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.

Molecular epidemiology of Marek’s disease virus in central Pennsylvania, USA

The evolution of Marek’s disease virus (MDV, Gallid herpesvirus 2) has threatened the sustainability of poultry farming in the past and its continued evolution remains a concern. Genetic diversity is key to understanding evolution, yet little is known about the diversity of MDV in the poultry industry. Here, we investigate the diversity of MDV on 19 Pennsylvanian poultry farms over a 3-year period. Using eight polymorphic markers, we found that at least twelve MDV haplotypes were co-circulating within a radius of 40 km. MDV diversity showed no obvious spatial clustering nor any apparent clustering by bird line: all of the virus haplotypes identified on the commercial farms could be found within a single, commonly reared bird line. On some farms, a single virus haplotype dominated for an extended period of time, while on other farms the observed haplotypes changed over time. In some instances, multiple haplotypes were found simultaneously on a farm, and even within a single dust sample. On one farm, co-occurring haplotypes clustered into phylogenetically distinct clades, putatively assigned as high and low virulence pathotypes. Although the vast majority of our samples came from commercial poultry farms, we found the most haplotype diversity on a noncommercial backyard farm experiencing an outbreak of clinical Marek’s disease. Future work to explore the evolutionary potential of MDV might therefore direct efforts toward farms that harbor multiple virus haplotypes, including both backyard farms and farms experiencing clinical Marek’s disease.

Comparative study on biological material for molecular diagnosis of Gallid herpesvirus 2

The present study was intended to investigate the sensitivity of different tissues of tumorous and non-tumorous birds to the polymerize chain reaction-baseddiagnosis of Marek’s disease. Tissue samples were collected from spleen, sciatic nerve, feather follicles and tumour parts (if any)of chickens during the routine necropsy examination. The presence of Marek’s disease virus was examined using primers for 132 bp repeats of BamHI-H regionand positive samples were sequenced for 132 bp repeats and Meq gene. In the birds showing tumours, various samples such as the spleen, feather pulp, sciatic nerve and tumour tissue yielded positive results in 92.31%, 88.46%, 100% and 100% cases respectively. Amongst non-tumorous birds, 11.53% spleen, 23.07% feather pulp and 11.53% sciatic nerve were found positive. This is the first comparative study between nerve and other organs samples for molecular diagnosis of Marek’s disease. Data generated shows that the probability of false negative in tumorous birds and false positive in non-tumorous birds was comparatively low while employing nerve tissue than spleen and feather follicles. Thus, it may be concluded from our study that nerves are a more reliable and robust diagnostic organ in comparison to the other organs used for conventional diagnosis.

Spontaneous spinal plexiform perineurioma in a juvenile chicken (Gallus domesticus)

A 2-mo-old pet chicken ( Gallus domesticus) was presented because of lameness and a hind limb mass of 1 mo duration. Radiographs revealed a soft tissue mass extending from the mid-femur into the body wall. Cytology of a sample obtained from a fine-needle aspirate was unremarkable. The bird was prepared for surgical investigation of the mass but died acutely at the time of pre-medication. Autopsy revealed an extradural mesenchymal neoplasm that focally compressed the lumbar spinal cord, extended into and along the adjacent nerve structures, and displaced the kidneys, skeletal muscle, and body wall. Histopathology was consistent with a perineurioma, which was positive for alcian blue and negative for S100, GLUT1, and neurofilament proteins on immunohistochemistry. PCR testing of tumor tissue was negative for gallid herpesvirus 2 (Marek’s disease virus) and avian leukosis virus. Although the etiology of the mass was not determined, our case highlights a severe manifestation of an uncommonly diagnosed tumor in the chicken. Though uncommon, perineurioma should be considered a differential diagnosis for lameness in the chicken.

Applying the isothermal DNA amplification method in identifying the Marek’s disease virus in poultry

Marek's disease is a viral disease, a type of poultry cancer. It is caused by MDV serotype 1 (Marek's disease virus, MDV) viruses, also referred to as Gallid herpesvirus 2 belonging to the family Herpesviridae. The complete, fully infectious virus particles are present in the feathers and the nodules months can survive in the environment for many months. The use of isothermal amplification methods of genetic material (Loop - Mediated Isothermal Amplification, LAMP) virus MDV enables rapid and precise identification of the virus in difficult diagnostic material, including dust. By optimizing the reaction conditions and using at least two pairs of primers which bind to specific sites in the genome of the virus, this technique has high sensitivity and specificity. The technique makes it possible to detect of single copies of the meq oncogene – a unique region of DNA of the first type of virus MDV.