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Title: CELLULAR ASSESSMENT OF CROP LYMPHOID TISSUE FROM SPECIFIC-PATHOGEN-FREE WHITE LEGHORN CHICKENS AFTER SALMONELLA ENTERITIDIS CHALLENGE

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

Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/23/2008
Publication Date: 8/19/2008
Citation: Vaughn, L.E., Holt, P.S., Gast, R.K. 2008. Cellular assessment of crop lymphoid tissue from specific-pathogen-free white leghorn chickens after salmonella enteritidis challenge. Avian Disease. 52:657-664.

Interpretive Summary: We have been conducting research on the immune response of the gastrointestinal (GI) tract of chickens against Salmonella Enteritidis (SE). An organ of study has been the crop located along the esophagus in the upper GI tract of the chicken. An IgA antibody response directed against SE-lipopolysaccharide (LPS) has been detected in the crop after SE challenge. Additionally, crop tissue sections have revealed speculative areas of lymphoid tissue when stained with hematoxylin & eosin (H&E) and evaluated with a light-microscope. Therefore, it appears that the crop organ of the chicken may have the capacity to generate an immune response locally along the upper GI tract. To further investigate this, we orally infected chickens with Salmonella Enteritidis (SE) and established the following objectives: (a) monitor progression of SE infection by culturing cecum and liver-spleen samples for SE; (b) collect fluid samples from the crop for assessment of a SE-LPS-specific IgA antibody response; (c) harvest crop tissue sections to look for presumptive lymphoid areas with H&E stain and microscopy; and then further evaluate the areas using histochemical and immunohistochemical (IHC) staining techniques to try to definitively identify lymphoid-lymphocytic cells and to characterize the cell populations. The culture results showed that SE was present in all of the ceca and liver-spleen samples at 1 week post-infection (pi), and then the number of SE positive samples declined as the weeks increased following the initial SE infection. IgA antibodies against SE-LPS were detectable in crop samples as early as 1 week post-SE-infection (pi), and a high antibody response was detected by 2-3 weeks pi. The SE-LPS-specific IgA antibody response remained elevated above the uninfected level at 10 weeks pi. Microscopy evaluation of H&E stained crop tissue sections identified presumptive lymphoid tissue areas. Those areas stained positive with a general leukocyte (white blood cell) marker, thus the areas were definitively identified as clusters of lymphoid-lymphocytic cells. Cellular characterization of the crop lymphoid sites revealed cells that stained positive for B-lymphocytes and plasma cells. The crop of the chicken appears to be an organ along the upper GI tract where lymphoid tissue may arise in response to SE oral infection, and a site where an antibody response may be generated against the SE pathogen.

Technical Abstract: The crop may be an important site along the upper alimentary tract where a humoral immune response against SE may perhaps be elicited locally. The mucosal immune response within the crop (ingluvies) of specific-pathogen-free (SPF) White Leghorn (WL) chickens against Salmonella Enteritidis (SE) was investigated. Three trials were conducted using SPF WL pullets at age 5-6 weeks. Trial 1 consisted of seventy-seven birds evaluated for 10 weeks post-SE infection (pi); trial 2 was comprised of seventy-two birds monitored through 8 weeks pi; and trial 3 was made up of thirty birds assessed for 5 weeks pi. Birds were challenged per os with 10e8 CFU/ml SE phage type 13 (PT13). Crop lavage samples, crop tissues, ceca and/or liver-spleen were collected pre-infection then at weekly intervals post-SE infection. Bacteriological examination of cecal contents and/or liver-spleen occurred weekly to monitor progression of SE infection. Crop lavages were analyzed for SE-lipopolysaccharide (LPS)-specific IgA by ELISA to assess humoral immune response. General histochemical staining [hematoxylin & eosin (H&E) and methyl green-pyronin (MGP)] and immunohistochemical (IHC) staining (monoclonal antibodies CD45 & Bu-1) were applied to serial sections of crop to evaluate lymphoid tissue via light microscopy, to grade isolated lymphoid follicles (ILFs) using score 0 (minimal) to 5 (sizable) scale, and to characterize the cellular population of ILFs. Results revealed that cecum samples and liver-spleen samples were 100% SE culture positive at 1 week pi, then the percentage of SE positives progressively declined over time. Markedly increased crop SE-LPS-specific IgA antibodies were detected in crop samples by 2-3 weeks pi, and the humoral response remained elevated above week 0 baseline for the duration of each trial. Crop ILFs of score 3 to 5 were observed in H&E stained tissues, with an increased proportion of ILFs in post-SE-infected crops versus uninfected. MGP staining showed plasma cells scattered within and at the periphery of ILFs. IHC staining revealed CD45 (pan-leukocyte) and Bu-1 (B-lymphocyte) positive cells within crop ILFs. The chicken crop appears to be an organ where lymphoid tissue may arise in response to enteric SE infection, and a site where a humoral response may be generated against the SE pathogen.