Detection of zoonotic Cryptosporidium parvum in invasive beavers from southern Tierra del Fuego, Chile

Detection of zoonotic Cryptosporidium parvum in invasive beavers from southern Tierra del Fuego, Chile | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Detection of zoonotic Cryptosporidium parvum in invasive beavers from southern Tierra del Fuego, Chile View ORCID Profile Paola Larrauri-Aguilar , View ORCID Profile Gabriela Contreras , View ORCID Profile Gonzalo Cabrera , View ORCID Profile Cristóbal Arredondo , View ORCID Profile Fernán Federici , View ORCID Profile Cristóbal Briceño doi: https://doi.org/10.1101/2025.11.13.688090 Paola Larrauri-Aguilar 1 Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Católica de Chile . Santiago, Chile 3 ANID - Millennium Science Initiative Program - Millennium Institute for Integrative Biology (iBio) Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Paola Larrauri-Aguilar Gabriela Contreras 6 Red de Observadores de Aves y Vida Silvestre de Chile, ROC . Santiago, Chile 7 Coastal Solutions Fellows Program, Cornell Laboratory of Ornithology . Ithaca, NY, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gabriela Contreras Gonzalo Cabrera 8 Institute of Biomedical Sciences. Faculty of Medicine, Universidad de Chile . Santiago, Chile Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gonzalo Cabrera Cristóbal Arredondo 5 Wildlife Conservation Society-Chile . Punta Arenas, Chile Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Cristóbal Arredondo Fernán Federici 1 Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Católica de Chile . Santiago, Chile 2 Department of Molecular Genetics and Microbiology. Faculty of Biological Sciences. Pontificia Universidad Católica de Chile . Santiago, Chile 3 ANID - Millennium Science Initiative Program - Millennium Institute for Integrative Biology (iBio) Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Fernán Federici For correspondence: cristobal.briceno{at}uchile.cl ffederici{at}bio.puc.cl Cristóbal Briceño 4 Department of Animal Preventive Medicine. Faculty of Veterinary and Livestock Sciences, Universidad de Chile. Santiago , Chile Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Cristóbal Briceño For correspondence: cristobal.briceno{at}uchile.cl ffederici{at}bio.puc.cl Abstract Full Text Info/History Metrics Preview PDF Abstract Invasive beavers, a species introduced to a remote area of Tierra del Fuego in 1946, have contributed to important environmental degradation. Here, we propose that invasive beavers may also act as reservoirs of Cryptosporidium spp. in Patagonia, affecting native species, wildlife, and human health. We describe zoonotic Cryptosporidium parvum in beavers. Given mammals’ susceptibility to this parasite, the influence of beavers extends across colonized watersheds, contributing to broader exposure. Our findings confirmed the presence of this water-borne parasite in Karukinka Park, Tierra del Fuego, underscoring the potential role of this emblematic invasive species in spreading Cryptosporidium . This research addresses the complexities of molecular identification of this parasite and presents a case study on pathogen monitoring in invasive species within the One Health framework. It emphasizes the consequences of human-introduced exotic species on pristine, remote, and vulnerable insular ecosystems. Introduction Cryptosporidium spp. is a water-borne pathogen causing gastrointestinal disease and deaths in developed and developing countries 1 . This parasite infects various animals, including humans, either broadly or in a species-specific manner. Transmission occurs through hosts like cattle, poultry, rodents, and domestic pets 2 . The infective stage is a thick-walled oocyst, 4-6 μm in diameter, highly resilient. Oocysts resist chemical and physical changes in water, except alkalinity, desiccation, and ozone exposure 3 . They endure conventional disinfectants like ultraviolet (UV) light and chlorine in high parasitic loads 4 . The trilaminar oocyst wall enables survival outside hosts, releasing sporozoites into