Richard A. Kane

Biomphalaria glabrata transcriptome: cDNA microarray profiling identifies resistant- and susceptible-specific gene expression in haemocytes from snail strains exposed to Schistosoma mansoni

BackgroundBiomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. B. glabrata/S. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. Examining differential gene expression between S. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences.ResultsWe have developed a 2053 element cDNA microarray for B. glabrata containing clones from ORESTES (Open Reading frame ESTs) libraries, suppression subtractive hybridization (SSH) libraries and clones identified in previous expression studies. Snail haemocyte RNA, extracted from parasite-challenged resistant and susceptible snails, 2 to 24 h post-exposure to S. mansoni, was hybridized to the custom made cDNA microarray and 98 differentially expressed genes or gene clusters were identified, 94 resistant-associated and 4 susceptible-associated. Quantitative PCR analysis verified the cDNA microarray results for representative transcripts. Differentially expressed genes were annotated and clustered using gene ontology (GO) terminology and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis. 61% of the identified differentially expressed genes have no known function including the 4 susceptible strain-specific transcripts. Resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin L, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as HSP70; proteins involved in signalling, such as importin 7 and copine 1, cytoplasmic intermediate filament (IF) protein and transcription enzymes such as elongation factor 1α and EF-2.ConclusionProduction of the first cDNA microarray for profiling gene expression in B. glabrata provides a foundation for expanding our understanding of pathways and genes involved in the snail internal defence system (IDS). We demonstrate resistant strain-specific expression of genes potentially associated with the snail IDS, ranging from signalling and inflammation responses through to lysis of proteinacous products (encapsulated sporocysts or phagocytosed parasite components) and processing/degradation of these targeted products by ubiquitination.

Molecular characterization of freshwater snails in the genus Bulinus: a role for barcodes?

BackgroundReliable and consistent methods are required for the identification and classification of freshwater snails belonging to the genus Bulinus (Gastropoda, Planorbidae) which act as intermediate hosts for schistosomes of both medical and veterinary importance. The current project worked towards two main objectives, the development of a cost effective, simple screening method for the routine identification of Bulinus isolates and the use of resultant sequencing data to produce a model of relationships within the group.ResultsPhylogenetic analysis of the DNA sequence for a large section (1009 bp) of the mitochondrial gene cytochrome oxidase subunit 1 (cox1) for isolates of Bulinus demonstrated superior resolution over that employing the second internal transcribed spacer (its2) of the ribosomal gene complex. Removal of transitional substitutions within cox1 because of saturation effects still allowed identification of snails at species group level. Within groups, some species could be identified with ease but there were regions where the high degree of molecular diversity meant that clear identification of species was problematic, this was particularly so within the B. africanus group.ConclusionThe sequence diversity within cox1 is such that a barcoding approach may offer the best method for characterization of populations and species within the genus from different geographical locations. The study has confirmed the definition of some accepted species within the species groups but additionally has revealed some unrecognized isolates which underlines the need to use molecular markers in addition to more traditional methods of identification. A barcoding approach based on part of the cox1 gene as defined by the Folmer primers is proposed.

Early differential gene expression in haemocytes from resistant and susceptible Biomphalaria glabrata strains in response to Schistosoma mansoni

The outcome of infection in the host snail Biomphalaria glabrata with the digenean parasite Schistosoma mansoni is determined by the initial molecular interplay occurring between them. The mechanisms by which schistosomes evade snail immune recognition to ensure survival are not fully understood, but one possibility is that the snail internal defence system is manipulated by the schistosome enabling the parasite to establish infection. This study provides novel insights into the nature of schistosome resistance and susceptibility in B. glabrata at the transcriptomic level by simultaneously comparing gene expression in haemocytes from parasite-exposed and control groups of both schistosome-resistant and schistosome-susceptible strains, 2 h post exposure to S. mansoni miracidia, using an novel 5K cDNA microarray. Differences in gene expression, including those for immune/stress response, signal transduction and matrix/adhesion genes were identified between the two snail strains and tests for asymmetric distributions of gene function also identified immune-related gene expression in resistant snails, but not in susceptible. Gene set enrichment analysis revealed that genes involved in mitochondrial electron transport, ubiquinone biosynthesis and electron carrier activity were consistently up-regulated in resistant snails but down-regulated in susceptible. This supports the hypothesis that schistosome-resistant snails recognize schistosomes and mount an appropriate defence response, while in schistosome-susceptible snails the parasite suppresses this defence response, early in infection.

