Transovarial transmission of Borrelia spirochetes by Ixodes scapularis: A summary of the literature and recent observations

Ticks and Tick-borne Diseases 4 (2013) 46–51

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Original article

Transovarial transmission of Borrelia spirochetes by Ixodes scapularis: A summary of the literature and recent observations Lindsay Rollend ∗ , Durland Fish, James E. Childs Yale School of Public Health, 60 College Street, New Haven, CT 06520-8034, USA

a r t i c l e

i n f o

Article history: Received 14 February 2011 Received in revised form 14 June 2012 Accepted 19 June 2012 Keywords: Transovarial transmission Borrelia miyamotoi Borrelia burgdorferi Ixodes scapularis Ticks

a b s t r a c t Transovarial transmission (TOT) of Borrelia burgdorferi (sensu lato), the agent of Lyme disease, by the Ixodes persulcatus group of hard ticks (Ixodidae) has frequently been reported in the literature since the discovery of Lyme disease 1982. Evidence for and against TOT by B. burgdorferi has led to uncertainty and confusion in the literature, causing misconceptions that may have public health consequences. In this report, we review the published information implicating B. burgdorferi as a bacterium transovarially transmitted among ticks of the Ixodes persulcatus group and present new data indicating the transovarially transmitted agent is actually Borrelia miyamotoi. B. miyamotoi, first described in 1995, is antigenically and phylogenetically related to B. burgdorferi, although more closely related to the relapsing fever-group Borrelia typically transmitted by soft ticks (Argasidae). Borrelia infections of unfed larvae derived from egg clutches of wild-caught Ixodes scapularis are demonstrated to result from transovarial transmission of B. miyamotoi, not B. burgdorferi. The presence of this second Borrelia species, apparently sympatric with B. burgdorferi worldwide also may explain other confusing observations reported on Borrelia/Ixodes relationships. © 2012 Published by Elsevier GmbH.

Introduction Transovarial transmission (TOT) is characteristic of many tick-borne pathogens (Burgdorfer and Varma, 1967) and has been reported in both ixodid and argasid ticks. It has also been shown that while ticks can pass infection from one stage to the next, transmission from female to offspring can be rare or non-existent (Parola and Raoult, 2001). Newer technologies, primarily PCR and DNA sequencing, have led to a better understanding of pathogen transmission properties within the tick. Field and laboratory evidence for TOT of Borrelia burgdorferi (sensu lato) has been repeatedly reported in the literature, as has evidence against it. The existence of TOT of B. burgdorferi would have significant epidemiological consequences. The presence of infected larvae in the environment would lengthen the transmission season well beyond the currently recognized nymphal activity period (June–August) into September. Larval I. scapularis do feed upon people (Falco and Fish, 1988), and their extremely small size would make them extremely difficult to detect. The presence of TOT would change the enzootic transmission dynamics of B. burgdorferi and provide alternative explanations for the observed infection prevalence in nymphal ticks. As an example, a sliding scale of the prevalence of transovarial transmission in

∗ Corresponding author. E-mail address: [email protected] (L. Rollend). 1877-959X/$ – see front matter © 2012 Published by Elsevier GmbH. http://dx.doi.org/10.1016/j.ttbdis.2012.06.008

Ixodes spp., which includes very low or zero values, will dramatically change the outcome of models which estimate the relative contribution of TOT to the basic reproduction number (R0 ) for B. burgdorferi; currently some models use 10% as model parameter (Hartemink et al., 2008). A related outcome of TOT would be errors in evaluating vertebrate reservoir competency in field studies which usually rely upon xenodiagnosis of naturally occurring larvae dropping from confined hosts (Fish and Daniels, 1990; Ostfeld et al., 2006). Fed larvae are allowed to molt and evaluated for infection prevalence as newly molted nymphs. Observations evaluating white-tailed deer competency, through xenodiagnostic and direct fluorescent antibody methods (DFA), indicate only sporadic and rare infection of nymphs occurs (Matuschka et al., 1993; Telford et al., 1998). Some of low prevalence (∼1%) of infected nymphs obtained from deer has been attributed to TOT (Telford et al., 1998). TOT of B. burgdorferi would also affect current theories on the underlying mechanism of spread of Lyme disease in the eastern US. The expansion of I. scapularis from relict endemic foci is thought to be facilitated by deer movements, population growth, and intentional releases during restocking efforts. However, the geographic distribution of deer exceeds that of endemic Lyme disease and theory suggests that while deer are critical for moving I. scapularis ticks, birds are important in establishing endemic foci by seeding new areas with ticks infected with B. burgdorferi (Madhav et al., 2004).

