Detection and differentiation of Marek's disease virus serotype by polymerase chain reaction (PCR) and characterization by DNA sequencing of the PCR product
Kalyani, Irsadullakhan Habibullakhan (2006) Detection and differentiation of Marek's disease virus serotype by polymerase chain reaction (PCR) and characterization by DNA sequencing of the PCR product. PhD thesis, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat, INDIA.
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Marek’s disease (MD) is a lymphoproliferative disorder of chicken characterized by oncogenic transformation of T cells that infiltrate lymphoid tissues, peripheral nerves and visceral organs, resulting in a complex pathogenesis that usually leads to death of the affected chicken. The causative agent of MD, Marek’s disease virus (MDV) a cell associated herpesvirus, is ubiquitous in almost all poultry population raised under commercial conditions. The present study was under taken to assess incidence of MD as per the farm records on the basis of clinical signs and post mortem lesions, detection of MDV from clinical cases exhibiting frank lesions of MD by PCR, comparing the efficacy of different primers in detecting the MDV as well as differentiating the serotypes in tissues and feather follicle and genetic characterization by sequencing of the PCR amplified fragment carrying 132 bp repeats. During June to November 2005 outbreaks of MD were suspected in flocks of egg type birds on farms situated in and around Anand district located of central Gujarat as well as from farms around Mahuva located in Saurastra region of Gujarat state. The strength of individual affected flocks varied from 4500 to 25000 birds with a total population of 4,26,991 birds. Of these, 14110 birds died due to MD (as per the farm records of history, symptoms and postmorterm lesions) with an over all mortality of 3.30 per cent during the months of June to November 2005. During the investigation, a total of thirty six commercial poultry farms and forty nine batches of layer birds suffered from mortality due to MD, in addition to Central Poultry Research Station (CPRS) Veterinary College Anand, where mortality occured in layer birds. Mortality in commercial poultry farms ranged between 0.6 to 7.3 per cent. A total of 34 clinical samples (17 feather follicles and 17 spleen ) from 27 birds suspected of MD were collected by making visits to the various commercial poultry farms and also from CPRS Veterinary College Anand. Suspected clinical samples and four MD vaccine strains, including three HVT (MDV- 3) vaccines and one SB-1(MDV-2) vaccine were processed for DNA extraction. DNA was extracted from samples of feather follicle using proteinase K mixture, where as DNeasy Tissue Kit (QIAGEN Pvt. Ltd) was used for tissue samples. Sample showing acceptable purity and concentration were subjected to PCR amplification using six different primers. Total of six pairs of primers were used, which were selected to amplify different gene segments. The primer pair BamH1/BamH2 specifically amplifying a 434 bp segment from BamHI fragment (of MDV-1) containing 132 bp repeats located in TRL and IRL region of MDV genome, detected 32 samples positive indicating presence of double 132 bp repeats in the amplified segment. All the three HVT vaccines and one SB-1 vaccine failed to produce the amplification as the selected primer is specific for MDV-1 only. Primer pair AGAM1/AGAM2 and P1/P2, both targeting the antigen A gene (UL44) produced 314 bp and 199 bp products respectively in 30 out of 34 clinical samples tested, however, none of the HVT vaccine or SB-1 vaccine yielded positive result, suggesting the specificity of these primers for MDV-1. Primer set M1.1/M1.8 amplified a 318 bp product as against expected 247 bp product in 30 samples. To confirm the result, these primers were subjected to NCBI BLAST, and it was found that the primer specific segment of 318 bp does exist in published sequence of Md5, and Md11BAC. None of the HVT as well as SB-1 vaccine produce any amplification indicating the specificity of primer in detecting MDV-1. Only two field samples produced expected amplicon of 505 bp when tested with MDV- 3 specific HVT1/HVT2 to detect the vaccine virus (HVT). All the three HVT vaccines produced the amplicon of 505 bp, whereas SB-1 vaccine was negative, proving specificity of this primer pair for HVT. Out of the six primer pairs, BamH1/ BamH2 and AGA1/AGA2 detected MDV in maximum number (32) of field samples. Primers AGAM1/AGAM2, P1/P2 and M1.1/M.1.8 detected the same number (30) of samples positive. Primer pair HVT-1/HVT-2 detected MDV- 3 in two feather follicle samples indicating possible excretion of the vaccine virus. PCR products (434 bp) of MDV genome amplified by BamH1/BamH2 primers from two field samples (M3, M4) having good quality DNA (i.e.1.7-1.9 OD at 260/280 nm) as determined by spectrophotometry were used for sequencing by ABI PRISM® 310 Genetic Analyzer (Applied Biosystems, USA), using BigDye® Terminator v3.1 Cycle sequencing kit (Applied Biosystems, USA). The sequence obtained revealed two 132 bp repeats in the 378 bp stretch indicating virulent nature of the field MDV. The alignment of 378 bp sequence of M3 and M4 with previously published sequences of the MDV isolates Md5, Md11, HPRS-16 and CDs of tumourogenicity associated m-RNA and CDs published from BamH gene family revealed that the field isolates of present study shared complete homology (100%). Results of sequencing along with history of vaccination in the affected birds and involvement of different organs on postmortem examination indicated about the increase in virulence of MDV circulating in field causing recent outbreaks.
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