Study Of Synergistic Pathogenesis Of Newcastle Disease Virus And Mycoplasma Gallisepticum By Establishing Co-infection Models Both In Vivo And In Vitro

Mycoplasma gallisepticum(MG)infection is one of the causes of chronic respiratory infectious diseases in chickens.Mixed infection of MG and Newcastle disease virus(NDV)is also common in clinical practice,which makes the prevention and treatment of respiratory diseases in chickens very difficult.Although there are many studies on the co-infection mechanism of multiple mycoplasmas and viruses,the co-infection mechanism of MG and NDV remains unclear.Therefore,it is very important to investigate the pathogenic mechanism of the mixed infection of MG and NDV.In this study,HD11 cells were used to construct a cell model of NDV and MG coinfection.The infection strain type,optimal inoculation dose,and imcubation time were determined based on the cytopathic effect(CPE).The results of indirect immunofluorescence directly reflected the distribution and localization of NDV and MG in HD11,and proved that the co-infection cell model had been successfully constructed.The expression levels of virulence related genes of NDV and MG in HD11 cells were detected by fluorescence quantitative PCR.The results showed co-infection could promote the replication of the pathogenic genes of both NDV and MG.Based on the well-established co-infection cell model,the co-infection of NDV and MG was studied in vitro.The nuclear morphological changes in HD11 cells after DAPI staining observed by fluorescence microscope and the quantitative detection results of flow cytometry proved that the co-infection of NDV and MG can cause and promote apoptosis of HD11 cells.The expression of apoptosis-related factors was detected at the m RNA level,and the results showed that co-infection of NDV and MG could promote the up-regulation of apoptosis-related genes of Cytc,Fas,Bak and Caspase families in HD11 cells,which indicated that NDV and MG infection mainly induce cell apoptosis through the caspases-dependent mitochondrial endogenous pathway.The HD11 cells are avian immune cells,which can participate in natural and adaptive immune response.Therefore,we further investigated the inflammatory response mediated by HD11 cells after co-infection.The results showed that the infection of MG could cause the expression of IL-1β,IL-6,IL-8,IL-18,CCL-4,CCL-20 and other cytokines in HD11 cells,while NDV and MG co-infection could up-regulate or down-regulate the expression of these factors,depending on the duration of co-infection and the sequence of infection.In addition,we also found that MG can suppress the expression of IFN-αand IFN-β antiviral genes,thereby promoting the pathogenic replication of NDV.In this study,an animal co-infection model of NDV and MG was also successfully constructed.According to the clinical observation of the exacerbation of respiratory disease symptoms and increased mortality of chickens co-infected with NDV and MG,there is a synergistic effect of NDV and MG co-infection.The pathological change of chicken air sacs is one of the important indicators of MG infection.Therefore,after the end of co-infection,we conducted autopsy on the experimental animals in each group,counted the pathological damage of the air sacs and scored them.Based on pathologic changes and scores of air sacs,NDV and MG co-infection caused more serious damage to air sacs of chickens.The observation results of pathological tissue sections showed that the co-infection of NDV and MG caused more obvious pathological changes in the lungs and trachea of chickens than infection of either of the two pathogens.We also investigated the effect of NDV and MG co-infection on the body’s immune response.The differentiation of chicken spleen T lymphocytes was detected by flow cytometry,and the results showed that NDV and MG co-infection could increase the number of CD8+ T cells and decrease the number of CD4+ T cells.The CD4+ to CD8+ ratio reflected a decreased overall cellular immune level in the chickens co-infected with NDV and MG.To evaluate the humoral immune level,we monitored the changes in NDV and MG antibodies within 15 days after the end of the infection cycle.The results of the indirect ELISA experiment showed that both MG and NDV antibodies could be produced during the early stage of co-infection.However,by the 6th day,the MG antibody level began to decrease continuously,while the NDV antibody level remained relatively high.The decrease of MG antibody level indirectly explained the reason why the clinical respiratory system symptoms and pathological damage of chickens were exacerbated by the proliferation of MG.In addition,we detected the expression of some immune-related factors,including interleukins,interferons,growth factors and chemokines,in the lung of chickens co-infected with NDV and MG by q PCR.The results showed that NDV infection can cause the up-regulated expression of most cytokines The the mixed infection of NDV and MG can significantly down-regulate the expression of these cytokines,whether it is co-infection or mixed infection in different order.It shows that in the co-infection of MG and NDV,MG exhibits immunosuppressive activity and inhibits the trend of increased expression of cytokines induced by NDV infection.In summary,this paper established a cell model and an animal model of NDV and MG co-infection,studied the synergistic pathogenic mechanism of the two in vivo and in vitro,and proved: 1.MG infection can inhibit type I interferon secretion and suppress the increase of immune-related factors brought on by NDV infection,thereby promoting NDV infection;2.NDV can promote the apoptosis of MG-induced HD11 cells through the caspases-dependent mitochondrial endogenous pathway and inhibit the MG-mediated inflammatory response in the late stage of infection,thereby promoting the proliferation of MG;3.NDV promotes MG infection by inhibiting MGmediated innate and acquired immune responses,aggravating chronic respiratory diseases in chickens caused by MG.The above results not only provide a new theoretical basis for the study on the pathogenic mechanism of the mixed infection of the two pathogens,but also provide a scientific basis for the joint prevention and control of Newcastle disease and chicken chronic respiratory infectious diseases.