Abstract
As an alternative to oral prophylaxis for the prevention of tick transmission of Borrelia burgdorferi, we tested antibiotic cream prophylactic formulations in a murine model of spirochete infection. A 4% preparation of doxycycline cream afforded no protection, but a single application of 4% azithromycin cream was 100% protective when applied directly to the tick bite site at the time of tick removal. Indeed, the azithromycin cream was 100% effective when applied at up to 3 days after tick removal and protected 74% of mice exposed to tick bite when applied at up to 2 weeks after tick removal. Azithromycin cream was also protective when applied at a site distal to the tick bite site, suggesting that it was having a systemic effect in addition to a local transdermal effect. Mice that were protected from tick-transmitted infection did not seroconvert and did not infect larval ticks on xenodiagnosis. Azithromycin cream formulations appear to hold promise for Lyme disease prophylaxis.
INTRODUCTION
Lyme disease is the most commonly reported vector-borne disease in the United States, with >30,000 cases reported to CDC annually (1). In fact, since Lyme disease became reportable in 1992, the number of cases reported to CDC has increased dramatically despite attempts at the federal, state, and local level to prevent this epidemic (2). The Lyme disease spirochete, Borrelia burgdorferi, is transmitted principally by the nymphal stage of the blacklegged tick, Ixodes scapularis, from mid-May to mid-June along the Atlantic seaboard from northern Virginia to Maine, with a smaller focus in the North Central United States (3). Approximately 80 million people live in states in the eastern half of the United States that contain regions where Lyme disease is highly endemic.
The first line of defense against tick-borne disease is certainly education regarding the risk of disease and the personal protection measures that are available (4). Unfortunately, avoidance of tick-infested areas, wearing of protective clothing, and using repellents and frequent tick checks are practiced inconsistently by the public; overall, these strategies have not been shown to be effective in blunting the Lyme disease epidemic, except under carefully controlled circumstances (5, 6). Despite a plethora of tick control tools developed by researchers, including area-wide acaricides, host-targeted acaricides, landscape modification, and least-toxic biological agents (4), actual proof that tick control can lead to a reduction in the incidence of Lyme disease is limited to a few efforts at deer eradication on islands (7, 8, 9).
More-direct approaches to preventing Lyme disease are those targeting humans, including vaccines and prophylactic use of antibiotics for tick bite. A Lyme disease vaccine based on recombinant outer surface protein A (OspA) was tested and shown to be 76% effective (10). This vaccine was released onto the U.S. market in 1999. Despite high hopes for the use of this tool to prevent Lyme disease, it was withdrawn from the market in 2002, ostensibly for market-related reasons. A variety of scientific, market, and legal issues may have contributed to the vaccine's withdrawal (11, 12). Antibiotic prophylaxis for tick bite in regions of high Lyme disease endemicity may be cost-effective (13). In a large clinical trial, Nadelman et al. (14) found a single dose of 200 mg of oral doxycycline to be 87% effective in preventing erythema migrans during a 6-week period of observation. The confidence intervals around this observation, however, were quite large (25% to 98%). The frequency of use of oral antibiotic prophylaxis for tick bite in the United States is currently unknown.
In order to better understand the dynamics of antibiotic prophylaxis to prevent Lyme disease, various researchers have conducted numerous experiments using a murine model and tick-transmitted spirochetal infection. Antibiotic prophylaxis has been delivered to mice via oral gavage (15, 16, 17), an injectable slow release formulation (15, 16), or topical applications (18, 19, 20). In the current study, the efficacy of topical creams containing doxycycline was compared to that of creams containing azithromycin. For the efficacious formulation (azithromycin), the time when, post-tick feeding, the cream must be delivered for maximum benefit was determined. In addition, whether the action of the cream is solely transdermal or whether it includes systemic action against tick-transmitted spirochetes was evaluated.
MATERIALS AND METHODS
Tick and bacterial strains.
