碩士 / 國立高雄大學 / 生命科學系碩士班 / 107 / This study investigated the epizootic of Enterocytozoon hepatopenaei (EHP) infection from Litopenaeus vannamei cultured in Taiwan. In addition, the diagnostic methods for EHP infection were established using biotechnology. The diseased L. vannamei were collected in southern Taiwan, mainly in Pingtung, Kaohsiung and Tainan. The PCR and nested PCR were carried out with the specific primers (ENF779/ENR779 and ENF176/ENR176) in order to confirm the EHP infection. Furthermore, the tissues tropism and histopathological changes of EHP infection were evaluated by traditional histological method. The amplicon of EHP SSU rDNA partial gene fragments (779 bp) by PCR reaction were cloned, sequencing, and sequence alignment and phylogenetic analysis. The DIG-probe of EHP SSU rDNA partial gene fragments were prepared by PCR method. The dot blot hybridization and in situ hybridization detective methods for EHP were developed. The results showed EHP microsporidia were detected in L. vannamei in Tainan using PCR and Nested PCR. In Pingtung and Kaohsiung, EHP infection were confirmed by nested PCR. Histopathological study, the epithelial cells of hepatopancreas of L. vannamei were target tissue infected by EHP using H& E staining. Except for hepatopancreas, EHP infections were not observed in other organs in the diseased shrimp. The EHP SSU rDNA partial gene fragment of EHP isolated from Taiwan were found more than 99% nucleotide sequence similarities with isolates of Thailand, Vietnam, India, and China. The phylogenetic tree analysis, Taiwan isolate should be same as EHP isolated from L. vannamei cultured in Southeast Asia. The sensitivity of DIG-probe for EHP was 13 pg/ul using dot blot hybridization. By Dot blot hybridization, the EHP infection was detected in the diseased shrimp using the DNA extracted from EHP infected L. vannamei and confirmed it is suffered from EHP microsporidian. In situ hybridization test also successfully detected the EHP infection of the hepatopancreas of L. vannamei by DIG-probe. The sensitivity was excellent than that of traditional histopathology. Conclusion, the Enterocytozoon hepatopenaei (EHP) microsporidian infection was confirmed in the L. vannamei cultured in Taiwan using PCR, nested PCR, histopathology, dot blot hybridization, in situ hybridization methods. In future, the preventive and treatment methods for EHP should be further developed in Taiwan.
| Identifer | oai:union.ndltd.org:TW/107NUK00105002 |
| Date | January 2019 |
| Creators | LIU, YU-HSUAN, 劉于瑄 |
| Contributors | Wang, Chun-Shun, 王俊順 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 71 |
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