Are glial cells of the Digenea (Platyhelminthes) muscle cells?

Muscle cells of a digenean fish blood fluke, Aporocotyle simplex, aggregate along the periphery of the cerebral ganglia. Solitary myocytons and sarcoplasmic processes with muscle fibres give rise to long, narrow lamellate projections, which are visible along the periphery and within ganglia. These ultrastructural observations suggest a switching of glial functions to muscle cells and represent additional evidence of the phylogenetic lability of glial cells in bilaterians.

Unique ultrastructural characteristics of the tegument of the digenean blood fluke Aporocotyle simplex Odhner, 1900 (Digenea: Aporocotylidae), a parasite of flatfishes.

This paper includes the first transmission electron microscopical (TEM) study of the tegument of a member of the basal digenean family Aporocotylidae. Scanning electron microscopical investigations of the fish blood fluke Aporocotyle simplex show that each boss on the lateral body surface bears 12-15 simple, uniform spines which extend from 0.5-2.7 µm above the surface of the boss. TEM observations revealed that these spines reach deep beneath the distal cytoplasm of the tegument for much of their length (9-12 µm) and are surrounded by a complex of diagonal muscles in each boss. This is the first record of any digenean with so-called 'sunken' spines. The results suggest that aporocotylid spines arise from within the sarcoplasm of the boss diagonal muscles. The sunken cell bodies (perikarya) of the tegument are connected to the distal cytoplasm via ducts (specialised processes lined by microtubules); this in contrast to other digeneans studied, where they are connected via non-specialised cytoplasmic processes. Within the distal cytoplasm, the tegumental ducts of A. simplex are surrounded by invaginations of the basal membrane and release their cytoplasmic inclusions into the distal cytoplasm. These apparently unique morphological features of the tegument, especially the deep origin of the spines, may represent useful characteristics for understanding aporocotylid relationships, especially in view of the known variation in the spine patterns of aporocotylids.

Trichobilharzia anseri n. sp. (Schistosomatidae: Digenea), a new visceral species of avian schistosomes isolated from greylag goose (Anser anser L.) in Iceland and France.

Parasitological investigations carried out on birds in Iceland and France highlight the presence of four species of avian schistosomes from greylag geese (Anser anser L.): the european nasal species Trichobilharzia regenti and three visceral species, among which an unknown species isolated from blood vessels of the large intestine and liver. Morphological and molecular analyzes of different parasite stages (eggs, adults) revealed new species of Trichobilharzia genus ­ Trichobilharzia anseri sp. nov. Studies on host-parasite relationship under natural conditions, showed that the life-cycle includes the snail Radix balthica (syn. R. peregra) as intermediate host. The cercariae, already isolated in Iceland from two ponds of the Reykjavik capital area ­ the Family park and Tjörnin Lake ­ are the same as those isolated in 1999 by Kolárová et al. during the first study on Icelandic parasitic agents of cercarial dermatitis.

Influence of Trichobilharzia regenti (Digenea: Schistosomatidae) on the defence activity of Radix lagotis (Lymnaeidae) Haemocytes.

Radix lagotis is an intermediate snail host of the nasal bird schistosome Trichobilharzia regenti. Changes in defence responses in infected snails that might be related to host-parasite compatibility are not known. This study therefore aimed to characterize R. lagotis haemocyte defence mechanisms and determine the extent to which they are modulated by T. regenti. Histological observations of R. lagotis infected with T. regenti revealed that early phases of infection were accompanied by haemocyte accumulation around the developing larvae 2-36 h post exposure (p.e.) to the parasite. At later time points, 44-92 h p.e., no haemocytes were observed around T. regenti. Additionally, microtubular aggregates likely corresponding to phagocytosed ciliary plates of T. regenti miracidia were observed within haemocytes by use of transmission electron microscopy. When the infection was in the patent phase, haemocyte phagocytic activity and hydrogen peroxide production were significantly reduced in infected R. lagotis when compared to uninfected counterparts, whereas haemocyte abundance increased in infected snails. At a molecular level, protein kinase C (PKC) and extracellular-signal regulated kinase (ERK) were found to play an important role in regulating these defence reactions in R. lagotis. Moreover, haemocytes from snails with patent infection displayed lower PKC and ERK activity in cell adhesion assays when compared to those from uninfected snails, which may therefore be related to the reduced defence activities of these cells. These data provide the first integrated insight into the immunobiology of R. lagotis and demonstrate modulation of haemocyte-mediated responses in patent T. regenti infected snails. Given that immunomodulation occurs during patency, interference of snail-host defence by T. regenti might be important for the sustained production and/or release of infective cercariae.

