Liana K. Jannotti-Passos

Preliminary analysis of miRNA pathway in Schistosoma mansoni.

RNA silencing refers to a series of nuclear and cytoplasmatic processes involved in the post-transcriptional regulation of gene expression or post-transcriptional gene silencing (PTGS), either by sequence-specific mRNA degradation or by translational arrest. The best characterized small RNAs are microRNAs (miRNAs), which predominantly perform gene silencing through post-transcriptional mechanisms. In this work we used bioinformatic approaches to identify the parasitic trematode Schistosoma mansoni sequences that are similar to enzymes involved in the post-transcriptional gene silencing mediated by miRNA pathway. We used amino acid sequences of well-known proteins involved in the miRNA pathway against S. mansoni genome and transcriptome databases identifying a total of 13 putative proteins in the parasite. In addition, the transcript levels of SmDicer1 and SmAgo2/3/4 were identified by qRT-PCR using cercariae, adult worms, eggs and in vitro cultivated schistosomula. Our results showed that the SmDicer1 and SmAgo2/3/4 are differentially expressed during schistosomula development, suggesting that the miRNA pathway is regulated at the transcript level and therefore may control gene expression during the life cycle of S. mansoni.

PCR amplification of the mitochondrial DNA minisatellite region to detect Schistosoma mansoni infection in Biomphalaria glabrata snails

Schistosoma infection in Biomphalaria glabrata can be detected by either exposing snails to light to induce cercarial shedding or by squeezing them between glass slides to detect parasites in the digestive gland and other regions. The methods available are inefficient for identification of prepatent infections and do not allow the diagnosis of infection in snails that die before arriving in the laboratory. Furthermore, infection is undetectable after migration of sporocysts from the head-foot region of the snail. We examined the use of polymerase chain reaction (PCR) amplification of minisatellite repeats from Schistosoma mansoni mitochondrial DNA (mtDNA) to identify snail infection. We found that amplification of mtDNA under low stringency conditions (LS-PCR) allowed for the identification of specific S. mansoni infection in Biomphalaria snails. To confirm these results, specific amplification reactions were performed using 2 sets of primers that allowed for the diagnosis of infection and an internal control of the reaction (multiplex PCR). Results obtained using multiplex PCR demonstrated the ability of the assay to detect S. mansoni-specific infection. Thus, LS-PCR as well as specific multiplex PCR allow for the detection of prepatent infections and show high specificity for S. mansoni in comparison with other trematode infections in either living or dead snails.

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% 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.

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 (http://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 x 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.

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.

Degree of host-parasite compatibility between Schistosoma mansoni and their intermediate molluscan hosts in Brazil

The compatibility of Biomphalaria tenagophila, B. straminea and B. glabrata from Minas Gerais with different strains of Schistosoma mansoni was evaluated using the method of Frandsen (1979b) in standardized experiments. One hundred and fifty of each species of snail were individually exposed in the laboratory to 50 miracidia of S. mansoni lines LE, SJ and AL. The cercariae from the infected snails were counted and used to calculate TCP/100 indices, which were compared with those of Frandsen (1979b). For B. tenagophila the TCP/100 indices varied from 37,996 to 74,266 (class II and III). The snail was poorly compatible with LE (class II) and compatible with SJ and AL (class III). For B. straminea the indices varied from 9,484 to 20,508. The snail was not very compatible with SJ (class I) and poorly compatible with LE and AL (class II). For B. glabrata the indices varied from 588,828 to 1,039,065. The snails was extremely compatible (class VI) with the three lines of S. mansoni. These results confirm the epidemiological importance of B. glabrata in Brazil followed by B. tenagophila and B. straminea.

Molecular epidemiology of Brazilian Biomphalaria: a review of the identification of species and the detection of infected snails.

The three vector species of Schistosoma mansoni in Brazil, Biomphalaria glabrata, B. tenagophila and B. straminea show different susceptibility levels to the trematode besides a wide geographical distribution. The identification of such molluscs is important to further understand the disease epidemiology. Considering the fact that morphological identification may become difficult or even impossible under particular circumstances, the use of molecular-based methods have permitted the generation of more consistent information concerning the population structure of Biomphalaria furthering knowledge on taxonomy and diagnosis of infection. We have developed methodologies in order to provide simultaneous species identification of the intermediate host and diagnosis of infection with S. mansoni.

