Project Number: 6612-32000-055-01
Project Type: Nonfunded Cooperative Agreement

Start Date: Aug 01, 2005
End Date: Jul 31, 2009

Objective:
The objective of this cooperative research project is to complete immunohistochemistry utilizing affinity purified antibodies to the major outer membrane protein of Campylobacter jejuni for detecting this agent in chicken tissues. This will help to determine the presence of this agent in tissues of the chicken gastrointestinal tract, reproductive tract and potentially among internal organs.

Approach:
Affinity purified polyclonal antibodies have been produced in rabbits (Sigma-GenosysTM) by standard procedures as part of ongoing research at the Poultry Microbiological Safety Research Unit, RRC, ARS, USDA. These antibodies were directed to peptide antigens representing the major outer membrane protein of Campylobacter jejuni. Tissues have been collected from chickens considered positive and negative for Campylobacter jejuni by aseptic methods. The following tissues were collected and fixed by immersion in 10% neutral buffered formalin for 48 hours: spleen, thymus, bursa, ceca, proventriculus, pancreas, small intestine, cecal tonsils, large intestine, caudal thoracic air sac, trachea, lung, heart, liver, kidney, Harderian gland, and reproductive tracts. Tissues have been further processed into paraffin. Tissues ill be further examined by investigators at the Department of Pathology, College of Veterinary Medicine, University of Georgia by sectioning tissue samples for hematoxylin and eosin staining followed by immunohistochemistry (IHC) utilizing standard pathology techniques. Following deparaffinization, sections will be rehydrated, and antigen sites exposed by microwaving (10 minutes at full power) in Vector antigen unmasking solution (Vector Laboratories, Burlingame, CA). Sections will be blocked (Universal Blocking Reagent; Biogenex, San Ramon, CA), and then incubated overnight at 4 C (or for 2 hr at 37 C) with primary anti-peptide (anti-MOMP protein) antibody diluted 1:8,000. After a brief wash, sections will be incubated with biotinylated goat anti-rabbit antibody, and then with avidin-biotin alkaline phosphatase (Vector Laboratories, Burlingame, CA). Substrate development will be with Vector Red (Vector Laboratories, Burlingame, CA). Sections will be counterstained lightly with hematoxylin and coverslipped for a permanent record.