Differentiating Borrelia burgdorferi strains

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
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Differentiating Borrelia burgdorferi strains

Post by RitaA » Sun 7 Oct 2012 1:09

I previously posted the following abstract in another thread, however I just skimmed the full article, and discovered that the abstract barely touches upon the possible significance and potential implications.

Perhaps those folks with a scientific background can help to put the full article into some sort of perspective. Some of what I read seems truly astounding:

Infect Immun. 2012 Apr;80(4):1519-29. Epub 2012 Jan 30.

Detection of established virulence genes and plasmids to differentiate Borrelia burgdorferi strains.

Chan K, Casjens S, Parveen N.


Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey, USA.


Borrelia burgdorferi sensu stricto is the major causative agent of Lyme disease in the United States, while B. garinii and B. afzelii are more prevalent in Europe. The highly complex genome of B. burgdorferi is comprised of a linear chromosome and a large number of variably sized linear and circular plasmids. Many plasmids of this spirochete are unstable during its culture in vitro. Given that many of the B. burgdorferi virulence factors identified to date are plasmid encoded, spirochetal plasmid content determination is essential for genetic analysis of Lyme pathogenesis. Although PCR-based assays facilitate plasmid profiling of sequenced B. burgdorferi strains, a rapid genetic content determination strategy for nonsequenced strains has not yet been described. In this study, we combined pulsed-field gel electrophoresis (PFGE) and Southern hybridization for detection of genes encoding known virulence factors, ribosomal RNA gene spacer restriction fragment length polymorphism types (RSTs), ospC group determination, and sequencing of the variable dbpA and ospC genes. We show that two strains isolated from the same tick and both originally named N40 are in fact very distinct. Furthermore, we failed to detect bbk32, which encodes a fibronectin-binding adhesin, in one "N40" strain. Thus, two distinct strains that show different plasmid profiles, as determined by PFGE and PCR, were isolated from the same tick and vary in their ospC and dbpA sequences. However, both belong to group RST3B.

[PubMed - indexed for MEDLINE]
Free PMC Article
The full article is here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318418/



The B. burgdorferi strain N40 was originally isolated from an Ixodes scapularis tick from Westchester County of New York by Durland Fish (12). This uncloned culture was found to be highly infectious in the mouse model and was later cloned independently by different laboratories. N40 has been important in the study of Lyme disease pathogenesis for the past 3 decades, and its use in animal infection models has led to a number of important findings. For example, it was used to determine the differential response of various strains of mice and primates to B. burgdorferi (2, 5, 9–11, 13, 17, 18, 23, 24, 37, 38, 44, 68). In addition, many of the discoveries on the roles of different virulence factors during mammalian and tick infection were initially made using N40, genetic stability of the culture was reported in this strain when recovered from chronically infected immunocompetent mice (78), and, in particular, immunological responses to B. burgdorferi have also been thoroughly investigated using this strain. We have been studying N40 clone D10/E9 and have examined the roles of several virulence factors in this clone (32, 39, 56, 72, 75, 76, 86). However, we recently began to suspect that published work from various laboratories using the strain name “N40” may in reality refer to several different strains or at least different clones of the same strain. For example, very different sequences have previously been reported for the same N40 genes (3) and also observed by us (see below), and various genes, including bbk32, have been reported to be present and absent from N40 (38, 79). We report here that it is likely that the original “N40”-carrying tick was infected with multiple B. burgdorferi strains, which led to independent cloning and selection of different clonal isolates that were given the same N40 designation.

In order to clarify the past literature on this important strain and to avoid more confusion in the future, we obtained four N40 cultures from different laboratories and compared them in detail in this report. We anticipate that this study will also help other researchers evaluate the published literature on the pathogenesis of different N40 cultures. Furthermore, the set of techniques described herein could be useful for characterization of newly isolated B. burgdorferi strains during epidemiological studies. They can also be exploited for developing a PCR assay for the established virulence factors to detect the presence of specific plasmids, characterize new B. burgdorferi strains, and, thus, expand genetic studies to a large repertoire of unsequenced spirochete strains.


http://www.ncbi.nlm.nih.gov/pmc/article ... figure/F5/
2D gel electrophoresis of total proteins from cN40 and N40D10/E9 strains of B. burgdorferi by. 2D gel electrophoresis of total proteins shows significant differences in the protein profiles of these two strains.


Lyme spirochetes can be isolated from Ixodes ticks in regions of endemicity, and several studies have shown that more than one strain often exists in individual ticks and that all of these strains are then transmitted to the mammalian hosts (33, 49). In the early days of the study of Lyme disease, this multistrain infection of ticks was not fully appreciated. Thus, mixed cultures were disseminated among laboratories, and when strain cloning became possible, different laboratories isolated different clones from the same culture, which were subsequently given the identical strain designation as the original culture, thus confusing the outcome of some research. We began to suspect that this was the scenario for the “N40” strain that has been studied extensively by various laboratories. Since our research primarily uses a clone, N40D10/E9, from the original N40 isolate, we decided to investigate whether we could differentiate various N40 cultures from one another using a series of tests, with the aim of using this information as a tool to reinterpret the results with “N40” that have been reported in the literature from a number of laboratories. The laboratory of Stephen Barthold cloned cN40 independently, and our results strongly indicate that N40D10/E9 and cN40 do indeed represent different strains cloned from the original N40 isolate. We suggest that the specific designations of various N40 clonal cultures, probably as defined here, be used in future studies to avoid confusion.


Some characteristics and virulence mechanisms are restricted to particular strains of any pathogen, and although such studies for Lyme disease have been severely limited in the past, B. burgdorferi studies involving multiple strains will be required to provide a complete picture of Lyme disease pathogenesis and will help determine the complete repertoire of the virulence factors contributing to this disease.


Summary and conclusions.

A comprehensive study of four B. burgdorferi N40 isolates that have been used in the study of Lyme disease pathogenesis allowed us to differentiate these isolates and establish that they are derivatives of two different strains. This information will help to clarify the confusion that is due to the use of two different strains that were thought to be the same but that are in fact very different, and it will allow this research field to avoid future confusion on this point. We also propose that the use of PFGE and PCR of virulence factor genes followed by Southern hybridization is a useful strategy to discriminate different strains of B. burgdorferi sensu stricto. A PCR-based assay can then help determine the plasmid content of the strains.

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Re: Differentiating Borrelia burgdorferi strains

Post by lou » Sun 7 Oct 2012 21:29

Impressive, Rita. Thanks for dissecting this and laying it out so it is easily comprehended.

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Re: Differentiating Borrelia burgdorferi strains

Post by inmacdonald » Sun 7 Oct 2012 22:12

Dear Rita<

Love that 2D SDSPAGE image showing the distinct Protein Mass differences
in 2 strains of ND40.

We can only imagine the potential for B31 based Diagnostic Kits
to completely or partially miss significant antibodies to
the patient's actual strain of pathogenic Borrelia strain
when the Diagnostic ruler is the protein repertoire of B31 strips on WB reagents.

It is prudent to recall that if you are searching for serologic confirmation
of a Babesia POSITIVE SMEAR case ,
and you are using a Babesia MICROTI manufactured test kit,
ANY AND ALL Babesia DUNCANI infecions will be SEROGENATIVE.

The Same rule of Reagent kit Test specificty applies to the Misssouri Babesia
Strain MO1 and one of the many European Babesia strains.

Anatomy is destiny,

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