Development and analytical validation of a novel quantitative PCR assay for the detection of Trachemys herpesvirus 1.

Emerging infectious diseases are a threat that contributes to the decline of global chelonian species. Herpesviruses are among the most impactful pathogens described in chelonians and are frequently associated with a range of presentations across hosts with the potential for severe morbidity and mortality. Trachemys herpesvirus 1 (TrHV1) has been reported in red-eared and yellow-bellied sliders (Trachemys scripta elegans and Trachemys scripta scripta, respectively) but is largely understudied. Invasive red-eared sliders may serve as a reservoir for transmission to sympatric native species. This study aimed to develop a sensitive and specific quantitative real-time PCR (qPCR) assay for the detection of TrHV1 DNA to aid in the characterization of the epidemiology of this virus in aquatic turtles. Two TaqMan-MGB FAM-dye labeled primer-probe sets were designed and evaluated using plasmid dilutions. The higher performing assay was specific for TrHV1 DNA and had a linear dynamic range of 1.0 × 107 to 1.0 × 101 copies per reaction with an R2 of 0.999, slope of -3.386, and efficiency of 97.39%. The limit of detection was 101 copies per reaction, and there was no loss of reaction efficiency in the presence of TrHV1-negative chelonian oral-cloacal DNA. Overall, the Trachemys herpesvirus 1 assay meets established criteria for acceptable qPCR assays and will be a valuable tool in characterizing the epidemiology of Trachemys herpesvirus 1 in chelonians.

In-nest mortality and pathology of hawksbill sea turtle (Eretmochelys imbricata) embryos and hatchlings in St. Kitts and Nevis.

Strategies to improve the hatch success and survival of critically endangered hawksbill turtles (Eretmochelys imbricata) require knowledge of threats to them including pathological conditions. The objective of this study was to describe the mortality and pathology of embryos and dead-in-nest hatchlings on St. Kitts and Nevis. Over the 2019/20 and 2020/21 nesting seasons, the combined mean (SD) hatch success for the two islands was 81.9% (13.2%) and deceased individuals within excavated nests were early-stage embryos (70.7%), late-stage embryos (17.7%), pipped-hatchlings (8.2%) or dead-in-nest hatchlings (3.4%). From 2017 to 2021, a post-mortem examination was performed on 183 turtles, including histology for 116. Anatomical malformations affected 77 (42.1%) examined turtles and included abnormal scute shape or number (22.4%), dysmelia (8.7%), schistosomus reflexus (7.7%) and compressed carapace (7.7%). Microscopic lesions were found in 49.1% of turtles and included tissue mineralization (26.7%, including renal, fetal membrane, liver, heart or muscle), chorioallantoitis (16.2%) and skeletal muscle degeneration and necrosis (10%). Inflammatory lesions associated with fungal or bacterial infections were in the skin (n = 3), chorioallantois (n = 4), lung (n = 3) or yolk sac (n = 1). These lesions may reflect non-specific terminal conditions but their presence in-nest helps explain some of the mortality and pathology documented in hatchlings that die during rehabilitative care. All of the gonads adequately represented for histological determination of sex were female (n = 62), supporting concern for feminization of Caribbean hawksbill turtle nests. The study identifies lesions that could affect hatch and emergence success. The high frequency of skeletal malformations indicates the need for investigations addressing regional impact and pathogenesis, especially genetic and environmental aetiologies including nest temperature. Immediate examination of live hatchlings on nest emergence is warranted to better determine the prevalence of non-fatal malformations that could impact fitness and population genetics.

Morphobiometry of the scleral ossicle rings in Chelonia mydas sea turtles.

