Immunogenicity of Rcombinant Type 1 Pilus Oil-emulsified Vaccine of Avian Escherichia coli in Chickens

[ Objective] To prepare recombinant type 1 pilus vaccine of avian Escherichia coil and to detect its immunogenicity in chickens. [Meth- od] The type 1 pilus was respectively isolated from the recombinant bacteria CZYR10 strain and wild avian Eschedchia co/i YR（ O18 ） strain and used to prepare oil-emulsified vaccine. The immunogenicity of the developed vaccine was detected in chickens using the challenge test. [ Result] The recombinant type 1 pilus protein had immunoprotective efficacy. The recombinant type 1 pilus protein had weaker protective efficacy than the isolated type 1 pilus protein, but the difference was not significant. [ Conclusion] The study provides a reference for immunization and control of chicken colibacillosis.

The virulence regulator AbsR in avian pathogenic Escherichia coli has pleiotropic effects on bacterial physiology

Avian pathogenic Escherichia coli(APEC)belonging to extraintestinal pathogenic E.coli(ExPEC)can cause severe infections in extraintestinal tissues in birds and humans,such as the lungs and blood.MprA(microcin production regulation,locus A,herein renamed AbsR,a blood survival regulator),a member of the MarR(multiple antibiotic resistance regulator)transcriptional regulator family,governs the expression of capsule biosynthetic genes in human ExPEC and represents a promising druggable target for antimicrobials.However,a deep understanding of the AbsR regulatory mechanism as well as its regulon is lacking.In this study,we present a systems-level analysis of the APEC AbsR regulon using ChIP-Seq(chromatin immunoprecipitation sequencing)and RNA-Seq(RNA sequencing)methods.We found that AbsR directly regulates 99 genes and indirectly regulates 667 genes.Furthermore,we showed that:1)AbsR contributes to antiphagocytotic effects by macrophages and virulence in a mouse model for systemic infection by directly activating the capsular gene cluster;2)AbsR positively impacts biofilm formation via direct regulation of the T2SS(type II secretion system)but plays a marginal role in virulence;and 3)AbsR directly upregulates the acid tolerance signaling system EvgAS to withstand acid stress but is dispensable in ExPEC virulence.Finally,our data indicate that the role of AbsR in virulence gene regulation is relatively conserved in ExPEC strains.Altogether,this study provides a comprehensive analysis of the AbsR regulon and regulatory mechanism,and our data suggest that AbsR likely influences virulence primarily through the control of capsule production.Interestingly,we found that AbsR severely represses the expression of the type I-F CRISPR(clustered regularly interspaced short palindromic repeats)-Cas(CRISPR associated)systems,which could have implications in CRISPR biology and application.

Drug Susceptibility Test of Volatile Oil of Artemisiaargyi to Avian Pathogenic Escherichia coli

The volatile oil of Artemisia argyi was extracted by ultrasonic assisted extraction, and the extraction rate of volatile oil was 0.68%. Thevolatile oil of A. argyi was emulsified with 1% Tween-80, and drug susceptibility test was conducted with avian Escherichia coli. The results showedthat the volatile oil of A. argyi had antibacterial effect against avian E. coli, and the minimal inhibitory concentration was 50 mg/mL. Taking sixcommon antibiotics as the control, drug susceptibility test was conducted with volatile oil of A. argyi. The results showed that 10 strains of E. coliwere sensitive to the volatile oil of A. argyi, three of which had different degrees of resistance and one had the tendency of resistance.

