| Cryptosporidium is a worldwide distributed zoonotic parasite which can cause watery diarrhoea, and even death in human and animal. At present, there is still no effective drug and vaccine available for cryptosporidiosis. The prevention and control of the disease is mainly based on sanitary control practice to minimize susceptive host contacting with infection sources and resonabl treatment of faecals of infective man and animals to reduce the pollution of oocyst to foods, drinking water and surrounding area. The infections of Cryptosporidium in animals not only bring severe damage to themselves health, but also become an important source of human infection. It is very important to develop a sensitive, effective, inexpecsive molecular detection method and survey the molecular epidemiology of cryptosporidiosis in animals. These basicly work will help us to understand their infection state, prevelace feature, species/genotype of Cryptosporidium, and then contribute to produce effective prevention methods to reduce the harm of the disease. On the other hand, MicroRNAs (miRNA) can regulate gene expression posttranscriptionally in much kind of organisms and participate in the regulation of many physiological processes. The research of miRNA helps us to understand the organisms'gene fuctions, developmental mechanism, correlations of parasites and their hosts. But until now there is no miRNA was reported in Cryptosporidium.In order to develop a sensitive PCR detection method for Cryptosporidium spp., the nested PCR amplification effect were compared with genomic DNA template extracted from Cryptosporidium baileyi oocysts with ten kind of methods. The results of 3 times duplicate test showed that the sensitive of nested PCR detection with the DNA template extracted by Chelex 100, FTA filter paper, Wizard DNA Clean-Up System were higher than other methods. They could get stably amplification results with template extracted from 1×102 Cryptosporidium oocysts and more. Same sensitivity was found on the detection of Cryptosporidium parvum isolated from dairy cattle and Cryptosporidium mouse genotype isolated from mice with the nested PCR using the DNA templates extracted with Chelex-100 method. The detection results of yak field samples showed that the detection rate of optimized nested PCR methods was much higher than that of Sheather's sucrose flotation protocol, although it is still insufficient in simple times with only 33.3%-44.4% detection rate. With the detection results of Sheather's sucrose flotation method as gold standards, the coincidence rate of optimized nested PCR detection results of positive samples and total samples of 192 field fecal samples collected from goats, pigs and ducks were 89.1% and 67.7%, respectively. The results indicated that the optimized nested PCRs were suitable for large number sample detections in molecular epidemiology research of all kind of animal cryptosporidiosis.Surveys on prevalence of Cryptosporidium spp. in dairy cattles at 19 farms in 7 suburban districts/county, pigs at 12 farms in 9 districts/county, chickens at 4 farms in 3 districts, ducks at 7 farms in 4 districts and dogs at 14 sites in 7 districts of Shanghai were conducted under Sheather's sucrose flotation protocol and modified acid-fast stain methods. A total of 826, 2323, 683, 857 and 446 faecal samples were collected, respectively and Cryptosporidium spp. oocysts were detected in 184 (22.3%), 800 (34.4%), 201 (29.4%), 188 (21.9%) and 11 (2.5%) samples. The prevalence states and features were preliminary elucidated and the infection rate of Cryptosporidium spp. in pigs was correlated with pigs age and seasons. Pigs with ages less than 2 months were the most concerned population for the examination and prevention of cryptosporidiosis. On the other hand, spring and winter are the major seasons for examination and prevention of Cryptosporidium infection in pigs. The samples of quails, sika deers, New Zealand rabbits, Microtus fortis, laboratory mice, pigeons, dark swans and cats were also collected and detected for the Cryptosporidium infection. The results showed that positive rate were 63.3%, 36.6%, 11.0%, 54.5%, 2.9%, 0, 0 and 0, respectively. Cryptosporidium infection was also found in a yellow cattle faecals. These results indicated that Cryptosporidium infection was common in animals in Shanghai. The genotype analyses were carried out through PCR-RFLP and partial sequences analysis of small subunit ribosomal RNA (SSU rRNA) in total of 170 positive samples. C. parvum,C. andersoni,C. ryanae and C. bovis infection in dairy cattles, C. suis and pig genotypeⅡin pigs, C. baileyi in chickens, C. baileyi and Cryptosporidium duck genotype in Pekin ducks, C. canis, C. muris and mouse genotype in dogs, C. ubiquitum in goat, C. Meleagridis in quail, C. andersoni in sika deer, C. cuniculus in rabbit and mouse genotype in mice were found. In my knowledge, it was the first report for Cryptosporidium duck genotype in Pekin ducks, C. ubiquitum in goat and mouse genotype in dogs. It also havn't been found report that in China the dogs infected with C. muris and C. canis, the sika deer infected with C. andersoni. These results would help to develop effective preventive methods of the disease.Solexa, a large number sequencing protocol, was employed to sequence the small RNA of C. parvum. Total of 15025804 reads were obtained and in them there were 14753144 high quality sequences and 12566783 clean reads. The result of mapping genome of C. parvum showed that there were total 17877 (5.3%) kinds of smll RNA find their homologisation sequences in the chromosomes, but the.distribution was inequable from 0.5% to 48.0% of total mapping uniques in different chromosomes. There were total 30 unique miRNA were found their homologisation sequences in C. parvum chromosomes and their expression abundance were from 1 to 1405. Two candidate new miRNA and 4032 target gene site were predicted with bioinformatics soft. At least 12 miRNA were found expression in oocysts of 3-6 species/genotype among C. parvum, C. baileyi, Cryptosporidium mouse genotype, C. cuniculus, C. suis and C. andersoni using real time quantification PCR of miRNA by RNA-Tailing and primer-extension RT-PCR. The work enlarged the knowledge of protozoa miRNA and helped to research the biology and preventive methods of Cryptosporidium. In this research, the nested PCR detection method was optimized, the prevalence of Cryptosporidium spp. and thier species and genotypes in 13 kinds of animals in Shanghai area were surveyed, and the miRNAs of C. parvum were identified preliminarily. It will much helpful for research the biology and preventive methods of Cryptosporidium.
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