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Important indicators of population health needed for large-scale sea turtle population recovery efforts include demographics, disease and mortality trends, condition indices, and baseline blood data. With this comprehensive health assessment of adult female green sea turtles Chelonia mydas nesting on Juno Beach, Florida, USA, we (1) established comprehensive baseline health indices; (2) identified individuals with evidence of infection by chelonid alphaherpesviruses 5 and 6 (ChHV5, ChHV6), which are implicated in fibropapillomatosis and respiratory and skin disease, respectively; and (3) compared measured health indices between turtles that did versus those that did not test positive for ChHV5 and/or ChHV6. All 60 turtles included in the study were in good body condition with no external fibropapillomatosis tumors. Hematological and biochemical reference intervals were established. Via quantitative PCR (qPCR), 5/60 turtles (8%) tested positive for ChHV5, and all turtles were negative for ChHV6. Of 41 turtles tested for antibodies to ChHV5 and ChHV6, 29% and 15% tested positive, respectively, and 10% tested positive for antibodies to both viruses. Notably, there were no statistically significant differences between health variables for nesting turtles that tested positive for ChHV5 DNA versus those that tested negative; and also no differences between turtles that tested positive for ChHV5 or ChHV6 antibodies and those that did not. This suggests that these viruses are enzootically stable in Florida’s adult green turtles. This study provides a health profile of nesting green turtles in southeastern Florida applicable to temporal and spatial investigations of this and other populations.

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.

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.

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.

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 (∼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.

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 α-herpesvirinae . We propose the genus Chelonivirus for this monophyletic group of chelonian herpesviruses.

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 in94% 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 in94% of affected green turtles. Variant B was not detected outside of the Indian River system. Chi-square analysis strongly supported (P0.001) 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.

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.

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.

We are presenting a rebuttal letter to the following article that appeared recently on PeerJ: Van Houtan KS, Smith CM, Dailer ML, and Kawachi M. 2014. Eutrophication and the dietary promotion of sea turtle tumors. PeerJ 2:e602. This article is available at the following URL: https://peerj.com/articles/602/. We argue that the article lacks an inferential framework to answer the complex question regarding the drivers of the turtle tumor disease fibropapillomatosis in Hawaii. The article also contains procedural flaws and does not provide any compelling evidence of a link between algae, arginine, and turtle tumors.

Lung-eye-trachea disease-associated herpesvirus (LETV) is linked with morbidity and mortality in mariculture-reared green turtles, but its prevalence among and impact on wild marine turtle populations is unknown. An enzyme-linked immunosorbent assay (ELISA) was developed for detection of anti-LETV antibodies and could distinguish LETV-exposed green turtles from those with antibodies to fibropapillomatosis-associated herpesvirus (FPHV). Plasma from two captive-reared green turtles immunized with inactivated LETV served as positive controls. Plasma from 42 healthy captive-reared green turtles and plasma from 30 captive-reared green turtles with experimentally induced fibropapillomatosis (FP) and anti-FPHV antibodies had low ELISA values on LETV antigen. A survey of 19 wild green turtles with and 27 without FP (with and without anti-FPHV antibodies, respectively) identified individuals with antibodies to LETV regardless of their FP status. The seroprevalence of LETV infection was 13%. The presence of antibodies to LETV in plasma samples was confirmed by Western blot and immunohistochemical analyses. These results are the first to suggest that wild Florida green turtles are exposed to LETV or to an antigenically closely related herpesvirus(es) other than FPHV and that FPHV and LETV infections are most likely independent events. This is the first ELISA developed to detect antibodies for a specific herpesvirus infection of marine turtles. The specificity of this ELISA for LETV (ability to distinguish LETV from FPHV) makes it valuable for detecting exposure to this specific herpesvirus and enhances our ability to conduct seroepidemiological studies of these disease-associated agents in marine turtles.

