Location: Infectious Bacterial Diseases Research2018 Annual Report
Objective 1: Refine the experimental infection models to characterize pathologic and immunologic responses in elk, swine and bison including use of molecular and/or proteomic and transcriptomic techniques. Subobjective 1.1: Refine the experimental challenge model for elk. Subobjective 1.2: Characterization of immunologic mechanisms related to protection after booster vaccination. Subobjective 1.3: Characterization of immunologic mechanisms related to protection after vaccination of swine and elk with novel new vaccines. Subobjective 1.4: Characterize transcriptomic responses of host and Brucella spp. to in vivo infection. Objective 2: Using the models refined in Objective 1, develop new and/or improved diagnostic and intervention strategies to control Brucella infections in wildlife reservoirs responsible for infecting domestic production animals. Subobjective 2.1: Identify vaccination strategies that are protective for bison, elk, and/or cattle against experimental challenge with Brucella abortus strain 2308. Subobjective 2.2: Characterize efficacy of novel vaccines to protect swine from virulent B. suis. Subobjective 2.3: Development of new brucellosis serologic assays using novel epitope identification strategies.
The long-term goals of this project are to facilitate the completion of brucellosis eradication programs in domestic livestock, and prevent reintroduction of brucellosis into livestock from wildlife reservoirs. Specifically, fundamental knowledge on Brucella pathogenesis will be gained, efficacious vaccination systems will be identified, and sensitive and specific diagnostic tools will be developed to aid eradication programs. Immunogenicity of vaccination strategies in targeted hosts (cattle, bison, elk, and swine), including novel vaccine platforms, will be evaluated in targeted species and efficacy characterized by experimental challenge. In addition, the project will try to improve the standard experimental challenge model for elk to better replicate the clinical effects of brucellosis under field conditions. By simultaneously characterizing the in vivo transcriptome of B. abortus and natural host during infection, we will develop knowledge of molecular mechanisms involved in regulation of host responses to infection, and genes expressed by the pathogen under in vivo conditions. This basic knowledge will identify future targets for development of new vaccines, diagnostics, immunomodulation, and possibly therapeutics. New diagnostics will be developed and analyzed for their ability to detect brucellosis in swine and cattle, and may allow differentiation of which Brucella spp. is associated with infection. The research will help resolve the risk of re-infection of domestic livestock from wildlife reservoirs of brucellosis, protect the financial investment that has been made in the U.S. brucellosis eradication program, and provide public health benefits by reducing the risk of zoonotic infection.
USDA initiated control measures for brucellosis in the 1930’s and established an eradication program in the 1950’s. In support of these regulatory efforts, billions have been invested at the state and federal level to achieve eradication of brucellosis from cattle. However, persistence of Brucella in wildlife reservoirs (bison, elk, and feral swine) pose a risk for reintroduction of disease to domestic livestock. Development of new vaccines and diagnostics that can be applied to domestic livestock and/or wildlife under current field conditions are needed. During the past year, work has been conducted on new vaccines for livestock and wildlife hosts, new diagnostics to detect brucellosis, and basic research to understand mechanism that Brucella used to establish and maintain infection. Advances in vaccines and diagnostics will be useful for protecting domestic livestock and managing brucellosis in current wildlife reservoirs within the U.S. The overall goal of the project is to facilitate eradication of brucellosis from natural hosts and prevent reintroduction of this disease into livestock in the United States. During the past year, progress was made in vaccine development including studies evaluating the efficacy of a ballistic vaccine in bison and characterization of immunologic responses to new vaccine candidates. Work was also completed to improve the brucellosis challenge model for elk in support of Subobjective 1.1. Although our laboratory has obtained high levels of abortions in non-vaccinated cattle and bison after experimental challenge, previous studies in our laboratory have resulted in very few abortions when elk receive an experimental challenge in accordance with the standard challenge model. In the current experiment the effect of challenge dose and time of delivery was evaluated in an effort to increase infection and clinical effects (abortions). Work was also completed on validating inactivation of Brucella bacteria to meet Select Agent requirements. Research studies to develop new diagnostics were conducted including use of a unique molecular approach for antigen discovery and expression of Brucella antigens in phage in support of Subobjective 2.3.