Nymphal I. scapularis ticks infected with the B31 strain of B. burgdorferi were produced as previously described (21). The colony originated from female ticks collected in New Jersey and Connecticut. Animals used in these experiments were female CD-1 mice, 4 weeks of age, purchased from Charles River Laboratories (Wilmington, MA). A single nymphal I. scapularis tick (>2 months post-larval feeding) was placed into a capsule on the back of each mouse and allowed to attach and feed as previously described (22). Nymphal ticks were allowed to feed for 72 h (near repletion), at which point they were removed from the host. Feeding to repletion generally takes 72 to 96 h for nymphal I. scapularis in our murine model (23). Animals were handled according to approved protocols on file with the Centers for Disease Control and Prevention, Division of Vector-Borne Diseases Animal Care and Use Committee Protocol numbers 10-007 and 13-002.
PCR analysis.
Nymphal ticks were tested for infection with B. burgdorferi by PCR as previously described (24). Briefly, after ticks were removed from the host, DNA was extracted and tested for the presence of B. burgdorferi using a primer set directed against the fliD gene of B. burgdorferi. The cutoff limit for this quantitative PCR (qPCR) test was 20 spirochete equivalents per sample. All mice fed upon by PCR-negative ticks were removed from the experiment. Similarly, exposed mice upon which nymphal ticks did not attach and feed were removed from the experiment.
Borrelia culturing.
Mice were tested for infection with B. burgdorferi as previously described (25). At 1 month post-nymphal tick removal, mice were sacrificed and ears, urinary bladder, and heart were cultured in Barbour-Stoenner-Kelly (BSK) medium. Cultures were examined by dark-field microscopy weekly for 4 weeks for the presence of live spirochetes. In addition to the culture experiments, a subset of mice was tested for serological reaction to B. burgdorferi. Sera from experimental mice were tested at a dilution of 1:500 for antibody to B. burgdorferi utilizing the Marblot strip test system (MarDX Diagnostic Inc., Carlsbad, CA) per the manufacturer's instructions with the following modification: alkaline phosphatase-labeled goat anti-mouse IgG plus IgM (H+L) (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was utilized as the detection antibody at a dilution of 1:2,500. Mice were considered reactive if there were ≥5 visible bands on the blot, per the manufacturer's specifications.
Xenodiagnosis.
To further confirm the infection status of mice treated with azithromycin cream, we exposed mice to infected nymphs for 72 h and then treated them with azithromycin or control cream immediately upon nymphal tick removal. At ca. 3 weeks after the nymphal feeding, xenodiagnostic larval ticks from our uninfected colony were allowed to feed on these test mice, as well as on naive controls. Larval xenodiagnostic ticks were tested for the presence of spirochetes by PCR at 10 days post-larval feeding as previously described (24).
Antibiotic cream.
Antibiotic creams were prepared with the assistance of a local pharmacy (Good Day Pharmacy, Loveland, CO). A 4% azithromycin cream was prepared by taking 3 tablets (250 mg each) and crushing them in 18.75 gm of Lipoderm cream (PCCA, Houston, TX); a trace amount of ethoxy diglycol was added to help form a paste. A control (nonantibiotic) cream was made the same way but without the azithromycin tablets added. A 4% doxycycline cream was prepared by adding 0.6 gm of doxycycline hyclate to 14.4 gm of anhydrous pluronic lecithin organogel (PLO; PCCA, Houston, TX). The preparation was made by wetting the doxycycline with trace amounts of dimethyl sulfoxide (DMSO) before mixing in syringes with anhydrous PLO. A nonantibiotic control cream was made the same way without the doxycycline added. Both cream preparations were delivered to the mouse by taking 50 μl of cream into a syringe and expressing the cream onto the designated spot on the skin where the tick capsule had been placed. Mice had been shaved to glue capsules onto the spot where ticks fed. The cream was then spread with the tip of the syringe. A total of 2 mg of the antibiotic was delivered to each of the treated mice. All mice were treated on only one occasion; no multiple treatments were performed in the course of these experiments.
Statistics.
The percent protection provided by antibiotic applications was calculated as previously described (17). Briefly, the formula [1 − Rt/Rc] × 100 was used to calculate percent protection where Rt = the proportion of the treatment group infected and Rc = the proportion of the control group infected. Fisher's exact test was used to measure statistical significance.
