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Update on tick-borne rickettsioses in mainland Portugal: emerging threats and potential vectors

Parasites & Vectors volume 17, Article number: 538 (2024) Cite this article

Abstract

Background

Tick-borne rickettsioses (TBR) are emerging, neglected, zoonoses, caused by intracellular α-proteobacteria of the genus Rickettsia, that pose a growing public health concern. The aim of the present study was to evaluate rickettsial infections in questing ticks collected from four different ecological areas in mainland Portugal.

Methods

Over a two-year period, a total of 707 questing ticks were collected. Individual adult ticks and pooled nymphs were submitted to DNA extraction, followed by qPCR assays targeting the gltA rickettsial gene. Positive samples were then submitted to conventional PCR targeting the gltA and the ompA genes for phylogenetic analysis.

Results

In total, eight tick species were identified: Dermacentor marginatus, Haemaphysalis inermis, Haemaphysalis punctata, Hyalomma lusitanicum, Ixodes frontalis, Ixodes ricinus, Rhipicephalus pusillus, and Rhipicephalus sanguineus sensu lato. Additionally, rickettsial infection was associated with seven of these species, with I. frontalis being the exception. Notably, the prevalence of Rickettsia spp. was 26.35%, with phylogenetic validation confirming infections with R. helvetica, R. massiliae, R. monacensis, Candidatus R. rioja, and R. slovaca.

Conclusions

The present study highlights the necessity for ongoing surveillance to map and monitor both questing and feeding ticks, along with their vertebrate hosts. Effective control strategies are of utmost importance to mitigate the escalating threat of TBR. Additionally, the present study provides valuable epidemiological insights into TBR in Portugal, including the identification of R. slovaca infecting I. ricinus - an unconventional tick-pathogen relationship - and the first report of Candidatus R. rioja infecting D. marginatus in Portugal. In conclusion, this study contributes with valuable data regarding epidemiological results on ticks and TBR circulating in Portugal, emphasizing the importance of proactive measures to address this emerging public health challenge.

Graphical abstract

Background

Tick-borne rickettsioses (TBR) are emerging, neglected zoonoses caused by obligate intracellular α-proteobacteria that belong to the spotted-fever group (SFG) of the genus Rickettsia [1]. Mediterranean Spotted Fever (MSF) is the most prevalent TBR in Europe, while tick-borne lymphadenopathy (TIBOLA) and Mediterranean spotted fever-like (MSF-like) diseases are the other TBRs in Europe that also pose significant threats to human health [2, 3]. MSF is caused by Rickettsia conorii, which has four subspecies, with both R. conorii conorii and R. conorii israelensis found in the Iberian Peninsula [1]. Rhipicephalus sanguineus sensu lato ticks not only are the primary vector but also act as reservoirs due to transstadial and transovarial transmission, with dogs potentially contributing to the persistence of MSF in the environment [1,2,3]. This TBR is characterized by fever, rash and the presence of an eschar of inoculation at the tick bite site. Most cases occur in summer, aligning with the peak activity of R. sanguineus s.l. [1,2,3]. TIBOLA is primarily caused by Rickettsia slovaca, R. raoultii and Candidatus R. rioja, while Rickettsia helvetica, R. massiliae and R. monacensis are known agents of MSF-like disease [3]. The first molecularly confirmed human case of TIBOLA, caused by R. slovaca, was reported in 1997 [4]. Since then, R. raoultii and Candidatus R. rioja have been not only associated with human cases of TIBOLA but have also been isolated from ticks removed from patients with this syndrome [5, 6]. Dermacentor ticks, specifically D. marginatus and D. reticulatus, are natural vectors of these rickettsial pathogens [2,3,4]. TIBOLA syndrome is characterized by the presence of neck lymphadenopathy, often linked to Dermacentor spp. bites on the human scalp. Other symptoms include fever, facial edema, alopecia around the tick bite site and, rarely, macular lesions on the limbs. These symptoms are the same regardless of the species of Rickettsia causing the infection [3]. Through the years, the knowledge about TIBOLA has expanded, and other terms such as “Dermacentor-borne necrosis erythema lymphadenopathy” (DEBONEL) and “scalp eschar and neck lymphadenopathy” (SENLAT) are also used to describe similar clinical manifestations or syndromes [2, 7].

