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The excretory ducts of Diphyllobothrium dendriticum (Nitzsch 1824) plerocercoids: ultrastructure and marker distribution.

An electron dense marker, lanthanum nitrate, was injected into the excretory ducts of living plerocercoids of Diphyllobothrium dendriticum and the observations made by electron microscopy. The contractions of the plerocercoid spread the marker into the excretory system, and the distribution was found to be irregular both within the ducts and from one duct to another. Communication between the smallest ducts and the rest of the excretory system was noted. The marker was also introduced from the surrounding medium, in this case the fixative. It was found to flush into the excretory ducts through the pore in the tail and to be distributed into the ducts in the same way as the injected marker. No other pores were observed through the tegument other than the excretory pore in the tail by either method. In ultrastructure the duct wall is similar to that of other cestodes. The distributive role of the excretory system is discussed, including a comparison between the tegument and the duct wall.

Animals↗

Proliferation and metastases formation of larval Echinococcus multilocularis. II. Ultrastructural investigations.

The larval stage (metacestode) of Echinococcus multilocularis was studied by means of electron microscopy (SEM, TEM) before and after subcutaneous transplantation to jirds (Meriones unguiculatus) and in their lymph nodes and lungs with parasite metastases. It was found that the metacestode consists of a network of solid, cellular protrusions (buds) of the germinal layer which transform to tube-like and cystic structures devoid or with a laminated layer. Proliferation of the metacestode apparently occurs by protruding filamentous solid cell columns (buds) from the germinal layer. Their tips have diameters of only one cell: they are covered with a smooth syncytial tegument without microtriches and are filled with undifferentiated cells which contain a nucleus with a large nucleolus. The tegument is constantly enlarged by fusion with the underlying undifferentiated cells that divide repeatedly. At some distance from the tip a cavity develops inside the protrusion, thus finally giving rise to a tube-like structure which may transform to a cystic expansion. Simultaneously, the surface of the protrusion changes by the formation of microtriches and the occurrence of an amorphous laminated layer. The latter is concentrically covered by connective tissue cells and large amounts of collagen. Within cyst-brood capsules, finally protoscoleces are formed from accumulations of undifferentiated cells beneath the tegument. The study has unequivocally proven the cestode nature of the cellular protrusions, and it is assumed that detachment of cells from these structures and their subsequent distribution via the circulation may play a role in the formation of metastases. The origin of the laminated layer is discussed.

Animals↗

Collagen immunotyping of the hepatic fibrosis in human alveolar echinococcosis.

An extremely dense fibrosis is responsible for the main complications of alveolar echinococcosis (AE), a severe parasitic disease due to the development in the liver of a larval form of the cestode Echinococcus multilocularis. Immunotyping of collagens present in fibrous, granulomatous, and normal areas of the liver was performed in nine patients with AE. Immunofluorescent labelling of collagen types showed that dense fibrosis was entirely composed of thick bundles of type I and type III collagens, surrounding old parasitic vesicles with an autofluorescent laminated layer. In granulomatous areas, collagens were labelled as clusters consisting mainly of type III procollagen and collagen. Concentric bundles of type III collagen surrounded active parasitic vesicles. An enhancement of the normal labelling in portal spaces of the intact liver was present in all the patients. These results suggest that irreversible acellular keloid scar-like fibrosis observed in AE is the ultimate result of cytotoxic and fibrogenetic events related to the immune response of the host which are taking place initially in the granulomatous area surrounding the young parasite larvae.

Collagen↗

Helminth parasites of wild foxes (Vulpes vulpes L.) in The Netherlands.

To study the helminth fauna of wild foxes (Vulpes vulpes) in The Netherlands, material was collected from 139 foxes. The following parasites were found. Cestodes: Taenia spp. 53.3%, Hymenolepis spp. 1.5%; Trematodes: Alaria alata 10.9%, Cryptocotyle lingua 3.6%, Euparyphium melis 1.5%, Apophallus donicus 0.7%; Nematodes: Toxocara canis 73.7%, Uncinaria stenocephala 59.9%, Capillaria aerophila 46.8%, C. plica 23.5%, Molineus patens 5.1%, Crenosoma vulpis 4.5%, Strongyloides spp. 0.7%. The tapeworms Mesocestoides spp. and Echinococcus spp. were not seen. No relationship was observed between worm burden and sex, time of year or place of origin.

