Identification of Bacteria Infecting Ixodes ricinus Ticks by 16S rDNA Amplification and Denaturing Gradient Gel Electrophoresis

Abstract

Ticks harbor a complex microbial population, which they acquire while feeding on a variety of mammalians and birds. Zoonotic diseases transferred by ticks are an increasing problem and have become a burden to the community. 16S rDNA amplification and denaturing gradient gel electrophoresis (DGGE) enables detection of the broad spectrum of bacteria that settles in the ticks. Profiling the complete microbial population in ticks may provide a better understanding of the ticks' potential to harbor and disperse pathogens. Separation of pathogenic species by DGGE is based on variation in %GC content within the 16S rDNA genetic region. Sequencing of these fragments allows identification of bacterial species. Present study identified some well-known tick-infecting bacteria, such as members of genus Borrelia, Rickettsiales, and Pseudomonas, but also less described tick-infecting bacteria such as Rhodococcus erythropolis, Spiroplasma spp., and an endosymbiont of the microarthropod Folsomia candida. This is the first report of Segniliparus rugosus–infected Ixodes ricinus ticks. Also, it is the first report of several of these pathogens in the Norwegian tick population.

Introduction

M ild winters, suitable vegetation, and high wildlife population densities along the Norwegian coastline secure a good habitat for Ixodes ricinus ticks. A variety of mammalians and birds can host ticks, and during a three-stage lifecycle, this diverse repertoire of hosts may influence the ticks' microbial content. Due to lack of enzymatic digestion, microbes may survive the digest of the blood meal and settles in the ticks. This enables ticks to transmit their acquired pathogens while feeding on their next host (Lindgren et al. 2000, Gray 2002, 2003). A large number of bacteria and viruses have been detected in tick samples (Swanson et al. 2006, Tokarz et al. 2010). For instance, the etiological agent of Lyme borreliosis, Borrelia burgdorferi sensu lato, may be transmitted from tick to host during feeding, and constitute a serious health risk for humans. Lyme borreliosis may result in skin, heart, joint, and neurological infections, and clinical symptoms may occur within days or weeks (Aguero-Rosenfeld et al. 2005). A widespread disease among sheep is tick-borne fever, an Anaplasma phagocytophilum infection that affects the immune system, making the infected sheep susceptible to life-threatening secondary infections (Stuen 2007). Other known bacterial species that harbor in ticks, Babesia spp., Pseudomonas spp., Rickettsia spp., and Francisella tularensis, are also associated with tick-borne infections (Sparagano et al. 1999, Duh et al. 2001, Schabereiter-Gurtner et al. 2003, Nilsson et al. 2005, Severinsson et al. 2010).

A broad-range molecular method that leads to genus-specific identification is a practical approach to identify the bacterial content in ticks (Schabereiter-Gurtner et al. 2003). Denaturing gradient gel electrophoresis (DGGE) separates polymerase chain reaction (PCR)-amplified genetic fragments based on their sequence composition, and the method can be used for bacterial profiling of complex microbial populations in environmental samples (Muyzer et al. 1993, Ferris et al. 1996). Bacteria have a 16S ribosomal DNA (rDNA) genetic region that has slight variations in %GC content (Head et al. 1998). Broad-range 16S rDNA primers generate 16S rDNA fragments, with a GC-clamp designed to prevent the double-stranded DNA fragment from separating during migration through the gel. Amplified DNA fragments of the same length are separated in a polyacrylamid gel with a linearly increasing gradient of denaturants. The double-stranded DNA fragments migrate in the gradient gel based on their melting profile. Each DGGE band can be reamplified and sequenced to determine the specific nucleotide sequence (Muyzer et al. 1993). Nucleotide sequence databases, such as basic local alignment search tool (BLAST), may be used to compare the sequences isolated in the DGGE analysis with known sequences from a range of bacterial species (Altschul et al. 1997). Schabereiter-Gurtner et al. (2003) applied this method to identify bacterial pathogens in adult ticks, revealing up until then unknown bacterial species in feeding ticks.

