Viral Prevalence in Galapagos Giant Tortoises Open Access

Over the past 2 decades, studies on reptiles, including captive and free-living turtles and tortoises, have resulted in the discovery of several parasitic, bacterial, fungal, and viral agents (Harrach et al. 2019; Jacobson and Garner 2020; Brugal et al. 2024). Along with these discoveries, advancements in molecular biology and veterinary diagnostics have improved the systematic classification of microbes while contributing to conservation medicine efforts (Okoh et al. 2021; Salzmann et al. 2021). These advancements have been key to understanding the prevalence and impact of infectious diseases in chelonians, which is crucial to help conserve species.

Herpesviruses and adenoviruses are infectious agents globally present in reptiles, including chelonians (Marschang 2011). Reptilian herpesviruses have been identified and characterized within the subfamily Alphaherpesvirinae (Marenzoni et al. 2018). Several studies have reported the presence of these viruses in clinically healthy chelonians as well as in individuals with a wide range of clinical signs (e.g., rhinitis, stomatitis–glossitis, tumors, and encephalitis; Okoh et al. 2021, 2023). The adenovirus family includes five genera: Mastadenovirus, Aviadenovirus, Atadenovirus, Siadenovirus, and Ichtadenovirus; Testadenovirus has been proposed as a sixth genus to include adenoviruses of Testudines order (Doszpoly et al. 2013). Siadenovirus and Atadenovirus genera may cause morbidity (e.g., lethargy, anorexia, stomatitis, and esophagitis) and mortality in free-living and captive individuals, whereas species in the Testadenovirus genus have been described in clinically healthy chelonians (Rivera et al. 2009; Okoh et al. 2023). In recent years, novel chelonian adenoviruses have been described that may have coevolved with their host, becoming genetically different; for example, adenoviruses associated with Eastern box turtles (Terrapene carolina carolina), Hermann’s tortoises (Testudo hermanni), and the saw-shelled turtle (Myuchelys latisternum; MacLachlan and Dubovi, 2016; Franzen-Klein et al. 2020; Salzmann et al. 2021).

The Galapagos Archipelago is home to the longest-lived terrestrial vertebrate, the Galapagos giant tortoise. Up to 16 species have been described; however, at least two are extinct because of human pressures, with the remaining 14 categorized as vulnerable, endangered, or critically endangered (IUCN 2024). San Cristobal Island, on the eastern side of the archipelago (0°52′3.30″S, 89°26′16.26″W), is home to the endangered Chelonoidis chathamensis species, with an estimated population of 2,950 individuals (Caccone et al. 2017). Isabela Island, on the western side of the archipelago, has five giant tortoise species, each one restricted to a different volcano. The critically endangered Chelonoidis guntheri is restricted to Sierra Negra Volcano (0°48′0.00″S, 90°56′47.40″W; Cayot et al. 2018), whereas the endangered Chelonoidis vicina is endemic to Cerro Azul Volcano (0°58′14.84″S, 91°25′52.00″W). At the single gap in the lava flow separating both volcanoes, a green corridor in the Cinco Cerros area allows the overlapping of Sierra Negra and Cerro Azul tortoises (Gibbs et al. 2020). According to a 2023 census, Sierra Negra held an estimated 704 C. guntheri; Cerro Azul held 5,275 C. vicina individuals (A.N.-C. pers. comm.).

Despite the conservation efforts conducted in the last decades, giant tortoises are still facing several challenges associated with climate change, introduced species, habitat degradation, and illegal trafficking (Deem et al. 2023; Blake et al. 2024). New threats to Galapagos tortoises include the presence of antimicrobial resistance genes in gut bacteria (Nieto-Claudin et al. 2021) and ingestion of plastics (Ramon-Gomez et al. 2023).

