Location: Animal Disease Research
Project Number: 2090-32000-039-15-T
Project Type: Trust Fund Cooperative Agreement
Start Date: May 1, 2016
End Date: Dec 15, 2018
1. Develop a continuous flow in vitro system for feeding of competent tick vector of T. parva. 2. Isolate Theileria parva sporozoites from infected adult ticks feeding on a continuous flow in vitro system. 3. Infect Rhipicephalus appendiculatus nymphs with T. parva using a continuous flow in vitro feeding system.
Immunization of cattle for East Coast Fever (ECF) using the infection and treatment method (ITM) elicits cross protective immunity to multiple strains of T. parva, and has been shown to protect >98% of vaccinated cattle from subsequent challenge in the field in Tanzania. Current production methods for the live sporozoite stabilate, the primary component of the vaccine, are labor and cost-intensive, requiring over 600,000 ticks, ~500 rabbits, and ~130 cattle to produce a batch sufficient to immunize one million cattle. This animal-based production system leads to large variability in sporozoite levels in the stabilate, differences in infectivity between stabilate batches, and requires significant expertise and specialized facilities for tick and animal handling. High costs for production and quality control significantly increase the price per immunization dose, limiting widespread adoption of ITM vaccine by smallholder farmers. This project will simplify and standardize the production process for ITM vaccine by developing methods to utilize a continuous flow in vitro tick feeding system for production of the vaccine. This innovative approach provides scalability for vaccine development against ECF in that it eliminates the need for large numbers of cattle and rabbits, making sporozoite generation more routine and easier to synchronize with vaccine demand. This will significantly reduce facilities and labor costs, resulting in lower per immunization dose costs incurred by smallholder farmers. The flow-through, artificial system will also eliminate animal-derived biological variation and enable standardization of piroplasm levels in the blood ingested by the ticks, maximizing infection efficiency. In the continuous flow in vitro feeding system, infected adult ticks can be allowed to feed to repletion so that mature sporozoites are secreted from the salivary glands into the medium. This will permit quantification using quantitative PCR that will accurately reflect only mature infectious units and will not over-estimate sporozoite yield by including immature sporozoites. In addition, viability of free sporozoites can be determined using flow cytometry to provide a better estimate of live parasite stabilate composition prior to preservation. Finally, the sporozoites will be collected in the context of the tick saliva proteins that are present during natural transmission, without additional, potentially confounding debris from homogenized, whole ticks, as is standard in the current ITM inoculum. This will increase the purity and uniformity of stabilate batches.