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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.)

Wild-caught European perch (Perca fluviatilis) were exposed in the laboratory to untreated bleached pulp and paper mill effluent in two separate experiments. The first experiment was conducted at 7-8°C using effluent concentrations of 5 and 10%, and the second experiment was conducted at ambient river temperature of 4-20°C using an effluent concentration of 1%. Trichodinid ciliates were identified and enumerated at the end of the exposure using a mucus subsampling technique from gill and skin as well as a formalin immersion technique, which provided total counts on each fish. Four different trichodinid species were identified on the fish. Prevalence of infection, mean number, and mean density of Trichodina spp. decreased on fish exposed to effluents compared with controls. Prevalence of infection, mean number, and mean density of Trichodinella epizootica decreased on fish exposed to 5% and 10% effluents but increased on fish exposed to 1% effluents compared with controls. These results demonstrate that trichodinid ciliates vary in their susceptibility to at least certain types of contaminants and cautions against using trichodinids as environmental indicators without delineating species.

The objectives of the present study were to standardize a reproducible infection procedure with Cryptocaryon irritans and to examine the effects of infectious dose level on the immune protection in Mozambique tilapia (Oreochromis mossambicus). This study demonstrated that direct enumeration of trophonts on the pectoral fin was useful to quantitatively assess immune protection against C. irritans. The number of trophonts on a pectoral fin was positively correlated with infectious dose of live theronts. Fish immunized by direct exposure under controlled laboratory conditions allowed for in depth examination of the effects of the degree of infectious dose on immune response. There was no significant positive correlation between the initial infectious dose and degree of immune responses. Mozambique tilapia initiated a strong immune protection by direct exposure with even a small number of parasites (e.g. 300 theronts per fish). Moreover, as the result of the protein analysis, we identified 28 kD proteins that could be responsible for the immobilizing antigen.

Cryptocaryon irritans is one of the most important ectoparasites of marine fish. To identify the potential role of immune-related genes in antiparasitic immune responses in fish, we monitored the expression change of IL-8, COX-2, C-type lectin and transferrin in local and systemic immune organs of orange-spotted grouper post-C. irritans infection. IL-8 expression was up-regulated during the course of infection in the skin, while COX-2 and transferrin expression was up-regulated in the gill. COX-2 expression was significantly down-regulated in the spleen (0·7-5% of its control) and head kidney (0·5-4% of its control) post-primary infection. Transferrin expression was also down-regulated in the spleen and head kidney from 6 h to 5 days post-primary infection. However, C-type lectin expression was up-regulated in all tested organs post-infection, with the exception of day 7 in the spleen post-primary infection where the expression level was slightly down-regulated (44% of its control). These results suggest that these four immune-related genes play an important role in grouper anti-C. irritans infection and that local immune organs as the active organs contribute more than systemic immune organs to this course., (© 2011 Blackwell Publishing Ltd.)

The scuticociliates Miamiensis avidus (syn. Philasterides dicentrarchi), Pseudocohnilembus persalinus, Pseudocohnilembus hargisi and Uronema marinum were cloned and identified using morphological characteristics and the small subunit ribosomal RNA gene (SSU rRNA). M. avidus strains YS1, WS1, YK1 and JJ3 from southern coastal areas and Jeju Island in Korea were pathogenic to olive flounder Paralichthys olivaceus (80 to 100% mortality in 8 to 10 g fish) when inoculated intraperitoneally (i.p.) with 1.0 to 1.4 x 10(6) ciliates fish(-1). Mortality was lower (10 to 45%) when the inoculum was 1.0 to 1.4 x 10(4) ciliates fish(-1) in the i.p.-injected group. The M. avidus strains of YS1, WS1, YK1 and JJ3 caused 60 to 100% mortality by immersion infection with 3.2 to 4.2 x 10(3) ml(-1) in 8 to 10 g fish and 3.0 to 4.0 x 10(3) ml(-1) in 30 to 40 g fish. M. avidus strain Mie0301 from the Mie prefecture in Japan caused 70% mortality by immersion infection with 4.4 x 10(3) ml(-1) in 30 to 40 g fish. The predominant sign was severe abdominal distension in i.p.-injected fish, and extensive ulcer lesions in the skeletal muscle in immersion-infected fish. Numerous ciliates were observed in the ascetic fluid, ulcers, haemorrhagic lesions, gills and brain of infected fish. However, P. persalinus (strain SCL-A), P. hargisi (strain SCL-B) and U. marinum (strain JK3) showed less than 30% mortality from both i.p. and immersion challenges, with no ciliate invasion in the skin, gills or brain. M. avidus-infected fish showed many ciliates in gills, fins, skin muscle, brain and intestine accompanied by necrosis and haemorrhages. However, no histological changes were observed in P. persalinus-, P. hargisi- or U. marinum-infected fish.

