The cathepsin-like cysteine peptidases of trematodes of the genus Fasciola.

Fasciolosis caused by trematode parasites of the genus Fasciola is a global disease of livestock, particularly cattle, sheep, water buffalo and goats. It is also a major human zoonosis with reports suggesting that 2.4-17 million people are infected worldwide, and 91.1 million people currently living at risk of infection. A unique feature of these worms is their reliance on a family of developmentally-regulated papain-like cysteine peptidases, termed cathepsins. These proteolytic enzymes play central roles in virulence, infection, tissue migration and modulation of host innate and adaptive immune responses. The availability of a Fasciola hepatica genome, and the exploitation of transcriptomic and proteomic technologies to probe parasite growth and development, has enlightened our understanding of the cathepsin-like cysteine peptidases. Here, we clarify the structure of the cathepsin-like cysteine peptidase families and, in this context, review the phylogenetics, structure, biochemistry and function of these enzymes in the host-parasite relationship.

Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay Kit and In-House Fasciola gigantica Cysteine Proteinases-Based Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Fascioliasis.

Fascioliasis, caused by Fasciola hepatica and Fasciola gigantica infection, is a major food-borne trematodiasis in many places of the world, with the central region of Vietnam being reported as a highly endemic area. Stool examination for Fasciola eggs is not a sensitive method, and immunodiagnostic methods are preferable. We investigated various enzyme-linked immunosorbent assays (ELISAs) to evaluate their efficacy for fascioliasis diagnosis. Test sera used are primarily screened using an ELISA kit produced in Vietnam (VN kit; Viet Sinh Chemical Producing & Trading Co. Ltd., Ho Chi Minh City, Vietnam): Seropositive individuals having symptoms compatible with fascioliasis were regarded as clinically diagnosed fascioliasis cases. A commercial Fasciola IgG ELISA kit from Diagnostic Automation/Cortez Diagnostics, Inc. (USA kit; Woodland Hills, CA), which has been commonly used in Vietnam, was assessed and compared with in-house ELISA systems, including a cystatin-capture (CC) ELISA using crude worm extract (CWE) and an indirect ELISA using a synthetic peptide Ac-TPTCHWECQVGYNKTYDEE-NHMe designed from the F. gigantica cathepsin B (FgCB5) molecule. The USA kit was suitable for routine diagnosis after recalibration of the manufacturer's suggested cutoff point. Cystatin-capture ELISA with CWE provided good sensitivity and specificity with perfect agreement to the results of the USA kit. In dot-blot ELISA, recombinant FgCB5 reacted more strongly with human antisera than did other F. gigantica antigens tested. Enzyme-linked immunosorbent assay using the synthetic peptide fragment of the FgCB5 exhibited nearly 80% sensitivity and specificity, but the test results showed low agreement with CC-ELISA or the USA kit. In conclusion, the commercially available Fasciola IgG ELISA kit from the United States and the in-house CC ELISA using CWE are suitable for practical diagnosis for fascioliasis.

Design and synthesis of a new peptide derived from Fasciola gigantica cathepsin L1 with potential application in serodiagnosis of fascioliasis.

