Progress 11/07/01 to 11/06/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Mastitis is a complex disease caused by numerous etiological agents, including Staphylococcus aureus, coagulase-negative Staphylococcus species, environmental Streptococci, Gram-negative bacteria (e.g., Escherichia coli), and Mycoplasma bovis. Mastitis is one of the most costly diseases in U.S. agriculture with economic losses to the dairy industry approaching $2 billion annually. Mastitis reduces milk production by 5-30%, which contributes to 65-70% of the total economic losses attributed to the disease. Additional economic losses resulting from mastitis include milk discarded due to antibiotic treatment, premature culling due to chronic infection and damage to the udder, and veterinary treatment costs. The dairy industry requires new tools to solve the mastitis problem. Antibiotic
therapy remains sub-optimal or ineffective for the treatment of many types of intramammary infections. In addition to problems of efficacy, the persistent use of antibiotics increases the risk for development of antibiotic-resistant strains of bacteria. Further, antibiotic contamination of milk is estimated to cost the U.S. dairy industry $50 million annually. Investigating the genetic, molecular, cellular, and immunological mechanisms that are operative during intramammary infection will provide insight into host defense mechanisms and disease resistance factors that contribute to the outcome of intramammary infection. This research will generate new genomic and immunologic tools and reagents for enhancing understanding of the mechanisms of disease resistance and tissue repair, and will lead to the development of biotherapeutics that reduce the incidence, severity, and/or duration of mastitis and the substantial economic losses associated with this disease. Specifically, this
research is expected to result in the: 1) development of immune-based interventions that reduce the incidence and severity of mastitis; 2) identification of disease resistance genes that can be used for the selective breeding of animals with enhanced resistance to mastitis; 3) development of immunological reagents necessary for further research into the host immune responses of cattle during bacterial-mediated diseases; and 4) development of strategies that prevent mammary tissue injury and enhance tissue repair following mastitis, and increase milk production efficiency. Development of interventions that reduce the severity and incidence of mastitis and that improve milk production efficiency will benefit dairy producers by deceasing economic losses associated with the disease. Consumers will benefit by having less expensive and higher quality milk. Because there are similarities in immune response to infection across species, scientists engaged in agricultural and human health
research will benefit from increased understanding of mechanisms of host innate immune responses to bacterial infection and generation of additional tools for research. Pharmaceutical companies will be able to use knowledge generated from this research to develop new biotherapeutics for the treatment of mastitis and other bacterial-mediated diseases. The outcomes of this research directly support Component 2 (Genetic and Biological Determinants of Disease Susceptibility), Problem Statement 2A: Mastitis, in the Animal Health NP 103 Action Plan. The research addresses Agency Performance Measure 3.2.1 - Provide scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health; Agency Performance Measure 3.2.2 - Identify, develop, and release to the U.S. agricultural community genetic markers, genetic lines, breeds, or germplasm that result in food animals with improved (either through traditional breeding or
biotechnology) pest- and disease-resistance traits; and Agency Performance Measure 3.2.3 - Develop and transfer tools to the agricultural community, commercial partners, and Federal agencies to control or eradicate domestic and exotic diseases that affect animal and human health. 2. List by year the currently approved milestones (indicators of research progress) Objective 1: Develop methods for modulating mammary gland cell-mediated and humoral immune responses. Milestone 1 (12 months) Evaluation of the repertoire of cytokines and chemokines produced by bovine mammary CD+8 lymphocytes. Identify the precise effector function and preferential trafficking of Th2 versus Th1 effector cells to the mammary gland. Milestone 2 (24 months) In vitro screening of CpG oligodeoxynucleotides to identify motifs for use in the bovine system. Evaluate bovine EST libraries for gene sequences of bovine chemokine reagents. Milestone 3 (36-48 months) In vivo studies to determine candidate CpGs for use as
mastitis vaccines adjuvants. Production of bovine chemokines using EST libraries. Milestone 4 (48-60 months) Formulation of mastitis vaccine adjuvants using candidate CpGs and determine immune responsiveness to CpGs as mastitis vaccine adjuvants. Continued production of bovine chemokines and novel cytokines. Objective 2: Enhance the role of phagocytic cells in preventing mastitis. Milestone 1 (12-36 months) Define the cytokine profile in E. coli infected mammary glands. Determine the role(s) and mechanism(s) of CD14 mediated responses in coliform mastitis. Production of transgenic mice expressing bovine CD14 and intramammary challenge with E. coli. Milestone 2 (48 months) Identify factors affecting cytokine secretion by epithelial cells. Production of anti-bovine CD14 monoclonal antibodies. Milestone 3 (60 months) Determination of the presence of CD14 in milk and serum following infection with E. coli. Production of CD14 in tobacco plants. Studies on in vitro and in vivo
neutralization of endotoxin. Production of BsAb and evaluation of effectiveness against mastitis. Objective 3: Develop a vaccine for the prevention and cure of S. aureus mastitis. Milestone 1 (12-23 months) Evaluation of the trivalent vaccine and antibiotics to cure chronic S. aureus infections. Initial studies on methods for immunizing heifers with the trivalent vaccine for prevention of S. aureus mastitis. Milestone 2 (36 months) Fingerprinting the S. aureus recovered from both chronic cows and heifers. Initial trial of S. aureus vaccine effectiveness in microspheres. Milestone 3 (12-60 months) Field trials and challenge studies to determine protectiveness of trivalent vaccine. Field trials using the trivalent vaccine to cure chronic S. aureus in individual farmers herds. Milestone 4 (36-60 months) Evaluation of endocytosis and phagocytosis of bacteria by epithelial cells. Test transport of antibacterial agents into and across the blood/milk barrier and their ability to kill
bacteria inside epithelial. 4a List the single most significant research accomplishment during FY 2006. The single most significant accomplishment of FY2006 was the completion of a study evaluating the efficacy of tobacco-produced recombinant bovine soluble (rbos) CD14 to enhance clearance of intramammary E. coli infections. Specifically, it was demonstrated that intramammary administration of plant-derived rbosCD14 reduced bacterial levels in E. coli-infected quarters. Since 300,000 animals die or are culled annually due to acute coliform mastitis, the development of sCD14 as a therapeutic has the potential to save the dairy industry ~$600 million annually in cattle replacement costs. This research also established an efficient and cost-effective method for producing CD14 in quantities necessary for therapeutic application. This accomplishment directly supports Component 2 (Genetic and Biological Determinants of Disease Susceptibility), Problem Statement 2A: Mastitis, in the
Animal Health NP 103 Action Plan. The research addresses: Agency Performance Measure 3.2.1 - provide scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health; and Agency Performance Measure 3.2.3 - develop and transfer tools to the agricultural community, commercial partners, and Federal agencies to control or eradicate domestic and exotic diseases that affect animal and human health. 4b List other significant research accomplishment(s), if any. The following accomplishments directly support Component 2 (Genetic and Biological Determinants of Disease Susceptibility), Problem Statement 2A: Mastitis, in the Animal Health NP 103 Action Plan. These accomplishments address: Agency Performance Measure 3.2.1 - provide scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health; Agency Performance Measure 3.2.2 -
identify, develop, and release to the U.S. agricultural community genetic markers, genetic lines, breeds, or Germplasm that result in food animals with improved (either through traditional breeding or biotechnology) pest- and disease-resistance traits; and Agency Performance Measure 3.2.3 - develop and transfer tools to the agricultural community, commercial partners, and Federal agencies to control or eradicate domestic and exotic diseases that affect animal and human health. Characterization of the innate immune response to intramammary infection. Characterized changes in the expression of the inflammatory-regulating cytokines, TGF-alpha, TGF-beta1, and TGF-beta2, during Staphylococcus aureus-induced mastitis and characterized the innate immune response to intramammary infection with Pseudomonas aeruginosa. This research represents a continuation of our efforts to identify the effect that differential cytokine expression has on the outcome of infection. It is expected that this
ongoing work will lead to the identification of genes involved in disease resistance and identify potential recombinant cytokine therapeutics that can be used in the treatment of chronic intramammary infection. Sequencing, mapping, and functional characterization of innate immune signaling molecules. We have sequenced, mapped, and cloned bovine flice-like inhibitory protein (FLIP) and identified an obligatory role for bovine FLIP in mediating bacterial lipopolysaccharide-induced bovine endothelial apoptosis. We have also established that similar to its human ortholog, bovine FLIP plays a regulatory role in the downregulation of NF-kappaB- dependent gene expression. We have also cloned and sequenced the following bovine TLR-4 signaling molecules: interleukin-1 receptor- associated kinase 1 (IRAK1), MD-2 protein (LY96), toll-like receptor adaptor molecule 2 (TICAM2), toll-interleukin 1 receptor domain containing adaptor protein (TIRAP), toll interacting protein (TOLLIP), TNF
receptor-associated factor 6 (TRAF6), and caspase 8 (CASP8). In addition, the chromosomal locations of these genes, as well as myeloid differentiation primary response gene 88 (MyD88) and toll-like receptor adaptor molecule 1 (TICAM1), were determined by radiation hybrid mapping. Results of this work indicate differences with a previously published bovine sequence for LY96 and a predicted sequence in the GenBank database for TIRAP based on the most recent assembly of the bovine genome. In addition, discrepancies between actual and predicted chromosomal map positions based on the Btau_2.0 genome assembly release were identified, although map positions were consistent with predicted locations based on the current bovine-human comparative map. Alignment of the bovine amino acid sequences with human and murine sequences showed a broad range in conservation, from 52 to 93%. Overall, this work should assist in the assembly and annotation of the bovine genome sequence, the identification
of variations in genes critically involved in host innate immunity, and facilitate the study of TLR-4 signaling pathways in cattle. 5. Describe the major accomplishments to date and their predicted or actual impact. The following accomplishments directly support Component 2 (Genetic and Biological Determinants of Disease Susceptibility), Problem Statement 2A: Mastitis, in the Animal Health NP 103 Action Plan. These accomplishments address: Agency Performance Measure 3.2.1 - provide scientific information to protect animals from pests, infectious diseases, and other disease-causing entities that affect animal and human health; Agency Performance Measure 3.2.2 - identify, develop, and release to the U.S. agricultural community genetic markers, genetic lines, breeds, or Germplasm that result in food animals with improved (either through traditional breeding or biotechnology) pest- and disease-resistance traits; and Agency Performance Measure 3.2.3 - develop and transfer tools to the
agricultural community, commercial partners, and Federal agencies to control or eradicate domestic and exotic diseases that affect animal and human health. Three hundred thousand cows suffer acute endotoxin shock and death annually due to mastitis caused by E. coli, which occurs primarily during the early periparturient period. Current therapeutic options for the treatment of E. coli mastitis remain sub-optimal. We have cloned, sequenced, and functionally characterized bovine sCD14, a protein that contributes to host detection of and the innate immune response to Gram- negative pathogens, including E. coli. Of greater significance and potential impact, we have produced recombinant bovine soluble CD14 (rbosCD14) and demonstrated that it enhances intramammary clearance of E. coli. We have also showed that in a murine model rbosCD14 can protect against endotoxin shock. This research has the potential to provide dairymen for the first time with a product that can be used to treat
mastitis caused by E. coli. Staphylococcus aureus vaccines currently available are of limited effectiveness because they include only 40% of the serotypes identified in the national dairy herd. Trivalent S. aureus vaccines, that cover 100% of the serotypes in the national dairy herd, were developed to immunize first calf heifers to prevent S. aureus mastitis and in conjunction with antibiotics to treat chronic S. aureus mastitis infections. This vaccine has the potential to significantly decrease dairy industry losses due to mastitis; to that end, these vaccines are being tested in commercial herds in Pennsylvania, Michigan, Maryland and North Carolina to cure existing chronic S. aureus infections. In addition, a large study evaluating the efficacy of a trivalent vaccine in preventing new intramammary infections in first calve heifers was completed in cooperation with a major animal health pharmaceutical company. This study required the development of a new challenge model and
involved a total of 72 animals over a one year period. Compared to a sham immunization, the trivalent vaccine demonstrated efficacy in protecting against S. aureus intramammary infection using a teat-dip challenge model. Since S. aureus is responsible for ~40% of all cases of mastitis and annual economic losses of ~$800 million, the development of an efficacious vaccine should substantially reduce these economic losses to producers. Identified differential cytokine expression profiles that correlate with successful bacterial clearance versus establishment of a chronic intramammary infection. Specifically, we established that E. coli elicits a profound pro-inflammatory response resulting in its clearance from the gland, whereas, S. aureus infection is characterized by a muted pro-inflammatory response and the establishment of a chronic infectious state. This work has identified potential recombinant cytokine therapeutics that can be used in the treatment of S. aureus infection.
