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Emerging infectious diseases represent a key problem for the survival of amphibian populations. Declines related to infections of the chytrid fungus Batrachochytrium dendrobatidis and Ranavirus have been described in several areas of Northern Spain, including protected areas. Co-infection—the simultaneous infection of a host by multiple pathogen species—can increase the susceptibility to subsequent infections. Here, we describe a case of triple dermocystid-ranavirus-chytrid fungus co-infection in a specimen of palmate newt (Lissotriton helveticus) from a protected wetland of North-western Spain. Five palmate newts and one Bosca’s newt (Lissotriton boscai) were found dead, and tissue samples were analysed by using qPCR methods. Ranavirus was detected in four out of five L. helveticus and in the L. boscai individual, whereas Bd was detected only in one of the five L. helveticus. Four out of six specimens tested positive for Dermocystidium. Sequencing was performed to double-check the presence of Dermocystidium in a qPCR-positive sample for that pathogen.

Amphibian parasites of the genus Amphibiocystidium are members of the class Ichthyosporea (=Mesomycetozoea), within the order Dermocystida. Most of the species in the Dermocystida fail to grow in ordinary culture media, so their life cycle has only been partially constructed by studies in host tissues. However, to date, there have been few reports on the life cycle of Amphibiocystidium parasites with respect to the developmental life stages of both Dermocystidium and Rhinosporidium parasites. In this study, we provide light and electron microscopic findings of developmental phenotypes of Amphibiocystidium sp., a parasite previously characterized in the Italian stream frog (Rana italica), which has caused an ongoing infection in a natural population of Central Italy. These phenotypes exhibited distinct morphological characteristics that were similar to A. ranae from the skin of R. temporaria, but showed histochemical properties particularly comparable with those of maturing phenotypes of Rhinosporidium seeberi, and compatible with fungal-like parasites. Therefore, for Amphibiocystidium sp. phenotypes, we suggest adopting the terminology used for maturing stages of R. seeberi, such as juvenile sporangia, early mature sporangia and mature sporangia. The characterization of these developmental stages will be useful to increase the understanding of the life cycle of parasites of the genus Amphibiocystidium and of the interactions with their amphibian hosts.

Thirty-six Brook Silversides, Labidesthes sicculus, and 15 Golden Silversides, Labidesthes vanhyningi, collected from the Ouachita and Red River drainage basins of Arkansas and Oklahoma, U.S.A., were examined for parasites. Six (17%) of the L. sicculus and 3 (20%) of the L. vanhyningi were infected with Dermocystidium sp., 1 (7%) L. vanhyningi harbored an immature tapeworm, Proteocephalus sp., and 1 (7%) L. vanhyningi was infected with the copepod Ergasilus funduli. We document new host records for all 3 parasites as well as 2 new state records for Dermocystidium sp. and E. funduli.

Murray cod (Maccullochella peelii (Mitchell, 1838)) is an iconic Australian species, initially farmed mostly for stocking but now also for human consumption. Here, we report an infection with Dermocystidium sp., class Mesomycetozoea, order Dermocystida in the gills of Murray cod, from a hatchery in south-eastern Australia. Based on phylogenetic analyses of the 18S ribosomal DNA the parasite was closely related to D. salmonis, however the size of the spores was smaller than that reported for D. salmonis in other parts of the world. Further studies using additional analyses including ultrastructure of the spores would be useful for elucidating the identification of the microorganism. Histopathology showed inflammatory response and epithelial hyperplasia resulting in lamellar fusion particularly in association with the parasite. The condition was diagnosed as branchitis caused by a co-infection with Dermocystidium and epitheliocystis, with Dermocystidium being the dominant pathogen.

Amphibian parasites of the Order Dermocystida (Ichthyosporea) are widespread pathogens known mainly from Europe and North America, which cause primarily a disease of skin and subcutaneous tissue in their hosts. The taxonomy of these organisms has been problematic given their conserved morphology, similar clinical disease and pathology. Currently recognized taxa belong to the three closely related genera, Amphibiocystidium, Amphibiothecum, and Rhinosporidium, whereas species of Dermocystidium and Sphaerothecum destruens include fish parasites. Here, we review the taxonomy of Dermocystida based on a molecular phylogenetic analysis, principally of amphibian parasites, including DNA sequences obtained from amphibian hosts collected in the central-eastern region of South America. A new taxonomic arrangement is proposed, which includes the designation of type material for Dermocystidium pusula, synonymization of Amphibiothecum with Dermocystidium, and the restriction of Amphibiocystidium to its type species A. ranae. We also review the taxonomic status of Dermosporidium hylarum until the present work included in the synonymy of the human and animal pathogen R. seeberi, and considered herein as a valid taxon, however. In addition, a new species of Sphaerothecum parasitic to amphibians is described, being the first record of this genus in the southern hemisphere and in an amphibian host. Fil: Borteiro, Claudio. Universidad de la República; Uruguay. Museo Nacional de Historia Natural; Uruguay Fil: Baldo, Juan Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Universidad Nacional de Misiones. Instituto de Biología Subtropical; Argentina Fil: Maronna, Maximiliano M.. Universidade de Sao Paulo; Brasil Fil: Pontes Baêta Da Costa, Délio. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Sabbag, Ariadne Fares. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Kolenc, Francisco. Museo Nacional de Historia Natural; Uruguay Fil: Debat, Claudio Martínez. Universidad de la República; Uruguay Fil: Haddad, Célio F. B.. Universidade Estadual Paulista Julio de Mesquita Filho; Brasil Fil: Cruz, Juan Carlos. Universidad de la República; Uruguay Fil: Verdes, José Manuel. Universidad de la República; Uruguay Fil: Ubilla, Martín. Universidad de la República; Uruguay

