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J R Stothard

Publications and source records attributed to J R Stothard.

29 records · Page 2Linked to original sources
PubMed1999

Genomic diversity in the Leishmania donovani complex.

The Leishmania donovani complex is considered to be composed of 3 species; L. donovani, L. infantum and L. chagasi, although this classification has been challenged. Genotypic relationships within the complex were evaluated at different levels by: binding of the probe Lmet9, specific for L. chagasi and Old World Leishmania spp.; partial sequencing of a constitutive major surface protease single gene (mspC) and random amplification of polymorphic DNA (RAPD). The Old World Leishmania spp. and the L. donovani complex have a monophyletic origin. Leishmania chagasi clearly belongs to the L. donovani complex but it is indistinguishable from L. infantum, which suggests introduction of L. chagasi into the New World in recent history. Leishmania infantum/L. chagasi was identified as a monophyletic group within the L. donovani complex but L. donovani may be paraphyletic. Diversity within L. donovani is substantial and phylogeographical patterns of association were found.

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PubMed1998

Temperature gradient gel electrophoresis (TGGE) analysis of riboprints from Trypanosoma cruzi.

To test the homogeneity of 18S sequences within Trypanosoma cruzi, riboprint profiles were separated by temperature gradient gel electrophoresis (TGGE). Upon interpretation of melting curves of fragments within a riboprint profile, there appeared to be two 18S sequence types within each stock examined. Two similar types were also observed within outgroup taxa Trypanosoma conorhini, Trypanosoma rangeli and Leishmania braziliensis. From DNA hybridization studies, these fragments were shown to have homology to the 18S V1 region. There are therefore two 18S V1 regions, differing in sequence, present in all taxa examined. When only a single 18S sequence is used to represent each taxa for phylogenetic inference, comparisons may be between paralogous and not orthologous copies of this region, such that, inferred relationships may merely reflect a gene history. This seriously questions the current molecular phylogeny of these protozoa using 18S data.

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PubMed1998

On the molecular taxonomy of Trypanosoma cruzi using riboprinting.

In order to investigate the molecular taxonomy within Trypanosoma cruzi, the ribosomal small subunit (18S) gene was amplified by polymerase chain reaction (PCR) from a selection of 21 stocks and 3 outgroup taxa. Amplification products were digested with 10 restriction enzymes; restriction fragments were separated by polyacrylamide gel electrophoresis and profiles were visualized by silver staining. Upon analysis of such riboprint profiles, an estimate of pairwise phenetic distance between stocks of T. cruzi was calculated. Upon principal coordinate analysis of this data matrix, a tendency towards a bi-polar grouping of stocks was observed. These 2 groups were predominantly either zymodeme 1 stocks or zymodeme 2 stocks. The position of zymodeme 3 stocks remained intermediate between the 2 groups but did not form a coherent group by themselves. It would therefore appear premature to warrant division of T. cruzi into 2 discrete taxa or subspecies until the relationships of further zymodeme 3 stocks are elucidated.

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PubMed1998

Random amplification of polymorphic DNA as a tool for taxonomic studies of triatomine bugs (Hemiptera: Reduviidae).

Eleven of 27 decameric primers were found to be suitable for random amplification of polymorphic DNA (RAPD) from triatomine bugs on the basis that they produced discrete profiles and distinguished among Panstrongylus megistus (Burmeister), Rhodnius prolixus Stål, and Triatoma infestans (Klug). The legs, or single leg segments, of individual bugs were used as the source of DNA so that the taxonomic value of the bug was conserved. Within the scope of the specimens studied, RAPD profiles allowed assignment to species even when bugs were kept dry for up to 12 mo. Profiles for individuals within a species were not identical. RAPD profiles, with the specimens tested, distinguished among species of 3 pairs considered to be morphologically similar and closely related, namely, Rhodnius ecuadorensis Lent & León and Rhodnius pictipes Stål; Rhodnius nasutus Stål, and Rhodnius neglectus Lent; Rhodnius prolixus Stål and Rhodnius robustus Larrousse. RAPD data conformed with the perceived affinities among these species. RAPD polymorphisms were seen with T. infestans from 3 different localities, but none of the polymorphisms was confined to 1 source. RAPD provided a molecular basis to reassess taxonomic relationships within the Triatomine subfamily. The accurate distinction of triatomine species and of intraspecific bug populations may contribute to elimination of vector-borne Chagas disease from the Americas.

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PubMed1998

Molecular characterisation of intermediate snail hosts and the search for resistance genes.

