First Record of Amblyomma fuscum (Acari: Ixodidae) on a Nine-Banded Armadillo (Dasypus novemcinctus) with Simultaneous Detection of Medically Important Pathogens: a case report

First Record of Amblyomma fuscum (Acari: Ixodidae) on a Nine-Banded Armadillo (Dasypus novemcinctus) with Simultaneous Detection of Medically Important Pathogens: a case report | 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 Research Article First Record of Amblyomma fuscum (Acari: Ixodidae) on a Nine-Banded Armadillo (Dasypus novemcinctus) with Simultaneous Detection of Medically Important Pathogens: a case report Willian Cardoso Ferreira Zorrer, Bibiana Rodrigues de Freitas, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7376330/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The nine-banded armadillo (Dasypus novemcinctus ) is a widely distributed wild mammal recognized as a reservoir for zoonotic pathogens like Mycobacterium leprae and Trypanosoma cruzi . This study reports the first record of the rare tick Amblyomma fuscum , a species historically associated with reptilian hosts, parasitizing a D. novemcinctus in Pelotas, Rio Grande do Sul, Brazil, detected through opportunistic sampling of a road-killed specimen. During necropsy, spleen, heart, liver, and blood samples were collected, and total genomic DNA was extracted using the TRIzol® method, which involves phenol–guanidine isothiocyanate-based cell lysis, chloroform phase separation, and ethanol DNA precipitation. PCR assays targeting the Mycobacterium spp., Rickettsia spp. and T. cruzi were performed on spleen tissue, confirming the presence of Mycobacterium spp. and T. cruzi . The tick was tested for Rickettsia spp., Spotted Fever Group Rickettsia spp., Mycobacterium leprae , Babesia spp., Borrelia spp. and B. burgdorferi , Theileria sp., Anaplasma phagocytophilum , and Ehrlichia spp., with M. leprae being the only detected pathogen. This finding expands the known host range of A. fuscum to mammals, challenging the hypothesis of its strict specificity to reptiles, and underscores the epidemiological relevance of D. novemcinctus as a sentinel species. The simultaneous detection of zoonotic agents in both host and ectoparasite highlights the value of systematic monitoring of road-killed fauna for understanding host–parasite–pathogen interactions and assessing public health risks at the wildlife–human interface. Ticks Wildlife Parasite-Host Interaction Mycobacterium Trypanosoma Figures Figure 1 Figure 2 Introduction The tick Amblyomma fuscum is a poorly known species originally described from specimens collected on Boa constrictor in South America (Neumann, 1907 ). Subsequent records include findings on the colubrid snake Drymarchon corais (DANTAS-TORRES et al. 2008 ) and sporadic reports from southern Brazil (Santa Catarina and Rio Grande do Sul states). Despite its historical documentation, A. fuscum has been omitted from some taxonomic catalogs (KEIRANS 1992 , HORAK et al. 2002 ), though recognized as valid by others (CAMICAS et al. 1998 , MARTINS et al. 2010 ). Wild mammals serve as important hosts for tick surveillance, often revealing novel parasite-host associations or range expansions. The nine-banded armadillo ( Dasypus novemcinctus ), a widely distributed xenarthran from the southern United States to northern Argentina, represents a particularly significant host due to its zoonotic potential as a reservoir for pathogens like Mycobacterium leprae and Trypanosoma cruzi (GOMES et al. 2016 ). While armadillos are known hosts for ticks such as Amblyomma auricularium , records of other Amblyomma species on these mammals remain scarce. Here, we document the first occurrence of a male A. fuscum parasitizing a road-killed D. novemcinctus found on Rio Grande do Sul, Brazil. This finding not only expands the known host range and geographic distribution of this rare tick species but also highlights the value of opportunistic sampling from roadkill for ectoparasite studies. Such findings are critical for tracking ectoparasite ecology, clarifying taxonomic uncertainties, and assessing potential zoonotic risks associated with emerging host-parasite interactions in changing environments where wildlife-vehicle collisions may facilitate parasite dispersal. Materials and Methods Sample Collection and Processing An adult male nine-banded armadillo ( Dasypus novemcinctus ) was found dead from a roadkill incident on highway BR-392, in the Cascata district (31°35'21.6"S 52°31'57.1"W). The specimen showed no signs of visceral expulsion or myiasis and was transported in a biological bag to the Pathology Laboratory of the Faculty of Veterinary Medicine at the Federal University of Pelotas for examination. Ectoparasite Collection and Morphological Identification During the necropsy, a single tick was carefully removed from the inter-band region near the tail using fine-tipped forceps, following the axial rotation technique (PEREIRA et al. 2012) to preserve taxonomically important structures. The ectoparasite was preserved in 70% ethanol and subsequently sent to Embrapa Gado de Corte (Campo Grande, MS) for analysis. Morphological identification was performed by stereomicroscopy, using standard dichotomous keys for Ixodidae (ARAGÃO & FONSECA 1961; GUIMARÃES et al. 2001; BARROS-BATTESTI et al . 