Molecular characterization of Marek’s disease virus detected in backyard chickens affected by classical nervous form.

Marek’s disease (MD) is a worldwide, contagious, lymphoprolipherative disease of chicken caused by a lymphotropic and oncogenic virus, Gallid alphaherpesvirus 2, also known as Marek’s Disease Virus serotype 1 (MDV-1). MDV-1 can cause neoplastic (lymphomas) and non neoplastic (neurological syndromes and immunosuppression) pathological syndromes. The nervous syndrome characterized mainly by peripheral nerve lesions accompanied by spastic paralysis of legs is termed classical MD. An outbreak of classical MD in unvaccinated Cochin chickens from an Italian backyard flock is reported. After suppression two birds, showing inability to stand upright and spastic paralysis of legs, were necropsied and organs were taken for histologic examination and molecular analysis. MDV-1 was detected from feathers and organs by PCR targeting the meq gene. Meq gene of the detected strain was fully sequenced along with commercial vaccines most commonly used in Italy. Using BioEdit Sequence Alignment Editor, nucleotide and amino acid sequences were edited and compared to meq sequences of selected MDV-1 reference strains, retrieved from GenBank, having different degrees of pathogenicity. Phylogenetic analysis of the amino acid sequences was performed using neighbor- joining clustering method. The high number of PPPP repeats found in the transactivation domain of the Meq protein and the phylogenetic tree obtained showed that the detected virus was closely related to mild or attenuated MDV-1 strains, but different from the analyzed vaccines. The molecular results were supported by clinical, macroscopic and microscopic findings. A- and B-type lesions, highly suggestive of a mild MDV- 1 infection, were observed in enlarged peripheral nerves (brachial plexus, lumbosacral plexus and sciatic, vagus and intercostal nerves). Major Histocompatibility Complex (MHC) haplotype B19, known to be correlated with high susceptibility to Marek’s disease, was identified in affected chickens by PCR and sequencing of B-L II family genes. Because sequencing of the meq gene is not a sufficient criteria to determine the pathotype, virus isolation will be attempted in order to confirm the molecular results by in vivo experimental trials.