Changes in surface glycosylation and glycocalyx shedding in Trichobilharzia regenti (Schistosomatidae) during the transformation of cercaria to schistosomulum.

The invasive larvae (cercariae) of schistosomes penetrate the skin of their definitive hosts. During the invasion, they undergo dramatic ultrastructural and physiological transitions. These changes result in the development of the subsequent stage, schistosomulum, which migrates through host tissues in close contact with host's immune system. One of the striking changes in the transforming cercariae is the shedding of their thick tegumental glycocalyx, which represents an immunoattractive structure; therefore its removal helps cercariae to avoid immune attack. A set of commercial fluorescently labeled lectin probes, their saccharide inhibitors and monoclonal antibodies against the trisaccharide Lewis-X antigen (LeX, CD15) were used to characterize changes in the surface saccharide composition of the neuropathogenic avian schistosome Trichobilharzia regenti during the transformation of cercariae to schistosomula, both in vitro and in vivo. The effect of various lectins on glycocalyx shedding was evaluated microscopically. The involvement of peptidases and their inhibitors on the shedding of glycocalyx was investigated using T. regenti recombinant cathepsin B2 and a set of peptidase inhibitors. The surface glycocalyx of T. regenti cercariae was rich in fucose and mannose/glucose residues. After the transformation of cercariae in vitro or in vivo within their specific duck host, reduction and vanishing of these epitopes was observed, and galactose/N-acetylgalactosamine emerged. The presence of LeX was not observed on the cercariae, but the antigen was gradually expressed from the anterior part of the body in the developing schistosomula. Some lectins which bind to the cercarial surface also induced secretion from the acetabular penetration glands. Seven lectins induced the shedding of glycocalyx by cercariae, among which five bound strongly to cercarial surface; the effect could be blocked by saccharide inhibitors. Mannose-binding protein, part of the lectin pathway of the complement system, also bound to cercariae and schistosomula, but had little effect on glycocalyx shedding. Our study did not confirm the involvement of proteolysis in glycocalyx shedding.

Phylogenetic analysis of nasal avian schistosomes (Trichobilharzia) from aquatic birds in Mazandaran Province, northern Iran.

Nasal schistosomes are trematodes in the family Schistosomatidae, many members of which are causative agents of human cercarial dermatitis (HCD). Little is known about the species diversity and distribution of nasal dwelling schistosomes of water birds, particularly in countries outside of Europe; even less is known in countries like Iran. Nasal schistosomes are of particular interest since these species migrate via the central nervous system to the nasal cavity once they penetrate their host. Thus, there must be efforts to determine the incidence of HCD due to nasal schistosomes. HCD outbreaks are reported seasonally in Mazandaran Province, northern Iran, an area well known for rice cultivation leading to increased person contact with water and infected snails. Such places include favorable habitat for both domestic ducks year round, and wild migratory ducks in the winter through spring. Recent reports have detected the presence of both nasal and visceral schistosomes in ducks in this area but with little species characterization. In this study, we examine a diversity of aquatic birds to determine the distribution, prevalence and bird host use of nasal schistosomes. We apply for the first time a molecular identification and phylogenetic analysis of these schistosomes. From 2012 to 2014, the nasal cavity of 508 aquatic birds from Mazandaran Province were examined that included species in Anseriformes, Gruiformes, Charadriiformes and Phoenicopteriformes. Nasal schistosomes were found in 45 (8.9%) birds belonging to Anseriformes (Anas platyrhynchos and Anas clypeata). Phylogenetic analysis of the nuclear internal transcribed spacer 1 rDNA and the mitochondrial cytochrome oxidase1 gene of isolated eggs revealed that all samples grouped in a sister clade to the European Trichobilharzia regenti. However, Trichobilharzia from this study were more similar to a unique haplotype of Trichobilharzia, isolated from the nasals of an A. clypeata in France. The genetic and phenotypic differences between the species found herein and T. regenti from Europe, may prove with additional data to be a distinct species of Trichobilharzia.

In vitro stimulation of penetration gland emptying by Trichobilharzia szidati and T. regenti (Schistosomatidae) cercariae. Quantitative collection and partial characterization of the products.

Induction of penetration gland emptying by cercariae of the bird schistosomes Trichobilharzia szidati and T. regenti employing linoleic acid, linolenic acid, praziquantel and calcium ionophore A23187 showed that both postacetabular and circumacetabular cells released their content at chosen stimulant concentrations. The gland secretions consisted of soluble and insoluble parts. The former one adhering to the ground seemed to have different saccharide composition from the glands of Schistosoma mansoni. It bound labelled saccharides, thus exhibiting lectin-like activity. Protein profiles of the latter one were identical after stimulation by all four stimulants in T. szidati. The soluble secretions contained several proteolytic enzymes; 31 kDa and 33 kDa cysteine proteases were identified in E/S products of T. szidati and T. regenti, respectively. The circumacetabular glands contained a significant amount of calcium. Immunohistochemistry revealed that the origin of E/S products after in vitro stimulation is in both penetration glands and tegumental structures. No crossreactivity was observed between the bird schistosomes and a serum raised against S. mansoni elastase.

Biochemical comparison of the serine protease (elastase) activities in cercarial secretions from Trichobilharzia ocellata and Schistosoma mansoni.

