Protocol for cryopreservation of the turbot parasite Philasterides dicentrarchi (Ciliophora, Scuticociliatia).

Philasterides dicentrarchi is a free-living marine ciliate that can become an endoparasite that causes a severe disease called scuticociliatosis in cultured fish. Long-term maintenance of this scuticociliate in the laboratory is currently only possible by subculture, with periodic passage in fish to maintain the virulence of the isolates. In this study, we developed and optimized a cryopreservation protocol similar to that used for the long-term storage of scuticociliates of the genus Miamiensis. The cryogenic medium comprised ATCC medium 1651 and a combination of 11% dimethylsulfoxide and 5% glycerol. We have verified that the most important factor ensuring the efficiency of the cryopreservation procedure is the growth phase of the culture, and that ciliates should be cryopreserved at the stationary phase (around the sixth day of culture). The cryopreservation protocol described here can be used for all strains of P. dicentrarchi as well as commercial strains of Miamiensis and enables the virulence of the strains to be maintained. Finally, this cryopreservation protocol has been shown to be more effective than others routinely applied to scuticociliates, yielding a higher survival rate with a lower initial concentration of ciliates. The results obtained indicate that the cropreservation protocol enables the long-term storage of scuticociliate parasites while maintaining the virulence of the isolates. The protocol is therefore suitable for use in vaccine production and related studies.

Combined antiparasitic and anti-inflammatory effects of the natural polyphenol curcumin on turbot scuticociliatosis.

The histiophagous scuticociliate Philasterides dicentrarchi is the aetiological agent of scuticociliatosis, a parasitic disease of farmed turbot. Curcumin, a polyphenol from Curcuma longa (turmeric), is known to have antioxidant and anti-inflammatory properties. We investigated the in vitro effects of curcumin on the growth of P. dicentrarchi and on the production of pro-inflammatory cytokines in turbot leucocytes activated by parasite cysteine proteases. At 100 µm, curcumin had a cytotoxic effect and completely inhibited the growth of the parasite. At 50 µm, curcumin inhibited the protease activity of the parasite and expression of genes encoding two virulence-associated proteases: leishmanolysin-like peptidase and cathepsin L-like. At concentrations between 25 and 50 µm, curcumin inhibited the expression of S-adenosyl-L-homocysteine hydrolase, an enzyme involved in the biosynthesis of the amino acids methionine and cysteine. At 100 µm, curcumin inhibited the expression of the cytokines tumour necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1ß) produced in turbot leucocytes activated by parasite proteases. Results show that curcumin has a dual effect on scuticociliatosis: an antiparasitic effect on the catabolism and anabolism of ciliate proteins, and an anti-inflammatory effect that inhibits the production of proinflammatory cytokines in the host. The present findings suggest the potential usefulness of this polyphenol in treating scuticociliatosis.

Disentangling the determinants of symbiotic species richness in native and invasive gammarids (Crustacea, Amphipoda) of the Baltic region.

Dispersal of alien species is a global problem threatening native biodiversity. Co-introduction of non-native parasites and pathogens adds to the severity of this threat, but this indirect impact has received less attention. To shed light on the key factors determining the richness of microorganisms in native and invasive host species, we compared symbiotic (parasitic and epibiotic) communities of gammarids across different habitats and localities along the Baltic coast of Poland. Seven gammarid species, two native and five invasive, were sampled from 16 freshwater and brackish localities. Sixty symbiotic species of microorganisms of nine phyla were identified. This taxonomically diverse species assemblage of symbionts allowed us to assess the effect of host translocation and regional ecological determinants driving assembly richness in the gammarid hosts. Our results revealed that (i) the current assemblages of symbionts of gammarid hosts in the Baltic region are formed by native and co-introduced species; (ii) species richness of the symbiotic community was higher in the native Gammarus pulex than in the invasive hosts, probably reflecting a process of species loss by invasive gammarids in the new area and the distinct habitat conditions occupied by G. pulex and invasive hosts; (iii) both host species and locality were key drivers shaping assembly composition of symbionts, whereas habitat condition (freshwater versus brackish) was a stronger determinant of communities than geographic distance; (iv) the dispersion patterns of the individual species richness of symbiotic communities were best described by Poisson distributions; in the case of an invasive host, the dispersion of the rich species diversity may switch to a right-skewed negative binomial distribution, suggesting a host-mediated regulation process. We believe this is the first analysis of the symbiotic species richness in native and invasive gammarid hosts in European waters based on original field data and a broad range of taxonomic groups including Microsporidia, Choanozoa, Ciliophora, Apicomplexa, Platyhelminthes, Nematoda, Nematomorha, Acanthocephala and Rotifera, to document the patterns of species composition and distribution.