Animals↗

Nematospiroides dubius: response of the late fourth-stage larva to anthelmintics in vitro.

Approximately 60% of fourth-stage larvae of Nematospiroides dubius recovered from mice 6 days after infection, developed to the young adult stage when cultured over a 7-day period in a complex medium in vitro. Larvae at the late fourth stage of development were found to be highly susceptible to most broad spectrum anthelmintics under in vitro conditions, the benzimidazole, imidazothiazole, pyrimidine, isothiocyanate and macrocyclic lactone compounds all being active at very low concentrations. Narrow spectrum anthelmintics active only against ascarids, pinworms, filariae, cestodes or trematodes had little or no effect on these larvae. Ineffective also were those chlorinated hydrocarbon, substituted phenol and salicylanilide compounds known to affect Haemonchus but not trichostrongylid worms in general. It is concluded that in vitro assays employing larvae of N. dubius are useful for the stringent screening of compounds for broad spectrum antitrichostrongyle activity. Used in conjunction with in vivo screens employing N. dubius in mice, they also afford means for detecting intrinsic activity against the parasite in a system free from any complicating host pharmacokinetics.

Animals↗

The effect of trifluoperazine (Stellazine) on Hymenolepis diminuta in vitro.

The influence of the phenothiazine trifluoperazine (Stellazine) on the rat tapeworm Hymenolepis diminuta was examined. The parasite was incubated in glucose-containing Krebs-Ringer media (pH 7.4) at 37 degrees C which included Ca2+ or EGTA and a range of trifluoperazine concentrations (0-2 mM). Release of soluble protein and lactate dehydrogenase activity were taken as measures of release of cytosolic components. The release of lactate dehydrogenase depended on drug concentration, maximum levels occurring at 2 mM trifluoperazine, this corresponded to 2% of the total lactate dehydrogenase present in the cestode. The effect of phenothiazines of differing lipophilicity were compared, and for trifluoperazine sulfoxide only minimal amounts of lactate dehydrogenase activity and protein were released. These values were similar to those obtained when H. diminuta was incubated in drug-free media. Our findings suggest that the integrity of the parasite is related to its calmodulin content. The potential cestocidal properties of trifluoperazine are considered.

Animals↗

Cystathionine beta-synthase and gamma-cystathionase in helminths.

The activities of gamma-cystathionase and cystathionine beta-synthase were investigated in a range of gastrointestinal, free-living and entomophagous nematodes. Although nematode gamma-cystathionase used the same range of substrates as the mammalian hepatic enzyme, its activity was extremely low and there were significant interspecies variations with respect to the relative order of active substrates. Like the mammalian liver enzyme, nematode cystathionine beta-synthase showed activity in the directions of both cystathionine synthesis and the forward and reverse "L-serine sulphhydrase" reactions. However, the most important feature of the survey was the widespread ability of nematode cystathionine beta-synthase to catalyse the non-mammalian "activated L-serine sulphhydrase" reaction (L-cysteine + R-SH----cysteine thioether + H2S). Additional survey work revealed that the ability to catalyse the activated L-serine sulphhydrase reaction was almost universal amongst nematodes. Activated L-serine sulphhydrase activity was also demonstrated in the acanthocephalan Pomphorhynchus laevis but was absent from cestodes and digeneans.

Acanthocephala↗

Echinococcus granulosus: the influence of mebendazole therapy on the ultrastructural morphology of the germinal layer of hydatid cysts in humans and mice.

Upon ultrastructural examination it was found that prolonged mebendazole therapy of patients with hydatid disease often resulted in complete necrosis of the germinal layer of Echinococcus granulosus cysts. The affected cysts showed only remnants of degenerated germinal layer tissue including heterogeneous vesicular membranes, electron-dense amorphous structures, myeloid bodies, lipid globules, crystal-like precipitates and remnants derived from muscular tissue. Examination of the hydatid cysts of treated mice revealed the same time-related deteriorative effect of the anthelmintic as described previously for other cestode species. The advanced stage of degeneration of the germinal layer tissue observed in the murine cysts after treatment resembled the morphologic features present in the necrotic human cysts and gave additional information about the degeneration phenomenon.

Animals↗

Peptidergic nerve elements in three developmental stages of the tetraphyllidean tapeworm Trilocularia acanthiaevulgaris. An immunocytochemical study.

