Controversy brewing over Lyme disease testing Roxanne Nelson Available online 23 September 2005. Lyme disease, a bacterial infection transmitted by ticks, is difficult to diagnose because its symptoms mimic those of other disorders. Although diagnostic tests are currently available, there is a growing controversy over the unreliability of standard testing, as well as the use of new testing approaches. The US Centers for Disease Control and Prevention (CDC) has cautioned against using assays whose accuracy and clinical usefulness has not been adequately established. These include urine antigen tests, immunofluorescent staining for cell wall-deficient forms of Borrelia burgdorferi, and PCR tests that are done on inappropriate specimens such as blood and urine. “Based on calls and complaints that we get from patients, and also based on what we're hearing from colleagues in Europe, we are concerned that some patients are being misdiagnosed or potentially misdiagnosed and mistreated, as a result of unfounded reliance on these tests”, says Paul Mead, a medical epidemiologist at the CDC. CDC guidelines recommend a two-test approach using a sensitive enzyme immunoassay (EIA) or immunofluorescent assay, to be followed by a western immunoblot if results are positive. But Lyme disease is often mistaken for other ailments, and conversely, serious illnesses such as amyotrophic lateral sclerosis have been misdiagnosed as Lyme disease. Nick Harris, the founder and chief executive of IGeneX, a reference laboratory that does PCR testing for Lyme disease on a variety of specimens, is among those who feel that the CDC's guidelines are actually part of the reason for the high rate of misdiagnosis. “The CDC says the two-tiered system works for Lyme victims within 3 to 4 months of a tick bite and for those who have an erythema migrans rash”, he points out. “But more than 50% of the patients do not remember being bitten by a tick and more than 50% do not get the rash.” Any screening test, according to laboratory experts, needs to be sensitive to 90–100%, and the EIA is less than 70% sensitive in early Lyme disease, Harris adds. In chronic or late stage Lyme disease, the percentage of positive EIA is much lower. “The CDC claims that the PCR is not useful in the diagnosis of Lyme because of its low predictive value,” says Harris. “The PCR's specificity is 100%, and it does have a high predictive value if positive.” New method for detection of Borrelia burgdorferi antigen complexed to antibody in seronegative Lyme disease Michael Brunner , The Children’s Hospital of Philadelphia, Department of Rheumatology, Abramson Research Center 1104D, 3516 Civic Center Blvd., Philadelphia, PA 19104-4318, USA Received 22 August 2000; revised 21 November 2000; accepted 30 November 2000. Available online 20 February 2001. Abstract Serologic tests for Lyme disease are problematic. Because of cross-reactive antigens Borrelia burgdorferi (Bb) shares with other organisms, Lyme disease can be overdiagnosed. However, in addition to specificity problems, serologic tests for early Lyme disease can be falsely negative due to lack of sensitivity of ELISAs and Western blots. Most routine antibody tests are designed to detect free antibodies, and in early, active disease, circulating antibodies may not be free in serum but sequestered in complexes with the antigens which originally triggered their production. This difficulty may be overcome by first isolating immune complexes (IC) from the serum and using this fraction for testing. Free Borrelia-specific antibodies can then be liberated from the immune complexes which may enhance test sensitivity in patients with active disease. We developed a technique that captures the antibody component of IC on immunobeads, and subsequently releases the antigen component of IC. Immunoblotting with monoclonal antibody detected at least one antigen to be OspA, thus definitively demonstrating a Borrelia-specific antigen in circulating IC in early Lyme disease. This test is also useful in demonstrating Bb antigen in otherwise seronegative Lyme disease patients. Author Keywords: Lyme disease ; Borrelia burgdorferi; Immune complexes; Borrelia-specific antigens; OspA; Serologic tests Abbreviations: IC, immune complex; Bb, Borrelia burgdorferi; OspA, outer surface protein A; PEG, polyethylene glycol; Mab, monoclonal antibody Article Outline 1. Introduction 2. Materials and methods 2.1. Detection of antigen from IC of a patient sample 2.2. Preparation of Bb sonicate 2.3. Commercial serologic assays 3. Results 4. Discussion Acknowledgements References 1. Introduction Lyme disease (LD), a common seasonal illness caused by the tick-borne spirochete, Borrelia burgdorferi (Bb) (Steere et al., 1983), is often diagnosed clinically by the presence of an erythema migrans rash. However, this ‘bullseye rash’ occurs in only 60 to 80% of patients ( Centers for Disease Control, 1997), and can be atypical in appearance. Other symptoms, such as summer flu-like illness or joint pains, commonly bring the patient to the physician for a diagnosis within a few weeks of the tick bite, often when the rash is absent or no longer present. A potential gold standard for making the diagnosis is a positive culture from a biopsy (usually skin) of the affected area, whereupon Borrelia burgdorferi can be detected. Typically, this is only done in certain circumstances at equipped facilities, and it is not practical for broad scale testing. Usually, indirect antibody tests are performed as a two-tier approach (Centers and Dressler) with an ELISA, or immunofluorescent IFA ( Lane et al., 1990) screening assay, followed by a confirmatory Western blot ( Centers and Dressler). The Western blot is performed on samples from equivocal or ELISA positive patients, and known consensus bands for IgM (early cases) or IgG are ascertained ( Centers and Dressler) before diagnosis is made. These tests can be problematic due to subjective interpretation of the banding pattern ( Liang et al., 2000), or that there are cross-reactive antigens shared by Bb (e.g., the flagellin 41 kD antigen of Treponema pallidum or other flagellin bearing organisms, rheumatoid factor or other serum contaminants, or 23 kD antigen from Helicobatcer pylori), which may give rise to false positive bands. Hence, LD can be easily overdiagnosed (Steere and Steere). In proficiency testing programs, Bakken and Bakken and others have found poor sensitivity, specificity and reproducibility ( Hedberg; Hedberg and Luger) in repeated testing which did not improve with time ( Bakken et al., 1997). Even the two-tier approach has not helped, and they ( Bakken et al., 1997) concluded that more stringent criteria are needed to approve commercially available kits for the serodiagnosis of Lyme disease. The potential problem of poor sensitivity in testing for LD may be due to an assay performed at an early time in disease when antibodies detected by these tests are not yet present in circulation, and may only be present as immune complexes (IC). Since routine serodiagnostic tests rely on free antibody, those antibodies tied up in complexes are unavailable and therefore missed (Schutzer and Schutzer), leading to false negative results. There have been academic laboratory examples of using IC to improve Lyme testing ( Schutzer and Coyle), and ICs have helped both in ELISAs ( Brunner and Brunner) and in Western blots ( Brunner and Schutzer) to detect LD in seronegative patients ( Schutzer and Coyle). Outer surface protein A (OspA), shed in outer surface membrane blebs by Bb into surrounding body fluids (Barbour; Dorward and Katona), has been used before in assays for early LD involving IC ( Schutzer et al., 1994). The importance of OspA is noted by the fact that recombinant OspA is used in a preventative vaccine for LD ( Steere and Sigal). In this report we looked for OspA because even though it has been shown to be down-regulated when Bb enters the mammalian host ( Schwan et al., 1995), there has been much work done on OspA expression and its antibodies found early and late in LD ( Akin et al., 1999; Kalish et al., 1995; Montgomery et al., 1996). Expression of OspA in early and late disease ( Akin; de; Schutzer; Montgomery and Krause), as well as its association with IC ( Brunner; Brunner and Schutzer), has possible implications for disease pathogenesis. The technique reported here is practical for detecting a Bb antigen in an otherwise seronegative LD patient (positive for Bb culture and presenting with an erythema migrans rash) by routine antibody testing. Moreover, this is the first clear demonstration that a specific Bb antigen, OspA, is part of an IC purified from a patient’s serum. This was accomplished by first capturing the IC by its antibody component on an immunobead, releasing the attached antigen, and confirming the identity of the OspA antigen using an OspA-specific monoclonal antibody in an immunoblot. Although reporting the results from this particular patient was considered noteworthy to illustrate the technique, the assay has proven to be sensitive and highly specific in correctly determining infectivity status in more that 20 patients with various manifestations of LD, as well as in numerous endemic area negatives and other disease controls (Brunner and Sigal, 2000). 2. Materials and methods 2.1. Detection of antigen from IC of a patient sample The patient’s serum (0.6 ml) was first precipitated with an equal amount of 7% PEG in 0.1 M borate buffer, pH 8.4, overnight at 4°C. High molecular weight components of serum are known to precipitate at this PEG concentration (Digeon et al., 1977). The pellet was washed several times with 3.5% PEG in 0.1 M borate buffer, and the final pellet was resuspended in half the volume (0.3 ml) of 0.1 M borate buffer, pH 10.2, to concentrate and dissociate the ICs. A small aliquot (20 μl) of this was frozen at −70°C to be analyzed later (diluted 1:2 in reducing buffer, boiled, and 30 μl added to well) and is referred to as dissociated PEG precipitate in Fig. 1, lane 3. The remainder (280 μl) was neutralized with (22.5 μl of 3 M sodium acetate pH 5.27) acid to re-associate the antigen–antibody complex and optimize it for subsequent binding through the antibody component. This was accomplished by sequential addition of commercial preparations of protein conjugated agarose beads used for immunoprecipitation. Specifically, 0.1 ml of GammaBind G Sepharose (Pharmacia, Piscataway, NJ, USA) was added to the neutralized sample, which was placed on a reciprocal shaker in the cold (4°C) for 1 h. Theoretically, the conjugated sepharose will bind any IC containing IgG antibody subclass. This process was repeated with 0.1 ml of immobilized Mannan binding protein (Ultralink, Pierce, Rockford, IL, USA) to remove all complexes containing IgM antibody. Finally, protein -agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to remove all remaining IgM, IgG, and IgA subclasses of antibody that might be contained in ICs. To insure complete removal, beads were shaken overnight at 4°C and centrifuged at 10,000 RPM (8600×g) for 15 min. After aspiration of supernatant, the bead pellet was resuspended in 90 μl of reducing buffer, and boiled for 10 min (to release antigen). The supernatant (30 μl/well) was then electrophoresed on 12% precast gels (Bio-Rad, Hercules, CA, USA), transferred to PVDF (Polyscreen, NEN, Boston, MA, USA) membranes and probed with anti-OspA mAb H5332 (from Alan Barbour, UCI) to detect the 31 kD OspA protein (Fig. 1, lane 2). The second antibody was peroxidase labelled goat anti-mouse IgG Fab-specific (A2304 Sigma, St. Louis, MO, USA) adsorbed with bovine, horse, and human serum proteins. This was followed by reaction with enhanced luminol chemiluminescent substrate (Renaissance Plus, NEN), and exposure to X-ray film (BioMax, NEN). (3K) Fig. 1. OspA detection in immune complexes of a seronegative, culture positive, EM positive Lyme disease patient. A Western blot was performed using the OspA-specific monoclonal antibody, H5332. Lane 1 contains Bb sonicate. The patient’s serum was treated as described in the Materials and methods, and the boiled immunobead eluate and the dissociated PEG precipitate are depicted in lanes 2 and 3, respectively. 2.2. Preparation of Bb sonicate This was done as reported previously (Brunner et al., 1998). Briefly, B. burgdorferi B31 was grown at 32°C in BSK-H medium supplemented with 6% rabbit serum (Sigma) to late log phase (5–6 days), harvested, and washed four times with phosphate buffered saline (PBS), pH 7.2, containing 5 mM MgCl2. The final pellet was resuspended in PBS and sonicated (medium setting; Braun-Sonic 2000) for four pulses of 30 s each with 1 min between pulses. The sonicate was diluted in reducing buffer, boiled, and 5 μg in 30 μl volume was added to the well (Fig. 1, lane 1). 2.3. Commercial serologic assays Patient serum was tested for Lyme antibodies by commercial ELISA and Western blot (MarDx diagnostics, Carlsbad, CA, USA). Immunofluorescence assay (IFA) for IgM in patient serum was done as described (Mitchell et al., 1996) using substrate slides containing B. burgdorferi B31 (Bion Enerprises, Chicago, IL, USA) and FITC-conjugated goat anti-human IgM (Kallestad Diagnostics, Chaska, MN, USA) diluted 1:100. 3. Results The serum from a patient who was seronegative on standard Lyme assays, including ELISA, IFA, and immunoblot, but who presented with EM and was culture positive from biopsy taken at the time of the serum sample, was tested for Lyme antigen as part of IC. The patient’s serum was first PEG precipitated at a concentration known to bring down high molecular weight circulating immune complexes if present (Digeon et al., 1977) but not free antibodies. When the dissociated precipitate was electrophoresed, blotted and probed with monoclonal antibody against OspA (H5332), a single 31 kD band was obtained ( Fig. 1, lane 3). Antigen migrated identically to the single band obtained when a B. burgdorferi sonicate was used as the starting material (Fig. 1, lane 1). When the dissociated PEG precipitate of the patient’s serum was first treated with antibody-binding beads, boiled to release antigen which was in a complex with the antibody, and similarly probed with mAb H5332, a band with the same electrophoretic mobility as the 31 kD OspA antigen was observed ( Fig. 1, lane 2). Therefore, Lyme-specific antigen (OspA) complexed to antibody was detected in the serum of a patient with LD at a time when routine Lyme testing (ELISA, IFA, immunoblot) was negative. 4. Discussion OspA has been previously detected in immune complexes of patients with Lyme disease, and has been shown to be useful both for ELISA (Brunner et al., 1997) and Western blot assays ( Schutzer et al., 1994). Specific IC containing LD antigens have been isolated in acid fractions off anti-C1q affinity columns and demonstrated in dot blots ( Zhong et al., 1997) where it was suggested that detection of IC may be a reliable parameter for monitoring a patient’s response to antibiotic treatment. In Fig. 1, lane 3, we demonstrate that the dissociated PEG precipitate yields a 31 kD band reactive with an OspA-specific monoclonal antibody. This shows that OspA antigen may be present in dissociated IC from a LD patient, as was seen previously with polyclonal anti-OspA ( Schutzer et al., 1999). Even though this suggested that the antigen could have been complexed with the antibody in IC, it could also have been associated with the high molecular weight fraction ( Digeon et al., 1977) and trapped inadvertently in the PEG precipitate. Fig. 1, lane 2, shows the boiled bead fraction, which releases antigen from captured IC on various antibody (Fc portion)-binding beads, and produces one 31 kD band on reaction with anti-OspA Mab, H5332. This represents a more compelling argument that Lyme-specific OspA antigen was originally present in IC, as demonstrated by capture through its antibody component on immunobeads. Therefore, this study gives more convincing evidence than previous work ( Schutzer and Zhong) that Bb antigen is actually present in IC of a patient with active Lyme disease. ICs are also thought to be useful to distinguish between active LD and residual titer of free antibody, as is sometimes seen with Lyme-specific IgG or IgM for months to years after LD is cured (Hammers-Berggren et al., 1994). In this regard, ICs are also useful in conjunction with IgM assays ( Brunner and Brunner) to demonstrate early disease. Since IC is present in numerous diseases, for example systemic lupus erythematosus (Herrmann et al., 1978), infectious hepatitis ( Carella et al., 1977) and multiple sclerosis ( Coyle, 1987), a variation of this technique may be useful as a generalized procedure to demonstrate or isolate antigen when appropriate. We have also seen antibodies to the other expected antigens in ICs of LD patients previously (e.g., flagellin, OspC, P39, even OspB) in ELISAs (Brunner et al., 1998) and in Western blots ( Schutzer et al., 1994). To date, using the method reported here and in related work ( Brunner and Sigal, 2000), we found OspA containing ICs, although we also probed with H9724 anti-flagellin, 41 kD (from Alan Barbour), and L22 1F8 anti-OspC, 23 kD (from Betina Wilske) monoclonal antibodies. This could be due to the ubiquitous nature of OspA as a result of its shedding (even though its expression may be down-regulated) when the Bb organism enters the mammalian host, ( Schwan et al., 1995). This could also possibly be due to a technical anomaly, that the OspA epitope recognized by H5332 is more stable to boiling than that for the flagellin or OspC, and/or that H5332 is a better Mab (higher affinity or avidity) than the others. Although OspA was thought to be down-regulated upon infection of the mammalian host (Schwan et al., 1995), a brisk early OspA antibody response occurs after needle inoculation of mice with OspA ( Fikrig et al., 1990). In another study, one-third of spirochetes inoculated into mice continued to express OspA ( de Silva et al., 1996), and in a mouse model of persisting Bb infection, OspA was expressed persistently ( Schwan et al., 1991). Human LD patients showed early OspA T cell responses ( Krause et al., 1992) as well as humoral antibodies by analyzing ICs from those patients on recombinant OspA immunoblots ( Schutzer et al., 1994). OspA and OspC were coexpressed in CSF in early neurologic Lyme patients ( Schutzer et al., 1997). By direct fluorescent staining of uncultured spirochetes ex vivo and by PCR amplification of spirochetal mRNA, Montgomery et al. (1996) found that spirochetes expressing OspA could be detected within the first 2 weeks of infection, and mRNA was present at day 14 of infection but not at day 30, suggesting that expression of OspA is transient. During periods of maximal Lyme arthritis, Akin et al. (1999) found that IgG antibody response to OspA correlated with severe and prolonged Lyme arthritis, and that this activity was directed against a C-terminal epitope of OspA. However, when Kalish et al. (1995) measured responses to epitopes in full length recombinant OspA and three recombinant OspA fragments, early IgM responses were found to epitopes in all three fragments of OspA. The conclusions from all of the above work are that OspA can be found early in disease, may be abundant and shed from the spirochete, and may be sequestered in IC. Therefore, the antibody would not be readily available to assays for free antibody at this time, and its complexed antigen (OspA) would be difficult to find (in free antigen assays) in addition to its reported transient appearance at that time ( Montgomery et al., 1996), giving it an unjustified reputation for being only a late antigen giving rise to a late antibody response. However, since the antigen has been shown to be present early, and to have multiple epitopes for reactivity at that time ( Kalish et al., 1995), it is not inconceivable that OspA would likely be found by the method described in this paper. Acknowledgements I would like to thank Dr. Alan Barbour for monoclonal antibodies H5332 and H9724, and Dr. Betina Wilske for monoclonal antibody L22 1F8. I would also like to thank Dr. Randy Cron for reading the manuscript and making helpful suggestions. References Akin, E., McHugh, G.L., Flavell, R.A., Fikrig, E. and Steere, A.C., 1999. 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Abstract-MEDLINE | Abstract-EMBASE | Order Document Sigal, L.H., Zahradnik, J.M., Lavin, P. et al., 1998. A vaccine consisting of recombinant Borrelia burgdorferi outer-surface protein A to prevent Lyme disease. New Engl. J. Med. 339, p. 216. Abstract-EMBASE | Abstract-Elsevier BIOBASE | Order Document | Full Text via CrossRef de Silva, A.M., Telford, S.R., Brunet, L.R. et al., 1996. Borrelia burgdorferi OspA is an artropod-specific transmission-blocking Lyme disease vaccine. J. Exp. Med. 183, p. 271. Abstract-MEDLINE | Abstract-Elsevier BIOBASE | Order Document | Full Text via CrossRef Steere, A.C., Sikand, V.K., Meurice, F. et al., 1998. Vaccination against Lyme disease with recombinant Borrelia burgdorferi outer-surface lipoprotein A with adjuvant. New Engl. J. Med. 339, p. 209. Abstract-EMBASE | Abstract-Elsevier BIOBASE | Order Document | Full Text via CrossRef Steere, A.C., Taylor, E., McHugh, G.L. and Logigian, E.L., 1993. The overdiagnosis of Lyme disease. J. Am. Med. Assoc. 269, p. 1812. Abstract-EMBASE | Order Document Steere, A.C., Grodzicki, R.L., Kornblatt, A.N., Craft, J.E., Barbour, A.G., Burgdorfer, W., Schmid, G.P., Johnson, E. and Malawista, S.E., 1983. The spirochetal etiology of Lyme disease. New Engl. J. Med. 308, p. 733. Abstract-MEDLINE | AbstractEMBASE | Order Document Zhong, W., Oschmann, P. and Wellensiek, H.-J., 1997. Detection and preliminary characterization of circulating immune complexes in patients with Lyme disease. Med. Microbiol. Immunol. 186, p. 15. Abstract-Compendex | Abstract-Compendex | Abstract-INSPEC | Order Document Tel.: +1-215-590-3792; fax: +1-215-590-1258; email: brunner@email.chop.edu Journal of Immunological Methods Volume 249, Issues 1-2 , 1 March 2001, Pages 185-190 The American Journal of Medicine Volume 110, Issue 3 , 15 February 2001, Pages 217-219 This Document SummaryPlus Full Text + Links ·Full Size Images PDF (57 K) External Links Actions Cited By Save as Citation Alert E-mail Article Export Citation doi:10.1016/S0002-9343(00)00701-4 Copyright © 2001 Excerpta Medica Inc. All rights reserved. Brief observation Intralaboratory reliability of serologic and urine testing for Lyme disease *1 Mark S. Klempner MD , a, Christopher H. Schmid PhDa, Linden Hu MDa, Allen C. Steere MDa, Gary Johnsona, Bilaal McCloud BSa, Richard Noring BSa and Arthur Weinstein MDb a Department of Medicine (MSK, CHS, LH, ACS, GJ, BM, RN), New England Medical Center, Boston, Massachusetts, USA b Department of Medicine (AW), George Washington University Medical Center, Washington, DC, USA Received 1 May 2000; revised 16 October 2000; accepted 16 October 2000. Available online 13 February 2001. Referred to by: Intralaboratory reliability of serologic and urine testing for lyme disease, The American Journal of Medicine, Volume 111, Issue 6, 15 October 2001, Pages 502-503 Boyd G. Stephensa and Nick S. Harrisa SummaryPlus | Full Text + Links | PDF (46 K) Article Outline • Material and methods • Study subjects • Sample collection • Serologic testing • Lyme urine antigen test • Statistical analysis • Results • Serologic test • Lyme urine antigen test • Discussion • References Laboratory testing for Lyme disease is controversial because of problems with test sensitivity and specificity, the lack of standardized reagents, and interlaboratory and intralaboratory variability [1, 2, 3, 4 and 5]. We determined the reliability of a serologic test and a urine test for Lyme disease, each performed in a reference laboratory, in control subjects and patients with Lyme disease who had posttreatment symptoms. Material and methods Study subjects We studied 10 healthy control subjects who had never had Lyme disease and 21 patients with a history of acute Lyme disease, as defined by the Centers for Disease Control and Prevention [6], who had chronic (>6 month’s duration) fatigue, musculoskeletal pain, or neurocognitive impairment despite treatment with recommended antibiotics. The study protocol was approved by the Human Investigation Institutional Review Boards of New England Medical Center and New York Medical College. All subjects gave written, informed consent. Sample collection Serum samples were obtained from all 21 patients and the 10 control subjects. One aliquot was immediately analyzed; duplicate aliquots were frozen at −70°C and tested within 6 months after collection. Midstream clean-catch urine samples were collected from the 10 control subjects into Vacutainer urine collection kits (Becton Dickinson, no. 36-4962) supplied by the manufacturer of the Lyme urine antigen test (IGeneX, Palo Alto, California). Each specimen was immediately aliquoted into five gray-top Vacutainer urine collection tubes, agitated to mix the sample with the preservative, and either packaged on ice and sent immediately for testing or stored at −70°C and tested within 6 months after collection. Aliquots of each sample were assayed separately. Frozen urine samples for offsite testing were packaged in insulated boxes containing refrigeration packets and shipped by overnight courier. All duplicate samples were sent blinded to the reference laboratories. Serologic testing Immunoglobulin G western blot assays for antibodies against Borrelia burgdorferi antigens were performed at New England Medical Center. In all cases, the immunoglobulin G Marblot strip test system kit (MarDx Diagnostics, Carlsbad, California) was used according to the manufacturer’s instructions. As recommended by the Centers for Disease Control and the Second National Conference on Serologic Diagnosis of Lyme Disease [7], strips that had 5 or more of the 10 significant bands were considered positive for specific immunoglobulin G antibody to B. burgdorferi. Lyme urine antigen test Urine testing was performed by IgeneX, the manufacturer of the Lyme urine antigen test. This test is based on an antigen capture-inhibition enzyme-linked immunosorbent assay that uses adsorbed polyclonal antibodies that bind to several antigenic moieties (31kDa, 34 kDa, 39 kDa, and 93kDa) of B. burgdorferi [8]. The test results are interpreted according to the antigen level in the sample: negative, <20 ng/mL; borderline, 20 to 31 ng/mL; positive, 32 to 45 ng/mL, and highly positive, >45 ng/mL. Statistical analysis Between-sample agreement was assessed with the kappa statistic for categories and the intraclass correlation for numeric values [9]. Kappa values >0.75 indicated excellent agreement; values <0.40 indicated poor agreement. Results Serologic test In all 10 control subjects, the initial western blot analysis yielded negative results. In three of four duplicate specimens analyzed, the same immunoreactive bands seen in the original aliquot were present; 1 duplicate specimen contained a 41-kDa band that was not present in the original aliquot. In the 21 patients with Lyme disease, the results of the initial western blot analysis were positive in 14 cases and negative in 7. Analysis of duplicate specimens yielded identical results in all 21 patients (κ = 1.0, Table 1). The same immunoreactive bands identified in the first analysis were present in 7 of the 14 seropositive duplicate samples; 5 samples had 1 additional band, and 2 samples had 2 additional bands. Repeat testing of the 7 seronegative samples showed fewer than 5 reactive bands in all samples. Table 1. Western Blot Testing for Immunoglobulin G Antibodies to B. burgdorferi in 21 patients with Posttreatment Symptoms of Lyme Disease legend Lyme urine antigen test The results of urine antigen testing in the 10 control subjects were not reliable (Table 2). The standard deviation of the antigen level in the five aliqots of each specimen ranged from 18 to 150 ng/mL. According to the manufacturer, the maximal standard deviation of a test result should be <9 ng/mL. The interpretation of these test results was also unreliable. It ranged from negative to highly positive for 6 specimens, from negative to positive for 1, from borderline to highly positive for 1, and from positive to highly positive for 2. The multiple category (negative, borderline, positive, highly positive) agreement was poor (κ = 0.10). Agreement was also poor when negative results were compared with all other categories (κ = 0.12) and when negative and borderline results together were compared with the other categories (κ = 0.18). Table 2. Results of Lyme Urine Antigen Testing in 10 Control Subjects without Lyme Disease Discussion Nonculture-based testing is the mainstay for laboratory diagnosis of Lyme disease. In patients with chronic symptoms of at least 6 months’ duration, the most appropriate serologic test for prior infection with B. burgdorferi is the immunoglobulin G western blot, which is recommended by the Centers for Disease Control as the final basis for determination of seroreactivity [7]. In a 1997 review of serologic testing for Lyme disease, there were no reports on the interlaboratory or intralaboratory consistency of this test [10]. Our study showed that testing of duplicate serum specimens from 21 patients with Lyme disease and 10 healthy controls by a single reference laboratory using a commercially available immunoglobulin G western blot kit gave 100% concordant results for seroreactivity and highly reproducible results for the identification of individual bands. In contrast, Lyme urine antigen testing of 10 healthy control subjects gave contradictory results on aliquots of the same specimen in 8 of 10 cases and yielded consistently false-positive results in the other 2. At least one aliquot of each specimen was falsely positive. IGeneX, the manufacturer of the test, claims a 3% false-positive rate and a 95% ability to distinguish between a positive and negative population with an antigen cut-off level of ≥32 ng/mL. Our results do not support these claims and indicate that this test should not be used for the laboratory diagnosis of active or suspected Lyme disease. References 1. H. Hofmann, Lyme borreliosis—problems of serological diagnosis. Infection 24 (1996), pp. 470–472. Abstract-EMBASE | Abstract-MEDLINE | Order Document 2. S.L. Brown, S.L. Hansen and J.J. Langone, Role of serology in the diagnosis of Lyme disease. JAMA 282 (1999), pp. 62–66. Abstract-MEDLINE | Order Document | Full Text via CrossRef 3. L.L. Bakken, S.M. Callister, P.J. Wand and R.F. Schell, Interlaboratory comparison of test results for detection of Lyme disease by 516 participants in the Wisconsin State Laboratory of Hygiene/College of American Pathologists proficiency testing program. J Clin Microbiol 35 (1997), pp. 537–543. Abstract-EMBASE | Abstract-MEDLINE | Order Document 4. L.L. Bakken, K.L. Case, S.M. Callister et al., Performance of 45 laboratories participating in a proficiency testing program for Lyme disease serology. JAMA 268 (1992), pp. 891–895. Abstract-MEDLINE | Abstract-EMBASE | Order Document 5. S.W. Luger and E. Krauss, Serologic tests for Lyme disease. Interlaboratory variability. Arch Intern Med 150 (1990), pp. 761–763. Abstract-EMBASE | Order Document 6. Centers for Disease Control and Prevention, Case definitions for infectious conditions under public health surveillance: Lyme disease. MMWR 46 suppl RR-10 (1997), pp. 20–21. 7. Recommendations for test performance and interpretation from the Second International Conference on Serologic Diagnosis of Lyme Disease. MMWR. 1995;44:590–591. 8. N.S. Harris and B.G. Stephens, Detection of Borrelia burgdorferi antigen in urine from patients with Lyme borreliosis. J Spirochetal Tick-Borne Dis 2 (1995), pp. 37– 41. 9. J. Fleiss. Statistical Methods for Rates and Proportions, Wiley & Sons, New York (1981). 10. P. Tugwell, D.T. Dennis, A. Weinstein et al., Laboratory evaluation in the diagnosis of Lyme disease. Ann Intern Med 127 (1997), pp. 1109–1123. AbstractMEDLINE | Abstract-EMBASE | Order Document *1 Supported by grants from the National Institute of Allergy and Infectious Diseases (AI-65308) and from the Division of Research Resources supporting the General Clinical Research Center (RR-00054) at New England Medical Center. Correspondence should be addressed to Mark S. Klempner, MD, Department of Medicine, New England Medical Center, 750 Washington Street, Boston, Massachusetts 02111 The American Journal of Medicine Volume 110, Issue 3 , 15 February 2001, Pages 217-219 Intralaboratory reliability of serologic and urine testing for Lyme disease *1 Mark S. Klempner MD , a, Christopher H. Schmid PhDa, Linden Hu MDa, Allen C. Steere MDa, Gary Johnsona, Bilaal McCloud BSa, Richard Noring BSa and Arthur Weinstein MDb a Department of Medicine (MSK, CHS, LH, ACS, GJ, BM, RN), New England Medical Center, Boston, Massachusetts, USA b Department of Medicine (AW), George Washington University Medical Center, Washington, DC, USA Received 1 May 2000; revised 16 October 2000; accepted 16 October 2000. Available online 13 February 2001. Referred to by: Intralaboratory reliability of serologic and urine testing for lyme disease, The American Journal of Medicine, Volume 111, Issue 6, 15 October 2001, Pages 502-503 Boyd G. Stephensa and Nick S. Harrisa SummaryPlus | Full Text + Links | PDF (46 K) Article Outline • Material and methods • Study subjects • Sample collection • Serologic testing • Lyme urine antigen test • Statistical analysis • Results • Serologic test • Lyme urine antigen test • Discussion • References Laboratory testing for Lyme disease is controversial because of problems with test sensitivity and specificity, the lack of standardized reagents, and interlaboratory and intralaboratory variability [1, 2, 3, 4 and 5]. We determined the reliability of a serologic test and a urine test for Lyme disease, each performed in a reference laboratory, in control subjects and patients with Lyme disease who had posttreatment symptoms. Material and methods Study subjects We studied 10 healthy control subjects who had never had Lyme disease and 21 patients with a history of acute Lyme disease, as defined by the Centers for Disease Control and Prevention [6], who had chronic (>6 month’s duration) fatigue, musculoskeletal pain, or neurocognitive impairment despite treatment with recommended antibiotics. The study protocol was approved by the Human Investigation Institutional Review Boards of New England Medical Center and New York Medical College. All subjects gave written, informed consent. Sample collection Serum samples were obtained from all 21 patients and the 10 control subjects. One aliquot was immediately analyzed; duplicate aliquots were frozen at −70°C and tested within 6 months after collection. Midstream clean-catch urine samples were collected from the 10 control subjects into Vacutainer urine collection kits (Becton Dickinson, no. 36-4962) supplied by the manufacturer of the Lyme urine antigen test (IGeneX, Palo Alto, California). Each specimen was immediately aliquoted into five gray-top Vacutainer urine collection tubes, agitated to mix the sample with the preservative, and either packaged on ice and sent immediately for testing or stored at −70°C and tested within 6 months after collection. Aliquots of each sample were assayed separately. Frozen urine samples for offsite testing were packaged in insulated boxes containing refrigeration packets and shipped by overnight courier. All duplicate samples were sent blinded to the reference laboratories. Serologic testing Immunoglobulin G western blot assays for antibodies against Borrelia burgdorferi antigens were performed at New England Medical Center. In all cases, the immunoglobulin G Marblot strip test system kit (MarDx Diagnostics, Carlsbad, California) was used according to the manufacturer’s instructions. As recommended by the Centers for Disease Control and the Second National Conference on Serologic Diagnosis of Lyme Disease [7], strips that had 5 or more of the 10 significant bands were considered positive for specific immunoglobulin G antibody to B. burgdorferi. Lyme urine antigen test Urine testing was performed by IgeneX, the manufacturer of the Lyme urine antigen test. This test is based on an antigen capture-inhibition enzyme-linked immunosorbent assay that uses adsorbed polyclonal antibodies that bind to several antigenic moieties (31kDa, 34 kDa, 39 kDa, and 93kDa) of B. burgdorferi [8]. The test results are interpreted according to the antigen level in the sample: negative, <20 ng/mL; borderline, 20 to 31 ng/mL; positive, 32 to 45 ng/mL, and highly positive, >45 ng/mL. Statistical analysis Between-sample agreement was assessed with the kappa statistic for categories and the intraclass correlation for numeric values [9]. Kappa values >0.75 indicated excellent agreement; values <0.40 indicated poor agreement. Results Serologic test In all 10 control subjects, the initial western blot analysis yielded negative results. In three of four duplicate specimens analyzed, the same immunoreactive bands seen in the original aliquot were present; 1 duplicate specimen contained a 41-kDa band that was not present in the original aliquot. In the 21 patients with Lyme disease, the results of the initial western blot analysis were positive in 14 cases and negative in 7. Analysis of duplicate specimens yielded identical results in all 21 patients (κ = 1.0, Table 1). The same immunoreactive bands identified in the first analysis were present in 7 of the 14 seropositive duplicate samples; 5 samples had 1 additional band, and 2 samples had 2 additional bands. Repeat testing of the 7 seronegative samples showed fewer than 5 reactive bands in all samples. Table 1. Western Blot Testing for Immunoglobulin G Antibodies to B. burgdorferi in 21 patients with Posttreatment Symptoms of Lyme Disease legend Lyme urine antigen test The results of urine antigen testing in the 10 control subjects were not reliable (Table 2). The standard deviation of the antigen level in the five aliqots of each specimen ranged from 18 to 150 ng/mL. According to the manufacturer, the maximal standard deviation of a test result should be <9 ng/mL. The interpretation of these test results was also unreliable. It ranged from negative to highly positive for 6 specimens, from negative to positive for 1, from borderline to highly positive for 1, and from positive to highly positive for 2. The multiple category (negative, borderline, positive, highly positive) agreement was poor (κ = 0.10). Agreement was also poor when negative results were compared with all other categories (κ = 0.12) and when negative and borderline results together were compared with the other categories (κ = 0.18). Table 2. Results of Lyme Urine Antigen Testing in 10 Control Subjects without Lyme Disease Discussion Nonculture-based testing is the mainstay for laboratory diagnosis of Lyme disease. In patients with chronic symptoms of at least 6 months’ duration, the most appropriate serologic test for prior infection with B. burgdorferi is the immunoglobulin G western blot, which is recommended by the Centers for Disease Control as the final basis for determination of seroreactivity [7]. In a 1997 review of serologic testing for Lyme disease, there were no reports on the interlaboratory or intralaboratory consistency of this test [10]. Our study showed that testing of duplicate serum specimens from 21 patients with Lyme disease and 10 healthy controls by a single reference laboratory using a commercially available immunoglobulin G western blot kit gave 100% concordant results for seroreactivity and highly reproducible results for the identification of individual bands. In contrast, Lyme urine antigen testing of 10 healthy control subjects gave contradictory results on aliquots of the same specimen in 8 of 10 cases and yielded consistently false-positive results in the other 2. At least one aliquot of each specimen was falsely positive. IGeneX, the manufacturer of the test, claims a 3% false-positive rate and a 95% ability to distinguish between a positive and negative population with an antigen cut-off level of ≥32 ng/mL. Our results do not support these claims and indicate that this test should not be used for the laboratory diagnosis of active or suspected Lyme disease. References 1. H. Hofmann, Lyme borreliosis—problems of serological diagnosis. Infection 24 (1996), pp. 470–472. Abstract-EMBASE | Abstract-MEDLINE | Order Document 2. S.L. Brown, S.L. Hansen and J.J. Langone, Role of serology in the diagnosis of Lyme disease. JAMA 282 (1999), pp. 62–66. Abstract-MEDLINE | Order Document | Full Text via CrossRef 3. L.L. Bakken, S.M. Callister, P.J. Wand and R.F. Schell, Interlaboratory comparison of test results for detection of Lyme disease by 516 participants in the Wisconsin State Laboratory of Hygiene/College of American Pathologists proficiency testing program. J Clin Microbiol 35 (1997), pp. 537–543. Abstract-EMBASE | Abstract-MEDLINE | Order Document 4. L.L. Bakken, K.L. Case, S.M. Callister et al., Performance of 45 laboratories participating in a proficiency testing program for Lyme disease serology. JAMA 268 (1992), pp. 891–895. Abstract-MEDLINE | Abstract-EMBASE | Order Document 5. S.W. Luger and E. Krauss, Serologic tests for Lyme disease. Interlaboratory variability. Arch Intern Med 150 (1990), pp. 761–763. Abstract-EMBASE | Order Document 6. Centers for Disease Control and Prevention, Case definitions for infectious conditions under public health surveillance: Lyme disease. MMWR 46 suppl RR-10 (1997), pp. 20–21. 