FACULTY OF VETERINARY MEDICINE DOCTORAL PROGRAMME IN CLINICAL VETERINARY MEDICINE UNIVERSITY OF HELSINKI Porcine Mycobacteriosis Caused by Mycobacterium avium subspecies hominissuis TANELI TIRKKONEN DISSERTATIONES SCHOLA DOCTORALIS SCIENTIAE CIRCUMIECTALIS, ALIMENTARIAE, BIOLOGICAE. UNIVERSITATIS HELSINKIENSIS 14/2017 YEB mycobacteriosis mycobacteriosis mycobacte mycobacteriosis MYCOBACTERIOSIS MYCO MYCO MYCO MYCOBACTERIOSIS mycobacteriosis mycobacteriosis MYCO mycobacteriosis MYCOBACTERIOSIS mycobacteriosis mycobacteriosis MYCOBACTERIOSIS mycobacteriosis mycobacteriosis mycobacteriosis mycobacteriosis mycobacteriosis M Y C O MYCO
Transcript
FACULTY OF VETERINARY MEDICINEDOCTORAL PROGRAMME IN CLINICAL VETERINARY MEDICINE UNIVERSITY OF HELSINKI
Porcine Mycobacteriosis Caused by Mycobacterium avium subspecies hominissuis
TANELI TIRKKONEN
dissertationes schola doctoralis scientiae circumiectalis, alimentariae, biologicae. universitatis helsinkiensis 14/2017
14/2017
Helsinki 2017 ISSN 2342-5423 ISBN 978-951-51-3507-0
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Faculty of Veterinary Medicine
University of Helsinki
Helsinki, Finland
Porcine mycobacteriosis caused by Mycobacterium avium subspecies hominissuis
Taneli Tirkkonen
Academic dissertation
To be presented, with the permission of the Faculty of Veterinary Medicine of the University of Helsinki, for public examination in Auditorium XV
of the Helsinki University Main Building, Fabianinkatu 33, Helsinki, on 16 June 2017, at 12 noon.
Helsinki 2017
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ISBN 978-951-51-3507-0 (paperback)ISBN 978-951-51-3508-7 (PDF)
Dissertationes Schola Doctoralis Scientiae Circumiectalis, Alimentariae, BiologicaeISSN 2342-5423 (print)
ISSN 2342 5431 (online)
Hansaprint Turenki 2017
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Porcine mycobacteriosis caused by Mycobacterium avium subspecies hominissuis
Taneli Tirkkonen, DVM, Specialist in production animal medicine,
Faculty of Veterinary Medicine, University of Helsinki
Director of Studies: Prof. Timo Soveri, Faculty of Veterinary Medicine,
University of HelsinkiEmeritus Prof. Hannu Saloniemi, Faculty of Veterinary Medicine,
University of Helsinki
Supervised by: Prof. Olli Peltoniemi, Faculty of Veterinary Medicine,
University of HelsinkiDos. Faik Atroshi, Faculty of Veterinary Medicine,
University of HelsinkiProf. Anna-Maija Virtala, Faculty of Veterinary Medicine,
University of HelsinkiDos. Hanna Soini, Mycobacterial Reference Laboratory,
National Institute for Health and Welfare Turku, FinlandDos. Terhi Ali-Vehmas, Faculty of Veterinary Medicine,
University of Helsinki, Finland
Reviewers: David J Taylor, Professor Emeritus, School of Veterinary Medicine,
College of Medical, Veterinary and Life Sciences, University of Glascow, Scotland, The United Kingdom
Joaquim Segales, Associate Professor, CReSA, The Veterinary School of the Universitat Autònoma de Barcelona, Spain
Opponent: Per Wallgren, Professor, National Veterinary Institute (SVA),
Uppsala, Sweden
Dedicated to my dear wife Ilona
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Abstract ................................................................................................................ 6List of Original Publications ................................................................................... 8The author’s contribution ....................................................................................... 9Abbreviations ......................................................................................................... 91. Literature Review............................................................................................. 10 1.1. Description of the mycobacteria ............................................................ 10 1.1.1. Environmental mycobacteria ...................................................... 10 1.1.2. Porcine-related mycobacteria ...................................................... 11 1.2. Disease risk of mycobacteria from an epidemiological viewpoint .......... 12
......................................... 13 1.4. Current methods and recommendations in pork meat control ............... 16 1.5. Serological response and monitoring of porcine mycobacterial infections ................................................................................................ 172. Aims of the study ............................................................................................. 203. Materials and methods .................................................................................... 20 3.1. Samples and experimental design ........................................................ 20 3.1.1. Piggery environmental samples and experimental design .......... 20 3.1.2. Pig organs and humans samples ................................................ 21 3.2. Typing of mycobacteria ......................................................................... 22 3.3. .................................................. 224. Results and discussion .................................................................................... 23 4.1. Viable mycobacteria in piggeries ........................................................... 23 4.2. Typing of mycobacteria by IS1234 RFLP and MIRU-VNTR methods ... 26
Mycobacterium subspecies in pig tissues using real-time quantitative PCR. .................................................................... 285. Conclusions ..................................................................................................... 296. Acknowledgements.......................................................................................... 297. References ...................................................................................................... 31 Original Publication 1 .................................................................................... 44 Original Publication 2 .................................................................................... 53 Original Publication 3 .................................................................................... 61 Original Publication 4 .................................................................................... 68
Contents
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Abstract
More than 150 species of mycobacteria are described, most being opportunistic path-ogens and all representing a risk for human and animal health. Human infections de-rived from environmental mycobacteria are increasing in both industrialized and de-veloping countries. The most susceptible groups are children, the elderly and those, including animals, with immunocompressive conditions. Drug therapy for myco-bacteriosis is dif cult and not always successful. nfections caused by drug-resistant mycobacteria can be life threatening also for healthy adults and thus represent a real risk for humans. Environmental mycobacterial infections of pigs are usually without clinical signs and the lesions are mainly detected at slaughter. Mycobacterium-infect-ed pork can pass for human consumption due to the poor sensitivity of visual meat control at slaughterhouses, and mycobacteria in pigs also cause economic losses due to condemnation of carcasses. The main challenge is represented by evaluation of the hygiene risk associated with using mycobacteria-contaminated pork.
