Progress 01/15/04 to 01/14/09
Outputs
OUTPUTS: The most significant outputs from this award were clearly the contributions made toward the generation of transgenic cattle that have the ability to produce lysostaphin in their milk and thus are resistant to mastitis caused by Staphylococcus aureus. This contagious pathogen is a significant cause of bovine mastitis and is notorious for causing a chronic form of the disease. Affected cows are often culled from the herd due to the persistent infection, and to prevent its spread to herdmates. This work was based on the continuous expression of lysostaphin in milk under the direction of the beta-lactoglobulin (a major milk protein) regulatory region and served as a proof of principle for this strategy. This regulatory region directed the synthesis of lysostaphin specifically to lactating mammary epithelial cells. We went on to generate a bovine mammary cell culture model to identify a large number of genes that are rapidly induced in response to infection. The regulatory regions of these genes could potentially serve as components of an infection-inducible expression system. The mammary cell model and a murine infection model were evaluated with microarray-based technology to elucidate the acute genomic response to infection. The regulatory region of one of these genes was then cloned and evaluated. Finally, in consideration that lysostaphin is not effective against other mastitis-causing pathogens, we went on to isolate a novel enzyme that has considerable activity against Streptococcus uberis, which is another significant cause of bovine mastitis. This bacteriophage lysin gene has been distributed to collogues at two separate institutions and the actual lysin protein is being used at a third location to study its crystal structure. The bovine mammary cells have been distributed to colleagues at four separate institutions. In addition to nine peer-reviewed publications there were ten abstracts resulting from this award that were presented in oral or poster format at national and international meetings. Two patents were awarded, and the DNA sequence of the bovine lactoferrin promoter and S. uberis PLY700 phage lysin gene were submitted to Genbank. The award led to the graduation of one Ph.D. and one M.S. student, and two post-doctoral fellows further developed their research careers. PARTICIPANTS: Principle Investigator: David E. Kerr was the PI responsible for general oversight of all aspects of the project. This included recruiting graduate students and post-doctoral fellows, identifying collaborators, coordinating research activities, and overseeing the publication and presentations of research findings. Graduate students: Former graduate students with major contributions to the research conducted with this award include Jiamao Zheng (obtained his Ph.D.) and Laura K. Celia (obtained her M.S.). Dr. Zheng is currently pursuing post-graduate studies at North Western University, while Ms. Celia is employed at the American Tissue Type Collection (ATCC). Post-doctoral fellows: Dr. Olga Wellnitz and Dr. R. Pareek enhanced their professional development during this award. Major collaborators: Drs. Robert J. Wall, and Kevin D. Wells (ARS-USDA) were instrumental in generating the transgenic animals. Dr Daniel D. Nelson (Rockefeller University) played a major role in evaluating the activity of the Ply700 bacteriophage lysin. TARGET AUDIENCES: The dairy agricultural community was the key target audience. The new research findings were delivered through publication of results in key, peer-reviewed scientific journals and presentation of results at various national and international scientific meetings. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A major impact was in the use of transgenic animal technology to develop a line of dairy cows that are highly resistant to S. aureus mastitis. This proof of concept opens the door to additional uses of transgenic animal technology to modify animals for agricultural purposes. Another outcome is the discovery of a bacteriophage enzyme that has killing activity against S. uberis. This enzyme contains both a cell binding domain and an enzymatic domain. Each domain has potential for further modification to enhance activity or form a component of a newly designed molecule. Finally, the genomic analysis of the mammary response to infection has identified numerous cytokine and chemokine genes that are rapidly induced. This work has also verified the microarray technology as a powerful technique to potentially examine differences between animals in the responses to a particular pathogen, or differences in how animals respond to different mastitis-causing pathogens.
Publications
- JOURNAL ARTICLES: Celia, L.K., Nelson, D., and Kerr D.E. 2008. Characterization of a bacteriophage lysin (Ply700) from Streptococcus uberis. Veterinary Microbiology, 130:107-117.
- Powell, A.M., Kerr, D.E., Guthrie, D., and Wall, R.J. 2007. Lactation induction as a predictor of post-parturition transgene expression in bovine milk. Journal of Dairy Research, 74:247-254.
- Zheng, J., Watson, A.D., and Kerr, D.E. 2006. Genome-Wide Expression Analysis of Lipopolysaccharide-Induced Mastitis in a Mouse Model. Infection and Immunity, 74:1907-1915.
- Donavan, D.M., Kerr, D.E., and Wall, R.J. 2005. Engineering disease resistant cattle. Transgenic Research, 14:563-567.
- Wall, R.J., Powell, A.M., Paape, M.J., Kerr, D.E., Bannerman, D.D., Pursel, V.G., Wells, K.D., Talbot, N., and Hawk, H.W. 2005. Genetically enhanced cows resist intramammary Staphylococcus aureus infection. Nature Biotechnology, 23:445-451.
- Zheng J., Ather, J.L., Sonstegard, T.S., and Kerr, D.E. 2005. Characterization of the infection-responsive bovine lactoferrin promoter. Gene, 353:107-117.
- Fleming, J.M., Leibowitz, B.J., Kerr, D.E., and Cohick, W.S. 2005. IGF-I differentially regulates IGF binding protein expression in primary mammary fibroblasts and epithelial cells. Journal of Endocrinology, 186:165-78.
