12th
International Conference on Lyme Disease
and Other Spirochetal and Tick-Borne
Disorders
Day 1 -
April 9, 1999
Keynote Address - The Complexity of
Vector-borne Spirochetes (Borrelia
spp)
The keynote speaker at this
conference was Willy Burgdorfer, PhD, of
the National Institutes of Health. In
the late 1970s, Dr. Burgdorfer and I
began debating whether the lone star
tick, Amblyomma americanum,
transmits pathogenic spotted fever group
rickettsia to humans. In 1981, Dr.
Burgdorfer was looking for rickettsia in
New York ticks when he detected
spirochetes -- the organisms,
subsequently named Borrelia
burgdorferi, that we now know to be
the etiologic agents of Lyme disease. In
his lecture, Dr. Burgdorfer reviewed the
scientific literature past (much of it
no longer written in English) and
present, dealing with spirochetal cysts,
blebs, and spherules.
Because of the seminal nature of his
work, Dr. Burgdorfer's presentation is
posted here in its entirety. As you read
his review of work in this area to date,
keep in mind much remains to be done. It
is possible that spirochetal bleb
formation signals the organism's
impending death. The research reviewed
here, however, suggests that it is a
survival mechanism, which of course, has
implications regarding patient
management.
-- Julie Rawlings, MPH
The Complexity of Arthropod-borne
Spirochetes (Borrelia spp)
Speaker: Willy Burgdorfer,
PhD
Today's investigators, eager to apply
their sophisticated microscopic,
immunochemical, molecular and genetic
methodologies often are not aware that
their research objectives are similar if
not identical to those of earlier
workers whose publications unfortunately
may no longer be available or are
published in foreign journals.
My talk today identifies the highly
controversial historical findings
related to the biology and vector(s)/host
relationships of borreliae, and
emphasizes their importance to our
current investigations of Lyme disease
and its spirochetes. Let us briefly
recall that the first discovery of
spriochetes pathogenic to humans is
credited to the German physician Dr.
Otto Obermeier (Fig. 1) who as early as
1868 during an epidemic in Berlin
detected in the blood of relapsing fever
patients highly motile threadlike
microorganisms (Fig. 2) that in
morphology were similar to the water
spirochetes Spirocheta plicatilis
-- a spirochete detected in 1835 by Dr.
Ehrenberg.
Figure 1
Figure 2
In 1878, the physician Gregor Münch
was the first to express the idea that
recurrent or relapsing fever may be
transmitted by the bite of blood-sucking
arthropods such as lice, fleas and bugs.
The theory of lice being the vector was
confirmed later by the French
microbiologists Sergent and Foley in
1910.
At the turn of the century, 1903
through 1905, the British physicians
Dutton (Joseph Everett) and Todd (John
Lancelot) working in the Congo, and
independently Ross (Philip Hedorland)
and Milne (Arthur Dawson) active in
Uganda, found that the disease referred
to as "human tick disease" by Livingston
(David) as early as 1857, was caused by
a spirochete transmitted by the African
soft-shelled or argasid tick, Orhithodoros moubata (Fig. 3). Both
Dutton and Todd contracted the disease.
Dutton, a pathologist, infected himself
accidentally during a post mortem and
died. He is remembered by having had
named the East African relapsing fever
spirochete Borrelia duttonii.
Figure 3
Also playing an important role in
relapsing fever research was the German
microbiologist Robert Koch. At the end
of 1904, he was called to East Africa to
investigate the widely distributed East
Coast Fever in cattle. He soon learned
that most Europeans traveling into the
interior regions had been suffering of
recurrent fever first thought to be
malaria. Although Koch was not aware of
the British findings in the Congo and
Uganda, he confirmed the vector role of
the Ornithodoros moubata. He
was the first to demonstrate that
spirochetes were transmitted via eggs (transovarial
transmission) to the progeny of infected
female ticks.
Ever since it was demonstrated that
the body louse (Pediculus humanus
humanus) and the African O
moubata were the vectors of the
relapsing fever spirochetes known today
as Borrelia recurrentis and B duttonii, respectively, intense
studies have been carried out on the
development of these microorganisms in
their vectors, and on the mode of
transmission to humans. Thus, in 1912,
the French worker Charles Nicolle and
coworkers studied the behavior of B
recurrentis in lice and noted that
the spirochetes had disappeared from the
midgut 24 hours after they had been
ingested; they were no longer detectable
until days 6 to 8 when they suddenly
reappeared in the hemolymph.