As part of a long-term health assessment led by the Galapagos Tortoise Movement Ecology Program, we previously described two novel viral sequences of adenoviruses and two of herpesviruses in Galapagos giant tortoises from Santa Cruz Island and on Alcedo Volcano, Isabela Island (Nieto-Claudin et al. 2022). With the aim of better understanding the presence and prevalence of these viruses and potentially novel viral strains in other Galapagos tortoise species, we tested conjunctival, oral, and cloacal swabs from 99 free-living individuals of three species: 40 tortoises from San Cristobal Island (C. chathamensis) and 59 from Isabela Island (16 of C. guntheri in Sierra Negra, 18 of C. vicina in Cerro Azul, and 25 tortoises in Cinco Cerros, an area where both species may coexist; Fig. 1). We used sterile cotton swabs, placed them in 2-mL cryovials, and preserved them at −20 C until analysis. We recorded morphometric measurements and the body weight of each individual and identified sex in adult animals on the basis of tail length and plastron concavity (Nieto-Claudin et al. 2022). Samples were collected under the Galapagos National Park Directorate (GNPD) annual research permit PC-28-20 and with the support and collaboration of the GNPD.

Genomic DNA was extracted using the DNeasy blood & tissue extraction kit (Qiagen, Valencia, California, USA) following the manufacturer's instructions. All DNA extractions were performed at the Charles Darwin Research Station (CDRS) in Galapagos, Ecuador. For adenovirus detection, we selected cloacal swab samples and performed broad-spectrum nested PCR with consensus primers that amplify a 330-base-pair (bp) region of the adenovirus DNA polymerase-dependent gen (Wellehan et al. 2004). To improve the quality of amplicons, we modified thermal cycles as follows: 94 C for 12 min; 45× (94 C for 30 min, 56 C for 60 min, 72 C for 30 min); 72 C for 10 min. We selected oral and conjunctival swab samples for herpesvirus detection using a nested PCR assay with degenerate primers amplifying a 250-bp region of the DNA polymerase gene (Nieto-Claudin et al. 2022). All PCR products were resolved on 1% agarose gels (120 V, 90 min) and positive results were confirmed by Sanger sequencing. Positive sequences, forward and reverse, were aligned, visually inspected, and subjected to basic local alignment search tool searches. We calculated p-distances for nucleotide and amino acid sequences using MEGAX Software version 11.0.13 (Kumar et al. 2018). Phylogenetic trees were constructed using the maximum likelihood criterion v.2 and based on the DNA polymerase-dependent gen with nucleotide and amino acid sequences using 1,000 bootstrap iterations.

All sampled tortoises were considered clinically healthy on the basis of visual examination. A total of 27 individuals (27.3%; confidence interval [CI] 18.50–36.05) tested positive for adenovirus, whereas no animals tested positive for herpesvirus. We obtained three different nucleotide sequences, classified in three nucleotide sequence types (ntST): AdVntST 1 was detected in tortoises from San Cristobal, AdVntST 2 was detected in Cinco Cerros individuals, and AdVntST 3 in Cerro Azul and Sierra Negra tortoises. Two nucleotide sequences (AdVntST 2, AdVntST 3) translated into the same amino acid sequence type (AdVaaST 2). The identities between AdVntST 1 and AdVntST 2 and AdVntST 1 and AdVntST 3 were 95.1% and 95.6%, respectively, whereas the identities between AdVaaST1 and AdVaaST2 were 95.1%. The closest sequence to the AdVntST1 found in Genbank was the Chelonoidis adenovirus 1 (Genbank accession no. OU508386) with an identity of 99.0%. Regarding AdVntST 2 and AdVntST 3, the closest sequence found was the Chelonoidis adenovirus 2 (Genbank accession no. OU508387), with an identity of 99.0% and 99.5%, respectively.

The phylogenetic analysis revealed that AdVntST 1 and AdVaaST 1 clustered in a clade with CheAdV1 (GenBank accession no. OU508386). The AdVntST 2, AdVntST 3, and AdVaaST 2 clustered in another clade with CheAdV2 (GenBank accession no. OU508387; Fig. 2), suggesting that the sequences obtained in our study could be classified as Chelonoidis adenovirus 1 for ntST 1/aaST1 and Chelonoidis adenovirus 2 for ntST 2, ntST 3/aaST2. Clustered by species, eight of 40 tortoises (20%; 95% CI 7.60–32.4) from San Cristobal Island tested positive to Chelonoidis adenovirus 1 (CheAdV1). In Isabela Island, one of 16 tortoises (6.2%; 95% CI 0.00–18.11) from Sierra Negra (C. guntheri), four of 18 tortoises (22.2%; 95% CI 3.02–41.43) in Cerro Azul (C. vicina), and 14 of 25 tortoises (56%; 95% CI 36.54–75.46) from Cinco Cerros (C. vicina or C. guntheri) tested positive to Chelonoidis adenovirus 2 (CheAdV2; Fig. 3).

