应用DNA芯片技术筛选禽致病性大肠杆菌和尿道致病性大肠杆菌的差异表达基因

为研究禽致病性大肠杆菌强毒株E058的毒力相关基因在鸡体内、体外表达情况以及E058和尿道致病性大肠杆菌HEC4在LB和尿液中培养的表达情况,本研究分别提取E058株在SPF鸡体内及E058株和HEC4株在LB和尿液中静置培养的总RNA,与构建的DNA芯片杂交,检测和分析RNA的差异表达情况。芯片的检测结果表明:E058株在鸡体内和LB中培养差异表达基因共有9个,上调基因为5个,分别为neuC、iutA、cvaC、aes-15和iucCD;下调基因为4个,分别为aes-8、gyrB、aec-30和mdh。另外,芯片检测结果也显示E058株和HEC4株在LB和尿液中静置培养,具有相似的基因表达情况。     

Duplex real-time PCR assays for rapid detection of virulence genes in E. coli isolated from post-weaning pigs and calves with diarrhoea

Duplex real-time PCR assays were used as modules to cover partially automated detection of 12 genes encoding adhesins, enterotoxins and Shiga toxins in faecal E. coli isolates. For this a total of 194 E. coli isolates from pigs suffering from post-weaning diarrhoea (PWD), including 65 isolates with haemolytic activity, and 83 isolates from calves with diarrhoea were examined. Data obtained by PCR were compared with O-typing and with haemolytic activity as indirect virulence markers. E. coli O-types O139:K82, O141:K85, and O149:K91 accounted for 43.8% (n = 85) of all porcine strains and for 55.4% (n = 36) of the porcine strains, which exhibited haemolytic activity. These strains carried virulence genes by 65.9% (n = 56) and 80.6% (haemolytic E. coli, n = 29), respectively. The E. coli O-types O139:K82 and O141:K85 were significantly associated with the adhesin gene F18, and O149:K81 with the F4 gene. In this context, detection of the gene encoding F18 was coupled predominantly with the genes responsible for the production of the toxins ST-I, ST-II and Stx2, and the F4 gene with those of the enterotoxins ST-I, ST-II and LT. Both virulence patterns were detected more pronounced in E. coli strains with haemolytic activity. Fifty-six of a total of 83 E. coli isolates originating from calves were O-typed as O101 (O101:K28, O101:K30, O101:K32; n = 29), O78:K80 (n = 23), and O9:K35 (n = 4). Most of the E. coli O78:K80 strains carried the F17 gene (69.6%, n = 16). Virulence genes encoding for F4, F5 or ST-I were detected only in single cases. Intimin and Shiga toxin genes that are present in enterohaemorrhagic E. coli (EHEC) were not detected.     

The role of RTX toxins in host specificity of animal pathogenic Pasteurellaceae

RTX toxins are bacterial pore-forming toxins that are particularly abundant among pathogenic species of Pasteurellaceae, in which they play a major role in virulence. RTX toxins of several primary pathogens of the family of Pasteurellaceae are directly involved in causing necrotic lesions in the target organs. Many RTX toxins are known as haemolysins because they lyse erythrocytes in vitro, an effect that is non-specific, but which serves as a useful marker in bacteriological identification and as an easily measurable signal in vitro in experimental studies. More recent studies have shown that the specific targets of most RTX toxins are leukocytes, with RTX toxins binding to the corresponding β-subunit (CD18) of β2 integrins and then exerting cytotoxic activity. After uptake by the target cell, at sub-lytic concentrations, some RTX toxins are transported to mitochondria and induce apoptosis. For several RTX toxins the binding to CD18 has been shown to be host specific and this seems to be the basis for the host range specificity of these RTX toxins. Observations on two very closely related species of the Pasteurellaceae family, Actinobacillus suis, a porcine pathogen particularly affecting suckling pigs, and Actinobacillus equuli subsp. haemolytica, which causes pyosepticaemia in new-born foals (sleepy foal disease), have revealed that they express different RTX toxins, named ApxI/II and Aqx, respectively. These RTX toxins are specifically cytotoxic for porcine and equine leukocytes, respectively. Furthermore, the ApxI and Aqx toxins of these species, when expressed in an isogenetic background in Escherichia coli, are specifically cytotoxic for leukocytes of their respective hosts. These data indicate the determinative role of RTX toxins in host specificity of pathogenic species of Pasteurellaceae.     

