1株羊源多杀性巴氏杆菌的分离鉴定及病原特性研究

doi:10.16431/j.cnki.1671-7236.2024.01.035

摘要/Abstract

摘要： 【目的】确定齐齐哈尔市某羊场育成绵羊发病和死亡的病原菌，并探究其病原学特性，为该病的有效防治提供科学依据。【方法】无菌采集病死羊肺脏组织，结合临床病征进行实验室诊断，分离培养病原菌。通过形态学观察、生化试验、16S rRNA PCR扩增并测序、kmtⅠ基因鉴定、荚膜血清分型、毒力基因检测、药敏试验、耐药基因检测和小鼠致病性试验对分离菌进行鉴定和病原特性分析。【结果】致病菌株为多杀性巴氏杆菌，分离菌在血平板上长出表面光滑、灰白色的圆形凸起菌落，革兰染色可见聚集成线状或散在单个的革兰阴性短杆菌。生化试验结果显示，该菌能发酵麦芽糖、阿拉伯糖、蔗糖、木糖、葡萄糖、果糖、海藻糖、甘露醇、尿素酶、山梨醇和β-半乳糖苷酶，精氨酸、靛基质试验呈阳性。荚膜血清分型结果显示，分离株为多杀性巴氏杆菌荚膜D型。药敏试验结果显示，分离株对恩诺沙星、杆菌肽和洛美沙星3种药物耐药，对新霉素等4种药物表现为中介，对氨苄西林等16种药物敏感。分离株携带14种毒力基因和6种耐药基因。小鼠感染分离菌后全部死亡。【结论】本研究成功分离到1株D型多杀性巴氏杆菌菌株，其携带多种毒力基因和耐药基因，对小鼠具有较强致病性，研究结果为探究羊源多杀性巴氏杆菌的生物学信息和病原学特征提供了数据。

关键词: 多杀性巴氏杆菌; 分离鉴定; 毒力基因; 耐药性; 致病性

Abstract: 【Objective】 This study was aimed to identify the pathogenic bacteria that causing the disease and death of breeding sheep in a sheep farm in Qiqihar city, and to explore its etiological characteristics, so as to provide scientific basis for effective prevention and control of the disease.【Method】 The lungs from dead sheep were aseptically taken, and laboratory diagnosis was conducted based on clinical symptoms.Pathogenic bacteria were isolated and cultured.Morphological observation, biochemical test, 16S rRNA PCR amplification and sequencing, kmtⅠ gene identification, capsule serotyping, virulence gene detection, drug susceptibility test, drug resistance gene detection and mouse pathogenicity test were used to identify and analyze the pathogenic characteristics of the isolated bacteria.【Result】 The pathogenic strain was Pasteurella multocida.The isolated bacteria showed round raised colonies with smooth, grayish white surface on the blood plate, and Gram staining showed that the Gram-negative brevibacterium was clustered into lines or scattered in single. Biochemical test results showed that the strain could ferment maltose, arabinose, sucrose, xylose, glucose, fructose, trehalose, mannitol, urease, sorbitol and and ONPG, and arginine acid hydrolysis and indigo test were positive.Serotyping of the capsule showed that the isolate was Pasteurella multocida capsular serotype D. The drug sensitivity test showed that the isolates were resistant to enrofloxacin, bacitracin and lomefloxacin, intermediary to 4 drugs such as neomycin, and sensitive to 16 drugs such as ampicillin.The isolates carried 14 virulence genes and 6 resistance genes.All mice died after infection with the isolated bacteria.【Conclusion】 One Pasteurella multocida serotype D strain was successfully isolated, carrying a variety of virulence genes and drug resistance genes, and was pathogenic to mice.This study provided data for exploring the biological information and etiological characteristics of the sheep origin Pasteurella multocida.

Key words: Pasteurella multocida; isolation and identification; virulence gene; drug resistance; pathogenicity

中图分类号:

S852.61⁺2

张蕾, 陈亮, 金振华, 冯万宇, 苗艳, 兰世捷, 秦平伟, 沈思思, 李丹, 张备, 董佳强, 黄宣凯, 李旭业, 江波涛, 王洪宝, 史同瑞. 1株羊源多杀性巴氏杆菌的分离鉴定及病原特性研究[J]. 中国畜牧兽医, 2024, 51(1): 347-356.

ZHANG Lei, CHEN Liang, JIN Zhenhua, FENG Wanyu, MIAO Yan, LAN Shijie, QIN Pingwei, SHEN Sisi, LI Dan, ZHANG Bei, DONG Jiaqiang, HUANG Xuankai, LI Xuye, JIANG Botao, WANG Hongbao, SHI Tongrui. Isolation, Identification and Pathogenic Characteristics of One Pasteurella multocida Isolate from Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(1): 347-356.

