Apoptosis Induction And Immune-related Gene Expression In The Oviduct Of Egg-laying Hens Experimentally Infected With H9N2 Avian Influenza Virus

The H9N2 subtype avian influenza virus(AIV) can cause serious damage to the reproductive tract of egg-laying hens, leading to reduced egg production and poor eggshell quality. The egg-laying hens infected with H9N2 AIV showed mild cough, diarrhea, depression symptoms as well as egg production dropping(30%-80%), which result in huge economic loss for the poultry industry. The oviduct is an important organ of egg-laying hens that can produce each element of eggs and it is a potential valid target organ for H9N2 AIV infection. However, previous studies in relation to the oviductal-dysfunction resulted from this agent have not been clearly elucidated. In the present study, the apoptosis and pathologic changes in the oviduct of egg-laying hens infected with H9N2 AIV were evaluated to fully understand the immune response in the pathogenic processes. Thirty-week old egg-laying hens were inoculated with H9N2 AIV through combined intra-ocular and intra-nasal routes. After infection, pathological changes and tissue apoptosis of challenged hens were detected by haematoxylin and eosin(H&E) and TUNEL staining as well as caspase-3 enzye activity detection, respectively. Kinetics of virus replication in challenged hens were also examined by Real-time PCR and NP protein staining of H9N2 AIV. Real-time PCR was further carried out for quantitation of mRNA expression of cytokine, chemokine, TLRs and MDA5. Immunofluorescence was employed to detect the CD3+CD4+ and CD3+CD8α+ lymphocytes in magnum. The results as follows:1. Birds were randomly divided into two groups. The first group of birds was used as uninfected controls, and birds in the second group were inoculated with H9N2 AIV. At 1, 3, 5, and 7 days post inoculation(p.i.), six hens were selected at random from each group, euthanased, and oviduct samples, including infundibulum, magnum, isthmus, uterus, and vaguna were collected and processed for further analysis. Real-time RT-PCR and nucleoprotein(NP) staining indicated that H9N2 AIV had spread through blood circulation and that the virus is capable of replication in the oviduct, especially in magnum. HE staining showed degeneration, necrocytosis or fallen off in the magnum and isthmus, glandular epithelial cells in lamia propria. In uterus, the mucosal epithelial cell layer became thinner, with cells showing signs of degeneration, necrocytosis or had fallen off. In the vagina, the mucosal epithelial cells also showed cellular degeneration, necrocytosis and edema, or fluid collection within tissues. In addition, scattered lymphocytes were found in the lamina propria of the infected infundibulum and uterus.2. TUNEL detection and caspase-3 enzyme activity examination were employed to detect apoptosis in infected egg-laying hens. TUNEL detection showed H9N2 AIV induced cellular apoptosis in the lamina propria of the five oviductal segments, and it is obvious in magnum and uterus. Caspase-3 enzyme activities of each of experimental groups were higher than the control group. Caspase-3 enzyme activity examined and the TUNEL assay confirmed H9N2 AIV induced apoptosis in the oviducts of hens. Moreover, apoptosis was predominant in the magnum and uterus.3. Real-time RT-PCR was carried out to exam the Toll like receptor(TLR3/TLR7/TLR21), MDA5, cytokines(IL-2, IFN-α, and IFN-β), chemokines and chemokine recetors(CXCLi1, CXCLi2, XCL1, XCR1, CCR5) mRNAs. Different mRNA expressions of TLRs and MDA5 in five oviductal segments were detected in this study. IFN-β, IL-2 was up-regulated with fluctuating mRNA levels while the IFN-α was significantly decreased at different time points in the five segments. IFN-β was significantly increased in five parts of the oviduct. The mRNA expression of CXCLi1, CXCLi2, XCL1, XCR1, and CCR5 increased in five parts of the oviduct during the whole infection process, especially significant in magnum.4. Alterations of the lymphocyte subpopulations during the infection of H9N2 in magnum were investigated by immunofluorescent detection. Infiltration of CD3+CD4+ and CD3+CD8α+ cells in the magnum was greater in the treatment groups. The CD3+CD8α+ cells were more aggregated than that of CD3+CD4+ cells in the bottom layer of the mucosal epithelium and in lamina propria.In conclusion, this study confirmed the virus replication in hens’ oviduct. Also, AIV infection lead to oviductal celluar apoptosis and pathological changes. Futhermore, H9N2 AIV induced differential immune-related genes expression and CD3+CD4+, CD3+CD8α+ lymphcytes infiltration. These results shed more light on theoretical foundation for the oviductal dysfunction of egg-laying hens infected with H9N2 AIV.