Monitoring the efficacy of drugs for neglected tropical diseases controlled by preventive chemotherapy.

In the last decade, pharmaceutical companies, governments and global health organisations under the leadership of the World Health Organization (WHO) have pledged large-scale donations of anthelmintic drugs, including ivermectin (IVM), praziquantel (PZQ), albendazole (ALB) and mebendazole (MEB). This worldwide scale-up in drug donations calls for strong monitoring systems to detect any changes in anthelmintic drug efficacy. This review reports on the outcome of the WHO Global Working Group on Monitoring of Neglected Tropical Diseases Drug Efficacy, which consists of three subgroups: (i) soil-transmitted helminthiases (ALB and MEB); (ii) onchocerciasis and lymphatic filariasis (IVM); and (iii) schistosomiasis (PZQ). Progress of ongoing work, challenges and research needs for each of the four main drugs used in helminthic preventive chemotherapy (PC) are reported, laying the ground for appropriate implementation of drug efficacy monitoring programmes under the co-ordination and guidelines of the WHO. Best practices for monitoring drug efficacy should be made available and capacity built as an integral part of neglected tropical disease (NTD) programme monitoring. Development of a disease-specific model to predict the impact of PC programmes, to detect outliers and to solicit responses is essential. Research studies on genetic polymorphisms in relation to low-efficacy phenotypes should be carried out to identify markers of putative resistance against all NTD drugs and ultimately to develop diagnostic assays. Development of combination and co-administration of NTD drugs as well as of new drug entities to boost the armamentarium of the few drugs available for NTD control and elimination should be pursued in parallel.

Recent advances in Schistosoma genomics.

Schistosome research has entered the genomic era with the publications reporting the Schistosoma mansoni and Schistosoma japonicum genomes. Schistosome genomics is motivated by the need for new control tools. However, much can also be learned about the biology of Schistosoma, which is a tractable experimental model. In this article, we review the recent achievements in the field of schistosome research and discuss future perspectives on genomics and how it can be integrated in a usable format, on the genetic mapping and how it has improved the genome assembly and provided new research approaches, on how epigenetics provides interesting insights into the biology of the species and on new functional genomics tools that will contribute to the understanding of the function of genes, many of which are parasite- or taxon specific.

Cloning and characterization of a cDNA fragment encoding a Schistosoma mansoni actin-binding protein (Smfilamin).

To identify vaccine candidates for Schistosoma mansoni, the IgG fraction of rabbit antiserum raised against immature female worms affinity purified over a NP-40 extract of 3-h schistosomula was used to immunoscreen a cercarial lambdagt11 cDNA library. One clone with a 1.5-kb cDNA insert revealed an encoded peptide of 479 amino acids, which bears homology to human actin-binding protein (ABP-280=filamin). Northern blot analysis revealed a transcript of about 8.6 kb, indicating that the complete gene was not cloned. Overlapping clones, which encode a composite sequence of 983 amino acids (45% identity with filamin), were subsequently isolated from the cDNA library. The 1.5-kb insert was cloned into pGEX, overexpressed, and the 479 amino acid peptide purified. Western blot analysis using polyclonal antisera specific to the peptide identified a 280-kDa molecule in adult worm extracts. RT-PCR demonstrated that Smfilaimin is expressed in various stages. Immunofluorescence studies with specific antisera revealed a tegument-associated fluorescence in adult worms. IgG specific to the Smfilamin fragment showed 36.6% killing of schistosomules in an in vitro killing assay.

DNA probes for identifying chromosomes 5, 6, and 7 of Schistosoma mansoni.

Schistosoma mansoni has a genome of 270 Mb contained on 8 pairs of chromosomes. C-banding has been a useful technique in identifying the 7 autosomal and sex chromosomes. However, even with C-banding, S. mansoni chromosomes 5, 6, and 7 are difficult to discriminate from each other, because of their small sizes, morphological similarity, and poor banding patterns. We have identified probes that specifically paint chromosomes 5, 6, and 7 of S. mansoni with the use of chromosome microdissection and the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). Exact chromosome identification is required for accurate chromosome mapping of genomic clones and genetic elements, which is an essential component of the schistosome genome project.

