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Immunochemical studies on a Brucella ovis specific protein antigen
l ~,terinaty Microbiology, 27 ( 1991 ) 125-131 Elsevier Science Publishers B.V., Amsterdam
125
Immunochemical studies on a Brucella ovis specific protein antigen Carlos E. Suarez, Gabriel A. Pacheco and Ana M. Vigliocco Comision Nacional de Energia Atomica, Avda del Libertador 8250-(1429), Capital Federal. Buenos A ires, A rgen tina (Accepted 19 September 1990)
ABSTRACT Suarez, C.E., Pacbeco, G.A. and Vigliocco, A.M., 1991. Immunochemical studies on a Brucella ovis specific protein antigen. Vet. Microbiol., 27:125-131. A Brucella ovis surface protein antigen ( P-II ), obtained by gel filtration with Sepharose 4B of a hot saline extract was characterized. The analysis of P-II over gradient sodium dodecylsulfate electrophoresis yielded an 18.5 and a 20 kDa band. In a radioimmunoprecipitation assay using P-If labeled with ~251, the antigen reacted specifically only with sera from rams experimentally infected with a naturally occurring rough strain of B. ovis and did not react with sera from rams experimentally infected with other smooth Brucella strains ( B. abortus and B. melitensis ).
INTRODUCTION
Brucella ovis, a causative agent of ram epidydimitis constitutes an important limiting factor for the development of sheep production in many parts of the world. Infection results in reduction of fertility of rams, and abortion has also been reported in infected ewes (McGowan and Schultz, 1956; McGowan and Devine, 1960 ). Differentiation of infection due to B. melitensis from that due to B. ovis by serological means is now essential, not only because of the natural infectivity of this strain, but also because of the introduction of live, attenuated B. melitensis strains as a vaccine to control B. ovis infection in sheep (Blasco et al., 1987; Plackett et al., 1989; Gamazo et al., 1989). Brucella ovis saline extract (SRA) (Myers et al., 1972) is composed of several antigenic components: ASR-pp, which contains LPS; P-III, a polysaccharide antigen, and P-II, a protein antigen (Vigliocco et al., 1982; Suarez et al., 1988 ). In enzyme-linked immunosorbent assays SRA reacts with sera from rams infected with B. melitensis (Riezu-Boj et al., 1986). However, in agar ~Present address: Dept. of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, 99163-7040, USA.
0378-1135/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
l~'6
, [ '~t \ R [ / I t
\1
gel diffusion tests, P-I1 and P-Ill react specifically only with sera t'rom rams experimentally infected with B. ovis. These results suggest that these coextracted and purified antigens are suitable candidates to bc used for the dcvelopment of a new and more specific serodiagnosis test. In this report additional immunochemical characterization of P-II is described. Radioiodinated P-II was employed in radioimmunoprecipitation ( R I P ) test to study its serological specificity and immunological relationships with other SRA components. MATERIALS
AND
METHOI)S
Antigen preparation The antigens used in the present work, ASR, ASR-pp, P-III and P-11 were obtained as previously described (Suarez et al., 1988). Briefly, P-II was obtained by gel filtration of SRA (0.4 mg protein ml -~ ) over a Sepharose 4B column ( 4 0 X 1 c m ) , eluted with saline phosphate buffer, pH 7.4. P-II appeared as a 280 nm absorbance peak with an apparent molecular weight similar to the ovalbumin standard ( M w 43 k D a ) . Radioiodination P-II (40 #g in protein ) was radioiodinated with ~esI (1 mCi, New England Nuclear, NEZ 0 3 3 / H ) by the chloramine T method ( McConahey and Dixon. 1980). S('rgl Four rams were experimentally infected with rough B. ovis (strain No. II ). two rams with smooth B. melitenis (Rev 1 ) and two rams with B. abortus ( S19) strains (Suarez et al., 1988 ). The seroagglutination titre for the rams infected with both smooth strains of Brucella used in the present work was 1:200.
