Study On The Effects And Mechanism Of Lactobacillus Johnsonii BS15 On Preventing Subclinical Necrotic Enteritis In Broilers

Necrotic enteritis(NE)is considered as a complicated gut disease mainly caused by type A of Clostridium perfringens(CP)in broilers.The ban on antibiotic growth promoters made NE one of the most threatening diseases of poultry.The subclinical NE(SNE)has proved to cause more damaging influences in poultry industry and food safety as no obvious lesions could be easily observed.Probiotics are considered as one of the most important methods to prevent SNE and worth deep understanding in post-antibiotic era.Probiotic strains with clear mechanism are also needed.Lactobacillus johnsonii BS15,a probiotic strain isolated by our research team,has been proven to exert beneficial effects in different animals including broilers.Based on abovementioned information,the present project aimed to study on the effect and possible mechaism of Lactobacillus johnsonii BS15 on preventing SNE in order to explore the application worth of BS15 as a probiotic.Part 1:Study on the growth promoting effect of BS15 and its underlying mechanism in broilersThe present project firstly determined the beneficial effects on growth performance of BS15 in broilers and then analyzed the mechaism.A total of 180 one-day-old broilers were randomly divided into 3 groups.One group was fed with common diet and the other two were fed with BS15 diet(one of them with disrupted BS15).The birds were sampled and determined on day 21 and 42.Based on the results,BS15 could improve intestinal development and digestive abiliy through enhancing gut microbiota,and thus improve function of lipid metabolism including mRNA expression levels in the liver and adipose tissue and biochemical indexes in serum.Therefore,the growth performance was increased and abdominal fat percentage was decreased by BS15.The present study also found that only live BS15 could exert these beneficial effects.Part 2:Study on the preventive effects of BS15 on damaged growth performance and meat quality in SNE-infected broilersAfter the beneficial effect was proven by the first experiment,further study was undertaken on the preventive effects of BS15 on decreased growth performance and damaged meat quality in SNE-infected broilers.A total of 480 one-day-old broilers were randomly divided into 4 groups(control group,SNE group and two preventive groups with different doses of BS15).Birds were sampled and determined on day 42.Based on the results,BS15 could effectively control the lower growth performance,increased gut permeability and damaged palatability and nutritional ingredients in chicken meat caused by SNE,especially pH values,water-holding ability,tenderness and oxdative damaging indexes.Moreover,fatty acids including EPA and DHA were influenced by CP infection and BS15 prevention.The abovementioned results suggested that BS15 a feed supplementation could be able to promote grow performance and protect the broilers from damages on growth and meat quailty in poultry industry.The finding provided theoretical basis for further exploring the mechanism of BS15 on preventing SNE.Part 3:Study on the effects and mechanism of BS15 on preventing SNE in broilersAccording to the abovementioned studies,a total of 300 one-day-old broilers were randomly divided into 5 groups:control group,SNE group,BS15 group,preventive group(fed with BS15 before SNE induction)and treatment group(fed with BS15 after SNE induction).The experiment lasts for 42 days.We found that BS15 could significantly promote body weight gain and feed conversion ratio against SNE.On day 28,we comprehensively analyzed the changes of intestinal mucosal immunity and blood parameters related to immunity in order to reveal the mechanisms of BS15 in local and systemic immunity while preventing SNE.The results are as below:1.Effects on intestinal histologic structureHistological examination showed intact villi structure in the control group,whereas those in the SNE group were destroyed and mainly presented the following features:shed epithelial cells,congested lamina propria,and increased number of lymphocytes.In the BS15 group,lamina propriae were lightly congested.No remarkable differences were observed in the cecal tonsil among the three groups.2.EffectsonnumberofIgA+Bcellsandimmunoglobulinlevels Immunohistochemistry and ELISA were used to detect the IgA+B cells numbers and immunoglobulin contents.The number of IgA+B cells in lamina propria of ileum was higher compared with that of the SNE group.Levels of IgG and IgA in ileum were significantly lower in SNE group than those in other groups,but no significant difference was noted between BS15 and control groups.In the BS15 group,sIgA level in the ileum was significantly lower than that of the control group.3.Effects on the T lymphocytes subsetsThe T-cell subsets in the peripheral blood,ileum and cecal tonsil were detected by flow cytometry methods.Results showed that no significant changes were observed in IELs of ileum and cecal tonsil,but in LPLs of ileum percentages of CD3+,CD4+,and CD8+T cells and CD4+/CD8+ratio were significantly lower in BS15 group.Meanwhile,changes of LPLs in ileum were consistent with those in the peripheral blood.4.Effects on apoptosisAntioxidant ability including MDA levels and SOD ability and levels of apoptosis-related proteins such as caspase-3 and Bax in ileum were damaged in SNE group and protected by BS15.Meanwhile,changes on apoptosis in ileum were consistent with those in the peripheral blood,and the trend was consistent in protein levels and mRNA expression levels in the same determined index.5.Effects on protein contents and mRNA expression levels of cytokines By the methods of ELISA and RT-qPCR,protein levels and mRNA expression levels of cytokines were both determined in ileum and serum.IL-2,IL-8,IL-10 and IFN-?in the ileum as well as were IL-2,IL-4 and IFN-?in the serum were found to be damaged by SNE and prevented by BS15 supplementation.To sum up the above results,we conclude that SNE could reduce the intestinal mucosal immune function and also cuase damages in blood immunity.The supplementation of Lactobacillus johnsonii BS15 could protect the animals from side influences of phenotypic changes and immunity.Taking advantages of the undeveloped intestinal tract of broilers at the starter phase,BS15 inhibits the oxidative damages and the subsequent apoptosis to prvent SNE,which could be considered as the molecular basis.Moreover,the possible direct reason and pathway may be related to the beneficial effects of BS15 on the lowered T lymphocyte subsets in LPLs of ileum and peripheral blood as well as IgA~+B cell numbers in ileum.They could also be related to the preventive effects on levels of immunoglobulins and cytokines decreased by SNE.