SCHIZANGIELLA INFECTIONS IN AN EASTERN RATSNAKE (PANTHEROPHIS ALLEGHANIENSIS) AND A TIMBER RATTLESNAKE (CROTALUS HORRIDUS).

This report describes Schizangiella infections in colubrid and viperid snakes. A captive eastern ratsnake (Pantherophis alleghaniensis) was presented for a large intraoral mass associated with the mandible. The mass was debulked and histologic examination revealed severe, granulomatous stomatitis with intralesional fungi exhibiting morphologic features consistent with Schizangiella serpentis. PCR and sequencing of affected tissues confirmed S. serpentis. Because of declining health, the ratsnake was euthanized and postmortem examination identified a disseminated S. serpentis infection involving the skeletal musculature, lung, kidney, mesentery, and mandible. A wild-caught timber rattlesnake (Crotalus horridus) was presented for cutaneous lesions, weakness, and lethargy and later died. Postmortem examination revealed a mass-like structure in the esophagus characterized by high numbers of Schizangiella-like fungi associated with extensive granulomatous inflammation; the snake also had cutaneous mycosis suggestive of ophidiomycosis. This is the first report to document the unique morphologic features of S. serpentis in tissues and the presentation of schizangiellosis in snakes. Schizangiellosis should be considered as a differential diagnosis for nodular lesions involving the oral cavity and/or the gastrointestinal tract of snakes.

Complexity in the timing of the first postnatal ecdysis in snakes.

Lepidosaurian reptiles, particularly snakes, periodically shed the outer epidermal layers of their skin (ecdysis) to restore or enhance vital functions such as regulating water and gaseous exchange, growth, and protection against insult, infection or physical injury. Although many studies have focused on the nature and mechanisms of skin shedding, little attention has been paid to the timing of the first ecdysis in neonates following birth or hatching. A recent study investigated patterns of the time to first postnatal ecdysis in snakes based on a large dataset taken from the literature. The analysis demonstrated patterns in the time to first postnatal ecdysis related to phylogeny as well as several life history traits. While this assessment provides important advances in our knowledge of this topic, data on known biophysical drivers of ecdysis - temperature and humidity - were largely unavailable and were not evaluated. The first postnatal ecdysis of neonatal snakes can be viewed as an adaptive adjustment to the transition from the aqueous environment of the embryo to the aerial environment of the newborn. Hence, the timing of the first postnatal ecdysis is logically influenced by the aerial environment into which a newborn snake or hatchling finds itself. Therefore, in this Commentary, we first emphasize the putative plasticity of ecdysis with respect to epidermal lipids that structure the water permeability barrier and are established or renewed during ecdysis to reduce transepidermal evaporative water loss. We then discuss the likely importance of biophysical variables as influential covariates that need future investigation as potential co-determinants of the timing of first postnatal ecdysis.

Bilateral palpebral reduction and concurrent mycoplasmosis in a wild Agassiz's desert tortoise (Gopherus agassizii).

A wild Agassiz's desert tortoise, Gopherus agassizii, with bilateral eyelid reduction and plaques of tissue covering the superior surface of both corneas was examined in the field and subsequently submitted to the University of Florida for diagnostics. Polymerase chain reaction (PCR), from a swab of both corneas, was positive for Mycoplasma agassizii. Two months later, the tortoise was euthanatized and necropsied. There was increased bulbar exposure associated with dermal excoriation of periocular scales in both superior and inferior palpebra resulting in an increased palpebral fissure opening. Concurrently, there was bilateral conjunctivitis of the nictitating membranes and squamous metaplasia of the bulbar conjunctiva. Using PCR, Mycoplasma testudineum, another pathogen of tortoises, was identified in both nasal cavities, and the upper respiratory tract histopathological findings were consistent with those described for M. testudineum in Agassiz's desert tortoises. Although eye disease has been reported in desert and gopher (Gopherus polyphemus) tortoises with mycoplasmosis, widespread loss of palpebral tissue, conjunctivitis of the nictitans, and squamous metaplasia of the bulbar conjunctiva have not been reported in tortoises.

A NEW COCCIDIAN (APICOMPLEXA: EIMERIIDAE) IN THE CRITICALLY ENDANGERED CENTRAL AMERICAN RIVER TURTLE (DERMATEMYS MAWII) IN BELIZE.

