Willy
Burgdorfer, PhD, MD(hon)
Scientist Emeritus
Laboratory of Pathobiology, Rocky Mountain Laboratories
Tick-Borne
Diseases of North America
The
list of the most important tick-borne diseases in the United
States includes Lyme disease (Borrelia burgdorferi), Rocky
Mountain spotted fever (rickettsia ricketsii), Tick-borne
relapsing fevers (Borrelia spp.), tularemia (Pasteurella
tularensis), babesiosis (Babesia microti), human ehrlichiosis
(Ehrlichia chaffeensis), canine ehrlichiosis (Ehrlichia
canis), Potomac horse fever (Ehrlichia resticii),
Anaplasmosis (Anaplasma marginale), Colorado tick fever
(CRF virus), and tick paralysis (caused by a toxin).
Lyme
disease is the most prevalent illness affecting thousands of
persons, children and adults alike. Occurrence of Lyme disease
in areas where the common tick vectors (Ixodes scapularis
and I. pacificus) are absent, suggests involvement of
different tick species or the existence of enzootic foci maintained
by ticks or other blood-sucking arthropods.
Rocky
Mountain spotted fever is still a major public health problem
even though the yearly totals of cases have dropped from 1,192
in 1981 to less than 500 in 1992. Mortality rates continue to
vary between 3 and 8 percent.
Human
babesiosis and its protozoan agent, Babesia microti, share
the ecological factors with those of Lyme disease but affect
a considerably smaller segment (10 to 20 cases per year) of our
population.
Human
ehrlichiosis caused by E. chaffeensis and Potomac horse
fever caused by E. risticci are recently emerged illnesses
for which the transmitting vectors have not as yet been established.
Ticks, particularly the lone star tick, Amblyomma americanum
and the American dog tick, Dermacentor variabilis are
suspected vectors. Similarly, the claim that the newly described
spirochete, Borrelia coriaceae is the cause of bovine
epizootic abortion (BEA) in the western parts of the country,
needs confirmation.
Tularemia
continues to be an important health problem in the southwest-central
region (Arkansas, Kansas, Louisiana, Missouri, Oklahoma, Texas)
that reports 200 to 250 cases every year.
Finally,
brief reference is made to tick-borne relapsing fevers, Colorado
tick fever, and tick paralysis.
James
H. Oliver, Jr., PhD
Institute of Arthropology and Parasitology
Georgia Southern University
Cospecificity
of Ixodes scapularis and Ixodes dammini
Reciprocal
crosses between Ixodes dammin Spielman, Clifford, Piesman
& Corwin from Massachusetts and Ixodes scapularis
Say from Georgia produced offspring through the F3 generation
when the experiment was discontinued. Reciprocal I. dammini
x Ixodes pacificus Cooley & Kohls (California) and I.
scapularis x I. pacificus crosses produced F1 progeny; however,
all progeny were sterile. Assortative mating experiments between
I. dammini and I. scapularis indicated that males
and females of both species mated with the opposite sex of heterospecific
or conspecific ticks when there was a choice. Conventional discrimination
analysis of morphometric measurements of ticks from Georgia,
North Carolina, Maryland, Massachusetts, and two populations
of F1 hybrids indicated that there were recognizable differences.
However, size-free (sheared) discriminant analysis indicated
that these differences were largely size dependent, with much
overlap of the four eastern and two hybrid populations but no
overlap with I. pacificus from California. Analysis of
chromosomes (morphology and C band) indicated no differences
between the Georgia and Massachusetts populations but showed
a difference between them and the California population of I.
pacificus. Analysis of isozymes showed that the genetic identity
value for the Georgia and Massachusetts populations was within
the normal range for conspecific populations, whereas the California
population indicated cogeneric but not conspecific relatedness
laboratory conditions showed no difference in length of feeding
and molting periods among Georgia, Massachusetts, and California
populations. These data and results of the work of other authors
on tick host preferences and vector competence indicates that
I dammini is not a valid species separate from I. scapularis
Say, 1821, has priority over the name Ixodes dammini Spielman,
Clifford, Piesman & Corwin, 1979, I. dammini is regulated
to a junior subjective synonym of I. scapularis (based
on Article 23 of the International Code of Zoological Nomenclature).
John
P. Mays, PhD
Microbiology and Research Administration
Connaught Laboratories, Inc.
Evaluation
of a Human Lyme Disease Vaccine for Safety and Immunogenicity
Lyme
disease is a tick-borne disease caused by the spirochete Borrelia
burgdorferi. In humans, chronic infection produces diverse
dermatologic, neurologic, rheumatic, and cardiac manifestations.
Prompt and immediate intervention can prevent many of these severe
sequellae, however, early clinical diagnosis is not always possible.
For several years, research and development has been directed
towards a vaccine for prevention of this debilitating disease.
Numerous animal studies demonstrate that pre-existing antibodies
to the outer surface proteins of Borrelia burgdorferi
can prevent infection and disease by this organism. Using recombinant
DNA technology, genes from B. burgdorferi were inserted
into E. coli-expression vectors and the outer surface
proteins from the B31 strain of B. burgdorferi were produced
and biochemically characterized. Animal studies demonstrated
the proteins were highly immunogenic and unaffected by the method
of isolation and purification. Using this technology, two vaccines
were formulated under cGMP conditions, characterized, and released
for clinical trial use. In September 1992, the FDA granted an
IND for Phase 1 clinical studies of a candidate Lyme disease
protein vaccine. The primary objective of this study was to evaluate
the safety of this candidate protein vaccine in healthy young
adults. In addition, the immunogenic response to the vaccine
was evaluated. The vaccine is composed of outer surface protein
A (OspA) of Borrelia burgdorferi.
Irwin
T. Vanderhoof, FSA, PhD, ACAS, CFA, CLU, BS
Stern School of Business
New York University
Symptoms
Based on Physician Speciality and Geographic Distribution:
Similar or Differing Presentations
This
discussion is based on an analysis of the data base of Lyme disease
cases developed jointly by the Lyme Disease Foundation and The
Society of Actuaries. The data base includes detailed information
on the symptoms reported by 645 physician diagnosed cases of
Lyme disease. Since the average time from infection to diagnosis
was 21 months, these may be considered to be cases deep seated
in the patients.
