Canine babesiosis among working dogs of organised kennels in India: A comprehensive haematological, biochemical, clinicopathological and molecular epidemiological multiregional study.

Canine babesiosis is a serious disease among tick-borne haemoprotozoan diseases, globally. The present study was envisaged for carrying out thorough investigation of the disease among working dogs of organised kennels situated in different agro-climatic zones of India as comprehensive understanding of the disease from this country was pertinently lacking. During the study period of three years (2012-2014), 330 dogs suspected for babesiosis were examined for clinicopathology by their physical examination, haematological and biochemical parameters estimation, while the detection of apicomplexan parasites was confirmed by using various diagnostic techniques i.e. by conventional microscopy, by two different Babesia specific 18S rRNA based PCR protocols (conventional/simple PCR and nested PCR assays) followed by sequencing of obtained PCR amplicons for Babsesia spp. identification. Out of 330 clinical cases screened 5.15% (17/330), 9.09% (30/330) and 15.45% (51/330) were found to be positive in microscopic examination, simple- and nested- PCR assay, respectively. Comparative statistical analyses of these diagnostic assay results revealed that significant difference exists among the three diagnostic methodologies and thus it is recommended that the nested PCR technique be relied upon as a screening molecular assay and also for epidemiological studies of the disease in this country. Phylogenetic analysis based on 18S rRNA depicted the monophyletic nature and clonal expansion among all the B. gibsoni, under study. Sequencing results of PCR amplicons revealed that B. gibsoni has predominantly established itself over B. vogeli as former was incriminated in 47 cases while latter was confirmed in only four animals. Based on the clinical severity, these 51 affected animals were classified into three main groups' of 17 animals each viz., apparently healthy-, simple or uncomplicated babesiosis- and atypical or complicated babesiosis- group. Haematological and biochemical profiling of these dogs confirmed the characteristics findings of infection by both the Babesia spp. It was observed that the infection by small form of Babesia (B. gibsoni) is posing a significant therapeutic challenge and chemosterilization by commonly prescribed anti-protozoal drugs was not achieved as clinical relapses were often observed. The clinical signs, sequence based confirmation and severity of the infection suggested that there is a positive selection of B. gibsoni (smaller form) over B. vogeli (larger form) in this country and raises serious concerns as prognosis in former is considered to be poor compared to latter. Thus, these findings have opened new paradigms for planning of pragmatic control strategies against this emerging canine health problem.