The aim of our study was to identify bacterial species in ticks of the species I. ricinus (Acarina: Ixodidae). Ticks may harbor several pathogens (Swanson et al. 2006), which emphasizes the importance of profiling the total bacterial content in ticks. So far our knowledge of tick-infecting bacteria and their role in transmission of disease is limited because only a few of the bacteria actually present in ticks are identified (Schabereiter-Gurtner et al. 2003). The Norwegian county Møre and Romsdal have several challenges related to tick-borne infections. In 2010, about 47 (18 cases:100,000 inhabitants) patients were found to have Lyme borreliosis according to the Norwegian Surveillance System for Communicable Diseases (MSIS 2010). In addition to this, farmers loose income due to severe illness among grazing livestock in tick-infested areas.

Materials and Methods

Tick collection

A total of 177 I. ricinus ticks were collected from Sunnmøre, which is located in the southern part of Møre and Romsdal county, in two separate turns. The first turn was in May/June and the second in September 2010. Host-seeking ticks were collected by flagging method as previously described (Kjelland et al. 2010b), and feeding ticks were individually picked from cats and dogs using tweezers. (As described in DNA extraction, all ticks are rinsed before DNA extraction, and sterile tweezers were not used.) In this study we do not distinguish between feeding tick from cats and dogs. Ticks collected in spring (May/June) were separated into group 1, consisting of 33 host-seeking ticks, and group 2, consisting of 83 feeding ticks. Ticks collected in autumn (September) were separated into group 3, consisting of 24 host-seeking ticks, and group 4, consisting of 37 feeding ticks (Table 1).

Table 1.

Origin of Ticks in Present Study

	Ixodes ricinus ticks			Collected ticks	DGGE profiles obtained
Spring	Group 1	Host seeking	Nymph	33	16
			Adult	0	0
	Group 2	Feeding^a	Nymph	12	10
			Adult	71	56
Autumn	Group 3	Host seeking	Nymph	8	8
			Adult	16	16
	Group 4	Feeding^a	Nymph	15	12
			Adult	22	10

Feeding ticks collected from cats and dogs.

DGGE, denaturing gradient gel electrophoresis.

DNA extraction

Ticks were placed into sterile tubes labeled with date of collection. Fully engorged ticks were dissected according to the DNA extraction protocol, to reduce PCR inhibition by blood components. Individual ticks were washed in 70% ethanol, placed individually into sterile tubes with sterile double-distilled (dd) H₂O, and homogenized using 5-mm steel beads and Qiagen TissueLyser (Qiagen GmbH). DNA was extracted from each sample using DNeasy blood and tissue kit (Qiagen GmbH) according to manufacturers' protocol.

PCR amplification of 16S rDNA fragments

Bacterial 16S rDNA fragments were amplified by nested PCR with primers 341f/907r and 341fGC/518r of the Esherichia coli 16S rDNA sequence (Table 2) (Schabereiter-Gurtner et al. 2003). The reaction was carried out in 25 μL volumes, containing 12.5 μL Red'y gold PCR master mix (Eurogentec), 1.0 μL of each primer (10 μM), 9.0 μL ddH₂O, and 1.5 μL template DNA. Amplification was performed in a 2720 thermal cycler (Applied Biosystems) with 1 cycle of denaturation (10 min, 95°C), followed by 30 cycles of denaturation (1 min, 95°C), annealing (1 min, 55°C), and extension (1 min, 72°C) and a final cycle of extension (10 min, 72°C) as previously described (Schabereiter-Gurtner et al. 2001), but with some modifications.

Table 2.

List of Primers in 5′ to 3′ Orientation

Primer		Primer sequence	Reference
341f	Outer primer	5′-CCTACGGGAGGCAGCAG-3′	Schabereiter-Gurtner et al. (2003)
907r	Outer primer	5′-CCCCGTCAATTCATTTGAGTTT-3′
341fGC	Inner primer with GC clamp	5′-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCCTACGGGAGGCAGCAG-3′	Schabereiter-Gurtner et al. (2003)
518r	Inner primer	5′-ATTACCGCGGCTGCTGG-3′

f, forward primer; r, reverse primer.