1. Brucella challenge model for elk. The prevalence of brucellosis in free-ranging elk in the Greater Yellowstone Area (area surrounding Yellowstone National Park) has been epidemiologically linked to infections in cattle herds. Previous work indicated that elk do not demonstrate high levels of clinical effects of brucellosis infection (i.e. abortion) when compared to clinical effects in cattle or bison hosts. In an effort to develop a better challenge model for assessment of vaccine efficacy, ARS scientists in Ames, Iowa, experimentally infected pregnant elk cows with standard or high dosages of virulent B. abortus early in the third trimester (standard challenge), or approximately 2 months prior in the early part of the second trimester. Experimental challenge earlier in gestation did not increase abortion rates although in both challenge times there appeared to be a slight increase in abortion rate when a higher challenge dose was delivered. This data demonstrates that elk do not abort as frequently after experimental infection with B. abortus as compared to cattle or bison natural hosts. This work suggests that pathogenesis of disease differs in elk as compared to other ruminant hosts of B. abortus and have implications for development of protective vaccines.
2. Optimization of sampling for gene expression. Evaluating gene expression in response to bacterial infection can provide insights into disease pathogenesis in natural hosts. However, measuring the transcribed RNA can be difficult due to its tendency to be highly degradable. In an effort to develop methods to sample tissues for measurement of gene expression, ARS researchers at Ames, Iowa, assessed procedures for isolating RNA from bovine tissues for the quality and quantity of recovered RNA. This data provides guidelines on appropriate procedures to recover high quality RNA from bovine tissues that will allow accurate assessment of gene expression differences under disease conditions.
3. Validation of inactivation methods for Brucella. Because of publicized failures involving Select Agent pathogens, the regulatory program has emphasized the need to validate methods for inactivation of bacterial and viral agents. In an effort to provide validated methods of inactivation of Brucella spp., ARS scientists at Ames, Iowa, tested a number of inactivation methods (heat, methanol, acetone, filtration, and formalin) under various conditions in a temporal manner. These experiments demonstrated that 95C heating for 1 hour, 67 percent methanol for 5 days, or formalin treatment for 30 minutes were sufficient to prevent recovery of Brucella from spiked samples. Filtration of sera through a 0.22 um filter removed all viable Brucella from spiked samples. This published data is of great interest to regulatory personnel and laboratories that work with Select Agents and provides validated procedures that are effective for inactivating Brucella from sera and tissue samples.
Vrentas, C.E., Schaut, R.G., Boggiatto, P.M., Olsen, S.C., Sutterwala, F.G., Moayeri, M. 2018. Inflammasomes in livestock and wildlife: Insights into the intersection of pathogens and natural host species. Veterinary Immunology and Immunopathology. 201:49-56. https://doi.org/10.1016/j.vetimm.2018.05.008.
Vrentas, C.E., Boggiatto, P.M., Schaut, R.G., Olsen, S.C. 2018. Collection and processing of lymph nodes from large animals for RNA analysis: preparing for lymph node transcriptomic studies of large animal species. Journal of Visualized Experiments. 135:e57195. doi:10.3791/57195.
Olsen, S.C., Boggiatto, P.M., Vrentas, C.E. 2017. Inactivation of virulent Brucella species in culture and animal samples. Applied Biosafety. 22(4):145-151. https://doi.org/10.1177/1535676017734202.
Olsen, S.C., Boggiatto, P.M., White, D.M., McNunn, T.B. 2018. Biosafety concerns related to Brucella and its potential use as a bioweapon. Applied Biosafety. 23(2)77-90. https://doi.org/10.1177/1535676018771983.