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United States Department of Agriculture

Agricultural Research Service

Research Project: Development of Strategies to Control Anaplasmosis

Location: Animal Diseases Research

2013 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. Replacing 5348-32000-027-00D (October 2011).


3.Progress Report:
Objective 1: Identify the molecular determinants of efficient tick-borne transmission of Anaplasma marginale through comparison of pathogen strains with distinctly different transmission phenotypes. For 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. However, through an assessment of the variability of outer membrane proteins we 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 Anaplasma strain rather than multiple strains. 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.

Objective 2: Identify the molecular determinants of vector competence with the goal of blocking tick transmission of Anaplasma marginale. A central step in meeting this objective is to characterize the transcriptome of Dermacentor andersoni during all stages of feeding. All samples have been collected, high quality RNA extracted, and high throughput sequencing is in progress. Because the genome of D. andersoni has not been sequenced, the resulting database will serve as an essential tool for the identification of tick proteins required for successful colonization of A. marginale within the tick. Additionally these experiments have been designed to identify the tick genes which are up-regulated in response to A. marginale colonization. These genes will serve as targets for RNAi experiments to determine if these genes are important for A. marginale colonization of the tick.

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 cross protective vaccine. Six primary vaccine candidates (Am202, Am368, Am1041, Oma87, Am854 and Am936) have been evaluated. These A. marginale genes have been sequenced from strains isolated from separate geographic locations in the United States, including an outbreak strain from the western United States, Mexico, and Puerto Rico. Sequencing of the two most promising candidates, Am854 and Am936, from Ghanian strains of A. marginale is in progress. The four most highly conserved proteins Am202, Am854, Am936 and Oma87 have been expressed as recombinant proteins. We have used these recombinant proteins to determine that Am854 and Am936 are strongly recognized in protectively immunized animals. An immunization and challenge experiment using these two proteins is in progress.


4.Accomplishments
1. Identification of two proteins which could serve as the primary components of a vaccine to prevent anaplasmosis. Anaplasmosis is a tick-borne bacterial pathogen of cattle which causes economic losses to cattle industries throughout the world due to a lack of safe and effective control measures. ARS researchers in Pullman, Washington with their collaborators at Washington State University are developing a vaccine to prevent this disease. Toward this end, they have identified two highly conserved, immunogenic proteins which, which when included in a vaccine, could induce protective immunity against most strains of anaplasma. This is significant progress because one of the major limitations of developing a vaccine against this disease is identifying vaccine candidates that could be universally effective.

2. Development of methods to block transmission of tick borne pathogens. One potentially highly effective and broadly applicable method to control tick-borne disease is to target the pathogen within the tick, thus preventing transmission of the pathogen to a new host. The first step in accomplishing this goal is to identify genes within the pathogen that are required for colonization of the tick vector. Toward this goal, ARS scientists in Pullman, Washington, (with additional funding from NIH) in collaboration with colleagues at Washington State University have identified four genes involved in tick transmission. Identification of these genes serves as the foundation for the development of chemotherapeutics or vaccines which will prevent transmission of tick borne diseases.


Review Publications
Herndon, D.R., Ueti, M.W., Reif, K.E., Noh, S.M., Brayton, K.A., Agnes, J.T., Palmer, G.H. 2013. Identification of multi-locus genetic heterogeneity in anaplasma marginale ss. centrale and its restriction following tick-borne transmission. Infection and Immunity. 81(5):1852-8.

Albarrak, S.M., Brown, W.C., Noh, S.M., Reif, K.E., Scoles, G.A., Turse, J.E., Norimine, J., Ueti, M.W., Palmer, G.H. 2012. Subdominant antigens in bacterial vaccines: Am779 is subdominant in the anaplasma marginale outer membrane vaccine but does not associate with protective immunity. PloSONE 7(9):e46372.

Noh, S.M., Turse, J., Brown, W.C., Normine, J., Palmer, G.H. 2013. Cross-linking of anaplasma marginale outer membrane proteins enhances immunogenicity, but is not required for protection from challenge. Vaccine. Clin.Vacc.Immun. 2013. 20(5):651-656.

Last Modified: 9/2/2014
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