Systematics and life cycles of four avian schistosomatids from Southern Cone of South America.

Relative to the numerous studies focused on mammalian schistosomes, fewer include avian schistosomatids particularly in the southern hemisphere. This is changing and current research emerging from the Neotropics shows a remarkable diversity of endemic taxa. To contribute to this effort, nine ducks (Spatula cyanoptera, S.versicolor, Netta peposaca), 12 swans (Cygnus melancoryphus) and 1,400 Physa spp. snails from Chile and Argentina were collected for adults and larval schistosomatids, respectively. Isolated schistosomatids were preserved for morphological and molecular analyses (28S and COI genes). Four different schistosomatid taxa were retrieved from birds: Trichobilharzia sp. in N. peposaca and S. cyanoptera that formed a clade; S.cyanoptera and S. versicolor hosted Trichobilharzia querquedulae; Cygnus melancoryphus hosted the nasal schistosomatid, Nasusbilharzia melancorhypha; and one visceral, Schistosomatidae gen. sp., which formed a clade with furcocercariae from Argentina and Chile from previous work. Of the physid snails, only one from Argentina had schistosomatid furcocercariae that based on molecular analyses grouped with T. querquedulae. This study represents the first description of adult schistosomatids from Chile as well as the elucidation of the life cycles of N.melancorhypha and T. querquedulae in Chile and Neotropics, respectively. Without well-preserved adults, the putative new genus Schistosomatidae gen. sp. could not be described, but its life cycle involves Chilina spp. and C. melancoryphus. Scanning electron microscopy of T. querquedulae revealed additional, undescribed morphological traits, highlighting its diagnostic importance. Authors stress the need for additional surveys of avian schistosomatids from the Neotropics to better understand their evolutionary history.

Molecular evidence of new freshwater turtle blood flukes (Digenea: Spirorchiidae) in the intermediate snail host Biomphalaria occidentalis Paraense, 1981 in an urban aquatic ecosystem in Brazil.

In this study, two potentially new species of turtle blood flukes (TBFs) (Digenea: Spirorchiidae) have been recorded from South Brazil. The spirorchiid parasites infect the vascular system of turtles, thereby compromising their health. The life cycle of these parasites is not well studied. The larval stage of cercaria is found in intermediate gastropod hosts, with some species presenting similar morphological characteristics, which can result in misinterpretations when using only morphological taxonomy for species identification. In this study, we recorded a single morphotype belonging to the family Spirorchiidae in Biomphalaria occidentalis in an urban aquatic ecosystem in Brazil. However, molecular data (28S rDNA and cytochrome oxidase subunit I) confirmed the presence of two species of Spirorchiidae in the sampled environment; both phylogenetically close to genera previously studied in freshwater turtles from the Peruvian Amazon. In this study, species characterization was possible because of molecular tools. We recommend using more than one molecular marker in future studies focusing on TBFs, which need attention about their evolutionary history and ecology to understand their distribution in South America.

Temperature and light effects on Trichobilharzia szidati cercariae with implications for a risk analysis.

BACKGROUND: Cercarial dermatitis (swimmer's itch) caused by bird schistosome cercariae, released from intermediate host snails, is a common disorder also at higher latitudes. Several cases were observed in the artificial Danish freshwater Ringen Lake frequently used by the public for recreational purposes. The lake may serve as a model system when establishing a risk analysis for this zoonotic disease. In order to explain high risk periods we determined infection levels of intermediate host snails from early spring to late summer (March, June and August) and elucidated the effect of temperature and light on parasite shedding, behavior and life span. RESULTS: Field studies revealed no shedding snails in March and June but in late summer the prevalence of Trichobilharzia szidati infection (in a sample of 226 pulmonate Lymnaea stagnalis snails) reached 10%. When investigated under laboratory conditions the cercarial shedding rate (number of cercariae shed per snail per day) was positively correlated to temperature raising from a mean of 3000 (SD 4000) at 7 °C to a mean of 44,000 (SD 30,000) at 27 °C). The cercarial life span was inversely correlated to temperature but the parasites remained active for up to 60 h at 20 °C indicating accumulation of cercariae in the lake during summer periods. Cercariae exhibited positive phototaxy suggesting a higher pathogen concentration in surface water of the lake during daytime when the public visits the lake. CONCLUSION: The only causative agent of cercarial dermatitis in Ringen Lake detected was T. szidati. The infection risk associated with aquatic activities is low during spring and early summer (March-June). In late summer the risk of infection is high since the release, behavior and life span of the infective parasite larvae have optimal conditions.