The genus Dermocystidium comprises fungal pathogens of the order Dermocystida in the class Ichthyosporea (http://www.ncbi.nlm.nih.gov/taxonomy). Several different Dermocystidium species have been described, infecting a variety of fish hosts and producing gill infections, skin lesions and visceral disease all over the world (Wildgoose 1995; Pekkarinen & Lotman 2003; Pekkarinen et al. 2003; Feist et al. 2004; Zhang & Wang 2005). To our knowledge, there are two reports on Dermocystidium infections in cardinal tetra, Paracheirodon axelrodi and neon tetra, Paracheirodon innesi (Reichenbach-Klinke 1982; Lewisch 2010). However, these reports did neither include histological and ultrastructural examinations nor molecular genetic investigations to confirm the diagnosis and identify the aetiologic species. In January 2013, increased mortalities of cardinal tetra, P. axelrodi of a 350-L aquarium were reported, occurring after purchase of additional fish. During the clinical examination, most of the fish were swimming in normal active condition, but some were lethargic or displayed a transparent mass on the skin of the head or body. The masses were up to 5 mm in diameter and contained a central, white tubular structure. Similar masses were located on the fins of some fish, but were considerably smaller in these locations. A parasitic infestation of the skin of these fish was excluded via microscopic examination, and normal results were obtained after analysis of the water values and inspection of the pump and filter system. Six cardinal tetra, P. axelrodi and two firehead tetra, Hemigrammus bleheri were submitted for pathological examination in formalin and for molecular genetic examination in ethanol. Only two cardinal tetra displayed skin lesions. Before embedding, one tetra was post-fixed in Davidson’s fixative and sections of glycolmethacrylate/methylmethacrylate-embedded samples were routinely processed for histological examination and stained with haematoxylin and eosin (HE), Giemsa, silver impregnation and periodic acid Schiff (PAS) reaction according to standard protocols. Samples of the mass were also routinely processed for transmission and scanning electron microscopy on a transmission electron microscope (Zeiss EM 10) or on a digital scanning electron microscope (Zeiss DSM 950), respectively. The macroscopic examination of the cardinal tetra revealed a focally extensive, bulging, hemispherical, transparent oedema (4 mm in diameter) of the ventral skin on the head. Central, within Correspondence M C Langenmayer, Institute of Veterinary Pathology at the Centre for Clinical Veterinary Medicine, LMU Munich, Veterin€ arstrasse 13, 80539 Munich, Germany (e-mail: langenmayer@patho.vetmed.uni-muenchen.de) Langenmayer and Lewisch contributed equally to this work.

SUMMARYOutbreaks of cutaneous infectious disease in amphibians are increasingly being attributed to an overlooked group of fungal-like pathogens, the Dermocystids. During the last 10 years on the Isle of Rum, Scotland, palmate newts (Lissotriton helveticus) have been reportedly afflicted by unusual skin lesions. Here we present pathological and molecular findings confirming that the pathogen associated with these lesions is a novel organism of the order Dermocystida, and represents the first formally reported, and potentially lethal, case of amphibian Dermocystid infection in the UK. Whilst the gross pathology and the parasite cyst morphology were synonymous to those described in a study from infectedL. helveticusin France, we observed a more extreme clinical outcome on Rum involving severe subcutaneous oedema. Phylogenetic topologies supported synonymy between Dermocystid sequences from Rum and France and as well as their distinction fromAmphibiocystidiumspp. Phylogenetic analysis also suggested that the amphibian-infecting Dermocystids are not monophyletic. We conclude that theL. helveticus-infecting pathogen represents a single, novel species;Amphibiothecum meredithae.

Goldfish, common carp and Nile tilapia were sampled between September 2002 and May 2003 to investigate lesions induced by viral diseases and ectoparasites. Goldfish exhibited neoplasms, Dermocystidium sp., Ichthyophthirius multifiliis (Ich), Trichodina reticulata, Lernaea cyprinacea and systemic infections. Neoplastic and systemic infections in goldfish were associated with viral infection. Oreochromis niloticus and Cyprinus carpio were mainly infested with several species of Trichodina and monogeneans. The presence of Dermocystidium sp. in carp, and viral particles in the liver and spleen of O. niloticus and C. carpio , was uncommon. Microscopically, intracytoplasmic inclusion bodies in the hepatic cells and lymphocytic infiltration in other internal organs of the three fish species were seen. Ultrastructural study showed enveloped viral particles in some cells in all fish species. The recorded parasitic infections were associated with degenerative, necrotic, inflammatory and proliferative changes in the skin and underlying muscles. Imported ornamental goldfish may be a source of infection to carp and native tilapia. Histopathology and transmission electron microscopy demonstrated the etiology and pathogenesis of infection and the preliminary diagnosis of viral infection, which is not common in Egypt. It is recommended that goldfish be excluded from edible-fish aquaculture ponds. Strict regulations regarding the transfer and quarantine of imported fish should be implemented. Keywords: Carassius auratus ; Cyprinus carpio ; Dermocystidium sp.; electron microscopy; histopathology; Ichthyophthirius multifiliis ; Ich; Lernaea cyprinacea ; Oreochromis niloticus ; Trichodina reticulata ; viruses African Journal of Aquatic Science 2009, 34(2): 111–121

