Differentiation of Borreliacidal Activity Caused by Immune Serum or Antimicrobial Agents by Flow Cytometry

Transcription

1 CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Mar. 1994, p Vol. 1, No X/94/$4.+ Copyright (C 1994, American Society for Microbiology Differentiation of Borreliacidal Activity Caused by Immune Serum or Antimicrobial Agents by Flow Cytometry YU-FEN LIU,1'2 LONY C. L. LIM,'2* KATHLEEN SCHELL,3 STEVEN D. LOVRICH,4 STEVEN M. CALLISTER,5 AND RONALD F. SCHELL' 2'4 Wisconsin State Laboratory of Hygiene,' Departments of Bacteriology2 and Medical Microbiology and Immunology,4 and University of Wisconsin Comprehensive Cancer Center Flow Cytometry Unit,3 University of Wisconsin, Madison, Wisconsin 5376, and Microbiology Research Laboratory, Gundersen Medical Foundation, LaCrosse, Wisconsin Received 8 September 1993/Returned for modification 6 October 1993/Accepted 18 October 1993 We demonstrated that borreliacidal activity caused by immune serum and complement can easily be differentiated by flow cytometry from killing activity caused by antimicrobial agents that are commonly used for the treatment of Lyme disease. Assay suspensions containing normal or immune serum were incubated with Borrelia burgdorferi in the presence or absence of ceftriaxone, doxycycline, penicillin, and phosphomycin for 2, 8, 16, and 24 h. Samples containing killing activity were identified by using flow cytometry and acridine orange. In 3 min, the effects of immune serum and complement were easily distinguished from the killing of spirochetes by antimicrobial agents by adding fluorescein isothiocyanate-conjugated goat anti-hamster immunoglobulin. This simple procedure greatly enhanced the usefulness of the borreliacidal assay by eliminating a major source of false-positive reactions. The detection of borreliacidal antibody is important for the serodiagnosis of Lyme disease (4, 5). We have shown that the level of borreliacidal antibody increases in sera from patients with Lyme disease with the duration and severity of infection (4). The mean percent killing of Borrelia burgdorferi was 23% for sera from patients with a single erythema migrans lesion, 42% for sera from patients with multiple lesions, 58% for sera from patients with Lyme arthritis of short duration, and 83% for sera from patients with Lyme arthritis of long duration. Furthermore, borreliacidal activity was highly specific (4, 5). Sera from humans not infected with B. burgdorferi and from patients with relapsing fever, Rocky Mountain spotted fever, syphilis, mononucleosis, rheumatoid factor, or DNA antibodies failed to kill the Lyme disease spirochete. Recently, we showed that borreliacidal antibody could be rapidly and accurately detected by flow cytometry (1, 13). Borreliacidal antibody was detected as early as 4 h after the start of incubation of B. burgdorferi organisms with immune serum and complement. By 16 and 24 h of incubation all flow cytometric parameters evaluated were significantly different from those obtained for B. burgdorferi organisms incubated in normal serum and complement. A major limitation of the borreliacidal assay, however, is that serum containing antimicrobial agents can kill B. burgdorferi. Therefore, serum free of antimicrobial agents is required to perform the assay (4, 5). In this study, we demonstrated that borreliacidal activity caused by immune serum and complement can be differentiated by flow cytometry from killing activity caused by antimicrobial agents that are commonly used for the treatment of Lyme disease. * Corresponding author. Mailing address: Wisconsin State Laboratory of Hygiene, 465 Henry Mall, Madison, WI Phone: (68) Fax: (68) MATERIALS AND METHODS Animals. Inbred LSH/Ss Lak hamsters, 6 to 8 weeks old, were obtained from our breeding colony at the Wisconsin State Laboratory of Hygiene (Madison). Hamsters weighing 6 to 12 g were housed three per cage at an ambient temperature of 21 C. Food and water were available ad libitum. Organism. Low-passage B. burgdorferi 