Sequenced, mapped, and cloned bovine FADD, and identified an obligatory role for bovine FADD in mediating bacterial lipopolysaccharide-induced endothelial apoptosis. We have also established that in contrast to its human ortholog, bovine FADD lacks the ability to downregulate NF-kappaB- dependent gene expression. Since NF-kappaB promotes the expression of pro-inflammatory gene products, this finding may explain the enhanced sensitivity and inflammatory response of cattle to Gram-negative infection and may assist in the selection of animals that are less prone to the deleterious effects of Gram-negative infection. Established a protective role for lysostaphin in protecting against S. aureus intramammary infection. Using a transgenic approach, cattle expressing mammary-derived lysostaphin were demonstrated to be resistant to S. aureus infection. This study is the first of its kind to demonstrate the effectiveness of transgenics at preventing disease in food production animals.
Developed methods for measuring bovine milk levels of various inflammatory mediators, including bacterial lipopolysaccharide binding protein (LBP); transforming growth factors (TGF)-alpha, beta1, and beta2; tumor necrosis factor-alpha (TNF-alpha); interleukin-10 (IL-10); interleukin-12 (IL-12); and soluble CD14. This accomplishment has provided a means for measuring key regulators of immunity and inflammation in the bovine mammary gland and has identified sensitive marker for mastitis and underlying infection in cattle. Scientists in laboratories around the world are using these protocols to characterize bovine immune responses during infection. A phenolic teat dip was developed and shown to be effective in preventing new intramammary infections. This teat dip is now available to dairymen. We have shown that treatment of beef cows at weaning by intramammary infusion of antibiotics will reduce incidence of mastitis and improve calf weaning weights. Adoption of this technology will
increase beef cattlemen's profits. A patent, No. 0108.01,"Use of recombinant bovine CD14 in the treatment and prevention of coliform mastitis in dairy cows" was prepared and submitted to the Patent Office through the USDA Office of Technology Transfer. A patent, No. 0076.04, "Use of DEAE-dextran for prevention of mastitis in dairy cows during the dry period" was prepared and submitted. CRADA, 58-3K95-3-1008, production of recombinant proteins in tobacco plants. Invention Disclosure (Draft April 1, 2004) Title of Invention: Production of a functional bovine CD14 protein in plants. Research Agreement with a major animal health pharmaceutical company (Protocol #3131R-03-60-312), "S. aureus challenge model to evaluate the efficacy of selected inactivated S. aureus vaccines when tested against placebo control". A patent, No. 20040028688, Vaccine for the prevention of bacterial infection of the bovine mammary gland" was prepared and submitted to the Patent Office through the USDA office of
Technology Transfer. Technology Transfer, TEDCO Federal Lab Program, Bioscience for Today and Beyond: Innovative Technologies for a Growing World Technology Showcase April 15, 2003. Recent Advances in the Control of Mastitis in Dairy Cows. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A TRUST (#58-1265-5-0102F) was expanded with a pharmaceutical company to evaluate a novel anti-inflammatory compound for reducing the complications associated with acute coliform mastitis. This TRUST will fund further in vivo studies investigating the anti-inflammatory effects of this compound in the setting of E. coli-induced mastitis. 7. List your most important publications in the popular press and presentations to organizations and articles written about your
work. (NOTE: List your peer reviewed publications below). Suszkiw, J. 2006. New mastitis treatment may offer alternative to antibiotics. Agricultural Research Magazine. 54(2):20.
Impacts
(N/A)
Publications
- Angelini, D.J., Hasday, J.D., Goldblum, S.E., Bannerman, D.D. 2005. Tumor necrosis factor-alpha-mediated pulmonary endothelial barrier dysfunction. Current Respiratory Medicine Reviews. 1(3):233-246.
- Bannerman, D.D., Chockalingam, A., Paape, M.J., Hope, J.C. 2005. The bovine innate immune response during experimentally-induced pseudomonas aeruginosa mastitis. Veterinary Immunology and Immunopathology. 107(3-4) :201-215.
- Bannerman, D.D., Paape, M.J., Chockalingam, A. 2006. Staphylococcus aureus intramammary infection elicits increased production of transforming growth factor-alpha beta1, and beta2. Veterinary Immunology and Immunopathology. 112(304):309-315.
- Bannerman, D.D., Paape, M.J., Baldwin, R.L., Rice, C., Bialek Kalinski, K. M., Capuco, A.V. 2006. Effect of mastitis on milk perchlorate concentrations in dairy cows. Journal of Dairy Science. 89(8):3011-3019.
- Bannerman, D.D. 2006. Natural defenses: Different responses for different bacteria. Mastitis Council(NMC)45th Annual Meeting Proceedings. pp.88-96.
- Burvenich, C., Kehrli, Jr., M.E., Paape, M.J., Bannerman, D.D. 2005. Role of neutrophils in the pathogenesis of bovine coliform mastitis. In: Proceedings of the 29th National Buiatrics Congress, August 11-13, 2005, Puebla, Pue, Mexico. p. S1.
- Burvenich, C., Kehrli, Jr., M.E., Paape, M.J. 2006. Individual cow responses to Escherichia coli mastitis. In: Proceedings of the 45th National Mastitis Council, January 22-25, 2006, Tampa, Florida. 45:61-87.
- Capuco, A.V., Rice, C., Baldwin, R.L., Bannerman, D.D., Paape, M.J., Hare, Jr., W.R., Kauf, A.C., McCarty, G.W., Hapeman, C.J., Sadeghi, A.M., Starr, J.L., McConnell, L.L., Van Tassell, C.P. 2005. Fate of dietary perchlorate in lactating dairy cows: Relevance to animal health and levels in the milk supply. Proceedings of the National Academy of Sciences. 102(45):16152- 16157.
- Goff, J.P., Springer, H.R., Bannerman, D.D., Paape, M.J. 2006. Bacteremia not detected during experimental coliform mastitis infection [abstract]. American Dairy Science Association-American Society of Animal Science 2006 Joint Annual Meeting. p. 11.
- Harp, J.A., Waters, T.E., Goff, J.P., Bannerman, D.D., Paape, M.J. 2006. Expression of lympocyte homing and adhesion molecules during intramammary infection of cows with Serratia marcescens or Streptococcus uberis: Correlation with bacterial colonization and clinical signs. Veterinary Immunology and Immunopathology. 109(1-2):13-21.
- Wei-Lee, J., Bannerman, D.D., Paape, M.J., Huang, M., Zhao, X. 2006. Characterization of Cytokine Expression in Milk Somatic Cells During Intramammary Infections with Escherichia Coli or Staphylococcus Aureus by Real-Time PCR. Veterinary Research. 37(2):219-229.
- Nemchinov, L.G., Paape, M.J., Sohn, E.J., Bannerman, D.D., Zarlenga, D.S., Hammond, R. 2006. Bovine cd14 receptor produced in plants reduces severity of intramammary bacterial infection.
- Paape, M.J., Wiggans, G.R., Bannerman, D.D., Thomas, D.L., Sanders, A., Contreas, A., Moroni, P., Miller, R.D. 2006. Effect of Tear, Stage of Lactation, Parity, Breed and Region on Goat Milk Somatic Cell Counts. National Mastitis Council (NMC) 45th Annual Meeting Proceedings. pp. 256- 257.
- Sohn, E.J., Connor, E.E., Fetterer, R.H., Peters, R., Bannerman, D.D. 2006. Release of CD14 by bovine neutrophils results in down regulation of IL-8. [abstract]. J. of Dairy Sci. 89(Suppl. 1):163.
- Springer, H.R., Goff, J.P., Bannerman, D.D., Paape, M.J. 2006. Lymphocyte and neutrophil response to Staphylococcus aureus and Escherichia coli mastitis [abstract]. American Dairy Science Association-American Society of Animal Science 2006 Joint Annual Meeting. p. 164.
- Szperka, M.E., Connor, E.E., Paape, M.J., Williams, J.L., Bannerman, D.D. 2006. Sequencing, chromosomal mapping, and functional characterization of bovine flice-like inhibitory protein (flip). Cytogenetics and Genome Research. 112(1-2):90-97.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Mastitis is the most costly of all dairy cattle diseases, resulting in losses of over $2,000,000,000 annually. Frequency of mastitis increases as milk production rises. Mastitis reduces milk production 5 30% per infected gland and this decrease in milk production contributes to 65-70% of the total economic losses attributed to mastitis. Significant losses also occur in beef cattle, goats, sheep and pigs. Additional losses connected with mastitis include milk discarded due to antibiotic treatment, premature culling due to chronic infection, increased labor, costs of surveillance of milk supply for antibiotics and general udder health costs (including veterinary and drug expenses). In the absence of effective vaccines, present control relies heavily upon antibiotics and topical germicidal chemicals.
