Genetic diversity and recruitment pattern of Schistosoma mansoni in a Biomphalaria glabrata snail population: a field study using random-amplified polymorphic DNA markers.

Random-amplified polymorphic DNA markers have been used to assess the amount and the distribution of the genetic diversity of Schistosoma mansoni within a natural population of Biomphalaria glabrata at a transmission site of the murine schistosomiasis focus of Guadeloupe. Despite high infection rate and heavy schistosome load within the definitive hosts (Ratus rattus), prevalences within intermediate snails ranged from 0.2 to 4.8%. Whatever the transmission season may be (rainy vs. dry), most of the infected snails were spatially aggregated and 88.4% of them harbored a single parasite genotype indicative of a monomiracidial infection; 4.7% had dual sex infections and a parasite intensity not exceeding 3 miracidia per snail. A substantial resistance level toward the parasite and recruitment regulatory process within snails may explain in part the observed low parasite prevalences and intensities. Considering such a distribution pattern of larval S. mansoni genetic diversity among B. glabrata, mobility of the definitive hosts, or rapid turnover of infected snails, or both, are required to maintain genetic heterogeneity within adult schistosome populations.     

A Schistosoma mansoni tri- and tetramer microsatellite catalog for genetic population diversity and differentiation

All Schistosoma mansoni tri- and tetranucleotide repeat microsatellites published as of December 2018 were identified. All 52 were evaluated for autosomal location, strength of amplification, scorability and behavior as single-copy loci by polyacrylamide and capillary gel electrophoresis. Of these, 27 were unique, autosomal, polymorphic, easily scored and single copy as assessed on pooled adult worm DNA from two different continental origins and adult worm clones. These microsatellites were distributed across all seven autosomal chromosomes. On laboratory strains their heterozygosity ranged from 0.22 to 0.77. Individual markers had 5–13 alleles, allelic richness of 2–10 and an effective allele number of 1.3–8.14. Those infected by Schistosoma mansoni carry many genetically distinct, sexually reproducing parasites, therefore, for an individual infection the complete allele frequency profile of their progeny consists of a pool of DNA from multiple diploid eggs. Using a set of 25 microsatellites, we calculated allele frequency profiles of eggs in fecal samples from people in two Brazilian communities separated by 6 km: Jenipapo (n = 80) and Volta do Rio (n = 38). There were no a priori characteristics that could predict the performance of markers in natural infections based on their performance with laboratory strains. Increasing marker number did not change accuracy for differentiation and diversity but did improve precision. Our data suggest that using a random set of 10–20 microsatellites appears to result in values that exhibit low standard deviations for diversity and differentiation indices. All identified microsatellites as well as PCR conditions, allele size, primer sequences and references for all tri- and tetramer microsatellites markers presented in this work are available at: https://sites.google.com/case.edu/cwru-and-fiocruz-wdrc/home.     

Genetic diversity of a population of Schistosoma haematobium derived from schoolchildren in east central Zimbabwe

To characterize the extent of genetic diversity of Schistosoma haematobium within and among its definitive host (intra- and interhost parasite diversity), 133 individual isolates from 25 infected schoolchildren were compared using randomly amplified polymorphic DNA markers. With 4 primers, 53 unambiguous loci were identified, and of these, 22 were polymorphic. Mean heterozygosity in the population was 0.116 +/- 0.043. Analysis of molecular variance showed the majority of variance occurred within, rather than between, hosts. Frequencies of certain alleles segregated the parasite population into 13 distinct clusters of associated genotypes, with 4 of these first appearing 10 mo after the initial survey. Considering the level of diversity within this limited geographical area and the possibility of rapid turnover of genotypes, parasite variance may impact acquired immunity and clinical outcome of the infection.     

Susceptibility of a Brazilian wild rodent isolate of Schistosoma mansoni to praziquantel in mice

