![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Previous Article | Next Article 
Journal of Clinical Microbiology, February 2001, p. 494-497, Vol. 39, No. 2
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.494-497.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Lone Star Tick-Infecting Borreliae Are Most Closely
Related to the Agent of Bovine Borreliosis
Stephen M.
Rich,1
Philip M.
Armstrong,2,3
Ronald D.
Smith,4 and
Sam R.
Telford III2,*
Division of Infectious Disease, Tufts
University School of Veterinary Medicine, North
Grafton,1 and Department of Immunology
and Infectious Diseases, Harvard School of Public Health,
Boston,2 Massachusetts; Southwest
Foundation for Biomedical Research, San Antonio,
Texas3; and Department of Veterinary
Pathobiology, University of Illinois School of Veterinary Medicine,
Urbana, Illinois4
Received Recevied 15 May 2000/Returned for modification 5 September
2000/Accepted 9 November 2000
 |
ABSTRACT |
Although Borrelia theileri, the agent of bovine
borreliosis, was described at the turn of the century (in 1903), its
relationship with borreliae causing Lyme disease or relapsing fever
remains undescribed. We tested the previously published hypothesis that spirochetes infecting Lone Star ticks (Amblyomma
americanum) may comprise B. theileri by analyzing the
16S ribosomal DNAs (rDNAs) and flagellin genes of these spirochetes.
B. theileri, the Amblyomma agent, and B. miyamotoi formed a natural group or clade distinct from but most
closely related to that of the relapsing fever spirochetes. B. theileri and the Amblyomma agent were 97 and 98%
similar at the nucleotide level within the analyzed portions of the 16S
rDNA and the flagellin gene respectively, suggesting a recent
divergence. The agent of bovine borreliosis might be explored as a
surrogate antigen for the as-yet-uncultivatable Amblyomma
agent in studies designed to explore the etiology of a Lyme
disease-like infection associated with Lone Star ticks.
| |
INTRODUCTION |
Ticks comprise two major natural
groups, or clades: the Ixodidae (hard ticks) and the more primitive
mite-like Argasidae (soft ticks) (4, 14). The spirochetes
associated with these morphologically and biologically divergent kinds
of ticks have generally been referred to as either hard or soft
tick-borne borreliae (16). Interestingly, of the speciose
hard ticks, solely the Lyme disease-like spirochetes (Borrelia
burgdorferi sensu lato), transmitted by Ixodes spp.
(subfamily Prostriata), have been extensively studied. Although
B. theileri, associated with Rhipicephalus and
Boophilus (both classified within the ixodid subfamily
Metastriata), was described during the early part of the 20th century
(17), its relationship to other borreliae remains poorly
described. Spirochetes have been detected within the metastriate Lone
Star ticks (Amblyomma americanum) (1, 2, 10, 18,
24), but only recently has their taxonomic status been analyzed.
Their identity has assumed prominence because Lone Star ticks are
associated with a syndrome of undescribed etiology (referred to as
southern tick-associated rash infection or Masters' disease) that is
confused with Lyme disease (3, 8, 22; P. M. Armstrong, L. Rosa Brunet, A. Spielman, and S. R. Telford III,
submitted for publication). Because deer served as reservoir hosts for
Boophilus spp. in the south central United States during the
19th and early 20th centuries, we previously suggested that spirochetes
infecting Amblyomma, a deer-dependent tick, may represent a
host shift of B. theileri (26). We tested this
hypothesis by analyzing the nucleotide sequences of the 16S ribosomal
DNAs (rDNAs) and flagellin (fla) genes of B. theileri and the Amblyomma agent, and we determined the
genetic relationship of these previously uncharacterized spirochetes to
others of the genus Borrelia.
| |
MATERIALS AND METHODS |
Sources of borreliae.
