Transcriptome analysis of grass carp (Ctenopharyngodon idella) and Holland's spinibarbel (Spinibarbus hollandi) infected with Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is a protozoan ciliate that causes white spot disease (also known as ichthyophthiriasis) in freshwater fish. Holland's spinibarbel (Spinibarbus hollandi) was less susceptible to white spot disease than grass carp (Ctenopharyngodon Idella). In this study, grass carp and Holland's spinibarbel are infected by I. multifiliis and the amount of infection is 10,000 theronts per fish. All grass carp died within 12 days after infection, and the survival rate of Holland's spinibarbel was more than 80%. In order to study the difference in sensitivity of these two fish species to I. multifiliis, transcriptome analysis was conducted using gill, skin, liver, spleen and head kidney of Holland's spinibarbel and grass carp at 48 h post-infection with I. multifiliis. A total of 489,296,696 clean reads were obtained by sequencing. A total of 105 significantly up-regulated immune-related genes were obtained by Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis in grass carp. Cluster of differentiation 40 (CD40), cluster of differentiation 80 (CD 80), tumor necrosis factor-alpha (TNF-α), toll-like receptor 4 (TLR-4), interleukin 1 beta (IL-1ß) and other inflammatory-related genes in grass carp were enriched in the cytokine-cytokine receptor interaction pathway and toll-like receptor pathway. In Holland's spinibarbel, a total of 46 significantly up-regulated immune-related genes were obtained by GO classification and KEGG pathway enrichment analysis. Immune-related genes, such as Immunoglobin heavy chain (IgH), cathepsin S (CTSS), complement C1q A chain (C1qA), complement component 3 (C3) and complement component (C9) were enriched in phagosome pathway, lysosome pathway and complement and coagulation concatenation pathway. C3 was significantly up-regulated in gill and head kidney. Fluorescence in situ hybridization (FISH) showed that the C3 gene was highly expressed in gill tissue of Holland's spinibarbel infected with I. multifiliis. A small amount of C3 gene was expressed in the gill arch of grass carp after infected with I. multifiliis. In conclusion, the severe inflammatory response in vivo after infecting grass carp with I. multifiliis might be the main cause of the death of grass carp. The extrahepatic expression of the gene of Holland's spinibarbel might play an important role in the immune defense against I. multifiliis.

MORPHOLOGY OF ADULTS AND IMMATURES OF THE ACANTHOCEPHALAN, POMPHORHYNCHUS FUHAIENSIS YUE, 1998 (ACANTHOCEPHALA: POMPHORHYNCHIDAE) FROM CYPRINID FISH IN NORTHWEST CHINA.

Adult specimens of Pomphorhynchus fuhaiensis were identified from common carp (Cyprinus carpio L.) in Ulungur Lake of northwest China, and acanthors, acanthellae, cystacanths dissected from Gammarus lacustris in a small tributary of Ulungur River for the first time. The acanthocephalans were also found in crucian carp (Carassius carassius L.), tench (Tinca tinca L.), oriental bream (Abramis brama orientalis Berg), and ide (Leuciscus idus L.) in the lake. This species is distinguished from other species in Pomphorhynchus by its large, spherical bulb and very long neck as well as by a cylindrical proboscis armed with 15-17 longitudinal rows of 9-12 hooks each. The anterior proboscis hooks are almost uniform in size and shape, the sixth hook in a short row and the seventh hook in long row decrease abruptly in size posteriorly with the last end hook being a little larger than the prebasal hook, and in a ring; posterior proboscis hooks much more widely spaced. Furthermore, the lemnisci are claviform. The mean neck:trunk ratio is about 0.5, which is larger than most other species in Pomphorhynchus. Females are larger than males. In males, the testes are in one-third to one-half of the trunk, equal, ovoid-spheroid, usually contiguous, and small relative to the body size, and there are 6 ovoid cement glands. Pomphorhynchus fuhaiensis is similar to Pomphorhynchus laevis but can be distinguished by the number of longitudinal rows of hooks. Pomphorhynchus laevis is armed with 18-20 longitudinal rows of 11-13 hooks, P. fuhaiensis is armed with 15-17 longitudinal rows of 9-12 hooks.