Molecular characterization and transcriptional regulation of two types of H+-pyrophosphatases in the scuticociliate parasite Philasterides dicentrarchi.

Proton-translocating inorganic pyrophosphatases (H+-PPases) are an ancient family of membrane bound enzymes that couple pyrophosphate (PPi) hydrolysis to H+ translocation across membranes. In this study, we conducted a molecular characterization of two isoenzymes (PdVP1 and PdVP2) located in respectively the alveolar sacs and in the membranes of the intracellular vacuoles of a scuticociliate parasite (Philasterides dicentrarchi) of farmed turbot. We analyzed the genetic expression of the isoenzymes after administration of antiparasitic drugs and after infection in the host. PdVP1 and PdVP2 are encoded by two genes of 2485 and 3069 bp, which respectively contain 3 and 11 exons and express proteins of 746 and 810 aa of molecular mass 78.9 and 87.6 kDa. Topological predictions from isoenzyme sequences indicate the formation of thirteen transmembrane regions (TMRs) for PdVP1 and seventeen TMRs for PdVP2. Protein structure modelling indicated that both isoenzymes are homodimeric, with three Mg2+ binding sites and an additional K+ binding site in PdVP2. The levels of identity and similarity between the isoenzyme sequences are respectively 33.5 and 51.2%. The molecular weights of the native proteins are 158 kDa (PdVP1) and 178 kDa (PdVP2). The isoenzyme sequences are derived from paralogous genes that form a monophyletic grouping with other ciliate species. Genetic expression of the isoenzymes is closely related to the acidification of alveolar sacs (PdVP1) and intracellular vacuoles (PdVP2): antiparasitic drugs inhibit transcription, while infection increases transcription of both isoenzymes. The study findings show that P. dicentrarchi possesses two isoenzymes with H+-PPase activity which are located in acidophilic cell compartment membranes and which are activated during infection in the host and are sensitive to antiparasitic drugs. The findings open the way to using molecular modelling to design drugs for the treatment of scuticociliatosis.

Enumeration of Clostridium perfringens spores in groundwater samples: comparison of six culture media.

In order to investigate the ability of Fluorocult-supplemented TSC agar (TSCF (Fluorocult supplemented TSC-agar): prepared from Tryptose Sulfite Cycloserine Agar Base (Merck), D-cycloserine (Fluka Chemika, USA), and fluorocult TSC-Agar supplement (Merck)) for detecting spores of Clostridium perfringens in water, we analyzed groundwater samples, pretreated by heating to 80 degrees C/5 min, using this fluorogenic medium together with five other media: mCP agar (Panreac; Cultimed), TSC agar (Merck, Germany), TSN agar (Merck), and SPS agar (BBL, USA) by the membrane filtration technique, and Wilson-Blair agar (WB) following the still-in-force Spanish official method. Variance analysis of the data obtained shows statistically significant differences in the counts obtained between media employed in this work. The C. perfringens spore counts on mCP agar were significantly lower (P<0.05) than the corresponding values of TSC, TSCF, SPS, and WB media. No statistically significant differences were found between C. perfringens spore counts on TSCF compared with those of other methods used. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that fluorogenic TSC agar was the most specific medium for C. perfringens spore recovery in groundwater samples. Additionally, the results obtained indicate that mCP agar, which is the reference method in the European Union, is not suitable medium for recovering C. perfringens spores from groundwater samples.

Genotoxicity of the coating lacquer on food cans, bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE.

