Development of recombinant BgP12 based enzyme linked immunosorbent assays for serodiagnosis of Babesia gibsoni infection in dogs

Indirect ELISA and dot-ELISA using recombinant BgP12 (rBgP12) were developed for the diagnosis of Babesia gibsoni infected dogs. The complete open reading frame of BgP12 gene (378 bp) was cloned in pET-32a(+) expression vector and expressed in Escherichia coli as a soluble thioredoxin (Trx) fusion protein. The purified rBgP12 was used for production of anti-rBgP12 rabbit serum, which recognized a native 12-kDa protein in B. gibsoni infected erythrocyte by Western blot analysis. To evaluate the potential of rBgP12 for the serodiagnosis of B. gibsoni, a panel of serum/plasma samples from dogs infected with B. gibsoni (n = 13), uninfected sera (n = 13) and sera from dogs infected with other haemoparasites viz., Babesia canis vogeli (n = 3), Ehrlichia canis (n = 3), Hepatozoon canis (n = 1) and Dirofilaria immitis (n = 1) were used in ELISA formats. In addition, the performance of rBgP12 based indirect ELISA and dot-ELISA were evaluated using 75 serum/plasma samples collected from suspected dogs, in respect to the nested PCR as reference test. The diagnostic sensitivities of indirect ELISA and dot-ELISA were 94.59% and 89.18%, respectively, while their specificities were 84.21% and 81.57%, respectively. Moreover, both the assays using rBgP12 showed no cross reaction with sera from dogs infected with other common haemoparasites indicating their high specificity. High kappa values of indirect ELISA and dot-ELISA indicated the potentials of these assays with substantial agreement at 95% confidence level. It is concluded that indirect ELISA and dot ELISA using rBgP12 might be used in large scale epidemiological surveys and clinical diagnosis of B. gibsoni infection in dogs.