Y-box binding protein from Schistosoma mansoni: interaction with DNA and RNA.

A Schistosoma mansoni homologue of the human Y-box binding protein (SMYB1), as well as truncated proteins containing its N-terminal Cold Shock Domain (CSD) or its C-terminal domain (TAIL) were cloned into the p-MAL-c2 expression vector and produced in Escherichia coli. In order to characterize the interactions of these proteins to an inverted CCAAT motif present in a number of gene promoters, their binding to DNA was measured by Electrophoretic Mobility Shift Assays. SMYB1 bound to single- and double-stranded DNA containing the CCAAT motif and could bind also to RNA. The truncated CSD and TAIL domain proteins bound to dsDNA and RNA, but exhibited distinct binding patterns. Protein-DNA interaction was also investigated in vivo, using the Yeast One-Hybrid System. The plasmid constructs were GSTTRI, a DNA fragment composed of three copies of the CCAAT motif of the S. mansoni glutathione S-transferase gene promoter and four oligonucleotides spanning different regions of the S. mansoni p14 gene promoter. None of the yeast clones transformed with the above plasmids was able to grow in selective medium or to activate the transcription of the HIS3 reporter gene, suggesting that SMYB1 could not interact with these promoters in vivo.

Additive host genetic factors influence fecal egg excretion rates during Schistosoma mansoni infection in a rural area in Brazil.

This study quantifies the influence of shared household and kinship on egg counts during Schistosoma mansoni infection in a sample from rural Brazil. Detailed genealogic information allowed assignment of 597 individuals to 6 multihousehold pedigrees residing in 145 households. A variance component method was used to partition egg counts into shared household, additive genetic, and individual-specific environmental effects. Host additive genetic effects consistently accounted for a large proportion of the variation in egg counts: 43% in an unadjusted model and 40% in model adjusted for covariates. In a model that examined the confounding of shared household with kinship, additive genetic effects still accounted for 27% of the variation in egg counts and shared household only 12%. The consistently important role for host additive genetic factors on the variation in egg counts points to new ways of modeling and understanding the mechanisms that contribute to trait variation during infection with S. mansoni.

Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries.

In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3%) sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds). Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8%) contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds). The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds). From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci.

Characterization of new Schistosoma mansoni microsatellite loci in sequences obtained from public DNA databases and microsatellite enriched genomic libraries

In the last decade microsatellites have become one of the most useful genetic markers used in a large number of organisms due to their abundance and high level of polymorphism. Microsatellites have been used for individual identification, paternity tests, forensic studies and population genetics. Data on microsatellite abundance comes preferentially from microsatellite enriched libraries and DNA sequence databases. We have conducted a search in GenBank of more than 16,000 Schistosoma mansoni ESTs and 42,000 BAC sequences. In addition, we obtained 300 sequences from CA and AT microsatellite enriched genomic libraries. The sequences were searched for simple repeats using the RepeatMasker software. Of 16,022 ESTs, we detected 481 (3 percent) sequences that contained 622 microsatellites (434 perfect, 164 imperfect and 24 compounds). Of the 481 ESTs, 194 were grouped in 63 clusters containing 2 to 15 ESTs per cluster. Polymorphisms were observed in 16 clusters. The 287 remaining ESTs were orphan sequences. Of the 42,017 BAC end sequences, 1,598 (3.8 percent) contained microsatellites (2,335 perfect, 287 imperfect and 79 compounds). The 1,598 BAC end sequences 80 were grouped into 17 clusters containing 3 to 17 BAC end sequences per cluster. Microsatellites were present in 67 out of 300 sequences from microsatellite enriched libraries (55 perfect, 38 imperfect and 15 compounds). From all of the observed loci 55 were selected for having the longest perfect repeats and flanking regions that allowed the design of primers for PCR amplification. Additionally we describe two new polymorphic microsatellite loci

