| Intestinal tract is not only the main site for digestion and absorption in pigs,but also the first line of defense of the body against foreign pathogens.The intestinal barrier can prevent intestinal harmful substances such as bacteria and toxins from entering the body’s blood circulation through the intestinal mucosa.The damage of the intestinal barrier is closely related to many diseases.Porcine epidemic diarrhea(PED)is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV)and presents particularly high morbidity and mortality in neonatal piglets.Studies have showed that the damage in the intestinal barrier is one of the important factors leading to high incidence of diarrhea and intestinal inflammation in piglets.Genetic variations of PEDV sequences result in an increase in the virulence of mutant PEDV strain and make it difficult to effectively vaccinate piglets with the current vaccines.In the long run,it is likely to be an efficacious strategy to genetically enhance the resistance of piglets against PEDV.Therefore,genetic screening of key candidate genes related to intestinal barrier of piglets will greatly contribute to implementing pig resistant breeding to PEDV.A total of 43 Duroc-Landrace-Yorkshire 8-day-old ternary crossbred pigs were used in this study,which included 28 diarrhea piglets and 15 normal piglets.Firstly,PEDV sequences were amplified by RT-PCR and the intestinal permeability of diarrhea and normal piglets was initially identified by using ELISA assays of serum D-lactic acid and DAO.At the same time,the intestinal tissues were collected and visually observed by scanning electron microscopy and transmission electron microscopy.Combined with paraffin sections,the intestinal barrier integrity of piglets was further validated to obtain intestinal barrier intact and damaged individuals.These samples were analyzed by RNA-seq sequencing to screen important regulatory pathways and differentially expressed genes.Preliminary functional verification was performed for the important candidate genes at the individual and cellular levels.The main results were as follows:1.Based on the sequence of the M gene,we established the phylogenetic tree by using the neighboring method.As a result,it was found that the PEDV strain had high homology with the epidemic PEDV strains worldwide confirming that the pigs with diarrheal diseases in this study were infected by PEDV virus.We then removed 15 piglets from all samples according to the results of the serum D-lactate and DAO levels and the remaining samples were preliminarily identified as intact and damaged intestinal barrier groups.Scanning and transmission electron microscopy and paraffin section staining analyses showed that the typical intestinal barrier intact piglets had intact and distinct structure of the intestinal mucosa,and the length and width of the villi were significantly higher than those of the damaged individuals(P<0.05).Mucosal epithelial cells have clear outlines,regular rules,and a large number of goblet cells.The typical intestinal barrier damaged piglets showed obvious lesions including shed and damaged villi,exposed lamina propria,atrophied intestinal glands,and lower number of goblet cells.In the end,this study successfully obtained 10 confirmed piglets with impaired intestinal barrier and 12 piglets with intact intestinal barrier.In this study,we established a system for evaluating piglets intestinal mucosal barrier integrity by a series of experiments including serum D-lactic acid and DAO ELISA assay,scanning and transmission electron microscopy visual observation,paraffin section detection,and finally successfully obtained corroborativepiglets with intact and impaired intestinal barriers.These can act as valuable materials for further investigations on intestinal diseases in piglets and provide an effective and feasible method for obtaining experimental materials of studies on pig intestinal barriers.2.In this study,RNA-seq technique was used for transcriptome sequencing of eight jejunal mucosal tissues derived from the piglets with intact or damaged intestine barriers,yielding 110.62,101.07,129.27,100.18,and 113,57,108.03,99.84,111.55 million raw reads,respectively.Alignment analysis with the reference genome using HISAT2revealed that the total mapped reads was between 90.75%and 95.41%,with more than 70%expected results.3.11%-6.78%of reads exist multiple alignment positions on the reference sequence,and 85.43%-91.76%of reads have unique alignment positions on the reference sequence.A total of 3721 differentially expressed genes(DEGs)were screened out between the intestinal barrier intact and damaged groups,of which 2151 were up-regulated and 1570 were down-regulated.GO enrichment analysis identified 57 significant GO classes(22 BPs,11 CCs,24 MFs).CCs mainly included intracellular parts,intracellular organelles,and Organelles,etc.,and MF mainly included nucleotide binding domain,nucleoside phosphate binding,and small molecule binding etc.KEGG analysis showed that DEGs were mainly enriched in Glycerolipid metabolism,Phosphatidylinositol signaling system,and Inositol phosphate metabolism pathways.3.12 important candidate genes including SELE,LRRK1,ITPR2,EDA,KIAA1429,PRAP1,ALPI,SDR16C5,CLDN10,ASAH2,AGPAT3,and KCNJ12 were screened based on gene expression fold changes and biological functions.The Real-time fluorescence quantitative PCR was conducted to verify the transcriptomic analysis results.The expression levels of these genes were consistent between the two quantitative methods,indicating that the results of transcriptome sequencing were accurate and reliable.Furthermore,the expression of these 12 candidate genes was detected in PEDV-infected IPEC-J2 cells at the cellular level.As a result,it was found that there was no significant differential expression of these genes between the infected and control cells,suggesting that these genes may be involved in maintaining or increasing the integrity of the intestinal barrier to resist PEDV infection instead of directly participating in immune responses. |
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