The bacteria causing the greatest loss to the dairy industry are Staphylococcus aureus and Gram-negative species, such as Escherichia coli. S. aureus and Gram-negative bacteria are ubiquitous in the environment and result in 85% of all cases of mastitis. S. aureus adhere to and penetrate tissues lining the gland to form abscesses that are impervious to antibiotics and host defense mechanisms, resulting in the establishment of chronic infection and necessitating culling of valuable milking stock. Infections by Gram-negative bacteria are especially debilitating, especially if contracted soon after calving, and often result in death of the cow. Death is due to the failure of the udder to control the inflammation caused by endotoxin produced by the rapidly multiplying coliform organisms in the milk. It is estimated that 300,000 dairy cows either die or are culled annually because of acute coliform mastitis. The dairy industry requires new tools to solve the mastitis problem. Use of
antibiotics and other drugs and chemicals in the dairy industry is one of the greatest threats to food safety. Surveys indicate that at least 5% of bulk milk shipments and 30% of milk sold to consumers contains detectable amounts of antibiotics and drugs. This presents a significant human health hazard. Also, the antibiotics approved for treating mastitis are increasingly ineffective, largely due to the appearance of resistant strains. This poses the possibility that the time is rapidly approaching when strains of bacteria will be produced that are resistant to currently approved antibiotics. In addition to the risks associated with antibiotic use, current antibiotic therapy remains sub-optimal against chronic cases of S. aureus- induced mastitis and ineffective against mastitis induced by Gram- negative pathogens. Thus, new interventions/therapeutics are necessary for the prevention and treatment of mastitis. There is evidence to suggest that economic losses to the dairy
industry resulting from mastitis can be reduced by enhancing the cow's natural defenses to bacterial infection of the mammary gland. Thus, means of improving the cow's natural defenses are urgently needed to prevent new infections and to minimize the use of antibiotics both prophylactically (e.g., dry-off therapy) and for treatment of existing intramammary infections. Current investigations are focused on determining the nature of S. aureus and Gram-negative bacteria virulence factors and means of shoring up the animal's defenses to combat them. Virulence factors under investigation are: 1) means of entrance of the bacteria into the gland, 2) mechanism(s) of avoiding host cellular and humoral defenses and 3) mechanism(s) of adherence and penetration into the tissue. Means of shoring up the animal's defenses to combat bacterial infection and neutralization of bacterial toxins under current investigation include: 1) strain variation in the various virulence factors, 2) strain
distribution in the national herd, 3) incorporation of the predominant virulence factors in a vaccine(s), 4) methods of delivery of the vaccine(s), 5) immunomodulators to enhance the immune defenses, 6) nutritional factors effecting immune function and 7) cloning of genes responsible for neutralization of endotoxin. The goal of this research is to increase the understanding of the immune response to mastitis- causing pathogens and to develop strategies to modulate this immune response so as to reduce the incidence and severity of mastitis and the substantial economic losses associated with this disease. 2. List the milestones (indicators of progress) from your Project Plan. Objective 1: Develop methods for modulating mammary gland cell-mediated and humoral immune responses. Milestone 1 (12 months) 1. Evaluation of the repertoire of cytokines and chemokines produced by bovine mammary CD+8 lymphocytes. 2. Identify the precise effector function and preferential trafficking of Th2
versus Th1 effector cells to the mammary gland. Milestone 2 (24 months) 1. In vitro screening of CpG oligodeoxynucleotides to identify motifs for use in the bovine system. 2. Evaluate bovine EST libraries for gene sequences of bovine chemokine reagents. Milestone 3 (36-48 months) 1. In vivo studies to determine candidate CpGs for use as mastitis vaccines adjuvants. 2. Production of bovine chemokines using EST libraries. Milestone 4 (48-60 months) 1. Formulation of mastitis vaccine adjuvants using candidate CpGs. 2. Determine immune responsiveness to CpGs as mastitis vaccine adjuvants. 3. Continued production of bovine chemokines and novel cytokines. Objective 2: Enhance the role of phagocytic cells in preventing mastitis. Milestone 1 (12-36 months) 1. Define the cytokine profile in E. coli infected mammary glands. 2. Determine the role(s) and mechanism(s) of CD14 mediated responses in coliform mastitis. 3. Production of transgenic mice expressing bovine CD14 and intramammary
challenge with E. coli. Milestone 2 (48 months) 1. Identify factors affecting cytokine secretion by epithelial cells. 2. Production of anti-bovine CD14 monoclonal antibodies. Milestone 3 (60 months) 1. Determination of the presence of CD14 in milk and serum following infection with E. coli. 2. Production of CD14 in tobacco plants. 3. Studies on in vitro and in vivo neutralization of endotoxin. 4. Production of BsAb and evaluation of effectiveness against mastitis. Objective 3: Develop a vaccine for the prevention and cure of S. aureus mastitis. Milestone 1 (12-23 months) 1. Evaluation of the trivalent vaccine and antibiotics to cure chronic S. aureus infections. 2. Initial studies on methods for immunizing heifers with the trivalent vaccine for prevention of S. aureus mastitis. Milestone 2 (36 months) 1. Fingerprinting the S. aureus recovered from both chronic cows and heifers. 2. Initial trial of S. aureus vaccine effectiveness in microspheres. Milestone 3 (12-60 months)
1. Field trials and challenge studies to determine protectiveness of trivalent vaccine. 2. Field trials using the trivalent vaccine to cure chronic S. aureus in individual farmers' herds. Milestone 4 (36-60 months) 1. Evaluation of endocytosis and phagocytosis of bacteria by epithelial cells. 2. Test transport of antibacterial agents into and across the blood/milk barrier and their ability to kill bacteria inside epithelial. Milestone 5 (48-60 months) 1. Test effectiveness of the vaccine(s) developed in the above Milestones in a large field trial in California. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. In vivo studies to determine candidate CpGs for use as mastitis vaccines adjuvants. Milestone Not Met Critical SY Vacancy 2. Production of bovine chemokines using EST libraries. Milestone Not Met Critical SY Vacancy 3. Identify factors affecting
cytokine secretion by epithelial cells. Milestone Fully Met 4. Production of anti-bovine CD14 monoclonal antibodies. Milestone Fully Met 5. Determinatino of the presence of CD14 in milk and serum folloiwng infection with E. coli. Milestone Fully Met 6. Production of CD14 in tobacco plants. Milestone Fully Met 7. Studies on in vitro and in vivo neutralization of endotoxin. Milestone Fully Met 8. Field trials and challenge studies to determine protectiveness of trivalent vaccine. Milestone Fully Met 9. Field trials using the trivalent vaccine to cure chronic S. aureus in individual farmers' herds. Milestone Fully Met 10. Evaluation of endocytosis and phagocytosis of bacteria by epithelial cells. Milestone Not Met Critical SY Vacancy 11. Test transport of antibacterial agents into and across the blood/milk barrier and their ability to kill bacteria inside epithelial. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008).
What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY2006: Objective 1: Develop methods for modulating mammary gland cell-mediated and humoral immune responses. Milestone 4 (48-60 months) #3. Continued production of bovine chemokines and novel cytokines. Objective 2: Enhance the role of phagocytic cells in preventing mastitis. Milestone 1 (12-36 months) #1. Define the cytokine profile in E. coli infected mammary glands. (Although this milestone was completed in FY2004, it will be expanded to look at the cytokine profile elicited by other major mastitis pathogens.) #3. Production of transgenic mice expressing bovine CD14 and intramammary challenge with E. coli. Milestone 2 (48 months) #1. Identify factors affecting cytokine secretion by epithelial cells. Milestone 3 (60 months) #2. Production of CD14 in tobacco plants. (Although this objective was completed in FY2005, it will be expanded to investigate both in vitro and in vivo
effectiveness against neutralizing LPS and enhancing host innate immune responses.) #3. Studies on in vitro and in vivo neutralization of endotoxin. Objective 3: Develop a vaccine for the prevention and cure of S. aureus mastitis. Milestone 3 (12-60 months) #1. Field trials and challenge studies to determine protectiveness of trivalent vaccine. #2. Field trials using the trivalent vaccine to cure chronic S. aureus in individual farmers' herds. Milestone 4 (36-60 months) #2. Test transport of antibacterial agents into and across the blood/milk barrier and their ability to kill bacteria inside epithelial. Milestone 5 (48-60 months) #1. Test effectiveness of the vaccine(s) developed in the above Milestones in a large field trial in California. FY2007 and FY2008 (Pending OSQR Review and Approval of New Project): Objective 1. Characterize the immune response to intramammary infection and identify genes that confer resistance to the development of chronic infection. Milestone 1.
Characterize the cytokine and inflammatory response to Mycoplasma bovis and coagulase-negative Staphlyococcus. Milestone 2. Utililize microarrays to identify differential gene expression patterns in milk somatic cells and mammary gland tissue during acute and chronic intramammary infections. Milestone 3. Identify breed differences in the host immune response to intramammary infection. Objective 2. Develop and evaluate new strategies and therapeutics for the treatment of mastitis and associated systemic complications. Milestone 1. Develop anti-microbial peptides with efficacious minimum inhibitory concentrations (MIC)/minimum bactericidal concentrations (MBC) against major Gram-negative mastitis pathogens. Milestone 2. Evaluate the efficacy of several pro-inflammatory compounds, as an alternative to prophylactic antibiotic use, in preventing new intramammary infections during the dry-period. Milestone 3. Evaluate the effect of Toll-like receptor immunomodulation on the
course/outcome of mastitis. 4a What was the single most significant accomplishment this past year? The single most significant accomplishment of FY2005 was the completion of a large study evaluating the efficacy of a trivalent vaccine in preventing new intramammary infections in first calve heifers. This study required the development of a new challenge model and involved a total of 72 animals over a one year period. Compared to a sham immunization, the trivalent vaccine demonstrated efficacy in protecting against S. aureus intramammary infection using a teat-dip challenge model. Since S. aureus is responsible for 40% of all cases of mastitis and annual economic losses of $800 million, the development of an efficacious vaccine should substantially reduce these economic losses to producers. 4b List other significant accomplishments, if any. Another significant accomplishment was the establishment of the bioactivity of tobacco-produced recombinant bovine soluble (rbos) CD14. During
FY2004, the methods for successful production and purification rbosCD14 from tobacco plants were developed. In FY 2005, the bioactivity of the compound was evaluated. In comparison to endogenous sCD14 and rbosCD14 produced in an insect cell system using a labor-intensive and costly procedure, the bioactivity of plant-derived rbosCD14 was determined to be comparable. Plant-derived rbosCD14 was demonstrated to be able to facilitate host cell recognition of LPS in vitro and to enhance leukocyte recruitment to the mammary gland in response to LPS in vivo in cattle. Plant-derived rbosCD14 was also established to have therapeutic efficacy in the treatment of cows with intramammary E. coli infections. Relative to saline, administration of plant-derived rbosCD14 into E. coli-infected quarters reduced the number of bacteria in the infected glands. Since 300,000 animals die or are culled annually due to acute coliform mastitis, the development of sCD14, which we have now shown can be
produced economically in plants and retain its bioactivity, has the potential to save the dairy industry $600 million annually in cattle replacement costs. Another major accomplishment in FY2005 was the establishment of a protective role for lysostaphin in protecting against S. aureus intramammary infection. Using a transgenic approach, cattle expressing mammary-derived lysostaphin were demonstrated to be resistant to S. aureus infection. This study is the first of its kind to demonstrate the effectiveness of transgenics at preventing disease in food production animals. Developed methods for measuring milk levels of TGF-a, TGF-B1, and TGF-B2 and characterized changes in the expression of these inflammatory- regulating cytokines during E. coli mastitis. Characterized the innate immune response to intramammary infection with Streptococcus uberis and Serratia marcescens in continuation with our strategy to identify the effect that differential cytokine expression has on outcome of
infection. It is expected that this ongoing work will lead to the identification of genes involved in disease resistance and identify potential recombinant cytokine therapeutics that can be used in the treatment of chronic intramammary infection. We have sequenced, mapped, and cloned bovine FADD, and identified an obligatory role for bovine FADD in mediating LPS-induced EC apoptosis. We have also established that in contrast to its human ortholog, bovine FADD lacks the ability to downregulate NF-kappaB-dependent gene expression. Since NF-kB promotes the expression of pro-inflammatory gene products, this finding may explain the enhanced sensitivity and inflammatory response of cattle to Gram-negative infection and may assist in the selection of animals that are less prone to the deleterious effects of Gram-negative infection. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Three hundred thousand cows suffer acute
endotoxin shock and death annually due to mastitis caused by E. coli, which occurs primarily during the early periparturient period. Current therapeutic options for the treatment of E. coli mastitis remain sub-optimal. We have cloned, sequenced, and functionally characterized bovine sCD14, a protein that contributes to host detection of and the innate immune response to Gram- negative pathogens, including E. coli. Of greater significance and potential impact, we have produced recombinant bovine soluble CD14 (rbosCD14) and demonstrated that it enhances intramammary clearance of E. coli. We have also showed that in a murine model it can protect against endotoxin shock. This research has the potential to provide dairymen for the first time with a product that can be used to treat mastitis caused by E. coli. Staphylococcus aureus vaccines currently available are of limited effectiveness because they include only 40% of the serotypes identified in the national dairy herd. Trivalent S.
