The project will investigate the mechanisms that underlie the observed suppression of adaptive, and in particular cell mediated immunity at the onset of sexual maturity of laying hens and the effect these changes have on immunity to Salmonella enterica serovar Enteritidis. Salmonella Enteritidis may be transmitted vertically through eggs and thereby poses a risk to public health through the consumption of poorly cooked contaminated eggs. Although vaccination is employed in the UK in the control of Salmonella in laying hens, the immune mechanisms that underpin protection are not known. It is also unclear whether there is a gap in immunity at point-of-lay in vaccinated animals that may compromise protection to salmonellosis. The first aim of the project is to characterise changes in the immune system in developing hens. Flow cytometry will be used to determine the levels of T and B lymphocytes in the spleens of hens. Further characterisation of CD4 and CD8 positive, along with T cell receptor type (TCR1, 2 or 3)will also be performed. Immunocytochemical staining will be performed on tissues, including the oviduct and ovaries which are the main sites of Salmonella infection. Whilst these assays will indicate changes in cell numbers and population distributions, analysis of T cell function will be determined by proliferation to stimulation with the mitogens ConA and PHA. Immunoglobulin levels will be determined by ELISA. In a second series of experiments, we will characterise the immune response to Salmonella infection and to vaccination with currently available live and killed vaccines. Cellular changes will be determined as outlined above. T cell proliferation to Salmonella antigens will be determined and specific antibody responses to Salmonella (IgM, IgG and IgA) determined by ELISA. Expression of key cytokines, IL-2, Il- 4, IL-10, IL-12 and interferon-gamma will be performed on tissues by qRT-PCR. The use of magnetic cell sorting will be done to obtain specific cell populations. The expreession profile of these cells will also be determined by qRT-PCR to allow a more detailed analysis of the cell types involved in initiating and maintaining the immune response. These studies will also allow us to determine the relative role of Th1/Th2/Treg cells in the immune response. On the basis of the findings of the first two groups of experiments, we will conduct oral challenge experments to determine whether any reduction or gap in immunity in vaccinated birds around point-of-lay makes the chickens more susceptible to infection than in vaccinated birds prior to lay, or in birds where egg laying is well established. This will be assesed by qunatitative bacteriology of caecal contents, spleen, liver, oviduct, ovaries and developing or laid eggs. The final part of the project will determine whether changes in vaccine strategy can be employed that lead to more effective protection. We will determine whether non-specific stimulation of the immune system just prior to the onset of lay may be utilised to boost protection. We intend to achieve this through DNA vaccination of a plasmid vector containing the chicken interferon gene under control of an appropriate promoter, including immunostimulatory CpG motifs in the plasmid backbone. This should stimulate a Th1 dominated response and stimulate cell-mediated immunity. It is hoped such stimulation may be sufficent to increase protection to Salmonella, and potentially other pathogens, through the start of the egg laying period and reducing the risk of transmission of Salmonella to eggs.