Morphological and genomic characterisation of the Schistosoma hybrid infecting humans in Europe reveals admixture between Schistosoma haematobium and Schistosoma bovis.

Schistosomes cause schistosomiasis, the world's second most important parasitic disease after malaria in terms of public health and social-economic impacts. A peculiar feature of these dioecious parasites is their ability to produce viable and fertile hybrid offspring. Originally only present in the tropics, schistosomiasis is now also endemic in southern Europe. Based on the analysis of two genetic markers the European schistosomes had previously been identified as hybrids between the livestock- and the human-infective species Schistosoma bovis and Schistosoma haematobium, respectively. Here, using PacBio long-read sequencing technology we performed genome assembly improvement and annotation of S. bovis, one of the parental species for which no satisfactory genome assembly was available. We then describe the whole genome introgression levels of the hybrid schistosomes, their morphometric parameters (eggs and adult worms) and their compatibility with two European snail strains used as vectors (Bulinus truncatus and Planorbarius metidjensis). Schistosome-snail compatibility is a key parameter for the parasites life cycle progression, and thus the capability of the parasite to establish in a given area. Our results show that this Schistosoma hybrid is strongly introgressed genetically, composed of 77% S. haematobium and 23% S. bovis origin. This genomic admixture suggests an ancient hybridization event and subsequent backcrosses with the human-specific species, S. haematobium, before its introduction in Corsica. We also show that egg morphology (commonly used as a species diagnostic) does not allow for accurate hybrid identification while genetic tests do.

Evidence of a Putative Novel Species of Avian Schistosome Infecting Planorbella trivolvis.

Freshwater gastropods of the genera Lymnaea Lamarck, 1799, Physa Draparnaud, 1801, Gyraulus Charpentier, 1837, Radix Montfort, 1810, and Stagnicola Jeffreys, 1830 are considered suitable intermediate hosts for avian schistosomes. A large trematode biodiversity survey performed across 3 yr on 6 lakes in Alberta confirmed 3 already-reported snail hosts for 7 North American avian schistosomes; however, the cytochrome c oxidase subunit 1 (COI) nucleotide sequence from 1 cercarial sample (from a single specimen of Planorbella trivolvis) was distinct from all other COI schistosome sequences. As part of a simultaneous, comparable study of P. trivolvis by us in Michigan, we collected another cercarial type from 6 lakes that was 99% similar (COI) to the aforementioned cercarial type. Phylogenetic analyses of the COI and 28S rDNA genes recovered the former cercaria in a clade of avian schistosomes. In Michigan, the feces of a Canada goose (Branta canadensis Linnaeus, 1758) had a miracidium with an identical COI nucleotide sequence. Preliminary swimmer's itch and cercarial emergence studies were performed to determine if the cercariae could cause swimmer's itch and to study the emergence pattern as compared with species of Trichobilharzia Skrjabin and Zakharow, 1920.

Migratory routes, domesticated birds and cercarial dermatitis: the distribution of Trichobilharzia franki in Northern Iran.

BACKGROUND: One of the major migration routes for birds going between Europe and Asia is the Black Sea-Mediterranean route that converges on the Volga Delta, continuing into the area of the Caspian Sea. Cercarial dermatitis is a disorder in humans caused by schistosome trematodes that use aquatic birds and snails as hosts and is prevalent in areas of aquaculture in Northern Iran. Before the disorder can be addressed, it is necessary to determine the etiological agents and their host species. This study aimed to document whether domestic mallards are reservoir hosts and if so, to characterize the species of schistosomes. Previous work has shown that domestic mallards are reservoir hosts for a nasal schistosome. RESULTS: In 32 of 45 domestic mallards (Anas platyrhynchos domesticus) (71.1%), the schistosome Trichobilharzia franki, previously reported only from Europe, was found in visceral veins. Morphological and molecular phylogenetic analysis confirmed the species designation. These findings extend the range of T. franki from Europe to Eurasia. CONCLUSION: The occurrence of cercarial dermatitis in Iran is high in areas of aquaculture. Previous studies in the area have shown that domestic mallards are reservoir hosts of T. regenti, a nasal schistosome and T. franki, as shown in this study. The genetic results support the conclusion that populations of T. franki from Iran are not differentiated from populations in Europe. Therefore, the schistosomes are distributed with their migratory duck hosts, maintaining the gene flow across populations with compatible snail hosts in Iran.