Most environmental mycobacteria species have been isolated from sources such as water, swimming pools, soil, plants and bedding material. n our study mycobacterial growth in piggeries was identi ed in all bedding materials, sawdust, straw, peat and wood chips in most cases, and water and food samples in many cases, and only oc-casionally in dust and on wall surfaces. The maximum number of mycobacteria was almost 1 billion (109) per gram of bedding, which is close to the maximum concen-tration in any growth media. Mycobacteria can multiply in piggeries and contami-nate feed and water. solation of mycobacteria from pig faeces can be considered an indicator for risk of human infection.
Environmental mycobacteriosis in humans and pigs is mainly caused by M. avi-um subsp. hominissuis. There is little evidence of direct transmission from animals to humans, but particular strains can be recovered from both humans and pigs. n our studies, identical mycobacteria and M - T ngerprints of porcine and human origins were evident. nterspecies clusters were more common than intraspe-cies clusters using both methods. Therefore, we concluded that pigs act as a reservoir for virulent M. avium strains and the vector for transmission of infections in humans to pigs, and vice versa, may have an identical source of infection. Culturing mycobacteria is the gold standard for diagnosis, but detection of environ-mental mycobacteria based on cultivation and biochemical methods can take several weeks. Culture-independent, rapid and accurate techniques for detecting mycobac-teria in food and feed chains are urgently needed. n this work we developed a rapid and accurate real-time quantitative PCR for detecting environmental mycobacteria in bedding materials and pig organs.
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Conclusion: Mycobacteria can multiply in bedding materials and the consequent heavy contami-nation can cause simultaneous infections in pigs. Mycobacterial D was found in pig organ samples, including those without lesions, and similar strains were found from humans and pig organ samples, which suggests that mycobacteria can be trans-mitted between humans and pigs.
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List of Original Publications
. Pakarinen , ieminen T, Tirkkonen T, Tsitko , li- ehmas T, eubauer P, Salkinoja-Salonen M, 2007: Proliferation of mycobacteria in a piggery environmentrevealed by Mycobacterium-speci c real-time quantitative PCR and 1 S rR sandwich hybridization. Veterinary Microbiology 120, 105-112.
. Tirkkonen T, Pakarinen , Moisander -M, M kinen , Soini H, li- ehmas T, 2007: High genetic relatedness among Mycobacterium avium strains isolated from pigs and humans revealed by comparative S 1245 RFLP analysis. Veterinary Micro-biology 125, 175-181.
. Tirkkonen T, Pakarinen , Rintala E, li- ehmas T, Marttila H, Peltoniemi , M kinen , 2010: Comparison of ariable- umber Tandem-Repeat Markers typing and S 1245 Restriction Fragment Length Polymorphism ngerprinting of Mycobac-terium avium subsp. hominissuis from human and porcine origins. cta eterinaria Scandinavica 52:21.
. Tirkkonen T, ieminen T, li- ehmas T, Peltoniemi , ellenberg , Pakarinen , 201 : uanti cation of Mycobacterium avium subspecies in pig tissues by real-
time quantitative PCR. cta eterinaria Scandinavica 55:2 .
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The author’s contribution
Paper : TT planned and performed the relevant sampling procedure and participated in writing the paper, particularly regarding veterinary medicine and zoonotic aspects.
Paper : TT performed strain selection for the porcine originating strains for the RFLP typing, interpreted the results and wrote the paper together with the co-authors.
Paper : TT participated in the study design, sampling, analysis and interpretation of the results, TT wrote the paper and was the corresponding author.
Paper : TT participated in the study design, sampling, analysis and interpretation of the results, TT wrote the paper and was the corresponding author.
AbbreviationsDS acquired immune de ciency syndrome
D discriminatory indexD deoxyribonucleic acidDSM Deutsche Sammlung von MikroorganismenEL S -test Enzyme-Linked mmunosorbent ssay EXP exponentIS1245 insertion sequence 1245IS 1245 RFLP IS 1245 Restriction Fragment Length Polymorphism ngerprintingM Mycobacterium avium subsp. aviumM C Mycobacterium avium complex M H Mycobacterium avium subsp. hominissuisM R genetic interspersed repetitive units of Mycobacteria
The genus Mycobacterium comprises over 150 species of aerobic, non-spore-forming, non-motile, rod-shaped acid-fast bacilli. The mycobacteria include diverse species, ranging from environmental saprophytes and opportunistic invaders to obligate path-ogens. M. africanum, M. bovis, M. canettii, M. caprae, M. microti, M. pinnipedii, M. tuberculosis, M. leprae and M. lepraemurium are obligate pathogens of humans and animals (Portaels 1995, aerewijck et al. 2005). lthough some pathogenic mycobac-teria exhibit a particular host preference, they can occasionally infect other species. Mycobacteria can multiply intracellularly and diseases in humans and animals are usually chronic granulomatous and progressive infections (Hibiya et al. 2011, Koh et al. 2002). bligate pathogens, shed by infected animals, can also survive in the envi-ronment for extended periods (Portaels 1995, aerewijck et al. 2005).
