High prevalence of the Mediterranean spotted fever agent Rickettsia aeschlimannii in Hyalomma marginatum ticks from Pianosa island, Italy

High prevalence of the Mediterranean spotted fever agent Rickettsia aeschlimannii in Hyalomma marginatum ticks from Pianosa island, Italy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report High prevalence of the Mediterranean spotted fever agent Rickettsia aeschlimannii in Hyalomma marginatum ticks from Pianosa island, Italy Patricia Alba, Valentina Tagliapietra, Alessia Franco, Leonardo Forbicioni, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7996641/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Feb, 2026 Read the published version in Parasites & Vectors → Version 1 posted 7 You are reading this latest preprint version Abstract Tick-borne rickettsioses, caused by bacteria of the spotted fever group (SFG) Rickettsia , are emerging diseases influenced by climate and land-use changes. Hyalomma spp. ticks in the protected area of Pianosa island, Tuscany (Italy), a renowned touristic location, were monitored and investigated in their bacterial community composition (575 specimens, composed in 120 pools) through metabarcoding sequencing. In our study, we report the high prevalence of Rickettsia aeschlimannii (58% glt-, and 75% 17kDa-Ag protein-positive pools), an agent of the SFG group in H. marginatum . These findings rise the warning on the risk of transmission of Spotted Fever rickettsiosis to residents and to international travellers and underline the need for enhanced surveillance, public awareness, and preventive measures. Rickettsia aeschlimannii Hyalomma marginatum Pianosa island metabarcoding genomics Figures Figure 1 Figure 2 Introduction Tick-borne rickettsioses are caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) of the Rickettsia genus [1]. The distribution of SFG rickettsioses varies geographically and correlates with the distribution of tick vectors. In Italy and Europe, Mediterranean Spotted Fever (MSF) has been associated with different Rickettsia spp . other than R. conorii such as: R. monacensis , R. massiliae , R. slovaca , R. helvetica , R. sibirica, R. raoulti, R. rioja and R. aeschlimannii [2,3]. Land use and climate changes are considered important drivers of rickettsiosis emergence altering the habitats and behaviours of ticks and their hosts [4]. In the frame of an eco-epidemiological monitoring programme focusing on Hyalomma spp.ticks carried out in the protected area of Pianosa island in Tuscany (Italy), a renowned touristic location, we investigated their bacterial microbiota through metabarcoding sequencing. The sampling site Pianosa island (Long. 10° 04' 44” E and Lat. 42° 35' 07” N) is the fifth, by extension, of the seven islands of the Archipelago Toscano National Park in Italy (https://www.parcoarcipelago.info/), with a total area of 10.25 km2. The territory of this island has witnessed intensive land use change since historical times. It was used as a penal colony from 1856 until 1998, with intense farming and breeding activities of various species, such as pigs, cows, sheep and chickens. Since 1996, it has become a natural protected area, and therefore most of the farming and breeding activities were removed. In 2014 a LIFE Nature project [5] was devoted to habitat restoration including the eradication of non-indigenous vertebrate species such as domestic farmed animals, rodent pests, feral cats, hedgehogs and game birds. The LIFE project also helped to highlight and clarify the genetic status of the local population of Lepus europaeus subsp. meridiei (Hilzheimer, 1906), considered the autochthonous form of the more widespread L. europaeus (European brown hare) in much of peninsular Italy. Pianosa island is one of the few refuge locations where this lineage still persists [6]. Wild rabbits and hares are known to play a significant ecological role in the life cycle of Hyalomma ticks in parts of Europe, including Italy [7]. Pianosa island is also known as an important stop-over and nesting site for migratory birds which are known crucial vertebrate hosts for ticks and tick-borne pathogens [8]. This island nowadays has become a highly attractive touristic site with 15-20,000 visitors per year [9] although, to protect the fragile ecosystem and uncontaminated nature of this site, the number of visitors is restricted to a maximum of 250 people per day. Surveillance of Hyalomma spp. Hyalomma spp. ticks were collected from the environment with CO 2 traps (Fig. 1B) activated with dry ice for 24 hours during eleven trapping sessions, from August 2021 to February 2023. In total 21 sampling points with one CO 