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United States Department of Agriculture

Agricultural Research Service

Research Project: Identification of Disease Mechanisms and Development of Improved Diagnostics and Vaccines for Brucellosis in Livestock and Wildlife
2013 Annual Report


1a.Objectives (from AD-416):
Objective 1: Identify specific factors including tissue or cell tropism, gene regulation, immune evasion mechanisms, and protective antigens through use of transcriptome and proteosome technologies to provide information on the pathophysiology of Brucella species and the host-pathogen interaction. Subobjective 1.1: Characterize transcriptome responses of cattle and pathogen (Brucella abortus) associated with experimental infection. Subobjective 1.2: Engineer site-directed mutants of Brucella spp. to be used as potential live attenuated vaccine candidates. Objective 2: Develop improved diagnostic assays with increased sensitivity and specificity that will differentiate the various Brucella species and allow determination of phylogenetic relationships. Subobjective 2.1: Development of more sensitive and specific B. suis serologic tests for swine. Subobjective 2.2: Improvements in cattle diagnostics to allow serologic differentiation of B. abortus and B. suis infections. Subobjective 2.3: Characterize molecular markers that clarify phylogenetic linkages among isolates with similar DNA fingerprints. Objective 3: Develop improved vaccines using new and novel delivery systems and platforms. Subobjective 3.1: Identify safe and efficacious vaccination strategies to protect targeted hosts against brucellosis caused by Brucella abortus. Subobjective 3.2: Identify safe and efficacious vaccination strategies to protect swine (including feral swine) against infection with Brucella suis.


1b.Approach (from AD-416):
The three objectives of this project include a basic research component (Obj 1), a diagnostic component (Obj 2), and a vaccine efficacy component (Obj.
3)as exemplified in Fig 3. The basic research portion is designed to develop basic knowledge of gene expression in the host or pathogen, or modify a Brucella gene which the pathogen may use to subvert immune recognition, in an effort to provide approaches for improved vaccines or diagnostics that could eventually be evaluated in other objectives (Obj 2 & 3). The vaccine efficacy component (Obj.
3)builds on previous experiments by expanding RB51 vaccination approaches that directly support the proposed approaches in the Bison Remote Vaccination EIS, and building on previous data using a rough vaccine strain (353-1) and Salmonella RASV strains. Other experiments will use a novel vaccine approach in elk that may modify the non-protective host response to intracellular bacteria. Experiments with B. suis and B. abortus in Obj 1 may also identify targets that may lead to novel approaches for diagnostics in Obj 2. Objectives 1 and 3 will also provide samples to assist in diagnostic development experiments in Obj 2. Scientific advances in diagnostics and vaccines will support National Brucellosis Eradication programs and provide scientific support to other agencies with responsibilities for managing brucellosis in wildlife.


3.Progress Report:
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 of dollars 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, collaborative projects evaluating brucellosis vaccines in elk, bison, and swine have been conducted; including efficacy trials conducted under Biolevel 3 containment. Data collected has indicated that booster vaccination of bison with RB51 (calfhood plus yearling vaccination) induces greater protection against experimental challenge than a single vaccination administered to calves. Data also suggests that a new B. suis vaccine developed in our laboratory is effective in protecting swine against brucellosis. Limited data suggests that feral swine may be more susceptible to infection with B. suis than domestic swine. The initial assembly of the bison genome is completed and further work is being done to polish and improve the annotated sequence. Initial studies of host and pathogen gene expression when B. abortus infects cattle are being completed. The availability of the bison genomic sequence combined with the studies in cattle will allow comparative analysis of the interaction between natural hosts and Brucella abortus, and may identify genes important for disease pathogenesis. The project is also evaluating new vaccine strategies and exploring new diagnostic approaches for detection and prevention of brucellosis in domestic livestock and wildlife. Advances in vaccines and diagnostics will be useful for protecting domestic livestock and managing brucellosis in current wildlife reservoirs within the U.S. Overall, work conducted by the project will facilitate eradication of brucellosis from natural hosts and prevent reintroduction of this disease into livestock in the United States.


4.Accomplishments
1. Booster vaccination of bison calves with the commercial RB51 vaccine. There is a high prevalence of brucellosis in free-ranging bison in Yellowstone National Park. Because of its risk for causing human infection, billions of dollars of federal and state funds were spent to eradicate brucellosis in domestic livestock. There is concern that brucellosis in this bison population is a risk for transmission of this disease into domestic livestock. As any vaccination program will be difficult and expensive, the most efficacious brucellosis vaccine for bison is needed. ARS researchers in Ames, Iowa, evaluated the safety, immunity, and protection after bison were booster vaccinated with RB51 (calfhood and yearling) and compared it to responses of bison inoculated once with RB51 (calfhood) or controls (non-vaccinates). Experimental protection was similar for the booster vaccination treatment and colonization of Brucella in tissues was reduced. This data suggests that booster vaccination of bison calves with RB51 will enhance protection against brucellosis, thereby providing a tool to reduce disease prevalence and prevent transmission of brucellosis to domestic livestock and protect public health.

2. Bison genome sequenced. Obtaining and annotating the genomic sequence of a natural host of Brucella will allow detailed understanding of gene expression and regulation in the host and pathogen during natural infections. Greater knowledge of these interactions will allow identification of host and pathogen genes that regulate or modulate infection. ARS researchers in Ames, Iowa, used new generation sequencing technology to obtain the sequence of the bison genome. Currently, the first assembly of the bison genome has been completed. Further work is ongoing to obtain additional sequence data to refine the first assembly. Advances in basic molecular knowledge of host and pathogen gene expression will be of benefit in developing new species-specific approaches for advancement of vaccines and diagnostics which will benefit regulatory personnel, livestock owners, and public health by facilitating control or eradication of brucellosis.

3. Analysis of gene expression of Brucella abortus in cattle. Characterizing increases or decreases in gene expression in the natural host (cattle) or the pathogen (B. abortus) during in vivo infection is an effective way to understand molecular mechanisms involved in disease pathogenesis. ARS researchers in Ames, Iowa, infected pregnant cattle with virulent B. abortus or the RB51 vaccine strain in midgestation, the time when cattle are most susceptible to Brucella infection. Infection in tissues was characterized by microbiologic methods and host and pathogen RNA was isolated from tissues for sequencing. Host and pathogen genes which are increased in expression during infection and genes that are upregulated during infection were identified. These genes will undergo further characterization and be evaluated for their potential use as diagnostic or vaccine targets. Development of new vaccines and diagnostics will benefit regulatory personnel, livestock owners and public health by facilitating control or eradication of brucellosis. This study will provide fundamental information on the interaction between a natural host and the Brucella pathogen, and genes of importance in the pathogenesis of infection.


Review Publications
Olsen, S.C. 2013. Biosafety considerations for in vivo work with risk group 3 pathogens in large animals and wildlife in North America. Animal Health Research Reviews. 14(1):2-10.

Olsen, S.C. 2013. Recent developments in livestock and wildlife brucellosis vaccination. World Organization for Animal Health Scientific and Technical Review. 32(1):207-217.

Last Modified: 8/19/2014
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