Comparative evaluation of Schistosoma mansoni, Schistosoma intercalatum, and Schistosoma haematobium alkaline phosphatase antigenicity by the alkaline phosphatase immunoassay (APIA).

To know if alkaline phosphatase (AP) from schistosomes other than Schistosoma mansoni can be used as diagnostic marker for schistosomiasis in alkaline phosphatase immunocapture assay (APIA), we comparatively tested n-butanol extracts of adult worm membranes from a Venezuelan (JL) strain of S. mansoni (Ven/AWBE/Sm); a Cameroonian (EDEN) strain of Schistosoma intercalatum (Cam/AWBE/Si) and a Yemeni strain of Schistosoma haematobium (Yem/AWBE/Sh). APIA was evaluated with sera of patients from Venezuela, Senegal, and Gabon infected with S. mansoni, from Gabon infected with S. intercalatum or S. haematobium, from Chine infected with Schistosoma japonicum and from Cambodian patients infected with Schistosoma mekongi. Results indicate that 92.5% (37/40) of Venezuela sera, 75% (15/20) of Senegal sera, 39.5% (17/43) of S. haematobium sera, and 19.2% (5/26) S. intercalatum sera were APIA-positive with the Ven/AWBE/Sm preparation. APIA with the Cam/AWBE/Si preparation showed that 53.8% of S. intercalatum-positive sera had anti-AP antibodies, and 51.2% S. haematobium-positive sera cross-immunocapturing the S. intercalatum AP. APIA performed with Yem/AWBE/Sh showed that 55.8% S. haematobium sera were positive. Only two out of nine S. japonicum sera were APIA-positive with the Ven/AWBE/Sm and Cam/AWBE/Si, and no reaction was observed with Cambodian S. mekongi-positive sera. AP activity was shown to be present in all the schistosome species/strains studied. The use of APIA as a tool to explore the APs antigenicity and the presence of Schistosoma sp. infections through the detection of anti-Schistosoma sp. AP antibodies in a host, allowed us to demonstrate the antigenicity of APs of S. mansoni, S. intercalatum, and S. haematobium.

Detection of the Sm31 antigen in sera of Schistosoma mansoni- infected patients from a low endemic area.

Schistosoma mansoni cathepsin B (Sm31) is a major antigen from adult worms that circulates in the blood of infected patients (Li et al., Parasitol Res 1996; 82: 14-18). An analysis of the Sm31 sequence (Klinkert et al., Mol Biochem Parasitol 1989; 33: 113-122) allowed the prediction of seven hydrophilic regions that were confirmed to be exposed on the surface of a 3D model of Sm31; the species specificity of these regions was checked using BLAST analysis. The corresponding peptides were chemically synthesized in polymerazible forms using the t-Boc technique. Rabbits developed a high humoral response against these peptides as tested by a multiple antigen blot assay; it recognized native Sm31 in crude S. mansoni extracts and as circulating antigen in sera of S. mansoni-infected patients by western blot. Relevant antigenic determinants were located at the N- and C-terminus sequences. Antibodies against these regions recognized the native enzyme in an ELISA-like assay called cysteine protease immuno assay in which the immunocaptured enzyme was revealed by the intrinsic cathepsin B hydrolytic activity of Sm31. The method successfully and specifically detected Sm31 in sera of infected individuals, most of them (83.3%) with light infections, offering a rationale for the development of parasite enzyme capture assays using anti-synthetic peptide antibodies for possible use in the diagnosis of schistoso,iasis.

Immunogenicity of synthetic peptides from the Sm31 antigen (cathepsin B) of the Schistosoma mansoni adult worms.

The chemical synthesis of peptides may simplify the production of molecules for diagnosis of Schistosoma mansoni. Seventeen polymeric, 20-amino acids long, peptides comprising the entire Sm31 molecule of the adult worm, were synthesized under the t-boc strategy and their immunogenicity was evaluated. Of these, 10 peptides were immunogenic in rabbits. The peptides containing the sequence Gly74-Ser93 (peptide IMT-172) and the sequence Val154-Ala173 (peptide IMT-180) were responsible for the recognition of the Sm31 molecule by Western blot. This was confirmed by the specific inhibition of recognition of each molecule with the homologous peptide. Additionally, antibodies against these peptides strongly fixed to the adult worm gut. The present results, together with the strong immunogenicity shown for the adult worm 31 kDa antigen, establish the basis for the development of an immunodiagnostic method using synthetic peptides.

Enzyme activities in Schistosoma mansoni soluble egg antigen.

Schistosoma mansoni egg antigens are mostly responsible for the granulomatous pathology in human intestinal schistosomiasis. Several previous studies have indicated that the induction of an immune response against some parasite enzymes may protect against pathology. The present work was designed to identify enzyme activities present in a standard soluble egg antigen (SEA) preparation. Simple colorimetric analyses were performed incubating SEA with 2-naphthyl, 2-naphthylamide (2NA), or p-nitrophenyl substrates at different pHs in the absence of added effectors. Results showed prominent acid phosphatase (pH 5.4), alkaline phosphatase (pH 8.5), and N-acetyl-beta-glucosaminidase (pH 5.4) activities. Relevant peptidase activities were also detected at pH 6.5-7.5 against 2NA derivatives of (1) aliphatic (alpha-Ala > beta-Ala > Leu > Met > S-benzyl-Cys), polar (Ser > Gln), basic (Arg > Lys > ornithine), and acidic (Glu) amino acids; (2) dipeptides: X-Ala (X = Gly > Leu > Lys > Asp), X-Arg (X = Ala > Arg > Phe > Gly > Pro > Asp), Ser-Met, and Phe-Pro; and (3) tripeptides (Ala-Phe-Pro > Phe-Pro-Ala). The data demonstrated that S. mansoni SEA contains a rich set of hydrolases with different specificities that might play a role in the egg physiology and possibly also in the host-parasite relationships.