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Schistosomiasis was diagnosed in two Portuguese soldiers who had been deployed to Portuguese colonies in Africa. The first veteran was diagnosed as having schistosomiasis 34 years after returning from Angola, and the second veteran was found with Schistosoma haematobium infection 40 years after returning from Mozambique. The patient with Schistosoma mansoni had an active infection, because eggs were recovered with living miracidia. The second patient had developed urothelial cancer, but eggs recovered were calcified. [ABSTRACT FROM AUTHOR]

Genotype analysis has revealed a high genetic diversity in strains of Toxoplasma gondii , isolated from a wide range of intermediate hosts and different geographic origins. Diversity is notably striking for parasites from wild hosts in South America, generally referred as non-archetypal genotypes. Those genotypes are implicated in the etiology of severe clinical disease, multivisceral toxoplasmosis, associated with high rate of mortality in immunocompetent individuals. Can we accept specific antibodies produced during T. gondii infection as biomarkers to identify infecting genotypes? Scientific evidence supports a positive response to this question; however, the genetic diversity of T. gondii genotypes organized into 16 haplogroups and collectively defined in 6 major clades, provides a reminder of the complexity and difficulty for the purpose. This review discusses serological approaches to genotyping T. gondii ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sousa, Fernandes and Correia da Costa.)

Toxoplasmosis is the most reported parasitic zoonosis in Europe, with implications in human health and in the veterinary field. There is an increasing need to develop serotyping of Toxoplasma gondii ( T. gondii ) in view of greater sensitivity and efficiency, through the definition of new targets and new methodologies. Nanotechnology is a promising approach, with impact in the development of point-of-care devices. The aim of this work was to develop a simple but highly efficient method for Toxoplasma gondii serotyping based on gold nanoparticles. A simple colorimetric method was developed using gold nanoparticles modified with the synthetic polymorphic peptide derived from GRA6 antigen specific for type II T. gondii . The method of preparation of the gold nanoprobes and the experimental conditions for the detection were found to be critical for a sensitive discrimination between positive and negative sera. The optimized method was used to detect antibodies anti-GRA6II both in mice and human serum samples. These results clearly demonstrate that a biosensor-based immunoassay using AuNPs conjugated with polymorphic synthetic peptides can be developed and used as a serotyping device.

Schistosomiasis is a major neglected tropical disease. Treatment for schistosomiasis with praziquantel (PZQ), which is effective against the parasite, by itself is not capable to counteract infection-associated disease lesions including hepatic fibrosis. There is a pressing need for novel therapies. Due to their biological properties, antioxidant biomolecules might be useful in treating and reverting associated pathological sequelae. Here, we investigated a novel therapy approach based on a combination of anthelmintic drugs with antioxidant biomolecules. We used a host-parasite model involving Bioamphalaria glabrata and newly transformed schistosomula (NTS) of Schistosoma mansoni . For in vitro drug screening assays, was selected several antioxidants and evaluated not only antischistosomal activity but also ability to enhance activity of the anthelmintic drugs praziquantel (PZQ) and artesunate (AS). The morphological alterations induced by compounds alone/combined were assessed on daily basis using an inverted and automated microscope to quantify NTS viability by a fluorometric-based method. The findings indicated that not only do some antioxidants improve antischistosomal activity of the two anthelmintics, but they exhibit activity per se, leading to high mortality of NTS post-exposure. The combination index (CI) of PZQ + Mel (CI = 0.80), PZQ + Resv (CI = 0.74), AS + Resv (CI = 0.34), AS + NAC (CI = 0.89), VDT + Flav (CI = 1.03) and VDT + Resv (CI = 1.06) reveal that they display moderate to strong synergism. The combination of compounds with discrete mechanisms of action might provide a valuable adjunct to contribution for treatment of schistosomiasis-associated disease.

