Free-ranging Eastern Box Turtles Research Articles

CUTANEOUS MYIASIS AND ITS RELATIONSHIP TO WELLNESS IN EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) IN CAPE COD, MASSACHUSETTS.

Eastern box turtles (Terrapene carolina carolina) face a variety of anthropogenic, infectious, and environmental threats and have been affected by high morbidity and mortality disease events. Wellness parameters in free-ranging eastern box turtles with a high prevalence of myiasis on Cape Cod, MA, were documented to identify epidemiologic trends or associations with several health parameters. There were 109 samples collected from 59 individual box turtles over the course of 4 mon. Six turtles died over the course of this study. Fly larvae infestations varied in severity and were observed in the cutaneous and subcutaneous tissue (n = 18; 30.5%). Animals with myiasis had fewer plastron abnormalities than those without (P = 0.034), and all turtles found in bogs had evidence of fly larvae infections (P < 0.0001). Individuals with myiasis also had lower body condition index (P = 0.014), lower total white blood cells (P = 0.031), lower PCV (P < 0.0001), lower total solids (P < 0.0001), higher erythrocyte sedimentation rate (P < 0.0001), lower calcium (P = 0.018), and lower phosphorus (P = 0.017). Three turtles tested positive for terrapene herpesvirus 1, but presence was not associated with myiasis. Heavy metal analysis revealed no significant differences between turtles with and without myiasis. This study examined the health of a population of eastern box turtles, and continued health assessments will be beneficial in determining the impact of myiasis on future conservation plans.

PREVALENCE AND ANTIMICROBIAL RESISTANCE PATTERNS OF SALMONELLA SPP. IN TWO FREE-RANGING POPULATIONS OF EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA).

Salmonellosis is an important zoonotic infection, and exposure to pet reptiles has been implicated in several human outbreaks. Although several studies report a low prevalence of salmonellae in free-ranging chelonians, they may serve as a reservoir. In spring and summer of 2013 and 2019, free-ranging eastern box turtles (Terrapene carolina carolina) from populations in Illinois (rural) and Tennessee (urban) were collected through canine and visual search. Cloacal swab samples were collected from each turtle, selectively enriched with tetrathionate broth, then plated on selective and differential media to isolate Salmonella spp. Genus was confirmed via MALDI-TOF MS and antibiotic sensitivities were performed. Isolates were serotyped by the National Veterinary Services Laboratory. Of the 341 turtles sampled, Salmonella spp. were detected in nine individuals (2.64%; 95% CI: 1.2-5.0%). The isolates included five different serovars: Anatum (n = 2), Newport (n = 2), Thompson (n = 1), Bareilly (n = 2), and Hartford (n = 2). Salmonella spp. were detected from six animals in 2013 (3.19%, 95% CI: 1.2-6.8%) and three in 2019 (1.96%, 95% CI: 0.4-5.6%). There was no significant difference in prevalence between state, (P = 0.115), Illinois locations (P = 0.224), season (P = 0.525), year (P = 0.297), sex (P = 0.435), or age class (P = 0.549). The health of Salmonella-positive and -negative turtles was not significantly different, as assessed through hematology and plasma biochemistry (P > 0.05), indicating asymptomatic carrier status. The low prevalence detected in this study likely concludes that free-ranging eastern box turtles play a minimal role in the spread of salmonellae. However, the identified serotypes are potentially human- and animal-pathogenic. Documenting the prevalence of Salmonella serotypes in animal indicators furthers our understanding of their spread between humans, animal agriculture, and the environment.

