Otariid gammaherpesvirus 1 in South American fur seals (Arctocephalus australis) and a novel related herpesvirus in free-ranging South American sea lions (Otaria byronia): Prevalence and effects of age, sex, and sample type.

Otariid gammaherpesvirus 1 (OtGHV1) is associated with high rates of urogenital carcinoma in free-ranging California sea lions (Zalophus californianus; CSL), and until recently was reported only in the Northern Hemisphere. The objective of this study was to survey free-ranging South American sea lions (Otaria byronia; SASL) and South American fur seals (Arctocephalus australis: SAFS) in Punta San Juan, Peru for OtGHV1 and to determine prevalence characteristics. Twenty-one percent (14/67) of urogenital swabs collected over three years (2011, 2014, 2015) from live pinnipeds of both species tested positive with a pan-herpesvirus conventional PCR. Sequencing of SAFS amplicons revealed 100% homology to OtGHV1 at the DNA polymerase, glycoprotein B, and viral bcl2-like genes. Sequencing of SASL amplicons revealed a novel related virus, herein called Otariid gammaherpesvirus 8 (OtGHV8). For comparison of sample sites, urogenital, conjunctival, and oropharyngeal swabs collected from 136 live pinnipeds of both species at Punta San Juan between 2011-2018 were then assayed using quantitative PCR for a segment of the OtGHV1/8 DNA polymerase gene using a qPCR assay now determined to cross-react between the two viruses. In total, across both species, 38.6% (51/132) of urogenital swabs, 5.6% (4/71) of conjunctival swabs, and 1.1% (1/90) of oropharyngeal swabs were positive for OtGHV1/8, with SASL only positive on urogenital swabs. Results from SASL were complicated by the finding of OtGHV8, necessitating further study to determine prevalence of OtGHV1 versus OtGHV8 using an alternate assay. Results from SAFS suggest a potential relationship between OtGHV1 in SAFS and CSL. Though necropsy surveillance in SAFS is very limited, geographic patterns of OtGHV1-associated urogenital carcinoma in CSL and the tendency of herpesviruses to cause more detrimental disease in aberrant hosts suggests that it is possible that SAFS may be the definitive host of OtGHV1, which gives further insight into the diversity and phyogeography of this clade of related gammaherpesviruses.

Environmental temperature influences ophidiomycosis progression and survival in experimentally challenged prairie rattlesnakes (Crotalus viridis).

Ophidiomycosis is a prevalent and intermittently pervasive disease of snakes globally caused by the opportunistic fungal pathogen, Ophidiomyces ophidiicola. Host response has yet to be fully explored, including the role of temperature in disease progression and hematologic changes. This study enrolled twelve adult prairie rattlesnakes (Crotalus viridis) in an experimental challenge with O. ophidiicola at two temperatures, 26°C (n = 6) and 20°C (n = 6). Each temperature cohort included four inoculated and two control snakes. Assessments involving physical exams, lesion swabbing, and hematology were performed weekly. Differences were observed between inoculated and control snakes in survival, behavior, clinical signs, ultraviolet (UV) fluorescence, hematologic response, and histologic lesions. All inoculated snakes held at 20°C were euthanized prior to study end date due to severity of clinical signs while only one inoculated animal in the 26°C trial met this outcome. In both groups, qPCR positive detection preceded clinical signs with regards to days post inoculation (dpi). However, the earliest appearance of gross lesions occurred later in the 20°C snakes (20 dpi) than the 26°C snakes (13 dpi). Relative leukocytosis was observed in all inoculated snakes and driven by heterophilia in the 20°C snakes, and azurophilia in the 26°C group. Histologically, 20°C snakes had more severe lesions, a lack of appropriate inflammatory response, and unencumbered fungal proliferation and invasion. In contrast, 26°C snakes had marked granulomatous inflammation with encapsulation of fungi and less invasion and dissemination. The results of this study identified that O. ophidiicola-infected rattlesnakes exposed to lower temperatures have decreased survival and more robust hematologic change, though minimal and ineffective inflammatory response at site of infection. Ophidiomycosis is a complex disease with host, pathogen, and environmental factors influencing disease presentation, progression, and ultimately, survival. This study highlighted the importance of temperature as an element impacting the host response to O. ophidiicola.