2006). Sample Collection and DNA Extraction For molecular investigation, biological samples were collected from an armadillo ( Dasypus novemcinctus ) and a tick specimen. During the armadillo's necropsy, 4 mL of whole blood were collected by cardiac puncture, along with fragments of the heart, liver, and spleen. The tick specimen was stored for subsequent taxonomic identification and molecular analysis. Genomic DNA was extracted from all samples (blood, tissues, and tick) using TRIzol® Reagent, a method based on a phenol and guanidine isothiocyanate solution that promotes cell lysis and protein denaturation. Following homogenization in the reagent, chloroform was added to the lysate, and subsequent centrifugation separated the mixture into an upper aqueous phase containing a DNA-rich phase and a lower organic phase. The DNA was then specifically precipitated from the interphase and organic layer with ethanol. Finally, the resulting DNA pellet was washed to remove impurities, air-dried, and resuspended in an appropriate buffer to be used as a template in PCR reactions. Molecular Detection by Polymerase Chain Reaction (PCR) Molecular analyses were conducted at two distinct institutions: the Federal University of Pelotas (UFPel) and Embrapa Gado de Corte. All PCR assays included positive and negative controls to ensure the validity of the results. The reactions were performed on a T100™ Thermal Cycler (Bio-Rad Laboratories). Table 1: Molecular protocols used for pathogen investigation in the nine-banded armadillo and its tick ectoparasite Pathogen Targeted Gene/Sequence Fragment Size Primers Reference Analyses performed at the Veterinary Molecular Biology Laboratory (UFPel) Mycobacterium spp. 16S rRNA gene 1030 bp MYCGEN-F and MYCGEN-R WILTON et al. (1992) Rickettsia spp. gltA gene 401 bp CS78 and CS323 LABRUNA et al. (2004) Trypanosoma cruzi Kinetoplast DNA (kDNA) 330 bp 121 and 122 SCHJIMAN et al. (2011) Analyses performed at Embrapa Gado de Corte (Campo Grande, MS) - in tick specimen Rickettsia spp. gltA gene 401 bp CS78 and CS323 LABRUNA et al. (2004) Spotted Fever Group Rickettsia spp. ompA gene 530 bp Rr190.70p and Rr190.602n LABRUNA et al. (2004) Mycobacterium leprae RLEP repetitive sequence 129 bp LP1 and LP2 DA SILVA et al. (2018) Babesia spp. 18S rRNA gene 600 bp KB-16 and KB-17 GUERRERO et al. (2007) Borrelia spp. and B.burgdorferi Flagellin B (flaB) and flagellar hook (flgE) genes 353 bp flaLL-flaRL and flgE BARBOUR et al. (1996); SAL et al. (2008) Theileria sp. and Babesia sp. 18S rRNA gene 426 bp ThF and ThR SLEMENDA et al . (2005) Anaplasma phagocytophilum 16S rRNA gene 526 bp Ge3a and Ge10 SILAGHI et al . (2011) Ehrlichia spp. 16S rRNA gene 389 bp ECC and ECB DAWSON et al. (1996) Results and Discussion The physical examination of a road-killed Dasypus novemcinctus in Pelotas, Rio Grande do Sul, Brazil, revealed the presence of a male tick identified as Amblyomma fuscum . Tissue samples from the armadillo were subjected to PCR assays targeting Mycobacterium spp., Rickettsia spp., and Trypanosoma cruzi . The analyses confirmed the presence of Mycobacterium spp. and T. cruzi . The tick specimen was also screened by PCR for Rickettsia spp., Spotted Fever Group Rickettsia spp., Mycobacterium leprae , Babesia spp., Borrelia spp. and B. burgdorferi , Theileria sp., Anaplasma phagocytophilum , and Ehrlichia spp. Among these, only M. leprae was detected. The identification of A. fuscum on D. novemcinctus is of particular scientific interest, as this tick is historically considered rare and strongly associated with reptilian hosts (ARAGÃO 1936; GUIMARÃES et al . 2001). This finding adds to growing evidence that A. fuscum exhibits broader host plasticity than previously assumed, challenging the notion of strict specificity for squamates (GUGLIELMONE et al. 2003). Morphological ambiguities in the taxonomy of A. fuscum , including inconsistent descriptions of hypostomal dentition (ROBINSON 1926) and overlooked ornamentation in preserved specimens, highlight the importance of integrative morpho-molecular approaches. The scarce records of the species in southern Brazil (from São Paulo to Rio Grande do Sul) and a single report in Pernambuco (CUNHA et al . 