Tick-borne pathogens in ticks from urban and suburban areas of north-western Spain: importance of Ixodes frontalis harbouring zoonotic pathogens

ORIGINAL ARTICLE Tick-borne pathogens in ticks from urban and suburban areas of north-western Spain: Importance of Ixodes frontalis harbouring zoonotic pathogens S. Remesar | R. Matute | P. Díaz | N. Martínez-Calabuig | A. Prieto | J. M. Díaz-Cao | G. L opez-Lorenzo | G. Fernández | C. L opez | R. Panadero | P. Díez-Baños | P. Morrondo | D. García-Dios Investigaci on en Sanidad Animal: Galicia (Grupo INVESAGA), Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain Correspondence P. Díaz, Facultade de Veterinaria, Enfermedades Parasitarias, Pabell on I, Planta Baja, Campus Universitario s/n, 27002 Lugo, Spain. Email: [email protected] Funding information Xunta de Galicia, Grant/Award Number: GRC2019/04 Abstract To identify the questing tick populations in urban and suburban areas from the city of Lugo (NW Spain), ticks were collected monthly by flagging. The presence of Borrelia spp., Rickettsia spp. and Anaplasma phagocytophilum also was determined by polymerase chain reaction (PCR) and sequence analysis. Overall, 342 questing ticks were collected; the tick abundance was higher in suburban (95.9%) than in urban areas (4.1%). Ixodes frontalis was the most abundant (86.5%); 88.5% were larvae, 11.1% nymphs and 0.3% adults. All development stages of I. ricinus (7.3%) and adults of Rhipicephalus sanguineus sensu lato (5.8%) and Dermacentor reticulatus (0.3%) were found. Rickettsia spp. (31.9%) was more prevalent than Borrelia spp. (2.7%); no ticks were positive to A. phagocytophilum. Six Rickettsia species were identified (R. slovaca,R. monacensis,R. massiliae,R. raoultii,R. sibirica subsp. mongolitimonae and R. aeschielmanii); Candidatus Rickettsia rioja and two novel Rickettsia species also were detected. In addition, Borrelia turdi (1.8%) and B. valaisiana (0.9%) were identified in Ixodes ticks. This is the first report of R. slovaca in R. sanguineus s.l. and of R. monacensis,R. raoultii,R. slovaca, R. sibirica subsp. mongolitimonae and Ca. R. rioja in I. frontalis. Since most of the pathogens detected are zoonotic, their presence in these areas may have implications for public health. KEYWORDS Anaplasma phagocytophilum,Borrelia,Dermacentor,Ixodes,Rhipicephalus,Rickettsia, urban areas INTRODUCTION Ticks are one of the most important vectors of pathogens in the northern hemisphere (Jongejan & Uilenberg, 2004). In the last decades, the incidence of tick-borne diseases has increased posing important economic and medical consequences (Lippi et al., 2021). Lyme borreliosis (LB), tick-borne encephalitis and rickettsiosis are considered the most prevalent tick-borne diseases in Europe (EstradaPeña et al., 2018; Oteo & Portillo, 2012). In addition, some tick-borne pathogens are considered emergent such as Anaplasma phagocytophilum, the causative agent of the granulocytic anaplasmosis (Stuen et al., 2013). These pathogens circulate in complex cycles involving different tick and vertebrate species, which can have different roles in the maintenance, transmission and dispersion of the pathogens (Estrada-Peña et al., 2018). The causative agents of LB are spirochetes within the group Borrelia burgdorferi sensu lato (s.l.) (Baranton et al., 1992). Among them, three genospecies are primarily responsible for LB in Europe, namely Received: 14 July 2022 Accepted: 26 February 2023 DOI: 10.1111/mve.12648 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2023 The Authors. Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society. Med Vet Entomol. 2023;1–12. wileyonlinelibrary.com/journal/mve 1 B. afzelii,B. garinii and B. burgdorferi sensu stricto (s.s.) (Margos et al., 2011). The pathogenicity of other genospecies such as B. valaisiana,B. lusitaniae and B. spielmani remains unknown (EstradaPeña et al., 2018). In Europe, Ixodes ricinus is their major vector (Margos et al., 2009), although other Ixodes species might be important for the maintenance of this pathogen in animal-tick-animal cycles (Estrada-Peña et al., 2017; Heylen et al., 2014). It has been suggested that transovarial transmission is rather inefficient for B. burgdorferi s.l. in the tick or of low epidemiological importance (Hauck et al., 2020); thus, ticks are not considered reservoirs of these pathogens, a role mainly played by vertebrate species (Estrada-Peña et al., 2018). Most cases of rickettsiosis diagnosed in Europe