Determining ACTB, ATP5B and RPL32 as optimal reference genes for quantitative RT-PCR studies of cryopreserved stallion semen.

Equine germplasm bank management involves not only the conservation and use of semen doses, in addition it can also be a resource to study stallion semen quality and after thawing semen properties for reproductive purposes. A possible criterion to measure quality may be based on differential gene expression of loci involved during spermatogenesis and sperm quality maturation. The rapid degradation of sperm after thawing affects the integrity and availability of RNA. In this study we have analyzed genes expressed in equine cryopreserved sperm, which provided an adequate amplification, specificity, and stability to be used as future reference genes in expression studies. Live spermatozoa were selected from cryopreserved semen straws derived from 20 stallions, through a discontinuous concentration gradient. RNA purification followed a combination of the organic and column extraction methods together with a deoxyribonuclease treatment. The selective amplification of nine candidate genes was undertaken using reverse transcription and real-time polymerase chain reaction (qPCR) carried out in a one-step mode (qRT-PCR). Specificities were tested by melting curves, agarose gel electrophoresis and sequencing. In addition, gene stabilities were also calculated. Results indicated that five out of the nine candidate genes amplified properly (ß-Actin, ATP synthase subunit beta, Protamine 1, L32 ribosomal protein and Ubiquitin B), of which ß-Actin and the L32 Ribosomal protein showed the highest stability thus being the most suitable to be considered as reference genes for equine cryopreserved sperm studies, followed by the ATP synthase subunit beta and Ubiquitin B.

Resveratrol promotes an inhibitory effect on the turbot scuticociliate parasite Philasterides dicentrarchi by mechanisms related to cellular detoxification.

The mechanisms involved in antiparasitic activity of the natural nonflavonoid polyphenol resveratrol (RESV) on the turbot (Psetta maxima) scuticoliate parasite Philasterides dicentarchi were investigated. At concentrations higher than 50microM, RESV caused significant inhibition of the in vitro growth of the ciliates, which was apparent on the third day of culture and, at the same concentration, RESV caused significant inhibition of O(2) consumption. RESV, at a concentration of 100microM, produced a significant increase in the production of intracellular reactive oxygen species (ROS), which were inhibited by the addition of 1mM of L (+) ascorbic acid. RESV (100microM) also caused significant inhibition of peroxidase, catalase and superoxide dismutase activities, but stimulated the activity of the redox regulating enzyme glutathione S-transferase. Confocal microscopy with the mitochondria-sensitive dye MitoTracker Orange CMTMRos revealed that RESV at concentrations higher than 50microM significantly increased the levels of fluorescence inside mitochondria and, at the same concentration, also caused an increase in the vacuolization of the trophozoites. The results obtained in the present study suggest that the inhibitory activity of RESV on the ciliate P. dicentrarchi is related to the induction of oxidative stress and to the inability of the parasite to eliminate ROS as a result of modified activity of antioxidant enzymes.

Complement-mediated killing of Philasterides dicentrarchi (Ciliophora) by turbot serum: relative importance of alternative and classical pathways.

The present study was carried out to elucidate the in vitro killing activity of turbot complement and specific antibodies against the ciliate parasite Philasterides dicentrarchi. Fresh serum from nonimmunized fish showed a moderate ability to kill the parasite, which indicates that P. dicentrarchi is able to activate the alternative complement pathway (ACP). Fresh serum from immunized fish, which contained high levels of specific antibodies, showed greater killing activity. Heat-inactivated serum, with or without antibodies, and antibodies alone did not have any effect on parasite viability, which indicates that serum mainly kills P. dicentrarchi through the antibody-mediated classical complement pathway (CCP). Ascitic fluid from infected fish, but containing low levels of specific antibodies, showed a low ability to kill the parasite, and fresh serum from nonimmunized infected fish did not kill the parasite. The latter serum contained some specific antibodies but lower levels of complement than serum from control and vaccinated fish, and the lack of ability of this serum to kill the parasite was probably related to low levels of complement. In addition, serum and ascitic fluid from infected turbot showed high proteolytic activity which degraded fish Igs. The proteolytic activity generated may favour survival of the parasite during infection.

Resveratrol modulates innate and inflammatory responses in fish leucocytes.

