Ma, Liang (National Institutes of Health Clinical Center) Liang Ma Kovacs, Joseph A. (National Institutes of Health Clinical Center) Joseph A. Kovacs Rothenburger, Jamie L. (University of Calgary) Jamie L. Rothenburger Cisse, Ousmane H. (National Institutes of Health Clinical Center) Ousmane H. Cisse Weissenbacher-Lang, Christiane (University of Veterinary Medicine Vienna) Christiane Weissenbacher-Lang Danesi, Patrizia (Istituto Zooprofilattico delle Venezie) Patrizia Danesi Blasi, Barbara (University of Veterinary Medicine Vienna) Barbara Blasi Lin, Isabella (National Institutes of Health Clinical Center) Isabella Lin Hunter, Summer T. (University of Calgary) Summer T. Hunter MDPI 2024 Despite its ubiquitous infectivity to mammals with strong host specificity, our current knowledge about Pneumocystis has originated from studies of merely 4% of extant mammalian species. Further studies of Pneumocystis epidemiology across a broader range of animal species require the use of assays with high sensitivity and specificity. To this end, we have developed multiple universal Pneumocystis primers targeting different genetic loci with high amplification efficiency. Application of these primers to PCR investigation of Pneumocystis in free-living hares (Lepus townsendii, n = 130) and rabbits (Oryctolagus cuniculus, n = 8) in Canada revealed a prevalence of 81% (105/130) and 25% (2/8), respectively. Genotyping analysis identified five and two variants of Pneumocystis from hares and rabbits, respectively, with significant sequence divergence between the variants from hares. Based on phylogenetic analysis using nearly full-length sequences of the mitochondrial genome, nuclear rRNA operon and dihydropteroate synthase gene for the two most common variants, Pneumocystis in hares and rabbits are more closely related to each other than either are to Pneumocystis in other mammals. Furthermore, Pneumocystis in both hares and rabbits are more closely related to Pneumocystis in primates and dogs than to Pneumocystis in rodents. The high prevalence of Pneumocystis in hares (P. sp. 'townsendii') suggests its widespread transmissibility in the natural environment, similar to P. oryctolagi in rabbits. The presence of multiple distinct Pneumocystis populations in hares contrasts with the lack of apparent intra-species heterogeneity in P. oryctolagi, implying a unique evolution history of P. sp. 'townsendii' in hares. PDFDocument en 2024-06-10T08:57:54.121Z Serial-Thin Sections; 3-Dimensional Reconstruction; Dihydropteroate Synthase; Dihydrofolate-Reductase; Mitochondrial Genomes; Phylogenetic Analysis; Carinii Infection; Dna; Rats; Transmission 1853218 b application/pdf CC BY 4.0 International