engleski

Detection of HVT FC 126 in lung tissue of chickens vaccinated by means of nebulization against Marek’ s disease

Marek's disease (MD) is one of the most important diseases that endanger poultry production worldwide today. Every chicken, fattening, parent or layer, which leaves the hatchery must be vaccinated. Never the less, because of the specific characteristics of the virus, protection cloud be broken as vaccination force the viral evolution to more virulent strains. The virulent wild strains despite vaccination could replicate in chicken organism and spread into environment and contaminate it for further generations (Kingham et al., 2001). Further efforts are going toward immunoprotection using recombinant or DNA vaccines that are in faze of development (Baigent et al., 2006) and basics for such research will be shown in this paper. Chicks were divided into four groups with 70 chicks per group. Freeze dried vaccine HVT FC126 against MD, Lyomarex® ; ; , Merial Select Inc., USA, was used for vaccination of newly-hatched chicks. Group A was given vaccine by means of nebulization during 60 seconds using SONOVAC® ; ; 096 (Mazija and Štimac, 1999), while group B received the same vaccine subcutaneously. Control group C received physiological solution by means of nebulisation, and group D was not vaccinated. Lung tissue samples were taken at one, two and four day interval after vaccination for isolation of HVT DNA using DNeasy® ; ; Tissue Kit (Qiagen, USA). Viral DNA, SORF1 region of HVT, was detected by RealTime PCR with specific primers described by Islam et al. (2004) using Brilliant® ; ; SYBR® ; ; Green QPCR Master Mix (Stratagene, USA) on Mx3005P (Stratagene, USA) device and thermal profile as described by the same authors. Data were analyzed using MxPro software (Stratagene, USA). We detected replication in group A in all sampling days with specific pattern of dissociation curve as in positive control (vaccine) with melting temperature (Tm) about 80.5°C. In group B that received vaccine subcutaneously we didn't detect any level of viral replication during first four days after vaccination, as well as in group C and D. Replication in group A started in the late stage of the reaction, between 40 and 50 cycles, getting to plateau after 50 cycles because of low quantity of specific DNA. As vaccine virus is applied deeply into the respiratory system by means of nebulization it is delivered to receptors and cells which are the primary entry of Marek’ s disease virus (MDV) (Schat, 2004). Mimicking natural infection vaccine virus blocks the cells and induces immune reaction in the very early stage which enables protection in the early period of life of chicks from the virulent MDV strains. This research confirms that nebulization could be used as massive vaccination technique already in the hatchery using freeze dried HVT vaccine which is the base for further efforts in developing recombinant vaccines with HVT as vector virus. In this research RealTime PCR is used for detection and quantitation of small amounts of viral DNA and will be used for the control of an early immune reaction detecting expression of cytokine and viral mRNA’ s in the following researches.

Marek's disease ; HVT FC 126 ; RealTime PCR ; poultry

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