Heterologous expression of a gene encoding a 35 kDa protein of Mycobacterium avium paratuberculosis in Escherichia coli

Veterinary Research Communications, 28, 209-224 The full-length open reading frame coding for a potentially immunogenic 35 kDa protein of Mycobacterium avium paratuberculosis was generated using polymerase chain reaction technology. The gene was inserted in-frame into Escherichia coli expression plasmid pQE32. The resulting recombinant plasmid pPMP35 was transformed into E. coli M15. Analysis of the E. coli induced with isopropyl-beta-D-thiogalactopyranoside revealed that the protein accumulated into the cytoplasm as insoluble inclusion bodies. The level of expression of the recombinant 35 kDa protein (P35) was more than 30% of the total

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protein of E coli cells. Expression of the recombinant protein was confirmed by immunoblotting. The P35 reacted with a rabbit antiserum raised against a sonicate of M. a. paratuberculosis. The protein was also recognized by serum from a goat with clinical paratuberculosis. Further, a polyclonal antiserum against P35 recognized a 35 kDa band in a membrane fraction of M. a. paratuberculosis. Also, the protein provoked a significant skin reaction in outbred guinea pigs sensitized with M. a. paratuberculosis, as well as in those sensitized with Mycobacterium avium. The results indicate that the 35 kDa protein of M. a. paratuberculosis is a membrane protein, having a role in the cellular immune response 35 kDa protein/antigens/AVIUM/CELL/CELLS/cellular immune response/CLONING/CROHNS-DISEASE/CULTURE/ESCHERICHIA-COLI/EXPRESSION/GENE/goat/heterologous expression/HIGH-LEVEL EXPRESSION/IDENTIFICATION/IMMUNE-RESPONSE/JOHNES-DISEASE/MEMBRANE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/POLYMERASE-CHAIN-REACTION/PROTEIN/PURIFICATION/rabbit/role

151 Deutz, A., Spergser, J., Rosengarten, R., Kofer, J. (2003) First detection of intrauterine transmission of paratuberculosis in red deer and chamois

Zeitschrift fur Jagdwissenschaft, 49, 314-319 The present paper describes to our knowledge the first detection of intrauterine transmission of M. avium subsp. paratuberculosis in red deer and chamois. A sharp increase of clinical cases of paratuberculosis in wild ruminants has been observed in southern Austria since 2002,which has been linked to an increase in suckler cow farming and a lack of feed hygiene in wildlife feeding. The paper discusses potential control measures against paratuberculosis in wild ruminants AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/chamois/control/deer/ FECES/INFECTION/Intrauterine/intrauterine transmission/JOHNES DISEASE/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/POLYMERASE-CHAIN-REACTION/RED DEER/RUMINANT PARA-TUBERCULOSIS/RUMINANTS/SCOTLAND/SURVIVAL/Transmission/WILD RUMINANTS/WILDLIFE

152 Uhart, M.M., Vila, A.R., Beade, M.S., Balcarce, A., Karesh, W.B. (2003) Health evaluation of pampas deer (Ozotoceros bezoarticus celer) at Campos del Tuyu Wildlife Reserve, Argentina

Journal of Wildlife Diseases, 39, 887-893 Samples from 14 free-ranging pampas deer (Ozotoceros bezoarticus celer) were collected in 1995 and 1998, at Campos del Tuyu Wildlife Reserve, Buenos Aires, Argentina. Hematology, serum chemistries, minerals and metals, and fecal parasites were analyzed. In addition, fecal ova and parasites were evaluated seasonally during 1998-2000. Serology for infectious diseases included bluetongue, brucellosis, bovine respiratory syncytial virus infection, bovine viral diarrhea/mucosal disease, infectious bovine rhinotracheitis, Johne's disease (paratuberculosis), foot and mouth disease (FMD), leptospirosis (eight serovars), epizootic hemorrhagic disease, and parainfluenza-3 (PI-3). Three (21%) pampas deer had antibodies to Leptospira spp. and six (43%) to PI-3 virus. Serologic results for all other infectious agents were negative. Domestic cattle (n=27) included in this study for comparison had antibodies to Leptospira, infectious bovine rhinotracheitis virus, bovine viral diarrhea virus, and PI-3 virus (74-100% of tested animals) and one animal (4%) to Brucella sp. All cattle had antibodies to FMD virus attributable to vaccination. This study provides the first data on the health status of the southernmost subspecies of pampas deer ANIMALS/ANTIBODIES/ANTIBODY/ARGENTINA/BOVINE/Bovine viral diarrhea/CATTLE/deer/Diarrhea/DISEASE/DISEASES/hematology/INFECTION/infectious disease/Johne's disease/LEPTOSPIRA/Ozotoceros bezoarticus celer/pampas deer/parasites/paratuberculosis/PREVALENCE/Serology/survey/VACCINATION/VIRAL DIARRHEA VIRUS/VIRUS/VIRUS-INFECTION/white-tailed deer/WILDLIFE

153 Stabel, J.R., Palmer, M.V., Whitlock, R.H. (2003) Immune responses after oral inoculation of weanling bison or beef calves with a bison or cattle isolate of mycobacterium avium subsp Paratuberculosis

Journal of Wildlife Diseases, 39, 545-555 Paratuberculosis is endemic in domestic and wild ruminants world-wide. We designed the following study to compare host immune responses and pathologic changes in beef calves and bison calves after challenge with either a cattle or bison (Bison bison) strain of Mycobacterium avium subsp. paratuberculosis. In the first part of the study, six bison and six beef calves were orally inoculated with a cattle isolate of M. avium, subsp. paratuberculosis over a 2 wk period. In the second part, an additional six bison and six beef calves were similarly inoculated with a bison strain of M. avium subsp.

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paratuberculosis. Throughout each of the studies, blood and fecal samples were taken monthly for a 6 mo infection period. Tissue samples were obtained at necropsy for culture and histopathologic analyses. Results from this study demonstrated that bison calves were more susceptible to tissue colonization than beef calves after challenge with the cattle isolate and, conversely, that beef calves were more susceptible to the bison strain of M. avium subsp. paratuberculosis. Although lesions were minimal they were most apparent in the jejunum and distal ileum. Interferon-gamma (IFN-gamma) responses were noted in some calves by 1 mo post-inoculation and were sustained longer in beef calves after challenge with the bison isolate. Antibody was not detected in either beef or bison calves during the 6 mo infection period. These results indicate that the host response to strains of M. avium subsp. paratuberculosis may differ between ruminant species AMERICAN WILD RUMINANTS/ANTIBODIES/ANTIBODY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BISON/bison bison/CALVES/CATTLE/challenge/CULTURE/deer/DIAGNOSTIC-TESTS/EXPERIMENTAL-INFECTION/FRAGMENT-LENGTH-POLYMORPHISM/IFN-gamma/immune responses/IMMUNE-RESPONSE/IMMUNE-RESPONSES/INFECTION/INOCULATION/interferon-gamma/JOHNES-DISEASE/LESIONS/LYMPHOCYTES/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberctilosis/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/ORAL INOCULATION/PARA-TUBERCULOSIS/paratuberculosis/PATHOLOGY/RESPONSES/RUMINANTS/STIMULATION/STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/TISSUE/WILD RUMINANTS

154 Enosawa, M., Kageyama, S., Sawai, K., Watanabe, K., Notomi, T., Onoe, S., Mori, Y., Yokomizo, Y. (2003) Use of loop-mediated isothermal amplification of the IS900 sequence for rapid detection of cultured Mycobacterium avium subsp. paratuberculosis

Journal of Clinical Microbiology, 41, 4359-4365 We evaluated the usefulness of loop-mediated isothermal amplification (LAMP) in detecting specific gene sequences of Mycobacterium avium subsp. paratuberculosis (MAP). A total of 102 primer sets for LAMP was designed to amplify the IS900, HspX, and F57 gene sequences of MAP. Using each of two primer sets (P-I and P-2) derived from the IS900 fragment, it was possible to detect MAP in a manner similar to that used with nested PCR. The sensitivity of LAMP with P-1 was 0.5 pg/tube, which was more sensitive than nested PCR. When P-2 was used, 5 pg/tube could be detected, which was the same level of sensitivity as that for nested PCR. LAMP with P-1 was specific. Although only 2 Mycobacterium scrofulaceum strains out of 43 non-MAP mycobacterial strains were amplified, the amplification reaction for these strains was less efficient than for MAP strains, and their products could be distinguished from MAP products by restriction digestion. LAMP with P-2 resulted in very specific amplification only from MAP, the same result obtained with nested PCR. Our LAMP method was highly specific, and the white turbidity of magnesium pyrophosphate, a by-product of the LAMP reaction, allowed simple visual detection. Our method is rapid, taking only 2 h, compared with 4 It for nested PCR. In addition, the LAMP method is performed under isothermal conditions and no special apparatus is needed, which makes it more economical and practical than nested PCR or real-time PCR. These results indicate that LAMP can provide a rapid yet simple test for the detection of MAP AMPLIFICATION/AVIUM/AVIUM SUBSP-PARATUBERCULOSIS/BOVINE FECES/CATTLE/CROHNS-DISEASE/CULTURE/DETECT MYCOBACTERIUM-PARATUBERCULOSIS/f57/GENE/IS900/JOHNES DISEASE/MAP/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/Nested PCR/NESTED-PCR/PARA-TUBERCULOSIS/paratuberculosis/PCR/POLYMERASE-CHAIN-REACTION/real time PCR/REAL-TIME/RESTRICTION/SENSITIVITY/SEQUENCE/STRAINS/WILD RABBITS

155 Muller, M. , Weber, A., Reith, B., Kratzer, R. (2003) Diseases of fallow deer from enclosures in Northern Bavaria. Results of post mortem examinations from 1988 to 2002

Tierarztliche Umschau, 58, 476-481 The present study describes the result of 288 post mortem examinations of fallow deer in Northern Bavaria during the years 1988 till 2002. The main causes of illness and death were alimentary diseases (28%), bacterial infections, mycoses (24%) and endoparasitism (22%). Eighteen percent of the cases remained unclear. Ruminal acidosis turned out to be the most important alimentary disease, whereas necrobacillosis, paratuberculosis, pneumonia, neonatal infections, listeriosis and yersiniosis were the main bacterial diseases. Among the endoparasites mixed infections with different intestinal nematodes and large lung worms dominated. In 8% of the examinations other causes of death, mainly traumatic injuries due to fights for the social rank, were found bacterial infection/causes of death/DAMA-DAMA/deer/DISEASE/DISEASES/disection results/enclosure/endoparasitism/FALLOW DEER/INFECTION/INFECTIONS/LISTERIA-

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MONOCYTOGENES/MORTALITY/NECROBACILLOSIS/NEMATODES/Northern Bavaria/paratuberculosis/postmortem examinations/ruminal acidosis

156 Deutz, A., Spergser, J., Wagner, P., Steineck, T., Kofer, J., Rosengarten, R. (2003) Paratuberculosis in wild animals - an observed increase in the clinical incidence

Tierarztliche Umschau, 58, 482-+ An apparent increase in the incidence of clinical paratuberculosis in six wild animal species (red deer, roe deer, chamois, mouffon, fallow deer, yellow necked mouse) in spring/summer of 2002 in Styria, a province of South Austria, is described. Of particular note was the occurrence of clinical disease in young animals. The first extraintestinal detection of M. avium subsp. paratuberculosis in wild animals and the isolation of the bacteria from roe deer, chamois, and mouffon in the wild in Austria are reported. The possible reasons for the observed increase in the incidence of clinical cases, the clinical pictures of paratuberculosis in wild animals, the potential for improved diagnostic methods and control and prevention measures together with meat hygiene issues are discussed ANIMALS/AVIUM/BACTERIUM/CATTLE/chamois/control/deer/DEER CERVUS-ELAPHUS/DISEASE/FALLOW DEER/HIPPELAPHUS/INFECTION/JOHNES-DISEASE/MOUSE/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/prevention/RED DEER/roe deer/SCOTLAND/SHEEP/Styria/WILD ANIMALS

157 White, S.N., Taylor, K.H., Abbey, C.A., Gill, C.A., Womack, J.E. (2003) Haplotype variation in bovine Toll-like receptor 4 and computational prediction of a positively selected ligand-binding domain

Proceedings of the National Academy of Sciences of the United States of America, 100, 10364-10369 Toll-like receptor 4 (TLR4) is a cell-surface receptor that activates innate and adaptive immune responses. Because it recognizes a broad class of pathogen-associated molecular patterns presented by lipopolysaccharides and lipoteichoic acid, TLR4 is a candidate gene for resistance to a large number of diseases. in particular, mouse models suggest TLR4 as a candidate gene for resistance to major agents in bovine respiratory disease and Johne's disease. The coding sequence of bovine TLR4 is divided into three exons, with intron/exon boundaries and intron sizes similar to those of human TLR4 transcript variant 1. We amplified each exon in 40 individuals from 11 breeds and screened the sequence for single-nucleotide polymorphisms (SNPs). We identified 32 SNPs, 28 of which are in the coding sequence, for an average of one SNP per 90 bp of coding sequence. Eight SNPs were nonsynonymous and potentially alter specificity of pathogen recognition or efficiency of signaling. To evaluate the functional importance of these SNPs, we used codon-substitution models to detect diversifying selection in an extracellular region that may physically interact with ligands. One nonsynonymous SNP is located within this region, and other substitutions are in adjacent regions that may interact with coreceptor molecules. The 32 SNPs were found in 20 haplotypes that can be assigned to geographic ranges of origin. Haplotype-tagging SNP analysis indicated that 12 SNPs need to be genotyped to distinguish these 20 haplotypes. These data provide a basic understanding of bovine TLR4 sequence variation and supply haplotype markers for disease association studies BOVINE/CELL-SURFACE/COMPLEX/DENDRITIC CELLS/DISEASE/DISEASES/DNA/GENE/GENES/immune responses/IMMUNE-RESPONSE/IMMUNE-RESPONSES/INFECTION/Johne's disease/LIPOPOLYSACCHARIDE/MODEL/MOUSE/POLYMORPHISMS/RECOGNITION/RESISTANCE/RESPONSES/SEQUENCE/SPECIFICITY/TLR4

158 Bannantine, J.P., Huntley, J.F.J., Miltner, E., Stabel, J.R., Bermudez, L.E. (2003) The Mycobacterium avium subsp paratuberculosis 35 kDa protein plays a role in invasion of bovine epithelial cells

Microbiology-Sgm, 149, 2061-2069 Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) enters intestinal epithelial cells of cattle and other ruminants via a mechanism that remains to be fully elucidated. This study showed that a gene encoding the M. paratuberculosis 35 kDa major membrane protein (MMP) is expressed at a higher level in low-oxygen and high-osmolarity conditions that are similar to the environment of the intestine. In addition, cattle with Johne's disease produced antibodies against MMP, suggesting that the protein is present during infection. The gene encoding MMP was cloned and expressed as a fusion protein with the maltose-binding protein (MBP-MMP) in Escherichia coli. Rabbit antisera were raised against a M. paratuberculosis whole-cell sonicate and MMP-specific antibodies were purified from these sera by affinity chromatography. MMP was localized to the surface of M. paratuberculosis by immunoelectron microscopy and by immunoblot analysis of fractionated protein lysates. Both anti-MMP antibodies and MBP-MMP protein inhibited M. paratuberculosis invasion of cultured Madin-Darby bovine kidney cells by 30 %. In similar invasion experiments with M. paratuberculosis incubated in low oxygen tension, these

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antibodies and protein decreased invasion by 60 %. Collectively, these data show that the 35 kDa MMP is a surface exposed protein that plays a role in invasion of epithelial cells. The authors suggest that the MMP is a virulence factor of M. paratuberculosis that may be important in the initiation of infection in vivo 35 kDa protein/35-KILODALTON PROTEIN/ANTIBODIES/ANTIBODY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/CATTLE/CELL/CELLS/DISEASE/ENVIRONMENT/EPITHELIAL-CELLS/ESCHERICHIA-COLI/GENE /IDENTIFICATION/INFECTION/INTESTINAL-MUCOSA/INVASION/Johne's disease/JOHNES-DISEASE/LEPRAE/MEMBRANE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/OXYGEN-TENSION/paratuberculosis/PEYERS-PATCHES/PROTEIN/rabbit/role/RUMINANTS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/VIRULENCE

159 Vecerek, V., Kozak, A., Malena, M., Tremlova, B., Chloupek, P. (2003) Veterinary meat inspection of bovine carcasses in the Czech Republic during the period of 1995-2002 Veterinarni Medicina, 48, 183-189 The results of veterinary meat inspection classification of 4 000 372 bovine carcasses reflect long-term aspects of health status in cattle herds and the quality of transport and handling of animals at slaughterhouses. Veterinary inspectors recorded the data obtained from meat inspection classification of bovine carcasses at slaughterhouses in the Czech Republic during the period of 1995-2002 together with the reasons for classification. The trends were evaluated by a comparison of two periods (Period I, 1995-1998, and Period II, 1999-2002) by means of calculating the indexes of values from Period II compared to those of Period I. Bovine carcasses classified as capable for human consumption (edible) were found in 87.87% of cases (88.83% during Period I and 86.58% during Period II, index 0.97), while those classified as capable for processing (conditionally edible) were found in 7.53% of cases (7.38% during Period I and 7.71% during Period II, index 1.04), and those condemned in 4.60% of cases (3.79% during Period I and 5.71% during Period II, index 1.51). The most important reason for classifying the carcasses as condemned was the finding of sensorial changes in meat, which occurred in 2.56% of cases (2.23% during Period I and 3.00% during Period II, index 1.35), followed by lesions due to non-infectious diseases - 1.00% (0.81% during Period I and 1.25% during Period II, index 1.53), added deleterious substances - 0.88% (0.60% during Period I and 1.27% during Period II, index 2.11), lesions due to respiratory infections - 0.03% (0.02% during Period I and 0.04% during Period II, index 1.74), and lesions due to miscellaneous infectious diseases - 0.10% (0.10% during Period I and 0.10% during Period II, index 1.05). Other reasons to condemn the carcasses included improper identification, lesions due to digestive infections, lesions due to tuberculosis, lesions due to paratuberculosis, lesions due to salmonellosis, leucosis and parasitic diseases. The occurrence of these conditions was on the level of mere hundredths of per cent. According to the results of meat inspection classification, the risk of food-borne diseases originating from bovine carcasses tends to be greater in the lesions due to non-infectious conditions with a long-term increasing trend. A considerable increase in the numbers of bovine carcasses condemned because of lesions due to paratuberculosis (index 4.62) represents an alarming finding with regard to potential food safety hazards ANIMALS/BOVINE/bull/CATTLE/CATTLE HERDS/CENTRAL-EUROPEAN COUNTRIES/cow/Crohn's disease/DISEASE/DISEASES/food/food safety/heifer/HERD/IDENTIFICATION/INFECTION/INFECTIONS/infectious disease/Johne's disease/LESIONS/meat inspection classification/MYCOBACTERIAL INFECTIONS/paratuberculosis/PIGS/POLAND/risk/risk assessment/SLAUGHTERHOUSE/Tuberculosis/WILD/zoonosis

160 Greenstein, R.J. (2003) Is Crohn's disease caused by a mycobacterium? Comparisons with leprosy, tuberculosis, and Johne's disease

Lancet Infectious Diseases, 3 , 507-514 ANTIBIOTIC REGIMEN/ANTIMYCOBACTERIAL THERAPY/AVIUM SUBSP PARATUBERCULOSIS/CONTROLLED TRIAL/Crohn's disease/DISEASE/IN-SITU HYBRIDIZATION/INFLAMMATORY-BOWEL-DISEASE/INTESTINAL TISSUES/Johne's disease/MOLECULAR EVIDENCE/mycobacteria/Mycobacterium/POLYMERASE-CHAIN-REACTION/Tuberculosis/WILD RUMINANTS

161 Manning, E.J.B., Kucera, T.E., Gates, N.B., Woods, L.M., Fallon-McKnight, M. (2003) Testing for Mycobacterium avium subsp Paratuberculosis infection in asymptomatic free-ranging tule elk from an infected herd Journal of Wildlife Diseases, 39, 323-328

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Forty-five adult tule elk (Cervus elaphus nannodes) in good physical condition were translocated from a population located at Point Reyes National Seashore, Marin County (California, USA), to a holding pen 6 mo prior to release in an unfenced region of the park. Because infection with Mycobacterium avium subsp paratuberculosis (Mptb) had been reported in the source population, the translocated elk underwent extensive ante-mortem testing using three johne's disease assays: enzyme linked immunosorbent assay (ELTSA); agar gel immunodiffusion assay (AGID), and decal culture. Isolation of Mptb was made from fecal samples in six of 45 elk (13%). All AGID results were negative while ELISA results for 18 elk (40%) were considered elevated. Elevated ELISA results or Mptb isolation from fecal samples were obtained for 22 of 45 elk (49%); these elk were euthanized and necropsied. Mycobacterium avium subsp. paratuberculosis was isolated from tissue in 10 of 22 euthanized elk (45%); of these 10 cases of confirmed infection, eight had elevated ELTSA results (80%) and four were fecal culture positive (40%). One of 10 cases had histopathologic lesions consistent with Mpth infection. Mycobacterium avium subsp. paratuberculosis was also isolated from tissue from one of eight fetuses sampled. The number of tule elk found to be infected was unexpected, both because of the continued overall health of the source herd and the normal clinical status of all study animals ANIMALS/ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/California/Cervus elaphits nannodes/Cervus elaphus/CERVUS-ELAPHUS/CULTURE/deer/DIAGNOSIS/DISEASE/ELISA/FECAL CULTURE/FETUSES/HERD/INFECTION/Johne's disease/JOHNES DISEASE/LESIONS/LINKED-IMMUNOSORBENT-ASSAY/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/paratuberculosis/PARATUBERCULOSIS INFECTION/POPULATION/SUBSP PARATUBERCULOSIS/SUBSP PARATUBERCULOSIS INFECTION/SUBSP-PARATUBERCULOSIS/TISSUE/tule elk

162 Manning, E.J.B., Steinberg, H., Krebs, V., Collins, M.T. (2003) Diagnostic testing patterns of natural Mycobacterium paratuberculosis infection in pygmy goats

Canadian Journal of Veterinary Research-Revue Canadienne de Recherche Veterinaire, 67, 213-218 Thirteen pygmy goats (Capra hircus) from a herd naturally infected with Mycobacterium avium ss. paratuberculosis (MPTB) were monitored with 4 diagnostic assays for 2 to 15 mo. Cellular and humoral immune responses to the infection were assessed with assays of gamma interferon (IFN-gamma), serum antibody [enzyme-linked immunosorbent assay (ELISA) and agar gel diffusion (AGID)], and radiometric fecal culture. Microscopic examination and radiometric culture of tissue from 12 sites were performed at necropsy. Goats were considered infected if MPTB was isolated from any tissue sample collected at necropsy. Mycobacterial isolates were confirmed as MPTB with an IS900 polymerase chain reaction assay. Ten goats whose antemortem tests indicated infection carried heavy organism burdens at necropsy, both within and beyond the gastrointestinal system. False-negative ELISA, AGID, and/or culture results were obtained in 5 of the 10 confirmed cases during the study period. In 3 goats with sporadic fecal shedding of MPTB or detectable IFN-gamma response, or both, no abnormalities were detected at necropsy and no MPTB was isolated from the tissue samples; the antemortem fecal-culture and IFNgamma results were thus considered false-positive. Diagnosticians should be alert to the possibility of both false-positive and false-negative test results for Johne's disease in goats. False-positive fecal-culture results may occur when a high prevalence of infection exists in the herd and the premises are likely to be heavily contaminated. The diverse antemortem testing patterns seen in these goats underscore the importance of using varied diagnostic assays serially or in parallel to increase the likelihood of identifying all infected goats ANTIBODIES/ANTIBODY/ASSAY/AVIUM/BOVINE PARATUBERCULOSIS/CATTLE/CULTURE/deer/DISEASE/ELISA/false positive/FECAL CULTURE/GAMMA/GAMMA-INTERFERON/goat/goats/HERD/IFN-gamma/immune responses/IMMUNE-RESPONSE/IMMUNE-RESPONSES/INFECTION/INTERFERON/IS900/Johne's disease/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/ORGANISM/paratuberculosis/PARATUBERCULOSIS INFECTION/POLYMERASE-CHAIN-REACTION/PREVALENCE/radiometric culture/RESPONSES/SERUM ANTIBODIES/SUBCLINICAL PARATUBERCULOSIS/TESTS/TISSUE

163 Machackova, M., Matlova, L., Lamka, J., Smolik, J., Melicharek, I., Hanzlikova, M., Docekal, J., Cvetnic, Z., Nagy, G., Lipiec, M., Ocepek, M., Pavlik, I. (2003) Wild boar (Sus scrofa) as a possible vector of mycobacterial infections: review of literature and critical analysis of data from Central Europe between 1983 to 2001

Veterinarni Medicina, 48, 51-65 Infected animals in the wild, which can act as a reservoir and/or vector for the origin of bovine tuberculosis, are a great problem for national programmes seeking to free herds of cattle from the infection. The circulation of Mycobacterium bovis in the wild animal population might cause a slow-down

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in the progress of control programmes through the reinfection of herds of livestock. The Eurasian badger (Meles meles) and red deer (Cervus elaphus) living in the wild in Great Britain and Ireland, brushtail possum (Trichosurus vulpecula), ferrets (Mustela putorius f. furo) in New Zealand and wild buffalo (Bubalus arnee) in Australia are among already known reservoirs and vectors of bovine tuberculosis. In 7 countries of Central Europe (Bosnia and Herzegovina, Croatia, Czech Republic, Hungary, Poland, Slovakia and Slovenia) bovine tuberculosis in cattle was controlled as part of national control programmes more than 20 years ago. In the last decade M. bovis has been diagnosed extremely sporadically in cattle and other domestic animals as well as in wild animals held in captivity or living in the wild. This favourable situation could be threatened by the mycobacteria spreading via the wild boar (Sus scrofa) which is susceptible to mycobacterial infection and very abundant in Central Europe. According to available literary data, mycobacteria were detected in 361 wild boar originating from countries other than those of Central Europe, such as Australia, Bulgaria, Germany, the Hawaiian island of Molokai, Italy and Spain. M. tuberculosis complex (33.9%) and M. bovis complex (39.8%) isolates were most frequently detected in the faeces and/or parenchymatous organs of wild boar. Of other mycobacterial species, M. intracellulare (3.8%), M. avium subsp. avium (3.8%), M. terrae (2.4%), M. fortuitum (2.2%), M. scrofulaceum (2.2%), M. gordonae (0.8%), M. simiae (0.5%), M. szulgai (0.5%), M. xenopi (0.5%), M. smegmatis (0.2%), M. vaccae (0.2%), fast-growing, further unspecified species (0.2%) and unidentified mycobacteria (8.8%) were isolated. Following the analysis of literary data and our own results, it was found that, in the area covered by the above-mentioned 7 countries of Central Europe, a total of 431 wild boar were examined for mycobacterial infections in the years 1983-2001. Tuberculous lesions in parenchymatous organs were found in 43 (10.0%) animals. M. bovis was identified in 22 (5.1%) animals, M. a. avium in 2 (0.4%), M. a. paratuberculosis in 1 (0.2%) animal and atypical mycobacteria in 27 (6.3%) animals. The wild boar may therefore represent, under certain unfavourable epizootio-logical conditions, a vector of some mycobacterial infections in not only animals, but also humans ANIMALS/ATYPICAL MYCOBACTERIA/AUSTRALIA/AVIUM/BOVINE/BOVINE TUBERCULOSIS/BOVIS/ CATTLE/Cervus elaphus/CERVUS-ELAPHUS/COMPLEX/control/deer/domestic animal/DOMESTIC-ANIMALS/faeces/FERAL PIGS/ferret/GREAT-BRITAIN/HERD/INFECTION/INFECTIONS/INTRACELLULARE/Italy/Johne's disease/LESIONS/ LYMPH-NODES/MOLECULAR EPIDEMIOLOGY/mycobacteria/MYCOBACTERIAL INFECTIONS/Mycobacterium/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/NEW-ZEALAND/NORTHERN-TERRITORY/paratuberculosis/POLAND/POPULATION/possum/RED DEER/reservoir/Review/risk assessment/SMEGMATIS/STRAINS/Tuberculosis/TUBERCULOSIS COMPLEX/TUBERCULOUS LESIONS/vector/veterinary epidemiology/WILD/WILD ANIMALS/WILD BOAR

164 Daniels, M.J., Henderson, D., Greig, A., Stevenson, K., Sharp, J.M., Hutchings, M.R. (2003) The potential role of wild rabbits Oryctolagus cuniculus in the epidemiology of paratuberculosis in domestic ruminants Epidemiology and Infection, 130, 553-559 Mycobacterium avium subspecies paratuberculosis, the organism responsible for paratuberculosis in cattle and sheep has been found in wild rabbits (Oryctolagus cuniculus) in the east of Scotland. Few studies have investigated either the level of faecal contamination by rabbits on farms, or the potential infectivity of rabbit excreta. The rate of rabbit faecal contamination deposited and the numbers encountered were estimated for 21 fields on 4 farms with a paratuberculosis problem. 7357 +/- 2571 S.E.M. rabbit faecal pellets were deposited per hectare per day and up to 81000 pellets/ha ('standing crop') were encountered in October/November 1998. Where access to rabbits was restricted, the standing crop of faeces encountered fell to 22 000 pellets/ha. The prevalence of infection with M. a. paratuberculosis was assessed for 83 rabbits from the four farms. M. a. paratuberculosis was isolated from rabbits on all farms with an overall prevalence of 17%. Out of 17 rabbits from which urine was available, M. a. paratuberculosis was isolated from two - the first reported isolation from urine in wild rabbits. The mean number of colony-forming units per gram of infected rabbit faeces was 7(.)6 x 10(5) +/- 5.2 x 10(5). A relative estimate of the input of M. a. paratuberculosis onto pasture, at the stocking levels found on the four farms, showed that sheep and cattle potentially contributed 4 and 125 times more organisms/ha per day respectively than rabbits. However, rabbits could still contribute millions of M. a. paratuberculosts organisms per ha per day. Existing rabbit control measures on farms may be inadequate in reducing the risk of transmission to livestock AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/CATTLE/control/Epidemiology/faeces/FARMS/INFECTION/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/ORGANISM/ORGANISMS/ORYCTOLAGUS-CUNICULUS/paratuberculosis/pasture/PELLETS/POTENTIAL ROLE/PREVALENCE/rabbit/RABBITS/risk/role/RUMINANTS/SCOTLAND/SHEEP/Transmission/WILD/WILD RABBIT/WILD RABBITS

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165 Daniels, M.J., Hutchings, M.R., Greig, A. (2003) The risk of disease transmission to livestock posed by contamination of farm stored feed by wildlife excreta Epidemiology and Infection, 130, 561-568 Livestock feed is susceptible to contamination from wildlife excreta during on farm storage. Pathogens associated with diseases such as paratuberculosis, salmonella and cryptosporidiosis are present in wild rodent and bird excreta. Feed stores on four farms in the east of Scotland were monitored monthly over the winter of 1998/9 to quantify the levels of wildlife faecal contamination. A mean of 79(.)9 rodent (95% confidence interval: 37(.)5-165(.)9) and 24(.)9 (14(.)3-41(.)7) bird faeces were deposited per m(2) of stored feed per month. It was estimated that individual cattle and sheep could encounter 1626 and 814 wildlife faeces over the winter. A model based on the numbers of infected faeces consumed per annum was used to estimate 'infectious probabilities' (P-inf) required to account for the reported prevalence of paratuberculosis, salmonella and cryptosporidiosis in sheep and cattle in the cast of Scotland in 1998. Based on empirical data for input variables [the number of faeces encountered (F-i) the number ingested (F-i) and the prevalence of infection in wildlife species (I-p)], P-inf estimates ranged from 1(.)6 x 10(-8) for cryptosporidiosis in sheep to 8(.)2 x 10(-6) for paratuberculosis in cattle. The model suggested that ingestion of feed contaminated by wildlife faeces could account for the prevalence of all three diseases. Wildlife faecal contamination of stored feed should be given serious consideration as a potential source of infection to livestock CATTLE/DISEASE/disease transmission/DISEASES/faeces/FARMS/INFECTION/MAMMALS/MODEL/paratuberculosis/PREVALENCE/RABBITS/risk/SALMONELLA/SCOTLAND/SHEEP/Transmission/WILD/WILDLIFE

166 Corner, L.A.L., Pfeiffer, D.U., Abbott, K.A. (2003) Unanswered questions about the transmission of Mycobacterium avium subspecies paratuberculosis Veterinary Journal, 165, 182-183 AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/CATTLE /INFECTION/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/Transmission/WILD RABBITS

167 Daniels, M.J., Lees, J.D., Hutchings, M.R. , Greig, A. (2003) The ranging behaviour and habitat use of rabbits on farmland and their potential role in the epidemiology of paratuberculosis Veterinary Journal, 165, 248-257 Grazing herbivores avoid grass swards contaminated with faeces as the ingestion of faeces is a common route of micro- and macro-parasite transmission. The recent novel finding that herbivores do not avoid grass swards contaminated with rabbit faeces suggests that disease risk posed to herbivores by rabbits is determined by rabbit ranging and excretory behaviour. Using as a case study rabbits and the risk Mycobacterium avium sub-species paratuberculosis (M. a. paratuberculosis) poses to cattle, the interaction between rabbits and grazing pasture was studied on an infected farm in the east of Scotland in spring and autumn 2000. Radio telemetry, burrow surveys and faecal pellet count data were collected on two areas (Areas 1 and 2) of the farm with different habitat mosaics, to study the potential effects of season and habitat on the spatial distribution of rabbits faeces and thus disease in the environment. Twenty one rabbits were radio tracked and a total of 902 fixes collected. Mean home range sizes (100% minimum convex polygons) were between 2.0 and 7.1 ha per rabbit per season. Home ranges were significantly larger in spring, and in Area 1 which had more moor and woodland and less rough pasture. Rabbits used rough pasture most in Area 1 and gorse scrub in Area 2. In both areas, significantly more burrows were located in gorse scrub than in any other habitat. Most faecal pellets were deposited on the moorland habitat of Area 2 in autumn. In habitats to which grazing livestock had access, the mean rate of faecal deposition was 8571 pellets per ha per day. The greatest risk of disease transmission occurred in habitats of poor grazing quality (e.g., gorse scrub) which were used by rabbits for burrowing and thus contained high concentrations of faeces. The findings of the study are discussed in relation to management practices aimed at reducing disease risk to livestock, including the fencing of scrub and the reduction of rabbit population size to prevent expansion of rabbit burrows from scrub into grazing pastures. (C) 2002 Elsevier Science Ltd. All rights reserved 2 LAGOMORPHS/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/CATTLE/DIFFERENT FEEDING STRATEGIES/DISEASE/disease transmission/ecology/ENVIRONMENT/Epidemiology/faeces/home-range/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/ORYCTOLAGUS-