TITLE: Routes migratoires, oiseaux domestiques et dermatite cercarienne : répartition de Trichobilharzia franki dans le nord de l'Iran. ABSTRACT: Contexte : L'une des principales voies de migration des oiseaux à destination et en provenance de l'Europe et de l'Asie est la route mer Noire-Méditerranée qui converge vers le delta de la Volga et se poursuit dans la région de la mer Caspienne. La dermatite cercarienne est une affection causée chez l'homme par des trématodes Schistosomatidae utilisant des oiseaux aquatiques et des mollusques comme hôtes, qui est répandue dans les zones d'aquaculture du nord de l'Iran. Avant de pouvoir lutter contre cette parasitose, il est nécessaire de connaître les agents étiologiques et leurs espèces hôtes. Ce travail vise à documenter si les canards domestiques sont des hôtes réservoirs et si oui, à caractériser les espèces de schistosomes. Des travaux antérieurs ont montré que les canards domestiques sont des hôtes réservoirs pour un schistosome nasal. Résultats : Chez 32 de 45 canards domestiques (Anas platyrhynchos domesticus) (71,1 %), le schistosome Trichobilharzia franki, précédemment signalé uniquement en Europe, a été trouvé dans les veines viscérales. L'identification de l'espèce a été vérifiée par une analyse morphologique et phylogénétique moléculaire. Cela étend l'aire de répartition de l'Europe à l'Eurasie. Conclusion : La dermatite cercarienne est répandue dans les zones d'aquaculture en Iran. Des travaux antérieurs dans la région ont montré que les canards domestiques sont des hôtes réservoirs de T. regenti, un schistosome nasal et de T. franki, comme le montre cette étude. Les résultats génétiques soutiennent que les populations de T. franki d'Iran ne sont pas différenciées des populations d'Europe. Par conséquent, les schistosomes se dispersent avec leur hôte canard lors de la migration, maintenant un flux génétique entre les populations avec des mollusques hôtes compatibles en Iran.

The Tao survivorship of schistosomes: implications for schistosomiasis control.

Schistosomiasis, caused by blood flukes of the genus Schistosoma, is a major public health problem which contributes substantially to the economic and financial burdens of many nations in the developing world. An array of survival strategies, such as the unique structure of the tegument which acts as a major host-parasite interface, immune modulation mechanisms, gene regulation, and apoptosis and self-renewal have been adopted by schistosome parasites over the course of long-term evolution with their mammalian definitive hosts. Recent generation of complete schistosome genomes together with numerous biological, immunological, high-throughput "-omics" and gene function studies have revealed the Tao or strategies that schistosomes employ not only to promote long-term survival, but also to ensure effective life cycle transmission. New scenarios for the future control of this important neglected tropical disease will present themselves as our understanding of these Tao increases.

Structure-function analysis of apical membrane-associated molecules of the tegument of schistosome parasites of humans: prospects for identification of novel targets for parasite control.

Neglected tropical diseases are a group of some 17 diseases that afflict poor and predominantly rural people in developing nations. One significant disease that contributes to substantial morbidity in endemic areas is schistosomiasis, caused by infection with one of five species of blood fluke belonging to the trematode genus Schistosoma. Although there is one drug available for treatment of affected individuals in clinics, or for mass administration in endemic regions, there is a need for new therapies. A prominent target organ of schistosomes, either for drug or vaccine development, is the peculiar epithelial syncytium that forms the body wall (tegument) of this parasite. This dynamic layer is maintained and organized by concerted activity of a range of proteins, among which are the abundant tegumentary annexins. In this review, we will outline advances in structure-function analyses of these annexins, as a means to understanding tegument cell biology in host-parasite interaction and their potential exploitation as targets for anti-schistosomiasis therapies.

The schistosome oesophageal gland: initiator of blood processing.

