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1.
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.  相似文献   

2.
The present study reports colibacillosis of layer chickens in a commercial egg-producing farm in western Japan. Three flocks of chicken at 18-21 weeks of age were affected during the initiation of egg lay. Postmortem examination revealed pericarditis, perihepatitis, airsacculitis, subcutaneous inguinal lesion, and injured cloaca. Escherichia coli was isolated from the lesions of the affected birds. Twenty-two of 26 E. coli isolates (84.6%) obtained from 18 birds in the 3 flocks showed pulsed-field gel electrophoresis (PFGE) patterns that were considered to be closely associated to each other and arbitrarily designated as pattern A. All the 22 isolates with the PFGE pattern A harbored the putative virulence genes, astA, iss, iucD, tsh, and cva/cvi. Additional 2 PFGE patterns (B and C) were also found in E. coli isolates obtained from the affected flocks and had the putative virulence genes in combinations different from those in the pattern A strains. The results suggested that certain E. coli virulence genes and host factors, such as initiation of egg lay may be associated with occurrence of colibacillosis.  相似文献   

3.
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.  相似文献   

4.
食品动物源产超广谱β-内酰胺酶大肠杆菌的流行状况调查   总被引：1，自引：1，他引：0  
本研究旨在了解产超广谱β-内酰胺酶(ESBLs)大肠杆菌和CTX-M型耐药基因在食品动物的流行状况。对分离自养殖场的1273株大肠杆菌(健康动物源755株,患病动物源518株),采用双纸片协同试验筛选ESBLs阳性菌,并通过PCR法和基因测序鉴定阳性菌CTX-M型的基因型。1273株食品动物源大肠杆菌中筛选出产ESBLs阳性菌181株,阳性率为14.2%,其中患病动物阳性率比健康动物高,分别为19.9%和10.3%。151株(83.4%)产ESBLs大肠杆菌中检测到CTX-M型基因,占所有食品动物源大肠杆菌的11.8%,其中以CTX-M-9G为主,阳性率为79.5%(120/151),远高于CTX-M-1G的携带率25.8%(39/151),其中8株同时携带了CTX-M-1G和CTX-M-9G。产ESBLs的大肠杆菌在食品源动物中流行普遍,CTX-M是产ESBLs阳性菌中的主要流行基因型。  相似文献   

5.
The presence of the astA gene responsible for production of enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) was examined in E. coli strains isolated from pigs with postweaning diarrhoea. Two hundred and seven isolates were tested using PCR for the astA marker and for heat-labile I (LTI), heat-stable I (STI), and heat-stable II (STII) enterotoxin genes. Moreover, the isolates were also analysed for their serotypes (O and K antigens) as well as for fimbrial adhesins using agglutination methods. It was shown that 96 (46.4%) of the isolates possessed the astA genetic determinant. The most common EAST1-positive E. coli serotype was O149:K91 and these strains were mostly LTI/STII-positive. A close correlation between the presence of F4 fimbriae and the EAST1 gene was also observed: 88 of 96 (91.7%) astA(+) isolates tested possessed the F4 antigen. Thus, EAST1 enterotoxin may represent an additional virulence determinant playing a role in the pathogenesis of porcine colibacillosis.  相似文献   

6.
Five clinically normal chickens from three farms (farm A, farm B, and farm C), for a total of 15 clinically normal chickens, were examined bacteriologically. In a similar manner, five dead chickens with lesions of peritonitis from each of the same three commercial egg-laying operations were selected for bacterial culturing. Escherichia coli were isolated from the cloaca in 14 of 15 healthy chickens and from all 15 chickens with peritonitis. Oviducts of normal chickens did not contain E. coli (0/15) whereas oviducts from 13 of 15 hens with peritonitis were positive for this pathogen. No lesions and no E. coli (0/15) were found in the peritoneal cavity of healthy hens, but peritonitis lesions from 13 of 15 dead chickens yielded E. coli. On farm A and farm B, a flock consisted of all chickens within a single house and all chickens in each flock were of the same age and same genetic strain. In flock 1 from farm A, all five E. coli isolates from the oviduct and all five isolates from the peritoneal cavity were serogrouped as O78; contained the virulence genes iroN, sitA, iutA, tsh, and iss; and belonged to phylogenetic group A. In flock 2 from farm B, all four E. coli isolates from the oviduct and all four isolates from the peritoneal cavity were serogrouped as O111; contained virulence genes iroN, sitA, iutA, traT, iss, and ompT; and belonged to phylogenetic group D. These data suggest that all chickens with peritonitis in a single flock on farms A and B were likely infected by the same E. coli strain. Escherichia coli isolates from the magnum and peritoneum had the same serogroup, virulence genotype, and phylogenetic group, which is consistent with an ascending infection from the oviduct to the peritoneal cavity.  相似文献   