参考文献

[1] MOSTAAN S, GHASEMZADEH A, SARDARI S, et al.Pasteurella multocida vaccine candidates:A systematic review[J].Avicenna Journal of Medical Biotechnology, 2020, 12(3):140.
[2] HURTADO R, MATURRANO L, AZEVEDO V, et al.Pathogenomics insights for understanding Pasteurella multocida adaptation[J].International Journal of Medical Microbiology, 2020, 310(4):151417.
[3] PENG Z, LIU J, LIANG W, et al.Development of an online tool for Pasteurella multocida genotyping and genotypes of Pasteurella multocida from different hosts[J].Frontiers in Veterinary Science, 2021, 8:771157.
[4] PENG Z, WANG X, ZHOU R, et al.Pasteurella multocida:Genotypes and genomics[J].Microbiology and Molecular Biology Review, 2019, 83(4):e00014-19.
[5] KANDIMALLA K, AWATI B, PUTTY K, et al.Evaluation of biofilm formation capacity of Pasteurella multocida and its relationship with antibiotic resistance[J].Indian Journal of Veterinary Sciences & Biotechnology, 2022, 18(5):68-74.
[6] WILSON B A, HO M.Pasteurella multocida:From zoonosis to cellular microbiology[J].Clinical Microbiology Reviews, 2013, 26(3):631-655.
[7] 刘海燕, 杨有文, 王远微, 等.6株山羊源多杀性巴氏杆菌血清型、耐药性及致病性分析[J].黑龙江畜牧兽医, 2020, 16:65-68. LIU H Y, YANG Y W, WANG Y W, et al.Analysis of serotypes, drug resistance, and pathogenicity of six Pasteurella multocida isolates from goats[J].Heilongjiang Animal Science and Veterinary Medicine, 2020, 16:65-68.(in Chinese)
[8] SARANGI L N, THOMAS P, GUPTA S K, et al.Virulence gene profiling and antibiotic resistance pattern of Indian isolates of Pasteurella multocida of small ruminant origin[J].Comparative Immunology Microbiology and Infectious Diseases, 2015, 38:33-39.
[9] TANG X, ZHAO Z, HU J, et al.Isolation, antimicrobial resistance, and virulence genes of Pasteurella multocida strains from swine in China[J].Journal of Clinical Microbiology, 2009, 47(4):951-958.
[10] 王镭.猪源多杀性巴氏杆菌A型、D型分离株病原学特性比较研究[D].合肥:安徽农业大学, 2022. WANG L.Comparison of pathogenicity characteristics of Pasteurella multocida serotype A and D in swine[D].Hefei:Anhui Agricultural University, 2022.(in Chinese)
[11] DAYAO D A E, GIBSON J S, BLACKALL P J, et al.Antimicrobial resistance genes in Actinobacillus pleuropneumoniae, Haemophilus parasuis and Pasteurella multocida isolated from Australian pigs[J].Australian Veterinary Journal, 2016, 94(7):227-231.
[12] WANG Z, KONG L C, JIA B Y, et al.Aminoglycoside susceptibility of Pasteurella multocida isolates from bovine respiratory infections in China and mutations in ribosomal protein S5 associated with high-level induced spectinomycin resistance[J].Journal of Veterinary Medical Science, 2017, 79(10):1678-1681.
[13] 陈国权, 张旭, 阎朝华, 等.鸭源多杀性巴氏杆菌的分离鉴定及耐药性分析[J].中国畜牧兽医, 2020, 47(2):620-628. CHEN G Q, ZHNAG X, YAN Z H, et al.Isolation, identification and drug resistance analysis of Pasteurella multocida from ducks[J].China Animal Husbandry & Veterinary Medicine, 2020, 47(2):620-628.(in Chinese)
[14] UJVARIJ B, MAKRIA L, MAGYAR T.Virulence gene profiling and ompA sequence analysis of Pasteurella multocida and their correlation with host species[J].Veterinary Microbiology, 2019, 233:190-195.
[15] SMITH E, MILLER E, AGUAYO J M, et al.Genomic diversity and molecular epidemiology of Pasteurella multocida[J]. PLoS One, 2021, 16(4):e0249138.
[16] ALDEEWAN A B, SAYHOOD M H, ABBAS B A.Isolation and identification of Pasteurella multocida from healthy and clinical cases of sheep and study their antimicrobial sensitivity[J].Journal of Wassit for Science & Medicine, 2019, 12(1):56-67.
[17] MOMBENI E G, GHARIBI D, GHORBANPOOR M, et al.Toxigenic and non-toxigenic Pasteurella multocida genotypes, based on capsular, LPS, and virulence profile typing, associated with pneumonic pasteurellosis in Iran[J].Veterinary Microbiology, 2021, 257:109077.
[18] PENG Z, WANG H, LIANG W, et al.A capsule/lipopolysaccharide/MLST genotype D/L6/ST11 of Pasteurella multocida is likely to be strongly associated with swine respiratory disease in China[J].Archives of Microbiology, 2018, 200(1):107-118.
[19] LI R C, FANG C, LUO W, et al.Capsular types, LPS genotypes, and virulence-associated genes of Pasteurella multocida strains isolated from pigs[J].Medycyna Weterynaryjna, 2020, 76(8):5.
[20] SMITH E, MILLER E, AGUAYO J M, et al.Genomic diversity and molecular epidemiology of Pasteurella multocida[J].PLoS One, 2021, 16(4):e0249138.
[21] PETROCCHI-RILO M, GUTIERREZ-MARTIN C B, PEREZ-FERNANDEZ E, et al.Antimicrobial resistance genes in porcine Pasteurella multocida are not associated with its antimicrobial susceptibility pattern[J].Antibiotics (Basel), 2020, 9(9):614.
[22] VU-KHAC H, TRINH T T H, NGUYEN T T G, et al.Prevalence of virulence factor, antibiotic resistance, and serotype genes of Pasteurella multocida strains isolated from pigs in Vietnam[J].Veterinary World, 2020, 13(5):896.