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The aim of the present study was to investigate the environmental dispersal of Cryptosporidium spp. and Giardia duodenalis in two distinct areas (coastal and inland) in Galicia (NW Spain). Faecal samples were collected from healthy asymptomatic domestic (cows and sheep) and wild animals (deer and wild boars) in the selected areas. In each of the selected areas, samples of untreated water (influent) and of treated water (final effluent) were collected from each of the 12 drinking water treatments plants (DWTPs) and 12 wastewater treatment plants (WTPs) under study. Analysis of a single sample from each of the 635 (coastal) and 851 (inland) domestic and wild animals selected at random revealed that the prevalences of cryptosporidiosis and giardiosis in coastal area were 9.2% and 15.9% respectively, and in inland area, 13.7% and 26.7% respectively. In the coastal area, Cryptosporidium spp. oocysts were detected in influent and effluent samples from 2/12 (16.6%) DWTPs and 8/12 (66.6%) WTPs, while G. duodenalis cysts were detected in influent and effluent samples from 3/12 (25.0%) DWTPs and 12/12 (100%) WTPs. The concentrations were notably higher in WTPs; the mean parasite concentrations in the final treated effluent were 10 oocysts per litre and 137.8 cysts per litre for Cryptosporidium and Giardia, respectively. The mean concentration of G. duodenalis cysts per litre was significantly higher (P<0.05) than the mean concentration of Cryptosporidium spp. oocysts per litre in both the influent and the effluent samples from all the treatment plants. In the coastal area, C. parvum, C. hominis and G. duodenalis assemblages A (I and II) and E were most repeatedly detected. In the inland area, C. parvum, C. andersoni and G. duodenalis assemblages A (I and II), B and E were most frequently identified., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Thesafety characteristics of feed used in fish and crustacean aquaculture systems are an essential tool to assure the productivity of those animal exploitations. Safety of feed may be affected by different hazards, including biological and chemical groups. The aim of this preliminary study was to evaluate fungi contamination and the presence of aflatoxins in 87 samples of feed for sea bass, collected in Portugal. Molds were found in 35 samples (40.2%) in levels ranging from 1 to 3.3 log10 CFU∙g(-1). Six genera of molds were found. Aspergillus flavus was the most frequent, found in all positive samples, with a range from 2 to 3.2 log(10) CFU∙g(-1). Aspergillus niger was found in 34 samples (39.1%), ranging from 1 to 2.7 log(10) CFU∙g(-1). Aspergillus glaucus was found in 26 samples (29.9%) with levels between 1 and 2.4 log(10) CFU∙g(-1). Penicillium spp. and Cladosporium spp. were both found in 25 samples (28.7%). Fusarium spp. was found in 22 samples (25.3%), ranging from 1 to 2.3 log(10) CFU∙g(-1). All feed samples were screened for aflatoxins using a HPLC technique, with a detection limit of 1.0 μg∙kg(-1). All samples were aflatoxin negative.

The organization of public clinical laboratories is experiencing changes without, however, an organizational assessment of its effectiveness. The study aimed to determine a parameter of effectiveness for public clinical laboratories of Belo Horizonte, Minas Gerais State, and set cut-off points for the sections of these laboratories. In order to do so, the total production and number of hours worked during a period of 7 months in the year 2008 were consolidated. Due to the entrance of the workers in the mode of production in the laboratories network, it could be observed a variability regarding the performance of these workers. The effectiveness parameter of the network was established in 29.90 tests per hour. As a consequence of this first analysis, the cut-off points are: 15.50 for the hematology section; 67.29 for chemistry; 6.45 for parasitology; 11.35 for urinalysis; 4.94 for microbiology and 19.03 for immunology. From these results, it was concluded that the working process in laboratories can generate a decrease in effectiveness.

We have previously identified the expression of an estradiol (E2)-related molecule by Schistosoma haematobium total antigen (Sh). We now show that this molecule has an antagonistic effect of estradiol in vitro. Our results are consistent with the existence of an estrogenic molecule that antagonizes the activity of estradiol. We found evidence for this molecule as we identified and characterized by mass spectrometry new estrogenic molecules previously unknown, present in schistosome worm extracts and sera of Schistosoma-infected individuals. We also show that Sh is able to interact in vitro with estrogen receptor (ER), explaining how host endocrine system can favor the establishment of schistosomes. These findings highlight the exploitation of the host endocrine system by schistosomes and represent an additional regulatory component of schistosome development that defines a novel paradigm enabling host-parasite interactions. The identification of these molecules opens new ways for the development of alternative drugs to treat schistosomiasis.

To understand the situation of water contamination with Cryptosporidium spp. and Giardia spp. in the northern region of Portugal, we have established a long-term program aimed at pinpointing the sources of surface water and environmental contamination, working with the water-supply industry. Here, we describe the results obtained with raw water samples collected in rivers of the 5 hydrographical basins. A total of 283 samples were analyzed using the Method 1623 EPA, USA. Genetic characterization was performed by PCR and sequencing of genes 18S rRNA of Cryptosporidium spp. and beta-giardin of Giardia spp. Infectious stages of the protozoa were detected in 72.8% (206 of 283) of the water samples, with 15.2% (43 of 283) positive for Giardia duodenalis cysts, 9.5% (27 of 283) positive for Cryptosporidium spp. oocysts, and 48.1% (136 of 283) samples positive for both parasites. The most common zoonotic species found were G. duodenalis assemblages A-I, A-II, B, and E genotypes, and Cryptosporidium parvum, Cryptosporidium andersoni, Cryptosporidium hominis, and Cryptosporidium muris. These results suggest that cryptosporidiosis and giardiasis are important public health issues in northern Portugal. To the authors' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in raw water samples in the northern region of Portugal.

Golden hamsters were inoculated with Schistosoma haematobium cercariae to examine histological lesions at different time points over an 18 month period of infection. Hamsters were sacrificed 26 weeks and 82 weeks after inoculation. The parasite was found in the blood and in the liver of infected animals as was expected, but we found exclusively male worms, no female worms nor eggs. Interestingly we observed unexpected hepatic lesions induced by S. haematobium adult male worms alone in the golden hamster, characteristic of schistosome eggs. Samples from liver, kidneys, lungs, bladder and gastrointestinal tract were collected during necropsy to evaluate injuries induced by S. haematobium. Notably we observed hepatitis in the liver of infected hamsters, no lesions were found in other organs. We also found liver fibrosis in infected hamsters. This study provides further experimental evidence for the role that schistosome worms, and their derived antigens, may play in the pathology of the infection and modulation of liver chronic inflammation in the murine model of schistosomiasis.

