Multilocus genetic analysis of Cryptosporidium in naturally contaminated bivalve molluscs

Abstract

To evaluate the application of discriminatory multilocus PCR procedures for the characterization of Cryptosporidium in samples of naturally contaminated bivalve molluscan shellfish. Nucleic acid was extracted from 22 shellfish previously identified as contaminated with Cryptosporidium spp. and subjected to PCR-based analysis for two independent fragments of the Cryptosporidium oocyst wall protein (COWP) gene, three microsatellite markers (ML 1, GP 15 and MS 5) and an extra-chromosomal small double-stranded RNA (dsRNA). Overall, at least one COWP gene fragment was amplified from all 22 samples, 21 amplified the dsRNA and 14 at least one of the three microsatellite loci. More than one dsRNA or microsatellite allele was detected in 50% of samples. The majority of samples were contaminated with Cryptosporidium parvum types circulating in both humans and livestock. A novel dsRNA element was identified in one sample, which did not amplify any of the three microsatellite loci investigated. Multilocus analysis of Cryptosporidium can be applied to DNA extracted from naturally contaminated shellfish. This multilocus genetic analysis highlights that filter feeder molluscs are a potential source of cryptosporidial oocysts, which may be infectious to humans.