The
analysis was done in several levels. How many systems (of a total
of seven) were involved? This seems to be very similar for a
variety of indicators such as: state, reporting of rash, and
positive or negative serology. The second level of analysis was
the specific systems involved. Again the pattern seems uniform
geographically.
Finally,
specific systems are delineated. No geographical differences
are noted. The questions raised as to whether the observed pattern
of symptoms is consistent with CFS or fibromyalgia.
Paul
Duray, MD
Department of Pathology
Brighams & Women's Hospital
Parallels
in Murine and Human Borrelia Pathology
No
abstract available
Mark
S. Klempner, MD
Professor of Medicine, Microbiology and Molecular Biology
New England Medical Center
Tufts University School of Medicine
Interactions
of B. burgdorferi with Skin Fibroblasts
Borrelia
burgdorferi
is inoculated into the skin where it causes the characteristic
erythema migrans lesion of early Lyme disease. As part of our
studies on the pathogenesis of early Lyme disease we have investigated
the interaction of Borrelia burgdorferi with elements
found in the skin including human dermal fibroblasts. To examine
whether fibroblasts might provide a protected niche for the spirochete
we determined whether spirochetes could be isolated from co-cultures
with human fibroblasts after treatment with the antibiotic ceftriaxone.
This antibiotic, which is commonly used for treatment of Lyme
disease, is cidal for B. burgdorferi in growth media.
Results from these in vitro experiments will be reviewed demonstrating
the recovery of viable B. burgdorferi from infected fibroblast
co-cultures despite treatment of the cell cultures with ceftriaxone
at > 10 X MBC. Both infectious and non-infectious strains
of B. burgdorferi were recovered from the antibiotic treated
fibroblast monolayers. Viable fibroblast were required for antibiotic
protection. To further study the interaction of B. burgdorferi
with primary cultures of human skin fibroblasts we used scanning
electron microscopy (SEM), laser scanning confocal microscopy
(LSCM), and immunofluorescent laser scanning confocal microscopy
(ILSCM). Using SEM we have observed Borrelia burgdorferi
adherence to human fibroblasts after co-culture for 24 hours
followed by extensive washing and fixation. SEM demonstrated
the complete absence of adherent extracellular spirochetes after
co-culture of fibroblasts with spirochetes followed by antibiotic
treatment. Despite the absence of surface organisms in these
monolayers viable spirochetes could be recovered from parallel
co-cultures with fibroblasts after antibiotic treatment. Using
LSCM, B. burgdorfer were observed within human foreskin
fibroblasts. ILSCM further demonstrated intracellular B. burgdorferi
labeled with fluoresce in conjugated monoclonal anti-flagellin
antibodies. Recent studies further characterizing the adherence
of B. burgdorferi to dermal fibroblast will be presented.
Finally, using a novel skin blistering technique, we have begun
to characterize the inflammatory mediators in the erythema migrans
lesion and to determine the contribution of dermal fibroblasts
to the pathogenesis of this lesion. Preliminary findings will
be presented.
Claude
F. Garon, PhD
Chief, Laboratory of Vectors and Pathogens
Rocky Mountain Laboratories, National Institute of Allergy and
Infectious Diseases
Extracellular
Components of Borrelia burgdorferi - Possible Role in
the Pathogenesis of Lyme Disease
Claude F Garon, David W Dorward, and William M Whitmire
Borrelia
burgdorferi,
the causative agent of Lyme disease, appears during periods of
growth to shed membranous materials from its surface. Using an
antigen capture/detection method developed in the laboratory,
this material could be demonstrated on the surface of spirochetes,
free in culture medium and in infected animals and ticks. Similar
results were obtained using various tissues and fluids from human
patients with Lyme disease. Although the captured antigens were
initially assayed by immune electron microscopy, other methods
have been used recently to characterize the nature these bioproducts
and to assess their possible role in the pathogenesis of Lyme
disease. Researchers in the laboratory have been able to demonstrate
that extra cellular components of B. burgdorferi: 1) appear
to be present wherever active growth of the organism is taking
place and therefore, may be useful as a diagnostic indicator
of active infection and/or treatment effectiveness; 2) are involved
in the packaging and protection of intact DNA molecules containing
a few known and many unknown genes and gene products; 3) appear
to specifically interact with immunoglobulin M molecules in a
unique fashion, perhaps to escape immune surveillance; and 4)
possesses potent, non-specific mitogenic activity which may cause
an inappropriate and non effective stimulation of the immune
system triggering autoimmune disease components. The Laboratory
of Vectors and Pathogens continues to apply a multidisciplinary
approach to these and other problems with the aim of providing
a rational solution to the improved prevention, treatment and
diagnosis of Lyme disease.
Steven
E. Schutzer, MD
Division of Allergy and Immunology, UMDNJ - Department of Medicine
The
Immune Response and Its Application Toward Diagnosis
The
immune response to an infectious agent involves complex interaction
among differentiate into antibody producing cells, macrophages,
and B cells. The B cells which antigenic components of the agent.
Initially, and in certain other instances, the predominance of
the antibody may be found bound to the agent in an antigen-antibody
or immune complex. Serologic diagnosis of infection with the
spirochete Borrelia burgdorferi (Bb), the cause of Lyme
disease has been hampered by the variability among existing tests
as well as the prolonged time needed for the humoral response
to reach threshold of detection by conventional assays. As specific
antibody (Ab) made be found bound to an infectious agent, especially
early in the infection, we hypothesized that this could be occurring
in Lyme disease. We isolated and dissociated serum immune complexes
from Lyme disease patients, fulfilling modified CDC criteria,
and controls. Immune complexes were first collected by polyethylene
glycol (PEG). Following dissociation by high pH, the dissociated
constituents were analyzed by ELISA and Western blots. Specificity
of the reactive Ab was evaluated by probing for the target antigen
using monoclonal and polyclonal Abs, as well as recombinant proteins.