Distribution of Virulence-Associated Genes of Avian Pathogenic Escherichia coli Isolates in China

216 avian pathogenic Escherichia coli （APEC） isolates were obtained from poultry with colibacillosis in different areas of China. Among them, 195 were serotyped as 078, 088, and 093. Thirteen virulence-associated genes, including fimC, iucD, iss, tsh, fyuA, irp2, eaeA, hlyE, colV, papC, stx2f, vat, and astA, were submitted to PCR amplification. The fimC gene was the most prevalent with a detection rate of 93.6%, followed by iucD （70.8%）, iss （58.8%）, and tsh （51.4%） in APEC isolates. The detection rate of high pathogenicity islands （HPI）-associatedfyuA and irp2 genes were both 44.9%, with no LEE （the locus of enterocyte effacement） island-associated gene eaeA detected. In terms of distribution patterns of the 13 virulence-associated genes, 5 isolates harborbed 10 genes, 19 isolates contained onlyfimC gene, and only 4 isolates had no virulence-associated gene detected. Different correlations of the virulence-associated genes with O serotypes were also investigated and 50% 078 isolates had a gene distribution patterns of fimC^＋iucD^＋irp2^＋fyuA^＋iss^＋colV^＋tsh^＋.

Differentiation of Avian Pathogenic <i>Escherichia coli</i>Strains from Broiler Chickens by Multiplex Polymerase Chain Reaction (PCR) and Random Amplified Polymorphic (RAPD) DNA

We examined 50 Escherichia coli (E. coli) strains isolated from broiler chickens between January 2013 to March 2014 in order to evaluate the epidemiological prevalence of avian pathogenic E. coli (APEC) in Jordan by multiplex PCR and random amplification of polymorphic DNA (RAPD) tests. The multiplex polymerase chain reaction (PCR) which was used as tentative criteria of APEC targets 8 virulence associated genes;enteroaggregative toxin (astA), Type 1 fimbria adhesion (fimH), iron-repressible protein (irp2), P fimbriae (papC), aerobactin (iucD), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), and colicin V plasmid operon (cva/cvi) genes. The number of detected genes could be used as a reliable index of their virulence. E. coli strains already typed as an APEC always harbor 5 to 8 genes, but non-APEC strains harbor less than 4 genes. Assuming the criteria of an APEC is possession of 5 or more virulence associated genes;we found that all 50 E. coli strains were classified as APEC strains. The RAPD analysis showed that the E. coli strains could be grouped into 35 of RAPD types by using these two different RAPD primer sets, RAPD analysis primer 4 5'AAGAGCCCGT5', and RAPD analysis primer 6 5'CCCGTCAGCA3'. The current study confirmed the endemic nature of APEC in broiler flocks in Jordan. It is essential that the biosecurity on poultry farms should be improved to prevent the introduction and dissemination of APEC and other agents. Furthermore, farmers need to be educated about the signs, lesions, and the importance of this agent.

Fosfomycin Resistance in Avian Pathogenic Escherichia coli Isolates

Fosfomycin, a broad-spectrum antibiotic against both Gram-positive and Gram-negative bacteria, is very important in the clinic but many fosfomycin-resistant bacteria have been isolated from patients. In this study, the resistance mechanism of three fosfomycin-resistant avian pathogenic Escherichia coli （APEC） strains （JE1, IF7 and CD11） isolated from septicemic chickens were analyzed. The results showed that their fosfomycin-resistance mechanisms were different. An alteration in the glpT transport system was the main reason of the fosfomycin-resistance mechanisms of strain IF7. Compared with the control stain BL21, the capacity of fosfomycin-uptake was low in all these three stains （JE 1 〉IFT〉CD 11）. Sequence results of murA showed that there were more than 10 sites of nucleotide mutation, but only one amino acid mutation T116A showed in CD11. Real-time detection test showed that the expression level of the murA gene of the three stains was significantly increased （four times increase in strain CD11 and two times increase in strains JE1 and IFT）. The transformation and recombinant test showed that the recombinant bacteria with the murA of JE1 and CD11 showed high minimal inhibitory concentration （MIC） against fosfomycin. From the results of this research, it showed that most of the fosfomycin- resistance mechanisms once showed in patient bacteria have appeared in the APEC strains and the fosfomycin-resistance mechanism of the three APEC isolates was different.