Resveratrol (RESV; trans-3,5,4'-trihydroxystilbene), a phytoalexin that is produced by some plants, among other effects has well-known antioxidant, anti-inflammatory and immunomodulatory activities in mammals. In the present study, the effects of RESV on several functions of turbot, Psetta maxima (L.), kidney leucocytes (KLs) related to the innate and inflammatory responses were investigated. RESV exerted a dose-dependent inhibitory effect on the migratory response and on the production of reactive oxygen species in KL, after stimulation of the respiratory burst activity with phorbol myristate acetate (PMA). RESV also significantly inhibited the generation of the pro-inflammatory mediator prostaglandin E(2) (PGE(2)) in the supernatant of KL cultures stimulated with acidic sulphated polysaccharides (ASPs) from the seaweed Ulva rigida. The effects of the polyphenol on enzymatic activity and on myeloperoxidase (MPO) gene expression in neutrophils were also tested. It was found that RESV strongly inhibited intracellular and extracellular MPO activity, behaving as a noncompetitive and reversible inhibitor, and also induced a decrease in MPO mRNA levels in turbot neutrophils. These findings indicate that RESV exerts important modulatory effects on inflammatory responses in fish, and considering the importance of innate immunity in these vertebrates and the similarities with mammals, it may be possible to use fish for analysis of the effects of different substances on inflammatory responses.

Vaccination of turbot, Psetta maxima (L.), against the protozoan parasite Philasterides dicentrarchi: effects on antibody production and protection.

The efficacy of a vaccine against the fish pathogen Philasterides dicentrarchi was evaluated in turbot by measuring the production of specific antibodies and duration of protection. Four groups of turbot were vaccinated twice, on days 0 and 30, with phosphate-buffered saline, mineral oil adjuvant, antigen or antigen plus adjuvant. Specific serum antibodies were determined on day 0 and 1 month after the first and the second vaccinations. Protection was evaluated 1 month after the first vaccination and 1 and 5 months after the second vaccination. Serum antibody titres, measured by enzyme-linked immunosorbent assay, and protection, assessed by challenges, increased significantly 1 month after the second vaccination in the group injected with antigen plus adjuvant and the protection lasted for at least a further 5 months in this group. The relative protection was 77% and 66% 1 and 5 months after the second vaccination, respectively. Administration of antigen or adjuvant separately had no effect on antibody response or protection. The results indicate that emulsion containing antigen plus adjuvant induced durable protection against P. dicentrarchi after the administration of the two vaccinations, and that this preparation can be used as a vaccine against the pathogen.

Scuticociliate cysteine proteinases modulate turbot leucocyte functions.

The effects exerted by cysteine proteinases isolated from the histiophagous ciliate Philasterides dicentrarchi on the phagocytic functions of turbot pronephric leucocytes (PL) were investigated. The enzymes were tested at concentrations of 125, 250 and 500 microg ml(-1), and it was found that the viability of the leucocytes was not affected after treatment for 24h. Leucocyte migration was inhibited by the cysteine proteinases in a dose-dependent manner, whereas the ascitic fluid obtained from turbot experimentally infected with P. dicentrarchi induced high chemotactic activity in the turbot PL. The proteinases did not affect yeast cell phagocytosis but increased intracellular production of the superoxide anion (O2(-)). Stimulation with the proteinases did not alter the PGE2 levels in supernatants from 24-h cultures of PL, however, beta-glucans (100 microg ml(-1)) provoked a large increase in PGE2 levels, which were inhibited after addition of 10 microg ml(-1) of indomethacin, a non-selective inhibitor of COX2 enzymatic activity. The mean PGE2 level in ascitic fluid from turbot, experimentally infected with P. dicentrarchi, was 500 pg ml(-1), and the addition of low levels of PGE2 (62.5 pg ml(-1)) to PL cultures stimulated O2(-) production, although addition of PGE2 at concentrations higher than 250 pg ml(-1) blocked the increase in stimulation. Addition of cysteine proteinases to 24-h cultures of PL also increased mRNA levels in the pro-inflammatory cytokine interleukin-1beta. The results revealed the capacity of cysteine proteinases isolated from P. dicentrarchi to modulate the innate immune response of turbot, which together with the inflammation mediators produced during infection, may play an important role in pathogenesis of the disease and in the survival of the parasite.

Scuticociliate proteinases may modulate turbot immune response by inducing apoptosis in pronephric leucocytes.