Radioimmunoprecipitation Sera ( 2 0 0 / t l ) diluted in veronal-saline buffer ( V B D ) , pH 7.4, was incubated overnight in glass tubes with about 30 X l0 s counts per minute ( c p m ) of radiolabeled P-II, at 4°C. Inhibitors, when present were also added in this step. Rabbit anti-sheep antiserum ( 2 0 0 / t l ) was added and the tubes were reincubated for 30 min at 37°C, centrifuged at 3500 g for 15 min at 4°C and the supernatant was discarded. The radioactivity present in the pellet was measured in a g a m m a counter. A blank assay containing buffer VBD instead of diluted serum was always performed in each series of assays. The results of the test are expressed as the fraction o f c p m present in the precipitate (% pp). % pp was calculated as follows: % pp = (cpm pellet/total cpm a d d e d ) X 100. % of inhibition was calculated as follows: % inhibition= 100% pp with addition of inhibitor X 100 % pp without additions
BRUCELLA OVIS SPECIFIC PROTEIN ANTIGEN
127
Other methods: Proteins were quantified by the Lowry procedure (Lowry et al., 1951 ), with bovine serum albumin as the standard, carbohydrates by the phenol-sulfuric acid method (Dubois et al., 1956) with glucose as the standard. Gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a 7.5 to 17.5 % gel as described by Takacs (1979). The molecular weight standards used were: albumin bovine (Mw 66 kDa), glyceraldehyde 3-phosphate dehydrogenase (Mw 36 kDa) and B-lactoglobulin (Mw 18.4
~ ] ~ i ~ i ~ iiiiiii~!i!!!!!ili!~!iiiiii~iiiii!i~; i~!!i!~i~iiiii~!!iiii!~ii!!tii~
~7 ¸ ¸ ~ ¸ ii~ i ¸¸ ~!
i~i~i i i ~i!iji i~ iiiilj!iii!i ~ii¸¸! i!i~ ~ iiii~iiiiiiiiii!ii~iiii~
i~¸¸¸¸¸¸¸¸
"~66
~iii!~!~
i
~
!
-~36
kD
kD
18,5kD
A
B
Fig. 1. Gradient SDS-PAGE ofSRA (A) and P-II (B).
' I ~I \t,:[/I I ~!
I-~N
kDa ). Proteins in the gels were detected with Coomassie-blue. ('omplenaenl fixation tests were performed as described by Laboratory Branch Task Force (Anon., 1965). RESULTS
Fig. 1 shows the S D S - P A G E gradient pattern of P-If. A 20 and 18.5 kDa polypeptide component was detected. P-II was radioiodinated with ~eSl and TABLE I Radioimmunoprecipitation lest using iodinatcd P-II antigen against scra from a non-infected ( ( ' F titrc: 0 ) and a experimentall? infected rain ( C F titrc: 1:320). ( ' F lilre I
Serum dilution
RIP (% p p ) :
0
1:100 1: 1000 1:100
8.2 8.1 8.0 33.7
1: 1000
34.7
I : 10000
28.3
1 : 10000
1:320
t('F : complement fixation titre ofsera. ~RIP for blank assa?: 7. I%. "FABLE 2 ( o m p e t i t i v e RIP assay using iodinated P-ll antigen against sera from non-infected (('t~ tin-e:0) or experimentally infected (CF tilre: 1:320) rams without and with the addition of antigenic flaclions purified from ASR ("F titrc ~~"
Addition
RIP ( % p p ) ~
'Mnhibition
ti 1:320 1:320 1:320 1:320 1:320 1:320 1:320 1:320 1:320 1:32(I
none none P-lit 15 jig )4 P - l i t 1.5/lg) P-ll ( 0.15 ltg ) ASR-pp( 10/lg) ASR-pp ( 1.0 fig ) ASR-pp(0.1 ltg ) P-Ill( 1()/lg) ~ P-Ill( 1.0 itg) P-III(O.I /~g)
7.6 38.0 24.7 29.7 32.0 33.0 35.6 37.8 37.6 36.(/ 36.0
~,5 ~2 I~ I 6 [).2 0.2 [).6 I).~
t('F : complement fixation titre of the sera. ~Serum dilution I:100. ~RIP 1or blank assay : 7%. 4Mass of inhibitor expressed in ]lg of protein. ~Mass of inhibitor expressed in/Lg of total carbohydrates.
