The Mechanism Of Co-Infection With Porcine Circovirus 2 And Streptococcus Suis Serotype 2 In Piglets

Porcine circovirus 2(PCV2)is primarily responsible for porcine circovirus-associated diseases(PCVAD),including postweaning multisystemic wasting syndrome,porcine dermatitis and nephropathy syndrome,porcine respiratory disease complex,reproductive disorders and enteritis.PCVAD are emerging globally and have caused considerable economic damage to the global swine industry.In clinical,PCV2 infection alone is rarely resulted in desease.Co-infections can often be detected among PCVAD cases in recent years.Streptococcus suis serotype 2(SS2)is a pathogen of swine streptococcosis,causing septicemia,meningitis,pneumonia,endocarditis and arthritis in piglets.PCV2 and SS2 are two of the main contributors to porcine respiratory disease complex.In recent years,PCV2 and SS2 co-infection cases have been frequently detected in clinical cases,characterized by high fever,difficulty breathing and septicemia,resulting in higher morbidity and mortality rates than single infection.However,there is poor understanding of whether co-infection with PCV2 and SS2 enhances the pathogenicity of SS2 and how co-infection promotes the development of disease.Viruses predispose the host to infection by bacterial pathogens by various mechanisms,including suppression of the immune response,disruption of the epithelium barrier,upregulation of bacterial adhesion receptors and changement of the micro-environment.The present study established PCV2 and SS2 co-infected piglet model,co-infected porcine alveolar macrophage cell(3D4/21)model and co-infected swine tracheal epithelial cell(STEC)model.The pathogenicity of the pathogens and immune responses in co-infected piglets were assessed;the mechanism of co-infection exacerbating inflammatory response in 3D4/21 cells was studied;the mechanism by which PCV2 promoting SS2 infection through disrupting the tracheal epithelial barrier and the effects of co-infection on SS2 clearance in STEC were clarified.The results above are helpful to elucidate the pathogenic mechanism of PCV2 and SS2 co-infection.1.Establishment of a PCV2 and SS2 co-infection piglet model and the immune responses in pigletsThree-week-old,Duroc× Long White× Large White,crossbred piglets were selected from a healthy pig farm.All piglets were confirmed seronegative for PCV2,porcine reproductive and respiratory syndrome virus(PRRSV),classical swine fever virus(CSFV),Haemophilus parasuis(HPS)and SS2 by commercial ELISA detection kits.Piglets were housed in clean-grade laboratory animal room.To establish a PCV2 and SS2 co-infected piglet model,the four-week-old piglets were infected with the PCV2 strain(106.5 TCID50/mL)through intranasal(2 mL)and intramuscular(3 mL)inoculation.5 days after PCV2 challenge,piglets were inoculated intranasally(2 mL)and intramuscularly(3 mL)with SS2(5×108 CFU/mL).Compared to single-infected piglets,the co-infected pigs had higher rectal temperatures at 8 days post-inoculation(dpi)and 17 dpi of PCV2 infection(over>39.5 ?),the co-infected pigs showed lower weight gains at 8 dpi,21 dpi,24 dpi and 28 dpi.Moreover,co-infected piglets showed more severe pneumonia,myocarditis and arthritis.The amounts of SS2 in the co-infected group were higher than those in the SS2-infected group at 8 dpi,11 dpi and 17 dpi.Further research found that the expression levels of TLR2 and TLR4,IL-6,IL-8 and TNF-? were significantly higher in the co-infected group than in the SS2-infected group and the PCV2-infected group at 8 dpi of PCV2.In addition,the transcriptional levels of CD4,CD8 and MHC II in co-infected pigles were down-regulated,the production of SS2 antibodies in coinfected piglets were inhibited.Taken together,this study establishes a PCV2 and SS2 co-infected piglet model;co-infected piglets shows more severe clinical symptoms,higher SS2 bacterial load,and a stronger inflammatory response,and appears immunosuppression.2.Effects of PCV2 and SS2 co-infection on the immune response of 3D4/21 cellsMacrophages are important immune cells,involved in phagocytosis and killing of pathogenic microorganisms,antigen processing and presentation,and secretion of various cytokines.Macrophages are one of the target cells of PCV2 in pigs.The porcine alveolar macrophages 3D4/21 cells were infected with PCV2 at a MOI of 1.Immunofluorescence assays were performed at different time points to detect viral infection,3D4/21 were confirmed infected by PCV2 at 24 h post-infection.For co-infection,3D4/21 cells were pre-infected by PCV2 for 24 h and then infected with SS2 at an MOI of 10.Compared to SS2 infection alone,co-infection with PCV2 and SS2 inhibited the proliferative