1. Surveillance for Borrelia miyamotoi in the Chicagoland Area
Mallory Orth
Abstract
Borrelia miyamotoi is a species within the Borrelia genus, which has the
potential to cause tick-borne relapsing fever. This species is similar to another
species within the Borrelia genus, Borrelia burgdorferi, which has the potential to
cause Lyme Disease. B. miyamotoi, however, does not have the ability to infect its
host with Lyme Disease. The tick population in the Chicagoland area was surveyed
for Borrelia miyamotoi by extracting the midguts of deer ticks (Ixodes scapularis),
the vector that transmits said pathogen to its host. The tick samples were screened
using nested PCR to target a specific DNA fragment within the genome of B.
miyamotoi. From the 175 tick samples tested from the Fort Sheridan area, one came
back with a potential positive for B. miyamotoi as well as B. burgdorferi, indicating a
possible coinfection.

2. Introduction
Borrelia miyamotoi is a spiral-shaped bacteria species. The Borrelia genus
has many different species, one of which is Borrelia burgdorferi sensu stricto, which
is the causative agent of Lyme Disease. Borrelia miyamotoi is a completely different
species, which causes influenza-like symptoms, but has the potential to cause tick-
borne relapsing fever5.
The vector for Borrelia miyamotoi is Ixodes scapularis, more commonly
known as the deer tick. The life cycle of Ixodes scapularis explains how humans can
obtain the pathogen. There are three stages: the larval stage, nymph stage and adult
stage. In each of these, the tick takes a blood meal. Within the first blood meal as a
larva, the tick has its first opportunity to acquire the pathogen from the reservoir,
but the pathogen can also be acquired in the nymph or adult stages. The reservoirs
that are most common are the white-footed mouse, Peromyscus leucopus, and the
grey squirrel, Sciurus carolinensis. If the tick is infected with the pathogen in the
nymph and adult stages, said pathogen can be transmitted to humans. The nymph
stage presents the greatest risk for human infection due to the small size of the tick;
it is not easily detected on the human body.
Borrelia miyamotoi was first discovered in an infected tick in Japan in 19951.
Since then, additional infected ticks with Borrelia miyamotoi have been found in the
United States (California and states in the Northeast), Europe (Germany and the
Czech Republic) and Russia2. The first human infection of Borrelia miyamotoi came
from Yekaterinburg City, Russia in 20093. Other human infections have come from
within the United States in New Jersey and California. One of which was an

3. immunocompromised patient in the northeastern U.S. 4. When a patient is treated
for Borrelia miyamotoi infection, doctors use doxycycline for a two-week period of
time.
Materials and Methods
Tick Collection and DNA Isolation
The tick samples were collected in forest preserves in the Chicagoland area
using flannel drags. After the specimens have been collected, the midguts of the ticks
were removed and the DNA was isolated using QIAGEN DNeasy Blook & Tissue kit.
PCR Process
After the DNA was isolated, a series of PCR (polymerase chain reactions)
were preformed to target a genus-specific DNA fragment within the B. miyamotoi
genome. Nested PCR was used in the screening of B. miyamotoi (see tables 1 and 2).
Nested PCR utilizes four different primers, Oligos 115-118. The first two primers,
Oligos 115 and 116, were used in the first step of PCR with the pooled DNA samples.
When five different tick’s DNA are combined into one tube, this is a tick pool, or
pooled DNA. This enables a quicker screening process, instead of testing one tick at
a time, five ticks can be screened at once. The second two primers, Oligos 117 and
118, were used in the second step of PCR which utilized the PCR product of the fist
reaction. This allows for a greater yield of product and targets a more specific area
within the genome. Potential positives for Borrelia miyamotoi are detected at
approximately 450 bp6,7. Due to the genus specific first set of primers, Borrelia
burgdorferi could also be detected at approximately 1,000 bp7. If both bands are
visualized, a potential coinfection could be possible within the tick population.