The therapeutic effects of praziquantel (PZQ) against a Schistosoma mansoni isolate derived from Nectomys squamipes (isolate R) and a susceptible isolate (BH) were analyzed in Swiss mice by fecal egg counting, adult worm reduction and oogram pattern. Infected mice were orally administrated with 62.5mg/kg (group 1), 125mg/kg (group 2), 250mg/kg (group 3) and 500mg/kg (group 4), each dose divided over 3 days (49, 50 and 51 days after infection). The data were analyzed using one-way analysis of variance (ANOVA). In regard to isolate R, no fecal eggs were observed with 250 mg/Kg and 500 mg/kg (p<0.05), whereas BH excretion reached zero with all doses. Mean worm burden reduction was significantly (p<0.05) higher at the two highest concentrations, regardless of isolate. At 62.5mg/kg, the percentage of immature eggs varied from 17% (isolate R) to 38% (isolate BH). At 125 mg/kg, the percentage of immature eggs varied from 20% (isolate R) to 16% (isolate BH). At 250 mg/kg, immature eggs dropped significantly to 1% (isolate R) and 4% (isolate BH). At 500 mg/kg, no immature eggs were found in isolate R, whereas in BH was 8%. No dosage significantly (p>0.05) affected the percentage of mature eggs, regardless of isolate. There was a large increase (p<0.001) in the percentages of dead eggs in all treated groups of 62% and 64% in groups 3 and 4, respectively (isolate R). The percentage of dead eggs rose from 34% (group 1) to 58% (group 3) in isolate BH. Although group 4 showed lowest increase in the percentage of dead eggs (46%), it was higher (p<0.001) compared to the 8% in the control. Our findings indicate that the wild isolate from N. squamipes is susceptible to PZQ.     

Isoenzyme studies in one Brazilian and two Venezuelan strains of Schistosoma mansoni.

1. Enzyme polymorphism, analyzed by starch gel electrophoresis, was found to be zero for acid phosphatase, phosphoglucomutase, phosphoglucose isomerase, glucose 6-phosphate dehydrogenase, lactate dehydrogenase, malate dehydrogenase and malic enzyme, in one Brazilian and two Venezuelan strains of Schistosoma mansoni. 2. All loci studied were monomorphic within strains, but the isoenzymic patterns were, however, different among the strains. 3. Results suggest a drastic loss of the genetic variability usually found in natural populations.     

Random spatial distribution of Schistosoma mansoni and hookworm infections among school children within a single village

Schistosomes and soil-transmitted helminths currently infect a third of the world's human population. An important feature of these parasitic infections is their focal distribution, which has significant implications for control. Only a few studies have been carried out at the microepidemiological scale, comparing infection levels among individuals or households within a single village. In this study, data are presented from a cross-sectional survey, examining all children attending a primary school in rural C?te d'Ivoire over several consecutive days for Schistosoma mansoni, soil-transmitted helminths, and intestinal protozoa. All houses in the main village were mapped, and school children were linked to these households for small-area spatial analyses. Comparison between the 260 school children who live within the main village and the 89 children who reside in nearby settlements revealed significant differences in the overall prevalence and intensity of infections with S. mansoni and hookworm, confirming the focal nature of these 2 parasites. On the other hand, S. mansoni and hookworm infections exhibited random spatial patterns within the main village. The validity of these results is discussed in the context of this epidemiological setting, drawing attention to the issue of scale. Our findings have direct implications for intervention because they call for a uniform, community-wide approach to control schistosomiasis and soil-transmitted helminthiasis. Implementation can be relatively straightforward, and the proposed control approach might be cost-effective and prove sustainable.     

Praziquantel Treatment Decreases Schistosoma mansoni Genetic Diversity in Experimental Infections

Background

Schistosomiasis has a considerable impact on public health in many tropical and subtropical areas. In the new world, schistosomiasis is caused by the digenetic trematode Schistosoma mansoni. Chemotherapy is the main measure for controlling schistosomiasis, and the current drug of choice for treatment is praziquantel (PZQ). Although PZQ is efficient and safe, its repetitive large-scale use in endemic areas may lead to the selection of resistant strains. Isolates less susceptible to PZQ have been found in the field and selected for in the laboratory. The impact of selecting strains with a decreased susceptibility phenotype on disease dynamics and parasite population genetics is not fully understood. This study addresses the impact of PZQ pressure on the genetics of a laboratory population by analyzing frequency variations of polymorphic genetic markers.

Methodology

Infected mice were treated with increasing PZQ doses until the highest dose of 3×300 mg/Kg was reached. The effect of PZQ treatment on the parasite population was assessed using five polymorphic microsatellite markers. Parasitological and genetic data were compared with those of the untreated control. After six parasite generations submitted to treatment, it was possible to obtain a S. mansoni population with decreased susceptibility to PZQ. In our experiments we also observed that female worms were more susceptible to PZQ than male worms.

Conclusions

The selective pressure exerted by PZQ led to decreased genetic variability in S. mansoni and increased endogamy. The understanding of how S. mansoni populations respond to successive drug pressure has important implications on the appearance and maintenance of a PZQ resistance phenotype in endemic regions.     