HEALTH ASSESSMENT OF PAINTED TURTLES (CHRYSEMYS PICTA) IN A RESTORED WETLAND HABITAT IN NORTHWESTERN INDIANA, USA.

Freshwater turtles are sensitive to environmental changes and can serve as sentinel species for ecosystem health assessments. The Efroymson Restoration at Kankakee Sands in northwestern Indiana, USA has been restored in the past 25 yr from primarily agricultural land to a mosaic of prairie and wetland habitats. Health assessments of 40 free-ranging painted turtles (Chrysemys picta) at Kankakee Sands were performed in May 2021 to evaluate overall health status, screen for infectious diseases, and obtain baseline clinical pathology values in this population. Assessment of each turtle included a physical examination, complete blood count, plasma biochemistry panel, blood lactate level, venous blood gas analysis, serum trace mineral panel, serum vitamin D3 level, and plasma protein electrophoresis. Oral and cloacal swabs were tested for adenoviruses, herpesviruses, frog virus 3, and Mycoplasmopsis species by PCR in 39 painted turtles. Four turtles were positive for adenovirus, which shared 100% homology to Sulawesi tortoise adenovirus. Two turtles were herpesvirus-positive with 100% homology to emydid herpesvirus 1. No Mycoplasmopsis spp. or frog virus 3 was detected. Female turtles had significantly higher manganese, prealbumin, uric acid, triglycerides, and ionized calcium levels, while male turtles had significantly higher cholesterol, glutamate dehydrogenase, and CO2 levels. These baseline data can be used for future research into freshwater turtle health in restored wetland habitats.

PREVALENCE OF MULTIPLE REPTILIAN PATHOGENS IN THE OROPHARYNGEAL MUCOSA, CLOACAL MUCOSA, AND BLOOD OF DIAMONDBACK TERRAPIN (MALACLEMYS TERRAPIN) POPULATIONS FROM MARYLAND AND GEORGIA, USA.

The diamondback terrapin (Malaclemys terrapin) is a coastal turtle with a range from Massachusetts to Texas and is the only exclusively brackish water turtle in North America. Two populations of wild terrapins from Maryland (n=55) and Georgia (n=7) were examined and tested for potential reptile pathogens. Whole blood and a mucosal (combined oropharyngeal and cloacal) swab from each animal were evaluated by quantitative PCR for 15 potential pathogens including frog virus 3, box turtle Mycoplasmopsis, Mycoplasma agassizii, Mycoplasma testudineum, Salmonella Enteritidis, Salmonella Typhimurium, Borrelia burgdorferi, Anaplasma phagocytophilum, tortoise intranuclear coccidia, testudinid alphaherpesvirus 2, terrapene herpesvirus 1, and terrapene adenovirus. Swabs were positive for a DNA segment 100% homologous to M. testudineum in both populations, with Maryland animals 87% (48 of 55) positive and Georgia animals 86% (6 of 7) positive. Although Mycoplasmopsis spp. are important respiratory pathogens for members of the order Testudines, none of the animals in the study showed any sign of upper respiratory disease. Our data suggest that M. testudineum may survive in non-Testudinidae turtles without causing clinical sigs of disease and suggesting appropriate precautions should be taken in facilities that house multiple species of turtles simultaneously.

Enhancing Chelonian Health Assessments by Determining Agreement in Pathogen Detection between Sampling Sites Using Quantitative PCR.

