Isolation And Identification Of S. Enteritidis And Development Assays For Detection Of S. Enteritidis

Salmonellae are gram-negative facultative rod-shaped bacteria and a member of the family of Enterobacteriaceae. Salmonellae are important enteric pathogens and they cause one of the most common foodborne diseases. Many types of Salmonella bacteria cause Salmonellosis in kinds of animal and people, and they live in the intestinal tracts of warm and cold blooded animals. The elderly, infants, and those with impaired immune systems may have a more severe illness, and the adults infected with Salmonella usually remain symptomless and unnoticed. Foods of animal origin, such as meat, dairy products, and eggs, have been implicated in outbreaks of human Salmonellosis. The prevalence of S. enteritidis has dramatically increased worldwide, and infection caused by S. enteritidis are similar or exceed the number of S. typhimurium, and it has been reported as the most common serotype in the United States and other developed countries, and many people die of the disease every year. It is similar in proceeding of disease in S. enteritidis and S. pullorum, and sometimes we confused these diseases. The sperum of S. enteritidis is wider than S. pullorum, and it is harder to detect and control of S. enteritidis infection. In this study, we isolated and identified S. enteritidis strain from diseased chickens, and developed assays to detect S. enteritidis by PCR and indirect ELISA.1Isolation and identification of S. enteritidis from Young ChickensIn this study, we isoalated and identidicated a strain of S. enteritidis from diseased chickens in Jiangsu province by morphology observation, biochemical characterization, serological examination and PCR amplification. Animal tests were conducted in ICR mouse and SPF chickens to evaluate the virulence of the isolate S. enteritidis. Results showed that the isolate S. enteritidis was lethal to4-week-old mouse in72hours after injection with1×109cfu intraperitoneally, it also was lethal to3-day-old SPF chicklings with the same dose in48hours by subcutaneous injection, but all eight17-day-old SPF chickens survived after challenging with3×109cfu. Antibiotics susceptibility tests revealed that the isolate S. enteritidis was resistant to multiple antibiotics such as ciprofloxacin, norfloxacin, tetracycline, streptomycin, et al., which are ever efficacious to gram-negative bacteria, but retains high sensitivities to ampicillin, cephalosporins, et al. The result indicated that infection of S. enteritidis in early stage of breeding was an important factor of high mortality of young chickens.2Extraction, purification and immune activity analysis of SEF14fimbriae of S. enteritidisS. enteritidis could express SEF14fimbriae in CFA medium and statically in vitro. And fimbriae were separated from S. enteritidis at room temperature by shearing the bacteria in a blender, and in this study we extracted and purified the SEF14fimbriae in vitro successfully. Both the mouse sera with high titer of anti-rSEFA or anti-SEF14were raised and detected after being immunized with the purified rSEFA protein or the purified SEF14fimbriae, which could recognize the purified SEF14fimbriae from reference strain50336and rSEFA, respectively. This indicated that rSEFA protein was immunogenicity and reactogenicity.3Development of assays for detection of S. enteritidisIn this study we developed a PCR assay for detection of S. enteritidis according the sequence sdfl gene. This PCR assay just successfully decteted the S. enteritidis strains, and there was no PCR products from S. doublin, S. pullorum et al and other pathogens belong to Enterobacteriaceae. An indirect ELISA assay was developed for detection of S. enteritidis using a recombinant fusion antigen rSEFA. This assay was optimized for antigen coating centration of7.5μg/ml and a serum dilutin of1:100, with a standard incubation time60min. A reading of OD490≥0.346was scored as positive. The recombinant antigen has no cross reaction with sera of other relative disease. The result also showed the indirect ELISA is highly sensitive, specific and reproducible. The application of indirect ELISA would provide a simple and rapid method for monitoring serum antibodies against S. enteritidis infection.