MSF-like disease can be caused by different tick-borne rickettsiae within the SFG-rickettsiae, such as R. helvetica, R. massiliae and R. monancensis [3, 8]. Originally identified as the “Swiss agent” in 1979, and initially characterized as having unknown pathogenicity, R. helvetica was first isolated from Ixodes ricinus ticks in Switzerland [9]. Besides I. ricinus, Ixodes ovatus, I. persulcatus and I. monospinus are known vectors of this SFG-rickettsiae [10]. Additionally, I. ricinus is the natural reservoir of R. helvetica, as both transstadial and transovarial transmissions have been demonstrated in this tick species [10]. Until 1999, human pathogenicity was not confirmed; however, two fatal human cases were associated with this rickettsial species, in which Swedish patients showed perimyocarditis and vascular complications [11].

Another SFG rickettsia that causes MSF-like disease is R. massiliae [8]. It was initially isolated from Rhipicephalus turanicus and R. sanguineus s.l., both in France [12]. Later, it was confirmed that R. turanicus can transmit this rickettsial agent transovarially to its offspring [13]. Besides Rhipicephalus turanicus, R. muhsamae, R. lunulatus and R. sulcatus are additional known vectors of R. massiliae [10]. This pathogen was first isolated from humans in 1985 but was only identified in 2005, infecting an Italian patient displaying the symptom triad: fever, rash and necrotic eschar [14].

First isolated and identified in Germany infecting I. ricinus ticks, R. monacensis is one of several SFG-rickettsiae agents that cause MSF-like disease [15]. Although human cases are seldom reported, most symptoms are those typical of a rickettsiosis infection, including fever, headache and rash on the trunk and limbs [3]. Although I. ricinus has not yet been recognized as the natural vector of R. monacensis, previous studies have shown a high prevalence of this rickettsial agent infecting this tick species [16,17,18].

In Portugal, rickettsial agents responsible for TIBOLA and MSF-like disease have not only been reported in human cases, leading to hospitalization [19, 20], but have also been identified infecting their respective known vectors: Dermacentor spp. [21], I. ricinus [21, 22] and R. turanicus [23]. Additionally, putative vectors have been identified, with R. massiliae infecting R. sanguineus s.l. [24,25,26] and R. monacensis infecting I. ricinus [17, 27]. It is important to remember that ticks in southwestern Europe identified as R. turanicus based on morphological characteristics are genetically indistinguishable from R. sanguineus s.l., pointing to a high level of morphological polymorphism in this group [28,29,30]. Millán and colleagues [28] recently proposed and described Rhipicephalus hibericus as a new species in the R. sanguineus s.l. group, which would correspond to the tick species previously identified in this region as R. turanicus. Therefore, considering that the tick population and TBR are directly intertwined, effective campaigns concerning tick surveillance are of utmost importance for mapping and monitoring the distribution of these arthropods and the rickettsial agents they carry or can even transmit. Continuous surveillance not only helps in tracking threats to human and animal health but also in identifying new and emerging rickettsial agents and their tick association or putative vectors. In this context, the present study aims to evaluate rickettsial infections in questing ticks collected from four different ecological areas in mainland Portugal and provides the first molecular characterization of R. slovaca infecting I. ricinus tick species in mainland Portugal.

Methods

Study sites, tick sampling and morphological identification

Questing ticks were collected from four ecological areas in mainland Portugal from August 2018 until May 2021. The studied areas included Grândola (38º06′19.6"N, 8º33′59.7"W) with tick collections in February 2019, Mata Nacional do Choupal (40.2223ºN, 8.4439ºW) where sampling was conducted in June 2019, Mata do Bussaco (40.3771ºN, 8.3669ºW) where ticks were also collected in June 2019 and Tapada Nacional de Mafra (TNM) (38.9646ºN, 9.3027ºW) where tick collection occurred in December 2019 and May 2021 (Fig. 1), as previously described [31]. Each area was visited once, except TNM, which was visited twice. All ecological areas were selected based on previous reports that confirmed the presence of ticks and potential circulation of TBR [32]. During field campaigns, up to 20 tick specimens, collected by dragging or flagging methods, were deposited in a single 15-ml tube. To prevent tick dehydration, some green vegetation was placed inside the tube. All tick specimens were kept refrigerated in a box with ice packs until being brought to the laboratory for further analysis. Taxonomic classification of all specimens was carried out to species level based on morphological characters according to previously published taxonomic keys [33] using a Motic SMZ171 stereomicroscope.

