HAL Id: hal-00902528 https://hal.archives-ouvertes.fr/hal-00902528 Submitted on 1 Jan 1998 HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Brucella melitensis infection in sheep: present and future Bruno Garin-Bastuji, José-Maria Blasco, Maggy Grayon, Jean-Michel Verger To cite this version: Bruno Garin-Bastuji, José-Maria Blasco, Maggy Grayon, Jean-Michel Verger. Brucella melitensis infection in sheep: present and future. Veterinary Research, BioMed Central, 1998, 29 (3-4), pp.255- 274. <hal-00902528>
Transcript
HAL Id: hal-00902528https://hal.archives-ouvertes.fr/hal-00902528
Submitted on 1 Jan 1998
HAL is a multi-disciplinary open accessarchive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come fromteaching and research institutions in France orabroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, estdestinée au dépôt et à la diffusion de documentsscientifiques de niveau recherche, publiés ou non,émanant des établissements d’enseignement et derecherche français ou étrangers, des laboratoirespublics ou privés.
Brucella melitensis infection in sheep: present and futureBruno Garin-Bastuji, José-Maria Blasco, Maggy Grayon, Jean-Michel Verger
To cite this version:Bruno Garin-Bastuji, José-Maria Blasco, Maggy Grayon, Jean-Michel Verger. Brucella melitensisinfection in sheep: present and future. Veterinary Research, BioMed Central, 1998, 29 (3-4), pp.255-274. <hal-00902528>
brucellose ovine / Brucella meliteusis / mise au point / perspectives
1. INTRODUCTION
Brucella are Gram-negative, faculta-tive, intracellular organisms which causeserious diseases in both humans and ani-mals. Sheep brucellosis (excluding Bru-cella ovis infection) is a zoonotic infec-tion with important effects on both publichealth and animal production and iswidespread in many areas of the world,particularly in Mediterranean countries.Sheep brucellosis is primarily caused byB. melitensis, and rarely by B. abor-tu.s (Luchsinger and Anderson, 1979;Garin-Bastuji et al., 1994) or B. suis(Paolicchi et at., 1993). In this review, wehave attempted to include up-to-dateknowledge on B. melitensis infection insheep giving particular attention to thetaxonomy of the organism, the epidemi-ology, the control and the diagnosis of thedisease. Despite a considerable increasein knowledge in recent years, manyaspects of brucellosis in sheep remainunknown, unclear or controversial.
2. TAXONOMY OF BRUCELLASPECIES INVOLVED IN SHEEPBRUCELLOSIS
Considering their high degree of DNAhomology (> 90 % for all species), Bru-cella have been proposed as a monospe-cific genus in which all types should be
regarded as biovars of B. melitensis(Verger et al., 1985). Since this proposalhas not yet met with complete agreement,the old classification of the genus (and rel-evant nomenclature) into six species, i.e.B. nzelitensis, B. abortus, B. suis,B. neotomae, B. ovi.s and B. canis (Cor-bel and Brinley Morgan, 1984), is the clas-sification used world-wide. The first four
species are normally observed in thesmooth form, whereas B. ovis and B. cani.shave only been encountered in the roughform. Three biovars are recognised for
B. melitensis (1-3), seven for B. abortiis( 1-6 and 9) and five for B. sui.s ( 1-5).
Species identification is routinely basedon lysis by phages and on some simplebiochemical tests (oxidase, urease, etc.).For B. melitensis, B. abortus and B..sui.s,the identification at the biovar level is cur-
rently performed by four main tests, i.e.carbon dioxide (C02) requirement, pro-duction of hydrogene sulphide (H2S), dye(thionin and basic fuchsin) sensitivity, andagglutination with monospecific A and Manti-sera. Moreover, the recent develop-ment of a coagglutination test, using a pairof mAb-coated latex beads, directedagainst the rough lipopolysaccharide (R-LPS) and the 25 kDa outer membrane pro-tein (Omp 25), respectively (Bowden etal., 1997), makes it possible to differen-tiate B. ovis from B. cani.s and the occa-sional rough isolates of the smooth Bru-cella species accurately. The phenotypiccharacteristics of the three speciesinvolved in sheep brucellosis, i.e.B. meliten.si.s, B. ovis and occasionallyB. abortus, are presented in table I
(species) and table II (biovars).Intermediate strains are occasionally
found due to the instability reported forsome of the phenotypic characteristicsused for the current classification of Bru-cella. This situation sometimes impedesthe identification of the species and theirbiovars. Therefore, the identification ofstable DNA-specific markers is consid-ered a high priority for taxonomic, diag-nostic and epidemiological purposes.
