INFECTION AND IMMUNITY, Aug. 1992, p. 3224-3230 0019-9567/92/083224-07$02.00/0 Copyright ©3 1992, American Society for Microbiology

Vol. 60, No. 8

Hemolytic Activity of Borrelia burgdorferi LISA R. WILLIAMS AND FAYE E. AUSTIN*

Department of Microbiology and Immunology, School of Medicine, University of Louisville, Louisville, Kentucky 40292 Received 12 February 1992/Accepted 29 May 1992

Zones of beta-hemolysis occurred around colonies of Borrelia burgdorferi grown on Barbour-Stoenner-Kelly medium containing agarose and horse blood. Blood plates were inoculated with either the infective strain Sh-2-82 or noninfective strain B-31 in an overlay and incubated in a candle jar. Both strains of B. burgdorferi displayed beta-hemolysis after 1 to 2 weeks of incubation. The hemolytic activity diffused out from the borrelial colonies, eventually resulting in lysis of the entire blood plate. Hemolysis was most pronounced with horse blood and was less intense with bovine, sheep, and rabbit blood. Hemolysis was enhanced by hot-cold incubation, which is typical of phospholipase-like activities in other bacteria. Further characterization of the borrelial hemolysin by using a spectrophotometric assay revealed its presence in the supernatant fluids of stationary-phase cultures. Detection of the borrelial hemolytic activity was dependent on activation of the hemolysin by the reducing agent cysteine. This study provides the first evidence of hemolytic activity associated with B. burgdorferi. The hemolytic activity of spirochetes has been associated with members of the genera Leptospira and Serpula. Both pathogenic Leptospira interrogans and free-living L. biflexa are beta-hemolytic (1, 9, 23). Leptospiral hemolysins have been identified as the enzymes phospholipase A in L. biflexa and L. interrogans (8, 12) and sphingomyelinase C in L. interrogans (6, 10, 25, 33). In the genus Serpula, the spirochete which causes swine dysentery, Serpula hyodysenteriae (formerly classified as Treponema hyodysenteriae [28]), is strongly beta-hemolytic when grown on blood agar under anaerobic conditions (16). Serpula innocens, an intestinal spirochete of nondysentery origin, is weakly beta-hemolytic (15). The observed hemolysis by Serpula hyodysentenae is due to an oxygen-stable hemolysin which is produced only in the presence of a carrier molecule such as sodium ribonucleate or RNA-core (14, 17, 20, 22). There have been no reports of hemolytic activity in spirochetes of the genus Borrelia, in contrast to those of the genera Leptospira and Serpula. Borrelia burgdorfien is the causative agent of Lyme disease, a systemic inflammatory disease with symptoms ranging from erythema migrans to neurologic and cardiac abnormalities and migratory arthritis. While B. burgdorferi may be cultured readily in modified Barbour-Stoenner-Kelly (BSK-II) medium in microaerophilic conditions in vitro (2), very little is known about its growth requirements or physiological characteristics. Colony formation has been detected when the spirochete has been plated on BSK-II medium solidified with agarose (19). In this study, the ability of B. burgdorferi to grow in BSK-II medium with blood was investigated. The present paper gives the first description of hemolytic activity associated with B. burgdorfen.