2 trap each were randomly distributed on the island (Fig. 1A). From each trap a subsample of 30 ticks (if available) was collected in vials and sent alive to the Lazio and Tuscany Experimental Institute for Animal Health (IZSLT) in Rome. Among these 10 ticks were taken and placed in two vials of 5 individuals each. Occasionally, dead wild animals were also collected, whose bodies were brought to the same lab for further investigation. In total 1057 tick samples were collected and morphologically identified [14], 1055 belonging to Hyalomma marginatum and 2 belonging to Ixodes ventalloi species. Among these, 575 H. marginatum were analysed in 120 pools. Moreover, 30 samples of different organs (liver, spleen, lung, brain, kidney, heart, gut content, skin, nasal and tracheal swabs) from 4 wild dead animals (two Brown hares, Lepus europaeus subsp meridiei , one Yellow-legged gull, Larus michahellis, and one House mouse, Mus musculus ) were extracted. Metabarcoding analysis Total DNA from the samples was extracted using the QIA symphony Path/virus kit after mechanical homogenization and a 60 min (overnight for the tissues) incubation with proteinase K and lysozyme, according to manufacturer's instructions. The purified DNA was amplified with universal 16S rRNA gene primers for the V3-V4 regions. Amplicons were sequenced using Illumina technology (MiSeq). Raw reads were analysed using the Qiime2 suit-tool [10], in particular Dada tool [11] for the filtering and cleaning, and the taxonomical classification by using Silva-138 database [12] after a clusterization step of the obtained Amplicon Sequence Variants (ASVs) with a 99% of similarity. Rickettsiales species identification was carried out using amplicon sequencing for two specific genes used for the citrate synthase-encoding gene ( glt ) , and the 17 kDa antigenic protein-encoding gene (17 kDa-Ag protein) of R. rickettsii [13]. The most prevalent AVSs were identified by using BLAST with nr/nt database. Results and discussion The 16s rRNA gene amplicon results indicated a high prevalence of Rickettsiales order in the H. marginatum pools. The g lt positive pools were 70/120 (58.34%, 95% C.I. 48.98–67.26%) while the 17 kDa-Ag protein positive pools were 90/120 (75.00%, 95% C.I. 66.27–82.45%). Animal tissue samples resulted 100% positive for both targets. All the amplicons (from positive pools and tissue samples) were identified as Rickettsia aeschlimannii , one of the causal agents of the Mediterranean Spotted Fever group [ 2 , 15 ]. H. marginatum is a known vector of important emerging infectious diseases such as Crimean-Congo Haemorrhagic Fever (CCHF) or Mediterranean spotted fever (MSF), thus representing an emerging public health issue. To complete its life cycle, immature H. marginatum stages feed mainly on birds and small vertebrate species (Rodentia and Lagomorpha), while adults mostly prefer large ruminants of the Bovidae family [ 7 ]. At the end of the 90s the island became a natural protected area and most of the farming and breeding activities were removed. In addition, the Park initiated in 2017, and mostly achieved, an intense eradication programme of non-autochthonous species such as domestic farmed animals, rodent pests, pheasants, (hybrid) red-legged partridges, feral cats and hedgehogs [ 5 , 16 – 18 ], consequently lowering the biomass of vertebrate hosts left on the island. Since then, the presence of H. marginatum ticks has been reported to cause considerable disturbance by the few residents and tourists, especially during summer months. In this study we were able to assess the pervasive presence of this tick species by collecting samples from the ground and occasionally from carcasses. Notably, the life cycle of H. marginatum and its actual presence on the island seems to be mainly supported by the local population of the vertebrate host L. europaeus subsp. meridiei where all tick stages were detected (Fig. 2 ). This vertebrate host, which is considered a relic and unique endemic population of the European brown hare ( L. europaeus Pallas, 1778) due to its isolation and distinct genetic characteristics [ 19 ] is constrained in the limited space availability of the island (10 km2) and exhibits a high population density (about 260 hares: 0.26 hare/ha) [ 20 ], thus representing an abundant and easy source of food for ticks. Moreover, the typical Mediterranean ecosystem of the island is facing an increase in extreme drought and hot conditions in recent years [ 21 ], together with the re-colonization of abandoned agricultural land by natural vegetation (in particular Mediterranean scrub) [ 22 ], which together with the continuous influx