Molecular Identification of Borrelia miyamotoi in Ixodes ricinus from Portugal

Introduction

I xodes ricinus is the most widespread and abundant European tick species capable of transmitting pathogens of medical and veterinary importance. These include bacteria such as Borrelia burgdorferi sensu lato (s.l.), considered a prevalent pathogen in these ticks, and causing Lyme borreliosis, Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis, Francisella tularensis causing tularaemia, Rickettsia helvetica and R. monacensis causing spotted fever rickettsiosis; protozoa such as Babesia divergens and B. microti, causal agents of babesiosis, and Candidatus Neoehrlichia mikurensis, responsible for neoehrlichiosis. This tick species has also been associated with the transmission of a multitude of viruses, including tick-borne encephalitis, Louping ill, and Tribec viruses (Medlock et al. 2013).

Other Borrelia spp., such as Borrelia miyamotoi, belonging to the relapsing fever group have recently been detected in I. ricinus ticks. This species was first identified in 1995 by Fukunaga and collaborators in Ixodes persulcatus ticks and in a small mammal (Apodemus argenteus), at Hokkaido, Japan (Fukunaga et al. 1995). Since then, B. miyamotoi has been found in several ticks classified in the Ixodes genus in North America, Europe, and Asia, revealing an extensive geographic distribution (Geller et al. 2012, Cochez et al. 2014, Cosson et al. 2014, Dibernardo et al. 2014, Hansford et al., 2014, Kiewra et al. 2014, Takano et al. 2014).

The aim of the present study was to determine the presence of B. miyamotoi in questing I. ricinus ticks collected throughout Portugal and to evaluate the phylogenetic relatedness of these spirochetes to other species of the relapsing fever group.

Material and Methods

To assess the presence of B. miyamotoi in I. ricinus ticks from Portugal, 12 sites from six districts from the North (Braga and Vila Real), Center (Aveiro, Lisboa and Setúbal) and South (Faro) regions of Portugal were selected (www.mapas-portugal.com/Mapa_Distritos_Portugal.htm). Between May, 2012, and May, 2014, from spring to autumn, ticks were collected by flagging once a month, conserved in 70% ethanol, and identified at the species level using taxonomic keys (Estrada-Peña et al. 2004). For logistic reasons, given the proximity to our laboratory, the majority of ticks were collected in the Lisboa district.

Ticks' genomic DNA was extracted by adding 500 μL of ammonium hydroxide (NH₄OH) to each adult tick, and 100 μL to each immature stage (nymphs). Adult specimens and nymphs were processed individually. The lysates obtained were then stored at −20°C for further use.

Detection of Borrelia DNA was carried out using two different nested PCR protocols. The first protocol targeted the internal transcribed spacer (ITS) region between the 5S and 23S rDNA with the external primers 23SN1 and 23SC1 (amplifying a 320-bp fragment) and inner primers 23SN2 and 5SC (amplifying a 280-bp fragment). The PCR conditions were denaturation at 94.5°C for 1 min, 25 cycles of amplification at 94°C for 30 s, 52°C for 30 s (outer primers) or 55°C for 30 s (inner primers), and 72°C for 1 min, followed by a 5-min extension phase at 72°C.

The second nested PCR targeted the flagellin gene and included a first amplification with the outer primers 132f and 905r (amplifying a 774-bp fragment) and a second amplification using the inner primers 220f and 823r (amplifying a 604-bp fragment). The PCR conditions were denaturation at 94°C for 10 min, 40 cycles of amplification at 94°C for 30 s, 50°C for 45 s (outer primers) or 54°C for 45 s (inner primers), and 72°C for 1 min, followed by a 7-min extension phase at 72°C.

