Research Project: Control Strategies for Theileriosis

Location: Animal Disease Research

2022 Annual Report

Objectives
Objective 1: Perform prevalence estimation (Theileria haneyi), host-parasite interaction (T. haneyi vs. Theileria equi), and drug susceptibility profile (T. haneyi) of U.S. equine Theileria sp. isolates. Sub-objective 1.A: Using molecular and serologic assays and characterize (in collaboration with APHIS) the prevalence of T. haneyi in the United States. Sub-objective 1.B: Characterize the host-parasite interface (T. haneyi vs. T. equi) and drug susceptibility profile (T. haneyi) of U.S. equine Theileria sp. isolates. Objective 2: Determine competent, native U.S. tick vectors of Theileria orientalis Ikeda (2016 VA isolate), assess potential T. orientalis Ikeda treatment strategies, and develop improved diagnostic and vaccine strategies for control of T. orientalis Ikeda. Sub-objective 2.A: Characterize competence of native U.S. ticks to acquire and transmit Theileria orientalis Ikeda (2016 VA isolate) to cattle. Sub-objective 2.B: Identify potential diagnostic and subunit vaccine targets for T. orientalis Ikeda (2016 VA isolate) and develop an improved serologic assay for T. orientalis Ikeda. Sub-objective 2.C: Provide more complete characterization of the drug susceptibility profile and virulence of the U.S. T. orientalis Ikeda isolate.

Approach
Theileria orientalis Ikeda and Theileria haneyi recently emerged as novel threats to the United States cattle and horse industries, respectively. The development of improved treatment, diagnostic, and preventive strategies for bovine and equine theileriosis is urgently needed. In the first objective, focused on equine theileriosis, we will first work to assess the potential risk of Theileria haneyi to U.S. horses by performing a study to estimate its prevalence at the southern border and in high-risk populations throughout the U.S. USDA-APHIS Veterinary Services will assist in obtaining samples. We will utilize our newly developed T. haneyi ELISA, in concert with previously published PCR and western blot assays to test the equine samples. We will then work with USDA-APHIS Center for Epidemiology and Animal Health to perform statistical analysis. Using this sample pool, we will also perform basic validation of our new T. haneyi ELISA. Next, we will work to improve current control strategies for equine Theileriosis by determining immune correlates of protection via assessment of the equine immune response using immune cell phenotyping, cytokine assessment, and cell function assays. In concert, we will utilize genomics and transcriptomics to identify parasite virulence factors and potential vaccine candidate antigens. Finally, we will begin to characterize the drug susceptibility profile of T. haneyi by performing 1. In vivo assessment of several drugs approved to treat other pathogens of horses and 2. In vitro assessment of compound libraries to identify novel compounds with potential anti-Theileria efficacy. In the second objective, focused on bovine theileriosis, we will first perform studies to determine if additional U.S. tick species are competent vectors of T. orientalis Ikeda. This work will utilize tick colonies and cattle maintained by our research unit, and will provide information on the potential range of spread of T. orientalis Ikeda in the U.S. Next, we will work to improve current control strategies for bovine Theileriosis by determining immune correlates of protection via assessment of the bovine immune response using immune cell phenotyping, cytokine assessment, and cell function assays. In concert, we will utilize genomics and transcriptomics to identify parasite virulence factors and potential diagnostic and vaccine candidate antigens. Using a subset of these antigens, we will work with collaborators to develop and perform initial validation of a serologic assay to diagnose T. orientalis Ikeda. Finally, we will begin to characterize the drug susceptibility profile of T. orientalis by performing 1. In vivo assessment of several drugs approved to treat other pathogens of cattle and 2. In vitro assessment of multiple compound libraries to identify novel compounds with potential anti-Theileria efficacy. The data from this project will assist in the development and execution of theileriosis control strategies within the U.S. U.S. stakeholders to be served by the data generated by this project include the USDA-APHIS, veterinarians, beef and dairy producers, the equine industry, and the veterinary biomedical industry.

