J. R. Stothard

Variation within the internal transcribed spacer (ITS) of ribosomal DNA genes of intermediate snail hosts within the genus Bulinus (Gastropoda: Planorbidae).

Species within the genus Bulinus are responsible for transmission of schistosomes within the Schistosoma haematobium group. In order to provide a molecular insight into the species relationships within the genus, genetic variation between species representing the four species groups was assayed by Polymerase Chain Reaction (PCR) amplification of the ribosomal Internal Transcribed Spacer (ITS) region followed by Restriction Fragment Length Polymorphism (RFLP) analysis of this product with six restriction enzymes. This PCR-RFLP methodology detected considerable variation within the ITS region indicating that restriction profiles will be useful as genetic markers for identification purposes. The complete ITS1 spacer was sequenced for B. globosus, B. cernicus and B. truncatus. There were numerous nucleotide differences between taxa mainly insertions and deletions. Nucleotide divergence was calculated between species from the restriction profiles: the B. truncatus/tropicus complex and B. reticulatus group were most similar which were in turn more closely related to the B. africanus group than to the B. forskalii group. The nucleotide divergence between the species groups is substantial and questions the placement of these groups within the same genus.

Control of schistosomiasis in sub-Saharan Africa: progress made, new opportunities and remaining challenges.

Several other journal supplements have documented progress made in the control of schistosomiasis in Egypt, China and Brazil, however, with more than 97% of the schistosome infections now estimated to occur in Africa, the relevance of this special issue in Parasitology cannot be overemphasized. In total, 18 articles are presented, inclusive of a lead-editorial from the WHO highlighting a seminal resolution at the 54th World Health Assembly in 2001 that advocated de-worming. Facilitated by a US

Interactions between intermediate snail hosts of the genus Bulinus and schistosomes of the Schistosoma haematobium group.

30 million grant from the Bill and Melinda Gates Foundation in 2002, the Schistosomiasis Control Initiative subsequently fostered implementation of large-scale schistosomiasis (and soil-transmitted helminthiasis) control programmes in six selected African countries. From 2005, CONTRAST, a European union-funded consortium, was formed to conduct multi-disciplinary research pertaining to optimisation of schistosomiasis control. Progress made in schistosomiasis control across sub-Saharan Africa since the turn of the new millennium is reviewed, shedding light on the latest findings stemming from clinical, epidemiological, molecular and social sciences research, inclusive of public health interventions with monitoring and evaluation activities. New opportunities for integrating the control of schistosomiasis and other so-called neglected tropical diseases are highlighted, but more importantly, several opportune questions that arise from it frame the remaining challenges ahead for an enduring solution.

Molecular epidemiology of Schistosoma mansoni in Uganda: DNA barcoding reveals substantial genetic diversity within Lake Albert and Lake Victoria populations

Within each of the four species groups of Bulinus there are species that act as intermediate hosts for one or more of the seven species of schistosomes in the Schistosoma haematobium group, which includes the important human pathogens S. haematobium and S. intercalatum. Bulinus species have an extensive distribution throughout much of Africa and some surrounding islands including Madagascar, parts of the Middle East and the Mediterranean region. Considerable variation in intermediate host specificity can be found and differences in compatibility between snail and parasite can be observed over small geographical areas. Molecular studies for detection of genetic variation and the discrimination of Bulinus species are reviewed and two novel assays, allele-specific amplification (ASA) and SNaPshot, are introduced and shown to be of value for detecting nucleotide changes in characterized genes such as cytochrome oxidase 1. The value and complexity of compatibility studies is illustrated by case studies of S. haematobium transmission. In Senegal, where B. globosus, B. umbilicatus, B. truncatus and B. senegalensis may act as intermediate hosts, distinct differences have been observed in the infectivity of different isolates of S. haematobium. In Zanzibar, molecular characterization studies to discriminate between B. globosus and B. nasutus have been essential to elucidate the roles of snails in transmission. B. globosus is an intermediate host on Unguja and Pemba. Further studies are required to establish the intermediate hosts in the coastal areas of East Africa. Biological factors central to the transmission of schistosomes, including cercarial emergence rhythms and interactions with other parasites and abiotic factors including temperature, rainfall, water velocity, desiccation and salinity are shown to impact on the intermediate host-parasite relationship.

