The neuro-muscular system in fresh-water furcocercaria from Belarus. I Schistosomatidae.

The neuro-muscular system (NMS) in cercariae of the family Schistosomatidae from Belarus was studied with immunocytochemical methods and confocal scanning laser microscopy. The specimens of Bilharziella polonica were compared with Trichobilharzia szidati and Trichobilharzia franki. The patterns of F-actin in the musculature, 5-HT-immunoreactive (IR), FMRFamide-IR neuronal elements and α-tubulin-IR in sensory receptors and nerves were investigated. No indications of structural differences in the musculature, the 5-HT-IR, FMRF-IR neuronal elements and the general distribution of sensory receptors were noticed between cercariae of Trichobilharzia spp. The number of 5-HT-IR neurons in the cercarial bodies is 16. In cercaria B. polonica, the tail musculature is weaker than in Trichobilharzia spp. A detailed schematic picture of the NMS in the tail of Trichobilharzia spp. cercaria is given. The function of NMS elements in the tail is discussed.

Secretory glands in cercaria of the neuropathogenic schistosome Trichobilharzia regenti - ultrastructural characterization, 3-D modelling, volume and pH estimations.

BACKGROUND: Cercariae of schistosomes employ bioactive molecules for penetration into their hosts. These are released from specialized unicellular glands upon stimuli from host skin. The glands were previously well-described in the human pathogen Schistosoma mansoni. As bird schistosomes can also penetrate human skin and cause cercarial dermatitis, our aim was to characterize the architecture and ultrastructure of glands in the neurotropic bird schistosome Trichobilharzia regenti and compare it with S. mansoni. In the context of different histolytic enzymes used by these two species, we focused also on the estimations of gland volumes and pH in T. regenti. RESULTS: The architecture and 3-D models of two types of acetabular penetration glands, their ducts and of the head gland are shown here. We characterized secretory vesicles in all three gland types by means of TEM and confirmed accuracy of the models obtained by confocal microscopy. The results of two independent approaches showed that the glands occupy ca. one third of cercarial body volume (postacetabular glands ca. 15%, circumacetabular 12% and head gland 6%). The inner environment within the two types of acetabular glands differed significantly as evidenced by dissimilar ability to bind fluorescent markers and by pH value which was higher in circumacetabular (7.44) than in postacetabular (7.08) glands. CONCLUSIONS: As far as we know, this is the first presentation of a 3-D model of cercarial glands and the first exact estimation of the volumes of the three gland types in schistosomes. Our comparisons between T. regenti and S. mansoni implied that the architecture and ultrastructure of the glands is most likely conserved within the family. Only minor variations were found between the two species. It seems that the differences in molecular composition have no effect on general appearance of the secretory cells in TEM. Fluorescent markers employed in this study, distinguishing between secretory vesicles and gland types, can be useful in further studies of mechanisms used by cercariae for host invasion. Results of the first attempts to estimate pH within schistosome glands may help further understanding of regulation of enzymatic activities present within the glands.

Multiple cathepsin B isoforms in schistosomula of Trichobilharzia regenti: identification, characterisation and putative role in migration and nutrition.

Among schistosomatids, Trichobilharzia regenti, displays an unusual migration through the peripheral and central nervous system prior to residence in the nasal cavity of the definitive avian host. Migration causes tissue degradation and neuromotor dysfunction both in birds and experimentally infected mice. Although schistosomula have a well-developed gut, the peptidases elaborated that might facilitate nutrition and migration are unknown. This is, in large part, due to the difficulty in isolating large numbers of migrating larvae. We have identified and characterised the major 33 kDa cathepsin B-like cysteine endopeptidase in extracts of migrating schistosomula using fluorogenic peptidyl substrates with high extinction coefficients and irreversible affinity-labels. From first strand schistosomula cDNA, degenerate PCR and Rapid Amplification of cDNA End protocols were used to identify peptidase isoforms termed TrCB1.1-TrCB1.6. Highest sequence homology is to the described Schistosoma mansoni and Schistosoma japonicum cathepsins B1. Two isoforms (TrCB1.5 and 1.6) encode putatively inactive enzymes as the catalytic cysteine is substituted by glycine. Two other isoforms, TrCB1.1 and 1.4, were functionally expressed as zymogens in Pichia pastoris. Specific polyclonal antibodies localised the peptidases exclusively in the gut of schistosomula and reacted with a 33kDa protein in worm extracts. TrCB1.1 zymogen was unable to catalyse its own activation, but was trans-processed and activated by S. mansoni asparaginyl endopeptidase (SmAE aka. S. mansoni legumain). In contrast, TrCB1.4 zymogen auto-activated, but was resistant to the action of SmAE. Both activated isoforms displayed different pH-dependent specificity profiles with peptidyl substrates. Also, both isoforms degraded myelin basic protein, the major protein component of nervous tissue, but were inefficient against hemoglobin, thus supporting the adaptation of T. regenti gut peptidases to parasitism of host nervous tissue.