Serotyping was previously described as a promising method for typing strains of Toxoplasma gondii. The majority of precedent studies utilized serum samples collected from human patients with different T. gondii-associated pathologies. The aim of this work was to study the applicability of the same procedure for serotyping naturally infected meat-producing animals. An ELISA test based on GRA6 and GRA7 C-terminal polymorphic peptides was used. Peptide GRA6II has polymorphisms specific for the archetypal strains type II, GRA6I/III for strains type I and III, GRA7I for strains type I and GRA7III for strains type III. As reference material, and to validate this approach, serum samples from eleven free-range chickens and fifteen pigs used for Toxoplasma genotypes isolation were selected. These strains integrate the Biological Resource Centre (BRC) ToxoBS Bank. Three serum samples from chickens and two from pigs had serotyping results in agreement with genotyping. Thirty-five serum samples from chickens, twenty-nine from pigs and fifty from sheep, seropositive for T. gondii, from which no isolate was obtained, were also serotyped. Serotype III appeared significantly more frequent among sheep. Our results show that serotyping still need refinement, but may become a valuable tool for typing Toxoplasma strains from animal origin.

Schistosoma haematobium is a parasitic flatworm that infects millions of people, mostly in the developing world, and is associated with high incidence of bladder cancer although why is not clear. But our group was able to define the mechanistic relationship for the first time between infection of S. haematobium and cancer. We used in vitro models to demonstrate the presence of informative carcinogenesis-associated phenotypes in CHO cells exposed to Sh total antigen, in which we showed increased cell proliferation, decreased apoptosis, up regulation of the anti-apoptotic molecule Bcl-2, down regulation of the tumor suppressor protein p27, and increased cell migration and invasion. We further discuss the molecular and cellular events that might be responsible for schistosomiasis-related bladder cancer.

Objective: The aim of this study was to compare the behavior of the major chewing muscles (anterior temporalis, masseter and suprahyoid muscles) by using two commercial chewing gums (A and B) and two insipid materials (cotton ball and Parafilm M) through analysis of electromyographic signals., Method: Ten female young adult subjects, aged from 18 to 27 years, with normal occlusion and no history of craniomandibular disorder were studied The masticatory activity was performed with the subjects comfortably sat so that the Frankfurt plane was parallel to the floor. Electromyographic exams were carried out using bipolar surface passive mini-electrodes positioned on the anterior temporalis, masseter, and suprahyoid muscles. The subjects were guided to chew bilaterally and simultaneously each one of the materials, in aleatory sequence. For the study of the masticatory activity the electromyographic signals were processed through rectification, linear envelope and normalization, so that the coefficient of variation obtained from the procedure was comparatively analyzed. All data were submitted to analysis of variance (uni- and multi-varied)., Results: The results of this study indicated that the best materials for electromyographic studies on the chewing were Parafilm and cotton ball because they demonstrated the smallest coefficients of variation., Conclusion: In addition, Parafilm showed the best palatability. In a general way, these materials are indicated for the accomplishment of electromyographic exams, since they demonstrated a smaller coefficient of variation as compared to the other materials.

The objective of the present study was to determine the degree of contamination by Cryptosporidium spp. and Giardia duodenalis in a river basin in a livestock farming area in Galicia (NW, Spain). Water samples (50 l) were collected at 22 points in the main basin (including 5 recreational areas), and at the source and mouth of the 3 most important rivers and at the mouth of a smaller, secondary river. Faecal samples were collected from dairy cattle selected at random from 18 herds farmed in the area. A total of 139 neonatal calves, 480 heifers and 697 cows were sampled. The prevalence, intensity of infection and the risk associated with the spread of infection by both enteropathogens were determined. Water and faecal samples were collected in spring, summer, autumn and winter of 2007. The species and genotypes of these parasites present in the water samples were identified. In both water and faecal samples, more parasitic stages were collected in spring and summer than in autumn and winter. In spring, Cryptosporidium spp. oocysts were detected in 33 (9.4%) cows from 13 (72.2%) herds, and G. duodenalis cysts were detected in 56 (16.0%) cows from 15 farms (83.3%); the intensity of infection ranged from 5 to 7895 G. duodenalis cysts per gram of faeces. Infective stages of Cryptosporidium spp. and G. duodenalis were also detected in respectively 26 (89.6%) and 27 (93.1%) water samples, in spring. The mean concentrations of parasites ranged from 2 to 1200 Cryptosporidium spp. oocysts per litre and from 2 to 400 G. duodenalis cysts per litre. Cryptosporidium parvum, C. andersoni, C. hominis and assemblages A-I, A-II, E of G. duodenalis were detected. The presence of both protozoans must be monitored in cattle, in sources of water used for recreational purposes and in artificial waterways used by farmers (water channels, animal drinking water and drainage systems).

Schistosome worms are blood-dwelling flukes that cause chronic infection in more than 200 million people and are thought to be responsible for 500,000 deaths annually. During infection with Schistosoma haematobium, eggs are deposited in the mucosa and submucosa of the bladder and lower ureters. Squamous cell carcinoma (SCC) of the bladder is a long-term sequela of chronic infection. The mechanisms underlying the association between S. haematobium and SCC of the bladder are largely unknown, with all reports to date exclusively demonstrating epidemiological evidence linking S. haematobium infection with SCC of the bladder. We hypothesised that the parasite antigens might induce alterations in epithelial cells towards cancer. For this we used Chinese Hamster Ovary (CHO) cells and treated the cells in culture with S. haematobium total antigen (Sh). Our results showed increased proliferation, increased S-phase and decreased apoptosis, as well as down-regulation of tumour suppressor p27 and up-regulation of anti-apoptotic molecule Bcl-2 in Sh-treated cells compared with controls. We also found increased migration and invasion. To our knowledge, this is the first report demonstrating alterations of normal epithelial cells as a direct effect of S. haematobium antigens.

