Molecular evidence of polymorphism in Pneumocystis from wild mice and shrews in the eastern Alps, Italy

INTRODUCTION: Pneumocystis organisms are considered as major opportunistic fungal pathogens that infect humans and a wide range of other mammalian host species. The genus comprises uncultured, host-specific parasites adapted to the alveolar epithelial cells type I where they may proliferate provoking severe pneumonitis. Thus far, five species have been described: P. carinii and P. wakefieldiae in brown rats, P. jirovecii in humans, P. murina in common mice and P. oryctolagi in rabbits. In the present study, we detected and characterised Pneumocystis organisms in wild mice and shrews from eastern Alpes. Our objective was to assess whether Pneumocystis organisms could be used as phylogeographic markers for these mammalian species. MATERIALS AND METHODS: A total of 96 lung tissue samples from wild rodents and insectivores were analyzed. Specimens of Apodemus flavicollis (n=30), Apodemus sp (n=1), Myodes glareolus (n=3), Sorex antinorii (n=61), and Sorex sp (n=1) were captured as part of an large collaborative European project (EDENext) by Fondazione Edmund Mach. After DNA extraction, samples were analyzed by nested PCR, using primers for the gene encoding the mitochondrial large subunit rRNA (mtLSU rRNA) (Derouiche et al. 2009) and sequenced. The mtLSU sequences were aligned with already known Pneumocystis sequences using Clustal X (version 1.63b) and a phylogenetic analysis was performed using Neighbour Joining integrated in Mega 4 software (version 4.0). RESULTS: Pneumocystis DNA was detected in 58 of 96 samples (60%). Lung tissues from Apodemus flavicollis (12/30; 40%), Sorex antinorii (44/61, 72%) and Myodes glareolus (2/3; 67%) were positive. The mtLSU sequences showed marked polymorphism with seven Pneumocystis sequence types representing three distinct major clades according to the three animal species. Among Sorex genus all sequences presented a similarity of 100%, whereas six different sequence types were obtained for the genera Apodemus (n=4) and Myoedes (n=2). CONCLUSIONS: These preliminary observations are in agreement with previous findings, which reported similar prevalence of Pneumocystis in Apodemus and Myodes animals as well as the highest prevalence in Sorex spp., when compared with other wild mammals (Laakkonen et al., 1995, Can J Zoology, 73:961-966). The genetic polymorphism in Apodemus and Myodes has already been described and seem to be a general tendency for rodent-derived Pneumocystis. The unique sequence type detected among Sorex shrews from the eastern Alpes suggests that Pneumocystis from insectivores display a lower level of genetic polymorphism.