Rapid detection of virulence-associated genes in avian pathogenic Escherichia coli by multiplex polymerase chain reaction

Based on recently published prevalence data of virulence-associated factors in avian pathogenic Escherichia coli (APEC) and their roles in the pathogenesis of colibacillosis, we developed a multiplex polymerase chain reaction (PCR) as a molecular tool supplementing current diagnostic schemes that mainly rely on serological examination of strains isolated from diseased birds. Multiple isolates of E. coli from clinical cases of colibacillosis known to possess different combinations of eight genes were used as sources of template DNA to develop the multiplex PCR protocol, targeting genes for P-fimbriae (papC), aerobactin (iucD), iron-repressible protein (irp2), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), enteroaggregative toxin (astA), increased serum survival protein (iss), and colicin V plasmid operon genes (cva/cvi). In order to verify the usefulness of this diagnostic tool, E. coli strains isolated from fecal samples of clinically healthy chickens were also included in this study, as were uropathogenic (UPEC), necrotoxigenic, and diarrhegenic E. coli strains. The application of the multiplex PCR protocol to 14 E. coli strains isolated from septicemic poultry showed that these strains harbored four to eight of the genes mentioned above. In contrast, those isolates that have been shown to be nonpathogenic for 5-wk-old chickens possessed either none or, at most, three of these genes. We found only one enterohemorrhagic (EHEC), one enteropathogenic (EPEC), and two enterotoxic (ETEC) E. coli strains positive for irp2, and another two ETEC strains positive for astA. As expected, UPEC isolates yielded different combinations of the genes iss, papC, iucD, irp2, and a sequence similar to vat. However, neither the colicin V operon genes cva/cvi nor tsh were amplified in UPEC isolates. The multiplex PCR results were compared with those obtained by DNA-DNA-hybridization analyses to validate the specificity of oligonucleotide primers, and the protocol was concluded to be a useful, sensitive, and rapid assay system to detect avian pathogenic E. coli and differentiate them from nonpathogenic strains and those belonging to other pathotypes.     

Host cell specific activity of RTX toxins from haemolytic Actinobacillus equuli and Actinobacillus suis

We assessed and compared host cell specificity of the haemolytic and cytotoxic activity of the RTX toxins from Actinobacillus equuli, an equine pathogen, and Actinobacillus suis, which is pathogenic for pigs. The two bacterial species are closely related, phenotypically as well as phylogenetically, sharing the same 16S rRNA gene sequence. Both species contain specific protein toxins from the family of pore-forming RTX toxins, however, the two species differ in their RTX toxin profiles. Haemolytic A. equuli contains the operon for the Aqx toxin, whereas A. suis harbours genes for ApxI and ApxII. We tested the toxic activity of the corresponding proteins on erythrocytes as well as on lymphocytes isolated from horse and pig blood. The strength of the haemolytic activity for each of the toxins was independent of the origin of erythrocytes. When testing cytotoxic activity, the Aqx protein showed a higher toxic effect for horse lymphocytes than for porcine lymphocytes. On the other hand, ApxI and ApxII showed a strong cytotoxic effect on porcine lymphocytes and a reduced toxicity for horse lymphocytes; the toxicity of ApxII was generally much lower than ApxI. Our results indicate a host species specificity of the toxic activity of RTX toxins Aqx of A. equuli and ApxI and ApxII of A. suis.     

Uropathogenic virulence factor FimH facilitates binding of uteropathogenic Escherichia coli to canine endometrium

Pyometra is a potentially life-threatening condition in bitches and is often caused by Escherichia coli infection. Both pathogenic and non-pathogenic E. coli strains commonly carry the genes for type 1 fimbriae that mediate bacterial adhesion onto host epithelium. To investigate whether the type 1 fimbrial adhesin, FimH, facilitates the binding of uropathogenic E. coli to canine endometrium, the fimH gene was insertionally inactivated in a pathogenic E. coli strain. The ability of E. coli to bind to canine endometrial epithelial cells was determined in vitro using canine uterine biopsies. Binding of the fimH mutant was only 0.3% of that of the wild type. Complementation of the mutation restored the phenotype to that of the parent. This study has developed an in vitro model that allows quantitative and qualitative assessment of bacterial binding to canine endometrium and has demonstrated that the fimH gene plays a role in adherence of pathogenic E. coli to canine endometrium.     

