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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 (β-Actin, ATP synthase subunit beta, Protamine 1, L32 ribosomal protein and Ubiquitin B), of which β-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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Research on intraspecific variation in ciliates is scarce, and in scuticociliate parasite of fish, virtually nonexistent. In this study, seven isolates obtained from turbots affected by scuticociliatosis in different parts of the Iberian Peninsula (northwest Spain and southwest Portugal) were morphologically and genetically characterized to investigate the intraspecific divergence in these amphizoic ciliates. The isolates were stained with ammoniacal silver carbonate and examined in an optical microscope; all were found to have the typical morphological characteristics described for Philasterides dicentrarchi (syn. Miamiensis avidus). Sixteen biometric characteristics of the seven isolates were used in a canonical discrimination analysis (CDA) to select a subset of those that best identified each isolate. Discriminant analysis indicated that the OPK3 width, length of the PM2, length of the buccal field, the body width, L:W ratio, the body length, the OPK1 width and the distance between OPK2 and OPK3 were the most important morphological variables for discriminating the isolates. The first three canonical functions accounted for 86% of the total variance. The scatter plots of the first two canonical variables grouped and separated the P. dicentrarchi isolates into five clusters. Flow cytometry analysis of isolates also indicated intraspecific polymorphisms among P. dicentrarchi isolates. Nuclear markers (a 349-bp and a 390-bp fragment of 18S rRNA and β-tubulin genes) and a 398-bp of the mitochondrial cytocrome oxidase subunit I (Cox1) gene were then used to investigate the intraspecific genetic variation in P. dicentrarchi. Haplotype analysis and neighbour-joining phylogenies of nucleotide sequences of seven isolates revealed a high degree of intraspecific genetic variation among the isolates. Analysis of Cox1 and β-tubulin genes revealed six haplotypes (and clusters) in both cases; however, analysis of the 18S rRNA gene revealed only two haplotypes. The results show clear intraspecific variation at morphological and genetic levels in the scuticociliate P. dicentrarchi, and verify the suitability of mitochondrial (Cox1) and nuclear (β-tubulin) genes for detecting intraspecific genetic variation within populations of scuticociliates that infect cultured turbot. The existence of this intraspecific variation must be taken into account in the design of an effective vaccine to control scuticociliatosis., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The phytoalexin resveratrol (RESV), a defensive substance produced by plants in response to infection by pathogenic microorganisms, displays a wide range of biological effects in mammalian cells. In the present study, we analysed the in vitro effect of RESV on the amphizoic ciliate Philasterides dicentrarchi and demonstrated for the first time that this polyphenol causes cellular and metabolic abnormalities that generate an autophagic process and a state similar to cryptobiosis in the ciliate. At concentrations between 50 and 100 microM, RESV had a cytocidal effect when the ciliate was grown in medium with low levels of nutrients, and a cytostatic effect when the parasite was grown in culture media rich in nutrients. At these concentrations, RESV induced alterations in mitochondria, generated autophagy, provoked a reduction in the cell volume, and also drastically reduced the ciliate endocytic activity in small ciliates, generating a state compatible with cryptobiosis. The results demonstrate that RESV is a potent inducer of autophagy in the scuticociliate P. dicentrarchi. The ciliate may therefore be a good experimental organism for identifying autophagy-inducing drugs with therapeutic potential in diseases in which autophagy plays a protective role.

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.

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.

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.

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.

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.