A disease affecting larval Pacific oysters (Crassostrea gigas) was observed during a 3-year period at a hatchery in Washington State. It was also detected at 3 other hatcheries and in wild stocks. The clinical course of the acute phase of the disease in the hatchery varied from one to 3 days. Larvae that completed metamorphosis in less than 12 days survived the infection. Under intensive cultural conditions, the mortality rate exceeded 95%. The disease occurred at water temperatures of 20 to 30°C and salinities of 13 to 28 ppt. The earliest sign of the disease was the presence of numerous zoo-spore-like bodies on the cilia of the velum. These bodies progressively increased in number and the velum and mantle became eroded. Numerous thick-walled spherical bodies, 4 to 24 μm in diameter, were found in the stomachs of apparently healthy and sick larvae that were at least 7 days of age. Moribund larvae regurgitated fascicular collections of zoospore-like and thick-walled spherical bodies from the stomach. The regurgitated masses became entwined on and were rotated by the velar cilia, further traumatizing the surface of the velum. The terminal stage of infection was characterized by severe velar erosion, numerous enlarged spherical bodies in the stomach, and dilated darkened intestines. Histologic and electron microscopic study of affected larvae revealed fascicular masses on velar and mantle epithelia associated with adjacent cellular destruction. Spherical bodies were impacted in the stomach and frequently obstructed the gastro-diverticular opening. In some cases the intestines were dilated and ruptured. Based upon observations, a mycotic etiology is hypothesized. The etiologic agent exhibits similarities to the Dermocystidium group of organisms. The relationship between the wide distribution of the diseast and reported difficulties in culturing Pacific oyster larvae is discussed.

Perkinsus (= Dermocystidium) marinus is a major cause of mortality in eastern oysters, Crassostrea virginica. Because initiation of infection and progression of disease are favored by high temperature and high salinity, we hypothesized that climatic cycles influence cycles of disease. Analyses of a 10-y time series of disease prevalence and intensity, chlorophyll a, suspended sediments, water temperature and salinity from a Louisiana site, using a wavelet technique, show a teleconnection between the El Nino-Southern Oscillation (ENSO) and oyster disease in the northern Gulf of Mexico. Salinity increases precede increased disease prevalence by several months. The changes in salinity that trigger changes in disease prevalence and intensity are strongly driven by ENSO events. Interannual variation is important in the initiation and intensification of disease, and salinity is the primary driving factor. The patterns in the environmental and disease time series suggest that epizootics can be initiated...

A species of Dermocystidium was discovered on the skin and fins of reared southern catfish Silurus meridionalis Chen. The parasite only appeared and caused disease in juvenile catfish at a water temperature of 18 to 23 degrees C. Marked external macroscopical clinical signs of the disease were sluggish movement of the fish, and the appearance of white filiform dermal cysts varying in size (3-20 mm in length and 0.15-0.35 mm in width). Examination of both fixed and fresh mounts for light microscopy and of samples for transmission electron microscopy (TEM) showed spherical spores (3.2-15 microm in diameter) with a peripheral nucleus (1.1-1.8 x 0.5-1.6 microm in diameter) and a prominent refractile body (2.08-10.83 microm in diameter) which occupied most of the volume of a mature spore. Three types of spore were identified, and are presumed to represent various developmental stages. Meanwhile, TEM showed the remnant nuclei in the residual plasmodium of a cyst, revealing its degenerative process. This paper represents the first observation and description of Dermocystidium sp. parasitizing catfishes.

Bullheads, Cottus gobio, with macroscopic external cysts on the skin and fins measuring up to 3 mm in diameter were detected in the River Allen and its tributaries in southern England between 1992 and 1998. The prevalence of these cysts was up to 50% at some sites. Examination of cyst contents revealed the presence of numerous spores, typical of the genus Dermocystidium, measuring 8 microm in diameter. The parasite developed within well-defined cysts, which were located in the hypodermal connective tissues of the host. No cysts were present on the fins of any of the fish examined. Histological examination revealed a cyst wall consisting of an inner layer of dense eosinophilic material similar to that reported for Dermocystidium spp. forming coenocytic hyphae. No evidence was found of systemic infection or hyphal formation. Spores contained a prominent refractile body, which gave a weakly positive reaction for polysaccharides with the periodic-acid Schiff reaction and was positively stained with acidic dyes. Several examples of ruptured cysts were seen in histological sections and in some of these cases the host epithelial layer was breached, allowing release of the spores to the environment. Morphological features of, and host response towards, the Dermocystidium sp. in bullheads are compared with similar infections in salmonids and other freshwater fish species.

In Finland Dermocystidium percae Reichenbach-Klinke was first recorded by Pekkarinen in the fins of a perch in 1988. Because it is a poorly known parasite, its occurrence and life stages are studied here. In occasional sampling during 1995-1998 and 2001, and more systematic sampling during 1999 and 2000 in two different lakes (one oligotrophic, the other slightly eutrophic), it was found to occur almost continuously, although sometimes very sparsely, in both lakes. The life cycle is maintained from year to year by overwintering cysts. Young perch acquire first infections in their first summer of life, and fish over 3 years possibly develop some immunity. In Estonia, in the Kasari River and the Matsalu Bay, the parasite seems to be very rare. Cysts of D. percae, capable of sporogenesis, can occasionally occur in the ruff, too. In perch in Finland, cysts were found differing from the longish and thin-walled cysts typical of D. percae. These roundish to dumbbell-shaped cysts with thicker walls are here sugge...