A similar "negative phase" had
previously been reported for B
duttonii in O moubata by
Dutton and Todd (1905-1907), Leishman
and other investigators (1907-1920),
Fantham (1911-1915), Hindle (1911), and
later also by Hatt (1929) and Nicolle
and associates (1930). According to
these investigators, ingested
spirochetes invade the gut epithelium
where they lose motility and after 3 to
4 days develop into cysts (blebs,
vesicles) that contain granules or
chromatin bodies (Fig. 4). Duton and
Todd postulated that these spherules are
formed by protuberance of the
spirochetes periplasmic membranes; they
may occur at any point along the
spirochete. At some time during their
development, these spherules or cysts
were said to burst and release their
granules. By the 10th day after
infectious feeding, Dutton and Todd no
longer found morphologically typical
spirochetes, but instead large numbers
of granules from which eventually new
spirochetes developed provided the ticks
were maintained at temperatures above
25° C.
Figure 4
Hindle, in 1911, reported similar
observations. In infected ticks held at
21° C, the spirochetes had disappeared
from the midgut by the 10th day after
infectious feeding. They could no longer
be detected either in the gut or in the
tissues. However, triturates of such
ticks injected into mice regularly
proved infective, and an increase in
temperature to 35° C resulted in the
reappearance of morphologically typical
spirochetes.
This "granulation theory" -- as it
was referred to -- received a
significant boost in 1950 when Edward
Hampp of the National Institute of
Dental Research in Bethesda showed by
stained smears, darkfield and electron
microscopy that oral treponemes and Borrelia vincenti in cultures
produce blebs and granules that were
considered "germinative units." His
hypothesis was supported by the
observation that 31 month-old cultures
containing only granules invariably
resulted in typical spirochetes upon
transfer to fresh medium.
Similar observations were also
reported by DeLamater and coworkers from
the University of Pennsylvania Medical
School. They provided evidence for the
occurrence of a complex life cycle in
the pathogenic and nonpathogenic strains
of Treponema pallidum.
Accordingly, these spirochetes multiply
by (1) transverse or binary fission, and
(2) by producing gemmae (cysts in which
a single or more granules appeared to be
the primordia of daughter spirochetes).
In contrast, there were many
investigators, including Wittrock
(1913), Kleine and Eckard (1913), Kleine
and Krause (1932), Feng and Chung
(1936-39), and your speaker (Burgdorfer,
1951), who conducted dynamic
investigations on the development of
various species of borreliae in lice or
ticks and found no evidence of a
negative phase or complex life cycle.
It is now generally accepted that B recurrentis spirochetes in the
body louse, P humanus humanus,
following ingestion with a patient's
blood, arrive in the midgut where most
are destined to die (Fig. 5). Those that
survive, within a few hours after
ingestion pass through the gut wall into
the hemolymph where they undergo massive
multiplication by binary fission. As a
result, large concentrations of
spirochetes are found in the hemolymph
surrounding the various tissues as early
as 8 to 10 days after ingestion.
Figure 5
Similarly, O moubata and
other relapsing fever ticks, during
their short feeding (10 to 30 minutes),
ingest spirochetes into the midgut where
they can be demonstrated in gradually
decreasing numbers for about 14 days but
not longer (Fig. 6). Within hours after
the ticks had engorged, spirochetes
accumulate in the intercellular spaces
of the gut epithelium (Fig. 7) from
where as early as 24 hours after
ingestion, they penetrate the basement
membrane to enter the body cavity where
they undergo massive multiplication by
binary fission (Figs. 8, 9).
Figure 6
Figure 7
Figure 8
Figure 9
Although most of the above mentioned
opponents of the "granulation theory"
verified the formation and existence of
cysts, blebs, spherules associated with
spirochetes, they considered them as
degeneration products.
Thus the question of "negative phase"
and "complex developmental cycle"
appeared to be settled until in 1981
your speaker discovered the Lyme disease
spirochete -- now known as Borrelia
burgdorferi -- associated with
ticks of the Ixodes ricinus/persulcatus
complex (Fig. 10). This relatively large
Borrelia is not readily
detectable in blood smears or thick
drops of Lyme disease patients and
susceptible host animals, yet
engorgement on infected hosts results in
up to 100% infected ticks.