the host intestine upon ingestion. This adaptation allows oocyst to remain viable for up to 16 months 1 . Microscopy is the standard method for oocyst visualization, whereas molecular techniques determine potential hosts. Oocyst transmission occurs orally via contact with infected hosts, contaminated water and food, and zoonotic pathways 2 . Sewage and watercourses in livestock-dense areas show higher oocyst levels than regions with minimal human intervention 3 . This is influenced by flow, turbidity, seasonal precipitation, and runoff events 3 , 5 . Another key factor is the thriving exotic species acting as pathogen reservoirs and linked to zoonotic episodes, possibly exacerbated by climate change 6 , 7 . Invasive alien species (IAS) contribute to biodiversity loss 8 and species extinction 9 . IAS richness can be predicted by anthropogenic factors and native species diversity 8 , 9 . They cause biotic homogenization and ecosystem transformation, particularly in isolated environments like archipelagos 10 . Such changes may facilitate diseases onset by establishing novel links between emerging pathogens and wildlife hosts 6 , 8 , 11 . As IAS expand, pathogen-host interactions may increase zoonotic spillovers, influenced by environmental factors, epidemiology, host susceptibility, and pathogen ecology 3 , 8 , 11 . The American Beaver ( Castor canadensis ) is a semi-aquatic mammal and the second-largest rodent. In 1946, ten reproductive pairs from Canada were introduced to the Argentinean side of Tierra del Fuego (TDF) Island. Its population has expanded onto the continent and northward, reaching over 100,000 individuals 12 . TDF is a fragile ecosystem with high endemicity. IAS represents two-thirds of the archipelago’s species, with beavers threatening peatlands, mature sub-Antarctic forests, and watercourses 13 . Beavers inhabit watersheds, building dams at dawn and dusk. Kits defecate in water–2-3 days post-birth, contributing to watershed pollution and waterborne pathogens dissemination 5 . Residents, trekkers, farmers, fly-fishers, and expeditioners in Patagonia may be at risk due to misleading perceptions of safety in pristine environments, given low human population densities 14 . Beaver activity also creates habitats suitable for invasive species, including exotic plants and salmonids 13 , and semi-aquatic mammals like muskrats ( Ondatra zibethica ), prey for American minks ( Neovison vison ), an opportunistic carnivore 13 . The interactions between these sympatric species in North America and Patagonia mirror synergistic dynamics, prone to pathogen transmission 15 . We assessed Cryptosporidium presence in beavers introduced to TDF, one of Earth’s least populated places. Fecal samples were collected during beaver population control in the 300,000-hectare Karukinka Park, a natural laboratory in TDF, managed by the Wildlife Conservation Society (WCS). Three beavers (n = 3) tested positive for Cryptosporidium spp. by optical microscopy (3/105) using the Ziehl-Neelsen staining technique. To identify this parasite species, we amplified and sequenced a fragment of 18S ribosomal DNA. Our findings confirm zoonotic Cryptosporidium parvum in beaver feces, and we discuss its implications for public, productive, and environmental health ( Figure 1 ). Download figure Open in new tab Figure 1. The influence of beaver activity on animal and environmental health in Tierra del Fuego. For decades, beavers have impacted watercourses in Tierra del Fuego. They expose native species ([1]-[3]), livestock, domestic animals, and humans ([4]-[6]), and other invasive ([7],[8]) to contaminated water, potentially facilitating Cryptosporidium spp. dissemination throughout the region. Legend: [1]: Traro ( Caracacara plancus ); [2]: Culpeo fox ( Lycalopex culpaeus lycoides ); [3]: Guanaco (Lama guanicoe); [4]: livestock; [5]: domestic animals; [7]: humans; [7]: Muskrat ( Ondatra zibethicus ); [8]: American Mink ( Neovison vison ). Created in https://BioRender.com Materials and methods As part of the GEF program between FAO (UN Food and Agriculture