1.1.1. Environmental mycobacteria
Environmental mycobacteria are a heterogeneous group of slow-growing species that include saprophytes and opportunistic pathogens (El-Sayed et al. 201 , Portaels 1995, Salem et al. 2012, aerewijck et al. 2005). lycopeptidolipids in cell walls render mycobacteria hydrophobic and resistant to adverse environmental in uences and dis-infectants, including chlorine. Some Mycobacterium spp. are resistant to, and even multiply at, high temperatures ( aerewijk et al. 2005). The lipid-rich layer of the cell wall increases bio lm formation in some mycobacteria species (Freeman et al. 200 , ohansen et al. 2009, Recht et al. 2000, Recht et al. 2001, amazaki et al. 200 ). The
ability to form bio lms is linked with virulence and resistance in bacteria (Carter et al. 200 , ohansen et al. 2009). Mycobacteria have been isolated from various envi-ronments, including soil, water, aerosols, protozoa, deep litter and fresh vegetation from all over the world ( iet et al. 2005, Eisenberg et al. 2010). pportunist envi-ronmental mycobacteria can cause tuberculous lesions and disseminate infections in humans and animals (Kunze et al. 1992). Human infections due to environmental my-cobacteria are increasing in both industrialized and developing countries. The most susceptible risk groups are children, the elderly and those, including animals, with immunosuppressive conditions (Falkinham 199 , Hiller et al. 201 , aerewijk et al. 2005). Drug therapy of mycobacteriosis is dif cult and not always successful. nfec-tions caused by drug-resistant mycobacteria can be life threatening also for healthy
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adults and thus they represent a relevant risk for humans (Eriksson et al. 2001, Hiller et al. 201 , ylen et al. 2000). eneralized disease in birds and poultry (Pavlic et al. 2000), pigs (Eisenberg et al. 2012), cattle (M bius et al. 200 ), cats and dogs (Thorel et al. 2001), horses, foxes, cervids, game (Moser et al. 2011) small rodents, insecti-vores (Fischer et al. 2000) and other species caused by members of the Mycobacte-rium avium complex have been reported (Pavlik et al. 2005, Thorel 1997).
1.1.2. Porcine-related mycobacteria
Mycobacterium avium subsp. hominissuis belongs to the Mycobacterium avium com-plex and is the most common environmental mycobacterium in infections of humans and pigs ( gdestein et al. 2011, gdestein et al. 2012, Domingos et al. 2009, wa-moto et al. 2012, ohansen et al. 2007, Koh et al. 2002, Mijs et al. 2002, Pavlik et al. 200 , Stepanova et al. 2012). The condition of the host can differ between humans and swine: human hosts often have the infection in their lungs ( shford et al. 2001, arzembowski et al. 2008, ohansen et al. 2009) whereas, pigs are infected usually
through oral ingestion. Tubercles can usually be found in the retropharyngeal, sub-maxillary and cervical lymph nodes of pigs (Matlova et al. 2005, Thorel et al. 2001). nfections in aborted foetuses and in the genital organs of pigs, and decrease in growth
rate as well as increased mortality, have been reported ( ille et al. 197 , Eisenberg et al. 2012, ellenberg et al. 2010). Hepatic lesions are observed in systemically in-fected pigs. However, mycobacterial pathogenesis is poorly understood (Hibiya et al. 2008, Hibiya et al. 2010). ohansen et al. 2009 speculated that pigs could become infected only when a large infective dose of M. avium strains occurs in their living environment. Mycobacteria in a single pig were reported to be multiple variants be-cause mycobacteriosis is of environmental origin (Eisenberg et al. 2012, ellenberg et al. 2010). n pigs M. avium infections can be persistent without any clinical signs, but may nonetheless represent economic losses for the farmer because meat from in-fected animals is considered unsuitable for human consumption and is condemned (Pavlik et al. 200 ). During the last decade, the prevalence of M. avium subsp. homi-nissuis in slaughtered pigs increased worldwide (El-Sayed et al. 201 , M bius et al. 200 ). There is also a recent study of Mycobacterium bovis in domestic free range pigs in Spain (Cardoso-Toset et al. 2017), but so far there is very limited knowledge on this theme from orth-European pigs. Prevalence of mycobacterial-like lesions in Finnish slaughter pigs in the period 1998-2012 is shown in Fig. 1.
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Figure 1. Prevalence of mycobacterial-like lesions in Finnish slaughter pigs during 1998-2012. Bars illustrating cumulative annual slaughter pigs and the line % mycobacteria. Finnish Food Safety Authority Evira meat control results database 2013.
1.2. Disease risk of mycobacteria from an epidemiological viewpoint
The reservoirs of infective environmental mycobacteria remain in doubt, but the ma-jority of the species have been isolated from various aquatic and terrestrial environ-ments ( rqueta et al. 2000, Engel et al. 1978, Falkinham 199 , Falkinham 2002, Matlova et al. 200 , Matlova et al. 2005, von, Reyn et al. 199 , ajko et al. 1995,
aerewijk et al. 2005). Drinking water was shown to be a possible source of environ-mental mycobacteria that contributed to both human and animal infections (Falkin-ham et al. 2001, Hilborn et al. 2008, ohansen et al. 2009, ishiuchi et al. 2007). Environmental mycobacteria survive in water distribution systems because they are usually resistant to treatment with ozone and chlorine, especially when growing as multicellular aggregates (Hilborn et al 200 , ohansen et al. 2009, Steed et al. 200 , Taylor et al. 2000, aerewijck et al. 2005). Environmental mycobacteria may also survive in water within amoebae (Hilborn et al. 200 , ohansen et al. 2009, aere-wijck et al. 2005).
