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

Location: Animal Disease Research Unit

2021 Annual Report

Objectives
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.

Approach
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
This is the final report for project plan 2090-32000-039-00D which terminates in September 2021. Overall, the project provided important scientific information to prevent dissemination of tick-borne parasites that affect cattle or horses, including the discovery of parasite proteins for vaccine development and diagnostics, therapeutics, and determination of a biological vector responsible for parasite transmission. The scientific information acquired in the project plan allowed for the development of the FY21-26 project plan. In support of Objective 1, significant research progress was made on all sub-objectives. For Sub-objectives 1A and 1B, cattle parasite genes and proteins were discovered which can be used to prevent the spread of the parasite via ticks. These genes and proteins were found in different life stages of the parasite during infection of cattle or ticks. Results of vaccinating cattle showed production of specific antibodies that disrupted the life cycle of the parasite within tick vectors and, consequently, blocked parasite transmission to cattle. In support of Sub-objective 1C, tick and parasite proteins were tested to control tick infestation and disease manifestation. Results provided important information regarding the strategy and demonstrated its potential to prevent animal death and tick feeding. For Sub-objective 1D, our laboratory developed a serological test to fill a gap in diagnosing infection. The serological test can be used to limit parasite spread by treatment or removal of infected animals from the herd. This will provide a strategy to keep the United States free of bovine babesiosis. In support of Objective 2, significant research progress was made on all sub-objectives. In support of Sub-objective 2A, our laboratory sequenced a horse parasite genome and identified proteins to develop a serological assay to detect infected horses. In addition, unique parasite genes were identified that can be used as additional markers to detect infected horses. For Sub-objective 2B, several ticks that feed on horses were tested to determine their ability to transmit the parasite. The testing showed that one tick species became infected with the new parasite suggesting its ability as a biological vector. Results obtained in this sub-objective add to the knowledge regarding the risk of parasite dissemination by biological vectors present in the United States. For Sub-objective 2C, our laboratory treated horses infected with parasites and demonstrated drug susceptibility or resistance of the parasite. This information is critically important to keep the United States free of parasites that infect horses. In addition, significant progress was made on subordinate projects toward understanding pathogen transmission by ticks. Using whole gene replacement, a promoter with a fluorescent reporter gene was appended into the parasite genome. We demonstrated the entire life cycle of the genetically modified parasite in the vertebrate and invertebrate hosts (2090-32000-039-12-I). As well, we isolated secreted parasites from infected ticks using the in vitro tick feeding system and showed they infected bovine lymphocytes demonstrating that isolated parasites remained viable and infectious (2090-32000-039-23-T). In support of Sub-objectives 1A, 1B, and 2B, these subordinate projects helped us understand the interactions between pathogen, mammalian, and tick to develop strategies to prevent parasite dissemination.

Accomplishments
1. Identification of genes that are essential for the parasite life cycle. Cattle tick fever causes an economic burden to the livestock industry in tropical and subtropical regions. These cattle pathogens are considered a foreign disease and if these parasites are introduced into the United States, it could cause high mortality to naïve cattle. ARS scientists in Pullman, Washington, are developing vaccines that will control disease or prevent transmission of the parasite by the biological vector. Proteins were used to vaccinate cattle with promising results showing prevention of tick infection and, consequently, disrupting parasite transmission to cattle. Other proteins are being investigated to determine their roles in parasite development during infection of cattle.

2. Development of treatment to control parasites that infect cattle and horses. Control of diseases that affect cattle and horses are continuously needed to prevent the spread of tick-borne parasites in the United States. Identification of efficient drug therapies to kill parasites would immediately help to control the dissemination of the parasites to naïve animals. ARS scientists in Pullman, Washington, tested novel drugs to control several parasites that infect cattle and horses. These drugs efficiently killed parasites in in vitro condition. In addition, these drugs were not toxic to mammalian cells. The next step is to determine the efficacy of these drugs in in vivo infected animals.

3. Development of a serological tool to identify infected horses. Parasites that infect horses cause significant economic loses for the horse industry. However, a new parasite that infects horses was identified along the U.S.-Mexico border. There are no available diagnostic tools to detect horses infected with the new parasite. ARS scientists in Pullman, Washington, developed a test to detect antibodies against the new parasite. This test identifies horses infected with the new parasite. This test may help identify animals infected with this parasite and thus provides opportunities for treatment.

