Six male New Zealand White rabbits, 8 weeks old, each weighing about 1.5kg, were procured from your Pasteur Institute, Tehran-Iran. ferric enterobactin binding protein may lead to its application in the restriction of Enterobacteriaceae propagation. O157:H7 causes sporadic outbreaks of intestinal disease in man. It has achieved notoriety as the cause of a number of large food- and water-borne outbreaks (1C3). Enterohemorrhagic (EHEC) O157:H7 is an emerging pathogen that causes acute human gastroenteritis and hemorrhagic colitis (4, 5). EHEC also causes disease in newborn calves and asymptomatically colonizes the gut mucosa of adult bovines, constituting the main reservoir for food and environmental contamination (6, 7). Cattle as Gefitinib (Iressa) well as other ruminants serve as a reservoir for O157:H7. In particular, surveys of beef and dairy cattle have exhibited carriage rates less than 0.5 to greater than 2.0% (8). As a result of O157:H7 carriage in cattle, beef Rabbit polyclonal to NOTCH4 and dairy products often Gefitinib (Iressa) become contaminated and serve as a source of contamination in outbreaks of O157:H7. In addition, it is associated with Shiga toxins (Stx), which can cause systemic complications including hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) affecting the kidneys and the central nervous system that may lead to death (9). The ability of the organism to survive in feed, water, soil, and manure has important implications for its persistence in cattle herds and contamination of water materials and crops. Effective measures to reduce or eliminate O157:H7 in cattle will reduce not only food borne illness but also the risk of transmission of the organism into the environment (10). A variety of treatment and prevention strategies to protect against O157:H7 are currently in development; these include toxin receptor analogues, passive antibody therapy, and vaccines to protect humans against the systemic effects of the toxin. Since an O157:H7 vaccine has not been developed or licensed for immunization of humans except for two vaccines that are currently in use in cattle, the most encouraging prevention strategies for O157:H7 focus on minimizing exposure to this pathogen (11C14). Iron is an important micronutrient for virtually all living organisms, except lactic acid bacteria where manganese and cobalt are used in place of iron. The low solubility of the ferric form is usually a serious obstacle to microorganisms that require iron. Possession of iron uptake systems is known to be important in bacterial pathogenesis. Targeting surface-exposed, specific antigenic proteins that can induce antibody production to block uptake of vital elements such as iron into the bacterial cell is usually a strategy to combat or control pathogens. The iron-regulated outer membrane proteins (IROMPs) are appealing candidates as vaccine antigens because they are surface-exposed, antigenic, and may induce production of antibodies to block uptake of iron into the bacterial cell (15). Our previous statement on immunogenicity of the membrane protein, FepA from O157:H7, indicated that this antibody produced therein could efficiently recognize and bind ferric enterobactin binding protein in the immunized mice challenged with higher doses of bacteria (16). Passive immunization is an appropriate therapeutic option against many organisms. Passive immunization with specific antibodies may have the potential to reduce O157:H7 pathogenesis in hosts (17). In order to practically exercise the application scope of the immunity brought about by FepA, this study was designed to evaluate active and passive immunity against that share gene homology of above 52%. MATERIALS AND METHODS Bacterial strains, plasmids, and culture media Ol57:H7 (ATCC 43889), (NTCC 12678), (ATCC 13883), and (PTCC 1609) were used. The bacteria were cultured Gefitinib (Iressa) in LuriaCBertani (LB) broth or LB agar Gefitinib (Iressa) at 37C for 24 hours media were supplemented, when required, with kanamycin (SIGMA, 40g/ml). Sorbitol-MacConkey agar made up of 0.5 mg/ml cefixime and 2.5 mg/ml potassium tellurite was used to test shedding of O157:H7. The plasmid pET28a(+) harbouring gene was constructed in our laboratory (16) was utilized for expression of the protein. Bacterial strains were produced in LB broth at 37C and were stored at 70C in 20% glycerol. NickelCnitrilotriacetic acid (NiCNTA) agarose was from Qiagen (Valencia, USA). All other analytical grade chemicals and Gefitinib (Iressa) reagents were from Merck (Darmstadt, Germany). Animals and animals husbandry Animal isolation rooms experienced individual floor drains. Floors and walls were washed once daily with.