B. theileri was derived
from naturally infected Boophilus microplus originally
collected from a Mexican site, as described previously and under an
existing U.S. Department of Agriculture permit (25). A
colony of infected Boophilus ticks was produced by allowing
nymphs to engorge on infected cattle. The resulting adult ticks were
examined by the hemolymph test (5) to determine whether
they were infected, allowed to engorge and oviposit to maintain the
infected colony, and stored frozen at 
70°C until they were analyzed.
Nymphal and adult A. americanum ticks were collected by
sweeping vegetation on Gibson Island, Md., during 1994 and
nondestructively assayed for spirochetal infection by dark-field
microscopy of hemolymph samples taken from the amputated stump of a leg
(5). Of 388 ticks, 2 contained spirochetes. Their gut
contents were removed and homogenized in 100 µl of lysis buffer (4 M
guanidine thiocyanate, 25 mM sodium citrate, 0.5% sarcosyl), and total
DNA was extracted by a standard phenol-chloroform extraction protocol. Attempts to cultivate the agent in BSK II and Kelly's medium failed.
DNA amplification and sequencing of borrelial genes.
PCR was
used to amplify an approximately 350-bp portion of the fla
gene (primers FLA-1 [5'-GATGATGCTGCTGGCATGGGAGTTGCGGG-3'] and FLAG-5 [5'-CCTGAAAGTGATGCTGGTG-3']), as well as
an 886-bp fragment of the 16S rDNA (primers BOR-16SA
[5'-TGTTAATTGATGAAAGGAAGCC-3'] and BOR-16SE
[5'-TTTACATGCTGGTAACAGAC-3']). Each 50-µl reaction mixture contained 5 µl of EXPAND 10× PCR buffer (Boehringer
Mannheim), 0.2 µl of each deoxynucleoside triphosphate (10 µM), 0.2 µl of EXPAND Taq polymerase (Boehringer Mannheim), 1 µl
of each primer (25 µM), and about 10 ng of template. Each of the 35 cycles consisted of denaturation at 94°C for 30 s, annealing at
50°C for 30 s, and extension at 72°C for 2 min in a
Perkin-Elmer (model 480) thermal cycler.
Double-stranded amplification products were electrophoresed in a 1.5%
agarose gel in 1× Tris-borate-EDTA buffer and were extracted from
excised gel bands using the GeneClean kit (Bio 101). Cycle sequencing
was performed using the Applied Biosystems dye-labeled dideoxy
termination kit, and the products were purified and analyzed on a 6%
polyacrylamide gel in an ABi 373A DNA sequencer (Applied Biosystems),
as recommended by the manufacturer.
Sequences were edited using the SeqEd 675 DNA sequence editor program
(Applied Biosystems). Each sequence was verified in duplicate by
analyzing both strands of the amplification product.
Phylogenetic analysis.
Borrelia gene sequences
were initially aligned with the CLUSTALW program (28).
Corrections to alignments were made manually, particularly in the case
of the fla gene, where the algorithm failed to preserve the
codon reading frame. The sequences that we derived from B. theileri and the Amblyomma agent were compared with
representative Borrelia 16S rDNA and fla gene
sequences accessioned in GenBank (Table
1). When the Harvard laboratory submitted
the sequences for the Amblyomma agent to GenBank in October
1995 (1), those for B. lonestari
(2) were under embargo and unavailable for comparison. At
the time we had provisionally named this spirochete B. barbouri, in honor of Alan Barbour's many contributions to the
study of Borrelia biology. The sequences accessioned under the names B. lonestari and B. barbouri are
identical and derive from the same entity; pending a formal designation
in Bergey's Manual, we here use the term
Amblyomma agent to refer to it.
Two 50% majority rule consensus phylograms, one for each of the two
genes examined, were constructed by performing 1000 bootstrap replications of the PAUP 4.0 (27) program's "Fast"
stepwise addition heuristic tree search method. In the 16S rDNA-based
analysis, Cristispira sp. served as the outgroup, whereas
the fla-based phylogeny is an unrooted network
Nucleotide sequence accession numbers.
