2012 Annual Report
1a.Objectives (from AD-416):
Objective 1: Identify the molecular determinants of efficient tick-borne transmission of Anaplasma marginale through comparison of pathogen strains with distinctly different transmission phenotypes.
Subobjective 1.A: Compare the tick colonization and transmission efficiency phenotypes of A. marginale subsp. centrale before and after tick selection.
Subobjective 1.B: Identify genetic markers that are predictive of the tick transmission phenotype of A. marginale field strains.
Objective 2: Identify the molecular determinants of vector competence with the goal of blocking tick transmission of Anaplasma marginale.
Subobjective 2.A: Define the proteome of the A. marginale-containing vacuole in cultured tick cells.
Subobjective 2.B: Identify tick proteins that are required for A. marginale replication in tick cell culture.
Subobjective 2.C: Identify tick-specific genes that are required for A. marginale transmission.
Objective 3: Assess the capacity to induce protective immunity to challenge by identifying and testing subdominant Anaplasma marginale antigens with the goal of developing a crossprotective vaccine.
Subobjective 3.A: Identify the widely conserved outer membrane proteins that induce broadly cross-reactive antibody.
Subobjective 3.B: Test the ability of the proteins identified in subobjective 3.A to induce protection to homologous and heterologous A. marginale challenge.
1b.Approach (from AD-416):
Anaplasma marginale, the causative agent of anaplasmosis, is the most prevalent tick-borne pathogen of livestock worldwide. This bacterial pathogen causes a significant disease burden to cattle in the United States and is a barrier to trade. The tools currently available to control this disease are limited and rely on treatment of clinically affected animals and tick control. The work proposed here is designed to fill knowledge gaps required for development of more effective control strategies. In the proposed experiments we target two points of control, one aimed at preventing infection of the bovine host, and the other aimed at preventing tick transmission. Using a comparative approach, we will identify genetic markers of highly efficient tick transmission in A. marginale, thus allowing for the development of a vaccine targeting potential outbreak strains. Concurrently, we will identify and test conserved subdominant antigens for the ability to induce protection against homologous and heterologous challenge in cattle. Together these data will guide the development of an effective vaccine. Using a proteomics approach followed by RNAi experiments to knock-down specific gene function, we will identify the molecules unique to the tick that are required for A. marginale transmission. Identification of these molecules will lay the foundation for development of novel methods to block transmission of A. marginale at the level of the tick vector.
Objective 1, we have rejected our first hypothesis that tick transmission selects for variants of A. marginale subsp. centrale that are more efficiently transmitted than the parent population. Consequently, the genetic comparison that was planned to identify molecular determinants of tick transmission will not be possible. However, an assessment of the variability of outer membrane proteins has led to insight into the requirements of an effective vaccine (see accomplishment 1).
Objective 2,we are in the process of acquiring reagents and developing assays for this objective. We have acquired and are successfully maintaining DAE100 tick cells derived from Dermacentor andersoni, a vector of A. marginale. We have developed an immunofluorescence assay, which will be used to characterize the A. marginale vacuole in the tick cells. Preliminary data suggest that the A. marginale vacuole within the tick resembles an early endosome.
Objective 3, Five primary vaccine candidates (Am202, Am368, Am1041, Oma87, Am854 and Am779) are in the process of being assessed and tested. Am202, Am368 and Am1041 from several widely separate geographic locations in the United States, including an outbreak strain from the western United States, Australia, Mexico, and Puerto Rico have been sequenced. All have >80% amino acid identity, with Am 1041 being the most highly conserved. Total IgG directed against Am202 in 15 animals protectively immunized with three different outer membrane based formulations has been measured. Ten of fifteen animals had antibody to Am202 with titers that ranged from 300-10,000 (reciprocal of the end-point dilution). We are in the process of expressing AM368, AM1041, OMA87, AM854 as protein. These recombinant proteins will then be used to determine if immunization induces a more or less robust immune response to these proteins as compared to Am202.
Testing of Am779 is complete and a manuscript is in preparation. Am779 is highly conserved and is recognized by protectively immunized animals. However, this protein was not protective in an immunization and challenge study.
Development of tools to assess the risk of exposure to tick-borne disease. A. marginale can cause severe disease and death in cattle, though such events tend to be episodic and difficult to predict. The abundance and distribution of the tick vector, parameters which are difficult to estimate, are two of the major determinants of disease outbreaks. ARS scientists in Pullman, Washington, in collaboration with colleagues at the Research Centre, Agriculture and Agri-Foods in Lethbridge, Canada have developed a set of recommendations for sampling populations of Dermacentor andersoni ticks under rangeland conditions. These findings will be essential for building models which can accurately asses risk of exposure to vector-borne disease in general and A. marginale, specifically.
Toward development of methods to block tick transmission. Many vector-borne pathogens that affect both humans and animals are obligate intracellular bacteria and are poorly amenable to genetic manipulation. Consequently, our ability to identify the molecular requirements of tick transmission is limited. The identification of these requirements is necessary to develop, novel, widely applicable tools, such as vaccines, to block the transmission of tick-borne diseases. To address this deficiency, ARS scientists in Pullman, Washington, (with additional funding from NIH) in collaboration with colleagues at Washington State University have determined that Francisella tularensis subsp. novicida colonizes and is transmitted by Dermacentor sp. ticks similar to tick borne pathogens. Because of Francisella sp. are amenable to genetic manipulation, this model can and is currently being used to identify genes that are required for tick transmission.
Toward developing a vaccine to prevent anaplasmosis. Anaplasma marginale is a tick-borne bacterial pathogen of cattle which continues to threaten and cause economic losses to cattle industries throughout the world due to a lack of safe effective control measures. While there is no recent analysis of the economic impact of anaplasmosis in the U.S., a 2006 study by the Canadian Food Inspection Agency estimated costs of endemic anaplasmosis in Canada, should it become established there, would be 10 to 30 million US dollars per year. This estimate is likely low for the U.S. situation as the Canadian cattle industry is approximately one-sixth the size of the U.S. industry. Currently, ARS researchers in Pullman, Washington with their collaborators at Washington State University are working to develop a vaccine to prevent this disease. Recent studies have determined that the A. centrale vaccine strain, a live attenuated organism used as a vaccine in many parts of the world, is composed of a single strain rather than multiple strains of bacteria. These findings are important because they indicate that inclusion of antigens from multiple strains within a vaccine is not necessary for the induction of protective immunity, thus simplifying the requirements for development of a vaccine to prevent anaplasmosis.
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