Evaluation of Immunocompetent Mouse Models for Borrelia miyamotoi Infection

Borrelia miyamotoi is a causative agent of hard tick relapsing fever, was first identified in the early 1990s, and was characterized as a human pathogen in 2011. Unlike other relapsing fever Borrelia species, B. miyamotoi spread by means of Ixodes ticks. ABSTRACT Borrelia miyamotoi is a relapsing fever spirochete that is harbored by Ixodes spp. ticks and is virtually uncharacterized, compared to other relapsing fever Borrelia vectored by Ornithodoros spp. ticks. There is not an immunocompetent mouse model for studying B. miyamotoi infection in vivo or for transmission in the vector-host cycle. Our goal was to evaluate B. miyamotoi infections in multiple mouse breeds/strains as a prelude to the ascertainment of the best experimental infection model. Two B. miyamotoi strains, namely, LB-2001 and CT13-2396, as well as three mouse models, namely, CD-1, C3H/HeJ, and BALB/c, were evaluated. We were unable to observe B. miyamotoi LB-2001 spirochetes in the blood via darkfield microscopy or to detect DNA via real-time PCR post needle inoculation in the CD-1 and C3H/HeJ mice. However, LB-2001 DNA was detected via real-time PCR in the blood of the BALB/c mice after needle inoculation, although spirochetes were not observed via microscopy. CD-1, C3H/HeJ, and BALB/c mice generated an antibody response to B. miyamotoi LB-2001 following needle inoculation, but established infections were not detected, and the I. scapularis larvae failed to acquire spirochetes from the exposed CD-1 mice. In contrast, B. miyamotoi CT13-2396 was visualized in the blood of the CD-1 and C3H/HeJ mice via darkfield microscopy and detected by real-time PCR post needle inoculation. Both mouse strains seroconverted. However, no established infection was detected in the mouse organs, and the I. scapularis larvae failed to acquire Borrelia after feeding on CT13-2396 exposed CD-1 or C3H/HeJ mice. These findings underscore the challenges in establishing an experimental B. miyamotoi infection model in immunocompetent laboratory mice. IMPORTANCE Borrelia miyamotoi is a causative agent of hard tick relapsing fever, was first identified in the early 1990s, and was characterized as a human pathogen in 2011. Unlike other relapsing fever Borrelia species, B. miyamotoi spread by means of Ixodes ticks. The relatively recent recognition of this human pathogen means that B. miyamotoi is virtually uncharacterized, compared to other Borrelia species. Currently there is no standard mouse-tick model with which to study the interactions of the pathogen within its vector and hosts. We evaluated two B. miyamotoi isolates and three immunocompetent mouse models to identify an appropriate model with which to study tick-host-pathogen interactions. With the increased prevalence of human exposure to Ixodes ticks, having an appropriate model with which to study B. miyamotoi will be critical for the future development of diagnostics and intervention strategies.

engorgement on SCID mice were infected; however, transstadial transmission was poor, with only approximately 20 to 30% of the molted nymphs and adults maintaining infection (41). I. scapularis that retained B. miyamotoi through the molts to adulthood were capable of transovarial transmission, which is not observed among Ixodes ticks harboring B. burgdorferi (42). Additional studies support this finding with field-collected Ixodes larvae being capable of transmitting B. miyamotoi to P. leucopus and NMRI mice (12,43).
Ornithodoros ticks that transmit STRF spirochetes are rapid feeders, being able to complete a blood meal within approximately 60 min. Additionally, these spirochetes reach high densities in the blood (approximately 10 6 to 10 7 cells/mL), and they are detectable via microscopy (44)(45)(46). To study STRF in the murine-tick model, mice are needle inoculated with spirochetes, and ticks are placed on the mice the following day and allowed to feed to repletion (22,(46)(47)(48). Alternatively, Ixodes ticks that transmit Lyme disease Borrelia (LDB) are slow feeders, taking 4 to 7 days to complete a blood meal. LDB do not reach high blood densities; rather, they disseminate throughout the mammalian host and are not detectable via microscopy (49). B. burgdorferi is studied in a mouse/tick model by needle inoculating mice with spirochetes and then feeding ticks to repletion on the mice at 14 to 28 dpi (50). Since B. miyamotoi is a relapsing fever spirochete that is transmitted by Ixodes ticks, we used a hybrid approach of LDB and STRF mouse models as a prelude to the development of an appropriate murine-tick infection model. We used two North American B. miyamotoi strains, namely, LB-2001 and CT13-2396, both with published genomes and vectored by I. scapularis, along with three immunocompetent mouse models, namely, CD-1, C3H/HeJ, and BALB/c. We evaluated the infectivity of both of the B. miyamotoi strains in the CD-1 and C3H/HeJ mice, and we evaluated that of B. miyamotoi LB-2001 in the BALB/c mice. We assessed acquisition and transmission by I. scapularis ticks from the CD-1 mice (inoculated with either LB-2001 or CT13-2396) and C3H/HeJ mice (inoculated with CT13-2396). Identifying an appropriate immunocompetent mouse model for B. miyamotoi is critical for future investigations into B. miyamotoi infection dynamics, including (i) spirochete localization and migration in Ixodes ticks, (ii) vector and reservoir host competency, and (iii) in vivo antigenic variation by gene conversion.

