In vitro efficacy of glutaraldehyde-crosslinked chitosan microspheres against the fish-pathogenic ciliate Philasterides dicentrarchi

Philasterides dicentrarchi is a protozoan ciliate which causes significant economic losses in fish aquaculture. This study investigated the effects of chitosan microspheres cross linked with glutaraldehyde and containing beta-cyclodextrin (betaCD) on the survival of this parasite in 7 d cultures. When used alone in assays, neither chitosan nor betaCD showed any activity, whereas free glutaraldehyde was strongly toxic to the parasite. Microspheres were likewise strongly toxic, at total glutaraldehyde concentrations much lower than with free glutaraldehyde: near-100% ciliate death was obtained (1) with 50 microg ml(-1) of microspheres prepared with 5% glutaraldehyde and no betaCD, or (2) with 10 microg ml(-1) of microspheres prepared with 0.15% glutaraldehyde and 0.1% betaCD. This suggests that the main active component is glutaraldehyde, but that the presence of small amounts of betaCD enhances efficacy. This high efficacy, together with the low toxicity to fish and rapid biodegradability of the individual components, suggest that these microspheres may be an attractive alternative to the formaldehyde baths traditionally used for the control of this parasite.     

Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW Spain)

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.     

CpG-ODN increases resistance of olive flounder (Paralichthys olivaceus) against Philasterides dicentrarchi (Ciliophora: Scuticociliatia) infection

Unmethylated cytosine-phosphate-guanine (CpG) dinucleotides flanked by specific bases in bacterial DNA induce a favorable immune response by acting as danger signals to the host. Synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODNs) also act like the unmethylated CpG oligonucleotides in bacterial DNA. In the present study, we investigated the effects of synthetic CpG-ODN on the protection of olive flounder (Paralichthys olivaceus) against infection by Philasterides dicentrarchi, a pathogen of scuticociliatosis, through two consecutive experiments (trial I and II). Fish were intraperitoneally (i.p.) injected with CpG-ODN 1668 or GpC-ODN 1720 at different doses (3 microg in trial I and 10 microg in trial II), and after one week the fish were i.p. challenged with P. dicentrarchi. In both trial I and II, fish injected with CpG-ODN 1668 showed significantly higher serum scuticocidal activity than fish injected with PBS alone, while the scuticocidal activity disappeared by heat-inactivation. This result suggests that CpG-ODN might activate an alternative pathway of complement of olive flounder, and complement-mediated killing might be an important innate immune factor in the resistance against P. dicentrarchi infection. Although the cumulative mortality was largely different between trials I and II, the relative survival rate of fish injected with a high dose of CpG-ODN 1668 was considerably higher than that of fish injected with a low dose of this ODN, while the relative survival rate was not different between fish injected with the high dose and low dose of GpC-ODN 1720. The results of the present study suggest that CpG-ODNs may be used as potential immunostimulants to lessen cultured fish loss caused by scuticociliates.     

Miamiensis avidus (Ciliophora: Scuticociliatida) causes systemic infection of olive flounder Paralichthys olivaceus and is a senior synonym of Philasterides dicentrarchi

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

Antiprotozoals effective in vitro against the scuticociliate fish pathogen Philasterides dicentrarchi

The histophagous ciliate Philasterides dicentrarchi causes fatal scuticociliatosis in farmed turbot Scophthalmus maximus and sea bass Dicentrarchus labrax. The present study screened 52 candidate antiprotozoals for activity against this pathogen in vitro. Of these compounds, 14 were effective (i.e. killed all ciliates within a 24 h assay period). In descending order of efficacy (minimum lethal concentration 100 to 0.8 ppm), these were niclosamide, oxyclozanide, bithionol sulfoxide, toltrazuril, N-(2'-hydroxy-5'-chloro-benzoyl) 2-chloro-4-nitroaniline, furaltadone, doxycycline hyclate, formalin, albendazole, carnidazole, pyrimethamine, quinacrine hydrochloride and quinine sulfate. Administration in filtered seawater rather than phosphate-buffered saline inactivated doxycycline hyclate and albendazole, and markedly reduced that of bithionol sulfoxide and toltrazuril, suggesting that these compounds may not be effective in bath administration. In view of these findings, we discuss the potential utility of chemotherapy as a strategy for the control of scuticociliatosis in farmed turbot and sea bass.     