The role of fish helminth parasites in monitoring metal pollution in aquatic ecosystems: a case study in the world's most productive platinum mining region.

Due to the increasing consumption of platinum (Pt), especially in automobile exhaust catalysts, environmental concentrations of Pt are of emerging concern worldwide. Limited information exists on environmental concentrations, particularly in Pt mining regions, while South Africa is the world's main supplier of Pt. Moreover, other metals are also released as by-products of Pt mining, which might also cause environmental concern. Certain fish parasite taxa have the ability to accumulate metals orders of magnitude higher than their hosts and can be used to reliably detect metals with naturally low abundance. Studies on Pt accumulation in parasite-host systems are limited. Therefore, the aims of the present study were (1) to determine the accumulation of a variety of metals (cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb), platinum (Pt), and zinc (Zn)) in helminth fish parasites compared with their hosts from a reference site and an impoundment impacted by Pt mining activities; (2) to assess whether there is a difference between bioaccumulation of metals in infected and uninfected hosts, as well as between hosts with different infection intensities; and (3) to compare the biomarker responses (acetylcholine esterase activity (AChE), metallothionein content (MT), catalase activity (CAT), reduced glutathione content (GSH), malondialdehyde content (MDA), protein carbonyls induction (PC), superoxide dismutase activity (SOD), and cellular energy allocation (CEA)) between infected and uninfected hosts. The cestode Atractolytocestus huronensis accumulated significantly higher concentrations of Cr, Ni, and Pt than their host Cyprinus carpio, while the nematode Contracaecum sp. accumulated significantly higher concentrations of Pt and Zn than their host Clarias gariepinus. Infected fish showed lower metal concentrations compared to uninfected fish, while the parasites had no significant effects on their hosts' biomarker responses. The parasites demonstrated the bioavailability of metals derived from Pt mining activities and their ability to resist its toxic effects. Thus, these parasites are promising sensitive accumulation indicators for Cr, Ni, Pb, and Pt contaminations from Pt mining activities.

Digenean Holostephanus (Trematoda: Digenea: Cyathocotylidae) metacercariae in common carp (Cyprinus carpio Linnaeus, 1758) muscle: zoonotic potential and sensitivity to physico-chemical treatments.

Metacercariae of various species within the genus Holostephanus Szidat, 1936 (Trematoda: Digenea: Cyathocotylidae) occur in muscles of both farmed and wild fish, including common carp (Cyprinus carpio Linnaeus, 1758). The life cycle includes a snail as first intermediate host, fish as second intermediate host and birds or mammals as final hosts. We studied the zoonotic potential and the viability of Holostephanus metacercariae from common carp following exposure to various physical and chemical treatments. Muscle tissue samples of common carp specimens from a fish farm in the north-eastern part of Hungary were examined and metacercariae recovered. The zoonotic potential was evaluated experimentally by using small mammals as models (albino mice, n = 2; and Syrian hamsters, n = 4) infected per os with Holostephanus cysts. Parallelly, Metagonimus metacercariae were used as positive controls. We could not confirm the zoonotic potential of Holostephanus metacercariae as they did not survive in the mammalian intestine whereas Metagonimus metacercariae developed to the adult stage. We assessed the viability of metacercariae isolated from common carp specimens during exposure to different physical treatments (temperatures of -18°C, +20°C, +40°C and +60°C) and chemical agents (5% and 10% acetic acid and 10% sodium chloride (NaCl)). Metacercariae lost viability by freezing at -18°C (2 h), heating at 60°C (20 min), incubation in 5% and 10% acetic acid (5 min) and 10% NaCl (2 h). These methods served as models to investigate the effectiveness of food preparation techniques (such as cold and hot smoking, freezing, salting and pickling) on the survival of metacercariae.