RESULTS
Experiments with B. miyamotoi strain LB-2001. (i) Assessment of B. miyamotoi LB-2001 acquisition and transmission by I. scapularis on CD-1 mice. Our lab has previously shown that CD-1 mice that are needle inoculated with LB-2001 elicit a robust humoral response against B. miyamotoi antigens and that spirochetes could occasionally be observed in blood smears and detected in organs via PCR (51,52). Based on our findings and on those of other studies conducted on B. miyamotoi and STRF spirochetes, we attempted to generate infected ticks using CD-1 mice inoculated with LB-2001 (40,41,48,51). The experimental design is illustrated in a flow chart in Fig. 1, and it is summarized in Table 1. Prior data indicated that spirochetes were detectable at 2 to 3 dpi; therefore, we placed ticks at 2 days post inoculation so as to give them time to attach before the spirochetes were detectable ( Fig. 1A and B) (40,41,51). A real-time PCR analysis was run on individual larvae and on pools of 10 fed larvae to ascertain the levels of B. miyamotoi LB-2001 DNA (Fig. 1D). The remaining larvae were allowed to molt to nymphs (Fig. 1E). All of the individual samples, pooled larval samples, and molted nymphs were real-time PCR negative, indicating that the larvae did not acquire B. miyamotoi from feeding, which suggests that the spirochetes were cleared from the mice via an immune response ( Table 2).
Organs and sera were collected from the mice at 6 dpi, after the completion of the larval feed, to assess the infections of the mice via serology and PCR of tissues (Fig. 1C). DNA extracted from organs were real-time PCR negative for B. miyamotoi (Table 3). Immunoblots using mouse sera that were collected at the feed termination showed IgM seroconversion, including a seroreactive band at approximately 25 kDa, which was likely Vsp1 (Fig. 2A). These results indicate that although B. miyamotoi LB-2001 was able to stimulate humoral immune responses from mice, there was no evidence for an established infection via organ colonization.
Furthermore, while the cohort of the molted flat nymphs also tested negative for B. miyamotoi via real-time PCR after the acquisition blood meal (Table 2), we proceeded with feeding on uninfected CD-1 mice to assess potential transmission, thereby ruling out the possibility of the B. miyamotoi concentrations being below the level of realtime PCR detection in the ticks (Fig. 1F). DNA was isolated from cohorts of nymphs 1 week after drop off and post molt ( Fig. 1H and I). Both cohorts were real-time PCR negative for B. miyamotoi DNA ( Table 2). The mice were terminally bled at 30 days after the nymphal drop off, and organs were collected (Fig. 1G). All of the tissues were real-  (Table 3). These results confirmed that the larval ticks failed to become infected during the acquisition feed and were therefore unable to transmit B. miyamotoi LB-2001 as nymphs to naive mice.
Since the mice could not be evaluated for active B. miyamotoi after inoculation at 2 dpi or during the tick feed, (via either microscopy or real-time PCR) we could not confirm whether the lack of acquisition by I. scapularis was because the bacteria were cleared from the mice prior to the establishment of an infection or because the spirochetes were not at a high enough density in the blood.
(ii) Assessment of B. miyamotoi LB-2001 infections via needle inoculation in C3H/HeJ and BALB/c mice. Since B. miyamotoi LB-2001 was not acquired by the I. scapularis larvae in the needle-inoculated, outbred CD-1 mice, we evaluated the ability of the strain to establish an infection in two inbred immunocompetent mouse strains, namely, C3H/HeJ and BALB/c, prior to the placement of the ticks. C3H/HeJ mice have a mutation in the gene for toll-like receptor 4 (Tlr4), which detects pathogen associated molecular patterns, particularly lipopolysaccharides. However, C3H/HeJ mice retain the functional genes for all of the other toll-like receptors, and they produce a fully functional immune response to both Lyme and relapsing fever Borrelia, although their initial bacterial burdens are usually higher (53,54). A flow chart illustrating the experimental design is shown in Fig. 3. Staggered tail nicks were performed on 2 mice/day at 4 dpi (C3H/HeJ) and at 10 dpi (BALB/c) from the LB-2001 needle-inoculated mice (n = 4 for each strain) to assess spirochetemia via microscopy and either conventional or real-time PCR (Table 4). Spirochetes were not observed in the blood of the mice from either strain; however, B. miyamotoi DNA was detectable via real-time PCR at 3 and 7 to 10 dpi from  the BALB/c mice, whereas all of the C3H/HeJ mice were PCR negative ( Table 4). The C3H/HeJ mice were only bled up to 4 dpi. Therefore, it is unknown whether B. miyamotoi DNA could have been detected in blood samples that were collected at later time points. Additionally, conventional PCR was used to detect B. miyamotoi DNA in the C3H/HeJ mice, and conventional PCR is less sensitive than real-time PCR, which was used for the BALB/c samples. The I. scapularis larvae were not fed on the BALB/c mice because we could not confirm the presence of live spirochetes. We could only confirm the presence of B. miyamotoi DNA.
