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Title: Flow Cytometric Characterization of Peyer’s Patch and Cecal Tonsil T Lymphocytes in Laying Hens Following Challenge with Salmonella enterica serovar Enteritidis

item Holt, Peter
item Vaughn, Lara
item Gast, Richard

Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/1/2009
Publication Date: 2/1/2010
Citation: Holt, P.S., Vaughn, L.E., Gast, R.K. 2010. Flow Cytometric Characterization of Peyer’s Patch and Cecal Tonsil T Lymphocytes in Laying Hens Following Challenge with Salmonella enterica serovar Enteritidis. Veterinary Immunology and Immunopathology. 133:276-281.

Interpretive Summary: Salmonella remains a serious human food safety thereat and much attention has been focused on identifying sources of the organism and then taking steps to eliminate it from the food chain. Poultry and their products were identified early-on as an important Salmonella reservoir (Rabsch et al, 2001). Much time, research and resources have been devoted to developing methods to reduce the incidence of Salmonella in poultry flocks and therefore improve the microbial safety of the products leaving the farm. An important tool used for reducing Salmonella in poultry is to increase the resistance of the bird to the organism through stimulation of the immune system. Vaccination (Lillehoj et al., 2000; Van Immerseel et al., 2005), cytokine/chemokine administration (Lowenthal et al., 2000), and immunomodulators (He et al., 2005; Lowry et al., 2005) have all been used to enhance resistance to infection and therefore reduce Salmonella flock presence. A more thorough understanding of the avian immune system increases the potential for developing more effective methods to stimulate immunity and therefore improve the chances for creating Salmonella-free flocks. Peyer’s patches (PP) are organized lymphoid tissues located at various sites along the small intestine (Makala et al., 2002). Generally characterized as a secondary lymphoid organ, the PP has been shown to be an important regulator and amplifier of mucosal immune responses to antigenic stimuli such as infection (Keren et al., 1978). Because the mammalian PP can be radily observed via the serosal surfaces of the bowel, a large volume of research has been conducted to define the role of this tissue in providing mammalian mucosal immune protection. This is in contrast to the avian PP which is much less grossly apparent in unfixed tissue. As a result, minimal information is available regarding PP in poultry especially with respect to its role in responding to and regulating the mucosal immune response against infection by intestinal pathogens. The development of a staining method for rapid identification of PP on the serosal surface of unfixed chicken ileum (Vaughn et al., 2007) has enabled researchers to pursue a wide range of studies to delineate the role of the chicken PP in the elicitation of mucosal immune responses of the intestinal tract. Two PP were shown to predominate along the chicken lower alimentary tract, the proximal PP which was approximately 3-6 cm caudal to the Meckel’s diverticulum and the distal PP which was 7-10 cm cranial to the ileocecal junction. The location of the two PP tissues in the upper and lower portions of the ileum, respectively, could potentially impact the degree of interaction with intestinal pathogens and therefore may exhibit different cellular dynamics during challenge. Salmonellae are considered intracellular pathogens and, as a result, T cell immunity plays an important role in providing protective immunity against infection. T cell immunity is mediated and regulated by both CD4+ helper T cells (TH) and CD8+ cytotoxic T cells (TC) and within these two T cell populations the cells may express one of three antigen receptors: TCR'' (TCR1), TCRv'1 (TCR2) and TCRv'2 (TCR3). Different T cell subpopulations have been shown to be more prevalent in certain tissues and infection can change this cellular prevalence in tissues over time (Vervelde and Jeurissen, 1995; Yun et al., 2000). The current study was undertaken to analyze, via flow cytometric analysis, the T cell repertoire of proximal and distal PP prior to and following Salmonella enterica serovar Enteritidis (SE) challenge and compare these populations with those found in cecal tonsils, another intestinal lymphoid tissue.

Technical Abstract: Specific-pathogen-free Single Comb White Leghorn hens, 34 weeks old, were challenged with 6.9 X 107 phage type 13 Salmonella enterica serovar Enteritidis (SE) per os. Each week, crop lavage samples were obtained from four hens, the birds were then sacrificed and their intestinal tracts aseptically excised. A portion of one cecum from each bird was removed and that, along with the crop sample was cultured for the presence of SE. The lumen of the intestinal tract was flushed thoroughly with distilled water and the Peyer's patches (PP) were visualized by infusing the lumen with eosin stain followed by crystal violet stain (Avian Diseases 50:298-302, 2006). Proximal and distal PP were extirpated along with both cecal tonsils (CT), the cells purified and then aliquots of cells (2 X 105) were incubated on ice with antibodies to different T cell markers, washed and then T subsets identified via flow cytometric analysis. Crop and cecal levels of SE exceeded 1 X 103 SE/g by day 8 post infection (PI), decreasing thereafter. CD4/CD8 ratios decreased at one week PI in the CT and distal PP and at two weeks PI in the proximal PP, then ratios increased in each instance back to control levels one week later. CD4+ T cells ranged from 19-22% in the PP and 17-19% in the CT while CD8+ T cells ranged from 12-15% in the PP and 10-15% in the CT. The predominant TCR was TCR2 (v'1) followed by TCR3 (v'2). Low numbers of TCR1 ('') cells were observed. These results indicate that differences in cell populations occur in the PP and CT cell populations and SE infection can affect the cellular dynamics of these lymphoid tissues. Abbreviations: FITC, fluoresceine isothiocyanate; PBS, phosphate buffered saline; PP, Peyer’s patch; R-PE, R-phycoerythrin; S., Salmonella; TCR, T cell receptor; Cy5, Cyanine 5; XLT4, xylose lysine tergitol-4 agar; SE, Salmonella enterica serovar Enteritidis; BSL-2; biosafety level-2