Figure 10
Unlike the other louse and tick-borne
borreliae that leave the midgut of their
vectors shortly after ingestion to cause
a hemolymph-limited or systemic
infection, B burgdorferi in
most of its tick vectors remains in the
midgut where it aggregates near the
microvillar brushborder and in the
intercellular spaces of the gut
epithelium. From there, it may penetrate
the gut wall during and after
engorgement and may initiate a systemic
infection particularly in the tissues of
the ticks' central ganglion, ovary and
Malpighian tubules. Regardless of such
generalized infections, however, the
midgut remains infected throughout the
life span of the tick.
Of particular interest to our
discussion is the presence in freshly
engorged Lyme disease ticks of
spirochetes with outer
membrane-associated cysts, blebs or
spherules that often contain numerous
granules with surrounding trilaminar
membranes (Figs. 11,12). Because the
internal material of these granules is
similar in appearance and electron
density to that of typical spirochetes
and because these cysts (blebs,
spherules) strongly react when treated
with FITC-labeled conjugates, the
questions concerning a complex life
cycle of borreliae have again been
raised and induced investigators to
critically examine the nature and
function of these formations -- a
research problem referred to for the
first time almost 100 years ago by
Dutton and Todd.
Figure 11
Figure 12
Thus, RML scientists Dave Dorward and
Claude Gron using silver staining,
transmission and scanning electron
microscopy investigated the nature of
naturally elaborated membrane blebs on
the surface of cultured B
burgdorferi or free in the medium,
and found both linear and circular DNA
(Fig. 13). The fact that his material
was packaged within the membrane-derived
vesicles suggested that it might play a
role in the protection of genetic
markers. In vivo and in
vitro exposure of B burgdorferi
to antibiotics (penicillin G,
ceftriaxon) were shown by Preac-Mursic
and associates to produce cytomorphic
atypical but motile spirochetes with
numerous membrane-derived vesicles
(spheroblast -- L-forms) (Fig. 14).[1]
Figure 13
Figure 14
In their recent publication, Brorson
and Brorson reported on the "In
vitro Conversion of Borrellia
burgdorferi to Cystic Forms in
Spinal Fluid, and on the Transformation
to Mobile Spirochetes by Incubation in
BSK-H Medium."[2]
Accordingly, B burgdorferi
converted rapidly to cystic forms when
transferred to spinal fluid. No normal
spirochetes were left after 24 hours of
incubation at 37° C; all were converted
to cysts. When these cystic forms were
transferred to a rich (BSK-H) medium,
the cysts were converted back to normal,
mobile spirochetes after incubation for
9 to 17 days.
These most recent findings do confirm
the development of membrane-derived
cysts, blebs, spherules, vesicles and
the potential transformation to motile,
helical spirochetes, not as part of a
complex developmental cycle -- as
postulated by Dutton and associates --
but rather as a "survival mechanism" of
spirochetes to overcome or escape
unfavorable conditions. Such conditions
prevail during early phases of infection
when spirochetes ingested into the
midgut of ticks or lice become exposed
to the vectors' digestive enzymes and
tissue barriers (peritrophic membrane,
gut epithelium). As a result, most
detectable spirochetes produce numerous
cysts often filled with granular
material.
Other in vitro and in
vivo factors shown to induce
development of cysts include
unsatisfactory culturing conditions,
presence of antibodies and the effects
of antibiotics.
Using silver impregnations and
immunochemical staining, cystic material
has been demonstrated in every animal
and human tissue infected by B
burgdorferi. As yet, it is not
known whether these forms of Borrelia represent products of
degenerated spirochetes or of surviving
organisms capable of transforming to
typical spirochetes once the faborable
environmental conditions are restored.
It is tempting to speculate, however,
that the survival mechanism of
spirochetes is responsible for the
diverse pathology of these organisms as
well as for their ability to survive as
cystic forms thereby producing
prolonged, chronic and periodically
recurrent disease.
References
- Preac-Mursic V, Wanner G,
Reinhardt S, et al: Formation and
Cultivation of Borrelia
burgdorferi Spheroplast-L-Form
Variants. Infection 24(3):218-225,
1996.
- Brornson O, Brornson SH: In
Vitro Conversion of Borrelia
burgdorferi to Cystic Forms in
Spinal Fluid, and Transformation to
Motile Spirochetes by Incubation in
BSK-H Medium. Infection
26(30):218-225, 1996.Infection
26(3):144-150, 1998.