Identification of a major causative agent of human cercarial dermatitis, Trichobilharzia franki (Müller and Kimmig 1994), in southern England and its evolutionary relationships with other European populations.

BACKGROUND: Trichobilharzia is the most species rich and widely distributed genus of schistosomes and is known throughout Europe and North America as an agent of human cercarial dermatitis. The disease is caused by an acute allergic reaction in the skin that develops as a consequence of repeated contact with water containing schistosomatid cercariae. However, despite historical outbreaks of the disease, there are no published records of accurately identified Trichobilharzia species from the UK. METHODS: Two hundred Radix auricularia (L.) were sampled from a recreational fishing lake in Hampshire and emerging schistosomatid cercariae were collected for microscopy and DNA extraction. General morphological description of the cercariae was performed, alongside sequencing and phylogenetic analysis of the 28S ribosomal DNA for accurate species identification as well as comparisons of ITS1 in order to identify evolutionary affinities with other European populations. All molecular comparisons were performed using published sequences. RESULTS: The phylogenetic analysis of 28S sequences identified the cercariae as Trichobilharzia franki. Two unique British ITS1 haplotypes were identified which were most closely related to haplotypes of T. franki populations from France. Haplotype network analysis indicated the mixing of T. franki populations throughout Europe. It is suggested that parasite distribution is the probable result of the movement of migratory waterfowl. CONCLUSIONS: This is the first accurate record of T. franki in the UK. The movement of T. franki with waterfowl could pose a considerable human health risk, as in mainland Europe, and signifies T. franki-associated human cercarial dermatitis as a re-emerging disease in the UK.

Morphology and surface topography of the schistosome Bivitellobilharzia nairi from the Asian elephant (Elephas maximus maximus) in Sri Lanka.

Bivitellobilharzia nairi was first recorded from an Indian elephant (Elephas maximus) in Berlin. Infections with this parasite have become increasingly important in E. maximus maximus populations in Sri Lanka. The present work is the first morphological description of this schistosome from Sri Lanka. A number of adult worms were recovered from a dead Asian elephant near the elephant orphanage, Pinnawala, in Sri Lanka. The observed clinical features of the infected elephant included emaciation, subventral oedema and anaemia. Post-mortem results indicated that the liver was enlarged and adult schistosomes were found in the blood vessels of the liver parenchyma. The total number of worms recovered from a portion of the liver was 129,870, which is an average of 22 worms per 100 g of liver. The present study uses both light microscopic and scanning electron microscope (SEM) techniques for the morphological and topographical characterization of this parasite and to permit comparison with other species of schistosomes. Morphologically, these worms correspond very well to the description of B. nairi by Dutt & Srivastava (1955). Moreover, it is clear that B. nairi is a distinctive species easily differentiated from other schistosomes. The SEM study of the tegument of male worms shows that the surface of B. nairi is smoother than in other schistosomes.

[Trematodes of birds (Aves) from the Middle Volga region. 2. Orders Plagiorchiida, Renicolida, Strigeida, and Schistosomatida].

Data on trematodes of Plagiorchiida, Renicolida, Strigeida, and Schistosomatida, parasitizing in birds of the Middle Volga region are given. Proceedings of the different authors are supplemented by results of our research. Two species of trematodes (Brachylaima mesostoma and Mosesia amplavaginata) are specified for birds of the middle Volga region for the first time. New hosts were revealed for 2 trematode species: the great tit for Plagiorchis laricola and the common chaffinch for M. amplavaginata. The following characteristics for each parasite are given: it's systematic position, the spectrum of hosts, localization, collecting site, biology, the degree of host specificity and geographical range. The morphological description and original figures of 11 species of trematodes are presented.

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.

Trichobilharzia regenti (Digenea: Schistosomatidae): changes of body wall musculature during the development from miracidium to adult worm.

