Correction: Gross anatomy, histology and blood vessel topography of the alimentary canal of the Inland Bearded Dragon (Pogona vitticeps).

[This corrects the article DOI: 10.1371/journal.pone.0234736.].

Rhabdomyosarcoma on the Forelimb of a Common Musk Turtle (Sternotherus odoratus).

Malignant mesenchymal tumours are only rarely reported in turtles. In the present case, an 8-year-old female common musk turtle (Sternotherus odoratus) was presented with a solid, reddened, non-mobile mass on the right forelimb. The mass had a thin, membranous lining and a grey-white cut surface. Histological examination revealed a cell-rich, focally infiltrative neoplasm consisting of spindloid tumour cells arranged in bundles and streams. Rarely, indistinct cytoplasmic cross-striations were seen in the neoplastic cells. Transmission electron microscopy revealed cytoplasmic, disorganized muscle fibrils and haphazardly arranged, attenuated Z-lines in the neoplastic cells. The histological, histochemical and ultrastructural findings led to the diagnosis of a rhabdomyosarcoma. This is the first description of rhabdomyosarcoma in a freshwater turtle.

PLASMA CAPILLARY ZONE ELECTROPHORESIS AND PLASMA CHEMISTRY ANALYTES IN TORTOISES (TESTUDO HERMANNI, TESTUDO GRAECA) AND TURTLES (TRACHEMYS SCRIPTA ELEGANS, GRAPTEMYS SPP.) IN FALL.

Clinical health checks including blood testing before brumation in fall are an important tool in reptile medicine and help to reduce the risk of complications during brumation. Electrophoresis may be useful for the evaluation of liver cell function and the detection of antigenic stimulation. The goal of this study was to compare species-specific reference intervals for plasma chemistry analytes and capillary zone electrophoresis (CZE) for a variety of pet chelonian species in fall. Lithium heparinized samples were collected from 68 clinically healthy spur-thighed tortoises (Testudo graeca), 79 red-eared sliders (Trachemys scripta elegans), and 30 map turtles (Graptemys spp.) from September to November 2016 and 2017. During the same period, 128 equivalent samples were collected from Hermann's tortoises (Testudo hermanni) and the obtained data was used for comparison. Chemistry analytes were measured from plasma using an autoanalyzer and electrophoresis was carried out by CZE. Significant variations (P < 0.0001) between the species were found for several analytes including total protein, percent albumin, and albumin: globulin ratio, α-, ß-, and γ-globulin, alkaline phosphatase, glutamate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, bile acids, creatine kinase, urea, uric acid, inorganic phosphorus, total calcium, and potassium. The variations in albumin (g/l) measured by CZE were also significant (P = 0.0064). No significant variations were detected for sodium levels. The results demonstrate the importance of species-specific reference intervals and provide reference intervals for the plasma chemistry and capillary zone electrophoresis in three chelonian species.

Gross anatomy, histology and blood vessel topography of the alimentary canal of the Inland Bearded Dragon (Pogona vitticeps).

Imaging techniques have proved to be crucial for diagnosis in reptile species. The topography of the internal organs of bearded dragons has been described in recent studies as meeting the small animal practitioners´ demand for knowledge concerning their anatomy. However, the nomenclature in the respective literature is not uniform, which could lead to misunderstandings concerning the respective and/or affected parts of the alimentary canal. Therefore, the aim of this study was to provide clear information on anatomy and histology of the alimentary canal of bearded dragons including supplying blood vessels. For the dissection of the alimentary canal, 11 Inland Bearded Dragons (Pogona vitticeps) were used (five males, six females), which had been euthanised for clinical reasons other than those concerning the digestive tract or had died spontaneously. The supplying arteries were demonstrated by injecting red latex into the aorta, while the intestinal veins were filled with blue latex via the portal vein. Microscopic examination was carried out on specimens of seven additional bearded dragons using routine histologic procedures. Macroscopically, the sections of the alimentary canal from oral to aboral were distinguished into oesophagus, stomach, small intestine, colic ampulla, colic isthmus, rectum and cloaca. Differentiation of the duodenum, jejunum and ileum was only possible when considering the bile duct, the vasculature and the histology of the organ wall. Arteries supplying the oesophagus and the final straight part of the large intestine originated from the aorta in a segmental manner. Between these, three unpaired arteries arose from the aorta. Their branches supplied stomach and intestine excluding its last part. Based on the findings of the present study, a nomenclature for the different parts of the alimentary canal and the supplying blood vessels of bearded dragons is suggested which is well understandable for veterinary practitioners and is based on zoological knowledge of reptiles.

