The Mechanism Of PEDV Vertical Transmission From Sows To Neonatal Piglets Via Breast Milk

Porcine epidemic diarrhea(PED)is an acute,highly infectious gastrointestinal disease caused by the porcine epidemic diarrhea virus(PEDV),which has disastrous effects on the global pig industry.PEDV primarily infects neonatal piglets,and is characterized by wide prevalence,fast transmission,and high fatality rate.As the immune system of neonatal piglets is immature,immune protection cannot be generated in time through direct immunization.The immunity generated by active immunization of sows can be transferred to neonatal piglets through breast milk,and the protection of neonatal piglets can be achieved through passive immunization.Therefore,in many large pig farms,active immunity in sows is induced by‘feeding-back’(feeding the intestinal tissue of piglets infected with PEDV or live PEDV isolated from feces).In this way,passive immunity can be transmitted to neonatal pigs via breast milk to achieve PED control.Although this method allows PED to be controlled in pig farms in the short term,it can pose a massive risk for pig farms by making sows the source of virulent infections.In recent years,the investigation results of our laboratory on some pig farms have confirmed that the outcome of this strategy has been satisfactory,as the incidence of clinical PED has markedly declined;however,the feedback has not been perfectly effective.After several investigations,PEDV was found to be present in sow milk.Therefore,we hypothesized that PEDV could be vertically transmitted from sows to neonatal piglets via breast milk.To verify this hypothesis,we investigated PEDV in sows and suckling piglets from multiple feeding-back farms and virus-breast milk cells challenge experiments were performed on neonatal piglets via oral inoculation.The results showed that both sows and neonatal piglets exhibited typical PED symptoms,and viruses were able to infect the small intestine.Therefore,this study will focus on this phenomenon to explore the mechanisms of PEDV transmission into milk in sows and PEDV transmission from sows to neonatal piglets via breast milk.The experimental results showed that PEDV colonizing the intestinal epithelial cells of orally infected sows could be transferred to CD3~+T cells located just beneath the intestinal epithelial cells.Subsequently,the CD3~+T cells carrying PEDV migrate from the intestine to the mammary gland via blood circulation,with an increase in homing receptor integrin α4β7 and chemokine receptor CCR10.Finally,the CD3~+T cells carrying PEDV enter the intestinal tract of neonatal piglets via breast milk,and subsequently transmit the virus to intestinal epithelial cells through direct contact,causing intestinal infection in neonatal piglets.This study is thus divided into the following three parts:1.Investigation and pathogenicity study of PEDV in sow milkIn this study,we investigated the colostrum of sows with diarrhea in multiple feeding-back farms and found PEDV in 44.5%of the milk samples,in breast milk-derived CD3~+T cells.To confirm that PEDV could infect suckling neonatal piglets via breast milk,we collected the small intestine,mammary glands,and milk of sows with diarrhea,and the small intestines of their suckling neonatal piglets.Western blotting,immunohistochemical analysis,and immunofluorescence showed that PEDV primarily infected the jejunum of sows and their suckling neonatal piglets,and both western blot and immunohistochemical analysis showed that PEDV could be detected in the mammary glands of sows.In addition,western blotting and flow cytometry results showed viral presence in breast milk-derived CD3~+T cells.To further verify PEDV vertical transmission from sows to suckling piglets via breast milk cells,the breast milk cells carrying PEDV(10~7/m L)were fed to neonatal piglets.The results of this challenge experiment showed that neonatal piglets had typical PED symptoms,and viruses were present in the small intestinal epithelium.The above results suggested that CD3~+T cells in breast milk may mediate vertical transmission of PEDV.2.The study of the T cells carrying PEDV migration from the intestine to the mammary gland in sowsAfter orally infecting sows with PEDV,the virus primarily replicates in the intestinal epithelial cells of sows.This raises several questions:how can CD3~+T cells acquire PEDV?How do CD3~+T cells carrying PEDV reach the mammary gland?The investigation of clinical feedback from pig farms found that PEDV was detected in the CD3~+T cells of the intestine and blood and in the CD3~+T cells of the mammary