Progress Report
In support of Sub-objective 1A, collection and storage of blood samples from horses at the southern border of the United States began and those samples were assayed for Theileria haneyi using polymerase chain reaction (PCR) and serologic assays. We have received and tested 1,000 equine samples so far. We continue to work on optimization of diagnostic assays for T. haneyi, including a quantitative PCR assay that can diagnose both T. haneyi and its more virulent counterpart, Theileria equi, and a sensitive serologic assay using the theileria rhoptry-associated proteins (RAP)-1 antigen as a target, based on data obtained and published by our group this year (Onzere et al 2021). In support of Sub-objective 1B, we have identified and purchased a subset of the horses. We have also completed analysis of the cytokine response to acute T. equi (manuscript in process) and published a manuscript on the virulence profile of T. haneyi in splenectomized horses (Sears et al, 2022), and have demonstrated clearance of T. haneyi by splenectomized horses (manuscript in process). Also, in support of Sub-objective 1B, we have almost completed in vivo studies on the efficacy of the drugs tulathromycin and Protazil against T. haneyi, and have begun work on the manuscripts describing this work. Finally, we reached an agreement with Bimeda Inc. to obtain 30 L of buparvaquone at no charge to complete a study on the efficacy of buparvaquone against T. haneyi and are currently working to import the drug from Africa. We have T. haneyi-infected horses ready for use in this buparvaquone efficacy study. In support of Sub-objective 2A, we obtained and established Rhipacephalus sanguineous and Dermacenter variabilis tick colonies for assessment of potential vector competence, and also obtained and splenectomized a group of calves for use in acquisition and transmission studies to begin in August of 2022. In support of Sub-objective 2B, we have completed genomic comparison of the Theileria orientalis Ikeda, Chitose, and Buffali strains and generated a list of conserved proteins. We have also generated a list of other probable T. orientalis candidate antigens based on comparative genomic analysis with other Theileria species paired with in silico antigenic prediction algorithms. A manuscript describing this work is in process. Based on our work on the Theileria rhoptry associated protein (RAP) antigens, we have begun in silico analysis of the T. orientalis RAP protein for potential use in a diagnostic assay and have begun work to express the protein recombinantly for assay development. We established a non-assistance cooperative agreement (NACA) with a collaborator at a veterinary diagnostic laboratory within the T. orientalis endemic area to obtain T. orientalis Chitose and Buffeli infectious stabilates and serum from field-infected cattle for use in our experiments. We generated the first batches of T. orientalis-infected tick salivary gland stabilate and T. orientalis infected erythrocyte stabilate, which will be used to infect cattle in subsequent experiments. We also continued work on monoclonal antibody development to enable more complete characterization of the bovine immune response. Finally, to assist stakeholders and obtain serum from cattle naturally infected with T. orientalis in the United States for use in experiments outlined in Objective 2, we worked with the Tennessee State Veterinarian to characterize the extent and strain type of a T. orientalis outbreak on a ranch there (the first outbreak in the state). In support of Sub-objective 2C, we have reached an agreement to obtain 30 liters of buparvaquone at no charge to complete a study on the efficacy of buparvaquone against T. orientalis and are currently working to import the drug from Africa.

Accomplishments
1. Developed and validated a monoclonal antibody to bovine CD138 molecule. CD138 is a protein expressed on the surface of bovine plasma cells, which make antibodies against infectious agents. Until now, isolation and characterization of the bovine plasma cell repertoire generated in response to various infectious diseases and candidate vaccines was not possible due to a lack of monoclonal antibody reagents to bovine plasma cell proteins. The monoclonal antibody generated by ARS researchers in Pullman, Washington, will enable more complete characterization of the bovine immune response to infectious diseases and vaccines, thereby increasing our ability to control these pathogens.

2. Characterized virulence of Theileria haneyi in splenectomized horses. Characterized virulence of Theileria haneyi in splenectomized horses. Theileria haneyi is a tick-transmitted blood parasite of horses recently discovered by scientists in our ARS Pullman, Washington, laboratory. These scientists have demonstrated that it is resistant to the gold-standard anti-theilerial therapy, imidocarb dipropionate, and that it interferes with clearance of the related parasite, Theileria equi, by imidocarb dipropionate in co-infected animals. However, the severity of infection with T. haneyi was not fully characterized prior to this study. ARS researchers in Pullman, Washington, demonstrated that T. haneyi is significantly less virulent than T. equi, and that even horses that have undergone removal of the spleen (splenectomy), an organ crucial to blood parasite control, survive infection. This is in stark contrast to T. equi, which is almost always fatal in splenectomized horses. Furthermore, a majority of splenectomized horses spontaneously cleared T. haneyi after months of persistent, asymptomatic infection. This data provides critical information on the potential impact of this disease for our U.S. equine stakeholders. Although there is not yet an effective treatment for T. haneyi, and T. haneyi interferes with drug clearance of T. equi in horses infected with both parasites, infection with T. haneyi alone will likely cause minimal disease in most horses.

