Location: Animal Disease Research Unit
2025 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
This report documents the FY 2025 progress of project 2090-32000-044-000D, titled, “Control Strategies for Theileriosis”, which began in November 2021.
In support of Sub-objective 1.A, research continued on the analysis of horse serum samples collected at the U.S.-Mexico border using the newly developed Theileria haneyi indirect enzyme-linked immunosorbent assay (ELISA), which is based on the parasite equi merozoite antigen 11 (EMA11). This work is part of a collaborative investigation with the USDA Animal and Plant Health Inspection Service (APHIS) to assess the prevalence of Theileria equi and/or T. haneyi infections in horses imported into the United States. Additionally, ARS researchers in Pullman, Washington, made significant progress in collaboration with APHIS to design and implement protocols for managing Theileria-infected horses imported into the United States and currently in quarantine, as well as in continuing to evaluate the overall risk of T. equi and T. haneyi infections within the country.
For Sub-objective 1.B, ARS researchers completed and published a study evaluating the efficacy of the therapeutic drug buparvaquone for the radical cure of T. haneyi in chronically/persistently infected horses. The results demonstrated that, although buparvaquone had a significant transient effect on the level of parasites circulating in the blood, it was ineffective in clearing T. haneyi from chronically/persistently infected animals (Onzere et al., 2024. 17(1):337. doi: 10.1186/s13071-024-06397-0). Additionally, an experiment to infect a splenectomized horse with the T. haneyi Eagle Pass isolate was conducted in order to produce blood parasite stocks, which are currently being used in our research as well as by collaborators in Japan (MTA # 19818).
Also, in support of Sub-objective 1.B, ARS researchers completed and published a study demonstrating that Haemaphysalis longicornis, the Asian longhorned tick (ALHT), is unable to transstadially transmit T. haneyi to horses (Poh et al., Frontiers in Veterinary Science. 2025. 12:1572944. doi: 10.3389/fvets.2025.1572944). In addition, a study was conducted to evaluate the humoral and cellular immune responses of horses during the acute phase of infection with the Florida strain of T. equi. Scientists are currently analyzing the data from this study and preparing a manuscript for publication.
In support of sub-ojective 2B, cattle were innoculated with a recombinant version of the immunodominant antigen major piroplasm surface protein (MPSP) from the U.S. isolate of T. orientalis Ikeda. The results demonstrated that the inoculated animals developed significant antibody responses against MPSP. Scientists are currently evaluating the cellular immune response against MPSP. Collectively, the data from this study will set the rationale for using MPSP as a subunit vaccine against the U.S. isolate of T. orientalis Ikeda, and for its application as an antigen in serological assays to detect cattle infected with or exposed to the parasite.
Also, in support of Sub-objective 2B, ARS researchers completed sequencing the full genome of the U.S. isolate of T. orientalis Ikeda in collaboration with APHIS and are currently working on genome annotation. The information obtained from the genome sequence will help fill critical knowledge gaps regarding the biology of the U.S. isolate of T. orientalis Ikeda, and support the development of effective diagnostics, drug treatments, and vaccines against this parasite.
For Sub-objective 2C, ARS resarchers completed and published a study evaluating the efficacy of the therapeutic drug buparvaquone for the radical cure of the U.S. isolate of T. orientalis Ikeda. The results demonstrated that buparvaquone significantly decreased the level of parasites circulating in the blood; however, it was ineffective in eliminating the parasite from chronically infected cattle (Bastos et al., 2024. doi: 10.3389/fvets.2024.1421710). Given that buparvaquone failed to clear the parasite, we have partnered with a private company to evaluate an alternative quinoline compound as a new drug treatment for the radical cure of the U.S. isolate of T. orientalis Ikeda. Scientists are currently conducting safety and efficacy studies of the new drug in cattle.
Also, in support of Sub-objective 2C, a study was conducted on cattle to evaluate the dynamics of infection following inoculation of tick salivary gland stages of the U.S. isolate of T. orientalis Ikeda. Preliminary results demonstrated a significant correlation between the increase in parasite load in peripheral blood and the development of anemia in acutely infected cattle. Scientists are currently analyzing the data from this study and preparing a manuscript for publication.
Accomplishments
1. A full genome made available to battle Theileria orientalis Ikeda. Theileria orientalis Ikeda is a virulent, tick-borne blood parasite that poses a significant threat to the cattle industry in both the United States and globally. Recent outbreaks of Theileria orientalis Ikeda in the United States have coincided with the spread of the invasive Asian longhorned tick—a primary vector of the parasite—and represent a growing concern for the nation's cattle industry. In addition to causing anemia, Theileria orientalis Ikeda infection is characterized by late-term abortions and mortality. Currently, no effective therapeutic drugs or vaccines are available to control Theileria orientalis Ikeda, and a deeper understanding of the parasite’s biology is needed to develop comprehensive control and eradication programs. ARS Researchers in Pullman, Washington, partnered with National Veterinary Services Laboratories (NVSL-USDA) to sequence the full genome of the U.S. isolate of Theileria orientalis Ikeda. The data generated a high-quality genome sequence, which will help fill critical knowledge gaps in the parasite’s biology and support the development of effective diagnostics, drug treatments, and vaccines, thereby assisting stakeholders in controlling Theileria orientalis Ikeda in the United States.
