Morphological and molecular characteristics for the identification of Eimeria spp. in Eurasian red squirrels (Sciurus vulgaris).

Coccidiosis caused by Eimeria spp. is one of the major causes of morbidity and mortality in captive squirrels. To conduct morphological and molecular characterization of Eimeria spp. in Sciurus vulgaris and to determine their prevalence in Henan Province, China, we devised a novel single oocyst amplification method, integrating morphological and molecular analyses. Fecal samples from 30 Eurasian red squirrels (S. vulgaris) were examined using microscopy which revealed a 93.3 % (28/30) Eimeria spp. infection rate. Morphometric analysis identified three Eimeria spp. namely E. sciurorum (73.3 %, 22/30), E. ontarioensis (50.0 %, 15/30), and E. lancasterensis (30.0 %, 9/30), with E. sciurorum being predominant. The nuclear 18S ribosomal RNA (18S rRNA) gene and mitochondrial cytochrome c oxidase subunit I (COI) gene of all three species were successfully amplified and sequenced. Phylogenetic analysis showed that these three species shared 96.2 % to 98.8 % sequence identity of the 18S rRNA gene and 91.5 % to 98.0 % sequence identity of the COI gene (bootstrap support ≥95 %, 1000 replicates). These values confirm their close affinity and support their recognition as distinct species. The analysis of the evolutionary tree revealed that the genetic relationship between Eimeria spp. is consistent with the genetic relationship between host animals. This is the first molecular report of Eimeria spp. in Chinese squirrels, summarizing their global prevalence by comparing newly obtained data with published records and offering critical insights for coccidiosis surveillance and Eimeria spp. taxonomy.