Diagnostic of Biomphalaria snails and Schistosoma mansoni: DNA obtained from traces of shell organic materials

Freshwater snails belonging to the genus Biomphalaria act as intermediate hosts for the parasite trematode Schistosoma mansoni in Africa and in the neotropical region. Identification of such molluscs is carried out based on morphological characters and the presence of cercariae is verified through squeezing snails between two glass slides or by exposing them to artificial light. However, sometimes, the material collected includes molluscs with decomposed bodies or, yet, only empty shells, which precludes their identification and S. mansoni detection. Due to these difficulties, we have developed a methodology in which DNA may be extracted from traces of organic material from inside shells in order to identify molluscs through polymerase chain reaction and restriction fragment length polymorphism and to detect S. mansoni into these snails, by using low stringency polymerase chain reaction. Species-specific profiles obtained from B. glabrata, B. straminea, and B. tenagophila snails and their shells, maintained in laboratory for ten years, showed the same profiles. S. mansoni profiles showed to be present in shell specimens as far as the eighth week after being removed from aquarium.

Vaccination with antioxidant enzymes confers protective immunity against challenge infection with Schistosoma mansoni

Schistosoma mansoni, an intravascular parasite, lives in a hostile environment in close contact with host humoral and cellular cytotoxic factors. To establish itself in the host, the parasite has evolved a number of immune evasion mechanisms, such as antioxidant enzymes. Our laboratory has demonstrated that the expression of antioxidant enzymes is developmentally regulated, with the highest levels present in the adult worm, the stage least susceptible to immune elimination, and the lowest levels in the larval stages, the most susceptible to immune elimination. Vaccination of mice with naked DNA constructs containing Cu/Zn cytosolic superoxide dismutase (CT-SOD), signal-peptide containing SOD or glutathione peroxidase (GPX) showed significant levels of protection compared to a control group. We have further shown that vaccination with SmCT-SOD but not SmGPX results in elimination of adult worms. Anti-oxidant enzyme vaccine candidates offer an advance over existing vaccine strategies that all seem to target the larval developmental stages in that they target adult worms and thus may have therapeutic as well as prophylactic value. To eliminate the potential for cross-reactivity of SmCT-SOD with human superoxide dismutase, we identified parasite-specific epitope-containing peptides. Our results serve as a basis for developing a subunit vaccine against schistosomiasis.

Identification of immunodominant epitopes of Schistosoma mansoni vaccine candidate antigens using human T cells

Paramyosin and Sm14 are two of the six antigens selected by the World Health Organization as candidates to compose a subunit vaccine against schistosomiasis. Both antigens are recognized by individuals naturally resistant to Schistosoma mansoni infection and induced protective immunity in the murine model. Three Sm14 epitopes and eleven paramyosin epitopes were selected by their ability to bind to different HLA-DR molecules using the TEPITOPE computer program, and these peptides were synthetically produced. The cellular recognition of Sm14 and paramyosin epitopes by peripheral blood mononuclear cells of individuals living in endemic area for schistosomiasis was tested by T cell proliferation assay. Among all Sm14 and paramyosin epitopes studied, Sm14-3 was preferentially recognized by individuals naturally resistant to S. mansoni infection while Para-5 was preferentially recognized by individuals resistant to reinfection. These two peptides represent promising antigens to be used in an experimental vaccine against schistosomiasis, since their preferential recognition by resistant individuals suggest their involvement in the induction of protective immunity.

Hypertensive portal colopathy in schistosomiasis mansoni: proposal for a classification

Portal hypertension is a frequent complication of chronic liver disease, detected not only in schistosomiasis, but also in cirrhosis of any etiology. Vascular alterations in the colonic mucosa are a potential source for acute or chronic bleeding and have been observed in patients with portal hypertension. The purpose of this prospective study was to describe and propose a classification for the vascular alterations of portal hypertension in the colonic mucosa among patients with hepatosplenic schistosomiasis mansoni. One or more alterations of portal colopathy were observed in all patients and they were classified according to their intensity, obeying the classification proposed by the authors. Portal colopathy is an important finding in hepatosplenic schistosomiasis and might be the cause of lower gastrointestinal bleeding in patients with severe portal hypertension.

Biomphalaria tenagophila/Schistosoma mansoni interaction: premises for a new approach to biological control of schistosomiasis

Biomphalaria tenagophila is very important for schistosomiasis transmission in Brazil. However its mechanisms of interaction with Schistosoma mansoni are still scantly studied. Since this snail displays strains highly susceptible or completely resistant to the parasite infection, the knowledge of that would be a useful tool to understand the mechanism of snail resistance. Particularly, the Taim strain consistently shows absolute resistance against the trematode, and this resistance is a dominant character. A multidisciplinary research group was created aiming at studying B. tenagophila/S. mansoni interaction. The possibility for applying the knowledge acquired to obtain a biological model for the control of S. mansoni transmission in endemic areas is discussed.

