Morphologic and physiologic characteristics of green sea turtle (Chelonia mydas) hatchlings in southeastern Florida, USA.

The ability of sea turtle hatchlings to survive into adulthood is related, in part, to their individual health status. Documenting a variety of health data is essential for assessing individual and population health. In this study, we report health indices for 297 green sea turtle (Chelonia mydas) hatchlings that emerged from 32 nests deposited on Juno Beach, Florida, USA in June-July, 2017. Results of physical examination, morphometrics, and infectious disease testing (chelonid alphaherpesvirus 5, ChHV5), and blood analyte reference intervals (hematology, plasma protein, glucose) are presented. Carapacial scute abnormalities were observed in 36% (108/297) of all hatchlings, including abnormal vertebral (86/297, 29%), lateral (72/297, 24%), and both vertebral and lateral (50/297, 17%) scutes. Hatchlings from nests laid in July, which was ~ 1.6 °C warmer than June, had significantly shorter incubation periods, and higher body mass, straight carapace length, body condition index, packed cell volume, and heterophil:lymphocyte ratios compared to hatchlings from nests laid in June. These results suggest that incubation temperatures are linked to hatchling developmental factors and size, nutritional and/or hydration status, and/or blood cell dynamics. Blood samples from all 297 hatchlings tested negative for ChHV5 DNA via quantitative PCR, including 86 hatchlings from the nests of 11 adult females that tested positive for ChHV5 via qPCR or serology in a separate study, lending support to the hypothesis that ChHV5 is horizontally (rather than vertically) transmitted among green turtles. Information resulting from this study represents a useful dataset for comparison to future health assessment and population monitoring studies of green turtle hatchlings in the northwestern Atlantic Ocean.

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.

Potential Noncutaneous Sites of Chelonid Herpesvirus 5 Persistence and Shedding in Green Sea Turtles Chelonia mydas.

Chelonid herpesvirus 5 (ChHV5), the likely etiologic agent of sea turtle fibropapillomatosis (FP), is predicted to be unevenly distributed within an infected turtle, in which productive virus replication and virion shedding occurs in cutaneous tumor keratinocytes. In this study, we measured and compared ChHV5 DNA quantities in tumors, skin, urine, major organs, and nervous tissue samples from green turtles Chelonia mydas. These samples were taken from the carcasses of 10 juvenile green turtles with and without clinical signs of FP that stranded in Florida during 2014. Quantitative PCR for ChHV5 UL30 was used to identify ChHV5 DNA in tumors, skin, heart, kidney, nerves, and urine sampled from five out of five FP-positive and three out of five FP-free turtles. The most frequently co-occurring sites were cutaneous tumor and kidney (n = 4). Novel data presented here include the identification of ChHV5 DNA in kidney, heart, and nerve samples from three FP-free turtles. These data support candidate nontumored anatomic sites of ChHV5 DNA localization and mobilization during two different disease states that may be involved in the ChHV5 infection cycle. Received September 8, 2016; accepted April 17, 2017.

Small-animal research imaging devices.

The scientific study of living animals may be dated to Aristotle's original dissections, but modern animal studies are perhaps a century in the making, and advanced animal imaging has emerged only during the past few decades. In vivo imaging now occupies a growing role in the scientific research paradigm. Imaging of small animals has been particularly useful to help understand human molecular biology and pathophysiology using rodents, especially using genetically engineered mice (GEM) with spontaneous diseases that closely mimic human diseases. Specific examples of GEM models of veterinary diseases exist, but in general, GEM for veterinary research has lagged behind human research applications. However, the development of spontaneous disease models from GEM may also hold potential for veterinary research. The imaging techniques most widely used in small-animal research are CT, PET, single-photon emission CT, MRI, and optical fluorescent and luminescent imaging.

Imaging devices for use in small animals.