Complexes Ab to Bb was found in 10 of 11 very early cases
(p=2 x 10-7), 55 of 56 (p=<10-8) symptomatic patients with
Lyme disease, 0 of 50 healthy controls, 2 of 50 patients from
the endemic areas with other disease including those likely to
have elevated levels of immune complexes, 13 of 13 (p=<10-8)
persistently seronegative patients who had erythema migrans and
a subset of 4 of 4 who were also positive on a T cell proliferation
assay to Bb, and 0 of 8 patients who had recovered. In
the early acute cases complexes IgM was the first antibody to
be detected. Predictive values (PV), based upon a sensitivity
of 98% and a specificity of 98% were PV+ of 36% (prevalence of
1%) to 98.6% (prevalence of 50%) and PV- of 99.9% to 99.7% between
these prevalences. The data suggests that this relatively simple
technique has potential to support or exclude a clinical diagnosis
of early as well as active Lyme disease.
Dorothy
M. Pietrucha, MD
Neurologist, Jersey Shore Medical Center, Cornell New York Medical
Center
Pediatric
Neuroborreliosis
No
abstract available
Martin
D. Fried, MD
Director of Pediatric Gastroenterology and Nutrition, Jersey
Shore Medical Center
Lyme
Disease Abdominal Pain and the Gastrointestinal Tract
Martin D. Fried, MD; Paul Duray, MD; and Dorothy Pietrucha, MD
Twelve
children with clinical and laboratory evidence of Lyme disease
were evaluated for abdominal pain. The abdominal pain in most
cases preceded the diagnosis and initiation of antibiotic therapy.
Gastrointestinal endoscopy with biopsy was significant for inflammation
in 8 out of 12 patients. In two patients with duodenitis, spirochetes
consistent with Borrelia burgdorferi were found on tissue
biopsy by the dieterle stain. In one patient who subsequently
has bloody diarrhea, colonic biopsies were consistent with Crohn's
disease. A gastrointestinal evaluation prior to endoscopy was
negative for occult blood, stool infections, parasites and clostridium
difficile toxin. Amylase, sedimentation rate, liver function
tests were normal. An ultrasound of the gallbladder, pancreas
and pelvis was non revealing. An upper gastrointestinal series
with small bowel follow through failed to reveal an ulcer or
terminal ileum disease.
Patients
with gastritis and duodenitis were treated with Omeprazole (acid
blocker therapy) for two months while also being treated with
antibiotics for Lyme disease. The pain improved in those patients
whose biopsy showed gastritis or duodenitis. The abdominal pain
persisted after treatment with Omeprazole although it was not
as severe as prior to treatment.
We
conclude that gastritis and duodenitis occurs in children with
Lyme disease and abdominal pain.
Mitchel
B. Alpert, MD, FAAP, FACC
Pediatric cardiologist, Jersey Shore Medical Center
Pediatric
Cardiac Involvement with Lyme Disease
No
abstract available
Patricia
K. Coyle, MD
University of New York at Stony Brook, School of Medicine
Evidence
for Rapid Nervous System Invasion by Borrelia burgdorferi
The
nervous system is involved in a significant number of people
with clinical Lyme disease. characteristic syndromes occur in
both the early and late stages of infection. Unusual neurologic
syndromes are also reported, as well as several post treatment
("postinfectious") syndromes. One of the major diagnostic
problems in Lyme disease has been the lack of a reliable assay
for active infection. This presentation will review the neurologic
manifestations and cerebrospinal fluid (CSF) findings in a group
of patients with early (²3 months) B. burgdorferi
infection. CSF was examined not only for routine studies but
also for a specific B. burgdorferi antigen (OspA) as well
as Borrelia specific immune complexes.
Twenty-five
patients were evaluated over a consecutive 9 month period. The
most common neurologic syndrome was headache accompanying erythema
migrans (68%), followed by Bell's palsy (16%), tick bite associated
with headache and stiff neck (8%), meningitis (4%), and acute
polyradiculopathy (4%). CSF contained OspA antigen in 36% of
patients, and Borrelia specific immune complexes in 72%. In contrast,
intrathecal production of Lyme antibodies was present in only
17%, elevated CSF protein in 24%, and increased CSF cell count
in 20%.
These
findings support early central nervous system invasion by B.
burgdorferi. In particular, headache is a suggestive symptom.
CSF shows frequent abnormalities with regard to B. burgdorferi
antigen and specific complexes, but not with regard to routine
studies. This is in contrast to what has been reported from Europe,
and suggests clinical differences for north American Lyme disease.
Robert
Lesser, MD
Neuro-Ophthalmologist
Ophthalmic Surgical Associates
Yale University School of Medicine
Eye
Findings in Lyme Disease
Eye
findings in Lyme disease have been reported to include conjunctivitis,
keratitis, uveitis, and neuroretinitis. Neuro-ophthalmologic
findings include papilledema, cranial nerve palsies, pupillary
abnormalities, and optic neuropathy.
Rudolph
J. Scrimenti, MD
Medical School of Wisconsin
Dermatologic
Manifestations of Borreliosis
Lyme
borreliosis is a multi-system disease with protean clinical manifestations.
Amongst these, the cutaneous manifestations appear to be fairly
specific, erythema migrans, borrelial lymphocytoma and acrodermatitis
chronica atrophican are dermatoses unequivocally associated with
borrelial infections. The clinical features, histopathological
findings and differential diagnoses will be reviewed in this
presentation.
Brian
Fallon, MD, MPH, MEd
The NYS Psychiatric Institute
Psychiatric
Aspects of Lyme Disease in Adults and Children: New Research
When
Lyme disease affects the central nervous system, a variety of
neuropsychiatric symptoms may emerge. This talk will present
an overview of the neuropsychological and the psychiatric effects
of Lyme disease upon adults and children. Data will be drawn
from clinical experience, published reports and as yet unpublished
data. Results from a large epidemiologic catchement area study
of psychiatric disorders among children in Westchester will be
presented. Finally, the psychological impact of Lyme disease
will be discussed, focusing in particular on the effect upon
doctors and patients of having an illness characterized by so
much uncertainty.