Screening of Chinese Herbal Medicines against Avian Infectious Laryngotracheitis Virus and Escherichia coli

[ Objective] This study aimed to screen Chinese herbal medicines against avian infectious laryngotracheitis virus and Escherichia coli. [ Method ] Conventional Chinese medicine plate dilution method, plate punching method and test tube method were applied to screen Chinese herbal medicines against avian infectious laryngotracheitis virus based on chicken embryo inoculation experiment. [ Result ] Forsythia suspensa, Radix lsatidis, Isatis iadigotica, Lonicera japonica, Codonopsis pilosula, Astragalus membranaceus, Rhizoma Atractylodis Macrocephalae, C, tyeyrrhiza uralensis and Pericarpium granati had relatively strong anti-ILTV effect; among the Chinese herbal medicines against avian E. coli, Sanguisorba offwinalis, Fructus Mume, Rheum palmatum, Anemarrhena asphodeloides, Radix Scutellariae and Fagopyrum cymosum had relatively strong effect against avian E. coli Os, while other Chinese herbal medicines had relatively weak or no inhibitory effect on avian E. coli 0s and 05. [ Conclusion] This study laid the foundation for further development of Chinese herbal medicine compound preparations to treat avian infectious laryngotracheitis, avian colibacillosis and other viral diseases and bacterial diseases.

The Pathogenicity of Chicken Pathogenic <i>Escherichia coli</i>Is Associated with the Numbers and Combination Patterns of Virulence-Associated Genes

Various virulence-associated genes or pathogenicity island are responsible for determining the pathogenicity of Escherichia coli strains. However, the correlation of the number and combination patterns of virulence-associated genes in Escherichia coli strains with their pathogenicity remains largely unknown. In this work, 581 chicken Escherichia coli strains were isolated from 1045 liver samples of dead chickens from 50 chicken farms at four provinces in China during 2007-2012. Based on the pathogenic test of SPF chickens, 320 chickens pathogenic Escherichia coli isolates were identified as highly (n = 193), intermediate (n = 98) and low pathogenic (n = 29) strains, respectively. Furthermore, the number of virulence genes in the 320 chicken pathogenic and 50 non-pathogenic Escherichia coli strains was examined. Our results reveal that thirteen virulence genes in Escherichia coli strains were detected, and all strains carried at least two or more than two virulence-associated genes. This study also suggests that highly pathogenic E. coli strains simultaneously carried at least 8 to13 virulence genes while intermediate pathogenic strains carried at least 5 to 8 virulence genes. The number of virulence-associated genes detected in highly pathogenic strains showed there were more significant differences than that in low pathogenic strains (P irp2, fyuA, and colV in high pathogenic strains was significantly higher than that in low and non-pathogenic strains (P irp2, fyuA, iucA, iucD, iutA, papC, iss, tsh, and colV were more often detected in highly and intermediate pathogenic E. coli strains. Taken together, our results provide evidences demonstrating that the pathogenicity of Escherichia coli strains is closely associated with the number and combination patterns of virulence-associated genes.

携带bla_(CTX-M)禽大肠杆菌的多重耐药特征和体外致病性研究

【目的】探究携带bla_(CTX-M)禽大肠杆菌的分子流行特点。【方法】检测76株携带bla_(CTX-M)禽大肠杆菌的多重耐药谱、系统进化分群及毒力基因,并分析毒力基因与耐药特点、系统进化分群之间的关联性。【结果】药敏结果显示,携带bla_(CTX-M)禽大肠杆菌对氟苯尼考的耐药率最高,达85.53%(65/76);其次是恩诺沙星,为48.68%(37/76);最常见的多重耐药谱为氟苯尼考+恩诺沙星+乙酰甲喹,达10.53%(8/76)。系统进化分群结果显示,主要的亚群为C群和B1群;其中,C群检出率最高,达53.95%(41/76);其次是B1群,为30.26%(23/76);E群、A群、D群和F群的检出率分别为9.21%(7/76)、2.63%(2/76)、2.63%(2/76)和1.32%(1/76);未检测出B2群和分支I群。毒力基因检测结果显示,76株携带bla_(CTX-M)禽大肠杆菌均检出了至少4种毒力因子,且有11株菌同时携带至少11种毒力基因;其中,有13株菌同时检出禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)的5个标志性毒力基因(iroN、iutA、hly、OmpT和iss),检出率为17.11%;有11株菌检出ColV质粒的标志基因(cva/cvi、iroN、iucD/iutA、etsAC、OmpT/hlyF和sitA),检出率为14.47%;有28株菌检出耶尔森菌强毒力岛(high pathogenitility island,HPI)的标志基因irp-2和fyuA,检出率为36.82%。【结论】携带bla_(CTX-M)禽大肠杆菌多为多重耐药菌,且多重耐药菌株携带ColV毒力质粒可能性更高;携带bla_(CTX-M)禽大肠杆菌的多重耐药与致病性有关联,即携带bla_(CTX-M)禽大肠杆菌具有多重耐药的同时,具有致病的可能性更高。

