B. burgdorferi clones used in this study were derived from strain B31. For genetic manipulations infectious low-passage clone A3-68Δbbe02 was utilized, which lacks cp9, lp56, and gene bbe02 on lp25 (Rego et al., 2011), and herein referred to as wild type. Spirochetes were cultivated in liquid Barbour-Stoenner-Kelly (BSK) II medium supplemented with gelatin and 6% rabbit serum (Barbour, 1984) and grown at 35°C with 2.5% CO₂. Luciferase plasmids were engineered in DH5α E. coli, grown in LB broth or on LB agar plates containing 300 μg/ml spectinomycin when appropriate, and transformed into B. burgdorferi as previously described (Samuels, 1995). Transformants were selected by plating in solid BSKII medium as previously described (Rosa and Hogan, 1992), in the presence of 50 μg/ml streptomycin and/or 200 μg/ml kanamycin, when applicable. All transformants were verified by PCR to contain the plasmid content of the parent clone (Elias et al., 2002; Jewett et al., 2007).

The Renilla reniformis luciferase gene (Lorenz et al., 1991) was codon-optimized for B. burgdorferi (rluc_Bb) with the OptimumGene™ algorithm, synthesized, and cloned into the E. coli vector pUC18 (Genscript) (Genebank accession number MF043582). All primer sequences are listed in Table 1. The rluc_Bb gene was PCR amplified from pUC18-rluc_Bb plasmid DNA using Phusion High-fidelity DNA polymerase (NEB) and primer pair 1732 and 1733. This also resulted in the addition of 27 bp of DNA from the 3′ of the flaB promoter to the 5′ of rluc_Bb. Concurrently, a DNA fragment containing the flaB promoter sequence with a 24 bp overhang from the 5′ of the rluc_Bb gene was Phusion-PCR amplified using B31 A3 genomic DNA and primer pair 1730 and 1731. The PCR fragments were ligated together by combining Gibson Assembly® Master Mix (NEB) and 0.16 pmol of each PCR fragment and incubating the reaction at 50°C for 1 h. One microliter of assembled product (flaBp-rluc_Bb) was Phusion-PCR amplified using primers 1730 and 1733 and the DNA fragment gel extracted using the QIAquick Gel Extraction kit (Qiagen), and cloned into pCR-Blunt using the Zero Blunt PCR cloning kit (Invitrogen) according to the manufacturer's instructions. The sequence of the flaBp-rluc_Bb cassette was verified by Sanger sequencing.

The flaBp-rluc_Bb cassette was Phusion-PCR amplified from the pCR-Blunt flaBp-rluc_Bb plasmid using primer pair: 1850 and 1910, introducing BamHI and KpnI restriction sites. B. burgdorferi shuttle vectors containing the promoterless B. burgdorferi optimized Photinus pyralis luciferase gene (fluc_Bb), flaBp-fluc_Bb, ospAp-fluc_Bb, or ospCp-fluc_Bb (Blevins et al., 2007; Adams et al., 2017) were digested with BamHI and KpnI high fidelity enzymes (NEB), gel extracted using the QIAquick Gel Extraction kit (Qiagen), and ligated to the BamHI/KpnI-digested flaBp-rluc_Bb cassette using T4 DNA ligase (NEB), generating plasmids pCFA701, pCFA801, pCFA802, and pCFA803. All plasmid constructs were confirmed by PCR, restriction digest, and Sanger sequencing.