Ed
Masters, MD
Family Physicians Group
Lyme
Disease in the Midwest
This
presentation focuses on Lyme disease in the Midwest. Examples
of erythema migrans and serological findings are presented. Tick
vectors are discussed, especially the association of Amblyomma
americanum (lone star) ticks with erythema migrans. Examples
of late sequelae are presented along with comparisons to other
geographic areas. Implications of the declassification of Ixodes
dammini as a separate tick species are discussed as they
relate to the Midwest.
Kenneth
B. Liegner, MD
New York Medical College
Persistent
Infection Despite Extensive Treatment
No
abstract available
James
Katzel, MD
Ukiah Valley Medical Center
Public
Forum
A
program for the public in New Jersey and surrounding area
W.M.
Whitmire, PhD
Laboratory of Vectors and Pathogens, Rocky Mountain Laboratories
National Institute of Allergy and Infectious Diseases
B
Cell Mitogenic Activity of Borrelia burgdorferi Surface
Components
W.M. Whitmire, PhD, C.F. Garon, PhD
The
ability of Borrelia burgdorfer to cause nonspecific proliferation
(mitogenesis) of murine B cells has been recently described in
several reports. Moreover, it has been shown that two spirochetal
lipoproteins (outer surface protein [Osp]A and OspB) are mitogenic
by themselves, and that OspC, spirochetal preparations (Sh-2-82
strain) were applied to two-dimensional (2-D) polyacrylamide
gel electrophoresis, electrophoretically transferred to nitrocellulose
and probed with rabbit antiserum that reacts with OspC. The protein
spot (23 kiloDalton) that reacted strongly with the antiserum
was then excised from 16 additional 2-D gels, pooled and subjected
to protein elution. Eluted protein was exhaustively dialyzed
to remove sodium dodecyle sulfate, precipitated in cold acetone,
air-dried and applied to lymphocyte blastogenesis assays that
included pooled spleen cells from three naive C57BL/10 female
mice. A protein spot (41 kiloDalton) that reacted with an anti-flagellin
monoclonal antibody on immunoblots was also excised from the
gels, processed and assayed in a similar fashion. Blastogenic
responses to flagellin were low, whereas responses to OspC were
significantly greater (p or=0.01) and 8-fold higher than flagellin
at identical protein concentrations. Flow cytometry identified
responding lymphocytes as B cells. These results indicate that,
like OspA or OspB, OspC is mitogenic for murine B cells and that
gel conditions of processing did not contribute to the mitogenic
effect. Mitogenic proteins may contribute to the pathogenesis
of Lyme disease.
David
W. Dorward, PhD
Laboratory of Vectors and Pathogens, Rocky Mountain laboratories
National Institute of Allergy and Infectious Diseases
Transferrin
as an Iron Source for Growth of Borrelia burgdorferi
David W. Dorward, Van E. Tamplin, William M. Whitmire
Iron
acquisition by pathogenic bacteria is a growth limiting requirement
for colonization and systemic infection in mammals. Mechanisms
by which B. burgdorferi acquires and utilizes ferric iron
are not currently understood. In order to investigate the iron
acquisition system(s) of B. burgdorferi in vitro cultures
were assayed for the production of catechol and hydroxylamine
siderophores. The ability of B. burgdorferi to grow in
cultures containing iron chelators and various sources of supplemental
iron, and the effects of chelation on spirochetal protein expression
were also examined. Neither catechols nor hydrxylamines were
detected in spent culture medium. The complexity of BSK II medium
may be a complicating factor in such assays. Cultures were inhibited
by the iron chelators dipyridyl and ethylenediamine di(0-hydroxyphenylacetic
acid) (EDDA). Addition of ferric chloride to chelated culture
allowed growth in the presence of EDDA but not in the presence
of dipyridyl, suggesting the latter is toxic to B. burgdorferi.
Cell growth was inhibited with 5 mM EDDA. Addition of human transferrin
at a final concentration of 1 mg per ml of culture enabled cell
growth in 10 mM EDDA. Electron microscopy showed that transferrin-colloidal
gold conjugates labeled cell surfaces. Labeling was enhanced
on cells grown in iron-limited medium (4mM EDDA), and labeling
was prevented by prior incubation of cells with unlabeled transferrin.
Two proteins which appeared to be up-regulated by iron stress
were detected by two dimensional gel electrophoresis. These proteins
migrate at approximately 45 and 92 kDa , and their functions
are unknown. Although the sub-cellular location of the 92kDa
protein has not been determined, the 45 kDa protein appears to
be associated with the outer sheath. These results indicate that
B. burgdorferi may be capable of growth using transferrin
directly as a sole source of available iron. Furthermore, iron
acquisition from transferrin may be inducible, and may involve
at least two different gene products.
Charles
S. Pavia, PhD
Assoc. Professor of Medicine
Division of Infectious Disease
New York Medical College
Sub-unit
Vaccine Development - New Discoveries
The
impact of the adjuvants QS-21 and aluminum hydroxide (alum) on
the immunogenicity of recombinant outer surface proteins A (OspA)
and B (OspB) of Borrelia burgdorferi was investigated. both OspA
and OspB derived from strain B31 were expressed in Escherichia
coli and purified by reversible citroconylation and anion exchange
chromatography. Antisera to OspA or OspB were prepared in mice
with antigens formulated with ZS-21 or alum, and evaluated for
specific immunoglobulin G isotypes, agglutination, and borreliacidal
activity. QS-21 enhanced IgG1 antibody responses to OspA 3-fold,
IgG2a 64-fold, and IgG2b 11-fold when compared with the formulation
containing antigen alone. a similar enhancement of antibody responses
to OspB by QS-21 was also observed. Alum also increased IgG1
response about 3-fold, but, unlike QS-21, had no measurable effect
on IgG2a and IgG2b responses. Antisera to OspA or OspB formulated
with QS-21 possessed higher titers of agglutinating antibody
than antisera to OspA or OspB alone, respectively. Borreliacidal
activity was 8-t0 64-fold higher in antisera to OspA formulated
with QS-21 than in antisera to OspA formulated with or without
alum. These antisera were highly borreliacidal to New York strain
B31, a California isolate CA-2-87, German isolate Fr, and Swedish
isolate G25. Antisera to OspB formulated with either adjuvant
had low or no borrelicidal activity against the 4 strains. This,
OspA was superior to OspB and QS-21 superior to alum at eliciting
functional antibody responses. The vaccine containing OspA and
OspB formulated with QS-21 was protective in mice against infection
with 10 infectious spirochetes of strains B31 or CA-2-87. This,
this formulation may be a promising vaccine candidate, and QS-21
may serve as a critical component in the subunit vaccine against
Lyme disease.