7. Recommendations for test performance and interpretation from the Second International Conference on Serologic Diagnosis of Lyme Disease. MMWR. 1995;44:590–591. 8. N.S. Harris and B.G. Stephens, Detection of Borrelia burgdorferi antigen in urine from patients with Lyme borreliosis. J Spirochetal Tick-Borne Dis 2 (1995), pp. 37– 41. 9. J. Fleiss. Statistical Methods for Rates and Proportions, Wiley & Sons, New York (1981). 10. P. Tugwell, D.T. Dennis, A. Weinstein et al., Laboratory evaluation in the diagnosis of Lyme disease. Ann Intern Med 127 (1997), pp. 1109–1123. AbstractMEDLINE | Abstract-EMBASE | Order Document *1 Supported by grants from the National Institute of Allergy and Infectious Diseases (AI-65308) and from the Division of Research Resources supporting the General Clinical Research Center (RR-00054) at New England Medical Center. Correspondence should be addressed to Mark S. Klempner, MD, Department of Medicine, New England Medical Center, 750 Washington Street, Boston, Massachusetts 02111 The American Journal of Medicine Volume 110, Issue 3 , 15 February 2001, Pages 217-219 Cardiovascular manifestations of Lyme disease • ARTICLE American Heart Journal, Volume 122, Issue 5, November 1991, Pages 1449-1455 Jafna Cox and Mel Krajden Abstract | Abstract + References Intrathecal antibody production in a mouse model of Lyme neuroborreliosis Libin Li1, Kavitha Narayan1, Elena Pak1 and Andrew R. Pachner , Department of Neurosciences, University of Medicine and Dentistry of New Jersey– New Jersey Medical School, 185 S. Orange Ave., Newark, NJ 07103, United States Received 27 September 2005; accepted 21 November 2005. Available online 4 January 2006. Abstract Intrathecal antibody (ITAb) production is a common feature of neurological diseases, yet very little is known about its mechanisms. Because ITAb is prominent in human Lyme neuroborreliosis (LNB), in the present study we established a mouse model of LNB to study ITAb production. We injected different strains of Borrelia burgdorferi into a variety of mouse strains by the intracerebral (i.c.) route to develop the model. Spirochetal infection and ITAb production were identified by complementary methods. This study demonstrates that the mouse model of LNB can be utilized to test hypotheses related to the mechanisms of ITAb production. Keywords: Antibody; Lyme ; Intrathecal Article Outline 1. Introduction 2. Materials and methods 2.1. Spirochetes 2.2. Mice 2.3. Antibody quantitation by enzyme-linked immunosorbent assay (ELISA) 2.4. Antibody deposition in brain parenchyma by immunoblotting 2.5. RNA isolation, reverse transcription (RT) and real-time reverse transcription– polymerase chain reaction (RT–PCR) 2.6. Quantitation of spirochetes in tissue by 16S RNA TaqMan RT–PCR 2.7. IgG RT–PCR for measurement of in situ antibody production 2.8. CXCL13 RT–PCR for measurement of in situ chemokine production 2.9. Number of antibody-secreting cells (ASCs) by ELISPOT and image analysis 2.10. Statistical analysis 3. Results 3.1. Experimental design 3.2. Quantitation of spirochetal infection 3.2.1. In naïve mice 3.2.2. In sensitized mice 3.3. Total IgG concentration in CSF and serum 3.3.1. After i.c. inoculation of naïve mice 3.3.2. After i.c. inoculation of sensitized mice 3.4. ITAb production of spirochete-specific IgG – measured by AI 3.4.1. WCS as antigen 3.4.2. DbpA as antigen 3.5. Antibody deposition in the brain 3.6. ITAb production of immunoglobulin as measured by in situ detection of IgG expression using IgG RT–PCR 3.6.1. In naïve mice 3.6.2. In sensitized mice 3.7. Detection of ASCs by ELISPOT 3.7.1. Total ASCs 3.7.2. B. burgdorferi-specific ASCs 3.8. In situ detection of CXCL13 expression using RT–PCR 4. Discussion Acknowledgements References 1. Introduction ITAb production is a hallmark of a variety of human CNS inflammatory and infectious diseases, occurring in more than 90% of multiple sclerosis (MS) patients and very frequently in chronic neurological infections such as Lyme neuroborreliosis (LNB) (Kaiser et al., 1993, Andersson et al., 1994, Pachner et al., 1998 and Yao et al., 2001). Several approaches have been used to study ITAb production (Wilske et al., 1991 and Picha et al., 2000). The most commonly used clinical measurement for ITAb production in infections of the CNS is that of assaying cerebrospinal fluid (CSF). CSF and serum are analyzed for either total immunoglobulin G (IgG) or antigen-specific IgG, and antibody index (AI), the comparison of antigen-specific immunoglobulin present in CSF relative to serum, is a standard measurement of ITAb (Steere et al., 1990, Kaiser and Lucking, 1993, Kaiser, 1995 and Picha et al., 2000) in clinical practice. However, given the fact that deposited immunoglobulin is detected in both peripheral tissues during chronic infection with Borrelia burgdorferi (Cadavid et al., 2003 and Pachner et al., 2004) and neural tissues after ovalbumin infusion in the brain (Knopf et al., 1998), it is possible that the immunoglobulin produced in the brain may reside in local parenchyma and interstitial fluids and not appear in CSF. Thus, CSF analysis alone might underestimate the magnitude of ITAb production. Our understanding of humoral immunity within the brain is fragmentary. Although some other basic mechanisms in neuroimmunology have been unraveled in the past few decades, a grasp of mechanisms of B cell activation, recruitment, differentiation and maintenance in CNS remains elusive (Reiber and Peter, 2001). Studies of rodent models utilizing infection with neurotropic viruses suggest that only activated, but not resting B cells, can infiltrate into the brain and induce local humoral immune responses (Tyor and Griffin, 1993 and Tschen et al., 2002). There is also evidence that peripheral lymphoid tissues, particularly in the deep cervical lymph nodes (DCLNs), might be an integral component of CNS antigen delivery and B cell priming (Knopf et al., 1998 and Harling-Berg et al., 1999). Knopf and his colleagues proposed the following hypothesis: after CNS infection, antigens in the brain drain to lymph nodes and are there presented to lymphocytes for priming. Activated B lymphoblasts then migrate back into the brain and differentiate into antibodysecreting cells (ASCs) when encountering the same antigen. This scenario might be accelerated if activated B cells already exist in the periphery. In human LNB (Pachner and Steere, 1984) and its animal model in Rhesus manaques (Pachner et al., 2001), chronic infection and inflammation usually occur in the nervous system after the dissemination of the causative spirochete to the CNS. The arm of the immune system primarily responsible for spirochetal clearance is the humoral immune response (Johnson et al., 1986 and McKisic and Barthold, 2000), a hypothesis supported by our previous report that in situ immunoglobulin and chemokine (CXCL13) production were identified in the sites of infection within tissues in the non-human primate (NHP) model (Narayan et al., 2005). These immunologically active areas found in nonlymphoid tissues are called ectopic germinal centers (Wolniak et al., 2004), and are related to the classic germinal center in secondary lymphoid tissues. Interactions between the pathogen and humoral immune response in the CNS need to be studied in greater depth. To address these questions, we established a mouse model of LNB, developed novel methods to measure ITAb production, and investigated the humoral mechanisms in this model. B. burgdorferi was injected intracerebrally to induce a CNS infection in various mouse strains. American or European strains of B. burgdorferi were used to infect either naïve or sensitized mice, and the results of ITAb production were compared among the strains. Mice with intracerebral (i.c.) inoculation of heat-killed spirochetes or intradermal (i.d.) inoculation of the bacteria served as controls. 2. Materials and methods 2.1. Spirochetes N40 and 297 are two Borrelia burgdorferi sensu stricto strains. N40 was originally isolated from ticks, subsequently passaged in mice, and isolated from infected mouse brain (Pachner and Itano, 1990); 297 is an American human CSF isolate (Goodman et al., 1991 and Fung et al., 1994). Both Pli and Pbi belong to the subspecies Borrelia garinii, and are CSF isolates from humans in Germany with LNB (Jauris-Heipke et al., 1993). Spirochetes were grown, counted, sonicated, and injected as previously described (Pachner and Itano, 1990). Heat-killed spirochetes were prepared by incubating live spirochetes in BSKII medium for 30 min at 57 °C. Heat-killed spirochetes were intact but non-mobile under dark-field microscopy. 2.2. Mice Outbred male Swiss–Webster mice were purchased from Taconic laboratories (Germantown, NY). Inbred C3H/HeJ and C57BL/6 mice were from Jackson Laboratories (Bar Harbor, ME). All mice were 4- to 8-week-old males, and were housed in isolated cages in the Research Resource Facility. Mice were allowed to adjust to the new environment for at least 1 week before infection. Mice were inoculated with B. burgdorferi either intradermally or intracerebrally. Both routes were reviewed and approved by the University Animal Care and Use Committee. Mice were first anesthetized with intraperitoneal (i.p.) injection of Syntex mouse cocktail (10 mg/ml ketamine, 1 mg/ml xylazine, 0.25 mg/ml acepromazine, 0.9% NaCl). I.d. injection was performed along the dorsal thoracic midline of mice, in multiple aliquots, with a total volume of 0.2 ml BSKII medium containing 106 spirochetes. For i.c. injection, 20 μl BSKII medium containing 106 spirochetes were injected using a 30-gauge needle. The needle was inserted 2 mm under the external surface of scalp skin in the right hemisphere 1 mm away from the bregma. For sensitization, mice were first inoculated by i.p. injection with B. burgdorferi whole cell sonicate (WCS) (20 μg WCS in 200 μl PBS per mouse). Two weeks later, the same mice were challenged by i.c. injection of the live agent. Mouse CSF was obtained by cisternal puncture performed by a modification of Ronald's method (DeMattos et al., 2002 and Fleming et al., 1983). After the animal was anesthetized, meninges overlying the cisterna magna were exposed, and the surrounding area was gently washed as clean as possible to prevent blood contamination. The animal was then placed on a platform in an inverted position. A 30-gauge needle was used to puncture the arachnoid membrane covering the cisterna. A polypropylene pipette was used to collect CSF by gentle pipetting. Depending on the age, about 20–30 μl of CSF was obtained from each mouse. Blood was obtained by cardiac puncture, and was allowed to clot to isolate sera. Perfusions were performed with 30 ml of PBS per mouse to remove the remaining blood from tissues. Brain, DCLNs, spleen, heart and bladder were collected in Fast Prep green tubes (Savant Instruments Inc, Holbrook, NY), each containing 1 ml of Trizol (Life Technologies, Grand Island, NY). Tissue samples were homogenized using the Fast Prep System (Savant Instruments Inc.) and stored at − 80 °C for RNA isolation. 2.3. Antibody quantitation by enzyme-linked immunosorbent assay (ELISA) Both total IgG and B. burgdorferi-specific IgG were determined in CSF and sera by ELISA as previously described (Pachner et al., 2002a). Capture ELISA was used to quantitate total IgG. Donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as coating antibody, and horseradish peroxidaseconjugated donkey anti-mouse IgG (Jackson) was used as detecting antibody. A purified murine IgG (Jackson) was serially diluted as a standard positive control on each plate. In quantitation of IgG specific for spirochete, coating antigens used in the experiments included B. burgdorferi WCS and decorin binding protein A (DbpA). To determine ITAb production, a serum sample with positive specific IgG response was serially diluted and used as reference standard on each plate. Using the upper titer as 100 units of IgG reactivity to the antigen, a linear standard curve was generated. Each sample was assigned an activity of arbitrary units (AU) according to the standard curve. Total IgG in sera and corresponding CSF were diluted to the same levels before anti-B. burgdorferi AU were determined. AI = AUCSF/AUserum. 2.4. Antibody deposition in brain parenchyma by immunoblotting Immunoblotting was performed as previously described (Cadavid et al., 2003). In brief, samples were collected, after thorough perfusion, from the mouse brain ipsilateral and contralateral to the injecting site. Cell lysis and protein extraction were performed using CelLytic MT (Sigma Saint Louis, MS). Protein concentrations were determined with the bicinchoninic acid protein assay (Pierce, Rockford, IL). Dot blots were prepared by spotting 0.3 to 3 μg in duplicate from each protein extract onto polyvinylidene difluoride membranes (BioRad, Hercules, CA). Goat anti-mouse polyvalent Ig (Sigma, 1:5000) and alkaline phosphatase-conjugated rabbit anti-goat IgG (Sigma, 1:5000) were used as primary and secondary antibodies respectively. A mouse IgG1 from the hybridoma line CRL-1605 (ATCC, Manassas, VA) and normal brain samples served as positive and negative control respectively. After incubation in fluorescence substrate enzyme-catalyzed fluorescence (Amersham Life Science, Piscataway, NJ) for 5 min, the membranes were scanned with a Typhoon 8600 scanner (Amersham). The results were analyzed by densitometry and were expressed as relative expression of ratios to normal brain. 2.5. RNA isolation, reverse transcription (RT) and real-time reverse transcription–polymerase chain reaction (RT–PCR) These methods were performed as previously described (Pachner et al., 2004, Bai et al., 2004, Cadavid et al., 2003 and Pachner et al., 2002b). In brief, total RNA was isolated from frozen tissue samples using the Trizol One Step Isolation method, and was incubated with DNAse. RT was performed in a Gene Amp PCR system 9700 (Perkin-Elmer Applied Biosystems, Foster City, CA). TaqMan RT–PCR was performed in an ABI 7000 Sequence Detection System (PE Applied Biosystems). 2.6. Quantitation of spirochetes in tissue by 16S RNA TaqMan RT–PCR Spirochetes in mouse tissues were quantitated using TaqMan RT–PCR as previously described. The 16S ribosomal of B. burgdorferi, found in all strains, was used as a target (Barbour et al., 1996, Wilske et al., 1992, Dever et al., 1992, Pachner et al., 2004 and Cadavid et al., 2003). For quantification of the spirochetal load in tissues, N40 spirochetes were cultured in vitro and the concentration was determined by counting under microscope. A solution with 106/ml spirochetes was then serially prepared with log10 dilutions, including a fresh medium as negative controls. Each diluted sample with known numbers of spirochetes was added to 100 mg of fresh brain tissues from noninfected mice and RNA was extracted from the mixtures. cDNA of these samples were run on each TaqMan test plate. A standard curve with a linear range coefficient of 0.95 was obtained when the threshold cycle (CT) values of the samples were plotted against known spirochetal concentration. Spirochetal load in tissue was estimated by referring the CT values of experimental samples to the standard curve. Results were expressed as estimated spirochetal numbers in each 100 mg tissue. Mouse GAPDH mRNA levels were used as a measure of RNA quality. GAPDH CT values which were greater than the mean ± 2 standard deviation (S.D.) of the CT values for the tissues in each assay were felt to be degraded and were not included in data analysis. Using normal mouse brain as negative control samples, mean ± 3 S.D. of calculated spirochetal number of these samples was used as a cut off value for negative spirochetal load. 2.7. IgG RT–PCR for measurement of in situ antibody production TaqMan RT–PCR was used to measure IgG mRNA expression in mouse tissues. This assay was validated in our prior studies in vitro and in vivo system (Narayan et al., 2005), in which the primers were derived from studies of IgG in baboons (Attanasio et al., 2002). Since IgG1 is one of the subtypes for murine Lyme borreliosis (Bockenstedt et al., 2003), we chose for our target sequence an IgG1 heavy chain (AF542525 on GenBank) for IgG RT–PCR. Primer and fluorogenic probe sequences are: upstream, CCTTGCATATGTACAGTCCCAGAA; downstream, CAGAGTAATGGTGAGCACATCCTT; probe, 6FAM, ATCTGTCTTCATCTTCCCCCCCAAAGCC–TAMRA. Mouse house keeping gene GAPDH (upstream, GGGAAGCCCATCACCATCTT; downstream, ACATACCCGGCCTC; probe, 6FAM, AGCGAGACCCCACTAACATCAAATGGG–TAMRA) was run with IgG in each test. IgG mRNA level was expressed as relative expression index of 2 to the power of − ΔCT, where − ΔCT = − (CT, IgG − CT, GAPDH). 2.8. CXCL13 RT–PCR for measurement of in situ chemokine production In situ CXCL13 expression was determined by RT–PCR. Primer and probe sequences are: upstream, AAATGTGAACTTGTAGCTCGTACTAACAA; downstream, ATTTTGGAAGCCTGCGTTTTTAC; probe, 6FAM, AGGTTTGCGATGGACTTCAGTTATTTTGCA–TAMRA. 2.9. Number of antibody-secreting cells (ASCs) by ELISPOT and image analysis We used ELISPOT to determine the ASCs in mouse tissues. Thorough perfusion was performed before tissue collection as described above. For spleen, tissue pieces were minced on frosted glass slides to obtain single cell suspensions. Ammonium chloride was used to lyse red blood cells in splenocytes. For the brain and heart, Percoll gradient procedures were used to isolate cerebral mononuclear cells (CMCs) as described previously (Tschen et al., 2002). CMCs were then counted, diluted and ELISPOT was performed according to manufacture's directions (KPL, Gaithersburg, MD). The ELISPOT plates were air-dried overnight before image analysis (Guerkov et al., 2003). Total ASCs were determined by using goat anti-mouse polyclonal IgG + IgM (KPL) to coat the plates and using Biotinylated goat anti-mouse Ig (Jackson) as detecting antibodies. B. burgdorferi WCS was used as coating antigen to detect B. burgdorferi-specific ASCs. We used a Series 1 Immunospot Image Analyzer (Cellular Technology, Cleveland, OH) to evaluate spots generated in ELISPOT assay. The Analyzer initially captured the images to an image file, following which the images were analyzed. A spot was considered an ASC when its color density exceeded background limits set based on the comparison between experimental and control wells, utilizing custom software (Cellular Technology). Data were recorded as spots per well. Results were expressed as number of ASCs in each mouse organ. 2.10. Statistical analysis Differences in mean concentration or density between groups were compared for statistical significance with Student's t test and Excel software. Statistical significance was established at p < 0.05. 3. Results 3.1. Experimental design Table 1 summarizes the experimental design of this study. We used different strains of B. burgdorferi, including American sensu stricto and European B. garinii strains, and a variety of mouse strains, both in- and outbred, to establish a mouse LNB model. Heat-killed spirochetes were also used in control groups. I.c. as well as i.d. injections were performed to infect mice. Infection of the brain and other tissues was documented by detection of Borrelia rRNA by RT–PCR. The analyses for ITAb production included: AI calculation, measurements of antibody deposition by immunoblot, IgG mRNA by RT–PCR, and ASCs by ELISPOT. In some experiments, sensitization of the peripheral immune system to spirochetal antigens before CNS infection was performed as a means of hastening the kinetics of B cell activation, as previously described (Knopf et al., 1998). Table 1. Experimental design Group Sensitized? Mouse straina Route of injection Spirochete live/heatkilled Spirochete strain Analysesb I-A No SW/C3/C57 i.c. Live N40/297 AI, 16S, IgG, E I-B No SW i.c. Heat-killed N40 AI, 16S, IgG I-C No SW i.d. Live N40 AI, 16S, IgG I-D No SW i.d. Heat-killed N40 AI, 16S, IgG I-E No SW i.c. Live Pli/Pbi AI, 16S, IgG II Yes C57 i.c. Live N40 AI, 16S, IgG, BLC, I, E a b Mouse strain: SW = Swiss–Webster, C3 = C3H/HeJ, C57 = C57BL/6. Analyses: AI = antibody index (CSF/sera); 16S = 16S RT–PCR for Borrelia quantitation; IgG = IgG mRNA by RT–PCR; BLC = BLC mRNA by RT–PCR; I = immunoblotting assay for antibody deposition analysis; E = ELISPOT for ASC detection. 3.2. Quantitation of spirochetal infection 3.2.1. In naïve mice Spirochetal distribution was determined by real-time RT–PCR of the 16S Borrelial rRNA in mouse tissues after inoculation. The concentration immediately after i.c. injection of live spirochetes was approximately 105 spirochetes in each 100 mg tissue (Table 2, Group I-A). Infection was then detected in other organs in the following weeks, predominantly in the DCLNs, heart and bladder. Although most injected spirochetes left the brain after 1 week, a low level of spirochetes were still detectable 3 weeks after injection. In contrast, heat-killed spirochetes injected intracerebrally were eliminated from the mouse brain after 1 week (Table 2, Group I-B). When the mice were inoculated with live spirochetes via the i.d. route, infection of the heart and bladder resulted; no spirochetes could be detected in the brain (Table 2, Group I-C). I.d. injection of heat-killed spirochetes resulted in no spirochetal accumulation in any tissues (Table 2 Group I-D). Interestingly, the i.c. route resulted in earlier and higher levels of spirochetes in the heart and bladder than the i.d. route. The i.d. route had previously been shown to result in significantly greater disseminated infection than the i.p. or intravenous (i.v.) routes (Pachner et al., 1992). Table 2. Spirochetal distribution in mouse organs after spirochetal inoculation Group I-A I-B I-C Tissues Weeks after inoculation 0 1 3 6 9 Brain 154,540 ± 11,922a 118 ± 12 86 ± 15 < 20b < 20 DCLN < 20 3339 ± 507 175 ± 25 < 20 < 20 Spleen < 20 118 ± 19 105 ± 1 22 ± 10 < 20 Heart 53 ± 3 3085 ± 215 1374 ± 404 1569 ± 349 1147 ± 512 Bladder < 20 2045 ± 155 861 ± 190 928 ± 279 698 ± 133 Brain 196,896 ± 39,893 46 ± 19 < 20 < 20 < 20 Heart < 20 < 20 < 20 < 20 < 20 Bladder < 20 < 20 < 20 < 20 < 20 Brain < 20 20 < 20 < 20 < 20 Heart < 20 < 20 1231 ± 691 1869 ± 387 291 ± 1 Group I-D I-E II Tissues Weeks after inoculation 0 1 3 6 9 Bladder 20 58 ± 21 72 ± 44 140 ± 25 < 20 Brain < 20 < 20 < 20 < 20 < 20 Heart < 20 32 < 20 < 20 < 20 Bladder < 20 < 20 < 20 < 20 < 20 Brain 27,935 ± 749 < 20 < 20 < 20 NDc Heart 92 ± 0 156 ± 74 1419 ± 231 355 ± 138 ND Bladder < 20 211 ± 0 316 ± 76 < 20 ND Brain 54,810 ± 4371 < 20 < 20 < 20 ND Heart < 20 < 20 < 20 < 20 ND Bladder < 20 < 20 < 20 < 20 ND Spirochetal distribution in each 100 mg mouse tissues determined by real-time TaqMan PCR. a Numbers are the calculated spirochetal load referring to the standard curve, representing the mean of two to six separated experiments for each time point ± standard error of the mean (S.E.M.). n > 4 animals per time point. b Cut off value for negative spirochetal load (see ""Materials and methods""). c ND = not done. The American strains of Borrelia, N40 and 297, were more infective in mice than the European strains Pli and Pbi. After injection, European strains were cleared from the mouse brain and systemic tissues earlier than American strains (Table 2, Groups I-A and I-E). This observation was also consistent with our previous report (Pachner et al., 2004). 3.2.2. In sensitized mice In Group II, mice were sensitized by i.p. injection with spirochetal WCS 2 weeks before i.c. injection with live spirochetes. In this group, the spirochetes were cleared from the mouse brain and systemic tissues earlier (Table 2, Group II), indicating accelerated clearance of infection and prevention of dissemination. 3.3. Total IgG concentration in CSF and serum 3.3.1. After i.c. inoculation of naïve mice Total IgG, quantitated by capture ELISA, increased more than 300% both in sera (Fig. 1A) and in CSF (Fig. 1B) of Group I-A mice. The combination of live spirochetes and the i.c. route resulted in significantly higher IgG concentrations in both CSF and serum than any other combination (p < 0.05). American strains induced a higher IgG response than European strains. (74K) Fig. 1. Increased total IgG concentrations in mouse sera and CSF after spirochetal infection. Data represent mean ± S.E.M. (A) Total IgG in sera of Group I mice. aTotal IgG was significantly higher in Group I-A than in Group I-B, I-C, and I-D for each time point after infection. bTotal IgG was significantly higher in Group I-E than in Group I-B, I-C, and I-D at week 6 after inoculation. (B) Total IgG in CSF of Group I-A was significantly higher than in Groups I-B, I-C, and I-D mice. (C) Accelerated IgG response in sera of Group II mice. *p < 0.05. n > 4 animals per time point. 3.3.2. After i.c. inoculation of sensitized mice The increase in total IgG after infection was accelerated in sensitized animals relative to that of naïve animals (Fig. 1C). While the IgG level continued to increase in naïve mice after the second week of infection, sensitized mice reached a plateau 2 weeks after i.c. infection and then started to decrease after 4 weeks. 3.4. ITAb production of spirochete-specific IgG – measured by AI 3.4.1. WCS as antigen AI values were calculated from the CSF/serum quotients for antibody reactive to B. burgdorferi WCS. The AI was < 0.6 when infection was systemic, as in Groups I-C and I-D. Levels above 1.0 were consistent with ITAb production. This level of AI was observed only in animal Group I-A (Fig. 2A), with the AI being 1.0 3 weeks after infection, and then continuing to rise to a peak of 1.4. I.c. inoculation of European strains also resulted in ITAb (AI > 1.0 at week 6). (64K) Fig. 2. Spirochete-specific IgG responses after spirochetal infection in mice, as expressed by AI. Data represent mean ± S.E.M. (A) AI was analyzed in Group I mice using WCS as antigen. ITAb was identified in Group I-A and I-E by elevated AI values. AI values were significantly higher in Group IA than in Group I-B, I-C, and I-D at week 3 and week 9. (B) AI was analyzed in Group II mice using WCS as antigen. AI reached 1.0 at day 5 in sensitized mice, but at day 21 in naïve mice, indicating accelerated ITAb production in sensitized mice. (C) AI was analyzed in Group I mice using DbpA as antigen. AI > 1 was found at week 9 of Group I-A mice only. *p < 0.05. n > 4 animals per time point. ITAb generation was accelerated in sensitized mice with AI values above 1 occurring by 5 days after i.c. injection of spirochete. ITAb production persisted throughout the duration of the experiments (i.e. 42 days) in these animals (Fig. 2B). 3.4.2. DbpA as antigen DbpA, a lipoprotein of B. burgdorferi predominantly expressed in vivo (Cassatt et al., 1998 and Pachner et al., 2002a) was tested in this model in order to determine whether DbpA was a major antigen driving the intrathecal antibody response. As predicted from previous data showing that DbpA is expressed in vivo and not during in vitro growth, when recombinant DbpA was used in the ELISA to measure B. burgdorferi-specific IgG, no anti-DBPA antibody was detected in mice inoculated with heat-killed spirochetes (Fig. 2C, Group I-B and I-D). In contrast, an increasing level of anti-DbpA antibody was observed in both serum and in CSF in animals infected with live spirochetes. However, the magnitude of the AI for DbpA was not as high as for WCS, indicating that DbpA does not appear to be a selectively important antigen relative to other spirochetal antigens in driving the intrathecal antibody response within the first few months of infection. 3.5. Antibody deposition in the brain We used quantitative densitometry of fluorescent dot blots to determined whether there were antibody deposited in the parenchyma of mouse brain, which may not appear in CSF. The tissue samples were collected from the mouse brain 2 weeks after i.c. infection, either on the injecting site (ipsilateral) or away from injecting site (contralateral). The whole protein extracts were dot blotted and probed with IgGspecific antibodies. Fig. 3 showed the result of relative fluorescence of each dot measured by densitometry. The amounts of IgG were 3.1 fold higher (p = 0.003) in the infected brain tissue ipsilateral to injecting and 2.3 fold higher (p = 0.008) in tissue contralateral to injecting site than in normal brain, respectively. This analysis confirmed our hypothesis that antibody produced locally in the brain can be deposited in parenchyma, and thus may not be measurable by analysis of the CSF compartment. (25K) Fig. 3. IgG deposition in perfused brain parenchyma from spirochetal-infected mice, as determined by quantitative dot blot analysis. Bars represent standard error. Relative fluorescence of each dot was measured by densitometry with a Typhoon 8600 scanner. Background readings were subtracted from the density values of experimental samples. **P < 0.01 vs. normal brain by Student's t test. 3.6. ITAb production of immunoglobulin as measured by in situ detection of IgG expression using IgG RT–PCR 3.6.1. In naïve mice We also performed quantitative real-time RT–PCR to examine IgG mRNA levels during the course of infection, an assay of IgG production in tissues validated in prior studies in human and non-human primate Lyme neuroborreliosis (Narayan et al., 2005). The data was expressed as a relative expression index (see detail in Materials and methods). In mice injected with live spirochetes via the i.c. route (Group I-A), IgG mRNA increased in the brain about 2 weeks after inoculation, and remained elevated for about 9 weeks post infection (p.i.) (Fig. 4A). IgG expression was also up-regulated in nonneural tissues. In DCLNs, IgG production began increasing by 1 weeks p.i., plateaued, and then returned to baseline by week 6 (Fig. 4B), a process consistent with the hypothesis of earlier immune activity in DCLNs than in the brain during CNS infection. In contrast, spleen and heart IgG production remained elevated through 14 weeks (Fig. 4C and D). As expected, IgG mRNA content in spleen and lymph node at baseline, prior to infection, was substantially higher, by 3–4 logs, than in brain or heart, since IgG expression is a feature of the baseline state in lymphoid, but not non-lymphoid tissues. (41K) Fig. 4. IgG expression in tissues of Group IA mice after spirochetal infection. The organs tested include (A) brain, (B) DCLN, (C) spleen and (D) heart. Open circles (○) represent relative IgG expression in each mouse organ, expressed as 2− CT(IgG − GAPDH). Mean mRNA values are represented as horizontal bars. n > 4 animals per group. 3.6.2. In sensitized mice As with other measures of immunoglobulin production, IgG expression was accelerated in infection of sensitized (Group II) mice (Fig. 5) relative to infection of naïve (Group I) mice. In contrast to naïve mice in which elevated IgG mRNA was not detected in the brain until 2 weeks after the i.c. injection, in sensitized mice, IgG mRNA increased rapidly, and was elevated 5 days p.i. (30K) Fig. 5. IgG expression in the brain of Group II mice after spirochetal infection. Accelerated IgG upregulation was found in sensitized mice. Open circles (○) represent relative IgG expression in each mouse brain, expressed as 2− CT(IgG − GAPDH). Mean mRNA values are represented as horizontal bars. n > 4 animals per time point. 3.7. Detection of ASCs by ELISPOT 3.7.1. Total ASCs We used ELISPOT to determine the number of ASCs in the tissues of sensitized and naïve mice. Splenocytes and CMCs were isolated from infected as well as normal tissues respectively, and their release of Ig was imaged and evaluated in the ELISPOT assay. Fig. 6 gives an image result of spots generated from CMCs of infected and normal brains. In sensitized animals, ASCs in the brain in the absence of i.c. inoculation were nearly undetectable, but high numbers of ASCs were then detected in the brain by day 7 (the first time point we measured after the i.c. infection), after which the numbers declined (Table 3). ASC numbers in the spleen peaked with a delay relative to the brain, with the maximal number observed at week 3. In infected hearts of naïve animals, high level of ASCs persisted for the duration of the experiment, i.e. 14 weeks, correlating with high concentration of spirochetes in this target tissue throughout the course of infection. (39K) Fig. 6. ELISPOT results of ACSs in mouse brain. Spots caught by image system from each ELISPOT plates, showing positive ACSs in infected brains (4 weeks after infection) vs. normal control in duplicates. Table 3. Number of total ASCs in mouse tissues Tissues Weeks after i.c. infection 0 1 2 3 4 6 14 Tissues Weeks after i.c. infection Group II Group I-A 0 1 2 3 4 6 14 Brain 8a 589 177 315 225 61 86 Spleen (×1000) 141 257 324 714 449 434 340 Heart 8 NDb 24 ND ND 865 451 Total ASCs in mouse tissues determined by ELISPOT. Samples from Group II mice were used to detect total ACSs in the brains and spleens; ACSs in the hearts were detected from mice without sensitization. To obtain adequate CMCs, at least 4 brains were pooled from same group in each test. Numbers represent total ACSs per organ. a Number represent total ACSs in each mouse organ. b ND = not done. 3.7.2. B. burgdorferi-specific ASCs B. burgdorferi WCS was used as the antigen in the ELISPOT assay to determine the B. burgdorferi-specific ASCs in the brain and spleen in sensitized mice. Table 4 showed the percentage of ASCs producing Ig specific for the WCS of B. burgdorferi within 19 days after i.c. infection. There were no B. burgdorferi-specific ASCs found in the brain prior to i.c. inoculation. The percentage then increased sharply and peaked at 1 week after i.c. infection. Only a small percentage of ASCs was B. burgdorferi-specific in the spleen. The percentages in other nonneural organs, such as lymph nodes and bone marrow, were also low (data not show). Table 4. The percentage of B. burgdorferi-specific ASC in the brain and spleen of sensitized mice Tissues Days after i.c. infection 0 6 11 13 19 Brain 0 45.3 21.7 18.6 17.2 Spleen 1.6 4.3 5.5 2.2 7.4 The percentage of ASCs that were B. burgdorferi-specific determined by ELISPOT. Samples from Group II mice were used. n > 4 mice per time point. 