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In vitro evaluation of live attenuated vaccines against Salmonella enteritidis: humoral immune response

Abstract Salmonella enteritidis (SE) vaccination is one of the major tool to reduce the infection in commercial poultry. In this work, techniques, evaluating the presence of Ig-G and Ig-A in serum, tears and intestinal secretions, were studied to improve the knowledge of the humoral immune response following SE live attenuated vaccination. The Ig-G and Ig-A ELISA are important and easy tests to plan and optimize SE vaccination programs.


Introduction
Salmonellosis is one of major food-borne disease worldwide. Much effort has been made to develop effective control strategies against SE infection in laying hens, including immunization with vaccines. Several studies have suggested that live attenuated vaccines are more effective than killed vaccines in preventing SE infection (Okamura et al., 2004). Attenuated vaccines con-tain modified live bacteria that are no longer capable of causing clinical disease but still capable to multiply and immunize the animal (Dee Grifin et al., 2002). Immunization of animals with live attenuated Salmonella vaccine is usually associated with stimulation of mucosal, systemic and cell-mediated immune response. Vaccination by killed vaccines or bacterins does not lead to the induction of either mucosal or cellular immune response, and humoral immunity may be relatively short lived (Curtiss et al., 1993).
The aim of this study was to evaluate the efficacy of in vitro test to optimize the use of a live commercial SE vaccine checking the humoral immune response.

Animals and immunization
Twenty 1 day-old commercial laying hens were housed and eyedrop vaccinated at 14 and 28 days following the label instructions (Gallivac SE, Merial, France). Ten birds were reared separately as negative controls.
Every week tears, sera and intestinal secretions were collected.

ELISA Test
Salmonella enteritidis antigen was purified as described by Beal et al. (2004).
ELISA Salmonella enteritidis Test Kit (IDEXX) was compared with the above "home made" ELISA SE Ig-G; respectively the OD (Optical Density) and the titres had the same trend (results not shown).

Results and discussion
Humoral immunity contributes to eliminate S. enteritidis from the intestine (Berthelot-Herault et al., 2003) and to lesser extend from liver and spleen (Desmidt et al., 1998). Antibodies may not protect against the intracellular stage of infection, but may provide an efficient macrophage-mediated opsonization and a final destruction of bacteria (Lillehoj et al., 2003). One week post vaccination (PV), we observed that the Ig-G titres were negative and reached the peak two weeks after the second vaccination ( Figure 1).
Secreted Ig-A antibodies play a significant role by inhibiting the adherence of microorganism to mucosal cell to preventing the translocation from the gastrointestinal tract (Desmit et al., 1998) to the GALT (Gut-Associated Lymphoid Tissue). Berthelot-Herautlt et al. (2003) suggest that IgA antibody response also contribute to the later elimination of the pathogen from the intestine.
In this study Ig-A antibodies were detected in tears, intestinal secretions and sera, showing a Ig-A peak during one week post booster vaccination. Tears had significantly higher ELISA OD values than intestinal secretions and sera. Intestinal secretions, obtained through mucosal washing, showed the lowest OD titres because they were more diluted than other samples (Figure 2).

Conclusions
Salmonella live vaccine programs are influenced by immunity status, somministration route and infection pressure; for these reasons, useful information about humoral immunity is needed to optimize the use of these vaccines in layer hens. Ig-G and Ig-A ELISA tests are easy techniques for this purpose. These antibodies are reliable to assay the animal resistance in the long period of time, because humoral immunity acts later during the infection while cell mediat-   (Poli et al., 1999).