Effects of Brucella ovis infection on semen characteristics of 16-month-old red deer stags

AIM: To determine the effects of Brucella ovis infection on semen characteristics of 16-month-old red deer stags (Cervus elaphus). METHODS: At monthly intervals during March, April and May, semen was collected using electro-ejaculation from 9 yearling red deer stags that had been artificially infected with B. ovis 3 months previously. In March, semen was also collected from 6 non-infected stags from the same peer group for comparison. Semen was evaluated for gross appearance, percentage of sperm showing forward motility, sperm morphology and sperm density/ml of semen, and the presence of white blood cells was determined. In addition, at the time of semen collection, the epididymes of each stag were palpated and lesions recorded. RESULTS: Grossly visible purulent material was present in semen from 6/9 infected stags and the percentage of sperm showing forward motility did not exceed 20% in any of these samples. Increased numbers of white blood cells and cellular debris were evident in semen from 8/9 infected stags. Compared with non-infected stags, sperm motility in semen from infected stags was significantly reduced (p<0.05). There were no differences in percentage of morphologically normal sperm between groups, but infected stags had more detached sperm heads present in semen than non-infected stags (p<0.05). Only 2 of the infected stags had lesions of epididymitis evident on scrotal palpation. CONCLUSIONS: B. ovis infection is likely to have a detrimental effect on stag semen quality. While there was variation between individual animals, overall sperm motility was decreased and semen from infected stags had increased numbers of white blood cells and detached sperm heads. CLINICAL RELEVANCE: The fertility of breeding stags may be reduced if they are infected with B. ovis and this possibility should be considered when investigating reproductive problems of deer.     

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.     

TESTICULAR PATHOLOGY OF MERINO RAMS

SUMMARY The scrotal contents of 2,281 Merino rams of wide age range examined at 3 abattoirs in Perth, Western Australia, showed a 40% prevalence of rams with 1 or more gross lesions. The percentage prevalence of gross lesions found on autopsy were: adhesions 21, testicular atrophy/hypoplasia 14, testicular calcification 13, congenital cyst of the epididymis 6, cryptorchidism 4, chronic epididymitis/scrotal abscesses 2, varicocoele 2, testicular aplasia1, seminoma1. The prevalence of lesions increased with age of rams. Testicular atrophy was often present when varicocoele, chronic epididymitis and scrotal abscess affected that side of the scrotal sac. Corynebacterium spp was the most frequent isolate from chronic epididymitis. Actinobacillus seminis was isolated once and Brucella ovis was not isolated. About 20% of the rams showed lesions compatible with a diagnosis of reproductive unsoundness. A large proportion of lesions were revealed only after removal of the scrotum and most of these did not appear to affect normal spermatogenesis except for an estimated 9% of rams, which showed mild atrophy/hypoplasia. Samples of these testes showed mild to moderate changes in spermatogenesis on histopathology. It was postulated that the latter group of rams might represent those ill-defined clinical cases which show equivocal results on appraisal of semen samples despite the presence of palpably normal testes.     

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.     

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.     

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 lymphnodes. 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. ovisin 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.     

Immunoglobulins and immunoglobulin-containing cells in the reproductive tracts of rams naturally infected with Brucella ovis

SUMMARY Thirteen rams with serological evidence of Brucella ovls exposure (CFT of 1:8 or greater), but with no or only mild epididymitis, were selected from a ram flock. Serum, semen, preputial washings and fluids from the accessory sex glands (ASGF) and testis and epididymis (TEF) were examined and immunoglobulin (lg) concentrations estimated. Genital tissues were examined histologically and the percentages of class specific immunoglobulin containing cells (ICC) determined. Eleven of these rams had histological evidence of active inflammation consistent with B. ovis infection; the organisam was cultured from the semen of 7. IgA concentration was high in semen (mean ± standard deviation of 5.03 ± 1.78 mg/ml) and ASGF (9.18 ± 7.28 mg/ml). These levels were much higher than those recorded in noninfected rams. IgA concentration was low in serum (0.78 ± 0.55 mg/ml) and TEF (0.59 ± 0.78 mg/ml). The concentrations of IgG¹, IgG², and IgM were low in all genital fluids sampled and not significantly different from those recorded in noninfected rams. This indicated that infection with B. ovis results in a pronounced IgA response in secretions, mostly from the accessory sex glands. Examinations of ICC, however, revealed that the plasma cell infiltrates of the epididymis, vas deferens, ampulla and seminal vesicle were predominantly IgG-containing (92.4, 97.2, 79.4 and 91.9% respectively). Fewer IgM-containing cells were scattered throughout these tissues, constituting 3.9, 6.3, 0.3 and 6.5% of all ICC, respectively. IgA-containing cells were most frequently seen in the ampulla (9.6% of ICC) where they were located directly beneath the epithelium, suggesting the ampulla as the most prominant location for the local production of IgA. These results indicate that although the tissue ICC response was predominantly IgG, IgA was selectively transferred into secretions with the exclusion of most IgG and IgM. ICC percentages in bulbourethral and prostate glands, and beneath pelvic urethral and preputial epithelia, were similar to those found in noninfected rams.     