B. burgdorferi clones were grown to logarithmic phase (3–7 × 10⁷ spirochetes/ml) or stationary phase (1–1.2 × 10⁸ spirochetes/ml) in 15 ml of BSKII medium and pelleted at 3,210 × g for 10 min. Cells were washed with phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) and resuspended in 300 μl of PBS. Eighty microliters of each sample was used to measure the optical density at 600 nm (OD₆₀₀) using a BioTek Synergy 4. This resulted in an average OD₆₀₀ value of ~0.25 for logarithmic phase spirochetes and ~0.36 for stationary phase spirochetes. One hundred microliters of each sample was loaded into a black, solid bottom 96-well plate (Corning) and combined with 700 μM D-luciferin (Regis) in PBS or 3.5 mM water soluble native coelenterazine (NanoLight Technology) in PBS. For samples containing coelenterazine, one well was left empty, in all directions around each sample, to decrease signal overlap between samples. For determining B burgdorferi Photinus luciferase (Fluc_Bb) and Renilla luciferase (Rluc_Bb) sensitivity, spirochetes containing pCFA801 were grown to logarithmic phase in 15 ml of BSKII medium, cell density determined using a Petroff Hauser counting chamber, washed with PBS, and resuspended in PBS to a density of 2 × 10⁶ cells/μl. Samples were serial diluted 10-fold and 100 μl of each dilution was loaded into a black, solid bottom 96-well plate (Corning) and combined with 700 μM D-luciferin or 3.5 mM coelenterazine. The relative luciferase units (RLUs) for Fluc_Bb and Rluc_Bb were determined by measuring photon emission in each well for 1 s, 10 times using the EnVision 2104 Multilabel Reader (PerkinElmer), following the addition of luciferin or coelenterazine substrate, respectively. Background relative Fluc_Bb or Rluc_Bb units, the average RLUs of the PBS control for either substrate, was subtracted from all experimental measurements, as appropriate. Background-subtracted relative Fluc_Bb units were then normalized to the OD₆₀₀ value or 10⁸ background-subtracted relative Rluc_Bb units of the same sample, when applicable (e.g., 4 × 10⁴ Fluc_Bb units/0.1 OD₆₀₀ value = 4 × 10⁵ relative Fluc_Bb units/OD₆₀₀; 4 × 10⁴ Fluc_Bb units/0.06 10⁸ Rluc_Bb units = 6.4 × 10⁵ relative Fluc_Bb units/10⁸ Rluc_Bb units). The limit of detection (LoD) and quantification (LoQ) for Fluc_Bb and Rluc_Bb were established as the average RLUs for PBS alone plus 3 or 10 standard deviations, respectively. All experiments were conducted in biological triplicate.

The University of Central Florida is accredited by the International Association for Assessment and Accreditation of Laboratory Animal Care. Protocols for all animal experiments were prepared according to the guidelines of the National Institutes of Health and were reviewed and approved by the University of Central Florida Institutional Animal Care and Use Committee.

One week prior to inoculation, and throughout the duration of the study, mice were treated with 5 mg/ml streptomycin and 1 mg/ml Equal® sweetener in their water to maintain selection for the luciferase plasmid in the B. burgdorferi clones. Using B. burgdorferi carrying pJSB175, pCFA701, pCFA801, pCFA802, or pCFA803, groups of two 6–8 week old female C3H/HeN mice (Envigo) per clone were inoculated with 1 × 10⁵ spirochetes per mouse 80% intraperitoneally and 20% subcutaneously. The inoculum doses were verified by colony forming unit (CFU) counts in solid BSKII medium. All inoculum were PCR verified to contain the endogenous B. burgdorferi plasmids of the parent clone as previously described (Elias et al., 2002; Jewett et al., 2007). Three weeks post inoculation mouse infection was confirmed by positive seroreactivity against B. burgdorferi protein lysate as previously described (Schwan et al., 1989; Jewett et al., 2007). Groups of approximately 200 naïve Ixodes scapularis larvae each (Centers for Disease Control, BEI resources) were fed to repletion on the B. burgdorferi infected mice (Jewett et al., 2009). Mice were further confirmed for infection by reisolation of spirochetes from bladder and joint tissues, as described (Showman et al., 2016). Larvae were analyzed for infection (Grimm et al., 2005; Jewett et al., 2009). Briefly, ticks were individually surface sterilized by sequential washes in 100 μl of 3% H₂O₂, 70% ethanol, and sterile H₂O. Subsets of larvae were analyzed for infection by reisolation of spirochetes in BSKII medium containing RPA cocktail (60 μM rifampicin, 110 μM phosphomycin, and 2.7 μM amphotericin B), immediately post feeding to repletion. Approximately 2 weeks following feeding, additional subsets of larvae were crushed and plated in solid BSKII containing RPA cocktail and 50 μg/ml streptomycin to determine CFU counts/larva. The remaining larvae were maintained and allowed to molt into nymphs. Two groups of 10–18 infected nymphs per B. burgdorferi clone were fed to repletion on naïve 6–8 week old female C3H/HeN mice (Envigo). These mice were treated with 5 mg/ml streptomycin and 1 mg/ml Equal® sweetener in their water 1 week prior to the feeding, to help sustain the luciferase plasmids in B. burgdorferi within the feeding nymphs. Throughout the duration of the study, ticks were stored in glass desiccation jars containing saturated potassium sulfate for to maintain appropriate humidity.