Fibroblast cell lines derived from normal skin and experimentally induced fibropapillomas of green turtles (Chelonia mydas), were propagated in vitro and tested for tumorigenicity in immunodeficient mice. Differential display RT-PCR was used to identify differences in messenger RNA expression between normal and tumorigenic fibropapillomatosis (FP)-derived fibroblasts from the same individual. Four unique products that were apparently overexpresed in FP and three that were apparently underexpressed were cloned and sequenced. Differential expression was confirmed for three products by Northern blotting. Two overexpressed products showed extensive sequence matches to the known mammalian cellular genes, beta-hexosaminidase and chain termination factor. The product that was underexpressed in FP showed homology with mammalian thrombospondin, a known tumor-suppressor gene and an inhibitor of angiogenesis. All of the partial gene sequences identified are novel and will require full length cDNA sequencing to further analyze their identities. These results, however, provide the foundation for further investigation to determine the role of each of these gene products in FP pathogenesis and cellular transformation. The potential for some of these products to serve as biomarkers for FP is discussed.

A recently developed enzyme-linked immunosorbent assay (ELISA) was used to assess exposure of Florida wild green turtles Chelonia mydas to LETV, the herpesvirus associated with lung-eye-trachea disease (LETD). Plasma samples from 329 wild juvenile green turtles netted in the Indian River lagoon, along the Sebastian reef, or in the Trident basin (Indian River and Brevard Counties, Florida) were tested by ELISA for the presence of antibodies to LETV. Plasma samples from 180 wild juvenile green turtles were tested from these study sites to compare the prevalence of anti-LETV antibodies. While some plasma samples from each site contained anti-LETV antibodies (confirmed by Western blot analysis), plasma samples collected from the Indian River lagoon had statistically higher optical density values measured in the ELISA. No statistical differences were observed when these same plasma samples were analyzed for changes in the level of anti-LETV antibodies over 3 years (1997, 1998, and 1999). To explore the relationship between anti-LETV antibodies and fibropapillomatosis (FP), plasma from 133 green turtles scored for fibropapilloma tumor severity were tested by ELISA. There was no correlation between tumor severity and the presence of antibodies against LETV. Additional plasma samples collected from 16 tagged green turtles captured and sampled more than once (recaptures) were also tested to monitor antibody levels to LETV relative to the FP status of individual turtles over time. Again there was no clear relationship between FP tumor status and the presence of antibodies to LETV. Finally, ELISA tests on plasma from 13 nesting female turtles (9 green and 4 loggerhead) revealed high levels of anti-LETV antibodies in 11 individuals, including 2 loggerhead turtles. These results provide strong evidence that wild Florida green turtle populations at these 3 study sites are exposed to LETV or a closely related virus and that loggerhead turtles may be exposed as well. Based on a cutoff optical density value of 0.310, 71 out of the 329 wild Florida green turtles tested were seropositive for LETV antibodies (seroprevalence = 21.6%). In addition, no relationship between FP tumor severity or status and the presence of anti-LETV antibodies was found, further supporting the hypothesis that LETV and the FP-associated herpesvirus (FPHV) are separate infections of marine turtles.

Herpesviruses are associated with several diseases of marine turtles including lung-eye-trachea disease (LETD) and gray patch disease (GPD) of green turtles (Chelonia mydas) and fibropapillomatosis (FP) of green, loggerhead (Caretta caretta), and olive ridley turtles (Lepidochelys olivacea). The stability of chelonian herpesviruses in the marine environment, which may influence transmission, has not been previously studied. In these experiments, LETD-associated herpesvirus (LETV) was used as a model chelonian herpesvirus to test viral infectivity after exposure to seawater. The LETV virus preparations grown in terrapene heart (TH-1) cells were dialyzed for 24 to 120 hr against aerated artificial or natural seawater or Hank's balanced salt solution (HBBS). Fresh TH-1 cells were inoculated with dialyzed LETV, and on day 10 post-infection cells were scored for cytopathic effect. Virus samples dialyzed up to 120 hr were positive for the herpesvirus DNA polymerase gene by polymerase chain reaction. Electron microscopy revealed intact LETV nucleocapsids after exposure of LETV to artificial seawater or HBSS for 24 hr at 23 C. LETV preparations remained infectious as long as 120 hr in natural and artificial seawater at 23 C. Similar results were obtained with a second culturable chelonian herpesvirus, HV2245. LETV infectivity could not be detected after 48 hr exposure to artificial seawater at 30 C. Since LETV and HV2245 remain infectious for extended periods of time in the marine environment, it is possible that FP-associated and GPD-associated herpesviruses also may be stable. These findings are significant both for researchers studying the epidemiological association of herpesviruses with diseases of marine turtles and for individuals who handle turtles in marine turtle conservation efforts.

