J. S. Hogan1, J. Lin, and K. L. Smith
The Ohio State University Department of Animal Sciences
Purified bovine and mouse antibodies directed against the iron receptor protein FepA inhibited growth of coliform bacteria in vitro. Growth of Escherichia coli was significantly inhibited by the addition of monoclonal antibody to synthetic medium containing apolactoferrin. Antigenic variation in the ferric enterobactin binding site resulted in a low percentage of Klebsiella pneumoniae isolates that were inhibited by the monoclonal antibody. Purified bovine immunoglobulin G (IgG) directed against FepA inhibited growth of E. coli but not K. pneumoniae isolates cultured in dry-cow secretion. Exogenous ferric chloride completely reversed the inhibitory effects of antibodies and lactoferrin. Immunoglobulin G from cows immunized with FepA apparently inhibited the growth of coliform bacteria by interfering with the binding of ferric enterobactin complex to the cell surface receptor FepA.
Controlling coliform mastitis during the dry period is based on the knowledge that bacterial growth can be controlled by preventing the uptake of essential nutrients. A limiting nutritional factor for many coliform bacteria in secretion from involuted mammary glands is iron. Iron is essential for most coliform bacteria to fulfill normal metabolic processes. The protein lactoferrin binds iron and makes the element unavailable to bacteria. Data from our laboratory have demonstrated that the ability of E. coli and K. pneumoniae to cause mastitis is related to the ability of these isolates to overcome the inhibitory properties of lactoferrin. Coliforms may overcome the inhibitory effects of lactoferrin with one of several iron acquisition systems, including enterochelin, aerobactin, citrate, and ferrichrome systems. Coliform isolates that infect involuted mammary glands probably do so as a result of one or more of these systems.
The primary objective of the project was to determine the feasibility of developing a mastitis vaccine that would inhibit growth of coliform bacteria in mammary secretion by preventing the uptake of iron. The first series of trials investigated the prevalence of specific iron uptake mechanisms among coliforms isolated from intramammary infections. All these isolates shared a specific enterochelin-iron retrieval system that included a protein on the cellular surface named FepA. The FepA was determined to be an excellent protein from which to formulate the vaccine because FepA was expressed on all isolates, and FepA caused an antibody response when injected in rabbits and mice. More importantly, the antibody produced in mice and rabbits blocked the uptake of iron by Escherichia coli.
Trials in the second phase of the project concentrated on adapting the technology we used in laboratory animals for use in dairy cows. The FepA vaccine caused an immune response in cows, and the antibody blocked growth of E. coli in synthetic medium and dry-cow secretion. While vaccination with FepA was effective against E. coli, the immune response was not effective against Klebsiella. Antibody from cows vaccinated with E. coli J5 did not alter growth responses of either E. coli or Klebsiella. Addition of excess iron to the media reversed the inhibitory activity of the antibody directed against FepA but had no effect on bacteria treated with antibody against E. coli J5.
Antibodies from cows immunized with FepA apparently inhibited the growth of coliform bacteria by interfering with the binding of ferric enterobactin complex to the cell surface receptor FepA. Antibody against FepA starve the bacteria of iron, thus preventing their ability to multiply. The mode of action for E. coli J5 vaccines is to coat bacteria, whereby the white blood cells can more effectively digest and kill the bacteria. Therefore, the actions of the two vaccines could be additive. A combination of FepA plus E. coli J5 vaccine may have a greater efficacy than the currently marketed E. coli J5 vaccines.
1 For more information, contact at: The Ohio State University, Ohio Agricultural Research and Development Center, 302 Pounden Hall, 1680 Madison Avenue, Wooster, OH 44691; (330) 263-3792, Fax (330) 263-3949; email:hogan.4@osu.edu