We report the enigmatic parasite Dermocystidium ranae in a green frog population (Solomeo, Umbria, Italy) of the Rana esculenta complex, consisting of the parental species R. lessonae (L) and hybrid form R. esculenta (E). In this population a rapid 50% decline of the parental form L was observed. Large dermal U-shaped cysts of D. ranae were found primarily on the ventral aspect of infected individuals, with a significantly higher incidence of infection in the parental species com- pared to the clonal hybrid. In each form, however, there was little pathological change associated with infection, and the cause of the recent declines of R. lessonae at this site remains unknown. In this paper we present the first ultrastructural description of an amphibian Dermocystidium sp. and we review the taxonomy of Dermocystidium, Dermosporidium and Dermomycoides spp. from amphib- ians. We conclude that Dermosporidium multigranulare Bro= & Kulda, 1954 is synonymous with Der- mocystidium ranae Guyenot & Naville, 1922 and, due to lack of sufficient differences between gen- era and significant dissimilarities with fish Dermocystidium spp., the 3 amphibian genera are synonymous. We propose that they should be designated to a new genus, Amphibiocystidium n. gen., and Dermocystidium retained for those species parasitic in fish.

During the early 1950s, Sammy M. Ray discovered that his high-salt modification of fluid thioglycollate sterility test medium caused dramatic in vitro enlargement of Perkinsus marinus (=Dermocystidium marinum) cells that coincidentally infected several experimentally cultured oyster gill tissue explants. Subsequent testing confirmed that the enlarged cells among some oyster tissues incubated in Ray's fluid thioglycollate medium (RFTM) were those of that newly described oyster pathogen. Non-proliferative in vitro enlargement, cell wall thickening, and subsequent blue-black iodine-staining of hypertrophied trophozoites (=hypnospores=prezoosporangia) following incubation in RFTM are unique characteristics of confirmed members of the protistan genus Perkinsus. A number of in vitro assays and manipulations with RFTM have been developed for selective detection and enumeration of Perkinsus sp. cells in tissues of infected molluscs, and in environmental samples. RFTM-enlarged Perkinsus sp. cells from tissues of infected molluscs also serve as useful inocula for initiating in vitro isolate cultures, and cells of several Perkinsus spp. from both in vitro cultures and infected mollusc tissues may be induced to zoosporulate by brief incubations in RFTM. DNAs from RFTM-enlarged Perkinsus sp. cells provide useful templates for PCR amplifications, and for sequencing and other assays to differentiate and identify the detected Perkinsus species. We review the history and components of fluid thioglycollate and RFTM media, and the characteristics of numerous RFTM-based diagnostic assays that have been developed and used worldwide since 1952 for detection and identification of Perkinsus spp. in host mollusc tissues and environmental samples. We also review applications of RFTM for in vitro manipulations and purifications of Perkinsus sp. pathogen cells.

In autumn 2011 in cyprinid farms located in Iasi on the Jijia river, several infections with bacterial strains and macroscopical external cysts on the skin were diagnosedwhich developed as a result of the stress induced by biotic and abiotic factors. On the examination of the cyst contents the presence of numerous spores was observed, mostly of the Dermocystidium spgenusThe samples were taken from the common carp (Cyprinus carpio) and crucian carp (Carassius auratus gibelio)species from the fish farm as well as from the Jijia River. 35 fish were examined, all of them showing cysts, fragmentation of their dorsal fin and congestion of the gills. Histological examination of the skin showed a field of multiple dermal cysts with round light eosinophilic formations (14-16µm) containing a central refractable body similar to that reported for Dermocystidium sp.Gills samples were taken from the affected areas for the SEM examination with the purpose of evaluating not only aspects of normal morphology, but also aspects of some modifications of the affected areas as well as the presence of the etiologically incriminated bacteria Pseudomonas fluorescens. The isolates were identified through phenotypic methods. All the strains that showed mobility and oxidase-positivity were tested using API 20 NE strip. Consequently, they were taxonomically grouped into the species Pseudomonas fluorescens.The scanning electron microscope (SEM) was used for the first time in the characterization of the bacterial lesions produced by Pseudomonasstrains on Cyprinus carpioand Carassius auratus gibeliogills. The diagnosis of septicemia with conditional pathogen species of Pseudomonas fluorescens was correlated with the results of the physico-chemical investigations of water and the data concerning the breeding conditions of the investigated livestock.

For the past 100 years the phylogenetic affinities of Rhinosporidium seeberi have been controversial. Based on its morphological features, it has been classified as a protozoan or as a member of the kingdom Fungi. We have amplified and sequenced nearly a full-length 18S small-subunit (SSU) ribosomal DNA (rDNA) sequence from R. seeberi . Using phylogenetic analysis, by parsimony and distance methods, of R. seeberi ’s 18S SSU rDNA and that of other eukaryotes, we found that this enigmatic pathogen of humans and animals clusters with a novel group of fish parasites referred to as the DRIP clade ( Dermocystidium , rossete agent, Ichthyophonus , and Psorospermium ), near the animal-fungal divergence. Our phylogenetic analyses also indicate that R. seeberi is the sister taxon of the two Dermocystidium species used in this study. This molecular affinity is remarkable since members of the genus Dermocystidium form spherical structures in infected hosts, produce endospores, have not been cultured, and possess mitochondria with flat cristae. With the addition of R. seeberi to this clade, the acronym DRIP is no longer appropriate. We propose to name this monophyletic clade Mesomycetozoa to reflect the group’s phylogenetic association within the Eucarya .

