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.     

Isolation of Actinobacillus seminis from rams in the United Kingdom.

Actinobacillus seminis was isolated from the semen of five rams on four farms. Four of the rams had abnormal semen and three were also infertile. The isolates of A seminis showed similar phenotypic profiles and electrophoretic protein patterns to the type strain of A seminis but were distinct from Histophilus ovis previously isolated from rams with epididymitis in Scotland. The infection appeared to be subclinical but two of the five rams had palpable abnormalities of their testes. Three rams were treated with antibiotics but the infection persisted. No gross lesions were found in the genitalia of two of three rams examined post mortem but one had necrotic abscesses in the testes and epididymis. A seminis was isolated from the seminal vesicles and epididymis of one ram without gross lesions but not from the genitalia of the other two. On one farm the infection in a recently purchased ram led to the detection of another case as a result of the bacteriological screening of 11 stock rams not in contact with the initial case. These five subclinical cases, which included a supposedly healthy stock ram, suggest that A seminis infection may be widespread and should be considered in cases of infertility.     

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.     

Evaluation of a PCR test for the diagnosis of Brucella ovis infection in semen samples from rams

The sensitivity and specificity of a PCR assay with primers derived from the insertion sequence IS6501 was compared with that of bacteriological culture and serological tests for the diagnosis of Brucella ovis infection in rams. No amplifications were detected with DNAs from the strains phylogenetically related to Brucella and from the seven bacterial species considered as the main etiologic agents of epididymitis in rams. In addition, the specificity of the PCR was 100% when testing semen samples from Brucella-free rams. The comparison of the semen culture and PCR results from 192 semen samples tested, showed a proportion of agreement of 0.91 between both tests. The PCR-based test described has sensitivity similar to that of semen culture and could be used as a complementary test for the direct diagnosis of Brucella ovis in semen samples of rams.     

Identification of an immunogenic protein of Actinobacillus seminis that is present in microvesicles

Actinobacillus seminis is a gram-negative bacterium of the Pasteurellaceae family that is involved in ovine epididymitis. Looking for a protein specific to this species, we determined the protein profile of subcellular fractions of A. seminis (American Type Culture Collection number 15768): proteins from the outer membrane (OMPs), inner membrane (IMPs), and cytoplasm (CPs). These profiles provide the first data, to our knowledge, regarding subcellular fractions of A. seminis. In the OMP fraction, we identified a protein with a molecular mass of 75 kDa that proved to be immunogenic and apparently specific for A. seminis. This conclusion was based on the reaction of hyperimmune serum of rabbits inoculated with whole cells of A. seminis that was tested against sonicated complete cells of reference strains and field isolates of Brucella ovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. No protein of these bacteria cross-reacted with the 75-kDa protein of A. seminis. Furthermore, when each type of hyperimmune serum was tested against the sonicated cells and each of the subcellular fractions of A. seminis, it did not recognize the A. seminis 75-kDa protein. We also isolated and identified this protein in microvesicles released to the culture supernatant. The results suggest that the 75-kDa protein could be used to establish a diagnostic test specific for ovine epididymitis caused by A. seminis.     

Detection of Brucella melitensis in semen using the polymerase chain reaction assay

An evaluation of the polymerase chain reaction (PCR) for detection of Brucella melitensis DNA in bovine and ovine semen was performed. Since semen contains different components that inhibit PCR amplification, a protocol was used to purify Brucella-DNA from bovine and ovine semen samples prior to conducting amplification of the targeted DNA. When separated fractions of naturally Brucella contaminated semen were analyzed by the PCR, most of B. melitensis DNA were present in the seminal fluid and non-sperm fractions.The PCR examination results for detection of B. melitensis DNA in different semen fractions were compared with the results for traditional cultural methods of Brucella from semen. The PCR was more sensitive than the traditional cultural methods since it detected Brucella-DNA in 12 (10%) out of 120 semen samples while direct culture detected only 7 (5.8%) in the same semen samples. The limit of detection by PCR was 100 CFU/ml of semen. In addition, the results of PCR were available in one day, whereas isolation and identification of Brucella organisms required days or even weeks. The PCR may be used as a supplementary test for detection of B. melitensis in semen.     

