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The serological response induced by Brucella abortus strain 19 was evaluated in 52 Holstein females from a brucellosis-free herd using seven serological tests. Each calf was vaccinated at an age of 4 and 8 months old with 3 x 10(10) CFU B. abortus S19 and the antibody response was determined as the proportion of positive results to each test. The antibody dynamics, measured with the buffered plate antigen (BPA) test and the rapid automated presumptive (RAP) test, were similar. The proportion of positive reactions in these tests reached 100% one week after vaccination and remained at this level for seven weeks, after which the proportion of positive samples slowly declined to 8% (BPA) and 2% (RAP) at week 50. The response in the indirect enzyme immunoassay (i-ELISA) was similar, but shorter than that observed with the BPA/RAP. The antibody dynamic, measured using the seroagglutination test (SAT) in parallel with the 2-mercaptoethanol (2-Me) test and the complement fixation test (CFT) were similar to the RAP/BPA, but of slightly shorter duration. The competitive ELISA (c-ELISA) was positive in all animals for 3 weeks, followed by a rapid decline. The fluorescence polarization assay (FPA) reached a maximum of 68.5% positive animals at week 4 and then declined. Based on these data, the c-ELISA and FPA discriminated residual antibody activity due to vaccination more efficiently than the other tests.

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Detection of antibodies to Anaplasma spp. using commercial competitive enzyme-linked immunosorbent assay (ccELISA) is based on the recombinant major surface protein 5 fused to maltose binding protein (MBP-MSP5) or glutathione S-transferase (GST-MSP5). To avoid false positive reactions due to the presence of antibodies against E. coli MBP in cattle, previous sera absorption is required. This study evaluated the replacement of MBP-MSP5 or GST-MSP5 antigens by the truncate MSP5 (residues 28–210) of A. marginale (tMSP5m), A. centrale (tMSP5c) and fusion protein MSP5 (tMSP5cm), expressed without N-terminus transmembrane helix in the ccELISA test. Immunoreactivity was evaluated by western blot using monoclonal antibodies against the tMSP5 and by in-house cELISA (hcELISA) with purified tMSP5m, tMSP5c or tMSP5cm using sera from cattle infected with A. marginale (n = 226) or vaccinated with A. centrale (n = 173) and uninfected cattle (n = 216). Results of hcELISA were compared with those of ccELISA. Recombinant protein was expressed highly soluble (> 95%) in E. coli without a molecular chaperone. Specificity of the hcELISA-tMSP5m, -MSP5c or -tMSP5cm was identical to (99.5%) and greater than that in ccELISA (96.3%). Sensitivity of hcELISA-tMSP5m and ccELISA was identical (95.5%), but lower than that of hcELISA-tMSP5cm (96.2%) and -tMSP5c (97.2%). The analysis of vaccinated cattle by hcELISA-tMSP5c showed sensitivity of 99.4%. In summary, the generation of fusion MSP5 A. marginale-A. centrale protein without transmembrane helix was a very effective method to express the recombinant protein highly soluble in the bacterial cytoplasm and contributed to an increased test performance for detecting antibodies in cattle naturally infected with A. marginale or vaccinated with A. centrale.

Bovine anaplasmosis is a worldwide infectious disease caused by the intraerythrocytic bacterium Anaplasma marginale, which is transmitted by ticks and fomites. A. centrale is a less virulent subspecies used as a live vaccine in cohorts of 8- to 10-mo-old calves that did not naturally reach enzootic stability. We developed 3 variants of a double-antigen sandwich ELISA (dasELISA) using a recombinant major surface protein 5 (MSP5) from A. marginale (dasELISAm) or from A. centrale (dasELISAc) or using MSP5 from both organisms (dasELISAmc). Each dasELISA was tested for the detection of antibodies against A. marginale and A. centrale. The tests were validated using serum samples from cattle not infected with Anaplasma spp. ( n = 388), infected with A. marginale ( n = 436), and vaccinated with A. centrale ( n = 358), confirmed by nested PCR. A total of 462 samples were compared with a commercial competitive ELISA (cELISA). For dasELISAm, dasELISAc, and dasELISAmc, specificities were 98.7%, 98.7%, and 97.4%, and overall sensitivities were 92.6%, 85.7%, and 97.4%, respectively. For A. marginale–infected and A. centrale–vaccinated cattle, sensitivities were 97.7% and 86.3% for dasELISAm, and 77.7% and 95.5% for dasELISAc, respectively. Sensitivity of dasELISAmc was similar for both groups (>96%). The agreement rate between dasELISAmc and cELISA was 96.3% (κ = 0.92); the former test allowed earlier detection of seroconversion of vaccinated cattle than did cELISA. Based on these results, the test could be used to 1) determine the enzootic stability or instability of anaplasmosis in calves, 2) conduct epidemiologic studies, and 3) evaluate the immunogenicity of A. centrale live vaccine.