B. burgdorferi strain B31-A3 (Elias et al., 2002) was propagated to mid-logarithmic stage (1 × 10⁶ cells/ml) in BSK-II media at 34 °C in capped tubes providing microaerophilic conditions. A full complement of plasmids (except for cp9, which is missing in the B31-A3 strain) was demonstrated based on a multiplex PCR (Bunikis et al., 2011).

HTS Transwell^®–96 Well Permeable Supports plate (Corning, Corning, NY) with an integral tray insert containing a 0.3 μm pore size polycarbonate membrane in each well were overlaid (50 μl/well) with 6% porcine gelatin (Sigma Aldrich, Billerica, MA) heated to 50 °C. The plate was placed at 4 °C for 5 min to let the gelatin solidify before introducing other liquids. B. burgdorferi were spun, washed in 1X phosphate buffered saline (PBS), resuspended in PBS or Hanks Balanced Salt solution (HBSS) and 2 × 10⁵ in 200 μl was added to the bottom of each control or sample well. Transwell inserts were positioned into the lower compartment of the well in contact with the PBS cell suspension. Test protein or chemical (100 μl) was added to the upper compartment of the transwell insert. All test recombinant proteins were added at equivalent concentrations (450 μg/ml) in 1X PBS. Saliva, SGE, and rabbit serum (PelFreeze, Rogers, AR) were tested undiluted. Transwell plates were sealed and incubated at 34 ° C in 5% CO₂ for 18–20 h. The transwell experimental setup is shown (Fig. 1A).

Following the incubation period, sample from the upper transwell chamber (100 μl) was carefully removed to avoid contact with the gelatin layer on the transwell membrane. This procedure ensured that only spirochetes that migrated through the membrane/gelatin layer would be counted. Care was taken to ensure the pipet tip did not press against the gelatin/membrane surface as damage could occur resulting in contamination of liquid from the lower compartment. Samples were placed in microfuge tubes containing 0.5 μl of SYTO61 Red Fluorescent Nucleic Acid Stain (Invitrogen, Carlsbad, CA) and mixed gently for 10 min at room temperature in a light-blocking container. Tubes were centrifuged (16,000 × g, 2 min), the supernatant removed and the pellet resuspended in PBS plus 2 mM EDTA (100 μl) with addition of 1 mM paraformaldehyde followed by gentle mixing for 10 min at room temperature in a light-blocking container. Samples were centrifuged (16,000 × g, 2 min), supernatant removed, and pellets resuspended in 1X PBS plus 2 mM EDTA (100 μl). Samples were placed in 96 well flat bottom microtiter plate for flow cytometry with plate covered to avoid light bleaching of samples.

Samples were analyzed by flow cytometry with BD Accuri^™ C6 Plus Benchtop Flow cytometer. Samples were run at a volume rate of 40–75 μl and gated to remove debris. Flow data was analyzed via the BD Accuri^™ C6 Plus software package and graphed with GraphPad Prism Version 8.

Animal experiments were approved and conducted in accordance with guidelines and regulations as established by the Division of Vector-Borne Diseases Institutional Animal Care and Use Committee (IACUC).

CD-1 mice (Charles River, Worcester, MA) were needle inoculated subcutaneously with 100 μl of log-phase B. burgdorferi B31-A3 (1 × 10⁵ cells/ml) in BSK-II media. Two weeks post-inoculation, ear biopsies were collected and cultured to determine infection status of mice. Three weeks post-inoculation, approximately 100 larval ticks or 50 nymphal ticks were placed on individual B. burgdorferi-infected mice. A total of 7 mice were utilized for each larval or nymphal feeding in two separate replicates. At 6-hour intervals, 2–3 nymphs or 8–10 larvae from each mouse were removed and placed into 70% ethanol at 4 °C prior to genomic DNA isolation.

Female New Zealand White rabbits were tranquilized with acepro-mazine delivered intramuscularly or subcutaneously using a 26-gage needle at a dose of 0.75–10 mg/kg (typically 0.75–1.0 mg/kg range) and fitted with a plastic e-collar to prevent direct oro-anal coprophagy. One-inch wide cotton athletic tape was applied around the base of each ear. 20–50 adult uninfected Ixodes scapularis adult ticks were placed into a cloth ear bag affixed to the tape at the base of the ear. A second piece of tape was placed around the base of the ear bag to hold it in place. The feeding period of adult ticks lasts approximately 7 days (range 4–10). Replete ticks were collected for saliva collection.

