© [NSTITUTPASTEUR/ELsEVIER Paris 1992
Res. Microbial. 1992, 143, 513-518
A rapid and sensitive method for the identification of Brucella species with a monoclonal antibody N . Vizcalno and L. Fermindez-Lago (')
Depa[tamenlo de &li~,obiolog(a y Gendtica. Facullad de Farmacia. Universidad de Salamanca, 37007 Salamanca (Espaha)
SUMMARY A coagglutination test usi~'.g monoclonal antibody BmE10-5 with specificity for the M antigen of Brucella melitensis 1 6 M has been developed for the rapid identification of the smooth Bruce/is species. All reference strains or several biovars of B. melitensis, B. abortus, B. suis and B. neotomae tested were positive in this assay. No significant differences in reaction intensity were observed in relation to the different distribution of the A and M antigens among the Brucella serovars analysed. Conversely, rough Bnacefla species, with the exception of B. abortus 45120. were negative in the assay. Among the diffe,ant organisms tested not belonging to the genus Bruce/Is, serovar 0 9 of Yer~dnia enterocolitica was the only one that gave a weak positive reaction of coagglutination. Thus, in view of its rapidity. ;ifr=plicity, specificity and low costs, this technique could be highly useful for rapid identification of smooth Btuceda strains in diagnostic laboratories.
Key-words: Coao01utination, Bruce#a, mAb; Rapid identification, Smooth strains.
INTRODUCTION Brucellosis, a zoonitic disease responsible for abortion and infertility in certain species of domestic animals, mainly sheep, cattle, goats and swine, is caused by three main bacterial species : Brucella abortus, B. melitensis and B. suis (Thoen and Enright, 1986). These microorganisms can be transmitted to humans by direct contact with contaminated material or, more often, through consumption o f insufficiently treated milk and milk products from infected animals (Banal et aL, 1990). The diagnosis of brucello-
Submitted January 14, 1992~ ~ccepted March 25. 1992. (=) Corresponding au~hnr.
sis in humans and animals is often difficult to establish. Isolation of the ae¢iological agent from clinical samples by conventional methods is n o t always possible(Alton etaL, 1988). Accordingly, the detection and quantification of Brucella antibodies in serum samples using different serological tests plays a major role in the routine diagnosis of brucellosis ( F A O / W H O . , 1986). Despite this, apart from their diagnostic value, the isolation and identification of Bruce]In spp. are cons;~dered t_-zbe necessary stages for .:he design of ep;demiological and eradication programs (Plommet, t986)~ ThP identification of
514
N. VIZCA[NO A N D L. F E R N A N D E Z - L A G O
new isolates is a relatively tedious process, based largely on the use o f certain biochemical tests and on slide agglutination reactions with test sera (Corbel and Brinley-Morgan, 1984). Thus, the development of new techniques that permit rapid identification of t'reshly isolated Brucella strains is o f great practical importance.
mAb
The M-antigen.specific BmE 10-5 mAb used in this work was produced as previously described (Vizcaino el aL, 1991). The binding profile of this mAb was established by a competitive enzyme-linked immunosorbent assay (ELISA) using chemically defined Brueellu antigens (Vizcaino et al., 1991). The isotype was determined by ELISA using purified S-LPS as the antigen and isotype-specifie peroxidase conjugates (Nordic immunological Laboratories, Tilburg, The Netherlands). The isotype of this mAb was IgG3 (Vizeatno et aL, 199!).
Monoclonal antibodies (mAb) to Brucella spp. have been reported with specificity for A, M or both A and M antigens (Bundle et aL, 1989; Dubray and Limet, 1987 ; Garin-Bastuji et aL. I990; Greiser-Wilke and Moennig, 1987; Meikle ef al., 1989; Vizcaino et al., 1991). Recently, using mouse mAb and synthetic oligosaceharides, the structures o f Brucella A and M epitopes were postulated and the relative distribution o f the two antigens among O polysaceharides o f the different Brucella biovars was established (Bundle et al., 1989; Dubray and Limet, ]987; Garin-Bastuji et aL, 1990).
Coagglutination test
The stabilization and coating of protein-Acontaining staphylococci (Stap#tylococcus aureus Cow•n [. ATCC 12598) to make the coagglutination reagent followed the procedures described previously (Fernfi.ndez-Lago et aL, 1988; Kronvall, 1973). A l-ml sample of a 10 ~70(vol/vol) cell suspension was coated with 0.1 ml of ascitic fluid containing the BmEI0-5 mAb. After mixing of suspension for 3 h at room temperature, the staphylococci were washed twice and suspended at 1 % in phosphate-buffered saline (PBS) containing 0 . 1 % sodium aside. Coagglutination tests were performed by mixing on a slide, for 15-30 s, one drop of the staphylococcal reagent with one drop of the corresponding microorganism in PBS. Reactions were considered negative if no clumping occurred within that time.