of ticks carried by migratory birds might have sustained and favoured the abundance of Hyalomma ticks. R. aeschlimannii may have spread across Europe through infected ticks carried by several migratory bird species [ 23 – 26 ]. The presence of R. aeschlimannii in ticks belonging to the genus Hyalomma has been already reported in Italy [ 23 , 27 ] and in other European countries [ 28 ], but this is the first time that such a high prevalence is detected in a consistent number of host seeking specimens collected from the field. Indeed, another population study recently conducted in the Mediterranean area (Occitanie region, France) by Joly-Kukla and colleagues [ 29 ] has also revealed a high prevalence of R. aeschlimanni in Hyalomma ticks, and a total positivity in egg pools from infected females. These features were considered suggestive by those authors of its possible role as a tick symbiont in H. marginatum . Nonetheless, the 100% positivity of the samples from the dead animal tissues/organs examined in our study, suggests a transmission cycle of the pathogen through infected ticks. Apparently, MSF cases due to R. aeschlimannii have been increasing in southern Europe and in other regions [ 2 , 30 ]. Indeed, the role of R. aeschlimannii infection and MSF disease occurrence in humans deserves more studies, due to the limited number of so far reported cases where the aetiology of the MSF has been deeply investigated and correctly attributed. Usually, symptoms range from fever and sore throat to maculopapular rashes, and in one case of R. aeschlimanni infection described by Tosoni and colleagues, to acute hepatitis [ 15 ]. Factors like climate change, animal migration routes and land use practices changes may have been contributing to an expanding geographic range of Hyalomma ticks, favouring potential increase in human exposure and infection. In view of the above, the abundance of H. marginatum coupled with the high prevalence rates of R. aeschlimanni could increase the risk of transmission of the agent to the residents and the tourists visiting Pianosa island. Occasionally, local social media reported the overwhelming presence of “giant ticks” on the island, and even tourists attacked by them. Although the Park authorities have already produced some informational material about the risks of tick exposure, a dedicated action plan aimed at increasing awareness regarding the presence of ticks should be implemented. It may consist of information campaigns using social media, trained staff, local tour operators and the national park website, informing also on the best practices to reduce the risk of infection (i.e. use of repellents, protective clothing, restrictions to walk out of cleared paths, daily tick body check for people and pets). Targeted control action through acaricides, since its use in protected areas is often controversial, could be also evaluated and limited to selected areas, such as the beach open to the public, recreational areas, the small village and meeting areas. All these prevention measures may help reduce the risk of tick exposure and of R. aeschlimanni infection for locals and visitors. In conclusion, this study highlights the high prevalence of R. aeschlimanni , a known and emerging human pathogen, in a population of H. marginatum ticks on Pianosa island and in dead vertebrate hosts there collected. In the light of the importance of the Tuscan Archipelago National Park islands for the avian migratory routes and of the pressure of climate change and tourism heavily affecting its fragile ecosystem, the information provided by this study is crucial for raising awareness on the potential health risk caused by arthropod vectors and arthropod-borne disease agents. The value of small-scale studies should not be overlooked when considered within a wider context. The unexpected adaptation of Hyalomma ticks to the only available vertebrate host due to contingent external factors deserves more attention, together with a longer monitoring effort to evaluate variation in Hyalomma tick abundance and R. aeschlimannii prevalence in relation to ecological, environmental and climatic factors. Particular attention should also be devoted to the role of migratory birds in the continuous introduction of infected ticks, especially in view of their potential role to carry other dangerous infectious diseases. Declarations Acknowledgements We thank the Park authorities and its personnel and the Carabinieri Nucleo Forestale - Reparto Arcipelago Toscano for their fundamental support and collaboration in facilitating logistic and fieldwork on Pianosa Island. Funding This study was funded by the Tuscan