Plant- and Bacteria-Derived Compounds with Anti-Philasterides dicentrarchi Activity.

Philasterides dicentrarchi is a scuticociliate that causes high mortalities in farmed fish. Although vaccination is an effective method to prevent scuticociliatosis caused by the homologous serotype, a universal vaccine has not been developed yet. Many compounds have been shown to be toxic to this ciliate species; moreover, most of them are toxic to aquatic life and cannot be used to prevent the disease. We have evaluated the toxicity to P. dicentrarchi of several compounds of natural origin to be used to reduce parasite levels in the seawater. Ciliates were exposed to several compound concentrations, and the mortality was determined at several incubation times. Tomatine, plumbagin and 2',4'-dihydroxychalcone displayed the highest anticiliate activity, with a dose-dependent response. The effects of these compounds on the EPC cell line were also evaluated, finding that 2',4'-dihydroxychalcone displayed the lowest toxicity to fish cells. At 7.54 µM, 2',4'-dihydroxychalcone inhibited 50% parasite growth but only killed about 10% of EPC cells after 24 h incubation. Finally, we evaluated the toxicity of Pseudomonas H6 surfactant (PS) to P. dicentrarchi, finding that PS was toxic to the ciliate but showed lower toxicity to EPC cells. At a concentration of 7.8 µg/mL (LC50 for the ciliate after 3 h incubation), PS killed 14.9% of EPC cells. We conclude that 2',4'-dihydroxychalcone, and PS could be used to reduce parasite levels in seawater, thus decreasing the risk of scuticociliatosis infection in cultured fish.

Characterization of the turbot Scophthalmus maximus (L.) myeloperoxidase. An insight into the evolution of vertebrate peroxidases.

We have completed the characterization of the turbot (Scophthalmus maximus) myeloperoxidase (mpx) gene and protein, which we partially described in a previous study. The turbot mpx gene has 15 exons that encode a protein of 767 aa, with a signal peptide, propeptide and light and heavy chains, and also with haem cavities, a Ca+2-binding motif and several N- and O-glycosylation sites. The mature protein forms homodimers of about 150 kDa and is very abundant in turbot neutrophils. In addition to the mpx (epx2a) gene, another three peroxidase genes, named epx1, epx2b1 and epx2b2, were identified in the turbot genome. Epx1, Epx2b1 and Epx2b2 proteins also have signal peptides and many structural characteristics of mammalian MPO and eosinophil peroxidase (EPX). Mpx was strongly expressed in head kidney, while epx2b1 and epx2b2 were strongly expressed in the gills, and epx1 was not expressed in any of the tissues or organs analysed. In vitro stimulation of head kidney leucocytes with the parasite Philasterides dicentrarchi caused a decrease in mpx expression and an increase in epx2b1 expression over time. In turbot infected experimentally with P. dicentrarchi a significant increase in mpx expression in the head kidney was observed on day 7 postinfection, while the other genes were not regulated. However, mpx, epx2b1 and epx2b2 were downregulated in the gills of infected fish, and epx1 expression was not affected. These results suggest that the four genes responded differently to the same stimuli. Interestingly, BLAST analysis revealed that Epx1 and Mpx showed greater similarity to mammalian EPX than to MPO. Considering the phylogenetic and synteny data obtained, we concluded that the epx/mpx genes of Gnathostomes can be divided into three main clades: EPX1, which contains turbot epx1, EPX2, which contains turbot mpx (epx2a) and epx2b1 and epx2b2 genes, and a clade containing mammalian EPX and MPO (EPX/MPO). EPX/MPO and EPX2 clades share a common ancestor with the chondrichthyan elephant shark (Callorhinchus milii) and the coelacanth (Latimeria chalumnae) peroxidases. EPX2 was only found in fish and includes two sister groups. One of the groups includes turbot mpx and was only found in teleosts. Finally, the other group contains epx2b1 and epx2b2 genes, and epx2b1-2b2 loci share orthologous genes with other teleosts and also with holosteans, suggesting that these genes appeared earlier on than the mpx gene.

Emergence of Asiatic Vibrio diseases in South America in phase with El Niño.

BACKGROUND: The seventh pandemic of Vibrio cholerae unexpectedly reached the coast of Peru in 1991, causing an explosive emergence of infections throughout the American continents. The origin and routes of dissemination are as yet unknown. A new Vibrio epidemic arose in 1997 in South America (northern Chile) when the pandemic clone of Vibrio parahaemolyticus was for the fist time detected outside of Asia. These 2 cases were concurrent with 2 episodes of El Niño. METHODS: We carried out a survey of records of V. parahaemolyticus infection and of strains existing in the Instituto Nacional de Salud of Peru between 1994 and 2005. Association between the El Niño event and the V. parahaemolyticus disease was analyzed through generalized additive models applied to time-series data with negative binomial response, selecting some oceanographic factors distinctive of the movement of the El Niño waters. RESULTS: Epidemiologic data and laboratory investigations of the strains showed that V. parahaemolyticus infections caused by the pandemic clone emerged in the coasts of Peru linked to the 1997 El Niño episode. The epidemic dissemination of this clone matched the expansion and dynamics of the poleward propagation and the receding of the El Niño waters. This pattern was similar to previously reported onset of cholera epidemic in 1991. CONCLUSIONS: These findings identify the El Niño episodes as a reliable vehicle for the introduction and propagation of Vibrio pathogens in South America. The movement of oceanic waters seems to be one of the driving forces of the spread of Vibrio diseases.

