1株牛源D:L3:L5型多杀性巴氏杆菌的分离鉴定及病原特性评价

doi:10.16431/j.cnki.1671-7236.2024.07.037

摘要/Abstract

摘要： 【目的】通过探讨青海省西宁市某规模化奶牛场病死牛源多杀性巴氏杆菌的荚膜血清型、耐药性和毒力基因分布情况,为多杀性巴氏杆菌引起的牛呼吸道疾病的治疗和预防提供参考。【方法】对病死牛肺脏样本进行细菌分离,通过菌落观察和染色镜检、生化鉴定、特异性引物扩增、16S rRNA测序分析及系统发育树构建、荚膜血清及脂多糖分型、毒力基因检测、致病性试验和药敏试验对分离菌进行鉴定和病原特性评价。【结果】肺脏样本中分离得到的菌落呈现灰白色中间微凹陷的露珠样,未出现溶血环;革兰染色可见粉红色球杆菌;瑞氏染色可见两端浓染的蓝色球杆菌。生化鉴定结果显示,分离菌株D-葡萄糖、D-甘露醇、D-甘露糖、酪氨酸芳胺酶、磷酸酶、COURMARATE和ELLMAN反应结果为阳性;16S rRNA测序结果显示,分离菌株与多种多杀性巴氏杆菌相似性＞95%,鉴定分离得到的菌株为多杀性巴氏杆菌,命名为XN222。荚膜血清分型及脂多糖分型结果显示,分离菌株XN222的荚膜血清分型为D型,其脂多糖分型为L3和L5型。毒力基因扩增结果显示,分离菌株XN222携带ptfA、fimA、hsf-2、sodA、tbpA、sodC等共计19种毒力基因。小鼠致病性试验结果显示,注射0.2 mL 1.5×10⁸ CFU/mL菌液,可导致100%(10/10)小鼠死亡,说明分离菌株XN222具有较强的致病能力。药敏试验结果显示,分离菌株XN222对头孢噻呋、左氧氟沙星、恩诺沙星、马波杀星、四环素和青霉素等15种抗菌药均敏感,对泰乐菌素中度敏感。【结论】本研究分离获得1株致病力较强的D型多杀性巴氏杆菌,该菌株的发现丰富了国内牛源多杀性巴氏杆菌亚型,可为多杀性巴氏杆菌引起的牛呼吸道疾病的防治、病原学调查和巴氏杆菌多价苗研制等提供生物素材。

关键词: D型多杀性巴氏杆菌; 分离鉴定; 病原特性

Abstract: 【Objective】 The experiment was aimed to explore the distribution of capsule serotypes,drug resistance and virulence genes of Pasteurella multocida from a large-scale dairy farm in Xining city,Qinghai province,in order to provide reference for the treatment and prevention of respiratory diseases caused by Pasteurella multocida in cattle.【Method】 Bacteria were isolated from the lung sample of a dead cattle.And then,through the methods of colony observation,dyeing microscopy,biochemical identification,specific primer amplification,16S rRNA clone sequencing analysis,phylogenetic tree construction,capsular serum and lipopolysaccharide type,virulence gene detection,pathogenicity test and drug sensitivity test,the isolated Pasteurella multocida was identified and its pathogen characteristics were evaluated.【Result】 The colonies isolated from the lung samples were dewdrop-like with a slight depression in the gray middle,and no hemolysis ring appeared.Gram staining showed coccidia pink,Wright’s staining showed blue coccidia with dense staining at both ends.Biochemical identification showed that the results of D-glucose,D-mannitol,D-mannose,tyrosine arylaminase,phosphatase,COURMARATE and ELLMAN reaction were positive.16S rRNA sequencing results showed that the strain was more than 95% similar to a variety of Pasteurella multocida,and the isolated strain was identified as Pasteurella multocida and named XN222.The results of capsule serum typing and lipopolysaccharide type showed that XN222 was classified as type D,and its lipopolysaccharide types were L3 and L5.The results of virulence gene amplification showed that XN222 carried 19 virulence genes,including ptfA,fimA,hsf-2, sodA, tbpA, sodC,and so on.The results of pathogenicity test in mice showed that injection of 0.2 mL 1.5×10⁸ CFU/mL bacterial solution resulted in 100% (10/10) death of mice,indicating that the isolated strain XN222 had strong pathogenicity. The results of drug sensitivity test showed that XN222 was sensitive to 15 kinds of antimicrobial agents,such as ceftiofur,levofloxacin,enrofloxacin,marbofloxacin,tetracycline and penicillin,and was moderately sensitive to tylosin.【Conclusion】 In this study,a highly pathogenic Pasteurella multocida type D strain was isolated.The discovery of this strain enriched the domestic bovine subtype of Pasteurella multocida,and provided biological materials for the prevention and control of bovine respiratory diseases caused by Pasteurella multocida,etiological investigation and development of Pasteurella multocida multivalent vaccine.

