Observations on the eradication of Brucella ovis infection from a ram flock

The measures taken to eradicate Brucella ovis infection from a naturally infected flock of 64 rams are described. Lesions of epididymitis were detected in 18 rams, all of which gave either positive or suspicious reactions in the complement fixation test. A further 20 rams gave serological reactions in the complement fixation test. Subsequently, semen was collected from 14 of these 20 rams and B. ovis was cultured from the semen of all 14 rams. Serum samples from two rams failed to react in the complement fixation test. However, they were identified as infected with the aid of an enzyme-linked immunosorbent assay and the subsequent culture of semen samples. It is suggested that, when eradicating B. ovis infection from ram flocks, the enzyme-linked immunosorbent assay be used in addition to both the complement fixation test and the physical examination. Using a combination of tests as described can increase the likehood of an earlier eradication of B. ovis infection.     

Multiplex PCR for the detection of Brucella ovis, Actinobacillus seminis and Histophilus somni in ram semen

OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the rapid detection of Brucella ovis, Actinobacillus seminis, Histophilus somni in fresh ram semen samples. DESIGN: The multiplex assay was based on the single PCR assays published for the detection of A seminis and B ovis, and the forward primer published for the detection of H somni; an alternative reverse primer for H somni was designed in this study. PROCEDURE: Culture and PCR of 295 fresh semen samples were carried out. RESULTS: The multiplex PCR was far more successful in the detection of H somni (45/295) than culture (23/295). A seminis was also detected in more semen samples by multiplex PCR (29/295) than culture (13/295) and B ovis was detected in three samples using both PCR and culture. No amplifications were detected with DNA from a range of bacterial isolates including species associated with epididymitis in rams. CONCLUSION: This PCR could be used as a complementary test, or alternative to culture of ram semen and other biological samples for the detection B ovis, H somni and A seminis.     

The complement fixation test for the diagnosis of Brucella ovis infection in rams--an Animal Health Division Report

Complement fixation tests using three B. ovis antigen preparations in warm fixation tests (WCFT) and cold fixation (CCFT) tests were done on 541 ram sera. Semen samples from the same rams were examined culturally to identify B. ovis excretors. The CCFT, using an antigen prepared by heat extraction of B. ovis cells, had a sensitivity of 97% in 124 rams which were shedding B.ovis. The specificity was 99% in 144 rams from non-infected flocks. Seventy-seven per cent of 156 rams which reacted to this test were shedding B. ovis in their semen. Tests with other antigens were inferior in sensitivity and/or specificity. The WCFT gave lower titres than CCFT. Vaccination caused large numbers of false positive reactions in 4 flocks.     

Comparison of three serological tests for Brucella ovis infection of rams using different antigenic extracts

The sensitivity and specificity of the complement fixation, gel diffusion and ELISA tests for the diagnosis of Brucella ovis infection of rams have been compared using three different antigenic preparations. The antigens obtained by petroleum ether - chloroform - phenol, or cold saline extractions gave poorer diagnostic results than those obtained by hot saline extraction in all the tests. The best sensitivity was obtained with the ELISA (97.6 per cent) followed by the gel diffusion (96.4 per cent) and complement fixation tests (92.7 per cent). The gel diffusion test detected as positive the two rams negative in the ELISA, while the complement fixation test did not improve the sensitivity of the other tests. Under these conditions all the tests were 100 per cent specific when testing sera from rams free of B ovis.     

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.     

Transmission of Brucella ovis from rams to red deer stags

AIM: To determine whether B. ovis will transmit from infected rams to non-infected red deer stags (Cervus elaphus) grazing together in the same paddock. METHODS: Six rams artificially infected with B. ovis were grazed with six non-infected 14-month-old red deer stags for a four and a half month period from March 4 to July 20, 1999. Stags were blood sampled at one- to six-weekly intervals to test for B. ovis antibodies using a complement fixation test. Stags that seroconverted were semen sampled to test for B. ovis infection by bacteriological culture. RESULTS: Between day 92 and day 124 of grazing together (June 4 and July 6), sera from five of the six stags became positive in the B. ovis complement fixation test. B. ovis was cultured from semen samples from four of the seropositive stags. CONCLUSIONS: Brucella ovis can be transmitted from infected rams to non-infected stags grazing in the same paddock, suggesting that B. ovis infection in farmed deer in New Zealand initially came from infected rams. Whether transmission occurs from direct contact between rams or stags, or indirectly by environmental contamination needs to be established.     

