Molecular detection and characterization of Bartonella spp. and Rickettsia spp. in Ctenocephalides felis fleas collected from dogs in Northeast Brazil

Abstract

The flea Ctenocephalides felis felis is a competent vector for several bacterial pathogens, such as Bartonella henselae, the main causative agent of cat scratch disease (CSD), and Rickettsia felis and Rickettsia asembonensis, causative agents of acute febrile rickettsial diseases. Despite their wide worldwide distribution and common parasitism in domestic dogs, studies on the occurrence and genetic diversity of zoonotic bacterial pathogens, especially Bartonella spp. and Rickettsia spp. in fleas are scarce in Brazil. This study aims to investigate the molecular occurrence and genetic diversity of Bartonella spp. and Rickettsia spp. in C. felis fleas collected from dogs in the state of Bahia, northeastern Brazil. To this end, approximately 400 fleas will be collected from dogs in the cities of Ilhéus and Itabuna. The siphonapter specimens will be identified morphologically, individually submitted to DNA extraction and, initially, to conventional PCR based on the cox-1 gene as an endogenous control. Samples positive in this PCR will be subjected to quantitative real-time PCR for Bartonella spp. based on the nuoG gene, and for Rickettsia spp. based on the gltA gene. For molecular characterization purposes, samples positive in the qPCR for Bartonella spp. will be submitted to additional PCR assays based on the gltA, ftsZ, groEL, nuoG, pap-31, rpoB, ribC and 16-23S rRNA intergenic region (ITS) genes; those positive in the qPCR for Rickettsia spp. will be submitted to PCR assays based on the gltA, htrA, ompA, ompB, sca5 and sca4 genes. The amplicons will be purified and sequenced using the Sanger method. The phylogenetic positioning of the sequences obtained will be carried out by constructing dendrograms. This study will contribute to knowledge about the genetic diversity of Bartonellaceae and Rickettsiaceae agents circulating in C. felis fleas from dogs in Brazil, contributing to the understanding of possible transmission cycles of zoonotic bacterial pathogens between fleas and human and animal hosts.