Location: Animal Disease Research Unit
2024 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 FY 2024 progress for project 2090-32000-044-000D, “Control Strategies for Theileriosis”, which began in November 2021.
In support of Sub-objective 1A, ARS scientists in Pullman, Washington, finished analyzing more than 1,000 blood samples collected from horses at the southern border of the United States. Samples were examined by molecular and serological assays for Theileria haneyi and Theileria equi. A manuscript describing the prevalence of the parasites in the target samples has been prepared and is currently under revision by co-authors. In addition, substantial progress was made in cooperation with external collaborators to plan a future experiment to assess the risk of T. equi and T. haneyi infections for horses in the United States.
For Sub-objective 1B, after increasing the horse breeding herd last year, ARS scientists began performing experiments on host-parasite interactions. They demonstrated that horses experimentally infected with blood stage T. equi parasites express significant levels of regulatory cytokines, and this is associated with antibody production without modulating the pro-inflammatory responses (Onzere et al., 2024. Frontiers in Immunology. 2024. 15:1370255. doi: 10.3389/fimmu.2024.1370255.). Also, in support of Sub-objective 1B, ARS scientists completed an in vivo study to evaluate the efficacy of buparvaquone for the radical cure of T. haneyi in horses. Results demonstrated that buparvaquone has a robust but transient parasiticidal effect on T. haneyi in chronically infected horses, and treated animals showed parasite recrudescence approximately seven to nine weeks post-treatment. Therefore, buparvaquone proved to be ineffective in the clearance of T. haneyi infection in persistently infected animals. A manuscript reporting these findings is currently under revision. Scientists are currently working to identify and test novel drug candidates for the radical cure of T. haneyi in horses.
Under Sub-objective 2A, ARS scientists completed and published a controlled acquisition and transmission tick study demonstrating that Dermacentor variabilis is unable to transstadially transmit the U.S. isolate of Theileria orientalis Ikeda (Onzere et la., 2023. Pathogens. 12(4):559. doi: 10.3390/pathogens12040559). Scientists are working to identify, obtain, and test vector competence of additional tick species for T. orientalis Ikeda through the next fiscal year.
For Sub-objective 2B, studies were performed on T. orientalis Ikeda experimental infection using tick salivary gland (SG) and blood stage stabilates. This strategy allowed ARS scientists to compare the development of infection following two different infectious stages of the parasite: sporozoites (SG stabilate) and merozoites (blood stabilate). Results are currently under analysis, and a manuscript is in preparation. Also, in support of Sub-objective 2B, ARS scientists made considerable progress in developing a serological assay (ELISA- enzyme-linked immunosorbent assay) based on the major piroplasm surface protein (MPSP) of T. orientalis Ikeda, U.S. isolate. The MPSP ELISA has shown promising results in detecting T. orientalis Ikeda acute and chronic experimental infection in cattle. In addition, they identified and started performing immunogenic analysis of novel members of the rhoptry-associated proteins of T. orientalis Ikeda. These newly identified proteins will be evaluated as potential targets for serological assays and subunit vaccines to control T. orientalis Ikeda.
In support of Sub-objective 2C, ARS scientists completed an in vivo study to assess the efficacy of buparvaquone for the clearance of T. orientalis Ikeda in chronically infected cattle. Results demonstrated that buparvaquone has a significant but transient parasiticidal effect on T. orientalis Ikeda, and treated animals showed parasite recrudescence approximately 9-11 weeks post-treatment. Therefore, buparvaquone proved to be ineffective in eliminating T. orientalis Ikeda infection in persistently infected cattle. A manuscript reporting these findings is currently under revision. ARS scientists are working to identify and evaluate novel drug candidates for the radical cure of T. orientalis Ikeda in cattle.
Accomplishments
1. Demonstrated the prevalence of Theileria equi and Theileria haneyi in blood samples collected from horses at the southern border of the United States. Theileria equi and Theileria haneyi cause equine theileriosis, an economically important tick-transmitted infectious disease of horses. Although the United States is currently free of equine theileriosis, recent detection of infected horses in Florida and Texas emphasizes the importance of developing, applying, and maintaining animal health surveillance programs regarding these parasites. ARS researchers in Pullman, Washington, partnered with collaborators to generate data on the prevalence of T. equi and T. haneyi in blood samples collected from horses at the southern border of the United States as part of the process for animal importation. Findings demonstrated the presence of Theileria DNA and antibodies in the target samples, providing evidence of active infections and previous exposure to the parasites. These results will help state and federal regulatory agencies to design improved surveillance strategies to keep the U.S. horse herd free of equine theileriosis.