Fascioliasis is a global parasitic disease that affects domestic animals and causes considerable economic losses in the process of domestic animal breeding in endemic regions. The cause of the disease involves a liver trematode of the genus Fasciola, which secretes materials into a host's body (mainly proteins) in order to protect it from the host's immune system. These materials can be involved in the migration, growth, and nutrition of the parasite. Among the expressive proteins of Fasciola, proteases have been introduced as the appropriate targets for diagnosis, treatment, and vaccination against parasites. Cathepsin L (CL) is a member of cysteine proteases; it is widely expressed in the Fasciola species. The aim of this study was to evaluate two synthetic peptides from F. gigantica CL1 for improving serological diagnosis of the Fasciola infection. Therefore, the potential diagnostic value of the surface epitopes of CL1 was assessed using ELISA. In the current study, bioinformatics tools were applied to select two appropriate epitopes of Fasciola Cathepsin L1 as synthetic antigens. Their diagnostic values were evaluated by two methods of indirect ELISA and dot blot analysis. The findings revealed that the first peptide at a dilution ratio of 1:400 and the second peptide at a dilution ratio of 1:100 had the best results and the best concentration of antigens was introduced at 4 µg/ml. Moreover, 191 sera samples were analyzed by both peptides by using the ELISA method, including fascioliasis sera, other parasitic sera and negative sera. The sensitivity of the peptides 1-ELISA and peptide 2-ELISA for the diagnosis of the various cases was 100%. The specificity of the first peptide was 87.3% and its efficacy was determined to be 93.65%. The specificity and the efficacy of the second peptide were 79% and 89.5%, respectively. The positive predictive values of the first and second peptides were obtained to be 86.27% and 79.27% respectively, and the negative predictive values of both peptides was calculated as 100%. In conclusion, the results of this study indicated that the peptide 1 from CL1 may be used as an appropriate antigen for the diagnosis of fascioliasis if the findings are backed up by using other serodiagnostic methods for checking serological cross-reactivity linked to other parasites.

Biochemical and thermodynamic comparison of the selenocysteine containing and non-containing thioredoxin glutathione reductase of Fasciola gigantica.

The thiol-disulfide redox metabolism in platyhelminth parasites depends entirely on a single selenocysteine (Sec) containing flavoenzyme, thioredoxin glutathione reductase (TGR) that links the classical thioredoxin (Trx) and glutathione (GSH) systems. In the present study, we investigated the catalytic and structural properties of different variants of Fasciola gigantica TGR to understand the role of Sec. The recombinant full-length Sec containing TGR (FgTGRsec), TGR without Sec (FgTGR) and TGRsec without the N-terminal glutaredoxin (Grx) domain (∆NTD-FgTGRsec) were purified to homogeneity. Biochemical studies revealed that Sec597 is responsible for higher thioredoxin reductase (TrxR) and glutathione reductase (GR) activity of FgTGRsec. The N-terminal Grx domain was found to positively regulate the DTNB-based TrxR activity of FgTGRsec. The FgTGRsec was highly sensitive to inhibition by auranofin (AF). The structure of FgTGR was modeled, and the inhibitor AF was docked, and binding sites were identified. Unfolding studies suggest that all three proteins are highly cooperative molecules since during GdnHCl-induced denaturation, a monophasic unfolding of the proteins without stabilization of any intermediate is observed. The Cm for GdnHCl induced unfolding of FgTGR was higher than FgTGRsec and ∆NTD-FgTGRsec suggesting that FgTGR without Sec was more stable in solution than the other protein variants. The free energy of stabilization for the proteins was also determined. To our knowledge, this is also the first report on unfolding and stability analysis of any TGR.

Fasciola gigantica cathepsin B5 is an acidic endo- and exopeptidase of the immature and mature parasite.

Cysteine proteases of the liver fluke Fasciola have been described as essential molecules in the infection process of the mammalian host. Destinct cathepsin Bs, which are already expressed in the metacercarial stage and released by the newly excysted juvenile are major actors in this process. Following infection their expression is stopped and the proteins will not be detectable any longer after the first month of development. On the contrary, the novel cathepsin B5 of Fasciola gigantica (FgCB5) described in this work was also found expressed in later juvenile stages and the mature worm. Like all previously described Fasciola family members it was located in the cecal epithelium of the parasite. Western blot analysis of adult antigen preparations detected procathepsin B5 in crude worm extract and in small amounts in the ES product. In support of these data, the sera of infected rabbits and mice were reactive with recombinant FgCB5 in Western blot and ELISA. Biochemical analysis of yeast-expressed FgCB5 revealed that it has properties of a lysosomal hydrolase optimized for activity at acid pH and that it is able to efficiently digest a broad spectrum of host proteins. Unlike previously characterized Fasciola family members FgCB5 carries a histidine doublet in the occluding loop equivalent to residues His110 and His111 of human mature cathepsin B and consequently showed substantial carboxydipeptidyl activity which depends on these two residues.

Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica.