Nitric oxide debilitates the neuropathogenic schistosome Trichobilharzia regenti in mice, partly by inhibiting its vital peptidases.

BACKGROUND: Avian schistosomes, the causative agents of human cercarial dermatitis (or swimmer's itch), die in mammals but the mechanisms responsible for parasite elimination are unknown. Here we examined the role of reactive nitrogen species, nitric oxide (NO) and peroxynitrite, in the immune response of mice experimentally infected with Trichobilharzia regenti, a model species of avian schistosomes remarkable for its neuropathogenicity. METHODS: Inducible NO synthase (iNOS) was localized by immunohistochemistry in the skin and the spinal cord of mice infected by T. regenti. The impact of iNOS inhibition by aminoguanidine on parasite burden and growth was then evaluated in vivo. The vulnerability of T. regenti schistosomula to NO and peroxynitrite was assessed in vitro by viability assays and electron microscopy. Additionally, the effect of NO on the activity of T. regenti peptidases was tested using a fluorogenic substrate. RESULTS: iNOS was detected around the parasites in the epidermis 8 h post-infection and also in the spinal cord 3 days post-infection (dpi). Inhibition of iNOS resulted in slower parasite growth 3 dpi, but the opposite effect was observed 7 dpi. At the latter time point, moderately increased parasite burden was also noticed in the spinal cord. In vitro, NO did not impair the parasites, but inhibited the activity of T. regenti cathepsins B1.1 and B2, the peptidases essential for parasite migration and digestion. Peroxynitrite severely damaged the surface tegument of the parasites and decreased their viability in vitro, but rather did not participate in parasite clearance in vivo. CONCLUSIONS: Reactive nitrogen species, specifically NO, do not directly kill T. regenti in mice. NO promotes the parasite growth soon after penetration (3 dpi), but prevents it later (7 dpi) when also suspends the parasite migration in the CNS. NO-related disruption of the parasite proteolytic machinery is partly responsible for this effect.

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.

Some facts on south asian schistosomiasis and need for international collaboration.

In this review, we are discussing South Asian schistosomiasis; more specifically species which are responsible for schistosomiasis in India or South Asia -Schistosoma indicum, S. spindale, S. nasale, S. incognitum, S. gimvicum (S.haematobium), Bivitellobilharzia nairi, Orientobilharzia bomfordi, O. dattai, O. turkestanicum and O.harinasutai, their survival strategies such as mild pathology to the host, producing low egg number and utilizing fresh water snails (Indoplanorbis exustus and Lymnaea luteola) in stagnant water bodies like ponds, lakes, ditches, low laying areas, marshy lands and rice fields. Presently, correct identification of blood fluke species, their immature stages, male schistosomes and their intermediate host details like strain variations, susceptibilities, ecologies are not well studied. Species like B. nairi, O. bomfordi, O. harinasutai (Lymnaea rubiginosa intermediate host for O.harinasutai in Thailand) are also not well studied. Moreover, snail species like Oncomalania spp are not from South Asia, but species of Tricula or Neotricula are reported from this geography, which gives indications of S. mekongi like blood fluke presence in the area. Although in humans, cercarial dermatitis is rampant in rural population with occasional reporting of schistosome eggs in stools, human schistosomiasis is considered absent from this region, despite finding a foci (now dead) of urinary schistosomiasis in Gimvi village of Ratnagiri district, Maharashtra, India. There is great difficulty in diagnosing the infection in man and animals due to low egg production, hence development of a single step antigen detection test is the need of the hour. Interestingly, lethal effect of praziquantel was seen against S.haematobium and S.mansoni. However, this drug failed to cause significant reduction of S. incognitum and S. spindale experimentally suggesting some differences in the biology of two groups of the schistosomes. Triclabendazole showed adulticidal effect at a dose rate of 20 mg/kg body against female schistosome worms, but at lower dose (10 mg/kg body wt) of the drug, a dose that is used in treating bovine fascioliasis, it is providing chances of drug resistance of the persisting schistosomes against triclabendazole. Though the South Asian institutes have all the facilities to tackle issues related to existing schistosomes, it is recommended to develop an international collaboration by establishing an international centre on schistosomiasis in India.

Comparative Transcriptomic Exploration Reveals Unique Molecular Adaptations of Neuropathogenic Trichobilharzia to Invade and Parasitize Its Avian Definitive Host.

