Genetic diversity within dominant Enterocytozoon bieneusi genotypes in pre-weaned calves

Cattle are commonly infected with the microsporidian parasite Enterocytozoon bieneusi. Sequence characterization of E. bieneusi in these animals at the ribosomal internal transcribed spacer (ITS) locus had identified I, J and BEB4 as the dominant genotypes. However, current studies on E. bieneusi in dairy cattle are mostly on infection rates and genotype distribution. This study aims to examine the intragenotypic diversity within dominant E. bieneusi genotypes in pre-weaned dairy calves in Shanghai, China. Enterocytozoon bieneusi genotypes and subtypes were identified by PCR sequence analysis of ITS and multilocus sequence typing (MLST), based on material from farms. Chi-square test was used to examine differences in E. bieneusi infection rates between farms or age groups. The overall infection rate of E. bieneusi was 26.5% (214/809), ranging from 12.6% (Farm 5) to 38.5% (Farm 4). Infection rates increased with age during early life, with the peak infection rate (43.0%; 43/100) occurring at six weeks. Four genotypes were present, including J (n = 145, 67.8%), BEB4 (n = 59, 27.6%), CHN4 (n = 4, 1.9%) and CHN15 (n = 1, 0.5%), with the former two belonging to Group 2 and the latter two belonging to Group 1. Differences were detected in the distribution of the dominant genotypes J and BEB4 among five study farms. Altogether, 10 multilocus genotypes (MLGs) were identified in the two dominant ITS genotypes, including MLG-J1 to MLG-J8 of genotype J and MLG-B1 to MLG-B2 of genotype BEB4. MLG-B1 and MLG-B2 were recovered in Farms 1, 2 and 5, whereas MLG-J1 to MLG-J5 and MLG-J6 to MLG-J8 were found in Farms 3 and 4, respectively. There is extensive genetic heterogeneity within the dominant E. bieneusi genotypes J and BEB4 in dairy calves in Shanghai, China, and MLST should be used in molecular epidemiological studies of E. bieneusi in cattle.