Simple SummaryThis study first reported the different occurrences ofCryptosporidiumspp.,G. duodenalis, andE. bieneusiinfection between diarrheic and non-diarrheic calves in Ningxia, China. TheCryptosporidiumspp. infection rates were 54.0% and 38.3% in diarrheic and non-diarrheic calves, which was an extremely significant difference (p <0.01). However, there was no statistical correlation between the prevalence ofG. duodenalisinfection as well asE. bieneusiinfection and calf diarrhea (p >0.05). FourCryptosporidiumspecies (C. parvum,C. bovis,C. ryanae, andC. andersoni), an assemblage E ofG. duodenalis, and fourE. bieneusigenotypes (BEB4, N, J, and NX1) were observed. The results showed thatCryptosporidiumspp. is closely associated with calf diarrhea, and the identification of zoonotic species/assemblage/genotypes in calves suggests that these animals may play an important role in the transmission of zoonosis.AbstractCryptosporidiumspp.,Giardia duodenalis, andEnterocytozoon bieneusiare significant parasitic gastrointestinal pathogens with global distribution in humans and domestic animals, including calves. The main symptoms of calf infection are severe diarrhea, dehydration, growth retardation, and sometimes even death. To date, there has been limited information on the prevalence ofCryptosporidiumspp.,G. duodenalis, andE. bieneusiinfections in calves in Ningxia, China, especially between diarrheic and non-diarrheic calves. A total of 438 fecal samples were collected from diarrheic (201) and non-diarrheic (237) calves in Ningxia. PCR and DNA sequencing were used to find the prevalence ofCryptosporidiumspp. at 46.8% (205/438),G. duodenalisat 16.9% (74/438), andE. bieneusiat 10.0% (44/438). The prevalence ofCryptosporidiumspp. infection in diarrheic and non-diarrheic calves was 54.0% (128/237) and 38.3% (77/201), respectively, and statistical analysis showed a positive correlation between the prevalence ofCryptosporidiumspp. infection and calf diarrhea (p< 0.01). However, in this study, there was no statistical correlation between the prevalence ofG. duodenalisinfection as well asE. bieneusiinfection and calf diarrhea (p >0.05). Furthermore, four knownCryptosporidiumspecies were successfully identified by comparing them with SSU rRNA gene sequences, includingC. parvum,C. bovis,C. ryanae,andC. andersoni. In addition, all 74G. duodenalis-positive samples were identified as assemblage E by comparative analysis ofbggene sequences. Among the 44E. bieneusi-positive samples sequenced in the present study, 4 distinctE. bieneusigenotypes were successfully identified by comparative analysis of ITS sequences, including 3 known genotypes (J, BEB4, and N) and 1 novel genotype, the latter of which was identified and designated as NX1. These findings indicated that the high genetic diversity and complex population structures ofCryptosporidiumspp.,G. duodenalis, andE. bieneusiin Ningxia diarrhea calves and non-diarrhea calves, which provide new data for understanding the epidemiological status ofCryptosporidiumspp.,G. duodenalis, andE. bieneusiin Ningxia calves.