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CUNICULUS/paratuberculosis/pasture/PELLETS/POPULATION/POTENTIAL ROLE/rabbit/RABBITS /risk/role/SCOTLAND/SIZES/STRATIFIED UPLAND LANDSCAPE/survey/Transmission/WILD RABBITS

168 de Lisle, G.W., Yates, G.F., Montgomery, R.H. (2003) The emergence of Mycobacterium paratuberculosis in farmed deer in New Zealand - a review of 619 cases

New Zealand Veterinary Journal, 51, 58-62 AIM: To review cases in which Mycobacterium paratuberculosis was identified in farmed deer in New Zealand. METHODS: Case histories were reviewed where M. paratuberculosis was identified in deer by either culture or a polymerase chain reaction (PCR) test using primers from IS900. RESULTS: Between 1986 and 2000, M. paratuberculosis was identified by bacterial culture and/or PCR in 619 farmed deer from 299 herds, representing approximately 6% of deer herds in New Zealand. Over 85% of cases were identified during the last 6 years. In 60% of the infected herds, only one infected animal was identified. The maximum number of cases identified in a single deer herd was 47, and these were identified over a period of 8 years. Only 36 (5.8%) cases came from clinically affected animals identified on farms by veterinarians. The majority (89.7%) of the 619 cases were identified from lesions in mesenteric lymph nodes, including the ileocaecal lymph nodes, identified at meat inspection as being macroscopically either typical or equivocal of bovine tuberculosis (M. bovis). While the overwhelming majority of lesions were identified in mesenteric lymph nodes, M. paratuberculosis was also identified in 27 lesions in lymph nodes of the head, especially the retropharyngeal lymph node. CONCLUSIONS: The figures presented underestimate the true prevalence of infection with M. paratuberculosis, especially since not all suspect cases were submitted for culture or PCR. However, they do show that M. paratuberculosis appears to be spreading in farmed deer in New Zealand and highlight the possibility that Johne's disease is emerging as a potential major problem affecting this species. Identification of the organism by bacterial culture or PCR is required in many cases to distinguish lesions in mesenteric lymph nodes and lymph nodes of the head caused by M. paratuberculosis from those caused by M. bovis and M. avium ANIMALS/AVIUM/BACTERIAL CULTURE/BOVINE/BOVINE TUBERCULOSIS/BOVIS/CERVUS-ELAPHUS/CULTURE/deer/DISEASE/FARMED DEER/FARMS/HERD/IDENTIFICATION/INFECTION/IS900/Johne's disease/JOHNES-DISEASE/LESIONS/LYMPH-NODES/mycobacteria /Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/NEW-ZEALAND/ORGANISM/paratuberculosis/PCR/polymerase chain reaction/POLYMERASE CHAIN-REACTION/POLYMERASE-CHAIN-REACTION/PREVALENCE/RED DEER/Review/SEQUENCE/Tuberculosis

169 Daniels, M.J., Hutchings, M.R., Beard, P.M., Henderson, D., Greig, A., Stevenson, K., Sharp, J.M. (2003) Do non-ruminant wildlife pose a risk of paratuberculosis to domestic livestock and vice versa in Scotland? Journal of Wildlife Diseases, 39, 10-15 Paratuberculosis (Johne's disease) was long considered only a disease of ruminants. Recently non-ruminant wildlife species have been shown to harbor Mycobacterium avium subsp. paratuberculosis, the causative organism of paratuberculosis. We review the known non-ruminant wildlife host range of M. avium subsp. paratuberculosis and consider their role in the epidemiology of paratuberculosis in domestic ruminant livestock. Mycobacterium avium subsp. paratuberculosis has been isolated from lagomorph, canid, mustelid, corvid, and murid species. In agricultural environments domestic ruminants may contact wildlife and/or their excreta when grazing or feeding on farm-stored feed contaminated with wildlife feces, opening up the possibility of inter-species transmission. Of the wildlife species known to harbor M. avium subsp. paratuberculosis in Scotland, the rabbit is likely to pose the greatest risk to grazing livestock. Paratuberculosis in domestic ruminants is a notoriously difficult disease to control; the participation of non-ruminant wildlife in the epidemiology of the disease may partially account for this difficulty AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/control/DISEASE/ENVIRONMENT/Epidemiology/FECES/INFECTION/Johne's disease/JOHNES-DISEASE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/non-ruminant wildlife/ORGANISM/PARA-TUBERCULOSIS/paratuberculosis/rabbit/RABBITS/Review/risk/role/RUMINANTS/SCOTLAND/Transmission/tule elk/WILDLIFE

170 Nettles, V.F., Quist, C.F., Lopez, R.R., Wilmers, T.J., Frank, P., Roberts, W., Chitwood, S., Davidson, W.R. (2002) Morbidity and mortality factors in key deer (Odocoileus virginianus clavium)

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Journal of Wildlife Diseases, 38, 685-692 The population health of endangered Key deer (Odocoileus virginianus clavium) was monitored from 10 February 1986 to 28 September 2000 by necropsy of animals that were killed by vehicles, euthanized because of terminal injuries or disease conditions, or found dead. The predominant mortality factor during the period was collision with motor vehicles; however, several infectious diseases were diagnosed, including infections with Arcanobacterium pyogenes, Haemonchus contartus, Salmonella spp., and Mycobacterium avium subsp. paratuberculosis. During the period monitored, the only infectious disease that was thought to have affected population dynamics was haemonchosis. Nevertheless, several of the observed diseases have potential to impact viability of the Key deer population under appropriate environmental conditions ANIMALS/Arcanobacterium pyogenes/AVIUM/CLAVIUM/deer/DISEASE/ DISEASES/Haemonchus contortus/IMPACT/INFECTION/INFECTIONS/infectious disease/Johne's' disease/Key deer/MORTALITY/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/Odocoileus virginianus/Odocoileus virginianus clavium/ODOCOILEUS-VIRGINIANUS/ODOCOILEUS-VIRGINIANUS-CLAVIUM/paratuberculosis/POPULATION/SALMONELLA/salmonellosis/white-tailed deer

171 Quist, C.F., Nettles, V.F., Manning, E.J.B., Hall, D.G., Gaydos, J.K., Wilmers, T.J., Lopez, R.R. (2002) Paratuberculosis in key deer (Odocoileus virginianus clavium)

Journal of Wildlife Diseases, 38, 729-737 Paratuberculosis was diagnosed in an endangered Key deer (Odocoileus virginianus clavium) in November 1996. Between 10 April 1997 and 28 September 2000, the Key deer population was monitored for infection with Mycobacterium avium subsp. paratuberculosis by necropsy of available carcasses (n=170), fecal cultures, and serology. One additional clinically affected Key deer was discovered in July 1998, and M. avium subsp. paratuberculosis was cultured from the feces of one live, asymptomatic deer. The results of this study provided sufficient evidence to consider the Key deer herd infected with M. avium subsp. paratuberculosis at very low prevalence AMERICAN WILD RUMINANTS/AVIUM/CLAVIUM/CULTURE/deer/DIAGNOSIS/FECAL CULTURE/FECES/HERD/INFECTION/Johne's disease/Key deer/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/Odocoileus virginianus/Odocoileus virginianus clavium/ODOCOILEUS-VIRGINIANUS/ODOCOILEUS-VIRGINIANUS-CLAVIUM/PARA-TUBERCULOSIS/paratuberculosis/PATHOGENESIS/PATHOLOGY/POPULATION/PREVALENCE/Serology/TESTS/tule elk

172 Feng, Z.Y. , Barletta, R.G. (2003) Roles of Mycobacterium smegmatis D-alanine : D-alanine ligase and D-alanine racemase in the mechanisms of action of and resistance to the peptidoglycan inhibitor D-cycloserine

Antimicrobial Agents and Chemotherapy , 47, 283-291 D-Cycloserine (DCS) targets the peptidoglycan biosynthetic enzymes D-alanine racemase (Air) and D-alanine:D-alanine ligase (Ddl). Previously, we demonstrated that the overproduction of Air in Mycobacterium smegmatis determines a DCS resistance phenotype. In this study, we investigated the roles of both Air and Ddl in the mechanisms of action of and resistance to DCS in M. smegmatis. We found that the overexpression of either the M. smegmatis or the Mycobacterium tuberculosis ddl gene in M. smegmatis confers resistance to DCS, but at lower levels than the overexpression of the alr gene. Furthermore, a strain overexpressing both the alr and ddl genes displayed an eightfold-higher level of resistance. To test the hypothesis that inhibition of Air by DCS decreases the intracellular pool Of D-alanine, we determined the alanine pools in M. smegmatis wild-type and recombinant strains with or without DCS treatment. Alr-overproducing strain GPM14 cells not exposed to DCS displayed almost equimolar amounts Of L- and D-alanine in the steady state. The wild-type strain and Ddl-overproducing strains contained a twofold excess Of L- over D-alanine. In all strains, DCS treatment led to a significant accumulation Of L-alanine and a concomitant decease Of D-alanine, with approximately a 20-fold excess Of L-alanine in the Ddl-overproducing strains. These data suggest that Ddl is not significantly inhibited by DCS at concentrations that inhibit Air. This study is of significance for the identification of the lethal target(s) of DCS and the development of novel drugs targeting the D-alanine branch of mycobacterial peptidoglycan biosynthesis AVIUM/CELL/CELLS/CHLORO-D-ALANINE/EFFICIENT/ENVELOPE/GENE/GENES/IDENTIFICATION/INHIBITION/MUTANT/mycobacteria/Mycobacterium/Mycobacterium

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smegmatis/paratuberculosis/RESISTANCE/role/SMEGMATIS/STRAINS/TRANSFORMATION/Tuberculosis/VANCOMYCIN

173 Collins, D.M., De Zoete, M., Cavaignac, S.M. (2002) Mycobacterium avium subsp paratuberculosis strains from cattle and sheep can be distinguished by a PCR test based on a novel DNA sequence difference

Journal of Clinical Microbiology, 40, 4760-4762 A DNA sequence differing between sheep and cattle types of Mycobacterium avium subsp. paratuberculosis was identified and used to develop a PCR test. The test unequivocally distinguished all sheep types from cattle types and was negative for a wide range of other strains from the Mycobacterium avium-Mycobacterium intracellulare complex. The test will be useful for epidemiological purposes, particularly in hosts such as deer that can be easily infected with either type ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/COMPLEX/deer/DNA/FRAGMENT-LENGTH-POLYMORPHISM/GENE/IDENTIFICATION/INTRACELLULARE/IS1311/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PCR/RESTRICTION ENDONUCLEASE ANALYSIS /SEQUENCE/SHEEP/STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS

174 Frolich, K., Thiede, S., Kozikowski, T., Jakob, W. (2002) A review of mutual transmission of important infectious diseases between livestock and wildlife in Europe Domestic Animal/Wildlife Interface: Issue for Disease Control, Conservation, Sustainable Food Production, and Emerging Diseases, 969, 4- 13 Oral vaccination of red foxes against rabies has been practiced in Europe since 1978 and has succeeded in greatly reducing the occurrence of this disease in foxes: this is an example of coordinated activity against a disease that affects both wild and domestic animals as well as humans. Some examples of diseases that affect both domestic and wild animals in Europe are: classical swine fever (hog cholera) in wild boars and domestic swine; myxomatosis and rabbit hemorrhagic disease in domestic and wild rabbits; bovine viral diarrhea (BVD) in cattle and roe deer; contagious ecthyma in domestic sheep and goats and also in, e.g., chamois, muskox, and reindeer; Mycobacterium bovis in cattle, wild boars, badgers, and deer; and brucellosis in a broad range of livestock and wildlife in all European countries. In addition, serological surveys performed in different free-ranging ungulate species revealed the presence of alphaherpesviruses related to bovine herpesvirus-1 in 7 European countries; and a study of malignant catarrhal fever in deer in Germany might indicate that in this case sheep are the main reservoir species. Although many data on infectious diseases are available in various European countries, there is more need for systematic surveillance and coordinated research ALPINE IBEX/ANIMALS/BADGERS/BOVINE/Bovine viral diarrhea /BOVIS/BROWN HARE SYNDROME/BRUCELLA-MELITENSIS/CAPTIVE DEER CERVIDAE/CATTLE/chamois/classical swine fever/deer/Diarrhea/DISEASE/DISEASES/domestic animal/DOMESTIC SHEEP/DOMESTIC-ANIMALS/Europe/FRANCISELLA-TULARENSIS/goat/goats/infectious disease/infectious diseases/LEPUS-EUROPAEUS/livestock/MALIGNANT CATARRHAL FEVER/mycobacteria/Mycobacterium/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/MYCOBACTERIUM-PARATUBERCULOSIS/MYXOMA VIRUS/ORAL VACCINATION/rabbit/RABBIT HEMORRHAGIC-DISEASE/RABBITS/reservoir/Review/roe deer/SHEEP/sheep and goats/survey/Transmission/two-way transmission/Ungulate/VACCINATION/WILD/WILD ANIMALS/WILD BOAR/wild boars/WILD RABBIT/WILD RABBITS/WILDLIFE

175 Estes, D.M., Brown, W.C. (2002) Type 1 and type 2 responses in regulation of Ig isotype expression in cattle

Veterinary Immunology and Immunopathology, 90, 1-10 Regulation of humoral immune responses is multifactorial involving appropriate activation, costimulation and the presence of specific soluble factors. Polarized type 1 or type 2 humoral responses in the laboratory mouse have been linked to expression of specific cytokines and thus can be used to provide insight into the type of response generated by infection. For example, IFNgamma has been linked to IgG2a and IgG3 production, IL-4 to IgG1 and IgE production and TGF-beta to IgA production. Unlike the laboratory mouse, generally housed under defined conditions, highly skewed isotype expression patterns generally occur in cattle in chronic infections. A few examples of polarized responses have been noted in chronic experimental or naturally occurring infections including F. hepatica, M. paratuberculosis, C. parvum and B. abortus. In vitro studies using purified bovine B cells and various forms of costimulation

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and cytokines have demonstrated that isotype responses can be polarized under certain experimental conditions in vitro. That is, IgG1 expression is positively regulated by IL-4 and IgG2 expression is positively regulated by IFNgamma. Other as yet unidentified factors may play pivotal roles in regulating humoral immune responses in large ruminant species in vivo. This possibility is best exemplified by recent studies using DNA vaccines in cattle that have been demonstrated in the mouse to be generally polarizing to a type 1 response. Surprisingly, studies in cattle using plasmid-DNA as vaccination material show an almost exclusive IgG1 response. Based on a number of studies using T cell clones and various biological assays, it is clear that the classical roles of many cytokines in the laboratory mouse do not extrapolate entirely or at all to cattle. Thus, the design of adjuvants and immune modulators should be based on studies done in cattle or using bovine cells. Based on studies to date, several "holes" in the cytokine repertoire exist and these roles may be assumed by unique factors or activities of other known cytokines. (C) 2002 Elsevier Science B.V. All rights reserved ASSAY/BOVINE/CATTLE/CELL/CELLS/costimulation/cytokine/CYTOKINE GENE-EXPRESSION/DNA/DNA vaccine/DNA vaccines/EXPRESSION/GROWTH-FACTOR-BETA/humoral response/IFN-GAMMA PRODUCTION/immune responses/IMMUNE-RESPONSE/IMMUNE-RESPONSES/IMMUNOGLOBULIN/IMMUNOGLOBULIN CLASS SWITCH/IN-VITRO/INFECTION/INFECTIONS/interferon-gamma/isotype/MOUSE/MURINE B-CELLS/paratuberculosis/PATHOGEN ANAPLASMA-MARGINALE/PLASMID DNA/RESPONSES/role/T cell/T-CELLS/VACCINATION

176 Pavlik, I. , Dvorska, L., Bartos, M., Parmova, I., Melicharek, I., Jesenska, A., Havelkova, M., Slosarek, M., Putova, I., Martin, G., Erler, W., Kremer, K., Van Soolingen, D. (2002) Molecular epidemiology of bovine tuberculosis in the Czech Republic and Slovakia in the period 1965-2001 studied by spoligotyping

Veterinarni Medicina, 47, 181-194 Spoligotyping was used to examine IS6110-positive DNA of 26 Mycobacterium bovis, M. bovis BCG and M. bovis subsp. caprae non-viable isolates stored up to 10 years. All of these isolates were previously identified by biochemical tests and all 17/17 tested isolates were earlier found virulent for guinea pigs. In total seven spoligotypes, designated S1-S7, were detected and compared with the spoligotypes of 3 176 isolates in the database of the National Institute of Public Health and the Environment (RIVM) in Bilthoven, the Netherlands. A Neotype M. bovis strain, isolated in 1965 in the USA and thereafter stored in The Czechoslovak National Collection of Type Cultures (My 310/87) since 1987 was of an identical spoligotype S4 with the original reference M bovis strain from the USA. The M. bovis isolates from capybara's (Hydrocboerus hydrochaeris) imported from Germany to the Czech Republic in 1989, as well as cattle isolates from 1966, 1991 and 1994, were of the most common type S1. Also a human isolate from 1981, a M. bovis BCG vaccine strain and clinical M. bovis BCG isolates from three children with post-vaccinal complications were of this most predominant spoligotype. The four unique spoligotypes S2, S3, S5 and S6 were identified in M. bovis isolates from cattle in the years 1965, 1996 and 1967 in the Czech Republic, respectively, but also in isolates from farmed red deer (Cervus elaphus) from 1991 and in cattle isolates from Slovakia from the year 1992. The scarcely occurring spoligotype S7, which is typical for M. b. caprae was detected in the Czech Republic from farmed red deer (1999), cattle isolates (1966, 1991, 1995) and in a strain isolated from an 80-year-old man (1999). Several strains isolated in each of three outbreaks in cattle herds were examined. Identical spoligotypes were detected in two outbreaks and different causal agents (M. bovis of spoligotype S 1 and M. b. caprae of spoligotype S7) were identified in two cows from the third outbreak. The results confirm an effective control of bovine tuberculosis in the Czech Republic and Slovakia during 1959-1968, because previously circulating spoligotypes were successfully eradicated. The data also suggest other reservoirs of bovine tuberculosis may exist among free-living wild animals ANIMALS/BCG/BOVINE/BOVINE TUBERCULOSIS/BOVIS/capybara/CATTLE/CATTLE HERDS/Cervus elaphus/CERVUS-ELAPHUS/COMPLEX STRAINS/control/cow/COWS/CULTURE/deer/DIFFERENTIATION/DNA/ELEMENT/ENVIRONMENT/Epidemiology/FARMED RED DEER/FRAGMENT-LENGTH-POLYMORPHISM/GENETIC DIVERSITY/HERD/IS6110/MOLECULAR EPIDEMIOLOGY/mycobacteria/Mycobacterium/Mycobacterium bovis/Mycobacterium bovis BCG,cattle/Mycobacterium bovis subsp caprae/MYCOBACTERIUM-BOVIS/MYCOBACTERIUM-TUBERCULOSIS/paratuberculosis/PIGS/post-vaccinal complications in children/RED DEER/reservoir/STRAINS/TANDEM DNA REPEATS/TESTS/Tuberculosis/WILD/WILD ANIMALS

177 Waters, W.R., Palmer, M.V., Whipple, D.L. (2002) Mycobacterium bovis-infected white-tailed deer (Odocoileus virginianus): detection of immunoglobulin specific to crude mycobacterial antigens by ELISA Journal of Veterinary Diagnostic Investigation, 14, 470 -475 White-tailed deer (Odocoileus virginianus) have recently emerged as a source of Mycobacterium bovis

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infection for cattle within North America. The objective of this study was to evaluate the antibody response of M. bovis-infected deer to crude mycobacterial antigens. Deer were experimentally inoculated with M. bovis strain 1315 either by intratonsilar instillation or by exposure to M. bovis-infected (i.e., in contact) deer. To determine the time course of the response, including the effects of antigen administration for comparative cervical skin testing, serum was collected periodically and evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (i.e., IgG heavy and light chains) reactivity to mycobacterial antigens. The reactivity to M. bovis purified protein derivative (PPDb) exceeded (P < 0.05) the reactivity to M. avium PPD (PPDa) only after in vivo administration of PPDa and PPDb for comparative cervical testing of the infected deer. The mean immunoglobulin response, as measured by ELISA, of intratonsilar-inoculated deer to a proteinase K-digested whole-cell sonicate (WCS-PK) of M. bovis strain 1315 exceeded (P < 0.05) the mean of the prechallenge responses to this antigen at approximately 1 month after inoculation and throughout the remainder of the study (i.e., similar to11 months). This response also exceeded (P < 0.05) that of the uninfected deer. Although this is encouraging, further studies are necessary to validate the use of the proteinase K-digested M. bovis antigens in the antibody-based assays of tuberculosis ANTIBODIES/ANTIBODY/ANTIBODY-RESPONSES/ANTIGEN/antigens/ARABINOMANNAN/ASSAY/AVIUM/BOVIS/CATTLE/deer /DIAGNOSIS/ELISA/enzyme-linked immunosorbent assay/IMMUNOGLOBULIN/INFECTION/INOCULATION/LINKED-IMMUNOSORBENT-ASSAY/LIPOARABINOMANNAN/MODEL/mycobacteria/Mycobacterium/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/Odocoileus virginianus/ODOCOILEUS-VIRGINIANUS/paratuberculosis/PROTEIN/RESPONSES/skin testing/TESTS/Tuberculosis/white-tailed deer

178 Stabel, J.R., Ackermann, M.R. (2002) Temporal Mycobacterium paratuberculosis infection in T-cell receptor (TCR)-alpha and TCR-delta-deficient mice Veterinary Immunology and Immunopathology, 89, 127-132 Mycobacterium paratuberculosis is the causative agent of paratuberculosis (Johne's disease), a chronic inflammation of the terminal portion of the ileum in ruminants. The predominance of cell-mediated immunity in early stages of the disease suggests that T lymphocytes are essential to protect the host from infection with M. paratuberculosis. In this study, we investigated the role of alphabeta and gammadelta T cells in resistance to M. paratuberculosis infection using a T-cell receptor (TCR) knockout mouse model. Weanling TCR-alpha-deficient, TCR-delta-deficient, and C57BL/6 control mice (5-6 weeks of age) were acclimated for 2 weeks and then inoculated intraperitoneally with 108 CFU/ml of M. paratuberculosis (either strain 19698 or strain Ben). Groups of mice within each treatment group were euthanized at 1, 3 and 6 months post-inoculation. Sections of spleen, liver, ileum and mesenteric lymph node were prepared for bacterial culture and histologic examination. At all time points of infection and regardless of bacterial strain, TCR-alpha-deficient mice had higher levels of M. paratuberculosis colonization in their tissues compared to TCR-6-deficient mice or C57BL/6 control mice. Lesions were located predominately in the liver and the ileum, depending upon period of infection, and lesion scores were higher for TCR-a-deficient mice compared to the other treatment groups. These results suggest that alphabeta T cells play a major role in resistance to infection with M. paratuberculosis and that gammadelta T cells may play a lesser role and potentially confound protective immune responses. (C) 2002 Elsevier Science B.V. All rights reserved BACTERIAL CULTURE/CELL/cell-mediated immunity/CELLS/control/CULTURE/DISEASE/GENES/immune responses/IMMUNE-RESPONSE/IMMUNE-RESPONSES/INFECTION/Johne's disease/LESIONS/ LYMPHOCYTES/MICE/MODEL /MOUSE/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/Mycobacterium paratuberculosis infection/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/PARATUBERCULOSIS INFECTION/RESISTANCE /RESPONSES/role/RUMINANTS/T cell/T-cell receptor/T-CELLS/TISSUE/TISSUES/Tuberculosis

179 Marco, I., Ruiz, M., Juste, R., Garrido, J.M., Lavin, S. (2002) Paratuberculosis in free-ranging fallow deer in Spain

Journal of Wildlife Diseases, 38, 629-632 Paratuberculosis was diagnosed in a population of approximately 1,000 free-ranging fallow deer (Dama dama) sampled from 1997-98 in the Regional Hunting Reserve of El Sueve (Asturias, Spain). Five of eight animals observed with diarrhea were diagnosed as having paratuberculosis on the basis of gross lesions at postmortem examination and histopathology. In two deer, Mycobacterium avium subsp. paratuberculosis was cultured and identified by polymerase chain reaction. Indirect enzyme-linked immunosorbent assay and immunodiffusion tests were used to evaluate sera from 33 adult deer from this population. All fallow deer tested were seronegative

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ANIMALS/ASSAY/AVIUM/Dama dama/Dama dana/DAMA-DAMA/deer/Diarrhea/enzyme-linked immunosorbent assay/FALLOW DEER/INFECTION/LESIONS/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/paratuberculosis/polymerase chain reaction/POLYMERASE-CHAIN-REACTION/POPULATION/Spain/TESTS

180 Feng, Z.Y. , Caceres, N.E., Sarath, G., Barletta, R.G. (2002) Mycobacterium smegmatis L-alanine dehydrogenase (Ald) is required for proficient utilization of alanine as a sole nitrogen source and sustained anaerobic growth

Journal of Bacteriology, 184, 5001-5010 NAD (H)-dependent L-alanine dehydrogenase (EC 1.4.1.1) (Ald) catalyzes the oxidative deamination of L-alanine and the reductive amination of pyruvate. To assess the physiological role of Aid in Mycobacterium smegmatis, we cloned the ald gene, identified its promoter, determined the protein expression levels, and analyzed the combined effects of nutrient supplementation, oxygen availability, and growth stage on enzyme activity. High Aid activities were observed in cells grown in the presence of L- or D-alanine regardless of the oxygen availability and growth stage. In exponentially growing cells under aerobic conditions, supplementation with alanine resulted in a 25- to 50-fold increase in the enzyme activity. In the absence of alanine supplementation, 23-fold-higher Aid activities were observed in cells grown exponentially under anaerobic conditions. Furthermore, M. smegmatis ald null mutants were constructed by targeted disruption and were shown to lack any detectable Aid activity. In contrast, the glycine dehydrogenase (EC 1.4.1.10) (Gdh) activity in mutant cells remained at wild-type levels, indicating that another enzyme protein is responsible for the physiologically relevant reductive amination of glyoxylate. The ald mutants grew poorly in minimal medium with L-alanine as the sole nitrogen source, reaching a saturation density 100-fold less than that of the wild-type strain. Likewise, mutants grew to a saturation density 10-fold less than that of the wild-type strain under anaerobic conditions. In summary, the phenotypes displayed by the M. smegmatis ald mutants suggest that Aid plays an important role in both alanine utilization and anaerobic growth ALLELIC EXCHANGE/BACILLUS-SUBTILIS/BOVIS BCG/CELL/CELLS/DEPLETION-INDUCED DORMANCY/EFFICIENT/EXPRESSION/GENE/GROWTH/IN-VITRO/MUTANT/mycobacteria/Mycobacterium/Mycobacterium smegmatis /NONREPLICATING PERSISTENCE/paratuberculosis/PROTEIN/role/SMEGMATIS/SOYBEAN NODULE BACTEROIDS/Tuberculosis

181 Mutwiri, G.K., Rosendal, S., Kosecka, U., Yager, J.A., Perdue, M., Snider, D., Butler, D.G. (2002) Adoptive transfer of BALB/c mouse splenocytes reduces lesion severity and induces intestinal pathophysiologic changes in the Mycobacterium avium subspecies paratuberculosis beige/scid mouse model Comparative Medicine, 52, 332-341 Successful immune reconstitution would enhance resistance of beige/scid mice to chronic infection with mycobacterium avium subspecies paratuberculosis, but may cause damage to intestinal tissue. Therefore, we investigated the effect of adoptive transfer of BALB/c mouse splenocytes on lesion severity and intestinal physiology in beige/scid mice infected with M. paratuberculosis. Mice were inoculated intraperitoncally (i.p.) with M. paratuberculosis, and two weeks later were inoculated i.p. with viable spleen cells from immune-competent BALB/c mice. Mice were necropsied 12 weeks after infection when engraftment of lymphocytes, clinical disease, pathologic lesions, and intestinal electrophysiologic parameters were evaluated. Lymphocytes were rare in control beige/scid mice not inoculated with spleen cells. In contrast, high numbers of CD4(+), CD8(+), and B220(+) lymphocytes were detected in the spleen of all beige/scid mice (n = 24) inoculated with spleen cells, indicating that adoptive transfer resulted in successful engraftment of donor lymphocytes (immune reconstitution). Immune reconstitution of M. paratuberculosis-infected beige/ scid mice significantly reduced the severity of clinical disease and pathologic lesions, and numbers of bacteria in the liver. However, intestinal electrophysiologic parameters studied in vitro indicated that intestinal tissues from reconstituted beige/scid mice had reduced short-circuit current responses (due to reduced ion secretion) following electrical, glucose, and forskolin stimulation. These abnormal responses suggested that neural or epithelial cells in the intestine were damaged. We conclude that successful immune reconstitution of beige/scid mice enhance their resistance to M. paratuberculosis infection, but may cause pathophysiologic changes associated with intestinal inflammation AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BACTERIUM/BOVINE PARATUBERCULOSIS/CELL/CELLS/CELLULAR IMMUNOLOGY/control/CROHNS-DISEASE/DISEASE/EPITHELIAL-CELLS/IMMUNITY/IN-VITRO /INFECTION/INFLAMMATORY-BOWEL-DISEASE/INTESTINAL PATHOPHYSIOLOGIC CHANGES/INTESTINAL TISSUES/JOHNES-

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DISEASE/LESIONS/LYMPHOCYTES/MICE/MODEL/MOUSE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PARATUBERCULOSIS INFECTION/RESISTANCE/RESPONSES/SCID MICE/STIMULATION/T-CELLS/TISSUE/TISSUES

182 Pavlik, I. , Machackova, M., Ayele, W.Y., Lamka, J., Parmova, I., Melicharek, I., Hanzlikova, M., Kormendy, B., Nagy, G., Cvetnic, Z., Ocepek, M., Lipiec, M. (2002) Incidence of bovine tuberculosis in wild and domestic animals other than cattle in six Central European countries during 1990-1999

Veterinarni Medicina, 47, 122-131 The study was undertaken in Croatia, Czech Republic, Hungary, Poland, Slovakia and Slovenia laying between Baltic and Adriatic seas on 610 402 km(2). Mycobacterium bovis infection was diagnosed in 70 animals belonging to 17 species other than cattle. The set of wild animals comprised 12 European bison (Bison bonasus), one red deer (Cervus elaphus), five wild boars (Sus scrofa), and one European wild goat (Capra aegagrus) bred in a game park. Further positive animals included two farmed red deer (Cervus elaphus) and one bactrian camel (Camelus ferus) owned by a circus. The infection was also demonstrated in 18 domestic animals belonging to 3 species living on farms where bovine tuberculosis was diagnosed in cattle. This set included 12 domestic pigs (Sus scrofa f. domestica), two domestic sheep (Ovis ammon f, aries), and four dogs (Canis lupus f. familiaris). The set of animals bred in zoological gardens consisted of 30 animals belonging to 9 species as follows: three bison (Bison bison), four tapirs (Tapirus terrestris), one cassowary (Casuarius casuarius - isolate identified by the biological assay in guinea pigs only), eight sitatungas (Tragelaphus spekei), three elands (Taurotragus oryx), one gnu (Connochaetes taurinus), eight reticulated giraffes (Giraffa cameloparadlis reticulata), one Puma (Puma concolor), and one Vietnamese pot-bellied pig (Sus bucculentus). Although, considering the population sizes, absolute numbers of the infected individuals are rather low, wild animals or such animals bred in captivity should be regarded as possible reservoirs of the causative agent of bovine tuberculosis. Tests for bovine tuberculosis are therefore necessary before transportation of all wild animals. Any lesion arousing suspicion of tuberculosis found on necropsy of wild animals must be laboratory examined for the presence of mycobacteria ANIMALS/ASSAY/BADGERS/BISON/bison bison/BOVINE/BOVINE TUBERCULOSIS/BOVIS/CATTLE/Cervus elaphus/CERVUS-ELAPHUS/deer/domestic animal/DOMESTIC SHEEP/DOMESTIC-ANIMALS/Epidemiology/FARMED DEER/FARMED RED DEER/FARMS/game park/game parks/goat/INFECTION/MICHIGAN/mycobacteria/Mycobacterium/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/paratuberculosis/PIGS/POLAND/POPULATION/RED DEER/reservoir/SHEEP/SIZES/TESTS/Tuberculosis/veterinary epidemiology/WILD /WILD ANIMALS/WILD BOAR/wild boars/zoological gardens

