Tuberculosis Surveillance in Free-Living Pinnipeds of Patagonia, Argentina

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Roca, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8831472/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Tuberculosis is a disease caused by bacteria belonging to the Mycobacterium tuberculosis complex (MTBC), which includes zoonotic agents capable of infecting a wide range of domestic and wild species, including marine mammals. Although tuberculosis in pinnipeds has been documented worldwide, evidence is largely restricted to post-mortem findings, with limited information from active surveillance of live populations. In this study, We conducted an exploratory field-based assessment of tuberculosis exposure in free-ranging South American sea lions ( Otaria byronia ) along the Patagonian coast of Argentina. During the austral summer of 2025, biological samples were collected from 28 neonate pups from four colonies located in Chubut and Santa Cruz provinces, using minimally invasive field procedures. Blood and nasal swab samples were obtained and processed using a combination of serological, bacteriological, and molecular diagnostic approaches. Serological testing was performed using a commercial rapid assay (Lionex Animal TB®) to detect antibodies against MTBC antigens, while nasal swabs were analyzed by bacteriological culture and PCR targeting the IS6110 sequence. Serological reactivity to at least one MTBC antigen was detected in 25.0% (7/28) of the sampled pups, indicating indirect serological evidence of exposure at the colony levelin three of the four colonies. In contrast, all samples were negative by bacteriological culture and PCR. Considering the neonatal age of the individuals and the chronic nature of tuberculosis, the serological findings most likely reflect passive transfer of maternal antibodies rather than active infection in pups. This study offers new evidence on tuberculosis surveillance in live free-ranging pinniped populations, emphasizing the relevance of combining serological, bacteriological, and molecular tools for comprehensive wildlife health monitoring under a One Health approach. Overall, the results reinforce the need for active and integrated surveillance strategies to address zoonotic risks in high-risk wildlife–human interface settings. Health sciences/Diseases Biological sciences/Microbiology Antibodies detection Mycobacterium tuberculosis complex tuberculosis South American sea lions zoonoses Figures Figure 1 Figure 2 Figure 3 Introduction Human activities, the overexploitation of resources and environmental pollution lead to the fragmentation and degradation of natural habitats. In this context, the use of wildlife as a tourist attraction, coupled with the inevitable scalating interaction between wildlife, domestic animals and humans, zoonotic diseases is becoming progressively more relevant due to its implications for both public health and biodiversity conservation (Garoma & Diba, 2022 ; Karesh et al., 2012 ; Uhart, 2021 ). Within this framework, the One Health approach has gained critical importance as a tool to understand, analyze, interpret, and ideally prevent future scenarios of infectious disease emergence and spread. In the domain of public health, tuberculosis (TB) is recognised as one of the most significant infectious diseases on a global scale (WHO, 2025). According to the World Health Organization (WHO), most prevalent infectious diseases in recent years, with a notable increase in reported cases (WHO, 2025). Nevertheless, the actual contribution of zoonotic tuberculosis to these alarming figures remains unknown. Zoonotic tuberculosis (zTB) refers to a form of tuberculosis in humans caused by members of the Mycobacterium tuberculosis complex (MTBC) that are primarily associated with animal hosts. The primary species, M. bovis , infects a broad range of host, including marine mammals, with cattle being its main reservoir (Kiers et al., 2008 ; Miller & Olea-Popelka, 2013 ; Vågene et al., 2022, Winter et al., 2024 ). In pinnipeds, however, the predominant associated species is M. pinnipedii , although M. bovis infection has also been reported in previous studies (see Sosa-Drouville et al., 2025 ). Mycobacterium pinnipedii is a zoonotic MTBC member and has the capacity to spill over to other animals and humans (Kiers et al., 2008 ; Miller & Olea-Popelka, 2013 ; Vågene et al., 2022). Since more than 100 years cases of MTBC infection in pinnipeds have been documented on a global scale (Sosa-Drouville et al., 2025 ). In Argentina, TB has been reported in post mortem individuals of three pinniped species: Arctocephalus australis , A. tropicalis , and Otaria byronia (Romano et al., 1995 ; Bernardelli et al., 1996; Zumárraga et al., 1999 ., Bastida et al., 2020 ., Cousins et al., 2003 ; Fiorito et al., 2020 ). Predominantly, the detection of tuberculosis in pinnipeds has relied on animals examined post mortem, based on the observation of compatible lesions and sample collection during necropsies (Sosa-Drouville et al., 2025 ). Accordingly, the most common diagnostic approaches applied have been bacteriological culture isolation followed by molecular confirmation using PCR techniques (Sosa-Drouville et al., 2025 ). Although MTBC infections in pinnipeds have been documented worldwide, detection has predominantly occurred in dead individuals, and few studies have attempted active surveillance in live, free-ranging populations. This limitation impedes our capacity to comprehensively grasp the genuine incidence and geographical distribution of tuberculosis within pinniped colonies. Consequently, it restricts our ability to evaluate the potential for spillover risks to livestock, humans, and other human-wildlife interactions that are precipitated by tourism, fisheries, and coastal development. From a One Health perspective, it is imperative to establish whether TB exposure is present in the wild, in order to inform surveillance, management and public health strategies. To address this gap, we conducted field-based screening of free-ranging South American sea lion ( Otaria byronia) breeding colonies along Argentina’s Atlantic coast to assess evidence of tuberculosis exposure and generate baseline data for future One Health surveillance and targeted follow-up investigations. Methodology Sampling area During the austral summer of 2025, a total of 28 South American sea lion pups (16 males and 12 females) were sampled in Patagonia, Argentina (Fig. 1). The individuals were obtained from four breeding colonies distributed along the Argentine Atlantic coast, Punta Buenos Aires (n = 7; 42° 14' 19'' S) and Leones Island (n = 5; 45° 03' 02''S) in Chubut Province, and Larga Island (n = 5; 47° 45' 16'' S) and Monte León (n = 11; 50° 18' 40'' S) in Santa Cruz Province (Fig. 1). These colonies were selected as they are key reproductive sites within the species’ southern distribution range. Capture of specimens and sample collection Upon arrival at the South American sea lion colony, pups located at the periphery were selected and captured using a telescopic pole with a rope at the end. Once an individual was restrained, it was carefully removed from the colony and moved several meters away to reduce disturbance to the group and collective stress. The individuals sampled were neonates aged 1–7 days, as estimated from the condition of the umbilical cord (Ameghino, 2012 ). Physical restraint did not exceed 10 minutes and was carried out by a team of three handlers: one held the fore flippers and head, another restrained the hind flippers, and the third performed sample collection (nasal swabbing and blood collection) while the rest of the team prepared the equipment and labeled the samples. In order to minimize stress, the animal’s eyes were covered, and all procedures were conducted quietly and efficiently (Fig. 2). All procedures described herein have been approved by the CICUAE (resolution N° 009/25) of the CCT CONICET-CENPAT. Following the measurement of physical parameters, biological samples were collected for laboratory analyses. For blood collection, approximately 10 mL of blood was obtained from the interdigital veins of the hind flippers of 28 individuals (Gulland et al., 2018 ) and stored in tubes containing anticoagulant (e.g., Vacutainer tubes®) (Fig. 2). Blood samples were centrifuged in the field, and a drop of plasma was used to perform a serological commercial animal tuberculosis assay (Lionex Animal TB®). The Lionex Animal TB Rapid Tuberculosis Test was originally developed for use in other animal species and has not been specifically validated in otariids; therefore, results should be interpreted as exploratory evidence of reactivity to MTBC-associated antigens.. The manufacturer does not disclose the specific antigens included in the test. Following collection, samples were maintained at − 20 °C until processing. Nasal swabs were inserted into the nostrils, gently rotated, and placed into transport media (i.e., AMIES) (Fig. 2). One swab was designated for bacteriological culture, and the other was used for direct DNA extraction and PCR. Bacteriological culture was conducted using Löwenstein-Jensen and Stonebrink medium. Prior to inoculation, the Petroff decontamination method was applied, as described by Jorge et al. ( 2005 ). Cultures were incubated at 37°C for up to 12 weeks, and those showing no bacterial growth were recorded as negative. The Ziehl-Neelsen stain (Biopack®) was used to evaluate the presence of acid-fast bacilli (AFB) in positive cultures. The DNA was extracted directly from the nasal swab using the commercial DNA Puriprep S-kit (INBIO HIGHWAY), according to the manufacturer’s protocol. Subsequently, molecular detection of MTBC was performed using a conventional PCR assay targeting the IS 6110 insertion sequence. The reaction included the primers INS1 and INS2, as previously described by Hermans et al. ( 1990 ), and was amplified using the Touch-Down cycle described by Zumarraga et al., (2005). PCR products were revealed by horizontal electrophoresis on a 2% agarose gel stained with ethidium bromide (0.5 µg/mL) and visualized under UV light. Results Serological-test Serological analysis was performed using the LIONEX test on 28 blood samples collected from South American sea lion pups (Fig. 3). Overall, 25% of the samples analyzed (7/28) were seropositive for at least one antigen of the MTBC (95% CI: 12.7–43.4%). Seroprevalence varied by location: at the Punta Buenos Aires colony, three out of seven samples (42.9%) were positive (95% CI: 15.8–74.9%); at Larga Island, one out of five samples (20.0%) showed reactivity (95% CI: 3.6–62.4%); and at Monte León, three out of eleven samples (27.3%) tested positive (95% CI: 9.7–56.6%). In contrast, no seropositive reactions were detected among five samples from Leones Island (0/5; 95% CI: 0–43.4%) (Table 1 ). Table 1 Id, colony, sex, kit, culture and PCR results of the sampled individuals. Id Colony Sex Kits result Culture result PCR result 1 Punta Bs As M Control negative - 2 Punta Bs As M control + T2 negative Negative 3 Punta Bs As F Control negative - 4 Punta Bs As F control + T2 negative negative 5 Punta Bs As F control + T2 negative negative 6 Punta Bs As F Control negative - 7 Punta Bs As M Control negative - 8 Leones Island M Control negative - 9 Leones Island M Control negative - 10 Leones Island M Control negative - 11 Leones Island M control negative - 12 Leones Island M control negative - 13 Larga Island F control negative - 14 Larga Island M control negative - 15 Larga Island M control + T2 negative negative 16 Larga Island F control negative - 17 Larga Island M control negative - 18 Monte León M control + T2 negative negative 19 Monte León M control negative - 20 Monte León M control negative - 21 Monte León F control negative - 22 Monte León M control + T2 + T1 negative negative 23 Monte León M control negative - 24 Monte León F control negative - 25 Monte León F control negative - 26 Monte León F control negative - 27 Monte León F control + T2 negative Negative 28 Monte León F control negative - Culture and PCR After 12 weeks of incubation, no bacterial growth was observed in the culture media. Similarly, direct DNA extractions from nasal swabs did not yield amplification products for the MTBC specific target (Table 1 ). Discussion The present study provides serological evidence suggesting exposure to members of the Mycobacterium tuberculosis complex (MTBC) at the population level in free-living South American sea lions from breeding colonies along Argentina’s Patagonian coast. These findings most likely reflect mother-to-pup passive transfer of maternal antibodies. Importantly, we have collected biological samples in live pinnipeds for processing through etiological and serological tests using minimally invasive procedures. Although bacteriological culture and direct PCR techniques are the most widely used and reliable tools to diagnose tuberculosis in wildlife, serological tests represent a valuable alternative for monitoring population health status (Thomas et al. 2021, Kelley et al. 2020 ). In this study, both bacterial culture and PCR yielded negative results for all samples, an outcome expected given that the individuals sampled were neonates aged 1–7 days, as estimated from the condition of the umbilical cord (Ameghino, 2012 ). Tuberculosis is a chronic disease, and once the pathogen has infected the host, a period of progression required before it can be detected through etiological tests. We used the Lionex-test for the first time in living pinnipeds. This test was originally developed for the diagnosis of bovine tuberculosis as a low-cost, minimally invasive tool (Lionex, 2015 ). While the test is designed to detect antibodies against M. bovis , it employs purified mycobacterial