SYBR Green based duplex real-time PCR assays for molecular detection of tick-borne haemoparasites of dogs from Northern India

Concurrent haemoparasitic tick-borne infections like babesiosis, ehrlichiosis and hepatozoonosis may occur in dogs following exposure to brown dog tick, Rhipicephalus sanguineus. In the present study, SYBR Green based duplex real-time PCR assays were evaluated to detect the infections with Babesia gibsoni, B. vogeli, Ehrlichia canis and Hepatozoon canis from selected north Indian states (Himachal Pradesh, Punjab, Uttarakhand and Uttar Pradesh) and union territories (Jammu and Kashmir and New Delhi). Evaluation of 343 blood samples by microscopy revealed the incidence of B. gibsoni, B. vogeli, E. canis and H. canis as 4.4%, 0.3%, 0.3% and 0.6%, respectively. However, the incidence rates for the respective parasites by duplex real-time PCR assays were 17.2%, 0.6%, 0.6% and 17.8%, along with co-infections infections of B. gibsoni and H. canis (4.4%), B. gibsoni and B. vogeli (0.3%) and B. gibsoni, B. vogeli and H. canis (0.3%). The diagnostic sensitivity and specificity of duplex real-time PCR assays, as compared to microscopy, for detection of B. gibsoni, B. vogeli, E. canis and H. canis ranged as 2.5%-100.0% and 78.26%-99.99%, respectively. The strength of agreement between the two tests for detection of these parasites ranged from ″slight″ to ″substantial″ by kappa value statistics and the data was statistically significant. A statistically significant association was recorded by duplex real-time PCR assays for incidence of B. gibsoni (age and location) and H. canis (age, location and season) as associated risk factors. These in-house validated PCR assays, after wider studies and validation from other regions of India, offer a great potential for detection and monitoring the response of treatment therapies for these haemoparasites under field conditions.