Imaging devices for small animals have emerged in the past 10 years as extraordinarily useful tools in translational research and drug development. The Food and Drug Administration requires animal testing after in vitro drug discovery but before human application. Many small animal instruments have been developed in analogy to human scale devices, including positron emission tomography, single-photon emission computed tomography, computed tomography, magnetic resonance imaging, and ultrasound. Conversely, optical imaging with fluorescent and bioluminescent tracer technology, originating in single-cell in vitro studies, has been scaled up to whole-body animal imaging. Imaging that uses multiple devices permits a comparison of different aspects of function, anatomy, gene expression, and phenotype by the use of software algorithms or more recently with hybrid instruments. Animal imaging facilitates "bench-to-bedside" drug development in 2 ways. Longitudinal imaging improves the science of animal research through the benefit of paired statistics with the use of animals as their own controls while it simultaneously reduces animal sacrifice. In addition, imaging makes explicit the development of diagnostic and therapeutic agents on nearly identical molecular synthesis platforms, therefore linking drug discovery to the development of imaging tracers. This powerful paradigm, now known as diagnostic/therapeutic pairing or theranostics, is already familiar from the use of (123)I used for thyroid diagnosis and (131)I for therapy of benign and malignant thyroid conditions. Many newer examples exist, such as "cold" or "hot" octreotide and meta-iodobenzylguanidine in neuroendocrine tumors; and rituximab in pharmaceutical doses, or with beta emitter tags, for therapy of indolent non-Hodgkin's lymphoma. Theranostic agents are also rapidly emerging that use nanoparticles, aptamers, peptides, and antibodies for magnetic resonance imaging/positron emission tomography/single-photo emission computed tomography/computed tomography imaging devices in animals with subsequent therapeutic drug development for translation to human use.

Spirorchiidiasis in stranded loggerhead Caretta caretta and green turtles Chelonia mydas in Florida (USA): host pathology and significance.

Spirorchiid trematodes are implicated as an important cause of stranding and mortality in sea turtles worldwide. However, the impact of these parasites on sea turtle health is poorly understood due to biases in study populations and limited or missing data for some host species and regions, including the southeastern United States. We examined necropsy findings and parasitological data from 89 loggerhead Caretta caretta and 59 green turtles Chelonia mydas that were found dead or moribund (i.e. stranded) in Florida (USA) and evaluated the role of spirorchiidiasis in the cause of death. High prevalence of infection in the stranding population was observed, and most infections were regarded as incidental to the cause of death. Spirorchiidiasis was causal or contributory to death in some cases; however, notable host injury and/or large numbers of parasites were observed in some animals, including nutritionally robust turtles, with no apparent relationship to cause of death. New spirorchiid species records for the region were documented and identified genera included Neospirorchis, Hapalotrema, Carettacola, and Learedius. Parasites inhabited and were associated with injury and inflammation in a variety of anatomic locations, including large arteries, the central nervous system, endocrine organs, and the gastrointestinal tract. These findings provide essential information on the diversity of spirorchiids found in Florida sea turtles, as well as prevalence of infection and the spectrum of associated pathological lesions. Several areas of needed study are identified with regard to potential health implications in the turtle host, and findings caution against over-interpretation in individual cases.

Detection of spirorchiid trematodes in gastropod tissues by polymerase chain reaction: preliminary identification of an intermediate host of Learedius learedi.

Marine spirorchiid trematodes are associated with morbidity and mortality in sea turtles worldwide. The intermediate hosts remain unknown, and discovery efforts are hindered by the large number and great diversity of potential hosts within sea turtle habitats, as well the potential for low prevalence and overdispersion. A high-throughput DNA extraction and polymerase chain reaction-based method was developed to detect the internal transcribed spacer 2 (ITS2) region of the ribosomal gene of 2 spirorchiid genera, Learedius and Hapalotrema , within pooled samples of gastropod tissues. A model system consisting of freshwater snail ( Pomacea bridgesii ) tissues and DNA extracts spiked with adult Learedius learedi and known quantities of spirorchiid DNA was used to develop and test the technique. Threshold of detection was found to be equivalent to an early prepatent infection within 1.5 g of gastropod tissue. This technique was used to screen approximately 25 species of marine gastropods at a captive facility where green turtles ( Chelonia mydas ) become infected by L. learedi . The parasite was detected in a sample of knobby keyhole limpet ( Fissurella nodosa ), thus providing the first evidence of an intermediate host for a marine spirorchiid trematode. This technique has many potential applications in trematode life cycle discovery studies.