OBJECTIVE: Scleral ossicle rings of reptiles have endoskeletal functions that are not completely understood. Moreover, descriptive reports on the anatomy of those rings are scarce. We tried to make an anatomical description that could contribute to a better understanding of their functions. ANIMAL STUDIED AND PROCEDURES: We quantified, histologically characterized and evaluated the morphobiometry of the scleral ossicles, and measured the aditus orbitae of 25 sea turtle (Chelonia mydas) heads. RESULTS: The aditus orbitae represented about one-third of the total head length and the mean area of the internal opening of each ring was up to 8.37% of the aditus orbitae area. The mean internal diameter of the rings (6.32 mm) was characteristic of scotopic species and the most frequent number of ossicles per ring varied between 11 and 12. Two new classifications were proposed for the ossicle types: plus-Verzahnung (+V) and minus-Verzahnung (-V). The bone tissue revealed a lamellar arrangement typical of compact and resistant bones. CONCLUSION: The obtained data may be used to support and expand the understanding of functions, animal activity patterns, distinctions between taxa and taphonomic interpretations.

Ranavirus (Frog Virus 3) Infection in Free-Living Three-Toed Box Turtles (Terrapene mexicana triunguis) in Missouri, USA.

Frog virus 3 (FV3) and related ranaviruses are emerging infectious disease threats to ectothermic vertebrate species globally. Although the impact of these viruses on amphibian health is relatively well studied, less is understood about their effects on reptile health. We report two cases of FV3 infection, 11 mo apart, in three-toed box turtles (Terrapene mexicana triunguis) from a wildlife rehabilitation center. Case 1 had upper respiratory signs upon intake but had no clinical signs at the time of euthanasia 1 mo later. Case 2 presented for vehicular trauma, had ulcerative pharyngitis and glossitis, and died overnight. In case 1, we detected FV3 nucleic acid with qPCR in oral swabs, kidney, liver, spleen, and tongue. In case 2, we detected FV3 in an oral swab, an oral plaque, heart, kidney, lung, liver, spleen, and tongue. We also detected FV3 nucleic acid with in situ hybridization for case 2. For both cases, FV3 was isolated in cell culture and identified with DNA sequencing. Histopathologic examination of postmortem tissue from case 1 was unremarkable, whereas acute hemorrhagic pneumonia and splenic necrosis were noted in case 2. The difference in clinical signs between the two cases may have been due to differences in the temporal course of FV3 disease at the time of necropsy. Failure to detect this infection previously in Missouri reptiles may be due to lack of surveillance, although cases may also represent a novel spillover to box turtles in Missouri. Our findings reiterate previous suggestions that the range of FV3 infection may be greater than previously documented and that infection may occur in host species yet to be tested.

Anthropogenic uranium signatures in turtles, tortoises, and sea turtles from nuclear sites.

Chelonians (turtles, tortoises, and sea turtles) grow scute keratin in sequential layers over time. Once formed, scute keratin acts as an inert reservoir of environmental information. For chelonians inhabiting areas with legacy or modern nuclear activities, their scute has the potential to act as a time-stamped record of radionuclide contamination in the environment. Here, we measure bulk (i.e. homogenized scute) and sequential samples of chelonian scute from the Republic of the Marshall Islands and throughout the United States of America, including at the Barry M. Goldwater Air Force Range, southwestern Utah, the Savannah River Site, and the Oak Ridge Reservation. We identify legacy uranium (235U and 236U) contamination in bulk and sequential chelonian scute that matches known nuclear histories at these locations during the 20th century. Our results confirm that chelonians bioaccumulate uranium radionuclides and do so sequentially over time. This technique provides both a time series approach for reconstructing nuclear histories from significant past and present contexts throughout the world and the ability to use chelonians for long-term environmental monitoring programs (e.g. sea turtles at Enewetok and Bikini Atolls in the Republic of the Marshall Islands and in Japan near the Fukushima Daiichi reactors).

Parasitism by Amblyomma rotundatum Koch, 1844 (Acari: Ixodidae) associated with blefaritis on a red-footed tortoise (Chelonoidis carbonarius) in Rio de Janeiro state - case report.