antigens, including cell wall components such as lipoarabinomannan (LAM). LAM is conserved across members of the Mycobacterium tuberculosis complex (MTBC) and widely used as a diagnostic target (Chatterjee & Khoo, 1998 , Minion et al., 2011 ). This shared antigenic structure provides a biological basis for potential cross-reactivity among MTBC species, possibly including M. pinnipedii . According to the manufacturer’s specifications, the test is intended for use in multiple animal species; however, validation studies to date have been conducted primarily in cattle, and it has not been specifically validated in otariids. This assay demonstrated its potential as a practical approach for health surveillance in free-ranging pinniped populations, particularly when combined with other diagnostic approaches. We considered an individual as seropositive to the MTBC when it reacted to at least one of the three antigens included in the test. According to the assay’s design, there are seven possible combinations of seropositive results: three single-antigen (1, 2, or 3) and four combined (1 + 2, 1 + 3, 2 + 3, or 1 + 2+3). Despite these seven possible outcomes, only two positivity patterns were observed in our samples (2 and 1 + 2). The latter pattern was also reported in a study conducted on milk samples from cattle herds infected with M. bovis (Kelley et al., 2020 ). These consistent patterns may suggest a functional relationship among the antigens targeted by the test, potentially reflecting a shared immune response to M. bovis or other members of the MTBC (Kelley et al., 2020 ). We detected serological reactivity in 25% of the sampled individuals. These findings should be interpreted as indirect evidence of MTBC exposure at the population level. Further studies integrating additional diagnostic approaches will be necessary to better understand the epidemiological dynamics of tuberculosis in these populations. - Our findings support the passive transfer of maternal antibodies, mainly acquired through colostrum and milk during the first days of lactation, rather than an active infection in neonates. This mechanism is explained by pinnipeds placentation: the endotheliochorial and zonary, typical of carnivores, permits moderate exchange and minimal immunoglobulin transfer, which explains why pups dependent on colostrum to acquire passive humoral immunity (Ross et al., 1994 ). Accordingly, several studies have shown that the highest antibody concentrations in pinniped pups occur during the first day post-partum (Ross et al., 1994 ; Castinel et al., 2008 ; Meza Cerda et al., 2022 ). In harbor seals ( Phoca vitulina ), for instance, pups are born with very low IgG levels (3% of maternal levels), which increase rapidly after colostrum ingestion, reaching approximately 65% within only 15 days (Ross et al., 1994 ). Furthermore, the seroconversion timeline in other wildlife species reinforces this interpretation. In experimentally infected badgers, antibody-based tests only begin to yield positive results around 45 days post-infection (Infantes-Lorenzo et al., 2019 ). Given that the pups in our study were sampled at 1–7 days of age, the detection of antibodies is highly unlikely to stem from postnatal infection, but instead robustly indicates epidemiological relevance. This serological evidence in pups serves as a valuable, indirect marker of MTBC circulation in the population. Highlighting the importance of considering the maternal origin of antibodies when interpreting serological results in newborn pinnipeds for population TB surveillance. We detected antibodies against MTBC in three geographically distinct colonies along the Patagonian coast, namely Punta Buenos Aires, Larga Island, and Monte León. In contrast, no seropositive individuals were detected at Leones Island, a colony characterized by its relative isolation, which likely reduces interactions with other species and human activities. Conversely, the three seropositive colonies are located in areas with higher ecological connectivity, involving interactions with other wildlife species and varying degrees of anthropogenic influence. These spatial patterns suggest that environmental connectivity and interspecific interactions may play a role in shaping the risk of MTBC exposure in these colonies. This observation is consistent with evidence from other systems showing that MTBC members can circulate at complex wildlife–livestock–human interfaces in the region (Barandiaran et al., 2025 ; Kiers et al., 2008 , Winter et al., 2024 ). In this context, potential exposure pathways may involve environmental contamination or indirect contact among species sharing overlapping habitats. Collectively, our findings highlight the importance of a One Health perspective to better understand pathogen circulation dynamics across interconnected ecosystems. Historically, the diagnosis of TB in free-ranging pinnipeds has relied almost exclusively on post-mortem findings from necropsies (Sosa-Drouville et al., 2025 ). While this approach is essential for the isolation of pathogens and the establishment of a definitive diagnosis, it constricts inferences about infection dynamics, pathogen circulation and true prevalence within living colonies (Silva-Pereira et al., 2019 ; Sacristán et al., 2021 ; Martins Melo et al., 2019 ; Castro Ramos et al., 2006). The present study proposes a minimally invasive survey of live neonatal pups, employing a priori, site-stratified sampling. This approach complements mortality-based surveillance, reduces sampling bias, and provides representative, field-feasible data on colony health. The serological results obtained in this study provide indirect evidence consistent with exposure to members of the Mycobacterium tuberculosis complex (MTBC) in South American sea lions from southern Patagonia.. These results demonstrate the practicality and epidemiological utility of the Lionex®, which could may represent a complementary approach for exploratory health surveillance in live pinnipeds. In order to strengthen the inferences made on transmission and zoonotic risk, it is recommended that paired mother-pup sampling is utilised, validation studies, that longitudinal follow-up of cohorts is conducted, and confirmatory diagnostics. In this context, the present results provide baseline information that may inform the development of One Health surveillance strategies and contribute to future research addressing risks at the wildlife–livestock–human interface. Declarations Acknowledgements We thank the Wildlife Rangers and Park Rangers of the provinces of Río Negro, Chubut, and Santa Cruz for their collaboration and support during fieldwork. We also thank Julio Rua and Ricardo Vera for their invaluable assistance with fieldwork and logistics. We thank the Clinical and Bacteriological Analysis Laboratory (LACyB) for support with clinical and bacteriological analyses, and the Universidad Nacional de la Patagonia Austral (UNPA) and Por el Mar Foundation for providing accommodation during fieldwork. Partial funding for this research was obtained from the Vienna Zoo. This research was the result of several years of Argentine investment in science and technology. Argentine science is now being defunded and dismantled, threatening the continuity of this and many other lines of research. Funding statement This work was partially supported by Agencia Nacional de Promoción de la Investigación, Desarrollo Tecnológico y la Innovación [PICT 2020-0400], and the Vienna Zoo. 