Fig. 1
figure 1

Geographical distribution of tick species in mainland Portugal from August 2018 until May 2021

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Tick nucleic acid extraction and detection of PCR inhibitors

Ticks were rinsed in pH 7.0 sterile phosphate-buffered saline solution (PBS) and pooled according to species, collection site, development stage and sex. All adult specimens were placed individually in a sterile 1.5-ml tube, while nymphs were pooled in groups of up to five specimens. Each sample was frozen in liquid nitrogen and crushed with sterile pestles. Genomic DNA (gDNA) was extracted using TRIzoltm reagent (ThermoFisher Scientific, Carlsbad, CA), according to the manufacturer's protocol for isolation of nucleic acids from tissues. gDNA was resuspended in nuclease-free water, and concentrations were measured using a Qubit4 fluorometer (ThermoFisher Scientific, Carlsbad, CA). Afterwards, gDNA was stored at – 20 ºC until being used as a template for polymerase chain reaction (PCR) amplifications. Prior to any TBR screening, approximately 20% of gDNA samples were randomly selected to amplify the 18SrDNA tick gene fragment to confirm the absence of PCR inhibitors [34].

Molecular detection of rickettsial DNA

Samples were screened targeting a 74-bp fragment of the gltA rickettsial gene by probe-based real-time PCR (qPCR). To amplify this rickettsial gene fragment, reactions were prepared in triplicate in 96-well plates using primers CS-F and CS-R with probe CS-P (Table 1), as previously described [35]. The positive control for qPCR reactions consisted of an eight-fold serial dilution of a synthesized fragment of the Rickettsia rickettsii gltA gene. This fragment, encompassing a 74-bp sequence (GenBank accession no. U59729), was synthesized as a gBlocks® gene fragment by Integrated DNA Technologies (IDT, Leuven, Belgium). Samples were considered positive in these qPCR screening assays if at least two or more of their replicates yielded the expected amplification result. To confirm positive results obtained from the qPCR screening assays and for phylogenetic purposes, samples were subsequently tested by conventional PCR for both the outer membrane protein A gene (ompA) and gltA gene [36] Table 1.

Table 1 Primers used for amplification of rickettsial gene fragments
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Statistical analysis

The standard prevalence of adult ticks was calculated based on individually tested samples. The prevalence of rickettsial infection in nymphs was estimated using the minimum infection rate (MIR) [37] and adjusted for pools of varying sizes and individual samples. MIR was calculated by dividing the number of positive pools, confirmed by qPCR when two or more replicates produced the expected amplification, by the total number of ticks tested. The overall prevalence of infection, combining adult ticks (tested individually) and nymphs (tested in pools using the MIR), was determined by weighting the prevalence of each group according to the number of ticks tested in each category. Here, the total number of ticks tested was calculated by summing the number of ticks across all pools and individual samples to account for different pool sizes and individual samples.

DNA sequencing and phylogenetic analysis

All the gltA and ompA amplicons, obtained by conventional PCR, were purified using the NZyGelpure kit (NZYtech, Lisbon, Portugal) according to the user guide protocol. Afterwards, they were sent to StabVida (Caparica, Portugal) for Sanger sequencing. All obtained sequences were aligned and compared to those already deposited at the NCBI (National Center for Biotechnology Information) nucleotide database (https://blast.ncbi.nlm.nih.gov/). Obtained sequences corresponding to the citrate synthase gene (gltA) were deposited at GenBank under accession numbers PP935122–PP935137, while those corresponding to the outer membrane protein A gene (ompA) were assigned accession numbers PP935138-PP935154.

