Strongyloides venezuelensis is a parasitic nematode of rodents that is frequently used to obtain heterologous antigens for immunological diagnosis of human strongyloidiasis. The aim of this study was to identify antigens from filariform larvae of S. venezuelensis for immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from filariform larvae of S. venezuelensis were obtained in phosphate saline (SS and SM) and in Tris–HCl buffer (TS and TM), and were analysed by Western blotting. Different antigenic components were recognized by IgG antibodies from the sera of strongyloidiasis patients. Highest recognition was observed for a 30–40 kDa mass range present in all antigenic fractions. The band encompassing this mass range was then excised and subjected to mass spectrometry for protein identification. Immunoreactive proteins identified in the soluble fractions corresponded to metabolic enzymes, whereas cytoskeletal proteins and galectins were more abundant in the membrane fractions. These results represent the first approach towards identification of S. venezuelensis antigens for use in immunodiagnostic assays for human strongyloidiasis.

Definitive diagnosis of strongyloidiasis in humans is typically achieved by detection of larvae in fecal samples. However, limitations on sensitivity of parasitological methods emphasize the need for more robust diagnostic methods. The aim of this study was to compare the diagnostic value of three methods: eggs per gram of feces (EPG), coproantigen detection by enzyme linked immunosorbent assay (ELISA), and DNA detection by conventional polymerase chain reaction (PCR). The assays were performed at 0 and 5, 8, 13, 21 and 39 days post-infection (dpi) using fecal samples from experimentally infected immunocompetent and immunosuppressed rats. In immunocompetent rats, eggs were detected in feces on days 5, 8 and 13 dpi; coproantigen detection and PCR amplification were successful at all post-infection time points (5, 8, 13, 21 and 39 dpi). In immunosuppressed rats, eggs were detected at 5, 8, 13 and 21; coproantigen detection and PCR amplification were successful at all post-infection time points. In conclusion, these results suggest that coproantigen detection and PCR may be more sensitive alternatives to traditional methods such as EPG for diagnosis of Strongyloides venezuelensis infection.

An estimated quarter of the world's population possesses an infection caused by gastrointestinal nematodes, which induce a Th2 type immune response. Concomitant infection of nematodes with Mycobacterium tuberculosis, which induces a predominantly Th1 type response, is very frequent in tropical and subtropical regions. This study examined immune responses of BALB/c mice infected with Strongyloides venezuelensis and then co-infected with Mycobacterium bovis. The number of worms in the intestine, eggs in feces, cytokine production in lungs and intestine and the expression of CD80, CD86, CTLA-4 and CD28 cell markers on pulmonary cells were analysed. Our results indicate that co-infected mice had an increased parasite burden, which correlates with elevated IFN-γ and IL-10 cytokine production and decreased IL-4 and IL-13. Moreover, decreased expression of CD80 and increased expression of CTLA-4 were observed in co-infected mice. Our data point out that susceptibility to Strongyloides venezuelensis infection is increased by Mycobacterium bovis co-infection, resulting in higher parasite survival.

Development of exact doses (<100) of Strongyloides venezuelensis third-stage larvae in adult Wistar rats was insignificant (mean proportion of 0·076 of the dose at day 8, n = 16) compared with a homogonic strain of S. ratti (0·538, n = 6; 0·726, n = 6) and heterogonic S. ratti (0·681, n = 6). Newly-weaned Wistars allowed development of a mean proportion of S. venezuelensis of 0·298 (n = 4) compared with 0·013 (n = 4) of the same sample of larvae in adult hosts. Experiments with 75Se-labelled larvae established that S. venezuelensis effectively failed to migrate from skin to intestine in adult animals, while mean proportions of 0·141 (n = 5) and 0·138 (n = 4) of the label was found in the intestines of newly-weaned rats 72 h after skin application. Labelled larvae of homogonic S. ratti migrated equally well in both age groups of host (0·350 and 0·358 in 12- and 3-week-olds respectively). Adult S. venezuelensis transferred surgically to the intestines of previously uninfected full-grown Wistars survived over a 21-day period to the same extent as either strain of S. ratti. Resistance of Wistar rats to S. venezuelensis therefore appears to affect the migratory stage preferentially. S. venezuelensis developed better in mature PVG inbred rats (mean = 0·301, n = 20). Studies of S. ratti showed that infections of both strains initiated by exact (<100) doses in Wistar rats had decayed to insignificance between days 26 and 32. The rate of loss of adults of the heterogonic strain was significantly greater than that for the homogonic. The egg content of worms declined as infection progressed and rats were idiosyncratic in their influence on parasite reproduction from the earliest time of sampling (8 d). It was established that ‘autoinfection’ was an unlikely feature of the biology of homogonic S. ratti following the surgical transfer of 450 first-stage larvae to the intestines of 8 adult Wistar rats. No evidence of infection appeared in the guts of these animals 8 days post-transfer. The significance of these results in terms of the biology of Strongyloides spp. naturally occurring in the rat is discussed.