Tumor biopsy samples from 25 Floridian and 15 Hawaiian green turtles (Chelonia mydas) with spontaneous green turtle fibropapillomatosis (GTFP) and from 27 captive-reared green turtles with experimen- tally induced GTFP were examined microscopically to differentiate the histologic features that result from GTFP pathogenesis and those that result from incidental factors that may vary according to geographic region. Com- mon histologic features for spontaneous and experimentally induced tumors included fibroblast proliferation in the superficial dermis, epidermal acanthosis and hyperkeratosis, epidermal basal cell degeneration with dermal- epidermal cleft formation, spinous layer degeneration with intraepidermal vesicle and pustule formation, and ulceration. Visceral tumors, found in eight of 10 (80%) free-ranging turtles with cutaneous disease that were examined after death, had extensive interstitial fibrous proliferation. The presence of spirorchid trematode eggs and associated foreign body granulomas, common secondary findings within spontaneous tumors, varied by geographic location, and these findings were not observed in experimentally induced tumors. Eosinophilic intranuclear inclusions and intranuclear herpesvirus-associated antigen immunoreactivity were found in 18 of 38 (47%) experimentally induced cutaneous tumors and nine of 119 (7.5%) spontaneous tumors from Floridian but not Hawaiian turtles. The possible involvement of GTFP-associated herpesvirus in the pathogenesis of epidermal degenerative changes and GTFP pathogenesis is discussed. Green turtle fibropapillomatosis (GTFP) is a disease of green turtles (Chelonia mydas) that is characterized by multiple cutaneous papillomas, fibromas, and fibro- papillomas, as well as occasional visceral fibromas. Concern over recent increases in the prevalence of GTFP around the world has focused attention on iden- tifying the cause and understanding the pathogenesis of this disease so that management strategies can be developed to minimize its impact on populations of endangered green turtles. 18

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

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.

Monoclonal antibodies (Mabs) were developed against the known immunoglobulin classes of the green turtle, Chelonia mydas. Plasma protein fractions enriched for 5.7S IgY, 7S IgY, and IgM turtle immunoglobulins were used to immunize Balb/c mice for hybridoma production and for hybridoma screening. Fifteen hybridomas produced Mabs with specificity for turtle immunoglobulins and for affinity purified dinitrophenol (DNP) specific turtle antibodies. Three Mabs specific for either turtle 5.7S IgY heavy chain (HL814), 7S IgY heavy chain (HL857), or IgM heavy chain (HL846) were purified and used in an enzyme-linked immunosorbent assay (ELISA) to measure antibody responses in two turtles immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) over a 10 month period. In both turtles the 7S IgY antibody response developed within 5 weeks of the first inoculation and remained high over the following 9 months. The 5.7S IgY antibody response was detected in one turtle at 3-4 months and in the other at 8 months, and reached high levels in both individuals by 10 months. The IgM responses were difficult to interpret. One turtle had pre-inoculation anti-DNP IgM antibody in its plasma and the other developed only a weak, transient response at about 4 months. The class-specific antibody activity in immune turtle plasma could be strongly inhibited by soluble DNP or by rabbit anti-DNP specific antiserum, showing that these antibody responses were directed predominantly to the DNP hapten on the DNP-BSA antigen. Antibody responses to the BSA carrier could not be detected in either turtle over the course of the immunization. Mab HL814, specific for an epitope on the 5.7S green turtle immunoglobulin heavy chain, will be useful for characterizing the molecular relationships of 5.7S, 7S and IgM heavy chains and the role of 5.7S antibody in humoral immunity in this species. All anti-turtle Ig Mabs were screened against the plasma globulins of Loggerhead (Caretta caretta), Olive Ridley (Lepidochelys olivacea), Kemp's Ridley (Lepidochelys kempi), Hawksbill (Eretmochelys imbricata), and Leatherback (Dermochelys coriacea). While the Mabs specific for IgM and 5.7S IgY reacted only with the green turtle, two Mabs specific for light chain reacted with all species except the leatherback, and nine mabs specific for 7S IgY heavy chain reacted with all five species. Thus, these Mabs may be useful for immunodiagnostic applications in these endangered species as well.

