Application of an enzyme-linked immunosorbent assay in the final stages of a bovine brucellosis eradication program

Bovine field serums from the Australian brucellosis eradication program were used to compare 2 enzyme linked immunosorbent assays (ELISA) with the complement fixation test (CFT) and Rose Bengal test (RBT). One ELISA used an anti-bovine IgG horseradish peroxidase conjugate (ELISA 1) and the other a monoclonal anti-bovine Ig alkaline phosphatase conjugate (ELISA 2). When compared with the CFT, the ELISA 2 like the ELISA 1 lacked specificity in B. abortus vaccinated herds but the ELISA 2 was more specific than the ELISA 1 in previously infected herds and equally as specific as the ELISA 1 in nonvaccinated Brucella free herds. In this study the ELISA 2 proved more sensitive than the CFT, RBT and ELISA 1 particularly in herds where B. abortus biotype 2 was present. The value of using the ELISA 2 in conjunction with the CFT in an eradication program is discussed.     

Early history of bovine brucellosis eradication in Australia

The first Australian bovine brucellosis eradication campaign on a state-wide basis was initiated and established in Tasmania. The procedures involved in drawing up a suitable scheme together with a summary of the difficulties encountered in setting up and operating the scheme are described, leading to the successful eradication of brucellosis from Tasmania. Our observations that milk was not a major factor in the transmission of brucellosis infection from cattle to man are recorded. Recognition is accorded to veterinarians, some eminent in the profession, who, in the absence of Australian research into the disease, gave freely of their advice in helping to pioneer the eradication program. Within one year of commencing the scheme 4 herds had been accredited; by 3 years 85 herds had been accredited.     

Surveillance and risk management during the latter stages of eradication: experiences from Australia

The paper reviews the eradication of bovine tuberculosis from Australia with special reference to surveillance and managing the risk of animals exposed to tuberculosis infected animals during the latter stages of eradication. The successful eradication was based on a sound technical program with strong industry and government support. The model of joint industry and government funding and decision-making first used during the brucellosis and tuberculosis eradication campaign (BTEC) has been successfully incorporated within subsequent livestock disease control programs in Australia. An overview of the history of tuberculosis eradication in Australia provides a background to the surveillance approach. Australia was fortunate that there were no wildlife reservoir hosts. Feral animal reservoir hosts were removed during the eradication program. Surveillance to detect rare diseases is recognised to be statistically challenging with high resource requirements. Australian veterinary authorities have a high level of confidence that the combination of increasing sensitivity of abattoir surveillance systems by the submission of all granulomas detected at slaughter with increasing risk management of animals exposed to tuberculosis infected animals during the final stages of eradication provides a high level of assurance that Mycobacterium bovis has been eradicated.     

How to substantiate eradication of bovine brucellosis when aspecific serological reactions occur in the course of brucellosis testing

Collaborative work was financed by the EU to develop and assess new diagnostic tools that can differentiate between bovine brucellosis and bovine infections due to Yersinia enterocolitica O:9 either in conjunction with, or as an alternative to, the classical serological, bacteriological or allergic skin tests. Sixteen heifers were experimentally infected with Brucella abortus biovar 1 (five heifers), Brucella suis biovar 2 (two heifers), Y. enterocolitica O:9 (six heifers) and Y. enterocolitica O:3 (three heifers). Four heifers, naturally infected with Y. enterocolitica O:9 that presented aspecific brucellosis serological reactions were also included in the experiment. A self-limited infection was induced in cattle by B. suis biovar 2. All the brucellosis serological tests used, i.e. the slow agglutination test (SAW), the Rose Bengal test (RB), the complement fixation test (CFT), indirect and competitive ELISA’s, lacked specificity when used to analyze sera from Y. enterocolitica O:9 infected animals. A Yersinia outer membrane proteins (YOPs)-ELISA was also used and although the test is able to detect a Yersinia group infection, it provided no evidence of whether or not there is a possible brucellosis infection when dual infections are present. The brucellergen IFN-γ test showed a lack of specificity also. The only test that was proven to be specific is the brucellergen skin test. All brucellosis serological tests, except the indirect ELISA, were limited in their ability to detect B. abortus persistently infected animals.

Based on these experimental studies, a strategy was implemented as part of the year 2001 Belgian Brucellosis Eradication Program to substantiate the eradication of bovine brucellosis. Epidemiological inquiries have identified risk factors associated with aspecific serological reactions, possible transmission and infection of cattle by B. suis biovar 2 from infected wild boars; and both legal and administrative measures taken by the veterinary services. No cases of bovine brucellosis have been confirmed in Belgium since March 2000.     

