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Neospora caninum is a protozoan parasite that causes abortion and important economic losses in cattle worldwide. There are no treatments or vaccines available; disease control is based on diagnosis and herd management strategies. We developed, validated, and evaluated under field conditions a competitive inhibition ELISA based on the truncated SAG1 protein (tSAG1), expressed in Escherichia coli , and the RafNeo5 monoclonal antibody (ciELISA tSAG1 ). A criterion based on the 3-y sequential serologic analysis of 230 dairy cows by IFAT was used as the gold standard. The assay was validated using 860 serum samples from cows that were consistently positive or negative by IFAT throughout the study period. ciELISA tSAG1 was then used to evaluate the prevalence of neosporosis in 16 beef cow herds (22 samples per herd, 352 total samples). The results were compared with those from IFAT and a commercial cELISA (cELISA VMRD ). The ciELISA tSAG1 cutoff was ≥ 29%I, with a diagnostic sensitivity of 98.7% (95% CI = 96.8-99.7%) and a diagnostic specificity of 97.9% (95% CI = 96.4-99.0%). Concordance among IFAT, cELISA VMRD , and ciELISA tSAG1 was 90.3%. The agreement (κ) between ciELISA tSAG1 and the other 2 tests was ≥ 0.81. The overall prevalence of neosporosis in the 16 beef herds was 30% (range: 5-60%). The ciELISA tSAG1 could be useful for large-scale detection of anti- N. caninum antibodies in cattle and seroepidemiologic investigations, given its appropriate sensitivity and specificity, and the simplicity of production.

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.