Genomic characterization of two novel reptilian papillomaviruses, Chelonia mydas papillomavirus 1 and Caretta caretta papillomavirus 1.

In this paper we describe the characterization of the genomes of two sea turtle papillomaviruses, Chelonia mydas PV (CmPV-1) and Caretta caretta PV (CcPV-1). The isolation and sequencing of the first non-avian reptilian PVs extend the evolutionary history of PVs to include all amniotes. PVs have now been described in mammals, birds and non-avian reptiles. The chelonian PVs form a distinct clade most closely related to the avian PVs. Unlike the avian PVs, both chelonian PVs have canonical E6 and E7 ORFs, indicating that these genes were present in the common ancestor to mammalian and non-mammalian amniote PVs. Rates of evolution among the non-mammalian PVs were generally slower than those estimated for mammalian PVs, perhaps due to lower metabolic rates among the ectothermic reptiles.

Use of baculovirus-expressed glycoprotein H in an enzyme-linked immunosorbent assay developed to assess exposure to chelonid fibropapillomatosis-associated herpesvirus and its relationship to the prevalence of fibropapillomatosis in sea turtles.

Chelonid fibropapillomatosis-associated herpesvirus (CFPHV) is an alphaherpesvirus believed to cause marine turtle fibropapillomatosis (FP). A serodiagnostic assay was developed for monitoring sea turtle populations for CFPHV exposure. CFPHV glycoprotein H (gH) expressed in recombinant baculovirus was used in an enzyme-linked immunosorbent assay (ELISA) to detect virus-specific 7S turtle antibodies. Using captive-reared green turtles (Chelonia mydas) with no history of virus exposure as "known negatives" and others with experimentally induced FP as "known positives," the assay had 100% specificity but low sensitivity, as seroconversion was detected in only half of the turtles bearing experimentally induced tumors. Antibodies were detected only in samples collected after cutaneous fibropapillomas appeared, consistent with observations that tumors are significant sites of virion production and antigen expression and the possibility that prolonged/repeated virus shedding may be required for adequate stimulation of 7S antibody responses to gH. Natural routes of infection, however, may produce higher seroconversion rates. High gH antibody seroprevalences ( approximately 80%) were found among wild green turtles in three Florida localities with different FP prevalences, including one site with no history of FP. In addition, all eight loggerhead turtles (Caretta caretta) tested were seropositive despite FP being uncommon in this species. The possibility that CFPHV infection may be common relative to disease suggests roles for environmental and host factors as modulators of disease expression. Alternatively, the possibility of other antigenically similar herpesviruses present in wild populations cannot be excluded, although antibody cross-reactivity with the lung/eye/trachea disease-associated herpesvirus was ruled out in this study.

Two herpesviruses associated with disease in wild Atlantic loggerhead sea turtles (Caretta caretta).

Herpesviruses are associated with lung-eye-trachea disease and gray patch disease in maricultured green turtles (Chelonia mydas) and with fibropapillomatosis in wild sea turtles of several species. With the exception fibropapillomatosis, no other diseases of wild sea turtles of any species have been associated with herpesviral infection. In the present study, six necropsied Atlantic loggerhead sea turtles (Caretta caretta) had gross and histological evidence of viral infection, including oral, respiratory, cutaneous, and genital lesions characterized by necrosis, ulceration, syncytial cell formation, and intranuclear inclusion bodies. Nested polymerase chain reaction targeting a conserved region of the herpesvirus DNA-dependent-DNA polymerase gene yielded two unique herpesviral sequences referred to as loggerhead genital-respiratory herpesvirus and loggerhead orocutaneous herpesvirus. Phylogenetic analyses indicate that these viruses are related to and are monophyletic with other chelonian herpesviruses within the subfamily alpha-herpesvirinae. We propose the genus Chelonivirus for this monophyletic group of chelonian herpesviruses.

Distribution of chelonid fibropapillomatosis-associated herpesvirus variants in Florida: molecular genetic evidence for infection of turtles following recruitment to neritic developmental habitats.

