Know your enemy - transcriptome of myxozoan Tetracapsuloides bryosalmonae reveals potential drug targets against proliferative kidney disease in salmonids.

The myxozoan Tetracapsuloides bryosalmonae is a widely spread endoparasite that causes proliferative kidney disease (PKD) in salmonid fish. We developed an in silico pipeline to separate transcripts of T. bryosalmonae from the kidney tissue of its natural vertebrate host, brown trout (Salmo trutta). After stringent filtering, we constructed a partial transcriptome assembly T. bryosalmonae, comprising 3427 transcripts. Based on homology-restricted searches of the assembled parasite transcriptome and Atlantic salmon (Salmo salar) proteome, we identified four protein targets (Endoglycoceramidase, Legumain-like protease, Carbonic anhydrase 2, Pancreatic lipase-related protein 2) for the development of anti-parasitic drugs against T. bryosalmonae. Earlier work of these proteins on parasitic protists and helminths suggests that the identified anti-parasitic drug targets represent promising chemotherapeutic candidates also against T. bryosalmonae, and strengthen the view that the known inhibitors can be effective in evolutionarily distant organisms. In addition, we identified differentially expressed T. bryosalmonae genes between moderately and severely infected fish, indicating an increased abundance of T. bryosalmonae sporogonic stages in fish with low parasite load. In conclusion, this study paves the way for future genomic research in T. bryosalmonae and represents an important step towards the development of effective drugs against PKD.

Integrated field, laboratory, and theoretical study of PKD spread in a Swiss prealpine river.

Proliferative kidney disease (PKD) is a major threat to wild and farmed salmonid populations because of its lethal effect at high water temperatures. Its causative agent, the myxozoan Tetracapsuloides bryosalmonae, has a complex lifecycle exploiting freshwater bryozoans as primary hosts and salmonids as secondary hosts. We carried out an integrated study of PKD in a prealpine Swiss river (the Wigger). During a 3-year period, data on fish abundance, disease prevalence, concentration of primary hosts' DNA in environmental samples [environmental DNA (eDNA)], hydrological variables, and water temperatures gathered at various locations within the catchment were integrated into a newly developed metacommunity model, which includes ecological and epidemiological dynamics of fish and bryozoans, connectivity effects, and hydrothermal drivers. Infection dynamics were captured well by the epidemiological model, especially with regard to the spatial prevalence patterns. PKD prevalence in the sampled sites for both young-of-the-year (YOY) and adult brown trout attained 100% at the end of summer, while seasonal population decay was higher in YOY than in adults. We introduce a method based on decay distance of eDNA signal predicting local species' density, accounting for variation in environmental drivers (such as morphology and geology). The model provides a whole-network overview of the disease prevalence. In this study, we show how spatial and environmental characteristics of river networks can be used to study epidemiology and disease dynamics of waterborne diseases.

Field study indicating susceptibility differences between salmonid species and their lineages to proliferative kidney disease.

Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea) is the causative agent of proliferative kidney disease (PKD), which affects both wild and farmed salmonid fish. The objective of this study was to outline differences in susceptibility to PKD in different salmonid species, hybrids and breeding lineages. Susceptibility to T. bryosalmonae infection was established based on cumulative mortality, pathological findings and detection of T. bryosalmonae in the kidney using immunohistochemistry and molecular methods. Determination of pure and hybrid individuals of different species in the genus Salvelinus, and dissimilarity of rainbow trout lineages, was performed using traditional polymerase chain reaction (PCR) and microsatellite analyses. Rainbow trout displayed higher disease severity compared with brook trout and Alsatian charr. Moreover, the results indicated differences in infection susceptibility, not only among different salmonid species but also among different lineages of charr and rainbow trout. Our study indicated that some salmonid species and even different lineages of the same species are more suitable for farming under PKD pressure.

Transcriptome Analysis Elucidates the Key Responses of Bryozoan Fredericella sultana during the Development of Tetracapsuloides bryosalmonae (Myxozoa).