Approximately 2 weeks post feeding to repletion triplicate groups of 24 fed larvae or 8 fed nymphs per B. burgdorferi clone were crushed with a sterile pestle in 250 μl of PBS to generate tick extracts. For tick extracts, which were also plated for CFU counts, the ticks were first surface sterilized as described above, with a final wash in sterile PBS instead of H₂O. Tick debris was allowed to settle and 100 μl of sample was removed and incubated with 700 μM D-luciferin (Regis) in PBS or 3.5 mM water soluble native coelenterazine (NanoLight Technology) in PBS. RLUs were measured as described for in vitro grown spirochetes. The limit of quantification (LoQ) for Fluc_Bb was established as the average relative Fluc_Bb units for PBS alone plus 10 standard deviations. The LoQ for Rluc_Bb was established as the average relative Rluc_Bb units for infected tick extracts with spirochetes containing pJSB175, which lacks the rluc_Bb gene, plus 10 standard deviations. Samples with relative Fluc_Bb units below the LoQ were given a value of zero; whereas, samples with relative Rluc_Bb units below the LoQ were removed from the analysis. Relative Fluc_Bb units were normalized to 10⁸ relative Rluc_Bb units for each sample. One microliter of each fed nymph extract was also plated for CFUs in solid BSKII containing RPA cocktail and 50 μg/ml streptomycin.

GraphPad Prism version 7.02 was used for all statistical analyses. One-way ANOVA was used for analysis of all luciferase assays. For statistical comparison of the relative Fluc_Bb units normalized to OD₆₀₀ or 10⁸ relative Rluc_Bb units, which had an extremely wide distribution (~10¹–10⁷), all values were first square root transformed prior to statistical analysis. Following ANOVA, all samples were compared to the B. burgdorferi clones carrying the promoterless fluc_Bb control plasmid pJSB161 or pCFA701 using Dunnett's multiple comparison test. To compare two groups (i.e., the same clone in logarithmic versus stationary phase) following ANOVA, Bonferroni's multiple comparison test was applied to determine significance. For association analysis, Pearson correlation coefficient (r) was determined. p ≤ 0.05 was considered statistically significant for all statistical tests.

The B. burgdorferi shuttle vector pJSB161 (Blevins et al., 2007) contains a promoterless B. burgdorferi codon-optimized Photinus pyralis luciferase gene (fluc_Bb) with a BlgII restriction site upstream of a ribosome binding site (RBS) for fluc_Bb (Figure 1A). This reporter plasmid allows a cloned promoter of interest to be analyzed for activity in a strand-specific manner via a bioluminescence detection method (Blevins et al., 2007; Skare et al., 2016; Adams et al., 2017). However, this approach does not allow for quantitative comparative analysis of promoter activity in different environments or between multiple promoters in the same environment due to the lack of an endogenous means to control for spirochete number across samples and conditions. Therefore to improve upon this technique for quantitative applications, we engineered a dual luciferase reporter system to constitutively express Renilla reniformis luciferase (rluc) (Lorenz et al., 1991), while maintaining fluc_Bb for quantifying the activity of a promoter of interest. Codon usage in B. burgdorferi is biased (Fraser et al., 1997; Nakamura et al., 2000), as the A/T nucleotide frequency is at 71.8% across the genome (Fraser et al., 1997; Adams et al., 2017). Codon optimization has been shown to improve production and activity of non-B. burgdorferi proteins expressed in B. burgdorferi (Blevins et al., 2007; Hayes et al., 2010). Therefore to prevent rare codons interfering with the Renilla luciferase reporter, the OptimumGene™ algorithm (GenScript) was used to refine the codon adaption index (CAI) of rluc (Lorenz et al., 1991) for B. burgdorferi from 0.64 to 0.85 (where a CAI value of 1.0 indicates the highest proportion of the most abundant codons) and synthesized (GenScript). This codon-optimized rluc gene (rluc_Bb) (Genebank accession number MF043582) was cloned into pJSB161 (Blevins et al., 2007), for use in the dual luciferase reporter system under control of the constitutive promoter flaBp and corresponding ribosome binding site, generating pCFA701 (Figure 1B).

