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Author Topic: B. LONESTARI RESEARCH
cjnelson
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Journal of Medical Entomology
Article: pp. 590-592 | Full Text | PDF (61K)

DNA Evidence of Borrelia lonestari in Amblyomma americanum (Acari: Ixodidae) in Southeast Missouri
Rendi Murphree BaconA, C, Robert D. Gilmore JrC, Miquel QuintanaB, C, Joseph PiesmanC, and Barbara J. B. JohnsonC

A. CDC, NCID, DVBID, P.O. Box 2087, Fort Collins, CO 80521 (E-mail: [email protected]), B. United States Army Center for Health Promotion and Preventive Medicine-West, Fort Lewis, WA 98433, C. Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Ft. Collins, CO 80521

Amblyomma americanum collected near Lake Wappapello, Missouri, tested positive for Borrelia lonestari using polymerase chain reaction and sequence analyses of B. lonestari 16S rRNA and flagellin (flaB) genes. Twelve pools containing a total of 214 nymph or adult ticks contained evidence of infection with B. lonestari (minimum prevalence 5.6%). These

data suggest that persons in southeast Missouri are at risk for exposure to B. lonestari after A. americanum tick bite, a possible cause of erythema migrans-like rash illness in this region. Derivation of the complete coding sequence for B. lonestari flaB is also reported.

Keywords: Borrelia, flagellin, 16S rRNA, Amblyomma, Missouri
-----------------------------------------------------------------------------

Solitary Erythema Migrans in Georgia and South Carolina
Michael W. Felz, MD; Francis W. Chandler, Jr, DVM, PhD; James H. Oliver, Jr, PhD; Daniel W. Rahn, MD; Martin E. Schriefer, PhD


Arch Dermatol. 1999;135:1317-1326.

Objective To evaluate the incidence of Borrelia burgdorferi infection in humans with erythema migrans (EM) in 2 southeastern states.

Design Prospective case series.

Setting Family medicine practice at academic center.

Patients Twenty-three patients with solitary EM lesions meeting Centers for Disease Control and Prevention (CDC) criteria for Lyme disease.

Interventions Patients underwent clinical and serologic evaluation for evidence of B burgdorferi infection. All lesions underwent photography, biopsy, culture and histopathologic and polymerase chain reaction analysis for B burgdorferi infection. Patients were treated with doxycycline hyclate and followed up clinically and serologically.

Main Outcome Measures Disappearance of EM lesions and associated clinical symptoms in response to antibiotic therapy; short-term and follow-up serologic assays for diagnostic antibody; growth of spirochetes from tissue biopsy specimens in Barbour-Stoenner-Kelly II

media; special histopathologic stains of tissue for spirochetes; and polymerase chain reaction assays of tissue biopsy specimens for established DNA sequences of B burgdorferi.

Results The EM lesions ranged from 5 to 20 cm (average, 9.6 cm). Five patients (22%) had mild systemic symptoms. All lesions and associated symptoms resolved with antibiotic therapy. Overall, 7 patients (30%) had some evidence of B burgdorferi infection. Cultures from 1 patient (4%) yielded spirochetes, characterized as Borrelia garinii, a European strain

not known to occur in the United States; 3 patients (13%) demonstrated spirochetallike forms on special histologic stains; 5 patients (22%) had positive polymerase chain reaction findings with primers for flagellin DNA sequences; and 2 patients (9%) were seropositive for B burgdorferi infection using recommended 2-step CDC methods. No late clinical sequelae were observed after treatment.

Conclusions The EM lesions we observed are consistent with early Lyme disease occurring elsewhere, but laboratory confirmation of B burgdorferi infection is lacking in at least 16 cases (70%) analyzed using available methods. Genetically variable strains of B burgdorferi, alternative Borrelia species, or novel, uncharacterized infectious agents may account for most of the observed EM lesions.


From the Departments of Family Medicine (Dr Felz), Pathology (Dr Chandler), and Internal Medicine (Dr Rahn), Medical College of Georgia, Augusta; the Institute of Arthropodology and Parasitology, Georgia Southern University, Statesboro (Dr Oliver); and the Centers for Disease Control and Prevention, Fort Collins, Colo (Dr Schriefer). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the Centers for Disease Control and Prevention.

-------------------------------------------------------------------------------------------
Journal of Clinical Microbiology, May 2004, p. 2326-2328, Vol. 42, No. 5
0095-1137/04/$08.00+0 DOI: 10.1128/JCM.42.5.2326-2328.2004


Glycerophosphodiester Phosphodiesterase Gene (glpQ) of Borrelia lonestari Identified as a Target for Differentiating Borrelia Species Associated with Hard Ticks (Acari:Ixodidae)
Rendi Murphree Bacon,1* Mark A. Pilgard,1 Barbara J. B. Johnson,1 Sandra J. Raffel,2 and

Tom G. Schwan2
Bacterial Zoonoses Branch, Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado 80521,1 Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana 598402

Received 23 September 2003/ Returned for modification 7 January 2004/ Accepted 27 January 2004

A glpQ ortholog was identified in DNA from Borrelia lonestari-positive Amblyomma americanum, providing further evidence that B. lonestari is more closely related to the relapsing fever group spirochetes than to borreliae that cause Lyme disease. This finding provides a basis for developing diagnostic assays to differentiate species of borrelia transmitted by hard ticks.
--------------------------------------------------------------------------------------------------------
http://health.state.ga.us/pdfs/epi/gers/ger0801.pdf

(Released Prior to the detection in one human - Cjnelson)

August 2001 volume 17 number 08
Division of Public Health
http://health.state.ga.us
Kathleen E. Toomey, M.D., M.P.H.
Director
State Health Officer
Epidemiology Branch
http://health.state.ga.us/epi
Paul A. Blake, M.D., M.P.H.
Director
State Epidemiologist
Mel Ralston
Public Health Advisor

Georgia Epidemiology Report
Editorial Board
Carol A. Hoban, M.S., M.P.H. - Editor
Kathryn E. Arnold, M.D.
Paul A. Blake, M.D., M.P.H.
Susan Lance-Parker, D.V.M., Ph.D.
Kathleen E. Toomey, M.D., M.P.H.
Angela Alexander - Mailing List
Jimmy Clanton, Jr. - Graphic Designer
Georgia Department of
Human Resources
Division of Public Health
Epidemiology Branch
Two Peachtree St., N.W.
Atlanta, GA 30303-3186
Phone: (404) 657-2588
Fax: (404) 657-7517
Please send comments to:
[email protected]
The Georgia Epidemiology Report is a
publication of the Epidemiology Branch,
Division of Public Health, Georgia
Department of Human Resources

Lyme disease is caused by the spirochete Borrelia burgdorferi and is the most commonly
reported vector-borne disease in the United States. Endemic foci for Lyme
disease occur in the Northeast, Upper Midwest, and Pacific Coast regions of the U.S.;
however, there is much controversy over the risk of acquiring Lyme disease in the
Southeast. Clinicians in this region frequently report erythema migrans (EM) and a

clinical syndrome that resembles Lyme disease, but B. burgdorferi has never been isolated
from these lesions and patients are typically seronegative when tested for Lyme
disease by standard methods. So, is there indigenous transmission of Lyme disease
to humans in Georgia?

