Vertical toxoplasmosis in a murine model. Protection after immunization with antigens of Toxoplasma gondii incorporated into liposomes

Distinct Toxoplasma gondii antigens were entrapped within liposomes and evaluated for their ability to protect Balb/c mice against congenital transmission: soluble tachyzoite antigen (L/STAg), soluble tissue cyst antigen (L/SCAg), soluble tachyzoite plus tissue cyst (L/STCAg) or purified 32kDa antigen of tachyzoite (L/pTAg). Soluble tachyzoite antigen alone in PBS (STAg) or emulsified in Freund's Complete Adjuvant (FCA/STAg) was also evaluated. Dams were inoculated subcutaneously with these antigens 6, 4 and 2 weeks prior to a challenge with four tissue cysts of the P strain of T. gondii orally between 10 and 14 days of pregnancy. Significant diminution differences were observed between the frequency of infected pups born of the dams immunized with the antigens incorporated into liposomes and that of pups born of the dams immunized with antigen emulsified in FCA or non immunized group (p<0.05). There was a significant decrease in the number of pups born dead in the groups L/STAg, L/SCAg and L/pTAg when compared with pups from all other groups (p <0.05). All dams immunized with or without adjuvant showed an antibody response and a proliferation of T-cells. However, no correlation was found between immune response and protection against the challenge.

Immunological follow-up of hydatid cyst cases

Hydatid disease is caused by Echinococcus granulosus. In this study, we aimed to investigate the benefit of monitoring cases with hydatid cyst by means of immune components in patients in a long-term follow-up after surgery. Eighty-four preoperative and postoperative serum samples from 14 cases undergoing surgery for hydatid disease were evaluated in terms of immune parameters, such as total and specific IgE, IgG, IgM, IgA and complement. Total and specific IgE were determined by ELISA. Specific IgG levels were measured by indirect hemaglutination.Total IgG, IgM, IgA and complement (C3 and C4) were detected by nephelometry. Imaging studies were also carried out during the follow-up. In none of the patients hydatid cysts were detected during the follow-up. Total IgE levels in the sera of the patients decreased to normal six months after surgery. Although specific IgE against echinococcal antigens decreased one year after operation, levels were still significantly high. There were no changes in the levels of anti-Echinococcus IgG and total IgG in follow-up period. Additionally, other parameters, such as IgA, IgM, C3 and C4, were not affected.

A simple modification of the Baermann method for diagnosis of strongyloidiasis

The diagnosis of Strongyloides stercoralis infections is routinely made by microscopic observation of larvae in stool samples, a low sensitivity method, or by other, most effective methods, such as the Baermann or agar culture plate methods. We propose in this paper a practical modification of Baermann method. One hundred and six stool samples from alcoholic patients were analyzed using the direct smear test, agar culture plate method, the standard Baermann method, and its proposed modification. For this modification the funnel used in the original version of the method is substituted by a test tube with a rubber stopper, perforated to allow insertion of a pipette tip. The tube with a fecal suspension is inverted over another tube containing 6 ml of saline solution and incubated at 37°C for at least 2 h. The saline solution from the second tube is centrifuged and the pellet is observed microscopically. Larva of S. stercoralis were detected in six samples (5.7%) by the two versions of the Baermann method. Five samples were positive using the agar culture plate method, and only in two samples the larva were observed using direct microscopic observation of fecal smears. Cysts of Endolimax nana and Entamoeba histolytica/dyspar were also detected in the modification of Baermann method. Data obtained by the modified Baermann method suggest that this methodology may helps concentrate larvae of S. stercoralis as efficiently as the original method.

Myxobolus absonus sp. n. (Myxozoa: Myxosporea) Parasitizing Pimelodus maculatus (Siluriformes: Pimelodidae), a South American Freshwater Fish

A new myxoporean species is described from a freshwater fish in Brazil. Myxobolus absonus sp. n. was found infecting Pimelodus maculatus captured in the river Piracicaba, State of São Paulo, Brazil. Cysts were found free in the opercular cavity. The spores are large (length-15.7 ± 1.5 µm, width-10.2 ± 0.7 µm; mean ± S.D.) and oval in shape, with the anterior end slightly pointed. The spore valves are relatively thin, smooth, and asymmetrical in a frontal view. The polar capsules are pyriform in shape, and unequal in size; the largest are 6.4 ± 0.7 µm long and 3.6 ± 0.5 µm wide, while the smallest are 4.2 ± 0.6 µm long and 2.5 ± 0.5 µm wide.