Use of an enzyme-linked immunosorbent assay in a bovine brucellosis eradication program

An enzyme-linked immunosorbent assay (ELISA) was developed and was compared with the complement fixation test (CFT) in a bovine brucellosis eradication program. The ELISA detected significantly more reactors than the CFT in both strain 19 vaccinated infected herds (1.79% versus 1.14%) and non-vaccinated infected herds (4.2% versus 3.59%) but not in either vaccinated or non-vaccinated brucella-free herds. The specificity for both tests in brucella-free herds was greater than 0.998. The specificity and sensitivity of the ELISA were compared with those of 3 other tests (the Rose Bengal test; the indirect haemolysis test [IHLT] and the CFT) on serum from 151 animals cultured at slaughter. The calculated specificity of the ELISA in this infected group was lower than for both the CFT and the IHLT (0.58 versus 0.67 versus 0.75). The sensitivity however was much greater (1.0 versus 0.73 versus 0.71). The value of the ELISA when used in an eradication program is discussed.     

Diagnosis of brucellosis by serology

Serological diagnosis of brucellosis began more than 100 years ago with a simple agglutination test. It was realized that this type of test was susceptible to false positive reactions resulting from, for instance, exposure to cross reacting microorganisms. It was also realized that this test format was inexpensive, simple and could be rapid, although results were subjectively scored. Therefore, a number of modifications were developed along with other types of tests. This served two purposes: one was to establish a rapid screening test with high sensitivity and perhaps less specificity and a confirmatory test, usually more complicated but also more specific, to be used on sera that reacted positively in screening tests. This led to another problem: if a panel of tests were performed and they did not all agree, which interpretation was correct? This problem was further compounded by the extensive use of a vaccine which gave rise to an antibody response similar to that resulting from field infection. This led to the development of an assay that could distinguish vaccinal antibody, starting with precipitin tests. These tests did not perform well, giving rise to the development of primary binding assays. These assays, including the competitive enzyme immunoassay and the fluorescence polarization assay are at the apex of current development, providing high sensitivity and specificity as well as speed and mobility in the case of the fluorescence polarization assay.     

The relationship between the microtitration serum agglutination and complement fixation tests in bovine brucellosis serology

The relationship between antibody titres in the microtitration serum agglutination test and the complement fixation test in bovine brucellosis is described. For low and high MSAT values there is good agreement between the 2 tests. This is not the case for MSAT values between 54 and 338 IU/ml. For practical reasons, results falling into this category cannot all be repeated. Repetitions are so structured that less than 4% of the tests need to be repeated. If the level of repetitions should show an increase above 4%, it is assumed that technical or human error has occurred.     

The reproducibility of results in bovine brucellosis serology and their correlation with the isolation of Brucella abortus

In both the complement fixation test (CFT) and the serum agglutination test (SAT) titres were reproducible for the most part within a twofold range. They seldom exceeded these limits and never a fourfold range. Brucella abortus was successfully isolated in 86% of serologically positive cases and evidence is presented to confirm the use of the 30 International Units/ml level in the CFT as being diagnostically significant. The SAT, when done in microtitration plates, is even more reproducible than when done in tubes. The incidence of infected animals aborting or calving down with negative titres was found to be low.     

Investigation of bovine brucellosis in the Northeastern Turkey

Bovine brucellosis, caused by Brucella abortus, is a significant problem for both public and animal health in Turkey. This study was conducted on the calving seasons between 2001 and 2006. A total of 626 serum samples of cattle obtained from 27 herds with a history of abortions was examined for Brucella antibodies by RBPT, SAT and ELISA. Of the cattle sera analysed, 221 (35,30%) and 206 (32, 92%) and 247 (39,45%) were found to be positive by RBPT , SAT and ELISA, respectively. B. abortus was isolated from 48 (32,21%) of 149 lung samples and stomach contents of the aborted fetuses. Based on the biochemical tests and the agglutination tests with monospecific A and M antisera, only 3 of the isolates were found to be B. abortus biotype 1 and the remaining 45 were biotype 3. This study also revealed that the dominant biotype of B. abortus was biotype 3 in this region. The determination of the agents responsible for bovine brucellosis and serosurvey of this disease are expected to help better understanding of this zoonotic infection in this region and neighbouring countries.