As part of a biannual health examination, coprological samples from 3-mo-old Central American river turtles, Dermatemys mawii (Gray, 1847) in a breeding program in Belize, Central America, revealed a previously undescribed coccidian (Apicomplexa) in 17 of 46 (37%) samples. Of 3 positive fecal samples transported to the University of Florida, coccidian oocysts were observed in 1 sample. Sporulated oocysts were measured and described, and using polymerase chain reaction (PCR), an approximately 400-base pair (bp) region of both the small subunit (18S) ribosomal RNA gene and 1,200-bp region of the internal transcribed spacer (ITS) gene were amplified in all 3 samples and their products were sequenced. For comparative value, the same PCR reactions and amplifications were performed on a fecal sample containing oocysts of Eimeria mitraria obtained from a red-eared slider, Trachemys scripta elegans. Results indicated a new eimerian in D. mawii, Eimeria grayi n. sp.

Pharmacokinetics of ponazuril after administration of a single oral dose to green turtles (Chelonia mydas).

The coccidian protozoan, Caryospora cheloniae, has been associated with severe enteritis and encephalitis in immature farm-raised green turtles (Chelonia mydas) in the Cayman Islands, immature green turtles off the coast of Florida, and immature stranded sea turtles in Australia. An effective anti-coccidial drug that is both orally absorbed and well-distributed throughout the body is needed for treatment of turtles diagnosed with coccidiosis in rehabilitation facilities. Ponazuril is a triazine antiprotozoal drug that is approved in the USA for the treatment of another Apicomplexan, Sarcocystis neurona, and has also been successfully used in the therapy of other coccidian parasites. The objective of this study was to perform an oral dose-ranging pilot study (10-100 mg/kg of body weight ponazuril) in green turtles (N = 9), followed by oral administration of ponazuril at 100 mg/kg body weight (N = 8) to assess its disposition. Another goal of this study was to optimize the method of oral drug administration to green turtles. Plasma ponazuril concentrations were quantified using high performance liquid chromatography (HPLC). Standard compartmental models were fit to the data. Ponazuril was absorbed after oral administration at 100 mg/kg BW, with a maximum plasma concentration of 3.3 µg/ml. Dose-dependent pharmacokinetic parameters only weakly correlated with the dose rate, apparently due to considerable pharmacokinetic variability observed between turtles. Administration of ponazuril in gelatin capsules using a balling gun was deemed the least variable and most successful method of drug administration. Further studies are needed to evaluate the safety and efficacy of ponazuril in sea turtles with coccidiosis.

Newly recognized Anaplasma sp. in erythrocytes from Gopher Tortoises (Gopherus polyphemus).

BACKGROUND: In 2015, a previously unrecognized intracytoplasmic erythrocytic inclusion was discovered in anemic wild-caught adult gopher tortoises (Gopherus polyphemus). Subsequently, molecular diagnostics revealed this inclusion to be a novel Anaplasma sp. OBJECTIVES: The goal of this study was to morphologically characterize these erythrocytic inclusions by light and transmission electron microscopy (TEM). METHODS: Blood samples were taken from two car-injured wild-caught gopher tortoises for the preparation of Wright-Giemsa stained smears and TEM specimens. CBC data were serially performed and morphologically examined during treatment periods. RESULTS: Studies revealed a moderate to severe anemia with moderate regeneration as indicated by polychromasia and the presence of immature erythroid precursors. In addition, on light microscopy, one to two variably-sized round basophilic stippled paracentral erythrocytic inclusions were present per cell in both animals and involved 10%-25% of erythrocytes. TEM identified the intraerythrocytic inclusions as discrete membrane-bound cytoplasmic vacuoles (morulae) containing membrane-bound bacterial subunits that were of variable size, shape, and electron density. Serial hematologic data indicated complete remission of the infection in response to a single long-term course of doxycycline. CONCLUSIONS: The presence of a regenerative anemia in gopher tortoises from Florida revealed a newly recognized bacterial species that has morphologic characteristics similar to members of the genus Anaplasma.

Longitudinal and Cross-Sectional Sampling of Serpentovirus (Nidovirus) Infection in Captive Snakes Reveals High Prevalence, Persistent Infection, and Increased Mortality in Pythons and Divergent Serpentovirus Infection in Boas and Colubrids.