Datasets were created with short reads obtained as a result of Sanger sequencing, reference sequences previously deposited in GenBank and sequences returned from Megablast research [Nucleotide BLAST: Search nucleotide databases using a nucleotide query (nih.gov)], which demonstrated the best “query cover” and “identity percentage” rates, always from different studies. All sequences in each gene-specific dataset were aligned using MAFFT (https://mafft.cbrc.jp/alignment/server/), and the obtained multiple sequence alignments were edited GBlocks 0.91b (http://phylogeny.lirmm.fr/phylo_cgi/one_task.cgi?task_type=gblocks). Two different approaches were used to construct the phylogenetic trees to minimize the biases that may be introduced by the methods used. At first, phylogenetic trees were constructed based on neighbor-joining (NJ) analysis with genetic distance matrixes corrected with Kimura two-parameter (K2P) substitution model using MEGA v.10 [38]. Afterwards, another set of phylogenetic trees was constructed based on the maximum-likelihood (ML) optimization criterion, using the best-fit model for each sequence dataset, according to BIC (Bayesian Information Criterion), as defined by IQ-TREE web server model selection tool (http://iqtree.cibiv.univie.ac.at/). In both cases, the topological soundness of the obtained trees was assessed by bootstrapping with 1000 resampling of the original sequence data. The graphic representation of phylogenetic trees selected for the current study was that from the NJ analysis once the results of both phylogenetic analysis approaches (NJ and ML) demonstrated identical topological characteristics, i.e. the same nodes, branches and bootstrap values.

Results

A total of 707 questing ticks (538 nymphs, 76 males, 93 females) were gathered during field collection, attaining a total of eight species: D. marginatus (n = 3; 0.42%), Haemaphysalis inermis (n = 31; 4.38%), Haemaphysalis punctata (n = 39; 5.52%), Hyalomma lusitanicum (n = 3; 0.42%), Ixodes frontalis (n = 1; 0.15%), I. ricinus (n = 603; 85.29%), Rhipicephalus pusillus (n = 16; 2.26%) and R. sanguineus s.l. (n = 11; 1.56%). Among the I. ricinus specimens collected, there were 86 adults (n = 52 males and n = 34 females) and 538 nymphs (Table 2).

Table 2 Prevalence and phylogenetic confirmation of tick-borne rickettsiae in collected tick species across different ecological areas
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The overall prevalence of rickettsial infection in the 277 samples where the presence of rickettsial DNA was investigated by qPCR assays (for detection of the gltA gene) was 10.31%. Among nymphs, the prevalence determined by MIR was 4.64%, while in individually tested adult samples, the obtained prevalence was 28.4% (Table 2). Moreover, all tick species, except for I. frontalis collected from Mata do Bussaco, yielded positive amplification results.

Phylogenetic analysis based on the citrate synthase (gltA) gene revealed that R. helvetica infected one male (PP935134) and one pool of nymphs (PP935135) from I. ricinus ticks. On the other hand, R. massiliae was found in association with two R. sanguineus s.l. female specimens (PP935136, PP935137), while R. monacensis was found exclusively in I. ricinus ticks. This included six males (PP935122, PP935123, PP935124, PP935125, PP935126, PP935128), one female (PP935127) and five pools of nymphs (PP935129, PP935130, PP935131, PP935132, PP935133) (Table 2) (Fig. 2).

Fig. 2
figure 2

Phylogenetic tree constructed and displayed by the neighbor-joining method with Kimura’s two-parameter evolution model from partial sequences of the gltA gene. The same topographic representation was obtained by the maximum likelihood method with transition-intermediary model (TIM) + F evolution model from partial sequences of the gltA gene according to the BIC (Bayesian information criterion), as defined by IQ-TREE web server model selection. Bootstrap values were obtained from 1000 replications and are indicated at the nodes of the respective branches (only values ≥ 75%). All Rickettsia spp. sequences obtained during this work are highlighted with a triangle and its respective accession, both in bold format, and clone names are underlined

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Regarding the phylogenetic analysis based on the outer membrane protein A (ompA) gene, the findings were as follows: Candidatus R. rioja infecting one female of D. marginatus (PP935154); R. slovaca infecting one pool of I. ricinus nymphs (PP935153); R. monacensis was detected exclusively in I. ricinus ticks, including one female (PP935144), six males (PP935138, PP935139, PP935140, PP935141, PP935142, PP935143) and eight pools of nymphs (PP935145, PP935146, PP935147, PP935148, PP935149, PP935150, PP935151, PP935152) (Table 2) (Fig. 3).

