High frequency and high genetic diversity of Pneumocystis sp. in foals

Twenty-two foals, aged 1-7 months, which died from interstitial pneumonia (15 foals) or unrelated illnesses and injuries (7 foals), were enrolled in this study. Sampling of the lungs was performed post-mortem for a total of 52 specimens. Pneumocystis was detected in 21/22 foals (48/52 specimens) using a Polymerase Chain Reaction (PCR) assay targeting the gene encoding the small subunit of the mitochondrial rRNA (mtSSUrRNA) of the horse-related Pneumocystis (Pneumocystis sp. 'Equus ferus caballus'). The sequencing of the PCR products was successful in 34/48 specimens. The sequences were identical to the horse-related Pneumocystis reference sequence (Genbank accession number OP738808) in 7 specimens (5 foals). Single Nucleotide Polymorphisms (SNPs) were detected in the sequences from 24 specimens (16 foals). Three alleles were determined according to these SNPs. Allele identification was successful in 10 specimens (7 foals) and unsuccessful in 21 specimens (14 foals) because of a mix of alleles. The presence of a mix of alleles highly suggests the presence of more than one strain able to infect foals. Three sequences from 2 foals did not align correctly to the reference sequence, with a percentage of identity of only 86.12%. Ultradeep sequencing of the gene encoding the large subunit of the mitochondrial rRNA (mtLSUrRNA) was performed on a subset of 5 specimens. The issued sequences were concatenated with mtSSUrRNA sequences and used for phylogenetic analysis. The phylogenetic analysis showed a shorter genetic distance of these sequences of Pneumocystis sp. 'Equus ferus caballus' with that of another species close to Pneumocystis sp. 'Sus scrofa domesticus', suggesting a putative other species able to infect foals. These results provided original and additional data on genetic diversity of Pneumocystis sp. in foals.