Captive Tortoises Research Articles

Development and validation of a quantitative PCR assay for detection of Emydoidea herpesvirus 1 in free-ranging Blanding’s turtles (Emydoidea blandingii)

Blanding’s turtles (Emydoidea blandingii), an endangered species in Illinois, have experienced range-wide declines because of habitat degradation and fragmentation, predation, and road mortality. While ongoing studies are crucial to a thorough understanding of the natural history and demographics in these disjointed Illinois populations, infectious disease threats have been largely unevaluated. Herpesvirus outbreaks have been associated with high morbidity and mortality in populations of captive tortoises and turtles worldwide, including the family Emydidae (pond and box turtles). However, novel herpesviruses including Terrapene herpesvirus 1, Emydid herpesvirus 1 and 2, and Glyptemys herpesvirus 1 and 2, have recently been identified in apparently healthy free-ranging freshwater turtles. In 2015, 20 free-ranging Blanding’s turtles in DuPage County, Illinois were screened for a herpesvirus using consensus PCR. A novel herpesvirus species (Emydoidea herpesvirus 1; EBHV1) was identified in two animals and shared a high degree of sequence homology to other freshwater turtle herpesviruses. Two quantitative real-time PCR assays, using EBHV1 primer-1 and primer-2, were developed to target an EBHV1-specific segment of the DNA-dependent DNA polymerase gene and validated. Both assays performed with high efficiency (slope = −3.2; R2 = 1), low intra-assay variability, and low inter-assay variability (coefficient of variation <2% at all dilutions). However, EBHV1 primer-2 displayed less variation and was selected to test clinical samples and five closely related herpesvirus control samples. Results indicate that this assay is specific for EBHV1, has a linear range of detection from 108 to 101 viral copies per reaction, and can categorically detect as few as 1 viral copy per reaction. This qPCR assay provides a valuable diagnostic tool for future characterization of EBHV1 epidemiology.

Biochemistry and hematology parameters of the San Cristóbal Galápagos tortoise (Chelonoidis chathamensis).

As part of a planned introduction of captive Galapagos tortoises (Chelonoidis chathamensis) to the San Cristóbal highland farms, our veterinary team performed thorough physical examinations and health assessments of 32 tortoises. Blood samples were collected for packed cell volume (PCV), total solids (TS), white blood cell count (WBC) differential, estimated WBC and a biochemistry panel including lactate. In some cases not all of the values were obtainable but most of the tortoises have full complements of results. Despite a small number of minor abnormalities this was a healthy group of mixed age and sex tortoises that had been maintained with appropriate husbandry. This work establishes part of a scientific and technical database to provide qualitative and quantitative information when establishing sustainable development strategies aimed at the conservation of Galapagos tortoises.

Hematologic Variation Values of Captive Red-footed Tortoise (Chelonoidis carbonaria) in South Brazil

