河西走廊地区犊牛腹泻致病性大肠杆菌分离鉴定、毒力因子及耐药性检测

[目的]为了分析河西走廊地区犊牛腹泻致病性大肠杆菌携带毒力基因和耐药性情况,[方法]2020—2021年在河西走廊地区采集患腹泻病犊牛的粪便、肛拭子及肝脏等病料组织279份,采用人工感染试验动物、PCR方法和K-B药敏纸片法分别检测犊牛腹泻性大肠杆菌致病性、毒力因子和耐药性。[结果]结果表明,分离得到了126株大肠杆菌,其中79株犊牛腹泻性大肠杆菌能引起小鼠死亡;分离的79株致病性大肠杆菌的毒力基因crl、irp2、fimH、papC、K88、K99、stx1、stx2检测率在40.5%～100%之间,其他毒力基因检测率在15.2%～34.2%之间;分离的79株致病性大肠杆菌对氨苄西林、阿莫西林、新霉素等8种药物的耐药率在49.4%～96.2%之间,对其他药物的耐药率在5.1%～32.9%之间。[结论]从河西走廊地区患腹泻病犊牛病料组织中分离得到79株致病性大肠杆菌,这些菌株携带多种毒力基因,对临床中常用的抗菌药物产生了耐药性。     

禽致病性大肠杆菌Hcp2b对雏鸡气管黏膜细胞因子-细胞因子受体相互作用通路的影响

T6SS(type Ⅵ secretion system)是革兰阴性菌中常见的一种分泌系统,其效应蛋白Hcp2b作用机制迄今仍未明晰.本研究以禽致病性大肠杆菌(avian pathogenic Escherichia coli,APEC)Hcp2b蛋白为研究主体,旨在探究Hcp2b蛋白在APEC感染鸡气管黏膜过程中发挥...     

Development of Monoclonal Antibodies against the cOmpT Protein of Avian Pathogenic Escherichia coli and Identification of Epitopes Recognized by Monoclonal Antibodies

To development monoclonal antibodies against cOmpT of avian pathogenic Escherichia coli (APEC), the recombinant cOmpT of APEC origin expression plasmid pET-28a-compT was employed, and cOmpT protein with a molecular weight about 36 kD in the form of inclusion bodies was obtained after induction with IPTG, and then renatured by urea gradient dialysis. BALB/c mice were immunized with the purified cOmpT. An indirect enzyme-linked immunosorbent assay (iELISA) was developed, the optimal coating concentration of the antigen was 0.625 μg·mL^-1 and the optimal serum dilution was 1:6 400. After the fourth immunization, the spleen of immunized mice was collected for cell fusion, three monoclonal hybridomas that can secrete antibody specific to cOmpT were obtained after multiple screenings, named 1G8, 2C3 and 2G3 respectively. And all of their immunoglobulin subclasses were IgG2b. The titers of monoclonal antibodies in the cell culture supernatant were 1:200, 1:3 200 and 1:3 200 determined by iELISA, respectively. All three monoclonal antibodies were confirmed to react with cOmpT in Western blot, without cross reaction with other tested bacteria. The antigenic epitopes recognized by the three monoclonal antibodies were identified by using a series of E. coli strains harboring expression plasmids recombined with truncated fragments from compT gene. The results revealed that the antigenic epitope required for reactivity with the 1G8 was ⁸³DQDWMDS⁸⁹, and ⁹⁰SNPGTW⁹⁵, ¹⁹⁷TFKYSGW²⁰³were recognized by 2C3 and 2G3, respectively. In this study, three monoclonal antibodies against cOmpT were successfully developed and the antigenic epitopes recognized by the antibodies were identified. The cOmpT specific monoclonal antibodies obtained in this study are potentially useful tools for both the functional study of cOmpT and the development of APEC epitope vaccines.     