Robert
Quackenbush, PhD
Division of Microbiology and Infectious Diseases
Natl. Institute of Allergy and Infectious Diseases, National
Institutes of Health
National
Institutes of Health 1993 Lyme disease grants
Presentation
of recent grant announcements.
James
H. Oliver, Jr., PhD
Institute of Arthropology and Parasitology
Georgia Southern University
Epizootiology
of Lyme Borreliosis in the Southeast
We
now have 16 spirochetal isolates in culture from several areas
of Georgia and Florida. In cooperation with Dr. Francis Chandler,
Medical College of Georgia, the isolates have been characterized
to various degrees. Four isolates are from Sapeio Island, Georgia
(2 from cotton mice, Peromyscus gossypinus; one from cotton
rat, Sigmodon hispidus; one from an adult male tick, Ixodes
scapularis), one isolate from the mainland area of St. Mary's,
Georgia (from a cotton mouse), nine isolates are Merritt Island,
Florida (3 from cotton mice and 6 from cotton rats), one from
Faver-Dykes state park, Florida (from a cotton rat), and one
from Amelia Island, Florida (from a cotton rat). Fifteen of the
sixteen isolates have been tested by IFA (courtesy of Dr. Robert
Lane, Univ. of California, Berkeley) and reacted positively using
five different monoclonal antibodies including two Osp A, two
Osp B, and one Borrelia (genus)-specific one. The isolates also
were identified as B. burgdorferi based on SDS-PAGE and
PCR analysis, and comparisons to three B. burgdorferi
strains from northeastern U.S. and the B-31 reference strain.
The most conspicuous difference seen in some strains from Georgia
and Florida is a heavily stained band of approximately 22.5 kDa
on SDS-PAGE gels. This band is especially prominent when enhanced
by silver nitrate staining. Several B. burgdorferi strains from
California and France have shown a similar low molecular weight
band. All cotton mouse and tick isolates consistently amplified
Osp-A, Fla (primers supplied by Dr. Barbara Johnson, CD) and
conserved chromosomal(primers supplied by Patricia Rosa, NIH)
gene sequences of B. burgdorferi by PCR assay, as did
the B. burgdorferi controls from New England and B.
burgdorferi B-31 strain. All of the cotton rat isolates consistently
amplified specific B. burgdorferi flagellin and chromosomal
gene sequences by PCR, but these isolates did not amplify Osp-A
gene sequences except for the Merritt Island- 6 strain. By SDS-PAGE,
the cotton rat isolates showed a molecular shift in the Osp-A
proteins with noted absence of an Osp-A band. Transmission of
three Sapelo island, Georgia, isolates (1, 2, and 4) has been
successful via inoculation into hamsters and mice, and spirochetes
were subsequently reisolated. Sapelo Island isolate 1 (from a
cotton mouse) was transmitted from inoculated hamsters to mice
by I. scapularis, but attempts via Amblyomma americanum
failed. Currently, transmission experiments are underway involving
additional isolates (SI-3, MI-4, MI-6) from cotton rats and SI4
from I. scapularis.
Tom
G. Schwan, PhD
Laboratory of Vectors and Pathogens, Rocky Mountain Laboratories
National Institute of Allergy and Infectious Diseases
Distribution
and Molecular Analysis of Lyme Disease Spirochetes Borrelia
burgdorferi, Isolated from Ticks throughout California
T.G. Schwan and R.H. Karstens, Arthropod-borne diseases section,
Laboratory of Vectors and Pathogens, Rocky Mountain Laboratory,
NIAID; J.R. Clover, California Department of Health Services;
J. Wong, M. Daugherty, and M. Struthers, Divisions of Laboratories,
Microbial Diseases Laboratory, California Department of Health
Services; P.A. Rosa, Laboratory of Microbial Structure and Function,
RML, NIAID
Previous
studies describing the occurrence and molecular characteristics
of Lyme spirochetes from California have been restricted primarily
to the north coastal region of this large and ecologically diverse
state. Our objective was to look for and examine Borrelia
burgdorferi isolated from Ixodes pacificus ticks collected
from numerous regions spanning most of the state where this tick
is found. Thirty-one isolates of B. burgdorferi were made
from individual or pooled I. pacificus ticks collected
from 25 counties throughout the state. One isolate from an I.
neotomae tick from an additional county was also examined.
SDS-polyacrylamide gel electrophoresis, immunblot analysis, agarose
gel electrophoresis, Southern blot analysis, and the polymerase
chain reaction (PCR) were used to examine molecular and genetic
determinants of the uncloned, low passaged isolates; 21 of the
isolates were inoculated into mice. All of these isolates were
identified as B. burgdorferi by their protein profiles
and reactivities with monoclonal and polyclonal antibodies and
all the isolates were typed by PCR as North American type spirochetes
(B. burgdorferi sensu stricto). All of the isolates expressed
flagellin, outer surface protein (Osp) A, OspC, P22A, and P39;
all the isolates probably expressed OspB also but this could
not be confirmed for a few by immunoblotting. Although ops
locus products were identified in protein analysis in all of
the isolates, the osp locus of four isolates could not be amplified
by PCR, and several other isolates contained deleted forms of
this locus that would result in the expression of chimeric Osp
A/B proteins. The analysis of OspC demonstrated that this protein
was widely conserved among the isolates but was also quite variable
in its molecular mass and the amount expressed. Plasmid profiles
were quite diverse among the 32 uncloned isolates with no obvious
pattern associated with their geographic origin. Isolates inoculated
into mice were rarely reisolated from triturates of the urinary
bladder, suggesting the Lyme spirochetes in California may be
either less infectious than isolates from other regions of North
America of that these spirochetes are less likely to reside in
the urinary bladder compared to other isolates. One isolate was
made from ticks collected at Wawona campground in Yosemite National
Park, documenting the occurrence of Lyme spirochetes in an area
of intense human recreational use.