are caused by spotted fever group Rickettsia species (Parola & Raoult, 2001). In this way, Rickettsia conorii subsp. conorii is the main aetiological agent of the Mediterranean Spotted Fever (MSF), endemic in southern areas; R. slovaca and R. raoultii are associated with the Dermacentor-Borne Necrosis Erythema Lymphadenopathy (DEBONEL), which is the second most prevalent rickettsiosis in Europe (Oteo & Portillo, 2012). Rhipicephalus sanguineus sensu lato (s.l.) and Dermacentor spp., respectively, are considered the main vectors, reservoirs and amplifying hosts of the Rickettsia species involved in both syndromes (Parola & Raoult, 2001). However, other tick species such as I. ricinus are recognized vectors and reservoirs of other less-known rickettsiosis such as MSF-like syndrome caused by R. helvetica and R. monacensis (Oteo & Portillo, 2012). Although LB and SF rickettsiosis are the most common tick-borne diseases in Spain, the impact of other tick-borne pathogens such as A. phagocytophilum should also be considered due its potential pathogenicity for human and other animals (Stuen et al., 2013). As no transovarial transmission of A. phagocytophilum has been demonstrated in I. ricinus, its main vector in Europe (Stuen et al., 2013), vertebrate reservoir hosts are necessary for maintaining its sylvatic cycle (Di Domenico et al., 2016). Tick-borne pathogens circulate in enzootic cycles, involving different tick species and suitable animal hosts (Parola & Raoult, 2001). However, other variables such as climate and social factors are involved in the epidemiology of these pathogens (Estrada-Peña et al., 2012). For this reason, effective surveillance for monitoring those tick-borne pathogens affecting humans and other animals is needed (Parola & Raoult, 2001). It is worth noting that most studies on questing ticks to date were performed in woodland areas far away from the major population centres; in consequence, the detection of high prevalences in ticks from these areas does not always involve a real risk of infection for humans and domestic animals (Estrada-Peña & de la Fuente, 2014). Previous studies on questing ticks collected from the vegetation in urban and suburban areas reported a high risk of acquiring tick-borne pathogens in cities where micromammals and birds may play an important role in the maintenance of tick populations (Hansford et al., 2022); this could be related to the high density of these animals in urban areas when compared to natural areas as they have few or no natural predators in urban areas (Estrada-Peña et al., 2017). Although data on the presence of ticks and tick-borne pathogens in urban and suburban areas from European countries are available, information from Spain is limited. For these reasons, the main objectives of the present study were: (i) to identify the questing tick populations in urban and suburban areas from the city of Lugo (northwestern Spain) and to establish tick phenology during a one-year period and (ii) to determine the prevalence of Borrelia spp., Rickettsia spp. and A. phagocytophilum in the collected ticks. MATERIALS AND METHODS Study area and tick collection Field studies were conducted in eight green areas of the city of Lugo (430004200 N7 3302600 W), located in Galicia (north-western Spain), from November 2020 to October 2021. Four sampling points were located in urban areas and four in suburban areas, which are primarily residential areas less-densely populated than urban commercial areas (Figure 1). Urban areas three and five and suburban areas one, six and eight were public parks. Urban areas four and seven corresponded to a green area close to the street and a dog park in the city centre, respectively. All these areas were mainly composed by grasses. Finally, suburban area two was a not-urbanized unused wasteland mainly composed by grass species (Poa pratensis,Dactylis glomerata,Lolium perenne and Festuca pratensis), ferns (Polypodium vulgare) and shrubs (Genista hirsuta and Calluna vulgaris). Ticks were collected monthly by flagging method along a constant single 300 m transect in each sampling area. The 1 m 2 flannel flag (1 1 m) was checked every 2–5 m depending on the number of ticks collected; if at least one specimen was observed when the flag was checked, the flag was checked every 2 m; if no specimens were present, the flag was checked every 5 m. Collected ticks were removed from the flag using tweezers and stored in 70% ethanol. All larvae adhering to