An Autochthonous Case of Echinococcus vogeli Rausch & Bernstein, 1972 Polycystic Echinococcosis in the State of Rondônia, Brazil

The present case report refers to a patient from the State of Rondônia, North region of Brazil, attended with clinical suspicion of hepatic echinococcosis. Examination by imaging (ultrasonography and computerized tomography) revealed a conglomerate of cystic lesions, with mobile contents within the cyst. The serology (immunoblot) for Echinococcus sp. was positive (21 and 31 kDa bands). This case is the first reported in Rondônia, suggesting the need to investigate the polycystic echinococcosis in individuals with hepatic cysts from areas of tropical forest and hunting habits where wild life was present as wild dogs, cats and rodents, particularly Agouti paca (paca) and Dasyprocta aguti (agouti).

Humoral immune responses induced by Kudoa sp. (Myxosporea: Multivalvulida) antigens in BALB/c mice

The majority of Kudoa species infect the somatic muscle of fish establishing cysts. As there is no effective method to detect infected fish without destroying them these parasited fish reach the consumer. This work was developed to determine whether this parasite contains antigenic compounds capable of provoking an immune response in laboratory animals, in order to consider the possible immunopathological effects in man by the ingestion of Kudoa infected fish. BALB/c mice were injected by the subcutaneous route with the following extracts suspended in aluminium hydroxide: group 1 (black Kudoa sp. pseudocyst extract), group 2 (white Kudoa sp. pseudocyst extract), and group 3 (non-infected hake meat extract). Specific antibody levels were measured by ELISA against homologous and heterologous antigens. The highest responses were obtained from the black Kudoa sp. pseudocyst extract (group 1).The low optic density levels detected in group 3 proved that the results obtained in groups 1 and 2 were a consequence of the parasitic extract injection. The IgG1 was the predominant subclass. IgE detected in groups 1 and 2 showed the possible allergenic nature of some of the components of the parasitic extract. High IgA levels and medium IgG2a and IgG3 levels were obtained in groups 1 and 2. Low IgG2b responses were shown. No cross-reactions between Kudoa sp. pseudocyst extracts and the non-infected hake meat extract were observed.

Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate). One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 µg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%); specificity, 95% (95% CI: 88.6-97.6%); positive predictive value, 91% (95% CI: 81.4-95.9%); and negative predictive value, 100% (95% CI: 96.1-100%). This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

Morphologic and morphometric analysis of Hepatozoon spp. (Apicomplexa, Hepatozoidae) of snakes

Hepatozoon species are the most abundant hemoparasites of snakes. Its identification has been based mainly on the morphologic characterization of the gamonts in the peripheral blood of the vertebrate host and also of the cysts found in the internal organs of the vertebrate and invertebrate hosts. Using a computerized image analysis system, we studied five species of Hepatozoon from recently captured snakes in Botucatu, State of São Paulo, Brazil, to evaluate the importance of the morphology and morphometry of the gamonts for the characterization of Hepatozoon species and to analyze the morphologic changes induced in the erythrocytes by the parasite. The studied species were H. terzii of Boa constrictor amarali, Hepatozoon sp. of Crotalus durissusterrificus, H. philodryasi of Philodryas patagoniensis, and H. migonei and H. cyclagrasi of Hydrodynastes gigas. We observed three different groups, one of them including the species H. terzii, H. philodryasi and Hepatozoon sp. of C. durissus terrificus; and the other two consisting of H. migonei and H. cyclagrasi. Degree of alterations in the erythrocytes was variable and it may be useful for characterization of Hepatozoon species.

