Screening And Identification Of Bacillus Subtilis And Protective Experimental Study On Salmonella

Salmonellosis is a worldwide zoonosis,which poses a serious threat to the reproduction of livestock and poultry as well as the health of young animals.Some strains are also pathogenic to humans.At present,the prevention of salmonellosis in livestock and poultry breeding in China mainly depends on the addition of antibiotics in feeds.The long-term use of antibiotics leads to the emergence of drug-resistant strains and the continuous improvement of drug resistance,which gradually increases the incidence of salmonellosis.Therefore,the study of antibiotic substitutes has attracted much attention.Probiotics belong to one of them.Bacillus in probiotics has great advantages in commercial processing,transportation and storage because of its high resistance to severe environment.In order to acquire the Bacillus with good characteristics,we used honey as sample to screen out the Bacillus strains resistant to high temperature,acid and bile salt.The probiotic effect of Bacillus was studied by animal models of Caenorhabditis elegans and Salmonella pullorum infection in chickens.In this study,three suspected high temperature-resistant Bacillus subtilis strains were screened out from honey,and an acid-and bile-salt-resistant strain was screened from the three strains.B.subtilis was identified by Gram staining,biochemical test and 16 S rDNA sequencing.It was named BSH,and a gene evolution tree was established.Growth curve was drawn to determine its susceptibility to commonly used antibiotics.The results of feeding BSH to Caenorhabditis elegans showed that BSH could prolong the longevity of C.elegans.In vivo lipofuscin observation showed that BSH could reduce the accumulation of lipofuscin in intestinal cells,suggesting that BSH could delay the senescence of nematodes.The nematode was infected with Salmonella enteritidis LT2 after feeding BSH in advance.It was found that BSH had protective effect on S.LT2 infection of C.elegans.The chickens infected with S.pullorum(CVCC533)were fed with BSH strain.The pathological changes of liver,the number of live bacteria in liver and spleen were observed,and the death of chickens was recorded.Observation of paraffin section of chicken liver showed that BSH could alleviate the liver lesion of chickens.Live Salmonella count was carried out on chicken liver and spleen.The results showed that BSH could reduce the invasion of S.pullorum to the liver and spleen.In addition,BSH had protective effect on chickens infected with pullorum.Furthermore,fecal flora of 9-day-old chickens were analyzed by 16 S rRNA high-throughput sequencing.The results showed that Salmonella infection could cause intestinal mucosal damage and intestinal flora change,while BSH could slow down the change.Likewise,BSH can also promote the proliferation of L.salivarius in intestinal tract.The results showed that BSH screened in this study had anti-Salmonella infection effect on C.elegans and chickens,and could be used as a candidate strain of microecological agents.