Willy
Burgdorfer, PhD
Rocky Mountain Laboratories, National Institutes of Health
The
Tick: A Pandora's Box
No
abstract available
David
H. Persing, MD, PhD
Medicine and Pathology, Mayo Clinic
Naturally
Occurring Coinfections in Reservoir Mice
The
main interest of our laboratory over the past several years has
been to study the basis of the remarkable biological variation
in Lyme disease expression. One possible contributor to biologic
variation in the clinical presentation of LD may be unrecognized
coinfection with other tick-transmitted pathogens. In the past
few years, it has become clear that transmission of other pathogens,
including Babesia microti and granulocytic Ehrlichia
spp., may occur via the same tick vector; infection with
these organisms alone and in combination with Bb may occur
occasionally in humans. More commonly, such coinfections exist
within reservoir mice, and analysis of such mice may be able
to predict the emergence of human disease. In order to identify
other agents that may be involved in the LD transmission cycle,
we have carried out a systematic surveillance of white-footed
mice by using broad-range PCR and other methods.
As
a result of these studies, we have discovered that a novel Bartonella
species is present in LD reservoir mice collected from distinct
geographic locations in the upper Midwest and the northeastern
U.S. The organism was isolated in pure culture, and is infectious
for laboratory mice. Interestingly, the Bartonella species was
recovered exclusively from animals already harboring other tick-transmitted
agents (usually Babesia microti and Bb). Studies
are currently underway to determine the extent of the distribution
of this organism and its relatives, and to determine whether
it may also be tick-transmitted.
Jesse
L. Goodman, MD
Infectious Diseases, Department of Medicine, University of Minnesota
School of Medicine
Human
Granulocytic Ehrlichiosis:
Clinical and Biological features of an Emerging Infection
Human
granulocytic ehrlichiosis (HGE) is an acute febrile illness usually
accompanied by thrombocytopenia, leukopenia and elevations in
serum hepatic transaminases. The disease was first reported in
patients in Minnesota and Wisconsin by Bakken and colleagues
who noted intracellular inclusions in granulocytes suggestive
of an ehrlichial infection. Chen and colleagues utilized PCR
amplification of 16S rDNA sequences from the blood to show that
the causative organism was likely an ehrlichia closely related
to the agents of equine and ruminant ehrlichiosis, E. equi
and E. phagocytophila. Our laboratory then reported isolation
of the etiologic agent from patients with characteristic clinical
findings through utilization of the HL60 promyelocytic leukemia
cell line. Isolates from 3 patients had 16S sequences which differed
at only one nucleotide from a strain of E. equi we sequenced.
We also developed sensitive and specific PCR primers for improved
diagnosis and have since noted that approximately 50% of culture
positive patients, while also PCR positive, have blood smears
which do not show characteristic inclusions.
Isolation
and continuous cultivation of the agent has provided the basis
for further studies aimed at better understanding the pathogenesis
and cell biology of infection and at improved treatment. Detailed
antibiotic susceptibility studies revealed that, while sensitive
to tetracyclines, HGE is resistant to the other antibiotics commonly
used to treat LD such as B-lactams and macrolides. Agents with
promising in vitro activity against HGE include some of the quinolones,
in particular the developmental agent trovafloxacin, and the
rifamycins. Since coinfections with Lyme disease are not rare,
these findings emphasize that tetracyclines are currently the
preferred treatment for both early LD and HGE.
We
have recently documented that the HGE agent can also infect CD34
positive bone marrow progenitor cells, including CD14 positive
monocytic cells. This finding suggests that the bone marrow may
be an important target of infection in patients and that such
infection is likely to play a role in the cytopenias observed.
The agent's interaction with monocytes is also likely to be important
in early events in infection and in the host's response. The
development of a culture system utilizing normal primary human
cells provides an alternative to the use of HL60 or other transformed
cell lines that will be important in better understanding the
agents interactions with its natural target cells. In addition,
antigen prepared from the human isolates in HL60 cells has led
to the development of improved and reproducible serologic assays
which have been useful for diagnostic and epidemiologic purposes.
J.
Stephen Dumler, MD
Division of Medical Microbiology, The John Hopkins Medical Institutions
Clinical
Aspects of Ehrlichiosis
Human
monocytic ehrlichiosis (HME) and human granulocytic ehrlichiosis
(HGE) are emerging tick-borne infections that occur in the south
central through southeastern states and in the northeast, upper
Midwest, northern California, and Europe, respectively. The clinical
presentations of these two distinct, potentially life-threatening
infections are fever, headache, myalgia and other diagnostically
undifferentiated signs and symptoms. The laboratory findings
that include leukopenia, thrombocytopenia, and increased serum
hepatic transaminase activities are helpful diagnostic clues.
The elderly and male patients are affected more frequently and
severely than younger individuals, and immune compromise is a
risk factor for severe infection. The most severe complications
include meningoencephalitis and adult respiratory distress syndrome,
and fatalities have been documented with both HME and HGE. Most
affected patients present during the months of May through October
and report tick bites in 75%. The tick vector(s) and major reservoirs
for Ehrlichia chaffeensis (HME) and the agent of HGE are
Amblyomma americanum (lone star tick)/Odocoileus virginianus
(white-tailed deer) and Ixodes scapularis (black-legged
tick or deer tick)/Peromyscus leucopus (white-footed mouse),
respectively.
Although
the prevalence of these infections varies widely depending upon
geographic regions, focally high rates of clinical disease can
be found. Based upon seroprevalence data, asymptomatic infections
are suspected to occur often with both infections. The seroprevalence
of HGE is highest in regions endemic for LD and babesiosis, and
these patients are more likely to have evidence of infection
with two or more of these pathogens. Diagnosis should be base
upon clinical suspicion and can be confirmed by PCR during the
acute illness or serology in convalescence. Blood smears are
not informative in the majority of patients. Prompt early treatment
with doxycycline is warranted since delayed diagnosis and therapy
are associated with more severe disease.
Ellen
S. Shang, PhD
U.C.L.A. School of Medicine
Outer
Membrane Proteins of Pathogenic Borrelia Species
Ellen S. Shang*, Jonathan T. Skare, Maurice M. Exner, Tajib Mirzabekov,
Denise M. Foley, Cheryl I. Champion, Hediye Erdjument-Bromage,
David R. Blanco, Bruce L. Kagan, Paul Tempst, James N. Miller,
and Michael A. Lovett. Department of Microbiology and Immunology,
UCLA School of Medicine
In
order to identify Borrelia outer membrane spanning proteins (Oms),
the outer membranes of Borrelia burgdorferi and Borrelia
hermsii were isolated and purified. We recently reported
on the isolation of the Bb outer membrane by applying
a novel technique used for the isolation of the Treponema pallidum
outer membrane. The Bb outer membrane was released with
a low pH, hypotonic citrate buffer and subsequently purified
by isopynic sucrose gradient centrifugation. The outer membrane
nature of the preparation was visualized by electron microscopy
and demonstrated by the presence of outer membrane porin activities
with single channel conductances of 12 nS (Oms66/p66) and 0.6
nS (Oms28). The absence of §-NADH oxidase activity in the
outer membrane preparation demonstrated the lack of inner membrane
contamination. Two-dimensional isoelectric focusing and Western
immunoblot analysis of the Bb outer membranes demonstrated
the presence of approximately 30 proteins including the outer
surface protein A (Osp A), and the p66 (Oms66) porin protein.
Further analysis of the Bb outer membrane with antiserum
specific for virulent strain B31 resulted in the identification
of several proteins which may be correlated to virulence and
protective immunity in the rabbit LD model.
Based
on the successful isolation of both the T. pallidum and
Bb outer membranes, we have further extended our application
of this technique to the isolation of the B. hermsii outer
membrane. Western immunoblot analysis of the isolated B. hermsii
outer membrane indicated that the variable major protein (Vmp),
glycerophosphodiester phosphodiesterase (Gpd), an Oms66 porin
homolog, and a small amount of flagellin were among the 30 constituent
proteins identified in the preparation. The successful isolation
of the outer membrane proteins which may be important to virulence
and pathogenesis.
Janis
J. Weis, PhD
Associate Professor, Department of Pathology, University of Utah
Role
of Bb Lipoproteins in Localized Inflammation:
Activation of Human Endothelial Cells and Neutrophils
Janis
J. Weis*, R. Mark Wooten, and Tom B. Morrison. University of
Utah, Department of Pathology Lyme disease is caused by infection
with Borrelia burgdorferi, and is characterized by bacterial
persistence and inflammation in a number of host tissues. Bb
outer surface lipoproteins possess cytokine stimulatory properties
and the presence of the spirochete is correlated with severity
of disease and the pathology of tissues such as the arthritic
joint. Spirochete invasion of tissues requires interaction with
and penetration of vascular endothelium, suggesting endothelial
cells may participate in the inflammation of LD. A model Bb
lipoprotein, OspA, was found to be a potent stimulant of NF-kB
nuclear translocation in human endothelial cells. Nuclear NF-kB
was detectable within 15 min., indication the lipoprotein directly
triggers this signaling event. OspA also rapidly upregulated
endothelial cell production of several proteins whose transcription
is dependent on Nf-kB: the cytokine IL-6, the chemokine IL-8,
and the adhesion molecules E-selectin, VCAM-1 and ICAM-1. This
activation resulted in enhanced binding of neutrophils to OspA
stimulated endothelial monolayers. Neutrophils are the predominant
infiltrate into the inflamed arthritic joints, suggesting their
interaction with the vascular endothelium could be an important
event in arthritis development. They have also been implicated
in the control of Bb in the mammalian host.