Pathogen surveillance is common in chelonians and multiple anatomical sampling sites are used for pathogen detection, but agreement between these sites has not been assessed. The objective of this study was to assess agreement between three sampling sites, oral swab (OS), cloacal swab (CS), and combined oral/cloacal swab (OCS), for detecting three pathogens in eastern box turtles (Terrapene carolina carolina). Box turtles (n=88) were assayed for Terrapene adenovirus 1 (TerADV1), Terrapene herpesvirus 1 (TerHV1), and Mycoplasmopsis sp. using quantitative PCR. Agreement between pathogen status based on sampling site was assessed using the Cohen kappa. Agreement was highest for Mycoplasmopsis sp. between OCS and OS (k=0.941), whereas moderate and minimal agreement were noted between OCS and CS (k=0.64) and OS and CS (k=0.538). For TerADV1, agreement was weak between OCS and OS (k=0.559), minimal between OS and CS (k=0.283), and absent between OCS and CS (k=0.204). TerHV1 agreement was moderate between OCS and OS (k=0.783) and absent between OCS and CS (k=0.106) and OS and CS (k=0.052). All pathogens were most frequently detected in OCS samples and DNA concentrations differed between sampling sites (P<0.0001). If testing multiple samples is not possible, OCS sampling improves the detection of these three pathogens over OS and CS alone.

BLOOD LACTATE CONCENTRATIONS IN EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) FOLLOWING CAPTURE BY A CANINE SEARCH TEAM.

Studies to assess wildlife health commonly evaluate clinical pathology changes, immune responses, pathogen presence, and contaminant exposure, but novel modalities are needed to characterize the unique physiologic responses of reptiles. Lactate is an indicator of hypoperfusion and/or anaerobic respiration and can be quickly and easily measured using a point-of-care analyzer. This study evaluated baseline blood lactate concentrations in free-living eastern box turtles (Terrapene carolina carolina, n = 116) using a point of care analyzer and then determined the effect of handling time, physical examination (PE) abnormalities, and quantitative polymerase chain reaction pathogen detection (Terrapene herpesvirus 1, Mycoplasma sp., Terrapene adenovirus) on lactate concentrations. Blood lactate concentrations were higher in turtles with Terrapene herpesvirus 1 (n = 11), quiet mentation, and increased packed cell volume (P < 0.05). Lactate concentrations increased between initial capture and PE, with peak values reaching 129 min after capture. Lactate at PE was positively associated with baseline lactate concentrations. Turtles with Terrapene herpesvirus 1 may have alterations in blood flow, oxygen delivery, or activity patterns, driving increases in baseline lactate. Increased handling time likely leads to more escape behaviors and/or breath holding, causing turtles to undergo anaerobic metabolism and raising lactate concentrations. Overall, lactate measured by a point of care analyzer shows variability caused by capture and health factors in eastern box turtles and may be a useful adjunctive diagnostic test in this species after full methodologic validation.

Ultraviolet Fluorescence as a Field-Applicable Screening Tool for Lesions Consistent with Ophidiomycosis in Lake Erie Watersnakes (Nerodia sipedon insularum).

Ophidiomycosis, commonly called snake fungal disease, has been linked to significant morbidity of free-ranging snakes in North America and Europe. Diagnosis of ophidiomycosis currently requires detection of skin lesions via physical exam or characteristic histopathology as well as detection of the causative agent, Ophidiomyces ophidiicola, through quantitative (q)PCR or fungal culture of a skin swab or tissue sample. While reliable, these methods require specialized training, invasive procedures (e.g., biopsy), and several days or weeks to receive results. Additionally, screening entire populations can quickly become costly. A fast, easy-to-use, cost-efficient, and sensitive screening tool is needed to optimize conservation strategies and treatment intervention. Our objective was to investigate the association between skin fluorescence under long-wave ultraviolet (UV) light (365 nm) and the detection of Ophidiomyces ophidiicola DNA using qPCR. Fifty-eight Lake Erie watersnakes (Nerodia sipedon insularum) collected in June of 2018 and 2019 from islands in western Lake Erie, Ottawa County, Ohio, US were visually inspected for skin lesions, photographed under natural light and UV light, and swabbed for qPCR analysis. Fluorescence was highly associated with the presence of skin lesions, and the presence of at least one fluorescent skin lesion was 86% sensitive and 100% specific for identifying animals with apparent ophidiomycosis, with a positive predictive value of 100%. While we recommend performing standard diagnostics along with fluorescence, our study supports the use of visual UV fluorescence identification as a preliminary, affordable, noninvasive, and field-applicable method to screen populations for ophidiomycosis.