Background: Taenia solium and Taenia saginata are zoonotic parasites of public health importance. Data on their occurrence in humans and animals in western Europe are incomplete and fragmented. In this study, we aimed to update the current knowledge on the epidemiology of these parasites in this region., Methods: We conducted a systematic review of scientific and grey literature published from 1990 to 2015 on the epidemiology of T. saginata and T. solium in humans and animals. Additionally, data about disease occurrence were actively sought by contacting local experts in the different countries., Results: Taeniosis cases were found in twelve out of eighteen countries in western Europe. No cases were identified in Iceland, Ireland, Luxembourg, Norway, Sweden and Switzerland. For Denmark, Netherlands, Portugal, Slovenia, Spain and the UK, annual taeniosis cases were reported and the number of detected cases per year ranged between 1 and 114. Detected prevalences ranged from 0.05 to 0.27%, whereas estimated prevalences ranged from 0.02 to 0.67%. Most taeniosis cases were reported as Taenia spp. or T. saginata, although T. solium was reported in Denmark, France, Italy, Spain, Slovenia, Portugal and the UK. Human cysticercosis cases were reported in all western European countries except for Iceland, with the highest number originating from Portugal and Spain. Most human cysticercosis cases were suspected to have acquired the infection outside western Europe. Cases of T. solium in pigs were found in Austria and Portugal, but only the two cases from Portugal were confirmed with molecular methods. Germany, Spain and Slovenia reported porcine cysticercosis, but made no Taenia species distinction. Bovine cysticercosis was detected in all countries except for Iceland, with a prevalence based on meat inspection of 0.0002-7.82%., Conclusions: Detection and reporting of taeniosis in western Europe should be improved. The existence of T. solium tapeworm carriers, of suspected autochthonous cases of human cysticercosis and the lack of confirmation of porcine cysticercosis cases deserve further attention. Suspected cases of T. solium in pigs should be confirmed by molecular methods. Both taeniosis and human cysticercosis should be notifiable and surveillance in animals should be improved.

Schistosomiasis, a major neglected tropical disease, affects more than 250 million people worldwide. Treatment of schistosomiasis has relied on the anthelmintic drug praziquantel (PZQ) for more than a generation. PZQ is the drug of choice for the treatment of schistosomiasis; it is effective against all major forms of schistosomiasis, although it is less active against juvenile than mature parasites. A pyrazino-isoquinoline derivative, PZQ is not considered to be toxic and generally causes few or transient, mild side effects. Increasingly, mass drug administration targeting populations in sub-Saharan Africa where schistosomiasis is endemic has led to the appearance of reduced efficacy of PZQ, which portends the selection of drug-resistant forms of these pathogens. The synthesis of improved derivatives of PZQ is attracting attention, e.g., in the (i) synthesis of drug analogues, (ii) rational design of pharmacophores, and (iii) discovery of new compounds from large-scale screening programs. This article reviews reports from the 1970s to the present on the metabolism and mechanism of action of PZQ and its derivatives against schistosomes., (Copyright © 2017 American Society for Microbiology.)

In this study, we assessed the presence of IS26 in food-borne ASSuT-type Salmonella enterica isolates. A new genetic region (R3) was described, that included a C14 caspase gene between IS26 elements. R3 was present in two Salmonella Rissen isolates from a swine carcass and a meat handler, collected at the same abattoir. Furthermore, a new rearrangement of resistance region R1, harboring the blaTEM-1 gene flanked by IS26 elements, was identified in Salmonella Typhimurium and Salmonella 4,[5],12:i:-, from different samples. This study highlights the zoonotic potential of Salmonella spp. isolates and the possible role of IS26 in the mobilization of resistance genes., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Infection with helminth parasites remains a persistent public health problem in developing countries. Three of these pathogens, the liver flukes Clonorchis sinensis, Opisthorchis viverrini and the blood fluke Schistosoma haematobium, are of particular concern due to their classification as Group 1 carcinogens: infection with these worms is carcinogenic. Using liquid chromatography-mass spectrometry (LC-MS/MS) approaches, we identified steroid hormone like (e.g., oxysterol-like, catechol estrogen quinone-like, etc.) metabolites and related DNA-adducts, apparently of parasite origin, in developmental stages including eggs of S. haematobium, in urine of people with urogenital schistosomiasis, and in the adult stage of O. viverrini. Since these kinds of sterol derivatives are metabolized to active quinones that can modify DNA, which in other contexts can lead to breast and other cancers, helminth parasite associated sterols might induce tumor-like phenotypes in the target cells susceptible to helminth parasite associated cancers, i.e., urothelial cells of the bladder in the case of urogenital schistosomiasis and the bile duct epithelia or cholangiocytes, in the case of O. viverrini and C. sinensis. Indeed we postulate that helminth induced cancers originate from parasite estrogen-host epithelial/urothelial cell chromosomal DNA adducts, and here we review recent findings that support this conjecture.

