The first detection of Myxobolus lentisuturalis Dyková, Fiala et Nie, 2002, a highly pathogenic muscle-infecting parasite of gibel carp (Carassius auratus gibelio Berg, 1932) in Hungary.

Myxobolus lentisuturalis is a myxosporean parasite infecting the musculature both of goldfish (Carassius auratus auratus) and gibel carp (Carassius auratus gibelio). The species was originally described in China from gibel carp that is a common fish for sport fishing in Hungary meanwhile is one of the most popular farmed fish in China due to its high demand. Eighteen gibel carp with distortions were collected from a barrage pond in southern Hungary. All fish had large humps in the dorsolateral region due to infection of the muscle between the head and the dorsal fin. The swollen degenerated tissue was filled with myxozoan spores, which were collected for morphological and molecular studies. By size and morphology, the spores were consistent with morphological description of M. lentisuturalis. Histopathological examination showed that the formation of plasmodia containing myxospores leads to severe destruction of muscle tissue. The 18S ribosomal DNA and 28S ribosomal DNA data of the samples presented matched with previous sequences of M. lentisuturalis in GenBank. Phylogenetic analyses confirmed that our sequences belong to a monophyletic group with them supported by a high bootstrap. This study highlights the occurrence of a highly pathogenic myxozoan, M. lentisuturalis in Hungary as a new geographical location.

Molecular evidence of the absence of Metagonimus yokogawai (Katsurada, 1912) in Europe: report of Metagonimus sp. in cyprinoid fish from the River Danube in Hungary.

Trematodes of the genus Metagonimus Katsurada, 1912 (Digenea: Heterophyidae) are zoonotic parasites that cause infections in humans, with most cases reported in Southeast Asia. Larvae from the second intermediate host, called metacercariae, of one of human-infecting species, M. yokogawai (Katsurada, 1912), have been reported from cyprinoid fish in Europe. In the present study, we provided DNA-based evidence that metacercariae of Metagonimus, which are commonly found in the scales of various cyprinoids in Central Europe (Danube River in Hungary) do not belong to M. yokogawai. Sequence analysis of the ITS region, 28S rDNA, and cox1 genes showed that this species is clearly distinct from all Asian species, including M. yokogawai, which probably does not occur in Europe. Metacercariae from cyprinoids might belong to Metagonimus romanicus (Ciurea, 1915), an insufficiently known species described from Romania.

Extreme pathological symptom generated by Dermocystidium koi infection of common carp (Cyprinus carpio L.) in Hungary.

Dermocystidiosis in common carp (Cyprinus carpio L.) as a skin infection in the form of small, pea-sized bulging nodules is well-known in Hungary. Its occurrence is sporadic, and the pathogenic effect is generally moderate. Nonetheless, here we report an unusual case of the infection when a mass of dermocystid spore-filled hyphae-like cysts formed a large tumour-like nodule under the skin intruding deeply in the red muscle on one side occipital region of the fish body. Histological sections showed that the tumour-like nodule was composed of two layers, a pale peripheral part with a high mass of cysts surrounded by some cell debris of muscle cells and a dark central part with a less dense meshwork of tubular cysts which were embedded in the partially damaged red muscle tissue. No infection was found in the white muscle. Sequences of 18S rDNA of spores from the tumour-like nodule showed a complete identity to sequences from typical bulging nodules in skin. They were also highly similar to the sequences of D. anguillae clones (99.5%-99.4%), to some samples of D. salmonis (99.7% and 99.4%), and to the Dermocystidium sp. CM-2002 (99.4%) from European perch.

Mycoplasma infections in freshwater carnivorous fishes in Hungary.

