[Show abstract][Hide abstract] ABSTRACT: Abstract Morphological and molecular evidence suggest that specimens formerly described as Lacunovermis sp. from Nacella (Patinigera) spp. (Patellogastropoda: Patellidae) belong to a new species of Gymnophalloides Fujita, 1925. Based on the new information, they are identified as G. nacellae n. sp. The new species differs from Gymnophalloides tokiensis, G. seoi, and G. heardi mainly through the presence of a group of papillae located on the ventral surface between oral and ventral suckers. A detailed morphological study revealed the lack of pars prostatica, a character previously reported in G. seoi, which is why it was formerly placed in the subfamily Gymnophallinae. Molecular information proved that G. nacellae is close to G. seoi, being nestled together with Parvatrema representatives. This molecular information, along with the absence of pars prostatica, allows these 2 genera to be placed in Parvatrematinae. An amended diagnosis of Gymnophalloides is provided. Histological sections of mantle epithelium of the limpet show metacercariae attached by their oral and ventral suckers in a similar manner to G. seoi in its host, the oyster Crassostrea gigas. Tissue reaction includes cells of outer mantle epithelium being stretched by sucker attachment, hemocyte infiltration of connective tissue between mantle epitheliums, and abnormal calcareous deposition on the inner surface of the shell.
Journal of Parasitology 07/2012; · 1.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The morphology, ultrastructure, genetics, and morphometrics of a species of Diplostomum von Nordmann 1832 (Digenea: Diplostomidae), isolated from the European flounder (Platichthys flesus (L.)) caught off the northwest coast of Portugal, are characterized. The metacercarial stage was found unencysted in the lens capsule of the eye. Light microscopical observations revealed the existence of some variability in specimen shape and size, with two morphotypes, referred to as "round" and "long", being apparent. Scanning electron microscopy revealed a smooth, unarmed tegument, with the lappet region being the most irregular and porose. Both the oral and ventral suckers were provided with a series of papillae, which presented very distinctive ultrastructural features and were particularly conspicuous in the case of the ventral sucker. The two morphotypes detected were found to have 100% genetic correspondence in the 18S + ITS1 + 5.8S region of the rDNA. Since the genetic data for this metacercaria differed from those of the species of Diplostomum available in GenBank, a description of a new genotype (accession number GQ370809) is provided. The molecular phylogenetic analyses, in conjunction with principal components and cluster analyses based on morphometric data, revealed the existence of consistent differences between the Diplostomum sp. metacercariae from flounder compared with Diplostomum spathaceum, Diplostomum mergi, Diplostomum pseudospathaceum, and Diplostomum paracaudum. The latter of these species was found to be the most similar to the present material. Our results do not support an evolutionary separation of the European and North American species of Diplostomum.
Parasitology Research 05/2011; 110(1):81-93. · 2.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study describes the anatomy and surface topography of the metacercaria of Microphallusprimas (Jägerskiöld, 1909) infecting the shore crab Carcinus maenas (L.) in Aveiro estuary, northern Portugal. The metacercaria species identification resulted from the combined use of morphological and molecular data, particularly the 28S rDNA gene. The metacercariae encysted preferentially in the host's hepatopancreas and also in the gonads. Isolated cysts were present in two distinct forms, spherical and oval, and were shown to be the identical species by the internal transcribed spacer 1 (ITS1) sequence. Chemically excysted metacercariae were studied by light (LM) and scanning electron microscopy (SEM). Their specific characteristics observed include the particular aspect of the vesiculo-prostatic pouch surrounded by a very thin membrane, the presence of a prominent muscular papilla, and an obvious metraterm. The dorsal and ventral tegumental surfaces of the metacercaria were densely packed with similar squamous spines, which decreased in number and size towards the hindbody. The edges of the posterior and ventral face of the body were coated with numerous microvilli, whose function remains unknown. In order to identify the species of metacercariae, we compared a 28S partial rDNA sequence of the two forms of cysts with the same 28S partial region of M. primas available in GenBank. With this comparison, we determined that the sequences had a 100% similarity and therefore belonged to the same species, i.e., M. primas.