Scrotal enlargement in rams and bucks in Qassim region,central of Saudi Arabia: clinical and ultrasonographic findings and seroprevalence of brucellosis

The aim of this study was to clarify the causes of scrotal enlargement in rams and bucks in Qassim region of Saudi Arabia. Enlarged scrotal contents of rams and bucks (n = 153) were examined by visual inspection, palpation, and ultrasonography. Blood samples were obtained and tested for Brucella sp. infection. Clinical and ultrasonographic findings showed that scrotal enlargement was mainly associated with orchitis, peri-orchitis, and epididymitis. Miscellaneous findings were scrotal hernia, scrotal hematoma, and hydrocele. The frequencies of orchitis, peri-orchitis, and epididymitis were 47.4, 21.1, and 14.1% in Awassi rams; 54.5, 21.7, and 8.7% in Najdi rams; 52.3, 20.5, and 9.1% in Ardi bucks; and 50, 16.7, and 16.7% in Damascus bucks, respectively. Orchitis was associated with no-abscess formation (23%), single-abscess formation (15.4%), and multiple-abscesses formation (61.6%). Peri-orchitis was characterized by hard consistency, atrophy of the testes, and extensive connective tissue formation. Epididymitis was observed mainly at the tail of the epididymis (82.4%) but rarely at the head (17.6%). Epididymitis was associated in many cases with abscessation (70.6%). Males with orchitis, peri-orchitis, and epididymitis were positive for Brucella melitensis and Brucella ovis in the frequency of 21.3% and 48.8%, respectively. In conclusion, scrotal enlargement in rams and bucks in Qassim region is caused mainly by inflammation of the testis and/or epididymis and associated tremendously with brucellosis seropositivity.

     

Observations on Brucella ovis CFT results

Three commercial flocks, deriving replacement rams from 15 parent studs, had respectively 33 of 71,13 of 31 and 8 of 82 rams positive to the Br. ovis CF test. In the first flock, clinical findings supported a diagnosis of active Brucella infection. In the other two flocks, positive results were attributed to vaccinational titres persisting for up to 3 years after vaccination. It is concluded that permanent identification of vaccinated rams would assist in Br. ovis flock-testing programmes.     

The complement fixation test for the diagnosis of Brucella ovis infection in rams

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.     

Ovine brucellosis in alberta

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

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

     

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.     

Lack of evidence of Brucella ovis infection in rams in Quebec

A study was conducted to estimate the seroprevalence of Brucella ovis infection in rams in the Estrie and Bas-Saint-Laurent regions (Quebec). Rams sera (n = 258) were serologically evaluated from 224 rams in 30 commercial flocks and from 34 rams at 2 slaughterhouses by using an enzyme linked immunosorbent assay. Epididymides and testes were examined by palpation on farms and microscopically for culled rams. No ram was seropositive to Brucella ovis or had lesions suggestive of brucellosis from the farm or slaughterhouse surveys.     

Effects of vaginal Brucella ovis infection of red deer hinds on reproductive performance, and venereal transmission to stags

AIMS: To investigate the effects of vaginal Brucella ovis infection on the reproductive performance of red deer (Cervus elaphus) hinds. To determine whether stags may become infected with B. ovis by venereal transmission from mating infected hinds. METHODS: Thirty mixed-age red deer hinds serologically negative for B. ovis antibodies were synchronised for oestrus on 22 March 2000. B. ovis was inoculated into the vagina of each hind at oestrus and again, 18 days later. At oestrus, hinds were randomly allocated to six groups, each joined with a 16 month-old red deer stag seronegative for B. ovis, for 55 days. Hinds were blood sampled and scanned for pregnancy using rectal ultrasonography at monthly intervals. Six pregnant and four non-pregnant hinds were slaughtered pre-calving and three hinds were slaughtered post-calving. Reproductive tracts and foetuses were examined grossly, histologically and microbiologically. Calves were identified and blood sampled within 3 days of birth. Hinds and calves were blood sampled in February and May 2001 and vaginal swabs were collected from hinds for B. ovis culture. Blood was collected from stags, 5 and 19 days after mating and semen was collected for B. ovis culture. The 17 remaining hinds were mated in 2001 to two mixed-age wapiti (Cervus canadensis) stags. Both stags were blood sampled after mating. Sera were tested in a B. ovis complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA). RESULTS: All 30 hinds developed B. ovis antibody levels, measurable using either the CFT or ELISA, but these did not remain elevated. There was no evidence of infection, either by gross pathology, histopathology or microbiological culture in the ten hinds or six foetuses slaughtered pre-calving. All remaining 20 hinds produced normal calves, 15 of which survived until weaning. Three hinds experienced dystocia and gave birth to dead calves and two calves died within 4 days of birth. One hind which had dystocia was euthanased. Samples from this hind and from 3/5 dead calves showed no evidence of B. ovis infection. B. ovis was cultured from the vagina of 1/19 hinds 48 weeks after inoculation, at which time B. ovis CFT and ELISA results for this hind were negative. Most calves had B. ovis serum antibodies at 1-3 days of age but levels were negligible when sampled at 10-15 weeks of age. Foetuses and dead calves were all seronegative. Three of the five red deer stags used for mating became infected with B. ovis. The two wapiti stags used to mate the remaining 17 hinds the following year remained seronegative. CONCLUSIONS: B. ovis is unlikely to have significant detrimental effects on the reproductive performance of red deer hinds. Venereal transmission via the vagina of hinds is a possible route of transmission between stags. It is possible that survival of the organism in the vagina of some hinds could create difficulties in disease control programmes. CLINICAL RELEVANCE: B. ovis infection of hinds at the time of mating is unlikely to cause significant reproductive losses. Venereal transmission of B. ovis between stags via the hinds may occur when groups of hinds are joined with more than one stag.     

