致狐狸眼炎大肠杆菌的生物学特性检测与致病性分析
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摘要
为确定辽宁省某狐狸养殖场致患病狐狸眼炎的病原菌,从患病狐狸眼部分离病原菌,并对分离株进行形态观察、生化试验、16S rRNA基因序列测定、血清分型及进化分群,在此基础上完成了毒力基因检测、药敏试验以及人工感染小鼠试验等。镜检结果显示,该细菌为革兰阴性短杆菌,最终鉴定结果可以确定此分离株为O25型大肠杆菌,并且2株菌均属于系统发育群B2群。毒力基因检测结果显示,其含有CNF-Ⅰ基因,无LT、K99、STa、SLT-Ⅰ、CNF-Ⅱ及EAE等毒力基因。人工感染小鼠试验显示,2株分离菌株均可引起小鼠眼睛发炎,化脓等疾病,其对小鼠的半数致死量(LD50)分别为3.60×10~6CFU/只、1.79×10~6 CFU/只。药敏试验表明,其对庆大霉素、环丙沙星、黏菌素及氯霉素耐药性较强,但对头孢噻肟、磺胺甲恶唑、四环素、左氧氟沙星、氟苯尼考及大观霉素等抗生素较为敏感。本研究结果为临床治疗由大肠杆菌引起的狐狸眼炎提供了重要依据。
In order to determine the pathogens of the diseased foxes in Liaoning Province,the pathogens were isolated from the diseased eyes of foxes,and the isolates were subjected to morphological observation,biochemical test,16 S r RNA identification,molecular typing and phylogenetic group analysis.Basis on this,the virulence genes test,drug susceptibility test and artificial infection were completed.In result,the isolates were gram-negative,and the serogroups were O25,and both strains belonged to B2 group.The virulence genes test showed the isolates had CNF-Ⅰgene,and had no LT,K99,STa,STb,SLT-Ⅰ,CNF-Ⅱand EAE virulence genes.Tests on artificial infection showed that the isolates could cause eye inflammation,suppuration and other diseases in mice.The LD50 were 3.60 ×10~6 CFU/mice and1.79×10~6 CFU/mice.The drug sensitivity test showed that the isolates were highly resistant to gentamicin,ciprofloxacin,colistin and chloramphenicol,but sensitive to cefotaxime,sulfamethoxazole, tetracycline,levofloxacin,flufenicol and macroamycin.These study results provide an important basis for clinical treatment of ocular inflammatory disease caused by E.coli in foxes.
In order to determine the pathogens of the diseased foxes in Liaoning Province,the pathogens were isolated from the diseased eyes of foxes,and the isolates were subjected to morphological observation,biochemical test,16 S r RNA identification,molecular typing and phylogenetic group analysis.Basis on this,the virulence genes test,drug susceptibility test and artificial infection were completed.In result,the isolates were gram-negative,and the serogroups were O25,and both strains belonged to B2 group.The virulence genes test showed the isolates had CNF-Ⅰgene,and had no LT,K99,STa,STb,SLT-Ⅰ,CNF-Ⅱand EAE virulence genes.Tests on artificial infection showed that the isolates could cause eye inflammation,suppuration and other diseases in mice.The LD50 were 3.60 ×10~6 CFU/mice and1.79×10~6 CFU/mice.The drug sensitivity test showed that the isolates were highly resistant to gentamicin,ciprofloxacin,colistin and chloramphenicol,but sensitive to cefotaxime,sulfamethoxazole, tetracycline,levofloxacin,flufenicol and macroamycin.These study results provide an important basis for clinical treatment of ocular inflammatory disease caused by E.coli in foxes.
引文
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[28]李清泉,王忠.林可霉素与大观霉素复方制剂的临床应用[J].兽医导刊,2012(S1):94-95.LI Qing-quan,WANG Zhong.Clinical application of lincomycin and macroamycin compound preparation[J].Veterinary Orientation,2012(S1):94-95.(in Chinese)
[2]尹姣姣,何敏,翟晶晶,等.晋中地区鸡致病性大肠杆菌分离株的鉴定及其16S rDNA序列的同源性分析[J].中国兽医科学,2019,49(3):341-346.YIN Jiao-jiao,HE Min,ZHAI Jing-jing,et al.Identification and 16S rDNA sequence homology analysis of pathogenic Escherichia coli isolates of chickensinJinzhong District[J].ChineseVeterinaryScience,2019,49(3):341-346.(in Chinese)
[3]王海永.毛皮动物大肠杆菌病防治[J].中国畜禽种业,2017,13(3):126.WANG Hai-yong.Prevention and treatment of Escherichia coli disease in fur animals[J].The Chinese Livestock and Poultry Breeding,2017,13(3):126.(in Chinese)
[4]DEBORY C,FRATAMICO P M,YAN X,et al.Comparison of O-antigen gene clusters of all O-serogroups of Escherichia coli and proposal for adopting a new nimenclature for O-typing[J].PLoS One,2016,11(1):e0147434.
[5]WANG S,MENG Q,DAI J,et al.Deveiopment of an allele-specific PCR assay for simultaneous sero-typing of avian pathogenic Escherichia coli predominant O1,O2,O18 and O78 strains[J].PLoS One,2014,9(5):e96904.
[6]李天芝,于新友.仔猪水肿病大肠杆菌O8型的分离与鉴定[J].养猪,2016(4):118-120.LI Tian-zhi,YU Xin-you.Isolation and identification of Escherichia coli serotype O8 from edema disease of pigs[J].Swine Production,2016(4):118-120.(in Chinese)
[7]JOHNSON J R,OSWALD E,O’BRYAN T T,et al.Phylogentic distribution of virulence-associated genes among Escherichia coli isolates associated with neonatal bacterial meningitis in the Netherlands[J].The Journal of Infectious Diseases,2002,185(6):774-784.
