Fasciola hepatica hemoglobin: isolation, characterisation and induction of protective immune response in cattle

A hemoprotein released in vitro by Fasciola hepatica was purified by gel filtration chromatography on Sephacryl S200 followed by ion exchange chromatography on DEAE sepharose. Absorption spectra studies characterised the molecule as hemoglobin. N-terminal amino acid sequence analysis revealed no similarity between the F. hepatica hemoglobin and other vertebrate or invertebrate hemoglobins. Immunolocalisation studies demonstrate that the hemoglobin is present in the vitelline glands and excretory tubules of mature flukes. The hemoglobin was shown to be highly immunogenic in F. hepatica infected bovines. Hemoglobin was included in a cattle vaccine trial to determine its immunoprophylactic potential. Vaccination with partially purified hemoglobin (Hf) yielded a significant level of protection (43.8%) against challenge infection. Protective immunity was increased to 72.4% when Hf was combined with the liver fluke cysteine protease CL2. Vaccination with Hf and CL2 also resulted in reduced liver damage as assessed by serum GLDH and 7GT. Furthermore, eggs recovered from both vaccine groups showed reduced viability. This anti-embryonation effect of vaccination was particularly evident in the group that received Hf / CL2 where >98% of recovered eggs did not embryonate to miracidia. Although both vaccine preparations induced high antibody titres which were boosted following the challenge infection, there was no correlation between antibody titre and protection. The results of these trials demonstrate that Hf and CL2 could form the basis of a molecular vaccine that would not only reduce parasite burden but would also prevent transmission of liver fluke disease. Using sera from cattle vaccinated with Hf to screen an adult F. hepatica cDNA library, genes encoding p tubulin and the novel antioxidant, peroxiredoxin were isolated. The presence of these proteins in the immunising fraction may have contributed towards the induction of the protective response. Peroxiredoxin activity was demonstrated in fluke extracts as the ability to protect glutamine synthetase from oxidative damage by mixed iron thiol oxidation systems. The antioxidant enzyme may play an important role in protecting against oxidative stress in the fluke