The localization and distribution of seven neuropeptides in the nervous system of the plerocercoid, adult and free proglottis stages of the tetraphyllidean tapeworm Trilocularia acanthiaevulgaris have been determined by an indirect immunofluorescence technique. Six of the peptides are vertebrate-derived, namely, pancreatic polypeptide (PP), peptide tyrosine tyrosine (PYY), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI), substance P (SP) and somatostatin (SRIF); the seventh is the invertebrate neuropeptide, FMR Famide. This is the first demonstration of VIP and SP immunoreactivity in a cestode parasite, and for SRIF this is its first description in any parasitic platyhelminth. Cell bodies and nerve fibres immunoreactive to PP, PYY, VIP, SP and FMRFamide are present throughout the CNS; the distributions of PHI and SRIF were more restricted. In the PNS, nerve fibres immunoreactive to PP occur in the bothridia, whilst in the free proglottis nerve fibres immunoreactive to PYY and VIP innervate the gonads; VIP-immunoreactive nerve elements also supply the reproductive ducts. Extra-neuronal sitings of peptide immunoreactivities were evident for PHI, in association with the excretory system, and for SRIF, in presumed tegumental cell bodies in the free proglottis. The results are discussed in relation to the possible roles of the peptides in the neurophysiology and developmental biology of the worm.

Animals↗

Scanning electron microscopic study of four Diphyllobothrium species.

Three-dimensional observation was carried out on plerocercoids and adults of Diphyllobothrium dendriticum, D. ditremum, D. latum, and D. vogeli using scanning electron microscopy. The species-specific differences between plerocercoids were recognized in the shapes of the whole body, scolex, and bothrium and the wrinkle pattern on the body surface. The differences between adult worms were also observed in the shapes of the scolex, neck, and genital papillae around the genital pore and the pattern on the egg surface. The significance of species specificity in the three-dimensional morphology of diphyllobothriid cestodes is briefly discussed.

Animals↗

Ultrastructure of the developing protonephridial system of the cercaria of Philophthalmus sp. (Trematoda, Digenea).

The fully developed flame bulb of Philopthalmus exhibits the structure characteristic of Trematoda and Monogenea: external and internal ribs forming a weir, external and internal leptotriches, and two longitudinal cytoplasmic cords connected by a septate junction. The proximal canal has a septate junction and surface lamellae. In developing cercariae, perikarya of terminal and proximal canal cells are close together, and sheet-like outgrowths of the terminal cell are externally surrounded by cytoplasm of the proximal canal cell containing a septate junction. Internal outgrowths and external cytoplasm are connected by many "membranes", i.e. desmosome-like structures. Internal sheets break up into internal ribs, and the external cytoplasm breaks up into external ribs, external and internal ribs connected by the filtration "membrane". The developing distal excretory duct possesses a septate junction and many branching and looping lamellae. A comparison of Philopthalmus with the cestode Austramphilina elongata, the only other platyhelminth species in which the development of the protonephridia has been studied at the ultra-structural level, revealed that the two species differ in the presence and absence, respectively, of a septate junction in the flame bulb at an early stage of development.

Animals↗

Spermatogenesis and the fine structure of the mature spermatozoon in the free proglottis of Trilocularia acanthiaevulgaris (Cestoda, Tetraphyllidea).

Spermatogenesis and the structure of the mature spermatozoon of Trilocularia acanthiaevulgaris have been studied by light microscopic histochemistry and transmission and scanning electron microscopy. The primary spermatogonia occupy the periphery of the testis and undergo four mitotic divisions; incomplete cytokinesis results in two secondary, four tertiary and eight quaternary spermatogonia and sixteen primary spermatocytes. The quaternary spermatogonia and successive stages develop in a plasmodial rosette with their nuclei at the periphery and connected to the cytophore by cytoplasmic bridges. The primary spermatocytes undergo two meiotic divisions, resulting in 64 spermatids that develop into spermatozoa. Intranuclear synaptonemal complexes in primary spermatocytes confirm the first meiotic division. The onset of spermiogenesis is marked by an elongation of the spermatid nucleus and the appearance of "arching membrane-bound clefts" (after Rosario 1964) that delimit the differentiation zone. The latter contains a pair of axonemes with their associated basal bodies and rootlets and, as it extends, the nucleus migrates into it. At first, the axonemes grow at right angles to the extension, but during development they rotate through 90 degrees and come to fuse with the cytoplasmic extension. The mature spermatozoon has a broad head region, a middle piece and a narrow tail region. It consists of a highly coiled nucleus, paired axonemes of the 9 + 1 pattern typical of platyhelminths, one of which extends the full length of the tail and the other ends part-way along the tail, particles of beta-glycogen and peripheral microtubules. A crested body spirals around the outside of the sperm body in the tail region. T. acanthiaevulgaris is placed in context within the phylogenetic schemes for cestodes based on sperm ultrastructure.