Nymphal ticks (n = 20) were placed on eight individual CD-1 mice (Charles River, Worcester, MA) and allowed to feed for 42 h. Ticks were removed with fine forceps and placed in microcentrifuge tubes on ice. For salivary gland dissection, individual ticks were placed in a drop of phosphate buffered saline (PBS) on a silane coated slide (Scientific Device Laboratory, Des Plaines, IL), and a scalpel was used to cut the basis capitulum and the contents of the tick were pushed out from the bottom through the cut using the side of fine tip forceps. The salivary glands were sorted from the midgut, washed in a clean drop of PBS, and placed into PBS containing protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) on ice. The salivary glands in PBS + protease inhibitors were sonicated at a 50% output for a total of 1 min to separate the cells and generate the SGE. Material from 115 salivary gland pairs (230 total glands) were utilized for fractionation. Collection of I. scapularis saliva was performed as described previously (Patton et al., 2012) and stored at −80 °C.

B. burgdorferi quantification by qPCR from the collected larvae and nymphs was performed as previously described (Patton et al., 2011) with the following modifications: genomic DNA was prepared from pooled ticks by time point per mouse and homogenizing in lysis buffer (Qiagen, Germantown, MD) by bead beating in a Biospec Mini (Biospec, Bartlesville, OK), subsequently processed with DNeasy Blood and Tissue Kit (Qiagen), and eluted in 50 μl of elution buffer (10 mM Tris pH 8.5). For qPCR, 1 μl of genomic DNA was utilized. The B. burgdorferi flaB Taqman primers have been previously described (Gilmore et al., 2007). A parallel reaction was performed to amplify a portion of the tick actin gene using 1 μM 5′ IscapactinSEN primer (5′-CGTGAAATTGTCCGTGACATC-3′), 1 μM 3′ IscapactinASN primer (5′-TCTCGTTGCCGATTGTGATG-3′), 0.15 μM tick actin probe (5′FAM-CTACGTCGCCCTGGACTTCGAGC-3′BHQ), and 2X TaqMan^® Universal PCR Master Mix (Applied Biosystems, Grand Island, NY) in a total volume of 20 μl. Amplification conditions were 1 cycle at 95 °C for 10 min and 40 cycles of 95 °C for 30 s and 60 °C for 1 min, with data collection after each cycle. Amplification of each DNA sample was performed in triplicate. To calculate copies of tick actin, a standard curve was generated by amplifying the portion of the tick actin gene which had been cloned into pCR2.1 with the TOPO TA Cloning kit (Invitrogen, Carlsbad, CA).

Tick salivary gland extract was filtered using 0.22 μm Ultrafree MC GV spin column (EMD Millipore), followed by a wash with 100 μl 30% HPLC grade acetonitrile. The filtered solution and the 30% acetonitrile wash were combined and fractionated by FPLC using AKTA Purifier (GE Healthcare, Pittsburgh, PA), size exclusion column Superdex 200 10/300 GL (GE Healthcare, USA), buffer of 10 mM KH₂PO₄, 150 mM NaCl, pH 7, 0.5 ml loop, flow rate of 0.5 ml/min at 25 °C and 0.5 ml fractions collected.

Ribosomal S15A, Sialostatin L, Salp 9- and 11-like genes were amplified by PCR using primers listed in Table 1 from I. scapularis genomic DNA. Amplicons were cloned into pETite C–His-vector (Lucigen, Middleton, WI) and transformed into E. coli BL21 (DE3) according to the manufacturer’s instructions. Recombinant proteins were expressed and purified using a QiaExpress Ni-NTA FastStart kit (Qiagen, Valencia, CA) as described previously (Brandt et al., 2019)

The assay was designed for borrelial migration to proceed upward from the bottom well to the upper transwell insert cup containing the test reagent (Fig. 1A). We initially found that borrelial movement through the transwell membrane proceeded within short timeframes that did not enable accurate counting by darkfield microscopy. We added a layer of 6% gelatin on the membrane for borreliae to gradually pass thereby allowing counting to be performed hours instead of minutes later which ensured a more precise enumeration.

