Molecular epidemiology of Schistosoma mansoni: A robust, high-throughput method to assess multiple microsatellite markers from individual miracidia

Abstract

Schistosomiasis is one of the major unconquered infectious diseases afflicting people of developing countries, particularly in Africa. A deeper understanding of the epidemiology of schistosomes is complicated by the intravascular location of adult worms which makes them routinely unavailable for study. Their progeny, miracidia, which are hatched from eggs that are passed in feces, are available and can provide valuable insights about human infections, but they are small in size, hindering robust molecular analyses. Here we present a new high-throughput technique to assess the genotypes at 21 previously published microsatellite loci for individual miracidia of S. mansoni. The 21 loci can be amplified in four multiplexed PCR reactions; however, enough template is produced for approximately six PCR reactions, which allows for additional PCR reactions for resampling or obtaining additional data. We validated this technique using a pedigree study employing laboratory crosses of S. mansoni from Kenya to obtain sets of parents and offspring. Of 23 loci examined, 21 loci were found to be reliable: false alleles were rare and missing alleles due to allelic dropout occurred at only two loci in approximately 5% of the offspring. The latter type of error can be further reduced by reamplification which is possible with our method. This technique is amenable to a 96-well format thus facilitating analysis of larger samples of miracidia, allowing more robust molecular epidemiological studies of S. mansoni to infer population size, population structure, gene flow, mating systems, speciation, and host race formation.

Multiple near-identical genotypes of Schistosoma japonicum can occur in snails and have implications for population-genetic analyses

“…Clearly, adult worms cannot be obtained from infected people, but miracidia are accessible. Gower et al (2007) and Steinauer et al (2008) were able to obtain MLGs from individual miracidia of S. mansoni, but this was technically difficult. Characterisation of MLGs from larval stages of S. japonicum has not been reported to date.…”

Non-Invasive Sampling of Schistosomes from Humans Requires Correcting for Family Structure

“…The miracidia were lysed individually in the wells of a 96 well plate and genotyped at 12 microsatellite loci as described by Steinauer et al [40]. GenBank accession numbers of the loci include the following: AF325695, AF202965, AF202966, AF202968, L46951, AF325698, AF325694 (Multiplex panel P17) and M85305, R95529, AI395184, BF936409, AI067617 (multiplex panel P22).…”

Approaches to genotyping individual miracidia of Schistosoma japonicum

“…An alternative is to genotype directly from miracidia themselves. Freshly hatched miracidia can be held briefly in water before being taken directly on to the genotyping stage if laboratory facilities are immediately available (Beltran et al 2008; Steinauer et al 2008; Valentim et al 2009). However, some sort of storage method is generally required since most samples are field-collected.…”

“…Some have PCR-amplified only a single locus for each miracidium (Shrivastava et al 2005), while others have multiplexed several loci within a single reaction per miracidium, and these studies have all used FTA cards (Rudge et al 2008; Gower et al 2007, 2011). A few studies, extracting DNA from freshly hatched miracidia in water or saline, have reported successful amplification of up to 21 loci in four reactions (Steinauer et al 2008; Valentim et al 2009). …”