禽致病性大肠杆菌cOmpT单克隆抗体的研制及其识别抗原表位的鉴定

旨在制备禽致病性大肠杆菌（APEC）染色体编码外膜蛋白（cOmpT）的特异性单克隆抗体，本研究利用实验室已构建的APEC cOmpT重组表达质粒pET-28a-compT，经IPTG诱导表达后，获得以包涵体形式存在的约36 ku的重组蛋白cOmpT，利用尿素浓度梯度透析复性获得纯化蛋白cOmpT，并以此免疫BALB/c小鼠。建立间接ELISA检测方法，最适抗原包被浓度为0.625 μg·mL^-1，最适血清稀释度为1：6 400。4次免疫后取小鼠脾进行细胞融合，采用有限稀释法多轮筛选后得到3株能稳定分泌针对cOmpT蛋白的单克隆抗体，分别命名为1G8、2C3和2G3，均为IgG2b亚类。3株杂交瘤细胞上清ELISA抗体效价分别为1：200、1：3 200和1：3 200。Western blot结果显示，3株单抗均能与cOmpT发生特异性反应，而不与其他受检菌发生交叉反应。运用原核表达系统对compT基因进行截短表达，对单克隆抗体针对的cOmpT抗原表位进行鉴定，结果显示单抗1G8、2C3和2G3识别的抗原表位分别是⁸³DQDWMDS⁸⁹、⁹⁰SNPGTW⁹⁵和¹⁹⁷TFKYSGW²⁰³。本研究成功制备了3株抗cOmpT蛋白的单克隆抗体，并对其识别的抗原表位进行了鉴定，为cOmpT蛋白功能研究和APEC新型表位疫苗研发奠定了基础。     

The Escherichia coli type III secretion system 2 Is involved in the biofilm formation and virulence of avian Pathogenic Escherichia coli

The Escherichia coli type III secretion system 2 (ETT2) is found in most pathogenic E. coli strains. Although many ETT2 gene clusters carry multiple genetic mutations or deletions, ETT2 is known to be involved in bacterial virulence. To date, no studies have been conducted on the role of ETT2 in the virulence of avian pathogenic Escherichia coli (APEC), which harbours ETT2. Thus, we deleted the ETT2 of APEC strain and evaluated the phenotypes and pathogenicities of the mutant. The results showed that deletion of ETT2 had no effect on APEC growth, but significantly promoted biofilm formation. In addition, as compared to the wild-type （WT） strain, the ETT2 deletion significantly promoted adherence to and invasion of DF-1 chicken fibroblasts and facilitated survival in the sera of specific-pathogen-free chickens. Analysis of the role of ETT2 in animal infection models demonstrated that the distribution of viable bacteria in the blood and organs of chicks infected with the ΔETT2 was significantly higher than those infected with WT. The results of RNA sequencing indicated that multiple genes involved in biofilm formation, lipopolysaccharide components, fimbrial genes and virulence effector proteins are regulated by ETT2. Collectively, these results implicated ETT2 is involved in the biofilm formation and pathogenicity of APEC.     

禽致病性大肠埃希氏菌与金色葡萄球菌的药物敏感性调查

从新疆阿克苏地区多个鸡场分离出大肠埃希氏菌51株，金色葡萄球菌18株，按世界卫生组织推荐的K-B法，用10种临床上常用的抗菌药物对其敏感性进行了测定。结果：对大肠埃希氏菌作用最强的依次是庆大霉素、红霉素、磺胺甲基异恶唑、磺胺嘧啶，抑菌作用较差的是青霉素、四环素、头孢唑啉；对金色葡萄球菌作用最强的依次是头孢唑啉、红霉素、庆大霉素，押菌作用较差的是青霉素、氟哌酸、环丙沙星。     

鸡致病性大肠杆菌菌毛fimC基因的表达及重组fimC蛋白卵黄抗体的免疫保护效力

在表达鸡致病性大肠杆菌菌毛fimC基因的基础上,对fimC蛋白卵黄抗体的免疫保护效力进行测定。对鸡致病性大肠杆菌O2血清型菌株fimC基因序列进行扩增,扩增产物克隆至pMD18-T载体并测序。测序结果显示,fimC基因全长657 bp,编码218个氨基酸,与人源大肠杆菌fimC基因进行同源性比较,核苷酸序列同源性达97.9%,氨基酸序同源性为96%。从重组质粒pMD18-T-fimC上将fimC基因亚克隆到表达载体质粒pET-30c上,转化大肠杆菌BL21（DE3）,表达出预期的25 ku融合蛋白,采用Western Blot对表达产物进行反应原性分析,结果显示fimC融合蛋白具有较好的抗原反应特异性。用表达的fimC蛋白免疫产蛋母鸡,制备fimC高免卵黄抗体。用制备的fimC高免卵黄抗体免疫1日龄健康雏鸡,1 d后注射鸡致病性大肠杆菌进行攻毒,结果显示,fimC卵黄抗体免疫组死亡率为40%,对照组分别为60%、70%,证明所制备的fimC蛋白卵黄抗体能在一定程度上抵抗鸡致病性大肠杆菌的攻击。     