t has been suggested that drinking water may be an important reservoir of infec-tive mycobacteria, especially for humans ( ronson et al. 1999, Falkinham 199 ), whereas bedding material has proved to be an important source of porcine infections ( lvarez et al. 2011, Matlova et al. 200 , Matlova et al. 2005). Mycobacterial infec-tions have a zoonotic character and similar types of mycobacteria strains have been found in pigs and humans (Komijn et al. 1999, M bius et al. 200 ). t cannot be ruled out that mycobacterial infection of humans may originate from piggeries (Komijn et al. 1999, Komijn et al. 2000, M bius et al. 200 ). Contaminated faecal material in bedding and mycobacteria in raw pork represent potential food safety hazards ( lva-rez et al. 2011, Komijn et al. 1999, Matlova et al. 2005, Matlova et al. 2004, Möbius et al. 200 , ohansen et al. 2014).
More knowledge about the modes of transmission in both animals and humans is required for the control of mycobacterioses ( gdestein et al. 2012, iet et al. 2005, Thegerström et al. 2005). ew methods for the identi cation, genetic pro ling, and rapid real-time quanti cation of environmental mycobacteria are needed to trace en-vironmental reservoirs of human and animal mycobacteriosis and for assessing the risk they may represent.
cid-fast staining is used to differentiate mycobacteria from other bacteria. n pigs, acid-fast bacilli can be found from caseous malformations in lymph nodes, kidneys, liver and spleen (Eisenberg et al. 2012, Hiller et al. 201 , an nger et al. 2010, ffer-mann et al. 1999), but also from Rhodococcus equi and infections due to other acid-fast bacilli (Dvorska et al. 1999, Eisenberg et al. 2012, Hiller et al. 201 , Komijn et al. 2007, Pate et al. 2004). However, mycobacteria can be present in porcine lymph nodes without any visible lesions (Dvorska et al. 1999, Hiller et al. 201 ). Differ-entiation of pathogenic mycobacteria relies on cultural characteristics, biochemical tests, animal inoculation, chromatographic analyses and molecular techniques. n addition, mycobacteria associated with opportunistic infections can be differentiated on the basis of pigment production, optimal incubation temperature and growth rate. Pathogenic mycobacteria grow slowly on solid media and colonies are not evident until cultures have been incubated for at least three weeks (Fig. 2).
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Figure 2. Mycobacterium avium growing for three weeks on Löwenstein-Jensen agar.
n contrast, the colonies and growth in broth of rapidly growing saprophytes are visible within days (Fig. ).
Figure 3AM. chelonae
time (h)
turb
idity
CFU kpl/ml
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4x107
4x106
4x105
4x104
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Figure 3 (A & B). M. chelonae growth curve in Middlebrook broth (Fig. 3A). The linear
intervals) (Fig. 3B), Ali-Vehmas 1998 (personal communication, method described in Ali-Vehmas 1998).
However, identi cation of the members belonging to the M. avium complex us-ing biochemical testing can take up to several weeks (Slana et al. 2010, Springer et al. 199 ). Some mycobacterial isolates cannot be identi ed using biochemical dif-ferentiation because their biochemical pro les are very dif cult to interpret ( unn-Moore et al. 199 ).
Culturing mycobacteria is the gold standard of diagnosis, but molecular tests are also used ( gdestein et al. 2011, nderlied et al. 199 , Thorel et al. 2001, Turenne et al. 2007). D probes, complementary to species-speci c sequences of rR , are commercially available for identifying some mycobacterium species. ucleic acid ampli cation procedures, including PCR, were and are being developed for the detec-tion of mycobacteria in environmental and tissue samples (Khan et al. 2004, Kox et al. 1995, Pakarinen 2008, Shrestha et al. 200 , Talaat et al. 1997, Telenti et al. 199 ) and D restriction endonuclease analyses (D ngerprinting) have been used in epidemiological studies over recent decades by several authors (Bauer et al. 1999, Collins et al. 1994, ohansen et al. 2007). However, these techniques are laborious, expensive or not suitable and not sensitive enough for mycobacteria.
Figure 3BM. chelonae
turb
idity
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1.4. Current methods and recommendations in pork meat control
European law (E 854 2004) describes the procedure for meat inspection at slaugh-ter, including palpation and incision of the lymph nodes, including the procedures for detection of porcine mycobacterial-like infections (Fig. 4, Fig. 5).
Figure 4. Palpation of porcine livers at a slaughterhouse meat control facility.
A presumptive mycobacterial lesion in a slaughtered pig liver.
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isual detection is not sensitive or accurate (Hiller et al. 201 , Komijn et al. 2007, isselink et al. 2010). Similar lesions can be caused by other hazardous food safety
microbes (Faldyna et al. 2012, Hamilton et al. 2002, Hiller et al. 201 , Komijn et al. 2007, Pavlik et al. 200 ) and mycobacteria can also be isolated from lymph nodes that do not exhibit visible lesions ( ffermann et al. 1999, isselink et al. 200 ). The tuberculin test has been established as a screening method for the detection of myco-bacteria-positive pigs prior to slaughter, but this method has low sensitivity (Faldyna et al. 2012, Francis et al. 1978). Therefore, there is a great need to develop new ap-proaches for the detection of mycobacteriosis in pigs (Faldyna et al. 2012).