We
now report that neutrophils are directly responsive to Bb
lipoproteins. Picomolar concentrations of OspA induced surface
markers associated with neutrophil activation: increased CD10
and CD11b expression; decreased CD62-L expression; and an increased
adherence to extracellular matrix. These events were similar
in kinetics and magnitude to those induced by the strong inflammatory
agonists lipopolysaccharide and formylated peptides. These classic
activation events involve the fusion of granules containing pre-formed
markers with the plasma membrane, and occur with rapid kinetics
indicating direct responses. OspA was also found to induce granule
fusion events associated with bacterial killing and tissue damage.
These depended on the ability of OspA to prime neutrophils to
respond to a secondary inflammatory stimulus, formylated peptide,
resulting in the release of elastase containing lysosomal granules
and production of superoxide. Thus, Bb lipoproteins may
directly participate in the recruitment of neutrophils into inflammatory
sites and their subsequent activation through combined effects
on vascular endothelium and resting neutrophils. These events
could contribute to the destruction of the spirochete and to
the pathological developments of Lyme arthritis.
John
Leong, MD, PhD
Department of Molecular Genetics and Microbiology, University
of Massachusetts Medical Center
Borrelia
burgdorferi
- Host Cell Interactions
Attachment
of the Lyme disease spirochete to host cells is likely to be
important for the colonization of diverse tissues. In previous
studies, we identified the platelet-specific integrin aIIbß:3
and heparan sulfate proteoglycans as host cell molecules that
mediate Bb attachment. We have now analyzed Bb
recognition of different integrins and different classes of proteoglycans
by different Lyme disease spirochete strains. An infectious Bb
strain bound to purified preparations of integrin aVß3
and a5ß1, two ubiquitously expressed integrins that are
also known as the vitronectin and fibronectin receptors, respectively.
This binding could be inhibited by RGD peptides and by the appropriate
anti-integrin antibodies. Binding to a cell line that expresses
aVß3 was shown to be mediated by this receptor. When a
diverse group of Lyme disease spirochetes was tested for binding
to aIIbß3, aVß3 and a5ß, all of the strains
bound to at least one integrin. Interestingly, binding to one
integrin was not always predictive of binding to other integrins,
so that individual strains showed different integrin binding
profiles.
We
have also analyzed proteoglycan-mediated bacterial attachment
to multiple cells types, including primary neural and endothelial
cells. LD spirochetes bound to proteoglycans expressed by all
of the cell lines tested-bacterial binding could be inhibited
by soluble proteoglycans, or by treatments that alter or remove
host cell proteoglycans. The particular class of proteoglycan
that was recognized by the bacteria, as determined by the effect
of specific lyases on bacterial attachment, varied with the cell
line. On most cells, heparin/heparan sulfate-related proteoglycans
played the most important role in bacterial attachment, while
on others, dermatan sulfate appeared to contribute significantly
to binding as well. Most of the strains tested showed a strong
binding preference to heparin over dermatan sulfate, but one
strain displayed the opposite binding profile. Thus, Bb
recognizes multiple integrins and multiple classes of proteoglycans.
However, the particular integrin and proteoglycan binding preference
profile varies with bacterial strain. This, combined with the
fact that different host cell types express different sets of
integrins and proteoglycans, means that the relative contribution
of any single integrin or proteoglycan to bacterial attachment
by Bb depends both on the bacterial strain and on the
host cell type.
David
W. Dorward, PhD
Microbiologist, Rocky Mountain Laboratories, National Institute
of Allergy and Infectious Diseases
Interaction
of Bb with Human Lymphocytes
Recent
studies have shown that Lyme disease spirochetes including Borrelia
burgdorferi and B. garinii can actively invade and
kill both primary and cultured human B and T lymphocytes. To
help understand the progression and effects of such interactions
we used temperature regulation techniques to synchronize spirochete-lymphocyte
co-incubation mixtures, then examined the mixtures by electron
microscopy. We found that low passage (<8) Bb Sh-2-82 readily
attached to SKW 6.4 B cells maintained at 4 degrees C. Coincidental
with warming the mixtures to 37 degrees C (time 0), intracellular
spirochetes were observed within endocytic pits and vacuoles.
No evidence of lysosomal fusion to endosomes containing spirochetes
was apparent. Intracellular invasion was transient, rapidly progressing
to lytic or non-lytic emergence. By 60 minutes less than 1 percent
of spirochetes remained associated with host cells. During non-lytic
emergence, spirochetes became enveloped in one or more layers
of host cell membrane and cytosol. Immune electron microscopy
showed that the outer-most surface of such enveloped spirochetes
contained human B cell-specific antigens, but lacked detectable
quantities of surface-exposed spirochetal OspA protein. Furthermore,
co-incubation of spirochetes with SKW 6.4 cells inhibited classic
complement-mediated killing in a time-dependent fashion. Such
results suggest that invasion and emergence of spirochetes into
and from human lymphocytes may mask surface-exposed antigens,
effectively protecting spirochetes from antibody-mediated recognition
and clearance. If active in natural infections, such a process
of membrane cloaking could represent a previously unrecognized
virulence strategy.
Fred
S. Kantor, MD
Yale University School of Medicine
Tick
Immunity as a Strategy for Prevention of Transmission of Spirochetes
and Other Tick-borne Pathogens
Repeated
infestations of guinea pigs with Ixodes scapularis nymphs
or larvae lead to a reduction in duration of nymphal tick attachment
and weight of recovered ticks consistent with the development
of tick immunity. Guinea pigs sensitized to I. scapularis
larvae also became immune to challenge with nymphs. Passive transfer
of serum from guinea pigs previously infested three times with
I. scapularis ticks conferred immunity on naive recipients. Two
cDNA libraries, constructed from RNA obtained from whole ticks
from tick salivary glands, were screened with sera from tick-immune
and salivary-gland-immune guinea pigs. Of the several clones
identified, one has been sequenced and identified as coding for
a salivary protein. We examined the effect of repeated infestation
of guinea pigs with I. scapularis on the capacity of infected
ticks to transmit Bb infection. Only 1/18 I. scapularis-immune
guinea pigs challenged with Bb-infected nymphal ticks
became infected, whereas 10/18 naive guinea pigs similarly challenged
became infected. We conclude that tick immunity interfered with
borrelial transmission.
Charles
S. Pavia, PhD
Associate Professor, New York Medical College
Infectivity
and Serologic Responses of Rabbits Fed upon by B. burgdorferi
Infected Deer Ticks
Charles S. Pavia*, Susan Bittker and Thomas Daniels. NY Medical
College and NYCOM Immunodiagnostic Lab
In
an effort to further characterize a rabbit model for studying
modes of borrelial transmission, we expose New Zealand white
rabbits to Ixodes scapularis ticks collected from an area
in Westchester County, NY, that is highly endemic for Lyme disease.
Ten to 15 adult deer ticks were placed onto the ears of 24 rabbits
(attachment rate averaged 8 ticks/rabbit) and the ability of
the ticks to transmit infection was based on the recovery of
live organisms from the draining cervical nodes and by serologic
testing. At 2 to 6 weeks post tick exposure, the rabbits were
sacrificed, bled out and cultures, in BSK media, were established
for the excised, hypertrophic cervical nodes. Spirochetes were
culturable from the nodes of 75% of the rabbits. Reactivity with
a specific panel of monoclonal antibodies identified all of the
isolates as Bb. Borrelial antibodies were detectable in
67% of these tick-exposed rabbits. Most of the seropositive animals
had high titers (greater than or equal to 4096) and immunoblot
analysis revealed a multi-protein antibody response. Passively
transferred high titer rabbit serum fully protected mice against
Borrelia challenge and this protection was associated with in
vitro killing of various strains of Bb by immune rabbit
serum. These data show that this model may be useful for understanding
tick-Borrelia-host interactions in acquiring LD and in
the generation and expression of protective mechanisms.
Reinhard
K. Straubinger, DVM, PhD
James A. Baker Institute for Animal Health, College of Veterinary
Medicine
The
Canine Model of LD: Migration of Bb in Tissues and its
Consequences
Reinhard K. Straubinger, Alix F. Straubinger, Luc HŠrter,
Richard H. Jacobson, Yung-Fu Chang, Brian A. Summers, Hollis
N. Erb, and Max J. G. Appel. Cornell University
Lyme
disease is not only found in humans, but it is also of significant
importance in veterinary practices located in endemic areas.
LD in dogs resembles the disease seen in man in many aspects.
Ticks initiate infection, clinical signs develop after a long
incubation period and include stiffness, depression, and lameness.
The investigation of the pathogenesis of the disease has been
the main focus of our laboratory during recent years. We have
observed the dissemination of spirochetes through tissues occurs
by migration and not by the hematogenous route.
In
this study, specific-pathogen-free beagles were infected by tick
challenges (Ixodes scapularis) at the age of six weeks.