Organization), SAG (Fish and Wildlife Service), CONAF (Protected Natural Park Service), and WCS Chile, 105 beavers from three locations on TDF Isla Grande (54°S 69°W) in Chile ( Figure 2 ) were captured in spring and summer from 2017 to 2019. Necropsies were carried out, and fecal content from the rectum was collected. Download figure Open in new tab Figure 2. Beaver sampling locations. Upper right inset shows South America with a red rectangle marking the Tierra del Fuego Archipelago, shared by Chile and Argentina. In the bottom image, beaver sampled locations are depicted with circles, being blue for negatives and red for positive samples of Cryptosporidium parvum collected between 2017 and 2019 in Chile. 3 cm 3 stool samples were stored at 4°C in 70% ethanol until processing at FAVET (Faculty of Veterinary Sciences, University of Chile). Samples were centrifuged, and fecal sediment was prepared on slides and stained using the modified Ziehl-Neelsen method. This technique facilitated Cryptosporidium oocysts detection through light microscopy, as described. For DNA extraction, ethanol was decanted, and 200mg of fecal samples were resuspended in 850μL Lysis Buffer (NucleoSpin® DNA Stool Mini Kit, Macherey-Nagel). Due to Cryptosporidium oocysts’ resistance, samples were treated with ten freeze-thaw cycles using liquid nitrogen and a 65°C water bath for one minute each per cycle, vortexed with 0.6–0.8 mm ceramic beads (2mL MN Bead Tube Type A) for 25 minutes to ensure disruption 16 . Genomic DNA was extracted using the above kit and stored at −20°C for analysis. A nested PCR protocol was developed to amplify the 18S ribosomal RNA (rRNA) gene, a marker distributed across the Cryptosporidium genus. This genetic marker features semi-conserved and hypervariable regions used for genus and species identification. Selected primers targeted a conserved region. This PCR technique enhances detection sensitivity, given the low oocyst loads in water and stool. Target sequences were used to build a synthetic template containing regions of the small (SSU/18S) and large (LSU/28S) subunits of the Cryptosporidium rRNA gene. Three sequences were selected (GenBank Accession Numbers: MZ892388.1 , AF108862.1, AF040725.1), keeping only relevant DNA regions for primer compatibility. Sequences were merged using Benchling, synthesized as a gBlock, housed in a pL0R plasmid, and cloned into E. coli TOP10 cells for supply. A nested PCR was performed using primers described by Briceño (2023) 16 . 50 μL-volume PCR reactions contained 10 μL of High-Fidelity 5X Buffer, 200 μM of each dNTP, 0.4 μM of each primer, 0.5 μL (20 units/ml) of Phusion High-Fidelity DNA Polymerase, 1.66 μL of DNA solution, and molecular-grade water. For the secondary reaction, 1.66 μL of primary product served as template under identical conditions. Thermocycling included initial denaturation at 98°C for 30 s, followed by 38 cycles consisting of 20 s at 98°C, 45 s at 65°C, and 1 min at 72°C, with final extension of 7 min at 72°C for the primary reaction. Secondary reactions used 38 cycles of 20 s at 98°C, 30 s at 65°C, and 40 s at 72°C. PCR products were visualized in 1% agarose gels, using SYBR ® SAFE dye (Invitrogen, S33102) and an open-source blue LED Mini Transilluminator. Nested PCR products were inserted into a pL0R-lacZ vector using Type IIS Golden Gate assembly, with SapI and T4 ligase enzymes. Plasmids were cloned using E. coli TOP10 chemo-competent cells, then purified for sequencing and storage. Results Rectum-fecal samples were collected from 105 captured beavers. Ziehl-Neelsen-stained fecal matter was examined through optical microscopy at 100X magnification ( Figure 3 ). Suspect bodies showed four main features: subspherical morphology, 4 – 6 μm size, fuchsia color, and visible content (four sporozoites). Three samples presented Cryptosporidium spp. oocysts; however, quantification was not conducted due to the low oocyst numbers observed. Download figure Open in new tab Figure 3. Detection of Cryptosporidium using optical microscopy and Nested PCR. [Left] A Ziehl-Neelsen-stained