engleski

Induction of cell mediated immunity with NDV strain ZG1999HDS in nebulised newly hatched chickens

Newcastle disease (ND) is highly contagious and economically important viral infection of domestic poultry and other avian species with global distribution. Live attenuated and then inactivated vaccines are commonly used in ND control. Industrial poultry production employs strict vaccination programme for parental flocks. Present maternal antibodies interfere with a development of acquired immunity in chickens upon primary vaccination applied in hatchery. Therefore, revaccination with inactivated vaccines at the age of 2-3 weeks is required. Development of a new NDV vaccine strain with capability to induce long-term protection upon primary vaccination despite presence of maternal antibodies is a prerequisite. New NDV strain ZG1999HDS originates from the outbreak in July of 1999 at chicken farm in North West Croatia. It is well characterised in respect to MDT, ICPI, IVPI, whole genome and proteome sequence at the cleavage site of fusion protein. In comparison with La Sota strain currently used for vaccination, humoral response to ZG1999HDS NDV strain in one-day old nebulised chickens is higher as confirmed by hemagglutination inhibition (HI) assay and ELISA. The strain causes significantly stronger lysis of tumour cells in vitro than La Sota strain and represents a suitable candidate for the further vaccine development. Two different ways of NDV strain ZG1999HDS delivery, conventional oculo-nasal route or nebulisation, were applied in commercial layer one-day old male chickens. The development of specific cell-mediated immunity (CMI) was evaluated ex vivo by consecutive blood sampling of chickens in intervals before vaccination, and then 3, 5, 7 and 14 days after the treatment. Peripheral blood lymphocytes (PBLs) were enriched from blood samples by modified gradient centrifugation method. In the end, PBLs aquired at 14 days post immunisation were stimulated for antigen-recall response in vitro and assessed for virus-specific CD4+ and CD8+ T cell proliferation on a flow cytometer. Modified slow-speed density gradient centrifugation with addition of dextran to whole blood samples has removed contamination of thrombocytes from the PBLs population and yielded more leukocytes in comparison to ficoll density gradient centrifugation. Consistent with previously confirmed better humoral response to the NDV strain ZG1999HDS following nebulisation, higher level of proliferation of NDV – specific CD4+ and CD8+ T cells was observed than in chickens treated by conventional route of vaccination. Nebulisation of newly hatched chickens with vaccines against ND at commercial hatchery is one of approved methods for ND control in Croatia. Immunogenicity of NDV strain ZG1999HDS following nebulisation of one-day-old chickens was confirmed by serum conversion as induced better humoral immune response than commercially available vaccine. This could be attributed to lack of stress by avoidance of individual chicken manipulation and to additional local response to vaccine particles, 3 to 5 µm in size, delivered deeper into respiratory tract than after conventional vaccination. Additionally, the specific CMI could be detected 2-3 days after the vaccination and contributes to development of humoral immunity and overall protection. Therefore, an assessment of virus-specific T cell proliferation is a valuable mean for correlation of protection achieved by vaccination. Future evaluation of cytokine production (e.g. IFN-gamma, IL-2 and TNF-alpha) in stimulated lymphocyte culture media will contribute to better characterisation of achieved specific cell-mediated immunity.

Newcastle disease ; nebulisation ; chicken ; cell immunity ; flow cytometry

*Equally contributing first authors.