Amblyomma rotundatum is a tick species commonly found on reptiles and amphibians. In Brazil, investigations of this ectoparasite have indicated its distribution across all Brazilian biomes, although it is concentrated in certain locations. In this context, the objective of the present study was to describe a case of parasitism by A. rotundatum in a red-footed tortoise (Chelonoidis carbonarius) in the state of Rio de Janeiro. A female red-footed tortoise (C. carbonarius), seized by the environmental police, was parasitized by a tick in the left periocular region. The tick was manually removed, preserved in 70º GL ethanol, and sent for identification at the Federal Rural University of Rio de Janeiro (UFRRJ). Based on the morphological evaluation Dantas-Torres et al. (2019), Barros-Battesti et al. (2006) and Lampo et al. (1997), a diagnosis of a female specimen of A. rotundatum was made. In conclusion, this study reports the first case of A. rotundatum parasitizing C. carbonarius in the state of Rio de Janeiro, highlighting the presence of periocular blepharitis associated with this parasitism.

Amblyomma rotundatum é uma espécie de carrapato comumente encontrada em répteis e anfíbios. No Brasil, as investigações desse ectoparasito indicam sua distribuição em todos os biomas brasileiros, embora concentrado em algumas localidades. Nesse contexto, o objetivo do presente estudo foi descrever um caso de parasitismo por A. rotundatum em jabuti-piranga (Chelonoidis carbonarius) no estado do Rio de Janeiro. Uma fêmea de jabuti-piranga (C. carbonarius), apreendida pela Polícia Ambiental, estava parasitada por carrapato na região periocular esquerda. O carrapato foi removido manualmente, preservado em etanol 70º GL e encaminhado para identificação na Universidade Federal Rural do Rio de Janeiro (UFRRJ). Com base na avaliação morfológica Dantas-Torres et al. (2019), Barros-Battesti et al. (2006) e Lampo et al. (1997), foi feito o diagnóstico de um espécime fêmea de A. rotundatum. Em conclusão, este estudo relata o primeiro caso de A. rotundatum parasitando C. carbonarius no estado do Rio de Janeiro, destacando a presença de blefarite periocular associada a este parasitismo.

Reproductive cycles of alligator snapping turtles (Macrochelys temminckii).

The alligator snapping turtle (Macrochelys temminckii) is a species for which captive propagation and reintroduction programs are well established; however, little is known about its reproductive behavior and physiology. In this study, we measured monthly plasma sex steroid hormone concentrations of androgen (T + DHT) estradiol-17B (E2), and progesterone (P4), and used ultrasonography to monitor annual reproductive cycles of a captive population of alligator snapping turtles that is maintained under semi-natural conditions in southeastern Oklahoma. Concurrently, we used automated radio telemetry to measure the relative activity levels of male and female alligator snapping turtles and examine these activity patterns in the context of their reproductive cycles. We also measured monthly concentrations of the glucocorticoid (GC) corticosterone (CORT). Seasonal variation was only detected for T in males, but was observed for T, E2, and P4 in females. Vitellogenesis began in August and ended in April and coincided with elevated E2. Ovulation took place 10-29 April and the nesting period lasted from 11 May - 3 June. Males exhibited greater relative activity levels than females in the fall, winter, and early spring, which coincided with the period when mature sperm would be available for mating. Females were more active than males during the peri-nesting period in the spring. Seasonal changes in CORT were detected and did not differ between males and females. CORT concentrations were elevated in the late spring and summer, coincident with the foraging season, and depressed in the fall, and winter, and at their nadir in the early spring.

A quantum leap in the evolution of platyhelminths: Host-switching from turtles to hippopotamuses illustrated from a phylogenetic meta-analysis of polystomes (Monogenea, Polystomatidae).

While monogenean worms are mainly parasites of the gills and skin of fish, and to a lesser extent parasites of the oral cavity, urinary bladder, and/or conjunctival sacs of amphibians and freshwater turtles, Oculotrema hippopotamiStunkard, 1924 is the single monogenean polystome reported from a mammal, the common hippopotamus (Hippopotamus amphibius Linnaeus). Several hypotheses have been suggested in the last decade to explain the origin of this enigmatic parasite which infects the conjunctival sacs of H. amphibius. Based on a molecular phylogeny inferred from nuclear (28S and 18S) and mitochondrial (12S and COI) sequences of O. hippopotami and chelonian polystomes, we found a sister group relationship between O. hippopotami and Apaloneotrema moleri (Du Preez & Morrison, 2012). This result suggests lateral parasite transfer between freshwater turtles and hippopotamuses, thus likely reflecting one of the most exceptional known examples of host-switching in the course of vertebrate evolution. It also demonstrates that the proximity in the ecological habitat of parasites within host species is an important feature for their speciation and diversification. Because A. moleri and its host, the Florida softshell turtle (Apalone ferox (Schneider)), are restricted to the USA, we suggest that an ancestral stock of parasites may have been isolated on primitive African trionychids after they diverged from their American relatives, and then switched to hippopotamuses or anthracotheres in Africa.