Archipelago National Park, (contract n. 9232707ED4). The work by Istituto Zooprofilattico Sperimentale del Lazio e della Toscana “M. Aleandri” (IZSLT), Italy was partially supported by the European Union funding from the NextGeneration EU-MUR PNRR Extended Partnership Initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT, PE13 INF-ACT, Nodes 2 and 4). Availability of data and materials The sequences have been submitted to the European nucleotide archive on 26th August 2025 under a single project with PRJEB96402 accession number. Authors’ contributions PA, AB, AR and VT conceived the manuscript. PA, AF, FS, AI, RO, and AB generated the laboratory data. VT, DA, SC and MC performed data collection. PA, AB and VT prepared a draft of the manuscript. PA, VT, AF, LF, SC, MC, FS, RO, AI, DA, CD, AB and AR critically revised the manuscript and all authors reviewed it. Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing interests Not applicable References Piotrowski M, Rymaszewska A. Expansion of Tick-Borne Rickettsioses in the World. 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Cite Share Download PDF Status: Published Journal Publication published 03 Feb, 2026 Read the published version in Parasites & Vectors → Version 1 posted Editorial decision: Revision requested 08 Nov, 2025 Reviews received at journal 07 Nov, 2025 Reviewers agreed at journal 03 Nov, 2025 Reviewers invited by journal 02 Nov, 2025 Editor assigned by journal 02 Nov, 2025 Submission checks completed at journal 01 Nov, 2025 First submitted to journal 31 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7996641","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":542040598,"identity":"6c53d792-ff90-4b04-973d-75a1a7add9bb","order_by":0,"name":"Patricia Alba","email":"","orcid":"","institution":"Istituto Zooprofilattico Sperimentale del Lazio e della Toscana “M. 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15:11:59","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":81537,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7996641/v1/38d610c16ecba6e99fac33f3.html"},{"id":95748269,"identity":"8d832e8f-57c7-4ca1-934b-5e6b97379bf8","added_by":"auto","created_at":"2025-11-12 15:11:58","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1193478,"visible":true,"origin":"","legend":"\u003cp\u003eA-Map of Pianosa island (Archipelago Toscano National Park, Italy) and monitoring sites (green circles). B- Example of CO\u003csub\u003e2 \u003c/sub\u003etraps for \u003cem\u003eHyalomma\u003c/em\u003e ticks collection.\u003c/p\u003e","description":"","filename":"Figure1AB.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7996641/v1/fa26703e1a91b719d7f420ad.jpeg"},{"id":95748272,"identity":"b1e38bc8-e594-4e16-94c6-7ca9a69a95d7","added_by":"auto","created_at":"2025-11-12 15:11:59","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3071081,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHyalomma \u003c/em\u003espp. ticks on the ear of a dead \u003cem\u003eL. europaeus \u003c/em\u003esubsp. \u003cem\u003emeridiei\u003c/em\u003e(Pianosa Island, Italy) (Photo V. Tagliapietra).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7996641/v1/61955a50c696c6493be28c43.jpg"},{"id":102234170,"identity":"c55b0464-144e-459e-a451-e709a076dd39","added_by":"auto","created_at":"2026-02-09 16:07:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4772700,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7996641/v1/c317b5d2-6b31-47a3-84a8-3c7f93513da8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"High prevalence of the Mediterranean spotted fever agent Rickettsia aeschlimannii in Hyalomma marginatum ticks from Pianosa island, Italy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTick-borne rickettsioses are caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) of the \u003cem\u003eRickettsia\u0026nbsp;\u003c/em\u003egenus [1]. The distribution of SFG rickettsioses varies geographically and correlates with the distribution of tick vectors. In Italy and Europe, Mediterranean Spotted Fever (MSF) has been associated with different \u003cem\u003eRickettsia\u0026nbsp;\u003c/em\u003espp\u003cem\u003e.