Borrelia burgdorferi Migration Assays for Evaluation of Chemoattractants in Tick Saliva
Abstract
:1. Introduction
2. Results
2.1. Assessment of Motility Using the U-Tube
2.2. The Gelatin Well Assay of Spirochete Migration
2.3. The Capillary Tube Migration Assay
3. Discussion
4. Materials and Methods
4.1. Ethics Statement
4.2. Bacterial Strain, Media and Growth Conditions
4.3. Chemoattractants and Matrices
4.4. Saliva Collection
4.5. Chemotaxis Assays
4.5.1. U-Tube Assay
4.5.2. Gelatin Well Assay
4.5.3. Capillary Tube Assay
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Appendix A.1. Experimental Model and Subject Details
Appendix A.2. Method Details
Bacterial Strain, Media and Growth Conditions
Appendix A.3. Quantification and Statistical Analyses
Appendix A.4. Key Resources Table
Reagent or Resource | Source | Identifier |
Bacterial and Virus Strains | ||
Borrelia burgdorferi, strain B31.5A19 | S. Norris | Purser, J. E. and S. J. Norris (2000). “Correlation between plasmid content and infectivity in Borrelia burgdorferi.” Proc. Natl. Acad. Sci. U.S.A. 97. |
Biological Samples | ||
Ixodes scapularis saliva | B. Grasperge | Produced in-house |
Chemicals, Peptides, and Recombinant Proteins | ||
N-acetyl-D-glucosamine (D-GlcNAc) | Sigma chemical | A3286 |
N,N’-Diacetylchitobiose (chitobiose) | Sigma chemical | D1523 |
Bovine Serum Albumin (Probumin) | Millipore | 82047 |
Type A gelatin from porcine skin | Sigma chemical | G2500 |
Experimental Models: Organisms/Strains | ||
Ixodes scapularis ticks, adult, female | BEI resources | NR-42510 |
Ixodes scapularis ticks, adult, female | Wild caught | Covington, LA, USA |
CD IGS Albino Rats, female, six to eight weeks old | Charles River | Strain Code: 001 |
New Zealand White Rabbits | Charles River | Strain Code: 052 |
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Matrix: Gelatin (%) | Well Diameter (mm) | Well Depth (mm) | Distance between Wells (mm) | Cells | [Cell] (×107/mL) | Lateral Well Inocula | [CA] | Volume per Well (µL) | Incubation Time (Hours) | Final Cell Count * | Ratio (CA:Control) |
---|---|---|---|---|---|---|---|---|---|---|---|
3 | 6 | 2 | 4 | in vitro Bb | 8 | chitobiose | 10 mM | 35 | 21 | 0.45/field | |
PBS | 0 | ||||||||||
3 | 6 | 2 | 4 | in vitro Bb | 8 | chitobiose | 10 mM | 35 | 21 | 0.9/field | (-) |
BSK-II | 1/field | ||||||||||
5 | 6 | 2 | 2 | in vitro Bb | 8 | chitobiose | 10 mM | 35 | 21 | 1.5/field | |
PBS | 0 | ||||||||||
5 | 6 | 2 | 2 | in vitro Bb | 8 | chitobiose | 10 mM | 35 | 21 | 14/cs | 7:1 |
BSK-II | 2/cs | ||||||||||
5 | 6 | 2 | 2 | in vitro Bb | 8 | tick saliva | 5% | 40 | 24 | 1/cs | 1:1 |
PBS | 1/cs | ||||||||||
5 | 6 | 2 | 2 | in vitro Bb | 4.6 | chitobiose | 10 mM | 40 | 21 | 1/cs | 1:1 |
PBS | 1/cs | ||||||||||
5 | 6 | 2 | 2 | in vitro Bb | 4.6 | tick saliva | 5% | 40 | 21 | 3/cs | (-) |
PBS | 8/cs | ||||||||||
5 | 6 | 2 | 1 | in vitro Bb | 7 | chitobiose | 10 mM | 40 | 26 | 20/cs | 10:1 |
PBS | 2/cs | ||||||||||
5 | 6 | 2 | 1 | in vitro Bb | 7 | tick saliva | 9% | 45 | 27 | 2/cs | 1:1 |
PBS | 2/cs | ||||||||||
5 | 6 | 2 | 1 | in vitro Bb | 7 | chitobiose | 10 mM | 45 | 27 | 24/cs | 3.5:1 |
PBS | 7/cs | ||||||||||
5 | 6 | 2 | 1 | in vitro Bb | 7 | chitobiose | 10 mM | 45 | 27 | 25/cs | 13:1 |
PBS | 2/cs | ||||||||||
5 | 6 | 2 | 1 | in vitro Bb | 7 | D-GlcNAc | 100 mM | 45 | 26 | 64/cs | 4:1 |
PBS | 16/cs | ||||||||||
