Three isolates of the genus Cryptosporidium, namely, Guangdong isolate, Anhui isolate and Jiangsu isolate from MainlandChina, were identified and characterized genetically utilizing nuclear DNA regions of the small subunit of ribosomal RNA(SSU rRNA) and heat shock protein 70 gene (HSP70) as genetic markers. These two regions were amplified by PCR fromDNA extracted from oocysts and amplicons of approximately 290 bp and 450 bp were produced, respectively. The ampliconswere purified, cloned and sequenced. Sequences of 446 bp and 290-292 bp were obtained for the SSU rRNA and HSP70regions, respectively. The obtained SSU rRNA and HSP70 sequences representing the three Cryptosporidium isolateswere compared with those retrieved from the DNA database. Genetic analyses using either DNA region revealed thatmembers of Cryptosporidium formed two clusters, with C. parvum, C. wariri, C. felis and C. meleagridis clusteredtogether, while C. andersoni, C. muris and C. serpentis belong to the other cluster. Based on SSU rRNA and HSP70sequences, both Guangdong and Anhui isolates of Cryptosporidium were identified as C. muris of the calf genotype (i.e., C. andersoni), whereas the Jiangsu isolate was identified as C. parvum of the calf genotype. The findings of thepresent study should have important implications for the diagnosis and control of Cryptosporidium infections in bothhumans and animals in China.
To develop a nested PCR assay for the detection of cattle-derived Cyclospora sp., two pairs of primers were designed on the basis of the cattle-derived Cyclospora sp. sequences. These primers selectively amplified a 168-bp DNA fragment of the 18S rRNA gene of cattle-derived Cyclospora sp. With these primers, a nested PCR assay for the detection of cattle- derived Cyclospora sp. was developed. The nested PCR assay was specific and there is no cross-reaction with other parasites, such as Eimeria spp., Cryptosporidium spp., Giardia sp., Toxoplasma sp., Trichuris sp. and cattle ciliate. The assay was able to detect as low as 2.85 × 10-2 fg of the control positive DNA. The results of the detection of clinical samples indicated that the assay coincided with microscopic examination. The results show that the nested PCR assay will be an effective tool for the detection of Cyclospora sp. in cattle feces.