Borrelia burgdorferi is maintained in a variety of rodent reservoirs by Ixodes scapularis
(the ``black-legged tick'' or ``deer tick'') in the Northeast and Upper Midwest regions
of the U.S. and by Ixodes pacificis (the ``western black-legged tick'') in the Pacific Coast
region. Ixodes scapularis is present in the Southeast. However, the prevalence of B.
burgdorferi in this vector species collected from southern states is low compared to its

prevalence in ticks collected from Lyme-endemic areas of the northeast. Recent studies
have estimated the prevalence of B. burgdorferi in adult I. scapularis collected from the
Northeast to be as high as 52-56% while only 0-3% of ticks in southern states have
been found to be infected with the spirochete. Furthermore, immature I. scapularis
ticks in the south do not commonly feed rodents or other warm-blooded mammals

that are known reservoirs of B. burgdorferi in endemic areas; rather, they feed on a
wider variety of hosts that includes several species of lizards. Thus, while the potential
risk of human exposure to B. burgdorferi in the Southeast does exist, it is
considerably lower than in other regions of the US.

The incidence of human Lyme disease or Lyme-like illness in GA cannot
currently be estimated. The diagnosis of Lyme disease is complicated and although

Lyme disease is physician and laboratory reportable in GA, the submission of clinical
data as well as laboratory data is required to meet the surveillance case definition.
During 2000, the Georgia Division of Public Health (GDPH) received 245 laboratory

reports of positive serological tests for B. burgdorferi. Of these 245 reports, 4 were
accompanied by physician reports, but still did not meet the surveillance case definition

set forth by the Centers for Disease Control and Prevention (CDC). An additional
3 physician reports had presumptive diagnoses based upon clinical presentations

without laboratory testing. Most of the laboratory reports did not list a physician
name or any patient contact information, making additional follow-up nearly
impossible.

The tick bite-associated EM lesions that occur in the southern U.S. appear to
be associated with bites of Amblyomma americanum (the ``Lone Star tick''), which is the
most common human-biting tick in the region but is not a competent vector of B.

burgdorferi. The etiology of this ``Southern tick-associated rash illness'' (STARI) is
unknown, but studies to date have failed to implicate B. burgdorferi. A novel tick transmitted

spirochete called Borrelia lonestari is being evaluated as a possible etiologic
agent. B. lonestari has been detected by molecular methods in two Amblyomma
americanum ticks collected as part of an investigation of a cluster of STARI in Alabama.

Tick Bites and Erythema Migrans in
Georgia: It Might NOT be Lyme Disease!

-2 -

CLINICAL SYMPTOMS OF LYME DISEASE AND STARI

Table 1. Lyme disease: Surveillance Case Definition (Centers for Disease Control and Prevention)
Clinical Case Definition A case with Erythema Migrans or a case with at least one late manifestation (as defined below) that is laboratory confirmed

Erythema Migrans (EM)

EM is a skin lesion that typically begins as a red macule or papule and expands over a period of days to weeks in an annular
manner, often with partial central clearing. A solitary lesion must reach at least 5 cm in diameter to meet the surveillance

definition. The primary lesion occurs at the site of a bite of an infected tick and multiple (secondary) lesions may also occur.
Annular erythematous lesions that occur within several hours after a tick bite represent hypersensitivity reactions and are not indicators of disease transmission. EM is usually

accompanied by nonspecific symptoms that may include fever, malaise,
fatigue, headache, stiff neck, myalgia, and migratory arthralgia and/or lymphadenopathy. The incubation period from infection to onset of symptoms is typically 7 to 10 days (range 3 to 32 days). If left untreated, EM will fade spontaneously after several weeks.

Late Manifestations

(when an alternate
explanation is not found)

Musculoskeletal System

Recurrent brief attacks (lasting weeks or months) of objective joint swelling in one or a few joints, sometimes followed by
chronic arthritis in one or a few joints. Manifestations not considered criteria for diagnosis include chronic progressive arthritis

not preceded by brief attacks and chronic symmetrical polyarthritis. Arthralgia, myalgia, or fibromyalgia syndromes alone are
not criteria for musculoskeletal involvement.

Nervous System

Any of the following, alone or in combination: lymphocytic meningitis; cranial neuritis, particularly facial palsy (may be
bilateral); radiculoneuropathy; or, rarely, encephalomyelitis. Encephalomyelitis must be

confirmed by showing antibody
production against B. burgdorferi in the CSF, demonstrated by a higher titer of antibody in CSF than in serum. Headache, fatigue,
paraesthesia, or mild stiff neck alone are not criteria for neurologic involvement.

Cardiovascular System

Acute-onset, high-grade (2nd or 3rd degree) atrioventricular conduction defects that resolve in days to weeks and are sometimes associated with myocarditis. Palpitations, bradycardia, bundle-branch block, or myocarditis alone are not criteria for cardiovascular involvement.

Laboratory Criteria for
Diagnosis and Specimen

Requirements
* Serum: 2-step process for early disease: 1) EIA or IFA - IgG
and IgM 2) if positive, do Western blot - IgG and IgM. If late
disease: IgG Western blot. If neuroborreliosis: EIA-IgG serum
and CSF. Calculate index if EIA elevated.

* Skin biopsy: isolation and culture of B. burgdorferi OR

* CSF: isolation and culture of B. burgdorferi OR

* Synovial fluid: isolation and culture of B. burgdorferi

Serum: 5 mL serum (red-top tube) collected both at
disease onset and 4-6 weeks later

Skin biopsy: 2-4 mm skin lesion in transport medium
collected in acute phase of the illness

CSF: 5-10 mL in sterile tube

Synovial fluid: 5-10 mL in sterile tube

Note: This surveillance case definition is a public health tool intended for the surveillance of health events in populations. It is not intended for use in clinical diagnosis or management decisions in individual cases. In geographic areas where Lyme disease occurs infrequently or not at all, the positive predictive value of this case definition is low (i.e. cases that meet or appear to meet the definition will have a low likelihood of being true cases).

Southern Tick-Associated Rash Illness (STARI) is characterized
by an expanding annular erythema, mild constitutional symptoms, a springsummer
seasonality, a recent antecedent tick bite at the site of the skin
rash in many cases, an absence of antibodies to Borrelia burgdorferi using

standardized methods, and negative results of skin biopsy cultures. STARI
is believed to be a self-limited disease in most cases; late sequelae or
disseminated disease are rare.

