Intracytoplasmic inclusions in circulating leukocytes from an eastern box turtle (Terrapene carolina carolina) with iridoviral infection

A free-ranging adult female eastern box turtle (Terrapene carolina carolina) was presented to the University of Tennessee in October 2003 because of suspected trauma and blindness. Physical examination revealed lethargy, clear ocular and nasal discharges, and white oral and laryngeal plaques. Intracytoplasmic inclusions within heterophils and large mononuclear leukocytes were observed on routine blood smear examination. Postmortem findings included necrosis of epithelial and parenchymal cells with intracytoplasmic inclusions. Ultrastructurally, the leukocyte inclusions consisted of variably electron-dense granular material and viral particles consistent with the Iridoviridae family of viruses. The virus shared 100% sequence identity to a 420-base pair sequence of frog virus 3 (family Iridoviridae, genus Ranavirus) as determined by polymerase chain reaction and gene sequencing targeting a portion of the Ranavirus major capsid protein gene.     

Ranavirus infection of free-ranging and captive box turtles and tortoises in the United States

Iridoviruses of the genus Ranavirus are well known for causing mass mortality events of fish and amphibians with sporadic reports of infection in reptiles. This article describes five instances of Ranavirus infection in chelonians between 2003 and 2005 in Georgia, Florida, New York, and Pennsylvania, USA. Affected species included captive Burmese star tortoises (Geochelone platynota), a free-ranging gopher tortoise (Gopherus polyphemus), free-ranging eastern box turtles (Terrapene carolina carolina), and a Florida box turtle (Terrepene carolina bauri). Evidence for Ranavirus infection was also found in archived material from previously unexplained mass mortality events of eastern box turtles from Georgia in 1991 and from Texas in 1998. Consistent lesions in affected animals included necrotizing stomatitis and/or esophagitis, fibrinous and necrotizing splenitis, and multicentric fibrinoid vasculitis. Intracytoplasmic inclusion bodies were rarely observed in affected tissues. A portion of the major capsid protein (MCP) gene was sequenced from each case in 2003-2005 and found to be identical to each other and to Frog virus 3 (FV3) across 420 base pairs. Ranavirus infections were also documented in sympatric species of amphibians at two locations with infected chelonians. The fragment profiles of HindIII-digested whole genomic DNA of Ranavirus, isolated from a dead Burmese star tortoise and a southern leopard frog (Rana utricularia) found nearby, were similar. The box turtle isolate had a low molecular weight fragment that was not seen in the digestion profiles for the other isolates. These results suggest that certain amphibians and chelonians are infected with a similar virus and that different viruses exist among different chelonians. Amphibians may serve as a reservoir host for susceptible chelonians. This report also demonstrated that significant disease associated with Ranavirus infections are likely more widespread in chelonians than previously suspected.     

Phaeohyphomycosis in a free-living eastern box turtle (Terrapene carolina carolina)

A free-ranging eastern box turtle (Terrapene carolina carolina) was referred to the Wildlife Center of Virginia with a three-month history of marked swelling of the right hind limb initially diagnosed as chromomycosis by histopathology. Hematology revealed severe anemia (9%), leukocytosis (12.8 cells x 10(3)/microl), heterophilia (6.14 cells x 10(3)/microl), and monocytosis (0.51 cells x 10(3)/microl). Gross necropsy revealed a firm, encapsulated 3 x 1 cm subcutaneous mass filled with dark brown-black, friable necrotic material of the distal right hind limb. Microscopically, the mass was characterized by a granulomatous inflammatory process with numerous multinucleated histiocytic giant cells. Fungal elements were present within necrotic centers and associated with multinucleated cells. Special stains revealed numerous phaeoid hyphae and yeast; Exophiala jeanselmei was isolated by routine mycologic culture. Phaeohyphomycosis was diagnosed based on the histologic appearance of the fungal elements within the mass and culture results. There was no histopathological evidence of systemic infection. This is the first report of phaeohyphomycosis caused by fungi of the genus Exophiala in free-living reptiles.     