DGGE analysis

Amplified 16S rDNA fragments were separated in polyacrylamide gels (0.5×TAE [Tris-acetate-EDTA buffer] with 8% wt/vol) with a linear denaturant gradient from 30% to 60%, where 100% denaturant contained 7 M urea and 40% formamide. Sample (25 μL) was placed into each well, after thoroughly removing casting residues by washing with 0.5×TAE. DGGE was performed in a V20 CDC Dual Vertical Unit System (Scie-Plas) at 60°/20V for 10 min, and then 60°/60 V for 16 h in 0.5×TAE. The gels were stained by SYBR^®GREEN (Sigma-Aldrich) (30 μL 10,000×SYBR GREEN in 300 mL 1×TAE) for 30 min, and photographed. Individual bands from each gel were excised with scalpel and placed into tubes containing 20 μL ddH₂O. Bands representing each different migration rate were cut out from each DGGE gel. The tubes were stored at +4°C over night and then frozen at −20°C.

Sequence analysis

16S rDNA fragments from DGGE bands were reamplified using 341f/518r primers. PCR products were purified in a two-step process by ExoSAP-IT^® (Affymetrix) according to manufacturers' protocol. The sequencing reaction was carried out in 10 μL volumes, containing 1 μL Big Dye Terminator 3.1 (Applied Biosystems), 1 μL 5×Sequencing Buffer (Applied Biosystems), 0.32 μL 341f primer (10 μM), 4.68 μL ddH₂O, and 3 μL template DNA. Amplification was performed in a 2720 thermal cycler (Applied Biosystems) with 1 cycle of denaturation (6 min, 96°C), followed by 25 cycles of denaturation (10 s, 96°C), annealing (5 s, 50°C), and extension (4 min, 60°C). ddH₂O (10 μL) was added to each well upon completed sequencing reaction and stored at −20°C until sequencing.

Each sequence was compared to sequences of known bacterial species in the BLAST database (Altschul et al. 1997).

Results

DGGE profile was obtained from a total of 128 ticks. Figure 1A shows the DGGE fingerprint of group 1, host-seeking nymphs, whereas Figure 1B shows the DGGE fingerprint of group 2, feeding adult ticks. Figure 2A shows the DGGE fingerprint of group 3, host-seeking ticks (nymphs; lane 1–8, adults; lane 9–23), whereas Figure 2B shows the DGGE fingerprint of group 4, feeding ticks (nymphs; lane 1–7, adults; lane 8–17).

FIG. 1.

(A) SYBR^® GREEN stained 16S rDNA DGGE profile of bacteria found in ticks from group one. Numbering indicates which bacteria the DGGE band has been identified according to Table 3. (B) SYBR GREEN stained 16S rDNA DGGE profile of bacteria found in ticks from group two. Numbering indicates which bacteria the DGGE band has been identified according to Table 3. DGGE, denaturing gradient gel electrophoresis.

FIG. 2.

(A) SYBR GREEN stained 16S rDNA DGGE profile of bacteria found in ticks from group three. Numbering indicates which bacteria the DGGE band has been identified according to Table 3. (B) SYBR GREEN stained 16S rDNA DGGE profile of bacteria found in ticks from group four. Numbering indicates which bacteria the DGGE band has been identified according to Table 3.

DGGE profiles were studied to identify bands with different migration rates. A total of 96 bands were excised and sequenced. Each DGGE 16S rDNA fragment was reamplified, purified, and used as template in a sequencing reaction. Each sequence was identified by BLAST search and the results are displayed in Table 3. Host-seeking ticks collected in spring resulted in a fingerprint dominated by a solid band identified as the I. ricinus 18S rDNA sequence. The other DGGE bands were identified as Pseudomonas spp. and Rickettsiales bacterium. The endosymbiont Folsomia candida was present in three of the samples. The bacteria indentified from DGGE profiles of feeding ticks collected in spring were members of the genus Pseudomonas, Rickettsiales bacterium, and Stenotrophomonas rhizophila. These samples also included an I. ricinus endosymbiont (IricES1) and an uncultured sheep mite bacterium, Llangefni 3. In addition, Segniliparus rugosus was identified in I. ricinus ticks for the first time in this study.

