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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

2020 Annual Report

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 intervention strategies to minimize the impact of bovine babesiosis outbreaks to include vaccines and therapeutic development targeting both the babesia pathogen and tick host. 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. Objective 3: Develop predictive models of potential babesia disease spread in the U.S. to assist in mitigating potential future outbreaks.

Objective 1: Develop diagnostic assays and intervention strategies to minimize the impact of bovine babesiosis outbreaks to include vaccines and therapeutic development targeting both the babesia pathogen and tick host. 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.

Progress Report
In support of Objective 1, significant progress has been made on all sub-objectives. For Sub-objective 1A, cattle were vaccinated with an antigen found on the surface of parasites. The results showed appropriate antibody responses against the molecule. However, live parasite challenge failed to show protection against severe disease. We discovered a group of proteins that can be used as potential vaccine candidates for controlling disease. We have synthesized the proteins to test if these proteins can protect against infection. For Sub-objective 1B, cattle were vaccinated with a protein found on the parasite during its development within the arthropod vector. Antibody responses against parasites were detected. The animals were infected with live parasites. Ticks were applied to determine if antibodies affected transmission. The results demonstrated that antibodies reduced the transmission and all the animals survived after tick transmission. In support of Sub-objective 1C, proteins were synthesized to vaccinate cattle. For Sub-objective 1D, validating a new diagnostic test to detect bovines infected with the parasite was complete and reported in a peer-reviewed journal. In support of Objective 2, progress has also been made in all sub-objectives. For Sub-objective 2A, progress was made towards developing both serological and molecular assays targeting a conserved gene present in the new horse parasite. For the serologic assay, recombinant protein was generated and subsequently purified and tested in serological assays. Results showed that the serum from infected horses recognized the recombinant protein and that assay background was minimal following protein purification. Subsequently, sera from horses infected with another parasite species was tested to assess potential cross-reactivity. We have developed a multiplex real-time qPCR assay capable of identifying and differentiating parasite infections from a blood sample. The assay was tested using blood from horses infected with different blood parasites and preliminary data suggests the test has high specificity. The assay has also been tested using blood from horses with two different parasites and results show that the assay successfully detected both parasites. For Sub-objective 2B, several tick species were fed on infected horses. However, only a few species were able to acquire the new parasite from infected horses. For Sub-objective 2C, the efficacy of drug therapy to clear parasite infection was assessed. Horses were infected with the new parasite and treated using a standard equine piroplasmosis dosing regimen. Following two rounds of treatment, all horses remained positive, indicating the lack of efficacy of the drug against this new horse parasite.

1. Proteins expressed by Babesia bigemina kinetes identified. Parasite transmitted by ticks causes severe disease in cattle and more than 90% of infected adult animals die. This parasite is spread in tropical and sub-tropical regions, including Mexico. There are live vaccines available in a few countries but not licensed in the United States. Should an outbreak occur, the U.S. cattle industry's ability to produce meat and milk will be severely affected. ARS scientists at Pullman, Washington, identified parasite proteins during tick infection that can be used as antigen candidates to develop vaccines for preventing parasite transmission. These vaccines will mitigate losses for the U.S. livestock industry if disease outbreaks occur in the United States.

2. Improvement of parasites detection infecting cattle. Parasites that cause infection in cattle present a significant economic concern for the cattle producers. Current diagnostic techniques are prone to human error and expensive and time-consuming to perform. Vibrational spectroscopic technologies can record a chemical snapshot of the entire organism and the surrounding cell, thereby providing a phenotype of the organism and the host infected cell. ARS scientists in Pullman, Washington, demonstrated the applicability of sensitive and specific vibrational spectroscopic imaging techniques including atomic force microscopy, infrared and confocal Raman microscopy to discover new biomarkers for the diagnosis of parasites that infect cattle. This technology has provided a valuable new tool for preventing parasite dissemination in the United States.

3. Evolution and diversity of the equi merozoite antigen (EMA) families of the divergent equid parasites. Tick-transmitted parasites impact equine health, as well as commerce, by restricting the movement of horses globally. The parasites persist in infected animals indefinitely, creating a reservoir for additional infections. The role of a group of parasite surface membrane proteins in maintaining the persistence of the parasite was investigated. ARS scientists in Pullman, Washington, used computer-based bioinformatic analysis to determine that the parasite proteins do not contribute to antigenic variation to escape the immune response, as is seen in related human and animal parasites. The proteins are predicted to interact with structural components of the horse’s red blood cells, and it is likely these proteins aid in blood cell entry and exit. These findings provide a valuable resource for vaccine development and eventual prevention of persistent infections.

