Molecular identification of Borrelia and SFG Rickettsia spp. in hard ticks parasitizing domestic and wild animals in southeastern Spain

Lyme disease and the spotted fever group rickettsiosis, involve bacteria belonging to the genus Borrelia and Rickettsia, respectively. These infections are the most important tick-borne zoonotic diseases involving ticks as vectors. Descriptive and epidemiological studies are essential to determine the animal hosts involved in the maintenance of these diseases. In the present study, 94 tick pool samples from 15 different host species located in the Region of Murcia (southeastern, Spain) were analysed. Ticks were morphologically identified as: Dermacentor marginatus, Hyalomma lusitanicum, Ixodes Ricinus, and Rhipicephalus sanguineus. Our results showed that 5.3% of the tick pool samples carried Borrelia spp. DNA, and 20.2% carried SFG Rickettsia DNA. In every hard tick pool Spot Fever Group (SFG) Rickettsia spp. DNA were detected, except for H. lusitanicum. Likewise, D. marginatum was the only species in which Borrelia spp. DNA was not detected. Barbary sheep and wild boar were the host species in which tick pools showed DNA presence of both pathogens. This study increases the knowledge about the presence of Borrelia spp. DNA and SFG Rickettsia spp. DNA in different hard tick species from this geographical area. Supplementary Information The online version contains supplementary material available at 10.1007/s11259-023-10292-x.


Introduction
Emerging tick-borne zoonotic diseases are a major global public health risk that has been increasing in recent decades (Parola et al. 2013).Ticks can act as vectors of dangerous bacterial, parasitic, and viral zoonotic pathogens, so epidemiologic studies in them are essential to identify zoonotic agents and their host animals in different geographical areas.
Lyme disease (LD) is one of the most prevalent zoonotic tick-borne infectious diseases caused by species or members of the Borrelia burgdorferi sensu lato (Bbsl) (Wolcott et al. 2021).The main vector is Ixodes ricinus, widely distributed N. Ortega and I. Arcenillas-Hernández contributed equally.
N. Ortega nortega@um.esal. 2022).Animal host species can play an important role as carriers of these microorganisms in the environment (Parola et al. 2013), due to the increase of contact between domestic and wild species, and the human-wildlife interaction occurring in anthropized areas.
The aims of this study were to determine the DNA presence of Bbsl and SFG Rickettsia spp. in different hard tick species collected from domestic and wild animals in southeastern Spain.

Study area, animals, and tick samples
A total of 463 ticks were collected from the Region of Murcia (SE, Spain) a semi-arid geographical area.Ninety-four tick pool samples (with 1-10 individuals/pool) were collected from 76 individuals of 15 different animal species (Table 1) during the spring (March-April) and autumn (September-October) seasons of 2022.Tick pools were made according to their species, without differentiating males (n = 195) from females (n = 268) or any life stage (340 were adults and 123 were nymphs).Ticks were properly removed with tweezers, and specimens were classified using a taxonomic key (Estrada-Peña et al. 2017).Finally, ticks were preserved in absolute ethanol until processing.Only ticks identified morphologically as genus Ixodes were additionally subjected to molecular identification by DNA Barcode, using for insect cell lines, the primers LepFl and LepRl (Cooper et al. 2007) these analyses were performed by the Molecular Biology Service (ACTI) of the University of Murcia (Spain).

Molecular detection of Bbsl complex and SFG rickettsia spp
For DNA extraction ticks were pooled according to species and individual host and they were left overnight at room temperature to allow complete evaporation of the ethanol.Subsequently, Extracted Genomic DNA Tissue kit (BLIRT S.A. Germany) was used, following the manufacturer's instructions.The extracted DNA was stored at -20ºC until their use.
Bbsl 23 S rRNA real-time qPCR was performed with Fast Gene Probe 2xqPCR Universal Mix (Nippon Genetics Europe GmbH) using the primers Bb23Sf and Bb23Sr and Bb23Sp-FAM probe (Courtney et al. 2004) to generate 75 bp fragment, PCR reactions were performed in duplicate and specific DNA from B. burgdorferi B31 strains and pure water were used as positive and negative controls, respectively.
Two different PCRs were used for genotypic identification of Rickettsiae, the first one was specific for the gltA gene and allows amplification of Rickettsia spp.380-397 bp fragment, using the primers RpC5.877p and RpC5.1258n (Regnery et al. 1991), and Zestaw NXT Taq kit hot start PCR (EURx.Poland).Samples that tested positive for the gltA gene were used for a second PCR, specific for gene encoding the protein rOmpA that allows amplification of the SFG Rickettsia 629-632 bp fragment using primers Rr190.70p and Rr190.701n(Roux et al. 1996), and Zestaw NXT Taq kit hot start PCR (EURx.Poland).For both PCR, specific DNA from SFG Rickettsia spp.and pure water were used as positive and negative controls, respectively.PCR products are analysed by gel electrophoresis on a 1.5% agarose gel stained with Midori Green (Nippon Genetics).
For the nucleotide sequencing study 19 PCR-amplified Rickettsiae gene protein rOmpA sequences and 5 PCRamplified B. burgdorferi 23 S rRNA sequences were selected and used.Amplicons of the expected size were sequenced on an Applied Biosystems 3500 Genetic Analyzer following the manufacturer's protocol.The sequences obtained were aligned with MEGA11: Molecular Evolutionary Genetics Analysis version 11 (Tamura et al. 2021) and compared with those sequences of other Rickettsia and Borrelia species downloaded from the GenBank database to identify known sequences with a high degree of similarity.

