The use of homologous antigen in the serological diagnosis of brucellosis caused by Brucella melitensis

In the European Union the serological diagnosis of brucellosis caused by Brucella melitensis is performed using the heterologous antigen of B. abortus S99. The possible higher sensitivity or ability of an early detection of antibodies by a homologous antigen may prove very useful in the final phases of an eradication programme. Results obtained in sheep experimentally infected by B. melitensis biovar 3 were compared using B. abortus S99, B. melitensis M1, M2 and M3 antigens in the Rose Bengal plate test (RBPT), the complement fixation test (CFT) and an enzyme-linked immunosorbent assay (ELISA) test. Forty-six sheep from an officially brucellosis-free flock were experimentally infected intraconjunctivally with B. melitensis biovar 3. Prior to infection, all animals were tested first against Brucella antibodies, weekly for 2 months post-infection (PI) and then monthly for a further 7 months. All sera were tested against the antigens listed above using RBPT, CFT and ELISA. Using a Bayesian approach, test sensitivities were estimated and compared. Their ability for the early detection of antibodies was evaluated through a regression model based on a logit response model, using the number of days PI as the independent variable and the logit of the fraction of positive animals as the dependent variable. No significant differences were detected among the various antigens used, either in terms of sensitivity or in terms of antibody kinetics; however, the CFT was significantly less sensitive than the RBPT and ELISA and it also showed a lower rate of increase of percentage positive animals (beta-coefficient of regression analysis).     

Evaluation of the cold complement fixation test for diagnosis of ovine brucellosis

SUMMARY A cold complement fixation test for ovine brucellosis in which fixation of complement occurs for 16 h at 4°C was compared with the conventional warm complement fixation test in which fixation takes place for 40 minutes at 37°C. The cold complement fixation test detected experimentally infected animals up to one week sooner than the warm complement fixation test and the titres were approximately one twofold dilution higher. Similar results were obtained when the 2 tests were compared using 11,922 serum samples collected from 700 ram flocks over a 2-year period. False positive reactions were observed in less than 0.1% of cases.     

Evaluation of different enzyme-linked immunosorbent assays for the diagnosis of brucellosis due to Brucella melitensis in sheep

Six serological assays for the diagnosis of ovine brucellosis, due to Brucella melitensis were evaluated. Reference serum samples from sheep of known B. melitensis infection status (n = 118) were assessed using the Rose Bengal test (RBT), complement fixation test (CFT) and four commercial enzyme-linked immunosorbent assays (ELISAs), including two indirect ELISAs (iELISAs), one competitive ELISA (cELISA) and one blocking ELISA (bELISA).The highest differential positive rates (DPR) were obtained with the cELISA and bELISA, while the lowest DPR was estimated using iELISAs. A latent class analysis was performed to estimate the accuracy of the CFT, RBT and bELISA using 1827 sera from sheep undergoing testing as part of a surveillance and control programme. Lower sensitivity and specificity were obtained for the three serological tests when the field samples were used. A higher DPR was achieved by the CFT, compared to bELISA and RBT. The results suggest that ELISAs, and particularly the bELISA, might be suitable for inclusion in the European Union legislation on intra-community trade for diagnosing B. melitensis infection in sheep, as it has a similar test performance compared to the RBT.     

Antibodies to the CP24 protein of Brucella melitensis lack diagnostic usefulness in ovine brucellosis

The potential diagnostic usefulness of antibodies to the ribosome recycling factor of Brucella melitensis (CP24) was assessed in sheep by an indirect ELISA with purified recombinant CP24. Sera from uninfected animals from the UK (n=44) and from local flocks (n=42), from sheep naturally infected with B. melitensis (n=12) or B. ovis (n=12), and from lambs (n=7) or pregnant ewes (n=6) vaccinated with B. melitensis Rev-1, were assayed. High specific optical densities (OD(with antigen) - OD(without antigen)) were obtained with both the groups of normal sera, which resulted in high cut-off values (1.414 and 1.267, respectively). Only two infected sheep yielded specific OD higher than these cut-off values. No significant difference was found between mean specific OD from B. melitensis- or B. ovis-infected sheep (0.574 and 0.472, respectively), those from vaccinated animals (0.396 and 0.400 for pregnant ewes and lambs, respectively), and those from Brucella-free animals. An inhibition ELISA with soluble CP24 confirmed the specificity of the antibodies detected in normal sera by the indirect ELISA; these antibodies belonged to the IgG class as revealed by the use of a specific conjugate. Sera from infected sheep were all positive for antibodies against lipopolysaccharides and lumazine synthase from Brucella. These results show that anti-CP24 antibodies have no diagnostic role in ovine brucellosis.     

Evaluation of a modified Rose Bengal test and an indirect enzyme-linked immunosorbent assay for the diagnosis of Brucella melitensis infection in sheep

A modified Rose Bengal test (mRB) and an indirect ELISA (iELISA) with Protein G as the conjugate, were evaluated for the diagnosis of Brucella melitensis infection in unvaccinated sheep with a known bacteriological status, and their diagnostic efficacy was compared with that of the standard Rose Bengal (RB) and Complement Fixation (CF) tests used in the current eradication campaign in EU countries. All tests showed 100% specificity when testing the sera from 212 Brucella-free sheep. When testing the sera from 219 Brucella melitensis culture-positive sheep, both the mRB and iELISA tests were more sensitive (98.6% and 96.8%, respectively) than the RB and CF tests (95.0% and 92.7%, respectively). These results were similar when testing the sera from 181 animals belonging to infected flocks but found bacteriologically negative, suggesting that the mRB or iELISA tests could advantageously replace the current RB procedure used as the screening test.     

