Location: Animal Disease Research
Project Number: 2090-32000-044-030-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2022
End Date: Jun 30, 2025
Objective:
The tick-borne parasite Theileria orientalis recently emerged as a novel threat to the United States (U.S.) cattle industry. The emergence of T. orientalis occurred in concert with the invasion of the U.S. by its primary tick vector, Haemaphysalis longicornis in 2017, and infection has become endemic in two states, where it causes significant losses due to abortion, neonatal death, and reduced meat and mild production. To prevent eventual widespread Theileria sp. endemnicity in the U.S., the development of improved treatment, diagnostic, and preventive strategies for bovine theileriosis is urgently needed. The objectives of this agreement are as follows: 1. Facilitate screening of cattle within endemic areas to both determine baseline prevalence in those areas and to create a bank of serum samples from positive cattle for use in diagnostic assay validation; and 2. Identify cattle infected with Theileria orientalis Ikeda, Chitose, and Buffeli strains for generation and transfer of infectious blood stabilate. These stabilates will then be used to infect cattle in order to perform tick competency studies to characterize the potential range of spread of T. orientalis in the U.S., and to perform comparative genomic and transcriptomic analyses for identification of conserved candidate diagnostic assay target antigens.
Approach:
1) Screen cattle throughout the eastern U.S. for Theileria orientalis using published assays, determine baseline prevalence in the eastern U.S., and bank serum and whole blood from at least 1000 positive animals. The cooperator has published a real-time PCR assay for use in diagnosis of T. orientalis in U.S. cattle, leads screening and diagnostic efforts for T. orientalis in the U.S., and has access to producers and samples as faculty the veterinary diagnostic laboratory at his institution, which is within the T. orientalis endemic area of the U.S. Upon development of a serologic assay to diagnose T. orientalis, these banked serum samples will be used for assay validation.
2) Identify cattle infected with T. orientalis Ikeda, Chitose, and Buffeli strains, generate cryopreserved, infected erythrocyte stabilate, and transfer stabilate. Using published PCR assays and subsequent sequencing, animals infected with each strain will be identified. With producer permission, a larger volume of blood will be collected, defibrinated, and the plasma removed via centrifugation. The remaining cells will be mixed with PVP cryopreservative, aliquoted into 1 mL vials, and cryopreserved at -80 degrees C. Vials will then be transferred, and cattle infected via intravenous inoculation. Once infected, cattle will be used in tick acquisition and transmission studies to determine the subset of U.S. tick species capable of spreading T. orientalis. Blood will be collected from infected cattle, parasites isolated, and comparative genomic and transcriptomic analyses performed to identify candidate diagnostic assay antigenic targets conserved between all strains of T. orientalis. Candidate antigens will be screened using serum from infected cattle to verify immunogenicity and optimize a serologic assay. The resultant assay will subsequently be validated using banked serum samples as described in 1) above.