Moluscos límnicos da microrregião de Belo Horizonte, MG, com ênfase nos vetores de parasitoses

A malacological survey to detect foci of transmission of schistosomiasis and other parasitic diseases was undertaken into water-courses from 13 municipalities of microregion of Belo Horizonte, MG, Brazil. From 1990 to 1996, 22,066 snails were collected. From those, 378 (1.7%) were found infected by trematodes: Biomphalaria glabrata (7,920), infected by Schistosoma mansoni (1.9%), Echinostomatidae (1.2%), Strigeidae (0.6%), Cercaria minense (0.1%) and Derogenidae (-0.1%); B. straminea (4,093) infected by Strigeidae (0.6%), Echinostomatidae (0.2%), Clinostomatidae (-0.1%) and two unidentified cercariae; B. tenagophila (1,338), infected by Strigeidae (0.1%) and Physa marmorata (1,776) by Echinostomatidae (1.6%). The snails Biomphalaria peregrina, B. occidentalis, B. schrammi, Drepanotrema depressissimum, D. lucidum, D. cimex, Physa cubensis, Lymnaea columella, Melania tuberculata, Idiopyrgus souleyetianus, Pomacea sp, Anodontites sp and Ancylidae were found noninfected. Snails from 9 municipalities were infected by S. mansoni and from 11 by other trematodes.

Isolation and detection of Fasciola hepatica DNA in Lymnaea viatrix from formalin-fixed and paraffin-embedded tissues through multiplex-PCR

Detection of Fasciola hepatica infection in Lymnaea viatrix through analysis of histological cuts is based upon morphological characters of the parasite during the intra-mollusk phase of parasitism. At this stage, trematode forms are very similar and, thus, very difficult to differentiate. Specific detection may also be impaired by the presence of other helminthes in the mollusk. Histological samples are usually fixed in formalin, embedded in paraffin, sectioned and HE stained. In the current study, a method for the extraction of DNA from formalin-fixed, paraffin-embedded tissues was standardized by means of deparaffinizing with xylol and digesting with proteinase K. Extracted DNA was amplified in a multiplex-PCR, by using simultaneous primers in a single reaction under high stringency conditions. Results showed specific amplification of DNA from the trematode and the snails. The technique was sensitive enough to detect F. hepatica infections in L. viatrix, in histological sections in which the presence of larval stages could not be observed through brightfield microscopy. The profiles generated were: stair bands referring to F. hepatica DNAmt amplification; a band of 1200 bp referring to L. viatrix ITS and another of 1300 bp referring to F. hepatica ITS and other trematodes. Multiplex-PCR has shown to be a fast, safe, highly sensitive and specific method, which is able to amplify DNA from fixed tissues, despite a low DNA quantity and its degradation caused by fixation processes. Such methodology may be useful in studies on fascioliasis epidemiology, enabling the use of material from histological collections.

BIPARENTAL MITOCHONDRIAL DNA INHERITANCE IN THE PARASITIC TREMATODE SCHISTOSOMA MANSONI

The maternal inheritance of mitochondrial DNA (mtDNA) in eukaryotic organisms occurs because of the selective destruction of paternal mtDNA molecules that may be present in the zygote. The elimination of sperm mtDNA is less efficient in interspecific crosses, and biparental inheritance of mtDNA has been observed in a variety of species. Because interspecific crosses are likely to be extremely rare in nature, parental inheritance of mtDNA has been deemed of little relevance to population genetics. The mtDNA of the parasitic trematode Schistosoma mansoni was examined for its utility in addressing epidemiological questions related to the transmission and spread of schistosomiasis. Prior to embarking on such experiments, we sought to confirm the mode of inheritance of this molecule using the highly polymorphic mtDNA minisatellite as a marker. In 3 separate crosses, mtDNA apparently identical to paternal DNA was observed in some individuals of the F2 and F3 generations. These observations thus suggest the intraspecific paternal inheritance of mtDNA across multiple generations in Schistosoma mansoni.