The scuticociliatosis produced by the endoparasite Philasterides dicentarchi is a severe parasitic infection of farmed turbot (Scophthalmus maximus) characterized by several histopathological effects including extensive inflammation. Indomethacin is a nonsteroidal anti-inflammatory drug that specifically inhibits synthesis of the proinflammatory mediator prostaglandins. The effect of indomethacin on the in vitro growth of P. dicentrarchi was investigated. In vitro growth of the scuticociliate was significantly inhibited by treatment with 100 microM indomethacin for 48 h. Higher concentrations of indomethacin (mM levels) did not affect the gelatinolytic activity of the cysteine proteinases of P. dicentrarchi. In vitro treatment with 25, 50 or 100 microM indomethacin for 3 days did not significantly affect the enzymatic activity of cysteine proteinases, as assayed with p-nitroanilide as substrate. Immunoblot analysis with anti-cysteine proteinase antibodies revealed an increase in proteinase expression (molecular weights of 80, 32 and 40-45 kDa) in parasite lysates originating from in vitro cultures incubated with 25 microM indomethacin for 72 h. Degradation of genomic DNA of the ciliates was observed in cultures incubated with 100 microM indomethacin for 1, 3 and 7 days. The results suggest that indomethacin is capable of inhibiting in vitro growth of the scuticociliate P. dicentrarchi by a mechanism related to the induction of programmed cell death, without affecting the enzymatic activation of parasite proteinases, which demonstrates the potential therapeutic use of this drug in the control of turbot scuticociliatosis.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Philasterides dicentrarchi is an opportunistic histiophagous ciliate parasite causing systemic scuticociliatosis in cultured turbot (Scophthalmus maximus L.). This study investigated the effects of inoculation with live or killed trophozoites of this ciliate (plus 3% thioglycollate) on the in vitro phagocytic activity and respiratory-burst responses of inflammatory peritoneal leucocytes obtained from the fish thus treated. The phagocytic activity of leucocytes from fish inoculated with killed P. dicentrarchi was higher in the presence than in the absence of infected turbot serum (ITS). The effect of ITS was smaller in fish inoculated with live P. dicentrarchi, indicating modulation of the opsonic activity of ITS. Inoculation with live ciliates led to a significant increase in subsequent in vitro extracellular ROS production, but only when normal turbot serum (NTS) or ITS was included in the assay medium. Inclusion of live P. dicentrarchi in the medium abolished this increase, suggesting ROS-scavenging activity. Inoculation with live P. dicentrarchi led to a significant decline in subsequent in vitro intracellular ROS production; when NTS was included in the medium, there was a significant increase in intracellular ROS production, but no such increase was observed when ITS was included in the medium. Inoculation with live P. dicentrarchi alone did not increase subsequent in vitro NO? production in response to LPS; a significant increase was observed when NTS or ITS was included in the assay medium, but this increase was not affected by prior inoculation with P. dicentrarchi. These results suggest that the amphizoic nature of this parasite may reflect the ease with which it can develop mechanisms of evasion of the host immune response.

Microsporidians of the genus Encephalitozoon are an important cause of disease in immunocompromised patients, and there are currently no completely effective treatments. The present study investigated the viability and infectivity of spores of Encephalitozoon cuniculi that had been exposed to resveratrol (RESV), a natural phytoalexin found in grapes and red wine. RESV at 50 microM showed significant sporicidal activity, and at 10 to 50 microM it reduced the capacity of the spores to infect dog kidney epithelial cells of the MDCK line. At 10 microM RESV also significantly inhibited intracellular development of the parasite, without affecting host cell viability. These results suggest that RESV may be useful in the treatment of Encephalitozoon infections.

