Sequence and PCR-RFLP analysis of the internal transcribed spacers of the rDNA repeat unit in isolates of Cryptosporidium from different hosts

The Cryptosporidium ITS1, 5.8S and ITS2 rDNA regions from a number of Cryptosporidium isolates from different hosts and geographical areas were cloned and sequenced in order to investigate the extent of sequence heterogeneity between human and cattle-derived isolates from different geographical locations and also between isolates of Cryptosporidium from different hosts such as cats, pigs, mice and a koala. Calf-derived isolates from different continents were virtually identical as were human-derived isolates from the UK and Australia. Genetic differences between Cryptosporidium isolates were extensive and were in fact greater than the level of nucleotide divergence between Toxoplasma gondii and Neospora caninum rDNA sequences. Based on the sequence information derived from this study, PCR-RFLP of the ITS1 region was undertaken in order to directly amplify and genotype Cryptosporidium isolates from different hosts. This PCR-RFLP approach can now be used for molecular epidemiology studies, circumventing the need for costly sequencing and allowing a wider range of genetically different isolates to be examined.