Attempted definition by immunoblotting of the causes of reactivity in suspected false-positive sera in the Brucella ovis complement fixation test

Seventy-nine suspected false-positive sera, obtained over 1 year from routine submissions for Brucella ovis serological testing, were used in this study. These sera, which exhibited titres in the complement fixation test, but which because of their epidemiological history and their reactions in the enzyme-linked immunosorbent assay and gel diffusion test wereisuspected to be false positives, were further analysed by immunoblotting. In blots, using B. ovis antigens, rough lipopolysacchride was identified as the major, immuno-reactive bacterial component. Antibodies against this macromolecule were present in 46.8% of the suspected falsepositive sera.

In order to find out if rough lipopolysaccharides from other bacterial species could be the possible cause for the suspected false positivity, 23 sera with highest complement fixation titres were reacted in blots with cell extracts from Escherichia coli, Yersinia enterocolitica, Yersinia pseudotuberculosis, Bortedella bronchiseptica, Actinobacillus setninis, Catnpylobacter fetus fetus, Campylobacter jejuni, Mycobacterium paratuberculosis, Mycobacterium phlei, Corynebacterium pseudotuberculosis and pure lipopolysaccharides from Escherrichia coli and Salmonella typhimurium. Despite high frequencies of antibody reaction with proteins in most of these bacterial cell extracts, which reflect the presence of infections with these bacteria, immuno-staining in the rough lipopolysaccharide region was not observed.     

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.     

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.     

Comparison of the enzyme-linked immunosorbent assay and complement fixation test for detecting Brucella ovis antibodies in sheep

A solid-phase, indirect, enzyme-linked immunosorbent assay (ELISA) was compared with the microtitre complement fixation test for detecting Brucella ovis antibodies in 220 ram sera. The ELISA was more sensitive than the complement fixation test; it demonstrated antibodies in 11 sera from known infected or vaccinated rams that were complement fixation test negative. No false positives were recorded with the ELISA and, in 36 sera positive to both tests, the ELISA titres were consistently higher than the corresponding complement fixation test titres.     

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.     

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.     

Comparison of an enzyme-linked immunospecific assay (ELISA) with the cold complement fixation test for the serodiagnosis of Brucella ovis infection

An enzyme-linked immunospecific assay (ELISA) for the serodiagnosis of Brucella ovis infection in sheep is described and compared with the cold complement fixation (CF) test. ELISA was performed in microtiter plates, using horse-radish peroxidase conjugated to anti-normal sheep serum globulins, and hydrogen peroxide plus o-phenylenediamine as substrate. A heated, cell-free B. ovis extract was used as antigen in both tests. ELISA was easier to perform, distinguished better between positive and negative sera, and did not need heat-inactivated sera.     

Use of the double immuno gel diffusion test and the enzyme-linked immunosorbent assay to distinguish false from true reactors in the complement fixation test for Brucella ovis

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 Brucella 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.     

Effect of Eperythrozoon ovis on the lysis of sheep erythrocytes in the complement fixation test

When erythrocytes from sheep experimentally infected with Eperythrozoon ovis were used in the titration of reagents for a standardised complement fixation test, increased amounts of both haemolysin and complement were required for erythrocyte lysis compared with preinfection titrations. The haemolysin requirement increased by up to 125% at 55 days post-infection and complement requirement increased by up to 40% at 40 days post-infection. These changes appeared to correlate with the development of a macrocytic anaemia in affected sheep rather than E. ovis parasitaemia. The results emphasise the need to carefully monitor the haematological parameters of sheep used as sources of erythrocytes for the complement fixation test.     

Attempted transmission of Brucella ovis between red deer stags by successive grazing or adjacent-paddock grazing

AIM: To determine whether Brucella ovis can be transmitted from stag to stag by successive grazing of infected and noninfected stags in the same paddock, or by grazing infected and non-infected stags in adjacent paddocks. METHODS: Six red deer stags (Cervus elaphus) were artificially infected with B. ovis and 5 were confirmed to be shedding the organism in their semen. Infected stags alternated paddocks, and therefore grazing and wallows (successive grazing), once or twice weekly with 6 non-infected stags from 3 March to 18 August, 1999. Direct contact between the 2 groups of animals was prevented. The 2 groups alternated paddocks 32 times. Six other non-infected stags were grazed in a paddock adjacent to the infected stags throughout this period, separated by a standard deer fence. Non-infected stags were blood sampled at 2to 6-week intervals to test for B. ovis antibodies using a complement fixation test and an enzyme linked immunosorbent assay. RESULTS: No stag from either non-infected group became infected with B. ovis. CONCLUSIONS: The risk of stags becoming infected with B. ovis by successive grazing of the same paddock as, or by grazing in paddocks adjacent to, infected stags appears to be low. We conclude from this result, and similar experimental evidence and experience of this disease in sheep, that transmission of B. ovis requires animals to be grazed or confined together in a way that allows direct contact between animals. CLINICAL RELEVANCE: It is likely that infected and non-infected stags can be managed on the same property without transmission occurring between the groups, provided that they do not come into direct contact with one another.