禽病原性大肠杆菌tsh突变株的构建及分离株tsh基因的检测

运用基因重组方法将庆大霉素（Gentamycin,GM）抗性基因连接到PCR扩增的tsh两端区域产生的2个目的基因片段之间,并共同插入到pUC18载体的多克隆位点中,构建出带GM标志的载体pUC18-tshFRGM,从中切下此tshF-GM-tshR片段,再将之克隆到pMEG-375自杀性载体中,构建出自杀性载体pMEG375-tshFRGM,将突变载体转化到含tsh基因的受体APEC E037株中,根据同源重组原理,筛选出tsh基因缺失的E037突变株,命名为E037（Δtsh）。E037和E037（Δtsh）株对1日龄鸡的LD50分别为105.6CFU和109.0CFU,动物感染性试验表明E037（Δtsh）株在内脏器官和血液中的感染能力和大肠杆菌病变程度均有明显下降。在243株禽源分离株中,有167株为tsh＋菌株,其中高致病株、中等致病株和低致病株分别为87.4%（146/167）、12.6%（21/167）和0%（0/167）;O1、O2和O78血清型的高致病株占所在血清型分离株的89.5%-100%,而其它血清型的高致病株仅占其它分离株的53.3%,差异极显著（P〈0.01）。结果显示tsh＋株大多数为高致病性菌株,且其致病性与血清型的种类有一定的相关性,温度敏感性血凝素为APEC重要的致病因子。     

禽病原性大肠杆菌温度敏感性血凝素(Tsh)突变株的构建

运用基因重组方法将庆大霉素抗性基因(GM)连接到PCR扩增的tsh两端区域产生的2个目的基因片段之间,并共同插入到pUC18载体的多克隆位点中,构建出带GM标志的载体pUC18-tshFRGM,从中切下目的片段,再将之克隆到pMEG-375自杀性载体中,构建出自杀性载体pMEG375-tshFRGM,将突变载体转化到含tsh基因的受体APECE037株中,根据同源重组原理,筛选出tsh基因缺失的E037突变株。E037和E037(Δtsh)株的LD50分别为105.6CFU和109.0CFU,动物感染性试验表明,E037(Δtsh)株在内脏器官和血液中的感染能力和大肠杆菌病变程度均有了明显降低。     

嘉兴地区禽病原性大肠杆菌的分离鉴定

2005年,笔者从嘉兴地区临诊有典型大肠杆菌病病变的病、死鸡中分离到60株大肠杆菌,除9株未能定型、1株自凝外,鉴定出50个分离株的血清型,这些分离株覆盖了12个血清型(以O78为最主要血清型)。对其中28株禽源性大肠杆菌进行致病性试验,有85.7%(24/28)为高致病株,10.7%(3/28)为中等致病株和3.6%(1/28)为低致病株。同时测定28个分离株对20种药物的敏感性。结果表明:壮观霉素、阿米卡星的抑菌作用最强,高敏菌株占71.4%~85.7%;痢特灵、庆大霉素、多粘菌素、卡那霉素、环丙沙星为中敏,高敏菌株为28.6%~64.3%;氧氟沙星、新霉素、甲基氟哌酸、依诺沙星、复方新诺明、磺胺甲基异垩唑、羧苄青霉素、青霉素、诺氟沙星、罗美沙星、强力霉素、四环素的抑菌作用较低,高敏菌株低于20%。     

山东省禽源性大肠杆菌耐药性研究

为了解山东地区禽大肠杆菌对常用抗生素的耐药情况,试验采集疑似患病鸡进行大肠杆菌分离鉴定,并对分离株进行药物敏感性分析。结果表明,从山东各地区采集的59份样品中分离得到45株大肠杆菌,对泰乐菌素的耐药率达100%,对克林霉素的耐药率高达95.6%,对阿莫西林钠、盐酸土霉素、磷霉素钠的耐药率超过80%;对环丙沙星的敏感度相对较高,高敏菌株比例占20%;对庆大霉素、安普霉素、粘杆菌素、利福平、痢菌净、环丙沙星有一定的敏感度,中高敏菌株的比例超过60%。试验结果提示山东地区禽大肠杆菌耐药性较严重,临床上需要科学合理使用抗生素。     