Behavioural and physiological responses to simulated predator-induced stress in the eastern box turtle, Terrapene carolina carolina

Abstract In spite of the fully-hinged shell being the defining characteristic of box turtles, few studies have quantified the behavioural use of the shell in response to predator encounters. While it is often assumed that box turtles simply withdraw into their shell in response to threats, we expected variability among individuals in shell usage and in the degree of head and limb retraction because withdrawal is likely to be costly. We tested the behavioural and physiological responses of free-ranging eastern box turtles to stimuli of varying intensity: observation only, a visual stimulus, a poke on the leg, or a pinch on the leg before being flipped. The more intense the simulated predatory stimulus, the more likely turtles were to fully close their shell. However, stimulus intensity did not affect plasma corticosterone or lactate levels. This may be due to too brief or weak of a stimulus, increased parasympathetic activity, or seasonal effects. Upon capture and during handling, almost all turtles withdrew into their shells and closed their anterior plastron, although fewer closed their posterior plastron, and a few engaged in other behaviours such as biting and urinating. Older, larger turtles were able to close their shells with greater strength than younger and smaller turtles. Our results indicate that in spite of their fully closable shell, box turtles exhibit a graded response to perceived threats. This suggests that full closure of the shell is likely to be costly and occurs only in response to the most intense stimuli.

BODY CONDITION OF EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) EVALUATED BY COMPUTED TOMOGRAPHY.

Conservation efforts determining the health status of individuals can aid in assessing population health and sustainability. Body condition was determined in 65 free-ranging Eastern box turtles (Terrapene carolina carolina) from four locations in Vermilion County, Illinois, in the months of May, July, and September 2014, 2015, and 2016 using computed tomography (CT). Physical examinations were performed to determine morphometric measurements, and CT scans measured body fat. Twenty-three linear models were constructed to determine which morphometric measurements best correlated to fat content. The top two models of body fat included a relationship between mass and carapace width. Fat increases as both mass and carapace width increase. CT, while impractical for daily use, improved on previous methods of measuring body condition and created a calculation that can be applied broadly to Eastern box turtles. By understanding how measurements of mass and carapace width correlate to body condition, practitioners, researchers, and conservationists can evaluate chelonians with increased confidence in the accuracy of their assessment.

Seasonal changes in baseline corticosterone, association with innate immunity, and effects of confinement in free-ranging Eastern Box Turtles, Terrapene carolina carolina

Seasonal changes in glucocorticoids and their influence on the immune system are widespread in vertebrates, but whether this occurs in lesser studied taxa like turtles is unclear. The purpose of this study was to test for seasonal changes in baseline corticosterone (CORT), innate immunity, body condition, and metabolic factors (triglycerides, uric acid) in free-ranging Eastern Box Turtles (Terrapene carolina carolina), a species which is in decline across its range. In addition, the effect of handling and confinement on CORT levels was measured. In both years of the seasonal study, baseline CORT concentrations were significantly higher in fall than in spring, but summer CORT levels differed between the years. The annual variation in CORT levels may be related to weather conditions or associated with opportunistic mating. CORT levels of turtles confined for one hour were higher than turtles bled as quickly as possible, and unlike the seasonal study, females had higher baseline and post-stressor CORT levels than males. Baseline CORT levels were positively correlated with hemolysis titer, indicating a possible immunoenhancing effect of CORT. Triglycerides were positively correlated with body condition and were higher in females than males. The higher triglyceride level in females was likely associated with the energetic demands of reproduction and nesting. Males had a lower body condition in the fall than in both spring and summer, while females exhibited no seasonal differences in body condition. Uric acid and innate immunity measures failed to exhibit significant seasonal or sex related differences. Overall our findings of substantial annual, seasonal, and sexual variation in turtle physiology are consistent with the findings reported for other vertebrates and indicate the importance of incorporating these sources of variation into the design of future studies.

CHARACTERIZING THE 25-HYDROXYVITAMIN D STATUS OF TWO POPULATIONS OF FREE-RANGING EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA).