Ticks were collected in North Salem, Westchester County, New
York, and 60-80% of the tick population carried Bb at
the time of collection. After an incubation median period of
66 days, 75% of all infected dogs developed severe clinical lameness.
Lameness and the localization of arthritis were highly correlated
to the site of infection. In a group of 13 dogs, twelve beagles
developed arthritis in the limb closest to the tick bites, in
our model mostly in the shoulder and elbow at the side of exposure.
Only one dog developed lameness in the shoulder and elbow of
the opposite side after a long incubation period of 154 days.
At
the time of lameness or five months after infection, 25 different
tissues from each dog were examined for the presence of live
spirochetes by culture. Similar tissues from the four body quadrants
were compared and the proportions of positive tissues were calculated.
In the tick exposed front quadrant, 75% of tissues harbored live
spirochetes. In the opposite front quadrant, only 60% of tissues
were positive. The infection rate dropped down to 26% in the
hind quadrant on the side of tick exposure and only 16% of tissues
of the ipsilateral hind quadrant contained viable spirochetes.
However, the number of positive tissues depended also on the
time of sampling or health status of the animal. When tissues
were examined during the episode of lameness (55-82 days after
infection), a median of 10 tissues per dog were culture-positive.
In contrast, tissues of beagles with no clinical signs of lameness
examined five months after infection contained less live spirochetes.
Only a median of 2 out of 25 possible tissues released viable
spirochetes into the culture medium. With a hematogenous spread
of the agent an equal distribution would be expected. Additionally,
the success rate for isolation Bb from pericardium, peritoneum,
and meninges was 65%, 60%, and 35%, respectively.
The
increase of serum antibody titers against Bb seems to
predict the outcome of the disease. Dogs, which fail to produce
clinical evident disease in our model showed a slower increase
of antibody titers than dogs with clinical, manifested arthritis.
The latter reached maximal antibody titers during 50 to 90 days
after infection. Infected dogs, which appear healthy, reached
the plateau of their antibody titers after 90 days, which could
indicate a low-level infection.
Taken
together, these data suggest the Bb spreads through the
body by migration and not by the hematogenous route. The distribution
of the spirochetes and frequency of culture-positive tissues
implies that a local overgrowth of organisms may trigger a strong
host response, which ultimately leads to the inflammation.
Paul
Duray, MD
Special Expert, Laboratory of Pathology
National Cancer Institute, National Institutes of Health
Acute
and Long Term Pathologic Lesions in Human Lyme Borreliosis
Review
of tissue biopsies and necropsies from both hemispheres have
provided information on the histopathology of inflammatory lesions
in documented cases. This report summarizes data on cases fulfilling
criteria for positive serology. The majority of submitted cases
either had not prior antibiotic therapy, or had therapy generally
not efficacious for Lyme borreliosis. Because multi-agent infection
may occur in Lyme borreliosis from a given tick vector (ehrlichiosis,
babesiosis), the following are summarized as borrrelia-associated
by the histochemical detection of Bb or reactive PCR for
OspA DNA sequences.
Acute
and recent infection (occurring days to 3-4 weeks): Erythema
migrans, hemorraghic attachment skin papule, lupus-like dermal
plaque, plasmacellular panniculitis, lymphoplasmacellular myocarditis,
lymphadenosis benigna cutis.
Short
term to extended (weeks to months): Myocarditis, fibrinous pericarditis,
meningoencephalitis, radiculitis/ganglionitis, splenitis, lymphadenitis,
diffuse fasciitis, tendonitis, myositis.
Long
term (months to years): Arthritis, synovitis, myositis, acrodermatitis
chronica atrophicans, late peripheral neuritis, ulnar fibrous
nodules, uveitis.
Janice
Soreth, MD
Medical Officer, Division of Anti-Infective Drug Products, Center
for Drug Evaluation and Research, Food and Drug Administration
Regulatory
Points-to-Consider in Clinical Trial Design for LD
Well-designed
clinical trials for LD are essential to define optimal treatments.
The Division of Anti-Infective Drug Products in the FDA is responsible
for reviewing data from sponsors seeking approval for drugs to
treat LD. Suggestions for trial design are offered.
Early
Lyme Disease: Double blind, prospective, randomized, multicenter
studies should be done whenever possible. At a minimum, an investigator-blinded
study can be considered if dosing schedule or mode of administration
of either test or comparator drug make double blinded trials
impractical. Placebo-controlled trials for acute erythema migrans
(EM)-associated Lyme disease are inappropriate but are encouraged
for trials studying chronic LD.
Comparative
Agent: The choice of comparator drug should be an agent shown
in peer-reviewed journals to be adequate.
Late
(Chronic) Lyme Disease: Patients enrolled in theses studies should
have previous physician-documented EM or well documented seroconversion.
At least one objective finding of late LD relating to musculoskeletal,
neurologic, and/or cardiovascular systems should be present as
a requirement for entry into the trial.
Conclusion:
A detailed "Points-to-Consider" document further elaborating
suggested clinical trial designs for LD will be available. The
Division encourages all sponsors who seek a claim for LD, early
or late, to discuss their study proposals with the Anti-Infective
Division of the FDA prior to initiation.
Jeffrey
J. Collins, PhD
Senior Clinical Program Head, Glaxo Wellcome Inc.
Cefuroxime
axetil: First Approved Antimicrobial for LD
Objective:
To compare the efficacy of cefuroxime axetil (Ceftin¨; CAE)
and doxycycline (DOC) in the treatment of patients with LD associated
with erythema migrans (EM).
Methodology:
Two identical randomized, multicenter, investigator-blinded clinical
trials were conducted 1 year apart. Patients with physician-documented
EM were eligible for enrollment and were randomized to receive
20 days of oral treatment with either CAE, 500mg b.i.d. or DOC,
100mg t.i.d. Clinical evaluations were conducted during treatment
(8-12 days) and at 1-5 days and 1, 3, 6, 9 and 12 months posttreatment.
Patients were assessed as to the resolution of EM and of signs
and symptoms related to early LD, as well as prevention of late
LD.
Results:
A total of 355 patients were enrolled in the two studies, 182
and 173 of whom were treated with CAE or DOC, respectively. A
satisfactory clinical outcome (success or improvement) was achieved
in 137 of 151 (91%) evaluable patients treated with CAE and in
133 of 144 (92%) evaluable patients treated with DOC (difference,
-1%, 95% confidence interval [CI], -9 to 9%). Of the patients
with satisfactory outcomes at 1 month posttreatment who were
evaluable at 1 year posttreatment, a satisfactory outcome was
achieved in 105 of 113 (93%) and in 88 of 91 (97%) patients treated
with CAE and DOC, respectively (difference, -4%, 95% CI, -11
to 6%). Drug-related adverse events were reported in 23% of patients
treated with CAE and in 32% of those treated with DOC (p=0.072).
DOC was associated with more photosensitivity reactions (9% vs.
0%; p<0.001), and CAE was associated with more cases of diarrhea
(11% vs. 3%; p=0.005). Jarisch-Herxheimer reactions occurred
in 6% of the patients in each treatment group.
Conclusion:
Twice daily CAE is well tolerated and appears to be equally as
effective as DOC given three times daily not only in the treatment
of early LD, but also in preventing the subsequent development
of late LD. CAE (Ceftin®) was approved by the FDA on December
19, 1996, for the treatment of "Early Lyme disease (erythema
migrans) caused by Borrelia burgdorferi."
Gerold
Stanek, MD
Hygiene Institut Der U. Wein, Austria
Infectious
Risk after Tick-bite in Middle Europe
No
abstract available
Bettina
Wilske, MD
Max Von Pettenkofer Institute, Germany
Molecular
and Immunological Variability of European Borrelia burgdorferi
Strains and the Implications for Diagnosis and Prophylaxis of
Lyme Borreliosis
Borrelia
burgdorferi sensu lato, the etiological agent of Lyme
borreliosis is considerably heterogeneous in Europe. Strains
isolated from humans belong at least to three different species,
B. burgdorferi sensu stricto, B. afzelii and B. garinii
(Wilske et al., 1993a and 1996). All three species have been
isolated also from Ixodes ricinus the main tick vector.
Two additional species designated groups VS116 and PotiB respectively
have been isolated from ticks in Europe (Postic et al., 1994).
Since
the outer surface proteins OspA and OspC are the most promising
candidates for a Borrelia vaccine (Schaible et al. 1990, Preac-Mursic
et al, 1992) the immunological heterogeneity of these proteins
was investigated. By immunological analysis with monoclonal antibodies
and sequence analysis of PCR-amplified ospA and ospC we previously
identified at least seven OspA-(Wilske et al., 1993a) and 15
different OspC-types respectively (Wilske et al., 1995, Jauris-Heipke
et al. 1995). The high OspC heterogeneity may be due to immune
selection of OspC variants (at least partially due to recombination
events among ospC genes as demonstrated by OspC gene sequence
analyses) in the mammal host where OspC is the predominantly
expressed Osp. OspA-serotyping of a large panel of isolates revealed
the following: Whereas human skin isolates (n=68) were quite
homogeneous (84% belonged to OspA-serotype 2 or B. afzelii),
isolates from human cerebrospinal fluid and from ticks (n=43
and n=90 respectively) were considerably heterogeneous in their
OspA-serotypes with prevalence of types 3-7 associated with B.
garinii (about 70%). OspA-type 4 was often found among isolates
from cerebrospinal fluid (28%), but only in 1% of the skin isolates.
