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Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula migrating to final localization. During invasion, cercariae employ histolytic and other bioactive products of specialized holocrine secretory cells – postacetabular (PA) and circumacetabular (CA) penetration glands. Although several studies attempted to characterize protein composition of the in vitro induced gland secretions in Schistosoma mansoni and Schistosoma japonicum, the results were inconsistent and dependent on the method of sample collection and processing. Products of both gland types mixed during their secretion did not allow localization of identified proteins to a particular gland. Here we compared proteomes of separately isolated cercarial gland cells of the avian schistosome Trichobilharzia szidati employing laser-assisted microdissection and shotgun LC-MS/MS, thus obtaining the largest dataset so far concerning the representation and localization of cercarial penetration gland proteins. We optimized the methods of sample processing with cercarial bodies (heads) first. Alizarin-pre-stained, chemically non-fixed samples provided optimal results of MS analyses, and enabled distinguishing PA and CA glands for microdissection. Using 7.5 × 106 μm3 sample volume per gland replicate, we identified 3347 peptides assigned to 792 proteins, from which 461 occurred in at least 2 of 3 replicates in either gland type (PA = 455, 40 exclusives; CA = 421, 6 exclusives; 60 proteins differed significantly in their abundance between the glands). Peptidases of 5 catalytic types accounted for ca. 8 % and 6 % of reliably identified proteins in PA and CA glands, respectively. Invadolysin, nardilysin, cathepsins B2 and L3, and elastase 2b orthologs were the major gland endopeptidases. Two cystatins and a serpin were highly abundant peptidase inhibitors in the glands. CA glands were rich in venom allergen-like proteins. The assembled total cercarial body proteome included 1631 identified proteins and revealed additional interesting factors possibly related to tissue invasion.HighlightsProteomes of two penetration gland types in schistosome cercariae greatly differPostacetabular glands possess 40 unique proteins and are abundant in hydrolasesCircumacetabular glands posses 6 unique proteins and are rich in VAL proteinsPeptidases make up 8 % of postacetabular and 6 % of circumacetabular gland proteinsCercarial elastase is unique to circumacetabular glands of Trichobilharzia szidatiNote: Supplementary data associated with this article All supplementary data files can be accessed from the following link: http://www.helminthology.cz/supplementary_files.html

In the present study, we analyzed the morphology of three genetic types of the bird-infecting acanthocephalan Polymorphus cf. minutus (PspT1, PspT2, PspT3), mainly based on the cystacanth-stage obtained from amphipods (Gammarus fossarum, Gammarus pulex, Gammarus roeselii, Echinogammarus spp.). Males and females were pooled as there was no considerable difference between the sexes concerning the hook measurements. Additionally, we conducted a laboratory infection of one domestic duck for each Polymorphus type, to compare their performance and localization in this host species, and to obtain adult specimens for morphological comparison. The recovery rate from the ducks 4 weeks after infection was 16% for PspT1, 23.8% for PspT2, and 25% for PspT3. The adult worms were gravid, and the females contained mature eggs. Hook size did not differ considerably between cystacanths and adults of the respective type. The three Polymorphus types could be distinguished based on the cystacanth stage by a linear discriminant analysis that included hook measurements, proboscis length, proboscis width, and number of longitudinal hook rows and hooks per row. Furthermore, PspT3 was more different from PspT1 and PspT2 than the latter types from each other. Mainly the number of longitudinal hook rows differed in PspT3 from the existing descriptions of P. minutus (mainly 14 vs. mainly 16 rows). Potentially, PspT3 could be a non-indigenous parasite that was introduced with G. roeselii and that adapted to use the indigenous G. pulex as a host, while PspT2 might have been introduced to central Europe together with Echinogammarus spp.

No effective method has yet been developed to prevent the threat posed by the emerging disease—cercarial dermatitis (swimmer’s itch), caused by infective cercariae of bird schistosomes (Digenea: Schistosomatidae). In our previous studies, the New Zealand mud snail—Potamopyrgus antipodarum (Gray, 1853; Gastropoda, Tateidae)—was used as a barrier between the miracidia of Trichobilharzia regenti and the target snails Radix balthica. Since the presence of non-indigenous snails reduced the parasite prevalence under laboratory conditions, we posed three new research questions: (1) Do bird schistosomes show totally perfect efficacy for chemotactic swimming behavior? (2) Do the larvae respond to substances emitted by incompatible snail species? (3) Do the excretory-secretory products of incompatible snail species interfere with the search for a compatible snail host? The experiments were carried out in choice-chambers for the miracidia of T. regenti and T. szidati. The arms of the chambers, depending on the variant, were filled with water conditioned by P. antipodarum, water conditioned by lymnaeid hosts, and dechlorinated tap water. Miracidia of both bird schistosome species chose more frequently the water conditioned by snails—including the water conditioned by the incompatible lymnaeid host and the alien species, P. antipodarum. However, species-specific differences were noticed in the behavior of miracidia. T. regenti remained more often inside the base arm rather than in the arm filled with water conditioned by P. antipodarum or the control arm. T. szidati, however, usually left the base arm and moved to the arm filled with water conditioned by P. antipodarum. In conclusion, the non-host snail excretory-secretory products may interfere with the snail host-finding behavior of bird schistosome miracidia and therefore they may reduce the risk of swimmer’s itch.