2. Demonstrated a significant correlation between acute infection with the U.S. isolate of T. orientalis Ikeda and anemia in cattle. Theileria orientalis Ikeda is an emerging, tick-borne, and virulent cattle parasite in the United States, transmitted by the invasive Asian longhorn tick. Following transmission by ticks, the parasite first replicates in white blood cells, then in red blood cells. Infections with Theileria orientalis Ikeda have been associated with anemia in cattle; however, no information is currently available on the ability of the U.S. isolate to cause anemia. ARS researchers in Pullman, Washington, conducted a study to evaluate the progression of infection in cattle following inoculation with the U.S. isolate of Theileria orientalis Ikeda. The results demonstrated a significant correlation between increased parasite load in the peripheral blood of cattle and the development of anemia during acute infection. This study enhances our understanding of the infection dynamics of the U.S. isolate of Theileria orientalis Ikeda and provides valuable insights for veterinarians and cattle producers.
3. Demonstrated that the invasive Asian longhorned ticks (Haemaphysalis longicornis) are unable to transmit the blood parasite Theileria haneyi to horses. The Asian longhorned tick (ALHT) is native to several East Asian countries, including China, South Korea, and Japan. From the Asia-Pacific region, ALHT has spread to New Zealand and Australia, where it has become a major vector for the transmission of economically significant tick-borne blood pathogens, including Theileria species. Theileria haneyi is a recently discovered tick-transmitted blood parasite and one of the causative agents of equine piroplasmosis, an economically impactful disease that adversely affects the global horse industry. Given the recent emerging of ALHT in the United States and the limited data on tick vectors capable of transmitting Theileria haneyi, ARS researchers in Pullman, Washington conducted a study to evaluate the competence of ALHT in transmitting Theileria haneyi to horses. The results showed that ALHT is not capable of transmitting Theileria haneyi to horses. This study provides relevant information to equine stakeholders and highlights the importance of maintaining robust livestock and wildlife health surveillance programs as essential for controlling tick-borne pathogens.
4. Demonstrated that a novel 8-aminoquinoline compound is effective in controlling the growth of Theileria equi in vitro. The availability of safe and effective drug treatments for Theileria equi and Theileria haneyi in horses, as well as Theileria orientalis Ikeda in cattle, is critical for controlling and eradicating these parasites. In vitro culture systems provide a rational platform for screening novel compounds to combat blood parasites. Currently, no in vitro culture systems exist for Theileria orientalis Ikeda and Theileria haneyi. However, since an in vitro culture system is available for Theileria equi, ARS researchers in Pullman, Washington, conducted a study to evaluate the efficacy of a novel 8-aminoquinoline compound against this parasite. The results demonstrated that the compound inhibited the in vitro growth of Theileria equi while exhibiting low toxicity to blood cells. This new 8-aminoquinoline compound shows promise as a much-needed and effective treatment against Theileria parasites that infect cattle and horses.
Review Publications
Poh, K.C., Oyen, K.J., Onzere, C., Kappmeyer, L.S., Bastos, R.G. 2025. Haemaphysalis longicornis ticks are unable to transstadially transmit Theileria haneyi to horses. Frontiers in Veterinary Science. 12. Article 1572944. https://doi.org/10.3389/fvets.2025.1572944.
Onzere, C.K., Hassan, A., Sears, K., Kappmeyer, L.S., Villarino, N.F., Fry, L.M., Bastos, R.G. 2024. Transient efficacy of buparvaquone against Theileria haneyi in chronically infected horses. PLOS Neglected Tropical Diseases. (17). Article 337. https://doi.org/10.1186/s13071-024-06397-0.
Bastos, R.G., Hassan, A., Onzere, C.K., Herndon, D.R., Villarino, N.F., Laughery, J.M., Fry, L.M. 2024. Transient efficacy of buparvaquone against the US isolate of Theileria orientalis Ikeda genotype in sub-clinically infected cattle. Frontiers in Veterinary Science. 11. Article 1421710. https://doi.org/10.3389/fvets.2024.1421710.
Mendoza, F.J., Pérez-Écija, A., Kappmeyer, L.S., Suarez, C.E., Bastos, R.G. 2024. New insights in the diagnosis and treatment of equine piroplasmosis: Pitfalls, idiosyncrasies, and myths. Frontiers in Veterinary Science. 11. Article 1459989. https://doi.org/10.3389/fvets.2024.1459989.