Clinical and immunological consequences of human T cell leukemia virus type-I and Schistosoma mansoni co-infection

Human T cell leukemia virus type-I (HTLV-I) infection is associated with spontaneous T cell activation and uncontrolled lymphocyte proliferation. An exacerbated type-1 immune response with production of pro-inflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) is significantly higher in patients with myelopathy associated to HTLV-I than in HTLV-I asymptomatic carriers. In contrast with HTLV-I, a chronic Schistosoma mansoni infection is associated with a type-2 immune response with high levels of interleukin (IL-4, IL-5, and IL-10) and low levels of IFN-gamma. In this study, clinical and immunological consequences of the HTLV-I and S. mansoni infection were evaluated. The immune response in patients with schistosomiasis co-infected with HTLV-I showed low levels of IL-5 (p < 0.05) in peripheral blood mononuclear cells cultures stimulated with S. mansoni antigen (SWAP) and decreased SWAP-specific IgE levels when compared with patients with only schistosomiasis (p < 0.05). Liver fibrosis was mild in all HTLV-I co-infected patients. Immunological response was also compared in individuals who had only HTLV-I infection with those who were co-infected with HTLV-I and helminths (S. mansoni and Strongyloides stercoralis). In patients HTLV-I positive co-infected with helminths the IFN-gamma levels were lower than in individuals who had only HTLV-I. Moreover, there were fewer cells expressing IFN-gamma and more cells expressing IL-10 in individuals co-infected with HTLV-I and helminths. These dates indicate that HTLV-I infection decrease type 2-response and IgE synthesis and are inversely associated with the development of liver fibrosis. Moreover, helminths may protect HTLV-I infected patients to produce large quantities of pro-inflammatory cytokines such as IFN-gamma.

Sativa seeds against Schistosoma mansoni different stages

The schistosomicidal properties of Nigella sativaseeds were tested in vitro against Schistosoma mansoni miracidia, cercariae, and adult worms. Results indicate its strong biocidal effects against all stages of the parasite and also showed an inhibitory effect on egg-laying of adult female worms. In the present work we also studied the effects of crushed seeds on some antioxidant enzymes; which have a role in protection of adult worms against host oxidant killing; as well as some enzymes of glucose metabolism; which have a crucial role in the survival of adult worms inside their hosts. The data revealed that the used drug induce an oxidative stress against adult worms which indicated by a decrease in the activities of both antioxidant enzymes, superoxide dismutase, glutathione peroxidase, and glutathione reductase and enzymes of glucose metabolism, hexokinase and glucose-6-phosphate dehydrogenase. Disturbing of such enzymes of adult worms using N. sativa seeds could in turn render the parasite vulnerable to damage by the host and may play a role in the antischistosomal potency of the used drug.

Isolation and characterization of the full-length cDNA encoding a member of a novel cytochrome p450 family (CYP320A1) from the tropical freshwater snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni

Cytochrome p450s (cyp450s) are a family of structurally related proteins, with diverse functions, including steroid synthesis and breakdown of toxins. This paper reports the full-length sequence of a novel cyp450 gene, the first to be isolated from the tropical freshwater snail Biomphalaria glabrata, an important intermediate host of Schistosoma mansoni. The nucleotide sequence is 2291 bp with a predicted amino acid sequence of 584aa. The sequence demonstrates conserved cyp450 structural motifs, but is sufficiently different from previously reported cyp450 sequences to be given a new classification, CYP320A1. Initially identified as down-regulated in partially resistant snails in response to S. mansoni infection, amplification of this gene using RT-PCR in both totally resistant or susceptible snail lines when exposed to infection, and all tissues examined, suggests ubiquitous expression. Characterization of the first cyp450 from B. glabrata is significant in understanding the evolution of these metabolically important proteins.

Compatibility of Biomphalaria tenagophila with Schistosoma mansoni: a study of homologous plasma transference

This study aims to investigate the importance of the serum factors present in the plasma of resistant Biomphalaria tenagophila snails, when transferred to susceptible conspecific. Susceptible B. tenagophila (CF) received plasma from resistant B. tenagophila (Taim), and both were later infected with Schistosoma mansoni. We noticed that the plasma transfer showed an increase on the resistance of susceptible snails of about 86% when compared to the non-immunized group (p < 0.001).

