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

Location: Animal Disease Research

2018 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 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.

Approach
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.

Progress Report
Progress was made on both objectives and their sub-objectives to address critical gaps in our knowledge concerning pathogens transmitted by tick vector to cattle and horses. Objective 1: Progress towards development of diagnostic assays and vaccines for bovine babesiosis by targeting antigens from multiple parasite stages were advanced by: identifying Babesia proteins expressed during infection in both tick vectors and cattle; vaccinating cattle with recombinant proteins to produce antibodies; and development of a serological test to identify infected cattle. Sub-objective 1A: Five proteins expressed by parasites were identified during its development in red blood cells. Sub-objective 1B: Ten stage specific parasite proteins were identified during parasite development in the gut and hemolymph of tick vectors. Protein expression and vaccination were performed. Sub-objective 1C: These proteins have been tested to determine if bovine antibodies impact parasite infection of red blood cells and tick vectors and, consequently, prevent disease manifestation in animals or prevent transmission by ticks. Bioinformatic analysis assisted in the identification of a conserved protein for serological assay development. Sub-objective 1D: A more reliable serological assay using a conserved parasite protein was developed to identify naturally infected cattle. This assay can consistently identify infected animals and can be used as part of strategies that seek to control parasite dissemination. Objective 2: Substantial progress was made towards identification of proteins expressed by the Theileria-like parasite, an emerging pathogen of horses, as part of diagnostic assay development. Ticks indigenous to the U.S./Mexico border regions where the new Theileria was discovered were collected and laboratory tick colonies established. Drug therapy was performed to determine drug efficacy against the Theileria-like parasite. Horses were infected with Theileria-like parasite and during acute infection, blood samples were collected and parasites isolated. Isolated parasites were used for the development of diagnostic assays and genomic DNA was extracted and submitted for sequencing. Sequencing the genome has been completed and potential genes were identified for the development of diagnostic assays. Sub-objective 2A: A recombinant protein was generated to detect antibodies from horses experimentally infected with the Theileria-like parasite. Sub-objective 2B: Three different laboratory colonized tick species were fed on high parasitemic horses to determine their potential as vectors for the Theileria-like parasites. After feeding, the ticks were collected, dissected and salivary glands isolated to determine if they were infected and thus capable of transmitting the parasite. Only one species of tick had detectible levels of parasites in the salivary glands, a follow-up experiment is planned to actually demonstrate transmission. Sub-objective 2C: Drug therapy was performed in horses experimentally infected with the Theileria-like parasites to determine drug efficiency to kill this parasite in the blood. An additional drug was tested in an in-vitro system to determine if it killed parasites that infect horses and cause equine piroplasmosis. Transfected Babesia parasites expressing exogenous antigens were developed. These transfected parasites expressed either green or red florescent proteins. This is the first step in development of a transfection system that can express exogenous antigens such as toxins or proteins that can kill the tick. The transfected parasites will be used to deliver a toxin to ticks during parasite development in the hemolymph and, consequently, kill competent tick vectors. This work is part of a National Institute of Food and Agriculture (NIFA), Agriculture and Food Research Initiative (AFRI) awarded grant, "A Pioneering Approach to Tick Control: Anti-tick Toxins Delivered via a Tick Transmissible Pathogen", 2090-32000-039-12I. In addition, these parasites can express exogenous proteins during parasite replication in the blood of the cattle. These proteins can induce an antibody response which may result in the prevention of tick attachment or feeding. This research is part of International Development Research Centre (IDRC) awarded grant, “Utilizing Bioinformatics and Genetic Transformation Approaches to Develop Novel Vaccines to Prevent Babesia bovis Acute Infection and Transmission” 2090-32000-039-20T.

Accomplishments
1. Identified proteins expressed by Babesia for vaccine development. Cattle tick fever is a disease caused by parasites that infect cattle red blood cells. The parasites cause severe disease and significant negative impact for the cattle industry in tropical and subtropical regions. Vaccines are not available to prevent disease or parasite transmission. ARS scientists in Pullman, Washington, discovered proteins expressed by multiple stages of the parasites during infection of cattle and tick vectors. Fifteen proteins expressed by parasites were identified, five during bovine infection and ten during tick infection. These parasite proteins can be used as candidate vaccines to protect cattle or block tick infection, potentially preventing parasite transmission via tick vectors.

2. Developed diagnostic tests to detect horses infected with the Theileria-like parasite. The lack of detection methods to identify horses infected with the Theileria-like parasite may allow entrance of infected horses to the U.S. ARS scientists in Pullman, Washington, identified potential genes and proteins that can be used for the development of diagnostic assays. These genes and proteins were tested to determine if they could be used in molecular or serologic assays to detect horses infected with the Theileria-like parasite. Results demonstrated that all horses infected with the Theileria-like parasite were consistently detected by these newly developed assays. This information will provide diagnostic assays that can be used by the Animal and Plant Health Inspection Service (APHIS) for the development of strategies to control the newly emerging pathogen.