Characterisation of avian pathogenic Escherichia coli (APEC) associated with colisepticaemia compared to faecal isolates from healthy birds

A total of 114 avian pathogenic Escherichia coli (APEC) isolates were collected from cases of colisepticaemia occurring in broilers (77) and layers (37) within Ireland. In addition 45 strains isolated from faeces of healthy birds were included for comparison. All isolates were serogrouped, and examined for known virulence factors, mostly by PCR. The O78 serogroup represented 55 and 27% of broiler and layer colisepticaemic isolates respectively. All isolates were positive for curli fimbriae (crl, csg) and negative for afimbrial adhesin (afa). S-fimbrial (sfa) sequences were present in 8.8% of septicaemic isolates and 8.9% of healthy bird isolates. The majority of E. coli from cases of colisepticaemia (97.4%) and healthy bird (95.6%) isolates were positive for aerobactin (aer), and temperature sensitive haemagglutinin (tsh) was similarly detected in high numbers in 93.9 and 93.3%, respectively. In comparison to E. coli isolates from the faeces of healthy birds, a significantly higher percentage of isolates from septicaemic cases possessed Type 1 fimbriae (fimC) and increased serum survival (iss) gene sequences. Forty-seven (41.2%) isolates from septicaemic birds possessed P-fimbriae (pap) gene sequences, compared with only 15.6% from E. coli isolated from healthy birds. Haemolysin (hlyE) sequences were detected in 46.7% of isolates from healthy birds in comparison with 6.1% of septicaemic isolates. Sequences encoding colicin V (cvaC) were detected in 99.1% of septicaemic isolates and 82.2% of isolates from healthy birds. The K1 capsule was only present in two septicaemic isolates, both taken from layers. Motility was detected in 36.8% of E. coli isolated from cases of septicaemia, compared with 93.3% of isolates from healthy birds. These results demonstrate the presence of 11 virulence genes in E. coli isolated from cases of colisepticaemia within Ireland, and indicate the prevalence of iss and fimC.     

Advances on the Experimental Infectious Models of Avian Pathologenic Escherichia coli

Avian pathogenic Escherichia coli(APEC) is an important pathogen that causes localized and systemic infection in avian species of all ages.It is also an important reservoir or source of virulence genes of human extraintestinal pathogenic Escherichia coli (ExPEC).In order to understand deeply the infection progresses,pathogenesis,host immune responses and genetic resistance mechanism of APEC,and evaluate the efficacy of drugs and vaccines,several experimental infection models were established to evaluate the virulence of APEC through different approaches.According to the different systems involved,it can be divided into respiratory system,vascular system,musculoskeletal system,dermatological system,reproductive system,gastrointestinal system and chicken embryo system.In addition,there are infection experiments in mice and rats,tissue culture cells and explants infection experiments in vitro.The author highlights the establishment,pathogenesis,host responses and application of the different APEC experimental infection models.     

禽致病性大肠杆菌实验感染模型研究进展

禽致病性大肠杆菌（avian pathogenic Escherichia coli,APEC）是导致不同日龄禽类局部和全身感染的重要病原菌,也是人肠外致病性大肠杆菌（extraintestinal pathogenic Escherichia coli,ExPEC）毒力基因的重要储存宿主或来源。为深入理解APEC的感染过程、致病机理、宿主免疫应答及遗传抵抗机制、评估药物和疫苗防治效果,研究者通过不同途径建立了多种评估APEC毒力的实验感染模型。根据所涉及的系统不同可分为呼吸系统、脉管系统、肌肉系统、皮肤系统、生殖系统、消化系统及鸡胚系统等。此外,尚有小鼠与大鼠感染试验及组织培养细胞和外植块感染试验等。作者重点介绍了不同APEC实验感染模型的建立、致病机制、宿主应答及应用。     

大肠埃希菌强毒力岛致病机制研究进展

大肠埃希菌(Escherichia coli)常引起婴儿和幼畜(禽)严重腹泻和败血症.近年来对于大肠埃希菌强毒力岛(HPI)致病机制的研究已有大量文献记载,但大肠埃希菌具体的致病机制目前尚不明确.研究表明耶尔森菌强毒力岛与一些肠道致病菌的致病性密切相关,如大肠埃希菌、沙门菌、克雷伯菌等.引起大肠埃希菌致病的因素多种多样,如气候变化,应激,机体本身状况等.论文就大肠埃希菌HPI的结构基因及其分子致病机制的研究现状进行综述.     