In the present wo#;, we developed a coagglutination technique using a m A b , BmE10-5, with specificity against toe M antigen f r o m B. melitensis 16M (Vizcaino et aL, 1991) for rapid, sensitive and specific identification o f smooth Brucella species.
MATERIALS AND METHODS
RESULTS
Bacterial strains and growth conditions
Results obtained by studying different Brucel/~ reference strains using the coagglutination technique with reagent staphylococci coated with BmEI0-5 m A b are shown in table I, A n intense positive reaction occurred with all the naturally occurring smooth Brucella strains assayed: B. melitensis, B, abortus, B, suis and B. neotomae. No significant differences irr reaction irttensity were evidenced when comparing the different distributions of the A and M epitopes on the LPS molecule of the various Brucella sero,'ars aria-
Bacterial strains employed and their sources are listed in tables I, II and tit. Cultures were stored as cell suspensions at -20~C in 50 % glycerol and grown at 37°C under a 5 % CO 2 atmosphere for 24-48 h on tryptic soy agar (BBL Microbiology Systems, Cockeysvil.~e, MD, USA) enriched with 0.3 % yeast extract (Difeo). Brucefla isolates were characterized by conventional methods (Alton et al., 1988). The colony type phase was checked using standard procedures (Alton et aL, 1988 ; Corbel and BrinleyMorgan, [ 984).
EL|SA LPS mAb
= = =
enzyme-linked imm'~rlOsorbehl lipop~ty,,accharide. m o n o c l o n a l alltibc.dy.
o~,~ay.
] I I
r3S S-LPS
= -
phosphate-buffered LPS from smooth
saline. ~train,;.
IDENTIFICATION OF BRUCELLA SPECIES
515
Table I. Reactions of BruceUa strains in the coagglutination test using the BmEI0 5 mAb. Brucella species
Biovar
Strain
Colony type
Reaction in coagglutination
B, B. B. B. B.
melitensis metitensis metitensis melitensis melitensis
1 1 2 3
16M (~) Revl (b) 63/9 tb) Ether (~1 ]8115 tD~
Smooth Smooth Smooth Smooth Rough
+ + + + -
B. B. B. B. B. B. B. B. B. B. B. B,
abortus abortus abortus abortu$ abortus abortus abortus ebortus abortus abortus abortus abortus
1 1 l 2 3 4 5 6 9 1
S 19 (b~ 544 (o 99 (c~ 86/8/59 to) Tuiy.a l° 292 t~, B3196 ~'~ 870 le) C68 re) 230g ~b~ 45/20 (bj RB51 ~b~
Smooth Smootla Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Rough Rough
+ + + + ~+ + + + + + -
B. B. B. B. B.
suis suis suis suis neotomae
l 2 3 4
1330 (o Thomsen ~ 686 ~c) 40 ~¢~ 5K33 ~o
Smooth Smooth Smooth Smooth Smooth
+ + + + +
BOW 63/290 (o RM-6/66 t~)
Rough Rough
-
B. ovis B. cams
t~ Obtained from the National Collection of Type Cultures, London, UK. '~ Obtained from G. Sehurtg, Regional College of Veterinary Medicine, Blaeksburg. VA, 24061, USA. L~IObtained from j . M . Verger, Station de Pathologic de la Reproduction, 37380 Nouziity, France.
lysed, in contrast, rough mutants o f B. metitensis (BI t5) and B. abortus (RB5 i) and all strains o f B. ovis a n d B. cauls assayed, which occur naturally in the rough f o r m , were negative in this assay (table I). U n d e r the same conditions, the specificity o f the assay was d e t e r m i n e d by studying d f f f c r c m bacterial species characterized by their crossreaction with Brucella spp. in other serological tests, Only a weak positive reaction was observed with strains belonging to serovar O9 o f Yersinia enterocolitica (table 11). Furthermore, a total o f 67 clinical isolates a n d field strains previously identified by conventional laboratory tests as be-
longing to the genus Brucella were investigated (table III). Results d e m o n s t r a t e d the high sensitivity of the assay, since all s~rains analysed gave a clear positive reaction. Finally, the specificity o f the coaggtutinatioa test using m A b BmE10-5 was investigated in a total o f 123 cl::nical isolates not belonging to the Brucella genus. N o false-positive reactions were noted. An antibody-free s~aphylococcal cell suspension and a suspension coated with purified lgG-class antibodies obtained from normal m o u s e serum were used as controls ; a o :tgglutination with any bacterial isolate occurred with these controls.
