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Cryptocaryon irritans infection could cause huge economic losses to the marine fish industry. Larimichthys crocea, a special economic species in China, suffered from the threat of serious infection, and L. crocea could enhance the level of piscidin 5-like to defense against the infection. This study set out to observe the main histopathological changes of some key tissues caused by infection, and determineed how an ectoparasite affected the expression of piscidin-5 like in its hosts. Pathological changes and immune response were assessed using histological and in situ hybridization (ISH) technologies. The infection induced inflammation occurring, especially in the gill where epithelium cells swell, hyperplasia, necrosis shedding adjacent to the parasites attachment sites. Infected hepatic cells grown big vacuoles in the cytoplasm. The boundary between red pulp and white pulp turned indistinct, splenic corpuscle lost the normal structure, the number and size of melano-macrophage centers increased apparently in the infected spleen. The whole structure of head kidney became loose. Immunostaining with RNA probes against piscidin 5-like showed subpopulations of mast cells (MCs) were positive. Piscidin 5-like-positive MCs existed mainly in the head kidney where they distributed around melano-macrophage center, followed in the gill located at different positions they also distributed in the margin of spleen, and randomly and sparsely existed in the liver. After being infected by C. irritans, the gill arch arose positive MCs groups, and they also migrated to spleen, while the positive staining deepen in other detected tissues. Therefore, organism enhanced the expression level through improving expression ability of positive MCs, or increasing the number of positive MCs., (Copyright © 2020. Published by Elsevier Ltd.)

Scuticociliatosis, caused by an opportunistic ciliate protozoan, is responsible for significant economic losses in marine ornamental fish. This study reports the occurrence of Uronema spp., parasitizing ten species of marine reef fish at an ornamental fish wholesaler: Blue green damselfish (Chromis viridis), Vanderbilt's Chromis (Chromis vanderbilti), Pennant coralfish (Heniochus acuminatus), Threespot angelfish (Apolemichthys trimaculatus), Goldspotted angelfish (Apolemichthys xanthopunctatus), Sea goldie (Pseudanthias squamipinnis), Orchid dottyback (Pseudochromis fridmani), Threadfin butterflyfish (Chaetodon auriga), Vagabond butterflyfish (Chaetodon vagabundus), and Bluecheek butterflyfish (Chaetodon semilarvatus). Diseased fish showed disorders such as hemorrhages and ulcerative lesions on the body surface. Histopathological analysis of the muscle, liver, gut, kidney, spleen, gills, and stomach revealed hemorrhages and degeneration of muscle fiber, vacuolar degeneration of hepatocyte, inflammatory process and granuloma in the liver, atrophy of intestinal villi, inflammatory process and granuloma in the kidney, melanomacrophage centers, as well as inflammatory process in the spleen, epithelial cells hyperplasia and granuloma formation in the gills, and vacuolar degeneration and eosinophils in the stomach. Due to the severity of the disease, it is necessary to implement biosecurity measures with rapid and accurate diagnosis to minimize the risk of economic losses caused by Uronema spp.

The genus Chilodonella includes free-living ciliated protozoa as well as pathogenic species for freshwater fish, with Chilodonella hexasticha and Chilodonella piscicola being the most important ones. These parasites cause outbreaks with high mortalities among farmed freshwater fishes with great economic losses. There are few reports of these species in China, and their identification has been based mostly on their morphological characteristics. In the present work, the parasites causing five outbreaks occurring in China between 2014 and 2017 have been identified by morphological and genetic analysis. We provide the first records of Ctenopharingodon idella and Siniperca chuatsi as hosts of C. hexasticha, and of Procypris rabaudi and Schizothorax wangchiachii as hosts of C. piscicola. There are no differences in the gross pathological findings produced by C. hexasticha and C. piscicola, consisting in desquamation and necrosis of epithelial cells in the skin and gills and in severe fusion of gill lamellae. However, both species differ in their geographic distribution: C. piscicola was found in farms located at altitudes over 1500 m above sea level and with a water temperature ≤18 °C, while C. hexasticha was found in farms located at altitudes under 50 m above sea level and with a water temperature ≥21 °C. Present results confirm that C. hexasticha and C. piscicola are two different species that can be differenced by their morphology; however, their biological variability may lead to erroneous identifications and the diagnosis should be preferably based in genetic analysis including nuclear LSU rDNA and mitochondrial SSU rDNA sequences., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Freshwater fish farming contributes to more than two-thirds of global aquaculture production. Parasitic ciliates are one of the largest causes of production loss in freshwater farmed fishes, with species from the genus Chilodonella being particularly problematic. While Chilodonella spp. include 'free-living' fauna, some species are involved in mortality events of fish, particularly in high-density aquaculture. Indeed, chilodonellosis causes major productivity losses in over 16 species of farmed freshwater fishes in more than 14 countries. Traditionally, Chilodonella species are identified based on morphological features; however, the genus comprises yet uncharacterized cryptic species, which indicates the necessity for molecular diagnostic methods. This review synthesizes current knowledge on the biology, ecology and geographic distribution of harmful Chilodonella spp. and examines pathological signs, diagnostic methods and treatments. Recent advances in molecular diagnostics and the ability to culture Chilodonella spp. in vitro will enable the development of preventative management practices and sustained freshwater fish aquaculture production., (© 2016 John Wiley & Sons Ltd.)