DIAGNOSIS OF LYME DISEASE AND STARI
The diagnosis of Lyme disease should be based primarily upon clinical
findings that are supported by serologic testing. When Lyme disease is
suspected based on clinical findings, a sensitive screening test such as an
enzyme-linked immunosorbent assay (ELISA) or immunofluorescent

antibody (IFA) test should be performed. Positive test results should
be interpreted with caution because cross-reacting ELISA and
IFA antibodies may produce false positive reactions in patients
with syphilis, relapsing fever, leptospirosis, HIV infection, Rocky
Mountain spotted fever (RMSF), infectious mononucleosis, lupus,
or rheumatoid arthritis. Samples with positive or equivocal ELISA or

IFA results should be further tested with the more specific Western
immunoblot (WB) test. Although antibiotic treatment in early-localized
disease may blunt or abrogate the antibody response, patients with early
disseminated or late disseminated disease usually have strong serological
reactivity and demonstrate expanded IgG banding patterns on WB.

Antibodies often persist for months or years following successfully treated
or untreated infection. Thus, seroreactivity alone cannot be used as a
marker of active disease. Repeated infection with B. burgdorferi has been
documented.

Although Borrelia burgdorferi can be cultured from 80% or more of
biopsy specimens taken from early EM lesions, the diagnostic usefulness of
this procedure is limited because of the need for a special bacteriologic
medium and protracted observation of cultures. Polymerase chain reaction

(PCR) has been used to amplify genomic DNA of B. burgdorferi in skin,
blood, CSF, and synovial fluid, but PCR has not been standardized for routine
diagnosis of Lyme disease. A Lyme urinary antigen test (LUAT) is commercially

available, but it has been shown to give contradictory results and it yields
a high rate of false-positives. The LUAT has not been approved by the FDA
and should not be used for diagnosing active or suspected Lyme disease.

There is currently no diagnostic test available for STARI. The agent of
STARI, presumably B. lonestari, has not been successfully cultivated from
Amblyomma ticks or from biopsy specimens of EM lesions, or from other
human tissues or blood. An experimental PCR test to be used on skin biopsy

specimens is available from the CDC. Serologic specimens obtained from
patients with STARI might yield a positive or equivocal Lyme disease (B.
burgdorferi) ELISA screening test, but will probably be negative when tested by
the more specific WB.

TREATMENT

Early Lyme disease responds to antibiotic therapy in almost all patients.
Oral antibiotic therapy shortens the duration of rash and generally prevents
development of late sequelae. While it is appropriate to treat patients with
early disease solely on the basis of objective signs and a known exposure, the

prophylactic treatment of tick bites is NOT recommended since the incidence
of infection after a tick bite is low and treatment of early disease is very
effective. There are currently no recommendations specific to STARI;
however, rash and other constitutional symptoms resolved quickly after the
initiation of doxycycline therapy in almost all published case reports

SUMMARY

There is much controversy over the etiology of EM rashes in the
Southeast. It is plausible that a newly recognized species of Borrelia is
transmitted by Amblyomma americanum ticks and is responsible for a Lyme-like

syndrome called STARI. Healthcare providers in GA are urged to keep an
open mind and consider the following as they evaluate and treat patients with
potential tick exposure:

1. Campbell GL, WS Paul, ME Schriefer, et al. Epidemiologic and diagnostic studies of patients with suspected early Lyme disease, Missouri, 1990-1993. J Infect Dis 1995;172:470-80.
2. Kirkland KB, TB Klimko, RA Meriwether, et al. Erythema migrans-like rash illness at a camp in North Carolina. A new tick-borne disease? Arch Intern Med 1995;157:2635-2641.
3. Masters E, S Granter, P Duray, P Cordes. Physician-diagnosed erythema migrans and erythema migrans-like rashes following lone star tick bites. Arch Dermatol 1998;134:955-60.
4. TR Burkot, GR Mullen, R Anderson, et al. Borrelia lonestari DNA in adult Amblyomma americanum ticks, Alabama. Emerg Infect Dis 2001; In Press.
5. Barbour AG. Does Lyme disease occur in the South?: a survey of emerging tick-borne infections in the region. Am J Med Sci 1996;311:34-40.
6. Barbour AG, Maupin GO, Teltow GJ, et al. Identification of an uncultivable Borrelia species in the hard tick Amblyomma americanum: possible agent of a Lyme disease-like illness. J Infect Dis 1996;173:403-9.
7. Melski, JW. Language, logic, and Lyme disease. Arch Dermatol 1999;135:1398-1400
8. Oliver JH. Lyme borreliosis in the southern United States: a review. J Parasitol 1996;82(6):926-35.
9. American College of Physicians. Guidelines for laboratory evaluation in the diagnosis of Lyme disease: clinical guideline parts 1 and 2. Annals of Internal Medicine 1997; 128(12):1106-1123.
10. Klempner MS, Schmid CH, Hu L, et al. Intralaboratory reliability of serologic and urine testing for Lyme disease. Am J Med 2001;110:217-19.
11. Goodman, JL. The diagnosis of Lyme disease: good news, bad news. Editorial. Am J Med 2001;110:236-38.
12. Nadelman, RB, et al. Prophylaxis with Single-Dose Doxycyline for the Prevention of Lyme Disease after an Ixodes scapularis Tick Bite, http://www.nejm.org/earlyrelase/index.asp.
13. Klempner, MS, et al. Two Controlled Trials of Antibiotic Treatment in Patients with Persistent Symptoms and a History of Lyme Disease, http://www.nejm.org/earlyrelase/index.asp.
14. Steere, AC. Medical Progress: Lyme Disease, http://www.nejm.org/earlyrelase/index.asp.
15. Shapiro, ED. Doxycycline for Tick Bites, http://www.nejm.org/earlyrelase/index.asp.
Authors | Catherine Rebmann, M.P.H. and Susan Lance-Parker, D.V.M., Ph.D.

