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The protozoan parasite Cryptocaryon irritans causes marine white spot disease in a wide range of fish hosts, including gilthead seabream, a very sensitive species with great economic importance in the Mediterranean area. Thus, we aimed to evaluate the immunity of gilthead seabream after a severe natural outbreak of C. irritans . Morphological alterations and immune cell appearance in the gills were studied by light microscopy and immunohistochemical staining. The expression of several immune-related genes in the gills and head kidney were studied by qPCR, including inflammatory and immune cell markers, antimicrobial peptides (AMP), and cell-mediated cytotoxicity (CMC) molecules. Serum humoral innate immune activities were also assayed. Fish mortality reached 100% 8 days after the appearance of the C. irritans episode. Gill filaments were engrossed and packed without any space between filaments and included parasites and large numbers of undifferentiated and immune cells, namely acidophilic granulocytes. Our data suggest leukocyte mobilization from the head kidney, while the gills show the up-regulated transcription of inflammatory, AMPs, and CMC-related molecules. Meanwhile, only serum bactericidal activity was increased upon infection. A potent local innate immune response in the gills, probably orchestrated by AMPs and CMC, is triggered by a severe natural outbreak of C. irritans .

The ciliated protozoan Cryptocaryon irritans infects a wide range of marine fish and causes the highly lethal white spot disease. This parasite possesses three morphologically and physiologically distinct life stages: an infectious theront, a parasitic trophont, and an asexually reproductive tomont. In the past few years, several attempts have been made to help elucidate how C. irritans transforms from one stage to another using transcriptomic or proteomic approaches. However, there has been no research studying changes in transcription profiles between different time points of a single C. irritans life stage-the development of this parasite. Here we use RNA-seq and compare gene expression profiles of theront cells collected by 1 and 10 hrs after they emerged from tomonts. It has been shown that infectivity of theront cells declines 6-8 hours post-emergence, and we used this characteristic as a physiological marker to confirm the aging of theront cells. We identified a total of 41 upregulated and 90 downregulated genes that were differentially expressed between young and aging theront cells. Using Blast2Go to further analyze functions of these genes, we show that genes related to energy production are downregulated, but quite surprisingly many genes involved in transcription/translation processes are upregulated. We also show that expression of all nine detectable agglutination/immobilization antigen genes, with great sequence divergence, is invariably downregulated. Functions of other differentially expressed genes and indications are also discussed in our study., Competing Interests: The authors have declared that no competing interests exist.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

A ciliate protozoan was discovered whose presence coincided with a rapid decrease in the viability (i.e. ability to close valves) of glochidia of the freshwater mussel Lampsilis siliquoidea. Microscopic examination showed it to be a histophagous tetrahymenine ciliate. Small subunit (SSU) rRNA and cytochrome c oxidase subunit 1 (cox1) barcode sequences from cultured cells showed that it belongs to the same new species isolated from water samples as a free-living ciliate. Phylogenetic analyses place this new ciliate in the same clade with the macrostome species Tetrahymena paravorax, and we propose the name T. glochidiophila n. sp. for this new species. The phylogeny provides further support for the hypothesis that histophagy was a life history trait of the ancestor of Tetrahymena.

A parasitological investigation on trichodinid ciliates from several freshwater fishes in the Baikka Beel of Moulvibazar district of Sylhet was carried out between January and December 2015. During the investigation period, two parasitic trichodinid ciliates (Ciliophora: Peritrichia), Trichodina pseudoheterodentata Tang et al. 2017 and Trichodina hafizuddini Asmat, 2005 were morphologically studied and described with using the silver nitrate impregnation technique. Trichodina pseudoheterodentata was isolated from the gills of Mystus bleekeri, while Trichodina hafizuddini from the gills of Amblypharyngodon mola. This investigation has been revealed that these two Trichodina species fall within the range of morphometry and agree closely in the overall appearance of the adhesive disc with the original populations. Geographical variation was also observed by considering the body size and number of the denticles. Depending on country’s available taxonomical survey on trichodinid ciliates, this study is the first formal report on these trichodinids from any part of Bangladesh.