183 Simpson, V.R. (2002) Wild animals as reservoirs of infectious diseases in the UK

Veterinary Journal, 163, 128-146 This review aims to illustrate the extent to which wildlife act as reservoirs of infections agents that cause disease in domestic stock, pet and captive animals and humans. Morc than 40 agents are described. In the case of some of these, e.g. Cryptosporidium spp., Escherichia coli O157 and malignant catarrhal fever, the current evidence is that wildlife either does not act as a reservoir or is of limited importance However, in the case of many important diseases, including bovine tuberculosis, Weil's disease, Lyme disease, avian influenza, duck virus, enteritis and louping ill, wild animals are considered to be the principal source of infection. Wildlife may be involved in the epidemiology of other major diseases, such its neosporosis. Johne's disease, mucosal disease and foot and mouth disease, but further studies are needed. The UK would benefit from a more positive approach to the study of wildlife and the infections they harbour. (C) 2002 Published by Elsevier Science Ltd ANIMALS/AVIAN INFLUENZA/BOVINE/BOVINE TUBERCULOSIS/BOVINE VIRUS DIARRHEA/DEER CAPREOLUS-CAPREOLUS/DISEASE/DISEASES/ENTERITIS/Epidemiology/ESCHERICHIA-COLI/FOXES VULPES-VULPES/INFECTION/INFECTIONS/infectious disease/infectious diseases/Johne's disease/LYME-DISEASE/MALIGNANT CATARRHAL FEVER/MUCOSAL DISEASE/NEOSPORA-CANINUM/neosporosis/RATS RATTUS-NORVEGICUS/RED DEER/reservoir/Review/roe deer/TOXOPLASMA-GONDII/Tuberculosis/UNITED-KINGDOM/veterinary education/VIRUS/WILD/WILD ANIMALS/WILDLIFE/zoonoses

184 Waters, W.R., Sacco, R.E., Fach, S.J., Palmer, M.V., Olsen, S.C., Kreeger, T.J. (2002) Analysis of mitogen-stimulated lymphocyte subset proliferation and nitric oxide production by peripheral blood mononuclear cells of captive elk (Cervus elaphus)

Journal of Wildlife Diseases, 38, 344-351

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Elk (Cervus elaphus) are reservoirs for Brucella abortus, Mycobacterium bovis, and Mycobacterium avium subsp. paratuberculosis, each a serious pathogen of domestic livestock. An understanding of the basic immune responsiveness of elk would aid efforts to develop methods to diagnose and prevent these diseases of elk. Peripheral blood mononuclear cells (PBMC) isolated from captive elk were examined for phenotype, lymphocyte subset proliferative capacity, and ability to produce nitric oxide (NO) upon pokeweed mitogen (PWM) stimulation. Although gammadelta TCR+ cells represented a high percentage of the peripheral blood lymphocyte pool, these cells responded poorly to PWM stimulation. B cells (i.e., sIgM(+) cells), conversely, were responsive to PWM stimulation. Addition of PWM to PBMC cultures also resulted in a significant production of nitrite, the stable oxidation product of NO. Similar to other ruminant species, the majority of elk peripheral blood sIgM(+) cells co-expressed MHC class II and B-B4, a B cell lineage marker that varies in expression during B cell development. Findings from the present study provide basic information on several parameters of cellular immunity of elk ABILITY/AVIUM/B cells/BOVIS/BRUCELLA-ABORTUS/CATTLE/CELL/CELLS/cellular immunity/Cervus elaphus/CERVUS-ELAPHUS/CULTURE/deer/DISEASE/DISEASES/EXPRESSION/FLOW CYTOMETRIC ANALYSIS/IMMUNITY/livestock/lymphocyte subsets/MACROPHAGE FUNCTION/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium bovis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-BOVIS/nitric oxide/NITRIC-OXIDE/paratuberculosis/PERIPHERAL-BLOOD/reservoir/RESPONSES/STIMULATION/SUBPOPULATIONS

185 Mackintosh, C., Haigh, J.C., Griffin, F. (2002) Bacterial diseases of farmed deer and bison Revue Scientifique et Technique de l Office International des Epizooties, 21, 249-263 The most important aerobic bacterial diseases of farmed deer and bison include bovine tuberculosis, Johne's disease (paratuberculosis), yersiniosis, leptospirosis, brucellosis, pasteurellosis, anthrax, salmonellosis and colibacillosis. Anaerobic bacterial infections affecting the same animals include necrobacillosis and a number of clostridial diseases such as tetanus, blackleg, malignant oedema and pulpy kidney. The relative importance of these diseases will vary throughout the world according to timing and circumstance, but bovine tuberculosis and Johne's disease are likely to present the most significant problems with respect to diagnosis, control, trade in live animals and the establishment of wildlife reservoirs of infection. The authors summarise the aetiology, the principal species of animal affected, geographical distribution, transmission, clinical signs, pathology, diagnosis, treatment and control of these diseases ANIMALS/bacterial diseases/bacterial infection/BISON/BOVINE/BOVINE TUBERCULOSIS/CATTLE/control/DAMA-DAMA/deer/DIAGNOSIS/DISEASE/DISEASES/ELK CERVUS-ELAPHUS/Epidemiology/FARMED DEER/INFECTION/INFECTIONS/Johne's disease/MYCOBACTERIUM-BOVIS/NECROBACILLOSIS/paratuberculosis/PATHOLOGY/RED DEER/reservoir/salmonellosis/Transmission/Tuberculosis/WILDLIFE/wildlife reservoir

186 Naser, S.A., Shafran, I., Schwartz, D., El-Zaatari, F., Biggerstaff, J. (2002) In situ identification of mycobacteria in Crohn's disease patient tissue using confocal scanning laser microscopy

Molecular and Cellular Probes, 16, 41-48 The diversity in the methodology employed to investigate Crohn's disease (CD) etiology has added significantly to the controversy of the mycobacterial role in this chronic inflammatory bowel disease. Mycobacterium avium subsp para tuberculosis (MAP), a proposed and suspected agent in many CD patients, is a fastidious and very slow grower bacillus, which causes Johne's disease (JD) in cattle. The methodology that has been widely and successfully used for isolation and identification of MAP from and in JD animals is not reliable and has proven to be unsuccessful in achieving the same objectives for CD diagnosis. In this study, a Confocal Scanning Laser Microscopy (CSLM) system has been employed in an attempt to detect MAP in CD patient. In situ hybridization was performed on full thickness tissue using rabbit anti-MAP polyclonal antibody that was adsorbed with E. coli protein extracts. Consequently, MAP was detected in the microvilli region in tissue specimens from CD patient and not in the controls. In the same CD tissue specimen, MAP was not detected when isotype normal rabbit sera was employed. The polyclonal antibody marker may be replaced with monoclonal antibodies, if available, or with MAP-specific-DNA or RNA probes. This technique adds an additional approach to investigate MAP role in CD etiology especially when the culture approach is long and inconsistent. (C) 2002 Elsevier Science Ltd ANIMALS/ANTIBODIES/ANTIBODY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/COMPLEX/confocal microscopy/control/Crohn's disease/CULTURE/DIAGNOSIS/DISEASE/etiology/HYBRIDIZATION/IDENTIFICATION/ INFECTION/INFLAMMATORY BOWEL-DISEASE/INFLAMMATORY-BOWEL-DISEASE/INTERLEUKIN-

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12/isotype/Johne's disease/MAP/MONOCLONAL-ANTIBODIES/ mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/POLYMERASE CHAIN-REACTION/PROTEIN/QUANTITATION/rabbit/RESPONSES/role/SARCOIDOSIS/TISSUE/Tuberculosis

187 Olsen, I., Siguroardottir, O.G., Djonne, B. (2002) Paratuberculosis with special reference to cattle - A review

Veterinary Quarterly, 24, 12-28 Paratuberculosis is a chronic, granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis affecting domestic and wild ruminants. The symptoms of clinical paratuberculosis are chronic diarrhoea and progressive weight loss while subclinically infected animals mainly have decreased production. The infection is widespread throughout the world and causes substantial financial losses for the farming industry. One of the major obstacles in the control of this disease, is the difficulty of identifying subclinically infected animals. This review gives a summary of several aspects of paratuberculosis including clinical importance, pathology, immunology and properties of the infectious agent. Special emphasis will be on the available diagnostic methods, their use and limitations ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/AVIUM SUBSPECIES PARATUBERCULOSIS/BOVINE FECAL SAMPLES/CATTLE/control/CROHNS-DISEASE/DELTA-T-CELLS/diagnostic methods/DISEASE/ENTERITIS/HEAT-SHOCK PROTEIN/immunology/INFECTION/interferon-gamma/Johne's disease/LINKED-IMMUNOSORBENT-ASSAY/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium paratuberculosis/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/ MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/PATHOLOGY/POLYMERASE-CHAIN-REACTION/Review/RUMINANTS/TUBERCULOSIS JOHNES DISEASE/WILD/WILD RUMINANTS

188 Daniels, M.J., Hutchings, M.R., Allcroft, D.J., McKendrick, I.J., Greig, A. (2002) Risk factors for Johne's disease in Scotland - the results of a survey of farmers Veterinary Record, 150, 135-139 The reported incidence of Johne's disease has been increasing in the east of Scotland since 1993. A postal questionnaire survey was sent to 127 farms to identify potential risk factors for Johne's disease in relation to wildlife and farm management practices, and 86 returns were obtained. Of 22 farms which had been assumed to be free of the disease, on the basis of information held by local veterinary centres, seven (32 per cent) reported cases of Johne's disease in the 1990s, indicating that the disease is under-reported. Logistic regression analyses showed that eight of 63 potentially explanatory variables were significant at the 5 per cent level in affecting the likelihood of farms reporting Johne's disease. of these, large numbers of livestock and rabbits, and access of wildlife to feed stores were the clearest and most consistent risk factors associated with the disease. The application of manure to grazing pasture, the type of water supply for the cattle and the numbers of crows were also related to the presence of Johne's disease but the nature of these relationships was less clear. only 38 per cent of the farms reported taking any control measures to combat Johne's disease, but three of the control measures were relevant to risk factors identified as significant by the survey, namely maintaining a clean water supply, controlling rabbits and not spreading manure on to grazing pasture CATTLE/control/DISEASE/FARMS/HERD/INFECTION/Johne's disease/livestock/MYCOBACTERIUM-PARATUBERCULOSIS/pasture/rabbit/RABBITS/risk/RISK-FACTORS/SCOTLAND/survey/WATER/WILD RABBITS/WILDLIFE

189 Verna, A., Morsella, C., Zumarraga, M.J., Gioffre, A., Casaro, A., Romano, M., Cataldi, A., Paolicchi, F. (2001) Typing of Mycobacterium avium subsp paratuberculosis from isolations of feces and organs of deer Acta Bioquimica Clinica Latinoamericana, 35, 531-540 It is important to develop the P.C.R. and the restriction fragments length polymorphism (R.F.L.P.) technologies to identify genome segments since they constitute quick, sensitive, and specific methodologies for the detection of Mycobacterium avium subsp. paratuberculosis (Mptbc), a microorganism that produces animal paratuberculosis and that is related to Crohn's disease in humans, Our Objectives were: 1. To isolate Mptbc strains from feces and organs and to apply a P.C.R. protocol to confirm the IS900 insertion sequence. 2. To establish, by R.F.L.P., the genetic pattern of the strains isolated and 3. To analyze cellular and extracellular proteins expressed in Mptbc, Feces and organs of deer were cultured in culture media (yolk-egg Herrold, with and without mycobactin, adding pyruvate and antibiotics) for the isolation of Mptbc. The strains isolated were amplified by P.C.R. and studied by R.F.L.P. and Immunoblotting (IB). Twelve Mptbc strains, 6 from feces and 6 from organs, were isolated. The strains isolated developed only in mycobactin Herrold medium, showing their characteristic

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dependence. The analysis by P.C.R. was positive starting from strains developed in the culture medium. When feces were inoculated with a Mptbc strain, the detection rate by R CA was of 100%, demonstrating the highest specificity for IS900 in a 1:1000 dilution of the problem sample, but it was negative starting from the raw sample (feces). The isolations revealed an identical "A" type genetic pattern of R.F.L.P.,R, with the probe of 217 base pairs (endonuclease BstEII and PstI). The immunoblotting detected protein antigens of 65 KDa (thermal shock), 42 ADa, 35 KDa, and 28 KDa, corresponding to Mptbc from an animal with clinical symptoms and characteristic histologic lesions in organs. 1. A single "A" genetic pattern was identified in the population studied. 2. The IS900 sequence specific of Mptbc was amplified. The result was not successful when feces without previous isolation were used. 3. Proteins excreted and contained in the bacterial soma of an animal were identified, but it is necessary to characterize Mptbc specific antigens better to increase the sensitivity of immunological tests ANTIBIOTICS/ANTIBODY REACTIVITIES/ANTIGEN/antigens/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/Crohn's disease/CROHNS-DISEASE/CULTURE/deer/DISEASE/ DNA HYBRIDIZATION/FECES/IDENTIFICATION/immunoblotting/IS900/JOHNES-DISEASE/LESIONS/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium avium subsp paratuberculosis polymerase chain reaction/MYCOBACTERIUM-AVIUM/paratuberculosis/POPULATION/PROBES/PROTEIN/PROTEINS/RESTRICTION/restriction fragments of longitude polymorphism/RESTRICTION-ENDONUCLEASE ANALYSIS/SENSITIVITY/SEQUENCE/SPECIFICITY/STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/TESTS

190 Szeredi, L., Makrai, L., Denes, B. (2001) Rapid immunohistochemical detection of Rhodococcus equi in impression smears from affected foals on postmortem examination

Journal of Veterinary Medicine Series B-Infectious Diseases and Veterinary Public Health, 48, 751-758 The first objective of this study was to develop an immunohistochemical procedure for rapid detection of Rhodoroccus equi in impression smears from affected organs of foals on Postmortem examination. The second aim was to demonstrate whether R. equi can be detected in smears of tracheal exudates collected from the same foals using an immunohistochemical method. Impression smears and cryostat and paraffin-embedded sections were made from the lungs and mediastinal lymph nodes of three foals (A, B and C) that had died of respiratory disease caused by R. equi, and also from the caudal mesenteric lymph node of foal A. Impression smears were made from the tracheal exudates of all foals. An affinity purified rabbit IgG was used for the immunohistochemical demonstration of R. equi. This antibody reacted with serotype 1 of R. equi in Ouchterlony's immunodiffusion and in the passive haemagglutination test, but not with other serotypes or with Streptococcus equi ssp, equi or Staphylococcus aureus, and failed to give an immunohistochemical reaction with Mycobacterium bovis or M. paratuberculosis. The immunohistochemical method proved to be of identical sensitivity to bacterial culture; moreover, from the lungs and mediastinal lymph nodes of one foal, R. equi could only be detected by this method. R. equi was demonstrated in smears of the tracheal exudates of all three foals. The results of this study indicate that the immunohistochemical method may be used for the rapid detection of R. equi in impression smears from the affected organs, especially abscesses, obtained postmortem, and possibly as a tool for diagnosing R. equi pneumonia in live foals by examining smears of tracheal aspirates AIDS/ANTIBODIES/ANTIBODY/ASPIRATION/BACTERIAL CULTURE/BOVIS/CORYNEBACTERIUM-EQUI/CULTURE/DIAGNOSIS/DISEASE/immunohistochemical/INFECTIONS/LESIONS/LYMPH-NODES/mycobacteria/Mycobacterium/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/paratuberculosis/PNEUMONIA/rabbit/SENSITIVITY/SEROTYPES/STAPHYLOCOCCUS-AUREUS/VIRULENCE-ASSOCIATED ANTIGENS

191 Ayele, W.Y., Machackova, M., Pavlik, I. (2001) The transmission and impact of paratuberculosis infection in domestic and wild ruminants

Veterinarni Medicina, 46, 205-224 Mycobacterium avium subsp. paratuberculosis (M paratuberculosis) infects domestic cattle, sheep, goats, deer, camelids and wild ruminants leading to chronic enteritis known as paratuberculosis (Johne's disease). The infection is chronic, progressive and unresponsive to treatment. Most infected animals do not develop clinical disease but may excrete the bacteria. Clinically sick animals suffer emaciation and in some species diarrhoea, followed by eventual death. During the course of the disease, excretion of M. paratuberculosis in faeces and milk occurs, and the organism spreads through the blood and lymph vessels of infected animals to multiple internal organs. The infection disseminates to both the female and male reproductive organs. Though M paratuberculosis is not classified as a human pathogen, current opinions on the possible role of this mycobacteria in public health is discussed. This article attempts to review the ways and circumstances by which M. paratuberculosis is transmitted within an animal

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population and the importance of the disease on animal production, Published reports concerning the transmission and epidemiology of the disease are reviewed herein, and preventive and control measures are summarised ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BACTERIUM/BOVINE FECAL SPECIMENS/CATTLE/CONDITIONS SIMULATING PASTEURIZATION/control/CROHNS-DISEASE/deer/DISEASE/DNA HYBRIDIZATION/domestic and wild ruminants/emaciation/ENTERITIS/Epidemiology/faeces/FARMED RED DEER/ goat/goats/IMPACT/INFECTION/INSERTION ELEMENT IS900/Johne's disease/MILK/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/ Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/ORGANISM/paratuberculosis/PARATUBERCULOSIS INFECTION/POPULATION/RESTRICTION-ENDONUCLEASE ANALYSIS/Review /role/RUMINANTS/SEMEN/SHEEP/Transmission/TUBERCULOSIS JOHNES DISEASE/WILD/WILD RUMINANTS

192 Pavlik, I. , Bolske, G., Englund, S., Dvorska, L., du Maine, R., Svastova, P., Viske, D., Parmova, I., Bazant, J. (1999) Use of DNA fingerprinting for epidemiological studies of paratuberculosis in Sweden and the Czech Republic Proceedings of the Sixth International Colloquium on Paratuberculosis, 176-187 Due to liberalisation of trade in farm animals, paratuberculosis is rapidly spreading among ruminants in many European countries. Two countries with different situations concerning Johne's disease are Sweden and the Czech Republic. Sweden has a very low disease prevalence and imports few cattle. The Czech Republic has imported massive numbers of cattle since 1992. Isolates of Mycobacterium avium subsp. paratuberculosis from the two countries were examined using RFLP (Restriction Fragment Length Polymorphisms) with restriction endonucleases PstI and BstEII. Twenty Swedish isolates of M. a. paratuberculosis from cattle herds were investigated. Five isolates from animals imported from Denmark (four Blonde d'Aquitaine, one Limousine) and one isolate from a cow imported from Finland (Aberdeen Angus). Six isolates came from herds within a domestic chain of infection in the Limousine breed, originating from cattle importation from France via Denmark in 1975. One isolate came from a Swedish Friesian cow apparently without any contact with imported animals. All Swedish isolates were of one RFLP type: B-C1. This finding supports the notion that the infection was spread from imported animals to cattle reared in Sweden. Isolations made from 24 cattle herds originally reared in the Czech Republic were RFLP types A-C10, B-C1, B-C9, C-S1 and D-C12. In all 44 infected cattle herds (Blonde d'Aquitaine, Charolais, Holstein, Hereford, Jersey, Limousine, Mont Belliarde, Sarers) imported From Denmark, France, Germany and Hungary, RFLP type B-C1 was the most prevalent. In ten of these herds, animals were infected with other RFLP types (A-C10, B-C5, B-C9, D-C12, E-C1, H-C1, I-C13 and M-C16). Among other ruminants with paratuberculosis, domestically reared sheep were infected with RFLP types A-C8, A-C10, B-Ci, B-C2, and E-C1, goats B-C1, B-C9 and C-S1 and wild ruminants RFLP types A-C10, B-C1, B-C9 and M-C16. In imported wild goats from Estonia RFLP type B-C1, in imported goats from Denmark RFLP type B-C1, in imported sheep from Poland and France RFLP type A-C1 and in red deer imported from Scotland RFLP type M-C26 were found ANIMALS/AVIUM/BOVINE/CATTLE/CATTLE HERDS/cow/deer/DISEASE/DNA/DNA fingerprinting/ENGLAND/FRAGMENT-LENGTH-POLYMORPHISM/goat/goats/HERD/HYBRIDIZATION/INFECTED CATTLE/INFECTION/Johne's disease/JOHNES-DISEASE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/PARA-TUBERCULOSIS/ paratuberculosis/POLAND/POLYMORPHISMS/PREVALENCE/RED DEER/RESTRICTION/RESTRICTION-ENDONUCLEASE ANALYSIS/RFLP/RUMINANTS/SCOTLAND/SHEEP/STRAINS/WILD/WILD RUMINANTS

193 Pavlik, I. , Horvathova, A., Dvorska, L., Svastova, P., du Maine, R., Fixa, B., Rychlik, I. (1999) Homogeneity/heterogeneity of Mycobacterium avium subsp paratuberculosis strains: Correlation between RFLP-type and source (animal, environment, human) Proceedings of the Sixth International Colloquium on Paratuberculosis, 321-329 We recently proposed a method of standardising RFLP (Restriction Fragment Length Polymorphisms) analysis of Mycobacterium avium subspecies paratuberculosis using IS900 and restriction endonucleases PstI and BstEII (http://www.vri.cz/wwwrflptext.htm). Based on a study of 1,008 strains, 13 RFLP types were detected after digestion with restriction endonuclease Pstl, (designated as A - M) and 20 RFLP types after digestion with restriction endonuclease BstEII(designated as C1-3, C5, C7-20, S1, and Il). After a parallel examination of DNA from individual strains, a total of 28 different RFLP types were detected. DNA fingerprints were scanned by CCD camera and analysed by software Gel Manager (Biosystematika, Tavistock, UK) and later by software Gel Compar(Applied Maths, Kortrijk, Belgium).

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Strains of M. a. paratuberculosis from 13 different species of animals (cattle, sheep, goat, rhinoceros, wild goat, mouflon, fallow deer, roe deer, red deer, bison etc.) from 29 laboratories and 22 countries were analysed. In addition, 9 strains isolated from patients with Crohn's disease, two strains from cow's milk and 25 strains from the environment were included. From a total of 28 RFLP types, 20 RFLP types were found in 17 European countries, in Australia and New Zealand 5 RFLP types were identified and in the USA 10 RFLP types were found. RFLP type B-CI was the most common RFLP type in the 3 continents; it was found in 16 European countries. The second most frequently identified was RFLP type E-C 1, identified in six countries. The third was RFLP type A-C 10, found in five European countries. 22 RFLP types in cattle, 10 RFLP types in sheep, 9 RFLP types in goats, 5 RFLP types in wild ruminants, two RFLP types in rabbits, one RFLP type in swine, goats and rhinoceros were identified. Strains isolated from patients with Crohn's disease were classified as four RFLP types ANIMALS/AUSTRALIA /AVIUM/AVIUM SUBSP PARATUBERCULOSIS/AVIUM SUBSPECIES PARATUBERCULOSIS/BISON/CATTLE/cow/ Crohn's disease/deer/DISEASE/DNA/DNA HYBRIDIZATION/ENVIRONMENT/FALLOW DEER/FRAGMENT-LENGTH-POLYMORPHISM/goat/goats/IDENTIFICATION/IS900/MILK/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/NEW-ZEALAND/paratuberculosis/POLYMORPHISMS/rabbit/RABBITS/RED DEER/RESTRICTION/RESTRICTION ENDONUCLEASE ANALYSIS/RFLP/ roe deer/RUMINANTS/SHEEP/STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/WILD/WILD RUMINANTS

194 Stabel, J.R. (1999) Transitions in immune responses to M-a. paratuberculosis infection

Proceedings of the Sixth International Colloquium on Paratuberculosis, 579-584 The host immune response to infection with M. a. paratuberculosis is paradoxical, with strong cell-mediated immune responses during the early, subclinical stages of infection and strong humoral responses during the late clinical stages of the disease. Cell-mediated immune responses modulated by various T-cell subsets are essential to provide protective immunity and prevent progression of the disease. Secretion of cytokines by T-cell populations serves to activate macrophages to kill ingested M. a. paratuberculosis as well as activate other T-cell subsets to contain the infection. This paper reviews the current knowledge of T-cell immune responses in M, a. paratuberculosis infection based upon clinical studies and research using mouse models BOVINE PARATUBERCULOSIS/CELLULAR IMMUNOLOGY/cytokine/DELTA T-CELLS/DISEASE/GAMMA/humoral response/immune responses/IMMUNE-RESPONSE/IMMUNE-RESPONSES/IMMUNITY/INFECTION/lymphocyte subsets/macrophage/MACROPHAGES/MODEL/MOUSE/MYCOBACTERIUM-PARATUBERCULOSIS/OVINE PARATUBERCULOSIS/paratuberculosis/PARATUBERCULOSIS INFECTION/POPULATION/POPULATIONS/RESPONSES/Review/T cell/TNF-ALPHA/Tuberculosis

195 Kennedy, D.J., Allworth, M.B. (1999) Progress in national control and assurance programs for bovine Johne's disease in Australia Proceedings of the Sixth International Colloquium on Paratuberculosis, 25-32 Cattle strains of M. avium subsp. paratuberculosis are known to infect cattle, goats and alpaca in south-eastern Australia, where there are also significant numbers of farmed deer. Although sheep strains have recently been identified in some cattle in Australia, epidemiological evidence to date supports the distinction for the purposes of control and assurance programs between bovine Johne's disease (caused by cattle strains in cattle, goats and alpaca) and ovine Johne's disease (caused by sheep strains in sheep and goats). The National Johne's Disease Control Program is coordinated by the Australian Animal Health Council Ltd (AAHC) working with the livestock industries and with the Commonwealth, State and Territory governments. The Council also brokers industry and government funding for the program. The National Johne's Disease Market Assurance Program for cattle was launched in 1996 as the first of a suite of voluntary national market assurance programs (MAPs) to assess and certify herds as test negative for Johne's disease. By December 1998, over 550 herds had achieved an assessed negative status. A MAP was also launched for alpaca in 1998 and a GoatMAP should be finalised in early 1999. National standards for State control of Johne's disease through zoning, movement controls and procedures in infected and suspect herds have also been developed. The paper covers factors affecting development and implementation, uptake of and improvements to national control and assurance programs for bovine Johne's disease in Australia AUSTRALIA/AVIUM/BOVINE/CATTLE/control/deer/DISEASE/FARMED DEER/goat/goats/ HERD/Johne's disease/livestock/MAP/OVINE/Ovine Johne's disease/paratuberculosis/SHEEP/sheep and goats/STRAINS

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196 Mackintosh, C.G., Reichel, M.P., Griffin, J.F.T., Montgomery, H., de Lisle, G.W. (1999) Paratuberculosis and avian tuberculosis in red deer in New Zealand: Clinical syndromes and diagnostic tests Proceedings of the Sixth International Colloquium on Paratuberculosis, 449-457 Paratuberculosis (caused by Mycobacterium avium subsp. paratuberculosis) and to a lesser extent avian tuberculosis (caused by Mycobacterium avium subsp. avium), are emerging as problems in red deer (Cervus elaphus) farmed in New Zealand. To date, the majority of reported cases have been subclinically affected animals detected at slaughter, usually with lesions in mesenteric lymph nodes. These lesions are a nuisance to the venison industry and result in financial loss to the farmers due to their gross and histological similarity to lesions caused by Mycobacterium bovis. Recently however, there have been a small number of outbreaks of severe clinical disease and fatalities in 8 to 15 month old deer. Studies were undertaken to determine which of the currently available tests were the most useful for diagnosing clinical paratuberculosis and detecting subclinically infected red deer. These tests were evaluated in a herd of red deer that had a severe outbreak of clinical paratuberculosis in yearling animals. An agar gel diffusion test (GD) and an Enzyme-linked Immunosorbent Assay (ELISA) were useful in confirming disease in clinically affected animals, although they could not differentiate between infections due to M. a. paratuberculosis and M. avium. Neither a GD test, various ELISA tests, a complement fixation test, a leukocyte transformation (LeT) test nor a comparative skin test were able to accurately diagnose subclinical paratuberculosis in deer ANIMALS/ASSAY/Avian tuberculosis/AVIUM/BOVIS/ Cervus elaphus/CERVUS-ELAPHUS/deer/DIAGNOSTIC-TESTS/DISEASE/ELISA/ELK/enzyme-linked immunosorbent assay/HERD/INFECTION/INFECTIONS/LESIONS/LYMPH-NODES/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium bovis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-BOVIS/NEW-ZEALAND/paratuberculosis/RED DEER/SKIN-TEST/SUBCLINICAL PARATUBERCULOSIS/TESTS/TRANSFORMATION/Tuberculosis

197 Whitlock, R.H., West, S.R., Layton, B., Ellingson, J., Stabel, J., Rossiter, C., Buergelt, C., Ginn, P., Pavlik, I., Collins, M.T., Juste, R., Habecker, P. (1999) Paratuberculosis in bison: A comparison of PCR, culture, serology and histopathology Proceedings of the Sixth International Colloquium on Paratuberculosis, 424-438 Multiple tissues, blood and fecal samples were collected from forty-nine adult bison, all with clinical signs suggestive of Johne's disease, including chronic weight loss. Tissues were harvested for culture, histopathology, immunohistochemistry, and PCR. AGID, total plasma protein (TPP) concentrations and ELISA tests were done on the serum samples. One serum sample was positive on the AGID test while three were positive on the CSL/IDEXX ELISA test. PCR using the IS-900 sequence detected M; a. paratuberculosis in 19 of 24 fecal samples. Tissues from 21 of 24 bison were PCR-positive when one or more tissues were tested. Tissues from the other three bison in the group of 24 were classified as PCR suspicious, since a faint band was visible by agarose gel. Tissues were culture-positive from 15 of the 24 bison tested. One fecal sample was culture-positive while the tissues from that bison were culture-negative. The colonies isolated grew better on HEYM without pyruvate than on HEYM with pyruvate (4.1 gm/l. Several cultures were first recorded as positive well beyond the typical 14 weeks of culture. Based on experience with M; a. paratuberculosis-infected tissues from cattle, the specific strain of M. a. paratuberculosis from bison tissues seemed much more difficult to isolate. The tissues were examined histopathologically using the hematoxylin & eosin and Ziehl-Neelsen stains. Histopathologic evaluation of the same tissues examined by culture and PCR identified 18 animals positive by finding giant cells containing acid-fast rods with morphology compatible with M. a. paratuberculosis. The histologically negative animals were also culture-negative but PCR-positive. Immunohistologic staining with specific polyclonal antibody stains were positive in each of the 25 bison AMERICAN WILD RUMINANTS/ANIMALS/ANTIBODIES/ANTIBODY/BISON/BOVINE FECES/CATTLE/CELL/ CELLS/COMMERCIAL DNA PROBE/CULTURE/DISEASE/DOMESTIC SHEEP/ELISA/EXPERIMENTAL-INFECTION /FECAL CULTURE/insertion sequences /IS900/Johne's disease/ JOHNES-DISEASE/MYCOBACTERIUM-PARATUBERCULOSIS /PARA-TUBERCULOSIS/paratuberculosis/PCR/PROTEIN/SEQUENCE/Serology/TESTS/TISSUE/TISSUES/Ziehl-Neelsen

198 Tobin, F.M. (1999) The Victorian ovine Johne's disease eradication program: Effect on sheep owners

Proceedings of the Sixth International Colloquium on Paratuberculosis, 147-152 In December 1996 the Department of Natural Resources and Environment commenced an eradication program of Ovine Johne's Disease in the state of Victoria, Australia. The program was based on compulsory slaughter and destocking for a minimum of 2 summers of all sheep, deer, goats and alpaca

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on properties where OJD was identified. This paper describes the effects of this program on sheep owners, their families and rural communities. The effects on farm profitability, the costs and logistics of changing to an alternative enterprise for the destocked period, the costs and problems faced in restocking the farm following the destocked period, and the hidden costs of eradication are examined. The effectiveness of any disease eradication program such as this OJD control program is clearly dependent on the cooperation of all sheep owners. The role of affected sheep owners and their cooperation in this control program is discussed. This program has provided a unique opportunity to evaluate the effects of an eradication program which requires total destocking of all susceptible species for a minimum of 15 months AUSTRALIA/control/ deer/DISEASE/ENVIRONMENT/eradication program/FAMILY/goat/goats/Johne's disease/OVINE/Ovine Johne's disease/role/SHEEP/victoria

199 Pavlik, I. , Bartl, J., Horvathova, A., Dvorska, L., Matlova, L., Fischer, O., du Maine, R., Rozsypalova, Z. (1999) Study of differing Mycobacterium avium subsp paratuberculosis DNA fingerprints from farm and wild ruminants in the Czech Republic during 1995-1998

Proceedings of the Sixth International Colloquium on Paratuberculosis, 188-199 Since 1989 paratuberculosis has frequently been diagnosed in ruminants imported to the Czech Republic. In December 1992, 19 pregnant Charolais heifers were imported through Hungary from France. One eleven month old heifer escaped and roamed in the wild over 15 to 20 km for seven months from November 1993 to May 1994. After having been captured, the animal showed clinical signs of paratuberculosis (emaciation and diarrhea). Consequently, 4 other animals infected with the same DNA type B-C1 of Mycobacterium avium subsp. paratuberculosis were detected. In the period 1995-1996 sampling of the small intestine and corresponding lymph nodes from 84 red deer (Cervus elaphus) and roe-deer (Capreolus capreolus) from 44 different locations in the same district were examined. Five M. a. paratuberculosis strains were isolated. one strain of DNA type B-C1 from a stag; three strains of DNA type B-C1 and one strain of DNA type B-C9 from a roe-deer. Three wild ruminants (one stag and two roe-deer) infected with the same DNA type B-C1 of strain were detected in the same area as the heifer. The infection source was likely this infected heifer. In one roe-deer infected with strain DNA type B-C9 the infection source was different: stags that escaped from the farm were purchased from an area infected with this latter DNA type. In a subsequent survey carried out during 1997-1998 throughout the entire Czech Republic (76 districts), more than 596 ruminant game animals were examined from 90% of the districts. M; a. paratuberculosis has been isolated from 28 (4.1%) of these animals: red deer, fallow deer, roe-deer and moufflon kept in farms and game parks. In nature, the infection has been found in roe-deer and a fellow deer (Dama dama) in the districts where two cattle herds were found to have been infected with the same DNA type ANIMALS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/CATTLE/CATTLE HERDS/Cervus elaphus/CERVUS-ELAPHUS/Dama dama/DAMA-DAMA/deer/Diarrhea/DNA/ELK/emaciation/FALLOW DEER/FARMS/game park/game parks/heifer/HERD/HYBRIDIZATION/IDENTIFICATION/INFECTION/JOHNES-DISEASE/LYMPH-NODES/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/paratuberculosis/RED DEER/RESTRICTION-ENDONUCLEASE ANALYSIS/roe deer/RUMINANTS/STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/survey/WILD/WILD RUMINANTS

200 Godfroid, J., Boelaert, F., Heier, A., Clavareau, C., Wellemans, V., Desmecht, M., Roels, S., Walravens, K. (1999) First evidence of Johne's disease in farmed red deer (Cervus elaphus) in Belgium