Several methods, mainly PCR-RFLPand Southern blot analysis of variousgenes or loci, have been employed to findDNA polymorphism which would enablethe molecular identification and typing ofthe Brucella species and their biovars(Allardet-Servent et al., 1988; Ficht et al., .,
1990, 1996; Halling and Zehr, 1990;Halling et al., 1993; Fekete et al., 1992b;Grimont et al., 1992, Herman and De Rid-der, 1992; Bricker and Halling, 1994,
1995; Cloeckaert et al., 1995, 1996c;Mercier et al., 1996; Ouahrani et al., 1993;Ouahrani-Bettache et al., 1996; Vizcainoet al., 1997). Among these methods, detec-tion of polymorphism by PCR-RFLP,since it is easier to perform and is lesstime-consuming when applied to largenumbers of samples, is considered to havean advantage over Southern blotting.
Of all the DNA sequences investigatedby PCR-restriction, the major outer-mem-brane protein (OMP) genes of Brucellaare the most interesting as they exhibitsufficient polymorphism to allow differ-entiation between Brucella species andsome of their biovars (Cloeckaert et al.,1996d). Studies of the RFLP patterns oftwo closely related genes, omp2a andomp2b, encoding and potentially express-ing the Brucella spp. major OMP of 36kDa (Ficht et al., 1988, 1989), showed thatthe type strains of the six Brucella speciescould be differentiated on this basis (Fichtet al., 1990). More recently using PCR-RFLP and a greater number of restriction
enzymes, Cloeckaert et al. (1995) detectedBrucella species-, biovar- or strain-spe-cific markers for the omp25 gene, encod-ing the Brucella 25 kDa major OMP (deWergifosse et al., 1995), and for theomp2a and omp2b genes. The omp31 gene(Vizcaino et al., 1996), encoding a majorouter-membrane protein in B. melitensis,is also an interesting gene for the differ-entiation of Brucella members. Using acombination of omp31 PCR-RFLP pat-terns and Southern blot hybridization, pro-files of Brucella species were differenti-ated with the exception of B. neotomaewhich was indistinguishable from B. suisbiovars 1, 3, 4 and 5. It was also shownthat B. abortus lacks a large DNA frag-ment of about 10 kb containing omp31 Jand its flanking DNA (Vizcaino et al.,1997).
More highly conserved Brucella genesmay also be useful for taxonomic and epi-demiological purposes, even if they detect
less polymorphism than the OMP genes.In this respect, the dnaK locus whichallows the identification of B. melitensis,the main Brucella pathogen for sheep, is ofparticular interest. All B. melitensis bio-vars showed a specific PCR-RFLP pat-tern with EcoRV, consistent with the pres-ence of a single site instead of two for theother Brucella species (Cloeckaert et al.,1996c).A selection of PCR-RFLP patterns
allowing the clear differentiation of Bru-cella species involved in sheep brucellosisis presented in table I. The electrophoreticanalysis of PCR-amplified omp25 and rel-evant EcoRV-restriction products forB. abortus, B. melitensis and B. ovis isillustrated in.figure l. The PCR patternsallow the differentiation of B. ovis, the
omp25 amplicon of which is shorter due toa deletion of 36 bp (figure 1 a), and theEcoRV-RFLP patterns the differentiationof B. melitensis that lacks the EcoRV site
(figure I b).
Clearly, taxonomic knowledge of Bru-cella has progressed a great deal since thetechniques of molecular biology have beenapplied to these bacteria. A number ofmolecular tools (nucleic probes, primers,etc.) are now available which make theelaboration of a more objective and reli-able classification of the genus possible.Judging by the emergence of new Bru-cella types from marine mammals, thegenus is far from being closed. In the nearfuture, efforts should be concentrated onthe harmonization of these tools to pro-pose the most suitable method for themolecular identification and typing of Bru-cella.
3. EPIDEMIOLOGYAND CLINICAL ASPECTS
B. melitensis infection in sheep appearsto occur naturally in the Mediterraneanregion, but the infection is widely spread.
However, North America is believed tobe free, as are Northern Europe, South-east Asia, Australia and New Zealand(FAO/OIE/WHO, 1997).
The main clinical manifestations of bru-cellosis in sheep are, as in all ruminants,reproductive failure, i.e. abortion and birthof weak offspring, in females, and orchi-tis and epididymitis in males. Arthritis isalso observed occasionally.
