博士 / 國立屏東科技大學 / 獸醫學系 / 95 / Over the course of the past 14 years, an unrecognized intracellular organism has emerged from fresh- and brackish- cultured diseased tilapia and causing high mortality, up to 95% in some cases in Taiwan. The causative agent is a cocco-bacillus with a polymorphous shape, and can be cultured in Chinook salmon embryo cell line (CHSE-241) and Tilapia ovary (TO-2) cells, but does not grow on artificial synthetic media, which was named rickettsia-like organism (RLO) or tilapia intracellular organism (TIO). Therefore, many researchers suggest it is an obligate intracellular bacterium, such as Piscirickettsia salmonis. Based on electron microscopic examination, the sequences of the 16S rRNA gene and phylogenetic analysis, RLO/TIO was a unique Francisella-like bacterium that was most closely related to Francisella spp. The intracellular organism associated with granulomas in tilapias might represent an undescribed Francisella species. The general context of this thesis is formal methods for characterization and identification of a novel bacterial microorganism, commonly referred to as Francisella-like bacterium (FLB), induced systemic granulomas in tilapias. It is weakly Gram-negative and polymorphic, and can be cultured in Chinook salmon embryo cell line (CHSE-214). The thesis is be roughly divided into three major parts. The first is dedicated to present the PCR assays for 16S rRNA
gene sequence analysis, electronmicroscopical examination and phenotypic characteristics for ten isolated strains of Francisella-like bacterium from the visceral granulomas of diseased tilapia in Taiwan. FLB colonies developed slowly, requiring 3 to 6 days incubation at 23℃ on the Thayer-Martin agar. Smooth colonies with a grayish pigment were observed. They showed little or no growth at 30℃ and none at 35℃. All the isolates only grow on Thyer-Martin agar. The organisms were strict aerobes, non-motile, and had negative reactions for reduction of nitrate, beta-galactosidase and resazurin, and hydrolysis of glucoside, ONPG, L-arabinoside, leucine and glycine. They were weakly positive for catalase, but strongly positive for β-lactamase, proline and sucrose. Compared the nucleotide sequences of the whole 16S rRNA gene, they had high sequence identities to F. philomiragia (98.7%), F. tularensis subsp. novicida (97.4%) and F. tularensis subsp. tularensis (96.2%), respectively. They might belong to Francisella species. In additional, we were confirmed by the in situ hybridization technique. The second part is to distinguish the FLB strains at the genus, species and subspecies levels using PCR assays that were applied using the universal primer, Francisella genus-specific primers and FLB-specific primers. These results allowed sensitive identification of FLB as a new species of the genus Francisella and could discriminate it from Francisella tularensis, F. novicida and F. philomiragia. The third part is to distinguish to archived formalin-fixed, paraffin-embedded tissues from diseased ornamental fish associated with visceral granulomas that were examined by histopathology, polymerase chain reaction (PCR) and in situ hybridization (ISH) for detection of Francisella-like bacterium (FLB). ISH was more sensitive than PCR or routine histopathological examination, based on the examination of archived formalin-fixed, paraffin-embedded tissues in this study.
Keyword: Cichlid Fish, Francisella-like bacterium (FLB), Rickettsia-like organism (RLO), polymerase chain reaction (PCR), in situ hybridization (ISH)
| Identifer | oai:union.ndltd.org:TW/095NPUST541001 |
| Date | January 2006 |
| Creators | Chia-Yu Hsieh, 謝嘉裕 |
| Contributors | Shinn-Shyong Tsai, 蔡 信 雄 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | en_US |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 109 |
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