The polyphenol mangiferin (MA) has been shown to have various effects on macrophage function, including inhibition of phagocytic activity and of free radical production. To further characterize the immunomodulatory activity of MA, this study investigated its effects on expression by activated mouse macrophages of diverse genes related to the NF-kappaB signaling pathway, using a DNA hybridization array containing 96 NF-kappaB-related genes and on cytokine levels using a cytokine protein array. MA at 10 microM significantly inhibited the expression of (a) two genes of the Rel/NF-kappaB/IkappaB family, RelA and RelB (=I-rel), indicating an inhibitory effect on NF-kappaB-mediated signal transduction; (b) TNF receptor-associated factor 6 (Traf6), indicating probable blockage of activation of the NF-kappaB pathway by lipopolysaccharide (LPS), tumor necrosis factor (TNF), and interleukin 1 (IL-1); (c) other proteins involved in responses to TNF and in apoptotic pathways triggered by DNA damage, including the TNF receptor (TNF-R), the TNF-receptor-associated death domain (TRADD), and the receptor interacting protein (RIP); (d) the extracellular ligand IL-1alpha, again indicating likely interference with responses to IL-1; (e) the pro-inflammatory cytokines IL-1, IL-6, IL-12, TNF-alpha and RANTES (CCL5), and cytokines produced by monocytes and macrophages, including granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF); (f) other toll-like receptor proteins (in addition to Traf6), including JNK1, JNK2 and Tab1; (g) Scya2 (small inducible cytokine A2=monocyte chemoattractant protein 1); and (h) various intracellular adhesion molecules (ICAMs), and the vascular cell adhesion molecule VCAM-1, which is locally increased in atheromas. The inhibition of JNK1, together with stimulation of c-JUN (i.e. the Jun oncogene) and the previously reported superoxide-scavenging activity of MA, suggests that MA may protect cells against oxidative damage and mutagenesis. Taken together, these results indicate that MA modulates the expression of a large number of genes that are critical for the regulation of apoptosis, viral replication, tumorogenesis, inflammation and various autoimmune diseases, and raise the possibility that it may be of value in the treatment of inflammatory diseases and/or cancer., (Copyright 2004 Elsevier B.V.)

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.

This is a survey of the helminth fauna of 285 individuals of 14 species of birds of prey (Falconiformes and Strigiformes) from Galicia (northwest Spain), namely Buteo buteo, Accipiter nisus, A. gentilis, Milvus migrans, M. milvus, Pernis apivorus, Circus pygargus, Falco tinnunculus, F. peregrinus, F. subbuteo, Tyto alba, Strix aluco, Asio otus and Athene noctua. A total of 15 helminth species were detected, namely 8 nematodes ( Eucoleus dispar, Capillaria tenuissima, Synhimantus laticeps, Microtetrameres sp., Physaloptera alata, Procyrnea leptoptera, Hovorkonema variegatum and Porrocaecum angusticolle), 4 cestodes ( Cladotaenia globifera, Paruterina candelabraria and Mesocestoides sp.), 2 trematodes ( Neodiplostomum attenuatum and Strigea falconis), and 1 acanthocephalan ( Centrorhynchus globocaudatus). The helminth communities observed were basically similar, although there were marked differences in species richness, which was higher in falconiforms (except for A. gentilis) than in strigiforms. More specifically, species richness was highest in B. buteo (13 species), followed by A. nisus (11 species). In the falconiforms, the helminth species present generally exhibited a clear relationship with host diet. In the strigiforms, by contrast, species richness was lower than expected given the host's diet, suggesting that a different explanation is needed.

The effect of time on genetic differentiation was studied among infrapopulations of mature specimens of the hemiurid fluke, Lecithochirium fusiforme, a parasite of marine fishes. Genetic distances and genetic structure within and among different temporal samples of a geographical population were investigated using starch gel electrophoresis, by screening 6 polymorphic loci in 2 groups of infrapopulations corresponding to different sampling data, i.e., winter 1997-1998 and autumn 1998. The genetic distance among infrapopulations was low (D = 0.000-0.058 +/- 0.041). However, genetic divergence among infrapopulations from the same geographic location was clearly lower within each temporal sample (G(ST) = 0.021 and 0.034) than the corresponding value obtained for 12 infrapopulations sampled at different seasons of the year (G(ST) = 0.067). These results suggest the existence of a relatively important temporal effect that accounts for the differences in genetic variability among adult infrapopulations of L. fusiforme. Therefore, a hypothetical temporal gene flow favored by the existence of persistent life-cycle stages of this species in paratenic hosts is not sufficient to mask the temporal differentiation caused by genetic drift.

