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.     

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.     

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.

     

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.     

Pathology of Brucella ovis infection in red deer stags (Cervus elaphus)

Abstract

AIM: To describe the pathology of the reproductive tract of red deer stags with active Brucella ovis infection and in stags in which B. ovis infection had resolved.

METHODS: Twenty-three red deer stags of varying history were slaughtered and their epididymides and accessory sex glands examined grossly and by histopathology. At the time of slaughter five of the stags had an active B. ovis infection of 24–55 days duration following exposure to infected rams, 10 stags had been experimentally infected with B. ovis by intravenous inoculation 649 days previously and had developed an active infection but the bacterial infection had resolved at least 308 days prior to slaughter, and eight stags had not been exposed to B. ovis at any time.

RESULTS: Of the five stags with an active infection, one had gross enlargement of the epididymides that could be detected by scrotal palpation. Histological lesions in all five stags included mild to severe, predominantly non-suppurative epididymitis, vesiculitis, prostatitis and ampullitis, with neutrophil exudation in associated glandular ducts. Additional lesions in the epididymides were spermatic granulomas and epithelial hyperplasia with intra-epithelial cyst formation. Of the 10 stags in which the bacterial infection had resolved, two had gross enlargement of the epididymides. The histological lesions were similar to those in stags with active infection but were generally milder, with increased periductal scar tissue in the epididymides. The lesions seen in stags resembled those seen in rams with B. ovis infection but they were usually less florid and had fewer plasma cells. No gross abnormalities or histopathological lesions were detected in the non-infected stags.

CONCLUSIONS: Only a small percentage of red deer stags infected with B. ovis develop lesions of epididymitis that can be detected by scrotal palpation. Gross and histological lesions of the genital tract of stags associated with B. ovis infection are similar to the lesions seen in rams. Lesions in stags persist for >300 days after the bacterial infection has resolved.

CLINICAL RELEVANCE: Brucella ovis infection should be considered when there are gross lesions of epididymitis or histological evidence of inflammation in the epididymides or accessory sex glands of red deer stags. Retrospective diagnosis of B. ovis in stags could be achieved by histological examination of the reproductive organs.     

Rough virulent strain of Brucella ovis induces pro- and anti-inflammatory cytokines in reproductive tissues in experimentally infected rams

The ovine brucellosis caused by Brucella ovis has tropism for reproductive tissues but until now the mechanism of bacterial persistence is not understood. Cytokine expression profiles were studied for 8 months in rams after being experimentally infected with the rough virulent strain of B. ovis (R-B. ovis) to study the pathogenesis of B. ovis and immune mechanism possibly associated to bacteria tropism and persistence. The messenger RNA (mRNA) expression levels of interleukin-1α (IL-1α), IL-1β, IL-6, IL-10, IL-12, interferon-γ (INF-γ) and tumour necrosis factor-α (TNF-α) cytokines were quantified by real-time quantitative RT-PCR (qRT-PCR) in reproductive tissues (epididymus, testicles, ampolae, vesicular glands and bulbourethral glands), and non-reproductive (liver, spleen and kidneys) tissues at 30, 60, 120 and 240 days post infection (dpi). During the acute phase of infection at 30 dpi, the host immune response was most notable demonstrating an up-regulation of several cytokines in reproductive tissues, including the epididymus (IL-6, IL-1β and IL-1α), testicles (INF-γ and IL-12), bulbourethral glands (IL-6 and TNF-α) and ampolae (INF-γ, IL-10, IL-1β and IL-1α). During the development of infection, cytokine gene expression levels decreased, providing evidence of immunosuppression and evidence of immune evasion that favoured persistence of chronic R-B. ovis infection. During the chronic phase of R-B. ovis infection (120 and 240 dpi), cytokine production was down-regulated in the epididymus (IL-1β and IL-1α), testicles (INF-γ and IL-12), and ampolae (INF-γ, IL-10, IL-1β and IL-1α), with the exception of the bulbourethral glands (IL-6 and TNF-α) and epididymus (IL-6); in these tissues, R-B. ovis infection resulted in up-regulation of the pro-inflammatory cytokine IL-6. Herein, we report cytokine expression profiles in tissues of rams experimentally infected with the rough strain of B. ovis, which are associated with bacterial persistence and macrophage activation.     

Brucella Ovis Eradication

Extract

Sir:- I am concerned about the validity of the Complement Fixation Test (C.F.T.) for Brucella ovis in rams.     

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.     

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.

