tick borne haemoparasites and haemorickettsiales pose a major health risk to animals worldwide. the present study reports the development and validation of multiplex PCr to simultaneously detect the most prevalent tick borne pathogens infecting dogs in kerala, South India. the assay targeting the small subunit ribosomal rNa genes of the organisms could amplify well demarcated amplicons of B. canis vogeli, B. gibsoni and E. canis. In the study population, which included both healthy dogs as well as those with clinical symptoms suggestive of the three infections under study, 46.6% animals were infected with one of the three pathogens, amongst which the occurrence of B. gibsoni was significantly the highest. Natural co-infections were also detected in nine dogs, which suggests the suitability of the assay to assist in the selection of pathogen specific treatment protocols.

DNA was extracted from tick pools by salting out method [14]. Real-time PCR protocol targeting the small subunit ribosomal RNA gene was standardized separately for B. vogeli and B. gibsoni using a gradient thermal cycling program in an Eco™ Real-Time PCR System (Illumina).

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