Short report: Immunodiagnosis of human fascioliasis using recombinant Fasciola hepatica cathepsin L1 cysteine proteinase.

Abstract

Abstract. Our laboratory recently developed a diagnostic test (ELISA) for human fascioliasis based on the detection of serum IgG4 antibodies reactive with Fasciola hepatica cathepsin L1 (CL1). In the present study, we have used recombinant CL1, generated by functional expression of the cDNA in Saccharomyces cerevisiae, in this immunodiagnostic test and compared its performance with native CL1. Sera obtained from 64 individuals living in Cutusuma village in the northern Altiplano of Bolivia, a region with a high prevalence of human fascioliasis, were analyzed by the IgG4-ELISA. A highly statistically significant correlation (r 2 ϭ 0.751, P Ͻ 0.001) was demonstrated between the absorbances obtained using the recombinant and native proteins. These assays showed that 38 (59%) of the individuals tested were seropositive for fascioliasis, whereas only 26 of them were coprologically positive for F. hepatica eggs. All seronegative patients were also coprologically negative. Serum from individuals infected with schistosomiasis mansoni, cysticercosis, hydatidosis, and Chagas disease did not contain antibodies reactive with the recombinant or native CL1. Therefore, recombinant CL1 shows excellent potential for the development of the first standardized assay for the sensitive and specific diagnosis of human fascioliasis. Finally, our data supports earlier reports on the high prevalence of human fascioliasis in the Bolivian Altiplano, which collectively suggest that the disease has been endemic there for more than a decade.Fascioliasis is caused by liver flukes of the genus Fasciola. Although the disease is predominantly one of domestic animals such as sheep and cattle, it is now emerging as an important chronic disease of humans. 1-3 Recent estimates suggest that as many as 2.4 million people are infected with the parasite worldwide and 2.4 million are at risk.3,4 The disease is particularly prevalent among the Aymaran people of the northern Altiplano region of Bolivia, with incidences of up to 60% being reported. 5-8 Our laboratory recently developed a simple ELISA for the detection of serum IgG4 antibodies reactive with F. hepatica cathepsin L1 (CL1) proteinase. 7 The use of purified CL1 antigen in this ELISA provided a more specific immunodiagnosis of human fascioliasis than tests that use parasite somatic or excretory/secretory (ES) antigen.We have also recently reported the expression of functionally active recombinant F. hepatica CL1 in the yeast Saccharomyces cerevisiae. 9 In the present study, we examined the potential of this recombinant protein as a diagnostic reagent for human fascioliasis in the IgG4-ELISA and compared the performance of the antigen with native CL1 antigen. Recombinant CL1 was purified from yeast culture medium by gel filtration chromatography on Sephacryl S200 HR, 9 and native CL1 was purified from ES products by a combination of gel filtration chromatography on Sephacryl S200 HR and ion exchange chromatography on QAE-Sephadex. 10 The concentration of each antigen preparation was measured using...

Detection of Fasciola hepatica and Fasciola gigantica common and uncommon antigens, using rabbit hyper immune serum raised against their excretory–secretory and somatic antigens

“…The use of specific antigens, purified from somatic or ES products of Fasciola spp. in ELISA or immunoblotting system, yielded satisfactory sensitivity and specificity as reported in different studies (O'Neill et al 1999;Rokni et al 2003;Espinoza et al 2007).…”

“…Among the subunit antigens, the Cathepsin-L, a component of Fasciola ES antigens, has received the most attention (O'Neill et al 1999;Rokni et al 2002). It is obvious that nature and quality of purified antigen contribute to the validity and value of serological diagnostic tests.…”

Preliminary antigenic characterisation of an adult worm vomit preparation of Fasciola hepatica by infected human sera

“…Other antigens are gut-associated highly glycosylated proteins 1 and yet others are cysteine proteinases 3,5,6,9,26,27 successfully used in immunodiagnosis 4,11,21,29 . However, specific bands within the range of 23-30 kDa were not detected in our FhTSE, perhaps due to non use of purification procedures.…”

Humoral response (IgG) of goats experimentally infected with Fasciola hepatica against cysteine proteinases of adult fluke

“…A protein band of 28 kDa present in F. hepatica adult preparations was immunodominant in cattle naturally exposed to the parasite [25]. Purified cysteine proteases of F. hepatica have been used in highly sensitive and specific ELISA for detecting human fasciolosis [6,24].…”

“…They have been used as antigens in the serodiagnosis of human and animal fasciolosis (in cattle, sheep and some camelids such as alpacas) [6,8,9,23], with an excellent potential for the sensitive and specific detection of the disease. In fact, serum from patients infected with Schistosoma mansoni, cysticercosis, hydatidosis and Chagas disease does not cross-react with Fasciola hepatica proteinases [24].…”