the flag in a clumped manner were defined as a ‘nest’ originating from a single female (Hauck et al., 2020). Tick species identification In the laboratory, ticks were identified to species level using previously described morphological keys (Estrada-Peña et al., 2017). A subset of each tick species was analysed using molecular methods to confirm the morphological identifications. DNA was extracted from individual ticks using a commercial kit (High Pure PCR Template Preparation Kit, Roche Diagnostics GmbH ® ) following the manufacturer’s instructions. Before DNA extraction, tick tissues were disrupted using a MagNaLyser Instrument (Roche Diagnostic) at 6000 rpm during 60 s. A 460 bp fragment of the 16S rRNA gene of ticks was amplified using previously reported PCR protocols (Norris et al., 1996). Selected fragments were purified and sequenced on an ABI 3730xl sequencer (Applied Biosystems) at the Sequencing and Fragment Analysis Unit of the Santiago de Compostela University. Sequences were aligned 2REMESAR ET AL. 13652915, 0, Downloaded from https://resjournals.onlinelibrary.wiley.com/doi/10.1111/mve.12648 by Universidade de Santiago de Compostela, Wiley Online Library on [15/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License and edited using ChromasPro (Technelysium) and consensus sequences were then compared to the sequences available in GenBank database using BLAST. DNA extraction and pathogen detection DNA was individually extracted from 26 adults, 56 nymphs and 17 larvae using a commercial kit as previously indicated. In addition, 49 I. frontalis larvae originating from two larvae nests composed of 14 and 232 ticks, respectively, were analysed in 14 pools (seven pools of two larvae for the first larvae nest and seven pools of five larvae for the second). DNA from larvae pools was extracted as previously indicated. ThepresenceofBorrelia spp., Rickettsia spp. and A. phagocytophilum in tick samples was detected molecularly using primers and protocols previously described. The presence of Borrelia spp. and A. phagocytophilum was studied using PCR assays targeting the genes encoding for the flagellin protein (Stromdahl et al., 2003)andgroEL heat-shock protein, respectively (Liz et al., 2002). Those samples positive to Borrelia spp. were further analysed at the glpQ gene (Hovius et al., 2013). In addition, Rickettsia DNA was detected by amplifying fragments of two genes encoding for the major outer membrane proteins rOmpA and rOmpB (Choi et al., 2005; Regnery et al., 1991). DNA of B. burgdorferi s.l., A. phagocytophilum and R. amblyommii and nuclease free water were included as positive and negative controls in each PCR assay. PCR products were processed and sequenced as previously indicated; finally, the obtained sequences were scanned against the GenBank database. Unique sequences identified in this study were deposited in GenBank under accession numbers ON859976ON859999. A phylogenetic analysis was carried out using MrBayes 3.2.7 software by Bayesian approach with Markov Chain Monte Carlo sampling (10,000,000 generations sampling every 1000 steps). A General Time Reversible substitution model with gamma-distributed rate variation across sites (GTR +G) and a Hasegawa–Kishino–Yano substitution model with gamma-distributed rate variation across sites (HKY +G) were used for the analysis of tick sequences at the rOmpA and rOmpB partial genes, respectively. The models were selected based on Akaike information criterion (AIC) value using the free software jModelTest v.2.1.10. The tree was visualized and edited using FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). Statistical analysis The influence of the sampling season and the studied area (urban and suburban) on the total number of collected nymphs and adults was assessed using a test of equal or given proportions. The possible influence of some variables (tick development stage, sampling area and season of sampling) on the prevalence of Rickettsia spp. was analysed by multivariate analysis using a multiple logistic regression model FIGURE 1 Location of the eight tick sampling points in the city of Lugo (north-western Spain). TICK-BORNE PATHOGENS FROM URBAN AREAS IN NW SPAIN 3 13652915, 0, Downloaded from https://resjournals.onlinelibrary.wiley.com/doi/10.1111/mve.12648 by Universidade de Santiago de Compostela, Wiley Online Library on [15/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License (Hosmer et al., 1989). Factors were eliminated