Sequences of nuclear-encoded small-subunit rRNA genes have been determined for representatives of the enigmatic genera Dermocystidium, Ichthyophonus, and Psorospermium, protistan parasites of fish and crustaceans. The small-subunit rRNA genes from these parasites and from the "rosette agent" (also a parasite of fish) together form a novel, statistically supported clade. Phylogenetic analyses demonstrate this clade to diverge near the animal-fungal dichotomy, although more precise resolution is problematic. In the most parsimonious and maximally likely phylogenetic frameworks inferred from the most stably aligned sequence regions, the clade constitutes the most basal branch of the metazoa; but within a limited range of model parameters, and in some analyses that incorporate less well-aligned sequence regions, an alternative topology in which it diverges immediately before the animal-fungal dichotomy was recovered. Mitochondrial cristae of Dermocystidium spp. are flat, whereas those of Ichthyophonus hoferi appear tubulovesiculate. These results extend our understanding of the types of organisms from which metazoa and fungi may have evolved.

Summary Dermocystidium koi Hoshina and Sahara, 1950 is characterized by formation of a web of aseptate hyplye which pervade the subcutaneous tissue of the host, Cyprinus carpio var. koi. Within the hyphae, multinucleate cytoplasmic contents eventually produce a large number of spores with a typical central refractile inclusion. They are of extremely variable size (diameter from 6.5 to 15 μm); there is some indication that the size range may reflect tle gradual growth of spores. Hyphae formation confirms the assumption of the fungal nature of this organism, yet its precise osition remains unsolved. The host reaction is first reflected in oedema formation, later with cellular infiltrate leading to proliferative inflammation followed by the formation of granulation tissue. Zusammenfassung Ein neuer Beweis der pilzartigen Natur von Dermocystidium koi Hoshina und Sahara, 1950 Dermocystidium koi Hoshina und Sahara, 1950 wird durch ein Netz von aseptaten Hyphen charakterisiert, die das subkutane Gewebe seines Wirtes, Cyprinus carpio var. koi durchdringen. Die Hyphen enthalten ein vielkerniges Cytoplasma, woraus schlieslich eine grose Zahl von Sporen entsteht, die durch einen grosen neutralen lichtbrechenden Korer gekennzeichnet werden. Sorengrose ist auserordentlich veranderlich mit einem Bereich von 6.5 bis 15 μm. Aus den durchefusrten Beobachtungen kann man vermuten, das es sich urn ein progressives Wachstum der Sporen tandelt. Die Bildung van Hyphen bestatigt die Annahrne der Angehorigkeit von Dermocystidium zu den pilzartigen Organismen, eine genaue taxonomische Stellung kann man aber leider nicht beurteilen. Die Wirtsreaktion wird zuerst als Oedema zum Ausdruck gebracht, spater als zellulares Infiltrat und proliferative Entzundung. Der Prozes wird durch Erscheinung von Granulationsgewebe vollendet.

Oysters from 48 Gulf of Mexico sites were examined for presence and infection intensity of the endoparasite, Perkinsus (= Dermocystidium) marinus (Mackin, Owen and Collier, 1950) as part of NOAA's Status and Trends Mussel Watch Program. Prevalence exceeded 75% at 25 sites. Infection intensity did not vary with sex or reproductive stage. Latitude, total polynuclear aromatic hydrocarbon (PAH) content and industrial and agricultural land use significantly affected the parasite's distribution. PAH and pesticide concentrations were latitudinally dependent, suggesting an impact of spawning frequency on uptake and depuration. P. marinus analysis complements the use of pollutant body burden for determining change in environmental quality because it responds differently than pollutant body burden to the biology and ecology of the oyster.

SUMMARYGiven the worldwide decline of amphibian populations due to emerging infectious diseases, it is imperative that we identify and address the causative agents. Many of the pathogens recently implicated in amphibian mortality and morbidity have been fungal or members of a poorly understood group of fungus-like protists, the mesomycetozoans. One mesomycetozoan, Amphibiocystidium ranae, is known to infect several European amphibian species and was associated with a recent decline of frogs in Italy. Here we present the first report of an Amphibiocystidium sp. in a North American amphibian, the Eastern red-spotted newt (Notophthalmus viridescens), and characterize it as the new species A. viridescens in the order Dermocystida based on morphological, geographical and phylogenetic evidence. We also describe the widespread and seasonal distribution of this parasite in red-spotted newt populations and provide evidence of mortality due to infection.

A visceral form of a Dermocystidium-like organism was found at 13% prevalence in a carp and in 7·4% in a roach population from Greece. Successful experimental transmission was recorded only in carp. Pathology of the parasite in experimental fish and naturally infected fish was similar. ©1998 The Fisheries Society of the British Isles

The pathogen of frogs Amphibiocystidium ranae was recently described as a new genus. Due to its spherical shape, containing hundred of endospores, it was thought to be closely related to the pathogens of fish, mammals, and birds known as Dermocystidium spp., Rhinosporidium seeberi , and Sphaerothecum destruens in the Mesomycetozoea, but further studies were not conducted to confirm this relationship. To investigate its phylogenetic affinities, total genomic DNA was extracted from samples collected from infected frogs containing multiple cysts (sporangia) and endospores. The universal primers NS1 and NS8, used to amplify the 18S small-subunit rRNA by PCR, yielded ≈1,770-bp amplicons. Sequencing and basic local alignment search tool analyses indicated that the 18S small-subunit rRNA of A. ranae from both Rana esculenta and Rana lessonae was closely related to all of the above organisms. Our phylogenetic analysis placed this pathogen of frogs as the sister group to the genus Dermocystidium and closely related to Rhinosporidium . These data strongly supported the placement of the genus Amphibiocystidium within the mesomycetozoeans, which is in agreement with the phenotypic features that A. ranae shares with the other members of this class. Interestingly, during this study Dermocystidium percae did not group within the Dermocystidium spp. from fish; rather, it was found to be the sister group to Sphaerothecum destruens . This finding suggests that D. percae could well be a member of the genus Sphaerothecum or perhaps represents a new genus.