Virulence gene regulation in pathogenic Escherichia coli.

The ability to regulate gene expression throughout the course of an infection is important for the survival of a pathogen in the host. Thus, virulence gene expression responds to environmental signals in many complex ways. Frequently, global regulatory factors associated with specific regulators co-ordinate expression of virulence genes. In this review, we present well-described regulatory mechanisms used to co-ordinate the expression of virulence factors by pathogenic Escherichia coli with a relative emphasis on diseases caused by E. coli in animals. Many of the virulence-associated genes of pathogenic E. coli respond to environmental conditions. The involvement of global regulators, including housekeeping regulons and virulence regulons, specific regulators and then sensor regulatory systems involved in virulence, is described. Specific regulation mechanisms are illustrated using the regulation of genes encoding for fimbriae, curli, haemolysin and capsules as examples.     

鸡源致病性大肠杆菌1型菌毛pilA基因的PCR扩增、克隆及序列分析

研究选择从四川规模化鸡场分离鉴定的5株优势血清型鸡源致病性大肠杆菌代表株(O89,O119,O141,O127)的1型菌毛pilA基因的PCR扩增片段分别定向克隆到pUC18质粒的多克隆位点,并转化到DH5 α株大肠杆菌载体菌中,对目的基因的序列测定结果表明：5个克隆片段均含有1型菌毛pilA基因的全序列,全长459bp,编码信号肽和结构蛋白。用生物软件(DNASTAR)对9株大肠杆菌1型菌毛pilA基因序列、氨基酸序列、菌毛蛋白质二级结构预测及抗原性进行了分析比较,结果显示：鸡源致病性大肠杆菌1型菌毛间具有同源性,1型菌毛间存在一定的相同抗原位点。     

IbeB is involved in the invasion and pathogenicity of avian pathogenic Escherichia coli

The ibeB gene in neonatal meningitis Escherichia coli (NMEC) contribute to the penetration of human brain microvascular endothelial cells (HBMECs). However, whether IbeB plays a role in avian pathogenic E. coli (APEC) infection remains unclear. Thus, this study was conducted to investigate the distribution of the ibeB gene in Chinese APEC strains and examine whether IbeB is involved in APEC pathogenicity. The ibeB gene was found in all 100 detected E. coli isolates with over 97% sequence homology. These results indicated that ibeB is a conserved E. coli gene irrelevant of pathotypes. To determine the role of ibeB in APEC pathogenicity, an ibeB mutant of strain DE205B was constructed and characterized. The inactivation of ibeB resulted in reduced invasion capacity towards DF-1 cells and defective virulence in animal models as compared to the wild-type strain. Animal infection experiments revealed that loss of ibeB decreased APEC colonization and invasion capacity in brains and lungs. These virulence-related phenotypes were partially recoverable by genetic complementation. Reduced expression levels of invasion- and adhesion-associated genes in ibeB mutant could be major reasons as evidenced by reduced ibeA and ompA expression. These results indicate that IbeB is involved in APEC invasion and pathogenicity.     

一株O1血清型鸡大肠埃希菌iss基因的序列分析

采用1日龄雏鸡对1株O1血清型鸡源大肠埃希菌(E.coli)的致病性进行测定,并扩增iss基因.结果表明,鸡E.coli O1对1日龄雏鸡具有较强的毒力.iss基因在致病性鸡E.coli O1中的序列与已知禽大肠埃希菌iss基因序列同源性为100%,与人源大肠埃希菌iss基因核苷酸,序列同源性为90.9%,显示了此基因具有保守性.     

Construction and evaluation of a delta cya delta crp Salmonella typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens.

