Absence of Lyme Disease Spirochetes in Larval Ixodes ricinus Ticks

Abstract

To determine which kind of spirochete infects larval Ixodes ricinus, we examined questing larvae and larvae derived from engorged females for the presence of particular spirochetal DNA that permitted species differentiation. Borrelia miyamotoi was the sole spirochete detected in larval ticks sampled while questing on vegetation. Questing nymphal and adult ticks were infected mainly by Borrelia afzelii, whereas larval ticks resulting from engorged females of the same population were solely infected by B. miyamotoi. Since larvae acquire Lyme disease spirochetes within a few hours of attachment to an infected rodent, questing larvae in nature may have acquired Lyme disease spirochetes from an interrupted host contact. Even if transovarial transmission of Lyme disease spirochetes may occasionally occur, it seems to be an exceedingly rare event. No undisputable proof exists for vertical transmission of Lyme disease spirochetes, whereas B. miyamotoi appears to be readily passed between generations of vector ticks.

Introduction

L yme disease spirochetes are horizontally transmitted between vector ticks of the Ixodes ricinus complex and various reservoir hosts. An infected nymphal tick transmits such spirochetes to the host on which it feeds. If a vertebrate species is competent for the particular genospecies (synonymously used with genomospecies) of Lyme disease spirochetes, it maintains the pathogen and serves as a reservoir for vector ticks subsequently feeding on it (Matuschka and Spielman 1986). Thus, larval ticks acquire spirochetal infection from infected hosts. Early observations suggested that they may also inherit spirochetes from their mothers, because on rare occasions, questing larvae or larvae that have been derived from field-collected females harbored spirochetes (Magnarelli et al. 1986, Piesman et al. 1986). An experimental approach, however, failed to prove the inheritance of Borrelia afzelii (Matuschka et al. 1998).

Besides Lyme disease spirochetes, their vector ticks may harbor an additional spirochete Borrelia miyamotoi, which is more closely related to those spirochetes that cause relapsing fever (Fukunaga and Koreki 1995, Fukunaga et al. 1995, Scoles et al. 2001, Richter et al. 2003, Mun et al. 2006). Several relapsing fever spirochetes cause generalized infections in their vectors and are vertically transmitted (Schwan and Piesman 2002). Since detection by various microscopic methods fails to differentiate these morphologically similar spirochetes, it remains uncertain as to which spirochetes had infected questing or hatched larvae of the I. ricinus complex in the early studies.

To determine which kind of spirochete infects I. ricinus larvae, we collected questing larvae and larvae derived from engorged females and examined them for the presence of particular spirochetal DNA that permitted us to differentiate them at the species level. In addition, we determined how rapidly larvae acquire Lyme disease spirochetes from an infected host.

Materials and Methods

The Czech study sites were located near the cities of Vranov and Valtice, in South Moravia, close to the border with Austria. Questing larval ticks were collected by dragging a flannel flag over the ground vegetation once a month from April through October of 2008 and in June of 2009 at Vranov and from July through October of 2008 and in May of 2009 in Valtice. Larval ticks were preserved in 80% ethanol and microscopically identified to species.

Engorged I. ricinus females were collected off various dogs that were walked near the town of Eberdingen, about 30 km northwest of Stuttgart in Southern Germany, from 2002 through 2008. To permit egg laying, these ticks were individually confined in screened vials stored at 20°C±2°C in sealed desiccator jars containing supersaturated MgSO_4. Once hatched, 30 to 40 progeny of each egg batch were preserved in 80% ethanol. At the same site, questing nymphs and adults were collected by flagging in 2003 and 2009.

