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Lactococcus garvieae has recently been identified and listed as one of the causative agents of hyperacute hemorrhagic sepsis in fish. In intensive recirculating aquaculture systems where there are high fish densities and minimal water changes, not only will it be conducive to the growth of bacteria, but Cryptocaryon irritans as a marine protozoan fish parasite is also prone to appear. This study reports the disease status of Trachinotus ovatus in an aquaculture area in Yangjiang City, Guangdong Province. Through the diagnosis of clinical symptoms of the diseased fish, identification of specific primers, 16s rRNA sequences phylogenetic tree analysis, physiological and biochemical identification, and observation of histopathological sections, the result of the experiment is that the mass death of T. ovatus is caused by a mixture of L. garvieae and C. irritants infections. Subsequently, regression infection experiments were performed to verify Koch's law. It was confirmed that the pathogen had strong virulence to T. ovatus. This is the first time that the co-infection of L. garvieae and C. irritans to T. ovatus was found in South China. The research results of this experiment have certain enlightenment significance for the epidemic trend of fish diseases in relevant sea areas., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Huang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cryptocaryon irritans is a highly detrimental parasite in mariculture, causing significant economic losses to the aquaculture industry of Larimichthys crocea . In recent years, copper and copper alloy materials have been used to kill parasites. In this study, the effect of copper plates on the tomont period of C. irritans was explored. The findings indicated that copper plates effectively eradicated tomonts, resulting in a hatching rate of 0. The metabolomic analysis revealed that a total of 2,663 differentially expressed metabolites (1,032 up-regulated and 1,631 down-regulated) were screened in the positive ion mode, and 2,199 differentially expressed metabolites (840 up-regulated and 1,359 down-regulated) were screened in the negative ion mode. L-arginine and L-aspartic acid could be used as potential biomarkers. Copper plate treatment affected 25 metabolic pathways in the tomont, most notably influencing histidine metabolism, retinol metabolism, the biosynthesis of phenylalanine, tyrosine, and tryptophan, as well as arginine and proline metabolism. It was shown that high concentrations of copper ions caused a certain degree of disruption to the metabolome of tomonts in C. irritans , thereby impacting their metabolic processes. Consequently, this disturbance ultimately leads to the rapid demise of tomonts upon exposure to copper plates. The metabolomic changes observed in this study elucidate the lethal impact of copper on C. irritans tomonts, providing valuable reference data for the prevention and control of C. irritans in aquaculture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Guo, Huang, Xu, Zhan, Sun and Hu.)

Cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) is a distinctive member of the serine–threonine protein AGC kinase family and an effective kinase for cAMP signal transduction. In recent years, scuticociliate has caused a lot of losses in domestic fishery farming, therefore, we have carried out morphological and molecular biological studies. In this study, diseased guppies ( Poecilia reticulata ) were collected from an ornamental fish market, and scuticociliate Philaster apodigitiformis Miao et al ., 2009 was isolated. In our prior transcriptome sequencing research, we discovered significant expression of the β-PKA gene in P. apodigitiformis during its infection process, leading us to speculate its involvement in pathogenesis. A complete sequence of the β-PKA gene was cloned, and quantified by quantitative reverse transcription-polymerase chain reaction to analyse or to evaluate the functional characteristics of the β-PKA gene. Morphological identification and phylogenetic analysis based on small subunit rRNA sequence, infection experiments and haematoxylin–eosin staining method were also carried out, in order to study the pathological characteristics and infection mechanism of scuticociliate. The present results showed that: (1) our results revealed that β-PKA is a crucial gene involved in P. apodigitiformis infection in guppies, and the findings provide valuable insights for future studies on scuticociliatosis; (2) we characterized a complete gene, β-PKA , that is generally expressed in parasitic organisms during infection stage and (3) the present study indicates that PKA plays a critical role in scuticociliate when infection occurs by controlling essential steps such as cell growth, development and regulating the activity of the sensory body structures and the irritability system.

The protozoan parasite Ichthyophthirius multifiliis is an economically important parasite for the aquaculture- and ornamental fish industry. The parasite is abundant worldwide and infects the skin, gills and fins of freshwater fish species. For approximately the last fifty years the innate and protective immune mechanisms induced by I. multifiliis have been in focus in different fish hosts. By utilizing transgenic zebrafish, new tools to investigate this have emerged. The aim of this study was therefore to elucidate early immune responses in zebrafish larvae by using gene expression and in vivo imaging of neutrophil and macrophage behavior during infection. For the first time, zebrafish larvae were infected with the parasite and infection dynamics, parasite size and host-parasite interactions were investigated. Results showed that the larvae responded with mild inflammation and that the 12 compared to 5 days post fertilization larvae were significantly less susceptible. It was furthermore observed that neutrophils and macrophages were attracted to the parasites and that neutrophils reacted with neutrophil extracellular traps (NETs) when fighting the parasite. The parasite was rotating vigorously, presumably to impede the neutrophils and macrophages from attaching to it but on rare occasions, neutrophils and macrophages were able to kill the parasite. Based on these observations, we concluded that the parasite uses the rotation as an immune evasive strategy and that the zebrafish larvae respond with high activity from neutrophils and macrophages locally but systemically only with mild inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mathiessen, Kjeldgaard-Nintemann, Gonzalez, Henard, Reimer, Gelskov, Marana and Jørgensen.)

Background: Recently, an increasing number of ichthyophthiriasis outbreaks has been reported, leading to high economic losses in fisheries and aquaculture. Although several strategies, including chemotherapeutics and immunoprophylaxis, have been implemented to control the parasite, no effective method is available. Hence, it is crucial to discover novel drug targets and vaccine candidates against Ichthyophthirius multifiliis. For this reason, understanding the parasite stage biology, host-pathogen interactions, molecular factors, regulation of major aspects during the invasion, and signaling pathways of the parasite can promote further prospects for disease management. Unfortunately, functional studies have been hampered in this ciliate due to the lack of robust methods for efficient nucleic acid delivery and genetic manipulation. In the current study, we used antisense technology to investigate the effects of targeted gene knockdown on the development and infectivity of I. multifiliis. Antisense oligonucleotides (ASOs) and their gold nanoconjugates were used to silence the heat shock protein 90 (hsp90) of I. multifiliis. Parasite stages were monitored for motility and development. In addition, the ability of the treated parasites to infect fish and cause disease was evaluated., Results: We demonstrated that ASOs were rapidly internalized by I. multifiliis and distributed diffusely throughout the cytosol. Knocking down of I. multifiliis hsp90 dramatically limited the growth and development of the parasite. In vivo exposure of common carp (Cyprinus carpio) showed reduced infectivity of ASO-treated theronts compared with the control group. No mortalities were recorded in the fish groups exposed to theronts pre-treated with ASOs compared with the 100% mortality observed in the non-treated control fish., Conclusion: This study presents a gene regulation approach for investigating gene function in I. multifiliis in vitro. In addition, we provide genetic evidence for the crucial role of hsp90 in the growth and development of the parasite, suggesting hsp90 as a novel therapeutic target for successful disease management. Further, this study introduces a useful tool and provides a significant contribution to the assessing and understanding of gene function in I. multifiliis., (© 2023. The Author(s).)