From ticks OspA-type 4 strains have not been cultured so far.
However, as reported in a previous study, type 4 - ospA could
be detected in ticks by the highly sensitive PCR. OspA-type 4
strains - notably isolated so far only from human specimens -
are possibly present in the tick only in mixed infection and
overgrown in culture by other types. The OspA-type 4 strains
so far analyzed (n=7) were nearly identical in their OspC sequences
(Wilske, unpublished results). In contrast, OspC from other strains
of identical OspA-type were rather heterogeneous. Thus OspA-type
4 strains may represent a newly emerged variant possibly associated
with higher pathogenicity since type 4 strains revealed higher
resistance than other serotypes (van Dam et al., 1997).
Since
it has been shown that recombinant OspA protects mice only against
homologous strains the heterogeneity of OspA and OspC (inter-species
sequence identities are only 77-83% and 71-75% respectively)
need to be considered in respect to development of a borrelia
vaccine. Higher inter-species sequence identities (approximately
87-90%) were observed for the membrane associated p39 (BmpA),
another candidate for a borrelia vaccine (Rossler et al, 1996).
However systematic cross protection studies and analysis for
neutralizing paratopes are still needed. The OspC heterogeneity
plays a minor role for serodiagnostic purposes since mainly conserved
epitopes are recognized by the sera from patients (Wilske et
al., 1994). In contrast serodiagnostic relevant epitopes appear
to be more heterogeneous in BmpaA. The flagellin genes are considerably
conserved (inter-species sequence identities 95-98%). Surprisingly,
for serodiagnosis, the use of truncated internal flagellin fragments
from different strains results in significant differences in
sensitivity (Wilske et al., 1994). When different strains of
B. burgdorferi s.l. are used for the conventional western
blot interpretation criteria must be developed for individual
strains (Hauser et al., 1997).
Joseph
J. Burrascano, Jr., MD
East End Medical Associates, Southampton Hospital
Diagnosis
and Treatment of Early Lyme Disease
The
widening availability of sensitive antigen capture assays have
demonstrated how prevalent and sever late, disseminated LD can
be. Data shows that as more time elapses after exposure to Borrelia
burgdorferi (Bb) before effective treatment is begun, the
more difficult it becomes to eradicate this infection. The most
severe cases do not at this time appear to be curable with currently
available medications. Therefore, it follows that the best way
to treat Lyme is to do so as early in it's course as possible.
Data also shows that effective treatment involves the proper
dosing of antibiotics, for Bb disseminates early and blood
levels of orally administered antibiotics is unreliable. Recognition
of early Lyme and the proper management of this entity is reviewed,
with the conservative goal of preventing all cases of late disseminated
Lyme utilizing aggressive therapy specific to the patient's presentation.
Franc
Strle, MD
University Clinical Center, Department of Infection, Yugoslavia
Clinical
Presentation and Treatment of Skin and Nervous System Infection
in Lyme Disease
No
abstract available
Patricia
K. Coyle, MD
Department of Neurology, SUNY at Stony Brook School of Medicine
Neurologic
Lyme Disease: Diagnosis and Treatment
The
nervous system is one of the major target organs of Lyme disease.
Neurologic involvement can occur at any stage of infection. A
variety of central (CNS) and peripheral (PNS) nervous system
syndromes are seen. The diagnosis and treatment of neurologic
LD face major current issues which need to be resolved.
Diagnosis
of neurologic Lyme disease is hampered by several problems. They
include specific properties of the organism, lack of a readily
available active infection assay, inappropriate reliance on host
immune response, incomplete data on the spectrum of clinical
syndromes, lack of a universal pathognomonic infection marker,
failure to evaluate patients, and incomplete data on the role
of tick coinfection agents
Treatment
of neurologic LD is also hampered by several problems. They include
lack of reliable antimicrobial cure criteria, discrepancy between
in vitro and in vivo studies, lack of reliable
controlled trials data, persistence of symptoms post treatment,
and incomplete data on the role of tick coinfection agents as
well as host immune response.
This
presentation will discuss current state of the art in the diagnosis
and treatment of neurologic LD, will highlight current controversies,
and will outline areas for future study.
Jorge
L. Benach, PhD
Department of Pathology, SUNY Health Science Center
Subtle
Injury to Transformed Neural Cell Lines by Bb
Lyme
disease is a chronic infection caused by the spirochete Borrelia
burgdorferi. The clinical manifestations of Lyme disease
include a wide spectrum of acute and chronic neurological disorders
which begin with alterations of the blood brain barrier. The
acute neurological manifestations include cranial neuritis, radiculoneuritis,
and meningitis. The Garin-Bujadoux-Bannwarth (GBBS) syndrome
is a lymphocytic meningoradiculitis seen more in Europe than
in the United States. Later manifestations include peripheral
neuritis, an encephalopathy with impaired cognitive functions
and memory loss, and a rare encephalitis (leukoencephalitis)
and encephalomyelitis. Magnetic resonance imaging of the brain
has disclosed damage to the white matter in some patients. In
addition, electromyographic studies have also shown axonal damage
and provided evidence for demyelination. Brain and peripheral
nerve biopsies have disclose both axonal and myelin disruption
as well as microgliosis. Peripheral nerve biopsies have disclosed
neuropathies of vasculitic origin. In the CNS, the neurons as
well as glial cells could be involved in the pathogenesis of
this disease. Evidence for Bb invasion of the CNS include
the detection of spirochetal antigens by immunological methods
as well as by PCR amplification of spirochetal DNA. Using primary
cultures of neonatal rat brains, we demonstrated that spirochetes
could adhere to cell of the CNS and damage oligodendrocytes.
In
this study, it was found that time in culture was an important
but not the sole factor for greater adherence of spirochetes
to monolayers of C6 rat glioma and PC-12 rat pheochromocytoma
cells than high passage strains. Polar adhesion was demonstrated
using scanning and transmission electron microscopy. The tips
of Bb spirochetes could partially penetrate the plasma
membrane of neural cells resulting in the formation of cavities
and surface blebs. Internalization of spirochetes by the neural
cells was not observed. Adhesion of spirochetes to C6 glioma
and to undifferentiated PC-12 cells resulted in sublethal and
reversible injury. Adhesion of spirochetes to PC-12 cells differentiated
with nerve growth factor resulted in a loss of the integrity
of the monolayer and in cell death. These results demonstrate
that Bb can injure neural cells directly.
Brian
A. Fallon, MD, MPH, MEd
The NYS Psychiatric Institute
Psychiatric
Aspects of LD and the Use of SPECT Imaging
Several
controlled studies have demonstrated that adults with LD experience
depression or irritability more commonly than patients with other
diseases or normal controls. In addition reports have associated
LD with a vast array of psychiatric disorders, such as psychoses,
mania, panic attacks, obsessive compulsive disorder, dementia,
and anorexia nervosa. In children with neurologic LD, disturbances
of behavior or mood are frequently reported, second in frequency
only to headaches. Results from our center and others indicate
that although mood disorders are common, so too are disorders
of attention in both adults and children. In this talk, the results
of four studies will be presented: an epidemiologic community
study of psychiatric disorders among children; a clinical study
of adults and children with LD using structured diagnostic interviews;
a series of adults with LD tested with SPECT imaging at two points
in time; and a series of adults with Lyme encephalopathy tested
with Xenon regional cerebral blood flow at two points in time.
The diagnostic and therapeutic implications of these findings
will be discussed.
Sousan
Sayahtaheri Altaie, PhD
Clinical Microbiology Review Officer, U.S. Food and Drug Administration
Transplacental
Transmission of Bb in a Murine Model
Sousan Sayahtaheri Altaie, Sologna Mookherjee, Ezzat Assian,
Ferdose. Al-Taie, Shaheen Nakeeb, and Saeeda Siddiqui. SUNY at
Buffalo.
Congenital
B. burgdorferi infections have been reported in the literature
in three neonates whose mothers had Lyme borreliosis during the
first trimester of pregnancy. In an effort to facilitate studying
Lyme borreliosis during pregnancy a murine model was developed.
Splenectomized
mating pairs, 6-8 week-old C3H/HeJ mice, were divided into groups
A-D.
- Group
A: infected females, uninfected males;
- Group
B: infected males, uninfected females:
- Group
C: infected males and females; and
- Group
D (control): uninfected males and females.
The
infectious dose was 106-107 Bb (strains 297, W18, and
Son-1) in 250 ul SKB II media administered subcutaneously. The
control group receivedsterile SKB II. The studies were performed
in two phases. In phase one the males were infected and immediately
mated. The day of coital plugging was established as day 1 of
pregnancy. The pregnant mice were then infected during early-
[day 6-7 post copulation (PC)], middle-(day 9-10 PC) and late-
(day 12-13 PC) gestation periods. Period mice were sacrificed
6 days post infection. Fetuses and their placentas were harvested
and cultured for nine weeks in SKB II. No Bb was detected
by culture, thus, PCR was performed on the cultures for detection
of Bb DNA. There were no appreciable differences observed
in transmission rates among the three Bb strains, therefore,
the data were pooled. No Bb was detected in samples from
group B. In groups A and C combined, during early-gestation Bb
was detected in 4/30 (13%) fetuses and 3/30 (10%) placentas.