We planned to develop predator-prey models using Paramecium and yeast, but they have not been empirically examined since work by Gause in the 1930s. Therefore, we evaluated if Paramecium aurelia ingests and grows on eight yeasts. Recognising that it ingested yeasts but could not grow, we assessed if it might grow on other yeasts, by empirically parameterising a predator-prey model that relies on ingestion, not growth. Simulations were compared to P. aurelia-yeast time-series data, from Gause. We hypothesised that if the model simulated predator-prey dynamics that mimicked the original data, then possibly P. aurelia could grow on yeast; simulations did not mimic the original data. Reviewing works by Gause exposed two issues: experiments were undoubtedly contaminated with bacteria, allowing growth on bacteria, not yeast; and the population cycle data cannot be considered a self-sustaining time series, as they were manipulated by adding yeast and ciliates. We conclude that past and future work should not rely on this system, for either empirical or theoretical evaluations. Finally, although we show that P. aurelia, P. caudatum, Euplotes patella, and Blepharisma sp. cannot grow on yeast, Tetrahymena pyriformis and Colpidium striatum can; these may provide models to explore predator-prey dynamics., (© 2016 The Author(s) Journal of Eukaryotic Microbiology © 2016 International Society of Protistologists.)

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.)

Selective antibiotic treatment of white syndrome (WS)-affected corals (Acropora muricata) from Fiji was used to identify 3 potential bacterial pathogens of the disease. Interestingly, the suite of bacterial associates of the disease was different to that recently identified using identical primer sets for WS on the GBR and in the Solomon Islands. In addition to the three bacterial pathogenic candidates and as previously shown for WS and more recently for white band disease (WBD) in the Caribbean, all samples of the disease were specifically associated with the histophagous ciliate Philaster lucinda. From the pattern of disease progression and histopathology in relation to the selective elimination of microbial groups, we conclude that these 'white' diseases are a result of a nonspecific bacterial infection and a 'secondary' infection by the P. lucinda ciliate. Although we have not observed the initiation of infection, a nonspecific, multispecies bacterial infection appears to be a corequirement for WS lesion progression and we hypothesize that the bacterial infection occurs initially, weakening the defences of the host to predation by the ciliates. Such ciliate histophagy gives rise to the characteristic white band of denuded coral skeleton that gives these diseases their names. The characteristics of the microbial communities of WBD and WS appear identical, and since the bacterial associates of WS vary geographically (and/or temporally), there appears to be no logical distinction between WS in the Indo-Pacific and WBD in the Caribbean., (© 2015 John Wiley & Sons Ltd.)

Outbreaks of an unidentified ciliate have occurred on several occasions in blue crabs from Chesapeake Bay held during winter months in flow-through systems. The parasite was initially thought to be Mesanophrys chesapeakensis, but molecular analysis identified it as Orchitophyra stellarum, a facultative parasite of sea stars (Asteroidea). We investigated the host-parasite association of O. stellarum in the blue crab host. Crabs were inoculated with the ciliate, or they were held in bath exposures after experimentally induced autotomy of limbs in order to determine potential mechanisms for infection. Crabs inoculated with the ciliate, or exposed to it after experimental autotomy, rapidly developed fatal infections. Crabs that were not experimentally injured, but were exposed to the ciliate, rarely developed infections; thus, indicating that the parasite requires a wound or break in the cuticle as a portal of entry. For comparative purposes, fiddler crabs, Uca minax, were inoculated with the ciliate in a dose-titration experiment. Low doses of the ciliate (10 per crab) were sometimes able to establish infections, but high intensity infections developed quickly at doses over 500 ciliates per crab. Chemotaxis studies were initiated to determine if the ciliate preferentially selected blue crab serum (BCS) over other nutrient sources. Cultures grown on medium with BCS or fetal bovine serum showed some conditioning in their selection for different media, but the outcome in choice experiments indicated that the ciliate was attracted to BCS and not seawater. Our findings indicate that O. stellarum is a facultative parasite of blue crabs. It can cause infections in exposed crabs at 10-15°C, but it requires a portal of entry for successful host invasion, and it may find injured hosts using chemotaxis., (Copyright © 2013 Elsevier Inc. 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.

Aquatic organisms are exposed to pollution which may make them more susceptible to infections and diseases. The present investigation evaluated effects of nickel contamination and parasitism (ciliates Ophryoglena spp. and intracellular bacteria Rickettsiales-like organisms), alone and in combination, on biological responses of the zebra mussel Dreissena polymorpha, and also the infestation abilities of parasites, under laboratory controlled conditions. Results showed that after 48 h, more organisms were infected in nickel-exposed groups, which could be related to weakening of their immune system. Acting separately, nickel contamination and infections were already stressful conditions; however, their combined action caused stronger biological responses in zebra mussels. Our data, therefore, confirm that the parasitism in D. polymorpha represents a potential confounding factor in ecotoxicological studies that involve this bivalve.