Relationships among aural abscesses, organochlorine compounds, and vitamin a in free-ranging eastern box turtles (Terrapene carolina carolina)

Aural abscesses are a common health problem in free-ranging eastern box turtles (Terrapene carolina carolina), and they have been associated with high body burdens of organochlorine (OC) compounds, which are known disruptors of vitamin A. The objective of this study was to determine if the presence of pathologic lesions in box turtles were correlated with increased and decreased levels of hepatic OC compounds and vitamin A, respectively. A graded scale for the pathologic changes observed in tissue samples collected from abscessed and nonabscessed box turtles over a 2-yr period (2003-04) was developed, and the levels of OC compounds and vitamin A in livers collected from the same turtles were determined through chemical analysis. Sixty-eight turtles (40 with aural abscesses and 28 without) were included in the study. Relationships between variables were analyzed using Spearman's Rank Correlation Test, where P相似文献   

Characterization of the bacterial microflora of the tympanic cavity of eastern box turtles with and without aural abscesses

Aerobic bacterial cultures of the tympanic cavity of the middle ear were performed in eight eastern box turtles (Terrapene carolina carolina) with aural abscesses and 15 eastern box turtles without aural abscesses (controls) that were admitted to The Wildlife Center of Virginia, Virginia, USA during 2003. Twenty-two bacterial isolates were identified from 17 turtles including 10 gram-negative and 12 gram-positive bacteria. Ten of 15 control animals had bacterial growth, resulting in identification of 13 bacteria, including six gram-negative and seven gram-positive agents. Seven of eight turtles with aural abscesses had bacterial growth, and 10 isolates were identified, including four gram-negative and six gram-positive organisms. The most frequently isolated bacteria from control animals were Micrococcus luteus (n = 3) and Pantoea agglomerans (n = 2). Morganella morganii (n = 2) was the only species isolated from the tympanic cavity of more than one turtle with aural abscesses. Staphylococcus epidermidis (n = 2) was the only species isolated from both groups. A trend toward greater bacterial growth in tympanic cavities of affected turtles compared with turtles without aural abscesses was noted. No single bacterial agent was responsible for aural abscesses in free-ranging eastern box turtles in this study, an observation consistent with the hypothesis that aerobic bacteria are not primary pathogens, but secondary opportunistic invaders of environmental origin.     

Temporal variance in hematologic and plasma biochemical reference intervals for free-ranging eastern box turtles (Terrapene carolina carolina)

Eastern box turtle (Terrapene carolina carolina) populations are in decline, likely due to anthropogenic forces and disease, necessitating hematologic and biochemical data from healthy individuals for evaluation of wild populations. We repeatedly sampled 21 free-ranging eastern box turtles from May to September 2009 in the spring, summer, and fall to establish temporal hematologic and biochemical reference intervals. Packed cell volume, aspartate aminotransferase, and potassium levels declined significantly as the active season progressed. High levels of albumin, globulin, and calcium coincided with the presence of eggs in females. These reference intervals should provide baseline data for the clinical evaluation of wild box turtles presented for veterinary care or for studies of wild populations.     

A novel mycoplasma detected in association with upper respiratory disease syndrome in free-ranging eastern box turtles (Terrapene carolina carolina) in Virginia

