General Introduction

General introduction Marek’s disease (MD) is a common lymphoproliferative disease of chickens worldwide, caused by Marek’s disease virus (MDV) an oncogenic highly cell- associated alphaherpesvirus that induces T-cell lymphomas in poultry. Originally described in 1907 as a classical paralytic disease associated with polyneuritis (Marek, 1907), MD has shown significant increases in severity in its clinical picture over the last 100 years, particularly in the latter half of this period (Witter, 1997). The earlier classification of Marek’s disease viruses (MDVs) into three serotypes, which was based on antigenic (von Bülow, 1975) properties, has been revised since representative strains of the three serotypes have been sequenced (Lee et al., 2000; Afonso et al., 2001;Izumiya et al., 2001; Kingham et al., 2001; Tulman et al., 2000). The three viruses certainly represent individual and clearly distinct virus species, which correspond to the previous serotypes. A new classification has been proposed and serotype 1 (MDV1) is now also referred to as Gallid herpesvirus 2 (GaHV-2), serotype 2 (MDV2) as Gallid herpesvirus 3 (GaHV-3) and serotype 3 of MDV, also known as Herpesvirus of Turkeys (HVT) is now referred to as Meleagrid herpesvirus 1 (MeHV-1) (Osterrieder and Vautherot, 2004). However, the serotype classification is still commonly used and is convenient, so is used throughout this thesis. MDV1 is the only pathogenic/oncogenic serotype of MDV. MD vaccines are live, often cell- associated, and comprise attenuated MDV1 or naturally occurring MDV2 and HVT strains, or combinations of these. Despite the widespread use of MD vaccines since 1970, MD remains an important disease threat, partly because of the cost of vaccination and also because of the emergence of new MDV pathotypes of increased virulence over time. Vaccination against MD may protect chickens against clinical MD but it does not preclude infection with wild type MDV or shedding of co-infecting MDV from the feather follicle epithelium (FFE) (Witter, et al., 1971). This is thought to have contributed to the evolution towards higher virulence of MDV since the advent of widespread vaccination. These new pathotypes represent a continuum of 1 increased virulence and based on extensive pathotyping studies, Witter (1997) proposed a classification of these isolates in mild (m), virulent (v), very virulent (vv) and very virulent plus (vv+). In addition to the increased oncogenic potential of these isolates, the most virulent recent isolates produced acute cytolytic disease with heavy mortality and massive atrophy of the immune organs well before the onset of lympomas. The primary targets of MDV infection are lymphocytes and consequently, diagnosis, isolation and monitoring of MDVs has been historically based around lymphocytes or lymphocyte-rich tissues, e.g. spleen material or peripheral blood lymphocytes (PBL). These materials are not easily accessible, are of fragile nature and require immediate and laborious processing after sampling. As MDV is a highly cell-associated virus, it is essential that material for in vitro isolation of MDV consists of viable cells and this has posed significant technical barriers to the development of routine monitoring systems for MD. However, production of fully infectious virus occurs in the FFE and MDV is shed with feather debris and dander which is the source of natural transmission of MDV and contamination of the environment (Calnek et al., 1970). This material is easily accessible and retains infectivity for long periods, under a wide range of conditions, but is unsuitable for direct inoculation to cell culture without ultra centrifugation to separate virus and additional treatments (Calnek et al. 1970). However, material from the FFE provides a good source of MDV antigens and viral DNA and the quantification of viral load in this material has shown to provide a good indicator of flock MD status in broiler chickens (Walkden- Brown et al., 2005; Islam and Walkden-Brown, 2007). Recent progress with immunological and molecular biology techniques has enabled more rapid, sensitive and accurate detection of MDV in biological samples, with clear differentiation of the different serotypes and enumeration of viral copy number. In particular, quantitative real-time PCR (qPCR) for differentiation and quantitation of viral nucleic acids offers the opportunity to routinely monitor the MD status from a wide variety of sample materials, e.g. spleen, PBL, feather tips or poultry dust. The latter is an excellent sample material as it integrates information from a whole flock or group of chickens, and is easy to sample, store and process for subsequent qPCR analysis. 