Free-living infective larvae Strongyloides venezuelensis were cultured in Dulbecco's modified Eagle's medium at 25 and 37 °C, and development to the parasitic stage was evaluated using morphological, protein and antigenicity criteria. Few larvae cultured at 25 °C showed development whereas, in most of the larvae cultured at 37 °C, there appeared characteristic changes such as a bulb-like head and droplets under the cuticle with an increase of body width of the larvae. The results obtained from two-dimensional polyacrylamide gel electrophoresis(2D–PAGE) revealed that the protein spot patterns of the larvae cultured at 25 and 37 °C were differentiated by 17 specific spots. In addition, Western blot analysis combined with 2D–PAGE for reaction with serum obtained from an infected rat revealed that protein spots showing immunodominant antigen at 37 °C were almost the same as those of the larvae recovered from the rats rather than those of the larvae at 25 °C. These results strongly suggested that a temperature shift from 25 to 37 °C has an important role in the development of free-living infective larvae to the parasitic stage of S. venezuelensis. The culture system established in the present study was useful for biological and biochemical studies in the development from/of the free-living to the parasitic stage of Strongyloides species.

The cuticle of adult forms of Strongyloides venezuelensis was studied by routine transmission electron microscopy, conventional freeze-fracture and also using quick-freeze and deep-etch techniques. In routine thin sections the cuticle of S. venezuelensis comprises 7 layers: epicuticle, outer cortical, inner corticcal, external medial, internal medial, fibrous and basal. Observation of replicas of specimens fractured across the thickness of the body wall, revealed at the epicuticle an ordered array of particles accompanying the cuticular annulations. At the level of the cortical and medial layers we observed few scattered particles embedded in an amorphous matrix without a particular arrangement. The fibrous layer was represented by several parallel lines of ordered particles of similar size. In tangentially fractured specimens, the epicuticle cleaves readily exposing 2 faces, one exhibiting intramembranous particles without any particular arrangement, immersed in a smooth matrix (P face), and the other showing depressions and very few particles (E face). In replicas of fractures submitted to etching, we observed at the level of the cortical, medial fibrous and basal layers an interconnecting fibrous and globous structure which was organized in a different direction at the fibrous layer. The association of freeze-fracture to deep-etch technique revealed the internal structural organization of the cuticle layers showing details that were not seen before using conventional freeze–fracture technique.

Infective larvae of an intestinal nematode, Strongyloides venezuelensis, enter rodent hosts percutaneously, and migrate through connective tissues and lungs. Then they arrive at the small intestine, where they reach maturity. It is not known how S. venezuelensis larvae develop during tissue migration. Here we demonstrate that tissue invasion ability of S. venezuelensis larvae changes drastically during tissue migration, and that the changes are associated with stage-specific protein expression. Infective larvae, connective tissue larvae, lung larvae, and mucosal larvae were used to infect mice by various infection methods, including percutaneous, subcutaneous, oral, and intraduodenal inoculation. Among different migration stages, only infective larvae penetrated mouse skin. Larvae, once inside the host, quickly lost skin penetration ability, which was associated with the disappearance of an infective larva-specific metalloprotease. Migrating larvae had connective tissue migration ability until in the lungs, where larvae became able to settle down in the intestinal mucosa. Lung larvae and mucosal larvae were capable of producing and secreting adhesion molecules.

The parasitic female of Strongyloides venezuelensis keeps invading the epithelial layer of the host intestinal mucosa. Upon invasion, it adheres to the surface of the intestinal epithelial cells with adhesion molecules secreted from the mouth. It has been demonstrated that S. venezuelensis are expelled from the intestine because mucosal mast cells inhibit the attachment of adult worms to the mucosal surface. In the present study, we generated specific antibodies against secreted adhesion molecules to investigate their function in vivo, because these molecules have been demonstrated only in vitro in spite of the importance in the infection processes. A mouse monoclonal antibody specific to S. venezuelensis adhesion molecules inhibited the attachment of adult worms to plastic dishes and the binding of adhesion molecules to rat intestinal epithelial cells. Immunohistochemical study revealed that adhesion molecules were produced by oesophageal glands and were continuously secreted in vivo to line the wall of the tunnels formed by adult worms in the intestinal mucosa. Our findings indicate that adhesion molecules play essential roles in the infection processes of S. venezuelensis in the host intestine.

The time-course of differentiation/proliferation of mast cells in gut epithelium was investigated in mice infected with the nematode Strongyloides venezuelensis. After infection, expression of proliferating cell nuclear antigen increased in gut intraepithelial mast cells on days 7 to 11, followed by an increase in the number of intraepithelial mast cells from days 11 to 14. Mast cell precursors were defined as cells that formed mast cell colonies in methylcellulose culture. After infection, the numbers of mast cell precursors in the population of gut intraepithelial mononuclear cells (IEMNC) increased significantly on day 3 and returned to the pre-infection level by day 7. Mast cell precursors in Peyer's patches, mesenteric lymph nodes (MLN), and spleen also increased from day 7 p.i. Production of IL-3 and IL-4 in MLN and spleen were increased between 7 and 11 days p.i. These results show that murine intestinal mastocytosis is initiated by an early increase in mast cell precursor number in the gut epithelium followed by proliferation/differentiation of mast cells. Mast cell precursor numbers increased even before the production of IL-3 and IL-4 in MLN and spleen, suggesting that some local factors might be involved in this phenomenon.