Location: Invasive Insect Biocontrol & Behavior Laboratory
Project Number: 8042-10400-001-010-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 13, 2026
End Date: Sep 30, 2027
Objective:
The objectives of the proposed research are to (1) characterize the in vitro activities of several bioactive compounds against Borrelia burgdorferi under controlled conditions; (2) determine the ability of those compounds to reduce bacterial burden, limit dissemination, and attenuate disease pathology in a well-established murine model of Lyme disease, and (3) select the most effective compound for further evaluation in white-footed mice, the reservoir host of the pathogen.
Approach:
This work will be conducted by the cooperator. Research protocols will be jointly developed by USDA-ARS In-house tick control project. Appropriate permits for animal and pathogen work will be obtained by the cooperator.
(1) In vitro evaluation against B. burgdorferi. To characterize the in vitro activities of test compounds against Borrelia burgdorferi under controlled laboratory conditions, a low-passage fully virulent B. burgdorferi strain will be cultured in a cell culture medium and maintained at 33°C under microaerophilic conditions. Each compound will be tested across a broad concentration range to establish dose-response relationships. Initial screening will involve monitoring spirochete growth and motility using dark-field microscopy at 24-hour intervals over a period of up to 7 days. Growth curves will be generated by enumerating spirochetes using a Petroff-Hausser counting chamber. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) will be determined.
(2) In vivo assessment of therapeutic efficacy in a murine model. Following the in vitro characterization, researchers will transition to an in vivo murine model to assess therapeutic efficacy under physiologically relevant conditions. C3H/HeJ mice will be used to B. burgdorferi infection and their ability to develop hallmark features of Lyme disease, including carditis and arthritis. Bacterial burden in mouse tissues will be quantified using quantitative PCR (qPCR) targeting B. burgdorferi flaB gene. This approach provides a sensitive and quantitative measure of spirochete load across different tissues and treatment conditions. Data analysis will involve comparing bacterial burden, serological responses, and histopathological scores across treatment groups using appropriate statistical methods, such as analysis of variance (ANOVA) followed by post hoc testing. Correlations between in vitro bactericidal activity and in vivo efficacy will be examined to identify predictive markers of therapeutic success. Throughout the study, careful attention will be paid to experimental reproducibility and rigor. All in vitro assays will be performed in biological triplicates, and in vivo experiments will include a sufficient number of animals to ensure statistical power. Randomization and blinding will be employed where appropriate to minimize bias. This integrated experimental approach will provide a comprehensive evaluation of the antimicrobial properties of the candidate compounds. By linking detailed in vitro analyses with robust in vivo outcomes, the study is designed to identify compounds with true bactericidal activity and meaningful therapeutic potential against B. burgdorferi. Data generated from this study will help us select the most promising compound for further evaluation in white-footed mice and development of potentially new commercial pest control disease mitigation product. Knowledge generated from this study will also help us understand the anti-Borrelia mechanisms of those compounds.