Typing identification and biological characterization of Clostridium perfringens toxins associated with lamb dysentery in Xizang sheep

Abstract

The objectives of this study were to investigate the etiology of morbidity and mortality in Tibetan sheep from a township in Shannan, Tibet, and to characterize the biological characteristics of the causative pathogen, thereby providing a scientific basis for effective disease prevention and control. Pathological specimens were aseptically collected from deceased animals, and the pathogen was isolated and cultured based on clinical presentations. Identification of the isolate involved morphological observation, 16S rRNA PCR amplification and sequencing, biochemical assays, toxin gene typing, Kirby-Bauer disk diffusion antimicrobial susceptibility testing, and antibiotic resistance genes screening. On tryptose sulfite cycloserine (TSC) agar, the organism formed black, circular colonies. Gram staining revealed short, straight, Gram-positive bacilli arranged singly or in pairs, consistent with the Clostridium perfringens . 16S rRNA gene sequence alignment showed >99 % identity to reference C. perfringens sequences in the NCBI database.

Confirming the strain asC. perfringens. Biochemical tests indicated positive reactions for glucose, Biochemical assays were positive for glucose, lactose, and maltose fermentation; gelatin hydrolysis; oxidase activity; nitrate reduction;hydrogen sulfide production; and ornithine decarboxylase, but negative results for indole production, mannitol and sorbitol fermentation, and urea hydrolysis, matching known traits of C. perfringens. . Toxin gene PCR detected cpa,cpb, and etx, but not iap, cpe, or netB, identifying the isolate as C. perfringens type B. Antimicrobial susceptibility testing revealed high sensitivity to sulfadiazine, norfloxacin, nitrofurantoin, and cefazolin, but resistance to amoxicillin, oxacillin, carbenicillin, and tetracycline.Among the resistance genes screened, only aac(6′)-Ib-cr was detected.This study reports the first isolation of C. perfringens type B in Shannan, Tibet; elucidates its biological characteristics and toxin profile; identifies effective antibiotics for targeted intervention; and provides foundational data for controlling lamb dysentery caused by this pathogen and for understanding its pathogenicity in sheep.