Effects Of Lung Colonization With Aspergillus Fumigatus On Innate Immunity And Avian Influenza Antibody Levels In Chickens

Aspergillus is a saprophytic fungus widely existing in the natural environment.Aspergillus fumigatus(A.fumigatus)and Aspergillus flavus are common pathogens causing avian aspergillosis,especially A.fumigatus.Clinically,A.fumigatus as an important conditional pathogen,spread the pathogen by releasing a large number of conidia(φ≈2-3 um)into the air.These conidia can cause serious respiratory diseases after inhaled into bronchi and alveoli by the host,especially chickens who were sensitive to fungi.It has been reported that there are airborne fungi in the microbial aerosols of livestock and poultry houses.When a small amount of A.fumigatus conidia in the air is inhaled by chickens,it may not cause infection,but can this part of the inhaled conidia be colonized in the lungs?Can local innate immune response of lung tissue be induced after colonization?And whether it affects the formation of avian influenza(AI)antibodies?Current studies mainly report the prevention and diagnosis of Aspergillus fumigatus infection in poultry.The innate immune response to Aspergillus fumigatus colonization in chickens and the immune effect on AIV infection remain to be study.In order to study the effects of A.fumigatus colonization on innate immunity and AI antibody levels in chickens lungs,We established chickens lung colonization model of A.fumigatus.In this study,200 9-day-old SPF chickens were selected as experimental animals and randomly divided into six groups:blank control group(A),Aspergillus fumigatus colonization group(B),H9N2 AIV infection group(C),Aspergillus fumigatus colonization+H9N2 AIV infection group(D),AI vaccine immunization group(E),AI vaccine immunization+Aspergillus fumigatus colonization group(F).At 9 and 23 days of age,groups E and F were subcutaneously inoculated with 0.2 mL H9 subtype AI vaccine;at 14 days of age,groups B,D and F were inoculated with Aspergillus fumigatus agar beads,group A was replaced by 0.3 mL saline;at 17 days of age,groups C and D were inoculated with 0.2 mL H9N2 AIV by intranasal drip.GMS stain,fungal culture and microscopic examination were used to determine whether group B chickens successfully colonized Aspergillus fumigatus.The expression of TLRs(TLR1,TLR2,TLR3,TLR4)and cytokines(IL4,IL6,IFN-α,IFN-β,TNF-α)in lung and spleen tissues of groups A and B chickens were detected by qRT-PCR at1,3,5,7 days after inoculation with Aspergillus fumigatus agar beads.The transcriptome expression in lung tissue of groups A and B chickens was detected by transcriptome sequencing on the second day after inoculation with Aspergillus fumigatus agar beads.Some innate immune factors(TLR1,TLR2,TLR3,TLR4,TLR7,IL-1β,IL6,IL8,IL10,IFN-α,IFN-β,IFN-γ)were verified by qRT-PCR.The titers of AI antibodies in groups C,D,E and F were detected by HA and HI tests.The expression of innate immune factors(TLR3,TLR7,IL-1β,IL4,IFN-α,IFN-γ)in lung and spleen tissues of groups C and D chickens were detected by qRT-PCR.The results showed that Aspergillus fumigatus agar beads inoculated with 0.3 mL concentration of 5.0×10~6 CFU/mL could colonize Aspergillus fumigatus in the lungs of chickens for 28 days.The expression of TLR1 and TLR4 were down-regulated in group B compared with group A,at the 1,3,5 and 7 days after inoculation of Aspergillus fumigatus agar beads,Inflammatory cytokine IL6 was down-regulated in lung tissue,up-regulated in spleen,TNF-αwas up-regulated on day 3 and 5,IFN-α、IFN-βwere up-regulated in lung and spleen as a whole,and IL4 was up-regulated on day 1 and 3.The total number of genes identified by transcriptome sequencing was 17,732,of which 980 differentially expressed genes(p<0.05,doubling 1.0 or more),767 up-regulated and 213 down-regulated genes.There were 107 differentially expressed genes in the immune system of KEGG biological pathway;the AI antibody titers of groups C and group D were basically the same during the experiment,and the difference was not significant(p>0.05);the AI antibody titers of groups E and group F were not significant(p>0.05);the expression levels of TLR3,TLR7,IL-1β,IL4,IFN-αin lung and spleen of group D chickens were generally higher than those of group C chickens.The results showed that the colonization model of Aspergillus fumigatus in chicken lung was successfully established in this study.After the colonization of Aspergillus fumigatus,the expression of lung tissue mRNA have changed,and the local immune response of lung tissue was activated by innate immune factors.Aspergillus fumigatus and AIV had synergistic effect,which could activate the innate immune factors in chickens,but had no significant effect on the specific immunity of AIV.In conclusion,this study successfully established a colonization model of Aspergillus fumigatus in chickens lungs,and sequenced the lung tissues of Aspergillus fumigatus colonized chickens.The innate immune response of Aspergillus fumigatus colonization and its effect on AI antibody levels in chickens were preliminarily studied.It laid a foundation for further study on the interaction between Aspergillus fumigatus and avian host and its influence on other pathogenic bacteria.