Comparison of european Trichobilharzia species based on ITS1 and ITS2 sequences.

Schistosomes are parasites of considerable medical and veterinary importance and, therefore, all aspects of their biology have been intensively studied. In contrast, our knowledge of species of the largest genus, Trichobilharzia, is insufficient. Because morphological characterization of Trichobilharzia species provides a limited number of criteria for species determination, molecular data are required. In the present paper, we sequenced internal transcribed spacers ITS1 and ITS2, and 5.8S ribosomal RNA (rRNA) genes of 3 European Trichobilharzia species (T. regenti, T. szidati and T. franki). We showed that ITS1 and ITS2 sequences can be used in species identification. Repetitive elements were found in ITS1 of all 3 Trichobilharzia species; their number and length varied depending on the species. Phylogenetic analysis showed that the visceral T. franki is more related to the nasal T. regenti, than to the visceral T. szidati. The newly designed primer, which is specific for T. regenti, might be used as a tool for diagnosis of this potential pathogen.

Developmentally regulated expression of surface carbohydrate residues on larval stages of the avian schistosome Trichobilharzia szidati.

Except other functions, surface saccharide residues on trematode larvae are supposed either to be the targets of the intermediate (molluscan) and final host immune systems, or to represent candidates for molecular mimicry. Therefore, changes in surface saccharide patterns during the development of the avian schistosome Trichobilharzia szidati were characterized. Whole parasite larval stages and their tissue sections were examined using FITC- conjugated lectins. Marked surface differences were found among larval stages (miracidia, mother sporocysts, daughter sporocysts, cercariae, schistosomula). Staining by some lectins reflected known ultrastructural changes of the outer tegument. Reaction of lectins with cercarial embryos was almost negative. In case of other developmental stages, binding of at least one member from each carbohydrate-specificity group of lectins (Man/Glc-, GlcNAc-, Gal/GalNAc- and Fuc-specific) occurred. One exception is represented by mother and daughter sporocysts which practically failed to react with Fuc-specific lectins. Besides other lectins which recognized larval surfaces, alpha-L-fucose-specific lectins (LTA, UEA-1) and (GlcNAc beta 1-->4)n-specific WGA bound very strong to certain stages. The comparison of mature intrasporocystic cercariae with those emerged from snails brought the indication that some snail glycosylated molecules adhere to the surface of schistosome larvae or that emerged cercariae express some new carbohydrate epitopes under changed environmental conditions. The result partially supports the theory of parasite mimicry/ masking strategies and immune evasion in the host.

Immunocytochemical study on biologically active neurosubstances in daughter sporocysts and cercariae of Trichobilharzia ocellata and Schistosoma mansoni.

Immunocytochemical techniques applied to sections and whole-mount preparations of cercariae from two species of trematodes, Trichobilharzia ocellata and Schistosoma mansoni, revealed the occurrence of immunoreactivity (IR) to several neurosubstances in the nervous system (NS). Immunostaining was localized in cerebral ganglia, in the main commissure, in anterior and posterior nerve trunks, as well as in a pair of nerve fibres running along the tail. In T. ocellata, immunoreactivity (IR) was observed with antisera raised against: glutamate, FMRFamide, catch-relaxing peptide (CARP), small cardiac peptide B (SCPB), arg-vasotocin (AVT), arg-vasopressin (AVP), and substance P. In S. mansoni antisera raised against glutamate, FMRFamide, CARP, SCPB, alpha-caudodorsal cell peptides (alpha-CDCP), and cholecystokinin (CCK) showed neuronal IR. With the other 51 antisera tested no IR was observed. With anti-APGWamide, IR was observed outside the NS in cells of the wall of the daughter sporocyst and in flame cells of cercariae of T. ocellata. IR to FMRFamide was present in the escape glands of the intrasporocystic cercariae of T. ocellata and S. mansoni. IR to somatostatin was observed in subtegumental parenchymal cells of cercariae of S. mansoni. IR to met-enkephalin was present in cells of the cercarial embryos and in undifferentiated cells in developing cercariae. Trematodes are, together with cestodes, phylogenetically the oldest classes in which glutamate-like material and immunopositivity to a number of neuropeptides isolated from invertebrates has been demonstrated. The results are discussed in relation to immunocytochemical data obtained for other platyhelminths, to endogenous functions of the immunopositive materials, and to their possible role in parasite-host interactions.