Comparative diagnostic performance of microscopy and PCR assays with preliminary mitochondrial sequence analysis of Babesia species infecting dogs in Jabalpur, central India.

Abstract

BACKGROUND: The present study evaluated the diagnostic performance of conventional microscopy and polymerase chain reaction (PCR) based assays for the detection of Babesia infections in dogs, including semi-nested PCR (SN-PCR) targeting the 18&#xa0;S rRNA gene and single-round PCR (SR-PCR) assays targeting the mitochondrial cytochrome b (cytb) and cytochrome c oxidase subunit 1 (cox1) genes for B. gibsoni and B. vogeli, respectively. Mitochondrial sequence variation was further assessed by integrating newly generated sequences from Jabalpur, Madhya Pradesh (central India), with global reference datasets. METHODS AND RESULTS: A total of 100 blood samples from dogs suspected of having haemoprotozoan infections were analysed between June 2022 and May 2023. Microscopic examination of Giemsa-stained smears detected Babesia parasites in 13% of the samples, whereas the 18&#xa0;S rRNA SN-PCR assay identified infections in 29%, comprising B. gibsoni (25%) and B. vogeli (4%). Representative sequences showed 98-99% identity with corresponding GenBank reference sequences. Representative sequences showed 98-99% identity with corresponding GenBank reference sequences. Compared with SN-PCR, microscopy demonstrated moderate sensitivity but perfect specificity, resulting in an overall diagnostic accuracy of 84.0% (p&#x2009;<&#x2009;0.01). Mitochondrial SR-PCR assays detected B. gibsoni and B. vogeli in 5% and 4% of the samples, respectively. The cytb-based assay showed higher sensitivity and a significant diagnostic association (p&#x2009;<&#x2009;0.01) than the cox1 assay, whereas the cox1 assay demonstrated lower sensitivity with a non-significant association (p&#x2009;>&#x2009;0.05). All PCR assays showed 100% specificity and positive predictive value. Bayesian phylogenetic and haplotype analyses indicated that B. gibsoni cytb sequences formed a monophyletic lineage with limited regional structuring, with Indian isolates clustering within a distinct sub-lineage. In contrast, B. vogeli cox1 sequences exhibited low global diversity with a dominant shared haplotype across geographic regions. CONCLUSIONS: The 18S rRNA SN-PCR assay showed the highest sensitive method for detecting Babesia infections in dogs. Mitochondrial markers (cytb and cox1) supported species confirmation and comparative phylogenetic assessment, highlighting the complementary value of nuclear and mitochondrial gene targets for molecular surveillance and control of canine babesiosis in India.