To detect and identify the various spirochetes that may be present in questing or derived larvae and in questing nymphal and adult ticks, the opisthosoma of each was opened, transferred to a tube containing 180 μL lysis buffer (ATL Tissue Lysis Buffer; Qiagen, Hilden, Germany) and 20 μL proteinase K (600 mAU/mg), and lysed at 56°C overnight. DNA was extracted by using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's instructions, DNA of larval and nymphal ticks or adult ticks was eluted with 50 or 75 μL elution buffer, respectively, and stored at −20°C until polymerase chain reaction (PCR) was performed. Borrelia genospecies were characterized by amplifying and sequencing a 600-nucleotide fragment of the gene encoding the 16S rRNA (16S-PCR). To increase the sensitivity for detection of spirochetal DNA in ticks, we used nested PCR, as previously described (Richter et al. 2003). This method detects as few as a single spirochete even in the presence of tick DNA. DNA was extracted, reaction vials were prepared for amplification, templates were added, and products were electrophoresed in separate rooms. As additional precaution, the reaction mixtures were prepared in a designated PCR workstation (Labcaire Systems, North Somerset, United Kingdom), and templates were added to the mixtures in a second PCR workstation. Benches and equipment were wiped down with a DNA decontamination solution (DNAerase; MP Biomedicals, Eschwege, Germany) after each use. In each sixth reaction mix, water was added instead of extracted DNA to serve as negative control. Each PCR amplification product was purified by using a QIAquick-Spin PCR column (Qiagen) according to the manufacturer's instructions. Amplified DNA fragments were directly sequenced in both directions using the inner primers by the dideoxynucleotide chain-termination method on a Licor DNA4200 sequencer (Licor Biosciences, Bad Homburg, Germany). Each resulting sequence was compared with sequences of the same gene fragment representing various spirochetal genospecies. The following sequences served for comparison: Accession numbers X85196 and X85203 for Borrelia burgdorferi s.s.; X85190, X85192, and X85194 for B. afzelii; X85193, X85199, and M64311 for Borrelia garinii; X98228 and X98229 for Borrelia lusitaniae; X98232 and X98233 for Borrelia valaisiana; AY147008 for Borrelia spielmanii; and AY253149 for B. miyamotoi. A complete match, permitting no more than two nucleotide changes, was required.

To determine the earliest time point at which a feeding larva acquires Lyme disease spirochetes from an infected host, we exposed two mice each of the hairless strain Crl: SKH1-Hr^hr to four nymphal ticks infected by either B. afzelii or B. burgdorferi s.s. and permitted larval ticks to attach to these mice 2 weeks later. About 20 larvae were removed from the mice by means of forceps at 2-h intervals for as long as 8 h after attachment. Ten removed larvae from each mouse and time point were preserved in 80% ethanol, and another 10 were immediately dissected on glass slides and acetone fixed for a subsequent immunofluorescence assay. Ethanol-preserved larvae were individually analyzed by nested 16S-PCR, as just described, and individual acetone-fixed larvae were stained by direct immunofluorescence by using an affinity-purified FITC-labeled goat anti-B. burgdorferi antibody (BacTrace, Kirkegaard & Perry Laboratories, Gaithersburg, MD).

Results

We first determined the prevalence and species of spirochetes in questing larval ticks collected in the two Czech sites. In the Vranov site, one of the 156 larvae that had been individually analyzed harbored a spirochetal infection (Table 1). Of the 99 random pools of five larvae each, spirochetal DNA was detected in six pools. In contrast, none of the 29 individually analyzed larvae and of the 13 pools of five larvae collected in the Valtice site harbored spirochetes. In each of the seven samples containing spirochetal DNA, B. miyamotoi was identified. Prevalence of B. miyamotoi in larval ticks in Vranov ranged between a minimum infection rate of 1.1%, if only one larva of each infected pool harbored spirochetes (with maximum likelihood confidence intervals of 0.5 and 2.2, determined according to Biggerstaff 2006) and a maximum infection rate of 4.8% (=31 infected larvae of 651 sampled ticks), if each larva in the six pool would have been infected. B. miyamotoi was the sole spirochete detected in larval ticks sampled while questing on the vegetation.

Table 1.

Spirochetal Prevalence in Ixodes ricinus Ticks

	Ticks			% ticks infected with Borrelia
Site	Source	Stage	No. examined	miy	afz	gar	val	bur	bis	>1 genospecies
Vranov	Flagging	Larva	156	0.6	0.0	0.0	0.0	0.0	0.0	0.0
		Larval pool^a	99	6.1	0.0	0.0	0.0	0.0	0.0	0.0
Valtice	Flagging	Larva	29	0.0	0.0	0.0	0.0	0.0	0.0	0.0
		Larval pool^a	13	0.0	0.0	0.0	0.0	0.0	0.0	0.0
Eberdingen	Flagging	Nymph	293	2.0	22.5	2.0	2.4	1.7	0.0	1.4
		Adult	272	3.3	13.6	6.6	8.5	2.6	3.7	2.6
	Egg batch	Larval pool^b	510	3.1	0.0	0.0	0.0	0.0	0.0	0.0
		Larva^c	136	92.6	0.0	0.0	0.0	0.0	0.0	0.0

Spirochetal prevelance in questing larval Ixodes ricinus ticks collected in two Czech study sites and comparison of spirochetal prevalence in questing nymphal and adult Ixodes ricinus ticks collected at a German study site with that in larvae derived from females which had attached and engorged naturally on dogs at the same site.

