FIRST ISOLATION AND CULTIVATION OF BORRELIA BURGDORFERI

BACTERIAL METHODS REPORT

VANESSA Student

Student@aol.com

BIOLOGY 240

MARCH 12,2001

PROFESSOR DR. NICK KAPP

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

First Isolation and Cultivation of Borrelia Burgdorferi

Introduction

There have been a lot of controversies towards the isolation of Borrelia Burgdorferi sensu lato. Various methods have been used to determine its presence in our environment or in certain species. One method used was the PCR which analyzed isolates of  Borrelia Burgdorferi sensu lato with the use of six pairs of primers known to amplify selected DNA target sequences specifically found in the reference strain Borrelia Burgdorferi sensu lato. Amplification was performed with a Perkin-Elmer/Cetus thermal cycler (model 9600). PCR-amplified products were electrophoresed in 2% agarose gels, then stained with ethidium bromide and finally visualized by ultraviolet light. The amplification products were seen as distinct bands and photographed by Polaroid for permanent documentation. The chromosomal target sequences was amplified repeatedly but could not detect most of the DNA of the other spirochetal isolates.  

However, the most effective method used in the isolation of  Borrelia Burgdorferi sensu lato is Spirochetal  isolation from ticks or hosts in nature in Barbour-Stoenner-Kelly(BSK) culture medium. These spirochetal isolates were collected for over a 2-year period from ticks feeding on a variety of rabbits at several locations on the same farm. The five spirochetal isolates used in this method  are from a farm in Bollinger, Mo.

Method Authors: J. H. Oliver Jr., T. M. Kollars Jr., F. W. Chandler Jr., A. M. James,

                              E. J. Masters, R. S. Lane, and L. O. Huey

 

 

 

 

Method Flow Chart:

1.      Eastern cottontail rabbits (Sylvilagus floridanus) were collected from September 1993 through September 1995 on a single farm in Bollinger County, which is in southeastern Missouri.

2.      Larval and nymphal Ixodes dentatus and larval Amblyomma americanum ticks were removed from the rabbits.

3.      These ticks were surface sterilized, triturated, and inoculated into BSK II medium containing 0.023% L-cysteine hydrochloride, 0.015% DL-dithiothreitol, 1 ug of L-glutamine per ml., 0.15% soft agarose, 50 ug of rifampin per ml, 20 ug of phosphomycin per ml, and 2.5 ug of amphotericin B per ml.

4.      These cultures were incubated in a 5% Carbon dioxide atmosphere at 33 to 34’C.

5.      Then they were examined for spirochetes by dark-field microscopy twice weekly for 2 weeks and weekly thereafter for six weeks.

6.      The spirochetal isolates were screened immunologically by indirect fluorescent antibody analysis with a series of monoclonal bodies.

7.      This showed Borrelia Burgdorferi specific anti-outer surface protein A (OspA) monoclonal bodies(H5332) and a Borrelia  Burgdorferi genus-specific antiflagellin monoclonal antibody (H9724).

8.      All of the Missouri isolates reacted positively to the Borrelia genus-specific antibody (H9724) but only one reacted to the OspA monoclonal antibodies (H5332).

 

 

 

Typical Results:

            Anderson et al also isolated rabbits and all of his isolates reacted with monoclonal antibody (H9724) which identifies the spirochetes as borreliae but more than half of the isolates did not bind with the antibody (H5332).

            Feir et al documented the presence of Borrelia Burgdorferi in ticks from rabbits also in southeastern Missouri. The spirochetes were detected and identified as Borrelia Burgdorferi by the immunofluorescent antibody tests using the monoclonal antibody (H5332) in 1.9% Amblyomma but not the monoclonal antibody (H9724).

Equipment used:

BSK II medium

See recipe

incubator

In laboratory

Dark-field microscopy

In laboratory

 

BSK II medium

l-cysteine hydrochloride

Sigma Chemical Co.

0.023%

dl-dithiothreitol

Sigma Chemical Co.

0.015%

l-glutamine

GIBCO Laboratories

1 ug per ml

Soft agarose

Seakem

0.15%

rifampin

Sigma Chemical Co.

50 ug per ml

phosphomycin

Sigma Chemical Co.

20 ug per ml

Amphotericin B

Fungizone

2.5 ug per ml

 

 

 

 

 

 

 

Bibliography:

1.      Oliver Jr., J. H., Kollars Jr., T.M, Chandler Jr., F. W., James, A. M., Masters, E. J.,       Lane, R. S., and Huey, L. O. 1998. First Isolation and Cultivation of Borrelia Burgdorferi sensu lato from Missouri. J. Clin. Microbiol. Volume 36, pp. 1-5.

2.      Anderson, J. F., Magnarelli, L. A., Lefebvre, R. B., Andreadis, T. G., McAnich, J. B., Perng, G. C., Johnson, R. C. 1989 . Antigenetically variable Borrelia Burgdorferi isolated from cottontail rabbits and Ixodes dentatus in rural and urban areas. J. Clin. Microbiol. Volume 27, pp. 13-20. [Pubmed]

3.      Feir, D., Santanello, C. R., Li, B. W., Xie, C. S., Masters, E., Marconi, R., and Weil, G. 1994. Evidence supporting the presence of Borrelia Burgdorferi in Missouri. Am J. Trop Med Hyg. Volume 51, pp. 475-482.