The role of proteinases of the histiophagous ciliate Philasterides dicentrarchi, purified by affinity chromatography in bacitracin-Sepharose, on apoptosis (programmed cell death) of turbot pronephric leucocytes (PL) was investigated. The results showed that more than 90% of proteinases purified by bacitracin-Sepharose were cysteine proteinases, which lacked significant caspase-3-like activity and generated three main gelatinolytic bands of molecular weights 36, 45 and 77 kDa as determined by gelatine-SDS-PAGE and immunoblot. Viability of PL cells after 24 h stimulation with P. dicentrarchi cysteine proteinases did not differ from that of non-stimulated cells. Apoptosis was confirmed by: (i) caspase activity, (ii) DNA fragmentation, and (iii) nucleus fragmentation. The caspase-3-like activity in PL incubated for 4h in the presence of 125, 250 and 500 microg/ml of proteinases increased in a dose-dependent fashion. The PL DNA was fragmented following 24-h exposure to P. dicentrarchi cysteine proteinases and characteristic DNA ladders consisting of multimers of approximately 180-200 pb were produced. Morphological changes, such as chromatin condensation and nucleus fragmentation, were observed under fluorescence microscopy after DAPI staining of the PL cells incubated with cysteine proteinase-incubated for 24 h. The results suggest that the pathogenic scuticociliate P. dicentrarchi may induce host leucocyte programmed cell death via the production of cysteine proteinases, as a mechanism of pathogenesis and evasion of the turbot innate immune response.

Influence of host age and sex on the helminth fauna of the yellow-legged gull (Larus michahellis) in Galicia (northwestern Spain).

We studied the influence of host age and sex on the helminth fauna of 324 Larus michahellis captured in different locations in the region of Galicia (northwestern Spain). Gulls were grouped into prefledglings, first-year immature birds, second- and third-year immature birds, and adults. Second-year, third-year, and adult birds were grouped by sex. Thirty-six helminth species were recorded. Total species richness and mean infracommunity species richness were both significantly lower for pre-fledglings than for the other age groups. Prevalence increased significantly with age for Brachylecithum microtesticulatum, probably reflecting changing feeding habits. Likewise, 8 species (Cardiocephaloides longicollis, Microphallus similis, Maritrema gratiosum, Gynaecotyla longiintestinata, Brachylecithum microtesticulatum, Himasthla elongata, Parorchis acanthus, and Renicola sp.) were absent or had very low prevalence in prefledglings. At least 5 of these 8 species are transmitted to gulls through ingestion of molluscs or crustaceans, which suggests that these types of prey are seldom fed to prefledglings. In Gymnophallus deliciosus, G. longiintestinata, and Cosmocephalus obvelatus, mean intensity, and in the latter case prevalence, declined with age, suggesting that protective immunity against these species increase with age. Only G. deliciosus, Microphallus similis, and G. longiintestinata presented significant differences between the sexes.

Macroparasites of five species of ray (genus Raja) on the northwest coast of Spain.

A parasitological study of rays captured on the Atlantic continental shelf off the estuary Muros-Noia in NW Spain (42 degrees 35' to 42 degrees 41' N, 9 degrees 2' to 9 degrees 10' W; mean capture depth 11.6 +/- 4.1 m) was performed. A total of 128 rays were examined: 52 specimens of Raja microocellata, 60 of R. brachyura, 6 of R. montagui, 3 of R. undulata and 7 of an unidentified Raja species, known locally as 'fancheca'. A total of 23 macroparasite species were detected: 5 monogeneans (Acanthocotyle sp., Calicotyle kroyeri, Empruthotrema raiae, Merizocotyle undulata, Rajonchocotyle emarginata), 11 cestodes (Acanthobothrium sp., Crossobothrium sp., Echeneibothrium sp., Echinobothrium brachysoma, Grillotia erinaceus, Grillotia sp., Lecanicephalum sp., Nybelinia lingualis, Onchobothrium uncinatum, Phyllobothrium lactuca, Tritaphros retzii), 6 nematodes (Anisakis simplex, Hysterothylacium sp., Histodytes microocellatus, Piscicapillaria freemani, Proleptus sp., Pseudanisakis baylisi) and a copepod (Holobomolochus sp.). All parasite species were present in several ray species, except for Acanthocotyle sp. and G. erinaceus (detected only in R. brachyura), H. microocellatus (detected only in R. microocellata) and T. retzii (detected only in R. montagui). Three species (C. kroyeri, M. undulata, E. brachysoma) have not been reported previously from Spain. The host with the highest parasite species richness was R. brachyura (18 species), followed by R. microocellata (17) and the unidentified Raja species (14). The parasite with the highest prevalence in R. microocellata was M. undulata, followed by R. emarginata, Acanthobothrium sp. and Echeneibothrium sp. The species with the highest prevalence in R. brachyura was R. emarginata, followed by C. kroyeri and P. baylisi. Some differences in parasite prevalence were detected between sexes and among size classes in both R. brachyura and R. microocellata.

