Borrelia Spirochete Morphology in Peripheral Blood

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Borrelia Spirochete Morphology Introduction
Borrelia Spirochete Morphology Results
Borrelia Spirochete Morphology Extended Results
Borrelia Spirochete Morphology Supporting Evidence
Borrelia Spirochete Morphology Fluorescent Antibody Experiment
Borrelia Spirochete Morphology Discussion, Limitations, Conclusion and Acknowledgments
Borrelia Spirochete Morphology References

Summary
Various morphological forms of spirochetes can be observed in blood. This experiment aimed to document some of these forms observed within 5 days of preparing a mini-culture on a microscope slide. Eleven donors with Lyme borreliosis and/or Myalgic Encephalomyelitis provided a fingertip blood drop which was mixed with BSK-H culture medium. Spirochetal forms were observed from each donor mostly within in 48 to 72 hours. Supporting evidence for the spirochetal nature of the agents is provided including micrographs from long-term culture experiments that included 5 of the current donors; and additionally, results from polyclonal fluorescent antibody staining specific for Borrelia species and Borrelia garinii from 3 donors.

Introduction
There is controversy surrounding some aspects of Lyme borreliosis, a spirochete infection which is transmitted by the bite of a tick and caused by Borrelia burgdorferi and related strains. The areas of disagreement include testing: which often depends upon a Western Blot detecting antibodies whose presence some believe may be variable (1), treatment: 100mg doxycycline twice daily is considered to eradicate the infection in one month (though alternatives, intravenous and longer treatments are sometimes given) (2); persistence: it is claimed that ongoing symptoms after treatment are not due to continued infection but are related to tissue damage, reinfection or other explanations (3); location of spirochetes: some consider that borrelia spirochetes are rarely found in blood (4).

Direct detection of spirochetes is possible using darkfield microscopy, a method that uses oblique illumination sometimes described as 'light staining' and in which the subject shows brightly against a dark background. Darkfield microscopy is used for the diagnosis of syphilis which is caused by another spirochete, Treponema pallidum, by observing the very thin spirochetes in tissue samples (5). Though microscopy alone cannot identify the exact species of a spirochete, it can locate their presence and allows unusual species or variants to be observed. In a study of 90 patients which compared testing methods, Tylewska-Wierzbanowska and Chmielewski (6) concluded that: "There is no correlation between the level of antibodies (ELISA), the number of protein bands (Western blot) and the presence of spirochetes in body fluids (culture and PCR), indicating that in addition to serological testing the use of PCR and cultivation in the diagnosis of Lyme borreliosis should be recommended."

Lyme borreliosis (LB) and myalgic encephalomyelitis (M.E.) patients have active communities sharing knowledge and support through online groups. Friends made through these resources were invited to participate by providing a few drops of fingertip blood to see if spirochetal forms could be cultured and observed with darkfield microscopy. Two different kits were designed to be sent by mail allowing donors to provide samples with minimal inconvenience.

The dimensions given for borrelia burgdorferi spirochetes vary considerably. Russian researchers found that the length varied from 3 to 74 microns with average size ranging from 10.7 to 24.8 microns (7). The bacteria are generally too thin for observation with conventional light microscopy (8). Spirochetes are often thought of as long bacteria with a helical or waveform appearance, though other forms have been identified. There are various spheroplast forms whose size and shape can vary considerably. Mursic et al state: "The role of different atypical bacterial forms, spheroplasts and L-forms in infectious disease is considered an important problem in microbiology. Spheroplasts and growth into L-forms occur in many bacterial species in liquid and on various solid media. These forms are products of partial or complete removal of the cell wall by enzymatic digestion (..) and partial or incomplete inhibition of cell wall synthesis..." (9)

As well as L-forms with a reduced or absent cell wall there are 'mutant' types of borrelia spirochetes. Some have lost their flagella and motility and are seen as straighter than wild types and may have lower metabolic activity (10). Atypical forms of borrelia include elongated bodies that are comparatively thick (11), they can appear bacillus-like, may be long and thin with a thicker region, can be looped or twisted or have a looped end (12).

Spirochetes can undergo transformation into a chain of round-bodies sometimes referred to as a 'string of pearls'. Prior to transforming into this distinctive form, spirochtetes can sometimes be seen to have signs of segmentation along their length. Lyme Info (www.lymeinfo.net) cite Hindle, E. (1912) who wrote on 'spirochaeta gallinarum' (13): "The spirochaete gradually assumes the appearance of a chain of beads (…) contained within the transparent cell-wall." And further cite Ewing (1907) on Spirochaete pallida: (14): "The organism may appear as a chain of granules which outline a complete spirochaete."

These possible variations do not mean that everything that is long and thin in a blood-drop is a spirochete. Amongst other possibilities to consider are fibrin strands, which can appear to wriggle with Brownian motion - BSK does not prevent the development of fibrin strands and neither does EDTA completely. Basket and smudge cells are sometimes seen and these disintegrating lymphocytes can create a lot of detritus of unusual shapes. A skin puncture can result in samples containing damaged cells and non-blood fluids as well as external contamination. Collagen fibers and decaying cell membranes have been suggested as an explanation for some spirochete-like agents (15).

