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Medical Hypotheses
Volume 67, Issue 4 , 2006, Pages 810-818

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doi:10.1016/j.mehy.2006.03.028    How to Cite or Link Using DOI (Opens New Window)  
Copyright © 2006 Elsevier Ltd All rights reserved.

A life cycle for Borrelia spirochetes?

Alan B. MacDonaldCorresponding Author Contact Information, 1, a, E-mail The Corresponding Author

aSt. Catherine of Siena Medical Center, Department of Pathology, 50 Rte 25 A, Smithtown, NY 11787, USA

Received 20 March 2006;  accepted 21 March 2006.  Available online 22 May 2006.


Summary

Subsequent to Schaudinn and Hoffman’s visualization of Treponema pallidum in 1905, many distinguished syphilologists proposed that spirochetes have a life cycle. What is the “essence” of a life cycle? Simply put, life cycles are diverse arrays of life forms, which emerge in an ordered sequence; which are “connected” to one another across primary and secondary hosts, and constitute a cycle with “circular” relationship between hosts. Fecal-oral life cycles and blood-to-blood life cycles are exemplary of host parasite relationships in this realm. The “blood-to-blood” begins and ends with an insect taking a blood “meal”. In this operatic scenario, a “blood-less” insect functions simultaneously as a hypodermic needle and as an incubator for some of the infectious components. The initial phase is inside the body fluid compartment of an insect. The second phase is in the blood or body fluid of a warm-blooded mammal. Third, is the phase inside the cell of a mammalian host. And a final portion of the “life” marked by “death” of the parasitized mammalian cells and the release of infectious parasites which return to the “warm” blood where the “cold blooded” vector again takes a blood meal. The cycle then begins again. In each phase of a blood to blood life cycle, the infectious agent changes its shape. Blood phase “profiles” look different from “tissue phase” profiles. Some of the tissue phase profiles may be “invisible”. Borrelia spirochetes offer an excellent example of a life cycle, by virtue of the insect vector to mammalian “piece”, the blood and intracellular residence “pieces” and the morphologic diversity “piece”. Stereotypes of what a spirochete “should “ look like, have actually produced a state of “perseveration” in spirochetal pathobiology. We have been “stuck” like a broken record, on the corkscrew form, and have failed to see the rest of the life cycle. Cystic, granular, and cell wall deficient spirochetal profiles, which were well known in the 19th and 20th centuries by such titans as Schaudinn, Hoffman, Noguchi, Delamater, Steiner, and Mattman, have been repudiated by professional microbiologists, and by pathologists who practice and who confer the status of 21st century truths in microbiology matters. Proper microscopic study, as is required by Dr. Robert Koch’s second postulate, for establishing links between microbes and disease, presupposes that the microscopist be aware of the complete array of morphologic repertoires of the alleged pathogen. (Morphologies, which are herein introduced.)



