Protective Effects Of Lactobacillus Zeae On The Gut Health Of Piglets Challenged With Enterotoxigenic Escherichia Coli

Enterotoxigenic Escherichia coli（ETEC）is a common enteric pathogen that can cause diarrhea in humans and animals.Lactobacillus zeae（LB1）has been shown to reduce ETEC infection of Caenorhabditis elegans.The present study was to evaluate the effects of L.zeae LB1 on the gut health of lactating piglets that were challenged with ETEC.In this test,64 healthy 7-day-old piglets of similar weight were selected and divided into 8 groups:1)NC group（basic diet,phosphate buffer solution（PBS））;2)PC group（basic diet+40mg/kg/BW Colistin（CT））;3)LL group（basic diet+1×10⁷ CFU LB1/pig/day）;4)HL group（basic diet+1×10⁸ CFU LB1/pig/day）;5)ETEC group(Basic diet+1×10¹⁰ CFU ETEC/pig/day);6)CT+ETEC group(basic diet+CT+1×10¹⁰ CFU ETEC/pig/day);7)LL+ETEC group(basic diet+1×10⁷ CFU LB1/pig/day LB1+1×10¹⁰ CFU ETEC/pig/day);8)HL+ETEC group(basic diet+1×10⁸ CFU LB1/pig/day LB1+1×10¹⁰CFU ETEC/pig/day).One repeat per group,one pig per repeat,PBS,CT,LL and HL were orally administered on the fourth to eighth days of the experiment,ETEC was administered on the seventh to eighth days,Slaughter samples were taken on days 9 and 10.1.Effect of LB1 on growth performance and blood biochemical parameters of piglets infected with ETECThe results showed that:（1）ETEC infection of piglets significantly reduced the ADG of piglets（P<0.05）,significantly increased the diarrhea rate of piglets（P<0.001）,and had a tendency to increase the mortality rate of seven-day-old piglets（P<0.10）.HL can significantly improve the ADG and ADFI of piglets（P<0.05）,feeding CT significantly improves ADG（P<0.05）,there is a tendency to improve ADFI（P<0.10）,feeding LL has a tendency to improve ADG and ADFI（P<0.10）.（2）Feeding ETEC significantly increased the levels of TP and TB in the plasma of suckling piglets（P<0.05）,feeding HL significantly increased the levels of TP and TC in the plasma of piglets（P<0.05）,and feeding LL increased the plasma of piglets The trend of TP and TC content（P<0.10）.Compared with the ETEC group,LL+ETEC group ALT,ALP,GLU,CREA were significantly increased（P<0.05）,HL+ETEC group ALT,ALP were significantly increased（P<0.05）.（3）ETEC significantly increased Higher WBC and LYMPH in the blood of suckling piglets（P<0.05）;HL significantly reduced the content and MONO%of MONO（P<0.05）,LL significantly reduced MONO%（P<0.05）Content trend（P<0.10）.In summary,Lactobacillus zeae can relieve the diarrhea of piglets caused by ETEC infection and improve the growth performance of piglets;at the same time,according to the results of plasma biochemistry and blood cell count,ETEC infection caused inflammatory reactions in piglets,and Lactobacillus zeae can alleviate this effect.2.Effect of LB1 on intestinal barrier function of piglets infected with ETECThe results showed that:（1）Compared with the NC group,the DAO content of the ETEC group increased significantly（P<0.05）.Compared with the ETEC group,the DAO content of the CT+ETEC group,LL+ETEC group,and HL+ETEC group decreased significantly（P<0.05）.（2）ETEC infection significantly reduced the activity of T-SOD in duodenum and jejunum（P<0.05）,and significantly increased the contents of duodenal MDA,colon MDA and MPO（P<0.05）.Both LL and HL had a tendency to increase plasma GSH-PX activity and duodenal CAT activity（P<0.10）,and significantly reduce the content of duodenal H2O2 and ileal MDA（P<0.05）,of which LL Can significantly reduce the content of MDA and H2O2 in the colon（P<0.05）;HL has a tendency to reduce the content of MDA and H2O2 in the colon（P<0.10）.Compared with the ETEC group,the plasma and jejunum MDA content,duodenal MPO content,ileal MPO and H₂O₂ content in the LL+ETEC group were significantly reduced（P<0.05）,and colon CAT activity was significantly increased（P<0.05）;The content of jejunum MDA and ileum MPO and H₂O₂ in the+ETEC group were significantly reduced（P<0.05）.（3）Infusion of ETEC significantly reduced duodenal VH,VSA and VH/CD（P<0.05）,significantly increased duodenal,ileal and colon CD（P<0.05）,significantly reduced jejunum VH,VW,VSA and VH/CD（P<0.05）,significantly reduced ileal VH,VSA and VH/CD（P<0.05）;oral administration of LL significantly reduced duodenal CD（P<0.05）;oral administration of HL increased The trend of high ileal VSA（P<0.10）.Compared with the NC group,the jejunum VH and VH/CD in the ETEC group were significantly reduced（P<0.05）,the ileum VH was significantly reduced（P<0.05）,and the colon CD was significantly increased（P<0.05）.Compared with the ETEC group,the jejunum VH was significantly increased in the LL+ETEC group（P<0.05）,and the colon CD was significantly reduced（P<0.05）;the ileum VH was significantly increased in the HL+ETEC group（P<0.05）,and the colon CD was significantly reduced（P<0.05）.