Ultraviolet B radiation is recommended for captive reptiles to stimulate production of adequate levels of vitamin D; however, little is known regarding the vitamin D status in many free-ranging populations. Current reference ranges for vitamin D in eastern box turtles have not yet been established. Sixty free-ranging eastern box turtles (Terrapene carolina carolina) from two well-studied populations in Illinois (n = 24) and Tennessee (n = 36) were assayed for plasma vitamin D concentration in 2014. There were no significant differences in concentrations between individuals in Illinois (mean: 117.5 nM/L) and Tennessee (mean: 98.7 nM/L) (P = 0.129) populations. Similarly, there were no differences in concentrations based on age class (P = 0.533) or sex (P = 0.532). There was a significant correlation between UV at the time of capture and vitamin D concentrations (R = 0.301, P = 0.030). Vitamin D was not correlated with total calcium (R = 0.018, P = 0.89) or Ca : P ratio (R = 0.025, P = 0.85). Diseases in captive individuals, including secondary nutritional hyperparathyroidism, may commonly be associated with vitamin D deficiencies, and clinical intervention relies on reference data. Vitamin D supplementation may be recommended if animals are deemed to be deficient. Data obtained can be used to improve the care of captive and free-ranging turtles by providing reference ranges, as well as better characterize the health of wild populations.

MULTI-DRUG RESISTANCE PATTERNS OF ENTERIC BACTERIA IN TWO POPULATIONS OF FREE-RANGING EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA).

Gram-negative isolates (n = 84) from 71% of free-ranging Eastern box turtles (Terrapene carolina carolina) in Illinois and Tennessee, United States, demonstrated resistance to at least one antibiotic while 30% of isolates demonstrated resistance to two or more antibiotics. Resistance was observed against cefoxitin, amoxicillin-clavulanic acid, cefazolin, ampicillin, ticarcillin, cefovecin, and ceftiofur. Gram-positive bacteria were isolated from 49 turtles, and all were observed to be resistant to two or more antibiotics. Gram-positive isolate resistance was observed to penicillin, cefoxitin, oxacillin, clindamycin, amikacin, enrofloxacin, cefovecin, ceftiofur, cefazolin, marbofloxacin, gentamicin, erythromycin, trimethoprim-sulfamethoxazole, and chloramphenicol. Health parameters including packed cell volume, total white blood cell count (WBC), total solids (TS), and weight were not significantly different based on antibiotic resistance patterns; however, decreasing WBC and TS were observed when the number of antibiotic-resistant detections in Gram-positive bacteria increased.

PREVALENCE OF TERRAPENE HERPESVIRUS 1 IN FREE-RANGING EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) IN TENNESSEE AND ILLINOIS, USA.

Diseases affecting the upper respiratory tract, such as herpesviruses, are well described in captive chelonians worldwide, but their importance in free-ranging populations is less well known. To characterize the disease epidemiology of terrapene herpesvirus 1 (TerHV1), 409 free-ranging eastern box turtles ( Terrapene carolina carolina) in Tennessee and Illinois, US were tested for TerHV1 in 2013 and 2014 using TaqMan quantitative PCR. Whole blood and swabs of the oral mucosa were collected from 365 adults (154 females, 195 males, 16 unknown sex) and 44 juveniles. The prevalence of detection was 31.3% (n=128). Turtles were more likely to be positive for TerHV1 in July (50%; n=67) compared to September (38%; n=44) and May (11%; n=17). Turtles sampled in 2014 had a significantly higher prevalence (50%; n=98) than in 2013 (14%; n=30). In a multivariate model, only season, year, and the interaction between season and year were maintained; turtles were most likely to be positive in July (odds ratio: 30.5) and September (odds ratio: 41.8) 2014 compared to May 2013. The prevalence was not statistically different by state of collection, sex, or age class. Packed cell volume (25.5%) and total solids (4.8 mg/dL) in positive turtles were significantly higher than in negative turtles (23.0%; 4.3 mg/dL). Positive turtles had increased eosinophil concentrations, fewer lymphocytes, and fewer monocytes. No clinical sign was associated with detection of herpesvirus. Widespread DNA evidence of TerHV1 infection was detected in eastern box turtles, and knowledge of the epidemiology of this virus may aid in management of free-ranging and captive individuals.