Our data on the presence and prevalence of adenoviruses in free-living tortoises from San Cristobal and southern Isabela islands contributes to baseline health data for these iconic species and provides pathogen distribution information to be used by local conservation authorities and scientists. According to our results, CheAdV1 is present in tortoises from San Cristobal, whereas CheAdV2 is found in southern Isabela. These findings agree with previous studies that had found that CheAdV1 was more prevalent in Santa Cruz tortoises and CheAdV2 in Alcedo Volcano (Isabela Island) tortoises (Nieto-Claudin et al. 2022). On the basis of genetic analyses (Poulakakis et al. 2020), San Cristobal tortoises (C. chathamensis) are most closely related to eastern Santa Cruz tortoises (Chelonoidis donfaustoi), in which CheAdV1 was also detected. In contrast, southern Isabela tortoise species (including those from Alcedo, Cerro Azul, and Sierra Negra Volcanoes) clustered together and are genetically more closely related to western Santa Cruz tortoises (Chelonoidis porteri), in which both CheAdV1 and CheAdV 2 were detected (Nieto-Claudin et al. 2022), than to eastern Santa Cruz tortoises (Poulakakis et al. 2020).

The highest prevalence (56%) of CheAdV2 was found in the Cinco Cerros location, an area where giant tortoises with a distinct morphology (so-called “flat”) have been described and a potential geographic overlap between C. guntheri and C. vicina occurs. Despite this location geographically corresponding to the slopes of Cerro Azul (C. vicina), we cannot rule out that some of the sampled tortoises might belong to C. guntheri. In Cerro Azul, the prevalence of adenovirus was 22%, which is higher than in both Sierra Negra and Alcedo Volcano tortoises (6% and 9%, respectively). Despite the geographic isolation of Cinco Cerros, this location has historically been visited by humans searching for tortoise meat and oil, as well as scientists looking for evolutionary answers related to the flat tortoise morphology (Gibbs et al. 2020). In present times, occasional poaching persists in the area, which is also a location used for tortoise releases from the breeding centers. It is possible that the influx of released tortoises may be behind the higher prevalence of adenovirus, although we cannot exclude a greater susceptibility of the species. Both CheAdV1 and CheAdV2 have been detected in tortoises from the Santa Cruz breeding center, as well as in Galapagos tortoises confiscated from the illegal trade (Nieto-Claudin et al. 2022). Herpesvirus was not detected in southern Isabela but it was reported in the northern volcanoes of Alcedo and Wolf. The relatively low prevalence found in both locations (1.3% and 3.6%, respectively) suggests that a bigger sample size would be needed in southern Isabela to assess the presence or absence of herpesvirus in these giant tortoise species.

Adenoviruses found in Galapagos tortoises appear to have coevolved with their hosts. Therefore, we surmise that they do not pose a high risk of morbidity without further virus mutation or jump to a novel species (Bean et al. 2013). Nevertheless, disease has been described in adenovirus-positive Galapagos tortoises under human care (Nieto-Claudin et al. 2022), which suggests that the virus might be pathogenic under certain conditions such as stress, coinfection, or other causes of poor health (Franzen-Klein et al. 2020; Okoh et al. 2023). More research is needed to better understand host–virus coevolution and viral distribution across reptile species in the archipelago. We recommend long-term health surveillance, including detection of viruses, in both tortoises and other wildlife species to help inform conservation actions in the Galapagos involving captive-breeding and rewilding programs such as the Floreana Ecological Restoration Project, which aims to perform several translocations and reintroductions of wildlife species including giant tortoises.

We thank our donors, the Saint Louis Zoo, Galapagos Conservation Trust, and Houston Zoo. We thank the CDRS and the GNPD for logistics and permits. This work was conducted under GNPD research permit PC-28-20. A special acknowledgment for their contributions goes to Freddy Cabrera, Jose Haro, Carlos Sacristán, Irene Peña, Kyana Pike, Anne Guezou, Boris Herrera, Paúl León, Byron Delgado, Johnny Mazón, and Stephen Blake. This publication is contribution 2694 of the Charles Darwin Foundation for the Galapagos Islands.