---------------------------------------------------------------------------------------
http://www.liebertonline.com/doi/abs/10.1089/vbz.2005.5.146


Vector-Borne and Zoonotic Diseases
Rapid Detection Methods and Prevalence Estimation for Borrelia lonestari glpQ in Amblyomma americanum (Acari: Ixodidae) Pools of Unequal Size
________________________________________
To cite this paper:
Rendi Murphree Bacon, Mark A. Pilgard, Barbara J.B. Johnson, Joseph Piesman, Brad J. Biggerstaff, Miguel Quintana. Vector-Borne and Zoonotic Diseases. June 1, 2005, 5(2): 146-156. doi:10.1089/vbz.2005.5.146.
________________________________________
Full Text PDF: * HiRes for printing (95.8 KB) * PDF Plus w/ links (143.2 KB)


Rendi Murphree Bacon
Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado.
Mark A. Pilgard
Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado.
Barbara J.B. Johnson
Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado.
Joseph Piesman
Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado.
Brad J. Biggerstaff
Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado.
Miguel Quintana
United States Army Center for Health Promotion and Preventive Medicine-West, Fort Lewis, Washington.
DNA was extracted from pools of Amblyomma americanum ticks collected from vegetation at two sites in Fort Leonard Wood, Missouri and tested for the presence of Borrelia spp. Two new methods were developed to detect Borrelia lonestari DNA by targeting the glycerophosphodiester phosphodiesterase (glpQ) gene. The first method detected B. lonestari DNA using a SYBR green I
melting curve analysis of the PCR product obtained with glpQ gene primers. The second method, a glpQ TaqMan assay, detected and confirmed the presence of B. lonestari glpQ-specific sequences. Twenty-two of 95 tick pools collected at site A148 contained B. lonestari DNA. None of 19 pools from site A241 contained B. lonestari DNA. No B. burgdorferi sensu lato DNA was detected using a SYBR green I melting curve analysis of the PCR product obtained with outer
surface protein A (ospA) primers. The overall B. lonestari infection prevalence (with 95% confidence interval) at site A148 was estimated using two algorithms: minimum infection rate 4.14% (2.45, 5.84) and maximum likelihood with correction 4.82% (3.11, 7.16). The merits of each are discussed. Sequencing of the entire B. lonestari glpQ and partial 16S rRNA genes revealed two genetic variants circulating in this population of A. americanum from Missouri. Vector-Borne Zoonotic Dis. 5, 146-156.
http://www.journals.uchicago.edu/doi/abs/10.1086/432935


Clinical Infectious Diseases 2005;41:958-965
2005 by the Infectious Diseases Society of America. All rights reserved.
1058-4838/2005/4107-0005$15.00
DOI: 10.1086/432935
MAJOR ARTICLE
Prospective Clinical Evaluation of Patients from Missouri and New York with Erythema Migrans-Like Skin Lesions
Gary P. Wormser,1
Edwin Masters,3
John Nowakowski,1
Donna McKenna,1
Diane Holmgren,1
Katherine Ma,1
Lauren Ihde,1
L. Frank Cavaliere,2 and
Robert B. Nadelman1
1Divisions of Infectious Diseases and 2Rheumatic Diseases, Department of Medicine, New York Medical College, Valhalla; and 3Family Practice of Dr. Edwin Masters, Cape Girardeau, Missouri
Background. The most common and most recognizable feature of Borrelia burgdorferi infection (Lyme disease) is the skin lesion erythema migrans (EM). An illness associated with an EM-like skin lesion, but which is not caused by B. burgdorferi,
occurs in many southern states in the United States (southern tick-associated rash illness [STARI], also known as Masters disease).
Methods. Clinical features of 21 cases of EM-like skin lesions in 21 patients from Missouri were compared in a prospective study with those of 101 cases in 97 patients with EM-like skin lesions from New York.
Results. Among Missouri cases, the peak incidence of EM-like skin lesions occurred earlier in the year than it did among New York cases ( ). Case patients from Missouri were more likely to recall a tick bite than were case patients from New York (85.7% and 19.8%, respectively; ), and the time period from tick bite to
onset of the skin lesion was shorter among Missouri case patients ( days and days, respectively; ). Missouri case patients were less likely to be symptomatic than were New York case patients (19.0% and 76.2%, respectively; ), and Missouri case patients were less likely to have multiple skin lesions
(4.8% and 26.7%, respectively; ). EM-like lesions in Missouri cases were smaller in size than those in New York cases ( cm and cm, respectively; ), more circular in shape ( ), and more likely to have central clearing (76.2% and 21.6%, respectively; ). After antibiotic treatment, Missouri case patients recovered more rapidly than did New York case patients ( ).
Conclusion. Cases of EM-like skin lesion in patients from Missouri and New York have distinct clinical presentations.
Received 8 February 2005; accepted 25 May 2005; electronically published 31 August 2005.
Reprints or correspondence: Dr. Gary P. Wormser, Div. of Infectious Diseases, New York Medical College, Munger Pavilion, Rm. 245, Valhalla, NY 10595 ([email protected]).
Cited by
Robert R Mllegger, Martin Glatz. (2008) Skin Manifestations of Lyme Borreliosis. American Journal of Clinical Dermatology 9:6, 355-368
Online publication date: 1-Feb-2008.
CrossRef
Eric Hoppa, Richard Bachur. (2007) Lyme disease update. Current Opinion in Pediatrics 19:3, 275???280
Online publication date: 1-Jul-2007.
CrossRef
Gary P. Wormser, Raymond J. Dattwyler, Eugene D. Shapiro, John J. Halperin, Allen C. Steere, Mark S. Klempner, Peter J. Krause, Johan S. Bakken, Franc Strle, Gerold Stanek, Linda Bockenstedt, Durland Fish, J. Stephen Dumler, and
Robert B. Nadelman. (2006) The Clinical Assessment, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and Babesiosis: Clinical Practice Guidelines by the Infectious Diseases Society of America. Clinical Infectious Diseases 43:9, 1089-1134
Online publication date: 1-Nov-2006.
Abstract-Full Text-PDF Version (3192 kB)
Edwin J. Masters. (2006) Lyme-Like Illness Currently Deserves Lyme-Like Treatment. Clinical Infectious Diseases 42:4, 580-581
Online publication date: 15-Feb-2006.
Citation-Full Text-PDF Version (268 kB)
David T. Dennis. (2006) Reply to Masters. Clinical Infectious Diseases 42:4, 581-582
Online publication date: 15-Feb-2006.
Citation-Full Text-PDF Version (268 kB)
David T. Dennis. (2005) Editorial Commentary: Rash Decisions: Lyme Disease, or Not?. Clinical Infectious Diseases 41:7, 966-968
Online publication date: 1-Oct-2005.
Citation-Full Text-PDF Version (60 kB)
* The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the Centers of Disease Control and Prevention.
* (See the editorial commentary by Dennis on pages 966-8)

http://archderm.ama-assn.org/cgi/content/full/134/8/955


Physician-Diagnosed Erythema Migrans and Erythema Migrans-like Rashes Following Lone Star Tick Bites
Edwin Masters, MD; Scott Granter, MD; Paul Duray, MD; Paul Cordes, MD
Arch Dermatol. 1998;134:955-960.
ABSTRACT


Objective To differentiate cases of physician-diagnosed erythema migrans and erythema migrans-like rashes associated with Lone Star tick (Amblyomma americanum) bites.
Design Retrospective case series.
Setting Private primary care clinic in rural Missouri.
Patients Seventeen patients with physician-diagnosed erythema migrans following a definite Lone Star tick bite at the rash site.
Interventions A biopsy was performed on all rash sites. All patients were treated with oral antibiotics.
Main Outcome Measures Rash appearance, size, body location, multiple lesions, incubation time, associated symptoms, seasonal occurrence, histopathological features, tick stage and sex, patient age and sex, treatment response, growth in BSK II culture media, and serologic evaluation.
Results Rashes associated with Lone Star ticks were similar to erythema migrans vectored by other Ixodes ticks. Differences were noted in Lyme disease serology results, especially flagellin-based enzyme immunoassays, and failure to yield spirochetes in BSK II cultures. Lyme serology results were often negative, but were also frequently inconsistent with results of controls without Lyme disease.
Conclusions Lone Star ticks are associated with rashes that are similar, if not identical, to erythema migrans associated with borrelial infection. The recent isolation and cultivation of Borrelia burgdorferi from ticks (including 1 Lone Star tick) from the farm of a patient included in this report has raised the possibility that Lone Star ticks are "bridge vectors" for human borrelial infection. Although further investigation is needed, these rashes may be secondary to spirochetal infection.


