Evaluation of Ctenocephalides felis felis fleas (Bouché, 1835) (Siphonaptera: Pulicidae) to infection by Bartonella henselae using transcriptomic and microbiome analysis

Abstract

Ctenocephalides felis felis (Bouché, 1835) fleas are important ectoparasites of dogs and cats worldwide and competent vectors for zoonotic species of Bartonella (B. henselae, B. clarridgeiae, B. koehlerae) and Rickettsia (R. typhi and R. felis). Bartonella henselae is the most pathogenic species of the genus Bartonella for humans and it is the main etiological agent of Cat Scratch Disease (CSD). Prevention of B. henselae transmission is currently based on controlling flea infestation in cats through the use of ectoparasiticides, for which reduced efficacy and resistance have been observed. Environmental friendly alternatives, such as vaccination against both flea infestation and for blocking the transmission of B. henselae by siphonapterans, have been gaining attention. However, the understanding of the interaction between B. henselae and the gut of fleas is still limited. The present study aims to investigate the interaction between B. henselae and the digestive tract of the flea C. felis through "-OMICS" approaches, in order to identify the expression of genes in flea gut against infection by B. henselae through transcriptomic analysis, as well as the possible alteration of the microbial community of the flea gut when infected by B. henselae. For this purpose, six domestic cats will be experimentally infected with B. henselae (Jaboticabal sample) and two cats will be inoculated with sterilized saline solution. Once peak bacteremia is reached, the animals will be infested with C. felis fleas which will feed for 24 hours or nine days. Subsequently, the fleas will be removed, their guts dissected and subjected to DNA and RNA extraction for transcriptomic and microbiome analysis. DNA samples extracted from fleas will be subjected to amplification of the V4-V6 region of the 16S rRNA gene and the constructed libraries to sequencing via Illumina. RNA samples extracted from the flea guts will be submitted to the RNA-Seq technique and the results of the transcriptomic analysis will be validated by RT-qPCR. Five treatments will be proposed for transcriptomics and microbiome analyses: non-fed fleas; fed for 24 hours in B. henselae negative cats; fed for nine days on B. henselae negative cats; fed for 24 hours in cats experimentally infected with B. henselae; and fed for nine days to cats experimentally infected with B. henselae. The present study will enable the identification of genes related to C. felis gut that are overexpressed in the face of infection by B. henselae, as well as provide information about changes in the gut microbiome of fleas in the face of infection by B. henselae. The results obtained will open doors for future vaccine development studies both to control the infestation by C. felis fleas and to block the transmission of B. henselae. (AU)