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- Bastos, Reginaldo
- Chung, Chungwon
- Noh, Susan
- Kappmeyer, Lowell
- Herndon, David
- Oyen, Kennan
- Poh, Karen
- 2023
- 2022
- Publications
- Animal Health (103)
- Food Safety, (Animal and Plant Products) (108)

Research Project: Control Strategies for Theileriosis **Location:
                            Animal Disease Research** 2023 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 fiscal year (FY) 2023 progress for project 2090-32000-044-000D, titled, “Control Strategies for Theileriosis”. 

In support of Sub-objective 1A, ARS researchers in Pullman, Washington, finished assaying their first batch of 1,000 samples from horses at the southern border of the United for Theileria haneyi using PCR. Researchers continue to work on optimization of a serological diagnostic assays for T. haneyi, a quantitative PCR that can detect both T. haneyi and its more virulent counterpart, Theileria equi, and a sensitive serologic assay using the Theileria RAP-1 antigen as a target, based on data obtained and published by our group (Onzere et al 2021). They also began working with our new unit scientist who has infectious disease modeling expertise to formulate a plan for future sampling and statistical analysis. 

In support of Sub-objective 1B, ARS researchers continued to expand their horse breeding herd. They have also completed analysis of the cytokine response to acute T. equi (manuscript in process) and worked to characterize a panel of monoclonal antibodies specific for equine leukocyte antigens for use in immune response characterization (manuscript in progress). Also, in support of Sub-objective 1B, ARS researchers completed in vivo studies on the efficacy of the drugs tulathromycin and Protazil, against T. haneyi, and successfully published that work. They also generated T. haneyi-infected erythrocyte stabilate for use in future infections. ARS researchers successfully imported the drug, buparvquone, from Africa (donated by Bimeda Inc.) and have treated their T. haneyi infected horses with buparvaquone and its efficacy against T. haneyi is currently under investigation.

In support of Sub-objective 2A, ARS researchers completed tick competency studies using colonized Dermacentor variabilis and Rhipicephalus microplus ticks. One of these studies has been published, and the other manuscript is under revision. They performed a preliminary tick competency study using Rhipicephalus sanguineous ticks; however, the tick colony obtained for this study did not feed well on cattle. Thus, ARS researchers are working to identify and obtain a new R. sanguineous colony to repeat this study. They are also working to identify and obtain colonies of additional tick species to test during the next fiscal year.

In support of Sub-objective 2B, ARS researchers utilized expertise garnered in other Theileria spp. to generate Theileria orientalis Ikeda-infected tick salivary gland inoculum and successfully inoculated cattle. This is an important breakthrough for the upcoming work, as it will enable experimental animals to be exposed to all parasite life stages, instead of only the merozoite stage. Researchers have selected a set of candidate antigens based on genomic comparison of Theileria orientalis Ikeda, Chitose, and Buffali, as well as other Theileria species. Candidate antigens were also selected using in silico antigenic prediction algorithms and predicted MHC class I and class II binding patterns. A manuscript describing this work is in process. ARS Researchers have also generated several batches of T. orientalis-infected erythrocyte inoculum, which, along with the infected tick salivary gland stabilate, can be used to infect cattle in future experiments. They published their first manuscript describing the use of their anti-bovine CD138 monoclonal antibody demonstrating its utility in identifying bovine plasma cells. Finally, to assist stakeholders in the United States, ARS researchers worked with the Texas State Veterinarian to test a group of cattle for T. orientalis and updated our stakeholders regarding our progress in T. orientalis at stakeholder meetings. 

