Epididymitis in rams: studies on vaccine efficacy.

Simultaneous inoculation with Br. ovis bacterin plus Br. abortus strain 19 produced the greatest immunity. This procedure was not permitted, however, as widespread use of Br. abortus in sheep might pose a hazard to the brucellosis eradication program in cattle. Subsequent work demonstrated that an aluminum hydroxide adsorbed bacterin, administered as two injections spaced 3-6 weeks apart, conferred a significant and acceptable level of immunity.     

Histopathology of experimental brucellosis in rams following vaccination with Brucella ovis

Lesions induced by inoculation of Brucella ovis into the epididymis were compared in rams previously vaccinated with B. ovis bacterin and unvaccinated rams. Inoculation of killed B. ovis did not produce significant lesions in either group whereas prior vaccination exacerbated epididymal lesions following inoculation of live B. ovis. Increased numbers of neutrophils, macrophages and lymphocytes were present in the interstitium and neutrophilic infiltration of the epididymal duct epithelium and intraepithelial cyst formation was more prominent. The inflammatory response surrounding extravasated spermatozoa was more severe in vaccinated rams but it was not determined if the response was directed at spermatozoa or intermixed brucellae, or both.     

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.     

The response of vaccinated rams to experimental challenge using Brucella ovis

One hundred and thirty eight rams were allocated to four experimental groups. An inactivated Br. ovis vaccine was administered either once by the intraperitoneal route (1 i/p), twice by the intraperitoneal route (2 i/p), or twice by the subcutaneous route (2 s/c), and the last group was left unvaccinated. They were then challenged by the intravenous inoculation of between 123 and 1.23 x 108 live Br. ovis bacteria. The number of rams that succumbed to infection within the four groups was 4135 (11%) for the 2 s/c, 7133 (21%) for the 2 i/p, 9135 (26%) for the 1 i/p and 18135 (51%) for the unvaccinated rams. Vaccination reduced the number of rams that succumbed to experimental challenge and although there were differences between the vaccinated groups, these were not statistically significant. Following challenge, unvaccinated rams were the first to excrete Br. ovis in their semen three weeks following inoculation. Next, those vaccinated by either one or two doses by the intraperitoneal technique began to excrete the organism (five weeks) and then finally those rams vaccinated twice by the subcutaneous route (seven weeks).     

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.     

Epidemiological Observations in a Corriedale Flock affected by Brucella ovis

Brucellosis in sheep, caused by Brucella ovis, is primarily a chronic infectious disease of rams with epididymitis as its most characteristic lesion. Six hundred rams from an infected farm were clinically and serologically examined once a year, over a 3-year period. An increase from 2.1% to 6.3% in the prevalence of animals serologically positive to B. ovis occurred over the 3 years. However, the prevalence of rams with lesions in the reproductive tract declined from 14.2% to 6.5% in the third year following one year of strict culling of clinically affected and rams that were serologically positive for B. ovis. Clinical lesions found in the 179 affected rams fell into two main categories: rams with epididymitis and rams with affected lymph nodes. These results suggest that the prevalence of the disease relates mainly to the sexual activity of the animal and not to age in itself. A single cull based on the results of clinical examination and serological test results was unable to decrease the prevalence of B. ovis in an extensive Corriedale sheep flock.     

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.     

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.     

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.     

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.     

Serodiagnosis of Histophilus ovis-associated epididymitis in rams

An ELISA for the detection of antibodies to Histophilus ovis was used to evaluate the association of epididymal lesions in rams with serologic response to His ovis. Comparison of ELISA results for His ovis in groups of rams with epididymal lesions with ELISA results of clinically normal rams (control group) revealed a significant difference (P less than 0.01) between the control group and those rams from which His ovis was isolated. A significant difference (P less than 0.01) was noticed between the control group and rams with lesions from which an organism other than His ovis or Brucella ovis was isolated. Additionally, a significant difference (P less than 0.01) was noticed in ELISA results between the control group and affected rams from which no organism was recovered and in which the epididymal lesion was not limited to the head of the epididymis. A difference was not detected in the His ovis ELISA results between control rams and rams with lesions associated with a B ovis infection or rams from which no organism was recovered and in which the epididymal lesion was limited to the head of the epididymis. The serologic findings in our study suggest that His ovis is more important in the development of epididymitis in rams than culture results alone would indicate.     

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.     

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.     

