Screening Analysis Of Gut Microbes Associated With Diarrhea Resistance In Early-weaned Piglets From Guizhou Congjiang Miniature Pig Resources And Dissecting Their Regulatory Mechanism

China is one of the largest country for pig production in the world with a total output value about 1.3 trillion.China raises the largest pig stock in the word with an annual pig slaughter about 700 million in recent five years.In intensive modern pig production,early-weaning technology is one of key application which contributes to improve the utilization of pens,improve reproductive performance of sow,shorten the feeding time,and prevent mother-to-child transmission disease.However,early-weaning technology can stimulate the piglets in terms of nutrition,environment and psychological effect and aggravate the weaning stress in piglets.The weaning stress can lead to anorexia,indigestion,intestinal inflammation,and intestinal microbial dysbiosis,causing diarrhea even death in piglets.Currently,the use of antibiotics in creep feed is widely used to prevent early-weaning stress-induced diarrhea in piglets.However,the spread of antibiotic-resistant pathogens and antibiotic residues in food have become serious problems.As a consequence,the use of antibiotics in animal husbandry was gradually forbidden.The development of new and effective probiotics is an important breakthrough to reduce the use of antibiotics in pig feed.Guizhou Congjiang miniature(CM)pig is one of Chinese native pig breed,which has the characters of strong resistance to disease,resistance to crude feed,and high meat quality.Our data showed that CM piglets have stronger resistance to early-weaning stress一induced diarrhea than that in eommercial crossbred(Landrace×Yorkshire)(LY)piglets.What are the eharacteristics of the composition of intestinal flora in CM piglets after early-weaning and how it changed?Whether there are differentially abundant intestinal microbes between LY piglets and CM piglets can influence the diarrhea resistance of early-weaned piglets?What are the underlying mechanisms?These important issues deserve further study.Thus,the aim of this study is to investigate the intestinal microbial composition and development in CM piglets after early-weaning.This study is also aimed to screen the specific intestinal microbes that provide resistance to early-weaning stress-induced diarrhea via the fecal microbiota transplantation(FMT),microbial omics,and the sources of CM pig and then elucidate mechanisms.We also investigated the regulatory roles of diarrhea resistance-associated intestinal microbes in intestinal epithelial barrier functions in early-weaned piglets.These findings will provide scientific basis and important data for improving intestinal health in piglets via targeted manipulation of gut microbiota.The main experimental idea and results were as follows:1.Analyses of intestinal microbial compositions and shifts in piglets after early-weaning(1)Bacterial 16S rDNA gene amplicon sequencing analyses of intestinal bacterial communities in CM piglets during the two weeks after early-weaning showed that the bacterial alpha diversities decreased,relative abundances of 24 bacterial genera decreased,and relative abundances of 7 bacterial genera increased with the age of piglets.Prevotella copri was the signature species predominated in gut bacterial communities of CM piglets.The relative abundances of 17 bacterial species(including Lactobacillus coleohominis,Lactobacillus frumenti,Eubacterium hallii,and Lactobacillus gasseri LA39)significantly increased with the age of weaned piglets.(2)Fungal internal transcribed spacer(ITS)gene amplicon sequencing analyses of intestinal fungal communities in CM piglets during the two weeks after early-weaning showed that fungal alpha diversities did not change significantly,whereas the relative abundances of 2 fungal genera decreased,and relative abundances of 4 fungal genera increased with the age of piglets.Kazachstania telluris was the signature species predominated in gut fungal communities of CM piglets.The relative abundances of 3 fungal species(Aspergillus sp.,Aspergillus penicillioides,and Simplicillium sp.)significantly increased with the age of weaned piglets.(3)PICRUSt analyses suggested that the functional maturation of the gut bacterial community was characterized by the significantly increased carbohydrate and protein digestion and absorption,glycan biosynthesis and metabolism,and vitamin B biosynthesis as the piglets aged,suggesting that colonization of gut microbiota in pig during the early life is vital to host physiological function of nutrient digestion.2.Screening analysis of gut microbes associated with diarrhea resistance in early-weaned piglets and dissecting their regulatory mechanisms(1)The results showed that the diarrhea incidences and indexes in CM piglets were lower than that in commercial crossbred LY piglets,suggesting that CM piglets have stronger resistance to early-weaning stress-induced diarrhea than that of LY piglets.Our data demonstrated that transferring fecal microbiota from CM piglets into the LY piglet recipients before early weaning significantly decreased the diarrhea incidences and diarrhea indexes of early-weaned piglets,indicating that FMT can transfer the resistance to early-weaning stress-induced diarrhea in piglets.