Proceedings of the Sixth International Colloquium on Paratuberculosis, 242-247 In 1998 a 4 year old hind died in poor condition after suffering chronic diarrhoea. The animal had been bought from a supplier with a history of paratuberculosis (Johne's disease). Johne's disease was suspected in this animal. Ziehl-Neelsen staining of the faeces of this hind were positive for clumps of small acid-fast bacilli, but faecal cultures remained negative. In order to assess the presence of M. a. paratuberculosis infection on this deer farm, direct and indirect tests were performed on 24 hinds and stags (yearlings, 2 and 4 years old animals). The following tests where performed: serology (Mycobacterium paratuberculosis Antibody ELISA, HerdChek(TM), IDEXX), Comparative Cervical Skin test (CCT) and Lymphoproliferation tests (LT) using M. bovis purified protein derivative (PPD) and M. avium PPD as antigens. Three positive serological results, 3 positive CCT and 8 positive LT were observed in hinds and stags older than 2 years. No positive serological results were observed in the yearling group whereas some sensitization was observed in the CCT as well as in the LT for the same group of animals. The degree of concordance among these indirect tests was poor. This can be explained partly by the pathogenesis of the disease. The lack of baseline data for these tests in wild

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farmed species also hampers diagnosis. The three seropositive animals were slaughtered and subjected to post-mortem examination. Histopathology was performed on mesenteric lymph nodes and the terminal ileum. Visible changes in some mesenteric lymph nodes were observed, no gross lesions were seen in the intestine. Although Ziehl-Neelsen staining did not reveal acid-fast organisms, a catarrhal focal necrotic enteritis associated with a granulomatous lymphadenitis compatible with Johne's disease was evidenced. The mycobacterial cultures on tissue samples from slaughtered animals were positive after two months for Mycobacterium avium subspecies paratuberculosis and negative for M. bovis and M. avium.. The intra-herd prevalence of Johne's disease has still to be assessed. This is the first description of Johne's disease in a deer farm in Belgium ANIMALS/ANTIBODIES/ANTIBODY/ANTIGEN/antigens/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BOVIS/Cervus elaphus/CERVUS-ELAPHUS/CULTURE/deer/DIAGNOSIS/DISEASE/ELISA/ENTERITIS/faeces/FARMED RED DEER/INFECTION/Johne's disease/LESIONS/LYMPH-NODES/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/ORGANISM/ORGANISMS/paratuberculosis/PARATUBERCULOSIS INFECTION/PATHOGENESIS/PREVALENCE/PROTEIN/RED DEER/Serology/SKIN-TEST/TESTS/TISSUE/Tuberculosis/WILD/Ziehl-Neelsen

201 Martin, H.M., Sumar, N., Bull, T.J., Sheridan, J., Tizard, M.L., Hermon-Taylor, J. (1999) Expression, purification and seroreactivity of GS proteins of Mycobacterium avium subsp paratuberculosis

Proceedings of the Sixth International Colloquium on Paratuberculosis, 311-320 A series of six contiguous open reading frames (gsa, gsb(A), gsb(B), gsc, gsd and mpa), isolated from a low G+C content genetic island has been investigated. Analysis of DNA and protein databases show that components of the GS element, which possess homologues in the pathogen M. tuberculosis but not M. bovis BCG, may encode functions related to extracellular polysaccharide synthesis or modification, and as such may influence the pathogenicity of M. avium subsp. paratuberculosis. We have designed constructs that facilitate the expression of the GS genes in bacterial and insect cell expression systems and the purification of the recombinant proteins utilising metal-chelate chromatography. Purification of GS components from E. coli and insect cells will provide reagents to test the diagnostic capability of the GS element. The reactivity of purified recombinant GS proteins with sera from Johne's disease cattle, Crohn's patients and appropriate controls can then be evaluated. In addition, GS specific anti-peptide rabbit polyclonal antibodies have been produced for use as reagents for further analysis of the GS element ANTIBODIES/ANTIBODY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BCG/BOVIS/BOVIS BCG/CATTLE/CELL/CELLS/control/DISEASE/DNA/ELEMENT/EXPRESSION/GENE/GENES/Johne's disease/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PROTEIN/PROTEINS/PURIFICATION/rabbit/RECOMBINANT PROTEINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/Tuberculosis

202 Ellingson, J.E.L., Stabel, J.R., Whitlock, R.H. (1999) Detection of Mycobacterium avium subspecies paratuberculosis in free-ranging bison by PCR

Proceedings of the Sixth International Colloquium on Paratuberculosis, 420-423 Bacterial culture is considered the "gold standard" method for diagnosing paratuberculosis infection (Johne's disease). However, bacterial culture of Mycobacterium avium subspecies paratuberculosis (Map) is time consuming and laborious and the success of bacterial culture varies with the species of animals tested. Improved diagnostic tests are needed that can be used in domestic and wild ruminants. PCR amplification has been used for the detection of Map DNA in feces and tissues. However, elaborate specimen preparation protocols are required that may result in reduced sensitivity of these assays. We applied the PCR amplification technique to detection of Map DNA in twenty-five free-ranging North American bison (Bison bison). We report the performance of preassembled PCR amplification mixtures to detect Map DNA in frozen ileum, jejunum, and lymph node samples collected from bison. Specific oligonucleotide primers used in the PCR amplification assay were derived from the 16S rRNA (M. avium) sequence and insertion element IS900 (Map). Crude genomic DNA samples were prepared from the bison tissues using a simple boiling technique and these DNA samples were tested by PCR amplification. An animal was considered positive if Map DNA was detected by PCR amplification in at least two separate tissue samples using the IS900 primer set or by detection of DNA in a single tissue sample from an animal using both the 16S rRNA and IS900 primer sets. Using these criteria, 15 of 25 (60%) bison tested were positive for Map DNA. The data indicate that these free-ranging bison have been infected by Map

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AMPLIFICATION/ANIMALS/ASSAY/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BACTERIAL CULTURE/BISON/bison bison/CULTURE/DIAGNOSTIC-TESTS/DISEASE/DNA/domestic and wild ruminants/ELEMENT/FECES/INFECTION/INSERTION ELEMENT/INSERTION ELEMENT IS900/IS900/Johne's disease/JOHNES DISEASE/MAP/MAP-DNA/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PARATUBERCULOSIS INFECTION/PCR/RUMINANT PARA-TUBERCULOSIS/RUMINANTS/SENSITIVITY/SEQUENCE/TESTS/TISSUE/TISSUES/WILD/WILD RUMINANTS

203 Lambeth, M., Griffin, F., Crawford, A., Mackintosh, C. (1999) Isolation and Sequencing of Insertion Element IS1311 from Mycobacterium avium subsp paratuberculosis

Proceedings of the Sixth International Colloquium on Paratuberculosis, 472-475 We describe a screening strategy for a M. a. paratuberculosis genomic library resulting in the isolation of a clone bearing IS1311. Sequencing of this clone has revealed an insertion element with very high homology to the M: avium sequence. Sequence differences in this element between sheep and cattle strains were used to examine M. a. paratuberculosis from deer. Both sheep and cattle strains could be isolated from deer AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/deer/ELEMENT/INSERTION ELEMENT/IS1311/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/SEQUENCE/SHEEP/STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS

204 Beard, P.M., Stevenson, K., Pirie, A., Rudge, K., Buxton, D., Rhind, S.M., Sinclair, M.C., Wildblood, L.A., Jones, D.G., Sharp, J.M. (2001) Experimental paratuberculosis in calves following inoculation with a rabbit isolate of Mycobacterium avium subsp paratuberculosis

Journal of Clinical Microbiology, 39, 3080-3084 The role of wildlife species in the epidemiology of paratuberculosis has been the subject of increased research efforts following the discovery of natural paratuberculosis in free-living rabbits from farms in east Scotland. This paper describes the experimental inoculation of young calves with an isolate of Mycobacterium avium subsp. paratuberculosis recovered from a free-living rabbit. After a 6-month incubation period, all eight calves inoculated with the rabbit isolate had developed histopathological and/or microbiological evidence of M. avium subsp. paratuberculosis infection. Similar results were obtained from a group of calves infected with a bovine isolate of M. avium subsp. paratuberculosis. The virulence of the rabbit isolate for calves demonstrated in this study suggests that rabbits are capable of passing paratuberculosis to domestic ruminants and that wildlife reservoirs of M. avium subsp. paratuberculosis should therefore be considered when formulating control plans for the disease AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BOVINE/CALVES/CATTLE/control/DISEASE/Epidemiology/FARMS/incubation period/INFECTION/INOCULATION/JOHNES-DISEASE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/ paratuberculosis/PARATUBERCULOSIS INFECTION/PATHOLOGY/rabbit/RABBITS/reservoir/role/RUMINANTS/SCOTLAND/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/VIRULENCE/WILD RABBITS/WILDLIFE/wildlife reservoir

205 Jones, P.H. (2001) Bovine paratuberculosis: ongoing challenges, renewed concerns

In Practice, 23, 402-+ PARATUBERCULOSIS (Johne's disease) is a chronic wasting disease of ruminants that was first described in cattle in 1895. it is generally accepted that Mycobacterium avium subspecies paratuberculosis (MAP) is the causal agent although many of the details regarding the mechanisms of pathogenesis remain unknown. Paratuberculosis is of principal economic importance in cattle, sheep and goats but infection with MAP has been reported in many other species including deer, South American camelids, bison, stumptail macaques, rabbits, foxes and stoats. Bovine paratuberculosis is a significant disease for a number of reasons. As well as causing economic losses as a result of reduced production, culling of infected animals and the costs of testing procedures and control measures, it has been suggested that MAP plays a role in the aetiology of Crohn's disease in humans - although this hypothesis remains highly controversial. This article discusses the challenges posed by bovine

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paratuberculosis, particularly with regard to diagnosis and control, and briefly reviews the evidence concerning the potential zoonotic role of MAP ANIMALS/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BISON/BOVINE/BOVINE PARATUBERCULOSIS/BOVINE-PARATUBERCULOSIS/CATTLE/challenge/control/Crohn's disease/deer/DIAGNOSIS/DISEASE/economic losses/ECONOMIC-LOSSES/ENGLAND/goat/goats/INACTIVATION/INFECTION/INFECTIONS/Johne's disease/JOHNES-DISEASE/MAP/MILK/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/ PASTEURIZATION TEMPERATURES/PATHOGENESIS/PREVALENCE/rabbit/RABBITS/Review/role/RUMINANTS/SHEEP/sheep and goats

206 Corn, J.L. , Nettles, V.F. (2001) Health protocol for translocation of free-ranging elk

Journal of Wildlife Diseases, 37, 413-426 When considering an elk (Cervus elaphus) restoration program, wildlife managers must evaluate the positive and negative elements of translocation. We prepared this protocol to give an overview of health considerations associated with translocation of elk, with an emphasis on movement of free-ranging elk from western North America to the southeastern USA. We evaluated infectious agents and ectoparasites reported in elk from two perspectives. First, we made a qualitative estimate of the ability of the agent to be introduced and to become established. This was done using a selected set of epidemiologic factors. Second, if there was a good possibility that the organism could become established in the release area, the potential pathological consequences for elk and other wildlife, domestic animals, and humans were assessed via examination of the literature and consultation with other animal health specialists. The results of these evaluations were used to classify infectious agents and ectoparasites as low risk (n = 174), unknown risk (n = 10), and high risk (n = 9). We classified Anaplasma marginale, Anaplasma ovis, Mycobacterium paratuberculosis, Pasteurella multocida serotype 3, Elaphostrongylus cervi, Dicrocoelium dendriticum, Fascioloides magna, Echinococcus granulosus, Dermacentor albipictus, and Otobius megnini as unknown risks. High risk infectious agents and ectoparasites were the agent of chronic wasting disease, Brucella abortus, Mycobacterium bovis, Dermacentor andersoni, Ixodes pacificus, and Psoroptes sp. Parelaphostrongylus tenuis, Elaeophora schneideri, and a Babesia sp. are parasites endemic in the southeastern USA that may present a "reverse risk" and adversely affect elk if released in some parts of the region. We developed a five-component protocol to reduce the risk of introduction of high risk infectious agents and ectoparasites that included: (1) evaluation of the health status of source populations, (2) quarantines, (3) physical examination and diagnostic testing, (4), restrictions on translocation of animals from certain geographic areas or populations, and (5) prophylactic treatment ABILITY/ANIMALS/BABESIA-ODOCOILEI/BOVINE TUBERCULOSIS/BOVIS/BRUCELLA-ABORTUS/Cervus elaphus/CERVUS-ELAPHUS/CERVUS-ELAPHUS-NELSONI /DISEASE/domestic animal/DOMESTIC-ANIMALS/ectoparasites/ELEMENT/ELK/importation/infectious agents/mycobacteria/Mycobacterium/Mycobacterium bovis/Mycobacterium paratuberculosis/MYCOBACTERIUM-BOVIS/MYCOBACTERIUM-PARATUBERCULOSIS /ODOCOILEUS-VIRGINIANUS/ORGANISM/parasites/paratuberculosis/POPULATION/POPULATIONS/restoration/RESTRICTION/risk /ROCKY-MOUNTAIN ELK/SPICULOPTERAGIA-SPICULOPTERA/SPONGIFORM ENCEPHALOPATHY/translocation/UNITED-STATES/white-tailed deer/WILDLIFE

207 Beard, P.M., Rhind, S.M., Buxton, D., Daniels, M.J., Henderson, D., Pirie, A., Rudge, K., Greig, A., Hutchings, M.R., Stevenson, K., Sharp, J.M. (2001) Natural paratuberculosis infection in rabbits in Scotland Journal of Comparative Pathology, 124, 290-299 Natural paratuberculosis infection of rabbits (Oryctolagus cuniculus) was recently diagnosed in Scotland, and an investigation into the pathology of the disease in wild rabbits is reported in this paper. Evidence of Mycobacterium avium subsp. paratuberculosis (M.a. paratuberculosis) infection was detected in 22% of 110 rabbits: the organism was cultured from 17 of 110 rabbits, Land histopathological lesions consistent with many. paratuberculosis infection were noted in 18 of 98 rabbits examined. No macroscopical lesions suggestive of M.a. paratuberculosis infection were observed. The histopathological le,ions were either severe or mild. Severe lesions consisted of extensive macrophage granulomata and numerous giant cells, with many intracellular acid-fast bacteria in the small intestine. For the examination formalin-fixed, paraffin wax-embedded tissues, neither immunohistochemistry nor the polymerase chain reaction was as sensitive a method of diagnosis as histopathology. (C) 2001 Harcourt Publishers, Ltd

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AVIUM/BACTERIUM/CELL/CELLS/DIAGNOSIS/DISEASE/EXTRACTION/FIXATION/INFECTION/IS900/LESIONS/macrophage/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS /ORGANISM/ORYCTOLAGUS-CUNICULUS/PARAFFIN-EMBEDDED TISSUES/paratuberculosis/PARATUBERCULOSIS INFECTION/PATHOLOGY/polymerase chain reaction/POLYMERASE-CHAIN-REACTION/rabbit/RABBITS/SCOTLAND/TISSUE/TISSUES/WILD/WILD RABBIT/WILD RABBITS

208 Paolicchi, F.A., Vagnozzi, A., Morsella, C.G., Verna, A.E., Massone, A.R., Portiansky, E.L., Gimeno, E.J. (2001) Paratuberculosis in red deer (Cervus elaphus): an immunohistochemical study

Journal of Veterinary Medicine Series B-Infectious Diseases and Veterinary Public Health, 48, 313-320 In the present study, we compared the utility of immunohistochemistry with serological and histological results for the characterization of Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) in tissues of affected red deer. Bacterial isolation was considered the standard reference. Samples were taken from seven clinically affected animals with typical macroscopic lesions. The enzyme linked immunosorbent assay (ELISA) and the gel diffusion tests (GD) were used for serological determinations. Samples from intestine and mesenteric lymph nodes were processed for bacterial isolation and histology. M. paratuberculosis was isolated from all the animals. Histologically, lymph notes displayed necrosis and mineralization at the cortical and medullar areas. Ziehl-Neelsen stained bacteria were numerous inside macrophages and Langhans-type giant cells. Giant and epithelioid cells and lymphocytes were prominent at the ileal mucous membrane. The immunostaining of M. paratuberculosis was very clear inside epithelioid and giant cells. Image analysis was carried out to determine the immunostained area. There was total agreement among the methods employed. Immunohistochemistry can be very useful when the microorganism cannot be recovered from tissues or faeces ANIMALS/ASSAY/AVIUM/BACTERIAL ISOLATION/BACTERIUM/CELL/CELLS/Cervus elaphus/CERVUS-ELAPHUS/deer/ELISA/faeces/immunohistochemical/LESIONS/LINKED-IMMUNOSORBENT-ASSAY/LYMPH-NODES/LYMPHOCYTES/macrophage/MACROPHAGES/MEMBRANE/mycobacteria/Mycobacterium/Mycobacterium avium/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/PARA-TUBERCULOSIS/paratuberculosis/RED DEER/SHEEP/TESTS/TISSUE/TISSUES/Ziehl-Neelsen

209 Whittington, R.J., Marsh, I.B., Whitlock, R.H. (2001) Typing of IS1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with a strain of Mycobacterium avium subsp paratuberculosis distinct from that occurring in cattle and other domesticated livestock

Molecular and Cellular Probes, 15, 139 -145 Isolates of Mycobacterium avium subsp. paratuberculosis from nine bison (Bison bison) from Montana, United States of America, were compared with those from other species from the United States of America. All 16 isolates had a novel IS1311 genotype in which all copies of the element possessed a thymidine-cytosine nucleotide variation at base position 223. Isolates from bison were termed B strain. Thirteen isolates from cattle and goats were polymorphic at this locus, a status previously recognised in isolates from cattle which are termed C strains. Differences in cultural phenotype between B and C strains which were noted upon primary isolation did not manifest upon subculture to Herrold's egg yolk medium or modified Middlebrook 7H10 agar. Rapid differentiation of these strains from each other and from S strains from sheep is possible using polymerase chain reaction and restriction endonuclease analysis. The epidemiology of paratuberculosis in bison in Montana appears to be distinct from that in cattle and other farmed livestock investigated thus far. (C) 2001 Academic Press AMERICAN WILD RUMINANTS/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BISON/bison bison/CATTLE/deer/DIAGNOSIS/DIFFERENTIATION/ELEMENT/ELK/Epidemiology/goat/goats/HYBRIDIZATION/IDENTIFICATION/IS1311/IS900/livestock/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/PARA-TUBERCULOSIS/paratuberculosis/PCR/polymerase chain reaction/POLYMERASE-CHAIN-REACTION/POLYMORPHISMS/RESTRICTION/RESTRICTION ENDONUCLEASE ANALYSIS/SHEEP/STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/United States/UNITED-STATES

210 Cheville, N.F., Hostetter, J., Thomsen, B.V., Simutis, F., Vanloubbeeck, Y., Steadham, E. (2001) Intracellular trafficking of Mycobacterium avium ss. paratuberculosis in macrophages

Deutsche Tierarztliche Wochenschrift, 108, 236-+ The granulomatous enteric lesions of cattle with Johne's disease are composed of infected

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macrophages, and grow by accumulation, re-infection, and expansion of macrophage populations in the intestinal wall. We have examined the growth of bacteria in macrophages to define characteristics of intracellular trafficking for exocytosis, replication, and antigen presentation. Using immunocytochemical markers for light, confocal and electron microscopy, we have examined potential pathway tropisms using data for bacterial attachment, phagosomal acidification, phagolysosomal degradation and apoptosis. Our hypotheses are that pathogenic/wild-type strains block phagosomal acidification so that the phagosome fails to obtain markers of the late phagosome and phagolysosome, and this leads to the replication pathway within bacteriophorous vacuoles. Non-pathogenic strains appear to be processed to exocytosis, and avirulent mutant strains may be degraded and have preference of antigen processing pathways that involve transport vesicles bearing MHC II antigens. Pathogenicity in a nude mouse model of intestinal infection reveals lesion development and confirms pathway preferences of virulent strains for bacteriophorous vacuole formation ACIDIFICATION/ANTIGEN/antigens/APOPTOSIS/ATTENUATION/AVIUM/BACTERIUM/BEIGE MICE/CATTLE/DISEASE/EXPRESSION/GROWTH/INFECTION/intracellular trafficking/Johne's disease/LESIONS/macrophage/MACROPHAGES/MODEL/MOUSE/MURINE MACROPHAGES/MUTANT/mycobacteria/Mycobacterium/Mycobacterium avium/MYCOBACTERIUM-AVIUM/NITRIC-OXIDE/nude mouse/paratuberculosis/POPULATION/POPULATIONS/RESISTANCE/STRAINS/TNF-ALPHA/TUBERCULOSIS PHAGOSOME

211 Daniels, M.J., Ball, N., Hutchings, M.R., Greig, A. (2001) The grazing response of cattle to pasture contaminated with rabbit faeces and the implications for the transmission of paratuberculosis Veterinary Journal, 161, 306-313 Transmission of Mycobacterium avium subspecies paratuberculosis, the organism responsible for paratuberculosis (or Johne's disease) in ruminants, occurs through the faecal-oral route. As M. a. paratuberculosis has been isolated from rabbit faeces, cattle grazing rabbit faecal contaminated pasture may thus be at risk. A herd of 57 beef cattle was monitored on a farm in Perthshire, throughout the 1999 'grazing year', to investigate whether the cattle avoided rabbit faecal contaminated pasture and thus the potential for disease transmission. Grazing was measured every two days over eight rotations by sward heights on 40 marked treatment plots (0.5 m x 0.5 m) to which 0, 10, 50 and 250 rabbit faecal pellets were added. Cattle were also monitored by an active transponder system which enabled individual animals contacting two plots per field rotation tone control and one contaminated) to be recorded. During the monitored grazing year, grazing pressure was low with a net mean sward offtake of 18% of sward height per rotation. There were no significant differences between rabbit faecal treatments (0, 10, 50 and 250 pellets) with respect to the height or proportion of sward removed, or between the numbers of contacts made by cattle on contaminated and uncontaminated plots. Over 90% of all the cattle contacted contaminated plots, indicating that the potential for disease transmission was widespread among the herd. To our knowledge, this is the first reported instance of a lack of avoidance by grazing cattle towards swards contaminated with faeces, and implies that the potential for transmission of paratuberculosis from rabbit contaminated pasture is high. (C) 2001 Harcourt Publishers Ltd ANIMALS/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BOVINE TUBERCULOSIS/CATTLE/cattle grazing/control/DISEASE/disease transmission/DUNG/faecal contamination/faecal-oral route/faeces/FECES/HERD/Johne's disease/mycobacteria/ Mycobacterium/Mycobacterium avium/ Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/ODORS/ORGANISM/ORYCTOLAGUS-CUNICULUS/paratuberculosis/pasture/PELLETS/rabbit/risk/RUMINANTS/SHEEP/STRATEGIES/ Transmission/URINE/WILD RABBITS

212 Glawischnig, W., Khaschabi, D. (2001) Paratuberculosis in a free living red deer (Cervus elaphus hippelaphus) from Vorarlberg

Wiener Tierarztliche Monatsschrift, 88, 66-69 Paratuberculosis in an eight year old free living red deer stag from the region Vorarlberg is reported. For a period of several weeks in January the emaciated stag was detected with symptoms of diarrhea near a feeding station. The animal was shot and a post mortem examination performed. Pathomorphological lesions, acid-fast organisms detected in faeces and smears from the intestinal mucosa, the growth of Ziehl-Neelsen-positive bacteria on media containing mycobactin, and serological positive results revealed an infection with Mycobacterium avium supsp. paratuberculosis AUSTRIA/AVIUM/BACTERIUM/Cervus elaphus/Cervus elaphus hippelaphus/CERVUS-ELAPHUS/deer/Diarrhea/Epidemiology/faeces/GROWTH/HIPPELAPHUS/INFECTION/INTESTINAL-MUCOSA/JOHNES-DISEASE/LESIONS/mycobacteria/Mycobacterium/Mycobacterium avium/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-

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PARATUBERCULOSIS/ORGANISM/ORGANISMS/ORYCTOLAGUS-CUNICULUS/paratuberculosis/RED DEER/WILD RABBITS

213 Beard, P.M., Daniels, M.J., Henderson, D., Pirie, A., Rudge, K., Buxton, D., Rhind, S., Greig, A., Hutchings, M.R., McKendrick, I., Stevenson, K., Sharp, J.M. (2001) Paratuberculosis infection of nonruminant wildlife in Scotland

Journal of Clinical Microbiology, 39, 1517-1521 Recent reports of natural paratuberculosis (or Johne's disease) in rabbits, foxes, and stoats has focused debate on the presence and importance of wildlife reservoirs in the epidemiology of this disease. This paper describes an extensive study investigating 18 nonruminant wildlife species for evidence of paratuberculosis. Using both culture and histopathological analysis, fox, stoat, weasel, crow, rook, jackdaw, rat, wood mouse, hare, and badger were found to harbor Mycobacterium avium subsp, paratuberculosis, the causative organism of paratuberculosis, suggesting that the epidemiology of this disease is more complex than previously realized AVIUM/BIRDS/BOVIS/California/COMPLEX/CULTURE/DISEASE/Epidemiology/INFECTION/Johne's disease/JOHNES-DISEASE/MOUSE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/ORGANISM/PARA-TUBERCULOSIS/paratuberculosis/PARATUBERCULOSIS INFECTION/PATHOLOGY/rabbit/RABBITS/reservoir/SCOTLAND/WILDLIFE/wildlife reservoir

214 Thorel, M.F., Huchzermeyer, H.F., Michel, A.L. (2001) Mycobacterium avium and Mycobacterium intracellulare infection in mammals

Revue Scientifique et Technique de l Office International des Epizooties, 20, 204-218 Mycobacterium avium subsp. avium and M. intracellulare are ubiquitous organisms in the environment. The reservoir of M. avium subsp. avium is generally accepted to be environmental, in particular, water and soil are sources of the organism. In contrast to M. avium infection in wild and domestic birds, M. avium infection in mammals occurs only sporadically and is rarely transmissible. Generalised disease is usually uncommon, owing to the non-progressive, chronic character of the infection. However, some cases of disseminated disease have been reported, e.g. in captive non-domestic hoofed animals as well as in immunosuppressed dogs and cats. The majority of M. avium and M. intracellulare infections in livestock are detected at slaughter and the diagnosis is confirmed by bacteriological procedures. Condemnation of affected portions of the carcass can result in significant economic losses, although gross lesions are mostly restricted to lymph nodes close to the alimentary tract. Successful treatment with antibiotics in combination with surgery has been reported in some affected domestic cats, but is not considered to be effective or economical in other species. In the past, differentiation of M. avium bacteria from the closely related M. avium su bsp. paratuberculosis was ba sed on the mycobactin dependence a nd prolonged incubation period of the latter. More recently, amplification of the genomic insertion sequence IS900 has proved to be a powerful tool for identification of M. avium subsp. paratuberculosis. The potential zoonotic importance of M. avium infections has been indicated, but requires clarification AMPLIFICATION/ANIMALS/ANTIBIOTICS/ATYPICAL MYCOBACTERIOSIS/AVIUM/BACTERIUM/BIRDS/COMPLEX INFECTION/DIAGNOSIS/DIFFERENTIATION/DISEASE/domestic mammals/economic losses/ECONOMIC-LOSSES/ENVIRONMENT/FARMED DEER/GENERALIZED AVIAN TUBERCULOSIS/GRANULOMATOUS ENTERITIS/IDENTIFICATION/incubation period/INFECTION/INFECTIONS/INTRACELLULARE/IS900/LESIONS/livestock/LYMPH-NODES/MAMMALS/MINIATURE SCHNAUZER/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium intracellulare/MYCOBACTERIUM-AVIUM/non-domestic mammals /ORGANISM/ORGANISMS/paratuberculosis/reservoir/RESTRICTION ENZYME ANALYSIS/SEQUENCE/STRAINS/SWINE/Tuberculosis/WATER/WILD

215 Bonenberger, T.E., Ihrke, P.J., Naydan, D.K., Affolter, V.K. (2001) Rapid identification of tissue micro-organisms in skin biopsy specimens from domestic animals using polyclonal BCG antibody

Veterinary Dermatology, 12, 41-47 Immunostaining with polyclonal anti-Mycobacterium bovis (BCG) was evaluated as a single screening method for the histological identification of micro-organisms in skin biopsy specimens from various veterinary species. Confirmed archival cases infected with Mycobacteria, Nocardia, Actinobacillus, Actinomyces, Streptococcus/Staphylococcus, Dermatophilus, spirochetes, Blastomyces, Coccidioides, Cryptococcus. Histoplasma, dermatophytes, Malassezia, Sporothrix, Leishmania, Pythium, phaeohyphomycetes and Prorotheca organisms were selected. A total of 70 skin biopsy specimens from the dog, cat, horse. ox and llama were evaluated. The anti-BCG immunostain labelled bacteria and fungi with high sensitivity and minimal background staining but did not label spirochetes and protozoa

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(Leishmania). Differences were not noted between veterinary species. The results indicate that immunostaining with polyclonal anti-BCG is a suitable screening technique for the rapid identification of most common bacterial and fungal organisms in paraffin-embedded specimens. Also, mycobacterial and nocardial organisms were identified more readily with the anti-BCG immunostain in comparison to the histochemical stains ANIMALS/ANTIBODIES/ANTIBODY/antigens/BACTERIUM/BCG/BIOPSY SPECIMENS/BOVIS/domestic animal/DOMESTIC-ANIMALS/IDENTIFICATION/immunohistochemistry/MICROORGANISMS/mycobacteria/Mycobacterium/MYCOBACTERIUM-PARATUBERCULOSIS/Nocardia/ORGANISM /ORGANISMS/pathology-veterinary/polyclonal antibody/SENSITIVITY/TISSUE

216 Chamberlin, W., Graham, D.Y., Hulten, K., El-Zimaity, H.M.T., Schwartz, M.R., Naser, S., Shafran, I., El-Zaatari, F.A.K. (2001) Review article: Mycobacterium avium subsp paratuberculosis as one cause of Crohn's disease

Alimentary Pharmacology & Therapeutics, 15, 337-346 A number of theories regarding the aetiology of Crohn's disease have been proposed. Diet, infections, other unidentified environmental factors and immune disregulation, all working under the influence of a genetic predisposition, have been viewed with suspicion. Many now believe that Crohn's disease is a syndrome caused by several aetiologies. The two leading theories are the infectious and autoimmune theories. The leading infectious candidate is Mycobacterium avium subspecies paratuberculosis (Mycobacterium paratuberculosis), the causative agent of Johne's disease, an inflammatory bowel disease in a variety of mammals including cattle, sheep, deer, bison, monkeys and chimpanzees. The evidence to support M. paratuberculosis infection as a cause of Crohn's disease is mounting rapidly. Technical advances have allowed the identification and/or isolation of M. paratuberculosis from a significantly higher proportion of Crohn's disease tissues than from controls. These methodologies include: (i) improved culture techniques; (ii) development of M. paratuberculosis-specific polymerase chain reaction assays; (iii) development of a novel in situ hybridization method; (iv) efficacy of macrolide and anti-mycobacterial drug therapies; and (v) discovery of Crohn's disease-specific seroreactivity against two specific M. paratuberculosis recombinant antigens. The causal role for M. paratuberculosis in Crohn's disease and correlation of infection with specific stratification(s) of the disorder need to be investigated. The data implicating Crohn's as an autoimmune disorder may be viewed in a manner that supports the mycobacterial theory. The mycobacterial theory and the autoimmune theory are complementary; the first deals with the aetiology of the disorder, the second deals with its pathogenesis. Combined therapies directed against a mycobacterial aetiology and inflammation may be the optimal treatment of the disease ANTIGEN/antigens/ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/AVIUM SUBSPECIES PARATUBERCULOSIS/BISON/CATTLE/control/CONTROL TISSUES/Crohn's disease/CULTURE/deer/DISEASE/DNA/etiology/HYBRIDIZATION/IDENTIFICATION/INFECTION/INFECTIONS/INFLAMMATORY BOWEL-DISEASE/INFLAMMATORY-BOWEL-DISEASE/Johne's disease/MAMMALS/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium avium subspecies paratuberculosis/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/PARATUBERCULOSIS INFECTION/PATHOGENESIS/ polymerase chain reaction/POLYMERASE CHAIN-REACTION/POLYMERASE-CHAIN-REACTION/Review/role/SEROREACTIVITIES/SERUM ANTIBODIES/SHEEP/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/THERAPY/TISSUE/TISSUES

217 Olsen, I., Reitan, L.J., Wiker, H.G. (2000) Distinct differences in repertoires of low-molecular-mass secreted antigens of Mycobacterium avium complex and Mycobacterium tuberculosis Journal of Clinical Microbiology, 38, 4453-4458 Antigens in a 4-week-old culture filtrate (CF) of Mycobacterium avium subsp, avium were separated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by Western blotting. The culture had minimal lysis of bacilli, giving a CF preparation consisting mainly of secreted proteins. Comparison with a similar CF of Mycobacterium tuberculosis with almost no contamination with intracellular proteins showed the presence of cross-reactive antigens homologous to the four components of the antigen 85 complex, as well as MPT32. These were major constituents of the M. avium subsp, avium CF, In addition, there were several low-molecular-mass bands (<15 kDa) in both species that did not cross-react with polyclonal and polyvalent rabbit antibodies in Western blotting. Furthermore, these bands were not detected in corresponding sonicate preparations, indicating high localization indexes, which is typical of soluble secreted proteins. A 14-kDa protein was selected for purification and more detailed characterization. The N-terminal amino acid sequence was determined, and a matching gene was found within the genomic sequence of M avium subsp. avium which was