Statistical analysis
R software 3.3.0was used to statistical analysis, where Fisher's test was used to evaluate statistical differences between tick pools species including in our study, considering as a significant difference the threshold p < 0.05.
The concurrent DNA presence of Borrelia spp.and SFG Rickettsia spp.were detected in tick pools from 3.2% animals (3/94; 95%CI -0.4-6.7):two pools of I. ricinus collected from two barbary sheep, respectively, and one pool of R. sanguineus from a dog.

Discussion
The findings described in this study suggest the presence of Borrelia an SFG Rickettsia in natural and urban environments in southeastern Spain.Although the detection of a microorganism in a tick collected on an animal do not demonstrate the vector capacity of this tick species, some of the results found in this study have been quite novel, either due to the species of tick where the DNA is detected or because the host species in which these ticks were found.
All tick species detected in this study had been previously identified in the Iberian Peninsula (Estrada-Peña et al. 2017;Remesar et al. 2022).Rhipicephalus sanguineus and H. lusitanicum are generally present in semi-arid environments and are the most abundant tick species in this study (Estrada-Peña et al. 2017).Indeed, H. lusitanicum, has suffered an increase in its population along northeastern Spain (Castillo-Contreras et al. 2021).This species could also be spreading throughout southeastern Spain, since in this study H. lusitanicum was the second most abundant tick species.
Interestingly, our results also showed the presence of I. ricinus and D. marginatus, ticks frequently associated with more humid environments (Estrada-Peña et al. 2017).Although Ixodes spp.preferably lives in moist environments with cover vegetation that helps to maintain humidity during warmer periods, their presence in our studied territory may be associated to the scrub or wooded areas available that could create an optimal microclimatic condition that favour the occurrence of this tick (Kahl and Gray 2023).
In the life cycle of Borrelia, larvae and nymphs feed on small mammals, but large mammals and reptiles are used as primary hosts for mating adults to maintain tick population (Gern et al. 1998).This is consistent with our results, as tortoise, dog and barbary sheep were the host species in which positive Borrelia spp.DNA tick pools were detected.Results from barbary sheep were the most expected, as this ungulate species tends to inhabit areas with denser vegetation cover and, therefore, are more likely to encounter Ixodes spp.ticks.Our sequencing data, performed on a fragment of only 75 bp, showed that Borrelia species detected in this study could be either B. garinii or B. afzelii, it was not possible to discriminate between the two species based on the small size of the amplified fragment.However, both species are related to LD in the European countries (Mendoza-Roldan et al. 2019).In the Mediterranean basin, the most notable prevalences have been associated with B. lusitaneae where certain lizard species haven been identified as the main reservoir host of this species (Dsouli et al. 2006;Norte et al. 2015).Although our study did not detect this species or include samples from lizards, B. garinii or B. afzelii have been detected in another reptile, the Mediterranean tortoise, so further research would be interesting to evaluate the reported in a study conducted in northern Spain (40%; 10/25) (Remesar et al. 2023).Nevertheless, our percentage was higher compared with other European studies carried out, for example, in Italy (14%; 30/215) (Cafiso et al. 2021) or France (1.4%; 10/696) (Akl et al. 2019).The differences found between these studies could be explained by the bias caused by the low number of ticks analysed.Rickettsia monacensis was the only SFG Rickettsia species identified from I. ricinus tick pools, and this same association has also been recently reported in a French study (Akl et al. 2019).Therefore, I. ricinus presence should be considered by the health authorities since it may represent an epidemiological risk.
Despite the low number of ticks pools in our study, D. marginatus was the tick species in which SFG Rickettsia DNA was mostly detected and the only tick species with a statistically significant result.A similar percentage was reported in Barcelona (NE, Spain) by Castillo-Contreras et al. (2021), while other studies in natural and urban environments in Italy obtained a significantly lower frequency (Sgroi et al. 2020).In accordance with other studies conducted in Europe (Pereira et al. 2018;Sgroi et al. 2020), D. marginatus ticks from host species such as deer and wild presence of Bbsl in different reptile species in this region.Our finding could explain the increasing LD cases reported in the Region of Murcia, where LD incidence by Borrelia spp.has been triplicated in the last 15 years in this region, being one of the Spanish regions where a greater increase has been observed since 2007 (238%) (Amores et al. 2022).
Moreover, SFG Rickettsia DNA was detected in 30.7% of I. ricinus tick pools of this study.A similar rate was (5/94) (*) Statistically significant differences (p < 0.05) Fig. 1 SFG Rickettsia and Borrelia species identified in different pools of tick species and the host from which they were extracted.Number of pools used for each tick species is included Acknowledgements This work was supported by Consejería de Salud de la Comunidad Autónoma de Murcia, Dirección General de Salud Pública y Adicciones: Servicio de Seguridad Alimentaria y Zoonosis, Murcia (Spain), through the transference of knowledge and research project with the University of Murcia, entitled "Highly specific diagnostic services for the purpose of extending the food safety and zoonosis service's surveillance programs to certain emerging and re-emergent pathogens" (Ref. OTRI 33329,Head Researcher: Maria Rosa Caro).The authors gratefully acknowledge Raquel Escudero Nieto from Instituto Carlos III (CNM, Spain) for kindly supplying the DNA of B. burgdorferi B31 strain for its use as positive control in the real time-qPCR reaction and to Maria Carmen Vieira Lista from CIETUS (Salamanca University, Spain) for kindly supplying DNA from Rickettsia spp.and SFG Rickettsia as positive controls for gltA and gene encoding the protein rOmpA PCR, respectively.We are also grateful to the Molecular Biology Service of the University of Murcia for their help in the genomic sequencing of the samples.Funding This work was supported by Consejería de Salud de la Comunidad Autónoma de Murcia, Dirección General de Salud Pública y Adicciones: Servicio de Seguridad Alimentaria y Zoonosis, Murcia (Spain), through the transference of knowledge and research project with the University of Murcia, entitled "Highly specific diagnostic services for the purpose of extending the food safety and zoonosis service's surveillance programs to certain emerging and re-emergent pathogens" (Ref.OTRI 33329, Head Researcher: Maria Rosa Caro).Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature.

