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.     

Economics of Brucella ovis control in sheep: computerized decision-tree analysis

The epidemiology and economics of Brucella ovis control in a hypothetical, commercial sheep flock (100 rams and 2,500 ewes) were investigated. The investigation consisted of an epidemiologic simulation model, reported in a companion paper, and a decision-tree analysis, reported here. It was predicted from the simulation model that B ovis could be eradicated successfully in 2 test periods (less than 1 year) from a flock by using intensive screening and culling. A computerized decision-tree program was used to determine the economically optimal control strategy among several alternatives. Two versions of the program were used to determine the optimal alternative, based on minimizing the expected monetary loss (deterministic) and minimizing the associated risk (stochastic). The economically optimal alternative was to screen the rams by means of palpation, semen testing, and ELISA prior to the mating season. Rams positive to any test were culled. After the mating season was completed, the optimal action was to use ELISA for the remaining rams and to cull all that were ELISA positive. The cost of this alternative was approximately $6,150, or less than one half the annual cost of a vaccination program ($12,800) or no program ($13,550). Continuing palpation and semen testing were considered worthwhile on the basis of detecting new cases of B ovis infection and in maintaining high flock fertility. Similarly, the cost of annual use of ELISA was small (approximately $100), compared with the potential cost of not detecting a new case of B ovis infection.     

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.     

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.     

Immunization with Brucella melitensis Rev 1 against Brucella ovis infection of rams

The efficacy of Brucella Melitensis Rev 1 vaccine (Rev 1) for the prophylaxis of Brucella ovis ram epididymitis was evaluated. Twenty-nine 3-month-old rams were vaccinated with 2 X 10(9) Rev 1 and 14 were revaccinated with 5 X 10(8) at 14 months of age. Six rams remained unvaccinated as a control group. All rams were challenged with 5 X 10(8) B. ovis at 21 months of age. Before being slaughtered 8 weeks later, only one vaccinated ram developed epididymitis while four of the six control rams developed testicular alterations. Genital and selected extragenital organs and lymph nodes were removed at slaughter and inoculated on selective media. B. ovis was isolated from 26.6% of the vaccinated rams, 21.4% of the revaccinated rams and 100% of control rams. Portions of epididymis, testes and vesicular glands were also used for pathological studies. More severe lesions were observed in control rams than in vaccinated ones. In conclusion, these results show that vaccination of young lambs, followed or not by revaccination, is a suitable method for the prophylaxis of B. ovis infection of rams.     

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.     

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.     

Factors associated with failure in breeding soundness examination of Western USA rams

Breeding-soundness examination (BSE) and eradication of Brucella ovis infection in rams are critical components of flock-health programs. The aims of this retrospective, cross-sectional study were to describe the results of BSE in a large sample of rams in the Western USA and to determine the association between BSE outcome and the semen collection method (penis manually extended vs. retained in the preputial cavity), ram body-condition score (BCS), the presence of ulcerative posthitis, and the size of the flock of origin. We evaluated the first BSE in a given year for rams from Colorado, Wyoming, and Utah, USA, from 2000 through 2007. Breeding-soundness examination consisted of physical examination, scrotal circumference and BCS measurement, semen collection by electroejaculation, and microscopic examination of semen motility, morphology, and leukocyte concentration. We assigned a reason for failure to each failed BSE and used multivariable logistic and Poisson regressions to measure associations between ram and flock variables and the risk or reason for failure on BSE. A non-random, owner-selected subset of rams was tested for antibodies to B. ovis by serum indirect ELISA (iELISA). The Rogan-Gladen corrected B. ovis seroprevalence was measured. Of the 14,667 BSEs performed on 11,804 rams, 29.0% were classified as "failed;" the most common reason for failure was substandard semen parameters (43.8%). Breeding-soundness examinations were more likely to have been categorized as failure for inflammatory causes when performed on rams from medium-sized flocks (OR 1.6; 95% CI 1.1, 2.3) and large flocks (OR 1.4; 95% CI 1.0, 1.9) (P=0.02), suggesting that larger flocks are at higher risk of contagious diseases. The adjusted seroprevalence of B. ovis antibodies among tested rams in this study was 10.0%. Of 233 rams seropositive to B. ovis, 125 (53.6%) were subclinical, a finding that supports the importance of this test in ram BSE. We found that emaciation in rams was associated with an increased risk of BSE failure from substandard semen parameters (P<0.001), but ulcerative posthitis and the semen collection method were not (P=0.09 and 0.34, respectively). However, collection of semen with the penis retained in the preputial cavity resulted in greater odds of leukospermia relative to semen collection with the penis extended (OR 4.1; 95% CI 2.9, 5.9; P<0.001), presumably from contamination of the semen sample with preputial leukocytes. For ram BSE, therefore, semen collection with the penis manually extended from the sheath is recommended to limit leukocyte contamination of the sample.     