B. burgdorferi survival in the tick vector is essential for maintenance of the pathogen in its enzootic cycle. The spirochete is known to change its transcriptional profile at different stages of tick colonization including: acquisition, persistence during the molt, and transmission to the mammalian host (Iyer et al., 2015; Caimano et al., 2016). Because of our interest in applying the dual luciferase reporter system to quantitative analysis of B. burgdorferi promoter activities in the tick, we selected three well characterized promoters with distinct patterns of expression in the tick environment for proof of principle analysis. The flagellar protein promoter, flaBp, is constitutively active (Ge et al., 1997). The promoter for outer surface protein A (ospAp) is active during in vitro culture and in the tick during acquisition and persistence. In contrast, the promoter for outer surface protein C (ospCp) is active in the feeding tick during transmission and the mammalian host during the early stages of infection (Schwan et al., 1995; Schwan and Piesman, 2000; Schwan, 2003; Srivastava and de Silva, 2008). The flaBp-rluc_Bb cassette was cloned into three previously constructed plasmids, each containing one of these promoters driving the expression of fluc_Bb (Blevins et al., 2007; Adams et al., 2017), generating plasmids pCFA801, pCFA802, and pCFA803, respectively (Figure 1B). Spirochetes carrying pCFA701, pCFA801, pCFA802, or pCFA803 had no observed in vitro growth defect in BSKII medium compared to the wild type or B. burgdorferi carrying flaBp-fluc_Bb alone (pJSB175) (Blevins et al., 2007, Figure 1C).

Photinus pyralis luciferase (Fluc) and Renilla reniformis luciferase (Rluc) are compatible for a dual reporter due to the specificity of each enzyme for distinct substrates (Bhaumik and Gambhir, 2002; McNabb et al., 2005). Therefore, we verified the selectivity of the Fluc_Bb and Rluc_Bb enzymes to recognize luciferin and coelenterazine, respectively. Based on our previous work using the flaBp-fluc_Bb reporter (Adams et al., 2017), we performed these analyses with approximately 3 × 10⁸ spirochetes harvested during log phase growth. As expected, the negative control, spirochetes not expressing fluc_Bb or rluc_Bb (+pJSB161), demonstrated no significant relative luciferase units for either substrate compared to wild type. Spirochetes expressing fluc_Bb alone (+pJSB175) demonstrated robust activity when incubated with luciferin, but no significant activity above the background of B. burgdorferi carrying pJSB161 when exposed to coelenterazine (Figure 2A). Conversely, spirochetes expressing rluc_Bb alone (+pCFA701) demonstrated strong activity when incubated with coelenterazine, but no significant activity above the negative control background when exposed to luciferin (Figure 2A). Spirochetes which express both fluc_Bb and rluc_Bb (+pCFA801) demonstrated significant relative luciferase units compared to spirochetes containing pJSB161 for both luciferin and coelenterazine. The background relative luciferase units for wild type and negative control spirochetes exposed to coelenterazine were found to be approximately 10-fold higher than those of the same spirochetes incubated with luciferin. Collectively, these data validated the ability of the codon-optimized Rluc_Bb enzyme to effectively oxidize coelenterazine and confirmed the specificity of the Fluc_Bb and Rluc_Bb enzymes for their respective substrates.

The utility of the dual luciferase reporter system not only depends on the substrate specificity of Fluc_Bb and Rluc_Bb, but also the sensitivity of detecting and quantifying spirochetes expressing rluc_Bb. The limit of detection (LoD) and limit of quantification (LoQ) were established as the number of spirochetes required to achieve relative Rluc_Bb units greater than that of phosphate-buffered saline (PBS) alone plus three standard deviations and 10 standard deviations, respectively. Analysis of triplicate samples of 10-fold serially diluted spirochetes, 2 × 10⁸ to 2 × 10⁰, harvested during log phase growth and incubated with coelenterazine, demonstrated 2 × 10³ spirochetes to be the lowest detectable number of flaBp-rluc_Bb expressing spirochetes in the assay (Figure 2B). However, the LoQ fell between 2 × 10³ and 2 × 10⁴ spirochetes. Saturation of the bioluminescence signal was never reached under the conditions examined, with a linear increase in relative Rluc_Bb units from 2 × 10³ to 2 × 10⁸ spirochetes (y = 0.0404x; R² = 0.9997). Extrapolating from this linear equation, the LoQ was calculated to be 4.8 × 10³ spirochetes. These data indicate that a minimum of ~1 × 10⁴ flaBp-rluc_Bb expressing spirochetes are needed to achieve quantifiable relative Rluc_Bb units in the assay. Similar to what has been reported previously (Hyde et al., 2011), 2 × 10³ spirochetes was also found to be the lowest detectable number of flaBp-fluc_Bb expressing spirochetes (data not shown).