Effect of cysteine peptidase inhibitor of Eudiplozoon nipponicum (Monogenea) on cytokine expression of macrophages in vitro.

The gills of the common carp, whose mucosal surface belongs to the key defence mechanisms of piscine immunity, can be infested with both the larval and adult stage of Eudiplozoon nipponicum (Monogenea). Although on their own, monogeneans do not considerably compromise their hosts' health status, fish with epithelial barriers damaged in parasite feeding and attachment sites are at an increased risk of bacterial challenge with possible harmful consequences. Several studies suggest that helminth parasites of teleost fish evade and manipulate host immune system via their excretory-secretory products, but our knowledge of these processes in the monogeneans is limited. Cysteine peptidase inhibitors (CPI), which are found in the secretions of numerous parasites, often induce immunosuppression by subverting Th1 mechanisms and drawing the immune system towards a Th2/Treg response. We employed the qPCR to test the effect of recently characterised CPI of E. nipponicum (rEnStef) on the mRNA expression of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 produced by porcine macrophages in vitro. After an initial preincubation with rEnStef, we stimulated the macrophages using LPS. By inducing a Th1 pro-inflammatory response, we imitated the immune reaction during a bacterial challenge in tissue damaged by the feeding and attachment of E. nipponicum. We observed a significant dose-dependent downregulation of the expression of TNF-α and IL-10 cytokines. The observed suppression of TNF-alpha expression by rEnStef could result in decreased pathogen control, which might in turn lead to increased rates of secondary bacterial infections in fish infected by E. nipponicum.

Pathological and immunological analyses of Thelohanellus kitauei (Myxozoa:Myxosporea) infection in the scattered mirror carp, Cyprinus carpio.

Thelohanellus kitauei is a spore-forming myxosporean parasite prevalent in scattered mirror carp (Cyprinus carpio) that generates numerous cysts in the intestine and causes mass mortality in fish. To investigate the infection and mortality induced by T. kitauei in pond-reared farms in Luo-Jiang (104°51'N, 31°31'E), southwest China, morphological and molecular analyses of infected fish were conducted. Natural and specific immune indicators were further evaluated to determine the immunological effects of response to parasitic infection. The infectious parasite was identified as Thelohanellus kitauei based on morphological, 18S rDNA and infectious characteristics. Scattered mirror carp was determined as the specific intermediate host of the parasite. However, T. kitauei still caused considerable damage to the fish, in particular, injury and blockage of the intestines, resulting in malnutrition and even death. The mature spores of T. kitauei colonize the intestinal submucosa of carp and form cysts of various sizes that block the intestinal tract and release spores into the enteric cavity upon rupture, leading to the next phase of T. kitauei growth. Moreover, T. kitauei-infected carp showed weaker innate immunity. IgM is involved in the fight against parasitic infection while cytokines, such as IL-6, IL-1ß and TNF-α, had an impact on infection processes. To our knowledge, this is the first report to show that T. kitauei infects and causes death in scattered mirror carp. Our collective findings from systematic pathology, morphology and immunology experiments provide a foundation for further research on infections by this type of parasite and development of effective treatment strategies.

Differential survival of 3rd stage larvae of Contracaecum rudolphii type B infecting common bream (Abramis brama) and common carp (Cyprinus carpio).

The main fish host reaction to an infection with third stage anisakid nematode larvae is a response in which host immune cells (macrophages, granulocytes, lymphocytes) in affected internal organs initially are attracted to the parasite whereafter fibroblasts may enclose the parasite forming granuloma. Generally, the reaction is non-lethal to the parasite which may survive for years in the fish host retaining infectivity to the final host. This may also apply for the anisakid nematode Contracaecum rudolphii (having the adult stage in cormorants, using copepods as first intermediate/paratenic host and zooplankton feeding fish as paratenic hosts). The present study has shown that most Contracaecum rudolphii larvae survive in bream (Abramis brama) (from Lake Balaton, Hungary) whereas the majority of the nematode larvae die in Cyprinus carpio (from Lake Hévíz, directly connected to Lake Balaton). Both cyprinid host species interacted with the nematode larvae through establishing a marked cellular encapsulation around them but with different effects. The differential survival in common carp and bream may theoretically be explained by ecological factors, such as the environmental temperature which either directly or indirectly affect the development of nematode larvae, and/or intrinsic host factors, such as differential immune responses and host genetics.