This study describes a co-infection of Kudoa islandica (Myxozoa) and Nucleospora cyclopteri (Microsporida) in farmed lumpfish, Cyclopterus lumpus L., in Norway. Several other parasites (Cryptocotyle sp., protozoan ciliates and Gyrodactylus sp.) were also found in gills. In June 2013, the mortality in a farmed lumpfish population increased to 65%. Lumpfish showed erratic swimming behaviour and loss of weight. At necropsy, nodules in the kidney were the only visible lesions. Histologically, all fish showed severe changes with gill inflammation and necrosis in the spleen, kidney and liver. Haemorrhages and necrosis were observed in some hearts. Intracellular microsporidians associated with the lesions were detected in most organs using histological examination and Calcofluor White. Kudoa spores were diagnosed in the skeletal muscle, but no inflammatory response was associated with the presence of the plasmodia. Comparison of 18S ribosomal DNA sequences showed 100% similarity to Kudoa islandica and Nucleospora cyclopteri. Kudoa islandica and N. cyclopteri have previously been described associated with lesions in wild lumpfish in Iceland. In the present case, N. cyclopteri is believed to be the main cause of systemic pathology. This is the first description of K. islandica and N. cyclopteri causing pathology in farmed lumpfish in Norway., (© 2015 John Wiley & Sons Ltd.)

Trichodina heterodentata Duncan, 1977 and Ichthyophthirius multifiliis Fouquet, 1876 obtained from gills during a parasitological survey conducted for the protozoan parasitic fauna of Schizothorax niger a snow trout in Dal Lake, Kashmir, India during the period October 2013 and March 2015. Thirty out of 180 fish were found infected with protozoan parasites. During the study of their pathogenecity the most common deteriorating signs observed in gill tissue were necrosis, hypertrophy, hyperplasia and fusion of secondary lamellae. Prevalence of infection was found to be 16.66%. This is the first record of the protozoan fauna of the schizothoracines from Kashmir valley, India., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Scuticociliatosis is an economically important, frequently fatal disease of marine fish in aquaculture, caused by histophagous ciliated protozoa in the subclass Scuticociliatida of the phylum Ciliophora. A rapidly lethal systemic scuticociliate infection is described that affected aquarium-captive zebra sharks (Stegostoma fasciatum), Port Jackson sharks (Heterodontus portusjacksoni), and a Japanese horn shark (Heterodontus japonicus). Animals died unexpectedly or after a brief period of lethargy or behavioral abnormality. Gross findings included necrohemorrhagic hepatitis and increased volumes of celomic fluid. Histologically, 1 or more of a triad of necrotizing hepatitis, necrotizing meningoencephalitis, and thrombosing branchitis were seen in all cases, with necrotizing vasculitis or intravascular fibrinocellular thrombi. Lesions contained variably abundant invading ciliated protozoa. Molecular identification by polymerase chain reaction from formalin-fixed tissues identified these as the scuticociliate Philasterides dicentrarchi (syn. Miamiensis avidus), a novel and potentially emergent pathogen in sharks.

