Pathological Observation And Intestinal Transcriptomics Study Of Porcine Epidemic Diarrhea Virus Naturally Infected Suckling Piglets

Porcine epidemic diarrhea(PED)is a multiple infectious disease caused by porcine epidemic diarrhea virus(PEDV),which can infect pigs of all ages,and suckling piglets are the most serious with a mortality rate of up to 90%.Once infected PEDV,even if cured,they will be also seriously affected the growth,caused stunting,and finally eliminated.As a big pig-raising country,China maintains a relatively high stock of pigs each year.Due to the frequent occurrence of diarrhea in pigs,huge economic losses have been caused,and the control of the disease is imminent.At present,the prevention and control of PED is mainly based on the strategy of adopting biosecurity measures and vaccine immunization,but the effect is poor,because of the severe dehydration of diarrhea in pigs,which greatly increases the difficulty of treatment.It is urgent to explore the developmental exact mechanism of this disease to find effectively drugs for alleviating the dehydration of diseased pigs in the future.In this study,3 to 5 days old healthy piglets and diseased piglets with clinical signs suspected of PEDV infection were selected from Danish Landrace in a large-scale pig farm in Guigang,Guangxi.The diseased piglets were identified as PEDV infection alone by the detection of common pathogen.The pathological changes and virus distribution characteristics of various tissues and organs were observed and analyzed,and the transcriptome sequencing analysis was performed to screen differentially expressed genes related to water and salt metabolism on the small intestine,aiming to determine the target genes of diarrhea caused by the water and salt metabolism disorders and lay the foundation for the study of the effects of water and salt metabolism disorders on diarrhea.The research results were as follows:1.Gross pathological changes of piglets infected with PEDVWith infected PEDV,piglets mainly presented with vomiting and watery diarrhea,and those may even die with severe diarrhea and dehydration.The anatomical results revealed the inflated and thin of intestinal wall,yellow and yellow-green contents in the intestinal cavity,undigested milk clot adhering on the stomach wall,mesenteric congestion,and mesenteric lymph nodes swelling.2.Histopathological changes of piglets infected with PEDVIn piglets infected with PEDV,the duodenal villus atrophied,and the mucosal epithelial cells necrotized and shed;Jejunal villus atrophied severely,and the cytoplasm was obviously vacuolated;The villous epithelial cells of the ileum were necrotic and the intestinal lumen was filled with exfoliated epithelial cells;The cecal mucosa was covered with an incomplete flat epithelium with a small amount of lymphocytes infiltration;Some mucosal epithelial cells of the colon shed,and the capillaries of lamina propria were congested and loosely structured;The boundary was unclear between the cortex and the medulla in mesenteric lymph nodes,and lymphocytes were decreased locally;The boundary was unclear between the red pulp and the white pulp in the spleen,accompanied by a large number of eosinophils infiltration;The localized alveolar wall was thickened in the lungs,and alveolus occurred rupture and fusion;The focal myocardial necrosis and myocardial fibrosis were observed in the heart.3.Viral distribution characteristics of piglets infected with PEDVImmunohistochemical staining showed that PEDV was distributed in all intestine except the rectum,among which the jejunum and ileum had the highest viral load.The virus was mainly distributed in the epithelial cells of the intestinal mucosa,appearing only in the cytoplasm rather than the nucleus,and no virus distribution was detected in other tissues and organs.4.Ultrastructural changes of jejunum in piglets infected with PEDVIn the jejunum of piglets infected with PEDV,under the scanning electron microscope,the local epithelial cells were shed,and the microvilli were generally disorganized,the tips of some microvilli swelled,and some microvilli were almost completely shed and disappeared on the intestinal villi,and the cell membrane was naked.Under the transmission electron microscope,the tight junctions of the epithelial cells were destroyed,and the nucleus of some epithelial cells was ruptured,and the electron density of the cytoplasm was reduced with appearing the various sizes vesicles,and the mitochondria swelled and became round,and the endoplasmic reticulum ruptured and constricted,and the typical Golgi bodies structure was notobserved.There were scattered coronavirus particles in the cytoplasm,and the cell membrane was destroyed.5.Transcriptomics analysis of small intestine of piglets infected with PEDVWith the diarrhea group infected with PEDV compared with the healthy group,the results of transcriptome analysis showed that there were 170 differentially expressed genes(DEGs),including 9 genes related to water and salt metabolism in duodenum;There were 1337 DEGs,including 58 genes related to water and salt metabolism in jejunum;There were 209 DEGs,including 13 genes related to water and salt metabolism in the ileum;The number of DEGs was most in the cellular processes,the biological regulation,the cell composition,the organelles,the binding,the catalytic activity,TNF signaling pathway,chemokine signaling pathway,and cytokine-cytokine receptor interaction in three intestines;All DEGs were significantly enriched in the positive regulation of c AMP metabolic process,TNF signaling pathway,cytokine-cytokine receptor interaction,and the protein digestion and absorption;The important DEGs related to water and salt metabolism were SLC9A3(down-regulated),SLC26A6(up-regulated),and KCNE3(up-regulated),KCNN4(up-regulation),AQP5(up-regulation)and AQP9(down-regulated).In summary,PEDV mainly invaded small intestinal mucosal epithelial cells of piglets and caused a certain degree of pathological damage.The diarrhea process caused by the virus was mainly affected by the cellular processes,the biological regulation,the cell composition,the organelles,the binding,the catalytic activity,the positive regulation of c AMP metabolic process and it was mainly related to TNF signaling pathway,chemokine signaling pathway,cytokine-cytokine receptor interaction,the protein digestion and absorption.In addition,the water and salt metabolism related genes SLC9A3,SLC26A6,KCNE3,KCNN4,AQP5 and AQP9 play an important role in the diarrhea process.This research lay a foundation for the study of the effect of water and salt metabolism disorders on diarrhea.