5 | 6 | 2 | 1 | in vitro Bb | 7 | chitobiose | 10 mM | 45 | 28 | 62/cs | 1.6:1 |
PBS | 40/cs | ||||||||||
5 | 6 | 4 | 1 | in vitro Bb | 7 | chitobiose | 10 mM | 46 | 27 | 27/cs | 2.5:1 |
PBS | 11/cs | ||||||||||
4 | 6 | 2 | 1 | in vitro Bb | 7 | chitobiose | 10 mM | 40 | 23 | 37/cs | 5.3:1 |
4 | 4 | 3 | 1 | in vitro Bb | 7 | D-GlcNAc | 100 mM | 30 | 22 | 57/cs | 3.2:1 |
PBS | 18/cs | ||||||||||
5 | 4 | 3 | 1 | in vitro Bb | 7 | D-GlcNAc | 100 mM | 30 | 22 | 21/cs | 7:1 |
PBS | 3/cs | ||||||||||
4 | 4 | 3 | 1 | Host-adapted Bb | 2 | chitobiose | 10 mM | 30 | 22 | 3/cs | |
PBS | 0 | ||||||||||
4 | 4 | 3 | 1 | Host-adapted Bb | 2 | chitobiose | 10 mM | 30 | 22 | 3/cs | |
PBS | 1/cs | ||||||||||
4 | 4 | 3 | 1 | Host-adapted Bb | 2 | tick saliva | 5% | 30 | 27 | 15/cs | 7.5:1 |
PBS | 2/cs | ||||||||||
4 | 4 | 3 | 1 | Host-adapted Bb | 2 | tick saliva | 10% | 30 | 28 | 17/cs | 17:1 |
PBS | 1/cs | ||||||||||
4 | 4 | 3 | 1 | Host-adapted Bb | 2 | D-GlcNAc | 100 mM | 30 | 26 | 3/cs | 1:1 |
Matrix | Cells | Cap Tube Contents: CA or Control | [CA] | Cap Tube Volume (µL) | No. Replicates | Incubation Environment | Incubation Time (Hours) | AVG. Cell Count per Field | Ratio (CA:Control) |
---|---|---|---|---|---|---|---|---|---|
Motility | Host-adapted | D-GlcNAc | 100 mM | 20 | 1 | 1.4 | 3.5:1 | ||
Buffer | Bb | tick saliva | 10% | 20 | 3 | RT | 2 | 3.1 | 7.8:1 |
MB buffer | 20 | 3 | 0.4 | ||||||
Motility | Host-adapted | D-GlcNAc | 100 mM | 20 | 1 | 4.8 | 5.3:1 | ||
Buffer | Bb | tick saliva | 10% | 20 | 4 | RT | 2 | 6.6 | 7.3:1 |
MB buffer | 20 | 3 | 0.9 | ||||||
Motility | in vitro Bb | D-GlcNAc | 100 mM | 20 | 3 | RT | 2 | 20.8 | 14.9:1 |
Buffer | MB buffer | 20 | 3 | 1.4 | |||||
Motility | in vitro Bb | D-GlcNAc | 100 mM | 20 | 3 | 22.3 | 9.7:1 | ||
Buffer | tick saliva | 10% | 20 | 3 | RT | 2 | 3.9 | 1.7:1 | |
MB buffer | 20 | 3 | 2.3 | ||||||
Motility | in vitro Bb | D-GlcNAc | 100 mM | 20 | 3 | RT | 2 | 13 | 26:1 |
Buffer | MB buffer | 20 | 3 | 0.5 | |||||
Motility Buffer+ | in vitro Bb | D-GlcNAc | 100 mM | 20 | 3 | 34 °C trigas | 3 | 4.9 | 5.5:1 |
3% gelatin | MB/gelatin | 20 | 3 | 0.9 | |||||
Motility Buffer+ | in vitro Bb | D-GlcNAc | 100 mM | 20 | 3 | 34 °C trigas | 3 | 9.4 | 5.9:1 |
3% gelatin | tick saliva | 10% | 20 | 3 | 2.1 | 1.3:1 | |||
MB/gelatin | 20 | 3 | 1.6 | ||||||
Motility Buffer+ | in vitro Bb | D-GlcNAc | 100 mM | 20 | 3 | 34 °C trigas | 3 | 7.4 | 5.7:1 |
3% gelatin | MB/gelatin | 20 | 3 | 1.3 | |||||
Motility Buffer+ | in vitro Bb | D-GlcNAc | 100 mM | 20 | 3 | 27 | 1.5 | 1.5:1 | |
3% gelatin | tick saliva | 10% | 20 | 3 | RT | 1.1 | 1.1:1 | ||
MB/gelatin | 20 | 3 | 1 |
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Jacobs, M.B.; Grasperge, B.J.; Doyle-Meyers, L.A.; Embers, M.E. Borrelia burgdorferi Migration Assays for Evaluation of Chemoattractants in Tick Saliva. Pathogens 2022, 11, 530. https://doi.org/10.3390/pathogens11050530
Jacobs MB, Grasperge BJ, Doyle-Meyers LA, Embers ME. Borrelia burgdorferi Migration Assays for Evaluation of Chemoattractants in Tick Saliva. Pathogens. 2022; 11(5):530. https://doi.org/10.3390/pathogens11050530
Chicago/Turabian StyleJacobs, Mary B., Britton J. Grasperge, Lara A. Doyle-Meyers, and Monica E. Embers. 2022. "Borrelia burgdorferi Migration Assays for Evaluation of Chemoattractants in Tick Saliva" Pathogens 11, no. 5: 530. https://doi.org/10.3390/pathogens11050530