RESULTS
The first trial conducted was a direct comparison of the protection provided by the doxycycline cream to that provided by azithromycin cream (Table 1). Animals in this trial were treated with the test cream right at the time of tick removal, after the ticks had been attached for 72 h. In the group receiving azithromycin cream, 0/12 mice developed spirochetal infection, representing a protection level of 100%. In sharp contrast, 11/12 of the mice receiving doxycycline cream developed spirochetal infection, representing only 8% protection. In this trial, all 11 control mice exposed to cream without antibiotics developed spirochetal infection. In subsequent experiments, we decided to test the limits of the azithromycin cream's efficacy rather than try to come up with a doxycycline formulation that gave greater protection.
TABLE 1.
Efficacy of topical antibiotic cream preparations for prophylaxis against tick-transmitted spirochetesa
| Drug | No. of spirochete-positive mice/no. of mice exposed | % protection |
|---|---|---|
| Azithromycin | 0/12 | 100b |
| Doxycycline | 11/12 | 8 |
| Control cream | 11/11 | NAc |
Mice were treated on the day of tick removal.
P < 0.0001 compared to control by Fisher's exact test.
NA, not applicable.
The question of when post-tick-removal azithromycin cream had to be delivered to have maximal benefit was evaluated. A group of mice was treated on the day of tick removal (considered day 0 for the purpose of this experiment) or on subsequent days for up to 2 weeks following tick removal. Azithromycin cream treatment was 100% protective if delivered on day 0, day 1, day 2, or day 3 following tick removal (Table 2). Although the level of protection fell off somewhat to 74% if delivered ≥4 days following tick removal, it was still at the 74%-protective level even at 2 weeks following tick removal.
TABLE 2.
Effect of timing of delivery of topical azithromycin on the efficacy of prophylaxis against tick-transmitted spirochetes
| Day post-tick bite | No. of spirochete-positive mice/no. of mice exposed | % protection |
|---|---|---|
| 0 | 0/12 | 100a |
| 1 | 0/11 | 100a |
| 2 | 0/10 | 100a |
| 3 | 0/12 | 100a |
| 4 | 3/12 | 74a |
| 5 | 2/8 | 74a |
| 7 | 2/8 | 74a |
| 14 | 3/12 | 74a |
| Control | 19/20 | NAb |
P < 0.0005 compared to controls by Fisher's exact test.
NA, not applicable.
The next series of experiments was aimed at determining whether the azithromycin cream had to be delivered directly to the site of the tick bite or could be delivered to the skin at a distal site. In experiments where a distal site was treated, mice were shaved on the belly and cream was delivered to this ventral location rather than to the dorsal location where the tick capsule had originally been placed. We also tested whether ingestion of the antibiotic cream during mouse grooming played a role in the efficacy of treatment. In these experiments, a capsule was left in place and the cream was placed inside the capsule where it could not be groomed off by the mice. The capsule was left for 24 h either at the tick bite site or the distal location on the ventral side of the mouse. It did not matter whether the treatments were placed at the tick bite site or the distal location (Table 3). Where grooming was allowed, 100% of mice treated at the tick bite site and 100% of mice treated at the distal location were protected. Where grooming was not allowed, 92% of mice were protected when treated at the tick bite site and 100% of mice were protected when treated at the distal location. In all, only 1 of 38 treated mice in this experiment was not protected from infection.
TABLE 3.
Effect of treatment site and grooming on the efficacy of azithromycin cream treatments for prophylaxis against tick-transmitted spirochetes
| Treatment site or treatment | Grooming allowed | No. of spirochete-positive mice/no. of mice exposed | % protection |
|---|---|---|---|
| Bite site | Yes | 0/15 | 100a |
| Bite site | No | 1/18 | 92a |
| Distal | Yes | 0/10 | 100a |
| Distal | No | 0/12 | 100a |
| Control cream | NA | 7/10 | NAb |
P < 0.0001 compared to controls by Fisher's exact test.
NA, not applicable.
