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.     

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.     

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.     

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   

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.     

PCR prevalence of Ranavirus in free-ranging eastern box turtles (Terrapene carolina carolina) at rehabilitation centers in three southeastern US states

Ranaviruses (genus Ranavirus) have been observed in disease epidemics and mass mortality events in free-ranging amphibian, turtle, and tortoise populations worldwide. Infection is highly fatal in turtles, and the potential impact on endangered populations could be devastating. Our objectives were to determine the prevalence of ranavirus DNA in blood and oral swabs, report associated clinical signs of infection, and determine spatial distribution of infected turtles. Blood and oral swabs were taken from 140 eastern box turtles (Terrapene carolina carolina) that were presented to the wildlife centers at the University of Tennessee (UT; n=39), Wildlife Center of Virginia (WCV; n=34), and North Carolina State University (NCSU; n=36), as well as a free-ranging nonrehabilitation population near Oak Ridge, Tennessee (OR; n=39) March-November 2007. Samples were evaluated for ranavirus infection using polymerase chain reaction (PCR) targeting a conserved portion of the major capsid protein. Two turtles, one from UT and one from NCSU, had evidence of ranavirus infection; sequences of PCR products were 100% homologous to Frog Virus 3. Prevalence of ranavirus DNA in blood was 3, 0, 3, and 0% for UT, WCV, NCSU, and OR, respectively. Prevalence in oral swab samples was 3, 0, and 0% for UT, WCV, and NCSU, respectively. Wildlife centers may be useful in detection of Ranavirus infection and may serve as a useful early monitoring point for regional disease outbreaks.     

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.     

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.     

Lesions associated with a novel Mycoplasma sp. in California sea lions (Zalophus californianus) undergoing rehabilitation

From July 1999 to November 2001, Mycoplasma sp. was cultured from lesions in 16 California sea lions (Zalophus californianus) undergoing rehabilitation. The Mycoplasma sp. was the likely cause of death of four animals in which it was associated with either pneumonia or polyarthritis. The most common lesion associated with this bacterium was subdermal abscessation, found in 12 animals. Other lesions included intramuscular abscesses, septic arthritis, and lymphadenopathy. Infection was associated with a leukocytosis and left shift in 12 animals. Animals with abscesses improved clinically after surgical lancing, irrigation, and systemic antibiotic therapy. The mycoplasma isolates had a consistent 16S rRNA sequence dissimilar from other Mycoplasma spp. and represent a novel species, Mycoplasma zalophi proposed sp. nov.     

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.     

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.     

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.     

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.     

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.     

Seed dispersal by the Florida box turtle (<Emphasis Type="Italic">Terrapene carolina bauri</Emphasis>) in pine rockland forests of the lower Florida Keys,United States

Seed dispersal by animals is one of the most important plant-animal mutualisms, but saurochory, the dispersal of seeds by reptiles, has received little attention. We investigated the role of the Florida box turtle (Terrapene carolina bauri) as a seed dispersal agent in pine rockland forests of the lower Florida Keys and examined the effect of turtle digestion on seed germination. We obtained seeds of 11 species with fleshy fruits and 2 species with non-fleshy fruits (a grass and legume) from the feces of 145 box turtles collected on Key Deer National Wildlife Refuge from 1999 to 2000. We planted the seeds of nine species and germination percentage (percentage of seeds that germinated during the experiment) varied from 10% to 80%. Comparative germination experiments were conducted with Thrinax morrissii, Serenoa repens, and Byrsonima lucida. We compared the germination percentage and germination rate (number of days from planting to seedling emergence) of seeds from three treatments (seeds recovered from feces, control seeds with pulp, and control seeds without pulp) and continued these experiments for up to 2 years. Passage through the box turtle digestive tract greatly enhanced the germination percentage and germination rate of S. repens, but decreased the germination percentage of B. lucida and T. morrissii, and decreased germination rate for T. morrissii. Subsequent destructive seed viability tests revealed that many ungerminated T. morrissii seeds remained viable, suggesting long-term seed dormancy may occur, even after passage through the turtle digestive system. In addition, the proportion of ungerminated seeds which remained viable was greater for seeds recovered from turtle feces than from control seeds with pulp. Furthermore, removal of fleshy pulp either manually or by the turtle digestive system may allow T. morrissii to escape insect predation.     

