Detection and characterisation of Giardia and Cryptosporidium in Hungarian raw, surface and sewage water samples by IFT, PCR and sequence analysis of the SSUrRNA and GDH genes

Abstract

We investigated the prevalence of Giardia and Cryptosporidium species and analysed the genotypes in 36 samples collected from different water sources and various geographic areas in Hungary. Samples were collected from drinking water and sewage treatment plants and from the recreation area of Lake Balaton. The (oo)cysts were purified according to the US EPA 1623 method and they were detected by immunofluorescence test (IFT). Genomic DNA was extracted from all samples and then the GDH target gene for Giardia and the SSUrDNA for both Giardia and for Cryptosporidium species were amplified by PCR. 24 out of 36 samples (67%) were Giardia positive and 15 (42%) were Cryptosporidium positive by IFT. PCR confirmed that 13 out of 36 samples (36%) were Giardia positive and 10 (28%) contained Cryptosporidium. Twelve Giardia and two Cryptosporidium PCR products were successfully sequenced. In seven samples G. lamblia Assemblage A and in one sample Assemblage B and in four cases Assemblages A and B have been found. In one sample C. parvum and in the other separate sample C. meleagridis were detected. Sequence analysis revealed a new subtype of G. duodenalis complex, clustered close to the Assemblage A group. This study provides the first report on simultaneous detection and genotyping of G. duodenalis and Cryptosporidium species from water supplies in Hungary.