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)
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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.
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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.
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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
spirochetal 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].