To date, most molecular investigations of schistosomatids have focused principally on blood flukes (schistosomes) of humans. Despite the clinical importance of cercarial dermatitis in humans caused by Trichobilharzia regenti and the serious neuropathologic disease that this parasite causes in its permissive avian hosts and accidental mammalian hosts, almost nothing is known about the molecular aspects of how this fluke invades its hosts, migrates in host tissues and how it interacts with its hosts' immune system. Here, we explored selected aspects using a transcriptomic-bioinformatic approach. To do this, we sequenced, assembled and annotated the transcriptome representing two consecutive life stages (cercariae and schistosomula) of T. regenti involved in the first phases of infection of the avian host. We identified key biological and metabolic pathways specific to each of these two developmental stages and also undertook comparative analyses using data available for taxonomically related blood flukes of the genus Schistosoma. Detailed comparative analyses revealed the unique involvement of carbohydrate metabolism, translation and amino acid metabolism, and calcium in T. regenti cercariae during their invasion and in growth and development, as well as the roles of cell adhesion molecules, microaerobic metabolism (citrate cycle and oxidative phosphorylation), peptidases (cathepsins) and other histolytic and lysozomal proteins in schistosomula during their particular migration in neural tissues of the avian host. In conclusion, the present transcriptomic exploration provides new and significant insights into the molecular biology of T. regenti, which should underpin future genomic and proteomic investigations of T. regenti and, importantly, provides a useful starting point for a range of comparative studies of schistosomatids and other trematodes.

The Early Worm Catches the Bird? Productivity and Patterns of Trichobilharzia szidati Cercarial Emission from Lymnaea stagnalis.

Digenean trematodes are common and abundant in aquatic habitats and their free-living larvae, the cercariae, have recently been recognized as important components of ecosystems in terms of comprising a significant proportion of biomass and in having a potentially strong influence on food web dynamics. One strategy to enhance their transmission success is to produce high numbers of cercariae which are available during the activity peak of the next host. In laboratory experiments with 13 Lymnaea stagnalis snails infected with Trichobilharzia szidati the average daily emergence rate per snail was determined as 2,621 cercariae, with a maximum of 29,560. During a snail's lifetime this summed up to a mass equivalent of or even exceeding the snail's own body mass. Extrapolated for the eutrophic pond where the snails were collected, annual T. szidati biomass may reach 4.65 tons, a value equivalent to a large Asian elephant. Emission peaks were observed after the onset of illumination, indicating emission synchronizing with the high morning activities of the definitive hosts, ducks. However, high cercarial emission is possible throughout the day under favorable lightning conditions. Therefore, although bird schistosomes, such as T. szidati constitute only a fraction of the diverse trematode communities in the studied aquatic ecosystem, their cercariae can still pose a considerable risk for humans of getting cercarial dermatitis (swimmer's itch) due to the high number of cercariae emitted from infected snails.

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.

Cathepsins B1 and B2 in the neuropathogenic schistosome Trichobilharzia regenti: distinct gene expression profiles and presumptive roles throughout the life cycle.

The neurotropic bird schistosome Trichobilharzia regenti possesses papain-like cysteine peptidases which have also been shown to be crucial enzymes in various developmental stages of the related human parasites Schistosoma spp. In this paper, we present data obtained by real-time polymerase chain reaction on the temporal distribution of transcripts of two cathepsins in different developmental stages of T. regenti: cathepsin B1 originally described from the gut lumen of schistosomula with presumptive role in nutrient digestion and cathepsin B2 originally found in penetration glands of cercariae with probable involvement in invasion of the final host. In spite of their mutual resemblance at the sequence level, the mRNA expression profiles clearly show distinct expression of cathepsins B1 and B2 during the development from eggs to cercariae. In the case of both cathepsins, the highest level of transcription was detected in intravertebrate stages. Putative functions of cathepsins B1 and B2 in schistosome developmental stages are discussed.

Methodical approaches in the identification of areas with a potential risk of infection by bird schistosomes causing cercarial dermatitis.

Larval stages (cercariae) of schistosomatid flukes represent the causative agents of swimmer's itch (cercarial dermatitis), a waterborne allergic disease. Cercariae of bird schistosomes are the most frequently reported agent. Recent studies on parasite behaviour in mammals showed that infections by cercariae can be linked to more than skin syndromes. Despite the failure of complete development in mammals, bird schistosomes can escape from the skin and migrate transitorily in the hosts. These findings brought novel insights into the fate and potential pathogenic effect of the parasites in non-compatible hosts, including humans. Cercarial dermatitis occurs globally and recently is considered to be re-emerging; however, there are no data on the number of afflicted persons per year. This might be explained by a relatively low interest in human skin infections arising after bathing in fresh water. In addition, the real occurrence of bird schistosomes in the field is known only for a few areas. The paucity of epidemiological/biogeographical data is probably caused by difficulties associated with detection of the parasites in intermediate and definitive hosts. Therefore, based on personal experience and data available in the literature, we have summarized methodological approaches enabling the detection of bird schistosomes in various hosts and environments.