Key words: Pasteurella multocida type D; isolation and identification; pathogen characteristics

中图分类号:

S852.61

赵奕朵, 张康, 王磊, 路晓荣, 张景艳, 程佳, 郭志廷, 徐国伟, 黄莉萍, 牛宇辉, 马婷, 冯晓微, EGIDE, 刘明超, 李建喜. 1株牛源D:L3:L5型多杀性巴氏杆菌的分离鉴定及病原特性评价[J]. 中国畜牧兽医, 2024, 51(7): 3128-3141.

ZHAO Yiduo, ZHANG Kang, WANG Lei, LU Xiaorong, ZHANG Jingyan, CHENG Jia, GUO Zhiting, XU Guowei, HUANG Liping, NIU Yuhui, MA Ting, FENG Xiaowei, EGIDE, LIU Mingchao, LI Jianxi. Isolation,Identification and Pathogen Characteristics Evaluation of a Strain of Pasteurella multocida D:L3:L5 from Cattle[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(7): 3128-3141.

参考文献

[1] SMITH E,MILLER E,AGUAYO J M,et al.Genomic diversity and molecular epidemiology of Pasteurella multocida[J].PLoS One,2021,16(4):e249138.
[2] DIGIACOMO R F,XU Y M,ALLEN V,et al.Naturally acquired Pasteurella multocida infection in rabbits:Clinicopathological aspects[J]. Canadian Journal of Veterinary Research,1991,55(3):234-238.
[3] CALDERÓN BERNAL J M,SERNA C,GARCÍA MUÑOZÁ,et al.Genotypic comparison of Pasteurella multocida from healthy animals at entry to the feedlots with that and from bovine respiratory disease-affected animals during the fattening period[J]. Animals,2023,13(17):2687.
[4] PIVA M M,SCHWERTZ C I,BIANCHI R M,et al.Pasteurella multocida polyserositis in growing-finishing pigs[J].Journal of Comparative Pathology,2023,202:16-22.
[5] HARPER M,JOHN M,TURNI C,et al.Development of a rapid multiplex PCR assay to genotype Pasteurella multocida strains by use of the lipopolysaccharide outer core biosynthesis locus[J].Journal of Clinical Microbiology,2015,53(2):477-485.
[6] 伍澜,李佑君,梁雄燕,等.一株鸡源多杀性巴氏杆菌的分离鉴定[J].黑龙江畜牧兽医,2022,13:71-76. WU L,LI Y J,LIANG X Y,et al.Isolation and identification of a chicken-derived Pasteurella multocida[J].Heilongjiang Animal Science and Veterinary Medicine,2022,13:71-76.(in Chinese)
[7] HARPER M,COX A D,ADLER B,et al.Pasteurella multocida lipopolysaccharide:The long and the short of it[J].Veterinary Microbiology,2011,153(1-2):109-115.
[8] 邬琴,张星星,顾晓晓,等.牛源多杀性巴氏杆菌血清分型及毒力相关基因的检测研究[J].中国畜牧兽医,2020,47(6):1910-1920. WU Q,ZHANG X X,GU X X,et al.Study on serotyping and detection of virulence-associated genes of Pasteurella multocida isolated from bovine[J].China Animal Husbandry&Veterinary Medicine,2020,47(6):1910-1920.(in Chinese)
[9] LIVINGSTONE M,JORGENSEN P,MCCALL M,et al.De novo whole-genome sequencing of two pathogenic Pasteurella multocida type D:6 strains isolated from pigs[J].Microbiology Resource Announcements,2023,12(5):e0009823.
[10] 贡嘎,王一飞,格桑卓玛,等.1株藏猪源荚膜血清D型多杀性巴氏杆菌的分离鉴定及其生物学特性[J].浙江大学学报(农业与生命科学版),2020,46(5):611-617. GONGGA,WANG Y F,GESANGZHUOMA,et al.Identification of capsular serotype D Pasteurella multocida isolated from Tibetan swine and its biological characteristics[J].Journal of Zhejiang University(Agriculture and Life Sciences),2020,46(5):611-617.(in Chinese)
[11] 唐军林,付磊,荆婷婷,等.1株山羊源荚膜D型多杀性巴氏杆菌的分离鉴定[J].畜牧与兽医,2022,54(9):90-96. TANG J L,FU L,JING T T,et al.Isolation and identification of a capsular D-type Pasteurella multocida from goat[J].Animal Husbandry&Veterinary Medicine,2022,54(9):90-96.(in Chinese)
[12] 周海琴,梁晏,孙琼飞,等.1株羊源多杀性巴氏杆菌的分离鉴定及生物学特性研究[J].畜牧与兽医,2022,54(7):88-95. ZHOU H Q,LIANG Y,SUN Q F,et al.Isolation,identification and biological characteristics of Pasteurella multocida from sheep[J].Animal Husbandry&Veterinary Medicine,2022,54(7):88-95.(in Chinese)
[13] DU S,XU F,LIN Y,et al.Detection of Porcine circovirus type 2a and Pasteurella multocida capsular serotype D in growing pigs suffering from respiratory disease[J].Veterinary Sciences,2022,9(10):528.
