Molecular characterization of Marek's disease virus in a poultry layer farm from Colombia
Sara López-OsorioDiego PiedrahitaMaria A. Espinal-RestrepoGloria RamírezVenugopal NairSusan M. WilliamsSusan J. BaigentCésar Ventura-PoliteDiego A. Aranzazu-TabordaJenny J. Chaparro-Gutiérrez
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Abstract:
Marek's disease (MD) is a lymphoproliferative disease caused by an Alphaherpesvirus, genus Mardivirus, serotype 1 (Gallid Herpesvirus 2, GaHV-2) that includes all known pathogenic strains. In addition to Marek's disease virus (MDV) serotype 1, the genus includes 2 distinct nonpathogenic serotypes: serotype 2 (GaHV-3) and serotype 3 (Meleagridis Herpesvirus 1, MeHV-1) which are used in commercially available vaccines against MD. As a result of vaccination, clinical signs are not commonly observed, and new cases are usually associated with emerging variant strains against which the vaccines are less effective. In this study, a commercial layer farm showing clinical signs compatible with MDV infection was evaluated. Histological lesions and positive immunohistochemistry in the sciatic nerve and thymus were compatible with cytolytic phase of MD. GaHV-2, GaHV-3 and MeHV-1 were identified by PCR and qPCR in blood samples from 17 birds with suspected MD. Analysis of the Meq gene of the Colombian GaHV-2 isolate revealed a 99% sequence identity with Asian strains, and in the phylogenetic analysis clustered with vv+ MDV. The analysis of amino acid alignments demonstrated an interruption of the proline rich region in P176A, P217A and P233L positions, which are generally associated with vv+ strains. Some of these changes, such as P233L and L258S positions have not been reported previously. In addition, primary cell cultures inoculated with lymphocytes isolated from the spleen showed typical cytopathic effect of GaHV-2 at 5 d post infection. Based on the molecular analysis, the results from this study indicate the presence of vv+ MDV infection in commercial birds for the first time in Colombia. It is recommended to perform further assays in order to demonstrate the pathotype characteristics in vivo.Keywords:
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Summary Antisera produced in rabbits against Marek's disease lymphoma cells and against four lymphoma‐derived cell lines (HPRS lines 1 and 2, MSB‐1 and RPL‐1) appeared, after extensive absorption with normal chicken cells, to react specifically by indirect immunofluorescent staining with antigens unique to Marek's disease tumour cells. The antigens present on the four cell lines were shown by antiserum titration and absorption tests to be related but not identical. The possibility is discussed that cellular transformation by Marek's disease virus involves the modification of a normal lymphocyte antigen and the modification of different allotypes of this antigen results in related but not identical neoantigens.
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Source of isolation. The source of isolation was two White Leghorn hens (85 weeks old) from a commercial flock with typical ocular form of MD. Only one eye of each bird was affected. Aqueous humor was collected aseptically with a 1-ml syringe and a 23-gauge needle from the affected eye of each bird following electrocution. The cornea was swabbed with alcohol cotton and then flamed prior to collection of aqueous humor.
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The investigated 16th and 45th in vitro passages of non-pathogenic variant 83 of the Kekava strain Marek's disease virus have led in chickens to resistance to Marek's disease by introduction of the above-mentioned virus 14 days before application of pathogenic variant 55 of the Kekava strain Marek's disease virus. Simultaneous administration of both variants of the Kekava strains Marek's disease virus did not protect chickens from the disease. Presence in those variants of the Kekava strain Marek's disease virus of genetic markers manifesting themselves on passaging the virus in chicken fibroblast cultures created the possibility to investigate interrelations between them in the organism of chickens, utilizing in isolation of the virus the method of infecting cultures with chicken fibroblasts. The results of isolation of the virus from the blood cells of vaccinated chickens have shown that in their organism there is interference between those virus variants since the frequency of isolation of the pathogenic virus variant was 3-times lower than that of the apathogenic Kekava strain Marek's disease virus, and both virus variants persisted in various cells. After simultaneous administration of both virus variants to chickens equal amounts of the pathogenic and of the apathogenic Kekava strain Marek's disease virus were isolated from their blood cells. In that case also persistance of both virus variants in one cell may occur.
