Wildlife fleas and ticks in Wisconsin, USA: unrecognized vectors of bacterial pathogens

Small wildlife species host flea and tick species that can also infest or transmit pathogens to domestic animals and humans, including Anaplasma, Babesia, Bartonella, Borrelia, Ehrlichia, and Rickettsia species. Despite their zoonotic potential, little is known regarding the prevalence, diversity, and epidemiology of these pathogens. Therefore, we aimed to survey the ectoparasites found on Eastern Cottontail Rabbits (rabbits), Eastern Grey Squirrels (squirrels), and Virginia Opossums (opossums) in south-central Wisconsin, and describe the prevalence of select pathogens. Ectoparasites were opportunistically collected from small mammals, then identified to the species level, pooled, washed, and DNA extracted for quantitative PCR (qPCR) to detect Anaplasmataceae, Apicomplexa, Bartonella, hemotropic Mycoplasma, and Rickettsia. To analyze the genomic diversity of uncharacterized Bartonella, three flea pools were subject to metagenomic sequencing. Cediopsylla simplex and Haemaphysalis leporispalustris were the most common ectoparasites on rabbits, while Orchopeas howardi was most common on squirrels and opossums. Bartonella species were detected in C. simplex pools (n = 52), most commonly two distinct Bartonella alsatica-like bacteria (38 %; 20/52). Bartonella durdenii, definitively identified by metagenomic sequencing, was detected in 42 % (13/31) of O. howardi pools from squirrels. From metagenomic sequencing, B. alsatica-like species displayed a 4.8 % dissimilarity rate while B. durdenii displayed a 0.4 % dissimilarity rate. Sequencing of one B. alsatica-like flea pool also identified phage-associated genes not found in the B. alsatica genome. Rickettsia felis (n = 1) and opossum-associated hemotropic Mycoplasma sp. (n = 2) were detected in O. howardi from opossums. Rickettsia bellii and Anaplasma sp. were detected in Haemaphysalis leporispalustris from rabbits. These findings reinforce the value of metagenomic sequencing, facilitating the correct identification of B. durdenii and identifying genes not found in the type strain, specifically phage related genes. Due to the known zoonotic potential of B. alsatica, further examination of B. alsatica-like and B. durdenii pathogenicity is warranted.