禽致病性大肠杆菌HlyE蛋白的免疫原性研究

为评估禽致病性大肠杆菌(APEC)溶血素HlyE蛋白的免疫原性,本研究对APEC溶血素HlyE蛋白进行原核表达和纯化,并对表达的重组蛋白进行SDS-PAGE和western blot分析,将纯化蛋白利用透析袋在4℃透析后,利用血琼脂平板对其溶血活性进行检测。SDS-PAGE和western blot结果显示,表达并纯化到分子量约为36 ku的重组HlyE蛋白(rHlyE),经ND-2000超微量核酸蛋白测定仪测定纯化后蛋白浓度为0.65 mg/mL;溶血活性检测结果显示,rHlyE具有溶血活性。以透析后的rHlyE作为抗原,按照50μg/只的剂量免疫小鼠,对照组于相同时间点注射等量PBS,共免疫3次间隔14 d,并于首免后不同时间采血,采用间接ELISA方法检测两组小鼠血清特异性抗体水平,并于三免后18 d以2 LD_(50)的APEC菌液攻毒小鼠,观察7 d内小鼠的死亡情况;于首免后28 d剖杀各组小鼠取其脾脏制备脾淋巴细胞,采用流式细胞术分别检测CD3^(+)、CD4^(+)、CD8^(+)T淋巴细胞亚型比率,对rHlyE的免疫原性进行评估。间接ELISA检测结果显示,该蛋白能够诱导机体产生体液免疫应答,分泌高表达量的IgG抗体,抗体水平于三免后15 d达到最高水平。rHlyE免疫攻毒保护试验结果显示,免疫组小鼠基本无明显临床症状,7 d内存活率达80%;而对照组小鼠表现明显临床症状,于攻毒后3 d内全部死亡。流式细胞术结果显示,与对照组相比,免疫组小鼠的CD3^(+)、CD4^(+)和CD8^(+)T淋巴细胞亚型比率均升高。上述结果表明,rHlyE在大肠杆菌BL21中为部分可溶性表达,将其免疫小鼠后可诱导小鼠产生较高水平的体液免疫应答,并且可对小鼠产生较好的免疫保护效果。本研究明确了APEC HlyE蛋白的免疫原性,为APEC免疫保护蛋白的筛选以及疫苗研发提供了借鉴与参考。

