Development of hexaplex PCR assay for the detection and characterization common tick-borne pathogens in dogs, and analysis of risk factors

• The developed hexaplex PCR assay using internal amplification control (IAC) successfully detected the five major canine tick-borne hemoparasites. • The analytical sensitivity of multiplex PCR assay was recorded to be 0.1 pg for detection of A. platys, H. canis and B. vogeli, and 10 pg for E. canis and B. gibsoni. • The strength of agreement for diagnostic sensitivity and specificity of multiplex PCR assay (n=61) for detection of A. platys, B. gibsoni, E. canis, H. canis and B. vogeli revealed to be ‘very good’ taking singleplex PCR as standard test. • The mPCR survey recorded 31.56% prevalence of tick-borne haemoparasitic infections in dogs with history/presence of tick infestation in 8 selected districts of Odisha (n=320). The present investigation was intended to develop and validate an internally controlled hexaplex PCR assay for concurrent detection and differentiation of the five major canine tick-borne pathogens (TBP) namely, Ehrlichia canis, Babesia gibsoni, Babesia vogeli, Hepatozoon canis and Anaplasma platy s in 8 selected districts of Odisha, India. The assay generated amplicons of 817 bp, 737 bp, 602 bp, 489 bp and 292 bp corresponding to E. canis (16S rRNA gene) , B. vogeli, B. gibsoni, H. canis (18S rRNA gene) and A. platy s (HSP GroEL gene), along with amplicon of 218 bp targeting canine β-actin gene as internal amplification control (IAC) to avoid false negative result. The kappa values for diagnostic specificity and sensitivity of multiplex PCR assay in detection of the pathogens revealed very good agreement with their singleplex PCR counterparts (n=61). The analytical sensitivity of multiplex PCR assay was recorded to be 0.1 pg for detection of A. platys, H. canis and B. vogeli, and 10 pg for E. canis and B. gibsoni . The epidemiological survey (n=320) revealed 31.56% of TBP infection and incidence of H. canis, B. gibsoni were high in summer season and A. platys in rainy. Co-infections exacerbated clinical affections. The generated sequence of the Odisha isolates of these pathogens in this study revealed 98-100% homology with various global isolates of these pathogens. The standardized multiplex PCR developed in the present study, can be used as a rapid, economical and sensitive diagnostic tool for identifying tick-borne haemoparasites in dogs.