aureus vaccines, that cover 100% of the serotypes in the national dairy herd, were developed to immunize first calf heifers to prevent S. aureus mastitis and in conjunction with antibiotics to treat chronic S. aureus mastitis infections. This vaccine has the potential to significantly decrease dairy industry losses due to mastitis; to that end, these vaccines are being tested in commercial herds in Pennsylvania, Michigan, Maryland and North Carolina to cure existing chronic S. aureus infections. Identified differential cytokine expression profiles that correlate with successful bacterial clearance versus establishment of a chronic intramammary infection. Specifically, we established that E. coli elicits a profound pro-inflammatory response resulting in its clearance from the gland, whereas, S. aureus infection is characterized by a muted pro-inflammatory response and the establishment of a chronic infectious state. This work has identified potential recombinant cytokine
therapeutics that can be used in the treatment of S. aureus infection. A phenolic teat dip was developed and shown to be effective in preventing new intramammary infections. This teat dip is now available (WestAgro, Emerge) to dairymen. We have shown that treatment of beef cows at weaning by intramammary infusion of antibiotics will reduce incidence of mastitis and improve calf weaning weights. Adoption of this technology will increase beef cattlemen's profits. A patent, No. 0108.01,"Use of recombinant bovine CD14 in the treatment and prevention of coliform mastitis in dairy cows" was prepared and submitted to the Patent Office through the USDA Office of Technology Transfer. A patent, No. 0076.04, "Use of DEAE-dextran for prevention of mastitis in dairy cows during the dry period" was prepared and submitted. CRADA, 58-3K95-3-1008, production of recombinant proteins in tobacco plants. Invention Disclosure (Draft April 1, 2004) Title of Invention: Production of a functional bovine
CD14 protein in plants. Research Agreement with a major animal health pharmaceutical company (Protocol #3131R-03-60-312), "S. aureus challenge model to evaluate the efficacy of selected inactivated S. aureus vaccines when tested against placebo control". A patent, No. 20040028688, "Vaccine for the prevention of bacterial infection of the bovine mammary gland" was prepared and submitted to the Patent Office through the USDA office of Technology Transfer. Technology Transfer, TEDCO Federal Lab Program, "Bioscience for Today and Beyond: Innovative Technonolgies for a Growing World Technology Showcase" April 15, 2003. "Recent Advances in the Control of Mastitis in Dairy Cows". 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A CRADA (#58-3K95-5-1070) was
developed with a major animal health pharmaceutical company to evaluate the "Use of CD14 in the Prevention of Mastitis". A TRUST (#58-1265-5-102F) was established with a pharmaceutical company to evaluate a novel anti-inflammatory compound for reducing the complications associated with acute mastitis. Connor, E.E., Szperka, M.E., Bannerman, D.D. 2005. Sequencing and mapping of the bovine flice-like inhibitory protein (flip) gene. Germplasm Release. Accession number AY882619. Connor. E.E., Szperka, M.E., Bannerman, D.D. 2004. Cloning and sequencing of the bovine fas-associated death domain protein (fadd) cDNA. Germplasm Release. Accession number AY725483. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Bannerman, D.D., Wall, R.J. 2005. A novel strategy for the prevention of Staphylococcus aureus-induced mastitis in dairy cows. Information
Systems for Biotechnology (ISB) News Report. May 2005 issue:1-4. http://www.isb.vt.edu/news/2005/news05.may.html#may0501 Log#179475
Impacts
(N/A)
Publications
- Bannerman, D.D., Eiting, K.T., Winn, R.K., Harlan, J.M. 2004. Flice-like inhibitory protein (flip) protects against apoptosis and suppresses nf-kb activation induced by bacterial lipopolysaccharide. American Journal of Pathology. 165(4):1423-1431.
- Bannerman, D.D., Paape, M.J., Goff, J.P., Kimura, K., Lippolis, J.D., Hope, J.C. 2004. Innate immune response to intramammary infection with serratia marcessens and streptococcus uberis. Veterinary Research. 35(6):681-700.
- Bannerman, D.D., Paape, M.J., Lunney, J.K. 2004. Immunity: innate IN Encyclopedia of Animal Science. W.G. Pond and A.W. Blel, editors. Marcell Dekker, Inc. New York pp. 552-554.
- Talbot, N.C., Paape, M.J., Sohn, E., Garrett, W.M. 2004. Macrophage population dynamics within fetal mouse fibroblast cultures derived from c57/bl/6, cd-1, cf-1 mice and il-6 and gm-csf knock out mice. In Vitro Cellular and Developmental Biology - Animals. 40(7):196-210.
- Wall, R.J., Powell, A.M., Paape, M.J., Kerr, D.E., Bannerman, D.D., Pursel, V.G., Wells, K.D., Talbot, N.C., Hawk, H.W. 2005. Genetically enhanced cows resist intramammary staphylococcus aureus infection. Nature Biotechnology. 23(4):445-451.
- Lee, J., Obrien, C.N., Guidry, A.J., Paape, M.J., Schafer-Weaver, K.A., Zhao, X. 2005. The effect of a trivalent vaccine against staphylococcus aureus mastitis on lymphocyte subpopulations, antibody production and neutrophil phagocytosis. Canadian Journal of Veterinary Research. 69(1):11- 18.
- Bannerman, D.D., Paape, M.J., Goff, J.P., Kimura, K., Lippolis, J.D., Hope, J.C. 2005. The systemic and localized innate immune response to intramammary infection with streptococcus uberis [abstract]. National Mastitis Council (NMC) 44th Annual Meeting Proceedings. pp. 211-212.
- Chockalingam, A., Paape, M.J., Bannerman, D.D. 2005. Increased milk levels of transforming growth factor-alpha, b-1 and b-2 during escherichia coli- induced mastitis. Journal of Dairy Science. 88(6):1986-1993.
- Connor, E.E., Szperka, M.E., Paape, M.J., Williams, J.L., Bannerman, D.D. 2005. Functional characterization of bovine flice-like inhibitory protein (flip): identification of a protective role against inflammatory mediator- induced apoptosis and nf-kb activation [abstract]. Plant and Animal Genome Conference XIII. p. 533.
- Diez-Fraile, A., Meyer, E., Duchateau, L., Paape, M.J., Burvenich, C. 2004. In vitro regulation of mac-1 expression on bovine polymorphonuclear leukocytes by endotoxin and tumor necrosis factor-alpha at different stages of lactation. Veterinary Research. 68(3):232-235.
- Kerr, D.E., Bannerman, D.D., Paape, M.J., Wall, R.J. 2005. Can we engineer genetic resistance to mastitis? NMC 2005 Regional Meeting Proceedings, July 12-13, 2005, Burlington, VT. p. 31-36.
- Kimura, K., Goff, J.P., Paape, M.J., Bannerman, D.D. 2004. Effect of Serratia marscens infection on intracellular cytokine expression in cells of cow milk [abstract]. Experimental Biology Meeting. Paper No. 93.1.
- Lunney, J.K., Paape, M.J., Bannerman, D.D. 2004. Immunity: acquired. In:Encyclopedia of Animal Science. W.G. bond and A.W. Bell, editors. Marcel Dekker, New York. pp. 548-551.
- Monfardini, E., Van Merris, V., Paape, M.J., Duchateau, L., Burvenich, C. 2004. L-selectin and chemotaxis throughout bone marrow granulocyte maturation in the bovine. Journal of Dairy Science. 87(10):3350-3357.
- Sohn, E.J., Paape, M.J., Peters, R.P., Fetterer, R.H., Talbot, N.C., Bannerman, D.D. 2004. The production and characterization of anti-bovine cd14 monoclonal antibodies. Veterinary Research. 35(5):597-608.
- Szperka, M.E., Connor, E.E., Paape, M.J., Williams, J.L., Bannerman, D.D. 2005. Characterization of bovine FAS-associated death domain gene. Animal Genetics. 36(1):63-66.
- Vangroenweghe, F., Duchateau, L., Boutet, P., Rainard, P., Paape, M.J., Lekeux, P., Burvenich, C. 2005. Effect of carprofen treatment following experimentally induced escherichia coli mastitis in primiparous cows. Journal of Dairy Science. 88(7):2361-2376.
- Butler, J., Richt, J., Harp, J., Young, A., Whipp, S., Richerson, H.B., Sinkora, M., Corbeil, L., Paape, M.J., Duncan, J. 2005. Collection, handling and analysis of specimens for studies of mucosal immunity in large animals. In: Mestecky, J., Bienenstock, J., Lamm, M., Strober. W., McGhee, J., Mayer, L. editors. Mucosal Immunology, 3rd edition, San Diego, CA. Academic Press, Inc. pp. 32-54.