How to become a successful invasive tapeworm: a case study of abandoned sexuality and exceptional chromosome diversification in the triploid carp parasite Atractolytocestus huronensis Anthony, 1958 (Caryophyllidea: Lytocestidae).

BACKGROUND: A cytogenetic analysis of the new local triploid population of the caryophyllidean tapeworm Atractolytocestus huronensis, a unique parthenogenetic species with the ability to colonise new regions, was performed to understand the inner structure of its chromosome complement. METHODS: A karyotype analysis was carried out using classical Giemsa staining and C-banding combined with fluorescent DAPI staining. A hypothesis that triplets are composed from three homologue chromosomes of approximately the same length and same centromere position was tested statistically for multiple dependent variables using a non-parametric Friedman's ANOVA. The chromosomal location of ribosomal DNA clusters within the nucleolar organization region (NORs) and telomeric (TTAGGG)n sequences were detected by fluorescent in situ hybridization (FISH). Chromosomes were subjected to AgNO3 staining in order to determine whether the rDNA sites represent active NORs. RESULTS: The cytogenetic analysis confirmed the karyotype composed from eight chromosome triplets (3n = 24) as well as the existence of a pair of NORs located on each chromosome of the second triplet. Six NORs varied their activity from cell to cell, and it was reflected in the numbers of nucleoli (from 1 to 5). A huge morphological diversification of homologue chromosomes was originally detected in six out of eight triplets; the homologue elements differed significantly either in length and/or morphology, and some of them carried discernible interstitial telomeric sequences (ITSs), while the end telomeres were minute. The heterochromatin bands with high AT content varied irregularly, and the course of aberrant spermatogenesis was evident. CONCLUSIONS: Diversification of homologues is a unique phenomenon very likely caused by the long-term absence of a recombination and consequential accumulation of chromosome rearrangements in the genome of A. huronensis during species evolution. Unalterable asexual reproduction of the tapeworm, along with international trade in its host (carp), is facilitating its ongoing spread.

Redescription and supplementary molecular characteristics of Aspidogaster ijimai Kawamura, 1915 (Trematoda, Aspidogastrea, Aspidogastridae), a parasite of Cyprinus carpio Linnaeus, 1758 s. lato (Actinopterygii) and freshwater bivalves in East Asia.

A detailed morphological description and ribosomal DNA (rDNA) sequence molecular data for Aspidogaster ijimai from Japan are provided. Morphological analysis, including a description of the cirrus-sac, indicate the conspecificity of Japanese and continental East Asian A. ijimai specimens. Analysis of ITS1-5.8S-ITS2 rDNA sequences of Japanese, Chinese and Russian specimens confirmed the morphological results. Phylogenetic analysis using ITS rDNA sequences confirmed that A. ijimai is a sister species for Aspidogaster chongqingensis. Median-joining network analysis showed an initial molecular differentiation step of Russian specimens from group of Japanese-Chinese samples. Our 28S rDNA results based on maximum likelihood (ML) and Bayesian (BI) phylogenetic analyses indicated well-supported monophyly of the Aspidogaster conchicola + A. ijimai group, a finding that indicates that these species are congeneric. At the same time, our data demonstrated that the genus Lobatostoma is paraphyletic and the family Aspidogastridae is polyphyletic, results that confirm previous studies.

Morphological and molecular characterization of Myxobolus dibombensis sp. n. (Myxozoa: Myxobolidae), a parasite of the African carp Labeobarbus batesii (Teleostei: Cyprinidae) from Dibombe River, Cameroon.

