Isolation And Identification Of Salmonella Isolated From Pigs And Effects Of Lactobacillus Rhamnosus In Preventing Weaned Diarrhea In Piglets

Post-weaning diarrhea is one of the important diseases affecting pig industry in China.Salmonella infection is one of the main causes of the diarrhea of weaned piglets.Salmo?nella infection is often mixed with other pathogens,and it is still difficult to be detected and prevented.The homeostasis of the intestinal microbiota has a positive effect on the prevention of post-weaning diarrhea.Lactobacillus rhamnosus GG(LGG),a potential al-ternative to antibiotics,can prevent post-weaning diarrhea.In this study,we isolated and identified the clinical pathogens,established the en-zyme-linked immuno sorbent assay(ELISA)kit for Salmonella detection,and investigated the effect of LGG on the gut microbiota in Salmonella-infected piglets,as well as the ef-fects on growth performance and immune function of weaned pigs.The main results are as follows:(1)The epidemiology and drug resistance of Salmonella were studied in large-scale pig farms.A total of 806 samples were collected from 123 farms in 26 provinces in China.A total of 173 Salmonella strains were mainly isolated from Hebei,Shandong,Hunan,Hubei and Chongqing and divided into 7 serotypes,which mainly were Salmonella typhi-murium(125 isolates).A total of 16 kinds of virulence factor were tested positive in all isolates.In addition,the antibiotic resistance rate of ampicillin,tetracycline,chloramphen-icol and florfenicol was more than 70%.All isolates were resistant to combination of neo-mycin,but were sensitive to amikacin.(2)The indirect ELISA method for FljB protein of S.Typhimurium was established and applied.The flagellin FljB protein of S.typhimurium isolates was expressed,and an indirect ELISA kit was established.The condition was 6 μg/mL with pET-32a-FljB recom-binant protein and incubated at 37℃ for 1 h and then 4℃ overnight,blocked by 5%BSA for 2 h,and 100 μL/well of an untested serum at 37Κ for 30 min,1:10000 dilutions of enzyme-labeled secondary antibody 100 μL/well,at 37℃ for 30 min,TMB for 10 min.The value was measured by microplate reader at OD450.The threshold of positive and neg-ative value was defined as 0.34 at OD450.The established ELISA method can specifically identify salmonella with high sensitivity.The variation coefficient of three repeated tests was 1.0%伍9.1%,and the coincidence rate with commercial kits reached 93.4%.(3)The effect of LGG on the gut microbiota of piglets infected with Salmonella isolate was investigated.High-throughput sequencing technology was used to analyze the ileal mucosal gut microbiota.The results showed that Firmicutes,Proteobacteria and Actino-bacteria were predominated in the gut microbiota,accounting for 85.00%,8.23%and 6.15%of the total abundance,respectively.Lactococcus piscium,Solibacillus silvestris and Lac-tococcus lactis were the most abundant species and accounted for 43.63%,8.50%and 8.00%,respectively.The relative abundance of Pseudomonas gessardii(2.06%),Bacillus oceanisediminis(1.49%),Exiguobacterium sp.of ATlb(1.27%)and Carnobacterium maltaromaticum(1.11%)was more than 1%of total abundance.Salmonella challenge led to increased levels of Psychrobacter,Chryseobacterium indoltheticum,and uncultured Corynebacteriaceae populations and formed a Prevotellaceae NK3B31 group-centric mi-crobiota network.The beneficial effect of LGG was correlated with attenuating the expan-sion of Prevotellaceae NK3B31 group and promoting symbiotic synergism of Fusobacte-rium,Lactobacillus animalis and Propionibacterium.(4)The effects of LGG on the growth performance and immune function were evalu-ated in large-scale farm.The weaned piglets were supplemented with diets containing 1 kg/1000 kg of LGG(1010 CFU/g)for three weeks.The piglets were recorded for additional two weeks after the diets with LGG were removed.Dietary supplementation of LGG in-creased the average daily gain and feed conversion ratio,and reduced the diarrhea rate.LGG increased the weight of pigs on week 4 and week 5,the average daily gain and feed conversion ratio on weeks 3,4 and 5,but significantly decreased the diarrhea rate on weeks 3,4 and 5.The concentrations of serum Immunoglobulin M(IgM)at week 1,IgG from week 3 to week 5 and IgA at week 5 were significantly elevated in pigs supplemented with LGG.In conclusion,our results show that the positive rate of Salmonella was 21.46%in piglets with diarrhea from 26 provinces in 2014～2018.Among,Salmonella typhimurium with multi-drug resistance was the most prevalent serotype.The indirect ELISA detection method for Salmonella F1jB has the characteristics of strong specificity,high sensitivity and good repeatability.LGG can modulate gut microbiota,improve growth performance and reduce diarrhea rate.