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.

Molecular detection of a novel cyprinid herpesvirus in roach (Rutilus rutilus) and asp (Leuciscus aspius) showing typical signs of carp pox disease.

In the early spring of 2018, in Lake Balaton (Hungary), a roach (Rutilus rutilus) and an asp (Leuciscus aspius) were found in an fish trap at the outlet of the river Sió showing typical signs of the so-called carp pox disease, such as foci of epidermal hyperplasia on the head and the whole body surface, including the fins. Molecular tests revealed the presence of the DNA of an unknown fish herpesvirus. Three genes encoding the DNA-dependent DNA polymerase, major capsid protein and ATPase subunit of terminase were amplified and sequenced from the alloherpesviral genome. The gene sequences of the viruses obtained from the two different fish species shared 94.4% nucleotide sequence identity (98.1% amino acid sequence identity), suggesting that they belong to the same virus species. Phylogenetic analysis based on the DNA polymerase (and the concatenated sequences of the amplified genes, as well) implied that the detected virus belongs to the genus Cyprinivirus within the family Alloherpesviridae. The sequences of the novel alloherpesvirus diverge from those of the five cyprinivirus species described previously, so it putatively represents the sixth virus species in the genus.

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.

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.

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 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.

Isolation and characterisation of flavobacteria from wild and cultured freshwater fish species in Hungary.

The objective of this study was to survey the incidence of Flavobacterium columnare in wild and cultured freshwater fish species in Hungary. This bacterium usually causes disease in waters of more than 25 °C temperature. However, with the introduction of intensive fish farming systems, infected fish exposed to stress develop disease signs also at lower temperatures; in addition, the temperature of natural waters rises to the critical level due to global warming. Twenty-five isolates from wild and cultured freshwater fishes were identified as F. columnare by specific PCR, although both the fragment lengths and the results of PCRRFLP genotyping with BsuRI (HaeIII) and RsaI restriction enzymes raised doubts regarding this species classification. Sequencing of the 16S ribosomal RNA gene revealed that 23 isolates belonged to the species F. johnsoniae and two represented Chryseobacterium spp. The isolates were found to have high-level multidrug resistance: all were resistant to ampicillin and polymyxin B, the 23 F. johnsoniae strains to cotrimoxazole, 88% of them to gentamicin, and 72% to chloramphenicol. The majority of the 25 isolates were sensitive to erythromycin (88%), furazolidone (76%), and florfenicol (68%).

Morphological and molecular characterisation of two closely related species: Myxobolus tihanyensis n. sp. and Myxobolus sandrae Reuss, 1906.

Based on spore morphology and small subunit ribosomal DNA sequences, we describe a new Myxobolus species, Myxobolus tihanyensis n. sp., parasitizing the European perch (Perca fluviatilis) from Lake Balaton in Hungary. The brownish plasmodia were found in various locations of the body, mainly in the muscle adjacent with fins and vertebrae. The spores were ovoid, and measured 9.84 ± 0.38 µm in length, 7.69 ± 0.23 µm in width, and 5.35 ± 0.21 µm in thickness, with 8-10 sutural (edge) markings. The polar capsules were mostly equal in size, with 4.91 ± 0.39 µm in length and 2.27 ± 0.24 µm in width. The polar tubule length is 38.15 µm ± 2.70, and coiled 6-7 times. In particular, these morphological data overlap with those of Myxobolus sandrae Reuss1906 infecting the European perch (Perca fluviatilis), the pikeperch (Sander lucioperca), and the Volga pikeperch (Sander volgensis) according to previous descriptions and the taxonomic data here described. However, the phylogenetic analyses separate the two species as sister clades with 16.8% genetic distance. This study has demonstrated that the two species of Myxobolus exhibit phenotypic similarity while displaying significant genetic divergence. Therefore, the importance of including molecular data in the taxonomic description of myxozoans is emphasized.

Molecular and SEM studies on Thaparocleidus vistulensis (Siwak, 1932) (Monopisthocotyla, Ancylodiscoididae).

Presenting new molecular and scanning electron microscope (SEM) features, this study gives additional data to the better knowledge of Thaparocleidus vistulensis (Siwak, 1932) (Monopisthocotyla, Ancylodiscoididae), a parasite of the European catfish Silurus glanis Linnaeus, 1758 (Siluriformes, Siluridae) cultured in a commercial fish farm in Hungary. In addition, notes on the early development of sclerotized anchors are also provided. The main morphological difference of T. vistulensis compared to other congeneric species is associated with the male copulatory organ, which exhibits 5-7 loops in the middle of the penis length and a long open V-shaped sclerotized accessory piece, dividing terminally into two parts, securing the terminal part of the penis tube. The present study provides for the first time molecular characterization data based on the 2694 bp long nucleotide sequence of rDNA (ITS1, 5.8S, ITS2, and flanked with partial 18S and partial 28S) submitted in GenBank with the accession number OR916383. A phylogenetic tree based on ITS1 sequences supports a well-defined clade including T. vistulensis, forming a sister group with T. siluri, a species-specific monopisthocotylan parasite to S. glanis. The morphological characterization of T. vistulensis, especially for the male copulatory organ, together with the molecular data in the present study, extends knowledge about this monopisthocotylan species and provides new information for future phylogeny studies.