516
N, VIZCA[NO A N D L. FERN.~NDEZ-LAGO
Table i1. Reactions in the coagglutination test with BmEI0-5 mAb of different bacterial species which crossreact with Brucella spp. in other serologic tests. Species and strain
Reaction in coagglutination
Yersinia enteroeolitica serovar 0 9 [P383 I~l W22708 ta~ WI024 ;a) W836 (a~ W830 ~'~ WI91 (at W227 ~a~ Eseherwhia colt sernvar O157 ATCC 35150 tbl Cut~t#ylvb,¢;~i f e t u s subsp, venerealis NCTC 10354 I:) Campylobaeterfetus subsp, fetus NCTC 10842 le) Salmonella urbana serovar 030 ATCC 9261 Ib) Vibrio eholerae biovar Inaba CECT 512,542 la~ ~,, Obtained ~ Obtained ~r~Obtained c~, Obtained
from from From from
G. Wauters, Universit¢ Catholiqu¢ de Loltvain, Brussels, Belgium. the American Type Culture Collection, Roekville, MD, USA. the Nationat Collection of Type Cultures, London, UK. the Colecci6n Espafiola de Cul~ivos Tipo, Valencia, EspafiaL
~lable III. Results of testing 67 Brucella spp. isolates by coagglutinat[on test using the BmEI0-5 mAb. Brucella species/ no. of strains B. B. B. B. B.
Binvar
O~igin
Colony type
Reaction in coagglutination
1 2 3 1 2
Human Human Human Bovine Bovine
Smooth Smooth Smooth Smooth Smooth
+ + + + +
melitensis/12 (°~ melitensis/3 melitensis/40 abortus/lO abonus/2
m Obtained from the Departamento de Microbio[ogia, Hospital Clinieo, Salamanca, Espafia.
DISCUSSION
R a p i d identification o f Brucella strains is o f practical i m p o r t a n c e for b o t h bacterial d i a g n o sis a n d epiderniologlcal studies. C o n v e n t i o n a l bacteriological tests c o m m o n l y u ~ d for laboratory identification o f Brucella spp. are relatively slow a n d laborious procedures that m a y take up to two days to complete ( A l t o n el a/,, 1988).
Accordingly, new a n d faster m e t h o d s have been designed to reduce this time, thus permittir~g a preliminary r e p o r t o n the same day (Fekete et aL, 1990; R o o p II et aL, 1987). In this sense, the data offered in this work underscore the usefulness o f the staphylococcal coagglutination test owing to its speed, simplicity and low costs, apart from its high sensitivity a n d specificity, to carry out preliminary identification of B. meliterr-
IDENTIFICATION OF BRUCELLA SPECIES sis, B. abortus, B. suis a n d B. neotomae in the diagnostic l a b o r a t o r y .
T h e e o a g g l u t i n a t i o n tests used in this work involve use o f the m A b BraE10-5, with specificity against the O polysaccharide chain (M antigen) o f the L P S f r o m B. melitensis 16M (Vizcaino e t a l . , 1991). T h e recognition by tltis m A b o f epitopes c o m m o n to b o t h A a n d M antigens w o u l d a c c o u n t for t h e positive coagglutination reaction also observed with all the smooth Brucella serovars t h a t preferentially express the A antigen o n the L P S molecule (Bundle et aL, 1989). By contrast, n o c o a g g l u t i n a t i o n reaction was o b s e r v e d with a n y o f t h e r o u g h Brucetla strains tested, with the exception o f the r o u g h B. abortus 4 5 / 2 0 strain, a m i c r o o r g a n i s m characterized by expression, o n its surface, o f variable a m o u n t s o f low molecular weight A antigen (Meikle et aL, 1989; $churig e t a L , 1984). T h e specificity o f the t e c h n i q u e was established by the use o f different bacterial strains that cross-react wire Brucella spp. ill o t h e r serological tests (Corhel~ tq75; Feeley~ 1.969; FernfindezLago etctL, 1982; Stuart a n d Corbel, 1982). Only a weak positive r e a c t i o n was observed with strains b e l o n g i n g t o serotype 0 9 of Y. enteroeolith:a (table 11). This can be explained by t h e close structural relationship between the O antigen f r o m Y. enteroeolitica 0 9 a n d the A antigen f r o m Brucella spp. (Bundle el eL, 1984, 1989; C a r o f f et aL, 1984). However, f r o m the diagnostic point o f view, these r,'sults have n o important drawbacks, since both bacterial strains can be readily differentiated in the l a b o r a t o r y o n t h e basis o f different biochemical tests (Corbel a n d Briniey-Morgan, 1984). In s u m m a r y , in view o f its speed, sensitivity a n d specificity, the a b o v e described coagglutin a t i o n test with m A b BraE10-5 m a y be particularly useful in b o t h h u m a n a n d veterinary diagnostic laboratories to carry out identification o f smooth B. abortus, B, melitensis, B, suis a n d B. neoLomae strains.
