A protein secreted in vivo by Echinococcus granulosus inhibits elastase activity and neutrophil chemotaxis.

A cDNA encoding the carboxy-terminal of the 12-kDa subunit of antigen B of Echinococcus granulosus has been cloned and sequenced. In addition, an amino acid sequence has been generated for the amino-terminal which is tentatively contiguous with the open reading frame of the DNA-derived sequence. Comparison of the inferred sequence of the 12-kDa antigen with other known sequences indicated a limited similarity to alpha-1 antitrypsin. In functional assays, gel-purified native 12-kDa antigen from natural infections inhibited elastase but not trypsin or chymotrypsin, providing further evidence that this antigen is a parasite protease inhibitor. Possibly unrelated to its anti-protease activity but a potentially important function of the 12-kDa antigen was its ability to inhibit recruitment of neutrophils. These functions may be important to the viability of the parasite in the face of the host immune response. In addition, the match between the DNA-derived sequence and the protein sequence was imperfect, with some residues having, according to the amino acid sequencing, two alternatives in approximately equal concentrations, and four DNA-derived residues failing to match with the protein sequence at all. The 12-kDa antigen may be expressed as isoforms from a polymorphic gene and, as far as aware, this observed sequence polymorphism has not, to date, been described for any other flatworm antigen.

Analysis of the monohexosylceramide fraction of Echinococcus multilocularis metacestodes.

Monohexosylceramides of Echinococcus multilocularis metacestodes have been isolated and analyzed by thin-layer chromatography, gas-liquid chromatography and mass spectrometry. 90.9% of the parasite fraction was galactosylceramide; glucosylceramide was present at only 9.1%. The most important fatty acids were normal C16:0 and C26:0 fatty acids. The hydroxylated fatty acids of the ceramide part constituted 20.1% of the total, their major constituents were C18:0 and C26:0. The sphinganine accounted for 70.4% of long-chain bases, phytosphingosine and sphingosine were also detected. The importance of the long chain fatty acids and the presence of sphinganine in the monohexosylceramide fraction were discussed.

Glycosphingolipids of Echinococcus multilocularis metacestodes.

Neutral and acid glycosphingolipids of Echinococcus multilocularis metacestodes that were obtained after intraperitoneal infection of Meriones unguiculatus have been analyzed by thin layer chromatography. Neutral and acid glycosphingolipids accounted for 95% and 5% of total glycosphingolipids, respectively. 12 different fractions were observed in the neutral glycosphingolipids extracts of the parasite. The most important was a monohexosylceramide fraction accounting for 56.4% of neutral glycosphingolipids. 9 different fractions were detected in gangliosides (acid glycosphingolipids). The fact that these glycosphingolipids were specific to the parasite was established by the analysis of different cell populations of the host. Glycosphingolipids were purified from control and parasite-infected gerbil blood cells as well as from peritoneal exudate cells of healthy gerbils after a non-specific immunostimulation. The chromatograms obtained with these extracts were totally different from the parasite. In addition, parasitosis was found to have no effect on the host blood cell glycosphingolipids.

Echinococcus granulosus: a comparison of free amino acid concentration in hydatid fluid from primary and secondary cysts and host plasma.

A total of 28 components were detected in the free amino acid (FAA) pool of hydatid fluid from primary and secondary equine cysts, secondary ovine cysts and host plasma. Examination of data from equine cysts revealed that the majority of FAAs were present in significantly greater concentrations in secondary cysts, glycine being over 30 times more concentrated. Values for total carbohydrates and glucose did not, however, differ significantly and total protein content was greater in primary cysts. Comparison of the (FAA) pool of secondary equine and ovine cysts revealed strain variation. It was demonstrated that most FAAs were more concentrated in hydatid fluid than in the corresponding host plasma, many concentration ratios exceeding 10. The possible contribution that mediated amino acid transport across the cyst wall and parasite amino acid metabolism makes to the composition of the FAA pool was discussed. No significant plasma aminoacidaemia was associated with infection.

Molecular cloning of Taenia solium genomic DNA and characterization of taeniid cestodes by DNA analysis.