The shell morphology of zebra mussels, Dreissena polymorpha, was analyzed to determine if alterations in shell shape and asymmetry between valves were related to its infection status, i.e. infected or not by microparasites like ciliates Ophryoglena spp. or intracellular bacteria Rickettsiales-like organisms (RLOs), and by macroparasites like trematodes Phyllodistomum folium and Bucephalus polymorphus. For microparasites, two groups of mussels were observed depending on shell measurements. Mussels with the more concave shells were the most parasitized by ciliates. This could be more a consequence than a cause and we hypothesized that a modification of the water flow through the mantle cavity could promote the infection with a ciliate. There were more RLOs present in the most symmetrical individuals. A potential explanation involved a canalization of the left-right asymmetry as a by-product of the parasite infection. Trematode infections were associated with different responses in valve width. Females infected by P. folium displayed significantly higher symmetry in valve width compared with non-infected congeners, whereas the infection involved an opposite pattern in males. B. polymorphus was also linked to a decrease in valve width asymmetry. This study suggested that a relationship exists between parasitism and shell morphology through the physiological condition of host zebra mussels., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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 scuticociliate Anophryoides haemophila, causes bumper car disease in American lobster (Homarus americanus) in commercial holding facilities in Atlantic Canada. While the parasite has been recognized since the 1970s and much has been learned about its biology, minimal molecular characterization exists. With genome consortiums turning to model organisms like the ciliates Tetrahymena and Paramecium, the amount of relevant sequence data available has made sequence surveys more attractive for gene discovery in related ciliates. We sequenced 9984 expressed sequence tags (ESTs) from a non-normalized A. haemophila cDNA library to characterize gene expression patterns, functional gene distribution and to discover novel genes related to the parasitic life history. The A. haemophila ESTs were grouped into 843 clusters and singletons with 658 EST clusters having identifiable homologs, while 159 ESTs were unique and had no similarity to any sequences in the public databases. Not unexpectedly, about 67% of the A. haemophila ESTs have similarity to annotated and hypothetical genes from the related oligohymenophorean ciliate, Tetrahymena. Numerous cysteine proteases, hypothetical proteins and novel sequences possess putative secretory signal peptides suggesting that they may contribute to the pathogenesis of bumper car disease in lobster. Real time RT-qPCR analysis of cathepsin L and two homologs of cathepsin B did not show any changes in gene expression under varying in vitro growth conditions or during a modified-in vivo infection which may be suggestive of the opportunistic life history strategy of this ciliate., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The American lobster fishery is a significant economic driver in coastal communities of North America. Increasingly, the impacts of infectious disease are recognized as important components and factors in the population ecology and subsequent management of the lobster fishery. Both environmental and anthropogenic factors impact marine diseases. The review herein highlights aspects of several important bacterial, fungal and protistan diseases, including gaffkemia, shell disease, vibriosis, disease caused by species of Lagenidium, Haliphthoros and Fusarium, paramoebiasis and Bumper Car disease. As the global environment continues to change, these diseases could more severely affect both wild caught and impounded lobsters., (Copyright © 2010 Elsevier Inc. All rights reserved.)

In environmental quality bioassessment studies, analysis of host-parasite interactions may well be a valuable alternative to classical macroinvertebrate sampling approaches. Herein, we investigated whether zebra mussel (Dreissena polymorpha) parasites could be useful biomonitoring tools. Mussel populations were sampled twice at two sites in northeastern France representing different levels of contamination and were characterized for parasite infection following standard histological methods. Our results indicated that sites of different environmental quality (i.e. chemical contamination) exhibited different parasite communities characterized by different trematode species and parasite associations. An additional significant finding was the positive correlation established between the prevalence of Rickettsiales-like organisms and metal contamination. Multivariate analyses were valuable in examining parasite communities., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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.