Allometric-kinetic model predictions of radionuclide dynamics across turtle taxa.

Chelonians (turtles, tortoises, and sea turtles; hereafter, turtles) inhabit a wide variety of ecosystems that are currently, or have the potential in the future to become, radioactively contaminated. Because they are long-lived, turtles may uniquely accumulate significant amounts of the radionuclides, especially those with long half-lives and are less environmentally mobile. Further, turtle shells are covered by scutes made of keratin. For many turtle taxa, each year, keratin grows sequentially creating annual growth rings or layers. Theoretically, analysis of these scute layers for radionuclides could provide a history of the radioactivity levels in the environment, yet there are few previously published studies focused on the dynamics of radionuclide intake in turtles. Using established biochemical and ecological principles, we developed an allometric-kinetic model to establish relationships between the radionuclide concentrations in turtles and the environment they inhabit. Specifically, we calculated Concentration Ratios (CRs - ratio of radionuclide concentration in the turtle divided by the concentration in the soil, sediment, or water) for long-lived radionuclides of uranium and plutonium for freshwater turtles, tortoises, and sea turtles. These CRs allowed prediction of environmental concentrations based on measured concentrations within turtles or vice-versa. We validated model-calculated CR values through comparison with published CR values for representative organisms, and the uncertainty in each of the model parameters was propagated through the CR calculation using Monte Carlo techniques. Results show an accuracy within a factor of three for most CR comparisons though the difference for plutonium was larger with a CR ratio of about 200 times for sea turtles, driven largely by the uncertainty of the solubility of plutonium in sea water.

PATHOLOGY OF HATCHLING HAWKSBILL SEA TURTLE (ERETMOCHELYS IMBRICATA) MORTALITIES OCCURRING WHILE UNDER REHABILITATIVE CARE, 2015-21.

Increasing hatchling survival is an important element of conservation of the critically endangered hawksbill sea turtle (Eretmochelys imbricata). Yet, there is little information regarding mortality-associated pathological states of hawksbill hatchlings. The aim of this study was to describe lesions affecting hawksbill hatchlings that died while under rehabilitative care. Forty-four turtles representing the nesting sites of two islands and a 7-yr study period were subjected to comprehensive postmortem examination. The most common lesions included dermatitis (34%), skeletal malformations (23%), and pneumonia (23%). Dermatitis and pneumonia were caused by a variety of presumptively opportunistic bacterial and fungal infections. Fungal infections affected 23% of study turtles, also causing rhinitis and esophagitis. Around half of the cases of dermatitis presented with history of skin lesions, and all those involving periocular areas had clinical history of eye lesions. Pneumonia was not predicted by clinical signs or time in rehabilitation. Malformations included carapace compressions, supra- or subnumerary scutes, and dysmelias with many of those affected having concurrent pathology involving other organs. Other lesions included bacterial yolk sacculitis (15%), skeletal muscle degeneration and necrosis (13%), and acute renal tubular necrosis (13%). The study population was female biased (93%), raising concern for skewed hatchling sex ratios and high incubation temperatures in the eastern Caribbean. The pathology described by this study improves our understanding of threats to hawksbill hatchlings and may be taken into consideration by clinicians when implementing strategies for rehabilitative care.

Pharmacokinetics and therapeutic efficacies of fenbendazole in comparison with levamisole in helminth-infected Caspian turtles (Mauremys caspica).