To further investigate whether B. miyamotoi LB-2001 established infection in the needle-inoculated mice, the C3H/HeJ mice underwent terminal cardiac sticks at 32 dpi, and organs were collected for DNA extraction (Fig. 3B). All of the organs from the C3H/ HeJ mice were real-time PCR negative for B. miyamotoi DNA ( Table 5). The BALB/c needle-inoculated cohort was split into two groups, an early time point post inoculation group (group 1: mouse 1 and mouse 2) and a later time point post inoculation group (group 2: mouse 3 and mouse 4), and they were terminally bled and necropsied at 11 (Fig. 3D) and 42 dpi (Fig. 3E), respectively. Their organs were real-time PCR negative for B. miyamotoi DNA at both time points (Table 5).
The IgM serology of BALB/c group 1 at 11 dpi showed an early seroconversion to strain LB-2001, whereas the IgG serology of BALB/c group 2 at 42 dpi demonstrated a more robust antibody response that was indicative of infection ( Fig. 2B and C). The IgG serology of the C3H/HeJ mice at 32 dpi demonstrated seroconversion against fewer immunoblot bands. The sera of both mouse strains were reactive against a 25 kDa band, which was presumably the immunogenic Vsp1 ( Fig. 2C and D). The serology results suggest that the mice that were exposed to B. miyamotoi LB-2001 mounted an immune response that may have prevented this strain from reaching densities that were detectable in the blood or organs or may have cleared the spirochetes altogether. Overall, these results did not convince us to proceed with tick acquisition experiments with strain LB-2001. Therefore, we turned our focus to strain CT13-2396.
A flow chart illustrating the the experimental design is shown in Fig. 4. B. miyamotoi CT13-2396 was needle inoculated into 5 CD-1 and C3H/HeJ mice (Fig. 4A). Blood from tail nicks was collected each day from 3 mice from each group, and it was examined via darkfield microscopy and real-time PCR for evidence of B. miyamotoi infection at up to 15 dpi (Fig. 4B).  CD-1 mouse 3 as well as CD-1 mouse 4 were real-time PCR positive at 1 and 4 dpi as well as 2 dpi, respectively, but no spirochetes were observed via microscopy in these mice. CD-1 mouse 5 was real-time PCR negative at all dpi, but spirochetes were observed via microscopy at 6 dpi (Table 6). In contrast, B. miyamotoi was detected more frequently in the C3H/HeJ mice, with all 3 mice demonstrating positivity via both microscopy and real-time PCR at multiple time points ( Table 6). The mice were terminally bled at 42 dpi, and their organs were collected for real-time PCR (Fig. 4C). All six mice seroconverted; however, all of the organs that were collected were real-time PCR negative for B. miyamotoi DNA (Table 7; Fig. 5A). Contrary to what we saw with the LB-2001 infected mice, we did not see a seroreactive band at 25 kDa. Rather, there was a strongly reactive band at approximately 35 kDa, which was likely an immunogenic variable large protein (Vlp) (Fig. 5A). These results presented evidence of mouse infection. Therefore, we proceeded to tick acquisition and transmission experiments.
(ii) Assessment of B. miyamotoi CT13-2396 acquisition and transmission by I. scapularis on CD-1 and C3H/HeJ mice. Two mice of each group were needle inoculated with CT13-2396 and were simultaneously infested with I. scapularis larvae, which were allowed to feed to repletion (4 dpi) (Fig. 4D). We could not monitor these mice for B. miyamotoi infection via tail nicks while the ticks were feeding, but the mice that were used for the tick feedings were inoculated with the same dose and passage as were the mice in "Assessment of B. miyamotoi CT13-2396 infection via needle inoculation in CD-1 and C3H/HeJ mice", thereby providing a comparison for the progression of the infection. The mice were terminally bled at the end of the tick feed (4 dpi) (Fig. 4E), and IgM antibodies were detected against B. miyamotoi protein lysate, indicating spirochete exposure with a strong seroreactive band at approximately 35 kDa, but not one at 25 kDa (Table 7; Fig. 5B). However, all of the organs from both of the Negative Negative mouse models were real-time PCR negative for B. miyamotoi DNA (Table 7). Ticks were collected for real-time PCR at drop off, 2 weeks post drop, and post molt (Fig. 4F-H).
All of the larvae and molted nymphs from the acquisition feed were negative for B. miyamotoi DNA via real-time PCR (Table 8).  Although the molted nymphs were PCR negative, we proceeded with a feed on naive CD-1 and C3H/HeJ mice to assess the transmission of Borrelia and rule out the possibility that spirochetes were present but undetectable (Fig. 4I). As a control, we broke the transmission feed into 4 groups with 10 mice each, based on the mouse model on which the ticks fed for their acquisition blood meals (i.e., nymphs that fed on C3H/HeJ mice as larvae were fed on C3H/HeJ or CD-1 mice for the assessment of transmission [ Fig. 4I]). Nymphs were collected after the transmission feed on the day of drop off ( Fig. 4J) and after they had molted (Fig. 4K). All of the ticks at both time points were real-time PCR negative for B. miyamotoi DNA, regardless of the mouse model used for acquisition and transmission (Table 8).