B. melitensis infection of sheep is quitesimilar from both pathological and epi-demiological standpoints to B. abortusinfection in cattle. B. melitensis biovar 3
appears to be the most frequently isolated
in Mediterranean countries. The preciserecognition of biovar 3, especially its dif-ferentiation from biovar 2 appears some-
times equivocal. Due to the use of insuf-ficiently discriminatory monospecific sera,a number of strains identified initially asbiovar 2 were later confirmed as biovar 3
by expert laboratories. There is no evi-dence that either the epidemiological orclinical features of B. melitensis infectionin sheep vary with the three different bio-vars (Fensterbank, 1987). In most cir-cumstances, the primary disseminationway of Brucella is the placenta, fetal flu-ids and vaginal discharges expelled by
infected ewes after abortion or full-term
parturition. Shedding of Brucella is alsocommon in udder secretions and semen,and Brucella may be isolated from vari-ous tissues, such as lymph nodes from thehead and those associated with reproduc-tion, and from arthritic lesions (Alton etal., 1988). Similarly to B. abortus infectionin cattle, B. melitensis can be transmittedfrom the ewes to lambs. A small propor-tion of lambs can be infected in utero, butthe majority of B. melitensis infections areprobably acquired through the colostrumor milk (Gri116 et al., 1997). It is also prob-able that a self-cure mechanism similar tothat suggested in cattle takes effect in mostof the infected lambs (Gri]16 et al., 1997).Despite this low frequency of transmis-sion, the existence of such latent infec-tions would greatly increase the difficultyof eradicating this disease, as B. rrceliten-sis persists without having a detectableimmune response. The exact mechanismof the development of latent B. meliten-sis infections remains unknown (Gril]6 etal., 1997).
4. DISEASE CONTROL
The strategies for the prevention andcontrol of brucellosis in sheep are mainlybased on the knowledge of the pathogen-esis and epidemiology of the infection.General non-specific measures should beimplemented, taking in account the longsurvival time of B. melitensis in the envi-ronment. Used exhaustively in whole-flock vaccination programmes, the liveB. melitensis Rev 1 vaccine greatlydecreases the prevalence of brucellosis inboth sheep and human populations(Elberg, 1981, 1996). Once the prevalencehas been diminished, a more efficient con-trol of the disease may be achieved
through the implementation of a pro-gramme based on the combination ofRev 1 vaccination of lambs with the test-
and-slaughter of adults. Finally, it may be
possible to use a test-and-slaughter pro-gramme only. This requires the exhaus-tive identification of animals and flocksand the control of animal movements. Italso requires enough economic means tobe implemented, but it could lead to theeradication of the disease (Garin-Bastuji,1996).
When B. melitensis strain Rev 1 vac-
cine is administered by the standardmethod (1-2 x 109 CFU injected subcu-taneaously), it may induce a long-lastingserological response. In contrast, whenthis vaccine is administered by the con-junctival route, the immunity conferred issimilar to that induced by the standardmethod but the serological responseevoked is significantly reduced (Fenster-bank et al., 1985). The classically recom-mended exclusive vaccination of youngreplacement animals has failed to controlbrucellosis in some developed countriesand is frequently inapplicable in the devel-oping world. As a result, whole-flock vac-cination appears to be the only feasiblealternative for controlling B. melite!sisinfection in small ruminants under theextensive management conditions of thesecountries. The vaccination of pregnantanimals with full standard doses of Rev 1administered subcutaneously or conjunc-tivally is followed by abortion in mostvaccinated animals (Zundel et al., 1992;Blasco, 1997). Reducing the dose of vac-cine has been suggested as a method ofavoiding this problem and accordingly, areduced dose vaccination has been widelyused and has been reported as a safe andeffective method of controlling smallruminant brucellosis (Elberg, 1981, 1996;Al Khalaf et at., 1992). However, fieldand experimental results support the factthat due to the induction of abortion in
pregnant animals and the low degree ofimmunity conferred, reduced doses ofRev 1 should not be recommended as analternative to the full standard doses (Zun-del et al., 1992; Blasco, 1997).
When tested in a mouse model, differ-ences in residual virulence and immuno-
genicity have been demonstrated betweenthe different Rev 1 vaccines producedworldwide. The differences could accountfor the discrepancies in safety resultsobtained in mass vaccination trials in dif-ferent countries (Blasco, 1997). The induc-tion of abortions when vaccinating preg-nant animals means that there is no entirelysafe strategy for Rev 1 vaccination. Con-
junctival vaccination is safer than subcu-taneous vaccination but is not safe enoughto be applied regardless of the pregnancystatus of the ewes and should be used onlyunder restricted conditions (Jim6nez deBago6s et al., 1989; Zundel et al., 1992).For sheep, conjunctival administration ofstandard doses of Rev 1 late in the lamb-
ing season or during lactation is recom-mended as a whole-flock vaccination strat-
egy (Blasco, 1997).