Marine turtle fibropapillomatosis is associated with chelonid fibropapilloma-associated herpesvirus (C-FP-HV) and commonly affects juvenile green turtles (Chelonia mydas) in neritic (nearshore) habitats. Green turtles have a complex life history, characterized by shifts in trophic level as well as habitat during ontogeny. Thus, several hypotheses can be proposed for when turtles become infected with C-FP-HV. They may acquire the virus at an early stage in the life cycle, including prenatal, hatchling, or the posthatchling pelagic stages. Alternatively, they may become infected later in life after they emigrate from the open ocean to neritic habitats. Each hypothesis generates predictions about the spatial distribution of genetic variants of C-FP-HV among nearshore sites within a region. Sequencing of polymerase chain reaction-amplified viral DNA from fibropapillomas of individual turtles was used to genotype the viral variants present in marine turtles from different coastal areas in Florida. We found four distinct virus variants (A, B, C, and D), two of which (A and C) were present in multiple turtle species. Green turtles in Florida were infected with variants A, B, and C. Variant A was found in green turtles from all three areas. Outside the Indian River Lagoon, variant A was most commonly detected and was found in >94% of diseased green turtles and 70% of loggerhead sea turtles (Caretta caretta) in the Florida Bay/Florida Keys. However, in the Indian River Lagoon, variant B was found in >94% of affected green turtles. Variant B was not detected outside of the Indian River system. Chi-square analysis strongly supported (P<0.001) an association between viral variant distribution in green turtles and location. On the basis of the assumption that juvenile green turtles found in Florida's west-central coast, Florida Keys, and Indian River Lagoon areas represented recruits from a mixed pelagic population, we expected that the distribution of viral variants in these turtles would be relatively homogeneous among locations; this would correspond to infection in the earlier phases of their life cycle. The heterogeneous distribution of viral variants in green turtle tumors from different Florida coastal locations strongly supports the hypothesis that, during epizootics, turtles are infected with specific C-FP-HV variants after they arrive as juveniles in neritic habitats. The conclusion that C-FP-HV is acquired after turtles recruit to nearshore habitats should help focus further research efforts on understanding the mechanisms of transmission and raises the possibility that the effect of fibropapillomatosis on turtle populations might be reduced by management strategies designed to break the cycle of transmission in these locations.

Induction of vitellogenesis by estradiol-17beta and development of enzyme-linked immunosorbant assays to quantify plasma vitellogenin levels in green turtles (Chelonia mydas).

Treatment of juvenile green turtles (Chelonia mydas) with estradiol-17beta resulted in the induction of a 200 kDa plasma protein, consistent with vitellogenin (Vtg). The N-terminal 15 amino acids of the anion exchange purified protein shared sequence homologies with vitellogenins of several vertebrate species. Rabbit antiserum raised against purified Vtg recognized the plasma protein as well as several yolk proteins. Monoclonal antibody (Mab) HL1248, produced by inoculating mice with turtle yolk granules, showed specificity for plasma Vtg as well as a set of yolk proteins 120, 82, 43 and 32 kDa in size. The N-terminal 22 amino acids of the 43 kDa yolk protein was similar to the lipovitellin I subunit of Vtg of several vertebrate species. The peptide mass map of the 82 kDa yolk protein shared enough ions with that of purified plasma Vtg to support the conclusion that this protein was derived from plasma Vtg. Taken together, these results validate the specificity of Mab HL1248 for Vtg. Using purified Vtg concentration standards, competition and antigen capture enzyme-linked immunosorbant assays (ELISAs) were shown to quantitatively detect Vtg in green turtle plasma. Pre-induced plasma of juvenile turtles had Vtg levels of 2-4 micrograms/ml whereas post-estradiol exposure samples had 38-40 mg/ml. The plasma Vtg concentration of a nesting female turtle was 4.6 mg/ml, approximately 20-fold higher than that of a non-nesting adult female. The antigen capture ELISA will be useful in population studies of this endangered species, to detect vitellogenesis in females that will nest in a given year and to detect inappropriate Vtg levels in turtles exposed to xenoestrogens.

Characterization of Mycobacterium montefiorense sp. nov., a novel pathogenic Mycobacterium from moray eels that is related to Mycobacterium triplex.