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highly homologous to Rv0455c of M. tuberculosis, The gene encoded a signal peptide typical of secreted mycobacterial proteins. A rabbit antiserum was raised against the purified protein, and the antigen was demonstrated by Western blotting in CFs of M. avium subsp. avium, Mycobacterium avium subsp, paratuberculosis, Mycobacterium intracellulare, and Mycobacterium scrofulaceum but was not detected in M tuberculosis. This is a new example of a highly homologous gene being differentially expressed by different mycobacterial species ANTIBODIES/ANTIBODY/ANTIGEN/antigens/AVIUM/BOVIS BCG/COMPLEX/CROHNS-DISEASE/CULTURE/CULTURE FILTRATE/GENE/IDENTIFICATION/INTRACELLULARE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium complex/Mycobacterium avium subsp/Mycobacterium intracellulare/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-TUBERCULOSIS/paratuberculosis/POLYACRYLAMIDE-GEL ELECTROPHORESIS/PROTEIN/PROTEIN ANTIGENS/PROTEINS/PURIFICATION/rabbit/SEQUENCE/SUBSP PARATUBERCULOSIS/Tuberculosis

218 Pavlik, I. , Bartl, J., Dvorska, L., Svastova, P., du Maine, R., Machackova, M., Ayele, W.Y., Horvathova, A. (2000) Epidemiology of paratuberculosis in wild ruminants studied by restriction fragment length polymorphism in the Czech Republic during the period 1995-1998

Veterinary Microbiology, 77, 231-251 In two studies carried out during the period 1995-1998, paratuberculosis was diagnosed in domestic and wild ruminants in the Czech Republic. The isolated Mycobacterium avium subspecies paratuberculosis strains were analysed by standardised restriction fragment length polymorphism (RFLP) [Pavlik, I., Horvarhova, A., Dvorska, L., Bartl, J., Svastova, P., du Maine; R., Rychlik, I., 1999. J. Microbiol. Methods 38, 155-167]. In December 1992, 19 late pregnant Charolais heifers were imported to the Czech Republic from Hungary (original import from France to Hungary). One 11-month-old heifer roamed in the wild in a range of approximately 15-20 km for 7 months from November 1993 to May 1994. Upon capture, the animal showed clinical signs of paratuberculosis (emaciation and diarrhoea). Seven other animals from the same herd were infected with the identical RFLP type B-C1 of M. paratuberculosis. During the period 1995-1996, samples were taken and examined from the small intestine and corresponding lymph nodes of 84 wild ruminants: 19 red deers (Cervus elaphus) and 65 roe-deers (Capreolus capreolus). These wild ruminants originated from 34 different locations within the same district from as the infected escaped heifer. Five M. paratuberculosis strains were isolated: one strain of RFLP type B-C1 from a stag and three strains of RFLP type B-C1 and one strain of RFLP type B-C9 from roe-deer. The three wild ruminants (one stag and two roe-deer) infected with the same RFLP type B-C1 were detected in the same area as the heifer, suggesting that this was the likely infection source. However, the infection source of the roe-deer infected with strain of RFLP type B-C9 was obviously different, and the stags that escaped from the farm were purchased from an area infected with this RFLP type. In the second study carried out during 1997-1998 in the whole Czech Republic (divided into 76 districts), 718 wild ruminants were examined from 90% of the districts. M. paratuberculosis was isolated from 25 (3.5%) animals from the wild, from farms and from game parks: 7.1% of 132 red deers, 1.5% of 336 roe-deers, 3.9% of 178 fallow deers (Dama dama), and 4.2% of 48 moufflons (Ovis musimon). This study discovered three RFLP types (B-CI, D-C12 and M-C16). A surprising finding was that of M. paratuberculosis (RFLP type B-C1) infection in roe-deer and a fallow deer in their natural habitat. The infection source was determined to have originated from two imported Holstein and Limousine cattle herds infected with the same strain. In the case of a mother and daughter roe-deer infected with RFLP type M-C16 and a fellow deer infected with RFLP type D-C12, all roaming in their natural habitat, the infection source was not discovered. The highest incidence of clinically ill wild ruminants was found in farmed red deer, and no relationship was found between the RFLP type or ruminant species and clinical status of animal. (C) 2000 Elsevier Science B.V. All rights reserved ANIMALS/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/CATTLE /CATTLE HERDS/Cervus elaphus/CERVUS-ELAPHUS/Dama dama/DAMA-DAMA/deer/deer farm/DNA HYBRIDIZATION/domestic and wild ruminants/emaciation/ENDONUCLEASE ANALYSIS/Epidemiology/FALLOW DEER/FARMED RED DEER/FARMS/FRAGMENT-LENGTH-POLYMORPHISM/game park/game parks/heifer/HERD/IDENTIFICATION/INFECTION/Johne's disease/JOHNES-DISEASE/LYMPH-NODES/MOLECULAR EPIDEMIOLOGY/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/nature/PARA-TUBERCULOSIS/paratuberculosis/RED DEER/RESTRICTION/RFLP/roe deer/RUMINANTS/STRAINS/tule elk/WILD/WILD RUMINANTS

219 Godfroid, J., Boelaert, F., Heier, A., Clavareau, C., Wellemans, V., Desmecht, M., Roels, S., Walravens, K. (2000)

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First evidence of Johne's disease in farmed red deer (Cervus elaphus) in Belgium

Veterinary Microbiology, 77, 283-290 In a deer farm, chronic diarrhoea was seen in a 4-year-old hind. This animal died in poor condition on the farm and Johne's disease was suspected. Ziehl-Neelsen staining of the faeces of this hind were positive for the presence of clumps of small acid-fast bacilli, but faecal cultures remained negative. Direct and indirect tests were performed on 24 hinds and stags (yearlings, 2- and 4-year-old animals). The indirect tests performed were serology (Mycobacterium paratuberculosis antibody ELISA, HerdChek(TM), Idexx), comparative cervical skin test (CCT) and lymphoproliferation test (LT) using Mycobacterium bovis purified protein derivative (PPD) and Mycobacterium avium PPD as antigens. Three positive serological results, three positive CCT and eight positive LT were observed in hinds and stags older than 2 years. No positive serological results were observed in the yearling group, whereas some sensitisation was observed in the CCT as well as in the LT for the same group of animals. The degree of concordance between these indirect tests was poor. The three seropositive animals were slaughtered and subjected to post-mortem examination. Histopathology was performed on mesenteric lymph nodes and on the terminal ileum. Visual changes in some mesenteric lymph nodes were observed, no gross lesion was seen in the intestine. Although Ziehl-Neelsen staining yielded no positive results, a catarrhal focal necrotic enteritis associated with a granulomatous lymphadenitis compatible with Johne's disease was evidenced. The mycobacterial cultures on organ samples from slaughtered animals were positive after 2 months for M. avium subspecies paratuberculosis and negative for M. bovis and M. avium. This is the first description of Johne's disease in a deer farm in Belgium. (C) 2000 Elsevier Science B.V. All rights reserved ANIMALS/ANTIBODIES/ANTIBODY/ANTIGEN/antigens/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/Belgium/BOVIS/Cervus elaphus/CERVUS-ELAPHUS/CULTURE/deer/deer farm/DISEASE/ELISA/ENTERITIS/faeces/FARMED DEER/FARMED RED DEER/HERD/Johne's disease/LYMPH-NODES/microbiological diseases/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium bovis/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-BOVIS/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/PROTEIN/RED DEER/Serology/SKIN-TEST/TESTS/Tuberculosis/Ziehl-Neelsen

220 Kennedy, D.J., Allworth, M.B. (2000) Progress in national control and assurance programs for bovine Johne's disease in Australia

Veterinary Microbiology, 77, 443-451 Cattle strains of Mycobacterium paratuberculosis are known to infect cattle, goats and alpaca in southeastern Australia, where there are also significant numbers of farmed deer. Although sheep strains have recently been identified in some cattle in Australia, epidemiological evidence to date supports the distinction (between bovine Johne's disease (JD), caused by cattle strains in cattle, goats and alpaca, and ovine JD, caused by sheep strains in sheep and goats) for the purposes of control and assurance programs. The National Johne's Disease Control Program is coordinated by the Australian Animal Wealth Council, working with the livestock industries and with the Commonwealth, state and territory governments. The council also brokers industry and government funding for the program, The National Johne's Disease Market Assurance Program for Cattle was launched in 1996 as the first of a suite of voluntary national market assurance programs (MAPs) to assess and certify herds as negative for JD. By December 1998, over 550 herds had achieved an assessed negative status. A MAP was also launched for alpaca in 1998 and a program for goats should be finalised in early 1999. National standards for state control of JD through zoning, movement controls and procedures in infected acid suspect herds have also been developed. The paper covers factors affecting development and implementation, uptake of and improvements to national control and assurance programs for bovine JD in Australia. (C) 2000 Elsevier Science B.V. All rights reserved assurance/AUSTRALIA/BOVINE/CATTLE/control/deer/DISEASE/FARMED DEER /goat/goats/HERD/Johne's disease/livestock/MAP/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/OVINE/paratuberculosis/SHEEP/sheep and goats/STRAINS

221 Stabel, J.R. (2000) Transitions in immune responses to Mycobacterium paratuberculosis Veterinary Microbiology, 77, 465-473 The host immune response to infection with Mycobacterium paratuberculosis is paradoxical, with strong cell-mediated immune responses during the early, subclinical stages of infection and strong humoral responses during the late clinical stages of the disease. Cell-mediated immune responses modulated by various T cell subsets are essential to provide protective immunity and prevent progression of the disease. Secretion of cytokines by T cell populations serves to activate macrophages to kill ingested M.

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paratuberculosis as well as activate other T cell subsets to contain the infection. This paper reviews the current knowledge of T cell immune responses in M. paratuberculosis infection based upon clinical studies and research using mouse models. Published by Elsevier Science B.V BOVINE PARATUBERCULOSIS/CELL/CELLULAR IMMUNOLOGY /cytokine/cytokines/DELTA T-CELLS/DISEASE/humoral response/immune responses/IMMUNE-RESPONSE/IMMUNE-RESPONSES/IMMUNITY/IN-VIVO/INFECTION/interferon-gamma/JOHNES-DISEASE/lymphocyte subsets/macrophage/MACROPHAGES/MODEL/MOUSE/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/OVINE PARATUBERCULOSIS/paratuberculosis/PARATUBERCULOSIS INFECTION/POPULATION/POPULATIONS/RESPONSES/Review/T cell/T lymphocytes/TNF-ALPHA/Tuberculosis

222 Ferroglio, E., Nebbia, P., Robino, P., Rossi, L., Rosati, S. (2000) Mycobacterium paratuberculosis infection in two free-ranging Alpine ibex

Revue Scientifique et Technique de l Office International des Epizooties, 19, 859-862 The authors report two cases of Mycobacterium paratuberculosis infection in free-ranging Alpine ibex (Capra ibex) from two different herds in the Western Alps, Italy. One ibex, found dead in October 1998, was in poor condition. The second animal died due to trauma following capture with a dart gun. The only gross lesions observed were the enlargement of the mesenteric and iliac lymph nodes. Samples from both ibex tested positive to polymerase chain reaction for a primer set specific for the M. paratuberculosis insertion sequence IS900 a nd one ibex also tested positive to the Zielh-Nielsen stain. Isolation by bacterial culture was not successful. The infected ibex originated from herds in which seroreactors to M. paratuberculosis had been found previously. Seroreactors to M. paratuberculosis were also detected in sympatric cattle ALPINE IBEX/Alps/BACTERIAL CULTURE/CATTLE/CULTURE/FARMED RED DEER/HERD/INFECTION/IS900/Italy/JOHNES-DISEASE/LESIONS/LYMPH-NODES/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/Mycobacterium paratuberculosis infection/MYCOBACTERIUM-PARATUBERCULOSIS/PARA-TUBERCULOSIS/paratuberculosis/PARATUBERCULOSIS INFECTION/polymerase chain reaction/POLYMERASE-CHAIN-REACTION/SEQUENCE/tule elk/WILD ANIMALS

223 Fischer, O., Matlova, L., Bartl, J., Dvorska, L., Melicharek, I., Pavlik, I. (2000) Findings of mycobacteria in insectivores and small rodents

Folia Microbiologica, 45, 147-152 The organs of 30 insectivorous mammals and 62 rodents from areas inhabited by people or livestock where cattle paratuberculosis or mycobacterial infections of swine had been found to occur were examined by cultivation during the monitoring of occurrence and spread of mycobacterioses in cattle and swine. Mycobacteria were found in the organs of 3 insectivores (10 %) and 6 rodents (9.7 %). Mycobacterium chelonae was isolated from the organs of the lesser white-toothed shrew (Crocidura suaveolens) and the common vole (Microtus arvalis), and M, vaccae and M. avium subsp. avium (IS901(+). serotype I) from the organs of the common shrew (Sorex araneus), M. avium subsp. avium (IS901(+), serotype 1) was also isolated from the organs of the yellow-necked mouse (Apodemus flavicollis). Slow-growing mycobacteria of group III (according to Runyon) were isolated from the organs of the mouse (Mus musculus sensu late) and the yellow-necked mouse (A. flavicollis). These findings had no connection with the epizootological situation in the nearby livestock. M. fortuitum was isolated from the organs of the common vole (M. arvalis) caught in a field within easy reach of a swine breeding herd. M. fortuitum was also identified in the lymph nodes and droppings of this swine herd, as well as in the straw, scrapings from the floor of stalls, troughs and banisters, as well as from larvae and imagoes of dipterous insects. These results demonstrate the possibility that insectivores and small rodents can spread the causative agents of mycobacteria in wild and domestic animals ANIMALS/AVIUM/BANK VOLES/CATTLE/domestic animal/DOMESTIC-ANIMALS/HERD/INFECTION/INFECTIONS/livestock/LYMPH-NODES/MAMMALS/MICE/monitoring/MOUSE/mycobacteria/MYCOBACTERIAL INFECTIONS/Mycobacterium/paratuberculosis/rodents/SWINE/WILD

224 Tanaka, S. , Itohara, S., Sato, M., Taniguchi, T., Yokomizo, Y. (2000) Reduced formation of granulomata in gamma delta T cell knockout BALB/c mice inoculated with Mycobacterium avium subsp paratuberculosis

Veterinary Pathology, 37, 415-421 The role of gamma delta T cells in the bovine immune response to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) infection is poorly understood. Accordingly, using BALB/c mice

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that are innately susceptible to M. paratuberculosis, we compared wild-type and gamma delta T cell knockout BALB/c mice to study the protective roles of gamma delta T cells in M. paratuberculosis infection. Ten-week-old mice were inoculated intraperitoneally with either a low dose (4 X 10(6) colony-forming units [CFU]/mouse) or a high dose (4 X 10(9) CFU/mouse) of M. paratuberculosis strain ATCC 19698. Histopathologic and morphometric examinations showed reductions in the number and area of granulomatous lesions in the liver of the knockout mice at 18 weeks after inoculation with either the low or the high dose of the mycobacteria. Furthermore, at 18 weeks after inoculation, the bacterial load in the spleens of the knockout mice inoculated with the high dose was significantly lower than that of wild-type mice. No differences were found in bacterial load between the knockout and the wild-type mice in the low-dose groups. In contrast, in the livers of wild-type mice inoculated with either the low or high mycobacterial dose, increased areas of epithelioid granulomata were observed and the granulomata became disseminated widely during the experimental period. These findings in model mice suggest that gamma delta T cells, rather than restricting mycobacterial growth, may play a crucial role in development of epithelioid granulomata similar to those seen consistently in bovine paratuberculosis. The results of this study may have relevance to our understanding of the pathogenesis of paratuberculosis in ruminants, in which a prominent number of gamma delta T cells exist in the lymphoid system ALPHA/APPEARANCE/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/BALB/c mice/BOVINE/BOVINE PARATUBERCULOSIS/BOVINE-PARATUBERCULOSIS/CELL/CELLS/DELTA-T-CELLS/GAMMA/gamma delta T cells/granuloma formation/GROWTH/IMMUNE-RESPONSE/INFECTION/INOCULATION/INTERLEUKIN-2/LESIONS/LISTERIA-MONOCYTOGENES/LYMPHOCYTES-T/MICE/MODEL/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PARATUBERCULOSIS INFECTION/PATHOGENESIS/role/RUMINANTS/SALMONELLA-CHOLERAESUIS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/T cell/T-CELLS/Tuberculosis/TUMOR-NECROSIS-FACTOR

225 Buergelt, C.D., Layton, A.W., Ginn, P.E., Taylor, M., King, J.M., Habecker, P.L., Mauldin, E., Whitlock, R., Rossiter, C., Collins, M.T. (2000) The pathology of spontaneous paratuberculosis in the North American bison (Bison bison)

Veterinary Pathology, 37, 428-438 Gross and histopathologic examinations were performed on 70 North American bison (Bison bison) from a Mycobacterium avium paratuberculosis culture-positive herd. The bison examined were part of a breeding herd totaling 2,800 animals. Eight of 70 (11%) animals had gross findings of intestinal mucosal thickening, and 16 of 70 (23%) of the animals had enlarged mesenteric lymph nodes. Histologic lesions compatible with Johne's disease were diagnosed in 30 of 70 (43%) bison on the basis of the demonstration of noncaseating granulomatous inflammatory infiltrates and of one or more acid-fast bacilli characteristic of Mycobacterium avium paratuberculosis. A suspicious diagnosis of Johne's disease was obtained in 11 of 70 (16%) bison on the basis of the observation of noncaseating granulomatous inflammatory infiltrates without demonstrable acid-fast bacteria. Twenty-nine of 70 (41%) animals were assessed as histologically paratuberculosis free. Histologic results were compared to Johne's disease tests such as culture, serology, and polymerase chain reaction, which were performed on some of the cohort animals ANIMALS/AVIUM/BACTERIUM/BISON/bison bison/CULTURE/DIAGNOSIS/DISEASE/ELK/gross and microscopic pathology/HERD/INFECTION/Johne's disease/JOHNES-DISEASE/LESIONS/LYMPH-NODES/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/North American bison/PARA-TUBERCULOSIS/paratuberculosis/PATHOLOGY/polymerase chain reaction/POLYMERASE-CHAIN-REACTION/RHODOCOCCUS-EQUI/Serology/TESTS/white-tailed deer

226 Bannantine, J.P., Stabel, J.R. (2000) HspX is present within Mycobacterium paratuberculosis-infected macrophages and is recognized by sera from some infected cattle

Veterinary Microbiology, 76, 343-358 A portion of the gene encoding HspX has been previously identified as a sequence specific to Mycobacterium avium subspecies paratuberculosis thereafter referred to as M. paratuberculosis) based on DNA hybridization experiments. In this study, rabbit antisera were raised against a recombinant protein of HspX fused to the Escherichia coli maltose binding protein (MBP/HspX). Immunoblots of lysates of M. paratuberculosis-infected macrophages probed with the rabbit antisera showed that HspX was present within infected macrophages of bovine and murine origin. This observation was confirmed by immunofluorescence microscopy of infected macrophages. Lysates of E. coli expressing HspX without the MBP fusion partner were loaded onto preparative SDS-PAGE gels and used to determine whether infected cattle generated a humoral immune response to the antigen. Sera from four of 24

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paratuberculous cows (17%) detected HspX. No reactivity was present in sera from control cows. While HspX may be immunogenic during infection in some cows, the protein is not secreted and it does not stimulate cell-mediated immunity. Collectively, these data give a preliminary characterization of the first described M. paratuberculosis protein identified within infected macrophages. Published by Elsevier Science B.V ANTIBODIES/ANTIGEN/antigens/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/AVIUM SUBSPECIES PARATUBERCULOSIS/BINDING PROTEIN/BOVINE/CATTLE/cattle-bacteria/cell-mediated immunity/control/cow/COWS/DIAGNOSIS/DNA/DNA HYBRIDIZATION/ESCHERICHIA-COLI/GENE/HYBRIDIZATION/IMMUNE-RESPONSE/IMMUNITY /INFECTED CATTLE/INFECTION/Johne's disease/JOHNES-DISEASE/LEPRAE/LONG-TERM CULTIVATION/ macrophage/MACROPHAGES/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/PROTEIN/rabbit/SEQUENCE/SILVATICUM/Tuberculosis

227 Singh, S.V., Singh, P.P., Singh, N., Gupta, V.K. (2000) Characterization of lipid pattern of Mycobacterium paratuberculosis isolates from goats and sheep

Indian Journal of Animal Sciences, 70, 899-903 Lipid pattern of 18 wild type Mycobacterium paratuberculosis isolates were determined and compared with standard M. paratuberculosis (Teps) strain used for commercial Johnin production. Fourteen of these isolates had typical pattern of alpha and keto mycolates in the ratio of 30: 70%, which resembled the Lipid pattern of M. avium, therefore providing evidence of their identity as M. paratuberculosis. Additional band of methoxy mycolate present in the M. paratuberculosis (Teps) strain and 3 other isolates, suggested major differences in their identity as M. paratuberculosis. PDIM patterns of these isolates were also determined ALPHA/AVIUM/chemical characterization/goat/goats/lipid analysis /methyl mycolates/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/SHEEP/WILD

228 Nebbia, P. , Robino, P., Ferroglio, E., Rossi, L., Meneguz, G., Rosati, S. (2000) Paratuberculosis in red deer (Cervus elaphus hippelaphus) in the Western Alps

Veterinary Research Communications, 24, 435-443 During the hunting seasons 1995-96 to 1997-98, 19 red deer from the Upper Susa Valley (Cottian Alps) were examined for paratuberculosis (Johne's disease). Specific DNA amplification on mesenteric lymph nodes detected Mycobacterium avium paratuberculosis in 17 animals. Ten of these red deer were tested for serum antibodies by the AGID and ELISA tests, nine being negative. Three isolates from infected deer were genetically characterized by an arbitrarily primed polymerase chain reaction, and showed similar genetic polymorphism to that of bovine strains isolated in different Italian areas. The study showed that paratuberculosis is present in red deer of the Upper Susa Valley and that serological tests are not an efficient means for monitoring this infection Alps /AMPLIFICATION/ANIMALS/ ANTIBODIES/ANTIBODY/AVIUM/BOVINE/BOVINE PARATUBERCULOSIS/CATTLE/Cervus elaphus/Cervus elaphus hippelaphus/CERVUS-ELAPHUS/CROHNS-DISEASE/deer/DIAGNOSIS/DISEASE/DNA/EFFICIENT/ELISA/HIPPELAPHUS/INFECTION/Italian Alps/Johne's disease/JOHNES-DISEASE/LYMPH-NODES/monitoring/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium paratuberculosis/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/PARA-TUBERCULOSIS/paratuberculosis/polymerase chain reaction/POLYMERASE CHAIN-REACTION/POLYMERASE-CHAIN-REACTION/RED DEER/SEROLOGICAL TESTS/Serology/SERUM ANTIBODIES/STRAINS/TESTS

229 Whittington, R.J., Hope, A.F., Marshall, D.J., Taragel, C.A., Marsh, I. (2000) Molecular epidemiology of Mycobacterium avium subsp paratuberculosis: IS900 restriction fragment length polymorphism and IS1311 polymorphism analyses of isolates from animals and a human in Australia

Journal of Clinical Microbiology, 38, 3240-3248 The distribution and prevalence of strains of Mycobacterium avium subsp, paratuberculosis were determined among sheep, cattle, and other species with Johne's disease in Australia. A total of 328 isolates were evaluated from numerous farms in New South Wales, Victoria, Tasmania, and South Australia, Australia. Restriction fragment length polymorphism (RFLP) analysis of genomic DNA using BstEII and an IS900 probe and IS1311 polymorphism analysis using PCR and restriction endonuclease

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analysis (PCR-REA) was used to classify isolates as cattle (C) or sheep (S) strains. IS1311 PCR-REA provided similar information to IS900 RFLP analysis but was more useful than RFLP analysis where DNA was degraded or scarce, Twelve IS900 RFLP types were found, Johne's disease in sheep was always due to S strains, while cattle were infected only with C strains. RFLP type S1 was the dominant strain in sheep in New South Wales (97% of isolates) and was the only strain found in sheep from Victoria. Seven RFLP types were present in cattle, RFLP types C3 and C1 were most common (collectively, 85% of isolates), but C1 was not found in New South Wales and C3 was present in dairy cattle but not in beef cattle in Victoria. These differences may be explained by restricted livestock trading patterns between different segments of the cattle industry, Up to five RFLP types were present in some geographic regions in Victoria, while up to three RFLP types were found among cattle on some farms. Individual cattle usually were infected with only one RFLP type, but one animal was infected with both C5 and CU4. Two isolates from goats were C type as were three from alpacas, one from a rhinoceros, and two from a human with Crohn's disease. The prevalences of specific RFLP types in Australia differ from those reported in Europe and elsewhere. Given the existence of geographical and farm enterprise differences in IS900 RFLP type, this technique may be applied selectively to trace the spread of Johne's disease, at least in the cattle industries. As these observations reflect past exposure of livestock to M. avium subsp, paratuberculosis, the monitoring of strains present in animals in Australia is continuing ANIMALS/AUSTRALIA/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/Crohn's disease/dairy cattle/DAIRY-CATTLE/DISEASE/DNA/DNA HYBRIDIZATION/ENDONUCLEASE ANALYSIS/Epidemiology/Europe/FARMS/FRAGMENT-LENGTH-POLYMORPHISM/goat/goats/INSERTION ELEMENT IS900/IS1311/IS900/Johne's disease/JOHNES-DISEASE/livestock/MOLECULAR EPIDEMIOLOGY/monitoring/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/MYCOBACTERIUM-AVIUM/NEW-SOUTH-WALES/PARA-TUBERCULOSIS/paratuberculosis/PCR/POLYMERASE-CHAIN-REACTION/PREVALENCE/RESTRICTION/RESTRICTION ENDONUCLEASE ANALYSIS/RFLP/SHEEP/ STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/victoria/WILD RABBITS

230 Horn, B., Forshaw, D., Cousins, D., Irwin, P.J. (2000) Disseminated Mycobacterium avium infection in a dog with chronic diarrhoea Australian Veterinary Journal, 78, 320-325 A 3-year-old Maltese-cross dog presented with a 4-month history of chronic diarrhoea and inappetence. Poorly regenerative anaemia, leukocytosis and hypoproteinaemia were evident on several occasions. Biopsies of stomach, duodenum and colon revealed marked infiltration of mucosae by macrophages containing many acid-fast bacilli. Similar organisms were numerous in a faecal smear. Melaena, haematochezia and severe abdominal pain developed and were unresponsive to therapy. Following euthanasia and necropsy, histiocytic cells containing acid-fast bacilli were found throughout the gastrointestinal tract, mesenteric and peri-pheral lymph nodes, spleen, liver, kidney and lungs. The organism was identified as Mycobacterium avium by bacterial culture and polymerase chain reaction testing acid-fast bacilli/AMPLIFICATION/AVIUM/BACTERIAL CULTURE/CELL/CELLS/COMPLEX INFECTION/CULTURE/diarrhoea/disseminated/dog/FECES/IDENTIFICATION/INFECTION/INTRACELLULARE/LYMPH-NODES/macrophage/MACROPHAGES/MINIATURE SCHNAUZER/mycobacteria/mycobacteriosis/Mycobacterium/Mycobacterium avium/MYCOBACTERIUM-AVIUM/ORGANISM/ORGANISMS/polymerase chain reaction/POLYMERASE CHAIN-REACTION/POLYMERASE-CHAIN-REACTION/SUBSP PARATUBERCULOSIS/THERAPY/Tuberculosis

231 O'Grady, D., Flynn, O., Costello, E., Quigley, F., Cogarty, A., McGuirk, J., O'Rourke, J., Gibbons, N. (2000) Restriction fragment length polymorphism analysis of Mycobacterium avium isolates from animal and human sources

International Journal of Tuberculosis and Lung Disease, 4, 278-281 Restriction fragment length polymorphism (RFLP) analysis using probes derived from the insertion sequences IS901, IS1245 and IS1311, was carried out on Mycobacterium avium isolates obtained from 18 human patients, 44 deer, 14 pigs and five cattle in the Republic of Ireland. Forty-two of the cervine isolates and two of the bovine isolates contained IS901, while this insertion sequence was absent from all of the human and porcine isolates. RFLP analysis with IS901 probe differentiated the 44 field isolates which contained this element into three types. All of the IS901-positive isolates had a characteristic three-band IS1245 hybridisation pattern and a characteristic single-band IS1311 hybridisation pattern. The IS902-negative isolates exhibited highly polymorphic IS1245 and IS1311 hybridisation patterns which differentiated the human and porcine isolates into a wide diversity of strain types

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AVIUM/BOVINE/CATTLE/COMPLEX/deer/DNA fingerprinting/ELEMENT/FRAGMENT-LENGTH-POLYMORPHISM/insertion sequences/IS1245/IS1311/IS901/mycobacteria/Mycobacterium/Mycobacterium avium/MYCOBACTERIUM-AVIUM/paratuberculosis/PIGS/PROBES/RELATEDNESS/RESTRICTION/RFLP/SEQUENCE/STRAINS

232 Olsen, I., Reitan, L.J., Holstad, G., Wiker, H.G. (2000) Alkyl hydroperoxide reductases C and D are major antigens constitutively expressed by Mycobacterium avium subsp paratuberculosis

Infection and Immunity, 68, 801-808 Antigens characteristic for Mycobacterium avium subspecies paratuberculosis were identified by crossed immunoelectrophoresis (CIE) and by absorbing out cross-reactive antigens by using a polyclonal and polyvalent Mycobacterium avium subspecies avium antiserum. Two antigens were present in M. avium subsp. paratuberculosis and not detected in Mycobacterium avium subsp. avium. They were identified as antigens 17 and 20 in a CIE reference system for M. avium subsp. paratuberculosis antigens. Purified antigen 20 was identified as alkyl hydroperoxide reductase C (AhpC) while the N-terminal part of purified antigen 17 showed 80% homology with alkyl hydroperoxide reductase D (AhpD) of Mycobacterium tuberculosis. AhpC had a nonreduced mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 45 kDa and is probably a homodimer linked with disulfide bridges in its native form. AhpD had a mobility corresponding to 19 kDa. Monospecific rabbit antiserum against AhpC and AhpD reacted with 9 strains of M. avium subsp. paratuberculosis but not with 20 other mycobacterial strains except for a Mycobacterium gordonae strain, against which a weak cross-reactive band was produced. Goats experimentally infected with M. avium subsp: paratuberculosis had strong gamma interferon (IFN-gamma) responses toward both AhpC and AhpD, and they also had antibodies against AhpC. The ability of AhpC and AhpD to induce IFN-gamma production shows that these proteins potentially could be used in future vaccines or in diagnostic assays. These results further show that AhpC and AhpD are immunologically important proteins which are constitutively and highly expressed in M. avium subsp. paratuberculosis without the bacteria being submitted to oxidative stress and that the specificities of antigens can be a matter of different levels of protein expression in various species as well as distinct structural differences ABILITY/ANTIBODIES/ANTIBODY/ANTIGEN/antigens/ASSAY/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/AVIUM SUBSPECIES PARATUBERCULOSIS/BACTERIUM/BOVIS BCG/CROHNS-DISEASE/CULTURE FILTRATE/ESCHERICHIA-COLI/EXPRESSION/GAMMA/GAMMA-INTERFERON/goat/goats/IFN-gamma/IFN-GAMMA PRODUCTION/INTERFERON/interferon-gamma/JOHNES-DISEASE/MAJOR ANTIGENS/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-TUBERCULOSIS/OXIDATIVE-STRESS/PARA-TUBERCULOSIS/paratuberculosis/PROTEIN/PROTEINS/rabbit/RESPONSES/SALMONELLA-TYPHIMURIUM/SPECIFICITY/STRAINS/STRESS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS/Tuberculosis

233 Beard, P.M., Henderson, D., Daniels, M.J., Pirie, A., Buxton, D., Greig, A., Hutchings, M.R., McKendrick, I., Rhind, S., Stevenson, K., Sharp, J.M. (1999) Evidence of paratuberculosis in fox (Vulpes vulpes) and stoat (Mustela erminea) Veterinary Record, 145, 612-613 CATTLE/INFECTION/MYCOBACTERIUM-PARATUBERCULOSIS/PARA-TUBERCULOSIS/paratuberculosis/SHEEP

234 Moreira, A.R., Paolicchi, F., Morsella, C. , Zumarraga, M., Cataldi, A., Fabiana, B., Alicia, A., Piet, O., Dick, V., Isabel, R.M. (1999) Distribution of IS900 restriction fragment length polymorphism types among animal Mycobacterium avium subsp paratuberculosis isolates from Argentina and Europe Veterinary Microbiology, 70, 251-259 Sixty-one Mycobacterium avium subsp. paratuberculosis isolates from cattle and deer from the Buenos Aires province, an important livestock region in Argentina, were typed by restriction fragment length polymorphisms (RFLP) analysis based on IS900. Four different RFLP patterns (designated 'A', 'B', 'C' and 'E') were identified in BstEII digests of genomic DNA. The most frequently observed type, pattern 'A', was found in 46 isolates (75%). The second, pattern 'E', included 8 isolates (13%), while the third, pattern 'B', included 6 isolates (10%). Pattern 'C' was found for only one isolate. All of the deer isolates were classified as pattern 'A', while cattle isolates represented all four RFLP patterns. Twenty-one

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isolates representing the four different BstEII-RFLP patterns were digested with PstI. Twenty isolates showed identical PsrI-RFLP pattern. BstEII-RFLP patterns from Argentine cattle and deer were compared with patterns found in cattle, goat, deer, rabbit, and human isolates from Europe. The most common pattern in Argentina, pattern 'A', was identical to a less frequently occurring pattern R9 (C17) from Europe. The other Argentine patterns 'B','C' and 'E', were not found in the Europe. These results indicate that the distribution of M, avium subsp. paratuberculosis genotypes in the Buenos Aires province of Argentina is different from that found in Europe. (C) 1999 Elsevier Science B.V. All rights reserved ARGENTINA/AVIUM/AVIUM SUBSP PARATUBERCULOSIS/CATTLE/deer/DNA/DNA HYBRIDIZATION/ENDONUCLEASE ANALYSIS/Europe/FRAGMENT-LENGTH-POLYMORPHISM/goat/IDENTIFICATION/IS900/IS900 RFLP /livestock/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/Mycobacterium avium subsp paratuberculosis/Mycobacterium avium subsp./MYCOBACTERIUM-AVIUM/paratuberculosis/POLYMORPHISMS/rabbit/RESTRICTION/RFLP/SEQUENCE/STRAINS/SUBSP PARATUBERCULOSIS/SUBSP-PARATUBERCULOSIS