Acknowledgements
This w~,rk was supported by gra~kt 1036/89 fror~ the Junta de Castilla y Le6n, Spain.
517
Une mdthode rapide el sensible u¢ilisant un anticorps monoelonal pour I'identifieation de I'esl~ce Bruceila
Nous avons dCvelopp~ un test de coagglutination utBisant un anticorps monoclonal dirig~ contre l'antig,~ne M de Brucella melitensis 16M pour l'identification rapide des esp&:es de Brucella ~smooth >>. Toutes Ies souehes de rCference de B. melitensis, B. abortus, B. suis et B, neotomae utilisdes ~taient positives darts ce test. Nous n'avons pas observ6 de diffCrence signiflcative de rintensit6 de la rCacrion en relation avec des diffdrences de distribution des antig~nes A e t M parmi los biovars de Brucella analys~s, Par contre, los aspires de Brace/In , r o u g h , , h l'exception de B, abortus 45/20, ont 6t~ u~gatives. Parmi los organismes analysCs qui n'appartiennent pas an genre Brucella, seule une ri'action de coagglutination faiblement positive a ~td observ~e avee Yetsinia enterocolitica serotype 0 9 . Grace/~ sa rapidit6 et sa zp~cificitC, cettc technique poarrait ~tre d'une grande ulilit6 pour I'identification rapide des souehes de Brucella, smooth~ dans les laboratoires de diagnostic. Mols-¢'l~5: Coagglutination, Brucelta, mAb; Identification rapide, Souches ~ smooth ~>.
References
Alton, G.G., Jones, L.M., Angus, R.D. & Verger, J.M (1988), Techniques for the brucellosis laboratory. Institut National de la Recherche Agrnnomique, Paris. Banal, M.B., Mayer, I, & Cohen, A. (1990), Isolation, identification and characterization in Israel cf Brucelia melitensis biovar 1 atypical strains susceptible to dyes and penicillin, indicating the evolution of a new variant. J. olin. MicrobioL. 28, 1057-1059. Bundle, D.R., Gidney, M.A.J., Perry, M.B., Duncan, J.R. & Cherwonogrodzky, J.W. 11984), Serological confirmation o f Bruce/f, abortus and Yersiniaenterocolitica 0:9 O-antisens by monoclonal antibodies. Infect. Imrnutt., 46, 389-393. Bundle. D.R.. Cherwonogrodzky, J.W., Gidney, M.A.J., Meikle~ P.J., Perry, M_B_& Pzters, T. (1989), Oet'i~:?.:.~,.lof Bc~celta A and M epitopes by monoelonal typing reagents and synthetic oligosaeeharides. Infect. [roman., 57, 2829-2836. Caroff, M.. Bundle, D.R. & Perry, M.B. (1934], Structure of the O-chain of the phenol-phase soluble cellular iipopolysaecharide of Yersinia enterocolitiea serotype O:9. Europ. 3. Biockem., t39, 195-200. Corbel, M.]. !1975L -[-he serological relationship between Brucella spp., YersiniaenterOcolitica serotype IX and Salmonella serotypes of Kauffmann-White group N. J. Hyg., 75, 151-171. Corbel. M. & Brinley-Morgan, W..I'. (1984}, Genus Brucelia Meyer and Shaw 1920, 173aL, in "Bergey's
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IV. V I Z C A / N O A N D L. F E R N A N D E Z - L A G O
manual of systemic bacteriology, vol. 1" (N.R, Krieg & J.G. Holt) (pp. 3"/7-388). Williams & Wiikins Co., Baltimore. Dubray, G. & Limet, J. (1987), Evidence of heterogeneity of lipopolysa¢charides among Bruce.lie biovars in rolelion to A and M specifici.'.ies, Ann. Inst. Pas. teur/MierobioL, I38, 27-37. FAO/WHO. Expert Committee on Brucellosis. (1986), Sixth report. W.H.O. Tech. Rep, Ser. 740. Feeley, J.C. (19691, Somatic 0 antigen relationship of Brucello and Vibrio eholerae. J. Beet., 99, 645-649. Fekete, A., Bantle, J.A., Hailing, S.M. & Sanborn, M.R. (1990~, Preliminary development of a diagnostic test for Brucella using polymerase chain reaction. Z appL Beet., 69, 216-227. Fern~mdez.Lago, L.. Moriy6n, 1., Toyos, J. & Dfaz, R. (1982), Immunological identity of Brucella native hapten, polysaccharide B, and Yersinia enterocoliliea seroiype 9 native hapten. Infect. Immun., 38, 778-780. Fernandez-Logo, L., Rodriguez-Nebreda, M.S. & Chordi, A. (1988), Rapid serotyping of enteropathogenic Yersinia enteroeolitica str~ns by coagglutination. Ann. Inst. Pasteur/MicrobioL, 139, 461-471. Garin-Bastuji, B., Bowden, R., Dubray, (3. & Limet, J. (1990), Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of smooth lipopolysaccharide heterogeneity among Brucelia biovars related te A and M speeificities. J. clio. MicrobioL. 28, 2169-2174. Gretser-Wilke, !. & Moenning, V. (1987), Monoclonal antibodies and characterization of epitopes of smooth Brucetla lipopoiysaccharides. Ann. Inst. Pas¢eur/M~crobio/., 138, 549-560.
