The Study Of Screen Of Probiotics As Substitute Antibiotics And Their Anti-salmonella Infection In Chickens

Probiotics are a class of active microorganisms that can be orally colonized in animals and change the structure and composition of the host’s flora in a certain part of the body.It promotes nutrient absorption and maintains intestinal health by regulating the mucosal immune function of the body or regulating the balance of flora in the intestinal tract.Since July 1,2020,China’s Ministry of Agriculture and Rural Affairs issued Notice No.194,which clearly requires that the addition of antibiotics in feed is strictly prohibited,meaning that we will enter the"antibiotic-free era".Many studies have shown that probiotics,as a safe and effective natural immunomodulatory substance or antimicrobial microorganism,can be the preferred target for"antibacterial replacement".In this study,we propose to screen the active candidate probiotic strains with broad-spectrum anti-pathogenic bacteria through in vitro antimicrobial experiments,and then use animal experiments to verify the effectiveness of the screened"antibiotic"probiotic strains against Salmonella infection in chickens,mainly by evaluating the antibacterial effect of oral candidate probiotics on infected chickens’organs and intestinal Salmonella to confirm the antibacterial effect of the candidate strains.The antibacterial effect of the candidate strains was confirmed by evaluating the antibacterial effect of oral probiotic candidates on Salmonella spp.This study provides an important reference for the prevention and control of Salmonella infection in chickens or the purification of chicken dysentery,and is of great significance for the promotion of green and healthy breeding and the development of"antibiotic-free"food safety.Objective:In vitro screening of candidate probiotic strains with broad-spectrum anti-pathogenic activity,animal experiments to verify their protective effects against Salmonella offensive infection and mimic natural infection in chickens,laying the foundation for the research of"alternative anti"probiotic formulations.Methods:(1)In vitro screening of candidate probiotic strains with broad-spectrum anti-pathogenic activity:identification of candidate probiotic bacteria by 16s r DNA;evaluation of probiotic properties of15 candidate probiotic strains by self-aggregation ability and in vitro co-aggregation ability with pathogenic bacteria;and screening of standard pathogenic Escherichia coli,Salmonella chicken dysentery,Staphylococcus aureus and 10 clinically isolated wild pathogenic bacteria as indicator bacteria by agar plate method.The candidate probiotic strains with the broadest antimicrobial spectrum and strongest antimicrobial activity were selected from 15 candidate probiotic strains.(2)Study of candidate probiotic strains against Salmonella typhimurium attack infection:The obtained candidate probiotic L.C332 was fed to three-week-old chicks at 1×10~9 CFU/day for 21 days,and 1×10~9CFU of Salmonella typhimurium C79-1 per chick was attacked by oral instillation on day 8,while an attack control group and a blank control group were established.Serum was collected from each group of chickens on days 3,7 and 14 after infection,and three chickens in each group were dissected to collect organs and intestinal contents.The expression of IL-1βin serum was measured by ELISA kit.The proliferation activity of T-lymphocytes and antigen-presenting cells in thymus and peripheral blood were measured by CCK-8 kit.The content of Salmonella spp.and E.coli bacteria in liver,spleen and intestinal contents were measured by using Salmonella spp.(3)Study on the candidate probiotic against Salmonella chicken dysentery mimicking natural infection:SPF chickens were randomly and equally divided into three groups:oral probiotic experimental group,fed1×10~9 CFU of the candidate strain L.C332 daily,and also mimicking natural infection with 1×10~4 CFU of the standard pathogenic strain of Salmonella chicken dysentery C79-1 orally daily;mimicking natural infection control group In the control group,1×10~4 CFU of Salmonella C79-1 was administered orally every day to mimic natural infection;in the blank control group,only the basal diet was fed.After 4 weeks,all chickens were dissected and killed,and the liver,spleen and intestinal contents were collected aseptically,and the bacterial contents of Salmonella spp.and E.coli spp.in the liver and intestine of each group were determined by plate count method.Results:(1)The candidate probiotics were identified by 16S r DNA,and the 15 strains were 12 strains of Lactobacillus mucosus,1 strain of Streptococcus enterica,1 strain of Enterococcus faecalis and 1 strain of Lactobacillus salivarius;the in vitro antibacterial screening results showed that L.salivarius L.C332had strong antibacterial activity against 3 standard pathogenic bacteria and 10 wild pathogenic strains isolated from clinically ill chickens(10 pathogenic bacteria were isolated from the intestinal tract of clinically diarrheic or chicken dysentery positive chickens).L.C332 showed strong antibacterial activity.(2)The results of animal experiments on Lactobacillus salivarius strain L.C332 against Salmonella dysentery attack infection in chickens showed that early feeding of Lactobacillus salivarius L.C332significantly reduced the colonization of Salmonella spp.or other pathogenic bacteria such as Escherichia coli in liver,spleen and intestinal contents;it significantly reduced the antibody conversion rate of chicken dysentery;feeding of Lactobacillus salivarius L.C332 significantly enhanced the proliferation activity of antigen-presenting cells and T lymphocytes in thymus and peripheral blood L.C332 significantly enhanced the proliferation activity of antigen-presenting cells and T lymphocytes in the thymus and peripheral blood,and significantly reduced the secretion of inflammatory cytokine IL-1βin the serum.(3)The results of animal experiments with the candidate probiotic Lactobacillus salivarius L.C332against Salmonella dysentery mimicking natural infection in chickens showed that feeding Lactobacillus salivarius L.C332 significantly blocked antibody transitivity in Salmonella dysentery mimicking natural infection in chickens,with a transitivity rate of only 10%(2/20);while the control group had an antibody transitivity rate of 80%(16/20);feeding the probiotic significantly reduced Salmonella spp.bacteria or other pathogenic bacteria such as E.coli in the liver,spleen and intestinal contents.