Genetic evolution of Marek's disease virus in vaccinated poultry farms

Background and Aim: The Marek's disease virus (MDV) is a neoplastic disease causing serious economic losses in poultry production. This study aimed to investigate MDV occurrence in poultry flocks in the Lower Egypt during the 2020 breakout and genetically characterized Meq, gL, and ICP4 genes in field strains of MDV. Materials and Methods: Forty samples were collected from different breeds from eight Egyptian governorates in 2020. All flocks had received a bivalent vaccine (herpesvirus of turkey FC-126 + Rispens CVI988). However, weight loss, emaciation, reduced egg production, paralysis, and rough/raised feather follicles occurred. Samples were collected from feather follicles, liver, spleen, and nerve tissue for diagnosis by polymerase chain reaction. MDV genetic characterization was then performed by sequencing the Meq, gL, and ICP4 genes of five positive samples representing different governorates and breeds. Results: A total of 28 samples were positive for MDV field strains, while two were related to MDV vaccinal strains. All samples tested negative for ALV (A, B, C, D, and J) and REV. Phylogenetic analysis of the Meq gene of sequenced samples revealed that all MDVs were related to the highly virulent European viruses (Gallid herpesvirus 2 ATE and PC12/30) with high amino acid (A.A.) identity 99.2-100%. Alternatively, there was low A.A. identity with the vaccine strains CVI988 and 3004 (up to 82.5%). These results indicate that further investigation of the efficacy of current Egyptian vaccines is required. The Egyptian strains also harbor a specific mutation, allowing clustering into two subgroups (A and B). By mutation analysis of the Meq gene, the Egyptian viruses in our study had R101K, P217A, and E263D mutations present in all Egyptian viruses. Furthermore, R176A and T180A mutations specific to our strains contributed to the high virulence of highly virulent strains. There were no mutations of the gL or ICP4 genes. Conclusion: Further studies should evaluate the protection contributed by current vaccines used in Egypt.

Gallid Alphaherpesvirus 2 in the Egyptian Turkeys: Molecular Characterization and Establishment of a Universal System for Phylogenetic Classification

<b><i>Introduction:</i></b> Gallid alphaherpesvirus 2 (GaHV-2) is a highly contagious oncogenic virus that causes Marek’s disease in chickens and occasionally in turkeys. Among 100 genes identified in GaHV-2 genome, the Meq gene appears to involve viral virulence, oncogenicity, and genetic diversity. Despite the use of Meq gene sequences in phylogenetic classification of GaHV-2 strains circulating in many countries worldwide, no integrated system exists yet. <b><i>Methods:</i></b> Turkeys from 2 commercial Egyptian farms were presented with signs of dullness, dehydration, and emaciation. Samples prepared from the internal organs were examined by histopathology and immunohistochemistry. Pools of the internal organs were analyzed by PCR for identification of GaHV-2, avian leucosis virus, and reticuloendotheliosis virus. The Meq gene of an Egyptian strain was sequenced and analyzed in comparison to 40 reference strains for generation of a universal system for phylogenetic classification of GaHV-2 strains. <b><i>Results:</i></b> Gross and histopathological examination revealed grayish-white soft masses in the internal organs characterized by diffuse infiltration of pleomorphic neoplastic cells. All lymphoma cells were identified as T-lymphocytes of CD3+ phenotype. Samples of both farms were only positive for GaHV-2 by PCR. Sequence analysis of the Meq gene has classified the current turkey strain as related to the Egyptian strains identified in chicken in 2012. A universal phylogenetic system for classification of GaHV-2 strains into 4 clusters was proposed. The vaccine strains were all grouped in cluster 2, and most of the classical American strains belonged to cluster 4. Cluster 1 was further divided into 3 subclusters (1.1–1.3). <b><i>Conclusion:</i></b> GaHV-2 was identified in turkeys for the first time in Africa and the Middle East. Sequence analysis of the Meq gene of the Egyptian strain along with a wide array of the global strains has enabled the construction of a novel phylogenetic classification system.

Pathological investigation of Marek’s disease in a pullet farm of Bangladesh and detection of Marek’s disease virus by pcr

Marek’s disease (MD) is a lymph proliferative disease of chickens, characterized by progressive emaciation, morbidity and mortality. The causative agent is a cell associated oncogenic alpha-herpes virus. This study investigated an outbreak of MD in a pullet farm (N=2200) of Ramu Upazilla, Cox’s bazaar during May 2016 vaccinated against MD. The infectivity was reported on day 45 of age. Birds (N=10) submitted to diagnose disease at necropsy in the Department of Pathology, Bangladesh Agricultural University showed prominent keel bone, asymmetric progressive paralysis of one or both of the legs and wings. At necropsy, the skeletal muscle appeared thinner and there was enlargement of liver, spleen, kidney and sciatic nerve. Impression smears prepared from the liver showed huge infiltration of lymphocytes. Sections of heart, lungs, liver, kidney, nerve, skin and spleen were stained with hematoxylin and eosin showed wide spread infiltrations and accumulation of lymphocytes. Lymphocytic infiltration was seen in the skin, nerves and all visceral organs and showed combined infectivity due to visceral and classical forms. The etiology of MD was confirmed by using polymerase chain reaction (PCR) targeting fragment of Meq gene of very virulent plus or very virulent MDV1. Results of PCR showed amplification of 317bp fragment of Meq gene suggestive for infectivity due to MDV1GA (Md/5) strain. It requires isolating viruses in culture to test further for its virulence and pathotype in vivo. Sequencing and phylogenetic analysis of Meq gene may unveil the pathotype of the virus involved.SAARC J. Agri., 16(1): 181-192 (2018)

Visceral Marek's disease in white-peafowl (Pavo cristatus)

ABSTRACT Marek's disease (MD) is a lymphoproliferative disorder caused by Gallid herpesvirus 2 (MDV) that infects mainly domestic gallinaceous birds although wild birds may occasionally be affected. The current report describes the anatomopathological and molecular findings of a case of MD in a white-peafowl (Pavo cristatus). The signs included apathy, hyporexia, and diarrhea. Grossly, 0.5 to 1.5cm in diameter, yellow, soft nodules were observed in the skeletal muscle, lung, kidney, air sacs, small intestine, heart, ovary, ventriculus, and proventriculus. Microscopically, numerous atypical round neoplastic cells were noted. The molecular detection of MDV DNA was implemented to amplify part of the meq gene and products were sequenced for the phylogenetic analysis. Template DNA was obtained from tissues of the affected bird and from blood of all the gallinaceous birds of the Zoo. The expected amplicon for the partial amplification of MDV meq gene was obtained and the amplicons were sequenced. Sequences obtained enabled grouping the strain (accession no. KT768121) with MDV serotype 1 strains from the GenBank. Based on the anatomopathological and molecular findings, the diagnosis of MD in a white-peafowl was reached, and to the authors' knowledge, no previous report regarding MD was published in Pavo cristatus.