Background: Bladder cancer is a significant health problem in rural areas of Africa and the Middle East where Schistosoma haematobium is prevalent, supporting an association between malignant transformation and infection by this blood fluke. Nevertheless, the molecular mechanisms linking these events are poorly understood. Bladder cancers in infected populations are generally diagnosed at a late stage since there is a lack of non-invasive diagnostic tools, hence enforcing the need for early carcinogenesis markers., Methodology/principal Findings: Forty-three formalin-fixed paraffin-embedded bladder biopsies of S. haematobium-infected patients, consisting of bladder tumours, tumour adjacent mucosa and pre-malignant/malignant urothelial lesions, were screened for bladder cancer biomarkers. These included the oncoprotein p53, the tumour proliferation rate (Ki-67>17%), cell-surface cancer-associated glycan sialyl-Tn (sTn) and sialyl-Lewisa/x (sLea/sLex), involved in immune escape and metastasis. Bladder tumours of non-S. haematobium etiology and normal urothelium were used as controls. S. haematobium-associated benign/pre-malignant lesions present alterations in p53 and sLex that were also found in bladder tumors. Similar results were observed in non-S. haematobium associated tumours, irrespectively of their histological nature, denoting some common molecular pathways. In addition, most benign/pre-malignant lesions also expressed sLea. However, proliferative phenotypes were more prevalent in lesions adjacent to bladder tumors while sLea was characteristic of sole benign/pre-malignant lesions, suggesting it may be a biomarker of early carcionogenesis associated with the parasite. A correlation was observed between the frequency of the biomarkers in the tumor and adjacent mucosa, with the exception of Ki-67. Most S. haematobium eggs embedded in the urothelium were also positive for sLea and sLex. Reinforcing the pathologic nature of the studied biomarkers, none was observed in the healthy urothelium., Conclusion/significance: This preliminary study suggests that p53 and sialylated glycans are surrogate biomarkers of bladder cancerization associated with S. haematobium, highlighting a missing link between infection and cancer development. Eggs of S. haematobium express sLea and sLex antigens in mimicry of human leukocytes glycosylation, which may play a role in the colonization and disease dissemination. These observations may help the early identification of infected patients at a higher risk of developing bladder cancer and guide the future development of non-invasive diagnostic tests.

Liquid chromatography in tandem mass spectrometry (LC-MS/MS) has emerged as an informative tool to investigate oxysterols (oxidized derivatives of cholesterol) in helminth parasite associated cancers. Here, we used LC-MS/MS to investigate in soluble extracts of the adult developmental stage of Opisthorchis viverrini from experimentally infected hamsters. Using comparisons with known bile acids and the metabolites of estrogens, the LC-MS data indicated the existence of novel oxysterol derivatives in O. viverrini. Most of these derivatives were ramified at C-17, in similar fashion to bile acids and their conjugated salts. Several were compatible with the presence of an estrogen core, and/or hydroxylation of the steroid aromatic ring A, hydroxylation of both C-2 and C-3 of the steroid ring and further oxidation into an estradiol-2,3-quinone., (© 2013. Published by Elsevier Ireland Ltd. All rights reserved.)

Mass spectrometric approaches have been fundamental to the identification of metabolites associated with steroid hormones, yet this topic has not been reviewed in depth in recent years. To this end, and given the increasing relevance of liquid chromatography-mass spectrometry (LC-MS) studies on steroid hormones and their metabolites, the present review addresses this subject. This review provides a timely summary of the use of various mass spectrometry-based analytical techniques during the evaluation of steroidal biomarkers in a range of human disease settings. The sensitivity and specificity of these technologies are clearly providing valuable new insights into breast cancer and cardiovascular disease. We aim to contribute to an enhanced understanding of steroid metabolism and how it can be profiled by LC-MS techniques., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

