Evaluation of recombinant BgSA3 protein based indirect-ELISA for sero-diagnosis and sero-surveillance of Babesia gibsoni in dogs

Abstract

Canine babesiosis a tick-borne haemoprotozoan disease of dogs is of significance globally due to its rapid spread. A precise confirmatory diagnosis is required to curtail the rapid spread of infection. Our study described the evaluation of recombinant BgSA3 protein based indirect ELISA for sero-diagnosis and sero-surveillance of Babesia gibsoni infection in dogs. A partial BgSA3 gene segment 1921 bp of B. gibsoni encoding for recombinant truncated BgSA3 75 k Da protein devoid of predicted signal peptide 23 aa at N-terminus and transmembrane region 20 aa at C-terminus, was expressed in E. coli using a pET28a positive vector. The rBgSA3 protein purified under native conditions using Ni-NTA super flow cartridge was confirmed by SDS-PAGE and Western blotting using sera from dogs infected/uninfected with B. gibsoni, and erythrocyte lysate/plasma from infected/uninfected dogs. The rBgSA3 protein was specific only to B. gibsoni antibodies but did not react with uninfected sera. Further, rBgSA3 protein was evaluated for sero-diagnosis/sero-surveillance using Indirect-ELISA format. There was no cross reactivity to B. vogeli, E. canis, H. canis and D. repens infected dogs serum samples. The diagnostic sensitivity and specificity of rBgSA3 based I-ELISA was found to be 86.4 and 93.1 % respectively, in comparison with cytb based PCR assay. Additionally, rBgSA3-ELISA evaluated using survey serum samples n =287, detected 11.85 percentage samples as positive. In conclusion, B. gibsoni infection, an emerging disease is prevalent in the present study area and the standardized rBgSA3 protein based indirect-ELISA was found to be a specific and sensitive test for large scale sero-diagnosis and sero-surveillance of B. gibsoni infection in dog.