Review Publications
Ueti, M.W., Johnson, W.C., Kappmeyer, L.S., Herndon, D.R., Mousel, M.R., Reif, K.E., Taus, N.S., Ifeonu, O.O., Silva, J.C., Suarez, C.E., Brayton, K.A. 2020. Comparative analysis of gene expression between Babesia bovis blood stages and kinetes allowed by improved genome annotation.. International Journal for Parasitology. 51(2-3):123-136. https://doi.org/10.1016/j.ijpara.2020.08.006.
Tirosh-Levy, S., Gottlieb, Y., Fry, L.M., Knowles, D.P., Steinman, A. 2020. Twenty years of equine piroplasmosis research: global distribution, molecular diagnosis, and phylogeny. Pathogens. 9(11). Article 926. https://doi.org/10.3390/pathogens9110926.
Mazuz, M.L., Laughery, J.M., Lebovitz, B., Yasur-Landau, D., Rot, A., Bastos, R.G., Edery, N., Fleiderovitz, L., Levi, M.M., Suarez, C.E. 2021. Experimental infection of calves with transfected attenuated Babesia bovis expressing the Rhipicephalus microplus Bm86 antigen and eGFP marker: Preliminary studies towards a dual anti-tick/Babesia vaccine. Pathogens. 10(2). Article 135. https://doi.org/10.3390/pathogens10020135.
Montenegro, V.N., Paoletta, M.S., Jaramillo Ortiz, J.M., Suarez, C.E., Wilkowsky, S.E. 2020. Identification and characterization of a Babesia bigemina thrombospondin-related superfamily member, TRAP-1: a novel antigen containing neutralizing epitopes involved in merozoite invasion. Parasites & Vectors. 13. Article 602. https://doi.org/10.1186/s13071-020-04469-5.
Ueti, M.W., Johnson, W.C., Kappmeyer, L.S., Herndon, D.R., Mousel, M.R., Reif, K.E., Taus, N.S., Ifeonu, O.O., Silva, J.C., Suarez, C.E., Brayton, K.A. 2020. Transcriptome dataset of Babesia bovis life stages within vertebrate and invertebrate hosts. Data in Brief. 33. Article 106533. https://doi.org/10.1016/j.dib.2020.106533.
Silva, M.G., Bastos, R.G., Stone, D.J., Riscoe, M.K., Pou, S., Winter, R., Dodean, R.A., Nilsen, A., Suarez, C.E. 2020. Endochin-like quinolone-300 and ELQ-316 inhibit Babesia bovis, B. bigemina, B. caballi and Theileria equi. Parasites & Vectors. 13. Article 606. https://doi.org/10.1186/s13071-020-04487-3.
Dinkel, K.D., Herndon, D.R., Noh, S.M., Lahmers, K.K., Todd, M.S., Ueti, M.W., Scoles, G.A., Mason, K.L., Fry, L.M. 2021. A U.S. isolate of Theileria orientalis, Ikeda genotype, is transmitted to cattle by the invasive Asian longhorned tick, Haemaphysalis longicornis. Parasites & Vectors. 14. Article 157. https://doi.org/10.1186/s13071-021-04659-9.
Ozubek, S., Bastos, R.G., Alzan, H.F., Inci, A., Aktas, M., Suarez, C.E. 2020. Bovine Babesiosis in Turkey: Impact, current gaps and opportunities for intervention. Pathogens. 9(12). Article 1041. https://doi.org/10.3390/pathogens9121041.
Sears, K.P., Knowles, D.P., Dinkel, K.D., Mshelia, P.W., Onzere, C., Silva, M., Fry, L.M. 2020. Imidocarb Dipropionate lacks efficacy against Theileria haneyi and fails to consistently clear Theileria equi in horses co-infected with T. haneyi. Pathogens. 9(12). Article 1035. https://doi.org/10.3390/pathogens9121035.
Cuy-Chaparro, L., Bohórquez, M.D., Arévalo-Pinzón, G., Castañeda-Ramírez, J.J., Suarez, C.F., Pabon, L., Ordoñez, D., Gallego-López, G.M., Suarez, C.E., Moreno-Pérez, D.A., Patarroyo, M.A. 2021. Babesia bovis ligand-receptor interaction: AMA-1 contains small regions governing bovine erythrocyte binding. International Journal of Molecular Sciences. 22(2). Article 714. https://doi.org/10.3390/ijms22020714.
Florin-Christensen, M., Rodriguez, A.E., Suarez, C.E., Ueti, M.W., Delgado, F.O., Echaide, I., Schnittger, L. 2021. N-glycosylation in piroplasmids: Diversity within simplicity. Pathogens. 10(1). Article 50. https://doi.org/10.3390/pathogens10010050.
Bastos, R.G., Sears, K.P., Dinkel, K.D., Kappmeyer, L.S., Ueti, M.W., Knowles, D.P., Fry, L.M. 2021. Development of an indirect ELISA to detect equine antibodies to Theileira haneyi. Pathogens. 10(3). Article 270. https://doi.org/10.3390/pathogens10030270.