Cathepsin L proteases are a major class of endopeptidases expressed at a high level in Fasciola parasites. Several isoforms of cathepsin L were detected and they may perform different functions during the parasite development. In this study, a complete cDNA encoding a cathepsin L protease was cloned from a newly excysted juvenile (NEJ) cDNA library of Fasciola gigantica and named FgCatL1H. It encoded a 326 amino acid preproenzyme which shared 62.8-83.1% and 39.5-42.9% identity to Fasciola spp. and mammalian cathepsins L, respectively. All functionally important residues previously described for cathepsin L were conserved in FgCatL1H. Phylogenetic analysis demonstrated that FgCatL1H belonged to a distinct group, clade 4, with respect to adult and other juvenile Fasciola cathepsin L genes. FgCatL1H expression was detected by RT-PCR, using gene specific primers, in metacercariae and NEJ, and the expression gradually decreased in advanced developmental stages. A recombinant proFgCatL1H (rproFgCatL1H) was expressed in the yeast Pichia pastoris, affinity purified, and found to migrate in SDS-PAGE at approximately 47.6 and 38.3kDa in glycosylated and deglycosylated forms, respectively. The molecular mass of the activated mature rFgCatL1H in glycosylated form was approximately 40.7kDa. Immunoblotting and immunohistochemistry using rabbit antibodies against rproFgCatL1H showed that FgCatL1H was predominantly expressed in epithelial cells of the digestive tract of metacercariae, NEJs and juveniles of F. gigantica. FgCatL1H could cleave the synthetic fluorogenic substrate Z-Phe-Arg-MCA preferentially over Z-Gly-Pro-Arg-MCA at an optimum pH of 6.5. It also showed hydrolytic activity against native substrates, including type I collagen, laminin, and immunoglobulin G (IgG) in vitro, suggesting possible roles in host tissue migration and immune evasion. Therefore, the FgCatL1H is a possible target for vaccine and chemotherapy for controlling F. gigantica infection.

Characterization and expression of cathepsin B2 in Fasciola gigantica.

Fasciola gigantica cathepsin B belongs to a family of cysteine proteases which is involved in invasion of host tissues. In this study, the recombinant cathepsin B2 (rFgCatB2), synthesized in Pichia pastoris, showed enzymatic activity on a fluorometric substrate Z-Phe-Arg-AMC and gelatin. Furthermore, this recombinant enzyme could degrade IgG and type I collagen. Mouse antiserum against rFgCatB2 reacted with the native FgCatB2 in whole body (WB) extracts of metacercariae (MET), newly excysted juveniles (NEJ) and 2week-old juveniles, but not in 3, 4 week-old juveniles and adult flukes. Immunolocalization showed the presence of cathepsin B2 only in the caecal epithelium of MET, NEJ and 2 week-old juveniles. Co-localization of FgCatB2 and a prominent antigen of NEJ, FgCatB3, revealed that these proteins were expressed at the same regions in the caecal epithelium. Anti-rFgCatB2 showed no cross reaction with the other parasites' antigens by Western blotting. These findings suggest that CatB2 is expressed only in early stages of the parasite and may be involved in digestion of host connective tissues and evasion of the host immune system during their penetration and migration. Thus, CatB2 could be considered as an immunodiagnostic and vaccine candidate for fasciolosis.

Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals.

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-ß, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.

Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes.

Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ∼ 90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ∼ 90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes.

Detoxifying and anti-oxidant enzymes of Fasciola gigantica worms under triclabendazole sulphoxide (TCBZ-SX): an in vitro study.

Triclabendazole (TCBZ) is an efficient fasciolicide that affects both juvenile and adult worms. After oral administration it is rapidly metabolized to TCBZ sulphoxide and sulphone that were found responsible for its fasciolicidal activity. Parasite defense mechanisms include detoxifying and anti-oxidant enzymes that would suppress its oxidative killing. The present work aimed at evaluating these enzymes under TCBZ-SX. Thirty juvenile and 30 adult F. gigantica worms collected from the liver parenchyma and bile ducts formed the subject of the study. Levels of superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), and glutathione (GSH) were measured in juvenile and adult worms incubated, without and with 15 and 50 microg/ml TCBZ-SX for 18 hours at 37 degrees C. Results demonstrated a significant decrease in SOD activity and an increase in GST level in both juvenile and adult worms after incubation in the two concentrations. The remaining enzymes and GSH revealed variable levels.