Genetic control of vaccine-induced immunity against a parasitic helminth, Schistosoma mansoni

Schistosoma mansoni is one of several species of trematode helminths responsible for schistosomiasis, a major parasitic infection of man. Genetic analysis in mice has revealed that the protective immunity induced against this parasite by an attenuated larval vaccine is strongly influenced by genes regulating the activation of macrophage effector cells. The latter finding suggests that the induction of cell-mediated immunity may be a successful strategy for a non-living vaccine against the human infection.     

Reappraisal of experimental infections with cercariae and schistosomula of a Brazilian strain of Schistosoma mansoni in mice.

The present investigation involves a reevaluation of previous results obtained after experimental infection of Swiss Webster mice with cercariae and schistosomula of the Schistosoma mansoni LE strain maintained under laboratory conditions. Three experimental groups of mice were considered: the animals of the first group were percutaneously (ring method) infected with cercariae, those of the second were subcutaneously inoculated with cercariae and the mice of the third were inoculated by the same route with schistosomula transformed in vitro. The data obtained so far indicated that the most effective method of infection is the subcutaneous injection with schistosomula, with a mean adult worm burden recovery of 54.1% when compared to the abdominal percutaneous and subcutaneous routes of infection with cercariae, in which the values were 36.7% and 32.4%, respectively. This suggests that, in experimental infections of SW mice with a LE S. mansoni strain, the skin is to be considered an effective attrition site in the percutaneous route, whereas in the case of inoculation with cercariae, a small amount of larvae fails to be transformed into viable schistosomula, possibly due to skin phase avoidance. A brief discussion about attrition sites and elimination of larval S. mansoni worms in mice is presented.     

Genetic diversity in Brazilian populations of Aedes albopictus

Random amplified polymorphic DNA (RAPD) analysis technique was undertaken in Aedes albopictus populations from three states in Brazil, Rio de Janeiro (RJ), Minas Gerais (MG) and Pernambuco (PE), to estimate the level of genetic variability and levels of genetic exchange between populations. Allele and genotype frequencies were measured on 47 RAPD loci. Average observed heterozigosity (Ho) ranged from 0.282 in MG to 0.355 in Casa Forte (PE) population. Genetic distances estimates indicated that RJ and MG were more genetically similar than populations from PE. Genetic variation observed in local Brazilian populations was attributed to genetic drift associated with restricted gene flow in recently established populations.     

A Multiplex-PCR approach to identification of the Brazilian intermediate hosts of Schistosoma mansoni

Due to difficulties concerning morphological identification of planorbid snails of the genus Biomphalaria, and given a high variation of characters and in the organs with muscular tissue, we designed specific polymerase chain reaction (PCR) primers for Brazilian snail hosts of Schistosoma mansoni from available sequences of internal transcribed spacer 2 (ITS2) of the ribosomal RNA gene. From the previous sequencing of the ITS2 region, one primer was designed to anchor in the 5.8S conserved region and three other species-specific primers in the 28S region, flanking the ITS2 region. These four primers were simultaneously used in the same reaction (Multiplex-PCR), under high stringency conditions. Amplification of the ITS2 region of Biomphalaria snails produced distinct profiles (between 280 and 350 bp) for B. glabrata, B. tenagophila and B. straminea. The present study demonstrates that Multiplex-PCR of ITS2-DNAr showed to be a promising auxiliary tool for the morphological identification of Biomphalaria snails, the intermediate hosts of S. mansoni.     

Vertebrate host protective immunity drives genetic diversity and antigenic polymorphism in Schistosoma mansoni

Schistosomes are gonochoric blood parasites with a complex life cycle responsible for a disease of considerable medical and veterinary importance in tropical and subtropical regions. Understanding the evolution of schistosome genetic diversity is clearly of fundamental importance to interpreting schistosomiasis epidemiology and disease transmission patterns of this parasite. In this article, we investigated the putative role of the host immune system in the selection of male genetic diversity. We demonstrated the link between genetic dissimilarity and the protective effect among male worms. We then compared the proteomes of three male clones with different genotypes and differing by their capacity to protect against reinfection. The identified differences correspond mainly to antigens known or supposed to be involved in the induction of protective immunity. These results underline the role played by host immune system in the selection of schistosome genetic diversity that is linked to antigenic diversity. We discuss the evolutionary consequences in the context of schistosome infection.     