Table 3.

Identification of Bacteria in Ticks Obtained by Sequencing Analysis of Reamplified Denaturing Gradient Gel Electrophoresis Bands

DGGE band	Closest related sequence ^a	Percent similarity (%)	GenBank ^b
1	Ixodes ricinus 18S rDNA	100%	GU074707.1
2	Borrelia burgdorferi s.s.	100%	HQ433690.1
3	Pseudomonas spp.	100%	HQ403142.1
		100%	HQ677222.1
		100%	HQ143608.1
		100%	GU939689.1
		100%	AY646430.1
		99%	HQ538785.1
4	Pseudomonas putida	99%	NC_010322.1
		99%	FN995246.1
		98%	GU191929.1
5	Pseudomonas fluorescens	100%	HQ704717.1
6	Pseudomonas rhizosphaerae	99%	GU585129.1
7	Uncultured sheep mite bacterium Llangefni 3	99%	AF289503.1
8	Serratia marcescens	100%	HQ680476.1
9	Stenotrophomonas rhizophila	97%	HQ689684.1
10	Endosymbiont of Folsomia candida	99%	AF327558.1
11	Segniliparus rugosus	99%	FJ593188.1
12	Rhodococcus erythropolis	98%	FR745420.1
13	Spiroplasma spp.	98%	M24477.1
14	Anaplasma phagocytophilum	99%	DQ426992.1
15	Rickettsiales bacterium	100%	AF525482.1
16	Rickettsiella spp.	97%	EU430250.1
17	Candidatus Nicolleia massiliensis	99%	DQ788562.1
18	Candidatus Midichloria spp.	100%	AM411600.1
19	I. ricinus endosymbiont (IricES1)	99%	AM087582.1
20	Bradyrhizobium spp.	99%	FN600563.2

Closest related sequence indentified by BLAST search (Altschul et al. 1997).

GenBank accession number of closest identified strain.

DGGE profiles from host-seeking ticks collected in autumn show four bands present in almost all samples. These were identified as the I. ricinus 18S rDNA sequence, Pseudomonas spp., Candidatus Nicolleia massiliensis, and Rickettsiales bacterium. The adult ticks have an additional repetitive band identified as A. phagocytophilum and the spirochete B. burgdorferi sensu stricto was identified in one of the adult ticks. Feeding ticks collected in autumn display a variety of bacteria, most of which are present on only a few of the DGGE profiles. The most common species in this group of ticks was Pseudomonas spp., which was identified in all samples.

Discussion

Feeding ticks can acquire several bacteria and endosymbionts during a three-phase lifecycle and tick-borne pathogens are etiological agents of multiple severe infections. A previous study revealed unexpected and so far unknown bacterial species in ticks and emphasized the need to map the total bacterial content in ticks (Schabereiter-Gurtner et al. 2003). The broad spectrum of bacteria that may harbor in ticks may be detected by a special molecular-based technique that enables identification of pathogens and endosymbionts based on amplification of the 16S rDNA gene.

We profiled the bacterial content in 128 I. ricinus ticks collected from Sunnmøre. The ticks were divided into four groups based on time of collection (spring/autumn) and whether they were host-seeking or feeding. DGGE fingerprinting gave distinctive profiling patterns that clearly separated the four groups. Host-seeking nymphs collected in spring gave a comparable pattern with one repeated dominating band and few other distinctive bands. Feeding ticks collected within the same period gave a quite diverse pattern with few dominating bands. In general, DGGE fingerprints from ticks collected in spring consist of two bands, usually one dominant and one weak band. The tick-collecting area was covered in snow until April and ground frost was registered in late May. This may indicate that winter hibernation could affect the bacteria and endosymbionts infecting ticks.