Myxobolus dibombensis sp. n. (Cnidaria: Myxosporea: Bivalvulida) is described from the fins of the African carp, Labeobarbus batesii, based on morphological and molecular data. Prevalence of infection was 51.9% (67/129). Ovoid to spherical cyst-like plasmodia were found in the intrasegmental region and among the fin rays. No pathological changes were found in the fish host tissue surrounding the cyst-like plasmodia. Mature myxospores were ovoid in frontal view and lenticular in lateral view, with slightly truncated anterior and rounded posterior ends. Myxospores measured 16.8 (15.8-18.0) µm long and 11.4 (10.0-13.0) µm wide. There was a triangular intercapsular appendix measuring 3.8 (2.6-4.5) µm long. Polar capsules were ovoid and slightly unequal in size, occupying approximately one-third of the myxospore length. The larger polar capsule measured 7 (6-8) µm long and 3.6 (3-4) µm wide, while the smaller one measured 5.8 (4.8-7.0) µm long and 3 (2-4) µm wide. The larger polar capsule contained nine to 11 filament coils, whereas the smaller one contained seven to nine coils. SSU rDNA gene sequence of M. dibombensis sp. n. did not match any sequences available in the GenBank. The similarity with available Myxobolus spp. sequences ranged from 65 to 81%. The novel species clustered with M. algonquinensis, which infects the cyprinid Luxilus cornutus from Canada.

Cysteine peptidases of Eudiplozoon nipponicum: a broad repertoire of structurally assorted cathepsins L in contrast to the scarcity of cathepsins B in an invasive species of haematophagous monogenean of common carp.

BACKGROUND: Cysteine peptidases of clan CA, family C1 account for a major part of proteolytic activity in the haematophagous monogenean Eudiplozoon nipponicum. The full spectrum of cysteine cathepsins is, however, unknown and their particular biochemical properties, tissue localisation, and involvement in parasite-host relationships are yet to be explored. METHODS: Sequences of cathepsins L and B (EnCL and EnCB) were mined from E. nipponicum transcriptome and analysed bioinformatically. Genes encoding two EnCLs and one EnCB were cloned and recombinant proteins produced in vitro. The enzymes were purified by chromatography and their activity towards selected substrates was characterised. Antibodies and specific RNA probes were employed for localisation of the enzymes/transcripts in tissues of E. nipponicum adults. RESULTS: Transcriptomic analysis revealed a set of ten distinct transcripts that encode EnCLs. The enzymes are significantly variable in their active sites, specifically the S2 subsites responsible for interaction with substrates. Some of them display unusual structural features that resemble cathepsins B and S. Two recombinant EnCLs had different pH activity profiles against both synthetic and macromolecular substrates, and were able to hydrolyse blood proteins and collagen I. They were localised in the haematin cells of the worm's digestive tract and in gut lumen. The EnCB showed similarity with cathepsin B2 of Schistosoma mansoni. It displays molecular features typical of cathepsins B, including an occluding loop responsible for its exopeptidase activity. Although the EnCB hydrolysed haemoglobin in vitro, it was localised in the vitelline cells of the parasite and not the digestive tract. CONCLUSIONS: To our knowledge, this study represents the first complex bioinformatic and biochemical characterisation of cysteine peptidases in a monogenean. Eudiplozoon nipponicum adults express a variety of CLs, which are the most abundant peptidases in the worms. The properties and localisation of the two heterologously expressed EnCLs indicate a central role in the (partially extracellular?) digestion of host blood proteins. High variability of substrate-binding sites in the set of EnCLs suggests specific adaptation to a range of biological processes that require proteolysis. Surprisingly, a single cathepsin B is expressed by the parasite and it is not involved in digestion, but probably in vitellogenesis.

A novel type I cystatin of parasite origin with atypical legumain-binding domain.