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Molecular characterization of mycobacteria isolated from seals. Microbiology 145 , 2519–2526 (1999). Zumárraga, M. J. et al. Use of touch-down polymerase chain reaction to enhance the sensitivity of Mycobacterium bovis detection. J. Vet. Diagn. Invest. 17 , 232–238 (2005). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 14 May, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers invited by journal 20 Apr, 2026 Submission checks completed at journal 14 Apr, 2026 First submitted to journal 12 Apr, 2026 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. 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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-8831472","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":633720515,"identity":"189d3274-2201-4752-8eda-4a755410b608","order_by":0,"name":"Maria Soledad Leonardi","email":"data:image/png;base64,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","orcid":"","institution":"Instituto de Biología de Organismos Marinos","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"Soledad","lastName":"Leonardi","suffix":""},{"id":633720516,"identity":"aacb7e19-ac97-4b8a-bbe0-b41a518e4a59","order_by":1,"name":"Ailin Sosa-Drouville","email":"","orcid":"","institution":"Instituto de Biología de Organismos Marinos","correspondingAuthor":false,"prefix":"","firstName":"Ailin","middleName":"","lastName":"Sosa-Drouville","suffix":""},{"id":633720517,"identity":"8f33ff25-f9c4-4670-aedc-988e53d5893d","order_by":2,"name":"Francisco J. Roca","email":"","orcid":"","institution":"Biomedical Research Institute of Murcia Pascual Parrilla","correspondingAuthor":false,"prefix":"","firstName":"Francisco","middleName":"J.","lastName":"Roca","suffix":""},{"id":633720518,"identity":"69996ef8-aed2-47a4-bd4b-bbeacd435719","order_by":3,"name":"Florencia Soto","email":"","orcid":"","institution":"Instituto de Biología de Organismos Marinos","correspondingAuthor":false,"prefix":"","firstName":"Florencia","middleName":"","lastName":"Soto","suffix":""},{"id":633720519,"identity":"58ac33de-ad9d-4541-8992-e17c0f96409f","order_by":4,"name":"Paula Olivera","email":"","orcid":"","institution":"Instituto de Biología de Organismos Marinos","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"","lastName":"Olivera","suffix":""},{"id":633720520,"identity":"da0d79de-362c-4032-bf00-b679af573c2c","order_by":5,"name":"David Ebmer","email":"","orcid":"","institution":"Vienna Zoo","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Ebmer","suffix":""},{"id":633720521,"identity":"31b37eef-abb8-4cf0-8350-51b4487c7eee","order_by":6,"name":"Philipp Sziderics","email":"","orcid":"","institution":"Vienna Zoo","correspondingAuthor":false,"prefix":"","firstName":"Philipp","middleName":"","lastName":"Sziderics","suffix":""},{"id":633720522,"identity":"6603efe9-7455-4a68-8287-a19e3ea34e46","order_by":7,"name":"Loreana Ponce","email":"","orcid":"","institution":"Instituto de Investigaciones en Producción Animal","correspondingAuthor":false,"prefix":"","firstName":"Loreana","middleName":"","lastName":"Ponce","suffix":""},{"id":633720523,"identity":"7e8d7ef7-cbb2-4d3a-9181-a2adb91b398c","order_by":8,"name":"Soledad Barandiaran","email":"","orcid":"","institution":"Instituto de Investigaciones en Producción Animal","correspondingAuthor":false,"prefix":"","firstName":"Soledad","middleName":"","lastName":"Barandiaran","suffix":""}],"badges":[],"createdAt":"2026-02-09 14:23:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8831472/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8831472/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108944306,"identity":"3441b44f-8cd8-44a9-8302-a7400b859679","added_by":"auto","created_at":"2026-05-11 05:58:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":403624,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Fig1SosaDrouvilleetal.png","url":"https://assets-eu.researchsquare.com/files/rs-8831472/v1/631817c8183c16fed3b2f90f.png"},{"id":108944312,"identity":"73cfff9f-bae7-4603-83fb-75da1e0392f8","added_by":"auto","created_at":"2026-05-11 05:58:25","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5006792,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Fig2SosaDrouvilleetal.png","url":"https://assets-eu.researchsquare.com/files/rs-8831472/v1/16a8873e2ff2511615d97492.png"},{"id":108944321,"identity":"ff795c57-f801-4f0e-a4a8-c32c9b5ee819","added_by":"auto","created_at":"2026-05-11 05:58:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2340772,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Fig3SosaDrouvilleetal.png","url":"https://assets-eu.researchsquare.com/files/rs-8831472/v1/3c641e83e4bc7152a4fee101.png"},{"id":108944457,"identity":"f5adcb02-577c-40bd-8c74-a35046c9dafa","added_by":"auto","created_at":"2026-05-11 05:59:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8716351,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8831472/v1/101cfd0e-af7b-4d8c-9257-84b93e03aa5a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tuberculosis Surveillance in Free-Living Pinnipeds of Patagonia, Argentina","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman activities, the overexploitation of resources and environmental pollution lead to the fragmentation and degradation of natural habitats. In this context, the use of wildlife as a tourist attraction, coupled with the inevitable scalating interaction between wildlife, domestic animals and humans, zoonotic diseases is becoming progressively more relevant due to its implications for both public health and biodiversity conservation (Garoma \u0026amp; Diba, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Karesh et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Uhart, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Within this framework, the \u003cem\u003eOne Health\u003c/em\u003e approach has gained critical importance as a tool to understand, analyze, interpret, and ideally prevent future scenarios of infectious disease emergence and spread.\u003c/p\u003e \u003cp\u003eIn the domain of public health, tuberculosis (TB) is recognised as one of the most significant infectious diseases on a global scale (WHO, 2025). According to the World Health Organization (WHO), most prevalent infectious diseases in recent years, with a notable increase in reported cases (WHO, 2025). Nevertheless, the actual contribution of zoonotic tuberculosis to these alarming figures remains unknown.