Ticks collected in autumn displayed a fingerprinting pattern consisting of an increased amount of bands and ticks displayed between four and eight bands, usually three or four dominating bands. Even though each single tick fingerprint contained multiple strong bands, the host-seeking ticks collected in autumn had the same comparable pattern as host-seeking ticks collected in spring whereas feeding ticks had a more diverse pattern. Feeding ticks may acquire new pathogens as well as secrete pathogens into the host while feeding (Gray 2003). This transmission of bacteria may result in the more diverse patterns obtained from the fingerprinting of feeding ticks.

Table 3 displayed a variety of bacteria identified by comparison of DGGE sequences to sequences of known bacteria in the BLAST database. One of the most prominent DGGE band was identified as I. ricinus 18S rDNA. This band is present in 65% of the samples (in 88% of the host-seeking ticks and 27% of the feeding ticks, respectively). The I. ricinus 18S rDNA sequence is comparative to the universal 16S rDNA primers, resulting in an amplification of the 18S rDNA fragment of I. ricinus (Schabereiter-Gurtner et al. 2003).

Two major bacterial orders were identified by the DGGE profiling. One was Gammaproteobacteria of the order Pseudomonadales and the other was proteobacteria of the order Rickettsiales.

Pseudomonadales

About 92% of the samples contained one or more DGGE bands, which were identified as various species of the order Pseudomonadales. Previous genetic studies have demonstrated the presence of Pseudomonas spp. in ticks (Schabereiter-Gurtner et al. 2003), but the presence of the species Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas rhizosphaerae found in this study have to our knowledge not been identified in ticks previously. Additionally, a strain that is genetically related to Pseudomonas, noted as uncultured sheep mite bacterium Llangefni 3 (DGGE band 7), was also identified in this study. This bacterium was first described by Hogg and Lehane (2001) in a study of microfloral diversity of cultured and wild strains of Psoroptes ovis infecting sheep (Hogg and Lehane 2001).

Rickettsiales

A commonly distributed tick-infecting bacterium of the order Rickettsiales is A. phagocytophilum and the species was identified in 26% in total and in 48% of the autumn samples. A. phagocytophilum is the causative agent of tick-borne fever, a widespread disease in Europe recorded in humans, wildlife, and domestic animals (Stuen 2007). Our genetic study has also demonstrated presence of a Rickettsiales bacterium in 25% of the samples. The species was first related to tick-borne infections by an Italian study of the genetic diversity of bacteria that harbor in ticks (Sanogo et al. 2003). Other species of this order detected in present study include C. N. massiliensis and Candidatus Midichloria spp. C. N. massiliensis was detected in 18% of the samples, whereas Candidatus Midichloria spp. was detected in 5% of the samples and they have both previously been described as tick-infecting bacteria (Matsumoto et al., unpublished work). A study revealed that C. Midichloria spp. was found as one of two bacteria that invade mitochondria in ticks (Epis et al. 2008).

The I. ricinus endosymbiont (IricES1) was present in 4% of the samples. The endosymbiont shown genetic relationship with Rickettsiales and was previously recognized as a tick-borne infection in a study of widespread distribution and high prevalence of an alpha-proteobacterial symbiont in I. ricinus (Lo et al., unpublished work).

Among the less prominent bacteria identified in this study are members of the order Spirochaetales, Actinomycetales, Legionellales, and Entomoplasmatales.

Spirochaetales

A member of the genus Borrelia (Spirochetaceae), B. burgdorferi sensu stricto, was identified in 4% of the samples. This prevalence is significantly lower than that found in other Norwegian studies (Kjelland et al. 2010a, 2010b). DGGE analysis is based on PCR amplification with general 16S rDNA primers, which makes it possible to amplify this genetic region of all bacterial species present in the sample. This competing situation can result in less efficient amplification of one species compared to another species based on DNA yield. Therefore, we have used DGGE analysis to study the bacterial diversity in ticks and not for prevalence studies.