Trichobilharzia regenti (Schistosomatidae, Digenea), a parasite of birds, exhibits a unique strategy among schistosomes, having affinity to the nervous system of vertebrate hosts. Migration of parasitic stages within hosts and/or swimming of non-parasitic larvae in water environment depend on the action of body wall muscles which were studied with confocal and electron microscopy. In all stages, body wall musculature is comprised of differently organized circular and longitudinal muscles. During the development, an extensive change of musculature characteristics and/or formation of new muscle structures were recorded; cercariae, schistosomula and adult worms produce additional underlying diagonal muscle fibers and inner plexus of radial musculature. Substantial changes of the outer environment during penetration of a host (osmotic values of water vs. host tissues) are accompanied by surface transformation of miracidia/mother sporocysts and cercariae/schistosomula. Contrary to that, changes of body musculature in these stages are characterized only by growth and re-organization of existing structures, and never by formation of new components of body musculature. Future studies in this field may contribute to a better knowledge of morphology and function of trematode muscles, including those of schistosomes that are important pathogens of humans and animals.

Cercariae developing in Lymnaea natalensis Krauss, 1848 collected in the vicinity of Pretoria, Gauteng Province, South Africa.

Freshwater snails are known to serve as first intermediate hosts for various parasitic diseases such as schistosomosis and fasciolosis. Snails were collected on several occasions in the proximity of Pretoria, South Africa and their cercarial sheddings were studied. This article describes three different types of cercariae shed by the freshwater snail, Lymnaea natalensis, viz. a fork-tailed cercaria of a Trichobilharzia sp., an avian parasite belonging to the family Schistosomatidae, an echinostomatid cercaria of the family Echinostomatidae, also avian parasites and a xiphidiocercaria of the family Plagiorchiidae which parasitise avians and amphibians. The morphology of these cercariae was studied by light and scanning electron microscopy.

An approach to revealing blood fluke life cycles, taxonomy, and diversity: provision of key reference data including DNA sequence from single life cycle stages.

Revealing diversity among extant blood flukes, and the patterns of relationships among them, has been hindered by the difficulty of determining if specimens described from different life cycle stages, hosts, geographic localities, and times represent the same or different species. Persistent collection of all available life cycle stages and provision of exact collection localities, host identification, reference DNA sequences for the parasite, and voucher specimens eventually will provide the framework needed to piece together individual life cycles and facilitate reconciliation with classical taxonomic descriptions, including those based on single life cycle stages. It also provides a means to document unique or rare species that might only ever be recovered from a single life cycle stage. With an emphasis on the value of new information from field collections of any available life cycle stages, here we provide data for several blood fluke cercariae from freshwater snails from Kenya, Uganda, and Australia. Similar data are provided for adult worms of Macrobilharzia macrobilharzia and miracidia of Bivitellobilharzia nairi. Some schistosome and sanguinicolid cercariae that we recovered have peculiar morphological features, and our phylogenetic analyses (18S and 28S rDNA and mtDNA CO1) suggest that 2 of the new schistosome specimens likely represent previously unknown lineages. Our results also provide new insights into 2 of the 4 remaining schistosome genera yet to be extensively characterized with respect to their position in molecular phylogenies, Macrobilharzia and Bivitellobilharzia. The accessibility of each life cycle stage is likely to vary dramatically from one parasite species to the next, and our examples validate the potential usefulness of information gleaned from even one such stage, whatever it might be.

Description and surface topography of the cercaria of Austrobilharzia sp. (Digenea: Schistosomatidae).

The cercaria of Austrobilharzia sp. from the marine prosobranch gastropod Planaxis sulcatus in Kuwait Bay is described. The surface microtopography and pattern of the tegumentary sensory receptors are examined using scanning electron microscopy. The general microtopography of the surface of the cercaria is similar to that previously observed in cercariae of mammalian schistosomes, although differences are recorded in the types, numbers and distribution of the sensory receptors. The study identified more than 13 types of receptors comprising aciliated, uniciliated and for the first time a multiciliated receptor in a strigeid cercaria. The ciliated receptor types differ in the cilium length and structure of the surrounding collar and tegumentary base. The receptor types are site specific: (1) the aciliated and pitlike on the anterior organ-neck region and ventral sucker; (2) the uniciliated with a long flexible cilium with or without collar or a tegumentary base on the body and tail; and (3) the uniciliated with a short rigid cilium and a robust collar and tegumentary base, and the multiciliated with 6 flexible cilia and a high cylindrical collar on the anterior organ tip. The reported SEM information on the sensory receptors may contribute to elucidating their functional role and to establishing morphological characters for the phylogeny of the family Schistosomatidae.

Observations on cercarial chaetotaxy as a means for the identification of European species of Trichobilharzia Skrjabin & Zakharow, 1920 (Digenea: Schistosomatidae).