Detection of a novel herpesvirus associated with squamous cell carcinoma in a free-ranging Blanding's turtle.

The spread of both infectious and noninfectious diseases through wildlife populations is of increasing concern. Neoplastic diseases are rarely associated with population-level impacts in wildlife; however, impacts on individual health can be severe and might reflect deteriorating environmental conditions. An adult male free-ranging Blanding's turtle (Emydoidea blandingii) originally captured in 2005 and deemed healthy, was recaptured in 2018 with a 1 × 1.5 cm intra-oral broad-based right mandibular mass. An excisional biopsy was performed, and histopathology revealed squamous cell carcinoma (SCC). Consensus herpesvirus PCR identified a novel herpesvirus (proposed name Emydoidea herpesvirus 2 [EBHV-2]) within the tumor. EBHV-2 shares 85% sequence homology with Terrapene herpesvirus 2 (TerHV-2), a herpesvirus linked to fibropapillomas in eastern box turtles (Terrapene carolina carolina). Virus-associated fibropapillomas have been identified in multiple marine turtle species and have had debilitating effects on their populations, but to date, virus-associated SCCs are rarely reported.

Development and validation of a quantitative PCR assay for detection of Terrapene herpesvirus 2 in eastern box turtles (Terrapene carolina carolina).

Herpesviruses are associated with disease in many chelonian species, resulting in pathology such as respiratory tract infection, stomatitis, conjunctivitis, hepatitis, and papillomatosis. Herpesvirus-associated fibropapillomas cause significant morbidity and mortality in marine turtles, and have been identified in an eastern box turtle (Terrapene carolina carolina) infected with Terrapene herpesvirus 2 (TerHV2). Further investigation is necessary to understand the impact of carcinogenic herpesviruses on chelonian health; however, reliable and specific methods for detection and quantitation of herpesviral load are lacking. The purpose of this study was to develop and validate a quantitative PCR assay for detection of TerHV2 in box turtles. TaqMan primer-probes were developed targeting the DNA polymerase gene. Inter- and intra-assay variability, linear range of detection, limit of detection, and specificity were assessed. The assay was highly specific for TerHV2, failing to amplify seven closely-related chelonian herpesviruses. It performed with high efficiency (slope = -3.52, R2 = 1, efficiency = 92.29 %), low intra-assay variability and low inter-assay variability (coefficient of variation ≤ 1.25 % at all standard dilutions). Reaction efficiency was not impacted in the presence of box turtle DNA from combined oral/cloacal swabs or whole blood. This qPCR assay has a linear range of detection from 107 to 101 viral copies per reaction and provides a valuable tool in the surveillance and characterization of TerHV2 in box turtles.

Erythrocyte sedimentation rate and hemoglobin-binding protein in free-living box turtles (Terrapene spp.).

The acute phase response is a highly conserved reaction to infection, inflammation, trauma, stress, and neoplasia. Acute phase assays are useful for wildlife health assessment, however, they are infrequently utilized in reptiles. This study evaluated erythrocyte sedimentation rate (ESR) in eastern (Terrapene carolina carolina) and ornate box turtles (Terrapene ornata ornata) and hemoglobin-binding protein (HBP) in T. ornata. Erythrocyte sedimentation rate in 90 T. carolina and 105 T. ornata was negatively associated with packed cell volume and was greater in unhealthy turtles (p < 0.05). Female T. ornata had higher ESR values than males (p < 0.05). Measurement of ESR with a microhematocrit tube proportionally overestimated values from a commercial kit (Winpette), though both methods may retain utility with separate reference intervals. Hemoglobin-binding protein concentration in 184 T. ornata was significantly increased in adults and unhealthy turtles (p < 0.05). Erythrocyte sedimentation rate values were similar between seasons and populations, and HBP values were consistent between years, indicating that these analytes may have more stable baseline values than traditional health metrics in reptiles. This study demonstrates that ESR and HBP are promising diagnostics for health assessment in wild box turtles. Incorporating these tests into wild herptile health assessment protocols may support conservation efforts and improve ecosystem health monitoring.

