The Establishment Of PCR Detection Methods For Marek’s Disease And Isolation Of SD2012-1Strain And Virlence Test

Marek’s disease is a contagious tumor disease caused by cell bindingherpesvirus. With the enhancement of Marek’s disease virus virulence, it causedgrowing threaten to poultry industry. In this study, a fast and accurate PCR methodhas been establishment for detection of Marek’s disease. In2012, a MDV isolate strainwas isolated from a chicken flock in Shandong Province, China, named SD2012-1. Inorder to understand the virulence and evolved features of SD2012-1strain, the studyby the sequencing, analysis and virulence testing, provided the basis of theevolutionary characteristics of part of Marek’s disease virus strains in China, and findthe reasons of immunization failure, provide recommendations for future preventionof Marek’s disease.Specificity and sensitivity comparative studies were carried out in a pair ofdesigned primers and four pairs of published PCR detection primers. All the primerswere found to be highly specific, but their sensitivities were different. A target bangcould be amplified when the DNA concentration reached3.81×10-3μg/mL, so thesensitivity of Meq5primer was better than other four pairs of primers. Detectionresult of tissue samples showed that Marek’s disease virus can be detected in featherpulp, heart, liver, spleen, kidney, glandular and duodenum. This study provides a basisfor epidemiological investigation and detection of Marek’s disease.The Meq gene and vIL-8gene of MDV SD2012-1strain，after the PCRamplified, cloning and sequencing, compared the nucleotide sequences and aminoacid sequences with other20reference strains. The result indicated that SD2012-1was found to have the characteristics of very virulent MDV-1, SD2012-1had thehighest Meq amino acid homology with LS, and had the lowest homology withCVI988. SD2012-1had the same amino acid mutation of Meq gene at position71,77,80,115,119,153,176and217sites, which displayed regularity of strains isolatedfrom China, except no mutation of SD2012-1at position139aa. SD2012-1had thehighest vIL-8nucleotide sequence homology with LS, RB1B and CU-2at99.6%, andhad the lowest homology with CVI988and584A at99.4%. SD2012-1had the amino acid mutation of vIL-8gene at position4and position31, which displayed regularityof strains isolated from China.Virulence tests showed, SD2012-1isolates has very strong virulence, it canbreak the immune protection of HVT vaccines and HVT+SB-1double seedlings,CVI988vaccine can protect chickens in limited extent, but not completely protectionof chickens. It shows SD2012-1isolates with characteristics of very virulent strain ofMarek’s disease. SD2012-1isolate seems to have a longer incubation periods, itusually caused chicken early death in45days after infected by viruses. Chickensvaccinated HVT vaccines first appeared early death in48days after infected byviruses. The main symptoms were swelling and degeneration of some of tissues andorgans, and then it will slowly appeare neoplastic lesions in tissues and organs.