Detection of tick-transmitted haemoparasites in naturally infected dogs of northern India using a multiplex PCR Assay

An in-house developed and optimized multiplex PCR (mPCR) assay was used to simultaneously detect tick-transmitted haemoparasites in dogs, including Babesia gibsoni, Babesia vogeli, Ehrlichia canis, and Hepatozoon canis. The assay was applied to 194 blood samples collected from various states and union territories in northern India. The target genes selected for the assay were 18S rRNA for Babesia species and H. canis, and VirB9 for E. canis. Canine actin was included as an internal control to enhance the diagnostic value of the test. The amplification resulted in specific products of 182 bp (B. gibsoni), 131 bp (B. vogeli), 268 bp (E. canis), and 407 bp (H. canis). The prevalence of haemoparasites detected by mPCR was 6.7% for B. gibsoni, 1.5% for H. canis, and 1.0% for B. vogeli, whereas microscopy detected 3.1% for B. gibsoni and 0.5% each for H. canis and B. vogeli. A significant variation (p < 0.05) was observed between the two tests in terms of the occurrence of babesiosis and hepatozoonosis. Comparative analysis revealed a sensitivity of 100% (95% CI: 2.50-100%) and specificity of 99.48% (95% CI: 92.48-99.99%). The kappa value statistics showed moderate to substantial agreement between the two tests. Except for season (for B. gibsoni), the risk factors were not significantly associated with the mPCR assay.