Bryozoans are sessile, filter-feeding, and colony-building invertebrate organisms. Fredericella sultana is a well known primary host of the myxozoan parasite Tetracapsuloides bryosalmonae. There have been no attempts to identify the cellular responses induced in F. sultana during the T. bryosalmonae development. We therefore performed transcriptome analysis with the aim of identifying candidate genes and biological pathways of F. sultana involved in the response to T. bryosalmonae. A total of 1166 differentially up- and downregulated genes were identified in the infected F. sultana. Gene ontology of biological processes of upregulated genes pointed to the involvement of the innate immune response, establishment of protein localization, and ribosome biogenesis, while the downregulated genes were involved in mitotic spindle assembly, viral entry into the host cell, and response to nitric oxide. Eukaryotic Initiation Factor 2 signaling was identified as a top canonical pathway and MYCN as a top upstream regulator in the differentially expressed genes. Our study provides the first transcriptional profiling data on the F. sultana zooid's response to T. bryosalmonae. Pathways and upstream regulators help us to understand the complex interplay in the infected F. sultana. The results will facilitate the elucidation of innate immune mechanisms of bryozoan and will lay a foundation for further analyses on bryozoan-responsive candidate genes, which will be an important resource for the comparative analysis of gene expression in bryozoans.

Identification and Expression Profiling of Toll-Like Receptors of Brown Trout (Salmo trutta) during Proliferative Kidney Disease.

Proliferative kidney disease is an emerging disease among salmonids in Europe and North America caused by the myxozoan parasite Tetracapsuloides bryosalmonae. The decline of endemic brown trout (Salmo trutta) in the Alpine streams of Europe is fostered by T. bryosalmonae infection. Toll-like receptors (TLRs) are a family of pattern recognition receptors that acts as sentinels of the immune system against the invading pathogens. However, little is known about the TLRs' response in salmonids against the myxozoan infection. In the present study, we identified and evaluated TLR1, TLR19, and TLR13-like genes of brown trout using data-mining and phylogenetic analysis. The expression pattern of TLRs was examined in the posterior kidney of brown trout infected with T. bryosalmonae at various time points. Typical Toll/interleukin-1 receptor protein domain was found in all tested TLRs. However, TLR13-like chr2 had a short amino acid sequence with no LRR domain. Phylogenetic analysis illustrated that TLR orthologs are conserved across vertebrates. Similarly, a conserved synteny gene block arrangement was observed in the case of TLR1 and TLR19 across fish species. Interestingly, all tested TLRs showed their maximal relative expression from 6 to 10 weeks post-exposure to the parasite. Our results suggest that these TLRs may play an important role in the innate defense mechanism of brown trout against the invading T. bryosalmonae.

Skin nodules associated with parasitism with Henneguya sp. (Cnidaria: Myxosporea) in the neotropical fish Cyphocharax modestus.

A new myxozoan species, Henneguya sp., is described based on material from skin of Cyphocharax modestus. Mature myxospores are were elongate and ellipsoidal, measuring 21.4 ±â€¯1.2 (19.4-23.2) µm in total length, 5.1 ±â€¯0.3 (4.5-5.8) µm in width, 11.9 ±â€¯0.5 (10.9-12.7) µm in body length and 9.6 ±â€¯0.7 (8.4-10.5) µm in length of the caudal process. The polar capsules were elongated and had unequal sizes, with length of 5.1 ±â€¯0.4 (4.5-6.0) µm and 5.6 ±â€¯0.4 (4.9-6.3) µm for smaller and larger respectively and width of 1.8 ±â€¯0.2 (1.4-2.0) µm. The larger polar capsule had 8 turns in polar filament while the smaller polar capsule had 5 turns in polar filament. The macroscopic analysis revealed the presence of large nodules, which were located before and after the dorsal fin of the hosts. The histopathological analysis showed the development of nodules filled with plasmodia, surrounded by loose connective tissue, developed in the dermis of the skin. Many cysts containing countless spores, as well as free spores, were located in the dermis and hypodermis of the hosts, causing the disorganization of the connective tissue that is responsible for the support. This is the first record of a Henneguya species in C. modestus.

Association mapping reveals candidate loci for resistance and anaemic response to an emerging temperature-driven parasitic disease in a wild salmonid fish.

Even though parasitic infections are often costly or deadly for the host, we know very little which genes influence parasite susceptibility and disease severity. Proliferative kidney disease is an emerging and, at elevated water temperatures, potentially deadly disease of salmonid fishes that is caused by the myxozoan parasite Tetracapsuloides bryosalmonae. By screening >7.6 K SNPs in 255 wild brown trout (Salmo trutta) and combining association mapping and Random Forest approaches, we identified several candidate genes for both the parasite resistance (inverse of relative parasite load; RPL) and the severe anaemic response to the parasite. The strongest RPL-associated SNP mapped to a noncoding region of the congeneric Atlantic salmon (S. salar) chromosome 10, whereas the second strongest RPL-associated SNP mapped to an intronic region of PRICKLE2 gene, which is a part of the planar cell polarity signalling pathway involved in kidney development. The top SNP associated with anaemia mapped to the intron of the putative PRKAG2 gene. The human ortholog of this gene has been associated with haematocrit and other blood-related traits, making it a prime candidate influencing parasite-triggered anaemia in brown trout. Our findings demonstrate the power of association mapping to pinpoint genomic regions and potential causative genes underlying climate change-driven parasitic disease resistance and severity. Furthermore, this work illustrates the first steps towards dissecting genotype-phenotype links in a wild fish population using closely related genome information.

