Mechanisms of the host immune response and helminth-induced pathology during Trichobilharzia regenti (Schistosomatidae) neuroinvasion in mice.

Helminth neuroinfections represent serious medical conditions, but the diversity of the host-parasite interplay within the nervous tissue often remains poorly understood, partially due to the lack of laboratory models. Here, we investigated the neuroinvasion of the mouse spinal cord by Trichobilharzia regenti (Schistosomatidae). Active migration of T. regenti schistosomula through the mouse spinal cord induced motor deficits in hindlimbs but did not affect the general locomotion or working memory. Histological examination of the infected spinal cord revealed eosinophilic meningomyelitis with eosinophil-rich infiltrates entrapping the schistosomula. Flow cytometry and transcriptomic analysis of the spinal cord confirmed massive activation of the host immune response. Of note, we recorded striking upregulation of the major histocompatibility complex II pathway and M2-associated markers, such as arginase or chitinase-like 3. Arginase also dominated the proteins found in the microdissected tissue from the close vicinity of the migrating schistosomula, which unselectively fed on the host nervous tissue. Next, we evaluated the pathological sequelae of T. regenti neuroinvasion. While no demyelination or blood-brain barrier alterations were noticed, our transcriptomic data revealed a remarkable disruption of neurophysiological functions not yet recorded in helminth neuroinfections. We also detected DNA fragmentation at the host-schistosomulum interface, but schistosomula antigens did not affect the viability of neurons and glial cells in vitro. Collectively, altered locomotion, significant disruption of neurophysiological functions, and strong M2 polarization were the most prominent features of T. regenti neuroinvasion, making it a promising candidate for further neuroinfection research. Indeed, understanding the diversity of pathogen-related neuroinflammatory processes is a prerequisite for developing better protective measures, treatment strategies, and diagnostic tools.

Differential proteomic analysis of laser-microdissected penetration glands of avian schistosome cercariae with a focus on proteins involved in host invasion.

Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula which migrate to the final location. 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 somewhat 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 of 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 us to distinguish 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 two of three replicates in either gland type (PA = 455, 40 exclusive; CA = 421, six exclusive; 60 proteins differed significantly in their abundance between the glands). Peptidases of five 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. While PA glands generally had rich enzymatic equipment, CA glands were conspicuously abundant in venom allergen-like proteins.

Isoforms of Cathepsin B1 in Neurotropic Schistosomula of Trichobilharzia regenti Differ in Substrate Preferences and a Highly Expressed Catalytically Inactive Paralog Binds Cystatin.

Schistosomula (the post-infective stages) of the neurotropic schistosome Trichobilharzia regenti possess multiple isoforms of cathepsin B1 peptidase (TrCB1.1-TrCB1.6) with involvement in nutrient digestion. The comparison of substrate preferences of TrCB1.1 and TrCB1.4 showed that TrCB1.4 had a very narrow substrate specificity and after processing it was less effective toward protein substrates when compared to TrCB1.1. Self-processing of both isoforms could be facilitated by sulfated polysaccharides due to a specific binding motif in the pro-sequence. Trans-activation by heterologous enzymes was also successfully employed. Expression profiling revealed a high level of transcription of genes encoding the enzymatically inactive paralogs TrCB1.5 and TrCB1.6. The transcription level of TrCB1.6 was comparable with that of TrCB1.1 and TrCB1.2, the most abundant active isoforms. Recombinant TrCB1.6wt, a wild type paralog with a Cys29-to-Gly substitution in the active site that renders the enzyme inactive, was processed by the active TrCB1 forms and by an asparaginyl endopeptidase. Although TrCB1.6wt lacked hydrolytic activity, endopeptidase, but not dipeptidase, activity could be restored by mutating Gly29 to Cys29. The lack of exopeptidase activity may be due to other mutations, such as His110-to-Asn in the occluding loop and Asp224-to-Gly in the main body of the mature TrCB1.6, which do not occur in the active isoforms TrCB1.1 and TrCB1.4 with exopeptidase activity. The catalytically active enzymes and the inactive TrCB1.6 paralog formed complexes with chicken cystatin, thus supporting experimentally the hypothesis that inactive paralogs could potentially regulate the activity of the active forms or protect them from being inhibited by host inhibitors. The effect on cell viability and nitric oxide production by selected immune cells observed for TrCB1.1 was not confirmed for TrCB1.6. We show here that the active isoforms of TrCB1 have different affinities for peptide substrates thereby facilitating diversity in protein-derived nutrition for the parasite. The inactive paralogs are unexpectedly highly expressed and one of them retains the ability to bind cystatins, likely due to specific mutations in the occluding loop and the enzyme body. This suggests a role in sequestration of inhibitors and protection of active cysteine peptidases.

