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Relative expression of interferon-gamma in the spleen and the bone marrow of SPF chicken embryos after in ovo stimulation with La Sota strain of Newcastle disease virus

Introduction: Newcastle disease (ND) is a highly contagious viral disease of poultry and wild birds and remains the most economically important viral disease of poultry in spite of effective vaccination regimes applied worldwide. The control of ND is performed by application of live and inactivated vaccines of various strains by different routes. Cellular and humoral response to NDV infection and vaccination has been well characterised. Currently the molecular signalling involved in stimulation and regulation of these responses is being intensively investigated by various molecular methods (e.g. imunohystochemistry, q RT-PCR). The immune system of birds is basically similar to the mammalian one, with T- and B- lymphocytes and antigen presenting cells being the main effectors. As in mammals, main signalling molecules involved in immune response are proteins, namely immunoglobulins and cytokines (interferons and interleukins). The principal cytokines involved in response to viral infections of chickens are interferon-alpha (IFN-alpha), IFN-gamma, IL-1beta, IL-2, IL-6 and IL-12. Interferon-gamma is produced by CD4 Th1, CD8 CTL, NK and NKT cells, but also in chicken fibroblasts as a response to viral antigens. In birds, spleen plays an important role in coordination of immune response and bone marrow is an important source of lymphocytes. However, the development of spleen and bone marrow function during embryogenesis and in early neonatal life is still poorly described. This study aims to reveal the NDV pathogenesis and possible molecular signalling involved in induction of immunocompetence development in chicken embryo. Methods: In this study the total of 15 SPF embryonated chicken eggs (ECE) were used. At the 11th day of incubation eggs were inoculated into their allantoic sacs with the live NDV (LS group of 5 ECE) at dose of 10^5.0 EID_50 or inactivated NDV (ILS group of 5 ECE). The eggs of control group (C) were not treated. Applied viruses were obtained from commercial live, lentogenic ND vaccine PESTIKAL® La Sota SPF (Genera, Croatia) and part was inactivated using UV. Following 72 hours incubation at 37 °C, the embryos were chilled and their spleens and bone marrow were collected. Total RNA was extracted from collected samples using TriReagent (Sigma-Aldrich, Germany) and the RNA pellet was dissolved in RNase-free water. Relative quantification of IFN-gamma mRNA in samples was done in duplicate by real-time RT-PCR using TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems, USA) and normalised with b-actin mRNA and mRNA from control samples. Calculations were done in silico. Results: Transcripts of IFN-gamma were detected in all spleen and bone marrow samples obtained at 72 hours post inoculation from both, ECE treated with live NDV La Sota virus as well as in samples obtained from ECE treated with inactivated NDV La Sota virus. The relative expression of mRNA for IFN-gamma at 72 hours post inoculation in both, spleen and bone marrow samples obtained from ECE treated with live NDV La Sota virus was significantly greater in comparison to control group and greater than in samples obtained from ECE treated with inactivated NDV La Sota virus. Conclusions: Based on the results of IFN-gamma relative expression, we can conclude that the chicken embryos are capable of an immune response after stimulating with a certain pathogen or immunogen and therefore are sufficiently developed already on the 14th day of embryogenesis. The abundance of relative IFN-gamma expression can be attributed to the stimulation of live NDV which seemed impaired by inactivation of virus. Further research of immune signalling during embryogenesis should contribute to an understanding of synergy in development of immunocompetence in chickens and other bird species.

Newcastle disease; in ovo; SPF chicken embryoes; relative expression; interferon-gama

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