新疆维吾尔自治区塔额垦区腹泻羔羊粪源大肠杆菌脂多糖核心型和致病性分析

doi:10.12160/j.issn.1672-5190.2024.06.017

摘要/Abstract

摘要： [目的] 了解新疆维吾尔自治区塔额垦区腹泻羔羊粪源大肠杆菌LPS核心型、毒力基因和致病性。[方法] 从塔额垦区采集415份羔羊腹泻样本,使用鉴别培养基分离大肠杆菌,通过大肠杆菌特异性基因phoA扩增和16S rDNA序列分析对分离菌株进行分子生物学鉴定;利用PCR技术确定分离株LPS核心型和毒力基因携带情况;采用Fisher′s exact test方法分析分离株LPS核心型与毒力基因之间的相关性;通过小鼠感染试验确定分离株的致病性。[结果] 从415份样本中分离、鉴定到73株大肠杆菌,分离率为17.6%（73/415）;73株大肠杆菌LPS核心型主要为R1型（57.53%）,其次为R3型（24.66%）,R4型（10.96%）、K12型（5.48%）、R2型（1.37%）均有检出;在检测的25种毒力基因中共检测出13种毒力基因,其中,mat（98.63%）、yijP（91.78%）、ibeB（90.41%）等基因的检出率较高,irp2（16.44%）、iucD（9.59%）、stx1（2.74%）基因的检出率较低;LPS核心型与毒力基因无明显相关关系;随机选取的9株菌株可导致小鼠腹泻和死亡,对小鼠具有较强的致病性。[结论] 塔额垦区腹泻羔羊粪源大肠杆菌LPS分型多样,携带多种毒力基因,具有一定的致病性,在生产中应当予以重视。

关键词: 羔羊, 大肠杆菌, LPS核心型, 毒力基因, 致病性

Abstract: [Objective] The aim of this study was to understand the core type, virulence genes and pathogenicity of Escherichia coli LPS of faecal origin in diarrhoeic lambs in Ta′e Reclamation Area, Xinjiang Uygur Autonomous Region. [Method] Four hundred and fifteen samples of diarrhoeic lambs were collected from Ta′e Reclamation Area, followed by isolation of Escherichia coli using identification medium. Then, the isolates were identified molecularly by amplification of the specific gene phoA of E. coli and 16S rDNA sequence analysis. PCR was used to determine the LPS core type and virulence gene carriage of the isolates. The correlation between LPS core type and virulence gene of the isolates was analyzed by Fisher′s exact test. The pathogenicity of the isolates was determined by mouse infection test. [Result] Seventy-three E. coli strains were isolated and identified from 415 samples with an isolation rate of 17.6% （73/415）. The Seventy-three E. coli LPS core types were mainly R1 （57.53%）, followed by R3 （24.66%）, R4 （10.96%）, K12 （5.48%） and R2 （1.37%）. A total of 13 virulence genes were detected among the 25 virulence genes tested, with higher detection rates for genes such as mat （98.63%）, yijP （91.78%), and ibeB （90.41%）, and lower detection rates for genes irp2 （16.44%）, iucD （9.59%）, and stx1 （2.74%）. No significant correlation was found between LPS core type and virulence genes. In addition, nine randomly selected strains were found to cause diarrhea and death in mice and were highly pathogenic to mice. [Conclusion] The above results indicate that LPS of E. coli from lambs in the Ta′e Reclamation Area is characterized by diverse phenotypes, carries various virulence genes, and has a certain pathogenicity, which should be considered in the production.

Key words: lambs, Escherichia coli, LPS core type, virulence genes, pathogenicity

中图分类号:

杨彬, 王继学, 邢国锋, 赵耀, 吴自豪, 吴静. 新疆维吾尔自治区塔额垦区腹泻羔羊粪源大肠杆菌脂多糖核心型和致病性分析[J]. 畜牧与饲料科学, 2024, 45(6): 122-128.

YANG Bin, WANG Jixue, XING Guofeng, ZHAO Yao, WU Zihao, WU Jing. Analysis of Fecal Source Escherichia Coli Lipopolysaccharide Core Type and Pathogenicity in Diarrhoeic Lambs from Ta′e Reclamation Area, Xinjiang Uygur Autonomous Region[J]. Animal Husbandry and Feed Science, 2024, 45(6): 122-128.