Prochilodus lineatus is a freshwater fish species found in South America. It is common in aquaculture, but few studies regarding diseases of this fish have been performed. This study presents data of the occurrence of Trichodina heterodentata Duncan, 1977, as well as the pathological alterations detected by light and scanning electron microscopy (SEM). Twenty 20-day-old larvae were harvested from an earth pond and examined. Larvae showed erratic swimming on the pond edges and some had a whitish tegument. Larval smears were either impregnated with silver nitrate or stained with Giemsa stain to observe the taxonomic features of the ciliates. Five larvae were fixed in formalin solution for histopathological analysis, and another five specimens were fixed in glutaraldehyde for SEM. All larvae were diagnosed with a severe infestation by trichodinid T. heterodentata. Histological sections showed discrete hyperplasia of the gill filaments with subepithelial oedema of the secondary lamellae. In the SEM, suction areas were observed on the skin, gill and eye; corrosion and ulceration of the fins were associated with the bacterial presence of cocci on the lesions. This is the first report of T. heterodentata in P. lineatus that is responsible for an acute disease that culminates in larval mortality.

Tetrahymena is a ciliated protozoan that can infect a wide range of fish species, although it is most commonly reported in guppies (Poecilia reticulata). The aim of this study was to compare the susceptibility to infection with Tetrahymena of five different ornamental fish species from two different super orders. The species examined were platy (Xiphophorus), molly (Poecilia sphenops) and angelfish (Pterophyllum scalare) of the Acanthopterygii super order (which also includes guppies) and goldfish (Carassius auratus auratus) and koi carp (Cyprinus carpio) of the Ostariophysi super order. These two super orders are phylogenetically distant from each other. Infection with Tetrahymena resulted in parasite invasion of internal organs, skin and muscle in all fish species. A relatively strong inflammatory response was observed in infected goldfish and koi, with negligible response in fish species of the Acanthopterygii super order. Guppies were the most susceptible to Tetrahymena infection, exhibiting a mortality rate of 87% and 100% in two separate experiments. A high mortality rate was also observed in platy (77%), while that of molly and angelfish was significantly lower (23% and 33%, respectively). Goldfish and koi carp were less susceptible to infection compared with guppies (24% and 59% mortality, respectively). Immunization studies revealed that the Tetrahymena are immunogenic, since infection of koi carp increased their Tetrahymena immobilization response by approximately three-fold at 3 weeks post infection, while immunization with Tetrahymena plus adjuvant increased their immobilization response by approximately 30-fold., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A retrospective study of the pathologic findings in weedy (Phyllopteryx taeniolatus) and leafy (Phycodurus eques) seadragons was performed on specimens submitted to 2 reference laboratories from 1994 to 2012 to determine the range and occurrence of diseases affecting aquarium-held populations. One hundred two and 94 total diagnoses were recorded in weedy and leafy seadragons, respectively. Two of the more common etiologic diagnoses in both species were mycobacteriosis and scuticociliatosis, whereas myxozoanosis was common in weedy seadragons. Metazoan parasite infections were less common etiologic diagnoses. There were no correlations between mycobacteriosis and ciliate protozoan infections in either species. Myxozoanosis was usually found in combination with other diseases and, except for 1 case, was restricted to weedy seadragons. Phaeohyphomycosis, nonmycobacterial bacterial infections, and trauma were also important but less frequent diagnoses. Intestinal coccidiosis was found in weedy but not leafy seadragons. Mineralization of the swim bladder was detected in 26 of 197 leafy seadragons and only 2 of 257 weedy seadragons. Although weedy and leafy seadragons share certain diseases of significance to exhibit populations, there are diseases unique to each species about which the veterinary pathologist, clinician, or diagnostician should be aware.