Kronvall, G. (1973), A rapid slide-agglutination method tor typing pneumococci by mea~s of specific antibody absorbed to protein-A-containing staphylococci. J. meal. MicrobioL, 6, 197-190. Meikle, P.J., Perry, M.B., Cherwonogrodzky, J.W. & BundIe, D.R. (1989)0 Fine structure of A and M anti= gens from Brucella biovars. Infect. lmmun., 57, 2820-2828. Plommet, M, (1986), Development of brucellosis control programmes. Principles and strategies for brucellosis control. Workshop on Brucellosis Control in Countries of the Mediterranean Area and the Arab Peninsula. Amman, 21 to 23 June ]986, Mediterranean Zoonoses Control Center. Keep II, M.R., Preston-Moore, D., Baggchi, T. & Schurig, G. (1987), Rapid identification of smooth Bru. celia species with a monoclonal antibody. J. clio. Mtcrobiol., 25, 2090-2093. Sehurig, G.G., Hammerberg, C. & Finkler, B.R. (1984), Monoelonal antibodies to Brucella surface antigens associated with the smooth lipopolysaccharide com~ plex. Amer. Jr. vet. Res., 45, 967-971. Stuart, F,A. & Corbel, M.J. (1982). Identification of a serological cross-reaction between Bru~ell¢ ebortus and Escheriehia coli O: 157. Vet. Rec., 110, 202-203. Thoen, C.O. & Enrigkt, F. (1986), Brueella, in "Pathogenests of bacterial infections in animals" (C.L. Gyles and C.O. Thoen) (pp. 160-171). Iowa State University Press, Ames. Vizea[no, N., Chordi, A. & Fermindez-Lago, L. (1991), Characterization of smooth Brucella lipopolysaccharides and polysaccharides bY monoclonal antibodies. Res. Microbiol.. 142, 971'-978.
Res. Microbial. 1992, 143, 513-518
A rapid and sensitive method for the identification of Brucella species with a monoclonal antibody N . Vizcalno and L. Fermindez-Lago (')
Depa[tamenlo de &li~,obiolog(a y Gendtica. Facullad de Farmacia. Universidad de Salamanca, 37007 Salamanca (Espaha)
SUMMARY A coagglutination test usi~'.g monoclonal antibody BmE10-5 with specificity for the M antigen of Brucella melitensis 1 6 M has been developed for the rapid identification of the smooth Bruce/is species. All reference strains or several biovars of B. melitensis, B. abortus, B. suis and B. neotomae tested were positive in this assay. No significant differences in reaction intensity were observed in relation to the different distribution of the A and M antigens among the Brucella serovars analysed. Conversely, rough Bnacefla species, with the exception of B. abortus 45120. were negative in the assay. Among the diffe,ant organisms tested not belonging to the genus Bruce/Is, serovar 0 9 of Yer~dnia enterocolitica was the only one that gave a weak positive reaction of coagglutination. Thus, in view of its rapidity. ;ifr=plicity, specificity and low costs, this technique could be highly useful for rapid identification of smooth Btuceda strains in diagnostic laboratories.
Key-words: Coao01utination, Bruce#a, mAb; Rapid identification, Smooth strains.