Actinomycetales

Two species from the order Acinomycetales were present in this study, Rhodococcus erythropolis and Se. rugosus. R. erythropolis has previously been described as a tick-infecting bacterium, but the presence of Se. rugosus was demonstrated for the first time in this study. The bacterium was first classified among mycobacterium, and described as a species in 2005 (Butler et al. 2005).

Entomoplasmatales

In 3% of the samples the bacterium Spiroplasma spp.was identified. The genus Spiroplasma is related to mycoplasmas, and separated into groups based on their vector association. Spiroplasma group VI is usually associated with ticks as their natural host (Weisburg et al. 1989).

Legionellales

The endosymbionts of F. candida was present in 6% of the samples. The endosymbiont is closely related to members of the Coxiella group, in the order Legionellales (Schabereiter-Gurtner et al. 2003). Present study also identified the species Rickettsiella spp. of the order Legionellales. Its presence in ticks was first discovered in an Australian study dedicated to detection of Rickettsia, Coxiella, and Rickettsiella in native Australian tick species (Vilcins et al. 2009).

DGGE profiling also resulted in identification of bacteria not previously described as tick-infecting bacteria. Serratia marcescens, St. rhizophila, and Bradyrhizobium spp. are all species that have been identified in soil samples. After feeding, ticks drops to the ground and digests the blood meal. On this stage of life, soil bacteria may attach to the outer surface of ticks, but we are not able to ascertain that these bacteria may also be present within the ticks. Although the ticks were rinsed with ethanol and ddH₂O, it is possible that some external soil bacteria are carried over into DNA isolation.

DGGE profiling has proven to be an efficient method to identify tick-infecting bacteria and endosymbionts (Schabereiter-Gurtner et al. 2003). The migration rate of each DGGE band on a gel is somewhat specific to a bacterial genus, but positive identification of DGGE 16S rDNA fragments can only be done by sequencing analysis (Muyzer et al. 1993). Even though some of the species are not yet proven to be clinically important, profiling the complete microbial population in ticks may give a better understanding of the potential ticks display to harbor pathogens and the role they play in dispersion of bacteria and endosymbionts. Weak bands proved to be difficult to cut out of the gel and some were not possible to reamplify, but the majority of DGGE bands were positively identified through sequencing analysis. The bacterial content of ticks would be somewhat defined by the environment and topography of its origin, but the sequencing analysis gave 100% identity to a bacterial strain found in ticks in Italy and 97% identity to a bacterial strain found in native Australian ticks.

Footnotes

Acknowledgments

We would like to thank Stein Erik Solevåg (Aalesund University College), Kristin Bjørdal (Aalesund University College), and Aalesund Veterinary Clinic with Ole Frøland for providing us with live ticks. We would also like to thank scientific assistant Anne Mari Simmones.

Disclosure Statement

No competing financial interest exists.

References

Aguero-Rosenfeld

, Wang

et al. Diagnosis of Lyme borreliosis. Clin Microbiol Rev, 2005; 18:484–509.

Altschul

, Madden

et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 1997; 25:3389–3402.

Butler

, Floyd

et al. Novel mycolic acid-containing bacteria in the family Segniliparaceae fam. nov., including the genus Segniliparus gen. nov., with descriptions of Segniliparus rotundus sp nov and Segniliparus rugosus sp nov. Int J Syst Evol Microbiol, 2005; 55:1615–1624.

Duh

, Petrovec

et al.

Diversity of Babesia infecting European sheep ticks (Ixodes ricinus)

J Clin Microbiol, 2001; 39:3395–3397.

Epis

, Sassera

et al. Midichloria mitochondrii is widespread in hard ticks (Ixodidae) and resides in the mitochondria of phylogenetically diverse species. Parasitology, 2008; 135:485–494.

Ferris

, Muyzer

et al. Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ Microbiol, 1996; 62:340–346.