Morphological and molecular analyses of Bipteria lusitanica n. sp. in wild white seabream, Diplodus sargus (Linnaeus, 1758) in Portugal.

The present study records the presence of Bipteria lusitanica n. sp. in wild white seabream, Diplodus sargus (Linnaeus, 1758) off the Portuguese coast. Myxosporean parasites were found in the interstitial tissue of kidney and free in urinary bladder of D. sargus with a 33.3% of prevalence of infection. Myxospores were triangular or inversely pyramidal in shape and anteriorly wider in frontal view, measuring 11.2 ± 1.0 µm in length, 12.6 ± 0.9 µm in width, and 11.6 ± 0.4 µm in thickness. The sinuous sutural line was parallel to the axis that connects the center of the two polar capsules. Spore valves were smooth without ridges. Wing-like appendages extended from the posterior part of each valve in sutural view, measuring 3.9 ± 1.1 µm in length. Spherical polar capsules measured 4.4 ± 0.2 µm in diameter and the polar filaments were helical arranged in 5-6 turns. Molecular data showed that this parasite clusters within species of the Sinuolineidae Shulman, 1959 family, and they all infect the urinary bladder. Using molecular and morphological characterization, we were able to identify this parasite as a novel species of the genus Bipteria Kovaleva, Zubtchenko, and Krasin, 1983.

Henneguya collaris sp. nov., (Myxosporidia), parasite of the Greenband Parrotfish Scarus collana Rüppell, 1835 (Actinopterygii, Scaridae) from the Red Sea, Egypt. A light and electron microscopic study.

The phylum Myxozoa comprises more than 2180 species, almost all of which are considered to be obligate parasites of aquatic fishes. In the present study, Henneguya collaris sp. nov. is the first described histozoic myxozoan species of the genus Henneguya infecting the kidney of the greenband parrotfish Scarus collana (Actinopterygii, Scaridae). One hundred and eighty specimens of fish were collected randomly during the period from September 2014 to October 2015 from boat landing sites and the market places at Hurghada City along the Red Sea in Egypt. Of these, 90 (50 %) specimens were infected. Light microscopic examination showed that the infection was detected as mature spores with two polar capsules regularly arranged at the anterior pole of each spore and extruded polar filaments free in the kidney tissue. The spore body was oval in shape, measured 7.1 ± 0.2 (6.2-8.4) µm in length and 6.3 ± 0.2 (5.8-7.0) µm in width, with a bifurcated caudal process of equal length, reaching 6.3 ± 0.2 (5.8-7.0) µm in length. Polar capsules were 3.4 ± 0.2 (3.0-4.2) µm in length and 1.9 ± 0.2 (1.6-2.4) µm in width with 6-8 (10) turns of polar filaments. Ultrastructural analysis showed that the spore development was asynchronous. Sporogenesis, capsulogensis, valvogenesis, and spore maturation of the present parasite were also described. The present species was compared morphologically with the spore characteristics of the most similar species of Henneguya spp. recorded previously from different geographical areas taking into account the stage and dimensions of the spore body, tails, and polar filament coils, including their number and the most important characteristic features that distinguish them from the present species. Considering the data obtained, the material described herein represents a new species and the name Henneguya collaris sp. nov. is proposed.

Seasonal fluctuation and histopathology of Henneguya ghaffari (Myxozoa: Myxosporea) infection in the gills of the Nile perch, Lates niloticus, in the River Nile: a new locality record.