\u003c/p\u003e \u003cp\u003eZoonotic tuberculosis (zTB) refers to a form of tuberculosis in humans caused by members of the \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e complex (MTBC) that are primarily associated with animal hosts. The primary species, \u003cem\u003eM. bovis\u003c/em\u003e, infects a broad range of host, including marine mammals, with cattle being its main reservoir (Kiers et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Miller \u0026amp; Olea-Popelka, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; V\u0026aring;gene et al., 2022, Winter et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In pinnipeds, however, the predominant associated species is \u003cem\u003eM. pinnipedii\u003c/em\u003e, although \u003cem\u003eM. bovis\u003c/em\u003e infection has also been reported in previous studies (see Sosa-Drouville et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). \u003cem\u003eMycobacterium pinnipedii\u003c/em\u003e is a zoonotic MTBC member and has the capacity to spill over to other animals and humans (Kiers et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Miller \u0026amp; Olea-Popelka, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; V\u0026aring;gene et al., 2022).\u003c/p\u003e \u003cp\u003eSince more than 100 years cases of MTBC infection in pinnipeds have been documented on a global scale (Sosa-Drouville et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In Argentina, TB has been reported in post mortem individuals of three pinniped species: \u003cem\u003eArctocephalus australis\u003c/em\u003e, \u003cem\u003eA. tropicalis\u003c/em\u003e, and \u003cem\u003eOtaria byronia\u003c/em\u003e (Romano et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Bernardelli et al., 1996; Zum\u0026aacute;rraga et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1999\u003c/span\u003e., Bastida et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e., Cousins et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Fiorito et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Predominantly, the detection of tuberculosis in pinnipeds has relied on animals examined post mortem, based on the observation of compatible lesions and sample collection during necropsies (Sosa-Drouville et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Accordingly, the most common diagnostic approaches applied have been bacteriological culture isolation followed by molecular confirmation using PCR techniques (Sosa-Drouville et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough MTBC infections in pinnipeds have been documented worldwide, detection has predominantly occurred in dead individuals, and few studies have attempted active surveillance in live, free-ranging populations. This limitation impedes our capacity to comprehensively grasp the genuine incidence and geographical distribution of tuberculosis within pinniped colonies. Consequently, it restricts our ability to evaluate the potential for spillover risks to livestock, humans, and other human-wildlife interactions that are precipitated by tourism, fisheries, and coastal development. From a One Health perspective, it is imperative to establish whether TB exposure is present in the wild, in order to inform surveillance, management and public health strategies.\u003c/p\u003e \u003cp\u003eTo address this gap, we conducted field-based screening of free-ranging South American sea lion (\u003cem\u003eOtaria byronia)\u003c/em\u003e breeding colonies along Argentina\u0026rsquo;s Atlantic coast to assess evidence of tuberculosis exposure and generate baseline data for future One Health surveillance and targeted follow-up investigations.\u003c/p\u003e"},{"header":"Methodology","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSampling area\u003c/h2\u003e \u003cp\u003eDuring the austral summer of 2025, a total of 28 South American sea lion pups (16 males and 12 females) were sampled in Patagonia, Argentina (Fig.\u0026nbsp;1). The individuals were obtained from four breeding colonies distributed along the Argentine Atlantic coast, Punta Buenos Aires (n\u0026thinsp;=\u0026thinsp;7; 42\u0026deg; 14' 19'' S) and Leones Island (n\u0026thinsp;=\u0026thinsp;5; 45\u0026deg; 03' 02''S) in Chubut Province, and Larga Island (n\u0026thinsp;=\u0026thinsp;5; 47\u0026deg; 45' 16'' S) and Monte Le\u0026oacute;n (n\u0026thinsp;=\u0026thinsp;11; 50\u0026deg; 18' 40'' S) in Santa Cruz Province (Fig.\u0026nbsp;1). These colonies were selected as they are key reproductive sites within the species\u0026rsquo; southern distribution range.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCapture of specimens and sample collection\u003c/h3\u003e\n\u003cp\u003eUpon arrival at the South American sea lion colony, pups located at the periphery were selected and captured using a telescopic pole with a rope at the end. Once an individual was restrained, it was carefully removed from the colony and moved several meters away to reduce disturbance to the group and collective stress. The individuals sampled were neonates aged 1\u0026ndash;7 days, as estimated from the condition of the umbilical cord (Ameghino, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Physical restraint did not exceed 10 minutes and was carried out by a team of three handlers: one held the fore flippers and head, another restrained the hind flippers, and the third performed sample collection (nasal swabbing and blood collection) while the rest of the team prepared the equipment and labeled the samples. In order to minimize stress, the animal\u0026rsquo;s eyes were covered, and all procedures were conducted quietly and efficiently (Fig.\u0026nbsp;2). All procedures described herein have been approved by the CICUAE (resolution N\u0026deg; 009/25) of the CCT CONICET-CENPAT.\u003c/p\u003e \u003cp\u003eFollowing the measurement of physical parameters, biological samples were collected for laboratory analyses. For blood collection, approximately 10 mL of blood was obtained from the interdigital veins of the hind flippers of 28 individuals (Gulland et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and stored in tubes containing anticoagulant (e.g., Vacutainer tubes\u0026reg;) (Fig.\u0026nbsp;2). Blood samples were centrifuged in the field, and a drop of plasma was used to perform a serological commercial animal tuberculosis assay (Lionex Animal TB\u0026reg;). The Lionex Animal TB Rapid Tuberculosis Test was originally developed for use in other animal species and has not been specifically validated in otariids; therefore, results should be interpreted as exploratory evidence of reactivity to MTBC-associated antigens.. The manufacturer does not disclose the specific antigens included in the test. Following collection, samples were maintained at \u0026minus;\u0026thinsp;20 \u0026deg;C until processing.