The Molecular Mechanism Of Artificial Infection Of Newcastle Disease Virus From Duck On The Immune Function Of Ducks

Newcastle Disease(ND) is one of Newcastle disease Virus(NDV) that causes an acute,highly contagious respiratory disease, which continues to have serious health and economic impacts on the poultry industry despite the existence of vaccination programs. Traditional theory considers that the duck is not sensitive to NDV, while with the NDV evolution, there is a risk to lead duck ND prevalent. Due to the irregulated immune procedure, it is difficult to control the disease. As a result, in order to improve duck immune condition, reduce the risk of the pathogen infection(such as NDV), and protect duck healthy, it is important for us to understand the signal transduction mechanism of host antiviral role.Thirty-nine 20-day-old SPF Chinese Shao ducks were used and separated randomly into two groups, 27 ducks in group one and the other 12 ducks in group two. Ducks in group one were inoculated intranasally with a NDV strain(108.38 EID50 of Md/CH/LGD/1/2005). Ducks in the group two were mock inoculated with phosphate-buffered saline(PBS) as the control. The ducks were monitored for clinical signs for up to 24 days post infection(dpi), and 6 ducks were killed at24 hpi and 48 hpi, respectively. Nine tissues, including spleen, lung, kidney, glandular stomach,bursa of Fabricius, cecal tonsil, trachea, Harderian glands, and marrow, were collected from ducks for quantitative PCR(q PCR) analysis of host genes. One Step real-time Prime Script reverse transcription-PCR(RT-PCR) was used to measure immune related genes, including toll-like receptors(TLRs), β-defensins(AvBDs), cytokine, apoptosis and other immune factors. In addition,serum were collected and specific antititres was evaluated at 4, 8, 12, 16, 20, and 24 dpi,respectively. Nasopharyngeal and cloacal swabs were collected and specific antititres was evaluated at 24, 48, 72 hpi and 5 dpi, respectively.The present result showed that similar to the control ducks, no obvious clinical signs were observed in the NDV infected ducks during the experiment. All ducks inoculated with the NDV strain showed a positive serum antibody response from 4 dpi to 24 dpi.To confirm the presence of NDV in the infected ducks, viral loads in nasopharyngeal and cloacal swabs of ducks inoculated with Md were detected from 1 dpi to 5 dpi.The results showed that AvBD1, AvBD5, AvBD9, AvBD16 were detectable in most tissues investigated from both the control and infected ducks. Furthermore, we detected significantly high level of AvBD5 expression in the cecal tonsil of NDV infected ducks at 24 hpi(P<0.05).For Av BD16, the expression level was increased in the lung and spleen of NDV infected ducks at 24hpi(P<0.05). It was shown that little TLR4 was detected from all of tissues of both the control and infected ducks. Compared with the control, the expression level of TLR2, TLR3, TLR7 were significantly increased in the spleen of NDV infected ducks(P<0.05). We observed that RIG-I was detectable in all of tissues investigated from both the control and infected ducks. Furthermore,high level of RIG-I expression was detected in the spleen, cecal tonsil and Harderian glands of NDV infected ducks at 24 hpi, compared with control group(P<0.05). MyD88 were detected in all of tissue samples, but only significantly upregulated in the spleen, cecal tonsil and Harderian glands in response to the NDV infection(P<0.05). Next, we evaluated the levels of Cytokine,Apoptosis and Immune factors. Fortunately, we found significantly higher level of Interleukin 2(IL-2) expression in the Harderian glands of NDV infected ducks than those of control ducks(P<0.05). IL-6 expression was increased in the spleen of NDV infected ducks(P< 0.05).Compared with the control, the expression level of IFN-γ was significantly increased in the kidney of NDV infected ducks(P<0.05). The expression level of extracellular regulated protein kinases 2(ERK2) and cysteinyl aspartate specific proteinase 3(Caspase-3) were increased in the spleen of NDV infected ducks(P<0.05).In conclusion, this study demonstrates that specific antibody could be induced as early as 4dpi by experimental infection with NDV. The mRNA expression levels of Av BD5 and Av BD16 were significantly increased in some tissues in response to the NDV infection.Compared to the control, expression of TLRs(2, 3 and 7) were significantly increased in spleen of infected ducks(P<0.05). Furthermore, the expression level of RIG-I was significantly increased in the spleen,cecal tonsil and Harderian glands by 24 hpi(P<0.05). Moreover, expression levels of IL-2, IFN-γ,IL-6, MyD88, ERK2, and Caspase 3were significantly increased in some tissues of ducks in response to NDV infection(P<0.05).