ERIC-PCR Based Fingerprint To Analyze The Structural Features Of Intestinal Microbial Communities Of Chicks Infected With Salmonella Gallinarum, Newcastle Disease Virus And The Healthy Chicks

In order to fully know the alteration of microbial community in chick, the influence of different kinds of pathogenic microorganism on chick, and the difference of intestinal flora, in this paper analysed the structure of intestinal flora of pullorum disease group, newcastle disease (ND) group and healthy group by ERIC-PCR based fingerprint on Molecular Ecology.63 Avian chicks on 7 day were randomly divided into nine groups of 7 each. 1-4 groups in turn orally supplemented with 1mL salmonella gallinarum concentration of bacteria (10⁸, 10⁹, 10¹⁰, 10¹¹ CFU/mL) one time a day, successively for 3 days, and faeces were sampled at the age of day 5, 9, 13 and 17. 5-8 groups were in turn dripped 0.2mL New castle disease strain F48E8 (dilluted 200,150,100,50 times) one time by eye drop and nostril drop, and faeces were sampled at the age of day 5, 9, 13 and 17. The control group was given orally 1 mL 0.9% physiologic saline one time a day, successively for 3 days, and stool speciments were collected at the age of day 5, 9, 13, 17, 23, 28, 33 and 38, 43.Ingredient of faece was complicated, which not only contained cast-off cells but also contained undigested food, digestive enzyme, mucus, bile salt, bilirubin, pigment, cellulose and polysaccharides so on. Therefore, in the experiment, before cell lysis, most of impurity in the faeces can be eliminated by differential centrifugation, and then washed 2-3 times with precooling acetone. In this way, considerable inhibitor of PCR like pigment, polysaccharides and some organo-salt were eliminated. High purity DNA was gained by schizolysis of protease K and extraction of phenol chloroform. Result of the templates electrophoresis and ERIC-PCR generate fingerprints both confirmed that the approach was reliable and efficient.The total DNA of microbial community was extracted from fecal samples, subsequently used as temples in ERIC-PCR to generate community fingerprints. BioNumeric(a software) was employed to analyze the fingerprints and found as follows: After chicks were inoculated salmonella gallinarum and NDV, influence of NDV on chicks was more serious than that of salmonella gallinarum on chicks generally. Accumulative curves of Cs values of the two types of samples were poltted and proportions of Cs lesst than 0.6 were 67% for ND group, and 50% for pullorum disease group. NDV (4×10⁵ ELD₅₀) resulted in disproportion of intestinal tract microbial population more serious than salmonella gallinarum (4×10¹¹ CFU). At the beginning of infection, NDV had a smaller influence on intestinal tract microbial population than salmonella gallinarum, but influence of NDV became more serious with time going.The were greater changes in pullorum disease group within each individual than ND group within each individual. It may be that ND group was more severe symptoms, more alteration of intestinal flora, and the structure of intestinal flora tend to tend to be simplified, therefore the individual similar probability was bigger.We had dynamic monitoring of intestinal tract microbial population structure, and found that microbial population was not stable at the age of 5 days old, number and site of major bands altered larger; after 9 days old, the tendency descended; the tendency was stable after 23 days old, by this, number and site of major bands altered smaller. Theses coincided with fixing planting of the main normal microbial Flora in chick, suggesting that ERIC-PCR based fingerprints can analyze the dynamic monitoring of Animal Intestinal Flora.