7.
One hundred four Escherichia coli isolates were collected from internal tissues and the cloacae of broilers with colibacillosis or from the cloacae of healthy birds. The isolates were tested for the presence of DNA sequences for temperature-sensitive hemagglutinin (tsh), for P (pap) and F1 (pil) fimbriae, and for aerobactin synthesis (iuc) by DNA/DNA hybridization. The isolates were also tested for O1, O2, and O78 serogroups, serum and antibiotic resistance, and virulence in day-old chickens. The Tsh/Pil/Iuc was the major pathotype detected in 53.8% of isolates from internal tissues, as compared with only 28.8% of isolates from the cloacae. The Tsh/Pap/Iuc pathotype was detected at a lower frequency (15.4%) but only in isolates from internal tissues. Among the virulence-associated marker genes, tsh and iuc were detected in most of the isolates from internal tissues (90.4% and 92.3%), as compared with only 51.9% and 63.5% of isolates from the cloacae, respectively, pap was detected to a lesser extent, in 25% of isolates but only from internal tissues. In contrast to the pil gene, the tsh-, pap-, and iuc-DNA sequences were more frequently detected in isolates from internal tissues than in isolates from the cloacae. O-antigen typing revealed that 25% of isolates belonged to serogroups O1 (4.8%), O2 (9.6%), and O78 (10.6%). Although most isolates appeared to be resistant to serum, only isolates from internal tissues were virulent in day-old chickens in contrast to isolates from the cloacae. More than 10% of isolates were resistant to most of the antibiotics used for the study. However, less resistance to enrofloxacin and norfloxacin was observed. Our data suggest that the Tsh/Pil/Iuc and Tsh/Pap/Iuc pathotypes and Tsh and Iuc virulence-associated markers are important factors of avian pathogenic E. coli. Enrofloxacin appeared to be the best choice for treatment of the infection.  相似文献   

8.
In the course of our molecular studies of virulence factors associated with invasive avian Escherichia coli infections, it was first necessary to distinguish between common E. coli and those that cause septicemia in poultry. We found a direct correlation between the ability of clinical isolates of E. coli to bind Congo red dye (CR) and their ability to cause septicemic infection in chickens. This finding was supported by bacteriological studies of 30 broiler flocks (26 sick and 4 healthy) and by virulence studies in chickens and mice. All 144 isolates of E. coli from internal tissues of diseased birds were determined to be CR-positive (red colonies). Congo-red-positive E. coli colonies were isolated from air sacs, pericardium, liver, lung, joint fluid, and heart blood of chickens with lesions of colisepticemia. In contrast, of 170 E. coli isolates from the poultry house environment and from the trachea and cloaca of healthy birds, more than half were CR-negative (white colonies). No CR-negative (white) E. coli colonies were found in internal organs from birds with typical lesions of colisepticemia. We feel that these preliminary findings suggest that the CR dye binding could be used as a phenotypic marker to distinguish between invasive and noninvasive isolates.  相似文献   

