Abstract
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.
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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 throughout the Iberian Peninsula, with a tendency to inhabit deciduous forests with shrubs. For an effective host-to-host transmission, a tick must acquire the bacteria from an infected animal, which allows it to survive and replicate while moulting, as well as be transmitted to another animal host during the next blood meal. The diversity of the Borrelia complex, which can cause human infection, is greater associated with B. burgdorferi sensu stricto (Bbss) species: Borrelia afzelii, Borrelia valaisiana and Borrelia garinii, being B. afzelii and B. garinii the most associated with human disease. This disease has an estimated global potential incidence of 12.3 million/year and a prevalence of 62.1 million cases, including a high percentage of undiagnosed and misdiagnosed cases due to failures and difficulties associated with diagnostic methods (Cook and Puri 2020).
Ticks are also considered the main vectors of SFG Rickettsia spp., being the main source of natural infection in tick-borne rickettsiosis (TBR) in Europe (Parola et al. 2013). In the Iberian Peninsula zoonotic species as Rickettsia conorii, Rickettsia monacensis, Rickettsia massiliae, Rickettsia aeschlimannii, Rickettsia raoultii or Rickettsia slovaca have been reported (Castillo-Contreras et al. 2021; Remesar et al. 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.
Materials and methods
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 PCR-amplified 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 3.3.0 was 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.
Results
Four different tick species: Dermacentor marginatus, Hyalomma lusitanicum, Ixodes ricinus and Rhipicephalus sanguineus, were identified. Borrelia spp. DNA were detected from 5.3% of tick pools (5/94; 95%CI 0.7–9.8): 2/5 from I. ricinus, 2/5 from R. sanguineus, and 1/5 from H. lusitanicum collected from 2 barbary sheep, one Mediterranean tortoise, and one dog, respectively (Table 1; Figure S1). Sequencing analysis of the 75 bp fragment identified: B. garinii (CP024316) and B. afzelii (CP075440), reaching 100% homology in all cases (Fig. 1; Table S1). Rickettsia spp. DNA were detected from 48.9% of tick pools (46/94; 95%CI 38.8–59.0), and specifically 41.3% of them (19/46; 95%CI 27.1–55.5) corresponding to DNA of SFG Rickettsia species; being present in tick pools of the majority of animal species sampled, except for Mediterranean tortoise, sheep, red fox, and eagle (Table 1) and mostly associated with R. sanguineus pools (Table 2). Sequence analysis identify five different SFG Rickettsia species: R. massiliae (KR401146, DQ212707 and KU498298), R. raoultii (KX506737, MT321626 and MH932055), R. slovaca (MF379311), R. monacensis (MK922659 and MN853333), and R. aeschlimanni (MF379306) (Fig. 1; Table S1). The homology between sequences ranged from 99.6 to 100%.
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 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 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 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 semi-arid 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.
Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
References
Akl T, Bourgoin G, Souq ML, Appolinaire J, Poirel MT, Gibert P, Rizk GA, Garel M, Zenner L (2019) Detection of tick-borne pathogens in questing Ixodes ricinus in the French Pyrenees and first identification of Rickettsia monacensis in France, vol 26. Parasite
Amores-Alguacil M, Estévez-Reboredo RM, Martínez de Aragón MV, Carmona R, Cano-Portero R (2022) Carga Hospitalaria De Enfermedad De Lyme en España (2005–2019). Boletín Epidemiológico Semanal 30(1):1–11
Cafiso A, Olivieri E, Floriano AM, Chiappa G, Serra V, Sassera D, Bazzocchi C (2021) Investigation of tick-borne pathogens in Ixodes ricinus in a peri-urban park in Lombardy (Italy) reveals the presence of emerging pathogens. Pathogens 10(6):732
Castillo-Contreras R, Magen L, Birtles R, Varela-Castro L, Hall JL, Conejero C, Aguilar XF, Colom-Cadena A, Lavin S, Mentaberre G, López-Olvera JR (2021) Ticks on wild boar in the metropolitan area of Barcelona (Spain) are infected with spotted Fever group rickettsiae. Transbound Emerg Dis 69(4):e82–e95
