Enzyme-linked immunosorbent assay for detecting herpesvirus exposure in Mediterranean tortoises (spur-thighed tortoise [Testudo graeca] and Hermann's tortoise [Testudo hermanni])

Abstract

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies to a herpesvirus associated with an upper respiratory tract disease in Mediterranean tortoises [spur-thighed tortoise (Testudo graeca) and Hermann's tortoise (Testudo hermanni)]. This serodiagnostic test was validated through a hyperimmunization study. The mean of the A(405) readings of the plasma samples collected at time zero of the hyperimmunization study plus three times the standard deviation was used as the cutoff for seropositivity in tortoises. ELISA results were compared to serum neutralization (SN) values for the same samples by using the McNemar test. The results obtained by SN and ELISA were not significantly different (P > 0.05). This new ELISA could be used as an important diagnostic tool for screening wild populations and private and zoo collections of Mediterranean tortoises.

FIG. 1

Western blot comparison of the antigens of HV4295/7R/95 and HV1976 using hyperimmune tortoise plasma. Lanes 1 to 3 were developed using tortoise plasma raised against HV4295/7R/95; lanes 4 to 6 were developed using tortoise plasma raised against HV 1976. Lanes 1 and 4, HV1976; lanes 2 and 5, HV4295/7R/95; lanes 3 and 6, TH-1 cell lysate. Few antibody reactions are detectable, and a very faint band of approximately 20 kDa is observable in the infected cells when stained using anti-HV1976 polyclonal tortoise sera. An antibody reaction with the same molecular mass was also detectable in the HV4295/7R/95-infected cells by using anti-HV4295/7R/95 polyclonal tortoise sera. The arrows show the approximate molecular masses of the different antibody reactions.

FIG. 2

Western blot comparison of the antigens of HV4295/7R/95 and HV1976 using rabbit polyclonal antibodies against HV1976 (STF) (lanes 1, 2, and 3) and HV4295/7R/95 (FOF) (lanes 4, 5, and 6). Lanes 1 and 4, HV1976; lanes 2 and 5, HV4295/7R/95; lanes 3 and 6, TH-1 cell lysate. Several low-molecular-mass antibody reactions can be seen in lanes 1, 2, 4, and 5 and are absent in lanes 3 and 4 (uninfected TH-1 cells). FOF serum was raised against HV4295/7R/95, while STF was raised against HV1976. The arrows show the approximate molecular masses of the different bands.

FIG. 3

ELISA results for tortoises hyperimmunized with HV1976 and HV4295/7R/95 i.n. (A) and i.m. (B). Data shown are A₄₀₅ readings obtained for the plasma samples collected from the tortoises in the hyperimmunization study from time zero to 36 and 34 weeks posthyperimmunization i.n. and i.m., respectively. Each bar corresponds to a single sampling. On the left side of the plotted area of each figure are the results from the ELISA when the tortoise plasma samples were tested against HV4295/7R/95 used as antigen, while on the right side the results are shown when the plasma was tested against HV1976. On the far right of each figure, outside the plotted area, the time sequence of the sampling is reported. The values reported on the y axis are A₄₀₅ readings. On the x axis, the tortoise animal numbers are reported along with the antigens used for the hyperimmunizations (HV4295/7R/95, tortoises no. 1 and 3; HV1976, tortoises no. 2 and 4; PBS, tortoise no. 5). On the top of the graphs, the antigens used in each ELISA are indicated.