The sequences
determined in this study have been deposited in GenBank under accession
numbers U38374, U38375, U42431, and U42432.
| |
RESULTS |
Relationships among spirochetes.
Maximum-parsimony
analysis was used to determine the relatedness of B. theileri, the Amblyomma agent, and B. recurrentis to several other Borrelia species, based on
the 16S rDNAs and fla genes. The low levels of nucleotide
polymorphism in either the flagellin gene or 16S rDNA limits the
information for resolving terminal taxa. However, reliable inferences
can be drawn from the two trees for the more broadly defined groupings.
Consistent with many other previous reports (9, 15, 20,
21, 23), the group of B. burgdorferi sensu lato
spirochetes (B. burgdorferi sensu stricto, B
garinii, and B. afzelii) group as a diverse monophyly (Fig. 1). Our analysis also places
B. japonica within the clade comprised of B. burgdorferi sensu lato.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 1.
Bootstrap consensus (1,000 times) of neighbor-joining
trees of Borrelia spirochetes using both 16S rDNA (left) and
flagellin (right) loci. The oral spirochete Cristispira sp.
was used as an outgroup. Distances were derived from the Tamura-Nei
algorithm (27a). Numbers indicate the bootstrap support
for each branch.
|
|
Phylograms based on both fla and 16S rDNA data sets (Fig. 1)
demonstrate that B. theileri, the Amblyomma
agent, and B. miyamotoi form a monophyletic group most
closely related to the two soft tick-transmitted spirochete clades.
Based on the fla DNA sequence, these Borrelia
spp. form a monophyletic sister group to the several relapsing fever spirochetes.
| |
DISCUSSION |
Our analysis supports Filippova's hypothesis that the vectors and
agents of Lyme disease comprise a monophyletic clade indicating evolution by descent within this group (11). The one
exception is B. miyamotoi, originally isolated from
Ixodes persulcatus (12, 13), the main Eurasian
vector of B. burgdorferi sensu lato. Although this
association may reflect a secondary host shift from the
metastriate-associated borreliae, it may be that I. persulcatus is not the main vector but that sympatric Japanese
ticks such as the metastriate Haemaphysalis longicornis
serve in this capacity. Further studies on the epizootiology of
B. miyamotoi may resolve this interesting exception to the
otherwise strongly supported congruence between Ixodes ticks
and Lyme disease-like borreliae.
The metastriate tick-transmitted B. theileri and the
Amblyomma spirochete clearly group outside the other
ixodid-borne borreliae, and certain features of their biology
corroborate this finding. Bovine borreliosis due to B. theileri is characterized by prominent spirochetemias in the
peripheral blood (7, 25), as is relapsing fever. Both
B. theileri and many of the argasid-transmitted spirochetes may be efficiently maintained by transovarial transmission. In the
vector, these spirochetes may easily be found in the hemolymph (25), in sharp contrast to the
Ixodes-transmitted borreliae, which less frequently produce
disseminated infections in the tick hosts (6). The
Amblyomma agent is detectable within ticks as frequently by
the hemolymph test as it is by indirect immunofluorescence of gut
contents (Fisher exact test, P = 0.376) (our
unpublished results), suggesting that other features of B. theileri's biology may be shared, such as the presence of
spirochetemia in the as-yet-undescribed reservoir and its possible
maintenance in ticks by transovarial transmission.
The degree of sequence similarity between the Amblyomma
agent and B. theileri is similar to that between different
isolates of B. hermsii and may reflect a very recent
divergence of the two metastriate-transmitted spirochetes. Because
A. americanum feeds mainly upon deer in all three instars,
we previously reasoned that any spirochete maintained by this tick
might be closely related to that maintained by the cattle tick
Boophilus (26). Indeed, deer served as
important enzootic hosts for the tick B. microplus, and the
eradication program for this economically important ectoparasite of
cattle (because of its role as a vector of Texas cattle fever or bovine
babesiosis) targeted cervids throughout the southern United States
(19). Because cattle and deer are sympatric in many sites,
Boophilus and the agents that it maintained could have
established parallel transmission cycles in deer.
