Morphological and molecular identification of Eimeria spp. (Apicomplexa: Eimeriidae) in dairy cattle, Bos taurus from intensive dairy cattle farms in some areas of China.

Coccidiosis is among the most prevalent diseases affecting dairy cattle globally, imposing significant economic losses on the dairy industry annually. To better understand the epidemiology and species distribution of Eimeria spp. in key dairy-producing regions of China, as well as to improve species-level identification, we developed an integrated approach combining morphological characterization with single-oocyst selection technology and multi-locus-nested PCR amplification. This method enables comprehensive genetic profiling of individual oocysts through multi-locus genotyping. A total of 900 fecal samples were collected from the rectums of dairy cattle, the overall prevalence of Eimeria spp. infection was 34.78 % (313/900). Thirteen Eimeria spp. were identified with the following distribution: Eimeria alabamensis (12.46 %, 39/313), E. auburnensis (28.43 %, 89/313), E. bovis (42.81 %, 134/313), E. bukidnonensis (5.75 %,18/313), E. canadensis (27.80 %,87/313), E. cylindrica (30.67 %,96/313), E. ellipsoidalis (37.06 %,116/313), E. pellita (0.30 %,1/313), E. subspherical (28.12 %,88/313), E. zuernii (50.48 %,158/313), E. illinoisensis (25.56 %,80/313), E. wyomingensis (3.83 %,12/313), E. ildefonsoi (1.28 %,4/313). Among these, E. bovis and E. zuernii were the predominant species. Our study contributes new data of Eimeria spp. infection in dairy cattle from key dairy-producing regions of China. We provide updated morphological characterizations for several Eimeria species, for the first time in China, the presence of E. ildefonsoi. Additionally, we performed comparative sequence analysis of the SSU rRNA and COI loci. The results revealed sequence homology ranges of 93.00 %-100.00 % (SSU rRNA) and 83.30 %-98.80 % (COI) among the 13 Eimeria species examined. Phylogenetic analysis based on these two loci effectively differentiated all 13 Eimeria species. This study represents the first report of single-oocyst-derived sequences for Eimeria spp. in dairy cattle at the SSU rRNA and COI loci, establishing a robust method for precise species identification and expanding the genetic database for bovine Eimeria.