Development of Highly Efficient Universal Pneumocystis Primers and Their Application in Investigating the Prevalence and Genetic Diversity of Pneumocystis in Wild Hares and Rabbits

Despite its ubiquitous infectivity to mammals with strong host specificity, our current knowledge about has originated from studies of merely 4% of extant mammalian species. Further studies of 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 primers targeting different genetic loci with high amplification efficiency. Application of these primers to PCR investigation of in free-living hares ( , = 130) and rabbits ( , = 8) in Canada revealed a prevalence of 81% (105/130) and 25% (2/8), respectively. Genotyping analysis identified five and two variants of 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, in hares and rabbits are more closely related to each other than either are to in other mammals. Furthermore, in both hares and rabbits are more closely related to in primates and dogs than to in rodents. The high prevalence of in hares ( sp. ' ') suggests its widespread transmissibility in the natural environment, similar to in rabbits. The presence of multiple distinct populations in hares contrasts with the lack of apparent intra-species heterogeneity in , implying a unique evolution history of sp. ' ' in hares.