1999) suggest either limited distribution or under-sampling. By documenting A. fuscum on a mammalian host within the Pampa biome, this study provides new ecological data on a poorly known tick species, while simultaneously reporting the co-occurrence of zoonotic agents in both host and ectoparasite. The detection of T. cruzi and Mycobacterium spp. in armadillo tissues, along with M. leprae in its tick, underscores the epidemiological significance of D. novemcinctus as a wildlife reservoir. Already recognized for its role in maintaining T. cruzi and M. leprae transmission cycles, this species emerges here as a sentinel for active pathogen circulation in the region. The use of a road-killed specimen demonstrates the effectiveness of opportunistic sampling for documenting ectoparasite diversity and detecting circulating pathogens in wildlife. Systematic application of such strategies, coupled with morphological and molecular identification of ectoparasites, is essential to advance our understanding of host–parasite–pathogen networks and to evaluate zoonotic risks in underexplored ecosystems. Declarations This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES). The authors have no relevant financial or non-financial interests to disclose. All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Willian Cardoso Ferreira, Bibiana Rodrigues de Freitas and Marcos Valério Garcia. The first draft of the manuscript was written by Willian Cardoso Ferreira and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. All procedures involving animals were approved by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) and conducted under license number 96569 and Animal Experimentation Ethics Committee (CEUA-UFPel), approval No. 182/2024/CEUA (Process No. 23110.038090/2024-45).. The study was performed in accordance with institutional and national guidelines for the ethical treatment of animals. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Author Contribution Willian Cardoso Ferreira: Wrote the main manuscript and molecular analysisBibiana Rodrigues de Freitas: Sample collectionMarcos Valério Garcia: Molecular analysisKauê Rodriguez Martins: Review and analysisRodrigo Casquero Cunha: Infrastructure and analysisFabio Raphael Pascoti Bruhn: Final review Acknowledgements We would like to thank the Veterinary Molecular Biology Laboratory, where sample processing and molecular analyses were carried out, and the Laboratory of Genetic Diversity and Evolution at the Federal University of Pelotas for their kind collaboration in capturing images of the ectoparasite - an essential component for the taxonomic assessment. References Aragão HB (1936) Ixodidas brasileiros e de alguns países limítrofes. Mem Inst Oswaldo Cruz 31:759-843 Barbour, Alan G. et al. Identification of an uncultivable Borrelia species in the hard tick Amblyomma americanum: possible agent of a Lyme disease-like illness. 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Re-description and molecular taxonomy of Babesia capreoli Enigk and Friedhoff, 1962 and Theileria capreoli Rukhlyadev, 1939-piroplasms of the European roe deer (Capreolus capreolus). 2005. Unpublished.[http://www. ncbi.nlm.nih.gov/nuccore/AY726008.1]. Szabó MPJ, Pinter A, Labruna MB (2007) Tick fauna from two locations in the Brazilian savannah. Exp Appl Acarol 43:73-84. https://doi.org/10.1007/s10493-007-9104-1 Wilton S, Cousins D (1992) Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. Genome Res 1(4):269-273. https://doi.org/10.1101/gr.1.4.269 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-7376330","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":525867109,"identity":"aa5568c7-1e3d-4d09-8fdc-da0f08edc309","order_by":0,"name":"Willian Cardoso Ferreira 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09:32:29","extension":"html","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":66217,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7376330/v1/0331d3af9aff3316766440ba.html"},{"id":93026540,"identity":"175c7c17-adbe-4f76-a35f-975530b1478d","added_by":"auto","created_at":"2025-10-08 09:32:28","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":429584,"visible":true,"origin":"","legend":"\u003cp\u003eCollection site of a nine-banded armadillo (\u003cem\u003eDasypus novemcinctus\u003c/em\u003e) specimen in the Cascata district of Pelotas, Brazil.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7376330/v1/d9a23501cca4c818ee0bfec1.jpeg"},{"id":93026548,"identity":"64ce7166-d3fa-48bb-9dc5-71c5d27e53b4","added_by":"auto","created_at":"2025-10-08 09:32:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":473497,"visible":true,"origin":"","legend":"\u003cp\u003eThe nine-banded armadillo, Dasypusnovemcinctus (1), and its ectoparasite, the tick Amblyommafuscum, highlighting morphological features: Spines (2), Detailed view of the spines on the tick’s leg (3), Dentition (4), Ventral view (5) and Dorsal view (6).