Rev Cubana Med Trop 1996;48(1)

Departament of Microbiology, Pasteur Institute, Tehran-Iran

Nuevo medio sólido para el crecimiento de Borrelia persica y Borrelia microti

A.R. BAHRMAND, H. NEKOUI y A.M. ARDEKANI

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RESUMEN

Se describe un nuevo medio sólido para la rápida detección de Borrelia persica y Borrelia microti. Corrientemente el cultivo y aislamiento de Borrelia demora alrededor de 21 días. El examen serológico más frecuentemente realizado demora menos tiempo pero está asociado con resultados falsos positivos relativamente altos. Sin embargo, nuestro nuevo medio sólido reduce el tiempo de cultivo a 72 horas, lo que permite un rápido diagnóstico de la enfermedad causada por Borrelia persica y Borrelia microti y el inicio temprano del tratamiento en estos pacientes.
Palabras clave: BORRELIA/aislamiento & purificación; INFECCIONES POR BORRELIA/diagnóstico; INFECCIONES POR BORRELIA/terapia, REACCIONES FALSO POSITIVAS.

INTRODUCTION

The genus Borrelia contains the spirochetes which cause relapsing fever and lyme disease in man1 and diseases in domestic animals and rodents.2,3 Borrelia are generally transmitted from its usual wildlife reservoirs to humans and domestic animals via ticks belonging to the class ornithodorous or class Ixodes which cause the lyme disease.4,5 The studies of these bacteria are very important due to their presence and rapid spread throughout the world including Iran.

Four species of Borrelia which have been isolated from man and animals in Iran are: B. persica, B. microti, B. baltazardi, B. latichewi. Two species, B. microti and B. persica have been identified to be the causing agents of relapsing fever in Iran6,7,8 rather than B. hermsii which is well known to be the cause of relapsing fever in the west. These bacterial species have been shown to vary in kind and have great antigenic variability.9-12 In order to study the antigenic variability of Borrelia species which are known to be caused by DNA rearrangements on their linear plasmids,13,14 isolation of single colonies is of special importance in molecular studies of the disease. For the first time after great efforts,15,16 kelly could prepare liquid medium for culturing B. hermsii. Then, Babour and his colleagues by changing the contents of Kelly's medium were able to culture B. burgdorferi.17 This method however, is not suitable for isolation of single Borrelia colonies for molecular studies. The growth of these bacteria on solid media has been very difficult when observed by inverted microscope after three weeks of growth.1,18 In this study we report a new solid medium for growing B. microti and B. persica known to cause relapsing fever17 in Iran. This medium allows a very rapid growth of the bacteria in 72 hours in comparison with the currently used media (solid or liquid) which takes about 10-21 days.

MATERIALS AND METHODS

The two species of Borrelia persica and Borrelia microti in our hands at Pasteur Institute were isolated from the ticks collected from the north western and north eastern regions of Iran. A stock of these bacteria are usually kept in the laboratory by culturing them in animal laboratories such as mice and guinea pigs.

SOLID CULTURE MEDIUM (SCM)

This medium is made up of 2 major components.

FRACTION I:

A) 50 gram of bovine serum albumin dissolved in 600 mL of ddH2O.

B) The following compounds were dissolved in 200 mL of ddH2O and its pH was adjusted to 7,6:

1. Neopeptone 5 g
2. HEPES 6 g
3. Sodium Citrate 0,7 g
4. Glucose 5 g
5. Sodium Pyruvate 0,8 g
6. N. acetylglucosamine0,4 g
7. Sodium bicarbonate 2,2 g
8. Yeast Extract 2,53 g
9. TC Medium 199 (10X) 100 mg

Items number 2,3,4,5,6,7 were all purchased from Sigma and Items 1,8,9, were purchased from Difco.

C) 28 grams of gelatin were dissolved in 100 mL of ddH2O then autoclaved at 121 oC for 15 min.