The aim of this study of serpentovirus infection in captive snakes was to assess the susceptibility of different types of snakes to infection and disease, to survey viral genetic diversity, and to evaluate management practices that may limit infection and disease. Antemortem oral swabs were collected from 639 snakes from 12 US collections, including 62 species, 28 genera, and 6 families: Pythonidae (N = 414 snakes; pythons were overrepresented in the sample population), Boidae (79), Colubridae (116), Lamprophiidae (4), Elapidae (12), and Viperidae (14). Infection was more common in pythons (38%; 95% CI: 33.1-42.4%), and in boas (10%; 95% CI: 5.2-18.7%) than in colubrids (0.9%, 95% CI: <0.01-4.7%); infection was not detected in other snake families (lamprophiids 0/4, 95% CI: 0-49%; elapids 0/12, 95% CI: 0-24.2%; and vipers 0/14, 95% CI: 0-21.5%), but more of these snakes need to be tested to confirm these findings. Clinical signs of respiratory disease were common in infected pythons (85 of 144). Respiratory signs were only observed in 1 of 8 infected boas and were absent in the single infected colubrid. Divergent serpentoviruses were detected in pythons, boas, and colubrids, suggesting that different serpentoviruses might vary in their ability to infect snakes of different families. Older snakes were more likely to be infected than younger snakes (p-value < 0.001) but males and females were equally likely to be infected (female prevalence: 23.4%, 95% CI 18.7-28.9%; male prevalence: 23.5%, 95% CI 18-30.1%; p-value = 0.144). Neither age (p-value = 0.32) nor sex (p-value = 0.06) was statistically associated with disease severity. Longitudinal sampling of pythons in a single collection over 28 months revealed serpentovirus infection is persistent, and viral clearance was not observed. In this collection, infection was associated with significantly increased rates of mortality (p-value = 0.001) with death of 75% of infected pythons and no uninfected pythons over this period. Offspring of infected parents were followed: vertical transmission either does not occur or occurs with a much lower efficiency than horizontal transmission. Overall, these findings confirm that serpentoviruses pose a significant threat to the health of captive python populations and can cause infection in boa and colubrid species.

Evidence of Diversity, Site, and Host Specificity of Sea Turtle Blood Flukes (Digenea: Schistosomatoidea: "Spirorchiidae"): A Molecular Prospecting Study.

Neospirorchis (Digenea: "Spirorchiidae") are blood flukes of sea turtles. Trematodes tentatively identified as Neospirorchis sp. infect various sites within sea turtles inhabiting waters of the southeastern United States, but efforts to obtain specimens adequate for morphologic study has proven difficult. Two genetic targets, the internal transcribed spacer region of the ribosomal RNA gene and the partial mitochondrial cytochrome c oxidase subunit I gene, were used to investigate potential diversity among parasite specimens collected from stranded sea turtles. Sequence data were obtained from 215 trematode and egg specimens collected from 92 individual free-ranging cheloniid sea turtles comprising 4 host species. Molecular analysis yielded more than 20 different genotypes. We were able to assign 1 genotype to 1 of the 2 recognized species, Neospirorchis pricei Manter and Larson, 1950 . In many examples, genotypes exhibited host and site specificity. Our findings indicate considerable diversity of parasites resembling Neospirorchis with evidence of a number of uncharacterized blood flukes that require additional study.

Development and validation of a competitive enzyme-linked immunosorbent assay for the measurement of total plasma immunoglobulins in healthy loggerhead sea (Caretta caretta) and green turtles (Chelonia mydas).

The quantification of circulating plasma immunoglobulins represents a valuable diagnostic tool in human and veterinary immunology, although its application is very limited in reptile medicine to date. The objectives of our study were the development and standardization of a competitive enzyme-linked immunosorbent assay (cELISA) for the measurement of total plasma immunoglobulins (Igs; both IgM and IgY) in loggerhead sea turtles (LST; Caretta caretta; n = 254) and green turtles (GT; Chelonia mydas; n = 111), the establishment of reference intervals for Ig for both species, and the examination of associations between Ig and total protein (TP), condition index, and water temperature. The cELISA for Ig was successfully developed and optimized. Reference intervals for Ig were 0.38-0.94 g/dL in LST (median: 0.59 g/dL; range: 0.16-2.15 g/dL) and 0.40-0.85 g/dL in GT (median: 0.58 g/dL; range: 0.18-1.80 g/dL). In LST, there were positive linear relationships of Ig with TP, and TP with Ig and condition index, and a negative relationship of Ig with condition index. The positive linear relationships of Ig with TP, and TP with Ig were also identified in GT. These positive associations of Ig and TP were expected, as Ig represents fractions of TP, and TP reportedly increases with straight carapace length and weight. The negative association of Ig with condition index may indicate potential biological variations. The cELISA and reference intervals for total Ig of LST and GT presented herein have the potential to be useful as a diagnostic and research tool for sea turtle immunology.