We report on serine protease activity in cercarial secretions (CSs) from the bird parasite Trichobilharzia ocellata. Using a colorigenic substrate, the biochemical properties of this enzyme were studied and its activity was compared to the homologous one in CSs from the human parasite Schistosoma mansoni. The specific serine protease activity was always 2- to 3-fold higher in CSs from T. ocellatacompared to S. mansoni. The enzyme has its optimal activity at pH 10.5, is Ca2+-dependent (inhibition with EDTA) and has a trypsin-like (inhibition with anti-pain) serine proteinase activity (inhibition with PMSF and aprotinin). The K(m) value of the serine protease from T. ocellatawas higher than that of S. mansoni, and the K(i) values for several inhibitors were generally lower for the enzyme of T. ocellatathan that of S. mansoni except for EDTA. The enzyme activities from both parasites had a molecular weight of 30 kDa in gelatin-SDS-polyacrylamide gels. The intensity of the gelatin digestion bands was stronger with the T. ocellata than with the S. mansoni enzyme.

Variations in immunofluorescent antibody response against Trichobilharzia and Schistosoma antigens in compatible and incompatible hosts.

Schistosome cercariae are able to penetrate into the skin of various vertebrate hosts. However, in contrast to the compatible host, the infection of incompatible hosts results in the death of parasites at various intervals post-infection. In order to compare the immune responses in both types of host infected with Trichobilharzia regenti, Trichobilharzia szidatior Schistosoma mansoni, antibody responses against various T. regenti, T. szidatiand S. mansonischistosome developmental stages were studied. Indirect immunofluorescence tests (IFAT) demonstrated no species-specific reactivity of human, mouse or duck immune sera with cercarial surfaces. Study of the cercarial glands also gave no significant results. However, differences were found in schistosomular and adult antigens: only the sera of compatible hosts recognised schistosomular and adult gut associated antigens in homologous as well as heterologous systems. Based on the presented data, our study supports the use of IFAT for the serological differentiation of schistosomiasis and cercarial dermatitis caused by bird schistosomes.

The suppressive excretory-secretory product of Trichobilharzia ocellata: a possible factor for determining compatibility in parasite-host interactions.

Factors which may determine trematode-snail interactions were assessed in the present study. Compatibility was examined using a bacterial clearance assay to detect the modulatory effects of both compatible and incompatible trematode infections on the activity of haemocytes from Lymnaea stagnalis, during the early stages of infection. Exposure to and injection with Trichobilharzia ocellata, a compatible trematode, or the incompatible Schistosoma mansoni, resulted in modulation of haemocyte activity. However, T. ocellata activated haemocytes 1.5 h post-infection (p.i.) and then suppressed activity 24-72 h p.i. whereas with S. mansoni no suppression, only activation of haemocytes was observed throughout the test period (1.5-72 h p.i.). In previous studies, modulation of the haemocyte clearance activity by T. ocellata was found to be mediated by 2 E-S fractions, an activating fraction and a suppressing one. Investigations to assess whether the lack of suppression of haemocyte activity, observed in the S. mansoni-L. stagnalis incompatible trematode-snail interaction studied, was due to either the absence or ineffectiveness of the suppressing E-S fraction, were performed on a second incompatible combination, T. ocellata-Planorbis corneus. Using this combination it was revealed that only the activating E-S fraction had modulatory effects on P. corneus haemocytes, indicating that the suppressing E-S fraction, which actively interferes with the clearance activity of haemocytes from L. stagnalis, appears to act in a host-specific manner. In conclusion, the suppressing E-S fraction determines, at least in part, compatibility in the trematode-snail association studied. This is also probably likely in other trematode-snail combinations.

Schistosoma mansoni and Trichobilharzia ocellata: comparison of secreted cercarial eicosanoids.

Schistosoma mansoni cercarial transformation and early stages of penetration can be correlated to specific eicosanoid products. Cercarial eicosanoids are suggested to play immunoregulatory roles during penetration. We examined production of cercarial eicosanoids by S. mansoni compared with that of the duck parasite Trichobilharzia ocellata after incubation with linoleate. Secretions were separated by reversed-phase high pressure liquid chromatography and also quantified by radioimmunoassay. The 2 parasites, differing in their immune evasion, synthesized the same types of prostaglandins, leukotrienes, and hydroxy-eicosatetranoic acids in similar quantities. Eicosanoid fractions of both species also showed a similar inhibition of superoxide production by human neutrophils. The coincidence of eicosanoid production suggests a function of eicosanoids in processes that are similar in both species.

Schistosomin: a pronase-sensitive agent in the hemolymph of Trichobilharzia ocellata-infected Lymnaea stagnalis inhibits the activity of albumen glands in vitro.

The schistosome parasite, Trichobilharzia ocellata, nearly completely inhibits the reproductive activity of its intermediate host, Lymnaea stagnalis. The synthetic activity of albumen glands of infected snails at day 35 postinfection (p.i.) is only 1% of the control value. The parasite acts by humoral means. We tested the hypothesis that (a) specific humoral agent(s) is (are) involved and refer to this (these) agent(s) as schistosomin. The presence of schistosomin in the hemolymph of infected snails was investigated by using galactogen synthesis in albumen glands as an in vitro bioassay. The synthetic activity of albumen glands of noninfected snails decreased by about 50% during a 1-h incubation in the hemolymph of infected snails. This inhibition is attributed to schistosomin. Based on these results, with the present bioassay schistosomin appears in the hemolymph between days 28-36 p.i. onwards. Schistosomin is heat-stable (100 degrees C) and pronase-sensitive, and therefore it might have a peptide nature. Schistosomin suppresses the stimulating action of the female, gonadotrophic dorsal body hormone at relatively low doses, which suggests that it may compete with this hormone for the same receptors. The development of two other bioassays for schistosomin in our laboratory is discussed.