from the initial model using a method based in AIC value until the best model was built. All pairwise interactions were evaluated. Odds ratio were computed by raising ‘e’to the power of the logistic coefficient over the first category of each factor (reference category). The level of significance was set at pvalues <0.05. All statistical analyses were performed using the statistical software R 4.1.2. RESULTS Questing tick species identified A total of 345 ticks were collected during the study (Table 1). Ticks were found in all suburban sampling areas and in two out of four urban areas (Table 1). A higher number of ticks was collected in suburban (331/345; 95.9%) than in urban areas (14/345; 4.1%). Significant differences in the total number of both nymphs and adults (χ 2 =122.95, p< 0.001) were detected between suburban (77/82; 93.9%) and urban (5/82; 6.1%) areas. Four tick species were identified, I. frontalis being the most abundant (296/345; 85.8%) followed by I. ricinus (25/345; 7.2%) and R. sanguineus s.l. (23/345; 6.7%); a single specimen of D. reticulatus was found (1/345; 0.3%). The number of the different development stages collected for each tick species is summarized in Table 1, including 246 I. frontalis larvae found in two nests, one in the suburban area 1 (14 larvae) and other in suburban area 8 (232 larvae). Morphological identification was confirmed molecularly in all cases. I. frontalis sequences showed a percentage of identity ranging from 99.8% to 100% when compared to the deposited sequences MF370645 and KJ414454 obtained from I. frontalis collected on vegetation in Portugal (Santos et al., 2018) and on birds in Belgium (Heylen et al., 2014), respectively. In addition, I. ricinus and D. reticulatus sequences were identical to MH645522 and MH645514, respectively, obtained from questing ticks captured in north-western Spain (Remesar, Díaz, Venzal, Pérez-Creo, et al., 2019). Finally, sequences of R. sanguineus s.l. were identical to MZ420717 obtained from that tick species feeding on wild rabbits in Spain (Remesar, Castro-Scholten, et al., 2021). A higher diversity of tick species was detected in suburban than in urban areas (Table 1); thus, all the species identified were found in suburban areas whereas only Ixodes spp. ticks were captured in urban areas. I. frontalis was the most abundant species since 33 nymphs and one adult were collected; this species was also widely distributed since it was detected in all suburban areas and in two urban areas (Table 1). In contrast, I. ricinus (23 nymphs and one adult) and R. sanguineus s.l. (23 adults) were only detected in two sampling areas (one urban and one suburban areas for I. ricinus and two suburban areas for R. sanguineus s.l.), and the only D. reticulatus specimen collected was found in one suburban area (Table 1). All development stages were only collected for Ixodes ticks, although no males were found. I. frontalis larvae were the most abundant stage of development followed by nymphs; only a single female was found (Table 1). It is worth noting that most I. frontalis larvae (93.9%) were found in two larval nests. For I. ricinus, nymphs were predominant and only one larvae and one female were identified (Table 1). Only adults of R. sanguineus s.l. and a single male of D. reticulatus were detected (Table 1). Ticks were found in all seasons, especially in autumn, when all the larvae were collected (Table 1and Figure 2). It is worth noting that no ticks were collected in June, August and September; in addition, only a single nymph of Ixodes spp. was detected in January, October and July (Table 1and Figure 2). I. frontalis nymphs were found from October to April; its density showed a bimodal pattern with peaks in November and February (Table 1and Figure 2). In contrast, I. ricinus nymphs were detected from February to April showing a unimodal pattern, peaking in April. The only adult specimen of I. frontalis was found in November and the only adult specimen of I. ricinus was found in May (Table 1and Figure 2). All the specimens of R. sanguineus s.l. TABLE 1 Number of tick specimens collected in the city of Lugo (north-western Spain) when considering the tick species, stage of development, sampling area and season. Area Ixodes frontalis Ixodes ricinus Rhipicephalus sanguineus s.l. Dermacentor reticulatus Season TotalL N FT LN FT F M T M T SpSuAu Wi 1 SU 20 11 - 31 - - - 0 5 3 8 - 0 9 - 22 8 39 2 SU 0 11 1 12 - - - 0 11 4 15 - 0 14 - 12 1 27 3U 1 2 - 3 - - - 0 - - 0 - 0 - - 1 2 3 4U --- 0---0 - 0 - 0 -- -- 0 5U 8 - - 8 - 21 3 - - 0 - 0 21 7 1 11 6 SU 1 1 - 2 1 21 - 22 - - 0 - 0 13 0 5 6 24 7U --- 0---0- - 0 - 0 -- -- 0 8 SU 232 8 - 240 - - - 0 - - 0 1 1 1 - 235 5 241 Total 262 33 1 296 1 23 1 25 16 7 23 1 1 39 1 282 23 345 Abbreviations: Au, autumn (samplings from October to December); F, females; L, larvae; M, males; N, nymphs; Sp, spring (including the samplings performed from April to June); SU, suburban; Su, summer (samplings from July to September); T, total; U, urban areas; Wi, winter (samplings from January to March). 