Fig. 3
figure 3

Phylogenetic tree constructed and displayed by the neighbor-joining method with Kimura’s two-parameter evolution model from partial sequences of the ompA gene. The same topographic representation was obtained by the maximum likelihood method with Tamura-Nei (TNe) evolution model from partial sequences of the ompA gene according to the BIC (Bayesian information criterion), as defined by IQ-TREE web server model selection. Bootstrap values were obtained from 1000 replications and are indicated at the nodes of the respective branches (only values ≥ 75%). All Rickettsia spp. sequences obtained during this work are highlighted with a triangle and its respective accession, both in bold format, and clone names are underlined

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Discussion

The present study provides molecular evidence for the circulation of several tick-borne rickettsiae, including, R. helvetica, R. massiliae, R. monacensis, Candidatus R. rioja and R. slovaca, in Ixodidae questing ticks collected from the field in mainland Portugal. It is important to emphasize that the overall prevalence of 10.31% was calculated using the minimum infection rate (MIR) for pooled tick samples (composed exclusively of nymphs), a conservative approach that assumes that there is only one infected tick per positive pool, even if multiple ticks may actually be infected. In contrast, the prevalence for adult ticks, which were individually analyzed, was determined through standard methods. Therefore, the estimated prevalence of Rickettsia spp. in ticks may be underestimated, and this value should be regarded as a lower bound for the actual infection rate [37]. Notably, the qPCR assay used in this study, targeting the gltA gene, was a probe-based method recognized for its high specificity and sensitivity [35]. This enhances confidence in the accuracy of the qPCR-positive results, even in cases where conventional PCR assays failed to amplify larger fragments of the gltA or ompA genes [35]. The inability of conventional PCR to confirm some qPCR-positive results can likely be attributed to its lower sensitivity, particularly in samples with degraded DNA or low target abundance. To ensure a comprehensive representation of Rickettsia spp.’s prevalence, all qPCR-positive samples were included in the calculations. This approach leverages the high sensitivity and specificity of the probe-based qPCR assay to provide a reliable estimation of Rickettsia spp. prevalence, while acknowledging the limitations of conventional PCR and the potential for minor overestimation in rare cases of nonspecific amplification [39]. The present report discloses the infection of I. ricinus with R. helvetica, one of the SFG-rickettsiae that causes MSF-like disease, via the association of species-specific gltA sequences obtained in the course of this work (PP935134-PP935135) to a topologically stable monophyletic group in a gene-specific phylogenetic tree (Fig. 2). Although there are no records of human MSF-like disease caused by R. helvetica in Portugal, previous reports have shown its presence in its known natural vector and reservoir, I. ricinus [22, 40]. Moreover, R. helvetica has also been detected infecting lizards (Teira dugesii) and I. ricinus ticks removed from the latter, suggesting that this vertebrate might act as a potential reservoir, maintaining R. helvetica throughout the enzootic cycle [22]. Additionally, Ixodes ventalloi has also been reported to be infected with R. helvetica in Portugal [41] although the competence of I. ventalloi as a vector for this bacterium remains unknown [42].

Two gltA gene sequences (PP935136, PP935137), obtained from infected R. sanguineus s.l. females, showed infection by R. massiliae forming a well-supported clade in the phylogenetic tree, indicating its clear genetic distinction within the SFG rickettsiae, another causative agent of MSF-like disease (Fig. 2). These outcomes align with previous studies conducted in Portugal [17, 24,25,26]. Additionally, R. massiliae has been reported infecting R. turanicus [41], a tick species recognized as both a vector and a reservoir for this SFG-rickettsiae [13]. While no human disease cases caused by R. massiliae have been reported in Portugal, infections have been documented in dogs (Canis lupus familiaris) [26], sheep (Ovis aries) [24] and ticks collected from these animals. Although not all infected dogs exhibited clinical signs, when they occurred, those most frequently observed were anorexia and jaundice. On the other hand, the most prevalent hematological abnormalities were moderate to mild thrombocytopenia, followed by monocytosis [26]. Conversely, none of the sheep exhibited relevant clinical signs in response to rickettsial infection [24]. As sheep are parasitized by the same tick species that accidentally parasitize humans [43], and since these domestic animals do not exhibit signs of a rickettsial infection, these intertwined scenarios not only elevate the transmission risk of R. massiliae to humans but also favor the circulation, as well as maintenance, of a possible interspecific domestic cycle of this pathogen.