The present study investigated the effects of orally administered Vimang (an aqueous extract of Mangifera indica) and mangiferin (the major polyphenol present in Vimang) on mouse antibody responses induced by inoculation with spores of microsporidian parasites. Inoculation induced specific antibody production with an exponential timecourse, peaking after about one month. Vimang significantly inhibited this antibody production from about three weeks post-inoculation, and most markedly by four weeks post-inoculation; by contrast, mangiferin had no significant effect. Determination of Ig isotypes showed that the IgM to IgG switch began about four weeks post-inoculation, with IgG2a predominating. Vimang significantly inhibited IgG production, but had no effect on IgM. Mangiferin did no affect either IgM or IgG2a, but significantly enhanced production of IgG1 and IgG2b. Neither Vimang nor mangiferin enhanced specific antibody secretion by splenic plasma cells from mice inoculated with microsporidian spores, whether administered in vivo before serum extraction or in vitro to the culture medium. Inoculation with spores induced splenomegaly, which was significantly reduced by Vimang and significantly enhanced by mangiferin. These results suggest that components of Mangifera indica extracts may be of potential value for modulating the humoral response in different immunopathological disorders., (Copyright 2003 John Wiley & Sons, Ltd.)

This study investigates the heterogeneity and immunogenicity of the Trichinella TSL-1 antigen gp53. Western blotting analysis of several Trichinella isolates with the gp53-specific monoclonal antibodies (mAbs) US5 and US9, produced in Btkxid mice, revealed that gp53 from the species T. britovi, T. murrelli and genotype T8 had higher MW (60 kDa) than gp53 from T. spiralis, T. nelsoni and genotype T6 (53 kDa) and from T. nativa (55 kDa). mAb US5 reacted only with gp53 from T. spiralis. Experiments including immunoassays of gp53 binding by sera from T. spiralis-infected mice, in the presence of different potential inhibitors (recombinant gp53, US5, T. britovi-crude larval extract (CLE), and CLE N- and O-glycans), indicate (i) that gp53 from T. spiralis bears specific epitopes that induce antibody formation during infection; (ii) that the protein epitopes of gp53 are much more important (76 or 68% of total antibody reactivity in BALB/c and Swiss CD-1 mice, respectively) than the corresponding glycan epitopes including tyvelose (11 or 32% of total reactivity) for the induction of anti-gp53 circulating antibodies; and (iii) that the species-specific epitopes present on gp53 are differentially recognized in different mouse strains. Whereas in BALB/c mice US5- and non-US5-recognized species-specific epitopes on gp53 bind about 84% of circulating antibodies on day 80 post-infection, this percentage was only 38% in Swiss CD-1 mice. These data on the antigenicity of gp53 contrast with data for Trichinella CLE antigens, in that most circulating antibodies reactive with CLE antigens recognized tyvelose-containing epitopes (57% and 58% of circulating antibodies in BALB/c and Swiss CD-1 mice, respectively). Together these results demonstrate that gp53 is recognized during infection but is antigenically different from other Trichinella TSL-1 antigens.

Allozyme markers were used to study genetic variation in Lecithochirium fusiforme within a natural population of Conger conger. Six of 16 enzyme-coding loci studied were found to be polymorphic. These loci were surveyed in 12 infrapopulations of adult flukes. High levels of genetic variation were detected (P = 0.375); Ho = 0.048; He = 0.085). However, the population did not conform to Hardy-Weinberg expectations, as it showed a significant deficit of heterozygotes. L. fusiforme also exhibited low differentiation between infrapopulations (FST = 0.064). Despite significant linkage disequilibrium at Pgm-1 and Pgm-2 (P < 0.05), mating system does not appear to be the principal reason for the deficit of heterozygotes detected, because some polymorphic loci were in Hardy-Weinberg equilibrium. Association between FIS and FST statistics suggests the existence of the Wahlund effect. However, all infrapopulations showed a strong deficit of heterozygotes for most polymorphic loci (FIS = 0.409). Detection of significant genetic differentiation among temporal samples and the existence of paratenic hosts in the life-cycle suggests the Wahlund effect, caused by the mixture of genetically distinct temporal samples in the infrapopulations. Occasional temporal gene flow also might explain the high estimated genetic polymorphism.