3. Characterized the Theileria equi rhoptry associated protein (RAP) antigen as a diagnostic assay target. Theileria equi is a tick-transmitted blood parasite that causes severe anemia in horses. As in related blood parasites, T. equi contains organelles called rhoptries whose contents have been implicated in host cell survival of the species within horse blood cells. Despite their importance, the composition of T. equi rhoptries and their role(s) in host cell invasion were largely unexplored until recently. ARS scientists in Pullman, Washington, recently characterized the expression, immunogenicity, and functional roles of two T. equi rhoptry-associated proteins (RAP): RAP-1a and RAP-1b. This work revealed that T.equi-infected horses generate a strong antibody response to RAP-1a that can prevent parasite invasion of host blood cells, and can be utilized to detect previous infection with T. equi. Scientists in this lab are now working to optimize this protein as both a diagnostic assay target for T. equi and potential vaccine antigen. Furthermore, the same scientists are exploring the use of RAP proteins from the related blood parasite of cattle, Theileria orientalis, as a diagnostic assay target for T. orientalis. This work has the potential to yield pen-side, rapid antibody tests for these blood parasites of horses and cattle, which will greatly aid in the control of these emerging diseases in the United States.

Review Publications
Sacco, R.E., Elnaggar, M.M., Abdellrazeq, G.S., Fry, L.M., Hulubei, V., Davis, W.C., Harsla, T., Mucci, M.L. 2022. Comparative analysis of the specificity of monoclonal antibodies developed against the bottlenose dolphin, Tursiops truncatus, TNF-a, IL1-ß, IL-6, IL-8, IL-10 with monoclonal antibodies made against ovine IFN-y bovine IL-17A. Veterinary Immunology and Immunopathology. 250. Article 110456. https://doi.org/10.1016/j.vetimm.2022.110456.
Sears, K.P., Knowles, D.P., Fry, L.M. 2022. Clinical progression of Theileria haneyi in splenectomized horses reveals decreased virulence compared to Theileria equi. Pathogens. 11(2). Article 254. https://doi.org/10.3390/pathogens11020254.
Vimonish, R., Dinkel, K.D., Fry, L.M., Johnson, W.C., Capelli-Peixoto, J., Bastos, R.G., Scoles, G.A., Knowles, D.P., Madder, M., Chaka, G., Ueti, M.W. 2021. Isolation of infectious Theileria parva sporozoites secreted by infected Rhipicephalus appendiculatus ticks into an in vitro tick feeding system. Parasites & Vectors. 14. Article 616. https://doi.org/10.1186/s13071-021-05120-7.
Salinas-Estrella, E., Ueti, M.W., Lobanov, V.A., Castillo-Payró, E., Lizcano-Mata, A., Badilla, C., Martínez-Ibáñez, F., Mosqueda, J. 2022. Serological and molecular detection of Babesia caballi and Theileria equi in Mexico: A prospective study. PLoS ONE. 17(3). Article e0264998. https://doi.org/10.1371/journal.pone.0264998.
Onzere, C.K., Fry, L.M., Bishop, R.P., Da Silva, M., Madsen-Bouterse, S.A., Bastos, R.G., Knowles, D.P., Suarez, C.E. 2022. Theileria equi RAP-1a and RAP-1b proteins contain immunoreactive epitopes and are suitable candidates for vaccine and diagnostics development. International Journal for Parasitology. 52(6):385-397. https://doi.org/10.1016/j.ijpara.2022.01.004.
Elnaggar, M.M., Knowles, D.P., Davis, W., Fry, L.M. 2021. Flow cytometric analysis of the cytotoxic T-cell recall response to Theileria parva in cattle following vaccination by the infection and treatment method. Veterinary Sciences. 8(6). Article 114. https://doi.org/10.3390/vetsci8060114.
Davis, W.C., Abdellrazeq, G.S., Mahmoud, A.H., Park, K., Elnaggar, M.M., Donofrio, G., Hulubei, V., Fry, L.M. 2021. Advances in understanding of the immune response to mycobacterial pathogens and vaccines through use of cattle and Mycobacterium avium subsp. paratuberculosis as a prototypic mycobacterial pathogen. Vaccines. 9(10). Article 1085. https://doi.org/10.3390/vaccines9101085.
Davis, W.C., Mahmoud, A.H., Abdellrazeq, G.S., Elnaggar, M.M., Dahl, J.L., Hulubei, V., Fry, L.M. 2022. Ex vivo platforms to study the primary and recall immune responses to intracellular mycobacterial pathogens and peptide-based vaccines. Frontiers in Veterinary Science. 9. Article 878347. https://doi.org/10.3389/fvets.2022.878347.