Ultrastructure and phylogeny of Philasterides dicentrarchi (Ciliophora, Scuticociliatia) from farmed turbot in NW Spain.

Several species of opportunistic histophagous scuticociliates have been implicated in systemic infections of farmed fish. In turbot, scuticociliatosis is an emerging disease, and the identification of the parasite species involved is controversial. We have previously isolated Philasterides dicentrarchi from farmed turbot scuticociliatosis outbreaks in northwest Spain. In the present study, we report detailed ultrastructural studies of this parasite, and investigate phylogenetic relations with other members of the order Philasterida on the basis of sequence comparison of the small-subunit rRNA (SSUrRNA) gene. Ultrastructural study indicates the presence of dikinetids in the anterior two-thirds of the body; micronucleus closely associated with the macronucleus, though not physically connected; numerous mitochondria located below the cell cortex, parallel to the surface; numerous spherical and fusiform extrusomes located close to the plasma membrane. We consider that these characteristics are useful for diagnosis of infections by this parasite. A nested 350-bp nucleotide sequence of the SSUrRNA gene of the turbot P. dicentrachi isolate showed high identity with previously reported SSUrRNA gene sequences from 2 scuticociliates isolated from olive flounder Paralichthys olivaceus in Korea, namely P. dicentrarchi (98%) and Miamiensis avidus (99%); conversely, our P. dicentrarchi sequence showed low identity (86%) with that of Uronema marinum, a scuticociliate that has also been implicated in scuticociliatosis outbreaks in turbot in Europe and olive flounder in Asia. Phylogenetic tree construction on the basis of the SSUrRNA gene sequences, using the neighbour-joining method, confirm that the different P. dicentrarchi isolates and M. avidus are closely related and a possible synonymy between both ciliates species should be considered.

Helminth fauna of the yellow-legged gull Larus cachinnans in Galicia, north-west Spain.

Thirty-six helminth species were found in 324 gulls examined during June 1994 to February 1996 from different localities of Galicia: 25 trematodes (Brachylaima sp., Brachylecithum microtesticulatum, Cardiocephaloides longicollis, Cryptocotyle lingua, Cryptocotyle concavum, Diplostomum spathaceum, Echinostephilla virgula, Galactosomum phalacrocoracis, Gigantobilharzia acotylea, Gymnophallus deliciosus, Gynaecotyla longiintestinata, Himasthla elongata, Himasthla quissetensis, Knipowitschiatrema nicolai, Levinseniella (Levinseniella) propinqua, Maritrema gratiosum, Maritrema linguilla, Microphallus primas, Microphallus similis, Ornithobilharzia canaliculata, Parorchis acanthus, Phagicola minuta, Psilostomum brevicolle, Renicola sp. and Stephanoprora denticulata), four cestodes (Alcataenia micracantha, Microsomacanthus ductilis, Tetrabothrius (Oriana) erostris and Wardium cirrosa), six nematodes (Anisakis simplex, Contracaecum rudolphii, Cosmocephalus obvelatus), Eucoleus contortus, Paracuaria adunca and Tetrameres (Tetrameres) skrjabini) and one acanthocephalan (Arhythmorhynchus longicollis). Tetrabothrius erostris was the most prevalent species (79.6%), followed by C. obvelatus (47.8%), C. lingua (37.4%), G deliciosus (30.9%), G. longiintestinata (22.8%), P. adunca (21.9%), B. microtesticulatum (17.6%), E. contortus (14.5%) and M. similis (9.3%). Microphallus similis was the dominant species, with a Berger-Parker index (BP) of 0.32, followed by T. erostris (BP=0.10). All species presented an aggregated dispersion except G. acotylea and G. phalacrocoracis, which showed a random dispersion. Species that seem to have the greatest predilection for specific sites along the intestine are: C. longicollis and A. micracantha (first third), Brachylaima sp., M. similis and G. longiintestinata (last third) and A. longicollis (second half). Eight species are known to be pathogenic to commercially important fish or molluscan species and several are pathogenic to humans.

Blood protozoans in elasmobranchs of the family Rajidae from Galicia (NW Spain).