Introduction

In the discussions of life cycles, diversities of shapes and sizes of parasites are not optional constructs. Entered as evidence “Exhibits A, B, C…” herein are images of the morphologic diversities of Borrelia spirochetes. Corkscrew shapes are “Exhibit A”. These are concentric coaxial cylinders with axial internal periplasmic flagellae in the outermost compartment Plasticity of forms for Exhibits B, C…) begins with the outer surface membrane of the Borrelia spirochete. Protrusions from the outer membrane may be small caliber “blebs”. Blebs from Borrelia are capable of “embolic” behavior. Cystic forms are a larger caliber variation on theme of rounded outer membrane invested derivatives from Borrelia spirochetes. Several cystic varieties exist. The first of these is the “inducible” cystic Borrelia, which is easily produced by placing the corkscrew forms into a “hostile environment” such as liquid media containing antibiotics, or acid pH, or starvation conditions. Under these circumstances, the corkscrews “round up” and are internalized. This “encystment” is a rapid event. The shortest elapsed time in video microscopy for encystment to begin is less than 1 min, but longer encystments have been noted experimentally. Like a marsupial in a pouch, the corkscrew is “infolded” into its own surface membrane. Darkfield microscopy and electron microscopy studies demonstrate within this variety of cystic Borrelia, that the “parent” form is still visible, and that the corkscrew form is capable of rapidly re-emerging from its “cocoon” if the adverse external environment is corrected. The simple concept of the retractable carpenter’s clip-on metal tape ruler facilitates the comprehension of this “early” cystic profile. Alternatively, another cystic profile may consist of an empty membrane bound “bag of fluid”. Varieties of the “bag of fluid” cyst may exist as either cyst forms of the “empty type” or cysts which are almost empty with little discernable internal filamentous or nucleoid-like central lumenal content. And finally, there is the “Aged” cyst form of Borrelia, which contains dense “nucleoid-like” granules within the cyst. Aged cysts are incapable of rapid reversion to motile corkscrew forms. Ruptured Aged Borrelia cysts discharge a shower of granules into the environment. Granular discharge via rupture recalls other “attack” mechanisms of Chlamydia and Toxoplasmosis. Treponemal spirochetal cysts described by Dr. Edward Delamater, show virtually identical structure with the Borrelia cysts; namely small, medium, and large profiles, granule and spirochetal content, and origin from the outer surface membrane.

A second sphere in the Borrelia life cycle is the granule. Granular spirochetal forms were known to Noguchi and his contemporaries [11]. Granular derivatives emerge from “senescent” spirochetes, by a process of “segmentation” of the inner DNA containing regions of the axial cylinder. The granular elements contain either DNA or RNA, derived from the “parent” and incrementally evolve from a “Morse Code” dot and dash profile within the cylinder. When released from the confines of the inner intact spirochetal cylinder, these granules are able to “round up” in liquid media. Some released granules have a small radius and others a larger radius. Jacquet and Sezary in their studies of Treponema pallidum in Chancre fluid under darkfield illumination described granules such as these. Small radius granular forms are denovo round forms, whereas large radius granular forms appear to begin as sausage shaped segments when they are confined inside the spirochetal cylinder. Rounding occurs when the sausages are released from the “corset” of the inner cylinder upon dissolution of the corkscrew form. It is not unusual to see small radius and larger radius granular forms in the same microscopic field in tissue or in chancre darkfield preparations. Dr. Gabriel Steiner [12] correctly implicated granular spirochetal profiles as agents of tissue injury in his landmark studies of human autopsy tissues from Multiple Sclerosis. In Alzheimer’s disease the granules within diseased and dying nerve cells of the hippocampus may be internalized Borrelia spirochetal granular forms, based on DNA in situ hybridization data.

The third sphere consists of L forms of spirochetes. L forms describe any bacterium which has lost its cell wall, but which has not lost its bacterial viability in the process. Spirochetal L forms are just as reasonable and equally contentious as cell wall deficient forms of any other microbe. If the Lister Institute conceptual model is embraced, then morphologic plasticity of Borrelia L forms is not a political “hot button”. Cell wall deficient microbes share with the “snowflake” the constraints that are implicit with any “soap bubble”, namely an ephemeral existence. Horizontal DNA transfer from Borrelia to other prokaryote or to eukaryotic cells might be mediated by L forms.

Additional special types of spirochetal morphology are addressed in the last two “bullets”, namely liberated periplasmic flagellae (undocked from their parent corkscrew forms) and finally, genetic mutants of spirochetes which do not demonstrate the corkscrew profile.

Naked or “sheathed” periplasmic flagellae, isolated from the parent intact corkscrew shaped Borrelia offer yet another morphologic variation on the theme of Borrelia spirochetal morphologic diversity. These forms are rigid, nonmotile, very small in caliber and very regular in the pattern of their coiling.