The rosette agent is an obligate intracellular parasite that causes morbidity and mortality in salmonid fish. In laboratory cultures, the spore stage (2-6 microm diam.) replicates in a salmonid cell line by sequential asexual division, giving rise to daughter cells. If infected cell cultures are transferred to distilled water, the spore stage undergoes internal division to give rise to at least 5 cells each of which develops into a uniflagellated zoospore with a body of approximately 2 microm and a flagellum approximately 10 microm long. Zoosporulation does not occur in cell culture medium alone, artificial seawater, or phosphate-buffered saline. This parasite is currently classified as a member of the Class Mesomycetozoea (formerly Ichthyosporea) based on phylogenetic analyses of the small subunit ribosomal DNA of three different isolates from fish. Given these new morphological observations combined with the available molecular phylogenetic data on other mesomycetozoeans, we propose to classify the rosette agent as Sphaerothecum destruens, n. g., n. sp. This new genus has unique features including (1) intracellular development of spore stages in various organs eliciting a host granulomatous response; and (2) the differentiation of mature spores into multiple, flagellated zoospores. Taken together, these characteristics clearly distinguish it from the closely related genera Dermocystidium and Rhinosporidium.

▪ Abstract When the enigmatic fish pathogen, the rosette agent, was first found to be closely related to the choanoflagellates, no one anticipated finding a new group of organisms. Subsequently, a new group of microorganisms at the boundary between animals and fungi was reported. Several microbes with similar phylogenetic backgrounds were soon added to the group. Interestingly, these microbes had been considered to be fungi or protists. This novel phylogenetic group has been referred to as the DRIP clade (an acronym of the original members: Dermocystidium, rosette agent, Ichthyophonus, and Psorospermium), as the class Ichthyosporea, and more recently as the class Mesomycetozoea. Two orders have been described in the mesomycetozoeans: the Dermocystida and the Ichthyophonida. So far, all members in the order Dermocystida have been pathogens either of fish (Dermocystidium spp. and the rosette agent) or of mammals and birds (Rhinosporidium seeberi), and most produce uniflagellated zoospores. Fish pathogens also are found in the order Ichthyophonida, but so are saprotrophic microbes. The Ichthyophonida species do not produce flagellated cells, but many produce amoeba-like cells. This review provides descriptions of the genera that comprise the class Mesomycetozoea and highlights their morphological features, pathogenic roles, and phylogenetic relationships.

The biological feature that characterizes fungi, above all else, is their osmotrophic (absorptive) mode of nutrition. They can neither ingest their food nor manufacture it in the way that plants do. This chapter discusses saprolegniasis, branchiomycosis, ichthyophonus, dermocystidium and harmful algae.

An outbreak of Dermocystidium infection in cultured eels, Anguilla anguilla L., from Scotland is described. The disease appeared in the winter months but did not cause significant mortalities or adversely affect the growth of the eels. Infected eels had large, elongate white cysts on the gills. The cysts consisted of a homogeneous layer of parasite origin surrounded by a fibrous capsule and hyper-plastic epithelium. Within the cysts were small spherical parasite cells with a densely staining central area and peripheral granular, vacuolated cytoplasm. The cell wall appeared to be broken down in many cases and the cells appeared degenerate. The cysts eventually collapsed and the cavity was filled with epidermal tissue.

An epizootic of dermocystidiosis occurred among pond-cultured European eels, Anguilla anguilla for the first time at a fish farm in Shizuoka Prefecture. The fungus was identified as Dermocystidium anguillae which was first reported by SPANGENBERG (1975) in East Germany. The cysts were parasitic in the epithelial and connective tissues of the gills. Attempts were made to culture the parasite on some media, but without success.When the water temperature of tanks with the diseased eels was experimentally raised from 20°C to 30°C, the cysts disappeared from the gills of all the eels within a few days, which suggested that this disease could be cured by warm-water bathing.

A new species of Dermocystidium is reported to be the causative agent of three outbreaks of gill disease in carp observed in recent years in Czechoslovakia. No other parasites or influences could be held responsible for the disease. It is suggested that at least some of gill disease of unknown aetiology in carp is due to dermocystidia, and the disease might be distributed in many regions of Europe. A description of a greater part of the developmental cycle of the species D. cyprini n.sp. Cervinka et Lom, is presented, supplemented by a discussion of other species of the genus. A summary of known facts on the host-parasite relation in these parasites is also given.

Electron-microscope studies demonstrate that presporangial cells (hypnospores) of the fungus Dermocystidium marinum are characterized by an apparent lack of mitochondria, by a cell wall homogeneous with respect to electron density, and by an electron-light nucleus. In precleavage sporangia, morphogenesis of mitochondria appears to occur from myelin figures, the cell walls acquire a three-layered appearance, and the nuclei become electron-dense with evidences of condensed chromatin. A discharge pore and tube are formed by rupture of the cell wall and subsequent unfolding of the tube from the innermost part of the cell wall. The pore is blocked by a plug which is a thickened section of the inner cell wall. Sporulation proceeds by successive bipartition of the protoplast involving alternating karyokinesis and cytokinesis. The nuclear membrane persists during karyokinesis. The process terminates with the formation of billagellated planonts, enclosed by two unit membranes. Evaginations of one or both of the membranes form a row of tinsels along one side of the anterior flagellum. Golgi bodies and a previously undescribed, electrondense body are found next to the planont nucleus. D. marinum is concluded to be a member of the Saprolegniales on the basis of planont morphology and previously described stages.

