China Animal Husbandry & Veterinary Medicine ›› 2025, Vol. 52 ›› Issue (11): 5465-5475.doi: 10.16431/j.cnki.1671-7236.2025.11.041

• Basic Veterinary Medicine • Previous Articles    

Isolation,Identification and Drug Sensitivity Analysis of Clostridium perfringens from Tibetan Sheep

WANG Dongjing1, ZENG Jiangyong1, SU Zhonghua2, BASANG Ciren3, NIMA Panduo4, WANG Dengyun5, MA Hongcai1   

  1. 1. Tibet Livestock Research Institute, Tibet Academy of Agricultural and Animal Husbandry Science, Lhasa 850009, China;
    2. Animal Disease Prevention and Control Center of Tibet Autonomous Region, Lhasa 850000, China;
    3. Biru County Agricultural and Animal Husbandry Science and Technology Service Station, Nagqu 852200, China;
    4. Suo County Agricultural and Animal Husbandry Science and Technology Service Station, Nagqu 852200, China;
    5. Nierong County Agricultural and Animal Husbandry Science and Technology Service Station, Nagqu 852200, China
  • Received:2025-03-18 Published:2025-10-30

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Abstract: 【Objective】 Pathogen detection was conducted on the samples collected from the suspected death of Clostridium perfringens infection in Qushui county,Lhasa city,Tibet,and in vitro drug sensitivity analysis was performed. 【Method】 Sterilely collected 12 samples of pathological specimens,including lungs,livers,hearts,spleens,kidneys,blood,stomach contents,and intestinal contents from dead sheep.The isolation,cultivation and purification of pathogenic bacteria were carried out through anaerobic culture.The isolated and purified strains were subjected to Gram staining microscopy,biochemical identification,16S rRNA gene PCR identification,and genetic sequence determination and genetic evolution analysis.The toxin genes of the isolates was detected by PCR and toxin typing was carried out,and the drug sensitivity of the isolates in vitro was studied. 【Result】 Ten suspected strains were isolated from 12 Tibetan sheep samples.The isolates grew vigorously in RMC medium,and showed white colonies with black edges in the middle on TSC medium.They showed double hemolytic rings on 5% defibrinated sheep blood plates.Gram staining was positive for bacteria.They were preliminarily determined to be Clostridium perfringens,named QS-1 to QS-10.The biochemical identification results showed that the isolates were positive for lactose,sucrose,D-ribose,rhamnose,glucose,maltose,xylose,mannitol,sorbitol,gelatin,urea,and milk fermentation test,while the results of the tests for sophorose,carobose,arabinose,M-R,V-P,glycerol,salicin,hydrogen sulfide,peroxide,urea,and indole were negative.The similarity of the 16S rRNA gene sequence of the isolates to the reference strain of Clostridium perfringens was 98.30% to 99.90%,and they were in the same branch as the reference strain of Clostridium perfringens,while they were in different branches from Escherichia coli,Salmonella,etc.The toxin gene test results showed that 8 isolates only carried cpa gene and were of the A type of Clostridium perfringens，2 isolates carried both cpa and etx genes and were of the D type of Clostridium perfringens.The isolates were sensitive or intermediate to norfloxacin,ofloxacin,ciprofloxacin,ampicillin,carbenicillin and piperacillin,and had different degrees of drug resistance to the other 15 drugs. Among them,9 isolates exhibited multi-drug resistance,and 80.00% of the isolates had resistance to more than 5 drugs.The isolates QS-3 exhibited resistance to 13 drugs. 【Conclusion】 In this study,10 strains of Clostridium perfringens were isolated from the sudden death of Tibetan sheep,including 2 strains of type D and 8 strains of type A.The drug resistance of the isolates was serious.This results provided data guidance for the treatment and prevention of Clostridium perfringens disease of Tibetan sheep in local area.

Key words: Tibetan sheep; Clostridium perfringens; isolation and identification; drug resistance

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