Blood smears from 132 skates Raja spp. captured on the continental shelf off Galicia (NW Spain) were examined for blood parasites. The skate species were Raja brachyura (n=60), R. microocellata (n=52) and a total of 20 specimens belonging to R. undulata, R. montagui and another 2 unidentified Raja species, all captured between March 1999 and March 2000. Two blood parasite species were found, Trypanosoma giganteum and Haemogregarina delagei. Of the 132 skates, 16% were infected only by T. giganteum, 17% only by H. delagei, and 5% by both T. giganteum and H. delagei. Both parasites showed highest prevalence in R. brachyura (22% T. giganteum only, 38% H. delagei only, 12% T. giganteum and H. delagei). Mean leucocyte percentages (n=132 fish) were lymphocytes (43%), eosinophils (35%), neutrophils (20%) and monocytes (2%); basophils were not found. As far as we are aware, this eosinophil percentage is the highest reported to date for elasmobranchs. We did not detect any statistically significant differences in leucocyte percentages between infected and uninfected fish, between male and female fish, among species or among weight groups.

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.

Glugea vincentiae n. sp. (Microsporidia: Glugeidae) infecting the Australian marine fish Vincentia conspersa (Teleostei: Apogonidae).

A parasite of the marine fish Vincentia conspersa was examined by light microscopy and transmission electron microscopy. This parasite develops in the subcutaneous tissue of the body and fins, forming spherical xenomas about 1-2 mm in diameter surrounded by a layer of amorphous material. The observed characteristics of the new parasite are in line with those of the other Glugea species; merogony takes place in the outer zone of the cytoplasm of the host cell, sporogony takes place in sporophorous vesicles, and mature spores are located in the central part of the xenoma. Meronts were cylindrical uninucleate or occasionally triradiate multinucleate, with plasmodia in direct contact with the host cytoplasm. Sporogonic plasmodia divided by multiple cleavage to produce sporoblast mother cells, which after binary fission became sporoblasts. Two types of spores were recognized, both uninucleate, i.e., ovoid or slightly ovoid microspores with a mean size of 5.1 x 2.2 microm and much less frequent as elongated oval macrospores with a mean size of 8.9 x 3.1 microm. The polar tube has between 12 and 14 coils arranged in 1, 2, or 3 layers. Taken together, these characteristics suggest that this microsporidian infecting V. conspersa is a new species of Glugea, which we have named Glugea vincentiae.

Effect of cis-resveratrol on genes involved in nuclear factor kappa B signaling.

This study investigated for the first time the effects of the cis isomer of RESV (c-RESV), a polyphenol present in red wine, on an array of genes whose expression is controlled by nuclear factor kappa B (NF-kappaB) and whose transcriptional activation is critical in a number of pathologies (including some cardiovascular diseases). In inflammatory peritoneal macrophages stimulated with lipopolysaccharide (LPS) and gamma interferon (IFN-gamma), c-RESV significantly blocked the expression of genes related to the REL/NF-kappaB/IkappaB family, adhesion molecules and acute-phase proteins; however, the greatest modulatory effect was obtained on the expression of genes related to the pro-inflammatory cytokines. c-RESV down-regulated the nuclear factor of kappa light chain gene enhancer in B-cells 1 (NFkappaBL1) gene product p105 and up-regulated the nuclear factor of kappa light chain gene enhancer in B-cells inhibitor alpha (IkappaBalpha) gene. c-RESV also significantly inhibited intercellular adhesion molecule-1 (ICAM-1) gene expression and the transmembrane receptors RIP (receptor TNFRSF) and TLR3 (toll-like receptor 7). At 100 muM, c-RESV significantly inhibited transcription of Scya2 (chemokine MCP-1), the chemokine RANTES (regulated on activation, normal T cell expressed and secreted), pro-inflammatory cytokines that attract monocyte-granulocyte cells such as M-CSF (colony-stimulating factor 1), GM-CSF (colony-stimulating factor 2) and G-CSF (colony-stimulating factor 3), the cytokine tumor growth factor beta (TGF-beta) and the extracellular ligand IL-1alpha. In contrast, c-RESV stimulated transcription of the pro-inflammatory cytokines IL-6 and tumor necrosis factor alpha (TNF-alpha), the extracellular ligand IL-1beta, and the IFN regulatory factor (IRF)-1. In conclusion, c-RESV has a significant modulatory effect on the NF-kappaB signaling pathway and, consequently, an important antioxidant role that may partially explain the cardioprotective effects attributed to long-term moderate red wine consumption.

