The complement fixation test for the diagnosis of Brucella ovis infection in rams--an Animal Health Division Report

Complement fixation tests using three B. ovis antigen preparations in warm fixation tests (WCFT) and cold fixation (CCFT) tests were done on 541 ram sera. Semen samples from the same rams were examined culturally to identify B. ovis excretors. The CCFT, using an antigen prepared by heat extraction of B. ovis cells, had a sensitivity of 97% in 124 rams which were shedding B.ovis. The specificity was 99% in 144 rams from non-infected flocks. Seventy-seven per cent of 156 rams which reacted to this test were shedding B. ovis in their semen. Tests with other antigens were inferior in sensitivity and/or specificity. The WCFT gave lower titres than CCFT. Vaccination caused large numbers of false positive reactions in 4 flocks.     

The detection of Brucella ovis infection in rams

Abstract

Extract

Each year in New Zealand, many rams are palpated for lesions of the epididymes and testes. Although regarded initially as a method of detecting infertile rams (Crawford and James, 1950 Biberstein, E. L. and McGowan, B. 1958. Cornell Vet., 48: 31–31.  [Google Scholar]), the technique is now regarded primarily as a method of detecting infection with the organism for which Buddie (1956 Buddle, M. B. 1956. J.Hyg., 54: 351–351.  [Google Scholar]) proposed the name Brucella ovis.     

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.     

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.     

Performance of an enzyme-linked immunosorbent assay for the diagnosis of Brucella ovis infection in rams

AIM: To describe the performance characteristics (sensitivity and specificity) of an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of Brucella ovis infection in rams. METHODS: Sera from a negative (n = 2535) and a positive (n = 589) reference population were tested in an ELISA for anti-B. ovis antibodies and cut-off values calculated from the raw, log10-transformed and fitted data. Statistical methods were used to fit curves to the frequency distribution of the data and receiver-operated characteristics analysis used to optimise the cut-off values. RESULTS: Analysis of the frequency distribution of the positive ELISA values suggested a normal distribution of the data, whereas, in the case of the negative population, a Pearson type IV curve appeared to best fit the data. The cut-off values calculated as the mean plus 1.96 standard deviations (s.d.) from the raw, log-transformed and fitted ELISA data did not differ markedly. The differences were much greater at the mean plus 3.09 s.d. cut-off, with the cut-off value calculated from the log-transformed data giving a much better estimate of specificity. Optimisation (minimisation of classification error) of the cut-off calculated from the fitted curves suggested varying cut-off values, depending on the prevalence of B. ovis infection. Discussion: Calculation of cut-off values from curves that were fitted from the observed data give more accurate estimations of the performance characteristics of an assay than traditional calculations from observed values. They also allow the calculation of optimal cut-off values taking into account the prevalence of B. ovis infection and give additional information about the performance of the assay at cut-off values varied according to the epidemiological situation.     

Evaluation of an enzyme linked-immunosorbent assay for the diagnosis of Brucella ovis infection in rams

A simple enzyme linked immunosorbent assay (ELISA) was developed for the serological diagnosis of Brucella ovis infections in rams. Serums from brucellosis accredited-free flocks and flocks known to be infected with B. ovis were tested and the results correlated with warm complement fixation (CF) test and bacteriological examination of semen. Both the ELISA and the CF test detected 0.5% false positive reactions in rams from clinically negative flocks. However the ELISA detected significantly more positive reactors in infected flocks and the CF test failed to detect some rams excreting B. ovis. The ELISA proved to be a valuable test in eradicating brucellosis from infected flocks.     

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.     

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.     

Production of immunity in rams against Brucella ovis infection

Extract

During earlier investigations in New Zealand into the bacterial and mycotic flora of hedgehogs (Erinaceus europaeus), strains of Salmonella typhimurium were obtained from the gut of a number of animals. It was therefore decided to examine these animals more thoroughly for the presence of salmonellae. This paper embodies the results of a small survey subsequently carried out in the Hamilton suburban area during February, 1964.     

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.     

Brucella ovis vaccination of rams

Abstract

Extract

An infectious epididymitis of rams caused by Brucella ovis (Buddie, 1956 Buddle, M. B. 1956. J. Hygiene, 54: 351–351.  [Google Scholar]) infection, first described in Australia (Simmons and Boyes, 1953 Simmons, C. G. and Hall, W. J. K. 1953. Aust.vet.J., 29: 33–33. [Crossref] , [Google Scholar]) and New Zealand (Buddie and Boyes, 1953 Buddle, M. B. and Boyes, B. W. 1953. Aust.vet. J., 29: 145–145. [Crossref] , [Google Scholar]) was recognized subsequently in Czechoslovakia (Gdovin et al, 1955 Gdovin, T., Hrudka, F., Chladecky, E. and Koppel, Z. 1955. Shorn, ces. Akad. zemedelsk Ved., 28: 617–617.  [Google Scholar]), the United States (McGowan and Shultz, 1956 McGowan, B. and Shultz, G. 1956. Cornel Vet., 46: 277–277.  [Google Scholar]), South Africa (Van Rensburg et al, 1958 Van Rensburg, S. W. J., Van Heerden, K. M., Le Roux, D. J. and Snyders, A. J. 1958. J.S.Afr. vet. med. Ass., 29: 223–223.  [Google Scholar]), Rumania (Tudoriu, et al, 1958 Tudoriu, C. D., Andrei, M., Draghici, D. and Moldoveanu, P. 1958. Anu. Inst. Pat.Igien. anim. Bucaresti, 8: 5–5.  [Google Scholar]), and South America (Dr Justo Zomara B, 1961, pers. comm.). As the infection can affect ram fertility and, further, can be responsible for abortion in ewes and perinatal mortality in lambs, attention has been directed to the development, evaluation, and application of control measures in a number of important sheep-raising countries.