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%.     

Persistence, serodiagnosis and effects on semen characteristics of artificial Brucella ovis infection in red deer stags

AIMS: To investigate the persistence of infection and serum antibody titres after infection of red deer (Cervus elaphus) stags with Brucella ovis, and compare these with those of rams. To assess the effects of recent and chronic infection on semen characteristics of stags. METHODS: Fourteen stags and eight rams were artificially infected with B. ovis by intravenous inoculation. Semen and blood samples were collected at approximately monthly intervals for 649 days. Semen samples were subjected to bacterial culture, and sera were tested for B. ovis antibodies using a complement fixation test (CFT) and an enzyme-linked immunosorbent assay (ELISA). At the end of the study, animals were slaughtered and reproductive organs subjected to bacterial culture. During the first and second breeding seasons, three and five semen samples, respectively, were evaluated from each stag for sperm motility and morphology. RESULTS: Twelve of 14 (86%) stags and 6/8 (75%) rams developed a patent B. ovis infection and shed the organism in semen. All six infected rams continued to shed B. ovis in semen throughout the 649-day study period, and at slaughter B. ovis was isolated from the reproductive tract and urinary bladder. In contrast, 10/12 (83%) infected stags stopped shedding B. ovis in semen 103-342 days after inoculation, and the organism could not be isolated from their reproductive tracts at slaughter. The remaining two infected stags shed B. ovis in semen throughout the study period and the organism was isolated from their reproductive tracts at slaughter. All inoculated animals initially developed serum antibody titres detectable using the B. ovis CFT and ELISA. For infected stags, the diagnostic sensitivity of these tests was 100% for the first 166 days, but decreased to 50-90% after this. The diagnostic sensitivity for the infected rams was 100% throughout the study period. Infection in stags resulted in variable effects on semen characteristics. Eight of 12 (67%) infected stags had a mean sperm motility of < 50%, and < 60% mean normal sperm in the first year of infection. Seven of these stags had resolved the infection by the following breeding season, and there was a significant improvement in sperm motility and morphology. CONCLUSIONS: Stags are as susceptible as rams to experimental B. ovis infection. However, the majority of infected stags resolved the infection within a year, whereas rams remained infected for at least 649 days (22 months). Serology, using CFT and ELISA, was effective at detecting infection during the first 166 days in both species, but after this time was less effective at detecting infection in stags than in rams. Infection with B. ovis had variable but generally deleterious effects on the semen characteristics of stags, which resolved following resolution of the infection. Differences in the characteristics of the disease in stags compared with rams mean that different control methods are warranted for the two species. CLINICAL RELEVANCE: Most stags infected with B. ovis are likely to resolve the infection within a year, and semen characteristics return to levels acceptable for breeding. Serology is useful for detection of infection in the early stages of the disease, but once disease has been present in the herd for some time false-negative reactions are likely to occur in individual stags.     

Evaluation of surface components of Brucella ovis as antigens for the detection of precipitin antibody in serums from artificially exposed rams

Surface components of Brucella ovis obtained by gentle physical shearing were tested as a potentially useful source of reagent for selective serological diagnosis. These antigens were used in a radial immunodiffusion (RID) test against serum from rams which had been inoculated with infective semen containing B. ovis by one of 4 routes namely mating rams with ewes previously inoculated intravaginally with infective semen, or by direct inoculation in the prepuce, rectum or nasal passage. Loosely attached surface antigens in the RID test formed precipitin bands with serums collected from rams 2 and 10 weeks after inoculation. In contrast, a detergent extracted membrane antigen B developed precipitin bands only with serum collected 10 weeks after inoculation from rams confirmed bacteriologically to be infected with B. ovis in the genital tract. The route by which the rams were artificially exposed did not affect the outcome of the RID test using the membrane B antigen. However, all experimentally exposed rams had demonstrable CF titres when a heat extracted antigen was used.     