5 mice in each transmission feed group were bled at 15 days post tick drop off (Fig. 4L), and their sera were negative for IgM antibodies against B. miyamotoi ( Table 7). All of the mice were sacrificed at 41 to 42 days after nymphal feeding, and their blood, sera, and organs were collected (Fig. 4M). The sera of the mice were negative for IgG antibodies against B. miyamotoi. Additionally, all of the mouse tissues that were collected post transmission feed were negative for B. miyamotoi DNA via realtime PCR. Regardless of the mouse model that the ticks initially fed on as larvae, all of the mice failed to become infected via nymphal tick bites. This outcome supports the lack of detectable B. miyamotoi DNA in ticks, indicating that the I. scapularis larvae failed to become infected during the acquisition feed and were therefore unable to transmit B. miyamotoi CT13-2396 to naive mice as nymphs. These results demonstrate that despite spirochetes likely being present in the mice as the larvae infested them, there was a yet unknown barrier to acquisition.
(iii) Reisolation of B. miyamotoi CT13-2396 from mice in coculture with ISE6 cells. In addition to detecting spirochetes via microscopy and real-time PCR, we also sought to confirm infection through the reisolation of B. miyamotoi CT13-2396 from needle-inoculated mice. Additionally, reisolation would allow us to assess the plasmid content and variable membrane protein expression, both of which are factors that influence the ability of the pathogen to infect a host and evade immune responses. Initial efforts to culture B. miyamotoi from mouse blood directly into MKP-F or BSK-R were unsuccessful. On day 7, C3H/HeJ mouse 8 had visible spirochetes in its blood (Table 6). Therefore, we cocultivated the mouse blood with ISE6 cells, and, after 17 days, we passaged the supernatant from the infected ISE6 cells into MKP-F and BSK-R. This procedure was successful in culturing a blood-borne reisolate of CT13-2396 in both media. In addition, using this method, we were able to reisolate B. miyamotoi from the blood of CD-1 mouse 3 on day 7, which was negative for B. miyamotoi via real-time PCR and microscopy, indicating an infection below the level of detection. We were unable to reisolate B. miyamotoi directly from the mouse blood into the MKP-F and BSK-R cultures, indicating the importance of the tick cells in the process of reisolation. Additionally, this result demonstrates that there can be infectious B. miyamotoi in the blood of mice that are below the level of detection via microscopy or real-time PCR but are capable of expansion in the right environment. Reisolated CT13-2396 maintained the same plasmid profile as the parent CT13-2396 isolate that was used for the needle inoculations (data not shown).

DISCUSSION
An immunocompetent experimental animal model is needed to understand how host immune responses affect the interactions between B. miyamotoi, the host, and the tick vector. Previous studies have shown that while immunocompromised mice can develop high levels of B. miyamotoi spirochetemia, immunocompetent mice clear the infection within 15 days (40,41,55). The aim of this study was to evaluate two B. miyamotoi strains with three immunocompetent mouse breed/strains to develop a mouse-tick-B. miyamotoi infection model.
Two Connecticut B. miyamotoi strains, namely, LB-2001 and CT13-2396, were used in this study, as they each have published genomes, and previous work has been performed using them (40,41,56,57). B. miyamotoi LB-2001 was identified in a field-collected I. scapularis tick and was maintained in SCID mice through intraperitoneal injection prior to isolation in MKP-F medium (39). B. miyamotoi CT13-2396 was cultured out of an I. scapularis nymph descendant from a lab-reared egg clutch of a field-collected female tick (56). Despite both strains coming from infected ticks in Connecticut, there are differences in their genome structures and in vitro culturing characteristics. Previous work has found that B. miyamotoi LB-2001 grows well in MKP-F (39), whereas we have observed inconsistencies in B. miyamotoi CT13-2396 growth in MKP-F. Alternatively, we have seen the consistent growth of B. miyamotoi CT13-2396 in BSK-R (58). These differences may be linked to their method of isolation and individual, distinct genomic rearrangements. Borrelia genomes consist of a chromosome and several linear and circular plasmids. While LDB have been shown to lose plasmids through prolonged in vitro passaging, STRF Borrelia retain their plasmids but are prone to rearrangements (48,59,60). Additionally, we have shown that B. miyamotoi LB-2001 retained its plasmids through prolonged in vitro cultivation and elicited an immune response from CD-1 mice (51).