Miamiensis avidus is a parasitic pathogen that causes the disease scuticociliatosis in teleost fish species. It is a ciliate and a free-living marine protozoan belonging to the order Philasterida, subclass Scuticociliatida, class Oligohymenophorea, and phylum Ciliophora. The complete mt-genome of M. avidus was linear and 38,695 bp in length with 47 genes, including 40 protein-coding genes, two ribosomal RNA (rRNA) genes, and five transfer RNA (tRNA) genes. Of these, 20 genes typically belong to the clusters of orthologous groups, playing roles in energy production and conversion, translation, ribosomal structure and biogenesis, and defense mechanisms. This is the first report of sequencing and characterization of the mt-genome of M. avidus, which was observed to be linear and possessing the typical ciliate mitochondrial genome organization and phylogenetic relationships. Remarkable differences were observed between M. avidus and other ciliates in the mitochondrially encoded rRNAs, extensive gene loss in ribosomal genes and tRNAs, terminal repeat sequences, and stop codon usage. A comparative and phylogenetic analysis of M. avidus and Uronema marinum of the order Hymenostomatida, which is most closely related to the order Philasterida, signified the promise of the mitogenome data of M. avidus as a valuable genetic marker in species detection and taxonomic research. The present study has potential applications in epidemiological studies and host-parasite interaction investigations facilitating disease control., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Ichthyophthirius multifiliis , a ciliated parasite causing ichthyophthiriasis (white spot disease) in freshwater fishes, results in significant economic loss to the aquaculture sector. One of the important predisposing factors for ichthyophthiriasis is low water temperature (i.e., below 20°C), which affects the health and makes freshwater fishes more susceptible to parasitic infections. During ichthyophthiriasis, fishes are stressed and acute immune reactions are compromised, which enables the aquatic bacterial pathogens to simultaneously infect the host and increase the severity of disease. In the present work, we aimed to understand the parasite-bacteria co-infection mechanism in fish. Later, Curcuma longa (turmeric) essential oil was used as a promising management strategy to improve immunity and control co-infections in fish. A natural outbreak of I. multifiliis was reported (validated by 16S rRNA PCR and sequencing method) in Pangasianodon hypophthalmus from a culture facility of ICAR-CIFRI, India. The fish showed clinical signs including hemorrhage, ulcer, discoloration, and redness in the body surface. Further microbiological analysis revealed that Aeromonas hydrophila was associated (validated by 16S rRNA PCR and sequencing method) with the infection and mortality of P. hypophthalmus , confirmed by hemolysin and survival assay. This created a scenario of co-infections, where both infectious agents are active together, causing ichthyophthiriasis and motile Aeromonas septicemia (MAS) in P. hypophthalmus . Interestingly, turmeric oil supplementation induced protective immunity in P. hypophthalmus against the co-infection condition. The study showed that P. hypophthalmus fingerlings supplemented with turmeric oil, at an optimum concentration (10 ppm), exhibited significantly increased survival against co-infection. The optimum concentration induced anti-stress and antioxidative response in fingerlings, marked by a significant decrease in cortisol and elevated levels of superoxide dismutase (SOD) and catalase (CAT) in treated animals as compared with the controls. Furthermore, the study indicated that supplementation of turmeric oil increases both non-specific and specific immune response, and significantly higher values of immune genes (interleukin-1β, transferrin, and C3), HSP70, HSP90, and IgM were observed in P. hypophthalmus treatment groups. Our findings suggest that C. longa (turmeric) oil modulates stress, antioxidant, and immunological responses, probably contributing to enhanced protection in P. hypophthalmus . Hence, the application of turmeric oil treatment in aquaculture might become a management strategy to control co-infections in fishes. However, this hypothesis needs further validation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kumar, Das, Swain, Chowdhury, Roy, Bera, Das, Parida, Dhar, Jana and Behera.)

Cryptocaryon irritans are the main pathogens of white spot disease in marine teleost. However, the occurrence of cryptocaryoniasis is influenced by several abiotic factors including the pH. To explore the effect of pH on the life cycle of C. irritans (encystment, cleavage, and hatchability), protomonts and tomonts of C. irritans were incubated in seawater of 10 different pH levels (2-11). pH 8 was used as the control. The change in morphology and infectivity of theronts that hatched from tomonts against Larimichthys crocea were then recorded. We found that pH 6-9 had no significant effect on the encystment, cleavage, and hatching of the parasites. However, pH beyond this limit decreased the cleavage and hatching of the tomonts. Furthermore, extreme pH decreased the number of theronts hatched by each tomont and the pathogenicity of the theronts, but increased the aspect ratio of the theronts. Infectivity experiments further revealed that extreme pH significantly decreased the infectivity of C. irritans against L. crocea . In conclusion, the C. irritans can survive in pH of 5 to 10, but pH 6-9 is the optimal range for the reproduction and infectivity of C. irritans . However, extreme pH negatively affects these aspects. IMPORTANCE Cryptocaryon irritans is a ciliate parasite that causes "white spot disease" in marine teleosts. The disease outbreak is influenced by hosts and a range of abiotic factors, such as temperature, salinity, and pH. Studies have shown that change in pH of seawater affects the structure (diversity and abundance of marine organisms) of marine ecosystem. However, how pH affects the life cycle and survival of C. irritans , and how future ocean acidification will affect the occurrence of cryptocaryoniasis, are not well understood. In this study, we explored the effect of pH on the formation and hatching of C. irritans tomonts. The findings of this study provide the foundation of the environmental adaptation of C. irritans , the occurrence of cryptocaryoniasis, and better management of marine fish culture.

Ichthyophthirius multifiliis is a major pathogen that causes a high mortality rate in trout farms. However, systemic responses to the pathogen and its interactions with multiple organs during the course of infection have not been well described. In this study, dual-organ transcriptomic responses in the liver and head kidney and hemato-serological indexes were profiled under I. multifiliis infection and recovery to investigate systemic immuno-physiological characteristics. Several strategies for massive transcriptomic interpretation, such as differentially expressed genes (DEGs), Poisson linear discriminant (PLDA), and weighted gene co-expression network analysis (WGCNA) models were used to investigate the featured genes/pathways while minimizing the disadvantages of individual methods. During the course of infection, 6,097 and 2,931 DEGs were identified in the head kidney and liver, respectively. Markers of protein processing in the endoplasmic reticulum, oxidative phosphorylation, and the proteasome were highly expressed. Likewise, simultaneous ferroptosis and cellular reconstruction was observed, which is strongly linked to multiple organ dysfunction. In contrast, pathways relevant to cellular replication were up-regulated in only the head kidney, while endocytosis- and phagosome-related pathways were notably expressed in the liver. Moreover, interestingly, most immune-relevant pathways (e.g., leukocyte trans-endothelial migration, Fc gamma R-mediated phagocytosis) were highly activated in the liver, but the same pathways in the head kidney were down-regulated. These conflicting results from different organs suggest that interpretation of co-expression among organs is crucial for profiling of systemic responses during infection. The dual-organ transcriptomics approaches presented in this study will greatly contribute to our understanding of multi-organ interactions under I. multifiliis infection from a broader perspective., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Roh, Kim, Lee, Park, Kim, Lee, Park and Kim.)

Epibionts often colonize the exoskeleton of crustaceans, which sometimes results in the development of a long-term relationship between them. Our present work confirmed that a specific epibiont is closely associated with the pelagic calanoid copepod Eurytemora lacustris, regardless of the region, which suggests a preserved interaction between these species. Molecular analyses revealed that the epibiont belongs to the genus Tokophrya. We also found that the level of basibiont colonization is related to its size and identified that the most intensely inhabited body parts are those located near the center of the copepod body. We hypothesize that the relationship between Eurytemora (basibiont) and Tokophrya (epibiont) was established during the Quaternary period, following which these two populations were fragmented into lakes where they survived in close interaction. In addition, we suppose that the close relationship between the two species indicates the coevolution of stenotherms. Further studies on the interactions between Eurytemora lacustris and Tokophrya are required in order to gain insight into the long-term relationship between the copepods and the epibionts.