Collected larvae were randomly assigned to a pool, consisting of at least five ticks, that is, larvae may not be related.

Each larval pool, consisting of five ticks, derived from one egg batch, that is, larvae were related.

Individual larvae from each of the 16 infected egg batches (line above) were analyzed.

miy, miyamotoi; afz, afzelii; gar, garinii; val, valaisiana; bur, burgdorferi s.s.; bis, bissettii-like.

Next, we compared the prevalence of various spirochetes in questing nymphal and adult ticks to that in larvae which hatched from egg batches derived from female ticks collected in the same German site. B. afzelii constituted the most prevalent spirochete genospecies in both nymphal and adult ticks, infecting a fifth and an eighth of them, respectively (Table 1). In contrast, in larval ticks that had been derived from 510 females that had naturally attached to and fully engorged on dogs in the same site, no Lyme disease spirochetes were detected, when pools of five larvae from each egg batch were analyzed. Larvae from 3% of these egg batches, however, harbored B. miyamotoi spirochetes. We determined the rate of infection in each infected egg batch by analyzing between 5 and 18 individual larvae. More than 90% of larval ticks derived from egg batches infected with B. miyamotoi harbored this spirochete. B. miyamotoi infected questing nymphal and adult ticks at about the same rate as it did egg batches laid by females engorging on dogs in the same site. Questing nymphal and adult ticks were infected mainly by B. afzelii, whereas larval ticks resulting from engorged females of the same population were solely infected by B. miyamotoi.

To determine how rapidly larvae may acquire Lyme disease spirochetes when feeding on an infected rodent, we removed larval ticks during the blood meal from their hosts at 2-h intervals and examined them individually for the evidence of spirochetes, either by an immunofluoresence assay or by nested PCR. Two mice each had been infected by tick-borne B. afzelii or B. burgdorferi s.s. 2 weeks before spirochete-free ticks were permitted to attach. As early as 2 h after initial attachment, nearly half of the larvae had acquired spirochetes when feeding on B. afzelii-infected mice (Fig. 1). Four hours after attachment, almost all larvae had imbibed such spirochetes. Acquisition of B. burgdorferi s.s. spirochetes by larval ticks occurred somewhat delayed, about a sixth of the larvae acquired spirochetes within a 2-h blood meal and three quarters at an 8-h blood meal. No hemoglobin was evident in the larvae at that time. Evidently, larvae rapidly acquire Lyme disease spirochetes from infected rodents.

FIG. 1.

Rate of acquisition of Borrelia afzelii (filled squares) or Borrelia burgdorferi s.s. (open squares) spirochetes by larval Ixodes ricinus ticks during the first 8 h of their blood meal.

Discussion

Spirochetes appear to rarely infect questing larval ticks of the I. ricinus complex. On average, 2.5% of more than 3600 field-derived larvae that had been analyzed in the course of 17 studies using various methods of detection harbored spirochetes (Table 2). This includes four studies in which no spirochetes were detected and one study in which as much as a fifth of all larvae contained spirochetes (Steere et al. 1983, Magnarelli et al. 1986, Mejlon and Jaenson 1993, Rijpkema et al. 1994, Tälleklint and Jaenson 1996). Our observation that spirochetes infected between 1% and 5% of the questing larvae collected in one of our Czech study sites corroborates earlier studies. All spirochetes that we detected by PCR were identified by sequencing as B. miyamotoi. Both B. miyamotoi and Lyme disease spirochetes are morphologically indistinguishable when observed by dark-field microscopy (Piesman et al. 1986, Matuschka et al. 1992, Halouzka et al. 1995). Likely, immunofluorescence microscopy applying polyclonal rabbit or human serum raised against Lyme disease spirochetes (Wilske et al. 1987, Doby et al. 1990, Miserez et al. 1990, Matuschka et al. 1992, Rijpkema et al. 1994, Zhioua et al. 1994) may similarly have visualized B. miyamotoi spirochetes, because such polyclonal antibodies cross react (Scoles et al. 2001). Similarly, monoclonal antibodies against flagellin cross react (Marquez and Constan 1990, unpublished observation). In fact, most spirochetes that have been detected in questing larvae described in earlier studies may not have been Lyme disease spirochetes, but B. miyamotoi.

Table 2.

