| Rabbit is very susceptible to intestinal infection,which often leads to serious inflammation and subsequent high mortality rate.Intestinal bacteriaincluding commnensal bacteria can induce inflammatory response through their metabolite and cellular components.Studies showed that gram-negative bacteria have stronger effect on inducing inflammatory response than gram-postive bacteria.Unlike pig and chicken,the dominant phylum in every segment of rabbit's intestine after supplementation with solid food is gram-negative Bacteroidetes.To dertermine whether the intestinal flora of rabbit can cause higher inflammatory level in the intestinal tract than that of pig and chicken,and whether lactic acid bacteria can alleviate the inflammatory response and exhibit speices speciality,the present paper aimed to compare the effect of the total bacteria from ileal content of healthy adultpigs,chicken and rabbits and lactic acid bacteriafrom rabbit on inducing inflammatoryresponses inrabbit's crypt cells and DCs.Our study provided theoretical basis for improving the ability of anti-inflammatory response in rabbit through screening and applying probiotics for rabbits.The main research contents and results are as follows:1.The ileal contents from the6 month Guanzhong black pig,42 day broilers and 60 day rabbits were collected.The ileal contents of 3 individuals of each animal were mixed before thegenomic DNA was extracted.The structure of the ileal microflora from pig,chicken and rabbit were analyzed by Illumina HiSeq sequencing technique.The results showed that the ileal flora of rabbit wasdifferent from that of pig and poultry,the firstdominant phylum in rabbit'sileum is Bacteroidetes bacteria(89.32%),but it isFirmicutes in pig's(80.79%)and chicken's ileum(88.50%).The abundance of Lactobacillus in ileum of the three animals was 19.19%,81.19% and 0.72%,respectively.The beta diversity index showed that the structure of ileal microflora of pig and chicken was similar,but was different from that of rabbit.2.(1)The isolated total ileal bacteria from pigs,chickens and rabbits were heat-treated,and then used to stimulate rabbit crypts and mice DCs.The blank control group wastreated with PBS.The crypt cellswerecollected after incubationwith total bacteria for 6 h.The expression of therelated genes was determined by RT-qPCR.The results showed as follows: Compared with the control group,the inactivated bacteria in ileum of pig and chicken significantly increased the expression of TLR2,TLR3 and TLR4 in rabbit crypt cells(P< 0.05),and the inactivated bacteria in ileum of rabbits significantly increased TLR4 expression(P < 0.01).The three treatments had no significant effect on TLR9 expression(P> 0.05).Expression of TNF-?and IL-6 were induced by treatments(P< 0.05).The inactivated ileal bacteria of chicken had strongerstimulatory effect on the expression of IL-10 than that of other(P< 0.05).The inactivated ileal bacteria of pigtended to increase the expression of IL-10 than that of rabbit(P<0.1).Inactivated ilealtotal bacteria of pig and chicken induced IFN-? expression(P< 0.05),butthat of rabbit had no stimulatory effect on expression of IL-10(P>0.05)andtended to decrease the expression of IFN-?(P<0.1).(2)Strains of theLactobacillus plantarum 2(L.p2),Lactobacillus casei(L.a1),and Enterococcus faecium(E.f2)separatedpreviously fromgrowing rabbit and purchased E.coli K88 were inactivated by heat and then were used to stimulate the crypt cells for 6 h.The results shown as follows: Crypt cells:Three strains of lactic acid bacteriasignificantly increased the expression ofTLR2,TLR3 and IFN-?(P < 0.05).E.coli K88 significantly inducedTLR4 expression(P < 0.05).Expression ofTNF-?was significantly stimulated by L.p2,E.f2 and E.coli K88(P < 0.05).All treatments significantly improved the expression of IL-6(P < 0.05).L.p2 and E.f2 obviouslyinduced expression of IL-10(P < 0.05),while E.coli K88 inhibited expression of IL-10(P < 0.05).3.The ilealtotal bacteria of pig,chicken and rabbit and threeLactic acid bacteria isolated from rabbit were used to stimulate mouse DC2.4.The results shownas follows:(1)DC2.4 cells were stimulated by inactivated L.p2,the expression of TLR2 and TLR4increased during 0-2 hafter stimulation and then decreased.The expression of TLR3 and TLR9 was not affected by treatment(P< 0.05),but the expression of TNF-?and IL-10 increased during 0-2 hafter stimulation and then decreased but still higher than control group.There was no significant difference inIL-6 andIFN-?between treatment and control(P> 0.05).(2)DC2.4 cells have stimulated by the ilealtotal bacteria of pig,chicken and rabbitfor2 h.Compared with the rabbit treatment group,the ileum total bacteria of pig and chicken had significant TLR3 expression(P< 0.05).The ilealtotal bacteria of pig and chicken induced expression of TLR2 and TLR4(P< 0.05).The ilealtotal bacteria of rabbits trended to stimulate expression of TLR2(P< 0.1),but had no obvious effect on expression of TLR3(P> 0.05).Treatmentsdidn'taffect TLR9 expression(P> 0.05),but significantly increased the expression ofTNF-? and IL-10(P< 0.05).The ilealtotal bacteria of rabbits hadmore obvious effect on expression of TNF-?than that of pig and chicken.The expression of IL-6and IFN-?was significantly increased by ileal bacteria from chicken(P < 0.05),and ileal bacteria of pig trended to stimulateIL-6and IFN-?(P< 0.1).However,there was no significant difference in IFN-?mRNA levelbetween the control group and rabbit group(P> 0.05)and ileal bacteria of rabbit trended to increasethe expression of IL-6(P< 0.1).(3)Compared with the control group,L.p2,L.a1 and E.f2 significantly increased the expression of TLR2 in DC2.4 cell after 2 h incubation(P< 0.05).L.a1 and E.f2 significantly increased the expression of TLR3(P <0.05).E coli k88 induced the expression of TLR4(P <0.05),and L.p2,L.a1 and E.f2 had no effect on the expression of TLR4((P> 0.05).Treatment groups didn't affect the expression ofTLR9(P > 0.05).The expression of TNF-?was increased by E coli k88(P <0.05).L.p2 and E.f2 significantly increased the expression ofTNF-?(P <0.05).L.a1 and E.f2 tended to increase the expression of IFN-?(P <0.1).E.f2 and E coli k88 significantly increased the expression of IL-10(P <0.05)In summary,the optimal phylum in ileal total bacteria of rabbit is bacteroidetes,but it is Firmicutes in pig's and chicken's ileum,and the abundance of lactobacillus in rabbit ileum is much lower than that of pig and chicken.Compared with pig's and chicken's,rabbit's ileal flora of rabbit showed higher potential of inducing crypt cells and DC2.4 into a state of inflammation.Heat-killed lactobacillus can contribute to inflammatory heomostatis by stimulating expression both pro-inflammatory factors and anti-inflammatory factors.Taken the expression of pro-inflammatory factors and anti-inflammatory factors together,Enterococcus faecium and Lactobacillus casie were better than Lactobacillus plantarum in inflammatory regulation in rabbit's crypt,but evidences didn't showed this conclusion in DC2.4. |
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