泰安及周边地区禽大肠杆菌分离鉴定及耐药分析

对泰安及周边地区鸡大肠杆菌病的流行情况和致病性大肠杆菌的耐药性状况进行了比较系统的调查分析：对25株鸡源性大肠杆菌进行了分离纯化、生化试验，同时对所分离的菌株进行了耐药性测定及β内酰胺药物耐药基因TEM的检测等研究。结果表明：大肠杆菌对多种抗生素均产生了耐药性，对β－内酰胺类药物的耐药性也相当严重，对在药敏试验中检测出对头孢菌素类药物不敏感的菌株进行TEM基因检测，发现这些菌株均含有耐β-内酰胺酶耐药基因，与Genebank上登录的其他耐β-内酰胺酶耐药基因序列的同源性达到97％以上，仅存在个别的碱基突变。通过研究以期探明地区鸡源性大肠杆菌病的流行情况和细菌的耐药现状．从而为有效控制鸡大肠杆菌病的发生流行提供可靠的理论依据，以便制订出较为科学合理的预防和治疗措施，以提高养殖效益。     

山东省部分地区禽源致病性大肠杆菌的血清流行病学调查与耐药性检测

为调查山东省禽源致病性大肠杆菌流行的血清型及耐药性,从山东部分地区的45家养禽场分离到致病性大肠杆菌96株,应用微量平板凝集试验进行了血清型鉴定,共鉴定出18种血清型,其中优势血清型6种,分别为O78、O2、O15、O18、O143、O88,占定型致病性菌株的64％。抗菌药物敏感性试验发现,96株大肠杆菌对20种药物有不同程度的耐药性。75％以上的菌株对氨苄青霉素、阿莫西林、土霉素等5种抗菌药耐药,50％以上的菌株对卡那霉素、多西环素、环丙沙星等7种抗菌药表现为耐药;所有分离株存在多重耐药现象,75％的受检菌对9种或9种以上的被测药物耐药。结果表明,O78、O2、O15、O18、O143、O886种血清型是山东省部分地区近年来禽源致病性大肠杆菌的优势血清型,且禽源致病性大肠杆菌的耐药现象严重,有必要加强耐药性检测,以指导兽医临床合理使用抗菌药物。     

Dynamics of shiga-toxin producing Escherichia coli (STEC) and their virulence factors in cattle

Starting at birth, twenty Holstein calves were housed individually, in groups of five and finally in one large freestall while fecal samples were collected weekly for 25 weeks. From each sample, twenty isolates of Escherichia coli were screened for 6 virulence markers including shiga-toxin 1, 2, intimin, enterohemolysin, the fimbrial antigen efa1 and the adhesin saa. Dynamic models of transmission of E. coli were used to model the transmission of different virulotypes between calves and the loss of the same virulotypes from the calves. It was found that, once E. coli encoding shiga-toxins in combination with enterohemolysin were transmitted and established in a calf, they tended to be eliminated less efficiently compared to E. coli without this combination of virulence markers. It was concluded that the presence of certain combinations of virulence markers coincided with persistence of E. coli in the bovine gastrointestinal tract. In addition, the combinations of stx with either eae or ehxA in E. coli have a greater impact on the loss rates than on the transmission rates.     

鸭致病性大肠杆菌外膜蛋白型研究

测定了从我国分离到的130株鸭病原性大肠杆菌外膜蛋白型（Outer membrane protein patterns,OMP型）,其中O93、O92、O78和O76优势血清型53株,O46等30个其他血清型分离株77株,这些分离株共产生了4个OMP型,其中OMP-1型81株,占62.3%（81/130）,覆盖O93、O92、O78和O76等29个血清型,占85.3%（29/34）,为主要的OMP型;根据GenBank中大肠杆菌K-12外膜蛋白A基因（ompA）的核苷酸序列设计引物,从9株优势血清型和1株O46血清型的鸭大肠杆菌中分别扩增得到ompA基因,进行序列测定及分析,发现优势血清型9个菌株的ompA均由1 171个碱基组成,均只有1个大的开放阅读框（ORF）,长1 053 bp,编码由350个氨基酸组成的前外膜蛋白A（pro-OmpA）,前21个氨基酸残基组成信号肽,成熟的OmpA由329个氨基酸残基组成;O46菌株的ORF全长1 041 bp,在400-411 bp位缺失了12个碱基,成熟的OmpA由325个氨基酸残基组成;10个鸭致病性E.coli的ompA基因核苷酸序列高度同源,相似性达95.8%~100%。     