Prevalence of Spirochetes in Field-Derived Questing Larval Ixodes Ticks Using Various Methods of Detection

Larvae						References
Species	Sample size	No. examined	% infected	Method of detection	Serum source or target gene	First author	Year
Ixodes dammini ^a	Individual	148	0.0	DFA	Rabbit serum	Steere	1983
I. dammini ^a	Individual^b	274	0.7	Dark-field	N/A	Piesman	1986
Ixodes scapularis	Individual	85	0.0	DFA	Rabbit serum	Magnarelli	1986
I. ricinus	Individual	367	1.1	DFA	Rabbit serum	Wilske	1987
I. ricinus	Individual	355	4.8	IFA	Human serum	Doby	1990
I. ricinus	Individual	57	10.5	IFA	mAb ospA/flagellin	Marquez	1990
I. ricinus	Individual	216	3.2	DFA	Undefined serum	Miserez	1990
I. ricinus	Individual	150	0.7	Dark-field, IFA	Rabbit serum	Matuschka	1992
I. ricinus	Individual	421	0.0	Phase-contrast	N/A	Mejlon	1993
I. ricinus	Individual	84	21.4	IFA	Human serum	Rijpkema	1994
I. ricinus	Individual	652	3.1	DFA	Rabbit serum	Zhioua	1994
I. ricinus	Individual	142	6.3	Dark-field	N/A	Halouzka	1995
I. ricinus	Individual	57	5.3^c	PCR	IGS	Rijpkema	1996
I. ricinus	Individual	260	0.0	Phase-contrast	N/A	Tälleklint	1996
I. ricinus	Individual	136	1.5^d	PCR	ospA	Fingerle	2008
I. ricinus	Individual	60	1.7^f	PCR	IGS	Strube	2010
I. ricinus	Pools of 10	210	0.5^e	Real-time PCR	16S	Kjelland	2010
I. ricinus	Individual and pools	745	0.9–4.2^g	PCR	16S	This study	2011

Now synonymous with I. scapularis.

Fed on hamsters, examined as nymphs.

One harbored Borrelia afzelii, one Borrelia valaisiana, and one B. afzelii with Borrelia garinii.

One harbored Borrelia burgdorferi s.s., one B. garinii serotype 6.

Harbored B. burgdorferi s.s.

Species was not determined.

Harbored Borrelia miyamotoi.

PCR, polymerase chain reaction; IFA, indirect immunofluorescence assay; DFA, direct immunofluorescence assay; mAb, monoclonal antibody; ospA, outersurface protein A; IGS, intergenic spacer; 16S, 16S rRNA; N/A, not applicable.

In more recent studies, Lyme disease spirochetes have been definitively identified in questing larval I. ricinus ticks, because detection by PCR methods permits to distinguish the spirochetes. Five larvae and one larval pool infected by B. afzelii, B. valaisiana, B. garinii, or B. burgdorferi s.s. spirochetes were identified by targeting the outersurface protein A (ospA), the 16S rRNA gene, or the intergenic spacer region (Rijpkema and Bruinink 1996, Fingerle et al. 2008, Kjelland et al. 2010, Strube et al. 2010). It remains undetermined whether these larvae acquired their spirochetal infection transovarially or from a host. A brief interrupted blood meal on an infected host may be sufficient for a larva to acquire Lyme disease spirochetes. Numerous Ixodes dammini (now synonymous with Ixodes scapularis) larvae that had been removed from field-caught rodents and appeared “nonengorged,” because no blood was evident, harbored spirochetes (Bosler et al. 1983). I. dammini larvae permitted to feed on experimentally infected rodents for a specified interval appeared to acquire spirochetes only after a 2-day blood meal (Nakayama and Spielman 1989); whereas, in another study, such larvae imbibed spirochetes as early as 8 h after attachment to infected hamsters (Piesman 1991). Our observation that the majority of I. ricinus larvae became infected with B. afzelii and B. burgdorferi s.s. spirochetes after a blood meal lasting for only four and 8 h, respectively, suggests that they acquire Lyme disease spirochetes before imbibing blood. Only after a 12-h blood meal, hemoglobin became microscopically evident in partially fed larvae. After an interrupted blood meal, a partially fed larva may readily quest for another host, because all larvae detached from their euthanized hosts and more than half of them successfully continued their meal when exposed to another host (Nakao and Sato 1996). A standard guaiac test to detect heme in infected field-derived larvae may proof host contact (Schulze et al. 1986), although it should be taken into account that Lyme disease spirochetes may already be acquired with lymph fluid. Since questing larvae collected in the field cannot be considered devoid of previous host contact, presence of Lyme disease spirochetes in questing larvae is no proof of transovarial transmission.