PREVALENCE OF BOX TURTLE ADENOVIRUS IN EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) PRESENTED TO A WILDLIFE REHABILITATION CENTER IN VIRGINIA, USA.

Eastern box turtles (Terrapene carolina carolina) are a native North American species with a declining population trend that may be attributable to habitat fragmentation, vehicle collisions, and disease. Adenoviral infections can cause significant morbidity and mortality in captive reptile populations. Adenoviruses have been documented in box turtles, but their occurrence and impact in wild populations are unknown. A disease survey was performed at The Wildlife Center of Virginia, USA, to assess the prevalence of box turtle adenovirus (BTAdV) in wild eastern box turtles and evaluate potential associations with clinical disease. Swabs from the oral cavity, including the choanal slit, and the cloaca were collected from 106 eastern box turtles from July 2015 through June 2016. The quantitative polymerase chain reaction (qPCR) primer detected both ornate box turtle adenovirus 1 and eastern box turtle adenovirus. The resulting qPCR adenovirus prevalence was 55.7% (n = 59). Most animals (99.3%) that tested positive for BTAdV had fewer than 100 viral copies/ng DNA. This study did not find a statistically significant association between cause of admission, age, sex, outcome, and BTAdV qPCR status. However, the probability of BTAdV detection was 1.5 times higher in rehabilitation turtles compared with wild turtles (P = 0.01). Albumin was significantly lower in qPCR BTAdV-positive turtles (P = 0.007). Hypoalbuminemia is not generally associated with adenovirus infections in other species, and no obvious clinical cause for this abnormality was identified. The results of this study suggest that eastern box turtles may harbor BTAdV infections at low levels and that infection is rarely associated with clinical disease, potentially identifying BTAdV as a host-adapted pathogen. Future studies should focus on this pathogen's ability to induce clinical disease and its potential impact on recovery efforts for this species.

Characterization of Dermotheca sp. Infection in a midwestern state-endangered salamander (Ambystoma platineum) and a co-occurring common species (Ambystoma texanum).

Ichthyosporean parasites (order Dermocystida) can cause morbidity and mortality in amphibians, but their ecology and epidemiology remain understudied. We investigated the prevalence, gross and histologic appearance, and molecular phylogeny of a novel dermocystid in the state-endangered silvery salamander (Ambystoma platineum) and the co-occurring, non-threatened small-mouthed salamander (Ambystoma texanum) from Illinois. Silvery salamanders (N = 610) were sampled at six ephemeral wetlands from 2016 to 2018. Beginning in 2017, 1-3 mm raised, white skin nodules were identified in 24 silvery salamanders and two small-mouthed salamanders from five wetlands (prevalence = 0-11.1%). Skin biopsy histology (N = 4) was consistent with dermocystid sporangia, and necropsies (N = 3) identified infrequent hepatic sporangia. Parasitic 18S rRNA sequences (N = 5) from both salamander species were identical, and phylogenetic analysis revealed a close relationship to Dermotheca viridescens. Dermocystids were not identified in museum specimens from the same wetlands (N = 125) dating back to 1973. This is the first report of Dermotheca sp. affecting caudates in the Midwestern United States. Future research is needed to determine the effects of this pathogen on individual and population health, and to assess whether this organism poses a threat to the conservation of ambystomatid salamanders.