INTRODUCTION Brucellosis, a zoonitic disease responsible for abortion and infertility in certain species of domestic animals, mainly sheep, cattle, goats and swine, is caused by three main bacterial species : Brucella abortus, B. melitensis and B. suis (Thoen and Enright, 1986). These microorganisms can be transmitted to humans by direct contact with contaminated material or, more often, through consumption o f insufficiently treated milk and milk products from infected animals (Banal et aL, 1990). The diagnosis of brucello-
Submitted January 14, 1992~ ~ccepted March 25. 1992. (=) Corresponding au~hnr.
sis in humans and animals is often difficult to establish. Isolation of the ae¢iological agent from clinical samples by conventional methods is n o t always possible(Alton etaL, 1988). Accordingly, the detection and quantification of Brucella antibodies in serum samples using different serological tests plays a major role in the routine diagnosis of brucellosis ( F A O / W H O . , 1986). Despite this, apart from their diagnostic value, the isolation and identification of Bruce]In spp. are cons;~dered t_-zbe necessary stages for .:he design of ep;demiological and eradication programs (Plommet, t986)~ ThP identification of
514
N. VIZCA[NO A N D L. F E R N A N D E Z - L A G O
new isolates is a relatively tedious process, based largely on the use o f certain biochemical tests and on slide agglutination reactions with test sera (Corbel and Brinley-Morgan, 1984). Thus, the development of new techniques that permit rapid identification of t'reshly isolated Brucella strains is o f great practical importance.
mAb
The M-antigen.specific BmE 10-5 mAb used in this work was produced as previously described (Vizcaino el aL, 1991). The binding profile of this mAb was established by a competitive enzyme-linked immunosorbent assay (ELISA) using chemically defined Brueellu antigens (Vizcaino et al., 1991). The isotype was determined by ELISA using purified S-LPS as the antigen and isotype-specifie peroxidase conjugates (Nordic immunological Laboratories, Tilburg, The Netherlands). The isotype of this mAb was IgG3 (Vizeatno et aL, 199!).
Monoclonal antibodies (mAb) to Brucella spp. have been reported with specificity for A, M or both A and M antigens (Bundle et aL, 1989; Dubray and Limet, 1987 ; Garin-Bastuji et aL. I990; Greiser-Wilke and Moennig, 1987; Meikle ef al., 1989; Vizcaino et al., 1991). Recently, using mouse mAb and synthetic oligosaceharides, the structures o f Brucella A and M epitopes were postulated and the relative distribution o f the two antigens among O polysaceharides o f the different Brucella biovars was established (Bundle et al., 1989; Dubray and Limet, ]987; Garin-Bastuji et aL, 1990).
Coagglutination test
The stabilization and coating of protein-Acontaining staphylococci (Stap#tylococcus aureus Cow•n [. ATCC 12598) to make the coagglutination reagent followed the procedures described previously (Fernfi.ndez-Lago et aL, 1988; Kronvall, 1973). A l-ml sample of a 10 ~70(vol/vol) cell suspension was coated with 0.1 ml of ascitic fluid containing the BmEI0-5 mAb. After mixing of suspension for 3 h at room temperature, the staphylococci were washed twice and suspended at 1 % in phosphate-buffered saline (PBS) containing 0 . 1 % sodium aside. Coagglutination tests were performed by mixing on a slide, for 15-30 s, one drop of the staphylococcal reagent with one drop of the corresponding microorganism in PBS. Reactions were considered negative if no clumping occurred within that time.
In the present wo#;, we developed a coagglutination technique using a m A b , BmE10-5, with specificity against toe M antigen f r o m B. melitensis 16M (Vizcaino et aL, 1991) for rapid, sensitive and specific identification o f smooth Brucella species.
MATERIALS AND METHODS
RESULTS
Bacterial strains and growth conditions
Results obtained by studying different Brucel/~ reference strains using the coagglutination technique with reagent staphylococci coated with BmEI0-5 m A b are shown in table I, A n intense positive reaction occurred with all the naturally occurring smooth Brucella strains assayed: B. melitensis, B, abortus, B, suis and B. neotomae. No significant differences irr reaction irttensity were evidenced when comparing the different distributions of the A and M epitopes on the LPS molecule of the various Brucella sero,'ars aria-
Bacterial strains employed and their sources are listed in tables I, II and tit. Cultures were stored as cell suspensions at -20~C in 50 % glycerol and grown at 37°C under a 5 % CO 2 atmosphere for 24-48 h on tryptic soy agar (BBL Microbiology Systems, Cockeysvil.~e, MD, USA) enriched with 0.3 % yeast extract (Difeo). Brucefla isolates were characterized by conventional methods (Alton et al., 1988). The colony type phase was checked using standard procedures (Alton et aL, 1988 ; Corbel and BrinleyMorgan, [ 984).
EL|SA LPS mAb
= = =
enzyme-linked imm'~rlOsorbehl lipop~ty,,accharide. m o n o c l o n a l alltibc.dy.
o~,~ay.
] I I
r3S S-LPS
= -
phosphate-buffered LPS from smooth
saline. ~train,;.