To confirm culture results, sera from 10 mice judged to be infected by culture and 27 mice judged to be negative by culture were selected from the overall data set and tested for evidence of antibodies to B. burgdorferi by MarBlot. All 10 mice judged to be positive via culture had a positive serological result, and all 27 mice judged to be negative via culture had a negative serological result (data not shown).
A total of 10 mice treated with azithromycin were subjected to feeding by xenodiagnostic larval ticks; 118 xenodiagnostic larvae derived from these mice were all negative for spirochete DNA upon PCR examination (Table 4). Similarly, 4 naive control mice produced a total of 39 ticks negative for spirochetes on PCR examination. In contrast, 47/47 (100%) larval ticks fed on 4 positive mice treated with control cream proved positive for spirochetal DNA on PCR examination.
TABLE 4.
Spirochetal infections in xenodiagnostic larval ticks fed on mice treated with antibiotic creamsa
| Treatment group | No. of mice | No. of spirochete-positive ticks/no. of ticks examined |
|---|---|---|
| Azithromycin | 10 | 0/118b |
| Naive controls | 4 | 0/39b |
| Control cream | 4 | 47/47b |
A total of 8 to 12 ticks from each mouse were tested by PCR at 10 days postrepletion.
P < 0.0001 compared to controls by Fisher's exact test.
DISCUSSION
Previous experiments with azithromycin have demonstrated that ≥4% topical creams can provide 100% protection when applied to mice twice daily for 3 days (19). The current work extends these observations and demonstrates that a single application of 4% azithromycin cream can be 100% protective when applied within the first 3 days following a tick bite in a murine model system. Interestingly, a commercially available 2% erythromycin topical ointment failed to protect mice against tick-transmitted infection with B. burgdorferi (20). At present, we do not know whether azithromycin is much more effective than the closely related macrolide antibiotic erythromycin or whether the difference in the concentration of the drug is a key element in their respective levels of success. Azithromycin has long been known to be more active than erythromycin against certain Gram-negative bacteria (26).
Our results with doxycycline prophylaxis in our murine model system have been mixed. Single-dose oral doxycycline given by gavage has resulted in disappointing levels of prophylaxis, demonstrating 20% to 43% protection (15, 16). In contrast, a slow-release injectable doxycycline gel that continues to release doxycycline into the bloodstream over several weeks gave 100% protection (15). Protection afforded by oral doxycycline in our murine model was increased to 74% only by delivering the drug twice daily on the day ticks were removed (17). Results with topical applications of doxycycline and tetracycline have been inconsistent. Shih and Spielman (18) found doxycycline to be completely protective but did not give details of the experiment. They found tetracycline to be protective only when delivered for 3 days compared to the ineffective results found when tetracycline was given for a single day. Wormser et al. (20) found a 3% tetracycline preparation to be ineffective in providing prophylaxis for tick-transmitted B. burgdorferi in a murine model. In addition, our experiments in the current study found single topical applications of 4% doxycycline to provide no protection against tick-transmitted infection with B. burgdorferi, even when delivered on the day of tick removal. The tendency of azithromycin to collect in tissues may offer an advantage over doxycycline when applied as a cream to skin. Overall, it appears that azithromycin holds more promise than doxycycline for development of cream preparations for prophylaxis for Lyme disease spirochetes.
The fact that azithromycin cream gave 100% protection when applied at any time during the first 3 days following tick removal, but continued to give high levels of protection (74%) even after 2 weeks post-tick removal, suggests that the action of the cream is both prophylactic and curative. The spirochete infection is well established in the rodent host by 2 weeks post-tick removal (27) and disseminates from the site of tick attachment by ≥3 days after spirochete transmission (28). Azithromycin must be curing well-established infections as well as preventing the infection from establishing in the first place. Experiments conducted in this study also support the suggestion that the action of the cream is transdermal (19) since mice that could not ingest the cream were protected. But, since cream placed distal to the site of tick feeding was also protective, the azithromycin must have been having a systemic effect in addition to a local effect. It is questionable whether the systemic effect of azithromycin cream seen in mice would be also observed in much larger hosts (such as humans) if the drug were to be applied topically to skin, since the drug would be diluted in the greater blood volume. Experiments with larger hosts might help determine whether the systemic effect of azithromycin for protection against Lyme disease spirochetes is essential or whether the transdermal effect suffices to give protection. Larger hosts from rabbits (29) to rhesus monkeys (30) have been used to study the dynamics of tick-transmitted Lyme disease spirochete infection. Finally, the fact that Knauer et al. (19) observed that the concentrations of azithromycin in the skin (mg/liter) was >100,000 × MIC of azithromycin for B. burgdorferi in dermal treatment sites suggests that spirochetes are being affected locally within the skin at the bite site.