In order to evaluate the importance of cercarial chaetotaxy in the identification of European avian schistosomatids of the genus Trichobilharzia Skrjabin & Zakharow, 1920, papillary patterns of T. franki Müller & Kimmig, 1994 and T. ocellata (La Valette, 1855) Brumpt, 1931 were examined and compared. Scanning electron microscopy (SEM) was also employed to confirm the number and distribution of the sensory papillae. The chaetotaxy of T. franki and T. ocellata were identical, which suggests a close kinship between the two species. Some sensory papillae stained insufficiently with silver nitrate or were difficult to examine using light microscopy. Thus, the use of SEM in future examinations of Trichobilharzia chaetotaxy will ensure the collection of data that are comparable between studies, even though some argentophilic structures are not visible using SEM.

Invasion of the vertebrate skin by cercariae of Trichobilharzia ocellata: penetration processes and stimulating host signals.

The penetration of Trichobilharzia ocellata cercariae into the skin of their duck hosts was described using electron microscopy and histology. The behavior patterns of the cercariae on their exposure to human skin differed only little from those known for Schistosoma mansoni cercariae. After their attachment to living human skin the cercariae crept to wrinkles within a mean of 8 s, and full penetration was achieved within a mean of 4.0 min (83 s to 13.3 min). Tail shedding occurred as early as within a mean of 6.5 s of the first penetration attempts. It was supported by a muscular sphincter at the cercarial hindbody. The skin-surface stimuli for cercarial penetration were contained in the lipid fraction of the duck and human skin surface; hydrophilic components were effective only in some T. ocellata isolates. The penetration-stimulating components of duck-skin lipids were exclusively free fatty acids with the same chemical characteristics known to stimulate penetration of Schistosoma species. Skin-surface lipids of the abnormal human host, with their higher fatty acid contents, stimulated higher cercarial penetration rates than did skin lipids of the natural duck host. Fatty acids as penetration stimuli may offer advantages for T. ocellata cercariae by increasing the specificity for an invasion of terrestrial vertebrates, which is additionally determined by cholesterol and ceramides as signals for attachment and enduring contact behavior.

Investigation of the life cycle and adult morphology of the avian blood fluke Austrobilharzia variglandis (Trematoda: Schistosomatidae) from Connecticut.

This study was undertaken to expand the current knowledge of the life cycle and adult morphology of the avian schistosome Austrobilharzia variglandis, which causes a marine cercarial dermatitis in New England. The specific objectives were to: (1) investigate the seasonality of the infection in the molluscan intermediate host, Ilyanassa obsoleta; (2) determine which bird species are acting as natural definitive hosts for the parasite; and (3) characterize the morphology of the parasite using scanning electron microscopy (SEM). One-thousand individuals of I. obsoleta were collected each month for 14 consecutive months and examined for the parasite. Ten to 15 specimens of each of the following avian species, Larus argentatus, Larus delawarensis, Larus marinus, Phalacrocorax auritus, and Branta canadensis, and 2 individuals of Larus atrilla, were collected and examined for schistosomes. Twenty adult male and 10 adult female specimens of A. variglandis were processed for SEM. Ilyanassa obsoleta was found to maintain a relatively low prevalence of infection (0.7-5.1%) throughout the 14-mo study, but no fully developed cercaria were visible in sporocysts recovered from snails collected in winter months. The Larus species had both the highest prevalence (85-92.8%) and highest mean intensity (12.1-34.5 male worms) of infection with A. variglandis. These data suggest that overwintering snail populations can harbor viable infections and in the spring infect shore birds (or humans) with cercaria. The snail and definitive host data suggest that A. variglandis is a year-round resident of the state.(ABSTRACT TRUNCATED AT 250 WORDS)

[Trichobilharzia franki n. sp.--the cause of swimmer's dermatitis in southwest German dredged lakes]. / Trichobilharzia franki n. sp.--die Ursache für Badedermatitiden in südwestdeutschen Baggerseen.