     

Evaluation in mice of Brucella ovis attenuated mutants for use as live vaccines against B. ovis infection

Brucella ovis causes ram contagious epididymitis, a disease for which a specific vaccine is lacking. Attenuated Brucella melitensis Rev 1, used as vaccine against ovine and caprine brucellosis caused by B. melitensis, is also considered the best vaccine available for the prophylaxis of B. ovis infection, but its use for this purpose has serious drawbacks. In this work, two previously characterized B. ovis attenuated mutants (Δomp25d and Δomp22) were evaluated in mice, in comparison with B. melitensis Rev 1, as vaccines against B. ovis. Similarities, but also significant differences, were found regarding the immune response induced by the three vaccines. Mice vaccinated with the B. ovis mutants developed anti-B. ovis antibodies in serum of the IgG₁, IgG_2a and IgG_2b subclasses and their levels were higher than those observed in Rev 1-vaccinated mice. After an antigen stimulus with B. ovis cells, splenocytes obtained from all vaccinated mice secreted similar levels of TNF-α and IL12(p40) and remarkably high amounts of IFN-γ, a crucial cytokine in protective immunity against other Brucella species. By contrast, IL-1α -an enhancer of T cell responses to antigen- was present at higher levels in mice vaccinated with the B. ovis mutants, while IL-10, an anti-inflammatory cytokine, was significantly more abundant in Rev 1-vaccinated mice. Additionally, the B. ovis mutants showed appropriate persistence, limited splenomegaly and protective efficacy against B. ovis similar to that observed with B. melitensis Rev 1. These characteristics encourage their evaluation in the natural host as homologous vaccines for the specific prophylaxis of B. ovis infection.     

Discrimination between sheep antibodies to Brucella melitensis and to Brucella ovis

When preparations containing smooth Brucella abortus lipopolysaccharide (LPS) were used as antigens in an ELISA, strong positive reactions were obtained with sera from sheep infected with Brucella melitensis or with Brucella ovis. Oxidation of the LPS with sodium metaperiodate greatly reduced the extent of the cross-reactions with antisera to B. ovis, with little effect on the reactions with antisera to smooth B. melitensis. Periodate oxidation of hot saline extract (HSX) antigen of B. ovis markedly reduced its reactivity in ELISA with anti-B. ovis sera and eliminated cross-reactivity with anti-B. melitensis sera. The reactivity of HSX was maintained after treatment with proteinase K.A simple ELISA system, in which replicate samples from a single serum dilution were tested in parallel against both B. ovis HSX antigen and periodate-oxidised smooth phase B. abortus LPS, was evaluated. It was found to discriminate well between antibodies induced by vaccination or virulent infection with B. melitensis strains and those induced by infection with B. ovis.     

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).     

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.     

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.     

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.

     

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.     

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.     

Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR

Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell’s serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific (Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk (n?=?5), aborted fetuses (n?=?13), and vaginal swabs (n?=?12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan.     

Pelvic dimensions of the romney ewe

A gel diffusion test with sonicated Brucella ovis antigen and an enzyme-linked immunosorbent assay based on heat-extracted antigen were used to distinguish false from true reactions in a complement fixation test based on heat-extracted antigen. Of 142 complement fixing reactors (occurring in supposedly Brucellu ovis-free, accredited flocks), the gel diffusion test correctly identified the status of 139 animals as compared to 128 with the enzyme-linked immunosorbent assay. A combination of the two methods resulted in a correct identification of 141 animals. The procedures provide an easy, cheap and quick way to determine the true status of reactors that show up during routine use of the complement fixation test in Brucella ovis re-accreditation procedures.     

Local tissue reaction to the administration of an inactivated Brucella ovis saline-in-oil vaccine by the intraperitoneal route

Abstract

An inactivated Brucella ovis saline-in-oil vaccine? was administered to 14 adult ewes using both the intraperitoneal route and the subcutaneous route. Pairs of animals were necropsied at intervals between 24 hours and ten weeks after injection. The nature of the local inflammatory reaction to the administration of the vaccine was similar at all sites. The lesion consisted of granulomatous inflammation arranged around droplets of oily vaccine. Diffuse peritonitis was seen at necropsy in 12 of the 14 animals. A local extraperitoneal infammatory response at the injection site was present in four animals despite careful attempts to deposit the vaccine within the abdominal cavity. A second study of 30 rams vaccinated by the intraperitoneal technique confirmed that extraperitoneal deposition of vaccine commonly occurred and that approximately 20% of animals vaccinated by the intraperitoneal method still had peritonitis six months later.