Green turtle fibropapillomatosis (GTFP) is a growing threat to the survival of green turtle (Chelonia mydas) populations worldwide. Recent transmission studies point to an infectious etiology. Several field studies suggest that high GTFP prevalence is associated with marine habitats that have been impacted by agricultural, industrial, or urban development. Environmental contaminants could be involved in GTFP through several plausible mechanisms including cocarcinogenesis and contaminant-induced immune suppression. However, an association of contaminants with GTFP has not been established. A broader perspective is needed when studying infectious diseases such as GTFP in complex ecosystems. Alternative explanations for high GTFP prevalence in some near-shore habitats include the following: a) these habitats provide an optimum physical environment for survival and transmission of the infectious agent; b) these habitats attract a high density of susceptible turtles or harbor a higher density of potential vectors, facilitating transmission of the pathogen in a density-dependent fashion; and c) these habitats may contain other stressors that render turtles more susceptible to GTFP. Application of scientifically rigorous criteria in the epizootiology of GTFP in free-ranging populations remains a formidable challenge. Images Figure 1.

Cutaneous fibropapillomatosis in green sea turtles, Chelonia mydas (GTFP), was first reported over 50 years ago. In the last decade, GTFP has emerged as a significant worldwide epizootic with prevalences as high as 92% in some green turtle populations. Lesions similar to GTFP have been observed in other marine turtle species including olive ridleys, Lepidochelys olivacea , flatbacks, Natator depressus , and loggerheads, Caretta caretta , but disease in these species occurs at lower frequencies and is less well documented. The etiology of GTFP is unknown, and a variety of hypotheses concerning the possible etiology and pathogenesis of GTFP have been proposed and are discussed in this paper. Possible etiologies include viruses, metazoan parasites, ultraviolet radiation, and chemical carcinogens. Recent evidence from controlled transmission experiments implicates a filterable infectious agent as the primary etiology of GTFP. A herpesvirus has been identified in some lesions but has not been isolated and cultured; consequently, Koch's postulates have not yet been fulfilled for this agent. The epizootiology and pathogenesis of GTFP are poorly understood. Epizootiologic evidence, while limited to a few field studies, suggests that environmental conditions in certain near-shore marine habitats favor a high prevalence of disease expression. The possibility that immune system modulators play a role in the persistence and severity of this disease is discussed. Detailed investigations of the epizootiology of GTFP must await identification of the etiologic agent and development of specific diagnostic tests. In addition, until immune function tests can be developed and validated for free-ranging turtles, hypotheses about the role of immune system dysfunction in GTFP epizootics cannot be tested.

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.

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.

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, includ- ing the southeastern United States. We examined necropsy findings and parasitological data from 89 log- gerhead 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 preva- lence of infection in the stranding population was observed, and most infections were regarded as inci- dental 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 nutri- tionally 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, Caretta- cola, 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.

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.

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.

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.

An outbreak of granulomatous dermatitis was investigated in a captive population of moray eels. The affected eels had florid skin nodules concentrated around the head and trunk. Histopathological examination revealed extensive granulomatous inflammation within the dermis and subcutaneous fascial plane between the fat and axial musculature. Acid-fast rods were detected within the smallest lesions, which were presumably the ones that had developed earliest. Eventually, after several months of incubation at room temperature, a very slowly growing acid-fast organism was isolated. Sequencing of the 16S rRNA gene identified it as a Mycobacterium species closely related (0.59% divergence) to M. triplex , an SAV mycobacterium. Intradermal inoculation of healthy green moray eels with this organism reliably reproduced the lesion. Experimentally induced granulomatous dermatitis appeared within 2 weeks of inoculation and slowly but progressively expanded during the 2 months of the experiment. Live organisms were recovered from these lesions at all time points, fulfilling Koch's postulates for this bacterium. In a retrospective study of tissues collected between 1993 and 1999 from five spontaneous disease cases, acid-fast rods were consistently found within lesions, and a nested PCR for the rRNA gene also demonstrated the presence of mycobacteria within affected tissues.