Nonfatal Traumatic Gastric Evisceration in Two Box Turtles (Terrapene carolina carolina)

Abstract Two Eastern box turtles (Terrapene carolina carolina) presented within a few hours of traumatic gastric evisceration; one had been struck by a lawn mower and the other had sustained a crush injury by automobile. In both cases, physical examination revealed severe carapacial fractures and a portion of the stomach exteriorized and entrapped by the carapacial fragments. Gastric perforation was identified and repaired in the turtle struck by lawn mower; the stomach of the second turtle was intact. The exposed gastric serosa of each turtle was irrigated and replaced within the coelom, and the carapacial fractures were reduced and stabilized. Following treatment and rehabilitation, the carapacial fractures healed, no complications occurred secondary to the gastric exteriorization, and both were released to the wild within 3 months of presentation. Treatment and rehabilitation of free-ranging Eastern box turtles following urban trauma may contribute to maintaining the wild populations of this species. T...

Development and validation of quantitative PCR for detection of Terrapene herpesvirus 1 utilizing free-ranging eastern box turtles (Terrapene carolina carolina)

Diseases that affect the upper respiratory tract (URT) in chelonians have been well described as a significant contributor of morbidity and mortality. Specifically, herpesviruses are common pathogens in captive chelonians worldwide, but their importance on free-ranging populations is less well known. Historical methods for the diagnosis of herpesvirus infections include virus isolation and conventional PCR. Real-time PCR has become an essential tool for detection and quantitation of many pathogens, but has not yet been developed for herpesviruses in box turtles. Two quantitative real-time TaqMan PCR assays, TerHV58 and TerHV64, were developed targeting the DNA polymerase gene of Terrapene herpesvirus 1 (TerHV1). The assay detected a viral DNA segment cloned within a plasmid with 10-fold serial dilutions from 1.04×107 to 1.04×101 viral copies per reaction. Even though both primers had acceptable levels of efficiency and variation, TerHV58 was utilized to test clinical samples based on less variation and increased efficiency. This assay detected as few as 10 viral copies per reaction and should be utilized in free-ranging and captive box turtles to aid in the characterization of the epidemiology of this disease.

Select metal and metalloid surveillance of free-ranging Eastern box turtles from Illinois and Tennessee (Terrapene carolina carolina).

The Eastern box turtle (Terrapene carolina carolina) is a primarily terrestrial chelonian distributed across the eastern US. It has been proposed as a biomonitor due to its longevity, small home range, and reliance on the environment to meet its metabolic needs. Plasma samples from 273 free-ranging box turtles from populations in Tennessee and Illinois in 2011 and 2012 were evaluated for presence of heavy metals and to characterize hematologic variables. Lead (Pb), arsenic (As), zinc (Zn), chromium (Cr), selenium (Se), and copper (Cu) were detected, while cadmium (Cd) and silver (Ag) were not. There were no differences in any metal detected among age class or sex. However, Cr and Pb were higher in turtles from Tennessee, while As, Zn, Se, and Cu were higher in turtles from Illinois. Seasonal differences in metal concentrations were observed for Cr, Zn, and As. Health of turtles was assessed using hematologic variables. Packed cell volume was positively correlated with Cu, Se, and Pb in Tennessee. Total solids, a measure of plasma proteins, in Tennessee turtles were positively correlated with Cu and Zn. White blood cell count, a measure of inflammation, in Tennessee turtles was negatively correlated with Cu and As, and positively correlated with Pb. Metals are a threat to human health and the health of an ecosystem, and the Eastern Box Turtle can serve as a monitor of these contaminants. Differences established in this study can serve as baseline for future studies of these or related populations.

PLASMA ELECTROPHORETIC PROFILES AND HEMOGLOBIN BINDING PROTEIN REFERENCE INTERVALS IN THE EASTERN BOX TURTLE (TERRAPENE CAROLINA CAROLINA) AND INFLUENCES OF AGE, SEX, SEASON, AND LOCATION