\u003c/p\u003e \u003cp\u003eNasal swabs were inserted into the nostrils, gently rotated, and placed into transport media (i.e., AMIES) (Fig.\u0026nbsp;2). One swab was designated for bacteriological culture, and the other was used for direct DNA extraction and PCR. Bacteriological culture was conducted using L\u0026ouml;wenstein-Jensen and Stonebrink medium. Prior to inoculation, the Petroff decontamination method was applied, as described by Jorge et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Cultures were incubated at 37\u0026deg;C for up to 12 weeks, and those showing no bacterial growth were recorded as negative. The Ziehl-Neelsen stain (Biopack\u0026reg;) was used to evaluate the presence of acid-fast bacilli (AFB) in positive cultures. The DNA was extracted directly from the nasal swab using the commercial DNA Puriprep S-kit (INBIO HIGHWAY), according to the manufacturer\u0026rsquo;s protocol. Subsequently, molecular detection of MTBC was performed using a conventional PCR assay targeting the IS\u003cem\u003e6110\u003c/em\u003e insertion sequence. The reaction included the primers INS1 and INS2, as previously described by Hermans et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), and was amplified using the Touch-Down cycle described by Zumarraga et al., (2005). PCR products were revealed by horizontal electrophoresis on a 2% agarose gel stained with ethidium bromide (0.5 \u0026micro;g/mL) and visualized under UV light.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSerological-test\u003c/h2\u003e \u003cp\u003eSerological analysis was performed using the LIONEX test on 28 blood samples collected from South American sea lion pups (Fig.\u0026nbsp;3). Overall, 25% of the samples analyzed (7/28) were seropositive for at least one antigen of the MTBC (95% CI: 12.7\u0026ndash;43.4%). Seroprevalence varied by location: at the Punta Buenos Aires colony, three out of seven samples (42.9%) were positive (95% CI: 15.8\u0026ndash;74.9%); at Larga Island, one out of five samples (20.0%) showed reactivity (95% CI: 3.6\u0026ndash;62.4%); and at Monte Le\u0026oacute;n, three out of eleven samples (27.3%) tested positive (95% CI: 9.7\u0026ndash;56.6%). In contrast, no seropositive reactions were detected among five samples from Leones Island (0/5; 95% CI: 0\u0026ndash;43.4%) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eId, colony, sex, kit, culture and PCR results of the sampled individuals.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eId\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eColony\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eKits result\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCulture result\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePCR result\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePunta Bs As\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePunta Bs As\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u0026thinsp;+\u0026thinsp;T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePunta Bs As\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePunta Bs As\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u0026thinsp;+\u0026thinsp;T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePunta Bs As\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u0026thinsp;+\u0026thinsp;T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePunta Bs As\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePunta Bs As\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeones Island\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeones Island\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeones Island\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeones Island\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLeones Island\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarga Island\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarga Island\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarga Island\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u0026thinsp;+\u0026thinsp;T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarga Island\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLarga Island\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonte Le\u0026oacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u0026thinsp;+\u0026thinsp;T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonte Le\u0026oacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonte Le\u0026oacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonte Le\u0026oacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonte Le\u0026oacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u0026thinsp;+\u0026thinsp;T2\u0026thinsp;+\u0026thinsp;T1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonte Le\u0026oacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eM\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonte Le\u0026oacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonte Le\u0026oacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonte Le\u0026oacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonte Le\u0026oacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u0026thinsp;+\u0026thinsp;T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMonte Le\u0026oacute;n\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003econtrol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003enegative\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCulture and PCR\u003c/h3\u003e\n\u003cp\u003eAfter 12 weeks of incubation, no bacterial growth was observed in the culture media. Similarly, direct DNA extractions from nasal swabs did not yield amplification products for the MTBC specific target (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study provides serological evidence suggesting exposure to members of the \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e complex (MTBC) at the population level in free-living South American sea lions from breeding colonies along Argentina\u0026rsquo;s Patagonian coast. These findings most likely reflect mother-to-pup passive transfer of maternal antibodies. Importantly, we have collected biological samples in live pinnipeds for processing through etiological and serological tests using minimally invasive procedures. Although bacteriological culture and direct PCR techniques are the most widely used and reliable tools to diagnose tuberculosis in wildlife, serological tests represent a valuable alternative for monitoring population health status (Thomas et al. 2021, Kelley et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In this study, both bacterial culture and PCR yielded negative results for all samples, an outcome expected given that the individuals sampled were neonates aged 1\u0026ndash;7 days, as estimated from the condition of the umbilical cord (Ameghino, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Tuberculosis is a chronic disease, and once the pathogen has infected the host, a period of progression required before it can be detected through etiological tests.