Brucella melitensis infection in dog: a critical issue in the control of brucellosis in ruminant farms

Canine brucellosis is a contagious disease associated with health implications for humans as well as for a wide range of wild and domesticated animals. In this study, 173 dog blood specimens were sampled from herding dogs in three different provinces including Tehran (n = 127), Qom (n = 40) and Alborz (n = 6) provinces. The presence of Brucella antibodies was determined using Rose Bengal plate test (RBPT), slow agglutination test (SAT) and 2-mercaptoethanol (2-ME), respectively. The seropositive samples were further screened using blood culture and PCR tests to identify and differentiate the implicated Brucella species. According to our results, 24.3% (42/173), 13.8% (24/173) and 6.3% (11/173) of blood samples were tested positive using RBPT, SAT and 2-ME, respectively. However, among 42 seropositive samples, only 38.1% (16/42) and 14.2% (6/42) were positive by PCR and culture, respectively. Brucella melitensis biovar 1 and biovar 2 was isolated from the bacterial cultures of 6 blood samples and confirmed by biotyping, AMOS PCR and Bruce-ladder PCR assays. These findings highlight the potential risk of Brucella transmission from dog to humans along with other livestock and reflect the critical role of infected dogs in the persistence of Brucella infections among ruminant farms. This study stresses the need for further epidemiological investigations on canine brucellosis among herding dogs and suggests the systematic screening of the disease among companion animals such as dogs in order to improve brucellosis surveillance and control programs.     

Evaluation of a delayed-type hypersensitivity test for the diagnosis of Brucella abortus infection in cattle

The delayed-type hypersensitivity (DTH) test was used to diagnose brucellosis in two cows experimentally induced with brucellosis, and 176 dairy cows from a farm suspected of brucellosis. DTH test results were compared with results of the milk ring test, the serum agglutination test, the complement fixation test and the Coombs test. Cows positive in the DTH test and in one of the other tests were examined bacteriologically. In experimentally infected animals the DTH test was positive 10 days after infection, 1-4 weeks before serologic tests indicated brucellosis. Although the DTH test was positive during the whole experiment, on the one occasion when serologic titres were high, it was negative. Of the 176 dairy cows, 45 were positive in one or more serologic tests. In twelve cows (29%) the diagnosis was inconclusive because they were positive in only one of the serologic tests. In these cases the DTH test confirmed the infection. Three cows with high serologic response tested negative in the DTH test. B. abortus was isolated from 13 of 15 cows examined. We conclude that when serologic results are ambiguous, the DTH test is a useful additional technique for diagnosing brucellosis.     

Evaluation of a microplate agglutination test (MAT) for serological diagnosis of canine brucellosis

A microplate agglutination test (MAT) was compared with the tube agglutinin test (TAT), a standard test for the diagnosis of Brucella canis, in terms of the sensitivity and specificity. The results showed that MAT was more sensitive, simpler to perform and easier to read the results than TAT. On top of that the MAT allows us to handle a larger number of samples at once. Using this method we conducted sero-surveillance of the prevalence of B. canis in dogs kept in an Animal Shelter located in Kanagawa Prefecture. Twelve of 485 (2.5%) showed seropositive against B. canis. These results indicate that B. canis infection in dogs is still occurring in Japan.     

Validation of a competitive ELISA for diagnosis of Brucella melitensis infection in sheep and goats

A competitive enzyme-linked immunosorbent assay (cELISA) was validated for the serodiagnosis of Brucella melitensis infection in small ruminants using 2108 positive and 2154 negative reference sera from sheep and goats. The optimum cut-off values, offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp), determined by receiver operating characteristic analysis, were at 23.6%, 21.8% and 25.0% inhibition of the conjugate control for sheep, goats and both species, respectively. The DSns of the cELISA for sheep, goats and both species at these cut-off values were 89.2% (95% confidence interval 87.1-91.1%), 74.0% (95% CI 71.4-76.5%) and 77.9% (95% CI 76.1-79.7%), whereas DSps were 96.4% (95% CI 95.2-97.4%), 92.9% (95% CI 91.1-94.3%) and 97.2% (95% CI 96.4-97.8%), respectively. Compared to cELISA, indirect ELISA and fluorescence polarisation assay have higher DSns and DSps. However, the results obtained with the cELISA were in good agreement with those of the complement fixation test (CFT) under field conditions using 5735 sheep and goat sera. The cELISA can be used as an alternative to the CFT for diagnosing B. melitensis infection in small ruminants.     

Comparison of milk-ELISA and serum-ELISA for the diagnosis of Brucella melitensis infection in sheep

Milk and blood samples from 704 lactating ewes were examined for the diagnosis of Brucella melitensis infection by milk-ELISA, serum-ELISA, RBPT, SAT and culture of milk. Of these ewes, 209 were from brucellosis free sheep flock, 443 from brucellosis infected sheep flock and 52 were from private sheep flocks of which status for brucellosis was not known. All the 209 ewes belonging to uninfected sheep flock were found negative in all the tests and of the remaining 495 ewes 105 were positive in serum-ELISA, 103 in milk-ELISA, 92 in RBPT, 85 in SAT, and B. melitensis biovar-1 was isolated from the milk of 29 ewes. Of the 105 serum-ELISA positive ewes, 99 were positive and 6 were negative in milk-ELISA, whereas of the 103 milk-ELISA positive ewes, 4 were negative in serum-ELISA. All together, 99 ewes were positive and 386 were negative in both the assays while 10 ewes yielded variable results. The specificity of milk-ELISA in brucellosis free flock was 100% and sensitivity and positive predictive value were 96.11% and 94.28%, respectively, in infected flocks. The Brucella antibody levels in milk and serum samples as determined by milk-ELISA and serum-ELISA were correlated significantly. The milk-ELISA for brucellosis appears to be an attractive alternative of serum-ELISA particularly in the lactating ewes.     

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.