beaver feces specimen from Tierra del Fuego (spherical fuchsia body, observed with 100X lens). [Right] Molecular confirmation of the Cryptosporidium 18S rRNA gene (552bp bands on agarose gel) in three beaver samples (CC52, CC53, CC55). Cryptosporidium spp 18S rDNA was amplified and sequenced from positive beaver fecal samples, with data in GenBank (460 bp; PV442491 ), showing 100% homology to Cryptosporidium parvum . Discussion Invasive wildlife affects disease transmission dynamics by creating novel host-parasite assemblages. Evaluating these risks requires examining pathogen occurrence in natural environments 3 , 17 . Our research shows invasive beavers carry C. parvum oocysts, which may spread as they successfully expand in TDF. Beavers’ dam construction contributes to sediment and fecal accumulation, establishing transmission pathways for this zoonotic parasite. Oocysts were detected in three beaver samples using microscopy, nested PCR, and sequencing ( Figure 3 ). Among 105 fecal samples from beavers captured over two years, prevalence was low (2.85%). However, even 1-10 oocysts can cause infection 2 , 4 , 5 . Increasing sample size and trapping locations could provide more conclusive prevalence data. Still, our study does not establish beavers as Cryptosporidium hosts, as we did not conduct immunohistochemical assays on intestinal tissue to verify infection in this species. Invasive beavers in TDF may play a subordinate role in parasitic transmission. Although our findings neither confirm active infection nor indicate outbreak-level prevalence, detecting Cryptosporidium even in low quantities, emphasizes an underestimated risk within such a conducive environment. Moreover, this prompts monitoring of other waterborne pathogens showing higher prevalence within TDF fauna. This report emphasizes the need for context-specific pathogen epidemiology 5 . Attention must broaden to native species such as guanacos, traros, and Fuegian culpeo foxes 18 , and invasive species chilla foxes ( Lycalopex griseus ), American minks, and muskrats 15 ( Figure 1 ). These species may encounter water or grass contaminated by beaver dams or prey upon beaver carcasses, which lack natural predators 15 . Trapped beavers could be oocyst sources for carnivores like foxes or scavengers 19 . Feral dogs and sheep may overlap with these species, exposing humans. Infected hosts may defecate near streams or within dams, becoming reservoirs for ongoing exposure. Furthermore, assessment of environmental water quality is essential. Although watercourses provide valuable data, methodologies for Cryptosporidium sample collection and filtration in remote settings need enhancement 5 . Transporting large samples to laboratories poses significant constraints, particularly in isolated locations. Field processing of water samples, using locally manufactured open-source instrumentation, may be more advantageous 20 . This report on Cryptosporidium in TDF shows beavers pose conservation threats to sub-Antarctic ecosystems and health risks. Our findings emphasize the need for governmental collaboration in alerting people and stakeholders in wildlife monitoring, invasive species management, ecotourism, and economic conflicts 14 . WCS Chile began warning against drinking water from beaver-intervened streams, which may disseminate Cryptosporidium oocysts to Karukinka Park visitors. These efforts aim to enhance understanding of wildlife-pathogen interactions, societal perceptions, and policy influence. As beaver populations migrate northward, coordinated One Health-based surveillance is imperative 12 . Author contributions Project administration: Cristóbal Briceño, Fernán Federici. Resources: Cristóbal Arredondo, Catherine Dougnac, Bárbara Saavedra, Cristóbal Briceño, Fernán Federici. Supervision: Cristóbal Briceño, Fernán Federici. Validation: Paola Larrauri-Aguilar, Séverine Cazaux, Fernando Fredes. Writing – original draft: Paola Larrauri-Aguilar, Cristóbal Briceño. Writing – review & editing: Paola Larrauri-Aguilar, Séverine Cazaux, Valentina Ferrando, Dominique Schwend, Cristóbal