Ewers Christa, Janssen Traute, Kiessling Sabine, Philipp Hans-C, Wieler Lothar H
Institut für Mikrobiologie und Tierseuchen, Freie Universität Berlin, Philippstrasse 13, 10115 Berlin, Germany.
Avian Dis. 2005 Jun;49(2):269-73. doi: 10.1637/7293-102604R.
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.
基于最近公布的禽致病性大肠杆菌（APEC）中毒力相关因子的流行率数据及其在大肠杆菌病发病机制中的作用，我们开发了一种多重聚合酶链反应（PCR）作为一种分子工具，以补充目前主要依赖于对从患病禽类分离的菌株进行血清学检查的诊断方案。从已知具有八个基因不同组合的大肠杆菌病临床病例中分离出的多个大肠杆菌菌株用作模板DNA来源，以开发多重PCR方案，其靶向P菌毛（papC）、气杆菌素（iucD）、铁抑制蛋白（irp2）、温度敏感血凝素（tsh）、空泡自转运毒素（vat）、肠聚集毒素（astA）、血清存活增强蛋白（iss）和大肠杆菌素V质粒操纵子基因（cva/cvi）的基因。为了验证这种诊断工具的实用性，从临床健康鸡的粪便样本中分离出的大肠杆菌菌株以及尿路致病性（UPEC）、产坏死毒素和致腹泻性大肠杆菌菌株也被纳入本研究。将多重PCR方案应用于从败血性家禽中分离出的14株大肠杆菌，结果表明这些菌株含有上述四至八个基因。相比之下，那些已被证明对5周龄鸡无致病性的分离株要么不含有这些基因，要么最多含有其中三个基因。我们发现只有一株肠出血性（EHEC）、一株肠致病性（EPEC）和两株肠毒素性（ETEC）大肠杆菌菌株的irp2呈阳性，另外两株ETEC菌株的astA呈阳性。正如预期的那样，UPEC分离株产生了iss、papC、iucD、irp2基因的不同组合以及一个与vat相似的序列。然而，在UPEC分离株中既未扩增出大肠杆菌素V操纵子基因cva/cvi，也未扩增出tsh。将多重PCR结果与通过DNA-DNA杂交分析获得的结果进行比较，以验证寡核苷酸引物的特异性，该方案被认为是一种用于检测禽致病性大肠杆菌并将其与非致病性菌株及其他致病型菌株区分开来的有用、灵敏且快速的检测系统。