Upon the receipt of Sh-2-82 by this laboratory, it was passaged one time and then frozen with 20% glycerol in liquid nitrogen. New cultures were initiated from the frozen stock. B. burgdorfen type strain B-31 was obtained from C. D. Cox, University of Massachusetts, Amherst. This strain has been maintained in our laboratory for several years. Staphylococcus aureus, Enterococcus durans, and Escherichia coli HB101 were part of the laboratory stocks. Culture media. For the detection of hemolytic activity on plates, a solid medium containing 5% blood and 1% agarose was prepared by a modification of the method of Kurtti et al. (19). Briefly, 7% agarose (SeaPlaque, low gelling and melting temperature; FMC Bioproducts, Rockland, Maine) in Milli-Q water (resistivity, 18 megohm/cm; Millipore Corp., Bedford, Mass.) was sterilized by autoclaving. The agarose solution was kept molten at 43°C. To 7 ml of agarose were added 83 ml of warm BSK-II medium without rabbit serum, 5 ml of heat-inactivated rabbit serum (Pel-Freez Biologicals, Rogers, Ark.), and 5 ml of either defibrinated animal blood (Cleveland Scientific, Bath, Ohio) or citrated human blood. Aged blood tended to give variable results; therefore, blood was always used within 1 week of being collected. After mixing, 18 ml was dispensed to polystyrene petri dishes (100 by 15 mm). The plates were incubated at 34°C overnight to allow them to dry before use. For the detection of hemolytic activity in culture supernatant fluids, it was necessary to remove phenol red from BSK-II medium because the dye interfered with spectrophotometric assays. Therefore, borreliae were grown in BSK-A medium, which is a modification of BSK-II medium that has a salts solution substituted for the CMRL 1066 medium. The final concentrations of salts in BSK-A medium were 97 mM NaCl, 4.5 mM KCl, 1.5 mM CaCl2- 2H20, 0.8 mM NaH2PO4. H20, and 0.7 mM MgSO4. 7H20. Borreliae were grown routinely in 10 ml of BSK-II or BSK-A medium in screw-cap culture tubes (16 by 125 mm). The tubes were sealed under ambient oxygen concentrations and incubated statically at 34°C. All other bacteria were grown in Trypticase soy broth at 37°C. Cultivation on blood plates. Exponentially growing borreliae from liquid cultures in BSK-II medium were enumerated

MATERIALS AND METHODS Bacterial strains. B. burgdorferi Sh-2-82 was obtained from Warren J. Simpson, Rocky Mountain Laboratories, Hamilton, Mont. This strain had been isolated from Ixodes dammini ticks and passaged fewer than 10 times in vitro. *

Corresponding author. 3224

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FIG. 1. Time course of hemolytic activity of B. burgdorferi Sh-2-82 on BSK-II medium with 1% agarose and 5% horse blood. Plates were incubated in a candle jar at 34°C and photographed at 8 (A), 12 (B), and 15 (C) days.

in a Petroff-Hausser counting chamber by using dark-field microscopy. Cultures were diluted in BSK-II medium to a cell density of 2 x 103 borreliae per ml. A sample (0.1 ml) of the dilution was mixed with 8 ml of the blood-agarose medium described above and poured onto the surface of each plate. After the overlay solidified, each plate was inverted and incubated in a candle jar at 34°C. After 1 to 3 weeks of incubation, the plates were examined for the presence of colonies and hemolysis. The presence of borreliae was verified by dark-field examination of colonies which had been picked with sterile Pasteur pipettes and mixed with BSK-II medium to disperse the agarose. Efficiency of plating was calculated by dividing the average number of colonies visible on a plate by the number of borreliae in the initial inoculum. Plates were inoculated in triplicate in all experiments. Photography of blood plates. Plates were placed on a tungsten (600-W) transilluminating light source within a circle cut in black construction paper. Thus, a black background for photographs was achieved while the plates were illuminated from below. Photographs were taken on Kodak TMAX 100 film and printed on Kodak Polycontrast III paper. Spectrophotometric assay of hemolysis. At various times of incubation, cultures of borreliae grown in BSK-A medium were centrifuged at 10,000 x g for 20 min at 4°C. Culture supernatant fluids were collected aseptically and then stored at -20°C until analyzed. For analysis, 30 ,ul of 1 M cysteine hydrochloride was added to 1 ml of culture supernatant fluid. The mixture was incubated for 30 min at room temperature. Horse erythrocytes were washed three to four times in phosphate-buffered saline (PBS) containing 0.68 mM CaCl2. 2H20, 0.49 mM MgCl2- 6H20, and 5.0 mM glucose. Then, 0.5 ml of a 5% suspension of erythrocytes was added to an equal volume of culture supernatant fluid in small screw-cap vials. The final concentrations in this reaction mixture were 2.5% erythrocytes and 15 mM cysteine. The reaction mixture was incubated for 2 to 3.5 h in a water bath at 37°C with frequent inversion of the vials. The reaction was stopped by centrifugation at 500 x g for 5 min at room temperature. The released hemoglobin was measured at 540 nm in a Beckman DU-50 spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.). Reaction mixtures with optical density (OD) readings greater than 1 were diluted 1:5 in PBS. The readings were corrected for turbidity of the culture supernatant fluid

as well as for nonspecific hemolysis due to BSK-A medium alone. Total lysis was achieved by the addition of 0.02% saponin (wtlvol) to the reaction mixture. All reactions were performed in duplicate.