3.8. In situ detection of CXCL13 expression using RT–PCR CXCL13 is an important chemokine for specific anti-spirochetal immunity in target tissues of Lyme borreliosis in the non-human primate (Pachner et al., 2002b and Narayan et al., 2005). We next tested whether CXCL13 was involved in ITAb production and IgG production in infected tissues by measuring CXCL13 expression in infected mouse brains and hearts. In both naïve and sensitized animals after infection, no significantly elevated CXCL13 mRNA levels were observed relative to baseline expression. This finding was different from our prior findings in the nonhuman primate system in which infected tissues had very high levels of CXCL13. In order to investigate these negative results in mice, we stimulated mouse splenocytes with spirochetal WCS, a potent inducer in vitro expression of a variety of cytokines (Habicht et al., 1991 and Wooten et al., 1996), and a stimulus markedly increasing CXCL13 expression in human PBMCs (Narayan et al., 2005). CXCL13 mRNA levels in the mouse cells did not vary after stimulation, confirming the results from the measurement of CXCL13 in vivo. 4. Discussion The goal of this study was to establish a mouse model of ITAb production. Our studies used a variety of techniques: ELISA to measure spirochete-specific antibody in CSF, immunoblotting to detect antibody deposition in brain parenchyma, IgG RT– PCR to measure in situ IgG expression in mouse brain, and ELISPOT to detect ASCs in the brain. These studies confirm that ITAb production is a feature of murine Lyme neuroborreliosis (mLNB), and the ITAb response is driven by the CNS pathogen. ITAb production is the most consistent diagnostic finding of neurological infectious and inflammatory diseases, yet very little is known about it. To date, few rodent models for CNS inflammation are suitable for the study of ITAb production. In the most commonly used rodent model for neurological inflammation, EAE (experimental allergic encephalomyelitis), ITAb production is not clearly demonstrable: antibody present in the CSF is likely synthesized in the periphery, not in the CNS (Whitacre et al., 1982, Whittaker and Whitacre, 1984, Gallo et al., 1989 and Rostrom et al., 2004). Although standard rodent models of Lyme disease have been developed (Barthold et al., 1988, Barthold et al., 1993 and Pachner, 1990), and inoculation of animals with B. burgdorferi usually results in chronic infection and systemic involvement such as carditis and arthritis (Garcia-Monco et al., 1990, Barthold et al., 1990, Moody and Barthold, 1991, Cadavid et al., 1994 and deSouza et al., 1993), these models are not suitable to study ITAb production, because the CNS is not usually infected after spirochete infection in the periphery. Our development of this model in mice was prompted by the high incidence of ITAb production in human LNB and the fact that mice infected intracerebrally with neurotrophic viruses have provided good models of ITAb production (Tyor et al., 1992, Lin et al., 1999 and Tschen et al., 2002). While viruses are difficult to clear once systemically disseminated, B. burgdorferi is a treatable pathogen, capable of being cleared by antibiotic treatment. Thus, mLNB is a potentially excellent model for addressing the issues of interactions between ITAb production and the pathogen. In order to optimize mLNB model for ITAb production, we first investigated the conditions for chronic infection induced by the spirochetes. A surprising finding in our study was that i.c. injection outperformed the i.d. route in leading to the maximal dissemination and growth of spirochetes. This correlates with the antibody quantitation results which demonstrated that the i.c. route resulted in higher systemic levels of spirochete-specific antibodies (p < 0.05). This finding, that antigen or pathogen delivered into the brain provokes a higher level of infection or a higher level of anti-pathogen antibodies, has been previously seen with other antigens and pathogens (Gordon et al., 1992, Kovacevic-Jovanovic et al., 1997 and Cserr et al., 1992). Some variables other than route of infection did not substantially affect the magnitude of ITAb production. Varying the mouse strains infected in this study did not appear to affect the outcome. Since C57BL/6 and C3HeJ are very commonly used strains in the murine models of Lyme disease (Barthold et al., 1993), we used these two inbred mice, as well as outbred Swiss–Webster in this study, and comparable spirochete distribution and ITAb production were found among these strains. Given previous data that European strains of B. burgdorferi are more neuroinvasive than American strains (Pachner et al., 2004, Lin et al., 2001 and van Dam et al., 1993), we tried infecting mice with the European B. burgdorferi strains Pbi and Pli in this mouse model, but direct injection of these strains into the brain did not produce a more neuropersistent disease than the American strains. I.c. injection of B. burgdorferi induced humoral immune responses not only in the CNS but also in infected peripheral tissues such as heart, bladder, and in professional immune organs such as the spleen, lymph nodes, and bone marrow. In all of these tissues, the IgG produced during bacterial infection included antigen-specific antibody as well as antibodies that had no specificity to the antigens. Studies on patients with LNB have indicated that the intrathecally produced IgG fraction accounted for more than 90% of total IgG in CSF (Reiber and Peter, 2001). In the blood compartment, in contrast, total IgG might increase only a few percent in response to an infection. In our mLNB model, however, serological results revealed over a 3-fold increase in total IgG both in CSF and in blood. These data are consistent with the observation that B. burgdorferi infection of mice induces polyclonal B cell activation, which results in production of a broad array of antibodies (Schoenfeld et al., 1992, Yang et al., 1992 and Tai et al., 1994). We detected ITAb production in mice initially by measurement of the antibody index, AI. IgG AI is a standard measurement of ITAb production and is used in Europe as the primary diagnostic assay for LNB (Hansen and Lebech, 1992) (Steere et al., 1990). In our mouse model, the AI value was 1.4 at week 9 in Group I-A mice (Fig. 2A), whereas in control groups this number was 0.3–0.7. Our data suggests that ITAb synthesis occurred in mice with i.c. infection, while the antibody detected in CSF of control mice was transferred passively from blood. The AI values induced by spirochetal infection in the mouse brain are relatively lower than that of other models (Knopf et al., 1998), likely due to the lack of persistence of large numbers of spirochetes in the brain. We also noticed that the AI curves of intracerebrally infected mice were still sloping up at week 9, and elevated IgG mRNA level persisted too. This suggests that ITAb is not able to completely clear the brain of spirochetes and low concentrations of B. burgdorferi or B. burgdorferi antigens are present persistently to drive the antibody production in the brain. Our experimental procedure for spirochetal distribution measurement may also have underestimated the actual spirochetal load in the CNS. Our previous experiments found spirochete localized to leptomeninges and duramater of non-human primates (Bai et al., 2004), areas which we did not include in mouse tissue analysis because they are difficult to isolate. Our results support the hypotheses about the importance of humoral immunity in virus infection within the CNS in which low levels of virus persist in the brain during chronic infection and drive antibody production (Ramakrishna et al., 2002, Tyor et al., 1992 and Tyor and Griffin, 1993). More detailed study of long term spirochetal infection is in progress in our laboratory. Since IgM has been reported as the dominant intrathecal antibody class in LNB (Reiber and Peter, 2001), studies on IgM in mLNB are in progress in the lab. Our data suggest that analysis of CSF for AI significantly underestimates the degree of ITAb production. Evidence comes from the immunoblotting results for antibody deposition analysis in the brain. IgG deposited in the infected brain parenchyma is significantly higher than in normal brain (p < 0.01), and the amount is highest in the site of injection. This finding indicates that some IgG produced locally in the brain may be deposited in brain parenchyma and would thus not be detected in CSF analysis. This reasoning prompted us to develop new methods in the laboratory to analyze ITAb production in the model. The Ig RT–PCR and the ELISPOT data from our studies are highly consistent with the hypothesis that these direct measures are more robust indicators of ITAb production than the indirect measurement of ITAb production by CSF analysis. IgG mRNA level was up-regulated in the brain during the early course of spirochetal infection, and started to drop off after 9 weeks. In the systemic target organ of heart, in contrast, IgG mRNA kept increasing even after 14 weeks. The similar patterns of responses were also observed when ASCs were detected by ELISPOT. These observations support our hypothesis that pathogen drives the antibody responses in this mLNB model. The ELISPOT data also confirms the results of Bergmann and Stohlman and co-workers (Tschen et al., 2002 and Ramakrishna et al., 2002) who studied ITAb in mouse hepatitis virus infection. They found ELISPOT to be an excellent measure of ITAb production, which developed slowly over weeks. We detected a high percentage of ASCs specific to B. burgdorferi WCS in the early phase of infection in the brain, indicating that an antigen-specific humoral immune response is involved in pathogen clearance from the CNS. Further studies in our laboratory will continue to use this sensitive technique to enumerate antigen-specific ASCs, and to identify which isotypes are being produced. We also are investigating whether measuring oligoclonal banding of CSF immunoglobulin may be a better measure than AI for analysis of ITAb production. Under normal circumstances of CNS infection, ITAb production can be a very slowly developing process. Initial antigen processing within the brain is considered to be poor, and lymphatic drainage from the brain into cervical lymph nodes is not highly efficient (Cserr et al., 1992). Our studies in mLNB certainly were consistent with this, with antibody production slowly increasing in animals naïve to the spirochete. It is hypothesized that in animals with i.c. infection, an initial Th2-type antibody response to CNS-derived antigen slowly develops first in cervical lymph nodes and spleen. Antigen-specific B lymphoblasts from these lymph nodes then can traffic back into CNS and develop their effector function (Knopf et al., 1998). Our data confirms Knopf's previous observations that this immune process can be substantially accelerated if the animal is preimmunized peripherally. However, one finding we could not confirm in this murine study was the importance of CXCL13 in the establishment of antibody production outside of lymphoid tissue in Lyme borreliosis, which in the initial observation involved work in humans and non-human primates (Narayan et al., 2005). Given our findings in mice, it appears likely that mice and primates have different uses for this chemokine in chronic B. burgdorferi infection. Thus, ITAb production is a feature of mLNB, induced by i.c. infection. To our knowledge, this is the first demonstration of ITAb in a mouse model by an agent other than viruses. Our initial identification of ITAb by CSF measurement was confirmed by the detection of parenchymal antibody, elevated IgG expression and ASCs in the brain. 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Available online 6 March 2006. Abstract The manifestations of Lyme disease, caused by Ixodes spp. tick-transmitted Borrelia burgdorferi, range from skin infection to bloodstream invasion into the heart, joints and nervous system. The febrile infection human granulocytic anaplasmosis is caused by a neutrophilic rickettsia called Anaplasma phagocytophilum, also transmitted by Ixodes ticks. Previous studies suggest that co-infection with A. phagocytophilum contributes to increased spirochetal loads and severity of Lyme disease. However, a common link between these tick-transmitted pathogens is dissemination into blood or tissues through blood vessels. Preliminary studies show that B. burgdorferi binds and passes through endothelial barriers in part mediated by host matrix metalloproteases. Since neutrophils infected by A. phagocytophilum are activated to release bioactive metalloproteases and chemokines, we examined the enhanced B. burgdorferi transmigration through vascular barriers with co-infection in vitro. To test whether endothelial transmigration is enhanced with co-infection, B. burgdorferi and A. phagocytophilum-infected neutrophils were co-incubated with EA.hy926 cells (HUVEC-derived) and human brain microvascular endothelial cells in Transwell™ cultures. Transmigration of B. burgdorferi through endothelial cell barriers was determined and endothelial barrier integrity was measured by transendothelial electrical resistivity. More B. burgdorferi crossed both human BMEC and EA.hy926 cells in the presence of A. phagocytophilum-infected neutrophils than with uninfected neutrophils without affecting endothelial cell integrity. Such a mechanism may contribute to increased blood and tissue spirochete loads. Keywords: Lyme disease; Borrelia burgdorferi; Anaplasma phagocytophilum; Endothelial cells; Neutrophils; Co-infection Article Outline 1. Introduction 2. Materials and methods 2.1. Cultivation and preparation of cell-free A. phagocytophilum 2.2. Preparation of A. phagocytophilum-infected neutrophils 2.3. Spirochetes 2.4. Human brain microvascular endothelial cells (human BMEC) 2.5. Human systemic endothelial cells 2.6. Endothelial monolayer transmigration assay 2.7. Quantitative PCR 2.8. Statistical tests 3. Results 3.1. Effects of co-incubation of B. burgdorferi with A. phagocytophilum-infected neutrophils on the integrity of endothelial cell barriers 3.2. Borrelia burgdorferi traversal of endothelial cells in the presence of A. phagocytophilum infected-neutrophils or uninfected neutrophils 4. Discussion 5. Uncited references Acknowledgements References 1. Introduction Lyme disease is now the most frequently reported arthropod-borne infection in North America and Europe (CDC, 2002). The bacteria, Borrelia burgdorferi, which are transmitted to humans by the bite of infected ticks of the Ixodes persulcatus complex, can infect many tissues including the skin, heart, joints, eye and in addition, the peripheral and central nervous systems (Steere, 1989 and Kalish, 1993). As the diversity of clinical presentations for Lyme disease has been recognised, it has been suggested that concurrent infections by other tick-borne pathogens may influence the natural course of disease, leading to more severe infection, persistence, even refractoriness to effective therapies (Belongia, 2002 and Krause et al., 2002). Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (HGA) is also transmitted by I. persulcatus-complex tick bites and accumulating data suggest that co-infection is not infrequent. In North America and also in Europe, approximately 10% of all patients with Lyme disease or HGA have evidence of co-infection with other pathogens (Thompson et al., 2001). Although it is unclear whether co-infections contribute to the comorbidity, in mice commitment of immunity toward Th1 reactions directed against the obligate intracellular A. phagocytophilum allows higher spirochetemia, tissue loads, longer persistence and increased disease with simultaneous B. burgdorferi infections (Thomas et al., 2001). Penetration into the blood and out of the bloodstream into sites of infection is a necessary component for dissemination of pathogens of Lyme disease and HGA, hence interactions of both pathogens at the level of blood–endothelial cell interface are critical determinants of dissemination and disease. Borrelia burgdorferi penetration of endothelial cells results in part from the actions of endothelial cellderived metalloproteases, after binding of spirochetes to intracellular junctions. Anaplasma phagocytophilum-infected neutrophils are induced for protracted degranulation, resulting in the elaboration of biologically active molecules, including chemokines, cytokines and metalloproteases (Choi et al., 2003). Owing to the production of metalloproteases from A. phagocytophilum-infected neutrophils, we hypothesised that concurrent infection with B. burgdorferi enhances penetration of the spirochete through blood–brain barrier (BBB) and systemic endothelial cell barriers. We tested this non-immunological mechanism using in vitro models of the human BBB and systemic endothelial cells. 2. Materials and methods 2.1. Cultivation and preparation of cell-free A. phagocytophilum The A. phagocytophilum strain Webster was cultivated in HL-60 cells in RPMI 1640 medium (GIBCO-BRL) supplemented with 5% FCS and 2 mM L-glutamine (GIBCOBRL) at 37 °C (Choi et al., 2003; Choi and Dumler, 2004). Cell-free A. phagocytophilum was prepared by sonication of heavily infected HL-60 cells (Branson Sonifier, VWR Scientific) at duty cycle of 70 and output control of 3 (Choi et al., 2003 and Garyu et al., 2005). These bacteria were washed and used immediately to infect 5×105 to 106 human peripheral blood neutrophils. Mockinfected cells were incubated with medium only. 2.2. Preparation of A. phagocytophilum-infected neutrophils Human peripheral blood neutrophils and monocytes were isolated from EDTAanticoagulated blood of healthy donors by dextran sedimentation followed by Ficoll density gradient centrifugation (Histopaque; Sigma). The contaminating residual erythrocytes present in the neutrophil preparations were lysed by exposure to hypotonic saline (0.2% NaCl) for 30 s and then adjusted back to isotonic conditions with hypertonic saline (1.8% NaCl) prior to washing in tissue culture medium. Neutrophil purity was confirmed to be ≥95% by Ramanowsky staining (Hema-3; Biochemical Sciences, Inc., Swedesboro, NJ), and the viability of cells was determined to be >98% by trypan blue dye exclusion. Human neutrophils were obtained with the approval of the Johns Hopkins University School of Medicine Institutional Review Board and in compliance with all relevant federal guidelines and institutional policies. Some of the neutrophils were suspended in medium M199 supplemented in 20% FCS and then incubated at 37 °C in 5% CO2 in a humidified environment. 2.3. Spirochetes Low-passage (less than five in vitro passages) B. burgdorferi was cultured at 34 °C in BSK II medium containing 10% rabbit serum as described by Barbour (1984). In our study, we used B. burgdorferi 297, a strain originally isolated from human cerebrospinal fluid (Leveris et al., 1995). The bacteria were examined for motility with dark-field microscope to verify viability and that the organisms were thoroughly dispersed at the start of all the assays. 2.4. Human brain microvascular endothelial cells (human BMEC) Human BMEC primary cultures used in this study were described previously (Persidsky et al., 1997 and Stins et al., 1997; Grab et al., 2004 and Grab et al., 2005). The cells were cultured in Medium 199 (GIBCO) supplemented with 20% heatinactivated FCS and 1×Glutamax (GIBCO). The cells were grown to confluence on 6.5-mm-diameter collagen-coated Costar inserts with a pore of size of 3.0 μm until transendothelial electrical resistance (TEER) measurements exceeded 25 Ω×cm2 (Grab et al., 2004). 2.5. Human systemic endothelial cells The EAhy926 endothelial cell line derived from fusion of A549 cells with primary human umbilical vein endothelial cells have also been described previously (Edgell et al., 1983). These were grown in high-glucose (4.5 g/l) DMEM supplemented with 20% heat-inactivated FCS, 1×HT supplement and 1×Glutamax (all from GIBCO). The cells were grown to confluence on 6.5-mm-diameter collagen-coated Costar Transwell™ inserts with a pore of size of 3.0 μm until transendothelial electrical resistance reached stable values <12 Ω×cm2 (Grab et al., 2004). 2.6. Endothelial monolayer transmigration assay Triplicate wells with and without endothelial cells received the following: medium only; B. burgdorferi only (ranging from 2×105 to 107); uninfected neutrophils only (2×105); A. phagocytophilum-infected neutrophils only (2×105); B. burgdorferi (2×105 to 107) and uninfected neutrophils (2×105); B. burgdorferi (2×105 to 107) and A. phagocytophilum-infected neutrophils (2×105). After 5 h incubation, the TEER was re-measured and aliquots were removed from the wells beneath the inserts. The quantity of transmigrating B. burgdorferi was determined for each well by either dark-field counting in a haemocytometer for initial experiments and then by quantitative PCR for subsequent experiments. The net transmigration of each barrier was calculated as a percentage of the number of spirochetes that crossing inserts with cells relative to the number crossing inserts without endothelial cells. 2.7. Quantitative PCR Borrelia burgdorferi DNA in transmigrated culture medium (below Transwells) was prepared using either the GenoM-48 DNA robot or using the Qiagen DNA extraction kit, both according to the manufacturers' instructions. Borrelia burgdorferi quantification was performed using quantitative real-time PCR targeting the single copy chromosomal flaB gene (Leutenneger et al., 1999) using the primers B.398f (GGGAAGCAGATTTGTTTGACA) and B.484r (ATAGAGCAACTTACAGACGAAATTAATAGA) with the fluorescent probe B.421p (FAM- ATGTGCATTTGGTTATATTGAGCTTGATCAGCAA-TAMRA). Amplifications were performed using either an ABI Taqman 7700 or a BioRad iCycler iQ5 Multicolor Real Time PCR detector. For standard curve, the flaB gene amplicon was cloned, plasmid DNA containing the insert was prepared and the DNA concentration was carefully measured to allow a precise determination of the copy number of flaB in each standard. Final concentrations of transmigrated B. burgdorferi were determined using the CT method as per software of the Taqman or iQ5 instruments. 2.8. Statistical tests Where appropriate, means of transmigration or TEER values were compared using one-tailed, unpaired or paired Student's t-tests; a P-value <0.05 was considered significant. 3. Results 3.1. Effects of co-incubation of B. burgdorferi with A. phagocytophilum-infected neutrophils on the integrity of endothelial cell barriers The integrity of EAhy.926 and the human BMEC barriers after co-incubation of B. burgdorferi with uninfected neutrophils or A. phagocytophilum-infected neutrophils was similar as measured by TEER (P=0.216; Fig. 1). However, when all of these conditions were compared with EAhy.926 cells incubated with medium only, there was a small but significant reduction in TEER at 5 h (P=0.018). (17K) Fig. 1. Change in transendothelial electrical resistance of human brain microvascular endothelial cells and EA.hy926 endothelial cell cultures after incubation with Borrelia burgdorferi (Bb) and neutrophils or Anaplasma phagocytophilum (Ap)-infected neutrophils. 3.2. Borrelia burgdorferi traversal of endothelial cells in the presence of A. phagocytophilum infected-neutrophils or uninfected neutrophils Borrelia burgdorferi transmigration of human BMEC was enhanced in the presence of A. phagocytophilum-infected neutrophils as compared with uninfected neutrophils. We conducted three replicated experiments. When 107 B. burgdorferi was used as inoculum, approximately two-fold enhanced transmigration of B. burgdorferi across the human BMEC was observed in the presence of A. phagocytophilum infectedneutrophils compared with uninfected neutrophils (P<0.045; Fig. 2). Incubation with lower spirochete numbers (2×105) revealed that three-fold (6.7 vs. 2.2%) more B. burgdorferi transmigrated the human BMEC in the presence of A. phagocytophiluminfected neutrophils (P=0.047) and this result was confirmed in a parallel experiment (Fig. 3). No enhanced transmigration through the EAhy.926 systemic endothelial cell barriers was observed with A. phagocytophilum-infected neutrophils vs. uninfected neutrophils (P=0.426). (16K) Fig. 2. Borrelia burgdorferi (Bb) transmigration of human brain microvascular endothelial cells monolayers with and without A. phagocytophilum (Ap)-infected neutrophils at 5 h quantitated by dark-field microscopy. (21K) Fig. 3. Borrelia burgdorferi (Bb) transmigration of endothelial cell monolayers with and without A. phagocytophilum (Ap)-infected neutrophils at 5 h. Transmigrated spirochetes were quantitated by real time PCR (Taqman) targeting the single copy chromosomal target flaB. Significant P values are shown comparing relevant bars. 4. Discussion While B. burgdorferi occasionally penetrates through the blood–brain barrier to cause infection in the central nervous system, including infection such as meningitis, A. phagocytophilum does not (Bakken and Dumler, 2000). However, co-infection of humans with B. burgdorferi and A. phagocytophilum is increasingly recognised and animal models suggest that spirochete titres and tissue injury are exacerbated with coinfection (Thomas et al., 2001, Thompson et al., 2001 and Belongia, 2002). Immunologic control of B. burgdorferi infection, in the absence of other co-infections, probably requires the active participation of both Th1 and Th2 responses (Thomas et al., 2001). The mechanism(s) that account for the increased borrelia loads and tissue inflammation with A. phagocytophilum co-infection is uncertain, although some studies imply the inappropriate induction of Th1 immunity that is effective for obligate intracellular pathogens, but may alter control of B. burgdorferi (Thomas et al., 2001). Our previous studies showed that B. burgdorferi transmigrate endothelial cell barriers, including the BBB, in part via the action of endothelial cell-derived matrix metalloproteases (MMPs) (Grab et al., 2005). With A. phagocytophilum infection, neutrophils are stimulated to degranulate a variety of vesicular components, including matrix metalloproteases (Choi et al., 2004). In fact, A. phagocytophilum-infected neutrophils do not adhere to activated endothelial cells because of the secretion of a sheddase metalloprotease that cleaves neutrophil surface platelet selectin glycoprotein ligand (PSGL-1, CD162), and because of MMP-induced loss of L-selectin (CD62L) (Choi et al., 2003). Based upon this information, we hypothesised that co-infection with both B. burgdorferi and A. phagocytophilum can result in increased exposure of endothelial cell barriers to MMPs would enhance B. burgdorferi penetration into tissues or the CNS. In keeping with this hypothesis, our results in three repeated experiments, measured by two different approaches, confirm that B. burgdorferi transmigrate across endothelial cells more in the presence of A. phagocytophilum-infected neutrophils than with uninfected neutrophils, even in the absence of any adaptive immune responses (in vitro). Although the most likely explanation for these observations is the production of metalloproteases, there are several additional possibilities that will require further investigation. First, A. phagocytophilum-infected neutrophils are markedly activated for production of chemokines and IL-6 (Klein et al., 2000; Choi et al., 2005). These biologically active compounds have multiple effects, including the potential to enhance changes in vascular permeability, perhaps related to alterations in the endothelial cell cytoskeleton. Second, A. phagocytophilum infection of neutrophils impairs their phagocytic capacity, and this could result in an increased availability of B. burgdorferi to transmigrate (Garyu et al., 2005). Regardless, the combined effects of enhanced protease, cytokine/chemokine release and impaired neutrophil phagocytosis with co-infection could lead to enhanced CNS entry of B. burgdorferi and worsened clinical manifestations of Lyme disease. Precedent for such enhanced clinical disease in the CNS exists in sheep infected with louping ill virus, a concurrently tick-transmitted flavivirus of the tick-borne encephalitis group. Louping ill virus alone initiates a mild clinical disease in sheep (Brodie et al., 1986 and Reid et al., 1986,); however, with A. phagocytophilum infection, louping ill virus typically results in severe or fatal meningoencephalitis, although the mechanism of enhanced CNS infection is not understood. In summary, our data show the differential enhancement of B. burgdorferi transmigration across a human BBB model. Further, investigation will be required to confirm the hypothesis that enhanced transmigration results from MMP-enhanced tight junction degradation. Importantly, these data provide an alternative explanation for the enhanced tissue distributions of Lyme disease spirochetes with co-infection in animal models and set the stage for further work if concurrent HGA proves to exacerbate Lyme disease and Lyme meningitis. 5. Uncited references Davenpeck et al. (2000), Roos and Law (2001). Acknowledgements EA.hy926 cells were kindly supplied by C. Edgell, University of North Carolina. This work was supported by a grant from the National Institutes of Health to D.J.G. (R21AI04894-01). References Barbour, 1984 A.G. Barbour, Isolation and cultivation of lyme disease spirochetes, Yale J. Biol. Med. 57 (1984), pp. 521–525. Abstract-EMBASE | AbstractMEDLINE | Order Document Belongia, 2002 E.A. Belongia, Epidemiology and impact of coinfections acquired from Ixodes ticks, Vector Borne Zoonotic Dis. 1 (2002), pp. 265–273. AbstractMEDLINE | Order Document Brodie et al., 1986 T.A. Brodie, P.H. Holmes and G.M. 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Fikrig, Coinfection with Borrelia burgdorferi and the agent of human granulocytic erhlichioses alters murine immune responses, pathogen burden, and severity of lyme arthritis, Infect. Immun. 69 (2001), pp. 3359–3371. Abstract-MEDLINE | Abstract-Elsevier BIOBASE | Abstract-EMBASE | Order Document | Full Text via CrossRef Thompson et al., 2001 C. Thompson, A. Spielman and P.J. Krause, Coinfecting deerassociated zoonoses: lyme disease, babesioses, erlichioses, Clin. Infect. Dis. 33 (2001), pp. 676–685. Abstract-EMBASE | Abstract-Elsevier BIOBASE | AbstractMEDLINE | Order Document | Full Text via CrossRef Corresponding author. Tel.: +1 410 955 8654; fax: +1 443 287 3665 International Journal for Parasitology Article in Press, Uncorrected Proof Comparison of isolation rate of Borrelia burgdorferi sensu lato in MKP and BSK-II medium Eva Ružić-Sabljića, , , Stanka Lotrič-Furlanb, Vera Maraspinb, Jože Cimpermanb, Mateja Logarb, Tomaž Jurcab and Franc Strleb Institute of Microbiology and Immunology, School of Medicine, Zaloška 4, SI-1000 Ljubljana, Slovenia b Department of Infectious Diseases, University Medical Centre Ljubljana, Japljeva 2 Ljubljana, Slovenia a Available online 10 March 2006. Abstract Different media have been utilized for borrelial cultivation. The aim of the present study was to evaluate the isolation rate of Borrelia burgdorferi sensu lato from two commonly used media, i.e. modified Kelly–Pettenkofer (MKP) and Barbour– Stoenner–Kelly II (BSK-II) medium, and to compare the isolated strains with regard to their phenotypic and genotypic characteristics. Skin biopsy specimens of 2×2×4 mm were taken from the peripheral site of human solitary erythema lesions and were divided in two pieces, one of which was inoculated into MKP and the other one into BSK-II medium. Species analysis of the obtained strains was performed and their plasmid and protein profiles were determined. Borrelia strains were isolated from 48/96 patients (50%) with erythema migrans. We obtained in 26/48 patients (54%) from MKP as well as from BSK-II, in 11 patients (23%) only from MKP, and in another 11 (23%) only from BSK-II medium a positive result. B. afzelii was isolated from 43 patients (23 were positive in both media, nine in MKP only and 11 in BSK-II medium only), while B. garinii was isolated from five patients (in three from both media, in two from MKP only). All strains of the obtained strain pairs were identical according to species and the type within the species. Plasmid profiles were identical in 17/21 B. afzelii strain pairs (81%) and in 1/3 B. garinii strain pairs; in 6/24 strain pairs, distinctions in the number of plasmids or in their molecular mass were present. Differences in the protein profile were found in 7/24 strain pairs (29%). The distinctions were uniform and were limited to the expression of OspC. In conclusion, our study showed comparable Borrelia isolation rates from MKP and BSK-II medium. The results of the present study indicate that human patients with Lyme borreliosis may simultaneously harbor heterogeneous B. burgdorferi s.l. strains. Keywords: Borrelia burgdorferi sensu lato; MKP medium; BSK-II medium; Isolation rate Article Outline Introduction Materials and methods Borrelia isolation from patients Culture media Patients Cultivation Analysis of Borrelia strains Identification of Borrelia species by pulsed-field gel electrophoresis (PFGE) Identification of Borrelia species by polymerase chain reaction (PCR) Analysis of plasmid profile Analysis of protein profile Results Isolation rate Species identification Plasmid profile Protein profile Discussion References Introduction Lyme borreliosis may exhibit different clinical manifestations, most frequently erythema migrans, which represents a typical clinical sign early in the course of the disease (Nadelman et al., 1996; Strle et al., 1996; Nadelman and Wormser, 1998; Arnež et al., 2003; Stanek and Strle, 2003). From this localized skin infection borreliae may disseminate to other parts of the human organism and cause different clinical manifestations (Lotrič-Furlan et al., 1999; Wormser et al., 1999;Maraspin et al., 2001; Oksi et al., 2001; Stanek and Strle, 2003). Isolation of the etiological agent from patient's material is the most reliable method for the diagnosis of borrelial infection (Pfister et al., 1984; Preac-Mursic et al., 1986; Picken et al., 1997; LotričFurlan et al., 1999; Wormser et al., 1999; Maraspin et al., 2002; Arnež et al., 2003). Numerous Borrelia strains obtained from human tissue and tissue fluids (skin, blood, cerebrospinal fluid, etc.) have been analyzed by different phenotypic and genotypic approaches (Wilske et al., 1988 and Wilske et al., 1993; Wang et al., 1999; RužićSabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b). In comparison to phenotypic assessment, analysis of borrelial genomic DNA provides more precise information on the genetic relationships among Borrelia strains (Belfaiza et al., 1993; Busch et al., 1995; Busch and Nitschko, 1999; Olive and Bean, 1999; Wang et al., 1999; Wormser et al., 1999; Ružić-Sabljić et al., 2000, RužićSabljić et al., 2001a and Ružić-Sabljić et al., 2001b). Many studies indicate that B. burgdorferi sensu lato populations are genetically divergent and phenotypically heterogeneous, and that Lyme borreliosis in humans is caused by at least three different species of the B. burgdorferi s.l. complex, viz. B. afzelii, B. garinii, and B. burgdorferi sensu stricto (Belfaiza et al., 1993; Burgdorfer, 1995; Rijpkema et al., 1995; Liveris et al., 1996; Picken et al., 1996; Mathiesen et al., 1997; Wang et al., 1999; Wormser et al., 1999; Ružić-Sabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b). Isolation of B. burgdorferi s.l. from clinical materials is a golden standard for confirming borrelial infection. Borrelia cultivation and isolation is quite a demanding procedure with limited sensitivity (Jobe et al., 1993; Nadelman et al., 1996; Strle et al., 1996; Kollars et al., 1997, Picken et al., 1997; Lotrič-Furlan et al., 1999; Wormser et al., 2000; Maraspin et al., 2001). Different media for borrelial cultivation have been reported. For routine work, modified Kelly–Pettenkofer (MKP), Barbour–Stoenner– Kelly II (BSK-II) medium and commercially available BSK-H (Sigma, USA) medium have been utilized most frequently (Barbour, 1984; Preac-Mursic et al., 1986; Pollack et al., 1993). The majority of the components of the three media are equivalent and present in similar concentrations (e.g. CMRL, N–acetyl glucosamine, bovine serum albumin, rabbit serum, etc.) but the origin of components may be dissimilar (e.g. bought from different commercial firms). However, media may differ in certain constituents. For example, MKP lacks yeast extract present in BSK–II and BSK-H, but–in contrast to BSK-II and BSK-H–contains gelatin (Barbour, 1984; Preac-Mursic et al., 1986; Pollack et al., 1993). To be useful for laboratory purposes, the medium must be stable, available and efficacious in supporting the growth of all human pathogenic Borrelia strains. In addition to medium ingredients, several other factors including temperature of incubation, pH of the medium, capacity of particular borrelial species to grow and number of Borrelia strains in the sample can influence borrelial growth and the outcome of cultivation (Hubalek et al., 1998; Ružić-Sabljić and Strle, 2004). The aim of the present study was to evaluate and compare the isolation rate of B. burgdorferi s.l. from the skin of patients with erythema migrans using MKP and BSK-II medium, and to compare phenotypic and genotypic characteristics of the strain pairs, i.e. the strains isolated in the two media from the skin biopsy of an individual patient. Materials and methods Borrelia isolation from patients Culture media In the present study MKP and BSK-II medium were used for Borrelia isolation. Both media were prepared in our laboratory after the original recipes (Barbour, 1984; Preac-Mursic et al., 1986). The two media differ with regard to the glucose concentration (3 versus 5 g/l in MKP and BSK-II, respectively), the origin and concentration of bovine serum albumin (35% solution with a final concentration of 1 g/ml in MKP versus fraction V powder with final concentration 4 g/ml in BSK-II), the concentration and preparation of rabbit sera (7.2% heat-inactivated versus 6% non-inactivated in MKP and BSK-II, respectively). Additionally, yeastolate (2 g/l) was added to BSK-II but not to MKP medium. After preparing and testing the quality of each medium lot, the media were distributed to clinicians. Patients Ninety-six consecutive adult patients diagnosed with erythema migrans at the Department of Infectious Diseases, University Medical Center Ljubljana, in spring of 2000 represented the basis for the present study. In each patient, a skin biopsy was taken from the peripheral site of erythema migrans after prior cleaning the skin with 70% alcohol and local anesthesia with 2% xylocaine (Jurca et al., 1998). Each specimen (2×2×4 mm) was dissected in two pieces: one was immediately inoculated into MKP, the other one into BSK-II medium. The inoculated media were sent to the laboratory at room temperature. Cultivation Samples were incubated at 33 °C and examined weekly for up to 9 weeks for the presence of spirochetes by dark-field microscopy. Isolated strains were grown in the same medium for 2–4 subcultures. Analysis of Borrelia strains For the large majority of isolates, strain identification was carried out by pulsed-field gel electrophoresis (PFGE). Identification by polymerase chain reaction (PCR) was performed only for cultures with borrelial concentration not high enough for PFGE. Identification of Borrelia species by pulsed-field gel electrophoresis (PFGE) Strain identification by PFGE was performed as described previously (Busch et al., 1995; Ružić-Sabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b). Briefly, Borrelia strains were mixed in agarose, and whole blocks were lysed in lysis buffer containing lysozyme (1 mg/ml) and ribonuclease (10 μg/ml), and digested in digestion buffer containing proteinase K (0.5 mg/ml). After exhaustive block washing, DNA was restricted with 30 U of MluI restriction enzyme. Restricted patterns were separated for 24 h with ramping pulse times of 1–40 s. Molecular mass marker of 50–1000 kb (Sigma, USA) was added to each electrophoresis. Borrelia species as well as the type within the particular species were determined by restriction fragment length polymorphism (RFLP) as described by Belfaiza et al. (1993) and Picken et al. (1996). Fragments interpreted as being species specific were those with molecular weights 440, 320 and 90 kb for B. afzelii, 220 and 80 kb for B. garinii and 145 kb for B. burgdorferi s.s. Types within species (Mla for B. afzelii, Mlg for B. garinii and Mlb for B. burgdorferi s.s.) were determined according to additional fragments of different molecular weights (Belfaiza et al., 1993; Picken et al., 1996). Identification of Borrelia species by polymerase chain reaction (PCR) Tubes with culture were centrifuged at 10,000 rpm and borrelial DNA was isolated from the sediment using commercial kit (QIAamp DNA mini kit, Qiagen cat. no. 51306, Germany). Interspace 5S-23S DNA region was amplified. The amplified products were restricted by restriction enzyme MseI and electrophoresed in 16% polyacrylamide gel. Borrelia species were determined by RFLP as described by Postic et al. (1994). Fragments interpreted as being species specific were those with molecular weights 108, 68, 50 and 20 bp for B. afzelii, 108, 95 and 50 bp for B. garinii and 108, 51, 38, 29 and 28 bp for B. burgdorferi s.s. Analysis of plasmid profile The procedure was limited to cultures with a sufficient Borrelia density to allow the execution of PFGE, and was determined only in patients in whom borreliae were isolated from an erythema migrans lesion concurrently in MKP and in BSK-II medium (when two isolates were obtained from an individual skin biopsy sample). To compare plasmid profiles within such strain pairs, we performed electrophoresis of both strains on the same gel. Plasmid profiles were determined by PFGE. Blocks with borrelial DNA were prepared identically as for species identification but electrophoresis was performed without previous restriction of DNA. Chromosomal and plasmid DNA were separated for 37 h in ramping pulse time of 0.9–3 s as described previously (Busch et al., 1995; Ružić-Sabljić et al., 1999, Ružić-Sabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b). Molecular mass marker of 0.1–200 kb (Sigma, USA) was added to each separation. Analysis of protein profile Protein profile analysis of borrelial strains was performed only in patients in whom borreliae were isolated in MKP as well as in BSK-II medium. To compare protein profiles of concurrently isolated strains, we performed electrophoresis of both strains on the same gel. For the determination of protein profiles, strains were harvested, washed and lysed in buffer containing sodium dodecyl sulfate (2.5%) and 2mercaptoethanol (2.5%), as described elsewhere (Wilske et al., 1988; Busch et al., 1995; Ružić-Sabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b). Electrophoresis was performed in 12% polyacrylamide gel. Low molecular mass marker of 21–106 kD (BioRad, Germany) was used as size marker in each protein profile determination. Results Isolation rate Skin biopsy specimens were obtained from 96 consecutive adult patients with typical solitary erythema migrans. Borrelia strains were isolated from 48/96 skin lesions (patients) (50%). There was no difference in the isolation rate when comparing MKP and BSK-II medium (37 positive cultures in each particular medium). In 26/48 patients (54%) borreliae were isolated in MKP as well as in BSK-II medium, while in 11/48 patients (23%) borreliae were isolated either in MKP or BSK-II (Table 1). Table 1. Borrelia burgdorferi sensu lato isolation rate from human erythema migrans skin biopsy sample divided in two specimens–comparison of modified Kelly–Pettenkofer (MKP) and Barbour–Stoenner– Kelly II (BSK-II) medium Culture-positive samples Culture negative Total In MKP and BSK-II In MKP only In BSK-II only 26 11 11 48 48 96 Species identification Out of the 74 isolated Borrelia strains, 69 (93%) were typed using PFGE, while in 5 strains (3 in MKP and 2 in BSK-II medium) that did not grow sufficiently, species identification was preformed by PCR. Sixty-six strains (89%) were identified as B. afzelii (in 23 patients, the strains were isolated in both media, in 9 patients in MKP only, and in 11 patients in BSK-II only) and 8 as B. garinii (in 3 patients isolated in both media, and in 2 patients isolated in MKP only). Data are shown in Table 2. Table 2. Species identification of Borrelia strains isolated from human erythema migrans skin biopsies divided in two specimens – comparison of modified Kelly–Pettenkofer (MKP) and Barbour–Stoenner–Kelly II (BSK-II) medium Positive culture in medium B. afzelii B. garinii No. of isolated strains PFGEa PCRb PFGE PCR MKP and 22 1 3 – 26 BSK-II 22 1 3 – 26 MKP only 7 2 2 – 11 BSK-II only 10 1 – – 11 Subtotal 61 5 8 0 Total 66 a b 8 74 Identification of Borrelia species with pulsed-field gel electrophoresis. Identification of Borrelia species with polymerase chain reaction. From 48 skin-culture-positive patients with erythema migrans (Table 1), there were 26 patients with borreliae isolated in both media. Of these 26 strain pairs (52 strains), 24 strain pairs were suitable for further PFGE identification and were used for analyzing and comparing PFGE types within the species as well as plasmid and protein profiles. Each individual strain pair was congruent according to the species as well as to the PFGE type. Regarding PFGE type, strain pairs consisted of either B. afzelii Mla1 (21 pairs) or B. garinii Mlg2 (3 pairs). Plasmid profile Identical plasmid profiles were found in 17/21 B. afzelii strain pairs (81%) and in 1/3 B. garinii strain pairs, while in 6/24 patients, the two strains differed either in the number of plasmids or their molecular mass. The plasmid profiles of some strain pairs are shown in Fig. 1. (50K) Fig. 1. Plasmid profile of Borrelia strains (1 and 2) isolated in both modified Kelly–Pettenkofer (MKP) and Barbour–Stoenner–Kelly II (BSK-II) medium. Strains were isolated from skin biopsies of patients with erythema migrans (A–J). MW: molecular mass marker; B. afzelii strains: A–F and J; B. garinii strains: G–I. Plasmid profile of strain pairs differs in patients A–D and G. Protein profile Protein profiles of strain pairs obtained from patients in whom borreliae were isolated in MKP as well as in BSK-II medium differed