2 This doctoral study was focussed on the development and optimization of MDV serotype-specific qPCR assays and their application under a variety of conditions to investigate aspects of MDV pathogenesis and epidemiology. The ultimate objective of this was to assess the usefulness of different samples in the monitoring of MD status. Another part of the study involved DNA sequence analysis of the MDV1 specific oncogene meq to find markers for MD virulence and examine phylogenetic relationships between Australian and international strains of MDV. A final experiment investigated the effect of two environmental enrichment strategies on the well-being of chickens involved in in vivo evaluation of MDV in isolators. The main objectives of this study were: 1. To develop and optimize absolute quantification of MDV2 serotype-specific quantitative real-time polymerase chain reaction (qPCR) assays. 2. To investigate the DNA sequence of the meq oncogene in recent Australian isolates of MDV and determine whether sequence variation correlates with virulence. 3. To determine the pathotype of recent Australian isolates of MDV in a pathotyping experiment using commercial layer chickens. 4. To investigate early predictors of subsequent clinical MD status 5. To measure the level of feather dust production in layer chickens, and the shedding pattern of virulent and vaccine isolates of MDVs in feather tips and dust. 6. To improve welfare of experimental layer chickens kept in isolators using environmental enrichment strategies. The results should advance our understanding of MD epidemiology and pathogenesis and will contribute to future efforts to place the ongoing control of MD onto a sustainable basis in the long term. Materials from this thesis have been published in international journals and several conference proceedings; a list of these publications is provided on page v. 3 Chapter 1 Review of literature 1.1 Introduction Marek’s disease virus (MDV) is the aetiological agent of Marek’s disease (MD) and a highly cell- associated alphaherpesvirus which was first isolated in 1967 (Churchill and Biggs, 1967). MD is a highly contagious lymphoproliferative disease in chickens inducing paralytic and lymphomatous syndromes, and causing significant losses in the poultry industry worldwide. Three MDV serotypes are classified: Marek’s disease virus serotypes 1 and 2 (MDV1 and MDV2) as well as herpesvirus of turkeys (HVT) (van Regenmortel et al., 2000). Soon after identification of MDV, the first vaccine against MD, an attenuated MDV1 was described in the UK (Churchill et al., 1969a). Almost simultaneously the efficacy of HVT as a vaccine was demonstrated in the USA (Okazaki et al., 1970) and the first HVT vaccine was licensed in USA proving to be a great success. In the following years, further serotype 1 and 2 vaccines were developed and shown to have synergistic effects when administered with HVT (Calnek et al., 1983). MD vaccines were the first successful vaccines to be used for protection against cancer (Davison and Nair, 2005) and MD is therefore often considered as a model for the study of viral oncology (Calnek, 1986). The vaccines protect to a greater or lesser extent against mortality, clinical signs and gross MD lesions but none prevent infection, replication and shedding of challenge virus. They are therefore unable to induce sterile immunity, and this is thought to have contributed to the evolution of MDV towards greater virulence (Witter, 1997). This evolution in virulence means that serious outbreaks of MD have occurred periodically around the world at different times, ensuring that MDV remains a persistent threat to the poultry industry worldwide. The last major epidemic of MD in Australia was between 1992 and 1997. 4 Chapter 1 The primary target of infection with MDV is the lymphocyte and early effects are therefore seen in lymphoid organs such as the bursa of Fabricius where B- lymphocytes are produced, the thymus which is the primary producer of T- lymphocytes and spleen (Venugopal and Payne, 1995; Calnek et al., 1998; Calnek, 2001). The tumour inducing oncogenic process in MD, which is characterized by an accumulation of transformed lymphocytes and other immune cells, can involve most organs and tissues including peripheral nerves. The most commonly affected organs and tissues are gonads, spleen, heart, lung, liver, muscle and peripheral nerves (Biggs, 2001). Cytolytic changes, early mortality, severe neurological and atherosclerotic syndromes can also be manifestations of MD as can a clinical syndrome described as transient paralysis. MD can occur at any age from a few weeks old, but is most common between 2 and 6 months of age (Biggs, 2001) although very virulent MDV can induce significant lesions and mortality before two weeks of age in susceptible chickens. In general, involvement of tissues and

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