MORTALITY INVESTIGATION OF MONITORED EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) IN CENTRAL ILLINOIS, USA, FROM 2016-18.

Mortality events in eastern box turtles (Terrapene carolina carolina) threaten conservation efforts across the species range. These events are often under-diagnosed and, when observed, predictive health factors are unavailable prior to death. At Kickapoo State Park in central Illinois, USA, ranaviruses caused observed mortality events in amphibians and chelonians in 2014 and 2015. Following these outbreaks, eastern box turtles (n=36) were affixed with radio transmitters and temperature data loggers to obtain repeated location and temperature data from spring 2016-spring 2018. Bimonthly, samples of blood and oral and cloacal swabs were collected to investigate health parameters (hematology and cytokine transcription) and presence of multiple pathogens. Deaths of instrumented turtles occurred in 2016 (n=5), 2017 (n=15), and 2018 (n=2). The largest single die-off occurred in February 2017 (n=7). Seventeen turtles were necropsied and multiple pathologic processes were identified, most frequently decreased adipose stores (n=6). Two turtles had pathologic findings consistent with multisystemic inflammation. In addition, infectious pathogens were identified in turtles prior to death, but no single agent was associated with each mortality event. Ranavirus was not detected in any turtle. Hot spot analysis revealed spatial clustering at the center and edges of the study area for body temperature as well as for relative cytokine transcription of interleukin-1 beta, tumor necrosis factor alpha, and interleukin-10 associated with turtle death. Though no single causal factor could be identified, the information from this mortality event can direct future chelonian mortality investigations by providing baseline longitudinal data prior to death and in surviving turtles.

EFFECTS OF OXYGEN AND ISOFLURANE ANESTHESIA ON HEMOLYMPH GAS ANALYSIS AND RIGHTING REFLEX OF ASIAN FOREST (HETEROMETRUS LONGIMANUS) AND DICTATOR SCORPIONS (PANDINUS DICTATOR).

Large arachnids are commonly managed under professional care, and anesthesia is occasionally required for physical examination and diagnostic and therapeutic procedures. Anesthetic responses and hemolymph gas analysis have been studied previously in spiders, but scorpions have yet to be investigated. This study measured hemolymph gas values with an i-STAT point of care blood gas analyzer in healthy adult Asian forest scorpions (Heterometrus longimanus = HL, n = 8) and dictator scorpions (Pandinus dictator = PD, n = 12) breathing: 1) room air (RA), 2) 100% oxygen for 10 min in a chamber (OX), and 3) 5% isoflurane and oxygen (ISO) in a chamber until induction or loss of righting reflex. All scorpions recovered without complications, and there were no cartridge failures. Analysis of hemolymph gas values revealed that pH was lower in OX compared with RA and ISO and was lower in PD compared with HL scorpions. The partial pressure of carbon dioxide did not differ between inhaled gases but was higher in PD compared with HL. The partial pressure of oxygen (pO2) was higher in ISO compared with OX, and both were higher than when breathing RA. Despite a lack of species difference in pO2, PD had a more dramatic increase in pO2 in ISO compared with HL (significant species and inhalant interaction). PD had a significantly shorter induction time than HL, but recovery time (return of righting reflex) did not differ between species. Subjectively, HL exhibited rough inductions compared with PD, characterized by violent whole-body and tail movements. The unexpected increase in pO2 in ISO compared with OX, along with the species-specific differences and anesthetic effects, emphasizes the unique respiratory physiology of scorpions and demonstrates that further species-specific studies of anesthetics are warranted.

FATAL RANAVIRUS INFECTION IN A GROUP OF ZOO-HOUSED MELLER'S CHAMELEONS (TRIOCEROS MELLERI).

