| Shiga toxigenic E. coli (STEC) strains have been known as important intestinal pathogens. There are more than 100 E. coli serotypes characterized by production of shiga toxins, and the most prominent serotype is O157:H7. Typical entero-hemorrhagic E. coli (EHEC) strains are shown to express the plasmid-encoded EHEC hemolysin (pO157) and the AE phenotype (attaching and effacing lesion). Typical EHEC strains, such as O157:H7, O26:H11 and O111 are spreading majorly through contaminated foodstuff, healthy domestic animals, especially ruminants, such as cattle and sheep, which are the most popular natural reservoirs of EHEC. EHEC can cause acute gastroenteritis, bloody diarrhoea, severe hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura (TTP), and to pose considerable threat for human health.The morbidity and mortality associated with several recent large outbreaks of STEC disease have highlighted the threat that these organisms pose to public health. For this reason, there is an increasing demand for improved diagnostic procedures for the detection of EHEC in fecal samples, and in particular, in foods such as meat and dairy products, and it becomes more and more important to carry out further study to clarify the molecular epidemiology, biological charcteristics, and causative agent, mechanism of infection and prevention of EHEC. Rapid clinical detection of EHEC accompanied with the methods of molecular biology and immunology, which is one of the most efficient solutions to prevent the infection of EHEC, especially EHEC O157:H7. Furthermore, investigation of the key virulence factors of EHEC could conduce to explore better new diagnostic methods to ensure the potential pathogenic bacteria to be successfully detected and isolated, and would show great significance in the inspection and public health.1. Establishment multiplex PCR to detect EHEC and its molecular epidemiologyIn order to gain the information of the distribution and epidemic status of O157,O26,O111 and other EHEC serotypes in cattle from Jiangsu area, we have developed multiplex PCR assays for the simultaneous detection of stx1, stx2, eaeA, and hlyA, and the techniques for enrichment and isolation of EHEC. In this study, 74 strains were successfully isolated from 1128 rectal samples of cattle in Jiangsu area, and the positive rate of E. coli culture was 6.56%. The diversity of virulence related genes of isolated strains was found, and 30 STEC strains were identified (30/74, 40.54%). The EHEC serogroups included O26 (23 isolates, 34.33% in identified 74 isolates; 2.04% in total samples) and O157 (2 isolates, 2.99% in identified 74 isolates; 0.18% in total samples). The results showed that other EHEC serogrups, such as O26, rather than EHEC O157, were the common causative agents in cattle species from Jiangsu area, which also indicated faeces might be the source of food contamination. Therefore, controlling the survival and transmission of EHEC in these farms and emphasizing the importance of beef and milk inspection might be the key route to prevent the infection of EHEC in human.2. Establishment of mice challenge model with EHECThe sensibility to EHEC was increased by the means of adding nalidixic acid (Nal) in drinking water and injecting mitomycin (MMC) into the abdominal cavity of mice. We enhanced the virulence of EHEC by in vivo passage. Mice model having part of the clinical symptoms and main histopathological changes as patients infected with EHEC O157 was obtained. These results confirmed that EHEC by intragastric administration could lead to more than 50% mortality of 3-week-old ICR mice. Furthermore, the EHEC challenge dose in mice was determined, and the total number of E. coli in mice faeces at post-challenge intervals and its persistence dynamics in different organs characterized was observed. The establishment of the mice challenge model is important for us to explore the mechanism of EHEC infection and virulence factors of this organism, which is also useful for vaccine evaluation.3. Development of monoclonal antibodies against the flagellin of EHEC O157:H7Spleen cells collected from BALB/c mice immunized with the whole cell and crude flagellin antigen of EHEC O157:H7 were fused with murine SP 2/0 myeloma cells. Ten of hybridoma cell lines specific to flagellin or other antigens of EHEC O157 were established after screening and subcloning. Six out of them reacted with all EHEC O157 reference strains or isolates but no other serogroup strains. Three hybridoma cell lines designated as 7E2, 4E2 and 1D7 have high titers and specificity juged by indirect ELISA test. One of them, designed as 1A6, reacted with not only all EHEC O157:H7 strains but also STEC H8 (O26) which habors H7 flagellum. The results of indirect ELISA and Western blot showed that the antigenic epitope of the monoclonal antibody (MAb) 1A6 located on H7 flagellin of EHEC strains. Consequently, the MAbs should be useful immunological reagents for EHEC O157:H7 detction. |
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