Clinical signs of upper respiratory tract disease-like syndrome (URTD-LS) were observed in free-ranging eastern box turtles (Terrapene carolina carolina) from Virginia, USA (May 2001-August 2003), some of which also had aural abscesses. After a Mycoplasma sp. was detected by polymerase chain reaction (PCR), a study was undertaken to better define the range of clinical signs of disease and to distinguish mycoplasma-associated URTD-LS from other suspected causes of URTD-LS and aural abscessation in box turtles. Nasal and/or ocular swabs (from turtles possessing URTD-LS) or nasal washes (from asymptomatic turtles) were collected from turtles May 2001-August 2003; samples were assayed for Mycoplasma spp., chelonian herpesvirus, and iridoviruses by PCR testing. A partial DNA sequence (933 bases) of the small ribosomal subunit (16S rRNA) of the box turtle Mycoplasma sp. was analyzed to determine its phylogenetic relatedness to other Mycoplasma spp. of veterinary interest. Mycoplasma sp. was detected in seven (six with clinical signs of URTD-LS; one asymptomatic) of 23 fortuitously collected animals from six of 11 Virginia counties. Clinical signs in Mycoplasma sp.-infected animals included unilateral to bilateral serous to mucopurulent nasal discharge, epiphora, ocular edema, and conjunctival injection. Five Mycoplasma sp.-positive animals possessed aural abscesses; two did not. Analysis of the mycoplasma 16S rRNA gene sequence from one asymptomatic and three symptomatic animals representing four counties revealed a consensus Mycoplasma sp. sequence closely related to, but distinct from, M. agassizii. None of the samples collected contained viral DNA of chelonian herpesviruses or invertebrate and vertebrate (including FV3) iridoviruses. In conclusion, a new Mycoplasma sp. was associated with URTD-LS in native box turtles from Virginia that was not codetected with other suspected causes of chelonian upper respiratory disease; there was no proof of a direct relationship between aural abscessation and the Mycoplasma sp.     

Diagnosis, treatment, and outcome of eastern box turtles (Terrapene carolina carolina) presented to a wildlife clinic in Tennessee, USA, 1995-2007

We performed a retrospective review of medical records for 574 eastern box turtles (Terrapene carolina carolina) that presented to the University of Tennessee College of Veterinary Medicine (UTCVM) wildlife clinic between 1995 and 2007. These cases were reviewed to document and investigate the presenting complaints, as identified by a good Samaritan, final diagnoses, treatment modalities, days in hospital, and final disposition. Presenting complaints were consistent with the final diagnoses in 81% of cases. Trauma was both the most common presentation and final diagnosis. Associations between final diagnosis and either environmental temperature or precipitation were not significant (P>0.05). However, precipitation had a statistically significant positive association (P<0.05) with aural abscessation. Approximately 5% (28/574) of the box turtles were evaluated for packed cell volume total protein and white blood cell count. No differences were observed between sick and healthy animals for any of these variables. Multiple treatment modalities were used in these cases and were often used in combination, rather than as individual therapies. The number of days spent in the hospital ranged from 0 to 66 days, with a median of 2 days. The wildlife clinic of UTCVM should continue to evaluate the health status of eastern box turtles. Our findings indicate that the high caseload, as well as case variety of eastern box turtles in eastern Tennessee, suggests potential use as a biomonitor for this species.     

Helminth parasites of eastern box turtles (Terrapene carolina carolina) from southern Indiana, USA

Very little is known about parasitic diseases of eastern box turtles (Terrapene carolina carolina). The objective of this study was to examine the parasitic fauna of eastern box turtles collected from southern Indiana, USA. Turtles (n = 40) were salvaged mostly as road kills from southern Indiana between May and October 2009. Seven species of helminths in total were found parasitizing the gastrointestinal tract, including two digenean trematodes (Brachycoelium salamandrae and Telorchis robustus) and five nematodes (Oswaldocruzia pipiens, Cosmocercoides dukae, Falcaustra affinis, F. chelydrae and Serpinema trispinosus). We report prevalence, abundance and mean intensity of infection for all helminths. Helminths were not found in any other organs examined (heart, gonads, liver, heart, kidney and urinary bladder) and no ectoparasites were found. Overall, mean intensity of infections was low (1-14 parasites/host), suggesting that these parasites are unlikely to be associated with negative health impacts. This constitutes the first study of this kind for Indiana.     

Epidemiologic determinants of aural abscessation in free-living eastern box turtles (Terrapene carolina) in Virginia

Epidemiologic determinants of 46 cases of aural abscessation in free-living eastern box turtles (Terrapene carolina) admitted to the Wildlife Center of Virginia (Virginia, USA) from 1991 to 2000 were evaluated. County human population density, year and season of admission, weight, and sex did not affect the risk for box turtles to develop aural abscessation. Counties with cases of aural abscessation were not randomly distributed, but rather were clustered into two multi-county regions. Geographic location was the only risk factor associated with aural abscessation in box turtles found in this study. Possible etiologies could include chronic infectious disease, malnutrition, or chronic exposure to environmental contamination with organochlorine compounds.     