Escherichia coli ghosts or live E. coli expressing the ferri-siderophore receptors FepA, FhuE, IroN and IutA do not protect broiler chickens against avian pathogenic E. coli (APEC)

The aim of this study was to investigate if immunization with the ferri-siderophore receptors FepA, FhuE, IroN and IutA could protect chickens against avian pathogenic Escherichia coli (APEC) infection. The antigens were administered as recombinant proteins in the outer membrane (OM) of E. coli strain BL21 Star DE3. In a first immunization experiment, live E. coli expressing all 4 recombinant ferri-siderophore receptors (BL21(L)) were given intranasally. In a second immunization experiment, a mixture of E. coli ghosts containing recombinant FepA and IutA and ghosts containing recombinant FhuE and IroN was evaluated. For both experiments non-recombinant counterparts of the tentative vaccines were administered as placebo. At the time of challenge, the IgG antibody response for BL21(L) and a mixture of E. coli ghosts containing recombinant FepA and IutA and ghosts containing recombinant FhuE and IroN was significantly higher in all immunized groups as compared to the negative control groups (LB or PBS) confirming successful immunization. Although neither of the tentative vaccines could prevent lesions and mortality upon APEC infection, immunization with bacterial ghosts resulted in a decrease in mortality from 50% (PBS) to 31% (non-recombinant ghosts) or 20% (recombinant ghosts) and these differences were not found to be significant.     

Characterization of a plasmid-encoded adhesin of an avian pathogenic Escherichia coli (APEC) strain isolated from a case of swollen head syndrome (SHS)

An avian pathogenic Escherichia coli (APEC) strain designated SHS4, isolated from a chicken with clinical signs of swollen head syndrome (SHS), adhered to but did not invade Hep-2 and tracheal epithelial cells. The PCR amplified fimA, csgA and tsh gene sequences. It produced Ia, Ib, E1, E3, K, and B colicins, but not colicin V and aerobactin. It harboured two plasmids of 60 and 98MDa and was resistant to streptomycin and tetracycline. Conjugation with a nalidixic acid (Na) resistant K-12 recipient strain (MS101) showed that the 98MDa plasmid did not transfer, whereas transfer of the 60MDa plasmid resulted in concomitant transfer of adhesion to Hep-2 and tracheal epithelial cells, production of the colicins Ia, E1, E3, and K, and the tsh-related DNA sequence. Transposon (TnphoA) mutagenesis of strain TR4 gave rise to strain Mut23, which lost its adhesive capacities, but was still able to express the same colicins as did strain TR4. PCR was able to amplify the tsh-related DNA sequence in this strain and a molecular probe based on transposon TnphoA indicated that the transposon was inserted in the 60MDa plasmid. Based on these results, we suggest that the 60MDa plasmid have adhesion genes, which may be responsible for the initial colonization of the upper respiratory tract of chickens.     

实验性鸡大肠杆菌病病理学动态变化

用致病性大肠杆菌O18分离株和／或低致病性禽流感病毒（Mildly pathogenic avian influenza virus ,MPAIV)接种10－12日龄SPF鸡。在接种后1－96h进行临床症状与大体病理变化、组织学观察发现：大肠杆菌接种组、MPAIV接种组和健康接种组除扑杀鸡外未见鸡死亡，MPAIV与大肠杆菌混合接种组除扑杀鸡外死亡率为24％。混合接种组的病变比大肠杆菌接种组出现的时间早，恢复也慢，各脏器的病理变化更严重。MPAIV主要引起各实质器官的坏死，结果表明，大肠杆菌经气管内接种后试验鸡主要表现为呼吸道的炎症反应；MPAVI可使鸡大肠杆菌病严重化。     

实验性鸡大肠杆菌病试验鸡细菌的动态分布

用致病性大肠杆菌O18分离株和或低致病性禽流感病毒（mildly pathogenic avian influenza virus,MPAIV)接种10－12日龄SPF鸡，细菌接种后1－96小时鸡鸡的血液、气管、肺、脾、肝和肾进行细菌学检查，发现大肠杆菌接种组、MPAIV与大肠杆菌混合接种组的气管、肺在整个试验过程均分离到细菌，血液、脾、肝、肾中细菌数随接种时间延长而减少，直至消失，MPAV与大肠杆菌混合接种组比大肠杆菌接种组从气管、肺中分离到细菌的频率更高，数量更多，表明气管、肺是鸡大肠杆菌定居的场所，MPAIV可延长细菌在气管、肺中定居的时间。