Avian pathogenic strains of Escherichia coli cause a number of extraintestinal diseases in poultry, including airsacculitis and colisepticemia. Expression of O78 lipopolysaccharide (LPS) is frequently associated with pathogenic isolates. Salmonella, a common poultry contaminant, is a major public health concern. The purpose of this work was to develop an E. coli vaccine for poultry with the use of an attenuated Salmonella typhimurium carrier that would benefit both the bird and the consumer. Orally administered attenuated S. typhimurium delta cya delta crp strains have been shown to provide excellent protection against wild-type Salmonella challenge in chickens. This work describes the construction of a delta cya delta crp derivative of an avian pathogenic S. typhimurium that expresses both the homologous group B determinants (O1,4,5,12) and the heterologous E. coli O78 LPS O antigens. This was accomplished by inserting the E. coli rfb region, which encodes the genes required for O78 expression, into the chromosomal cya gene of S. typhimurium, creating a defined deletion/insertion mutation. A delta crp mutation was introduced in a subsequent step. Expression of both O antigens was stable in vitro and in vivo. Vaccination of white leghorn chicks at day of hatch and 14 days with the recombinant vaccine strain induced serum immune responses against both S. typhimurium and E. coli LPS and protected the birds against subsequent challenge with an avian pathogenic E. coli O78 strain. Introduction of a mutation in rfc, which encodes the O antigen polymerase, reduced the chain length of the S. typhimurium LPS without affecting the expression of O78. The rfc mutation further enhanced the ability of the vaccine strain to protect chickens against E. coli challenge.     

鸡致病性大肠杆菌1型菌毛fimI基因克隆与序列测定

对鸡O2血清型致病性大肠杆菌1型菌毛fimI基因进行了扩增并与国外同源菌株基因序列进行了比较。结果表明：两者核苷酸同源性达98．42％，预测氨基酸顺序同源性达98．92％。fimI基因的预测氨基酸序列中仅第46位氨基酸由ALA变为THR，第76位氨基酸由SER变为ALA，其余核苷酸的变化未影响氨基酸的翻译，推测，这种改变是由分离株的差异所引起的。     

Genotypic and phenotypic characterisation of Escherichia coli strains associated with porcine pyelonephritis

Urinary tract infection (UTI) is a severe problem in humans as well as in many domestic animals like pigs. The most frequent infectious agent in UTI is uropathogenic Escherichia coli. Such strains have been extensively characterised with respect to virulence and fitness factors as well as clonal type when it comes to human isolates. However, relatively little has been done to characterise the corresponding porcine strains. On this background we have analysed 20 porcine pyelonephritis E. coli strains isolated from infected pig kidneys. The strains were quite distinct from that of human uropathogenic strains with regards to adhesion profile and haemolysin production. Also, the clonal profiles differed from that of human infections since our strains all belonged to the E. coli clonal groups A and B1.     

鸡致病性大肠杆菌I型菌毛交叉保护性研究及其结构基因 FimA 的序列分析

从山东省各地分离到107株鸡致病性大肠杆菌，选择了其中2株高致病性大肠杆菌，血清型分别为O78和OM，经增菌培养后提取菌毛制备为单价菌毛油乳苗，分别接种于1日龄和14日龄雏鸡，于4周龄攻毒。结果二者之间有一定的交叉保护作用。根据Genbank收录的人源大肠杆菌I型菌毛FimA基因和P型菌毛papA基因序列，分别设计了2对引物。通过PCR对上述2株大肠杆菌进行扩增，结果只有FimA的一对引物扩增出相应的条带，经测序证明为FimA基因。papA基因的引物未扩出任何条带，证明这2株大肠杆菌表达I型菌毛。通过对2株大肠杆菌结构基因FimA进行分析，发现二者具有高度的同源性。本研究的目的是探讨菌毛亚单位之间的交叉保护性与其菌毛的结构基因之间是否存在相关性。     

鸡源致病性大肠埃希菌菌毛基因研究进展

菌毛是鸡大肠埃希菌的重要致病因子之一。文章对鸡源致病性大肠埃希菌菌毛的特点及分类,各型菌毛亚单位基因结构及其功能与控制,鸡源致病性与其他动物源大肠埃希菌菌毛之间的同源性进行了综述。这对了解菌毛亚单位基因结构与致病力的关系,探讨鸡源大肠埃希菌的致病力与分布有重要意义。     

Virulence factors of avian pathogenic Escherichia coli strains isolated from chickens with colisepticemia in Japan

The virulence factors of avian pathogenic Escherichia coli (APEC) isolated in Japan were investigated. Serogroups O, serotypes K1 and K5, and genes cva C, iss, iutA, papA, tsh, and usp, which have been thought to be related to virulence, were examined for their association with E. coli strains isolated from diseased and healthy chickens. The frequently recognized serogroups O1, O2, and O78 were found in 56 of 125 (44.8%) strains of diseased chickens (APEC) versus 13 of 100 (13.0%) strains of healthy chickens (commensal E. coli), a significant difference at risk ratio < 0.01. Although iss, iutA, and tsh were widely distributed in the APEC irrespective of O serogroup, papA, usp, and the K1 serotype were detected in serogroup O2 of APEC. The kfiD gene related to the K5 capsule and VT, LT, and ST genes related to exotoxins were not detected in any strains examined.     