Finally, various mutations of the flagellin genes of Borrelia, have provided a “molecular rationale” for the “straightened spirochetes” which were illustrated in papers from the 19th and 20th centuries. Spiral morphology is, in some but not in all cases, “tethered” to the periplasmic flagellae. In the simplest case, no flagellae due to a mutation in the flagellin complex of genes, translates into a Nonmotile spirochete which looks like a bacillus form; that is straight, rigid, and in the current vernacular… “totally NOT a Spirochete” (sic). Electron microscopic studies of Flagellin-less spirochetal mutants prove that these bacillary nonmotile forms are indeed spirochetal. Such straightened spirochetal forms were tabulated by Jacquet and Sezary in the year 1907.

A different set of “molecular rules” for mutant forms of treponemes of the oral cavity, shows the converse case. Some oral Treponemal mutant forms which are “flagellin-less” become more regularly coiled and more perfect corkscrews, when compared with their species cohorts who have an intact flagellin gene expression, and demonstrate Noncoiled Axial “Bulbosities” which are in all respects identical to the Atypical T. pallidumorganisms which Warthin and Olsen depicted in 1930 in their Matrix montage of morphologic variations of syphilis in autopsy cases of Syphilitic Aortitis [14].

A tolerance for spirochetal morphologic “plasticity” is indeed prudent. An awareness of a spirochetal life cycle is an intellectual asset. Repudiation of the spirochetal life cycle nosology is a medical and scientific liability. In a court of law, it might be said “ignorance of a life cycle for spirochetes is no excuse”. We must get it right for the sake of the patient.

Evaluation of the hypothesis

Peer reviewed scientific reports from the published literature dating back to 1900 must be scrutinized for images of Borrelia and Treponema which might “populate” the scaffolds of a tripartite life cycle consisting of intersecting but autonomous “rings” of replicating, DNA containing spirochetal forms of the following types: granules (a la Hindle’s infective Granule life cycle), cysts (a la Delamater’s myriad cystic profiles for Treponema species), corkscrews and their Cell wall deficient progeny. An image database must be compiled and collated. Pure laboratory cultures of Borrelia burgdorferi shall be evaluated in vitro and in vivo after various incubations ranging from 1 day to beyond 1 year to accommodate slow to replicate individual components. Spirochetal morphology should be photographed in preparations stained with 19th and 20th century methods, such as silver stains (Warthin Starry and Krajian methods), and 21st century technologies to demonstrate DNA [Acridine orange, Picogreen (Invitrogen Inc.) (a reagent for double strand DNA staining) and Oligreen (Invitrogen Inc.) (a reagent for single strand DNA staining)] and specific Borrelia specific DNA markers such as Molecular beacons which are optimized to find only Borrelia specific DNA sequences in laboratory models. Darkfield microscopy, and Phase contrast microscopy illumination shall be used to identify cell wall deficient L forms.

Empirical data

Cystic Borrelia forms (at least three categories are proposed)

1. “Primary” type cysts – rapidly reversible to motile corkscrew shaped forms: cystic Borrelia rapidly emerge from the motile corkscrew shaped forms as a response to “stressful conditions” in their environment (see Figure 1 and Figure 2).


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Figure 1. Cystic forms of Borrelia burgdorferi (American Type Culture Collection 35210), darkfield image 1000×, original, Alan MacDonald, MD unpublished, Photograph date 1988.


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Figure 2. Emerging – Bleb and Cystic profiles from Borrelia burgdorferi, strain B31, electron micrograph from Claude Garon PhD, Rocky Mountain Laboratory, National Institutes of Allergy and Infectious Disease, National Institutes of Health, Hamilton Montana, gifted to Alan MacDonald MD 1988.

2. “Secondary” type Cysts of Borrelia – no residual internal corkscrew remnants are seen. (“Empty” type cysts or nearly empty cyst content but uniformly positive for single strand type spirochetal DNA) (see Figure 3, Figure 4 and Figure 5).