297, originally isolated from human spinal fluid (19), was obtained from R. C. Johnson (University of Minnesota, Minneapolis). This strain was cultured once in modified Barbour-Stoenner-Kelly medium (BSK) (3) at 32 C to a concentration of 5 x 17 spirochetes per ml. Five-hundred-microliter samples were dispensed into 1.5-ml screw-cap tubes (Sarstedt, Newton, N.C.) containing 5 pl of BSK supplemented with 3% glycerol (Sigma, St. Louis, Mo.), and then the tubes were sealed and stored in liquid nitrogen. When needed, a tube containing a suspension of B. burgdorferi was thawed and an aliquot was used to inoculate fresh BSK. The culture was incubated at 32 C for 3 days and diluted with fresh BSK to yield 5 x 16 or 5 x 17 spirochetes per ml. Hamster sera. Hamster sera were obtained as previously described (16). Briefly, immune sera were obtained by injecting hamsters subcutaneously in each hind paw with.2 ml of BSK containing 17 viable spirochetes. Three weeks after infection, the hamsters were anesthetied by inhalation of ether from a nose-and-mouth cup and bled by intracardiac puncture. The blood was allowed to clot, and the serum was separated by centrifugation at 5 x g, pooled, dispensed in 1.-ml aliquots into 1.5-ml screw-cap tubes, and froen at - 2 C until used. The pooled serum had a borreliacidal-antibody titer of 1:1,28. Concomitantly, pooled normal serum was obtained from normal uninfected hamsters. Antimicrobial agents. Stock solutions (2,,ug/ml) of ceftriaxone, doxycycline, penicillin, and phosphomycin (Sigma) were prepared in phosphate-buffered saline (PBS) (ph 7.4) and dispensed into 1.5-ml screw-cap tubes, which were then sealed and stored at - 2 C until used.

2 146 LIU ET AL. Borreliacidal assay in the presence of antimicrobial agents. Normal and immune sera were heat inactivated at 56 C for 3 min, diluted 1:1 with fresh BSK, and filter sterilied with a.22-,um-pore-sie filter apparatus (Acrodisc; Gelman Sciences, Ann Arbor, Mich.). A froen suspension of B. burgdorferi was thawed, inoculated into fresh BSK, and incubated at 32 C for 72 h. Spirochetes were enumerated by using dark-field microscopy and a Petroff-Hausser counting chamber. One hundred microliters of the B. burgdorferi culture containing 15 spirochetes per ml was added to round-bottom wells of a 96-well microtiter plate (GIBCO Laboratories, Grand Island, N.Y.). Subsequently, 5 [li of sterile hamster serum, 5 RI of BSK containing various concentrations (.1 to 1,ug/ml) of each antimicrobial agent, and 2,u of sterile guinea pig complement (Sigma) were added to each well of the microtiter plate. The microtiter plates were sealed, shaken gently, and incubated at 32 C for 2, 8, 16, and 24 h. Controls included normal hamster sera with and without antimicrobial agents, B. burgdorferi alone, and heat-killed B. burgdorferi (56 C for 3 min). All assays were performed in duplicate or triplicate. Flow cytometric data acquisition. After each incubation period, 1 RI of each assay suspension was diluted fivefold with PBS (ph 7.4) and 5 RI of acridine orange (AO) (5.4 x 1-' M; Sigma) was added. The diluted samples were then analyed with a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, Calif.) with Lysis II software for data acquisition and analysis. Initially, viable and heat-killed B. burgdorferi organisms were detected and differentiated from BSK and serum particles by forward scatter, side scatter, and AO fluorescence. Data were acquired for spirochetes only and not for BSK and complement particles. Data were acquired for 1 min. Detection of bound anti-b. burgdorferi antibody by flow cytometry. After samples with killing activity were identified, anti-b. burgdorferi antibody bound to B. burgdorferi was detected by setting up samples of B. burgdorferi organisms in the presence or absence of immune serum without complement and antimicrobial agents. After incubation for 3 min at 32 C, 2 RI of fluorescein isothiocyanate (FITC)-conjugated goat anti-hamster immunoglobulin (heavy and light