The relationship between schistosomes and their intermediate hosts is an extremely intricate one with strains and species of the parasite depending on particular species of snail, which in turn may vary in their susceptibility to the parasites. In order to gain a better understanding of the epidemiology of the disease we have been investigating the use of molecular markers for snail identification and for studying host-parasite relationships. In this paper we will draw on examples concerning schistosomiasis in West and East Africa to illustrate how a molecular analysis can be used as part of a "total evidence" approach to characterisation of Bulinus species and provide insights into parasite transmission. Particular emphasis is given to ribosomal RNA genes (rRNA), random amplified polymorphic DNA (RAPDs) and the mitochondrial gene cytochrome oxidase I (COI). Snails resistant to infection occur naturally and there is a genetic basis for this resistance. In Biomphalaria glabrata resistance to Schistosoma mansoni is known to be a polygenic trait and we have initiated a preliminary search for snail genomic regions linked to, or involved in, resistance by using a RAPD based approach in conjunction with progeny pooling methods. We are currently characterising a variety of STSs, (sequence tagged sites) associated with resistance. These can be used for local linkage and interval mapping to define genomic regions associated with the resistance trait. The development of such markers into simple dot-blot or specific PCR-based assays may have a direct and practical application for the identification of resistant snails in natural populations.

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PubMed1997

Use of polymerase chain reaction-based single strand conformational polymorphism and denaturing gradient gel electrophoresis methods for detection of sequence variation of ribosomal DNA of Trypanosoma cruzi.

Polymerase chain reaction (PCR) was used to amplify the V1 region of the small sub-unit (18S) ribosomal DNA gene from representative strains of Trypanosoma cruzi. In order to screen for sequence variation, amplification products were subsequently analysed by single strand conformational polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) techniques. SSCP could not detect sequence variation within T. cruzi, although electrophoretic profiles were clearly distinct from both Leishmania donovani and Leishmania braziliensis. DGGE could differentiate strains of T. cruzi and it appears that there are at least 2 18S V1 sequences of this multi-gene family within each strain examined, contrasting with Leishmania spp. where only 1 was identified. This is the first application of PCR-linked SSCP and DGGE analysis for differentiating parasitic protozoa.

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PubMed1996

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.

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PubMedDate not supplied

New insights into the transmission biology of urinary schistosomiasis in Zanzibar.

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.

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PubMedDate not supplied

Urinary schistosomiasis in schoolchildren on Zanzibar Island (Unguja), Tanzania: a parasitological survey supplemented with questionnaires.

The distribution of urinary schistosomiasis in schoolchildren on Zanzibar Island (Unguja) was surveyed in May 2001 to test a potential correlation with the distribution of snail species of the Bulinus africanus group and to record contemporary baseline epidemiological data. Quasi-random samples of 40 schoolchildren of mixed sexes were selected from each of 10 schools. Schistosoma haematobium infections were detected upon the basis of micro-haematuria with subsequent confirmation by microscopy examination for schistosome eggs. At the time of urine collection, each child was interviewed with a suite of 12 questions prepared as a standardized questionnaire. Total prevalence of urinary schistosomiasis (known locally as kichocho) was 12% although schistosome infections were absent in 5 schools. Schools located west of 39 degrees 19'E and north of 6 degrees 10'S harboured nearly all of the infections; the highest prevalence (55%) was found at Kinyasini where many B. globosus habitats occur nearby. The general level of understanding of kichocho was low (24%) and individual self-diagnosis was poor (sensitivity, 8.5%; specificity, 85%). Grouped freshwater-contact patterns of schoolchildren differed significantly between schools and correlated well with prevalence of infections within schools. Across the island the area of active transmission of S. haematobium to humans appears confined within the distribution of B. globosus. There was no epidemiological evidence to suggest any involvement of B. nasutus in local transmission, confirming previous laboratory findings. In areas where B. globosus occurs, targeted snail control should be considered, to reduce schistosome transmission.

Adolescent↗
PubMedDate not supplied

Analysis of genetic diversity of Trypanosoma cruzi: an application of riboprinting and gradient gel electrophoresis methods.

Analysis of restriction fragment length polymorphism (RFLP) profiles derived from digestion of polymerase chain reaction (PCR) products of the ribosomal 18S from Trypanosoma cruzi yields a typical 'riboprint' profile that can vary intraspecifically. A selection of 21 stocks of T. cruzi and three outgroup taxa: T. rangeli, T. conorhini and Leishmania braziliensis were analysed by riboprinting to assess divergence within and between taxa. T. rangeli, T. conorhini and L. braziliensis could be easily differentiated from each other and from T. cruzi. Phenetic analysis of PCR-RFLP profiles indicated that, with one or two exceptions, stocks of T. cruzi could be broadly partitioned into two groups that formally corresponded to T. cruzi I and T. cruzi II respectively. To test if ribosomal 18S sequences were homogeneous within each taxon, gradient gel electrophoresis methods were employed utilising either chemical or temperature gradients. Upon interpretation of the melting profiles of riboprints and a section of the 18S independently amplified by PCR, there would appear to be at least two divergent 18S types present within T. cruzi. Heterogeneity within copies of the ribosomal 18S within a single genome has therefore been demonstrated and interestingly, this dimorphic arrangement was also present in the outgroup taxa. Presumably the ancestral duplicative event that led to the divergent 18S types preceded that of speciation within this group. These divergent 18S paralogues may have, or had, different functional pressures or rates of molecular evolution. Whether or not these divergent types are equally transcriptionally active throughout the life cycle, remain to be assessed.

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