During middle-gestation Bb was detected in 3/57 (5%) fetuses
and 4/57 (7%) placentas. No Bb was detected in fetuses
or placenta during late-gestation period.
In
phase two studies mating pairs were assigned to groups A-D and
were infected immediately prior to mating. The pregnancies were
allowed to go to term and the pups were sacrificed at 1, 7, 14,
and 21 days of age. The milk content of the stomach, sections
from ear, skin, heart, liver, spleen, brain, bladder, and kidney
of all pups were cultured for Bb. Milk was not cultured
from sacrificed 21 day-old weanlings. Transmission to offspring
was indicated when Bb was isolated from any tissue. Of
25 infected females, 2 (8%) transmitted Bb to their pups
on day one via their milk. No transmission was detected via milk
on days 7 or 14. Among 49 infected females from groups A and
C, 5 (10.2%) transmitted Bb to their pups either in utero
or intrapartum. Two of the transmissions were detected on day
1, two on day 7, and one on day 14. From the 132 pups at risk
for close contact infection in group B, 9 pups were infected
resulting in a close contact transmission rate of 6.8%. This
transmission model suggests that Bb can be transmitted
in utero. Increasing the inoculum size and/or changing the route
of inoculation to intrauterine or intra-amniotic may enhance
infection rates. This model has the potential to be used to study
intervention strategies for gestational LD.
Irwin
T. Vanderhoof, PhD, FSA, ACAS, CFA, CLU, BS
NYU Stern School of Business
Lyme
Disease - Cost to Society and Symptoms
The
Lyme Disease Foundation and Society of Actuaries have jointly
built a data base of victims of severe symptoms of this disease.
A questionnaire was used to develop information on the costs
of the disease based on medical expenses, loss of earnings etc.
The data base also includes extensive data on symptoms of these
patients.
The
presentation summarizes this information and demonstrates the
justification for greater research fund allocation to reduce
the costs of this disease to society. It also demonstrates that
the disease is characterized by multiple system involvement in
each of the various areas of the country to which it is endemic.
Steven
E. Schutzer, MD
Department of Medicine, UMDNJ
Present
and Future Testing for Lyme Disease
Laboratory
diagnosis of Lyme disease has been hampered by a combination
of biological and technical factors related to infection with
B. burgdorferi. The organism grows slowly and is difficult
to find except in erythema migrans lesions. Commercial serologic
tests require a long lag phase of up to 4-6 weeks before seropositivity
to any of the Bb proteins reaches a detectable threshold.
Eventually in most cases antibodies are produced against Bb
proteins. Some antigens are shared with other infectious agents
and therefore , by themselves are not discriminating of a Bb
infection. Present interim recommendations of ELISA followed
by Western blot, counting the number of positive bands, which
include non Bb specific ones has not been universally
effective. Future serologic diagnostic testing, regardless of
the approach, will likely involve identification of specific
antibody or antigen related to unique Bb proteins that
are expressed in vivo such as some of the Osp Ags, p35, p37,
p39, p93 as well as unique combinations and sequences of these.
Certain ones may have relevance at difference stages of disease
and in different body compartments.
Raymond
J. Dattwyler, MD
Professor of Medicine, Chief of Allergy & Immunology
Director of LD Center, SUNY at Stony Brook School of Medicine
New
Serologic Approaches to the Diagnosis of LD
B.
burgdorferi
expresses a large number of proteins. Outer surface proteins
(Osp) A through F have been described and others may be defined
in the future. Three proteins, OspA, OspB and OspC are best characterized
and appear to be the most important immunologically. OspA and
OspB are encoded on a 49 kilobase (kB) linear plasmid and OspC
is encoded on a 28kB circular plasmid. OspA is the major surface
protein expressed by this spirochete in the tick and when cultured.
However, once tick feeding begins, OspA expression stops and
OspC becomes the dominant outer surface protein. Thus, in the
initial phase of mammalian infection, OspC is the major surface
protein.
Definitive
diagnosis of Bb infection is complicated by the following
four key factors:
- The
varied nature of the presentation of the clinical symptoms
- The
overlap of these symptoms with numerous other infections and
non-infectious diseases.
- The
difficulty of isolating and culturing the infectious agent from
clinical samples.
- The
organism's extensive cross-reactivity with other infectious agents
and immunopathologic factors.
Testing
for Bb infection is frequently an early step in the differential
diagnosis of patients with rheumatologic or neurologic symptoms.
Definitive diagnosis or exclusion requires one or more serologic
assays. With more than 5 million LD tests performed annually
in the US, accurate, reliable assays are essential both to ensure
early treatment of infected individuals and to exclude the large
majority of uninfected patients with "Lyme-like" symptoms.
Except
in patients with EM, Bb is rarely observed in clinical
samples. Direct diagnosis via microbiological techniques is not
practical at present. Instead, Bb infection is defined
indirectly by detection of a humoral immune response to the organism.
Antibody responses in the infected host follow the usual pattern.
IgM appears first, followed by increasing levels of IgG and IgA
during the second and third months of infection. Once the latter
responses are established, they may remain detectable for years.
In most patients, the earliest immune response to Bb is
directed against the proteins flagellin (p41 or Fla) and OspC
(25kd): the 35-37 kd and 39 kd proteins also elicit an early
response in many individuals. The repertoire of antibody responses
continues to expand as the disease progresses.
One
serious issue in detecting antigens is that many Bb antigens
comprise epitopes that cross-react with antibodies directed against
other common infectious agents. For example, Bruckbauer et al.
demonstrated extensive cross-reactivity between Bb and
the bacterial pathogens B. hermsii, T. pallidum, L. interrogans,
N. meningitidis, H. influenzae, Y. enterocolitica,, C. jejuni,
L. monocytogens, P. aeruginosa, E. coli, S. typhimurium, S. flexeri,
and L. micdadei. The Borrelia proteins in the 60-75 kd
range, p41, p33, and two proteins of about 20kd are among the
most highly cross-reactive. The immunodominant 41 kd flagellin
antigen is not cross-reactive with non-flagellated bacteria,
but it is highly cross reactive with similar protein from other
spirochetes: greater than 50% of normal healthy adults with no
history of Bb infection have circulating anti-41 kd IgG.
Many individuals also have antibodies directed against other
spirochetal antigens, most commonly the 60 kd common bacterial
and 66 kd antigens. In addition, the accuracy of indirect assays
for Bb infection can be compromised by immunopathogical
conditions such as rheumatoid arthritis and systemic lupus erythematosus.
Hence the positive predictive value of commercially available
serological assays is poor because of the relatively high incidence
of false positives.
Currently,
the serological tests commonly used to detect antibodies against
Bb are indirect immunofluorescence assays (IFA) or enzyme-linked
immunosorbent assays (ELISA). These tests use whole cell Bb
preparations. Bb are used as the antigen substrate for
IFA and crude fractions of sonicated organisms are used for most
ELISA tests. The use of whole Borrelia as the source of antigen
introduces a high degree of variability and a large number of
proteins that contain cross-reactive epitopes. In addition, since
the assays are not standardized, there are significant differences
in how the tests are performed and reported. For example, ELISA
tests of Bb performed at five academic research centers
and the CDC was recently evaluated. It was noted that there was
only limited concordance among the results from laboratories
that did not use immunoblotting to supplement the ELISA results.
Moreover, the group in the study that used a commercial test
had the poorest results by virtually all criteria. This study,
together with other reports of poor results in terms of the accuracy,
precision, and concordance among the various test kits on the
market, has led to recommendations that all positive whole cell
Borrelia ELISA and IFA test results be confirmed by immunoblotting
assays. While immunoblotting has been shown to improve the accuracy
of LD testing, the procedure is cumbersome and adds significantly
to the time and cost of definitive diagnosis of LD.
We
have identified a series of highly specific epitopes on several
Bb antigens. Recombinant DNA techniques have then been
used to isolate the genomic sequences coding for these epitopes
and to incorporate them into vectors that can be expressed in
E. coli, Gerber et al. demonstrated that an ELISA using
recombinant OspC is more sensitive than a whole cell ELISA in
detecting Borrelia antibodies in patients with erythema migrans.
The use of single recombinant protein, however, may limit the
utility of the assay. By combining specific portions of key proteins,
into unique chimeric recombinant proteins, specific proteins
have been developed that contain a higher ration specific to
non-specific epitopes than the native proteins.
Stephen
Straus, MD
National Institutes of Health
Chronic
Fatigue Syndrome and Chronic LD: Living with Diagnostic Uncertainty
Clinical
experience and published reports indicate that persistent fatigue
is common following acute and chronic Lyme infection. Fatigue
often persists for prolonged periods after antibiotic treatment
and can be severe and debilitating. Many such patients meet criteria
for chronic fatigue syndrome (CFS). What are the implications
of this finding?
The
mechanism of persistence of fatigue and other symptoms after
treatment for LD is not understood. Possible explanations include
a reactive, post-infectious type process or the persistence of
borrelia infection causing a nonspecific syndrome.