235 Dvorska, L., Havelkova, M., Bartos, M., Bartl, J., Pavlik, I. (1999) Insertion sequences of mycobacteria and their use in the study of epidemiology of mycobacterial infections

Veterinarni Medicina, 44, 233-251 The current studies on human and veterinary epidemiology have highly contributed to the detection of specific, small and mobile elements of the DNA - insertion sequences (IS). These sequences were: IS900 in Mycobacterium avium subspecies paratuberculosis, IS6110 in M. tuberculosis, and IS901 in M. avium complex. It has been ten years since the discovery of mycobacterial insertion Sequences. Accordingly, the aim of this abstract was to present a summary of various research aspects and utilization of insertion sequences in mycobacteriology. The discovery, composition and function of IS are described in the introduction part. Priority has been given to the insertion sequences found in M. avium complex: IS902, IS1245, IS1311, IS1110, IS1141, IS1613; in M. tuberculosis: IS1081; and in some opportunic pathogenic mycobacteria (M. smegmatis, M. kansasii and M. fortuitum). The last section of the article deals with the use of insertion sequences in epidemiological and epizootological studies of mycobacterial infections caused by M. tuberculosis, M. bovis, M. a. paratuberculosis, and M. avium complex AVIUM/AVIUM SUBSP PARATUBERCULOSIS/AVIUM SUBSPECIES PARATUBERCULOSIS/BOVINE TUBERCULOSIS/BOVIS/COMPLEX/DNA/DNA HYBRIDIZATION/DNA-typing/ELEMENT/Epidemiology/FARMED DEER/FRAGMENT-LENGTH-POLYMORPHISM/INFECTION/INFECTIONS/insertion sequence/insertion sequences/IS1245/IS1311/IS3 FAMILY/IS6110/IS900/IS901/mycobacteria/MYCOBACTERIAL INFECTIONS/Mycobacterium/ Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/POLYMERASE CHAIN-REACTION/RESTRICTION-ENDONUCLEASE ANALYSIS/SEQUENCE/SMEGMATIS/STRAINS/Tuberculosis/TUBERCULOSIS COMPLEX/veterinary epidemiology

236 Lugton, I.W. (1999) Mucosa-associated lymphoid tissues as sites for uptake, carriage and excretion of tubercle bacilli and other pathogenic mycobacteria

Immunology and Cell Biology, 77, 364-372 Pathogenic mycobacteria, including those that cause tuberculosis and paratuberculosis, cross mucosal barriers by endocytosis within mucosal lymphoepithelial sites. These entry sites commonly include oropharyngeal and nasopharyngeal tonsils and Peyer's patches. Bacilli discharged at the basolateral surfaces of engulfing epithelial M cells are taken up by professional antigen-presenting cells associated with T lymphocytes of the parafollicular area. Dendritic cells and macrophages in these sites allow mycobacterial replication, due to the permissive immunological environment in lymphoepithelial tissues. Abrogation of local delayed-type hypersensitivity reactions generally ensures continuing integrity and function of these tissues. Phagocytes containing intracellular mycobacteria disseminate infection to other parts of the body and also probably migrate back onto the mucosal surface to shed bacilli BOVINE TUBERCULOSIS/BRUSHTAIL POSSUMS/CELL/CELLS/DENDRITIC CELLS/ENTRY/ENVIRONMENT/EPITHELIAL-CELLS/EXPERIMENTALLY INFECTED CATTLE/IMMUNE-SYSTEM/INFECTION/leprosy/LYMPHOCYTES/M cells/M-CELLS/macrophage/MACROPHAGES/mucosa-associated lymphoid tissue/mycobacteria/Mycobacterium/NASAL-MUCOSA/NEW-ZEALAND/paratuberculosis/Peyer's patch/Peyer's patches/PEYERS-PATCHES/RED DEER/T lymphocytes/TISSUE/TISSUES/tonsil/TUBERCLE-BACILLI/Tuberculosis

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237 Rauzier, J., Gormley, E., Gutierrez, M.C., Kassa-Kelembho, E., Sandall, L.J., Dupont, C., Gicquel, B. , Murray, A. (1999) A novel polymorphic genetic locus in members of the Mycobacterium tuberculosis complex Microbiology-Uk, 145, 1695-1701 It has previously been shown that the P-AN promoter from Mycobacterium paratuberculosis can be used as a DNA probe to identify an RFLP between wild-type Mycobacterium bovis and the vaccine strain Mycobacterium bovis BCC. To investigate the genetic basis of this phenomenon, DNA fragments from a New Zealand M. bovis cattle strain and M. bovis BCG Pasteur, containing the P-AN-binding region, were isolated from gene libraries, sequenced and characterized. Sequence analysis and comparison with database sequences showed that the P-AN region in M. bovis, M. bovis BCG sind Mycobacterium tuberculosis is identical and shares 70% similarity to the P-AN sequence from M. paratuberculosis. The Shine-Dalgarno sequence and the -10 and -35 promoter regions are conserved between the different species. Analysis of the flanking sequences of the P-AN region revealed that less than 1 kb downstream of P-AN is a 2405 bp fragment that is present in M. bovis BCG but absent in the M: bovis wild-type strain. The distribution of the 2405 bp fragment in members of the M. tuberculosis complex was investigated and found to be present in 70 out of 70 M. tuberculosis strains, and 7 out of 7 M. bovis BCC daughter strains, 2 out of 2 Mycobacterium africanum strains, 2 out of 2 Mycobacterium microti strains and 7 out of 25 114. bovis strains. This is the first report of a genetic region of M. bovis BCG that is not universally present in M. bovis strains. The fragment does not appear to be present in any mycobacterial species outside the IM. tuberculosis complex. It does not possess any characteristics of known transposable elements and the flanking sequences do not have any obvious features to suggest a deletion mechanism. The genetic location of this region is close to the 3' end of the RD1 region of M. bovis and 114. tuberculosis. The polymorphic nature of this locus in 114. bovis will provide an additional genetic marker for strain differentiation BCG/BOVIS/BOVIS BCG/CATTLE/COMPLEX/DIFFERENTIATION/DNA/ELEMENT/Epidemiology/GENE/IDENTIFICATION/mycobacteria/Mycobacterium/Mycobacterium bovis/Mycobacterium paratuberculosis/Mycobacterium tuberculosis/MYCOBACTERIUM-BOVIS/MYCOBACTERIUM-PARATUBERCULOSIS/MYCOBACTERIUM-TUBERCULOSIS/nature/NEW-ZEALAND/paratuberculosis/PCR/polymorphic locus/RFLP/SEQUENCE/STRAINS/TB complex/Tuberculosis/TUBERCULOSIS COMPLEX

238 Greig, A., Stevenson, K., Henderson, D., Perez, V., Hughes, V., Pavlik, I., Hines, M.E., McKendrick, I., Sharp, J.M. (1999) Epidemiological study of paratuberculosis in wild rabbits in Scotland

Journal of Clinical Microbiology, 37, 1746-1751 A survey of 22 farms confirmed the presence of paratuberculosis in wild rabbits in Scotland. Regional differences were apparent in the prevalence of the disease in rabbits, with a significantly higher incidence occurring in the Tayside region. Statistical analysis showed a significant relationship between a previous history or current problem of paratuberculosis in cattle and the presence of paratuberculosis in rabbits on the farms. Molecular genetic typing techniques could not discriminate between selected rabbit and cattle isolates from the same or different farms, suggesting that the same strain may infect and cause disease in both species and that interspecies transmission may occur. The possibility of interspecies transmission and the involvement of wildlife in the epidemiology of paratuberculosis have important implications for the control of the disease BOVINE ORIGIN/CATTLE/control/DIFFERENTIATION/DISEASE/DNA/Epidemiology/EXPERIMENTAL-INFECTION/FARMS/GEL-ELECTROPHORESIS/MYCOBACTERIUM-PARATUBERCULOSIS/ORYCTOLAGUS-CUNICULUS/paratuberculosis/PREVALENCE/rabbit/RABBITS/RUMINANT PARA-TUBERCULOSIS/SCOTLAND/SOLID-PHASE DISPERSION/survey/Transmission/WILD/WILD RABBIT/WILD RABBITS/WILDLIFE

239 Stabel, J.R., Goff, J.P., Ackermann, M.R. (1998) Dietary calcium modulates Mycobacterium paratuberculosis infection in beige mice

Veterinary Immunology and Immunopathology, 66, 377-390 A 6-month study was conducted to evaluate the effects of feeding different levels of dietary calcium (Ca) on the persistence of Mycobacterium paratuberculosis infection using a mouse model. Beige mice, averaging 8 weeks of age, were randomly assigned to one of the following dietary treatments: 1) 0.02% Ca, 2) 0.15% Ca, 3) 0.45% Ca, and 4) 1.0% Ca. Mice were infected intraperitoneally with 10(8) CFU viable M. paratuberculosis for 1, 3, and 6 month periods. Plasma Ca levels was unaffected by dietary Ca (x = 7.3 mg/dl). Plasma levels of 1,25(OH)(2)D-3 was elevated significantly in 0.02% and 0.15% Ca groups compared to other treatments at the end of each period, with the highest levels observed for

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0.02% Ca mice and intermediate values for 0.15% Ca mice. One month after infection, numbers of viable M. paratuberculosis cultured from the spleen were significantly reduced for 0.15% Ca mice, whereas the number of bacteria isolated from the liver and mesenteric lymph node (MLN) were higher for the 0.02% Ca group. There were no differences in bacterial numbers in the ileum although they tended to be higher for the 0.02% Ca group. Three months after infection, bacterial numbers in the spleen, ileum, and MLN did not differ across treatments, however, significantly lower numbers were found in the liver of 1.0% Ca mice. Reduced bacterial counts were also observed in the liver of 0.15%, 0.45%, and 1.0% Ca mice after a 6-month infection period compared to the 0.02% Ca group, with the lowest numbers isolated from the 1.0% Ca mice. Numbers of viable bacteria cultured from the ileum and MLN after 6 months of infection were also significantly reduced in 1.0% Ca mice. These results suggest that Ca metabolism is an important modulator of M. paratuberculosis infection. (C) 1998 Published by Elsevier Science B.V. All rights reserved 1,25(OH)2D3/1,25-DIHYDROXYVITAMIN-D /ALVEOLAR MACROPHAGES/BACTERIUM/BEIGE MICE/calcium/DISEASE/HYPERCALCEMIA/INFECTION/METABOLISM/MICE/MODEL/MOUSE/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/Mycobacterium paratuberculosis infection /MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/PARATUBERCULOSIS INFECTION/PATIENT/SARCOIDOSIS/Tuberculosis/VITAMIN-D

240 de Lisle, G.W., Wilson, C.A., Yates, G.F., Wards, B.J., Collins, D.M. (1997) The post-mortem diagnosis of paratuberculosis in farmed deer

Fifth International Colloquium on Paratuberculosis - Meeting of the International Association for Paratuberculosis, 175-179 During the last 10 years, 186 Mycobacterium paratuberculosis-infected deer from 105 herds have been identified by culture. Tie overwhelming majority of these animals were discovered at meat inspection as having lesions resembling those caused by Mycobacterium bovis. Unlike cattle and sheep, many deer infected with M. paratuberculosis have necrotic lesions in mesenteric lymph nodes. In a small number of herds, multiple cases of clinical paratuberculosis were recorded in deer less than two years old. Two polymerase chain reaction (PCR) rests were used to identify M. paratuberculosis in post-mortem tissue samples. These tests were a rapid and practical means of establishing a post-mortem diagnosis of paratuberculosis in deer. PCR tests proved valuable in detecting M. paratuberculosis in tissues containing unculturable mycobacteria ANIMALS/BOVIS/CATTLE/ CULTURE/deer/DIAGNOSIS /FARMED DEER/HERD/LESIONS/LYMPH-NODES/mycobacteria/Mycobacterium/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/paratuberculosis/PCR/polymerase chain reaction/POLYMERASE-CHAIN-REACTION/SHEEP/TESTS/TISSUE/TISSUES

241 Sharp, J.M., Stevenson, K., Challans, J.A., Ramage, C., Hitchcock, D., Reid, H.W. (1997) Mycobacterial infections of free-living deer in Scotland Fifth International Colloquium on Paratuberculosis - Meeting of the International Association for Paratuberculosis, 180-182 A survey was conducted of mycobacterial infections in 1235 free-living deer from different regions of Scotland. Selected lymph nodes and spleen were pooled and examined by Ziehl-Neelson, culture and PCR. The overall prevalence of mycobacterial infection was 6.6% Mycobacterium paratuberculosis was found in 2.1% and IS901(+)M. avium in 3.1%. Na evidence of infection by M. bovis was forthcoming. The PCR procedure was the most sensitive technique, with a greatly reduced delay between sample preparation and result AVIUM/BOVIS/ CULTURE/deer/INFECTION /INFECTIONS/LYMPH-NODES/mycobacteria/MYCOBACTERIAL INFECTIONS/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/PCR/PREVALENCE/SCOTLAND/survey

242 Stehman, S.M., Rossiter, C.A., Shin, S.J., Chang, Y.F., Lein, D.H. (1997) Johne's disease in a fallow deer herd: Accuracy of fecal culture and results of environment sampling

Fifth International Colloquium on Paratuberculosis - Meeting of the International Association for Paratuberculosis, 183-189 In a farmed fallow deer herd, 51 of 52 deer sampled were determined to be infected with M. paratuberculosis by at least one of the following diagnostic methods; fecal culture, tissue culture, and/or histopathology. As a diagnostic test,fecal culture detected 32 of 51 infected animals cultured with an apparent sensitivity of 62%. Sensitivity of the fecal culture varied from a low of 45% in the most mildly infected deer to 91% in the most severely affected animals. The ileocecal junction was positive in 82%

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(range 71 to 100%) of cases. In this herd, the palatine tonsil was positive by culture in 88% of 17 deer tested. Culture isolates were confirmed to be M. paratuberculosis by a polymerase chain reaction technique using printers targeting the IS900 insertion sequence. Paratuberculosis infection and fecal shedding of organism were found in deer as young as 6 months of age. In clinical cases, disseminated infections were occasionally observed. Affected tissues included the intestine, liver; lymph nodes including those from the thorax, head and neck, and occasionally lung. Samples of pooled lymph nodes (head neck, thoracic, and mesenteric LN) from 31 deer were submitted to the National Veterinary Service Laboratory for M. bovis culture, M. bovis was not isolated. However, 15 of the 31 deer sampled were positive for M. paratuberculosis by the culture methods used including infections in 3 deer which were not detected by routine methods used for M. paratuberculosis isolation. Paddocks, on the affected farm, were divided into equal quadrants and the environment was sampled to determine the level of environmental contamination. A total of 43 areas were sampled including the pond within the enclosure. Of the pasture quadrants sampled 26 of 36 (72%) were positive for M. paratuberculosis by culture. The inflow sediment from the pond was also positive. Intact fecal pellets were Sound on re-examination of the paddock 13 months after all deer were removed. The follow-up environmental samples were negative for M. paratuberculosis by routine culture ANIMALS/BOVIS/CULTURE/CULTURE METHODS/deer/diagnostic methods/DISEASE/disseminated/enclosure/ENVIRONMENT/FALLOW DEER/FECAL CULTURE/HERD/INFECTION/INFECTIONS/insertion sequence/IS900/Johne's disease/LYMPH-NODES/ORGANISM/paratuberculosis/PARATUBERCULOSIS INFECTION/pasture/PELLETS/polymerase chain reaction/POLYMERASE-CHAIN-REACTION/SENSITIVITY/SEQUENCE/TISSUE/tissue culture/TISSUES/tonsil

243 Greig, A., Stevenson, K., Perez, V., Pirie, A.A., Grant, J.M., Sharp, J.M. (1997) Paratuberculosis in wild rabbits (Oryctolagus cuniculus)

Fifth International Colloquium on Paratuberculosis - Meeting of the International Association for Paratuberculosis, 190-195 A preliminary survey of wild rabbits on four farms in the Tayside Region of Scotland where Johnes disease has an above average incidence revealed that 67 per cent of healthy adult rabbits were infected with Mycobacterium avium subsp. paratuberculosis. Gross changes in the intestine were minimal but histologically the changes int he mesenteric lymph nodes and intestines were consistent with those seen with paratuberculosis in ruminants. A more extensive rabbit survey covering 22 animal units scattered throughout Scotland revealed that the findings of the preliminary survey were largely confined to the Tayside Region. These surveys raise the possibility that wildlife could be involved in the epidemiology of paratuberculosis which has important implications for the control of this disease AVIUM/control/DISEASE/Epidemiology/FARMS/JOHNES DISEASE/JOHNES-DISEASE/LYMPH-NODES/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subsp/MYCOBACTERIUM-AVIUM/ORYCTOLAGUS-CUNICULUS/paratuberculosis/rabbit/RABBITS/RUMINANTS/SCOTLAND/survey/WILD/WILD RABBIT/WILD RABBITS/WILDLIFE

244 Burrells, C., Palmarini, G., Porter, J., Stevenson, K., Sharp, J.M. (1997) Experimental infection of red deer with Mycobacterium avium subspecies paratuberculosis and IS901(+) Mycobacterium avium

Fifth International Colloquium on Paratuberculosis - Meeting of the International Association for Paratuberculosis, 196-201 Experiments were conducted to investigate reports that deer are more susceptible than sheep, cattle to goats to infections with M. paratuberculosis and M. avium (Nyange, 1990). Groups of 4 red deer calves (Cervus elaphus) were infected orally with either IS901, M. avium or M. paratuberculosis. Two calves served as non-infected controls. Regular examinations were made of blood cell-mediated immune (CMI) reactions, antibody responses and fecal excretion of mycobacteria throughout the 41 week term of the experiment. Clinical illness was observed only in deer inoculated with M. avium and fecal excretion of mycobacteria was demonstrated between weeks 3 and 25 post-inoculation. In contrast, fecal excretion of M. paratuberculosis was detected much less frequently between weeks 13 and 21 post-inoculation. Differences in immune responses to the two infections were obvious. Essentially, the group of M. paratuberculosis-infected deer exhibited low antibody and transient CMI reactivity, whereas both responses were evident early in M. avium-infected deer and were sustained throughout the 41-week period of the experiment ANTIBODIES/ANTIBODY/ANTIBODY-RESPONSES/AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/CALVES/CATTLE/Cervus elaphus/CERVUS-ELAPHUS/control/deer/experimental infection/EXPERIMENTAL-INFECTION/goat/goats/immune responses/IMMUNE-RESPONSE/IMMUNE-

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RESPONSES/INFECTION/INFECTIONS/IS901/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/paratuberculosis/RED DEER/RESPONSES/SHEEP

245 Naser, S.A., Gillespie, R.F., Naser, N.A., El-Zaatari, F.A.K. (1998) Effect of IS900 gene of Mycobacterium paratuberculosis on Mycobacterium smegmatis

Current Microbiology, 37, 373-379 Mycobacterium paratuberculosis is mycobactin dependent and contains multiple copies of the IS900 gene that encodes for p43 (46.5K protein). The correlation between the two characteristics has been investigated. A3.2-kb BamHI fragment from M. paratuberculosis containing the 1.451 kb IS900 gene was cloned in Escherichia coli and Mycobacterium smegmatis with pcDNA II and pNEZ6.3 plasmids, respectively. Surprisingly, the recombinant M, smegmatis grew poorly and slower in 7H9 broth supplemented with OADC (12 day) compared with M. smegmatis wild type or to M. smegmatis transformed with pNEZ6.3 (2 day). The growth rate of the recombinant M. smegmatis was restored by the addition of 2.4 mu M ferric mycobactin J to the media. There was no effect on the growth rate of E. coli recombinants. Western blot analysis with p43-specific anti-peptide antibodies resulted in the expression of 46.5K and a cleaved form of 33.5K protein bands in the recombinant E. coli. There was no expression in the recombinant M. smegmatis. A lower expression of 33.5K protein band was detected in the native M. paratuberculosis protein. The nucleotide sequence of the 3.2-kb fragment confirmed the presence of p43-encoded ORE There was no additional encoding sequence in the fragment. This suggests that the IS900 gene and/or its encoding products are involved in mycobactin dependency and possibly the slow growth rate of M. paratuberculosis ANTIBODIES/ANTIBODY/CROHNS-DISEASE/ESCHERICHIA-COLI/EXPRESSION/GENE/GROWTH/IRON TRANSPORT/IS900/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/Mycobacterium smegmatis/MYCOBACTERIUM-PARATUBERCULOSIS/ NUCLEOTIDE-SEQUENCE/paratuberculosis/PROTEIN/SEQUENCE/SMEGMATIS/WILD

246 Manning, E.J.B., Steinberg, H., Rossow, K., Ruth, G.R., Collins, M.T. (1998) Epizootic of paratuberculosis in farmed elk

Journal of the American Veterinary Medical Association, 213, 1320-+ After multiple cases of chronic diarrhea and weight loss in a farmed elk herd, 3 yearlings and 1 adult elk with similar clinical signs were euthanatized and necropsied. Gross and histologic evidence of paratuberculosis were found in the yearlings. Evidence of serum antibody to Mycobacterium paratuberculosis was detected in the 2 elk with the most disseminated infection. Acid-fast organisms were isolated from multiple organs in all It elk and identified as M paratuberculosis by DNA probe. Thirty-five percent of 1 calf crop (n = 31) died or were euthanatized because of paratuberculosis before they were 2 years old. The organism was believed to have been spread by standing water that was used by calves as a wallow and a source of drinking water. The water was believed to have been contaminated by an infected adult female elk introduced to the herd just before calving season ANTIBODIES/ANTIBODY/CALVES/CERVUS-ELAPHUS/COWS/Diarrhea/disseminated/DNA/ELK/HERD/INFECTION/JOHNES-DISEASE/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/ORGANISM/ORGANISMS/paratuberculosis/PATHOLOGY/RED DEER/SERUM ANTIBODIES/Tuberculosis/WATER

247 Hines, M.E., Frazier, K.S., Baldwin, C.A., Cole, J.R., Sangster, L.T. (1998) Efficacy of vaccination for Mycobacterium avium with whole cell and subunit vaccines in experimentally infected swine

Veterinary Microbiology, 63, 49-59 Mycobacterium avium infections are a common problem in large swine producing states and cause substantial financial losses at slaughter inspection due to carcass condemnation. once the infection is established in a swine herd it is difficult to effectively prevent or eliminate the disease. Previous mouse studies in our laboratory suggested that Macrophage Inhibitory Factor-A3 (MIF-A3) is a virulence factor of M. avium and potential antigen for vaccine development. In this study we evaluated the efficacy of a killed 'whole cell' M. avium serovar 2 bacterin and conjugated MIF-A3 subunit vaccine in preventing infection and disease in swine challenged with virulent M. avium serovar 2. Gross and microscopic pathology, acid-fast staining, culture and polymerase chain reaction (PCR) for the M. avium specific insertion sequence IS902 were utilized in evaluation. Results indicated that neither vaccine prevented infection in challenged animals; however, a 47% reduction in severity of disease was found in swine vaccinated with the 'whole cell' M, avium serovar 2 bacterin. Reduction in severity of disease was not detected in animals vaccinated with the subunit MIF-A3 vaccine. (C) 1998 Elsevier Science B.V. All

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rights reserved ANIMALS/ANTIGEN/AVIUM/CELL/CHAIN-REACTION DETECTION/COMPLEX/CULTURE/DISEASE/gross and microscopic pathology/HERD/INFECTION/INFECTIONS/insertion sequence/macrophage/macrophage inhibitory factor-A3/MACROPHAGES/MOUSE/mycobacteria/mycobacteriosis/Mycobacterium/Mycobacterium avium/MYCOBACTERIUM-AVIUM/paratuberculosis/PATHOLOGY/PCR /pig-bacteria/polymerase chain reaction/POLYMERASE-CHAIN-REACTION/SEQUENCE/SUBSP SILVATICUM/SWINE/swine tuberculosis/VACCINATION/VIRULENCE

248 Grant, I.R., Ball, H.J., Rowe, M.T. (1998) Isolation of Mycobacterium paratuberculosis from milk by immunomagnetic separation Applied and Environmental Microbiology, 64, 3153 -3158 An immunomagnetic separation (IMS) technique was developed to facilitate selective isolation of Mycobacterium paratuberculosis cells from milk Rabbit polyclonal antibodies against radiation-killed intact M. paratuberculosis cells were produced and used to coat sheep anti-rabbit immunoglobulin G (IgG) type M-280 Dyna-beads. The rabbit anti-ill. paratuberculosis IgG-coated beads (IMB) reacted strongly with laboratory strains of M. paratuberculosis as determined by slide agglutination, and microscopic examination confirmed that M. paratberculosis cells attached to the IMB. The IMB were found to have a maximum binding capacity of 10(4) to 10(5) CFU of M paratuberculosis. Studies showed that IMS selectively recovered M paratuberculosis from inoculated milk containing as few as 10 CFU of M. paratuberculosis per mi when 10 mu l of IMB (ca. 10(6) beads) was added to 1 mi of milk and the preparation was incubated for 30 min at room temperature with gentle agitation. Larger volumes of milk (10 and 50 mi) were centrifuged and resuspended in 1 mi of phosphate-buffered saline-0.05% Tween 20 prior to IMS in order to increase the sensitivity of the method. Currently, primary isolation of M paratuberculosis from a milk sample relies on chemical decontamination, followed by culturing on Herrold's egg yolk medium, which must be incubated at 37 degrees C for up to 18 weeks. The potential value of our IMS method is as an aid for rapid detection of ill. paratuberculosis in milk when it is used in conjunction with end point detection methods, such as IS900 PCR or an enzyme-linked immunosorbent assay ANTIBODIES/ANTIBODY/ASSAY/CELL/CELLS/CHAIN-REACTION/COWS MILK/CROHNS-DISEASE/DECONTAMINATION/detection methods/ENRICHMENT/enzyme-linked immunosorbent assay/FOODS/IMMUNOGLOBULIN/IS900/MILK/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/PCR/polyclonal antibody/rabbit/RAPID DETECTION/SALMONELLA/SAMPLES/SENSITIVITY/SHEEP/STRAINS

249 Karesh, W.B., Uhart, M.M., Dierenfeld, E.S., Braselton, W.E., Torres, A. , House, C., Puche, H., Cook, R.A. (1998) Health evaluation of free-ranging guanaco (Lama guanicoe) Journal of Zoo and Wildlife Medicine, 29, 134-141 Twenty free-ranging guanaco (Lama guanicoe) in Chubut Province, Argentina, were immobilized for health evaluations. All but two animals appeared to be in good condition. Hematology, serum chemistry, and vitamin and mineral levels were measured, and feces were evaluated for parasites. Serology tests included bluetongue, brucellosis, bovine respiratory syncitial virus, bovine viral diarrhea/mucosal disease, equine herpesvirus 1, infectious bovine rhinotracheitis, Johne's disease (Mycobacterium paratuberculosis), foot and mouth disease, leptospirosis (17 serovars), para influenza-3, and vesicular stomatitis. Blood samples from 20 domestic sheep (Ovis aries) maintained in the same reserve with the guanaco were also collected at the same time for serology tests. No guanaco had positive serologic tests. Sheep were found to have antibody titers to bovine respiratory syncytial virus, Johne's disease, leptospirosis, and parainfluenza-3. There was no apparent difference in external appearance or condition, or statistical difference in blood test values, between the animals that were positive or negative for parasite ova ALPHA-TOCOPHEROL/ANIMALS/ANTIBODIES/ANTIBODY/APPEARANCE/ARGENTINA/BOVINE/DISEASE/DISEASES/DOMESTIC SHEEP/FECES/flumazenil/guanaco/hematology/immobilization/Johne's disease/Lama guanicoe/LEPTOSPIRA/Minnesota/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/NATIONAL-PARK/parasites/paratuberculosis/PREVALENCE/SAMPLES/SEROLOGICAL REACTIONS/Serology/SHEEP/TESTS/VIRUS/white-tailed deer

250 Mackintosh, C.G. (1998) Deer health and disease

Acta Veterinaria Hungarica, 46, 381-394

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This paper describes the most significant diseases of farmed deer which have emerged over the last 30 or so years. It describes their characteristic signs, how control measures have evolved, their current status and gives an indication of future diagnostic and control measures. Overall, it shows that wild deer brought into a farming environment have developed some of the production limiting diseases which affect sheep and cattle, such as parasitism and trace element deficiencies. In addition, farmed deer are susceptible to potentially fatal diseases such as tuberculosis, malignant catarrhal fever and yersiniosis. A disease which has recently emerged and has the potential to be more serious than any of the above is Johne's disease. In North America, Chronic Wasting Disease occurs in captive and wild deer in only two states but has the potential to be a serious threat to wild and farmed deer elsewhere if it spreads. The zoonotic risks of diseases affecting deer are discussed, as well as stress, welfare and deer restraint. The productivity of farmed deer can be maximised by using a well-designed deer health programme integrated with good management and feeding CATTLE/Chronic Wasting Disease/control/copper deficiency/deer/DISEASE/DISEASES/ELEMENT/ENVIRONMENT/FARMED DEER/health programmes/Johne's disease/MALIGNANT CATARRHAL FEVER/parasitism/restraint/risk/SHEEP/STRESS/Tuberculosis/WILD/yersiniosis/zoonoses

251 Wahlstrom, H., Englund, L., Carpenter, T., Emanuelson, U., Engvall, A., Vagsholm, I. (1998) A Reed-Frost model of the spread of tuberculosis within seven Swedish extensive farmed fallow deer herds

Preventive Veterinary Medicine, 35, 181-193 The within-herd transmission of tuberculosis, after introduction of infection, was evaluated in seven Swedish herds of farmed fallow deer. The evaluation was based on a subset of data obtained from a previous epidemiological investigation, comprising 13 tuberculosis-infected deer herds, with the purpose of tracing the source of infection. A computer spreadsheet model based on the Reed-Frost method was developed to estimate the number of new infections. For each herd, a k-value (the number of effective contacts made by an individual during a time period) was estimated through fitting the model to the observed incidence in each herd. We concluded that, despite the relatively short observation periods and uncertain tuberculosis incidence estimates for the observed herds, the k's obtained could be used to quantify the estimated spread of tuberculosis in extensive deer herds in Sweden. (C) 1998 Elsevier Science B.V BOVINE TUBERCULOSIS/CATTLE/deer/Epidemiology/FALLOW DEER/HERD/INFECTION/INFECTIONS/MELES-MELES POPULATIONS/MODEL/modelling/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/paratuberculosis/SIMULATION-MODEL/SOUTHWEST ENGLAND/Transmission/Tuberculosis/zoonoses

252 Faldyna, M. (1998) Differential antigens of leukocytes of dog, cat, horse, pig and ruminants

Veterinarni Medicina, 43, 55-66 Differential antigens or clusters of differentiation (CD antigens) are molecules on the surface of cells, which are bound with immunity system, having different functions. Two important diagnostic tools - cell fusion techniques and flow cytometry - helped to develope the knowledge about these antigens. Knowledge of CD antigens and subpopulations of animal leukocytes is employed for the study of diseases' pathogenesis. Results from literature about the distribution of CD antigens of leukocytes of dog, cat, horse, pig and ruminants are summarized in this paper ADHESION PROTEIN-DEFICIENCY/ANTIGEN/antigens/BOVINE PARATUBERCULOSIS/BRONCHOALVEOLAR LAVAGE FLUID/cat/CELL/CELLS/CELLULAR IMMUNOLOGY/DIFFERENTIATION/DISEASE/DISEASES/dog/FELINE IMMUNODEFICIENCY VIRUS/FIRST INTERNATIONAL WORKSHOP /horse/IMMUNITY/LYMPHOCYTE SUBSET ALTERATIONS/MONOCLONAL-ANTIBODIES/PATHOGENESIS/PERIPHERAL-BLOOD/pig/PORCINE T-LYMPHOCYTES/ RUMINANTS/SUBPOPULATIONS

253 Overnes, G., Matre, T., Sivertsen, T., Larsen, H.J.S., Langseth, W., Reitan, L.J., Jansen, J.H. (1997) Effects of diets with graded levels of naturally deoxynivalenol-contaminated oats on immune response in growing pigs

Journal of Veterinary Medicine Series A-Physiology Pathology Clinical Medicine, 44, 539-550 A trial was conducted to evaluate the effect of including different levels of deoxynivalenol (DON)-contaminated oars in the complete diets of growing pigs on immune response and performance. The diets contained 0.6, 1.8 and 4.7 mg DON/kg, and both restricted and ad libitum feeding were used.