Enumeration of live borreliae was initially performed by microscopy but proved onerous when counting multiple replicate wells of timepoints in real time. We turned to flow cytometry of fixed cells which had advantages over microscopy: i) borreliae were paraformaldehyde-fixed and stained enabling counts to be performed for multiple timepoints in a single run; and ii) quantification could be performed more rapidly by software. Enumeration of borreliae between the two methods was compared and found to be equivalent (Fig. 1B). The R²=0.95 indicates a good linear trend with a low proportion of variance. The small discrepancy between counts between microscopy and flow cytometry may be due to sample loss while staining, washing, and fixing the cells during flow cytometry sample prep. Pellets prepared for flow cytometry were small and flaky, therefore, to ensure minimal sample loss, gentle removal of supernatant after each step was critical.

We found that BSK-II culture media was the best positive control for borrelial attraction (Fig. 2). Other studies have used BSK-II, N-acetyl glucosamine, chitobiose, rabbit serum, and glucosamine, as positive controls when assaying borrelial motility (Bakker et al., 2007; Motaleb et al., 2007, 2005; Shi et al., 1998). N-acetyl glucosamine and rabbit serum are components of BSK media, and we found that their chemoattractant ability was diminished compared to BSK, but N-acetyl glucosamine activity was significantly higher than controls (Fig. 2). Therefore, in our assays, B. burgdorferi were suspended in PBS in bottom wells and BSK was used in upper chambers as a positive control. We observed that B. burgdorferi were stable, viable, and motile in PBS for 24 h, therefore we performed the migration assays with both B. burgdorferi and the test proteins suspended in PBS.

The small size of nymphs and larvae, together with low yield, logistically precluded our collection of saliva. Therefore, intact salivary glands which provided ample test material, were dissected for whole extract preparation that included saliva. Salivary glands were harvested from nymphs after attachment for 42 h on mice. This timepoint was chosen to coincide with the stage at which borrelial acquisition began to increase with the premise that putative chemoattractants would be prominent in saliva. Borrelial acquisition was determined by feeding uninfected larvae and nymphs on B. burgdorferi-infected mice and calculating the spirochete burden by PCR in ticks removed every 6 h during the feed (Fig. 3A). Primary acquisition of B. burgdorferi was observed approximately 42–48 h post-attachment with the highest densities measured from 54 to 66 h post-attachment for both tick stages (Fig. 3B). The physical limitation of dissecting larvae for salivary glands was a factor, therefore we confirmed that both stages demonstrated similar time course acquisition of borreliae and collected salivary glands from nymphs. Saliva was collected from replete adults 5–6 days post-attachment on rabbits.

Unfractionated SGE and saliva were tested for B. burgdorferi chemoattractant activity in the transwell assay. Both SGE and saliva demonstrated statistically significant positive borrelial migratory activity compared to the PBS control, with saliva producing levels close to that of the BSK positive control and significantly better than SGE (Fig. 4).

SGE proteins were subjected to size exclusion chromatography and collected in multiple fractions, each tested for borrelial migration activity by the transwell assay. Positive and negative chemoattractant activity were demonstrated by the SGE fractions when compared to the PBS control (Fig. 5). Each fraction contained limited volume, therefore could only be tested once in the transwell assay.

Mass spectrometry analysis from designated SGE fractions 17, 26, and 38 that demonstrated chemoattractant activity allowed us to select individual proteins for preliminary testing in the transwell assay. From fraction 17, we chose Sialostatin L, and Salp-like proteins 9 and 11 for recombinant protein expression and observed chemoattractant activity with these proteins using the transwell assay. The I. scapularis protein S15A, a ribosomal binding protein, was negative (Fig. 6).

We performed the transwell borrelial migration assay in a dose response experiment with a dilution series of each recombinant Sialostatin L, Salp-like 9, and Salp-like 11 proteins. The results revealed a decline in borrelial migration with a concomitant decrease in the protein concentration for all three proteins (Fig. 7). These results represented additional confirmation for the consistency of the transwell assay and proof of principle to measure borrelial migration to chemotactic test proteins.