The pathology associated with an intracellular ciliate infection in the digestive gland of pearl oysters Pinctada maxima (Jameson, 1901) is described. Histopathological and transmission electron microscopic examination were used to characterise the organism and its location within host cells. The parasite is tear-drop shaped measuring 5.53 microm (range of 2.73-7.47 microm, n=9) in width and 11.15 microm (range of 9.02-16.2 microm) in length with a centrally located lobulated nucleus and a large nucleus:cytoplasmic ratio. The ciliate has nine evenly spaced rows of cilia running obliquely along the length of cell, converging on the pointed end. Infected digestive glands typically had a moderate to severe infiltration with mononuclear hemocyte. A strong correlation existed between the burden of ciliates and the host response; (p<0.001, C=0.315 Pearson Correlation). The use of a single tissue section upon microscopic examination was found to detect only 38-50% of the infections. However, examination of serial haematoxylin and eosin stained sections improved the reliability of detecting infection.

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.

The article considers morpho-functional organization of the cilia, locomotor organelle of the infusoria, and demonstrates the complicity of locomotor behavior of these protista. The problem of control of locomotion of infusoria is whole organism in discussed; and conclusion is drawn that system of control of movements could be multilevel and include receptor, afferent, central, efferent and effector units. In this context the macronucleus, could act as a central integrator and coordinator of the locomotor behavior being closely connected with periphery by dynamic elements of cytoskeleton. The eradication of infusoria parasitizing in humans and animals by interrupting of locomotion of the protista is also discussed.

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.

An apostome ciliate, Collinia oregonensis n. sp., is reported inhabiting the cephalothorax and abdomen of 3 euphausiid species from the Oregon-Washington coast: Euphausia pacifica Hansen, 1911, Thysanoessa spinifera Holmes, 1900, and Thysanoessa gregaria G.O. Sars, 1883. This ciliate is the 7th species described for the genus Collinia and the 2nd species known to infect euphausiids. Disease progression and ciliate morphology are described using (1) modified protargol stain, (2) hematoxylin counterstained with Fast Green, and (3) Scanning Electron Microscopy (SEM). All endoparasitic developmental stages (trophont, tomont, tomitogenesis, protomite, and tomite) of C. oregonensis are astomatous and possess between 14 and 22 kineties. C. oregonensis is smaller than C. beringensis Capriulo & Small, 1986, which infects the euphausiid Thysanoessa inermis Krøyer, 1846 in the Bering Sea and which possesses between 24 and 80 kineties. The ciliate is a parasitoid because it must kill the host to complete its life cycle. Infections and mortalities in multiple host species likely reflect the virulent nature of the ciliate. Adult euphausiids infected with this parasitoid possess a swollen and bright orange cephalothorax. C. oregonensis feeds and proliferates inside euphausiids, producing fulminating infections that rupture the cephalothorax and release large numbers of tomites into the surrounding water. After several hours in the free swimming stage under shipboard conditions in the present study, the tomites adhered to each other, forming filaments. Infection rates ranged between 3 and 20% within individual euphausiid aggregations, but infected aggregations were randomly and sparingly distributed. Infected euphausiids were found at 6.7% of 316 stations sampled during 3 summer cruises. No infected euphausiids were collected in winter. Because E. pacifica and T. spinifera account for about 90% of the euphausiid standing stock in the northern California Current System, this parasitoid ciliate may have a significant impact on euphausiid population abundance, distribution and secondary productivity.

We investigated the swimming activity of 70 European eels Anguilla anguilla in relation to natural infection with 2 parasite species: the eel-specific swimbladder nematode Anguillicola crassus and the non-specific skin and gill protozoan Ichthyophthirius multifiliis. We measured how long individual eels exposed to a water current in a swimming channel with a steady-stream profile could withstand the water current. The parasites affected the swimming behaviour of eels in different ways. The maximum period of time the fish were able to swim against the current was not correlated with infection by A. crassus. In contrast, infection with I. multifiliis reduced the swimming time. The protozoan has a higher pathogenicity than the swimbladder nematode, at least in closed systems, where I. multifiliis is able to spread within a few days. Reduction in swimming capacity after infection with the ciliate averaged 47 % compared to capacity prior to infection. Thus, our results do not support the previously suggested strong negative relation between swimming activity of eels and intensity of A. crassus infection, at least in the short-term. However, there are indications in the literature that the pathological effects of A. crassus on the eel swimmbladder may involve a higher energy demand, possibly manifested in a prolonged spawning migration. As a result, eels heavily infected with this parasite may arrive too late at the spawning site to participate in mating. This could ensure a selection of 'good genes'.