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7376330/v1/a176bf72704b07dc3f797f47.png"},{"id":93027585,"identity":"07980ef4-30a4-4833-b62d-2388bae4c283","added_by":"auto","created_at":"2025-10-08 09:40:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1585446,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7376330/v1/16e43092-36b7-4513-95fb-186a00a37ea8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"First Record of Amblyomma fuscum (Acari: Ixodidae) on a Nine-Banded Armadillo (Dasypus novemcinctus) with Simultaneous Detection of Medically Important Pathogens: a case report","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe tick \u003cem\u003eAmblyomma fuscum\u003c/em\u003e is a poorly known species originally described from specimens collected on \u003cem\u003eBoa constrictor\u003c/em\u003e in South America (Neumann, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1907\u003c/span\u003e). Subsequent records include findings on the colubrid snake \u003cem\u003eDrymarchon corais\u003c/em\u003e (DANTAS-TORRES et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) and sporadic reports from southern Brazil (Santa Catarina and Rio Grande do Sul states). Despite its historical documentation, \u003cem\u003eA. fuscum\u003c/em\u003e has been omitted from some taxonomic catalogs (KEIRANS \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, HORAK et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), though recognized as valid by others (CAMICAS et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, MARTINS et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWild mammals serve as important hosts for tick surveillance, often revealing novel parasite-host associations or range expansions. The nine-banded armadillo (\u003cem\u003eDasypus novemcinctus\u003c/em\u003e), a widely distributed xenarthran from the southern United States to northern Argentina, represents a particularly significant host due to its zoonotic potential as a reservoir for pathogens like \u003cem\u003eMycobacterium leprae\u003c/em\u003e and \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e (GOMES et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). While armadillos are known hosts for ticks such as \u003cem\u003eAmblyomma auricularium\u003c/em\u003e, records of other Amblyomma species on these mammals remain scarce.\u003c/p\u003e\u003cp\u003eHere, we document the first occurrence of a male \u003cem\u003eA. fuscum\u003c/em\u003e parasitizing a road-killed \u003cem\u003eD. novemcinctus\u003c/em\u003e found on Rio Grande do Sul, Brazil. This finding not only expands the known host range and geographic distribution of this rare tick species but also highlights the value of opportunistic sampling from roadkill for ectoparasite studies. Such findings are critical for tracking ectoparasite ecology, clarifying taxonomic uncertainties, and assessing potential zoonotic risks associated with emerging host-parasite interactions in changing environments where wildlife-vehicle collisions may facilitate parasite dispersal.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch3\u003e\u003cem\u003eSample Collection and Processing\u003c/em\u003e\u0026nbsp;\u003c/h3\u003e\n\u003cp\u003eAn adult male nine-banded armadillo (\u003cem\u003eDasypus novemcinctus\u003c/em\u003e) was found dead from a roadkill incident on highway BR-392, in the Cascata district (31\u0026deg;35\u0026apos;21.6\u0026quot;S 52\u0026deg;31\u0026apos;57.1\u0026quot;W). The specimen showed no signs of visceral expulsion or myiasis and was transported in a biological bag to the Pathology Laboratory of the Faculty of Veterinary Medicine at the Federal University of Pelotas for examination.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eEctoparasite Collection and Morphological Identification\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eDuring the necropsy, a single tick was carefully removed from the inter-band region near the tail using fine-tipped forceps, following the axial rotation technique (PEREIRA \u003cem\u003eet al.\u003c/em\u003e 2012) to preserve taxonomically important structures. The ectoparasite was preserved in 70% ethanol and subsequently sent to Embrapa Gado de Corte (Campo Grande, MS) for analysis. Morphological identification was performed by stereomicroscopy, using standard dichotomous keys for Ixodidae (ARAG\u0026Atilde;O \u0026nbsp; \u0026amp; FONSECA 1961; GUIMAR\u0026Atilde;ES\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e 2001; BARROS-BATTESTI \u003cem\u003eet al\u003c/em\u003e. 2006).