Detection of Tickborne Relapsing Fever Spirochete, Austin, Texas, USA

Jack D. Bissett; Suzanne Ledet; Aparna Krishnavajhala; Brittany A. Armstrong; Anna Klioueva; Christopher Sexton; Adam Replogle; Martin E. Schriefer; Job E. Lopez

Disclosures

Emerging Infectious Diseases. 2018;24(11):2003-2009. 

In This Article

Results

Initial Diagnosis of Relapsing Fever Borreliosis

The patient was afebrile on initial presentation to the emergency department, but a fever quickly developed (temperature 103.3°F). Her health deteriorated, and she became tachycardic (heart rate 125 beats/min) and mildly hypotensive (blood pressure 92/38 mm Hg). An initial complete blood count showed a leukocyte count of 6,700 cells/mm3, a hemoglobin level of 11.1 g/dL, and a platelet count of 145,000/mm3. Levels of liver enzymes, bilirubin, blood urea nitrogen, and creatinine were within reference limits.

At the hospital visit, the patient reported a travel history to Cancun, Mexico, during February 14–20, 2017, but she had no illness during or upon return from that trip. Also, she had no extensive travel history outside Austin, during February–March 29, 2017, when her illness began. However, during March 24–26, the patient spent a weekend at a rustic conference center in Austin where attendees from throughout the United States converged. Austin Public Health had previously investigated the conference center as the exposure site for an outbreak of TBRF the previous month, but tick trapping efforts were unsuccessful.

During the conference, the patient noticed several insect bites on her legs. However, she did not report seeing ticks. Given her travel history to Cancun, the emergency department physician suspected malaria and requested a peripheral blood smear. Instead of malaria parasites, spirochetes were visualized (Figure 1) and a diagnosis of relapsing fever was made. Treatment with doxycycline was initiated on the evening of hospital admission, and her fever resolved within 24 hours. Modest leukopenia (leukocyte count 3,300 cells/mm3) and thrombocytopenia (71,000 platelets/mm3) then developed. Her other signs and symptoms rapidly resolved over the next 3 days, and she was discharged on April 12, 2017.

Figure 1.

Giemsa-stained peripheral blood smear of a 34-year-old woman with tickborne relapsing fever, Austin, Texas, USA, showing 2 spirochetes (arrows). Scale bar indicates 20 μm.

Diagnosis of Exposure to B. Turicatae

Because blood was not collected during the initial hospitalization of the patient to identify the infectious agent, we retrospectively implemented 2 molecular approaches to identify the causative agent. First, we extracted DNA from a portion of a blood smear and performed a real-time PCR with primers and probe specific for B. turicatae glpQ gene. This assay detected B. turicatae but did not detect B. hermsii. For this real-time PCR, the amplicon was 67 bp, and given the small size, we did not evaluate the sequence or submit it to GenBank.

The average cycle threshold of the assay was 28.81, and use of primers and probe specific for B. hermsii showed negative results. A no template control also showed negative results.

Second, we assessed a serum sample against B. turicatae protein lysates and rBipA. Results also indicated that the patient was infected with B. turicatae (Figure 2). Immunoblotting detected reactivity to 7 protein bands in the B. turicatae protein lysate and to rBipA (Figure 2, panel A). A positive control serum sample was used from a previous case report,[15] and the negative control human serum sample indicated no serologic cross-reactivity (Figure 2, panels B, C).

Figure 2.

Serologic responses to Borrelia turicatae protein lysates and rBipA in 34-year-old woman with tickborne relapsing fever, detected by immunoblotting, Austin, Texas, USA. A) Serum sample from the case-patient; B) positive control serum sample from another case-patient; C) negative control sample. Lane 1, B. turicatae; lane 2, rBipA. Values on the left are in kilobases. Asterisks (*) indicate the size of native BipA. rBipA, recombinant Borrelia immunogenic protein A.

Evaluation of Infected O. Turicata Ticks From a Public Park

We collected O. turicata ticks in a public park near the rustic conference center from the base of a tree that had 2 dens containing rodent waste, which suggested rodent activity in the area (Figure 3). Ticks emerged from leaf litter ≈20 min after we placed CO2 traps, and we collected ticks ≈30 cm from a cavity opening. Within 5 days after feeding ticks from each location on naive mice, spirochetes were visualized by microscopy in the blood. After we inoculated mBSK medium with a serum sample, spirochetes propagated. Once spirochetes entered the late logarithmic growth phase, we passaged them, which confirmed maintenance of these isolates by vitro cultivation. The 3 isolates were designated BRP1, BRP1a, and BRP2.

Figure 3.

Location of collection sites for Ornithodors turicata ticks, Austin Texas, USA. Two rodent dens (insets) were located at the base of an oak tree. Carbon dioxide traps were placed at the openings until ticks emerged. Borrelia turicatae isolates BRP1 and BRP1a originated from ticks that were collected from the den shown at bottom left, and isolate BRP2 originated from the den shown at the bottom right.

Sequence Analysis and Plasmid Diversity of BRP1, BRP1a, and BRP2 Isolates of B. Turicatae

We performed multilocus sequencing to characterize the 3 spirochete isolates that originated in Austin. Sequences of 1,400, 566, and 432 bases were generated for the flaB, rrs, and gyrB genes, respectively (GenBank accession nos. MH503949–51, MH507599–601, and MH507602–04, respectively). Assessment of DNA sequences by BLAST analysis indicated 100% nucleotide identity to the 91E135 and BTE5EL isolates of B. turicatae. Isolates 91E135 and BTE5EL originated from field-collected ticks and a febrile soldier from Texas, respectively.[3,9,11]

We performed reversed-field gel electrophoresis and identified variation in plasmid diversity between the BRP1, BRP1a, BRP2, and 91E135 isolates of B. turicatae (Figure 4). The 3 isolates from Austin contained an ≈40-kb linear plasmid that was absent from 91E135. In addition, BRP1 contained an ≈60-kb linear plasmid that was absent from 91E135, BRP1a, and BRP2.

Figure 4.

Reversed-field gel electrophoresis of Borrelia turicatae isolates collected from Ornithodors turicata ticks, Austin, Texas, USA (BRP1, BRP1a, and BRP2) and an isolate from previously field-collected ticks (91E135) (9). Lane 1, 91E135; lane 2, BRP1; lane 3, BRP1a; lane 4, BRP2. White boxes indicate a plasmid in BRP1 that is absent from the other strains (top); plasmids unique to BRP1, BRP1a, and BRP2 (middle); and a plasmid in 91E135 strain that is absent in isolates from Austin (bottom).

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