Chronic infection with the blood fluke, Schistosoma haematobium, is associated with squamous cell carcinoma of the bladder. Previously, it has been shown that soluble extracts of mixed sex adult S. haematobium worms (SWAP) are tumourigenic, both in vitro and in vivo. In addition, oestrogen-related molecules in SWAP of S. haematobium down-regulate oestrogen receptors (ERs) alpha and beta in oestrogen responsive cells. Moreover, schistosome oestrogens occur in sera of persons with schistosomiasis haematobia and repress transcription of ERs in urothelial cells. Given that eggs of S. haematobium are the developmental stage directly responsible for urogenital disease during schistosomiasis haematobia, we suspected that soluble antigens from S. haematobium eggs exhibit similar or more potent tumorigenic capacity. Here we investigated the tumorigenic potential of soluble egg antigens (Sh-SEA) of S. haematobium and the endocrine system in favouring parasitism by schistosomes. The findings confirmed that 6.25μg/ml of Sh-SEA was enough to stimulate cell proliferation, reduce apoptosis and increase oxidative stress of Sh-SEA-exposed urothelial cells. In addition, genotoxic effects of Sh-SEA on these cells were determined by using alkaline single-cell gel electrophoresis (Comet). Furthermore, Liquid Chromatography Diode Array Detection Electron Spray Ionisation Mass Spectrometry indicated the presence of catechol-oestrogens in S. haematobium SEA. A prospective oestrogen-DNA adduct mediated pathway in S. haematobium egg induced bladder cancer is also discussed., (Copyright © 2012 Australian Society for Parasitology Inc. All rights reserved.)

We recently reported the expression of an estradiol-like molecule by a trematode parasite Schistosoma haematobium. We further established that this estradiol-like molecule is an antagonist of estradiol, repressing the transcriptional activity of the estrogen receptor (ER) in estrogen-responsive MCF7 cells and also that S. haematobium total antigen (Sh) contains estrogenic molecules detected by mass spectrometry. In the present study, we used HCV29 cells, a cell line derived from normal urothelial cells, as well as an in vivo model to evaluate the expression of ER in the bladders of Sh-instilled animals. We show that, similarly to MCF7 cells, Sh down-regulates the transcriptional activity of ER in HCV29 cells and also in the bladders of Sh-treated mice. The antiestrogenic activity of the S. haematobium extract and its repressive role in ER could have implications in the carcinogenic process in bladders with S. haematobium infection.

Background: Human fasciolosis is a re-emerging disease worldwide and is caused by species of the genus Fasciola (F. hepatica and F. gigantica). Human fasciolosis can be diagnosed by classical coprological techniques, such as the Kato-Katz test, to reveal parasite eggs in faeces. However, although 100% specific, these methods are generally not adequate for detection of acute infections, ectopic infections, or infections with low number of parasites. In such cases immunological methods may be a good alternative and are recommended for use in major hospitals where trained personnel are available, although they are not usually implemented for individual testing., Methodology/principal Findings: We have developed a new lateral flow test (SeroFluke) for the serodiagnosis of human fasciolosis. The new test was constructed with a recombinant cathepsin L1 from F. hepatica, and uses protein A and mAb MM3 as detector reagents in the test and control lines, respectively. In comparison with an ELISA test (MM3-SERO) the SeroFluke test showed maximal specificity and sensitivity and can be used with serum or whole blood samples., Conclusions/significance: The new test can be used in major hospitals in hypoendemic countries as well as in endemic/hyperendemic regions where point-of-care testing is required.

Since 1911 epidemiological evidence indicates that S. haematobium is associated with squamous cell carcinoma of the bladder. However, the mechanisms of this interaction are not clearly defined. Using normal epithelial cells, S. haematobium parasite extracts were able to induce cancer-like phenotypes such as proliferation, apoptosis, migration, invasion and tumorigenesis. The parasite extracts on normal urothelium also presented carcinogenic and mutagenic ability. To further elucidate the biological effects of this parasite, new estrogenic molecules were identified in its extracts. These estrogens are also present in the sera of Schistosoma-infected patients, and they have the ability to repress ER transcriptional activity both in estrogen-responsive MCF7 cells and normal urothelial HCV29 cells. This review will present some of the recent studies of mass spectrometry of S. haematobium extracts and sequence analysis of bladder tissue treated with the same extracts. Finally the molecular and cellular events that might be responsible for schistosomiasis-related bladder cancer will be discussed.

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.)

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.

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.

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.

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.

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.

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.

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.

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.

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.)