New generation vaccines can be clas-sified by the method by which they wereobtained, by classical techniques, or muta-genesis or genetic engineering. Amongthe classically obtained Brucella strainswith smooth LPS, there is B. suis S2 whichwas apparently successful in field experi-ments in China and Lybia (Mustafa andAbusowa, 1993), but showed no protec-tion in controlled experiments againstB. melitensis (Verger et al., 1995). B. abor-tus RB51, a rough stable strain, was pro-tective against all Brucella species in amouse model (Jim6nez de Bagues et al.,1994). The strain RB51 was also protec-tive against B. abortus in cattle in USAwithout inducing levels of anti-O chainantibodies capable of being measured byserological tests (Palmer et al., 1997) butwas not protective against B. ovis in con-trolled experiments in sheep (Jim6nez deBagues et al., 1995). Up to now, there is noreport on the efficacy of RB51 as a vaccineagainst B. rrcelitensis in sheep. VRTM1 Iand VTRS I are two live strains obtained
by transposon mutagenesis from
B. melitensis 16M and B. suis 2579,respectively. Both strains showed growthcurves similar to those of the Rev 1 vac-cine and were protective in the Balb/cmodel against B. melitensis biovar 1 (strain16M) and B. suis biovars 1 (strain 750)and 4 (strain 2579) (Winter et al., 1996).Further studies are needed to characterizethe immunity conferred by these new livestrains against B. melitensis in small rumi-nants particularly.
5. DIAGNOSIS
5.1. Direct diagnosis
The most reliable and the only unequiv-ocal method for diagnosing animal bru-cellosis is based on the isolation of Bru-cella spp. (Alton et al., 1988). Thebacteriological diagnosis of B. melitensiscan be made by means of the microscopicexamination of smears from vaginalswabs, placentas or aborted fetuses stainedwith the Stamp modification of the Ziehl-Neelsen method. However, morphologi-cally related microorganisms such asB. ovis, Chlamydia pyittaci or Coxiellaburnetii can cause misleading diagnoses.Therefore, the isolation of B. melitensison appropriate culture media is recom-mended for an accurate diagnosis. Vaginalexcretion of B. meliten.sis is usually copi-ous and persists several weeks after abor-tion (Alton, 1990). Moreover, the mam-mary gland is the main target of infectionin small ruminants (Marin et al., 1996a).Thus, taking vaginal swabs and milk sam-ples is the best way to isolate B. meliten-si.s from sheep. The spleen and lymphnodes (iliac, mammary and prefemoral)are the best areas for samples for isola-tion purposes in necropsied animals(Marin et al., 1996a).
B. melitensis does not require serum orC02 for growth and can be isolated on
ordinary solid media under aerobic con-ditions at 37 °C. However, the use of non-selective media cannot be recommendedbecause of the overgrowing contaminantsusually present in field samples, and selec-tive media are needed for isolation pur-poses. The Farrell selective medium,developed for the isolation of B. abortusfrom milk (Farrell, 1974), is also recom-mended for the isolation of B. melitensis
(Alton et al., 1988). However, nalidixicacid and bacitracin, at the concentrationused in this medium, have inhibitoryeffects for some B. melitensis strains
(Marin et al., 1996b). Thus, its sensitiv-ity for the isolation of B. melitensis fromnaturally infected sheep is sometimeslower than that obtained with the lessselective Thayer-Martin modified medium(Marin et al., 1996a). The sensitivityincreases significantly by the simultaneoususe of both the Farrell and the modified
Thayer-Martin media (Marin et al.,1996b). Additional work should be car-ried out to develop a new selectivemedium that is more efficient and suitablefor isolating all Brucella species.
While culturing is a specific method,its sensitivity depends on the viability ofBrucella within the sample, the kind ofsample (fetus organs, fetal membranes,lymph nodes, etc.) and the number of spec-imens tested from the same animal (Hor-nitzky and Searson, 1986). The timerequired for culturing field specimens canbe long and tissues or fluids that are onlycontaminated with a low number of Bru-cella may not be detected. PCR assay hasbeen shown to be a valuable method for
detecting DNA from different microor-ganisms and provides a promising optionfor the diagnosis of brucellosis. Severalauthors reported good sensitivity of PCRfor detecting of Brucella DNA with purecultures (Fekete et al., 1990a, b; Baily etal., 1992; Herman and De Ridder, 1992;Romero et al., 1995a; Da Costa et a].,1996). Others showed that PCR could be
a potentially useful tool when used alone(PCR, AP-PCR, rep-PCR, ERIC-PCR) orin combination with labelled probes to dif-ferentiate some Brucella species and bio-vars (Fekete et al., 1992b; Bricker andHalling, 1994, 1995; Cloeckaert et al.,1995; Mercier et al., 1996; Ouahrani-Bet-tache et al., 1996; Tcherneva et al., 1996).However few studies have been performedwith clinical or field samples (Fekete etal., 1992a; Leal-Klevezas et al., 1995;Romero et al., 1995b; Matar et al., 1996;Rijpens et al., 1996). The possibility ofusing the PCR technique to detect theDNA of dead bacteria, or in paucibacil-lary samples and even in samples highlycontaminated with other microorganisms,could increase the rate of detecting ani-mals infected by Brucella. However, upto now, no technique is sensitive enough toreplace classical bacteriology on all kindsof biological samples.
