Porcine Circovirus Type 2 Promotes The Survival Of Actinobacillus Pleuropneumoniae In Alveolar Macrophages By Inhibiting ROS

Actinobacillus pleuropneumoniae(APP)and porcine circovirus type 2(PCV2)are important pathogens causing porcine respiratory disease syndrome(PRDC).Being infected with PCV2 could damage the pig’s immune system,cause the pig’s lymphocyte depletion,and result in immunosuppression,which make the body more susceptible to secondary infection with other viruses or bacteria.APP is a respiratory pathogen with extremely strong virulence factors.Pigs infected with APP were mainly characterized by fibrinous hemorrhage and necrotizing pleural pneumonia,accompanied with severe respiratory distress and extremely high mortality.Recently,several coinfections of PCV2 and APP have been found in pig farms around the world,and mixed infection could cause more serious and complicated diseases.Serious influence raised the development of pig industry,causing tremendous economic loss.Porcine alveolar macrophage(PAM)is the first line of defense against pathogen invasion to the lungs.It first contacts the pathogen and exerts powerful natural immune functions such as phagocytosis and the production of cytokines.Macrophages can also directly kill pathogens by producing reactive oxygen species(ROS).When PCV2 and APP enter the lung through the respiratory tract,PAM will perform its function first.In order to promote their growth and reproduction,PCV2 and APP must resist the natural immunity of PAM.In this process,PAM may be damaged to a certain extent.The pathogenesis of PAM infection with APP and PCV2 alone has been studied,but the research on the pathogenesis of their coinfection is still a close blank domain.The purpose of this study was to investigate the pathogenesis of PAM coinfection with APP and PCV2.Firstly,the changes of phagocytic function and cytokines release function of PAM were detected after PCV2 and APP coinfected PAM.Next,experiments in vitro were used to detect the APP adhesion to and invasion of PAM with successively PCV2 participation,the changes of ROS production in PAM infection with APP and PCV2 alone and coinfection with APP and PCV2,the changes of cytotoxicity after PAM infection with APP and PCV2 alone and coinfection with APP and PCV2,and the changes in the main pathways of ROS production.Finally,the two pathogens coinfected ICR mice intranasally.The time points of 6h,12 h and 24 h after infection were used to simulate early infection,intermediate infection and late infection stages,respectively.The changes of ROS production of mice alveolar macrophages,and the changes of bacteria burden of APP in alveolar macrophages and lung homogenates were detected to verify the results in vitro.At the same time,the changes of body weight,lung index and cytokines release in the lungs were measured;the clinical signs and the lung lesion were observed.The results showed that the phagocytosis function of macrophages was affected by APP,and their function of releasing cytokines was inhibited by PCV2 when APP and PCV2 coinfected.It was also found that PCV2 promoted APP adhesion to and invasion of PAM.In further investigation,it was found that PCV2 reduced the production of ROS by inhibiting the NADPH oxidase activity which is on the cell membrane of macrophage,thereby weakening the bactericidal effect of PAM,which would be conducive to the survival of APP in PAM and eventually cause more severe cytotoxicity to PAM.The results of cellular experiments were validated in the mouse.At the early stage,the coinfected mice had severer lung lesions than the separately infected mice,and PCV2 increased the bacteria burden of APP in the alveolar macrophages and lung homogenates by inhibiting the ROS production of alveolar macrophages.During coinfection,PCV2 also inhibited the release of cytokines such as TNF-α,IFN-γ and IL-4 in lung homogenates.This discovery provides a new perspective for revealing the pathogenesis of coinfection and lays the foundation for solving this major problem.