4REMESAR ET AL. 13652915, 0, Downloaded from https://resjournals.onlinelibrary.wiley.com/doi/10.1111/mve.12648 by Universidade de Santiago de Compostela, Wiley Online Library on [15/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License were found from March to May and the male of D. reticulatus in April (Table 1and Figure 2). Pathogens detected in questing ticks Three out of 113 (2.7%) individual tick samples were positive to Borrelia spp. Two Borrelia species were identified in Ixodes ticks: B. turdi was detected in two (1.8%; 2/113) I. frontalis specimens (one female and one nymph) and B. valaisiana in a single (0.9; 1/113) I. ricinus nymph (Table 2). Both fla and IGS sequence data of Borrelia-positive isolates are summarized in Table S1. In addition, 31.9% of samples were positive to Rickettsia spp. (36/113). Positive samples to this pathogen genus were detected in 17 (26.6%) I. frontalis (two individual and two larvae pools and 13 nymphs), 10 (40%) I. ricinus nymphs and in nine (39.1%) R. sanguineus s.l. adults (Table 2). Most Rickettsia-positive ticks were detected in winter (34.8%; 8/23) and autumn (34.0%; 17/50), whereas a percentage of 28.2% was detected in spring (11/39); no Rickettsiapositive ticks were detected in summer, but it must be noted that only one tick was collected. Positive ticks to A. phagocytophilum were not found. Logistic regression results showed that the prevalence of Rickettsia spp. was significantly higher in I. ricinus than in I. frontalis and in nymphs than in larvae (Table 3). In addition, significant differences also were observed regarding the sampling season; thus, the risk of being infected by Rickettsia spp. was 8.1 and 24.2-fold higher in winter and autumn, respectively, than in spring (Table 3). rOmpA and rOmpB sequence data of Rickettsia-positive isolates is summarized in Table S1; sequence analysis allowed the identification of six validated and one Candidatus00 Rickettsia species (Table 2). R. slovaca was the most prevalent, followed by R. monacensis,R. massiliae, R. raoultii and Candidatus Rickettsia rioja (Table 2). R. sibirica mongolitimonae and co-infections R. massiliae/R. aeschielmanii and R. massiliae/R. monacensis were detected in one sample each (0.9%; 1/113). In addition, two novel Rickettsia species were detected: one novel species was detected in a single I. ricinus nymph (4.35%; 1/113) and the other in a single R. sanguineus female in co-infection with R. massiliae (4.35%; 1/113). Phylogenetic analysis at rOmpA and rOmpB genes showed that both novel Rickettsia species were located outside the clusters including the main Rickettsia species (Figures 3and 4). The highest diversity of Rickettsia species was detected in I. ricinus,whichwaspositivetoallthe species identified, with the exception of R. sibirica subsp. mongolitimonae. Five and three Rickettsia species were identified in I. frontalis and R. sanguineus s.l., respectively, and co-infections were only detected in I. ricinus and R. sanguineus s.l. (Table 2). It is also worth noting that two Rickettsia species were detected in I. frontalis larvae, R. slovaca and Ca. R. rioja (Table 2). DISCUSSION The detection of ticks and tick-borne pathogens in urban and suburban areas is of increasing concern. Although tick densities are usually lower in urbanized areas than in non-urban woodland environments (Hauck et al., 2020), the presence of tick populations in urban and suburban areas could pose an increased risk of tick-bites since people and companion animals usually develop their activities in these areas (EstradaPeña et al., 2017). Therefore, the presence of zoonotic pathogens in ticks from urbanized areas may involve implications for public health. Questing tick species identified Our data reveal the presence of a considerable number of ticks in urban and suburban areas of the city of Lugo, although tick abundance was lower than