Detection of schistosomes polymerase chain reaction amplified DNA by oligochromatographic dipstick

The applications of highly specific and sensitive molecular techniques based on polymerase chain reaction (PCR) have constituted a valuable tool for the diagnosis of schistosomiasis and also for the detection of schistosome infections in the snail intermediate hosts. The common method of detecting PCR amplicons is gel electrophoresis in the presence of ethidium bromide, a carcinogen, which is followed by UV transillumination. Other methods, which are available for detecting PCR products, are real-time PCR, PCR-enzyme-linked immunosorbent assay (PCR-ELIZA) and mass spectrometry but they are cumbersome while they are sometimes complex and expensive. Therefore, a simple method of PCR product detection would be a welcome idea and a most valuable tool particularly in disease endemic countries with limited research facilities and resources. In this study, we applied a simple and rapid method for the detection of Schistosoma haematobium and Schistosoma mansoni PCR amplified DNA products using oligochromatographic (OC) dipstick. The amplicons are visualized by hybridization with a gold conjugated probe, while a control for the chromatographic migration is incorporated in the assay. The lower detection limit observed was 10fg of genomic DNA from each of the two species, while the dipstick was also specific for each of the species used in this study.

Molecular approaches to the identification of Bulinus species in south-west Nigeria and observations on natural snail infections with schistosomes.

The current study considers the distribution of a small sample of 138 Bulinus snails, across 28 localities within eight Nigerian states. Snails were identified using a combination of molecular methods involving both DNA sequencing of a partial cytochrome oxidase subunit 1 (cox1) fragment and restriction profiles obtained from ribosomal internal transcribed spacer (its) amplicons. The results showed that the majority of Bulinus samples tested belonged to the species Bulinus truncatus while only two were Bulinus globosus. The use of RsaI restriction endonuclease to cleave the ribosomal its of Bulinus, as a method of species identification, was adopted for the majority of samples, this being a quicker and cheaper method better suited to small laboratory environments. Polymerase chain reaction (PCR) amplification of the schistosome Dra1 repeat within each of the collected Bulinus samples was employed to determine the extent and distribution of infected snails within the sample areas. Successful amplification of the Dra1 repeat demonstrated that 29.7% of snails were infected with schistosomes. Sequencing of the partial schistosome its from a small subset of snail samples suggested that some snails were either penetrated by both Schistosoma haematobium and Schistosoma bovis miracidia or hybrid miracidia formed from the two species.

Use of sentinel snails for the detection of Schistosoma haematobium transmission on Zanzibar and observations on transmission patterns

Urogenital schistosomiasis is an important public health issue in Zanzibar. Transmission of the parasite to the human population is related to the distribution of the intermediate snail host, Bulinus globosus. We measured B. globosus abundance and Schistosoma haematobium prevalence within snails in a series of naturally occurring populations and compared prevalence detected by observing cercarial shedding for patent infections, and by PCR using DraI repeat. A total of 2146 B. globosus were collected throughout the study period from 2003 to 2007; of these 85 (3.96%) were shedding cercariae. The levels of infection detected by PCR were consistently higher (40-100 percent). Levels of exposure to miracidia in the field were measured using sentinel snails. B. globosus (a susceptible host) and B. nasutus (a non-susceptible host) were placed in cages at transmission sites for 72h to observe rates of penetration by miracidia. Both B. globosus and B. nasutus tested positive for S. haematobium by PCR (up to 24% infected) indicating frequent contamination of the waterbodies with S. haematobium miracidia. The use of sentinel snails coupled with PCR diagnostics could be a sensitive tool for mapping and monitoring transmission of schistosomiasis in areas of low prevalence.

Confirmed Infection with Intestinal Schistosomiasis in Semi-Captive Wild-Born Chimpanzees on Ngamba Island, Uganda

BACKGROUND Intestinal schistosomiasis, caused by Schistosoma mansoni, is endemic to Lake Victoria, with high prevalence of the disease observed in human lakeshore communities. However, nonhuman primates have recently been overlooked as potential hosts of the disease, despite known susceptibility. METHODS Using a variety of stool, urine, and serological diagnostic methods, 39 semi-captive wild-born chimpanzees and 37 staff members at Ngamba Island Chimpanzee Sanctuary, Lake Victoria, Uganda, were examined for S. mansoni infection. Miracidia recovered from stool were DNA barcoded to investigate cross-over between humans and chimpanzees. The island was also surveyed for Biomphalaria intermediate host snails, which were examined for infection with S. mansoni. RESULTS Chimpanzees were unequivocally shown to be infected with intestinal schistosomiasis with a seroprevalence in excess of 90%. Three egg-positive cases were detected, although the sensitivity of the diagnostic tests varied due to earlier prophylactic praziquantel treatment. Miracidia hatched from chimpanzee stool revealed three DNA haplotypes commonly found in humans living throughout Lake Victoria, including staff on Ngamba Island, as well as two novel haplotypes. At one site, a snail was observed shedding schistosome cercariae. CONCLUSIONS The anthropozoonotic potential of intestinal schistosomiasis on Ngamba Island is greater than previously thought. Moreover, the ability of chimpanzees to void schistosome eggs capable of hatching into viable miracidia further suggests that these nonhuman primates may be capable of maintaining a local zoonotic transmission of schistosomiasis independently of humans. The implications for management of captive and wild primate populations at risk of exposure are discussed.