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.     

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.     

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.     

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.     

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.     

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.     

Epididymitis Caused by Brucella ovis in a Southern Ontario Sheep Flock

Epididymitis was diagnosed in three rams in a commercial sheep flock in southern Ontario. The affected rams had palpably enlarged epididymides and two rams had semen which contained inflammatory cells and was of poor quality. Serum compliment fixation titers for Brucella ovis were 1:20, 1:80 and 1:90. Five other rams in the flock were clinically normal and without titers. Two of the affected rams had lesions similar to those produced by experimental infection with B. ovis. The infection in the rams had no apparent affect on ewe performance. The source of the infection remains unknown, but the rams were purchased from a flock which had imported ewes from the western U.S.A.     

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.     

Assessment of ram fertility in hill flocks in Argyll

Examination of 1260 rams, involving 1540 semen examinations, revealed that 9 per cent of rams in hill flocks in Argyll were subfertile as assessed by semen evaluation. Although 34 lesions of the genitalia were identified, only 13 were associated with poor semen quality. A five year survey of one blackface flock resulted in the elimination, after three years, of a 17 per cent level of infertility following routine examination of all rams, with an increase of 2.27 per cent in the lamb crop. Based on the 1981 lamb prices for this flock an increased income of 1085 pounds was achieved.     

Economics of Brucella ovis control in sheep: epidemiologic simulation model

The epidemiology and economics of Brucella ovis control in a hypothetical, commercial sheep flock (100 rams and 2,500 ewes) were investigated. The investigation consisted of an epidemiologic simulation model, reported here, and a decision-tree analysis, reported in a companion paper. The epidemiologic model was designed to simulate the transmission and persistence of B ovis in a ram flock during the mating season as well as the nonmating (isolation) season. A constant contact rate was selected for the nonmating season and a varying contact rate was selected for the mating season to reflect changes in numbers of ewes in estrus. These contact rates were used to evaluate all possible combinations of 5 control alternatives for flock infection rates ranging from 0% to 38%. Vaccination was found to be more effective as a control strategy when the prevalence of flock infection was high (greater than 15%); however, it did not substantially reduce B ovis transmission when the prevalence of flock infection was low (less than 10%). The effect of increasing vaccine efficacy from 40% to 80% had minimal effect on incidence of new cases. The speed with which B ovis could be eradicated depended on the initial prevalence of infection and the screening tests used (palpation, semen testing for leukocytes, and ELISA). All combinations of screening tests verified the usefulness of palpation. Simulation model results indicated that it may be feasible to eradicate B ovis from flocks with moderate to high (10% to 38%) prevalence of infection by culling on the basis of 2 sequential tests.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Seroprevalence of Brucella ovis infection in commercial ram flocks in the Tamworth area

Commercial ram flocks in the Tamworth area of northern New South Wales were surveyed to estimate the proportion of flocks, and rams, infected with Brucella ovis. The flock prevalence (percentage of flocks containing seropositive rams) of 9.1% for Merino flocks was significantly lower than that for British-breed flocks (43.8%, p=0.006) and mixed-breed flocks (46.7%, p=0.017). The mean flock prevalence over all flock types was 32.9%. These estimates were supported by data obtained from diagnostic testing for brucellosis carried out during the previous 6 years. The seroprevalence in rams was 10.8% overall, 2.5% for Merinos, 19% for Border Leicester and 26% for Dorset rams. Within infected flocks, the estimated prevalences were 21%, 65% and 67% for Merinos, Border Leicester and Dorset rams respectively. The seroprevalence in Merino rams was significantly lower than that for both other breeds (p<0.001) for all flocks and in infected flocks. There was no apparent association between age and serological status, or age and the prevalence of epididymitis.