Presentation of Case
In June 2013, a Caucasian male from Kerr county, Texas, with an extensive history
of outdoor activity working with sheep, goats, and exotic game became ill, displaying
fever, chills, uveitis, headache, retrobulbar pain, severe malaise, and weakness.
Myalgia was centered on upper extremities, most notably the shoulders, arms, and hands,
and by July 2013 the patient had experienced two febrile episodes (39°C). Numerous
insect bites were reported, most notably on areas of the body that were in direct
contact with the ground, but offending arthropods were not found. The patient was
initially tested for Coxiella burnetti exposure, but repeated serological results
demonstrated that phase I and phase II immunoglobulin G (IgG) antibody titers failed
to increase. Antibiotic treatment was administered, and symptoms improved. A retrospective
evaluation of the patient’s history and clinical summary led to suspicion of tick-borne
relapsing fever borreliosis. The patient did not have a history of traveling outside
of Texas prior to the onset of symptoms, and Borrelia turicatae was suspected as the
causative agent. A serum sample was sent to us four months after antibiotic treatment,
and we implemented a molecular approach to determine seroreactivity against two diagnostic
antigens for relapsing fever spirochetes, recombinant glycerophosphodiester phosphodiesterase
(rGlpQ) and Borrelia immunogenic protein A (rBipA).
Verbal and written informed consent was obtained from the patient to test the serum
sample. The study was submitted to the Mississippi State University Institutional
Review Board, protocol #13–369, and approved by designated review.
Protein Analysis, Production of Diagnostic Antigens, and Serological Testing
rGlpQ has been used to evaluate mammalian exposure to species distributed in the western
United States and East and West Africa [1–4]. The gene is absent from other pathogenic
spirochetes, and Schwan and colleagues first characterized rGlpQ as an antigen that
could discriminate between exposure to relapsing fever and Lyme-causing Borrelia .
Amino acid alignments of GlpQ using Vector NTI 11.5 software (Life Technologies, Foster
City, California, US) indicated the B. turicatae homologue is highly conserved between
New (B. turicatae, Borrelia parkeri, and Borrelia hermsii) and Old (Borrelia recurrentis)
World species of relapsing fever spirochetes (Table 1).
Percent amino acid identity of GlpQ between species of relapsing fever spirochete.
To produce B. turicatae GlpQ as a recombinant fusion protein, the gene was amplified
using genomic DNA from the 91E135 isolate with 5ʹ-CACCATGAAATTAATTAAAACAA AATTATTAAT
GCTTACAATGAATATTTTT-3ʹ and 5ʹ-TTGTTTTACAAACTTCACTAC TGTATCA GTAAAATCTGTAAAT-3ʹ forward
and reverse primers, respectively. The amplicon was cloned into the pET102 expression
vector, and the absence of nucleotide errors was confirmed by sequencing analysis
using Vector NTI Advanced 11.5 software (Life Technologies). rGlpQ was produced as
a six-histidine and thioredoxin fusion protein and purified by immobilized metal affinity
chromatography using HisTrap FF Crude columns precharged with Ni2+ (GE Healthcare
Life Sciences, Pittsburgh, Pennsylvania, US). Thioredoxin was removed by incubating
the purified protein with 0.01 U of EKMax Enterokinase (Life Technologies), following
the manufacturer’s protocol. The molecular mass of native GlpQ and rGlpQ are 40 and
45 kDa, respectively.
Previous studies reported the identification and antigenicity of BipA and indicated
a homologue was absent outside of relapsing fever Borrelia [5,6]. BipA also shares
24%–76% amino acid identity between relapsing fever spirochete homologues (Table 2)
and was demonstrated to differentiate between infections caused by B. turicatae and
B. hermsii . B. turicatae rBipA was produced using the pET102 expression system
as previously described , and thioredoxin was left attached to maintain protein
solubility. The molecular mass of native and rBipA are 60 and 75 kDa, respectively.
Percent amino acid identity of BipA between species of relapsing fever spirochete.