BRUCELLA OVIS SPECIFIC PROTEIN ANTIGEN
129
TABLE 3 Specificity of ~25I-P-II in RIP assay with sera from r a m s non-infected a n d experimentally infected with B. ovis, B. melitensis a n d B. abortus. Each sample was processed by duplicate. % pp in RIP for the blank assay was 7% Type of s e r u m ~
R I P ( X % pp_+ S D ) 2
Non-infected B. OVi33 B. melitensis 4 B. abortus 4
8.0_+0.2 22.0_+0.5 9.9 _+0.4 8.0 _+0.2
~Serum dilution 1:100. 2~ represents the average in % pp in RIP o f two r a m s tested for each group; SD: s t a n d a r d deviation. 3 C o m p l e m e n t fixation titre: 1: 180. 4Seroagglutination titre: 1:200.
further tested in a RIP test against sera of rams experimentally infected with B. ovis at several dilutions. A typical result is shown in Table 1, were the % pp in RIP obtained using serum from one experimentally infected ram (CF titre 1:320) is compared with the % pp obtained with serum from a non-infected ram (CF titre 0). Even at high dilutions ( 1: 10 000), the labeled antigen reacted only with the sera from experimentally infected rams. Sera from noninfected rams (CF titre: 0), as the one showed in Table 1, usually gave RIP values which did not differ from those obtained from the blank assay. The possibility of cross-reactions between P-III and SRA-pp with P-II, which are co-extracted with P-II from the saline extract, was studied. A competitive RIP assay was performed using P-II, P-III and SRA-pp as inhibitors. The results obtained are shown in Table 2. Only P-II gave significant inhibition. Neither ASR-pp nor P-III inhibited the binding of iodinated P-II to antisera, except of SRA-pp at a high protein concentration. The immunological specificity of iodinated P-II was also studied in RIP assays using sera from rams experimentally infected with B. abortus, B. melitensis and B. ovis. The results are shown in Table 3. Sera from rams experimentally infected with smooth Brucella strains failed to react with radiolabeled P-II in this assay, given RIP values which did not differ with those obtained for sera from non-infected rams or in the blank assay. DISCUSSION
We have previously demonstrated that P-II, a protein antigen, reacts specifically with sera from rams experimentally infected with B. ovis in agar gel diffusion test (Suarez et al., 1988 ). This study tested suitability and specificity of P-II for the differential diagnosis between B. ovis, a naturally occurring infective rough strain and B. melitensis and B. abortus, both smooth strains, in a radioimmunoprecipitation test. The data in Table 1 show that the RIP
f "ill
I
'-,I
\1,~1/1
! \!
assay is highly sensitix.e when used to detect infected rams. l h e RIP data in Tables 1 and 3 suggest the possible development of a primary interaction test for serodiagnosis of ovine brucellosis. P-II was shown to be free of P-Ill and LPS contamination bv S D S - P A G E data and the inhibition assay (Fig. I and Table 2 ). The minor degree of inhibition obtained when ASR-pp was used as inhibitor (Table 2 ) is likely due to P-II contamination of this fraction. This contamination was visualized in gel filtration of ASR-pp (data not shown ). The results presented here suggest that P-II could be a valuable tool for serodiagnosis of ovine brucellosis, particularly in those cases involving infection or vaccination which require a differentiation between infection due to B. ovis and B. melitensi.~. ACKNOWLEDGEMENTS
The authors thanks to Guy H. Palmer and Melissa Swan for revision of the manuscript.