chains; Organon Teknika, Durham, N.C.) was added to yield a final concentration of 1:25. After a second incubation of 3 min at 32 C, 1 RI of each sample was diluted fivefold with PBS (ph 7.4) and analyed by flow cytometry. Forward scatter, side scatter, and FITC fluorescence were used to detect B. burgdorferi organisms and differentiate them from BSK and serum particles. Live gating was performed only on profiles of B. burgdorferi during data acquisition to exclude all BSK particles. Controls included normal serum and FITC-conjugated goat anti-hamster immunoglobulin (heavy and light chains; Organon Teknika). Samples were then analyed by histogram profiles of FITC fluorescence. Gates were established for normal serum and FITC-conjugated goat anti-hamster immunoglobulin controls. Measurements of percent shift in fluorescence and mean channel fluorescence were obtained for all samples and controls. Flow cytometric statistical analysis. Single-parameter analysis of AO fluorescence was performed on assay samples with Lysis II software. Gates were established for viable and heat-killed spirochetes on the basis of their incorporation of AO (1). Two parameters were measured, percent shift in fluorescence and mean channel fluorescence. All measurements correlated. Assessment of viability of B. burgdorferi. To confirm borreliacidal activity by immune serum or antimicrobial agents, assay suspensions containing B. burgdorferi were sterilely O 4 2- CLIN. DIAGN. LAB. IMMUNOL CONCENTRATION (pg/mn) FIG. 1. Effects of different concentrations of ceftriaxone (), doxycycline (C1), penicillin (A), and phosphomycin (-) on uptake of AO by B. burgdorferi. All assay suspensions, including the BSK control (+), were incubated for 24 h before analysis by flow cytometry. sorted with the FACStar flow cytometer (Becton Dickinson Immunocytometry Systems). By using the established gates of histogram profiles of AO-labeled viable and dead spirochetes, B. burgdorferi organisms were sterilely sorted into fresh BSK and incubated at 32 C. Sorted samples were observed weekly for 3 weeks by dark-field microscopy for growth of B. burgdorferi. In other experiments assay suspensions with or without antimicrobial agents were diluted 1,-fold with fresh BSK, incubated at 32 C, and examined weekly for 3 weeks by dark-field microscopy. Statistics. Values obtained were tested by the analysis of variance. The Fisher least-significant-difference test (18) was used to examine pairs of means when a significant F ratio indicated reliable mean differences. The alpha level was set at.5 before the experiments were started. RESULTS In vitro susceptibilities of B. burgdorferi. We showed previously (1) that AO labeled both live and dead B. burgdorferi organisms; however, the intensity of fluorescence was enhanced for dead spirochetes. Figure 1 shows the effects of different concentrations of ceftriaxone, doxycycline, penicillin, and phosphomycin on the uptake of AO by B. burgdorferi organisms. A significant increase (P c.1) in AO uptake by B. burgdorferi organisms occurred at.1,ug/ml or more for ceftriaxone and penicillin compared with AO uptake by spirochetes labeled with AO after incubation with phosphomycin or in drug-free BSK. Doxycycline at 1,ug/ml also enhanced the uptake of AO by spirochetes. At ceftriaxone, penicillin, and doxycycline concentrations of 1,ug/ml or more approximately 95% of the B. burgdorferi organisms were intensively labeled with AO. To confirm that the spirochetes were killed at 1,ug/ml of ceftriaxone, doxycycline, and penicillin, assay suspensions were diluted 1,-fold with fresh BSK, incubated at 32 C, and examined weekly for 3 weeks by dark-field microscopy. No viable spirochetes were detected except in cultures of the BSK control and the medium containing phosphomycin. Kinetics of borreliacidal activity in the presence or absence of antimicrobial agents. Normal and immune sera with complement in the presence or absence of 1,ug of ceftriaxone, doxycycline, penicillin, or phosphomycin per ml were incu-