A group of five juvenile Meller's chameleons (Trioceros melleri) experienced 100% mortality over a period of 1 mo due to ranavirus infection. The index case was found dead without premonitory signs. The three subsequent cases presented with nonspecific clinical signs (lethargy, decreased appetite, ocular discharge) and were ultimately euthanatized. The final case died after initially presenting with skin lesions. Postmortem examination revealed thin body condition in all five animals and mild coelomic effusion and petechiae affecting the tongue and kidneys of one animal. Microscopically, all animals had multifocal necrosis of the spleen, liver, and kidney; four of five animals had necrosis of the nasal cavity; and two of five had necrosis of adrenal tissue, bone marrow, and skin. Numerous basophilic intracytoplasmic inclusions were present in the liver of all animals and nasal mucosa of three of the five animals. Consensus polymerase chain reaction for herpesvirus and adenovirus were negative, whereas ranavirus quantitative polymerase chain reaction was positive. Virus isolation followed by whole genome sequencing and Bayesian phylogenetic analysis classified the isolates as a strain of frog virus 3 (FV3) most closely related to an FV3 isolate responsible for a previous outbreak in the zoo's eastern box turtle (Terrapene carolina carolina) group. This case series documents the first known occurrence of ranavirus-associated disease in chameleons and demonstrates the potential for interspecies transmission between chelonian and squamate reptiles.

Venous blood gas in free-living eastern box turtles (Terrapene carolina carolina) and effects of physiologic, demographic and environmental factors.

Sustainable wildlife populations depend on healthy individuals, and the approach to determine wellness of individuals is multifaceted. Blood gas analysis serves as a useful adjunctive diagnostic test for health assessment, but it is uncommonly applied to terrestrial reptiles. This study established reference intervals for venous blood gas panels in free-living eastern box turtles (Terrapene carolina carolina, N = 102) from Illinois and Tennessee, and modeled the effects of environmental and physiologic parameters on each blood gas analyte. Blood gas panels included pH, partial pressure of oxygen (pO2), partial pressure of carbon dioxide (pCO2), total carbon dioxide (TCO2), bicarbonate (HCO3-), base excess (BE) and lactate. Candidate sets of general linear models were constructed for each blood gas analyte and ranked using an information-theoretic approach (AIC). Season, packed cell volume (PCV) and activity level were the most important predictors for all blood gas analytes (P < 0.05). Elevations in PCV were associated with increases in pCO2 and lactate, and decreases in pH, pO2, HCO3-, TCO2 and BE. Turtles with quiet activity levels had lower pH and pO2 and higher pCO2 than bright individuals. pH, HCO3-, TCO2 and BE were lowest in the summer, while pCO2 and lactate were highest. Overall, blood pH was most acidic in quiet turtles with elevated PCVs during summer. Trends in the respiratory and metabolic components of the blood gas panel tended to be synergistic rather than antagonistic, demonstrating that either (1) mixed acid-base disturbances are common or (2) chelonian blood pH can reach extreme values prior to activation of compensatory mechanisms. This study shows that box turtle blood gas analytes depend on several physiologic and environmental parameters and the results serve as a baseline for future evaluation.

Investigation of multiple mortality events in eastern box turtles (Terrapene carolina carolina).

Wildlife mortality investigations are important for conservation, food safety, and public health; but they are infrequently reported for cryptic chelonian species. Eastern box turtles (Terrapene carolina carolina) are declining due to anthropogenic factors and disease, and while mortality investigations have been reported for captive and translocated individuals, few descriptions exist for free-living populations. We report the results of four natural mortality event investigations conducted during routine health surveillance of three Illinois box turtle populations in 2011, 2013, 2014, and 2015. In April 2011, over 50 box turtles were found dead and a polymicrobial necrotizing bacterial infection was diagnosed in five survivors using histopathology and aerobic/anaerobic culture. This represents the first reported occurrence of necrotizing bacterial infection in box turtles. In August 2013, paired histopathology and qPCR ranavirus detection in nine turtles was significantly associated with occupation of moist microhabitats, identification of oral plaques and nasal discharge on physical exam, and increases in the heterophil count and heterophil to lymphocyte ratio (p < 0.05). In July 2014 and 2015, ranavirus outbreaks reoccurred within a 0.2km radius of highly-disturbed habitat containing ephemeral ponds used by amphibians for breeding. qPCR ranavirus detection in five individuals each year was significantly associated with use of moist microhabitats (p < 0.05). Detection of single and co-pathogens (Terrapene herpesvirus 1, adenovirus, and Mycoplasma sp.) was common before, during, and after mortality events, but improved sample size would be necessary to determine the impacts of these pathogens on the occurrence and outcome of mortality events. This study provides novel information about the causes and predictors of natural box turtle mortality events. Continued investigation of health, disease, and death in free-living box turtles will improve baseline knowledge of morbidity and mortality, identify threats to survival, and promote the formation of effective conservation strategies.