L. Rollend et al. / Ticks and Tick-borne Diseases 4 (2013) 46–51

For these above-stated reasons, the question of TOT by B. burgdorferi should not remain unresolved. Herein, we summarize previous studies on this subject, provide evidence for confusion with a more recently described species, B. miyamotoi, and present new data, which we believe should help to resolve the issue. Borrelia burgdorferi: evidence for transovarial transmission In the US, TOT of B. burgdorferi has been sporadically reported among field-collected larval I. scapularis ticks (Table 1), with prevalence ranging from 2 of 374 (0.53%) from Massachusetts (Piesman et al., 1986) to 7 of 18 (38.8%) larval progeny obtained from egg clutches derived from wild-caught I. scapularis from New York and Connecticut (Magnarelli et al., 1987). Bosler et al. (1983) reported that larval I. scapularis collected from Shelter Island, New York, were infected with B. burgdorferi and concluded TOT. Burgdorfer et al. (1988) reported systemic spirochetal infection (Malpigian tubules, salivary glands, and genital system) among 3.9% of 102 adult I. scapularis (dammini) collected from Shelter Island, New York. Among 130 spirochete-free I. scapularis larvae fed on Peromyscus leucopus, experimentally infected with B. burgdorferi, then raised to the adult stage, 103 had midgut infections and 3 (2.8%) had disseminated infections. Examination of 100 eggs from each of 5 systemically infected I. scapularis indicated TOT with a prevalence ranging from 0 to 100% (median = 25%) of individual clutches. The prevalence of TOT to larval progeny ranged from 0 to 40% (median = 8%), but no conclusive evidence of infection was found among nymphal progeny (Burgdorfer et al., 1988). A similar situation occurs in Europe. The reported range of presumptive TOT by B. burgdorferi among larval I. ricinus ticks varies from 0% [several locations (Hubalek and Halouzka, 1998)] to 21% of 84 larval I. ricinus collected from Ameland Island in the Dutch North Sea (Rijpkema et al., 1994). In an extensive review of tick studies in Europe, the average prevalence of TOT by B. burgdorferi among larval I. ricinus was reported as 1.9% (Hubalek and Halouzka, 1998). Lane and Burgdorfer (1987) described only “moderate” staining of spirochetes with an anti-B. burgdorferi polyclonal serum and with monoclonal anti-B. burgdorferi H9724. However, attempts to stain this Borrelia with monoclonal antibody H5332 were negative (Lane and Burgdorfer, 1987). H9724 is Borrelia genus-specific, and therefore would be expected to cross with B. miyamotoi, while monoclonal H5332 is specific to the outer surface protein A of B. burgdorferi (Barbour et al., 1983, 1986). Subsequent to this study, B. miyamotoi was demonstrated not to express the outer surface membrane A, as determined by monoclonal antibody testing (Hulínská et al., 2007), explaining the previous negative results. Of note, several authors have questioned whether TOT was actually due to B. burgdorferi. Lane and Burgdorfer (1987) were suspicious of ascribing the observed TOT, revealed only by weak fluorescence of spirochetes among I. pacificus, to B. burgdorferi. Their suspicion that an additional Borrelia could be the cause of these infections was later supported by findings of Scoles et al. (2001) and Mun et al. (2006). Reports of presumed observations of TOT by B. burgdorferi among other ixodid ticks has also met with skepticism by researchers working in eastern Europe (Nefedova et al., 2004).