When spirochetal forms become visible in a wet-drop blood slide, they can usually be observed for a few days before the majority have developed into the characteristic 'string of pearls' which then break-up into shorter chains and individual coccoids. The resulting forms would then require techniques such as PCR or antibody staining to identify them as being spirochetal. Even though the window of opportunity for observation is reasonably broad its starting point can be quite variable. Sometimes spirochetal forms can be observed immediately upon preparation of a slide, yet even with the same donor on different occasions it may take hours or days for them to grow. If a donor is taking antibiotics this might also be a factor that could impede growth.

Therefore it is desirable to create conditions which encourage the growth of any spirochetal forms present to improve the chances of observation. Borrelia burgdorferi is microaerophilic preferring the presence of oxygen at low levels (16). Mysterud and Laane (17) achieved good results in isolating spirochetes in blood culture by using a solution of sodium citrate which encouraged the growth of spirochetes. Additionally, the mini-culture environment that they created on the slide gradually developed regions where oxygen levels were favourable for spirochete growth.

Materials and Methods
Donors gave Informed Consent to participate in the experiments and for their data to be published. All experiments were conducted in accordance with the Declaration of Helsinki 1975.

For these experiments Barbour-Stoenner-Kelly Medium (BSK) with 6% rabbit serum (Sigma-Aldrich) was chosen as an additive to encourage spirochete growth, dilute blood components that could impede spirochete growth and separate the blood cells for ease of observation with a darkfield microscope.

For the first phase of the experiment a kit was sent to donors consisting of a hardboard slide mailer containing 3 pre-cleaned glass microscope slides and 50mm coverslips, a lancet and a cleaning wipe, along with a return stamped and addressed envelope (SAE). Donors put a small fingertip drop of blood in the centre of each slide, placed the coverslip over it and returned the slides in the mail. Upon return of the slides a drop of BSK was added at the edge of the coverslip which was drawn into the sample by surface tension. The long sides of the coverslip were then sealed with adhesive. First observations with the microscope were made 24 to 48 hours after the sample was prepared and again on following days for up to a total of 5 days.

The second kit tested additionally included a sterile pipette and a tiny tube of BSK as well as a tube of adhesive. To prepare the sample the donor produced a blood drop as with Kit 1, then added a small drop of BSK to the sample. This was stirred for a few seconds with the pipette tip before the coverslip was placed over it and then blotted. The long sides of the coverslip were then sealed with adhesive.

The stock BSK was tested for contamination with control slides in which the culture medium was exposed to the atmosphere on a slide for one minute and stirred with a pipette-tip (as occurred with Kit 2) before a 50mm coverslip was placed and sealed; then stored for 48 hours and examined for contamination. Some particles were noted, a few very sparse bacteria and a very few elongated agents were observed which did not have spirochete characteristics.

Videos of 640x480 pixels at 30 frames per second were recorded from the microscope as well as still images at 1600x1200 pixels with a Fuji Finepix F10 digital camera mounted onto the microscope eyepiece. Images and videos were carefully edited by filtering noise and adjusting levels and some were cropped. Clips from each individual donor's sample were compiled into short videos which comprise the main data for the experiment.

Of eleven donors in total, seven donors provided samples for Kit 1. Ten donors provided samples for Kit 2 including 6 of the donors of Kit 1. Two donors additionally provided samples for fluorescence microscopy experiments.

Donor Demographics
6 women and 5 men donated samples. Ages ranged from 37 to 70 years. All donors have had long-term health problems with 8 of 11 being ill for 19 years or longer. 8 donors have at some time been diagnosed with M.E. 10 of 11 donors had been tested for Lyme borreliosis through the NHS and the result was negative (1 not tested). 9 of 11 had private tests that were positive; (2 not tested privately). 7 of 11 had taken antibiotics within one week of preparing their culture sample. The impact of illness varied widely amongst donors with average self-rated physical ability at 32% and mental ability at 44%. See Table 1.

Table 1.

Gender Male 5 Female 6
Age range / average 37 to 70 average 57
Years of illness range / average 5 to 29 average 20
Diagnosis M.E. 1, LB 3 ME + LB 7
NHS Lyme borreliosis test Neg 10 N/A 1
Private Lyme borreliosis test Positive 9 N/A 2
Antibiotics taken within last week Yes 7 No 4
Self-rated physical ability range / average 10% to 70% average 32%
Self-rated mental ability range / average 10% to 85% average 44%

Donor demographics. LB = Lyme borreliosis. M.E. = Myalgic Encephalomyelitis. NHS laboratory test for Lyme borreliosis is a 2 tier test, part one being an ELISA which if positive or equivocal is followed by a Western Blot. Self rated abilities compared to former health or, if ill for more than 10 years, compared to peers.

Next: Results

Navigate this article:
Borrelia Spirochete Morphology Introduction
Borrelia Spirochete Morphology Results
Borrelia Spirochete Morphology Extended Results
Borrelia Spirochete Morphology Supporting Evidence
Borrelia Spirochete Morphology Fluorescent Antibody Experiment
Borrelia Spirochete Morphology Discussion, Limitations, Conclusion and Acknowledgments
Borrelia Spirochete Morphology References

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