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The role of pp38 in the pathogenesis of Marek's disease (MD) has not been fully elucidated. Previously, we reported the presence of two splice variants (Spl A and Spl B) for pp38. We also reported that the wild-type pp38 (WT), as well as the mutated pp38 (MUT), altered the oxidative phosphorylation pathway in infected cells. To determine whether the different forms of pp38 are important for the pathogenesis of MD, we generated RB-1B-based bacterial artificial chromosome (BAC) clones expressing pp38MUT, pp38Sp1 A, and pp38Spl B. Infectious viruses were recovered from these BAC clones in chick kidney cells (CKC). The Spl A and Spl B viruses had significantly smaller plaque sizes and replicated to a lesser degree in CKC than the WT and MUT viruses. Two in vivo experiments were conducted by inoculating 7-day-old P2a chicks with 1000 plaque-forming units of each virus. In the first experiment, chicks were sacrificed at 4, 8, 11, and 15 days postinfection (PI). WT and MUT viruses had similar viremia levels using virus isolation and quantitative real-time PCR (qPCR) assays, whereas Spl A and Spl B viruses had significantly lower viremia levels than WT and MUT viruses. In the second experiment, we showed that tumor development and MD mortality were similar in the WT- and MUT-infected chickens, with all birds MD positive at 5 wk PI. In contrast, chickens infected with Spl B and Spl A had a significantly lower MD incidence at 11 wk PI, when the experiment was terminated.
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Skin tissues were obtained from 12 chickens showing Marek's disease (MD) skin tumour induced by a very virulent MD/5 strain of Marek's disease virus serotype 1 (MDVl) and taken from 24 field broiler chickens (8 to 9-weeks-old) manifesting MD skin tumour. They were examined by the immunohistochemical method in order to examine the topographic expression of MDV1-specific phosphorylated proteins (PP) and by the reverse-transcription polymerase chain reaction following Southern hybridization to analyse their gene transcriptional activity. PP were massively detected and their gene transcripts were abundantly expressed only in the necrotic areas of some experimentally-induced MD cutaneous lymphoma lesions. Strong PP-positive cells were degenerate or necrotic lymphoblasts. The other field or experimentally-induced MD cutaneous lymphoma lesions, even those consisting almost completely of tumorous lymphoblasts, had only a few PP-positive lymphoid cells and few gene transcripts. Necrotic areas of feather pulp MD lymphoma lesions consisting mainly of degenerate or necrotic lymphoblasts and cytolytic feather follicle epithelium (FFE) were also strongly positive for PP. PPs and their gene transcripts were detectable only in the cytolytically infected cells such as FFE and tumorous lymphoblasts converting to the abortic state.
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Vaccination with herpesvirus of turkey (HVT) vaccine provides protection against clinical Marek's disease (MD) but does not preclude infection with wild-type MD virus (MDV). The quantity of MDV detected in circulating lymphocytes during the early period after infection may be a useful predictor of subsequent clinical MD later in the life. A study was designed to quantify MDV and HVT copy number in peripheral blood lymphocytes (PBL) using real-time polymerase chain reaction between days 5 and 35 post-challenge and to relate this to subsequent development of gross MD lesions. Female commercial broiler chickens were vaccinated with HVT or were sham-vaccinated at hatch, then challenged with MDV strain MPF-57 at day 2 post-vaccination and reared in positive-pressure isolators up to 56 days post-challenge, when all survivors were euthanized. All dead and euthanized chickens were examined post mortem for gross MD lesions. Birds were scored for MD lesions and mortality. MDV and HVT genome copy numbers were determined for each PBL sample. There was an increase in HVT load in PBL between days 7 and 37 post-vaccination, with marked increases between days 7 and 16 and again between days 30 and 37. There was a steady increase in MDV load to 35 days post-challenge. The mean MDV copy number (log(10)) was greater in chickens subsequently exhibiting gross MD lesions (5.05 +/- 0.21) than in those that did not (2.88 +/- 0.223), with the largest difference at 14 and 21 days post-challenge (P < 0.001). Quantification of MDV during early infection is therefore a potential tool for monitoring MD in broiler flocks.
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The results of 50 clinical trials conducted in 7 countries with a cell-associated Marek’s disease vaccine* have been summarized. Over 93,000 controls and more than 70,000 vaccinated layer chickens have been observed for the first 16 weeks, or more, of age. The median Marek’s disease mortality was 6.0% in the controls and 0.9% in the vaccinates giving a reduction in mortality of 85%. The route of administration, whether subcutaneous, intraperitoneal or intramuscular, appeared to have no effect on efficacy. Mortality due to causes other than Marek’s disease was decreased in the vaccinated birds in 25 of 30 trials in which this observation was recorded. The median decrease in mortality due to causes other than Marek’s disease was 35%. DEPTAVAC-HVT has also been used in broilers. In 18 field trials involving over 217,000 controls and 225,000 vaccinates, the median percentage decrease in condemnations due to Marek’s disease in the vaccinates was 79%. In the same trials the median weight gain and corrected feed conversion (corrected for condemnations) were improved in the vaccinates by 1.7% and 6.8%, respectively. In addition to providing an excellent vaccine for the poultry industry, the development of a vaccine for Marek’s disease opens a new area for research in oncology. A key question which remains to be answered is: why are most chickens infected with Marek’s disease virus while only a relatively few show manifestations of the disease.
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