禽致病性大肠杆菌生物被膜的形成及其影响因素

为确定影响细菌生物被膜(BF)的形成条件,本研究采用BF体外定性观察和定量粘附性检测两种方法对1株野生禽致病性大肠杆菌(E.coli)在不同环境(培养时间、培养基类型、引导载体类型、营养条件)下产生BF的差异进行了研究。结果显示野生禽致病性E.coli其宿主体外BF最适形成条件是培养时间为48h,培养基为5%TSB、引导载体为平底96孔聚苯乙烯微孔板(美国Corning Costar)。其生长周期为8h时开始起始粘附、24h形成若干微菌落、36h微菌落粘连、48h形成完整的BF、72h细菌脱落开始下一轮的BF生长。糖分和适量的无机盐都可以在一定程度上促进BF的形成和被膜内细菌的粘附性提高。该研究表明禽致病性E.coliBF的形成周期,并为抑制其形成提供了实验依据。     

不同地区101个禽源性大肠杆菌分离株的致病性试验

从国内１８个省、市、自治区大肠杆菌病疑似病禽中分离并鉴定出大肠杆菌５９５株,鉴定出其中４４０个分离株的血清型。采用Ｒｏｓｅｎｂｅｒｇ氏报道的方法,测定了来自不同地区的６０个优势血清型分离株对ＳＰＦ鸡、３０个其他常见血清型及１１个国内少见血清型分离株对普通易感鸡的致病性。结果表明：所测定的１０１个分离株均为致病株,且９０％以上属高、中度致病株。     

禽源性大肠杆菌对14种抗生素的敏感性

禽大肠杆菌病是指部分或全部由禽源性大肠杆菌所引起的局部或全身性感染的疾病,包括大肠杆菌性败血症、大肠杆菌肉芽肿、气囊病（慢性呼吸道病）、禽蜂窝组织炎、肿头综合征、腹膜炎、输卵管炎、滑膜炎、全眼球炎、脐炎/卵黄囊感染。随着养禽业的集约化、产业化的不断发展,药物使用越来越多,分离细菌对抗生素的耐药性也表现得越来越强,给养殖业带来巨大的损失。     

53株禽致病性大肠杆菌的耐药表型及耐药基因的检测

为了解禽致病性大肠杆菌耐药表型及耐药基因的情况,选取江苏、安徽等地分离的53株禽致病性大肠杆菌,采用药敏纸片法对9种抗菌药物进行药敏试验,并对四环素类tet(A)、tet(B)、tet(C)、tet(W)、tet(M)、tet(O)、tet(K)、tet(L)耐药基因,喹诺酮类GryA、ParC耐药基因,磺胺类sulⅠ、sulⅡ、sulⅢ耐药基因,β-内酰胺类SHV、CTX-M、ompCA耐药基因,氨基糖苷类aac(3)-Ⅰ、aac(3)-Ⅱ、aac(6')-Ⅰb、aac(6')-Ⅱ、ant(3″)-Ⅰ、armA、rmtA、rmtB、rmtC、rmtD、npmA耐药基因进行PCR检测。结果显示:53株禽致病性大肠杆菌对磺胺异噁唑、环丙沙星、氨苄西林、多西环素的耐药率较高,分别为88.68%(47/53)、71.71%(38/53)、86.79%(46/53)、75.47%(40/53)。其中50株禽致病性大肠杆菌表现为多重耐药,耐4、5、6种药物的现象最为普遍,且不同地区菌株存在差异。tet(A)是四环素耐药基因中最为流行的一种耐药基因(52.83%,28/53),喹诺酮类耐药基因主要由gryA(94.33%,50/53)、parC(94.33%,50/53)基因编码,耐磺胺类药物sulⅠ、sulⅡ、sulⅢ基因均有检出,分别为96.23%(52/53)、98.11%(48/53)、86.79%(46/53),耐β-内酰胺类药物中仅检出ompC A基因(30.19%,16/53),在检测的11种耐氨基糖苷类耐药基因中,最为流行为aac(3)-Ⅱ、aac(6')-Ⅰb、ant(3″)-Ⅰ基因,分别为92.59%(49/53)、98.11%(52/53)、100%(53/53)。耐药基因与相关耐药菌株检出率基本呈正相关。试验结果表明:53株禽致病性大肠杆菌耐药性高,耐药谱广,耐药基因流行现象十分普遍。本试验结果能为禽致病性大肠杆菌的耐药现状与临床用药提供理论指导。     