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Performance was recorded as weight gain, feed intake, efficiency of feed utilization and carcass quality. Immune response parameters recorded included primary and secondary antibody titres after injections of five different antigens: Human serum albumin (HSA), sheep red blood cells (SRBC), paratuberculosis vaccine (MPT), tetanus toroid (TT) and diphteria toroid (DT). A johnin test was also performed. Lymphocyte stimulation response was measured with three different mitogens (PWM, ConA and PHA). A significant, DON dose-dependent reduction in secondary antibody response to tetanus toroid was observed. A slightly higher mitogen response after PHA stimulation in lymphocytes from the medium and high DON groups compared to the low DON group after 9 weeks was considered inconclusive. No other indication of dose-dependent immune response inhibition or stimulation was found. Significantly reduced feed intake with increased levels of DON was observed in groups fed restricted rations according to weight, but not in animals fed an libitum ANIMALS/ANTIBODIES/ANTIBODY/ANTIGEN/antigens/B-CELLS/B6C3F1 MOUSE/BLOOD PARAMETERS/CELL/CELLS/EXPOSURE/FUSARIUM MYCOTOXINS/IMMUNE-RESPONSE/INDUCED IGA NEPHROPATHY/INHIBITION/intake/LYMPHOCYTES/OCHRATOXIN-A/paratuberculosis/pig/PIGS/SHEEP/STIMULATION/TRICHOTHECENE VOMITOXIN/VOMITOXIN DEOXYNIVALENOL/ZEARALENONE

254 Cetinkaya, B., Erdogan, H.M., Morgan, K.L. (1997) Relationships between the presence of Johne's disease and farm and management factors in dairy cattle in England

Preventive Veterinary Medicine, 32, 253-266 The data collected by a postal questionnaire sent to 3772 randomly selected dairy farmers in England and the border regions in Wales were used to estimate the relationships between the presence of clinical Johne's disease and farm and management factors associated with that disease. Two binary outcomes (case reported in 1993, case reported in 1994) and 27 predictor variables were considered. Only two variables were consistently and significantly associated with clinical disease in multivariable analysis. Farms on which Channel Island breeds were predominant were associated with an increased risk of reporting disease (odds ratios (ORs) ranged from 10.9 to 12.9), The presence of farmed deer on the farm also increased the risk of reporting disease (ORs ranged from 15.2 to 209.3). There were other significant but inconsistent associations involving the source of replacements, age of first-offering hay, type of concentrate feed to calves, and calving in individual pens when the cows were at grass. Since Johne's disease is predominantly subclinical, these contributing factors may play important roles in switching subclinical infection to overt disease. (C) 1997 Elsevier Science B.V CALVES/CATTLE/cattle microbiological diseases/cow/COWS/CROHNS/dairy cattle/DAIRY-CATTLE/deer/DISEASE/ENGLAND/farm and management variables/FARMED DEER/FARMS/HERD/INFECTION/Johne's disease/logistic regression/paratuberculosis/PREVALENCE/RED DEER/risk/role/RUMINANT PARA-TUBERCULOSIS/SOUTH-WEST ENGLAND

255 Collins, D.M., Cavaignac, S., deLisle, G.W. (1997) Use of four DNA insertion sequences to characterize strains of the Mycobacterium avium complex isolated from animals Molecular and Cellular Probes, 11, 373-380 The Mycobacterium avium complex (MAC) includes the closely related species M. avium, M. intracellulare and M. paratuberculosis. The insertion elements IS900, IS901, lS1245 and IS1311 were used as DNA probes to characterize by restriction fragment polymorphisms (RFLPs) eight reference strains, three animal isolates of M. paratuberculosis from outside New Zealand and 61 selected New Zealand MAC isolates from cattle, deer, pigs, sheep and humans. IS900 was found only in strains of M. paratuberculosis. All MAC strains contained IS1311 and the RFLPs associated with this insertion element divided M. paratuberculosis strains into the same groups as IS900 RFLPs. Except for M. paratuberculosis, all MAC strains contained IS 1245 and the majority of those from lesions in cattle, deer and pigs also contained IS901. All animal strains containing IS901 had the same RFLPs with IS901, IS1245 and lS1311. In three cases, these apparently identical strains could be differentiated by restriction fragment analysis with BstEII. IS901 was not present in four human isolates or in isolates from deer without lesions. These results indicate that a very closely related group of strains causes the majority of non-paratuberculosis MAC lesions in animals in New Zealand. (C) 1997 Academic Press Limited ANIMALS/AVIUM/CATTLE/COMPLEX/deer/DNA/DNA PROBES/ELEMENT/ELEMENT IS900/FARMED DEER/HYBRIDIZATION/IDENTIFICATION/INSERTION ELEMENT/insertion sequence/insertion sequences/INTRACELLULARE/IS1245/IS1311/IS900/IS900 RFLP/IS901/LESIONS/MOLECULAR-LEVEL/mycobacteria/ Mycobacterium/Mycobacterium avium/ Mycobacterium avium complex/Mycobacterium paratuberculosis/MYCOBACTERIUM-AVIUM/NEW-

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ZEALAND/paratuberculosis/pig/PIGS/POLYMERASE CHAIN-REACTION/POLYMORPHISMS/PROBES/RESTRICTION/RESTRICTION ENDONUCLEASE ANALYSIS/RFLP/SEQUENCE/SHEEP/STRAINS/SUBSP SILVATICUM

256 Cetinkaya, B., Erdogan, H.M., Morgan, K.L. (1997) Risk factors for bovine paratuberculosis .1. The univariate analysis of risk factors for bovine paratuberculosis

Turkish Journal of Veterinary & Animal Sciences, 21, 297-302 Data collected by a postal questionnaire sent to 3772 randomly-selected dairy farmers in England and the border regions in Wales were used to estimate the univariate relationships between the presence of clinical paratuberculosis and farm and management factors associated with that disease. Eight variables were determined to be risk factors for clinical disease. These were carving place, group size of the calves, the length of keeping calves in the groups, type of concentrate feeds to calves, breed, source of replacements, the presence of farmed deer and region. Since paratuberculosis is predominantly subclinical, these contributing factors may play important roles in switching subclinical infection to overt disease BOVINE /BOVINE PARATUBERCULOSIS/BOVINE-PARATUBERCULOSIS/CALVES/CATTLE/CROHNS/deer/DISEASE/ENGLAND/farm and management variables/FARMED DEER/FARMED RED DEER/INFECTION/JOHNES-DISEASE/paratuberculosis/risk/RISK-FACTORS/role/RUMINANT PARA-TUBERCULOSIS/SOUTH-WEST ENGLAND/univariate analysis

257 Cetinkaya, B., Erdogan, H.M., Morgan, K.L. (1997) Risk factors for bovine paratuberculosis .2. The multiple analysis of risk factors for bovine paratuberculosis

Turkish Journal of Veterinary & Animal Sciences, 21, 303-306 Farm and management variables that had a P value of 0.25 or less and those that were considered biologically important were offered to logistic regression models. Two different models were constructed : Model I included all the variables and replacement rate was left out of Model II. Channel Island breeds (Jersey and Guernsey), the purchase of replacements privately, the presence of farmed deer and calving in groups were determined to be risk factors for bovine paratuberculosis in logistic regression BOVINE/BOVINE PARATUBERCULOSIS/BOVINE-PARATUBERCULOSIS/CATTLE/deer/farm and management variables/FARMED DEER/logistic regression/MODEL/paratuberculosis/risk/RISK-FACTORS/RUMINANT PARA-TUBERCULOSIS/TUBERCULOSIS JOHNES DISEASE

258 Caceres, N.E., Harris, N.B., Wellehan, J.F., Feng, Z.Y., Kapur, V., Barletta, R.G. (1997) Overexpression of the D-alanine racemase gene confers resistance to D-cycloserine in Mycobacterium smegmatis Journal of Bacteriology, 179, 5046-5055 D-Cycloserine is an effective second-line drug against Mycobacterium avium and Mycobacterium tuberculosis. To analyze the genetic determinants of D-cycloserine resistance in mycobacteria, a library of a resistant Mycobacterium smegmatis mutant was constructed, A resistant clone harboring a recombinant plasmid with a 3.1-kb insert that contained the glutamate decarboxylase (gadA) and D-alanine racemase (alrA) genes was identified, Subcloning experiments demonstrated that alrA was necessary and sufficient to confer a D-cycloserine resistance phenotype. The D-alanine racemase activities of wild-type and recombinant M. smegmatis strains were inhibited by D-cycloserine in a concentration-dependent manner, The D-cycloserine resistance phenotype in the recombinant clone was due to the overexpression of the wild-type alrA gene in a multicopy vector. Analysis of a spontaneous resistant mutant also demonstrated overproduction of wild-type AlrA enzyme. Nucleotide sequence analysis of the overproducing mutant revealed a single transversion (G-->T) at the alrA promoter, which resulted in elevated beta-galactosidase reporter gene expression, Furthermore, transformants of Mycobacterium intracellulare and Mycobacterium bovis BCG carrying the M. smegmatis wild-type alrA gene in a multicopy vector were resistant to D-cycloserine, suggesting that AlrA overproduction is a potential mechanism of D-cycloserine resistance in clinical isolates of M. tuberculosis and other pathogenic mycobacteria. In conclusion, these results show that one of the mechanisms of D-cycloserine resistance in M. smegmatis involves the overexpression of the alrA gene due to a promoter-up mutation AVIUM/BACILLUS-STEAROTHERMOPHILUS/BCG/BOVIS/BOVIS BCG/CELL-WALL/DNA-SEQUENCE/ESCHERICHIA-COLI GENOME/EXPRESSION/GENE/gene expression/GENE-EXPRESSION/GENES/INTRACELLULARE/MUTANT/MUTATION/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium bovis/Mycobacterium intracellulare/Mycobacterium smegmatis/Mycobacterium tuberculosis/MYCOBACTERIUM-AVIUM /MYCOBACTERIUM-

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BOVIS/MYCOBACTERIUM-TUBERCULOSIS/NUCLEOTIDE-SEQUENCE/paratuberculosis/PROTEIN-SEQUENCE DETERMINATION/RESISTANCE/SALMONELLA-TYPHIMURIUM/SEQUENCE/SMEGMATIS/STRAINS/TRANSFORMATION/Tuberculosis/vector

259 Rhyan, J.C., Aune, K., Ewalt, D.R., Marquardt, J., Mertins, J.W., Payeur, J.B., Saari, D.A., Schladweiler, P., Sheehan, E.J., Worley, D. (1997) Survey of free-ranging elk from Wyoming and Montana for selected pathogens

Journal of Wildlife Diseases, 33, 290-298 From December 1991 through January 1995, a disease survey was conducted on herds of free-ranging, hunter-killed elk (Cervus elaphus nelsoni) From three areas in proximity to Yellowstone National Park. (YNP), Wyoming (USA), after tuberculosis caused by Mycobacterium bovis was discovered in a captive herd of elk in the area. Complete or partial sets of specimens from 289 elk collected between December 1991 and January 1993 were examined histologically; no mycobacterial lesions were observed. Lesions of tuberculosis were not detected in tonsils or lymph nodes of the head from an additional 99 hunter-killed, adult elk from one area (area 2) collected in January 1995. Neither M. bovis nor M. paratuberculosis were isolated from any of the specimens cultured. Antibodies to Brucella abortus mere detected in serum samples from 0%, 1%, and 1% of elk from three areas sampled (areas 1, 2, and 3), respectively. Brucella abortus biovar 1 was isolated from multiple tissues from one seropositive animal from area 3. Larvae with morphology consistent with Dictyocaulus sp. were found in 12%, 14%, and 0% of fecal specimens tested from areas 1, 2, anti 3, respectively. Pasteurella multocida and Actinomyces pyogenes were isolated from a lung with purulent bronchopneumonia and abscesses ANTIBODIES/ANTIBODY/BOVINE TUBERCULOSIS/BOVIS/Brucella/BRUCELLA-ABORTUS/brucellosis/Cervus elaphus/CERVUS-ELAPHUS/CERVUS-ELAPHUS-NELSONI/deer/DISEASE/ELK/HERD/INFECTION/LESIONS/LYMPH-NODES/Montana/mycobacteria/Mycobacterium/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/NATIONAL-PARK/Neolipoptena ferrisi/paratuberculosis/SAMPLES/survey/TISSUE/TISSUES/tonsil/Tuberculosis/UNITED-STATES/Yellowstone National Park/YELLOWSTONE-NATIONAL-PARK

260 Greig, A., Stevenson, K., Perez, V., Pirie, A.A., Grant, J.M., Sharp, J.M. (1997) Paratuberculosis in wild rabbits (Oryctolagus cuniculus) Veterinary Record, 140, 141-143 A survey of wild rabbits in Tayside, Scotland revealed that 67 per cent were infected with Mycobacterium avium subspecies paratuberculosis. In general, the infected rabbits had histopathological changes within the lymph nodes and intestines which were consistent with the changes due to paratuberculosis in ruminants, The survey raises the possibility that rabbits and other wildlife may be involved in the epidemiology of paratuberculosis, a possibility which has important implications for the control of the disease AVIUM/AVIUM SUBSPECIES PARATUBERCULOSIS/BOVINE ORIGIN/control/deer/DISEASE/Epidemiology/EXPERIMENTAL-INFECTION/LESIONS/LYMPH-NODES/MICE/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium avium subspecies paratuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-PARATUBERCULOSIS/ORYCTOLAGUS-CUNICULUS/PARA-TUBERCULOSIS/paratuberculosis/rabbit/RABBITS/RUMINANTS/SCOTLAND/survey/WILD/WILD RABBIT/WILD RABBITS/WILDLIFE

261 Whittington, R.J., Saunders, V.F., Egerton, J.R. (1996) Antigenic cross-reactions between the causative agent of ovine footrot, Dichelobacter nodosus, and other bacteria

Small Ruminant Research, 22, 55-67 Lack of test specificity for the causative agent of ovine footrot, Dichelobacter nodosus, currently precludes routine application of serological tests. To investigate this problem antigenic cross-reactivity between D. nodosus and other bacteria was studied using double immunodiffusion and enzyme-linked immunosorbent assay (ELISA). Sera from sheep and rabbits immunised with D. nodosus reacted with other taxa but particularly Yersinia pseudotuberculosis, Y. enterocolitica, Cardiobacterium hominis, Suttonella indologenes, Corynebacterium pseudotuberculosis, Brucella ovis, Proteus vulgaris and Histophilus ovis. Sheep sera were then adsorbed with bacterial antigens and reacted in ELISA with cell envelope antigens of D. nodosus to determine whether non-specific reactivity could be reduced, C. pseudotuberculosis and the antigenically related organisms Nocardia asteroides and Mycobacterium phlei, as well as Bacteroides ruminicola and several other taxa reduced the signal in the ELISA, Antibodies reactive with many taxa increased with age of sheep, Sheep sera were more cross reactive than rabbit sera and distinguished relatively poorly between the antigens of different bacteria.

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Unfortunately the simple expedient of immunoadsorption was not effective in reducing non-specific reactions to the extent required for practical application of a serological test for ovine footrot ANTIBODIES/ANTIBODY/ANTIGEN/antigens/ASSAY/BACTERIUM/BACTEROIDES-NODOSUS/Brucella/CATTLE/CELL/COMMON ANTIGEN/cross-reactions/CROSS-REACTIVITY/Dichelobacter nodosus/ELISA/ENVELOPE/enzyme-linked immunosorbent assay/ESCHERICHIA-COLI/footrot/JOHNES DISEASE/LINKED-IMMUNOSORBENT-ASSAY/LYME-DISEASE/mycobacteria/Mycobacterium/Nocardia/ORGANISM/ORGANISMS/OVINE/PROTEIN/rabbit/RABBITS/SEROLOGICAL DIAGNOSIS/SEROLOGICAL TESTS/Serology/SHEEP/SPECIFICITY/TESTS/YERSINIA-PSEUDOTUBERCULOSIS

262 Heatley, J.J., Boothe, D.M., Jensen, J.M. (1996) Disposition of amikacin in fallow deer (Dama dama) following a single intramuscular dose

Journal of Zoo and Wildlife Medicine, 27, 227-233 Single-dose pharmacokinetics of amikacin following i.m. administration were determined in six adult fallow deer (Dama dama). A dose of 7.47 +/- 0.71 mg/kg was determined by metabolic scaling. Serial blood samples were collected through an indwelling jugular catheter at 0, 5, 15, 30, 45, 60, 75, and 90 min and 2, 4, 5, 6, 8, 10, and 12 hr postadministration. Drug concentration vs. time curves were best fit to a biexponential equation, which was interpreted as a single compartment open model with first-order input. Maximal serum concentrations of 34.99 +/- 9.93 mu g/ml were reached at 0.40 +/- 0.21 hours based on a disappearance rate constant of 0.57 +/- 0.11 hr(-1) and a model-dependent disappearance half-life of 1.14 +/- 0.051 hr. Model-independent parameters were mean residence time (1.62 +/- 0.32 hr), area under the curve (70.32 +/- 8.14 mu g[hr]/ml), area under the moment curve (115.8 +/- 8.29 mu g[hr](2)/ml), average concentration at steady state (20.41 +/- 1.37 mu g/ml), and effective half-life (1.16 +/- 0.20 hr). Based on the minimum inhibitory concentration of typical organisms, the suggested dosing regimen for fallow deer is 7.5 mg/kg every 4-6 hr, depending on the organism encountered AMBIENT-TEMPERATURE/amikacin/AMINOGLYCOSIDE/BACTERICIDAL ACTIVITIES/Dama dama/DAMA-DAMA/deer/EFFICACY/FALLOW DEER/GENTAMICIN/GRAM-NEGATIVE INFECTIONS/minimum inhibitory concentration/MODEL/MYCOBACTERIUM-PARATUBERCULOSIS/NEPHROTOXICITY/ORGANISM/ORGANISMS/PHARMACOKINETIC PARAMETERS/PHARMACOKINETICS/REGIMEN/SAMPLES

263 Stehman, S.M. (1996) Paratuberculosis in small ruminants, deer, and South American Camelids

Veterinary Clinics of North America-Food Animal Practice, 12, 441-& Paratuberculosis in sheep, goats, deer, and South American Camelids presents with weight loss as the primary clinical sign of disease. This article focuses on the unique features of Johne's disease in farmed species other than cattle, with an emphasis on the differences in clinical presentation, diagnosis, management, and control AGAR-GEL IMMUNODIFFUSION/ CATTLE/COMPLEMENT-FIXATION TEST/control/deer/DIAGNOSIS/DISEASE/DOMESTIC SHEEP/goat/goats/Johne's disease/JOHNES-DISEASE/LARGE GOAT HERD/LINKED-IMMUNOSORBENT-ASSAY/MYCOBACTERIUM-PARATUBERCULOSIS/PARA-TUBERCULOSIS/paratuberculosis/RESTRICTION ENDONUCLEASE ANALYSIS/RUMINANTS/SHEEP/white-tailed deer

264 Stabel, J.R., Goff, J.P., Whipple, D.L., Ackermann, M.R., Reinhardt, T.A. (1996) Low calcium diet and 1,25-dihydroxyvitamin D-3 infusion modulate immune responses during Mycobacterium paratuberculosis infection in beige mice

Veterinary Immunology and Immunopathology, 50, 127-143 A 12-month study was conducted to evaluate the effects of feeding a low calcium (Ca) diet or 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) infusion on the persistence of Mycobacterium paratuberculosis infection using a mouse model, Male beige mice 6-8 weeks of age were assigned to one of the following treatments: (1) non-infected, (2) infected, (3) non-infected/1,25(OH)(2)D-3, (4) infected/1,25(OH)(2)D-3, and (5) infected/low Ca (0.15%) diet, Infected mice were inoculated intravenously with live M. paratuberculosis. At 1, 6 and 12 months postinfection, mice in Treatments 3 and 4 were implanted subcutaneously with mini-osmotic pumps to deliver 1,25(OH)(2)D-3. Infusion with 1,25(OH)(2)D-3 exacerbated M. paratuberculosis infection in most tissues at all time points, Mice infused with 1,25(OH)(2)D-3 had higher bacterial counts in spleen, liver, and ileum compared with control infected mice after 1 month of infection. In contrast, feeding a low Ca diet reduced the number of viable organisms cultured from the liver and ileum of infected mice, Plasma Ca and 1,25(OH)(2)D-3 were increased in mice infused with 1,25(OH)(2)D-3 at all time points but values for low Ca mice were not different than for non-infused mice. Splenocyte production of TNF, IL-1 and IL-6 was higher for mice fed

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the low Ca diet compared with control infected mice after 1 month of infection, Inducible IL-6 activity remained higher for this treatment at 6 months postinfection. These results suggest that feeding a low Ca diet to mice chronically infected with M. paratuberculosis appears to enhance their ability to clear the infection in a manner distinct from any effect of 1,25(OH)(2)D-3 ABILITY/ALVEOLAR MACROPHAGES/BEIGE MICE/calcium/CELLS/control/GROWTH/HYPERCALCEMIA/immune responses/IMMUNE-RESPONSE/IMMUNE-RESPONSES/INFECTED MICE/INFECTION/INTERLEUKIN-6/LYMPHOCYTE-T/MICE/MODEL/MOUSE/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/Mycobacterium paratuberculosis infection/MYCOBACTERIUM-PARATUBERCULOSIS/ORGANISM/ORGANISMS/PARATHYROID-HORMONE/paratuberculosis/PARATUBERCULOSIS INFECTION/PROLIFERATION/PULMONARY TUBERCULOSIS/RESPONSES/TISSUE/TISSUES/vitamin D-3/VITAMIN-D

265 Cross, M.L., Chinn, N.D., Griffin, J.F.T., Buchan, G.S. (1996) T cell responses to Mycobacterium bovis in red deer, a large animal model for tuberculosis Immunology and Cell Biology, 74, 32-37 Red deer (Cervus elaphus) represent an appropriate large animal model to study the immunology of tuberculosis, being naturally susceptible to Mycobacterium bovis infection. Cell-mediated immune responses were investigated in deer displaying protective- or disease-type reactions, following immunization with M. bovis bacille Calmette-Guerin (BCG) or infection with virulent M. bovis, respectively. T cell responses were measured as antigen-dependent cell proliferation and production of T cell growth factor (TCGF) following in vitro stimulation with M. bovis antigens (live or heat-killed BCG, or PPD). T cells from immunized deer proliferated less in response to soluble denatured culture antigen (purified protein derivative, PPD) than to particulate BCG, although there were no differences in the magnitude of these responses between the two groups of animals. Cells derived from immunized deer produced less TCGF than cells from infected deer when stimulated with PPD in vitro, although responses to BCC antigens were similar between the two groups. The majority of TCGF activity was neutralized by anti-IL-2 antibodies, regardless of the animal group or source of antigen used for in vitro stimulation. After 7 days in vitro culture with antigen, blast cells staining positively for alpha beta (CD4, CD8) and gamma delta T cell receptors were recorded. The majority of blasts were CD4(+), although in immunized deer fewer CD4(+) blasts were produced following in vitro stimulation with PPD than with BCG antigens. These results, together with previous reports from our laboratory, represent the only detailed examinations of T cell responses to M. bovis in this naturally-susceptible ruminant species ALPHA/animal model/ANIMALS/ANTIBODIES/ANTIBODY/ANTIGEN/antigens/BCG/BLOOD MONONUCLEAR-CELLS/BOVIS/CALMETTE-GUERIN/CATTLE/CELL/CELLS/Cervus elaphus/CERVUS-ELAPHUS/CHRONIC PARATUBERCULOSIS/CULTURE/CYTOTOXICITY/deer/GAMMA/GROWTH/IL-2/immune responses /IMMUNE-RESPONSE/IMMUNE-RESPONSES/immunology/IN-VITRO/INFECTION/LYMPHOBLAST PROLIFERATIVE CAPACITY/LYMPHOCYTES/MODEL/mycobacteria/Mycobacterium/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/PROLIFERATION/PROTEIN/RED DEER/RESPONSES/STIMULATION/T cell/T cells/T-CELLS/Tuberculosis

266 Rohonczy, E.B., Balachandran, A.V., Dukes, T.W., Payeur, J.B., Rhyan, J.C., Saari, D.A., Whiting, T.L., Wilson, S.H., Jarnagin, J.L. (1996) A comparison of gross pathology, histopathology, and mycobacterial culture for the diagnosis of tuberculosis in elk (Cervus elaphus) Canadian Journal of Veterinary Research-Revue Canadienne de Recherche Veterinaire, 60, 108-114 Using the isolation of Mycobacterium bovis as the reference standard, this study evaluated the sensitivity, specificity and kappa statistic of gross pathology (abattoir postmortem inspection), histopathology, and parallel or series combinations of the two for the diagnosis of tuberculosis in 430 elk and red deer. Two histopathology interpretations were evaluated: histopathology I, where the presence of lesions compatible with tuberculosis was considered positive, and histopathology II, where lesions compatible with tuberculosis or a select group of additional possible diagnoses were considered positive. In the 73 animals from which M. bovis was isolated, gross lesions of tuberculosis were most often in the lung (48), the retropharyngeal lymph nodes (36), the mesenteric lymph nodes (35), and the mediastinal lymph nodes (16). Other mycobacterial isolates included: 11 M. paratuberculosis, 11 M. avium, and 28 rapidly growing species or M. terrae complex. The sensitivity estimates of gross pathology and histopathology I were 93% (95% confidence limits [CL] 84,97%) and 88% [CL 77,94%], respectively, and the specificity of both was 89% [CL 85,92%]). The sensitivity and specificity of histopathology II were 89% (CL 79,95%) and 77% (CL 72,81%), respectively. The highest sensitivity estimates (93-95% [CL 84,98%]) were obtained by interpreting gross pathology and histopathology in parallel (where an animal had to be positive on at least one of the two, to be classified as combination positive). The highest specificity estimates (94-95% [CL 91-97%]) were generated when the two tests were interpreted in

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series (an animal had to be positive on both tests to be classified as combination positive). The presence of gross or microscopic lesions showed moderate to good agreement with the isolation of M. bovis (Kappa = 65-69%). The results show that post-mortem inspection, histopathology and culture do not necessarily recognize the same infected animals and that the spectra of animals identified by the tests overlaps ANIMALS/ AVIUM/BOVIS/CATTLE/Cervus elaphus/CERVUS-ELAPHUS/COMPLEX/CULTURE/deer/DIAGNOSIS/ELK/LESIONS/LYMPH-NODES/mycobacteria/Mycobacterium/Mycobacterium bovis/MYCOBACTERIUM-BOVIS/paratuberculosis/PATHOLOGY/RED DEER/SENSITIVITY/SPECIFICITY/TESTS/Tuberculosis

267 Tanaka, S. , Sato, M., Taniguchi, T., Yokomizo, Y. (1996) Relationship of acid phosphatase activity to ultrastructural features in mice inoculated with Mycobacterium paratuberculosis

Journal of Comparative Pathology, 114, 81-91 Macrophage activation, measured as increased acid phosphatase (AcPase)-positive areas by image analysis, and ultrastructural features were examined in granulomatous mycobacterial lesions of mice innately susceptible (BALB/c mice; Bcg(s)) and innately resistant (C3H/HeJ mice; Bcg(r)) to Mycobacterium paratuberculosis strain ATCC 19698. In the liver and spleen of BALB/c mice 3 weeks after intraperitoneal inoculation with M. paratuberculosis, AcPase activity detected in epithelioid cell nodules was high; it had decreased, however, in the liver and spleen after a further 3 and 6 weeks, respectively. In C3H/HeJ mice, the size of epithelioid cell nodules in the liver and spleen was smaller than in BALB/c mice, and infiltrating macrophages, which had increased by week 9 after inoculation, showed high AcPase activity. Ultrastructurally, by week 32 in BALB/c mice, small phagolysosomes (SPLs) had greatly increased in number in the epithelioid cells. These SPLs contained a few AcPase-positive areas and a small number of bacteria, most of which were surrounded by an electron-translucent space (or electron-transparent zone [ETZ]). In contrast, only a few SPLs were observed in C3H/HeJ mice at week 32; in the liver and spleen, large phagolysosomes (LPLs) showed high AcPase activity and contained many degenerated bacteria, which also had an ETZ. These results suggest that the enzymatic and ultrastructural differences in phagolysosomes between BALB/c mice and C3H/HeJ mice reflect the susceptibility of these mouse strains to M. paratuberculosis. (C) 1996 W.B. Saunders Company Limited AVIUM/BACTERIUM/BALB/c mice/BCG GENE/CELL/CELLS/ELECTRON-TRANSPARENT ZONE/INBRED MICE/INFECTION/INOCULATION/LESIONS/macrophage/MACROPHAGES/MICE/MOUSE/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/STRAINS/susceptibility

268 Giacometti, M., Tolari, F., Mannelli, A., Lanfranchi, P. (1995) Seroepidemiologic Investigations in Alpine Ibex (Capra-I-Ibex) of Albris Colony (Grisons, Switzerland)

Schweizer Archiv fur Tierheilkunde, 137, 537-542 Sero-epidemiological investigations in wild animals may allow to assess distribution of selected pathogens that sometimes seem to be involved in sanitary interrelationships between wild and domestic ungulates sharing the same areas. Sero-logical studies were carried out to investigate the prevalence of antibody against 8 pathogens in Alpine ibex of Albrix colony (Grisons, Switzerland). Investigated sera came from 89 animals shot by gamekeepers in 1990-1991. Antibody against smooth Brucella, Coxiella burnettii, Leptospira interrogans, Borrelia burgdorferi, Mycobacterium paratuberculosis, BHV-1 and ovi-caprine lentiviruses were not detected in the tested sera. However, 31% of sera analysed were found to be positive for Chlamydia psittaci. Three sera showed high antibody tires (greater than or equal to 1/128) suggestive of active infection in the animals. Any influence of Chlamydia psittaci in reproductive performance of free-ranging alpine ibex should be investigated through isolation of the agent. Results are discussed with reference to methods used and with epidemiological picture in Switzerland and were compared with results of serological investigations carried out in ibex in Italy and France ALPINE IBEX/Alps/ANIMALS/ANTIBODIES/ANTIBODY/ARTHRITIS-ENCEPHALITIS/Brucella/CAPRA I IBEX/CHLAMYDIOSIS/Epidemiology/goats/IMMUNODIFFUSION TEST/INFECTION/Italy/LEPTOSPIRA/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/PREVALENCE/SEROLOGICAL STUDY/Serology/SHEEP/SWITZERLAND/Ungulate/VIRUS/WILD/WILD ANIMALS

269 Deshpande, R.G., Khan, M.B., Bhat, D.A., Navalkar, R.G. (1995) Immunoaffinity Chromatographic Isolation of A High-Molecular-Weight Seroreactive Protein from

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Mycobacterium-Leprae Cell Sonicate

Fems Immunology and Medical Microbiology, 11, 163-169 The purpose of this study was to isolate Mycobacterium leprae antigen(s) by immunoaffinity chromatography using immunoglobulins from leprosy patients and from rabbit anti-M. leprae hyperimmune serum coupled to CNBr-Sepharose 4B. A high molecular weight (M(r)) M. leprae protein (MLP) with a subunit M(r) of 22 000 was isolated. MLP was recognized by monoclonal antibody MMPII1G4 which is known to react with MMPII, a 22 kDa protein of M. leprae. The N-terminal sequence of the 22 kDa subunit (Met-gln-gly-asp-pro-asp-val-leu-arg-leu-leu-asn-glu-gln-leu-thr) was identical to MMPII and to antigen D (bacterioferritin) of M. paratuberculosis. It showed 44% homology with N-terminal end of E. coli bacterioferritin. In ELISA, MLP showed 100% and 60% positivity with leprosy and TB sera respectively as compared to normal healthy sera. The role of bacterioferritin in M. leprae and the importance of MLP as an immunogen has been discussed ANTIBODIES/ANTIBODY/ANTIGEN/ANTIGEN-D/CELL/CONTACTS/ELISA/IMMUNOAFFINITY CHROMATOGRAPHY/IMMUNOGLOBULIN/LEPRAE/leprosy/LEPROSY PATIENTS/MONOCLONAL-ANTIBODIES/mycobacteria/Mycobacterium/MYCOBACTERIUM LEPRAE/paratuberculosis/PATIENT/ PROTEIN/rabbit/RESPONSES/role/SEQUENCE/SERA/SEROREACTIVE/Tuberculosis

270 [Anon] (1995) Johnes-Disease Organism Found in Wild Rabbits

Veterinary Record, 136, 452-454 JOHNES DISEASE/JOHNES-DISEASE/ORGANISM/rabbit/RABBITS/WILD/WILD RABBIT/WILD RABBITS

271 deLisle, G.W., Collins, D.M. (1995) Johnes-Disease in Red Deer

Veterinary Record, 136, 336-336 deer/JOHNES DISEASE/JOHNES-DISEASE/RED DEER

272 Fawcett, A.R., Goddard, P.J., Mckelvey, W.A.C., Buxton, D., Reid, H.W., Greig, A., Macdonald, A.J. (1995) Johnes-Disease in A Herd of Farmed Red Deer

Veterinary Record, 136, 165-169 An outbreak of Johne's disease in a herd of farmed red deer was studied for four years. Serological, histopathological and cultural techniques were used to monitor the progress of the disease, and delayed type hypersensitivity skin tests were also applied. The results of the serological tests showed that they were poor predictors of future clinical cases and did not consistently identify animals harbouring mycobacteria. The histopathological methods provided a sensitive and specific means of confirming the infection. The skin tests had a low sensitivity and the results were poorly correlated with the serological results in seropositive animals. A vaccination policy was instituted which was accompanied by a change in the pattern of disease. la!though the histopathological evidence suggested that the infection was still occurring, there was a marked reduction in the incidence of clinical disease. Vaccinated animals showed a good response to the skin test ANIMALS/CATTLE/deer/DIAGNOSIS/DISEASE/FARMED RED DEER/HERD/INFECTION/Johne's disease/JOHNES DISEASE/JOHNES-DISEASE/mycobacteria/Mycobacterium/PARA-TUBERCULOSIS/paratuberculosis/RED DEER/SENSITIVITY/SEROLOGICAL TESTS/SKIN-TEST/TESTS/VACCINATION

273 Mutwiri, G.K., Butler, D.G., Rosendal, S., Woodward, B. (1995) The Role of Restricted Food-Intake in the Pathogenesis of Cachexia in Severe Combined Immunodeficient Beige Mice Infected with Mycobacterium-Paratuberculosis

Canadian Journal of Veterinary Research-Revue Canadienne de Recherche Veterinaire, 59, 40-45 A paired feeding experiment was conducted to investigate if reduced food intake is a reason for the body weight loss previously observed in severe combined immunodeficient beige (SCID bg) mice infected with Mycobacterium paratuberculosis. Mice were paired on the basis of age, litter and sex. One of each pair was injected intraperitoneally with 10(5) viable M. paratuberculosis organisms. The remainder served as uninfected pairfed mates. Each uninfected mouse was restricted to the amount of food (per gram body weight) that its infected paired mate ate in the previous 24 hour period starting at four weeks postinfection until 12 weeks postinfection when the mice were necropsied. The mean body weights of the two groups were not significantly different (p < 0.05) at the start of the experiment (infected 27.6 +/- 2.1 g, pairfed 27.3 +/- 3.4 g) but the pairfed group weighed less after 12 weeks of restricted food intake.