CFS
is diagnosed by exclusion using case criteria such as those developed
by the CDC in 1994. They require debilitating fatigue for at
least 6 months, unrelieved by rest, and accompanied by 4 or more
of 8 symptoms including neuropsychological problems, muscle and
joint aches. Physical findings and laboratory tests do not support
the diagnosis. Evidence of chronic infection excludes consideration
of CFS. CFS is often precipitated, through, by an acute infectious
episode. It peaks in incidence in the 4th decade and is more
prevalent in women.
For
most chronic infections, microbiologic testing confirms an organism,
or the injury inflicted by that organism reveals itself in an
objective manner. Unfortunately, these can not yet be said of
chronic LD. Until diagnostic tests are proven to reliably confirm
persistent borreliosis, one is not able to exclude a reactive
or postinfectious process in individuals who otherwise meet CFS
criteria after treatment for LD. Management of the patient as
one with CFS may prove beneficial, and should not be rejected
through a strict reliance on empirical antibiotic treatment.
Lesley
A. Fein, MD, MPH
Private Practice, Rheumatology
Lyme
Disease versus Fibromyalgia
The
clinical definition of fibromyalgia and Lyme disease will be
presented. Clinical and other diagnostic criteria (laboratory
testing, radiology studies, etc.) will be examined to differentiate
between the two. Examination of recent literature on fibromyalgia
suggests a neurochemical etiology. These studies will be discussed.
Brian
A. Fallon, MD, MPH, MEd
The NYS Psychiatric Institute
Lyme
Disease versus Somatoform Disorders
The
category of somatoform disorders includes somatization disorder,
conversion disorders, pain disorder, hypochondriasis, and body
dysmorphic disorder. The diagnostic criteria and treatment for
these disorders will be reviewed. Because the essential aspect
of a somatoform disorder is the presence of physical symptoms
or complaints in the absence of objective evidence, physicians
may mistakenly label a patient as having a somatoform disorder
when in fact the symptoms are signs of an underlying medical
illness. This mistake often occurs among patients with Lyme disease
because of the multisystem nature of the illness, the problems
with diagnostic tests, and the common concurrent mood or anxiety
disorders. Physicians may also mistakenly diagnose a patient
with a somatoform disorder as having LD. The utility of self-report
questionnaires, such as the MMPI or the Whiteley Index, in the
identification of hypochondriasis will be reviewed. Clues to
aid in the differential diagnosis will be presented, along with
vignettes drawn from clinical practice.
Carrie
A. Hughes, PhD
Department of Microbiology and Immunology, Georgetown University
School of Medicine
Complement
Resistance in Borrelia burgdorferi
Borrelia
burgdorferi (Bb)
the causative agent of Lyme disease, must evade host innate immunity
to establish infection. In the infected host, the spirochete
must resist the bacterial action of the alternative complement
system to survive, invade and establish infection prior to the
production of a specific immune response directed toward the
spirochete. This study investigates the resistance of Bb
to killing by the antibody-independent alternative complement
pathway.
Virulent
wild-type Bb strain 297 (WT297) is resistant to killing
by normal human serum (NHS) in bactericidal assays. In contrast,
mutant Bb strain 297 (MUT297), which lacks the plasmid
that encodes outer surface protein (Osp) A and OspB, is killed
following incubation with NHS. Heat-inactivation of NHS abrogates
killing of MUT297. In addition, MUT297 is killed by NHS in the
presence of EGTA and MgCl2, which inhibits the classical complement
pathway but not the alternative pathway. This suggests that killing
of MUT297 occurs via the alternative complement pathway in the
absence of Bb-specific antibody.
The
deposition of the membrane attack complex (MAC) on the surface
of Bb following incubation with NHS was examined by indirect
ELISA using MAb specific for the neoepitope expressed on activated
MAC. The deposition of MAC is detected with both variants. However,
the formation of MAC was significantly more rapid with MUT297
than with WT297. Therefore, WT297 activates complement but is
resistant to lysis, suggesting that the improper insertion of
MAC to the Bb membrane may confer complement resistance
to the spirochete.
The
ability of Bb to resist complement-mediated killing may
be a mechanism of invasion used by the spirochete to establish
infection. The identification of factors associated with complement
resistance will contribute to a better understanding of the multifactorial
process of spirochete pathogenesis and the interaction of the
spirochete with its host.
Max
J. G. Appel, DVM, PhD James A. Baker Institute for Animal Health
Cornell University College of Veterinary Medicine
Resistance
of Bb to Antibiotic Treatment in Beagles
There
appears to be a general agreement that Borrelia burgdorferi
persists in humans and animals for months or years, and perhaps
for life despite a strong humoral immune response. The question
remains unresolved whether antibiotic treatment eliminates the
persistent infection. Although Bb is susceptible to antibiotics
in vitro, the in vivo efficacy of antibiotics is still debated.
Spirochetes tend to invade poorly vascularized connective tissues,
where they may be protected from antibiotics. In that respect,
borrelias behave similar to other members of the order Spirochitaceae
like Leptospira (causing leptospirosis) and Treponema (causing
syphilis) which are known to persist despite treatment with certain
antibiotics.
We
have addressed the unresolved question whether Bb persists
in man and animal after conventional antibiotic treatment. The
canine model appears to be valuable because LD in dogs has many
similarities with Lyme disease in humans. We have shown that
treatment with high doses of amoxicillin or doxycycline for a
30 day period was not sufficient to eliminate persistent infection
in dogs. The dogs had been exposed to Bb infected ticks
(Ixodes scapularis) approximately two months before antibiotic
treatment was initiated. Less borrelias were found after treatment,
antibody levels declined, and joint lesions were prevented or
cured. However, some spirochetes persisted in dog tissues for
up to 6 months after treatment and antibody levels began to rise
again at that time. By isolation and/or PCR Bb was demonstrated
in tissues (lymph node, synovium, fascia, muscle) of 3 of 5 dogs
that were treated with amoxicillin and 4 of 6 dogs that were
treated with doxycycline. The possibility of clinical and pathological
relapses, therefore, remain.
Catherine
J. Luke, PhD
Department of Microbiology & Molecular Genetics
University of California at Irvine College of Medicine
Expression
of Borrelia DNA in Mammals
Catherine J. Luke, Kristin Carner*, Charles D. Sohaskey, Xiaowu
Liang* and Alan G. Barbour. Department of Microbiology &
Molecular Genetics, *Vical Incorporated.
Expression
of Borrelia DNA in humans and other animals has relevance for
both immunoprophylaxis and pathogenesis. We showed that immunization
of mice with plasmid DNA bearing the ospA coding sequence from
Borrelia burgdorferi strain B31 resulted in the expression
of immunogenic OspA protein and the production of OspA-specific
antibodies that protected mice against infectious challenge.
In this plasmid the ospA gene was under the transcriptional control
of the cytomegalovirus (CMV) immediate early promoter, and the
prokaryotic signal sequence was replaced by the human tissue
plasminogen activator (hTPA) signal sequence. Further characterization
of the OspA protein expressed by mammalian cells, including immunoblot
analysis, phase separation and glycosidase digestion, revealed
that the protein is very likely glycosylated. Simon and co-workers
reported that the ospA gene can also be expressed in mammalian
cells under the control of its endogenous promoter, and that
this can occur even when the prokaryotic signal sequence is present
(Simon et. al, Eur. J. Immunol. 26:2831-2840, 1996).
These
findings have important implications in the light of the studies
by Persing and colleagues (Persing et at, J. Infect. Dis.
169:668-72, 1994) in which ospA sequences but not sequences from
chromosomal genes were amplified by polymerase chain reaction
of synovial fluid from patients with Lyme arthritis, suggesting
that Borrelia genes may be expressed in the mammalian host in
the absence of intact organisms. To address this possibility,
mammalian cells were transfected with plasmid DNA in which a
chloramphenicol transferase (CAT) reporter gene was placed downstream
of the ospA promoter. There was no measurable expression of CAT
when transfected cell lysates were assayed for CAT activity.
In contrast, high CAT activity was detected in lysates from cells
transfected with a eukaryotic expression vector bearing the CMV
immediate early promoter and the same reporter gene. In conclusion,
we have shown that Borrelia genes can be expressed in mammalian
cells under the control of a eukaryotic promoter and a eukaryotic
post-translation modification signal sequence and that the expressed
protein is capable of eliciting a specific, protective antibody
response. Evidence for the expression of Borrelia genes in the
mammal under the control of endogenous prokaryotic elements,
however, remains equivocal.
Adriana
Rodriguez Marques, MD
National Institutes of Health
Intramural
Clinical Program on Chronic Lyme Disease
Lyme
disease has become a highly controversial illness. The issue
that has probably generated the most controversy today is the
mechanism underlying persistent signs and symptoms of disease,
despite the administration of what is currently considered to
be adequate antibiotic therapy. Determining whether chronic LD
is caused by persistent infection or is a post-infectious disorder
is a fundamental issue. Finding the answer to this question for
any individual patient will have an important bearing on his
or her treatment, as our approach to the disease would be different
depending on the underlying mechanism.
To
try to answer some of this questions, we developed a new study
in collaboration with scientists in National Institute of Allergy
and Infectious Disease, in the National Institute of Neurological
Disorders and Stoke (NINDS), in the National Institute on Deafness
and Other Communication Disorders (NIDCD), in the National Institute
of Mental Health (NIMH) and with leading LD specialists at Stony
Brook, New England Medical Center and the Mayo Clinic. The study
is now open for accrual.