\u003c/em\u003e other than \u003cem\u003eR. conorii\u0026nbsp;\u003c/em\u003esuch as: \u003cem\u003eR. monacensis\u003c/em\u003e, \u003cem\u003eR. massiliae\u003c/em\u003e, \u003cem\u003eR. slovaca\u003c/em\u003e, \u003cem\u003eR. helvetica\u003c/em\u003e, \u003cem\u003eR. sibirica, R. raoulti, R. rioja\u003c/em\u003e and \u003cem\u003eR. aeschlimannii\u003c/em\u003e [2,3]. Land use and climate changes are considered important drivers of rickettsiosis emergence altering the habitats and behaviours of ticks and their hosts [4]. In the frame of an eco-epidemiological monitoring programme focusing on \u003cem\u003eHyalomma\u0026nbsp;\u003c/em\u003espp.ticks carried out in the protected area of Pianosa island in Tuscany (Italy), a renowned touristic location, we investigated their bacterial microbiota through metabarcoding sequencing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe sampling site\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePianosa island (Long. 10° 04' 44” E and Lat. 42° 35' 07” N) is the fifth, by extension, of the seven islands of the Archipelago Toscano National Park in Italy (https://www.parcoarcipelago.info/), with a total area of 10.25 km2. The territory of this island has witnessed intensive land use change since historical times. It was used as a penal colony from 1856 until 1998, with intense farming and breeding activities of various species, such as pigs, cows, sheep and chickens. Since 1996, it has become a natural protected area, and therefore most of the farming and breeding activities were removed. In 2014 a LIFE Nature project [5] was devoted to habitat restoration including the eradication of non-indigenous vertebrate species such as domestic farmed animals, rodent pests, feral cats, hedgehogs and game birds. The LIFE project also helped to highlight and clarify the genetic status of the local population of \u003cem\u003eLepus europaeus\u003c/em\u003e subsp. \u003cem\u003emeridiei\u003c/em\u003e (Hilzheimer, 1906), considered the autochthonous form of the more widespread \u003cem\u003eL. europaeus\u003c/em\u003e (European brown hare) in much of peninsular Italy. Pianosa island is one of the few refuge locations where this lineage still persists [6]. Wild rabbits and hares are known to play a significant ecological role in the life cycle of \u003cem\u003eHyalomma\u003c/em\u003e ticks in parts of Europe, including Italy [7]. Pianosa island is also known as an important stop-over and nesting site for migratory birds which are known crucial vertebrate hosts for ticks and tick-borne pathogens [8]. This island nowadays has become a highly attractive touristic site with 15-20,000 visitors per year [9] although, to protect the fragile ecosystem and uncontaminated nature of this site, the number of visitors is restricted to a maximum of 250 people per day.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSurveillance of\u003cem\u003e\u0026nbsp;Hyalomma\u0026nbsp;\u003c/em\u003espp.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHyalomma\u003c/em\u003e spp. ticks were collected from the environment with CO\u003csub\u003e2\u003c/sub\u003e traps (Fig. 1B) activated with dry ice for 24 hours during eleven trapping sessions, from August 2021 to February 2023. In total 21 sampling points with one CO\u003csub\u003e2\u003c/sub\u003e trap each were randomly distributed on the island (Fig. 1A). From each trap a subsample of 30 ticks (if available) was collected in vials and sent alive to the Lazio and Tuscany Experimental Institute for Animal Health (IZSLT) in Rome. Among these 10 ticks were taken and placed in two vials of 5 individuals each. Occasionally, dead wild animals were also collected, whose bodies were brought to the same lab for further investigation. In total 1057 tick samples were collected and morphologically identified [14], 1055 belonging to \u003cem\u003eHyalomma marginatum\u0026nbsp;\u003c/em\u003eand 2 belonging to \u003cem\u003eIxodes ventalloi\u0026nbsp;\u003c/em\u003especies. Among these, 575 \u003cem\u003eH. marginatum\u003c/em\u003e were analysed in 120 pools. Moreover, 30 samples of different organs (liver, spleen, lung, brain, kidney, heart, gut content, skin, nasal and tracheal swabs) from 4 wild dead animals (two Brown hares, \u003cem\u003eLepus europaeus\u003c/em\u003e subsp \u003cem\u003emeridiei\u003c/em\u003e, one Yellow-legged gull, \u003cem\u003eLarus michahellis,\u0026nbsp;\u003c/em\u003eand one House mouse, \u003cem\u003eMus musculus\u003c/em\u003e) were extracted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabarcoding analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal DNA from the samples was extracted using the QIA symphony Path/virus kit after mechanical homogenization and a 60 min (overnight for the tissues) incubation with proteinase K and lysozyme, according to manufacturer's instructions. The purified DNA was amplified with universal 16S rRNA gene primers for the V3-V4 regions. Amplicons were sequenced using Illumina technology (MiSeq). Raw reads were analysed using the Qiime2 suit-tool [10], in particular Dada tool [11] for the filtering and cleaning, and the taxonomical classification by using Silva-138 database [12] after a clusterization step of the obtained Amplicon Sequence Variants (ASVs) with a 99% of similarity. \u003cem\u003eRickettsiales\u0026nbsp;\u003c/em\u003especies identification was carried out using amplicon sequencing for two specific genes used for the citrate synthase-encoding gene (\u003cem\u003eglt\u003c/em\u003e)\u003cem\u003e,\u0026nbsp;\u003c/em\u003eand the 17 kDa antigenic protein-encoding gene (17 kDa-Ag protein) of \u003cem\u003eR. rickettsii\u003c/em\u003e [13]. The most prevalent AVSs were identified by using BLAST with nr/nt database.