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Mycobacterium paratuberculosis was isolated from the spleen, liver, gut and fecal pellets of the infected but not the uninfected mice. Acid-fast bacilli were seen histologically in the liver, spleen and intestines of the infected mice only. Analysis of carcass compositions indicated that both infected and pairfed mice lost dry matter. Despite the loss in dry matter, the infected mice appeared to have maintained their body weights due to an increased retention of body water (presumably due to edema of inflammation). These results suggest that infection of SCID bg mice with M. paratuberculosis causes a reduction in their food intake (presumably due to reduced appetite) which, in turn, contributes to a loss in dry matter. We suggest that this loss in dry matter is one of the initial events that eventually lead to cachexia, and that it precedes the body weight loss that inevitably occurs in SCID bg mice chronically infected with il M. paratuberculosis acid-fast bacilli/BCG/BEIGE MICE/food/INFECTED MICE/INFECTION/intake/MALNOURISHED RATS/MICE/MOUSE/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/ORGANISM/ORGANISMS/paratuberculosis/PATHOGENESIS/PELLETS/role/TUMOR NECROSIS FACTOR/WATER

274 Hutchings, D.L., Wilson, S.H. (1995) Evaluation of Lymphocyte Stimulation Tests for Diagnosis of Bovine Tuberculosis in Elk (Cervus-Elaphus) American Journal of Veterinary Research, 56, 27-33 Lymphocyte stimulation tests (LST), performed using 6 antigen preparations, were compared individually and in pairs. The tests were performed on 433 blood samples collected from elk in Mycobacterium bovis-infected herds. These elk were killed as part of Agriculture and Agri-Food Canada's bovine tuberculosis eradication policy, and mycobacterial culture results were obtained from tissues of each animal. The LST, which had the highest total sum of sensitivity and specificity, was a comparative test that used M bovis purified protein derivative (PPD) and M paratuberculosis (johnin) PPD. This test had a sensitivity of 76%, with confidence limits (CL) of 63 to 85% for this estimate, and specificity of 77% (cr, 72 to 81%). The LST, using only M bovis PPD antigen, had a sensitivity of 70% (ct, 57 to 80%) and specificity of 74% (CL, 69 to 79%); when it was compared with culture results, using the kappa statistic, agreement was only 32%. This indicated that the LST identified different elk than did M bovis isolation tests ANTIGEN/BLASTOGENESIS/BOVINE/BOVINE TUBERCULOSIS/BOVIS/CATTLE/Cervus elaphus/CERVUS-ELAPHUS/CULTURE/DIAGNOSIS/ELK/FARMED DEER/HERD/interferon-gamma/INVITRO/mycobacteria/Mycobacterium/paratuberculosis/PROTEIN/RED DEER/RESPONSES/SAMPLES /SENSITIVITY/SPECIFICITY/STIMULATION/TESTS/TISSUE/TISSUES/Tuberculosis

275 deLisle, G.W., Wards, B.J., Collins, D.M. (1991) The Diagnosis of Johnes-Disease in Farmed Deer Proceedings of the Third International Colloquium on Paratuberculosis - A Meeting of the International Association for Paratuberculosis, 164-171 deer/DIAGNOSIS/FARMED DEER/JOHNES DISEASE/JOHNES-DISEASE

276 Smokvina, T., Henderson, D.J., Melton, R.E., Brolle, D.F., Kieser, T., Hopwood, D.A. (1994) Transposition of Is117, the 2.5 Kb Streptomyces-Coelicolor A3(2) Minicircle - Roles of Open Reading Frames and Origin of Tandem Insertions

Molecular Microbiology, 12, 459-468 IS117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of ORF1 of IS117, presumed to encode a transposase, abolished transposition. Deletion or mutation of ORF2 and ORF3, which overlap each other on opposite strands of IS117, caused a c. 20-fold reduction in integration frequency of the circular form of IS117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. ORF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild-type IS117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS117 derivatives yielded tandem chromosomal insertions, which arose by co-transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration

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CLONING/CONSTRUCTION/DNA/ELEMENT/INACTIVATION/LIVIDANS/MINI-CIRCLE/MUTANT/MUTATION/MYCOBACTERIUM-PARATUBERCULOSIS/NUCLEOTIDE-SEQUENCE/role/TARGET SITE/TRANSFORMATION/VECTORS

277 Gilot, P., Misonne, M.C. (1994) Mycobacterium-Paratuberculosis and Escherichia-Coli Share Common Antigenic Determinants

Veterinary Microbiology, 39, 353-360 Paratuberculosis (Johne's disease) is a chronic enteritis of ruminants, which is due to infection by Mycobacterium paratuberculosis. By comparative analysis of Western blots of M. paratuberculosis components incubated with sera from paratuberculous cows (either pre-absorbed or not on an Escherichia coli sonicate), we have shown the presence of cross-reactive antigens between M. paratuberculosis and E. coli. Components in the range of 22 to 75 kDa are recognized by sera from paratuberculous cows, but only some components in the range of 20 to 40 kDa are endowed with specific B-cell epitopes to M. paratuberculosis with respect to E. coli. Cross-reactions were further stressed by the fact that rabbit immunoglobulins to E. coli recognized some ten M. paratuberculosis components (in the range of 45 to 66 kDa). The importance of cross-reactivity between the two bacterial species was evaluated by enzyme-linked immunosorbent assay (ELISA) using soluble sonic-extract components from M. paratuberculosis as antigens. Pre-absorption of series of bovine sera with E. coli resulted in the diminution of ELISA mean optical density readings by 16.1% and 62.0% for paratuberculous or healthy animals, respectively ANIMALS/ANTIBODY REACTIVITIES/ANTIGEN/ANTIGENIC DETERMINANT/antigens/ASSAY/BOVINE/BOVINE PARATUBERCULOSIS/CATTLE/COMPLEX /cow/COWS/cross-reactions/CROSS-REACTIVITY/DIAGNOSIS/DISEASE/ELISA/ENTERITIS/enzyme-linked immunosorbent assay/ESCHERICHIA COLI/ESCHERICHIA-COLI/IMMUNOGLOBULIN/immunology/INFECTION/Johne's disease/JOHNES DISEASE/LINKED-IMMUNOSORBENT-ASSAY/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/PARA-TUBERCULOSIS/paratuberculosis/PROTEINS/rabbit/RUMINANTS/SERA

278 Ellis, J.A., Russell, H.I., Du, C.W. (1994) Effect of Selected Cytokines on the Replication of Corynebacterium-Pseudotuberculosis and Ovine Lentiviruses in Pulmonary Macrophages

Veterinary Immunology and Immunopathology, 40, 31-47 Opportunistic bacterial pathogens that induce monokine secretion by pulmonary alveolar macrophages (PAM) are frequently encountered complicating factors in lentivirus-associated pneumonias in ungulates and man. We examined the effect of selected cytokines on the replication of Corynebacterium pseudotuberculosis and ovine lentivirus (OvLV) in ovine PAM. Recombinant bovine (rBo) IL 1beta, rBoIL-2, rBo interferon-gamma (IFNgamma) and rBoTNFalpha, alone and in combination at physiological doses had no apparent effect on the extracellular growth of C. pseudotuberculosis, compared with the growth of the pathogen in medium alone. Untreated ovine PAM, derived from bronchoalveolar lavage, were found to substantially reduce, but not eliminate the growth of C pseudotuberculosis in culture. This bactericidal effect was neither enhanced nor inhibited by pretreatment of PAM with the recombinant bovine cytokines or low doses of LPS that induce monokines. In contrast, addition of rBoTNFalpha or rBoIL-1beta, at physiological doses, at the initiation of, or on Day 4, after OvLV infection resulted in a significant increase in viral replication in PAM, as measured in an antigen capture assay for OvLV p25, compared with untreated infected cells. This effect was more pronounced with lower levels of infecting OvLV, and, in the case of TNFalpha, was abrogated by preincubation of the cytokine with specific anti-serum. Conversely, in most instances, inclusion of rBoIFNalpha in OvLV-infected PAM cultures resulted in a significant decrease in viral replication. These results suggest that these soluble mediators that are probably secreted in response to C. pseudotuberculosis infection may have little direct effect on the extra- or intracellular survival of the bacteria in the lung, but may modulate lentiviral replication and, by extension, disease expression, in sheep with dual infection ACQUIRED IMMUNODEFICIENCY SYNDROME /ALVEOLAR MACROPHAGES/ANTIGEN/ASSAY/BACTERIAL PATHOGENS/ BACTERIUM/BOVINE/caseous lymphadenitis/CELL/CELLS/CULTURE/cytokine/cytokines/DISEASE/EXPRESSION/FACTOR-ALPHA/GAMMA-INTERFERON/GROWTH/INFECTION/interferon-gamma/INTRACELLULAR SURVIVAL/LYMPHOID INTERSTITIAL PNEUMONIA/macrophage/MACROPHAGES/MOUSE PERITONEAL-MACROPHAGES /MYCOBACTERIUM-PARATUBERCULOSIS/OVINE/PNEUMONIA/SHEEP/SURVIVAL/TUMOR-NECROSIS-FACTOR/Ungulate/VIRUS TYPE-1

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279 deLisle, G.W., Yates, G.F., Collins, D.M. (1993) Paratuberculosis in Farmed Deer - Case-Reports and Dna Characterization of Isolates of Mycobacterium-Paratuberculosis Journal of Veterinary Diagnostic Investigation, 5, 567-571 Mycobacterium paratuberculosis was isolated from farmed deer in New Zealand on 21 occasions over a 7-year period. The number of cases increased during the last 3 years of the study. Clinical paratuberculosis was observed in 5 deer, 3 other cases came from grossly normal animals that reacted to a tuberculin skin test, and the remaining 13 animals had tuberculous lesions that were identified at meat inspection. Pathologic features of the lesions in these 13 cases included necrosis and mineralization in lymph nodes draining the gastrointestinal tract. The histologic changes in these lesions were very similar to those caused by Mycobacterium bovis and members of the M. avium complex. Characterization of 20 of the isolates of M. paratuberculosis by restriction endonuclease analysis and DNA hybridization revealed that 3 of these isolates were identical to New Zealand sheep isolates and 17 were the same as cattle isolates. The source of the cervine infections was not determined ANIMALS/AVIUM/BOVIS/CATTLE/COMPLEX/deer/DNA/DNA HYBRIDIZATION/ENDONUCLEASE ANALYSIS/FARMED DEER/HYBRIDIZATION/INFECTION/INFECTIONS/LESIONS/LYMPH-NODES/mycobacteria/Mycobacterium/Mycobacterium bovis/Mycobacterium paratuberculosis/MYCOBACTERIUM-BOVIS/MYCOBACTERIUM-PARATUBERCULOSIS/NEW-ZEALAND/paratuberculosis/RESTRICTION /RESTRICTION ENDONUCLEASE ANALYSIS/SHEEP/SKIN-TEST/TUBERCULOUS LESIONS

280 Hughes, M.S., Skuce, R.A., Beck, L.A., Neill, S.D. (1993) Identification of Mycobacteria from Animals by Restriction Enzyme Analysis and Direct Dna-Cycle Sequencing of Polymerase Chain Reaction-Amplified 16S Ribosomal-Rna Gene-Sequences

Journal of Clinical Microbiology, 31, 3216-3222 Two methods, based on analysis of the polymerase chain reaction-amplified 16S rRNA gene by restriction enzyme analysis (REA) or direct cycle sequencing, were developed for rapid identification of mycobacteria isolated from animals and were compared to traditional phenotypic typing. BACTEC 7H12 cultures of the specimens were examined for ''cording,'' and specific polymerase chain reaction amplification was performed to identify the presence of tubercle complex mycobacteria. Combined results of separate REAs with HhaI, MspI, MboI, and ThaI differentiated 12 of 15 mycobacterial species tested. HhaI, MspI, and ThaI restriction enzyme profiles differentiated Actinobacillus species from mycobacterial species. Mycobacterium bovis could not be differentiated from M. bovis BCG or Mycobacterium tuberculosis. Similarly, Mycobacterium avium and Mycobacterium paratuberculosis could not be distinguished from each other by REA but were differentiated by cycle sequencing. Compared with traditional typing, both methods allowed rapid and more accurate identification of acid-fast organisms recovered from 21 specimens of bovine and badger origin. Two groups of isolates were not typed definitively by either molecular method. One group of four isolates may constitute a new species phylogenetically very closely related to Mycobacterium simiae. The remaining unidentified isolates (three badger and one bovine) had identical restriction enzyme profiles and shared 100% nucleotide identity over the sequenced signature region. This nucleotide sequence most closely resembled the data base sequence of Mycobacterium senegalense AMPLIFICATION/ANIMALS/AVIUM/BCG/BOVINE/BOVIS/BOVIS BCG/CHAIN-REACTION/COMPLEX/CULTURE/DIFFERENTIATION/GENE/IDENTIFICATION/MOLECULAR-LEVEL/mycobacteria/Mycobacterium/Mycobacterium avium/Mycobacterium bovis/Mycobacterium paratuberculosis/Mycobacterium tuberculosis/MYCOBACTERIUM-AVIUM/MYCOBACTERIUM-BOVIS/MYCOBACTERIUM-PARATUBERCULOSIS/MYCOBACTERIUM-TUBERCULOSIS/NUCLEOTIDE-SEQUENCE/ORGANISM/ORGANISMS/paratuberculosis/PHYLOGENY/polymerase chain reaction/POLYMERASE-CHAIN-REACTION/RESTRICTION/RESTRICTION ENZYME ANALYSIS/RIBOSOMAL-RNA/SEQUENCE/ Tuberculosis

281 Reid, J.D. , Chiodini, R.J. (1993) Serologic Reactivity Against Mycobacterium-Paratuberculosis Antigens in Patients with Sarcoidosis

Sarcoidosis, 10, 32-35 Although sarcoidosis has clinical and histopathologic similarities to some forms of tuberculosis and other mycobacterial infections, attempts to establish a mycobacterial etiology have not been successful. Using cytoplasmic antigens derived from a wild strain of Mycobacterium paratuberculosis in an enzyme-linked immunosorbent assay, patients with sarcoidosis were found to have immunoglobulin levels significantly higher than those found in a control population in the IgG, but not in IgA or IgM antibody classes. Results were comparable to those reported from patients with Crohn's disease, where M. paratuberculosis has

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been intensively studied as a possible etiologic agent. To elucidate these relationships, examination of DNA from sarcoid tissues for possible homology with DNA from M. paratuberculosis and closely related organisms, as well as cultural attempts with techniques and media appropriate for M. paratuberculosis may be warranted ANTIBODIES/ANTIBODY/ANTIGEN/antigens/ ASSAY/control/Crohn's disease/CROHNS DISEASE/CROHNS-DISEASE/DISEASE/DNA/ELISA/ELISA ASSAY/enzyme-linked immunosorbent assay/etiology/IMMUNOGLOBULIN/INFECTION/INFECTIONS/INFLAMMATORY BOWEL-DISEASE/LINKED IMMUNOSORBENT-ASSAY/mycobacteria/MYCOBACTERIAL INFECTIONS/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/ORGANISM/ORGANISMS/paratuberculosis/PATIENT/POPULATION/SARCOIDOSIS/TISSUE/TISSUES/Tuberculosis/WILD

282 Markesich, D.C., Elzaatari, F.A.K., Graham, D.Y. (1993) Activity of Clarithromycin Against Intracellular Mycobacterium-Paratuberculosis - Putative Agent of Crohns-Disease European Journal of Gastroenterology & Hepatology, 5, 613-615 Objective: To test whether Mycobacterium paratuberculosis, the putative etiologic agent of Crohn's disease, was senisitive to clarithromycin. Design: To test the susceptibility of two mycobactin-dependent mycobacteria, M. paratuberculosis and M. avium subsp. silvaticum, to clarithromycin grown in mouse monocyte/macrophage cell line J774A.1. Results: Clarithromycin, 2 and 4 mug/ml, inhibited M. paratuberculosis (80 and 25% of control, respectively) but was less effective against M. avium subsp. silvaticum (80 and 82% of control, respectively) growing intracellularly in the murine macrophage cell line J-774A.1. Conclusions: Clarithromycin shows promise for use in double-blind controlled trials for testing the hypothesis that M. paratuberculosis is etiologically related to Crohn's disease AVIUM/CELL/CLARITHROMYCIN/control/CONTROLLED TRIAL/Crohn's disease/CROHNS DISEASE/CROHNS-DISEASE/DISEASE/macrophage/MOUSE/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/SILVATICUM/susceptibility

283 Lal, H., Ashraf, N.V.K. (1993) Paratuberculosis in A Wild Gaur (Bos-Gaurus Smith) in Palamau Tigere Reserve, Bihar

Indian Veterinary Journal, 70 , 463-464 paratuberculosis/WILD

284 Power, S.B., Haagsma, J., Smyth, D.P. (1993) Paratuberculosis in Farmed Red Deer (Cervus-Elaphus) in Ireland

Veterinary Record, 132, 213-216 Paratuberculosis was diagnosed in one 18-month-old and two 30-month-old hinds in a herd of 70 red deer (Cervus elaphus) in Ireland. Loss of condition and intermittent diarrhoea were the main clinical findings. Clumps of acid-fast organisms were found in the faeces of the three deer. Post mortem examination of one deer showed a slight swelling and pallor of the intestinal tract and associated lymph nodes. Histopathology showed a severe, granulomatous enteritis and lymphadenitis, with extensive cellular infiltration, notably with epithelioid macrophages containing numerous acid-fast organisms. Mycobacterium paratuberculosis was isolated from intestinal and lymph node samples. Paratuberculosis was also confirmed in one of nine clinically normal, yearling stags, sampled at slaughter. Complement fixation tests and enzyme-linked immunosorbent assays gave higher readings for clinically affected deer than healthy ones. Acid soil on the farm was believed to be a contributory cause ASSAY/Cervus elaphus/CERVUS-ELAPHUS/deer/diarrhoea/ENTERITIS/enzyme-linked immunosorbent assay/faeces/FARMED RED DEER/FIXATION/GRANULOMATOUS ENTERITIS/HERD/LYMPH-NODES/macrophage/MACROPHAGES/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/ORGANISM/ORGANISMS/paratuberculosis/RED DEER/RUMINANT PARA-TUBERCULOSIS/SAMPLES/TESTS

285 Mondal, D. , Sinha, R.P. (1992) Pathogenicity of Caprine Strain of Mycobacterium-Paratuberculosis in Rabbit

Indian Journal of Animal Sciences, 62 , 1117-1120 Pathogenicity and immunologic property of caprine strain of Mycobacterium Paratuberculosis was studied in 30 growing rabbits for 75 days. On oral exposure the incubation period of the disease varied

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from 42 to 70 days in rabbits. In the infected animals diarrhoea and subsequently emaciation occurred. Positive cell-mediated immunity was observed as seen through leukocyte migration inhibition test. The small and large intestines, mesenteric lymphnodes, spleen. kidneys, lungs and liver took heavy infection of M. paratuberculosis ANIMALS/caprine/cell-mediated immunity/diarrhoea/DISEASE/emaciation/EXPOSURE/IMMUNITY/incubation period/INFECTION/INHIBITION/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/rabbit/RABBITS

286 Mutwiri, G.K., Butler, D.G., Rosendal, S., Yager, J. (1992) Experimental-Infection of Severe Combined Immunodeficient Beige Mice with Mycobacterium-Paratuberculosis of Bovine Origin

Infection and Immunity, 60, 4074-4079 Severe combined immunodeficient beige mice were inoculated orally and intraperitoneally with a bovine strain of Mycobacterium paratuberculosis to explore their potential as laboratory animal models in the study of paratuberculosis (johne's disease). Control animals were similarly inoculated with heat-killed M. paratuberculosis. In the mice inoculated intraperitoneally, focal lesions and acid-fast bacilli were first detected in the livers (4 weeks postinfection) and later in the spleens and intestines of the test but not the control animals. No bacteria were seen in the hearts, kidneys, or lungs. At 12 weeks postinfection, all test mice had significant losses in body weight compared with those in controls (P < 0.05), a characteristic sign of bovine paratuberculosis. Tumor necrosis factor alpha was not detected in the serum. Histologic lesions were seen in the intestines, livers, and spleens of the animals in the orally inoculated test group after 26 weeks of infection. Our results suggest that the severe combined immunodeficient beige mouse may be a useful model for the investigation of paratuberculosis and cachexia and the evaluation of antimycobacterial drugs acid-fast bacilli/ALPHA/animal model/animal models/ANIMALS/BACTERIUM/BEIGE MICE/BOVINE/BOVINE ORIGIN/BOVINE PARATUBERCULOSIS/BOVINE-PARATUBERCULOSIS/CATTLE/CELLS/control/DISEASE/experimental infection/EXPERIMENTAL-INFECTION/FACTOR-ALPHA/INFECTION/Johne's disease/LESIONS /MICE/MODEL/MOUSE/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/PARA-TUBERCULOSIS/paratuberculosis/PATHOLOGY/SERA/TUMOR NECROSIS FACTOR/TUMOR-NECROSIS-FACTOR

287 Weber, A., Gurke, R., Bauer, K., Schreyer, K. (1992) Bacteriological Investigations on the Presence of Mycobacterium-Paratuberculosis in Fecal Samples of Zoo Ruminants

Berliner und Munchener Tierarztliche Wochenschrift, 105, 161-164 Mycobacterium (M.) paratuberculosis was isolated from fecal samples of 3 (21.4 %) from 14 mufflons, of 10 (20.4 %) from 49 dwarf goats, of 5 (14.3 %) from 35 camerun sheep and of 1 (9.1%) from 11 alpine ibex. M. paratuberculosis could not found by cultural method in fecal samples of 22 Pinzgauer goats, of 15 bantengs, of 9 wild goats, of 9 skuddens, of 6 four-horned sheep, of 3 red-head sheep, and of 1 chamois. From all 19 animals with cultural positive fecal samples complement binding antibodies against M. paratuberculosis could not be found in the corresponding serum samples. The results confirm that M. paratuberculosis is more frequently in small zoo ruminants than up to now was suspected. The cultural examination of fecal samples has been proved to be a better method for detecting animal excretors than serological investigations by means of the complement fixation test ALPINE IBEX/ANIMALS/ANTIBODIES/ANTIBODY/chamois/FIXATION/goat/goats/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/PARA-TUBERCULOSIS/paratuberculosis/RUMINANTS/SAMPLES/SERA/SHEEP/ WILD

288 Dukes, T.W., Glover, G.J., Brooks, B.W., Duncan, J.R., Swendrowski, M. (1992) Paratuberculosis in Saiga Antelope (Saiga-Tatarica) and Experimental Transmission to Domestic Sheep

Journal of Wildlife Diseases, 28, 161-170 Mycobacterium paratuberculosis was isolated in low numbers from the small intestine and associated mesenteric lymph nodes of a saiga antelope (Saiga tatarica) using routine culture techniques in spite of histologic evidence of high numbers of acid-fast bacteria in these tissues. Two newborn domestic sheep were fed the ground intestinal tissue containing acid-fast bacteria and the progression of the experimental disease was followed by fecal culture, immunodiffusion (AGID) and lymphocyte stimulation

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(LST) tests. One experimentally infected sheep developed progressive clinical illness 1 vr postinoculation. Few M. paratuberculosis were isolated from feces or tissues although an extensive granulomatous mycobacterial enteritis, lymphadenitis and lymphangitis were observed containing large numbers of typical acid-fast organisms. No clinical illness was observed in the second inoculated sheep after 18 mo of observation, although infection was demonstrated at necropsy. Both sheep developed AGID and LST reactions indicative of paratuberculosis. This study demonstrated that a difficult to culture isolate of M. paratuberculosis was responsible for paratuberculosis in captive wild ruminants and was transmissible to domestic sheep. Diagnosis of paratuberculosis in four of eight of the imported saiga antelope and in eleven of their 18 offspring indicates the importance of this disease in management of captive wild ruminants and the ease with which this organism can be transmitted AMERICAN WILD RUMINANTS/BACTERIUM/BOVINE PARA-TUBERCULOSIS/CULTURE/DIAGNOSIS/DISEASE/DNA/DOMESTIC SHEEP/ENTERITIS/EXPERIMENTAL-INFECTION/FECAL CULTURE/FECES/INFECTION/JOHNES DISEASE /LYMPH-NODES/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/NATURAL INFECTION/ORGANISM/ORGANISMS/paratuberculosis/PATHOLOGY/RESTRICTION ENDONUCLEASE ANALYSIS/RUMINANT PARA-TUBERCULOSIS/RUMINANTS/SAIGA ANTELOPE/SAIGA-TATARICA/SHEEP/STIMULATION/STRAINS/TESTS/TISSUE/TISSUES/Transmission/WILD/WILD RUMINANTS

289 Weber, A., Gurke, R. (1992) Bacteriological Investigations on the Presence of Mycobacterium-Paratuberculosis in Fecal Samples of Fallow Deer (Dama-Dama L)

Zeitschrift fur Jagdwissenschaft, 38, 55-59 Mycobacterium (M.) paratuberculosis, the causal agent of paratuberculosis (Johne's disease) was bacteriologically detected in 10 (16.1%) fecal samples of 62 fallow deer (Dama dama L.) investigated from 4 enclosures in northern Bavaria. This agent was isolated from fallow deer from 2 enclosures with an isolation rate of respectively 27% and 60%. The investigation results confirm that M. paratuberculosis is more frequent in native fallow deer than was formerly suspected Dama dama/DAMA-DAMA/deer/DISEASE/enclosure/FALLOW DEER/Johne's disease/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/Northern Bavaria/paratuberculosis/SAMPLES

290 Brumbaugh, G.W., Frelier, P.F., Roussel, A.J., Thomson, T.D. (1992) Prophylactic Effect of Monensin Sodium Against Experimentally Induced Paratuberculosis in Mice

American Journal of Veterinary Research, 53, 544-546 Monensin sodium (0, 15, or 30 mg/kg of complete feed) was fed ad libitum for 1 week to female mice (strain C57BL6/J) that were genetically susceptible to infection with Mycobacterium paratuberculosis. Ten mice in each of the 3 groups were inoculated intraperitoneally with M paratuberculosis (10(9) organisms). Sterile saline solution was injected intraperitoneally into 10 other mice in each group. Rations were continued for 50 days, then mice were euthanatized, and body weight, splenic weight, and hepatic weight for each mouse were recorded. Ratios of body weight to splenic weight and of body weight to hepatic weight were calculated for each mouse. Hepatic granulomas in 50 light microscopic fields were counted, and presence of acid-fast organisms in those granulomas was recorded. Infected mice given monensin had higher body weight and fewer hepatic granulomas than did mice not given monensin. Although hepatic granulomas were fewer in these mice, they contained acid-fast organisms. Effects of 15 mg of monesin and those of 30 mg of monensin/kg of complete feed were not different DISEASE/INFECTED MICE/INFECTION/MICE/MOUSE/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/ORGANISM/ORGANISMS/paratuberculosis

291 Hamilton, H.L., Cooley, A.J., Adams, J.L., Czuprynski, C.J. (1991) Mycobacterium-Paratuberculosis Monoassociated Nude-Mice As A Paratuberculosis Model

Veterinary Pathology, 28, 146-155 In this study, a paratuberculosis (Johne's disease) model was developed by intragastrically dosing gnotobiotic athymic nude mice with Mycobacterium paratuberculosis. The mice infrequently shed bacilli from their intestinal tracts during the first 4 months after inoculation. Following this time, increasing numbers of M. paratuberculosis (greater than 4.0 log10 bacilli per fecal pellet by 40 weeks) were recovered from the feces of the 12 mice that remained in the isolator. A similar pattern of recovery of M.

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paratuberculosis was obtained from the ileum, cecum, colon, and liver. Histopathologic lesions and acid-fast bacilli were rare during the first 4 months of infection and then, with time, increased in prevalence and severity. Mice maintained for 7 months or longer exhibited severe granulomatous inflammation and large numbers of acid-fast bacilli in the gastrointestinal tract and liver (up to 10(8) log10 colony forming units per gram wet weight). Five mice maintained for 7 months or more developed clinical signs consistent with those seen in paratuberculosis (weight loss, chronic diarrhea); three of these mice eventually died or became moribund and were euthanatized. M. paratuberculosis monoassociated mice released increased levels of tumor necrosis factor activity into their sera, as compared to uninfected control mice, when they were injected with bacterial lipopolysaccharide. The clinical signs, fecal shedding of M. paratuberculosis, granuloma formation, and progressive bacillary multiplication observed with these mice are consistent with naturally occurring M. paratuberculosis infection of ruminants (Johne's disease). This model will be useful for future studies of immunoregulation and antimicrobial therapy of paratuberculosis acid-fast bacilli/AVIUM COMPLEX/CACHECTIN/CACHEXIA/control/CROHNS-DISEASE/Diarrhea/DISEASE/DNA PROBES/FECES/granuloma formation/GRANULOMA-FORMATION/INFECTION/INFLAMMATORY BOWEL-DISEASE/INOCULATION/Johne's disease/JOHNES DISEASE/LESIONS/LIPOPOLYSACCHARIDE/MICE/MODEL/MOUSE/MULTIPLICATION/mycobacteria/Mycobacterium/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/paratuberculosis/PARATUBERCULOSIS INFECTION/PREVALENCE/RUMINANTS/SERA/THERAPY/TUBERCULOSIS JOHNES DISEASE/TUMOR NECROSIS FACTOR/ TUMOR-NECROSIS-FACTOR

292 Dedek, J., Witt, W., Loepelmann, H., Nattermann, H., Knopke, C. (1991) Results Obtained from Serological Investigations of Red, Roe, and Fallow Deer and Moufflon for Selected Infections

Monatshefte fur Veterinarmedizin, 46, 101-104 Haematoserological investigations of red, roe, and fallow deer as well as of moufflon provided evidence to the presence of reagents to brucellosis and of agglutinins to various Yersinia enterocolitica serovars in roe, red, and fallow deer or of reagents to paratuberculosis in roe deer, chlamydial antibodies in roe, red, and fallow deer as well as of reagents to Q-fever in roe deer. Examinations for tularaemia and LCM were negative in all species. The above findings are discussed in some detail under epizootiological aspects ANTIBODIES/ANTIBODY/brucellosis/CATTLE/CLOVEN-HOOFED GAME/DAMA-DAMA/deer/FALLOW DEER/INFECTION/INFECTIONS /JOHNES DISEASE/PARA-TUBERCULOSIS/paratuberculosis/PREVALENCE/Q-FEVER/roe deer/WILD-LIVING RUMINANTS/YERSINIA-ENTEROCOLITICA

293 Angus, K.W. (1990) Intestinal Lesions Resembling Paratuberculosis in A Wild Rabbit (Oryctolagus-Cuniculus)

Journal of Comparative Pathology, 103, 101-105 LESIONS/ORYCTOLAGUS-CUNICULUS/paratuberculosis/rabbit/WILD/WILD RABBIT

294 Mokresh, A.H., Czuprynski, C.J., Butler, D.G. (1989) A Rabbit Model for Study of Mycobacterium-Paratuberculosis Infection

Infection and Immunity, 57, 3798-3807 INFECTION/MODEL/Mycobacterium paratuberculosis/Mycobacterium paratuberculosis infection/MYCOBACTERIUM-PARATUBERCULOSIS/MYCOBACTERIUM-PARATUBERCULOSIS INFECTION/rabbit

295 Shulaw, W.P., Gordon, J.C., Bechnielsen, S., Pretzman, C.I., Hoffsis, G.F. (1986) Evidence of Paratuberculosis in Ohio White-Tailed Deer, As Determined by An Enzyme-Linked-Immunosorbent-Assay

American Journal of Veterinary Research, 47, 2539-2542 deer/paratuberculosis/white-tailed deer

296 Williams, E.S., Snyder, S.P., Martin, K.L. (1983) Experimental-Infection of Some North-American Wild Ruminants and Domestic Sheep with Mycobacterium-Paratuberculosis - Clinical and Bacteriological Findings

Journal of Wildlife Diseases, 19, 185-191

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DOMESTIC SHEEP/experimental infection/EXPERIMENTAL-INFECTION/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/RUMINANTS/SHEEP/WILD/WILD RUMINANTS

297 Williams, E.S., Snyder, S.P., Martin, K.L. (1983) Pathology of Spontaneous and Experimental-Infection of North-American Wild Ruminants with Mycobacterium-Paratuberculosis

Veterinary Pathology, 20, 274-290 experimental infection/EXPERIMENTAL-INFECTION/Mycobacterium paratuberculosis/MYCOBACTERIUM-PARATUBERCULOSIS/PATHOLOGY/RUMINANTS/WILD/WILD RUMINANTS

298 Vogel, O. (1977) Paratuberculosis in A Dog

Berliner und Munchener Tierarztliche Wochenschrift, 90, 419-421 dog/paratuberculosis

299 Hillerma, K. (1966) A Disease Resembling Paratuberculosis (Johnes Disease) in Roe Deer (Capreolus Capreolus L) - An Aetiological and Patho-Anatomical Study Acta Veterinaria Scandinavica, 7, 330-& deer/DISEASE/JOHNES DISEASE/JOHNES-DISEASE/paratuberculosis/roe deer

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badger, 1, 73, 94, 120 boar, 1, 3, 4, 5, 23, 32, 49, 73 deer, 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 31, 32, 33, 36, 37, 38, 40, 41, 42, 44, 45, 47, 49, 50, 51, 52, 53, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 66, 67, 68, 69, 70, 72, 73, 75, 76, 77, 78, 79, 80, 81, 82, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 97, 98, 100, 101, 102, 103, 106, 107, 109, 110, 111, 112, 113, 114, 115, 117, 118, 120, 121, 123, 124, 125

dog, 1, 15, 95, 102, 110, 125 elephant, 1 fox, 1, 7, 94, 103

hare, 1, 7, 45, 53, 94 hedgehog, 1 hippo, 1 mole, 1, 65 mouse, 1, 13, 14, 28, 34, 35, 36, 42, 45,

47, 51, 53, 54, 59, 70, 78, 79, 80, 86, 93, 94, 98, 99, 105, 108, 114, 116, 117, 121, 122, 123

possum, 1, 51, 73 rabbit, 1, 3, 7, 10, 14, 21, 24, 26, 37, 39,

40, 48, 50, 51, 53, 54, 57, 58, 59, 68, 71, 73, 74, 76, 77, 83, 84, 86, 89, 90, 91, 92, 93, 94, 96, 100, 103, 105, 107, 109, 113, 117, 119, 122, 124

wild, 1 wolf, 1