参考文献

[1] SHAHZAD A, ULLAH F, IRSHAD H, et al.Molecular detection of Shiga toxin-producing Escherichia coli (STEC) O157 in sheep, goats, cows and buffaloes[J].Molecular Biology Reports,2021,48(8):6113-6121.
[2] 刘正明,李金泉,黄德浩,等.内蒙古地区羊源大肠杆菌耐药性研究[J].中国畜牧兽医,2017,44(3):839-846.
[3] 陈家露,蔡重振,次旦,等.西藏那曲市羊源大肠杆菌分离鉴定及耐药性研究[J].中国畜牧兽医,2019,46(12):3759-3767.
[4] 宋晓莉,成大荣,刘晓明,等.临床腹泻肉羊源大肠杆菌流行病学调查及耐药性分析[J].黑龙江畜牧兽医,2019(23):71-75,80.
[5] 张学勇,简莹娜,李秀萍,等.腹泻羔羊中大肠杆菌的分离鉴定及耐药基因检测分析[J].青海畜牧兽医杂志,2017,47(5):27-34.
[6] 张宇曦,韩先干,左佳坤,等.禽致病性大肠杆菌脂多糖核心型分布与毒力基因的相关性分析[J].微生物学通报,2015,42(8):1619-1625.
[7] AMOR K, HEINRICHS D E, FRIRDICH E, et al.Distri bution of core oligosaccharide types in lipopolysaccharides from Escherichia coli[J].Infection and Immunity,2000,68(3):1116-1124.
[8] LECLERCQ S O, BRANGER M, SMITH D G E, et al. Lipopolysaccharide core type diversity in the Escherichia coli species in association with phylogeny, virulence gene repertoire and distribution of type Ⅵ secretion systems[J].Microbial Genomics,2021,7(9):000652.
[9] SKIDMORE B J, MORRISON D C, CHILLER J M, et al.Immunologic properties of bacterial lipopolysaccharide (LPS). Ⅱ. The unresponsiveness of C3H/HeJ Mouse spleen cells to LPS-induced mitogenesis is dependent on the method used to extract LPS[J].The Journal of Experimental Medicine,1975,142(6):1488-1508.
[10] EWERS C, JANSSEN T, KIESSLING S, et al.Rapid detection of virulence-associated genes in avian pathogenic Escherichia coli by multiplex polymerase chain reaction[J].Avian Diseases,2005,49(2):269-273.
[11] 孟庆美,王少辉,韩先干,等.禽致病性大肠杆菌毒力基因多重PCR方法的建立和应用[J].微生物学报,2014,54(6):696-702.
[12] LORENZ S C, SON I, MAOUNOUNEN-LAASRI A, et al.Prevalence of hemolysin genes and comparison of ehxA subtype patterns in Shiga toxin-producing Escherichia coli (STEC) and non-STEC strains from clinical, food, and animal sources[J].Applied and Environmental Microbiology,2013,79(20):6301-6311.
[13] DOMINGOS M O, MELO K C M, NEVES I V, et al. Potential for colonization of O111∶H25 atypical enteropathogenic E. coli[J].Journal of Microbiology,2016,54(11):745-752.
[14] JOHNSON T J, WANNEMUEHLER Y M, NOLAN L K.Evolution of the iss gene in Escherichia coli[J].Applied and Environmental Microbiology,2008,74(8):2360-2369.
[15] BRANDAL L T, SEKSE C, LINDSTEDT B A, et al.Norwegian sheep are an important reservoir for human-pathogenic Escherichia coli O26∶H11[J].Applied and Environmental Microbiology,2012,78(12):4083-4091.
[16] 董贵,于月通,马志远,等.新疆维吾尔自治区和田地区规模化羊场腹泻羔羊源大肠杆菌的分离鉴定及毒力基因和耐药基因分析[J].畜牧与饲料科学,2023,44(6):116-122.
[17] 高海慧,张明艳,汪洁,等.腹泻绵羔羊源大肠杆菌耐药性及LEE毒力岛部分相关基因的检测[J].黑龙江畜牧兽医,2023(6):89-92,96,142.
[18] 张兴民,张焕容,毛从剑,等.四川部分地区山羊腹泻病主要病原菌的分离鉴定及耐药性检测[J].黑龙江畜牧兽医(下半月),2019(2):78-82.
[19] 尚立宏,张啸,许伟,等.甘肃部分羊场羔羊腹泻病原菌分离鉴定及其耐药性研究[J].中兽医医药杂志,2022,41(4):7-11.
[20] WU Z H, CHI H M, HAN T T, et al.Differences of Escherichia coli isolated from different organs of the individual sheep:Molecular typing, antibiotics resistance, and biofilm formation[J].Folia Microbiologica,2024,69(3):567-578.
[21] CURRIE C G, POXTON I R.The lipopolysaccharide core type of Escherichia coli O157∶H7 and other non-O157 verotoxin-producing E. coli[J].FEMS Immunology and Medical Microbiology,1999,24(1):57-62.
[22] GIBBS R J, STEWART J, POXTON I R.The distribution of, and antibody response to, the core lipopolysaccharide region of Escherichia coli isolated from the faeces of healthy humans and cattle[J].Journal of Medical Microbiology,2004,53(10):959-964.
[23] DISSANAYAKE D R A, WIJEWARDANA T G, GUNAWARDENA G A, et al. Distribution of lipopolysaccharide core types among avian pathogenic Escherichia coli in relation to the major phylogenetic groups[J].Veterinary Microbiology,2008,132(3/4):355-363.
[24] 王显峰,白金英.羊源大肠杆菌血清型鉴定、毒力基因检测及耐药性分析[J].畜牧与兽医,2020,52(3):69-72.
[25] ZHAO X L,LV Y Y, ADAM F E A, et al. Comparison of antimicrobial resistance, virulence genes, phylogroups, and biofilm formation of Escherichia coli isolated from intensive farming and free-range sheep[J].Frontiers in Microbiology,2021,12:699927.
[26] 李广西,韩婷婷,吴自豪,等.新疆维吾尔自治区阿克苏地区某屠宰场羊源大肠杆菌MLST分型、耐药性分析和毒力基因检测[J].畜牧与饲料科学,2024,45(2):95-102.