The Distribution Of Newcastle Disease Virus, Toll-Like Receptors Expression And Lymphocyte Infiltration In Hens Oviduct Infected With Velogenic Genotype Ⅶd NDV

Newcastle disease(ND) is an important disease hazardous to poultry industry, and ND virus(NDV) is the etiological agent. It not only causes the death of chickens but also causes serious damage to the reproductive tracts of egg-laying hens. This leads to an overall decrease in egg production and increases the percentage of deformed, sand-shelled, and soft-shelled eggs. Furthermore, the reproductive tract is a target of both the vaccine strain and field isolates of NDV. Epidemiological studies have revealed that genotype VII viruses circulating predominantly in China and in some other Asian counties in the past decade are responsible for disease outbreaks in chicken and goose flocks. In recent years, most of the researchs dedicated to genotype ⅦNDV cause immune organ disorders. Studies investigating the mechanism underlying the more severe pathological damages in the oviducts of egg-laying hens after NDV infection are limited, so the innate immunity roles of local oviduct infected with the velogenic genotype VIId NDV strain played a critical role in viral pathogenesis. These were investigated using 40-week old egg-laying hens inoculated with the velogenic genotype VIId NDV strain through combined intra-ocular and intra-nasal routes. Clinical signs and pathological lesions in the oviducts of egg-laying were observed infected with the velogenic genotype VIId NDV; The real-time RT-PCR was used to detect the NDV viral loads in different sections in oviduct; Real-time PCR was carried out for quantitation of mRNA expression of TLRs and MDA5; Immunofluorescence was employed to detect the CD3+CD4+ and CD3+CD8α+ lymphocytes in magnum during viral infection. The results as follows:1. Forty-week old specific-pathogen-free(SPF)White Leghorn egg-laying hens inoculated with genotype VIId NDV. Five birds from each group were sacrificed on days 1, 3, 5, 7, 9, 11 and 15 post-infection(dpi), and various parts of the oviduct were sectioned. Standard curves of NDV M gene were construsted by real-time RT-PCR, the correlation coefficient(R2) was 0.9979. According to standard curve, the differences in viral replication in the oviduct of egg-laying hens were evaluated after NDV infection. NDV were detected in every section of oviduct after NDV infection. The viral load reached the peak at 5 dpi. The magnum presented the highest amount of virus load(8918.6 copies/μL), and followed by the uterus(2934.6 copies/μL). Positive staining of the NDV HN protein was observed in five part of the oviducts. HN proteins were observed in the glandular epithelial cells of the lamina propria and in the submucosa.2. Histopathological examinations found that obvious pathological changes were presented in magnum and uterus infected with genotype VIId NDV. In the magnum,the gland showed congestion, hemorrhage or dissolution. The mucosal epithelial cell layer became thinner. In the uterus, edema of the tubular glands, scattered lymphocytes in lamia propria. In addition, the mucosal epithelial cells showed necrocytosis and fallen off. Real-time quantitative PCR assay was used to quantify the expression profiles of TLR3(7, 21)and MDA5 genes in the magnum and uterus of the oviduct of SPF egg-laying hens infected with genotype VIId NDV. The mRNA levels of TLR3, 7, and 21 were altered in the oviducts of the infected laying hens and were upregulated in the magnum and uterus tissues. The fold changes in their mRNA expression levels in the magnum segments peaked at 5 dpi(42.6-, 98.9-, and 27.1-fold, respectively).3. Immunofluorescence was employed to detect the CD3+CD4+ and CD3+CD8α+ lymphocytes in magnum during viral infection. The accumulation of CD3+CD4+ and CD3+CD8α+ lymphocytes in the magnum was greater in the experimental than in the control group throughout the course of infection. The CD3+CD4+ and CD3+CD8α+ lymphocytes began to accumulate 1 dpi and peaked 5 and 3 dpi, respectively. CD3+CD4+ and CD3+CD8α+ lymphocytes were distributed in the lamina propria of the magnum. Moreover, CD8α+ cells were recruited into the magnum more deeply than the CD4+ cells. The frequency of CD4+ cells in the lamina propria was significantly higher compared to the bottom of the mucosal epithelium. In contrast, the CD8α+ cells localized predominantly at the bottom of the mucosal epithelium.In conclusion, this study confirmed that the virus replication and survival in hens’ oviduct after genotype VIId NDV infection, which lead to oviductal pathological changes. These result laid a theoretical foundation for the oviductal dysfunction and of egg-laying hens infected by genotype VIId NDV.