(2)Bacterial 16S rDNA and fungal ITS gene amplicon sequencing analyses of intestinal microbiota in piglets showed that FMT induced the shifts from LY(saline)piglets to CM piglets in the intestinal microbiota,including beta diversity,alpha diversity,bacterial functional profiles(Kyoto Encyclopedia of Genes and Genomes(KEGG)and Cluster of Ortholog Genes(COG)),and microbial taxonomic compositions(phylum,class,order,family,and genus levels),indicating that FMT can transfer the intestinal microbiota in piglets effectively.(3)By comparing the relative abundance of intestinal microbiota in saline and microbiota transferred LY piglets,we identified five microbes(including L.frumenti,L.gasseri LA39,Butyricicoccus pullicaecorum,E.hallii,and Blautia hansenii)as key candidate species that may mediate diarrhea resistance.(4)The results of animal experiment indicated that L.gasseri LA39 and L.frumenti conferred the resistance to early-weaning stress-induced diarrhea in piglets.Further data indicated that L.gasseri LA39 and L frumenti-mediated diarrhea resistance depends on secretory gassericin A.(5)The results of further functional experiments indicated that gassericin A bound to the Keratin 19(KRT19)protein in the intestinal epithelial cell plasma membrane and then decreased cellular cyclic nucleotides levels by activating the mechanistic target of rapamycin(mTOR)-mediated phosphodiesterases(PDEs)activity,thereby increasing the expression of proteins associated with intestinal fluid absorption and decreasing the expression of proteins associated with intestinal fluid secretion.3.Study on regulatory roles of gut microbes associated with diarrhea resistance in intestinal epithelial barrier functions in early-weaned piglets(1)The results showed that beforehand oral administration of L.frumenti prior to early weaning significantly increased the villi heights and the ratios of villi heights to crypts depths in the jejunum and ileum in early-weaned piglets.The L.frumenti also significantly decreased the serum endotoxin and D-lactic acid levels in early-weaned piglets.These data suggested that L.frumenti facilitates improve the intestinal epithelial barrier function in early-weaned piglets on a whole.(2)The results demonstrated that the intestinal tight junction proteins(including ZO-1,Occludin,and Claudin-1)were significantly up-regulated by L.frumenti administration.The relative mRNA expression levels of intestinal mucins and the numbers of intestinal goblet cells were not significantly changed by L.frumenti administration.The serum immunoglobulin G(IgG)levels,intestinal secretory imnunoglobulin A(slgA)levels,and interferon-γ(IFN-γ)levels were significantly increased by L.frumenti administration.These data revealed that oral administration of L.frumenti improved the intestinal physical barrier and immunological barrier functions,whereas had no significant effect on intestinal chemical barrier function.(3)The data showed that oral administration of L.frumenti significantly increased the relative abundances of health-promoting microbes(including L.frumenti,L gasseri LA39,Parabacteroides distasonis,and K.telluris)and decreased the relative abundances of opportunistic pathogens(including Desulfovibrio desulfuricans and Candida humilis),suggesting that L frumenti facilitates the intestinal epithelial microbial functions in early-weaned pigets.In sum:(1)The intestinal microbial communities(especially bacterial communities)significantly change with the age of CM piglets after early weaning.P.copri and K.telluris are the most abundant intestinal bacteria and fungus,respectively.These microbes(including L.coleohominis,L.frumenti,E.hallii,and L.gasseri LA39)whose relative abundances increase with the age of piglets may be potential probiotics.(2)We identified and validated L.gasseri LA39 and L.frumenti as two bacterial species that confer the diarrhea resistance.Diarrhea resistance depends on the bacterial secretory gassericin A.The binding of gassericin A to KRT19 on the plasma membrane of intestinal epithelial cells is essential for enhancement of fluid absorption and decreased secretion.L.frumenti facilitates improve the intestinal epithelial barrier function in early-weaned piglets on a whole,mainly via improving the intestinal physical barrier,immunological barrier,and microbial barrier functions.