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THE ROLE of the Lone Star tick (Amblyomma americanum) in the transmission of Lyme disease, erythema migrans, or Lymelike illness has been controversial since first reported in 1984.1 The recent isolation of Borrelia burgdorferi sensu lato from ticks, including a Lone Star tick, collected at the Missouri farm of a patient with a physician-diagnosed erythema migrans rash following a witnessed Lone Star tick bite raises the possibility that the Lone
Star tick may act as a "bridge vector" much like Ixodes pacificus in the western United States.2 Recently, 14 cases of erythema migrans-like rashes were reported in North Carolina where a tick survey implicated Lone Star ticks as the likely vector since they accounted for 95% of the human tick bites. Borrelia burgdorferi were cultured from 1 local white-footed mouse. Two of the 14 patients accurately described a Lone Star tick bite as preceding their rash. In 1 case, the Lone Star tick was positively identified. The study results argued against a B31 B burgdorferi-like cause.3 In one study of suspected early Lyme disease in Missouri, several patients with erythema migrans-like rashes accurately described the adult female
Lone Star tick as the one that bit them.4 A Lone Star nymph that did stain positive to H5332 immunofluorescent assay after being removed from a human has been previously reported.5 The increasing heterogeneity of B burgdorferi in North America may relate to the possibility that strain variants may have adapted to the Lone Star tick.6-9 Additionally, the identification of Borrelia lonestari in Lone Star ticks10 and the findings of other researchers of spirochetes
consistent with B burgdorferi or Borrelia DNA in Lone Star ticks8, 11-15 have warranted a closer look at Lone Star ticks as a possible vector of a borreliosis. The exact cause of physician-diagnosed erythema migrans in the South, including Missouri, is currently being investigated. To our knowledge, this is the first published series of physician-diagnosed erythema migrans rashes associated with definite Lone Star tick bites.

PATIENTS AND METHODS

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Seventeen patients who presented to a primary care clinic in Cape Girardeau, Mo, from May 6, 1990, to September 15, 1993, with rashes similar, if not identical, to erythema migrans and documented Lone Star tick (Amblyomma americanum) bites were studied. For inclusion in this study, positive identification of the arthropod as a Lone Star tick required confirmation of the distinctive white dot on the back of the adult female. Witnessed nymph
tick bites were excluded unless the tick was saved for definitive identification. Some patients have been subjects of other studies. Five patients (patients 3, 5, 6, 8, and 9) were enrolled in a double-blind, randomized controlled trial comparing azithromycin with amoxicillin in the treatment of erythema migrans.16 Remarkably, these patients were not distinguishable in this study from patients in accepted Lyme disease endemic areas. Six patients (patients 1, 3, 4, 5, 7, and 9) were in a Centers for Disease Control and Prevention (CDC) retrospective
study of Missouri patients with suspected early Lyme disease.4 Fourteen of the rashes had biopsy specimens of the peripheral margins available for study. All biopsy specimens were formalin fixed and paraffin embedded by standard techniques. Sections were also stained using a modified Dieterle silver stain to identify spirochetes. This stain was performed as previously described.17

RESULTS

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CLINICAL FINDINGS
The clinical data are summarized in Table 1. Seventeen patients, 9 male and 8 female, were studied. Their ages ranged from 12 to 69 years (median, 33 years).


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Table 1. Clinical Characteristics of Study Patients*


All rashes were measured and photographed. They had a median incubation time of 7 days, with a range from 2 to 15 days. The relationships among the tick sizes and sex, incubation times, and rash diameters are illustrated in Figure 1. The nymph tick bites were associated with rash incubation times ranging from 3 to 14 days; 3 of 4 had incubation times of 11 days or longer. Rash location included the back (10 cases), anterior thorax or abdomen (4
cases), groin (2 cases), and leg (1 case). The median rash diameter was 7.5 cm, with the largest being 15 cm (range, 2.5-15 cm). All rashes were similar to, and often indistinguishable from, erythema migrans in patients from areas with endemic Lyme disease. Examples of rashes are shown in Figure 2, Figure 3, Figure 4, and Figure 5. Fourteen of the 17 rashes had central clearing. Sixteen of these rashes were solitary
erythemas. Patient 6 had multiple lesions. This is consistent with the experience at the study site clinic in which approximately 15% of cases show multiple lesions. None was significantly pruritic or painful. Five patients (patients 1, 6, 9, 13, and 14) had mild associated flulike constitutional symptoms. The rashes occurred from April 18 to September 5 (median, May 31) during the years 1990 to 1993. There was no correlation between tick stage or sex and
rash diameter or incubation time. Nymph-associated rash incubation time ranged from 3 to 14 days and adult female-associated rash incubation time ranged from 2 to 15 days. Most patients have been followed up since, and although many have had additional tick bites, none has developed an erythema migrans-like rash.

