Genetic Diversity Of Clostridium Perfringens And Effects Of Bacillus Licheniformi On The Ileum Flora Of Chicken Necrotizing Enterocolitis

To validate an investigation method for the epidemiology of Clostridium perfringens in chicken, genetic diversities of Clostridium perfringens isolated from healthy chicken of in cecum and crop, on days 21,28,35,42 and 49 were analyzed by REP-PCR and ERIC-PCR methods. To isolate beneficial Bacillus spp. from the gastrointestinal tracts of healthy chickens,and to screen strains to against Clostridium perfringens, a causative agent of necrotic enteritis in poultry. Prevention of Bacillus and treatment of necrotic enteritis caused by Clostridium perfringens and to evaluate the effect of Bacillus spp. on growth performance, immune organ index, ileal flora chicken. The main contents as follows:1. Isolation and identification of Clostridium perfringens:Samples of healthy chickens were collected from different days.27 strains were selected by culture with selective medium. The isolating rate of Clostridium perfringens is 9.0%.2. Genetic diversity of Clostridium perfringens:By multi-PCR genotype of the isolates for toxin detection, PCR products were sequenced and the results show that the separation of Clostridium perfringens type A were serotype. The results revealed that the genetic types were correlated closely with the origins of the isolates. The difference of genetic types was significant among different organic and days. A subtype of Clostridium perfringens was main on any one organic and days with a few other subtypes.3. Screening of Bacillus:The strain H2 were screened for further study due to their tolerate acid, bile and bacteriostatic test.The result suggested that H2 had higher antimicrobial activity. The main antibacterial component of strain H2 was acid-dependent substance based on the result of biol-performance testing of metabolite.4. Animal testing:Health 1 day old chickens were randomly assigned into nine groups, Group1,2,9 were fed the diet,3,4,5 basal diet group were 0.05%,0.1%,0.2% FMⅠ(1.0×109 CFU/g),15-day feeding insect (1.0×107/mL 1mL),17-20 days feeding of Clostridium perfringens (1.0×108 CFU/mL 1mL),experimental study on the pathogenicity of necrotic enteritis in chicken, Group 3 after 21 days shared equally the three groups of 6,7,8, Basic diet were added in 0.1%,0.2%,0.4% FMⅡ(1.0×1010 CFU/g), the group of 9 based diets supplemented with 0.1% salinomycin. (1) Growth performance:Bacillus adding different doses of prevention, treatment, infection of the chicken model and the end of the control group body weight, average daily gain than those in the high infection group (P<0.01), and decreased F/G (P<0.01),of which the most obvious effect of 4,8 group.(2) Immune organ index(IOG):28 days of age gap, the group of 3,4,5 thymus index, spleen index and index of infection bursal significant difference (P<0.01), spleen index of group 4 had significantly lower with 3,5 (P<0.05). This shows that adding 0.1% in powder-based diet I in raising chickens in the effective index of immune organs.6,7,8 Group thymus index and spleen index and the infection group, significant difference (P<0.01),bursal index infection significant difference (P<0.05), The antibiotic group of thymus index, spleen index and bursa of Fabricius index significant difference in infection (P<0.01), With the control group no significant difference.(3)Ileal flora(IF):28 days old when the Bacillus prevention group, Add the powder of the t group of 3,4,5 it is increased that the number of Lactobacillus, Bifidobacterium, Bacteroides and total anaerobic bacteria (P<0.05), The same time, it is decreased that the number of Clostridium perfringens, E. coli, enterococci, and total aerobic bacteria significantly (P<0.05), Bacillus treatment group, add the powder first 6,7,8 group II Lactobacillus, Bifidobacterium, it is increased that the number of Bacteroides and total anaerobic bacteria (P<0.05), it is decreased that the number of Clostridium perfringens, E. coli, enterococci, and total aerobic bacteria significantly (P<0.01). In addition to the total aerobic antibiotic group, Bifidobacterium, no significant difference in the total anaerobic bacteria. indicated that the ecological environment balance between normal flora in ileum has recovered.(4)Applied PCR-DGGE to analysis of structure of the diversity and ileum:28-day-old, the blank control group, fed Bacillus prevention, treatment groups with the infected group stripe pattern difference among the groups were highly significant differences(P<0.01), Infection group, the average of three repeated only 19 algebraic, it hows that chickens have taken place in the gut flora imbalance somewhat eased, but the lower gut microbes polymorphism. White control group, the average of algebra 26.3,3,4,5, the average of algebra, respectively 23.7,31,30.3, Description fed Bacillus gut microflora can protect and improve the polymorphism of the chicken intestinal microbes, and the group of 4 prevention group the best results.6,7,8,9the average of algebra, respectively 23.6,25.3,33.7,21. In the feeding of Bacillus prevention is based on, and then add the high-dose group of 8 treatment of Bacillus gut microflora can protect and improve the polymorphism of the chicken intestinal microbes.(5) Ileum morphology test:Blank control group, infection group, the group of 4 to prevent and the group of 8 treatment of ileal villus height was significantly different (P<0.01), Compared with the control group, the group of 4 ileum villus height increased 19.52%, the group of 8 reduced compared with the control group 17.25%, still higher than the infected group. This shows that one-day-old to add the prevention of Bacillus subtilis could play a role in protection of intestinal tissue.