Development of Hepatozoon caimani (Carini, 1909) Pessôa, De Biasi & De Souza, 1972 in the Caiman Caiman c. crocodilus, the frog Rana catesbeiana and the mosquito Culex fatigans

The sporogony of Hepatozoon caimani has been studied, by light microscopy, in the mosquito Culex fatigans fed on specimens of the caiman Caiman c. crocodilus showing gametocytes in their peripheral blood. Sporonts iniciate development in the space between the epithelium of the insect gut and the elastic membrane covering the haemocoele surface of the stomach. Sporulating oocysts are clustered on the gut, still invested by the gut surface membrane. Fully mature oocysts were first seen 21 days after the blood-meal. No sporogonic stages were found in some unidentified leeches fed on an infected caiman, up to 30 days following the blood-meal. When mosquitoes containing mature oocysts were fed to frogs (Leptodactylus fuscus and Rana catesbeiana), cysts containing cystozoites developed in the internal organs, principally the liver. Feeding these frogs to farm-bred caimans resulted in the appearance of gametocytes in their peripheral blood at some time between 59 and 79 days later, and the development of tissue cysts in the liver, spleen, lungs and kidneys. Transmission of the parasite was also obtained by feeding young caimans with infected mosquitoes and it is suggested that both methods occur in nature. The finding of similar cysts containing cystozoites in the semi-aquatic lizard Neusticurus bicarinatus, experimentally fed with infected C. fatigans, suggests that other secondary hosts may be involved.

A new species of Henneguya, a gill parasite of Astyanax altiparanae (Pisces: Characidae) from Brazil, with comments on histopathology and seasonality

A new species of Myxosporea, Henneguya chydadea, is described parasitizing the gills of Astyanax altiparanae collected from a lake on Rio das Pedras farm near Campinas, state of São Paulo, Brazil. Of the fish examined, 88.3% had gills parasitized by myxosporeans. The prevalence of the parasite ranged from 80% in the spring and fall, 93% in the summer and 100% in the winter. The parasite induced the formation of white, oval-shaped cysts measuring 40-64 µm x 64-80 µm which deformed the gill lamellae, compressed the capillaries, and caused retraction of the neighboring lamellae. The mature spores were elongated and had two identical, parallel elongate polar capsules. Each capsule contained a polar filament with 9-10 turns. There was no mucous envelope or iodinophilous vacuole. Morphometric differences between this parasite and other species of the genus Henneguya indicated, that he parasite observed in A. altiparanae is a new species. This is the first report of a myxosporeanparasitizing A. altiparanae.

Detection of Toxoplasma gondii DNA by polymerase chain reaction in experimentally desiccated tissues

Despite toxoplasmosis being a common infection among human and other warm-blooded animals worldwide, there are no findings about Toxoplasma gondii evolutionary forms in ancient populations. The molecular techniques used for amplification of genetic material have allowed recovery of ancient DNA (aDNA) from parasites contained in mummified tissues. The application of polymerase chain reaction (PCR) to paleoparasitological toxoplasmosis research becomes a promising option, since it might allow diagnosis, acquisition of paleoepidemiological data, access to toxoplasmosis information related origin, evolution, and distribution among the ancient populations.Furthermore, it makes possible the analysis of parasite aDNA aiming at phylogenetic studies. To standardize and evaluate PCR applicability to toxoplasmosis paleodiagnostic, an experimental mummification protocol was tested using desiccated tissues from mice infected with the ME49 strain cysts, the chronic infection group (CIG), or infected with tachyzoites (RH strain), the acute infection group (AIG). Tissues were subjected to DNA extraction followed by PCR amplification of T. gondii B1 gene. PCR recovered T. gondii DNA in thigh muscle, encephalon, heart, and lung samples. AIG presented PCR positivity in encephalon, lungs, hearts, and livers. Based on this results, we propose this molecular approach for toxoplasmosis research in past populations.

Bicc1 links the regulation of cAMP signaling in polycystic kidneys to microRNA-induced gene silencing.