Antimicrobial susceptibility and molecular characterization of avian pathogenic Escherichia coli isolates

Ninety-five avian pathogenic Escherichia coli (APEC) isolates recovered from diagnosed cases of avian colibacillosis from North Georgia between 1996 and 2000 were serotyped and examined for typical virulence-factors, susceptibility to antimicrobials of human and veterinary significance, and genetic relatedness. Twenty different serotypes were identified, with O78 being the most common (12%). The majority of the avian E. coli isolates (60%), however, were non-typeable with standard O antisera. Eighty-four percent of isolates were PCR positive for the temperature-sensitive hemagglutinin (tsh) gene and 86% positive for the increased serum survival (iss) gene. Multiple antimicrobial-resistant phenotypes (> or =3 antimicrobials) were observed in 92% of E. coli isolates, with the majority of isolates displaying resistance to sulfamethoxazole (93%), tetracycline (87%), streptomycin (86%), gentamicin (69%), and nalidixic acid (59%). Fifty-six E. coli isolates displaying resistance to nalidixic acid were co-resistant to difloxacin (57%), enrofloxacin (16%), gatifloxacin (2%), and levofloxacin (2%). DNA sequencing revealed point mutations in gyrA (Ser83-Leu, Asp87-Tyr, Asp87-Gly, Asp87-Ala), gyrB (Glu466-Asp, Asp426-Thr), and parC (Ser80-Ile, Ser80-Arg). No mutations were observed in parE. Twelve of the quinolone-resistant E. coli isolates were tolerant to cyclohexane, a marker for upregulation of the acrAB multi-drug resistance efflux pump. Quinolone-resistant isolates were further genetically characterized via ribotyping. Twenty-two distinct ribogroups were identified, with 61% of isolates clustering into four major ribogroups, indicating that quinolone resistance has emerged among multiple avian pathogenic E. coli serogroups and chromosomal backgrounds.     

Phenotypic and genotypic characterization of Paenibacillus larvae isolates

Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a severe disease of honeybees (Apis melifera). The aim of this work was to develop a strategy for the subtyping and the epidemiological analysis of P. larvae. Phenotypic characterisation, susceptibility to several antibiotics, electrophoresis of whole bacterial proteins, rep-PCR, ribotyping and DGGE were assessed using a collection of P. larvae isolates from different Uruguayan and Argentinean locations. Results indicated that there are two P. larvae genotypes circulating in Uruguay ERIC I-BOX A (worldwide distributed) and ERIC I-BOX C (exclusively detected in Argentina until this study). These results suggest that P. larvae isolates had moved between Argentina and Uruguay, probably through the Uruguay River. Patterns of whole bacterial proteins, DGGE and ribotyping did not improve the P. larvae intraspecific discrimination. Antibiotic susceptibility assays showed that 100% isolates were OTC-sensitive and 22% (belonging to ERIC I-BOX A group) were sulfisoxazole-resistant. This work may contribute to the elucidation of basic aspects related to the epidemiology of AFB in Uruguay and in the region.     