Genetic epidemiology of fecal egg excretion during Schistosoma mansoni infection in an endemic area in Minas Gerais, Brazil

There is considerable variation in the level of fecal egg excretion during Schistosoma mansoni infections. Within a single endemic area, the distribution of egg counts is typically overdispersed, with the majority of eggs excreted coming from a minority of residents. The purpose of this study was to quantify the influence of genetic factors on patterns of fecal egg excretion in a rural study sample in Brazil. Individual fecal egg excretions, expressed in eggs per gram of feces, were determined by the Kato-Katz method on stool samples collected on three different days. Detailed genealogic information was gathered at the time of sampling, which allowed assignment of 461 individuals to 14 pedigrees containing between 3 and 422 individuals. Using a maximum likelihood variance decomposition approach, we performed quantitative genetic analyses to determine if genetic factors could partially account for the observed pattern of fecal egg excretion. The quantitative genetic analysis indicated that between 21-37 percent of the variation in S. mansoni egg counts was attributable to additive genetic factors and that shared environment, as assessed by common household, accounted for a further 12-21 percent of the observed variation. A maximum likelihood heritability (h²) estimate of 0.44 ± 0.14 (mean ± SE) was found for the 9,604 second- and higher-degree pairwise relationships in the study sample, which is consistent with the upper limit (37 percent) of the genetic factor determined in the variance decomposition analysis. These analyses point to the significant influence of additive host genes on the pattern of S. mansoni fecal egg excretion in this endemic area

Evaluation of Schistosoma mansoni retinoid X receptor (SmRXR1 and SmRXR2) activity and tissue distribution.

Recently, we reported the identification of cDNA's encoding retinoid X receptor (RXR) homologues in Schistosoma mansoni. RXRs are known to be involved in the regulation of genes important for homeostasis and development. Previous studies indicated that SmRXR1 plays a role in the regulation of the female-specific gene, p14. Herein, we report that SmRXR2 also binds to cis-elements present in the p14 upstream region when evaluated in yeast reporter strains. SmRXR2 shows a pattern of recognition of cis-sequences present in the p14 gene upstream region different than SmRXR1. However, the SmRXR2 C (DNA binding) domain binds promiscuously in electrophoretic mobility shift assays to cis-elements of the p14 upstream region. The SmRXRs differ in their ability to activate transcription. The N-terminal A/B domain of SmRXR1 is necessary and sufficient for autonomous transcription activation function (AF) in yeast. SmRXR2 does not exhibit an equivalent autonomous AF. SmRXR1 and SmRXR2 fail to dimerize when investigated both in the yeast two-hybrid system and in immunoprecipitation experiments. In situ hybridization experiments using paraffin sections of adult worms demonstrate that SmRXR1 and SmRXR2 exhibit both common and unique cell type distribution which indicates that SmRXR1 and SmRXR2 both play a role in regulating gene expression in certain cells, yet each plays a distinct role in modulating the expression of genes in other cell types. Both SmRXR1 and SmRXR2 localize to vitelline cells. These studies provide a solid basis for improving our understanding of RXRs and their importance in female-specific gene regulation.

Exposure to Schistosoma mansoni infection in a rural area of Brazil. I: water contact.