Dermocystidium koi sp. nov. This species is found as a parasite; in one case it is parasiting to the integument and in another to the muscle of each separate Cyprinus carpis L. The specimen of the former was captured at the Yamada Fish Culture Farm (Locality-Yamada Village, Hyogo Pref., Date-July 23, 1940), and that of the latter obtained from the Seto Fish Culture Farm. (Locality-Vicinity of Odawara City, Kanagawa Pref., Date-May 11, 1959). In the case of integumentary parasitim, the parasites grow between the epidermis and the cutis in uarious places of the body surface without scales; and the characteristic lesions a e formed arising from the integument, due to the increase of the parasites and host's tissues such as fibrous connective tissueand blood vessels. (see text-fig. 1 and 2, A, B.) The muscular parasitism occurs when the parasites are parasitic to the scaley host; and in this case the parasites grow in the muscle near the body surface; on the infected parts, the large and hard tumor like inflammatory swelling arise from the increase of the parasites and the same host's tissues as the former. The parasite is filiform in external character; its transuerse section is round; the diameter 0.04-0.30mm., and it contains innumerable spares. Spare subspherical, 6-14μ in diameter; the diameter of the large characteristic spherical enclosure is 4.5-10.0μ. The details are compiled in the table 1. The spares are figured in text-fig. 3, NOS. 1-9. The present species is distinguishable from all other known species, in external form of the parasite or the cyst, the modus parasitic, and the dimension of the spare etc. The lesions may be ruptured naturally in a certa'n period; and the contents fall out from them; and then they seem to be healed gradually. Therefore, the host should not suffer great influence from the parasite. But the histological reactions of the host are marked, which are chiefly proliferocis inflammation, rarely accompanied with phagocytosis. (Text.-fig. 3, No. 10).

PAGE INTRODUCTIONa. Studies in the Gulf of Mexicox. Discovery of Dermocystidiumn marinurm in Virginia 2 METHODS OF STUDY 2 The Thioglycollate Culture Technique 2 Rating the Intensity of Infections 3 Definitions of Terms 3 THE OCCURRENCE OF Dermocystidium marinuim IN LIVE OYSTERS 3 Seasonal Incidence of Infections 3 Intensity of Infections 5 Recapitulation ........ 6 THE OCCURRENCE OF Der-mocystidium marinumn IN GAPERS 6 Collection of Gapers 6 Seasonal Incidence ..... 6 Incidence in Gapers from Outlying Trays and Natural Habitats ..... 8 Recapitulation ........ 8 DISTRIBUTION OF Dermocystidium marinum IN CHESAPEAKE BAY 11 FUNGUS INFECTIONS IN RELATION TO AGE AND SouRCE OF OYSTEST.11 Data from Live Oysters . ..... 11 Data from Gapers ......... 12 Recapitulation. 13 EXPERIMENTAL INFECTION OF OYSTERS 14 Infection of Old Oysters ...... 14 Infection of Young Oysters I ........ 14 PAGE

MSX, a pathogen of oysters (Crassostrea virginica), produced a drastic epizootic in high—salinity areas of Chesapeake Bay from 1959 to 1963. The patterns of infection and mortality were determined by imports from disease—free seed—oyster areas. Winter and spring imports became infected in early summer and began dying in late summer. Late—summer imports apparently became infected promptly but infections remained subclinical until the following May. Death rates were highest during warm months but losses occurred throughout the year. MSX was assigned as the cause of a high percentage of deaths by sampling live and dead oysters. Mortality for the first and second years after import was usually 50 to 60% annually. A fungus disease caused by Dermocystidium marinum was also prevalent in some areas. Prevalence of MSX did not decline as oyster populations were decimated; hence nearly half of Virginia's private beds have been forced out of production.

A serious outbreak of mortalities occurred among adult salmon held in the Priest Rapids Dam spawning channel in November 1965. Gross examination revealed small, white cysts on and in the gill filaments of otherwise normal-appearing fish. Approximately 25% of the 5000 mature adult salmon in the channel died prior to spawning from this disease caused by a fungus identified as the genus Dermocystidium. This is the first record of an epidemic among fish of North America associated with Dermocystidium. The primary cause of death appeared to be anoxia due to subacute inflammation and granulation of the gill tissue. A description of the histopathology associated with these parasites is given. Chondrococcus columnaris, a pathogen of warm-water fishes, was present as a secondary invader on the gill filaments of several salmon and an unknown fungal or yeast-like organism was present in the testes of the male fish examined.

1. 1. Fatty acids of four marine fungi Thraustochytrium roseum, T. aureum, Schizochytrium aggregatum and Dermocystidium sp. and one fresh-water fungus Phlyctochytrium punctatum were determined. 2. 2. These fungi synthesized ω 3 and ω 6 polyunsaturated fatty acids. They contained large quantities of C20 and C22 polyenes; three of the marine fungi contained unusually large amounts of 22:6 fatty acid. 3. 3. Major fatty acids for T. roseum and S. aggregatum were palmitic, oleic and docosahexaenoic acids; for T. aureum, palmitic, docosapentaenoic and docosahexaenoic acids; for Dermocystidium, palmitic, palmitoleic and eicosadienoic acids; for P. punctatum, palmitic, stearic, oleic and linoleic acids. 4. 4. The pattern of biosynthesis of polyunsaturated fatty acids by these aquatic fungi as well as their morphology supports the hypothesis that they evolved from a primitive phytoflagellate (monad) ancestor.