天蚕素对H_(9)N_(2)亚型禽流感病毒和禽致病性大肠杆菌共感染肉鸡的干预作用

试验旨在探讨天蚕素对H_(9)N_(2)亚型禽流感病毒(AIV)与禽致病性大肠杆菌(APEC)共感染致炎性渗出的干预效果,为临床H_(9)N_(2)亚型AIV与APEC共感染的有效防治提供参考。试验选取150只14日龄白羽肉鸡,随机分为5组,每组3个重复,每个重复10只鸡。对照组肉鸡使用无菌生理盐水0.2 mL滴鼻,APEC感染组肉鸡使用含2.09×109 CFU APEC O2菌株肉汤0.2 mL滴鼻,H_(9)N_(2)感染组肉鸡使用H_(9)N_(2)病毒尿囊液(约105 EID50 H_(9)N_(2)病毒)0.2 mL滴鼻,H_(9)N_(2)+APEC感染组肉鸡使用0.2 mL APEC O2菌株肉汤离心后的沉淀与0.2 mL H_(9)N_(2)病毒尿囊液混匀得到的病毒尿囊液滴鼻,H_(9)N_(2)+APEC干预组肉鸡采用与H_(9)N_(2)+APEC感染组相同处理后,使用天蚕素饮水(300 mg/kg)+基础饲粮饲喂,其余各组正常饮水+基础饲粮饲喂。正式试验期21 d。结果显示,H_(9)N_(2)+APEC感染组肉鸡临床症状极为明显,死亡率显著升高(P<0.05),气管和肺脏病变最为严重。与H_(9)N_(2)+APEC感染组相比,H_(9)N_(2)感染组和APEC感染组在感染后第3、7、14、21 d时的H_(9)N_(2)病毒拷贝数和APEC载量均显著降低(P<0.05);H_(9)N_(2)+APEC干预组在感染后第3、7、14、21 d时的H_(9)N_(2)病毒拷贝数均显著降低(P<0.05),H_(9)N_(2)+APEC干预组在感染后第7、14、21 d时的APEC载量均显著降低(P<0.05)。研究表明,天蚕素可以缓解H_(9)N_(2)亚型AIV、APEC以及两者共感染引起的肉鸡呼吸道症状,降低死亡率,其机制与天蚕素降低病原在组织内的载量及修复呼吸系统、改善呼吸机能有关。

山东省禽源产超广谱β-内酰胺酶大肠杆菌的分子流行病学分析

【目的】分析山东省禽源产超广谱β-内酰胺酶(extended-spectrum β-lactamases, ESBLs)大肠杆菌的耐药情况、耐药机制、毒力基因、多位点序列分型(multilocus sequence typing, MLST)、亲缘性和Inc型质粒,为禽临床合理使用抗菌药物防治此类细菌病提供参考。【方法】复苏2021年4月-2021年7月分离自山东省禽泄殖腔拭子样本中的38株产ESBLs大肠杆菌,采用微量肉汤稀释法测定其对16种抗菌药物的敏感性,通过全基因组测序检测其携带的耐药基因、毒力基因、MLST、Inc型质粒,使用Parsnp构建菌株系统发育树。【结果】药敏试验结果显示,所有菌株对氨苄西林、头孢噻呋和头孢他啶耐药,对磺胺异噁唑(97.4%)、甲氧苄啶/磺胺甲噁唑(94.7%)、四环素(89.5%)、氟苯尼考(86.8%)、大观霉素(84.2%)耐药严重。全基因组分析结果显示,38株产ESBLs大肠杆菌的blaCTX-M型中blaCTX-M-55基因携带率为34.2%,blaNDM型中blaNDM-5和blaNDM-4基因携带率分别为21.1%和10.5%,其携带大量介导临床中常用抗菌药物的耐药基因和黏附类、侵袭类、血清抗性、铁转运类毒力基因。MLST结果显示,ST10(13.2%)为产ESBLs大肠杆菌最流行的ST型,其次是ST616(10.5%)和ST746(10.5%)。系统发育树分析表明,ST10、ST616和ST746分别属于同一起源。Inc型质粒结果显示,IncFIB(AP001918)携带率最高(68.4%),其次为IncHI2(42.1%)。【结论】山东省禽源产ESBLs大肠杆菌耐药状况严峻,blaCTX-M基因是导致大肠杆菌对超广谱β-内酰胺类抗菌药物耐药的主要原因,且该类菌株携带大量毒力基因,亟需对其加强监测。

禽致病性大肠埃希氏菌生物被膜形成的调控机制研究进展

禽致病性大肠埃希氏菌能够引起禽类感染性细菌病,给禽类养殖业造成了重大的经济损失,并严重限制了禽类养殖业的健康发展。为了了解禽致病性大肠埃希氏菌的感染机制,论文简述了生物被膜的形成过程及其危害。细菌生物被膜的形成不但增强细菌对抗菌药物的耐药性,而且导致宿主持续和反复性感染,同时还很难被预防、控制和清除。论文还分析了普遍存在于各类细菌中的重要调控系统——群体感应、双组份系统和第二信使的信号转导途径及其调控机制,并阐述了群体感应、双组份系统和第二信使以及两系统间的互作网络对细菌生物被膜的影响。因此,了解多系统共同调控生物被膜形成的分子机制或许可以作为破坏生物被膜形成的新策略,从而为控制由生物被膜引起的感染和抗菌药物耐药性提供研究方向。