Gray

. Biology of Ixodes species ticks in relation to tick-borne zoonoses. Wiener Klinische Wochenschrift, 2002; 114:473–478.

Gray

. The biology of Ixodes ticks, with special reference to Ixodes ricinus. Society for Zoonotic Ecology and Epidemiology. 2003. www.zooeco.org. 2010 December 3.

Head

, Saunders

et al. Microbial evolution, diversity, and ecology: a decade of ribosomal RNA analysis of uncultivated microorganisms. Microb Ecol, 1998; 35:1–21.

10.

Hogg

J.C.

, Lehane

. Microfloral diversity of cultured and wild strains of Psoroptes ovis infesting sheep. Parasitology, 2001; 123:441–446.

11.

Kjelland

, Stuen

et al. Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from migratory birds in Southern Norway. Acta Veterinaria Scandinavica, 2010a. 52:59.

12.

Kjelland

, Stuen

et al. Prevalence and genotypes of Borrelia burgdorferi sensu lato infection in Ixodes ricinus ticks in southern Norway. Scand J Infect Dis, 2010b. 42:579–585.

13.

Lindgren

, Tälleklint

et al. Impact of climatic change on the northern latitude limit and population density of the disease-transmitting European tick Ixodes ricinus. Environ Health Perspect, 2000; 108:119–123.

14.

MSIS. MSIS; the Norwegian Surveillance System of Public health: Individual disease; Lyme Borreliosis. 2010. http://www.msis.no. 2010 December 3.

15.

Muyzer

, Dewaal

et al. Profiling of complex microbial-populations by Denaturing Gradient Gel-Electrophoresis analysis of polymerase chain reaction-amplified genes-coding for 16S ribosomal-RNA. Appl Environ Microbiol, 1993; 59:695–700.

16.

Nilsson

, Lukinius

et al. Evidence of Rickettsia spp. infection in Sweden: a clinical, ultrastructural and serological study. APMIS, 2005; 113:126–134.

17.

Sanogo

, Parola

et al. Genetic diversity of bacterial agents detected in ticks removed from asymptomatic patients in northeastern Italy. Rickettsiology: Present Future Dir, 2003; 990:182–190.

18.

Schabereiter-Gurtner

, Lubitz

et al. Application of broad-range 16S rRNA PCR amplification and DGGE fingerprinting for detection of tick-infecting bacteria. J Microbiol Methods, 2003; 52:251–260.

19.

Schabereiter-Gurtner

, Maca

et al. 16S rDNA-based identification of bacteria from conjunctival swabs by PCR and DGGE fingerprinting. Invest Ophthalmol Visual Sci, 2001; 42:1164–1171.

20.

Severinsson

, Jaenson

et al. Detection and prevalence of Anaplasma phagocytophilum and Rickettsia helvetica in Ixodes ricinus ticks in seven study areas in Sweden. Parasites Vectors, 2010; 3:66.

21.

Sparagano

OAE

, Allsopp

MTE

et al. Molecular detection of pathogen DNA in ticks (Acari: Ixodidae): a review. Exp Appl Acarol, 1999; 23:929–960.

22.

Stuen

. Anaplasma Phagocytophilum—the most widespread tick-borne infection in animals in Europe. Vet Res Commun, 2007; 31:79–84.

23.

Swanson

, Neitzel

et al. Coinfections acquired from Ixodes ticks. Clin Microbiol Rev, 2006; 19:708–727.

24.

Tokarz

, Jain

et al. Assessment of Polymicrobial Infections in Ticks in New York State. Vector-Borne Zoonot Dis, 2010; 10:217–221.

25.

Vilcins

IME

, Old

et al. Molecular detection of Rickettsia, Coxiella and Rickettsiella DNA in three native Australian tick species. Exp Appl Acarol, 2009; 49:229–242.

26.

Weisburg

, Tully

et al. A Phylogenetic Analysis of the mycoplasmas—basis for their classification. J Bacteriol, 1989; 171:6455–6467.