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.

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.

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.

Myxobolus sp. and Henneguya sp. (Cnidaria: Myxobolidae) natural co-infection in the kidney of Piaractus mesopotamicus (Characiformes: Serrasalmidae).

This study evaluated the myxozoan infection and histopathology of the kidney of freshwater fish Piaractus mesopotamicus from intensive fish farming in Brazil. A total of 55 fish were examined for myxozoan infection. Infected organs were processed by usual histology and stained with hematoxylin-eosin (H&E) and Ziehl-Neelsen (ZN). From the total of 55 fish analyzed, 47 (85.45%) presented myxospores, being 9.09% (5/55) only with Myxobolus sp., 5.45% (3/55) only with Henneguya sp., and 70.91% (39/55) presenting both parasites. The presence of myxospores was associated with histological alterations in both stromal and renal parenchyma. Myxospores were found mostly in the peritubular interstitial tissue and in low intensity in the glomerulus which caused nuclear hypertrophy and loss of Bowman space. An increase in the glomerular tuft and a reduction in the lumen of the collector tubules were also observed, besides the high number of melanomacrophage cells in the glomerulus. This study reports for the first time detection of myxozoan mixed infection in one organ of pacu and discuss the possible transportation of myxospores in the circulating blood.

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.

A synopsis of records of myxozoan parasites (Cnidaria: Myxozoa) from shrews, with additional data on Soricimyxum fegati from common shrew Sorex araneus in Hungary and pygmy shrew Sorex minutus in Slovakia.

Myxozoans (Cnidaria: Myxozoa) are almost exclusively endoparasites of aquatic vertebrates and invertebrates, with the notable exception being two species of Soricimyxum Prunescu, Prunescu, Pucek et Lom, 2007 described from terrestrial shrews (Soricidae) in central Europe. Myxospores of the two parasites are morphologically indistinguishable, but have SSU rDNA sequences that differ by about 4%. Herein, we report additional molecular and histology data from Soricimyxum fegati Prunescu, Prunescu, Pucek et Lom, 2007 from common shrew (Sorex araneus Linnaeus) from Hungary, and add a new geographic record for S. fegati in pygmy shrew (Sorex minutus Linnaeus) from Slovakia. A limited survey of shrews from the northern United States, Blarina brevicauda Say and Sorex sp. from New York, and Sorex spp. from Oregon, did not discover any infections, which is in stark contrast to the relatively high infection rates (up to 66%) in European shrew populations. We also provide a summary and discussion of literature records of species of Soricimyxum and a host survey. Given the lack of distinguishing morphological or morphometric characters between Soricimyxum spp., and the overlap in vertebrate hosts and geographic ranges, unambiguous identification of these closely related shrew parasites can presently only be achieved through sequence comparison of one or more variable SSU rDNA regions.

Description of two new species of Myxobolus Bütschli, 1892, M. peleci n. sp. and M. cultrati n. sp., detected during an intensive mortality of the sichel, Pelecus cultratus (L.) (Cyprinidae), in Lake Balaton, Hungary.

In the summer of 2014, mass mortality of the sichel, Pelecus cultratus (L.), was observed in Lake Balaton, Hungary. Parasitological examination conducted in the framework of a complete diagnostic survey revealed myxozoan infections. Two species of Myxobolus Bütschli, 1892 were found, one in the gill lamellae and another in the eyes. Following this mass mortality, 113 sichel specimens were examined during a 14-month period. Gill infection with scattered spores in the lamellae was found in 51 fish, while infection in the eyes was recorded in three specimens only. Based upon the morphological and molecular biological data the species from the gills is described here as Myxobolus peleci n. sp. and the species from the eye as M. cultrati n. sp. The 18S rDNA sequences of the two species proved that they differ from all known Myxobolus spp. with sequence data available in the GenBank database. Histological examinations revealed that the spores found in the gill lamellae were derived from plasmodia developing in and around the afferent branchial arteries of the gill arches. No mortality of sichel was recorded in 2015. Infection with these two Myxobolus spp. does not seem to play a role in the mortality of the host fish.

Histological investigation on Ancyrocephalus paradoxus (Dactylogyridea: Ancyrocephalidae) infection causing mortalities in an intensively cultured pikeperch [Sander lucioperca (L.)] stock.

In a cultured pikeperch (Sander lucioperca) stock the monopisthocotylean monogenean gill parasite Ancyrocephalus paradoxus caused heavy infection and mortalities. The gills of the affected fish specimens were infected by 50 to 800 monogenean parasites. Severe pathological changes were found in areas where the worms attached to the gills. At the attachment sites the haptoral discs of the worms formed a deep depression in the epithelium of the filaments, and the anchors pierced into and fixed themselves to the connective tissue of the cartilaginous gill rays. At these attachment sites red blood cells released from injured capillaries were found among the damaged epithelial cells. Around the hooks, anchors and body sections coming into contact with the gill filaments a proliferative tissue developed in which only a remnant of the damaged lamellae was found. Due to the damage caused by the worms the tips of the heavily infected gill filaments fused, formed clubs and were composed of epitheloid-type regeneration tissue lacking respiratory lamellae. In the basal parts of the filaments, where most of the worms attached to the gill, only denuded filaments deprived of lamellae were observed among the cross-sectioned worms in histological sections.