Progress 06/01/10 to 05/31/13
Outputs
Target Audience: Livestockproducers; State Department of Agriculture; State Game and Fish; wildlife managers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Results of the goat study will be presented at the December 2013, Conference of Research Workers on Animal Diseases (CRWAD) annual meeting. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Goal 1: A pilot immunization/challenge study in the gravid caprine model for brucellosis was completed. Three sub-unit vaccine formulations adjuvanted with aluminum hydroxide (Alhydrogel were used to vaccinate 50 female mixed-breed goats in a two-dose immunization regimen. Immune take was assessedserologically by Western blot. Animals were subsequently impregnated, challenged in the third trimester with Brucella abortus 2308, and and allowed to come to full gestational term. Control groups consisted of adjuvant-only immunized animals and a group that received the RB51 vaccine. Abortion rates in the sub-unit vaccine groups were no different than the adjuvant-only negative control groups (best case, p = .084, Fisher's Extact). More remarkably, RB51 failed to protect animals, suggesting that the established live attentuated vaccine is effective at abrogating brucellosis in this host species. Bacterial loads in milk and tissues of the dams and kids were also assessed. Again, no differences were noted either in the distribution of the pathogen in different tissues (uterus, lymph nodes, lung, abomason), nor in bacterial numbers. Goal 2: Weobserved the ability of Brucella abortus to avoid amoebal predation, and consequently exploited this phenomenon as a simple, high-throughput screen of amoebal consumption preference to mine for virulence determinants based on gain-of-toxicity phenotypes in a B. abortus cosmid library. Our results show that when B. abortus RB51 is challenged with predatory Acanthamoeba castellanii, the bacteria avoid consumption and display an unusual escape phenotype. Using a previously described method, we screened a library of over 400 RB51 cosmid clones and found that none conferred the ability to avoid amoebal consumption to the E. coli host. In addition, none of the fosmids granted E. coli the ability of B. abortus to become internalized by amoebae and to survive for up to 45 minutes in a gentamicin protection assay. Our results provide evidence for a relevant interaction between Brucella spp. and free-living amoebae, and show promise for the use of A. castellanii as a model host for virulence gene discovery in Brucella.
Publications
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Progress 06/01/11 to 05/31/12
Outputs
OUTPUTS: Project 1: Evaluation of protective efficacy of selected B. abortus recombinant protein formulations in a caprine challenge model for brucellosis. Purified large quantities of two key brucella virulence proteins, Hia and D15, for use in the goat immunization/challenge study. Immunized 50 mixed-bred female goats with three different vaccine formulations. Immunized two control groups with RB51 live vaccine strain and adjuvant (aluminum hydroxide) respectively, in a two-dose regimen. Bred all immune animals. PARTICIPANTS: Gerard P. Andrews, PI/Project Director, University of Wyoming; Richard Bowen, co-PI, Colorado State University; Jack Leonhardt, graduate student, University of Wyoming TARGET AUDIENCES: State and regional veterinarians, epidemiologists, WY Game and Fish Department, Wyoming Department of Public Health, cattle producers in WY PROJECT MODIFICATIONS: Location of off-site goat vaccine study has changed from LSU to CSU.
Impacts
In a related study, we demonstrated that two B. abortus proteins, Hia and D15, are able to induce clearance immunity in a BLAB/c mouse model for fully virulent B. abortus infection. In this regard, we have chosen these two proteins to evaluate in a gravid caprine model for their ability to abrogate disease (abortogenicity). If successful, one or both of these proteins, formulated with a non-toxic adjuvant, may represent a next-generation veterinary vaccine against brucellosis.
Publications
- Andrews, G.P., Leonhardt, J.A., Dougherty, A.M., Lowry, J.E., and R. Bowen. 2012. Brucella abortus recombinant outer membrane proteins induce clearance immunity against virulent challenge in BALB/c mice. The 93rd Annual Meeting of the Conference of Research Workers in Animal Diseases (CRWAD), Chicago, IL.
