Isolation and molecular characterization of chicken Marek’s disease virus in selected districts of Ethiopia
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Date
2017-06
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Abstract
A cross sectional study was conducted on clinically diseased chickens suspected of Marek's disease (MD) virus infection in a backyard and commercialized poultry farms from November, 2016 to May, 2017 in selected districts of Ethiopia. Clinical and post-mortem examination was conducted on 33 clinically diseased chickens suspected of MD virus infection and thus virus isolation on chicken embryo fibroblast (CEF) cell and molecular and phylogenetic characterization of Marek‟s disease virus were conducted. Typical clinical signs of MD were observed in all of the 33 clinically examined chickens. Out of the 10 autopsied chicken gross lesions were observed on 8(80%) chicken on the liver and heart as well as enlargement of spleen were observed. 165 feather follicles and 33 spleen samples were collected from the 33 clinically examined chickens under sterile condition and virus isolation was performed using CEF cell culture. Out of 7 pooled spleen samples, viruses were isolated from 5 (71.4%) preparations and also out of 10 pooled feather samples, viruses were isolated from 9 (90%). All the isolates that were grown and developed CPEs shows visible small plaques on CEF cell cultures starting from the 4th days of 3rd blind passage. Further confirmation of the virus was conducted by conventional polymerase chain reaction using MD virus specific primers targeting to amplify the ICP4 gene and Meq gene on 7 and 5 pooled samples respectively. The expected band sizes of 318bp as well as 600bp were amplified by 6 and 5 of the pooled samples out of 7 and 5 pooled samples respectively. Positive PCR products were further subjected to sequencing using the amplification MDV1OP forward and backward primers and the sequence analysis result using MEGA6 bioinformatics software confirmed that the chickens were infected with MD virus. Phylogenetic tree construction revealed that the current sequenced isolates were clustered together with Gallid herpes virus 2 serotype 1 MDV. Further study on the isolation and molecular characterization of chicken Marek‟s disease virus in all regions of the country were recommended.
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Keywords
Marek’s disease, Marek’s disease virus, molecular characterization, polymerase chain reaction