Our initial goal was to generate infected I. scapularis ticks via feeding on needle-inoculated mice and to work toward a reproducible tick-mouse infection model. Prior data indicated that there were spirochetes in the blood of mice at 2 to 3 dpi, which was the reason why we proceeded with the larval feed (51). In the first pilot experiment, larvae that fed on B. miyamotoi LB-2001 inoculated CD-1 mice failed to acquire spirochetes. However, we could not determine whether the unsuccessful acquisition was due to a failure of the needle-inoculated mice to establish an infection or whether the spirochetes were cleared by the innate immune system. The CD-1 outbred mice that were used for the larval feed produced an IgM response against B. miyamotoi, indicating exposure, but the lack of organ colonization suggested that the mice cleared the spirochetes prior to an infection being established.
Therefore, we took a step back and evaluated LB-2001 infectivity in two inbred mouse strains, namely, BALB/c and C3H/HeJ, prior to tick feeding. We chose to use inbred mice because they are genetically homogenous and would therefore likely produce more consistent immune responses to B. miyamotoi than would outbred mice. We did not observe spirochetes in the blood of these mice, but we were able to detect B. miyamotoi DNA in the BALB/c mice past 2 dpi. However, unlike the BALB/c mice, the C3H/HeJ mice were only monitored out to 4 dpi, which may have been a limitation for our detection of spirochetes in these mice. Both the BALB/c and C3H/HeJ mice generated antibody responses to B. miyamotoi, but there was no evidence of organ colonization. Therefore, we did not feed larvae on either mouse strain, as we did not observe live bacteria in the blood. Combined with the results that we saw in the CD-1 mice that were inoculated with LB-2001, the data suggest that this strain has become attenuated or that the laboratory mice that were used were not optimal hosts for infection. The prolonged passaging of the original B. miyamotoi LB-2001 isolate in SCID mice (around 10 passages) with the lack of immune pressures may have led to attenuation or a loss of mechanisms for antigenic switching that are necessary for establishing or maintaining infections in immunocompetent hosts (39). This hypothesis is supported by immunoblot results, which show IgM and IgG seropositive bands primarily against a 25 kDa protein, likely Vsp1, which is known to be highly immunogenic. A lack of serotype switching would severely inhibit B. miyamotoi immune evasion. Previous work in our lab and in the labs of others has shown detectable B. miyamotoi LB-2001 in mice, contrary to what we observed in this study (40,51). These inconsistencies with LB-2001 could be linked to variability between and within mouse models or to the culture passage of LB-2001 used (passage of ,10 but .4). Additionally, Wagemakers et al. was able to observe spirochetes in C3H/HeN mice that were inoculated with 10-fold more LB-2001 spirochetes than we used, and those inoculations were performed via intraperitoneal injections, rather than being subcutaneous inoculations (40). Either of these factors could contribute to differences in observable B. miyamotoi LB-2001 in mice.
Needle inoculations of CT13-2396 into the CD-1 and C3H/HeJ mice produced better results, as spirochetes were observed in the blood, and DNA was detectable via realtime PCR at several dpi. Moreover, C3H/HeJ mouse 8 showed evidence of a relapse, as B. miyamotoi DNA was detectable in the blood from this mouse on days 1 and 7, with live spirochetes being observed on day 7 but not on day 4 ( Table 6). We were unable to detect the colonization of B. miyamotoi in the organs of any of the mice. However, all of the mice had a robust IgG serological response, suggesting a phenotype of nondissemination to organs or clearance from the organs prior to necropsy. It should be noted that LB-2001 and CT13-2396 have predominant serotypes that express Vsp1 and VlpC2, respectively, with these genes residing in the expression locus for Vmp, according to data from the GenBank databases (GCA_000445425.4, GCF_017748095.1, and GCA_001767415.1). Accordingly, our immunoblots indicated seroreactivity against these proteins when the mice were inoculated with the respective strain. Perhaps, the Vmp serotype variation between strains accounts for the phenotypic differences that we observed in vivo and the differences in vitro in their preferred culture media.
Since CT13-2396 spirochetes were observed in CD-1 and C3H/HeJ mouse blood ( Fig. 4A and B), we proceeded to tick feeding with a second cohort of needle inoculated mice to assess acquisition ( Fig. 4A and D). However, I. scapularis larvae that were fed on CT13-2396 needle inoculated mice failed to acquire and subsequently transmit spirochetes as nymphs to naive mice. The mice used in the acquisition feed were serologically positive to B. miyamotoi, confirming that they had been exposed to B. miyamotoi. However, there was no evidence for organ colonization via real-time PCR, and the mice could not be bled via tail nick to confirm an active infection during the larval feed. These findings align with our results with LB-2001 and suggest that sustained B. miyamotoi infections at relatively high densities may be necessary for Borrelia acquisition by feeding ticks. Lynn et al. were able to infect I. scapularis larvae and nymphs by feeding them on B. miyamotoi infected SCID mice, but the transstadial transmission to adulthood was low (25 to 30%) (41). This finding, coupled with our results, suggests that larvae, and potentially nymphs, are not ideal for the acquisition of B. miyamotoi via bloodmeal. Previous investigations have shown that B. miyamotoi has high transovarial transmission rates (12,61,62). Therefore, infection maintenance in ticks may not depend entirely on the acquisition of B. miyamotoi via bloodmeal engorgement from reservoir hosts. Lynn et al. found that there is a correlation between a high spirochete burden in the female I. scapularis and successful transovarial transmission (42). Field collections of Ixodes ticks have shown that infected larvae, nymphs, and adults persist in nature, but the primary route of infection is unknown (43,(62)(63)(64)(65). Different tick life stages may be more conducive to B. miyamotoi acquisition and maintenance due to the larger bloodmeal imbibed. Depending on Ixodes spp. and environmental factors, engorged adult female ticks oviposit 2 to 4.5 weeks after repletion (66). It is unknown how long it takes for B. miyamotoi to disseminate from the midgut to other tick tissues; however, other STRF Borrelia are known to migrate to the salivary glands within 7 to 14 days post feed (20,67). If this dissemination speed is similar with B. miyamotoi, then the pathogen would have time to escape the midgut and potentially infect eggs prior to oviposition.