that previously reported, using the same collection FIGURE 2 Number of ticks collected in the city of Lugo (north-western Spain) when considering the tick species, stage of development and month of sampling. The data series of nymphs and adults was aligned to the primary Yaxis and larvae data series to the secondary Yaxis. TICK-BORNE PATHOGENS FROM URBAN AREAS IN NW SPAIN 5 13652915, 0, Downloaded from https://resjournals.onlinelibrary.wiley.com/doi/10.1111/mve.12648 by Universidade de Santiago de Compostela, Wiley Online Library on [15/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License TABLE 2 Percentage of individual ticks positive to Borrelia spp. and Rickettsia spp. and maximum likelihood estimation when considering the tick species and the development stage. Ixodes frontalis Ixodes ricinus Rhipicephalus sanguineus s.l. Dermacentor reticulatus TOTAL (%) [CI] Larvae (%) [CI] a larvae pools (%) Nymphs (%) [CI] Female (%) [CI] TOTAL (%) [CI] Larvae (%) [CI] Nymphs (%) [CI] Female (%) [CI] TOTAL (%) [CI] Female (%) [CI] Male (%) [CI] TOTAL (%) [CI] Male (%) [CI] Borrelia spp. Borrelia turdi 0/16 (0%) [0.00–24.07] a 0/14 (0%) [0.00–26.76] 1/33 (3.03%) [0.16–17.51] 1/1 (100%) [5.46–100] 2/64 (3.13%) [0.54–11.81] 0/1 (0%) [0.0–94.54] 0/23 (0%) [0.0–17.81] 0/1 (0%) [0.0–94.54] 0/25 (0%) [0.0–16.58] 0/16 (0%) [0.00–24.07] 0/7 (0%) [0.00–43.91] 0/23 (0%) [0.0–17.81] 0/1 (0%) [0.0–94.54] 2/113 (1.77%) [0.31–6.88] Borrelia valaisiana 0/16 (0%) [0.00–24.07] a 0/14 (0%) [0.00–26.76] 0/33 (0%) [0.0–12.98] 0/1 (0%) [0.0–94.54] 0/64 (0%) [0.0–0.75] 0/1 (0%) [0.0–94.54] 1/23 (4.35%) [0.23–23.97] 0/1 (0%) [0.0–94.54] 1/25 (4.0%) [0.21–22.32] 0/16 (0%) [0.00–24.07] 0/7 (0%) [0.00–43.91] 0/23 (0%) [0.0–17.81] 0/1 (0%) [0.0–94.54] 1/113 (0.88%) [0.05–5.55] TOTAL 0/16 (0%) [0.00–24.07] a 0/14 (0%) [0.00–26.76] 1/33 (3.03%) [0.16–17.51] 1/1 (100%) [5.46–100] 2/64 (3.13%) [0.54–11.81] 0/1 (0%) [0.0–94.54] 1/23 (4.35%) [0.23–23.97] 0/1 (0%) [0.0–94.54] 1/25 (4.0%) [0.21–22.32] 0/16 (0%) [0.00–24.07] 0/7 (0%) [0.00–43.91] 0/23 (0%) [0.0–17.81] 0/1 (0%) [0.0–94.54] 3/113 (2.65%) [0.69–8.14] Rickettsia spp. Rickettsia raoultii 0/16 (0%) [0.00–24.07] a 0/14 (0%) [0.00–26.76] 2/33 (6.06%) [1.06–21.62] 0/1 (0%) [0.0–94.54] 2/64 (3.13%) [0.54–11.81] 0/1 (0%) [0.0–94.54] 1/23 (4.35%) [0.23–23.97] 0/1 (0%) [0.0–94.54] 1/25 (4.0%) [0.21–22.32] 0/16 (0%) [0.00–24.07] 0/7 (0%) [0.00–43.91] 0/23 (0%) [0.0–17.81] 0/1 (0%) [0.0–94.54] 3/113 (2.65%) [0.69–8.14] Rickettsia monacensis 0/16 (0%) [0.00–24.07] a 0/14 (0%) [0.00–26.76] 2/33 (6.06%) [1.06–21.62] 0/1 (0%) [0.0–94.54] 2/64 (3.13%) [0.54–11.81] 0/1 (0%) [0.0–94.54] 1/23 (4.35%) [0.23–23.97] 0/1 (0%) [0.0–94.54] 1/25 (4.0%) [0.21–22.32] 2/16 (12.50%) [2.20–39.59] 1/7 (14.29%) [0.75–57.99] 3/23 (13.04%) [3.43–34.67] 0/1 (0%) [0.0–94.54] 6/113 (5.31%) [2.18–11.67] Rickettsia slovaca 1/16 (6.25%) [0.32–32.29] a 2/14 (14.29%) [2.51–43.85] 8/33 (24.24%) [11.74–42.63] 0/1 (0%) [0.0–94.54] 11/64 (17.19%) [9.29–29.10] 0/1 (0%) [0.0–94.54] 2/23 (8.70%) [1.52–29.51] 0/1 (0%) [0.0–94.54] 2/25 (8.0%) [21.81–61.11] 1/16 (6.25%) [0.33–32.29] 0/7 (0%) [0.00–43.91] 1/23 (4.35%) [0.23–23.97] 0/1 (0%) [0.0–94.54] 14/113 (12.39%) [7.19–20.24] Ca. R. rioja 1/16 (6.25%) [0.32–32.29] a 0/14 (0%) [0.00–26.76] 0/33 (0%) [0.0–12.98] 0/1 (0%) [0.0–94.54] 1/64 (1.56%) [0.08–9.54] 0/1 (0%) [0.0–94.54] 2/23 (8.70%) [1.52–29.51] 0/1 (0%) [0.0–94.54] 2/25 (8.0%) [21.81–61.11] 0/16 (0%) [0.00–24.07] 0/7 (0%) [0.00–43.91] 0/23 (0%) [0.0–17.81] 0/1 (0%) [0.0–94.54] 3/113 (2.65%) [0.69–8.14] Rickettsia massiliae 0/16 (0%) [0.00–24.07] a 0/14 (0%) [0.00–26.76] 0/33 (0%) [0.0–12.98] 0/1 (0%) [0.0–94.54] 0/64 (0%) [0.0–0.75] 0/1 (0%) [0.0–94.54] 1/23 (4.35%) [0.23–23.97] 0/1 (0%) [0.0–94.54] 1/25 (4.0%) [0.21–22.32] 4/16 (25.00%) [8.33–52.59] 0/7 (0%) [0.00–43.91] 4/23 (17.39) [57.24–39.55] 0/1 (0%) [0.0–94.54] 5/113 (4.43%) [1.64–10.53] Rickettsia sibirica 0/16 (0%) [0.00–24.07] a 0/14 (0%) [0.00–26.76] 1/33 (3.03%) [0.16–17.51] 0/1 (0%) [0.0–94.54] 1/64 (1.56%) [0.08–9.54] 0/1 (0%) [0.0–94.54] 0/23 (0%) [0.0–17.81] 0/1 (0%) [0.0–94.54] 0/25 (0%) [0.0–16.58] 0/16 (0%) [0.00–24.07] 0/7 (0%) [0.00–43.91] 0/23 (0%) [0.0–17.81] 0/1 (0%) [0.0–94.54] 1/113 (0.88%) [0.05–5.55] Rickettsia sp. 0/16 (0%) [0.00–24.07] a 0/14 (0%) [0.00–26.76] 0/33 (0%) [0.0–12.98] 0/1 (0%) [0.0–94.54] 0/64 (0%) [0.0–0.75] 0/1 (0%) [0.0–94.54] 1/23 (4.35%) [0.23–23.97] 0/1 (0%) [0.0–94.54] 1/25 (4.0%) [0.21–22.32] 0/16 (0%) [0.00–24.07] 0/7 (0%) [0.00–43.91] 