Identification of free and conjugated ecdysteroids in cercariae of the schistosome Trichobilharzia ocellata.

Extracts of cercariae of the avian schistosome Trichobilharzia ocellata were analysed for the presence of ecdysteroids by radioimmunoassay, high-performance liquid chromatography monitoring fractions by radioimmunoassay, and gas chromatography/mass spectrometry (selected ion monitoring). Both free ecdysteroids and polar conjugated ecdysteroids were detected in the cercarial extracts. The free ecdysteroid fraction, as well as the hydrolysed polar conjugated ecdysteroid fraction, contained both ecdysone and 20-hydroxyecdysone in approximately equal amounts. The amount of ecdysteroids detected is comparable to those found in other platyhelminths. A possible role for the ecdysteroids in the development of the parasite and/or the interactions between the parasite and its intermediate host, the freshwater snail Lymnaea stagnalis, is discussed.     

Carbohydrate epitopes on Haemonchus contortus antigens

Extracts of infective larvae and adults of the trichostrongylid Haemonchus contortus were studied for the presence of carbohydrate moieties. Several different lectin-binding sites were demonstrated in both stages using a panel of nine lectins. The carbohydrate specificity of the lectins used strongly suggests that α-D-mannose, α-D-glucose, and D-N-acetylglucosamine are the most important carbohydrate epitopes present on H. contortus proteins. Thus, N-linked oligosaccharides form the major part of the carbohydrate moieties on these glycoproteins. Treatment with sodium periodate was performed to investigate the immunoreactivity towards the carbohydrate moieties. This treatment resulted in a reduction in the immunoreactivity of these antigens as demonstrated by enzyme-linked immunosorbent assay (ELISA) and immunoblotting, suggesting that a substantial part of the host immune response against H. contortus is directed against the carbohydrate epitopes on the parasite antigens.     

Quantitative nucleic acid sequence-based assay as a new molecular tool for detection and quantification of Leishmania parasites in skin biopsy samples

Currently available methods for the diagnosis of cutaneous leishmaniasis (CL) have low sensitivities or are unable to quantify the number of viable parasites. This constitutes a major obstacle for the diagnosis of the disease and for the study of the effectiveness of treatment schedules and urges the development of improved detection methods. In this study, quantitative nucleic acid sequence-based amplification (QT-NASBA) technology was used to detect and quantify Leishmania parasites in skin biopsy samples from CL patients. The assay is based on the detection of a small subunit rRNA (18S rRNA), which may allow for the detection of viable parasites. The QT-NASBA assay was evaluated using in vitro-cultured promastigotes and amastigotes and 2-mm skin biopsy samples from Old and New World CL patients. The study demonstrated that the lower detection limit of the QT-NASBA was two parasites per biopsy sample. Parasites could be quantified in a range of 2 to 11,300,000 parasites per biopsy sample. The QT-NASBA could detect levels of parasites 100-fold lower than those detected by conventional PCR. Test evaluation revealed that the QT-NASBA had a sensitivity of 97.5% and a specificity of 100% in the present study. The QT-NASBA is a highly sensitive and specific method that allows quantification of both Old and New World Leishmania parasites in skin biopsy samples and may provide an important tool for diagnosis as well as for monitoring the therapy of CL patients.     

Schistosomin,a peptide present in the haemolymph ofLymnaea stagnalis infected withTrichobilharzia ocellata,is produced only in the snail's central nervous system

A peptide, schistosomin, is present in haemolymph ofLymnaea stagnalis infected withTrichobilharzia ocellata. There are indications that schistosomin is produced by the central nervous system (CNS) of the snail. Schistomin inhibits the effects of the snail's gonadotropic hormones, e.g. calfluxin (CaFl). CaFl stimulates Ca²⁺ influx into the mitochondria of the albumen gland, as shown using the ultracytochemical potassium pyroantimonate precipitation technique. We investigated the question as to whether schistosomin is produced only by the snail or by both the snail and the parasite. Several types of extract of stages of the parasite and of the CNS of the snail were tested for their capability to inhibit the CaFl response. Acid extracts of cercariae and of CNS showed an inhibitory effect, suggesting that both contain schistosomin. From these extracts, material was obtained that showed the same HPLC characteristics as schistosomin. This material was tested again. The hormone response was inhibited only by material derived from the CNS of the snail, indicating that the parasite does not produce schistosomin.     