\u003c/p\u003e \u003cp\u003eWe used the Lionex-test for the first time in living pinnipeds. This test was originally developed for the diagnosis of bovine tuberculosis as a low-cost, minimally invasive tool (Lionex, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). While the test is designed to detect antibodies against \u003cem\u003eM. bovis\u003c/em\u003e, it employs purified mycobacterial antigens, including cell wall components such as lipoarabinomannan (LAM). LAM is conserved across members of the \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e complex (MTBC) and widely used as a diagnostic target (Chatterjee \u0026amp; Khoo, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, Minion et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). This shared antigenic structure provides a biological basis for potential cross-reactivity among MTBC species, possibly including \u003cem\u003eM. pinnipedii\u003c/em\u003e. According to the manufacturer\u0026rsquo;s specifications, the test is intended for use in multiple animal species; however, validation studies to date have been conducted primarily in cattle, and it has not been specifically validated in otariids. This assay demonstrated its potential as a practical approach for health surveillance in free-ranging pinniped populations, particularly when combined with other diagnostic approaches. We considered an individual as seropositive to the MTBC when it reacted to at least one of the three antigens included in the test. According to the assay\u0026rsquo;s design, there are seven possible combinations of seropositive results: three single-antigen (1, 2, or 3) and four combined (1\u0026thinsp;+\u0026thinsp;2, 1\u0026thinsp;+\u0026thinsp;3, 2\u0026thinsp;+\u0026thinsp;3, or 1\u0026thinsp;+\u0026thinsp;2+3). Despite these seven possible outcomes, only two positivity patterns were observed in our samples (2 and 1\u0026thinsp;+\u0026thinsp;2). The latter pattern was also reported in a study conducted on milk samples from cattle herds infected with \u003cem\u003eM. bovis\u003c/em\u003e (Kelley et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These consistent patterns may suggest a functional relationship among the antigens targeted by the test, potentially reflecting a shared immune response to \u003cem\u003eM. bovis\u003c/em\u003e or other members of the MTBC (Kelley et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe detected serological reactivity in 25% of the sampled individuals. These findings should be interpreted as indirect evidence of MTBC exposure at the population level. Further studies integrating additional diagnostic approaches will be necessary to better understand the epidemiological dynamics of tuberculosis in these populations.\u003c/p\u003e \u003cp\u003e-\u003c/p\u003e \u003cp\u003eOur findings support the passive transfer of maternal antibodies, mainly acquired through colostrum and milk during the first days of lactation, rather than an active infection in neonates. This mechanism is explained by pinnipeds placentation: the endotheliochorial and zonary, typical of carnivores, permits moderate exchange and minimal immunoglobulin transfer, which explains why pups dependent on colostrum to acquire passive humoral immunity (Ross et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Accordingly, several studies have shown that the highest antibody concentrations in pinniped pups occur during the first day post-partum (Ross et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Castinel et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Meza Cerda et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In harbor seals (\u003cem\u003ePhoca vitulina\u003c/em\u003e), for instance, pups are born with very low IgG levels (3% of maternal levels), which increase rapidly after colostrum ingestion, reaching approximately 65% within only 15 days (Ross et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Furthermore, the seroconversion timeline in other wildlife species reinforces this interpretation. In experimentally infected badgers, antibody-based tests only begin to yield positive results around 45 days post-infection (Infantes-Lorenzo et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Given that the pups in our study were sampled at 1\u0026ndash;7 days of age, the detection of antibodies is highly unlikely to stem from postnatal infection, but instead robustly indicates epidemiological relevance. This serological evidence in pups serves as a valuable, indirect marker of MTBC circulation in the population. Highlighting the importance of considering the maternal origin of antibodies when interpreting serological results in newborn pinnipeds for population TB surveillance.\u003c/p\u003e \u003cp\u003eWe detected antibodies against MTBC in three geographically distinct colonies along the Patagonian coast, namely Punta Buenos Aires, Larga Island, and Monte Le\u0026oacute;n. In contrast, no seropositive individuals were detected at Leones Island, a colony characterized by its relative isolation, which likely reduces interactions with other species and human activities. Conversely, the three seropositive colonies are located in areas with higher ecological connectivity, involving interactions with other wildlife species and varying degrees of anthropogenic influence. These spatial patterns suggest that environmental connectivity and interspecific interactions may play a role in shaping the risk of MTBC exposure in these colonies.\u003c/p\u003e \u003cp\u003eThis observation is consistent with evidence from other systems showing that MTBC members can circulate at complex wildlife\u0026ndash;livestock\u0026ndash;human interfaces in the region (Barandiaran et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Kiers et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e, Winter et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this context, potential exposure pathways may involve environmental contamination or indirect contact among species sharing overlapping habitats. Collectively, our findings highlight the importance of a One Health perspective to better understand pathogen circulation dynamics across interconnected ecosystems.