الفهرس 
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Abstract
Marek’s disease (MD) is a highly destructive lymphoproliferative disease to which all chicken genotypes are susceptible. This disease is caused by an alphaherpes DNA virus called Marek’s disease virus (MDV). The disease is characterized by development of various tumors in infected chickens. Those tumors are usually associated with appearance of the disease clinical symptoms including blindness, paralysis and death. Furthermore, the immune suppression due to cell cytolysis and lymphomas development ( Calnek B W, 2001) results in major economic consequences. MD infection is associated with great economic losses, and it represents a threat to poultry industry due to the sever immune suppressive nature of MDV. Usually, the great economic losses associated with MDV infection are owing to the cost of vaccination, morbidities, mortalities and the increased susceptibility of the infected birds to other infections due to MD induced immune suppression. MDV is highly cell associated and usually targets both B and T lymphocytes during the cytolytic and latent phases of infection respectively. It primary harbors the bursa of Fabricius during the early stage of infection resulting in B cells cytolysis (Schat et al.,1981), then targets the thymocytes resulting in thymic atrophy (Morimura et al., 1996).Thus, those two organs were our target of investigation due to their reported role in MD pathogenesis.
Due to the highly- cell associated nature of MDV, the T cell mediated responses ((Precisely, cytotoxic T – lymphocytes (CTLs)) are proposed to play a more significant role than antibody mediated responses in controlling MDV infection. The cytokines are the secreted products of CTLs and are proposed to play a significant role against MDV infection, shaping the disease pathogenesis (Xing and Schat., 2000a ; Kaiser et al., 2003; Jarosinski et al., 2005; Quéré et al., 2005 and Abdul-Careem et al., 2007) and may possess protective responses following vaccination. Additionally, cytokines are one of the sensitive means in examining the immune responses. Although MD has been controlled by vaccination against MDV, the post vaccines immune mechanisms are still vague. In Egypt, even with intensive vaccination practice in broiler breeder flocks, cases of tumor development still reported. Therefore, in this study, the transcripts of the cellular immune responses of three commercial vaccines used in Egypt (the monovalent Rispens and HVT vaccine and the bivalent Vaccine) were examined, through characterization of their cytokine responses. Studying of cytokines responses following mono and bivalent vaccination help in understanding the role of bursal and thymic cytokines in vaccines-induced immunity. Also, the effect of the bivalent MD- vaccine ”as the most widely used practice in major commercial flocks” on humoral responses of Newcastle LaSota vaccine has been determined. We examined the responses ND as a major threat to the local poultry industry so we used it as an indicator virus for MD-vaccines. A total of One hundred and eighty Avian 48 broilers chick one day old chicks, free from hatchery vaccination were used in the study. One hundred and seventy were assigned into 4 groups. The vaccinated groups were as following; one vaccinated with CVI988; Rispens vaccine, other received HVT vaccine and the third received the bivalent (CVI988 and HVT) vaccine at one day old. The fourth group was mock vaccinated and received only the vaccine diluent. The bivalent group received the LaSota NDV vaccine at 7 days of age (and additional group of only 10 chicks kept as control receiving only the LaSota NDV vaccine). Tissue samples (the bursa of Fabricius and thymus) were taken at 7, 14, and 21 days P.1., were sampled for RNA extraction from MD vaccinated chickens and their mock control only. Also, whole blood was collected and serum separation was done for HI anti-LaSota Ab levels detection at different times post immunizations. Total RNA extraction was carried out from control and vaccinated groups using Trizol reagent (Life Technologies, Ambion). Then the quality of the RNA yield was assessed by agrose gel electrophoresis. DNase treatment and the reverse transcription into single stranded cDNA was done following the manufacturer’s protocols using RQ1 RNase-Free DNase (Promega) and High Capacity cDNA Reverse Transcription kit (Applied Biosystems) respectively. The relative cytokines analysis was determined by real-time polymerase chain reaction (RT-PCR). RT-PCR amplification of different pro-inflammatory and anti-inflammatory cytokines (including IFN gamma, iNOS, IL-1B, IL6 and IL-10) genes were carried out a long with GADPH gene (as internal control) using previously published primers.
The reaction condition for each gene was optimized using a Powerup SYBR®Green PCR master Mix (Life Technologies) in Mx3005P Real-time PCR system (Agilent Technologies, Santa Clara, CA, USA). The cycle threshold (CT) values obtained were used to determine the fold changes in cytokine genes expressions, and the variations in the transcripts expression among different groups were expressed as the fold change compared to the control group using the Livak; 2−ΔΔCT method. Data presented showed up-regulation of the pro-inflammatory cytokines; IFN-γ and IL-1B, and the inflammatory cytokine IL-6, at different time points, with a maintained expression of the signaling molecule iNOS was observed in the treated groups. Meanwhile, up-regulated expression of the anti-inflammatory cytokine, IL-10 was only noticed by 21 days P.I. Those findings indicate that the vaccine strains mimic the natural infection by inducing early inflammatory cell mediated, while devoid its immune suppressive effect. Additionally, vaccination against MD results in persistence of vaccinal strains to interfere with later natural infection and trigger anti-tumor cell mediated responses that hinder the wild virus latency and interfere with tumor development. Additionally, the fold changes in cytokine gene expression were higher at the bivalent vaccinated group followed by those at the Rispens recipient in most of examination times. These observations support previous investigation that indicted the gold standard protective efficacy of the attenuated CVI988 MD viral strain. Also, the non-significant differences between the Rispens alone and the bivalent combination at most of the tested cytokines indicate that the bivalent vaccine can be administered if possibilities of natural infection are high otherwise the Rispens alone can be very efficacious. Similarly, results showed differences in cytokines expression following vaccination depending on the examined tissues, where the thymic tissue showed higher levels of different cytokines than those in bursal tissue. <Finally, HI Anti- LaSota Ab levels in serum of ND vaccinated chicks in the presence and absence of Bivalent MD-vaccination, indicates that the MD vaccination does not interfere or improve the Anti-ND humoral Ab responses in the absence of MDV natural infection.