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cp\u003eThe 16s rRNA gene amplicon results indicated a high prevalence of \u003cem\u003eRickettsiales\u003c/em\u003e order in the \u003cem\u003eH. marginatum\u003c/em\u003e pools. The g\u003cem\u003elt\u003c/em\u003e positive pools were 70/120 (58.34%, 95% C.I. 48.98\u0026ndash;67.26%) while the 17 kDa-Ag protein positive pools were 90/120 (75.00%, 95% C.I. 66.27\u0026ndash;82.45%). Animal tissue samples resulted 100% positive for both targets. All the amplicons (from positive pools and tissue samples) were identified as \u003cem\u003eRickettsia aeschlimannii\u003c/em\u003e, one of the causal agents of the Mediterranean Spotted Fever group [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eH. marginatum\u003c/em\u003e is a known vector of important emerging infectious diseases such as Crimean-Congo Haemorrhagic Fever (CCHF) or Mediterranean spotted fever (MSF), thus representing an emerging public health issue. To complete its life cycle, immature \u003cem\u003eH. marginatum\u003c/em\u003e stages feed mainly on birds and small vertebrate species (Rodentia and Lagomorpha), while adults mostly prefer large ruminants of the Bovidae family [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAt the end of the 90s the island became a natural protected area and most of the farming and breeding activities were removed. In addition, the Park initiated in 2017, and mostly achieved, an intense eradication programme of non-autochthonous species such as domestic farmed animals, rodent pests, pheasants, (hybrid) red-legged partridges, feral cats and hedgehogs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], consequently lowering the biomass of vertebrate hosts left on the island. Since then, the presence of \u003cem\u003eH. marginatum\u003c/em\u003e ticks has been reported to cause considerable disturbance by the few residents and tourists, especially during summer months.\u003c/p\u003e\u003cp\u003eIn this study we were able to assess the pervasive presence of this tick species by collecting samples from the ground and occasionally from carcasses. Notably, the life cycle of \u003cem\u003eH. marginatum\u003c/em\u003e and its actual presence on the island seems to be mainly supported by the local population of the vertebrate host \u003cem\u003eL. europaeus\u003c/em\u003e subsp. \u003cem\u003emeridiei\u003c/em\u003e where all tick stages were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This vertebrate host, which is considered a relic and unique endemic population of the European brown hare (\u003cem\u003eL. europaeus\u003c/em\u003e Pallas, 1778) due to its isolation and distinct genetic characteristics [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] is constrained in the limited space availability of the island (10 km2) and exhibits a high population density (about 260 hares: 0.26 hare/ha) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], thus representing an abundant and easy source of food for ticks. Moreover, the typical Mediterranean ecosystem of the island is facing an increase in extreme drought and hot conditions in recent years [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], together with the re-colonization of abandoned agricultural land by natural vegetation (in particular Mediterranean scrub) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], which together with the continuous influx of ticks carried by migratory birds might have sustained and favoured the abundance of \u003cem\u003eHyalomma\u003c/em\u003e ticks.