BACKGROUND: Although the ultrastructure of the schistosome esophageal gland was described >35 years ago, its role in the processing of ingested blood has never been established. The current study was prompted by our identification of MEG-4.1 expression in the gland and the observation of erythrocyte uncoating in the posterior esophagus. METHODOLOGY/PRINCIPAL FINDINGS: The salient feature of the posterior esophagus, characterized by confocal and electron microscopy, is the enormous increase in membrane surface area provided by the plate-like extensions and basal invaginations of the lining syncytium, with unique crystalloid vesicles releasing their contents between the plates. The feeding process was shown by video microscopy to be divided into two phases, blood first accumulating in the anterior lumen before passing as a bolus to the posterior. There it streamed around a plug of material revealed by confocal microscopy as tethered leucocytes. These were present in far larger numbers than predicted from the volume of the lumen, and in varying states of damage and destruction. Intact erythrocytes were detected in the anterior esophagus but not observed thereafter, implying that their lysis occurred rapidly as they enter the posterior. Two further genes, MEGs 4.2 and 14, were shown to be expressed exclusively in the esophageal gland. Bioinformatics predicted that MEGs 4.1 and 4.2 possessed a common hydrophobic region with a shared motif, while antibodies to SjMEG-4.1 showed it was bound to leucocytes in the esophageal lumen. It was also predicted that MEGs 4.1 and 14 were heavily O-glycosylated and this was confirmed for the former by 2D-electrophoresis and Western blotting. CONCLUSIONS/SIGNIFICANCE: The esophageal gland and its products play a central role in the processing of ingested blood. The binding of host antibodies in the esophageal lumen shows that some constituents are antibody targets and could provide a new source of vaccine candidates.

Bird schistosomes in planorbid snails in the Czech Republic.

Bird schistosomes have been in focus as causative agents of cercarial dermatitis of humans in the last years; however, our knowledge of their species spectrum and intermediate host specificity is still insufficient. Our study focused on bird schistosomes developing in planorbid snails that have been less studied so far. From 2001 to 2010, cercariae of bird schistosomes were found in four snail species (Gyraulus albus, Segmentina nitida, Anisus vortex and Planorbis planorbis) from seven localities in the Czech Republic. Based on morphology and results of molecular analysis, the isolates found belong to at least six species. Five of them are probably undescribed species, and one species appears to be identical with Gigantobilharzia vittensis Reimer, 1963 (syn. G. suebica Dönges, 1964). The finding from S. nitida represents the first report of a bird schistosome from this snail.

Comparison of microscopy and Alamar blue reduction in a larval based assay for schistosome drug screening.

BACKGROUND: In view of the current widespread use of and reliance on a single schistosomicide, praziquantel, there is a pressing need to discover and develop alternative drugs for schistosomiasis. One approach to this is to develop High Throughput in vitro whole organism screens (HTS) to identify hits amongst large compound libraries. METHODOLOGY/PRINCIPAL FINDINGS: We have been carrying out low throughput (24-well plate) in vitro testing based on microscopic evaluation of killing of ex-vivo adult S. mansoni worms using selected compound collections mainly provided through the WHO-TDR Helminth Drug Initiative. To increase throughput, we introduced a similar but higher throughput 96-well primary in vitro assay using the schistosomula stage which can be readily produced in vitro in large quantities. In addition to morphological readout of viability we have investigated using fluorometric determination of the reduction of Alamar blue (AB), a redox indicator of enzyme activity widely used in whole organism screening. A panel of 7 known schistosome active compounds including praziquantel, produced diverse effects on larval morphology within 3 days of culture although only two induced marked larval death within 7 days. The AB assay was very effective in detecting these lethal compounds but proved more inconsistent in detecting compounds which damaged but did not kill. The utility of the AB assay in detecting compounds which cause severe morbidity and/or death of schistosomula was confirmed in testing a panel of compounds previously selected in library screening as having activity against the adult worms. Furthermore, in prospective library screening, the AB assay was able to detect all compounds which induced killing and also the majority of compounds designated as hits based on morphological changes. CONCLUSION: We conclude that an HTS combining AB readout and image-based analysis would provide an efficient and stringent primary assay for schistosome drug discovery.

Schistosoma kisumuensis n. sp. (Digenea: Schistosomatidae) from murid rodents in the Lake Victoria Basin, Kenya and its phylogenetic position within the S. haematobium species group.

