Schistosoma mansoni: a method for inducing resistance to praziquantel using infected Biomphalaria glabrata snails

To elucidate the mechanisms of antischistosoma resistance, drug-resistant Schistosoma mansoni laboratory isolates are essential. We developed a new method for inducing resistance to praziquantel (PZQ) using successive drug treatments of Biomphalaria glabrata snails infected with S. mansoni. Infected B. glabrata were treated three times with 100 mg/kg PZQ for five consecutive days with a one-week interval between them. After the treatment, the cercariae (LE-PZQ) produced from these snails and the LE strains (susceptible) were used to infect mice. Forty-five days after infection, mice were treated with 200, 400 or 800 mg/kg PZQ. Thirty days post-treatment, we observed that the mean number of worms recovered by perfusion was significantly higher in the group of mice infected with the LE-PZQ isolate treated with 200 and 400 mg/kg in comparison to the LE strain with the same treatment. Moreover, there was a significant difference between the ED50 (effective dose required to kill 50 percent of the worms) of the LE-PZQ isolate (362 mg/kg) and the LE strain (68 mg/kg). In the in vitro assays, the worms of the LE-PZQ isolate were also less susceptible to PZQ. Thus, the use of infected snails as an experimental model for development of resistance to S. mansoni is effective, fast, simple and cheap.

Antibody reactivity against potato apyrase, a protein that shares epitopes with Schistosoma mansoni ATP diphosphohydrolase isoforms, in acute and chronically infected mice, after chemotherapy and reinfection

Schistosoma mansoni ATP diphosphohydrolase isoforms and potato apyrase share conserved epitopes. By enzyme-linked immunosorbent assays, elevated levels of IgM, IgG2a and IgG1 antibody reactivity against potato apyrase were observed in S. mansoni-infected BALB/c mice during the acute phase of infection, while only IgM and IgG1 antibody reactivity levels maintained elevated during the chronic phase of infection. Antibody reactivity against potato apyrase was monitored over an 11-month period in chronically-infected mice treated with oxamniquine. Eleven months later, the level of seropositive IgM decreased significantly (~30 percent) compared to the level found in untreated, infected mice. The level of seropositive IgG1 decreased significantly four months after treatment (MAT) (61 percent) and remained at this level even after 11 months. The IgG2a reactivity against potato apyrase, although unchanged during chronic phase to 11 MAT, appeared elevated again in re-infected mice suggesting a response similar to that found during the acute phase. BALB/c mouse polyclonal anti-potato apyrase IgG reacted with soluble egg antigens probably due to the recognition of parasite ATP diphosphohydrolase. This study, for the first time, showed that the IgG2a antibody from S. mansoni-infected BALB mice cross-reacts with potato apyrase and the level of IgG2a in infected mice differentiates disease phases. The results also suggest that different conserved-epitopes contribute to the immune response in schistosomiasis.

Phylogenetic analysis of Biomphalaria tenagophila (Orbigny, 1835) (Mollusca: Gastropoda)

Mitochondrial DNA of Biomphalaria tenagophila, a mollusc intermediate host of Schistosoma mansoni in Brazil, was sequenced and characterised. The genome size found for B. tenagophila was 13,722 bp and contained 13 messenger RNAs, 22 transfer RNAs (tRNA) and two ribosomal RNAs (rRNA). In addition to sequencing, the mitochondrial DNA (mtDNA) genome organization of B. tenagophila was analysed based on its content and localization of both coding and non-coding regions, regions of gene overlap and tRNA nucleotide sequences. Sequences of protein, rRNA 12S and rRNA 16S nucleotides as well as gene organization were compared between B. tenagophila and Biomphalaria glabrata, as the latter is the most important S. mansoni intermediate host in Brazil. Differences between such species were observed regarding rRNA composition. The complete sequence of the B. tenagophila mitochondrial genome was deposited in GenBank (accession EF433576). Furthermore, phylogenetic relationships were estimated among 28 mollusc species, which had their complete mitochondrial genome deposited in GenBank, using the neighbour-joining method, maximum parsimony and maximum likelihood bootstrap. B. tenagophila was positioned at a branch close to B. glabrata and Pulmonata molluscs, collectively comprising a paraphyletic group, contrary to Opistobranchia, which was positioned at a single branch and constituted a monophyletic group.