Henneguya ghaffari Ali (Dis Aquat Org 38:225-230, 1999), which was originally described in Lake Wadi El-Rayan in the western desert of Egypt, has been discovered in the gills of the Nile perch, Lates niloticus, sourced from the River Nile at Beni-Suef governorate. The species identification was based on the spore morphometry. Of 180 Nile perch, 68 were found to be naturally infected with H. ghaffari (37.7%). A significant seasonal fluctuation in the prevalence was discerned, with the maximum rate occurring in the winter (68.8%) and the minimum rate in the summer (8.8%). The plasmodia of the parasite were evident as white rods, occupying almost a third of the gill filament and with mean dimensions of 0.7 × 0.2 mm. Histological investigations revealed that the present plasmodia were potentially compatible with the intrafilamental type. Infection with H. ghaffari initiated epithelial hyperplasia and curling and atrophy of the respiratory lamellae, which underpin its deleterious effect on the host by decreasing the functional respiratory surface of the gills. The present study concluded that infection with H. ghaffari originated in the River Nile before moving to the new ecosystem of Lake Wadi El-Rayan through drainage water.

Thermotactic behaviour in lacustrine and riverine forms of Salmo trutta and its relevance to an emerging parasitic disease (PKD) in the wake of climate change.

The thermotactic response of brown trout (Salmo trutta) was examined with the goal to investigate potential effects of the emerging temperature-dependent fatal trout disease PKD (proliferative kidney disease). First the differences in cold-water preferences of two forms of brown trout, lacustrine (migratory) and riverine, were determined. Second, it was studied whether this preference was changed in fish infected with PKD. The experiment involved a one-week habituation period at 14 °C in a two-chamber runway followed by a week of 3 °C temperature difference between the two runways. The fish could freely move between lanes via an opening at the end where food was provided. The temperature manipulation was repeated twice, and there were 3 trials per experimental group. All fish developed a clear spatial preference in the test. Lacustrine trout demonstrated a preference for warmer water, while riverine trout preferred cooler water. This may increase the risk to PKD in the lacustrine form. Most strikingly, riverine trout experimentally exposed to Tetracapsuloides bryosalmonae, the parasite that causes PKD, demonstrated stronger cold-seeking behaviour than control fish. Cold seeking behaviour suggests the occurrence of a disease-induced behavioural chill response, which may play an important role in disease recovery. This demonstrates the significance of protecting river connectivity and cold-water sanctuaries as management strategies for preserving salmonid populations in a warming climate.

Effects of Enteromyxum leei (Myxozoa) infection on gilthead sea bream (Sparus aurata) (Teleostei) intestinal mucus: glycoprotein profile and bacterial adhesion.

The intestinal myxosporean parasite Enteromyxum leei causes severe desquamative enteritis in gilthead sea bream (Sparus aurata) (Teleostei) that impairs nutrient absorption causing anorexia and cachexia. In fish, as in terrestrial vertebrates, intestinal goblet cells are responsible for the adherent mucus secretion overlying epithelial cells, which constitutes a first line of innate immune defense against offending microorganisms but serves also as substrate and nutrient source for the commensal microflora. The secreted intestinal mucus of parasitized (n = 6) and unexposed (n = 8) gilthead sea bream was isolated, concentrated, and subjected to downward gel chromatography. Carbohydrate and protein contents (via PAS and Bradford stainings), terminal glycosylation (via lectin ELISA), and Aeromonas hydrophila and Vibrio alginolyticus adhesion were analyzed for the isolated intestinal mucins. Parasitized fish, compared with unexposed fish, presented intestinal mucus mucins with a lower glycoprotein content and glycosylation degree at the anterior and middle intestine, whereas both glycoprotein content and glycosylation degree increased at the posterior intestine section, though only significantly for the total carbohydrate content. Additionally, a slight molecular size increase was detected in the mucin glycoproteins of parasitized fish. Terminal glycosylation of the mucus glycoproteins in parasitized fish pointed to an immature mucin secretion (N-acetyl-α-D-galactosamine increase, α-L-fucose, and neuraminic-acid-α-2-6-galactose reduction). Bacterial adhesion to large-sized mucus glycoproteins (>2,000 kDa) of parasitized fish was significantly lower than in unexposed fish.

Food poisoning associated with Kudoa septempunctata.

BACKGROUND: Kudoa septempunctata is a recently identified cause of food poisoning. We report three cases of food poisoning due to ingestion of this parasite. CASE REPORTS: Among the 358 people exposed during the same catered meal, 94 (including our 3 patients) developed vomiting and diarrhea within 1-9 h after ingestion of raw muscle from contaminated aquacultured olive flounders (Paralichthys olivaceus). These symptoms occurred frequently but were temporary; only 1 patient was hospitalized for dehydration and was discharged 2 days later. CONCLUSION: In Japan, cases of food poisoning due to eating olive flounder have increased during recent years. This increase should prompt heightened awareness among clinicians diagnosing food poisoning.