0/23 (0%) [0.0–17.81] 0/1 (0%) [0.0–94.54] 1/113 (0.88%) [0.05–5.55] R. massiliae + R. aeschielmanii 0/16 (0%) [0.00–24.07] a 0/14 (0%) [0.00–26.76] 0/33 (0%) [0.0–12.98] 0/1 (0%) [0.0–94.54] 0/64 (0%) [0.0–0.75] 0/1 (0%) [0.0–94.54] 1/23 (4.35%) [0.23–23.97] 0/1 (0%) [0.0–94.54] 1/25 (4.0%) [0.21–22.32] 0/16 (0%) [0.00–24.07] 0/7 (0%) [0.00–43.91] 0/23 (0%) [0.0–17.81] 0/1 (0%) [0.0–94.54] 1/113 (0.88%) [0.05–5.55] R. massiliae + R. monacensis 0/16 (0%) [0.00–24.07] a 0/14 (0%) [0.00–26.76] 0/33 (0%) [0.0–12.98] 0/1 (0%) [0.0–94.54] 0/64 (0%) [0.0–0.75] 0/1 (0%) [0.0–94.54] 1/23 (4.35%) [0.23–23.97] 0/1 (0%) [0.0–94.54] 1/25 (4.0%) [0.21–22.32] 0/16 (0%) [0.00–24.07] 0/7 (0%) [0.00–43.91] 0/23 (0%) [0.0–17.81] 0/1 (0%) [0.0–94.54] 1/113 (0.88%) [0.05–5.55] R. massiliae +R. sp. 0/16 (0%) [0.00–24.07] a 0/14 (0%) [0.00–26.76] 0/33 (0%) [0.0–12.98] 0/1 (0%) [0.0–94.54] 0/64 (0%) [0.0–0.75] 0/1 (0%) [0.0–94.54] 0/23 (0%) [0.0–17.81] 0/1 (0%) [0.0–94.54] 0/25 (0%) [0.0–16.58] 1/16 (6.25%) [0.33–32.29] 0/7 (0%) [0.00–43.91] 1/23 (4.35%) [0.23–23.97] 0/1 (0%) [0.0–94.54] 1/113 (0.88%) [0.05–5.55] TOTAL 2/16 (12.50%) [2.20–39.59] a 2/14 (14.29%) [2.51–43.85] 13/33 (39.39%) [23.43–57.76] 0/1 (0%) [0.0–94.54] 17/64 (26.56%) [16.65–39.31] 0/1 (0%) [0.0–94.54] 10/23 (43.48%) [23.88–65.13] 0/1 (0%) [0.0–94.54] 10/25 (40%) [21.81–61.11] 8/16 (50%) [28.00–72.00] 1/7 (14.29%) [0.75–57.99] 9/23 (39.13%) [20.47–61.22] 0/1 (0%) [0.0–94.54] 36/113 (31.86%) [23.59–41.38] Note: Ticks were collected from eight urban and suburban sites in the city of Lugo (north-western Spain). a Pooled I. frontalis larvae. 6REMESAR ET AL. 13652915, 0, Downloaded from https://resjournals.onlinelibrary.wiley.com/doi/10.1111/mve.12648 by Universidade de Santiago de Compostela, Wiley Online Library on [15/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License protocol in constant 300 m transects, in forest areas from the same region (Remesar, Díaz, Venzal, Prieto, et al., 2019); thus, the number of specimens yearly collected in each sampling area ranged from 0 to 241 in the present study, but increased to 500–2000 in forest areas (Remesar, Díaz, Venzal, Prieto, et al., 2019). Previous studies carried out in Europe indicated that overall tick densities are low in urban areas, but noticeable variations mainly related to habitat types and urban gradient can be detected (Hansford et al., 2022). In this regard, a significantly higher number of ticks were found in suburban areas than in urban areas. These results are consistent with previous investigations performed in urban environments reporting the highest tick densities in woodland habitats such as wide centrally located parks or urban fringes (Hauck et al., 2020; Sta nczak et al., 2015). Although the tick density is influenced by the urban gradient, some results are conflicting and suggest that other variables such as the tick species and the presence of wild animals acting as maintenance hosts for tick populations must be considered (Hansford et al., 2022). Although the urban zones sampled in the present study have bird and micromammal populations, they are isolated from the main woodland areas and therefore wild large mammals have no access to these zones. This fact may have a major role in the presence and number of I. ricinus collected in a particular area; thus, wild large mammals such as roe deer (Capreolus capreolus) and wild boar (Sus scrofa), which can host all the stages of development of this tick species (Estrada-Peña et al., 2017), are common in the forest area close to suburban sampling area 6 where I. ricinus was mainly found. Nevertheless, the presence and abundance of wild ungulates cannot explain the predominance and abundance of I. frontalis, which is considered a passerine tick (Estrada-Peña et al., 2017). In this regard, it has been suggested that the abundance of I. frontalis could be higher in areas acting as roost sites for birds (Plantard et al., 2021). In the present study, this tick species was mostly found in suburban areas with high vegetation and tree density (sampling area 8) or with a high bramble density (sampling areas 1 and 2) where birds can nest and are protected from predators. With regard to R. sanguineus s.l. and D. reticulatus, only adults were collected since their immature stages show endophilic behaviour and, therefore, are not usually captured by flagging (Estrada-Peña et al., 2017). Rhipicephalus sanguineus s.l. was only captured in the two suburban