Terry
L. Schulze, PhD
Research Scientist
New Jersey State Department of Health
Suppression
of Ixodes Tick Populations in Large Residential Communities
Ground
and aerial applications of several rates of granular carbaryl
to the shrub layer and wooded buffers of a forested residential
community during the peak activity period of Ixodes dammini
nymphs significantly reduced their abundance on Peromyscus
leucopus. Ground applications of liquid and granular carbaryl
formulations to similar residential habitats yielded comparable
levels of control. At the maximum recommended rates, carbaryl
granules provided better control, but required significantly
more active ingredient compared to the liquid formulation. Although
both formulations effectively suppressed populations of this
important tick vector, factors such as logistics, depth of leaf
litter, and available application equipment must be considered
in the design of the control program. The use of properly timed
acaricide applications to I. dammini habitat within residential
communities provides a reliable means of reducing exposure to
I. dammini nymphs, the stage which is chiefly responsible
for transmitting Borrelia burgdorferi to humans.
Elizabeth
Burgess, DVM, PhD
Associate Professor
University of Wisconsin
School of Veterinary Medicine
Epidemiologic
Findings in 4 Midwest County Farms - Variations in Animal and
Tick Infection Rates
No
abstract available
Borrelia
burgdorferi
Infection in Dairy Cows, Rodents, and Birds from Four Wisconsin
Dairy Farms
Burgess, E.C., Wachal, M.D., and Cleven, T.D.
A
combination of culture and subsequent spirochete identification
with the polymerase chain reaction technique was used to identify
cows, rodents, and birds infected with Borrelia burgdorferi.
Animals were trapped on four Wisconsin dairy farms during the
summer of 1990. Farms 1 and 2 were located in counties nonendemic
for Lyme disease and Farms 3 and 4 were located in counties endemic
for Lyme disease. The results of the rodent and bird samples
were as follows given as the number yielding organisms number
tested: Farm 1, 1/17 Mus musculus and 2/52 P. domesticus;
Farm 2, 4/49 M. musculus, 1/2 Peromyscus maniculatus,
1/1 P. leucopus, and 1/35 P. domesticus; Farm 3,
0/27 M. musculus, 0/5 P. leucopus, 0/12 P. maniculatus
and 3/58 P. domesticus; and Farm 4, 1/24 M. musculus,
2/19 P. leucopus, 1/12 Microtus pennsylvanicus,
and 0/17 P. domesticus. One P. leucopus and M.
musculus from Farm 2 were pregnant and fetal tissues form
both were positive. Cow blood sample results were as follows:
Farm 1, 7/47 in July, and 2/45 in august; Farm 2, 0/28 in August
and 0/23 in October; Farm 3, 0/13 in July and 1/18 in August
29; and Farm 4, 3/45 in August. Ticks were found on rodents on
Farm 4 and on one bird on Farm 3. Spirochetemic cows, rodents
and birds were found in non-Lyme endemic counties suggesting
that alternate modes of transmission other than by ticks may
be important. Transplacental transmission was shown in M.
musculus and P. leucopus.
Sandra
Lee Bushmich, MS, DVM
Asst. Professor of Pathobiology
University of Connecticut
Infection
of Calves with Borrelia burgdorferi
Two
studies will be discussed. In the first study, symptomatic and
asymptomatic cows from a New England dairy herd with clinical
history and serological evidence consistent with Lyme Borreliosis
were evaluated using several diagnostic techniques. Eleven dairy
cows with clinical signs of lameness, erythema, and/or joint
swelling along with 15 healthy herd mate controls were tested
serologically by Immune Fluorescent Antibody (IFA) test and Western
blot analysis. Presence of Borrelia burgdorferi in blood
and urine samples from these cows determined by using 3 techniques
of varying sensitivity and specificity: dark field examination,
fluorescent antibody (FA) staining and polymerase chain reaction.
Serological results showed no significant difference between
symptomatic and asymptomatic cow groups in 1. percent seropositivity
(IFA) or 2. antibody response to specific proteins of B. burgdorferi
(Western blot). Most cows (both symptomatic and asymptomatic)
appeared to have been exposed to B. burgdorferi. Symptomatic
cows were significantly (p=.003) more likely to shed spirochetes
in the urine compared to asymptomatic control cows using the
sensitive and specific PCR technique. A similar, but not significant,
trend was seen when FA was utilized for detection of Borrelia
in the urine. Preliminary results of experimental infection of
calves with Borrelia burgdorferi will also be discussed.
Elizabeth
Burgess, DVM, PhD
Associate Professor
University of Wisconsin School of Veterinary Medicine
Animal
Findings Regarding in utero Lyme borreliosis
To
determine if in utero transmission of B. burgdorfer
could occur in dogs, 10 female Beagles were inoculated intradermally
with approximately 1000 B. burgdorferi on day 1 of proestrus
and repeated every 2 weeks during the gestation period. Ten female
controls were similarly inoculated with phosphate-buffered saline
solution. Prior to the start of the experiment, all of the females
and 3 males were used for breeding seronegative for B. burgdorferi
on the basis of results of the indirect immunofluorescent antibody
test (IFA) and the western blot test (WB). Similarly results
of blood culture of B. burgdorferi were negative. All
20 of the females were bred naturally. Blood was collected weekly
for serologic tests and culture. Live pups were bled on day 1
of life and then weekly until 6 weeks of age when they were humanely
euthanitized and tissues cultured and tested by the polymerase
chain reaction test (PCR). Eight of 10 spirochete inoculated
females (SI) became infected with B. burgdorferi as evidenced
by culture and/or PCR detected B. burgdorferi DNA in the
tissues of the females or their pups. Eight of the 10 SI females
delivered litters (3 to 7 pups) that had at least 1 pup with
PCR positive tissues for B. burgdorferi DNA in neonatal
or 6 week old pups and pups from 2 litters were also culture
positive. Three pups from 2 separate litters (a stillborn, a
neonate that survived to 30 minutes of age, and a 20 hr. old)
had PCR positive tissues demonstrating in utero infection.