New antiprotozoals active against Philasterides dicentrarchi, the causative agent of scuticociliatosis in farmed turbot and Black Sea bass-bream, have been synthesised and tested. The most active compounds posses a piperazine ring, generally N-bonded to the heterocycle, and are the 1,8-naphthyridines, 2f and 5o, the pyridothienopyrimidine (7), and the pyridothienotriazines, 8, 9, 12d, 12f, 12h, 12m and 12k. Pyridothienotriazine (12k) presents the same activity (Lethal Dose, LD=0.8/1.5 mg L(-1)) as the well-known antiparasitics niclosamide and oxyclozanide.

Philasterides dicentrarchi is a histophagous ciliate causing systemic scuticociliatosis in cultured turbot. This study demonstrates that turbot which survive this disease have serum antibodies that recognize ciliary antigens of this ciliate in ELISA and immobilize/agglutinate the ciliate in vitro. Mouse sera raised against ciliary antigens and integral membrane proteins are likewise capable of immobilizing/agglutinating the ciliates, indicating that P. dicentrarchi, like other ciliates, expresses surface immobilization antigens. Furthermore, the antigen agglutinating reaction induces the parasite to shed its surface antigens rapidly, replacing them with others with different specific serology. This antigen shedding and variation response is similar to that detected in other protozoan parasites. Immunization of turbot with ciliate lysate plus adjuvant or with formalin-fixed ciliates induced synthesis of agglutinating antibodies and conferred a degree of protection against challenge infection, suggesting that the response to surface antigens may play an important role in defence against this pathogen, SDS-PAGE and immunoblotting studies indicated the existence of a predominant polypeptide of about 38 kDa in the ciliary antigen and membrane protein fractions, and this may be the principal surface antigen of P. dicentrarchi.

Philasterides dicentrarchi is a scuticociliate causing fatal disease in farmed turbot and sea bass. In view of its high virulence and endoparasitic location, this parasite cannot be effectively controlled by formalin baths, and no systemic chemotherapeutic treatments have yet proved effective; immunoprophylaxis may thus be an attractive alternative approach. Since vaccine development is greatly facilitated by axenic culture of the pathogen, we have developed a simple axenic culture system based on commercially available Leibovitz L-15 medium, supplemented with fetal bovine serum, lipids (lecithin and Tween 80), nucleosides and glucose. After 1 week's culture under optimal conditions (salinity 10 per thousand, pH 7.2, temperature between 18 and 23 degrees C), yields of 1-2 x 10(5)cells/ml were obtained. Even cultures with seeding densities as low as 20 cells/ml were found to produce a good yield of ciliates (about 6 x 10(4)cells/ml) after 11 days of incubation. The ciliates thus obtained were free of contamination by other microorganisms, enabling preparation of pure P. dicentrarchi antigens for vaccine development studies.

Three sympatric species of the genus Lecithochirium, Lecithochirium fusiforme, Lecithochirium rufoviride and Lecithochirium musculus, parasites of Conger conger and Anguilla anguilla, were compared morphologically and electrophoretically. The three species can be discriminated by enzyme analysis, and differentiation can also be made by the analysis of several morphometric features, in particular body size and sucker ratio. Fourteen enzyme systems representing 15 loci were examined by starch gel electrophoresis. Two of the enzyme systems studied (ALD and GOT) were totally diagnostic among Lecithochirium species. Fixed allelic differences between L. fusiforme and L. musculuswere observed at five loci, between L. fusiforme and L. rufoviride at nine loci, and between L. musculusand L. rufoviride at ten loci. The percentage of fixed differences among the species under study ranged from 33 to 77%. The results show that the three taxa can be clearly differentiated, and that L. fusiforme is genetically more similar to L. musculus than to L. rufoviride.

We describe a new genus, Histodytes, within the family Guyanemidae (Nematoda: Spirurida: Camallanina: Dracunculoidea). The type species, Histodytes microocellatus n. sp., is found in the gill, heart, kidney, spleen and gonad tissues of Raja microocellata from the continental shelf off the estuary of Muros y Noia (north-western Iberian Peninsula). Histodytes differs morphologically from the three other genera described to date in this family ( Guyanema, Travassosnema, Pseudodelphys) because the vulva is situated a long way back from the oesophageal-intestinal union, and the anterior uterine branch almost reaches to the level of this union. In addition, it can be distinguished from Guyanema and Travassosnema by the absence of caudal alae in the male, and from Travassosnema by the much greater length of the glandular oesophagus and the lack of an oesophageal appendix. Histodytes is the only guyanemid genus described to date from an elasmobranch and the first one to be found on the European Atlantic coast.