A comparison of thyroid hormone levels and plasma capillary zone electrophoresis in red-eared sliders (Trachemys scripta elegans) and map turtles (Graptemys spp.) depending on season and sex.

BACKGROUND: Thyroid hormones and capillary zone electrophoresis are rarely studied in reptile medicine. OBJECTIVES: The goal of this prospective study was to establish RIs for plasma proteins and thyroid hormones, total tetraiodothyronine (tT4), free T4 (fT4), total triiodothyronine (tT3), and free T3 (fT3), as well as iodine blood levels in red-eared sliders (Trachemys scripta elegans) and map turtles (Graptemys spp.). METHODS: Variables were measured over 1 ½ years to determine variations depending on sex and season, as well as any correlations between the parameters. A total of 131 blood samples from red-eared sliders and 51 blood samples from map turtles were collected from May 2016 to November 2017. The levels of tT4, fT4, and fT3 were measured using standardized autoanalyzer test systems; tT3 was measured by ELISA and iodine inductively coupled plasma mass spectrometry. Total protein was quantitated using the biuret method, and plasma protein fractions were measured using capillary zone electrophoresis. RESULTS: The results showed significant variations in thyroid hormone levels and plasma protein fractions depending on sex and season. Total T4 and fT4 concentrations were significantly (P < .05) correlated in both turtle species and sexes in all seasons. Thyroid hormone levels correlated with blood proteins in a few seasons, including a positive correlation between tT4 and fT4 in female red-eared sliders in fall and female map turtles in summer and fall. CONCLUSIONS: This study demonstrates the importance of considering species, season, and sex variations when interpreting thyroid hormone and plasma protein levels. It also shows that thyroid hormone levels are not always influenced by total protein and albumin.

Parasite burden in a short-lived chameleon, Furcifer labordi.

Life history theory predicts that species with shorter lifespan should show higher investments into growth and reproduction at the expense of immune defenses. Labord's chameleon (Furcifer labordi) is the tetrapod with the shortest known life span. To investigate to which extent immunosenescence influences the die-off of these chameleons when they are only about 6 months old, we examined the gastrointestinal-, blood- and ectoparasite burden in F. labordi in Kirindy Forest (western Madagascar) and compared them with sympatric and longer living F. cf. nicosiai. Moreover, we included data from wild F. labordi that were singly housed under ambient conditions with daily food and water supply. Gastrointestinal parasite prevalence of wild F. labordi increased dramatically during the last 3 months of their lives, which include the reproductive period. Furcifer cf. nicosiai was found to have a belated increase in gastrointestinal parasites compared to F. labordi. In F. cf. nicosiai higher prevalence of blood parasites were found, which probably result from the longer exposure to the arthropod intermediate host. Both species showed infestations with ectoparasites, which peaked in the rainy season but disappeared towards the dry season. Male F. labordi showed a significantly higher prevalence of gastrointestinal - and ectoparasites and higher intensities of coccidians and ectoparasites than females. Males of F. cf. nicosiai exhibited higher prevalence of blood- and ectoparasites, as well as higher intensities in ectoparasites. Caged individuals of both sexes showed delayed senescence, reduced parasite burden and lived longer than their wild conspecifics. Overall, the increase in the prevalence in gastrointestinal - and blood parasites towards the disappearance of the wild population of F. labordi indicates that this species invests comparatively less energy in efficient immune system function, supporting the prediction of life history theory.