Briceño, Fernán Federici. Conceptualization: Paola Larrauri-Aguilar, Cristóbal Briceño, Fernán Federici. Data curation: Paola Larrauri-Aguilar, Séverine Cazaux. Formal analysis: Paola Larrauri-Aguilar, Gabriela Contreras, Cristóbal Briceño. Funding acquisition: Fernán Federici, Cristóbal Briceño. Investigation: Paola Larrauri-Aguilar, Gabriela Contreras, Cristóbal Briceño, Fernán Federici. Methodology: Paola Larrauri-Aguilar, Gabriela Contreras, Alejandra Sandoval, Matilde Larraechea, Claudio Abarca, Galia Ramírez-Toloza, Fernando Fredes, Gonzalo Cabrera, Cristóbal Briceño, Fernán Federici. Conflict of interest statement The authors declare that they have no conflict of interest. Data availability statement The data supporting the findings of this study are available from the co-corresponding authors upon reasonable request. Ethics statement All protocols were approved by the Institutional Committee for Animal Care and Use (CICUA) of the University of Chile (Protocol N° 14-2019). Acknowledgements We extend our gratitude to Bárbara Saavedra, Catherine Dougnac, Cristóbal Arredondo, and the park ranger team of the Wildlife Conservation Society-Chile for granting us access to Karukinka Park, as well as for their guidance and continuous assistance throughout the research process and the primary collection of biological samples. We also acknowledge the ongoing training, methodological validation, and confirmation of findings, and significant support provided by Alejandra Sandoval and Matilde Larraechea from the ConserLab; Claudio Abarca and Galia Ramírez-Toloza from the Laboratory of Parasitology, and Fernando Fredes at the Department of Animal Preventive Medicine. Faculty of Veterinary and Livestock Sciences, Universidad de Chile. Santiago, Chile. We are grateful for the invaluable support provided by Séverine Cazaux, Valentina Ferrando, Dominique Schwend, Alejandro Aravena, Javier Santiago, Alfredo L’Homme, Tomás Oliva, Javiera Avilés, Ariel Cerda, Tamara Matute, Isaac Núñez, and Daniel Núñez in protein purification, design of synthetic positive controls and PCR/LAMP primers, molecular assays standardization and sample processing. Similarly, we thank the ongoing assistance in 3D-printing learning, assembly and validation of the OFM by Pierre Padilla-Huamantinco, along with the suggestions, and observations from Fernando Castro, Octavio Duarte, Joe Knapper, and Freya Whiteford. Finally, we acknowledge the conceptual and experimental contributions of Frank Katzer, Hayder Al Mshelesh, Ghulam Musaddaq, Linda Marriott, and Maïwenn Kersaudy-Kerhoas in working with Cryptosporidium . Funder Information Declared Agencia Nacional de Investigación y Desarrollo , ANID Millennium Science Initiative Program (ICN17_022) , National Doctorate Scholarship 2021-2024 Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo, https://ror.org/03myqtf56 , ‘RELARUS – Red Latinoamericana de Reactivos de Libre Acceso para Una Salud’ (225RT0168) Footnotes Funding Information This work was supported by the National Agency of Research and Development (ANID) through the ANID Millennium Science Initiative Program (ICN17_022), the National Doctorate Scholarship 2021-2024, provided by ANID, Ministry of Science, Technology, Knowledge and Innovation - Government of Chile, and the Ibero-American Program for Science and Technology for Development (CYTED), through the thematic network ‘RELARUS – Red Latinoamericana de Reactivos de Libre Acceso para Una Salud ’ (225RT0168). Bibliography 1. ↵ Luka , G. et al. 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Agonistic behavior between introduced beaver (Castor canadensis) and endemic culpeo fox (Pseudalopex culpaeus lycoides) in Tierra del Fuego Island and implications . Acta Ethologica 21 , 29 – 34 ( 2018 ). OpenUrl 20. ↵ Hill , A. P. et al. Leveraging conservation action with open-source hardware . Conserv. Lett . 12 , e12661 ( 2019 ). OpenUrl View the discussion thread. Back to top Previous Next Posted November 14, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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