Schistosoma haematobium and Schistosomiasis mansoni: production of an estradiol-related compound detected by ELISA.

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.

Determination of the more adequate modified agglutination test cut-off for serodiagnosis of Toxoplasma gondii infection in sheep.

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.

Analysis of the immune response to Neospora caninum in a model of intragastric infection in mice.

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.

Neospora caninum: high susceptibility to the parasite in C57BL/10ScCr mice.

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.

Characterization of Toxoplasma gondii isolates in free-range chickens from Portugal.

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.

Biologic and molecular characterization of Toxoplasma gondii isolates from pigs from Portugal.

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.

A recombinant antigen recognized by Fasciola hepatica-infected hosts.

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.

First isolation of Neospora caninum from an aborted bovine fetus in Spain.

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.

Determination of an optimized cut-off value for the Neospora agglutination test for serodiagnosis in cattle.

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.

First Portuguese isolate of Neospora caninum from an aborted fetus from a dairy herd with endemic neosporosis.

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.

Isolation of viable Toxoplasma gondii from naturally infected aborted bovine fetuses.

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.

Effects of carvedilol in heart failure due to dilated cardiomyopathy. Results of a double-blind randomized placebo-controlled study (CARIBE study).

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.

[Rheumatoid pannus of the wrist]. / Punho reumatóide.

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.

Antigenic components of partially purified antigens of Leishmania donovani infantum recognized by sera from dogs with asymptomatic or active visceral leishmaniasis.

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.

Antigenic components of excretory-secretory products of adult Fasciola hepatica recognized in human infections.

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.

Use of cross-reactive antigens of the microsporidian Glugea atherinae for the possible detection of Enterocytozoon bieneusi by western blot.

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.

Exploitation of parasite-derived antigen in therapeutic success against canine visceral leishmaniosis. Veterinary Group of Lupino.

In an attempt to obtain therapeutic success against canine visceral leishmaniosis, the potential of LiF2 antigen (Leishmania infantum-derived Fraction 2, 94-67 kDa), given alone or in combination with the chemotherapeutic agent N-methylglucamine antimonate, was compared with conventional chemotherapy with that drug. Absence of any parasite in direct microscopic examination of bone-marrow aspirates in treated dogs was considered a parasitological cure, i.e. therapeutic success. Results showed that the disappearance of clinical symptoms did not always indicate parasitological healing in dogs. The parasitological healing rates with chemotherapy and immunotherapy alone were 37.5% and 25% respectively, in contrast to the 100% cure rate observed with chemotherapy combined with immunotherapy. The development of a protective response in dogs, as measured by the in vitro leishmanicidal activity of monocyte-derived macrophages in the presence of autologous lymphocytes, was found to correlate well with the success of therapy. The overall findings of this study give an important insight into the immunotherapeutic strategy by which therapeutic success can be achieved in canine visceral leishmaniosis.

[Fixation and immunological reactivity of parasitic protozoa in sol-gel matrices]. / Fixation et réactivité immunologique de protistes parasites au sein de matrices sol-gel.

Parasitic protozoa have been entrapped within sol-gel silica matrices. Stationary phase promastigote cells of Leishmania donovani infantum are mixed with a silicon alkoxide solution. They remain trapped within the growing oxide network formed upon hydrolysis and condensation of the alkoxide. Electron microscopy shows the preservation of the cellular organization and the integrity of the plasma membrane of entrapped parasites. Specific antigen-antibody reactions have been performed within the sol-gel matrix via enzyme-linked immunosorbent assays (ELISA) currently used for the diagnostic of visceral leishmaniasis. A clear-cut difference in optical density was measured between positive and negative sera from dogs. Entrapped protozoa antigens are still able to react with specific antibodies through the open porosity of the silica network. These results suggest that sol-gel matrices could be used for the development of immunodiagnostic assays requiring whole cell parasites as antigens.

[Reflux esophagitis in an experimental model. Are the systemic consequences attributable to the esophagitis?]. / Esofagitis por reflujo en un modelo experimental. Son las repercusiones sistémicas atribuibles a las esofagitis?

The esophagojejunostomy model of reflux esophagitis has been shown to cause nutritional deterioration and alterations of hematologic parameters. However, these disturbances have been assumed rather than proved to be the consequence of esophagitis. Little attention has been paid to the anatomical and physiological alterations related to the reflux-inducing operation. We produced reflux esophagitis by an end-to-side esophagojejunostomy (EJ) in Sprague-Dawley rats. These rats were compared to a second group with a similar operation not leading to reflux esophagitis (Roux-en-Y esophagojejunostomy, R-Y). Sham operated animals were used as controls. Erosive esophagitis was produced in all but one of the EJ subjects at day 14, and in none of the R-Y animals. Significant weight loss (-21.7%), anemia (HCT, -17.3%) and hypoproteinemia (-14.2%) were seen in EJ model with respect to sham group. However, a similar variation of weight (-28.7%) and hematocrit (-26.2%) were also found in Y-R group; therefore, these parameters seemed to correlate more with the surgical challenge itself than with the esophagitis.

[Treatment of colonic atresia. Report of 3 cases]. / Tratamiento de la atresia de colon. A propósito de tres casos.

Atresia of the colon (AC) is an uncommon cause of neonatal intestinal obstruction requiring prompt surgery. However, its optimal operative management is matter of discussion. We report the surgical therapy and evolution of three cases successfully treated at our department. A type I case was submitted to a coloplasty plus resection of the diaphragm; two cases of type III underwent a two-staged procedure, i.e., colostomy with subsequent anastomosis without resection. After analysing the different operative procedures we suggest that a colostomy with later establishment of intestinal continuity may be an advantageous procedure even in the healthy patients with proximal lesions. A coloplasty is a safe alternative to type I atresias.