1.5. Serological response and monitoring of porcine mycobacterial infections
Several authors have suggested that M. avium may cause porcine infections and may be a potential food safety hazard for humans. Detection of mycobacterial disease in a live animal is often very dif cult. Therefore, must the presence of disease be deter-mined by post mortem examination. nfection in swine exposed to M.avium is usually associated with the lymph nodes of the head, liver and the digestive tract and rarely spreads to other locations Caseous lesions can also be found in porcine kidneys and spleen (Hiller et al. 201 , an ngen et al. 2010, ffermann et al. 1999, Thoen et al. 200 ), but the formation of lesions caused by M. avium infections may take several months in pigs ( isselink et al. 200 ). lceration and necrosis of the skin have also been observed in M. avium infections ( gdestein et al. 2012). Post mortem visual in-spection of lymph nodes and livers can give a high number of both false positive and negative results because it is a non-speci c and non-sensitive test respectively (Ei-senberg et al. 2012, Faldyna et al. 2012, Hiller et al. 201 , Komijn et al. 2007, is-selink et al. 200 , isselink et al. 2010). The sensitivity of visual meat inspection has been found to be highest in pigs infected by M. avium at an age of between 2.5 and 4.5 weeks, but is low in pigs infected at the age of 18 weeks ( isselink et al. 200 ). Mycobacterial infections without any visible lesions have been reported (Brown et al. 1979, Dvorska et al. 1999, Hiller et al. 201 , ffermann et al. 1999, isselink et al. 200 ). Repeated infections by M. avium may cause an altered immune response and inhibit the formation of lesions in pigs. t has been reported that pigs that were experimentally infected three times had low number of lesions in their lymph nodes ( isselink et al. 200 ). n such a case mycobacteria-infected pigs can pass the visual post mortem inspection ( isselink et al. 200 ). lso other bacteria, such as Rhodoc-occus, have been isolated from pathological, mycobacteria-like lesions in pigs (Dvor-ska et al. 1999, Faldyna et al. 2012, Hiller et al. 201 , Komijn et al. 2007, Pavlik et al. 200 ). Cross-contamination with other pathogens, for example salmonella (Hamilton et al. 2002), has also been reported. p to two thirds of the reported granulomatous
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malformations in porcine lymph nodes result for reasons other than mycobacterial infection (Hiller et al. 201 ).
The tuberculin skin test is the standard method for diagnosis of mycobacterial infections in living animals, but the sensitivity of the test is low (Monagham et al. 1994, Stepanova et al. 2011). mmunological responses detected with the tuberculin skin test can be applied at herd level only and they are relatively inaccurate in that case (Eisenberg et al. 2012). Rather poor correlations were reported between results of the EL S -test (Eisenberg et al. 2012), gamma interferon release assay (Faldyna et al. 2012) and the tuberculin skin test (Eisenberg et al. 2012, Faldyna et al. 2012).
Several methods based on serological response of mycobacterial infections have been published (Boadella et al. 2011, Faldyna et al. 2012). mmunological response, such as the interferon release assay, may be more sensitive than the tuberculin test, and the interferon assay can be used to diagnose M. avium infections in live and nat-urally-infected pigs (Faldyna et al. 2012). M. avium induced central memory cells in porcine infections, but a six month period at least was needed to detect cell-mediated immunity to M. avium in pigs (Stepanova et al. 2011). This is problematic because most nisher pigs are slaughtered around the age of six months. Moreover, the in vitro re-stimulation interferon gamma production was decreased (Stepanova et al. 2011). Some lymphocyte release may cause long-term immunity in pigs infected with M. avium (Stepanova et al. 2011). Recently, several mycobacteria-speci c tests were ap-plied to describe the correlations among abortions, re-breedings or stillbirths and my-cobacterial infections (Eisenberg et al. 2012.). Stepanova et al. (2011) concluded that interferon gamma and lymphocyte transformation may represent a speci c method for the identi cation of individual M. avium infections in pigs, but more detailed studies are needed (Stepanova et al. 2011). The results of Stepanova et al. (2011) indicate that the interferon gamma release assay and lymphocyte transformation test can, in some cases, be used for the identi cation of M. avium-infected pigs (Stepanova et al. 2011).
Hiller et al. (201 ) applied a mycobacterial-speci c enzyme-linked immunosorb-ent assay (EL S ) to test for the presence of M. avium antibodies in blood samples of slaughtered pigs in the etherlands and ermany. The presence of M. avium anti-bodies was detected to estimate the prevalence of mycobacterial infections at the herd level. The M. avium EL S test was validated to identify M. avium positive farms. The validation results in this research showed that the sensitivity of an individual test was low and only 20 of the bacteriologically positive herds could be identi ed when blood samples were tested. The low sensitivity at the herd level was sup-posed to be due to the presence of infections with other M. avium serotypes that have reduced immunity towards the antigens tested. Closely related M. avium subsp. avium and subsp. hominissuis showed different capacities to stimulate the porcine immune system. M. avium subsp. hominissuis showed low cell-mediated immunity with high individual variability (Dvorska et al. 2004, Hiller et al. 201 , Stepanova et al. 2012). However, Hiller et al. (201 ) concluded that serological screening using a M. avium-
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speci c EL S -test was able to identify bacteriological M. avium-positive herds and pig populations at risk of M. avium infections. The discriminatory power between infected and non-infected farms using the EL S test could be improved with addi-tional mycobacterial antigens (Hiller et al. 201 ).
