牦牛源A型产气荚膜梭菌的分离鉴定及生物学特性研究

doi:10.16431/j.cnki.1671-7236.2024.01.024

Abstract

Abstract: 【Objective】 From April 2023 to May 2023, yaks in multiple areas of Lhasa city, Tibet experienced severe diarrhea, bloody stools, and even death.This study was aimed to identify the pathogenic bacteria of yak diarrhea and explore their biological characteristics, so as to provide reference for the prevention and treatment of yak diarrhea disease in the region.【Method】 Fresh fecal samples of yak diarrhea were collected for anaerobic cultivation, and preliminary identification was conducted through culture characteristics and staining microscopy.The biochemical characterization verification, multiplex PCR toxin genotyping and cpe, netB, cpb2 toxin identification were performed on suspected positive strains, the genetic evolution analysis of cpa, cpb2 and 16S rRNA genes, the growth curve of some isolated strains and animal pathogenicity tests were also conducted.The in vitro drug sensitivity of isolated strains was measured using K-B method, and the multiple drug resistance results of different isolated strains were statistically analyzed.【Result】 Eight isolated bacteria were isolated and purified from the samples, which grew vigorously in anaerobic culture medium, and exhibited typical colonies similar to Clostridium perfringens on TSC medium, 5% defibrated sheep blood plate and egg yolk agar medium.Gram staining showed that they were purple bacteria, and were preliminarily identified as Clostridium perfringens, named XZ-1 to XZ-8.The biochemical test results of the isolates were consistent with those of Clostridium perfringens.The isolates all carried cpa and cpb2 genes, which were Clostridium perfringens type A.The 16S rRNA gene analysis results showed that the similarity between the 8 isolates and the reference strains of Clostridium perfringens were 97.3% to 100%, which were in the same branch, while the isolates were in the different branches with the reference strains of Enterococcus faecalis and Escherichia coli.There was some variability between cpa and cpb2 genes. The growth curve showed that the growth patterns of the three isolates(XZ-1, XZ-4 and XZ-7) were basically consistent, with 0 to 4 h being the delayed phase, 4 to 8 h being the logarithmic phase, and 8 to 22 h being the stable phase.After 22 h, the isolates began to enter the decay phase.The results of animal pathogenicity tests showed that the isolates had strong pathogenicity to Kunming mice.The isolates were all sensitive to oxacillin, erythromycin, chloramphenicol, ofloxacin, minocycline, cefoperazone, rifampicin and roxithromycin, while had varying degrees of resistance to 22 drugs such as penicillin, piperacillin, carbendazim, medimycin and ceftriaxone.The isolates had multiple drug resistance, with up to 11 strains of XZ-7 resistant, and 75.0% of strains with more than 6 types of resistance. 【Conclusion】 This study successfully isolated 8 strains of Clostridium perfringens type A from yak with severe diarrhea and bloody stools in Lhasa.The isolates had strong pathogenicity to mice and varying degrees of resistance to drugs such as penicillin, piperacillin and carbendazim.The results provided a reference basis for the prevention and control of yak diarrhea in the region.

Key words: yak; Clostridium perfringens; isolation and identification; growth curve; animal pathogenicity; drug resistance

CLC Number:

S852.61⁺6.3

WANG Dongjing, WU Jincuomu, ZENG Jiangyong. Isolation, Identification and Biological Characteristics Analysis of Clostridium perfringens Type A from Yaks[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(1): 229-241.

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Isolation, Identification and Biological Characteristics Analysis of Clostridium perfringens Type A from Yaks

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