Examining larvae derived from egg batches provides a more reliable indication of spirochetal inheritance. In 3 out of 9 studies, no spirochetes were detectable by immunofluorescence assays and/or PCR in the progeny of naturally infected female ticks of the I. ricinus complex that had engorged on various hosts (Schoeler and Lane 1993, Rijpkema et al. 1994, Patrican 1997) (Table 3). None of nearly 5000 larvae produced by 48 females that had been experimentally infected with B. afzelii inherited these spirochetes (Matuschka et al. 1998). In contrast, B. miyamotoi spirochetes infected 3% of larvae deriving from 52 field-collected I. scapularis female ticks, two females had passed these spirochetes to 6% and 73% of their offspring (Scoles et al. 2001). Since this study detected spirochetes by means of PCR targeting the flagellin gene, it permitted species identification that had not been possible in two earlier studies describing a similar infection rate of 1.3% and 1.9% in larvae from field-derived females (Magnarelli et al. 1986, 1987). Nonculturable spirochetes were visualized by Giemsa stain, but not detected by PCR, in 7% of progeny deriving from one Ixodes persulcatus female (Nefedova et al. 2004). B. miyamotoi, related more closely to spirochetes causing relapsing fever than to those causing Lyme disease, are likely to cause generalized infections in their vector ticks (Schwan and Piesman 2002). Indeed, in early studies that examined solely offspring of female ticks harboring generalized spirochetal infections, between 13% and 80% larvae inherited infection from these females (Burgdorfer et al. 1986, Lane and Burgdorfer 1987). Our observation that transovarial transmission of B. miyamotoi is so efficient that virtually all larvae inherited spirochetes from their infected mothers indicates that it may have been B. miyamotoi which was detected in those early studies. When questing larvae are collected from the vegetation, infection rates with B. miyamotoi may vary greatly (see range of prevalence in Table 2), because larvae tend to cluster after eclosion; sampling, pooling, and analyzing methods may need to take the efficient B. miyamotoi inheritance and the poor dispersal behavior of larvae into account. Even if transovarial transmission of Lyme disease spirochetes may occasionally occur, it seems to be an exceedingly rare event. No undisputable proof currently exists for vertical transmission of Lyme disease spirochetes, whereas B. miyamotoi appears to be readily passed between generations of vector ticks.

Table 3.

Prevalence of Spirochetes in Larval Ixodes Ticks Derived from Egg Batches of Naturally or Experimentally Infected Females, Analyzed by Various Methods of Detection

Larvae		Females from which larvae were derived				Larvae				References
Species	Sample size	Source	% infected	Fed on	No.	No. examined	% infected	Method of detection	Serum source or target gene	First author	Year
I. scapularis	Individual	naturally attached	nd	deer	6	152	1.3	DFA	rabbit serum	Magnarelli	1986
I. ricinus	No data	questing, field	21.9	rabbit	2	no data	80.0	DFA	rabbit serum	Burgdorfer	1986
I. dammini ^a	Individual	naturally attached	52.9	dog or deer	18	2297	1.9	DFA/IFA	rabbit/mAb ospA	Magnarelli	1987
I. pacificus	Individual	questing, field	3.0	rabbit	3	230	13.0	DFA/IFA	rabbit serum/mAb flagellin	Lane	1987
I. pacificus	Individual	questing, field	8.8	rabbit	119	∼6000	0.0	DFA	rabbit serum	Schoeler	1993
I. ricinus	Individual	questing, field	nd	sheep	15	168	0.0	IFA	human serum	Rijpkema	1994
I. scapularis	Individual and pools	questing, field	73.8	dog	48	∼44,700	0.0	IFA, PCR	rabbit serum, flagellin	Patrican	1997
I. ricinus	Individual and pools	experimentally infected	nd	rabbit	48	4932	0.0	IFA, PCR	rabbit serum, ospA	Matuschka	1998
I. scapularis	Pools of 50	questing, field	nd	rabbit or sheep	52	2700	3.0^b	PCR	flagellin	Scoles	2001
Ixodes persulcatus	Individual and pools	questing, field	80.0	mouse	17	2797	0.3^c	PCR, Giemsa, culture	16S	Nefedova	2004
I. ricinus	Pools of 5	naturally attached	nd	dog	510	2550	0.6–3.1^b	PCR	16S	This study	2011

Now synonymous with I. scapularis.

Harbored Borrelia miyamotoi.

Only by Giemsa stain.

nd, not done.

Footnotes

Acknowledgments

The authors thank Rainer Allgöwer and Juraj Pesko for collecting ticks in Germany and in the Czech Republic, respectively, and Udo Bischoff, Nicole Held, Mandy Marbler-Pötter, and Andrea Schäfer for expert technical assistance.

Disclosure Statement

No competing financial interests exist.

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