Total DNAs, isolated from a range of taeniid cestodes (Taenia solium, T. saginata, T. pisiformis, T. crassiceps, T. hydatigena, T. ovis, T. multiceps and T. taeniaeformis), have been subjected to restriction enzyme digestion, Southern transfer and hybridization analysis using cloned fragments of the ribosomal RNA gene of Schistosoma mansoni. Substantial inter-specific genetic differences have been revealed on the basis of characteristic hybridization patterns for each of the taeniid cestode species. Furthermore, a random genomic DNA library has been constructed in the vector plasmid pAT153 using DNA extracted from a pig isolate (Indian origin) of T. solium. A panel of taeniid cestode DNAs including DNA from Echinococcus granulosus, has been used in conjunction with hybridization and restriction enzyme analysis to identify in the library a single recombinant plasmid with a T. solium-specific insert (coded pTS10) and two recombinant plasmids with T. solium inserts having selective specificities for T. solium and T. ovis (coded pTS17) and T. solium, T. saginata, T. ovis and T. multiceps (coded pTS28). These recombinant plasmids and the cloned fragments of the ribosomal RNA gene of S. mansoni have been used in restriction endonuclease, Southern transfer and hybridization analysis to detect intra-specific genetic variation in cysticerci of T. solium from India, Mexico and Zimbabwe. In addition, pTS10 and pTS17 have been used in a simple dot-blot assay to distinguish T. solium from T. saginata.

Echinococcus multilocularis: comparative study of glycogen synthase in metacestodes and in the livers of infected and control Meriones unguiculatus.

A comparative study of the kinetic parameters of glycogen synthase was performed on Echinococcus multilocularis metacestodes and on the livers of infected and control host (Meriones unguiculatus). The enzyme of the parasite was found to be different from the enzyme of infected host liver. The apparent Km for UDP-glucose is 100 microM for the parasite and 400 microM for the host liver. The apparent Km for glucose 6-phosphate is 4 mM for the parasite and 2 mM for the host liver. The apparent Km for glycogen is 16 mg/ml for the parasite and 125 mg/ml for the host liver. The influence of glucose 6-phosphate and exogenous glycogen on the activity of glycogen synthase differs between the metacestode and the host liver. The enzyme of the metacestodes apparently does not need exogenous glycogen to work, contrary to the case for the liver host enzyme. The glycogen synthase of the parasite seems to be present in forms I and D, whereas the enzyme of the host liver appears in form I and that of the control liver in form D.

[Characterization of surface and intracellular glycoconjugates in larval forms of Echinococcus multilocularis using lectins]. / Caractérisation des glycoconjugués de surface et intracellulaires dans les formes larvaires d'Echinococcus multilocularis à l'aide de lectines.

Carbohydrate moieties have been detected with nine fluorescent lectins in Echinococcus multilocularis cysts, developed in liver and lung of infested jirds. Tegument and glycocalyx, and two types of medullary cells were found to selectively and strongly bind lectins which are specifically adsorbed by N acetyl glucosamine, N acetyl galactosamine, galactose or mannose. The significance of these features in the host-parasite interaction are discussed.

Echinococcus granulosus: occurrence of ecdysteroids in protoscoleces and hydatid cyst fluid.

Both free ecdysteroids and hydrolysable polar conjugated ecdysteroids were detected in protoscoleces of Echinococcus granulosus from the equine host, and in hydatid cyst fluid from the same source. Comparisons were made of hydatid cyst fluid from E. granulosus infections of three intermediate host species: horses, sheep and humans. Ecdysone and 20-hydroxyecdysone were identified in both protoscoleces and hydatid cyst fluids by high-performance liquid chromatography monitoring fractions by radioimmunoassay, and by capillary gas chromatography/mass spectrometry (selected ion monitoring). The free ecdysteroid fractions of hydatid cyst fluid from horses and sheep also contained several unidentified, chromatographically unique, immunoreactive compounds which were refractory to hydrolysis with a crude Helix pomatia aryl sulphatase enzyme preparation.

A comparison of two procedures for labelling the surface of the hydatid disease organism, Echinococcus granulosus, with 125I.

Living, intact protoscoleces of the British horse and sheep strains of Echinococcus granulosus were subjected to surface radioiodination procedures using 125I and Iodogen and 125I-Bolton Hunter reagent. Subsequent combined electron microscopy and autoradiography revealed specific surface membrane labelling with the Iodogen procedure, but significant tegumental labelling with the Bolton-Hunter reagent. The two parasite strains yielded different profiles of electrophoretically separated labelled proteins; the Iodogen method, not surprisingly, resulted in a less complex pattern of labelled polypeptides than the Bolton and Hunter reagent.

Isolation, fractionation and partial characterization of the tegumental surface from protoscoleces of the hydatid organism, Echinococcus granulosus.