Review Publications
Abdellrazeq, G.S., Fry, L.M., Elnaggar, M.M., Bannantine, J.P., Schneider, D.A., Chamberlain, W.M., Mahmoud, A.H.A., Park, K.T., Hulubei, V., Davis, W.C. 2020. Simultaneous cognate epitope recognition by bovine CD4 and CD8 T cells is essential for primary expansion of antigen-specific cytotoxic T-cells following ex vivo stimulation with a candidate Mycobacterium avium supsp. paratubeculosis peptide vaccine. Vaccine. 38(8):2016-2025.
Bastos, R.G., Sears, K.P., Dinkel, K.D., Knowles, D.P., Fry, L.M. 2019. Changes in the molecular and functional phenotype of bovine monocytes during Theileria parva infection. Infection and Immunity. 87(12).
Mosqueda, J., Hidalgo-Ruiz, M., Calvo-Olvera, D., Hernandez-Silva, D., Ueti, M.W., Mercado-Uriostegui, M.A., Rodriguez, A., Ramos-Aragon, J.A., Hernandez-Ortiz, R., Kawazu, S.I., Igarashi, I. 2019. RON2, a novel gene in Babesia bigemina, contains conserved, immunodominant B cell epitopes that induce antibodies that block merozoite invasion. Parasitology. 146(13):1646-1654.
Sears, K.P., Kappmeyer, L.S., Wise, L.N., Siva, M., Ueti, M.W., White, S.N., Reif, K.E., Knowles, D.P. 2019. Infection dynamics of Theileria equi and Theileria haneyi, a newly discovered apicomplexan of the horse. Veterinary Parasitology. 271:68-75.
Bohaliga, G.A.R., Johnson, W.C., Taus, N.S., Hussein, H.E., Bastos, R.G., Suarez, C.E., Scoles, G.A., Ueti, M.W. 2019. Identification of proteins expressed by Babesia bigemina kinetes. Parasites & Vectors. 12(1).
Rathinasamy, V., Poole, W.A., Bastos, R.G., Suarez, C.E., Cooke, B.M. 2019. Babesiosis vaccines: Lessons learned, challenges ahead, and future glimpses. Trends in Parasitology. 35(8):622-635.
Gallego-López, G.M., Cooke, B.M., Suarez, C.E. 2019. Interplay between attenuation-and virulence-factors of Babesia bovis and their contribution to the establishment of persistent infections in cattle. Pathogens. 8(3).
Bastos, R.G., Franceschi, V., Tebaldi, G., Connelley, T., Morrison, I.W., Knowles, D.W., Donofrio, G., Fry, L.M. 2019. Molecular and antigenic properties of mammalian cellexpressed Theileria parva antigen Tp9. Frontiers in Immunology. 10.
Elnaggar, M.M., Grandoni, F., Abdellrazeq, G.S., Fry, L.M., El-Naggar, K., Hulubei, V., Buttazzoni, L., Khaliel, S.A., Torky, H.A., Davis, W.C. 2019. Pattern of CD14, CD16, CD163 and CD172a expression on water buffalo (Bubalus bubalis) leukocytes. Veterinary Immunology and Immunopathology. 211:1-5.
Tretina, K., Pelle, R., Orvis, J., Gotia, H.T., Ifeonu, O.O., Kumari, P., Palmateer, N.C., Iqbal, S.B., Fry, L.M., Nene, V.M., Daubenberger, C.A., Bishop, R.P., Silva, J.C. 2020. Re-annotation of the Theileria parva genome refines 53% of the proteome and uncovers essential components of N-glycosylation, a conserved pathway in many organisms. BMC Genomics. 21.
Tretina, K., Haidar, M., Madsen-Bouterse, S.A., Sakura, T., Mfarrej, S., Fry, L.M., Chaussepied, M., Pain, A., Knowles, D.P., Nene, V.M., Ginsberg, D., Daubenberger, C.A., Bishop, R.P., Langsley, G., Silva, J.C. 2020. Theileria parasites subvert E2F signaling to stimulate leukocyte proliferation. Scientific Reports. 10.
Abdellrazeq, G.S., Mahmoud, A.H., Kun-Taek, P., Fry, L.M., Elnaggar, M.M., Schneider, D.A., Hulubei, V., Davis, W.C. 2020. relA is Achilles’ heel for mycobacterial pathogens as demonstrated with deletion mutants in Mycobacterium avium subsp. paratuberculosis and mycobacterium bovis bacillus Calmette-Guérin (BCG). Tuberculosis. 120.
Álvarez Martíneza, J.A., Figueroa Millán, J.V., Ueti, M.W., Rojas-Martínez, C. 2020. Innovative alternatives for continuous in vitro culture of Babesia bigemina in medium free of components of animal origin. Pathogens. 9(5).
Wise, L.N., Kappmeyer, L.S., Knowles, D.P., White, S.N. 2019. Evolution and diversity of the EMA families of the divergent equid parasites, Theileria equi and T. haneyi. Infection, Genetics and Evolution. 68:153-160.
Bishop, R.P., Odongo, D., Ahmed, J., Mwamuye, M., Fry, L.M., Knowles, D.P., Nanteza, A., Lubega, G., Gwakisa, P., Clausen, P., Obara, I. 2020. A review of recent research on Theileria parva: Implications for the infection and treatment vaccination method for control of East Coast fever. Transboundary and Emerging Diseases. 67(51):56-67.