Pathology of aural abscesses in free-living Eastern box turtles (Terrapene carolina carolina)

Aural abscess or abscess of the middle ear is common in free-living Eastern box turtles (Terrapene carolina carolina) of Virginia (USA) and elsewhere. Although its etiology remains unknown, hypovitaminosis A has been suggested on the basis of similar lesions occurring in captive chelonians fed diets that are deficient in vitamin A. This hypothesis was supported by significantly greater body burdens of organochlorine compounds (reported disruptors of vitamin A metabolism) and a nonsignificant trend toward lower serum and hepatic vitamin A levels in free-living box turtles with this lesion. The tympanic epithelium was evaluated in 27 box turtles (10 with aural abscesses and 17 without). Lesions of the tympanic epithelium of box turtles with aural abscesses included hyperplasia, squamous metaplasia, hyperemia, cellular sloughing, granulomatous inflammation, and bacterial infection. These changes were more severe in turtles with aural abscesses than in those without and were more severe in tympanic cavities that had an abscess compared to those without when the lesion was unilateral. Organs from 21 box turtles (10 with aural abscesses and 11 without) from the study population were examined for microscopic lesions, and minimal histopathologic changes were found, none of which were similar to those found in the tympanic epithelium. Histopathologic changes in box turtles with aural abscesses were consistent with a syndrome that may involve hypovitaminosis A.     

Morbidity and mortality of reptiles admitted to the Wildlife Center of Virginia, 1991 to 2000

Medical records from 694 reptiles admitted to the Wildlife Center of Virginia (WCV; Waynesboro, Virginia, USA) from 1991 to 2000 were reviewed to determine causes of morbidity and mortality. Eighteen species were represented but the majority of cases were four species; eastern box turtle (Terrapene carolina) (66%), eastern painted turtle (Chrysemys picta) (11%), common snapping turtle (Chelydra serpentina) (10%), and rat snake (Elaphe sp.) (6%). There was a significant increase in reptile cases during the study period both in absolute number and in proportion to the total caseload. Trauma (74%) was the most frequent cause of morbidity and mortality followed by unknown or undetermined (13%), aural abscessation (7%), infectious diseases (2%), and one nutritional disorder (0.1%). In addition, 3% of the cases were healthy animals that had been removed from the wild and consequently brought to the WCV. Causes of morbidity and mortality differed between the four most numerous species. Impact with a motor vehicle was the most frequent cause of trauma for eastern box turtles, eastern painted turtles, and common snapping turtles; however, garden-equipment-related trauma was the most frequent cause for rat snakes. Aural abscessation was only seen in eastern box turtles. Eighty percent of cases occurred between May and September and 65% occurred within the five counties closest to the WCV. The majority of morbidity and mortality was the result of human activities. The expanding human population in Virginia likely will continue to have an impact on the health of wild reptiles.     

Prevalence of infection by Batrachochytrium dendrobatidis and Ranavirus in eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) in eastern Tennessee

Hellbenders (n=97) were collected from the Little and Hiwassee Rivers in eastern Tennessee, USA, during 2009 and 2010. Location and morphometrics for each animal were recorded, and nonlethal tissue samples were collected to estimate the prevalence of infection with Batrachochytrium dendrobatidis (Bd) and Ranavirus in each watershed and year. Real-time polymerase chain reaction was performed on skin swabs for Bd and on tail clips for ranaviruses. Overall prevalences of DNA of Bd, Ranavirus, and coinfections (i.e., detectable DNA of both pathogens in the same individual) were 26%, 19%, and 5%, respectively. Differences in infection prevalence were detected between watersheds and years. Gross lesions were observed in 31 animals (32%), but the types of lesions were not consistent with chytridiomycosis or ranaviral disease. This is the first report of infection of eastern hellbenders with Bd and Ranavirus. Despite infection by both pathogens, it is unclear whether chytridiomycosis or ranaviral disease develops in wild populations of hellbenders. More research is needed to determine the susceptibility of hellbenders to Bd and ranaviruses and their role in the epidemiology of these pathogens.     

