Immunosuppression has a negative impact on bird welfare and represents a key economic challenge for the poultry industry. Environmental stressors and poor nutrition contribute to immunosuppression but the major cause in chickens are viruses, including immunosuppressive viruses, such as infectious bursal disease virus (IBDV) and oncogenic retroviruses such as avian leukosis virus (ALV) and reticuloendotheliosis virus (REV). Suppression of the immune system by these viruses results in either the destruction of immune cells, B cells, or their transformation into a cancerous tumour. There is a need for new control methods and vaccinations as new strains of viruses arise and vaccine failures are being reported. In vivo protocols to study these viruses typically require chickens to be infected and tissues or tumours removed for study, but these experiments have scientific limitations and cause significant morbidity to infected birds.
Why we funded it
This Project Grant aims to establish a chicken primary B cell culture model to replace the need for live birds in pathogenesis studies of immunosuppressive viruses.
Infecting birds with IBDV is listed as a severe procedure, as defined by the Animals (Scientific Procedures) Act, with symptoms of disease present between 24 and 48 hours post-inoculation. In severe cases, birds may be prostrated with laboured breathing and mortality rates can reach as high as 100% in some flocks. Studies with ALV and REV are typically conducted in cells derived from metastatic tumours that develop over several months in infected birds. Three timepoints post-infection are used in a typical in vivo study of viral pathogenesis, with birds culled for each time point. Dr Broadbent estimates his group requires over 300 birds per year in pathogenicity studies, with a further 60 needed for IBDV viral stock production. The use of the primary B cell culture model could replace up to two-thirds of birds needed in pathogenicity studies and all the birds needed for IBDV viral stock production.
Initial experiments aim to validate the use of chicken primary B cells for studying IBDV-host interactions. Chicken primary B cells will be infected with IBDV in triplicate and RNA extracted from the cultures. The amount of virus present in the cells can then be quantified using the extracted RNA and gene expression compared to mock-infected cells to identify any differentially expressed genes following infection. These results will be generated by RNA-Seq and compared to in vivo data as a validation of the chicken primary B cell model for studying IBDV-host interactions. Once the use of the cells is validated for IBDV-host interactions, similar experiments will be performed for ALV and REV. Finally, the suitability of the chicken primary B cells to be used to test the immunogenicity of vaccine candidates prior to challenge and for IBDV stock production will be evaluated. For the primary B cells to be suitable the virus must replicate to a high titre and not develop attenuating mutations during replication. These properties will be assessed in the primary B cells and compared to in vivo studies.
Dulwich KL et al. (2018). An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis. Journal of visualized experiments : JoVE (140):58489. doi: 10.3791/58489
Dulwich KL et al. (2017). Differential gene expression in chicken primary B cells infected ex vivo with attenuated and very virulent strains of infectious bursal disease virus (IBDV). The Journal of General Virology 98(12):2918-2930. doi: 10.1099/jgv.0.000979
Principal investigatorDr Andrew Broadbent
InstitutionThe Pirbright Institute
Co-InvestigatorProfessor Venugopal Nair
Dr Michael Skinner