Although specific names have been applied to the Amblyomma
agent (B. lonestari, B. barbouri, and a Borrelia
sp. similar to B. nr. miyamotoi), given its genetic
similarity to B. theileri, any formal description would
require justifying the application of a new name. More importantly,
because the Amblyomma agent is as yet uncultivatable,
determining its role in the etiology of southern tick-associated rash
infection (Masters' disease) may be facilitated by the use of B. theileri as a surrogate. These spirochetes attain a great density
within the blood of cattle and may be purified for use as an antigen.
In addition, the knowledge that B. theileri is maintained
transovarially and may be found as disseminated infections within the
vector, as well as producing transient spirochetemia in the ungulate
host, may help advance studies that support or refute the role of
borreliae in the epidemiology of Lone Star tick-associated, Lyme
disease-like infections of humans.
| |
ACKNOWLEDGMENTS |
This work was supported by NIH grants AI 37993, AI 39002, and AI 19693.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Immunology and Infectious Diseases, Harvard School of Public Health,
665 Huntington Ave., Boston, MA 02115. Phone: (617) 432-4079. Fax:
(617) 432-1796. E-mail: stelford{at}hsph.harvard.edu.
| |
REFERENCES |
| 1.
|
Armstrong, P. M.,
S. M. Rich,
R. D. Smith,
D. L. Hartl,
A. Spielman, and S. R. Telford.
1996.
A new Borrelia infecting Lone Star ticks.
Lancet
347:67-68[Medline].
|
| 2.
|
Barbour, A. G.,
G. O. Maupin,
G. J. Teltow,
C. J. Carter, and Piesman.
1996.
Identification of an uncultivable Borrelia species in the hard tick Amblyomma americanum: possible agent of a Lyme disease-like illness.
J. Infect. Dis.
173:403-409[Medline].
|
| 3.
|
Barbour, A. G.
1996.
Does Lyme disease occur in the south? A survey of emerging tick-borne infections in the region.
Am. J. Med. Sci.
311:34-40[Medline].
|
| 4.
|
Black, W. C. I.,
J. S. Klompen, and J. E. Keirans.
1997.
Phylogenetic relationships among tick subfamilies (Ixodida: Ixodidae: Argasidae) based on the 18S nuclear rDNA gene.
Mol. Phylogenet. Evol.
7:129-144[CrossRef][Medline].
|
| 5.
|
Burgdorfer, W.
1970.
Hemolymph test. A technique for detection of rickettsiae in ticks.
Am. J. Trop. Med. Hyg.
19:1010-1014.
|
| 6.
|
Burgdorfer, W.,
S. F. Hayes, and D. Corwin.
1989.
Pathophysiology of the Lyme disease spirochete, Borrelia burgdorferi, in ixodid ticks.
Rev. Infect. Dis.
11:S1442-S1450.
|
| 7.
|
Callow, L. L.
1967.
Observations on tick-transmitted spirochaetes of cattle in Australia and South Africa.
Br. Vet. J.
123:492-497[Medline].
|
| 8.
|
Campbell, G. L.,
W. S. Paul,
M. E. Schriefer,
R. B. Craven,
K. E. Robbins, and D. T. Dennis.
1995.
Epidemiologic and diagnostic studies of patients with suspected early Lyme disease, Missouri, 1990-1993.
J. Infect. Dis.
172:470-480[Medline].
|
| 9.
|
Canica, M. M.,
F. Nato,
L. du Merle,
J. C. Mazie,
G. Baranton, and D. Postic.
1993.
Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis.
Scand. J. Infect. Dis.
25:441-448[Medline].
|
| 10.
|
Feir, D.,
C. R. B. Santanello,
W. Li,
C. S. Xie,
E. Masters,
R. Marconi, and G. Weil.
1994.