Schistosomes in the north: a unique finding from a prosobranch snail using molecular tools.

Samples of schistosome cercariae from three different snail species (Lymnaea stagnalis, Radix auricularia and Valvata (Tropidina) macrostoma) collected from lakes in Central Finland were analyzed using molecular techniques. Based on sequences of ITS region of rDNA, the parasite isolates from L. stagnalis and R. auricularia belong to Trichobilharzia szidati and T. franki, respectively. This confirms a wide distribution of these two species in Europe. On the other hand, the isolates from V. macrostoma represent a unique finding--they belong to yet unknown schistosome species falling into the bird schistosome clade. Therefore, identification of natural final hosts and morphological characterization of particular developmental stages need to be performed in the future.

In vitro cultivation of early schistosomula of nasal and visceral bird schistosomes (Trichobilharzia spp., Schistosomatidae).

Cercariae of bird schistosomes (Trichobilharzia szidati and Trichobilharzia regenti) were mechanically stimulated to transform to schistosomula and kept in different cultivation media supplemented with duck red blood cells and/or homogenized nervous tissue. The development under in vitro conditions was compared with that in vivo, using the following characters: emptying of penetration glands, surface changes, food uptake, and growth of early schistosomula. The results show that the cultivation medium routinely used for human schistosomes is also suitable for mass production of early schistosomula of bird schistosomes, including the unique nasal species-T. regenti. The changes observed resemble those present in worms developing in vivo; therefore, the in vitro produced early schistosomula might be used for further studies of host-parasite interactions.

Molecular systematics of the avian schistosome genus Trichobilharzia (Trematoda: Schistosomatidae) in North America.

Trichobilharzia is a genus of threadlike schistosomes with a cosmopolitan distribution in birds. Species of Trichobilharzia achieve notoriety as major etiological agents of cercarial dermatitis, or swimmer's itch. There are 40 species described in the literature, for which the majority lacks molecular sequence information. To better understand the phylogenetic relationships, diversity, species boundaries, host use, and geographic distribution of this genus better, we surveyed 378 birds and over 10,000 snails from North America. The phylogenetic analysis was based on nuclear 18S, 28S rDNA, internal transcribed spacer region and mitochondrial cytochrome oxidase I sequence data. Specimens were recovered that could be related to 6 of the 14 described species of Trichobilharzia from North America (Trichobilharzia physellae, Trichobilharzia querquedulae, Trichobilharzia szidati, Trichobilharzia stagnicolae, Trichobilharzia franki, and Trichobilharzia brantae). An additional 5 lineages were found that could not be related directly to previously described species. Trichobilharzia brantae, transmitted by Gyraulus parvus, grouped outside the clade containing the recognized species of Trichobilharzia. A subgroup of the Trichobilharzia clade designated Clade Q was comprised of closely related species whose adults and eggs are similar, yet the European species use lymnaeids whereas the North American species use physids as snail hosts. This molecular phylogeny provides a useful framework (1) to facilitate identification of worms, including those involved in dermatitis outbreaks; (2) to test hypotheses about the evolution, diversification, host-parasite interactions and character evolution of Trichobilharzia; and (3) to guide future taxonomic revision of Trichobilharzia.

Peptidases of Trichobilharzia regenti (Schistosomatidae) and its molluscan host Radix peregra S. Lat. (Lymnaeidae): construction and screening of cDNA library from intramolluscan stages of the parasite.

Trichobilharzia regenti is a neurotropic bird schistosome,causing cercarial dermatitis in humans. In this study, ZAP cDNA expression library from Radix peregra s. lat. hepatopancreases containing intramolluscan stages of T. regenti was constructed and screened using PCR with specific and degenerate primers, designed according to previously described serine and cysteine peptidases of other parasite species. Full-length sequences of cathepsins B1 and L, and two serine peptidases, named RpSP1 and RpSP2, were obtained. The protein-protein BLAST analysis and parallel control reactions with template from hepatopancreases of T. regenti non-infected snails revealed that only cathepsin B1 was of parasite origin. The remaining sequences were derived from the snail intermediate host, which implies that the initial source of parasite mRNA was contaminated by snail tissue. Regardless of this contamination, the cDNA library remains an excellent molecular tool for detection and identification of bioactive molecules in T. regenti cercariae.