Characterisation of avian Pasteurella multocida strains with PCR-RFLP analysis of the ompH gene.

The 16 somatic serotype type strains and 60 field isolates of Pasteurella multocida, representing various avian species and geographic regions in Hungary, were characterised by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the ompH gene with DraI restriction endonuclease. The type strains yielded eight different (I-VIII) profiles. Strains whose PCR fragment was uncut by DraI (profile IV) could be differentiated with HindIII and PvuII restriction endonucleases. Five of the eight PCR-RFLP profiles (I, III, V, VI and VII) were detected among the field strains. Only a correlation of limited strength was found between the classical somatic serotypes and the PCR-RFLP profiles. However, the results confirmed that molecular methods could confidently distinguish serotype A:1 strains from the other serotypes. Moreover, the specific relationship between somatic serotypes and PCR-RFLP types among isolates from turkey raises the possibility of the existence of host-specific clones within the P. multocida population.

Reproductive strategies of the parasitic flatworm Thaparocleidus vistulensis (Siwak, 1932) (platyhelminthes, monogenea) infecting the European catfish Silurus glanis Linnaeus, 1758.

The life cycle of Thaparocleidus vistulensis (Siwak, 1932), a host-specific monogenean parasite of European catfish (Silurus glanis Linnaeus, 1758), was investigated by detailed observation of infection dynamics, egg development, hatching rate and in vitro survival rates of the parasite at different life stages at 23 °C. A total of 30 naive fingerlings were infected in three exposure trials by co-habitation with donor fish carrying adult parasites. Two fish were dissected every two days during the 10-day experimental period to explore the development of larvae and juvenile parasites on the host gills. Freshly laid eggs by adult monogeneans were collected and observed daily under a light microscope until hatching. A total of 445 eggs were collected and distributed into wells of 96-well microtiter plates containing filtered fish tank water to determine their hatching rates. A similar method was used to investigate the survival rates of isolated parasites at different developmental stages (larvae, juveniles, and adults). T. vistulensis populations on the European catfish in fish tanks increased markedly within ten days, dependent on the severity of the initial infection levels of the donor fish. The first eggs hatched three to four days after oviposition, and the hatching rate peaked on the fifth day (89.7%). The survival rate for freely swimming oncomiracidia without host was 7.4% after five days, whereas isolated juvenile and adult parasites showed a higher dependence of host contact (survival rates three days post-isolation of 0.9% and 1.6%, respectively). The data allows prediction of parasite-host dynamics and may improve control of gill-disease in cultured European catfish stocks in fish farms.

Juvenile Wels Catfish (Silurus glanis) Display Age-Related Mortality to European Catfish Virus (ECV) under Experimental Conditions.

We have limited knowledge about the course of the European catfish virus (ECV) infection in different age groups of wels catfish (Silurus glanis). The results of this study demonstrate that an ECV strain isolated from the brown bullhead (Ameiurus nebulosus) in Hungary could cause devastating losses among juvenile wels catfish. Furthermore, the age-related mortality rate following ECV infection was investigated in three virus challenge experiments at two different virus dosages. Eight-week-old (ca. 3 g), ten-week-old (ca. 8 g), and sixteen-week-old (ca. 55 g) catfish were infected with ECV at 21°C. In the youngest age group, 96% (at a 106 TCID50/mL dosage) and 100% (at 105 TCID50/mL) mortality rates were observed, while these rates were reduced to 56% and 68% in the ten-week-old groups, respectively. The mortality was significantly higher in the virus-exposed groups than in the control ones. In the sixteen-week-old group, 23% mortality was detected at a 105 TCID50/mL concentration of ECV. Here, a significant difference was not found between the exposed and control groups. The performed experiments show that different age groups of wels catfish may have various susceptibility to ECV. These findings draw attention to the importance of the prevention of/protection against virus infections in juvenile (up to 3-month-old) wels catfish in aquaculture.

Effect of 80% ethanol or 10% formalin fixation, freezing at -20 °C and staining on Myxobolus (Myxosporea) spores to be deposited in parasitological collections.