DETECTION OF COPATHOGENS IN FREE-RANGING EASTERN BOX TURTLES ( TERRAPENE CAROLINA CAROLINA) IN ILLINOIS AND TENNESSEE.

Conservation efforts are investigating the impact of diseases within a species of interest, including prevalence and transmission and morbidity and mortality rates. However, the majority of these studies focus solely on the characteristics of a single pathogen. Recently, the role of copathogens has been reported to impact disease susceptibility and mortality. To that effect, a survey was conducted including 318 eastern box turtles ( Terrapene carolina carolina) from populations in Illinois and Tennessee in 2014 and 2015. Blood samples and oral swabs were collected for quantitative polymerase chain reaction (qPCR) of 15 different pathogens performed in a multiplex format using Fluidigm array technology. Four pathogens were found with varying qPCR prevalence: ranavirus (FV3; n = 2, 0.6%), Terrapene herpesvirus 1 (TerHV1; n = 129, 40.7%), box turtle Mycoplasma sp. (BT Myco; n = 14, 4.6%), and box turtle adenovirus (BT Adv1; n = 18, 11%). Thirteen pathogens were not identified in any sample, including Mycoplasma agassizii, M. testudineum, Salmonella enteriditis, S. typhmirium, Borrelia burgdorferi, Anaplasma phagocyophilum, tortoise intranuclear coccidia, Ambystoma tigrinum virus, Bohle iridovirus, Epizootic hematopoietic necrosis virus, and testudinid herpesvirus 2. Copathogen occurrence was rare but was observed in eight individuals with TerHV1-BT Myco detection and two animals with TerHV1-Adv1. Significant differences were observed in pathogen detection across season (TerHV1, BT Adv1, BT Myco, and TerHV1-Myco) and year (TerHV1, BT Adv1, and TerHV1-Myco). The results of this survey highlight that a single pathogen model may not adequately explain pathogen dynamics and that conservation efforts need to be aimed at detecting multiple pathogens in order to fully characterize population health.

DETECTION OF RANAVIRUS USING BONE MARROW HARVESTED FROM MORTALITY EVENTS IN EASTERN BOX TURTLES ( TERRAPENE CAROLINA CAROLINA).

The causes of free-living chelonian mortality events are often unknown because of infrequent recovery of remains and rapid postmortem decomposition. This study describes a technique to harvest bone marrow and detect frog virus 3-like ranavirus (FV3) using quantitative polymerase chain reaction in skeletonized eastern box turtles ( Terrapene carolina carolina) ( N = 87), and assesses agreement with concurrent perimortem samples ( N = 14). FV3 was detected in bone marrow samples from 12 turtle shells (14%). Three of 14 turtles had detectable FV3 loads in both bone marrow and perimortem samples, two turtles had detectable FV3 in bone marrow only, and nine turtles tested FV3 negative in both bone marrow and concurrent perimortem samples. There was substantial agreement between FV3 testing of bone marrow and other tissues ( κ = 0.658). Harvesting bone marrow from shells is easily performed and can serve as a means for biologists and wildlife veterinarians to improve postmortem surveillance for systemically distributed pathogens, including FV3.