The solutions made in steps A,B,C were heated to 40 oC, mixed and filtered (0,22 m). Then the following antibiotic solutions were added to the solution: Kanamycin 8 mg/mL (Bristol laboratories, N.Y.), and 5-Fluorouracil 230 mg/mL (Roche laboratories, N.J.).

FRACTION II:

A) 1,7 g agarose with low electroendosmosis was dissolved in 20 mL of ddH2O and autoclaved at 121 oC for 15 min.

B) 4,6 mL inactive fresh and sterile rabbit serum (Sigma).

C) 5,3 mL of sterile 5 % sodium bicarbonate.

The solutions in steps A,B,C were mixed and kept at 4 oC.

FINAL CULTURE AND MEDIUM PREPARATION

Heat the 100 mL bottles containing fractions I & II to 40 oC, mix and then distribute to petri dishes under laminar hood.

BORRELIA PERSICA CULTURE

Four guinea pigs (300g each) were injected peritoneally with 0,5 mL spirochetemia blood collected in sterile test tube containing 0,1 mL of 4 % sodium citrate. After 5 days post injection time, 2 mL of septicimic spirochetemia guinea pig blood were drawn and 0,1 mL were put on slide (1 cm2 marked area) and viable cells were counted in triplicate experiments 30 times. The result indicated average of 10 bacteria under 40X magnification. From 2 mL of spirochetemia guinea pig blood containing 10 bacteria under 40X magnification, 0,2 mL of blood was spread on each petri-dish containing the SCM. Then they were put in a Jar along with a tube containing 20 mL of sterile distilled H2O for humidity purpose. In order to create an anaerobic or microaerophilic condition, the eandle method or a special gas pack was used.18 After the candle was out, they were put in a 34 oC incubator.

BORRELIA MICROTI CULTURE

0,5 mL of spirochetemia blood was taken from white mouse which contained 20 bacteria under 40X magnification. 0,1 mL of blood was spread on each petri-dish containing the SCM. The rest of the procedure used was the same as B. persica.18

COLONY COUNT AND GROWTH MEASUREMENT

BORRELLA GROWTH CURVE

For obtaining the growth curve of B. persica and B. microti the procedure used was as follows: 1 mL suspension of liquid culture containing of B. persica and B. microti containing 100 organisms were kept separately in two test tubes at 4 oC. To analyse their growth 30 agar plates containing SCM were used and 3 drops (0,1 mL each) of suspension were placed in 3 selected areas (approx 1 cm2 circle marked) on each plate for each Borrelia species. Then, the plates were put in an anaerobic Jar and incubated at 3-4 oC. Every 12 hours one plate was taken out from the Jar and the bacterial growth was determined. Every selected areas where the bacteria were grown, was isolated and completely suspended in sterile physiological serum. From this suspension serial dilutions of 1/10, 1/100, 1/1 000 were prepared and 0,1 mL from each dilution was taken to be put on the surface of slide (marked 1 cm2). After fixing with methanol, the slide was stained in 10 % Giemsa for 20 minutes. Finally the bacteria were counted under 100X objective lens. During the early hours when the number of bacteria were low the 1/10 dilution and with the increased in growth, 1/100 and 1/1 000 dilutions were used. As figures 1, and 2 show growth of the bacteria started from the 36th hour (lag phase), and maximized at 120th (log phase) hour. Then, the number of bacteria remained constant until 216th hour (stationary phase). Then, the number of bacteria started to decrease (decline phase). The viable cell counts of B. persica in this medium reached 1 x 106 and B. microti reached 1 x 107 and the generation time for B. persica was measured to be 9:55 hours and for B. microti 6:25 hours.

RESULTS

Tables 1 and 2 summarize the growth of B. persica and B. microti respectively. Subculturing both B. persica and B. microti after passages in guinea pigs, liquid culture, and white laboratory mice show a more than 2X viable cell increase if the rabbit serum was added in the SCM. Similar results were not obtained when guinea pig serum were used. Figures 1 and 2 indicate reduced microscopic detection time to observe B. persica and B. microti colonies to 72 hours.