3 VOL. 1, 1994 DIFFERENTIATION OF BORRELIACIDAL ACTIVITIES E NS O is 7 - * NS+CEF n IS+CEF A NS CONTROL PS=4.5±1.4 MCF=15.4±.1 IS CONTROL PS=49.7±18.8 MCF=93.9± W 4- u rit IL... -ip its, CEF NS PS=2.4±.7 MCF=14.8±.5 CEF IS PS=73.7±5.2 MCF=121.5±8.2 X V Z 1- O- V 7- eh 3 6' PS,5 4- M I1-2 8 NS O IS ONS+PEN IS+PEN A C NS l IS * NS+PHOS IS+PHOS INCUBATION TIME (HOURS) FIG. 2. Effects of normal sera (NS) and immune sera (IS) and complement in the presence or absence of ceftriaxone (CEF) (A), doxycycline (DOX) (B), penicillin (PEN) (C), and phosphomycin (PHOS) (D) on the uptake of AO by B. burgdorferi. Assay suspensions were incubated for 2, 8, 16, and 24 h before analysis by flow cytometry. Results are means ± the standard errors of the mean. bated with B. burgdorferi organisms for 2, 8, 16, and 24 h. In general, B. burgdorferi organisms incubated for 8 h or more in normal or immune sera in the presence of ceftriaxone, doxycycline, or penicillin had a significant increase (P c.5) in uptake of AO compared with spirochetes incubated in normal serum without antimicrobial agents (Fig. 2). In addition, the amount of AO uptake by B. burgdorferi increased after 16 and 24 h of incubation. The presence of ceftriaxone, doxycycline, and penicillin also enhanced the uptake of AO by B. burgdorferi organisms incubated in immune serum and complement. By contrast, phosphomycin had no effect on the uptake of AO by B. burgdorferi organisms in normal or immune sera. Detection of anti-b. burgdorferi antibody in the presence of antimicrobial agents. The purpose of this experiment was to determine if anti-b. burgdorferi antibody could be detected in the presence of antimicrobial agents. Normal and immune sera without complement in the presence or absence of 1,ug each of ceftriaxone, doxycycline, penicillin, and phosphomycin per ml were incubated with B. burgdorferi organisms for 3 min. FITC-conjugated goat anti-hamster immunoglobulin was then added to the assay suspensions and they were incubated for another 3 min. Figure 3 shows that anti-b. burgdorferi antibodies were readily detected in the presence of ceftriaxone, D u: E-4 _n LI IO= m1*1 l, ~~DOX NS PS=1.9_+1.2 MCF=14.8±O.5 PEN NS MCF=16.4±1.1-1 A11'd1It Ito tui a PHOS NS PS=3.3i.2 MCF=15.2±.1 I4L Ulle- --jt -l M.. S.._1.33_u FITC FLUORESCENCE E1... DOX IS PS=71.4±2.9 MCF=124.±IO.7 PEN IS PS=77.±8.5 MCF=12.3±7. PHOS IS PS=7.9±2.4 MCF=16.9±6.4 FIG. 3. Histogram profiles of FITC-conjugated goat anti-hamster immunoglobulin-labeled B. burgdorferi organisms in normal sera (NS) or immune sera (IS) in the presence or absence of ceftriaxone (CEF), doxycycline (DOX), penicillin (PEN), and phosphomycin (PHOS). Two values were obtained, the percent shift in fluorescence (PS) and the mean channel fluorescence (MCF). REsults are means ± the standard errors of the mean. doxycycline, penicillin, or phosphomycin. By contrast, no anti-b. burgdorferi antibodies were detected in normal serum with or without antimicrobial agents, even though normal serum containing ceftriaxone, doxycycline, and penicillin caused an increase in uptake of AO by spirochetes (Fig. 2). Figure 3 also shows that the percent shift in fluorescence and the mean channel fluorescence remained relatively constant in normal serum with or without antimicrobial agents, while these values were significantly (P s.5) increased in immune serum in the presence or absence of antimicrobial agents. DISCUSSION This paper confirms and extends our previous findings (1) that flow cytometry and the use of AO-labeled spirochetes provide a rapid means for the detection of borreliacidal antibody. Lim et al. (1) showed that borreliacidal antibody can be detected as early as 4 h (with optimal detection at 16 to 24 h) after the start of incubation of B. burgdorferi organisms with immune serum and complement. In this study we showed that borreliacidal activity caused by the effects of immune serum and complement can easily be distinguished from the killing of spirochetes by antimicrobial agents.