The allozyme variation between 2 sympatric populations of Lecithochirium furcolabiatum and L. rufoviride was examined with the aim to investigate if the 2, morphologically similar, helminths are reproductively distinct. Three enzyme loci, Gpd, Pgm-1, and Idh were found to be diagnostic for the 2 species. These results support the conclusion that the worms are different biological species. Only the Gpi and Gdh loci showed no differences. Marked genetic subdivision was detected at the Idh locus in the L. rufoviride population. Given that the genotypic distribution at Pgm-I showed no significant deviations from Hardy-Weinberg expectations, it does not appear that the absence of heterozygotes for Idh was caused by the reproductive structure of the population. Although there are several other possible reasons for the deficiency of heterozygotes in natural populations, the existence of a new noninterbreeding group within the population studied constitutes a plausible explanation.

This study found that a plasmid construct encoding the small-subunit ribosomal RNA (SSUrRNA) of the microsporidian Microgemma caulleryi generates a humoral response upon intramuscular inoculation in mice. The plasmid used was pCMV, following preliminary trials indicating efficient beta-galactosidase gene expression in mouse muscle cells transfected with pCMV/beta-Gal. The antibodies produced after inoculation with pCMV/SSUDNA recognized parasite spore antigens and reached maximum levels at 30 days postinoculation, subsequently remaining stable for at least 120 days. Due to the highly conserved sequence of the SSUrDNA in different microsporidian species, these results open up interesting prospects for broad-spectrum vaccination.

Isozyme variation in adenosine deaminase (ADA) and glutamate-oxalacetate transaminase (GOT) was examined in 401 individuals of Lecithochirium rufoviride and Lecithochirium fusiforme from Conger conger. The banding patterns obtained for ADA were consistent with a monomeric structure and with single-locus control. This enzyme was polymorphic in the population, with two allozymes being detected. However an additional band, which may correspond to a secondary isozyme, was also observed. In accordance with this interpretation, ADA showed a significant departure from Hardy-Weinberg predictions associated with heterozygote deficiencies. Only one activity zone for GOT was detected in a subset of individuals, whereas some specimens showed a second weakly stained band, which may have been due to host enzyme contamination, although other possible reasons are also discussed. The electrophoretic patterns for ADA and GOT in these species provide examples of how some problems in electrophoretic analysis, such as nonheritable variation, may lead to the misinterpretation of zymograms in systematic and population genetic studies of parasites.

This study investigated the spatial distribution of Tetramicra brevifilum spores in the musculature of infected turbot Scophthalmus maximus, with the aim of identifying the most appropriate body locations for diagnostic assays. A PCR protocol optimized for the detection of T. brevifilum spores in turbot muscle is also described. In fish showing low- and moderate-intensity infection, the spatial distribution of spores was best fitted by a negative binomial distribution, indicating a clumped spatial pattern; the negative binomial coefficient k was lower for fish with low-intensity infection, indicating a more markedly clumped pattern in these fish. In fish with high-intensity infection, the spatial distribution of spores was best fitted by the Poisson distribution, indicating a random pattern. In both low- and moderate-intensity infection, spores were present at highest density in the musculature adjoining the dorsal fins. Samples for PCR were therefore obtained from this location. PCR amplification was of the small subunit ribosomal DNA (SSUrDNA), using a pair of species-specific primers that amplify the 1250 bp product. The PCR protocol developed showed better sensitivity than microscopical techniques (detection rate by microscopy 25%, versus 42% by PCR), suggesting that it may be useful for routine screening for Tetramicra brevifilum infection in cultured turbot.