The study of water contact patterns in rural Brazil presents unique challenges due to widely dispersed settlement patterns, the ubiquity of water contact sites, and the privatization of water resources. This study addresses these challenges by comparing the two most widely used methods of assessing water contact behaviour: direct observation and survey. The results of a 7-day direct observation of water contact were compared with water contact surveys administered 1 week after and then 1 year after the direct observation study. The direct observation study recorded a water contact rate higher than reported by other investigators (3.2 contacts per person per day); however, 75% of these contacts were for females and consisted mainly of domestic activities occurring around the household. A comparison of the frequency of water contact activities between the direct observation and the two surveys revealed several important points. First, no significant differences were found between methods for routine water contact activities (e.g. bathing), indicating that participants were able to accurately self-report some types of water contact activities. Second, significant differences were found in the recording of water contact activities that took place outside the observation area, indicating that direct observation may under-report water contact activities in areas where contact sites are dispersed widely. Third, significant differences between the direct observation and the survey method were more common for males than for females, indicating that the combination of widespread water contact sites and gender-specific division of labour may result in under-reporting of male contacts by direct observation methods. In short, despite the limitations in the recording of duration and body exposure, the survey method may more accurately record the frequency of water contact activities than direct observation methods in areas of widely dispersed water contact sites. Hence, surveys may be more suitable for the unique challenges of water contact in rural areas of Brazil.

Exposure to Schistosoma mansoni infection in a rural area in Brazil. II: household risk factors.

A number of studies have pointed out the potential importance of the household in the transmission of schistosomiasis. The clustering of domestic activities associated with water collection, storage, and usage can result in the sharing of transmission sites and infective water contact behaviours. In this study, we employed a variance component method to estimate effects due to individual risk factors and shared residence on the variance in faecal egg counts during Schistosoma mansoni infection. A suite of covariates, which included demographic, socioeconomic, water supply, and water contact behaviour terms, contributed 15% to the variance in faecal egg counts. Shared residence alone accounted for 28% of the variance in faecal egg excretion. When both the suite of covariates and shared residence were considered in the same model, shared residence still contributed 22% to the variance in infection intensity. These results point to the importance of shared residence as a means of capturing the complex interrelationship between shared demographic, socioeconomic, physical environmental, and behavioural factors that influence transmission of schistosomiasis at the household level.

Identification and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor-beta signal transducer.

Smad proteins are essential intracellular signal transducers of the transforming growth factor-beta (TGF-beta) superfamily. The TGF-beta superfamily signals through phosphorylation and activation of R-Smad proteins, receptor-regulated Smads, by heteromeric complexes of ligand-specific type I and type II serine/threonine kinase receptors. R-Smads receive a signal from the activated receptor complex and transmit it to the nucleus. A cDNA was isolated that encodes a 649-amino acid protein found to be homologous to members of R-Smad subfamily with highest homology scored to clawed African frog and human Smad2. The Schistosoma mansoni homologue (SmSmad2) was overexpressed in bacteria as a Sj26-GST fusion protein and used to raise specific antibodies. The IgG fraction of the immunized rabbit serum identified 70- and 72-kDa protein bands in Western analysis of schistosome extracts. Treatment with alkaline phosphatase removed the 72-kDa band, which indicates that this band represents the phosphorylated form of schistosome Smad2. SmSmad2 was localized in the subtegument, parenchymal cells, and sex organs in both male and female worm cryosections. Similar results were also obtained from the analysis of the Smad2 mRNA distribution pattern revealed by in situ hybridization of adult worm pair paraffin sections. SmSmad2 mRNA levels were determined by reverse transcriptase-polymerase chain reaction in different mammalian host developmental stages and found to be constitutively expressed. SmSmad2 was also found to interact with a previously identified SmTbetaR-I, a serine/threonine type I kinase receptor. Furthermore, SmSmad2 was shown to undergo phosphorylation by constitutively active forms of SmTbetaR-I in vitro. In addition, SmSmad2 localized in the nuclei of mink lung epithelial cells upon treatment with TGF-beta(1). These data indicate that the SmSmad2 responds to the TGF-beta signals by interaction with receptor I, which phosphorylates it, whereupon it translocates into the nucleus presumably to regulate target gene transcription and consequently elicit a specific TGF-beta effect.

Identification and functional characterization of a member of the PUR-alpha family from Schistosoma mansoni.