The abundance of mycoplasma-like microorganisms seems to be common in the intestinal tract of fish, but their occurrence in other organs can be considered pathological. Isolation of piscine mycoplasmas is essential to prove their virulence. In this study, a total of 100 specimens of nine freshwater fish species collected from natural waters and fish farms were analysed for the prevalence of mycoplasmas in different extraintestinal organs. Our results demonstrate that mycoplasmas are present in carnivorous fish (pike, wels catfish, black bullhead and pike perch) than in cyprinids more frequently. Among the surveyed organs, they colonize mainly the gills, the kidney and the livers. Although mycoplasmas could be detected in one-fifth of samples by molecular assays, the isolation of mycoplasmas belonged to the M. sualvi cluster within the hominis group of Mycoplasmataceae, but was separate from M. mobile, has been successful only from some specimens originated from wels catfish that was feasible only by special preparation steps (prefreezing, filter sterilization and incubation conditions (long culturing period, etc.). The low-efficiency of isolation trials indicates an extensive gap in our knowledge about piscine mycoplasmas, especially in the field of their nutritional requirements.

Efficacy of silver nanoparticles to control flavobacteriosis caused by Flavobacterium johnsoniae in common carp Cyprinus carpio.

Flavobacterial infections are among the causes of fish losses in farms with the emergence of antibiotic-resistant isolates. Silver nanoparticles (AgNPs) are known for their potent antimicrobial activity against different types of bacteria. In this study, we evaluated the antibacterial properties of AgNPs (diameter: 23 nm) against Flavobacterium johnsoniae infection in common carp Cyprinus carpio. The assays included both in vitro and in vivo antibacterial tests in addition to evaluation of cell toxicity effects on the fish cell lines. The in vitro results revealed potent inhibitory effects of AgNPs on the growth of F. johnsoniae with a minimum inhibitory concentration of 34 µg ml-1. Fish cell (epithelioma papulosum cyprini and koi carp fin) viability was 95-100% after exposure to 500 ng ml-1 (and lower concentrations) of AgNPs. In the exposure experiment, mortality rates decreased from 45% in the infected non-treated group to 30 and 15% in the intraperitoneal injection and immersion-treated groups, respectively. Neither of the treated groups showed any clinical signs or histopathological lesions. The single-dose treatment with AgNPs during early infection with F. johnsoniae aided in minimizing fish losses.

Infection of the Carpathian brook lamprey (Eudontomyzon danfordi Regan, 1911) with a dermocystid parasite in the Tisza River Basin, Hungary.

The Carpathian brook lamprey (Eudontomyzon danfordi Regan, 1911) is an endemic protected species of Cephalaspidomorphi in the Carpathian Basin. No parasites have become known from these jawless vertebrates to date. Here, the authors describe an infection from a single specimen manifesting in protuberant skin cysts 7-10 mm in diameter, scattered on the body surface. Similar dermal infection was observed in 25 of the 274 lampreys recorded in the population survey. Skin cysts filled with round spore-like structures of a dermocystid parasite were found. These particles measured 8-14 µm in diameter and had an about 0.5 µm thick wall, and containing mainly a granular mass and a relatively scarce plasma. No hyphae were recorded. Despite conspicuous morphological changes in the skin, no inflammatory reactions were found. The molecular analysis of 18S rDNA showed similarity to dermocystid species of several fish species but differed from them approximately by 2%. This is the first record of a dermocystid parasite infecting a jawless vertebrate.

Henneguya (Cnidaria: Myxosporea: Myxobolidae) infections of cultured barramundi, Lates calcarifer (Perciformes: Latidae) in an estuarine wetlands system of Malaysia: description of Henneguya setiuensis n. sp., Henneguya voronini n. sp. and Henneguya calcarifer n. sp.