Ocellate furcocercariae from the lymnaeid snail Radix auricularia have been identified as causative agents of swimmer's itch, which has become increasingly common in the 'Tunisee' near Freiburg. On an average 11.0%, maximally 25.0%, of the snails were parasitized with these trematode larvae. The Trichobilharzia parasitizing rate in the intermediate hosts found here was thus approximately 10-100 times higher than the Trichobilharzia prevalence described so far. Dwarf mallards (Anas platyrhynchos, blood red) were experimentally infected with Trichobilharzia cercariae from the 'Tunisee'. From the 13th day p.i. miracidia could be found in their faeces by means of the hatching method. The trematode eggs had the shape of an elongated spindle, with a pointed and a rounded end. The sexually differentiated adult worms were localized only in the hepatic and enteric veins. They had an average length of 4.0 mm (3.2-4.6 mm) and a width of 125 microns. Their tegument was strongly folded and had tegumental spines in the oral and ventral suckers, in males in addition along the canalis gynaecophorus. The most striking internal organ was the gut filled with hematin and separated in 2 caeca at its anterior end. Its form and localisation as well as those of the sexual organs were important criteria for the differentiation of the different Trichobilharzia species. The Trichobilharzia species found in the 'Tunisee' clearly differed from Trichobilharzia szidati both morphologically and physiologically. The latter has been considered, until now, to be the main causative agent of swimmer's itch in Germany. Moreover, the adult trematodes from the 'Tunisee' could not be associated with any other species of this genus. We propose therefore the name Trichobilharzia franki n. sp. for the species from the Tunisee.

[The effect of praziquantel on Trichobilharzia (Digenea, Schistosomatidae), a cause of swimmer's dermatitis in humans]. / Die Wirkung von Praziquantel auf Trichobilharzia (Digenea, Schistosomatidae), einen Verursacher von Badedermatitiden beim Menschen).

Cases of swimmer's itch caused by cercariae of the genus Trichobilharzia have spread during the last years in Southwestern Germany and increased in intensity. The only consequence was to forbid swimming because no effective strategies are known which would be ecologically or financially acceptable. In this examination we therefore tried, by means of medication of ducks, the possible final hosts, to kill the different stages of Trichobilharzia in patency and prepatency in order to interrupt the parasitic cycle. For this the effectiveness of praziquantel on the parasitic stages in the final host (preadults, adults, eggs or fully developed miracidia) was tested in experimentally infected dwarf mallards (Anas platyrhynchos, hemerot.) and mallards (Anas platyrhynchos platyrhynchos). During therapy of ducks with patent Trichobilharzia infection, onefold or twofold application of this drug (1 x 500 mg/duck, 1 x 300 mg/duck, 2 x 200 mg/duck) only prevented the releasing of eggs with hatchable miracidia for up to 24 h. Only a threefold application of 200 mg/duck in 24 h intervals led to a permanent reduction of detectable miracidia. Application of praziquantel in low doses (30 or 40 mg per duck and day) did not reduce the number of released miracidia. Medication with praziquantel led to a strong shift of adult worms located in the enteric veins of the ducks to the liver veins in a little as 3 h; examinations by scanning electron microscopy revealed changes in the worms' surface and damage of the tegument. During prepatency doses of 22.5 mg praziquantel per duck and day, given continuously for one week, were sufficient to completely stop the release of miracidia. In this low dosage, a special preparation of the bitter drug was voluntarily swallowed by the ducks, thus providing in principal a simple, cheap and ecologically sound method of controlling cercarial dermatitis.

Trichobilharzia ocellata: interference with endocrine control of female reproduction of Lymnaea stagnalis.

Calfluxin (CaFl), one of the gonadotropic hormones of Lymnaea stagnalis, stimulates the influx of Ca2+ into the mitochondria of the cells of the albumen gland, one of the accessory sex organs of the snail. This effect is suppressed in glands of noninfected snails by an agent (schistosomin) present in the hemolymph of snails infected by Trichobilharzia ocellata as shown in in vitro experiments. The agent is present from 6 weeks postinfection onward. Ca2+ deposits in the mitochondria were demonstrated with the ultracytochemical antimonate precipitation technique. The percentage of Ca2+-positive mitochondria was taken as a measure for the effects of CaFl. This percentage appeared to be greatly reduced when glands were incubated in serum of infected snails (Sinf). The data showed that Ringer incubations can serve as controls for experiments with serum: no differences were found between Ringer incubations and incubations in either fresh or frozen serum of noninfected snails. Schistosomin was not affected by freezing, which enables cold storage of Sinf. The dose-response relationship of schistosomin shows that at a 1:2 dilution of Sinf with Ringer the response to CaFl was reduced more than 50%. Schistosomin is heat-stable and Pronase-labile, which indicates that it has a peptide nature. Probably schistosomin(s) is responsible for the reduction/cessation of fecundity in trematode-infected snails.