REFERENCES Anonimous, 1965. Standard diagnostis complement fixation. Method and adaptation to micro test. Laboratory Branch Task Force. Public Health Monograph (U.S.). No 74. Blasco. J.M., Marin. C.M., Barberan, M., Moriyon I. and Diaz, R., 1987. Immunization with Brucella melitensis REV- 1 against Brucella ovis infection of rams. Vet. Microbiol., 14:381 392. Dubois, M., Gilles, K.A., Hamilton, J.K.. Rebers, P.A. and Smith, F., 1956. ('olorimetric method for determination of sugars and related substances. Anal. Chem., 28: 350-356. Gamazo, C., Winter, A.J., Moriyon, I., Riezu-Boj, J.l. Blasco, J.M. and Diaz, R.. 1989. Comparative analyses of proteins extracted by hot saline or released spontaneously into outer membrane blebs from field strains ofBrucella ovis and Brucella melitensis. Infect. hnmun., 57: 1419-t426. Lowry, O.H., Rosebrough, N.J., Farr, L. and Rendall, R.J., 1951. Protein measuremcnl with the folin phenol reagent. J. biol. Chem., 193: 265-275. McConahey, P.J. and Dixon, F.J., 1980. Radioiodination of proteins by the use of the chloramine T methods Enzymol., 70:210-213. McGowan, B. and Devine, D.R., 1960. Epididimytis of rams: the effect of the naturally occurring disease upon fertility. Cornell Vet., 50:103-106. McGowam B. and Schultz, G., 1956. Epididymitis of rams: clinical description and field aspects. Cornell Vet., 46:277-281. Myers, D.M., Jones, L.M. and Varela Diaz, V.M., 1972. Studies of antigen for complement fixation and gel diffusion test in the diagnosis of infection caused by Bruce/la ovi~ and other Brucella. Appl. Microbiol., 23: 894-902. Plackett, P., Corner, L.A., Fifis, T., Radford, A.J., Ripper, J.k., Chen Naichang and Liu Yumei, 1989. Discrimination between sheep antibodies to Brucella melilen,~is and to Brucella ovi.v. Vet. Microbiol., 20: 339-348. Riezu-Boj, L., Moriyon, 1., Blasco, J.M., Marin, C M . and Diaz, R., 1986. Comparison oflipo-
BRUCELLA OVIS SPECIFIC PROTEIN ANTIGEN
131
polysaccharide and outer membrane protein-lipopolysaccharide extracts in an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection. J. Clin. Microbiol., 23: 938-942. Suarez, C.E., Pacheco, G.A. and Vigliocco, A.M., 1988. Characterization of Brucella ovis surface antigens. Vet. Microbiol., 18: 349-356. Takacs, B., 1979. Electrophoresis of proteins in polyacrylamide slab gels. In: T. Lefkovits and B. Pernis (Editors), Immunological Methods Academic Press, New York, p. 81. Vigliocco~ A.M., Suarez, C.E. and Pacheco, G.A., 1982. Aislamiento, purification caracterizacion y posterior marcacion con I de las distintas fracciones immunologicamente activas del antigeno superficial de Brucella ovis. Rev. Mil. Vet., 30: 87-99.
125
Immunochemical studies on a Brucella ovis specific protein antigen Carlos E. Suarez, Gabriel A. Pacheco and Ana M. Vigliocco Comision Nacional de Energia Atomica, Avda del Libertador 8250-(1429), Capital Federal. Buenos A ires, A rgen tina (Accepted 19 September 1990)
ABSTRACT Suarez, C.E., Pacbeco, G.A. and Vigliocco, A.M., 1991. Immunochemical studies on a Brucella ovis specific protein antigen. Vet. Microbiol., 27:125-131. A Brucella ovis surface protein antigen ( P-II ), obtained by gel filtration with Sepharose 4B of a hot saline extract was characterized. The analysis of P-II over gradient sodium dodecylsulfate electrophoresis yielded an 18.5 and a 20 kDa band. In a radioimmunoprecipitation assay using P-If labeled with ~251, the antigen reacted specifically only with sera from rams experimentally infected with a naturally occurring rough strain of B. ovis and did not react with sera from rams experimentally infected with other smooth Brucella strains ( B. abortus and B. melitensis ).
INTRODUCTION
Brucella ovis, a causative agent of ram epidydimitis constitutes an important limiting factor for the development of sheep production in many parts of the world. Infection results in reduction of fertility of rams, and abortion has also been reported in infected ewes (McGowan and Schultz, 1956; McGowan and Devine, 1960 ). Differentiation of infection due to B. melitensis from that due to B. ovis by serological means is now essential, not only because of the natural infectivity of this strain, but also because of the introduction of live, attenuated B. melitensis strains as a vaccine to control B. ovis infection in sheep (Blasco et al., 1987; Plackett et al., 1989; Gamazo et al., 1989). Brucella ovis saline extract (SRA) (Myers et al., 1972) is composed of several antigenic components: ASR-pp, which contains LPS; P-III, a polysaccharide antigen, and P-II, a protein antigen (Vigliocco et al., 1982; Suarez et al., 1988 ). In enzyme-linked immunosorbent assays SRA reacts with sera from rams infected with B. melitensis (Riezu-Boj et al., 1986). However, in agar ~Present address: Dept. of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, 99163-7040, USA.