Immunodiagnostic/protective role of cathepsin L cysteine proteinases secreted by Fasciola species.

Cathepsin L, a major cysteine proteinase secreted by the parasite plays a pivotal role in various aspects of its pathogenecity. The enzyme takes part in nutrient acquisition by catabolizing host proteins to absorbable peptides, facilitates the migration of the parasite through the host intestine and liver by cleaving interstitial matrix proteins such as fibronectin, laminin and native collagen and is implicated in the inactivation of host immune defenses by cleaving immunoglobulins. Recently, Cathepsin L has been shown to suppress Th1 immune response in infected laboratory animals making them susceptible to concurrent bacterial infections. Accordingly, the protease has been recognized as an important target at which parasite intervention strategies should be directed. Fluke Cathepsin L cysteine proteinases are also reported as sensitive and specific markers for the immunodiagnosis of fasciolosis in ruminants. Further, vaccination of laboratory or large animals with these proteases resulted in a significant reduction in fluke burden and/or fecundity.

Fasciola gigantica: purification and characterization of adenosine deaminase.

Nucleotidase cascades (apyrase, 5' nucleotidase, and adenosine deaminase (ADA) were investigated in the parasitic trematode Fasciola gigantica. ADA had the highest activity in the nucleotidase cascades. Adenosine deaminase was purified from F. gigantica through acetone precipitation and chromatography on CM-cellulose. Two forms of enzyme (ADAI, ADAII) were separated. ADAII was purified to homogeneity after chromatography on Sephacryl S-200. The molecular mass was 29 KDa for the native and denatured enzyme using gel filtration and SDS-PAGE, respectively. The enzyme (ADAII) had a pH optimum at 7.5 and a K(m) 1.0 mM adenosine, a temperature optimum at 40 degrees C and heat stability up to 40 degrees C. The order of effectiveness of metals as inhibitors was found to be Hg(2+)>Mn(2+)>Cu(2+)>Ca(2+)>Zn(2+)>Ni(2+)>Ba(2+).

Fasciola gigantica: enzymes of the ornithine-proline-glutamate pathway--characterization of delta1-pyrroline-5-carboxylate dehydrogenase.

Ornithine aminotransferase (OAT), proline oxidase (PO), Delta 1-pyrroline-5-carboxylate reductase (P5CR), and Delta 1-pyrroline-5-carboxylate dehydrogenase (P5CD) were assessed in Fasciola gigantica. All enzymes are involved in the conversion of ornithine into glutamate and proline. High levels of P5CD suggest that the direction of the metabolic flow from ornithine is more toward glutamate than proline. F. gigantica P5CD1 and P5CD2 were separated from the majority of contaminating proteins in crude homogenate using a CM-cellulose column. A Sephacryl S-200 column was employed for P5CD2 to obtain pure enzyme with increased specific activity. The molecular mass of P5CD2 was estimated to be 50kDa using a Sephacryl S-200 column and SDS-PAGE. It migrated as a single band on SDS-PAGE, indicating a monomeric enzyme. P5CD2 had Km values of 1.44mM and 0.37mM for NAD and P5C, respectively. P5CD2 oxidized a number of aliphatic and aromatic aldehydes, where the aromatic compounds had higher affinity toward the enzyme. All amino acids examined had partial inhibitory effects on the enzyme. While 3mM AMP caused 31% activation of enzyme, 3mM ADP and ATP inhibited activity by 18% and 23%, respectively. Apart from Cu2+, the divalent cations that were studied caused partial inhibitory effects on the enzyme.

Comparative molecular analysis of two asparaginyl endopeptidases and encoding genes from Fasciola gigantica.