The distribution of Fasciola hepatica and Fasciola gigantica within southern Tanzania - constraints associated with the intermediate host

Representative samples of Ugandan Schistosoma mansoni from Lake Albert and Lake Victoria were examined using DNA barcoding, sequence analysis of two partially overlapping regions - ASMIT (396 bp) & MORGAN (617 bp) - of the mitochondrial cytochrome oxidase subunit I (cox1). The Victorian sample exhibited greater nucleotide diversity, 1.4% vs. 1.0%, and a significant population partition appeared as barcodes did not cross-over between lakes. With one exception, Lake Albert populations were more mixed by sampled location, while those from Lake Victoria appeared more secluded. Using statistical parsimony, barcode ASMIT 1 was putatively ancestral to all others and analysis of MORGAN cox1 confirmed population diversity. All samples fell into two of five well-resolved lineages; sub-lineages therein broadly partitioning by lake. It seems that barcode ASMIT 1 (and close variants) was likely widely dispersed throughout the Nilotic environment but later diversified in situ, and in parallel, within Lake Albert and Lake Victoria. The genetic uniformity of Ugandan S. mansoni can no longer be assumed, which might better explain known epidemiological heterogeneities. While it appears plausible that locally evolved heritable traits could spread through most of the Lake Albert populations, it seems unlikely they could quickly homogenise into Lake Victoria or amongst populations therein.

New insights into the transmission biology of urinary schistosomiasis in Zanzibar

In East Africa, Fasciola gigantica is generally the causative agent of fasciolosis but there have been reports of F. hepatica in cattle from highland regions of Kenya, Ethiopia, Uganda and Zaire. The topography of the Southern Highlands of Tanzania provides an environment where the climatic conditions exist for the sustenance of lymnaeid species capable of supporting both Fasciola hepatica and F. gigantica. Theoretically this would allow interaction between fasciolid species and the possible creation of hybrids. In this report we present molecular data confirming the existence of the snail, Lymnaea truncatula, at high altitude on the Kitulo Plateau of the Southern Highlands, Tanzania, along with morphometric and molecular data confirming the presence of F. hepatica in the corresponding area. At lower altitudes, where climatic conditions were unfavourable for the existence of L. truncatula, the presence of its sister species L. natalensis was confirmed by molecular data along with its preferred fasciolid parasite, F. gigantica. Analysis based on a 618 bp sequence of the 28S rRNA gene did not reveal the presence of hybrid fasciolids in our fluke samples.

The transmission status of bulinus on zanzibar island (unguja), with implications for control of urinary schistosomiasis

A better understanding of the transmission biology of urinary schistosomiasis in Zanzibar, Tanzania was only possible after the development of molecular DNA markers for identification of Bulinus africanus group snails, the potential intermediate hosts of Schistosoma haematobium. Hitherto, identification of natural populations of B. globosus and B. nasutus was problematic and the intermediate host status and distribution of either species remained speculative. By recourse to molecular markers, snail distribution maps could be drawn, revealing an allopatric distribution and, more importantly, leading to the discovery that B. nasutus played no role in transmission. Indeed, in Unguja the area of active transmission of S. haematobium to humans is confined within the distribution of B. globosus. This strong relationship may prove useful for predicting the distribution of urinary schistosomiasis within Zanzibar and, if snail schistosome compatibilities persist, in other areas nearby, e.g. coastal Tanzania and Kenya. The transmission biology of urinary schistosomiasis in Zanzibar is reviewed, the paper reports on ongoing malacological studies in Zanzibar and Kenya and finally closes by posing the question whether medical malacology forms an essential component associated with mass-scale chemotherapy control programmes.

A spot-check of the efficacies of albendazole or levamisole, against soil-transmitted helminthiases in young Ungujan children, reveals low frequencies of cure

(2000). The transmission status of Bulinus on Zanzibar Island (Unguja), with implications for control of urinary schistosomiasis. Annals of Tropical Medicine & Parasitology: Vol. 94, No. 1, pp. 87-94.

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

On Unguja (Zanzibar Island), as elsewhere in sub-Saharan Africa, soil-transmitted helminthiases (STH) can be common within pre-school-aged children (PSAC). In line with the Millennium Development Goals, regular de-worming of PSAC is recommended and typically provided through mother-and-child-health clinics (Olsen, 2007; Albonico et al., 2008). In their recent epidemiological survey, Sousa-Figueiredo et al. (2008) found that up to 50% of Ungujan PSAC had at least one soiltransmitted helminth — 20.5% having concurrent ascariasis and trichuriasis — and that there was significant spatial heterogeneity in such diseases, by sampled village. In a subsequent, island-wide survey, Stothard et al. (2008) expanded upon these findings, observing lower general prevalences of STH — 8.6% for ascariasis, 18.9% for trichuriasis and just 1.7% for hookworm — but confirming the existence of significant spatial heterogeneity. Although all the villages included in this survey had similar access to mother-and-child-health clinics and albendazole, STH were much more common in PSAC from the more northern villages than in those from other study sites (Stothard et al., 2008). The main aim of the present study was to investigate whether this observation was the result of local heterogeneity in the efficacy of albendazole. As the protocols for the regular monitoring of drug efficacy, which is strongly advocated (Albonico, 2003; Albonico et al., 2008), are still being standardized (Scherrer et al., 2009), a spot-check equivalence comparison was deemed more appropriate than a placebo-blinded trial. In the spot-check, the efficacy of albendazole was compared with that of the second-line anthelminthic drug, levamisole.

Schistosoma bovis in western Uganda.

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.