[Show abstract][Hide abstract] ABSTRACT: The digenean trematode Diphtherostomum brusinae (Stossich, 1888) Stossich, 1903 presents a complex life cycle that may involve more than one intermediate host. The present study represents the first description of the metacercariae from D. brusinae infecting the labial palps of a new intermediate host, Mytilus galloprovincialis, in the Aveiro estuary, Portugal. The morphology of this parasitic stage was studied by light (LM) and scanning electron microscopy (SEM) and some differences were reported (body and sucker sizes, and spine distribution and shape). In this work, the 18S partial region of the ribosomal DNA was sequenced from D. brusinae metacercariae isolated from M. galloprovincialis collected in different localities of the Aveiro estuary. In addition, sequences from the same region of the 18S rDNA were obtained from D. brusinae cercariae and metacercariae, hosted by Nassarius reticulatus and Cerastoderma edule, respectively. No intraspecific polymorphism was detected in the 18S partial region, since there was 100% homology among all the sequences analysed. The same comparison was made for the ITS1, and we observed intraspecific polymorphism in this region. To our knowledge, this is the first report of D. brusinae metacercariae infecting the mussel M. galloprovincialis with support from morphological and molecular data.
Journal of Helminthology 08/2010; · 1.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diphtherostomum brusinae was first recorded by the present study in the north of Portugal. Sporocysts, containing cercariae and encysted metacercariae, were observed in the gonads and digestive gland of the gastropod Nassarius reticulatus. Metacercariae were also found infecting the foot, mantle border and gills of the cockle Cerastoderma edule. The adult form was lodged in the rectum of the definitive host Diplodus sargus. The morphology of the three parasitic stages was studied by light (LM) and scanning electron microscopy (SEM). Despite the close similarity between cercaria and metacercaria, SEM data provided information that allowed their differentiation, namely the presence of a dense crown of microvilli around the oral cavity of the cercariae, which was absent in the metacercariae. In addition, the metacercariae presented a specific pre-acetabular rectangular band with conspicuous triangular spines. The adult showed characteristics of D. brusinae species, in particular the presence of acetabular lips, compact vitellaria and large elliptical eggs. Sequenced ITS1 data clearly demonstrated that the cercariae and metacercarial cysts from N. reticulatus, the cysts from C. edule and the adult isolated from D. sargus were life cycle stages that belonged to the same species, i.e. D. brusinae. Two transmission strategies in the life cycle of this species were observed: (1) cercariae encyst within the sporocysts of N. reticulatus and await ingestion by the definitive host; and (2) N. reticulatus naturally emits cercariae; they encyst in C. edule or the environment and are ingested by the definitive host.
Journal of Helminthology 04/2009; 83(4):321-31. · 1.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cercaria of Bucephalus minimus infects the digestive gland and gonads of its first intermediate host, the edible cockle, Cerastoderma edule. Light microscopy (LM) and scanning electron microscopy (SEM) of the cercaria showed a tail formed by a central stem, with 2 long contractile arms presenting distinct morphological surfaces. The encysted metacercaria naturally infected the flathead grey mullet, Mugil cephalus. The cysts found in the heart, liver, and spleen were shown to be identical by the internal transcribed spacer (ITS 1) sequence and morphological features and were associated with encapsulation, recruitment of cell infiltrates, and presence of melanomacrophages and adipose tissue. To establish the life cycle, we compared the ITS1 sequence in an adult from the known definitive host, Dicentrarchus labrax; encysted metacercariae from the liver, heart, and spleen of M. cephalus; and a cercaria from C. edule. With this comparison, we determined that they had a 100% similarity. Therefore, the ITSI sequence data clearly indicate that these 3 parasitic stages belong to the same species, i.e., B. minimus.
Journal of Parasitology 09/2008; 95(2):353-9. · 1.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cercaria sevillana is the cercaria larval stage that infects the gonads and the digestive gland of its first intermediate host, Nassarius reticulatus. In this study the decapodous crustacean Carcinus maenas was used to determine if it would serve as second intermediate host in the parasite's life cycle. The latter hypothesis was based on the knowledge that C. maenas is the second intermediate host of several other digenean species. After dissection, it was possible to observe encysted metacercariae in the antennal glands of the green crab. After biochemical excystment, the metacercariae were processed for light and scanning electron microscopy. The morphological features observed led us to conclude that this species was a microphallid fluke, and it was identified as Gynaecotyla longiintestinata. To establish a possible relationship between C. sevillana and this metacercariae, the ITS1 region was analyzed. Thus, DNA was extracted from C. sevillana and from the cysts isolated from the antennal glands. The ITS1 region was amplified and sequenced, and the alignment clearly demonstrated that the cercaria and the metacercariae belonged to the same species, G. longiintestinata.