9.
In this study, we determined the occurrence of the tsh gene among 305 Escherichia coli isolates from chickens by means of the polymerase chain reaction and agglutination of chicken erythrocytes; 200 of those isolates were obtained from chickens with colisepticemia, 52 isolates were from lesions of cellulitis, and 53 were from feces of normal chickens. The tsh gene was found in 79 (39.5%) isolates from colisepticemia, in 10 (19%) cellulitis-derived E. coli isolates, and in two (3.8%) fecal isolates. Among the tsh+ strains, 68 (86%) isolates from colisepticemia and nine (90%) from cellulitis agglutinated chicken erythrocytes in the presence of mannose, after growing the strains on colonization factor antigen agar plates at 26 C, which confirms a correlation between mannose-resistant hemagglutination and expression of hemagglutinin Tsh. These results show, for the first time, the presence of the gene tsh in cellulitis-derived E. coli isolates; the high frequency of this gene among avian pathogenic E. coli isolates in Brazil indicates that its putative role as a virulence factor should be studied more thoroughly.  相似文献   

10.
为了解105株携带耶尔森菌强毒力岛(HPI)的大肠杆菌(E.coli)中相关毒力因子的流行情况和基因序列,根据GenBank中参考序列设计引物,采用PCR方法对广东地区养殖场来源的105分离株HPI+E.coli的fyuA、tsh、iucD、iss 4种毒力基因进行检测,统计基因类型;并对部分分离株的5种毒力基因(irp2和fuA、tsh、iucD、iss)进行了克隆与序列分析.结果显示105株HPI+E coli中4种毒力因子携带情况不尽一致,基因fyuA、tsh、iucD和iss的阳性率分别为55.24％、17.14％、49.52％和23.81％,105株HPI+E.coli共有13种基因型;分析表明,除iss基因与参考序列的同源性在88.0 ％～90.9％外,irp2、fyuA、tsh、iucD4种基因与GenBank中参考序列的同源性高达96％以上;广东省养殖场E.coli毒力因子基因型复杂,并以基因型irp2+ fyuA+ iucD+和仅含irp2+的菌株分离率最高,分别为17.14％和28％.  相似文献   

11.
Five distinct lipopolysaccharide (LPS) core types, namely R1-R4 and K12 have been identified in Escherichia coli. The aims of this study were to determine, primarily by means of PCR, the distribution of those oligosaccharide core types among avian pathogenic E. coli and their relationship to phylogenetic groups. To identify putative avian pathogenic E. coli, serum resistance and the presence of three virulence genes encoding temperature sensitive haemagglutinin (tsh), increased serum survival (iss) and colicin V (cvaC) were determined. Of the 143 clinical isolates examined 62% possessed the R1 core, 22% were R3, 13% were R4 and 3% were R2. Fifty commensal isolates consisted of 58% with R1 core, 38% with R3 core, 4% with R4 core, and none with R2. None of the isolates were of K12 core type. The distribution of core oligosaccharide types in clinical and commensal isolates were not statistically significant (P=0.51). Three genes, tsh, iss and cvaC were found in E. coli of all four core types. The genes tsh (P<0.001) and iss (P=0.03412) were significantly associated with the R4 core oligosaccharide type. The isolates containing R4 core type LPS were mainly confined to phylogenetic group D. The widespread R1 core type showed less ability to possess virulence genes and 83% were in the phylogenetic group A. Results of this study indicated that E. coli with R1, R2, R3 and R4 were important in causing infections in chickens and further, the E. coli with R4 core type were less common among commensals, possessed more virulence genes and were related to phylogenetic groups pathogenic for poultry.  相似文献   

12.
This study determined the prevalence of F4, F5, F6, F17 and F41 fimbriae and the genes for FedA (F18 fimbriae), LT and ST enterotoxins, and Shiga toxins Stx1, Stx2 and Stx2e among E. coli isolated from 372 weaned pigs with diarrhea and 46 healthy pigs of the same age. Agglutination tests showed that most isolates were negative for all five fimbrial antigens. The F4 antigen was found in 71 (19.1%) and the F5, F6, or F41 antigen was detected in 6.4% of isolates from diseased pigs. Genes for the F18 fimbriae were detected in 10 (2.7%) strains from diarrheic pigs and in 1 of 46 isolates from healthy pigs. Most isolates (213, 57.3%) from pigs with diarrhea were positive for LTI only or for LTI and STI or Stx2e toxin genes. Fifteen strains (13.7%) possessed only the STI or STII toxin genes. All F4-positive bacteria had genes for LTI or LTI and STI, whereas F18-positive isolates had genes for LTI, LTI/STI, or LTI/Stx2e. Of the strains isolated from diseased pigs, 264 (71.0%) were negative for the fimbrial antigens (genes) examined in this study. The fimbria-negative isolates frequently possessed genetic determinants for LTI (118, 31.7%) or for STII (16, 4.3%) enterotoxins.  相似文献   