M. avium subsp. hominissuis is a weaker pathogen than M. avium subsp. avium (Faldyna et al. 2012). The developed commercial serological tests are not suitable for detecting M. avium subsp. hominissuis (Domingos et al. 2009, Faldyna et al. 2012). The immunological parameters of M. avium subsp. avium and M. avium subsp. hom-inissuis have been compared and the systemic immune response during experimen-tal mycobacteriosis of pigs has been measured. Release of interferon gamma can be detected after ve weeks in M. avium subsp. avium and subsp. hominissuis-infected pigs, but not in every individual ( gdestein et al. 2012). The cell-mediated immuno-logical response of M. avium subsp. hominissuis is signi cantly weaker when com-pared with that of M. avium subsp. avium. The release of mycobacterial speci c an-tibodies or gamma interferon was low and variable in M. avium subsp. hominissuis infections compared with that in M. avium. subsp. avium infections (Stepanova et al. 2012). The release of pro-in ammatory cytokines from macrophages was nota-bly higher during in vitro experiments when the inductor was M. avium subsp. avium than with M. avium subsp. hominissuis. n addition, the quantity of M. avium subsp. hominissuis was at least 1000 fold lower than that of M. avium subsp. avium in in-fected gastro-intestinal tissues of pigs (Slana et al. 2010, Stepanova et al. 2012). The macrophage response to M. avium subsp. hominissuis infections was reported to be signi cantly weaker than the response to M. avium subsp. avium infections in vitro. M. avium subsp. hominissuis-infected macrophages also showed weaker induction of pro-in ammatory cytokines and chemokines (Stepanova et al. 2012). The immu-nological response also differed among different M. avium subsp. hominissuis geno-types (El-Sayed et al. 201 , Stepanova et al. 2012, Thegerström et al. 2012). t can be concluded that M. avium subsp. hominissuis can only induce a weak cell-mediat-ed immunity. However in some cases, positive results were obtained for the interfer-on gamma release assay of M. avium subsp. hominissuis infections of pigs without detecting speci c antibodies (Stepanova et al. 2012). The interferon gamma release assay may represent an effective tool for discrimination of M. avium subsp. avium-infected pigs, but be too inaccurate for detection of M. avium subsp. hominissuis in-fections (Stepanova et al. 2012).
Moreover, most of the immunological tests for mycobacterial infections in pigs have been applied to M. avium subsp. avium, but the majority of M. avium strains in mycobacteriosis of pigs are M. avium subsp. hominissuis (Domingos et al. 2009, ar-rido et al. 2010, Shitaye et al. 200 , Stepanova et al. 2011, Stepanova et al. 2012). n the porcine mycobacteriosis caused by M. avium subsp. hominissuis the infected pigs produced signi cantly lower levels of mycobacterial-speci c antibodies and interfer-on gamma as compared with M. avium. subsp. avium-infected pigs (Stepanova et al.
20
2012). The immunodominant antigens in different mycobacterial species may not be cross-reactive (Faldyna et al. 2012) and therefore more reliable detection methods are required for the identi cation of mycobacterial infections in routine diagnosis of pigs (Faldyna et al. 2012, isselink et al. 200 ).
2. Aims of the study
1. Examination and quanti cation of environmental samples from piggeries for the presence of mycobacteria in order to develop and apply D and rR -based methods for the detection of potentially hazardous mycobacteria in the piggery environment.
2. Sequencing (1 S rD ) and typing (RFLP, M R - TR) of the myco bacterial isolates of porcine origin and comparison of them with human isolates to examine the similarity of the M. avium strains originating from slaughter pigs and human cases regarding public health aspects in Finland.
. Parallel application of Restricted Fragment Length Polymorphism (RFLP) patterns and ariable- umber Tandem Repeat ( TR) typing of genetic inter-spersed repetitive units of mycobacteria (M R s).
4. uanti cation of Mycobacterium avium subspecies in pig tissue material using real-time quantitative PCR.
3. Materials and methods
3.1. Samples and experimental design3.1.1. Piggery environmental samples and experimental design
Piggery environmental samples were collected from birth to slaughter farms with high condemnation rates for environmental mycobacteria. ll laboratory work was done according to strict hazard class two working standards. The total viable mycobacteria contents were analyzed from environmental samples taken from ve piggeries, about 15-20 samples per piggery, totalling 94 samples, with 2 parallel samplings. Regard-
21
ing meat control, the prevalence of tuberculous lesions in the selected ve piggeries was more than 4% during 2002-2004.
The experimental design for piggery environmental samples is shown in Fig. . The results were con rmed using 1 S rR sandwich hybridization.
Figure 6. Experimental design for piggery environmental samples.
3.1.2. Pig organs and humans samplesPig organ samples were collected in Finland from the slaughter line after meat in-spection. 1 M. avium-positive pig organ samples from these were used (papers and ). Seven additional samples of pig organs from the etherlands were kindly provided by erard ellenberg (paper ).
Thirteen clinical M. avium isolates collected from Finnish human patients in 2001-2004 were randomly selected from the strain collection of the ational Public Health nstitute. The origin of the isolates was sputum, bronchial washings and lung biopsies (papers and )
M. avium strains from pigs and humans, in addition to reference strains, were stored at the culture collection of the Mycobacterial Reference Laboratory, The
Sample suspension (100 mg sample + 5 ml H2O) homogenized
Incubated for 30 min with 550 μl of 50% H2SO4
neutralized with NaOH centrifuged The pellet was washed twice with H2O
+H2O+sterile sand
qPCR
22
3.2. Typing of mycobacteria
Mycobacteria strains (1 pig and 1 human) were identi ed by direct sequence determination of 1 S rR gene fragments and genotyped both with IS1245 restric-tion fragment length polymorphism (RFLP) and ariable- umber Tandem Repeat ( TR) of genetic interspersed repetitive units of mycobacteria (M R s). denti ca-tion and RFLP and M R - TR typing were carried out in the mycobacterial refer-ence laboratory, ational Public Health institute in Turku, Finland. The discrimina-tory indices (D ) of RFLP and M R s were calculated.