Bishop, R.P., Kappmeyer, L.S., Onzere, C.K., Odongo, D.O., Githaka, N., Sears, K.P., Knowles, D.P., Fry, L.M. 2020. Equid infective Theileria cluster in distinct 18S rRNA gene clades comprising multiple taxa with unusually broad mammalian host ranges. Parasites & Vectors. 13. Article 261. https://doi.org/10.1186/s13071-020-04131-0.
Rüther, A., Perez-Guaita, D., Poole, W.A., Cooke, B.M., Suarez, C.E., Heraud, P., Wood, B.R. 2020. Vibrational spectroscopic based approach for diagnosing babesia bovis infection. Analytical Chemistry. 92(13):8784-8792. https://doi.org/10.1021/acs.analchem.0c00150.
Mahmoud, M.S., Kandil, O.M., Abu El-Ezz, N.T., Hendawy, S.H.M., Elsawy, B.S.M., Knowles, D.P., Bastos, R.G., Kappmeyer, L.S., Laughery, J.M., Alzan, H.F., Suarez, C.E. 2020. Identification and antigenicity of the Babesia caballi spherical body protein 4 (SBP4). Parasites & Vectors. 13. Article 369. https://doi.org/10.1186/s13071-020-04241-9.
Idoko, I.S., Edah, R.E., Adamu, A.M., Machunga-Mambula, S., Okubanjo, O.O., Balogun, E.O., Adamu, S., Johnson, W.C., Kappmeyer, L.S., Mousel, M.R., Ueti, M.W. 2021. Molecular and serological detection of piroplasms in horses from Nigerian. Pathogens. 10(5). Article 508. https://doi.org/10.3390/pathogens10050508.
Philip, M.W., Kappmeyer, L.S., Johnson, W.C., Kudi, C.A., Oluyinka, O.O., Balogun, E.O., Richard, E.E., Onoja, E., Sears, K.P., Ueti, M.W. 2020. Molecular detection of Theileria species and Babesia caballi from horses in Nigeria. Parasitology Research. 119:2955-2963. https://doi.org/10.1007/s00436-020-06797-y.
Alzan, H.F., Bastos, R.G., Ueti, M.W., Laughery, J.M., Rathinasamy, V.A., Cooke, B.M., Suarez, C.E. 2021. Assessment of Babesia bovis 6cys A and 6cys B as components of transmission blocking vaccines for babesiosis. Parasites & Vectors. 14. Article 210. https://doi.org/10.1186/s13071-021-04712-7.
Florin-Christensen, M., Schnittger, L., Bastos, R.G., Rathinasamy, V.A., Cooke, B.M., Alzan, H.F., Suarez, C.E. 2021. Pursuing effective vaccines against cattle diseases caused by apicomplexan protozoa. CABI Agriculture and Bioscience (CABI A&B). https://doi.org/10.1079/PAVSNNR202116024.
Onzere, C.K., Fry, L.M., Bishop, R.P., Silva, M.G., Bastos, R.G., Knowles, D.P., Suarez, C.E. 2021. Theileria equi claudin like apicomplexan microneme protein contains neutralization-sensitive epitopes and interacts with components of the equine erythrocyte membrane skeleton. Scientific Reports. 11. Article 9301. https://doi.org/10.1038/s41598-021-88902-4.
Scoles, G.A., Lohmeyer, K.H., Ueti, M.W., Bonilla, D., Lahmers, K.K., Piccione, J., Rogovskyy, A.S. 2021. Stray Mexico origin cattle captured crossing into Southern Texas carry Babesia bovis and other tick-borne pathogens. Ticks and Tick Borne Diseases. 12(5). Article 101708. https://doi.org/10.1016/j.ttbdis.2021.101708.
Álvarez Martíneza, J.A., Figueroa Millán, J.V., Ueti, M.W., Rojas-Martínez, C. 2021. Establishment of Babesia bovis in vitro culture using medium free of animal products. Pathogens. 10(6). Article 770. https://doi.org/10.3390/pathogens10060770.
Barbosa, I.C., André, M.R., Amaral, R.B.D., Valente, J.D.M., Vasconcelos, P.C., Oliveira, C.J.B., Jusi, M.M.G., Machado, R.Z., Vieira, T.S.W.J., Ueti, M.W., Vieira, R.F.C. 2020. Anaplasma marginale in goats from a multispecies grazing system in northeastern Brazil. Ticks and Tick Borne Diseases. 12(1). Article 101592. https://doi.org/10.1016/j.ttbdis.2020.101592.
Paoletta, M.S., Laughery, J.M., Arias, L.S.L., Ortiz, J.M.J., Montenegro, V.N., Petrigh, R., Ueti, M.W., Suarez, C.E., Farber, M.D., Wilkowsky, S.E. 2021. The key to egress? Babesia bovis perforin-like protein 1 (PLP1) with hemolytic capacity is required for blood stage replication and is involved in the exit of the parasite from the host cell. International Journal for Parasitology. 51(8):643-658. https://doi.org/10.1016/j.ijpara.2020.12.010.