Analysis of Schistosoma mansoni population structure using total fecal egg sampling

Abstract

Many parasite populations are difficult to sample because they are not uniformly distributed between several host species and are often not easily collected from the living host, thereby limiting sample size and possibly distorting the representation of the population. For the parasite Schistosoma mansoni, we investigated the use of eggs, in aggregate, from the stools of infected individuals as a simple and representative sample. Previously, we demonstrated that microsatellite allele frequencies can be accurately estimated from pooled DNA of cloned S. mansoni adults. Here, we show that genotyping of parasite populations from reproductively isolated laboratory strains can be used to identify these specific populations based on characteristic patterns of allele frequencies, as observed by polyacrylamide gel electrophoresis and automated sequencer analysis of fluorescently labeled PCR products. Microsatellites used to genotype aggregates of eggs collected from stools of infected individuals produced results consistent with the geographic distribution of the samples. Preferential amplification of smaller alleles, and stutter PCR products, had negligible effect on measurement of genetic differentiation. Direct analysis of total stool eggs can be an important approach to questions of population genetics for this parasite by increasing the sample size to thousands per infected individual and by reducing bias.

Figure 1

S. mansoni microsatellite specificity. Microsatellite amplification of laboratory strains of S. mansoni, and other organisms DNA. DNA templates are as follows: lanes 1-9, S. mansoni strains CWRU, BRI-1, BRI-2, BRI-3, BRI-4, CPGM, IPR, TBI, York; lane 10, human; lanes 11-14, hookworm isolates 1-4; lane 15, S. japonicum; lane 16, S. hematobium.

Figure 2

Microsatellite amplification of DNA extracted from S. mansoni total stool eggs from individual human infections analyzed on non-denaturing polyacrylamide gels. a. Kenyan samples 1-11. b. Brazilian samples 1-9.

Figure 3

Microsatellite analysis of stool egg samples by capillary electrophoresis. a. Inverted digital image of EtBr stained polyacrylamide gel. b. Band intensity peaks as interpreted by gel analysis software. Allele size in bp as calculated by SigmaGel software in a and b. c. Capillary electrophoresis analysis of fluorescently labeled products. Vertical scale, fluorescence intensity; horizontal scale, size in bp.