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

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

Widespread recombination, reassortment, and transmission of unbalanced compound viral genotypes in natural arenavirus infections.

Arenaviruses are one of the largest families of human hemorrhagic fever viruses and are known to infect both mammals and snakes. Arenaviruses package a large (L) and small (S) genome segment in their virions. For segmented RNA viruses like these, novel genotypes can be generated through mutation, recombination, and reassortment. Although it is believed that an ancient recombination event led to the emergence of a new lineage of mammalian arenaviruses, neither recombination nor reassortment has been definitively documented in natural arenavirus infections. Here, we used metagenomic sequencing to survey the viral diversity present in captive arenavirus-infected snakes. From 48 infected animals, we determined the complete or near complete sequence of 210 genome segments that grouped into 23 L and 11 S genotypes. The majority of snakes were multiply infected, with up to 4 distinct S and 11 distinct L segment genotypes in individual animals. This S/L imbalance was typical: in all cases intrahost L segment genotypes outnumbered S genotypes, and a particular S segment genotype dominated in individual animals and at a population level. We corroborated sequencing results by qRT-PCR and virus isolation, and isolates replicated as ensembles in culture. Numerous instances of recombination and reassortment were detected, including recombinant segments with unusual organizations featuring 2 intergenic regions and superfluous content, which were capable of stable replication and transmission despite their atypical structures. Overall, this represents intrahost diversity of an extent and form that goes well beyond what has been observed for arenaviruses or for viruses in general. This diversity can be plausibly attributed to the captive intermingling of sub-clinically infected wild-caught snakes. Thus, beyond providing a unique opportunity to study arenavirus evolution and adaptation, these findings allow the investigation of unintended anthropogenic impacts on viral ecology, diversity, and disease potential.

Ball python nidovirus: a candidate etiologic agent for severe respiratory disease in Python regius.

UNLABELLED: A severe, sometimes fatal respiratory disease has been observed in captive ball pythons (Python regius) since the late 1990s. In order to better understand this disease and its etiology, we collected case and control samples and performed pathological and diagnostic analyses. Electron micrographs revealed filamentous virus-like particles in lung epithelial cells of sick animals. Diagnostic testing for known pathogens did not identify an etiologic agent, so unbiased metagenomic sequencing was performed. Abundant nidovirus-like sequences were identified in cases and were used to assemble the genome of a previously unknown virus in the order Nidovirales. The nidoviruses, which were not previously known to infect nonavian reptiles, are a diverse order that includes important human and veterinary pathogens. The presence of the viral RNA was confirmed in all diseased animals (n = 8) but was not detected in healthy pythons or other snakes (n = 57). Viral RNA levels were generally highest in the lung and other respiratory tract tissues. The 33.5-kb viral genome is the largest RNA genome yet described and shares canonical characteristics with other nidovirus genomes, although several features distinguish this from related viruses. This virus, which we named ball python nidovirus (BPNV), will likely establish a new genus in Torovirinae subfamily. The identification of a novel nidovirus in reptiles contributes to our understanding of the biology and evolution of related viruses, and its association with lung disease in pythons is a promising step toward elucidating an etiology for this long-standing veterinary disease. IMPORTANCE: Ball pythons are popular pets because of their diverse coloration, generally nonaggressive behavior, and relatively small size. Since the 1990s, veterinarians have been aware of an infectious respiratory disease of unknown cause in ball pythons that can be fatal. We used unbiased shotgun sequencing to discover a novel virus in the order Nidovirales that was present in cases but not controls. While nidoviruses are known to infect a variety of animals, this is the first report of a nidovirus recovered from any reptile. This report will enable diagnostics that will assist in determining the role of this virus in the causation of disease, which would allow control of the disease in zoos and private collections. Given its evolutionary divergence from known nidoviruses and its unique host, the study of reptile nidoviruses may further our understanding of related diseases and the viruses that cause them in humans and other animals.

