Development of an indirect immunofluorescence technique for the diagnosis of toxoplasmosis in bottlenose dolphins

Abstract

The diagnosis of toxoplasmosis is often complicated by the lack of specific clinical symptoms or postmortem features, in humans and other animals. The only diagnostic test described so far for the serological diagnosis of Toxoplasma gondii in marine mammals is the modified agglutination test (Dubey et al., Am J Vet Res 48(8):1239-1243, 1987). The development of more sensible and specific immunological techniques requires specific antibodies, which are currently unavailable in the scientific market. Indirect immunofluorescence (IIF) is one of the most widely used methods for the diagnosis of toxoplasmosis in humans (Auer et al., Parasitol Res 12:965-970, 2000). In order to develop and apply this technique to the bottlenose dolphin (Tursiops truncatus), immunoglobulins were firstly purified using ion-exchange chromatography. The purified immunoglobulins were then injected in New Zealand rabbits in order to obtain polyclonal antibodies. These antisera were validated by the IIF technique, using as controls serum samples of dolphins infected by Toxoplasma. The results were visualized using antirabbit IgG labeled with fluorescein. This newly developed and specific serological assay was then tested with the dolphin collection of Loro Parque, Tenerife, Spain (group I), and L'Oceanogràfic of Valencia, Spain (group II). The obtained results in this study showed that none of the dolphins from group 1 were infected by T. gondii and two animals were positive in group 2. Furthermore, we conclude that this study has produced antibodies with high specificity against dolphin immunoglobulins and an IIF method which may be used as immunological diagnostic tools, especially for the serological diagnosis of toxoplasmosis.

Development of an indirect immunofluorescence technique for the evaluation of generated antibody titers against Erysipelothrix rhusiopathiae in captive bottlenose dolphins (Tursiops truncatus)

“…Part of this problem is likely to be the lack of secondary antibodies. The antidolphin IgG generated in rabbits (Bernal-Guadarrama et al 2014) and tested in this study did not show nonspecific or cross-reactions with other pathogens, and erysipelas is the second disease in dolphins with a specific IIF test for its diagnosis.…”

“…The obtained products from each wash were separated by electrophoresis in 10 % polyacrylamide sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis gels using the system Protean III (Bio-Rad) and stained for visualization using Coomassie blue (Merck). After that, tubes containing the higher concentrations of immunoglobulins were selected and were further purified by dialysis using a 16-mm-diameter cellulose membrane (dialysis tubing, cellulose membrane, Sigma, Tres Cantos, Madrid, Spain) with a retaining limit of 12 kDa and treated as indicated by Bernal- Guadarrama et al (2014). Dialysis continued with agitation for 24 h at 4 °C against a dialysis buffer of Tris-HCl 0.1 M (pH 6.5) 0.1 × , changing the buffer three times during the 1 3 process.…”

“…A third rabbit, used as control, was inoculated once with CFA and twice with IFA in a final total volume of 1 ml. Rabbits were sacrificed and bled 45 days after the last injection of immunoglobulin as previously described (Bernal-Guadarrama et al 2014). …”