In this study we describe the first cysteine proteinases of the MEROPS Clan CD family C13 in Fasciola gigantica. Family C13 contains asparaginyl endopeptidases and glycosylphosphatidylinositol-anchor transamidases and is also called the legumain family due to the discovery of the first asparaginyl endopeptidase in a legume. The cDNAs encoding two asparaginyl endopeptidases, FgLGMN-1 and FgLGMN-2, were cloned and used for the analysis of nucleic acid and protein properties. The deduced amino acid sequences showed 47.4% identity to each other and from 42.2 to 51.1% identity to homologs of other trematode species. The catalytic site residues histidine, cysteine and preceding hydrophobic residues, characteristic for the cysteine proteinase families C11, C13, C14, and C25, were found conserved. Northern and reverse transcription PCR analyses demonstrated that the transcriptional products are present in metacercariae, juveniles and adults. RNA in situ hybridization and immunohistochemistry revealed that RNA and protein products of the two genes are specifically expressed in the intestinal epithelium of juveniles and adults. Immune sera of mice infected with F. gigantica reacted with immunoblotted, bacterially expressed recombinant proteins starting 4 weeks after infection. Polyclonal antisera raised against the recombinant proteins detected 40 and 30 kDa antigens, respectively in crude worm protein extracts but not in the excretion-secretion products of adult parasites. Likewise, legumain-specific activity was found in crude worm protein extracts but not in excretion-secretion products. This study elucidates the molecular characteristics of these proteins in F. gigantica and demonstrates differences in the biology between Fasciola and Schistosoma which may prove useful for the development of vaccines against fasciolosis in domestic livestock.

Immuno-diagnosis of bubaline fasciolosis with Fasciola gigantica cathepsin-L and recombinant cathepsin L 1-D proteases.

Fasciola gigantica cathepsin-L cysteine proteinase and recombinant cathepsin L 1-D were assessed for their potential in the immuno-diagnosis of F. gigantica infection in buffaloes. A diagnostic ELISA, based on these two antigens, was developed to detect antibodies against F. gigantica in water buffaloes. Sensitivity of the ELISA was assessed using sera from buffaloes experimentally or naturally infected with F. gigantica from F. gigantica endemic areas and its specificity by probing the sera of the host from F. gigantica non-endemic area. Our earlier studies under experimental setting showed 100% sensitivity of cathepsin-L ELISA in the diagnosis of fasciolosis in buffaloes, with the earliest detection of infection at 4 weeks post-infection. However, under field situation of natural F. gigantica infection, this sensitivity declined to 97.1% but specificity of the test remained 100%. Cross-reactivity of the antigen was checked with Schistosoma indicum, S. spindale, Paramphistomum epiclitum, Gastrothylax spp., Gigantocotyle explanatum, hydatid and Strongyloides papilossus in the bubaline host, naturally infected with these helminths. F. gigantica cathepsin-L and the recombinant cathepsin L-1D does not cross-react with these helminth parasites in natural mono or mixed infection of the host. The present ELISA contributes a relatively sensitive and reliable tool for the early serodiagnosis of bubaline fasciolosis.

Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection.

Cathepsin L cysteine proteinase from Fasciola gigantica was evaluated for its potential in the early prepatent detection of this helminth infection in bovine calves. Five cross-bred bovine calves were experimentally infected with 400 metacercariae/calf and evaluated for anti-cathepsin L antibody response. F. gigantica infection in these calves could be detected 4 weeks post-infection using an ELISA, dipstick ELISA and Western blotting with 100% sensitivity. The antigen was also used to detect F. gigantica field infection in cattle, by screening 256 sera of these animals by an ELISA, which demonstrated an overall infection rate of 26.95%. Preliminary studies showed that F. gigantica cathepsin L cysteine proteinase does not cross-react with Paramphistomum epiclitum, Gigantocotyle explanatum and hydatid cyst antigens. However, extensive studies on the cross-reactivity of this antigen with related helminth parasites of cattle and buffaloes are required, before this antigen can be considered suitable for immuno-diagnosis of fasciolosis in these ruminants.

Evaluation of immunoglobulin G subclass antibodies against recombinant Fasciola gigantica cathepsin L1 in an enzyme-linked immunosorbent assay for serodiagnosis of human fasciolosis.