The epoxy resin bisphenol A diglycidyl ether (BADGE), its hydrolysis products and a chlorohydrin of BADGE (BADGE.2HCl), were examined for their genotoxicity in the micronucleus test (MNT) with human peripheral blood lymphocytes in vitro, in presence and in absence of an exogenous metabolizing system S9 rat liver. The treatment was done using different compound concentrations up to cytotoxic doses. The concentrations tested ranged between 12.5 to 62.5microg/ml of BADGE, 12.5 to 62.5microg/ml of first BADGE hydrolysis product (BADGE.H(2)O), 25.0 to 100.0microg/ml of second BADGE hydrolysis product (BADGE.2H(2)O) and 6.25 to 50.0microg/ml of BADGE.2HCl. These compounds are able to induce both cytotoxic and genotoxic effects, as revealed by the increases observed in cytokinesis block proliferation index (CBPI) and in micronuclei (MN) frequencies, respectively.

Genotypic mutation analysis in the p53 gene of benzo[a]pyrene-treated European flounder (Platichthys flesus).

We have applied a genotypic mutation detection system (the Restriction Site Mutation (RSM) assay) to detect mutations in the marine teleost flounder (Platichthys flesus). The aim of this study was to evaluate this species as an environmental indicator of genotoxic exposure. We have used the model genotoxin benzo[a]pyrene (B[a]P) to determine the limits of mutation detection in the p53 gene of flounder liver DNA. This study has revealed two important findings. Firstly, we were able to demonstrate that a polymorphism exists in the TaqI restriction site of exon 8 of the flounder p53 gene at codon 243. This polymorphic allele was present as a heterozygote at a mean frequency of 15%, whereas 85% carried the homozygous wild type sequence. Secondly, we established that B[a]P treatment resulted in specific mutational events at the adenine base of the same TaqI site, contrasting previous reports stating that there was a guanine preference for this chemical in mammalian DNA. This difference in mutation specificity may possibly be accounted for by sequence specific factors or by species differences in metabolic activation and/or DNA repair and are worthy of further study.

Evaluation of fluorogenic TSC agar for recovering Clostridium perfringens in groundwater samples.

Clostridium perfringens is widely recognised as a reliable water pollution indicator. Since several media can be employed for the membrane filtration enumeration of this microorganism, the main aim of this work was to investigate the ability of fluorocult-supplemented TSC-agar (Merck) for recovering Cl. perfringens from public springs used for direct human consumption. Cl. perfringens recovery was also performed on mCP agar (Cultimed) according to Directive 98/83 as well as on TSC-Agar (Merck), TSN-Agar (Merck) and SPS-Agar (BBL) media. Variance analysis of data obtained showed no statistically significant differences in the counts obtained among all media employed in this work. However, the Cl. perfringens recovery efficiencies with TSC and fluorogenic TSC agars were significantly greater (P = < 0.05) than the corresponding values of mCP and TSN media. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that fluorogenic TSC agar was the most specific medium for Cl. perfringens recovery in groundwater samples (85.3% of typical colonies and 82.8% of atypical colonies confirmed). In summary, the membrane filtration technique with fluorogenic TSC agar showed the best performance characteristics of all the media tested as judged by their recovery efficiency and specificity in these water samples.

Evaluation of Coli-ID and MUG Plus media for recovering Escherichia coli and other coliform bacteria from groundwater samples.

Several chromogenic media for detecting coliform bacteria in water are commercially available including Coli ID medium (ID) (bioMérieux) and MUG Plus cefsulodin agar (MP) (Laboratorios Microkit, S.L.). Since little is known about the performance of these media, we have evaluated their usefulness for recovering Escherichia coli and other coliform organisms in groundwaters used for direct human consumption. Variance analysis of obtained data showed that no statistically significant differences in counts of E. coli and other coliforms on ID and MP media compared with reference methods. However, the evaluation of sensitivity and recovery efficiency of both media showed that the two chromogenic media were more sensitive and significantly more efficient (P = < 0.05) than reference medium for detecting coliforms in groundwater. However, the identification of 400 typical and atypical colonies isolated from ID and MP media demonstrated a higher specificity when using ID for coliforms and E. coli. In summary, the two chromogenic media evaluated could be used as alternative methods to reference media for detecting and recovering coliform bacteria in groundwater samples. MP agar was more sensitive and efficient than ID agar whereas the latter was more specific and selective.

[Incidence of bacterial contamination in the Ares-Betanzos estuary (NW Spain)]. / Incidencia de la contaminación bacteriana en la ría de Ares-Betanzos (NW España).