Examination of 35 barramundi (Lates calcarifer) from aquaculture cages in Setiu Wetland, Malaysia, revealed a single fish infected with three Henneguya spp. (Cnidaria: Myxosporea). Characterization of the infections using tissue tropism, myxospore morphology and morphometry and 18S rDNA sequencing supported description of three new species: Henneguya setiuensis n. sp., Henneguya voronini n. sp. and H. calcarifer n. sp. Myxospores of all three species had typical Henneguya morphology, with two polar capsules in the plane of the suture, an oval spore body, smooth valve cell surfaces, and two caudal appendages. Spores were morphometrically similar, and many dimensions overlapped, but H. voronini n. sp. had shorter caudal appendages compared with H. calcarifer n. sp. and H. setiuensis n. sp. Gross tissue tropism distinguished the muscle parasite H. calcarifer n. sp. from gill parasites H. setiuensis n. sp. and H. voronini n. sp.; and these latter two species were further separable by fine-scale location of developing plasmodia, which were intra-lamellar for H. setiuensis n. sp. and basal to the filaments for H. voronini n. sp. small subunit ribosomal DNA sequences distinguished all three species: the two gill species H. setiuensis n. sp. and H voronini n. sp. were only 88% similar (over 1708 bp), whereas the muscle species H. calcarifer n. sp. was most similar to H. voronini n. sp. (98% over 1696 bp). None of the three novel species was more than 90% similar to any known myxosporean sequence in GenBank. Low infection prevalence of these myxosporeans and lack of obvious tissue pathology from developing plasmodia suggested none of these parasites are currently a problem for barramundi culture in Setiu Wetland; however additional surveys of fish, particularly at different times of the year, would be informative for better risk assessment.

Detection of cyprinid herpesvirus 1 (CyHV-1) in barbel (Barbus barbus): First molecular evidence for the presence of CyHV-1 in fish other than carp (Cyprinus carpio).

Two adult barbels (Barbus barbus) with visible skin tumours were subjected to histopathological and molecular examinations. The fish were caught in the River Danube near Budapest. Papillomas were found around their oral cavity, at the operculum and at the pectoral fins, while epidermal hyperplasias were seen on the body surface. Cyprinid herpesvirus 1 (CyHV-1) was detected in the kidney of the specimens by polymerase chain reaction (PCR), and barbel circovirus 1 (BaCV1) was found in all internal organs and in the tissues of the tumours. The whole genome of BaCV1 and three conserved genes from the genome of CyHV-1 were sequenced. Previously, BaCV1 had been reported only once from a mass mortality event among barbel fry. The whole genome sequence of our circovirus shared 99.9% nucleotide identity with that of the formerly reported BaCV1. CyHV-1 is known to infect common carp and coloured carp (Cyprinus carpio), and has been assumed to infect other cyprinid fish species as well. We found the nucleotide sequences of the genes of CyHV-1 to be identical in 98.7% to those of the previous isolates from carp. To the best of our knowledge, this is the first molecular confirmation of the presence of CyHV-1 DNA in cyprinid fish species other than carp.

New record of metacercariae of the North American Posthodiplostomum centrarchi (Digenea, Diplostomidae) in pumpkinseed (Lepomis gibbosus) in Hungary.

Two species of the genus Posthodiplostomum (Digenea: Diplostomatidae) (Posthodiplostomum brevicaudatum Nordmann, 1832 and Posthodiplostomum cuticola Nordmann, 1832) are known as parasites of Hungarian native fishes. Metacercariae of P. cuticola are widespread in Europe and cause black spot disease. Several species of Posthodiplostomum were described also from North America but none of them has been isolated in Hungary up to now. Posthodiplostomum centrarchi Hoffman, 1958 has been detected recently in pumpkinseeds (Lepomis gibbosus L., 1758) in several European countries. Posthodiplostomum centrarchi was isolated for the first time in Hungary from pumpkinseeds caught in the Maconka water reservoir in 2015. Thereafter, several natural waters (e.g. the River Danube, Lake Balaton and the Sió channel) were sampled in order to determine its presence and distribution. Only the native species P. cuticola was detected in Lake Balaton on cyprinids but a relatively high infection rate of P. centrarchi was observed in the Sió channel close to the lake. Pathological changes were absent, and metacercariae were mostly attached to the surface of the liver, kidney and heart. The phylogenetic analysis of the ITS and COI sequences of P. centrarchi and P. cuticola clustered into two distinct branches, which was in agreement with the morphological results.

Morphological, histological, and molecular description of Myxobolus ompok n. sp. (Myxosporea: Myxobolidae), a kidney myxozoan from Pabdah catfish Ompok pabda (Hamilton, 1822) (Siluriformes: Siluridae) in India.