[8]STANNARIUS C,ZWEIFEL C,REGULA G,et al.Antimicrobial resistance in Escherichia coli strains isolated from swiss weaned pigs and sows[J].Schweizer Archiv für Tierheilkunde,2009,151(3):119-125.
[9]TIBBETTS R J,WHITE D G,DYER N W,et al.Charaterization of Escherichichia coli isolates incriminated in colisepticaemia in mink[J].Veterinary Research Communications,2003,27(5):341-357.
[10]IGUCHI A,LYODA S,SETO K,et al.Esherichia coli O-genotyping PCR:a comprehensive and practical platform for molecular O serogroupin[J].Journal of Clinical Microbiology,2015,53(8):2427-2432.
[11]CLERMON O,CHRISTENSON J K,DENAMUR E,et al.The clermont Escherichia coli phylo-typing method revisited:improvement of specificity and detection of new phylo-groups[J].Environmental Microbiology Reports,2013,5(1):58-65.
[12]CLERMONT O,BONACORSI S,BINGEN E,et al.Rapid and simple determination of the Escherichia coli phylogenetic group[J].Applied and Environmental Microbiology,2000,66(10):4555-4558.
[13]CLERMONT O,BONACORSI S,BINGEN E.Characterization of an anonymous molecular marker strongly linked to Escherichia coli strains causing neonatal meningitis[J].JournalofClinicalMicrobiology,2004,42(4):1770-1772.
[14]OJENIYA B,AHRENS P,MEYLING A,et al.Detection of fimbrial and toxin genes in Escherishia coli and their prevalence in piglets with diarrhoea.The application of colony hybridization assay,polymerase chain reation and phenotypic assays[J].Journal of Veterinary Medicine.Series B,1994,41(1):49-59.
[15]GANNON V P,RASHAD M,KING R K,et al.Detection and characterization of the eae gene of shiga-like toxin-producing Escherichia coli using polymerase chain reaction[J].Journal of Clinical Microbiology,1993,31(5):1268-1274.
[16]BLANCO M,BLANCO J E,BLANCO J,et al.Polymerase chain reaction for detection of Escherichia coli strains producing cytotoxic necrotizing factor type 1 and type 2(CNF1 and CNF2)[J].Journal of Microbiological Methods,1996,26:95-101.
[17]LEIMBACH A,HACKER J,DOBRINDT U.E.coli as an all-rounder:The thin line between commensalism and pathogenicity[J].Current Topics in Microbiologyand Immunology,2013,358:3-32.
[18]WANG M C,TSENG C C,WU A B,et al.Different roles of host and bacterial factors in Escherichia coli extra-intestinal infections[J].European Journal of Clinical Microbiology&Infectious Diseases,2009,15(4):372-379.
[19]VAN TYNE D,CIOLINO J B,WANG J,et al.Novel phagocytosis-resistant extended-spectrumβ-lactamase-pro ducing Escherichia coli from keratitis[J].JAMAOphthalmology,2016,134(11):1306-1309.
[20]RANJITH K,ARUNASRI K,REDDY G S,et al.Global gene expression in Escherichia coli,isolated from the diseased ocular surface of the human eye with a potential to form biofilm[J].Gut Pathogens,2017,9:15.
[21]林漫亚.鸡眼炎型大肠杆菌病的诊治[J].浙江畜牧兽医,2017,42(6):45.LIN Man-ya.Diagnosis and treatment of corns type Escherichia coli[J].Zhejiang Journal Animal Science and Veterinary Medicine,2017,42(6):45.(in Chinese)
[22]MUSILLI M,CIOTTI M T,PIERI M,et al.Therapeutic effects of the Rho GTPase modulator CNF1 in a model of Parkinson’s disease[J].Neuropharmacology,2016,109:357-365.
[23]MALORNI W,FIORENTINI C.Is the Rac GTPase-activating toxin CNF1 a smart hijacker of host cell fate[J].Federation of American Societies for Experimental Biology Journal,2006,20:606-609.
[24]SMATI M,CLERMONT O,BLEIBRTIU A,et al.Quantitative analysis of commensal Escherichia coli populations reveals host-specific enterotypes at the intra-species level[J].Microbiology Open,2015,4(4):604-615.
[25]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.LIU Zheng-ming,LI Jin-quan,HUANG De-hao,et al.Study on the drug-resistantce of Escherichia coli isolated from sheep in Inner Mongolia[J].China Animal Husbandry&Veterinary Medicine,2017,44(3):839-846.(in Chinese)
[26]LANTOS J,HEGED譈S M,ZSIG魷M.Escherichia coli strains isolated from surface waters.Distribution by resistance to antibiotics and R-plasmid transfer[J].Acta Microbiologica Academiae Scientiarum Hungaricae,1982,29(3):161-171.
[27]曹军平,姜小勇,麦倍华,等.鸡眼炎型大肠杆菌分离鉴定与药敏试验[J].中国动物检疫,2006(9):36-37.CAO Jun-ping,JIANG Xiao-yong,MAI Bei-hua,et al.Isolation and identification of Escherichia coli with corns and drug susceptibility test[J].China Animal Health Inspection,2006(9):36-37.(in Chinese)
[28]李清泉,王忠.林可霉素与大观霉素复方制剂的临床应用[J].兽医导刊,2012(S1):94-95.LI Qing-quan,WANG Zhong.Clinical application of lincomycin and macroamycin compound preparation[J].Veterinary Orientation,2012(S1):94-95.(in Chinese)