in 7/24 cases (29%). All those strains were identified as B. afzelii and showed identical distinction–one strain of each pair expressed OspC, whereas the other did not. Protein profiles differed in six strain pairs which were identical according to the species and the plasmid profile, and in one strain pair both strains belonged to the same species but were distinct in their plasmid profile (patient D from the Fig. 1). Discussion Although erythema migrans represents a pathognomonic clinical sign of Lyme borreliosis, the isolation rate of Borrelia strains from the skin lesions was only 50% (48/96 patients) in the present study, corroborating previous findings from Slovenia (Strle et al., 1996; Jurca et al., 1998; Zore et al., 2002). The fastidious nature of the organism and changes that Borrelia must undergo to adapt the transition from living biological material to an artificial medium probably limit the isolation rate (Picken et al., 1996; Norris et al., 1997). Additionally, ingenious characteristics of the individual Borrelia species such as a tendency of better growth or overgrowth of certain Borrelia species over the other(s) (Ružić-Sabljić and Strle, 2004) might influence borrelial isolation rate. Several other factors, for example, the quantity of spirochetes in a given sample, previous antibiotic treatment, local anesthesia at the site of biopsy, conditions of sample transport to the laboratory, medium used for Borrelia cultivation, aseptic environment of culture maintenance, etc. can also influence the isolation rate (Kollars et al., 1997; Wormser et al., 2000). The relatively low isolation rate of Borrelia strains in our patients with erythema migrans might have been caused by the rather small size of skin biopsy specimens (e.g. 2×2×4 mm) that was additionally divided in two pieces. Some previous studies suggested that borreliae are present in erythema migrans lesions in only low concentration and are irregularly distributed (Pachner et al., 1993; Picken et al., 1997; Zore et al., 2002). With larger samples, the isolation rate would probably be higher (Wormser et al., 2000). The main interest of the present study was to compare the isolation rate of B. burgdorferi s.l. from erythema migrans skin samples in two commonly used media, MKP and BSK-II. We had anticipated that our approach of taking one skin biopsy from an individual patient, dissecting it in two pieces and inoculating each single piece in a particular medium, would lead to identical (positive or negative) results in the large majority of cases. The unexpected discrepancy may be explained by unequal distribution of Borrelia strains within individual samples. Our findings and proposed explanations are in accordance with the results of some previous studies which suggested that borreliae are present in erythema migrans lesions in low concentration and are irregularly distributed (Pachner et al., 1993; Picken et al., 1997; Zore et al., 2002). Sixty-six out of 74 isolates (89%) were typed as B. afzelii and 8 as B. garinii; no B. burgdorferi s.s. strain was isolated (Table 2). These findings corroborate the results of several European studies on the predominance of B. afzelii strains in skin manifestations of Lyme borreliosis (Picken et al., 1996; Rijpkema et al., 1997; Ružić-Sabljić et al., 2000; Ciceroni et al., 2001; Ornstein et al., 2001; Logar et al., 2004). Twenty-four strain pairs (out of 26 double isolates) grew well enough to allow RFLP analysis (Table 2) and to carry out a comparison of the phenotypic and genotypic characteristics of the isolates within the individual pairs (species identification, plasmid and protein profiles). Either strain of each strain pair contained the same Borrelia genospecies (21 pairs: B. afzelii; 3 pairs: B. garinii) and the same type (all B. afzelii strains were determined as Mla1, and all B. garinii strains as Mlg2), whereas some distinctions in plasmid and protein profiles were found. A plasmid profile analysis revealed 6/24 strain pairs (25%) composed of strains with distinct plasmid outlines. Some of these strains are shown in Fig. 1. The plasmid profile is an excellent tool for strain discrimination particularly when comparing strains within the same species. Many conditions can affect the borrelial plasmid content including the possibility that plasmids can be lost upon cultivation (Xu and Johnson, 1995; Busch et al., 1997; Ružić-Sabljić et al., 1999, Ružić-Sabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b; Wang et al., 1999; Iyer et al., 2003). We would like to stress that strains compared in our study were subjected to quite identical circumstances. They were isolated, multiplied and analyzed using analogous approaches and procedures but only differed in the culture medium. However, the potential impact of a particular medium (MKP versus BSK-II) on the plasmid content is not known. Differences in the plasmid content within particular strain pairs have been described previously (Ružić-Sabljić et al., 2005). We believe that the plasmid content of our strains most probably represents the natural situation and that differing plasmid profiles of strains may be quite safely interpreted as an infection with different clones of the same type of an identical Borrelia species. Differences in the protein profile were found within 7/24 strain pairs (29%). All strain pairs with dissimilar protein profiles belonged to B. afzelii. The distinctions were uniform and limited to the expression of OspC. It is of interest that 6/7 strain pairs with distinct protein profiles had congruent plasmid profiles. Besides the adaptive mechanisms of the spirochetes that influence protein expression, each strain has its own potential to express (or not to express) a particular protein. The latter statement is supported by the finding that populations of B. afzelii and B. garinii generally differ in their potential to express some important proteins such as OspB and OspC (Wilske et al., 1988 and Wilske et al., 1996; Ružić-Sabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b). Therefore, it is possible that proteins expressed by the strains presented in the present study reflect their own genuine status. In conclusion, our study showed comparable Borrelia isolation rates in MKP versus BSK-II medium indicating their similar suitability for routine clinical work. The results of the present study indicate that human patients with Lyme borreliosis may simultaneously harbor heterogeneous B. burgdorferi s.l. strains. References Arnež et al., 2003 M. Arnež, D. Pleterski-Rigler, T. Lužnik-Bufon, E. Ružić-Sabljić and F. 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AbstractMEDLINE | Order Document | Full Text via CrossRef Wormser et al., 1999 G.P. Wormser, D. Liveris, J. Nowakowski, R.B. Nadelman, L.F. Cavaliere, D. McKenna, D. Holmgren and I. Schwartz, Association of specific subtypes of Borrelia burgdorferi with hematogenous dissemination in early Lyme disease, J. Infect. Dis. 180 (1999), pp. 720–725. Abstract-MEDLINE | AbstractEMBASE | Abstract-Elsevier BIOBASE | Order Document | Full Text via CrossRef Wormser et al., 2000 G.P. Wormser, S. Bittker, D. Cooper, J. Nowakowski, R.B. Nadelman and C. Pavia, Comparison of yields of blood cultures using serum or plasma from patients with early Lyme disease, J. Clin. Microbiol. 38 (2000), pp. 1648–1650. Abstract-MEDLINE | Abstract-EMBASE | Abstract-Elsevier BIOBASE | Order Document Xu and Johnson, 1995 Y. Xu and R.C. Johnson, Analysis and comparison of plasmid profiles of Borrelia burgdorferi sensu lato strains, J. Clin. Microbiol. 33 (1995), pp. 2679–2685. Abstract-MEDLINE | Abstract-EMBASE | Order Document Zore et al., 2002 A. Zore, E. Ružić-Sabljić, V. Maraspin, J. Cimperman, S. LotričFurlan, A. Pikelj, T. Jurca, M. Logar and F. Strle, Sensitivity of culture and polymerase chain reaction for the etiologic diagnosis of erythema migrans, Wien. Klin. Wschr. 114 (2002), pp. 606–609. Abstract-MEDLINE | Abstract-EMBASE | Order Document International Journal of Medical Microbiology Article in Press, Corrected Proof Anaplasma phagocytophilum-infected neutrophils enhance transmigration of Borrelia burgdorferiacross the human blood brain barrier in vitro E. Nyarkoa, D.J. Graba and J.S. Dumlerb, , a Division of Infectious Diseases, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD, USA b Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Ross 624, Baltimore, MD 21205, USA Received 6 November 2005; revised 20 January 2006; accepted 30 January 2006. Available online 6 March 2006. Abstract The manifestations of Lyme disease, caused by Ixodes spp. tick-transmitted Borrelia burgdorferi, range from skin infection to bloodstream invasion into the heart, joints and nervous system. The febrile infection human granulocytic anaplasmosis is caused by a neutrophilic rickettsia called Anaplasma phagocytophilum, also transmitted by Ixodes ticks. Previous studies suggest that co-infection with A. phagocytophilum contributes to increased spirochetal loads and severity of Lyme disease. However, a common link between these tick-transmitted pathogens is dissemination into blood or tissues through blood vessels. Preliminary studies show that B. burgdorferi binds and passes through endothelial barriers in part mediated by host matrix metalloproteases. Since neutrophils infected by A. phagocytophilum are activated to release bioactive metalloproteases and chemokines, we examined the enhanced B. burgdorferi transmigration through vascular barriers with co-infection in vitro. To test whether endothelial transmigration is enhanced with co-infection, B. burgdorferi and A. phagocytophilum-infected neutrophils were co-incubated with EA.hy926 cells (HUVEC-derived) and human brain microvascular endothelial cells in Transwell™ cultures. Transmigration of B. burgdorferi through endothelial cell barriers was determined and endothelial barrier integrity was measured by transendothelial electrical resistivity. More B. burgdorferi crossed both human BMEC and EA.hy926 cells in the presence of A. phagocytophilum-infected neutrophils than with uninfected neutrophils without affecting endothelial cell integrity. Such a mechanism may contribute to increased blood and tissue spirochete loads. Keywords: Lyme disease; Borrelia burgdorferi; Anaplasma phagocytophilum; Endothelial cells; Neutrophils; Co-infection Article Outline 1. Introduction 2. Materials and methods 2.1. Cultivation and preparation of cell-free A. phagocytophilum 2.2. Preparation of A. phagocytophilum-infected neutrophils 2.3. Spirochetes 2.4. Human brain microvascular endothelial cells (human BMEC) 2.5. Human systemic endothelial cells 2.6. Endothelial monolayer transmigration assay 2.7. Quantitative PCR 2.8. Statistical tests 3. Results 3.1. Effects of co-incubation of B. burgdorferi with A. phagocytophilum-infected neutrophils on the integrity of endothelial cell barriers 3.2. Borrelia burgdorferi traversal of endothelial cells in the presence of A. phagocytophilum infected-neutrophils or uninfected neutrophils 4. Discussion 5. Uncited references Acknowledgements References 1. Introduction Lyme disease is now the most frequently reported arthropod-borne infection in North America and Europe (CDC, 2002). The bacteria, Borrelia burgdorferi, which are transmitted to humans by the bite of infected ticks of the Ixodes persulcatus complex, can infect many tissues including the skin, heart, joints, eye and in addition, the peripheral and central nervous systems (Steere, 1989 and Kalish, 1993). As the diversity of clinical presentations for Lyme disease has been recognised, it has been suggested that concurrent infections by other tick-borne pathogens may influence the natural course of disease, leading to more severe infection, persistence, even refractoriness to effective therapies (Belongia, 2002 and Krause et al., 2002). Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (HGA) is also transmitted by I. persulcatus-complex tick bites and accumulating data suggest that co-infection is not infrequent. In North America and also in Europe, approximately 10% of all patients with Lyme disease or HGA have evidence of co-infection with other pathogens (Thompson et al., 2001). Although it is unclear whether co-infections contribute to the comorbidity, in mice commitment of immunity toward Th1 reactions directed against the obligate intracellular A. phagocytophilum allows higher spirochetemia, tissue loads, longer persistence and increased disease with simultaneous B. burgdorferi infections (Thomas et al., 2001). Penetration into the blood and out of the bloodstream into sites of infection is a necessary component for dissemination of pathogens of Lyme disease and HGA, hence interactions of both pathogens at the level of blood–endothelial cell interface are critical determinants of dissemination and disease. Borrelia burgdorferi penetration of endothelial cells results in part from the actions of endothelial cellderived metalloproteases, after binding of spirochetes to intracellular junctions. Anaplasma phagocytophilum-infected neutrophils are induced for protracted degranulation, resulting in the elaboration of biologically active molecules, including chemokines, cytokines and metalloproteases (Choi et al., 2003). Owing to the production of metalloproteases from A. phagocytophilum-infected neutrophils, we hypothesised that concurrent infection with B. burgdorferi enhances penetration of the spirochete through blood–brain barrier (BBB) and systemic endothelial cell barriers. We tested this non-immunological mechanism using in vitro models of the human BBB and systemic endothelial cells. 2. Materials and methods 2.1. Cultivation and preparation of cell-free A. phagocytophilum The A. phagocytophilum strain Webster was cultivated in HL-60 cells in RPMI 1640 medium (GIBCO-BRL) supplemented with 5% FCS and 2 mM L-glutamine (GIBCOBRL) at 37 °C (Choi et al., 2003; Choi and Dumler, 2004). Cell-free A. phagocytophilum was prepared by sonication of heavily infected HL-60 cells (Branson Sonifier, VWR Scientific) at duty cycle of 70 and output control of 3 (Choi et al., 2003 and Garyu et al., 2005). These bacteria were washed and used immediately to infect 5×105 to 106 human peripheral blood neutrophils. Mockinfected cells were incubated with medium only. 2.2. Preparation of A. phagocytophilum-infected neutrophils Human peripheral blood neutrophils and monocytes were isolated from EDTAanticoagulated blood of healthy donors by dextran sedimentation followed by Ficoll density gradient centrifugation (Histopaque; Sigma). The contaminating residual erythrocytes present in the neutrophil preparations were lysed by exposure to hypotonic saline (0.2% NaCl) for 30 s and then adjusted back to isotonic conditions with hypertonic saline (1.8% NaCl) prior to washing in tissue culture medium. Neutrophil purity was confirmed to be ≥95% by Ramanowsky staining (Hema-3; Biochemical Sciences, Inc., Swedesboro, NJ), and the viability of cells was determined to be >98% by trypan blue dye exclusion. Human neutrophils were obtained with the approval of the Johns Hopkins University School of Medicine Institutional Review Board and in compliance with all relevant federal guidelines and institutional policies. Some of the neutrophils were suspended in medium M199 supplemented in 20% FCS and then incubated at 37 °C in 5% CO2 in a humidified environment. 2.3. Spirochetes Low-passage (less than five in vitro passages) B. burgdorferi was cultured at 34 °C in BSK II medium containing 10% rabbit serum as described by Barbour (1984). In our study, we used B. burgdorferi 297, a strain originally isolated from human cerebrospinal fluid (Leveris et al., 1995). The bacteria were examined for motility with dark-field microscope to verify viability and that the organisms were thoroughly dispersed at the start of all the assays. 2.4. Human brain microvascular endothelial cells (human BMEC) Human BMEC primary cultures used in this study were described previously (Persidsky et al., 1997 and Stins et al., 1997; Grab et al., 2004 and Grab et al., 2005). The cells were cultured in Medium 199 (GIBCO) supplemented with 20% heatinactivated FCS and 1×Glutamax (GIBCO). The cells were grown to confluence on 6.5-mm-diameter collagen-coated Costar inserts with a pore of size of 3.0 μm until transendothelial electrical resistance (TEER) measurements exceeded 25 Ω×cm2 (Grab et al., 2004). 2.5. Human systemic endothelial cells The EAhy926 endothelial cell line derived from fusion of A549 cells with primary human umbilical vein endothelial cells have also been described previously (Edgell et al., 1983). These were grown in high-glucose (4.5 g/l) DMEM supplemented with 20% heat-inactivated FCS, 1×HT supplement and 1×Glutamax (all from GIBCO). The cells were grown to confluence on 6.5-mm-diameter collagen-coated Costar Transwell™ inserts with a pore of size of 3.0 μm until transendothelial electrical resistance reached stable values <12 Ω×cm2 (Grab et al., 2004). 2.6. Endothelial monolayer transmigration assay Triplicate wells with and without endothelial cells received the following: medium only; B. burgdorferi only (ranging from 2×105 to 107); uninfected neutrophils only (2×105); A. phagocytophilum-infected neutrophils only (2×105); B. burgdorferi (2×105 to 107) and uninfected neutrophils (2×105); B. burgdorferi (2×105 to 107) and A. phagocytophilum-infected neutrophils (2×105). After 5 h incubation, the TEER was re-measured and aliquots were removed from the wells beneath the inserts. The quantity of transmigrating B. burgdorferi was determined for each well by either dark-field counting in a haemocytometer for initial experiments and then by quantitative PCR for subsequent experiments. The net transmigration of each barrier was calculated as a percentage of the number of spirochetes that crossing inserts with cells relative to the number crossing inserts without endothelial cells. 2.7. Quantitative PCR Borrelia burgdorferi DNA in transmigrated culture medium (below Transwells) was prepared using either the GenoM-48 DNA robot or using the Qiagen DNA extraction kit, both according to the manufacturers' instructions. Borrelia burgdorferi quantification was performed using quantitative real-time PCR targeting the single copy chromosomal flaB gene (Leutenneger et al., 1999) using the primers B.398f (GGGAAGCAGATTTGTTTGACA) and B.484r (ATAGAGCAACTTACAGACGAAATTAATAGA) with the fluorescent probe B.421p (FAM- ATGTGCATTTGGTTATATTGAGCTTGATCAGCAA-TAMRA). Amplifications were performed using either an ABI Taqman 7700 or a BioRad iCycler iQ5 Multicolor Real Time PCR detector. For standard curve, the flaB gene amplicon was cloned, plasmid DNA containing the insert was prepared and the DNA concentration was carefully measured to allow a precise determination of the copy number of flaB in each standard. Final concentrations of transmigrated B. burgdorferi were determined using the CT method as per software of the Taqman or iQ5 instruments. 2.8. Statistical tests Where appropriate, means of transmigration or TEER values were compared using one-tailed, unpaired or paired Student's t-tests; a P-value <0.05 was considered significant. 3. Results 3.1. Effects of co-incubation of B. burgdorferi with A. phagocytophilum-infected neutrophils on the integrity of endothelial cell barriers The integrity of EAhy.926 and the human BMEC barriers after co-incubation of B. burgdorferi with uninfected neutrophils or A. phagocytophilum-infected neutrophils was similar as measured by TEER (P=0.216; Fig. 1). However, when all of these conditions were compared with EAhy.926 cells incubated with medium only, there was a small but significant reduction in TEER at 5 h (P=0.018). (17K) Fig. 1. Change in transendothelial electrical resistance of human brain microvascular endothelial cells and EA.hy926 endothelial cell cultures after incubation with Borrelia burgdorferi (Bb) and neutrophils or Anaplasma phagocytophilum (Ap)-infected neutrophils. 3.2. Borrelia burgdorferi traversal of endothelial cells in the presence of A. phagocytophilum infected-neutrophils or uninfected neutrophils Borrelia burgdorferi transmigration of human BMEC was enhanced in the presence of A. phagocytophilum-infected neutrophils as compared with uninfected neutrophils. We conducted three replicated experiments. When 107 B. burgdorferi was used as inoculum, approximately two-fold enhanced transmigration of B. burgdorferi across the human BMEC was observed in the presence of A. phagocytophilum infectedneutrophils compared with uninfected neutrophils (P<0.045; Fig. 2). Incubation with lower spirochete numbers (2×105) revealed that three-fold (6.7 vs. 2.2%) more B. burgdorferi transmigrated the human BMEC in the presence of A. phagocytophiluminfected neutrophils (P=0.047) and this result was confirmed in a parallel experiment (Fig. 3). No enhanced transmigration through the EAhy.926 systemic endothelial cell barriers was observed with A. phagocytophilum-infected neutrophils vs. uninfected neutrophils (P=0.426). (16K) Fig. 2. Borrelia burgdorferi (Bb) transmigration of human brain microvascular endothelial cells monolayers with and without A. phagocytophilum (Ap)-infected neutrophils at 5 h quantitated by dark-field microscopy. (21K) Fig. 3. Borrelia burgdorferi (Bb) transmigration of endothelial cell monolayers with and without A. phagocytophilum (Ap)-infected neutrophils at 5 h. Transmigrated spirochetes were quantitated by real time PCR (Taqman) targeting the single copy chromosomal target flaB. Significant P values are shown comparing relevant bars. 4. Discussion While B. burgdorferi occasionally penetrates through the blood–brain barrier to cause infection in the central nervous system, including infection such as meningitis, A. phagocytophilum does not (Bakken and Dumler, 2000). However, co-infection of humans with B. burgdorferi and A. phagocytophilum is increasingly recognised and animal models suggest that spirochete titres and tissue injury are exacerbated with coinfection (Thomas et al., 2001, Thompson et al., 2001 and Belongia, 2002). Immunologic control of B. burgdorferi infection, in the absence of other co-infections, probably requires the active participation of both Th1 and Th2 responses (Thomas et al., 2001). The mechanism(s) that account for the increased borrelia loads and tissue inflammation with A. phagocytophilum co-infection is uncertain, although some studies imply the inappropriate induction of Th1 immunity that is effective for obligate intracellular pathogens, but may alter control of B. burgdorferi (Thomas et al., 2001). Our previous studies showed that B. burgdorferi transmigrate endothelial cell barriers, including the BBB, in part via the action of endothelial cell-derived matrix metalloproteases (MMPs) (Grab et al., 2005). With A. phagocytophilum infection, neutrophils are stimulated to degranulate a variety of vesicular components, including matrix metalloproteases (Choi et al., 2004). In fact, A. phagocytophilum-infected neutrophils do not adhere to activated endothelial cells because of the secretion of a sheddase metalloprotease that cleaves neutrophil surface platelet selectin glycoprotein ligand (PSGL-1, CD162), and because of MMP-induced loss of L-selectin (CD62L) (Choi et al., 2003). Based upon this information, we hypothesised that co-infection with both B. burgdorferi and A. phagocytophilum can result in increased exposure of endothelial cell barriers to MMPs would enhance B. burgdorferi penetration into tissues or the CNS. In keeping with this hypothesis, our results in three repeated experiments, measured by two different approaches, confirm that B. burgdorferi transmigrate across endothelial cells more in the presence of A. phagocytophilum-infected neutrophils than with uninfected neutrophils, even in the absence of any adaptive immune responses (in vitro). Although the most likely explanation for these observations is the production of metalloproteases, there are several additional possibilities that will require further investigation. First, A. phagocytophilum-infected neutrophils are markedly activated for production of chemokines and IL-6 (Klein et al., 2000; Choi et al., 2005). These biologically active compounds have multiple effects, including the potential to enhance changes in vascular permeability, perhaps related to alterations in the endothelial cell cytoskeleton. Second, A. phagocytophilum infection of neutrophils impairs their phagocytic capacity, and this could result in an increased availability of B. burgdorferi to transmigrate (Garyu et al., 2005). Regardless, the combined effects of enhanced protease, cytokine/chemokine release and impaired neutrophil phagocytosis with co-infection could lead to enhanced CNS entry of B. burgdorferi and worsened clinical manifestations of Lyme disease. Precedent for such enhanced clinical disease in the CNS exists in sheep infected with louping ill virus, a concurrently tick-transmitted flavivirus of the tick-borne encephalitis group. Louping ill virus alone initiates a mild clinical disease in sheep (Brodie et al., 1986 and Reid et al., 1986,); however, with A. phagocytophilum infection, louping ill virus typically results in severe or fatal meningoencephalitis, although the mechanism of enhanced CNS infection is not understood. In summary, our data show the differential enhancement of B. burgdorferi transmigration across a human BBB model. Further, investigation will be required to confirm the hypothesis that enhanced transmigration results from MMP-enhanced tight junction degradation. Importantly, these data provide an alternative explanation for the enhanced tissue distributions of Lyme disease spirochetes with co-infection in animal models and set the stage for further work if concurrent HGA proves to exacerbate Lyme disease and Lyme meningitis. 5. Uncited references Davenpeck et al. (2000), Roos and Law (2001). Acknowledgements EA.hy926 cells were kindly supplied by C. Edgell, University of North Carolina. This work was supported by a grant from the National Institutes of Health to D.J.G. (R21AI04894-01). References Barbour, 1984 A.G. Barbour, Isolation and cultivation of lyme disease spirochetes, Yale J. Biol. Med. 57 (1984), pp. 521–525. Abstract-EMBASE | AbstractMEDLINE | Order Document Belongia, 2002 E.A. Belongia, Epidemiology and impact of coinfections acquired from Ixodes ticks, Vector Borne Zoonotic Dis. 1 (2002), pp. 265–273. AbstractMEDLINE | Order Document Brodie et al., 1986 T.A. Brodie, P.H. Holmes and G.M. 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Fikrig, Coinfection with Borrelia burgdorferi and the agent of human granulocytic erhlichioses alters murine immune responses, pathogen burden, and severity of lyme arthritis, Infect. Immun. 69 (2001), pp. 3359–3371. Abstract-MEDLINE | Abstract-Elsevier BIOBASE | Abstract-EMBASE | Order Document | Full Text via CrossRef Thompson et al., 2001 C. Thompson, A. Spielman and P.J. Krause, Coinfecting deerassociated zoonoses: lyme disease, babesioses, erlichioses, Clin. Infect. Dis. 33 (2001), pp. 676–685. Abstract-EMBASE | Abstract-Elsevier BIOBASE | AbstractMEDLINE | Order Document | Full Text via CrossRef Corresponding author. Tel.: +1 410 955 8654; fax: +1 443 287 3665. International Journal for Parasitology Article in Press, Uncorrected Proof Functionality of Borrelia burgdorferi LuxS: The Lyme disease spirochete produces and responds to the pheromone autoinducer-2 and lacks a complete activated-methyl cycle Kate von Lackuma, 1, Kelly Babba, 1, Sean P. Rileya, 1, Rachel L. Wattiera, b, 2, Tomasz Bykowskia and Brian Stevensona, , a Department of Microbiology, Immunology, and Molecular Genetics, College of Medicine, University of Kentucky, MS 415 Chandler Medical Center, Lexington, Kentucky 40536-0298, USA b Agricultural Biotechnology Program, College of Agriculture, University of Kentucky, Lexington, Kentucky, USA Available online 10 March 2006. Abstract Borrelia burgdorferi produces Pfs and LuxS enzymes for breakdown of the toxic byproducts of methylation reactions, producing 4,5-dihydroxy-2,3-pentanedione (DPD), adenine, and homocysteine. DPD and its spontaneously rearranged derivatives constitute a class of bacterial pheromones named autoinducer-2 (AI-2). We describe that B. burgdorferi produces DPD during laboratory cultivation. Furthermore, addition of in vitro synthesized DPD to cultured B. burgdorferi resulted in altered expression levels of a specific set of bacterial proteins, among which is the outer surface lipoprotein VlsE. While a large number of bacteria utilize homocysteine, the other LuxS product, for synthesis of methionine as part of the activated-methyl cycle, B. burgdorferi was found to lack that ability. We propose that the main function of B. burgdorferi LuxS is to synthesize DPD and that the Lyme disease spirochete utilizes a form of DPD as a pheromone to control gene expression. Keywords: Borrelia burgdorferi; Quorum sensing; Pheromone; Bacterial gene regulation; Homocysteine; Methionine Article Outline Introduction Materials and methods Bacteria and growth conditions In vitro synthesis of DPD Vibrio harveyi bioassay of AI-2 Genomic analyses Borrelia burgdorferi genomic libraries construction and analyses Methionine synthase assay Results Borrelia burgdorferi synthesizes AI-2 during laboratory cultivation Borrelia burgdorferi modulates protein expression in response to DPD Borrelia burgdorferi lacks a complete activated-methyl cycle Many other pathogenic spirochete species lack activated-methyl cycles Discussion Acknowledgements References Introduction As do most other organisms, the Lyme disease spirochete Borrelia burgdorferi lives in a series of dynamic environments. This bacterium persists in nature through cycles requiring infection of both vertebrates and ticks. During these infectious cycles, the spirochete must not only adapt to life in two extremely different types of host, but must also manage efficient transmission between the two. These constraints require that for B. burgdorferi to survive, it must precisely control gene expression so that only appropriate proteins are expressed during each stage of infection (Schwan, 1996; Indest et al., 2001b; Seshu and Skare, 2001; Anguita et al., 2003). Since disruption of regulatory networks that control expression of infection-associated proteins is a key goal for development of novel therapies to prevent and treat Lyme disease, characterization of those control mechanisms will be of great value. A growing number of bacteria are known to utilize derivatives of 4,5-dihydroxy-2,3pentanedione (DPD) as a pheromone to control gene expression (Xavier and Bassler, 2003). DPD can spontaneously cyclize and/or interact with borate to form at least two different, interconvertible molecules collectively described as autoinducer-2 (AI-2, Fig. 1) (Chen et al., 2002; Miller et al., 2004). Although AI-2 was originally described as a quorum sensing molecule for measuring bacterial density (Surette et al., 1999), it appears that many bacteria instead use AI-2 as a pheromone during the exponential growth phase to signal metabolic status and fitness (Xavier and Bassler, 2003). In a previous study, our laboratory demonstrated that B. burgdorferi encodes a functional copy of LuxS, the enzyme that synthesizes DPD (Stevenson and Babb, 2002; Babb et al., 2005). (6K) Fig. 1. Composite diagram of metabolic pathways that constitute activated-methyl cycles. Abbreviations: SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; SRH, Sribosylhomocysteine; DPD, 4,5-dihydroxy-2,3-pentanedione; Me-THF, 5-methyltetrahydrofolate; THF, tetrahydrofolate. DPD can spontaneously cyclize and/or combine with borate to produce at least two different, interconvertible forms of AI-2 (Schauder et al., 2001; Chen et al., 2002; Miller et al., 2004). Most organisms contain either the SAH hydrolase pathway or the Pfs/LuxS pathway for homocysteine synthesis, although some organisms contain both (Sun et al., 2004). Figure adapted from Babb et al. (2005). Many bacteria utilize LuxS as part of the activated-methyl cycle (Fig. 1). This series of reactions generates a methyl donor molecule, S-adenosylmethionine (SAM) for use in a wide variety of methylation reactions, then detoxifies byproducts and regenerates SAM. The second product of LuxS is homocysteine, which many organisms metabolize to produce methionine. For bacteria with a complete activated-methyl cycle such as illustrated in Fig. 1, LuxS plays an important role in maintaining a pool of free methionine in the cell. For example, mutants of Escherichia coli defective in methionine synthase are unable to grow in minimal media that lack added methionine (Urbanowski et al., 1987; Matthews, 1996). This function of LuxS has led to suggestions that the primary role of that enzyme in bacteria is to synthesize homocysteine for recycling, rather than for synthesis of DPD, and that effects of luxS mutations on bacterial pathogenicity are due to methionine starvation (Winzer et al., 2002a, Winzer et al., 2002b and Winzer et al., 2003; Blevins et al., 2004). For this reason, we examined B. burgdorferi in detail to determine whether that bacterium can also regenerate methionine from homocysteine, and found that it lacks such an ability, indicating that the Lyme disease spirochete produces LuxS for a purpose other than homocysteine synthesis. Materials and methods Bacteria and growth conditions B. burgdorferi were cultured in Barbour–Stoenner–Kelly II broth (Barbour, 1984). B. burgdorferi strain 297 is an infectious, wild-type bacterium and was originally isolated from human cerebrospinal fluid in New York, USA (Steere et al., 1983). Strain AH309 is a luxS-deficient mutant of strain 297 (Hübner et al., 2003) and was obtained from Michael Norgard (University of Texas Southwestern, Dallas, USA). For analyses of AI-2 production by B. burgdorferi, cultures of strains 297 and AH309 were grown at 34 °C to densities of approximately 107 bacteria per ml. Those cultures were then placed at 23 °C, which greatly retards bacterial growth (Stevenson et al., 1995). Aliquots of each initial culture were diluted 1:1000 into fresh medium on 6 subsequent days, with each secondary culture incubated at 34 °C to allow optimal growth. On day 7, supernatant was removed from each secondary culture and assayed for AI-2 content. Through this technique, cultures with essentially the same starting densities could be grown under the same conditions for 1–7 days and then assayed simultaneously. Bacterial density of each culture was also determined at that time, using a Petroff–Hauser counting chamber and dark-field microscopy. E. coli strain GS162 is wild type for both methionine synthase genes, while GS472 is defective in both metH and metE. Both were obtained from George Stauffer (University of Iowa, USA) (Urbanowski et al., 1987). For attempted complementation of E. coli metE and metH, transformed GS472 was plated on M9 minimal salts agar supplemented with 1 μg/ml thiamin, with this same medium plus 100 μg/ml methionine serving as a positive control for growth (Urbanowski et al., 1987; Sambrook et al., 1989; Zeh et al., 2002). For use as a positive control for methionine synthase analyses, GS162 was cultured in M9 supplemented with 3.4 μg/ml hydroxocobalamin, 1 μg/ml thiamin and 100 μg/ml phenylalanine (Urbanowski et al., 1987; Jarrett et al., 1997). Vibrio harveyi strain BB170 (Bassler et al., 1997) was obtained from Bonnie Bassler (Princeton University, USA). V. harveyi were cultivated in modified autoinducer bioassay medium (Greenberg et al., 1979) containing 40 μM sodium borate (pH=6.8). Leptospira interrogans serovar pomona type kennewicki strain JEN4 (Nally et al., 2001) was cultured at 30 °C in Bovuminar PLM-5 medium (Intergen, Purchase, NY). In vitro synthesis of DPD DPD was synthesized from S-adenosylhomocysteine nucleosidase (SAH) (Sigma, St. Louis, MO, USA), using equimolar concentrations of recombinant B. burgdorferi LuxS and Pfs proteins. Reaction products were quantified using Ellman's reaction, as previously described (Schauder et al., 2001; Babb et al., 2005). Vibrio harveyi bioassay of AI-2 V. harveyi strain BB170 autofluoresces in the presence of AI-2 in a dose-dependent manner, enabling its use in a bioassay specific for AI-2 (Bassler et al., 1997; Taga, 2005). Bioassays were performed as previously described (Surette and Bassler, 1998; Stevenson and Babb, 2002). AI-2 activities are reported as average luminescence values of each strain 297 assay minus the average luminescence of equivalent cultures of strain AH309. Studies were repeated at least three times using independent cultures. Genomic analyses Genome databases of B. burgdorferi and three other spirochetal species were queried for presence of specific gene homologs. Complete genome sequences of B. burgdorferi strain B31 (Fraser et al., 1997; Casjens et al., 2000), Treponema pallidum Nichols strain (Fraser et al., 1998), T. denticola ATCC 35405 (Seshadri et al., 2004), L. interrogans serovar Lai strain 56601 (Ren et al., 2003), and L. interrogans serovar Copenhageni strain Fiocruz L1-130 (Nascimento et al., 2004a) were accessed through the Institute for Genomic Research microbial database at http://www.tigr.org. To search for homologs of enzymes involved in SAH metabolism, each genome was queried using BLAST-P with the amino acid sequences of the biochemically characterized V. harveyi and B. burgdorferi LuxS, E. coli Pfs, and Rhodobacter capsulatus SAH hydrolase proteins, and the hypothetical SAH hydrolase of L. interrogans (GenBank accession numbers AF120098, AAC66762, U24438, M80630, and AAN51667, respectively). To search for homologs of methionine synthase enzymes, the genomes were queried with protein sequences of the MetE homologs of E. coli, Mycobacterium tuberculosis, the archaeon Methanobacterium thermoautotrophicum, and the plant Solanum tuberosum; the MetH homologs of E. coli, L. interrogans, and Synechococcus sp. WH8102; the methylmethioninedependent YagD enzyme of the E. coli CP4-6 prophage; and the human betaine- dependent methionine synthase (GenBank accession numbers M87625, AAK45422, X92082, AF082893, P13009, AAN51667, CAE07753, AAC73364, and U50929, respectively). Each identified spirochetal gene was used to re-query all of the spirochete genomes, along with the complete GenBank database at http://www.ncbi.nlm.nih.gov/blast/. Borrelia burgdorferi genomic libraries construction and analyses Total genomic DNA of infectious, wild-type B. burgdorferi strain B31 was digested using either EcoRI or PstI, then ligated with appropriately digested pUC118 (Vieira and Messing, 1987). Each ligation mixture was used to transform E. coli strain GS472 and bacteria plated on solid M9 medium lacking methionine. Methionine synthase assay 5-methyltetrahydrofolate-homocysteine S-methyltransferase activity was determined as described by Jarrett et al. (1997). Produced 14C-labeled methionine was separated from the reactants by passage through AG 1-X8 columns (Bio-Rad), then measured using a scintillation counter. Both E. coli strains GS162 and GS472 were analyzed as controls, and all experiments included a negative control that lacked any bacterial lysate. Number of decays per minute in the column flow-through of the negative control reaction was then subtracted from each experimental value. Results Borrelia burgdorferi synthesizes AI-2 during laboratory cultivation Cultured B. burgdorferi appear to utilize SAM in a variety of methylation reactions (Hughes and Johnson, 1990; Fraser et al., 1997; Charon and Goldstein, 2002). Both immunoblot analysis and RT-PCR demonstrated that cultured B. burgdorferi also produce both Pfs and LuxS enzymes (Hübner et al., 2003; Babb et al., 2005). Although earlier assays failed to detect production of DPD by cultivated B. burgdorferi (Stevenson and Babb, 2002; Hübner et al., 2003), we have since refined analysis techniques and found that the spirochete does indeed synthesize DPD, as described below. The most sensitive assay available for detection of DPD uses the marine bacterium V. harveyi as a biosensor (Taga, 2005). V. harveyi recognizes a cyclic, borate derivative of DPD and responds by autofluorescing at levels proportional to the amount of added DPD. Generally, unused growth medium of the tested microorganism is utilized as a negative control, and values obtained for the control then subtracted from experimental results. However, B. burgdorferi is cultured in a very rich, complex medium. We observed that addition of unused B. burgdorferi culture medium to V. harveyi caused the reporter bacteria to grow far more rapidly than did addition of B. burgdorferi culture supernatant, yielding the artifactual result of greater bioluminescence levels being attained from unused medium than from spent medium (Babb et al., 