In vitro effects of the polyphenols resveratrol, mangiferin and (-)-epigallocatechin-3-gallate on the scuticociliate fish pathogen Philasterides dicentrarchi

This study investigated the in vitro effects of the polyphenols resveratrol, mangiferin and (-)-epigallocatechin-3-gallate (EGCG) on the histiophagous ciliate Philasterides dicentrarchi, which causes fatal scuticociliatosis in farmed turbot Scophthalmus maximus L. Of the 3 polyphenols, resveratrol showed strongest antiprotozoal activity, reducing ciliate density after 1 wk culture by, on average, 91% at 50 microM, and 96% at 500 microM. EGCG reduced ciliate density by, on average, 93% at 500 microM, with no significant effect at 50 microM. Mangiferin reduced ciliate density by, on average, 56% at 500 microM, again with no significant effect at 50 microM. In view of these findings, we discuss the potential utility of chemotherapy with polyphenols as a strategy for the control of scuticociliatosis in farmed turbot.     

海洋纤毛虫--冠帆口虫的口器发生

对海洋盾纤目纤毛虫,冠帆口虫(Pleuronema coronatum)无性分裂期间口器的发生过程做了跟踪观察。结果显示其口器发生与已知的同属种普氏帆口虫具相似的过程。其口器发生及演化的基本模式可简述如下：老口区的三片小膜经历一个分化和移行过程;前仔虫的口侧膜来源于老口区的口侧膜;后仔虫的小膜1～3与口侧膜均来源于老的口侧膜;在口器发生过程中,前、后仔虫口侧膜的后端均形成一个相应的盾片区,其内的毛基粒在进入营养期之前被完全吸收[动物学报49(6)：829～834,2003]。     

Uncovering QTL for resistance and survival time to Philasterides dicentrarchi in turbot (Scophthalmus maximus)

Disease resistance‐related traits have received increasing importance in aquaculture breeding programs worldwide. Currently, genomic information offers new possibilities in breeding to address the improvement of this kind of traits. The turbot is one of the most promising European aquaculture species, and Philasterides dicentrarchi is a scuticociliate parasite causing fatal disease in farmed turbot. An appealing approach to fight against disease is to achieve a more robust broodstock, which could prevent or diminish the devastating effects of scuticociliatosis on farmed individuals. In the present study, a genome scan for quantitative trait loci (QTL) affecting resistance and survival time to P. dicentrarchi in four turbot families was carried out. The objectives were to identify QTL using different statistical approaches [linear regression (LR) and maximum likelihood (ML)] and to locate significantly associated markers for their application in genetic breeding strategies. Several genomic regions controlling resistance and survival time to P. dicentrarchi were detected. When analyzing each family separately, significant QTL for resistance were identified by the LR method in two linkage groups (LG1 and LG9) and for survival time in LG1, while the ML methodology identified QTL for resistance in LG9 and LG23 and for survival time in LG6 and LG23. The analysis of the total data set identified an additional significant QTL for resistance and survival time in LG3 with the LR method. Significant association between disease resistance‐related traits and genotypes was detected for several markers, a single one explaining up to 22% of the phenotypic variance. Obtained results will be essential to identify candidate genes for resistance and to apply them in marker‐assisted selection programs to improve turbot production.     

First isolation of Pseudocohnilembus persalinus (Ciliophora: Scuticociliatida) from freshwater-reared rainbow trout, Oncorhynchus mykiss

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.     

Growth rate variability in geographically diverse clones of Uronema (Ciliophora: Scuticociliatida)

Abstract Growth rates in 15 clones from the bacterivorous ciliate Uronema and one from Parauronema acutum have been compared under similar culture conditions. Five species were identified on a morphological basis: U. marinum, U. nigricans, U. elegans, P. acutum , and an undescribed form designated here as SWi1 . Intra- and inter-specific distinct growth characteristics were observed. Net cellular production tended to decrease with lower growth rate in different species or morphotypes but increased in those clones belonging to the same species. Growth rate and cell volume were not significatly correlated although mean growth rate increased with mean cell volume. Growth rate was however significantly correlated to the relative variation in cell volume between different growth phases. Estimation of growth rates using different models indicated that the experimental growth rates observed were not significatly different from those expected for the cell volumes. These results indicate how effects of habitat or geographical variability, in this cosmopolitan genus, may account for trophodynamic variation of morphologically similar species.     

Growth, Fine Structure and Cyst Formation of a Microbial Mat Ciliate: Pseudocohnilembus pusillus (Ciliophora, Scuticociliatida)