Schistosoma mansoni p14 gene encodes an eggshell precursor that is expressed only in vitelline cells of mature female worms in response to a male stimulus. The upstream region of the p14 gene contains several potential cis-acting regulatory sequences. We used the upstream region of the p14 gene as bait in a yeast-one-hybrid screen of a S. mansoni cDNA library to identify interacting proteins. We report the identification and characterization of a cDNA (S. mansoni PUR-alpha (SmPUR-alpha)) encoding a protein homologous to single-stranded DNA transcription activator PUR-alpha, that binds to the p14 upstream region and activates transcription of the HIS3 reporter gene in yeast. SmPUR-alpha has a predicted molecular mass of 30 kDa and shares an overall homology of 63% with mammalian PUR-alpha. The DNA binding domain of SmPUR-alpha is highly conserved. We show by gel shift assays that GST-SmPUR-alpha binds to oligonucleotides comprising the p14 upstream region. SmPUR-alpha binds preferentially to single-stranded DNA and also binds RNA. Unlike the mammalian homologue, SmPUR-alpha exhibits little specificity for the PUR element GGn, but shows strong preference for a sequence containing alternating pyrimidines. Our data support that SmPUR-alpha is a single-copy gene and through reverse transcriptase-polymerase chain reaction and in situ hybridization, we show that SmPUR-alpha is constitutively transcribed in many cell types and thus likely plays a role as a general transcription activator in schistosomes.

Schistosoma mansoni phosphoenolpyruvate carboxykinase, a novel egg antigen: immunological properties of the recombinant protein and identification of a T-cell epitope.

In schistosomiasis mansoni, hepatic granulomatous inflammation surrounding parasite eggs is mediated by CD4(+) T helper (Th) cells sensitized to schistosomal egg antigens (SEA). We previously showed that a prominent lymphoproliferative response of CD4(+) Th cells from schistosome-infected C57BL/6 (BL/6) mice was directed against a 62-kDa component of SEA. A partial amino acid sequence of the 62-kDa component was found to be identical with one present in the enzyme phosphoenolpyruvate carboxykinase (PEPCK). Based on this sequence, a cDNA clone containing the entire coding region of PEPCK was identified, and the full recombinant Schistosoma mansoni PEPCK (rSm-PEPCK) of 626 amino acids was purified from a prokaryotic expression system. rSm-PEPCK strongly stimulated a specific T-cell hybridoma, 4E6, as well as CD4(+) Th cells from SEA-immunized BL/6 mice and from infected BL/6, CBA, and BALB/c mice. In the infected mice, rSm-PEPCK elicited significant gamma interferon production as well as, to a lesser extent, production of interleukin-2 and -5. In BL/6 and BALB/c mice, the CD4(+) Th cell response to rSm-PEPCK was greater than that directed against the egg antigen Sm-p40; conversely, CBA mice responded better to Sm-p40 than to Sm-PEPCK. A 12-amino-acid region (residues 398 to 409: DKSKDPKAHPNS) was demonstrated to contain a T-cell epitope; synthetic peptides containing this epitope significantly stimulated specific hybridoma 4E6 and polyclonal CD4(+) Th cells. The identification and characterization of immunogenic egg components will contribute to the understanding and possible control of T-cell-mediated schistosomal disease.

Construction and characterization of a Schistosoma mansoni bacterial artificial chromosome library.

A bacterial artificial chromosome (BAC) library has been established from genomic DNA isolated from the trematode parasite of human, Schistosoma mansoni. This library consists of more than 21,000 recombinant clones carrying inserts in the pBeloBAC11 vector. The mean insert size was 100 kb, representing an approximate 7.95-fold genome coverage. Library screening with eight chromosome-specific or single-copy gene probes yielded between 1 and 9 positive clones, and none of those tested was absent from the library. End sequences were obtained for 93 randomly selected clones, and 37 showed sequence identity to S. mansoni sequences (ESTs, genes, or repetitive sequences). A preliminary analysis by fluorescence in situ hybridization localized 8 clones on schistosome chromosomes 1 (2 clones), 2, 3, 5, Z, and W (3 clones). This library provides a new resource for the physical mapping and sequencing of the genome of this important human pathogen.

