| Muscovy duck reovirus(MDRV)infection can cause immune suppression,destroy the intestinal structures and mucosal immune function in Muscovy ducklings,and result in severe economic losses to Muscovy duck breeding industry.Our previous studies have showed that Hericium erinaceus polysaccharide(HEP)has the positive effect on the immunity of Muscovy ducklings,which can protect and repair the intestinal mucosa,and relieve the immune suppression caused by MDRV.The intestinal mucosal immune system includes inductive site and effective site,both of them are mainly linked by lymphocyte homing,which is the progress of lymphocytes migration to the inflammatory effective site,mainly involving the coaction of the integrin family and immunoglobulin superfamily adhesion molecules and chemokines and other cytokines.All the above factors participate together in the lymphocytes migration between inductive site and effective site to achieve lymphocyte recirculation in vivo,and promote the repair of mucosal damage.Therefore,the mechanism of lymphocyte homing is an explaination of the protective and repair mechanism of HEP on the body and intestinal mucosa of Muscovy ducks.In this study,we first constructed the gene library through high-throughput sequencing(RNA-seq)of the duodenal transcriptome from Muscovy ducklings,and then annotated the Unigenes generated from the experiment to provide a reference for the selection of lymphocyte homing-related genes in Muscovy ducks.Finally,to further confirm the results obtained from transcriptome analysis,we detected the lymphocyte homing-related factors with ELISA to investigate the effect of HEP on the expression of CCL28/CCR10,CCL25/CCR9,?4?7/MAdCAM-1,CCL21/CCR7,LFA-1/ICAM-1 in intestinal tract of Muscovy ducks infected with MDRV.This study will provide foundations for further study of the mechanism that HEP regulates the lymphocyte homing in MDRV-infected Muscovy ducklings,as well as the clinical application of HEP in the control of MDRV infection.Methods:In this study,we based on the estabilished MDRV natural infection model,a total of 340 1-day-old healthy Muscovy ducklings were randomly divided into five groups:normal control group(NCG),Hericium erinaceus polysaccharide control group(HCG),cohabitation infection control group(CICG),Hericium erinaceus polysaccharide prevention group(HPG),each group of 70 ducklings,and an additional Artificial infection group(AIG)including 60 ducklings,2-day-olds Muscovy ducklings were artificially infected with MDRV,three days later,the artificially challenged ducklings were randomly selected to join normal ducklings(CICG)or HEP-pretreated ducklings(HPG)with a 2:5 ratio for cohabitation infection.The duodenal samples were collected at 1 d,3 d,6 d,10 d,15 d,21 d,28 d and 36 d after post-infection(dpi),from 6 ducklings in each group.The samples were collected in duplicate at the 3 d,6 d and 21 d dpi,one copy for transcriptome high-throughput sequencing(RNA-seq)analysis;and the other copy and the rest samples were analyzed for integrins and their ligands,chemokines and their receptors in ELISA test.Results:(1)Transcriptional sequencing results showed:? The data evaluation results are reliable,47456230 original Reads were obtained,and 80950 Unigenes were assembled with the average length of 1054 bp and N50 length of 2523 bp;?The sequences in the Nr,Swissprot,KOG,KEGG databases were 26824,22250,15786 and 14726,respectively;?There were 8113 sequences were found similar with the Anas platyrhynchos,which accounted for 30.24%of the total number of sequences annotated in the Nr database,and the coding sequence-based BLAST analysis also showed that Muscovy duck had higher homology with the Anas platyrhynchos;?As for the GO annotation,numerous annotated genes were classified into the cellular process and the protein binding pathway.The Signal transduction mechanisms in the KOG annotation and the KEGG pathway mainly analysis involved cytokine-cytokine receptor interaction,cell adhesion molecules(CAMs),intestinal immune network for IgA production and leukocyte transendothelial migration,which are all related to the immune system and lymphocytes homing,the cytokine-cytokine receptor interaction were significantly enriched in the CICG group after MDRV infection,and this enrichment of the HPG group was also existence;?For differential comparison,the numbers of up-regulation genes in at 6 dpi were more than other time points over the same period.CICG group was significantly enriched(P<0.05)in the immune system process,cytokines and chemokine activity,and HPG group was significantly enriched(P<0.05),significantly enriched(P<0.05)in the immune system process as well as defense response.There are significantly(P<0.05)increased trend of lymphocyte surface adhesion molecules ?4?7,LFA-1 and chemokine receptors CCR10,CCR9,CCR7 in MDRV infected ducklings,while HEP also has this up-regulative trend.(2)The results of ELISA showed that:during the acute period of MDRV infection,the expression of ?4?7,LFA-1,chemokine CCL28,CCL25,CCL21,chemokine receptors CCR9 and CCR7 in the duodenums from the CICG group were significant(P<0.05)or extremely significant(P<0.01)higher than those in the NCG group,while HEP treatment induced varying degrees of up-regulation in the secretion of these factors from HPG group in comparison with CICG group.The secretion of LFA-1,MAdCAM-1,CCL28,CCR9,ICAM-1 and CCL21 in HPG group was significantly(P<0.05)or extremely significant(P<0.01)higher than those in CICG group at 21 dpi;due to the discontinuation of HEP treatment since 22 dpi,the secretion of ?4?7,MAdCAM-1 and CCL28 in HPG group consequently decreased,while the secretion of CCL25,CCR9 and CCR7 secretion jaggedly increased in comparison with CICG group at 28 dpi.Compared with expression at 28 dpi,the expression of above factors recovered up at 36 dpi,which was also extremely significant(P<0.01)higher than CICG group.Conclusions:(1)80950 Unigenes were obtained from the transcriptome analysis,and 8113 of which are similar with the similar species Anas platyrhynchos;they are mainly involved in the cytokine-cytokine receptor interaction,cell adhesion molecules(CAMs),intestinal immune network for IgA production and leukocyte transendothelial migration,which are related to the immune system and lymphocytes homing.The cytokine-cytokine receptor interaction pathway is activated in the CICG group,after HEP pretreatment,Cell activity still exists and HEP can stimulate defense response in Muscovy ducklings.(2)Muscovy ducklings increase the secretion of lymphoid homing receptors and corresponding ligands in the small intestine to resist the invasion of MDRV.The HEP can further up-regulate the secretion of intestinal lymphocyte homing-related adhesion molecules,chemokine receptors and their corresponding ligands in MDRV-infected Muscovy ducklings,and promote lymphocyte homing to achieve synergistic resistance to MDRV infection.Additionally,HEP still have certain protective effect on Muscovy ducklings after the discontinuation of HEP treatment during the late infection of MDRV. |
PDF Full Text Request