Specific anatomy and radiographic illustration of the digestive tract and transit time of two orally administered contrast media in Inland bearded dragons (Pogona vitticeps).

The aim of this study was to describe the specific gross and radiographic anatomy of the digestive tract of inland bearded dragons (Pogona vitticeps). Eleven bearded dragon cadavers of both sexes (6 females, 5 males) were dissected to examine, measure, and document the specific gross anatomy of the alimentary canal. Measurements collected from the cadavers included snout-vent length, total length of the alimentary canal, and the lengths of the individual sections of the gastrointestinal tract, including the esophagus, stomach, small intestine, ampulla coli, isthmus coli, rectum, and the distance from the coprodeum to the vent opening. Twenty-two healthy adult bearded dragons (13 females, 9 males) maintained under standardized husbandry conditions underwent a physical examination, blood collection, and whole-body dorsoventral and lateral survey radiographs; these animals were used to provide the radiographic images of the complete digestive tract. For the subsequent contrast passage studies, two different contrast media, barium sulfate (BaSO4, Barilux suspension) and an iodinated ionic radiocontrast agent (Sodium meglumine amidotrizoate [SMAT], Gastrografin), were used. Water-diluted Barilux suspension (dose 9 ml/kg) was administered orally to 5 bearded dragons, while Gastrografin (dose 5ml/kg) was administered orally to 21 bearded dragons. Four animals were used for both contrast media studies, but received a break of four weeks in between. Dorsoventral and laterolateral radiographs were collected at 0 (baseline), 15, 30, and 45 minutes and 1, 2, 3, 4, 5, 6, 8, 10, 12, 24, 30, and 36 hours after each contrast medium was administered. Both contrast media were found to illustrate the alimentary tracts in the adult bearded dragons. Transit time was substantially faster with SMAT, and SMAT illustrated the entire gastrointestinal tract within 36 hours; BaSO4 did not fully illustrate the gastrointestinal tract in 36 hours. These results might serve as a guideline for the interpretation of subsequent contrast studies in this lizard species.

REFERENCE INTERVALS FOR PLASMA CAPILLARY ZONE ELECTROPHORESIS IN HERMANN'S TORTOISES (TESTUDO HERMANNI) DEPENDING ON SEASON AND SEX.

Electrophoresis is a useful diagnostic tool for the detection of shifts in plasma protein fractions, which can be associated with a variety of diseases. This method is not often used in reptile medicine because there are only a few studies available and no reference intervals have been published for most species. The goal of this study was to establish reference intervals for plasma capillary zone electrophoresis in Hermann's tortoises (Testudo hermanni) under consideration of variations by season and sex. 251 samples from healthy adult Hermann's tortoises were collected from May 2016 to October 2017. Total protein was measured by the biuret method and albumin was measured twice by bromocresol green dye-binding method (BCG) and capillary zone electrophoresis (CZE) in order to compare the detection methods. The globulin fractions were analyzed by CZE. Seasonal variations were significant for total protein (P = 0.0055), albumin (P = 0.0322) and α-globulins (P = 0.0007). The variations between male and female Hermann's tortoises were highly significant for albumin (P < 0.0001), α-globulins (P < 0.0001) and ß-globulins (P < 0.0001). The differences between the measurements of albumin by CZE and BCG also differed depending on the sex and season. This study demonstrates that it is important to include the variations by sex and season when establishing reference intervals and interpreting results. Furthermore, it confirms the importance of using electrophoresis for the correct determination of albumin in tortoises.

Magnetic resonance imaging measurements of organs within the coelomic cavity of red-eared sliders (Trachemys scripta elegans), yellow-bellied sliders (Trachemys scripta scripta), Coastal plain cooters (Pseudemys concinna floridana), and hieroglyphic river cooters (Pseudemys concinna hieroglyphica).