LYME DISEASE SEROLOGY
Ten of the 17 case patients had Lyme serology results inconsistent with test-negative non-Lyme (uninfected) controls. (Control subjects were randomly selected from consenting office patients and emergency department patients having blood drawn for other purposes and volunteers with no history compatible with a borreliosis.) Eight Lyme serology results from
the 17 case patients had results suggestive of a borreliosis. Frozen serum samples were available on 3 patients and underwent multiple serologic evaluations. Extensive testing for other diseases and causes of possible cross-reactivity were negative with 1 exception: patient 7 tested positive for Coxiella burnetii (cause of Q fever). All tested patients had
negative test results for rheumatoid factor, antinuclear antibody, and syphilis serology. Patients 3, 5, and 7 did not have Western immunoblots. Patient 8 had multiple negative enzyme-linked immunoabsorbent assays (ELISAs). Patient 16 did not return after his initial visit when the biopsy was done and was unavailable for follow-up. Patient 9 seroconverted in the treatment study.16 A CDC whole-cell sonicated ELISA on patient 9 was also strongly
positive at 3.176 (positive >1.0). Results of serologic evaluations in patients 5, 6, and 7 are presented in Table 2. Additionally, 13 months after the tick bite, patient 6 (Figure 2) had a Western immunoblot with IgM of 59 and IgG of 20, 34, 38, 39, 41, 50, 60, 63, and 75 kd. Patient 6 was also the only patient in this series with multiple lesions (5) and associated
constitutional symptoms. She also had the most positive results of Lyme tests, including a positive biopsy result. Results of Lyme Western blots are presented in Table 3. These data show the unusually high frequency of 4 or more IgG Western blot bands, B burgdorferi- associated bands, and positive ELISAs, all of which are inconsistent with published data on non-Lyme controls.18-21 Six patients (patients 3, 5, 6, 7, 8, and 10) were enrolled in a
national erythema migrans treatment study, but patient 3 dropped out of the study. Test results of patient 8 were all negative, whereas the other 4 patients had 1 or more positive Lyme ELISAs.16
PATHOLOGICAL FINDINGS
Biopsies were performed on all 17 rashes at the peripheral margin and cultured in BSK II medium with negative results. All 14 rashes with biopsy specimens for histopathological evaluation revealed findings consistent with erythema migrans (Figure 6 and Figure 7). Six of 11 biopsy specimens examined with the modified Dieterle method17 showed silver-positive structures consistent with dermal spirochetes (Figure 8).
EPIDEMIOLOGY
In a separate tick survey, Lone Star ticks containing Borrelia -appearing spirochetes variably reactive to H5332 were found in the counties of 16 case patients.22 One patient was bitten in
a county in Southern Illinois where ticks have not yet been examined. Ticks (2 nymphs and 3 adult males) from 5 study patients were examined with midgut smears and stained negative to H5332 immunofluorescent assay at 1:100 dilution.
TREATMENT
All 17 patients were treated early and aggressively with oral antibiotics. The most common regimens were amoxicillin or doxycycline for 20 or more days. No sequelae or symptoms indicative of treatment failure were found in this small group.

COMMENT

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Our study shows that Lone Star ticks are associated with rashes similar to, or even indistinguishable from, erythema migrans rashes associated with Lyme disease in CDC-accepted endemic areas. Photographs of Missouri physician-diagnosed erythema migrans rashes have been published.23-26 A few points regarding the rashes we evaluated deserve
mentioning. It is not surprising that most of the rashes in this study were on the back since 10 of the 17 ticks were adults: this location would allow the larger adult tick to go unnoticed for a longer time and better transmit possible pathogens or antigens. Positive identification of the distinctive white dot on the back of the adult female tick was required for inclusion in this study; however, nymphal and adult male forms had to be saved for definitive
identification. This study has an obvious selection bias for patients bitten by adult female forms. Therefore, it is not surprising that only 4 of 17 study cases presented here involved nymphal ticks, whereas our experience in dealing with physician-diagnosed erythema migrans during the past decade in Missouri indicates that the majority of these rashes are associated with nymphal ticks.
The summer peak incidence, histological findings, treatment response, rash diameter, incubation time, patient age and sex, frequency of multiple lesions, and signs and symptoms were similar to that associated with Lyme disease reported nationally. Notably different was the inability to culture spirochetes in BSK II media. We did not necessarily expect good
culture results with a medium designed for spirochetes from other Ixodes ticks. If the rashes we encountered are indeed associated with borrelial infection, the BSK II media may not be satisfactory for isolation of potential spirochetes associated with the Lone Star tick. It has been shown that that BSK II culture media can select for specific genotypes of B burgdorferi.27
Lyme serology testing argues against a B31 B burgdorferi cause. However, the serology results are also inconsistent with a test-negative, uninfected control population.18-21,26 We know that different strain variants can have different test results28-29 and that B burgdorferi sensu lato in Europe can test negative with culture-proven disseminated disease.30 With more B burgdorferi sensu lato being cultured in the South (eg, the farm of patient 12),2 this
possibility needs to be explored. In 3 patients in our study there was a dramatic and unexplained difference in ELISA testing of Missouri patients using whole-cell sonicated antigens and flagellar antigens. This was observed in a serologic study by the CDC.4 Previously, the CDC whole-cell sonicated and flagellar ELISAs were highly concordant, but not in Missouri patients. The whole-cell sonicated ELISA tested positive in approximately
45% of Missouri patients with erythema migrans, but tested negative in 37 (96%) of 38 Missouri control subjects, whereas the flagellar ELISA was almost always negative. The discordant results were such that the odds of this occurring by chance were 1 in 25 million. These results are consistent with the possibility of a related Borrelia that frequently cross-reacts with the whole-cell sonicated ELISA, but rarely with flagellar ELISA.4, 18, 26 Similarly,
Missouri Western blot results are usually negative by the strict criteria of Dressler et al31 adopted by the CDC where neither outer surface protein A (31 kd) nor outer surface protein B (34 kd) are counted.31 The results are also inconsistent with test-negative non-Lyme controls.18-21,26
The treatment of patients with erythema migrans and erythema migrans-like rashes outside CDC-accepted endemic areas of Lyme disease is controversial. We believe, given the likelihood of a borrelial etiology, that these rashes should be treated with antibiotics as would an erythema migrans rash in an accepted endemic area. This is also the view of
others.3-4 No sequelae or symptoms indicative of treatment failure were found in this small group, which is similar to observations of others.32 Nationally, the erythema migrans treatment failure rate has been variously reported at between 5% and 10%.23 Until a borrelial cause is either proven or refuted for these cases, proper treatment and follow-up will be controversial.
In conclusion, we have presented evidence that rashes visibly similar or indistinguishable from other Ixodes tick-vectored Lyme erythema migrans can be associated with Lone Star tick bites. Collateral evidence suggests the possibility that Borreli a play a pathogenic role in these patients. If proven, the clinical and epidemiological implications of a Lone Star-
vectored borreliosis are great, especially in view of the prevalence of the tick in the South and south central United States, as well as evidence that it is becoming more widespread. For example, the increase in prevalence of the Lone Star tick from 2 New York counties in the 1970s to 46 of the 62 New York counties today has been documented.33 Also, there is the possibility in areas where there are both Ixodes scapularis (Ixodes dammini)34 and Lone
Star (Amblyomma americanum) ticks that frequently bite humans (eg, New Jersey), that a Lone Star-vectored borreliosis could result in physician-diagnosed or -suspected Lyme disease that could often be seronegative. Clearly, the pathogenic role of Borrelia in these patients needs further investigation.