13.
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.  相似文献   

14.
Avian pathogenic Escherichia coli, the causative agent of colibacillosis, harbors several putative virulence genes. In this study we examined by polymerase chain reaction (PCR) the presence of 16 of those genes in 200 colibacillosis isolates from our region. The seven virulence genes iutA, iss, cvaC, tsh, papC, papG and felA were detected significantly more often amongst colibacillosis isolates than in fecal isolates from healthy birds, thereby confirming their worldwide occurrence and possible pathogenic role in colibacillosis. However, several of those genes were not detected in many colibacillosis isolates, and none of them were detected in 27.5% of those isolates, which suggests that variants of those genes and yet undetected virulence factors should be searched for.  相似文献   

15.
Eight Escherichia coli isolates from ostriches with respiratory disease were investigated for the presence of genes encoding the following adhesins: type 1 pili (fim), pili associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin (afaI), temperature regulated adhesin, curli (crl, csgA) and temperature-sensitive hemagglutinin (tsh). Genes for heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf), alpha-haemolysin (hly) and aerobactin (aer) production were also investigated. Other characteristics investigated were the presence of hemagglutination activity, growth on an iron-deficient medium, aerobactin production, serum resistance, adherence to chicken tracheal cells, pathogenicity for day-old chicks, and serogroup. Serogrouping showed that four isolates belonged to serogroup O2, two to serogroup O78, one to serogroup O9, and one to serogroup O21. The virulence genes found were: fim in all eight isolates, csgA in seven, aer in six, and pap, crl and tsh in one isolate each. All isolates analyzed were positive for mannose-resistant hemagglutination, adhered in vitro to ciliated tracheal epithelium, grew on iron-deficient medium, and showed serum resistance. Pathogenicity tests on day-old chickens revealed one highly pathogenic isolate, three of low pathogenicity and four isolates with intermediate pathogenicity.  相似文献   

16.
In this study, we assessed the pathogenic potential of Escherichia coli associated with a commercial competitive exclusion (CE) product by examining the phenotypic characteristics associated with E. coli virulent for humans and domestic animals. Most E. coli isolates were capable of proliferating in iron-deplete chicken sera. Interestingly, none of the E. coli isolates from the commercial CE product contained the bacterial adhesin Tsh characteristic of avian pathogenic E. coli associated with airsacculitis and colisepticemia. In terms of virulence potential for humans, most E. coli isolates (78%) were sensitive to killing by 12.5% human sera. Because of their sensitivity to human sera, the E. coli in the CE product are not likely to cause a serious systemic infection in humans and, therefore, do not present a risk of causing septicemia in humans. Because these isolates also lack the gene tsh, they are also less likely to cause systemic disease or airsacculitis in poultry than pathogenic strains commonly isolated from diseased birds.  相似文献   

17.
Escherichia coli is commonly isolated in canine pyometra, but little is known of the virulence factors that may be involved in the precipitation of this disease. The aim of this study was to compare the prevalence of uropathogenic virulence factor (UVF) genes in E. coli isolates from canine pyometra and from feces of healthy bitches to evaluate their role in the pathogenesis of pyometra. E. coli from 23 cases of canine pyometra and from the feces of 24 healthy bitches were analyzed, by polymerase chain reaction, for UVF genes associated with canine and human urinary tract infections (UTIs). The prevalences of UVFs in E. coli from canine pyometra were similar to that in canine and human uropathogenic E. coli. The prevalence of pap was greater (P=0.036) for E. coli from pyometra (52%) than for fecal isolates (21%), and the papGIII allele was present in all pap-containing isolates. The prevalences of genes for alpha-haemolysin and cytotoxic necrotising factor 1 were not significantly higher (P=0.075) in E. coli from pyometra than from feces. The proportion of pyometra strains with >or=3 UVFs was higher (P=0.039) than that of fecal strains, suggesting that possession of >or=3 UVF genes enhances the pathogenicity of the strain. Our findings demonstrate that E. coli associated with canine pyometra are similar to uropathogenic strains, and that operons that encode P fimbriae, alpha-haemolysin and cytotoxic necrotising factor 1 probably enhance the virulence and pathogenicity of the strain in the canine genital tract.  相似文献   