\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eSample Collection and DNA Extraction\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eFor molecular investigation, biological samples were collected from an armadillo (\u003cem\u003eDasypus novemcinctus\u003c/em\u003e) and a tick specimen. During the armadillo\u0026apos;s necropsy, 4 mL of whole blood were collected by cardiac puncture, along with fragments of the heart, liver, and spleen. The tick specimen was stored for subsequent taxonomic identification and molecular analysis. Genomic DNA was extracted from all samples (blood, tissues, and tick) using TRIzol\u0026reg; Reagent, a method based on a phenol and guanidine isothiocyanate solution that promotes cell lysis and protein denaturation. Following homogenization in the reagent, chloroform was added to the lysate, and subsequent centrifugation separated the mixture into an upper aqueous phase containing a DNA-rich phase and a lower organic phase. The DNA was then specifically precipitated from the interphase and organic layer with ethanol. Finally, the resulting DNA pellet was washed to remove impurities, air-dried, and resuspended in an appropriate buffer to be used as a template in PCR reactions.\u003c/p\u003e\n\u003ch3\u003e\u003cem\u003eMolecular Detection by Polymerase Chain Reaction (PCR)\u003c/em\u003e\u003c/h3\u003e\n\u003cp\u003eMolecular analyses were conducted at two distinct institutions: the Federal University of Pelotas (UFPel) and Embrapa Gado de Corte. All PCR assays included positive and negative controls to ensure the validity of the results. The reactions were performed on a T100\u0026trade; Thermal Cycler (Bio-Rad Laboratories).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Molecular protocols used for pathogen investigation in the nine-banded armadillo and its tick ectoparasite\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"687\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePathogen\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 117px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTargeted Gene/Sequence\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 74px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFragment Size\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePrimers\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width: 173px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eReference\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"18\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd height=\"33\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"bottom\" style=\"width: 687px;\"\u003e\n \u003cp\u003eAnalyses performed at the Veterinary Molecular Biology Laboratory (UFPel)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"21\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cem\u003eMycobacterium\u0026nbsp;\u003c/em\u003espp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e16S rRNA gene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e1030 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eMYCGEN-F and MYCGEN-R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eWILTON \u003cem\u003eet al.\u0026nbsp;\u003c/em\u003e(1992)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"50\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cem\u003eRickettsia\u0026nbsp;\u003c/em\u003espp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003egltA gene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e401 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eCS78 and CS323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eLABRUNA \u003cem\u003eet al.\u003c/em\u003e (2004)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"35\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cem\u003eTrypanosoma cruzi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003eKinetoplast DNA (kDNA)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e330 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e121 and 122\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eSCHJIMAN \u003cem\u003eet al.\u0026nbsp;\u003c/em\u003e(2011)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"35\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" style=\"width: 687px;\"\u003e\n \u003cp\u003eAnalyses performed at Embrapa Gado de Corte (Campo Grande, MS) - in tick specimen\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"21\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cem\u003eRickettsia\u0026nbsp;\u003c/em\u003espp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003egltA gene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e401 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eCS78 and CS323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eLABRUNA\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e (2004)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"35\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003eSpotted Fever Group \u003cem\u003eRickettsia\u0026nbsp;\u003c/em\u003espp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003eompA gene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e530 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eRr190.70p and Rr190.602n\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eLABRUNA\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e (2004)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"35\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cem\u003eMycobacterium leprae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003eRLEP repetitive sequence\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e129 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eLP1 and LP2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eDA SILVA \u003cem\u003eet al.