Vaccine-induced modulation of gene expression in turbot peritoneal cells. A microarray approach.

We used a microarray approach to examine changes in gene expression in turbot peritoneal cells after injection of the fish with vaccines containing the ciliate parasite Philasterides dicentrarchi as antigen and one of the following adjuvants: chitosan-PVMMA microspheres, Freund́s complete adjuvant, aluminium hydroxide gel or Matrix-Q (Isconova, Sweden). We identified 374 genes that were differentially expressed in all groups of fish. Forty-two genes related to tight junctions and focal adhesions and/or actin cytoskeleton were differentially expressed in free peritoneal cells. The profound changes in gene expression related to cell adherence and cytoskeleton may be associated with cell migration and also with the formation of cell-vaccine masses and their attachment to the peritoneal wall. Thirty-five genes related to apoptosis were differentially expressed. Although most of the proteins coded by these genes have a proapoptotic effect, others are antiapoptotic, indicating that both types of signals occur in peritoneal leukocytes of vaccinated fish. Interestingly, many of the genes related to lymphocytes and lymphocyte activity were downregulated in the groups injected with vaccine. We also observed decreased expression of genes related to antigen presentation, suggesting that macrophages (which were abundant in the peritoneal cavity after vaccination) did not express these during the early inflammatory response in the peritoneal cavity. Finally, several genes that participate in the inflammatory response were differentially expressed, and most participated in resolution of inflammation, indicating that an M2 macrophage response is generated in the peritoneal cavity of fish one day post vaccination.

Ecological determinants of the occurrence and dynamics of Vibrio parahaemolyticus in offshore areas.

The life cycle of Vibrio parahaemolyticus has been conventionally associated with estuarine areas characterized by moderate salinity and warm seawater temperatures. Recent evidence suggests that the distribution and population dynamics of V. parahaemolyticus may be shaped by the existence of an oceanic transport of communities of this organism mediated by zooplankton. To evaluate this possibility, the presence of V. parahaemolyticus in the water column of offshore areas of Galicia was investigated by PCR monthly over an 18-month period. Analysis of zooplankton and seawater showed that the occurrence of V. parahaemolyticus in offshore areas was almost exclusively associated with zooplankton and was present in 80% of the samples. The influence of environmental factors assessed by generalized additive models revealed that the abundance and seasonality of V. parahaemolyticus in zooplankton was favoured by the concurrence of downwelling periods that promoted the zooplankton patchiness. These results confirm that offshore waters may be common habitats for V. parahaemolyticus, including strains with virulent traits. Additionally, genetically related populations were found in offshore zooplankton and in estuaries dispersed along 1500 km. This finding suggests that zooplankton may operate as a vehicle for oceanic dispersal of V. parahaemolyticus populations, connecting distant regions and habitats, and thereby producing impacts on the local community demography and the spread of Vibrio-related diseases.

Prevalence and genetic diversity of pathogenic populations of Vibrio parahaemolyticus in coastal waters of Galicia, Spain.

The natural reservoirs and biological characteristics of pathogenic populations of Vibrio parahaemolyticus in marine habitats remain unclear due to difficulties in obtaining pathogenic strains from the environment. The distribution and characteristics of pathogenic V. parahaemolyticus were investigated over 1 year in three coastal environments in Galicia (Spain), including areas of the major international ports in the region. Vibrio parahaemolyticus was present in 35.3% of the samples analysed, and 535 strains were isolated over the period of study. Virulence genes were detected in 94 strains with diverse genetic traits: 66 trh+/tdh-, 24 trh-/tdh+ and 4 trh+/tdh+. Different spatial and seasonal patterns were observed in relation to genetic traits. The trh+/tdh- strains were detected exclusively in northern areas and prevailed in the autumn, when seawater is warmer and less saline, whereas the trh-/tdh+ strains were found in all three areas throughout winter and spring. Characterization of potentially pathogenic strains from the environment revealed an unexpectedly diverse array of serotypes and pulsed-field gel electrophoresis (PFGE) profiles (pulsotypes) that were unrelated to clinical strains of V. parahaemolyticus that are prevalent in Spain. The results of the current study provide a novel view of V. parahaemolyticus in Europe, in which diverse pathogenic groups are constitutive components of the environmental populations in coastal habitats.