Schistosoma kisumuensis n. sp. is described based on 6 adult males and 2 adult females collected from the circulatory system of 3 murid rodent species, Pelomys isseli, Mastomys natalensis, and Dasymys incomtus. Specimens were collected from a single location, Nyabera Swamp, in Kisumu, Kenya in the Lake Victoria Basin. This new species is morphologically similar to members of the S. haematobium group, currently represented by 8 species parasitizing artiodactyls and primates, including humans. Schistosoma kisumuensis differs from these species by producing relatively small Schistosoma intercalatum-like eggs (135.2 x 52.9 microm) with a relatively small length to width ratio (2.55). Comparison of approximately 3000-base-pair sequences of nuclear rDNA (partial 28S) and mtDNA (partial cox1, nad6, 12S) strongly supports the status of S. kisumuensis as a new species and as a sister species of S. intercalatum. The cox1 genetic distance between these two species (6.3%) is comparable to other pairwise comparisons within the S. haematobium group. Separation of the Congo River and Lake Victoria drainage basins is discussed as a possible factor favoring the origin of this species.

Imaging schistosomes in vivo.

Schistosomes are intravascular, parasitic helminths that cause a chronic, often debilitating disease afflicting over 200 million people in over 70 countries. Here we describe novel imaging methods that, for the first time, permit visualization of live schistosomes within their living hosts. The technology centers on fluorescent agent uptake and activation in the parasite's gut, and subsequent detection and signal quantitation using fluorescence molecular tomography (FMT). There is a strong positive correlation between the signal detected and parasite number. Schistosoma mansoni parasites of both sexes recovered from infected experimental animals exhibit vivid fluorescence throughout their intestines. Likewise, the remaining important human schistosome parasites, S. japonicum and S. hematobium, also exhibit gut fluorescence when recovered from infected animals. Imaging has been used to efficiently document the decline in parasite numbers in infected mice treated with the antischistosome drug praziquantel. This technology will provide a unique opportunity both to help rapidly identify much-needed, novel antischistosome therapies and to gain direct visual insight into the intravascular lives of the major schistosome parasites of humans.

Schistosomes in the southwest United States and their potential for causing cercarial dermatitis or 'swimmer's itch'.

Cercarial dermatitis or swimmer's itch results when cercariae of schistosomes penetrate human skin and initiate inflammatory responses. The parasites typically die in the skin but in some cases may persist and infect other organs. Cercarial dermatitis is caused by a complex and poorly known assemblage of schistosome species, and can occur in any location where people come in contact with water bodies harbouring schistosome-infected snails. In North America, most cases are reported from the upper Midwest. In south-western USA, this phenomenon has not been well studied, and it is not known which schistosome species are present, or if cercarial dermatitis occurs with any regularity. As part of our ongoing studies of schistosome diversity, using morphological traits and sequence data to differentiate species, we have thus far identified eight schistosome genetic lineages from snails from New Mexico and Colorado. We have investigated two cercarial dermatitis outbreaks, one occurring in Stubblefield Lake in northern New Mexico, and one in Prospect Lake in the heart of Colorado Springs, Colorado. The New Mexico outbreak involved either one or two different avian schistosome species, both transmitted by physid snails. The Colorado outbreak was due to Trichobilharzia brantae, a species transmitted by geese and the snail Gyraulus parvus. These outbreaks are in contrast to those in northern states where schistosomes infecting snails of the family Lymnaeidae are more often responsible for outbreaks. Our survey suggests that dermatitis-causing schistosomes are not rare in the southwest, and that there are plenty of opportunities for dermatitis outbreaks to occur in this region.

[The structure and function of schistosome tegument and related proteomic study].

Schistosome is covered by a living syncytium, called tegument, which plays important role in nutrient uptake and immune evasion. Recently, tegumental proteomic research identified lots of proteins, of which there may be potential targets for diagnosis, drugs and vaccines.

Seasonal morphological variations in bird schistosomes.

The present work is a contribution to the systematics of Bilhorziello and Dendritobilharzio. Wildfowl was killed in hunting seasons or found dead in Champagne-Ardenne region, France, and autopsied with focus on schistosomes. Seven Anas plotyrhynchos (mallards), one Ardeo cinerea (grey heron) and two Cygnus olor (mute swans) were parasitized by Bilharziella. One C. olor was parasitized by Dendritobilharzia. Depending on season and hosts, various morphological forms of Bilharziello suggesting several species were observed. The differences in male and female worms concerned the morphology of genital apparatus, the spination on suckers, the body size and proportions. However, the comparison of DNA sequences led to a conclusion that these forms belonged to one species, Bilharziella polonica (Kowalewski, 1895). The morphological features and the body sizes of our samples of Dendritobilharzia seemed to differ from the type species of D. pulverulenta (Braun, 1901). Nevertheless, molecular analysis confirmed identity. We hypothesize that the differences in Bilharziella and Dendritobilharzia might be linked to internal host factors (e.g. hormonal levels), and influenced by season, host, and worm age. The definition of the genera Bilharziello and Dendritobilharzio was amended.