Genetic defects in autosomal-dominant polycystic kidney disease (ADPKD) promote cystic growth of renal tubules, at least in part by stimulating the accumulation of cAMP. How renal cAMP levels are regulated is incompletely understood. We show that cAMP and the expression of its synthetic enzyme adenylate cyclase-6 (AC6) are up-regulated in cystic kidneys of Bicc1(-)(/-) knockout mice. Bicc1, a protein comprising three K homology (KH) domains and a sterile alpha motif (SAM), is expressed in proximal tubules. The KH domains independently bind AC6 mRNA and recruit the miR-125a from Dicer, whereas the SAM domain enables silencing by Argonaute and TNRC6A/GW182. Bicc1 similarly induces silencing of the protein kinase inhibitor PKIα by miR-27a. Thus, Bicc1 is needed on these target mRNAs for silencing by specific miRNAs. The repression of AC6 by Bicc1 might explain why cysts in ADPKD patients preferentially arise from distal tubules.

Basic biology of Pneumocystis carinii: a mini review

Basic aspects of cell biology of Pneumocystis carinii are reviewed with major emphasis on its life cycle and the structural organization of the trophozoites and cyst forms. Initially considered as a protozoan it is now established that Pneumocystis belongs to the Fungi Kingdom. Its life cycle includes two basic forms: (a) trophozoites, which are haploid cells that divide by binary fission and may conjugate with each other forming an early procyst and (b) cysts where division takes place through a meiotic process with the formation of eight nuclei followed by cytoplasmic delimitation and formation of intracystic bodies which are subsequently released and transformed into trophozoites. Basic aspects of the structure of the two developmental stages of P. carinii are reviewed.

Multiple nodules of the scrotum: histopathological findings and surgical procedure. A study of five cases.

BACKGROUND: Multiple nodules of the scrotum are uncommonly reported. Their origin is controversial. Treatment is always surgical but the best procedure is still to be determined. MATERIALS AND METHODS: Five new cases are reported with description of the histopathological findings and surgical procedure. RESULTS: Nodules of the scrotum were more frequent in patients with dark skin suggesting an ethnic susceptibility. No other predisposing factors were noted. Screening for disturbances of phosphate or calcium balance was negative. The following histopathological findings were observed: non-calcified epidermoid cysts (3 patients), calcified epidermoid cysts (1 patient) and nodular calcifications without epithelial or glandular structures (1 patient). Subtotal excisions of the scrotum wall using tumescent anaesthesia were performed in all patients without any significant complications. Cosmetic results were excellent. No new lesions were observed during the 1-year follow-up period. CONCLUSIONS: Most cases of multiple nodules of the scrotum are due to non-calcified epidermoid cysts. The term scrotal calcinosis is therefore probably abusively used by many authors. Some cases of nodular calcifications may be due to dystrophic calcification of epidermoid cysts, but calcifications may also occur without any visible epithelial or glandular structure. Subtotal excision of the scrotum wall is a safe and effective surgical procedure to treat multiple nodules of the scrotum. Cosmetic results are excellent and recurrences are rare.

Comparative evaluation of different immunoassays for the detection of Taenia solium cysticercosis in swine with low parasite burden

Seven swine were experimentally infected with Taenia solium eggs and blood samples from each animal were periodically collected. At the end of the experiment (t140) the animals did not show clinical aspects of cysticercosis or parasites in tongue inspection. All animals were slaughtered and cut into thin slices in searching for cysts. The number of cysts found in each animal varied from 1 to 85. Enzyme-linked immunosorbent assay (ELISA) tests for antibody (Ab) detection and for antigen (Ag) detection were performed, which presented respectively 71 and 57% of positivity. By immunoblot (IB), using 18/14(T. crassiceps Ag) or lentil-lectin-purified glycoproteins from T. solium Ag (LLGP) as Ag, five (71%) and six (86%) animals were positive, respectively. The association between Ag-ELISA with any IB (18/14 or LLGP) allowed the detection of all animals at 140 days post-experimental infection (days p.e.i.). The use of IB 18/14 combined to the Ag-ELISA allowed the detection of all animals since 70 days p.e.i., and the association between IB LLGP and Ag-ELISA allowed the detection of all animals since 112 days p.e.i. While all animals could be considered healthy by conventional screening tests, the use of immunoassays for detecting Ab and Ag showed better accuracy; therefore it would be more useful than usual clinical examination for screening cysticercosis in slightly infected pigs.