A non-isotopic in situ hybridization (ISH) method was developed for detection of Tetramicra brevifilum, a commercially important parasite in farmed turbot Scophthalmus maximus. The probe relies on sequences from the small-subunit rRNA gene (SSUrDNA) of Tetramicra brevifilum and was obtained by polymerase chain reaction then labeled with digoxigenin. The results obtained demonstrate that the probe hybridizes well with genomic DNA of the spores; thus, it is an effective method for detecting multiorgan infections of turbot by T. brevifilum.

In vitro assays were performed to investigate microsporidian-induced intracellular and extracellular production of reactive oxygen species (ROS) by peritoneal-exudate adherent (PEA) cells from turbot. ROS production was quantified using the fluorescent reagents OxyBURST Green H2HFF BSA (extracellular) and OxyBURST Green H2DCFDA succinimidyl ester (intracellular). Five days before assay, the cells had been elicited in vivo by intraperitoneal injection of sodium thioglycollate or spores of Tetramicra brevifilum. Elicitation with spores led to a marked increase in the proportion of neutrophils among PEA cells. PEA cells from normal turbot showed considerable extracellular and intracellular ROS production in response to microsporidian spores. By contrast, PEA cells from microsporidian-infected turbot showed considerably reduced extracellular and intracellular ROS production in response to microsporidian spores. Extracellular ROS production was affected by the addition of infected turbot serum to the assay medium, regardless of whether the PEA cells had been obtained from normal or infected fish. The presence of microsporidian-infected turbot serum significantly reduced intracellular ROS production by PEA cells elicited with microsporidian spores. These results suggest that (a) microsporidian spores partially suppress the repiratory-burst response of turbot phagocytes; and (b) infected turbot serum contains substances capable of modulating the respiratory-burst response of turbot phagocytes to microsporidian spores.

Background: The number of allergic reactions to A. simplex reported in Spain has increased dramatically in the last decade. Nevertheless, there have been no studies of the prevalence of and possible risk factors for IgE sensitization to this parasite, possibly because suitably specific diagnostic methods have only recently become available. The objective was to investigate the prevalence of and risk factors for IgE sensitization to A. simplex in Galicia, a region of northwestern Spain with a population of about 3 million and high average fish consumption (78.5 g/person per day)., Methods: The study was performed with a random sample of 2801 healthy blood donors distributed in 53 geographic areas, proportional to the density of donors. IgE sensitization to A. simplex was tested by a capture ELISA method that has proved to be the most specific method currently available., Results: The results showed a total of only 12 positive subjects, of whom five also showed IgG1 sensitization. All positive subjects and 101 randomly selected seronegative subjects were then included in a case-control study of risk factors for sensitization to A. simplex, based on a telephone interview about fish consumption (especially raw and undercooked fish). All seropositive subjects (but only 25% of seronegative subjects) reported consumption of undercooked fish or homemade raw-fish products., Conclusions: Our results strongly suggest that sensitization to A. simplex is caused only by live larvae, and not by allergens contained in fish tissues, and that ingestion of homemade boquerones (anchovies [Engraulis encrasicholus] in vinegar), and to a much lesser extent of undercooked fish, are the main risk factors for IgE sensitization to Anisakis in this region.

Electrophoretic analyses of phosphoglucomutase (PGM) and fumarase (FH) in a population of Lecithochirium rufoviride parasitizing Conger conger, revealed 2 independent activity zones for each enzyme on starch gel electrophoresis. However, some individuals exhibited only 1 activity zone for 1 or both enzymes. The banding patterns observed strongly suggest that (1) PGM is coded by 2 polymorphic loci, Pgm-1 (expressed in all individuals) with allelic frequencies not significantly different from those expected under Hardy-Weinberg equilibrium, and Pgm-2 (expressed in a subset of individuals); and (2) FH is coded by 2 loci, Fh-2 (monomorphic and expressed in all individuals) and Fh-1 (expressed in a subset of individuals). A high degree of concordance (88.75%) was observed between the expression and nonexpression of Pgm-2 and Fh-1. The most likely explanations for these findings are either variation in enzyme expression with developmental stage or the presence of null alleles at high frequencies in the population.