Chilodonelids are small ciliated protozoans found worldwide and can be dangerous in culture conditions. This study presents morphometric data on the ciliate Chilodonella that is found in cultured Nile tilapia (Oreochromis niloticus), native bait fish tuvira (Gymnotus aff. inaequilabiatus) and native pacu (Piaractus mesopotamicus) and includes a histopathological assessment of the changes that occur in the pacu. For parasitic diagnosis, skin and gill samples were scraped onto slides, dried at room temperature, stained with Giemsa or impregnated with silver nitrate, and the measurements were obtained from photomicrographs. In the diseased pacu, the first gill arch was collected and fixed in a 10% buffered formalin solution for histopathological analysis. Parasite specimens from the different collection sites were identified morphologically as C. hexasticha Kiernik (1909). Diseased fish exhibited depigmentation, skin ulceration, scale loss, excessive mucus production and gill lesions. Histopathological analysis of pacu gills displayed epithelial proliferation with mononuclear inflammatory infiltrate, hemorrhages, and scattering necrosis. In Brazilian-farmed fish this is the first record of C. hexasticha, which has great pathogenic potential in cultured freshwater species. In addition, two new hosts are presented., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This study compared the susceptibility of three blue catfish strains (D&B, USDA 101 and USDA 102) to the parasite Ichthyophthirius multifiliis (Ich). In Trial I, a cohabitation study (all strains stocked communally) was conducted and fish were exposed to theronts at 0, 200, 1000, 5000 or 25 000 theronts fish(-1), respectively. All fish died when exposed to theronts at 5000 or 25 000 theronts fish(-1). When exposed to 1000 theronts fish(-1), USDA 102 strain of blue catfish showed significantly lower mortality (78.5%) compared to USDA 101 and D&B strains (92.7% and 100%). In Trial II, the same three strains of blue fish were evaluated for their susceptibility to Ich with strains challenged in separate tanks by adding Ich theronts at 0, 200 and 1000 theronts fish(-1), respectively. All D&B and USDA 101 blue catfish died; however, 42.3% of USDA 102 strain survived the infection when exposed to 1000 theronts per fish. The results indicate that there are differences among strains of blue catfish for susceptibility to Ich, and these differences will be useful in the development of improved catfish germplasm for commercial aquaculture., (Published 2012. This article is a US Government work and is in the public domain in the USA.)

Philasterides dicentrarchi is a histophagous scuticociliate infecting Korean olive flounder farms in Jeju Island, South Korea, where it causes significant economic losses. However, the route of entry of these parasites in olive flounder is currently unknown. In the present study, we attempted experimental infection with different doses (concentrations) of P. dicentrarchi by intraperitoneal (IP) injection; maximum cumulative mortality of 95% was recorded on the administration of 2.1×10(8) ciliates/ml. In small size group (3 cm length, 24±2 gwt) the ciliates were found in connective tissues of muscle, fins, and nervous tissue while in large size group (5 cm length, 47±3 gwt), the ciliates were predominantly occurred with associated symptoms of liquefaction in the central nervous system. The major clinico-pathological manifestations were loss of scales, appearance of bleached spots that coalesced to form brownish patches, hemorrhagic, and severe dermal necrotic lesion. In addition there was degeneration of muscle fibers, necrosis of the hepatic parenchyma, and severe edema of the intestinal walls, extensive fouling, necrotic degeneration and hyperplasia in the gill bronchial epithelium. In this study, many ciliates were found in the blood vessel, brain, dorsal fins, muscle, kidney, and vertebra of infected flounder. The histopathological investigations are of significant importance in view of possible routes of entry into the host and doses of ciliates that trigger fast infection for potential utility of drugs as a strategy for the control of P. dicentrarchi in farmed olive flounder., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Scuticociliates are extracellular histophagous parasites that affect farmed fish worldwide. One of the most common pathogenic species is Miamiensis avidus, a pathogen of New Zealand groper (Polyprion oxygeneios). The aim of this study was to characterise both the host (groper)-parasite (M. avidus) immune interactions and the possible protective role of dietary sodium ascorbate. Head-kidney leucocytes (HKLs) from naturally infected adult groper showed decreased respiratory burst response and peroxidase (Px) levels than healthy individuals. Infected groper also had significantly higher serum Px levels compared to controls. Myeloperoxidase (MPO) was inhibited in the head-kidney (HK) whereas MPO(+) cells were observed in the skin and muscle lesions. The inhibition of the innate immune responses was further studied in experimental infections with M. avidus, which confirmed depletion of Px inside leucocytes and marked increases in serum Px in infected individuals. Groper juveniles were fed a diet supplemented with sodium ascorbate (Vitamin C) (2g Kg(-1)) for 21 days and then challenged by subcutaneous injection or immersion exposure with live M. avidus cells. No protection was observed in the sodium ascorbate fed groper compared to the control diet following challenge by either injection or immersion. In vitro assays showed that sodium ascorbate itself results in the inhibition of Px and respiratory burst of groper HKLs, supporting the results obtained in vivo. Our results show that histophagous protozoa such as M. avidus hamper innate immune defences of fish hosts and that dietary sodium ascorbate does not protect groper against experimental infection with this parasite., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Japanese flounder (Paralichthys olivaceus) were experimentally infected with the highly pathogenic scuticociliate Miamiensis avidus (syn. Philasterides dicentrarachi) using the immersion method to clarify/identify the possible neural routes of entry and possible ways of dissemination of the scuticociliate in the fish body. Scuticociliates were observed on the skin and gills right from day 0-1 post-infection, muscle tissue on day 2 post-infection, reached the brain, and spinal cord on day 3 post-infection, and systemic infection was prominent afterwards. Brain lesions were observed in most of the examined fish from days 3 and 4 post-infection and considered to be the cause of the sudden increase in mortality. Affected fish showed varying degrees of tissue damage including severe epidermal and dermal necrotic lesions, necrotic myositis, encephalitis and myelitis. Whereas, scuticociliates were frequently observed along the optic and/or olfactory nerve in the fish which were accompanied by severe brain lesions but by minimum lesions in the gills and skin, suggesting that in addition to skin and/or gills, neural routes including periorbital and nasal routes may play a role in scuticociliate invasion to the brain. Scuticociliates were also observed in the peripheral nerve fibers in the muscle tissue, cranial and spinal nerves, cranial cavity and in the vertebral canal, suggesting that nerve fibers and/or cerebrospinal fluid circulation may be involved in the spread of the scuticociliate throughout the body in addition to the blood circulation and connective tissue.