Animals↗

Electron immunogold labeling of regulatory peptide immunoreactivity in the nervous system of Moniezia expansa (Cestoda: Cyclophyllidea).

An electron immunogold-labeling technique was used in conjunction with a post-embedding procedure to demonstrate for the first time the ultrastructural distribution of the parasitic platyhelminth neuropeptide, neuropeptide F (NPF), in the nervous system of the cestode Moniezia expansa. Two axon types, distinguished by their populations of different-sized electron-dense vesicles, were identified. Immunogold labeling demonstrated an apparent homogeneity of PP, FMRFamide and NPF (M. expansa) antigenic sites throughout the larger dense-cored vesicles within the central nervous system. Triple labeling clearly demonstrated the co-localisation of immunoreactivities (IR) for NPF, PP and FMRFamide within the same dense-cored vesicles. The presence of NPF-IR within the vesicles occupying the perikaryon of the neuronal cell body indicated that the peptides had undergone post-translational C-terminal amidation prior to entering the axon. Antigen pre-absorption experiments using NPF prevented labeling with either PP or FMRFamide antisera, and the failure of these antisera to block NPF-IR supports the view that some, if not all, of the PP/FMRFamide-IR is due to NPF-like peptides.

Animals↗

Serotonin and neuropeptide immunoreactivities in the intramolluscan stages of three marine trematode parasites.

Using an indirect immunofluorescence technique interfaced with confocal scanning laser microscopy, whole-mount preparations of three genera of marine trematode larvae, Cryptocotyle lingua, Cercaria emasculans and Himasthla leptosoma, were screened for 5-hydroxytryptamine (5-HT) and selected neuropeptide immunoreactivities (IRs). IRs for pancreatic polypeptide (PP), peptide YY (PYY) and FMRFamide were found in the central nervous systems of the three species of cercariae, immunostaining the paired ganglia and central commissure and the longitudinal nerve cords, with slight differences in both distribution and intensity of IRs being observed for the different antisera used. PP, PYY and FMRFamide IRs were evident in both central and peripheral components of the nervous system in the rediae of C. lingua. 5-HT IR was confined to the peripheral nervous systems of the cercariae of C. emasculans and the rediae of C. lingua, appearing in the form of a network of immunoreactive fibres and associated large cell bodies. A moderate substance P IR was observed in the nervous system of the cercariae of C. lingua. The patterns of immunostaining described were compared with those obtained using antiserum directed to the C-terminal decapeptide amide of neuropeptide F (NPF), a native parasitic peptide from the cestode Moniezia expansa. Results demonstrated that serotoninergic and peptidergic components were present in the nervous systems of all of the trematode larvae studied and that some, if not all, of the IR for PP, PYY and FMRFamide was due to the presence of a trematode NPF homologue.

Amino Acid Sequence↗

Establishment and survival of the strobilar stage of Taenia crassiceps in hamsters, gerbils, and mice, with reference to different helminth isolates.

Following the oral administration of metacestodes of two isolates of Taenia crassiceps, the enteral establishment and survival of the strobilar stage were examined in golden hamsters (Mesocricetus auratus), Mongolian gerbils (Meriones unguiculatus) and laboratory mice. The origin of the isolates was Microtus montebelli caught in Japan in 1985 or Clethrionomys rutilus captured on St. Lawrence Island, Bering Sea, in 1988 (abbreviated as JPN and SLI isolates), respectively. The enteral establishment of the SLI isolate was distinctly higher than that of the JPN isolate in golden hamsters and mice, whereas the difference was marginal in Mongolian gerbils. All initially-established parasites survived to become gravid adults in prednisolone-treated golden hamsters and Mongolian gerbils; the average recovery of cestodes of the SLI and JPN isolates were 55.8%-76.7% vs 11.7%-35.0% in the former and 28.0%-52.7% vs 25.8%-32.2% in the latter. The distinctly higher level of enteral establishment of the SLI isolate in golden hamsters makes available a model for quantitative studies on parasite-host relationships in experimental taeniasis.