The characterization of a novel Mycobacterium sp. isolated from granulomatous skin lesions of moray eels is reported. Analysis of the hsp65 gene, small-subunit rRNA gene, rRNA spacer region, and phenotypic characteristics demonstrate that this organism is distinct from its closest genetic match, Mycobacterium triplex, and it has been named M. montefiorense sp. nov.

A candidate metastasis-associated DNA marker for ductal mammary carcinoma.

BACKGROUND: Molecular genetic markers to identify the 13% lymph node-negative mammary carcinomas that are prone to develop metastases would clearly be of considerable value in indicating those cases in need of early aggressive therapy. METHODS: Representational difference analysis was used in an attempt to identify genetic alterations related to breast cancer metastasis by comparing genomic DNA from microdissected normal cells and from metastatic cells of ductal breast carcinoma patients. RESULTS: Representational difference analysis products yielded 10 unique metastasis-associated DNA sequences (MADS), i.e. products apparently lost in metastatic cell DNA. Of these sequences, MADS-IX was found to be lost in the transition from primary to metastasis in two out of five ductal breast carcinoma cases. This sequence was localized on chromosome 10q21 by radiation hybrid mapping and fluorescence in situ hybridization. The PTEN gene, which is also located on chromosome 10q, was detected to be present by PCR in all five cases. On the contrary, a breast carcinoma cell line, HCC-1937, which has homozygous loss of a region encompassing the PTEN gene, showed the presence of MADS-IX. PCR screening of three additional breast carcinoma cell lines with known losses in specific chromosomal regions also showed the presence of MADS-IX. CONCLUSION: These data suggest that MADS-IX possibly is part of a novel candidate metastasis-associated gene located close to the PTEN gene on chromosome 10q. The first set of PCR screening in five patient samples indicates that it could be used as a molecular marker for ductal mammary metastasis.

Administration of vaccinia virus to mice may cause contact or bedding sentinel mice to test positive for orthopoxvirus antibodies: case report and follow-up investigation.

Routine testing of bedding sentinels from a barrier room revealed one mouse seropositive to ectromelia virus (EV). Results of hemagglutination-inhibition testing and western blot analysis were confirmatory for orthopoxvirus antibodies. Additional seropositive animals were not identified. Interviews indicated that replication-competent vaccinia virus (VV), Western Reserve strain (VV-WR), recently had been given to mice. Although VV-WR was not expected to spread by contact or via fomites, the case evidence suggested transmission of vaccinia via soiled bedding. In a follow-up experiment, 15 index mice were inoculated with 10(7) plaque-forming units of VV by either subcutaneous or intrarectal instillation. A dedicated contact sentinel and a bedding sentinel were provided for each index mouse. All 15 index mice were positive for antibodies when tested 22 days after inoculation. One mouse, inoculated by the subcutaneous route, appeared ill and developed lesions on the proximal portion of the tail. The contact sentinel mouse housed with this index mouse was the only sentinel to seroconvert. We conclude that VV-WR can spread to contact sentinels and potentially to bedding sentinels. The ability of other VV strains to be transmitted horizontally and the susceptibility of different mouse strains to infection merit further investigation. The use of VV in animal facilities must be managed carefully since the available serologic tests do not distinguish between VV and EV, an exotic agent of major concern to laboratory animal facilities.

Identification and expression of immunogenic proteins of a disease-associated marine turtle herpesvirus.

Herpesviruses are associated with several diseases of marine turtles, including lung-eye-trachea disease (LETD) and fibropapillomatosis. Two approaches were used to identify immunodominant antigens of LETV, the LETD-associated herpesvirus. The first approach targeted glycoprotein B, which is known to be immunogenic and neutralizing in other species. The second strategy identified LETV proteins recognized on Western blots by antibodies in immune green turtle plasma. A 38-kDa protein was resolved by two-dimensional gel electrophoresis, sequenced, and identified as a scaffolding protein encoded by the overlapping open reading frames of UL26 and UL26.5. Glycoprotein B and the scaffolding protein were cloned and expressed in Escherichia coli. The expressed proteins were recognized on Western blots by antibodies in immune green turtle plasma. Phylogenetic studies based on UL26, DNA polymerase, and glycoprotein B revealed that LETV clusters with the alphaherpesviruses.