Morphological and molecular characterization of Apatemon sp. infecting killifish in Mozambique.

Strigeid trematodes of the genus Apatemon Szidat, 1928 are intestinal parasites of fish-eating birds, utilizing various fish species as second intermediate hosts. In this study, we report morphometrical and molecular characterization of Apatemon sp. metacercariae parasitizing killifish Nothobranchius furzeri (Cyprinodontiformes: Nothobranchiidae) in south-east Mozambique. Metacercariae obtained from the cerebral cavity of killifish and two adult individuals isolated from experimentally infected ducklings were used for detailed morphological and molecular description, both resulting in generic affiliation to Apatemon. This is the first molecularly confirmed record of this trematode genus in Africa. Considering the morphological variability and wide host range of individual Apatemon species, the combination of both morphological and molecular analyses is indispensable for valid identification of this parasite. The results of our molecular analysis together with phylogenetic reconstruction indicated the presence of a new African lineage, reflecting potentially high diversity within the genus Apatemon comparable with other digenean genera.

Molecular evidence for distinct modes of nutrient acquisition between visceral and neurotropic schistosomes of birds.

Trichobilharzia species are parasitic flatworms (called schistosomes or flukes) that cause important diseases in birds and humans, but very little is known about their molecular biology. Here, using a transcriptomics-bioinformatics-based approach, we explored molecular aspects pertaining to the nutritional requirements of Trichobilharzia szidati ('visceral fluke') and T. regenti ('neurotropic fluke') in their avian host. We studied the larvae of each species before they enter (cercariae) and as they migrate (schistosomules) through distinct tissues in their avian (duck) host. Cercariae of both species were enriched for pathways or molecules associated predominantly with carbohydrate metabolism, oxidative phosphorylation and translation of proteins linked to ribosome biogenesis, exosome production and/or lipid biogenesis. Schistosomules of both species were enriched for pathways or molecules associated with processes including signal transduction, cell turnover and motility, DNA replication and repair, molecular transport and/or catabolism. Comparative informatic analyses identified molecular repertoires (within, e.g., peptidases and secretory proteins) in schistosomules that can broadly degrade macromolecules in both T. szidati and T. regenti, and others that are tailored to each species to selectively acquire nutrients from particular tissues through which it migrates. Thus, this study provides molecular evidence for distinct modes of nutrient acquisition between the visceral and neurotropic flukes of birds.

Complete mitochondrial genome of the giant liver fluke Fascioloides magna (Digenea: Fasciolidae) and its comparison with selected trematodes.

BACKGROUND: Representatives of the trematode family Fasciolidae are responsible for major socio-economic losses worldwide. Fascioloides magna is an important pathogenic liver fluke of wild and domestic ungulates. To date, only a limited number of studies concerning the molecular biology of F. magna exist. Therefore, the objective of the present study was to determine the complete mitochondrial (mt) genome sequence of F. magna, and assess the phylogenetic relationships of this fluke with other trematodes based on the mtDNA dataset. FINDINGS: The complete F. magna mt genome sequence is 14,047 bp. The gene content and arrangement of the F. magna mt genome is similar to those of Fasciola spp., except that trnE is located between trnG and the only non-coding region in F. magna mt genome. Phylogenetic relationships of F. magna with selected trematodes using Bayesian inference (BI) was reconstructed based on the concatenated amino acid sequences for 12 protein-coding genes, which confirmed that the genus Fascioloides is closely related to the genus Fasciola; the intergeneric differences of amino acid composition between the genera Fascioloides and Fasciola ranged 17.97-18.24 %. CONCLUSIONS: The determination of F. magna mt genome sequence provides a valuable resource for further investigations of the phylogeny of the family Fasciolidae and other trematodes, and represents a useful platform for designing appropriate molecular markers.

Effect of Fascioloides magna (Digenea) on fecundity, shell height, and survival rate of Pseudosuccinea columella (Lymnaeidae).