In a parasitological survey of freshwater fishes near Meerut, Uttar Pradesh, India, myxozoan infections in Pabdah catfish Ompok pabda Ham. (Siluriformes: Siluridae) were found. Round plasmodia and scattered spores of Myxobolus ompok n. sp. were found in the kidney of the host. Plasmodia measuring 150-200 µm were located in the renal interstitium. Spores of Myxobolus ompok n. sp. were elongated pyriform, 13.6-14.4 (14.8 ± 0.42) µm long, 5.6-6.4 (6.5 ± 0.33) µm wide, and 5.2-6.4 (5.9 ± 0.43) µm thick with two equal polar capsules measuring 8.0-8.5 (8.2 ± 0.2) µm in length and 1.5-2.4 (1.8 ± 0.33) in width having six filamental turns. Both the morphology and DNA analysis of the 18S rRNA gene revealed that Myxobolus ompok n. sp. is distinct from previously described species of Myxobolus and shares no significant similarity with any other Myxobolus deposited in the GenBank database. Phylogenetic analysis inferred that this species showed the closest similarity to Myxobolus miyarii (KT001495). This is the first record of any Myxobolus sp. from O. pabda in India.

Description of Henneguya jaczoi sp. n. (myxosporea, myxobolidae) from Perca fluviatilis (L.) (pisces, percidae) with some remarks on the systematics of Henneguya spp. of european fishes.

A new Henneguya species, H. jaczoi sp. n., is described from perch (Perca fluviatilis) from Lake Balaton, Hungary. This species infects the palatal region of the fish, forming large plasmodia in the thickened caudal part of the buccal cavity and at the dorsal ends of the cartilaginous gill arches. The species differs from the gill-dwelling Henneguya species of perch and pike (Esox lucius) both morphologically and in molecular aspects. The authors conclude that the type species H. psorospermica Thélohan is a specific parasite of pike, while the species forming plasmodia in the gills of perch corresponds to H. texta Cohn, which was hitherto regarded as a synonym of H. psorospermica. Besides the above-mentioned species, H. creplini was frequently found in pikeperch (Sander lucioperca) and Volga pikeperch (Sander volgensis), but no Henneguya infection has been recorded in ruffe (Gymnocephalus cernua), which is a common percid fish of the lake and is known to be the type host species for H. creplini.

Cross section of gill filaments in histological preparations helps better identification of the location of myxosporean plasmodia in gill tissues.

Location and tissue preference of filamental-type myxosporean plasmodia in histological slides of the gills can be properly identified only in cross sections of the gill filaments. The authors selected three myxosporeans (Myxobolus rutili, M. dispar and Henneguya psorospermica, parasites of the roach, the common carp and the pike, respectively) for studying the problem. The plasmodia of these species studied in longitudinal sections were earlier regarded as developing inside the filamental arteries. Cross sections of the filaments showed that all the three species developed plasmodia in the dense connective tissue constituting the adventitia of gill arteries and covering the cartilaginous gill rays. Myxobolus rutili started its development close to the afferent branchial artery but attached to the cartilaginous gill ray. More developed plasmodia of this species surrounded the rays. Plasmodia of M. dispar were formed on the inner side of the afferent branchial artery, while those of H. psorospermica were located at the external side of the efferent branchial artery.

Myxobolus infection in the cornea of the roach (Rutilus rutilus) in Lake Balaton.

Infection of the cornea in fishes by Myxobolus plasmodia is a common but still little known site preference of myxosporeans. A sporadic but striking infection in the cornea of the roach (Rutilus rutilus) was observed in Lake Balaton, Hungary. Relatively small, round plasmodia 250 to 500 µm in diameter developed in the dense connective tissue of the cornea. Morphological and molecular biological examination of spores collected from cysts in the cornea demonstrated that this infection is caused by Myxobolus fundamentalis, a species hitherto reported only from the cartilaginous gill arch of the roach. The 18S rDNA sequences of spores from the cornea showed 99.9% identity to the sequences of spores from the gill arch, and they also shared 99.9% identity with the sequences of triactinomyxon actinospores obtained from the oligochaete Isochaetides michaelseni.

Epicellular coccidiosis in goldfish.

