Comparison of Blood and Feather Pulp Samples for the Diagnosis of Marek's Disease and for Monitoring Marek's Disease Vaccination by Real Time-PCR

Abstract

Comparison of blood and feather pulp (FP) samples for the diagnosis of Marek's disease (MD) and for monitoring Marek's diseases vaccination in chickens (serotypes 2 and 3 vaccines) by real time-PCR was evaluated. For diagnosis of MD, quantification of serotype 1 Marek's disease virus (MDV) DNA load was evaluated in 21 chickens suffering from MD. For each chicken, samples of blood and FP were collected and MDV DNA load was quantified. Solid tumors are the sample of choice for MD diagnosis by real time-PCR and, hence, 14 solid tumors were included in the study as positive controls. Load of MDV DNA in FP was equivalent to that detected in solid tumors (threshold cycle [Ct] ratio above 1.7). MDV DNA load in blood samples was lower than in solid tumors and FP samples. Nonetheless, there was a statistically significant correlation of the results obtained from FP and blood (r = 0.92). Results of the Pearson correlation test showed that Ct ratio values of 1.7 in FP correspond to Ct ratio values of 1.2 in peripheral blood. For monitoring vaccines, serotypes 2 and 3 MDV DNA load was evaluated in blood and FP samples of vaccinated chickens. Serotype 2 MDV DNA load was evaluated in samples of blood and FP from 34 chickens vaccinated with SB-1 strain. Serotype 3 MDV DNA load was evaluated in blood and FP samples from 53 chickens vaccinated with HVT strain. For both serotypes, frequency of positive samples and load of vaccine DNA was higher in FP than in blood samples. There was not a statistically significant correlation between the load of SB-1 DNA (r = 0.17) or HVT DNA (r = -0.04) in FP and blood. Our results show that the load of serotypes 1, 2, and 3 DNA is higher in FP than in blood. Diagnosis of MD could be done using both FP and blood samples. Monitoring of MD vaccination by real time-PCR required the use of FP samples. There were a high percentage of false negative samples when using blood to detect serotypes 2 and 3 MDV by real time-PCR.

Chicken skin virome analyzed by high-throughput sequencing shows a composition highly different from human skin

“…In order to verify these results, PCR assays were performed from extracted amplified DNA, with three different sets of primers specific for GaHV-2, GaHV-3, and HVT species [10]. PCR analysis confirmed the presence of GaHV-3 DNA (Fig.…”

“…For that, we used primers specific for GaHV-2, GaHV-3, and HVT already described by Cortes and colleagues [10] with 2 and 10 ng of amplified DNA extracted from skin swabs. PCR conditions used were as follows: 3 min at 94°C, then 30 cycles with 15 s at 94°C, 15 s at 60°C, and 15 s at 72°C, followed by 10 min at 72°C.…”

Marek’s disease virus and skin interactions

“…For the past years, feathers and dust have been considered the material of choice to follow the evolution and distribution of pathogenic and vaccine strains of Mardiviruses in poultry houses [59]. Four to five pulp-rich feathers, preferably collected on the axillary tract, are sufficient to detect viral DNA using qPCR (S. Baigent personal communication).…”

“…Indeed, it has been shown that 20 week-old birds vaccinated with HVT had an incidence of vitiligo 4 times higher than non-vaccinated birds [72]. This puzzling result raises various hypotheses regarding the impact of HVT vaccination on the development of vitiligo, knowing that the HVT vaccine also penetrates and replicates in feather follicles [51,59]. In the SL line, depigmentation is associated with melanocyte death and with the presence of anti-melanocyte auto-antibodies; therefore, one hypothesis would be that HVT infects melanocytes or their precursors, leading to their death and triggering an auto-immune response against melanocyte markers.…”

“…In poultry houses, vaccines are administered to 1-day-old chicks manually or in the embryo in ovo, 2–3 days before hatching using an automated injection system. All vaccinating strains replicate in feather follicles and their DNA is detectable in feather tips by qPCR [59,64]. The kinetics of detection of the genome of these strains is similar to that of pathogenic strains.…”