AUTHOR INFORMATION

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Accepted for publication May 6, 1998.
We thank Group Health Foundation of St Louis and the Lyme Disease Institute for financial support of our effort to study physician-diagnosed erythema migrans rashes in Missouri.
This article is an expansion of an abstract presented at the VI International Conference on Lyme Borreliosis in Bologna, Italy, June 19-22, 1994.
We acknowledge the assistance of Paul Spence, MD, Patrick Downey, MD, Rod Crist, MD, Charles Crist, MD, and David Catron, MD, for referring patients and supporting the study; Don Miles, PhD, for technical and microbiological assistance; Pam Burton, Bonnie Holmes, and Jackie Masters for secretarial and data collection work; Charles Darby for computer data expertise; and Brent Voszler, MD, for reviewing the manuscript.
Reprints: Edwin Masters, MD, Regional Primary Care, 69 Doctors Park, Cape Girardeau, MO 63703.
From Regional Primary Care, Cape Girardeau, Mo (Dr Masters); Brigham and Women's Hospital, Harvard Medical School, Boston, Mass (Dr Granter); National Institutes of Health, Bethesda, Md (Dr Duray); and Southeast Missouri Hospital, Cape Girardeau (Dr Cordes).

REFERENCES

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1. Schulze TL, Bowen GS, Bosler EM, Lakat MF, Parkin WE. Amblyomma americanum: a potential vector of Lyme disease in New Jersey. Science. 1984;224:601-603. FREE FULL TEXT

2. Oliver JH, Kollars TM, Chandler FW, et al. First isolation and cultivation of Borrelia burgdorferi sensu lato from Missouri. J Clin Microbiol. 1998;36:1-5. FREE FULL TEXT

3. Kirkland KB, Klimko TB, Meriwether RA, et al. Erythema migranslike rash illness at a camp in North Carolina. Arch Intern Med. 1997;157:2635-2641. ABSTRACT

4. Campbell GL, Paul WS, Schriefer ME, Craven RB, Robbins KE, Dennis DT. Epidemiologic and diagnostic studies of patients with suspected early Lyme disease, Missouri, 1990-1993. J Infect Dis. 1995;182:470-480.

5. Masters EJ. Case Histories in Missouri: Lyme Disease in the South Central United States. St Louis: The Missouri Academy of Science 1990;88:5-8. Occasional Paper No. 8.

6. Zingg BC, Anderson JF, Johnson RC, LeFever RB. Comparative analysis of genetic variability among Borrelia burgdorferi isolates from Europe and the United States by restriction enzyme analysis, gene restoration fragment length polymorphism, and pulsed field gel electrophoresis. J Clin Microbiol. 1993;31:3115-3122. FREE FULL TEXT

7. Picken RN, Cheng Y, Han D, et al. Genotypic and phenotypic characterization of Borrelia burgdorferi isolated from ticks and small animals in Illinois. J Clin Microbiol. 1995;33:2304-2315. ABSTRACT

8. Oliver JH. Lyme borreliosis in the Southern United States: a review. J Parasitol. 1996;82:926-935. FULL TEXT | PUBMED

9. Mathieson DA, Oliver JH, Kolbert CP, et al. Genetic heterogeneity of Borrelia burgdorferi in the United States. J Infect Dis. 1997;175:98-107. ISI | PUBMED

10. Barbour AG, Maupin GO, Tetlow GJ, et al. Identification of an uncultivable Borrelia species in the hard tick Amblyomma americanum: possible agent of a Lyme diseaselike illness. J Infect Dis. 1996;173:403-409. ISI | PUBMED

11. Levine JF, Sonenshine DE, Nicholson WL, Turner RT. Borrelia burgdorferi in ticks (Acari;Ixodidae) from coastal Virginia. J Med Entomol. 1991;28:668-674. ISI | PUBMED

12. Tetlow GJ, Fournier PV, Rawlings JA. Isolation of Borrelia burgdorferi from arthropods collected in Texas. Am J Trop Med Hyg. 1991;44:469-474.

13. Luckhart S, Mullen GR, Durden LA, Wright JC. Borrelia sp in ticks recovered from whitetailed deer in Alabama. J Wildl Dis. 1992;28:449-452. ABSTRACT

14. Rawlings JA, Teltow GL. Prevalence of Borrelia (Spirochaetaceae) spirochetes in Texas ticks. J Med Entomol. 1994;31:297-301. ISI | PUBMED

15. Levine JF, Apperson CS, Nicholson WL. The occurrence of spirochetes in Ixodes ticks in North Carolina. J Entomol Sci. 1989;24:594-602.

16. Luft BJ, Dattwyler RJ, Johnson RC, et al. Azithromycin compared with amoxicillin in the treatment of erythema migrans. Ann Intern Med. 1996;124:785-791. FREE FULL TEXT

17. Duray PH, Kusnitz A, Ryan J. Demonstration of the Lyme spirochete by a modification of the Dieterle stain. Lab Med. 1985;16:685-687.

18. Masters EJ, Donnell HD. Epidemiologic and diagnostic studies of patients with suspected early Lyme disease, Missouri, 1990-1993 [letter]. J Infect Dis. 1996;173:1527-1528. ISI | PUBMED

19. Fawcet PT, Gibney KM, Rose CD, Dubbs SB, Doughty RA. Frequency and specificity of antibodies that crossreact with Borrelia burgdorferi antigens. J Rheumatol. 1992;19:582-587. ISI | PUBMED

20. Ma B, Christen B, Leung D, Vigo-Pelfrey C. Serodiagnosis of Lyme borreliosis by Western immunoblot: reactivity of various significant antibodies against Borrelia burgdorferi. J Clin Microbiol. 1992;30:370-376. FREE FULL TEXT

21. Kowal K, Weinstein A. Western blot band intensity analysis. Arthritis Rheum. 1994;37:1206-1211. ISI | PUBMED

22. Feir D, Santanello CS, Li BW, et al. Evidence supporting the presence of Borrelia burgdorferi in Missouri. Am J Trop Med Hyg. 1994;51:475-482.

23. Masters EJ. Erythema migrans: rash as key to early diagnosis of Lyme disease. Postgrad Med. 1993;94:1334-1342.

24. Masters EJ, King LE. Differentiating loxoscelism from Lyme disease. Emerg Med. 1994;26:46-49.

25. Masters EJ, Donnell HD, Fobbs M. Missouri Lyme disease: 1989 through 1992. J Spirochetal Tickborne Dis. 1994;1:12-17.