0378-1135/91/$03.50
© 1991 - - Elsevier Science Publishers B.V.
l~'6
, [ '~t \ R [ / I t
\1
gel diffusion tests, P-I1 and P-Ill react specifically only with sera t'rom rams experimentally infected with B. ovis. These results suggest that these coextracted and purified antigens are suitable candidates to bc used for the dcvelopment of a new and more specific serodiagnosis test. In this report additional immunochemical characterization of P-II is described. Radioiodinated P-II was employed in radioimmunoprecipitation ( R I P ) test to study its serological specificity and immunological relationships with other SRA components. MATERIALS
AND
METHOI)S
Antigen preparation The antigens used in the present work, ASR, ASR-pp, P-III and P-11 were obtained as previously described (Suarez et al., 1988). Briefly, P-II was obtained by gel filtration of SRA (0.4 mg protein ml -~ ) over a Sepharose 4B column ( 4 0 X 1 c m ) , eluted with saline phosphate buffer, pH 7.4. P-II appeared as a 280 nm absorbance peak with an apparent molecular weight similar to the ovalbumin standard ( M w 43 k D a ) . Radioiodination P-II (40 #g in protein ) was radioiodinated with ~esI (1 mCi, New England Nuclear, NEZ 0 3 3 / H ) by the chloramine T method ( McConahey and Dixon. 1980). S('rgl Four rams were experimentally infected with rough B. ovis (strain No. II ). two rams with smooth B. melitenis (Rev 1 ) and two rams with B. abortus ( S19) strains (Suarez et al., 1988 ). The seroagglutination titre for the rams infected with both smooth strains of Brucella used in the present work was 1:200.
Radioimmunoprecipitation Sera ( 2 0 0 / t l ) diluted in veronal-saline buffer ( V B D ) , pH 7.4, was incubated overnight in glass tubes with about 30 X l0 s counts per minute ( c p m ) of radiolabeled P-II, at 4°C. Inhibitors, when present were also added in this step. Rabbit anti-sheep antiserum ( 2 0 0 / t l ) was added and the tubes were reincubated for 30 min at 37°C, centrifuged at 3500 g for 15 min at 4°C and the supernatant was discarded. The radioactivity present in the pellet was measured in a g a m m a counter. A blank assay containing buffer VBD instead of diluted serum was always performed in each series of assays. The results of the test are expressed as the fraction o f c p m present in the precipitate (% pp). % pp was calculated as follows: % pp = (cpm pellet/total cpm a d d e d ) X 100. % of inhibition was calculated as follows: % inhibition= 100% pp with addition of inhibitor X 100 % pp without additions
BRUCELLA OVIS SPECIFIC PROTEIN ANTIGEN
127
Other methods: Proteins were quantified by the Lowry procedure (Lowry et al., 1951 ), with bovine serum albumin as the standard, carbohydrates by the phenol-sulfuric acid method (Dubois et al., 1956) with glucose as the standard. Gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a 7.5 to 17.5 % gel as described by Takacs (1979). The molecular weight standards used were: albumin bovine (Mw 66 kDa), glyceraldehyde 3-phosphate dehydrogenase (Mw 36 kDa) and B-lactoglobulin (Mw 18.4
~ ] ~ i ~ i ~ iiiiiii~!i!!!!!ili!~!iiiiii~iiiii!i~; i~!!i!~i~iiiii~!!iiii!~ii!!tii~
~7 ¸ ¸ ~ ¸ ii~ i ¸¸ ~!
i~i~i i i ~i!iji i~ iiiilj!iii!i ~ii¸¸! i!i~ ~ iiii~iiiiiiiiii!ii~iiii~
i~¸¸¸¸¸¸¸¸
"~66
~iii!~!~
i
~
!
-~36
kD
kD
18,5kD
A
B
Fig. 1. Gradient SDS-PAGE ofSRA (A) and P-II (B).
' I ~I \t,:[/I I ~!
I-~N
kDa ). Proteins in the gels were detected with Coomassie-blue. ('omplenaenl fixation tests were performed as described by Laboratory Branch Task Force (Anon., 1965). RESULTS
Fig. 1 shows the S D S - P A G E gradient pattern of P-If. A 20 and 18.5 kDa polypeptide component was detected. P-II was radioiodinated with ~eSl and TABLE I Radioimmunoprecipitation lest using iodinatcd P-II antigen against scra from a non-infected ( ( ' F titrc: 0 ) and a experimentall? infected rain ( C F titrc: 1:320). ( ' F lilre I
Serum dilution
RIP (% p p ) :
0
1:100 1: 1000 1:100
8.2 8.1 8.0 33.7
1: 1000
34.7
I : 10000
28.3
1 : 10000
1:320
t('F : complement fixation titre ofsera. ~RIP for blank assa?: 7. I%. "FABLE 2 ( o m p e t i t i v e RIP assay using iodinated P-ll antigen against sera from non-infected (('t~ tin-e:0) or experimentally infected (CF tilre: 1:320) rams without and with the addition of antigenic flaclions purified from ASR ("F titrc ~~"
Addition
RIP ( % p p ) ~
'Mnhibition
ti 1:320 1:320 1:320 1:320 1:320 1:320 1:320 1:320 1:320 1:32(I
none none P-lit 15 jig )4 P - l i t 1.5/lg) P-ll ( 0.15 ltg ) ASR-pp( 10/lg) ASR-pp ( 1.0 fig ) ASR-pp(0.1 ltg ) P-Ill( 1()/lg) ~ P-Ill( 1.0 itg) P-III(O.I /~g)
7.6 38.0 24.7 29.7 32.0 33.0 35.6 37.8 37.6 36.(/ 36.0
~,5 ~2 I~ I 6 [).2 0.2 [).6 I).~
t('F : complement fixation titre of the sera. ~Serum dilution I:100. ~RIP 1or blank assay : 7%. 4Mass of inhibitor expressed in ]lg of protein. ~Mass of inhibitor expressed in/Lg of total carbohydrates.
BRUCELLA OVIS SPECIFIC PROTEIN ANTIGEN
129
TABLE 3 Specificity of ~25I-P-II in RIP assay with sera from r a m s non-infected a n d experimentally infected with B. ovis, B. melitensis a n d B. abortus. Each sample was processed by duplicate. % pp in RIP for the blank assay was 7% Type of s e r u m ~
R I P ( X % pp_+ S D ) 2
Non-infected B. OVi33 B. melitensis 4 B. abortus 4
8.0_+0.2 22.0_+0.5 9.9 _+0.4 8.0 _+0.2
~Serum dilution 1:100. 2~ represents the average in % pp in RIP o f two r a m s tested for each group; SD: s t a n d a r d deviation. 3 C o m p l e m e n t fixation titre: 1: 180. 4Seroagglutination titre: 1:200.
further tested in a RIP test against sera of rams experimentally infected with B. ovis at several dilutions. A typical result is shown in Table 1, were the % pp in RIP obtained using serum from one experimentally infected ram (CF titre 1:320) is compared with the % pp obtained with serum from a non-infected ram (CF titre 0). Even at high dilutions ( 1: 10 000), the labeled antigen reacted only with the sera from experimentally infected rams. Sera from noninfected rams (CF titre: 0), as the one showed in Table 1, usually gave RIP values which did not differ from those obtained from the blank assay. The possibility of cross-reactions between P-III and SRA-pp with P-II, which are co-extracted with P-II from the saline extract, was studied. A competitive RIP assay was performed using P-II, P-III and SRA-pp as inhibitors. The results obtained are shown in Table 2. Only P-II gave significant inhibition. Neither ASR-pp nor P-III inhibited the binding of iodinated P-II to antisera, except of SRA-pp at a high protein concentration. The immunological specificity of iodinated P-II was also studied in RIP assays using sera from rams experimentally infected with B. abortus, B. melitensis and B. ovis. The results are shown in Table 3. Sera from rams experimentally infected with smooth Brucella strains failed to react with radiolabeled P-II in this assay, given RIP values which did not differ with those obtained for sera from non-infected rams or in the blank assay. DISCUSSION
We have previously demonstrated that P-II, a protein antigen, reacts specifically with sera from rams experimentally infected with B. ovis in agar gel diffusion test (Suarez et al., 1988 ). This study tested suitability and specificity of P-II for the differential diagnosis between B. ovis, a naturally occurring infective rough strain and B. melitensis and B. abortus, both smooth strains, in a radioimmunoprecipitation test. The data in Table 1 show that the RIP
f "ill
I
'-,I
\1,~1/1
! \!
assay is highly sensitix.e when used to detect infected rams. l h e RIP data in Tables 1 and 3 suggest the possible development of a primary interaction test for serodiagnosis of ovine brucellosis. P-II was shown to be free of P-Ill and LPS contamination bv S D S - P A G E data and the inhibition assay (Fig. I and Table 2 ). The minor degree of inhibition obtained when ASR-pp was used as inhibitor (Table 2 ) is likely due to P-II contamination of this fraction. This contamination was visualized in gel filtration of ASR-pp (data not shown ). The results presented here suggest that P-II could be a valuable tool for serodiagnosis of ovine brucellosis, particularly in those cases involving infection or vaccination which require a differentiation between infection due to B. ovis and B. melitensi.~. ACKNOWLEDGEMENTS
The authors thanks to Guy H. Palmer and Melissa Swan for revision of the manuscript.
REFERENCES Anonimous, 1965. Standard diagnostis complement fixation. Method and adaptation to micro test. Laboratory Branch Task Force. Public Health Monograph (U.S.). No 74. Blasco. J.M., Marin. C.M., Barberan, M., Moriyon I. and Diaz, R., 1987. Immunization with Brucella melitensis REV- 1 against Brucella ovis infection of rams. Vet. Microbiol., 14:381 392. Dubois, M., Gilles, K.A., Hamilton, J.K.. Rebers, P.A. and Smith, F., 1956. ('olorimetric method for determination of sugars and related substances. Anal. Chem., 28: 350-356. Gamazo, C., Winter, A.J., Moriyon, I., Riezu-Boj, J.l. Blasco, J.M. and Diaz, R.. 1989. Comparative analyses of proteins extracted by hot saline or released spontaneously into outer membrane blebs from field strains ofBrucella ovis and Brucella melitensis. Infect. hnmun., 57: 1419-t426. Lowry, O.H., Rosebrough, N.J., Farr, L. and Rendall, R.J., 1951. Protein measuremcnl with the folin phenol reagent. J. biol. Chem., 193: 265-275. McConahey, P.J. and Dixon, F.J., 1980. Radioiodination of proteins by the use of the chloramine T methods Enzymol., 70:210-213. McGowan, B. and Devine, D.R., 1960. Epididimytis of rams: the effect of the naturally occurring disease upon fertility. Cornell Vet., 50:103-106. McGowam B. and Schultz, G., 1956. Epididymitis of rams: clinical description and field aspects. Cornell Vet., 46:277-281. Myers, D.M., Jones, L.M. and Varela Diaz, V.M., 1972. Studies of antigen for complement fixation and gel diffusion test in the diagnosis of infection caused by Bruce/la ovi~ and other Brucella. Appl. Microbiol., 23: 894-902. Plackett, P., Corner, L.A., Fifis, T., Radford, A.J., Ripper, J.k., Chen Naichang and Liu Yumei, 1989. Discrimination between sheep antibodies to Brucella melilen,~is and to Brucella ovi.v. Vet. Microbiol., 20: 339-348. Riezu-Boj, L., Moriyon, 1., Blasco, J.M., Marin, C M . and Diaz, R., 1986. Comparison oflipo-
BRUCELLA OVIS SPECIFIC PROTEIN ANTIGEN
131
polysaccharide and outer membrane protein-lipopolysaccharide extracts in an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection. J. Clin. Microbiol., 23: 938-942. Suarez, C.E., Pacheco, G.A. and Vigliocco, A.M., 1988. Characterization of Brucella ovis surface antigens. Vet. Microbiol., 18: 349-356. Takacs, B., 1979. Electrophoresis of proteins in polyacrylamide slab gels. In: T. Lefkovits and B. Pernis (Editors), Immunological Methods Academic Press, New York, p. 81. Vigliocco~ A.M., Suarez, C.E. and Pacheco, G.A., 1982. Aislamiento, purification caracterizacion y posterior marcacion con I de las distintas fracciones immunologicamente activas del antigeno superficial de Brucella ovis. Rev. Mil. Vet., 30: 87-99.