4. Table 1: Volumes of reagents for the first round of PCR to screen for B.
miyamotoi
Sample
dH2O 5.5 µL
Master Mix 12.5 µL
Oligos 115 1 µL
Oligos 116 1 µL
Tick Pool 5 µL
Table 2: Volumes of reagents for the second round of PCR to screen for B.
miyamotoi
Sample
dH2O 8.5 µL
Master Mix 12.5 µL
Oligos 117 1 µL
Oligos 118 1 µL
PCR Product from round 1 2 µL
The PCR process was carried out in the Thermo Scientific Piko Thermal
Cycler. The specifications are listed in tables 3 and 4.
Table 3: Cycle specifications for Borrelia miyamotoi round one PCR (for 35
cycles)
Temperature Duration
94 °C 30 seconds
56 °C 30 seconds
72 °C 60 seconds
Table 4: Cycle specifications for Borrelia miyamotoi round two PCR (for 40
cycles)
Temperature Duration
94 °C 30 seconds
60 °C 30 seconds
72 °C 60 seconds

5. The initial denaturing step was at 94°C and the final extension at 72°C. The
final PCR products were then held at 4°C until they were put into the freezer for
future use.
Gel Electrophoresis
The PCR products were then run through gel electrophoresis to visualize the
results on ImageLab software. Potential positives for Borrelia miyamotoi appear at
approximately 450 base pairs6,7. Potential positives for Borrelia burgdorferi appear
at 1,000 base pairs7. From the Fort Sheridan area, 35 tick pools (175 individual tick
samples) were tested for B. miyamotoi. One tick showed the potential for a
coinfection with bands indicative of both B. miyamotoi and B. burgdorferi.
Results
From the 35 pools, 175 individual tick samples, tested from the Fort Sheridan
area, one resulted in a potential positive for B. miyamotoi due to the bands at
approximately 450 base pairs. Since bands are also visualized just below 1,000 base
pairs, this suggests a potential coinfection with B. miyamotoi and B. burgdorferi. The
figures shows below show these potential positive bands within the pool and the
individual tick sample.

6. Discussion
Out of the 175 tick samples screened for B. miyamotoi, pool 43 came back as
a potential positive. After screening the individual ticks within the pool, tick 754
showed bands indicative of a potential coinfection with both B. miyamotoi and B.
burgdorferi, at approximately 450 bp and 1,000 bp.
It is possible that there is a presence of Borrelia miyamotoi in the
Chicagoland area. From this research, it is suggested that there is Borrelia
Figure 2: Replicate PCR reaction for T-754
with potential coinfection
Figure 1: Pools 38-44, with a
potential positive (pool 43)
DNA ladder
38, 39, 40, 41, 42, 43, 44
DNA ladder

7. miyamotoi presence in the Chicagoland area, specifically, Fort Sheridan. Further
research will be done on sequencing the potential positive in order to definitively
say it is either positive or negative.
From the results it appears, the prevalence is very low and is no extreme
concern for health care professionals. Continued screening for Borrelia miyamotoi
will continue to follow the prevalence of this newly emerging species in the
Chicagoland area.

8. References
1 Borrelia miyamotoi. Emerg Infect Dis [Internet]. 2014 Aug [March 13, 2015].
2 Crowder CD, Carolan HE, Rounds MA, Honig V, Mothes B, Haag H, et al. Prevalence
of Borrelia miyamotoi in Ixodesticks in Europe and the United States. Emerg Infect
Dis. 2014 Oct [April 1, 2015].
3 Platonov, Alexander E. et al. “Humans Infected with Relapsing Fever
Spirochete Borrelia Miyamotoi, Russia.” Emerging Infectious Diseases 17.10 (2011):
1816–1823. PMC. Web. 3 May 2015.
4 Joseph L. Gugliotta, M.D., Heidi K. Goethert, Sc.D., Victor P. Berardi, B.S., and Sam R.
Telford, III, Sc.D. N Engl J Med 2013; 368:240-245 January 17,2013.
5 Centers for Disease Control and Prevention. Centers for Disease Control and
Prevention, 02 July 2014. Web. 12 Apr. 2015.
6 Fraenkel, Carl-Johan, Ulf Garpmo, and Johan Berglund. "Determination of
Novel Borrelia Genospecies in Swedish Ixodes Ricinus Ticks." Journal of Clinical
Microbiology. American Society for Microbiology, Sept. 2002. Web. 12 May 2015.
7 Padgett, Kerry. "Large Scale Spatial Risk and Comparative Prevalence of Borrelia
Miyamotoi and Borrelia Burgdorferi Sensu Lato in Ixodes Pacificus."PLOS ONE:
Large Scale Spatial Risk and Comparative Prevalence of Borrelia Miyamotoi and
Borrelia Burgdorferi Sensu Lato in Ixodes Pacificus. PLOS, 21 Oct. 2014. Web. 8 May
2015.