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eR. aeschlimannii\u003c/em\u003e may have spread across Europe through infected ticks carried by several migratory bird species [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The presence of \u003cem\u003eR. aeschlimannii\u003c/em\u003e in ticks belonging to the genus \u003cem\u003eHyalomma\u003c/em\u003e has been already reported in Italy [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and in other European countries [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], but this is the first time that such a high prevalence is detected in a consistent number of host seeking specimens collected from the field. Indeed, another population study recently conducted in the Mediterranean area (Occitanie region, France) by Joly-Kukla and colleagues [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] has also revealed a high prevalence of \u003cem\u003eR. aeschlimanni\u003c/em\u003e in \u003cem\u003eHyalomma\u003c/em\u003e ticks, and a total positivity in egg pools from infected females. These features were considered suggestive by those authors of its possible role as a tick symbiont in \u003cem\u003eH. marginatum\u003c/em\u003e. Nonetheless, the 100% positivity of the samples from the dead animal tissues/organs examined in our study, suggests a transmission cycle of the pathogen through infected ticks.\u003c/p\u003e\u003cp\u003eApparently, MSF cases due to \u003cem\u003eR. aeschlimannii\u003c/em\u003e have been increasing in southern Europe and in other regions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Indeed, the role of \u003cem\u003eR. aeschlimannii\u003c/em\u003e infection and MSF disease occurrence in humans deserves more studies, due to the limited number of so far reported cases where the aetiology of the MSF has been deeply investigated and correctly attributed. Usually, symptoms range from fever and sore throat to maculopapular rashes, and in one case of \u003cem\u003eR. aeschlimanni\u003c/em\u003e infection described by Tosoni and colleagues, to acute hepatitis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Factors like climate change, animal migration routes and land use practices changes may have been contributing to an expanding geographic range of \u003cem\u003eHyalomma\u003c/em\u003e ticks, favouring potential increase in human exposure and infection. In view of the above, the abundance of \u003cem\u003eH. marginatum\u003c/em\u003e coupled with the high prevalence rates of \u003cem\u003eR. aeschlimanni\u003c/em\u003e could increase the risk of transmission of the agent to the residents and the tourists visiting Pianosa island.\u003c/p\u003e\u003cp\u003eOccasionally, local social media reported the overwhelming presence of \u0026ldquo;giant ticks\u0026rdquo; on the island, and even tourists attacked by them. Although the Park authorities have already produced some informational material about the risks of tick exposure, a dedicated action plan aimed at increasing awareness regarding the presence of ticks should be implemented. It may consist of information campaigns using social media, trained staff, local tour operators and the national park website, informing also on the best practices to reduce the risk of infection (i.e. use of repellents, protective clothing, restrictions to walk out of cleared paths, daily tick body check for people and pets). Targeted control action through acaricides, since its use in protected areas is often controversial, could be also evaluated and limited to selected areas, such as the beach open to the public, recreational areas, the small village and meeting areas. All these prevention measures may help reduce the risk of tick exposure and of \u003cem\u003eR. aeschlimanni\u003c/em\u003e infection for locals and visitors.\u003c/p\u003e\u003cp\u003eIn conclusion, this study highlights the high prevalence of \u003cem\u003eR. aeschlimanni\u003c/em\u003e, a known and emerging human pathogen, in a population of \u003cem\u003eH. marginatum\u003c/em\u003e ticks on Pianosa island and in dead vertebrate hosts there collected. In the light of the importance of the Tuscan Archipelago National Park islands for the avian migratory routes and of the pressure of climate change and tourism heavily affecting its fragile ecosystem, the information provided by this study is crucial for raising awareness on the potential health risk caused by arthropod vectors and arthropod-borne disease agents.\u003c/p\u003e\u003cp\u003eThe value of small-scale studies should not be overlooked when considered within a wider context. The unexpected adaptation of \u003cem\u003eHyalomma\u003c/em\u003e ticks to the only available vertebrate host due to contingent external factors deserves more attention, together with a longer monitoring effort to evaluate variation in \u003cem\u003eHyalomma\u003c/em\u003e tick abundance and \u003cem\u003eR. aeschlimannii\u003c/em\u003e prevalence in relation to ecological, environmental and climatic factors. Particular attention should also be devoted to the role of migratory birds in the continuous introduction of infected ticks, especially in view of their potential role to carry other dangerous infectious diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Park authorities and its personnel and the Carabinieri Nucleo Forestale - Reparto Arcipelago Toscano for their fundamental support and collaboration in facilitating logistic and fieldwork on Pianosa Island.