Evaluation of an enzyme linked-immunosorbent assay for the diagnosis of Brucella ovis infection in rams

A simple enzyme linked immunosorbent assay (ELISA) was developed for the serological diagnosis of Brucella ovis infections in rams. Serums from brucellosis accredited-free flocks and flocks known to be infected with B. ovis were tested and the results correlated with warm complement fixation (CF) test and bacteriological examination of semen. Both the ELISA and the CF test detected 0.5% false positive reactions in rams from clinically negative flocks. However the ELISA detected significantly more positive reactors in infected flocks and the CF test failed to detect some rams excreting B. ovis. The ELISA proved to be a valuable test in eradicating brucellosis from infected flocks.     

Serological, bacteriological and pathological changes in rams following different routes of exposure to Brucella ovis

Mature Merino rams were exposed to Brucella ovis by contact with infected semen, using either ewe transmission, intrapreputial, intranasal or intrarectal inoculation of infected semen or intrapreputial inoculation of B. ovis culture. Thirty-six of the 41 rams developed significant complement fixation (CF) test titres, but only 9 of these reactors showed clinical, bacteriological or pathological evidence of infection. Infection occurred in some of the rams from all groups. The results are discussed in relation to the transmission of the disease and the significance of CF titres in rams exposed to B. ovis.     

Antimicrobial therapy for rams with Brucella ovis infection of the urogenital tract.

Rams shedding Brucella ovis in semen but without palpable abnormalities of the epididymides were treated with long-acting oxytetracycline for 15 days and dihydrostreptomycin for 7 days (n = 9) or conventional oxytetracycline and dihydrostreptomycin (n = 9) for 7 days. Nine rams were not treated. More treated rams were considered to have satisfactory breeding soundness examination results at posttreatment weeks 3, 7, 12, and 19. Nontreated rams continued to shed B ovis in semen. After treatment, B ovis was not recovered from 78% of rams given long-acting oxytetracycline and dihydrostreptomycin or from 89% of rams given conventional oxytetracycline and dihydrostreptomycin. At week 21, all rams were euthanatized, and specimens of the testes and epididymides were bacteriologically cultured for B ovis. Brucella ovis was not recovered from the testes of rams or from the epididymides from rams not shedding the organism in the semen. In one treated ram, B ovis was recovered from the semen but not from other tissues. All rams remained ELISA-positive, with the exception of 2 treated rams that ceased shedding B ovis in semen immediately after treatment was started; both these rams became ELISA-negative on the last examination at week 19.     

Diagnostic sensitivity and specificity of an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection in rams.

An enzyme-linked immunosorbent assay (ELISA) for antibody to Brucella ovis was compared with a standard complement fixation test. Sera of 176 rams from uninfected flocks gave 175 negative and one suspect ELISA reaction (diagnostic specificity 99.4%) whereas the complement fixation test yielded 167 negative, seven suspect and two anticomplementary reactions (diagnostic specificity of 96.0%). Diagnostic sensitivity was evaluated on sera of 79 rams from which B. ovis had been isolated. The ELISA showed 75 positive and four suspect reactions, while complement fixation test revealed 64 positive, 13 suspect and two negative results. Considering both positive and suspect reactions, the diagnostic sensitivity was 100% for ELISA and 97.5% for complement fixation test. The ELISA method was considered more specific, more sensitive and technically more advantageous than complement fixation test as a serodiagnostic test for B. ovis infection in rams.     

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.     

SENSITIVITY AND SPECIFICITY OF TWO MICROTITRE COMPLEMENT FIXATION TESTS FOR THE DIAGNOSIS OF BRUCELLA OVIS INFECTION IN RAMS

SUMMARY: An investigation was made into microtitre complement fixation test (CFT) procedures suitable for the serological diagnosis of naturally occurring Brucella ovis infection in rams. A procedure similar to the Australian standard procedure for bovine brucellosis was unsatisfactory when applied to sheep. Modification of the procedure by use of an initial serum and anticomplementary control dilution of 1:8 and increasing complement fixation time to 60 minutes at 37°C, greatly improved the efficiency of the test. A sensitivity of 100% was recorded for 59 serums from known infected rams and a specificity of 99.9% for 1593 serums from rams known or believed to be free of infection. Some aspects of applying CF tests to sheep serums are discussed.     