Age-related protective immunity after vaccination with Haemonchus contortus excretory/secretory proteins

Protection against an experimental challenge infection by immunization with excretory/secretory products (ES) from Haemonchus contortus, containing predominantly proteins of 15 and 24 kDa, depends on the age of the sheep. Vaccinated sheep 9 and 6 months of age had reduced final worm burdens of 82 and 77, respectively. No reduction in worm burden was found in 3-month-old lambs. Nine-month-old sheep had significantly higher ES-specific serum immunoglobulin (Ig)G1 and IgA during immunizations and after challenge infection than 3-month-old lambs. There was no correlation within the 9-month-old sheep between ES-specific IgA or IgG1 levels and protection, measured as worm burden. However, when the different age groups were combined, negative correlations between percentage protection and ES-specific IgA and IgG1 levels after challenge were found. At the end of the experiment, peripheral blood eosinophils and mast cell counts in abomasal tissue were also significantly higher in the vaccinated and challenged 9-month-old sheep than in the vaccinated and challenged 3-month-old or than in the 9-month-old sheep with challenge, but without vaccination. The responses measured in young lambs were similar to the responses in sheep, but the height of these responses was in general of a lower magnitude.     

Protection in lambs vaccinated with Haemonchus contortus antigens is age related, and correlates with IgE rather than IgG1 antibody

The involvement of mucosal mast cells (MMC) in protection against infection with the murine nematode parasite Trichuris muris was studied in genetically mast cell-deficient WBB6F1-W/Wv mice and their normal littermates WBB6F1-+/+ mice. Expulsion of T. muris worms occurred in infected +/+ mice, whereas no worm expulsion was observed in infected W/Wv mice where the infection persisted until at least day 46 postinfection. No MMC responses were induced in either infected W/Wv or +/+ mice. Specific IgG1and IgG2a antibodies to T. muris excretory/secretory antigens were observed in infected W/Wv and +/+ mice, and antibody production showed similar kinetics. Interleukin 4 production by concanavalin A (Con A)-stimulated mesenteric lymph node cells (MLNC) was induced preferentially in infected +/+ mice. T. muris infection increased the levels of IFN-gamma produced by Con A-stimulated MLNC of infected W/Wv and +/+ mice, with the levels of IFN-gamma in infected W/Wv mice being higher than those in infected +/+ mice. Taken together, these results indicate that W/Wv and +/+ mice are susceptible and resistant to T. muris infection, respectively, and that MMC responses are not required for protective immunity.     

Accordance and concordance of PCR and NASBA followed by oligochromatography for the molecular diagnosis of Trypanosoma brucei and Leishmania

Objective To evaluate the repeatability and reproducibility of four simplified molecular assays for the diagnosis of Trypanosoma brucei spp. or Leishmania ssp. in a multicentre ring trial with seven participating laboratories. Methods The tests are based on PCR or NASBA amplification of the parasites nucleic acids followed by rapid read‐out by oligochromatographic dipstick (PCR‐OC and NASBA‐OC). Results On purified nucleic acid specimens, the repeatability and reproducibility of the tests were Tryp‐PRC‐OC, 91.7% and 95.5%; Tryp‐NASBA‐OC, 95.8% and 100%; Leish‐PCR‐OC, 95.9% and 98.1%; Leish‐NASBA‐OC, 92.3% and 98.2%. On blood specimens spiked with parasites, the repeatability and reproducibility of the tests were Tryp‐PRC‐OC, 78.4% and 86.6%; Tryp‐NASBA‐OC, 81.5% and 89.0%; Leish‐PCR‐OC, 87.1% and 91.7%; Leish‐NASBA‐OC, 74.8% and 86.2%. Conclusion As repeatability and reproducibility of the tests were satisfactory, further phase II and III evaluations in clinical and population specimens from disease endemic countries are justified.