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

Abstract

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.

Biotic concerns in generating molecular diagnosis matrixes for 4 avian viruses with emphasis on Marek’s disease virus

“…Moreover, primers and probes concentrations in the amplification reaction, in general, are in excess (Elfath et al, 2000). Many studies employed dual multiplex amplifications for two viruses, including our studies that amplified quantitatively the MDV and CAV (Davidson et al, 2013) and ILTV and the fowlpox virus (FPV) from cases of diphteric manifestations in chickens (Fatumbi et al, 1995;Davidson et al, 2015), or two genes of one virus, the TMEV (Davidson et al, 2017b), An originally approach was the development of the multiplex qPCR on the level of two TaqMan probes in one tube to increase the sensitivity of detection of the MDV vaccine and wild type viruses (Davidson et al, 2017c). Several other examples among numerous dual virus amplification assays are (a) the avian metapneumovirus (AMPV) quantitative multiplex subtype A or subtype B to assist in disease diagnosis and to carry out rapid surveillance with high levels of sensitivity and specificity (Cecchinato et al, 2013), (b) Abdelwhab et al (2010) developed therefore a versatile, sensitive and lineagespecific multiplex RT-qPCR for detection and typing of H5N1 viruses in Egypt.…”

“…The ILTV viremia could also be demonstrated in the feather shafts of commercial ILTV-infected chickens (Davidson et al, 2009c;Davidson et al, 2016). TMEV was the first turkey virus that could be detected in the turkey feather shafts (Davidson et al, 2017b) The correlation between sampling feathers or pooled visceral organs for the detection of MDV and for CAV in commercial flocks was analysed. We included samples submitted for diagnosis from various poultry flocks and of various lines and ages.…”

“…In the case of the turkey flavivirus TMEV, the DIVA development (Davidson et al, 2017b) was motivated by the need to differentiate between affected birds as a result of vaccination, or as a result of infection with a TMEV field strain, and relied on whole genomic sequences of the TMEV vaccine and field viruses (Fernández-Pinero et al, 2014).…”

Diagnosing Infectious Diseases in Poultry Requires a Holistic Approach: A Review

“…Specific application of PCR is a DIVA (differentiating infected from vaccinated animals)-PCR, a method used for screening a vaccinated flock for infections with a filed/virulent strain, as for example in Marek's disease virus (MDV), Mycoplasma gallisepticum, Mycoplasma synoviae or turkey meningoencephalitis virus [223][224][225][226]. In order to accomplish functionality, DIVA tests should be able to distinguish between a vaccine and a field strain even in situations when multiple strains are present within the sample.…”