Previously, we reported quantification of in vitro active B. burgdorferi promoters by normalizing relative luciferase units (RLUs) from fluc_Bb expressing cells to the optical density of the bacterial sample measured at 600 nm (OD₆₀₀) (Adams et al., 2017). In this manner, the OD₆₀₀ measurement reflects the number of spirochetes in the sample allowing normalization of RLUs across samples and assay conditions. To establish the flaBp-rluc_Bb reporter as an effective alternative for OD₆₀₀ readings in our assay, first, relative Rluc_Bb units were measured for all rluc_Bb-expressing B. burgdorferi clones and normalized to the number of spirochetes in the assay by OD₆₀₀ (Figure 3A). All clones demonstrated consistent, robust relative Rluc_Bb units, ranging from 5 × 10⁷ to 1.68 × 10⁸. There was no significant difference among clones except for B. burgdorferi carrying pCFA802, which demonstrated higher relative Rluc_Bbunits/OD₆₀₀ compared to all other clones as well as a difference between logarithmic and stationary phase growth. The same rluc_Bb-expressing clones, were also incubated with luciferin and relative Fluc_Bb units were determined by normalizing to OD₆₀₀ (Figure 3B). All fluc_Bb promoter fusions displayed the expected relative Fluc_Bb units/OD₆₀₀, given the known expression patterns of their corresponding mRNA during logarithmic and stationary phase growth (Arnold et al., 2016). Both the flaB (+pCFA801) and ospA (+pCFA802) promoters demonstrated significant activity above the promoterless fluc_Bb control (+pCFA701) for both logarithmic and stationary phase growth. The activity of the ospC promoter (+pCFA803) during logarithmic phase growth was no different than the promoterless fluc_Bb control (+pCFA701). Whereas, the ospC promoter activity underwent significant induction from logarithmic to stationary phase growth (Figure 3B). Normalization of the relative Fluc_Bb units to 10⁸ relative Rluc_Bb units for each clone demonstrated no difference in the trend of the data resulting from this method of analysis compared to the data resulting from Fluc_Bb units normalized to OD₆₀₀ (Figure 3B). Together these findings establish flaBp-rluc_Bb as an effective constitutive control reporter, whose quantitation is reflective of spirochete number and is a robust means to normalize data obtained from fluc_Bb promoter fusions using the dual luciferase reporter system.

Having established the dual luciferase reporter system for use with in vitro grown spirochetes, we examined the efficacy of the reporter system for measuring B. burgdorferi promoter activities in the tick vector. Naïve Ixodes scapularis larval ticks were infected with B. burgdorferi carrying the dual luciferase reporter plasmids or flaBp-fluc_Bb, lacking rluc_Bb (+pJSB175) by feeding on groups of mice infected with the reporter clones via needle inoculation. Immediately following feeding to repletion, the percent of infected larvae per experimental group was determined by spirochete reisolation in BSKII medium. This analysis revealed that 60–90% of each experimental group of larvae successfully acquired B. burgdorferi upon feeding on infected mice. As an additional means to determine the percentage of infected larvae and to quantitate the number of spirochetes per tick, individual fed larvae were crushed and plated in solid medium for colony forming units (CFUs). Similar to the spirochete reisolation analysis, the groups of fed larvae were found by CFU analysis to be 66–100% infected. Moreover, although a broad range of spirochetes per tick was detected, there was no statistical difference between the average spirochete load per tick for each of the B. burgdorferi clones (Figure 4). These data suggest that all of the clones were able to colonize the ticks with the same efficiency.

