1株羊源伪结核棒状杆菌的分离鉴定及耐药性分析

doi:10.16431/j.cnki.1671-7236.2025.03.041

摘要/Abstract

摘要： 【目的】确定云南宜良地区某养殖场疑似患伪结核病病羊的病原，并探究其耐药性及致病性，为该病的诊断和治疗提供理论依据。【方法】通过患病羊临床症状初步判断病因，无菌采集病料，分离纯化病原菌后对其形态特征、培养特性和生化特性进行鉴定；利用PCR扩增分离株16S rRNA基因序列并进行测序比对分析；通过药敏试验、耐药基因检测和小鼠致病性试验探究分离株的耐药性和致病性。【结果】分离株在血琼脂平板上生长状态良好，菌落呈黄白色、不透明样，在普通营养琼脂和普通营养肉汤内生长不良；革兰染色镜检结果显示，菌体为球杆状或棒状，为革兰阳性菌。生化鉴定结果显示，分离株的葡萄糖、尿素试验呈阳性，乳糖、麦芽糖、甘露醇、蔗糖、西蒙氏枸橼酸盐、硫化氢、靛基质、硝酸盐还原试验、甲基红(MR)试验和VP试验均呈阴性。16S rRNA序列比对结果显示，分离株与NCBI数据库中的伪结核棒状杆菌16S rRNA基因序列相似性＞99%，鉴定其为伪结核棒状杆菌。药敏试验结果显示，分离株对链霉素、庆大霉素、新霉素表现中介，对氨曲南、头孢他啶、多黏菌素表现耐药。耐药基因检测结果显示，该分离株携带耐药基因Sul2、Sul3、bla_TEM及bla_CTX-M。动物致病性试验结果显示，腹腔、皮下接种4.4×10⁸ CFU/mL分离菌后可引起小鼠死亡，死亡率达100%。皮下接种96 h后可见小鼠皮下出现脓肿，脓液中可分离出伪结核棒状杆菌。【结论】本试验成功分离鉴定出1株羊源伪结核棒状杆菌，明确了患病羊病原，该分离株对小鼠致病性较强。研究结果为伪结核棒状杆菌感染羊的诊断、流行病学调查以及治疗提供参考依据。

关键词: 山羊; 伪结核棒状杆菌; 分离鉴定; 耐药性

Abstract: 【Objective】 The purpose of this experiment was to identify the pathogen of goats suspected to be suffering from pseudotuberculosis in a farm in Yiliang,Yunnan province,and explore its drug resistance and pathogenicity,so as to provide theoretical basis for the diagnosis and treatment of the disease.【Method】 According to the clinical symptoms of the infected goats,the cause of the disease was preliminarily determined,the disease material was collected aseptically,and the pathogenic bacteria were isolated and purified to identify the morphological characteristics,culture characteristics and biochemical characteristics.The 16S rRNA gene sequences of the isolate was amplified by PCR and analyzed by sequencing.The drug resistance and pathogenicity of the isolate were investigated by drug susceptibility test,drug resistance gene test and pathogenicity test in mice.【Result】 The isolate grew well on the blood agar plate,the colonies were yellowish-white and opaque,and did not grow well in the common nutrient agar and the common nutrient broth.Gram staining microscopy showed that the bacteria were spheroidal or rod-shaped and Gram-positive bacteria.The results of biochemical identification showed that glucose and urea tests were positive,while lactose,maltose,mannitol,sucrose,Simon’s citrate,hydrogen sulfide,indigo matrix,nitrate reduction test,methyl red (MR) test and VP test were negative.16S rRNA sequence alignment results showed that，the 16S rRNA gene sequence similarity between the isolated and Corynebacterium pseudotuberculosis in NCBI database was more than 99%,and it was identified as Corynebacterium pseudotuberculosis.Drug sensitivity test results showed that，the isolate showed intermediation to streptomycin,gentamicin and neomycin,and resistance to amtronam,ceftazidime and polymyxins.The results of drug resistance gene detection showed that the isolate carried drug resistance genes Sul2,Sul3,bla_TEM and bla_CTX-M. The results of animal pathogenicity test showed that abdominal and subcutaneous inoculation with 4.4×10⁸ CFU/mL isolated bacteria could cause death of mice,and the mortality rate was 100%.Subcutaneous abscess appeared 96 h after subcutaneous inoculation,and Corynebacterium pseudotuberculosis could be isolated from the pus.【Conclusion】 In this experiment,a strain of Corynebacterium pseudotuberculosis of goats origin was successfully isolated and identified,and the pathogen of the infected goats was identified.The strain was highly pathogenic to mice.The results provided reference for the diagnosis,epidemiological investigation and treatment of Corynebacterium pseudotuberculosis infection in goats.

Key words: goats; Corynebacterium pseudotuberculosis; isolation and identification; drug resistance

中图分类号:

S852.6

刘泽武, 樊月圆, 袁嘉芮, 格桑卓嘎, 班旦, 白卫兵, 茶金龙, 四朗玉珍, 富国文. 1株羊源伪结核棒状杆菌的分离鉴定及耐药性分析[J]. 中国畜牧兽医, 2025, 52(3): 1405-1415.

LIU Zewu, FAN Yueyuan, YUANJiarui, GESANG Zhuoga, BAN Dan, BAI Weibing, CHAJinlong, SILANG Yuzhen, FU Guowen. Isolation，Identification and Drug Resistance Analysis of a Strain of Corynebacterium pseudotuberculosis from Goats[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(3): 1405-1415.

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