Allobilharzia visceralis gen. nov., sp. nov. (Schistosomatidae-Trematoda) from Cygnus cygnus (L.) (Anatidae).

In Iceland, the examination of whooper swans (Cygnus cygnus L.) viscera resulted in the detection of adult digenean flukes of the family Schistosomatidae. The mature worms occurring in the blood vessels of the large intestine and mesenterium caused vascular lesions, around the eggs deposited in the intestinal mucosa and liver granulomatous reactions developed. The morphology of the isolated schistosomes shows certain similarity with the flukes of the genus Trichobilharzia; in males reduced gynecophoral canal, and on both sexes both suckers and spatulate ends are present. However, the Icelandic flukes possess other morphological features which are distinct from the genus: the point of caecal reunion in males takes place posterior to gynecophoral canal and the genital pore is behind acetabulum and anterior to caecal reunion. In order to evaluate the identity of Icelandic schistosomes, sequencing of ITS region of DNA was performed, and the obtained sequence was deposited in GenBank under the accession no. DQ067561. Following phylogenetic analysis of relationship between the sequence of Icelandic flukes and database sequences of other bird schistosome genera (Trichobilharzia, Gigantobilharzia and Dendritobilharzia) showed different position of Icelandic worms in the phylogenetic tree. In conclusion, our study revealed new genus and species of schistosome flukes--Allobilharzia visceralis gen. et sp. n.

Proteins exposed at the adult schistosome surface revealed by biotinylation.

The human blood-dwelling parasite Schistosoma mansoni can survive in the hostile host environment for decades and must therefore display effective strategies to evade the host immune responses. The surface of the adult worm is covered by a living syncytial layer, the tegument, bounded by a complex multilaminate surface. This comprises a normal plasma membrane overlain by a secreted bilayer, the membranocalyx. Recent proteomic studies have identified constituents of the tegument, but their relative locations remain to be established. We labeled the most exposed surface proteins using two impermeant biotinylation reagents that differed only in length. We anticipated that the two reagents would display distinct powers of penetration, thereby producing a differential labeling pattern. The labeled proteins were recovered by streptavidin affinity and identified by tandem mass spectrometry. A total of 28 proteins was identified, 13 labeled by a long form reagent and the same 13 plus a further 15 labeled by a short form reagent. The parasite proteins included membrane enzymes, transporters, and structural proteins. The short form reagent additionally labeled some cytosolic and cytoskeletal proteins, the latter being constituents of the intracellular spines. Only a single secreted protein was labeled, implying a location between the plasma membrane and the membranocalyx or as part of the latter. Four host proteins, three immunoglobulin heavy chains and C3c/C3dg, a fragment of complement C3, were labeled by both reagents indicating their exposed situation. The presence of the degraded complement C3 implicates inhibition of the classical pathway as a major element of the immune evasion strategy, whereas the recovery of only one truly secreted protein points to the membranocalyx acting primarily as an inert protective barrier between the immune system and the tegument plasma membrane. Collectively the labeled parasite proteins merit investigation as potential vaccine candidates.

Description and classification of abnormal cercariae of Schistosoma mattheei and a method of concentration and collection.

