| Echinococcosis is a zoonosis caused by Echinococcus. Cystic echinococcosis (CE) caused by E. granulosus and alveolar echinococcosis (AE) caused by E. multilocularis are distributed all around the world. Shiqu County, on the East Qing-hai Tibet Plateau, is one of the most serious Echinococcus infection districts. Besides the two Echinococcus mentioned above, there is another special species, called E. shiquicus, which is only found existing on the Qing-hai Tibet Plateau. The taxonomy of Echinococcus has been a controversial issue for years:early identification has been based on morphological and ecological features, which were mainly used on adult worms and required high quality samples; as the development of molecular biology, lots of mtDNA and nuclear genes have been used to identify Echinococcus spp., but there has been no standard identification method so far. As a growing technology, DNA barcoding has been proved useful on150,250species (until Feb.2012). In this study, based on the three Echinococcus species, we originally tried to test the utility of DNA barcoding in the genus Echinococcus, with sequences of other Echinococcus spp. in the GenBank. Meanwhile, for the primers we used are family Taeniidae universal primers, we extended the range of our research to the whole family Taeniidae.The major results and conclusions are as follows:1.13E. granulosus isolates,15E. multilocularis isolates and17E. shiquicus isolates were successfully amplified and sequenced. These45Echinococcus sequences,136Echinococcus sequences and260Taenia sequences from GenBank were cut and calculated separately and integrally. In the genus Echinococcus, the mean K2P distance within species is0.62%,(SD,0.81%; range,0-8.9%). Mean K2P distance between species is9.86%,(SD,1.15%; range,4%-15.28%). In the genus Taenia, the mean K2P distance within species is1.30%,(SD,2.65%; range,0-11.71%). Mean K2P distance between species is16.06%,(SD,3.28%; range,3.39%-24.59%). In the family Taeniidae, the mean K2P distance within species is1.06%,(SD,2.17%; range,0-11.71%). Mean K2P distance between species is16.95%,(SD,4.12%; range,3.39%-30.69%). Mean K2P distance between genera is19.29%,(SD,2.46%; range,11.99%-30.69%). The COI DNA barcodes were successfully proved to be a powerful and reliable tool for species identification in the family Taeniidae.2. A polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) based on the444-bp COI gene was developed to give a rapid way to differentiate among E. granulosus, E. multilocularis, and E. shiquicus, for practical application in the Eastern Tibetan Plateau. The E. granulosus samples were yielded to two clear bands of210-bp and234-bp using AluI, and the samples from E. multilocularis were yielded to two clear bands of281-bp and163-bp using BfaI. E. shiquicus could be yielded by neither restriction endonuclease above. These results could be detected directly by agarose gel electrophoresis.The second part of this dissertation is a primary study on the Echinococcus infection of plateau pika (Ochotona curzoniae)Plateau pika is a kind of small mammal widely distributed on the Qing-hai Tibet Plateau. It can serve as an intermediate host for Echinococcus. Former studies mainly used autopsy and histopathological examinations to survey its infection and found it mainly infected with E. multilocularis and E. shiquicus. This present study used the autopsy method and molecular technology to investigate the Echinococcus infection rates of plateau pika from Shiqu County, Sichuan Province, by choosing specific primers for the three Echinococcus species. The major results and conclusions are as follows:1. Three out of ten published and designed specific primers for E. multilocularis, E. shiquicus and E. granulosus were chosen to evaluate the Echinococcus infection rates.2. In total, nine isolates were suspected infected out of105hunted plateau pikas. The molecular identification result is that zero pikas were infected with E. granulosus (infection rate0); two pikas were infected with E. multilocularis (infection rate1.9%); two pikas were infected with E. shiquicus (infection rate1.9%). Two pikas were mixed infected with E. multilocularis and E. shiquicus (infection rate1.9%)... |
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