Management of wounds in a loggerhead sea turtle (Caretta caretta) caused by traumatic bycatch injury from the spines of a spotted eagle ray (Aetobatus narinari).

A subadult female loggerhead sea turtle (Caretta caretta) was caught in a trawl net off the west coast of Florida with a spotted eagle ray (Aetobatus narinari) spine lodged in the left stifle. Surgical removal of the spine was performed and antibiotic treatment was initiated. Four weeks later, endoscopy revealed a second spine entering an intestinal lumen. The fistulous tract of the left prefemoral fossa was surgically excised and the intestinal perforation was repaired. Dehiscence occurred and a vacuum-assisted closure (VAC) system was used on the wound for approximately 18 days to help reduce infection and increase the rate of healing. The left stifle wound was treated to heal by second intention. The turtle remained in rehabilitation for 19 mo before being released off the west coast of Florida. This case describes stingray envenomation injuries as a complex and potentially life-threatening bycatch effect to sea turtles caught in trawl nets.

Mycoplasmosis and upper respiratory tract disease of tortoises: a review and update.

Tortoise mycoplasmosis is one of the most extensively characterized infectious diseases of chelonians. A 1989 outbreak of upper respiratory tract disease (URTD) in free-ranging Agassiz's desert tortoises (Gopherus agassizii) brought together an investigative team of researchers, diagnosticians, pathologists, immunologists and clinicians from multiple institutions and agencies. Electron microscopic studies of affected tortoises revealed a microorganism in close association with the nasal mucosa that subsequently was identified as a new species, Mycoplasma agassizii. Over the next 24 years, a second causative agent, Mycoplasma testudineum, was discovered, the geographic distribution and host range of tortoise mycoplasmosis were expanded, diagnostic tests were developed and refined for antibody and pathogen detection, transmission studies confirmed the pathogenicity of the original M. agassizii isolate, clinical (and subclinical) disease and laboratory abnormalities were characterized, many extrinsic and predisposing factors were found to play a role in morbidity and mortality associated with mycoplasmal infection, and social behavior was implicated in disease transmission. The translation of scientific research into management decisions has sometimes led to undesirable outcomes, such as euthanasia of clinically healthy tortoises. In this article, we review and assess current research on tortoise mycoplasmosis, arguably the most important chronic infectious disease of wild and captive North American and European tortoises, and update the implications for management and conservation of tortoises in the wild.

Weighing empirical and hypothetical evidence for assessing potential invasive species range limits: a review of the case of Burmese pythons in the USA.

Range expansion potential is an important consideration for prioritizing management actions against an invasive species. Understanding the potential for range expansion by invasive reptiles such as the Burmese python can be challenging, because the lack of knowledge on fundamental physiological and behavioral constraints initially forces reliance on modeling to predict hypothetical invasive range potential. Hypothetical predictions for Burmese python range limits in the USA have been highly divergent, from only extreme South Florida and the extreme southern Gulf edge of Texas to a broad swath over the southern third of the continental USA. Empirical observations on python thermal tolerances and behavioral abilities to cope with more temperate temperatures became evident during a cold spell in December 2009-January 2010. We review and highlight important considerations for improving invasive range estimation methodology, deciding between competing range predictions, and the importance of having, and applying, empirical data to aid in decision making.

Immunohistochemical detection of a unique protein within cells of snakes having inclusion body disease, a world-wide disease seen in members of the families Boidae and Pythonidae.

Inclusion body disease (IBD) is a worldwide disease in captive boa constrictors (boa constrictor) and occasionally in other snakes of the families Boidae and Pythonidae. The exact causative agent(s) and pathogenesis are not yet fully understood. Currently, diagnosis of IBD is based on the light microscopic identification of eosinophilic intracytoplasmic inclusion bodies in hematoxylin and eosin stained tissues or blood smears. An antigenically unique 68 KDa protein was identified within the IBD inclusion bodies, called IBD protein. A validated immuno-based ante-mortem diagnostic test is needed for screening snakes that are at risk of having IBD. In this study, despite difficulties in solubilizing semi-purified inclusion bodies, utilizing hybridoma technology a mouse anti-IBD protein monoclonal antibody (MAB) was produced. The antigenic specificity of the antibody was confirmed and validated by western blots, enzyme-linked immunosorbent assay, immuno-transmission electron microscopy, and immunohistochemical staining. Paraffin embedded tissues of IBD positive and negative boa constrictors (n=94) collected from 1990 to 2011 were tested with immunohistochemical staining. In boa constrictors, the anti-IBDP MAB had a sensitivity of 83% and specificity of 100% in detecting IBD. The antibody also cross-reacted with IBD inclusion bodies in carpet pythons (Morelia spilota) and a ball python (python regius). This validated antibody can serve as a tool for the development of ante-mortem immunodiagnostic tests for IBD.

