Identification of Ctenocephalides felis felis and the detection and characterization of associated Wolbachia endosymbiont in Pakistan.

Fleas are holometabolous, blood-feeding ectoparasites capable of transmitting diverse pathogens of significant veterinary and public health concerns. Their occurrence and abundance within a given habitat depend on environmental factors and the availability of suitable hosts. In Pakistan, research on flea fauna and their associated pathogens has been neglected. To date, no reports have documented the molecular characterization of Ctenocephalides felis felis and their associated bacteria in Pakistan. In the present study, three hundred and eighty morphologically identified C. felis felis specimens-comprising 184 collected from free roaming (stray) dogs (n = 69) and 196 from cats (n = 86), were subjected to DNA extraction followed by amplification of the cytochrome oxidase subunit 1 gene (cox1) and citrate synthase gene (gltA) for flea identity confirmation and screening for the presence of associated bacteria, respectively. Amplicons of appropriate base pair sizes were sequenced and submitted to BLASTn and subsequently subjected to phylogenetic analyses. The obtained cox1 sequence from the morphologically identified C. felis felis in this study showed 100% identity and phylogenetically clustered with C. felis felis sequences from India, Australia, Thailand, China, and Laos in GenBank. Similarly, gltA sequences showed 100% identity with the Wolbachia endosymbiont of C. felis reported from the United Kingdom. This study provides the first genetic characterization of C. felis felis infesting dogs and cats, and their associated Wolbachia endosymbiont in Khyber Pakhtunkhwa (KP), Pakistan. These findings provide baseline molecular data and highlight the need for systematic surveillance and management measures to mitigate any potential veterinary and public health threats.