A topical antibiotic cream could be prescribed to residents living in regions where Lyme disease is highly endemic in the beginning of the nymphal I. scapularis activity season (mid-May). Residents could self-apply the cream right at the time of tick removal. An extremely small amount of antibiotic would be used, reducing worries about selecting for antibiotic-resistant bacteria. Steps along the way toward such a product would include additional animal model work in larger animals, the development of a topical formulation of azithromycin by a commercial entity, and, eventually, human clinical trials. Interestingly, according to a personal communication from Reinhard Straubinger, Munich, Germany, and Gustave Huber, Zürich, Switzerland, “Ixodes AG* has performed clinical trials up to phase III. Phase I and II studies demonstrated safety and efficacy (surrogate endpoint based on azithromycin concentrations in different skin layers exceeded published MIC for borrelia). The phase III trial confirmed excellent safety and showed a positive trend to prevent clinical signs of Lyme borreliosis. Since the number of evaluable cases was not sufficient to test the null hypothesis with a statistical significance, a second phase III trial is being planned.” The outcome of that clinical trial should be of some public health interest.
ACKNOWLEDGMENTS
We thank Aaron McGrew for assistance with antibiotic formulations. Gabrielle Dietrich assisted with tick colony maintenance.
The findings and conclusions of this study are ours and do not necessarily represent the views of the Centers for Disease Control and Prevention. Mention of a method or product does not constitute an endorsement.
Footnotes
Published ahead of print 28 October 2013
REFERENCES
- 1.Centers for Disease Control and Prevention 2012Notice to readers: final 2011 reports of nationally notifiable infectious diseases. Morb. Mortal. Wkly. Rep. 61:624–637 http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6132a8.htm [Google Scholar]
- 2.Bacon RM, Kugeler KJ, Mead PS. 2008. Surveillance for Lyme disease—United States, 1992-2006. MMWR Surveill. Summ. 57(SS10):1–9 [PubMed] [Google Scholar]
- 3.Diuk-Wasser MA, Hoen AG, Cislo P, Brinkerhoff R, Hamer SA, Rowland M, Cortinas R, Vourc'h G, Melton F, Hickling GJ, Tsao JI, Bunikis J, Barbour AG, Kitron U, Piesman J, Fish D. 2012. Human risk of infection with Borrelia burgdorferi, the Lyme disease agent, in eastern United States. Am. J. Trop. Med. Hyg. 86:320–327. 10.4269/ajtmh.2012.11-0395 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Piesman J, Eisen L. 2008. Prevention of tick-borne diseases. Annu. Rev. Entomol. 53:323–343. 10.1146/annurev.ento.53.103106.093429 [DOI] [PubMed] [Google Scholar]
- 5.Daltroy LH, Phillips C, Lew R, Wright E, Shadick NA, Liang MH. 2007. A controlled trial of a novel prevention program for Lyme disease and other tick-borne illnesses. Health Educ. Behav. 34:531–542 [DOI] [PubMed] [Google Scholar]
- 6.Gould LH, Nelson RS, Griffith KS, Hayes EB, Piesman J, Mead PS, Cartter ML. 2008. Knowledge, attitudes, and behaviors regarding Lyme disease prevention among Connecticut residents, 1999-2004. Vector Borne Zoonotic Dis. 8:769–776. 10.1089/vbz.2007.0221 [DOI] [PubMed] [Google Scholar]