Further evidence of in utero exposure was the presence
of IgM antibodies to B. burgdorferi detectable by WB in
3/7 1 day old pups that did not receive colostrum, demonstrating
a primary immune response exposure.
Colin
R. Young, PhD
Associate Professor
The Texas Veterinary Medical Center, Texas A & M University
Unusual
Findings in Feline Lyme Borreliosis
Colin R. Young, Michael D. Gibson, M.Tawfik Omran, John Edwards,
Leon Russell, Kathy Palma, Julie Rawlings
Lyme
disease (LD) is a multisystem disease with mainly skin, neural,
cardiac, muscular and joint manifestations. The disease is caused
by the gram negative spirochete Borrelia burgdorferi (Bb)
and is transmitted by infected ticks. Experimental models
of Lyme disease have been demonstrated in species such as mice,
rats, hamsters, cats, and dogs. Recent experiments by Dr. Elizabeth
Burgess reported that cats are susceptible to infection with
LD but that clinical signs may or may not be apparent. We have
investigated further this feline model of LD using 20 uninfected
normal healthy cats. These cats were divided into 4 groups each
containing 5 cats. One group was used as a control group, whereas
each remaining of the 3 groups were injected intradermally in
a single site in the sacral region with 106 live Borrelia
burgdorferi spirochetes isolated from different arthropods.
The 3 different isolates of Borrelia burgdorferi used
were B31 (a reference strain isolated from the tick Ixodes
scapularis), Bb1579 (isolated from the lone star tick, A.
americanum), and Bb532 (isolated from a pool of 5 cat fleas,
Ctenocephalides from Fort Bend County in Texas). The cats were
examined daily, bled biweekly, and one cat per group was sacrificed
each month for serological and histological studies on all tissues
and organs. Clinically, in the test cats there was a variable
picture ranging form front or hind-limb lameness to hyperemia
in all joint at necropsy. Gross pathology at necropsy indicated
that Bb injected cats had liver degeneration, hyperplasia
of the spleen, plasmacytosis of regional lymph nodes and occasional
pneumonitis of the lungs. Control cats had no abnormal lesions.
Differential WBC counts indicated that infected cats had cycles
of reduction in the neutrophil count accompanied by an increase
in the lymphocyte and eosinophil counts. During the course of
infection of these cats we noted appearance of an "atypical
cell" in the blood films. Following staining with Hemacolor
this "atypical cell" has the following characteristics:
6-7 u in diameter, a single compact round dark blue nucleus 1.5-3
u in diameter, blue-gray cytoplasm. These "atypical cells"
differ from the enlarged immunoblastic cells (occasionally similar
to Reed-Sternberg cells of Hodgkin's disease) or atypical enlargement
plasmacytoid immunoblastic cells (sometime binucleate) reported
for certain cases of Lyme borreliosis. To the best of our knowledge
these "atypical cells" have not been reported before
any animal specifically infected with Bb. The presence
of this "atypical cell" in other animal species infected
with Bb is currently under study.
Pamela
Paparone, RN, MSN
Atlantic City Medical Center
Patient
Education
No
abstract available
Pamela
A. Lynxwiler, LPN
Lyme Coordinator, Family Physicians Group
Patient
Evaluations - Increasing Primary Care Giver Efficiency
Our
main goal in caring for the Lyme disease patient is to make their
visit as effective as possible. We want to get all the pertinent
information necessary in making a diagnosis and developing a
treatment plan for each individual patient. We send questionnaires
and lab request forms to be filled out by the patient before
they arrive for their initial appointment. This allows more time
to be spent on patient evaluation rather than paperwork. When
the treatment is started and the patient returns for a follow-up
visit, we use a patient evaluation form so we can assess their
condition since the last appointment. This form is filled out
by the patient upon arrival at the office. Using the forms, one
is able to address those problems that are most serious, urgent,
or that require additional attention. This facilitates making
necessary changes in the treatment program as well as providing
a more complete and objective medical record.
Patricia
Dennler, RN
East Hyde Park Internal Medicine
Management
of Treatment Options
Patricia
Dennler is a private practice nurse at East Hyde Park Internal
Medicine Group.
Sue
Jacobson, BA, MS, MA
Tolland Center for Individual and Family therapy
The
Value of Support Groups to Families Coping with Potentially Chronic
Disease
The
discussion will define the purpose of a support group. Further,
the usefulness of support groups for the physician, the patient,
and the family will be discussed. In addition, the various supports
available will be discussed.
Joseph
J. Burrascano, Jr., MD
Southampton Hospital
Failure
of Aggressive Antibiotic Therapy to Protect the Placenta from
Invasion by B. burgdorferi in a Pregnant Patient with
Lyme Borreliosis
A
34 year old patient with Lyme borreliosis remained symptomatic
despite seven months of therapy with amoxicillin. Her therapy
was therefore changed to cefotaxime, 6g IV daily. After ten weeks
of continuous treatments, she did not experience a remission.
Accordingly, treatment was switched to high dose, pulsed cefotaxime,
12g daily, given on two consecutive days each week. After four
weeks, the patient was found to have become pregnant; in retrospect,
conception was thought to have occurred during the initial course
of daily cefotaxime. Cefotaxime therapy was changed back to 6g
daily for a total of four more months, and was followed by oral
amoxicillin. Due to poor response, she then changed to cefixime,
which was continued through to delivery. Post partum, B. burgdorferi
(Bb) was found to be present in the placenta by histologic
staining.
Discussion:
It is now well known that Borrelia can survive several weeks
of antibiotic therapy; mechanisms include sequestration of the
spirochete in immunologically protected sites, including intracellular
locations, protection from immune and antibiotic attack by S-layers,
and periods of dormancy during which time this organism cannot
be killed by antibiotics. This case report confirms the ability
of Bb to evade host defenses and antibiotic therapy, and
extends this concept to resistance to even prolonged courses
of antibiotics. It also demonstrates that Bb can cross
into the placenta despite such ongoing therapy, and that there
must obviously be periods of spirochetemia even late into this
illness.