[14] 王蕾,邬琴,柴迎锦,等.羊源D型多杀性巴氏杆菌的分离鉴定及毒力基因检测[J].动物医学进展,2019,40(1):25-30. WANG L,WU Q,CHAI Y J,et al.Isolation and identification of Pasteurella multocida serotype D and detection of virulence genes from sheep[J].Progress in Veterinary Medicine,2019,40(1):25-30.(in Chinese)
[15] TOWNSEND K M,FROST A J,LEE C W,et al.Development of PCR assays for species-and type-specific identification of Pasteurella multocida isolates[J].Journal of Clinical Microbiology,1998,36(4):1096-1100.
[16] KHAMESIPOUR F,MOMTAZ H,AZHDARY MAMOREH M.Occurrence of virulence factors and antimicrobial resistance in Pasteurella multocida strains isolated from slaughter cattle in Iran[J].Frontiers in Microbiology,2014,5:536
[17] KONG L,WANG Z,WANG Y,et al.Antimicrobial susceptibility and molecular typing of Pasteurella multocida isolated from six provinces in China[J].Tropical Animal Health and Production,2019,51(4):987-992.
[18] ZHANG K,ZHANG J,QIU Z,et al.Prevalence characteristic of BVDV in some large scale dairy farms in Western China[J].Frontiers in Veterinary Science,2022,9:961337.
[19] KOFTERIDIS D P,CHRISTOFAKI M,MANTADAKIS E,et al.Bacteremic community-acquired pneumonia due to Pasteurella multocida[J].International Journal of Infectious Diseases,2009,13(3):e81-e83.
[20] SANTANIELLO A,GARZILLO S,AMATO A,et al.Occurrence of Pasteurella multocida in dogs being trained for animal-assisted therapy[J].International Journal of Environmental Research and Public Health,2020,17(17):6385.
[21] DEWHIRST F E,PASTER B J,OLSEN I,et al.Phylogeny of 54 representative strains of species in the family Pasteurellaceae as determined by comparison of 16S rRNA sequences[J].Journal of Bacteriology,1992,174(6):2002-2013.
[22] KORCZAK B,CHRISTENSEN H,EMLER S,et al.Phylogeny of the family Pasteurellaceae based on rpoB sequences[J].International Journal of Systematic and Evolutionary Microbiology,2004,54(4):1393-1399.
[23] 郭伟娜,赵霞,路振香.兔源多杀性巴氏杆菌的分离鉴定及耐药性分析[J].中国兽医杂志,2023,59(4):59-62. GUO W N,ZHAO X,LU Z X.Isolation,identification and drug resistance analysis of Pasteurella multocida from rabbits[J].Chinese Journal of Veterinary Medicine,2023,59(4):59-62.(in Chinese)
[24] ELSAYED M S A E,ELDSOUKY S M,ROSHDY T,et al.Virulence determinants and antimicrobial profiles of Pasteurella multocida isolated from cattle and humans in Egypt[J].Antibiotics,2021,10(5):480.
[25] CALDERÓN BERNAL J M,FERNÁNDEZ A,ARNAL J L,et al.Molecular epidemiology of Pasteurella multocida associated with bovine respiratory disease outbreaks[J].Animals,2023,13(1):75.
[26] HARPER M,BOYCE J.The myriad properties of Pasteurella multocida lipopolysaccharide[J].Toxins,2017,9(8):254.
[27] QIU R,WEI H,HU B,et al.Experimental pathogenicity and comparative genome analysis of high-and low-virulence strains of rabbit-origin Pasteurella multocida[J].Comparative Immunology,Microbiology and Infectious Diseases,2022,90-91:101889.
[28] 黄萃,伍思华,李钰,等.乌鸡源多杀性巴氏杆菌杀禽亚种16S rDNA鉴定及药敏分析[J].生物灾害科学,2019,42(3):243-247. HUANG C,WU S H,LI Y,et al.16S rDNA identification and drug sensitivity analysis of Pasteurella multocida subsp.Gallicida[J].Biological Disaster Science,2019,42(3):243-247.(in Chinese)
[29] VILARÓ A,NOVELL E,ENRIQUE-TARANCON V,et al.Susceptibility trends of swine respiratory pathogens from 2019 to 2022 to antimicrobials commonly used in Spain[J].Porcine Health Management,2023,9(1):47.
[30] TABATABAEI M,ABDOLAHI F.Molecular evaluation of sheep and goats isolates of Pasteurella multocida and their antibiotic resistance[J]. Veterinary Research Forum,2023,14(9):481-487.
[31] SAHOO M,BALONI S,THAKOR J C,et al.Pathology,virulence-associated gene profiling,antimicrobial susceptibility,and pathogenicity of untypeable capsular serotypes of Pasteurella multocida isolated from slaughtered pigs of India[J].Letters in Applied Microbiology,2023,76(10):ovad112.