\u003c/em\u003e (2018)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"35\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cem\u003eBabesia\u0026nbsp;\u003c/em\u003espp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e18S rRNA gene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e600 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eKB-16 and KB-17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eGUERRERO\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e (2007)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"35\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cem\u003eBorrelia\u0026nbsp;\u003c/em\u003espp. and\u003cem\u003e\u0026nbsp;B.burgdorferi\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003eFlagellin B (flaB) and flagellar hook (flgE) genes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e353 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eflaLL-flaRL and flgE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eBARBOUR\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e (1996); SAL\u003cem\u003e\u0026nbsp;et al.\u0026nbsp;\u003c/em\u003e(2008)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"65\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cem\u003eTheileria\u0026nbsp;\u003c/em\u003esp. and \u003cem\u003eBabesia\u0026nbsp;\u003c/em\u003esp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e18S rRNA gene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e426 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eThF and ThR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eSLEMENDA\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e. (2005)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"21\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cem\u003eAnaplasma phagocytophilum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e16S rRNA gene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e526 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eGe3a and Ge10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eSILAGHI\u003cem\u003e\u0026nbsp;et al\u003c/em\u003e. (2011)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"35\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 227px;\"\u003e\n \u003cp\u003e\u003cem\u003eEhrlichia\u0026nbsp;\u003c/em\u003espp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 117px;\"\u003e\n \u003cp\u003e16S rRNA gene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 74px;\"\u003e\n \u003cp\u003e389 bp\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003eECC and ECB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 173px;\"\u003e\n \u003cp\u003eDAWSON\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e (1996)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd height=\"35\" style=\"width: 0px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eThe physical examination of a road-killed \u003cem\u003eDasypus novemcinctus\u003c/em\u003e in Pelotas, Rio Grande do Sul, Brazil, revealed the presence of a male tick identified as\u003cem\u003e\u0026nbsp;Amblyomma fuscum\u003c/em\u003e. Tissue samples from the armadillo were subjected to PCR assays targeting \u003cem\u003eMycobacterium\u0026nbsp;\u003c/em\u003espp., \u003cem\u003eRickettsia\u0026nbsp;\u003c/em\u003espp., and \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e. The analyses confirmed the presence of \u003cem\u003eMycobacterium\u0026nbsp;\u003c/em\u003espp. and\u0026nbsp;\u003cem\u003eT. cruzi\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe tick specimen was also screened by PCR for \u003cem\u003eRickettsia\u0026nbsp;\u003c/em\u003espp., Spotted Fever Group \u003cem\u003eRickettsia\u0026nbsp;\u003c/em\u003espp.,\u003cem\u003e\u0026nbsp;Mycobacterium leprae\u003c/em\u003e, \u003cem\u003eBabesia\u0026nbsp;\u003c/em\u003espp., \u003cem\u003eBorrelia\u0026nbsp;\u003c/em\u003espp. and \u003cem\u003eB. burgdorferi\u003c/em\u003e, \u003cem\u003eTheileria\u0026nbsp;\u003c/em\u003esp., \u003cem\u003eAnaplasma phagocytophilum\u003c/em\u003e, and \u003cem\u003eEhrlichia\u0026nbsp;\u003c/em\u003espp. Among these, only \u003cem\u003eM. leprae\u003c/em\u003e was detected.