Widespread occurrence of ranavirus in pond-breeding amphibian populations

Ranaviruses are an emerging threat for many amphibian populations, yet their distribution in amphibian communities and the association of infection with possible stressors and species is not fully understood due to historically sparse surveillance. Agricultural practices that reduce the water quality of amphibian breeding habitats (e.g., cattle access to wetlands) and environmental stressors (e.g., lower temperatures) may contribute to ranavirus emergence. We tested larval amphibians for ranavirus infection across four seasons in farm ponds (n?=?40) located in Tennessee, USA. Cattle at various densities were allowed access to half of the sampled ponds. Ranavirus infections were detected in nine species and in 33 of the sampled ponds (83%), illustrating widespread occurrence of the pathogen. Species within the family Ranidae were the most frequently infected. In 13 of the ponds containing infected individuals, prevalence exceeded 40% during at least one season. Infections were detected in multiple seasons in 20 of the sampled ponds containing infections, suggesting that ranaviruses are relatively persistent in these systems. Cattle had negative effects on water quality (turbidity and ammonia) and there was a positive association between cattle abundance and ranavirus prevalence in the summer. Counter to previous field studies in North America, we found a significant positive association between water temperature and ranavirus prevalence in the fall sampling events. Despite these findings, the influences of cattle and temperature on ranavirus prevalence were not consistent across seasons. As such, the mechanisms driving high ranavirus prevalence across the landscape and over time remain unclear. Given the widespread occurrence of ranaviruses in wild amphibians, we encourage the implementation of surveillance programs to help identify potential drivers of emergence. Sites with high ranavirus prevalence should be monitored annually for outbreaks, and the long-term effects on population size determined.     

Assessment of hematologic and corticosterone response in free-living eastern box turtles (Terrapene carolina carolina) at capture and after handling

Hematology is a common tool for wildlife health assessments. Manual leukocyte counts are required in reptiles, however, disagreement between quantification methods has been observed in some chelonians. This study determined agreement between two methods of leukocyte quantification, eosinophilic leukopet, and blood film white blood cell (WBC) estimates, in free-living eastern box turtles (Terrapene carolina carolina). Simultaneously, we assessed the impact of handling duration on both leukocyte quantity and corticosterone levels. We collected blood at capture (<2 min from disturbance) and again before release 30–150 min later from 92 box turtles at six sites in Illinois and Tennessee. Constant and proportional error was present in the leukopet results for WBC, lymphocytes, and basophils compared to the estimate method. Both methods were in agreement for heterophils, monocytes, and eosinophils. Agreement between the methods was significantly more likely at WBC counts below 23,241/µl. All hematologic parameters were significantly higher at the final blood draw compared to the initial blood draw using both WBC determination methods, except relative eosinophil and basophil counts. Corticosterone levels varied with time, with maximum concentrations reached at 54 min postcapture, followed by a rapid return to baseline levels. Corticosterone level was not significantly associated with any hematologic parameter or sex. This study provides a framework for understanding the effects of animal handling methodology and diagnostic modality when evaluating hematologic health in box turtles.     

The influence of bacterial lipopolysaccharide on the thermoregulation of the box turtle Terrapene carolina

Ectotherms can adjust their thermoregulatory set points in response to bacterial infection; the result may be similar to endothermic fever. We examined the influence of dose on the set point of body temperature (T(b)) in Terrapene carolina. After acclimating postprandial turtles to 20 degrees C, we injected them with two doses of bacterial endotoxin (LPS; lipopolysaccharide from Escherichia coli), 0.0025 or 0.025 mg LPS/g nonshell body mass, or with reptilian saline (control group). We placed the animals singly in linear thigmothermal gradients and recorded their T(b)'s for 48 h. The turtles showed dose-influenced thermal selection. Turtles injected with the high dose had T(b)'s significantly higher than control turtles, whereas low-dose turtles had T(b)'s significantly lower than control turtles. Also, there was a low daily effect on the T(b) of the turtles injected with the high dose. High-dose turtles had significantly higher T(b)'s than the control turtles during the first day but not during the second. Our results support the prediction of Romanovsky and Székely that an infectious agent may elicit opposite thermoregulatory responses depending on quality and quantity of the agent and the host health status.     