areas closest to the main urbanized zone; these results are surprising since this tick is traditionally associated to dogs and humans and consequently it would be more likely to be found in urban areas (Shimada et al., 2003). It has been previously suggested that the use of ectoparasiticides may play a significant role in its absence in the selected urban areas (Lorusso et al., 2010). Our results also indicate that the tick species identified showed different activity patterns. In this way, I. frontalis showed a bimodal activity pattern with the highest peak in autumn (November) when the only I. frontalis adult as well as the highest number of nymphs were captured. However, it is worth noting that the pattern observed in the present study is based on reduced number of individuals (n=33 nymphs spread among two different sites). These data are consistent with previous studies indicating that all the development stages can be found from late autumn to early-winter showing an absence of activity in summer (Plantard et al., 2021). I. ricinus showed an activity peak in late spring-early summer; although different seasonal patterns have been observed in Europe (Estrada-Peña et al., 2017), our results are in line with previous studies carried out in forest areas from Spain (Remesar, Díaz, Venzal, Pérez-Creo, et al., 2019). Finally, R. sanguineus s.l. was only detected between March and May, agreeing with previous studies reporting a reduced activity of this tick during winter in southern Europe (DantasTorres, 2010). Pathogens detected in questing ticks Two out of the three analysed pathogens, Borrelia spp. and Rickettsia spp., were detected in the ticks collected in green areas from the city of Lugo. Borrelia DNA was only detected in a low percentage of I. ricinus and I. frontalis nymphs as well as in the only I. frontalis female analysed. It is worth noting that no data on the prevalence of Borrelia spp. in questing I. frontalis is currently available whereas this pathogen TABLE 3 Logistic regression model for the prevalence of Rickettsia spp. in questing ticks collected in green areas from the city of Lugo (NW Spain). Estimate Z-value p-Value OR 95% CI (Intercept) 5.0397 4.195 <0.001 - - Tick species Ixodes frontalis --- - - Ixodes ricinus 1.8535 2.252 0.024 6.38 1.40–37.86 Tick development stage Larvae - - - - - Nymph 1.8839 2.795 0.005 6.58 1.90–27.86 Sampling season Spring - - - - - Autumn 3.1845 3.033 0.00242 24.16 3.57–229.19 Winter 2.0853 2.229 0.02580 8.05 1.45–61.25 TICK-BORNE PATHOGENS FROM URBAN AREAS IN NW SPAIN 7 13652915, 0, Downloaded from https://resjournals.onlinelibrary.wiley.com/doi/10.1111/mve.12648 by Universidade de Santiago de Compostela, Wiley Online Library on [15/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 3 Phylogenetic tree clustering of the partial rOmpA of Rickettsia spp. isolated from ticks collected in the city of Lugo (north-western Spain). The tree was obtained using a General Time Reversible substitution model with gamma-distributed rate variation across sites (GTR +G) with the software MrBayes 3.2.7 by Bayesian approach with Markov Chain Monte Carlo sampling (10,000,000 generations sampling every 1000 steps). This analysis involved 66 nucleotide sequences. The nucleotide sequence of Rickettsia felis was used as an outgroup. Isolates identified in this study are shown in bold. 8REMESAR ET AL. 13652915, 0, Downloaded from https://resjournals.onlinelibrary.wiley.com/doi/10.1111/mve.12648 by Universidade de Santiago de Compostela, Wiley Online Library on [15/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License FIGURE 4 Phylogenetic tree clustering of the partial rOmpB of Rickettsia spp. isolated from ticks collected in the city of Lugo (north-western Spain). The tree was obtained using a Hasegawa– Kishino–Yano substitution model with gamma-distributed rate variation across sites (HKY +G) with the software MrBayes 3.2.7 by Bayesian approach with Markov Chain Monte Carlo sampling (10,000,000 generations sampling every 1000 steps). This analysis involved 47 nucleotide sequences. The nucleotide sequence of Rickettsia typhi was used as an outgroup. Isolates identified in this study are shown in bold. TICK-BORNE PATHOGENS FROM URBAN AREAS IN NW SPAIN 9 13652915, 0, Downloaded from https://resjournals.onlinelibrary.wiley.com/doi/10.1111/mve.12648 by Universidade de Santiago de Compostela, Wiley Online Library on [15/06/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License