Journal of Parasitology 05/2007; 93(2):318-22. · 1.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During the years 2004 and 2005 high mortalities in turbot Scophthalmus maximus (L.) from a fish farm in the north of Portugal were observed. Moribund fish showed darkening of the ventral skin, reddening of the fin bases and distended abdominal cavities caused by the accumulation of ascitic fluid. Ciliates were detected in fresh mounts from skin, gill and ascitic fluid. Histological examination revealed hyperplasia and necrosis of the gills, epidermis, dermis and muscular tissue. An inflammatory response was never observed. The ciliates were not identified to species level, but the morphological characteristics revealed by light and electronic scanning microscopes indicated that these ciliates belonged to the order Philasterida. To our knowledge this is the first report of the occurrence of epizootic disease outbreaks caused by scuticociliates in marine fish farms in Portugal.
Diseases of Aquatic Organisms 04/2007; 74(3):249-53. · 1.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepatic iron overload in hemochomatosis patients can be highly variable but in general it develops in older patients. The purpose of this study was to compare development of iron load in of beta2m-/- and Hfe-/- mice paying special attention to liver pathology in older age groups. Liver iron content of beta2m-/-, Hfe-/- and control B6 mice of different ages (varying from 3 weeks to 18 months) was examined. Additional parameters (haematology indices, histopathology, lipid content and ferritin expression) were also studied in 18-month-old mice. The beta2m-/- strain presents higher hepatic iron content, hepatocyte nuclear iron inclusions, mitochondria abnormalities. In addition, hepatic steatosis was a common observation in this strain. In the liver of Hfe-/- mice, large mononuclear infiltrates positive for ferritin staining were commonly observed. The steatosis commonly observed the beta2m-/- mice may be a reflection of its higher hepatic iron content. The large hepatic mononuclear cell infiltrates seen in Hfe-/- stained for ferritin, may point to the iron sequestration capacity of lymphocytes and contribute to the clarification of the differences found in the progression of hepatic iron overload and steatosis in older animals from the two strains.
International Journal of Experimental Pathology 09/2006; 87(4):317-24. · 2.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Despite heavy alcohol consumption, only a low percentage of heavy drinkers develop liver disease. Imbalances in T-cell subsets and iron metabolism parameters are common findings in heavy drinkers, yet the possible role played by discrete T-lymphocyte subsets under heavy alcohol consumption remains unclear.Methods: To gain new insights into the possible role played by T lymphocytes during alcohol consumption, characterization of CD28 expression and TcR repertoire in peripheral blood CD4+ and CD8+ T cells by two and three-color flow cytometry was performed. A group of heavy alcohol drinkers (AHD, n= 71) and a group of age-matched controls (n= 81), both HLA-phenotyped and HFE-genotyped, constituted the groups under study.Results: Marked expansions of CD28− T cells within the CD8+ but not the CD4+ T-cell pool were observed in AHD compared with controls. These CD8+CD28− expansions were paralleled by expansions of CD8+ T cells bearing specific TcR Vα/β chains, namely Vβ5.2. Moreover, AHD, but not controls, carrying the H63D mutation in the HFE gene showed significantly higher percentages of CD28− T cells within the CD8+ T-cell pool than AHD carrying the normal HFE gene. Finally, high numbers of CD8+CD28− T cells in AHD were associated with lower levels of the liver-related enzymes ALT and GGT.Conclusions: This study showed that under active ethanol consumption, expansions of discrete CD8+ T-cell subsets occur within the CD8+ T-cell pool, that molecules of the MHC-class I locus seem to influence the extent of the expansions, and that high numbers of CD8+CD28− T cells are associated with low levels of liver enzymes in AHD.
