The enzyme-linked immunosorbent assay (ELISA) technique can play an important role in the early detection of fascioliasis. However, they have some diagnostic limitations, including cross-reaction with other helminths. It seems that the combination of recombinant parasite proteins as antigen can reduce these problems. Hence, the present study was aimed to design and confirm the antigenic recombinant multi-epitope (rMEP) construct of three protein epitopes (linear and conformational B-cell epitopes) of the parasite using immunoinformatic tools. For this purpose, the tertiary structures of Fasciola hepatica cathepsin-L1, saposin-like protein 2 and 16.5-kDa tegument-associated protein were predicted using the I-TASSER server. Validation of the modelled structures was performed by Ramachandran plots. The antigenic epitopes of the proteins were achieved by analysing the features of the IEDB server. The synthesized gene was cloned into the pET-22b (+) expression vector and transformed into the Escherichia coli BL21. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to verify and analyse the expression of the rMEP protein. Western blotting was utilized to confirm rMEP protein immunogenicity in two forms, one using an anti-His tag antibody and the other with human pooled sera samples (fascioliasis, non-fascioliasis and negative control sera). Our results demonstrated that the rMEP designed for the three proteins of F. hepatica was highly antigenic, and immune-detection techniques confirmed the antigen specificity. In conclusion, the presented antigenic multi-epitope may be very helpful to develop serodiagnostic kits such as indirect ELISA to evaluate the proper diagnosis of fascioliasis in humans and ruminants.

Leishmaniasis has an important impact on global public health, and the common form of the disease is cutaneous form as well in Iran. Different species of Leishmania parasite make variable clinical manifestations, so prompt diagnosis and recognition at the species level are important due to their impact on the treatment and outcome of the disease. We aimed to examine the potential existence of the Leishmania parasite genome in the exudate materials derived from lesions of the cutaneous leishmaniasis suspected patients referred to Varamin Health Center Laboratory, that were reported negative microscopically. Regarding the object of the study, kDNA-Nested-PCR was used. A 570 bp band equal to what expected for Leishmania major was amplified in 18 out of 29 tested samples (62%). Findings indicate the effectivness of kDNA as a high copy number gene to avoid false-negative results.