IDENTIFICATION OF BRUCELLA SPECIES
515
Table I. Reactions of BruceUa strains in the coagglutination test using the BmEI0 5 mAb. Brucella species
Biovar
Strain
Colony type
Reaction in coagglutination
B, B. B. B. B.
melitensis metitensis metitensis melitensis melitensis
1 1 2 3
16M (~) Revl (b) 63/9 tb) Ether (~1 ]8115 tD~
Smooth Smooth Smooth Smooth Rough
+ + + + -
B. B. B. B. B. B. B. B. B. B. B. B,
abortus abortus abortus abortu$ abortus abortus abortus ebortus abortus abortus abortus abortus
1 1 l 2 3 4 5 6 9 1
S 19 (b~ 544 (o 99 (c~ 86/8/59 to) Tuiy.a l° 292 t~, B3196 ~'~ 870 le) C68 re) 230g ~b~ 45/20 (bj RB51 ~b~
Smooth Smootla Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth Rough Rough
+ + + + ~+ + + + + + -
B. B. B. B. B.
suis suis suis suis neotomae
l 2 3 4
1330 (o Thomsen ~ 686 ~c) 40 ~¢~ 5K33 ~o
Smooth Smooth Smooth Smooth Smooth
+ + + + +
BOW 63/290 (o RM-6/66 t~)
Rough Rough
-
B. ovis B. cams
t~ Obtained from the National Collection of Type Cultures, London, UK. '~ Obtained from G. Sehurtg, Regional College of Veterinary Medicine, Blaeksburg. VA, 24061, USA. L~IObtained from j . M . Verger, Station de Pathologic de la Reproduction, 37380 Nouziity, France.
lysed, in contrast, rough mutants o f B. metitensis (BI t5) and B. abortus (RB5 i) and all strains o f B. ovis a n d B. cauls assayed, which occur naturally in the rough f o r m , were negative in this assay (table I). U n d e r the same conditions, the specificity o f the assay was d e t e r m i n e d by studying d f f f c r c m bacterial species characterized by their crossreaction with Brucella spp. in other serological tests, Only a weak positive reaction was observed with strains belonging to serovar O9 o f Yersinia enterocolitica (table 11). Furthermore, a total o f 67 clinical isolates a n d field strains previously identified by conventional laboratory tests as be-
longing to the genus Brucella were investigated (table III). Results d e m o n s t r a t e d the high sensitivity of the assay, since all s~rains analysed gave a clear positive reaction. Finally, the specificity o f the coaggtutinatioa test using m A b BmE10-5 was investigated in a total o f 123 cl::nical isolates not belonging to the Brucella genus. N o false-positive reactions were noted. An antibody-free s~aphylococcal cell suspension and a suspension coated with purified lgG-class antibodies obtained from normal m o u s e serum were used as controls ; a o :tgglutination with any bacterial isolate occurred with these controls.
516
N, VIZCA[NO A N D L. FERN.~NDEZ-LAGO
Table i1. Reactions in the coagglutination test with BmEI0-5 mAb of different bacterial species which crossreact with Brucella spp. in other serologic tests. Species and strain
Reaction in coagglutination
Yersinia enteroeolitica serovar 0 9 [P383 I~l W22708 ta~ WI024 ;a) W836 (a~ W830 ~'~ WI91 (at W227 ~a~ Eseherwhia colt sernvar O157 ATCC 35150 tbl Cut~t#ylvb,¢;~i f e t u s subsp, venerealis NCTC 10354 I:) Campylobaeterfetus subsp, fetus NCTC 10842 le) Salmonella urbana serovar 030 ATCC 9261 Ib) Vibrio eholerae biovar Inaba CECT 512,542 la~ ~,, Obtained ~ Obtained ~r~Obtained c~, Obtained
from from From from
G. Wauters, Universit¢ Catholiqu¢ de Loltvain, Brussels, Belgium. the American Type Culture Collection, Roekville, MD, USA. the Nationat Collection of Type Cultures, London, UK. the Colecci6n Espafiola de Cul~ivos Tipo, Valencia, EspafiaL
~lable III. Results of testing 67 Brucella spp. isolates by coagglutinat[on test using the BmEI0-5 mAb. Brucella species/ no. of strains B. B. B. B. B.
Binvar
O~igin
Colony type
Reaction in coagglutination
1 2 3 1 2
Human Human Human Bovine Bovine
Smooth Smooth Smooth Smooth Smooth
+ + + + +
melitensis/12 (°~ melitensis/3 melitensis/40 abortus/lO abonus/2
m Obtained from the Departamento de Microbio[ogia, Hospital Clinieo, Salamanca, Espafia.