\u003c/p\u003e\n\u003cp\u003eThe identification of \u003cem\u003eA. fuscum\u003c/em\u003e on \u003cem\u003eD. novemcinctus\u003c/em\u003e is of particular scientific interest, as this tick is historically considered rare and strongly associated with reptilian hosts (ARAG\u0026Atilde;O 1936; GUIMAR\u0026Atilde;ES \u003cem\u003eet al\u003c/em\u003e. 2001). This finding adds to growing evidence that A. fuscum exhibits broader host plasticity than previously assumed, challenging the notion of strict specificity for squamates (GUGLIELMONE \u003cem\u003eet al.\u0026nbsp;\u003c/em\u003e2003).\u003c/p\u003e\n\u003cp\u003eMorphological ambiguities in the taxonomy of \u003cem\u003eA. fuscum\u003c/em\u003e, including inconsistent descriptions of hypostomal dentition (ROBINSON 1926) and overlooked ornamentation in preserved specimens, highlight the importance of integrative morpho-molecular approaches. The scarce records of the species in southern Brazil (from S\u0026atilde;o Paulo to Rio Grande do Sul) and a single report in Pernambuco (CUNHA \u003cem\u003eet al\u003c/em\u003e. 1999) suggest either limited distribution or under-sampling.\u003c/p\u003e\n\u003cp\u003eBy documenting \u003cem\u003eA. fuscum\u003c/em\u003e on a mammalian host within the Pampa biome, this study provides new ecological data on a poorly known tick species, while simultaneously reporting the co-occurrence of zoonotic agents in both host and ectoparasite. The detection of \u003cem\u003eT. cruzi\u003c/em\u003e and \u003cem\u003eMycobacterium\u0026nbsp;\u003c/em\u003espp. in armadillo tissues, along with \u003cem\u003eM. leprae\u003c/em\u003e in its tick, underscores the epidemiological significance of \u003cem\u003eD. novemcinctus\u003c/em\u003e as a wildlife reservoir. Already recognized for its role in maintaining \u003cem\u003eT. cruzi\u003c/em\u003e and \u003cem\u003eM. leprae\u0026nbsp;\u003c/em\u003etransmission cycles, this species emerges here as a sentinel for active pathogen circulation in the region.\u003c/p\u003e\n\u003cp\u003eThe use of a road-killed specimen demonstrates the effectiveness of opportunistic sampling for documenting ectoparasite diversity and detecting circulating pathogens in wildlife. Systematic application of such strategies, coupled with morphological and molecular identification of ectoparasites, is essential to advance our understanding of host\u0026ndash;parasite\u0026ndash;pathogen networks and to evaluate zoonotic risks in underexplored ecosystems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThis work was supported by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; Brasil (CAPES). The authors have no relevant financial or non-financial interests to disclose. All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Willian Cardoso Ferreira, Bibiana Rodrigues de Freitas and Marcos Val\u0026eacute;rio Garcia. The first draft of the manuscript was written by Willian Cardoso Ferreira and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. All procedures involving animals were approved by the Instituto Chico Mendes de Conserva\u0026ccedil;\u0026atilde;o da Biodiversidade (ICMBio) and conducted under license number 96569 and Animal Experimentation Ethics Committee (CEUA-UFPel), approval No. 182/2024/CEUA (Process No. 23110.038090/2024-45).. The study was performed in accordance with institutional and national guidelines for the ethical treatment of animals. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eWillian Cardoso Ferreira: Wrote the main manuscript and molecular analysisBibiana Rodrigues de Freitas: Sample collectionMarcos Val\u0026eacute;rio Garcia: Molecular analysisKau\u0026ecirc; Rodriguez Martins: Review and analysisRodrigo Casquero Cunha: Infrastructure and analysisFabio Raphael Pascoti Bruhn: Final review\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eWe would like to thank the Veterinary Molecular Biology Laboratory, where sample processing and molecular analyses were carried out, and the Laboratory of Genetic Diversity and Evolution at the Federal University of Pelotas for their kind collaboration in capturing images of the ectoparasite - an essential component for the taxonomic assessment.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eArag\u0026atilde;o HB (1936) Ixodidas brasileiros e de alguns pa\u0026iacute;ses lim\u0026iacute;trofes. 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Notes from the Leyden Museum 29:88-100\u003c/li\u003e\n \u003cli\u003ePereira JS, Gurgel-Gon\u0026ccedil;alves R, de Sousa Alencar C et al (2012) Ectoparasitos em pre\u0026aacute;s (Galea spixii Wagler, 1831) cativos no semi\u0026aacute;rido do Rio Grande do Norte. Pesq Vet Bras 32(8):789-793. https://doi.org/10.1590/S0100-736X2012000800017\u003c/li\u003e\n \u003cli\u003eRegitano LCA, Coutinho LL (2001) Biologia molecular aplicada \u0026agrave; produ\u0026ccedil;\u0026atilde;o animal. Embrapa Informa\u0026ccedil;\u0026atilde;o Tecnol\u0026oacute;gica, Bras\u0026iacute;lia\u003c/li\u003e\n \u003cli\u003eRobinson LE (1926) Ticks. A monograph of the Ixodoidea. Part IV. The genus \u003cem\u003eAmblyomma\u003c/em\u003e. Cambridge University Press, Cambridge\u003c/li\u003e\n \u003cli\u003eSal, Melanie S. et al. Borrelia burgdorferi uniquely regulates its motility genes and has an intricate flagellar hook-basal body structure. Journal of bacteriology, v. 190, n. 6, p. 1912-1921, 2008.