The preparation of myxosporeans for the description of myxospores and their preservation as type material in parasitological collections show great variations. Most frequently, formalin and ethanol are used for fixation and Giemsa solution for staining spores. In this work, authors studied the effect of 80% ethanol and 10% formalin fixation, freezing at -20 °C and staining on the size and transparency of two Myxobolus species of cyprinid fishes, M. bramae and M. bliccae spore, and recommended a new method for the deposition of type material to parasitological collections in museums. The studies have commended that fresh spores from mature plasmodia are the best material for measuring the size and studying the inner structures, the number of polar tubules in polar capsules and the morphological characters of the intercapsular appendix. The obtained quantitative data suggest that cryo- and chemical preservation do not have a notable negative effect on spores compared to fresh samples but they decrease the transparency of spores. Staining the spores with Ziehl-Neelsen has proved to be a useful method for studying the fine structure without size reduction, while Giemsa staining induced a shrinkage of spores so it seems to be not ideal for description of a new species. When treating spores of Myxobolus spp. with Lugol's solution, iodinophilous vacuoles in the sporoplasm were not recognised but visualisation of the coils of polar tubules was enhanced. As a type material for newly described species, authors suggest phototypes and spores fixed in 80% ethanol to be deposited into collections, as this preservation method is suitable for subsequent research, such as re-measurements and molecular analysis.

Morphological and molecular studies on two myxosporean infections of cyprinid fishes: Thelohanellus pyriformis from tench and Thelohanellus cf. fuhrmanni from nase.

During surveys on myxosporean parasites of Lake Balaton and River Danube fishes, two Thelohanellus spp. were found on tench (Tinca tinca) and on common nase (Chondrostoma nasus). They were identified as Thelohanellus pyriformis and Thelohanellus cf. fuhrmanni, respectively. Myxospores of T. pyriformis from tench were collected from arteria branchialis afferens of gill filaments. The mature myxospores of this species were pyriform in shape and 19 ± 0.6l (18-19.5) long, 8.2 ± 0.54 (7.5-9) wide, 7.3 ± 0.25 (7-7.5) thick containing polar tubules with 9-10 turns. The plasmodia of Thelohanellus cf. fuhrmanni were collected from under the skin of snout of the common nase. The myxospores were pyriform, 16.3 ± 0.39l (15.5-16.5) long, 6.5 ± 0.55 (6.3-7) wide, 6.3 ± 0.53 (5.8-7) thick containing polar tubules with 6 turns. Small subunit ribosomal DNA sequences of both Thelohanellus species differed from other known myxozoans. The myxospores morphology, histopathology and ssrDNA sequences supported a diagnosis of T. pyriformis from tench and T. cf. fuhrmanni from common nase.

Viruses Infecting the European Catfish (Silurus glanis).

In aquaculture, disease management and pathogen control are key for a successful fish farming industry. In past years, European catfish farming has been flourishing. However, devastating fish pathogens including limiting fish viruses are considered a big threat to further expanding of the industry. Even though mainly the ranavirus (Iridoviridea) and circovirus (Circoviridea) infections are considered well- described in European catfish, more other agents including herpes-, rhabdo or papillomaviruses are also observed in the tissues of catfish with or without any symptoms. The etiological role of these viruses has been unclear until now. Hence, there is a requisite for more detailed information about the latter and the development of preventive and therapeutic approaches to complete them. In this review, we summarize recent knowledge about viruses that affect the European catfish and describe their origin, distribution, molecular characterisation, and phylogenetic classification. We also highlight the knowledge gaps, which need more in-depth investigations in the future.

Housing, Husbandry and Welfare of a "Classic" Fish Model, the Paradise Fish (Macropodus opercularis).

Thanks to its small size, external fertilization and fecundity, over the past four decades, zebrafish (Danio rerio) has become the dominant fish model species in biological and biomedical research. Multiple lines of evidence, however, suggest that the reliance on only a handful of genetic model organisms is problematic, as their unique evolutionary histories makes them less than ideal to study biological questions unrelated to their historically contingent adaptations. Therefore, a need has emerged to develop novel model species, better suited for studying particular problems. The paradise fish (Macropodus opercularis) has a much more complex behavioral repertoire than zebrafish and has been a favored model animal in ethological research during the last decades of the previous century. We believe that with currently available, easily adaptable genetic toolkits, this species could be easily developed into a popular model of behavioral genetics. Despite its earlier popularity, however, the description of a detailed housing and husbandry protocol for this species is still missing from scientific literature. We present here a detailed description of how to raise and breed paradise fish successfully under laboratory conditions, and also discuss some of the challenges we faced while creating a stable breeding population for this species in our facility.