- 7.Wilson ML, Telford III SR, Piesman J, Spielman A. 1988. Reduced abundance of immature Ixodes dammini (Acari: Ixodidae) following elimination of deer. J. Med. Entomol. 25:224–228 [DOI] [PubMed] [Google Scholar]
- 8.Telford SR., III 2002. Deer tick transmitted zoonosis in the eastern United States, p 310–324 In Ostfeld RS, Tabor GM, House C, Pearl MC. (ed), Conservation medicine. Oxford University Press, Oxford, United Kingdom [Google Scholar]
- 9.Rand P, Lubelczyk C, Holman MS, Lacombe EH, Smith RP., Jr 2004. Abundance of Ixodes scapularis (Acari: Ixodidae) after the complete removal of deer from an isolated offshore island, endemic for Lyme disease. J. Med. Entomol. 41:779–784. 10.1603/0022-2585-41.4.779 [DOI] [PubMed] [Google Scholar]
- 10.Steere AC, Sikand VK, Meurice F, Parenti DL, Fikrig E, Schoen RT, Nowakowski J, Schmid CH, Laukamp S, Buscarino C, Krause DS. 1998. Vaccination against Lyme disease with a recombinant Borrelia burgdorferi outer-surface lipoprotein A with adjuvant. Lyme disease vaccine study group. N. Engl. J. Med. 23:209–215 [DOI] [PubMed] [Google Scholar]
- 11.Poland GA. 2011. Vaccines against Lyme disease: what happened and what lessons can we learn? Clin. Infect. Dis. 52:s253–s258. 10.1093/cid/ciq116 [DOI] [PubMed] [Google Scholar]
- 12.Shen AK, Mead PS, Beard CB. 2011. The Lyme disease vaccine—a public health perspective. Clin. Infect. Dis. 52:s247–s252. 10.1093/cid/ciq115 [DOI] [PubMed] [Google Scholar]
- 13.Magid D, Schwartz B, Craft J, Schwartz JS. 1992. Prevention of Lyme disease after tick bites. A cost-effectiveness analysis. N. Engl. J. Med. 327:534–541 [DOI] [PubMed] [Google Scholar]
- 14.Nadelman RB, Nowakowski J, Fish D, Falco RC, Freeman K, McKenna D, Welch P, Marcus R, Aguero-Rosenfeld ME, Dennis DT, Wormser GP. 2001. Prophylaxis with single-dose doxycycline for the prevention of Lyme disease after an Ixodes scapularis tick bite. N. Engl. J. Med. 345:79–84. 10.1056/NEJM200107123450201 [DOI] [PubMed] [Google Scholar]
- 15.Zeidner NS, Brandt KS, Dadey E, Dolan MC, Happ C, Piesman J. 2004. Sustained-release of doxycycline hyclate for prophylaxis of tick bite infection in a murine model of Lyme borreliosis. Antimicrob. Agents Chemother. 48:2697–2699. 10.1128/AAC.48.7.2697-2699.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Zeidner NS, Massung RF, Dolan MC, Dadey E, Gabitzsch E, Dietrich G, Levin ML. 2008. A sustained-release formulation of doxycycline hyclate (Atridox) prevents simultaneous infection of Anaplasma phagocytophilum and Borrelia burgdorferi transmitted by tick bite. J. Med. Microbiol. 57:463–468. 10.1099/jmm.0.47535-0 [DOI] [PubMed] [Google Scholar]
- 17.Piesman J, Hojgaard A. 2012. Protective value of prophylactic antibiotic treatment of tick bite for Lyme disease prevention: an animal model. Ticks Tick Borne Dis. 3:193–196. 10.1016/j.ttbdis.2012.01.001 [DOI] [PubMed] [Google Scholar]
- 18.Shih CM, Spielman A. 1993. Topical prophylaxis for Lyme disease after tick bite in a rodent model. J. Infect. Dis. 168:1042–1045. 10.1093/infdis/168.4.1042 [DOI] [PubMed] [Google Scholar]
