Comparison of IgM and IgG ELISAs using recombinant thrombospondin-related adhesive protein (BgTRAP) for the differentiation of early and late Babesia gibsoni infections in canines.

Abstract

Vector-borne illnesses pose a rising global threat to pet health, with Babesia gibsoni being the most common haemoprotozoan infection in canines of South India. Low parasitaemia either in early or late infection is a big hurdle in the diagnosis of canine babesiosis. The present study aimed to differentiate the early and late B. gibsoni infections in dogs using IgM/IgG ELISA based on recombinant thrombospondin-related adhesive protein (BgTRAP). The N-terminal BgTRAP gene was cloned into pET32a, expressed in BL21 Escherichia coli cells, and purified to get the recombinant protein. Using the recombinant antigen, IgM ELISA detected anti-B. gibsoni IgM antibodies in 73 out of 130 samples (56.15 per cent) while the IgG ELISA detected IgG antibodies in 77 out of 130 samples (59.23 per cent). Polymerase chain reaction of the 130 samples targeting the BgTRAP gene revealed 73.07 % positivity. When compared with the PCR, the sensitivity and a specificity of newly standardized indirect IgM ELISA were 36.54 % and 75.00 % while that of IgG ELISA were 42.31 % and 71.88 %. The accuracy, positive predictive value and negative predictive value of the IgM ELISA in comparison to PCR were 51.19 %, 70.4 %, 42.1 % while that of IgG ELISA were 53.57 %, 71.0 %, 43.4 % respectively. The area under the curve (AUC) and Youden Index in ROC curve for IgM and IgG ELISAs revealed a moderate diagnostic accuracy. The rBgTRAP antigen showed no cross-reactivity with common helminth parasites viz, Ancylostoma caninum, Dirofilaria immitis, D. repens, Spirometra spp., Toxocara canis and haemoparasites like Trypanosoma evansi, B.vogeli, Hepatozoon canis and Ehrlichia canis. The assay could clearly differentiate early (IgM) and late (IgG) infections, making it a valuable diagnostic tool for differentiating early and late infections with B. gibsoni.