Serious outbreaks of a pernicious disease caused by the fungus Dermocystidium are reported in both adult and juvenile salmon in the Columbia River and its tributaries. The disease evokes a characteristic histopathology and appears to cause death in a large percentage of the infected fish. The disease appears to be most virulent at water temperatures below 60 F. Some prophylactic relief is given by treatment with the organic herbicide, Diquat, at a dilution of 1:500,000 for 1 hr.

Twelve antifungal antibiotics were screened to determine their potential for controlling Dermocystidium marinum, a lethal fungus parasite of oysters. Two antibiotics, cycloheximide and streptimidone, completely inhibited enlargement of D. marinum in thioglycollate cultures at concentrations of 0.25–1.0 μg/ml. Cycloheximide shows promise for use in arresting D. marinum infections in oysters held in closed laboratory systems. Twenty-two antibacterial antibiotics were screened for their effect on the enlargement of D. marinum in thioglycollate cultures, and for their efficacy in retarding growth in bacteria associated with oyster tissues. Three tetracyclines (tetracycline, chlortetracycline, and oxytetracycline), bacitracin, and gramicidin showed effective antibacterial properties at levels of 250–500 units or mcg/ml. However, all of these agents except gramicidin markedly inhibited parasite enlargement at concentrations of 250–750 units or mcg/ml. Further studies proved gramicidin to be inferior to either chloramphenicol or the penicillin-dihydrostreptomycin combination for inhibiting bacterial growth and tissue putrefaction in the thioglycollate culture technique for detecting D. marinum.

Dermocystidium Gasterostei n. sp., A Parasite of Gasterosteus Aculeatus L. and Gasterosteus Pungitius L.

Dermocystidium has been recorded from oysters (Mackin et al., 1950), numerous species of freshwater fish and some Amphibia (Reichenbach-Klinke and Elkan, 1965). The taxonomic affinities of the group have been uncertain until recently when Perkins (1976) demonstrated that Dermocystidium marinum from oysters, previously considered by many workers to be a fungus is, in fact, a member of the protozoan phylum Apicomplexa, since it possesses a zoospore stage containing an apical complex. Subsequently Levine (1978) erected a new genus Perkinsus to distinguish the oyster parasite from those found in fish and Amphibia and placed it in a new class Perkinsea, within the Apicomplexa. The relationship between the invertebrate and vertebrate parasites is not clear because of the lack of information on developmental stages of the latter group.

The life cycle of a nonfilamentous marine phycomycete, Dermocystidium sp., is described from its growth in axenic culture under controlled conditions. Reproduction occurs by vegetative means involving a process of cell enlargement and ecdysis modifiable by the methods of cultivation. Zoospores and sexuality were not observed. The results of inoculation studies with algae, phanerogam debris, and molluscs suggest that the fungus may be a free-living saprophyte in coastal waters.

The microstructure of the protist Labyrinthomyxa marina as found in a host organism, Crassostrea virginica, is described. Most of the organelles were similar to those of many eucaryotic protists; however, a few unusual structural features were observed. Viruslike particles (460–530 A diameter) were found in the nucleus and cytoplasm of the Virginia, but not the Florida, pathogens. Cell-wall formation appeared to occur, in part, by formation of material in vesicles derived from the endoplasmic reticulum. The vesicles appeared to fuse with the plasmalemma and release material into the cell wall. Funguslike lomasomes were found between the cell wall and plasmalemma. Vacuoplast formation occurred by synthesis of droplets of electrondense material through the membranes of large vacuoles followed by coalescence to form one or two large vacuoplasts. Mitochondria were involved in the formation of vesicles which contained electron-dense material and which were delimited by two membranes. Since L. marina has previously been allied with the Labyrinthulia (Protozoa) and the Saprolegniales (Phycomycetes), comparisons are made with the ultra-structure of representatives from both groups. Affinities with the fungi seem to be the closest on the strength of lomasome formation and the repeated formation of cell walls in all vegetative stages.

In the skin of Rana temporaria from Czechoslovakia two unicellular parasites have been found: Dermocystidium ranae Guyénot & Naville and Dermosporidium granulosum n.sp.Dermocystidium ranae. Two stages were found, one of which is new. An account is given of the morphology, development and occurrence of both stages, including details which have hitherto not been recorded.Dermosporidium granulosum n.sp. This species is represented by cysts situated in the stratum spongiosum corii. They are divided by septa into small chambers containing groups of spherical spores with a central nucleus and with numerous small cytoplasmic inclusions. Within the cyst, the sporoblasts occupy the periphery, while the ripe spores are in the centre.

10 páginas, 3 tablas, 5 figuras, Parasites identified as Perkinsus atlanticus have been reported infecting carpet shell clams in Galicia (northwest Spain). We have sequenced the 18S ribosomal RNA gene of in vitro cultured Perkinsus atlanticuslike or hypnospores from diseased clams, and compared it with the same genomic region from P. marinus and Perkinsus sp. We have also compared the sequence of internal transcribed spacer (ITS) 1, ITS 2, and 5.8S rRNA from our isolate with the P. atlanticus GenBank sequence. The phylogenetic analysis of our cultured parasite based on the 18S gene led us to conclude that this isolate is not related to the genus Perkinsus but to the protists Anurofeca, Ichthyophonus, and Psorospermium, located near the animal-fungal divergence. These last two genera have been included, together with Dermocystidium, in the newly described DRIPs (Dermocystidium, rossete agent, Ichthyophonus, and Psorospermium) clade, recently named Mesomycetozoa., This work was partially funded by the Commission for Cultural, Educational and Scientific Exchange between the United States and Spain.