Further evaluation of an updated PCR assay for the detection of Schistosoma mansoni DNA in human stool samples

A previously reported sensitive PCR assay for the detection of Schistosoma mansoni DNA was updated and evaluated. Changes in the DNA extraction method, including the use of a worldwide available commercial kit and the inclusion of additional quality control measures, increased the robustness of the test, as confirmed by the analysis of 67 faecal samples from an endemic area in Brazil. The PCR assay is at hand as a proven, reliable diagnostic test for the control of schistosomiasis in specific settings.

Use of a saline gradient for the diagnosis of schistosomiasis

The development of novel methods for parasitological diagnosis that are both highly sensitive and low in cost has been strongly recommended by the World Health Organization. In this study, a new technique for diagnosis of schistosomiasis mansoni is proposed based on the differential sedimentation of eggs when subjected to a slow continuous flux of 3 percent saline solution through a porous plaque. This influx suspends low-density faecal material, effectively cleaning the sample. The remaining sediment covering the porous plaque surface is then transferred to a glass slide and examined under a bright field microscope. Twelve Kato-Katz slides were used for comparison in the present study. Our results suggest that the saline gradient method detects a signifi-cantly higher number of eggs than the 12 Kato-Katz slides (p < 0.0001). We also found microscopic inspection to be quicker and easier with our newly described method. After cleaning the sample, the obtained sediment can also be conserved in a 10 percent formaldehyde solution and examined for at least 45 days later without statistically significant egg count differences.

A new miracidia hatching device for diagnosing schistosomiasis

It is still imperative to develop a parasitological technique highly sensitive for diagnosing schistosomiasis in epidemiological and individual surveys. A simple and cheap hatching device with a collecting container was manufactured and tested under experimental conditions. Twelve Kato-Katz slides were performed as golden standard for comparison. Quantitative results can be carried out by counting miracidia in a plate and parasite load can be calculated (miracidia/gram of feces). Statistically significant values were higher in the hatching test. More sensitive results, with statistical significance, were achieved using 1.5 g of feces (which corresponds to 36 Kato-Katz slides) than by using the Kato-Katz method. Advantages of this technique and its limitations are presented.

A new method for fixing Biomphalaria glabrata for histologic studies, using shell perforation

A new technique for fixation of Biomphalaria glabrata for histologic studies is described. It consists in performing several external holes in the shell, before placing the entire snail into the fixative. It is a very practical and quick procedure that showed excellent results when compared to the usual techniques

Schistosoma mansoni: reinfections and concomitant immunity in mice. Importance of perfusion time after challenge infection for evaluation of immunoprotection

The concomitant immunity in the presence of repeated infections (with 15 cercariae) was studied in mice sacrificed on the 20th day after each infection. The comparison of the averages of immature worms, recovered from mice submitted to reinfection, with those of their respective controls (previously uninfected) showed a significantly lower worm recovery rate in the animals with previous infections (concomitant immunity). However, statiscally significant differences could not be detected among the various groups of animals, when the mice that accumulated worms in this mature stage were perfused. The theoretical projection based on the accumulation of young worms which developed to adult ones indicates a lower recovery rate of adult worms in the animals with concomitant immunity, but this projection was not corroborated by the experimental data. The visceral hemodynamic alterations that occurred in reinfections due to the pathogeny, favouring recirculation of the recent arriving worms to the other organs on the occasion of perfusion of the portal system. These results suggest that special care should be taken when one wants to investigate concomitant immunity in mice based on the distinction of the immature worms from challenge infection and the mature ones from primary infection.

Schistosoma mansoni in mice: modulation of granulomatous response after reinfection and chemotherapeutic treatment

Mice previously infected with Schistosoma mansoni, and cured by specific treatment (400 mg/kg oxamniquine,p.o.) in tire chronic phase of the disease, were infected 20 days after treated to assess their capacity for modulalion of the granulomatous response. Histopathologic examination of the animals'liver, at 60 days after reinfection, evidenced the presence of typical granulomas of the chronic phase in most animals. This infer that the capacity for modulalion of the granulomatous response had been maintained, thus preventing a new acute phase of lhe disease. Conversely, a group of previously infected mice, untreated and submitted to reinfection, showed reactivation of the granulomatous response in 50 per cent ofthe animals. The possible implications of these findings in human schistosomiasis mansoni are discussed.

