Nanopore sequencing of the mtSSUrRNA and mtLSUrRNA genes of Pneumocystis sp. in foals
Twenty-two foals, aged one to seven 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 out of 22 foals (48 out of 52 specimens) using a PCR assay targeting the gene encoding the small subunit of the mitochondrial rRNA (mtSSUrRNA) of the horse-related Pneumocystis (Pneumocystis sp. 'Equus ferus caballus'). Sanger sequencing of the PCR products was successful in 34 out of 48 specimens. The sequences were identical to the horse related Pneumocystis reference sequence (Genbank accession number OP738801) in seven specimens (five 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 two foals did not align correctly to the reference sequence, with a percentage of identity of only 86.12%. Ultradeep sequencing of the mtSSUrRNA and mtLSUrRNA genes of Pneumocystis sp. was performed on a subset of 12 and 5 specimens, for mtSSUrRNA and mtLSUrRNA, respectively. These results provided original and additionnal data on the genetic diversity of Pneumocystis sp. in foals.