羊呼吸道溶血性曼氏杆菌的分离鉴定及其致病性和耐药性研究

doi:10.16431/j.cnki.1671-7236.2025.05.044

摘要/Abstract

摘要： 【目的】了解内蒙古地区养殖场引起羊呼吸道疾病的主要病原菌，并探究其致病性及耐药性，为相关疾病防治药物的选择提供参考。【方法】对采集的病死羊肺脏组织进行病原菌分离培养、生化鉴定、分子生物学鉴定，并对其进行小鼠致病性试验，采用PCR法检测分离菌株毒力基因携带情况；利用K-B法检测分离菌株的药物敏感性，通过PCR法检测分离菌株耐药基因携带情况；用牛津杯法检测中药单体小檗碱、柠檬醛和百里香酚对分离菌株的体外抑菌效果。【结果】分离菌株在血琼脂平板培养基上形成β-溶血的灰白色菌落，共分离获得2株疑似菌株，分别命名为Mh1-1和Mh1-2。生化鉴定结果显示，分离株麦芽糖、甘露糖、蔗糖、木糖、山梨醇反应均呈阳性。PCR扩增出大小为453 bp的溶血性曼氏杆菌特异性基因及306 bp的荚膜血清A1型条带。相似性比对结果显示，分离株Mh1-1与溶血性曼氏杆菌GDZJcattle2014株(登录号：KM576848.1)的相似性为98.49%，分离株Mh1-2与溶血性曼氏杆菌38599株(登录号：CP017491.1)的相似性为98.14%。经鉴定2株分离株均为血清A1型溶血性曼氏杆菌。小鼠致病性试验结果显示，分离株Mh1-1和Mh1-2对小鼠的半数致死量(LD₅₀)分别为2.2×10⁷和1.0×10⁷ CFU/mL。2株分离菌株均检出毒力基因gs60、lktC、lktA、tbpB、adh和nmaA。药敏试验结果显示，分离株Mh1-1对四环素、林可霉素表现耐药，对链霉素表现中介；分离株Mh1-2对链霉素和林可霉素表现耐药，对四环素表现中介；2株分离菌株均对氧氟沙星、环丙沙星、恩诺沙星、复方新诺明、头孢噻肟、氟苯尼考等表现敏感。耐药基因检测结果显示，2株分离株均携带strA、strB、tetA基因。体外抑菌试验结果显示，小檗碱和柠檬醛对分离株均有较好的抑制作用。【结论】本研究从患呼吸道疾病的病死羊肺脏组织中分离获得2株荚膜血清A1型溶血性曼氏杆菌，分离株均有致病性并携带多种毒力基因，对抗菌药物林可霉素耐药并携带strA等耐药基因。试验结果为临床科学合理地选择抗菌药物提供参考依据，为后续治疗药物的开发和研究提供思路。

关键词: 溶血性曼氏杆菌; 分离鉴定; 致病性; 耐药性

Abstract: 【Objective】 The purpose of this experiment was to understand the main pathogens causing respiratory tract diseases in sheep in Inner Mongolia,and explore the pathogenicity and drug resistance,so as to provide reference for the control drugs selection of related disease.【Method】 The pathogenic bacteria were isolated,cultured,biochemical and molecular identified from the lung tissue of dead sheep,and the pathogenicity test was carried out in mice.The virulence genes of the isolates were detected by PCR.The drug susceptibility of isolates was detected by K-B method,and the drug resistance genes of the isolates was detected by PCR method.The in vitro inhibitory effects of berberine,citraldehyde and thymol on the isolated strains were determined by Oxford cup method.【Result】 The isolated strains formed β-hemolysis grey white colonies on the blood agar plate medium,and two suspected strains were isolated，and named Mh1-1 and Mh1-2,respectively.Biochemical identification showed that maltose,mannose,sucrose,xylose and sorbitol of the isolates were positive.PCR amplified the specific gene of Mannheimia haemolytica with the size of 453 bp and the capsular serotype A1 band with the size of 306 bp.The similarity comparison results showed that the similarity between the isolate Mh1-1 and Mannheimia haemolytica GDZJcattle2014 (accession No.:KM576848.1) was 98.49%,and the similarity between the isolate Mh1-2 and Mannheimia haemolytica 38599 (accession No.:CP017491.1) was 98.14%.The two isolates were identified as serotype A1 Mannheimia haemolytica.The results of pathogenicity test in mice showed that the LD₅₀ of Mh1-1 was 2.2×10⁷ CFU/mL,and the LD₅₀ of Mh1-2 was 1.0×10⁷ CFU/mL.Virulence genes gs60,lktC,lktA,tbpB,adh and nmaA were detected in both isolates.The results of drug sensitivity test showed that Mh1-1 was resistant to tetracycline and lincomycin,and intermediated to streptomycin.Mh1-2 showed resistance to streptomycin and lincomycin,and intermediated to tetracycline.The two isolates were sensitive to ofloxacin,ciprofloxacin,enrofloxacin,cotrimoxazole,cefotaxime and flufenicol.The results of drug resistance gene detection showed that the two isolates carried strA,strB and tetA genes.The results of in vitro bacteriostatic test showed that berberine and citraldehyde had good inhibitory effects on the isolates.【Conclusion】 In this study,two strains of serotype A1 Mannheimia haemolytica were isolated from the lung tissues of dead sheep with respiratory diseases.Two isolates were pathogenic and carried a variety of virulence genes，and resistant to lincomycin,and carried resistance genes such as strA.The results provided reference for rational selection of antibiotics in clinical,and provided ideas for the development and research of subsequent therapeutic drugs.

Key words: Mannheimia haemolytica; isolation and identification; pathogenicity; drug resistance

中图分类号:

S855.1

武佳鑫, 孙月, 毛伟, 刘淑英, 尹凯雯, 张志丹, 韩凯凡, 赵红霞. 羊呼吸道溶血性曼氏杆菌的分离鉴定及其致病性和耐药性研究[J]. 中国畜牧兽医, 2025, 52(5): 2421-2431.

WU Jiaxin, SUN Yue, MAO Wei, LIU Shuying, YIN Kaiwen, ZHANG Zhidan, HAN Kaifan, ZHAO Hongxia. Isolation and Identification of Mannheimia haemolytica from Sheep Respiratory Tract and Its Pathogenicity and Drug Resistance[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(5): 2421-2431.

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