Pneumocystis spp. diversity and host associations in wild golden jackals, Eurasian badgers, red foxes, and nutrias from Serbia

Abstract The aim of this study was to genetically characterize the mtLSU rRNA, mtSSU rRNA, dhps , and the ribosomal RNA (rSSU – ITS1 – 5.8S – ITS2 – rLSU) genes of Pneumocystis species derived from Serbian golden jackals ( Canis aureus ), Eurasian badgers ( Meles meles ), red foxes ( Vulpes vulpes ), and nutrias ( Myocastor coypus ). Analysis of the mtLSU and the mtSSU rRNA genes confirmed the presence of host-specific Pneumocystis lineages for each species. In addition to these host-specific strains, two golden jackals were infected with P. canis and one red fox carried a lineage specific for golden jackals, supporting interspecies transmission among closely related mammalian hosts. Nutria-derived Pneumocystis species displayed single nucleotide polymorphisms (SNPs) and insertions associated with the emergence of distinct genetic variants. Dhps PCR amplification was successful only for nutria-derived Pneumocystis and revealed a single variant. Based on ribosomal RNA loci, two variants were identified in golden jackal-derived, and one variant each in badger-, red fox-, and nutria-derived Pneumocystis . In the phylogenetic analysis, lineages isolated from carnivores were closely related to P. canis CK1, raising questions about whether they should be classified as separate species in the future or instead regarded as P. canis strains. Nutria-derived Pneumocystis clustered not only with other rodent-derived Pneumocystis species but also with Pneumocystis from primates, lagomorphs, and carnivores. Their genetic relationships therefore require further clarification based on large-scale genomic data. Given their greater genetic divergence from other species, nutria-derived Pneumocystis may warrant recognition as separate species.