The pharmacokinetics and bioavailability of fenbendazole and levamisole were determined in Caspian turtles after a single intravenous (i.v.) and subcutaneous (s.c.) administration. Thirty turtles diagnosed as naturally infected with Serpinema microcephalus and Falcaustra armenica nematodes received fenbendazole (50 mg/kg) or levamisole (10 mg/kg) by i.v. and s.c. administrations. Blood samples were collected at time 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 12, 24, and 48 h after drug administration. Plasma drug concentrations were determined by a validated high-performance liquid chromatography method. Data were analyzed by noncompartmental methods. The mean elimination half-life of levamisole was 5.16 h and 12.03 h for i.v. and s.c. routes, respectively, and for fenbendazole, the mean elimination half-life was 25.38 h (i.v.) and 29.77 h (s.c.). The total clearance and volume of distribution at steady-state for levamisole and fenbendazole following i.v. administration were 0.22, 0.44 ml/g/h, and 1.06 and 7.35 ml/g, respectively. For the s.c. route, the peak plasma concentration of levamisole and fenbendazole was 10.53 and 5.24 µg/mL, respectively. The s.c. bioavailability of levamisole and fenbendazole was complete. Considering high anthelmintic efficacy and bioavailability after s.c. administration of levamisole and fenbendazole, and the absence of adverse effects, this route of administration is an easy and efficacious way of treating nematodes in Caspian turtles.

Clinical Pathology of Freshwater Turtles.

Freshwater turtles are physiologically unique in their adaptations to life on both land and freshwater habitats. Appropriate interpretation of laboratory values specific to these species is important for both conservation efforts in free-ranging populations and in captive populations, especially because these animals become increasingly popular as pets. Although normal physiology has been well characterized, understanding of clinicopathologic changes in response to disease processes in freshwater chelonian species is relatively limited. This article reviews the current knowledge of hematology, plasma biochemistry, and urinalysis specific to freshwater turtles, with correlates to other chelonian species when specific data are unavailable.

Sea turtle hatchlings can distinguish between coastal and oceanic seawaters.

Following their emergence on land, sea turtle hatchlings need to travel through the open ocean. Whether hatchlings can detect ecologically and functionally relevant chemical cues released in the marine habitat is unknown. We collected seawater at 6 and 27 km off the Brazilian coast, i.e. within and beyond the continental shelf. In a two-choice flume, we exposed post-emergent (<24 h old) loggerhead (Caretta caretta) turtles to these seawaters. Based on their life history, we posited that if hatchlings could distinguish between the seawater from these regions, they should prefer the oceanic seawater and/or avoid the coastal seawater. Hatchlings were tested singly and could access any parts of the flume. We recorded the seawater plume first visited and the time spent in each plume. Of all the first choices and time spent in a plume, nearly 70% involved the oceanic seawater. The ability of hatchlings to distinguish between seawaters could provide goal-recognition information.

Case report: An innovative non-invasive technique to manage shell injuries in C. carbonarius.

Shell fractures are one of the most traumatic and recurrent injuries observed in chelonians during clinical practice. The most common causes of fractures are falling, being run over by automobiles, being burned, and wild animal bites. Epoxy, acrylic resin, polyester, fiber-grass blanket, and screw fixation are among the current techniques used to treat fractures. Regarding the difficulty of fracture repair in the carapace, this case report aimed to report a procedure that is effective, less time-consuming, accessible, affordable, and safe for shell fractures in C. carbonarius. During the physical examination, the animal showed two fractures, in the dorsal region of the carapace and right lateral side of the bridge, with subcutaneous tissue exposure and loss of a small piece of dorsocranial carapace. To treat these injuries, the animal was submitted to a resin application. The procedure consists of using ethyl-cyanoacrylate associated with sodium bicarbonate, which produces a more resistant resin that is bactericidal, non-toxic, and easy to apply in a low surgery time compared to the common methods used to fix shell fractures. The resin application was successfully done, and the animal was under care for a month after the fracture reduction. It was observed that the treatment was effective, presenting reduction of the fracture. A month after the procedure, the animal showed no intercurrence. Three years after the procedure, the animal still presents part of the material still fixed to the shell, normal growth, without interference in locomotor capacity. This resin proved to be an innovative and promising alternative way to treat fractures, suggesting the development of new non-invasive approaches for several tissues and different animal species.