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TOT of B. burgdorferi was observed. PCR primers specifically targeting B. miyamotoi have identified this species among Ixodes spp. in locations where previous serological analyses have implicated B. burgdorferi in TOT in Europe (Fraenkel et al., 2002; Hulínská et al., 2007) and in California (Mun et al., 2006). B. burgdorferi infection has been described as being limited to the midgut of I. scapularis. However, in 1989, generalized infections were detected by the use of Borrelia genus-specific antiserum within the central ganglion and ovary of ∼4% of I. scapularis and I. ricinus ticks collected from the United States and from Switzerland (Burgdorfer et al., 1988; Burgdorfer, 1989). This prevalence is within the range reported for B. miyamotoi in Ixodes spp. ticks (<1–4%) sampled from regions of the United States and Europe (Scoles et al., 2001; Hulínská et al., 2007) and could represent disseminated infection by B. miyamotoi. Concerns regarding methodologies Most of the above-cited reports of putative TOT of B. burgdorferi were made prior to the discovery of B. miyamotoi in 1995 (Fukunaga and Koreki, 1995), and its first documentation in the United States in 2001 (Scoles et al., 2001) and in Europe in 2002 (Fraenkel et al., 2002). Earlier reports of TOT of B. burgdorferi were based on the detection of spirochetes stained by DFA or IFA methods using polyclonal sera obtained from animals, naturally or experimentally infected with B. burgdorferi, or with sera obtained from humans displaying an erythema migrans rash, pathognomonic for the clinical diagnosis of Lyme borreliosis. Polyclonal sera raised against B. burgdorferi and anti-Borrelia genus antibody were subsequently shown to stain B. miyamotoi-like spirochetes (Scoles et al., 2001), in addition to B. burgdorferi. In contrast to reports based on antigen detection, no TOT of B. burgdorferi was observed when 14,700 larval I. scapularis, raised from 48 B. burgdorferi-infected females, were fed on dogs (Patrican, 1997). In contrast, when I. scapularis larvae suspected of harboring B. miyamotoi-like Borrelia were fed on mice, xenodiagnosis of nymphal ticks indicated that 73 of 96 (76%) were positive by anti-Borrelia genus DFA testing. Molecular probes targeting 4 gene sequences specific to B. burgdorferi were negative, while sequences related to B. miyamotoi were obtained from each of the DFA-positive nymphs (Scoles et al., 2001). TOT of B. burgdorferi sensu lato has been attributed to B. afzelii and B. garinii based on PCR amplification of the intergenic spacer (IGS) between the 5S and 23S rRNA (Rijpkema and Bruinink, 1996). These primers also amplify B. miyamotoi (Bunikis et al., 2004), but produce a smaller amplicon, requiring additional methods to differentiate species. Herein, we describe the results of large-scale and systematic testing of larval ticks, derived from egg clutches of wild-caught I. scapularis, by PCR. An IFA assay using B. miyamotoi spirochetes harvested from the blood of infected SCID mice is described and used to demonstrate the ‘one-way’ cross between antibodies derived from B. miyamotoi- or B. burgdorferi-infected mice. A DFA assay is also described to demonstrate the cross-reactivity of genus-specific anti-Borrelia FITC. These data indicate the occurrence of TOT of B. miyamotoi, but provide no evidence of TOT in B. burgdorferi.

Borrelia miyamotoi: evidence for transovarial transmission

Materials and methods

Scoles et al. (2001) documented TOT by B. miyamotoi among unfed larval progeny from 2 of 52 (3.8%) egg clutches obtained from wild-caught I. scapularis adults. The use of B. burgdorferi- and B. miyamotoi-specific PCR primers revealed the differential contribution of these organisms to the observed phenomenon of TOT in the northeastern USA. In this report and that of Scoles et al. (2001), no

Tick origins Egg clutches were obtained from adult I. scapularis ticks collected from the Southern Connecticut Regional Water Authority property in North Branford and Bridgeport and from the Audubon Center, Sharon, Connecticut.

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Table 1 Summary of publications reporting Borrelia spp. spirochetes in ticks. Publication date

Location

Observation

Methods

Authority

1983 1986 1987

Shelter Island, NY, USA Massachusetts, USA Mendocino County, California, USA

DFA DFA DFA

Bosler et al. (1983) Piesman et al. (1986) Lane and Burgdorfer (1987)

1987

New York and Connecticut, USA

23/306 (7.5%) I. scapularis unfed larvae infected 2/374 (0.5%) I. scapularis larvae infected 3/100 (3%) field-collected adult I. pacificus fed on rabbits have disseminated spirochetal infection 7/18 (39%) larvae from egg clutches derived from wild-caught I. scapularis removed from deer or dogs

DFA/IFA

Magnarelli et al. (1987)

1988

Shelter Island, NY, USA

0/77 and 4/102 (3.9%) wild unfed adult I. scapularis have generalized infection with spirochete 3/106 (2.8%) of experimentally infected, but unfed adult I. scapularis have generalized infection with spirochetes

DFA

Burgdorfer et al. (1988)