RESULTS A representative experiment on the cultivation of B. burgdorferi Sh-2-82 on horse blood is presented in Fig. 1. Borrelial colonies were visible in the overlay with the unaided eye after 8 days (range, 7 to 11 days) of incubation in a candle jar at 340C (Fig. 1A). At this time, an area of greening or incomplete hemolysis was evident at the leading edges of borrelial colonies. Clear zones of beta-hemolysis became apparent over and around the colonies upon further incubation (Fig. 1B). The clearing over the borrelial colonies made it difficult to see the center of the colonies, which were less dense at early time points. Within 13 to 15 days of incubation, the beta-hemolytic zones began to coalesce (Fig. 1C and 2A), and eventually, hemolysis of the entire plate occurred. The observed diffusion of hemolytic activity out from the colonies suggested the release of a soluble borrelial hemolysin into the medium. Control experiments with beta-hemolytic strains of Staphylococcus aureus and E. durans and the nonhemolytic strain HB101 of E. coli were performed. When these bacteria were grown for 24 h in the same medium as B. burgdorferi in a candle jar at 34°C, they exhibited the characteristic patterns of hemolysis (data not shown). Therefore, the observed hemolysis in Fig. 1 was due specifically to the growth of B. burgdorfen and not to the unique plating conditions or media. Strain Sh-2-82 was known to be infective when tested in rodent infection models, including the white-footed mouse, Peromyscus leucopus (24), and neonatal Lewis rats (la). Therefore, it became important to determine whether the observed hemolytic activity was characteristic of both infective and noninfective strains of B. burgdorferi. Available in the laboratory was B. burgdorfeni type strain B-31, which had been passaged extensively in vitro and was noninfective in neonatal Lewis rats (la). As shown in Fig. 2B, strain B-31 on horse blood also exhibited beta-hemolysis which diffused out from the colonies. The hemolytic activity of B-31 appeared and progressed more slowly than that of Sh-2-82 (Fig. 2A). The plating efficiencies of Sh-2-82 and B-31 on horse blood were 59 ± 6 and 64 ± 6%, respectively. These values




FIG. 2. Hemolytic activity of B. burgdorfen Sh-2-82 and B-31 grown on BSK-II medium with 1% agarose and either 5% horse blood (A and B, respectively) or 5% bovine blood (C and D, respectively). Plates were incubated in a candle jar at 34°C and photographed at 13 days for Sh-2-82 and at 22 days for B-31.

represent the mean plating efficiency + standard error; nine experiments with Sh-2-82 and 10 experiments with B-31 were performed. While both Sh-2-82 and B-31 produced discrete colonies on horse blood, the colonies appeared to be of different sizes and shapes. Both small, compact colonies and large, diffuse colonies were evident primarily with B-31 (Fig. 2B). Subculturing of either colony type in liquid BSK-II medium gave rise to colonies of all types when they were replated on solid medium. This observation suggested that colony morphology is not a stable phenotype. Similar observations had been reported previously for B-31 grown on solid BSK-II medium in the absence of blood (19). Dark-field examination of

material removed from the different colony types revealed typical spirochetes. No differences in hemolysis were noted for the different colony types. Four colonies of Sh-2-82 and six colonies of B-31 were picked at random with sterile Pasteur pipettes and inoculated into liquid BSK-II medium. The resulting single-colony clones were examined in order to determine their hemolytic phenotypes on horse blood. All clones produced hemolysis similar to that of the parental strain (data not shown). The growth and the hemolytic activity of B. burgdorferi on blood from other animal species were determined. A comparison of borrelial growth on horse and bovine blood plates showed similar plating efficiencies but smaller zones of