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.

Objective: To determine the prevalence of microorganisms that are potentially pathogenic for humans in horse/mule manure along the John Muir Trail (JMT)., Methods: Random samples of horse/mule manure were collected along sections of the JMT in Yosemite, Kings Canyon, and Sequoia national parks (NP), as well as in portions of the Pacific Crest Trail (PCT) and selected JMT/PCT access trails. Convenience samples of wild animal scat found within I mile of trails were also collected. The fresh specimens were individually preserved both in 0.9% saline and polyvinyl alcohol (PVA)-containing tubes and stored at 4 degrees C until time of analysis. Bacteriological analysis was performed using standard microbiology laboratory procedures. PVA samples were stained with trichrome and were then examined by a parasitologist., Results: Collection: A total of 186 trail miles were sampled, including 113 on the JMT (Yosemite 37, Kings 53, and Sequoia 23). The PCT samplings included 24 miles, and NP and wilderness area access trails added an additional 49 miles. A total of 102 samples were collected, which included 81 samples from pack animals and 21 identified as having come from wild animals. Pack Animal Bacteria: All plated specimens grew large numbers of commensal gut flora. Potential pathogenic bacteria were found in only 12 samples and included Hafnia alvei (4), Serratia odorifera (1), Citrobacter freundii (1), Escherichia vulneris (1), Clostridium clostridioforme (1), Yersinia enterocolitica (1), Sherwinella putraformus (1), and Enterobacter spp (4). No Escherichia coli O157, Salmonella, or Aeromonas were found. Microscopic examination for protozoal organisms revealed occasional commensal ciliates and I Giardia. Wild Animal Pathogens: One specimen grew Y enterocolitica, and another grew Enterobacter amnigenus., Conclusions: We found a low prevalence of human pathogens in pack animal manure on the JMT.

The Canadian lobster industry holds lobsters Homarus americanus in captivity for various periods to supply markets with live product year-round. Mortality during holding results in considerable losses, estimated at 10 to 15 % yr(-1) by the industry. This study examined the prevalence of Anophryoides haemophila and Aerococcus viridans, causative agents of 'bumper car' disease and gaffkemia, respectively, in lobsters freshly captured in the waters of Prince Edward Island during the spring and fall fishing seasons of 1997. A total of 116 lobsters were sampled in the spring, and 138 in the fall. A. haemophila was not detected in the spring, while the prevalence was 0.72 % in the fall with a 95% confidence interval (CI) of 0.02 to 3.97% and an overall prevalence of 0.39% (95% CI: 0.01 to 2.17%). The prevalence of A. viridans was estimated at 6.9% (95% CI: 3.0 to 13.14%) in the spring, 5.8% in the fall (95% CI: 2.54 to 11.10%), and 6.30% overall (95% CI: 3.64 to 10.03%). Because of the reduced interest in food of diseased lobsters, and compromised metabolism in the case of gaffkemia, these prevalence estimates are likely underestimates of the true prevalence of gaffkemia and 'bumper car' disease in the wild populations of lobster around Prince Edward Island.

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.

The ability of two aposymbiotic (algae-free) subclones of the same green clone of C. virens to establish a stable symbiotic association with Chlorella sp. has been studied by light and electron microscopy. Alga-free subclone No. 1 was obtained from the original green clone by a long-term cultivation in darkness, while subclone No. 2 originated from one cell that spontaneously lost the algae and was found among normal green cells during daily inspection. For infection, algae isolated from ciliates with chlorellae of parental clone of C. virens were used. 5-10 minutes after feeding with Chlorella, specimens of both subclones show numerous algae mostly inside food vacuoles, but some rare algae (3-4 per cell) may occur in individual perialgal vacuoles. Later on, the number of symbiotic chlorellae in ciliates of subclone No. 1 increased, and a stable symbiotic association was reestablished. Unlike, in specimens of subclone No. 2 all newly ingested algae were seen digested within food vacuoles. Within 24-28 h all the ciliates investigated appeared free of algae. However, obviously stable symbiotic ciliate-algae systems in this subclone were obtained after improving the microinjection technique. Injection of algae into alga-free ciliates resulted in maintenance of intact chlorellae in these ciliates. The algae were seen to be located individually within perialgal vacuoles, being presumably protected against host lytic enzyme attack. The endosymbiont population in ciliates was established from as many as 3-5 originally injected algae. The number of symbiotic chlorellae increased steadily reaching the value equal to that in the parental clone 28-30 days after the start of experiment.