Comparison of different antigenic preparations for the detection of ovine serum antibodies against Brucella ovis by ELISA

An enzyme-linked immunosorbent assay has been developed for the detection of antibodies against Brucella ovis using serum from control rams (Con-S), naturally infected rams (Inf-S), rams inoculated intravenously with B. ovis (IV-S) and rams vaccinated intramuscularly (IM-S). The serum was titrated by serial double dilutions from 1/25 to 1/25,600 against whole bacteria, B. ovis lipopolysaccharide and a detergent-extracted component of the outer membrane complex of B. ovis as antigens immobilised on microtitre plates. Sheep antibodies bound to antigen were assayed with rabbit anti-sheep gammaglobulin and alkaline phosphatase conjugated protein A. A high level of antibody activity against intact B. ovis cells was detected in Inf-S and IM-S. When lipopolysaccharide was the immobilised antigen, only IM-S yielded significant antibody activity. The component from detergent extracts of the outer membrane complex of B. ovis reacted best with serum (up to 1/6,400) from field-infected rams, while serum from vaccinated and intravenously inoculated rams registered significant titres up to a serum dilution of 1/800 and 1/200 respectively. These results indicate that ELISA is a very sensitive test but its value as a serodiagnostic procedure is dependent upon the choice of antigen used in the assay.     

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.     

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.     

Etiologic significance of bacterial isolates from rams with palpable epididymitis

Identified and partly identified bacterial isolates were obtained from 48 rams of various breeds that had unilateral or bilateral epididymitis. Most of the animals were approximately 1 year of age; a few were older. Brucella ovis, Actinobacillus spp, Corynebacterium spp, Haemophilus spp, Acinetobacter spp, Escherichia coli, Moraxella spp, Staphylococcus spp, Pasteurella spp, Streptococcus spp, and Chlamydia psittaci were isolated. A vaccine strain of B ovis, isolated species of bacteria, and mixtures of isolates of tissue homogenates containing all isolates except B ovis and C psittaci were inoculated via the mucous membranes of the eyes, nares, and prepuce. Palpable epididymitis was not produced by the inoculations. The vaccine strain of B ovis induced complement-fixation reaction in 11 of 20 rams.     

An improved immunoblotting technique for the serodiagnosis of Brucella ovis infections

Two antigen preparations, the routinely used Brucella ovis sodium dodecylsulfate-mercapto ethanol extract and a B. ovis Triton X-114-derived detergent-rich phase, were compared under standard conditions for their use in electrophoretic immunoblotting for confirmafory, serological testing for B. ovis infections, by using 88 sera from ram flocks with a history of freedom from B. ovis infections, 80 sera from chronically infected rams, which were shedding B. ovis in their semen at the time of sampling, and 104 sera from a naturally infected ram flock. Blots with the detergent-rich phase as antigen gave better correlation with the serological results from naturally infected rams, exhibited no non-specific staining with sera from the negative group, gave clearer visualisation of specific bands for positive sera, and were equally sensitive when compared to the standard antigen for sera from chronically infected rams.     

Merino sheep breed's genetic resistance to scrapie: genetic structure and comparison of five eradication strategies

Three thousand one hundred and ninety-three Merino-breeding males were genotyped for Scrapie-resistance gene. They showed a high frequency of ARQ allele while VRQ allele frequency, linked to the highest susceptibility, was close to 0. Alleles linked to Scrapie-susceptibility frequencies have been studied in different Merino flocks. Most of the variability is intraflock, not interflock. In addition, there is an excess of heterozygotes due to crossbreeding or Wahlund effect.

Five control and prevention strategies were studied: first genotyping males and females and eliminating VRQ carriers and ARQ/ARQ rams; second genotyping males and females and eliminating VRQ carriers; third genotyping males and eliminating VRQ carriers and ARQ/ARQ rams; fourth genotyping males and using only as breeders ARR/ARR, ARR/AHQ, AHQ/AHQ and ARQ/ARQ rams; fifth genotyping males and using only as breeders ARR/ARR, ARR/AHQ and AHQ/AHQ rams. They were simulated from allelic and genotypic frequencies to decide which animals had to be genotyped and which animals had to be eliminated due to their genotypes and risk levels. The third option, to genotype rams and to eliminate ARQ/ARQ and VRQ carriers, would be the best strategy to improve the resistance and would cause minimal cost and loss of animals.