Familial resemblance in humoral immune response to defined and crude Schistosoma mansoni antigens in an endemic area in Brazil.

This study addressed whether the humoral immune response to crude and defined Schistosoma mansoni antigens aggregates within families. The sample included 155 siblings from 42 nuclear families in Brazil. Sera examined by ELISA for antibody isotypes reactive to defined schistosome antigens and crude schistosome antigens (soluble adult worm antigen preparation and soluble egg antigen) demonstrated that there was a difference in sibling-pair correlations between defined and crude S. mansoni antigens. In contrast to the finding with crude antigens, egg-positive sibling pairs showed significant familial resemblance for all IgG subclasses and IgE to adult-stage antigens Smp20.8 and Smp50. Only the IgE and IgG4 isotypes showed familial resemblance to the egg-stage antigen, Smp40. Egg-negative sibling pairs showed significant familial resemblance only for IgE and IgG4 to Smp40. That both the IgE and IgG4 response to defined S. mansoni antigens showed familial resemblance is interesting in light of the converging evidence for the role of IgE and IgG4 in human susceptibility and resistance to reinfection.

Transient expression of DNA and RNA in parasitic helminths by using particle bombardment.

Parasitic helminths (worms belonging to several metazoan phyla) cause considerable morbidity and mortality in humans. They are an important veterinary problem, and they result in significant economic losses in animal grazing and agriculture. Experimental studies on parasitic helminths have been limited by a lack of parasite cell lines and methods for molecular genetic analyses. We evaluated particle bombardment (biolistics) as a strategy to introduce and express nucleic acids in these multicellular parasites. By using embryos of the parasitic nematode Ascaris as a model, we developed methods to introduce and express both DNA and RNA during several stages of Ascaris embryogenesis. Biolistic transfection will facilitate experimental strategies in Ascaris embryos complementing other biochemical tools available (e.g., in vitro whole-cell embryo extracts for transcription, RNA processing, and translation). Transfection experiments with adult schistosomes further suggest that the biolistic strategy should be applicable to a variety of other parasitic helminths. The development of these methods provides molecular genetic tools to study gene expression and the biology of a variety of types and developmental stages of important helminth parasites.

Characterization of the Ras homologue of Schistosoma mansoni.

Ras is a member of a super-family of guanine-binding or G-proteins. Ras functions as a molecular switch in the transduction of signals generated by the activation of a variety of cell surface receptors and relays the signals to downstream effectors. Little is known about signal transduction in schistosomes. In order for Schistosoma mansoni to survive different immune responses triggered by the host as well as to migrate from the site of penetration at the skin to the final destination in portal circulation, they must receive signals from the host environment and respond to them in a way that allows their survival. We have isolated the schistosome Ras cDNA by using sequence information of the schistosome Ras homologue submitted to the Genbank database. Analysis of the encoded peptide revealed 81% identity and 92% similarity with K-Ras from various species. Ras is a single copy gene as determined by quantitative hybridization experiments. The cDNA was cloned into pGEX-4T and the expressed peptide was used to generate specific antibody reagents. Affinity purified antibodies identified a 23 kDa native protein that localizes to the subtegument. Ras is not associated with the tegument. Ras is expressed in all the developmental stages of the parasite. However, Ras is over-expressed in female worms compared to males. Schistosome Ras was also shown to be post-translationally modified by addition of farnesyl isoprenoid moiety to the cysteine residue in the C-terminal box. Using a schistosome extract in vitro SmRas farnesylation was inhibited by the farnesyl transferase inhibitor, FTI-277, at concentrations comparable to those required to inhibit K-Ras processing. These initial studies on signal transduction in schistosomes should provide a solid basis for improving our understanding of schistosome-host interactions.