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United States Department of Agriculture

Agricultural Research Service

Research Project: STRESS EFFECTS ON IMMUNITY AND PHYSIOLOGY OF POULTRY Title: Use of Novel Staining Procedure to Visualize and Isolate Chicken Peyer’s Patches for Intestinal Immune Response Studies Against Salmonella Infection

Authors
item Holt, Peter
item Vaughn, Lara
item Moore, Randle
item Gast, Richard
item Kaiser, Pete - INST FOR ANIMAL HEALTH

Submitted to: Avian Immunology Group Meeting
Publication Type: Abstract Only
Publication Acceptance Date: May 22, 2006
Publication Date: October 21, 2006
Citation: Holt, P.S., Vaughn, L.E., Moore, R.W., Gast, R.K., Kaiser, P. 2006. Use of novel staining procedure to visualize and isolate chicken peyer’s patches for intestinal immune response studies against salmonella infection. 9th Avian Immunology Group Meeting.p. 57

Technical Abstract: The ileal Peyer's patches (Pp), secondary gut-associated lymphoid tissue of the mucosal immune system, may serve as an important site for monitoring inflammatory and immunological responses of the host against enteric pathogens. Chicken Peyer's patches are often difficult to observe grossly and a simple technique to enhance visualization of the Pp is lacking. Therefore, we designed a novel staining method that is quick, easy and accurate to aid in gross identification and recovery of the chicken Pp from fresh tissue specimens. The ileocecocolic region was excised intact from White Leghorn hens, flushed with deionized-water to remove ingesta, then a dilute Eosin-Y solution was infused. After 1 minute, the Eosin-Y was gently extruded. Modified-Crystal Violet was then injected into the GI segment, where upon the lymphoid tissue area became apparent at the serosal surface. The distal ileal Pp appeared as a pale whitish-pink ovoid focalized area with surrounding gut tissue stained light purple. The Pp site could be delineated at the serosal and mucosal surface by gross assessment. Light-microscopy evaluation of H&E stained tissue slides prepared from the excised Pp site revealed lymphoid tissue aggregations with multiple follicular units indicative of Pp. Using this staining technique, we collected Pp from adult Salmonella enterica serovar Enteritidis (SE)-challenged White Leghorn hens at days 1, 4, 11 and 19 post-infection (PI). The impact of infection on Pp cytokine mRNA expression was assessed via quantitative RT-PCR. IL-10 and IL-18 mRNA expression was largely unaffected by infection. At 24 hours PI, expression of IFN-gamma, IL-6, and IL-4 mRNA was upregulated 101-, 29-, and 5.5-fold, respectively, which dropped back to control levels by day 4 PI. IL-12 expression was slightly upregulated at 24 hours PI (2.7-fold) but was downregulated 10-fold at day 4 PI. This simple staining technique, combined with molecular-based assays, will enable researchers interested in poultry mucosal immunology to more effectively dissect the development of immunity in the intestinal tract and to analyze the role of GALT cytokine expression in response to infection.

Last Modified: 10/21/2014
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