Schistosoma haematobium is endemic in several regions of Africa and has been shown to be associated with predominantly squamous cell bladder carcinoma. The mechanisms underlying the association between S. haematobium and bladder squamous cell carcinoma is largely unknown. All the reports so far, demonstrate exclusively an epidemiological evidence linking S. haematobium infection with squamous cell bladder carcinoma. We hypothesized that these parasite antigens might induce tumourigenesis. For this, we used normal mammalian cells of Chinese hamster ovary (CHO) and treated the cells in culture with S. haematobium total antigen (Sh). Our results showed increased proliferation in Sh-treated cells in comparison with the controls. The CHO cells exposed to Sh were inoculated subcutaneously into male nude mice and formed sarcomas (n = 5/5). The cells from the sarcomas expressed vimentin filaments and were negative to cytokeratin. Our results demonstrate for the first time that S. haematobium antigens induce tumour development in nude mice.

Estradiol is a steroid hormone secreted principally by the ovarian follicles in vertebrate animals. We have identified the production of an estradiol-related molecule in the trematodes Schistosoma haematobium and Schistosomiasis mansoni. We show in this work that this molecule related to estradiol is present in schistosome worm extracts. The detection method ELISA specific for estradiol, revealed the expression of this estradiol-related molecule in schistosome worm extracts, but not in Fasciola hepatica worm extracts. Our results demonstrate for the first time the production of an estradiol-related compound by a human parasite of the genus Schistosoma.

Toxoplasmosis is one of the most common food borne disease world-wide. Among food animals, sheep seems to having higher prevalence of Toxoplasma gondii infection. However, there is no consensus about the best cut-off for serodiagnosis in sheep. To estimate the more adequate cut-off value of Modified Agglutination Test (MAT) for serodiagnosis in sheep, a commercial ELISA kit was used as a golden standard. Evaluation of the optimal sensitivity and specificity was calculated using Youden's J-statistics. Values obtained were used to estimate the prevalence of sheep toxoplasmosis. One thousand four hundred and sixty seven blood samples were collected randomly from 160 farms from northern Portugal, representing approximately 10% of the ovine population from the region. All sera were tested for anti-T. gondii antibodies using the MAT. One hundred nine sheep (7.4%) presented a MAT titer > or = 1:80; 45 (3.0%) a MAT titer of 1:40; 97 (6.6%) a MAT titer of 1:20 and 1216 (83.0%) a MAT titer < or = 1:20. The best Youden's J-statistic was obtained at 1:20 titer (0.752), with 86.15% of sensitivity and 89.09% of specificity with negative and positive predictive values of 90.32% and 84.48% respectively, suggesting that the 1:20 was the most appropriate cut-off for serodiagnosis of toxoplasmosis in sheep. Assuming this cut-off, the prevalence of toxoplasmosis in the studied population was 17.1% and 92 (57.5%) of the 160 studied flocks having one or more positive sheep. Those results indicate that toxoplasmosis in Portugal should be considered in the differential diagnosis of abortions in sheep and neurological signs in lambs. Furthermore, while Portugal produces ovine meat for internal consumption and for exportation, isolation of T. gondii from ovine meat and further characterization of the isolates will be needed to understand the risk that ovine toxoplasmosis may represent for human health.

Cashew apple and guava residues from fruit juice industry were prepared as dehydrated fruit powders and used at different levels of wheat flour substitution for cookies formulations. The effects of guava and cashew apple fruit powders supplementation on physicochemical and sensorial characteristics of the cookies were evaluated. The pH, fibre and protein content were significantly affected. Biscuits with 15 g and 20 g/100g cashew apple and guava fruit powders showed the highest scores for sensorial attributes, respectively. The supplementation seems to be suited for wheat flour substitution and it is possible to obtain cookies with value-added food ingredient within the standards.

The lipase activity of nine strains of six chromoblastomycosis agents (Fonsecaea pedrosoi, Phialophora verrucosa, Cladophialophora bantianum, Cladophialophora carrionii, Rhinocladiela aquaspersa and Exophiala jeanselmei) grown on solid medium was investigated using Fourier transform infrared spectroscopy and hierarchical clustering analysis. The data was quantified by p-nitrophenyl palmitate assay using partial least squares (PLS) regression. These methods allowed the correlation of six genera and species within the 1230-1650 and 2800+3000 cm(-1) spectral ranges among strains grown for 14 days from their respective lipolytic activity with RMSEV=0.048 and R2val=0.95 and ten latent variables. The lipolytic activity also was predicted using PLS models with 1230-1650 and 2800-3000 cm(-1) and 900-1450 cm(-1) spectral ranges for strains grown for 21 days. The separate analysis of F. pedrosoi strains yielded a prediction model for biomass at 21 days with RMSEV=0.065 and R2val=0.95 with eight latent variables using (1100-1300)+(1330-1460)+(1550-1650) cm(-1) spectral regions The best model obtained with the clustering of P. verrucosa, C. bantianum, C. carrionii, R. aquaspersa and E. jeanselmei strains was constructed with the same spectral ranges, but with RMSEV=0.074 and R2val=0.94 and ten latent variables. Infrared spectroscopy is suitable for the quantitation of extracellular lipase activity linked to the biomass of chromoblastomycosis agents.

To evaluate the presence of Cryptosporidium spp. and Giardia duodenalis in the influent and final effluent of sixteen drinking water treatment plants located in a hydrographic basin in Galicia (NW Spain) - in which the principal river is recognised as a Site of Community Importance (SCI) - estimate the efficiency of treatment plants in removing these protozoans and determine the species and genotypes of the parasites by means of a molecular assay. All plant samples of influent and final effluent (50-100 l) were examined in the spring, summer, autumn and winter of 2007. A total of 128 samples were analysed by method 1623, developed by US Environmental Protection Agency for isolation and detection of both parasites. To identify the genotypes present the following genes were amplified and sequenced: 18S SSU rRNA (Cryptosporidium spp.) and b-giardina (G. duodenalis). The mean concentrations of parasites in the influent were 0.0-10.5 Cryptosporidium spp. oocysts per litre and 1.0-12.8 of G. duodenalis cysts per litre. In the final treated effluent, the mean concentration of parasites ranged from 0.0-3.0 oocysts per litre and 0.5-4.0 cysts per litre. The distribution of results by season revealed that in all plants, the highest numbers of (oo)cysts were recorded in spring and summer. Cryptosporidium parvum, C. andersoni, C. hominis and assemblages A-I, A-II, E of G. duodenalis were detected. Cryptosporidium spp. and G. duodenalis were consistently found at high concentrations in drinking water destined for human and animal consumption in the hydrographic basin under study, in Galicia (NW Spain). It is important that drinking water treatment authorities rethink the relevance of contamination levels of both parasites in drinking water and develop adequate countermeasures.

