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Factors affecting the uptake and transmission of Mareks disease herpesvirus - OD0715

Description
Marek’s disease (MD) is a naturally-occurring and widespread immunosuppressive and neoplastic disease of the domestic chicken caused by a herpesvirus (MDV). Its re-emergence will bring serious economic, health and welfare problems for the UK poultry industry. In the past intensive use of MD vaccines directed against replication phase in the virus life cycle has led to the emergence of pathotypes of MDV of increasing virulence in many countries, including the UK. Current vaccine strains, obtained by attenuating pathogenic MDV, now border on being harmful in susceptible chickens. There is therefore a need to identify critical stages in MDV infection where vaccines, or other intervention strategies, can be targeted to block virus uptake and infection so reducing pressures on MDV to evolve to even greater virulence. This programme will investigate very early events in MDV infection with the natural form of infectious virus (MDV-infected dust) as well as the production and dissemination of infectious virus from the feather follicle epithelium, the only site of production of fully-infectious MDV. In this project we will investigate (1) the lung as a barrier to natural infection with cell-free MDV, (2) the cells involved in the uptake and transport of MDV across the respiratory tract and (3) the feasibility of improving protective immune responses to MDV in the respiratory tissues and (4) the production of MDV in the FFE and the immune responses associated with fully-productive MDV infection. The objectives are to provide a basis for improving resistance to MD in commercial poultry by developing strategies to block MDV infection, rather than its replication in lymphoid tissues, by (1) identifying and enhancing protective responses to the early events in MDV infection (2) devising intervention strategies that improve the lung as a barrier (3) identifying and exploiting immune responses that reduce production of fully-infectious MDV and shedding from the FFE and/or (4) identifying host genes associated with reduced MDV uptake and dissemination. This work is consistent with DEFRA’s research requirement R20: “Studies on the host/pathogen interactions with MDV in domestic chickens following infection and the mechanism of virus excretion with the aim of sustaining vaccine strategies and seeking alternative methods for control of the disease”. In addition this work will: (1) help improve the health, welfare and immunity of commercial poultry by improving the effectiveness of vaccination (2) reduce the need for pharmaceutical control of opportunistic infections resulting from MDV-induced immunosuppression and/or (3) help identify genetic mechanisms that can be harnessed for selective breeding programmes. The outcome will be increased competitiveness of the poultry industry in the UK through a reduction in economic losses caused morbidity and mortality from MD. It should lead to improved health and welfare of poultry as well as increased product quality for the consumer. It will also maintain and strengthen the base of expertise for work on an important oncogenic virus which is in a position to jump species barriers through the food chain.
Objective
1) Role of the lung in establishing MDV infectionThe lung may merely provide a portal of entry for MDV but it could also provide target cells in which the virus replicates before transport to the lymphoid tissues. We have established a reliable system for infecting chickens with MDV by the respiratory tract using a standard amount of MDV-infected dust administered as a dry aerosol. We propose to refine our model aerosol system infection system to use a cell-free MDV. This will allow us to determine the minimal infectious doses and to investigate the importance of the keratin coating in flakes of skin on MDV uptake and infection. We also plan to determine if MDV undergoes an early round of replication in specific lung leukocytes by employing sensitive PCR techniques to measure transcripts of immediate early (IE), early (E) and late (L) MDV genes. If this is so we will then consider the importance of chicken genotype on MDV uptake via the lung epithelium. 2) Lung as a lymphoid tissueThis objective will concern the role of the lung as a barrier to entry and the site where innate and acquired immune responses prevent virus uptake and infection. Once protective responses have been identified we can then determine if these responses can be enhanced by vaccination or if other intervention strategies can be developed to improve protection. This part of the work will involve determining the structure of the bronchiolar tissue and bronchus-associated lymphoid tissues (BALT), especially characterising the leukocyte sub-populations in uninfected chickens and pathological changes after infection with MDV by the respiratory route. We will also investigate changes after MD vaccination and challenge with vvMDV. A further question will be the importance of chicken genotype on immunity, determining if resistant genotypes present a more effective barrier to virus entry across the lung tissue. 3) Role of antigen-presenting cells in MDV infectionAntigen-presenting cells (APC) most likely play an important role in MDV infection, a view widely-discussed, though supported by few functional studies. We therefore plan to investigate the importance of macrophages and members of the dendritic cell family play in primary MDV infection and after vaccination and challenge by the natural route of MDV infection. This part of the project will involve isolating dendritic cells (DC) and macrophages from the lungs, using techniques we have already developed for chicken spleen populations, and characterising them using monoclonal antibodies against surface markers. We will also investigate the feasibility of culturing DC in vitro and inoculating them into recipients to determine their ability to protect naïve recipients. Possible differences in numbers and functions of APC in resistant and susceptible genotypes will also be addressed with a view to mapping any differences.Skin Langerhans cells (LC), an important member the DC family, have been neither identified nor isolated from chickens. Nevertheless, these APC are likely play a key role in the immune responses at the sites where fully-infectious MDV is produced in the FFE. Using our experience of techniques for isolating chicken splenic DC, we plan to isolate chicken LC, characterise them and study the changes that occur (1) after infection with vvMDV and (2) after vaccination and vvMDV challenge. We will also investigate the feasibility of culturing these cells and adoptively transferring them into naïve recipients to investigate their role in immunity to MDV.4) Fully-productive MDV infection in the FFEThe objective is to investigate the fully-productive MDV infection in the FFE to determine if it is subject to immunological controls. If this is so, to identify the key innate and/or acquired immune responses involved. Although there have been a few histological investigations of the structure of the FFE in MDV- infected chicken, differences in feather pulp lesions have been reported [18-20]. We plan to make a detailed study of the leukocyte sub-populations in the FFE using a panel of monoclonal antibodies and paying attention to the phenotypes of the cells infected with MDV. We will also investigate the feasibility of isolating leukocytes from the FFE and using quantitative RT-PCR methods to identify cytokine transcripts in order to gain insight into the immune responses involved. The question of differences in MDV production and virus load at the sites of fully-productive infection in resistant and susceptible chicken genotypes will also be addressed.5) Lung as a route for delivering vaccinesWe will also investigate the feasibility of infecting by the natural route utilising different carriers (eg. MDV BAC DNA, nanoparticles etc) to mimic a natural infection and to use MD vaccine viruses to determine if this represents a practical method for delivering an effective vaccine dose. We aim to gain insight into the nature of natural infectious MDV so that this can be exploited in the development of novel vaccine delivery systems that use the natural route for infection.General note concerning objectives Most of the work will be carried out using the resistant line 6 and susceptible line 7 birds for which a linkage map of a backcross population, (6 x 7) x 6, has already been produced. Consequently any clear differences in immunological or disease parameters identified in these two lines can be mapped. At this stage it is impossible to predict those factors that are mappable but once identified they may provide important information on the chicken genes associated with innate and acquired immune responses during the early stages of infection or in the fully-productive infection. In some cases we will compare the differences in response to MDV infection between lines 6 and 7 with those between lines that possess different genes associated with resistance and susceptibility to MD; the resistant line N (B21 haplotype) and line P (B19 haplotype).
Project Documents
• Final Report : Final Report SID5   (10242k)
Time-Scale and Cost
From: 2003

To: 2006

Cost: £393,888
Contractor / Funded Organisations
Institute for Animal Health (BBSRC)
Keywords
Animal Diseases              
Animal Health              
Biotechnology              
Control              
Epidemiology              
GM Non-Food              
Marek's              
Neoplastic              
Plants and Animals              
Poultry              
Transmission              
Fields of Study
Animal Health