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.

Massive occurrence of trichodinids is frequently accompanied by serious disease in fish farms. In this study, trichodinid species from the gills and skin of Nile tilapia (Oreochromis niloticus) farmed in the central-western region of Brazil (state of Goiás) were morphologically characterized. Dried slides were prepared from the parasites and were impregnated with silver nitrate (2%). Morphometric characteristics were determined and schematic drawings of the denticles were made using photomicrographs produced from the slides. Seven species of trichodinid ectoparasites (Protozoa: Ciliophora: Trichodinidae) were found parasitizing the gills: four of the genus Trichodina Ehrenberg, 1838; one of Tripartiella Lom, 1959; one of Paratrichodina Lom, 1963; and one of Trichodinella Ṧrámek-Huṧek, 1953. On the body surface, three specimens of the genus Trichodina were identified. This study presents new geographical records of trichodinids in Brazil, thus confirming that Trichodina centrostrigeata, Trichodina compacta, Trichodina heterodentata, Paratrichodina africana and Tripartiella orthodens are widely distributed worldwide. Additionally, the first record of the genus Trichodinella in Brazil is presented.

Treated wastewater (TWW) constitutes a sustainable water resource and has been used for fish culture in some countries around the world, although there are no comprehensive data on the effect of TWW on fish growth and health in the context of aquaculture production. Our objectives were to examine how fish culture in TWW affected fish growth and fitness, as well as compliance with the international standards for safe consumption. Guppy (Poecilia reticulata) fingerlings were reared in 0%, 50% and 100% tertiary TWW (TTWW), from the age of five days, for a period of four months. In water analyses, 33 out of 67 tested organic micropollutants (OMPs) were detected in the TTWW samples at least once, at concentrations that are typically reported in domestic TTWW. Fish survival ranged between 77-80% and did not differ between treatment groups. Fish growth and mortality following challenge infection with Tetrahymena sp. (which ranged between 64-68%), were similar among treatment groups. Of tested immunological parameters, lysozyme and anti-protease was similar among treatments while complement activity was highest in the 50% TTWW-reared fish. No abnormalities were observed in the histopathological analysis. Levels of heavy metals, polychlorinated-biphenyls (PCBs) and organochlorines (OCs) in fish were below the detection limit and below the Food and Agriculture Organization of the United Nations (FAO) and the European Union EU maximal permitted levels in food fish. Results suggest that the yield of fish grown in TTWW is potentially similar to that in freshwater, and the produced fish comply with the standards of consumer safety. The results are in line with previous studies that examined the feasibility of TWW-fed aquaculture., Competing Interests: The authors have declared that no competing interests exist.

Identification and characterization of protein complexes and interactomes has been essential to the understanding of fundamental nuclear processes including transcription, replication, recombination, and maintenance of genome stability. Despite significant progress in elucidation of nuclear proteomes and interactomes of organisms such as yeast and mammalian systems, progress in other models has lagged. Protists, including the alveolate ciliate protozoa with Tetrahymena thermophila as one of the most studied members of this group, have a unique nuclear biology, and nuclear dimorphism, with structurally and functionally distinct nuclei in a common cytoplasm. These features have been important in providing important insights about numerous fundamental nuclear processes. Here, we review the proteomic approaches that were historically used as well as those currently employed to take advantage of the unique biology of the ciliates, focusing on Tetrahymena , to address important questions and better understand nuclear processes including chromatin biology of eukaryotes., Competing Interests: The authors declare no conflict of interest.

Recently, the South American lungfish Lepidosiren paradoxa is being found inside aquaculture ponds, and even though there are a few studies on their parasite fauna, there is still much to be reported. Thus, the objective of this study is to report parasitism by trichodinids in L. paradoxa, as these ciliate protozoa are related to injuries and mortality in fish farming. The lungfish were collected from experimental tanks, had their tegument scraped and the resultant mucus was analyzed under an optical microscope for morphological and morphometrical analyses in Giemsa and silver nitrate stained slides. The species found was identified as Trichodina quelleni. This is the first report of this parasite in L. paradoxa, and the second report in cultivated fish in Brazil.

The parasitic ciliate Ichthyophthirius multifiliis (Ich), which infects almost all freshwater fish species, provides an optimal model for the study of immunity against extracellular protozoa. Ich invades the epithelia of mucosal tissues, forms white spots covering the whole body, and induces high mortality, while survivor fish develop both innate and adaptive immunity against Ich attack in systemic and mucosal tissues. Besides the protective roles of the Toll-like receptor (TLR)-mediated innate immune response, the critical immune functions of novel IgT in the skin, gut, gill, and olfactory organ of teleosts have been demonstrated in recent years, and all this information contributes to the ontogeny of the mucosal immune response in vertebrates. Especially in rainbow trout, Ich-infected fish exhibited higher IgT concentrations and titers in the mucosa and increased IgT + B-lymphocyte proliferation in mucosal tissues. IgM mainly functions in the adaptive immune response in the systemic tissues of rainbow trout, accompanied with increased IgM + B-lymphocyte proliferation in the head kidney of Ich-infected trout. However, little is known about the interaction between these mucosal tissues and systemic immune organs and the interaction between the inductive immune organs and functional immune organs. Immobilization antigens (Iags), located on the parasite cell and ciliary membranes, have been characterized to be targeted by specific antibodies produced in the host. The crosslinking of antigens mediated by antibodies triggers either an escape response or the immobilization of Ich. With more knowledge about the Iags of Ich and the immunity of teleosts, a more targeted vaccine, even a DNA vaccine, can be developed for the immune control strategy of Ich. Due to the high frequency of clinical fish ichthyophthiriasis, the study of fish immune responses to Ich provides an optimal experimental model for understanding immunity against extracellular protozoa., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

During March to August of 2017, hundreds of leopard sharks ( Triakis semifasciata) stranded and died on the shores of San Francisco Bay, California, US. Similar mass stranding events occurred in 1967 and 2011, but analysis of those epizootics was incomplete, and no etiology was confirmed. Our investigation of the 2017 epizootic revealed severe meningoencephalitis in stranded sharks, raising suspicion for infection. We pursued a strategy for unbiased pathogen detection using metagenomic next-generation sequencing followed by orthogonal validation and further screening. We showed that the ciliated protozoan pathogen, Miamiensis avidus, was present in the central nervous system of leopard ( n=12) and other shark species ( n=2) that stranded in San Francisco Bay but was absent in leopard sharks caught elsewhere. This ciliated protozoan has been implicated in devastating outbreaks in teleost marine fish but not in wild elasmobranchs. Our results highlight the benefits of adopting unbiased metagenomic sequencing in the study of wildlife health and disease.