Pathological findings associated with scuticociliatosis in farmed Japanese flounder in Japan are described. Ten moribund fishes, farmed in Tottori Prefectural Fisheries Experimental Station, showed cutaneous ulcers, darkened skin, fin and tail rot, exophthalmia and alterations in swimming behaviour. Histopathologically, severe epidermal degeneration and necrosis, hyperplasia of branchial epithelium, myositis, myelitis, encephalitis associated with heavy accumulation of scuticociliates in the periorbital cavity and optic nerve fiber were observed. Moreover, masses of ciliates were found to feed on the host tissues such as skeletal muscles, gills and brain, causing severe degenerative changes associated with abundant neutrophilic and lymphocytic infiltration. These findings suggest that the present scuticociliate, Miamiensis avidus, is a highly invasive and destructive pathogen infecting Japanese flounder and capable of developing systemic fatal infection.

Miamiensis avidus, a causative agent of scuticociliatosis in olive flounder Paralichthys olivaceus, was previously reported to proliferate the fastest in media with an osmolarity of 300 to 500 mOsm kg(-1). This suggests that hyposaline conditions can promote the development of the disease. In the present study, olive flounder constantly showed high mortalities when they were experimentally challenged with the parasite by immersion and subsequently reared in hyposaline conditions. Furthermore, affected flounder produced by the challenge showed symptoms identical to those in naturally infected flounder. It was experimentally demonstrated that hyposaline conditions can be a key factor for the development and outbreak of scuticociliatosis in olive flounder.

The parasitic ciliate Ichthyophthirius multifiliis (Ich) is one of the most important protozoan pathogens of freshwater fish worldwide. Examination of 4 species of Tilapia from the River Nile recorded highest rate infection in Oreochromis niloticus, followed by Oreochromis aureus, Sarotherodon galilaeus and Tilapia zilli. By electron microscopy, apoptotic cells collected from Tilapia species infected with "Ich" showed an aggregation on the apical and basal parts of the ciliated and non-ciliated endothelial lining the gill epithelium. Ultrastructural analysis showed that the chromatin in the liver of Ich-infected O. niloticus was highly condensed and massed at the center of the nucleus. UItrastructural analysis of "Ich" infected O. aureus showed that the nuclear membrane of hepatocytes was also shrunken. In S. galilaeus, chromatin was highly condensed, fragmented and massed at the nuclear center, occasionally forming crescent-shaped masses. Ultrastructural of O. niloticus spleen showed that the macrophage number was comparatively high as compared to control. In O. aureus, damaged splenic cell number did not change, and phagocytic capacity of macrophages was not very high. In Ich-infected S. galilaeus, a marked decrease in splenic cells number was seen. Analysis of DNA by agarose gel electrophoresis on gill, liver and spleen cells showed a ladder of DNA fragments in multiple of 180bp in length, pointing to an internucleosomal DNA cleavage.