Application of single strand conformational polymorphism (SSCP) analysis with fluorescent primers for differentiation of Schistosoma haematobium group species.

To assess the utility of single-stranded conformational polymorphism (SSCP) analysis for the differentiation of schistosomes, using methods adapted for a Perkin Elmer ABI Prism 377 automated sequencer, 3 isolates of Schistosoma haematobium, 2 of S. intercalatum and single isolates of S. curassoni and S. bovis were selected for study. Two fluorescently labelled, double-stranded polymerase chain reaction products, amplified from the mitochondrial cytochrome oxidase subunit 1 (CO1) gene and the nuclear ribosomal second internal transcribed spacer (ITS2), were generated from single male and female worms. Changes in electrophoretic mobility of fragments within an SSCP profile revealed variation at individual, isolate and species levels. The mutational basis between representative SSCP profiles was confirmed by direct sequencing, demonstrating that single point substitutions were detectable. SSCP analysis has considerable potential as an alternative molecular method of identification and characterization of schistosomes. More broadly, fluorescence-based SSCP analysis is applicable to almost any gene target from any species of parasite and is a powerful molecular tool for genetic profiling.

Parasitological and malacological surveys reveal urogenital schistosomiasis on Mafia Island, Tanzania to be an imported infection.

To confirm the local endemicity of Schistosoma haematobium on Mafia Island, Tanzania, conjoint parasitological and malacological surveys were undertaken in July 2006 with parasitological investigations supplemented with case-history questionnaires. A total of 238 children (125 girls and 113 boys, mean age of 13.9 years) across 9 primary schools were examined. The prevalence of micro-haematuria and egg-patent infection was 18.1% (CI95=9.6-33.6) and 4.2% (CI95=1.9-7.6), respectively but a strong female bias was observed for micro-haematuria (5.6F:1M) contrasting with a strong male bias for the presence of eggs (1F:4M). All egg-patent infections were of light-intensity (<10eggs/10ml). No clear associations between infection prevalence and local water-contact, by school, were found and all 10 of the egg-positive children had a travel history to the nearby mainland or Zanzibar. Inspection of community diagnostic registers at Kilindoni Hospital revealed a low proportion (<2%) of egg-patent infection for 20,306 samples tested in the 2000-2005 period. A total of 43 freshwater sites, a third of which were previously sampled in 1999 and 2002, were surveyed and 11 species of freshwater mollusc were found. Four species of Bulinus (B. nasutus, B. forskalii, B. barthi and B. sp.) were encountered across 13 sites with B. nasutus restricted to 3 of these towards the north of the island. No collected snail was observed to shed schistosome cercariae. Further characterisation of B. nasutus and S. haematobium included infection challenge on two occasions, with miracidia obtained from egg-patent children from Mafia and Unguja islands as well as DNA barcoding of snails and schistosomes. B. nasutus was shown refractory to infection. With the substantial travel to and from Mafia, the refractory nature of local snails and evidence from DNA barcoding in schistosomes and snails, we conclude that urogenital schistosomiasis is an imported infection.

Inter-specific parasite competition: mixed infections of Schistosoma mansoni and S. rodhaini in the definitive host

Competition between parasite species has been predicted to be an important force shaping parasite and host ecology and evolution, although empirical data are often lacking. Using the Mus musculus-Schistosoma mansoni and Schistosoma rodhaini host-parasite systems we characterized mate choice and inter-specific competition between these two schistosome species. Simultaneous infections revealed species-specific mate preferences for both species as well as suggesting mating competition, with male S. rodhaini appearing dominant over male S. mansoni. S. rodhaini homologous pairs were also shown to have increased reproduction per paired female in the presence of a competitor in simultaneous infections. Overall total reproductive success was, however, similar between the two species under conditions of direct competition due to the greater initial infectivity of S. mansoni in comparison to S. rodhaini. Inter-specific competition was also implicated as increased parasite virulence to the host. The potential effects of such interactions on parasite and host ecology and evolution in nature are discussed.