Migration of parasitic worms through the host tissues, which may occasionally result in fatal damage to the internal organs, represents one of the major risks associated with helminthoses. In order to track the parasites, traditionally used 2D imaging techniques such as histology or squash preparation do not always provide sufficient data to describe worm location/behavior in the host. On the other hand, 3D imaging methods are widely used in cell biology, medical radiology, osteology or cancer research, but their use in parasitological research is currently occasional. Thus, we aimed at the evaluation of suitability of selected 3D methods to monitor migration of the neuropathogenic avian schistosome Trichobilharzia regenti in extracted spinal cord of experimental vertebrate hosts. All investigated methods, two of them based on tracking of fluorescently stained larvae with or without previous chemical clearing of tissue and one based on X-ray micro-CT, exhibit certain limits for in vivo observation. Nevertheless, our study shows that the tested methods as ultramicroscopy (used for the first time in parasitology) and micro-CT represent promising tool for precise analyzing of parasite larvae in the CNS. Synthesis of these 3D imaging techniques can provide more comprehensive look at the course of infection, host immune response and pathology caused by migrating parasites within entire tissue samples, which would not be possible with traditional approaches.

Swimmer’s itch is a re-emerging human disease caused by bird schistosome cercariae, which can infect bathing or working people in water bodies. Even if cercariae fail after penetrating the human skin, they can cause dangerous symptoms in atypical mammal hosts. One of the natural methods to reduce the presence of cercariae in the environment could lie in the introduction of non–host snail species to the ecosystem, which is known as the “dilution” or “decoy” effect. The caenogastropodPotamopyrgus antipodarum—an alien in Europe—could be a good candidate against swimmer’s itch because of its apparent resistance to invasion by European bird schistosome species and its high population density. As a pilot study on this topic, we have carried out a laboratory experiment on howP. antipodaruminfluences the infestation of the intermediate hostRadix balthica(a native lymnaeid) by the bird schistosomeTrichobilharzia regenti. We found that the co–exposure of 200P. antipodarumindividuals per oneR. balthicato theT. regentimiracidia under experimental conditions makes the infestation ineffective. Our results show that a non–host snail population has the potential to interfere with the transmission of a trematode via suitable snail hosts.

Notocotylus attenuatus (Digenea: Notocotylidae) is a monostome fluke parasitizing the intestinal caeca of waterfowl that uses an injection apparatus to infect its intermediate snail host. Morphology of the invading larva (a sporocyst), and the intramolluscan larval development of this fluke have not been characterized extensively. In this study, experimental infections of Lymnaea stagnalis using N. attenuatus eggs resulted in the development of sporocysts containing one germ ball or mother redia between 12 and 21 days post exposure (p.e.) within the hepatopancreas. Independent mother rediae and developing daughter rediae were present between day 25 and day 42 p.e. Cercariae, within the body of rediae, were detected 42 days p.e. The development of daughter rediae and cercariae started posteriorly in the body of parent redia and these larvae migrated anteriorly during development towards the birth pore. A cercaria was also observed emerging from the birth pore and released cercariae maturated further within the snail hepatopancreas prior to leaving the snail. The intramolluscan development was completed 45 days p.e. when the first fully formed cercariae were shed into the outer environment. These data detail the fascinating post-embryonic development of N. attenuatus and highlight the intricate nature of larval transitions within its snail host.

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.

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.

Hepatozoon ayorgbor n. sp. is described from specimens of Python regius imported from Ghana. Gametocytes were found in the peripheral blood of 43 of 55 snakes examined. Localization of gametocytes was mainly inside the erythrocytes; free gametocytes were found in 15 (34.9%) positive specimens. Infections of laboratory-reared Culex quinquefasciatus feeding on infected snakes, as well as experimental infection of juvenile Python regius by ingestion of infected mosquitoes, were performed to complete the life cycle. Similarly, transmission to different snake species (Boa constrictor and Lamprophis fuliginosus) and lizards (Lepidodactylus lugubris) was performed to assess the host specificity. Isolates were compared with Hepatozoon species from sub-Saharan reptiles and described as a new species based on the morphology, phylogenetic analysis, and a complete life cycle.

Two experimental trials were performed to elucidate the role of rodents in the life cycle of Hepatozoon species using snakes as intermediate hosts. In one trial, two ball pythons, Python regius Shaw, 1802 were force fed livers of laboratory mice previously inoculated with sporocysts of Hepatozoon ayorgbor Sloboda, Kamler, Bulantová, Votýpka et Modrý, 2007. Transmission was successful in these experimentally infected snakes as evidenced by the appearance of intraerythrocytic gamonts, which persisted until the end of trial, 12 months after inoculation. Developmental stages of haemogregarines were not observed in histological sections from mice. In another experimental trial, a presence of haemogregarine DNA in mice inoculated with H. ayorgbor was demonstrated by PCR in the liver, lungs and spleen.