Antimicrobial susceptibilities, serogroups, and molecular characterization of avian pathogenic Escherichia coli isolates in Japan

In total, 83 avian pathogenic Escherichia coli (APEC) isolates from avian colibacillosis during a period from 2001 to 2006 in Japan were investigated for serogroups, typical virulence factors, antimicrobial susceptibility, and genetic relatedness. The most common serogroup was O78 (30.1%); 80.7% of isolates harbored the iss gene and 55.4% of isolates harbored the tsh gene. Antimicrobial resistance of the isolates was found for ampicillin (77.1%), oxytetracycline (75.9%), kanamycin (36.1%), fradiomycin (33.7%), trimethoprim (25.3%), enrofloxacin (21.7%), and florfenicol (6.0%). Although multiple antimicrobial-resistant phenotypes (three or more antimicrobials) accounted for 54.2% of isolates, no isolate exhibited resistance to all agents tested. The fluoroquinolone-resistant isolates had point mutations in GyrA (Ser83 --> Leu, Asp87 --> Asn) and ParC (Ser80 --> Ile, Glu84 --> Gly). Of 18 enrofloxacin-resistant E. coli isolates, nine isolates belonged to serotype O78. In PFGE analysis, eight of the nine enrofloxacin-resistant O78 isolates were classified into an identical cluster. This suggests that a specific genotype of fluoroquinolone-resistant O78 APEC may be widely distributed in Japan.     

禽致病性大肠杆菌pilA基因的克隆及其在乳酸菌中的表达

根据GenBank中禽致病性大肠杆菌pilA基因序列设计合成1对引物,以本实验室分离的禽致病性大肠杆菌基因组DNA为模板,采用PCR技术扩增得到pilA基因片段,经测序鉴定准确后将其克隆到乳酸乳球菌表达载体pMG36e中,构建重组质粒并将其电转入乳酸乳球菌MG1363,得到重组乳酸乳球菌。SDS-PAGE分析显示,表达的蛋白约为19 ku,与预期相符。Western blot进一步证实了该蛋白的免疫反应性。     

Phenotypic characterization of mastitic Prototheca spp. isolates

Bovine mastitis associated with Prototheca is considered a rare pathology, but is increasing in prevalence all over the world and therefore becoming more relevant to the dairy industry. The biochemical characterization of 47 Prototheca isolates retrieved from mastitic milk was performed in this study using API 20C Aux and two BBL Crystal Kits, followed by an analysis with InforBio software. The usage of this methodology, allowed the identification of discriminative phenotypic characteristics for the strains tested. The differential-character-finding algorithm used by this software permitted the identification of new phenotypic characteristics to discriminate between Prototheca zopfii, P. blaschkeae and P. wickerhamii, such as, citrate, phosphorycholine and arabinoside. The main objective of this study was to determine new phenotypic characteristics that allowed a better characterization of Prototheca spp. Usage of recent bioinformatic tools improved the analyses of several features that are important for a better characterization of Prototheca spp.     

Characterizing avian Escherichia coli isolates with multiplex polymerase chain reaction

Colibacillosis caused by Escherichia coli infections account for significant morbidity and mortality in the poultry industry. Yet, despite the importance of colibacillosis, much about the virulence mechanisms employed by avian E. coli remains unknown. In recent years several genes have been linked to avian E. coli virulence, many of which reside on a large transmissible plasmid. In the present study, a multiplex polymerase chain reaction (PCR) protocol to detect the presence of four of these genes is described. Such a protocol may supplement current diagnostic schemes and provide a rapid means of characterizing the E. coli causing disease in poultry. The targets of this procedure included iss, the increased serum survival gene; tsh, the temperature sensitive hemagglutinin gene; cvi, the ColV immunity gene; and iucC, a gene of the aerobactin operon. Organisms, known for their possession or lack of these genes, were used as a source of the template DNA to develop the multiplex PCR protocol. Identity of the amplicons was confirmed by size, DNA:DNA hybridization with specific gene probes, and DNA sequencing. When the multiplex PCR protocol was used to characterize 10 E. coli isolates incriminated in avian colibacillosis and 10 from the feces of apparently healthy birds, nine of the isolates from apparently healthy birds contained no more than one gene, while the 10th contained all four. Also, eight of the isolates incriminated in colibacillosis contained three or more genes, while the remaining two contained two of the target genes. Interestingly, the isolates of sick birds containing only two of the targeted genes killed the least number of embryos,and the isolate of healthy birds that contained all the genes killed the most embryos amongthis group. These genes were not found among the non-E. coli isolates tested, demonstrating the procedure's specificity for E. coli. Overall, these results suggest that this protocol might be useful in characterization and study of avian E. coli.     