2005). To avoid this artifact, we instead compared bioassay results of wild-type strain 297 and an isogenic luxS mutant, AH309, using results from the mutant as background. Equivalent cultures of each strain were simultaneously examined during early through late exponential phases of growth, as well as after reaching stationary phase (Fig. 2). Significant levels of DPD were found in the supernatants of cultured B. burgdorferi 297, reaching a maximum during the midexponential phase and then decreasing as cultures entered stationary phase. The reduced levels of DPD in stationary phase cultures suggest that B. burgdorferi actively removes the molecule from the medium, as do several other studied bacteria (Surette and Bassler, 1999; Taga et al., 2001 and Taga et al., 2003; Hardie et al., 2003; Xavier and Bassler, 2003). (20K) Fig. 2. Production of AI-2 by Borrelia burgdorferi. Strains 297 (wild-type) and AH309 (luxS) were diluted into fresh medium and grown for 1–7 days at 34 °C. Culture densities are illustrated as a growth curve (left Y-axis). Vibrio harveyi bioassays were performed for each culture, with luminescence values obtained for the negative control AH309 being subtracted from corresponding values obtained for strain 297. Statistically significant (>90% confidence interval by independent sample t-test) mean luminescence values are illustrated as rectangles (right Y-axis). Error bars represent standard deviations of 2–5 separate experiments. Since culture pH may affect bioassay results (DeKeersmaecker and Vanderleyden, 2003), the pH of the B. burgdorferi culture medium was examined at all time points and found to have dropped only slightly during the studies, from an initial 7.5 to a final pH of 6.8 after 3 days at stationary phase, so it is unlikely that the pH of the tested B. burgdorferi culture media influenced these results. However, V. harveyi also controls bioluminescence in response to cellular levels of cAMP, repressing light generation in the presence of PTS sugars such as glucose (Chatterjee et al., 2002; DeKeersmaecker and Vanderleyden, 2003). Unfortunately, all culture media capable of supporting B. burgdorferi growth include glucose as a primary carbon source, as well as other, essential PTS carbohydrates such as N-acetylglucosamine (Barbour, 1984; von Lackum and Stevenson, 2005). Furthermore, B. burgdorferi is capable of high growth rates for 3–4 days in media lacking glucose, due to energy provided by other medium components (von Lackum and Stevenson, 2005). Media capable of supporting growth of these fastidious spirochetes but lack trace carbohydrates do not exist. The unavoidable effects of catabolite repression on V. harveyi reporter strain bioluminescence lead us to suspect that the DPD synthesis results reported for B. burgdorferi may be artificially low (Babb et al., 2005). Borrelia burgdorferi modulates protein expression in response to DPD Results of the above-described studies indicated that B. burgdorferi can and does synthesize DPD. Two additional studies indicated that B. burgdorferi utilizes DPD (or a derivative of that molecule) as an AI-2 pheromone to control protein expression. First, a luxS-deficient mutant of E. coli was complemented with the B. burgdorferi luxS gene, and demonstrated to synthesize DPD (Stevenson and Babb, 2002). Addition of sterile culture supernatant from those complemented bacteria to B. burgdorferi influenced expression levels of more than 50 B. burgdorferi proteins, including the factor H-binding Erp outer surface proteins (Stevenson and Babb, 2002). Culture supernatants from the uncomplemented E. coli luxS mutant had no detectable effects. Second, DPD was synthesized in vitro and B. burgdorferi cultures examined for effects of the added compound (Babb et al., 2005). Again, expression levels of a specific subset of B. burgdorferi proteins were measurably affected by addition of reaction products to cultures (Babb et al., 2005). Furthermore, the effect of DPD addition was dose dependent, as would be expected for a pheromone. Control assays in which only homocysteine or SAH were added did not yield detectable changes to expression levels of any B. burgdorferi protein. The effects of adenine, the third product of the Pfs and LuxS reactions were not examined, since B. burgdorferi is an auxotroph for that molecule (Wyss and Ermert, 1996) and culture medium contains a substantial concentration of adenine (Barbour, 1984). These control experiments demonstrated that the effects of adding reaction products to culture medium were due to DPD alone. Parallel studies of wild-type strain 297 and its isogenic luxS mutant AH309 revealed that some proteins were detectable in lysates of uninduced strain 297, but were not visible in lysates of AH309, suggesting that the amounts of DPD produced by wild-type B. burgdorferi during laboratory cultivation cause significant effects on protein levels. Additionally, AH309 responded to addition of DPD in manners similar to the wild-type strain, indicating that responses to that molecule can occur independently of LuxS (Babb et al., 2005). Both proteomic and microarray analyses were utilized to identify B. burgdorferi proteins and genes that are controlled through DPD (Babb et al., 2005). Most of the tentatively identified proteins have yet to be characterized. We are in the process of developing reagents to confirm those preliminary data and further characterize DPDregulated genes and proteins. Previously characterized proteins whose expression levels have been demonstrated to be influenced by DPD include the Erp protein family (Stevenson and Babb, 2002). These outer membrane lipoproteins are expressed during transmission between ticks and mammals and during persistent mammalian infection, but not during tick infection (Lam et al., 1994; Akins et al., 1995; Stevenson et al., 1995 and Stevenson et al., 1998; Suk et al., 1995; Wallich et al., 1995; Das et al., 1997; El-Hage et al., 2001; Gilmore et al., 2001; Hefty et al., 2001 and Hefty et al., 2002; McDowell et al., 2001; Miller et al., 2003 and Miller et al., 2005; Miller and Stevenson, 2004). Many members of the Erp protein family have been demonstrated to bind the host complement regulator molecule factor H, suggestive of roles in establishment and maintenance of mammalian infection (Hellwage et al., 2001; Kraiczy et al., 2001a, Kraiczy et al., 2001b, Kraiczy et al., 2003 and Kraiczy et al., 2004; Alitalo et al., 2002; Stevenson et al., 2002; McDowell et al., 2003; Metts et al., 2003). Along with the effects of DPD, erp genes are also controlled by environmental temperature and uncharacterized chemical signals (Stevenson et al., 1995; Akins et al., 1998; Babb et al., 2001 and Babb et al., 2004; Hefty et al., 2001; El-Hage and Stevenson, 2002). In other studies, we identified DNA sequences involved in control of erp gene transcription (Babb et al., 2004) and are continuing to explore the mechanism by which DPD affects Erp expression. We also identified that B. burgdorferi increases expression of the polymorphic surface protein VlsE in directly proportional response to DPD concentration (Babb et al., 2005). VlsE is expressed during mammalian infection, where it protects the bacterium from host antibodies through an antigenic variation mechanism (Zhang et al., 1997; Zhang and Norris, 1998a and Zhang and Norris, 1998b; Liang et al., 1999). Regulatory mechanisms controlling vlsE expression are also quite complex, and include environmental signals such as temperature and pH and interactions with mammalian cell membranes (Hudson et al., 2001; Indest et al., 2001a; Ohnishi et al., 2003; K. von Lackum, K. Babb, S.P. Riley, R.L. Wattier, T. Bykowski, B. Stevenson, unpublished results). As with the erp loci, the vlsE promoter/operator region specifically binds multiple B. burgdorferi cytoplasmic proteins (T. Bykowski, K. Babb, and B. Stevenson, unpublished results), one or more of which may be responsible for the effects of DPD on gene expression. Borrelia burgdorferi lacks a complete activated-methyl cycle As noted above, evidence indicates that B. burgdorferi utilizes SAM as a methyl donor for many cellular reactions. We have demonstrated that the Lyme disease spirochete expresses functional Pfs and LuxS enzymes, and can therefore detoxify SAH to yield DPD, adenine, and homocysteine (Babb et al., 2005). Additional studies demonstrated that B. burgdorferi also encodes a functional MetK for synthesis of SAM from methionine (S.P. Riley and B. Stevenson, unpublished results). Almost all examined organisms, from bacteria to plants to animals, possess complete activatedmethyl cycles and regenerate methionine from homocysteine (Fig. 1) (Sun et al., 2004). For this reason, it has been suggested that the major, and possibly the only, reason bacteria possess LuxS is to produce homocysteine for re-synthesis of methionine (Winzer et al., 2002a, Winzer et al., 2002b and Winzer et al., 2003; Blevins et al., 2004). However, the following studies indicated that B. burgdorferi lacks the ability to synthesize methionine. The genome sequence of B. burgdorferi strain B31 was examined for an open reading frame (ORF) homologous to a previously characterized methionine synthase. The two major classes of this enzyme use 5-methyltetrahydrofolate or its derivatives as the methyl donor. The best characterized methionine synthases are the MetH and MetE proteins of E. coli. Proteins homologous to one or both these enzymes have been found in nearly every examined prokaryote and eukaryote (Sun et al., 2004). MetH and MetE enzymes are commonly referred to as cobalamin-dependent and cobalaminindependent methionine synthases, respectively (González et al., 1992). Two other identified types of methionine synthase use either betaine or methylmethionine as the methyl donor (Garrow, 1996; Neuhierl et al., 1999; Thanbichler et al., 1999). Both those enzymes share very similar amino acid sequences with cobalamin-dependent methionine synthases (Garrow, 1996; Thanbichler et al., 1999). BLAST-P queries against the predicted proteins of B. burgdorferi indicated that this bacterium lacks a protein with recognizable homology to any known methionine synthase. To examine the possibility that B. burgdorferi may encode a completely novel methionine synthase, we used plasmid libraries of B. burgdorferi DNA in attempts to complement an E. coli metE metH mutant. While this technique had previously been used to clone methionine synthase genes from organisms as evolutionarily distant from E. coli as the potato (Zeh et al., 2002), all attempts have failed to identify such a gene in B. burgdorferi. Finally, we biochemically analyzed cellular extracts of B. burgdorferi for methionine synthase activity. Consistent with data from our genomic and cloning analyses, no enzymatic activity could be detected in cell-free extracts of this spirochete (data not shown). Control studies with wild-type E. coli detected enzyme activity, as expected. Many other pathogenic spirochete species lack activated-methyl cycles Results of our studies on B. burgdorferi prompted us to examine other species of spirochetes for possible methionine synthases. Examination of the unpublished genome sequences of the relapsing fever spirochetes B. hermsii and B. turicatae indicates that both possess luxS homologs, yet lack homologs of any known methionine synthase (Tom Schwan, pers. comm.). The causative agent of syphilis, T. pallidum, contains a homolog of pfs, ORF TP0170. However, T. pallidum lacks genes for either LuxS or methionine synthase, and so probably does not encode a complete activated-methyl cycle (Fig. 3). The periodontal disease-associated spirochete T. denticola likewise contains a pfs homolog (ORF TDE0105) and lacks both luxS and a methionine synthase gene. Hence, none of these Treponema species is predicted to synthesize DPD. Related to the lack of methionine synthase genes in these spirochetes, neither B. burgdorferi nor T. pallidum encode homologs of any cobalamin-dependent enzyme or any proteins involved in either cobalamin synthesis or transport (Rodionov et al., 2003). These data indicate that an activated-methyl cycle is completely unnecessary for any of these pathogenic spirochetes to survive in nature. (5K) Fig. 3. Experimentally determined metabolic pathways found in Borrelia burgdorferi. The Lyme disease spirochete utilizes methionine to produce SAM and detoxifies SAH to DPD, adenine, and homocysteine via Pfs and LuxS. This bacterium is unable to regenerate methionine from homocysteine. Among the six spirochete species for which genome sequence data are available, only L. interrogans encodes a potential homocysteine salvage enzyme, an ortholog of MetH (Fig. 4). Consistent with that observation, analysis of L. interrogans lysates detected levels of methionine synthase activity that were comparable to wild-type E. coli controls (data not shown). L. interrogans contains a gene homologous to SAH hydrolases (ORF LB106), but lacks homologs of either pfs or luxS. Infectious leptospires also encode homologs of many other biosynthetic enzymes, as well as proteins involved with cobalamin metabolism (Picardeau et al., 2003; Ren et al., 2003; Rodionov et al., 2003; Nascimento et al., 2004a and Nascimento et al., 2004b; Sekowska et al., 2004). We conclude that L. interrogans, alone among the examined spirochete species, encodes a complete activated-methyl cycle, and can regenerate methionine from homocysteine (Fig. 5). It is also apparent that the leptospirosis spirochete cannot synthesize DPD. (4K) Fig. 4. Metabolic pathways of the syphilis spirochete Treponema pallidum and the peridontitisassociated spirochete T. denticola, as determined by genomic analyses. These spirochetes produce SAM from methionine for methylation reactions, then detoxify SAH to SRH and adenine via Pfs. Neither bacterium is predicted to regenerate methionine or to produce DPD. (6K) Fig. 5. Metabolic pathways of the leptospirosis agent, Leptospira interrogans, as determined by genomic analyses. This spirochete species is predicted to encode a complete activated-methyl cycle, with a single-step detoxification of SAH to homocysteine and adenosine. L. interrogans lacks a homolog of LuxS and is not predicted to synthesize DPD. Discussion Our studies demonstrated that B. burgdorferi detoxifies SAH by converting that molecule into DPD, adenine, and homocysteine. The Pfs- and LuxS-catalyzed reactions occur during laboratory cultivation, with DPD accumulating during the exponential phase of growth. However, biochemical and genomic analyses indicated that B. burgdorferi lacks a complete activated-methyl cycle, and is unable to regenerate methionine from homocysteine. These data lead us to conclude that the Lyme disease spirochete synthesizes LuxS for a purpose other than production of homocysteine for regeneration of methionine. We propose that B. burgdorferi produces a LuxS enzyme for the express purpose of synthesizing DPD. Several species of bacteria utilize DPD or a derivative as an AI-2 pheromone to control gene expression, and our studies indicate that B. burgdorferi is to be included among that list. The finding that relapsing fever Borrelia species also contain luxS raises the possibility that those infectious spirochetes may likewise use DPD as a signal. The mechanism by which B. burgdorferi senses DPD as a signal molecule is not yet known, as the sequenced genome does not encode recognizable homologs of proteins known to function in DPD sensing by other bacteria (Stevenson et al., 2003). However, we note that the two Gram-negative bacterial species for which the AI-2 sensors have been defined use extremely different mechanisms, so it is quite possible that the distantly related spirochetes use a completely novel sensor/regulatory mechanism (Bassler et al., 1994; Taga et al., 2001; Stevenson et al., 2003). We are continuing studies to characterize the mechanisms by which AI-2dependent regulation occurs in B. burgdorferi, as well as examining other spirochetes for use of this pheromone. Two recent publications reported that a luxS mutant of B. burgdorferi was capable of infecting both mice and ticks, leading those authors to suggest that neither LuxS nor AI-2 are necessary for infection processes (Hübner et al., 2003; Blevins et al., 2004). However, there are significant questions about how to interpret results of those experiments due to the process by which the mutant bacteria were derived: Bacteria were transformed by electroporation, briefly cultured in liquid medium with a selective antibiotic, placed in a dialysis bag that was then implanted in the peritoneum of a rat for 15 days, then removed from the dialysis bag, injected into a mouse, and, after 2 weeks of infection, a skin biopsy was cultured in liquid medium and, finally, plated in solid medium (Hübner et al., 2003). Bacteria from two resulting colonies were then tested for ability to infect mice. Since this complicated selection scheme mandated that bacteria maintain infectivity, it is impossible to know whether bacteria that survived the process contain only the introduced luxS lesion or if spontaneous mutations arose at additional loci to compensate for the loss of luxS. Moreover, none of those earlier studies compared relative levels of infectivity of mutant and wild-type bacteria. For these reasons, it is not yet known how critical AI-2-mediated genetic regulation is for B. burgdorferi pathogenesis. That B. burgdorferi does not regenerate methionine from homocysteine is consistent with other indications that it is an auxotroph for all amino acids. Culture media capable of supporting B. burgdorferi growth must contain all amino acids and many other nutrients (Barbour, 1984; Pollack et al., 1993). Growth of relapsing fever borreliae and oral treponemes also requires very complex culture media (Kelly, 1971; Wyss, 1992). The genome sequences of borreliae and treponemes indicate homologs of proteases and transporters of polypeptides and amino acids, but few or no biosynthetic enzymes (Fraser et al., 1997 and Fraser et al., 1998; Seshadri et al., 2004). This probably explains why all identified species of Borrelia or Treponema are found only in close association with host animals. In contrast, the sole spirochete known to possess a complete activated-methyl cycle, L. interrogans, is a water-borne pathogen capable of survival outside animal hosts. Additionally, L. interrogans encodes a great many amino acid biosynthetic enzymes (Picardeau et al., 2003; Ren et al., 2003; Nascimento et al., 2004a and Nascimento et al., 2004b). These observations indicate that while the free-living leptospires probably require a complete activated-methyl cycle in order to survive in methionine-deficient environments, the obligatory parasitic borreliae and treponemes do not need that pathway since they can scavenge sufficient quantities of methionine from their hosts for all metabolic processes. B. burgdorferi produces maximum levels of DPD during the exponential phase of growth, as is also found with many other bacteria (Xavier and Bassler, 2003). DPD synthesis increases during rapid bacterial growth, presumably due to increased number of methylation reactions, and is thereby thought to serve as a signal of bacterial fitness (Xavier and Bassler, 2003). DPD/AI-2 may thus function to coordinate growth-related processes throughout a population or serve as a form of positive feedback to the cell that produced it, or a combination of those two possibilities. Since DPD synthesis is often unrelated to culture density, it is probably more accurate to think of AI-2 as a pheromone than as a ‘quorum sensing’ molecule. In conclusion, B. burgdorferi can and does synthesize DPD. Furthermore, B. burgdorferi uses either DPD or a derivative thereof as an AI-2 pheromone to control expression levels of a specific subset of bacterial proteins. Those observations, along with the lack of a complete activated-methyl cycle, lead us to hypothesize that the Lyme disease spirochete produces LuxS for the express purpose of synthesizing DPD pheromone. Since maximal synthesis of DPD during cultivation was observed during the exponential phase of growth, it is likely that the pheromone also functions during periods of rapid growth in the natural infectious cycle. B. burgdorferi within the midguts of infected, feeding ticks experience dramatic increases in growth rate (Benach et al., 1987; de Silva and Fikrig, 1995; Piesman et al., 2001 and Piesman et al., 2003). Bacteria within feeding ticks also increase synthesis of LuxS (Narasimhan et al., 2002), consistent with our hypothesis and suggesting a role for AI-2 in coordinating bacterial gene expression during transmission (Stevenson and Babb, 2002; Miller et al., 2003; Stevenson et al., 2003; Miller and Stevenson, 2004). It is possible that B. burgdorferi may also use DPD/AI-2 to regulate gene expression during other stages of its infectious cycle, perhaps through self-induction (Redfield, 2002; Koerber et al., 2004). Acknowledgements This work was supported by National Institutes of Health Grants R01-AI53101 and 5T32-AI49795. We thank Bonnie Bassler, Michael Norgard, George Stauffer, and Xiaofeng Yang for providing bacterial strains; Tom Schwan for sharing unpublished results; Bonnie Bassler, Kenneth Cornell, Klaus Winzer, and Wolfram Zückert for helpful discussions; Sarah Wackerbarth for statistical analyses; and Sara Bair, Sarah Kearns, Natalie Mickelsen, Jennifer Miller, Ashutosh Verma, and Michael Woodman for assistance in this research. References Akins et al., 1995 D.R. Akins, S.F. Porcella, T.G. Popova, D. Shevchenko, S.I. Baker, M. Li, M.V. Norgard and J.D. Radolf, Evidence for in vivo but not in vitro expression of a Borrelia burgdorferi outer surface protein F (OspF) homologue, Mol. Microbiol. 18 (1995), pp. 507–520. Abstract-EMBASE | Abstract-MEDLINE | Abstract-Elsevier BIOBASE | Order Document Akins et al., 1998 D.R. Akins, K.W. Bourell, M.J. Caimano, M.V. Norgard and J.D. 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Norris, Antigenic variation in Lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes, Cell 89 (1997), pp. 1–20. Comparison of isolation rate of Borrelia burgdorferi sensu lato in MKP and BSK-II medium Eva Ružić-Sabljića, , , Stanka Lotrič-Furlanb, Vera Maraspinb, Jože Cimpermanb, Mateja Logarb, Tomaž Jurcab and Franc Strleb Institute of Microbiology and Immunology, School of Medicine, Zaloška 4, SI-1000 Ljubljana, Slovenia b Department of Infectious Diseases, University Medical Centre Ljubljana, Japljeva 2 Ljubljana, Slovenia a Available online 10 March 2006. Abstract Different media have been utilized for borrelial cultivation. The aim of the present study was to evaluate the isolation rate of Borrelia burgdorferi sensu lato from two commonly used media, i.e. modified Kelly–Pettenkofer (MKP) and Barbour– Stoenner–Kelly II (BSK-II) medium, and to compare the isolated strains with regard to their phenotypic and genotypic characteristics. Skin biopsy specimens of 2×2×4 mm were taken from the peripheral site of human solitary erythema lesions and were divided in two pieces, one of which was inoculated into MKP and the other one into BSK-II medium. Species analysis of the obtained strains was performed and their plasmid and protein profiles were determined. Borrelia strains were isolated from 48/96 patients (50%) with erythema migrans. We obtained in 26/48 patients (54%) from MKP as well as from BSK-II, in 11 patients (23%) only from MKP, and in another 11 (23%) only from BSK-II medium a positive result. B. afzelii was isolated from 43 patients (23 were positive in both media, nine in MKP only and 11 in BSK-II medium only), while B. garinii was isolated from five patients (in three from both media, in two from MKP only). All strains of the obtained strain pairs were identical according to species and the type within the species. Plasmid profiles were identical in 17/21 B. afzelii strain pairs (81%) and in 1/3 B. garinii strain pairs; in 6/24 strain pairs, distinctions in the number of plasmids or in their molecular mass were present. Differences in the protein profile were found in 7/24 strain pairs (29%). The distinctions were uniform and were limited to the expression of OspC. In conclusion, our study showed comparable Borrelia isolation rates from MKP and BSK-II medium. The results of the present study indicate that human patients with Lyme borreliosis may simultaneously harbor heterogeneous B. burgdorferi s.l. strains. Keywords: Borrelia burgdorferi sensu lato; MKP medium; BSK-II medium; Isolation rate Article Outline Introduction Materials and methods Borrelia isolation from patients Culture media Patients Cultivation Analysis of Borrelia strains Identification of Borrelia species by pulsed-field gel electrophoresis (PFGE) Identification of Borrelia species by polymerase chain reaction (PCR) Analysis of plasmid profile Analysis of protein profile Results Isolation rate Species identification Plasmid profile Protein profile Discussion References Introduction Lyme borreliosis may exhibit different clinical manifestations, most frequently erythema migrans, which represents a typical clinical sign early in the course of the disease (Nadelman et al., 1996; Strle et al., 1996; Nadelman and Wormser, 1998; Arnež et al., 2003; Stanek and Strle, 2003). From this localized skin infection borreliae may disseminate to other parts of the human organism and cause different clinical manifestations (Lotrič-Furlan et al., 1999; Wormser et al., 1999;Maraspin et al., 2001; Oksi et al., 2001; Stanek and Strle, 2003). Isolation of the etiological agent from patient's material is the most reliable method for the diagnosis of borrelial infection (Pfister et al., 1984; Preac-Mursic et al., 1986; Picken et al., 1997; LotričFurlan et al., 1999; Wormser et al., 1999; Maraspin et al., 2002; Arnež et al., 2003). Numerous Borrelia strains obtained from human tissue and tissue fluids (skin, blood, cerebrospinal fluid, etc.) have been analyzed by different phenotypic and genotypic approaches (Wilske et al., 1988 and Wilske et al., 1993; Wang et al., 1999; RužićSabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b). In comparison to phenotypic assessment, analysis of borrelial genomic DNA provides more precise information on the genetic relationships among Borrelia strains (Belfaiza et al., 1993; Busch et al., 1995; Busch and Nitschko, 1999; Olive and Bean, 1999; Wang et al., 1999; Wormser et al., 1999; Ružić-Sabljić et al., 2000, RužićSabljić et al., 2001a and Ružić-Sabljić et al., 2001b). Many studies indicate that B. burgdorferi sensu lato populations are genetically divergent and phenotypically heterogeneous, and that Lyme borreliosis in humans is caused by at least three different species of the B. burgdorferi s.l. complex, viz. B. afzelii, B. garinii, and B. burgdorferi sensu stricto (Belfaiza et al., 1993; Burgdorfer, 1995; Rijpkema et al., 1995; Liveris et al., 1996; Picken et al., 1996; Mathiesen et al., 1997; Wang et al., 1999; Wormser et al., 1999; Ružić-Sabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b). Isolation of B. burgdorferi s.l. from clinical materials is a golden standard for confirming borrelial infection. Borrelia cultivation and isolation is quite a demanding procedure with limited sensitivity (Jobe et al., 1993; Nadelman et al., 1996; Strle et al., 1996; Kollars et al., 1997, Picken et al., 1997; Lotrič-Furlan et al., 1999; Wormser et al., 2000; Maraspin et al., 2001). Different media for borrelial cultivation have been reported. For routine work, modified Kelly–Pettenkofer (MKP), Barbour–Stoenner– Kelly II (BSK-II) medium and commercially available BSK-H (Sigma, USA) medium have been utilized most frequently (Barbour, 1984; Preac-Mursic et al., 1986; Pollack et al., 1993). The majority of the components of the three media are equivalent and present in similar concentrations (e.g. CMRL, N–acetyl glucosamine, bovine serum albumin, rabbit serum, etc.) but the origin of components may be dissimilar (e.g. bought from different commercial firms). However, media may differ in certain constituents. For example, MKP lacks yeast extract present in BSK–II and BSK-H, but–in contrast to BSK-II and BSK-H–contains gelatin (Barbour, 1984; Preac-Mursic et al., 1986; Pollack et al., 1993). To be useful for laboratory purposes, the medium must be stable, available and efficacious in supporting the growth of all human pathogenic Borrelia strains. In addition to medium ingredients, several other factors including temperature of incubation, pH of the medium, capacity of particular borrelial species to grow and number of Borrelia strains in the sample can influence borrelial growth and the outcome of cultivation (Hubalek et al., 1998; Ružić-Sabljić and Strle, 2004). The aim of the present study was to evaluate and compare the isolation rate of B. burgdorferi s.l. from the skin of patients with erythema migrans using MKP and BSK-II medium, and to compare phenotypic and genotypic characteristics of the strain pairs, i.e. the strains isolated in the two media from the skin biopsy of an individual patient. Materials and methods Borrelia isolation from patients Culture media In the present study MKP and BSK-II medium were used for Borrelia isolation. Both media were prepared in our laboratory after the original recipes (Barbour, 1984; Preac-Mursic et al., 1986). The two media differ with regard to the glucose concentration (3 versus 5 g/l in MKP and BSK-II, respectively), the origin and concentration of bovine serum albumin (35% solution with a final concentration of 1 g/ml in MKP versus fraction V powder with final concentration 4 g/ml in BSK-II), the concentration and preparation of rabbit sera (7.2% heat-inactivated versus 6% non-inactivated in MKP and BSK-II, respectively). Additionally, yeastolate (2 g/l) was added to BSK-II but not to MKP medium. After preparing and testing the quality of each medium lot, the media were distributed to clinicians. Patients Ninety-six consecutive adult patients diagnosed with erythema migrans at the Department of Infectious Diseases, University Medical Center Ljubljana, in spring of 2000 represented the basis for the present study. In each patient, a skin biopsy was taken from the peripheral site of erythema migrans after prior cleaning the skin with 70% alcohol and local anesthesia with 2% xylocaine (Jurca et al., 1998). Each specimen (2×2×4 mm) was dissected in two pieces: one was immediately inoculated into MKP, the other one into BSK-II medium. The inoculated media were sent to the laboratory at room temperature. Cultivation Samples were incubated at 33 °C and examined weekly for up to 9 weeks for the presence of spirochetes by dark-field microscopy. Isolated strains were grown in the same medium for 2–4 subcultures. Analysis of Borrelia strains For the large majority of isolates, strain identification was carried out by pulsed-field gel electrophoresis (PFGE). Identification by polymerase chain reaction (PCR) was performed only for cultures with borrelial concentration not high enough for PFGE. Identification of Borrelia species by pulsed-field gel electrophoresis (PFGE) Strain identification by PFGE was performed as described previously (Busch et al., 1995; Ružić-Sabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b). Briefly, Borrelia strains were mixed in agarose, and whole blocks were lysed in lysis buffer containing lysozyme (1 mg/ml) and ribonuclease (10 μg/ml), and digested in digestion buffer containing proteinase K (0.5 mg/ml). After exhaustive block washing, DNA was restricted with 30 U of MluI restriction enzyme. Restricted patterns were separated for 24 h with ramping pulse times of 1–40 s. Molecular mass marker of 50–1000 kb (Sigma, USA) was added to each electrophoresis. Borrelia species as well as the type within the particular species were determined by restriction fragment length polymorphism (RFLP) as described by Belfaiza et al. (1993) and Picken et al. (1996). Fragments interpreted as being species specific were those with molecular weights 440, 320 and 90 kb for B. afzelii, 220 and 80 kb for B. garinii and 145 kb for B. burgdorferi s.s. Types within species (Mla for B. afzelii, Mlg for B. garinii and Mlb for B. burgdorferi s.s.) were determined according to additional fragments of different molecular weights (Belfaiza et al., 1993; Picken et al., 1996). Identification of Borrelia species by polymerase chain reaction (PCR) Tubes with culture were centrifuged at 10,000 rpm and borrelial DNA was isolated from the sediment using commercial kit (QIAamp DNA mini kit, Qiagen cat. no. 51306, Germany). Interspace 5S-23S DNA region was amplified. The amplified products were restricted by restriction enzyme MseI and electrophoresed in 16% polyacrylamide gel. Borrelia species were determined by RFLP as described by Postic et al. (1994). Fragments interpreted as being species specific were those with molecular weights 108, 68, 50 and 20 bp for B. afzelii, 108, 95 and 50 bp for B. garinii and 108, 51, 38, 29 and 28 bp for B. burgdorferi s.s. Analysis of plasmid profile The procedure was limited to cultures with a sufficient Borrelia density to allow the execution of PFGE, and was determined only in patients in whom borreliae were isolated from an erythema migrans lesion concurrently in MKP and in BSK-II medium (when two isolates were obtained from an individual skin biopsy sample). To compare plasmid profiles within such strain pairs, we performed electrophoresis of both strains on the same gel. Plasmid profiles were determined by PFGE. Blocks with borrelial DNA were prepared identically as for species identification but electrophoresis was performed without previous restriction of DNA. Chromosomal and plasmid DNA were separated for 37 h in ramping pulse time of 0.9–3 s as described previously (Busch et al., 1995; Ružić-Sabljić et al., 1999, Ružić-Sabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b). Molecular mass marker of 0.1–200 kb (Sigma, USA) was added to each separation. Analysis of protein profile Protein profile analysis of borrelial strains was performed only in patients in whom borreliae were isolated in MKP as well as in BSK-II medium. To compare protein profiles of concurrently isolated strains, we performed electrophoresis of both strains on the same gel. For the determination of protein profiles, strains were harvested, washed and lysed in buffer containing sodium dodecyl sulfate (2.5%) and 2mercaptoethanol (2.5%), as described elsewhere (Wilske et al., 1988; Busch et al., 1995; Ružić-Sabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b). Electrophoresis was performed in 12% polyacrylamide gel. Low molecular mass marker of 21–106 kD (BioRad, Germany) was used as size marker in each protein profile determination. Results Isolation rate Skin biopsy specimens were obtained from 96 consecutive adult patients with typical solitary erythema migrans. Borrelia strains were isolated from 48/96 skin lesions (patients) (50%). There was no difference in the isolation rate when comparing MKP and BSK-II medium (37 positive cultures in each particular medium). In 26/48 patients (54%) borreliae were isolated in MKP as well as in BSK-II medium, while in 11/48 patients (23%) borreliae were isolated either in MKP or BSK-II (Table 1). Table 1. Borrelia burgdorferi sensu lato isolation rate from human erythema migrans skin biopsy sample divided in two specimens–comparison of modified Kelly–Pettenkofer (MKP) and Barbour–Stoenner– Kelly II (BSK-II) medium Culture-positive samples In MKP and BSK-II In MKP only In BSK-II only 26 11 11 48 Culture negative Total 48 96 Species identification Out of the 74 isolated Borrelia strains, 69 (93%) were typed using PFGE, while in 5 strains (3 in MKP and 2 in BSK-II medium) that did not grow sufficiently, species identification was preformed by PCR. Sixty-six strains (89%) were identified as B. afzelii (in 23 patients, the strains were isolated in both media, in 9 patients in MKP only, and in 11 patients in BSK-II only) and 8 as B. garinii (in 3 patients isolated in both media, and in 2 patients isolated in MKP only). Data are shown in Table 2. Table 2. Species identification of Borrelia strains isolated from human erythema migrans skin biopsies divided in two specimens – comparison of modified Kelly–Pettenkofer (MKP) and Barbour–Stoenner–Kelly II (BSK-II) medium Positive culture in medium B. afzelii B. garinii No. of isolated strains PFGEa PCRb PFGE PCR MKP and 22 1 3 – 26 BSK-II 22 1 3 – 26 MKP only 7 2 2 – 11 BSK-II only 10 1 – – 11 Subtotal 61 5 8 0 Total 66 a b 8 74 Identification of Borrelia species with pulsed-field gel electrophoresis. Identification of Borrelia species with polymerase chain reaction. From 48 skin-culture-positive patients with erythema migrans (Table 1), there were 26 patients with borreliae isolated in both media. Of these 26 strain pairs (52 strains), 24 strain pairs were suitable for further PFGE identification and were used for analyzing and comparing PFGE types within the species as well as plasmid and protein profiles. Each individual strain pair was congruent according to the species as well as to the PFGE type. Regarding PFGE type, strain pairs consisted of either B. afzelii Mla1 (21 pairs) or B. garinii Mlg2 (3 pairs). Plasmid profile Identical plasmid profiles were found in 17/21 B. afzelii strain pairs (81%) and in 1/3 B. garinii strain pairs, while in 6/24 patients, the two strains differed either in the number of plasmids or their molecular mass. The plasmid profiles of some strain pairs are shown in Fig. 1. (50K) Fig. 1. Plasmid profile of Borrelia strains (1 and 2) isolated in both modified Kelly–Pettenkofer (MKP) and Barbour–Stoenner–Kelly II (BSK-II) medium. Strains were isolated from skin biopsies of patients with erythema migrans (A–J). MW: molecular mass marker; B. afzelii strains: A–F and J; B. garinii strains: G–I. Plasmid profile of strain pairs differs in patients A–D and G. Protein profile Protein profiles of strain pairs obtained from patients in whom borreliae were isolated in MKP as well as in BSK-II medium differed in 7/24 cases (29%). All those strains were identified as B. afzelii and showed identical distinction–one strain of each pair expressed OspC, whereas the other did not. Protein profiles differed in six strain pairs which were identical according to the species and the plasmid profile, and in one strain pair both strains belonged to the same species but were distinct in their plasmid profile (patient D from the Fig. 1). Discussion Although erythema migrans represents a pathognomonic clinical sign of Lyme borreliosis, the isolation rate of Borrelia strains from the skin lesions was only 50% (48/96 patients) in the present study, corroborating previous findings from Slovenia (Strle et al., 1996; Jurca et al., 1998; Zore et al., 2002). The fastidious nature of the organism and changes that Borrelia must undergo to adapt the transition from living biological material to an artificial medium probably limit the isolation rate (Picken et al., 1996; Norris et al., 1997). Additionally, ingenious characteristics of the individual Borrelia species such as a tendency of better growth or overgrowth of certain Borrelia species over the other(s) (Ružić-Sabljić and Strle, 2004) might influence borrelial isolation rate. Several other factors, for example, the quantity of spirochetes in a given sample, previous antibiotic treatment, local anesthesia at the site of biopsy, conditions of sample transport to the laboratory, medium used for Borrelia cultivation, aseptic environment of culture maintenance, etc. can also influence the isolation rate (Kollars et al., 1997; Wormser et al., 2000). The relatively low isolation rate of Borrelia strains in our patients with erythema migrans might have been caused by the rather small size of skin biopsy specimens (e.g. 2×2×4 mm) that was additionally divided in two pieces. Some previous studies suggested that borreliae are present in erythema migrans lesions in only low concentration and are irregularly distributed (Pachner et al., 1993; Picken et al., 1997; Zore et al., 2002). With larger samples, the isolation rate would probably be higher (Wormser et al., 2000). The main interest of the present study was to compare the isolation rate of B. burgdorferi s.l. from erythema migrans skin samples in two commonly used media, MKP and BSK-II. We had anticipated that our approach of taking one skin biopsy from an individual patient, dissecting it in two pieces and inoculating each single piece in a particular medium, would lead to identical (positive or negative) results in the large majority of cases. The unexpected discrepancy may be explained by unequal distribution of Borrelia strains within individual samples. Our findings and proposed explanations are in accordance with the results of some previous studies which suggested that borreliae are present in erythema migrans lesions in low concentration and are irregularly distributed (Pachner et al., 1993; Picken et al., 1997; Zore et al., 2002). Sixty-six out of 74 isolates (89%) were typed as B. afzelii and 8 as B. garinii; no B. burgdorferi s.s. strain was isolated (Table 2). These findings corroborate the results of several European studies on the predominance of B. afzelii strains in skin manifestations of Lyme borreliosis (Picken et al., 1996; Rijpkema et al., 1997; Ružić-Sabljić et al., 2000; Ciceroni et al., 2001; Ornstein et al., 2001; Logar et al., 2004). Twenty-four strain pairs (out of 26 double isolates) grew well enough to allow RFLP analysis (Table 2) and to carry out a comparison of the phenotypic and genotypic characteristics of the isolates within the individual pairs (species identification, plasmid and protein profiles). Either strain of each strain pair contained the same Borrelia genospecies (21 pairs: B. afzelii; 3 pairs: B. garinii) and the same type (all B. afzelii strains were determined as Mla1, and all B. garinii strains as Mlg2), whereas some distinctions in plasmid and protein profiles were found. A plasmid profile analysis revealed 6/24 strain pairs (25%) composed of strains with distinct plasmid outlines. Some of these strains are shown in Fig. 1. The plasmid profile is an excellent tool for strain discrimination particularly when comparing strains within the same species. Many conditions can affect the borrelial plasmid content including the possibility that plasmids can be lost upon cultivation (Xu and Johnson, 1995; Busch et al., 1997; Ružić-Sabljić et al., 1999, Ružić-Sabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b; Wang et al., 1999; Iyer et al., 2003). We would like to stress that strains compared in our study were subjected to quite identical circumstances. They were isolated, multiplied and analyzed using analogous approaches and procedures but only differed in the culture medium. However, the potential impact of a particular medium (MKP versus BSK-II) on the plasmid content is not known. Differences in the plasmid content within particular strain pairs have been described previously (Ružić-Sabljić et al., 2005). We believe that the plasmid content of our strains most probably represents the natural situation and that differing plasmid profiles of strains may be quite safely interpreted as an infection with different clones of the same type of an identical Borrelia species. Differences in the protein profile were found within 7/24 strain pairs (29%). All strain pairs with dissimilar protein profiles belonged to B. afzelii. The distinctions were uniform and limited to the expression of OspC. It is of interest that 6/7 strain pairs with distinct protein profiles had congruent plasmid profiles. Besides the adaptive mechanisms of the spirochetes that influence protein expression, each strain has its own potential to express (or not to express) a particular protein. The latter statement is supported by the finding that populations of B. afzelii and B. garinii generally differ in their potential to express some important proteins such as OspB and OspC (Wilske et al., 1988 and Wilske et al., 1996; Ružić-Sabljić et al., 2000, Ružić-Sabljić et al., 2001a and Ružić-Sabljić et al., 2001b). Therefore, it is possible that proteins expressed by the strains presented in the present study reflect their own genuine status. In conclusion, our study showed comparable Borrelia isolation rates in MKP versus BSK-II medium indicating their similar suitability for routine clinical work. The results of the present study indicate that human patients with Lyme borreliosis may simultaneously harbor heterogeneous B. burgdorferi s.l. strains. References Arnež et al., 2003 M. Arnež, D. Pleterski-Rigler, T. Lužnik-Bufon, E. Ružić-Sabljić and F. 