ABSTRACT Pseudocohnilembus species exhibit a polymorphic life cycle consisting of trophic cells, theronts, and cysts. Pseudocohnilembus pusillus isolated from the intertidal mats of Laguna Figueroa, Baja California Norte, Mexico, forms desiccation-resistant cysts in response to bacterial food depletion. This isolate is a euryhaline organism, able to grow at salinities from freshwater to 96 ppt total salinity and from pH 6–9. Electron micrographs show that oral and somatic cilia and kinetids are retained inside young cysts. Cyst walls are composed of a single layer (0.1 μm) of granular material. Under all conditions, as bacterial food was depleted, P. pusillus cells formed cysts, except for a small proportion (1–5%) that continued to swim. Changes in pH and salinity did not directly induce cyst formation. Salinity did greatly affect growth rate. Doubling times were shortest at 16 ppt salinity and at pH 7–8. Cyst formation occurred later in the growth cycle as more food bacteria were added. Additionally, ciliates grown in small culture volumes (10 ml) formed cysts sooner than cultures in larger volumes (100 ml), suggesting that crowding may influence cyst formation. Mature cysts may survive desiccation at least as long as one month at 37° C and for as long as one year at 20 ± 3° C. Although trophic cells did not survive desiccation or anoxia, encysted ciliates from liquid stationary phase cultures kept in anoxic seawater for one month excysted into swimming cells within 2.5 h after exposure to air. The adaptability of P. pusillus to extremes of salinity, pH, desiccation, and anoxia permits survival in its environmentally variable, microbial mat habitat.     

Occurrence of scuticociliatosis in olive flounder Paralichthys olivaceus by Phiasterides dicentrarchi (Ciliophora: scuticociliatida)

In the course of identifying scuticociliates recently obtained from systemically infected olive flounder Paralichthys olivaceus in Korea, we found a scuticociliate species whose small subunit ribosomal RNA (SS rRNA) gene was not amplified by species-specific primers previously designed for Uronema marinum and Pseudocohnilembus persalinus. By studying morphological characteristics of wet-mounted and stained specimens, we identified the species as Philasterides dicentrarchi, which has been reported to cause systemic infection in the European sea bass Dicentrarchus labrax and turbot Scophthalmus maximus. In this study, we compared morphological characteristics of our specimens with previously reported Philasterides species, including P. dicentrarchi, and sequenced the SS rRNA gene in order to design P. dicentrarchi specific primers. This is the first report on scuticociliatosis caused by P. dicentrarchi from marine fish in Asia.     

Description of Uronema marinum (Ciliophora,Scuticociliatida) from the Antarctica and observations on the nuclear events in conjugation

The morphology is described of a scuticociliate, isolated from a sediment from Terra Nova Bay, Ross Sea, in the Antarctica. The studies, using light microscopy of silver impregnated specimens and by scanning electron microscopy, permit an assignment to the species, Uronema marinum. The main steps of micronuclear events during conjugation, that appear to be similar to the general plan of Tetrahymena and Paramecium aurelia complex, are shown.     

Effects of bile acids on proliferation and production of proteinase activity of Uronema marinum (Ciliophora: Scuticociliatida)

Little is known about the effects of bile acids in relation to infectivity on the biological characteristics of Uronema marinum, a serious opportunistic parasite of farmed olive flounder Paralichthys olivaceus. In this study, we examined the effects of bile acids on the proliferation of U. marinum and on proteinase production in vitro. Proliferation of U, marinum was significantly enhanced by lithocholic acid (LCA) at 30 and 60 pmol, and by chenodeoxycholic acid (CDCA) at 0.06 pmol. In contrast, a significant decrease in proliferation was observed with cholic acid (CA) at 30 and 60 micromol, and with deoxycholic acid (DCA) at all amounts used. Proteinase production from live U. marinum was significantly increased by LCA, whereas CA significantly decreased proteinase production. CDCA and DCA had no effect on proteinase production. Although the types and concentrations of bile acids in the faeces of olive flounder are not known, the present results suggest that bile acids in the culturing water might influence the proliferation and production of proteinases in U. marinum, resulting in an increased possibility of scuticociliatosis in olive flounder farms.     

Gene Expression Profiles of Spleen, Liver, and Head Kidney in Turbot (Scophthalmus maximus) Along the Infection Process with Philasterides dicentrarchi Using an Immune-Enriched Oligo-Microarray

We evaluated the expression profiles of turbot in spleen, liver, and head kidney across five temporal points of the Philasterides dicentrarchi infection process using an 8x15K Agilent oligo-microarray. The microarray included 2,176 different fivefold replicated gene probes designed from a turbot 3' sequenced EST database. We were able to identify 221 differentially expressed (DE) genes (8.1% of the whole microarray), 113 in spleen, 83 in liver, and 90 in head kidney, in at least 1 of the 5 temporal points sampled for each organ. Most of these genes could be annotated (83.0%) and functionally categorized using GO terms (69.1%) after the additional sequencing of DE genes from the 5' end. Many DE genes were related to innate and acquired immune functions. A high proportion of DE genes were organ-specific (70.6%), although their associated GO functions showed notable similarities in the three organs. The most striking difference in functional distribution was observed between the up- and downregulated gene groups. Upregulated genes were mostly associated to immune functions, while downregulated ones mainly involved metabolism-related genes. Genetic response appeared clustered in a few groups of genes with similar expression profiles along the temporal series. The information obtained will aid to understand the turbot immune response and will specifically be valuable to develop strategies of defense to P. dicentrarchi to achieve more resistant broodstocks for turbot industry.     