- Vangroenweghe, F., Rainard, P., Paape, M.J., Duchateau, L., Burvenich, C. 2004. Increased escherichia coli intramammary inoculum dose induces an earlier innate immune response in primiparous dairy cows during the periparturient period. Journal of Dairy Science. 87(12):4132-4144.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Mastitis is the most costly of all dairy cattle diseases, resulting in losses of over $2,000,000,000 annually. Frequency of mastitis increases as milk production rises. Mastitis reduces milk production 5 30% per infected gland and this decrease in milk production contributes to 65 70% of the total economic losses attributed to mastitis. Significant losses also occur in beef cattle, goats, sheep and pigs. Additional losses connected with mastitis include milk discarded due to antibiotic treatment, premature culling due to chronic infection, increased labor, costs of surveillance of milk supply for antibiotics and general udder health costs (including veterinary and drug expenses). In the absence of effective vaccines, present control relies heavily upon antibiotics and topical germicidal chemicals.
The bacteria causing the greatest loss to the dairy industry are Staphylococcus aureus and Gram-negative species, such as Escherichia coli. S. aureus and Gram-negative bacteria are ubiquitous in the environment and result in 85% of all cases of mastitis. S. aureus adhere to and penetrate tissues lining the gland to form abscesses that are impervious to antibiotics and host defense mechanisms, resulting in the establishment of chronic infection and necessitating culling of valuable milking stock. Infections by Gram-negative bacteria are especially debilitating, especially if contracted soon after calving, and often result in death of the cow. Death is due to the failure of the udder to control the inflammation caused by endotoxin produced by the rapidly multiplying coliform organisms in the milk. It is estimated that 300,000 dairy cows either die or are culled annually because of acute coliform mastitis. The dairy industry requires new tools to solve the mastitis problem. Use of
antibiotics and other drugs and chemicals in the dairy industry is one of the greatest threats to food safety. Surveys indicate that at least 5% of bulk milk shipments and 30% of milk sold to consumers contains detectable amounts of antibiotics and drugs. This presents a significant human health hazard. Also, the antibiotics approved for treating mastitis are increasingly ineffective, largely due to the appearance of resistant strains. This poses the possibility that the time is rapidly approaching when strains of bacteria will be produced that are resistant to currently approved antibiotics. In addition to the risks associated with antibiotic use, current antibiotic therapy remains sub-optimal against chronic cases of S. aureus- induced mastitis and ineffective against mastitis induced by Gram- negative pathogens. Thus, new interventions/therarpeutics are necessary for the prevention and treatment of mastitis. There is evidence to suggest that economic losses to the dairy
industry resulting from mastitis can be reduced by enhancing the cow's natural defenses to bacterial infection of the mammary gland. Thus, means of improving the cow's natural defenses are urgently needed to prevent new infections and to minimize the use of antibiotics both prophylactically (e.g., dry-off therapy) and for treatment of existing intramammary infections. Current investigations are focused on determining the nature of S. aureus and Gram-negative bacteria virulence factors and means of shoring up the animal's defenses to combat them. Virulence factors under investigation are: 1) means of entrance of the bacteria into the gland, 2) mechanism(s) of avoiding host cellular and humoral defenses and 3) mechanism(s) of adherence and penetration into the tissue. Means of shoring up the animal's defenses to combat bacterial infection and neutralization of bacterial toxins under current investigation include: 1) strain variation in the various virulence factors, 2) strain
distribution in the national herd, 3) incorporation of the predominant virulence factors in a vaccine(s), 4) methods of delivery of the vaccine(s), 5) immunomodulators to enhance the immune defenses, 6) nutritional factors effecting immune function and 7) cloning of genes responsible for neutralization of endotoxin. The goal of this research is to increase the understanding of the immune response to mastitis- causing pathogens and to develop strategies to modulate this immune response so as to reduce the incidence and severity of mastitis and the substantial economic losses associated with this disease. 2. List the milestones (indicators of progress) from your Project Plan. Objective 1: Develop methods for modulating mammary gland cell-mediated and humoral immune responses. Milestone 1 (12 months) 1. Evaluation of the repertoire of cytokines and chemokines produced by bovine mammary CD+8 lymphocytes. 2. Identify the precise effector function and preferential trafficking of Th2
versus Th1 effector cells to the mammary gland. Milestone 2 (24 months) 1. In vitro screening of CpG oligodeoxynucleotides to identify motifs for use in the bovine system. 2. Evaluate bovine EST libraries for gene sequences of bovine chemokine reagents. Milestone 3 (36-48 months) 1. In vivo studies to determine candidate CpGs for use as mastitis vaccines adjuvants. 2. Production of bovine chemokines using EST libraries. Milestone 4 (48-60 months) 1. Formulation of mastitis vaccine adjuvants using candidate CpGs. 2. Determine immune responsiveness to CpGs as mastitis vaccine adjuvants. 3. Continued production of bovine chemokines and novel cytokines. Objective 2: Enhance the role of phagocytic cells in preventing mastitis. Milestone 1 (12-36 months) 1. Define the cytokine profile in E. coli infected mammary glands. 2. Determine the role(s) and mechanism(s) of CD14 mediated responses in coliform mastitis. 3. Production of transgenic mice expressing bovine CD14 and intramammary
challenge with E. coli. Milestone 2 (48 months) 1. Identify factors affecting cytokine secretion by epithelial cells. 2. Production of anti-bovine CD14 monoclonal antibodies. Milestone 3 (60 months) 1. Determination of the presence of CD14 in milk and serum following infection with E. coli. 2. Production of CD14 in tobacco plants. 3. Studies on in vitro and in vivo neutralization of endotoxin. 4. Production of BsAb and evaluation of effectiveness against mastitis. Objective 3: Develop a vaccine for the prevention and cure of S. aureus mastitis. Milestone 1 (12-23 months) 1. Evaluation of the trivalent vaccine and antibiotics to cure chronic S. aureus infections. 2. Initial studies on methods for immunizing heifers with the trivalent vaccine for prevention of S.aureus mastitis. Milestone 2 (36 months) 1. Fingerprinting the S.aureus recovered from both chronic cows and heifers. 2. Initial trial of S.aureus vaccine effectiveness in microspheres. Milestone 3 (12-60 months) 1.
Field trials and challenge studies to determine protectiveness of trivalent vaccine. 2. Field trials using the trivalent vaccine to cure chronic S.aureus in individual farmers' herds. Milestone 4 (36-60 months) 1. Evaluation of endocytosis and phagocytosis of bacteria by epithelial cells. 2. Test transport of antibacterial agents into and across the blood/milk barrier and their ability to kill bacteria inside epithelial. Milestone 5 (48-60 months) 1. Test effectiveness of the vaccine(s) developed in the above Milestones in a large field trial in California. 3. Milestones: A. List milestones that were scheduled to be addressed in FY 2004. Objective 1 Milestone 3 (36-48 months) 1. In vivo studies to determine candidate CpGs for use as mastitis vaccines adjuvants. 2. Production of bovine chemokines using EST libraries. (NOT MET - The scientist assigned to these projects, Kimberly Shafer- Weaver, resigned in 2001 and that vacancy was not filled) Objective 2 Milestone 1 (12-36
Months) 1. Define the cytokine profile in E. coli infected mammary glands.(FULLY MET) 2. Determine the role(s) and mechanism(s) of CD14 mediated responses in coliform mastitis. (FULLY MET) 3. Production of transgenic mice expressing bovine CD14. (PARTIALLY MET - RESEARCH IS ONGOING) Milestone 2 (48 months) 1. Identify factors affecting cytokine secretion by epithelial cells. (PARTIALLY MET - RESEARCH IS ONGOING) 2. Production of anti-bovine CD14 monoclonal antibodies. (FULLY MET) Milestone 3 (60 months) 1. Determine presence of CD14 in milk and serum. (FULLY MET) 2. Production of CD14 in tobacco plants. (FULLY MET) 3. Studies on in vitro and in vivo neutralization of endotoxin. (PARTIALLY MET - RESEARCH IS ONGOING) 4. Production of BsAb and evaluation of effectiveness against mastitis. (NOT MET -The scientist assigned to these projects, Kimberly Shafer- Weaver, resigned in 2001 and that vacancy was not filled) Objective 3 Milestone 2 (36 months) 1. Fingerprinting the S.
aureus recovered from both chronic cows and heifers. (FULLY MET) 2. Initial trial of S. aureus vaccine effectiveness in microspheres. (FULLY MET) Milestone 3 (12-60 months) 1. Field trials and challenge studies to determine protectiveness of trivalent vaccine. (FULLY MET FOR THIS YEAR - RESEARCH IS ONGOING) 2. Field trials using the trivalent vaccine to cure chronic S. aureus mastitis. (FULLY MET FOR THIS YEAR - RESEARCH IS ONGOING) Milestone 4 (36-60 months) 1. Evluation of endocytosis and phagocytosis of bacteria by epithelial cells. 2. Test transport of antibacterial agents across blood/milk barrier. (NOT MET - The scientist responsible for these projects Kimberley Shafer- Weaver, resigned in FY01 and the vacancy was not filled) B. List milestones that will be addressed over the next 3 years (FY 2005, 2006, 2007. Objective 1: Develop methods for modulating mammary gland cell-mediated and humoral immune responses. Milestone 4 (48-60 months) #3. Continued production of
bovine chemokines and novel cytokines. Objective 2: Enhance the role of phagocytic cells in preventing mastitis. Milestone 1 (12-36 months) #1. Define the cytokine profile in E. coli infected mammary glands. Although this objective was completed in FY2004, it will be expanded to look at the cytokine profile elicited by other major mastitis pathogens. #3. Production of transgenic mice expressing bovine CD14 and intramammary challenge with E. coli. Milestone 2 (48 months) #1. Identify factors affecting cytokine secretion by epithelial cells. Milestone 3 (60 months) #2. Production of CD14 in tobacco plants. Although this objective was completed in FY2004, it will be expanded to investigate both in vitro and in vivo effectiveness against neutralizing LPS and enhancing host innate immune responses. #3. Studies on in vitro and in vivo neutralization of endotoxin. Objective 3: Develop a vaccine for the prevention and cure of S. aureus mastitis. Milestone 1 (12-23 months) #1.