glands of sows orally infected with PEDV.These results indicate that CD3~+T cells carrying PEDV in the intestinal mucosa might migrate to the mammary gland via circulating blood.Therefore,in this study,an in vitro the co-culture model of CD3~+T cells and epithelial cells was first used to find that the epithelial cells infected with PEDV could transmit the virus to CD3~+T cells through direct contact.Secondly,PEDV infection resulted in an increase in CD3~+T cells homing receptor integrin α4β7 and chemokine CCR10 levels.Transwell migration assay revealed that more CD3~+T cells carrying PEDV could migrate under the action of the cytokine MAd CAM-1.Finally,a co-culture model of CD3~+T cells and mammary epithelial cells(MECs)in vitro and an autotransfusion assay in vivo showed that the T cells carrying PEDV could be transported across MECs into the lumen(colostrum),and this process primarily depends on the interaction between CCR10 and CCL28.The above results indicate that after oral infection of PEDV in sows,PEDV colonizing the intestinal epithelial cells of sows could be transferred to CD3~+T cells located just beneath the intestinal epithelial cells,and that the CD3~+T cells carrying PEDV migrated from the intestine to the mammary gland through blood circulation and were transported across MECs into the lumen(colostrum).3.Transmission of virus by T cells carrying PEDV in milk to neonatal piglet intestinal epithelial cellsIt has previously been proven that after oral infection of PEDV in sows,the CD3~+T cells carrying PEDV could migrate from the intestine to the mammary gland through blood circulation.Again,this raises the question:How do CD3~+T cells carrying PEDV in breast milk infect the intestinal epithelial cells of neonatal piglets?Confocal microscopy revealed that breast milk cells could be interspersed between the intestinal epithelial cells of neonatal piglets after oral inoculation,suggesting that the structures of cell-to-cell contact may provide an opportunity for viral transmission.To study the mechanism of virus-infected intestinal epithelial cells,a co-culture model of T cells and intestinal epithelial cells was established in vitro,and the results showed that the T cells carrying PEDV could transmit the virus to the intestinal epithelial cells,primarily depending on intercellular adhesion molecule-1(ICAM-1).In addition,we found high levels of the cytokine TGF-βin the breast milk of sows with diarrhea.TGF-βcan upregulate the expression of ICAM-1 in intestinal epithelial cells,thereby facilitating viral transmission.These results suggest that TGF-βin breast milk promotes viral transmission between T cells carrying the virus and intestinal epithelial cells by upregulating the expression of ICAM-1 in intestinal epithelial cells of neonatal piglets.4.PEDV infection in neonatal piglets via the nasal cavity is mediated by subepithelial CD3~+T cellsOur results suggest that feeding sows with infected small intestine tissue to control PEDV infection in neonatal piglets has the potential to spread the virus throughout the pig farm.PEDV can survive in the surrounding environment for a long time.Studies have shown that airborne transmission is one of the key modes of PEDV transmission.PEDV infects piglets through the nasal cavity,a process in which dendritic cells play an important role.However,this study found that subepithelial CD3~+T cells mediated PEDV invasion through the nasal cavity in neonatal piglets.PEDV can replicate in nasal epithelial cells(NECs)isolated from the nasal cavity of neonatal piglets.The infected NECs mediated the transfer of the virus to CD3~+T cells distributed in the subepithelium of the nasal cavity via cell-to-cell contact.Subsequently,CD3~+T cells carrying PEDV can enter the blood,providing a condition in which PEDV can survive for several hours.Finally,the infected CD3~+T cells could migrate to the intestine via blood circulation,causing intestinal infection in neonatal piglets.Thus,the findings of this study indicate the importance of CD3~+T cells in the dissemination of PEDV from the nasal cavity to the intestinal mucosa in neonatal piglets.To the best of our knowledge,this study provides the first evidence of the mechanisms of PEDV transmission in sows into milk and PEDV transmission to neonatal piglets via breast milk.In addition,this study demonstrates the mechanism of PEDV transnasal infection in neonatal piglets.This study not only provides a new strategy for preventing PEDV infection in neonatal piglets,but also provides an animal model for the study of vertical transmission of human infectious diseases.