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Progress 06/01/10 to 05/31/11
Outputs
OUTPUTS: Project 1. Evaluation of protective efficacy of selected B. abortus recombinant protein formulations in a caprine challenge model for brucellosis. Outputs: Scaled-up production of three selected B. abortus antigenic proteins previously shown to be relevant to infection in two different susceptible hosts. Completed protocol (reviewed and approved) for immunization/challenge study using formulations of the three Brucella abortus recombinant proteins in a gravid caprine model for brucellosis. Project 2. Identification of novel candidate virulence factors from B. abortus using Rapid Virulence Annotation (RVA), a high-throughput functional genomic screen. Construct cosmid libraries from B. abortus strains. Identify candidate genomic regions by screening cosmid libraries for gain-of-toxicity against host organisms/cell lines. Identify specific genes responsible for gain-of-toxicity by transposon mutagenesis of positive cosmid clones. Outputs: Isolated and purified high molecular weight genomic DNA from B. abortus RB51. Constructed a cosmid library of RB51 genomic DNA in the Epicentre cosmid vector pWEB. Effect of an improved brucellosis vaccine on cattle producers' incentives to use adult-booster vaccination. Project 3. Determine the potential for an improved brucellosis vaccine to increase the willingness of cattle producers in the Greater Yellowstone Area to use adult-booster vaccination. Outputs: Estimated the costs and revenues of a cow-calf-yearling operation with versus without adult-booster vaccination, combined with the presence versus absence of a brucellosis-infected animal. Constructed a worksheet in Excel to calculate expected profit with and without adult-booster vaccination assuming current versus improved vaccine efficacy. Defined distributions for relevant epidemiological and economic parameters. We are now using the software program @Risk to conduct a Monte Carlo simulation of expected profit and adoption of adult-booster vaccination under current versus improved vaccine efficacy. We will then compare simulation results to determine the effect of an improved vaccine on producers' willingness to adopt adult-booster vaccination. PARTICIPANTS: Gerard P. Andrews, Department of Veterinary Sciences, UW, co-PI and project director; Phil Elzer, School of Veterinary Medicine, LSU, co-PI; Naomi Ward, Department of Molecular Biology, UW, co-PI; Chris Vassallo, Department of Molecular Biology, UW, graduate student; Dannele E. Peck, Department of Agricultural and Applied Economics, UW, co-PI; Bryan A. Wilson, Department of Agricultural and Applied Economics, UW, graduate student; Trenton W. Roberts, Department of Agricultural and Applied Economics, UW, graduate student; John P. Ritten, Department of Agricultural and Applied Economics, UW, collaborator; Roger H. Coupal, Department of Agricultural and Applied Economics, UW, collaborator. TARGET AUDIENCES: State and regional veterinarians and epidemiologists; Wyoming Game and Fish Department; Wyoming Department of Public Health; cattle producers in the Greater Yellowstone Area. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Project 1. Our previous work in the murine model strongly suggest the potential for use of B. abortus malate dehydrogenase, as a recombinant subunit vaccine candidate for brucellosis. AfuA and D15 may also represent promising subunit vaccine candidates when used in combination with Mdh. In this regard, it is quite possible that immunization with a cocktail of these antigens will produce synergistic effects. To test this hypothesis, we have scaled up four B. abortus proteins for use in an ungulate model for evaluation of their ability to induce protective immunity against wild-type Brucella challenge. Project 2. Successful construction of a cosmid library requires availability of high-quality, high molecular weight genomic DNA. Thus we have expended considerable effort in obtaining such DNA from B. abortus strains. We began work with strain RB51, with the goal of optimizing DNA isolation and purification protocols in this strain, and subsequently applying them to a second B. abortus strain. The Andrews lab has successfully optimized protocols to obtain DNA of the requisite quality and quantity from strain RB51. This DNA has been used to generate a cosmid library. A library of over four hundred cosmid clones has been generated and preserved by preparation of glycerol freezer stocks. Production of this resource has been quite time-consuming, principally due to problems experienced with early attempts at cosmid cloning, and the troubleshooting required to resolve these problems. We are currently involved in end-sequencing the library to verify genome coverage. Other short-term goals include characterization of representative cosmid inserts by determination of insert size by pulsed-field gel electrophoresis, and testing for protein expression by polyacrylamide gel electrophoresis of E. coli culture supernatants. project 3. Our economic analysis reveals that if a 400-head herd contracts brucellosis, and is quarantined for one year without compensation, the owner will incur $134,000 in additional costs (feed and labor ). The total cost of administering adult-booster vaccination to all adult cows once in their ten-year useful life, and to incoming two-year old heifers each year over a ten-year period, is roughly $16,500. When this total cost is annualized over a 10-year period, adult-booster vaccination costs $1,695 per year. The producer incurs just $854 of this annual cost because of cost-shares with the Wyoming Livestock Board, which pays the remaining $841. We have constructed spreadsheets in Excel to calculate expected profit with and without adult-booster vaccination assuming current versus improved vaccine efficacy. The spreadsheets accommodate a range of values for the probability of a cow being exposed to brucellosis, and the effectiveness of calfhood and adult-booster vaccinations. The spreadsheets track sexually-intact cows through one year, during which time each individual cow has a given probability of contracting brucellosis. This probability depends on whether their calfhood vaccination was effective, whether they received an adult-booster vaccination, and whether the adult-booster vaccination was effective.
Publications
- Lowry, J.E., Isaak, D.D., Leonhardt, J., Vernati, G., Fluegel, A.M., Pate, J., and Andrews, G.P. 2011. Vaccination with recombinant antigens reduces Brucella abortus strain-19 colonization in a mouse model for infection. Plos ONE. March 11; 6(3): e17425.
- Roberts, T.W. 2011. Master's Thesis: Costs and Expected Benefits to Cattle Producers of Brucellosis Management Strategies in the Greater Yellowstone Area of Wyoming. Department of Agricultural and Applied Economics, University of Wyoming.
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