Evaluation of the trivalent vaccine and antibiotics to cure chronic S.aureus infections. #2. Initial studies on methods for immunizing heifers with the trivalent vaccine for prevention of S.aureus mastitis. Milestone 3 (12-60 months) #1. Field trials and challenge studies to determine protectiveness of trivalent vaccine. #2. Field trials using the trivalent vaccine to cure chronic S.aureus in individual farmers' herds. Milestone 4 (36-60 months) #1. Evaluation of endocytosis and phagocytosis of bacteria by epithelial cells. #2. Test transport of antibacterial agents into and across the blood/milk barrier and their ability to kill bacteria inside epithelial. Milestone 5 (48-60 months) #1. Test effectiveness of the vaccine(s) developed in the above Milestones in a large field trial in California. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment. The single most significant accomplishment was the elucidation of
differential cytokine profiles in response to major mastitis pathogens including E. coli and S. aureus. This research suggests that the elimination of intramammary infections is due, in part, to a heightened pro-inflammatory response. Specifically, E. coli elicits a profound pro- inflammatory response resulting in its clearance from the gland, whereas, S. aureus infection is characterized by a much lower level pro- inflammatory response and the establishment of a chronic infectious state. This work has identified potential candidate proteins that can be used as recombinant cytokine therapeutics that can be used in the treatment of S. aureus infection. Since S. aureus is responsible for 50% of the cases of mastitis and annual economic losses of 1 billion, identification and development of new treatments for mastitis have enormous potential to substantially reduce these economic losses. B.Other significant accomplishments. A biologically active form of recombinant bovine soluble
(rbos) CD14 was produced in tobacco plants in cooperative studies by scientists in the BFGL and PMPL, Beltsville. Three hundred thousand cows suffer acute endotoxin shock and death annually due to mastitis caused by E. coli, which occurs primarily during the early periparturient period. Intramammary injection of recombinant bovine soluble CD14 (rbosCD14) was 100% effective in preventing mastitis by E. coli in lactating dairy cows. It is now possible to produce large quantities of rbosCD14 for the treatment and prevention of acute mastitis caused by coliform bacteria. Since 300,000 animals die annually from acute coliform mastitis, the development of sCD14 as potential new therapeutic has the potential to save the dairy industry $600 million annually in cattle replacement costs. A non-antibiotic preventative of mastitis during the dry period of dairy cows was shown to be as effective as antibiotics. Widespread use of antibiotics results in contamination of the environment and the
human food chain, as well as development of antibiotic resistant strains of bacteria. At dry off, mammary quarters of 40 cows were injected with antibiotics and 40 cows were injected with Poly-X. At the time of calving, cows treated with Poly-X had less mastitis than cows treated with antibiotics. A patent application (D.N.0076.04) has been filed. Dairymen and organic farmers will have available a non-antibiotic compound for use during the dry period for dairy cows. The costs associated with Poly-X are estimated to be half that of currently used therapeutics translating into cost savings of $22.5 million annually (0.5 X $2.50/tube/quarter X 4 quarters X 4.5 million cows/annually = $22.5 million) C. Significant activities that support special target populations. None. D. Progress Report opportunity to submit additional programmatic information to the Area Office and NPS. None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual
impact. Three hundred thousand cows suffer acute endotoxin shock and death annually due to mastitis caused by E. coli, which occurs primarily during the early periparturient period. Current therapeutic options for the treatment of E. coli mastitis remain sub-opitmal. We have cloned, sequenced, and functionally characterizd bovine sCD14, a protein that contributes to host detection of and the innate immune response to Gram- negative pathogens, including E. coli. Of greater significance and potential impact, we have produced recombinant bovine soluble CD14 (rbosCD14) and demonstrated that it enhances intramammary clearance of E. coli. We have also showed that in a murine model it can protect against endotoxin shock. This research has the potential to provide dairymen for the first time with a product that can be used to treat mastitis caused by E. coli. Staphylococcus aureus vaccines currently available are of limited effectiveness because they include only 40% of the serotypes
identified in the national dairy herd. Trivalent S. aureus vaccines, that cover 100% of the serotypes in the national dairy herd, were developed to immunize first calf heifers to prevent S. aureus mastitis and in conjunction with antibiotics to treat chronic S. aureus mastitis infections. This vaccine has the potential to significantly decrease dairy industry losses due to mastitis; to that end, these vaccines are being tested in a Pfizer Animal Health-funded study at the USDA, Beltsville and in commercial herds in Pennsylvania, Michigan, Maryland and North Carolina to cure existing chronic S. aureus infections. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A phenolic teat dip was developed and shown to be effective in preventing new intramammary
infections. This teat dip is now available (WestAgro, Emerge) to dairymen. We have shown that treatment of beef cows at weaning by intramammary infusion of antibiotics will reduce incidence of mastitis and improve calf weaning weights. Adoption of this technology will increase beef cattlemen's profits. A patent, No. 0108.01,"Use of recombinant bovine CD14 in the treatment and prevention of coliform mastitis in dairy cows" was prepared and submitted to the Patent Office through the USDA Office of Technology Transfer. A patent, No. 0076.04, "Use of DEAE-dextran for prevention of mastitis in dairy cows during the dry period" was prepared and submitted. CRADA, 58-3K95-3-1008, production of recombinant proteins in tobacco plants. Invention Disclosure (Draft April 1, 2004) Title of Invention: Production of a functional bovine CD14 protein in plants. Research Agreement (Protocol #3131R-03-60-312), "S. aureus challenge model to evaluate the efficacy of selected inactivated S. aureus
vaccines when tested against placebo control". Pfizer Animal Health, Kalamazoo, Michigan. A patent, No. 20040028688, "Vaccine for the prevention of bacterial infection of the bovine mammary gland" was prepared and submitted to the Patent Office through the USDA office of Technology Transfer. Technology Transfer, TEDCO Federal Lab Program, "Bioscience for Today and Beyond: Innovative Technonolgies for a Growing World Technology Showcase" April 15, 2003. "Recent Advances in the Control of Mastitis in Dairy Cows." 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Bannerman, D.D., Paape, M.J. 2004. Recent advances in the control of mastitis in dairy cows. FeedInfo News Service. Available: http://www. feedinfo.com/console/PageViewer.aspx?page=55817 . Log #: 0000160196.
Impacts
(N/A)
Publications
- Bannerman, D.D., Paape, M.J., Hare Jr, W.R., Hope, J.C. 2004. Characterization of the bovine innate immune response to intramammary infection with klebsiella pneumoniae. Journal of Dairy Science. 87(8):2420- 2432.
- Bannerman, D.D., Paape, M.J., Lee, J., Zhao, X., Hope, J.C., Rainard, P. 2004. Escherichia coli and staphylococcus aureus elicit differential innate immune responses following intramammary infection. Clinical and Diagnostic Laboratory Immunology. vol. 11(3), pp. 463-72.
- Paape, M.J., Bannerman, D.D., Zhao, X., Lee, J. 2003. The bovine neutrophil; structure and function in blood and milk. Veterinary Research. Vol. 34, pp. 597-627.
- Paape, M.J., Contreras, A., Ledbetter, T.K. 2004. Variation among goats in the ability of their polymorphonuclear neutrophil leukocytes and mammary secretions to support phagocytosis: inhibitory effects of milk fat globules. Small Ruminant Research. 54:183-189.
- Sakarya, S., Rifat, S., Zhou, J., Bannerman, D.D., Stamatos, N.M., Cross, A.S., Goldblum, S.E. 2004. Mobilization of neutrophil sialidase activity desialyiates the endothelial surface and increases resting neutrophil adhesion to and migration across the endothelium. Glycobiology. vol. 14, pp. 481-494.
- Paape, M.J., Burvenich, C., Mehrzad, J., Monfardini, E., Capuco, A.V. 2004. Role of neutrophil polymorphonuclear leukocytes during bovine coliform mastitis: physiology or pathology? Veterinary Research. 66(2):97-153.
- Capuco, A.V., Ellis, S.E., Hale, S.A., Long, E., Erdman, R.A., Zhao, X., Paape, M.J. 2003. Lactation persistency: insights from mammary cell proliferation studies. Journal of Animal Science, 81, Supp. 3, pp.18-31.