We used a larger inoculum of B. miyamotoi (10 5 to 10 6 cells) than that which would be delivered by infected ticks to a host, which could generate an antibody response in the absence of spirochete proliferation in the mice. We chose this inoculum in an attempt to establish infections in mice. Future work evaluating immune responses to B. miyamotoi should include a control of dead spirochetes inoculated into mice to establish a baseline for antibody responses to B. miyamotoi antigens in the absence of live replicating bacteria. The 50% infectious dose (ID50) for various strains of B. miyamotoi should be a focus of future work.
Alternatively, laboratory mice, or at the least, the three models tested here, may not be competent experimental reservoir hosts for B. miyamotoi, although one strain of B. miyamotoi (FR64b) was isolated from the blood of a Japanese field mouse (Apodemus argenteus) (2). STRF Borrelia have various host competencies. Borrelia hermsii has been found in small rodents, including chipmunks and squirrels (24,68), whereas coyotes have been found to have high rates of seroconversion against B. turicatae, which has also been studied in dog models (25,26). B. miyamotoi DNA has been detected in nature in variety of species, including wild turkeys, voles, and other small rodents (2,28,31). It is possible that other animal models are better suited for these two North American B. miyamotoi strains.
Although we observed CT13-2396 in mouse blood, subsequent expansion in culture medium alone was unsuccessful. We have observed that B. miyamotoi does not grow well in culture medium when the inoculums are extremely dilute. Therefore, the low density of spirochetes in the blood could explain the growth deficiency in the medium. However, we successfully reisolated B. miyamotoi CT13-2396 from mouse blood via cocultivation with ISE6 cells. We have grown B. miyamotoi from culture on ISE6 cells and in cell free L15B-300 with 20% MKP-F. While B. miyamotoi grew well in the ISE6 coculture, the growth was poor in the cell-free media. ISE6 cells likely provided an important, but unknown, component(s) necessary for B. miyamotoi growth at low concentrations. Alternatively, since relapsing fever Borrelia are maintained in a sylvatic cycle between host and vector, B. miyamotoi may be primed for growth in the presence of tick cells while in the vertebrate host. The cocultivation of B. miyamotoi with tick cells for (i) growth expansion from low density sources, (ii) isolation from in vivo infections for the determination of serotype switching, (iii) the generation of clonal populations, and (iv) the improvement of the infection of mice via needle inoculation are the foci of current investigations.
In conclusion, while this study was by no means exhaustive to achieve the goal, we evaluated two B. miyamotoi strains in three immunocompetent mouse models and found that while these mice seroconverted after needle inoculation, an established infection in mice was undetermined. Furthermore, I. scapularis larvae failed to acquire either Borrelia strain, and this was confirmed by subsequent unsuccessful nymphal transmission to naive mice. This study showed that strain CT13-2396 spirochetes had a more consistent infection pattern than did strain LB-2001 and that C3H/HeJ appeared to be the better laboratory mouse strain, relative to CD-1. BALB/c mice also showed promise as a potential infection model and should be given consideration for future studies (58). Future work should evaluate other B. miyamotoi strains from Europe, Asia, or the Western U.S. in similar mouse models and should consider alternatives by which to obtain infected tick colonies, such as nymphal or adult tick acquisition feeds, tick immersion, and artificial membrane blood feeds.

