Invasion Of Human Shigella To Chicken And Intestinal Epithelial Cell And The Expression Of CD44 In Developing Chicken Intestine

Using the Biochemical and Serotyping test,Sereny test,pathogenicity on mice and PCR methodes to identify the 7 strains isolated from patients with acute diarrhea firstly, then the shigella strains identified were selected to be cultured continuously in order to research the stability of invasive plasmid by PCR method with the ipaH,ipaB,ipaC,set1,sen genes.As a result,ZD01,ZD02 straines were identified to be S.flexneri, positive of Sereny test, and had a strong pathogenicity to mices, virulence genes of ipaH,ipaB,ipaC,set1,sen can also be dectected..After 22 and 28 times of incubation,the isolates of ZD01,ZD02 lost some virulence genes .The ZD01,ZD02 strain isolated can be used for follow-up research.To investigate the pathogenesis of shigella isolated from patients,3 day specific pathogen-free chicken were infected by oral infection or peritoneal injection using in vivo model,and chicken intestinal epithelial cells to be the in vitro model. The results showed that the LD50 of peritoneal injection was 0.85×106 CFU/ml,which is much higher than that of oral infection.The bacteria were recorded in heart,liver,spleen,lung and intestine 6 hours after peritoneal injection.The lung had the highest bacteria count for both oral infection or peritoneal injection at 24 hours infection.The electronic microscropy observation on the infected IEC cells suggested shigella can invaded the cell and present in the cytoplasm. Conclusion,shigella isolated from patients can also invade the chicken intestine and make pathogenesy to chicken.CD44 molecular has been identified the receptor of shigella when invading the patients.To observe the chronological and apatial expression of CD44 molecular in developing chicken intestine ,reverse translate PCR was developed firstly to identified CD44 molecular existing in the intestine or not.Then fluorescent quantitative RT-PCR was developed to quantitate CD44 mRNA in chicken ,at last,1-28 day chicken were choosed to to observe chronological and apatial expression of CD44 molecular in chicken intestine.As a result,CD44 mRNA can be detected in chicken intestine . The results showed that the Real-time PCR assay developed was highly specific and the regression coefficient was 1.00 with 95%PCR amplification efficiency of CD44 and 101% PCR amplification of GAPDH.It had a detection threshold of 45 copies of CD44 plasmid DNA and 77 copies of GAPDH plasmid DNA .The fluorescent quantitative RT-PCR developed was highly sensitive,specific,and can be used as a useful tool for evaluating he chronological and apatial expression of CD44 molecular in developing chicken intestine.Results indicates that CD44 mRNA levels in developing chicken at day 5-10 day were increased from chicken cecum to rectum , Intestinal segments dropped afer 15day.In conclusion,we found that shigella isolated from patients canalso invade the chicken intestine and make pathogenesy to chicken. CD44 mRNA can be detected in chicken intestine, CD44 mRNA levels was much higher at cecum and rectum than other intestine section.