Copper and alloys containing >60% copper by weight are antimicrobial. In aquaculture, copper alloys are used as part of corrosion-resistant cages or as part of copper coating. To test whether a copper alloy surface prevents the outbreak of parasitosis in the aquaculture of Larimichthys crocea , we covered the bottom of the aquaculture tank with sheets of copper alloy containing 74% to 78% copper, and we cultured L. crocea juveniles that had been artificially infected with the protozoan parasite Cryptocaryon irritans Our results showed that these copper alloy sheets effectively blocked the infectious cycle of C. irritans within a 1-week period and significantly reduced the number of C. irritans trophonts and tomonts, thereby decreasing the mortality rate of L. crocea In in vitro assays, the cytoplasmic membranes of protomonts disintegrated and the cytoplasm overflowed after just 5 minutes of contact with copper alloy surfaces. Although the same cytoplasmic membrane disintegration was not observed in tomonts, the tomonts completely lost their capacity for proliferation and eventually died following direct contact with copper alloy sheets for 1 h; this is likely because C. irritans tomonts took in >100 times more copper ions following contact with the copper alloy sheets than within the control aquaculture environment. Exposure to copper alloy sheets did not lead to excessive heavy metal levels in the aquacultured fish or in the culture seawater. IMPORTANCE Cryptocaryon irritans , a parasitic ciliate that penetrates the epithelium of the gills, skin, and fins of marine fish, causes acute suffocation and death in cultured fish within days of infection. Much of the existing research centers around the prevention of C. irritans infection, but no cure has been found. Studies demonstrate that copper has strong antimicrobial properties, and fish grown in copper-containing cages have lower rates of C. irritans infection, compared to those grown in other currently used aquaculture cages. In this study, we found that an alloy containing 74% to 78% copper by weight effectively killed C. irritans cells and prevented cryptocaryoniasis outbreaks within a 1-week period. These findings offer a new perspective on the prevention and control of cryptocaryoniasis., (Copyright © 2019 American Society for Microbiology.)

Trichodinids, which are ciliate protists, are causative agents of an aquatic animal disease, trichodiniasis, especially among both captive and wild fish. This disease can adversely affect aquaculture and have economic impacts. The objectives of this study were to evaluate the prevalence and mean intensity of Trichodina unionis infection, describe qualitative and quantitative morphological characters, and perform a molecular phylogenetic analysis. The gastropod samples were randomly collected by hand-picking and a hand net. Trichodina unionis was collected by the crushing method under a stereomicroscope. Among all 4977 examined gastropods, 55 individuals of two gastropod species, Gyraulus siamensis and Physella acuta, were found to be infected by T. unionis, with overall prevalence and mean intensity of infection of 1.11% and 16.65, respectively. The characteristics of the denticles indicated T. unionis as having moderately wide blades and moderately curved blade margins, with distinctive bend angles near the distal end. The quantitative characters showed some variations, which could be due to food availability. Molecular phylogenetic analysis conducted with 18S rRNA provided a monophyletic tree of our specimens and previously identified T. unionis, confirming species identification. This study represents the first record of T. unionis in Thailand., (© P. Wiroonpan & W. Purivirojkul, published by EDP Sciences, 2019.)

The ciliate Ichthyophthirius multifiliis is an important pathogen of freshwater fish that occurs in both temperate and tropical regions around the world. The purpose of present study was to report an outbreak of I. multifiliis in Steindachneridium parahybae from the Paraiba do Sul River in state of São Paulo (Brazil). The fins, tegument and gills of S. parahybae were examined for the presence of I. multifiliis, and blood parameters (Red Blood Cell, thrombocytes and leukocytes) were evaluated. All the examined fish (100%) were parasitized with I. multifiliis and the mean infection intensity was 15.5 parasites per fish. All the fish died 72 hours as a consequence of ichthyophthiriasis with alterations to the tegument coloration and open opercula. Red Blood Cell counts and hemoglobin concentration were low, indicating an anemic process.

Teleost skin serves as the first line of defense against invading pathogens, and contain a skin-associated lymphoid tissue (SALT) that elicit gut-like immune responses against antigen stimulation. Moreover, exposed to the water environment and the pathogens therein, teleost skin is also known to be colonized by diverse microbial communities. However, little is known about the interactions between microbiota and the teleost skin mucosal immune system, especially dynamic changes about the interactions under pathogen infection. We hypothesized that dramatic changes of microbial communities and strong mucosal immune response would be present in the skin of aquatic vertebrate under parasite infection. To confirm this hypothesis, we construct an infected model with rainbow trout ( Oncorhynchus mykiss ), which was experimentally challenged by Ichthyophthirius multifiliis (Ich). H & E staining of trout skin indicates the successful invasion of Ich and shows the morphological changes caused by Ich infection. Critically, increased mRNA expression levels of immune-related genes were detected in trout skin from experimental groups using qRT-PCR, which were further studied by RNA-Seq analysis. Here, through transcriptomics, we detected that complement factors, pro-inflammatory cytokines, and antimicrobial genes were strikingly induced in the skin of infected fish. Moreover, high alpha diversity values of microbiota in trout skin from the experimental groups were discovered. Interestingly, we found that Ich infection led to a decreased abundance of skin commensals and increased colonization of opportunistic bacteria through 16S rRNA pyrosequencing, which were mainly characterized by lose of Proteobacteria and increased intensity of Flavobacteriaceae . To our knowledge, our results suggest for the first time that parasitic infection could inhibit symbionts and offer opportunities for other pathogens' secondary infection in teleost skin.

Many studies have shown that coagulation systems play an important role in the defence against pathogens in invertebrates and vertebrates. In vertebrates, particularly in mammals, it has been established that the coagulation system participates in the entrapment of pathogens and activation of the early immune response. However, functional studies investigating the importance of the fish coagulation system in host defence against pathogens are scarce. In the present study, injection of turbot (Scopthalamus maximus) with the pathogenic ciliate Philasterides dicentrarchi led to the formation of macroscopic intraperitoneal clots in the fish. The clots contained abundant, immobilized ciliates, many of which were lysed. We demonstrated that the plasma clots immobilize and kill the ciliates in vitro. To test the importance of plasma clotting in ciliate killing, we inhibited the process by adding a tetrapeptide known to inhibit fibrinogen/thrombin clotting in mammals. Plasma tended to kill P. dicentrarchi slightly faster when clotting was inhibited by the tetrapeptide, although the total mortality of ciliates was similar. We also found that kaolin, a particulate activator of the intrinsic pathway in mammals, accelerates plasma clotting in turbot. In addition, PMA-stimulated neutrophils, living ciliates and several ciliate components such as cilia, proteases and DNA also displayed procoagulant activity in vitro. Injection of fish with the ciliates generated the massive release of neutrophils to the peritoneal cavity, with formation of large aggregates in those fish with live ciliates in the peritoneum. We observed, by SEM, numerous fibrin-like fibres in the peritoneal exudate, many of which were associated with peritoneal leukocytes and ciliates. Expression of the CD18/CD11b gene, an integrin associated with cell adhesion and the induction of fibrin formation, was upregulated in the peritoneal leukocytes. In conclusion, the findings of the present study show that P. dicentrarchi induces the formation of plasma clots and that the fish coagulation system may play an important role in immobilizing and killing this parasite., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Rearranged V(D)J genes coding for T cell receptor α and β chains are integrated into the germline genome of channel catfish. Previous analysis of expressed TCR Vβ2 repertoires demonstrated that channel catfish express multiple public clonotypes, which were shared among all the fish, following infection with a common protozoan parasite. In each case a single DNA sequence was predominately used to code for a public clonotype. We show here that the rearranged VDJ genes coding for these expressed public Vβ2 clonotypes can be amplified by PCR from germline DNA isolated from oocytes and erythrocytes. Sequencing of the Vβ2 PCR products confirmed that these expressed public Vβ2 clonotypes are integrated into the germline. Moreover, sequencing of PCR products confirmed that all five Vβ gene families and Vα1 have rearranged V(D)J genes with diverse CDR3 sequences integrated into the germline. Germline rearranged Vβ2 and Vβ4 genes retain the intron between the leader and Vβ sequence. This suggests that the germline rearranged TCR Vβ genes arose through VDJ rearrangement in T cells, and subsequently moved into the germline through DNA transposon mediated transposition. These results reveal a new dimension to the adaptive immune system of vertebrates, namely: the expression of evolutionarily conserved, rearranged V(D)J genes from the germline.