Genetic variability of natural populations of trematodes of the genus Lecithochirium parasites of eels.

Allozyme variation within and among populations of 3 species of the genus Lecithochirium (Trematoda: Hemiuridae) was studied by starch gel electrophoresis. In total, 19 loci were analysed in 7 populations. The level of genetic variability was relatively high in all populations. The percentage of polymorphic loci (0.95 criterion) observed per population varied from 21.0% to 55.5%, and expected heterozygosity levels varied from 0.082 to 0.197. All populations showed significant heterozygote deficiencies. In Lecithochirium fusiforme most of the deviations from Hardy-Weinberg proportions were within the populations and this species showed moderate population structuring (F(IS)=0.486, F(ST)=0.142, Nm= 1.51) and accordingly low intraspecific genetic distances (D=0.003 to 0.027). A significant lack of heterozygotes for several polymorphic loci was revealed in Lecithochirium rufoviride and Lecithochirium musculus. The most probable cause of the population genetic subdivision in L. rufoviride is the presence of at least 1 cryptic species in the populations studied. Although the lowest percentage of fixed genetic differences was that between L. fusiforme and L. musculus, two different algorithms for the construction of evolutionary trees on a matrix of genetic distances confirmed that L. fusiforme and L. rufoviride are phenetically the most closely related species.

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.

Cysteine proteinase activities in the fish pathogen Philasterides dicentrarchi (Ciliophora: Scuticociliatida).

This study investigated protease activities in a crude extract and in vitro excretion/secretion (E/S) products of Philasterides dicentrarchi, a ciliate fish parasite causing economically significant losses in aquaculture. Gelatin/SDS-PAGE analysis (pH 4, reducing conditions) detected 7 bands with gelatinolytic activity (approximate molecular weights 30-63 kDa) in the crude extract. The banding pattern observed in analysis of E/S products was practically identical, except for 1 low-molecular-weight band detected in the crude extract but not in the E/S products. In assays with synthetic peptide p-nitroanilide substrates, the crude extract hydrolysed substrates characteristic of cysteine proteases, namely Z-Arg-Arg pNA, Bz-Phe-Val-Arg pNA and Z-Phe-Arg pNA. These activities were strongly inhibited by the cysteine protease inhibitor E-64 and by Ac-Leu-Val-Lys aldehyde, a potent inhibitor of cysteine proteases of the cathepsin B protease subfamily. The proteases present in the crude extract degraded both type-I collagen and haemoglobin in vitro, consistent with roles in tissue invasion and nutrition respectively. Again, E-64 completely (collagen) or markedly (haemoglobin) inhibited this degradation. Finally, the histolytic activity of the ciliate in turbot fibroblast monolayers was strongly reduced in the presence of E-64, confirming the importance of secreted cysteine proteinases in the biology of Philasterides dicentrarchi.

An Anacardiaceae preparation reduces the expression of inflammation-related genes in murine macrophages.

This study investigated the effects of an aqueous extract of the stem bark of Mangifera indica L. (Anacardiaceae; Vimang), which contains a defined mixture of components including polyphenols (principally mangiferin, MA), triterpenes, phytosteroids, fatty acids and microelements, on expression of inflammation mediators in inflammatory murine macrophages after stimulation in vitro with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). In vitro treatment with Vimang at 4 microg/ml reduced levels of NOS-2 mRNA and NOS-2, while treatment at 40 microg/ml also reduced levels of COX-2 mRNA, COX-2, and prostaglandin E2 (PGE2). Results suggested that MA is involved in these effects. In vitro treatment with Vimang at 40 microg/ml also inhibited mRNA levels of the proinflammatory cytokines interleukin 1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha) and colony-stimulating factor (GM-CSF), but did not affect mRNA levels of IL-6 or tumor growth factor-beta (TGF-beta). Extracellular release of TNF-alpha by inflammatory macrophages was inhibited by in vitro treatment with Vimang at the same concentrations that showed inhibition of TNF-alpha mRNA levels. The inhibition of TNF-alpha production appears to be at least partially attributable to MA. Vimang at 4 microg/ml decreased mRNA levels of nuclear factor-kappaB (NF-kappaB) but did not affect expression of the NF-kappaB inhibitor (IkappaB). These data indicate that the potent anti-inflammatory effects of Vimang are due to selective modulation of the expression of inflammation-related genes, leading to attenuation of macrophage activation.