Identification and expression of Fasciola gigantica thioredoxin

In the present study, a cDNA encoding Trx from F. gigantica (FgTrx) was cloned by polymerase chain reaction (PCR). The sequence of FgTrx, analyzed by BLAST, SignalP, and ClustralW programs, showed 315 bp of an open reading frame (ORF), 12 bp 5’UTR, 78 bp 3’UTR, and the putative FgTrx peptide comprising of 104 amino acids, with a molecular weight of 11.68 kDa, with the active site containing five amino acids (tryptophan, cysteine, glycine, proline, cysteine) with a conserved dithiol motif from the two cysteines, and pI 5.86. The peptide had no signal sequence; hence, it was not a secreted protein. The recombinant FgTrx was expressed in Escherichia coli BL21 (DE3) and used for production for a polyclonal antibody in rabbits (anti-rFgTrx). The FgTrx protein expression, estimated by indirect ELISA using the rabbit anti-rFgTrx as probe, showed high levels in eggs, 2- and 4-week-old juveniles, and adult parasite. In a functional test, the rFgTrx exhibited specific activity that could be suppressed by an inhibitor (PX12). When tested by immunoblotting and immunohistochemistry, rabbit anti-rFgTrx reacted with natural FgTrx at a molecular weight of 11.68 kDa from eggs, metacercariae, NEJ, 2- and 4-week-old juveniles, and adult F. gigantica. The FgTrx protein was distributed at high levels in the tegument of 2- and 4-week-old juveniles, and the tegument, parenchyma, eggs, and reproductive organs of adult parasites. FgTrx may be one of the major factors acting against oxidative stresses that can damage the parasite; hence, it could be considered as a novel vaccine or drug target.     

Vaccine potential of recombinant saposin-like protein 2 against Fasciolosis gigantica in mice

Saposin-like protein 2 (SAP-2) is a protein that adult of Fasciola spp. use to lyse plasma membrane of red blood cells, so that their contents can be digested by proteases for the parasites’ nutrients. Thus SAP-2 is a plausible target for vaccination against these parasites. Recombinant Fasciola gigantica saposin-like protein 2 (rFgSAP-2) was expressed in Escherichia coli BL21 (DE3). A vaccination was performed in ICR mice (n = 10) by subcutaneous injection with 50 μg of rFgSAP-2 combined with Freund's adjuvant. At 2 weeks after the second boost, mice were infected with 30 F. gigantica metacercariae by oral route. The percentages of protection of rFgSAP-2 vaccine against F. gigantica were estimated to be 76.4–78.5% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. The antibodies in immune sera of vaccinated mice were shown by immuno-blotting to react with native FgSAP-2 in the extract of 2- and 4-week-old juvenile parasites. By determining the levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune responses, it was found that both Th1 and Th2 humoral immune response were significantly increased in rFgSAP-2 immunized group compared with the control groups, with higher levels of Th2 (IgG1) than Th1 (IgG2a). The levels of serum aspartate aminotransferase (AST) and alanine transaminase (ALT) in rFgSAP-2-immunized group showed no significant difference from those of the non-immunized and infected group, indicating that early juvenile parasites induced liver parenchyma damage, even though the numbers of worm recoveries were significantly different. This study indicates that rFgSAP-2 has a high potential as a vaccine candidate against F. gigantica in mice, and this potential will be tested in larger economic animals.     

Expression and characterization of glutathione peroxidase of the liver fluke,Fasciola gigantica

Parasitology Research - Glutathione peroxidase (GPx) is a key member of the family of antioxidant enzymes in trematode parasites including Fasciola spp. Because of its abundance and central role as...     

Immunization with recombinant leucine aminopeptidase showed protection against Fasciola gigantica in mice

Leucine aminopeptidase (LAP) is expressed in all stages of Fasciola gigantica and, hence, is considered as a potential vaccine candidate. In this study, we have tested a vaccine potential of LAP and the types of immune responses it elicited in vaccinated mice. Recombinant F. gigantica leucine aminopeptidase (rFgLAP) was expressed in Escherichia coli, BL21 (DE3). The imprinting control region mice subcutaneously immunized with 50 μg of rFgLAP combined with Freund's adjuvant (n?=?10) exhibited a significant reduction in worm recoveries when compared with non-immunized and Freund's adjuvant controls at 60.8 and 64.3 %, respectively, and both T helper (Th)1 and Th2 humoral immune responses were elicited in the hosts as reflected by the levels of IgG1 and IgG2a, with Th2 predominating. The levels of IgG1- and IgG2a-specific antibodies to rFgLAP were inversely and significantly correlated with the numbers of worm recoveries. The rFgLAP-vaccinated mice showed significantly reduced levels of serum glutamic oxaloacetic transaminase and serum glutamic pyruvic transaminase and liver damage. These indicated that rFgLAP has a potential as a vaccine candidate against F. gigantica, whose efficacy will be studied further in economic animals including cattle, sheep, and goat.