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

摘要/Abstract

摘要： 【目的】2023年4月至2023年5月，西藏拉萨市多地出现牦牛严重腹泻、便血甚至死亡的现象。试验旨在确定牦牛腹泻的病原菌并研究其生物学特性，以期为该地区牦牛腹泻病的防治提供参考。【方法】采集新鲜牦牛腹泻粪便样品进行厌氧培养，通过培养特性及染色镜检进行初步鉴定；对可疑阳性菌株进行生化特性、多重PCR毒素基因分型及cpe、netB、cpb2毒素鉴定，并进行cpa、cpb2、16S rRNA基因的遗传进化分析、部分分离株生长曲线测定及动物致病性试验；采用K-B法测定分离株的体外药物敏感性，统计不同分离株的多重耐药结果。【结果】从样品中分离纯化得到8株分离菌，在厌氧培养基中生长旺盛，在TSC培养基、5%脱纤维绵羊血平板、卵黄琼脂培养基上均呈现与产气荚膜梭菌相似的典型菌落，革兰染色为紫色大杆菌，初步判定为产气荚膜梭菌，命名为XZ-1~XZ-8。分离株生化检测结果与产气荚膜梭菌相符；分离株均携带cpa和cpb2基因，为A型产气荚膜梭菌；16S rRNA基因分析结果显示，8株分离株与产气荚膜梭菌参考株的相似性为97.3%~100%，在系统进化树中处于同一分支，而与屎肠球菌、大肠杆菌参考株处于不同分支；分离株cpa和cpb2基因存在一定的变异性。生长曲线显示，3株分离株（XZ-1、XZ-4、XZ-7）生长规律基本一致，其中0~4 h为迟缓期，4~8 h为对数期，8~22 h为稳定期，22 h后开始进入衰亡期。动物致病性试验结果显示，分离株对昆明小鼠具有较强的致病性。分离株对苯唑西林、红霉素、氯霉素、氧氟沙星、米诺环素、头孢哌酮、利福平及罗红霉素敏感，对青霉素、哌拉西林、羧苄西林、麦迪霉素、头孢曲松等22种药物均存在不同程度的耐药性，存在多重耐药性，其中XZ-7株耐药数量多达11种，6重以上耐药的菌株占75.0%。【结论】本研究成功从拉萨严重腹泻、血便牦牛粪样中分离出8株A型产气荚膜梭菌，分离株对小鼠具有较强的致病性，对青霉素、哌拉西林、羧苄西林等药物存在不同程度的耐药性。研究结果为该地区牦牛腹泻病防控提供了参考依据。

关键词: 牦牛; 产气荚膜梭菌; 分离鉴定; 生长曲线; 动物致病性; 耐药性

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

中图分类号:

S852.61⁺6.3

王冬经, 吴金措姆, 曾江勇. 牦牛源A型产气荚膜梭菌的分离鉴定及生物学特性研究[J]. 中国畜牧兽医, 2024, 51(1): 229-241.

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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