Background: Reference hematological values for chelonians are uncommon, especially those that take account of seasonal variations. While there exists controversy over how climate influences hematological values in different reptile species, the identification of this influence would help with the diagnosis, treatment, and prognosis of diseases that affect these species, often kept as pets. Chelonians are ectotherms and, therefore, intrinsic and extrinsic factors may affect physiological and hematological values, affecting the interpretation of clinical information on these species. The aims of this study were to assess the hematological values of captive red-footed tortoises (Chelonoidis carbonaria) and to check their variation according to the cold and warmer seasons of the year.Materials, Methods &amp;Results: Blood samples were collected from nine red-footed tortoises (C. carbonaria) of indeterminate sex and different ages, bred in captivity without control over environmental variables during one year, one each season (summer, fall, winter and spring). A total of 36 samples were considered to study. The colder seasons were collapsed together into one group that holds winter and fall (colds) and the months whit warmer weather spring and summer were grouped as a class called (warm). The complete blood count was performed manually, and medians, maximum values, and minimum values were obtained from the animals in each season of the year. The samples were grouped in “cold” and “warm” classes as above. Repeated measures ANOVA were used in order to compare the influence of climate variation on hematologic variables. For this mater Bonferroni’s test was employed as post-hoc analysis. As results, the erythrocytes total count, hemoglobin value, hematocrit, mean corpuscular hemoglobin concentration, absolute numbers of heterophils, eosinophils, basophils, monocytes, and total plasma proteins varied significantly (P &lt; 0.05) between colder and warmer seasons. Erythrocyte values yielded significantly higher values in warmer seasons than in the other seasons. The seasonality was verified in erythrocytes total count (P &lt; 0.001), hemoglobin value (P &lt; 0.001), hematocrit values (P &lt; 0.001) and differential white blood cell count (P &lt; 0.001).Discussion: The hematological alterations detected in C. carbonaria were similar observed in similar studies in reptiles species and due to the distinct method and management used and different species, it was not possible to compare the statistical results. In previously studies, as a C. carbonaria, the hematological variations between climatic seasons were identified in other reptile species due particular ectothermic metabolism. The variation in total WBC count produced by seasons of the year had already been described in snakes, such as in South American rattlesnakes and in tropical snakes. Changes in metabolic activity may be influence in TPP values in reptiles when season of the year is included as a variable. In hibernating species, the variation in TPP values is attributed to hibernating behavior when the lower food intake led to a decrease in plasma protein levels. In the case of C. carbonaria, which does not hibernate, other variables could influence the difference between seasons. All hematological changes observed in studied animals confirm the influence of climate between cold and hot months, given the peculiar metabolism of chelonians. The results point out the importance is to consider the seasonal climate variations in the interpretation of hematological values of C. carbonaria under uncontrolled environmental conditions.

Efficacy of a Topical Formulation Containing Emodepside and Praziquantel (Profender®, Bayer) against Nematodes in Captive Tortoises

Abstract Gastrointestinal parasites are commonly diagnosed in captive tortoises. In response, fenbendazole has traditionally been used as an anthelmintic, either in single or repeated doses. However, fenbendazole requires oral administration and the process can be very challenging in some individuals. A topical preparation containing emodepside and praziquantel (Profender®, Bayer, Leverkusen, Germany) is promoted as effective against a broad range of nematodes, trematodes, and cestodes. Although this product is currently only licensed for administration to cats, previous studies have shown positive results with tortoises. The aim of this study was to determine the efficacy of Profender against oxyurid and ascarid parasites in captive tortoises. This was achieved by quantifying nematode eggs per gram (EPG) in feces using a modified McMaster technique before (Day 0) and after (Days 14 and 33) topical application of Profender at a dose rate of 21.5 mg emodepside and 85.5 mg praziquantel per kilogram. Twenty-...

Cues from a common predator cause survival-linked behavioral adjustments in Mojave Desert tortoises (Gopherus agassizii)

Animals are expected to engage in behavioral decision-making that minimizes their risk of predation; these decisions can cause non-lethal predator effects to behavior and spatial use. Our goal was to determine whether non-lethal effects of a common predator, coyotes (Canis latrans), could affect the behavior of a declining reptile, the Mojave Desert tortoise (Gopherus agassizii), hereafter tortoise. We exposed captive tortoises to olfactory and visual cues of coyotes and measured their resulting foraging behavior, burrow use, and other antipredator behaviors. We also measured risk of coyote predation in wild tortoises based on burrow use. Willingness to feed and time spent feeding were unaffected by the presence of coyote urine, but higher air temperatures at the start of feeding caused a decrease in time spent at their food dish. Captive tortoises chronically exposed to coyote urine did, however, spend more time in their burrows than when exposed to rabbit urine. When exposed to a coyote decoy, captive tortoises showed more general antipredator behaviors than those exposed to a control stimulus (deer decoy). Wild tortoises were less likely to be depredated by coyotes, if they were encountered in burrows more frequently. Predator olfactory and visual cues exerted non-lethal effects on captive tortoises through behavioral adjustments to their refuge-seeking behavior, which was a behavior linked to decreased coyote predation risk for wild tortoises. Human-subsidized predator populations may negatively affect declining species, including reptiles, as a result of behavioral modifications in response to predator cues. Predators can affect behavior of animals through olfactory and visual cues, which can have measurable effects on prey populations. Changes in prey behavior in response to predator presence can occur at the expense of opportunities to forage or reproduce, leading to decreased survival and reproduction. We tested behavioral responses of captive Mojave Desert tortoises to olfactory and visual cues of coyotes, which are a common predator in the Mojave Desert and subsidized by human food sources. Foraging behavior of captive tortoises was strongly affected by air temperatures at the start of feeding, but not coyote cues. Captive tortoises did, however, increase the amount of time they spent in burrows when coyote urine was present and showed greater fearfulness at the presentation of coyote urine combined with coyote models. Increased burrow use was associated with lower risk of coyote predation in wild tortoises, which suggested an advantage to increased refuge use when coyotes were detectable. Responses by tortoises suggest that coyote cues on the landscape affect tortoises through behavioral pathways that result in increased refuge use.