Epidemiological studies on ovine brucellosis in selected ram flocks

SUMMARY The epidemiology of ovine brucellosis was investigated in 6 ram flocks in which anomalous reactions to the complement fixation test was recorded. It was shown that rams can become infected as young as 4 months of age. Naturally infected animals have an epididymitis and excrete Brucella ovis in their semen or they may only show a serological response for a short time then recover. The base line titres of chronically infected animals stay relatively constant. The importance of abortive infection and possible reinfection is discussed.     

CLINICAL AND PATHOLOGICAL STUDIES OF BORDER LEICESTER RAMS NATURALLY INFECTED WITH ACTINOBACILLVS SEMINIS

Clinical examination of 31 rams with evidence of Actinobacillus seminis infection revealed 15 cases with marked lesions of the scrotal contents, two cases with very mild lesions, one with testicular atrophy but no abnormality of the epididymides, and another 13 without any palpable abnormalities of the genitalia. The rams were from a Border Leicester flock and included 27 un-mated rams from 6 to 12 months old. Histological examinations of affected epididymides showed a mixed purulent and non-purulent inflammation, with cystic hyperplasia of the ductus epididymis. Interstitial fibrosis and spermatic granulomas were seen in chronic lesions. Bilateral testicular atrophy and seminal degeneration were found in the nine rams that were autopsied. The clinical and pathological features of the disease are similar to those of ovine brucellosis. A pyelonephritis of unknown significance was found in four of the nine rams examined.     

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.     

Lameness in rams following vaccination against Brucella ovis infection

Extract

Since the introduction in 1957 of a vaccination procedure to control Brucella ovis infection in sheep, many thousands of rams have been vaccinated annually throughout New Zealand almost without complications.     

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 × 10⁸ live Br. ovis bacteria. The number of rams that succumbed to infection within the four groups was 4/35 (11%) for the 2 s/c, 7/33 (21%) for the 2 i/p, 9/35 (26%) for the 1 i/p and 18/35 (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).     

The Complement Fixation Test for Brucella Ovis

Abstract

Sir:- A project to investigate the efficacy of the complement fixation test (CFT) for the diagnosis of Brucella ovis infection in rams has recently been completed. The investigation was undertaken by field and laboratory staff of the Division of Animal Health and several practitioners. The results of CFT's done with 3 antigens by waim and cold fixation methods (WCFT and CCFT) were correlated with results of cultures done of semen from the same rams. It is our aim to publish a full report in this Journal at a later date. In view of the widespread interest in the subject some of the important findings are summarised below:

• ? Semen and blood samples were collected from 541 rams in 40 flocks.

• ? The type of antigen which has been used for a number of years in New Zealand was unsatisfactory. The CCFT using this antigen had a sensitivity of only 85% in 124 rams which gave positive semen cultures. These rams came from 16 infected flocks.

• ? The CCFT using the best of the 3 antigens proved to be a remarkably reliable test. In the same group of infected animals mentioned above, 120 of the 124 infected rams had positive or suspicious CFT's. The sensitivity of the test was therefore 97%. The specificity was > 99% in 144 rams in 16 uninfected flocks. Only 1 suspicious titre was found in the uninfected group.

• ? In 4 flocks in which B. ovis was never isolated, 18 of 58 sera gave positive or suspicious reactions. Recognition of this type of 'problem flock' is therefore important. A history and clinical picture which does not agree with serological results should always be thoroughly investigated. Semen should be collected from an appropriate number of cases for bacteriological examination. It is suspected that at least some ‘problem flocks’ may contain unreported vaccinated animals. Further investigation of ‘problem flocks’ will be an aim of the Animal Health Division in the future.