PNEUMOCYSTIS IN LUNGS OF SQUIRREL, CALLOSCIURUS FINLAYSONII (RODENTIA, SCIURIDAE)

Pneumocystis spp. are ubiquitous fungal commensals of the respiratory tract of many animals and represent a potential cause of life-threatening pneumonia in a wide range of mammals. All species show a restricted host-range, with a high degree of host-specificity. So far, only five Pneumocystis entities are formally described as species. The purpose of the present study was to investigate the occurrence of Pneumocystis in squirrels (Callosciurus finlaysonii) and the functionality of different markers for species recognition. Lung tissues of 54squirrels have been investigated for the presence of Pneumocystis DNA by amplification and sequencing of a portion of the mitochondrial large subunit (mtLSU), small subunit (mtSSU) and internal transcribed spacers 1 and 2 (ITS1/ITS2) of the rRNA genes according protocols previously reported (Danesi et al., 2016). Two gene datasets of Pneumocystis species, with mtSSU and ITS sequences, were analysed by Neighbor Joining with 1000 bootstrap replicates and conducted in MEGA6. Pneumocystis DNA was isolated from three squirrels. Amplification and sequences were obtained from mtSSU (n=3) and ITS (n=2) genetic targets, but not from mtLSU. In mtSSU and ITS trees, constructed without outgroup, all main clusters were well supported and consistent with mammal hosts. Mitocondrial SSU sequences compose a separate clade, which is polyphyletic with the only Pneumocystis carinii f. sp. squirrel (Sciurus aestuans) present in the database. The rDNA ITS tree confirmed Pneumocystis grouping according animal species. In general, squirrels were in apparently good condition, thus supposing animals were carrying Pneumocystis organisms without disease. The amplification of Pneumocystis mtLSU target did not succeed with these set of primers, suggesting this genetic target might be different in squirrels. Indeed, the detection of Pneumocystis spp. in a large variety of animals provides evidence of a highly diversified Pneumocystis genus. According to the Barcoding approach, ITS might be an excellent marker for species characterization.