Identification of Fasciola hepatica recombinant 15-kDa fatty acid-binding protein T-cell epitopes that protect against experimental fascioliasis in rabbits and mice.

Vaccination with fatty acid-binding proteins (FABPs) from Fasciola hepatica has been shown to confer significant levels of protection against challenge infection in mice, rabbits, and sheep. A recombinant 15-kDa FABP (rFh15) has been purified and also shown to be an immunoprotective molecule. From the rFh15 molecule sequence 2, 12- and 10-mer putative T-cell epitopes were identified, the first an Fh15Ta of amino acid sequence IKMVSSLKTKIT, and the second an Fh15Tb of amino acid sequence VKAVTTLLKA. The synthesized oligonucleotides were cloned individually into a pGEX-2TK expression vector. The overexpressed fusion protein was affinity purified using glutathione S-transferase (GST) by competitive elution with excess reduced glutathione. These GST fusion proteins were emulsified in Freund adjuvant for rabbit immunizations or further purified as peptides after digestion with thrombin. The purified 12- and 10-mer peptides were either emulsified in Freund adjuvant for immunizations in rabbits or used in an adjuvant-adaptation (ADAD) system, followed by challenge infection with F. hepatica metacercariae in mice and rabbits. In vaccinated-challenged rabbits, the highest levels of protection were found in those treated with GST-epitopes (Fh15Ta 48.2% and Fh15Tb 59.1% reduction, respectively), as compared to GST-immunized controls. Moreover, those immunized with Fh15Ta had higher (84%) numbers of immature flukes as compared with Fh15Tb (41%) or GST alone (64%). The rabbits immunized with the putative T-cell epitopes in adjuvant had a 13% reduction in flukes in those with Fh15Ta and also were highest with immature flukes (46%). In vaccinated mice challenged with a lethal number of metacercariae, both CD-1 and BALB/c mice treated with complete ADAD-GST-Ta had the highest (40%) survival rates of all groups by 47 days postinfection. Thus the Fh15Ta and Fh15Tb polypeptide epitopes warrant further study as a potential vaccine against F. hepatica. Antibody isotype studies in mice revealed a mixed Thl/Th2 response to vaccination.

Efficiency of the oral, intramuscular and subcutaneous routes for the experimental infection of hamster and sheep with Schistosoma bovis.

The percutaneous administration of cercariae is the usual method for experimental infections with Schistosoma bovis. These procedures are laborious and have important inconveniences when working with a large number of animals, especially if they are ruminants. In the present study, the efficiency of the oral, intramuscular and subcutaneous routes are evaluated by comparison with the percutaneous route in experimental infections with S. bovis. The infections developed in hamsters and sheep were evaluated taking as a basis the parasite burden, the concentrations of eggs in tissues and the levels of anti-Schistosoma antibodies. The oral infection failed in both hamsters and sheep. The administration of the cercariae by the intramuscular route was effective in sheep, developing infections of intensity similar to that of the infections acquired percutaneously. In hamsters, on the contrary, although all the animals developed the infection, they were very little intense. The injection of the cercariae by the subcutaneous route induces acceptable infections in hamsters and can also be an alternative route to percutaneous exposure. The levels of the anti-Schistosoma bovis antibodies detected in hamster and sheep were proportional to the number of worms present, which shows that the humoral response is a good indicator of the intensity of the infections. It can be concluded that the intramuscular route is a good alternative to the percutaneous route for experimental infections of sheep with S. bovis. Likewise, the subcutaneous route can also substitute, with some advantages, the percutaneous infections in hamsters.