- 19.Knauer J, Krupka I, Fueldner C, Lehmann J, Straubinger RK. 2011. Evaluation of the preventive capacities of a topically applied azithromycin formulation against Lyme borreliosis in a murine model. J. Antimicrob. Chemother. 66:2814–2822. 10.1093/jac/dkr371 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Wormser GP, Daniels TJ, Bittker S, Cooper D, Wang G, Pavia CS. 2012. Failure of topical antibiotics to prevent disseminated Borrelia burgdorferi infection following a tick bite in C3H/HeJ mice. J. Infect. Dis. 205:991–994. 10.1093/infdis/jir382 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Piesman J. 1993. Standard system for infecting ticks (Acari: Ixodidae) with the Lyme disease spirochete Borrelia burgdorferi. J. Med. Entomol. 30:199–203 [DOI] [PubMed] [Google Scholar]
- 22.Soares CAG, Zeidner NS, Beard CB, Dolan MC, Dietrich G, Piesman J. 2006. Kinetics of Borrelia burgdorferi infection in larvae of refractory and competent tick vectors. J. Med. Entomol. 43:61–67. 10.1603/0022-2585(2006)043[0061:KOBBII]2.0.CO;2 [DOI] [PubMed] [Google Scholar]
- 23.Hojgaard A, Eisen RJ, Piesman J. 2008. Transmission dynamics of Borrelia burgdorferi s.s. during the key third day of feeding by nymphal Ixodes scapularis (Acari: Ixodidae). J. Med. Entomol. 45:732–736. 10.1603/0022-2585(2008)45[732:TDOBBS]2.0.CO;2 [DOI] [PubMed] [Google Scholar]
- 24.Dolan MC, Schulze TL, Jordan RA, Dietrich G, Schulze CJ, Hojgaard A, Ullmann AJ, Sackal C, Zeidner NS, Piesman J. 2011. Elimination of Borrelia burgdorferi and Anaplasma phagocytophilum in rodent reservoirs and Ixodes scapularis ticks using a doxycycline hyclate-laden bait. Am. J. Trop. Med. Hyg. 85:1114–1120. 10.4269/ajtmh.2011.11-0292 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Piesman J, Happ CM. 1997. Ability of the Lyme disease spirochete Borrelia burgdorferi to infect rodents and three species of human-biting ticks (Blacklegged Tick, American Dog Tick, Lone Star Tick) (Acari: Ixodidae). J. Med. Entomol. 34:451–456 [DOI] [PubMed] [Google Scholar]
- 26.Retsema J, Girard A, Schelkly W, Manousos M, Anderson M, Bright G, Borovoy R, Brennan L, Mason R. 1987. Spectrum and mode of action of azithromycin (CP-62,993), a new 15-membered-ring macrolide with improved potency against gram-negative organisms. Antimicrob. Agents Chemother. 31:1939–1947. 10.1128/AAC.31.12.1939 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Donahue JG, Piesman J, Spielman A. 1987. Reservoir competence of white footed mice for Lyme disease spirochetes. Am. J. Trop. Med. Hyg. 36:92–96 [DOI] [PubMed] [Google Scholar]
- 28.Shih CM, Pollack RJ, Telford SR III, Spielman A. 1992. Delayed dissemination of Lyme disease spirochetes from the site of deposition in the skin of mice. J. Infect. Dis. 166:827–831. 10.1093/infdis/166.4.827 [DOI] [PubMed] [Google Scholar]
- 29.Piesman J, Maupin GO, Campos EG, Happ CM. 1991. Duration of adult female Ixodes dammini attachment and transmission of Borrelia burgdorferi, with description of a needle aspiration isolation method. J. Infect. Dis. 163:895–897. 10.1093/infdis/163.4.895 [DOI] [PubMed] [Google Scholar]
- 30.Bernardino AL, Kaushal D, Philipp MT. 2009. The antibiotics doxycycline and minocycline inhibit the inflammatory responses to the Lyme disease spirochete Borrelia burgdorferi. J. Infect. Dis. 199:1379–1388. 10.1086/597807 [DOI] [PMC free article] [PubMed] [Google Scholar]