Density of the waterborne parasite Ceratomyxa shasta and its biological effects on salmon.

The myxozoan parasite Ceratomyxa shasta is a significant pathogen of juvenile salmonids in the Pacific Northwest of North America and is limiting recovery of Chinook (Oncorhynchus tshawytscha) and coho (O. kisutch) salmon populations in the Klamath River. We conducted a 5-year monitoring program that comprised concurrent sentinel fish exposures and water sampling across 212 river kilometers of the Klamath River. We used percent mortality and degree-days to death to measure disease severity in fish. We analyzed water samples using quantitative PCR and Sanger sequencing, to determine total parasite density and relative abundance of C. shasta genotypes, which differ in their pathogenicity to salmonids. We detected the parasite throughout the study zone, but parasite density and genetic composition fluctuated spatially and temporally. Chinook and coho mortality increased with density of their specific parasite genotype, but mortality-density thresholds and time to death differed. A lethality threshold of 40% mortality was reached with 10 spores liter(-1) for Chinook but only 5 spores liter(-1) for coho. Parasite density did not affect degree-days to death for Chinook but was negatively correlated for coho, and there was wider variation among coho individuals. These differences likely reflect the different life histories and genetic heterogeneity of the salmon populations. Direct quantification of the density of host-specific parasite genotypes in water samples offers a management tool for predicting host population-level impacts.

Castrating parasites and colonial hosts.

Trajectories of life-history traits such as growth and reproduction generally level off with age and increasing size. However, colonial animals may exhibit indefinite, exponential growth via modular iteration thus providing a long-lived host source for parasite exploitation. In addition, modular iteration entails a lack of germ line sequestration. Castration of such hosts by parasites may therefore be impermanent or precluded, unlike the general case for unitary animal hosts. Despite these intriguing correlates of coloniality, patterns of colonial host exploitation have not been well studied. We examined these patterns by characterizing the responses of a myxozoan endoparasite, Tetracapsuloides bryosalmonae, and its colonial bryozoan host, Fredericella sultana, to 3 different resource levels. We show that (1) the development of infectious stages nearly always castrates colonies regardless of host condition, (2) castration reduces partial mortality and (3) development of transmission stages is resource-mediated. Unlike familiar castrator-host systems, this system appears to be characterized by periodic rather than permanent castration. Periodic castration may be permitted by 2 key life history traits: developmental cycling of the parasite between quiescent (covert infections) and virulent infectious stages (overt infections) and the absence of germ line sequestration which allows host reproduction in between bouts of castration.

Ceratomyxa shasta genotypes cause differential mortality in their salmonid hosts.

Ceratomyxa shasta is a myxozoan parasite of salmonid fish. In natural communities, distinct genotypes of the parasite are associated with different salmonid hosts. To test the hypothesis that genotypes of C. shasta cause differential mortality, the polychaete host was experimentally infected with different parasite genotypes. Genotype I was obtained from Chinook salmon, Oncorhynchus tshawytscha, and genotype II from either coho salmon, O. kisutch, or rainbow trout, O. mykiss, We then challenged four salmonid strains: Chinook and coho salmon that occur in sympatry with the parasite and allopatric Chinook salmon and rainbow trout. Parasite genotype I caused mortality only in Chinook strains, although mortality in the allopatric strain also occurred from exposure to genotype II. A second experiment demonstrated that genotype II could be separated into two biotypes based on differential mortality in rainbow trout and coho salmon. These differential patterns of mortality as a result of infection by certain genotypes of C. shasta support field observations and suggest a co-evolutionary relationship between these parasites and their hosts.

Supplementary studies on Henneguya doneci Schulman, 1962 (Myxozoa: Myxosporea) infecting the gill filaments of Carassius auratus gibelio (Bloch) in China: histologic, ultrastructural, and molecular data.