The PCR products were directly sequenced at GATC Biotech AG (Cologne, Germany), and the sequences were compared with those available in the GeneBank/EMBL/DDBJ database using the National Center for Biotechnology Information (NCBI) BLAST program. Additionally, phylogenetic trees were constructed using the neighbor-joining (NJ) clustering algorithm and a matrix of corrected genetic distances or inferred using the maximum likelihood (ML) criterion, starting from a NJ tree, using Mega 5.0 (www.megasoftware.net/). Correction of distances (for NJ analysis) and phylogenetic tree search based on the ML optimization criterion were carried out using the Tamura–Nei+Γ model, as suggested by Mega 5.0. In both cases, the topology of the trees obtained was assessed by bootstrapping, with 1000 random resamplings or the original sequence data. Phylogenetic analysis also included the construction of a tree following a Bayesian approach using the MrBayes v3.0b4 software (http://mrbayes.sourceforge.net/).

Results

A total of 640 I. ricinus ticks were collected and analyzed according to the following distribution by district: Braga, two adults; Vila Real, 16 adults; Aveiro, three adults; Lisboa, 447 nymphs and 118 adults; Setúbal, 19 adults; and Faro, 35 adults (Table 1).

Analysis of nucleotide sequences of the fla gene amplicons obtained from a nymph collected in the district of Lisboa (Tapada Nacional de Mafra) and deposited in the GenBank/EMBL/DDBJ databases under accession number LC006092 revealed 99% identity with several B. miyamotoi reference sequences (BLASTn). Phylogenetic trees constructed on the basis of the analysis of nucleotide sequence alignments of fla coding sequences (using multiple Borrelia spp. as references) clearly indicate that LC006092 (indicated by the arrow in Fig. 1), clustered with the group of relapsing fever–like Borrelia within the European sequence lineage (supported by bootstrap analysis of the NJ and ML trees). This lineage, found inside the B. miyamotoi monophyletic group, segregates from the Japan/Russia–Asia, and US monophyletic clusters.

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FIG. 1.

Borrelia phylogenetic analysis of fla nucleotide sequences (372 bp). B. miyamotoi clusters are denoted by species, accession number, and country of origin. At specific branch nodes bootstrap values (b; >75%) or posterior probabilities (pp; >0.90) are indicated (b_NJ/b_ML/pp_Bayes), “–” indicating either bootstrap values or posterior probabilities below the indicated limits of significance. The arrow indicates the position of the B. miyamotoi, and the asterisks indicate the positions of other B.b.s.l. species, all described in this article. The size bar indicates 5% divergence.

Finally, B. burgdorferi s.l. DNA was also amplified by the two nested PCR protocols, leading to an overall prevalence of 11% (71/640). These positive samples were collected from Vila Real, Lisboa, Setúbal, and Faro districts, and about 50% (33/71) of them were sequenced, B. lusitaniae being the predominant genospecies (18/33).

Discussion

In the current study, B. miyamotoi DNA was detected for the first time in Portugal in a questing nymph collected in Tapada Nacional de Mafra, in the outskirts of Lisboa. However, so far, no evidence has ever been found to indicate that this putative pathogen may be responsible for causing human clinical cases in the country. Nevertheless, studies from Europe have suggested that B. miyamotoi may cause human disease, because B. miyamotoi–specific antibodies and DNA have already been detected in patients presenting symptoms like fever, headache, and muscle aches, typical of tick-borne relapsing fever.

Current studies have shown a crucial role of several vector species in the spread of B. miyamotoi. The typical vectors of tick-borne relapsing fever spirochetes are soft ticks from the genus Ornithodoros, therefore the low prevalence of B. miyamotoi, revealed by this and other studies may indicate a recent emergence and spread of these bacteria in hard ticks. It is important to highlight that ticks could simultaneously transmit B. miyamotoi and Lyme disease spirochetes to humans, supporting the risk of possible co-infections to occur, with potential strong implications for public health. Therefore, further investigations are needed to understand the competence of I. ricinus for B. miyamotoi and predict a possible spread of this group of spirochetes in Portugal. This will, in turn, contribute to the determination of the risk of exposure (for humans) in areas where Ixodes ticks live.