5.2. Indirect diagnosis
5.2.1. Immune response
As mentioned before, the B. melitensisRev I strain is the best vaccine available,but when applied under standard condi-tions (i.e. full dose via the subcutaneousroute in young replacement animals) itinduces long lasting serological responsesthat seriously interfere with subsequentserological screening (Alton and Elberg,1967; Elberg, 1981, 1996; Alton, 1990;MacMillan, 1990). As no differences havebeen found between the diagnostic anti-gens, those from field strains of B. meliten-.sis and those from the Rev I vaccine, it isdifficult to find a serological test able todistinguish infected from vaccinated ani-mals. This problem currently impedes thecombined use of vaccination and test and
slaughter programmes for eradicating bru-cellosis.
5.2.2. Diagnostic azztigens
There is no scientific agreement onwhat should be the nature and character-istics of a universal antigen for diagnosingbrucellosis due to smooth Brtrw·Ila
(B. abortus, B. w!///<!;.s7.s and B..stri.s). Oneof the most critical and controversial pointsconcerning the serological diagnosis ofB. mclitensis infection in small ruminantsis rotated to which Brru’c llu species andbiovars are used in the production of thediagnostic antigens. The rose bengal test(RB) and the complement fixation test(CF) are the most widely used tests forthe serolo!!ical diagnosis of sheep bruccl-losis (Farina, 1985; MacMillan, 1990),and are currently the official tests used in nthe European Union countries (EuropeanCouncil Directive 64/432/EEC, 1964).The antigenic suspensions (whole cells) >used in both tests are made with B. abor-tus biovar I (an A-dominant strain) (Altonet al., 1988) which means that, theoreti-cally, infections due to M-dominant strains(B. melitensis biovar I ; B. abortus biovars4, 5 and 9; B. suis biovar 5) could be mis-diagnosed (Alton et a]., 1988; MacMil-lan, 1990). However, recent resultsshowed no significant difference in thesensitivity of the classical RB antigen pie-pared with B. a/wrn/.s biovar I (A-dotni-nant) between ovine populations infectedwith either biovar I (M-dominant) or bio-var 3 (A-dominant) of B. melitensis(Blasco et at., 1994b).
The outer membrane of the bacteriacontains the main antigens involved in thehumoral response against Brucella (Dfazet a]., 1968a). As in other Gram-negativebacteria, the outer membrane of smoothBrucella is composed of phospholipids,proteins and lipopolysaccharide (smoothlipopolysaccharide, S-LPS). The S-LPSis the immunodominant antigen and mostof serological tests, particularly those usingwhole-cell suspensions as antigen (suchas RB, CF), and most ELISA tests, have
been developed to detect antibodies to thisantigen (Dftz et al., 1968a). The S-LPSof smooth Brucellu is composed of aninner glycolipidic moiety (the core
oligosaccharide plus the lipid A) and anouter polysuccharide chain (O-chain). ThisO-chain is the relevant antigenic moietyand is chemically composed of a per-osamin homopolymer showing a-1,2 anda-1,3 linkages (Cherwonogrodxky et a].,1990). The O-chain polysaccharide ofB. <-</7!;’/;<.s biovar I (A-dominant) pos-sesses a fine structure with only a low-frequency (ca 2 °!°) of a-1,3 linked 4,6-dideoxy-4-for)T)amido-D-mannopyranoseresidues. In contrast, the O-chain polysac-charide of B. melitensi.s biovar I (M-dom-inant) contains repeated pcntasaccharideunits with one a-1,3 and four a-1,2 link-ages. As a result, the A and M antigeniccharacteristics depend on the O polysac-charides in which the frequency of a-1,3linked residues varies. Studies with mono-clonal antibodies (Douglas and Palmer,1988) show that the A epitope is related toportions of at least five sugars with a-1,2linkages and that the M epitope includessugars with a-1,3 linkages (thus its rele-vance in the 0-chain of B. abortus bio-var l should not be important). Therefore,all biovars assigned as A-dominant shouldexpress few or no a-1,3 linked residues,while M-dominant strains possess a uniqueM epitope as well as a di-, tri- or tetrasac-charides with a-1,2 linkages, and can thusbe considered to be contained within the A
epitope structure (Bundle et al., 1989;Meikle et al., I 989; Cherwonogrodzky et=11., 1990). The presence of commonoligosaccharides of four or less sugars isconsistent with the existence of a com-mon (C) epitope. Indeed, this C epitopehas been detected with the appropriatemonoclonal antibodies (Douglas andPalmer, 1988) and can account for the highsensitivity of the antigens made from A-dominant strains (i.e. B. abortus biovar 1 )at detecting M-dominant B. melitensis bio-var I infections and vice-versa (MacMil-
lan, 1990; Diaz-Aparicio et al., 1993). Infact, crude LPS extracts from eitherB. melitensis 16M (biovar 1, M-dominant)or B. abortus 2308 (biovar 1, A-dominant)are equally sensitive in an indirect ELISA(i-ELISA) for diagnosing brucellosis insheep infected by B. melitensis biovar 1(Marin et al., unpublished results). How-ever, the native hapten and the S-LPShydrolytic polysaccharides containing the0-chain and core sugars from B. abortusbiovar 1 fail to react in precipitation testswith a large proportion of B. melitensisinfected sheep, goats and cattle under con-ditions in which the same antigensobtained from B. melitensis biovar 1detected most of those animals (Diaz-Aparicio et al., 1993). Therefore, furtherresearch is needed to clarify the practicalimportance and interest of using species-specific diagnostic antigens for the dif-ferent serological tests.