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beta-hemolysis on bovine blood for Sh-2-82 (Fig. 2C) and B-31 (Fig. 2D). On sheep and rabbit blood plates, tiny zones of beta-hemolysis were observed for both Sh-2-82 and B-31 after 10 to 16 days of incubation (data not shown). These results indicated that the borrelial hemolysin was most active against horse erythrocytes, followed by bovine, sheep, and rabbit erythrocytes in order of decreasing sensitivity. In similar experiments with human blood, colonies of B-31 were observed after 3 to 4 weeks of incubation. Pinpoint zones of beta-hemolysis around the colonies were detected on human blood of groups A positive, B positive, and 0 positive (data not shown). Sh-2-82 did not grow on human blood under the same experimental conditions. The lack of growth of Sh-2-82 was not related to the source of blood used, since negative results were obtained with human blood from different donors as well as with blood pooled from a number of healthy donors. The sensitivity of human erythrocytes to the borrelial hemolysin cannot yet be determined because of the slow growth of B-31 and the lack of growth of Sh-2-82. Hemolytic activity that was potentiated by hot and then cold incubation had been reported previously for Leptospira strains (3, 23, 27). In order to test this possibility with B. burgdorfeni, blood plates were incubated at 34°C until zones of hemolysis appeared and then were transferred to the cold (4°C) overnight. As shown in Fig. 3, the size of the hemolytic zones of Sh-2-82 on bovine blood increased following the cold incubation. The size of the colonies did not appear to change. Similar results were obtained for Sh-2-82 grown on the blood of other animal species as well as for B-31 (data not shown). The control bacteria were Staphylococcus aureus and E. durans, which had been plated on Trypticase soy agar with 5% blood to confirm the presence of a hemolysin that was cold enhanced or not cold enhanced, respectively. When tested under the same conditions as B. burgdorferi, these bacteria exhibited those patterns of enhancement (data not shown). This control experiment confirmed that cold incubation specifically increased the zones of hemolysis around the borrelial colonies. This hot-cold enhancement of hemolytic activity suggested that the action of the hemolysin on the erythrocyte membrane at 34°C rendered the erythrocytes sensitive to lysis upon shifts in temperature. Further characterization of the borrelial hemolytic activity involved the development of a spectrophotometric assay to monitor the release of any soluble hemolysin into the growth medium. For these experiments, culture supernatant fluids were collected from borreliae grown in BSK-A medium. This medium was used because BSK-II contains phenol red, which interfered with spectrophotometric measurements. A representative growth curve of Sh-2-82 in BSK-A medium at 34°C is shown in Fig. 4. The average doubling time for the borreliae was 18 h, with a total cell yield of approximately 2 x 108 cells per ml. The cells reached stationary phase within 5 to 6 days of incubation. At this time, low levels of hemolytic activity were detected in culture supernatant fluids, as indicated by the increase in OD540. Upon further incubation of the borreliae, the level of hemolytic activity continued to increase and reached a maximum by day 10. These results suggested that the borreliae continuously produce and release a hemolysin, resulting in its accumulation in the culture medium over time. In the assay system, total lysis was achieved by the addition of 0.02% saponin to the reaction mixture. The observed maximal hemolysis in Fig. 4 represented approximately 45% of total lysis with 2.5% horse erythrocytes after


3.5 h of incubation at 37°C. In order to determine the optimal concentration of 2.5% horse erythrocytes, a range of concentrations, including 0.5, 1, 2.5, and 5%, had been tested with culture supernatant fluids collected on day 10 of incubation. Concentrations of 0.5 and 1% erythrocytes were limiting in the assay, while a concentration of 5% did not increase the sensitivity of the system. Similar experiments had been previously carried out in order to determine the optimal temperature of the reaction. While incubation at 0 and 25°C yielded no hemolytic activity, incubation at 37°C yielded levels that were fivefold higher than those observed at 34°C (data not shown). In this spectrophotometric assay, preliminary experiments using culture supernatant fluids had yielded consistently negative results, which conflicted with the evident diffusion of hemolysis across the plates. Oxidation of the hemolysin may have occurred during the manipulation of culture supernatant fluids. In order to reverse any oxidation, reducing agents at various concentrations were added to culture supernatant fluids of Sh-2-82 and incubated for 30 min at room temperature prior to the addition of erythrocytes. As shown in Table 1, no hemolytic activity was detected in the presence of P-mercaptoethanol and sodium thioglycolate. In contrast, hemolytic activity was observed in the presence of cysteine hydrochloride, which indicated that cysteine activated the hemolysin. With increasing concentrations of cysteine, there was a dramatic increase in hemolytic activity. A final concentration of 15 mM cysteine resulted in maximal activation, whereas concentrations greater than 15 mM resulted in the formation of a precipitate due to the reaction of cysteine with the released hemoglobin. These results indicated that cysteine is the most effective reducing agent in the activation of the borrelial hemolysin.