1989

Shelter Island, NY, USA

4/151 (3.9%) adult I. scapularis have generalized infection with spirochetes 6/112 (5.0%) adult I. ricinus have generalized infection with spirochetes; 2/37 (5.4%) infected adult female I. ricinus produce infected progeny 25/1687 (1.5%) adult I. pacificus have generalized infection with spirochetes; 1/3 infected adult female I. ricinus produced infected progeny

DFA

Burgdorfer (1989)

0/132 larvae and eggs produced from B. burgdorferi-infected I. pacificus females infected 17/81 questing larvae found to be positive 20/652 (3.1%) I. ricinus larvae infected 3/61 (5%) nymphal I. ricinus ticks infected by Borrelia spp. 0/48 infected female I. scapularis ticks positive for B. burgdorferi 108/5699 (1.9%) unfed larval I. ricinus infected 2/185 (1%) nymphs infected that developed from larvae collected from deer 2/301 (0.7%) adult I. ricinus had B. miyamotoi-like sequences by PCR 3/96 (3%) infected female I. persulcatus had spirochetes in ovaries 4/237 (1.7%) unfed nymphs and 2/282 (0.7%) adult ticks infected with B. miyamotoi-like spirochetes

DFA/culture

Schoeler and Lane (1993)

IFA IFA PCR

Rijpkema et al. (1994) Zhioua et al. (1994) Rijpkema and Bruinink (1996)

DFA/PCR

Patrican (1997)

Giemsa/DFA/IFA DFA

Hubalek and Halouzka (1998) Telford et al. (1998)

PCR

Fraenkel et al. (2002)

Giemsa/PCR

Nefedova et al. (2004)

PCR

Mun et al. (2006)

Staatswald, Switzerland

Northern California and southern Oregon, USA 1993

Northern California, USA

1994 1994 1996

Ameland Island, The Netherlands Switzerland Ameland Island, The Netherlands

1997 1998 1998

Westchester County, New York, USA Europe, Russia Massachusetts, USA

2002

Sweden

2004

Perm oblast, Russia

2006

California, USA

Larval development All female I. scapularis ticks were fed on either New Zealand white rabbits or sheep and held for oviposition. The females analyzed in this study were used to generate larvae that would be used in future experiments. Pools of 50 larvae from each egg clutch were tested by PCR for the presence of B. burgdorferi and B. miyamotoi and when a pool was found to be positive an additional 50 individual larvae were tested to establish filial infection prevalence.

elongation phase at 72 ◦ C completed the cycle. The samples were held at 4 ◦ C until amplicons were electrophoresed on a 2% agarose gel and viewed using a Kodak Gel Logic 200.B. burgdorferi was detected in ticks by performing a nested PCR, which amplifies the 941-bp 16S–23S rDNA IGS, as described in detail elsewhere (Liveris et al., 1999). The reaction mix was identical to the B. miyamotoi protocol; 22 ␮l of HotStar Taq mix was added, followed by 0.5 ␮l of each primer, and finally 2 ␮l of DNA template.

DNA extraction and PCR

DFA staining

The methods used to detect specific genetic signatures of B. burgdorferi or B. miyamotoi infection have been previously described in detail (Scoles et al., 2001). Beginning in 2007, DNA was extracted using proteinase K (Roche Applied Science, Indianapolis, IN) and a DNeasy tissue kit (Qiagen, Valencia, CA) as previously described in Beati and Keirans (2001). Two flagellin primers, FLA181F (5 -CCAGCATCATTAGCTGGAA-3 ) and FLA400R (5 -CACCTTGAACTGGAGCGGCT-3 ), were used to specifically amplify B. miyamotoi sequences (Scoles et al., 2001). The amplicon produced by these primers was a 219-bp gene fragment. The PCR mix was prepared using 22 ␮l of HotStar Taq (Qiagen, Valencia, CA), 0.5 ␮l of each primer at a 10-␮M concentration, and 2 ␮l of DNA template, for a total volume of 25 ␮l. PCR conditions consisted of an initial 12-min step at 94 ◦ C to activate the HotStar Taq, followed by 35 cycles of denaturation at 94 ◦ C for 30 s, annealing at 62 ◦ C for 30 s, and elongation at 72 ◦ C for 30 s. A final 5-min