Evidence supporting the presence of Borrelia burgdorferi in Missouri.
Am. J. Trop. Med. Hyg.
51:475-482.
|
| 11.
|
Filippova, N. A.
1990.
The taxonomic aspects of the transmission of the causative agent of Lyme disease.
Parazitologiia
24:257-267[Medline].
|
| 12.
|
Fukunaga, M.,
M. Sohnaka,
M. Nakao, and K. Miyamoto.
1993.
Evaluation of genetic divergence of borrelial isolates from Lyme disease patients in Hokkaido, Japan, by rRNA gene probes.
J. Clin. Microbiol.
31:2044-2048[Abstract/Free Full Text].
|
| 13.
|
Fukunaga, M.,
M. Sohnaka,
Y. Takahashi,
M. Nakao, and K. Miyamoto.
1993.
Antigenic and genetic characterization of Borrelia species isolated from Ixodes persulcatus in Hokkaido, Japan.
J. Clin. Microbiol.
31:1388-1391[Abstract/Free Full Text].
|
| 14.
|
Hoogstraal, H., and A. Aeschlimann.
1982.
Tick-host specificity.
Bull. Soc. Entomol. Suisse
55:5-32.
|
| 15.
|
Kawabata, H.,
T. Masuzawa, and Y. Yanagihara.
1993.
Genomic analysis of Borrelia japonica sp. nov. isolated from Ixodes ovatus in Japan.
Microbiol. Immunol.
37:843-848[Medline].
|
| 16.
|
Korenberg, E. I.
1996.
Infections of the Lyme borreliosis group ixodid tick-borne borrelioses in Russia.
Med. Parazitol. (Mosk)
3:14-18.
|
| 17.
|
Laveran, A.
1903.
Sur la spirillose des bovides.
C. R. Acad. Sci. Paris
136:939.
|
| 18.
|
Levine, J. F.,
C. S. Apperson, and W. L. Nicholson.
1989.
The occurrence of spirochetes in ixodid ticks in North Carolina.
J. Entomol. Sci.
24:594-602.
|
| 19.
|
MacKellar, W. M.
1942.
Keeping livestock healthy. Yearbook of Agriculture 1942. USDA, 77th Congress, 2nd Session, House Document 527.
United States Government Printing Office, Washington, D.C.
|
| 20.
|
Marconi, R. T.,
D. Liveris, and I. Schwartz.
1995.
Identification of novel insertion elements, restriction fragment length polymorphism patterns, and discontinuous 23S rRNA in Lyme disease spirochetes: phylogenetic analyses of rRNA genes and their intergenic spacers in Borrelia japonica sp. nov. and genomic group 21038 (Borrelia andersonii sp. nov.) isolates.
J. Clin. Microbiol.
33:2427-2434[Abstract].
|
| 21.
|
Marti Ras, N.,
D. Postic,
M. Foretz, and G. Baranton.
1997.
Borrelia burgdorferi sensu stricto, a bacterial species "made in the U.S.A."?
Int. J. Syst. Bacteriol.
47:1112-1117[Abstract/Free Full Text].
|
| 22.
|
Masters, E. J., and H. D. Donnell.
1995.
Lyme and/or Lyme-like disease in Missouri.
Missouri Med.
92:346-353.
|
| 23.
|
Postic, D.,
M. V. Assous,
P. A. Grimont, and G. Baranton.
1994.
Diversity of Borrelia burgdorferi sensu lato evidenced by restriction fragment length polymorphism of rrf (5S)rrl (23S) intergenic spacer amplicons.
Int. J. Syst. Bacteriol.
44:743-752[Abstract/Free Full Text].
|
| 24.
|
Schulze, T. L.,
G. S. Bowen,
E. M. Bosler,
M. F. Lakat,
W. E. Parkin,
R. Altman,
B. G. Ormiston, and J. K. Shisler.
1984.