- Wang, Y., Zarlenga, D.S., Paape, M.J., Dahl, G.E. 2003. Expression and functional analysis of recombinat bovine CD14. Vet. Research, vol. 34, pp. 413-421.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Economic loss to the dairy industry due to mastitis can be reduced by enhancing the cow's natural defenses to bacterial infection of the mammary gland. Thus, means of improving the cow's natural defenses are urgently needed to minimize the use of antibiotics in preventing mammary gland infections. Widespread use of antibiotics has resulted in the development of resistant strains of bacteria which has necessitated periodic development of new more potent antibiotics. This poses the awesome possibility that the time is rapidly approaching when strains of bacteria will be produced that are resistant to any and all antibiotics. The use of antibiotics also poses the ever present problem of antibiotic contamination of the milk supply. The bacteria causing the greatest loss to the dairy industry are Staphylococcus aureus and Gram-negative species. Staphylococcus aureus and gram-negative
bacteria are ubiquitous in the environment and result in 85% of all cases of mastitis. More importantly once inside the gland S. aureus adhere to and penetrate tissues lining the gland to form abscesses that are impervious to antibiotics and host defense mechanisms. This results in chronic cases of mastitis that necessitate culling of valuable milking stock. Infections by Gram-negative bacteria are especially debilitating if contracted soon after calving, usually resulting in death of the cow. Death is due to the failure of the udder to control the inflammation caused by endotoxin produced by the rapidly multiplying coliform organisms in the milk. It is estimated that 300,000 dairy cows either die or are culled from the herd because of acute coliform mastitis. Current investigations are focused on determining the nature of S. aureus and Gram-negative bacteria virulence factors and means of shoring up the animal's defenses to combat them. Virulence factors under investigation are: 1)
means of entrance of the bacteria into the gland, 2) mechanism(s) of avoiding host cellular and humoral defenses and 3) mechanism(s) of adherence and penetration into the tissue. Means of shoring up the animal's defenses to combat bacterial infection and neutralization of bacterial toxins under current investigation include: 1) strain variation in the various virulence factors, 2) strain distribution in the national herd, 3) incorporation of the predominant virulence factors in a vaccine(s), 4) methods of delivery of the vaccine(s), 5) immunomodulators to enhance the immune defenses, 6) nutritional factors effecting immune function and 7) cloning of genes responsible for neutralization of endotoxin. 2. How serious is the problem? Why does it matter? Mastitis is the most costly of all dairy cattle diseases, resulting in losses of over $2,000,000,000 annually. Frequency of mastitis increases as milk production rises. Mastitis reduces milk production 5 30% per infected gland,
contributing 65 70% of the total loss due to mastitis. Significant losses also occur in beef cattle, goats, sheep and pigs. Additional losses connected with mastitis include milk discarded due to antibiotic treatment, premature culling due to chronic infection, increased labor, costs of surveillance of milk supply for antibiotics and general udder health costs (including veterinary and drug expenses). In the absence of effective vaccines, present control relies heavily upon antibiotics and topical germicidal chemicals. The dairy industry requires new tools to solve the mastitis problem. Use of antibiotics and other drugs and chemicals in the dairy industry is one of the greatest threats to food safety. Surveys indicate that at least 5% of bulk milk shipments and 30% of milk sold to consumers contains detectable amounts of antibiotics and drugs. This presents a significant human health hazard. Also, the antibiotics approved for treating mastitis are increasingly ineffective, largely
due to the appearance of resistant strains. This presents the awesome danger of the development of strains of bacteria that are resistant to all current and future developed antibiotics. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program Area 103 Animal Health (100%). Addresses components: Animal Immunology and Strategies to Control Infectious and Non-Infectious Disease. This research allows scientists to characterize bacterial strains according to their virulence factors and to determine their distribution in the national herd. It also results in the development of environmentally sound control measures applicable to the national dairy herd. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY2003: Three hundred thousand cows suffer acute endotoxin shock and death annually due to mastitis caused by Escherichia coli, which occurs
primarily during the early periparturient period. Intramammary injection of recombinant bovine soluble CD14 (rbosCD14) was 100% effective in preventing mastitis by Escherichia coli in lactating dairy cows. Intraperitoneal injection of rbosCD14 together with endotoxin reduced fatality in mice. Dairymen will have for the first time a product that can be used to treat and prevent mastitis caused by Escherichia coli. B. Other significant accomplishments: New tools are needed to assess efficacy of treatment and prevention of coliform mastitis in dairy cows. Anti-rbosCD14 monoclonal antibodies were produced and an ELISA for measuring soluble CD14 (sCD14) in blood and milk developed. These assays confirmed that intramammary injection of endotoxin resulted in a rapid increase in sCD14 in milk that coincided with an increase in milk somatic cells, whereas intramammary injection of an endotoxin/rbosCD14 complex immediately induced recruitment of leukocytes into the mammary gland. These
studies will help highlight new biotherapeutic treatments for mastitis and help decrease dependence on antibiotic usage. BARC researchers have shown potential therapeutic value for rbosCD14 for mastitis treatment; studies were initiated to characterize the participation of other molecules, such as LBP (lipopolysaccharide binding protein), in the mammary gland response to mastitis. Researchers in the Immunology and Disease Resistance Laboratory at BARC infused the mammary glands of 5 cows with bacterial lipopolysaccharide (LPS) and were the first to detect and characterize changes in LBP in milk and blood during the course of mastitis. The finding that sCD14 and LBP levels rise in parallel during the course of mastitis, and prior knowledge that these molecules work in concert with one another, may suggest that co- administration of rbosCD14 with rbosLBP may enhance the function of rbosCD14 to initiate host innate immune responses. C: Significant accomplishments/activities that
support special target populations: None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Staphylococcus aureus vaccines currently available are of limited effectiveness because they include only 40% of the serotypes identified in the national dairy herd. A trivalent S. aureus vaccine, that covers 100% of the serotypes in the national dairy herd, was used to immunize first calf heifers to prevent S. aureus mastitis and was used in conjunction with antibiotics to treat chronic S. aureus mastitis infections. The vaccine was 100% effective in protecting heifers from S. aureus infections and 70% effective in curing chronic infections, thus, preventing at the very beginning of the cow's productive life. This vaccine could significantly decrease dairyman's losses due to mastitis; to that end it is being tested in commercial herds in Michigan, Maryland and North Carolina to cure existing chronic S. aureus infections. 6. What
do you expect to accomplish, year by year, over the next 3 years? During the next year, we will elucidate the mammary gland innate immune response to various Gram-negative organisms that are major contributors to mastitis. These organisms include Klebsiella pneumonia, Serratia marcescens, Pseudomonas aeruginosa and Escherichia coli. We will continue to test the efficacy of the trivalent vaccine. This will be conducted in cooperation with local commercial dairy herds in Maryland and through a Trust with Pfizer Animal Health. It will involve combining immunization with the trivalent vaccine in conjunction with antibiotics in the treatment of chronic cases of S. aureus mastitis. Serum samples will be tested for their ability to enhance phagocytosis and block S. aureus adherence to mammary epithelial cells. The trivalent vaccine will also be used to immunize first calf heifers beginning at various intervals from six months to first calving to determine its effectiveness in preventing
S. aureus infections around calving, a particularly vulnerable period. A 3rd study will be conducted to determine the effectiveness of the trivalent vaccine to produce an effective humoral and cell-mediated immune response and protection against a direct challenge with each of the S. aureus strains used in the vaccine. Studies on the interrelationships of rbosCD14 with endotoxin and TNF will be initiated to better understand the immunopathology of infections caused by Gram-negative bacteria. The CD14 gene, which produces sCD14 that neutralizes endotoxin, will be inserted into tobacco mosaic virus and tobacco plants will be infected with the virus. Once expression of the protein is verified by ELISA and Western blot, the protein will be purified and used in clinical trials to determine effectiveness in reducing symptoms of acute coliform mastitis. Transgenic mice producing rbosCD14 in their milk will be challenged with Escherichia coli to determine if the secreted rbosCD14 prevents
mastitis. In an effort to reduce the amount of antibiotics used in dairy cows, the use of a non-antibiotic formulation developed at BARC in place of antibiotics at dry-off will be evaluated. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A phenolic teat dip was developed and shown to be effective in preventing new intramammary infections. This teat dip will be made available to dairymen. We have shown that treatment of beef cows at weaning by intramammary infusion of antibiotics will reduce incidence of mastitis and improve calf weaning weights. Adoption of this technology will increase beef cattlemen=s profits. A patent, No. 0108.01, "Use of recombinant bovine CD14 in the treatment and prevention of coliform mastitis in dairy cows" was prepared
and submitted to the Patent Office through the USDA Office of Technology Transfer. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). Researchers develop effective mastitis treatments. Issue 15, July 2003. Healthy Animals. U.S. Department of Agriculture, Agricultural Research Service. http://www.ars.usda.gov/is/np/ha/han15.htm An udder solution for Bossie's woes. 2002. Agricultural Research. U.S. Department of Agriculture, Agricultural Research Service. June issue. p. 18-19.
Impacts
(N/A)
Publications
- Van Oostveldt, K., Paape, M.J., Dosogne, H., Burvenich, C. Effect of apoptosis on phagocytosis, respiratory burst and CD18 adhesion receptor expression of bovine neutrophils. Domestic Animal Endocrinology. 2003. v. 22. p. 37-50.
- Lee, J.-W., Paape, M.J., Zhao, X. Soluble CD14 prevents intramammary infection by Escherichia coli. FASEB Journal. 2003. v. 17. p. A670.
- Erwert, R.D., Eiting, K.T, Tupper, J.C., Winn, R.K., Harlan, J.M., Bannerman,D.D. Shiga toxin induces decreased expression of the anti- apoptotic protein Mc1-1 concomitant with the onset of endothelial apoptosis. Microbial Pathogenesis. 2003. v. 35. p. 87-93.
- Bannerman, D.D., Paape, M.J., Hare, W.R., Sohn, E.J. Increased levels of LPS-binding protein (LBP) in bovine blood and milk following bacterial lipopolysaccharide challenge. Journal of Dairy Science. 2003. v. 86(Suppl. 1). p.193.
- Lee, J.-W., Zhao, X., Paape, M.J. Characterization of soluble CD14 in bovine milk. Journal of Dairy Science. 2003. v. 86 (Suppl. 1.). p. 299.
- Diez-Fraile, A., Meyer, E.,Paape, M.J., Burvenich, C. Analysis of selective mobilization of L-selectin and Mac-1 reservoirs in bovine neutrophils and eosinophils. Veterinary Research. 2003, v. 34. p. 57-70
- Bannerman, D.D., Goldblum, S.E. Mechanisms of bacterial lipopolysaccharide- induced endothelial apoptosis. American Journal of Physiology. 2003. v. 284. p.L899-L914.
- Lee, J.-W., Paape, M.J., Elsasser, T.H., Zhao, X. Elevated milk soluble CD14 in bovine mammary glands challenged with Escherichia coli Lipopolysaccharide, Journal of Dairy Science. 2003. v. 86. p.2382-2389.
- Lee, J.-W., Paape, M.J., Zhao, X. Recombinent bovine soluble CD14 reduces severity of experimental Escherichia coli mastitis in mice. Veterinary Research. 2003. v. 34, p. 306-316.
- Lee, J-W., Paape, M.J., Elsasser, T.H., Zhao, Xin. 2003. Recombinant soluble CD14 reduces severity of intramammary infection by escherichia coli. Infection and Immunity. 71:4034-4039.
- Li, X., Tupper, J.C., Bannerman, D.D., Win, R.K., Rhodes, C.J., Harlan, J. M. Phosphoinositide 3 kinase mediates toll-like receptor 4-induced activation of NF-OB in endothelial cells. Infection and Immunity. 2003. v. 71, p. 4414-4420.