MATERIALS AND METHODS
Borrelia strains, ticks, tick cell lines, and mice. The B. miyamotoi strains LB-2001 and CT13-2396 were grown in modified Kelly-Pettenkofer medium with 7% gelatin (MKP-F) and modified Barbour-Stoenner-Kelly medium (BSK-R), respectively (12,39,56,58). Cultures were initiated from frozen glycerol stocks and cultivated at 34°C in capped tubes until they reached the late log phase, approximately 10 6 to 10 7 cells/mL. All of the cultures used for the experiments were of a low-passage-number (,10 passages in medium). Prior to the mouse inoculations, the cultures were enumerated using Cellometer disposable counting chambers (Nexcelom, Lawrence, MA). I. scapularis larvae were purchased from the Oklahoma State University Tick Rearing Facility and were kept in desiccators at $90% humidity and 22°C. Female CD-1 outbred mice from Jackson Laboratories (Bar Harbor, ME) and inbred C3H/HeJ, and BALB/c mice from Charles River Laboratories (Wilmington, MA) that were between 6 and 8 weeks old were used for these studies. All of the experiments utilizing mice were approved and conducted in accordance with the guidelines and regulations established by the Division of Vector-Borne Diseases Institutional Animal Care and Use Committee (IACUC). ISE6 embryonic tick cells were grown to confluence in L15B media on 12-well poly-D-lysine coated plates (Corning, Kennebunk, ME) at 34°C (69,70).
B. miyamotoi inoculations in mice and reisolation via cocultivation with ISE6 cells. Table 1 summarizes the B. miyamotoi experimental infections in the mice. All of the mice were subcutaneously needle inoculated with 10 5 to 10 6 cells of low-passage-number B. miyamotoi in their respective culture media, except for two of the C3H/HeJ mice, which were infected via intraperitoneal injection. Mouse tail nicks were performed to monitor the blood for spirochetes. Briefly, the mice were anesthetized with isoflurane, and the ends of their tails were cleaned with 70% ethanol. Sterile scissors were used to cut the tip of the tail to collect blood. A drop of blood was placed on a slide and examined under darkfield microscopy at 200Â magnification on a Nikon Eclipse 80i microscope (Nikon, Melville, NY). An additional 5 mL of blood were mixed with 45 mL 1Â PBS and frozen at -20°C until analysis via conventional PCR or real-time PCR. The mice were terminally bled via cardiac stick, and their organs were collected and frozen at 280°C until DNA extraction. The sera were separated from the blood using either a serum separator tube (BD, Franklin Lakes, NJ) spun at 6000 Â g for 10 min or a microcentrifuge tube spun at 8000 Â g for 15 min. The sera and blood clots were stored at 220°C until analysis via immunoblot and real-time PCR, respectively.
B. miyamotoi CT13-2396 was reisolated from the blood of the mice at 7 dpi by inoculating 10 mL of blood in 1Â PBS (as stated above) onto confluent ISE6 cells in 12-well plates with 2 mL of L15B-300 with either 5% BSK-R and 15% MKP-F or 10% BSK-R and 10% MKP-F, and this was followed by incubation at 34°C with 5% CO 2 (70,71). 1 week after the inoculation medium was removed, and after spinning at 8000 Â g for 5 min, the supernatant was decanted, and the pellet was resuspended in fresh L15B-300 with 20% MKP-F and returned to the original well of ISE6 cells. 17 days after the initial inoculation, B. miyamotoi spirochetes were observed in the media from the ISE6 cells under darkfield microscopy. 200 mL of supernatant from the infected ISE6 cells were inoculated into 1 mL of BSK-R or MKP-F, grown as stated above, and designated passage 1. Once the cultures reached the late log phase of growth, frozen stocks were made, with a final concentration of 20% glycerol. An analysis of the plasmid profiles was performed as previously described (51,57). Additional culturing attempts were made, inoculating 10 mL of blood in 1Â PBS into 450 mL of either MKP-F or BSK-R.
I. scapularis feedings. I. scapularis larvae were fed on CD-1 mice that had been inoculated with B. miyamotoi LB-2001 or CT13-2396 as well as on C3H/HeJ mice that had been inoculated with CT13-2396 (Table 1). Approximately 100 larvae were placed on the mice either 2 days after the needle inoculation (CD-1 mice infected with LB-2001) or on the same day as the inoculation. Larvae were collected at multiple time points during and after feeding, and they were stored at 280°C for DNA extraction and realtime PCR analysis. Engorged larvae were stored in desiccators at 24°C and $90% humidity through their molt. After molting, a subset of ticks were collected for real-time PCR, and the rest were transferred to a 22°C incubator until the nymphal feed. The mice were terminally bled at the end of the larval feed.
After molting, B. miyamotoi exposed nymphs underwent transmission feeds on naive mice. 6 to 13 ticks per mouse were fed to repletion and stored at 22°C in desiccators with $90% humidity. Replete nymphs were collected at several time points post feeding. The mice that were fed on by nymphs exposed to B. miyamotoi CT13-2396 were bled via cheek punch 15 days after the completion of the tick feed. All of the mice were terminally bled at 30 to 42 days after the tick drop off (Table 1).