Alcoholism Clinical and Experimental Research 04/2006; 24(4):519 - 527. · 3.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Iron overload in the liver may occur in clinical conditions such as hemochromatosis and nonalcoholic steatohepatitis, and may lead to the deterioration of the normal liver architecture by mechanisms not well understood. Although a relationship between the expression of ICAM-1, and classical major histocompatibility complex (MHC) class I molecules, and iron overload has been reported, no relationship has been identified between iron overload and the expression of unconventional MHC class I molecules. Herein, we report that parameters of iron metabolism were regulated in a coordinated-fashion in a human hepatoma cell line (HepG2 cells) after iron loading, leading to increased cellular oxidative stress and growth retardation. Iron loading of HepG2 cells resulted in increased expression of Nor3.2-reactive CD1d molecules at the plasma membrane. Expression of classical MHC class I and II molecules, ICAM-1 and the epithelial CD8 ligand, gp180 was not significantly affected by iron. Considering that intracellular lipids regulate expression of CD1d at the cell surface, we examined parameters of lipid metabolism in iron-loaded HepG2 cells. Interestingly, increased expression of CD1d molecules by iron-loaded HepG2 cells was associated with increased phosphatidylserine expression in the outer leaflet of the plasma membrane and the presence of many intracellular lipid droplets. These data describe a new relationship between iron loading, lipid accumulation and altered expression of CD1d, an unconventional MHC class I molecule reported to monitor intracellular and plasma membrane lipid metabolism, in the human hepatoma cell line HepG2.
[Show abstract][Hide abstract] ABSTRACT: Host factors that may influence progression of hepatitis C infection to chronic hepatitis include T-cell responses and iron accumulation. We evaluated the hepatic expression of immunological markers relevant for a cytotoxic response in relation to viral and HFE genotype.
Frozen liver biopsies were obtained at diagnosis from 28 HFE genotyped patients. Sections stained for CD8, MHC-I, beta(2)m, HFE and CD68 were analyzed blind by morphometry. Response to therapy was available in 12 cases.
A negative correlation was found between the number of CD8(+) cells and fibrosis. CD8(+) cells localized as clusters in portal tracts and sinusoids and were seen interacting with MHC-I positive lining cells. MHC-I and beta(2)m were expressed mainly in the endothelial and Kupffer cells. HFE was expressed in most, but not all, round and dendritic CD68(+) cells. Patients with virus genotype 3a had higher hepatic MHC-I and HFE expression, and a better-sustained response to IFN therapy than other patients.
In chronic hepatitis C virus infection MHC-I expression in the liver seems to relate to viral-genotype. In addition, the expression of MHC-I molecules by Kupffer cells places them as probable important players in the host response to HCV.
Journal of Hepatology 09/2004; 41(2):319-26. · 9.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Variability in T-lymphocyte numbers is partially explained by a genetic regulation. From studies in animal models, it is known that the Major Histocompatibility Complex (MHC) is involved in this regulation. In humans, this has not been shown yet. The objective of the present study was to test the hypothesis that genes in the MHC region influence the regulation of T-lymphocyte numbers. Two approaches were used. Association studies between T-cell counts (CD4(+) and CD8(+)) or total lymphocyte counts and HLA class I alleles (A and B) or mutations in the HFE (C282Y and H63D), the hemochromatosis gene, in an unrelated population (n = 264). A second approach was a sibpair correlation analysis of the same T-cell counts in relation to HLA-HFE haplotypes in subjects belonging to 48 hemochromatosis families (n = 456 sibpairs). In the normal population, results showed a strong statistically significant association of the HLA-A*01 with high numbers of CD8(+) T cells and a less powerful association with the HLA-A*24 with low numbers of CD8(+) T cells. Sibpair correlations revealed the most significant correlation for CD8(+) T-cell numbers for sibpairs with HLA-HFE-identical haplotypes. This was not observed for CD4(+) T cells. These results show that the MHC region is involved in the genetic regulation of CD8(+) T-cell numbers in humans. Identification of genes responsible for this control may have important biological and clinical implications.
