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

Abstract

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.

FIG. 1

Antibodies to LETV in plasma from captive-reared and wild green turtles by ELISA. Plasma from 42 captive-reared turtles with no known exposure to herpesviruses and from 30 captive-reared turtles with experimentally induced FP had low optical density values (seronegative). Plasma from 27 wild FP-positive and 19 wild FP-negative turtles had higher ELISA values, regardless of FP status. The cutoff of 0.310 (dashed line) was used to calculate seroprevalence. Plasma samples were tested at a 1:25 dilution. Positive control: mean A₄₀₅ = 1.874, SE = 0.059; negative control: mean A₄₀₅ = 0.007, SE = 0.005, 10 replicates.

FIG. 2

Western blot confirms presence of antibodies to LETV in green turtle plasma samples with the highest optical density values in the ELISA. LETV (10 μg) was used as the antigen in odd-numbered lanes. Cell lysate (10 μg) was used as the control antigen in even-numbered lanes. Lanes 1 and 2, preimmune 99C-1 plasma; lanes 3 and 4, 2-month postimmune plasma; lanes 5 and 6, FP-positive plasma; lanes 7 and 8, FP-negative plasma. Lane MW, molecular size markers (in kilodaltons).

FIG. 3

Immunohistochemistry confirms presence of antibodies to LETV in green turtle plasma samples with the highest optical density values in the ELISA. Plasma samples from FP-positive and FP-negative turtles and from the preimmune (negative control) and 2-month postimmunized (positive control) 99C-1 turtle were incubated with LETV-infected and uninfected TH-1 monolayers. Magnification, ×400.