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Tuscan Archipelago National Park, (contract n. 9232707ED4).\u003c/p\u003e\n\u003cp\u003eThe work by Istituto Zooprofilattico Sperimentale del Lazio e della Toscana \u0026ldquo;M. Aleandri\u0026rdquo; (IZSLT), Italy was partially supported by the European Union funding from the NextGeneration EU-MUR PNRR Extended Partnership Initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT, PE13 INF-ACT, Nodes 2 and 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequences have been submitted to the European nucleotide archive on 26th August 2025 under a single project with PRJEB96402 accession number.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePA, AB, AR and VT conceived the manuscript. PA, AF, FS, AI, RO, and AB generated the laboratory data. VT, DA, SC and MC performed data collection. PA, AB and VT prepared a draft of the manuscript.\u0026nbsp;PA, VT, AF, LF, SC, MC, FS, RO, AI, DA, CD, AB and AR critically revised the manuscript and all authors reviewed it.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePiotrowski M, Rymaszewska A. Expansion of Tick-Borne Rickettsioses in the World. Microorganisms. 2020;8(12):1906.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOteo JA, Portillo A. Tick-borne rickettsioses in Europe. 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Genomic characteristics of emerging human pathogen \u003cem\u003eRickettsia aeschlimannii\u003c/em\u003e isolated from two \u003cem\u003eHyalomma\u003c/em\u003e tick species. iScience. 2025;28(3):112080.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Rickettsia aeschlimannii, Hyalomma marginatum, Pianosa island, metabarcoding, genomics","lastPublishedDoi":"10.21203/rs.3.rs-7996641/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7996641/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTick-borne rickettsioses, caused by bacteria of the spotted fever group (SFG) \u003cem\u003eRickettsia\u003c/em\u003e, are emerging diseases influenced by climate and land-use changes. \u003cem\u003eHyalomma\u003c/em\u003e spp. ticks in the protected area of Pianosa island, Tuscany (Italy), a renowned touristic location, were monitored and investigated in their bacterial community composition (575 specimens, composed in 120 pools) through metabarcoding sequencing. In our study, we report the high prevalence of \u003cem\u003eRickettsia aeschlimannii\u003c/em\u003e (58% glt-, and 75% 17kDa-Ag protein-positive pools), an agent of the SFG group in \u003cem\u003eH. marginatum\u003c/em\u003e. These findings rise the warning on the risk of transmission of Spotted Fever rickettsiosis to residents and to international travellers and underline the need for enhanced surveillance, public awareness, and preventive measures.\u003c/p\u003e","manuscriptTitle":"High prevalence of the Mediterranean spotted fever agent Rickettsia aeschlimannii in Hyalomma marginatum ticks from Pianosa island, Italy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-12 15:11:54","doi":"10.21203/rs.3.rs-7996641/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-08T21:46:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-07T13:45:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75036251380509733527742101122919500603","date":"2025-11-03T11:05:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-02T21:10:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-02T15:32:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-01T13:40:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasites \u0026 Vectors","date":"2025-10-31T09:01:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"63036cd2-0782-48cd-aabf-d1d06b76b141","owner":[],"postedDate":"November 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:03:07+00:00","versionOfRecord":{"articleIdentity":"rs-7996641","link":"https://doi.org/10.1186/s13071-026-07281-9","journal":{"identity":"parasites-and-vectors","isVorOnly":false,"title":"Parasites \u0026 Vectors"},"publishedOn":"2026-02-03 15:57:00","publishedOnDateReadable":"February 3rd, 2026"},"versionCreatedAt":"2025-11-12 15:11:54","video":"","vorDoi":"10.1186/s13071-026-07281-9","vorDoiUrl":"https://doi.org/10.1186/s13071-026-07281-9","workflowStages":[]},"version":"v1","identity":"rs-7996641","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7996641","identity":"rs-7996641","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}