The protective immune response in zebrafish (Danio rerio) against the parasitic ciliate Ichthyophthirius multifiliis, targeting host skin, fins and gills, comprises an accelerated and manifold elevated immunoglobulin gene expression as well as a significantly elevated number of neutrophils at infected sites. Experimental fish were subjected to a primary I. multifiliis infection followed by a series of secondary exposures before they were challenged by a high dosage of infective theronts. Immunized fish responded immediately with a protective response suggesting existence of immunological memory whereas fish exposed to the parasite for the first time obtained a marked infection. The primary response to infection was dominated by expression of genes encoding acute phase reactants and inflammatory cytokines as well as recruitment of neutrophils at infected locations. Immunized fish showed a significantly upregulated immunoglobulin gene expression following challenge, which indicates existence of a secondary response effected by antibodies. Both responses induced a significantly elevated expression of the Th2 signature cytokine Il13. The increased presence of neutrophils in immunized fish suggests that innate cell mediated immunity supplements or influence the protective response against the parasite., Competing Interests: The authors have declared that no competing interests exist.

Ichthyophthirius multifiliis is a ciliated protozoan parasite recognized as one of the most pathogenic diseases of wild and cultured freshwater fish. Fish skin mucus plays a significant role against invading pathogens. However, the protein-based modulation against infection with I. multifiliis, of host fish at this barrier is unknown. Thus, we investigated the skin mucus proteome of common carp using a shotgun proteomic approach at days 1 and 9 after I. multifiliis exposure. We identified 25 differentially expressed proteins in infected carp skin mucus. Upregulated proteins were mainly involved in metabolism, whereas downregulated proteins were mainly structural. This is the first proteomic analysis of infected common carp skin mucus, and it provides novel information about proteome alteration caused by I. multifiliis. Furthermore, we identified novel proteins with yet unknown function in common carp following penetrating injuries such as olfactomedin 4, lumican, dermatopontin, papilin and I cytoskeletal 18. This analysis, therefore, represents a key for the search for potential biomarkers, which can help in a better understanding and monitoring of interactions between carp and I. multifiliis. This proteomic study not only provides information on the protein-level pathways involved in fish-ciliate interactions but also could represent a complementary system for studying tissue repair.

The objective of this study was to apply digital imaging to improve quantification of rumen protozoal biomass and distinguish treatment differences in cell motility and volume among ruminal protozoa. Observations of protozoa in rumen fluid treated with essential oils (CinnaGar, CIN; Provimi North America, Brookville, OH) or an ionophore (monensin, MON) indicated possible cell shrinkage. We hypothesized that MON would decrease protozoal motility and interact with CIN on cell volume. In addition, we hypothesized that analysis of still frames from video of swimming protozoa would improve volume prediction accuracy. Flocculated rumen fluid was incubated in batch culture dosed with N-free feed only (control), MON, CIN, or a combination of MON+CIN. Samples were taken at 0, 3, or 6 h post-treatment and wet-mounted on a microscope fitted with a high-definition camera. At 3 h post-inoculation, there was a treatment interaction for average speed such that CIN attenuated the effect of MON, with treatment means of 243, 138, 211, and 183 µm/s for control, MON, CIN, and MON+CIN, respectively. At 6 h post-inoculation, MON decreased average speed by 79.2 µm/s compared with the main effect mean without MON. We measured both minimum and maximum diameters (depth and width, respectively) perpendicular to the longitudinal axis of swimming protozoa, yielding a 3-dimensional estimate of protozoal volume. The ellipsoid formula (4/3)πabc, where a = 1/2 length, b = 1/2 width, and c = 1/2 depth, was compared with previously published volume estimations using genera-specific coefficients (genera-specific coefficient × length × width 2 ). Residuals (genera-specific coefficients - ellipsoid) were plotted against predicted (ellipsoid) and centered to the mean (X i -x¯) to evaluate both mean and slope biases. For Entodinium spp., Y = 0.248 (±0.037) (X i - 7.98 × 10 4 ) + 1.97 × 10 4 (±1.48 × 10 3 ); n = 100; r 2 [coefficient of determination (squared correlation coefficient)] = 0.31, with significant slope and mean biases. For family Isotrichidae, Y = -0.124 (±0.068) (X i - 2.54 × 10 6 ) - 1.21 × 10 4 (±4.86 × 10 4 ); n = 32; r 2 = 0.10, where slope tended to be different from zero but with no mean bias. For Epidinium spp., Y = 0.375 (±0.056) (X i - 2.45 × 10 5 ) + 6.65 × 10 4 (±0.28 × 10 4 ); n = 64; r 2 = 0.43, with both mean and slope biases. The present regression analyses demonstrate that the genera-specific coefficient-based method more likely overestimates volume for Entodinium and Epidinium than for the teardrop-shaped Isotrichidae. Based on simulations derived from previous literature reporting treatments that depress protozoal populations or among-animal changes in protozoal population structures, our proposed ellipsoid method offers potential to advance the prediction of treatment effects on protozoal volume and to shift focus from the number of cells present to the diversity, function, and biomass of protozoa under various treatment conditions., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Sixty suspected protozoan oocysts were demonstrated from 260 fecal samples collected from water buffaloes aged one month to seven years old with clinical signs of diarrhea in four provinces in the Philippines after conventional methods of isolation, sporulation, morphological characteristics and Kinyoun Acid Fast Staining techniques. The recovered protozoan oocysts were subjected to molecular analysis. Amplification of DNA extracted from recovered Eimeria oocysts using universal primers for the ITS-1 region of 18S rRNA revealed PCR products with 348 bp size demonstrated by samples collected from Benguet, La Union and Nueva Ecija provinces in the Philippines while DNA extracted from oocysts of suspected Cryptosporidium spp. samples that applied primers for the SSU of 18S rRNA registered PCR products but no genes were amplified from diarrheic water buffaloes from these provinces. Alignment of the DNA sequences of the suspected Eimeria and Cryptosporidium species revealed sequences for three isolates of Buxtonella sulcata with product lengths that varied from 235 to 252 bp. This is an initial observation on the involvement of B. sulcata in diarrhea condition of water buffaloes in the Philippines. Phylogenetic analysis of the three local isolates of B. sulcata revealed no similarity with other protozoan constructed according to Neighbor-Joining method.