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Progress 10/01/01 to 09/30/02
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Economic loss to the dairy industry due to mastitis can be reduced by enhancing the cow's natural defenses to bacterial infection of the mammary gland. Thus, means of improving the cow's natural defenses are urgently needed to minimize the use of antibiotics in preventing mammary gland infections. Widespread use of antibiotics has resulted in the development of resistant strains of bacteria which has necessitated periodic development of new more potent antibiotics. This poses the awesome possibility that the time is rapidly approaching when strains of bacteria will be produced that are resistant to any and all antibiotics. The use of antibiotics also poses the ever present problem of antibiotic contamination of the milk supply. The bacteria causing the greatest loss to the dairy industry are Staphylococcus aureus and Gram-negative species. Staphylococcus aureus and gram-negative
bacteria are ubiquitous in the environment and result in 85% of all cases of mastitis. More importantly once inside the gland S. aureus adhere to and penetrate tissues lining the gland to form abscesses that are impervious to antibiotics and host defense mechanisms. This results in chronic cases of mastitis that necessitate culling of valuable milking stock. Infections by Gram-negative bacteria are especially debilitating if contracted soon after calving, usually resulting in death of the cow. Death is due to the failure of the udder to control the inflammation caused by endotoxin produced by the rapidly multiplying coliform organisms in the milk. It is estimated that 300,000 dairy cows either die or are culled from the herd because of acute coliform mastitis. Current investigations are focused on determining the nature of S. aureus and Gram-negative bacteria virulence factors and means of shoring up the animal's defenses to combat them. Virulence factors under investigation are: 1)
means of entrance of the bacteria into the gland, 2) mechanism(s) of avoiding host cellular and humoral defenses and 3) mechanism(s) of adherence and penetration into the tissue. Means of shoring up the animals defenses to combat bacterial infection and neutralization of bacterial toxins under current investigation include: 1) strain variation in the various virulence factors, 2) strain distribution in the national herd, 3) incorporation of the predominant virulence factors in a vaccine(s), 4) methods of delivery of the vaccine(s), 5) immunomodulators to enhance the immune defenses, 6) nutritional factors effecting immune function and 7) cloning of genes responsible for neutralization of endotoxin. 2. How serious is the problem? Why does it matter? Mastitis is the most costly of all dairy cattle diseases, resulting in losses of over $2,000,000,000 annually. Frequency of mastitis increases as milk production rises. Mastitis reduces milk production 5-30% per infected gland,
contributing 65-70% of the total loss due to mastitis. Significant losses also occur in beef cattle, goats, sheep and pigs. Additional losses connected with mastitis include milk discarded due to antibiotic treatment, premature culling due to chronic infection, increased labor, costs of surveillance of milk supply for antibiotics and general udder health costs (including veterinary and drug expenses). In the absence of effective vaccines, present control relies heavily upon antibiotics and topical germicidal chemicals. The dairy industry requires new tools to solve the mastitis problem. Use of antibiotics and other drugs and chemicals in the dairy industry is one of the greatest threats to food safety. Surveys indicate that at least 5% of bulk milk shipments and 30% of milk sold to consumers contains detectable amounts of antibiotics and drugs. This presents a significant human health hazard. Also, the antibiotics approved for treating mastitis are increasingly ineffective, largely
due to the appearance of resistant strains. This presents the awesome danger of the development of strains of bacteria that are resistant to all current and future developed antibiotics. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? National Program Area 103 Animal Health (100%). This research addresses components of the Action Plan: Pathogen Detection and Diagnostics; Animal Immunology; Mechanism of Disease; and Strategies to Control Infectious and Non-Infectious Disease. This research allows scientists to characterize bacterial strains according to their virulence factors and to determine their distribution in the national herd. It also results in the development of environmentally sound control measures applicable to the national dairy herd. 4. What was your most significant accomplishment this past year? A. Single most significant accomplishment during FY2002: Three hundred thousand cows suffer acute endotoxin
shock and death annually due to mastitis caused by Escherichia coli, which occurs primarily during the early periparturient period. Intramammary injection of recombinant bovine soluble CD14 (rbosCD14) was 100% effective in preventing mastitis by Escherichia coli in lactating dairy cows. Intraperitoneal injection of rbosCD14 together with endotoxin reduced fatality in mice. Dairymen will have for the first time a product that can be used to treat and prevent mastitis caused by Escherichia coli. B. Other significant accomplishment(s), if any: Anti-rbosCD14 monoclonal antibodies were produced and an ELISA was produced for measuring soluble CD14 (sCD14) in blood and milk. Intramammary injection of endotoxin resulted in a rapid increase in sCD14 in milk that coincided with an increase in milk somatic cells. The gene for producing sCD14, which binds and neutralizes endotoxin, was cloned and the recombinant bovine (rbo)sCD14 produced. Intramammary injection of an endotoxin/rbosCD14 complex
induced recruitment of leukocytes into the mammary gland. A patent application No. 0108.01 "Use of rbosCD14 in the treatment and prevention of coliform mastitis in dairy cows was prepared and submitted to the patent office through the USDA Office of Technology Transfer. Staphylococcus aureus vaccines currently available are of limited effectiveness because they include only 40% of the serotypes identified in the national dairy herd. A S. aureus vaccine that covers 100% of the serotypes in the national dairy herd was used to immunize first calf heifers to prevent S. aureus mastitis and was used in conjunction with antibiotics to treat chronic S. aureus mastitis infections. The vaccine was 100% effective in protecting heifers from S. aureus infections and 70% effective in curing chronic infections. This vaccine could significantly decrease dairyman's losses due to mastitis. C: Significant accomplishments/activities that support special target populations: None 5. Describe your major
accomplishments over the life of the project, including their predicted or actual impact? Developed a trivalent S. aureus vaccine that represents the surface characteristics of 100% of the strains present in the national herd. This vaccine is currently being tested for its ability to prevent mastitis in first-calf heifers. Thus, preventing at the very beginning of the cow's productive life. It is also being tested in commercial herds in Michigan, Maryland and North Carolina to cure existing chronic S. aureus infections. Research in this project focused world-wide attention on the errors involved in the determination of somatic cell counts in cow and goat milk. These counts form the basis of national and international abnormal milk control programs which determines the price dairymen receive for milk. Reliable stains and accurate counting procedures for cells in milk were developed that are industry standards and currently used by regulatory agencies the world over. It was further
established that neither physiological nor environmental stressors caused alterations in the somatic cell content of milk. As a consequence, dairymen, leaders of industry and researchers throughout the world have accepted milk somatic cell counts as an index of milk quality. In vitro procedures for measuring neutrophil function and procedures for the isolation of white blood cells from blood and milk were developed, which are used by researchers throughout the world. The discovery that neutrophil function is depressed after parturition, a time of increased susceptibility of the gland to mastitis, stimulated industry interest in the use of cytokines as modulators of neutrophil function during the periparturent period. Were the first to demonstrate that differences existed among cows in the ability of neutrophils to ingest and kill bacteria. Further showed a relationship between neutrophil function and susceptibility to mastitis. These differences in phagocytic competence may
provide methods of capitalizing on genetic differences in natural resistance, which could be quantified at an early age. 6. What do you expect to accomplish, year by year, over the next 3 years? FY03: During the next year studies on the interrelationships of rbosCD14 with endotoxin and TNF will be initiated to better understand the immunopathology of infections caused by Gram-negative bacteria. Transgenic mice expressing rbosCD14 will be produced and will be used in intramammary E. coli challenge studies to determine if the secreted rbosCD14 prevents mastitis. In an effort to reduce the amount of antibiotics used in dairy cows, the use of dextran in place of antibiotics at dry-off will be evaluated. We will continue to test the efficacy of the trivalent vaccine. This will be conducted in cooperation with local commercial dairy herds in Maryland. It will involve combining immunization with the trivalent vaccine in conjunction with antibiotics in the treatment of chronic cases of S.
aureus mastitis. Serum samples will be tested for their ability to enhance phagocytosis and block S. aureus adherence to mammary epithelial cells. The trivalent vaccine will also be used to immunize first calf heifers beginning at various intervals from six months to first calving to determine its effectiveness in preventing S. aureus infections around calving, a particularly vulnerable period. A 3rd study will be conducted to determine the effectiveness of the trivalent to produce an effective humoral and cell-mediated immune response and protection against a direct challenge with each of the S. aureus strains used in the vaccine. FY04 - FY05: 1. The CD14 gene, which produces sCD14 that neutralizes endotoxin, will be inserted into potato mosaic virus and potato plants will be infected with the virus. Once expression of rbosCD14 is verified by ELISA and Western blot, the protein will be purified and used in clinical trials to determine effectiveness in reducing symptoms of acute
coliform mastitis. 2. Determine the role(s) and mechanism(s) of CD14 mediated responses in coliform mastitis. 3. Production of transgenic mice expressing bovine CD14 and intramammary challenge with Escherichai coli. 4. In vitro screening of CpG oligodeoxynucleotides to identify motifs for use in the bovine system. 5. Evaluate bovine EST libraries for gene sequences of bovine chemokine reagents. 7. What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? Developed the first in vitro model of the blood/milk barrier - a culture of cells lining the blood vessels and cells lining the mammary gland separated by interstitial cells and proteins characteristic of the interstitial tissues in the mammary gland. The model is currently being used by scientists in the US, Canada, Sweden and Belgium to study various
effects on blood/cells as they passage the blood/milk barrier. Trained and advised scientist in these laboratories in development and use of the model and donated cell cultures to get them started. A phenolic teat dip was developed and shown to be effective in preventing new intramammary infections. This teat dip will be made available to dairymen. We have shown that treatment of beef cows at weaning by intramammary infusion of antibiotics will reduce incidence of mastitis and improve calf weaning weights. Adoption of this technology will increase beef cattlemen=s profits. A patent, No. 0108.01 on "Use of recombinant bovine CD14 in the treatment and prevention of coliform mastitis in dairy cows" was prepared and submitted to the Patent Office through the USDA ARS Office of Technology Transfer. An application for a patent for the S. aureus vaccine that covers 100% of the serotypes in the national dairy herd was submitted through the ARS OTT In July 2002. This vaccine potentially
will not only help protect against chronic mastitis but also help prevent this disease in first calf heifers. This vaccine could significantly decrease dairyman's losses due to mastitis and is being actively developed with commercial and university partners. 8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) McBride,J. "An Udder Solution For Bossie's Woes". Agricultural Research. June 2002. p. 18-19.
Impacts
(N/A)
Publications
- Lents,C.A.,Wettemann,R.P.,Paape,M.J.,Vizcarra,J.A.,Looper,M.I.,Buchanan,D. S., Lusby,K.S. Efficacy of intramuscular treatment of beef cows with oxytetracycline to reduce mastitis and to increase calf growth. Journal of Animal Science. 2002. v.80. p.1405-1412.
- Van Oostveldt,K.,Paape,M.J.,Burvenich,C. Apoptosis of bovine neutrophils following diapedesis through a monolayer of endothelial and mammary epithelial cells. Journal of Dairy Science. 2002. v.85. p.1-9.
- Wang,Y.,Zarlenga,D.S.,Paape,M.J.,Dahl,G.E. Recombinant bovine CD14 sensitizes the mammary gland to lipopolysaccharide. Veterinary Immunology and Immunopathology. 2002. v.86. p.115-124.
- Long,E.,Capuco,A.V.,Wood,D.L.,Sonstegard,T.,Tomita,G.,Paape,M.J.,Zhao,X. Escherichia coli induces apoptosis and proliferation of mammary cells. Cell Death and Differentiation. 2001. v.8. p.808-916.
- Paape,M.J.,Rautiainen,P.M.,Lilius,E.M.,Malstron,C.E.,Elsasser,T.H. Development of anti-bovine TNF-alpha mAb and ELISA for quantitating TNF- alpha in milk after intramammary injection of endotoxin. Journal of Dairy Science. 2002. v.85. p.765-773.
- Paape,M.J.,Poutrel,B.,Contreras,A.,Marco,J.C.,Capuco,A.V. Milk somatic cells and lactation in small ruminants. Journal of Dairy Science. 2001. v. 84. p.E237-244.
- Duenas,M.I.,Paape,M.J.,Wettemann,R.P.,Douglass,L.W. Incidence of mastitis in beef cows after intramuscular administration of oxytetracycline. Journal of Animal Science. 2001. v.79. p.1996-2005.
- Van Oostveldt,K.,Tomita,G.,Paape,M.J.,Capuco,A.V.,Burvenich,C. Apoptosis of bovine neutrophils during mastitis experimentally induced with Escherichia coli or endotoxin. American Journal of Veterinary Research. 2002. v.63. p.448-453.
- Monfardini,E.,Paape,M.J.,Wang,Y.,Capuco,A.V.,Husheem,M.,Wood,L.,Burvenich, C. Evaluation of L-selectin expression and assessment of protein tyrosine phosphorylation in bovine polymorphonuclear neutrophil leukocytes around parturition. Veterinary Research. 2002. v.33. p.271-281.
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