OBJECTIVE To determine anatomic reference points for 4 turtle species and to evaluate data on relative anatomic dimensions, signal intensities (SIs), and position of selected organs within the coelomic cavity by use of MRI. ANIMALS 3 turtle cadavers (1 red-eared slider [Trachemys scripta elegans], 1 yellow-bellied slider [Trachemys scripta scripta], and 1 Coastal plain cooter [Pseudemys concinna floridana]) and 63 live adult turtles (30 red-eared sliders, 20 yellow-bellied sliders, 5 Coastal plain cooters, and 8 hieroglyphic river cooters [Pseudemys concinna hieroglyphica]). PROCEDURES MRI and necropsy were performed on the 3 turtle cadavers. Physical examination, hematologic evaluation, and whole-body radiography were performed on the 63 live turtles. Turtles were sedated, and MRI in transverse, sagittal, and dorsal planes was used to measure organ dimensions, position within the coelomic cavity, and SIs. Body positioning after sedation was standardized with the head, neck, limbs, and tail positioned in maximum extension. RESULTS Measurements of the heart, liver, gallbladder, and kidneys in sagittal, transverse, and dorsal planes; relative position of those organs within the coelom; and SIs of the kidneys and liver were obtained with MRI and provided anatomic data for these 4 turtle species. CONCLUSIONS AND CLINICAL RELEVANCE MRI was a valuable tool for determining the position, dimensions, and SIs of selected organs. Measurement of organs in freshwater chelonians was achievable with MRI. Further studies are needed to establish reference values for anatomic structures in turtles. Results reported here may serve as guidelines and aid in clinical interpretation of MRI images for these 4 species.

[The anatomy of the heart of tortoises (Testudinidae)]. / Die Anatomie des Herzens der Landschildkröte (Testudinidae).

25 formalin-fixed hearts of different tortoise species (Testudinidae) underwent gross-anatomical examination. The aim of the study was to illustrate the specific anatomy of the heart of these species in comparison to the data available in the literature. The examined tortoises showed the well-known basic structure of a reptile heart with two atria and a ventricle composed of three interconnected chambers. The right atrium was consistently slightly larger than the left atrium. The atrioventricular (AV-) valves emerged as double-flap valves, whereby the lateral leaflets were only present in a rudimentary form. Neither papillary muscles nor chordae tendineae could be detected macroscopically. A vertical septum in order to subdivide the dorsal chambers was missing. However, the muscular ridge between Cavum venosum and Cavum pulmonale was well developed. The Cavum pulmonale represented itself as the smallest chamber respectively rather as a small passageway to the Truncus pulmonalis. Apart from two-parted aortic valves also multicuspidated valves of the Truncus pulmonalis could be visualized.

[Echocardiopgraphy in European tortoises (Testudo spp.)]. / Echokardiografische Untersuchung bei Landschildkröten (Testudo spp.).

An echocardiographic examination was carried out in 71 European tortoises (Testudo spp.) via the cervical-brachial acoustic windows. Simultaneously an electrocardiographic examination was performed. The inflow- and outflow tract of the heart were presented in frontal and sagittal longitudinal sections in B-mode. Within B-mode the size (diameter and area) of the atria and the ventricle (Cavum dorsale), the ventricular wall thickness and the diameter of the origin of the right aorta and of the right Arteria pulmonalis were measured. Also, the fractional shortening (FS%) and a fractional area shortening (FAS%) were calculated for the Cavum dorsale. Standard values for these cardiac parameters were determined for four different tortoise groups (depending on their carapace lengths). The direction of blood flow within the heart could be assessed via colour flow Doppler. By using pulsed-wave Doppler examinations of the inflow- and outflow tract the velocities, pressure gradients, velocity-time-integrals and acceleration- and deceleration times could be determined from the recorded inflow and outflow patterns and standard values were established for these parameters as well.