Background: Cryptocaryon irritans is an obligate ectoparasitic ciliate pathogen of marine fishes. It can infect most marine teleosts and cause heavy economic losses in aquaculture. There is currently no effective method of controlling this disease, and little information is available regarding the genes involved in its development and virulence. We aimed to investigate the distinct features of the three major life-cycle stages of C. irritans in terms of gene transcription level, and identify candidate vaccines/drug targets. We established a reference transcriptome of C. irritans by RNA-seq., Methods: Three cDNA libraries using total poly(A) + mRNA isolated from trophonts, tomonts, and theronts was constructed and sequenced, respectively. Clean reads from the three stages were de novo assembled to generated unigene. Annotation of unigenes and transcriptomic comparison of three stages was performed., Results: Totals of 73.15, 62.23, and 109.57 million clean reads were generated from trophont, tomont, and theront libraries, respectively. After de novo assembly, 49,104 unigenes were obtained, including 9,253 unigenes with significant similarities to proteins from other ciliates. Transcriptomic comparisons revealed that 2,470 genes were differentially expressed among the three stages, including 2,011, 1,404, and 1,797 genes that were significantly differentially expressed in tomont/theront, tomont/trophont, and theront/trophont pairwise comparisons, respectively. Based on the results of hierarchical clustering, all differentially expressed genes (DEGs) were located in five major clusters. DEGs in clusters 1 and 2 were more highly expressed in tomonts than in other stages, DEGs in cluster 3 were dominant in the tomont and trophont stages, whereas clusters 4 and 5 included genes upregulated in the theront stage. In addition, Immobilization antigens (I-antigens) and proteases have long been considered major targets for vaccine development and potential drug targets in parasites, respectively. In the present study, nine putative I-antigens transcripts and 161 protease transcripts were found in the transcriptome of C. irritans., Conclusion: It was concluded that DEGs enriched in tomonts were involved in cell division, to increase the number of theronts and ensure parasite continuity. DEGs enriched in theronts were associated with response to stimuli, whereas genes enriched in trophonts were related to nutrient accumulation and cell growth. In addition, the I-antigen and protease transcripts in our transcriptome could contribute to the development of vaccines or targeted drugs. Together, the results of the present study provide novel insights into the physiological processes of a marine parasitic ciliate.

Trichodinid ciliophorans are opportunistic parasites of many species of fish, amphibians, and molluscs, but yet never reported in association with lesions in birds. Postmortem and histopathological evaluation of a commercial adult Toulouse gander and female goose, and a wild Mallard drake revealed the presence of severe pathological parasitic colonization of their reproductive tracts. Histopathological findings included moderate to severe granulocytic inflammation, acanthosis, accentuation of the rete pegs, and proliferative hyperplastic squamous metaplasia of the mucosa of the ejaculatory ducts and groove, sulcus spermaticus, glandular part of the phallus (cavum penis), and oviduct in association with large numbers of ciliated protozoa anchored to the tissues or free in the lumen. These protozoa had characteristic morphological features analogous to the family of Trichodinidae. The source of this parasitism could not be determined. To our knowledge, this is the first report of trichodinosis associated with pathology in birds.

Background: Cryptocaryon irritans, a species of obligatory ciliate ectoparasite, can infect various species of marine teleost fish. Cryptocaryon irritans that fall to the seabed or aquarium bottom in winter can form "dormant tomonts" and wake up when the temperature rises the next year. Abundant studies and analyses on the dormant tomonts were carried out at the transcriptome level, in order to investigate the molecular mechanism of C. irritans tomonts entering the dormant state under low-temperature conditions., Methods: The paired-end sequencing strategy was used to better assemble the entire transcriptome de novo. All clean sequencing reads from each of the three libraries (Group A: untreated blank control; Group B: treated for 24 h at 12 °C; and Group C: developed for 24 h at 25 °C) were respectively mapped back to the transcriptome assembly using the bioinformatics software., Results: In this study, 25,695,034, 21,944,467, and 28,722,875 paired-end clean reads were obtained respectively from the three cDNA libraries of the C. irritans tomont by Illumina paired-end sequencing technology. A total of 25,925 unique transcript fragments (unigenes) were assembled, with an average length of 839 bp. Differentially expressed genes (DEGs) were scrutinized; in Group B/A pairwise comparison, 343 genes presented differential expression, including 265 up-regulated genes and 78 down-regulated genes in Group B; in Group C/A pairwise comparison, there were 567 DEGs, including 548 up-regulated genes and 19 down-regulated genes in Group C; and in Group B/C pairwise comparison, 185 genes showed differential expression, including 145 up-regulated genes and 40 down-regulated genes in Group B., Conclusions: This is the first transcriptomic analytical study of the C. irritans tomonts under low temperature. It can be concluded that most of the genes required for its cell survival under low temperature, or for cell entry into a deeper dormancy state were discovered, and that they might be considered as candidate genes to develop the diagnostic and control measures for cryptocaryoniasis.

The aim of this study was to compare the parasite communities in two sympatric host populations, Trachelyopterus coriaceus andTrachelyopterus galeatus, which were caught in tributary of the Amazon River in Brazil. All the specimens of T. galeatusand T. coriaceus were infected by one or more parasites, such as Ichthyophthirius multifiliis, Tripartiella tetramerii, Trichodina nobilis, Cosmetocleithrum striatuli, Contracaecumsp., Cystidicoloides sp., Dadaytremoides parauchenipteri and Gorytocephalus spectabilis. Seven species were common to both host fish, and there were 1-5 parasite species per host. In both hosts, trichodinids were dominant. Aggregate dispersion of ectoparasites and endoparasites was observed, with greater aggregation among endoparasites. Only the ectoparasites species showed differences in intensity and/or abundance. However, the parasite communities of the two hosts were taxonomically similar (99%) and characterized by high prevalence and high abundance of ectoparasites, but with low diversity, prevalence and abundance of endoparasites. Trachelyopterus galeatus, the host with the larger body size, presented greater variation of Brillouin diversity and evenness, while T. coriaceus had higher Berger-Parker dominance values and total numbers of parasites. This first study on these parasites ofT. galeatus and T. coriaceus showed that the life mode, size of the hosts and the availability of infective forms of the parasites were the main factors that influenced the parasite communities structure.

Ichthyophthirius multifiliis, an external fish parasite, often causes significant economic damage to the aquaculture industry. Since the use of malachite green was banned, the search of alternative substance to control I. multifiliis infections becomes stringent. In present study, in vitro and in vivo anti-ich efficacies of isopsoralen and psoralidin, two active compounds isolated from methanol extract of Psoralea corylifolia by bioassay-guided fractionation based on the efficacy of anti-ich encysted tomonts, were evaluated. In vitro antiprotozoal efficacy of psoralidin is much better than that of isopsoralen. Psoralidin can kill all theronts at concentrations of 0.8 mg/L or more during 4 h exposure; and terminate reproduction of I. multifiliis post 6 h exposure of protomonts to 0.9 mg/L and encysted tomonts to 1.2 mg/L. In vivo trials showed that 5 h exposure of infected fish to 2.5 mg/L of psoralidin significantly reduced the number of theronts released from tomonts. Furthermore, we observed that a part of protomonts, collected from infected fish post treatment, presented characteristic morphological changes of apoptosis after staining with Annexin V-EGFP/propidium iodide, indicating the possible mechanism of psoralidin against I. multifiliis trophont in situ. On the basis of these results, psoralidin can be used as a potential lead compound for the development of commercial drug against I. multifiliis.