Based on our quantitation of the average number of spirochetes per tick (Figure 4), we estimated that pools of 24 fed larvae would equate to approximately 10⁴ spirochetes per sample, suggesting that the Rluc_Bb activity would be quantifiable by our assay (Figure 2B). Therefore, 2 weeks following the blood meal, 24 fed larvae per experimental group, in triplicate, were crushed in PBS and relative Fluc_Bb and Rluc_Bb units measured using the luciferin and coelenterazine substrates, respectively (Table 2). The remaining fed larvae were reserved and allowed to molt into nymphs. The unfed, infected nymphs were then fed to repletion on naïve mice. Approximately 2 weeks post feeding, groups of eight fed nymphs were crushed in PBS, in triplicate, and assessed for relative Fluc_Bb and Rluc_Bb units (Table 2). Under the assumption that the spirochete load per fed nymph is increased approximately 10-fold compared to that of fed larvae (Jewett et al., 2007, 2009), we estimated the average spirochete load per fed nymph to be approximately 10⁴. Therefore, a pool of eight fed nymphs was estimated to equate to approximately 8 × 10⁴ spirochetes, which is above both the LoD and LoQ of the in vitro assay (Figure 2B). The actual LoQ for the in vivo tick assay was established using the average Rluc_Bb units plus 10 standard deviations for tick extracts from fed ticks infected with B. burgdorferi lacking rluc_Bb but expressing flaBp-fluc_Bb, (+pJSB175), rather than PBS alone. This is due to the observation that this tick extract negative control resulted in lower background relative Rluc_Bb units compared to PBS alone (Table 2). In contrast, there was no observed difference in the background relative Fluc_Bb units between PBS and the tick samples containing B. burgdorferi with a promoterless fluc_Bb and expressing flaBp-rluc_Bb (+pCFA701) in the luciferin assay. Therefore, the LoQ for Fluc_Bb in the in vivo tick assay was determined using the average relative Fluc_Bb units for PBS plus 10 standard deviations. Samples that fell below the LoQ threshold for either luciferase enzyme were considered no different than background (Table 2). As expected, we detected quantifiable relative Rluc_Bb units for all fed larvae samples, and all but two fed nymph samples (Table 2), indicating that sufficient spirochetes were present in the samples for the assay. In the pools of fed larvae only samples containing B. burgdorferi carrying flaBp-fluc_Bb (+pCFA801) demonstrated quantifiable relative Fluc_Bb units. The activities of ospAp and ospCp were below the LoQ for Fluc_Bb (Table 2). In contrast, all three promoters produced quantifiable relative Fluc_Bb units in the fed nymphs. Although one of the extracts from the fed nymphs infected with B. burgdorferi carrying ospCp-fluc_Bb (+pCFA803) did not result in quantifiable relative Fluc_Bb units, this sample also failed to achieve quantifiable relative Rluc_Bb units (Table 2), indicating that the number of spirochetes in the sample was insufficient for the assay. The promoter activities of the spirochetes in the fed nymph samples were analyzed by subtracting the average relative Fluc_Bb units of PBS from the relative Fluc_Bb units of each sample and the average relative Rluc_Bbunits of the infected tick extracts containing the negative control plasmid (+pJSB175) from the relative Rluc_Bb units of each sample. Background-subtracted Fluc_Bb units were then normalized to the respective background-subtracted relative Rluc_Bb units, for all quantifiable values. The Rluc_Bb-normalized promoter activities reflected the expected corresponding B. burgdorferi transcript expression pattern during the fed nymph life stage (Figure 5A, Iyer et al., 2015).

As an additional means to validate the method as well as to demonstrate that relative Rluc_Bb units are directly reflective of spirochete numbers in the infected tick samples, a portion of each sample from the fed infected nymphs used for Rluc_Bb and Fluc_Bb quantitation (Table 2, Figure 5A), was plated in solid BSKII medium for determination of B. burgdorferi CFUs. The average CFUs per 100 μl of tick extract, the same volume used for the dual luciferase assay, across all clones, was found to be 3.72 × 10⁵ spirochetes. These data support our rationalization for the use of 8 fed nymphs in the assay. Raw relative Rluc_Bb units (Table 2) for these samples plotted against their corresponding CFU counts demonstrated a significant positive correlation (Figure 5B). Furthermore, this analysis indicated that 1.2 × 10³ spirochetes are sufficient to generate relative Rluc_Bb units above the LoQ for the in vivo tick assay, which is similar to the sensitivity we observed for the in vitro assay. Collectively, we have described a valuable new method to determine the activity of B. burgdorferi promoters of interest under in vitro growth conditions and in infected ticks. This is the first application of a dual reporter system for B. burgdorferi and, to the best of our knowledge, the first quantification of spirochete promoter activities in the tick vector.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.