Rainbow trout Oncorhynchus mykiss were immunised by intra-peritoneal injection using a live vaccine based on Ichthyophthirius multifiliis (Ich) theronts, which previously has shown protection against white spot disease. Samples were taken pre-vaccination and on Day 1, 7, 21 and 28 post-immunisation (p.i.). Expression of immune relevant genes in the liver, spleen and head kidney was monitored by qPCR. To describe the immune reaction following this immunisation, a series of genes encoding cytokines, complement factors, immunoglobulins and acute phase reactants were studied. Genes encoding acute phase reactants in the liver were up-regulated with serum amyloid A (SAA) as the most pronounced with a 2299-fold increase at 24 h p.i. Hepcidin and pre-cerebellin were also up-regulated in the liver 24 h p.i., by 7- and 4-fold, respectively. Complement factors C3, C5 and factor B (Bf) were up-regulated in the spleen and the head kidney 24 h and 28 d p.i. Genes encoding immunoglobulins were not up-regulated, but a specific low titer IgM response (titer 25) against parasite antigens was detected by a modified ELISA 4 wk p.i.

A medium for the in vitro culture of Cryptocaryon irritans, which is an obligatorily parasitic ciliate of marine teleosts and causes 'white spot disease', was developed. The medium consisted of a layer of cultured fish cells (FHM), with an agarose gel layer covering the cell layer. The agarose gel contained 0.22% agarose, 10% fetal calf serum, 100 I.U. ml(-1) Penicillin G potassium and 100 microg ml(-1) streptomycin sulphate. Theronts of C. irritans transformed to trophonts and grew to 180 microm in mean length in the medium, although they gradually decreased in number. When trophonts fully developed in medium were transferred into seawater 4 d after inoculation, approximately 70% of them transformed to encysted tomonts and released theronts. When fish were challenged with theronts obtained from in vitro-raised parasites, approximately 40% of the theronts were recovered from fish, indicating comparative infectivity of in vitro-raised theronts to those of in vivo-raised theronts. This is the first report that C. irritans fully developed in vitro and its entire life cycle was completed without a host fish.

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.

Rainbow trout Oncorhynchus mykiss Walbaum, 1792 fingerlings were vaccinated by intraperitoneal (i.p.) injection using live theronts of the skin parasitic ciliate Ichthyophthirius multifiliis Fouquet, 1876 at 2 temperatures (12 and 20 degrees C), and protection against challenge infections was subsequently evaluated by bath exposure to live theronts. Vaccination conferred a relative protection (evaluated as the decrease in the number of established theronts) at 12 degrees C and almost complete immunity at 20 degrees C. Significantly increased immobilisation titers (using plasma immobilisation of live theronts) were found in immunised fish at Week 2 and 4 post-vaccination. Lysozyme activity of plasma from vaccinated fish increased from Week 1 to 4. Both immobilisation titers and lysozyme activity were significantly higher at 20 degrees C. This study demonstrated that live theronts are good candidates for an antigen source for development of effective vaccines against white spot disease in this fish host, and further indicated that the protection of rainbow trout against I. multifiliis infection is highly temperature dependent and may be associated with both adaptive and innate response mechanisms.

Systemic ciliatosis caused by histophagous ciliates constitutes a serious disease of cultured turbot. Six ciliate isolates were obtained from parasitized turbot during six epizootics at four different farms located in Spain, France and Portugal. Axenic cultures of the six isolates were obtained by periodical subculturing in ATCC 1651MA or supplemented L-15 media. In basal media or seawater, the parasites could survive starving for long periods with no apparent proliferation. In adequate media, growth kinetics was found to be very similar for isolates A and B, with a clear influence of temperature. Morphological studies demonstrated that all isolates share common features that allows their assignment to either Philasterides Kahl, 1931 or Miamiensis Thompson et Moewus, 1964. However, statistically significant differences were evident in pairwise comparisons of the isolates from the four farm sites in 16 taxonomically relevant morphometric features. This could allow the discrimination of different species or strains. Virulence of isolates A and B for healthy turbot was tested in several experiments. Differences in the virulence were especially evident after long-term in vitro culturing, isolate A being clearly attenuated after 35-42 passages, whereas isolate B became more virulent after 20-42 passages. The need of further studies to confirm such virulence variability and its implications in pathogenesis and prevention of turbot scuticociliatoses is stressed.

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 mass infection of larvae and youngsters of carp fishes with the peritrichoid Ambiphrya ameiuri has been recorded in natural condition in the Rybinsk water reservoir. The uneven localisation of ambiphyrans on the body surface has been observed on the hosts Blicca bjorkna and Abramis brama. Most high density of parasites was on the breast, back and tail fins. The morphological variability of ambiphyrans in dependence upon their localisation on the fish body has been observed. Details being significant for the taxonomy have been studied by means of the scan electron microscope. It has been found that the scopulae (greatly developed attaching organ of A. ameiuri) in cases of heavy infection cover about 50-60% of the skin epithelium of fish larvae. In these cases the significant part of skin epithelium, which performs the respiratory function is excludes of the active oxygen supply that cause the illness and death of young fishes.