Amblyomma americanum: a potential vector of Lyme disease in New Jersey.
Science
224:601-603[Abstract/Free Full Text].
|
| 25.
|
Smith, R. D.,
G. S. Miranpuri,
J. H. Adams, and E. H. Ahrens.
1985.
Borrelia theileri: isolation from ticks (Boophilus microplus) and tick-borne transmission between splenectomized calves.
Am. J. Vet. Res.
46:1396-1398[Medline].
|
| 26.
|
Spielman, A.,
R. J. Pollack, and S. R. Telford, III
1992.
The origins and course of the present outbreak of Lyme disease, p. 83-96.
In
H. S. Ginsberg (ed.), Ecology and environmental management of Lyme disease. Rutgers University Press, New Brunswick, N.J.
|
| 27.
|
Swofford, D.
1998.
Phylogenetic analysis using parsimony, paup4d61.ppc ed.
Smithsonian Institution, Washington, D.C.
|
| 27a.
|
Tamura, K., and M. Nei.
1993.
Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees.
Mol. Biol. Evol.
10:512-526[Abstract].
|
| 28.
|
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22:4673-4680[Abstract/Free Full Text].
|
Journal of Clinical Microbiology, February 2001, p. 494-497, Vol. 39, No. 2
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.2.494-497.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Bacon, R. M., Pilgard, M. A., Johnson, B. J. B., Raffel, S. J., Schwan, T. G.
(2004). Glycerophosphodiester Phosphodiesterase Gene (glpQ) of Borrelia lonestari Identified as a Target for Differentiating Borrelia Species Associated with Hard Ticks (Acari:Ixodidae). J. Clin. Microbiol.
42: 2326-2328
[Abstract]
[Full Text]
-
Varela, A. S., Luttrell, M. P., Howerth, E. W., Moore, V. A., Davidson, W. R., Stallknecht, D. E., Little, S. E.
(2004). First Culture Isolation of Borrelia lonestari, Putative Agent of Southern Tick-Associated Rash Illness. J. Clin. Microbiol.
42: 1163-1169
[Abstract]
[Full Text]
-
Stromdahl, E. Y., Williamson, P. C., Kollars, T. M. Jr., Evans, S. R., Barry, R. K., Vince, M. A., Dobbs, N. A.
(2003). Evidence of Borrelia lonestari DNA in Amblyomma americanum (Acari: Ixodidae) Removed from Humans. J. Clin. Microbiol.
41: 5557-5562
[Abstract]
[Full Text]
-
Masters, E. J., Olson, G. S., Weiner, S. J., Paddock, C. D.
(2003). Rocky Mountain Spotted Fever: A Clinician's Dilemma. Arch Intern Med
163: 769-774
[Abstract]
[Full Text]
-
Schwan, T. G., Battisti, J. M., Porcella, S. F., Raffel, S. J., Schrumpf, M. E., Fischer, E. R., Carroll, J. A., Stewart, P. E., Rosa, P., Somerville, G. A.
(2003). Glycerol-3-Phosphate Acquisition in Spirochetes: Distribution and Biological Activity of Glycerophosphodiester Phosphodiesterase (GlpQ) among Borrelia Species. J. Bacteriol.
185: 1346-1356
[Abstract]
[Full Text]
-
Moore IV, V. A., Varela, A. S., Yabsley, M. J., Davidson, W. R., Little, S. E.
(2003). Detection of Borrelia lonestari, Putative Agent of Southern Tick-Associated Rash Illness, in White-Tailed Deer (Odocoileus virginianus) from the Southeastern United States. J. Clin. Microbiol.
41: 424-427
[Abstract]
[Full Text]
-
Fraenkel, C.-J., Garpmo, U., Berglund, J.
(2002). Determination of Novel Borrelia Genospecies in Swedish Ixodes ricinus Ticks. J. Clin. Microbiol.
40: 3308-3312
[Abstract]
[Full Text]
Top
Abstract
Introduction