[Survey on natural nidus of Trichobilharzia in Huainan area].

The life cycle of Trichobilharzia sp. can be completed in Radix auricularia and domestic or wild ducks, and people can contract cercarial dermatitis through water contact. Natural nidus of Trichobilharzia exists in Huainan area.

[Swimming in summer]. / Zwemmen in de zomer.

It happens every summer: 'Swimming prohibited because of cyanobacteria'. Blooming of these toxine producing bacteria in warm summer months is a well known risk for swimming human beings. But how big is this risk for swimming dogs? And how big are other risks? In this article these risks are assessed for dogs that occasionally swim in surface water during the summer in the Netherlands. Based on these findings an advice will be given for dog owners to keep these risks for waterborne infections as small as possible.

Identification of an avian schistosome recovered from Aythya novaeseelandia and infectivity of its miracidia to Lymnaea tomentosa snails.

A partial life cycle involving miracidia hatched from the eggs of Trichobilharzia sp. recovered from New Zealand scaup (Aythya novaeseelandia) to the release of furcocercariae by laboratory snails (Lymnaea tomentosa) was accomplished. Challenges with five and ten miracidia per snail were lethal. Challenge with three miracidia resulted in development to the daughter sporocyst stage and death in five, development to furcocercarial stage and death in one, and shedding of furcocercariae in one of seven snails. Observed lethality of schistosome miracidia to L. tomentosa may explain the low infection prevalence observed in the wild. Future work should plan challenge exposures of three or fewer miracidia to ensure snail survival and successful recovery of furcocercariae. The Trichobilharzia sp. found in the New Zealand scaup does not key morphologically to the literature. It may be a new species and further work is needed.

Molluscan and vertebrate immune responses to bird schistosomes.

There is a growing understanding of risks posed by human contact with the cercariae of bird schistosomes. In general, there are no fundamental biological differences between human and bird schistosomes in terms of their interactions with snail and vertebrate hosts. The penetration of host surfaces is accompanied by the release of penetration gland products and the shedding of highly antigenic surface components (miracidial ciliated plates and cercarial glycocalyx) which trigger host immune reactions. New surface structures are formed during transformation: the tegument of mother sporocysts and the tegumental double membrane of schistosomula. These surfaces apparently serve as protection against the host immune response. Certain parasite excretory-secretory products may contribute to immunosuppression or, on the other hand, stimulation of host immune reactions. Discovery of new species and their life cycles, the characterization of host-parasite interactions (including at the molecular level), the determination of parasite pathogenicity towards the host, the development of tools for differential diagnosis and the application of protective measures are all topical research streams of the future. Regularly updated information on bird schistosomes and cercarial dermatitis can be found at http://www.schistosomes.cz (web pages of Schistosome Group Prague).

Trichobilharzia regenti: the developmental differences in natural and abnormal hosts.

Trichobilharzia regenti is a bird nasal parasite causing human cercarial dermatitis. Schistosomula are able to migrate via the bird nervous system and then, they mature and lay eggs in the nasal cavity. To some extent they can also migrate and develop in mammals. The present study has shown the developmental differences of T. regenti in the natural (ducks) and the abnormal (mice; inbred strains BALB/c, SCID) hosts. The study describes the following parameters of developing worms: length and width of the body, length and content of the intestine, development of the reproductive organs and characterization of surface and intestinal epithelium by lectin probes. The differences in length and width of schistosomula localized in the spinal cord of various hosts cannot be simply explained and may depend on yet unknown host factors. Moreover, there must be several physiological changes during the migration through the skin, the nervous tissue and the nasal cavity, enabling uptake and digestion of different host components. For example the intestine of schistosomula was mostly filled with light-brown pigmented granules until 6 days p.i. (probably of nervous tissue origin) while the older schistosomula and adult intestine was mostly full of dark-brown pigment (probably of blood origin). Reproductive organs were observed from day 9 p.i. in worms from ducks. Whereas ConA and PSA specifically bound to the surface and intestinal epithelium of schistosomula and adults, only the labelled UEA-I lectin could be used as a surface marker of cercaria-schistosomulum transformation. The results confirmed retarded development of parasites in abnormal hosts; the factor responsible for this phenomenon should be clarified in the future.