Samples of the influent and final effluent from 12 wastewater treatment plants from Galicia (NW, Spain) were analyzed for the presence of Cryptosporidium spp. oocysts and Giardia duodenalis cysts. All of the plants discharge effluent to a hydrographic basin in which there are numerous recreational areas and fluvial beaches. The samples (25-50 liters) were collected in spring, summer, autumn and winter of 2007. A total of 96 samples were analyzed using techniques included in the US Environmental Protection Agency Method 1623. To identify the genotypes present, the following genes were amplified and sequenced: 18S SSU rRNA (Cryptosporidium spp.) and beta-giardina (G. duodenalis). Both parasites were detected in influent and effluent samples from all treatment plants (100%) throughout the year, and G. duodenalis always outnumbered Cryptosporidium spp. The mean concentration of G. duodenalis per liter of influent was significantly higher (P<0.05) than the mean concentration of Cryptosporidium spp. per liter of influent. The mean concentrations of parasites in influent samples ranged from 6 to 350 Cryptosporidium spp. oocysts per liter and from 89 to 8305 G. duodenalis cysts per liter. In final treated effluent, the mean concentration of parasites ranged from 2 to 390 Cryptosporidium spp. oocysts per liter and from 79 to 2469 G. duodenalis cysts per liter. The distribution of results per season revealed that in all plants, the highest number of (oo)cysts were detected in spring and summer. Cryptosporidium parvum, Cryptosporidium andersoni, Cryptosporidium hominis and assemblages A-I, A-II, E of G. duodenalis were detected. The risk of contamination of water courses by Cryptosporidium spp. and G. duodenalis is therefore considerable. It is important that wastewater treatment authorities reconsider the relevance of the levels of contamination by both parasites in wastewater, and develop adequate countermeasures.

Background and Objectives: The Brazilian Intensive Care Medicine Association (AMIB) has been stimulating the creation of undergraduate associations/study groups on Critical Care Medicine (CCM), considering them a useful instrument to fill gaps in professional formation. The aim of this study was to evaluate the activities developed by CCM undergraduate study groups in Brazil., Methods: The analyzed information was obtained on answers to a standard questionnaire. Contact by e-mail and/or telephone was made with representatives of all study groups registered on AMIB Committee of Undergraduate Study Groups on CCM (LIGAMI-AMIB) in September of 2007. In that period, there were 33 groups associated to LIGAMI, which 4 doesn't exist anymore, 17 answered to the sent questionnaire and the remaining 12 didn't answer the questionnaire or it was not obtained contact., Results: Most study groups were founded after 2005, coinciding with the LIGAMI-AMIB Committee creation, and they are linked to one or more medical schools. Among the group's activities, the most frequent was theoretical classes (100%), usually supplied by teachers or invited physicians (69%). Other activities include practices on Intensive Care Units (88%), organization of scientific events (77%) and research projects (65%). Most study groups (65%) had already organized some scientific event such as courses and symposia; however, only three had already accomplished the AMIB CCM Introductory Course., Conclusions: The growing number of CCM undergraduate study groups in Brazil demonstrates students' interest for this specialty. Besides, there is a necessity of larger integration between existing groups to change experiences, cooperate in the accomplishment of research projects and participation on national and international events.

To determine the prevalence and intensity of infection of Cryptosporidium spp. and Giardia duodenalis in healthy adult domestic ruminants, faecal samples were collected from 379 cattle of between 3 and 13 years old, 446 sheep and 116 goats selected at random from 60 dairy farms and 38 and 20 herds, respectively, in Galicia (NW Spain). Cryptosporidium spp. oocysts were detected in 32 cows (8.4%), 24 sheep (5.3%) and in nine goats (7.7%) from, respectively, 48.3% of the farms and 34.2 and 30.0% of the herds. The intensity of infection in cows ranged between 25 and 5,924 oocysts per gram of faeces (OPG), whereas in sheep and goats, the number of oocysts shed ranged from 8-515 OPG and from 17-782 OPG, respectively. Parasitization by Cryptosporidium spp. was significantly higher (P<0.05) in cows than in sheep and goats. G. duodenalis cysts were identified in 101 cows (26.6%), 86 sheep (19.2%) and 23 goats (19.8%) from, respectively, 96.6% of the farms and 92.1 and 90% of the herds. The number of cysts shed by cows ranged between 15 and 3,042 cyst per gram of faeces (CPG), whereas the intensity of infection in sheep and goats ranged from 16-3010 CPG and from 15-1845 CPG, respectively, and was significantly lower (P<0.05) than in cows and sheep. The number of Cryptosporidium spp. oocysts isolated from sheep and goats was insufficient for successful polymerase chain reaction analysis. Nevertheless, gene sequence analysis of the hsp70 and 18SrRNA genes of Cryptosporidium revealed the presence of only C. parvum in faecal samples from cows. Genotyping studies of the beta-giardin and glutamate dehydrogenase genes of G. duodenalis revealed mainly assemblage E of Giardia in cows, sheep and goat faecal samples. Assemblage B of G. duodenalis was also detected in one sheep sample. These animals should be considered as a possible source of cryptosporidiosis and giardiosis, thereby maintaining the infections on farms and in herds.

Fecal samples from 291 calves and 176 adult cattle in Northern Portugal were screened for Cryptosporidium and Giardia using a formalin-ethyl acetate concentration method. Acid-fast staining techniques for Cryptosporidium oocyst identification and direct microscopic observation of fecal smears for Giardia cyst identification were performed so as immunofluorescence microscopy examination. Polymerase chain reaction methods were employed to determine the genotype of each isolate. Molecular characterization was performed using amplification and sequencing of the hsp70 and 18SrRNA genes of Cryptosporidium and beta-giardin gene and glutamate dehydrogenase for assemblage determination of Giardia duodenalis. Seventy-four out of 291 calves (25.4%) and 8 out of 176 adult bovines (4.5%) were positive for Cryptosporidium. Forty-one out of 291 calf samples (14.1%) and 1 out of 176 adults samples (0.57%) were positive for Giardia. From the Cryptosporidium positive samples we obtained 63 isolates from calves samples and 7 isolates from adult samples. Additionally, Giardia was isolated in 13 out of 41 positive samples from calves and it was also possible to isolate Giardia from the positive adult sample. Molecular characterization of the Cryptosporidium and Giardia isolates showed us that C. parvum and G. duodenalis assemblage E were the prevalent species. C. parvum may infect humans, representing a potential public health risk. On the other hand, the assemblages B and A2 of Giardia, previously described in humans, were here identified in cattle. Further studies will be needed for determine the importance of cattle as carrier of zoonotic assemblages of G. duodenalis.