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Abstract Growth kinetic analyses of Borrelia burgdorferi indicated that this bacterium can utilize a limited number of carbon sources for energy: the monosaccharides glucose, mannose, and N-acetylglucosamine, the disaccharides maltose and chitobiose, and glycerol. All of these carbohydrates are likely to be available to B. burgdorferi during infection of either vertebrate and arthropod hosts, enabling development of a model describing energy sources potentially used by the Lyme borreliosis spirochete during its natural infectious cycle. Keywords: Borrelia burgdorferi; Lyme borreliosis; Lyme disease; Carbohydrate utilization; Spirochete Article Outline 1. Introduction 2. Materials and methods 2.1. Bacterial strains 2.2. Media preparation 2.3. Growth assays 2.4. Genomic analyses 3. Results 4. Discussion Acknowledgements References 1. Introduction While Lyme borreliosis ( Lyme disease) is a significant threat to human health in many parts of Europe, North America, and other parts of the globe, very few studies have been conducted to elucidate the metabolic properties of the causative agent, Borrelia burgdorferi. Such basic biological information may be useful in not only increasing the understanding of B. burgdorferi pathogenesis, but also in design of improved therapies for treatment of Lyme borreliosis. B. burgdorferi is an obligate parasite, persisting in nature only by infecting vertebrates and certain species of ticks. As is often observed in other parasitic organisms, the genome of B. burgdorferi is rather small and is predicted to encode a limited number of metabolic enzymes [1] and [2]. Cultivation in the laboratory is possible only with complex media that include undefined components, such as yeast extract [3] and [4]. Elimination of any of those undefined constituents results in significant inhibition of B. burgdorferi growth. Previously-conducted metabolic studies of this bacterium concluded that it is an auxotroph for adenine, spermidine and N-acetylglucosamine (GlcNAc), although the disaccharide chitobiose may substitute for GlcNAc [4], [5] and [6]. Studies conducted in the 1960s on the related bacterium B. recurrentis (formerly Spirochaeta recurrentis) demonstrated the presence of enzymes for Embden-Meyerhof fermentation of glucose to lactic acid [7], [8], [9] and [10]. Only one glycolytic enzyme of B. burgdorferi, the pyrophosphate-dependent phosphofructokinase, has been characterized in detail [11] and [12]. Radiolabeling studies with the related B. hermsii using 14C-glucose found incorporation into the glycerol components of phosphatidylcholine and other membrane constituents [13]. Consistent with those biochemical characterizations, analysis of a B. burgdorferi genome sequence revealed homologs of all glycolytic pathway enzymes, some components of the pentose phosphate cycle, but none for oxidative phosphorylation or gluconeogenesis [1] and [2]. Those genomic analyses also indicated numerous homologs of carbohydrate transporters. Based upon similarities with characterized carbohydrate transporters of other bacteria, it was predicted that B. burgdorferi can utilize a broad range of carbon sources [1]. To better understand the metabolism of the Lyme borreliosis spirochete, we assessed its ability to grow in media containing a variety of carbohydrates. Our results demonstrated that there are actually very few carbohydrates capable of supporting B. burgdorferi growth. 2. Materials and methods 2.1. Bacterial strains The B. burgdorferi type strain B31 was originally cultured from an infected Ixodes scapularis tick collected on Shelter Island, New York [14]. All experiments were performed with a subculture of strain B31 that is infectious to both mammals and ticks, and has a low in vitro passage history [15]. The genome of this subculture was recently sequenced [1] and [2]. 2.2. Media preparation A modified version of a standard B. burgdorferi culture medium, Barbour–Stoener– Kelly II (BSK-II) [4], was produced that lacked any specifically added carbohydrates. This sugar-free medium, which we designated BSK-Lite, is identical to BSK-II excepting the omission of free glucose and the use of glucose-free CMRL 1066 (product number C5900-02, United States Biologicals, Swampscott, MA). Carbohydrate solutions were prepared at 20% (w/v) in distilled water, sterilized by passage through a 0.2 μm filter, and individually added to BSK-Lite to final concentrations of 0.4% (w/v, or v/v in the case of glycerol). This concentration was used since it is the concentration of glucose specifically added to BSK-II [4], and is in excess of carbohydrate concentrations likely to be encountered during mammalian infection [16]. Each of the following carbohydrates were tested: the hexoses Dglucose, D-mannose, D-galactose, and D-fructose; the pentoses D-ribose, D-xylose, L-arabinose, D-arabitol, and adonitol (ribitol); the disaccharides maltose and melibiose; the methyl pentose L-fucose; the amino sugars D-glucosamine, GlcNAc, and chitobiose; and glycerol (all obtained from Sigma, St. Louis, MO). Due to its highly acidic nature, glucosamine was dissolved in 25 mM HEPES and the pH adjusted to 7.6 prior to use. Examined carbohydrates include all those predicted to be utilized by B. burgdorferi [1], plus several additional carbohydrates. Although B. burgdorferi ORFs BBB04, BBB05 and BBB06 were originally annotated as encoding a potential cellobiose transporter [1], it is highly unlikely that B. burgdorferi, as an obligate parasite of vertebrates and ticks, would ever encounter that cellulose derivative in nature. Research on those genes suggests they actually encode a transporter for chitobiose, the disaccharide building block of arthropod chitin [5] and [17]. For these reasons, we did not test the ability of cellobiose to support B. burgdorferi growth. 2.3. Growth assays B. burgdorferi was initially cultivated in complete BSK-II medium to midexponential phase (approximately 1 × 107 bacteria/ml). Aliquots were then diluted 1:100 into tubes of BSK-Lite plus each tested carbohydrate. As controls, bacteria were also inoculated into BSK-Lite without any added carbohydrate, and into complete BSK-II. Total numbers of intact bacteria in each culture were counted every 24 h using a Petroff–Hausser counting chamber and dark-field microscopy. Every carbohydrate was independently analyzed two times. Those carbon sources that supported growth during the initial two studies were further analyzed at least one additional time. 2.4. Genomic analyses Initial analysis of putative carbohydrate metabolism genes was based upon the annotation provided for B. burgdorferi strain B31 by Fraser et al. [1] and the web site http://www.tigr.org/tigr-scripts/CMR2/GenomePage3.spl?database=gbb. Identified open reading frames (ORFs) were compared with genes of other organisms using BLAST-P analysis of public databases through the web site http://www.ncbi.nlm.nih.gov/blast/. Additionally, sequences of proteins with known functions in other organisms were specifically compared with the B. burgdorferi B31 database through the web site http://tigrblast.tigr.org/cmr-blast/. 3. Results A significant complication of performing growth kinetic assays of a fastidious organism, such as B. burgdorferi is the necessary complexity of the culture medium. While these bacteria require addition of GlcNAc for cell wall synthesis, that compound can also serve as an energy source (see below). Other essential medium components, such as yeast extract, neopeptone, and rabbit serum, contain carbohydrates that may be utilized for energy production. Elimination of any of those ingredients results in significant inhibition of bacterial growth [4], which could possibly mask results of growth kinetic studies. For these reasons, BSK-Lite, a modified medium that eliminated all specifically added carbohydrates, was utilized as a base medium for the present studies. As described below, this medium was suitable for discrimination of carbon sources capable of supporting B. burgdorferi growth. Presumably because of the unavoidable carbohydrates, B. burgdorferi was capable of growth in BSK-Lite without additional carbohydrate (Fig. 1). Bacteria grew for approximately three days before culture densities abruptly stabilized, apparently due to depletion of energy sources. However, inclusion of any of several carbohydrates resulted in bacterial culture densities that significantly superceded those observed when using plain BSK-Lite (Fig. 1). In contrast, many other carbohydrates did not support bacterial growth any better than did unsupplemented BSK-Lite. We therefore defined a carbohydrate as being utilized for growth by B. burgdorferi if it supported culture densities greater than twofold higher than did BSK-Lite without added carbohydrate (Table 1). (33K) Fig. 1. Growth kinetics of B. burgdorferi in BSK-Lite medium supplemented with each of various carbohydrates. Data shown are representative of experiments performed at least three separate times per carbohydrate. Results for those carbohydrates that supported growth at levels greater than twofold higher than unsupplemented BSK-Lite are illustrated. Addition of all other tested carbohydrates to BSK-Lite yielded growth rates and maximal densities indistinguishable from those obtained using unsupplemented BSK-Lite, and are not illustrated. Table 1. Tested carbohydrates and their abilities to support growth of B. burgdorferi Carbohydrate Growtha Glucose + Maltose + Mannose + GlcNAc + Chitobiose + Glycerol + Galactose − Fructose − Melibiose − Ribose − Xylose − Arabinose − Arabitol − Adonitol − Fucose − Glucosamine − a Defined as greater than twofold higher final culture density relative to unsupplemented BSK-Lite (+), or culture density levels equal to those achieved in unsupplemented BSK-Lite (−). The standard culture medium used for growth of B. burgdorferi, BSK-II, contains glucose as the added carbon source [3] and [4]. As expected, addition of glucose to BSK-Lite yielded culture densities comparable to those obtained when using standard BSK-II (Fig. 1). Maltose, a disaccharide of glucose, also supported B. burgdorferi growth, indicating that this bacterium produces an α-glucosidase. Supplementation with mannose, glycerol, GlcNAc and the GlcNAc disaccharide chitobiose consistently resulted in final culture densities comparable to those obtained in either BSK-Lite plus glucose or in standard BSK-II. It is therefore concluded that B. burgdorferi expresses transporters and enzymes for utilization of these alternative energy sources. Although analysis of the B. burgdorferi genome suggested that it encodes homologs of galactose, fructose, and ribose transporters [1], none of those sugars supported bacterial growth. None of the other tested carbohydrates enabled growth at levels exceeding those obtained from unsupplemented BSK-Lite. 4. Discussion The Lyme borreliosis spirochete has long been known as a fastidious organism, with cultivation possible only in complex media. The reason for this became obvious upon sequence analysis of a B. burgdorferi genome, which revealed homologs of very few metabolic enzymes [1]. It appears that this bacterium is unable to synthesize amino acids, nucleotides, fatty acids, or most other cellular building blocks. Previous biochemical studies were supported by genomic data suggesting fermentation of sugars to lactate, and the absence of both a citric acid cycle and oxidative phosphorylation. The present metabolic studies determined that B. burgdorferi is capable of utilizing only a small number of different carbohydrates as energy sources, consistent with the general paucity of enzymes encoded by the spirochete. Fig. 2 indicates the probable metabolic pathways by which these carbohydrates are utilized for energy and for synthesis of cellular components. (98K) Fig. 2. Diagram of predicted carbohydrate transporters and enzymes of B. burgdorferi, based on results of these and other functional studies and genome sequence analyses [1], [5], [7], [8], [9], [10], [11], [12], [13] and [17]. Predicted proteins are noted by their conventional bacterial nomenclature, with encoding open reading frames designated by the ORF number assigned to the B. burgdorferi strain B31 genome [1] and [2]. Please see text for details. The B. burgdorferi genome sequence indicates three probable phosphotransferase system (PTS) glucose transporters (Fig. 2). This apparent redundancy suggests that glucose is a major energy source for B. burgdorferi, not altogether surprising considering the parasitic nature of this organism. A recent study demonstrated that deletion of one of the putative glucose transporters, malX2, resulted in a slight inhibition of growth in culture [18], consistent with the predicted overlap in transporter function. B. burgdorferi appears to also contain a fourth, non-PTS glucose transporter, homologous to the Mgl transporter of Escherichia coli (Fig. 2). The E. coli Mgl system is a high-affinity glucose transporter that can also transport methylgalactose and other sugars [19]. It was this latter characteristic of the E. coli Mgl system that led to annotation of the B. burgdorferi system as a potential galactose transporter [1]. The present studies suggest that the previous annotation is incorrect, as we were unable to find evidence that B. burgdorferi can utilize galactose for energy. The existence of a non-PTS glucose transporter in B. burgdorferi is supported by the earlier biochemical characterization of glucokinase activity in the related spirochete B. recurrentis [8] and [10]. Blast-P analyses of the B. burgdorferi genome sequence indicated strong similarities between ORF BB831 and the proven glucokinase enzymes of Streptomyces coelicolor (P = 7.5 × 10−16) [20] and Thermotoga maritima (P = 9.5 × 10−12) [21]. ORF BB693 exhibits slightly lower degrees of similarity with those glucokinases. While both ORFs were originally annotated as encoding putative xylose operon regulatory proteins [1], sugar kinases and transcriptional repressors often share significant homology [22]. The inability of B. burgdorferi to grow in xylose-containing media also supports the notion that both BB831 and BB693 were initially annotated incorrectly. B. burgdorferi grew quite well on medium containing maltose, confirming that this bacterium encodes an enzyme for breakdown of glucose polysaccharides. ORF BB166 encodes a protein with significant similarity to such enzymes (Fig. 2). Probable natural sources of α-glucosides are glycogen obtained from tissues within vertebrates hosts and from ingested blood within the tick vector. The only other hexose found to support B. burgdorferi growth was mannose, with mannose-modified host proteins as likely natural sources of this sugar. Many characterized bacteria import mannose through a PTS transporter, and we note that B. burgdorferi encodes two separate, single-subunit PTS sugar transporters encoded by ORFs BB408 and BB629 (Fig. 2). One or both of these transporters may function to bring mannose into the cell. Additionally, several characterized E. coli sugar transporters exhibit loose specificity in the carbohydrates they are able to import [23], raising the possibilities that these B. burgdorferi transporters may import more than just one type of sugar, and that other transporters may also import mannose. Genetic analyses, involving deletion and complementation of the six predicted B. burgdorferi PTS transporters, will be required to confirm each system’s substrate specificity. GlcNAc is an essential component of artificial media for cultivation of B. burgdorferi [3] and [4]. Genomic analysis suggests this is due to inability of the organism to synthesize GlcNAc de novo (Fig. 2). While this molecule is a building block of cell wall peptidoglycan, the present studies demonstrated that GlcNAc can also serve as an energy source, presumably following the catabolic pathway delineated in Fig. 2. Many other bacteria import GlcNAc into the cell through glucose PTS transporters [23], as may also be the case with B. burgdorferi. The finding that chitobiose, a disaccharide of GlcNAc, can likewise substitute for glucose to support growth of B. burgdorferi supported and extended upon earlier studies. It was previously demonstrated that chitobiose can substitute for GlcNAc in BSK-II medium [5], indicating that the spirochete can effectively transport chitobiose and cleave it into monosaccharides for use in cell wall biosynthesis. Tick cuticle is comprised of chitin, polymers of GlcNAc/chitobiose, leading to a hypothesis that chitobiose transport and catabolism play roles during tick infection [5]. Previous characterization of ORFs BBB04, BBB05, and BBB06 strongly suggested that these genes encode the chitobiose PTS transporter ChbCAB (Fig. 2) [5] and [17]. Disruption of this probable transporter did not disrupt ability of B. burgdorferi to infect ticks and mammals [17], suggesting that this source of energy is redundant to other catabolites. It is possible, however, that energy provided by chitobiose enhances survival of the bacteria during tick-associated stages. Glycerol can also serve as an energy source for B. burgdorferi, presumably being acquired from host blood. However, glycerol catabolism yields only one ATP per molecule, as opposed to two ATPs per hexose. This suggests that glycerol is more likely to be used for phospholipid and lipoprotein synthesis than as an energy source in nature. No other tested carbohydrate was capable of supporting B. burgdorferi growth. Although annotation of the B. burgdorferi genome suggested the presence of transporters for both galactose and fructose [1], the present studies strongly suggest that those annotations are incorrect. It should be remembered that original annotation of the B. burgdorferi genome was performed by in silico comparisons between its ORF sequences and those of proteins with known or postulated functions from other organisms [1]. Biological analyses of those predicted functions needs to be performed before one can conclusively attribute function to any ORF product. For example, B. burgdorferi ORFs BBB04, BBB05 and BBB06 were initially annotated as encoding a cellobiose transporter, based on their similarity with such transporters [1], while subsequent laboratory studies suggest that those ORFs actually encode a chitobiose transporter. As B. burgdorferi and well-characterized bacteria, such as E. coli are separated by untold millions of years of evolution, divergences in protein specificity or domain linkage are bound to have occurred. Thus, while the possibility remains that B. burgdorferi may utilize sugars, such as galactose or fructose under certain conditions in nature, it is important to note that there are no metabolic or biochemical data to support such hypotheses. Earlier genomic analysis also suggested that ORFs BB677, BB678 and BB679 may constitute a ribose transporter, based on similarities with both the E. coli Rbs and Mgl transporters [1]. Although our analyses indicated that B. burgdorferi cannot utilize ribose as a sole carbon source, it remains possible that the B. burgdorferi system may transport ribose for other uses. Examination of the B. burgdorferi genome sequence revealed that the spirochete encodes homologs of only part of the pentose-phosphate pathway, wherein glucose can be used to produce ribose, but ribose cannot be metabolized to form glucose or any other energy-producing molecule (Fig. 2). Thus, any ribose transported into the bacterial cell could be utilized only for nucleic acid synthesis and not for energy production. Our study represents the first analysis of carbohydrate utilization by B. burgdorferi, enabling construction of a model describing possible sources of energy throughout the bacterial infectious cycle. In nature, the Lyme borreliosis spirochete undergoes a period of rapid growth during transmission from a feeding tick to a vertebrate host [24], [25] and [26]. At that time, serum glucose and glycerol, along with mannose, GlcNAc and glucose polysaccharides associated with ingested host blood cells, provide energy to the growing bacteria. Chitobiose from the tick cuticle may provide additional energy and/or GlcNAc. Within the infected vertebrate, glucose, glycerol, mannose, GlcNAc, and glucose polymers continue to serve as energy sources for bacterial maintenance, as well as providing building blocks for peptidoglycan, phospholipids, lipoproteins, and nucleic acids. Those same carbohydrates fuel the bacteria during transmission from host to naïve, feeding ticks, with ingested blood providing nutrients during tick colonization. Mutagenesis of genes encoding specific transporters and catabolic enzymes will test this model and enable determination of the relative importance of each carbohydrate in the B. burgdorferi pathogenic cycle. Acknowledgements These studies were supported by US Public Health Service Grants R01-AI53101 and 5T32-AI49795. 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Abstract-EMBASE | AbstractMEDLINE | Order Document [25] A.M. Silva and E. Fikrig, de Growth and migration of Borrelia burgdorferi in Ixodes ticks during blood feeding, Am. J. Trop. Med. Hyg. 53 (1995), pp. 397–404. [26] J. Piesman, B.S. Schneider and N.S. Zeidner, Use of quantitative PCR to measure density of Borrelia burgdorferi in the midgut and salivary glands of feeding tick vectors, J. Clin. Microbiol. 39 (2001), pp. 4145–4148. Abstract-Elsevier BIOBASE | Abstract-EMBASE | Abstract-MEDLINE | Order Document | Full Text via CrossRef FEMS Microbiology Letters Volume 243, Issue 1 , 1 February 2005, Pages 173-179 Corresponding author. Tel.: +1 859 257 9358; fax: +1 859 257 8994 Evaluation of Venezuelan Equine Encephalitis (VEE) replicon-based Outer surface protein A (OspA) vaccines in a tick challenge mouse model of Lyme disease Clay L. Gipson, Nancy L. Davis, Robert E. Johnston and Aravinda M. de Silva Department of Microbiology and Immunology, University of North Carolina, CB# 7290, Chapel Hill, NC 27599, USA Received 13 January 2003; revised 2 April 2003; accepted 9 April 2003. ; Available online 14 May 2003. Abstract Venezuelan Equine Encephalitis (VEE) virus replicon particles (VRPs) encoding Borrelia burgdorferi Outer surface protein A (OspA) were evaluated for their ability to induce an immune response and provide protection from tick-borne spirochetes. VRPs expressing ospA that accumulated intracellularly (VRP OspA) or that was secreted from host cells (VRP tPA-OspA) were tested. Both VRP OspA and VRP tPA-OspA expressed ospA in immunized mice. Mice vaccinated with VRPs expressing secreted OspA produced significant amounts of anti-OspA antibodies, whereas VRPs expressing intracellular OspA were less immunogenic. The VRP method of delivery induced a Th1 type immune response unlike the recombinant OspA protein in Freund’s adjuvant, which induced a mixed (Th1 and Th2) immune response. The VRP tPA-OspA construct induced an immune response that reduced the bacterial load in feeding Ixodes scapularis and blocked transmission to the host. These results indicate that VRPs are capable of providing protection against tickborne B. burgdorferi, and potentially can be used for developing improved vaccines against Lyme disease. , Author Keywords: Venezuelan Equine Encephalitis; Virus replicon particles; Outer surface protein A; Vaccine Article Outline 1. Introduction 2. Materials and methods 2.1. Mice and ticks 2.2. Cells and plasmids 2.3. Production of VEE replicon particles (VRPs) containing ospA and tpA-ospA 2.4. Expression assays 2.5. Immunization 2.6. ELISA assays for determining total specific IgG and IgG isotype 2.7. Immunohistochemistry 2.8. Bacterial load in ticks 2.9. Evaluation of mice for B. burgdorferi infections 3. Results 3.1. OspA protein production by VEE mRNA and replicons 3.2. Vaccination of mice with VRP OspA and VRP tPA-OspA 3.3. Characterization of immune responses elicited by the different OspA-based vaccines 3.4. Challenge of immunized mice with B. burgdorferi infected ticks 3.5. Assessment of bacterial load within infected ticks 4. Discussion Acknowledgements References 1. Introduction Lyme disease is an emerging, infectious disease caused by the tick-borne spirochete, Borrelia burgdorferi [1]. The disease is now recognized as the most prevalent vectorborne illness in the United States and Europe. In 1999, a Lyme disease vaccine was approved for human use in the US [2]. This vaccine is based on recombinant, lipidated B. burgdorferi Outer surface protein A (OspA). The vaccine has been withdrawn from the market, with the manufacturer citing poor sales. Spirochetes within ticks produce OspA in abundance, whereas it is not a major antigen during early stages of host infection. In an unfed infected nymphal tick, the burden of spirochetes within the tick is low with the majority expressing OspA. As the nymphal tick engorges, the population of the spirochetes expands rapidly, and the phenotype of the population in the gut lumen shifts towards a more heterogeneous population of bacteria with many expressing OspA and Outer surface protein C (OspC) [3]. This vaccine confers protective immunity because OspA antibodies enter the gut of feeding ticks and bind to spirochetes, blocking transmission from the vector to the host [4]. Animals can be protected up to 24 h after infected tick attachment by passively receiving OspA antibodies [5]. Thus, the OspA vaccine is an arthropod specific transmission blocking vaccine. Because the OspA protein is mainly produced within the tick, immune cells of the vaccinated host do not encounter this antigen during challenge and the memory response of the host is not stimulated. In the absence of a memory response, the host has to maintain high circulating antibody levels for protection. With the current vaccine this can only be achieved through an initial vaccination, followed by two successive boosters over 6 months. In the OspA vaccine trials to date, the individuals who were not protected by vaccination had, on average, lower antibody titers, which further supports the notion that antibody titer is critical for protection [6]. Vaccination strategies that require fewer immunizations and lead to long lasting protective immunity are needed and will likely to lead to greater acceptance of the vaccine among high risk populations. Multiple vaccination strategies have been employed to elicit protective immunity using OspA in laboratory animals. The lipidated rOspA vaccine that was approved is produced in E. coli and the purified protein is used with an Alum adjuvant to produce an immune response [7 and 8]. Langermann et al. were able to show protection in mice immunized with recombinant BCG (bacillus Calmette-Guerin) mycobacteria expressing OspA [9]. DNA plasmid vaccines expressing OspA and/or OspC have been shown to induce protective antibodies in mice as well [10, 11 and 12]. Recently, different alphaviruses, including attenuated strains of Venezuelan Equine Encephalitis (VEE) virus, have been engineered as vaccine vectors [13, 14 and 15]. Vectors derived from VEE present many advantages to vaccine development. The replicons efficiently target lymphoid tissues, and direct expression of the heterologous protein in antigen presenting cells [16]. The heterologous protein is also placed downstream of the viral 26S promoter, which results in independent, high level production of the heterologous protein [17]. During production of the replicon particles, the structural genes are provided in trans, leading to a viral particle that contains only the nonstructural VEE-based genes and the vaccine gene of interest [13]. This results in a safe VEE vaccine, which does not lead to the production of new virus particles in the host. The VEE replicons are only capable of a single round of infection and the antigen of interest is only produced within host cells initially infected by the replicon particles. The vectors are also RNA based, which reduces the likelihood of integration into the host chromosome. The majority of the human population has not been exposed to VEE, reducing the chance of pre-existing immunity to the vector, which might compromise the efficacy of the vaccine [18, 19 and 20]. VEE replicon particles (VRPs) have provided protection against influenza in mice, Marburg virus in primates and Ebola virus in rodent models [13, 21 and 22]. Given the promising results with VEE replicons and the need for high circulating levels of OspA antibodies for preventing infections with B. burgdorferi, we decided to explore VEE replicons for delivering an OspA-based Lyme disease vaccine. Many Lyme vaccine models are based on syringe inoculation of cultured bacteria into animals. Syringe challenge is not a suitable surrogate for tick challenge because spirochetes delivered by ticks produce different antigens than those cultured in vitro. B. burgdorferi Decorin binding protein A (DbpA) was effective as a vaccine against cultured organisms but not against the same strain of spirochetes delivered by tick bite [23 and 24], emphasizing the importance of using tick challenge in Lyme vaccine investigations. Here we present the results of vaccine studies that were performed to test VEE replicons coding for intracellular or secreted forms of OspA in a tick challenge mouse model of Lyme disease. 2. Materials and methods 2.1. Mice and ticks Female C3H HenJ mice, 4–6 weeks of age, were obtained from the National Institutes of Health (Bethesda, MD). The ticks used in this study were raised by placing larval Ixodes scapularis on mice infected with B. burgdorferi strain B31 (from Shelter Island, NY). The ticks were kept in a humid chamber at 21 °C and allowed to molt. After the molt the nymphs were assessed for infection prevalence. Individual nymphs were homogenized in PBS, and spotted onto slides. The homogenates were acetone fixed, and blocked in 5% FBS/PBS at RT for 1 h. Twenty-five microliters of goat α-B. burgdorferi-FITC (1:200) was applied to each spot, and incubated at RT for 1.5 h. Slides were washed, and Anti-Fade (Molecular Probes, Eugene, OR) mounting media was applied. Eighteen of 20 nymphs assessed were positive for B. burgdorferi which results in an infection prevalence of 90%. 2.2. Cells and plasmids BHK cells obtained from the American Type Culture Collection in passage 53 were used between passages 54 and 64. Cells were maintained in alpha minimal essential media containing 10% fetal calf serum, 10% tryptose phosphate broth, and 0.29 mg of -glutamine per milliliter. For electroporation, cells were cultured overnight, harvested when subconfluent, and prepared for electroporation as previously described [15]. OspA was cloned from B. burgdorferi strain N40 using ospA forward primer (5′AGTCTAGTCCGCCAAGATGAAGCAAATGTTAGCAGCCTTGAACGAG-3′) and reverse primer (3′GTCATAATCGATTATCATTTTAAAGCGTTTTTAATTTCATC-5′) creating a 850 bp fragment that was cloned into a TOPO TA cloning vector as specified by the manufacturer (Invitrogen; Carlsbad, CA). To create the tpA-ospA construct, the tpA signal sequence was produced from pJW4304 (gift from James Arthos, NIH) using tpA forward primer (5′AGCCGAACTCTAGTCCGCCAAGATGAGCTTGCAATCA-3′) and reverse primer (3′-CGACGATCGATAGCTTGCAATCATGGATGCAATG-5′). The tpA and ospA PCR products were subjected to overlapping PCR, creating the tpA-ospA construct that was subsequently cloned into a TOPO TA vector. The sequences of both ospA and ospA-tpA constructs were confirmed by DNA sequencing. 2.3. Production of VEE replicon particles (VRPs) containing ospA and tpA-ospA OspA and tpA-ospA gene products were inserted into pVR21, the VEE replicon expression vector downstream of the 26S promoter by overlapping extension PCR [25]. Transcripts of pVR21-ospA and PVR21-tpA-ospA were generated in vitro using a mMessage mMachine T7 transcription kit (Ambion; Austin, TX). Transcripts were electroporated into 800 μl BHK cell suspensions at a concentration of 107 cells/ml in PBS using three electrical pulses of 0.85 kV at 25 μF with a Bio-Rad Gene Pulser II electroporator (Bio-Rad; Hercules, CA). VRPs were generated by co-electroporating the transcript containing ospA or tpA-ospA with two helper mRNA’s encoding the VEE capsid and attenuated glycoproteins [13]. The electroporated BHK cells were seeded into 75 cm2 flasks and incubated at 37 °C for 24 h. VRPs were harvested by clarifying the electroporated BHK supernatant by centrifugation at 10,000 rpm for 30 min, and then pelting the VRPs at 24,000 rpm for 3 h through a 20% sucrose cushion. VRPs were assayed by indirect immunofluorescence (IFA) of VRP-infected BHK cell monolayers and the titers of the resulting VRPs were 8.7×107 i.u./ml (infectious units/ml) (VRP OspA) and 3.1×108 i.u./ml (VRP tPA-OspA). 2.4. Expression assays BHK cells electroporated with RNA transcribed from pVR21-ospA and PVR21-tpAospA were incubated for 30 h at 37 °C. The culture media was separated from the cells and clarified by centrifuging at 2000×g for 10 min. The BHK cells were harvested with a cell scraper and resuspended in PBS. Cells were lysed in a 0.2% NP40/TAE buffer. For Western blot analysis, polypeptides from the cell supernatants and cell lysates were separated by 12% SDS-PAGE and transferred to nitrocellulose at 400 mA for 75 min. The membranes were probed with polyclonal mouse α-OspA sera at 1:400. The membranes were then incubated with goat α-mouse-alkaline phosphatase (1:1000) (KPL, Gaithersburg, MD) and developed with BCIP/NBT (KPL, Gaithersburg, MD). Treatment with PNGase (NEB, Beverly, MA) before PAGE was used to determine presence of N-linked glycosylation. 2.5. Immunization Mice were immunized subcutaneously (s.c.) via footpad inoculation with 1×106 i.u. of VRPs in 10 μl at days 0 and 21. Control mice were immunized s.c. (above the shoulder) with 100 μl containing 20 μg of purified recombinant protein in Freund’s complete adjuvant or Alum (Alumax, Pierce, IL) on day 0, and boosted with 20 μg protein in Freund’s incomplete adjuvant or Alum on day 21. 2.6. ELISA assays for determining total specific IgG and IgG isotype Sera from vaccinated mice at days 0, 14 and 35 were tested for α-OspA antibodies on 96 well ELISA plates (Griener, Lake Murray, FL). Each well on the plate was treated with 10 μg of OspA-glutathione-S-transferase fusion protein (OspA-GST) in 100 μl of carbonate binding buffer (pH 9.2) overnight at 4 °C to coat the well with OspA. Plates were washed with TBST (Tris buffer saline–0.2% Tween-20), and blocked with 0.25% BSA in TBS (Tris buffer saline) for 30 min at 37 °C. Sera from immunized mice were applied to ELISA plates in multiple dilutions in blocking buffer, and incubated at 37 °C for 90 min. The plates were washed three times in TBST, and incubated with goat α-mouse-alkaline phosphatase (1:1000) for 60 min at 37 °C. The plates were washed three times in TBST, developed with pNPP (Sigma, St. Louis, MO) before measuring absorbance at 405 nm. The isotyping ELISAs were similar to the total IgG ELISAs, except that the secondary antibodies were alkaline-phosphatase conjugated isotype specific antibodies (Bethyl Laboratories, Montgomery, TX) were used at dilutions of 1:1000 (IgG1), 1:800 (IgG2a, IgG2b) and 1:400 (IgG3). 2.7. Immunohistochemistry At 18 h after innoculation with VRPs mice were sacrificed via cervical dislocation, and draining popliteal lymph nodes were removed. The unfixed tissues were fresh frozen in a supercooled N-methylbutanol bath, cryosectioned, and fixed with 4% paraformaldehyde in PBS. Sections were blocked in 10% normal goat serum/PBS for 1 h, washed with PBS, and incubated with rabbit α-OspA Ab at 1:200 overnight at 4 °C. The sections were thoroughly washed, and incubated with goat α-rabbit-FITC at 1:500 for 1 h at room temperature (RT). 2.8. Bacterial load in ticks Ticks that had fed for 60 h were homogenized, spotted onto silylated slides and acetone fixed, and blocked in 5% FBS/PBS at RT for 1 h. 25 μl of goat-α-B. burgdorferi-FITC (1:200) was applied to each spot, and incubated at RT for 1.5 h. Slides were washed, and Anti-Fade (Molecular Probes, Eugene, OR) mounting media was applied. The spirochetes in each sample were counted by IFA as previously described [5]. 2.9. Evaluation of mice for B. burgdorferi infections Three weeks after tick detachment mice were sacrificed and serum, spleen and bladder samples were obtained. Samples were placed in BSK-II media, incubated at 34 °C, and checked weekly for evidence of spirochetes by dark field microscopy. The serum was probed for production of α-B. burgdorferi antibodies by Western blot as previously done [7]. 