4 148 LIU ET AL. In this study a major limitation of the borreliacidal assay was removed. When assay suspensions containing normal or immune sera were incubated with B. burgdorferi in the presence of antimicrobial agents and then incubated with FITC-conjugated goat anti-hamster immunoglobulin, borreliacidal activity due to antimicrobial agents or immune factors could be rapidly determined. Although it is uncommon, sera containing antimicrobial agents are received for Lyme disease serology. When the borreliacidal assay is performed with these sera, falsepositive results can occur. With FITC-conjugated goat antihamster immunoglobulin serum, spirochetes labeled with anti-b. burgdorferi antibodies can be detected rapidly, i.e., within 6 min. To prevent false-positive reactions due to naturally occurring cross-reactive antibodies, normal and immune sera were diluted 2-fold before testing. Similar studies are now being performed with case-defined human Lyme disease sera, normal sera from regions where B. burgdorferi is endemic, potentially cross-reactive sera, and sera from individuals not infected with B. burgdorferi. In addition, the effects of other substances or medications on borreliacidal antibody will be determined. These studies should further clarify the usefulness of the borreliacidal assay for the detection of human Lyme disease. The detection of borreliacidal antibody is important for determining the serodiagnosis of Lyme disease. Callister et al. (4, 5) showed that humans infected with B. burgdorferi develop borreliacidal antibodies. The levels of borreliacidal antibody in sera from patients with Lyme disease increased with the severity and duration of infection. The lowest levels of borreliacidal antibody were detected in patients with early disease, while the highest levels of borreliacidal antibody were detected in patients with chronic disease. Furthermore, detection of borreliacidal activity was specific for B. burgdorferi (4, 5), unlike detection in other serodiagnostic tests for Lyme disease (2, 7, 8, 11). Sera from patients with relapsing fever, Rocky Mountain spotted fever, syphilis, mononucleosis, rheumatoid factor, or DNA antibodies failed to kill B. burgdorferi organisms. Borreliacidal antibody is also important for the determination of immune status. In a study by Schmit et al. (17), borreliacidal activity was detected 7 days after infection of hamsters with B. burgdorferi, peaked at weeks 3 to 5, and thereafter decreased. The borreliacidal activity correlated with passive protection against challenge with B. burgdorferi (17). Schaible et al. (14, 15) and others (6) also demonstrated that immune serum and antibodies to OspA and OspB have protective capabilities. Recently, Philipp et al. (12) demonstrated that rhesus monkeys develop antibodies capable of killing B. burgdorferi. These studies (3, 12, 14, 15, 17) and those on the serodiagnosis of Lyme disease (4, 5) suggest that the production of borreliacidal antibody is an important response of humans and animals to infection with B. burgdorferi. Its role in protection and serodiagnosis is becoming clearer. These studies should also provide a more rapid means to assay the ability of different or new antimicrobial agents to kill B. burgdorferi. Presently, antimicrobial assays require several days of incubation and then subculturing in fresh BSK for determination of the MBC or MIC of the antimicrobial agent (1, 9). Our results and those of Lim et al. (1) show that borreliacidal activity can be detected between 4 and 24 h after the start of incubation of B. burgdorferi with immune serum or antimicrobial agents. The distinct difference in AO label ing between live and dead spirochetes detected by flow cytometry eliminates the time necessary for long incubation periods, subculturing, and reading of results by dark-field microscopy. CLIN. DIAGN. LAB. IMMUNOL. In summary, these studies have demonstrated that flow cytometry is a rapid and accurate means for distinguishing borreliacidal activity caused by immune serum and complement and activity caused by antimicrobial agents. ACKNOWLEDGMENTS This work was supported by funds from Public Health Service grants AI and AI-3736 from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. Agger, W. A., S. M. Callister, and D. A. Jobe In vitro susceptibilities of Borrelia burgdorferi to five oral cephalosporins and ceftriaxone. Antimicrob. Agents Chemother. 36: Bakken, L. L., K. L. Case, S. M. Callister, N. J. Bourdeau, and R. F. 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