First highly sensitive and specific competitive ELISA for detection of bovine besnoitiosis with potential as a multi-species test

Serological cross-reactions represent a serious problem in some currently available tests to diagnose Besnoitia infections in many species including cattle, caribou and donkeys. False-positive results are due to the low positive-predictive value (PPV) of these serological tests for besnoitiosis. These tests therefore have clear limitations if large herds are screened in areas with low prevalence, since increased numbers of false-positive reactions require confirmatory testing by alternative serological methods, e.g. immunoblotting, which are time-consuming and create extra costs. To overcome this problem, we aimed to develop a highly sensitive and specific competitive ELISA (cELISA) using a panel of 12 monoclonal antibodies raised against the tachyzoite stage of Besnoitia besnoiti. A cELISA set up with one of these antibodies (Bb-cELISA1) was screened with a large panel of B. besnoiti-positive bovine sera to estimate the diagnostic sensitivity of the test. Sera from herds with Neospora caninum- or Sarcocystis spp.-infected cattle were used to estimate its diagnostic specificity. Relative to a reference standard, which combined the results obtained in a previously established highly sensitive and specific ELISA, in the immunofluorescence antibody test and in B. besnoiti tachyzoite and bradyzoite immunoblots, the new Bb-cELISA1 revealed a diagnostic sensitivity of 99.2% (95% confidence interval: 97.1 - 99.9%) and a diagnostic specificity of 99.9% (95% confidence interval: 97.7 - 100%). This novel assay was tested on a variety of proven Besnoitia-positive sera from other species, including B. besnoiti-infected cats, rabbits or Besnoitia bennetti-infected donkeys or Besnoitia tarandi-infected caribou. The results obtained with the new Besnoitia-cELISA for these animal species also corresponded almost perfectly with those of the reference tests, which included immunoblot and immunofluorescence antibody tests. In conclusion, the novel Besnoitia-cELISA represents a valuable tool for the diagnosis and control of bovine besnoitiosis and for studies on the epidemiology of Besnoitia infections in a variety of host species, including naturally exposed wildlife and experimental hosts.