Recently, mass mortality due to histophagous scuticociliate, Philasterides dicentrarachi in olive flounder, Paralichthys olivaceus occurred in aquaculture farms of Jeju Island, South Korea. We have artificially induced infection through immersion on day 12, the density of ciliates slowly decreased in the experimental tanks; on day 28 to 2300cells/ml in 3cm group and 1800cells/ml in 5cm group. The survival rate of P. olivaceus on days 24 and 28 of infection in the 3cm group was decreased to 4 and 6, respectively; however, no mortality was recorded in the 5cm group. In the control group 100% survival in the chosen period of both groups occurred except in the 3cm group whereas on days 24 and 28 the death rate was 1 and 2, respectively. Many ciliates with ingested red blood cells were observed in the gills, skeletal muscle, skin, fins and brains of infected flounder. The parasites were also observed in the lamina propria of the digestive tract, pharynx, peritoneal fluid, cerebrospinal fluid and cornea. Histopathological and inflammatory changes were characterized by swelling and generalized erythema and muscle tissue necrosis. Affected fish showed varying levels of tissue damage including severe epidermal and dermal necrotic lesions. Loss of scales, appearance of bleached spots that coalesced to form brownish patches, haemorrhagic and severe dermal necrotic lesions were the major clinico-pathological manifestations. The parasite was also seen distributed extensively in the entire brain causing widespread nerve necrosis after a prolonged period of infection. These results appear to support the hypothesis that the P. dicentrarchi may be penetrating via the gills or the skin, and then travel via the bloodstream to other parts of the body, including the body cavity.

Scuticociliatosis is a disease of fish induced by ciliated parasites of the genus Scuticociliatida. It has been described in sea horses (Hippocampus sp.), flounders (Paralichthys olivaceus), and turbots (Scophthalmus maximus). Here we present a case study of a population of sea dragons chronically infected with scuticociliates identified as Philasterides dicentrarchi by histopathology and PCR. Beginning in 2004, over a period of 19 months, 10 sea dragons (Phycodurus eques and Phyllopteryx taeniolatus) were found dead in an aquarium of the Zoological Garden Basle, Switzerland. Clinically, the animals showed only faint symptoms of disease over a short period of time. At necropsy, macroscopic lesions were confined to the skin with multiple, often hemorrhagic, ulcerations. Histologically, epidermal ulcers were associated with necrosis and inflammation of the underlying dermis and musculature. Numerous ciliates, with a morphology consistent with scuticociliates, were present in these lesions. In several animals these ciliates had invaded blood vessels and were detected in gills and internal organs including kidney, thyroid gland, and central nervous system (CNS). In these organs, mild degenerative lesions and inflammatory reactions were evident. The ciliates were identified as Philasterides dicentrarchi based on small-subunit ribosomal RNA (SSUrRNA) gene sequences obtained by polymerase chain reaction (PCR) on DNA extracted from paraffin-embedded tissue sections. Our report shows that scuticociliate infections of sea dragons can develop into a systemic infection and that both species of sea dragons can be affected.

A scuticociliate strain (B-2), originally isolated from an outbreak in a turbot Scophthalmus maximus (= Psetta maxima) farm in Galicia (northwestern Spain) and maintained in axenic culture, was injected intracoelomically (lethal dose 80 equivalent, LD80) in healthy turbot (50 g). Ciliate-injected fish were kept under controlled conditions in a recirculating seawater system and sampled on Days 1 through 8, 10, 12 and 14 postinfection (PI). Necropsies were conducted and included blood collection from the caudal vein and samples of liver, spleen, heart, digestive tract, kidney, gills, abdominal wall and neurocranium taken for routine histology. Mortality occurred from Day 6 until Day 12 PI and reached 66.7% by the end of the experiment. Presence of ciliates in the coelomic fluid was scarce until Day 4 PI. Parasitaemia was only observed from Day 5 until Day 10 PI and its incidence was always low. Presence of scuticociliates in tissue sections followed a progressive pattern of diffusion, with ciliates showing preference for loose connective tissue and also a clear haematophagous activity. The most severely affected organs were the pancreas and digestive tract. No special tropism for nervous tissues was observed in this study. The inflammatory reaction was variable depending on the tissue. After 3 wk, survivors had apparently managed to extinguish the infection.