Free-living and captive turtles and tortoises as carriers of new Chlamydia spp.

A variety of Chlamydia species belonging to the Chlamydiaceae family have been reported in reptilian hosts but scarce data about their occurrence in turtles and tortoises are available. In this study, research was conducted to acquire information on invasive alien species (IAS) of turtles and indigenous turtles and tortoises, living both free and in captivity, as possible reservoirs of Chlamydiaceae. Analysis of specimens (pharyngeal and cloacal swabs and tissues) from 204 turtles and tortoises revealed an overall Chlamydiaceae prevalence of 18.3% and 28.6% among free-living and captive animals respectively, with variable levels of shedding. Further testing conducted with a species-specific real-time PCR and microarray test was unsuccessful. Subsequently sequencing was applied to genotype the Chlamydiaceae-positive samples. Almost the full lengths of the 16S rRNA and ompA genes as well as the 16S-23S intergenic spacer (IGS) and 23S rRNA domain I were obtained for 14, 20 and 8 specimens respectively. Phylogenetic analysis of 16S rRNA amplicons revealed two distinct branches. Group 1 (10 specimens), specific to freshwater turtles and reported here for the first time, was most closely related to Chlamydia (C.) pneumoniae strains and the newly described Candidatus C. sanzinia. Group 2 (four specimens), detected in Testudo spp. samples, showed highest homology to C. pecorum strains but formed a separate sub-branch. Finally, molecular analysis conducted on positive samples together with their geographical distribution in places distant from each other strongly suggest that Group 1 specimens correspond to a new species in the Chlamydiaceae family. In-depth studies of Chlamydia spp. from turtles and tortoises are needed to further characterise these atypical strains and address arising questions about their pathogenicity and zoonotic potential.

Chelonian haematology 1. Collection and handling of samples

Chelonians have a unique anatomy that causes problems when it comes to using common techniques for assessing health status and diagnosing disease; for example, palpation, auscultation, radiography and exploratory laparotomy....

Long-term memory of relative reward values.

Long-term memory can be adaptive as it allows animals to retain information that is crucial for survival, such as the appearance and location of key resources. This is generally examined by comparing choices of stimuli that have value to the animal with those that do not; however, in nature choices are rarely so clear cut. Animals are able to assess the relative value of a resource via direct comparison, but it remains unclear whether they are able to retain this information for a biologically meaningful amount of time. To test this, captive red-footed tortoises (Chelonoidis carbonaria) were first trained to associate visual cues with specific qualities and quantities of food, and their preferences for the different reward values determined. They were then retested after an interval of 18 months. We found that the tortoises were able to retain the information they had learned about the cues as indicators of relative reward values over this interval, demonstrating a memory for the relative quantity and quality of food over an extended period of time. This is likely to impact directly on an animal's foraging decisions, such as the exploitation of seasonally varying resources, with obvious fitness implications for the individual; however, the implications may also extend to the ecological interactions in which the animal is involved, affecting processes such as herbivory and seed dispersal.

SELECTED PLASMA BIOCHEMISTRY ANALYTES OF HEALTHY CAPTIVE SULCATA (AFRICAN SPURRED) TORTOISES (CENTROCHELYS SULCATA).