Protein lysates from 1 x 107 spirochetes and 1 μg of rGlpQ and rBipA were electrophoretically
separated and transferred to nitrocellulose membranes as previously described 
using TGX gels, Mini-PROTEAN Tetra cell, and the Mini Trans Blot system (BioRad, Hercules,
California, US). Immunoblots were probed with the patient’s serum sample at a 1:400
dilution, and Rec-Protein G-HRP (Life Technologies) diluted 1:4,000 was used as the
secondary molecule. Serological reactivity to multiple proteins in the B. turicatae
whole spirochete lysates, rGlpQ, and rBipA (Fig. 1A) was detected by chemiluminescence
and indicated exposure to relapsing fever spirochetes. A serum sample from a subject
living in a nonendemic region of the US without a history of exposure to relapsing
fever spirochetes was used as a negative control (Fig. 1B). The nitrocellulose membrane
was subsequently probed with a Monoclonal Anti-polyHistidine-Peroxidase antibody (Sigma-Aldrich,
St. Louis, Missouri, US), indicating the presence of the recombinant proteins (Fig.
1C and D). Additionally, the patient’s antibody titers to rGlpQ and rBipA were over
a 1:6,400 dilution.
Immunoblotting to evaluate antibody binding to B. turicatae protein lysates, rGlpQ,
and rBipA (A).
The asterisk and arrowhead represent the molecular masses for native GlpQ and BipA,
respectively (A). rBipA was produced as a thioredoxin fusion protein and is 15 kDa
larger than the native protein. Immunoblots were also probed with a serum sample from
an uninfected patient (B) and a monoclonal antibody against the six-histidine fusion
tag (C and D). Molecular masses are shown to the left of each immunoblot.
The ecological overlap and nonspecific symptoms caused by vector-borne bacterial,
viral, and parasitic pathogens signifies the importance of utilizing improved molecular
assays to accurately determine pathogen exposure. In this report, the patient’s extensive
outdoor activity and nonspecific clinical manifestation made it difficult to initially
determine the causative agent. Retrospective analysis of serum reactivity to rGlpQ
and rBipA suggested likely exposure to B. turicatae. A potential limitation of BipA
as a species-specific antigen is that B. turicatae and B. parkeri are closely related
, and the homologues share 76% amino-acid identity. However, we are unaware of
a report indicating the occurrence of Ornithodoros parkeri, the vector for B. parkeri,
in Texas, as the ticks have been collected throughout the western and midwestern US
[8,9], further causing us to suspect B. turicatae.
Accurate epidemiological studies for tick-borne relapsing fever borreliosis in Texas
have been challenging because of nonspecific symptoms and limited molecular diagnostic
assays. Human exposure has been evaluated retrospectively, by confirming spirochete
colonization of the tick vector, Ornithodoros turicata, collected from presumed contact
sites, which frequently included caves and manmade dugouts [10,11]. The disease has
also been misdiagnosed as Lyme borreliosis because of similar neurological symptoms
. Further complicating a correct diagnosis was serological cross-reactivity with
immunofluorescent assays (IFA) and enzyme-linked immunosorbent assays (ELISA), in
which patient serum samples were assessed for reactivity to fixed Borrelia burgdorferi
or total protein lysates, respectively . Given the number of conserved antigens
between Borrelia species and observed serological cross-reactivity [12,13], IFA and
ELISA may be misleading. Furthermore, Ixodid ticks that transmit B. burgdorferi and
feed for 5–7 days were not found to be attached on the patients .
Transmission of B. turicatae from the tick and the ecology and feeding behavior of
O. turicata indicate that outdoor enthusiasts, military ground personnel, and low-income
families living in primitive housing conditions are at-risk populations [14,15]. The
ticks complete their blood meal within 5–60 minutes and subsequently return to the
cave crevice, nest, or den in which they cohabit with small mammals [10,16]. Consequently,
O. turicata is rarely found on the host, and a full blood meal is not required for
transmission and infection . Moreover, mammalian hosts supporting the maintenance
of B. turicatae in nature are not completely known. Schwan and colleagues isolated
the spirochetes from symptomatic domestic dogs, while the tick vector has been collected
in caves [10,17,18]. These findings suggest a role of wild canids and bats in B. turicatae
maintenance. Our ecological studies in Texas utilizing rGlpQ and rBipA to determine
small mammal exposure to B. turicatae indicate coyotes and rodents may maintain the
pathogens (manuscript in preparation). As improved molecular assays are utilized to
evaluate mammalian exposure to relapsing fever spirochetes, we will further define
the ecology and human health burden in regions where the pathogens are overlooked.
Key Learning Points
Given nonspecific clinical symptoms, relapsing fever spirochetes are likely underdiagnosed.
Serological responses to rGlpQ and rBipA can indicate exposure to relapsing fever
Likely at-risk populations include outdoor enthusiasts, military ground personnel,
and those living in primitive housing conditions.