[Show abstract][Hide abstract] ABSTRACT: Beta2-microglobulin knockout (beta2m-/-) mice represent an instructive model of spontaneous iron overload resembling genetic hemochromatosis. The mechanism of iron accumulation in this mouse model may be more complex than involving the MHC class I-like protein HFE. We report that beta2m-deficient mice, like Hfe-/- mice, lack the adaptive hepatic hepcidin mRNA increase to iron overload. The inverse correlation of hepatic iron levels and hepcidin mRNA expression in six beta2m-/- mice underlines the importance of hepcidin in regulating body iron stores. In contrast to Hfe-/- mice, beta2m-deficient mice display increased expression of the duodenal iron transporters DMT1 and ferroportin 1. This result implicates a broader role of beta2m in mammalian iron metabolism, suggesting that (an) additional beta2m-interacting protein(s) could be involved in controlling iron homeostasis, and highlighting the emerging connection of iron metabolism with the immune system.
[Show abstract][Hide abstract] ABSTRACT: Hereditary hemochromatosis (HH) is classically associated with a Cys282Tyr (C282Y) mutation of the HFE gene. Non-C282Y HH is a heterogeneous group accounting for 15% of HH in Northern Europe. Pathogenic mutations of the transferrin receptor 2 (TfR2) gene have been identified in 4 Italian pedigrees with the latter syndrome. The goal of this study was to perform a mutational analysis of the TfR2 and HFE genes in a cohort of non-C282Y iron overload patients of mixed ethnic backgrounds. Several sequence variants were identified within the TfR2 gene, including a homozygous missense change in exon 17, c2069 A-->C, which changes a glutamine to a proline residue at position 690. This putative mutation was found in a severely affected Portuguese man and 2 family members with the same genotype. In summary, pathologic TfR2 mutations are present outside of Italy, accounting for a small proportion of non-C282Y HH.
[Show abstract][Hide abstract] ABSTRACT: The major histocompatibility complex (MHC) shows a remarkable conservation of particular HLA antigens and haplotypes in linkage disequilibrium in most human populations, suggesting the existence of a convergent evolution. A recent example of such conservation is the association of particular HLA haplotypes with the HFE mutations. With the objective of exploring the significance of that association, the present paper offers an analysis of the linkage disequilibrium between HLA alleles or haplotypes and the HFE mutations in a Portuguese population. Allele and haplotype associations between HLA and HFE mutations were first reviewed in a population of 43 hemochromatosis families. The results confirmed the linkage disequilibrium of the HLA haplotype HLA-A3-B7 and the HLA-A29 allele, respectively, with the HFE mutations C282Y and H63D. In order to extend the study of the linkage disequilibrium between H63D and the HLA-A29-containing haplotypes in a normal, random population, an additional sample of 398 haplotypes was analyzed. The results reveal significant linkage disequilibrium between the H63D mutation and all HLA-A29-containing haplotypes, favoring the hypothesis of a co-selection of H63D and the HLA-A29 allele itself. An insight into the biological significance of this association is given by the finding of significantly higher CD8(+) T-lymphocyte counts in subjects simultaneously carrying the H63D mutation and the HLA-A29 allele.
[Show abstract][Hide abstract] ABSTRACT: An earlier study of reference values of iron parameters in Portugal showed significant differences between populations from northern and southern villages. This study addresses the question of the geographical distribution in Portugal of the two main mutations (C282Y and H63D) of the hereditary hemochromatosis gene, HFE. For that purpose, a stratified sample of 640 anonymous dried blood spot samples was randomly selected from the major regions of Portugal: North, Center, Lisbon and the Tagus Valley, Alentejo and Algarve. Differences in the geographical distribution of these two mutations were observed thus confirming the presumed differences between the age of the two mutations which is compatible with the postulated Celtic/Nordic origin of the C282Y mutation. The finding of a significantly higher allelic frequency of the C282Y mutation in the North (0.058) than in the South (0.009) could also point to an effect of differential selective forces acting in the different geographical areas of the country. Data on archaeological, ethnographic and linguistic records and on the North/South distribution of Portuguese cattle breeds of European or African origin have also been reported. In addition to their interest for population genetics, the results represent a reminder of the need to take into account regional differences in the design of strategies for population screening of hereditary hemochromatosis.
European Journal of HumanGenetics 12/2001; 9(11):843-8. · 4.32 Impact Factor