There is limited information on thevalue of outer membrane and inner cyto-plasmic proteins for the diagnosis ofB. melitensis infection in sheep.
The immunoelectrophoretical patternsof cytoplasmic proteins show little differ-ences between Brucella species whenassayed with polyclonal sera (Diaz et al.,1967, 1968b). These inner antigens areconsidered specific for the genus, beinguseful to differentiate infections due toBrucella from those due to bacteria whoseLPS cross-reacts with the Brucella S-LPS,as is the case of Yersinia enterocolitica0:9 (Díaz and Bosseray, 1974). However,a cross-reactivity among cytosolic pro-teins of B. melitensis and those obtainedfrom Ochrobactrum anthropi, an oppor-tunist human pathogen, has been reportedrecently (Velasco et al., 1997). The Bru-cella cytoplasmic antigens, known also asbrucellin (Jones et al., 1973) have beenused successfully for the allergic diagno-sis of brucellosis in sheep and goats (Fen-sterbank, 1982, 1985; Ebadi and Zowghi,1983; Loquerie and Durand, 1984; Blasco
et al., 1994b). Moreover, these cytoplas-mic antigens have been reported to be sen-sitive and specific enough for the diagno-sis of brucellosis in sheep and goats whenused in precipitation tests (Muhammed etal., 1980; Trap and Gaumont 1982; Diaz-Aparicio et al., 1994). In contrast, whenthese cytoplasmic antigens are used in thei-ELISA, the sensitivity obtained is notadequate due to the high background IgGreactivities with sera from Brucella-freeanimals (Diaz-Aparicio et al., 1994; Salih-Alj Debbarh et al., 1996). An importantdrawback of diagnostic tests using unchar-acterized cytosolic proteins is the lack ofspecificity when testing Rev 1 vaccinatedsheep and goats. But a partially purifiedcytosoluble protein of 28 kDa (CP28) fromthe cytosoluble protein extract (CPE) ofB. melitensis has been reported as beingable to differentiate Rev 1 vaccinated fromB. melitensis infected ewes when used ini-ELISA (Debbarh et al., 1995). Howeverthis test is less sensitive than both the RBand CF tests for diagnosing B. melitensisinfected ewes (Salih-Alj Debbarh et al.,1996). The corresponding B. melitensis16M bp26 gene was expressed inEscherichia coli and monoclonal anti-bodies were produced (Cloeckaert et al.,1996a, b). Sequence analysis of the clonedgene revealed that it was nearly identicalto the recently published B. abortus bp26gene, coding for a periplasmic protein(Rossetti et al., 1996). A competitiveELISA (c-ELISA) using CPE as antigenand some of these monoclonal antibodies
improved the sensitivity for diagnosinginfected sheep, and no antibody responsewas detected in Rev 1 vaccinated sheep(Debbarh et al., 1996).
Several authors have attempted to iden-tify the main polypeptide specificities ofthe antibody response to outer-membraneprotein (OMP) extracts of B. melitensisby using either immunoblotting orc-ELISAs with specific monoclonal anti-bodies (Zygmunt et al., 1994a, b; Debbarh
et al., 1995; Hemmen et al., 1995; Tibor etal., 1996). While OMPs of 10, 17, 19,25-27 and 31-34 kDa were found thatwere suitable as potential antigens for thediagnosis of brucellosis in sheep byimmunoblotting or ELISA, the antibodyresponse to OMP was very low and het-
erogeneous in B. melitensi.s infected sheep(Zygmunt et a]., 1994a, b).
Further research is needed on the iden-
tification, isolation, characterization andcloning of both inner and outer membraneproteins which could be used as diagnos-tic antigens that are more sensitive andspecific. This should be followed by thedevelopment of subunit or live antigen-deleted vaccines, able to protect animalswithout interfering with diagnostic tests,and should be a major goal of research inthe near future.
