Isolation And Screening Of Probiotic Bacillus From Pigeons And Evaluation Of Their Effect On The Control Of Salmonellosis In Pigeons

Pigeons are the fourth largest poultry breeding industry in our country.Salmonellosis is the disease that causes the greatest harm to the pigeon breeding industry except for Newcastle disease.The main control measure for pigeon salmonellosis is to add antibiotics in feed.Due to the issuance of the"anti-antibiotic ban",the research on alternatives to antibiotics has become important.Studies have shown that probiotics has resistance to pathogenic bacteria by competitive exclusion,producing antibacterial substances and changing the living environment of pathogenic bacteria in the body,so it can be anfeed additives replace antibiotics,and homologous probiotics are more likely to play their role in inhibiting pathogenic bacteria in animal bodies.At present,there is no relevant research on pigeon-derived probiotic Bacillus subtilis.In this study,Bacillus subtilis with probiotic potential was isolated from the feces of healthy white-feathered king pigeons,and its prevention and control effect on pigeon salmonellosis was explored through the model of salmonella infection in pigeons.In this experiment,the acid and bile salt tolerance test was carried out on the isolated probiotic Bacillus subtilis,microscopic examination,biochemical tests,and 16S r DNA sequencing.After the candidate strains were determined through the above tests,it was carried out the vivo safety test.Ten 40days old male White Feather King pigeons were given 4 m L of F31 bacterial at 1.1×10¹⁰CFU/m L every day for 10 consecutive days.To determine the concentration of Salmonella to establish an animal model,Thirty 40 days old male White Feather King pigeons were randomly divided into three groups,ten pigeons in each group,and were given low concentration（5.0×10²CFU/m L）,medium concentration（5.0×10⁴CFU/m L）,and high concentration（5.0×10⁸CFU/m L）of Salmonella for 7 days,4 m L per day.Fifty40 days old male White Feather King pigeons were randomly divided into five groups with 10 pigeons in each group.Group A was the blank control group and was given 4 m L of normal saline for 14 days.Group B and Group C were the experimental groups to evaluate the preventive effect.Group B was given 5.5×10⁹CFU/m L of the the candidate strain for the first 7 days,and group C was given 4 m L of normal saline for the first 7 days.After B and C groups were fed with Salmonella bacterial solution 5.0×10⁴CFU/m L for 7 days.Groups D and E were the experimental groups to evaluate the treatment effect.Groups D and E were given Salmonella bacteria solution 5.0×10⁴CFU/m L for the first 7 days,After group D was given the candidate strain bacteria solution 5.5×10⁹CFU/m L for 7 days.m L,and group E was given 4 m L of normal saline for 7 days.The preventive and therapeutic effects of Bacillus subtilis F31 on pigeon salmonellosis were investigated by weight changes and clinical signs,serum cytokine IL-6,TNF-α,CRP and PCT concentrations,histological lesions of liver and duodenum,and intestinal pathogenic bacteria counts in child pigeons.The results of the study are as follows:1.Three gram-positive bacilli obtained by isolation and culture were identified as Bacillus subtilis and named as F21,F31 and F51.The three isolated strains had different degrees of Staphylococcus aureus,Escherichia coli and Salmonella,and none of the three strains had Hemolysis occurred,F21 and F31 were sensitive to most antibiotics,and F54 was not sensitive to many antibiotics.Based on the results of the growth curve test,acid resistance test,bile salt resistance test,in vitro antibacterial test,drug susceptibility test,and hemolytic test,the F31 with good tolerance and the strongest inhibitory effect on Salmonella was finally selected as the infection model of pigeons candidate strains.2.In the safety experiment,the pigeons had no adverse symptoms,and there were no pathological changes in the eyes after dissection.3.The medium concentration of Salmonella with higher diarrhea rate and no death of pigeons,it was selected as the concentration of Salmonella for establishing animal models.4.Compared with group C,the less weight of pigeons in group B was reduced after Salmonella infection,and the diarrhea rate was reduced,the serum IL-6 concentration was significantly lower（P<0.05）,the TNF-αconcentration was significantly lower（P<0.01）,and the PCT concentration was reduced but there has not significant difference.The CRP concentration decreased（P<0.05）,the pathological changes in the duodenum and liver were significantly alleviated,and the number of Salmonella in the rectum decreased.5.Compared with group E,group D had less weight and the rate of diarrhea was not reduced.There was no significant difference in IL-6 and TNF-αconcentrations,PCT and CRP concentrations were decreased,has not significantly different.Pathological changes in the duodenum and liver were alleviated,and the number of Salmonella in the rectum was reduced.In this study,the results showed that F21 and F31 of Bacillus subtilis isolated had good properties of probiotics.F54 was resistant to most antibacterial drugs,so F54 don’t meet the conditions of probiotics.The candidate strain F31 had a preventive effect on Salmonella infection in pigeons,but treatment is ineffective.