The presence of bacterial indicators of fecal pollution and V. parahaemolyticus in the estuary of Ares-Betanzos (ría de Ares-Betanzos, NW of Spain) was investigated. Resistance patterns of coliform bacteria to eight antibacterial agents were also determined. In general, high numbers of indicator bacteria were found; for instance, heterotrophic bacteria ranged between 1.82 x 10(2) to 1.9 x 10(4) CFU/ml and up to 4.6 x 10(3)/100 ml fecal coliforms in surface waters and 1.2 x 10(4)/100 ml fecal streptococci in sediment could be found. Surface waters of sampling points 2 and 7, located at the inner part of the estuary, were more polluted than the corresponding ones in the mouth (sampling points, 1, 3, 4 and 9), whereas the sediment showed just the opposite distribution. An 88.5% of isolated coliforms were resistant to one or more antibacterial agents. The MAR index points to urban wastewaters as the probable origin of pollution. The low incidence of V. parahaemolyticus and the lack of correlation with any of the fecal indicator bacteria determined, discard its use as indicative of fecal pollution in marine environments.

Underestimation of Fecal Coliform Counts in Shellfish-Growing Waters by the Spanish Official Method.

We compared two tube fermentation methods for the enumeration of fecal coliforms in mussels: the APHA method and the Spanish official method (CP method). In the study area (Galicia, northwest Spain), the regional authorities have proposed that the CP method be adopted as standard. Results showed that the APHA method gave significantly higher counts (P < 0.01) than the CP method. The mean difference between APHA-method counts and CP-method counts was particularly high when only those samples containing less than 500 fecal coliforms per 100 g (as determined by either method) were considered. A significantly higher number of samples were classified as unacceptable (more than 300 fecal coliforms per 100 g) by the APHA method than by the CP method. These results suggest that the CP method is inappropriate for the enumeration of fecal coliforms in mussels.

Mutagenic potential of bisphenol A diglycidyl ether (BADGE) and its hydrolysis-derived products in the Ames Salmonella assay.

The mutagenicity of bisphenol A diglycidyl ether (BADGE), its first and second hydrolysis products (the diol epoxide and bis-diol of BADGE, respectively) and the bis-chlorohydrin of BADGE were investigated using the Ames Salmonella assay with strains TA98, TA100, TA1535 and TA1537. The assays were performed in the absence and presence of various concentrations of rat liver S9 fraction. The results obtained confirm the mutagenic power of BADGE in strains TA100 and TA1535 and show a positive response to the diol epoxide of BADGE in these strains, although the latter compound was approximately 10 times less potent than the former. A lack of mutagenic activity of the bis-diol of BADGE and the chlorohydrin under study is also shown. These findings suggest that BADGE and, to a much lesser extent, the diol epoxide of BADGE may constitute a genotoxic hazard, but not the bis-diol or bis-chlorohydrin of BADGE.

The restriction site mutation assay: a review of the methodology development and the current status of the technique.

The restriction site mutation (RSM) assay has been employed in our laboratory, as a mutation detection system, since its first description in 1990. In principle the technique is capable of detecting mutations in ubiquitous restriction enzyme sites and is, therefore, readily applicable to any sequenced gene and/or organism. The RSM assay has been applied in our laboratory in various species, detecting rare mutations induced in mouse, rat, Xenopus, flatfish and human cells and tissues. This paper reviews the data accumulated by the RSM methodology in our hands and charts the developmental processes which have steadily improved the technique such that it is now applicable as a sensitive genotypic mutation detection system. This paper also includes PCR primer sequences and restriction enzymes employed in mutational analyses performed in the various species studied. We detail a variety of problems associated with the assay and the steps taken to solve them. The specific hurdles which have been overcome include the lack of quantitative data, the question of the contribution of DNA adducts to the induced mutation profile and the presence of false positives. Finally, the methods which have been developed to increase the sensitivity of the assay are also detailed. This paper describes our recommended RSM methodology, as it is routinely employed in our laboratory, which enables the analyses of mutations induced by chemical exposures and spontaneous endogenous processes. Our aim in presenting the developmental data on the RSM assay is to provide other researchers with sufficient information about the RSM methodology to facilitate its application in mutation analysis in other genes and organisms.