Correlation of the presence of seminal white blood cells and the prevalence of separated spermatozoal heads with subclinical Brucella ovis infection in rams

A breeding soundness evaluation was conducted on 824 Colorado range rams. These rams were determined to be free from epididymitis via testicular palpation. Semen evaluation included microscopic observation for the presence of WBC. Of the 824 rams, 15.5% failed the breeding soundness evaluation on the basis of the semen evaluation: 10.6% had WBC in the semen and 4.9% had poor sperm morphology. The prevalence of Brucella ovis isolation varied from 0% to 16.2% within flocks. The prevalence of subclinical B ovis infection was 10% in the control flocks. Brucella ovis was isolated from 71.9% of the rams that had WBC in their semen. From this study, it appeared that palpation and vaccination may be inadequate for control of ram epididymitis.     

Serological and necropsy findings for rams infected with Brucella ovis which were not identified by the complement fixation test

The eradication of Brucella ovis from a commercial flock of 36 Romney rams was complicated by four infected rams remaining undetected despite four successive flock examinations using the complement fixation test. These four rams were subsequently tested using an enzyme-linked immunosorbent assay and a gel diffusion test and shown to be infected by semen culture. All four rams could have been identified as infected at the initial test if the enzyme-linked immunosorbent assay had been used in addition to the complement fixation test. Although gross evidence of epididymitis was found in only one ram at necropsy, three had histological lesions of epididymitis and all four had a seminal vesiculitis.     

Trials with an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of subclinical genital infections in rams caused by Histophilus ovis and Actinobacillus seminis

The performance of an enzyme-linked immunosorbent assay (ELISA) was evaluated in the serological diagnosis of subclinical genital infection in 6 naturally infected ram flocks and 2 experimentally infected ram hoggets. The test employs lipopolysaccharide (LPS) antigen prepared by autoclaving from Actinobacillus seminis and Histophilus ovis. A total of 193 sheep (118 unmated virgin rams and 75 mature breeding rams) were examined clinically, serologically (by ELISA) and bacteriologically (semen bacteriology) at the same time. Serum samples from all animals were also tested by an ELISA employing LPS antigen prepared from Brucella ovis in the same way. Shedding of A. seminis and H. ovis did not show close correlation with serological positivity (Table 1), as only 9 (15.0%) out of the 60 A. seminis shedders were ELISA seropositive at the same time. As regards H. ovis only 10 (19.2%) out of the 52 H. ovis shedders were ELISA seropositive at the same time. The results indicate that, when used alone, the ELISA employing LPS antigen is unsuitable for diagnosing subclinical genital infection caused by H. ovis and A. seminis in rams. The authors discussed shortly the employing fields of this ELISA test in the diagnostic work.     

Brucella ovis excretion in semen of seronegative, clinically normal breeding rams

A commercial flock of Suffolk and Suffolk-cross breeding rams was monitored for 5 years in an effort to control epididymitis caused by Brucella ovis. Scrotal palpation, semen evaluations, and vaccination against B ovis were used the first 3 years. Serologic evaluation (complement fixation and ELISA) was added the fourth year, and bacteriologic culturing was added to the program the fifth year. Semen culturing in the fifth year revealed 9 (37.5%) of 24 rams were actively excreting B ovis; 6 of those 9 rams were seronegative. Neither semen quality nor the presence of WBC in the semen were dependable criteria to detect these seronegative carriers. In spite of the high percentage of B ovis excretors, few clinical signs of epididymitis were detected in the flock during the last 3 years of the study. It was hypothesized that vaccination protected rams against the clinical disease but not the carrier state. The importance of culturing semen for assessment of a control program was emphasized.     

Ovine brucellosis in alberta

Two parallel surveys of rams from Alberta sheep flocks were conducted to determine the presence of infection with Brucella ovis. In a retrospective study over a period of 24 months, using complement fixation test, 12 flocks out of 142 tested were considered infected. In another 17-month survey of slaughter rams by serology and culture methods 11 flocks out of 124 were found to be infected. The overall prevalence of ovine brucellosis was 8.6% of the flocks tested which represented 12.5% of the estimated sheep flocks in Alberta. Up to 67% of rams in infected flocks reacted to complement fixation test.

The complement fixation test was evaluated for its efficiency in the diagnosis of ovine brucellosis and compared with a limited number of an enzyme-linked immunosorbent assay (ELISA) results and clinical criteria. The complement fixation test as well as ELISA identified all culture positive rams. Both serological tests appeared satisfactory for the diagnosis of B. ovis epididymitis when the results could be interpreted in the light of flock history and clinical findings.