Study Of The Innate Immune Response In Chickens Infected With Aspergillus Fumigatus And Secondary Infection Of Escherichia Coli And H9N2 Avian Influenza Virus

Aspergillus fumigatus(A.fumigatus)is a kind of saprophytic mould,distributed in moist soil,animal bedding and moldy feed.Many studies show that A.fumigatus exists widely in fowlery environment.The spores produced by A.fumigatus can be inhaled into the respiratory tract and lung,causing respiratory disease.At present,the studies of A.fumigatus were focused on the diagnosis,prevention of the clinical cases and the pathogenic mechanism of mammalian hosts,but poultry immune responses to A.fumigatus infection remain unclear.Moreover,there is no study on the innate immune response to A.fumigatus mixed with bacteria and viruses.In this study,we first established the artificial infection model of A.fumigatus in chickens,and detected the expression of innate immune factors in the target organ and the main lymphoid organ.And then the experiment of A.fumigatus secondary infection of Escherichia coli and H9N2 subtype avian influenza virus were carried out.The mortality rate,bacteria content,virus content and natural immune index were detected in the host of A.fumigatus secondary infection of Escherichia coli and H9N2 subtype avian influenza virus.Thus,we provided some reference for the study of fungal immunity and fungal secondary infection of other pathogens from the perspective of innate immunity.This study is divided into the following 3 parts:First,we established an artificial infection model of the A.fumigatus in chickens.14 days old chickens were infected by A.fumigatus through the method of subcutaneous injection,irrigation,trachea injection and air sac injection,with the dose of 0.1 mL,0.5 mL,1.0 mL,respectively.We found that the best way of A.fumigatus artificial infection is air sac injection,and the optimal dose was 0.1 mL 107 CFU/m L spore suspension in chickens.Thus,we established an artificial infection model of the A.fumigatus in chickens successfully.Second,the innate immune response in chickens after infecting by A.fumigatus.In order to clarify the innate immune response induced by A.fumigatus infect in chickens,we detected the expression of Toll like receptors(TLR1、TLR2、TLR4、TLR7),cytokines(IL-2、IL-6、Cxcl-8、IFN-α、IFN-β、TNF-α)and 2 major histocompatibility complex(MHC-I、MHC-Ⅱ)at mRNA level by RT-PCR.Thirty 14-day-old SPF chickens were randomly divided into two groups: A.fumigatus infection group and experimental control group.At 1,3,5,7 dpi,each experimental group spleen,lung tissue were taken respectively.The results showed that the expression of TLR1,TLR2,TLR7 in the lung and spleen were significantly higher than those in control group at 3dpi(p < 0.05);and Cxcl-8,IL-6,IFN-α were up-regulated in lung and spleen significant at 3dpi(p < 0.05),Cxcl-8 in the lung raised the largest level,up to 33.9-fold.The expression of MHC-I and MHC-Ⅱ in lung and spleen were significantly higher than those in control group at 5dpi(p < 0.05),and MHC-I can reach 21.7-fold.Thus,it can be seen that TLR1,RLR2,TLR7 play important roles in the identification of A.fumigatus,Cxcl-8 and MHC-I can also play major effect.Third,the study of A.fumigatus secondary infection of Escherichia coli,H9N2 avian influenza virus and the innate immune response.The bacterial content in the heart,liver and spleen of chickens infected with Escherichia coli and A.fumigatus secondary infection of Escherichia coli were determined by plate counting method.We found that in the same time period,the bacterial contents in the three tissues of secondary infection group were significantly higher than those of the infection of Escherichia coli group(p < 0.05).At 1 dpi,the bacterial content of secondary infection group in the spleen was the highest at 106 CFU/g,which was significantly different from that in the single infection group(p < 0.05).At 2 dpi,the bacterial content of secondary infection group in the liver reached 109 CFU/g,which was significantly higher than that in the single infection group(p < 0.05).In addition,the innate immune responses in the heart and spleen were detected by RT-PCR in secondary infection group and simple infection group.At 2 dpi,the expression of TLR4 and IFN-β in the secondary infection group were significantly higher than that in the simple infection group(p < 0.05);the expression of TLR4,TLR2 and IL-1 in the spleen of secondary infection group were significantly higher than that of simple infection group(p < 0.05).It was found that the expression of TLRs and cytokines was stronger when the host secondary infection of A.fumigatus and Escherichia coli.The virus titer in the lung of chickens infected with H9N2 and A.fumigatus secondary infection of H9N2 were detected by hemagglutination titer assay.The results showed that the virus titer of the secondary infection group was higher than that of the single infection group,but the difference was not significant(p > 0.05).In the study of the innate immune response induced by H9N2 infection and A.fumigatus secondary infection of H9N2,the expression of TLR3,TLR7,IL-1β in the lung and spleen of the secondary infection group than simple H9N2 infection group increased significantly at 2dpi(p < 0.05).The trend of TLR3 and TLR7 expression in the lung of the two groups were similar,and the initial expression was not obvious,and gradually increased with the passage of time.The expression of IFN-β in the spleen and lung of the secondary infection group reached peak value at 3dpi,and the difference was significant(p < 0.05).Therefore,the innate immune response of the host of secondary infection group was significantly higher than that of the simple infection.In this study,this experiment has studied the innate immune response induced by A.fumigatus infection,A.fumigatus secondary infection of Escherichia coli,H9N2 to pave the way for the further study of the innate immunity.