Parasite inhibitors of cysteine peptidases are known to influence a vast range of processes linked to a degradation of either the parasites' own proteins or proteins native to their hosts. We characterise a novel type I cystatin (stefin) found in a sanguinivorous fish parasite Eudiplozoon nipponicum (Platyhelminthes: Monogenea). We have identified a transcript of its coding gene in the transcriptome of adult worms. Its amino acid sequence is similar to other stefins except for containing a legumain-binding domain, which is in this type of cystatins rather unusual. As expected, the recombinant form of E. nipponicum stefin (rEnStef) produced in Escherichia coli inhibits clan CA peptidases - cathepsins L and B of the worm - via the standard papain-binding domain. It also blocks haemoglobinolysis by cysteine peptidases in the worm's excretory-secretory products and soluble extracts. Furthermore, we had confirmed its ability to inhibit clan CD asparaginyl endopeptidase (legumain). The presence of a native EnStef in the excretory-secretory products of adult worms, detected by mass spectrometry, suggests that this protein has an important biological function at the host-parasite interface. We discuss the inhibitor's possible role in the regulation of blood digestion, modulation of antigen presentation, and in the regeneration of host tissues.

Anthelmintic activity of Rosmarinus officinalis against Dactylogyrus minutus (Monogenea) infections in Cyprinus carpio.

Monogenean parasites are important ectoparasites of fish, and are responsible for severe economic impacts in the aquaculture industry. They are usually treated with chemicals, but the chemicals can have harmful side effects in the fish and may pose threats to human health. Rosemary (Rosmarinus officinalis) is a common medicinal herb, with antimicrobial and antitumor properties. Here, we examined the anthelmintic activity of rosemary extract against the monogenean (Dactylogyrus minutus) in vitro and in vivo using bath treatment and oral administration. The in vitro experiments showed that parasite survival was affected by both rosemary extract concentration and the solvent (water and ethanol). Parasites were dead at 61.8±5.6 and 7.8±1.4min when exposed to 100 and 200g aqueous rosemary extract solution/L of water respectively. It took 166.7±48.2 and 5.4±1.01min to kill the parasites when exposed to 1 and 32g ethanol rosemary extract solution/L of water respectively. Moreover, pure component of rosemary extract obtained commercially used in in vitro experiments showed that 1,8-Cineole was the most toxic component of the main components tested. Parasite intensity and prevalence in fish exposed to 50 and 100g aqueous rosemary solution/L water for 30min were significantly lower than they were in controls (p<0.05). In oral treatment experiments, diets of Cyprinus carpio were supplemented with eight different concentrations of aqueous rosemary extract. The intensity of parasites was significantly less in fish fed for 30days with feed containing 60, 80 and 100ml aqueous extract/100g feed than in control (p<0.05). Together these results indicate that rosemary is a promising candidate for prevention and control of monogenean infection.

The life cycle of Thelohanellus kitauei (Myxozoa: Myxosporea) infecting common carp (Cyprinus carpio) involves aurantiactinomyxon in Branchiura sowerbyi.

Thelohanellus kitauei is a freshwater myxosporean parasite causing intestinal giant cystic disease of common carp. To clarify the life cycle of T. kitauei, we investigated the oligochaete populations in China and Hungary. This study confirms two distinct aurantiactinomyxon morphotypes (Aurantiactinomyxon type 1 and Aurantiactinomyxon type 2) from Branchiura sowerbyi as developmental stages of the life cycle of T. kitauei. The morphological characteristics and DNA sequences of these two types are described here. Based on 18S rDNA sequence analysis, Aurantiactinomyxon type 1 (2048 bp) and Aurantiactinomyxon type 2 (2031 bp) share 99.2-99.4 %, 99.8-100 % similarity to the published sequences of T. kitauei, respectively. The 18S rDNA sequences of these two aurantiactinomyxon morphotypes share 99.4 % similarity, suggesting intraspecific variation within the taxon, possibly due to geographic origin. Phylogenetic analyses demonstrate the two aurantiactinomyxon types clustered with T. kitauei. Regardless, based on 18S rDNA synonymy, it is likely that Aurantiactinomyxon type 1 and 2 are conspecific with T. kitauei. This is the fourth elucidated two-host life cycle of Thelohanellus species and the first record of T. kitauei in Europe.