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Figure 3. Cystic Borrelia burgdorferi from cultured human spinal fluid, 1000× original magnification, Oligreen stain for single strand DNA, Western Blot positive for antibodies to Borrelia burgdorferi in spinal fluid. Oligreen stained preparation (demonstrates single strand DNA – green signal – within the otherwise “empty cyst”). 1000× magnification, Alan MacDonald, MD 2006.


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Figure 4. Cystic Borrelia burgdorferi from spinal fluid culture for comparison, showing internal DNA granular foci, 1000× magnification, Oligreen stain for single strand DNA.


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Figure 5. Cystic Borrelia burgdorferi from spinal fluid culture, for comparison, showing a dense “nucleoid-like profile”, 1000× magnification, Oligreen stain for single strand DNA.

3. Tertiary (“Chronic” or “Latency” type) Borrelia cystic forms containing internal granular structures of variable caliber (see Fig. 6).


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Figure 6. Borrelia burgdorferi strain B31, from Rocky Mountain Laboratory, NIAID, NIH, Hamilton Montana, Conventional corkscrew and Concurrent Cystic forms, 1000× magnification, Oligreen stain (Invitrogen), Alan MacDonald, MD, unpublished image, 2006, Note: These cystic forms are NOT detected in conventional Acridine orange or in Picogreen (Invitrogen) stains for double strand spirochetal DNA, and therefore would be “invisible” with standard detection modalities. These profiles overturn the concept that only during times of stress will you expect to find Cysts of Borrelia. These cyst forms developed in BSK culture medium during log phase normal growth of the reference strain of Borrelia burgdorferi.

Granular and so called “Morse code” forms of Borrelia which are derived from corkscrew shaped spirochetes (see Figure 7 and Figure 8).


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Figure 7. Borrelia burgdorferi in transition to granular form, Murine Monoclonal antibody H5332 (Outer surface protein A), Indirect Immunofluorescence, 1000× magnification, Alan MacDonald, MD, (note variable caliber for the individual granular elements, in a “Morse code – dot and dash array”, 1000× magnification.


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Figure 8. Borrelia burgdorferi, ATCC 35210, Warthin Starry silver stain, 1000× magnification, from culture aged one year. Note the evolving segmentation of granular forms inside the cylinder and the evolving cystic profile at the apex.

Cell wall deficient forms of Borrelia – devoid of a cell wall – detectable only in hypersonic liquid media under dark field illumination using maximal and optimized optical resolutions (see Figure 9 and Figure 10).


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Figure 9. Cell wall deficient “L form” emerging from Borrelia burgdorferi ATCC 35210 (strain B31) culture after one year, Alan MacDonald, MD, unpublished image, darkfield 1000× with oil immersion objective outfitted with a correction collar. Note the beaded granular parent spirochete attached to the L form.


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Figure 10. L form of Borrelia burgdorferi strain B31 in 1-year-old culture, 1000× magnification darkfield, L form represented at 3 o’clock position by a circular profile within a circular “soap bubble”, conventional corkscrew form at 9 o’clock position.

Detached Periplasmic Flagellae of Borrelia and precursor “Extruded” periplasmic flagellae still attached to parental corkscrew shaped forms (see Figure 11 and Figure 12).


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Figure 11. Extruded periplasmic flagellae of Borrelia burgdorferi, at 3 o’clock, 7 o’clock, and 10 o’clock, 1000× darkfield, Alan B. MacDonald, MD (unpublished image 1988).


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Figure 12. Periplasmic Flagellae of Borrelia detached from parent spirochete, from Aged culture of Borrelia burgdorferi ATCC 35210 (Strain B31) darkfield 1000× oil immersion with correction collar objective. Note the diminutive cross sectional diameters when compared with diameter of intact parental spirochetal forms above. Alan MacDonald MD, unpublished image.