Mycoplasma testudineum in free-ranging desert tortoises, Gopherus agassizii.

We performed clinico-pathological evaluations of 11 wild Agassiz's desert tortoises (Gopherus agassizii) from a translocation project in the central Mojave Desert, California, USA. Group 1 consisted of nine tortoises that were selected primarily due to serologic status, indicating exposure to Mycoplasma testudineum (seven) or both M. agassizii and M. testudineum (two), and secondarily due to clinical signs of upper respiratory tract disease (URTD). Group 2 consisted of two tortoises that were antibody-negative for Mycoplasma and had no clinical signs of URTD, but did have other signs of illness. Of the Group 1 tortoises, M. testudineum, but not M. agassizii, was amplified by polymerase chain reaction and DNA fingerprinted from two tortoises. Using light microscopy, mild to severe pathologic changes were observed in one or more histologic sections of either one or both nasal cavities of each tortoise in Group 1. Our findings support a causal relationship between M. testudineum and URTD in desert tortoises.

Environmental temperatures, physiology and behavior limit the range expansion of invasive Burmese pythons in southeastern USA.

A well-established population of Burmese pythons resides in the Everglades of southern Florida. Prompted in part by a report that identified much of southern USA as suitable habitat for expansion or establishment of the Burmese python, we examined the plausibility of this snake to survive winters at sites north of the Everglades. We integrated daily low and high temperatures recorded from October to February from 2005-2011 at Homestead, Orlando and Gainesville, Florida; and Aiken, South Carolina, with minimum temperatures projected for python digestion (16 °C), activity (5 °C) and survival (0 °C). Mean low and high temperatures decreased northward from Homestead to Aiken and the number of days of freezing temperatures increased northward. Digestion was impaired or inhibited for 2 months in the Everglades and up to at least 5 months in Aiken, and activity was increasingly limited northward during these months. Reports of overwinter survivorship document that a single bout of low and freezing temperatures results in python death. The capacity for Burmese pythons to successfully overwinter in more temperate regions of the USA is seemingly prohibited because they lack the behaviors to seek refuge from, and the physiology to tolerate, cold temperatures. As tropical Southeast Asia is the source of the Everglades Burmese pythons, we predict it is unlikely that they will be able to successfully expand to or colonize more temperate areas of Florida and adjoining states due to their lack of behavioral and physiological traits to seek refuge from cold temperatures.

Serologic and molecular evidence for Testudinid herpesvirus 2 infection in wild Agassiz's desert tortoises, Gopherus agassizii.

Following field observations of wild Agassiz's desert tortoises (Gopherus agassizii) with oral lesions similar to those seen in captive tortoises with herpesvirus infection, we measured the prevalence of antibodies to Testudinid herpesvirus (TeHV) 3 in wild populations of desert tortoises in California. The survey revealed 30.9% antibody prevalence. In 2009 and 2010, two wild adult male desert tortoises, with gross lesions consistent with trauma and puncture wounds, respectively, were necropsied. Tortoise 1 was from the central Mojave Desert and tortoise 2 was from the northeastern Mojave Desert. We extracted DNA from the tongue of tortoise 1 and from the tongue and nasal mucosa of tortoise 2. Sequencing of polymerase chain reaction products of the herpesviral DNA-dependent DNA polymerase gene and the UL39 gene respectively showed 100% nucleotide identity with TeHV2, which was previously detected in an ill captive desert tortoise in California. Although several cases of herpesvirus infection have been described in captive desert tortoises, our findings represent the first conclusive molecular evidence of TeHV2 infection in wild desert tortoises. The serologic findings support cross-reactivity between TeHV2 and TeHV3. Further studies to determine the ecology, prevalence, and clinical significance of this virus in tortoise populations are needed.