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The over-arching goal of this project is to gather data to better compare groups of cattle that are given one, two or three doses of RB51 vaccine; calfhood vaccinated, calfhood and adult vaccinated while pregnant, or calfhood vaccinated, heifer booster vaccinated, followed by adult vaccination while pregnant. It is our hypothesis that RB51 booster vaccination will provide increased cellular or humoral immune responses than calfhood vaccination alone. We intend to observe the effects of RB51 boosting by measuring humoral (related partner grant) and cell-mediated immunity. These data may provide a scientific rationale for the addition of booster vaccines to at-risk cattle. <P>We have defined our objective as three specific aims: 1. Make clinical observations on pregnant cattle given RB51 boosts to determine the feasibility of a larger clinical study to refute the notion that RB51 boosting of pregnant cattle induces abortions directly. Additional funding for this objective has been requested by Dr. Schumaker. 2. Develop specific cell-based methodologies to assess the development of cell-mediated immunity induced by RB51 vaccination. 3. Down select the best cell mediated immunity assay and use this to follow the cattle's response to RB51 boosting. Make the data and methodology available to use as a rationale for veterinarians to decide when and if to boost at risk cattle. <P>Expected Significance: 1. Collection of controlled experimental data on the effects of RB51 boosts on pregnant cattle will provide a scientific basis for the inclusion or exclusion of pregnant cattle from vaccine regimens. In addition, the data collected during our study will serve as preliminary data for a larger clinical-type study on this phenomenon. When RB-51 boost studies are conducted in the field or combined with a live Brucella challenge they will provide powerful incentives to both producers and veterinarians to modify RB51 vaccine practices. This may result in increased protection from both Brucella infection and Brucella-induced abortions saving money, reducing cattle to cattle, cattle to wild-life and potentially cattle to human Brucella transmission. <br/>2. New bovine-specific T-cell assays and reagents will further Brucellosis research in general and Brucella vaccine research in particular. The availability of a qualified assay to assess cell mediated immunity in cattle is critical. This assay will provide a fundamental tool for predicting and eventually determining the benefits of RB51 or other related experimental vaccines in cattle. 3. Understanding the utility and risks of boosting cattle with RB-51 vaccine may lead to a more rationale vaccine policy for the state of Wyoming, reduce disease incidence in an economically important food source and help reduce/break the cycle of brucellosis transmission from infected wildlife such as elk. <P>We expect that the data will help producers and veterinarians make more rationale, cost-effective decisions on if/when to vaccinate at-risk cattle both within and outside the designated surveillance area. This proposal will provide a clear link between research and positive public/veterinary health problems.
Non-Technical Summary:<br/>
This project will test the idea that multiple does of RB51 vaccine will protect cattle against brucellosis better than single doses of RB51 vaccine by generation of a more robust immune response. We propose to measure specific aspects of the immune response to RB51 vaccine in cattle to provide a scientifically sound rationale for ranchers/veterinarians to provide RB51 vaccine boosts to cattle. A better immune response in the cattle may not only protect the cattle from disease caused by the brucella bacteria but also reduce the chance that infected cattle will pass the disease to other uninfected cattle. We also hope to demonstrate that the vaccine can be given to pregnant cattle without increasing the risk of spontaneous abortion that has previously been blamed on the vaccine. If we are successful this work will provide a scientifically sound alternative to the current RB51 vaccine schedule, and may dispel vaccine related myths. The ability to reduce disease transmission will also improve the overall health of cattle in and around the infected area of the greater Yellowstone park. Lastly, the reduction in brucellosis in cattle is an economically desirable goal, reducing disease related costs to both the produced and consumer.
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Approach:<br/>
Animal studies: To investigate the parameters of humoral and cellular immunity conferred by the RB51 vaccine, we will undertake a prospective collaborative study measuring both humoral and cellular responses using cattle kept in the vicinity of the Wyoming State Veterinary Laboratory. We will used a single group of vaccinate animals for all three studies. There will be three study groups: 1. Calfhood vaccinates without any subsequent vaccination (Controls), 2. Calfhood vaccinates with adult vaccination while pregnant (AV) 3. Calfhood vaccinates with booster vaccination as heifers and adult vaccination while pregnant (BV+AV). Our hypothesis is that the humoral and cellular immune response in BV+AV group will be statistically significantly higher than the titers in the AV or control groups. Measures of Cell Mediated Immunity (CMI): We will test multiple measures of CMI in preliminary studies to determine the best assays to pursue as measures for RB51 vaccinates in this study. The analyses we intend to pursue are briefly outlined below. Serologic cytokine analysis. Whole blood (WB) ex vivo stimulation is a useful tool to investigate cytokine responses to a variety of stimuli, including bacterial endotoxin (LPS), antigens, allergens, and antibiotics. Cytokine profiling by transcriptional analysis. AWB ex vivo stimulation assay is a two-step process: (1) antigenic or mitogenic stimulation of cells in whole blood and (2) analysis of either plasma or cells for resulting cytokine production using a variety of methods such as RT-PCR, ELISA, flow cytometry or multiplex analyses, such as the fluorescent bead-based Luminex x-Multi Analyte Profile (MAP) technology. Lymphocyte differentiation/proliferation. CD4+ and CD8+ T-cell populations will be differentially quantified from PBMC's using the methods of Olsen and others [19]. Brucellin Test (DTH) The Brucellin skin test has been used for screening unvaccinated cattle herds. In this test, a defined quantity of Brucellin (Synbiotics, Lyon France) antigen is injected into the caudal fold or side of the neck 7-14 days post vaccination. Macrophage Killing to assess the capacity of mouse macrophages, activated with either soluble cytokines or whole immune T lymphocytes, to control or reduce numbers of intracellular bacteria. "Measurement of killing" is inferred from a reduction in the number of colony-forming units (CFU) of bacteria at the end of a culture period, compared to the input numbers of CFU at initiation of culture, to the peak numbers of CFU measured during culture, or to a control group in which killing is expected to be poor. T cell Epitope Screening, a recent addition to the repertoire of epitope prediction methods is reverse vaccinology, a method that requires knowledge of the whole pathogenic genome sequence.
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Progress:<br/>
2012/01 TO 2012/12<br/>
OUTPUTS: No discernable outputs have been achieved as the study has not reached a definitive milestone as of December 2012.
<br/>PARTICIPANTS: Dr. Jeff Adamovicz, Ph.D., RBP, lead investigator; Dr. Brant Schumaker DVM; Dr. Walt Cook DVM; Dr. Scott Lake DVM; artifical insemination Ms. Alex Kesterson, MS candidate; Dr. Steve Olsen, Ph.D. ARS, USDA Collaborator for challenge studies.
<br/>TARGET AUDIENCES: Clinical veterinarians, ranchers, beef producers, wildlife managers.
<br/>PROJECT MODIFICATIONS: We have arranged to have our pregnant cattle challenged with virulent Brucella abortus in the ARS, USDA ABSL-3 facility in Ames, Iowa. This goal was not part of our original proposal but will significantly improve the relevance of our data and conclusions. This effort is separately funded.
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IMPACT: Our proposed cattle vaccinations are underway. We have written and received approval for our experimental animal use cattle protocol. We have bred and delivered 25 healthy calves. We have administered the first dose of RB-51 vaccine to these cattle along with other standard veterinary care. We have collected and stored white blood cells from these cattle per our experimental protocol. During this past year we have developed methodologies to measure cell mediated immunity in cattle. This development effort will allow us to achieve our specific aim two. In addition, the use of these methods will support future research on Brucellosis in cattle. Our research is proceeding per our proposal and our staff has procured straws to inseminate our test cattle.