18.
A total of 1002 Escherichia coli strains isolated from pre-weaned pigs with diarrhoea on 1114 swine farms were screened for the presence of the adhesin involved in diffuse adherence (AIDA) gene by polymerase chain reaction (PCR). Escherichia coli isolates that carried AIDA genes were also tested by PCR for the detection of five fimbriae (F4, F5, F6, F18 and F41), heat-stable (STa, STb) and heat-labile (LT) enterotoxin, enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1), and Shiga toxin 2 oedema disease (Stx2e) genes. Twenty-three (2.3%) of the 1002 E. coli isolates carried the gene for AIDA. Among 23 isolates shown to carry genes for AIDA, three carried the AIDA gene as the only shown virulence factor. Other isolates carried other virulence factor genes in addition to AIDA. Four isolates carried genes for at least one of the fimbrial adhesins and enterotoxins. Sixteen isolates carried genes for enterotoxins only. The AIDA may represent an additional virulence determinant in pre-weaned pigs with diarrhoea.  相似文献   

19.
In this study, 294 Escherichia coli isolates from birds with colibacillosis were collected from disease outbreaks throughout the United States and were compared with 75 fecal E. coli isolates of apparently healthy chickens by their possession of several purported virulence genes, resistance to rough-lipopolysaccharide-specific bacteriophages (rLPSr), and elaboration of capsule. Traits were selected for study on the basis of their association with complement resistance. The genes targeted in this study included those encoding colicin V (cvaC) and the outer membrane proteins TraT (traT), OmpA (ompA), and Iss (iss). No significant differences were found between the two groups of isolates in the occurrence of cvaC-, traT-, or ompA-homologous sequences or in rLPSr. Only a few isolates were encapsulated, and the isolates of healthy birds were significantly more likely to be encapsulated than were the isolates of sick birds. However, iss, whether detected through hybridization or amplification, was found in more of the disease-associated isolates than in those of healthy birds. This difference was highly significant. Further, iss sequences were widely distributed among isolates of different serotypes from various avian host species and sites within these hosts. Such results suggest that possession of the iss sequence by an avian E. coli isolate may be a good indicator of that isolate's potential to cause disease. This association warrants further study because iss and the protein it encodes may be useful targets of future colibacillosis control efforts.  相似文献   

20.
Coliform mastitis (CM) is not only a serious economical and animal welfare touching problem in dairy cattle, but also in sows after farrowing. Due to this disease, the essential adequate supply with colostrum for the growth and the health of the piglets is not ensured. Besides other influencing factors, Escherichia (E.) coli is of great importance as a causative agent of this multifactorial disease. In this study, E. coli isolates from milk samples of healthy and CM-affected sows were examined for the presence of virulence genes associated with extraintestinal E. coli strains, enterotoxigenic E. coli and other pathogenic E. coli. The isolated E. coli harbored mainly virulence genes of extraintestinal E. coli strains (especially fimC, ompA, traT, hra, kpsMTII, iroN). The virulence gene spectrum for both samples from CM-affected and healthy sows did not differ significantly. Particular virulence gene profiles of E. coli isolates from diseased sows were not detected. This study provides novel insights into the role of E. coli in association with mastitis in sows since it is the first time E. coli isolates from CM-affected sows' milk were analysed for virulence genes. Because there were no differences in the prevalence of E. coli and their virulence-associated genes between healthy and diseased sows, other causative factors seem to have greater influence on the pathogenesis of porcine CM.  相似文献   

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