Flagella-less mutants of Borrelia burgdorferi – strain HB19 – American Type Collection reference 55131 (see Fig. 13) [17].


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Figure 13. Borrelia burgdorferi mutant (Flagella-less) due to a naturally occurring spontaneous mutation in the Flagellin gene complex of Strain HB19.

Flagella-less mutants of Borrelia burgdorferi – strain HB19 – American Type Collection reference 55131 (see Fig. 14) [18].


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Figure 14. Borrelia burgdorferi mutant strain MC-1 that is nonmotile and also noncoiled due to a mutation in the flagellin gene complex. These Borrelia mutants are straight “bacillary forms” which would be mis- identified as Not spirochetal by 21st century pathologists who reject all but perfect corkscrews . Straightened forms such as these were correctly identified in 1907 by [15].

Spirochetal Images from the early 20th century demonstrating Diverse morphology for T. pallidum [15].

Warthin and Olsen’s 1930 tabulation of T. pallidum morphologies in autopsy cases of Syphilic aortitis [14].

Various examples of intracellular pathogenic spirochetes (see Figure 15 and Figure 16).


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Figure 15. Spirochete traversing nerve cell nucleus in Fetal Autopsy brain. Gestational Lyme borreliosis study, 1989.


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Figure 16. Head of Rabbit spermatozoan transected by Treponema pallidum. Image by Dr. Pierre Collart, Institut Alfred Fournier, Paris, France. Gifted to Alan MacDonald, MD by Dr. Collart, 1988.

Cystic diversity of the Nichols strain of T. pallidum in the works of Dr. Edward Delamater and colleagues, 1950 (see Fig. 17) [16].


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Figure 17. A montage of diverse spirochetal cystic profiles from the work of Dr. Edward Delamater, observations of the emergence of cystic forms from Treponemal spirochetes. (Nichols strain of Treponema pallidum.) [16]

Cystic diversity of the Nichols strain of T. pallidum in the works of Dr. Edward Delamater and colleagues, 1950 (see Fig. 18) [19].


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Figure 18. Montage of Cystic spirochetal forms derived from Treponemal spirochetes, from Dr. Edward Delamater and colleagues, 1950.

Consequences of the hypothesis

A life cycle for spirochetes based on the spirochetal infective granule was defended by Balfour [4], Breinl [3], Dutton and Todd [2], Leishman [5], Fantham [6], McDonagh [8], and Hindle [9]. The germinative ability of the minute dot-like granules and the contributions of the spherical cyst-like forms were implicated in life cycle diagrams which linked the development of corkscrew shaped spirochetes to infective granules. In the present 21st century “revision” the early 20th century models; cystic and L form spirochetal elements are added, and membrane Blebs containing parasitic DNA were integrated into the infective granule life cycle model. This amalgamation produces a tripartite “Cyst-Bleb-Granule life cycle” which consists of three “arms”. Each “arm” is capable of assaulting mammalian cells.

Pathologic studies correlate-45 degree rulespirochetal mediated structural alterations in diseased tissues. When infection is a diagnostic consideration for the actual etiology of a disease process, it is the incumbent responsibility of the histopathologist to either confirm or to exclude the “infection hypothesis”. Application of “special stains” which transform “invisible microbes” in diseased tissues into “visible” agents of tissue injury accomplishes the first step in the correct classification process. The second step, namely seeing with the “frontal cortex” and interpreting the images, which are projected onto the retina, is only accomplished by the mind, which is prepared to recognize and interpret the retinal images. This is the difficult step. Each potential microbial pathogen should have its picture in the Atlas of “alleged perpetrators”. A defined life cycle assists in this step.

Images contained within this manuscript offer a “new scaffold” namely a life-cycle concept. Hopefully the life-cycle gallery of “legitimate” spirochetal profiles which is here presented. will assist in the correct classification of Borrelia related infectious diseases in mammalian hosts. The images herein are only intended to serve as a “primer” of the truly protean microscopic profiles which must be duly considered in the microscopic quest for pathogenic spirochetes in tissue [13].