Neuregulin receptor degradation protein-1 (Nrdp1) was recently identified in humans as an important immune factor responding to the challenge of virus, LPS or cytokine. Its role in fish immune defense and whether it is involved in anti-parasite immunity have not been proven yet. In this report, the full-length cDNA sequence and genomic structure of Nrdp1 in the large yellow croaker Larimichthys crocea (LcNrdp1) were identified and characterized. The full-length cDNA of LcNrdp1 was 1248bp, including a 5' untranslated region (UTR) of 32bp, a 3' UTR of 259bp and an open reading frame (ORF) of 937bp, encoding a polypeptide of 318 amino acid residues. The full-length genomic DNA sequence of LcNrdp1 was composed of 2635 nucleotides, including four exons and three introns. The putative LcNrdp1 protein had no signal peptide sequence and contained a characteristic Nrdp1 consensus motif C3HC3D ring finger and a Coiled-coil domain. Phylogenetic analysis showed that Nrdp1 in fish was closer with that in other vertebrates (79%-90% amino acid identity) than in invertebrates and bacteria (27%-65%). In fishes, Nrdp1 in large yellow croaker was closer with that in Takifugu rubripes. The expression profile showed that LcNrdp1 was constitutively expressed in all tested tissues, especially highly expressed in brain, muscle and kidney. Post-infection (PI) with Cryptocaryon irritans, an increased expression of LcNrdp1 was induced in infection sites (skin and gill), whereas in immune organs, the expression of LcNrdp1 was up-regulated in spleen (except the 1st d and 10th d PI) but suppressed in head kidney. These results suggested that LcNrdp1 might play an important immune role in the finfish L. crocea in the defense against the parasite C. irritans., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Buxtonellosis is a disease caused by the ciliated protozoan Buxtonella sulcata (Jameson, 1926). B. sulcata is a common protozoan of ruminants and may result in subclinical infection or clinical disease including diarrhea. This study examined the prevalence of B. sulcata in cattle from Sanadaj province, Iran. Faecal samples were collected from cattle in the province from May 2013 to June 2014. A total of 217 cattle were selected randomly according to the age, sex, health, management system and season. In total, 99 cattle (45.63%) were found to be infected with B. sulcata. In adults, prevalence of B. sulcata infection (51.64%) was higher than calves (28.58%) and young cattle (40.55%). The prevalence of B. sulcata infection was also found to be higher in female (47.32%) than male (38.46) cattle. B. sulcata infection was significantly (p<0.05) higher in poor health cattle (body condition and weight) (79.54%) than healthy cattle (24.47%). This study demonstrates that cattle are highly susceptible to B. sulcata infection under a variety of housing and environmental conditions in this region of Iran. The study also demonstrates a significant effect of infection on cattle health.

Objective: The aim of this study was to investigate the morphological features of 7 endocommensal ciliates, B. (s. str.) postciliata, B. (s. str.) piriformis, B. (s. str.) nana, B. (C.) elongata, B. (C.) triangularis, B. (F.) dolichosoma, and B. (F.) inflata belonging to the genus Bundleia, which exist in the hindgut of 15 Rahvan horses and 15 racing horses living in the vicinity of Izmir and to investigate the ontogenesis in the Bundleia genus., Methods: Specimens were obtained from 30 horses and were fixed with 10% formalin. The morphological features of the species were investigated with the pyridinated silver carbonate impregnation and silver nitrate impregnation techniques at the level of light microscopy., Results: The morphological features of Bundleia spp. were found to be similar to the original descriptions, and it was detected that ontogenesis in the Bundleia genus exhibited suitability with other ciliate species in the Entodiniomorphida order., Conclusion: We conclude that the geographical variation and feeding habits of host animals are important for evaluating the biometric data of species.

Extensive DNA elimination occurs as part of macronuclear differentiation during Tetrahymena sexual reproduction. The identification of sequences to excise is guided by a specialized RNA interference (RNAi) machinery that targets the methylation of histone H3 lysine 9 (K9) and K27 on chromatin associated with these internal eliminated sequences (IESs). This modified chromatin is reorganized into heterochromatic subnuclear foci, which is a hallmark of their subsequent elimination. Here, we demonstrate that Lia4, a chromoshadow domain-containing protein, is an essential component in this DNA elimination pathway. LIA4 knockout (ΔLIA4) lines fail to excise IESs from their developing somatic genome and arrest at a late stage of conjugation. Lia4 acts after RNAi-guided heterochromatin formation, as both H3K9 and H3K27 methylation are established. Nevertheless, without LIA4, these cells fail to form the heterochromatic foci associated with DNA rearrangement, and Lia4 accumulates in the foci, indicating that Lia4 plays a key role in their structure. These data indicate a critical role for Lia4 in organizing the nucleus during Tetrahymena macronuclear differentiation., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Mycobacterial infections in laboratory zebrafish Danio rerio are common and widespread in research colonies. Mycobacteria within free-living amoebae have been shown to be transmission vectors for mycobacteriosis. Paramecium caudatum are commonly used as a first food for zebrafish, and we investigated this ciliate's potential to serve as a vector of Mycobacterium marinum and M. chelonae. The ability of live P. caudatum to transmit these mycobacteria to larval, juvenile and adult zebrafish was evaluated. Infections were defined by histologic observation of granulomas containing acid-fast bacteria in extraintestinal locations. In both experiments, fish fed paramecia containing mycobacteria became infected at a higher incidence than controls. Larvae (exposed at 4 d post hatch) fed paramecia with M. marinum exhibited an incidence of 30% (24/80) and juveniles (exposed at 21 d post hatch) showed 31% incidence (14/45). Adult fish fed a gelatin food matrix containing mycobacteria within paramecia or mycobacteria alone for 2 wk resulted in infections when examined 8 wk after exposure as follows: M. marinum OSU 214 47% (21/45), M. marinum CH 47% (9/19), and M. chelonae 38% (5/13). In contrast, fish feed mycobacteria alone in this diet did not become infected, except for 2 fish (5%) in the M. marinum OSU 214 low-dose group. These results demonstrate that P. caudatum can act as a vector for mycobacteria. This provides a useful animal model for evaluation of natural mycobacterial infections and demonstrates the possibility of mycobacterial transmission in zebrafish facilities via contaminated paramecia cultures.

A postmortem evaluation of a domestically bred, adult, female Xenopus laevis revealed the presence of a urinary bladder protozoan consistent with Trichodina xenopodus. T. xenopodus is considered an incidental finding, as its presence in the urinary bladder in frogs has not been correlated with disease or with urinary bladder epithelial lesions. Trichodina spp. are ciliated protozoa known to colonize many species of amphibians and fish. These protozoa frequently inhabit the skin and gills, but may also be present in the urinary bladder of infected animals. Their presence on the skin and gills in low numbers is not related to disease; however, large numbers may indicate poor water quality and overcrowding.

Background: Parasite peptidases have been actively studied as vaccine candidates or drug targets for prevention or treatment of parasitic diseases because of their important roles for survival and/or invasion in the host. Like other parasites, the facultative histophagous ciliate Miamiensis avidus would possess peptidases that are closely associated with the invasion into the host tissue and survival in the host., Results: The 17 genes encoding peptidases, including seven cathepsin-like cysteine peptidases, four serine carboxypeptidases, a eukaryotic aspartyl protease family protein, an ATP-dependent metalloprotease FtsH family protein, three leishmanolysin family proteins and a peptidase family M49 protein were identified from a Miamiensis avidus cDNA library by BLAST X search. Expression of genes encoding two cysteine peptidases, three leishmanolysin-like peptidases and a peptidase family M49 protein was up-regulated in the cell-fed ciliates compared to the starved ciliates. Especially, one cysteine peptidase (MaPro 4) and one leishmanolysin-like peptidase (MaPro 14) were transcribed more than 100-folds in the cell-fed ciliates., Conclusions: The genetic information and transcriptional characteristics of the peptidases in the present results would be helpful to elucidate the role of peptidases in the invasion of scuticociliates into their hosts.

