| Along with the increasing public attention on food safety and deeper research on animal microecology,probiotic microbes,as a kind of green and safe feed additives,has been widely used in feed industry.Bacillus subtilis(BS),as one of high quality and safe probiotics,has multiple functions including increasing animal growth performance,enhancing body immunity,antibacterial ability,improving intestinal flora,enhanceing antioxidant function and so on.Bacillus subtilis,as an alternative to antibiotic,has attracted wide interests in animal husbandry industry recently.Bacillus has been widely recognized for the broilers,and there are many literatures on this subject,but they are basically based on rapid white feather broiler.There are few studies on yellow broiler,and now the domestic yellow broiler accounts for nearly 50%the whole broiler.The purposes of this thesis were to elucidate the effects of BS048 on production performance and immune functions of Xueshan broiler and its mechanism,thus providing the theoretical instruction on the application of Bacillus subtilis in feed industry.The research contents are as follows:Trial 1.Evaluation of antibacterial effects of BS on Escherichia coli(E.coli)and Salmonella using bacteriostatic test in vitro.In this trial,eight BS strains including BS044,BS048,BS050,BS075,BS090,BS155,BS281 and BS306 were selected to evaluate their antibacterial ability.The bacteriostasis of Bacillus subtilis on two standard strains(E.coli K88 and Salmonella enteritis(SE)ATCC13076)and sixty-one clinical isolated stains(29 strains of E.coli and 32 strains of Salmonella)were detected using inhibition zone method.Results were showed that:(1)For the antibacterial activity against E.coli K88,BS048 and BS155 had larger inhibition zones(20.5±0.3 mm and 15.8± 1.1 mm)and stronger bacteriostatic function than other BS strains;while BS090 and BS306 had smaller inhibition zones(8.6 ± 0.7 mm and 10.1± 0.6 mm)and weaker antibacterial activity;(2)BS048 and BS155 had larger inhibition zones(19.2 士 0.7 mm and 17.3 士 0.9 mm),stronger antibacterial ability against SE ATCC13076;while BS090 had smaller inhibition zone(11.2 士 0.8 mm)and weaker antibacterial effect against SE ATCC13076;(3)The 20 and 12 strains of clinical isolated E.coli were inhibited by BS048 and BS155,respectively,which demonstrated their wide antibacterial spectrum against E.coli;However,only 2 and 5 strains of E.coli clinical isolates were inhibited by BS090 and BS306,respectively,indicating their weakly antibacterial activity against E.coli;(4)Both BS048 and BS155 strains inhibited 23 stains of clinical isolated Salmonella,showing their wide antibacterial spectrum against Salmonella.BS090 inhibited 7 strains of Salmonella clinical isolates,showing its narrow antibacterial spectrum against Salmonella.Based on the antibacterial test,it was concluded that BS048 and BS155 had strong antibacterial activity against E.coli and Salmonella.Hence,the effects of BS048 and BS155 on growth performance,immunity and anti-inflammation in Xueshan broilers were studied in the subsequent trials.Trial 2.Effects of BS on production performance,nutrient digestibility,cecal microflora and intestinal health of Xueshan broilers.In this trial,a total of 1470 Xueshan broilers(1-day-old)with similar weight(35.0g±1.0 g)were randomly divided into three groups,including control group(basal diet),BS048 group(basal diet supplemented with 0.1%(W/W)BS048)and BS155 group(basal diet supplemented with 0.1%(W/W)BS155),with 7 replicates per group and 70 broilers per replicate.The broilers were raised for 80 days to investigate the effects of BS048 and BS155 on their growth performance,death rate,viscosity and pH value of intestinal chyme,jejunal digestive enzyme activities,and cecal volatile fatty acid(VFA)content.Compared with the control group,the average daily gain(ADG),average daily feed intake(ADFI),jejunal digestive enzyme activities and cecal VFA contents were significantly increased(P<0.05),whereas the viscosity of intestinal chyme was remarkably decreased(P<0.01)in BS048 group.Feed conversion ratio and pH value of intestinal chime were not affected by BS supplementation(P>0.05).The BS155 supplementation significantly increased the ADFI,jejunamylase and VFA contents in respect to the control group(P<0.05).Hence,It was concluded that BS048 enhanced intestinal digestive function by reducing the viscosity of intestinal chyme and enhancing the jejunal digestive enzyme activities,and also improved intestinal health by promoting the production of cecal VFAs;there was no significant difference in death rate between the three groups.As a result,the BS048 supplementation stimulated the growth performance of Xueshan broilers.Although BS155 had the similar effects as BS048 on intestine health of Xueshan broilers,however,BS155 had no promoting effects on growth performance of Xueshan broilers.Therefore,the immune regulation and anti-infection mechanism of BS048 were studied in the next chapters.The apparent utilization efficiency of crude protein and energy of Bacillus subtilis were significantly increased at 80 days of age,and there was no significant difference between BS048 and BS155 groups.The apparent utilizationratio of dry matter,calcium and phosphorus were not affected by Bacillus subtilis supplementation.At the age