Molecular characterisation of a disseminated Cryptosporidium infection in a Koi carp (Cyprinus carpio).

Cryptosporidium is a protozoan parasite that infects a wide range of hosts, yet relatively little is known about the epidemiology of cryptosporidiosis in fish. Here we report a disseminated Cryptosporidium infection in a male Koi carp (Cyprinus carpio), with parasite stages identified deep within the epithelium of the intestine, kidneys, spleen, liver and gills causing severe granulomatous inflammatory lesions. Molecular characterization at two loci; 18S ribosomal RNA (rRNA) and actin, revealed this to be a novel Cryptosporidium genotype, most closely related to Cryptosporidium molnari.

Evaluation of medicated feeds with antiparasitical and immune-enhanced Chinese herbal medicines against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idellus).

Since malachite green was banned for using in food fish due to its carcinogenic and teratogenic effects on human, the search of alternative drug to treat Ichthyophthirius multifiliis becomes urgent. This study aimed to (1) evaluate the ethanol extracts of medicinal plants Cynanchum atratum, Zingiber officinale, Cynanchum paniculatum, immunostimulant (A), and immunostimulant (B) for their efficacy against I. multifiliis, and (2) determine effects of medicated feeds with C. atratum, Z. officinale, C. paniculatum, and immunostimulant (A) to treat I. multifiliis in grass carp. The results in this study showed that the minimum concentrations of C. atratum, Z. officinale, and C. paniculatum extracts for killing all theronts were 16, 8, and 16 mg/L, respectively. In vivo experiments, fish fed with medicated feeds of C. atratum for 10 days, or Z. officinale for 3 days, or combination of three plants for 10 days resulted in a significant reduction in the I. multifiliis infective intensity on grass carp after theronts exposure. Grass carp fed with medicated feeds of immunostimulant (A) for 21 days showed no infection and 100 % of survival 15 days post theronts exposure. Therefore, immunostimulant (A) is a promising feed supplement to treated I. multifiliis with good antiparasitic efficacy.

Henneguya doneci (Myxosporea: Bivalvulida) in the gill filaments of Prussian carp Carassius gibelio (Bloch) from the upper Yellow River running through Inner Mongolia, China.

We examined 11 Prussian carp, Carassius gibelio (Bloch), from the upper Yellow River running through Inner Mongolia (Wuhai City) to record myxosporean species. Between 6 and 15 elongated cysts of Henneguya doneci were located at the basal part of the gill filaments of 3 carp (27.3%); no more myxosporean plasmodia were found in other organs. Although the morphology and morphometric values of the spores (average measurements of 14 spores in µm: 11.4 long by 9.2 wide with 7.5 in thickness; 2 polar capsules, equal, 5.5 long by 3.2 wide; and a bifurcated caudal process, 51.6 long) with an evident intercapsular appendix were basically coincident with the species, the dimensions of the spore bodies were marginally larger, and the length of the caudal processes was distinctly longer than previously reported values for H. doneci (44.2-59.2 µm vs. 26.8-42.6 µm, respectively). Genetic analysis of the 18S ribosomal RNA gene (rDNA) found few nucleotide substitutions when compared with 3 deposited sequences of H. doneci collected around the Yangtze River (Sichuan and Hubei Provinces), China, indicating that the uniqueness of some of the morphological features exhibited by the present Wuhai isolate should be ascribed to intraspecific variation.

Myxozoan infections of the three Indian major carps in fish ponds around Meerut, UP, India, with descriptions of three new species, Myxobolus basuhaldari sp. n., M. kalavatiae sp. n. and M. meerutensis sp. n., and the redescription of M. catlae and M. bhadrensis.