Several approaches were adopted for the disruption and removal of the tegumental surface from protoscoleces of the horse strain of the hydatid organism, Echinococcus granulosus. The effectiveness of each method and the purity of subsequent microthrix-enriched fractions obtained by differential centrifugation were evaluated by electron microscopy, by the amount of protein released and by the degree of enrichment of surface plasma membrane marker enzymes. Incubation in saponin for 10 min produced the purest microtriche preparation, but in low yield; freeze/thawing, incubation in Triton X-100 for 10 min or in saponin for 20 min produced fractions containing significant amounts of relatively pure microtriches, but mild homogenization was a poor method for surface disruption and subsequent isolation of microtriches. Phosphodiesterase, adenosine triphosphatase (total and ouabain-inhibited), leucine aminopeptidase and glutamyltransferase were active in the protoscoleces but none were enriched in any of the microthrix fractions. In contrast, alkaline phosphatase, acid phosphatase, 5' nucleotidase and maltase were enriched significantly in all of the isolated microtriche preparations, which suggests that these enzymes are predominantly surface membrane bound. The protein profiles of the microthrix-enriched fractions, following SDS-PAGE, were basically similar, although there were some qualitative and quantitative differences in the proteins released by each isolation procedure. Three major PAS-staining components were present in all the preparations and these probably originated from the glycocalyx. One of these PAS-positive components, with an approximate molecular weight of 110 kDa, may be a glycoprotein specific to the horse strain of E. granulosus.

Echinococcus multilocularis: identification of proteins inducing antibody formation in metacestode infection.

An approach to the identification of parasite proteins which are immunogenic in natural infections is described, using the infection with the larval stage of Echinococcus multilocularis as a parasite model. Metacestode proteins were separated by SDS-polyacrylamide gel electrophoresis, and transferred electrophoretically to nitrocellulose sheets (Western blotting). Subsequently, immune recognition of the proteins was performed with various host sera and antigen-antibody complexes were detected enzymatically. Using homologous antisera, different patterns of immunogenic bands were revealed by sera of different host species. Cross-reactions with sera from individuals infected with unrelated helminths were analysed. Four proteins of E. multilocularis which failed to show any cross-reaction were identified.

A biochemical study of adult and cystic stages of Echinococcus granulosus of human and animal origin from Kenya.

A comparative biochemical study was performed on adult and cystic stages of Echinococcus granulosus. Basic quantitative differences in metabolism were apparent between the cystic forms of sheep origin from the UK and Kenya which suggest that each may represent a different geographical strain or sub-strain. The biochemistry of the human and sheep forms from Kenya was very similar, which probably reflects a certain close affinity between the two. The fact that the cattle, goat and camel forms of E. granulosus, from that country, were distinct biochemically, both from each other and from the sheep and human types, suggests the existence of an unusually complex strain picture there, and that these organisms are either non-infective or only poorly infective to man. The differences in biochemical composition and metabolism observed between adults produced experimentally and those obtained from naturally infected dogs, may have been as a result of their original hydatid source and/or their differing stage of development.

Cytotoxicity of Echinococcus granulosus cyst fluid in vitro.

In a cytotoxicity assay, using rat spleen cells as target cells, Echinococcus granulosus cyst fluid from cattle exerted a marked degree of cytotoxicity in vitro. When trypan blue exclusion or [3H]thymidine incorporation by concanavalin A stimulated spleen cells was used as a measure of cell viability, dialyzed cyst fluid showed maximum cell destruction up to a 1:8 dilution. The effect was dose and time dependent, cells being affected by 24 h after exposure to cyst fluid. The components responsible for cytotoxicity of cyst fluid were heat stable and could be recovered using gel chromatography on Sephadex G 50 and G 15 as a single low molecular weight fraction. It is assumed that the toxic products released by the living parasite can readily penetrate through the cyst wall into the surrounding host tissue. The interference of such substances with immunocompetent cells might account for the long-term survival of the parasite in the intermediate host.

Differences in the chemical composition and carbohydrate metabolism of Echinococcus granulosus (horse and sheep strains) and E. multilocularis.

Metabolic studies in vitro and studies on chemical composition indicate basic biochemical differences between the horse and sheep strains of Echinococcus granulosus and between these and the closely related species, E. multilocularis. The horse strain of E. granulosus has a similar level of DNA, but significantly more polysaccharides and lipids, with less RNA and protein than the sheep strain. E. multilocularis has significantly more lipids and DNA but less polysaccharides than the horse and sheep strains of E. granulosus, more RNA and protein than the horse strain but similar protein to and less RNA than the sheep strain. Incubations under air and under 95% N2-5% CO2 for 3 h show that only E. multilocularis takes up glucose, that all three forms consume different amounts of oxygen and endogenous glycogen and produce different concentrations of lactate, succinate, acetate, malate, pyruvate, propionate and ethanol as end products of carbohydrate metabolism.