Prevalence of Histophilus ovis and Actinobacillus seminis in the genital tract of sheep

Histophilus ovis and Actinobacillus seminis were isolated from the preputial cavity of 6-month-old rams and the vagina of 6-month-old ewes at a substantially higher rate than that in mature (greater than 2 years old) rams and ewes. These organisms appeared to be a transitory component of the ovine genital flora, the prevalence of which was associated with age regardless of gender. Additional evaluation of the recoverability of H ovis and A seminis from the preputial cavity of rams from birth to 1 year of age indicated that the isolation rate from rams and predominance of the organisms in the preputial cavity differed greatly over this age period. These organisms were not recoverable until ram lambs were 12 weeks of age and were most prevalent at 20 weeks of age, after which recoverability of H ovis and A seminis from the preputial cavity steadily decreased, continuing through the time of the last evaluation at 1 year of age. The time period with which these organisms can be isolated from the preputial cavity is closely correlated with the time period when epididymitis associated with these organisms develops, and may be an important factor in the pathogenesis of epididymitis.     

SEROLOGICAL COMPARISON BETWEEN HISTOPHILUS OVIS, ACTINOBACILLUS SEMINIS AND BRUCELLA OVIS

SUMMARY: The serological relationships between 4 strains of Histophilus ovis , the neotype strain of Actinobacillus seminis and Brucella ovis were examined using a cross-absorption complement-fixation technique.
It was found that the 4 strains of H. ovis were serologically homologous and that an incomplete relationship existed between these organisms and A. seminis. Anteriserums prepared against one strain of H. ovis and the strain of A. seminis gave a weak, apparently nonspecific cross-reaction with Br. ovis antigen.
The practical significance of these results is discussed.     

Actinobacillus seminis infection in sheep in the Republic of South Africa. I. Identification of the problem

A clinical palpation and semen smear examination of 647 rams submitted to the Regional Veterinary Laboratory during 1967 revealed that 42 (6,5%) of these animals had clinical epididymitis or orchitis, 6 (0,9%) showed other types of genital lesions and 98 (15,1%) suffered from subclinical genital infection. A. seminis and A. seminis-like organisms were isolated from semen specimens of 18 out of 35 rams with clinical epididymitis or orchitis, 25 out of 33 rams with subclinical infection and none out of 13 rams which showed no neutrophils in their semen. On 4 stud farms where Elberg Rev. 1 vaccine was meticulously applied and the complete absence of Brucella ovis infection was established, of a total of 327 rams examined, 10 (3,6%) were found to be clinically and 72 (22,0%) subclinically affected. A. seminis was isolated from 5 out of 6 of these rams with clinical lesions and 10 out of 15 of those which showed evidence of subclinical infection.     

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.     

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 polymerase chain reaction and cell culture for the detection of Chlamydophila species in the semen of bulls,buffalo-bulls,and rams

Two hundred and thirty six semen samples were collected from 120 bulls, 60 buffalo-bulls, and 56 rams located on farms of known history of infection with Chlamydophila species. All semen samples were examined by polymerase chain reaction (PCR) and cell culture techniques for detection of Chlamydophila species. The primers were selected to allow the amplification of all target species in a single reaction by identifying conserved sequences in the omp2 gene. PCR assay detected more positive samples (36) from the semen samples collected from different animal species than were detected by the culture method (21). The results indicated that all culture-positive semen samples (21) from different species were PCR positive. The detection limit of the PCR assay was determined with DNA extracted from fourfold serial dilution of C. abortus (B577) and C. pecorum (11/88) cultures and found to be 0.25 inclusion-forming units (IFU) per PCR, while the culture method could not detect less than 4 IFU. This is the first report using PCR for the detection of Chlamydophila species in buffalo-bulls' semen and the assay provides a simple, sensitive, rapid, and reliable means for the detection and identification of the organism.     