DISCUSSION Previous studies showed that B. burgdorferi formed colonies when plated onto BSK-II medium with agarose and incubated in a candle jar (7, 19). In these studies, the method of inoculation involved spreading a spirochetal suspension on the surface of the agarose medium. Plating efficiencies were highly variable, ranging from less than 1% (7) to greater than 100% (19). In the present study, blood plates were inoculated with B. burgdorferi suspended in an agarose overlay. The agarose used had a low gelling and melting temperature, so that spirochetes (and erythrocytes) were not exposed to the high temperatures required to keep standard agarose in a molten state. Well-isolated macroscopic colonies and reproducible plating efficiencies of approximately 60% were obtained with this method. Similar plating efficiencies had been obtained in preliminary experiments in which BSK-II medium was not supplemented with blood. Therefore, the improved plating efficiencies may be related directly to the inoculation of spirochetes within the agarose rather than on the surface. Since borreliae are microaerophilic, the agarose overlay probably reduces the diffusion of oxygen into the medium, which favors the viability of the spirochetes. A search of the literature has shown that this is the first report describing the hemolytic activity of B. burgdorferi. Plating experiments demonstrated that horse erythrocytes were most sensitive to lysis, while rabbit erythrocytes were relatively resistant. Erythrocytes from different animal species have been shown to vary in sensitivity to the hemolysins of other bacteria, including L. interrogans (23), Staphylococcus aureus (4), and certain Vibrio species (11, 18, 32, 34).




FIG. 3. Cold enhancement of hemolytic activity of B. burgdorferi. Strain Sh-2-82 was grown on BSK-II medium with 1% agarose and 5% bovine blood in a candle jar at 34WC for 11 days. The plate was photographed and transferred to the cold (4°C) overnight. The results shown are before (A) and after (B) incubation at 4°C.

With B. burgdorfen, the basis of the spectrum of erythrocyte sensitivity is not yet known. The borrelial hemolysin may bind to a specific receptor or act on a specific molecule in the erythrocyte membrane. The membranes of horse erythrocytes may contain a proportionally large amount of the target molecule, while the membranes of rabbit erythrocytes contain very little. Another characteristic of the hemolysin of B. burgdorfeni

is enhancement by hot and then cold incubation (Fig. 3). A similar enhancement of hemolytic activity has been described for the hemolysins of L. interrogans (3, 23, 27), the beta-hemolysin of Staphylococcus aureus (26), and the alpha-toxin of Clostridium perfringens (30). In all of these bacteria, the hemolysins are phospholipase A and/or phospholipase C (sphingomyelinase C). Therefore, the observed cold enhancement of hemolytic activity of B. burgdorfeni