3. Results 3.1. OspA protein production by VEE mRNA and replicons The full length OspA protein when produced in B. burgdorferi is a 273 AA protein, with a 16 AA signal sequence and a lipidation sequence at the N-terminus. The 16 AA signal sequence is cleaved and the protein is attached to the outer membrane by a lipid anchor attached to a cysteine residue at position 17. For the current study, two different VEE replicons expressing ospA were produced. We used B. burgdorferi N40 ospA as template but used B31 infected ticks to challenge immunized mice. They are 99% similar, and cross-protection between the two stains has been shown previously [26 and 27]. The first replicon was designed to produce the region of ospA encoding the mature protein (A.A. 18-273) only (Fig. 1). We expected ospA expressed from this replicon to accumulate in the cytoplasm of cells because it did not contain a signal sequence for secretion. The second replicon expressing ospA was designed to target the protein to the secretion pathway of eukaryotic cells by fusing a tissue plasminogen activator (tPA) signal sequence to the N-terminus of the ospA gene (Fig. 1). Each ospA gene was inserted into the plasmid pVR 21, which contains all the VEE nonstructural genes along with a strong promoter for expressing the foreign gene of interest [25]. When replicon RNAs transcribed in vitro from these plasmids (pVR 21OspA and pVR 21-tPA-OspA) were electroporated into cultured cells, both resulted in the synthesis of OspA ( Fig. 2A). As expected, the OspA expressed without the signal sequence was mainly present in the cell pellet whereas the OspA expressed with the tPA signal sequence was mainly secreted into the media (Fig. 2A). (19K) Fig. 1. Forms of OspA used in this study. Native full length OspA shown in relation to the three VRP expressed genes (OspA, tPA-OspA, and HA) along with purified recombinant proteins, OspA-GST and GST. (7K) Fig. 2. Production of OspA from RNA transcripts of pVR21-OspA and pVR21-tPA-OspA. (A) OspA produced by pVR21-OspA transfected BHK cell lysate (2) and supernatant (1). pVR21-tPA-OspA transfected BHK cell supernatant (3) and cell lysate (4). (B) OspA produced by pVR21-tPA-OspA displayed the ladder pattern consistent with N-linked glycosylation. Cell supernatants were incubated either without (1) or with (2) PNGase overnight at 37 °C prior to being resolved. The protein produced by the tPA-OspA replicon RNA was distributed among multiple bands indicating possible glycosylation during secretion. The ospA sequence has five potential N-linked glycosylation sites. When the protein produced by tPA-OspA was treated with N-glycosidase, the multiple bands disappeared leaving a single, faster migrating band corresponding to the size of the band produced by the replicon RNA lacking the signal sequence (Fig. 2B) confirming that the OspA expressed with the tPA signal sequence was, indeed, glycosylated in mammalian cells. Once we determined that each VEE-ospA replicon expressed OspA in BHK cells, VEE replicon particles were produced. The VRPs were capable of infecting BHK cells and driving the expression of ospA as determined by immunofluorescence using anti-OspA antibodies (data not shown). BHK cells were also used to titer each replicon. The VRPs expressing ospA alone had a titer of 8.7×107 i.u./ml whereas the replicons coding for tPA-ospA had a titer of 3.1×108 i.u./ml. 3.2. Vaccination of mice with VRP OspA and VRP tPA-OspA In order to assess the immunogenicity of the two OspA-expressing VRPs, vaccine trials were carried out as outlined in Fig. 3. In the first trial groups of four mice were immunized with VRP particles (1×106 i.u.) expressing ospA or tPA-ospA by footpad injection as previously described [28]. A control for the replicon system was included by inoculating a group with 1×106 i.u. of influenza hemaglutinin (HA) encoding replicons. The other groups consisted of mice inoculated subcutaneously with the recombinant OspA-GST fusion protein or GST alone in the presence of Freund’s adjuvant. In the second trial eight mice were immunized with the tPA-OspA VRPs and four mice each were immunized with recombinant OspA-GST or GST alone resuspended in Alum as an adjuvant. Sera were collected on days 0, 14 and 35 in order to determine serum antibody levels against OspA. (9K) Fig. 3. Schematic of immunization schedule. Groups of mice were immunized initially on day 0, and boosted on day 21. Sera were collected from the mice prior to initial vaccination on days 0, 14 and 35. After the initial vaccination, antibody responses were detected in the mice immunized with OspA-GST protein as well as with tPA-OspA VRPs (Fig. 4). After boosting, significantly (Student’s t-test, P<0.01) increased responses were detected in the mice immunized with OspA-GST and VRP tPA-OspA (Fig. 4). These results indicate that VRPs expressing tPA-OspA were capable of stimulating an antibody response, unlike the VRPs expressing OspA alone without a secretion signal sequence. Furthermore, the VRP tPA-OspA generated significantly (Student’s t-test, P<0.01) greater antibody levels than the OspA-GST recombinant protein in Alum adjuvant. (13K) Fig. 4. Induction of α-OspA IgG antibodies by different immunogens. Serum was collected at days 0 (not shown), 14 and 35 to determine amount of serum IgG against OspA. Each group is an average of four mice, except for VRP tPA-OspA which is an average of 12 mice. Standard deviation is indicated by error bars. (*) Indicates group significantly different from GST control at same time point (P<0.01). (**) Indicates significantly different from both GST and Alum OspA-GST at same time point (P<0.01). The different immune responses against VRP OspA and VRP tPA-OspA may be due to the intracellular and extracellular location of the OspA. Alternatively, only the VRP tPA-OspA may be expressed in vivo following footpad inoculation. VRP’s have previously been shown to infect and express foreign antigen in the popliteal lymph nodes of immunized mice [16]. To distinguish between these possibilities, mice were immunized with VRPs encoding ospA and tPA-OspA. The popliteal lymph nodes were removed 18 h post-footpad immunization, cryosectioned, and probed by IFA for cells expressing OspA. It was determined that both VRPs were capable of infecting and producing OspA in lymph node cells (Fig. 5). The lack of an antibody response to VRP OspA replicons was not due to the lack of in vivo expression of OspA, but more likely due to the cellular localization of the protein that it produced. (42K) Fig. 5. Detection of OspA in mouse popliteal lymph nodes. Individual mice were immunized with 5×105 i.u. of VRP OspA (1), VRP tPA-OspA (2), or PBS alone (3) in each hind footpad. Eighteen hours post-immunization, mice were euthanised, and popliteal lymph nodes were removed, snap frozen, and cryosectioned. The sections were probed with rabbit α-OspA polyclonal sera, and a goat-α-rabbitFITC 2° antibody. 3.3. Characterization of immune responses elicited by the different OspA-based vaccines The method of vaccine delivery can alter the host immune response against the vaccine antigen. Studies were done to characterize T helper cell subsets activated following immunization with VRPs or protein antigens. VRP tPA-OspA and OspAGST produced significant levels of serum antibodies, which allowed us to isotype the antibody profiles of these two immunogens. OspA-GST in Freund’s produced significant (Student’s t-test, P<0.01) levels of IgG1, IgG2a and IgG2b (Fig. 6B), indicating a mixed Th-1 and Th-2 response. The mice immunized with VRP tPAOspA and the OspA-GST protein in Alum produced significant (Student’s t-test, P<0.01) levels of IgG2a, with lesser levels of IgG2b indicating a mainly TH1 type response (Fig. 6). We confirmed that mice immunized with VRP constructs generated a TH1 type response by measuring cytokines secreted by spleenocytes harvested from immunized mice 21 days after their final boost. Cells from mice immunized with OspA and tPA-OspA VRPs produced higher levels of INF-γ than cells from control VRP HA animals (data not shown). Both the antibody isotype and cytokine data confirmed that mice immunized with VRPs developed a TH1 type response against OspA. (30K) Fig. 6. Serum IgG isotype profile of mice immunized with VRP tPA-OspA or OspA-GST in Alum or Freund’s adjuvant. Sera collected at day 35 were used in the OspA ELISA with isotype specific 2° antibodies. VRP tPA-OspA resulted in the productions of mainly IgG2a, with some IgG2b (A). OspAGST in Freund’s produced significant amounts of all IgG isotypes other than IgG3 (B). When Alum was used as the adjuvant, OspA-GST produced predominantly IgG2a (C). IgG3 was tested but not detected in any samples. (*) Indicates group level significantly different that GST control (P<0.01). 3.4. Challenge of immunized mice with B. burgdorferi infected ticks Protection of animals receiving OspA vaccines is mainly dependent on circulating levels of antibody because transmission is blocked within the tick gut where host cellular immunity is unlikely to be functional. Mice immunized with the different OspA vaccines were challenged with five to eight infected I. scapularis nymphs. Three weeks after tick challenge, the mice were tested for Borrelia infection by serology and by organ culture. When mouse sera were tested by Western blot, it was evident that the majority of mice immunized with OspA-GST/Freund’s (4/4), OspAGST/Alum (3/4), and VRP tPA-OspA (10/12) were protected from infection (Table 1). The serology results were confirmed by culture of Borrelia from mouse organs (Table 1). Table 1. Prevention of infection with tick-borne B. burgdorferi Three weeks post-tick detachment, serum was collected from each mouse to determine production of αB. burgdorferi antibodies other than OspA. Mice were then euthanised, and spleen and bladder were cultured in BSK-II media. Cultures were examined weekly for 4 weeks and scored for either positive or negative growth of the spirochetes. (One set of organ cultures from the VRP OspA and VRP HA were contaminated.) 3.5. Assessment of bacterial load within infected ticks Anti-OspA antibodies block transmission by binding to spirochetes within the tick gut. Infected ticks removed at 60 h from vaccinated animals were tested for Borrelia infection to determine if VRPs stimulated an antibody response that reduces the number of spirochetes within the tick gut. Spirochete numbers were significantly reduced (Student’s t-test, P<0.01) within ticks that fed on mice immunized with tPAOspA VRPs and OspA-GST fusion proteins in comparison to ticks that fed on a control mouse (Fig. 7). The highest level of killing was seen in ticks that fed on a mouse immunized with OspA-GST in Freund’s adjuvant, which is consistent with these mice having higher antibody levels. Mice immunized with VRP OspA and VRP HA showed no reduction in the number of B. burgdorferi within the ticks when compared with the negative controls (data not shown), which is consistent with the lack of protection observed among mice in these groups. (7K) Fig. 7. Percent survival of B. burgdorferi in partially engorged I. scapularis nymphs. Two to four ticks were removed from each mouse 60 h into the bloodmeal. Pairs were homogenized in 50 μl of PBS, and spotted onto silylated slides, and stained with α-B. burgdorferi-FITC antibodies to detect number of spirochetes. Five fields were counted in four spots at 400× for each sample. Numbers are expressed at percent of spirochetes in each sample with the number in the GST negative control set at 100. (*) Indicates group level significantly different that GST control (P<0.01). 4. Discussion Venezuelan Equine Encephalitis virus replicon particles are effective vaccine vectors for protecting against a number of viral pathogens. We have evaluated VEE replicon particles as a delivery system for the OspA vaccine against the extracellular bacterium, B. burgdorferi. During the life cycle of the Lyme disease spirochete, OspA is mainly produced by bacteria within the tick gut. The OspA vaccine confers protective immunity by targeting the bacteria in the tick gut and preventing transmission to the host. Robust antibody responses are vital to conferring protection in OspA-based vaccines. We were especially interested in the ability of VEE replicons to stimulate the production of antibodies because they are the effector molecules in the bloodmeal that block transmission. We evaluated VEE replicons with (VRP tPA-OspA) or without (VRP OspA) a eukaryotic secretion signal. OspA expressed without a signal sequence accumulated in the cytoplasm of VRP infected cells. OspA with a signal sequence was targeted to the secretion pathway as evidenced by the presence of N-linked sugars on the protein as well as its accumulation outside cells. When mice were immunized with the VRPs, both constructs infected lymph node cells and induced a specific cytokine (IFN-γ) response. However, only the tPA-OspA VRP induced an antibody response. Others have also observed that antigens that accumulate inside cells are prone to inducing a cellular immune response with little or no antibody production unlike secreted antigens that stimulate both humoral and cellular immunity [25]. Given the primary, if not exclusive, role of antibody in OspA mediated protection, we expected only the secreted construct to be effective. In fact, when VRP vaccinated animals were challenged, only the tPA-OspA VRP vaccinated animals were protected and none of the animals receiving the OspA VRPs were protected. The tPA-OspA produced within mammalian cells was different from the native Borrelia protein because tPA-OspA was glycosylated. The N-linked sugars might alter the epitopes on the protein compared to the native bacterial protein. Additionally, it has been shown that glycosylation can affect how a peptide is presented to the immune system. Manoury et al. showed that N-glycosylation of an antigen could block presentation by MHC II molecules [29]. Similarly, tPA-OspA may have also stimulated a TH1 type response because glycosylation blocked MHC class II presentation. Recently, an epitope of OspA has been found to have homology with human leukocyte function-associated antigen-1 (hLFA-1) [30]. Patients that have been diagnosed with treatment-resistant Lyme arthritis were found to generate a response to OspA when compared to other groups of chronic arthritis patients [30]. This raises concerns over vaccinating people with OspA, especially in individuals who carry the HLA-DRB1*0401 allele. However, both pre- and post-clinical evaluations show no signs that vaccinating with OspA induces this type of auto-immune response [31]. We have produced constructs with amino acid changes in the region of OspA suspected to mimic hLFA-1. In future studies we plan to assess how changes in this region affect immunogenicity and vaccine efficacy. In summary, we have used a tick challenge mouse model to demonstrate that VEE replicons can be used to deliver an OspA vaccine that is similar in efficacy to a recombinant OspA protein vaccine. In human challenge studies with the recombinant protein antigen in Alum, vaccine efficacy was 80% in people receiving three doses of the vaccine (0, 1 and 12 months). The vaccine failures were associated with lower antibody titers and it is likely that people would have to be boosted with the recombinant protein at regular intervals to maintain protective antibody levels. We are currently testing if the VEE replicon particle delivery method could be used to develop an OspA vaccine that generates longer lasting antibody levels and would require fewer boosts than the recombinant protein vaccine. Acknowledgements We thank Kevin Brown, Martha Collier and Gene MacDonald for advice and technical assistance. We also thank members of the de Silva Laboratory for their help. 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In: Proceedings of the IX International Conference on Lyme Borreliosis and Other Tick-Borne Diseases, New York; 2002. Vaccine Volume 21, Issues 25-26 , 8 September 2003, Pages 3875-3884 Corresponding author. Tel.: +1-919-843-9964; fax: +1-919-962-8103. Experimental infection of laboratory mice and rabbits with several isolates of Borrelia burgdorferi sensu lato; comparison of antigens from different genospecies in serological measurement of immune responses J. Tuomi, L. K. Rantamäki and R. Tanskanen , Faculty of Veterinary Medicine, Department of Basic Veterinary Sciences, University of Helsinki, PO Box 57, 00014 Helsinki, Finland Accepted 10 September 2001 Available online 14 December 2001. Abstract Infectivity of and immune responses to 28 Finnish Borrelia burgdorferi sensu lato isolates was studied in 3–4-week-old outbred NMRI and inbred BALB/c/Hy laboratory mice; rabbits were also inoculated. Twenty-one isolates were found to detectably infect mice. A variation among isolates in degree of infectivity was observed. Higher infection rate and higher average ELISA readings were recorded for intradermal than intraperitoneal inoculations. The results suggest differences between Borrelia genospecies in organotropism. The ear was frequently infected by representatives of all genospecies; among high infectivity experiments, this rate was highest, 100%, in infections by Borrelia afzelii. Further differences between genospecies specific organ distributions: B. burgdorferi sensu stricto and Borrelia garinii isolates seemed to infect the bladder relatively more frequently than B. afzelii did; B. afzelii isolates infected heart relatively more frequently than others did. Genospecies specific differences were demonstrated between antigens in reactivity, i.e. in their ‘sensitivity’ as reagents of ELISA and IFA methods to measure isolate specific immune responses. Antigens from two B. afzelii isolates differed clearly in sensitivity. Résumé Les réponses infectieuses et immunes des souris NMRI et BALB/c/Hy à 28 souches de Borrelia burgdorferi isolées en Finlande ont été étudiées. Des lapins ont également été inoculés. 21 souches ont pu infecter la souris. Une variation dans le degré d'infection a été observée. Un taux d'infection plus élevé et un taux d'anticorps plus élevé à l'ELISA ont été trouvés pour les inoculations intradermiques, que pour les inoculations intraperitonéales. Les résultats suggèrent des différences dans l'organotropisme des Borrelia. L'oreille était fréquemment infectée par les représentants de toutes les espèces types. Un taux de 100% d'infection a été décelé pour Borrelia afzelii. Des différences ont été notées dans la distribution des organes infectés: Borrelia burgdorferi et Borrelia garinii semblent infecter plus souvent la vessie que Borrelia afzelii; Borrelia afzelii a été isolée plus fréquemment dans le coeur que les autres espèces. Les différences entre espèces ont été observées en ce qui concerne la réactivité des antigènes c'est à dire leur "sensibilité" à l'ELISA et l'immunofluorescence. Author Keywords: Borrelia burgdorferi; Genospecies; Infectivity; Mice; ELISA; Antigen sensitivityAuthor Keywords: Borrelia burgdorferi; Les espèces types; Infectivité; Souris; ELISA; La réactivité des antigènes Article Outline 1. Introduction 2. Materials and methods 2.1. Experimental animals 2.2. Isolates of B. burgdorferi sensu lato used in the inoculations 2.3. Inoculation of animals and collection of blood samples 2.4. Isolation of Borreliae 2.5. ELISA 2.6. IFA 2.7. Sensitivity 3. Results 4. Discussion Acknowledgements References 1. Introduction In Europe, the three genospecies of Borrelia burgdorferi sensu lato known to be pathogenic for humans are B. burgdorferi sensu stricto (s.s.), Borrelia garinii, and Borrelia afzelii. Isolates representative of all three genospecies have been isolated from Ixodes ricinus ticks in Finland [1 and 2]. Part of the isolates have been subjected to some basic characterizations, especially immunological [2]. In addition, some isolates have been studied for their infectivity for cattle [3]. The present experiments were designed to study infectivity of various B. burgdorferi sensu lato isolates and to characterize infections and responses to infections caused by them, mainly in laboratory mice but also in rabbits. Responses in these laboratory animals were monitored by the isolation method and serologically by both ELISA and IFA techniques. Genospecies specific distribution of borreliae in infected mice was of major interest. As antigens in serology, isolates from all three genospecies were comparatively studied. 2. Materials and methods 2.1. Experimental animals The infection experiments were performed in the Unit for Studies of Infectious Diseases in Large Animals at the former Veterinary College. Mice were either outbred NMRI or inbred BALB/c/Hy mice from the Laboratory Animal Center of Helsinki University. The ages of mice at inoculation were 3–6-weeks. New Zealand White rabbits, 4–6-months of age, were purchased from a local commercial colony providing rabbits for experimental purposes. 2.2. Isolates of B. burgdorferi sensu lato used in the inoculations All 28 isolates used in the infection studies had been isolated from Ixodes ricinus ticks collected during the summer of 1992 in Finland [1]. Their genospecies identities were determined and are given with documentations in another study [2]. Of the isolates, Y8, Y19, Y26 and Ri5 represent genospecies B. burgdorferi s.s., M2, K3, H1, K5, Ri1, Ri3, Ri4, Ri8, P1, U2, U4, Y13, Y16, Y17, and Y29 are B. garinii isolates and K1, K4, H2, H7, Po1, Y12 are B. afzelii isolates. Isolates M1 and U1 are atypical B. afzelii strains and Y18 could not be classified. BSKII medium was used for culturing the isolates. Some isolates were passaged in mice before their use in the present experiments (see Table 1 and Table 2). Table 1. In comparative "large experiments"a, two groups of six laboratory mice, each divided in threemice subgroups, were inoculated, one intradermally, the other intraperitoneally, with one of Finnish isolates of Borrelia burgdorferi sensu lato. Two other types of experiments with inoculum size other than in large experiments were made each in one six-mice group only. The two subgroups within each six-mice group were sacrificed separately, at different times postinoculation. Experiments found positive by ELISA and/or IFA and/or isolation method are included in this Table. (Note. The 14 negative large experiments were inoculations with isolates (x refers to number of experiments; P=passages in culture; M=passages in culture after mouse passage): M2x2, p7 in both; Y13, p3; P1, p4; H7, p3; H1, p2; K1, p1; Po1, p14; Ri5x2, p5, p12; M1x2, p3, p6; U1, p6; Y19, m1.) Table 2. Organ specific infection rates in BALB/c and NMRI mice inoculated with various Borrelia burgdorferi sensu lato isolates. Included are experimentsa in which overall infection rate of mice was 50% or higher. All these experiments were also positive by ELISA 2.3. Inoculation of animals and collection of blood samples Inocula were prepared as described earlier [3]. In brief, borreliae were counted using the method of Stoenner [4] from undiluted or 1:10 diluted cultures. Isolates in their late exponential-early stationary phase—some primary cultures in late stationary phase—were removed from BSKII medium by centrifugation (9000×g for 30 min) and suspended in phosphate buffered saline, pH 7.4 (PBS). Borreliae to be inoculated were not washed, as in preliminary trials washing seemed to affect adversely borreliae. For the majority, 32 in all, of the mouse inoculations, called large experiments, a dilution containing 104 borreliae/0.3 ml was prepared, allowing a programme in which six mice each received 104 borreliae intraperitoneally (i.p.), and another six (rarely nine) received 3.3×103 borreliae intradermally (i.d.) (positive experiments are displayed in Table 1). This dilution was also used to perform a confirming viable count by growth titration with a dilution series in BSKII medium. In the four-mouse experiments of the second category, 3×105 to 5×105 borreliae were administered i.d. only (Table 1). The third category mouse experiments, in which the size of inoculum was unknown, were made (a) to study infectivity of several random isolates at various phases of their passage history, or, and especially, of some primary cultures kept first about 2 months at 34°C and then further stored for 2–5 months at 4°C, and (b) for the purpose of purifying contaminated cultures (third category positive experiments are displayed in Table 1 and Table 2). Rabbits were inoculated in pairs. The sizes of the inocula injected subcutaneously (sc) or intravenously (iv) varied from 5×102 to 108. Rabbit inoculations, and IFA results, positive or negative, of the studies of their immune responses, have been described previously only in a preliminary, limited way [3]. In [3] of that article, the isolates inoculated and sizes of inocula for the 20 rabbits included in the studies are given. In addition to all the rabbit inoculations, some of the present mouse inoculations were likewise partially designed to offer infectivity controls to cattle inoculations and have only to that effect, isolation + or −, been reported earlier [3]. For antibody studies, a combined serum from each three-mice group killed simultaneously was obtained. Blood was also collected from rabbits for both borrelia isolation attempts and for separation of serum. For Borrelia culturing attempts, the scheme of whole blood sampling was: twice weekly for 3 weeks and thereafter once weekly for 3 additional weeks. Blood for serum was collected once weekly throughout the experiments. 2.4. Isolation of Borreliae In the majority of experiments, half of each six-mice group, constituting the threemice ‘unit experiment’, were killed 3–4 weeks (occasionally 2 or 6 weeks, see Table 1) postinoculation, and the other half one week later (rarely 2 weeks, once 4 weeks later, Table 1). In the third category experiments, three to six mice were inoculated and then killed after 2–5 weeks, all at the same time or in two groups as in other experiments ( Table 1 and Table 2). Isolation was attempted from blood, heart, bladder and spleen according to Schwan et al. [5], with some modifications. For trituration of tissues in 1 ml of BSKII medium, mortar and pestle was used. Three portions of approximately 0.3 ml of each suspension were inoculated into three tubes containing 6 ml of BSKII medium, two of the tubes containing phophomycin (100 μg/ml) and rifampin (50 μg/ml) and the third being without antibiotics. Only two tubes, one with and the other without antibiotics, were inoculated with blood, 0.1 ml/tube. For many of the three-mice groups isolation was also attempted from a piece of the ear [6]. As with blood, only two tubes were inoculated. From rabbits, whole blood samples obtained according to the scheme described above were cultured as blood from mice. The culture tubes were incubated at 34°C and, for most of the experiments, examined by dark-field microscopy at least twice after about 1 and 2 months of culturing; some cultures were also studied in an earlier phase. For a few experiments, the first examination could be delayed with a few weeks and confirmative examinations could be made as late as 6 months after inoculation of tubes. 2.5. ELISA The antigens and ELISA plates were prepared as described earlier [3]. The mouse and rabbit sera were diluted 1:500, and 100 μl was applied per well and incubated for 2 h at 37°C. After washing the plates, the conjugate MIG5-POD [7 and 8], an IgGbinding region from Protein MIG fused to MAL and conjugated with horseradish peroxidase according to Wilson and Nakane [9], was used for the mouse sera. The conjugate was a kind gift from Dr Peter Mueller, Department of Microbiology, University of Agricultural Sciences, Uppsala, Sweden. For rabbit sera, the conjugate Protein G-POD was applied as described earlier [3], as were also the following steps. Each serum was tested as duplicate, and on each plate a negative serum and a twofold serially diluted positive serum (dilutions from 1:500 to 1:64,000) from the respective species were applied. The mean OD values of the duplicate measurements are given in the results. OD values >0.20 were considered as signs of immune responses to B. burgdorferi isolates. OD values in a group of negative control mice remained for all antigens used ≤0.10. 2.6. IFA The majority of mouse sera were first examined with B31 antigen. Later, selected sera were also tested with one or more of the antigens prepared from Finnish Borrelia isolates Y8, P1, M2, K3, K4, and H7. The antigen slides (Multitest Slides, Flow, Inc Biomedicals) were prepared and the test mainly performed as described earlier [2]. The fluorescein isothiocyanate conjugate was rabbit anti-mouse IgG,A,M (Serotec, Oxford, UK), which was used at a 1:40 dilution. The sera were diluted in two-fold steps and first screened in dilutions 1:2, 1:8, and 1:32. For sera of interest, further dilutions were examined against selected antigens to determine the titre. The large number of three-mice group sera from experiments negative both by ELISA and isolation and several sera from uninoculated mice had all been clearly negative by IFA at dilution 1:2. Consequently, that dilution was selected as the level at which to decide between positivity and negativity in the present studies. A positive reaction was recorded if >50% of the spirochetes were fluorescent with an intesity of ≥3 out of a possible score of 6 (very bright). When comparative measurements with three to four different antigens were made, the tests were run simultaneously. 2.7. Sensitivity With antigen sensitivity, as used here, is meant the relative ability of antigens, in comparison with others, to react with anti-borrelia antibodies of a serum tested. In ELISA testing of this study, the measure of sensitivity is the relative magnitude of the OD value. 3. Results In all, 28 Finnish isolates were subjected to experiments. Twenty-one of them were found to infect mice, infectivity of one (Ri5) was regarded as doubtful, and six isolates (M1, H1, U1, U4, K5 and Y17) were formally recorded as negative but remained, most of them, insufficiently studied. No reisolation was made from seven experiments with isolate Ri5; increased ELISA OD values were recorded in two experiments (Table 1). Genuine noninfectivity was suggested for the atypical B. afzelii isolate M1 [2] by four experiments of various categories. Of the five other ‘negative’ isolates, tested each only once, three (U4, K5 and Y17) were contaminated primary cultures preserved long at 4°C. Among the 21 isolates found infective, differences in infectivity were observed (see Table 1 and Table 2). Table 1 is a master table giving subgroup-specifically the results for positive large and second category experiments, and also for part of third category experiments. In the following text, these results are analysed and/or commented on selectively only. The 18 positive large experiments were inoculations with 10 different isolates. Demonstrating the infectivity variation of some isolates in the course of the studies, eight of the 14 negative large experiments (see footnote in Table 1) were inoculations with isolates found positive either in some other large experiments or in third category experiments. The experiments of the second category described in Table 1 were made with relatively highly passaged organisms. The responses are considered to have been induced with noninfectious organisms in large enough numbers. The titration results of inocula for positive and most of the negative large experiments were either 10−3 or 10−4, with even 10−5 recorded in four cases. In three negative large experiments (K1,p1; M1,p6; Po1,p14) the titre was, however, only 10−1. The latter finding very likely explains the negativity of the respective three infection trials. Experiments of the third category were 28, representing 17 different isolates, of which 14 experiments made with 11 different isolates were positive by isolation of borreliae (11 of the 14 positive experiments are shown in Table 1 and Table 2; of the rest, one was made with isolate Y18, 2 with Po1). The four original cultures, which infected mice ( Table 2), were all heavily contaminated with bacteria or with fungi and had been stored consecutively at 34 and 4°C for 4–7 months. The growth of the isolates in culture was in majority of cases relatively slow, and consequently the present culture times between passages were rather long, on the average more than one month. In these conditions, definite infectivity was not recorded for inoculations at higher than five passages. As an example of recorded pace of in vitro losses of infectivity, the case of M2 inoculations is referred to. The negative double experiment with this isolate (note, Table 1) was made with a higher passaged (p7) culture than the 3 positive experiments ( Table 1). As seen from Table 1, more mice were found infected among i.d. than i.p. inoculated ones. In comparisons concerning the 30 positive i.d.–i.p. pairs of three-mice groups (either one or both groups of the pair positive serologically and/or by isolation), and regarding individual mice and positivity by isolation, the chi-square test for paired samples gives between i.d. and i.p. inoculations a statistically significant difference, P<0.05. Comparison by ELISA results of i.d. and i.p. inoculations (included are data from 24 i.d.–i.p. pairs of three-mice units) gave mean OD values 1.22 and 0.86, respectively; the difference in Wilcoxon signed rank test was: P<0.02. In comparison between first and second sacrifice groups, without differentiation by inoculation mode, the ELISA OD values were in average clearly higher among the second groups. As shown in Table 2, the ear was the most frequent source of borreliae in experiments both with B. afzelii and B. garinii isolates (ear as the source for B. burgdorferi s.s. was only tested in one large experiment). Relatively, the isolation rate from the ear, in comparison with rates for other organs, was higher among B. afzelii than B. garinii infections presented in Table 2. The result that B. afzelii regularly, in all mice of the included experiments, caused detectable ear infection is conspicuous. Isolation rate for the urinary bladder, relative to rates for other organs, was very high for B. burgdorferi s.s. experiments, high for B. garinii and very low for B. afzelii experiments. Rate for isolation from heart was proportionally lowest in B. garinii experiments. Even in experiments in which all three mice, and one or more organs of each mouse, proved positive by isolation, all three culture tubes from the positive organ (either heart, bladder or spleen) being positive was more exceptional. Such cases were recorded especially for experiments with M2, K3 and K4 (Table 2; detailed data not given). No clear difference between tubes with or without antibiotics were observed. In successful isolations from ear, both two tubes being positive—a result most frequently recorded for B. afzelii infections—was relatively more frequent than positivity of two to three tubes in successful isolation attempts from heart, bladder or spleen. As seen in Table 3 and Fig. 1, in studies with ELISA of the mouse immune responses, the highest OD values were as a rule recorded when the antigen from the homologous genospecies was used. Within genospecies, the immune responses to different isolates or isolate groups could, however, differ as to the sensitivity spectrum of the measuring antigens. Among responses to B. garinii strains, the response to the isolate K3 clearly deviated from those to other isolates. Differences among B. afzelii responses are conspicuous in regard to the measuring sensitivity of the two B. afzelii antigens, H7 and K4. The Y12 immune response observed is quite atypical in its virtual nonreactivity with the K4 antigen—and with B. garinii and B. burgdorferi s.s. antigens. Responses to H7 are recorded (Table 3) to show, on average, as high OD values both by H7 and K4 antigens. The average result is, however, decisively influenced by the fact that in two clearly strongest H7 responses recorded in mice, higher OD values were, paradoxically, measured with antigen K4 than with the homologous H7 ( Table 1 and Fig. 1). Reactivity of H7 antibodies is atypical only in mice inoculated with passage 7;6(m1) inoculum ( Table 1), and among them only in NMRI, not in BALB/c mice. Table 3. Summary data of OD measurements with ELISA of mouse immunresponses to various Borrelia burgdorferi sensu lato isolates, by different isolates as antigens (13K) Fig. 1. Antibody responses in mice measured with ELISA using antigens of three genospecies, B. burgdorferi sensu stricto (B31 and Y8), B. garinii (P1 and M2) and B. afzelii (H7 and K4). Responses in 6 mice each inoculated with different B. garinii isolates (A), six mice inoculated with B. afzelii isolates (B), and three mice inoculated with B.burgdorferi s.s. isolates (C) are presented. Symbols for identities of the infecting isolates: A: P1 (○), M2 (•), U2 ( ), Ri1 (▪), Ri3 ( ), K3 (♦), Ri8 ( ), Ri4 ( ), Y16 ( ), Y29 ( ); B: K4 (○), H7 (•), Y12 ( ), Po1 (▪), K1 ( ), and H2 ( ); C: Y8 (○), Y19 (•), and Ri5 ( ). No borreliae were isolated from blood samples of rabbits. Fig. 2 shows ELISA results of immune responses of four rabbits as measured with four different antigens. For the response to the isolate Y8, the most sensitive antigen was B31, a representative of the same genospecies as Y8 (in a parallel ELISA test with both B. burgdorferi s.s. antigens, B31 and Y8, included—data not shown—their sensitivities were equal); for H7 response, the homologous antigen was slightly more sensitive than antigens from two other genospecies, whereas antigen K4, also a B. afzelii strain antigen, was the least sensitive; for M2 response the homologous antigen was clearly the most sensitive; and for K3 response, all the four antigens showed roughly similar sensitivities. The pairs to the rabbits depicted in Fig. 2 showed (data not given) similar patterns of antigen sensitivity in measurement of their immune responses (also the other M2 rabbit, which had earlier [3] been found IFA negative by B31 antigen), except for the other one of the rabbits inoculated with isolate H7, the response of which was measured with equal sensitivity with both antigen H7 and M2. The responses in rabbits (data not given) inoculated with B. garinii isolate U2 resembled quite closely those to M2, whereas responses to isolates P1 and Ri3 (for the latter, only one of the two rabbits had responded) represented in their antigen sensitivities types in the middle between those to M2 and K3. (12K) Fig. 2. Antibody responses in four rabbits inoculated once or twice (arrows) with borrelia isolates: A with B. burgdorferi sensu stricto isolate Y8; B with B. afzelii isolate H7; C and D with B. garinii isolates M2 and K3, respectively. Symbols for antigens used: B31 (•), M2 (▪), H7 (♦), and K4 ( ). 