5.2.3. Serological tests
No specific serological tests forB. melitensis infection of sheep have beendeveloped and it is widely assumed thatthe serological tests used for B. (ibortitsinfection in cattle are also adequate forthe diagnosis of B. melitensis infection insmall ruminants. Accordingly, the RB andCF are the most widely used tests for theserological diagnosis of brucellosis insheep and goats (Farina, 1985; Alton,1990; MacMillan, 1990). ).
The RB test was developed more than20 years ago for the diagnosis of bovinebrucellosis and, despite the scanty andsometimes conflicting information avail-able (Trap and Gaumont, 1975; Fenster-bank and Maquere, 1978; Farina, 1985;MacMillan, 1990; Alton, 1990; Blasco etal., 1994a, b), this test is internationallyrecommended for the screening of bru-cellosis in small ruminants (JointFAO/WHO expert committee on Brucel-losis, 1986; Garin-Bastuji and Blasco,1997). An important problem affectingthe sensitivity of the RB test concerns the
standardization of the antigens. The Euro-pean Union regulations require antigensuspensions in lactate buffer at pH 3.65 ±0.05 that are able to agglutinate at a dilu-tion of 1:47.5 (21 IU/mL) of the Interna-tional Standard anti-B. abortus serum
(ISaBS) but which give a negative reac-tion at a dilution of 1:55 ( I 8.2 IU/mL) ofthe same serum (European Council Direc-tive, 1964). These standardization condi-tions, which seem to be suitable for thediagnosis of B. ahortus infection in cattle(MacMillan, 1990), are not adequate forthe diagnosis of B. melitensis infection insheep (Blasco et a]., 1994a, b). Thisaccounts for the relatively low sensitivityof some commercial RB antigens at diag-nosing brucellosis in sheep and goats(Falade, 1978, 1983; Blasco et al., 1994a)and for the fact that a high proportion ofsheep and goats belonging to B. melitcllsis-infected areas give negative results in theRB but positive ones in the CF (Blasco etal., 1994a). These phenomena seriouslyquestion the efficacy of using the RB as anindividual test in small ruminants. At leastfor sheep, the sensitivity of the RB testimproves significantly when the antigensare standardized against a panel of serafrom several B. melitensis culture positiveand Brcrcello-free sheep (Blasco et al.,1994a).
The CF is the most widely used test forthe serological confirmation of brucellosisin animals. As in cattle brucellosis, despiteits complexity and the heterogeneity ofthe techniques used in the different coun-tries, there is agreement that this test iseffective for the serological diagnosis ofbrucellosis in sheep and goats (Farina,1985; MacMillan, 1990; Alton, 1990).When testing a limited number of seraobtained from B. meliten.sis culture posi-tive and Brucella-free goats, the CF test
provided the same sensitivity as those ofthe RB and i-ELISA (Diaz-Aparicio et al.,1994). However, under field conditions,the sensitivity of the CF test has been
reported to be somewhat lower (88.6 %)than those of the RB (92.1 %) andi-ELISA (100 %) for diagnosingB. meliten.sis infection in sheep (Blascoet al., 1994a, b). On the other hand, theCF test has many drawbacks such as com-
plexity, variability of reagents, prozones,anticomplementary activity of sera, diffi-culty to perform with hemolysed sera, andsubjectivity of the interpretation of lowtiters. Therefore, while the sensitivity ofRB is sufficient for the surveillance of freeareas at the flock level, RB and CF shouldbe used together in infected flocks toobtain accurate individual sensitivity intest-and-slaughter programmes. Moreover,an important drawback of both RB andCF tests is their low specificity when test-ing sera from sheep and goats vaccinatedsubcutaneously with Rev 1 (Fensterbank etal., 1982; Jim6nez de Bagues et al., 1992;Diaz-Aparicio et al., 1994). However,when the Rev I vaccine is applied con-junctivally (Fensterbank et al., 1982), theinterference problem is significantlyreduced in all serological tests (Jimdnezde Bagues et al., 1992; Diaz-Aparicio etal., 1994).