of 20 and 40 days,the diversity of cecal bacteria of broilers was increased in BS048 and BS155 group,and the bacterial abundances of BS048 broilers were higher than BS155 group.Based on the results in this trial,BS048 was more effective on broilers than BS155.Hence,the immune regulation effects and anti-inflammation mechanism of BS048 were evaluated in the subsequent trials.Trial 3.The antibacterial effect of BS048 to Salmonella in Xueshan broilers was investigated.Two hundred and forty SE-negative Xueshan broilers(1-day-old)with similar weight(35.0 g±1.0 g)were randomly divided into four groups with 3 replicates per group and 20 chickens per replicate.The four groups were control group and SE-infected group(Basal diet),BS-treated group and BS-treated SE-infected group(Basal diet supplemented with 0.1%(W/W)BS048).The experiment period lasted for 14 days.On day 5 to day 7,broilers in two SE-infected groups were infected with 1×108 CFU SE per bird.Variables including SE amount in cecal mucosa,liver and kidney,jejunal mucosal structure,serum Ig and ileal slgA levels,alkaline phosphatase(ALP)and myeloperoxidase(MPO)activities in jejunal mucosa,antioxidant functions and gene expression of tight junction proteins,Toll-like receptors(TLR)and inflammatory cytokines in jejunum mucosa of normal and SE-infected Xueshan broilers were investigated.Results were showed below:(1)For Xueshan broilers in Control group,BS048 significantly stimulated the development of jejunal mucosa and the secretion of intestinal slgA(P<0.05),increased the activity of ALP in jejunum mucosa(P<0.05)and the antioxidant function in small intestine(P<0.05).Meanwhile,the gene expression of tight junction proteins(CLDN-1,OCLN,ZO-1),TLR2,inflammatory cytokines(IL-6,IL-10)in jejunal mucosal cells(P<0.05)were up-regulated by BS048 supplementation;(2)For SE-infected Xueshan broilers,BS048 supplementation significantly reduced the SE amount in cecal mucosa,liver and kidney(P<0.01),promoted the injury recovery and development of small intestinal mucosa(P<0.05),increased the secretion of blood Ig and intestinal sIgA(P<0.05).Besides,BS048 supplementation increased the activity of ALP(P<0.05)but decreased the activity of MPO in jejunum mucosa(P<0.01),and enhanced the antioxidant function in small intestine(P<0.05).In addition,the gene expression of tight junction proteins(CLDN-1,OCLN,ZO-1)were up-regulated but the gene expression of TLRs(TLR2,TLR4)and inflammatory cytokines(IL-6,IFN-γ,IL-10)in jejunal mucosal were down-regulated.In conclusion,BS048 supplementation could help to enhance the intestinal mucosal barrier,body immunity and anti-SE infection ability in Xueshan broilers by the following pathways:(1)promoting the development of small intestinal mucosa and its injury recovery after infection;(2)enhancing the humoral and intestinal mucosal immunity of broilers;(3)enhancing the intestinal antioxidant function and attenuating the oxidative stress caused by bacterial infection;(4)enhancing the physical mucosal barrier of intestine;(5)enhancing the pathogen recognition and defense ability of the jejunal mucosa.Trial 4.The mechanism of BS048 on immunity and anti-inflammatory functions in HD11 chicken macrophage cell was investigated.In this study,HD11 chicken macrophage cell lines were divided into three treatments and cultured in DMEM/F12 medium,DMEM/F12 medium supplemented with 108 CFU/mL-BS048,and DMEM/F12 medium supplemented with 100 ng/mL LPS,respectively.The activation,phagocytosis and bactericidal ability,cytokine secretion and NO synthesis of HD11 cells were detected after 12-h culture.We found that:(1)BS048 supplementation had no significant effects on extracellular lactate dehydrogenase(LDH)activity(P>0.05),which demonstrated its good safety for broilers;(2)BS048 increased the acid phosphatase and LDH activities in HD11 cells(P<0.05),indicating its ability to activate HD11 cells;(3)BS048 significantly enhanced the phagocytic capacity of HD11 cells and their ability to kill intracellular SE(P<0.01);(4)The gene expression of pro-inflammatory cytokines(IL-1β and IL-6)and anti-inflammatory cytokines(IL-10,TGF-β1),as well as antiviral cytokine(IFN-β)in HD 11 cells(P<0.05)wereup-regulated by BS048,which suggested the strong anti-inflammatory effects of BS048 in broilers;(5)BS048 significantly up-regulated the gene expression of iNOS in HD11 cells(P<0.01)and increased extracellular NO content and iNOS activity(P<0.01),indicating that BS048 could enhance the NO production of HD 11 cells.In short,BS048 is safe to the chicken macrophages and it could activate macrophages to enhance the immune function and anti-SE ability of Xueshan broilers.In concusion,the results obtained in this thesis demonstrated that(1)BS048 has a good inhibitory effect on both standard and clinically isolated E.coli and Salmonella strains;(2)BS048 could help to the productive performances of Xueshan broilers by enhancing intestinal digestive function and improving intestinal health;(3)BS048 could improve the intestinal health and anti-Salmonella infection ability of Xueshan broilers by enhancing macrophage functions,intestinal mucosal barrier and body immunity.Thus,BS048 could be used as an alternative probiotic to improve the productive performance of Xueshan broilers. |
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