Infection with Fascioloides magna (Digenea) causes serious damage to liver tissue in definitive hosts represented by ruminants, especially cervids. The distribution of F. magna includes the indigenous areas in North America, and the areas to which F. magna was introduced-Central Europe, Southeast Europe, and Italy. The North American intermediate host of F. magna, the freshwater snail Pseudosuccinea columella (Lymnaeidae), is an invasive species recorded in South America, the Caribbean, Africa, Australia, and west and Southeast Europe. In Europe, Galba truncatula is the snail serving for transmission, but P. columella has potential to become here a new intermediate host of F. magna. Little is known about interactions between F. magna and P. columella. In this study, the susceptibility of P. columella (Oregon, USA) to the infection by a single miracidium of the Czech strain of F. magna and the influence of F. magna on snail fecundity, shell height, and survival were evaluated. The data show that the Oregon strain of P. columella is a highly suitable host for the Czech strain of F. magna, with the infection rate of 74 %. In addition, a negative effect on survival rate of infected snails was recorded only in the late phase of infection. The infection was accompanied by a major reduction in egg mass production and by a decrease in the number of eggs per egg mass. The shell height of infected snails did not significantly differ from that in unexposed controls.

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.

Highland cattle and Radix labiata, the hosts of Fascioloides magna.

BACKGROUND: Fascioloides magna is a pathogenic fluke introduced to Europe ca 140 years ago. As it is spreading over the continent, new intermediate and definitive hosts might be involved in transmission of the parasite. In Europe, several studies reported potential new intermediate snail hosts (Radix spp.) for F. magna, and also several cases of fascioloidosis of wild and domestic animals were published. However, the data based on molecular and histological analyses confirming these findings remained unreported. This study aims to refer to unique findings of F. magna in European snails and domestic animals (the first observation in the Czech Republic in the last 30 years) and demonstrate the use of molecular techniques in determination of F. magna. RESULTS: Two snails of R. labiata naturally infected with F. magna were found; mature cercariae and daughter rediae were observed. Maturity of cercariae was checked by histological methods, however, their ability to encyst was not confirmed. Co-infection of F. magna and Fasciola hepatica in the liver of two highland cattle bulls was proved. Adult fasciolid flukes producing eggs were found in the liver pseudocysts (F. magna) and the bile ducts (F. hepatica). Identification of intermediate hosts, intramolluscan stages, adult flukes and eggs was performed by sequencing the ITS2 region. Connection of F. magna pseudocysts with the gut (via the bile ducts) was not confirmed by means of histological and coprological examinations. CONCLUSIONS: For the first time, Radix labiata was confirmed as the snail host for F. magna under natural conditions and, together with the finding of F. magna infection in cattle, we can expect further transmission of F. magna from wildlife to livestock in localities shared by these hosts.

Radix spp.: Identification of trematode intermediate hosts in the Czech Republic.

Lymnaeid snails of the genus Radix serve as intermediate hosts of some schistosomes and fasciolids. In Europe, delineation of species within the genus Radix is unresolved and, therefore, spectrum of snail hosts susceptible to trematode infections is under discussion. We used and compared three criteria for species delineation using snails collected at 43 localities. (a) Sequence analysis of ITS-2 rDNA disclosed that the collected snails belong to four species - R. auricularia (Linnaeus, 1758), R. peregra (Müller, 1774), R. lagotis (Schrank, 1803) and R. labiata (Rossmaessler, 1835) (criteria and names are based on the work of Bargues et al. 2001). Occurrence of R. peregra in the Czech Republic was confirmed by molecular data for the first time. (b) Characterization of reproductive system disclosed differences in location, size and shape of bursa copulatrix and its ductus. Unfortunately, some R. labiata specimens shared morphological features of reproductive organs with R. lagotis. (c) Statistical analysis of shell morphology proved that significant differences exist among particular species. One prediction model showed that correct classification of species may be achieved in 82-84% of cases. However, identification of individual snails in the field (without knowledge of respective snail population and use of statistical tools) still remains a complicated issue due to overlaps of shell characteristics. Concerning the role in trematode transmission, R. lagotis, R. labiata and R. peregra are susceptible to Trichobilharzia regenti. Also, successful experimental infections of R. lagotis and R. labiata by Fascioloides magna were accomplished.