Serodiagnostic tests for detecting green turtle (Chelonia mydas) antibody responses were developed to test the strength of association between exposure to spirorchid trematode antigens or herpesvirus antigens and having green turtle fibropapillomatosis (GTFP). Plasma samples from 46 captive-reared green turtles, including paired pre- and 1-yr post-inoculation samples from 12 turtles with experimentally induced GTFP, were found by enzyme-linked immunosorbent assay (ELISA) to be negative for antibodies to adult spirorchid (Learedius learedi) antigens. In contrast, all 12 turtles that developed experimentally induced GTFP converted within 1 yr from having negative to positive antibody reactivity to GTFP-associated herpesvirus antigens, whereas the three controls and four turtles that failed to develop tumors remained negative. Plasma samples from 104 free-ranging green turtles from two Florida (USA) coastal feeding grounds with different GTFP prevalences were tested by ELISA for antibodies to L. learedi adult antigens; and there was no statistically significiant association between antibody prevalence and sampling site. When a low optical density cutoff value (0.15) was used to interpret ELISA results, 98% of the turtles from each site were spirorchid antibody-positive and there was no association between antibody reactivity to spirorchids and GTFP status. When a higher negative cutoff value was used, however, a statistically significant association between antibody reactivity to spirorchids and GTFP-free status was found. These results suggest that spirorchids do not have a role in GTFP pathogenesis. All 20 of the tumor-bearing lagoon turtles had antibodies to herpesvirus antigens whereas only two (10%) of the tumor-free reef turtles had detectable anti-herpesvirus reactivity. The strong association between antibody reactivity to herpesvirus antigens and GTFP status in both captive-reared and free-ranging turtles is consistent with the hypothesis that the transmissible agent that causes GTFP is a herpesvirus.

I compared gross pathological effects for 2 methods of placing intraperitoneal telemetry devices in female armadillos (Dasypus novemcinctus). Placing radio transmitters free in the peritoneal cavity resulted in ensheathment by the greater omentum without obvious detrimental effects. The use of retaining sutures required longer incision lengths and failed to hold the device in place in 2 cases. Retaining sutures also resulted in adhesions involving the gastrointestinal tract in 1 case and death following bowel entrapment in another. Two females successfully conceived while carrying free-floating devices and carried their pregnancies to term despite undergoing second surgeries

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.

Plasma and erythrocyte cholinesterase activities were determined for 40 free-living and 12 captive common long-nosed armadillos (Dasypus novemcinctus) in order to establish normal values for monitoring pesticide exposure. Plasma cholinesterase activity ranged from 105 to 549 U/liter with no sexual or seasonal differences. Plasma values from captive animals were significantly lower than those from wild armadillos. Erythrocyte cholinesterase activity ranged from 2,915 to 15,126 U/liter with no differences detected between captive and wild animals or between sexes. However, erythrocyte cholinesterase values varied seasonally. Erythrocyte and plasma cholinesterase activities were not significantly correlated. Packed cell volume ranged from 24 to 51% and did not vary significantly between captive and wild samples, between sexes or among seasons. However, both whole blood and erythrocyte cholinesterase activities showed significant negative correlations with packed cell volume. Controlled experiments are needed to find the factors responsible for the statistically significant difference between plasma cholinesterase activities of captive and wild armadillos. The seasonal variation in erythrocyte cholinesterase activity and the negative correlation between erythrocyte cholinesterase activity and packed cell volume can be explained by an hypothesis that relates the variation in erythrocyte cholinesterase activity to variation in erythrocyte turnover rate. Future work should involve experiments to test this hypothesis.