雷州半岛地区禽源致病性大肠杆菌和沙门氏菌分离鉴定及耐药分析

为了了解雷州半岛雷州市规模化家禽养殖场腹泻家禽的细菌感染情况,试验对采集的20余份病料(脾脏、肝脏、心包液)进行病原菌的分离培养、革兰氏染色和镜检、麦康凯和伊红美蓝培养基筛选,对筛选到的病原菌进一步进行生化试验和16S rRNA鉴定确定其种类,最后对病原菌进行药敏试验和动物回归试验。结果表明:从病料中分离出31株大肠杆菌和10株沙门氏菌。分离得到的大肠杆菌对多黏菌素B的敏感率仅为58.0%,对头孢噻肟的敏感率为100%,对其他14种药物的耐药率在67.7%~100%之间;沙门氏菌对环丙沙星、多黏菌素B和头孢噻肟的敏感率分别为50.0%、80.0%和100%,对其他13种药物的耐药率在50.0%~100%之间。大肠杆菌和沙门氏菌均对雏鸭具有致病性,病理解剖肝脏和脾脏肿大,表面分布白色坏死点和坏死灶,病原菌分离呈阳性。说明在雷州半岛雷州市规模化家禽养殖场可选择多黏菌素B和头孢噻肟防治致病性大肠杆菌和沙门氏菌的感染。

禽大肠杆菌病的非抗生素物质防控研究进展

禽大肠杆菌病是危害养鸡业发展的主要细菌性传染病之一,给家禽产业带来了巨大的经济损失。抗生素在预防和治疗禽大肠杆菌病中有重要作用。然而,随着世界范围内在养鸡领域治疗性抗生素限用和饲用抗生素禁用措施的实施,禽大肠杆菌病的发病率在全球范围内呈上升趋势,对家禽生长与健康造成严重威胁。因此,迫切需要研发新的技术和产品代替抗生素来预防和控制禽大肠杆菌病。本文首先简介了禽大肠杆菌病的危害,然后重点综述了饲用抗生素替代物在预防和控制禽致病性大肠杆菌感染中的效果和作用机制,为应用非抗生素物质预防禽大肠杆菌病提供参考,为研发防控禽大肠杆菌病的新型非抗生素物质的提供借鉴。

禽多病因气囊炎病原的分离鉴定及耐药性分析

为了解广东省四会地区某鸡场多病因气囊炎的主要致病菌及其耐药情况。采用形态学鉴定、16S rRNA基因测序等方法对分离株进行鉴定,选取3种常用抗生素进行药物敏感性试验,进行耐药性研究。结果表明:第1株分离株菌落呈典型的“荷包蛋”状,第2株分离株为革兰氏阴性杆菌,第3株分离株为革兰氏阳性球菌,经16S rRNA基因测序比对分析,3种病原的同源性均在99%以上,确定多病因气囊炎的主要致病菌为鸡毒支原体、大肠埃希氏杆菌和金黄色葡萄球菌,分别命名为SH-MG株、SH-E株和SH-S株。药物敏感性结果表明,SH-E株对阿莫西林、氟苯尼考和泰妙菌素产生了耐药性,SH-S株对阿莫西林产生了耐药性,SH-MG株对泰妙菌素敏感。说明禽多病因气囊炎很可能是金黄色葡萄球菌、大肠杆菌和鸡毒支原体共同感染造成的,其中大肠杆菌和金黄色葡萄球菌对一些常用的抗生素表现出耐药性,泰妙菌素是防治鸡毒支原体感染的首选药物。