Description of the samples in real-time qPCR is shown in Table 1. Samples were kindly provided by . ellenberg.
Sample Description
Pig 4, sample 1. Lesion
Pig 4, sample 2. Outside the lesion
Pig 9-5577, sample 1. Lesion
Pig 9-5577, sample 2. Outside the lesion
Pig Austria 3, sample 1. Lesion
Pig Austria 3, sample2. Outside the lesion
Pig 187, sample 1. Lesion
Pig 187, sample 2. Outside the lesion
solation of mycobacterial D from tissue specimens was originally attempted according to a standard protocol, but the quantity of isolated mycobacterial D was low. Therefore, the protocol was modi ed to increase the mycobacterial cell wall lysis by digesting the tissue at 5 C with Proteinase K under agitation at 1 0 rpm for 1 h. This novel modi cation improved markedly the amount of isolated myco- bacterial D . The detailed protocol is described in paper .
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4. Results and discussion4.1. Viable mycobacteria in piggeries
Mycobacterial growth was found in all bedding materials: sawdust, straw, peat and wood chips in most cases, water and food samples in many cases, but rarely in dust and spider webs. (Fig. 7( B), 8( B), 9( B), 10( B), 11( ,B C)), (Tab. 2).
Figure 7 (A&B). Mycobacterial growth was found in over 60% of used bedding material samples inside the piggeries.
material samples outside the piggeries.
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the piggeries.
Figure 10 (A&B). Mycobacteria grew in approximately 20 % of water samples inside piggery.
25
growth was found within eight weeks of cultivation in ventilation dust, colostrum or spider webs, but some growth was registered in spider webs and ventilation dust after three months of cultivation.
Result, mycobacterial growth
Used bedding inside piggery (7, A&B) >60 % of samplesUnused bedding outside piggery (8, A&B) appr. 35 % of samplesPig feed inside the piggery (9, A&B) appr. 25 % of samplesPig drinking water inside the piggery (10, A&B) appr. 20 % of samples
Ventilation dust and spider web (11, A&C) 8 weeks negative, 3 months some
Colostrum (11B) negative in 8 weeks and 3 months
Table 2. Mycobacterial growth in piggery samples.
Concentration of the mycobacteria in pig environmental samples was 105 to 107 in unused and 108 cells per gram of dry weight at its highest in used bedding materials when Mycobacterium-speci c hybridization probes were used for detection. Myco-bacterial strains contain usually one or two 2 1 S rR gene copies per cell (Klap-penbach et al. 2001, Pakarinen 2008). Since rR is found mainly in living cells, the results con rm that mycobacteria are viable and proliferate in bedding materials
26
during their use in the pig-rearing environment. The routes of infection for environmental mycobacteria are unclear, but several
previously publications reported that bedding materials may be the source of infection in pigs (Matlova et al. 200 , Matlova et al. 2004, Matlova et al. 2005, Pakarinen 2008,
indsor et al. 1984) and mycobacteria from farm originating faecal materials can be found in drinking water (Bland et al. 2005, Pakarinen 2008). uantitative meth-ods are required for the detection of the high concentrations of potentially infective mycobacteria in the environment ( ichols et al. 2004, Pakarinen 2008). n this work we quanti ed mycobacterial concentration from piggery bedding materials, some-thing that had not been done earlier.
4.2. Typing of mycobacteria by IS1234 RFLP and MIRU-VNTR methods
M. avium isolates obtained from pig livers and clinical human samples were compared using the IS1245 RFLP analysis to evaluate the similarity be-tween the strains of human and swine origin. Nearly identical IS1245 RFLP patterns were found from M. avium isolates from pigs and humans. The
analysis.
27
ne human and one porcine strain clustered identically using both typing meth-ods, indicating that the strains were clonal. The same M. avium strains are able to in-fect both humans and pigs. ur results concerning the high degree of similarity be-tween M. avium subsp. hominissuis of human and porcine origins are in agreement with those of other studies (Komijn et al. 1999, Möbius et al. 200 , Thorel et al. 2001). Similar RFLP-pro les for M. avium were also found in peat, human and swine samples ( gdestein et al. 2011, Bauer et al. 1999, ohansen et al. 2009, Matlova et al. 2005), supporting the hypothesis that human and pig infections derive from the same source in the environment. Their results are in accordance with those of other studies ( ohansen et al. 2007, ohansen et al. 2009, Möbius et al. 200 , rady et al. 2000).
Four different mycobacteria strains were simultaneously found in one pig, which could be the result of a heavy load of M. avium in the piggeries or high susceptibil-ity of some pigs to M. avium infections. Different M. avium strains in one pig were also reported by Eisenberg et al. (2010). ur study showed that piggery environ-ments could be important sources of mycobacterial infections for pigs and humans (Pakari nen et al. 2007). This is in accordance with several published reports ( rbeit et al. 199 , liveira et al. 2000, Pate et al. 2008, gdestein et al. 2011, gdestein et al. 2014).
