Kefei Shen

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.

Background: Paratuberculosis is a widespread chronic infection of Mycobacterium avium subspecies paratuberculosis (MAP) that causes significant economic losses to the sheep industry. The current study investigated this disease, which causes diarrhea in sheep, particularly, in Bayannaoer, Inner Mongolia, China. Diagnosis was based on clinical symptoms, pathological autopsy, histopathological inspection, and serological and molecular methods. Results: MAP was confirmed using polymerase chain reaction using DNA extracted from tissue and fecal samples. Serum samples from 472 individual sheep were obtained to detect antibodies against MAP using an enzyme-linked immunosorbent assay. MAP antibodies were separately detected in 17.86% (35/196) and 18.48% (51/276) of sheep herds at approximately 6 months and ≥ 1 year of age, respectively. The tissue lesion and pathological section results were consistent with paratuberculosis infection. Conclusions: To our knowledge, this is the first report of Mycobacterium avium subspecies paratuberculosis seroprevalence in Bayannaoer sheep in Inner Mongolia. Our findings show that MAP is not only prevalent, but also a potential threat to this region. Further investigations, including long-term epidemiological surveillance and isolation are needed for the awareness and effective treatment of paratuberculosis in sheep of Inner Mongolia.