Henneguya doneci Schulman, 1962 was collected from the gill filaments of Carassius auratus gibelio (Bloch) in Hubei Province, China. The plasmodia located on the surface of the gill arches deformed the neighboring gill filaments. The size of the plasmodia ranged from 0.6 to 4.5 mm in diameter in different months. The myxospores in the plasmodia measured 10.1 (9.2-11.5) µm long × 8.0 (7.5-8.5) µm wide × 7.5 (7-8) µm thick, with two equal capsules at 4.7 (4-5.5) µm long × 3.3 (2.5-4) µm wide, and two caudal processes 32.7 (24-38.5) µm long, respectively. Polar filaments were coiled 5-6 turns. Ultrastructural observation of the plasmodia showing the capsulogenesis of H. doneci is described briefly. The external tubule initially invaginated into the polar capsule. The rudimentary polar filaments were observed to undergo a series of considerable modification, finally developing into mature polar filaments. Molecular analysis demonstrated that although the myxosporean species were collected from different tissues of hosts in various geographic locations, they clustered with the Cyprinidae-infecting myxosporean species in the phylogenetic tree.

Morphology and host-parasite interaction of Henneguya azevedoi n. sp., parasite of gills of Leporinus obtusidens from Mogi-Guaçu River, Brazil.

Henneguya azevedoi n. sp. is described from the piava (Leporinus obtusidens). Between 2005 and 2007, 60 fish were collected from the Mogi-Guaçu River near Cachoeira de Emas Falls located in the municipality of Pirassununga, state of São Paulo, Brazil. A total of 70% had plasmodia of the parasite. The plasmodia were white, spherical, and measured 40-200 µm in diameter. Histopathological analysis revealed that the development of the parasite was intralamellar and caused stretching of the epithelium, with accentuated deformation, as well as compression of the capillary and adjacent tissues. Ultrastructural analysis revealed that the wall of the plasmodium was a single membrane in direct contact with the host cells and contained pinocytic canals that extended into the plasmodium. The development of the parasite was asynchronous, with the earliest stages at the periphery and mature spores in the central region. Mature spores were elongated in the frontal view [mean ± standard deviation (range)]: 45.2 ± 0.6 (45.0-47.0) µm in total length, 10.0 ± 0.07 (9.9-10.2) µm in body length, 35.6 ± 0.9 (34.9-36.5) µm in caudal process length, and 4.4 ± 0.4 (4.0-5.0) µm in body width. The polar capsules were elongated and equal in size: 3.8 ± 0.3 (3.5-4.0) µm in length and 1.0 µm in width. The polar filaments were coiled in six to seven turns and perpendicular to the axis of the capsule. Scanning electron microscopy revealed smooth valves and a conspicuous rim around the spore body. This is the first time that a myxosporean has been reported in L. obtusidens.

Low host specificity in the Kudoidae (Myxosporea: Multivalvulida) including seventeen new host records for Kudoa thalassomi.

Kudoid parasites are known to infect a large variety of fish. A significant proportion of Kudoa species have relatively low host specificity, with a single species able to infect multiple host species representing various host families even from different host orders. Since DNA sequences have been associated with myxosporean species characterisations, it has become far easier to determine host range of new species and validate host records from earlier descriptions. This study investigated the host specificity of a kudoid parasite, Kudoa thalassomi Adlard, Bryant, Whipps et Kent, 2005, from the Great Barrier Reef in Australia using DNA sequence analysis and morphology. The results revealed the host specificity to be broad, with K. thalassomi identified in 18 different fish species representing six different fish families. This study also compares current genetic information from different host isolates of Kudoa Meglitsch, 1947 to their host ranges recorded in existing literature. From this analysis, only half of the Kudoa species with multiple host records (27 Kudoa species) have half or more isolates that are genetically characterised, and thus specifically identified with a high confidence, from their known hosts. Only five kudoid species have genetically characterised isolates from all of their recorded hosts.

Xenoma-like formations induced by Soricimyxum fegati (Myxosporea) in three species of shrews (Soricomorpha: Soricidae), including records of new hosts.

In South Bohemia, Czech Republic, 178 shrews, including 98 common shrews, Sorex araneus L., 70 pygmy shrews, Sorex minutus L., and 10 lesser white-toothed shrews, Crocidura suaveolens (Pallas), were examined for Soricimyxum fegati Prunescu, Prunescu, Pucek et Lom, 2007 infections, using squash preparations of unfixed tissues, histological sections and molecular methods. The infection was found in 51 (52%) S. araneus, 14 (20%) S. minutus and 1 (10%) C. suaveolens. The records of the latter two species extend host range of S. fegati. Lesions associated with S. fegati infections in the liver, the organ of specific localisation of the parasite, were found to be induced by proliferative stages migrating toward lumina of bile ducts. In other organs of these three host species, xenoma-like formations (XLFs) were found that severely injured blood vessels. XLFs contained presporogonic stages of S. fegati, whose species identity was evidenced using molecular methods.