Chisu V, Leulmi H, Masala G, Piredda M, Foxi C, Parola P (2017) Detection of Rickettsia hoogstraalii, Rickettsia helvetica, Rickettsia massiliae, Rickettsia slovaca and Rickettsia aeschlimannii in ticks from Sardinia, Italy. Ticks Tick Borne Dis 8(3):347–352
Cook MJ, Puri BK (2020) Estimates for Lyme borreliosis Infections based on models using sentinel canine and human seroprevalence data. Infect Dis Model 16(5):871–888
Cooper JK, Sykes G, King S, Cottrill K, Ivanova NV, Hanner R, Ikonomi P (2007) Species identification in cell culture: a two-pronged molecular approach. In vitro Cell Dev Biol-Animal 43:344–351
Courtney JW, Kostelnik LM, Zeidner NS, Massung RF (2004) Multiplex real-time PCR for detection of Anaplasma phagocytophilum and Borrelia burgdorferi. J Clin Microbiol 42(7):3164–3168
Dsouli N, Younsi-kabachii H, Postic D, Nouira S, Gern L, Bouattour A (2006) Reservoir role of lizard Psammodromus algirus in transmission cycle of Borrelia burgdorferi Sensu Lato (Spirochaetaceae) in Tunisia. J Med Entomol 43:737–742
Estrada-Peña A, Mihalca A, Petney TN (2017) Ticks of Europe and North Africa. Springer International Publishing, Cham, Switzerland
Gern L, Estrada-Peña A, Frandsen F, Gray JS, Jaenson TG, Jongejan F, Kahl O, Korenberg E, Mehl R, Nuttall PA (1998) European reservoir hosts of Borrelia burgdorferi sensu lato. Zentralbl Bakteriol 287(3):196–204
Kahl O, Gray JS (2023) The biology of Ixodes ricinus with emphasis on its ecology. Ticks Tick Borne Dis, 102114
Mendoza-Roldan JA, Colella V, Lia RP, Nguyen VL, Barros-Battesti DM, Latta R, Dantas-Torres F, Otranto D (2019) Borrelia burgdorferi (Sensu Lato) in ectoparasites and reptiles in southern Italy. Parasit Vectors 12(1):35
Norte AC, Alves da Silva A, Alves J, da Silva LP, Núncio MS, Escudero R, Anda P, Ramos JA Lopes De Carvalho I 2015 the importance of lizards and small mammals as reservoirs for Borrelia lusitaniae in Portugal. Environ Microbiol Rep 7, 188–193
Parola P, Paddock CD, Socolovschi C, Labruna MB, Mediannikov O, Kernif T, Abdad MY, Stenos J, Bitam I, Fournier PE, Raoult D (2013) Update on tick-borne rickettsioses around the world: a geographic approach. Clin Microbiol Rev 26:657–702
Pereira A, Parreira R, Cotao AJ, Nunes M, Vieira ML, Azevedo F, Campino L, Maia C (2018) Tick-borne bacteria and protozoa detected in ticks collected from domestic animals and wildlife in central and southern Portugal. Ticks Tick Borne Dis 9(2):225–234
Regnery RL, Spruill CL, Plikaytis BD (1991) Genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes. J Bacteriol 173(5):1576–1589
Remesar S, Castro-Scholten S, Cano-Terriza D, Díaz P, Morrondo P, Jiménez-Martin D, Rouco C, García-Bocanegra I (2022) Molecular identification of zoonotic Rickettsia species in Ixodidae parasitizing wild lagomorphs from Mediterranean ecosystems. Transbound Emerg Dis 69(4):e992–e1004
Remesar S, Matute R, Díaz P, Martínez-Calabuig N, Prieto A, Díaz‐Cao JM, López-Lorenzo G, Fernández G, López C, Panadero R, Díez-Baños P, Morrondo P, García‐Dios D (2023) Tick‐borne pathogens in ticks from urban and suburban areas of north‐western Spain: importance of Ixodes frontalis harbouring zoonotic pathogens. Med Vet Entom 1:1–12
Roux V, Fournier PE, Raoult D (1996) Differentiation of spotted Fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of PCR-amplified DNA of the gene encoding the protein rOmpA. J Clin Microbiol 34(9):2058–2065
Sgroi G, Iatta R, Lia RP, D’Alessio N, Manoj RRS, Veneziano V, Otranto D (2020) Spotted Fever group rickettsiae in Dermacentor marginatus from wild boars in Italy. Transbound Emerg Dis 68(4):2111–2120
Tamura K, Glen S, Sudhir K (2021) MEGA11: Molecular Evolutionary Genetics Analysis version 11. Mol Biol Evol 38:3022–3027
Wolcott KA, Margos G, Fingerle V, Becker NS (2021) Host association of Borrelia burgdorferi sensu lato: a review. Ticks Tick Borne Dis 12(5):101766
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.
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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.
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Ortega, N., Arcenillas-Hernández, I., Villa, M. et al. Molecular identification of Borrelia and SFG Rickettsia spp. in hard ticks parasitizing domestic and wild animals in southeastern Spain. Vet Res Commun (2024). https://doi.org/10.1007/s11259-023-10292-x
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DOI: https://doi.org/10.1007/s11259-023-10292-x