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ARS Home » Pacific West Area » Pullman, Washington » Animal Disease Research » Research » Research Project #431742

Research Project: Development of Detection and Control Strategies for Bovine Babesiosis and Equine Piroplasmosis

Location: Animal Disease Research

2017 Annual Report

1a. Objectives (from AD-416):
The goals of this project are to develop multivalent bovine babesiosis subunit vaccines targeting antigens expressed in different Babesia bovis life cycle stages, to develop serological diagnostic assays to aid in bovine babesiosis and equine piroplasmosis diagnosis and surveillance, and to characterize vector competence and drug susceptibility for a new protozoan parasite of horses. These goals will be addressed in the following objectives: Objective 1: Develop diagnostic assays and vaccines for bovine babesiosis by targeting antigens from multiple parasite stages. Subobjective 1A: Determine if immunization with B. bovis blood stage subdominant antigens reduces disease severity and impacts tick infection. Subobjective 1B: Identify B. bovis tick stage specific targets for development of a vaccine to reduce or block tick infection. Subobjective 1C: Develop a multivalent vaccine targeting tick and B. bovis parasite proteins to decrease clinical disease and B. bovis transmission. Subobjective 1D: Develop a B. bovis serological assay to determine infection prevalence. Objective 2: Develop improved diagnostic assays and control strategies for emerging equine piroplasmosis organisms. Subobjective 2A: Identify diagnostic targets for detection of horses infected with Theileria-like parasite. Subobjective 2B: Determine competent tick vectors of the new Theileria-like parasite. Subobjective 2C: Determine Theileria-like parasite drug susceptibility.

1b. Approach (from AD-416):
Objective 1: Develop diagnostic assays and vaccines for bovine babesiosis by targeting antigens from multiple parasite stages. Goals: The goal of this objective is to develop preventive measures and diagnostic assays for bovine babesiosis in an effort to stop pathogen spread via tick vectors and to understand pathogen prevalence and distribution. Approach: Target B. bovis proteins expressed in vertebrate or invertebrate hosts which may provide control strategies to reduce disease severity in the mammalian host and block transmission of parasites via tick vectors. In addition, this project will provide diagnostic tools to determine vaccine efficacy and to assess pathogen prevalence and distribution in the U.S. Objective 2: Develop improved diagnostic assays and control strategies for emerging equine piroplasmosis organisms. Goals: Develop diagnostic assays for the newly discovered Theileria-like parasite (TLP) to discriminate between horses infected with this new parasite and those infected with T. equi, elucidate vector competency and determine the efficacy of drug therapy to prevent TLP spread in the U.S. horse population.

3. Progress Report:
Progress was made on both objectives and their sub objectives to address critical gaps in our knowledge concerning tick-borne pathogens of cattle and horses. For Objective 1, significant progress has been made towards the identification of Babesia bovis developmental proteins in both the tick vector and bovines. We developed serological tests to detect cattle infected with parasites. Progress has been made for the development of strategies to control bovine babesiosis by identifying five stage specific proteins that play significant roles for the B. bovis to complete its life cycle. These proteins are expressed by the parasite during its development in the gut and hemolymph of tick vectors (Sub objective 1a). Disrupting tick infection by targeting these proteins would prevent parasite transmission via tick vectors. Our laboratory also developed a more reliable serological assay for B. bovis (Sub objective 1d). This diagnostic assay allows us to understand the risks of spreading bovine babesiosis to the U.S. cattle papulation. An exogenous gene delivery system was developed in our laboratory by transfecting Babesia parasites. Stably transfected B. bovis expressing either green or red florescent proteins using a B. bovis kinete specific promoter were generated. These transfected B. bovis parasites will be used to deliver toxin proteins to ticks during B. bovis development in the hemolymph, and consequently, kill competent tick vectors as part of a National Institute of Food and Agriculture (NIFA) Agriculture and Food Research Inititative (AFRI) awarded grant, "A Pioneering Approach to Tick Control: Anti-tick Toxins Delivered via a Tick Transmissible Patogen", 2090-32000-039-12I(NIFA grant 2016-67015-24968). For objective 2, progress has been made towards identification of proteins expressed by the Theileria-like parasite, an emerging pathogen of horses, as part of diagnostic assay development. Genomic DNA of Theileria-like parasite was sequenced and potential proteins were identified to develop diagnostic assays (Sub objective 2a). In addition, our laboratory initiated drug therapy of experimentally infected horses with Theileria-like parasite to determine if imidocarb dipropionate kills this parasite in the blood. Blood samples are being collected weekly to determine the efficacy of imidocarb dipropionate to eliminate the parasites from infected horses (Sub objective 2c).

4. Accomplishments
1. Identified tick stage-specific proteins of Babesia bovis. Bovine babesiosis is a tick-borne disease caused by B. bovis, the parasite that infects red blood cells. This disease is one of the most deadly tick-borne diseases for the cattle industry in tropical and subtropical regions. There are no efficient vaccines available to prevent parasite transmission to animals. ARS scientists in Pullman, Washington, discovered proteins expressed by B. bovis during tick infection. These parasite tick-stage specific proteins can be used as candidate vaccines to block tick infection, potentially preventing parasite spread via tick vectors.

