Recipient Organization
OKLAHOMA STATE UNIVERSITY
(N/A)
STILLWATER,OK 74078
Performing Department
Entomology and Plant Pathology
Non Technical Summary
This is a USDA-NIFA-AFRI Seed Award that focuses on detecting three major blood-borne diseases that affect cattle and sheep in the United States and around the world. The major disease of focus is Anaplasma marginale which causes bovine anaplasmosis, an economically important disease that annually impacts the cattle industry in the United States at estimates above $300 million. While short-term disease in cattle produces the largest loss in cattle numbers, cattle that survive the initial infection with Anaplasmosis will stay infected the rest of their lives. This chronic infection can pose a risk to the other cattle in the herd because these infections often 'wake up' in the infected cow and can be transmitted to other cattle through unsterile equipment (needles, knives), ticks or horse flies. Two other disease-causing bacteria, Anaplasma phagocytophilum and Anaplasma ovis, can also occur in livestock and could possibly affect humans as well. To date, there is a commercially available test provided by certified diagnostic veterinary laboratories to test cattle blood for bovine anaplasmosis. However, because of the shifting nature of Anaplasma infections in cattle, these tests don't always catch the infected cattle in a herd that are carrying the disease at a low level. There is a need to develop a simple, but effective screening test for producers that will provide rapid results at a low cost, yet be easy-to-use and accurate. To be the most helpful, this test should also be able to detect more than one species of Anaplasma. The goal of this project, then, is to develop a develop a specific and sensitive field deployable Rapid Anaplasma Detection (RAD) kit for the economical, large scale, and rapid screening of livestock (mainly cattle and sheep) for the detection of A. marginale, A. phagocytophilum and A. ovis. The project will have two main objectives. Objective One will put all the pieces together for a field-testing kit that will tell the difference between different types of Anaplasma using blood obtained from laboratory-infected animals. The second objective will take blood samples from cattle in sale barns across Oklahoma where Anaplasmosis occurs and make sure the kit works using fresh blood samples.
Animal Health Component
50%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Goals / Objectives
Overall goal The overall goal of this research is to develop a specific and sensitive field deployable Rapid Anaplasma Detection (RAD) kit for the economical, large scale, and rapid screening of livestock (mainly cattle and sheep) for infection with Anaplasma marginale, A. phagocytophilum and A. ovis.Specific objectives Objective 1: To develop a RAD kit that will differentiate A. marginale, A. phagocytophilum, and A. ovis in cattle and sheep using defined blood isolates collected from cattle and sheep experimentally infected with regional strains of A. marginale and A. ovis, respectively, and cell culture stocks infected with A. phagocytophilum.Objective 2: To validate the RAD kit performance using blood samples collected from cattle in bovine anaplasmosis endemic areas of the U.S. and sheep in areas of the world where A. ovis is a problem.
Project Methods
Objective 1: To develop a RAD kit that will differentiate A. marginale, A. phagocytophilum, and A. ovis in cattle and sheep.The sample transport cartridge: An Elution-Independent Collection Device (EICD): The first component is a novel EICD for rapid (3-5 min) collection and sample preparation, and long-term storage at room temperature. Involving less time than the 15-30 minutes required using commercially available kits for sample preparation of microorganism's nucleic acid recovery, EICD is easy-to-operate, collects fluid specimens by contact and lateral flow filtration. This EICD will be an integral component of the RAD kit and will simplify the process of handling samples and assist operators who may not be familiar with DNA extractions for RPA or PCR assays.The DNA amplification assay: Replicase Polymerase Amplification (RPA) of DNA: RPA is a single tube, isothermal DNA amplification technology alternative to PCR. By adding a reverse transcriptase enzyme to an RPA reaction, it can detect RNA as well as DNA, without the need for a separate step to produce cDNA. Because it is isothermal, RPA reactions need much simpler equipment than PCR, which needs a thermal cycler. This ease of use and low-cost investment allows RPA technology to be used at local veterinary venues.Visualization component: We will use a rapid PCRD Nucleic acid lateral flow assay as an alternative to DNA agarose gel electrophoresis. PCRD is a rapid, safe and sensitive way to confirm the successful outcome of a DNA amplification exercise.Assay validation component: Artificial Positive Control (APC). The fourth technology refers to the development a safe APC, which will be multi-target, non-infectious and clonable for routine RPA and PCR-based assays.Objective 2: Validation of the RAD kit performance using blood samples collected from cattle and sheep. Once a functional assay capable of detecting varying parasitemias of the Anaplasma species in bovine and sheep blood is created, we will test it using blood samples from cattle at sale barns across Oklahoma and other Anaplasma endemic areas. We will also procure bovine and sheep blood samples where A. phagocytophilum and A. ovis is endemic. The final analysis will include a comprehensive analysis of sample costs, processing, and storage.