Quantitative profiling of the shedding rate of the three Marek’s disease virus (MDV) serotypes reveals that challenge with virulent MDV markedly increases shedding of vaccinal viruses Aminul Islam3 and Stephen W. Walkden-Brown Correspondence Aminul Islam Aminul.Islam@hnehealth.nsw. gov.au Centre for Animal Health and Welfare, School of Rural Science and Agriculture, University of New England, Armidale, NSW 2351, Australia Received 5 March 2007 Accepted 23 April 2007 The shedding profile of Marek’s disease virus serotype 1 (MDV1, virulent), serotype 2 (MDV2, vaccinal) and herpesvirus of turkeys (HVT, vaccinal) in commercial broiler chickens was determined by measuring the daily rate of production of feather dander from chickens housed in isolators and by quantifying the viral load of each of these serotypes in the dander using quantitative real-time PCR (qPCR). MDV1 and HVT viruses were detectable in dander filtered from isolator exhaust air from day 7 and MDV2 from day 12 after infection and thereafter until the end of the experiment at 61 days of age of the chickens. There was no difference in shedding rate among the three MDV1 isolates. Daily shedding of MDV1 increased sharply between days 7 and 28 and stabilized thereafter at about 10 9 virus copies per chicken per day, irrespective of vaccination status. Challenge with the three different MDV1 isolates markedly increased shedding of the vaccinal viruses HVT and MDV2 in dander by 38- and 75-fold, respectively. These results demonstrate the utility of qPCR for the differentiation and quantification of different MDV serotypes in feather dander and have significant implications for the routine monitoring of Marek’s disease using qPCR assays of dust, for epidemiological modelling of the behaviour and spread of MDVs in chicken populations and for studies into the evolution of virulence in MDV1 in the face of blanket vaccination with imperfect vaccines that ameliorate disease but do not prevent infection and replication of virulent virus. INTRODUCTION Marek’s disease viruses (MDVs) are cell-associated viruses belonging to the family Herpesviridae, the subfamily Alpha- herpesviridae and the genus Mardivirus (Fauqet et al., 2005). Antigenically, they are closely related and their classification into three serotypes, namely serotype 1 (MDV1), 2 (MDV2) and 3 (MDV3 or herpesvirus of turkeys, HVT) is widely used. MDV1 is oncogenic and the aetiological agent of Marek’s disease (MD), a lymphopro- liferative and neoplastic disease of poultry (Churchill & Biggs, 1967; Nazerian et al., 1968) with an estimated worldwide economic impact of US$1–2 billion (Morrow & Fehler, 2004). MDV1 isolates vary widely in their pathogenic and oncogenic potential (Witter, 1997). The other two serotypes (MDV2 and HVT) are non-oncogenic and are used in live vaccines against MD, either alone, in combination or in combination with attenuated MDV1 strains (Witter & Schat, 2003). Attenuated strains of MDV1 and naturally occurring vaccine strains of MDV2 or HVT produce non-sterile immunity against MD tumours and persist together with virulent MDV1 in the host (Witter et al., 1971; Witter & Schat, 2003). Whilst the primary target cell for MDV pathology is the lymphocyte, MDV1 is contagious and is readily trans- mitted by the airborne route (Biggs, 1985). This is due to replication of fully infectious virus in the epithelial cells of the keratinizing layer of the feather follicle epithelium, which then slough off and are shed as highly infective dander (Calnek et al., 1970). MDV2 is also contagious (Witter, 1987), but HVT does not spread readily between chickens infected early in life (Cho & Kenzy, 1975). Understanding the rate of shedding of the different MDVs, and factors influencing this, is a prerequisite to a detailed understanding of the epidemiology of MDV, but our current understanding of this is limited. Earlier reports suggested that shedding of MDV1 commences from 2 to 4 weeks after infection, well before the appearance of clinical signs, and may continue throughout the life of the chicken (Witter et al., 1971; Carrozza et al., 1973). Since then, studies using more sensitive molecular methods have 3Present address: Division of Microbiology, Hunter Area Pathology Service (HAPS), Locked Bag 1, Hunter Region Mail Centre, NSW 2310, Australia. Journal of General Virology (2007), 88, 2121–2128 DOI 10.1099/vir.0.82969-0 0008-2969 G 2007 SGM Printed in Great Britain 2121