The sulcata or African spurred tortoise (Centrochelys sulcata) is a large tortoise species that is commonly kept in zoologic collections and as a pet. The objectives of this study were to establish reference intervals for selected biochemical analytes in clinically healthy captive sulcata tortoises and to evaluate the impact of blood sampling site and sex. Blood samples were collected from 60 tortoises from either the dorsal coccygeal (tail) vein or the subcarapacial venous plexus based on their body size. The packed cell volume and refractometric total solids (TS) were determined. The concentrations of selected plasma biochemical analytes were determined using the VetScan VS2 analyzer and included albumin, aspartate aminotransferase, bile acids, calcium, creatine kinase, globulins, glucose, potassium, sodium, phosphorous, total proteins (TP), and uric acid. The calcium-to-phosphorous ratio was calculated. Reference intervals were determined and evaluated for the potential effects of blood sampling site and sex. There were significant differences (P < 0.05) associated with the blood sampling site in TS, TP, phosphorus, and globulins, with higher values in samples from the tail versus the subcarapacial sampling site. No significant statistical differences were noted in the plasma biochemistry analytes between the sexes. Reading of the globulins by the analyzer failed in 36 of 60 of the samples and was largely associated with the subcarapacial plexus sampling site. The reference intervals defined by the VetScan analyzer in this study can be used for clinical medicine and conservation of this tortoise species. Sampling site was identified as a factor significantly affecting some blood analytes in this study; this factor should thus be taken into consideration when assessing sulcata tortoise health status and using this testing methodology.

Negative impacts of invasive plants on conservation of sensitive desert wildlife

AbstractHabitat disturbance from development, resource extraction, off‐road vehicle use, and energy development ranks highly among threats to desert systems worldwide. In the Mojave Desert, United States, these disturbances have promoted the establishment of nonnative plants, so that native grasses and forbs are now intermixed with, or have been replaced by invasive, nonnative Mediterranean grasses. This shift in plant composition has altered food availability for Mojave Desert tortoises (Gopherus agassizii), a federally listed species. We hypothesized that this change in forage would negatively influence the physiological ecology, immune competence, and health of neonatal and yearling tortoises. To test this, we monitored the effects of diet on growth, body condition, immunological responses (measured by gene transcription), and survival for 100 captive Mojave tortoises. Tortoises were assigned to one of five diets: native forbs, native grass, invasive grass, and native forbs combined with either the native or invasive grass. Tortoises eating native forbs had better body condition and immune functions, grew more, and had higher survival rates (&gt;95%) than tortoises consuming any other diet. At the end of the experiment, 32% of individuals fed only native grass and 37% fed only invasive grass were found dead or removed from the experiment due to poor body conditions. In contrast, all tortoises fed either the native forb or combined native forb and native grass diets survived and were in good condition. Health and body condition quickly declined for tortoises fed only the native grass (Festuca octoflora) or invasive grass (Bromus rubens) with notable loss of fat and muscle mass and increased muscular atrophy. Bromus rubens seeds were found embedded in the oral mucosa and tongue in most individuals eating that diet, which led to mucosal inflammation. Genes indicative of physiological, immune, and metabolic functions were transcribed at lower levels for individuals fed B. rubens, indicating potential greater susceptibility to disease or other health‐related problems. This study highlights the negative indirect effects of invasive grasses, such as red brome, in desert ecosystems, and provides definitive evidence of a larger negative consequence to health, survival, and ultimately population recruitment for Mojave Desert tortoises than previously understood.

Host contact and shedding patterns clarify variation in pathogen exposure and transmission in threatened tortoiseGopherus agassizii: implications for disease modelling and management