Nutritional analyses were performed on several fruit species that are eaten by the neotropical frugivorous bat Carollia perspicillata. All essential amino acids assayed were found to be present in fruit pulp. Compared to the relative amounts of essential amino acids required for growth by laboratory rats, methionine and lysine were the most limiting amino acids in fruit protein. Captive bats were fed fruits of either Chlorophora tinctoria or Muntingia calabura so that the digestibilities of pulp nitrogen and gross energy could be determined. Digestibility estimates were lower than those found for Artibeus jamaicensis fed fruits of Ficus insipida, but this was probably caused by methodological differences. The measures of nutrient content and digestibility were used with estimates of daily nitrogen and energy requirements of bats to predict the amount of pulp needed to meet these requirements. Fruits were judged to be adequate nitrogen sources if the amount predicted to satisfy nitrogen requirements was less than that to satisfy energy requirements. By this criterion, most fruits were found to be adequate for maintenance metabolism; only fruit of Piper amalago was adequate for lactating bats. However, when amino acid deficiencies were considered, Piper amalago fruit was adequate only for maintenance metabolism, and no other fruits met this requirement. Insects found in fruit pulp were insufficient to constitute an important nitrogen source. Thus, if insects are important nitrogen sources they must be actively sought by bats. However, this analysis suggests that by selecting certain fruit species, frugivorous bats may not need to supplement their diets with insects. NEARLY ALL SPECIES OF NEOTROPICAL frugivorous bats (Phyllostomidae) studied include insects in their diets (Gardner 1977). The short-tailed fruit bat Carollia perspicillata also ingests insects (Arata et al. 1967, Fleming et al. 1972, Ayala & D'Alessandro 1973, Howell & Burch 1974), but their importance to the diet is unknown. Fleming et al. (1972) found that insects formed on average about 13 percent by volume of stomach contents in a sample of 272 bats whose stomachs contained food, but recently Thomas (1984a) has questioned some of the evidence for voluntary insect eating. An explanation for the inclusion of insects in the diet is that while fruits provide an often abundant and easily handled energy source, they do not provide sufficient nitrogen to meet the dietary requirements of bats (Morrison 1980, Thomas 1984b). Compared with insects, fruits contain very little nitrogen (Morton 1973, White 1974), however it is not clear that fruits are inadequate nitrogen sources. For example, Foster (1978) calculated that some fruits may be adequate nitrogen sources for nestling frugivorous birds. In this paper, I present the results of an investigation of the nutritional contents and digestibilities of some fruits eaten by Carollia perspicillata. The goal of this paper is to assess the value of fruit tissue as the only source of nitrogen for frugivorous bats. Specifically, I test the hypothesis that nitrogen requirements are met before energy requirements when bats ingest enough fruit to meet their energy requirements. MATERIALS AND METHODS The study was carried out from May to August, 1980 and 1981, in Parque Nacional Santa Rosa, Guanacaste Province, Costa Rica. This site was described in detail by Heithaus and Fleming (1978). Many fruit species that are available at this site during the wet season are eaten by Carollia perspicillata (Goodwin & Greenhall 1961, Gardner 1977). I collected sufficient fruit (>20 g dry pulp) for nutritional analyses from the following five plant species: Muntingia calabura (Eleocarpaceae), Chlorophora tinctoria (Moraceae), Cecropia peltata (Moraceae), Ficus ovalis (Moraceae), and Piper amalago (Piperaceae). These species represent roughly half of the wet season fruits reported to be eaten by Carollia in the park and account for about 60 percent of recorded fecal contents (Heithaus & Fleming 1978). Ripe fruits were collected daily and preserved in liquid nitrogen. Individual specimens were randomly sampled from the collected total and the following data were recorded: total fresh weight, seed number, total wet weight of seeds, and the number of insects present in the pulp. Pulp samples were prepared by removing seeds and insects from all fruit except Muntingia. Except for the skins of Muntingia, Ficus, and Cecropia, fruit parts not normally eaten by Carollia, such as the central stems of ' Received 12 November 1984, accepted 25 January 1985. 