Differential amplification of Marek's disease CVI988 vaccine and of wild-type isolates from organs of commercial chickens using single or duplexed probes in real-time PCR.

The differentiation of Marek's disease virus (MDV)-infected and vaccinated animal (DIVA) test, based on the MDV pp38 gene was described by Baigent et al. [(2016). Real-time PCR for differential quantification of CVI988 vaccine and virulent MDV strains. Journal of Virological Methods, 233, 23-36], using similar primers and alternate probes for virulent MDV-1 and the vaccine CVI988 virus. We explored the assay's applicability for commercial vaccines and commercial chickens, as the above-mentioned study employed tissue-cultured MDV strains and tissues from experimental trials. DNA of visceral organs and feathers of vaccinated or naturally infected chickens was used. Further, the applicability of the DIVA assay was evaluated using single or duplexed probes for the two viruses in the same amplification tube. Due to the high viral content in the commercial vaccines and in the clinical cases of MDV-1 infected commercial chickens, their examination by the MDV-1 DIVA real-time PCR was performed in one step. However, for the feather DNAs of commercially vaccinated birds, a step of pre-amplification was required. The MDV-1 DIVA real-time PCR performed as single probe in separate tubes using the Vir3 probe was very sensitive for virulent MDV-1 strains, but not very specific, as it also gave a clear signal with CVI988 vaccine virus. In contrast, the CVI vaccine probe was specific for CVI988, and did not recognize the MDV-1 strains. When both probes were present in one tube, the CVI probe showed a greater sensitivity for CV1988, while the Vir3 probe showed a much better specificity for virulent MDV-1.

Monitoring the uptake of live avian vaccines by their detection in feathers.

Protection against diseases caused by the avian viruses, Marek's disease, Infectious laryngotracheitis, chicken anemia and turkey meningoencephalitis is achieved by live vaccines. The application quality is important to assure proper uptake in commercial flocks. We describe a novel evaluation method for the vaccination process by sequential monitoring the vaccine viruses in feathers. Feather collection is easy, non-invasive and non-lethal for the birds, therefore advantageous for monitoring purposes. To demonstrate the vaccine virus presence, an innovative assay of nested real-time amplification was approached because vaccine viruses presence in vivo is less abundant comparing to virulent wild-type isolates. The Marek's disease virus vaccine virus, Rispens/CVI988, in feathers of commercial flock was detected from 4 to 7 days and for at least 3 months post-vaccination, until the survey stopped. As the drinking water route was newly adopted for Infectious laryngotracheitis vaccination, one or two vaccine doses/bird were administered. The virus uptake was detected in feathers between 2 and 20 days-post-vaccination. With a doubled vaccine dose the positivity bird rate was higher. For the first time the chicken anemia vaccine virus presence in chicken feathers was demonstrated between 14 and 35 days-post-vaccination. No previous studies were available, thus in parallel to feathers the vaccine virus was demonstrated in the livers and spleens. The turkey meningoencephalitis vaccine virus uptake in turkey feather-pulps is even more innovative because this is the first turkey virus amplified from feather-pulps. The vaccine virus presence resemble the kinetics of the other 3 viruses, 3-21 days-post-vaccination. Detecting the specific antibodies following vaccination possessed a lower sensitivity than vaccine virus demonstration in feathers. In summary, the presented assay can be adopted for the quality evaluation of the vaccination process in poultry.