In vitro and in silico antifungal efficacy of nitrogen-doped carbon nanohorn (NCNH) against Rhizoctonia solani

We have investigated in vitro antifungal efficiency of nitrogen-doped carbon nanohorn (NCNH) against Rhizoctonia solani (R. solani) plant pathogenic fungi. NCNH with size of 50–60 nm and concentrations of 10, 50, 100, and 150 μg mL^?1 were used. The results showed that growth of fungi in the presence of NCNH was significantly (p > .05) inhibited at 150 μg mL^?1 (85.13 ± .97) after 72 h. The results were validated through computational approaches. Molecular docking analysis of NCNH with endochitinase protein of R. solani was performed to validate the potential of antifungal activity of NCNH. Docking results showed different conformations of interaction of NCNH with endochitinase enzyme. The conformation with least binding energy ?13.54 kcal/mol was considered further. It is likely that NCNH interacts with the pathogens by mechanically wrapping, which may be one of the major toxicity actions of NCNH against R. solani. The analysis showed that NCNH might interwinds to endochitinase of R. solani leading to the deactivation of the enzyme. To best of our knowledge, this is the first report of antifungal efficacy of NCNH against R. solani and provides useful information about the application of NCNH in resisting crop disease.     

Chemotactic responses of the fish-parasitic scuticociliate Philasterides dicentrarchi to blood and blood components of the turbot Scophthalmus maximus, evaluated using a new microplate multiassay

This study describes a new capillary-type microplate multiassay for characterization of protozoal chemotactic responses, allowing up to 32 assays to be run simultaneously. We used the new multiassay to evaluate the chemoattractant activity of turbot blood components and turbot cells for the facultative parasite Philasterides dicentrarchi, which is responsible for significant losses in turbot farming. Preliminary tests indicated that the assay requires 3-4 h for detection of chemoattractant activity, that it can be performed effectively using the ciliate axenic culture medium, and that it distinguishes clearly between different concentrations of chemoattractant. Application of the assay indicated that whole blood and serum from normal turbot, and especially infected turbot, have strong chemoattractant activity for P. dicentrarchi trophozoites, whereas neither turbot blood cells nor other turbot cells nor bacteria were significant chemoattractants. These results raise the possibility that turbot serum components are involved in host detection and host invasion by P. dicentrarchi, in line with previous findings indicating that turbot with skin lesions show increased susceptibility to P. dicentrarchi infection.     

In vitro and in vivo efficacy of Monepantel (AAD 1566) against laboratory models of human intestinal nematode infections

Background

Few effective drugs are available for soil-transmitted helminthiases and drug resistance is of concern. In the present work, we tested the efficacy of the veterinary drug monepantel, a potential drug development candidate compared to standard drugs in vitro and in parasite-rodent models of relevance to human soil-transmitted helminthiases.

Methodology

A motility assay was used to assess the efficacy of monepantel, albendazole, levamisole, and pyrantel pamoate in vitro on third-stage larvae (L3) and adult worms of Ancylostoma ceylanicum, Necator americanus and Trichuris muris. Ancylostoma ceylanicum- or N. americanus-infected hamsters, T. muris- or Ascaris suum-infected mice, and Strongyloides ratti-infected rats were treated with single oral doses of monepantel or with one of the reference drugs.

Principal Findings

Monepantel showed excellent activity on A. ceylanicum adults (IC₅₀ = 1.7 µg/ml), a moderate effect on T. muris L3 (IC₅₀ = 78.7 µg/ml), whereas no effect was observed on A. ceylanicum L3, T. muris adults, and both stages of N. americanus. Of the standard drugs, levamisole showed the highest potency in vitro (IC₅₀ = 1.6 and 33.1 µg/ml on A. ceylanicum and T. muris L3, respectively). Complete elimination of worms was observed with monepantel (10 mg/kg) and albendazole (2.5 mg/kg) in A. ceylanicum-infected hamsters. In the N. americanus hamster model single 10 mg/kg oral doses of monepantel and albendazole resulted in worm burden reductions of 58.3% and 100%, respectively. Trichuris muris, S. ratti and A. suum were not affected by treatment with monepantel in vivo (following doses of 600 mg/kg, 32 mg/kg and 600 mg/kg, respectively). In contrast, worm burden reductions of 95.9% and 76.6% were observed following treatment of T. muris- and A. suum infected mice with levamisole (200 mg/kg) and albendazole (600 mg/kg), respectively.

Conclusions/Significance

Monepantel reveals low or no activities against N. americanus, T. muris, S. ratti and A. suum in vivo, hence does not qualify as drug development candidate for human soil-transmitted helminthiases.