DNA fragments of R. monacensis from both gltA or ompA were detected exclusively in I. ricinus ticks in a well-supported clade, confirming its status as a distinct species and common presence in these ticks (Figs. 2 and 3). These results align with previous studies in mainland Portugal and Madeira Island, which have shown not only the presence of this pathogen in questing I. ricinus [17, 27] but also in lizards (T. dugesii) and their parasitizing ticks [22]. Recently, the first confirmed human case of MSF-like disease caused by R. monacensis was reported in Portugal. The patient showed fever, rash, myalgia, fatigue and anorexia [19]. Moreover, an engorged female I. ricinus tick was removed from this patient, and both the tick and the eschar biopsy sample confirmed infection by this rickettsial agent [19].

Concerning the ompA rickettsial gene, the present study also confirms the presence of R. slovaca and Candidatus R. rioja in a pooled sample of I. ricinus nymphs and a D. marginatus female, respectively. Both, causative agents of TIBOLA reported here show that their phylogenetic placement is robust, confirming a clear genetic distinction from other rickettsial agents. Initially, R. slovaca was identified as the only causative agent of TIBOLA involving D. marginatus tick species [4]. Over the years, not only has D. marginatus been considered the natural vector of R. slovaca, but also D. reticulatus has been incriminated as a potential vector [2, 3]. Nevertheless, the present study reports I. ricinus infected with R. slovaca (PP935153), an uncommon but increasingly frequent finding, especially in areas where this SFG-rickettsiae has been reported infecting not only both questing and feeding ticks but also their respective vertebrate hosts [16, 44, 45]. Furthermore, the pooled sample, consisting of five I. ricinus nymphs, was collected at TNM, a forest-like region with rich fauna featuring large mammals, such as the red deer (Cervus elaphus), medium-sized mammals, like the European fallow deer (Dama dama), and smaller ones, such as foxes (Vulpes vulpes) and wild boar (Sus scrofa) (BNP-Tapada de Mafra. Available online: http://bibliografia.bnportugal.gov.pt/bnp/bnp.exe/registo?1687264). In this context, when different vertebrate hosts sympatrically coexist in such a rich environment, this complex and dynamic scenario not only facilitates the parasitism of different hosts by the same tick species but also contributes to the maintenance of the enzootic/sylvatic cycle of Rickettsia spp. within this region. Although scarce in Portugal, there have been at least three previously reported cases of human TIBOLA, all caused by R. slovaca [20].

The other causative agent of TIBOLA detected in this study was Candidatus R. rioja, infecting one female D. marginatus specimen. Although there are no records of humans, or ticks infected with this rickettsial agent in Portugal, its association with ticks has already been confirmed in Spain, where a patient exhibiting signs of TIBOLA syndrome had a D. marginatus tick attached to her scalp, where infection with Candidatus R. rioja was later confirmed [5]. Later, a biological sample from the same patient confirmed the first TIBOLA/DEBONEL/SENLAT human case caused by Candidatus R. rioja [6]. Most recently, once again in neighbouring Spain, Candidatus R. rioja was not only identified as the most frequent causative rickettsial agent of TIBOLA [46] but also, in two specific regions bordering Portugal, this SFG-rickettsiae was detected in both wild ungulates (C. elaphus and S. scrofa) and D. marginatus ticks parasitizing them [47]. The scenario presented in these regions on the opposite side of the Portuguese border raises some concern regarding their possible circulation within the Portuguese territory, despite the apparent low prevalence of Rickettsia sp. infection in wild animals, as these may support their maintenance in D. marginatus. Indeed, this scenario could potentially be extended to any of the regions within the Portuguese territory where D. marginatus, C. elaphus and S. scrofa have been found to coexist [47].