During the years 2004 and 2005 high mortalities in turbot Scophthalmus maximus (L.) from a fish farm in the north of Portugal were observed. Moribund fish showed darkening of the ventral skin, reddening of the fin bases and distended abdominal cavities caused by the accumulation of ascitic fluid. Ciliates were detected in fresh mounts from skin, gill and ascitic fluid. Histological examination revealed hyperplasia and necrosis of the gills, epidermis, dermis and muscular tissue. An inflammatory response was never observed. The ciliates were not identified to species level, but the morphological characteristics revealed by light and electronic scanning microscopes indicated that these ciliates belonged to the order Philasterida. To our knowledge this is the first report of the occurrence of epizootic disease outbreaks caused by scuticociliates in marine fish farms in Portugal.

The scuticociliate Miamiensis avidus was isolated from olive flounder Paralichthys olivaceus showing typical symptoms of ulceration and hemorrhages in skeletal muscle and fins. In an infection experiment, olive flounder (mean length: 14.9 cm; mean weight: 26.8 g) were immersion challenged with 2.0 x 10(3), 2.0 x 10(4) and 2.0 x 10(5) ciliates ml-1 of the cloned YS1 strain of M. avidus. Cumulative mortalities were 85% in the 2.0 x 10(3) cells ml-1 treatment group and 100% in the other 2 infection groups. Many ciliates, containing red blood cells in the cytoplasm, were observed in the gills, skeletal muscle, skin, fins and brains of infected fish, which showed accompanying hemorrhagic and necrotic lesions. Ciliates were also observed in the lamina propria of the digestive tract, pharynx and cornea. The fixed ciliates were 31.5+/-3.87 pm in length and 18.5+/-3.04 microm in width, and were ovoid and slightly elongated in shape, with a pointed anterior and a rounded posterior, presenting a caudal cilium. Other morphological characteristics were as follows: 13 to 14 somatic kineties, oral ciliature comprising membranelles M1, M2, M3, and paroral membranes PM1 and PM2, contractile vacuole at the posterior end of kinety 2, shortened last somatic kinety and a buccal field to body length ratio of 0.47+/-0.03. In addition, continuous PM1 and PM2, lack of M3 and variable kinetosome numbers in M2 and M3 were frequently observed. Specimens in the current study were compared with previous reports on M. avidus and Philasterides dicentrarchi and confirmed consistently that these 2 taxa are conspecific.

The ciliated protozoan parasite Cryptocaryon irritans infecting marine fishes in Taiwan is described. Developmental characteristics and sequences of the ribosomal DNA regions such as part of 18 S, the entire first internal transcribed spacer, and part of 5.8 S of various Taiwan isolates of C. irritans were investigated. A total of 5 isolates was obtained from different fish-host species and localities, the majority from cultured fish species. C. irritans from Taiwan is able to shift its developmental characteristics, i.e. from non-adherent to adherent tomonts, from individualistic to aggregate-forming tomonts, from infection of the gills only to infection of the gills and body. Thus, it is not possible to classify strains of C. irritans on the basis of these parameters. Premature tomonts that developed from dead fishes were able to produce theronts that could infect fish host. Isolates from Pingtung and the USA had identical nucleotide sequences while an isolate from Malaysia was identical to an Israel isolate. Percentage variation among pairs of Taiwan isolates showed a higher degree of variation than isolate sequences listed in GenBank. Sequence analysis revealed highly aberrant isolates in Taiwan, and a phylogenetic tree distinguished a marine and a low-salinity variant. C. irritans from marine fishes in Taiwan, therefore, display some characteristics not previously reported. Since manipulation of salinity in brackishwater ponds and marine cage sites is not feasible, there is a need to develop new strategies for the control and prevention of cryptocaryoniasis.

Two outbreaks of scuticociliatosis affecting farmed turbot Scophthalmus maximus in Galicia are described. Moribund fish showed cutaneous ulcers, darkened skin, swimming behaviour alterations, exophthalmos, and/or abdominal distension as a result of accumulation of ascitic fluid in the body cavity. Ciliates were detected in fresh mounts of practically all organs and tissues, including the blood and ascitic fluid. Histopathological studies revealed severe encephalitis and meningitis (associated with different degrees of softening or liquefaction of the brain), necrosis of the hepatic parenchyme, severe oedema of the intestinal wall, degeneration of muscle fibres, hyperplasia of the branchial epithelium, and/or vascular and perivascular inflammation. In some cases, parasites are surrounded by abundant monocytic and lymphocytic infiltrate. We report the morphological and biometric characteristics of this ciliate, which allow identification as Philasterides dicentrarchi. We discuss possible routes of entry into the host, and environmental factors possibly facilitating infection.