Transmission of Schistosoma mansoni under experimental conditions using the bovine--Biomphalaria glabrata-bovine model

Three calves experimentally infected with Schistosoma mansoni, and passing viable eggs in feces, as well as 5 normal calves (coming from a non-endemic area for schistosomiasis) kept as controls, were maintained in an enclosure (850 m2 in area). In this enclosure, a tank with water received 500 laboratory reared Biomphalaria glabrata. All the control calves were infected for a period ranging from 79 to 202 days after the beginning of the experiment, and afterwards presented viable S. mansoni eggs in feces. The mean worm recovery was 555. The snail population increased throughout the experimental period, showing a high number of B. glabrata infected with S. mansoni (42 on average). According to the present study, bovine has been suggested as having potentially a role in the maintenance of the life cycle of S. mansoni.

Dimensöes do granuloma hepático produzido por ovos de duas linhagens geográficas do Schistosoma mansoni, no camundongo / Size of hepatic granuloma produced by Schistosoma mansoni eggs (from 2 different geographic strains) in mice

Os autores determinaram o diâmetro médio dos granulomas de fígado de camundongos infectados com cercárias de duas cepas geográficas bem definidas do, Schistosoma mansoni (LE, Belo Horizonte e SJ, Säo Paulo). No total foram medidos 1.170 granulomas. Os granulomas com 60 dias eram de dimensöes maiores do que os de 90 dias. A modulaçäo da resposta imunopatológica foi significativamente mais eficiente na cepa LE e os granulomas, tanto aos 60 como aos 90 dias, da cepa SJ eram significativamente maiores. Os dados obtidos indicam uma maior patogenicidade da cepa SJ. Especula-se se o significado destes achados poderiam, em parte, explicar a ocorrência das variaçöes regionais das formas anatomo-clínicas da esquistossomose

Shistosoma mansoni: Acquired resistance in mice by implantation of young irradiated worms into the portal system

Em dois experimentos distintos, vermes imaturos de Schistosoma mansoni, com 20 dias de idade, obtidos do sistema porta de camundongos albinos, foram irradiados com 14 e 4 Krad e posteriormente inoculados diretamente na veia porta de camundongos normais. Cada animal, de cada experimento específico, recebeu 20 vermes irradiados. Decorridos 50 dias de inoculaçäo, os camundongos com os vermes irradiados com 4 e 14 Krad e seus respectivos controles foram infectados pela via transcutânea, com cercárias da cepa LE Schistosoma mansoni. No 20§ dia após esta infecçäo desafio, os camndongos foram sacrificados e perfundidos para as contagens dos vermes maduros irradiados (90 dias de idade) e imaturos (20 dias de idade). A análise dos resultados mostrou que ocorreu proteçäo, estatísticamente signifciativa, contra cercárias nos grupos previamente inoculados com vermes irradiados com as doses de 4 e 14 Krad

Pulmonary involvement in Schistosomiasis mansoni

The post-treatment pulmonary alterations were evaluated in patients (Study 1) and in mice (Study 2) infected with Schistosoma mansoni. Study 1: the patients were examined pre and post-treatment (with ora oxamniquine) and the following exams were performed: sputum for eosinophils and chest x-ray. Study 2: four groups of mice (total = 64) were studied; Group I (infected and treated with oxamniquine); II (infected and not treated); III (not infected and treated) and IV (not infected and not treated). All were x-rayed to check for pulmonary abnormalities pre and post-treatment and lung specimens were studied by optical microscopy and immunofluorescence. We have found abnormalities in the parameters checked in both studies and the results suggest an immunological reaction, probably due to deposition of immune complexes in the lungs, with subsequent activation of the complement system. The experimental study showed that the alterations are not dependent of the presence of eggs and/or worms of S. mansoni in the lungs, thus corroborating the hypothesis of deposition of circulating material.