\u003c/li\u003e\n \u003cli\u003eSchjiman AG, Altcheh J, Burgos JM et al (2011) Aetiological treatment of congenital Chagas disease diagnosed and monitored by the polymerase chain reaction. J Antimicrob Chemother 66(4):876-880. https://doi.org/10.1093/jac/dkr034\u003c/li\u003e\n \u003cli\u003eSilaghi, C1 et al. PCR detection of Anaplasma phagocytophilum in goat flocks in an area endemic for tick-borne fever in Switzerland. Parasite: journal de la Soci\u0026eacute;t\u0026eacute; Fran\u0026ccedil;aise de Parasitologie, v. 18, n. 1, p. 57, 2011.\u003c/li\u003e\n \u003cli\u003eSlemenda, S. B. et al. Re-description and molecular taxonomy of Babesia capreoli Enigk and Friedhoff, 1962 and Theileria capreoli Rukhlyadev, 1939-piroplasms of the European roe deer (Capreolus capreolus). 2005. Unpublished.[http://www. ncbi.nlm.nih.gov/nuccore/AY726008.1].\u003c/li\u003e\n \u003cli\u003eSzab\u0026oacute; MPJ, Pinter A, Labruna MB (2007) Tick fauna from two locations in the Brazilian savannah. Exp Appl Acarol 43:73-84. https://doi.org/10.1007/s10493-007-9104-1\u003c/li\u003e\n \u003cli\u003eWilton S, Cousins D (1992) Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube. Genome Res 1(4):269-273. https://doi.org/10.1101/gr.1.4.269\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ticks, Wildlife, Parasite-Host Interaction, Mycobacterium, Trypanosoma","lastPublishedDoi":"10.21203/rs.3.rs-7376330/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7376330/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe nine-banded armadillo \u003cem\u003e(Dasypus novemcinctus\u003c/em\u003e) is a widely distributed wild mammal recognized as a reservoir for zoonotic pathogens like \u003cem\u003eMycobacterium leprae\u003c/em\u003e and \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e. This study reports the first record of the rare tick \u003cem\u003eAmblyomma fuscum\u003c/em\u003e, a species historically associated with reptilian hosts, parasitizing a \u003cem\u003eD. novemcinctus\u003c/em\u003e in Pelotas, Rio Grande do Sul, Brazil, detected through opportunistic sampling of a road-killed specimen. During necropsy, spleen, heart, liver, and blood samples were collected, and total genomic DNA was extracted using the TRIzol\u0026reg; method, which involves phenol\u0026ndash;guanidine isothiocyanate-based cell lysis, chloroform phase separation, and ethanol DNA precipitation. PCR assays targeting the \u003cem\u003eMycobacterium\u003c/em\u003e spp., \u003cem\u003eRickettsia\u003c/em\u003e spp. and \u003cem\u003eT. cruzi\u003c/em\u003e were performed on spleen tissue, confirming the presence of \u003cem\u003eMycobacterium\u003c/em\u003e spp. and \u003cem\u003eT. cruzi\u003c/em\u003e. \u003cem\u003eThe\u003c/em\u003e tick was tested for \u003cem\u003eRickettsia\u003c/em\u003e spp., Spotted Fever Group \u003cem\u003eRickettsia\u003c/em\u003e spp., \u003cem\u003eMycobacterium leprae\u003c/em\u003e, \u003cem\u003eBabesia\u003c/em\u003e spp., \u003cem\u003eBorrelia\u003c/em\u003e spp. and \u003cem\u003eB. burgdorferi\u003c/em\u003e, \u003cem\u003eTheileria\u003c/em\u003e sp., \u003cem\u003eAnaplasma phagocytophilum\u003c/em\u003e, and \u003cem\u003eEhrlichia\u003c/em\u003e spp., with \u003cem\u003eM. leprae\u003c/em\u003e being the only detected pathogen. This finding expands the known host range of \u003cem\u003eA. fuscum\u003c/em\u003e to mammals, challenging the hypothesis of its strict specificity to reptiles, and underscores the epidemiological relevance of \u003cem\u003eD. novemcinctus\u003c/em\u003e as a sentinel species. The simultaneous detection of zoonotic agents in both host and ectoparasite highlights the value of systematic monitoring of road-killed fauna for understanding host\u0026ndash;parasite\u0026ndash;pathogen interactions and assessing public health risks at the wildlife\u0026ndash;human interface.\u003c/p\u003e","manuscriptTitle":"First Record of Amblyomma fuscum (Acari: Ixodidae) on a Nine-Banded Armadillo (Dasypus novemcinctus) with Simultaneous Detection of Medically Important Pathogens: a case report","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 09:32:24","doi":"10.21203/rs.3.rs-7376330/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5b167bf7-b2fa-4cb6-9a91-107adab22b79","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-21T15:08:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 09:32:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7376330","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7376330","identity":"rs-7376330","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}