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.     

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.     

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.     

Detection of Theileria ovis in naturally infected sheep by nested PCR

A nested polymerase chain reaction (PCR) for the detection of Theileria ovis in sheep using oligonucleotide primers designed from the small subunit ribosomal RNA (SSU rRNA) gene sequence of T. ovis from sheep in eastern Turkey is described. A 398-bp DNA fragment was specifically amplified from blood samples from sheep, naturally infected with T. ovis. No PCR products resulted from T. lestoquardi, T. annulata, T. parva, T. buffeli and Babesia spp. DNA using these specific primers. The sensitivity of the nested PCR for T. ovis, which was assessed showed that one infected cell in 10(7) sheep erythrocytes, equivalent to a blood parasitemia of 0.00001%, could be detected. This is more sensitive than examining 200 fields under light microscopy. In addition, of the 124 field samples obtained from sheep in eastern Turkey tested, 19.35% (24/124) were positive for the presence of Theileria spp. by microscopic examination compared to 54.03% (67/124) positive for T. ovis by nested PCR. The primer pairs described in this study will be useful for epidemiological studies on ovine theileriosis and for discrimination between T. lestoquardi and T. ovis infections in sheep.     

Temporal ELISA response of rams to Brucella ovis following experimental infection or vaccination

Sera from rams vaccinated with antigens extracted chaotropically from Brucella ovis by potassium thiocyanate treatment were used to optimise a whole-cell, enzyme-linked immunosorbent assay (CELISA) and to monitor the temporal serological response of rams which had been challenged with infected semen by the intranasal or intrapreputial route. Three patterns of CELISA response were detected. Thirteen of 15 rams intranasally challenged did not respond serologically (pattern 1 or nil response). Only one of 15 rams in the intranasal group exhibited a rise and fall response with CELISA (pattern 2), while another showed a rise and surge response (pattern 3). The numbers of rams in the intrapreputial group which displayed a pattern 1 or 2 or 3 response were four, nine and two, respectively. No ram with a pattern 2 response excreted B ovis in the semen or showed any other evidence of infection, whereas rams with a pattern 3 response excreted B ovis in the semen and developed palpable lesions. Intrapreputially challenged rams that were CELISA-positive consistently mounted an antibody response against B ovis about two to four weeks earlier than intranasally challenged rams.     

EVALUATION OF PROCEDURES FOR THE DIAGNOSIS OF BRUCELLA OVIS INFECTION IN RAMS

Procedures for the diagnosis of Br. ovis infection in rams were evaluated by examining 10 rams artificially infected by preputial inoculation. Observations were undertaken at weekly intervals for 1 year to follow changes in clinical, bacteriological and serological findings. Clinical lesions were detected in 1 ram 3 weeks after inoculation and in all rams by 8 weeks; lesions were undetectable in 3 rams at the completion of the trial. The presence of inflammatory cells in semen samples was the earliest indication of infection being demonstrated in 2 rams at 2 weeks and in all rams by 8 weeks; subsequently 86% of samples were positive. Br. ovis was detected in semen smears from 3 rams at 4 weeks but only once in all rams (at 27 weeks); overall 52% of semen smears were positive from 4 weeks onwards. Br. ovis was cultured from semen of 5 rams after 4 weeks and from all rams at 5 weeks; thereafter 97% of samples were positive. All rams developed significant titres to the CFT between 2 and 9 weeks; thereafter the CFT was a reliable indication of infection in 6 rams, highly suggestive in 3 and unreliable in one. By 8–10 weeks all rams developed significant titres to the IHA which were then maintained in all rams for the remainder of the trial.