Commercially available fluorescein isothiocyanate-conjugated genus-specific anti-Borrelia antibody [# 02-97-92 Kirkegaard and Perry Laboratories (KPL) Gaithersburg, MD] and a B. burgdorferispecific antibody (# 02-97-91, KPL), produced by cross-adsorption with proteins derived from other Borrelia species (B. hermsii, B. coriaceae, and B. anserina), were used at a 1:50 dilution to detect spirochetes. Larval ticks that had tested positive for B. miyamotoi were first frozen and then pulverized and suspended in 200 ␮l 1× PBS. Using 12-well microscope slides, 10 ␮l of suspension was added to each well. Following air-drying and acetone fixation, slides were incubated with the DFA conjugate for 2 h, washed, and cover slipped in a ProLong Gold Antifade reagent (Invitrogen Eugene, OR). Slides were examined using a Zeiss LSM 510. Extra DFA slides were prepared to be used in the IFA assay described below. Slides designated for IFA were wrapped in aluminum foil and held at –80 ◦ C until use.

L. Rollend et al. / Ticks and Tick-borne Diseases 4 (2013) 46–51

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Fig. 1. (A) IFA using B. miyamotoi antigen and polyclonal B. burgdorferi antiserum obtained from an infected P. leucopus. (B) IFA using B. miyamotoi antigen and serum obtained from an uninfected P. leucopus. (C) IFA using B. miyamotoi antigen and polyclonal B. miyamotoi antiserum obtained from an infected P. leucopus. (D) IFA using B. burgdorferi antigen and polyclonal B. miyamotoi antiserum obtained from an infected P. leucopus.

IFA staining Slides using infected larval antigens were removed from storage at −80 ◦ C and equilibrated to room temperature. After warming, 30 ␮l of a 1% blocking agent (50-61-00 KPL) was placed in each well; slides were incubated at room temperature for 5 min, rinsed in 1× PBS and allowed to dry. A 1:20 dilution of primary antibody in 6.6% blocking agent was added to each well in a volume of 20 ␮l; slides were incubated at 37 ◦ C for 1.5 h, then washed in 0.2% Tween20/1× PBS and dried. Primary antibody against B. miyamotoi was derived from infecting Peromyscus leucopus mice with B. miyamotoi-infected larvae. Similarly, primary antibody against B. burgdorferi was generated by infecting P. leucopus with B. burgdorferi-infected nymphs. After application of FITC secondary antibody (02-33-06 KPL), slides were incubated at 37 ◦ C for 1 h, washed in 0.2% Tween20/1× PBS, then cover-slipped in a ProLong Gold Antifade reagent (Invitrogen Eugene, OR). Slides were viewed using a Zeiss LSM 510 microscope. Results From 2000 to 2010, a total of 1214 pools of larval ticks, each derived from single females, were tested for the presence of B. burgdorferi and B. miyamotoi by PCR; 19 pools (0.016; 95% confidence interval: 0.9–2.4) were infected with B. miyamotoi. The site-specific numbers and prevalence of B. miyamotoi were 16/1104 (0.014; CI: 0.8–2.3) in Branford, 1/61 (0.016; CI: 0.04–8.8) in Bridgeport, and 2/89 (0.022; CI: 0.7–7.8) in Sharon (Table 2). No B. burgdorferi infections were detected from the same pools of ticks by PCR (95% CI: 0.0–0.3%).

IFA results in Fig. 1, panels A–D, validate the cross-reactivity properties of B. burgdorferi sera against B. miyamotoi antigen, which was responsible for initial confusion of B. burgdorferi infection. Fig. 1, panel A, shows a positive reaction of B. burgdorferi sera against B. miyamotoi antigen, whereas Fig. 1, panel D shows no cross-reactivity properties of B. miyamotoi sera against B. burgdorferi. Serum from an uninfected P. leucopus served as a negative control against B. miyamotoi antigen (Fig. 1, panel B), and serum from a B. miyamotoi-infected P. leucopus was used as a positive control against B. miyamotoi antigen (Fig. 1, panel C). DFA results conclude that when reacted against Borrelia genusspecific antiserum, B. miyamotoi will yield a positive result (Fig. 2, panel A). However, when B. miyamotoi is reacted against B. burgdorferi-specific antiserum, results are negative (Fig. 2, panel C). Panels B and D of Fig. 2 reveal that B. burgdorferi antigen will react with both genus (panel B) and B. burgdorferi (panel D)-specific antisera. Discussion The results from this study provide strong evidence that TOT by B. burgdorferi does not occur. This study also supports the Table 2 Results of larval Ixodes scapularis pool screening. Location

