Screening Of Probiotics From Swine Faeces And Its Protection Of Chicken From Salmonella Spp.Infection

Since the discovery of penicillin,antibiotics played an important role in a variety of common bacterial infectious diseases.However,the abuse of antibiotic leads to the emergence of drug-resistant strains,antibiotic residues in food products,disturbance of intestinal flora of patients,antibiotic contamination of water and soil ect.Nowadays the negative news of antibiotics has widespread around the world.Looking for green safe,non-toxic antibiotic alternatives is around the corner.A large number of studies have shown that probiotics may be an ideal substitute for antibiotics,which would promote animal growth,enhance immunity,anti-bacterial infection,regulate intestinal flora.In this study,the faece specimens were collected from local swine which never feed with manufactured feeds.The amylase,cellulose and protease-producing Bacillus spp,tolerance of acid and bile salt as well were isolated from fresh feces.The Lactobacillus strains were additionally chosen by antimicrobial activity of Escherichia coli O157: H7,Salmonella typhimurium and Shigella sonnei.The major aim of this study was to determine the effect of these isolated probiotic strains on the growth and immune performance of chicken under the challenge of Salmonella.The results are as follows:1.The fresh swine faeces which were collected in a backyard farm in Chongqing were separated on LB agar plates after withstanding at 80 ? for 15 min by water bath.the isolates were then treated with p H 3.0 for 2 h,and 0.5% with the bile salt for 24 h.Choose the strains of the survival rate higher than 90%.The ability of producing amylase,cellulase and protease was detected by transparent circle method.The candidates were identified by API biochemical assays and 16 S rDNA sequence alignment analysis.As a result,7 strains could secrete amylase,cellulose and proteinase were isolated and designated as P1.1-4,P1.1-10,P1.2-1,P1.2-3,P1.3-1,P1.3-5,P1.4-2.Among them,P1.1-4 had the strongest amylase production capacity(D/d=2.03).P1.1-10 and P1.3-1had high yield protease capacity with 7.90 and 6.56 of D/d values respectively.Cellulase production capacity is not very different.The D/d values were about 1.5.These strains were Bacillus stearothermophilus(P1.2-1),Bacillus licheniformis(P1.3-5)and Bacillus subtilis(P1.1-4,P1.1-10,P1.2-3,P1.3-1,P1.4-2)by biochemical identification and 16 S rDNA analysis.2.There were 6 strains which tolerance to acid and bile salt were isolated from the fresh faeces.They were identified as Lactobacillus delbrueckii(P3-a),Lactobacillus plantarum(P3-b),Lactobacillus acidophilus(P1.1-a,P1.1-c,P4-a,P5-a)by API biochemical assays and 16 S rDNA sequence alignment analysis.The susceptibility to 10 common antibiotics was determined by a standard method.The results showed that 6 Lacobacillus strains were resistant to ciprofloxacin and gentamicin and were sensitive to chloramphenicol.The sensitivity to clindamycin,metronidazole,benzylpenicillin and vancomycin was different between strains.There are no clear breakpoints to sulfa,sensitive for P3-b and P5-a.The other 4 strains were resistance to sulfa.P1.1-a and P1.1-cwere resistance to five kinds of antibiotics.Plate tests showed that the culture solution of 6 strains had inhibitory effects on E.coli O157: H7,S.typhimurium and S.Sonnei.Among them,Lactobacillus plantarum P3-b had the strongest inhibitory effect on the three pathogens.And the inhibition zone were 7.2 cm,6.3 cm and 8.8 cm,respectively.The pH value of P1.1-c and P3-a decreased to about 4.2 after incubation in MRS for 24 h.P1.1-a,P3-b,P4-a and P5-a decreased to3.5.The three pathogens were inoculated into MRS liquid medium with P3-b.And the growth curve showed that the lower the pH value,the slower the growth.Mice test showed that the Lactobacillus plantarum P3-b could reduce the weight loss caused by Citrobacter rodentium infection in mice.3.The effects of three probiotics strains,P1.1-4,P1.3-1,P3-b,on the protection chicken from infection of Salmonella were investigated.A 20-day-old alpine gray chicken were administrated with108 CFU Bacillus and/or 108 CFU Lactobacillus in the whole period of experiment,and infected with Salmonella(5�1010 CFU/m L)on the first week.The number of Salmonella in the faeces was measured every day after challenge.The weight and the feed were measured weekly,as to calculate the gain ratio.The chickens were sacrificed on the 21 day after challenge,and the duodenum,jejunum,ileum,cecum,rectum,pancreas,spleen and bursa of Fabricius were used to histopathology analysis.The results showed that the content of Salmonella in feace was not significantly different in each group on the first day after infection,it's about 106 CFU/g(P>0.05).Then the number of pathogens in each group decreased over time.There was no Salmonella detected in the faeces of the Bacillus group 6 days after infection and in control group it lasted to 12 days after infection.The average daily gain of group Bacillus was 7.36 g,and the gain ratio was 9.6 which was significantly better than that of the control group(P<0.05).The gain ratio of other groups were slightly better than control group.Histological observation showed that the spleen and the proliferation of lymphocytes in the bursa of Fabricius of group Bacillus augmented obviously.And the immune organ index was significantly higher than that of the control group(P<0.05).There was no significant change in bursa of Fabricius of group Lactobacillus(P>0.05).Intestinal mucosal lobular leukocyte infiltration and telangiectasia congestion or bleeding were the major lesions in chicken intestinal.The degree of intestinal mucosal capillaries expand congestion and bleeding of group Bacillus and group mixed bacteria was significantly lower than that of the control group(P<0.05).There was no significant difference in the degree of nuclear leukocyte infiltration.On a whole,7 Bacillus strains producing amylase,cellulase and protease,and 6 Lacobacillus strains were isolated from faece specimen of healthy swine.The addition 4 weeks of Bacillus and Lactobacillus in diet could increase the thickness of the intestinal muscle and the length of the villi,reduce the weight ratio,and protect chicken from Salmonella infection to certain extent.