The
objectives of this study include evaluation of diagnostic laboratory
abnormalities and their correlation with the various syndromes;
assessment of the extension of infection with Bb and its
consequences to patients; and the study of the role of immune-mediated
and other pathogenic mechanisms in injury to the nervous system,
including spirochete interactions with the immune system, auto-antibodies,
cytokines, cellular immune responses, and immune complexes.
The
first part of the study is an evaluation of patients suspected
to suffer from chronic neuroborreliosis who have documented positive
serology to Bb (confirmed by IgG Western blot). These
patients will be compared to other patients with LD (patients
with Lyme arthritis, people who recovered from LD and people
who are found to be seroposivtive for Bb), patients with
multiple sclerosis and healthy volunteers. The evaluation includes
lumbar puncture, high-resolution MRI, audiologic evaluation,
neuropsychological testing, immunological studies, and clinical
and laboratory assessments with parallel evaluations of multiple
available laboratory tests for Bb infection. Patients
with chronic neuroborreliosis who have evidence of persistent
infection (as defined in the study) are offered four weeks of
ceftriaxone therapy in the second part of the study. These patients
will then be serially reevaluated using a battery of tests similar
to the one in the first phase. An important part of both phases
of this study will be the preparation of a uniform and well-defined
bank of clinical specimens that will be available to help evaluate
new tests for LD.
We
realize that the patient group we are starting with may represent
only the apex of a pyramid of diseases that may be related to
Bb infection. However, our intention in this first study
is to develop answers for one well-defined patient group, that
might eventually be applicable to others. We think that these
initial studies involving very selected patients will provide
new information about chronic LD, and suggest additional avenues
for patient care and research.
Mark
Klempner, MD
Tufts New England Medical Center
Randomized,
Double-blinded, Placebo-controlled, Multicenter Trials of the
Safety and Efficacy of ceftriaxone and doxycycline in the Treatment
of Patients with Seropositive and Seronegative Chronic LD
A
description of the NIH supported clinical protocols for the characterization
and treatment of patients with chronic Lyme disease (CLD) will
be presented.
- The
objectives of these studies are to determine whether:
- intensive
antibiotic treatment benefits seropositive and seronegative patients
with CLD,
- evidence
of persistent infection with Bb can be found in patients with
CLD,
- evidence
of coinfection with other microorganisms can be found in patients
with CLD,
- specific
clinical or laboratory parameters improve in patients who receive
antibiotic therapy compared to patients who receive placebo,
and
- specific
parameters are predictive of a response to therapy should it
be observed.
These
studies are Phase III, randomized, double-blinded, placebo-controlled,
multicenter trials (two centers). 260 patients will be enrolled
in the studies and randomized to receive either antibiotic therapy
or placebo in a 1:1 ratio; antibiotics and placebo will be administered
both intravenously and orally. The antibiotic regimen will be
intravenous ceftriaxone 2 gms/day for 30 days followed by oral
doxycycline 200mg/day for 60 days. Placebos identical in appearance
to the intravenous and oral antibiotics will be administered
by the same routes and for the same duration to patients randomized
to the placebo group. Initial and serial analyses during treatment
will include PCR of plasma and CSF for borrelia and other organism
DNA, borrelia urinary antigen, and serum antibodies to organisms
transmitted by Ixodes ticks. Primary outcome analysis for the
efficacy of antibiotic therapy will be determined by improvement
in the patient's health related quality of life as measured by
the SF-36 Health Survey. Other assessments will include changes
in pain and cognition using scales from the Medical Outcomes
Study, the fibromyalgia impact questionnaire, neuropsychological
tests, and nerve conduction studies for patients with neuropathic
pain.
Kenneth
B. Liegner, MD, PC
New York Medical Center
Seronegative
Chronic Meningoencephalomyelitis in Lyme Disease
A
severe case of seronegative Lyme neuroborreliosis is presented
in detail. It is argued that the seronegative subset subsumes
individuals most severely affected by LD and that currently promoted
laboratory standards of diagnosis fail utterly to detect such
cases. Analogy is made to dichotomous disease expression in leprosy
which depends upon host T-cell and B-cell response.
Sam
T. Donta, MD
Professor of Medicine, Boston Univ. Medical School
Management
of Patients with Lyme Disease: Reactivation of Latent Infection
In
examining the various clinical courses of Lyme disease in patients,
several patterns can be observed that can be used as the basis
for developing models of chronic infection. The clinical result
of initial infection that is untreated or partially treated may
be the progressive development of symptoms, a period of asymptomatic
infection prior to the onset of symptoms, or no obvious clinical
infection. The mechanisms responsible for these patterns likely
involve both bacterial (e.g. initial inoculum size, antigenic
variation, intracellular localization, toxins) and host (e.g.
hormones, cytokines) factors.
Particularly
intriguing is the issue of reinfection vs. reactivation of latent
infection. Using examples of other chronic infectious diseases
(e.g. tuberculosis, salmonellosis, herpes virus, syphilis), and
analyses of patients with recurrent tick bites, patients who
received Lyme vaccines, as well as other patients, including
pregnant women, the evidence would seem to favor reactivation
of latent infection vs reinfection in patients who were previously
asymptomatic.
A
better understanding of the microbial and host factors responsible
for chronic infection should lead to better methods to diagnose
and treat the disease.
Edward
M. Bosler, PhD - SUNY at Stony BrookSchool of Medicine
Daniel E. Dykhuizen, PhD - SUNY at Stony Brook Department of
Ecology and Evolution
Genetic
Variation within Local Populations of Bb
Lyme
disease, which is caused by the spirochete Borrelia burgdorferi
sensu stricto in the United States, is endemic in eastern
Long Island, New York. A three-year population genetic study
of the Bb populations has been conducted to detect the
spatial and temporal dynamics of the genetic structure in this
area. The genetic diversity of Borrelia populations was determined
by cold single-strand conformation polymorphism (Cold SSCP) analysis
on the outer membrane protein A locus, OspA, and on the outer
membrane protein C locus, OspC , the genetic diversity at the
OspC locus is much greater than that at the OspA locus. The two
genes are in almost total linkage disequilibrium, so distinct
strains can be defined. The frequency of these strains is the
same across eastern Long Island in any particular year, but can
change from year to year. The strain frequencies of the spirochetes
infecting foraging nymphal ticks is different from the strain
frequencies infecting foraging adult ticks. This suggests possible
host preference of particular genotypes. This distinction and
an animal study using chipmunks suggest that ticks infected with
Bb as larvae have high mortality in the wild. Furthermore, Ewen-Watterson
tests of neutrality revealed that the high level of genetic diversity
within local Borrelia populations is maintained by balancing
selection. The pattern of the variation in OspC compared to that
of OspA in local populations implies that the balancing selection
is likely frequency dependent selection on the OspC alleles.
Patricia
A. Rosa, PhD
Rocky Mountain Laboratories, National Institutes of Health
Targeted
Gene Inactivations in Bb
Patricia A. Rosa (*1), James Bono (1), Brian Stevenson (1), D.
Scott Samuels (2), Sherewood Casjens (3), and Kit Tilly (1).
(1) Laboratory of Microbial Structure and Function, Rocky Mountain
Labs, NIAID, NIH; (2) Division of Molecular Biology and Genetics,
Department of Oncological Sciences, University of Utah Medical
Center.
Borrelia
burgdorferi
has a complex genome composed of a linear chromosome and multiple
linear and circular plasmids. We would like to investigate the
potential role of genes located on a ubiquitous 26 kb circular
plasmid (cp26) in environmental sensing and adaptation to the
tick vector and mammalian host. These include genes encoding
the differentially synthesized outer surface protein C (OspC),
the purine biosynthetic enzymes GMP synthetase (guaA) and IMP
dehydrogenase (guaB), and a homolog of the peptide binding component
of oligopeptide permease (oppA). Studies of the biology of Bb
and the pathogenesis of LD are severely limited by the current
lack of genetic tools . As an initial step toward facile genetic
manipulation of this pathogenic spirochete, we have investigated
gene inactivation by allelic exchange using a mutated borrelial
gene (gyrB) that confers resistance to the antibiotic coumermycin
A1 as a selectable marker. We have inactivated the OspC, guaB,
and oppA genes on cp26 by directed insertion of gyrB into each
of these loci, and describe mutant construction and preliminary
phenotypic characterization. These studies will allow us to determine
the roles of these, and ultimately other, cp26 genes on growth,
plasmid maintenance and tick-mammal transmission.
Catherine
L. Lawson, PhD
Biology Department, Brookhaven National Laboratory
Crystal
OspA and Genomic Sequencing of Bb Catherine L. Lawson
& John J. Dunn. Brookhaven National Laboratory.
Objectives:
High-resolution 3D models of major B. burgdorferi antigens
and their complexes with antibodies can serve as invaluable guides
for design of second-generation vaccines and diagnostics, as
well as provide structural insights into the natural biological
functions of the molecules. The completed sequence of the Bb
genome will greatly improve understanding of the biology of the
organism, providing new points of attack for medical intervention.
Methods:
Recombinant forms of LD antigens are overexpressed and crystallized
either by themselves or in complexes with antigen-combining fragments
(Fabs) of monoclonal antibodies. Structures are determined using
crystal diffraction techniques, and then analyzed by a variety
of methods. Novel DNA sequencing techniques generated at BNL
are being applied to determine the sequence of the entire bacterial
genome.