DISCUSSION

We have shown in this study that B. microti and B. persica can be grown in a newly developed SCM enabling the investigators to have single isolated colonies in 72 h. This medium can be easily obtained and prepared in a microbiology laboratory. As tables 1 and 2 show both species of Borrielia have grown well in SCM when rabbit serum was used. However, guinea pig serum used in SCM did not support the growth of B. persica. This finding was in support ofearlier studies which showed that guinea pigs and white laboratory mice are naturally sensitive to B. persica and B. microti respectively.8 In this study we have used TC medium 199 used in tissue culture. This medium contains not only amino acids of type D but also of type I, which are important in the synthesis of glucopeptides necessary for the formation of bacterial cell wall and rapid growth of the bacteria19 (Razavi M. Antigenic cross reactivation in Borrelia spp. in Iran [MS. Thesis]. 1988:88). The colonies formed on the SCM are detectable in 72 hours and characteristically are dome shape with the smooth periphery, colorless and 0,2-0,8 mm in diameter.

The pathogenic species of the genus Borrelia are antigenetically heterogeneous between strains and are variable in the expression of certain outer surface proteins within a strain. B. hermsii, is the most studied type of Borrelia species causing relapsing fever in the west. However, the species causing this disease in Iran are B. microti and B. persica.8 In order to study the plasmids of individual strains one has to be able to isolate the strain and the unique plasmids belonging to that strain. Therefore preparation of a solid medium for helping us to achieve this goal was an essential first step. In this study we have shown that our SCM has reduced the time required to grow and to observe B. persica and B. microti colonies on the surface of SCM to 72 hours rather than 3 weeks which is requiered for growth when other media are used.16 Our results as shown in figures 1 and 2 indicate that growth of the two Borrella species started from the 36th hour and maximized at 120th hour. Then, the number of bacteria remained constant until 216th hour and gradually decreased thereafter. The generation time for B. microti and B. persica was found to be 6:25 hours and 9:55 hours respectively. The generation time calculated when these two species are grown in liquid culture in our laboratory have shown to be 8:40 hours and 15:40 hours respectively. The shorter generation time observed between the two types of culture regarding each species is probably due to the richness of the SCM. The growth of these two species in SCM do not seem to cause any differences in virulency since injection of them into swiss white mouse and guinea pig cause spirochetemia in less than 3 days (unpublished data). Our laboratory does not grow Borrelia species other than B. microti and B. persica. However, we expect SCM to be used for the growth of other Borrelia species as well.

ACKNOWLEDGEMENT

We would to thang our colleagues M.R. Razavi, A. Amirkhani, M. Amiri and H. Norouzi for their assitance.

REFERENCIAS BIBLIOGRAFICAS

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2. Cuba. Ministerio de Salud Pública. Cuadro epidemiológico. La Habana; 1992:5-16.
3. Sierra VG, Campa C, Valcárcel M, Sotolongo F, Figueredo L, Izquierdo L. Efficacy evaluation of the Cuban vaccine VA- MENGOC BC against disease caused by serogroup B Nisseria meningitidis. En: Markachtman C, et al, eds. Neisseria 1990. Proceedings of the Seventh International Conference on Pathogen Neisserias; 1990 Sept 9-14; Berlin, Federal Republic of Germany. Berlin: Walter Dgruyter, 1992:129.
4. Halloran ME, Haber M, Longini I, Struchiner C. Direct and indirect effects in vaccine efficacy and effectiveness. Am J Epidemiol 1991;133(4):323-31.
5. Sáez-Nieto JA. Campos J, Latorre C, Juncosa T, Sierra M, García-Barrero B. Prevalence of Nisseria meningitidis in Family members of patients with meningococal infection. J Hyg Camb 1982;89:139-47.

Recibido: 18 de febrero de 1993. Aprobado: 6 de junio de 1993.

Dr. Osvaldo Rico Cordeiro. Instituto de Medicina Tropical "Pedro Kourí". Apartado 601, Marianao 13, Ciudad de La Habana, Cuba.