双组份系统调控禽致病性大肠杆菌生物被膜的研究进展

禽致病性大肠杆菌是一种典型的肠道外致病性大肠杆菌,能够引起各种肠道外疾病,给禽类养殖业造成重大的经济损失。生物被膜作为一种保护细菌的物理屏障用于抵御抗生素的损伤及逃避宿主的免疫系统,从而导致宿主持续和反复性感染。双组份系统是普遍存在于各类细菌中主要的信号传导系统,参与调控细菌抗生素耐药性和生物被膜的形成。为了解由生物被膜造成的禽致病性大肠杆菌感染机制,文章详述了生物被膜的形成过程,并综述了双组份系统调控生物被膜形成的分子机制,以期探寻阻断双组份系统信号转导的新方法以破坏生物被膜的形成作为控制禽致病性大肠杆菌感染的治疗策略,从而为防治由生物被膜引起的抗生素耐药性以及持续或反复性感染提供研究方向。

鸡源大肠杆菌对氟喹诺酮类药物的多重耐药性

利用药敏试验监测系统分析了 71株鸡源大肠杆菌临床分离菌株对 9种氟喹诺酮类药物的耐药性 ,结果表明临床分离菌株对沙拉沙星、恩诺沙星、环丙沙星、二氯沙星、单诺沙星、奥比沙星和萘定酸呈现很高的耐药性 ,耐药率分别为 6 9%、6 9%、5 5 %、76 %、6 9%、76 %和 99% ;对盖特沙星和左氟沙星有轻度的耐药性 ,耐药率分别为11%和 13%。在 71株分离菌株中 ,只有 1株没有耐药性 ,对 3种以上药物有耐药性的菌株占 74 % (5 2 / 70 ) ,对 6种以上药物有耐药性的菌株占 70 % (49/ 70 ) ,呈现出多重耐药性。对耐药菌株GyrA基因QRDR氨基酸变异分析表明 70株耐药菌株的第 83位氨基酸均发生了变异 ,由丝氨酸 (Serine)变为亮氨酸 (Leucine)。对 6种以上氟喹诺酮类药物有耐药性的 4 9株分离株除了 83位氨基酸发生变异外 ,其 87位氨基酸也发生了变异 ,4 1株 87位的氨基酸由天冬氨酸 (D)变为天冬酰胺 (N) ,6株 87位的氨基酸由天冬氨酸 (D)变为酪氨酸 (Y) ,由天冬氨酸 (D)变为甘氨酸和丙氨酸的各 1株 ;对 3种以下氟喹诺酮类药物有耐药性的 2 1株 87位的氨基酸没有发生变异。由此表明鸡源大肠杆菌GyrA基因QRDR区的变异与菌株对氟喹诺酮类药物的耐药程度密切相关 。

禽源性大肠杆菌HPI irp2基因序列的扩增及比较

从养殖场发病家禽中分离到多株禽源性大肠杆菌(E scherich ia coli),用其中Y ZG 040515、NTLFC 040402、CHZ031016菌株提取细菌高致病性染色体DNA,根据耶尔森菌高致病性毒力岛(HP I)irp 2基因设计引物,用PCR扩增出278 bp基因片段。将PCR产物克隆到pGEM-T-easy中,经E coRⅠ酶切鉴定为阳性者进行序列测定。结果表明:该基因片段与G eneB ank中发布的耶尔森菌HP I irp 2基因的同源性高达98%以上,证明禽源性大肠杆菌中具有耶尔森菌毒力岛HP I基因序列,结合临床发病情况和流行病学,提示该毒力岛与日趋严重的家禽大肠杆菌病可能有一定的相关性。

fimC基因与鸡源大肠杆菌致病性的相关

将20株鸡大肠杆菌分别腹腔接种1日龄健康雏鸡,检测其致病性。同时以该20株菌的基因组DNA为模板,应用PCR技术分别进行fimC基因的扩增,结果95%高致病力菌为fimC+,而其它非致病性或致病力极低的菌株均为fimC-。结合相关资料表明,fimC基因几乎只存在于高致病力鸡大肠杆菌中,可以作为高致病力鸡大肠杆菌鉴定的标志基因。将O2菌株PCR扩增产物克隆到pMD18-T载体上,并对其进行酶切鉴定和测序分析,测序结果与参考序列经同源性比较,核苷酸序列同源性为97.9%,氨基酸序列同源性为96.0%,证明所扩增基因是fimC基因。