Results:
A detailed atomic model for vaccine candidate outer surface protein
A (OspA) has recently been obtained, and work is in progress
towards structure determinations of OspB and OspC. Sequencing
of the Borrelia linear chromosome is nearing completion. Under
100 gaps remain to be closed and annotation is now in progress,
prior to release to a public database. Finishing the plasmid
sequences will take longer.
Conclusions:
The implications of the structural and sequencing results in
directing future research on LD will be discussed.
James
H. Oliver, Jr., PhD
Institute of Arthropodology & Parisitology, Georgia Southern
University
Bb
sensu lato
Isolates from Missouri
J.H. Oliver, Jr., T.M. Kollars, Jr., and F.W. Chandler, Jr. Institute
of Arthropodology and Parasitology, Georgia Southern University
A
total of 31 species of vertebrate animals were examined for presence
of ticks and spirochetes at 20 study sites in southeastern Missouri.
These included 22 mammals, 6 birds, and 3 reptile species. Ticks
were also obtained by flagging/dragging the vegetation. Tick
and/or host tissues were inoculated into BSKII medium in hopes
of establishing laboratory cultures of spirochetes.
A
total of 45 isolates were obtained from ticks attached to 15
eastern cottontail rabbits from 8 sites located in 5 counties.
All isolates were from various developmental stages of Ixodes
dentatus except one was from an Amblyomma americanum
larva and one from a Haemaphysalis leporispalustris larva.
The spirochetes were routinely screened by IFA monoclonal antibodies
specific for Bb and included OspA (H5332, H3TS), OspB
(H5TS,H6831, H614), and the Borrelia genus-specific H9724; two
B. hermsii specific antibodies (9826, H4825) were also
tested. The spirochete isolates were also analyzed by the PCR
using several Bb specific ospA primers (788/944, 149/319,
149/459,3'/5'), fla (245/855), and Rosa's chromosomal primer
(147/520). Several of the isolates were subjected to SDS-PAGE
analysis and pulsed-field gel electrophoresis (PFGE).
Based
on the above analyses all of the Missouri spirochete isolates
are considered to be Bb sensu lato but are phenotypically
and genetically different from Bb sensu stricto and in
fact, appear to be rather similar to the genospecies B. andersonii.
There are interesting differences among the Missouri isolates
themselves. There are at least 5 immunological types and a dendrogram
based on those data including 5 isolates from the Dowd farm and
B. burgdorferi s.s., B. garinii, and B. hermsii
suggests that MOD-5 is more closely related to B-31 B. burgdorferi
s.s. than to MOD-1, MOD-2, MOD-3, and MOD-6. This relationship
appears to be similar when a dendrogram is constructed based
on data from pulsed-field gel electrophoresis. There are also
at least 5 PCR types among the Missouri isolates thus far screened.
An
ELISA for detecting antibodies against Missouri isolate MOD-1
was developed and analyses of sera from mammals collected in
southeast Missouri thus far indicate high prevalences of antibodies
in several species of hosts of ticks with the highest in cottontail
rabbits followed by white-tailed deer, rodents, and raccoons.
Western blots are being conducted to confirm positive ELISA results.
Rabbits, deer, white-footed mice, cotton mice, and deer all show
a prevalence of 65% or more positive for presence of IgG antibodies
to the MOD-1 Bb isolate.
Five
of the Missouri isolates were obtained from the Dowd farm which
is the site of a human patient with a classical erythema migrans
(EM) lesion and clinically diagnosed with LD. Interestingly,
however, preliminary experiments involving needle injection of
2 of the isolates into mice failed to infect the mice.
Edwin
Masters, MD
Regional Primary Care, Inc.
Clinical
Aspects of Lyme and/or Lyme-like Disease in MO
Examples
of clinical erythema migrans in Missouri are presented along
with other clinical presentations and examples of sequelae. Etiological
and epidemiological theories are presented and discussed. The
possible role of the lone star tick (Amblyomma americanum)
in the transmission of this illness is explored.
Stephen
Barthold, DVM, PhD
Professor, Yale University School of Medicine
Humoral
Immune Response to in vivo Bb Antigens
Active
infection of mice with B. burgdorferi elicits an immune
response that can be measured by passive transfer. Passive transfer
of immune serum prior to challenge inoculation invokes strong
protection against challenge with Bb (protective immunity).
Immune serum will also cure mice of infection when passively
transferred up to 4 days after inoculation (post-infection immunity).
Beyond that point, passive transfer of immune serum will neither
cure mice of infection, nor prevent dissemination of spirochetes
in host tissues. However, transfer of immune serum to infected
mice with active arthritis will selectively induce arthritis
resolution (arthritis-resolving immunity). These three phenomena
are highly reproducible, and provide biologic markers for understanding
the host-parasite interaction.
Spirochetes
grown in culture differ antigenically from spirochetes within
ticks or the host. OspA, for example, is abundantly expressed
in culture and in the midgut of unfed ticks, but rapidly disappears
when ticks commence feeding and when spirochetes enter and invade
host tissue. OspC, on the other hand, is upregulated during tick
feeding and entry into the host. A number of Bb antigens
may be expressed both in culture and in the host, such as OspC,
P39, and decorin binding protein (DBP), whereas others may be
exclusively expressed in the host and are therefore not seen
on immunoblots (such as P21, PG, EppA, BbK2.10, P35 and P37).
Examination
of immune serum from mice inoculated with antigenically subliminal
doses of spirochetes or by tick-borne infection reveals strong
and early reactivity to a limited repertoire of 41, 39 and 22
kDa antigens on immunoblot. Such serum contains protective, post-infection
and arthritis-resolving activity. It has been assumed that the
reactive antigens represent flagellin, P39 and OspC, respectively,
but none of these antigens elicit protective, post-infection
or arthritis-resolving immunity. Other antigens may not be apparent
on immunblots for 2 possible reasons: co-migration, particularly
in the 22 kDa range, so that the actual antigen is masked and
presumed to be OspC, or expression of the antigen exclusively
in vivo. Both situations seem to be occurring. Serum from mice
is reactive against several 22 kDa range proteins co-migrating
with OspC. Serum form mice is reactive against several 22 kDa
range proteins co-migrating with OspC, including DBP and a novel
protein, P22-H3. Two recently discovered proteins, P35 and P37,
are expressed exclusively in vivo, and elicit strong early antibody
responses. P21 is also expressed in vivo, but antibody responses
appear late in the course of infection.
These
proteins may represent targets for different biologic effects
of humoral immunity during early and persistent infection. DBP
elicits both protective and post-infection immunity, but studies
to date indicate that it has no apparent effect upon arthritis
resolution. P35 and P37 elicit protective, but not post-infection
or arthritis-resolving immunity. P22-H3, and its partner on the
same operon, P20-H3, do not elicit protective or post-infection
immunity, but may have arthritis-resolving activity. No biologic
activity can be invoked with P21 or a number of other proteins.
Theses studies provide evidence that several novel Bb
proteins are primary immunogens for host immunity, and provide
indirect evidence that different antigens elicit different immunogenic
events. This suggests that there may be differential expression
of antigens during different phases of infection and in different
phases of infection in different tissues during infection. The
mouse model for LD is allowing insight into these complex events.
Mark
S. Hanson, PhD
Director, Molecular Microbiology, MedImmune, Inc.
Bb Decorin
Binding Proteins as Components of a Second Generation LD Vaccine
B.
burgdorferi
has been shown recently to bind decorin, a collagen-associated
extracellular matrix proteoglycan. Decorin is found in skin,
the site of entry for the spirochete, and in many other tissues.
Two candidate borrelial adhesions that recognize this proteoglycan
have recently been identified, decorin binding proteins A and
B (DbpA, DbpB). Antibodies against DbpA and DbpB were found to
be components of the early immune response of mice to low-dose
borrelia challenge, unlike OspA. We showed that rabbit antiserum
raised against recombinant DbpA from sensu stricto isolate 297
killed 19/35 and Bb sensu lato isolates of diverse geographic,
biological, and clinical origin including 2 B. afzelii
and 4 B. garinii isolates. Rabbit anti-OspA killed 17
of these same 35 isolates. Rabbit anti-DbpA passively protected
mice from Bb challenge. However, unlike OspA-specific
serum, anti-DbpA serum administered several days after challenge
still eliminated the borrelia infection. This is the first Bb
target described showing this effect.
Mice
immunized with DbpA were also protected from Bb challenge.
We found that sera from 31% (6/19) of patients in the early stages
of LD have DbpA-reactive antibodies, suggesting that DbpA is
expressed during the early stages of human Lyme borreliosis and
may be a target for protection. Evaluation of DbpB as a vaccine
for LD is still in the early stages. These findings support a
role for DbpA in the immunoprophylaxis of LD, and suggest that
vaccines based on DbpA, and possibly DbpB, would have the potential
to eliminate Bb infections during their early stages.
Denise
M. Foley, PhD
Department of Microbiology and Immunology, University of California,
LA, School of Medicine
Acquired
Resistance to Bb in the Rabbit: OpsA Vaccine-derived versus
Infection-Derived Immunity1
Denise M. Foley*, Yi-Ping Wang, Xiao-Yang