2. Developed a serological diagnostic test to determine cattle infected with B. bovis that cause bovine babesiosis. The discovery of acaricide resistant tick populations capable of transmitting protozoan parasites that cause bovine babesiosis on the U.S.-Mexico border and the lack of efficient vaccines to control infection and/or disease in the U.S. is a major concern for the U.S. livestock industry. Babesiosis is spread by tick vectors and cattle of any age, especially naive adults, are susceptible to infection that can result in 90 percent mortality. ARS scientists in Pullman, Washington, developed a modified diagnostic test with greater accuracy than previous competitive enzyme-linked immunosorbent assay (ELISA) to determine infection prevalence of bovine babesiosis on the U.S.-Mexico border. The diagnostic test will be useful to determine if there are potential risks of bovine babesiosis for the U.S. cattle population.

3. Identified proteins expressed by the Theileria-like parasite for the development of serological diagnostic tests. Theileria-like parasite in infected horse cannot be detected by any of the current official Theileria equi diagnostic tests. The lack of detection methods could allow this horse parasite to be unnoticed upon previous screening and to potentially be widespread within the susceptible U.S. horse population. ARS scientists in Pullman, Washington, completed the annotation of the Theileria-like parasite genome sequence and discovered genes that can be used to detect horses infected with this newly emerging pathogen. The serological diagnostic test will be useful to determine the distribution of the Theileria-like parasite in the U.S. horse population. The information will be critical for the development of control strategies to prevent pathogen spread to naïve U.S. horse population.

Review Publications
Silva, M.G., Knowles, D.P., Domingos, A., Esteves, A.M., Antunes, S., Suarez, C.E. 2017. Inhibition of the in vitro growth of babesia bigemina, babesia caballi and theileria equi parasites by trifluralin analogues. Ticks and Tick Borne Diseases. 8(4):593-597.

Johnson, W.C., Taus, N.S., Reif, K.E., Bohaliga, G., Kappmeyer, L.S., Ueti, M.W. 2017. Analysis of stage-specific protein expression during babesia bovis development within female rhipicephalus microplus. Journal of Proteome Research. 16(3):1327-1338.

Olds, C.L., Paul, T., Scoles, G.A. 2016. Detection of theileria parva in tissues of cattle undergoing severe east coast fever disease show significant parasite accumulation in the spleen. Veterinary Parasitology. 232:32-35.

Olds, C.L., Mwaura, S., Odongo, D., Scoles, G.A., Bishop, R., Daubenberger, C. 2016. Induction of humoral immune response to multiple recombinant rhipicephalus appendiculatus antigens and their effect on tick feeding success and pathogen transmission. Parasites & Vectors. 9(1):484.

Fry, L.M., Schneider, D.A., Frevert, C.W., Nelson, D.D., Morrison, I., Knowles, D.P. 2016. East coast fever caused by Theileria parva is characterized by macrophage activation associated with vasculitis and respiratory failure. PLoS One. doi:10.1371/journal.pone.0156004.

Elnaggar, M.M., Abdellrazeq, G.S., Mack, V., Fry, L.M., Davis, W.C., Park, T. 2016. Characterization and use of new monoclonal antibodies to CD11c,CD14, and CD163 to analyze the phenotypic complexity of ruminantmonocyte subsets. Veterinary Immunology and Immunopathology. 178:57-63.

Davis, W.C., Grandoni, F., Mahmoud, E.M., Abdellrazeq, G.S., Fry, L.M., Mack, V., Marchitelli, C., Barile, V.L. 2017. Characterization of leukocyte subsets in buffalo (Bubalus bubalis) with cross-reactive monoclonal antibodies specific for bovine MHC class I and class II molecules and leukocyte differentiation molecules. Developmental and Comparative Immunology. 74:101-109.

Alzan, H.F., Knowles, D.P., Suarez, C.E. 2016. Comparative bioinformatics analysis of transcription factor genes indicates conservation of key regulatory domains among babesia bovis, babesia microti and theileria equi. PLOS Neglected Tropical Diseases. doi:1371/journal.pntd.0004983.

Alzan, H.F., Lau, A., Knowles, D.P., Herndon, D.R., Ueti, M.W., Scoles, G.A., Kappmeyer, L.S., Suarez, C.E. 2016. Expression of 6-Cys gene superfamily defines babesia bovis sexual stage development within rhipicephalus microplus. PLoS One. doi:10.1371/journal.pone.0163791.

Suarez, C.E., Knowles, D.P., Sila, M. 2016. Identification of interchangeable cross-species function of elongation factor-1 alpha promoters in babesia bigemina and babesia bovis. Parasites & Vectors. doi:10.1186/s13071-016-1859-9.

Oldiges, D., Laughery, J., Tagliari, N., Leite Filho, R., Davis, W., Da Silva, V.I., Termignoni, C., Knowles, D.P., Suarez, C.E. 2016. Transfected babesia bovis expressing a tick GST as a live vector vaccine. PLOS Neglected Tropical Diseases. doi:10.1371/journal.pntd.0005152.

Chungwon, J.C., Suarez, C.E., Bandaranayake-Mudiya, N.L., Bandaranayake-Mudiya, N., Rzepka, J., Heiniger, T.J., Chung, G., Lee, S.S., Adans, E., Yun, G. 2017. A novel modified-indirect ELISA based on spherical body protein 4 for detecting antibody during acute and long-term infections with diverse babesia bovis strains. Parasites & Vectors. 10(1):77.

Alzan, A., Silva, M., Davis, W., Herndon, D.R., Schneider, D.A., Suarez, C.E. 2017. Geno- and phenotypic characteristics of a transfected babesia bovis 6-Cys-E knockout clonal line. Parasites & Vectors. 10(1):214.

Rogovskyy, A.S., Nebogatkin, I.V., Scoles, G.A. 2016. Ixodid ticks in the megapolis of Kyiv, Ukraine. Ticks and Tick Borne Diseases. 8(1):99-102.