An obligate parasite, Cryptocaryon irritans, which is responsible for the white spot disease of marine fish is known to develop in the temperature regime over 19 degrees C. Recently, however, we found white spot disease of olive flounder Paralichthys olivaceus during winter at water temperatures ranging between 12 and 16 degrees C in Korea. In the present study we isolated a C. irritans-like ciliate from the affected fish and investigated its reproductive characters to compare the newly found ciliate with typical C. irritans. The newly found ciliate had an additional process in the reproductive stage, characterized by a budding before palintomic division, and it showed a higher ability to carry out tomitogenesis at a low temperature (16 degrees C) than at a high temperature (24 degrees C). Nevertheless, the present ciliates still had much in common with typical C. irritans with respect to clinical, histopathological, and morphological characters, suggesting that it is a new strain of C. irritans, adapted to lower water temperature.

The dynamics of the densities of epidermal thionin-positive cells (putative mast cells) in the skin of brown trout fry were investigated during experimental infections with the skin parasites Ichthyophthirius multifiliis (Ciliophora) and Gyrodactylus derjavini (Monogenea). It was shown that the metachromatic thionin-stained cells were extremely sensitive to parasite exposure, as the density of cells in the skin of trout decreased markedly after exposure to the pathogens. As early as 7 d post infection the cell counts were significantly reduced and almost totally depleted following 9 d infection, which suggests that degranulation of the cells occurs following parasite exposure. No recruitment of new cells was seen during the study period. Some reduction in uninfected control groups indicates that the putative mast cells are sensitive to stress as well. A notable variation in densities of thionin-stained cells between different fins was found and the corneal surface was devoid of these cells. The possible implications of these cells in host-parasite interactions are suggested and discussed.

The present report demonstrates that a medicated food containing quinine kills the skin-inhabiting trophozoite stage of Ichthyophthirius multifiliis in ornamental fish. Artificially infected swordtails. (Xiphophorus helleri), black mollies (Poecilia sphenops), and black neons (Hyphessobrycon herbertaxelrodi) were used in the trials. The fish were maintained in groups of 10 or 20 inside aquaria (20 or 60 l) at 25 degrees C. Ultrastructure investigations by means of transmission electron microscopy revealed clear deleterious effects of quinine on the trophozoite stages. Following the initial application the outer limiting membrane of the trophozoite was broken at places. Within the nephridial plasma the plasma bridges were broken in part. After 2 days of treatment the lumen of the alveolar sac became enlarged. The food vacuoles in treated trophozoites were more electron-dense than those in the untreated controls. Numerous lipid droplets were found close to the vacuoles. The degree of damage in the nephridial plasma was intensified. When feeding was prolonged for 3 or more days, all kinds of damage became more extensive as seen in the trophozoites after 1 or 2 days of treatment. In addition food vacuoles in the final stages of digestion were no longer detectable. In long-term feeding tests, when typical ornamental fish species were fed three times daily ad libitum with the medicated food over a 12-week period, the animals showed no adverse clinical symptom. From the toxicological as well as the ecological and economical point of view the feeding of flakes containing quinine has considerable advantages as compared with conventional bath treatment.

O-group carp (Cyprinus carpio) which had been immunized against Ichthyophthirius multifiliis by controlled infections were challenged by topical application of theronts to the caudal fin. The parasites which established were examined ultrastructurally, and host leucocyte responses were compared with those observed in primary infections. In the primary exposure group eosinophils and (to a lesser extent) basophils were the predominant cells infiltrating infection sites. In contrast, parasite development in immunized fish initiated localized leucocytic infiltrations which were dominated by eosinophilic granular cells (EGCs) and basophils. Greater localized phagocytosis was recorded in immunized fish by neutrophils, macrophages and resident epidermal filament cells. In vitro studies indicated that pronephric leucocytes from immunized fish displayed enhanced non-specific phagocytosis. In the skin, leucocytes were observed in close proximity to the trophozoite surface in both immunized and primary exposure fish, often undergoing lysis and release of cellular contents. However, there was no evidence of active cell adherence nor of any cell-mediated damage incurred to the parasite in either case. These observations are discussed in relation to the possible role of leucocytes in mediating pathogenesis and immune responses.