Load of Challenge Marek's Disease Virus DNA in Blood as a Criterion for Early Diagnosis of Marek's Disease Tumors

Abstract

Outbreaks of Marek's disease (MD) in vaccinated flocks still occur sporadically and lead to economic losses. Unfortunately, adequate methods to predict MD outbreaks are lacking. In the present study, we have evaluated whether high load of challenge MD virus (MDV) DNA in peripheral blood could aid in the early diagnosis of MD and in monitoring efficacy of vaccines against MD. One experiment was conducted to simulate field conditions by combining various vaccines (turkey herpesvirus [HVT] and HVT + MDV serotype 2 [SB1]) and challenge viruses (GA, Md5, and 648A). Vaccine efficacy among our experimental groups ranged from 13.3% to 94.2%. Each chicken was sampled three times during the length of the experiment (3, 5, and 15 wk postchallenge [wpc]), and gross lesions were evaluated in chickens that died and at termination of the experiment. DNA was extracted from whole blood and buffy coats from each sample, and the load of challenge MDV DNA and HVT DNA were quantified by real-time polymerase chain reaction. Chickens that developed MD by the end of the experiment had higher load of challenge MDV DNA (threshold cycle [Ct] glyceraldehyde-3-phosphate dehydrogenase [GAPDH]/Ct glycoprotein B [gB] ratios of 1.0, 1.04, and 1.05 at 3, 5, and 15 wpc, respectively) than those that did not develop MD (Ct GAPDH/Ct gB ratios of 0.7, 0.69, and 0.46 at 3, 5, and 15 wpc, respectively). However, load of HVT DNA in blood was not correlated with the development of tumors (Ct GAPDH/Ct HVT ratios from 0.04 to 0.10 in both groups). Vaccinated groups with >75% protection had statistically significant less challenge DNA virus (Ct GAPDH/Ct gB ratios of 0.76, 0.70, and 0.45 at 3, 5, and 15 wpc, respectively) than less protected groups (Ct GAPDH/Ct gB ratios of 0.92, 0.97, and 0.85 at 3, 5, and 15 wpc, respectively). No differences in the load of HVT DNA could be found between protected and nonprotected groups at any time point of the study (Ct GAPDH/Ct HVT from 0.05 to 0.09 in both groups). Our results showed that load of challenge MDV DNA but not load of HVT DNA in blood can be used as criterion for early diagnosis of MD.

Quantification of rHVT-F genome load in feather follicles by specific real-time qPCR as an indicator of NDV-specific humoral immunity induced by day-old vaccination in SPF chickens

“…Additionally, serum and plasma were also tested, as types of samples conveniently collected in the field, and BF, as lymphoid tissue involved in humoral antibody responses in young chicks. In the present study, virus load in the tissues is expressed as number of rHVT-F PFU per amount of input organs weight, while it was previously reported as HVT genome copies per amount of input DNA (Abdul-Careem et al, 2008b;Haq et al, 2010), as number of DNA copies per 1 × 10 6 cells (Islam et al, 2006) or as Ct ratio (Gimeno et al, 2008(Gimeno et al, , 2011aCortes et al, 2011). A direct quantitative comparison to these previously reported results may therefore not be possible.…”

“…The results generated by the eight independent experiments provided consistent data to allow meaningful statistical analysis and inference. DNA extracted from spleen, lung, FF, PBL and whole blood were analysed by real-time qPCR for vaccine load, as previously studied after HVT vaccination against MD (Islam et al, 2006;AbdulCareem et al, 2008a, b;Gimeno et al, 2008Gimeno et al, , 2011aHaq et al, 2010;Cortes et al, 2011). Additionally, serum and plasma were also tested, as types of samples conveniently collected in the field, and BF, as lymphoid tissue involved in humoral antibody responses in young chicks.…”

“…HVT is non-oncogenic for chickens and has been used for a long time as live vaccine in vaccination programmes, alone or in combination with other MDV vaccine strains (like Rispens or SB-1), for protection against MD. Quantitative real-time polymerase chain reaction (real-time qPCR) specific to HVT has been developed for monitoring HVT genome load in blood (Gimeno et al, 2008(Gimeno et al, , 2011bCortes et al, 2011), buffy coat (Gimeno et al, 2008), spleen (Abdul-Careem et al, 2008a;Gimeno et al, 2011a), feathers follicles (FF; AbdulCareem et al, 2008b;Cortes et al, 2011;Gimeno et al, 2011a, b), lung (Haq et al, 2010;Gimeno et al, 2011a) and dust (Islam & Walkden-Brown, 2007) after experimental in ovo or day-old MD vaccination of specific pathogen free (SPF) chickens with HVT vaccine.…”

Characterizing the Molecular Basis of Attenuation of Marek's Disease Virus via In Vitro Serial Passage Identifies De Novo Mutations in the Helicase-Primase Subunit Gene UL5 and Other Candidates Associated with Reduced Virulence

“…DNA extracted from spleens of infected birds was used to assay the in vivo replication levels of serially passed replicates and recombinant viruses relative to control virulent Md5B40BAC-c1 p5. Extracted DNA was used to quantify the relative copy number of MDV genomes present via qPCR using primers for chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and MDV gB with the TaqMan Fast Universal PCR kit (Applied Biosciences, Foster City, CA) as described by Gimeno et al (21).…”

Virulent Marek's disease virus generated from infectious bacterial artificial chromosome clones with complete DNA sequence and the implication of viral genetic homogeneity in pathogenesis

“…Latent infection is considered to be a prerequisite for the transformation of T-cells by MDV (Osterrieder et al, 2006). This possibility might partly account for the unique characteristics of MD vaccines, which are protective for virulent virus challenge very soon after the vaccination (such as 5 days or earlier) without eliminating virulent virus from the host (Gimeno et al, 2008). One of the early effects of vaccine virus infection is that the infection of CVI988, a live vaccine for MDV, significantly reduces CD4 T-cells in the vaccinated birds for a certain period (Yamamoto et al, 1995).…”