Location: Cattle Fever Tick Research Unit
Project Number: 3094-32000-042-095-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2023
End Date: Jun 30, 2025
Objective:
1) Determine the impact of three organic compounds (lavender oil, Stop the Bites, and BioUD) on R. microplus survival and attachment in vitro;
2) Evaluate the repellent effect of four organic compounds (lavender oil, Stop the Bites, BioUD, and nootkatone) towards R. microplus in vitro;
3) Assess suitability of four organic compounds (lavender oil, Stop the Bites, BioUD, and nootkatone) for on-animal application to cattle, including monitoring animals for potential toxic side effects of exposure; and
4) For those deemed safe to use on-animal, evaluate effect of organic compound application on reduction of on-host tick populations.
Approach:
1) Define lethal concentrations of organic compounds.
Bioassays will be performed to establish the lethal concentration needed to kill 50% and 90% of R. microplus in different life stages. The Larval Immersion Test (LIT) will be used to determine the LC50 and LC90 of larva. Adult Immersion test will be used to define the LC50 and LC90 for adults. Tick mortality will be quantified 24 hr post exposure and difference to control (diluent) will be evaluated by One-way ANOVA.
2) Characterize effect on tick hatching, mortality, and fecundity.
Egg masses will be immersed in vials containing 2 ml of the determined larval LC90, and they will then be maintained in a humidified incubator. Control eggs treated with the diluent alone and untreated control eggs will also be maintained in parallel. Percentage of hatching will be calculated. To define impact on reproduction and molting, fed larvae and mated female R. microplus will be collected from a stanchioned infested calf. Engorged larvae and females will be immersed in the LC50 and LC90 and incubated until ticks die, molt, or oviposit. Hatch rate, attachment rate, molt success, and egg mass size will be recorded. Control ticks will be immersed in diluent alone and maintained in an incubator with untreated controls. The differences will be evaluated by One-way ANOVA.
3) Determine repellency towards R. microplus.
The repellent effect of compounds will be determined using trapeze bioassays. Filter paper strips will be suspended vertically. Strips will be treated with one of three dilutions of the compounds. The diluted compound will be added to the strip midline and R. microplus larvae will be applied to the bottom of the strips with a paint brush. The number of ticks crossing the midline will be counted at specific time intervals. The level of repellency will be calculated based on comparison to strips treated with the diluent and untreated strips.
4) Evaluate toxicity towards cattle.
Each organic compound will be applied to cattle every 2 weeks for 3 months. We will monitor the reaction of the animals to the application of the product and observe for 1 hour to monitor the animal's behavior. Temperature at time of application will be recorded, as will weight [pre- and post-tick infestation] to determine any effect on appetite. Blood samples will be obtained from each individual weekly and submitted to the Diagnostic Lab for analysis to screen for effects of toxicity using 1) complete blood count (CBC), and 2) a complete blood chemistry panel.
5) Assess on-host effectiveness.
On-host effectiveness will be assessed in a stall trial. Three cattle will be experimentally infested with 125 mg of R. microplus larvae weekly for three weeks, after which all life stages will be present on each animal. Animals will be treated at day 21 post-infestation with the determined LC90 for each compound. Thereafter, egressing females will be collected and counted, and efficacy will be estimated by calculating the index of fecundity.