- Pareek, R., Wellnitz, O., Van Dorp, R., Burton, J., and Kerr, D.E. 2005. Immunorelevant gene expression in LPS-challenged bovine mammary epithelial cells. Journal of Applied Genetics, 46:171-177.
- Wellnitz, O. and Kerr, D.E. 2004. Cryopreserved bovine mammary cells to model epithelial response to infection. Veterinary Immunology and Immunopathology, 101:191-202.
- ABSTRACTS: Kerr, D.E., Latshaw, M. and Parik, R. 2008. Genomic response of immune associated genes to LPS challenge in bovine mammary gland and epithelial cells. Journal of Dairy Science 91 (Suppl. 1).
- Kerr, D.E., Latshaw, M., Pareek, R., Zheng, J., and Bond, J.P. 2007. Comparison of genomic responses to acute LPS challenge of bovine mammary epithelial cells in vitro, and bovine and murine mammary glands in vivo. 8th International Veterinary Immunology Symposium, Ouro Preto, Brazil.
- Kerr, D.E., Pareek, R.S., Mcfadden, T.B., Bond, J.P., Watson, A.D., and Dowd, S.E. 2006. Affymetrix GeneChip-based analysis of the genomic response to acute LPS-induced bovine mastitis. 2nd International Symposium on Animal Functional Genomics, East Lansing, MI.
- Celia, L.K. and Kerr, D.E. 2005. Bacteriophage lysins to combat mastitis due to Streptococcus uberis. 105th General Meeting of the American Society for Microbiology. Atlanta GA.
- Pareek, R., Wellnitz, O., Burton, J.L., and Kerr, D.E. 2005. Microarray analysis of immunorelevant gene expression in LPS-challenged bovine mammary epithelial cells. 2005. Journal of Dairy Science 88 (Suppl. 1).
- Zheng, J., Watson, A., and Kerr, D.E. 2005. Microarray analysis of LPS-induced mastitis in a mouse model. Journal of Dairy Science 88 (Suppl. 1).
- Wellnitz, O. and Kerr, D.E. 2004. Mammary epithelial cell response to infection with different bacteria. 55th Annual Meeting of the European Association for Animal Production. Bled, Slovenia.
- Zheng, J., Ather, J.L., and Kerr, D.E. 2004. Functional characterization of the bovine lactoferrin gene promoter. 7th International Veterinary Immunology Symposium. Quebec City, Canada.
- Kerr, D.E. and Wellnitz, O. 2004. Cryopreserved bovine mammary cells to model epithelial response to infection. 7th International Veterinary Immunology Symposium. Quebec City, Canada.
- PAPERS IN PROCEEDINGS: Kerr, D.E., Bannerman, D.D., Paape, M.J., and Wall, R.J. 2005. Can we engineer genetic resistance to mastitis NMC Regional Meeting. Burlington, Vermont. pages 31-36.
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Progress 10/01/04 to 09/30/05
Outputs
This year, our successful collaborative efforts to produce transgenic cows with enhanced resistance were published in a high profile scientific journal. These cows produce the anti-staphylococcal enzyme, lysostaphin, in their milk and are highly resistant to mastitis caused by S. aureus. We have also made good progress on identifying and cloning an enzyme that has anti-streptococcal activity. It is anticipated that such an enzyme will be effective in prevention of streptococcal mastitis. This enzyme was discovered in a bacteriophage residing in a strain of Streptococcus uberis, a major mastitis pathogen. Another aspect of our research is to identify infection-responsive genes in the bovine mammary gland. The controlling elements of these genes may be effective to control transgenic production of anti-bacterial enzymes. We have used a genomic approach to simultaneously evaluate the endotoxin-responsiveness of approximately 20,000 genes in the bovine mammary gland. Our
preliminary data indicates that approximately 200 genes are activated by more than two-fold within four hours of exposure to E. coli endotoxin. The genomic results are now being validated by a more accurate technique (real-time RT-PCR).
Impacts
The long-term goal is to develop transgenic dairy cows with enhanced resistance to mastitis. We have shown that transgenic cows producing lysostaphin in their milk are highly resistant to S. aureus infection. The goals of the current project are to design, assemble, and test new constructs that will further our previous positive results by providing protection against other mastitis-causing pathogens, and also will offer protection in an infection responsive manner.
Publications
- Celia, L.K. 2005. Characterization of a Streptococcus uberis derived bacteriophage lysin. M.S. Thesis, University of Vermont. (D.E. Kerr Advisor).
- Donovan, D.M., D.E. Kerr, and R.J. Wall. 2005. Engineering disease resistant cattle. Transgenic Research 14:563-567
- Wall, R.J., A.M. Powell, M.J. Paape, D.E. Kerr, D.D. Bannerman, V.G. Pursel1, K.D. Wells, N. Talbot, and H.W. Hawk. 2005. Genetically enhanced cows resist intramammary Staphylococcus aureus infection. Nature Biotechnology 23:445-451.
- Zheng J., J.L. Ather, T.S. Sonstegard, and D.E. Kerr. 2005. Characterization of the infection-responsive bovine lactoferrin promoter. Gene 353:107-117.
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