In a goldfish stock held in a pet fish pond, heavy coccidian infection, caused by an epicellularly developing Goussia species, appeared in April of 3 consecutive years (2014 to 2016). The shape and size of the oocysts resembled those of an inadequately described species, Goussia carassiusaurati (Romero-Rodriguez, 1978). In histological sections, gamogonic and sporogonic stages infested mostly the second fifth of the intestine, where almost all epithelial cells were infected. Both gamonts and young oocysts occurred intracellularly but in an extracytoplasmal position, seemingly outside the cells. Oocysts were shed non-sporulated. Spheroid to ellipsoidal non-sporulated oocysts measured 12.4 × 13.5 µm on average, but after 48 h sporulation in tap water they reached a size of 16 × 13 µm. The 4 elliptical sporocysts located loosely within the sporulated oocysts measured 13 × 5.4 µm. The oocysts and sporocysts were smaller than those of the better-known Goussia species G. aurati (Hoffman, 1965).

An investigation of the host-specificity of metacercariae of species of Apophallus (Digenea: Heterophyidae) in freshwater fishes using morphological, experimental and molecular methods.

Metacercariae of species of the genus Apophallus Lühe, 1909, infecting the fins and skin of freshwater fishes, frequently cause black spot disease. Two species, Apophallus muehlingi (Jägerskiöld, 1899) and A. donicus (Skrjabin & Lindtrop, 1919), are known to occur in Hungarian fishes. It has generally been thought that metacercariae of A. muehlingi infect cyprinid fishes, whereas those of A. donicus develop in percids. As part of a morphological, experimental and molecular study, metacercariae were collected from 99 infected specimens of five cyprinid hosts (Abramis brama, Blicca bjoerkna, Chondrostoma nasus, Squalius cephalus, Scardinius erythrophthalmus) and 18 infected specimens of two percid hosts (Gymnocephalus cernua, Perca fluviatilis) in Hungarian natural waters (Lake Balaton, River Danube). Moreover, 1024 common carp (Cyprinus carpio) specimens collected from Hungarian fish ponds were investigated for Apophallus infection, but without positive results. For reliable species identification, experimental infections of chicks were carried in order to produce adult specimens from metacercariae collected from the fins and skin of the cyprinid and percid hosts. Within 8 days, adult specimens of both A. muehlingi and A. donicus developed in chicks infected with metacercariae from the cyprinid common bream (Abramis brama) and the white bream (Blicca bjoerkna) and the ruffe (Gymnocephalus cernua), a percid, respectively. The morphology of the collected metacercariae and adult individuals developed in the feeding experiments was characterised. A molecular analysis was extended to cercarial samples from the snail Lithoglyphus naticoides and to a single adult specimen of Apophallus from a fox. Sequences of 28 specimens were analysed using molecular methods (sequencing the internal transcribed spacer region and the cytochrome oxidase I subunit). Phylogenetic analysis was executed, and the Apophallus samples clustered into three distinct branches using both genes, A. muehlingi from cyprinids, A. donicus from percids and, a third, previously unknown, Apophallus clade, also from cyprinids.

Observations on non-random distribution of spores of Henneguya spp. (Cnidaria: Myxosporea: Myxobolidae) within plasmodia.

Species of the cnidarian genus Henneguya Thélohan, 1892 (Myxosporea: Myxobolidae) are histozoic parasites commonly found in freshwater and, more rarely, in marine fish. The development of these parasites in fish tissues includes the formation of plasmodia within which occurs the sporogony originating spores with two caudal processes, which are usually randomly distributed within the plasmodia. In this report the authors present some cases of non-random distribution of the spores of six species of Henneguya within their plasmodia. Two different patterns of non-random distribution were found based on a literature survey. These patterns and their origin are discussed. Apparently this non-random distribution of the spores is due to both internal and external factors.

Redescription of Henneguya chaudhuryi (Bajpai & Haldar, 1982) (Myxosporea: Myxobolidae), infecting the gills of the freshwater fish Channa punctata (Bloch) (Perciformes: Channidae) in India.

