Development of duplex dual-gene and DIVA real-time RT-PCR assays and use of feathers as a non-invasive sampling method for diagnosis of Turkey Meningoencephalitis Virus

The avian flavivirus Turkey Meningoencephalitis Virus (TMEV) causes a neuroparalytic disease of commercial turkeys, expressed in paresis, incoordination, drooping wings and mortality that is controlled by vaccination. The molecular diagnosis using brain tissue RNA has now been upgraded by the development of a diagnostic dual-gene multiplex real-time PCR targeting the envelope and the non-structural NS5 gene, increasing the sensitivity by 10-100-fold compared to the previously existing assays. Based on the recent complete sequences of five TMEV isolates we have now developed a Differentiating Infected from Vaccinated Animals (DIVA) assay, to distinguish between wild-type TMEV strains and the vaccine virus. The DIVA assay was evaluated on commercial vaccines produced by two manufacturers, on RNA purified from brains of experimentally infected turkeys with TMEV strains, and on clinical samples collected between the years 2009 and 2015. We also investigated turkey feather pulps for their suitability to serve for TMEV detection, to avoid invasive sampling and bird killing. The parallel TMEV diagnosis in brain and feather-pulp RNA were similarly useful for diagnosis, at least in experimentally infected turkeys and in three cases of disease encountered in commercial flocks.