DISCUSSION
R a p i d identification o f Brucella strains is o f practical i m p o r t a n c e for b o t h bacterial d i a g n o sis a n d epiderniologlcal studies. C o n v e n t i o n a l bacteriological tests c o m m o n l y u ~ d for laboratory identification o f Brucella spp. are relatively slow a n d laborious procedures that m a y take up to two days to complete ( A l t o n el a/,, 1988).
Accordingly, new a n d faster m e t h o d s have been designed to reduce this time, thus permittir~g a preliminary r e p o r t o n the same day (Fekete et aL, 1990; R o o p II et aL, 1987). In this sense, the data offered in this work underscore the usefulness o f the staphylococcal coagglutination test owing to its speed, simplicity and low costs, apart from its high sensitivity a n d specificity, to carry out preliminary identification of B. meliterr-
IDENTIFICATION OF BRUCELLA SPECIES sis, B. abortus, B. suis a n d B. neotomae in the diagnostic l a b o r a t o r y .
T h e e o a g g l u t i n a t i o n tests used in this work involve use o f the m A b BraE10-5, with specificity against the O polysaccharide chain (M antigen) o f the L P S f r o m B. melitensis 16M (Vizcaino e t a l . , 1991). T h e recognition by tltis m A b o f epitopes c o m m o n to b o t h A a n d M antigens w o u l d a c c o u n t for t h e positive coagglutination reaction also observed with all the smooth Brucella serovars t h a t preferentially express the A antigen o n the L P S molecule (Bundle et aL, 1989). By contrast, n o c o a g g l u t i n a t i o n reaction was o b s e r v e d with a n y o f t h e r o u g h Brucetla strains tested, with the exception o f the r o u g h B. abortus 4 5 / 2 0 strain, a m i c r o o r g a n i s m characterized by expression, o n its surface, o f variable a m o u n t s o f low molecular weight A antigen (Meikle et aL, 1989; $churig e t a L , 1984). T h e specificity o f the t e c h n i q u e was established by the use o f different bacterial strains that cross-react wire Brucella spp. ill o t h e r serological tests (Corhel~ tq75; Feeley~ 1.969; FernfindezLago etctL, 1982; Stuart a n d Corbel, 1982). Only a weak positive r e a c t i o n was observed with strains b e l o n g i n g t o serotype 0 9 of Y. enteroeolith:a (table 11). This can be explained by t h e close structural relationship between the O antigen f r o m Y. enteroeolitica 0 9 a n d the A antigen f r o m Brucella spp. (Bundle el eL, 1984, 1989; C a r o f f et aL, 1984). However, f r o m the diagnostic point o f view, these r,'sults have n o important drawbacks, since both bacterial strains can be readily differentiated in the l a b o r a t o r y o n t h e basis o f different biochemical tests (Corbel a n d Briniey-Morgan, 1984). In s u m m a r y , in view o f its speed, sensitivity a n d specificity, the a b o v e described coagglutin a t i o n test with m A b BraE10-5 m a y be particularly useful in b o t h h u m a n a n d veterinary diagnostic laboratories to carry out identification o f smooth B. abortus, B, melitensis, B, suis a n d B. neoLomae strains.
Acknowledgements
This w~,rk was supported by gra~kt 1036/89 fror~ the Junta de Castilla y Le6n, Spain.
517
Une mdthode rapide el sensible u¢ilisant un anticorps monoelonal pour I'identifieation de I'esl~ce Bruceila
Nous avons dCvelopp~ un test de coagglutination utBisant un anticorps monoclonal dirig~ contre l'antig,~ne M de Brucella melitensis 16M pour l'identification rapide des esp&:es de Brucella ~smooth >>. Toutes Ies souehes de rCference de B. melitensis, B. abortus, B. suis et B, neotomae utilisdes ~taient positives darts ce test. Nous n'avons pas observ6 de diffCrence signiflcative de rintensit6 de la rCacrion en relation avec des diffdrences de distribution des antig~nes A e t M parmi los biovars de Brucella analys~s, Par contre, los aspires de Brace/In , r o u g h , , h l'exception de B, abortus 45/20, ont 6t~ u~gatives. Parmi los organismes analysCs qui n'appartiennent pas an genre Brucella, seule une ri'action de coagglutination faiblement positive a ~td observ~e avee Yetsinia enterocolitica serotype 0 9 . Grace/~ sa rapidit6 et sa zp~cificitC, cettc technique poarrait ~tre d'une grande ulilit6 pour I'identification rapide des souehes de Brucella, smooth~ dans les laboratoires de diagnostic. Mols-¢'l~5: Coagglutination, Brucelta, mAb; Identification rapide, Souches ~ smooth ~>.
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