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.

Recent (1993) landings of American lobsters (Homarus americanus) were valued at $294 million (Can.) in Canada and $213 million (Can.) in the United States. However, post-harvest losses are estimated at $50-75 million (10-15%) annually. The lobster fishery is one of the few remaining viable traditional fisheries in eastern North America. "Bumper car" disease of lobsters, caused by the scuticociliate Anophryoides haemophila, can cause significant losses in coldwater impoundments. Apparently epidemics now occur more frequently and with greater severity; surprisingly the epidemiology and economic impacts of "bumper car" disease are not well documented. The ciliate A. haemophila is easily maintained in a cell-free, chemically defined, seawater-based medium at 5 degrees C. Cultured ciliates require longer and more parasites to kill lobsters than those transmitted by intrahaemocoelic injection from lobster-to-lobster. Regardless of source of ciliates, the larger the inoculum, the more rapid the death of lobsters. The pathogenesis of "bumper car" disease is unknown. Horizontal transmission could occur across the thin cuticle of gills or via wounds in the exoskeleton present during moulting of lobsters. Because ciliates are initially sequestered in lobster tissues for an extended period, they are detectable sooner by histological examination of tissues than by direct examination or culture of haemolymph. Additional to indirect fluorescent antibody testing and immunoperoxidase staining of tissues, utilizing monoclonal antibodies prepared to sonicated ciliates, the parasites are readily detected with oligonucleotide probes based on ssu-rDNA of A. haemophila. The prevalence of A. haemophila should be re-evaluated. Ciliates sequester in gill, heart and muscle tissues. Several disinfectants and chemotherapeutants, licensed in North America for veterinary use in food-producing animals, are efficacious against A. haemophila in vitro. A definition of healthy vs ciliate-infected lobsters is being prepared, based on haematology and clinical chemistry of haemolymph. Our novel bar-coded labelling system for aquatic organisms facilitates experimental design and randomization protocols of lobsters. The model of "bumper car" disease will aid study of health and infectious disease processes of lobsters and other crustaceans.

The transformation of Lambornella stegomyiae trophonts to theronts, the distribution of invasion cysts on larval Aedes albopictus cuticle, and the virulence of L. stegomyiae to Ae. albopictus and Aedes aegypti were studied in the laboratory. Transformation of trophonts into theronts was induced by a morphogenic agent released from larval Ae. albopictus homogenate. The first transformation was observed 4 hr after exposure to larval mosquito homogenate, but most transformations occurred between 12 and 16 hr. Distribution of invasion cysts on the cuticle of mosquito larvae was not uniform and most cysts were formed on the abdomen and head. L. stegomyiae was highly infective and virulent to Ae. albopictus (mortality rate: 99.53%) and Ae. aegypti (mortality rate: 90.83%) larvae.

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.

Tannin concentration, measured as total phenolics, varied widely in natural treehole water throughout the period of development for Aedes sierrensis (Ludlow), the western treehole mosquito. Tannic acid induced a parasitic response in free-living populations of the protozoan Lambornella clarki Corliss & Coats, an effect not entirely explained by acidity. Tannic acid concentration influenced mosquito developmental rates, survival, size, and proportion females more than food level, L. clarki parasite exposure, or any interaction of treatments for Ae. sierrensis populations reared in artificial microcosms exposed to natural conditions.

Lambornella clarki (Ciliophora: Tetrahymenidae), an endoparasite of Aedes sierrensis (Diptera: Culicidae), is dispersed by infected adult mosquitoes. Invasion of the ovaries induces parasitically castrated females to exhibit oviposition behavior and thereby actively disperse ciliates through deposition into water. Oviposition behavior of infected females is prolonged and mimics that of normal gravid females in their first gonotropic cycle. Adults of both sexes also passively disperse ciliates by dying on water surfaces, and infected adults are more likely to die on water than uninfected adults. Ciliates dispersed by infected adults can infect larvae and form desiccation-resistant cysts. Parasite-induced dispersal by hosts, desiccation-resistant cysts, an active host-seeking infective stage, and high infection and mortality rates all indicate significant biological control potential for these and related ciliates against container-breeding mosquitoes.