Over a period of about 12 years, 30 abnormal Schistosoma mattheei cercariae were found among a total of approximately 2.8 million examined. Initially seven were recovered from about 1.02 million (0.0007%), which were examined individually while being counted with the aid of a stereoscopic microscope. Subsequently, on the strength of relatively high percentages of abnormal individuals recovered when counting cercariae that failed to penetrate into oxen, it appeared that the morphologically abnormal cercariae were unable to swim and would mostly sediment out of a suspension while most of the normal cercariae would remain swimming. This surmise is supported by recovery of 23 morphologically abnormal cercariae (0.001%) from about 1.8 million, by examining the sediment after the cercarial suspension had been left standing undisturbed in glass measuring cylinders. The abnormalities ranged from aberrant tails only (e.g. an underdeveloped tail, or different degrees of schism) or aberrant heads only, to abnormalities of both the heads and tails. A suggested schematic classification of abnormal cercariae is presented. A young, adult hamster was exposed to eight S. mattheei cercariae with complete schism of the shaft of the tail, by pipetting the cercariae onto the shaved abdominal skin of the anaesthetised animal. Two underdeveloped females were subsequently encountered in squash preparations of the liver when the hamster was killed for worm recovery 10 weeks after infection, thus showing that some of the abnormal cercariae were viable. A method is also described for killing and fixing cercariae while retaining some of the shining brilliance of live cercariae, without them becoming shrivelled, granular and semi-opaque, as occurs when cercariae die spontaneously or are killed with heat. This is apparently the first report of abnormal cercariae of S. mattheei. In addition, a method of concentrating abnormal cercariae after emergence from a snail, a schematic classification of abnormal cercariae and a method for killing and fixing cercariae while retaining much of the shiny brilliance of live cercariae are also reported for the first time as far as is known.

Schistosoma ovuncatum n. sp. (Digenea: Schistosomatidae) from northwest Thailand and the historical biogeography of Southeast Asian Schistosoma Weinland, 1858.

Schistosoma sinensium Bao, 1958 was first isolated from an unidentified snail in Sichuan Province, PR China. This species was apparently rediscovered in Chiang Mai Province, northwest Thailand (Baidikul et al., 1984); the definitive host was the rat Rattus rattus and the intermediate host was the snail Tricula bollingi. In this paper S. sinensium is rediscovered in Sichuan Province and compared with worms recovered from experimentally infected mice, which had been exposed to cercariae shed by T. bollingi from Chiang Mai. Evidence is presented suggesting that the schistosome collected by Baidikul was not S. sinensium and that a new species is involved. The new species, named Schistosoma ovuncatum (etymology: ovum (egg) + uncatus (hooked)), is described and compared with related taxa. All previous papers on the Thai schistosome have used worms recovered from field-collected rodents only; this is the first account in which the life-cycle has been completed in the laboratory, using cercariae shed by T. bollingi, and the resulting worms described. S. ovuncatum differs from S. sinensium in terms of size and shape of body and egg, number of testes, size of ovary, length of vitellarium, intermediate host and biogeographical distribution. The relationships of the two taxa and their position with respect to the Schistosoma indicum- and S. japonicum-groups are discussed. The implications of the findings for the evolution of human schistosomiasis in the region are also commented upon.

Schistosomiasis in the Mekong region: epidemiology and phylogeography.

An account is given of progress made over the last 20 years in the study of Mekong schistosomiasis, causative agent Schistosoma mekongi (Trematoda: Digenea). Emphasis is given to the discussion of work concerning the origin and subsequent dispersal of S. mekongi and related taxa, including relevant snails. The role of such phylogeographical data in schistosomiasis control and the prediction of areas at risk is examined. New palaeogeographical models are reviewed in relation to traditional explanations for the biogeographic deployment of Southeast Asian Schistosoma and their intermediate hosts. The demographics and molecular ecology of Neotricula aperta (Gastropoda: Pomatiopsidae), the snail host of S. mekongi, are reviewed with particular reference to new models for the life cycle of this species and their importance in snail control. The use of population genetic data in the limitation of N. aperta populations is evaluated and strategies suggested for schistosomiasis control efforts directed against the intermediate host. Developments in the taxonomy of N. aperta, and related taxa, and changes in nomenclature are covered. The direction of future investigations into the problem of Mekong schistosomiasis is also discussed.

Schistosome cercariae as the causative agent of swimmer's itch in Iceland.

During late summer in 1995 to 1997, repeated outbreaks of maculopapular skin eruptions were noted on the legs of children after wading in the pond in the Family Park in Laugardalur, Reykjavík, Iceland. Clinical symptoms developing on the legs resembled those of cercarial dermatitis. An examination of Lymnaea peregra snails from this pond and from the adjacent Lake Tjornin resulted in detection of previously undescribed schistosome cercariae. This is the first report of schistosomes in Iceland and also the most northern occurrence of these parasites in Europe.