In the present work Trichodina reticulata and T. nobilis (Ciliophora: Trichodinidae) are morphologically characterised from ornamental freshwater fish culture in the State of Santa Catarina, Brazil. The prevalence of infection and a list of comparative measurements are discussed. We examined "southern platyfish" Xiphophorus maculatus (n = 35), "goldfish" Carassius auratus (n = 31), "guppy" Poecilia reticulata (n = 20), "sailfin molly" Poecilia latipinna (n = 6), "beta" Betta splendens (n = 2) and "spotted headstander" Chilodus punctatus (n = 1). After being anesthetised in a benzocaine solution, fishes were examined for parasitological evaluation. A total of 51.57% fishes were parasitised by Trichodina spp. Carassius auratus was the most parasitised species, followed by X. maculatus and P. reticulata. Beta splendens, C. punctatus and P. latipinna were not parasitised by any trichodinid species. Two species of Trichodina were collected from the skin of fish: T. nobilis was found in C. auratus, P. reticulata and X. maculatus and T. reticulata was only observed in C. auratus. The importance of adequate handling in ornamental fish culture are also discussed.

Objective: The aim of this study was to determine the cytological features of 2 endocommensal ciliates, P. colpoidea and P. minuta belonging to genus Paraisotricha found in the hindgut of 15 Turk rahvan and 15 English and Arabic horses from Izmir, compare the morphological characters of species with their original descriptions and previous reports and discuss the similarities and differences., Methods: The cytological features of two endocommensal ciliates were investigated with the pyridinated silver carbonate impregnation and silver nitrate impregnation techniques at the level of light microscopy., Results: Specimens from our horses were found to be similar to the original descriptions on the basis of cytological features, morphological characters and biometric data., Conclusion: We have concluded that the geographical variation and feeding habits of the host animals are important for evaluating the biometric data of species.

Objective: The aim of this study was to determine the cytological features of the rumen ciliates Entodinium palmare and E. okoppensis living in the rumen of domestic cattle in the vicinity of Kastamonu, compare the morphological characters of species with their original descriptions and discuss the similarities and differences., Methods: The specimens were obtained from 24 cattle, fixed with 10% formalin and stained with methylgreen formalin saline (MFS) solution., Results: Specimens were measured from cattle in our country, and were found to be similar to the original description on the basis of morphological characteristics and biometric data., Conclusion: With this investigation, E. palmare and E. okoppensis were firstly detected from our cattle and were secondly detected from cattle throughout the world. E. palmare and E. okoppensis have low frequency appearance and percentage composition in our cattle. This study concluded that the feeding habits and feeding frequencies of cattle, the physiological conditions of rumen or competition between species can be important reasons for this situation.

Archey's frogs (Leiopelma archeyi) are first on the list of evolutionarily distinct and globally endangered (EDGE) amphibians. Captive breeding is an important strategy for protection of the species, but programs are hampered by a lack of information on diseases present in wild and captive populations. Two novel nematodes (Koerneria sp. and Rhabditis sp.) were found separately in four captive Archey's frogs showing clinical signs of hemorrhagic purulent nasal discharge and weight loss. One of these frogs also had a novel protozoal infection (Tetrahymena) in the nasal cavity. Koerneria, Rhabditis, and Tetrahymena have not previously been reported in amphibians in New Zealand. One frog was treated successfully with oral moxidectin at 0.4 mg/kg for the nematode infection and topical metronidazole at 10 mg/kg for the protozoal infection. The clinical signs abated only after both infections were cleared. The second frog died before treatment could be established. The third and fourth frogs were found dead.

Ichthyophthiriasis, caused by Ichthyophthirius multifiliis, is an economically important worldwide parasitic disease that infects all freshwater fish. Since the banning of malachite green for use in food fish, there has been a great need for alternative therapeutants. The goal of this study was to investigate the efficacy of quinine against I. multifiliis. Parasite developmental stages from our laboratory-established life cycle in rainbow trout Oncorhynchus mykiss were exposed to quinine in vitro, and a dual fluorescent staining technique was used to allow a clear distinction between viable and damaged parasites. Furthermore, the effect of quinine was assessed in vivo by oral administration and intraperitoneal injections in common carp Cyprinus carpio. The results of the in vitro experiments proved quinine to be effective against the parasite. Quinine injected at a dosage of 60 mg kg(-1) body weight resulted in a significantly lower number of trophonts. In contrast, in-feed trials did not show a significant reduction of trophonts after treatment commencing 1 d after infection with concentrations of up to 20 g quinine kg(-1) feed for 3 d. After a 14-d treatment at concentrations of up to 10 g quinine kg(-1) feed prior to theront exposure, there was also no significant difference in parasite numbers between treated and control groups. The results of oral versus parenteral application of quinine indicate that the substance is not completely absorbed from the intestinal tract of common carp. However, medicated feed containing higher concentrations of quinine was less readily accepted by the fish, presumably due to the bitter taste.

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.

Ciliated protists were isolated from the ovarian fluid of apparently healthy adult rainbow trout (Oncorhynchus mykiss) maintained in freshwater. The organism was identified as Pseudocohnilembus persalinus based on morphometric and morphological analysis of silver-stained specimens obtained from culture and on analysis of ribosomal RNA gene sequences. The cytochrome c oxidase subunit 1 gene sequence of this organism also was characterized. This ciliate has been reported previously as free living only in saline environments and as an endosymbiont in a marine teleost, the olive flounder (Paralichthys olivaceus). A cyst-like stage may have facilitated the novel occurrence of this organism as an endosymbiont in rainbow trout.

We examined stool samples for trophozoites of the entodiniomorphid ciliate Troglodytella abrassarti Brumpt and Joyeux, 1912, from a habituated group of chimpanzees (Pan troglodytes schweinfurthii) at Mahale Mountains National Park in western Tanzania. In our study, fresh fecal samples from identified individuals were collected immediately after defecation and fixed in 10% formalin. In total, 52 samples from 38 chimpanzees (61% of 62 chimpanzees in the group) were examined using a direct smear method. A stool sample from an individual collection date from an individual chimpanzee was examined up to 3 separate times before it was called negative. Forty-eight (92%) of the 52 samples were positive, and stools from 37 (97%) of the 38 chimpanzees were positive for trophozoites of T. abrassarti. The high prevalence of T. abrassarti in these chimpanzees is consistent with previous reports of this organism in chimpanzees.

Hemiprorodon gymnoposthium was investigated in the intestine of horses, found in Cyprus. It was found in four of fifth horses examined and thus had a frequency of appearence of 80%. This study reports for the first time the presence of Hemiprorodon gymnoposthium in the intestine of Equids in Cyprus. In addition, it is the second report from the world. Specimens were found to be similar to the original description on the basis of morphological characters and biometric data.

Ciliates associated with fish mortalities in a New Zealand hatchery were identified by DNA sequencing of the small subunit ribosomal RNA gene (SSU rRNA). Tissue samples were taken from lesions and gill tissues on freshly dead juvenile groper, brain tissue from adult kingfish, and from ciliate cultures and rotifers derived from fish mortality events between January 2007 and March 2009. Different mortality events were characterized by either of 2 ciliate species, Uronema marinum and Miamiensis avidus. A third ciliate, Mesanophrys carcini, was identified in rotifers used as food for fish larvae. Sequencing part of the SSU rRNA provided a rapid tool for the identification and monitoring of scuticociliates in the hatchery and allowed the first identification of these species in farmed fish in New Zealand.

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.