2. Demonstrated that T. equi-infected horses express significant levels of regulatory cytokines, leading to antibody production without modulating pro-inflammatory immune responses. Theileria equi is the most virulent causative agent of equine theileriosis, an economically important tick-transmitted disease of horses that negatively affects the equine industry globally. Vaccines to control T. equi are currently unavailable in part due to the paucity of information on protective immune mechanisms and parasite virulent factors. ARS researchers in Pullman, Washington, performed T. equi blood stage experimental infection of horses and evaluated the development of immune responses. Results demonstrated that infected animals produce significant levels of regulatory cytokines, which is associated with antibody production without modulating pro-inflammatory responses. These findings challenge the previous paradigm on the profile of immune responses during T. equi infection and set the rationale for future vaccine trials. This information will help design the test of novel effective vaccine to control the parasite, which will assist our stakeholders to better manage eventual outbreaks of equine theileriosis in the U.S. horse population.
3. Demonstrated that the therapeutic drug buparvaquone has a robust parasiticidal effect on Theileria haneyi but is ineffective in the clearance of parasites in chronically infected horses. Theileria haneyi is a recently discovered tick-transmitted blood parasite and one of the causative agents of equine piroplasmosis, an economically important disease that negatively affects the horse industry globally. ARS researchers in Pullman, Washington, have previously demonstrated that T. haneyi is resistant to the gold-standard anti-theilerial therapy, imidocarb dipropionate, and to the approved veterinary drugs tulathromycin and diclazuril. Availability of safe and effective therapeutic drugs against T. haneyi will help horse veterinarians and stakeholders to control and eliminate this parasite. ARS researchers in Pullman, Washington, evaluated the efficacy of buparvaquone, a broad-spectrum anti-protozoal drug, in clearing the parasite during chronic infection in horses. Findings demonstrated that buparvaquone administration, at label and higher doses, is safe for horses. The drug showed a robust parasiticidal effect but was ineffective for the clearance of T. haneyi chronic infection in horses. This experiment advances our knowledge on the dynamics of T. haneyi infection and treatment in horses, providing relevant information for horse producers, veterinarians, and regulatory agencies.
4. Demonstrated that buparvaquone has a significant parasiticidal effect on Theileria orientalis Ikeda (U.S. isolate) but is ineffective in the clearance of parasites in chronically infected cattle. Theileria orientalis Ikeda is a tick-transmitted blood parasite that causes an economically important disease in cattle globally. Emerging outbreaks of T. orientalis Ikeda, followed by invasion of Haemaphysalis longicornis ticks, a major vector of the parasite, have been recently reported in the United States, posing a serious threat to the country’s cattle industry. Acute infection with T. orientalis Ikeda is characterized by anemia, abortions, and mortality, although subclinical infections are common and can lead to significant losses in animal production. Currently, no effective therapeutic drug is available to control and eliminate T. orientalis Ikeda, and the availability of such modality could help developing comprehensive programs aimed at controlling and eradicating this parasite. ARS researchers in Pullman, Washington, evaluated the efficacy of buparvaquone, a broad-spectrum anti-protozoal drug, against T. orientalis Ikeda in sub-clinically infected cattle. Buparvaquone treatment significantly reduced parasitemia to undetectable levels, but parasite recrudescence was observed in all treated cattle. These findings provide crucial information for stakeholders, veterinarians, and regulatory agencies involved in the control of T. orientalis Ikeda in the United States.
Review Publications
Furtado, A.P., Fry, L.M., Piel, L.M., Bastos, R.G., Schneider, D.A., Varvil, M.S. 2023. B-cell leukemia in an adult sheep. Veterinary Clinical Pathology. 52(4):716-721. https://doi.org/10.1111/vcp.13303.
Onzere, C.K., Hassan, A., Herndon, D.R., Oyen, K.J., Poh, K.C., Scoles, G.A., Fry, L.M. 2023. Dermacentor variabilis does not transstadially transmit the U.S. isolate of Theileria orientalis Ikeda: A controlled acquisition and transmission study. Parasitologia. 3(3):284-292. https://doi.org/10.3390/parasitologia3030029.
Onzere, C.K., Bastos, R.G., Bishop, R.P., Suarez, C.E., Fry, L.M. 2024. Expression of IL-10 and TGF-ß1 in horses experimentally infected with T. equi merozoites is associated with antibody production but not modulation of pro-inflammatory responses. Frontiers in Immunology. 15. Article 1370255. https://doi.org/10.3389/fimmu.2024.1370255.