Author contributions
boar, showed in this study DNA of two different Rickettsia species: R. slovaca and R. raoultii.
The most frequent Rickettsia species detected in this study was R. massiliae, which was only detected in R. sanguineus.This was also reported in Italian ticks collected from wild carnivores (Chisu et al. 2017).Rickettsia raoultii and R. aeschlimannii, were also sequenced from R. sanguineus ticks in our study, and they have been previously reported from southern Europe areas, including the Iberian Peninsula (Chisu et al. 2017;Pereira et al. 2018;Castillo-Contreras et al. 2021).Hyalomma spp.ticks have an important contribution to the maintenance of R. aeschlimannii in natural environments.Nevertheless, in our case, no DNA SFG Rickettsia species were detected in this tick species.In this sense, other studies conducted in Spain showed similar results suggesting that H. lusitanicum might not act as an optimal vector of Rickettsia species (Pereira et al. 2018;Castillo-Contreras et al. 2021).
Our results suggest that wild boar may be contributing to the dispersal and/or maintenance of D. marginatus and H. lusitanicum ticks in urban and peri-urban environments, increasing the risk of zoonotic diseases transmission (Sgroi et al. 2020;Castillo-Contreras et al. 2021).
The approach of wildlife to peripheral urban areas caused by habitat fragmentation has establish an interaction between wildlife, domestic animals, and humans (Cafiso et al. 2021) and could increase the risk of disease transmission in the wild-domestic-human interface.Hence, combined analysis considering environmental and epidemiological factors are essential to develop effective control strategies for pathogens such as SFG Rickettsia species or Borrelia spp.

Conclusions
DNA of both pathogens, SFG Rickettsia spp.and Borrelia spp.have been detected in I. ricinus and R. sanguineus ticks so they could be acting as carriers of both pathogens in the southeastern of Spain.In terms of host species, Bbsl was detected in H. lusitanicum and R. sanguineus, both ticks from a Mediterranean tortoise.This is the first time that Borrelia has been detected in this reptile in the studied area.In addition, this study provides information about the presence of I. ricinus that are not usually present in semiarid environments.In this sense, further studies would be necessary for a better understanding of emerging tick-borne diseases in Spain, as well as the tick species circulating in this territory.
All authors contributed to the study conception and design: Material preparation and data collection by M.I.V. and M.D.G.; and analysis were performed by N.O., I.A.H., and M.R.C.The first draft of the manuscript was written by N.O.and I.A.H. M.R.C. revised the manuscript, and all authors commented on previous versions of the manuscript.All authors read and approved the final manuscript.

Table 2
Percentage of Rickettsia spp., SFG Rickettsia spp.and Borrelia spp.tick pools with DNA presence based on the different tick