| In the present study, Cyclospora-like oocysts were found in feces of dairy cattle.Using wet mount slides, the oocyst walls appeared as well-defined non-refractile spheres measuring 6 to 10μm in diameter, and within each unsporulated oocyst was a central morula-like structure containing a variable number of inclusions. At a magnification of×1000, the inclusions were refractile, exhibiting a greenish tinge. Oocysts were stained variably using a modified acid-fast stain but colorless in saline wet mounts. Oocysts were concentrated and purified by sucrose flotation. Purified oocysts were suspended in equal volumes of 5% potassium dichromate solution to sporulate. The sporulated oocyst had 2 sporocysts and each sporocyst contained 2 sporozoites, i.e. each oocyst contained a total of 4 sporozoites. All the above phenotypic characteristics of the cattle-derived Cyclospora sp. were similar to those described previously from human being. To our knowledge, this is the first cyclosporan to be described in dairy cattle.To identify the cattle-derived oocysts, a molecular characterization based on both ITS-1 and partial 18S rDNA sequence was conducted to determine the phylogenetic relationships between these cattle-derived oocysts, primate-derived Cyclospora, Eimeria sp. and other protozoan. With the designed primers CYCF and CYCR, the ITS-1+ of the cattle-derived Cyclospora-like oocysts was amplified and sequenced. Sequencing result showed that the ITS-1+(GenBank accession No. AY876933) included partial 18S rDNA (371bp), the complete ITS-1 sequence (385bp) and partial 5.8S rDNA (109bp). BLAST analysis indicated that the ITS-1 sequence was unique and had little similiar to none of other known organisms, while the partial 18S rDNA was most closely related to that of the Eimeria species (EBU77084) and Cyclospra species (AF111183), with identities of 98% and 94%, respectively, and the partial 5.8S rDNA was most closely related to the Cyclospora species (AF301406, AF301390), which identities is 96%. These results showed that the cattle-derived Cyclospora sp. may represent a new species of Eimeriida, though it cannot still be identified based on the ITS-1 sequence.For further identification, partial 18S rDNA of the Cyclospora sp. from calfNos. 1 and 2 were amplified using newly developed nested PCR protocol with primers NesCycF and NesCycR, and the amplicons were sequenced. The two sequences (GenBank accession numbers: DQ082866 and DQ082867) obtained in this study and other 33 sequences (5 Cyclospora sp. and 28 species of apicomplexan protozoa) retrieved from the GenBank, were aligned using Clustal X (V1.83) sequences alignment program Phylogenetic trees constructed by neighbor-joining (NJ), minimum evolution (ME) and maximum parsimony (MP) algorithms had identical topology. The results revealed that the cattle-derived Cyclospora sp., the primate-derived Cyclospora, and Eimeria species formed a clade within the phylum Apicomplexa. Within this clade, all apicomplexan protozoa were divided into at least four taxonomic groups: one comprising the Cyclospora species, one comprising the avian-derived Eimeria species, one comprising the rodent-derived Eimeria species, and another encompassing the ruminant-derived Eimeria species. These cattle-derived Cyclospora sp. clustered with the group of Cyclospora together with human-derived Cyclospora species, but they were distinct from the latter. The cattle-erived Cyclospora sp. may represent an exclusive species of dairy cattle.Based on partial 18S rDNA of the cattle-derived Cyclospora sp., two pairs of primers were designed. 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 method for detection of cattle-derived Cyclospora sp. was developed. The results showed that the PCR method was specific and there was no cross-reaction with other parasites, such as Eimeria spp.,Cryptosporidium spp., Giardia sp., Toxoplasma sp., Trichuris sp. and cattle ciliate. It may provide an effective tool to detect Cyclospora sp. in cattle feces or in environment. Moreover,this study attempted to develop a sensitive, specific method for quantitative detection of Cyclospora sp. infection. Hence a nested PCR-liquid phase hybridization assay was developed. The PCR amplicon were biotin-labeled by a 5'-biotin-labled primer through sample amplification. After the labeled amplicon was mixed with a 5'-digoxin-labeled probe, hybridization was carried out in a thermocycler for 2 minutes at 90℃and for 15 second at 66.9℃. Then the hybridized product was captured on microplate wells coated with streptavidin and was detected with antibody labeled alkaline phosphatase and pNPP substrate. The sensitivity of the PCR-liquid phase hybridization assay was 10 times higher than that demonstrated by PCR-agarose gel electrophoreses. The results indicated that the assay is simple, rapid and suitable for clinical detection of cattle-derived Cyclospora sp.Cysts of Giardia sp. were firstly found and isolated from feces of cattle in China in this study. The isolate of Giardia sp. was putatively identified as G. lamblia based on the phenotypic characteristics by microscopy. To perform a molecular identification, two independent genetic loci in the triosephosphate isomerase (TPI) gene and the small subunit ribosomal RNA gene (16S rRNA) were amplified and sequenced. Based on the obtained sequences (GenBank accession numbers: DQ157270 for TPI and DQ157272 for 16S rRNA), A neighbor-joining tree was constructed based on the evolutionary distances calculated by Kimura-2-parameter analysis using the program MEGA 3.0. The results of the phylogenetic analysis confirm that the first cattle-derived Giardia isolate in China represent G. lamblia, Assemblages E.Based on the TPI sequence, a nested PCR assay was developed for the detection of G lamblia. This method was specific and there was no cross-reaction with genomic DNA of other parasites. The sensitivity of the nested PCR assay was 100 times higher than that of conventional PCR amplification. It was estimated that as few as 0.395 fg positive plasmid DNA, which contains the target fragment, could be detected by the nested PCR assay. These findings indicate that the specific nested PCR approach provides a useful tool for the detection of Giardia sp. in cattle feces or in environment, and should have important implications for studying the molecular epidemiology of giardiasis... |
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