4. Discussion Among the isolates tested, a variation in mouse infectivity was observed. Part of the variation seemed original, based on differences of freshly isolated borreliae and not resulting from changes during the growth in the artificial medium. Some isolates were of relatively high others of lower infectivity, whereas at the other end of the scale the isolates Ri5 and M1, subjected to repeated experiments, could not in the conditions of the present studies on any occasion be reisolated from the mice inoculated. Both Ri5 and M1 had earlier been found, within their respective genospecies, atypical in their protein and antigen structure [2]. In regard to present occurrence of noninfective or low infectivity isolates, it may be relevant that all the investigated strains were originally isolated from ticks. In literature, isolates noninfective to laboratory mice [10, 11 and 12] and rats [13] have been described, as are those of weak infectivity [14 and 15]. Decrease and subsequent loss of infectivity of B. burgdorferi isolates by the increasing length of in vitro culture has been well documented [16, 17, 18 and 19]. In the cited studies, the passage level at which infectivity was lost varied widely. A relatively late loss was that between passages 17 and 21, with a stay of more than 4 months in in vitro conditions, reported by Moody et al. [18]. Even preservation of infectivity up to the passage level 75 has, however, been reported [19]. In the conditions of the present studies, decrease of infectivity seemed to start earlier than usual: infectivity was demonstrated only for isolates at ≤5 passages (a very low grade infection was serologically suggested to have been caused by Y12 at the passage 7), high infectivity only at passages ≤4. As stated in the results, the passaging of the isolates was on the average slow in our studies. The total times in culture became long if compared with those reported in the literature. This probably is the major factor to explain the observed loss of infectivity at a relatively low passage level. Another aspect of the subject of phenotypic and genotypic changes in culture is effect of storage at 4°C. A rather unexpected result was that a months-long storage of some isolates at 4°C in heavily contaminated primary cultures did not seem to have affected notably the infectivity. Thus, any regular and decisive selection for noninfectivity in the population preserved relatively long in stationary phase at 4°C is not indicated by these data. There are few reports on the rapidity of loss of infectivity in comparable conditions. Norton Hughes et al. [20] describe one well documented case of the loss after 6 months storage of the isolate at 5°C. Differences have been demonstrated between the three pathogenic Borrelia genospecies in the organotropism in human infections and diseases caused by them, e.g. Refs. [21 and 22]. Affection of skin is typical of B. afzelii. In the mouse model, organotropism of infection has more thoroughly been investigated only for the B. burgdorferi s.s. genospecies. As organs of predilection for that infection, the urinary bladder and the heart have been identified [5 and 23]; also the rodent ear has been found frequently infected [6 and 24]. In our studies, B. afzelii and B. garinii isolates were subjected to comparative investigations by the isolation method of distribution of observable infection among selected organs in laboratory mice. Although the material of mouse infections was not large enough to allow more definitive statistical conclusions, the results, nevertheless, clearly suggest—as a hypothesis to be further studied—that for B. afzelii mouse ear (i.e. skin), in comparison with other organs, is a distinct predilection site. It is further suggested by culture tube and individual mouse specific rates that B. afzelii infects the ear of the mice with greater relative intensity than the other two species. Other hypotheses raised by the results include: a low infection rate of the urinary bladder by B. afzelii and relatively low rate for heart by B. garinii are features suggestively typical of the mouse infections by the respective species. A strong tropism by B. afzelii to the skin of rodents, its natural hosts, may also be suggestively inferred from the results of some other studies [25]. One of the goals of these studies was to compare i.d. and i.p. inoculations in low doses, to do it in larger scale and with several isolates, also of different genospecies. As shown by the titration results, sizes of inocula by viable count could vary to certain extent between experiments, but the relative sizes within each comparative experiment remained fixed. As expected from the results of the basic works by others using B. burgdorferi s.s. strain [26 and 27], both infection rate and mean ELISA titers at defined intervals postinoculation were, at doses used in these experiments, significantly higher among i.d. than i.p. inoculated mice and mouse groups, respectively. It appears that in critical cases of lower infectivity the infectious dose level remained more often unreached by the i.p. inocula of 104 borreliae than the i.d. inocula of 3.3×103; the observed lower level of ELISA antibodies in i.p. inoculated mice is thought to be associated with a delayed development due to smaller number of route specific infectious doses injected. Immune responses were recorded in mice inoculated with higher doses (≥3×105, with the titration results ≥106) of higher passaged (≥9 passages) isolates. Borreliae were, however, not reisolated from the mice. In these cases, responses are regarded to have been induced by nonmultiplicating, noninfectious borreliae. In literature, the inoculum size needed to cause detectable immune response in mice by noninfective borreliae has been estimated to be in the range of 106 to 107 [28 and 27]. In an earlier study with ELISA of experimental immune responses in cattle, we showed differences in reactivity of the antigens both by genospecies and, sometimes also, by isolate within a genospecies [3]; we speak in this connection of antigen sensitivity. As a rule, the homologous antigen was the most sensitive one in those ELISA measurements. A wide variation in sensitivity of antigens was recorded by the isolate identity of the immune response investigated. Within B. afzelii, two isolates K4 and H7 clearly differed from each other as antigens. A discussion of literature on antigen heterogeneity in ELISA studies is found in the above referred article. The results of the present studies in mice and rabbits confirm in general our earlier findings on antigen differences, i.e. ELISA serologic differences when the extent of cross-reacting between genospecies is investigated. Minor differences apparently caused by the host factor are recorded, among them those between members of each pair of rabbits—cf earlier findings on pairs of cattle—but the general pattern of antigen sensitivity relationships remains the same for isolate specific immune responses. A special emphasis is laid on how in mouse serum ELISA studies the antigen from the homologous genospecies was regularly the most sensitive one, and how, with the one exception of the responses to B. garinii strain K3, the sensitivity difference with antigens from other genospecies was very clear indeed. Responses to B. afzelii isolates were divided into two groups, each more reactive with one of the two B. afzelii type antigens used. 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Lobet, Tick transmission of Borrelia burgdorferi to inbred strains of mice induces an antibody response to P39 but not to outer surface protein A. Infect Immun 62 (1994), pp. 2625–2627. Abstract-EMBASE | Abstract-MEDLINE | Abstract-Elsevier BIOBASE | Order Document Comparative Immunology, Microbiology and Infectious Diseases Volume 25, Issue 2 , March 2002, Pages 109- Corresponding author. Tel.: +358-9-239-2951; fax: +358-9-191-49799; email: raili.tanskanen@helsinki.fi Functionality of Borrelia burgdorferi LuxS: The Lyme disease spirochete produces and responds to the pheromone autoinducer-2 and lacks a complete activated-methyl cycle Kate von Lackuma, 1, Kelly Babba, 1, Sean P. Rileya, 1, Rachel L. Wattiera, b, 2, Tomasz Bykowskia and Brian Stevensona, , a Department of Microbiology, Immunology, and Molecular Genetics, College of Medicine, University of Kentucky, MS 415 Chandler Medical Center, Lexington, Kentucky 40536-0298, USA b Agricultural Biotechnology Program, College of Agriculture, University of Kentucky, Lexington, Kentucky, USA Available online 10 March 2006. Abstract Borrelia burgdorferi produces Pfs and LuxS enzymes for breakdown of the toxic byproducts of methylation reactions, producing 4,5-dihydroxy-2,3-pentanedione (DPD), adenine, and homocysteine. DPD and its spontaneously rearranged derivatives constitute a class of bacterial pheromones named autoinducer-2 (AI-2). We describe that B. burgdorferi produces DPD during laboratory cultivation. Furthermore, addition of in vitro synthesized DPD to cultured B. burgdorferi resulted in altered expression levels of a specific set of bacterial proteins, among which is the outer surface lipoprotein VlsE. While a large number of bacteria utilize homocysteine, the other LuxS product, for synthesis of methionine as part of the activated-methyl cycle, B. burgdorferi was found to lack that ability. We propose that the main function of B. burgdorferi LuxS is to synthesize DPD and that the Lyme disease spirochete utilizes a form of DPD as a pheromone to control gene expression. Keywords: Borrelia burgdorferi; Quorum sensing; Pheromone; Bacterial gene regulation; Homocysteine; Methionine Article Outline Introduction Materials and methods Bacteria and growth conditions In vitro synthesis of DPD Vibrio harveyi bioassay of AI-2 Genomic analyses Borrelia burgdorferi genomic libraries construction and analyses Methionine synthase assay Results Borrelia burgdorferi synthesizes AI-2 during laboratory cultivation Borrelia burgdorferi modulates protein expression in response to DPD Borrelia burgdorferi lacks a complete activated-methyl cycle Many other pathogenic spirochete species lack activated-methyl cycles Discussion Acknowledgements References Introduction As do most other organisms, the Lyme disease spirochete Borrelia burgdorferi lives in a series of dynamic environments. This bacterium persists in nature through cycles requiring infection of both vertebrates and ticks. During these infectious cycles, the spirochete must not only adapt to life in two extremely different types of host, but must also manage efficient transmission between the two. These constraints require that for B. burgdorferi to survive, it must precisely control gene expression so that only appropriate proteins are expressed during each stage of infection (Schwan, 1996; Indest et al., 2001b; Seshu and Skare, 2001; Anguita et al., 2003). Since disruption of regulatory networks that control expression of infection-associated proteins is a key goal for development of novel therapies to prevent and treat Lyme disease, characterization of those control mechanisms will be of great value. A growing number of bacteria are known to utilize derivatives of 4,5-dihydroxy-2,3pentanedione (DPD) as a pheromone to control gene expression (Xavier and Bassler, 2003). DPD can spontaneously cyclize and/or interact with borate to form at least two different, interconvertible molecules collectively described as autoinducer-2 (AI-2, Fig. 1) (Chen et al., 2002; Miller et al., 2004). Although AI-2 was originally described as a quorum sensing molecule for measuring bacterial density (Surette et al., 1999), it appears that many bacteria instead use AI-2 as a pheromone during the exponential growth phase to signal metabolic status and fitness (Xavier and Bassler, 2003). In a previous study, our laboratory demonstrated that B. burgdorferi encodes a functional copy of LuxS, the enzyme that synthesizes DPD (Stevenson and Babb, 2002; Babb et al., 2005). (6K) Fig. 1. Composite diagram of metabolic pathways that constitute activated-methyl cycles. Abbreviations: SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; SRH, Sribosylhomocysteine; DPD, 4,5-dihydroxy-2,3-pentanedione; Me-THF, 5-methyltetrahydrofolate; THF, tetrahydrofolate. DPD can spontaneously cyclize and/or combine with borate to produce at least two different, interconvertible forms of AI-2 (Schauder et al., 2001; Chen et al., 2002; Miller et al., 2004). Most organisms contain either the SAH hydrolase pathway or the Pfs/LuxS pathway for homocysteine synthesis, although some organisms contain both (Sun et al., 2004). Figure adapted from Babb et al. (2005). Many bacteria utilize LuxS as part of the activated-methyl cycle (Fig. 1). This series of reactions generates a methyl donor molecule, S-adenosylmethionine (SAM) for use in a wide variety of methylation reactions, then detoxifies byproducts and regenerates SAM. The second product of LuxS is homocysteine, which many organisms metabolize to produce methionine. For bacteria with a complete activated-methyl cycle such as illustrated in Fig. 1, LuxS plays an important role in maintaining a pool of free methionine in the cell. For example, mutants of Escherichia coli defective in methionine synthase are unable to grow in minimal media that lack added methionine (Urbanowski et al., 1987; Matthews, 1996). This function of LuxS has led to suggestions that the primary role of that enzyme in bacteria is to synthesize homocysteine for recycling, rather than for synthesis of DPD, and that effects of luxS mutations on bacterial pathogenicity are due to methionine starvation (Winzer et al., 2002a, Winzer et al., 2002b and Winzer et al., 2003; Blevins et al., 2004). For this reason, we examined B. burgdorferi in detail to determine whether that bacterium can also regenerate methionine from homocysteine, and found that it lacks such an ability, indicating that the Lyme disease spirochete produces LuxS for a purpose other than homocysteine synthesis. Materials and methods Bacteria and growth conditions B. burgdorferi were cultured in Barbour–Stoenner–Kelly II broth (Barbour, 1984). B. burgdorferi strain 297 is an infectious, wild-type bacterium and was originally isolated from human cerebrospinal fluid in New York, USA (Steere et al., 1983). Strain AH309 is a luxS-deficient mutant of strain 297 (Hübner et al., 2003) and was obtained from Michael Norgard (University of Texas Southwestern, Dallas, USA). For analyses of AI-2 production by B. burgdorferi, cultures of strains 297 and AH309 were grown at 34 °C to densities of approximately 107 bacteria per ml. Those cultures were then placed at 23 °C, which greatly retards bacterial growth (Stevenson et al., 1995). Aliquots of each initial culture were diluted 1:1000 into fresh medium on 6 subsequent days, with each secondary culture incubated at 34 °C to allow optimal growth. On day 7, supernatant was removed from each secondary culture and assayed for AI-2 content. Through this technique, cultures with essentially the same starting densities could be grown under the same conditions for 1–7 days and then assayed simultaneously. Bacterial density of each culture was also determined at that time, using a Petroff–Hauser counting chamber and dark-field microscopy. E. coli strain GS162 is wild type for both methionine synthase genes, while GS472 is defective in both metH and metE. Both were obtained from George Stauffer (University of Iowa, USA) (Urbanowski et al., 1987). For attempted complementation of E. coli metE and metH, transformed GS472 was plated on M9 minimal salts agar supplemented with 1 μg/ml thiamin, with this same medium plus 100 μg/ml methionine serving as a positive control for growth (Urbanowski et al., 1987; Sambrook et al., 1989; Zeh et al., 2002). For use as a positive control for methionine synthase analyses, GS162 was cultured in M9 supplemented with 3.4 μg/ml hydroxocobalamin, 1 μg/ml thiamin and 100 μg/ml phenylalanine (Urbanowski et al., 1987; Jarrett et al., 1997). Vibrio harveyi strain BB170 (Bassler et al., 1997) was obtained from Bonnie Bassler (Princeton University, USA). V. harveyi were cultivated in modified autoinducer bioassay medium (Greenberg et al., 1979) containing 40 μM sodium borate (pH=6.8). Leptospira interrogans serovar pomona type kennewicki strain JEN4 (Nally et al., 2001) was cultured at 30 °C in Bovuminar PLM-5 medium (Intergen, Purchase, NY). In vitro synthesis of DPD DPD was synthesized from S-adenosylhomocysteine nucleosidase (SAH) (Sigma, St. Louis, MO, USA), using equimolar concentrations of recombinant B. burgdorferi LuxS and Pfs proteins. Reaction products were quantified using Ellman's reaction, as previously described (Schauder et al., 2001; Babb et al., 2005). Vibrio harveyi bioassay of AI-2 V. harveyi strain BB170 autofluoresces in the presence of AI-2 in a dose-dependent manner, enabling its use in a bioassay specific for AI-2 (Bassler et al., 1997; Taga, 2005). Bioassays were performed as previously described (Surette and Bassler, 1998; Stevenson and Babb, 2002). AI-2 activities are reported as average luminescence values of each strain 297 assay minus the average luminescence of equivalent cultures of strain AH309. Studies were repeated at least three times using independent cultures. Genomic analyses Genome databases of B. burgdorferi and three other spirochetal species were queried for presence of specific gene homologs. Complete genome sequences of B. burgdorferi strain B31 (Fraser et al., 1997; Casjens et al., 2000), Treponema pallidum Nichols strain (Fraser et al., 1998), T. denticola ATCC 35405 (Seshadri et al., 2004), L. interrogans serovar Lai strain 56601 (Ren et al., 2003), and L. interrogans serovar Copenhageni strain Fiocruz L1-130 (Nascimento et al., 2004a) were accessed through the Institute for Genomic Research microbial database at http://www.tigr.org. To search for homologs of enzymes involved in SAH metabolism, each genome was queried using BLAST-P with the amino acid sequences of the biochemically characterized V. harveyi and B. burgdorferi LuxS, E. coli Pfs, and Rhodobacter capsulatus SAH hydrolase proteins, and the hypothetical SAH hydrolase of L. interrogans (GenBank accession numbers AF120098, AAC66762, U24438, M80630, and AAN51667, respectively). To search for homologs of methionine synthase enzymes, the genomes were queried with protein sequences of the MetE homologs of E. coli, Mycobacterium tuberculosis, the archaeon Methanobacterium thermoautotrophicum, and the plant Solanum tuberosum; the MetH homologs of E. coli, L. interrogans, and Synechococcus sp. WH8102; the methylmethioninedependent YagD enzyme of the E. coli CP4-6 prophage; and the human betainedependent methionine synthase (GenBank accession numbers M87625, AAK45422, X92082, AF082893, P13009, AAN51667, CAE07753, AAC73364, and U50929, respectively). Each identified spirochetal gene was used to re-query all of the spirochete genomes, along with the complete GenBank database at http://www.ncbi.nlm.nih.gov/blast/. Borrelia burgdorferi genomic libraries construction and analyses Total genomic DNA of infectious, wild-type B. burgdorferi strain B31 was digested using either EcoRI or PstI, then ligated with appropriately digested pUC118 (Vieira and Messing, 1987). Each ligation mixture was used to transform E. coli strain GS472 and bacteria plated on solid M9 medium lacking methionine. Methionine synthase assay 5-methyltetrahydrofolate-homocysteine S-methyltransferase activity was determined as described by Jarrett et al. (1997). Produced 14C-labeled methionine was separated from the reactants by passage through AG 1-X8 columns (Bio-Rad), then measured using a scintillation counter. Both E. coli strains GS162 and GS472 were analyzed as controls, and all experiments included a negative control that lacked any bacterial lysate. Number of decays per minute in the column flow-through of the negative control reaction was then subtracted from each experimental value. Results Borrelia burgdorferi synthesizes AI-2 during laboratory cultivation Cultured B. burgdorferi appear to utilize SAM in a variety of methylation reactions (Hughes and Johnson, 1990; Fraser et al., 1997; Charon and Goldstein, 2002). Both immunoblot analysis and RT-PCR demonstrated that cultured B. burgdorferi also produce both Pfs and LuxS enzymes (Hübner et al., 2003; Babb et al., 2005). Although earlier assays failed to detect production of DPD by cultivated B. burgdorferi (Stevenson and Babb, 2002; Hübner et al., 2003), we have since refined analysis techniques and found that the spirochete does indeed synthesize DPD, as described below. The most sensitive assay available for detection of DPD uses the marine bacterium V. harveyi as a biosensor (Taga, 2005). V. harveyi recognizes a cyclic, borate derivative of DPD and responds by autofluorescing at levels proportional to the amount of added DPD. Generally, unused growth medium of the tested microorganism is utilized as a negative control, and values obtained for the control then subtracted from experimental results. However, B. burgdorferi is cultured in a very rich, complex medium. We observed that addition of unused B. burgdorferi culture medium to V. harveyi caused the reporter bacteria to grow far more rapidly than did addition of B. burgdorferi culture supernatant, yielding the artifactual result of greater bioluminescence levels being attained from unused medium than from spent medium (Babb et al., 2005). To avoid this artifact, we instead compared bioassay results of wild-type strain 297 and an isogenic luxS mutant, AH309, using results from the mutant as background. Equivalent cultures of each strain were simultaneously examined during early through late exponential phases of growth, as well as after reaching stationary phase (Fig. 2). Significant levels of DPD were found in the supernatants of cultured B. burgdorferi 297, reaching a maximum during the midexponential phase and then decreasing as cultures entered stationary phase. The reduced levels of DPD in stationary phase cultures suggest that B. burgdorferi actively removes the molecule from the medium, as do several other studied bacteria (Surette and Bassler, 1999; Taga et al., 2001 and Taga et al., 2003; Hardie et al., 2003; Xavier and Bassler, 2003). (20K) Fig. 2. Production of AI-2 by Borrelia burgdorferi. Strains 297 (wild-type) and AH309 (luxS) were diluted into fresh medium and grown for 1–7 days at 34 °C. Culture densities are illustrated as a growth curve (left Y-axis). Vibrio harveyi bioassays were performed for each culture, with luminescence values obtained for the negative control AH309 being subtracted from corresponding values obtained for strain 297. Statistically significant (>90% confidence interval by independent sample t-test) mean luminescence values are illustrated as rectangles (right Y-axis). Error bars represent standard deviations of 2–5 separate experiments. Since culture pH may affect bioassay results (DeKeersmaecker and Vanderleyden, 2003), the pH of the B. burgdorferi culture medium was examined at all time points and found to have dropped only slightly during the studies, from an initial 7.5 to a final pH of 6.8 after 3 days at stationary phase, so it is unlikely that the pH of the tested B. burgdorferi culture media influenced these results. However, V. harveyi also controls bioluminescence in response to cellular levels of cAMP, repressing light generation in the presence of PTS sugars such as glucose (Chatterjee et al., 2002; DeKeersmaecker and Vanderleyden, 2003). Unfortunately, all culture media capable of supporting B. burgdorferi growth include glucose as a primary carbon source, as well as other, essential PTS carbohydrates such as N-acetylglucosamine (Barbour, 1984; von Lackum and Stevenson, 2005). Furthermore, B. burgdorferi is capable of high growth rates for 3–4 days in media lacking glucose, due to energy provided by other medium components (von Lackum and Stevenson, 2005). Media capable of supporting growth of these fastidious spirochetes but lack trace carbohydrates do not exist. The unavoidable effects of catabolite repression on V. harveyi reporter strain bioluminescence lead us to suspect that the DPD synthesis results reported for B. burgdorferi may be artificially low (Babb et al., 2005). Borrelia burgdorferi modulates protein expression in response to DPD Results of the above-described studies indicated that B. burgdorferi can and does synthesize DPD. Two additional studies indicated that B. burgdorferi utilizes DPD (or a derivative of that molecule) as an AI-2 pheromone to control protein expression. First, a luxS-deficient mutant of E. coli was complemented with the B. burgdorferi luxS gene, and demonstrated to synthesize DPD (Stevenson and Babb, 2002). Addition of sterile culture supernatant from those complemented bacteria to B. burgdorferi influenced expression levels of more than 50 B. burgdorferi proteins, including the factor H-binding Erp outer surface proteins (Stevenson and Babb, 2002). Culture supernatants from the uncomplemented E. coli luxS mutant had no detectable effects. Second, DPD was synthesized in vitro and B. burgdorferi cultures examined for effects of the added compound (Babb et al., 2005). Again, expression levels of a specific subset of B. burgdorferi proteins were measurably affected by addition of reaction products to cultures (Babb et al., 2005). Furthermore, the effect of DPD addition was dose dependent, as would be expected for a pheromone. Control assays in which only homocysteine or SAH were added did not yield detectable changes to expression levels of any B. burgdorferi protein. The effects of adenine, the third product of the Pfs and LuxS reactions were not examined, since B. burgdorferi is an auxotroph for that molecule (Wyss and Ermert, 1996) and culture medium contains a substantial concentration of adenine (Barbour, 1984). These control experiments demonstrated that the effects of adding reaction products to culture medium were due to DPD alone. Parallel studies of wild-type strain 297 and its isogenic luxS mutant AH309 revealed that some proteins were detectable in lysates of uninduced strain 297, but were not visible in lysates of AH309, suggesting that the amounts of DPD produced by wild-type B. burgdorferi during laboratory cultivation cause significant effects on protein levels. Additionally, AH309 responded to addition of DPD in manners similar to the wild-type strain, indicating that responses to that molecule can occur independently of LuxS (Babb et al., 2005). Both proteomic and microarray analyses were utilized to identify B. burgdorferi proteins and genes that are controlled through DPD (Babb et al., 2005). Most of the tentatively identified proteins have yet to be characterized. We are in the process of developing reagents to confirm those preliminary data and further characterize DPDregulated genes and proteins. Previously characterized proteins whose expression levels have been demonstrated to be influenced by DPD include the Erp protein family (Stevenson and Babb, 2002). These outer membrane lipoproteins are expressed during transmission between ticks and mammals and during persistent mammalian infection, but not during tick infection (Lam et al., 1994; Akins et al., 1995; Stevenson et al., 1995 and Stevenson et al., 1998; Suk et al., 1995; Wallich et al., 1995; Das et al., 1997; El-Hage et al., 2001; Gilmore et al., 2001; Hefty et al., 2001 and Hefty et al., 2002; McDowell et al., 2001; Miller et al., 2003 and Miller et al., 2005; Miller and Stevenson, 2004). Many members of the Erp protein family have been demonstrated to bind the host complement regulator molecule factor H, suggestive of roles in establishment and maintenance of mammalian infection (Hellwage et al., 2001; Kraiczy et al., 2001a, Kraiczy et al., 2001b, Kraiczy et al., 2003 and Kraiczy et al., 2004; Alitalo et al., 2002; Stevenson et al., 2002; McDowell et al., 2003; Metts et al., 2003). Along with the effects of DPD, erp genes are also controlled by environmental temperature and uncharacterized chemical signals (Stevenson et al., 1995; Akins et al., 1998; Babb et al., 2001 and Babb et al., 2004; Hefty et al., 2001; El-Hage and Stevenson, 2002). In other studies, we identified DNA sequences involved in control of erp gene transcription (Babb et al., 2004) and are continuing to explore the mechanism by which DPD affects Erp expression. We also identified that B. burgdorferi increases expression of the polymorphic surface protein VlsE in directly proportional response to DPD concentration (Babb et al., 2005). VlsE is expressed during mammalian infection, where it protects the bacterium from host antibodies through an antigenic variation mechanism (Zhang et al., 1997; Zhang and Norris, 1998a and Zhang and Norris, 1998b; Liang et al., 1999). Regulatory mechanisms controlling vlsE expression are also quite complex, and include environmental signals such as temperature and pH and interactions with mammalian cell membranes (Hudson et al., 2001; Indest et al., 2001a; Ohnishi et al., 2003; K. von Lackum, K. Babb, S.P. Riley, R.L. Wattier, T. Bykowski, B. Stevenson, unpublished results). As with the erp loci, the vlsE promoter/operator region specifically binds multiple B. burgdorferi cytoplasmic proteins (T. Bykowski, K. Babb, and B. Stevenson, unpublished results), one or more of which may be responsible for the effects of DPD on gene expression. Borrelia burgdorferi lacks a complete activated-methyl cycle As noted above, evidence indicates that B. burgdorferi utilizes SAM as a methyl donor for many cellular reactions. We have demonstrated that the Lyme disease spirochete expresses functional Pfs and LuxS enzymes, and can therefore detoxify SAH to yield DPD, adenine, and homocysteine (Babb et al., 2005). Additional studies demonstrated that B. burgdorferi also encodes a functional MetK for synthesis of SAM from methionine (S.P. Riley and B. Stevenson, unpublished results). Almost all examined organisms, from bacteria to plants to animals, possess complete activatedmethyl cycles and regenerate methionine from homocysteine (Fig. 1) (Sun et al., 2004). For this reason, it has been suggested that the major, and possibly the only, reason bacteria possess LuxS is to produce homocysteine for re-synthesis of methionine (Winzer et al., 2002a, Winzer et al., 2002b and Winzer et al., 2003; Blevins et al., 2004). However, the following studies indicated that B. burgdorferi lacks the ability to synthesize methionine. The genome sequence of B. burgdorferi strain B31 was examined for an open reading frame (ORF) homologous to a previously characterized methionine synthase. The two major classes of this enzyme use 5-methyltetrahydrofolate or its derivatives as the methyl donor. The best characterized methionine synthases are the MetH and MetE proteins of E. coli. Proteins homologous to one or both these enzymes have been found in nearly every examined prokaryote and eukaryote (Sun et al., 2004). MetH and MetE enzymes are commonly referred to as cobalamin-dependent and cobalaminindependent methionine synthases, respectively (González et al., 1992). Two other identified types of methionine synthase use either betaine or methylmethionine as the methyl donor (Garrow, 1996; Neuhierl et al., 1999; Thanbichler et al., 1999). Both those enzymes share very similar amino acid sequences with cobalamin-dependent methionine synthases (Garrow, 1996; Thanbichler et al., 1999). BLAST-P queries against the predicted proteins of B. burgdorferi indicated that this bacterium lacks a protein with recognizable homology to any known methionine synthase. To examine the possibility that B. burgdorferi may encode a completely novel methionine synthase, we used plasmid libraries of B. burgdorferi DNA in attempts to complement an E. coli metE metH mutant. While this technique had previously been used to clone methionine synthase genes from organisms as evolutionarily distant from E. coli as the potato (Zeh et al., 2002), all attempts have failed to identify such a gene in B. burgdorferi. Finally, we biochemically analyzed cellular extracts of B. burgdorferi for methionine synthase activity. Consistent with data from our genomic and cloning analyses, no enzymatic activity could be detected in cell-free extracts of this spirochete (data not shown). Control studies with wild-type E. coli detected enzyme activity, as expected. Many other pathogenic spirochete species lack activated-methyl cycles Results of our studies on B. burgdorferi prompted us to examine other species of spirochetes for possible methionine synthases. Examination of the unpublished genome sequences of the relapsing fever spirochetes B. hermsii and B. turicatae indicates that both possess luxS homologs, yet lack homologs of any known methionine synthase (Tom Schwan, pers. comm.). The causative agent of syphilis, T. pallidum, contains a homolog of pfs, ORF TP0170. However, T. pallidum lacks genes for either LuxS or methionine synthase, and so probably does not encode a complete activated-methyl cycle (Fig. 3). The periodontal disease-associated spirochete T. denticola likewise contains a pfs homolog (ORF TDE0105) and lacks both luxS and a methionine synthase gene. Hence, none of these Treponema species is predicted to synthesize DPD. Related to the lack of methionine synthase genes in these spirochetes, neither B. burgdorferi nor T. pallidum encode homologs of any cobalamin-dependent enzyme or any proteins involved in either cobalamin synthesis or transport (Rodionov et al., 2003). These data indicate that an activated-methyl cycle is completely unnecessary for any of these pathogenic spirochetes to survive in nature. (5K) Fig. 3. Experimentally determined metabolic pathways found in Borrelia burgdorferi. The Lyme disease spirochete utilizes methionine to produce SAM and detoxifies SAH to DPD, adenine, and homocysteine via Pfs and LuxS. This bacterium is unable to regenerate methionine from homocysteine. Among the six spirochete species for which genome sequence data are available, only L. interrogans encodes a potential homocysteine salvage enzyme, an ortholog of MetH (Fig. 4). Consistent with that observation, analysis of L. interrogans lysates detected levels of methionine synthase activity that were comparable to wild-type E. coli controls (data not shown). L. interrogans contains a gene homologous to SAH hydrolases (ORF LB106), but lacks homologs of either pfs or luxS. Infectious leptospires also encode homologs of many other biosynthetic enzymes, as well as proteins involved with cobalamin metabolism (Picardeau et al., 2003; Ren et al., 2003; Rodionov et al., 2003; Nascimento et al., 2004a and Nascimento et al., 2004b; Sekowska et al., 2004). We conclude that L. interrogans, alone among the examined spirochete species, encodes a complete activated-methyl cycle, and can regenerate methionine from homocysteine (Fig. 5). It is also apparent that the leptospirosis spirochete cannot synthesize DPD. (4K) Fig. 4. Metabolic pathways of the syphilis spirochete Treponema pallidum and the peridontitisassociated spirochete T. denticola, as determined by genomic analyses. These spirochetes produce SAM from methionine for methylation reactions, then detoxify SAH to SRH and adenine via Pfs. Neither bacterium is predicted to regenerate methionine or to produce DPD. (6K) Fig. 5. Metabolic pathways of the leptospirosis agent, Leptospira interrogans, as determined by genomic analyses. This spirochete species is predicted to encode a complete activated-methyl cycle, with a single-step detoxification of SAH to homocysteine and adenosine. L. interrogans lacks a homolog of LuxS and is not predicted to synthesize DPD. Discussion Our studies demonstrated that B. burgdorferi detoxifies SAH by converting that molecule into DPD, adenine, and homocysteine. The Pfs- and LuxS-catalyzed reactions occur during laboratory cultivation, with DPD accumulating during the exponential phase of growth. However, biochemical and genomic analyses indicated that B. burgdorferi lacks a complete activated-methyl cycle, and is unable to regenerate methionine from homocysteine. These data lead us to conclude that the Lyme disease spirochete synthesizes LuxS for a purpose other than production of homocysteine for regeneration of methionine. We propose that B. burgdorferi produces a LuxS enzyme for the express purpose of synthesizing DPD. Several species of bacteria utilize DPD or a derivative as an AI-2 pheromone to control gene expression, and our studies indicate that B. burgdorferi is to be included among that list. The finding that relapsing fever Borrelia species also contain luxS raises the possibility that those infectious spirochetes may likewise use DPD as a signal. The mechanism by which B. burgdorferi senses DPD as a signal molecule is not yet known, as the sequenced genome does not encode recognizable homologs of proteins known to function in DPD sensing by other bacteria (Stevenson et al., 2003). However, we note that the two Gram-negative bacterial species for which the AI-2 sensors have been defined use extremely different mechanisms, so it is quite possible that the distantly related spirochetes use a completely novel sensor/regulatory mechanism (Bassler et al., 1994; Taga et al., 2001; Stevenson et al., 2003). We are continuing studies to characterize the mechanisms by which AI-2dependent regulation occurs in B. burgdorferi, as well as examining other spirochetes for use of this pheromone. Two recent publications reported that a luxS mutant of B. burgdorferi was capable of infecting both mice and ticks, leading those authors to suggest that neither LuxS nor AI-2 are necessary for infection processes (Hübner et al., 2003; Blevins et al., 2004). However, there are significant questions about how to interpret results of those experiments due to the process by which the mutant bacteria were derived: Bacteria were transformed by electroporation, briefly cultured in liquid medium with a selective antibiotic, placed in a dialysis bag that was then implanted in the peritoneum of a rat for 15 days, then removed from the dialysis bag, injected into a mouse, and, after 2 weeks of infection, a skin biopsy was cultured in liquid medium and, finally, plated in solid medium (Hübner et al., 2003). Bacteria from two resulting colonies were then tested for ability to infect mice. Since this complicated selection scheme mandated that bacteria maintain infectivity, it is impossible to know whether bacteria that survived the process contain only the introduced luxS lesion or if spontaneous mutations arose at additional loci to compensate for the loss of luxS. Moreover, none of those earlier studies compared relative levels of infectivity of mutant and wild-type bacteria. For these reasons, it is not yet known how critical AI-2-mediated genetic regulation is for B. burgdorferi pathogenesis. That B. burgdorferi does not regenerate methionine from homocysteine is consistent with other indications that it is an auxotroph for all amino acids. Culture media capable of supporting B. burgdorferi growth must contain all amino acids and many other nutrients (Barbour, 1984; Pollack et al., 1993). Growth of relapsing fever borreliae and oral treponemes also requires very complex culture media (Kelly, 1971; Wyss, 1992). The genome sequences of borreliae and treponemes indicate homologs of proteases and transporters of polypeptides and amino acids, but few or no biosynthetic enzymes (Fraser et al., 1997 and Fraser et al., 1998; Seshadri et al., 2004). This probably explains why all identified species of Borrelia or Treponema are found only in close association with host animals. In contrast, the sole spirochete known to possess a complete activated-methyl cycle, L. interrogans, is a water-borne pathogen capable of survival outside animal hosts. Additionally, L. interrogans encodes a great many amino acid biosynthetic enzymes (Picardeau et al., 2003; Ren et al., 2003; Nascimento et al., 2004a and Nascimento et al., 2004b). These observations indicate that while the free-living leptospires probably require a complete activated-methyl cycle in order to survive in methionine-deficient environments, the obligatory parasitic borreliae and treponemes do not need that pathway since they can scavenge sufficient quantities of methionine from their hosts for all metabolic processes. B. burgdorferi produces maximum levels of DPD during the exponential phase of growth, as is also found with many other bacteria (Xavier and Bassler, 2003). DPD synthesis increases during rapid bacterial growth, presumably due to increased number of methylation reactions, and is thereby thought to serve as a signal of bacterial fitness (Xavier and Bassler, 2003). DPD/AI-2 may thus function to coordinate growth-related processes throughout a population or serve as a form of positive feedback to the cell that produced it, or a combination of those two possibilities. Since DPD synthesis is often unrelated to culture density, it is probably more accurate to think of AI-2 as a pheromone than as a ‘quorum sensing’ molecule. In conclusion, B. burgdorferi can and does synthesize DPD. Furthermore, B. burgdorferi uses either DPD or a derivative thereof as an AI-2 pheromone to control expression levels of a specific subset of bacterial proteins. Those observations, along with the lack of a complete activated-methyl cycle, lead us to hypothesize that the Lyme disease spirochete produces LuxS for the express purpose of synthesizing DPD pheromone. Since maximal synthesis of DPD during cultivation was observed during the exponential phase of growth, it is likely that the pheromone also functions during periods of rapid growth in the natural infectious cycle. B. burgdorferi within the midguts of infected, feeding ticks experience dramatic increases in growth rate (Benach et al., 1987; de Silva and Fikrig, 1995; Piesman et al., 2001 and Piesman et al., 2003). Bacteria within feeding ticks also increase synthesis of LuxS (Narasimhan et al., 2002), consistent with our hypothesis and suggesting a role for AI-2 in coordinating bacterial gene expression during transmission (Stevenson and Babb, 2002; Miller et al., 2003; Stevenson et al., 2003; Miller and Stevenson, 2004). It is possible that B. burgdorferi may also use DPD/AI-2 to regulate gene expression during other stages of its infectious cycle, perhaps through self-induction (Redfield, 2002; Koerber et al., 2004). 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Norris, Antigenic variation in Lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes, Cell 89 (1997), pp. 1–20. International Journal of Medical Microbiology Article in Press, Corrected Proof Corresponding author. Tel.: +1 859 257 9358; fax: +1 859 257 8994. The first three authors contributed equally to these studies. 2 Present address: Yale University School of Medicine, 333 Cedar Street, New Haven, CT, USA. 1