Conclusions

The present study provides molecular characterization of several tick-borne rickettsiae circulating in Portugal, including R. helvetica, R. massiliae, R. monacensis, Candidatus R. rioja and R. slovaca, detected in naturally infected Ixodidae ticks. The findings regarding TBR that cause MSF-like disease, specifically, R. helvetica, R. massiliae and R. monacensis, confirm their presence in their respective vectors and reservoirs. Additionally, the detection in this work of Candidatus R. rioja and R. slovaca, two of the causative agents of TIBOLA, not only confirms their natural presence in association with their natural vector (D. marginatus) but also confirms the latter in an unusual interspecific relationship with an anthropophilic tick such as I. ricinus.

Overall, these outcomes highlight the need for a continued surveillance program and the implementation of effective prevention and control strategies to address the emerging threat of TBR in Portugal. Future studies should focus on the vector competence of tick species, particularly those associated with unusual TBR, as well as the ecological dynamics that influence the maintenance and transmission of rickettsial agents within enzootic or sylvatic cycles. Additionally, the collection of anthropophilic ticks is essential, as it provides valuable data for mapping and monitoring eco-epidemiological changes. This comprehensive data collection will enhance the development of predictive risk models, offering physicians updated information on the epidemiological situation and supporting the One Health approach.

To the best of our knowledge, this is the first report of R. slovaca infecting I. ricinus and the first detection of Candidatus R. rioja infecting D. marginatus, its natural vector, in Portugal.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

The authors acknowledge the financial support from Fundação para a Ciência e a Tecnologia (FCT) through the PhD scholarship 2022.14376.BD awarded to Leonardo Moerbeck. Additionally, we deeply appreciate Dr. Ana Sofia Santos for her invaluable support, manuscript review and expertise in tick surveillance during the field campaigns. We also extend our gratitude to the administrative and technical staff of Tapada Nacional de Mafra and Fundação Mata do Bussaco. Finally, we acknowledge Fundação para a Ciência e a Tecnologia (FCT) for funding GHTM-UID/04413/2020 and LA-REAL–LA/P/0117/2020.

Funding

L. M. is a recipient of a PhD grant supported by Fundação para a Ciência e a Tecnologia (FCT), under reference 2022.14376.BD.

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Authors and Affiliations

  1. Global Health and Tropical Medicine- Institute of Hygiene and Tropical Medicine, NOVA University of Lisbon, Rua da Junqueira 100, 1349-008, Lisbon, Portugal

    Leonardo Moerbeck, Ricardo Parreira, Gonçalo Seixas, Rita Velez, Ana Domingos & Sandra Antunes

  2. Instituto de Higiene e Medicina Tropical, Universidade NOVA de Lisbon, Rua da Junqueira 100, 1349-008, Lisboa, Portugal

    Leonardo Moerbeck, Ricardo Parreira, Gonçalo Seixas, Rita Velez, Ana Domingos & Sandra Antunes

  3. Associate Lab in Translation and Innovation, Towards Global Health, LA-REAL, Rua da Junqueira 100, 1349-008, Lisbon, Portugal

    Ricardo Parreira, Ana Domingos & Sandra Antunes

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  1. Leonardo Moerbeck
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Contributions

Conceptualization, L.M., A.D. and S.A.; analysis, L.M., R.P. and S.A.; interpretation of data, L.M., R.P., G.S., R.V. and S.A.; writing—original draft preparation, L.M. and S.A.; writing—review and editing, L.M., R.P., G.S., R.V., A.D. and S.A.; supervision, S.A. and A.D.; funding acquisition, L.M., A.D. and S.A. All authors have reviewed and agreed to the published version of the manuscript.

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Correspondence to Leonardo Moerbeck or Sandra Antunes.

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Moerbeck, L., Parreira, R., Seixas, G. et al. Update on tick-borne rickettsioses in mainland Portugal: emerging threats and potential vectors. Parasites Vectors 17, 538 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13071-024-06627-5

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Parasites & Vectors

ISSN: 1756-3305

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