Most directly transmitted infections require some form of close contact between infectious and susceptible hosts to spread. Often disease models assume contacts are equal and use mean field estimates of transmission probability for all interactions with infectious hosts. Such methods may inaccurately describe transmission when interactions differ substantially in their ability to cause infection. Understanding this variation in transmission risk may be critical to properly model and manage some infectious diseases. In this study, we investigate how varying exposure and transmission may be key to understanding disease dynamics in the threatened desert tortoise Gopherus agassizii. We created heterogeneity in Mycoplasma agassizii exposure (the putative bacterial agent of a respiratory disease) by varying the duration of interactions between naturally infected and uninfected captive desert tortoises. Using qPCR, we identified new infections and compared models of transmission probability as a function of contact duration and pathogen load. We then examined the contact patterns of a wild tortoise population using proximity loggers to identify heterogeneity in contact duration. The top-ranked model predicting M.agassizii transmission included a dose term defined as the product of the number of days in proximity to an infected host and the infection level of that host. Models predicted low transmission probability for short interactions, unless the infectious host had a high load of M.agassizii: such hosts were predicted to transmit infection at higher rates with any amount of contact. We observed predominantly short-lived interactions in a free-ranging tortoise population and thus, expect transmission patterns in this population to vary considerably with the frequency and duration of high infection levels. Mean field models may misrepresent natural transmission patterns in this and other populations depending on the distribution of high-risk contact and shedding events. Rapid outbreaks in generally solitary species may result from changes to their naturally low-risk contact patterns or due to increases in the frequency of severe infections or super-shedding events - population characteristics that should be further investigated to develop effective management strategies.

Persistence of distinctive morphotypes in the native range of the CITES-listed Aldabra giant tortoise.

Understanding the extent of morphological variation in the wild population of Aldabra giant tortoises is important for conservation, as morphological variation in captive populations has been interpreted as evidence for lingering genes from extinct tortoise lineages. If true, this could impact reintroduction programmes in the region. The population of giant tortoises on Aldabra Atoll is subdivided and distributed around several islands. Although pronounced morphological variation was recorded in the late 1960s, it was thought to be a temporary phenomenon. Early researchers also raised concerns over the future of the population, which was perceived to have exceeded its carrying capacity. We analyzed monthly monitoring data from 12 transects spanning a recent 15‐year period (1998–2012) during which animals from four subpopulations were counted, measured, and sexed. In addition, we analyzed survival data from individuals first tagged during the early 1970s. The population is stable with no sign of significant decline. Subpopulations differ in density, but these differences are mostly due to differences in the prevailing vegetation type. However, subpopulations differ greatly in both the size of animals and the degree of sexual dimorphism. Comparisons with historical data reveal that phenotypic differences among the subpopulations of tortoises on Aldabra have been apparent for the last 50 years with no sign of diminishing. We conclude that the giant tortoise population on Aldabra is subject to varying ecological selection pressures, giving rise to stable morphotypes in discrete subpopulations. We suggest therefore that (1) the presence of morphological differences among captive Aldabra tortoises does not alone provide convincing evidence of genes from other extinct species; and (2) Aldabra serves as an important example of how conservation and management in situ can add to the scientific value of populations and perhaps enable them to better adapt to future ecological pressures.

Prevalence of Haemoproteus spp. (Apicomplexa: Haemoproteidae) in tortoises in Brazil and its molecular phylogeny.

Captive terrestrial tortoises of the species Chelonoidis carbonaria (n = 17) and Chelonoidis denticulata (n = 37) in the state of Minas Gerais, southeastern Brazil, were examined for hematozoans by using a combination of microscopic and molecular methods. Microscopic examination revealed young intra-erythrocytic forms in blood smears from both species of tortoises. The results of PCR, sequencing, and phylogenetic analysis indicated that these parasites belonged to the Haemoproteus spp., whose observed prevalence was 17.6 % in C. carbonaria and 13.5 % in C. denticulata. Phylogenetic analysis indicated that these sequences formed a clade that was grouped with other sequences of Haemoproteus spp. parasites in birds, separate from the clade formed by Haemoproteus spp. of reptiles. This study expands the information regarding the occurrence and distribution of hemosporidia in turtles and is the first study of blood parasites in C. carbonaria.

A Genomic Approach to Unravel Host-Pathogen Interaction in Chelonians: The Example of Testudinid Herpesvirus 3.