Schistosoma mansoni major egg antigen Smp40: molecular modeling and potential immunoreactivity for anti-pathology vaccine development

The pathogenesis of Schistosoma mansoni infection is largely determined by host T-cell mediated immune responses such as the granulomatous response to tissue deposited eggs and subsequent fibrosis. The major egg antigens have a valuable role in desensitizing the CD4+ Th cells that mediate granuloma formation, which may prevent or ameliorate clinical signs of schistosomiasis.S. mansoni major egg antigen Smp40 was expressed and completely purified. It was found that the expressed Smp40 reacts specifically with anti-Smp40 monoclonal antibody in Western blotting. Three-dimensional structure was elucidated based on the similarity of Smp40 with the small heat shock protein coded in the protein database as 1SHS as a template in the molecular modeling. It was figured out that the C-terminal of the Smp40 protein (residues 130 onward) contains two alpha crystallin domains. The fold consists of eight beta strands sandwiched in two sheets forming Greek key. The purified Smp40 was used for in vitro stimulation of peripheral blood mononuclear cells from patients infected with S. mansoni using phytohemagglutinin mitogen as a positive control. The obtained results showed that there is no statistical difference in interferon-g, interleukin (IL)-4 and IL-13 levels obtained with Smp40 stimulation compared with the control group (P > 0.05 for each). On the other hand, there were significant differences after Smp40 stimulation in IL-5 (P = 0.006) and IL-10 levels (P < 0.001) compared with the control group. Gaining the knowledge by reviewing the literature, it was found that the overall pattern of cytokine profile obtained with Smp40 stimulation is reported to be associated with reduced collagen deposition, decreased fibrosis, and granuloma formation inhibition. This may reflect its future prospect as a leading anti-pathology schistosomal vaccine candidate.

Primary culture of the region of the amebocyte-producing organ of the snail Biomphalaria glabrata, the intermediate host of Schistosoma mansoni

Biomphalaria glabrata snails are major hosts for the digenetic trematoda Schistosoma mansoni, the causative agent of human schistosomiasis. The success or failure of the infection will be dependent on the mobilization of the molluskan internal defense system, where a major role will be played by circulating hemocytes produced by the APO (amebocyte-producing organ) of the snail. In this report, the primary culture of the APO region of B. glabrata was obtained for the first time, as well as a control culture of the ovotestis. Three different cell populations migrated easily from the explants in culture, with no need of any dispersion agent. The cells grew in suspension at an incubation temperature of 15ºC and the cultures were maintained viable for up to two weeks. Two of these cell populations obtained resembled cell types known to be present in the hemolymph of Biomphalaria. The availability of APO cells in culture may contribute to a better understanding of the internal defense in mollusks, in general, as well as the specific response of B. glabrata to S. mansoni infection.

Identification of a genetic marker associated with the resistance to Schistosoma mansoni infection using random amplified polymorphic DNA analysis

In schistosomiasis, the host/parasite interaction remains not completely understood. Many questions related to the susceptibility of snails to infection by respective trematode still remain unanswered. The control of schistosomiasis requires a good understanding of the host/parasite association. In this work, the susceptibility/resistance to Schistosoma mansoni infection within Biomphalaria alexandrina snails were studied starting one month post infection and continuing thereafter weekly up to 10 weeks after miracidia exposure. Genetic variations between susceptible and resistant strains to Schistosoma infection within B. alexandrina snails using random amplified polymorphic DNA analysis technique were also carried out. The results showed that 39.8% of the examined field snails were resistant, while 60.2% of these snails showed high infection rates.In the resistant genotype snails, OPA-02 primer produced a major low molecular weight marker 430 bp. Among the two snail strains there were interpopulational variations, while the individual specimens from the same snail strain, either susceptible or resistant, record semi-identical genetic bands. Also, the resistant character was ascendant in contrast to a decline in the susceptibility of snails from one generation to the next.

Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA

This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR) protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purified genomic DNA, that means less than the equivalent to one parasite cell (genome ~580 fg DNA). The efficiency was 0.99 and the correlation coefficient (R²) was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83ºC). The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden) in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water. Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.

Production of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from Schistosoma mansoni

The number of sequences generated by genome projects has increased exponentially, but gene characterization has not followed at the same rate. Sequencing and analysis of full-length cDNAs is an important step in gene characterization that has been used nowadays by several research groups. In this work, we have selected Schistosoma mansoni clones for full-length sequencing, using an algorithm that investigates the presence of the initial methionine in the parasite sequence based on the positions of alignment start between two sequences. BLAST searches to produce such alignments have been performed using parasite expressed sequence tags produced by Minas Gerais Genome Network against sequences from the database Eukaryotic Cluster of Orthologous Groups (KOG). This procedure has allowed the selection of clones representing 398 proteins which have not been deposited as S. mansoni complete CDS in any public database. Dedicated sequencing of 96 of such clones with reads from both 5' and 3' ends has been performed. These reads have been assembled using PHRAP, resulting in the production of 33 full-length sequences that represent novel S. mansoni proteins. These results shall contribute to construct a more complete view of the biology of this important parasite.

A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni

To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (biology.unm.edu/biomphalaria-genome.html), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 × coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ~ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI.