Use of Brucella canis antigen for detection of ovine serum antibodies against Brucella ovis

Brucella ovis causes a genital disease of sheep manifested by epididymitis in rams and placentitis in ewes producing reduced fertility in the flock. Clinical diagnosis is not sensitive enough and bacteriological testing is not feasible for detection of the disease in large numbers of animals. Indirect methods of serological testing are preferred for routine diagnosis, of which agar gel immunodiffusion (AGID), complement fixation (CF) and ELISA tests are recommended as the most efficient. Since B. ovis shares antigenic components with Brucella canis, it would seem that either strain could be used as antigen with the same results; however, the advantage of the B. canis (M-) strain variant is that it can be used to develop a satisfactory antigen for agglutination tests. We present data on AGID and IELISA tests using B. ovis antigen and rapid screening agglutination test (RSAT), 2-mercapto-ethanol RSAT (2ME-RSAT) and IELISA using B. canis antigen. We tested 225 animals. The cut-off values were adjusted by ROC analysis using 51 negative and 32 positive sera; the IELISA-B. canis cut-off value was 39 (%P) and IELISA-B. ovis, 51 (%P), with 100% sensitivity and specificity. Of the 32 positive sera from the infected flock RSAT detected 32 (100%), 2ME-RSAT 29 (91%) and AGID 31 (97%). Of the 142 sera from suspicious flocks, 46 were negative and 56 positive in all the tests; 16 were positive by RSAT, IELISA-B. canis and IELISA-B. ovis, 20 positive only with RSAT and 2 positive only by both IELISAs. RSAT is a very sensitive screening test that, because of its simplicity and easy interpretation, following a study in larger sample, could replace AGID as a screening test for diagnosis of ovine brucellosis caused by B. ovis. The IELISA-B. canis or IELISA-B. ovis could be used as confirmatory tests, since they show equal specificity and sensitivity.     

A comparison of three serological tests for the diagnosis of B. ovis infection in rams

The complement fixation test (CFT), the enzyme labelled immunosorbent assay (ELISA) and the gel diffusion precipitin test (GD) were compared, for the diagnosis of Brucella ovis infection in rams. The sensitivities of the tests in 109 rams which were shedding B. ovis in their semen were: CFT 96.3%; ELISA 97.2%; GD 91.7%. The specificities of the tests in 141 rams from non-infected flocks were: CFI 99.3%; ELISA 98.6%; GD 100%. Predictive values of the three tests were measured in 285 rams from infected flocks. Thirty-eight percent of these rams were shedding B. ovis in their semen. Predictive values of positive tests were: CFT 75.5%; ELISA 66.7%; GD 72.5%. Predictive values of negative tests were: CFI 97.1%; ELISA 97.6%; GD 93.8%.     

Detection of antibodies to Brucella ovis in sheep milk using B. ovis and B. canis antigen

The diagnostic techniques most widely used for detecting brucellosis caused by Brucella ovis are serological tests such as complement fixation (CFT), agar gel immunodiffusion (AGID), and ELISAs. However, to our knowledge, milk tests, with the advantage that samples may be taken in a non invasive manner, have not been investigated as diagnostic tools. We studied 144 samples of milk and sera from lactating ewes, comparing bacteriological studies, serological and milk tests using Brucella canis and B. ovis antigens. A group of 75 ewes in an uninfected flock were serologically negative to rapid slide agglutination test (RSAT), indirect ELISA (IELISA)-B. canis, AGID and IELISA-B. ovis. The milk of these ewes had an IELISA-B. canis mean (%P) value of 16.18 (S.D. 5.63), while the IELISA-B. ovis had a mean (%P) value of 12.52 (S.D. 4.94). A cut-off value of (%P) 27.44 (+2 S.D.) or (%P) 33 (+3 S.D.) was determined by milk-ELISA-B. canis and (%P) 22.4 (+2 S.D.) and (%P) 27.34 (+3 S.D.) by milk-IELISA-B. ovis. These cut-off values were adjusted by receiver-operator characteristics (ROC) analysis using 23 positive samples from infected ewes, which indicated a milk-IELISA-B. canis cut-off value of (%P) 33 and milk-IELISA-B. ovis of (%P) 26 with 100% sensitivity and specificity. Based on our results, we propose that, following a study of a larger number of samples, the milk-IELISA-B. canis could be considered a suitable test for the diagnosis of B. ovis brucellosis in lactating ewes.     

The use of homologous antigen in the serological diagnosis of brucellosis caused by Brucella melitensis

In the European Union the serological diagnosis of brucellosis caused by Brucella melitensis is performed using the heterologous antigen of B. abortus S99. The possible higher sensitivity or ability of an early detection of antibodies by a homologous antigen may prove very useful in the final phases of an eradication programme. Results obtained in sheep experimentally infected by B. melitensis biovar 3 were compared using B. abortus S99, B. melitensis M1, M2 and M3 antigens in the Rose Bengal plate test (RBPT), the complement fixation test (CFT) and an enzyme-linked immunosorbent assay (ELISA) test. Forty-six sheep from an officially brucellosis-free flock were experimentally infected intraconjunctivally with B. melitensis biovar 3. Prior to infection, all animals were tested first against Brucella antibodies, weekly for 2 months post-infection (PI) and then monthly for a further 7 months. All sera were tested against the antigens listed above using RBPT, CFT and ELISA. Using a Bayesian approach, test sensitivities were estimated and compared. Their ability for the early detection of antibodies was evaluated through a regression model based on a logit response model, using the number of days PI as the independent variable and the logit of the fraction of positive animals as the dependent variable. No significant differences were detected among the various antigens used, either in terms of sensitivity or in terms of antibody kinetics; however, the CFT was significantly less sensitive than the RBPT and ELISA and it also showed a lower rate of increase of percentage positive animals (beta-coefficient of regression analysis).     

Antibodies to the CP24 protein of Brucella melitensis lack diagnostic usefulness in ovine brucellosis

The potential diagnostic usefulness of antibodies to the ribosome recycling factor of Brucella melitensis (CP24) was assessed in sheep by an indirect ELISA with purified recombinant CP24. Sera from uninfected animals from the UK (n=44) and from local flocks (n=42), from sheep naturally infected with B. melitensis (n=12) or B. ovis (n=12), and from lambs (n=7) or pregnant ewes (n=6) vaccinated with B. melitensis Rev-1, were assayed. High specific optical densities (OD(with antigen) - OD(without antigen)) were obtained with both the groups of normal sera, which resulted in high cut-off values (1.414 and 1.267, respectively). Only two infected sheep yielded specific OD higher than these cut-off values. No significant difference was found between mean specific OD from B. melitensis- or B. ovis-infected sheep (0.574 and 0.472, respectively), those from vaccinated animals (0.396 and 0.400 for pregnant ewes and lambs, respectively), and those from Brucella-free animals. An inhibition ELISA with soluble CP24 confirmed the specificity of the antibodies detected in normal sera by the indirect ELISA; these antibodies belonged to the IgG class as revealed by the use of a specific conjugate. Sera from infected sheep were all positive for antibodies against lipopolysaccharides and lumazine synthase from Brucella. These results show that anti-CP24 antibodies have no diagnostic role in ovine brucellosis.     

Evaluation of electrophoretic immunoblotting for Brucella ovis infection in deer using ram and deer serum

AIMS: Recently the first case of natural infection of deer with Brucella ovis was discovered. The aim of this study was to develop and evaluate an electrophoretic immunoblotting method for testing deer serum for specific B. ovis antibodies. METHODS: An existing immunoblotting method for sheep serum was altered by using a recombinant protein G-alkaline phosphatase conjugate and Tris-buffered saline containing 3% non-fat dry milk powder for the blocking step and the serum and conjugate dilutions. The method was evaluated using 106 sheep sera from B. ovis - negative, accredited flocks, 69 sera from chronically infected rams shedding B. ovis in their semen, 110 sera from a B. ovis-infected flock, 18 sera from stags from which B. ovis was isolated, and 48 sera from deer flocks free from B. ovis infections. The immunoblotting method was applied to another 85 deer sera. RESULTS: The sensitivity of the new immunoblotting method was 98.6% for sheep and 94.4% for deer, and the specificity 99.1% for sheep and 100% for deer. Sixty-nine out of 97 deer sera, originating from the property from which the first B. ovis deer case had been reported, tested positive or suspicious in the complement fixation test. Of these, 53 sera exhibited staining patterns in blots typical for B. ovis infections and also one serum which was negative in the CFT. Only six out of 1498 deer sera. from throughout New Zealand had positive or suspicious reactions in the B. ovis complement fixation test. Of these, one exhibited a staining pattern in the blot suggestive of a B. ovis infection, while four showed patterns of suspicious reactions. CONCLUSION: The new immunoblotting technique is useful as a confirmatory serological test method for B. ovis infections in deer.     

Comparative performance of tests using cytosolic or outer membrane antigens of Brucella for the serodiagnosis of canine brucellosis

Although some ELISA tests using cytoplasmic or outer membrane antigens of Brucella have been developed to improve the diagnosis of canine brucellosis, the performance of these assays has not been compared. In the present study three ELISA tests using lipopolysaccharide (LPS)-free cytoplasmic proteins (CPs) of Brucella abortus, the lumazine synthase (LS) of Brucella spp. or a hot-saline (HS) extract of Brucella canis containing outer membrane antigens were used to test sera from dogs with suspected or confirmed brucellosis (n=36) and from dogs with pathological conditions other than brucellosis (n=212). In the first group the proportion of positive results was 92, 92 and 81% for the ELISAs with HS, CP and LS, respectively, and 94% of the samples were positive by at least one ELISA test. Three dogs that were negative by agglutination (2ME-RSAT) had a positive result by at least one ELISA, and this discrepancy was attributed to the lower analytical sensitivity of agglutination tests. This hypothesis was confirmed by a serological follow-up of seven dogs recently infected with B. canis in three of which the illness was diagnosed earlier by one or more ELISA tests than by 2ME-RSAT. Among dogs having pathological conditions other than brucellosis, specificities were 94.3, 96.7 and 96.7% for the ELISAs with HS, CP and LS, respectively. This study shows that HS-ELISA and CP-ELISA are highly specific and sensitive for the diagnosis of canine brucellosis and can detect the infection by B. canis shortly after the exposure to the pathogen.     

Validation of the Fluorescence Polarisation Assay (FPA) for the serological diagnosis of brucellosis

In order to evaluate suitability of Fluorescence Polarisation Assay (FPA) for serological Brucella diagnostic, 1739 samples of sera from cattle, pigs, sheep and goats (65 Brucella-positive, 960-negative and 714 false-positive sera) were investigated at a dilution of 1:10. The cut-off was adjusted by means of ROC analysis. Furthermore, the serum samples were examined for Brucella antibodies using SAT, CFT and ELISA and the results were evaluated regarding sensitivity and specificity. FPA, SAT, CFT and ELISA attained a sensitivity of 92.3, 98.5, 84.6 and 86.2%. In comparison, specificity varied with 87.8, 72.6, 92.5 and 85.8%, respectively. Accordingly, FPA is a suitable test for serodiagnosis of brucellosis.     

Enzyme-linked immunosorbent assay for Brucella ovis specific antibody in ram sera

An enzyme-linked immunosorbent assay (ELISA) for the detection of Brucella ovis specific antibody in ram serum was compared with the currently employed complement fixation test (CFT). Rabbit anti-sheep IgG coupled to horseradish peroxidase was used as the antibody-enzyme conjugate and 2,2'-azino-di[3-ethylbenzthiazolin sulphonate (6)] as the substrate. The ratio of the optical density at 414 nm for positive and negative control sera (P/N ratio) was used to optimise the parameters of the test. Ram serum samples (16,527) were tested using ELISA and CFT (warm and cold) over a one year period. The ELISA was more sensitive and provided a more reliable measure of B ovis specific antibody than did the CFT. Implications of employing ELISA as the sole test in an eradication scheme are discussed.     

Antigen-induced production of interferon-gamma in samples of peripheral lymph nodes from sheep experimentally inoculated with Mycobacterium avium subsp. paratuberculosis.

The production of interferon-gamma (IFN-gamma) in response to Johnin purified protein derivate was measured in samples of the prescapular lymph node (PLN) from 10 sheep, aged 2 years, and nine sheep, aged 1 year that had been inoculated orally with Mycobacterium avium subsp. paratuberculosis within their first month of life. Ten non-inoculated sheep, aged 1 year, constituted the negative control group. The results obtained in the PLN IFN-gamma assay were compared with those derived from serological tests: a complement fixation test (CFT), agar gel diffusion test (AGID) and enzyme-linked immunosorbent assay (ELISA), as well as an IFN-gamma test on samples of blood. Among the 19 inoculated sheep, 16 gave positive reactions in the PLN IFN-gamma assay on samples incubated overnight, and 18 tested positive when the assay was applied to PLN samples incubated for 48h. In comparison, three, four and seven inoculated sheep gave positive reactions in the ELISA, CFT and in the blood IFN-gamma assay on samples incubated overnight, respectively. The AGID and IFN-gamma assay on blood samples incubated for 48h detected eight inoculated animals. Twelve inoculated sheep, that tested positive in the PLN IFN-gamma assay were clinically normal, gave negative results in an IS900-based polymerase chain reaction (PCR) assay on samples of ileum and ileocaecal lymph node and had no histological evidence of paratuberculosis, but tested positive on more than two occasions in sequential serological testing before necropsy. None of the 10 non-inoculated sheep tested positive in the AGID, CFT, ELISA, blood IFN-gamma assay on samples incubated overnight and for 48h or the PLN IFN-gamma assay on samples incubated overnight, but one gave a positive result in the PLN IFN-gamma assay on samples stimulated for 48h. It is likely that the positive reactions obtained by the PLN IFN-gamma assay in the 12 inoculated sheep that tested negative in the PCR assay and histopathological examination represents immunological evidence of latent infection or previous exposure to M. paratuberculosis rather than active infection.     

Comparison of a maedi-visna virus CA-TM fusion protein ELISA with other assays for detecting sheep infected with North American ovine lentivirus strains.

A maedi-visna virus CA-TM fusion protein ELISA (MVV ELISA) was evaluated for the detection of antibody in sheep infected with North American ovine lentivirus (OvLV). The results of the MVV ELISA were compared with other assays for OvLV antibody and with viral infection in an intensively studied group of 38 sheep with a high prevalence of OvLV infection and disease. The sensitivity, specificity, and concordance of assays for OvLV antibody (MVV ELISA, indirect ELISA, Western blot, and AGID), virus (virus isolation, PCR, antigen ELISA), and OvLV-induced disease in each animal were compared with OvLV infection status as defined by a positive result in two or more of the assays. Five sheep met the criteria for absence of OvLV infection. The sensitivity of the MVV ELISA in detecting OvLV infected sheep was 64%, whereas the sensitivity of the other three tests for antibody ranged from 85 to 94%. All the antibody assays were 100% specific in this group of animals. Of the assays for virus, the PCR test had the highest sensitivity and the best concordance with OvLV infection, but it also had the lowest specificity of any of the virus or antibody assays. Among the antibody tests, the concordance of the MVV ELISA compared most favorably with the AGID test for detecting OvLV-infected sheep. Analysis of serum samples from 28 lambs experimentally-infected with one of three North American strains of OvLV suggested that there were no significant strain differences detectable by antibody assay. Twenty virus-inoculated lambs were positive by both the MVV ELISA and the AGID test, five lambs were MVV ELISA negative and AGID test positive, and three lambs were MVV ELISA positive and AGID test negative. No pre-inoculation samples were positive by either assay. In a longitudinal study involving seven lambs, antibodies to OvLV were detected by AGID 3-5 weeks post-inoculation, but were not detected by MVV ELISA until 5-10 weeks post-inoculation. Among 128 naturally and experimentally-infected sheep that were seropositive in the AGID test, the overall sensitivity of the MVV ELISA was higher in the naturally infected sheep (84%) than in the experimentally infected sheep (69%). The data indicated that the MVV ELISA represents a less sensitive, but specific alternative for the detection of OvLV antibodies.     

Serologic survey of brucellosis in captive neotropical wild carnivores in northeast Brazil

Abstract. This study reports the detection of antibodies against Brucella abortus and B. canis in wild neotropical carnivores kept in captivity in three zoos in northeastern Brazil. A total of 42 serum samples were examined, 17 from coatis (Nasua nasua), eight from crab-eating raccoons (Procyon cancrivorus), three from crab-eating foxes (Cerdocyon thous), three from hoary foxes (Lycalopex vetulus), two from little spotted cats (Leopardus tigrinus), five from tayras (Eira barbara), two from greater grisons (Galictis vittata), and two from neotropical river otters (Lontra longicaudis). The Rose-Bengal test and complement fixation test (CFT) were performed to detect anti-Brucella spp. antibodies, whereas the agar gel immunodiffusion test (AGID) was employed to detect anti-B. canis antibodies. The overall seroprevalence varied by species and by test; in addition, CFT and AGID seemed better able to detect antibodies against B. abortus and B. canis, respectively. This is the first study on the presence of anti-Brucella spp. antibodies in captive carnivores from Brazil, as well as the first report of antibodies to Brucella spp. in coatis, crab-eating raccoons, hoary foxes, little spotted cats, tayras, and greater grisons.     

Use of enzyme-linked immunosorbent assay for detection of antibodies to Brucella ovis in sheep: field trial

An enzyme-linked immunosorbent assay (ELISA), using an autoclave-extracted soluble antigen, for the detection of serum antibodies to Brucella ovis in sheep was developed. The test seemed to be both sensitive and specific, on the basis of the control groups studied. The antigen showed no deterioration in prepared plates stored at -70 C for up to a year. The ELISA was used in conjunction with palpation of rams for epididymal lesions as a means to detect and control B ovis infection in a naturally infected flock. All rams were evaluated by the ELISA. At the time that the first blood sample was obtained, all positive and suspicious reactors were removed. With subsequent blood sample collections, only the positive reactors were removed. Brucella ovis was not isolated from any rams during the following year, and none of the mature breeding rams developed epididymal lesions.     

Serology and semen culture for the diagnosis of Brucella ovis infection in chronically infected rams

The serological response to Brucella ovis and the shedding of the organism in semen was followed for a period of 13-14 months in 42 naturally infected rams. Most rams remained chronically infected and excreted the organism in their semen throughout the investigation. B. ovis was isolated from 87.9% of the semen samples from the infected rams. The most common sites from which B. ovis could be isolated at necropsy were the epididymides and accessory sexual glands. In one ram the organism was isolated from lung, spleen, kidney and iliac lymph nodes. Three rams ceased to shed B. ovis in their semen during the course of the investigation. Seventy-five (11%) of 686 sera from infected rams were negative in the complement fixation test (CFT) although 76% and 77% of CFT-negative sera were positive in the gel diffusion precipitin test (GDT) and enzyme labelled immunosorbent assay (ELISA) respectively. The high incidence of CFT-negative infected rams was due to the selection for the investigation of many rams with histories of negative or vacillating CFT titres. Sera from five rams which never shed B. ovis in their semen reacted erratically in the three serological tests. The five rams were from heavily infected flocks and were kept in contact with infected rams throughout the investigation.     

Seroprevalence of Oestrus ovis infection in sheep in southwestern Germany

The aim of the survey was to determine the seroprevalence of Oestrus ovis infection in flocks in southwestern Germany. Serum samples collected from 1497 sheep (>6 months of age) of 110 flocks in 1997 and 1998 were examined for antibodies to crude somatic antigens of O. ovis second-stage larvae using an ELISA test. Data on the farm management were obtained by a questionnaire. Overall, 76% of the flocks had at least one seropositive animal, and the seroprevalence of anti-Oestrus antibodies was 50% in sheep. Flock size was the only risk factor significantly associated with the detection of antibodies. Larger flocks (>50 ewes) were more likely to be seropositive than smaller ones. These results show that Oestrus infections are widespread in sheep in southwestern Germany. Further investigations are required to estimate the economic importance of oestrosis and the efficiency of control measures.     

A comparison of whole virus and recombinant transmembrane ELISA and immunodiffusion for detection of ovine lentivirus antibodies in Italian sheep flocks

Sera from two sheep experimentally infected with ovine lentivirus (OLV) and from 186 sheep selected from flocks with known high or low prevalence of infection or on the basis of virological or histopathological examination were simultaneously tested by whole virus (WV) ELISA, recombinant transmembrane (r-TM) ELISA and AGID assay. Antigens for both the WV ELISA and AGID were prepared from an Italian field isolate; recombinant antigen was derived from the N-terminal region of the transmembrane envelope protein of strain K1514. The WV ELISA detected the highest number of seropositives, followed by the r-TM ELISA and AGID test. The sensitivity and specificity of the r-TM ELISA relative to the WV ELISA were 0.66 and 0.95, respectively. Immunoblot analysis of 14 WV ELISA-positive and r-TM ELISA-negative sera showed that the major core protein was immunodominant on WV antigen. It is concluded that the r-TM ELISA was more sensitive than the AGID test but less sensitive that the WV ELISA, particularly for detecting antibodies in the early stages of infection.Abbreviations AGID agar gel immunodiffusion - bp base pair - ELISA enzyme-linked immunosorbent assay - FBS fetal bovine serum - IB immunoblot - kD kilodalton - OLV ovine lentivirus - MEM minimal essential medium - p.i. post-infection - r recombinant - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TM transmembrane - WV whole virus     

Comparison of an enzyme-linked immunospecific assay (ELISA) with the cold complement fixation test for the serodiagnosis of Brucella ovis infection

An enzyme-linked immunospecific assay (ELISA) for the serodiagnosis of Brucella ovis infection in sheep is described and compared with the cold complement fixation (CF) test. ELISA was performed in microtiter plates, using horse-radish peroxidase conjugated to anti-normal sheep serum globulins, and hydrogen peroxide plus o-phenylenediamine as substrate. A heated, cell-free B. ovis extract was used as antigen in both tests. ELISA was easier to perform, distinguished better between positive and negative sera, and did not need heat-inactivated sera.     

Brucellae through the food chain: the role of sheep, goats and springbok (Antidorcus marsupialis) as sources of human infections in Namibia

A confirmed case of human brucellosis motivated an investigation into the potential source of infection in Namibia. Since domestic animals are principal sources of Brucella infection in humans, 1692 serum samples were screened from sheep, goats and cattle from 4 presumably at-risk farms and 900 springbok (Antidorcas marsupialis) serum samples from 29 mixed farming units for Brucella antibodies by the Rose-Bengal test (RBT) and positive cases confirmed by complement fixation test (CFT). To assess the prevalence of human brucellosis, 137 abattoir employees were tested for Brucella antibodies using the standard tube agglutination test (STAT) and by enzyme linked immunosorbent assay (ELISA). Cattle and sheep from all 4 farms were negative by RBT and CFT but 2 of the 4 farms (Ba and C) had 26/42 and 12/285 seropositive goats, respectively. Post mortem examination of seropositive goats revealed no gross pathological lesions typical of brucellosis except enlarged mesenteric and iliac lymph nodes seen in a single buck. Culture for brucellae from organs of seropositive animals was negative. None of the wildlife sera tested positive by either RBT or CFT. Interviews revealed that besides the case that prompted the investigation, a family and another person from other farms with confirmed brucellosis shared a common history of consumption of unpasteurised goat milk, home-made goat cheese and coffee with raw milk and prior contact with goats, suggesting goats as the likely source of infection. All 137 abattoir employees tested negative by STAT, but 3 were positive by ELISA. The 3 abattoir workers were clinically normal and lacked historical connections with clinical cases. Although goats are often associated with B. melitensis, these studies could not explicitly implicate this species owing to cross-reactivity with B. abortus, which can also infect goats. Nevertheless, these data reinforce the need for a better National Control Programme for brucellosis in Namibia.     

布鲁氏菌表面抗原研究进展

布鲁氏菌表面最主要的抗原是脂多糖(LPS)和外膜蛋白(OMPs),R-LPS是R型布鲁氏菌主要的表面抗原,S型布鲁氏菌的S-LPS已被证明是一个主要的毒力因子,它的O链部分含有布鲁氏菌表面绝大多数的抗原位点,是一个很重要的保护性抗原;OMPs被认为是布鲁氏菌表面潜在的抗原结构,在光滑型布鲁氏菌表面,由于OMPs受到了LPS-O链的干扰,致使它的抗原性没有充分地表现出来,但在绵羊布鲁氏菌表面却出现了不同的情况。未来几年,对OMPs的研究将成为该领域的热点,尤其是对Omp25突变体的研究。     

Effects of vaginal Brucella ovis infection of red deer hinds on reproductive performance, and venereal transmission to stags

AIMS: To investigate the effects of vaginal Brucella ovis infection on the reproductive performance of red deer (Cervus elaphus) hinds. To determine whether stags may become infected with B. ovis by venereal transmission from mating infected hinds. METHODS: Thirty mixed-age red deer hinds serologically negative for B. ovis antibodies were synchronised for oestrus on 22 March 2000. B. ovis was inoculated into the vagina of each hind at oestrus and again, 18 days later. At oestrus, hinds were randomly allocated to six groups, each joined with a 16 month-old red deer stag seronegative for B. ovis, for 55 days. Hinds were blood sampled and scanned for pregnancy using rectal ultrasonography at monthly intervals. Six pregnant and four non-pregnant hinds were slaughtered pre-calving and three hinds were slaughtered post-calving. Reproductive tracts and foetuses were examined grossly, histologically and microbiologically. Calves were identified and blood sampled within 3 days of birth. Hinds and calves were blood sampled in February and May 2001 and vaginal swabs were collected from hinds for B. ovis culture. Blood was collected from stags, 5 and 19 days after mating and semen was collected for B. ovis culture. The 17 remaining hinds were mated in 2001 to two mixed-age wapiti (Cervus canadensis) stags. Both stags were blood sampled after mating. Sera were tested in a B. ovis complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA). RESULTS: All 30 hinds developed B. ovis antibody levels, measurable using either the CFT or ELISA, but these did not remain elevated. There was no evidence of infection, either by gross pathology, histopathology or microbiological culture in the ten hinds or six foetuses slaughtered pre-calving. All remaining 20 hinds produced normal calves, 15 of which survived until weaning. Three hinds experienced dystocia and gave birth to dead calves and two calves died within 4 days of birth. One hind which had dystocia was euthanased. Samples from this hind and from 3/5 dead calves showed no evidence of B. ovis infection. B. ovis was cultured from the vagina of 1/19 hinds 48 weeks after inoculation, at which time B. ovis CFT and ELISA results for this hind were negative. Most calves had B. ovis serum antibodies at 1-3 days of age but levels were negligible when sampled at 10-15 weeks of age. Foetuses and dead calves were all seronegative. Three of the five red deer stags used for mating became infected with B. ovis. The two wapiti stags used to mate the remaining 17 hinds the following year remained seronegative. CONCLUSIONS: B. ovis is unlikely to have significant detrimental effects on the reproductive performance of red deer hinds. Venereal transmission via the vagina of hinds is a possible route of transmission between stags. It is possible that survival of the organism in the vagina of some hinds could create difficulties in disease control programmes. CLINICAL RELEVANCE: B. ovis infection of hinds at the time of mating is unlikely to cause significant reproductive losses. Venereal transmission of B. ovis between stags via the hinds may occur when groups of hinds are joined with more than one stag.