We report the first de novo sequence assembly and analysis of the genome of Testudinid herpesvirus 3 (TeHV3), one of the most pathogenic chelonian herpesviruses. The genome of TeHV3 is at least 150,080 nucleotides long, is arranged in a type D configuration and comprises at least 102 open reading frames extensively co-linear with those of Human herpesvirus 1. Consistently, the phylogenetic analysis positions TeHV3 among the Alphaherpesvirinae, closely associated with Chelonid herpesvirus 5, a Scutavirus. To date, there has been limited genetic characterization of TeHVs and a resolution beyond the genotype was not feasible because of the lack of informative DNA sequences. To exemplify the potential benefits of the novel genomic information provided by this first whole genome analysis, we selected the glycoprotein B (gB) gene, for detailed comparison among different TeHV3 isolates. The rationale for selecting gB is that it encodes for a well-conserved protein among herpesviruses but is coupled with a relevant antigenicity and is consequently prone to accumulate single nucleotide polymorphisms. These features were considered critical for an ideal phylogenetic marker to investigate the potential existence of distinct TeHV3 genogroups and their associated pathology. Fifteen captive tortoises presumptively diagnosed to be infected with TeHVs or carrying compatible lesions on the basis of either the presence of intranuclear inclusions (presumptively infected) and/or diphtheronecrotic stomatitis-glossitis or pneumonia (compatible lesions) were selected for the study. Viral isolation, TeHV identification, phylogenetic analysis and pathological characterization of the associated lesions, were performed. Our results revealed 1) the existence of at least two distinct TeHV3 genogroups apparently associated with different pathologies in tortoises and 2) the first evidence for a putative homologous recombination event having occurred in a chelonian herpesvirus. This novel information is not only fundamental for the genetic characterization of this virus but is also critical to lay the groundwork for an improved understanding of host-pathogen interactions in chelonians and contribute to tortoise conservation.

USE OF COMPUTED TOMOGRAPHY FOR INVESTIGATION OF HEPATIC LIPIDOSIS IN CAPTIVE CHELONOIDIS CARBONARIA (SPIX, 1824).

Computed tomography is a sensitive and highly applicable technique for determining the degree of radiographic attenuation of the hepatic parenchyma. Radiodensity measurements of the liver can help in the diagnosis of hepatic lipidosis in humans and animals. The objective was to investigate the presence of hepatic lipidosis in captive red-footed tortoises (Chelonoidis carbonaria) using computed tomography. Computed tomography was performed in 10 male red-footed tortoises. Mean radiographic attenuation values for the hepatic parenchyma were 11.2±3.0 Hounsfield units (HU). Seven red-footed tortoises had values lower than 20 HU, which is compatible with C. carbonaria hepatic lipidosis. These results allowed an early diagnosis of the hepatic changes and suggested corrective measures regarding feeding and management protocols.

COMPARISON OF FRESH AND FROZEN FECAL SAMPLES FOR DETECTION OF ENTERIC SALMONELLA FROM CAPTIVE INDIAN STAR TORTOISES (GEOCHELONE ELEGANS).

The use of frozen fecal samples in enrichment media to detect Salmonella spp. strains was evaluated in Indian star tortoises (Geochelone elegans) to determine the utility of this test method for field collection. Fresh fecal samples were collected from 10 captive adult Indian star tortoises. Each sample was split, with one portion in enrichment media at room temperature for 1 or 2 days before submission to a reference laboratory for standard enteric culture. The other was placed in the same enrichment media and frozen at -20°C for 2 wk. Afterwards, it was transferred to a -80°C freezer for 1 mo before submission to the same reference laboratory. These freezer temperatures and holding times were selected to replicate typical field collection procedures. Salmonella enterica was isolated from all 10 fresh samples but from only six frozen samples. Statistically, results showed no significant difference between the two methods; however, this may be due to the limited sample size, and if so, utilization of frozen fecal samples to determine Salmonella spp. prevalence may underestimate the actual prevalence.

Multiple paternity and sperm storage in captive Hermann's tortoises, Testudo hermanni boettgeri determined from amniotic fluid adhering to the eggshell

We identified multiple paternity in 52.9% of the clutches of Hermann's tortoise Testudo hermanni boettgeri using polymorphic microsatellite markers. In addition we demonstrated sperm storage across seasons. DNA was extracted from the amniotic fluid adhering to the eggshell's inner surface, a procedure suitable for easy, non-invasive DNA sampling in conservation and breeding programs. To improve the informative value of monomorphic single tandem repeat (STR) markers we additionally analyzed single nucleotide polymorphism (SNP) variability.