activity of macrophages and promoted apoptosis;PCV2 and SS2 co-infection significantly upregulated the expression levels of IL-6,IL-8,TNF-? and TLR2,TLR4 in 3D4/21 cells;however,MHC ? expression in co-infected cells was down-regulated.The results above were consistent with the results of animal experiments.Further research found that PCV2 and SS2 co-infection activated both MAPK and NF-?B signaling pathways and up-regulated the expression of inflammatory cytokines.IL-8 is an important cytokine that causes tissue damage.TAK-242 was used to block the TLR4 signaling pathway,and the transcriptional level of IL-8 was down-regulated after TAK-242 treatment,indicating that TLR4 regulated IL-8 expression in co-infected 3D4/21 cells.In summary,this study establishes a PCV2 and SS2 co-infected 3D4/21 cell model;co-infection with PCV2 and SS2 activates MAPK and NF-?B signaling pathways and exacerbates the inflammatory response.Excessive inflammation could cause damage to tissues and organs,which is one of the main causes of host morbidity and mortality.3.Effects of PCV2 and SS2 co-infection on tracheal epithelial barrier functionThe airway epithelium plays a crucial role in defense against a wide range of pathogens.Tight junction proteins(TJs)ZO-1 and occludin are known to play a primary role in the maintenance of the epithelial barrier.Streptococcus suis can breach the epithelial barriers and cause infections.In this study,Piglets were infected with PCV2 for 28 days before being necropsied.The lung samples were collected for further analysis.The effect of PCV2 infection on TJs of lungs were analyzed using immunofluorescence staining,Western Blot and quantitative Real-Time PCR.Animal experiments found that PCV2 infection disrupted the continuity of TJs and down-regulated the expression of TJs in the lungs.Immortalized swine tracheal epithelial cells(STEC)were infected with PCV2 at a MOI of 1.Immunofluorescence assays were performed,STEC were confirmed infected by PCV2 at 24 h post-infection.For co-infection,STEC were pre-infected by PCV2 for 24 h,36 h or 48 h before being infected with SS2 at an MOI of 10.Meanwhile,an in vitro tracheal epithelial barrier model was also established using STEC.Our results showed that PCV2 infection in STEC decreased the expression levels of tight junction proteins and increased the permeability of the tracheal epithelial barrier through activating the JNK/MAPK signaling pathway,resulting in easier translocation of SS2.Together,this study establishes a PCV2 and SS2 co-infected STEC model;PCV2 promotes SS2 translocation across the tracheal epithelium through disrupting the tight junctions of the respiratory epithelium and increasing the permeability of the epithelial barrier.4.Effect of PCV2 and SS2 co-infection on the clearance of bacteria in STECReactive oxygen species(ROS),including superoxide,hydrogen peroxide,hypochlorous acid and other metabolites,are involved in many vital physiological processes in cells and play a critical role in the elimination of pathogens.ROS are also important signaling molecules for inflammatory response and participate in host immune response to infection.NADPH oxidase is widely expressed in airway epithelial cells,and is an important source of ROS,In the current study,STEC were infected with PCV2 at a MOI of 1 for 24 h or 48 h before being infected with SS2 at an MOI of 30.The ROS levels in PCV2 and SS2 co-infected STEC were evaluated;the roles of ROS in the clearance of SS2 and inflammatory cytokine expressions were studied.The results showed that SS2 infection induced ROS production through activating NADPH oxidase in STEC.ROS contributed to the clearance of SS2,antivated P38/MAPK signaling pathway and up-regulated the expression levels of IL-6?TNF-? and IL-1?.Compared to SS2-infected STEC,co-infection with PCV2 and SS2 suppressed the activity of NADPH oxidase and inhibited the generation of ROS,resulting in higher bacterial intracellular survival rate and lower cytokine expressions.The above research shows that PCV2 contributes to SS2 infection by promoting the intracellular survival of SS2 and suppressing the expression of inflammatory cytokines.In summary,co-infection with PCV2 and SS2 has a synergetic pathogenic effect.PCV2 and SS2 co-infection induces a strong inflammatory response and appears immunosuppression,causing serious tissue damage;PCV2 and SS2 co-infection disrupts the respiratory tract epithelial barrier function and inhibits bacterial clearance in respiratory epithelial cells,promoting SS2 infection.This study elucidates the pathogenic mechanism of PCV2 and SS2 co-infection and provides evidence for preventing and controlling PCV2 and SS2 co-infectious diseases.