3. Assessed if species of ticks indigenous to the U.S. can transmit the Theileria-like parasite. This newly emerging pathogen of horses potentially causes a significant economic loss for the horse industry. The introduction of horses infected with the Theileria-like parasite in the U.S., due to the lack of detection methods, and the potential presence of tick vectors could cause a tremendous setback for the equine piroplasmosis eradication program. ARS scientists at Pullman, Washington, demonstrated that ticks located at the U.S./Mexico border region are potential competent vectors for the Theileria-like parasite. The data highlight the importance for the U.S. to prevent the introduction of horses infected with this newly emerging pathogen by testing all imported horses prior to commingling with naïve U.S. horse population.

Review Publications
Levi, M.M., Tirosh-Levy, S., Dahan, R., Berlin, D., Steinman, A., Edery, N., Savistsky, I., Lebovich, B., Knowles Jr, D.P., Suarez, C.E. 2017. First detection of diffuse and cerebral theileria equi infection in neonatal filly. Journal of Equine Veterinary Science. 60:23-28.
Gallego-Lopez, G., Lau, A., Johnson, W.C., Ueti, M.W., Suarez, C.E. 2018. Spherical body protein 2 truncated copy 11 as a specific babesia bovis attenuation marker. Parasites & Vectors. 11:169. https://doi.org/10.1186/s13071-018-2782-z.
Silva, M.G., Villarino, N.F., Knowles, D.P., Suarez, C.E. 2018. Assessment of Draxxin® (tulathromycin) as an anti-babesia bovis, babesia bigemina and theileria equi chemotherapeutic in cultured parasites. International Journal for Parasitology: Drug and Drug Resistance. 17:8(2):265-270. https://doi: 10.1016/j.ijpddr.2018.04.004.
Scudiero, L., Mercado, W.J., Rudolph, A., Wang, J., Laughery, J.M., Suarez, C.E. 2018. Comparisons of the topographic characteristics and electrical charge distributions among Babesia-infected erythrocytes and extraerythrocytic merozoites using AFM. Journal of Microscopy. doi:10.1111/jmi.12697.
Hussein, H.E., Bastos, R.G., Schneider, D.A., Johnson, W.C., Adham, F.K., Davis, W.C., Laughery, J.M., Herndon, D.R., Alzan, H.F., Ueti, M.W., Suarez, C.E. 2017. The babesia bovis hap2 gene is not required for blood stage replication, but expressed upon in vitro sexual stage induction. PLOS Neglected Tropical Diseases. 11(10):e0005965. https://doi.org/10.1371/journal.pntd.0005965.
Suarez, C.E., Bishop, R.P., Alzan, H., Poole, W., Cook, B. 2017. Advances in the application of genetic manipulation methods to apicomplexan parasites. International Journal for Parasitology. https://doi.org/10.1016/j.ijpara.2017.08.002.
Wise, L.N., Kappmeyer, L.S., Dasilva, M., White, S.N., Grause, J.F., Knowles Jr, D.P. 2017. Verification of post-chemotherapeutic clearance of theileria equi through concordance of nested PCR and immunoblot. Ticks and Tick Borne Diseases. https://doi.org/10.1016/j.ttbdis.2017.08.007.
Mohamad, S.B., Alagib, A., Abdelkareim, T.B., Haasan, M.M., Johnson, W.C., Hussein, H.E., Taus, N.S., Ueti, M.W. 2018. Molecular detection and characterization of theileria spp infecting cattle in Sennar State, Sudan. Parasitology Research. 117(4):1271-1276. https://doi.org/10.1007/s00436-018-5775-0.
Olds, C.L., Mason, K.L., Scoles, G.A. 2018. Rhipicephalus appendiculatus ticks transmit theileria parva from persistently infected cattle in the absence of detectable parasitemia: implications for East Coast fever epidemiology. Parasites & Vectors. https://doi.org/10.1186/s13071-018-2727-6.
Hernández-Silva, D., Valdez-Espinoza, U., Mercado Uriostegui, M., Aguilar-Tipacamú, G., Ramos-Aragón, J., Hernández-Ortiz, R., Ueti, M.W., Mosqueda, J. 2018. Immunomolecular characterization of MIC-1, a novel antigen in babesia bigemina, which contains conserved and immunodominant B-cell epitopes that induce neutralizing antibodies. Journal of Veterinary Science. https://doi.org/10.3390/vetsci5020032.
Elnaggar, M.M., Abdellrazeq, G.S., Dassanayake, R.P., Fry, L.M., Hulubei, V., Davis, W.C. 2018. Characterization of alpha beta and gamma delta T cell subsets expressing IL-17A in ruminants and swine. Developmental and Comparative Immunology. 85:115-124. https://doi.org/10.1016/j.dci.2018.04.003.
Silva, M.G., Knowles, D.P., Mazuz, M.L., Cooke, B.M., Suarez, C.E. 2018. Stable transformation of Babesia bigemina and Babesia bovis using a single transfection plasmid. Scientific Reports. 8:6096. doi:10.1038/s41598-018-23010-4.
Gold, J.R., Knowles, D.P., Coffey, T., Bayly, W.M. 2018. Exercise-induced pulmonary hemorrhage in barrel racing horses in the Pacific Northwest region of the United States. Journal of Veterinary Internal Medicine. 32:839-845. https://doi.org/10.1111/jvim.15066.