2 Current address: Department of Ecology and Behavioral Biology, University of Minnesota, Minneapolis, Minnesota 55455, U.S.A. BIOTROPICA 18(1): 39-44 1986 39 This content downloaded from 40.77.167.28 on Fri, 27 May 2016 05:46:25 UTC All use subject to http://about.jstor.org/terms TABLE 1. Physical characteristics of five fruit species. Fresh weight (g) Water content Pulp: seed Fruit species Mean SD N Percenta dry weight ratioa Muntingia calabura 1.41 0.36 120 81.8 (20) 2.33b Chlorophora tinctoria 4.76 1.01 60 79.6 (20) 6.00 (20) Cecropia peltata 4.87 1.61 22 78.1 (22) 0.87 (10) Ficus ovalis 0.82 0.20 130 78.5 (22) 6.53 (22) Piper amalago 1.25 0.69 48 73.0 (20) 0.83 (20) a Overall mean of (N) samples. b Calculated from mean no. of seeds per fruit, mean seed dry weight, and mean whole fruit dry weight. Cecropia and Piper, were also removed (based on personal observations of bats' feeding in captivity and examinations of fruits dropped under night roosts). Samples of Muntingia seeds and skins were analyzed separately and used to correct results from analyses of whole fruits. Pulp and seed samples were dried in an oven to constant weight at 5 5?C. Dried pulp samples were pooled for each species, ground through size 20 mesh in a Wiley mill and analyzed by standard techniques (Association of Official Analytical Chemists 1980). Analyses included: gross caloric content, ash, total nitrogen (Kjeldahl), and neutral detergent fiber. In addition, subsamples (1 g) of all fruit except Muntingia were hydrolyzed and run through an automatic amino acid analyzer using an internal standard of n-leucine. This technique provided quantitative determinations of 17 of the 20 common amino acids. I used the amino acid analyses to calculate the proportion of total nitrogen that is actually in the form of amino acids by summing the amounts of nitrogen present in each amino acid quantity and dividing this by the total (Kjeldahl) nitrogen content. Results of amino acid analyses were also used to assess the quality of fruit protein by comparing the amounts of essential amino acids present (except tryptophan, which was not measured) with the amounts required for a balanced diet. Because no data exist on the amino acid requirements of bats, I used the levels recommended for growth in laboratory rats (Maynard et al. 1979). I conducted digestibility studies on captive bats in order to estimate the efficiency with which bats extract energy and nitrogen from fruit pulp. Eight male bats were captured and held in captivity for 4 days. Six bats were fed fresh fruits of Chlorophora tinctoria and two were fed fruits of Muntingia calabura ad libitum. Each bat was housed in a hardware cloth cage measuring 30 x 30 x 30 cm. To minimize contamination of food by feces, a food dish was suspended on a wire shelf about 15 cm above the cage bottom. Cages were set on aluminum baking pans for the collection of all feces and urine produced each night. The first day of captivity was used as an adjustment period and no feces were collected. On the following three mornings, the feces-urine mixture was collected, weighed to the nearest 0.1 g, and stored in airtight vials in liquid nitrogen. To estimate the wet weight of fruit ingested, uneaten portions of fruit were also weighed and subtracted from the weight of fruit offered. Feces samples were dried to constant weight at 5 50C, pooled for each bat, ground through a size 20 mesh, and subsampled for nitrogen and gross caloric content determinations. The remaining material was pooled among individuals to produce a sample large enough to complete the analysis. Seeds were either removed from the feces before drying (Chlorophora) or were included in analyses and corrected for in later calculations (Muntingia). Dry matter digestibility (DMD) was calculated (on a non-ash-free basis) using the ash tracer method (Johnson & Maxell 1966). Because urine was collected with the feces, corrections for urinary loss of minerals were not necessary. An assumption of this method is that the animals are in mineral balance. This method gave a lower, more conservative estimate than dividing the difference between ingested fruit and feces weights by ingested fruit weight. The partial apparent digestibility (PAD) of a nutritional component was calculated as follows: PD=A, (1 DMD)Af PAD Al(1A, )A (1) A, where A, and Af are the relative amounts of a nutrient in ingesta and feces respectively. These estimates do not yield digestibility per se because urinary losses are included in the feces. Because urinary loss of nitrogen is related to basal metabolic rate rather than intake rate (Pike & Brown 1967), the estimates of nitrogen partial apparent digestibility were corrected as follows: PADcorrected= PAD + EUN/I(N), (2) where PAD = estimated partial apparent digestibility of nitrogen, EUN = endogenous urinary nitrogen loss (g/ Kcal basal metabolic rate), I = observed intake of dry fruit pulp, and N, = the nitrogen content of fruit pulp. These digestibility estimates were used in conjunction with estimated requirements to predict minimum food intake levels for normal and lactating bats.

Three measurements on the left forewing, and head width, were used to predict functional proboscis length of honey bees (Apis mellifera ligustica). Functional length of the proboscis (FLP) is a measure of the depth that the proboscis can be extended to reach nectar in tubular flowers and is operationally the depth of a vertical capillary tube (inside diam = 1mm) emptied of sucrose solution. The morphometric characters were useful predictors of FLP, which may affect honey been foraging behavior.