C57BL/10ScCr mice, lack Toll-like receptor 4 and a functional Interleukin-12 receptor. Taking this into account, susceptibility of these mice to Neospora caninum infection was assessed comparatively to that of immunocompetent C57BL/10ScSn mice. C57BL/10ScCr mice inoculated intraperitoneally with 5x10(5)N. caninum tachyzoites showed a high susceptibility to this parasite. All infected C57BL/10ScCr mice were dead by day 8 post-infection whereas all control C57BL/10ScSn mice survived this parasitic challenge. Immunohistochemical analysis of infected C57BL/10ScCr mice showed N. caninum tachyzoites spread in the pancreas, liver, lung, intestine, heart and brain whereas no parasites were detected in similarly infected C57BL/10ScSn controls. The higher susceptibility of C57BL/10ScCr mice to neosporosis correlates with reduced interferon-gamma mRNA expression and increased IL-4 mRNA expression, comparatively to C57BL/10ScSn controls, detected in the spleen after the parasitic challenge. C57BL/10ScCr mice could thus be used as a new experimental model where to study immunobiological mechanisms associated with host susceptibility to neosporosis.

To study experimental Neospora caninum infection initiated at the gastrointestinal tract, Toll-like Receptor 4- and functional IL-12Rbeta2 chain-deficient C57BL/10 ScCr mice were challenged intragastrically with 5 x 10(6) N. caninum tachyzoites. All parasite-inoculated mice eventually died with disseminated infection. In contrast, immunocompetent BALB/c mice challenged with 1 x 10(7) N. caninum tachyzoites by the intragastric (i.g.) or the intraperitoneal (i.p.) route remained alive for at least 6 months. Expansion of splenic B- and T-cells, the latter displaying both activated and regulatory phenotypes, and increased levels of IFN-gamma and IL-10 mRNA were detected in both groups of infected BALB/c mice compared with non-infected controls, whereas in the Peyer's patches only IFN-gamma mRNA levels were found to be increased. Parasite-specific IgG1, IgG2a and IgA antibody levels were elevated in the sera of all infected mice, whereas increased N. caninum-specific IgA levels were detected in intestinal lavage fluids of i.g. challenged mice only. These results show that N. caninum infection can be successfully established in mice by i.g. administration of tachyzoites. They also show that the immune response elicited in i.g. or i.p. infected BALB/c mice, although conferring some degree of protection, was not sufficient for complete parasite clearance.

Objectives: We assessed the safety and efficacy of early administration of abciximab prior to percutaneous coronary intervention (PCI) in acute myocardial infarction (AMI) patients., Background: Research suggests that platelet glycoprotein IIb/IIIa receptor inhibitors, e.g. abciximab, may improve myocardial perfusion. In particular, early administration in the emergency department, prior to PCI, may result in more effective reperfusion., Methods: Eighty AMI patients with planned PCI were randomized in a double-blind fashion to receive a 0.25 mg/kg abciximab bolus either "early" in the emergency department or "late" in the catheterization laboratory after angiographic assessment. In total, 74 patients underwent PCI after diagnostic angiography, all of which then received an abciximab infusion of 0.125 microg/kg/min for 12 hr., Results: Prior to PCI, no significant differences were observed between the two groups regarding the angiographic endpoints or ST-segment resolution. After PCI, thrombolysis in MI (TIMI) frame count (TFC) was significantly improved in patients treated early rather than in those treated late (23 +/- 10 vs. 41 +/- 35; P = 0.02). Consistent trends, also favoring early treatment, were observed for TIMI flow grade 3 (TFG 3), corrected TFC (CTFC), and TIMI myocardial perfusion grade 3 (TMPG 3). Nine deaths (4 early, 5 late) and six significant bleeds (4 early, 2 late) were observed at 30 days after randomization., Conclusions: Early administration of abciximab is both feasible and safe in patients planned for primary PCI, increasing coronary flow and myocardial reperfusion after PCI, as demonstrated by significantly decreased TFC scores and trends toward improvements in TFG, CTFC, and TMPG.

We report here the first Portuguese case of acute fatal granulomatous encephalitis attributed to Balamuthia mandrillaris, initially thought to be a brain tumor, which had a progressive and fatal outcome. Balamuthia mandrillaris is a free-living amoeba recognized as an uncommon agent of granulomatous encephalitis. Infections have been identified in immunocompromised hosts and in immunocompetent pediatric patients. Balamuthia infections are very rare, with only two reported cases in Europe. The case presented here occurred in a previously healthy boy who died 5 weeks after the onset of the symptoms. No evidence of immunological deficiency was noted, and testing for human immunodeficiency virus antibodies was negative. The symptoms were initially thought to be the result of a tumor, but histopathologic examination showed evidence of amoebic infection. Immunofluorescence staining of brain tissue identified B. mandrillaris as the infectious agent. The diagnosis was confirmed with PCR by detecting Balamuthia DNA in formalin-fixed brain tissue sections. Despite initiation of empirical antimicrobial therapy for balamuthiasis, the patient died 3 weeks after being admitted to the hospital. No source of infection was readily apparent.