A cystatin capture enzyme-linked immunosorbent assay (ELISA) using recombinant Fasciola gigantica cathepsin L1 antigen was developed to detect specific immunoglobulin G (IgG) subclass antibodies (IgG1, IgG2, IgG3, and IgG4) and was evaluated for its diagnostic potential for human fasciolosis. In an analysis of the sera of 13 patients infected with F. gigantica, 209 patients with other parasitic infections, 32 cholangiocarcinoma patients, and 42 healthy controls, the IgG4-ELISA gave the highest diagnostic values. The sensitivity, specificity, accuracy, and positive and negative predictive values of this method based on the detection of IgG4 antibody were 100%, 99.3%, 99.3%, 86.7%, and 100%, respectively. The results revealed that restricting the ELISA to the detection of specific IgG4 antibody enhanced the specificity and accuracy for the serodiagnosis of human fasciolosis.

Potent epitopes derived from Fasciola gigantica cathepsin L1 in peptide-based immunoassay for the serodiagnosis of human fascioliasis.

A peptide-based enzyme-linked immunosorbent assay (ELISA) was developed and evaluated for its diagnostic ability to detect human IgG antibodies against Fasciola gigantica cathepsin L1. Two previously identified B-cell epitopes of cathepsin L1 were synthesized as single synthetic peptides (acetyl-DKIDWRESGYVTEVKDQGNC-carboxamide and acetyl-DKIDWRESGYVTELKDQGNC-carboxamide) and their diagnostic potential was evaluated. The peptide-based ELISA was compared with an indirect ELISA with crude excretory-secretory products or with partially purified specific 27-kDa (FG27) antigen from adult F. gigantica. In an analysis of the sera of 13 patients infected with F. gigantica, 212 patients with other parasitic infections, 32 patients with cholangiocarcinoma, and 57 healthy controls, the sensitivity, specificity, accuracy, and positive and negative predictive values of this peptide-based ELISA with both peptides had the same performance and were shown to be 100%, 97.3%, 97.5%, 61.9%, and 100%, respectively. When 4 different ELISAs were compared, the results revealed that the specificity, sensitivity, accuracy, and negative predictive values of all antigens were similar except for the positive predictive value that was highest in the ELISA with the FG27 antigen. These results demonstrated that peptide antigens can be used in the serodiagnosis of human fascioliasis with the additional advantage that they are relatively cheap and easy to produce. This rapid, highly sensitive and specific peptide-ELISA has the potential to be used in future large-scale prevalence surveys throughout Southeast Asia.

Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult.

Proteolytic activity of 0-12 day old eggs, miracidium and adult worm of Fasciola gigantica was assessed and proteases were partially purified by DEAE-Sepharose and CM-cellulose columns. Four forms of protease were separated, PIa, PIb, PIc and PII. Purifications were completed for PIc and PII using Sephacryl S-200 chromatography. A number of natural and synthetic proteins were tested as substrates for F. gigantica PIc and PII. The two proteases had moderate activity levels toward azoalbumin and casein compared to azocasein, while gelatin, hemoglobin, albumin and fibrin had very low affinity toward the two enzymes. Amidolytic substrates are more specific to protease activity. PIc had higher affinity toward BAPNA-HCl (N-benzoyl-arginine-p-nitroanilide-HCl) and BTPNA-HCl (N-benzoyl-tyrosine-p-nitroanilide-HCl) at pH 8.0 indicating that the enzyme was a serine protease. However, PII had higher affinity toward BAPNA at pH 6.5 in the presence of sulfhydryl groups (beta-mercaptoethanol) indicating that the enzyme was a cysteine protease. The effect of specific protease inhibitors on these enzymes was studied. The results confirmed that proteases PIc and PII could be serine and cysteine proteases, respectively. The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000, respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and K(M) of 2 and 5 mg azocasein/mL, respectively. For amidolytic substrates, PIc had K(M) of 0.3 mM BAPNA/mL and 0.5 mM BTPNA/mL at pH 8.0 and PII had K(M) of 0.6 mM BAPNA/mL at pH 6.5 with reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 degrees C and were stable up to 40 degrees C. All examined metal cations tested had inhibitory effects toward the two enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be designated as serine PIc and cysteine PII, respectively.