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.     

Association of herpesvirus with fibropapillomatosis of the green turtle Chelonia mydas and the loggerhead turtle Caretta caretta in Florida.

Sea turtle fibropapillomatosis (FP) is a disease marked by proliferation of benign but debilitating cutaneous fibropapillomas and occasional visceral fibromas. Transmission experiments have implicated a chloroform-sensitive transforming agent present in filtered cell-free tumor homogenates in the etiology of FP. In this study, consensus primer PCR methodology was used to test the association of a chelonian herpesvirus with fibropapillomatosis. Fibropapilloma and skin samples were obtained from 17 green and 2 loggerhead turtles affected with FP stranded along the Florida coastline. Ninety-three cutaneous and visceral tumors from the 19 turtles, and 33 skin samples from 16 of the turtles, were tested. All turtles affected with FP had herpesvirus associated with their tumors as detected by PCR. Ninety-six percent (89/93) of the tumors, but only 9% (3/33) of the skin samples, from affected turtles contained detectable herpesvirus. The skin samples that contained herpesvirus were all within 2 cm of a fibropapilloma. Also, 1 of 11 scar tissue samples from sites where fibropapillomas had been removed 2 to 51 wk earlier from 5 green turtles contained detectable herpesvirus. None of 18 normal skin samples from 2 green and 2 loggerhead turtles stranded without FP contained herpesvirus. The data indicated that herpesvirus was detectable only within or close to tumors. To determine if the same virus infected both turtle species, partial nucleotide sequences of the herpesvirus DNA polymerase gene were determined from 6 loggerhead and 2 green turtle samples. The sequences predicted that herpesvirus of loggerhead turtles differed from those of green turtles by only 1 of 60 amino acids in the sequence examined, indicating that a chelonian herpesvirus exhibiting minor intratypic variation was the only herpesvirus present in tumors of both green and loggerhead turtles. The FP-associated herpesvirus resisted cultivation on chelonian cell lines which support the replication of other chelonian herpesviruses. These results lead to the conclusion that a chelonian herpesvirus is regularly associated with fibropapillomatosis and is not merely an incidental finding in affected turtles.     

Morphological and molecular evidence indicates that the Gulf Coast box turtle (Terrapene carolina major) is not a distinct evolutionary lineage in the Florida Panhandle

Four extant subspecies of Terrapene carolina in eastern North America, Terrapene carolina bauri, Terrapene carolina carolina, Terrapene carolina triunguis, and Terrapene carolina major, are recognized based on morphological studies. A fifth subspecies, Terrapene carolina putnami, has been described from Pleistocene deposits but is very similar morphologically to T. c. major. Questions concerning the relationship of the Gulf Coast box turtle (T. c. major) to other box turtles have been pervasive ever since it was described. We used a combined morphological and genetic analysis to address the status of T. c. major and other T. carolina lineages. Terrapene c. bauri, T. c. carolina, and T. c. triunguis are distinct based on a discriminate function analysis of 25 morphological characters, including characters traditionally used to assign subspecies. The results of the present study confirm that box turtles phenotypically diagnosed as T. c. bauri, T. c. carolina, and T. c. triunguis all occur within the hypothesized range of T. c. major, and that the latter does not possess a diagnosable morphology. The three morphological lineages also possess divergent mitochondrial haplotypes that are present within the hypothesized range of T. c. major. In addition, a fourth distinct mtDNA lineage co‐occurs within the putative range of T. c. major. This unique lineage may include mitochondrial DNA variation from the Pleistocene T. c. putnami. Analysis of nine nuclear DNA microsatellites revealed no population structure in box turtles currently assigned to T. c. major from the Florida Panhandle, suggesting a complete admixture of lineages in this region. The results of the present study indicate that box turtles traditionally assigned to T. c. major based on phenotype are the result of introgression between eastern extant (predominantly T. c. carolina) and an extinct subspecies, T. c. putnami. Published 2011. This article is a US Government work and is in the public domain in the USA. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 889–901.