鸡大肠杆菌iss基因的克隆测序及原核表达

本实验对鸡大肠杆菌O2血清型菌株进行iss基因克隆测序，并在此基础上设计出两对套式引物，分别对含信号肽Miss基因序列和不含信号肽Miss基因序列进行扩增，并与原核表达载体pGEX-6p-1连接进行原核表达。iss基因克隆测序的结果与两组国外发表Miss基因序列进行比对，其同源性达100％。SDS—PAGE鉴定显示融合蛋白获得了较理想的表达，融合蛋白分子量分别约为36kD和33kD。     

Phenotypic and molecular characterization of isolates of Ornithobacterium rhinotracheale from Peru.

Twenty-five isolates of Ornithobacterium rhinotracheale were examined by agar gel precipitation, immunoperoxidase assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot analysis, and a polymerase chain reaction. All of the isolates were identified as serotype A. Protein profiles of whole cell extracts were similar for all the isolates, and a polypeptide with a molecular weight of 33 kD was a major component, being present in all the isolates. In the main, proteins of 33, 42, 52, and 66 kD were recognized in immunoblots with sera from chickens naturally exposed to O. rhinotracheale. A modified polymerase chain reaction assay identified O. rhinotracheale DNA from all the isolates and tracheal swabs, producing amplicons of 784 bp, and distinguished O. rhinotracheale from bacterial agents causing similar clinical signs.     

Virulence factors of Escherichia coli, with emphasis on avian pathogenic isolates

E. coli bacteria isolated from localized and systemic disease processes in poultry are designated as Avian Pathogenic E. coli (APEC). The disease-inducing potential of these isolates has been explained by the occurrence of specific virulence factors. Despite the extensive literature on virulence factors for E. coli, unambiguous markers of virulence have not been identified yet. The relationship between serotyping and virulence is not straightforward either and raises the question whether E. coli infections in poultry should mainly be considered as opportunistic. Investigations into the occurrence of certain (combinations of) virulence factors in APEC isolates as virulence markers should fulfil the molecular version of Koch's postulates if the former question is to be answered.     

Evaluation of the adhesive capacity of Escherichia coli isolates associated with avian cellulitis

It has been shown that Escherichia coli isolates from lesions of cellulitis belong to a limited number of clonal groups distinct from those of isolates found in the environment of these birds. In this study, different in vitro methods were used to evaluate adherence properties of E. coli isolates from cellulitis lesions and environments of high- and low-cellulitis prevalence broiler flocks. One hundred isolates were tested by hemagglutination. Adherence to frozen sections of chicken skin and binding to soluble fibronectin were examined for 40 of these 100 isolates by immunofluorescence and by immunocytofluorometry, respectively. Localization of bacterial adherence to skin tissues was confirmed by immunohistochemistry. It was demonstrated that O78:K80 isolates from cellulitis lesions adhered to skin sections to a much greater extent in deeper than in superficial tissue layers. A greater bacterial adherence following growth in TSB at 37 C was demonstrated for isolates from flocks with high prevalence of cellulitis than for isolates from flocks with low prevalence of cellulitis. MANOVA analysis results showed a significant difference between superficial and deep tissue layers only for one set of isolates from flocks with high prevalence of cellulitis. Hemagglutinating activity was variable among the O78:K80 isolates obtained from flocks with high prevalence of cellulitis. The results obtained for some O78:K80 isolates following growth in TSB suggest a role for type 1 fimbriae or F1 in adherence to skin sections. This was reinforced by the finding that adherence was inhibited by D-mannose. Poultry E. coli isolates that express F1 had no affinity for soluble fibronectin, although localization of the adherence in skin sections suggested a role for extracellular matrix components such as collagen and insoluble fibronectin.