Administration, Oral↗

A review of the taxonomy and speciation of the genus Echinococcus Rudolphi 1801.

The taxonomy of Echinococcus is reviewed and consideration given to speciation within the genus. The morphological and other biological characteristics of all species and subspecies so far described are given, and evidence for the existence of other intraspecific variants is presented. A total of 16 species have been described but only four have well-defined characteristics and are generally accepted as being taxonomically valid; E. granulosus, E. multilocularis, E. oligarthrus and E. vogeli. The taxonomic status of intraspecific variants is uncertain. Some authorities consider that the majority of described subspecies are invalid taxonomically. However, concern is expressed that previous taxonomic considerations of Echinococcus have not taken into account the nature of its reproductive mechanism which, like that of most cestodes, defies the application of traditional taxonomic concepts and definitions derived from dioecious cross-fertilising organisms. At the present time intraspecific variants of Echinococcus are referred to as strains. The existence of such strains, particularly of E. granulosus, in different parts of the world is firmly established. Their importance in the epidemiology of hydatidosis has high-lighted the needed to establish criteria for their differentiation and characterisation. The limitations of morphology as a sole differential criterion are emphasised. Attention is given to recent studies on strain variation which have demonstrated the value of applying several different criteria in which the developmental, physiological and biochemical characteristics of different strains are compared in conjunction with morphological investigations.

Animals↗

Development and dynamics of regional specialization within the syncytial epidermis of the rat tapeworm, Hymenolepis diminuta.

The epidermis of the tapeworm Hymenolepis diminuta is a highly organized syncytium, composed of an outer layer of continuous cytoplasm, or ectocytoplasm, and an inner layer of nucleated cell bodies, or perikarya. The perikarya are in direct cytoplasmic continuity with the ectocytoplasm via narrow plasmalemma-bound bridges called internuncial process. Although distinct structural and functional differences are apparent between ectocytoplasm and perikarya, all of the perikarya along the body of the cestode are morphologically similar, as are all regions of ectocytoplasm. However, immunocytochemically distinct subpopulations of perikarya and regionally defined areas of ectocytoplasm were identified along the tapeworm strobila by the use of monoclonal antibodies raised against a preparation of isolated tegument. The different types of perikarya and the regionally specialized areas of ectocytoplasm were organized in a topographically precise manner along the body of the parasite. Examination of labeling patterns after colchicine treatment suggests that different types of perikarya are specialized for biosynthesis of specific tegumental molecules and for turnover or recycling of tegumental material. Furthermore, it appears that a 52 kDa polypeptide synthesized by one population of perikarya is transported through the syncytium and ultimately resorbed by a different population of tegumental perikarya. These data suggest that the syncytial epidermis of parasitic platyhelminthes exhibits a more complex organization of function than previously appreciated.

Animals↗

Detection of Echinococcus coproantigens by enzyme-linked immunosorbent assay in dogs, dingoes and foxes.

An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Echinococcus coproantigens in fecal samples from dogs, dingoes or foxes infected with either E. granulosus or E. multilocularis. The ELISA was based on protein-A-purified polyclonal antibodies [anti-E. granulosus excretory/secretory (E/S) antigens]. The specificity of the assay as determined in 155 samples derived from carnivores that were free of helminth infection (n = 37) or infected with non-Echinococcus cestodes (n = 76) or with various nematodes (n = 42) was found to be 98% overall. The diagnostic sensitivity was strongly dependent on the homologous worm burden. All 13 samples from foxes harboring greater than 1,000 E. multilocularis worms and 13 of 15 (87%) samples from dogs or dingoes containing greater than 200 E. granulosus worms were ELISA-positive, whereas 34 of 46 samples from foxes harboring less than 1,000 E. multilocularis and 9 of 10 samples from dogs or dingoes bearing less than 200 E. granulosus tested negative. Experimental prepatent infections of dogs with E. granulosus revealed positive ELISA reactions within the prepatent period (10-20 days post-infection) for six animals bearing greater than 1,000 E. granulosus each; a low worm burden (less than 1,000 tapeworms/animal) resulted in ELISA positivity in only 2 of 3 animals at 30 days post-infection at the earliest. All five dogs that had been experimentally infected with E. multilocularis tested positive in the coproantigen ELISA as early as on day 5 post-infection.

Animals↗