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of incubation. The level of hemolysin necessary for detection cannot be determined until it is purified from the protein-rich medium. Once the hemolysin is purified, then specific probes will be developed in order to determine when it is synthesized during the growth of B. burgdorfen. The detection of hemolytic activity in the spectrophotometric assay required pretreatment of culture supernatant cccr a.2W0 fluids with a reducing agent prior to incubation with erythCD) rocytes. This observation suggested that the hemolysin may ~~~~~~~0 have been inactivated by oxidation. Such inactivation may 107 0 ~~~~~~~~~~1.2 not have occurred on plates because they were incubated in X a candle jar, which reduced the oxygen tension in the environment. The microaerophilic nature of B. burgdorfen as well as the requirement of a reducing agent for the detection of activity suggest that this organism synthesizes 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 an oxygen-labile hemolysin. Other oxygen-labile hemolDAYS OF INCUBATION ysins, such as streptolysin 0, are known to be activated by FIG. 4. Detection of hemolytic activity in stationary-phase culvarious reducing agents. The concentrations of reducing tures of B. burgdorferi. Strain Sh-2-82 was grown in BSK-A medium agents and the conditions for activation in Table 1 were at 34WC and enumerated by direct cell counts (- [cells per ml]), using similar to those described for streptolysin 0 (5). Of cysteine, dark-field microscopy. Culture supernatant fluids were activated by 1-mercaptoethanol, and sodium thioglycolate, only cysteine using 30 mM cysteine, and then they were incubated with 2.5% activated the borrelial hemolysin under these conditions. horse erythrocytes for 3.5 h at 37°C. Hemolysis (-) was determined by OD540 readings. However, 3-mercaptoethanol, sodium thioglycolate, and other reducing agents may also be effective under different activation conditions. The observed activation by cysteine suggests the involvement of a similar type of enzyme. suggests the presence of a sulfhydryl group that may be However, the involvement of other enzymes such as proteessential for the activity of the borrelial hemolysin. In other ases cannot be eliminated on the basis of this observation. oxygen-labile hemolysins, including streptolysin 0 (13), Data from both plating experiments and spectrophotometpneumolysin (31), perfringolysin 0 (29), and listeriolysin 0 ric assays indicated that B. burgdorfen produces and re(21), the essential sulfhydryl group is contained in a single leases a soluble hemolysin into the culture medium. Hemocysteine residue near the carboxy-terminal region of the lytic activity was detected only in stationary-phase cultures. molecule. On the basis of the observations with cysteine One possibility suggested by this observation is that the activation, the borrelial hemolysin may also contain a cysborreliae synthesize the hemolysin only during stationary teine residue that must be in a reduced state for lytic activity. phase. Another possibility is that the hemolysin must reach The hemolytic activity of B. burgdorferi is associated with a certain concentration in order to be detected in culture both infective strain Sh-2-82 and noninfective strain B-31 supernatant fluids. In this case, the hemolysin may be and with single-colony clones of each of these strains. synthesized continuously throughout growth, yet the hemoDifferent colony types were detected on the blood plates, but lysin is detected only after its accumulation in the medium. there was no correlation between colony phenotype and This latter possibility seems most likely, because B. burghemolytic activity. Since both Sh-2-82 and B-31 are hemodorfeni has a slow doubling time in BSK-A medium and does lytic, it is difficult to assess the role of the hemolysin of B. ml until after 5 to 6 not reach a density of 108 cells per days burgdorferi in the pathogenesis of Lyme disease on the basis of existing information. In swine-dysentery spirochetes, beta-hemolysis has been used in order to distinguish Serpula TABLE 1. Cysteine activation of Borrelia burgdorfeni hemolytic hyodysenteriae from nonpathogenic, intestinal spirochetes. activity in culture supernatant fluids In contrast, both free-living L. biflexa strains and some Hemolysis (OD540) pathogenic L. interrogans strains are beta-hemolytic. HowFinal concn Sodium (mM) Cysteine ever, they differ in their complements of hemolytic enzymes, ,-Mercaptoethanol hydrochloride thioglycolate with L. bifle-xa possessing only phospholipase A and L. interrogans possessing both phospholipase A and sphingo0 0 0 0 0 NDb 5.0 0.08 ± .03a myelinase C (12). It has been speculated previously that ND 10.0 0.26 ± .04 ND these enzymes that are involved in the alteration of erythro0.33 ± .02 ND ND 12.5 cyte membranes may also function to degrade phospholipids ± 0 15.0 1.88 .52 0 of other kinds of cells and thus contribute to the pathological 0 25.0 0 pptc changes seen in leptospirosis. As with Leptospira strains, it ND 0 35.0 0 is possible that infective and noninfective strains of B. ND 0 50.0 0 burgdorferi produce different hemolysins. Furthermore, a Mean OD ± standard deviation of hemolysis obtained with culture pathological changes may occur only in tissues in which the supernatant fluid incubated with 2.5% horse erythrocytes at 37'C for 2 h; three oxygen-labile hemolysin is in a reduced active form. Further experiments for cysteine hydrochloride and two experiments for 1-mercaptoethanol and sodium thioglycolate were performed. Reaction mixtures with study of the nature and activity of the hemolysin of B. OD readings of greater than 1 were diluted 1:5. burgdorferi and its mechanism of release from the cell may b Not determined. provide a better understanding of the factors which contribc Precipitate formed because of the reaction of cysteine with the released ute to the pathogenesis of Lyme disease. hemoglobin. Cl)









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Hemolytic activity of Borrelia burgdorferi.

Zones of beta-hemolysis occurred around colonies of Borrelia burgdorferi grown on Barbour-Stoenner-Kelly medium containing agarose and horse blood. Bl...
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