Prevalence of B. miyamotoi % (# positive/# tested)

Prevalence of B. burgdorferi % (# positive/# tested)

Branford, CT Bridgeport, CT Sharon, CT

1.4 (16/1104) 6 (1/16) 2 (2/89)

0 (0/1104) 0 (0/16) 0 (0/89)

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Fig. 2. (A) DFA using B. miyamotoi antigen and Borrelia genus-specific antiserum. (B) DFA using B. burgdorferi antigen and Borrelia genus-specific antiserum. (C) DFA using B. miyamotoi antigen and B. burgdorferi-specific antiserum. (D) DFA using B. burgdorferi antigen and B. burgdorferi-specific antiserum.

conclusion that B. miyamotoi is most likely responsible for earlier reports of TOT of B. burgdorferi by I. scapularis. In addition, B. miyamotoi occurs in European I. ricinus (Fraenkel et al., 2002) and I. persulcatus (Fukunaga et al., 1995; Platonov et al., 2011) necessitating further work to sort out the possible contributions of various Borrelia spp. to observed instances of TOT. As indicated previously and summarized in Table 1, the majority of previous studies of TOT of Borrelia have relied upon fluorescent staining of spirochetes in eggs (Burgdorfer et al., 1988), larvae (Piesman et al., 1986; Zhioua et al., 1994), unfed nymphs (Rijpkema et al., 1994; Telford et al., 1998), and adult Ixodes spp. (Magnarelli et al., 1987; Burgdorfer et al., 1988) with polyclonal or Borrelia genus-specific antiserum. Polyclonal sera raised against B. burgdorferi bind to both B. burgdorferi and B. miyamotoi antigens, indicating a heterologous cross, while polyclonal antiserum raised against B. miyamotoi appears species-specific, indicating a one-way crossover in antigen recognition (Figs. 1 and 2). In this way, paired assays utilizing Borrelia genus-specific and species-specific B. burgdorferi antisera can be used to distinguish between these spirochetes. Reports of potential TOT by B. burgdorferi identified by molecular methods still appear in the literature. Hamer et al. (2010) reported finding B. burgdorferi in 2 host seeking I. scapularis larvae collected in Michigan. Detection was made by PCR and confirmed by sequencing of the amplicons suggesting TOT by I. scapularis. However, Hamer et al. (2010) also reported finding B. burgdorferi sequences in a host-seeking larva of Dermacentor variabilis at the same site using the same detection methods. D. variabilis is not vector-competent for B. burgdorferi, although spirochetes have been previously detected in the larval stage (Piesman and Sinsky, 1988). Hemolymph obtained from D. variabilis ticks has been shown to be borreliacidal (Johns et al., 2000, 2001), suggesting

a mechanism for their incompetency as vectors of B. burgdorferi. Furthermore, engorged Dermacentor females injected with B. burgdorferi culture did not produce infected eggs (Johns et al., 2000). In light of these findings and our observations, and as suggested by the authors (Hamer et al., 2010), the best possible explanation for finding host-seeking larvae infected with B. burgdorferi would be that these larvae had partially fed upon a competent infected host and then resumed host-seeking after dislodging from the initial host. While this is possible, we were unable to locate any reference to support the notion that partially fed larvae have been found host-seeking in nature. Of interest, the infection load of B. miyamotoi in skin biopsies from the ears of infected P. leucopus is far lower than that of B. burgdorferi, while the prevalence and infection load of B. miyamotoi in the blood of infected P. leucopus is higher than that of B. burgdorferi (Barbour et al., 2009), suggesting systemic infection of B. miyamotoi in its mammalian host. The bimodal distribution of the infection load of B. miyamotoi among infected nymphal I. scapularis ticks is hypothesized to result from differences in transovarially and transtadially acquired infection (low load) as compared to high loads representing spirochetal amplification following larval feeding on infected P. leucopus (Barbour et al., 2009). Furthermore, the overall mean infection prevalence of B. miyamotoi in I. scapularis throughout the United States is 1.9%, a figure closely resembling the frequency of TOT occurrence reported in the literature (Barbour et al., 2009). The results reported herein and the review of the literature provides direct evidence for TOT of B. miyamotoi, but not for B. burgdorferi. In locations where data are inconsistent regarding TOT of B. burgdorferi among Ixodes spp., and where the contribution of B. miyamotoi to this phenomenon has not been evaluated, highly

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