Evaluation of plasma electrophoretic profiles and acute phase protein concentrations may play a valuable role in health assessment of reptiles; however, little is known about reference intervals in free-ranging eastern box turtles (Terrapene carolina carolina). The purpose of this study was to establish reference intervals of protein electrophoretic profiles and hemoglobin binding protein ([HBP] as determined by a haptoglobin assay) in free-ranging eastern box turtles and to assess any possible correlations between varying age class (adults vs. juvenile), sex (male, female, or unknown), season (spring, summer, or fall), or location (Tennessee vs. Illinois). Blood samples were obtained from 324 eastern box turtles from 2010 to 2012 at three sites in Illinois and one site in Tennessee, USA. Significant differences were observed with total protein (sex, season, state, Illinois location), albumin (age class, season, state, Illinois location), α-1 globulins (sex, season, Illinois location), α-2 globulins (sex, season, state, Illinois location), β globulins (age class, sex, season, state, Illinois location), γ globulins (sex, season state, Illinois location), and hemoglobin binding protein (age class, sex, state, Illinois location). The use of electrophoretic profiles and acute phase proteins is a relatively new concept in reptilian medicine, and this study allowed for establishment of references intervals in the eastern box turtle and emphasized differences that occured based on age, sex, season, and location. Future research in this area can now build on these data to determine changes in population health over time or alterations due to specific environmental or disease threats.

Seasonal variations in plasma vitellogenin and sex steroids in male and female Eastern Box Turtles, Terrapene carolina carolina

The Eastern Box Turtle (Terrapene carolina carolina) is a widespread species that has recently experienced precipitous declines throughout its range. Although many studies have documented aspects of reproduction in box turtles, reproductive physiology of free-ranging animals is unknown and can be crucial in this species’ recovery. Over a two-year period, we measured reproductive parameters, (vitellogenin [Vtg], estradiol-17β [E2], and total testosterone [TT]), in plasma of 116 free-ranging Eastern Box Turtles across their active season. We found similar seasonal variations of Vtg and E2 within females. Mid-season, females showed a sharp peak in E2 that correlates with the putative beginning of the ovarian cycle. Individual females lacking these expected peaks of both Vtg and E2 suggest that some female T. c. carolina may not reproduce annually. Females typically expressed undetectable levels of TT, yet there was a small peak in TT early in the active season. Male Eastern Box Turtles exhibited a dual peak in TT. Elevated TT in males was significantly associated with observed sexual behaviors and smaller home ranges. Body condition had no effect on the concentration of TT or E2 in either sex. This is the first study to (1) document Vtg and sex steroid hormones in free-ranging animals of this genus, and (2) relate those metrics to individuals, the population, the purported annual cycle, and to other chelonian species.

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.

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.

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</=0.05 was considered significant. Twenty-seven different OC compounds were identified. Mean+/-standard deviation (SD) total OC compound level for all turtles was 0.35+/-0.83 ppm (range 0-5.81 ppm), and mean+/-SD vitamin A level was 72.8+/-98.6 ppm (range 0-535.7 ppm). There was no correlation between pathologic score and total hepatic OC compound concentration (r = -0.18, P = 0.16). However, pathologic score was positively correlated with o,p'-DDT (r = 0.25, P = 0.05). Vitamin A was positively correlated with pathologic score (r = 0.32, P = 0.01), which was contrary to the expected result. There was no linear correlation between vitamin A and total hepatic OC compound concentration (r = -0.04, P = 0.75). However, a nonlinear regression provided a significant fit (r(2) = 0.12, P = 0.02), indicating an initial increase in vitamin A as the OC compound burden increased, followed by a decline as OC compound levels increased further. The hepatic OC compound residue concentrations in these box turtles were lower compared to levels found in freshwater aquatic turtles but similar to levels in other terrestrial reptile species. This study provides mixed results in support of a role of OC compounds, presumably of environmental origin, in the etiology of aural abscesses in free-ranging box turtles.

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.

Feldman SH, Wimsatt J, Marchang RE, et al. A novel mycoplasma detected in association with upper respiratory disease syndrome in free-ranging eastern box turtles ( Terrapene carolina carolina) in Virginia. J Wild Dis 42:279-289, 2006

Feldman SH, Wimsatt J, Marchang RE, et al. A novel mycoplasma detected in association with upper respiratory disease syndrome in free-ranging eastern box turtles ( Terrapene carolina carolina) in Virginia. J Wild Dis 42:279-289, 2006

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.

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.