[Osteolysis of the hip joint in an eastern river cooter (Pseudemys concinna concinna)]. / Osteolyse des Hüftgelenks bei einer Hieroglyphen-Schmuckschildkröte (Pseudemys concinna concinna).

Lameness in freshwater turtles is rarely seen and is mostly caused by bite injuries, other traumata or inflammatory processes. A 17 year old female eastern river cooter which was pretreated for disseminated shell necrosis a few months ago was presented due to acute lameness of the right hind limb. Diagnostic imaging revealed complete lysis of the hip joint and was classified as necrotizing osteomyelitis by histopathology. Septicemic spreading of pathogens and thus a link to the previous shell necrosis could not be identified. The patient developed wound complications and died a few days after the surgical procedure. Postmortem, a pronounced subacute pneumonia was diagnosed as presumed cause of death, which has not been clinically manifest during the whole medical history.

Measurement of intraocular pressure in healthy unanesthetized inland bearded dragons (Pogona vitticeps).

OBJECTIVE: To evaluate the use of rebound and applanation tonometry for the measurement of intraocular pressure (IOP) and to assess diurnal variations in and the effect of topical anesthesia on the IOP of healthy inland bearded dragons (Pogona vitticeps). ANIMALS: 56 bearded dragons from 4 months to 11 years old. PROCEDURES: For each animal following an initial ophthalmic examination, 3 IOP measurements were obtained on each eye between 9 AM and 10 AM, 1 PM and 2 PM, and 5 PM and 7 PM by use of rebound and applanation tonometry. An additional measurement was obtained by rebound tonometry for each eye in the evening following the application of a topical anesthetic to evaluate changes in the tolerance of the animals to the tonometer. Descriptive data were generated, and the effects of sex, time of day, and topical anesthesia on IOP were evaluated. RESULTS: Bearded dragons did not tolerate applanation tonometry even following topical anesthesia. Median daily IOP as determined by rebound tonometry was 6.16 mm Hg (95% confidence interval, 5.61 to 6.44 mm Hg). The IOP did not differ significantly between the right and left eyes. The IOP was highest in the morning, which indicated that the IOP in this species undergoes diurnal variations. Topical anesthesia did not significantly affect IOP, but it did improve the compliance for all subjects. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that rebound tonometry, but not applanation tonometry, was appropriate for measurement of IOP in bearded dragons. These findings provided preliminary guidelines for IOP measurement and ophthalmic evaluation in bearded dragons.

Ranavirus infections associated with skin lesions in lizards.

Ranaviral disease in amphibians has been studied intensely during the last decade, as associated mass-mortality events are considered to be a global threat to wild animal populations. Several studies have also included other susceptible ectothermic vertebrates (fish and reptiles), but only very few cases of ranavirus infections in lizards have been previously detected. In this study, we focused on clinically suspicious lizards and tested these animals for the presence of ranaviruses. Virological screening of samples from lizards with increased mortality and skin lesions over a course of four years led to the detection of ranaviral infections in seven different groups. Affected species were: brown anoles (Anolis sagrei), Asian glass lizards (Dopasia gracilis), green anoles (Anolis carolinensis), green iguanas (Iguana iguana), and a central bearded dragon (Pogona vitticeps). Purulent to ulcerative-necrotizing dermatitis and hyperkeratosis were diagnosed in pathological examinations. All animals tested positive for the presence of ranavirus by PCR and a part of the major capsid protein (MCP) gene of each virus was sequenced. Three different ranaviruses were isolated in cell culture. The analyzed portions of the MCP gene from each of the five different viruses detected were distinct from one another and were 98.4-100% identical to the corresponding portion of the frog virus 3 (FV3) genome. This is the first description of ranavirus infections in these five lizard species. The similarity in the pathological lesions observed in these different cases indicates that ranaviral infection may be an important differential diagnosis for skin lesions in lizards.