Transcriptomic analysis of preovipositional embryonic arrest in a nonsquamate reptile (Chelonia mydas).

After gastrulation, oviductal hypoxia maintains turtle embryos in an arrested state prior to oviposition. Subsequent exposure to atmospheric oxygen upon oviposition initiates recommencement of embryonic development. Arrest can be artificially extended for several days after oviposition by incubation of the egg under hypoxic conditions, with development recommencing in an apparently normal fashion after subsequent exposure to normoxia. To examine the transcriptomic events associated with embryonic arrest in green sea turtles (Chelonia mydas), RNA-sequencing analysis was performed on embryos from freshly laid eggs and eggs incubated in either normoxia (oxygen tension ~159 mmHg) or hypoxia (<8 mmHg) for 36 h after oviposition (n = 5 per group). The patterns of gene expression differed markedly among the three experimental groups. Normal embryonic development in normoxia was associated with upregulation of genes involved in DNA replication, the cell cycle, and mitosis, but these genes were commonly downregulated after incubation in hypoxia. Many target genes of hypoxia inducible factors, including the gene encoding insulin-like growth factor binding protein 1 (igfbp1), were downregulated by normoxic incubation but upregulated by incubation in hypoxia. Notably, some of the transcriptomic effects of hypoxia in green turtle embryos resembled those reported to be associated with hypoxia-induced embryonic arrest in diverse taxa, including the nematode Caenorhabditis elegans and zebrafish (Danio rerio). Hypoxia-induced preovipositional embryonic arrest appears to be a unique adaptation of turtles. However, our findings accord with the proposition that the mechanisms underlying hypoxia-induced embryonic arrest per se are highly conserved across diverse taxa.

Symmetrical Dimethylarginine as a Diagnostic Parameter in Hermann's Tortoises (Testudo hermanni).

Background: Despite improvements in habitational conditions, kidney disease is relatively common in tortoises. Objectives: Purpose of this study was the establishment of Symmetrical dimethylarginine (SDMA) reference values for clinically healthy Hermann's Tortoises. Animals: Clinically healthy Hermann's Tortoises (n = 131) were included in the period from October 2017 to September 2019. Methods: Creatinine and other biomarkers were tested at IDEXX Laboratories, Germany using residual blood samples from Hermann's tortoises. SDMA was measured with the IDEXX test and verified by liquid chromatography-mass spectrometry at IDEXX Laboratories, USA. Results: SDMA values ranged from 1 to 21 µg/dl (n = 131) for the IDEXX SDMA Test and SDMA values ranged from 1 to 17 µg/dl (n = 82) for LC-MS. For the comparison of the two measuring systems, the following results were obtained R 2 = 0.75 (p < 0.001). Conclusion and Clinical Importance: SDMA can be measured in Hermann's Tortoises and the reference values range in clinically healthy animals is comparable to that of dogs and cats.

The midday sun reveals what the waters hide: Podocnemis unifilis Troschel, 1848 (Testudines, Podocnemididae), another exotic species in the Pantanal of Mato Grosso state / O sol do meio-dia revela o que as águas escondem: Podocnemis unifilis Troschel, 1848 (Testudines, Podocnemididae), outra espécie exótica no Pantanal mato-grossense