Genetic diversity in a reintroduced swift fox population

Swift fox (Vulpes velox) were historically distributed in southwestern South Dakota including the region surrounding Badlands National Park (BNP). The species declined during the mid-1800s, largely due to habitat loss and poisoning targeted at wolves (Canis lupus) and coyotes (Canis latrans). Only a small population of swift foxes near Ardmore, which is located in Fall River County, South Dakota, persisted. In 2003, a reintroduction program was initiated at BNP with swift foxes translocated from Colorado and Wyoming. Foxes released in the years 2003, 2004 and 2005 were translocated from Colorado (BNP-Colorado) whereas in 2006, released foxes were translocated from Wyoming (BNP-Wyoming). Our objective was to evaluate genetic diversity and structure of the restored swift fox population in the area surrounding BNP compared to source fox populations in an area of Colorado and Wyoming, as well as the local swift fox population neighboring BNP near Ardmore in Fall River County, South Dakota. A total of 400 swift foxes (28 released in 2003, 28 released in 2004, 26 released in 2005, 26 released in 2006, 252 wild-born foxes, 40 individual foxes from the Ardmore area of South Dakota) was genotyped using twelve microsatellite loci. We report mean gene diversity values of 0.778 (SD = 0.156) for the BNP-Colorado population, 0.753 (SD = 0.165) for the BNP-Wyoming population, 0.751 (SD = 0.171) for the BNP population, and 0.730 (SD = 0.166) for the Fall River population. We also obtained F_st values ranging from 0.014 to 0.029 for pair-wise comparisons of fox populations (BNP, Fall River, BNP-Wyoming, BNP-Colorado). We conclude that the reintroduced fox population around BNP has high genetic diversity comparable to its source populations in Colorado and Wyoming. Although genetic diversity indicates that the reintroduction was successful, additional time is necessary to fully evaluate long-term genetic maintenance and interconnectivity among these populations.     

Genetic diversity and structuring of the grey wolf population from the Central Balkans based on mitochondrial DNA variation

The Dinaric-Balkan grey wolf population used to be at a border between the large remaining Eastern European populations and the largely eradicated Western European populations. During the last few decades we have witnessed the Western European wolf population recovery. Substantial genetic variation has previously been reported in the Balkan wolf population, but rigorous genetic characterization has not been done for its central parts. The aims of this research were to determine genetic diversity based on mtDNA sequence variability, to infer possible population structuring, to find genetic signals of population expansions or bottlenecks and to evaluate phylogenetic position of the grey wolf population from the Central Balkans. Six haplotypes were detected, of which three have only been found in the Balkan region. These haplotypes belong to both haplogroups previously determined in Europe. Based on our mtDNA sequence analyses, the Dinaric-Balkan wolf population is vertically differentiated into “western” (Croatia/Bosnia and Herzegovina) and “eastern” (Serbia/Macedonia) subpopulations. None of the results support assumption of population expansion. Instead, significantly positive values for Tajima's D and Fu's Fs may suggest recent population bottleneck. Obtained data may be helpful in observation to which extent gene pool from the Balkans contribute to newly founded populations in Western Europe.     

Long-term genetic stability and population dynamics of laboratory strains of Schistosoma mansoni

Measures of genetic differentiation between populations are useful tools for understanding the long-term dynamics of parasite communities. We followed the allele frequencies of microsatellite markers in samples taken over a period of 16 yr from the Case Western Reserve University-Naval Medical Research Institute (CWRU-NMRI) laboratory strain of Schistosoma mansoni. DNA was isolated from pooled samples of adults, eggs, or cercariae collected at 46 time points and genotyped for 14 tri- or tetranucleotide microsatellite markers. For comparison, 2 S. mansoni reference strains (Biomedical Research Institute-NMRI, which has a common origin with the CWRU line, and PR-1) were analyzed over shorter periods of time. We observed that the long-term allele frequencies are generally stable in large laboratory populations of this parasite, and a high degree of similarity was observed between the allele frequencies of consecutive samples from different developmental stages. The CWRU strain, however, showed 2 periods of marked deviation from stability as demonstrated using genetic differentiation measures. The first period corresponds to an admixture event with the BRI strain in which a new equilibrium was established as the "migrants" became blended into the existing CWRU population, consistent with 23% admixture from BRI. The second corresponds to a period of genetic drift when the CWRU population size was greatly reduced with an accompanying loss in genetic diversity. Having demonstrated the utility of pooled samples for the genetic analysis of population dynamics in laboratory strains of schistosomes, this approach will be useful for analyzing field samples to determine the impact of schistosomiasis control programs on parasite population structure. Accounting only for the intensity or prevalence of parasite populations may fail to register significant changes in population structure that could have implications for resistance, morbidity, and the design of control measures.