Neosporosis is a major cause of abortion in cattle all over the world. Congenital transmission as well as horizontal transmission by ingestion of oocysts has been described. The detection of Neospora caninum DNA in bull semen warrants the investigation of possible transmission through the use of contaminated semen. In this experiment four cows were artificially inseminated with frozen-thawed semen contaminated in vitro with viable N. caninum tachyzoites (group A) and four control cows were inseminated with tachyzoites-free frozen-thawed semen, from the same bull (group B). Serum samples were collected 15 days before the artificial insemination (AI) and at days 10, 14, 21, 28, 45, 60 and 75 post-insemination. All sera samples were tested for neosporosis by direct agglutination test (DAT). Three of the cows from group A had negative DAT titers (< or =1:20) in all of the samples, while the fourth cow from this group had a low titer of antibodies (1:80) at day 10, and became negative at day 45, suggesting a stimulation of the immune system by the tachyzoites placed in uterus, rather than the induction of an infection. All of the cows from group B had negative DAT titers (< or =1:20) in all of the samples. These results suggest that transmission of neosporosis by artificial insemination with frozen-thawed semen is an unlikely event.

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 225 free-range chickens (Gallus domesticus) from Portugal was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 61 chickens with titers of 1:5 in 8, 1:10 in 6, 1:20 in 3, 1:40 in 23, 1:80 in 5, 1:160 in 4, 1:320 in 8, and 1:640 or higher in 4. Hearts, leg muscles, and brains of 15 seropositive (MAT 1:10 or higher) chickens were bioassayed individually in mice. Tissue from 38 chickens with titers of 1:5 or less were pooled and fed to a T. gondii-free cat. Feces of the cat were examined for oocysts, but none was found. Toxoplasma gondii was isolated from 16 of 19 chickens with MAT titers of 1:10 or higher. Genotyping of 12 of these 16 isolates with polymorphisms at the SAG2 locus indicated that 4 were type III, and 8 were type II. None of the isolates was lethal for mice. Phenotypically, T. gondii isolates from chickens from Portugal were different from those of T. gondii isolates from chickens from Brazil.

Little is known of Toxoplasma gondii infections in animals in Portugal. In the present paper, we report the first isolation of viable T. gondii from pigs in Portugal. Antibodies to T. gondii were found in 52 (15.6%) of 333 pigs prior to slaughter using the modified agglutination test (MAT) at a serum dilution of 1:20. Attempts were made to isolate T. gondii from 37 seropositive pigs. Samples of brain and/or heart from each pig were digested in acid pepsin, and bioassayed into mice. Viable T. gondii was isolated from 15 pigs. Restriction fragment length polymorphism on products of SAG2 locus amplified by PCR and microsatellite analysis revealed that 11 isolates were Type II and four were Type III. The results indicate that phenotypically and genetically T. gondii are similar to isolates from pigs from the U.S.

Activation of B cells occurring in hosts infected with protozoan parasites has been implicated either in protective or parasite-evasion immune-mediated mechanisms. Intraperitoneal inoculation of Neospora caninum tachyzoites into BALB/c mice induces an acute response characterized by a rapid increase in the numbers of CD69-expressing peritoneal and splenic B cells. This early B-cell stimulatory effect preceded an increase in the numbers of total and immunoglobulin-secreting splenic B cells and a rise in serum levels of N. caninum-specific immunoglobulins, predominantly of the immunoglobulin G2a (IgG2a) and IgM isotypes. Increased numbers of B cells expressing the costimulatory molecules CD80 and CD86 were also observed in the N. caninum-infected mice. The B-cell stimulatory effect observed in mice challenged with N. caninum tachyzoites was reduced in mice challenged with gamma-irradiated parasites. Contrasting with the peripheral B-cell expansion, a depletion of B-lineage cells was observed in the bone-marrow of the N. caninum-infected mice. Intradermal immunization of BALB/c mice with diverse N. caninum antigenic preparations although inducing the production of parasite-specific antibodies nevertheless impaired interferon-gamma (IFN-gamma) mRNA expression and caused lethal susceptibility to infection in mice inoculated with a non-lethal parasitic inoculum. This increased susceptibility to N. caninum was not observed in naïve mice passively transferred with anti-N. caninum antibodies. Taken together, these results show that N. caninum induces in BALB/c mice a parasite-specific, non-polyclonal, B-cell response, reinforce previous observations made by others showing that immunization with N. caninum whole structural antigens increases susceptibility to murine neosporosis and further stress the role of IFN-gamma in the host protective immune mechanisms against this parasite.

Objective: To assess the role of myocardial contrast echocardiography (MCE) in early identification of myocardial viability in patients with residual segmental dysfunction after myocardial infarction and primary angioplasty (PA), in comparison with dobutamine stress echocardiography (DSE), using late functional recovery as gold standard., Design: Prospective study for comparison of the two methods., Setting: Hospital., Patients: 17 patients (11 male, 53 +/- 11 years old) were consecutively included, with a first myocardial infarction and PA, with residual segmental akinesis or dyskinesis and good echocardiographic window., Methods: All patients underwent: a) baseline echocardiographic study, MCE, and DSE obtained at 4.0 +/- 1.2 days after PA; b) late echocardiographic study performed at 4.4 +/- 0.8 months after PA. MCE was performed with Optison, administered as a slow infusion via a peripheral vein and a modality of real-time perfusion imaging with power pulse inversion and flash and subsequent data acquisition of triggered end-systolic images. Segmental contractility and perfusion were assessed using a 16-segment model. Perfusion assessment was qualitative (three perfusion patterns) and quantitative (ratio of maximal intensity between dysfunctional segments and contralateral normal segments). The viability criterion for MCE was defined as homogenous enhancement in 50% of wall thickness in each segment. The standard criterion for myocardial viability was defined as late functional recovery. 6., Results: Viability was present in 56 (63.6%, Group 1) of dysfunctional segments and was absent in the remaining 32 (36.4%, Group 2). The sensitivity of DSE for viability was 80.0%, while specificity was 86.5%. The positive and negative predictive values were, respectively, 91.8% and 69.6%. MCE yielded a sensitivity of 96.5% and specificity of 78.1%, while positive and negative predictive values were respectively 86.2% and 94.1%. With the two methods together, the positive predictive value was 90.3% and negative was 80.0%. The intensity ratio was higher for viable segments (Group 1) in comparison with non-viable ones (Group 2; p<0.005). 7., Conclusions: This study showed a potentially valuable role for MCE in assessing viability in patients with myocardial infarction and PA. In comparison with DSE, MCE yielded a higher negative predictive value as well as a high positive predictive value. The use of both methods together is promising as a useful tool for early assessment of viability after primary angioplasty.