Abstract The process of exotic species introduction is recognized as one of the greatest threats to the ecology and economy of the planet, due to changes in interactions in native biological communities. In this sense, reporting bioinvasions is the first step to understanding its introduction process and creating strategies to mitigate possible socio-environmental damages. Here we report the first records of Podocnemis unifilis introduced in the Northern Pantanal, Mato Grosso, Brazil. We obtained the species' first records occasionally during 2014 and 2015. In the following years from 2016 to 2019, we carried out systematic observations through nautical incursions on the Paraguay River, between the Sepotuba River mouth and the Taiamã Ecological Station (TES). We also included third part records, when it was possible to prove the species identification and registration sites. Podocnemis unifilis adults, hatchling and nests were recorded at different points along the Paraguay River. Our results indicate that P. unifilis was introduced close to the urban nucleus of Cáceres about 30 years ago. Currently, it has an established population, recording nests and hatchling for years in a row. A particular one is its occurrence at the TES, an important natural fish nursery and refuge for the wetland fauna. Thus, continuous monitoring is suggested from P.unifilis, since the Pantanal's environment with several different aquatic environments, offers favorable conditions for the species population increase in the Northern Pantanal.

Resumo O processo de introdução de espécies exóticas é reconhecido como uma das maiores ameaças à ecologia e economia do planeta, devido às mudanças nas interações em comunidades biológicas nativas. Nesse sentido, relatar as bioinvasões é o primeiro passo para entender seu processo de implantação e criar estratégias para mitigar possíveis danos socioambientais. Aqui relatamos os primeiros registros de P. unifilis introduzidos no Pantanal Norte, Mato Grosso, Brasil. Obtivemos os primeiros registros das espécies ocasionalmente durante 2014 e 2015. Nos anos seguintes, de 2016 a 2019, realizamos observações sistemáticas por meio de incursões náuticas no rio Paraguai, entre a foz do rio Sepotuba e a Estação Ecológica Taiamã (TES). Incluímos também registros de terceiros, quando foi possível comprovar os locais de identificação e registro das espécies. Adultos de Podcnemis unifilis, filhotes e ninhos foram registrados em diferentes pontos ao longo do rio Paraguai. Nossos resultados indicam que P. unifilis foi introduzido próximo ao núcleo urbano de Cáceres há cerca de 30 anos. Atualmente, ela tem uma população estabelecida, registrando ninhos e filhotes por anos consecutivos. Uma em particular é a sua ocorrência no TES, importante viveiro natural de peixes e refúgio da fauna pantaneira. Assim, sugere-se o monitoramento contínuo de P.unifilis, uma vez que o ambiente do Pantanal com diversos ambientes aquáticos distintos, oferece condições favoráveis para o aumento da população da espécie no Pantanal Norte.

Evaluation of a dexmedetomidine-midazolam-ketamine combination administered intramuscularly in captive ornate box turtles (Terrapene ornata ornata).

OBJECTIVE: To characterize the effects of a combination protocol of dexmedetomidine-midazolam-ketamine (DMK) administered intramuscularly (IM) in ornate box turtles (Terrapene ornata ornata). STUDY DESIGN: Prospective experimental trial. ANIMALS: A total of 16 apparently clinically healthy adult ornate box turtles (eight male, eight female). METHODS: Each turtle was treated with dexmedetomidine (0.1 mg kg-1), midazolam (1 mg kg-1) and ketamine (10 mg kg-1) administered IM. Time to first response, time to maximal effect, the plateau phase and time to recovery from reversal administration were recorded. Physiologic variables, muscle tone, reflexes and the ability to perform endotracheal intubation were recorded at 5 minute intervals. Movement in response to an IM injection of 0.1 mL sterile 0.9% NaCl administered in the left pelvic limb, using a 25 gauge needle to a depth of just past the bevel of the needle, was assessed every 15 minutes. Atipamezole (0.5 mg kg-1) IM and flumazenil (0.05 mg kg-1) SC were administered 60 minutes after the initial DMK injections. RESULTS: The mean time to first response, time to maximal effect, the plateau phase and time to recovery were 2.1, 14.9, 38.7 and 7.8 minutes, respectively. A respiratory rate was not observed in most turtles. The body temperature significantly increased over time. The palpebral reflex was persistent in 43% of turtles and the tail pinch reflex remained intact in 13% of turtles. All turtles recovered with no observed adverse effects. CONCLUSIONS AND CLINICAL RELEVANCE: In this study, this DMK protocol administered to ornate box turtles resulted in a rapid-onset, light anesthesia lasting approximately 40 minutes and a smooth recovery with no adverse effects noted.