Visceral leishmaniosis is one of the most important zoonotic diseases on the planet and dogs are the main reservoir of canine visceral leishmaniosis (CVL) in endemic areas. They play an important role in human infection because in dogs the disease appears long time after infection, and they can move uncontrollably, contributing to disperse the parasite. To take the decision to treat the animals or for euthanasia, in an elimination programme, in order to reduce the parasitic load, it is necessary to diagnose correctly, having more effective tools. Our group has developed a new recombinant antigen-based kinesin-related gene of Leishmania braziliensis (Lbk39), which shows 59% amino acid identity to the L. infantum homologue. The Lbk39 gene was synthesized, inserted into the pLEXSY-sat2 vector and transfected into L. tarentolae cells by electroporation. The recombinant protein was secreted in the culture with a C-terminal histidine marker, purified, generating a product at 337.68 μg mL−1. A total of 152 sera from dog's endemic and non-endemic areas were used, being 78 positives and 75 negatives. The antigen Lbk39 showed 100% sensitivity and 96.1% specificity. We compared this antigen with other antigens such as total extract of the parasite, TRDPP, and our data indicate that Lbk39 has potential application in the diagnosis of CVL through antibody detection.

Diagnosis of loiasis and analysis of the specific immune response are limited by a paucity of parasite material. To circumvent this problem, a Loa loa antigen has been expressed in a prokaryote vector (pTrcHis). Immunization of Balb/c mice with this soluble recombinant protein produced a strong antibody response, with antibodies recognizing 2 major bands of 38 and 20 kDa in a native crude extract of Loa loa adult worms and microfilariae on Western blots. The target molecule was located mainly in the hypodermis and cuticle of the adult worm. Analysis of human IgG subclasses against this antigen by enzyme-linked immunosorbent assay (ELISA) showed IgG1, IgG2 and IgG3 but not IgG4 reactivity. IgG2 against this recombinant antigen was 100% specific for loiasis when tested against samples from European donor individuals. The same IgG2 antibodies showed 91% specificity for loiasis by comparison with Wuchereria bancrofti, Onchocerca volvulus, Mansonnella perstans and other helminth infections. Furthermore, the IgG2 antibody level correlated with the density of Loa loa microfilariae (r=0·400; P=0·02). This recombinant 15r3 molecule and specific IgG2 assay may be useful for monitoring control programmes.

The present paper describes two repetitive proteins representing common antigens of African trypanosomes which are non-variant and which are recognized early in infection by the host immune system. These antigens were identified by their ability to immunoreact with bovine serum taken during the early phase of a cyclic trypanosomal infection. Screening of a cDNA library from T. b. gambiense with such early infection serum identified a protein which contains a repetitive motif consisting of 68 amino acid repeat units (GM6). Immunofluorescence and immunogold electron microscopy revealed that GM6 is located on fibres which connect the microtubules of the membrane skeleton with the flagellum. A second repetitive antigen detected by this serum is MARP1 (microtubule-associated repetitive protein 1), a protein previously characterized in this laboratory as a high-molecular weight component of the membrane skeleton, which consists of more than 50 tandemly repeated, near-identical 38 amino acid repeat units. Beta-galactosidase fusion products of both proteins demonstrated a strong immunoreactivity with sera from T. b. brucei and T. congolense-infected cattle. The result from this preliminary immunological evaluation indicates a high immunodiagnostic sensititivy (90%) of the two recombinant antigens which make them interesting candidates for immunodiagnosis of trypanosomiasis in cattle.

A recombinant Echinococcus multilocularis antigen (II/3–10), which had previously been shown to exhibit immunodiagnostic characteristics highly specific for human alveolar echinococcosis, and the corresponding native parasite antigen, were further characterized with immunochemical and molecular biological methods. Immunoblot analysis using a polyclonal antiserum raised in rabbits against the recombinant protein, and subsequent Northern hybridization analysis, revealed that the native antigen was expressed by E. multilocularis at the adult as well as at the metacestode stage. In metacestodes, the antigen was shown by using indirect immunofluorescence and the same antiserum to be localized within the germinal layer and membrane structures of developing protoscolices. Electrophoretic analyses revealed remarkable differences in the apparent molecular weight of the antigen under reducing and non-reducing conditions. In further immunoblot analyses, anti-II/3–10 antibodies identified the corresponding epitopes on bands with identical Mr in all E. multilocularis isolates investigated (European, Asian and North American). By Southern hybridization analyses of the respective gene, phylogenetically related sequences were shown to be present in other helminth species such as E. granulosus and several Taenia spp. In the same respect, immunoblotting revealed that anti-II/3–10 antibodies reacted with antigens of different Mr from various E. granulosus isolates and some other cestode species, indicating the presence of shared and thus potentially cross-reacting epitopes. The relevance of these findings for the immunodiagnostic performance of the recombinant antigen is discussed.

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1·8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.

A novel 28 kDa cysteine protease (Cs28CF) secreted by the hepatobiliary trematode, Clonorchis sinensis was identified. The protease was purified from the excretory-secretory products (ESP) of the adult worm using DEAE-ion exchange and Arginine-Sepharose 4B chromatography. It showed a high activity between pH 6·5 and 7·5 in a dithiothreitol (DTT)-dependent manner. Inhibitors specific to cysteine proteases down-regulated the activity. Addition of Cs28CF to monkey cholangiocyte cultures resulted in approximately 95% cell death after 7 days. The full-length cDNA (1078 bp) encoded a single peptide of 328 amino acids (aa) with an N-terminal hydrophobic sequence, an ERFNAQ motif in the propeptide and a mature domain. Expression of mRNA transcripts of Cs28CF was observed in both the metacercaria and adult stages. Bacterially expressed recombinant protein exhibited a specific antibody reaction with clonorchiasis sera. Deduced aa exhibited 52–76% sequence identity with the cathepsin F analogues from other organisms. A novel E/DXGTA motif was recognized in the propeptide region. Phylogenetic analysis of 63 papain family members revealed that the trematode cysteine proteases formed 2 major clades of cathepsins F and L. The trematode cysteine proteases classified as cathepsin F shared higher homology among themselves than those classified as cathepsin L. Cathepsin F is phylogenetically conserved in the trematode parasites as well as in mammals.

The burrowing mite Sarcoptes scabiei is the causative agent of the highly contagious disease sarcoptic mange or scabies. So far, there is no in vitro propagation system for S. scabiei available, and mites used for various purposes must be isolated from infected hosts. Lack of parasite-derived material has limited the possibilities to study several aspects of scabies, including pathogenesis and immunity. It has also hampered the development of high performance serological assays. We have now constructed an S. scabiei cDNA expression library with mRNA purified from mites isolated from red foxes. Immunoscreening of the library enabled us to clone a full-length cDNA coding for a 102.5 kDa protein. Sequence similarity searches identified the protein as a paramyosin. Recombinant S. scabiei paramyosin expressed in Escherichia coli was recognized by sera from dogs and swine infected with S. scabiei. We also designed a small paramyosin construct of about 17 kDa that included the N-terminal part, an evolutionary variable part of the helical core, and the C-terminal part of the molecule. The miniaturized protein was efficiently expressed in E. coli and was recognized by sera from immunized rabbits. These data demonstrate that the cDNA library can assist in the isolation of important S. scabiei antigens and that recombinant proteins can be useful for the study of scabies.