Analysis of Genetic Diversity of cytb gene from Babesia gibsoni Isolates from Naturally Infected Dogs in Karnataka, India.

PURPOSE: The study aimed to investigate genetic diversity in Babesia gibsoni, the causative agent of canine babesiosis, and to assess the presence of atovaquone-resistant isolates in naturally infected dogs. METHODS: A total of 24 blood samples confirmed for B. gibsoni infection was subjected to PCR amplification and sequencing based on cytb gene. Genetic characterization of B. gibsoni as well as attempts to detect the point mutation rendering atovaquone resistance was carried out based on the analysis of nucleotide sequence of cytb gene using bioinformatics software. RESULTS: The findings indicated that the B. gibsoni isolates in the investigation exhibited a high nucleotide identity with the Asian genotype, ranging from 98.41 to 98.69%. Notably, none of the isolates carried cytb gene variants associated with atovaquone resistance. Phylogenetic analysis revealed clustering of most isolates with those from Japan and China, except for one isolate forming a distinct subclade. Haplotype network analysis indicated a high diversity with 22 distinct haplotypes among the B. gibsoni isolates, emphasizing the genetic variability within the studied population. CONCLUSION: In conclusion, the cytb gene exhibited remarkable conservation among the twenty-four B. gibsoni isolates studied and the study represents the first genetic diversity assessment of B. gibsoni using the cytb gene in dogs from India. These findings shed light on the genetic characteristics of B. gibsoni in the region and provide valuable insight for addressing the challenges posed by this life-threatening disease in dogs.

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

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 kDa) 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(+) vector. The rBgSA3 protein purified under native conditions using Ni-NTA superflow 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 % 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 dogs.