26. Masters EJ, Donnell HD. Lyme and/or Lymelike disease in Missouri. Mo Med. 1995;92:345-353.

27. Norris DE, Johnson BJ, Piesman J, Maupin GO, Clark JL, Black WC. Culturing selects for specific genotypes of Borrelia burgdorferi in an enzootic cycle in Colorado. J Clin Microbiol. 1997;35:2359-2364. ABSTRACT

28. Bunikis J, Olden B, Westman G, Bergstrom S. Variable serum immunoglobulin responses against different Borrelia burgdorferi sensu lato species in a population at risk for and patients with Lyme disease. J Clin Microbiol. 1995;33:1473-1478. ABSTRACT

29. Hauser U, Krahl H, Peters H, Fingerle V, Wilske B. Impact of strain heterogeneity on Lyme disease serology in Europe: comparison of enzyme-linked immunosorbent assays using different species of Borrelia burgdorferi sensu lato. J Clin Microbiol. 1998;36:427-436. FREE FULL TEXT

30. Strle FS, Picken RN, Cheng Y, et al. Clinical findings for patients with Lyme borreliosis caused by Borrelia burgdorferi sensu lato with genotypic and phenotypic similarities to strain 25015. Clin Infect Dis. 1997;25:273-280. ISI | PUBMED

31. Dressler F, Whalen JA, Reinhardt BN, Steere AC. Western blotting in the serodiagnosis of Lyme disease. J Infect Dis. 1993;167:392-400. ISI | PUBMED

32. Nadelman RB, Nowakowski J, Forseter G, et al. Failure to isolate Borrelia burgdorferi after antimicrobial therapy in culture-documented Lyme borreliosis associated with erythema migrans: report of a prospective study. Am J Med. 1993;94:583-588. FULL TEXT | ISI | PUBMED

33. Means RG, White DJ. New distribution records of Amblyomma americanum (L.) (Acari: Ixodidae) in New York State. J Vector Ecol. 1997;22:133-145. ISI | PUBMED

34. Oliver JH, Owsley MR, Hutcheson JH, et al. Conspecificity of the ticks I. scapularis and I. dammini (Acari:Ixodidae). J Med Entomol. 1993;30:54-63. ISI | PUBMED
REPLY
Microbiologically Confirmed Early Lyme Disease
Robert P. Smith, MD, and Allen C. Steere, MD

15 October 2002 | Volume 137 Issue 8 | Page 698
________________________________________
IN RESPONSE:
Dr. Masters objects to our inclusion of the reference by Campbell and associates (1) in our brief allusion to an erythema migrans-like rash that has been associated with the bites of lone star ticks (Amblyomma americanum). Although the clinical characteristics of this illness, which has been reported predominantly in the southeastern and south-central United States, have been described by Masters and colleagues (2),
others, including Campbell and associates, have also contributed to the investigation of its epidemiology and possible etiology (1, 3-5). Attempts to demonstrate infection in these patients by Borrelia burgdorferi sensu stricto, the recognized cause of Lyme disease in the United States, have been unsuccessful. Nevertheless, as pointed out by Masters and colleagues, the characteristics of the rash and its apparent response to antibiotics suggest that this illness may be provoked by an infectious agent. The identification
of "Borrelia-like organisms" in lone star ticks provides tantalizing circumstantial evidence that these organisms may turn out to be the cause (4, 5). Campbell and associates refer to this possibility as "intriguing and deserving of further study." Their article, although unable to provide direct evidence for a
specific infectious cause for this illness, does provide a well-designed epidemiologic analysis of its occurrence in Missouri. It also describes an intense effort to search for an infectious cause. Given the wide distribution of lone star ticks in the United States, continued investigation into the clinical characteristics, epidemiology, and pathogenesis of this illness is certainly warranted.

of Clinical Microbiology, January 2003, p. 424-427, Vol. 41, No. 1
0095-1137/03/$08.00+0 DOI: 10.1128/JCM.41.1.424-427.2003
Copyright 2003, American Society for Microbiology. All Rights Reserved.
Detection of Borrelia lonestari, Putative Agent of Southern Tick-Associated Rash Illness, in White-Tailed Deer (Odocoileus virginianus) from the Southeastern United States
Victor A. Moore IV,1 Andrea S. Varela,1 Michael J. Yabsley,2 William R. Davidson,2,3 and Susan E. Little1*
Department of Medical Microbiology and Parasitology,1 Southeastern Cooperative Wildlife Disease Study, College of Veterinary Medicine,2 The D. B. Warnell School of Forest Resources, The University of Georgia, Athens, Georgia 306023
Received 20 May 2002/ Returned for modification 7 August 2002/ Accepted 12 October 2002
To determine if white-tailed deer may serve as a reservoir host for Borrelia lonestari, we used a nested PCR for the Borrelia flagellin gene to evaluate blood samples collected from deer from
eight southeastern states. Seven of 80 deer (8.7%) from 5 of 17 sites (29.4%) had sequence-confirmed evidence of a B. lonestari flagellin gene by PCR, indicating that deer are infected with B. lonestari or another closely related Borrelia species. Our findings expand the known geographic range of B. lonestari and provide the first evidence of this organism in a vertebrate other than humans.
Journal of Clinical Microbiology, March 2004, p. 1163-1169, Vol. 42, No. 3
0095-1137/04/$08.00+0 DOI: 10.1128/JCM.42.3.1163-1169.2004
Copyright 2004, American Society for Microbiology. All Rights Reserved.
First Culture Isolation of Borrelia lonestari, Putative Agent of Southern Tick-Associated Rash Illness
Andrea S. Varela,1 M. Page Luttrell,2 Elizabeth W. Howerth,3 Victor A. Moore,1 William R. Davidson,2,4 David E. Stallknecht,1,2 and Susan E. Little1*
Department of Medical Microbiology and Parasitology,1 Southeastern Cooperative Wildlife Disease Study,2 Department of Pathology, College of Veterinary Medicine,3 Warnell School of Forest Resources, University of Georgia, Athens, Georgia 306024
Received 25 August 2003/ Returned for modification 5 October 2003/ Accepted 5 November 2003
Southern tick-associated rash illness (STARI) is a Lyme disease-like infection described in patients in the southeastern and south-central United States, where classic Lyme disease is relatively rare. STARI develops following the bite of a lone star tick (Amblyomma americanum)
and is thought to be caused by infection with an "uncultivable" spirochete tentatively named Borrelia lonestari. In this study, wild lone star ticks collected from an area where B. lonestari is endemic were cocultured in an established embryonic tick cell line (ISE6). The cultures were examined by dark-field microscopy for evidence of infection, and spirochete identity and
morphology were evaluated by flagellin B and 16S rRNA gene sequence, by reaction to Borrelia-wide and B. burgdorferi-specific monoclonal antibodies, and by electron microscopy. Live spirochetes were first visualized in primary culture of A. americanum ticks by dark-field microscopy 14 days after the cell culture was inoculated. The sequences of the flagellin B and 16S rRNA genes of cultured spirochetes were consistent with previously reported sequences of B.
lonestari. The cultured spirochetes reacted with a Borrelia-wide flagellin antibody, but did not react with an OspA antibody specific to B. burgdorferi, by indirect fluorescent antibody testing. Electron microscopy demonstrated organisms that were free and associated with ISE6 cells, with characteristic Borrelia sp. morphology. This study describes the first successful isolation of B.
lonestari in culture, providing a much needed source of organisms for the development of diagnostic assays and forming a basis for future studies investigating the role of the organism as a human disease agent.

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Thanks!

Nice to have this together is a neat and orderly fashion. BIG help.

Good job!

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