This study was carried out to determine the prevalence of neosporosis in an area of intensive dairy production, in Portugal. Sera samples were obtained in a random basis from 114 cows in 49 herds (group A), and from 1237 cows in 36 herds with a history of abortion outbreaks (group B). All sera samples were tested for neosporosis by direct agglutination test (DAT). Additionally, attempts to isolate Neospora caninum in 42 aborted bovine fetuses from 38 dairy herds (group C) were carried out, utilizing a bioassay with immuno-depressed Swiss Webster mice. Parasitological confirmation was done by indirect fluorescent antibody test (IFAT). The prevalence of neosporosis in the group A was 28%. Group B had a significantly (P < 0.001) higher prevalence (46%) and Neospora caninum was isolated in 36% of the aborted fetuses (group C). These results indicate that neosporosis, a disease only recently (2001) diagnosed in Portugal, has a high prevalence in the country, particularly in populations with a story of abortion. Thus, neosporosis should systematically be considered in the differential diagnosis of abortion. In the context of embryo transfers, the importance of selecting Neospora-free embryo recipients is discussed, as well as the pertinence of assessing the Neospora status of traded and imported cattle.

Neospora caninum was isolated from the brain of a 6-mo-old aborted bovine fetus from Galicia, Spain. The fetal brain homogenate was inoculated intraperitoneally into cortisonized mice. The peritoneal exudate from the infected mice, along with mouse sarcoma cells (Tg180), was inoculated into a second group of mice, and parasites were harvested from the peritoneal exudate. The parasites were adapted to in vitro growth in Vero monolayers. The tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies on indirect fluorescent antibody test. The tachyzoites were lethal to interferon gamma gene knock out (KO) mice and could be identified immunohistochemically in the tissues. The identity of the parasite was also confirmed by polymerase chain reaction amplification of N. caninum-specific fragments. The sequences of the amplified gene 5 fragments (GenBank AY494944) were found to be identical to that of an Austrian isolate of N. caninum but not to that of NC-1. This is the first isolation of viable N. caninum from Spain.

This work reports the detection of specific immunoglobulins (Ig) against rFh8, a recombinant Fasciola hepatica adult worm excretion-secretion antigen, in sera from experimentally (rabbit, Wistar rat, cattle, and sheep), or naturally (human) infected hosts. In the case of laboratory experimental models the study revealed significant differences between rabbits, which recognized the recombinant antigen all along the infection, and Wistar rats, which showed high anti-rFh8 Ig levels only for a short period of the infection. Available sera from experimentally infected cattle and sheep, as well as sera from naturally F. hepatica-infected humans, also contained significant levels of Ig against rFh8, suggesting that Fh8 was produced by F. hepatica at a very early stage of infection in all hosts so far analyzed and that the rFh8 antigen could be used as a tool for the diagnosis of F. hepatica infections.

A definitive diagnosis of neosporosis in cattle implies the examination of the aborted fetus. However, in many instances fetal material is not available. Therefore, most diagnosis are based on serological tests. At the moment, there are no consensuses about the cut-off for serodiagnosis of neosporosis in cattle, for any test. The objective of the present study was to estimate the best cut-off for the Neospora agglutination test (NAT) for serodiagnosis in cattle. For that purpose, 246 serum samples from 4 groups of dairy cows (aborted Neospora positive; not aborted healthy; aborted other diseases and herds endemic neosporosis) were collected and antibodies anti-N. caninum were determined by NAT. Additionally, immunoblot (IB) was performed with sera from all cows of the endemic neosporosis group and the patterns of seroreactivity were contrasted with the NAT titers for this group of cows. Evaluation of the optimized sensitivity and specificity was calculated using Youden's J-statistics. The best Youden's J-statistic was obtained at 1:40 titer, presenting 100% of sensitivity and 90.4% of specificity with negative and positive predictive values of 100 and 75.0%, respectively. The comparison between NAT titers and the IB banding pattern support the results of the statistical analysis, i.e. titers of 1:40 and higher showed a complex pattern of bands, while titers lower than 1:40 did not precipitate any bands. These results indicate that 1:40 was an optimized NAT cut-off for serodiagnosis in cattle.

Neospora caninum was isolated from the brain of an aborted 4-month-old fetus from a dairy cow herd with endemic neosporosis in Porto, Portugal. The fetal brain homogenate was inoculated interperitoneally first into outbred Swiss Webster mice given dexamethasone and then the peritoneal exudates from these mice was co-inoculated with mouse sarcoma cells in the peritoneal cavity of mice given dexamethasone. N. caninum tachyzoites were seen in peritoneal exudate of the second passage. Tachyzoites from the peritoneal exudate reacted positively with anti-N. caninum antibodies and not with anti-Toxoplasma gondii antibodies and contained N. caninum specific DNA. This Portuguese isolate of N. caninum has been successfully maintained in cell culture. The dam of the aborted fetus had an antibody titer of 1:10240 in the Neospora agglutination test (NAT). Antibodies to N. caninum were found in 76 of 106 cows from this herd in titers of 1:40 in 31, 1:80 in 22, > or =1:160 or more in 23 in the Neospora agglutination test. This is the first isolation of a viable N. caninum-like parasite from any host in Portugal., (Copyright 2002 Elsevier Science B.V.)

Neospora caninum and Toxoplasma gondii are related parasites. The former is a common cause of abortion in dairy cattle. The latter has not been conclusively demonstrated in bovine fetuses. During the course of attempts to isolate N. caninum from aborted fetuses, T. gondii was isolated from 2 aborted fetuses, 1 from Portugal and 1 from the United States. Both isolates were made by bioassay of fetal brains in mice. The fetus from Portugal was about 5 mo in gestational age, and the fetus from the United States was a full-term stillborn.

A coprological sedimentation method is evaluated for quantification of egg shedding in bovine faeces. Through the inclusion of different number of Fasciola hepatica eggs in negative faeces, egg recovery rate and the sensitivity of the method were determined. The mean egg recovery rate of the technique was 76.72+/-15.42%. The sensitivity of the method was 33.3% whenever eggs/g of faeces (EPG) are less than 1.5 and 100% for higher values. To improve the diagnostic accuracy with this technique, it is advisable to increase the sample from 10 to 30g of faeces when manipulating low egg shedding, which allowed for a sensitivity of 83.3%. Regression equations were calculated to quantify the relationship between the number of recovered and incorporated eggs.