Location of increased serum survival gene and selected virulence traits on a conjugative R plasmid in an avian Escherichia coli isolate

Avian colibacillosis is a costly disease for the poultry industry. The mechanisms of virulence employed by the etiologic agent of this disease remain ill defined. However, accumulated evidence suggests that complement resistance and the presence of the increased serum survival gene (iss) in an avian Escherichia coli isolate may be indicative of its ability to cause disease. This association of iss with the E. coli implicated in avian disease may mean that iss and/or, perhaps, the genes associated with it are important contributors to avian E. coli virulence. For this reason, we have begun a search for iss's location in the bacterial genome. Thus far, iss in an avian E coli isolate has been localized to a conjugative R plasmid and estimated to be about 100 kilobase (kb) in size, encoding resistance to tetracycline and ampicillin. Hybridization studies have revealed that this plasmid contains sequences with homology to tsh, a gene associated with virulence of avian E coli; intI 1, a gene encoding the integrase of Class 1 integrons; and certain genes of the aerobactin- and CoIV-encoding operons. Sequences homologous to merA, a gene of the mercury resistance operon, were not identified on this R plasmid. This plasmid, when transferred into an avirulent, recipient strain by conjugation, enhanced the transconjugant's resistance to complement but not its virulence, in spite of the plasmid's possession of several putative virulence genes and traits. Such results may reflect the multifactorial nature of virulence, the degree of the recipient's impairment for virulence, or an inability of the embryo assay used here to detect this plasmid's contribution to virulence. Additionally, this plasmid contains genes encoding antimicrobial resistances, which may provide a selective advantage to virulent E. coli in the production environment. Further study will be needed to determine whether this plasmid is widespread among virulent E. coli and to ascertain the implications that this link between virulence and antimicrobial resistance genes may have for poultry management.     

禽大肠杆菌外膜蛋白基因C(ompC)的序列分析

根据 Gen Bank中人源大肠杆菌 K- 12外膜蛋白基因 C(omp C)的核苷酸序列设计引物 ,应用 PCR方法从禽大肠杆菌 O2 、O78株及它们的融合双价弱毒菌株 O2 ,78(Norr Chlr)中分别扩增得到 omp C基因 ,序列测定及分析比较发现 ,3个菌株的 om p C基因均由 170 2 nt组成 ,核苷酸序列完全相同 ,只有 1个大的开放性阅读框 (ORF) ,长 10 92 bp,编码由 36 3个氨基酸组成的前 Om p C蛋白 ,前 2 1个氨基酸残基组成信号肽 ,成熟的 Omp C蛋白由 342个氨基酸残基组成 ,Mr为 4 0 0 0 0。其氨基酸序列也完全相同。从基因水平上证明了禽大肠杆菌 O2 、O78株及融合双价弱毒菌株 O2 ,78(NorrChlr)存在相同的外膜蛋白 C抗原 ,从而为进一步研究 Omp C蛋白的免疫原性奠定了基础     

Characterisation of Escherichia coli strains associated with canine urinary tract infections

Of 33 Escherichia coli strains isolated from canine urinary tract infections, 22 were haemolytic and 27 were classified into O serogroups, the most common being O4, O6, O2 and O83. P-fimbriated strains were haemolytic and belonged mainly to serogroups O4 and O6. Twenty-nine strains possessed type-1 fimbriae but only small numbers possessed S fimbriae, type-1C fimbriae, X adhesins or the aerobactin system. It is postulated that P fimbriae and haemolysin production contribute to bacterial virulence in canine pyelonephritis and cystitis.     

新疆某牛场大肠杆菌的分离鉴定及16S rRNA序列分析

为了调查新疆某规模化奶牛场犊牛腹泻的原因并确定病原,无菌采集15份腹泻犊牛粪样进行病原的分离培养,采用形态学观察、生化试验、血清学和致病性分析等方法鉴定分离菌株,利用大肠杆菌16SrRNA通用引物进行基因序列扩增并测序,使用DNAStar软件将分离菌株测序结果与GenBank中的其他菌株序列进行同源性比对,采用Mega...