Immunoblot analysis of mouse sera. Mouse sera collected after the needle inoculation or tick feed were immunoblotted against B. miyamotoi whole-cell lysate. Whole-cell lysate from approximately 10 7 B. miyamotoi spirochetes was fractionated on Criterion TGX AnyKD gels (Bio-Rad Laboratories, Hercules, CA) and transferred to PVDF stacks (Thermo Fisher Scientific, Waltham, MA) using an iBlot 2 dry blotting system (Thermo Fisher Scientific, Waltham, MA). Membranes were blocked overnight at room temperature with SuperBlock blocking buffer in TBS (Thermo Fisher Scientific, Waltham, MA). The membranes were washed three times for 3 min each with 1Â Tris-buffered saline with 0.05% Tween 20 (TBS-T). Then, the membranes were incubated with the collected mouse sera at a 1:1,000 dilution for 1 h at room temperature. The secondary antibody incubation was with either KPL phosphatase labeled goat anti-mouse IgM (#15 days postinfection) or IgG (.15 days postinfection) antibodies (Seracare, Milford, MA) at a 1:5,000 dilution for 1 h at room temperature. The membranes were washed and then incubated with 1-step NBT/BCIP substrate solution (Thermo Fisher Scientific, Waltham, MA), washed with DI water, and allowed to dry.
DNA extraction from ticks, mouse blood, and mouse organs. Individual ticks were crushed with a Tenbroeck grinder (nymphs and adults) or disrupted with a dissection probe (larvae) and boiled in 50 mL of PBS. Another 200 mL of PBS were added to the sample, and DNA was extracted via phenol-chloroform as follows. An equal volume of phenol-chloroform-isoamyl alcohol (ratio of 25:24:1, Thermo Fisher Scientific, Waltham, MA), was added, and the sample was repeatedly inverted and spun at 16,250 Â g for 7 min at 4°C. The top aqueous layer was transferred to a clean tube, an equal volume of chloroformisoamyl alcohol (ratio of 24:1, Acros Organics, Waltham, MA) was added, and the sample was inverted and centrifuged as above. DNA was precipitated from the collected aqueous layer overnight in 0.2 volumes of 3 M sodium acetate (pH 5.2), GlycoBlue Coprecipitant (Thermo Fisher Scientific, Waltham, MA), and 3 volumes of 100% ethanol. The samples were spun at 16,250 Â g for 30 min at 4°C. The pellets were washed with 70% ethanol and spun again for 25 min at 4°C. The ethanol was decanted, and the pellets were air dried prior to their resuspension in water. The DNA concentration was determined using a Nanodrop One Spectrophotometer (Thermo Fisher Scientific, Waltham, MA).
Mouse hearts and spleens were thawed to room temperature, suspended in 200 mL of PBS, and disrupted by bead beating in a 220°C cassette with a mixer mill (Retsch, Verder Scientific, Newtown, PA) for 5 min at 30 Hz. The samples were centrifuged at 8,000 Â g for 10 min, and tissue lysate was transferred to a clean tube. Ears, bladders, and brains were cut into small pieces with scissors and resuspended in 200 mL of 1Â PBS. DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). The samples were eluted in water. The blood clots were thawed to room temperature and disrupted as described above. 100 mL of blood were aliquoted into 1Â PBS for a final volume of 200 mL and underwent DNA extraction using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany).
Conventional PCR and real-time PCR analysis. The primers and probes were synthesized by Integrated DNA Technologies (Coralville, IA) ( Table 9). Conventional PCR was performed on B. miyamotoi LB-2001-infected, C3H/HeJ tail-nicked blood samples. Frozen blood in PBS was thawed, and an aliquot was diluted 1:4 in water and boiled at 95°C for 5 min. The samples were spun briefly to pellet the cell debris, and 1 mL of supernatant was run in a PCR using Platinum SuperFi II Master Mix (Invitrogen, Waltham, MA). B. miyamotoi flaB and mouse actin primers were used at a final concentration of 0.5 mM, and the cycling conditions were 98°C for 30 s (initial denaturation), 98°C for 10 s (denaturation), 60°C for 10 s (annealing), 72°C for 30 s (extension), and 72°C for 5 min (final extension), with steps 2 to 4 repeated for 35 cycles. The amplicons were observed on a 1% Tris-Acetate-EDTA (TAE) with ethidium bromide precast gel (Bio-Rad Laboratories, Hercules, CA).
A 10 mL aliquot of all of the other tail-nicked blood samples was boiled as above and spun down. The supernatant was transferred to a clean tube with 5 mL of water, and it was then run on a real-time PCR, as described below. The tick and mouse DNA samples were evaluated for B. miyamotoi infection using real-time PCR with primers at a final concentration of 250 nM and probes at 150 nM. The presence of B. miyamotoi DNA was tested using B. miyamotoi flaB primers and probe, whereas the I. scapularis or mouse actin primers and probe were used as sample controls, depending on the sample DNA (Table 9). Assays were run using SsoAdvanced Universal Probe Supermix (Bio-Rad Laboratories, Hercules, CA), and the cycling conditions, executed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA), were 95°C for 3 min (initial denaturation), 95°C for 15 s, and 60°C for 15 s (extension and plate read), with the final two steps repeated for 40 cycles. 10 ng of DNA was used per reaction, except in cases of tail nick bleeds, in which 2 mL of the boiled supernatant were used. The mouse tail nick samples were run in duplicate with a cutoff Ct value of 40. The tick and mouse tissue DNA samples were run in triplicate with a cutoff Ct value of 35.