Study of BSK II and BSK H Media for Culturing Lyme Disease Spirochetes Yelena Polovinchik Department of Environmental Science University of California at Berkeley Abstract Borrelia burgdorferi the causative agent of Lyme disease can be grown in vitro in Barbour Stoenner Kelly BSK II and modified Barbour Stoenner Kelly BSK H growth media Two growth media BSK II and BSK H were compared with respect to their ability to support the growth of three Borrelia isolates B burgdorferi sensu stricto CA4 B bissettii sp nov CA389 and B corieaceae CA435 The growth kinetics of high passage P10 and low passage P7 B bissettii CA389 isolates were compared to determine if adaptation to cultural media facilitates faster growth of the isolates In addition effects of gelatin on the bacterial growth were studied All isolates tested grew significantly faster and appeared healthier in BSK H media The replication rate of B bissettii correlated with the number of passages in the cultural media The results of this study also suggested that gelatin inhibits growth of B burgdorferi CA4 and B bissettii CA389 in BSK H and BSK II media and has no significant effect on the growth rate of B corieaceae CA435 in BSK H Introduction With human population expansion and encroachment into wildlife habitats people have entered a dangerous enzootic cycle causing them to become frequent accidental hosts for ticks and the disease agents they carry including the bacterium Borrelia burgdorferi the agent of Lyme disease Daszak et al 2000 Consequently Lyme borreliosis has become the most frequently reported arthropod born human infection in Europe and North America Stanek et al 1993 As of 1995 cases of Lyme disease had been reported in 48 of the 50 states and appear to be increasing both in number of people affected and in geographic distribution Figure 1 Ostfeld 1997 Lyme disease is transmitted by several tick species and caused by spirochetes from the general taxon Borrelia burgdorferi sensu lato Postic et al 1998 At least three genospecies including B burgdorferi sensu stricto B garinii and B afzelii are known to be associated with human disease in Europe Baranton et al 1992 In the United States B burgdorferi sensu stricto has been the only genospecie known to cause Lyme borreliosis in the past however recent studies have shown substantial heterogeneity among Californian and other Northern American isolates Mathiesen et al 1997 Figure 1 Lyme disease prevalence United States 1998 CDC 1999 Using the nucleotide sequence analysis of ribosomal gene from 19 atypical strains isolated in the US Postic et al 1998 identified a new specie B bissettii sp nov and a cluster of other related but distinct strains which require further characterization e g the disease status of these isolates is unknown Borrelia corieaceae isolate is an example of an uncharacterized strain that was isolated from the Ornithodoros corieaceaous tick and may be more closely related to the relapsing fever pathogen than Lyme disease spirochetes The ability to isolate and culture the etiological agent of Lyme disease is essential in both research and clinical environments For instance the ability to produce a viable culture of B burgdorferi in vitro has facilitated production of spirochetes antigens which allow detection of Lyme pathogens in clinical specimens Steere et al 1983 In addition isolation of the pathogen from infected animals and ticks has been effective for defining endemic Lyme disease areas Anderson et al 1985 and 1989 Callister 1988 Attempts to culture Borrelia go back to the early 20th century when Kligler and Robertson 1922 defined the conditions for maintenance and growth of the relapsing fever pathogen in derivatives of Noguchi s medium Today variations of the complex BarbourStoenner Kelly BSK growth media are traditionally used for in vitro cultivation of Borrelia spirochetes Barbour 1984 Norris et al 1997 Picken et al 1997 BSK II and standardized BSK H are two media of interest in the following study Barbour 1984 Pollack et al 1993 Since BSK II medium is not available commercially it must be prepared in the individual laboratories from several components Consequently batch variations in media can influence growth kinetics morphology and antigenic characteristics of Lyme disease spirochetes Callister et al 1990 The latter medium BSK H was developed in order to create a source of readily available standardized medium Table 1 describes the specific differences in the formulation of two media Although both BSK II and BSK H media are routinely used to grow B burgdorferi in vitro each medium shows some selectivity for specific isolates Kleinjan 1999 pers comm The purpose of this study was to compare two growth media BSK II and BSK H based on their ability to support the growth of three Borrelia isolates B burgdorferi sensu stricto B bissettii sp nov and B corieaceae isolate and to compare the growth kinetics of high passage P10 and low passage P7 B bissettii CA389 isolates in order to evaluate if adaptation to cultural media facilitates faster growth in the isolates In addition the effects of increased media viscosity on bacterial growth via addition of gelatin to the media were studied BSK H Ingredient BSK II CMRL mg Ingredients mg Arginine 57 Arginine 70 Citric acid tetrasodium 700 Sodium citrate 800 2 Deoxycytidine HCL 11 6 2 Deoxycytidine 10 Flavin adenine dinucleotide 0 106 Flavin adenine dinucleotide 1 0 Gelatin 0 Gelatin 14000 Magnesium Sulfate 97 69 Magnesium Sulfate 200 Sodium Acetate 50 Sodium Acetate 83 Sodium Glucaronate 3 88 Sodium Glucaronate 4 2 Sodium Phosphate monobasic 122 Sodium Phosphate monobasic 140 BSK H contains endotoxin tested tissue culture grade water Table 1 Comparison of ingredients that differ between BSK H and BSK II media Materials and Methods Bacterial strains and Growth Kinetics The designations passage levels and origins of the Borrelia strains used in this study are given in Table 2 In order to evaluate the growth supporting capabilities of BSK II and BSK H media four replicate cultures of each Borrelia isolates were set up in both media and incubated for ten days To culture the spirochetes 0 5ml of isolates in glycerol stock solution were added to 5 ml of each medium in a sterile culture tube and sealed to maintain the gas balance The tubes were incubated in the dark at 34 C The spirochetes were sampled a total of five times once every other day during the ten day incubation period Each sample was taken
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