（4）Compared with the NC group,the ETEC group significantly increased the relative expression of the jejunum TRPV6 and ARG1 genes in seven-day-old piglets（P<0.05）,and significantly reduced the relative expression of the jejunum GPX2 gene（P<0.05）;the ETEC group significantly increased The relative expression of ileal TRPV6,Nrf2 and ARG1 genes（P<0.05）significantly reduced the relative expression of ileal GPX2 genes（P<0.05）.Compared with the ETEC group,the LL+ETEC group significantly reduced the relative expression of the jejunum Nrf2 and ARG1 genes（P<0.05）;significantly reduced the relative expression of the ileum Nrf2 gene（P<0.05）.HL+ETEC group significantly reduced the relative expression of jejunum Nrf2 and ARG1 genes（P<0.05）.（5）Compared with NC group,the relative expression of clajelin-1 protein in jejunum was significantly decreased in the ETEC group（P<0.05）,and the relative expression of AQP3,occludin,and claudin-1 protein in the HL group was significantly increased（P<0.05）.Compared with ETEC group:the relative expression of cladulin-1 protein in HL+ETEC group showed an upward trend（P<0.10）.In summary,ETEC infection will destroy the intestinal villi structure,inhibit the absorption of nutrients in the small intestine;at the same time disrupt the metabolism of oxidative products and the secretion of related antioxidant enzymes,produce a large amount of oxygen free radicals,so that the intestine is in an oxidative stress state;The tight connection of the tract eventually leads to impaired intestinal barrier function.LB1 can alleviate the oxidative stress and intestinal barrier function damage caused by ETEC infection.3.Effect of LB1 on intestinal immune function of piglets infected with ETECThe results showed:（1）Compared with NC group,CT group,LL group and HL group significantly reduced the content of IL-8（P<0.05）.The ETEC group significantly increased the content of IL-1βin plasma（P<0.05）,and the content of TNF-αshowed a tendency to increase（P<0.10）.Compared with the ETEC group,the LL+ETEC group and the HL+ETEC group significantly reduced the content of IL-1βin plasma（P<0.05）,and there was a tendency to reduce the content of TNF-α（P<0.10）.（2）Compared with the NC group,the PC group,LL group and HL group significantly reduced the relative expression of the jejunum S100A12 gene（P<0.05）,and the LL group significantly reduced the relative expression of the ileum HSP70,S100A12,Mx1 and Mx2genes（P<0.05）,HL group significantly reduced the relative expression of ileal HSP70 and Mx2 genes（P<0.05）,ETEC group significantly increased the relative expression of jejunum IL-8,HP and S100A12 genes（P<0.05）,significantly Decreased the relative expression of the Mx1 and Mx2 genes in the jejunum（P<0.05）;the ETEC group significantly increased the relative expression of the S100A12 gene in the ileum（P<0.05）and significantly reduced the relative expression of the ileum Mx1 and Mx2 genes（P<0.05）.Compared with the ETEC group,the LL+ETEC group significantly reduced the relative expression of the jejunum IL-8 and S100A12 genes（P<0.05）,and significantly increased the relative expression of the jejunum HSP70 and Mx2 genes（P<0.05）,significantly Reduced the relative expression of S100A12 gene in ileum（P<0.05）;HL+ETEC group significantly reduced the relative expression of jejunum IL-8 and S100A12genes（P<0.05）,significantly increased the relative expression of jejunum Mx1 and Mx2Expression（P<0.05）.（3）ETEC infection significantly increased the relative expression of NLRP3 and HSP70 proteins in piglet jejunum（P<0.05）.After ETEC infection:HL administration significantly reduced the relative expression of HSP70 protein in the jejunum of piglets（P<0.05）,and there was a tendency to decrease the amount of NLRP3 protein in jejunum（P<0.10）.In summary,ETEC infection activates the piglet’s intestinal immune system,promotes the expression of inflammatory related genes in the piglet’s small intestine,and releases a large number of pro-inflammatory factors,leaving the intestine in a state of immune stress and causing intestinal damage.LB1 can alleviate the intestinal immune stress of piglets caused by ETEC infection by inhibiting the expression of inflammatory factor related genes and proteins.ETEC infection disrupted the cell structure and biochemical indicators of blood,undermined the integrity of the intestinal tract,and induced oxidative stress,diarrhoea,intestinal damage,and death of piglets.The supplementation of L.zeae LB1 alleviated ETEC’s adverse effects by reducing pig diarrhoea,oxidative stress,and death,modulating cell structure and biochemical indicators of blood,improving the capacity of immunity and anti-oxidation of pigs,and restoring their intestinal integrity.At the molecular level,the beneficial effects of L.zeae LB1 appeared to be mediated by regulating functional related proteins（including HSP70,Caspase-3,NLRP3,AQP3,and AQP4）and genes（including RPL4,IL-8,HP,HSP70,Mx1,Mx2,S100A12,Nrf2,GPX2,and ARG1）.These results suggest that dietary supplementation of L.zeae LB1 improved the intestinal functions and health of piglets.