Acknowledgement

This work was supported by a Research Grant from the Turn the Corner Foundation, New York, New York and by generous Institutional support from St. Catherine of Siena Medical Center, Smithtown, New York .


References

[2] J.S. Dutton and J.L. Todd, A note on the morphology of Spirochaeta duttoni, Lancet 2 (1907), p. 1523. SummaryPlus | Full Text + Links | PDF (640 K)

[3] A. Breinl, The morphology and life history of Spirochaeta duttoni, Ann Trop Med Parasitol 3 (1907).

[4] A. Balfour, A peculiar blood condition, probably parasitic in Sudanese Fowls, Brit Med J (1907) (November), pp. 1330–1333.

[5] W.B. Leishman, An address on the mechanism of infection in tick fever, and on hereditary transmission of Spirochaeta duttoni in the tick, Lancet 133 (1911), pp. 11–14.

[6] H.B. Fantham, Some researches on the life cycle of spirochetes, Ann Trop Med Parasitol 5 (1911), pp. 479–496.

[8] J.E.R. McDonagh, The life cycle of the organism of syphilis, Lancet 2 (1912), p. 1011. SummaryPlus | Full Text + Links | PDF (419 K)

[9] E. Hindle, On the life cycle of Spirochaeta gallinarum, Parasitology IV (1912), pp. 463–477.

[11] Rubel Joanne. Spirochetal cysts, L forms, and Blebs, Observations from 1905 to 2005. Web access, http://www.lymeinfo.net/medical/LDBibliography.pdf, date visited March 15, 2006.

[12] G. Steiner, Morphology of Spirocheta myelopthora in Multiple Sclerosis, J Neuropathol Exp Neurol 13 (1954), p. 221. Abstract-MEDLINE  

[13] MacDonald AB. Plaques of Alzheimer’s disease originate from cysts of Borrelia burgdorferi, the Lyme disease spirochete. Med Hypotheses 2006, in press.

[14] A.S. Warthin and R.E. Olsen, The Granular transformation of Spirochaeta pallida in aortic focal lesions, Am J Syphilis 14 (1930), pp. 433–437.

[15] L. Jacquet and A. Sezary, Des Formes atypiques et degeneratives du Treponeme pale, Bull Soc Med Hopital Paris 24 (1907), pp. 114–116.

[16] E.D. DeLamater, R.H. Wiggall and M. Haanes, Studies on the life cycle of spirochetes: IV. The life cycle of the Nichols pathogenic Treponema pallidum in the rabbit testis as visualized by means of stained smears, J Exp Med 92 (1950), pp. 247–250.

[17] A. Sadziene, D.D. Thomas, V.G. Bundoc, S.C. Holt and A.G. Barbour, A flagella-less mutant of Borrelia burgdorferi, structural, molecular and in vitro functional characterization, J Clin Invest 88 (1991), pp. 82–92. Abstract-EMBASE | Abstract-MEDLINE | Abstract-ScienceDirect Navigator  

[18] A.M. Motaleb, L. Corum, J.L. Bono, P. Rosa, D.S. Samuels and N.Y. Charon, Borrelia burgdorferi periplasmic flagella have both skeletal and motility functions, Proc Nat Acad Sci 97 (2000), pp. 10899–10904.

[19] E.D. Delamater, M. Hanes and R.H. Wiggall, Studies on the life cycles of spirochetes: V. The life cycle of the Nichols nonpathogenic Treponema in culture, Am J Syphilis 35 (1951), pp. 164–179.



Corresponding Author Contact InformationTel.: +1 631 862 3764/7241335; fax: +1 631 862 3863.
1 1 Franciscan Lane, Smithtown, NY 11787, USA.


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Medical Hypotheses
Volume 67, Issue 4 , 2006, Pages 810-818


 
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