engleski

Assessment of cell mediated immunity to NDV strain ZG1999HDS applied to one-day old chickens by means of nebulisation or oculonasal instillation

Newcastle disease (ND) is highly contagious viral infection of domestic poultry and other avian species with global distribution. It has huge economic impact due to high costs of control measures. Inactivated or live attenuated vaccines containing lentogenic or attenuated mesogenic NDV strains are commonly used for immunisation. Industrial poultry production employs strict vaccination programme for parental flocks ; thus, resulting in passive protection of offspring at neonatal age. Present maternal antibodies neutralize vaccine virus but also interfere with development of acquired immunity upon primary vaccination applied in hatchery. Therefore, revaccination of chickens 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. Immunogenicity of ZG1999HDS NDV strain was evaluated in comparison with La Sota strain in one-day old vaccinated chickens by measuring level of antiviral antibodies in hemagglutination inhibition (HI) assay and ELISA (Bidin and Mazija, 2009). The strain causes significantly stronger lysis of tumour cells in vitro than La Sota strain currently used for vaccination (Mazija et al, 2011), and represents a suitable candidate for the further vaccine development. Specific cellular immune response was characterised after application of NDV strain ZG1999HDS in one-day old male chickens of commercial laying lines. The virus was applied by two different ways: either by conventional oculonasal instillation or by means of nebulisation. The development of specific cellular immune response was evaluated ex vivo by consecutive blood sampling of chickens in intervals before vaccination, and then 3, 5, and 7 days after the treatment. Peripheral blood lymphocytes (PBLs) were enriched and isolated from blood samples by modified gradient centrifugation method. Afterwards, PBLs were stimulated for antigen-recall response and assessed for virus-specific CD4+ and CD8+ T cell proliferation on a flow cytometer. Modified density gradient centrifugation with addition of dextran to whole blood samples has removed contamination of thrombocytes from the PBL population and yielded more leukocytes in comparison to ficoll density gradient centrifugation. Based on previously confirmed better humoral response following nebulisation, greater proliferation of virus – specific T cells is expected in chickens treated by means of nebulisation then by conventional route of infection. The application of vaccines against ND by means of nebulisation to one-day old chickens at commercial hatchery is one of approved methods for ND control in Croatia. Immunogenicity of NDV strain ZG1999HDS following nebulisation in one-day-old chickens has been confirmed by specific antibody production and it induced higher humoral immunity then commercially available vaccine when applied by means of nebulisation. Better humoral immunity could be attributed to lack of stress by avoidance of individual manipulation of chickens and additional local pulmonary response achieved by vaccine particles 3 to 5 µm in size reaching deeper into respiratory tract than by conventional vaccination. However, cell-mediated immune response is detectable 2-3 days after the infection and thus plays an important role in development of humoral immunity and overall protection. Therefore, an assessment of virus-specific T cell proliferation could be used as valuable correlation of protection of chickens achieved by vaccination. At the same time, evaluation of cytokine production by proliferated cells (i.e. levels of IFN-gamma, IL-2 and TNF-alpha in stimulated lymphocyte culture media) would contribute to better characterisation of achieved immunity.

Newcastle disease ; nebulisation ; dextran ; density gradient centrifugation

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