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.     

Identification of immunoreactive membrane antigens of Brucella ovis by ELISA profiling

Enzyme-linked immunosorbent assay (ELISA) profiling was used to identify the immunoreactive membrane antigens of Brucella ovis. Immunoreactive membrane antigens obtained after detergent extraction of the bacterial membrane complex (inner and outer membranes) were resolved into five peaks (A, B, B1, C and D) by gel permeation chromatography. Aliquots from each of the chromatographed fractions were coupled to 96-well microtitre plates and immunoreactive fractions identified with sera from two rams. Serum from ram 1 which had been vaccinated with a single injection of formalin-killed B ovis emulsified in incomplete Freund's adjuvant identified A and B as the major immunoreactive peaks. Serum from ram 2, which had been successfully infected with B ovis, reacted mainly against peaks A, B1, C and D. This observation facilitated the use of A, B, B1, C and D peak antigens as test reagents to examine the serological response of 12 other rams exposed to B ovis by vaccination or intraconjunctival or intravenous inoculation. Sera from rams which developed productive infections reacted strongly against peaks A, B1, C and D while vaccinated rams had preferential antibody activity against peaks A and B.     

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.     

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.     

Comparison of subcutaneous and conjunctival routes of Rev 1 vaccination for the prophylaxis of Brucella ovis infection in rams

The serological response and protection conferred against Brucella ovis by the Rev 1 vaccine was evaluated in both adult (experiment 1) and young rams (experiment 2) vaccinated either subcutaneously or conjunctivally. In experiment 1 the Rev 1 vaccine protected 55.5 per cent and 100 per cent, respectively, of subcutaneously and conjunctivally vaccinated rams against three consecutive challenges that infected 100 per cent of unvaccinated controls. In experiment 2, Rev 1 protected 100 per cent of rams vaccinated subcutaneously and 70 per cent of those vaccinated conjunctivally against a challenge dose able to infect all the unvaccinated controls. The serological response after vaccination was significantly lower in rams vaccinated conjunctivally than in those vaccinated subcutaneously.     

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.     

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.     

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.     

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.     

Efficacy of long-acting oxytetracycline alone or in combination with streptomycin for treatment of Brucella ovis infection of rams

Twenty-four rams inoculated with Brucella ovis by conjunctival and preputial routes were treated with a long-acting oxytetracycline alone or in combination with dihydrostreptomycin sulfate. The combined treatment eliminated Brucella ovis from 11 of 12 (91.6%) treated rams. Only 4 of 12 (33.3%) rams treated with oxytetracycline alone were bacteriologically negative. Neither treatment resolved clinical epididymitis in 2 rams affected before treatment. Many rams had pathologic lesions in the epididymis and ampullae, which limited the efficacy of antibiotic treatment.     

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.     

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.     

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.     

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.     

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.     

Immune response in rams experimentally infected with Brucella ovis

Cellular as well as humoral immune responses were detected in six rams experimentally infected with Brucella ovis. Specific antibodies were detectable by enzyme-linked immunosorbent assay by day 11 after infection in all the rams. The levels of IgM antibodies and total antibodies in the serum rose until 33 and 41 days after infection respectively, then levelled off. Antigen-induced blastogenic responses by lymphocytes developed as early as five days after infection in all rams but had decreased to low levels by day 63 in most. Blastogenesis induced by phytohaemagglutinin and concanavalin A varied among infected rams and did not differ significantly (P greater than 0.05) from control rams. All rams had developed delayed-type skin hypersensitivity by day 63 after infection. One ram which did not become infected as a result of exposure had low levels of B ovis serum antibodies and a detectable antigen-induced lymphocyte blastogenic response before infection, suggesting the involvement of cell-mediated immunity in protection against B ovis.     

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.     

Association of sexual experience with isolation of various bacteria in cases of ovine epididymitis

Testicles from rams in flocks experiencing ram epididymitis in Idaho and eastern Oregon were cultured. Twenty-six breeding rams from 6 flocks were cultured and only Brucella ovis was isolated. Virgin rams (65) harbored numerous species of small fastidious gram-negative rods, including Actinobacillus actinomycetemcomitans, Haemophilus spp, Moraxella spp, and Pasteurella spp. Thus, there appeared to be 2 separate disease entities, dependent on sexual experience of the animal.     

Association of age of ram with distribution of epididymal lesions and etiologic agent

A 2-year study of the frequency of isolation of various organisms from mature and yearling rams with epididymitis was conducted at the US Sheep Experiment Station at Dubois, Idaho. Investigation into the distribution of lesions in the epididymis in relation to age of the ram also was studied. Serologic or bacteriologic evidence of Brucella ovis infection was demonstrated in 79.5% of the mature rams with epididymal lesions. Actinobacillus seminis and Histophilus ovis were the 2 most frequently isolated organisms from yearling rams with lesions. The tail(s) of the epididymis was the most frequent site of lesion development in the mature rams (86.4%). Yearling rams developed lesions twice as frequently in the tail(s) of the epididymis as in the head(s) of the epididymis. When lesions were localized in the head(s) of the epididymis, an etiologic agent usually was not demonstrated.