Immunological changes at point-of-lay increase susceptibility to Salmonella enterica Serovar enteritidis infection in vaccinated chickens

Abstract

Chicken eggs are the main source of human Salmonella enterica serovar Enteritidis infection. S. Enteritidis infects the oviduct and ovary of the chicken leading to infection of developing eggs. Therefore, control in poultry production is a major public health priority. Vaccination of hens has proved successful in control strategies in United Kingdom leading to a 70% drop in human cases since introduced. However, as hens reach sexual maturity they become immunosuppressed and it has been postulated this leads to increased susceptibility to Salmonella infection. In this study we define the changes to the systemic and reproductive tract-associated immune system of hens throughout sexual development by flow cytometry and histology and determine changes in susceptibility to experimental S. Enteritidis challenge in naive and vaccinated hens. Changes to both systemic and local immune systems occur in chickens at sexual development around 140 days of age. The population of several leukocyte classes drop, with the greatest fall in CD4+ lymphocyte numbers. Within the developing reproductive tract there an organised structure of lymphocytic aggregates with γδ-T lymphocytes associated with the mucosa. At point-of-lay, this organised structure disappears and only scattered lymphocytes remain. Protection against Salmonella challenge is significantly reduced in vaccinated birds at point-of-lay, coinciding with the drop in CD4+ lymphocytes. Susceptibility to reproductive tract infection by Salmonella increased in vaccinated and naïve animals at 140 and 148 days of age. We hypothesise that the drop in γδ-T lymphocytes in the tract leads to decreased innate protection of the mucosa to infection. These findings indicate that systemic and local changes to the immune system increase the susceptibility of hens to S. Enteritidis infection. The loss of protective immunity in vaccinated birds demonstrates that Salmonella control should not rely on vaccination alone, but as part of an integrated control strategy including biosecurity and improved animal welfare.

Figure 1. Changes in splenic mononuclear leucocyte populations through development of and onset of sexual maturity in commercial laying hens determined by flow cytometry following specific single color staining with monoclonal antibodies to major chicken cell markers.

Fig. 1A shows the CD4+ population which falls significantly between 102 and 130 days of age as does the γδ population (Figure 1C). Figure 1B shows the CD8+ population (data for CD8α and CD8β combined), Figure 1D shows B lymphocyte (Bu-1) and Figure 1E macrophage (KUL01) stained populations. Data are shown as means of the percentage of positive cells (± SD). Data shown are based on five birds per time point.

Figure 2. Changes in cell populations in the chicken reproductive tract as determined by immunohistological examination.

2A shows T lymphocyte populations in the infundibulum (upper oviduct), 2B in the magnum (mid oviduct) and 2Cthe ovary. Figure 2D shows the changes in macrophage populations in the ovary and oviduct. Data are based on 10 fields of view for two sections of each tissue from each animal. Between five to seven birds were examined at each time point. Data are given as a mean (±SD).

Figure 3. Changes to CD4+ lymphocyte populations in the reproductive tract.

At 102 days of age CD4+ populations are primarily organised into aggregates in the ovary (Figure 3A) and oviduct. Birds lack lymph nodes and form more loose lymphoid structures. At 130 days (Figure 3B) these structures are no longer found, with smaller populations of cells found throughout the ovary. At 165 days (Figure 3C) CD4+ numbers increase slightly, but remain scattered throughout the tissue. Photomicrographs taken at a magnification of ×400.

Figure 4. Expression of Interleukin-4 (A) and Interleukin-6 (B) in the ovary.

Data is expressed as 40-Ct values in ovarian tissue in comparison to expression in breast muscle tissue as a control for no expression (40-Ct = 0). RNA levels are normalised between tissues using 28S RNA expression. Data are shown as means based on expression levels of 5 birds at each time point (±SD).