Detection of antibodies to a disease-associated herpesvirus of the green turtle, Chelonia mydas.

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.

Survey of Florida green turtles for exposure to a disease-associated herpesvirus.

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.

Persistent infectivity of a disease-associated herpesvirus in green turtles after exposure to seawater.

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.

Serological association between spirorchidiasis, herpesvirus infection, and fibropapillomatosis in green turtles from Florida.

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.

Cutaneous fibropapillomas of green turtles (Chelonia mydas).

Six juvenile green turtles (Chelonia mydas) from the Indian River Lagoon System, Florida, U.S.A., with multiple cutaneous fibropapillomas, were kept in isolation and examined over a 6-month period. Histologically, the fibropapillomas consisted of a slightly to moderately hyperplastic epidermis overlying a thickened hypercellular dermis. In the earliest lesions, ballooning degeneration was present predominantly in the stratum basale where rete ridges extended into the dermis; aggregates of mixed inflammatory cells were present around dermal vessels. As the lesions matured, they developed an arborizing, papillary pattern. More mature lesions had a less verrucous, often ulcerated surface, with the dermis composed primarily of large collagenous fascicles and relatively few fibroblasts. While numerous trematode eggs were present within dermal capillaries of a histologically similar biopsy specimen from an Hawaiian green turtle, no trematode eggs were observed in any of 28 biopsies examined from the six Florida green turtles in this study. Low stringency Southern blot hybridization and a reverse Southern blot failed to demonstrate papillomavirus DNA in any of the samples extracted. Ultrastructural evaluation of the earliest lesions demonstrated membrane-bound intracytoplasmic vacuoles within epidermal cells in the stratum basale. Similar vacuoles were also observed in the epidermal intercellular spaces and within the dermis. Occasionally, particles with electron-dense centres and measuring 155 to 190 nm were observed in these vacuoles.