Development and evaluation of serodiagnostic assays with recombinant BgSA1 of Babesia gibsoni

Abstract

Indirect ELISA, dot-ELISA and double antibody sandwich ELISA (DAS-ELISA) using truncated recombinant BgSA1 (rBgSA1) were developed for detecting Babesia gibsoni infection in naturally infected dogs. Truncated BgSA1 gene of 858bp, encoding 32kDa protein was cloned in pET-32a(+) expression vector, expressed in Escherichia coli and the recombinant protein was purified under native conditions. To evaluate the ability of the truncated rBgSA1as serodiagnostic reagent for B. gibsoni infection, a panel of sera/plasma samples from dogs infected with B. gibsoni (n=13), uninfected sera (n=13) and sera from dogs infected with other haemoparasites namely, Babesia canis vogeli (n=3), Ehrlichia canis (n=3), Hepatozoon canis (n=1) and Dirofilaria immitis (n=1) were used. Besides these, 75 samples collected from dogs suspected for babesiosis were used to evaluate the performance of rBgSA1 based serological assays in comparison to nested PCR. Based on the results, the diagnostic sensitivity of indirect ELISA, dot-ELISA and DAS-ELISA were 97.3%, 91.9% and 100%, respectively, when nested PCR was taken as a reference test, while their specificities were 81.6%, 84.2% and 97.4%, respectively. Further, DAS-ELISA had a quantitation limit of 0.03μg/ml of the rBgSA1. High kappa values of indirect ELISA, dot-ELISA and DAS-ELISA were recorded, indicating that these assays had substantial to almost perfect agreement at 95% confidence level. There was no cross-reactivity with sera from dogs infected with B. canis vogeli, E. canis, H. canis and D. immitis. The results suggest that the indirect ELISA, dot-ELISA and DAS-ELISA with rBgSA1 may be used in large scale epidemiological surveys and clinical diagnosis of B. gibsoni infection in dogs. DAS-ELISA has advantages over indirect or dot-ELISA in the detection of current infection as well as monitoring the parasite burden.