Coprological examination based on egg detection in stool samples is currently used as the gold standard for the diagnosis of human fascioliasis. However, this method is not effective during the acute phase of the disease and has poor sensitivity during the chronic phase. Serodiagnosis has become an excellent alternative to coprological examination in efforts to combat the effects of fascioliasis on human and animal health. Two novel recombinant Fasciola hepatica proteins, i.e., a ferritin (FhFtn-1) and a tegument-associated protein (FhTP16.5), were used as antigens to develop in-house enzyme-linked immunosorbent assay (ELISA) methods. The assays were optimized and validated using 152 serum samples from humans with a known infection status, including healthy subjects, patients with chronic fascioliasis, and patients with other parasitic diseases. The FhFtn-1 ELISA was shown to be 96.6% sensitive and 95.7% specific; the respective parameters for the FhTP16.5 ELISA were 91.4% and 92.4%. The performances of the FhFtn-1 and FhTP16.5 ELISAs were compared with that of an available commercial test (the DRG test) using a subset of serum samples. Our in-house tests were slightly more sensitive than the DRG test in detecting antibodies against F. hepatica, but the differences were not statistically significant. In conclusion, the present study provides evidence for the potential of the FhFtn-1 and FhTP16.5 ELISAs as diagnostic tools for human fascioliasis, as might be implemented in conjunction with standard assays for large-scale screenings in areas where the disease is endemic and for the detection of occasional cases in clinical laboratories.

ABSTRACTA cDNA encoding a 16.5-kDa protein termed FhTP16.5 was identified by immunoscreening of a cDNA library fromFasciola hepaticaadult flukes using pooled sera from rabbits infected withF. hepaticafor 4 weeks. Quantitative reverse transcriptase PCR (qPCR) analysis revealed that FhTP16.5 is not expressed in unembryonated eggs. It is poorly expressed in miracidia and highly expressed at the juvenile and adult stages; however, significant differences were found between the expression levels of FhTP16.5 in juveniles versus adult flukes. Recombinant FhTP16.5 was expressed at high levels inEscherichia coli, purified by affinity chromatography, and used to raise anti-FhTP16.5 polyclonal antibodies in rabbits. Immunoblot analysis using the anti-FhTP16.5 IgG antibody identified FhTP16.5 in crude and tegumental extracts and in excretory-secretory products ofF. hepatica. The protein was not detected in crude extracts ofSchistosoma mansoniorSchistosomajaponicum. Antibodies to FhTP16.5 were detected in the sera of rabbits at 3 to 12 weeks ofF. hepaticainfection as well as in the sera of humans with chronic fascioliasis; these findings suggest that FhTP16.5 could be a good antigen for serodiagnosis of fascioliasis. Immunohistochemistry demonstrated that FhTP16.5 localizes to the surface of the tegument of various developmental stages and in parenchymal tissues of the adult fluke. Such specific localization makes FhTP16.5 an attractive target for immunoprophylaxis or chemotherapy.

Ferritins are proteins that play a central role in maintaining intracellular iron balance. A cDNA clone of Fasciola hepatica (687 bp long) encoding a putative 228-amino acid polypeptide (FhFtn-1) homologous with ferritins of vertebrates and invertebrates was identified. FhFtn-1 contains a conserved motif of the ferroxidase center typical of vertebrate ferritins. Phylogenetic tree analysis showed that FhFtn-1 clusters with two ferritins of Paragonimus westermani, which suggests a common ancestry for the ferritins of these two trematodes. Recombinant FhFtn-1 protein expressed and purified from an Escherichia coli system showed iron-uptake ability. Moreover, FhFtn-1 showed strong reactivity with sera from rabbits infected with F. hepatica for 2–12 weeks, which suggests that this protein could be a potential antigen for immunodiagnosis of fascioliasis. qPCR analysis demonstrated that FhFtn-1-mRNA is expressed at significantly higher levels in adults and unembryonated eggs than in juveniles or miracidia. These results represent the first characterization of a ferritin protein from the liver fluke F. hepatica.

Fasciola hepatica, is a prevalent and economically important disease in the husbandry industry. A 17KDa protein termed Fh4.26 (Q5I5Y3) was recently identified by means of successive screenings of a cDNA library previously prepared from F. hepatica adult worms using a rabbit anti‐F. hepatica excretion and secretion (E/S) antigens serum and a serum from rabbit with 4 week of F. hepatica infection. Fh4.26 preserves two DM9 Domains of unknown function, this repeats are conserved in some methyltransferases protein. Recombinant Fh4.26 protein was purified by affinity chromatography. The immunodiagnostic potential of the antigen rFh4.26 was assessed by the antibody detection Enzyme‐Linked ImmunoSorbent Assay and Western blot using a large panel of sera from animals infected with the liver fluke F. hepatica. The assay was highly sensitive and revealed that animals infected with F. hepatica develop antibodies against Fh4.26 at early and late stages of infection. Confocal microscopy analysis demonstrated that Fh4.26 is localized on vitellaria cells of the liver fluke. Fh4.26 could be one of the antigens responsible for this maintenance which make it an attractive target for immunoprophylaxis or chemotherapy. This study was approved by IACUC #7870104.