Relatively little information is avail-able on the value of the ELISA for the
diagnosis of B. melitensis in small rumi-nants. The indirect ELISA, using more orless purified S-LPS of B. melitensi.s asantigen and polyclonal conjugate (anti-IgG H+L), has been reported to be sensi-tive enough for the diagnosis of infectionin sheep and goats (Jim6nez de Bagaes etal., 1992; Diaz-Aparicio et al., 1994;Blasco et al., 1994b; Delgado et al., 1995).A similar technique has been also pro-posed for diagnosing sheep brucellosis inindividual or pooled milk samples (Bian-cifiori et al., 1996), but due to the low rateand frequency of Brucella antibodies inmilk, the test lacks sensitivity, comparedwith tests performed on serum. One of theproblems of the i-ELISAs performed onserum is the high background reactivity
obtained when testing sera from Brucellafree animals (Jim6nez de Bago6s et al.,1992). The use of protein G as conjugatesignificantly reduces this problem, increas-ing the ELISA specificity (Diaz-Aparicioet al., 1994; Ficapal et al., 1995). Thisincreased specificity is also obtained whentesting Brucella-free sheep in the abovei-ELISA but using a monoclonal anti-ruminant IgGI conjugate. However, thesensitivity of i-ELISAs with either pro-tein G or monoclonal conjugates decreaseswith respect to that obtained with the poly-clonal conjugate (Blasco et al., unpub-lished results). Literature references avail-able on the use of competitive ELISA(c-ELISA) protocols for the diagnosis ofbrucellosis in sheep are scanty. In ourexperience, competitive protocols usingan anti-C epitope monoclonal antibodydid not outperform conventional i-ELISAsfor the diagnosis of B. melitensis in sheepand goats (Moreno et al., unpublishedresults). As happens with the other sero-logical tests, the specificity of the ELISAsis quite low when testing sera from Rev Ivaccinated animals (Jim6nez de Bagueset al., 1992; Diaz-Aparicio et al., 1994;Moreno et al., unpublished results). How-ever, as mentioned before, the use of apurified periplasmic protein (26 kDa) ini-ELISA or c-ELISA protocols could beuseful for differentiating B. meliten.ri.rinfections from Rev I vaccinated sheep(Debbarh et al., 1996; Salih-Alj Debbarhet al., 1996).
Further research is needed to developserological tests of improved sensitivityfor the diagnosis of brucellosis in sheep,which would be able to discriminatebetween infected and vaccinated animals.
5.2.4. Cell mediated immunity(CMl) based diagnosis
The detection of the delayed-typehypersensitivity (DTH) reaction (skin test)has been used for the diagnosis of sheep
brucellosis with variable success (Jonesand Marly, 1975; Fensterbank, 1982,1985; Loquerie and Durand, 1984; Blascoet al., 1994b). The allergens used in earlystudies were generally obtained from cul-ture supernatants or by acid extraction ofsmooth Brucella cells (Alton, 1990) and,therefore, contained S-LPS or its
hydrolytic polysaccharides. Since theS-LPS does not take part in DTH reac-tions (Jones et al., 1973; Jones, 1974) and,in contrast, its 0-chain elicits a strong anti-
body response, injection of minimalamounts of S-LPS in previously sensitizedanimals could cause an inflammatory reac-tion interfering with the interpretation ofthe skin test. Moreover, such extractscould cause a secondary antibody responseinterfering with future serological testing.These problems are practically solved byusing strains devoid of the 0-chainpolysaccharide, as is the case of the roughB. melitensis 115 (Jones et al., 1973). Theallergens can be obtained from this strainby several methods (Bhonghibhat et al.,1970; Jones et al., 1973; Dubray, 1985;Blasco et al., 1994b). Despite the exis-tence of quantitative and qualitative dif-ferences among the allergens obtained bythese different methods, the results showthat purified allergens would not offerpractical advantages in sensitivity andspecificity over complex protein mixtures(Blasco et al., 1994b). The site and route ofallergen inoculation are not important fac-tors affecting the sensitivity of skin testfor brucellosis (Fensterbank, 1985; Alton,1990; Blasco et al., 1994b). The methodconsidered more efficient and practicalfor sheep is the subcutaneous inoculationin the lower eyelid with readings 48 h afterinoculation (Jones et al., 1973; Jones andMarly, 1975; Fensterbank, 1985). How-ever, since mixed DTH-antibody medi-ated intradermal reactions are occasion-
ally observed, a reading time of 72 hseems advisable for a better assessmentof true DTH reactions (Blasco et al.,1994b). Anergy induced by repeated skin
testing is a well known phenomenon inbovine tuberculosis (Radunz and Lepper,1985). This phenomenon is not absolute inthe case of brucellosis in sheep, but theskin test responses to Brucella allergenslessen within the 24 days that follow apositive skin test (Blasco et al., 1994b). ).The skin test is considered as always beingnegative when testing unvaccinated Bru-cella-free animals (Fensterbank, 1982,1985; Loquerie and Durand, 1984; Blascoet al., 1994b). In contrast, the skin test is
positive in many Rev 1 vaccinated ani-mals (2 years or more after vaccination),thus lacking specificity for differentiatinginfected from vaccinated sheep (Fenster-bank, 1982; Pardon et al., 1989). No infor-mation has been published on the diag-nostic value of in vitro CMI tests (i.e.blastogenesis, IL and IFNy detection, etc.)for the diagnosis of brucellosis in smallruminants.
6. CONCLUSION
Clearly knowledge concerningB. melitensis infection in sheep has dra-matically progressed within the past 20years. Even though many aspects requireadditional research, several diagnostic andprophylactic tools have been sufficientlyvalidated and standardized, and are read-ily available to control the disease effi-ciently.
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