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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48489

Title: 兩屬石斑虹彩病毒共感染點帶石斑魚之致病相關研究
Pathogenic study of orange-spotted grouper (Epinephelus coioides) coinfected with two iridovirus genera
Authors: Huang,Ching-Ting
黃勁婷
Contributors: NTOU:Department of Aquaculture
國立臺灣海洋大學:水產養殖學系
Keywords: 臺灣石斑虹彩病毒;石斑虹彩病毒;共感染
TGIV;GIV;coinfection
Date: 2014
Issue Date: 2018-08-22T06:05:22Z
Abstract: 感染臺灣養殖石斑魚的虹彩病毒有兩類,分別為歸屬巨大細胞病毒屬之臺灣石斑虹彩病毒 (Grouper Iridovirus of Taiwan,TGIV),及歸屬於蛙病毒屬之石斑虹彩病毒 (Grouper Iridovirus, GIV)。本論文由養殖石斑魚分離出以上兩屬病毒之共感染檢體,TGIV高劑量和GIV低劑量 (簡稱T+g) ,以及TGIV高劑量和GIV高劑量(簡稱T+G),因此嘗試探討在點帶石斑魚體內此兩病毒共感染的可能交互作用。由於TGIV不易體外增殖,所以實驗的病毒來源皆由活體石斑魚苗增殖,目前的石斑魚苗常有GIV帶原的情形,因此本論文僅以單獨GIV組做為正對照組。首先進行病毒攻擊試驗,GIV組兩周後的累計死亡率如預期達100%,T+G組和T+g組分別為85% 和3%。將感染魚脾、頭腎和心臟與對照組進行臟體比分析,結果只有脾臟有顯著性腫大的差異。GIV單獨感染魚在感染初期即出現脾臟腫大情形,但T+G和T+g則是在感染後一周後才觀察到,T+g組較T+G組明顯。另外以即時聚合酶鏈鎖反應 (real-time PCR) 分析各組魚脾臟、頭腎、心臟、鰓、腎臟、腸道、肝臟和肌肉的病毒量,結果GIV組臟器病毒量在脾臟、腸道、肝臟和頭腎較高,第七天皆可高達4#westeur024#107 copies以上;T+g組各臟器中,TGIV在第1、7、11天皆維持在106 copies左右,第15天下降至103 copies,而GIV 在所有採樣天數皆維持在103~104 copies,沒有增殖情形。T+G組別中,各臟器中TGIV 的量會隨時間增加,脾臟在第7天達8.5#westeur024#106 copies,其他各臟器則在第11天病毒量達106~107 copies為最高值,第15天病毒量則下降至104~105 copies左右;而GIV趨勢與TGIV相似,脾臟在第7天達3.8#westeur024#106 copies,頭腎、心、腎也有105 copies以上,各臟器在第11天病毒量達最高為106~107 copies,第15天病毒量則下降至104~106 copies左右,只是下降幅度較TGIV少。GIV感染組的頭腎有明顯的組織壞死,T+G組第7、11、15天皆可觀察到巨大細胞,最後分別針對GIV和TGIV的主要外鞘蛋白 (Major Capsid Protein, MCP) 設計探針進行原位雜交染色,結果在各病毒攻毒組的頭腎和脾臟之組織壞死區域易觀察到陽性細胞反應。以上結果顯示,GIV會造成急性感染並導致高死亡率,反之TGIV病程發展緩慢,推論兩病毒共感染魚體時,可能相互干擾,且GIV增殖與否是魚隻致死的關鍵。 關鍵詞:臺灣石斑虹彩病毒、石斑虹彩病毒、共感染。 
Iridovirus infection in Taiwan grouper have been classified into two genera: Megalocytivirus (TGIV, grouper irridovirus of Taiwan) and Ranavirus (GIV, grouper irridovirus). To explore the probable interaction between these two iridovirus co-exist in grouper, we had separated the two irridovirus origins from orange-spotted grouper into two groups: (i) TGIV high dose and GIV low dose (T+g), and (ii) TGIV high dose and GIV high dose (T+G). TGIV is restricted to replicate in-vitro and thus it was obtained from grouper larvae for propagation. Recently the obtained grouper larvae are GIV-carrier therefore we used GIV group only as positive control in this study. In fish challenge experiment, the cumulative percent mortality was 100% in GIV group, and 85% and 3% in T+G and T+g group, respectively. We had compared the infected fish spleen, head kidney, and heart with the control group to analyze organ coefficient (ratio of organ to body weight), and found that there was only significant differences in spleen enlargement. GIV- infected fish appeared spleen enlargement in early stage but observed one week after infection in both T+G- and T+g- infected fish. The spleen of T+g- infected fish is bigger than T+G group. In addition, real time PCR was used to analyze the viral loads in spleen, head kidney, heart, gill, kidney, intestine, liver and muscle in each group of fish. The results revealed high viral loads were detected in spleen, intestine, liver and head kidney in GIV group where it can reach up to 4 #westeur024# 107 copies or above at 7th day. For T+g group, the TGIV loads in each organ was maintained at 106 copies in 1st, 7th and 11th day and decreased to 103 at 15th day. GIV was maintained at 103 ~104 copies in all sampling days, no proliferation was observed. For T + G group, the TGIV loads will increase over time in each organ; at 7th day it reach to 8.5 #westeur024# 106 copies in spleen, the other organs also reach to the highest value, 106 ~ 107 copies; and the viral loads was decline to 104 ~ 105 copies at 15th day. While GIV loads trends had similar with TGIV as it reached 3.8 #westeur024# 106 copies in spleen at 7th day. Head kidney, heart and kidney were reached to 105 copies above. At 11th day, the high GIV loads was detected in each organ where it reached up to 106~107 copies. At 15th day, the GIV load was dropped to about 104~106 copies but decline less than TGIV. For GIV-infected group, the head kidney expressed tissue necrosis whereas the mast cell was observed in T+G group at 7th, 11th and 15th days. Ultimately, major capsid protein (MCP), for GIV and TGIV, respectively was designed to explore the in-situ hybridization staining and the results revealed tissues necrosis near in head kidney and spleen area in each experimental group had positive reaction. The above results indicated the GIV caused acute infection and leads to high mortality whereas the progression of TGIV infection is slow. This had predicted that these two virus co-infected fish may interfere each other and the fish death may cause by GIV proliferation. keywords:TGIV、GIV、coinfection。
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010033020.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48489
Appears in Collections:[水產養殖學系] 博碩士論文

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