In support of Sub-objective 2C, ARS researchers successfully imported buparvaquone from Africa (donated by Bimeda Inc), obtained 14 cattle, infected them with T. orientalis Ikeda, and have initiated the buparvaquone treatment. Preliminary results from this study suggest that T. orientalis parasitemia is only transiently affected by the drug, used at the label dose. Thus, scientists are currently working with collaborators to perform pharmacokinetics analysis of buparvaquone and establish the rationale for testing additional drug doses and regimens. **Accomplishments**
                                

1. Rhipicephalus microplus and Dermacentor variabilis ticks are not able to transmit the United States isolate of Theileria orientalis Ikeda. The tick-transmitted blood parasite Theileria orientalis Ikeda recently emerged as novel threat to the United States cattle industry. Its emergence occurred in concert with the invasion of the United States by its primary tick vector, Haemaphysalis longicornis in 2017, and infected cattle herds have since been detected in multiple states. It is not yet known whether other tick species native to the United States and surrounding countries are also capable of transmitting T. orientalis. Since different species of ticks live in defined host geographic ranges, determination of tick species capable of transmitting T. orientalis will enable prediction of where the parasite is most likely to spread in the United States, thereby improving control of T. orientalis. Using controlled studies, ARS Scientists in Pullman, Washington, determined that Rhipicephalus microplus and Dermacentor variabilis ticks are unable to transmit T. orientalis to cattle, which provides important information for cattle producers, veterinarians, and regulatory personnel. 2. The drugs tulathromycin and diclazuril lack efficacy against Theileria haneyi, and provided supporting evidence that tulathromycin is safe to use in adult horses. Theileria haneyi is a tick transmitted blood parasite of horses recently discovered by ARS researchers in Pullman, Washington. These scientists have demonstrated that T. haneyi is resistant to the gold-standard anti-theilerial therapy, imidocarb dipropionate, and thus, discovery of efficacious new drug therapies against this parasite is an urgent need. Several in vitro studies suggested that the approved veterinary drugs, tulathromycin and diclazuril, may be efficacious against Theileria spp. However, using in vivo studies, ARS researchers in Pullman, Washington, determined that neither drug is efficacious against T. haneyi, thereby prioritizing testing of additional drug candidates. Importantly, these studies also demonstrated that administration of tulathromycin to adult horses does not cause any significant pathologic side effects. This finding will facilitate justification of the use of this antibiotic in horses to combat bacterial diseases, including respiratory infections, which is an important advancement for the equine industry. 3. A recombinant anti-bovine CD138 monoclonal antibody can be used to detect bovine plasma cells. CD138 is a protein expressed on the surface of plasma cells, which make antibodies against infectious agents. Previously, the lack of antibody reagents to bovine CD138 precluded isolation and characterization of bovine plasma cells generated in response to various infectious diseases and candidate vaccines. Scientists in Pullman, Washington, generated a monoclonal antibody to bovine CD138, and then worked with collaborators to generate a recombinant version of the antibody that can be readily produced. The scientists then tested this new antibody in a panel with other antibodies used to label various types of bovine white blood cells and verified that the monoclonal antibody can be used to successfully label bovine plasma cells. This recombinant monoclonal antibody will enable more complete characterization of the bovine immune response to infectious diseases and vaccines globally, thereby increasing our ability to control pathogens and improve livestock production. **Review Publications**
                            *Onzere, C.K., Hulbert, M., Sears, K.P., Williams, L.B., Fry, L.M. 2023. Tulathromycin and diclazuril lack efficacy against Theileria haneyi, but tulathromycin is not associated with adverse clinical effects in six treated adult horses. Pathogens. 12(3). Article 453. https://doi.org/10.3390/pathogens12030453.*
                                *Onzere, C.K., Herndon, D.R., Hassan, A., Oyen, K.J., Poh, K.C., Scoles, G.A., Fry, L.M. 2023. A U.S. isolate of Theileria orientalis ikeda is not transstadially transmitted to cattle by Rhipicephalus microplus. Pathogens. 12(4). Article 559. https://doi.org/10.3390/pathogens12040559.*
                                *Roos, E.O., Bonnet-Di Placido, M., Mwangi, W., Moffat, K., Fry, L.M., Waters, R., Hammond, J.A. 2022. OMIP-085: Cattle B-cell phenotyping by an 8-color panel. Cytometry Journal. 103(1):12-15. https://doi.org/10.1002/cyto.a.24683.*


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