New myxosporean species are described from Indian fishes cultured in pond farms of Meerut, Uttar Pradesh (UP) state. Based upon plasmodia found in the Indian major carps (Catla catla, Cirrhinus cirrhosus, Labeo rohita and their hybrids), three new Myxobolus spp., Myxobolus basuhaldari sp. n., Myxobolus kalavatiae sp. n. and Myxobolus meerutensis sp. n., are described, and two species, Myxobolus catlae and Myxobolus bhadrensis, are redescribed. Plasmodia of M. basuhaldari sp. n., M. kalavatiae sp. n., M. meerutensis sp. n. and M. catlae developed in small cysts in the gill lamellae, while plasmodia and scattered spores of M. bhadrensis were found in the muscles and kidney, respectively. Plasmodia and spores found in these fishes differed from each other with respect to their morphology, tissue tropism and 18S ribosomal DNA (18S rDNA) sequence. No major pathological changes were found, but severe infections were observed.

Thelohanellus wangi n. sp. (Myxozoa, Myxosporea), a new gill parasite of allogynogenetic gibel carp (Carassius auratus gibelio Bloch) in China, causing severe gill myxosporidiosis.

We describe here a new myxozoan, Thelohanellus wangi n. sp., infecting the allogynogenetic gibel carp, Carassius auratus gibelio (Bloch), in a fry nursery farm in Jiangsu Province, China. Polysporous gray white round or ovoid plasmodia, 500-1,500 µm in size, were found exclusively in the gill filaments. The diagnostic characteristics of the myxospores are as follows: spore melon seed shaped in frontal view with smooth surface and asymmetrical valves; convex-shaped in sutural view with straight or slightly bent and thick sutural line, averaging 20.2 (16.5-22.3) × 9.9 (9.1-10.8) × 9.3 µm (8.2-10.4) in size; and one elliptical polar capsules with subulate anterior end and round posterior end, averaging 10.1 (8.4-11.2) × 6.5 µm (6.1-7.0) in size. Polar filaments coil six to seven turns and are slightly oblique to the longitudinal axis of the polar capsule. An elongate melon seed-shaped mucus envelope surrounds almost completely the spore, but with largest distance in posterior end between spore valve and mucus envelope. Granular sporoplasm contains two slightly oval nuclei, and no iodinophilous vacuole is observed. Histopathological analysis showed that the plasmodia embedded in the gill filaments and occasionally extended into the connective tissue of the gill arch. No significant inflammatory responses were provoked by the infection. The development of parasite is asynchronous, with mature spores centrally locating and trophozoites and pre-sporogonic stages peripherally locating within the plasmodia. Overall prevalence was 74% (37/50). Infection intensity was usually high, with about 10-30 cysts per fish. Phylogenetical analysis shows that T. wangi n. sp. is most closely related to several Thelohanellus species infecting cyprinid fish and formed a Thelohanellus clade based on the ribosomal DNA data. The cysts of this myxosporean were only found from fry seed with body length below 7 cm, generally from late April to early June when the fry fish of allogynogenetic gibel carp were nursed in the epizootic areas.

Human laryngitis caused by Clinostomum complanatum.

A 64-year-old Japanese man visited our outpatient department complaining of an irritable sensation in the throat, occurring two days after eating raw freshwater fish (carp sashimi) at a Japanese-style inn. During laryngeal endoscopy, a slow-moving worm (fluke) was found attached to the surface of the right aryepiglottic fold. After inhalation of 4% lidocaine, the fluke was removed using endoscopic forceps. Patient's throat symptoms immediately improved. The worm was microscopically identified as Clinostomum complanatum. C. complanatum is a digenetic trematode that usually infects fish-eating water birds. Clinostomum infections in humans are rare, and only 21 cases have been described in Japan and Korea. C. complanatum infection is known to occur after eating raw freshwater fish, which is a secondary intermediate host. In humans, the metacercariae are released into the stomach and migrate through the esophagus before lodging in the throat. Primary therapy involves endoscopic removal of the worm.