The 1 S rR sequences are similar in M. avium strains from humans and pigs. They grouped together using different typing methods and are classi ed as M. avium subsp. hominissuis (Mijs et al. 2002). The S1245 insertion sequence is speci c to M. avium subsp. hominissuis and those strains have a high degree of S1245-based poly-morphism, which can be used to detect the genetic diversity among M. avium strains, ( uerrero et al. 1995, de Sequeira et al. 2005, ohansen et al. 2007, Eisenberg et al. 2012, El-Sayed et al. 201 ).
enotyping M. avium isolates has been done many times previously using RFLP (El-Sayed et al. 201 , Moravkova et al. 2008) and M R - TR typing (Despierres et al. 2012, El-Sayed et al. 201 , Pate et al. 2011, Romano et al. 2005). n our stud-ies the major RFLP clusters grouped together with M R - TR clusters. S1245 RFLP patterns and M R - TR mostly resulted interspecies rather than intraspe-cies clusters. The discriminatory index for S1245 RFLP was 0.98 and was 0.92 for M R - TR typing. ur results from the Finnish isolates are parallel with other publications (Eisenberg et al. 2012, nagaki et al. 2009, Pate et al. 2011). RFLP and M R - TR typing resulted in high levels of reproducibility and genetic diversity. RFLP and M R - TR typing methods show great potential for epidemiological mapping and determination of transmission pathways for M. avium subspecies (Ei-senberg et al. 2012, El-Sayed et al. 201 ).
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Mycobacterium subspecies
M. avium subspecies in pig tissues are dif cult, and sometimes impossible, to quantify using culture methods. To date there have been very few reports on the identi cation of M. avium strains directly from infected tissue without previous culture ( gdestein et al. 2011, Slana et al. 2010, Tell et al. 200 ). n this work we developed and applied culture-independent real-time quantitative PCR assays for detecting M. avium strains in pig tissues. Concentrations of mycobacteria cells per gram in organ tissues, with or without lesions, were from about 105. Similar results were reported earlier ( gdestein et al. 2011). The response of the qPCR assay to the logarithmic quantity of M. avium added to pig liver was linear, approximately in the range of 105 to 107 bacteria per gram (Fig. 1 .). The qPCR method results were con rmed with microscopy calcula-tion. Recently several qPCR methods have been developed for the detection of myco-bacteria strains from human, animal and environmental samples. However, less labo-rious and complex methods are needed (Kriz el al 2014). n this work we developed an accurate qPCR method for identifying mycobacterial infections in pigs. ur pro-tocol provides a novel, ef cient and simple protocol to detecting total mycobacterial cell numbers, including for samples from tissues lacking visible lesions.
4
107
4
of gene copies per gram for the parallel samples decreases when the number of
29
5. Conclusions
Environmental mycobacteria are regarded as a potential zoonotic risk and cause economic losses worldwide. The results reported in this thesis show that:
1. iable mycobacteria commonly occur in piggeries and can multiply in bedding materials, reaching concentrations that can represent a potential infection risk for humans and animals.
2. dentical M. avium subsp. hominissuis genotypes were obtained from human and porcine isolates, suggesting that the bacterium is transmitted between pigs and humans, or that pigs and humans share common environmental sources of infection.
. Several mycobacteria strains found in a single pig may be the result of a heavy load of mycobacteria in the piggery environment, or susceptibility of some pigs to mycobacteria infections.
4. The real-time qPCR method developed was suitable for the identi cation of tuberculous infections of pigs, including those without visible lesions.
Implications Future research on mycobacterial infections and epidemiology are needed to estimate the common sources and reservoirs of mycobacteria. However, the epidemiology of mycobacteria is very complex and challenging.
6. Acknowledgements
This work was carried out in the Faculty of eterinary Medicine of the niversity of Helsinki in collaboration with following laboratories: the Faculty of griculture and Forestry, Department of pplied Chemistry and Microbiology, niversity of Helsin-ki, Bioprocess Engineering Laboratory, Department of Process and Environmental Engineering and Biocenter, niversity of ulu, Mycobacterial Reference Labora-tory, ational Public Health nstitute, Turku, nimal Health Service ( D-Deventer) Deventer, The etherlands.
wish to express my gratitude to my supervisors: lli Peltoniemi, Hannu Salonie-mi, Timo Soveri, Faik troshi, nna-Maija irtala, Hanna Soini and Terhi li- ehmas.
30
want to thank all my co-authors: Mirja Salkinoja-Salonen, aakko Pakarinen, Timo ieminen, Hanna Soini, ohanna M kinen, lli Peltoniemi, nna-Maria Moi-sander, Elina Rintala, rina Tsitko, Harri Marttila, Peter eubauer, erard ellenberg and Terhi li- ehmas.
thank rmeli Sippola for the pig tissue samples and Prof. lav Tirkkonen for checking my discriminatory power calculations in IS1245 RFLP and M R - TR typing methods – and Sirpa Perkkiö, for the layout work and guiding me through the publication process.
My warmest thanks also to my supervisors and colleagues in the Finnish meat industry: eli-Matti ppil , Stefan Saaristo, eikko Tuovinen, Matti Per l , Sari Eskel , uti H lli, Elias ukola and Seija Pihlajaviita.
Finally and most, would like to thank my family for their love, tolerance and sup-port during this work. ithout you this work would never have been done.
This work was nanced by the by the cademy of Finland (5 05,1197 9), Mer-cedes achariassen s foundation, Finnish veterinary foundation, TEKES (the Finnish Funding gency for nnovation) grant no. 22 5 1 0 , the Finnish raduate School of pplied Bioscience and Finnish Meat ndustry ssociation.
31
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Original Publication 1
FACULTY OF VETERINARY MEDICINEDOCTORAL PROGRAMME IN CLINICAL VETERINARY MEDICINE UNIVERSITY OF HELSINKI
Porcine Mycobacteriosis Caused by Mycobacterium avium subspecies hominissuis
TANELI TIRKKONEN
dissertationes schola doctoralis scientiae circumiectalis, alimentariae, biologicae. universitatis helsinkiensis 14/2017
14/2017
Helsinki 2017 ISSN 2342-5423 ISBN 978-951-51-3507-0
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