\u003c/p\u003e \u003cp\u003eHistorically, the diagnosis of TB in free-ranging pinnipeds has relied almost exclusively on post-mortem findings from necropsies (Sosa-Drouville et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). While this approach is essential for the isolation of pathogens and the establishment of a definitive diagnosis, it constricts inferences about infection dynamics, pathogen circulation and true prevalence within living colonies (Silva-Pereira et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sacrist\u0026aacute;n et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Martins Melo et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Castro Ramos et al., 2006). The present study proposes a minimally invasive survey of live neonatal pups, employing a priori, site-stratified sampling. This approach complements mortality-based surveillance, reduces sampling bias, and provides representative, field-feasible data on colony health. The serological results obtained in this study provide indirect evidence consistent with exposure to members of the \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e complex (MTBC) in South American sea lions from southern Patagonia.. These results demonstrate the practicality and epidemiological utility of the Lionex\u0026reg;, which could may represent a complementary approach for exploratory health surveillance in live pinnipeds.\u003c/p\u003e \u003cp\u003eIn order to strengthen the inferences made on transmission and zoonotic risk, it is recommended that paired mother-pup sampling is utilised, validation studies, that longitudinal follow-up of cohorts is conducted, and confirmatory diagnostics. In this context, the present results provide baseline information that may inform the development of One Health surveillance strategies and contribute to future research addressing risks at the wildlife\u0026ndash;livestock\u0026ndash;human interface.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Wildlife Rangers and Park Rangers of the provinces of R\u0026iacute;o Negro, Chubut, and Santa Cruz for their collaboration and support during fieldwork. We also thank Julio Rua and Ricardo Vera for their invaluable assistance with fieldwork and logistics. We thank the Clinical and Bacteriological Analysis Laboratory (LACyB) for support with clinical and bacteriological analyses, and the Universidad Nacional de la Patagonia Austral (UNPA) and \u003cem\u003ePor el Mar\u003c/em\u003e Foundation for providing accommodation during fieldwork. Partial funding for this research was obtained from the Vienna Zoo. This research was the result of several years of Argentine investment in science and technology. Argentine science is now being defunded and dismantled, threatening the continuity of this and many other lines of research.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially supported by Agencia Nacional de Promoci\u0026oacute;n de la Investigaci\u0026oacute;n, Desarrollo Tecnol\u0026oacute;gico y la Innovaci\u0026oacute;n [PICT 2020-0400], and the Vienna Zoo.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll necessary permits for the field campaigns and studies were obtained from Subsecretar\u0026iacute;a de Turismo y \u0026Aacute;reas Protegidas and Direcci\u0026oacute;n de Fauna y Flora Silvestre, Chubut Province, the Administraci\u0026oacute;n de Parques Nacionales, and the Institutional Animal Care and Use Committee (CICUAE CCT CONICET-CENPAT), Argentina.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmeghino, S. 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Invest.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 232\u0026ndash;238 (2005).\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":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Antibodies detection, Mycobacterium tuberculosis complex, tuberculosis, South American sea lions, zoonoses","lastPublishedDoi":"10.21203/rs.3.rs-8831472/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8831472/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTuberculosis is a disease caused by bacteria belonging to the \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e complex (MTBC), which includes zoonotic agents capable of infecting a wide range of domestic and wild species, including marine mammals. Although tuberculosis in pinnipeds has been documented worldwide, evidence is largely restricted to post-mortem findings, with limited information from active surveillance of live populations. In this study, We conducted an exploratory field-based assessment of tuberculosis exposure in free-ranging South American sea lions (\u003cem\u003eOtaria byronia\u003c/em\u003e) along the Patagonian coast of Argentina. During the austral summer of 2025, biological samples were collected from 28 neonate pups from four colonies located in Chubut and Santa Cruz provinces, using minimally invasive field procedures. Blood and nasal swab samples were obtained and processed using a combination of serological, bacteriological, and molecular diagnostic approaches. Serological testing was performed using a commercial rapid assay (Lionex Animal TB\u0026reg;) to detect antibodies against MTBC antigens, while nasal swabs were analyzed by bacteriological culture and PCR targeting the IS6110 sequence. Serological reactivity to at least one MTBC antigen was detected in 25.0% (7/28) of the sampled pups, indicating indirect serological evidence of exposure at the colony levelin three of the four colonies. In contrast, all samples were negative by bacteriological culture and PCR. Considering the neonatal age of the individuals and the chronic nature of tuberculosis, the serological findings most likely reflect passive transfer of maternal antibodies rather than active infection in pups. This study offers new evidence on tuberculosis surveillance in live free-ranging pinniped populations, emphasizing the relevance of combining serological, bacteriological, and molecular tools for comprehensive wildlife health monitoring under a One Health approach. Overall, the results reinforce the need for active and integrated surveillance strategies to address zoonotic risks in high-risk wildlife\u0026ndash;human interface settings.\u003c/p\u003e","manuscriptTitle":"Tuberculosis Surveillance in Free-Living Pinnipeds of Patagonia, Argentina","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-11 05:56:06","doi":"10.21203/rs.3.rs-8831472/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-14T14:33:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"283659288563017730663170441557694427436","date":"2026-04-23T15:45:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-20T21:22:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-15T02:42:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-12T19:29:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5f1ea61b-b374-4c44-85a0-f57bf8f30456","owner":[],"postedDate":"May 11th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-14T14:33:06+00:00","index":77,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":67445912,"name":"Health sciences/Diseases"},{"id":67445913,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-05-11T05:56:11+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-11 05:56:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8831472","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8831472","identity":"rs-8831472","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}