Objectives: To evaluate the role of contrast Doppler echocardiography in the assessment of aortic stenosis severity, in comparison with the conventional method and using the catheterization study as the gold standard., Study Design: Prospective comparative study., Setting: Echocardiography Laboratory of Cardiology Department., Population: We included 36 consecutive patients, 20 male, aged 67 +/- 11 years, referred for catheterization study to evaluate aortic stenosis severity., Methods: All patients underwent conventional and contrast Doppler echocardiography and catheterization study. For contrast Doppler, we used Levovist (300 mg/ml infusion). We analyzed the following echocardiographic parameters: a) left ventricle dimensions, wall thickness and function; b) aortic valve morphology; c) post-stenotic aortic valve flow--peak velocity, velocity-time integral, peak gradient, mean gradient; d) left ventricle outflow tract flow--peak velocity, velocity-time integral; e) aortic valve functional area; f) acquisition time and Doppler signal intensity for post-stenotic aortic valve flow. Catheterization parameters analyzed: a) peak aortic valve gradient; b) mean aortic valve gradient., Results: Contrast Doppler yielded higher peak gradients than conventional Doppler (85.6 +/- 30.2 vs 72.6 +/- 26.1 mmHg, p < 0.001), as well as higher mean gradients (51.4 +/- 19.0 vs 44.2 +/- 15.9 mmHg, p < 0.001). Peak gradients obtained with contrast Doppler correlated with those obtained invasively (r = 0.88, p < 0.001), although the values were higher (85.6 +/- 30.2 vs 73.6 +/- 32.0 mmHg, p < 0.001). There was no difference between mean contrast Doppler gradients and mean catheterization gradients, which showed a high correlation (r = 0.89, p < 0.001). There was no difference between peak and mean gradients obtained by conventional Doppler and invasively, which yielded correlations of 0.73 and 0.75, respectively (p < 0.001). The sensitivity of contrast Doppler for detection of severe aortic stenosis was 100% for peak gradient and 84% for mean gradient, while for conventional Doppler it was 68% and 58%. The specificity of contrast Doppler was 65% for peak gradient and 88% for mean gradient, while for conventional Doppler it was, respectively, 58% and 88%. Acquisition time for aortic flow visualization was lower (p < 0.001) and flow intensity higher for contrast Doppler, in comparison with conventional Doppler., Conclusions: In this study, contrast Doppler yielded high correlations with invasive data and higher sensitivity and specificity for detection of severe aortic stenosis than conventional Doppler. It is a useful method for evaluation of aortic stenosis severity.

Objective: To assess the effects of carvedilol in patients with idiopathic dilated cardiomyopathy., Methods: In a double-blind randomized placebo-controlled study, 30 patients (7 women) with functional class II and III heart failure were assessed. Their ages ranged from 28 to 66 years (mean of 43 +/- 9 years), and their left ventricular ejection fraction varied from 8% to 35%. Carvedilol was added to the usual therapy of 20 patients; placebo was added to the usual therapy of 10 patients. The initial dose of carvedilol was 12.5 mg, which was increased weekly until it reached 75 mg/day, according to the patient's tolerance. Clinical assessment, electrocardiogram, echocardiogram, and radionuclide ventriculography were performed in the pretreatment phase, being repeated after 2 and 6 months of medication use., Results: A reduction in heart rate (p = 0.016) as well as an increase in left ventricular shortening fraction (p = 0.02) and in left ventricular ejection fraction (p = 0.017) occurred in the group using carvedilol as compared with that using placebo., Conclusion: Carvedilol added to the usual therapy for heart failure resulted in better heart function.

Presentation of the destructive mechanisms provoked by rheumatoid pannus at the level of ligaments, joints and bones of the wrist. Evaluation of the wrist problems requiring surgical solution, in particular at the radio-carpal and distal radio-ulnar joints. Review of the most useful surgical techniques for soft tissue and bone surgery.

The antigenic components of a semipurified fraction of Leishmania donovani infantum were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using 14 serum samples from dogs with symptomatic visceral leishmaniasis and 11 serum samples from apparently healthy dogs collected in an area endemic for canine visceral leishmaniasis (CVL). It was found that these antigens were composed of many polypeptides, among which seven components recognized by symptomatic CVL sera, had molecular weights of approximately 18, 28, 30, 33, 63, 70, and 72 kilodaltons (kD); two components of 63 and 70 kD were recognized by three of 11 healthly dog sera. These findings suggest that specific antigens induce humoral immune response in dogs with asymptomatic or active visceral leishmaniasis. Infected dogs are not readily identifiable by their symptoms. The potential interest of the immunoblot test for CVL diagnostic purposes is discussed.

The antigenic components of excretory-secretory products (ESP) of adult worms of Fasciola hepatica were revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis using sera from 20 patients infected with F. hepatica. Sera from 184 other parasitic infections and 20 healthy volunteers were also analyzed. It was found that the ESP were composed of more than 11 polypeptides; five components detected in fascioliasis sera had molecular weights of 12.4, 16.4, 19.4, 25, and 27 kilodaltons (kD). Only the 25- and 27-kD components were recognized by all 20 fascioliasis sera. Using the ESP as antigen, it was possible to perform an enzyme-linked immunosorbent assay with a sensitivity of 95% and a specificity of 97%. Sera from other parasitic infections had antibodies to antigenic components with apparent molecular weights of 37, 38.4, 52, 63, 73, 87, 109, and 116 kD that were also found in sera from fascioliasis patients. These findings suggested that the 25- and 27-kD antigenic components may be sensitive and specific for the diagnosis of human fascioliasis.

The microsporidia Enterocytozoon bieneusi is reported in 10-30% of those infected with the human immunodeficiency virus. The parasite appears to be a cause of gastralgia, malabsorption, and diarrhea. A Western blot technique using another microsporidian species, Glugea atherinae, has demonstrated an antigenic similarity between this parasite and E. bieneusi. Preliminary results show the variability of the antigenic profiles obtained from the sera of immunodeficient patients infected with E. bieneusi and also of the cross-reactivity to Glugea sp. antigens of some sera from patients with cryptosporidiosis. The origin of this cross-reactivity is undetermined. The possibility of coinfection with undetected microsporidia is not excluded. These results raise questions concerning the interpretation of serologic data and of the potential immunodiagnostic value of microsporidian antigens.