Direct and Indirect Effects of Fire on Eastern Box Turtles

Prescribed fire is an increasingly important management tool for eastern deciduous forests, but relativity little is known about the direct effects of fire on the eastern box turtle (Terrapene carolina carolina). We used very high frequency (VHF) transmitters to monitor mortality, movement, and spatial ecology of 118 box turtles in response to 17 prescribed fires across 4 seasons and 3 sites in east Tennessee, USA, during 2016–2018. Annual survival of box turtles that experienced a prescribed fire event was lower (0.87 ± 0.04 [SE]) than turtles that did not (0.98 ± 0.01) and was negatively correlated with fire intensity, fire temperature the turtle experienced, and litter depth. All prescribed fire-related mortalities occurred during the early (Apr–May, n = 5) or late growing season (Sep–Oct, n = 1). Fourteen percent of box turtles we captured exhibited damage to their carapace from previous fire events. Box turtles that survived prescribed fires were in microsites that did not burn, moved to unburned areas during the fire, or burrowed following ignition. Home range size was similar before and after burns and sinuosity of movements did not differ in burned or unburned areas. Our results indicate that though box turtles are susceptible to prescribed fire during their active season, they have behavioral and physical traits that reduce the direct effects of prescribed fire. Prescribed fire practitioners should be aware of the risks of fire, particularly during the active season. We suggest managers consider altering prescribed fire intensity, seasonality, and firing pattern to minimize risk of direct effects where box turtles are of concern. © 2020 The Wildlife Society.     

Spatial Ecology and Resource Selection of Eastern Box Turtles

Eastern box turtles (Terrapene carolina carolina) are widely distributed throughout the eastern United States. Although once common throughout much of its distribution, the species has experienced declines in local populations. Understanding resource selection is important for the conservation of this species; however, few data exist on resource selection for eastern box turtles in the southeastern United States. We estimated home range and resource selection for 100 individual turtles in the Blue Ridge, Ridge and Valley, and Cumberland Plateau and Mountains physiographic regions in Tennessee, USA, from 2016 to 2018. We used step-selection functions to investigate eastern box turtle resource selection during May–August 2017 and May–August 2018 at 2 spatial scales. We classified vegetation type, measured vegetation composition and structure, recorded time since fire, and measured coarse woody debris abundance at 1,225 used telemetry locations and 1,225 associated available points. Home range sizes averaged 9.3 ha ± 3.0 (SE) using minimum convex polygon analysis, 8.25 ha ± 2.88 using 95% kernel density analysis, and 1.50 ha ± 0.56 using 50% kernel density analysis. Box turtles selected areas with greater visual obstruction at the 0–0.25-m level, greater amounts of 10-hour and 100-hour fuels (timelag categories used in fire-danger ratings), and greater litter depths compared to available locations. Box turtles were more likely to select areas with greater cover of brambles and coarser woody debris and were less likely to select areas with less vegetation cover. Vegetation type and time since last fire did not affect selection. Our data suggest that management activities that encourage greater understory vegetation cover, greater visual obstruction at the 0–0.25-m level, and greater bramble cover will enhance habitat quality for eastern box turtles. © 2020 The Wildlife Society.     

Isolation and characterization of polymorphic microsatellite loci for the three-toed box turtle (Terrapene carolina triunguis) and cross-amplification in other Terrapene species

We isolated and characterized eight polymorphic microsatellite loci for a Texas population of three-toed box turtle, Terrapene carolina triunguis, using a refined hybridization capture procedure. All eight primer pairs amplified successfully at all loci in seven Texas ornate box turtles (T. ornata ornata). Due to the decline and conservation concerns of North American box turtles, these isolated microsatellites may be a most valuable tool for evaluating baseline population genetic structure for threatened box turtle populations.