Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are 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 of Cryptosporidium spp., G. duodenalis, and E. bieneusi infections 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 of Cryptosporidium spp. at 46.8% (205/438), G. duodenalis at 16.9% (74/438), and E. bieneusi at 10.0% (44/438). The prevalence of Cryptosporidium spp. 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 of Cryptosporidium spp. infection and calf diarrhea (p < 0.01). However, in this study, there was no statistical correlation between the prevalence of G. duodenalis infection as well as E. bieneusi infection and calf diarrhea (p > 0.05). Furthermore, four known Cryptosporidium species were successfully identified by comparing them with SSU rRNA gene sequences, including C. parvum, C. bovis, C. ryanae, and C. andersoni. In addition, all 74 G. duodenalis-positive samples were identified as assemblage E by comparative analysis of bg gene sequences. Among the 44 E. bieneusi-positive samples sequenced in the present study, 4 distinct E. bieneusi genotypes 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 of Cryptosporidium spp., G. duodenalis, and E. bieneusi in Ningxia diarrhea calves and non-diarrhea calves, which provide new data for understanding the epidemiological status of Cryptosporidium spp., G. duodenalis, and E. bieneusi in Ningxia calves.

Mannheimia haemolytica is a primary pathogen causing bacterial pneumonia in cattle and sheep. Multiple PCR targets exist for its identification, but no standardized primer pairs have been widely adopted. This study evaluated the universality of six reported primers (including rpt2) using clinical M. haemolytica isolates. Genus specificity was tested against standard strains of common ruminant pathogenic/commensal bacteria and other Mannheimia species. Four primer sets (gcp, artJ-lktC, lktD, sodA) failed to distinguish M. haemolytica from other Mannheimia species. The rpt2 primer showed suboptimal universality, with 3 of 22 tested isolates yielding negative results. The rpoB primer had favorable universality and specificity, but its 136 bp amplicon was unsuitable for constructing multiplex PCR with M. haemolytica serotyping primers. We identified design flaws in the original rpt2 primer and developed a novel rpt2-N primer. Evaluation confirmed its strong universality, specificity, and sensitivity, as well as compatibility with serotyping primers for multiplex PCR assays. Field testing on three goat farms in the Chongqing region showed markedly higher M. haemolytica positivity rates after sharp temperature drops and transportation. Serotype A2 was confirmed as the dominant serotype in goats in Chongqing. This study systematically assessed multiple PCR identification primers, confirmed the feasibility of rpoB, and resolved the universality limitation of rpt2, providing technical support for rapid and accurate M. haemolytica detection.

Clostridium perfringens type A causes neonatal clostridial enteritis, hemorrhagic enteritis, and sudden death syndrome in cattle. This study examined a fatal outbreak at a fattening farm housing 58 adult yaks in Southwest China, with an incidence of 22.41% (13/58) and a 100% case fatality rate. Histopathology of deceased yaks showed widespread multi-organ hemorrhage and severe hemorrhagic enteritis. A specific PCR assay amplified the alpha-toxin gene of Clostridium perfringens, while no other pathogens were detected. Clostridium perfringens was isolated from small intestinal contents using TSC agar, designated strain CQ1, and identified as type A through toxinotyping. Whole-genome sequencing revealed that strain CQ1 carries the core alpha-toxin gene along with key accessory virulence determinants (pfoA, colA, and cloSI). Phylogenomic analysis based on core single-nucleotide polymorphisms revealed that strain CQ1 is clustered into a distinct, independent evolutionary lineage with an existing historical yak isolate and a canine-origin isolate. Intraperitoneal challenge in mice confirmed its extreme lethality, causing 100% mortality within 36 h with severe intestinal necrosis. These findings provide fundamental genetic resources for understanding the virulence mechanisms and genetic background of this pathogen and offer critical insights for preventing and controlling clostridial diseases in high-altitude yak farming.