Conjunctival flora of clinically normal and diseased turtles and tortoises.

BackgroundIn captive breed turtles and tortoises conjunctival disease is common. Our aim was to investigate the bacterial and fungal flora present in the eyes of healthy and pathological chelonians and to compare findings in turtles with those in tortoises.ResultsSamples were taken from the conjunctival sacs of 34, diseased and healthy, chelonians (18 tortoises and 16 turtles) and submitted to bacterial and fungal investigation. All samples showed bacterial growth. Thirteen animals (38%), harboured a single bacterial species as sole isolate and twenty-one animals (62%) harboured more than one species. Detection of multiple bacterial infection was clearly greater in tortoises compared to turtles. Most frequently isolated bacterial species were Bacillus spp. (13 isolates), Staphylococcus xylosus (10 isolates), Sphingomonas paucimobilis (6 isolates), Staphylococcus sciuri and Aeromonas hydrophila/caviae (each 5 isolates), Ochrobactrum anthropi (3 isolates), Citrobacter freundii, Enterobacter cloacae and Pseudomonas luteola (each 2 isolates). Only one isolate of Kocuria varians/rosea, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus haemolyticus, Staphylococcus lentus, Morganella morganii, Pasteurella multocida, Pasteurella pneumotropica/haemolytica, Proteus spp., Pseudomonas putida, Salmonella enterica ssp. arizonae, Stenotrophomonas maltophilia and Vibrio parahaemolyticus was evidenced. The presence in 8 animals of Mycoplasma spp. and in 1 animal with severe conjunctivitis of Chlamydia spp. was detected by PCR. Candida spp. was also isolated from two healthy animals.ConclusionsA clear predominance of Gram positive isolates in tortoises and Gram negative isolates in turtles was found. However, we cannot ascribe the observed difference to the diversity of animal species, as other factors, including especially different characteristics of the living environments, may play a role. Almost all bacterial species isolated may have clinical significance, mostly as opportunistic pathogens, both for humans and animals. That chelonians are often carrier of bacteria with zoonotic potential is a well-known fact, in particular with regard to Salmonella spp. Therefore, it is not surprising the detection of a strain of Salmonella enterica ssp. arizonae in the eye of one of the animals tested. Worthy of note is the finding of Chlamydia spp. in a severe case of conjunctivitis, though we cannot epidemiologically assess a cause-effect relationship between presence of chlamydia and disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0405-x) contains supplementary material, which is available to authorized users.

Integrating Gene Transcription-Based Biomarkers to Understand Desert Tortoise and Ecosystem Health.

Tortoises are susceptible to a wide variety of environmental stressors, and the influence of human disturbances on health and survival of tortoises is difficult to detect. As an addition to current diagnostic methods for desert tortoises, we have developed the first leukocyte gene transcription biomarker panel for the desert tortoise (Gopherus agassizii), enhancing the ability to identify specific environmental conditions potentially linked to declining animal health. Blood leukocyte transcript profiles have the potential to identify physiologically stressed animals in lieu of clinical signs. For desert tortoises, the gene transcript profile included a combination of immune or detoxification response genes with the potential to be modified by biological or physical injury and consequently provide information on the type and magnitude of stressors present in the animal's habitat. Blood from 64 wild adult tortoises at three sites in Clark County, NV, and San Bernardino, CA, and from 19 captive tortoises in Clark County, NV, was collected and evaluated for genes indicative of physiological status. Statistical analysis using a priori groupings indicated significant differences among groups for several genes, while multidimensional scaling and cluster analyses of transcription C T values indicated strong differentiation of a large cluster and multiple outlying individual tortoises or small clusters in multidimensional space. These analyses highlight the effectiveness of the gene panel at detecting environmental perturbations as well as providing guidance in determining the health of the desert tortoise.

Draft Genome Sequence of Chelonobacter oris Strain 1662T, Associated with Respiratory Disease in Hermann's Tortoises.

Chelonobacter oris 1662T is a type strain of the recently described species of the Pasteurellaceae family. The strain was isolated from the choanae of a captive tortoise with signs of respiratory tract infection. The genome reported here is approximately 2.6 Mb in size and has a G+C content of 47.1%.