During a survey of myxosporean parasites of freshwater fishes in Meerut, Uttar Pradesh (UP), India, spores of Henneguya chaudhuryi (Bajpai & Haldar, 1982) were found in the gill lamellae of the spotted snakehead fish Channa punctata (Bloch) (Perciformes: Channidae). This species was described lacking several characteristics in the original description, which makes challenging the accurate diagnosis. Here, we supplemented its description based on morphological, histological and molecular data. Plasmodia of H. chaudhuryi are oval, measuring 60-100 × 40-68 µm, located intralamellarly. Mature spores are elongate, measuring 10.5-13.2 × 3.6-4.2 µm, with two slightly unequal polar capsules with 6-7 filamental turns and two straight, equal caudal appendages, 10-17 µm long. Scanning electron microscopy revealed a flat surface. The 18S rDNA sequence for H. chaudhuryi did not show a close relationship with those of any other Henneguya spp., represented in the GenBank.

Diverse Chlamydia-like agents associated with epitheliocystis infection in two cyprinid fish species, the common carp (Cyprinus carpio L.) and the gibel carp (Carassius auratus gibelio L.).

During a general annual fish health survey in natural waters and ponds, epitheliocystis infections were recorded in fingerlings of two cyprinid fish species, the cultured common carp and the wild gibel carp. Benign and heavy infections were equally observed without mortality. In addition to the general health inspection of fish, histopathological examinations of infected gills and molecular biological investigations of separated epitheliocysts were performed. Epitheliocysts were formed both in the interlamellar epithelial cells and in the lamella-free multilayered epithelium of the gill filaments. At the early stage of infection darkstaining inclusion bodies densely stuffed with some pathogenic agents were located at the centre of the cell, while in a progressive stage of the process inclusion bodies within the host cells were disseminated in the cytoplasm and stained pale. Molecular studies demonstrated three different agents related to Neochlamydia, Protochlamydia and Piscichlamydia based on sequence analysis of short regions of the 16S rRNA gene. Among them, Piscichlamydia is a primary fish pathogen, while Neochlamydia and Protochlamydia mostly infect free-living amoebae but have adapted thoroughly to fish.

Molecular biological studies of adult and metacercarial stages of Petasiger exaeretus Dietz, 1909 (Digenea: Echinostomatidae).

Molnár et al. (2015) reported two types of echinostomatid metacercariae in the lateral line organ of Hungarian fish species. Type 1 metacercariae possessed 27 collar spines and 16 uniform and three larger dorsal spines, whereas Type 2 metacercariae bore 27 collar spines and 19 equal-sized dorsal spines. In the recent work, molecular studies carried out on the ITS region and partial 28S rDNA sequences of two types of echinostomatid metacercariae and the sequences of adult stages of the species of Petasiger Dietz, 1909 collected from cormorants (Phalacrocorax carbo L.) showed that some of the Type 2 metacercariae corresponded to Petasiger exaeretus Dietz, 1909, whereas other morphologically similar metacercariae were identified as Petasiger phalacrocoracis (Yamaguti, 1939). The sequences of the Type 1 metacercariae with three larger dorsal spines could not be identified with any of the known sequences from echinostomatid trematodes.

Molecular detection and genome analysis of circoviruses of European eel (Anguilla anguilla) and sichel (Pelecus cultratus).

The prevalence and distribution of piscine circoviruses (CVs) were tested in a routine virus monitoring programme in Lake Balaton, Hungary. A high prevalence of European eel CV (EeCV) was found in the apparently healthy eel population (35.5%). The copy number of the viral DNA in different organs was determined by quantitative real-time PCR. The results suggested that some eel specimens were in active viraemic status despite their asymptomatic condition. Furthermore, a novel, previously undescribed CV was also detected in eel and sichel samples. Full genome characterisation confirmed that the virus represents a novel EeCV species (EeCV-2). The genome contains an integrated eel chromosome-derived fragment, suggesting that the original host of the virus was the eel and it probably emerged subsequently in the sichel by host switching. In some samples, an additional, 1,111-nt-long circular ssDNA was also observed involving a CV-like stem-loop structure and an ORF showing homology to CV capsid protein genes, without any sign of a replication initiator protein sequence.