Ronald
F. Schell, PhD
Department of Medical Microbiology/Immunology, University of
Wisconsin Medical School
Borreliacidal
Assay: Measuring the Immune Function
This
presentation will provide evidence for antibodies that kill Borrelia
burgdorferi in vivo and in vitro, demonstrate usefulness
for diagnosis of human Lyme disease and show that the flow cytometry
can accurately and reproducibly detect killing antibodies.
Charles
S. Pavia, PhD
Assoc. Professor of Medicine
Division of Infectious Disease, New York Medical College
Serologic
Findings in Culture - Positive EM Patients
Patients
with erythema migrans were followed prospectively for clinical
and laboratory markers of Lyme disease. In a significant number
of these patients, spirochetes were isolated and tissue specimens
were positive by PCR amplification after establishing cultures
of skin-biopsy or aspirate material. Serology was performed by
ELISA IgM/IgG (BioWhittaker), ELISA-IgM (Hillcrest) and by an
in-house indirect hemagglutination antibody (IHA) test. At initial
presentation, 42% were positive by BioWhittaker ELISA, 39% by
Hillcrest IgM and 45% by IHA. Subsequent testing on selected
evaluable patients, performed at follow-up visits (8,20, and
30 days post antibiotic treatment), revealed that borrelial antibodies
were detected best by the IHA test (76-88% reactive) followed
by the IgM/IgG ELISA (71-82%) and the IgM ELISA (63-70%). Based
on these results, selected high titer (pretreatment) sera were
reacted with live Borrelia burgdorferi organisms in vitro
and were found to inhibit spirochete growth partially in a dose-dependent
fashion. These findings show that the majority of patients with
early stage disease could be diagnosed by positive skin culture,
by PCR or by serologic methods or by combining all test procedures.
They also show that more patients produced antibodies shortly
after receiving therapy than before and their sera have some
anti-borrelial activity.
Ingeborg
S. Dziedzic, MD
Ophthalmologist
New York Medical College, Monteriore Medical Center
Ophthalmological
Findings of Persistent Infection: New Research
Dr.
Dziedzic will be presenting several cases of patients with Lyme
disease and concurrent ophthalmologic problems. All cases present
diagnosis and treatment dilemmas and pose questions as to the
consequences of infection with borreliosis.
Kenneth
B. Liegner, MD
New York Medical College
Diagnostic
Guidelines
No
abstract available
D.
Scott Samuels, PhD
Laboratory of Vectors and Pathogens
Rocky Mountain Laboratories, NIAID, National Institutes of Health
Coumarin
Susceptibility and Resistance in the Lyme Disease Agent
Coumermycin
A1 is an inhibitor of DNA gyrase, an enzyme that catalyzes super
coiling of DNA and is required for bacterial DNA replication.
We have recently shown that Borrelia burgdorferi, a spirochete
and a causative agent of Lyme disease, is more susceptible than
many other eubacteria to coumermycin, as well as novobiocin,
another coumarin antibiotic; this contrasted with its relative
resistance to the DNA gyrase inhibitors nalidixic acid, oxolinic
acid, and ciprofloxacin. Coumermycin at 0.2 ug/ml inhibited growth
(MIC) in BSK II medium and the slightest inhibitory dose of 0.003
ug/ml induced the reversible relaxation of two negatively-super
coiled circular plasmids within 2 hours (20% of the doubling
time). Because there are very few B. burgforferi mutants
of any sort derived from selection, we isolated 11 coumermycin-resistant
clones from approximately 1010 cells. All had a MIC
of at least 20 ug/ml and maintained coumermycin resistance after
at least 30 generations in the absence of selection. Two variants
produced proteins not found significant levels in parental cells.
CR9B had an outer surface protein with a molecular mass of approximately
27 kDa and CR9C overproduced OspC, a 23 kDa protein that is coded
for by the only gene mapped to a circular plasmid in B. burgdorferi.
None of the variants appeared to have lost the circular plasmids.
In the absence of coumermycin, three of the variants (CR8a, CR9C,
and CR9E) maintained circular plasmid super coiling while the
others had relaxed circular plasmids. Unfortunately, coumermycin
is not a clinically useful anti microbial agent; we hope that
further work on the coumarin drugs yields an effective treatment
for Lyme disease.
Mori
Schwartzberg, MD New Jersey Medical Center
Rheumatologic
Manifestations of Lyme Borreliosis
No
abstract available
J.
Joseph Burrascano, Jr., MD
Southampton Hospital
Rehabilitation
Therapy as a Support to Treatment
A
significant number of patients with chronic Lyme disease present
in very poor physical condition. They complain of exhaustion,
poor stamina, soreness of tendons, ligaments, and joints, and,
especially of the neck. On exam, many have gained weight, have
increased percentage of body fat, display generalized muscular
weakness, loss of range of motion, and elevated resting heart
rates, even in the absence of clinical myopathy or markers of
inflammation, such as elevated CPK and sedimentation rates. Because
of these symptoms and signs, many spend excessive amounts of
time in bed and have sharply curtailed their physical activities.
Antibiotic therapy alone cannot address this deconditioning.
A carefully designed program of progressive rehabilitation contributed
greatly to the patient's recovery. Such a program must be graded,
individualized, and done one-on-one at the outset to maximize
effectiveness and supervision. It begins with classic physical
therapy to relive pain and loosen muscle spasms, then evolves
to a comprehensive stretching program to restore range of motion
and regain flexibility. When the patient is ready, gentle whole-body
strengthening and conditioning is done in a progressive manner
for one hour at a time, every other day. This serves as the basic
mode or reconditioning that helps these patients the most. Finally,
aerobic conditioning is added in addition to the basic sessions.
Essential points that must be emphasized are the begin with one-on-one
sessions, run by credentialed physical therapists and exercise
physiologists, always involving a graded, whole-body approach,
in which it is better to gently exercise for the whole hour session,
rather than work too hard and be forced to end the session early.
Attention must also be paid to improved dietary habits and elimination
of caffeine and alcohol.
