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研究生:顧倩君
研究生(外文):chien-chun Ku
論文名稱:台灣石斑虹彩病毒防治對策之相關研究
論文名稱(外文):Studies on control strategy for iridoviral infection in grouper (Epinephelus sp.)
指導教授:周信佑周信佑引用關係
指導教授(外文):Hsin-Yiu Chou
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:水產養殖學系
學門:農業科學學門
學類:漁業學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:75
中文關鍵詞:虹彩病毒石斑魚防治疫苗
外文關鍵詞:iridovirusgroupercontrolvaccine
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本論文為建立本省石斑虹彩病毒(TGIV)的防治對策,首先進行了TGIV不活化實驗。物理因子部分選擇溫度、酸鹼度及紫外線照射三種處理方式,結果顯示TGIV以70℃作用5分鐘可完全將之不活化,此病毒於40℃下3小時後其力價可維持在106.0 TCID50/ml;於pH 11下72小時後仍有103.33 TCID50/ml的力價,於pH 3下1小時後以完全被不活化;以910 µW/cm2處理1小時後其力價仍有殘留。於化學因子方面,以氯25ppm處理10分鐘或50ppm處理10分鐘可完全不活化;碘以25ppm處理15分鐘或50ppm處理5分鍾可完全不活化TGIV;以4%福馬林處理24小時後可將TGIV完全不活化;以4% ß-乙丙酸內酯 (BPL) 處理12小時後病毒已被完全不活化。於活存實驗中,TGIV於無菌蒸餾水可存活10天以上;以有無添加2%血清的HESS中皆可存活40天之久;TGIV於乾燥環境下可維持近40天,於30天時仍有103.0 TCID50/ml的力價。
本省虹彩病毒疫情大多於高水溫期爆發,發病可能與水溫有關。將腹腔注射感染虹彩病毒的石斑魚分別飼養於25℃、30℃、18℃下,飼養於25℃下的實驗魚於第五天開始死亡,累積死亡率達100%;飼養於30℃下的實驗魚提前兩天開始死亡,累積死亡率達100%;飼養於18℃下的實驗魚死亡延遲至第七天開始死亡,兩週後累積死亡率僅有20%。以PCR檢測所有檢體,結果均有檢測到病毒的存在,顯示可能於18℃下有抑制死亡的情形發生。
於不活化疫苗開發方面,以福馬林與BPL分別於4℃及20℃下以1:200、1:400、1:800此三種濃度進行病毒的不活化4天,以注射、浸泡、餵食此三種方式進行免疫。注射法於保護30及40天後的累積存活率為100%,但於病毒再分離的測試中1:400的保護效果最好;保護40天後BPL組的保護效果增加,且與濃度有正相關。在浸泡實驗中以BPL組的微膠囊疫苗浸泡1小時累積存活率達75%,比福馬林組及浸泡2小時的保護效果佳。將已浸泡過一次的微膠囊疫苗再次使用浸泡1小時後,有65%的保護效果。於餵食免疫實驗中,將微膠囊疫苗與鰻粉混合製做成餵食疫苗,餵食20及30天後,並無保護效果。
This paper is the study of control strategy on grouper iridovirus of Taiwan (TGIV); first we did the deactivation tests of TGIV. Physical factors include heat, pH and UV irradiation. For the heat treatment, the result shows 5 minutes under 70℃ completely inactivation of TGIV can be achieved. This virus is also vulnerable to extreme acid and alkal environment, its resistance to acidity is even weaker than to the alkalinity. After 72 hours put into the pH11 solution, its titer indicator has been reduced to 103.33 TCID50/ml, but it only took 1 hour in pH3 solution to completely inactivate the virus. The TGIV has stronger resistance to UV irradiation. After 1 hour exposure to 910 µW/cm2 UV, the virus still has some residue titer. As far as the chemical treatment part, chloride can completely inactivate TGIV, either 25 ppm 20min or 50 ppm 10 min can reach the result; the virus are even more sensitive to iodine, 25 ppm 15 min or 50 ppm 5 min can inactivate the virus. For Formalin, it would take more time to reach the complete inactivation of TGIV, i.e., 4% formalin would take 24 hours to completely inactivate TGIV. ß-propiolactone (BPL) has the similar effect, 4% BPL need 12 hours to inactivate the TGIV. The survival test for TGIV at different environments show that the virus can last more than 10 days in distilled water, in Hank’s balanced salt solution (HBSS buffer), has added serum or not, all survived more than 40 days. TGIV has excellent tolerance to dry condition; its infection ability can keep up for more than 40 days.
The test of water temperature to TGIV infection of grouper, the result reveals that TGIV infection definitely related with water temperature. This agrees with the higher occurrences of TGIV infection at high temperature season in south Taiwan. Artificial infection TGIV groupers feed at 25℃ or 30℃, mortality rate reach 100% at the 9th day; but feed at 18℃, mortality would not start until the 7th day. After 2 weeks, mortality rate only reach 20%. Using PCR detection, all infection groupers have virus positive, so low temperature 18℃ inhibit TGIV burst.
The part of development for TGIV inactivation vaccine, three kind of concentrations (1:200, 1:400, 1:800) of Formalin and BPL treatment at 4℃, 20℃,37℃ has been conducted. After three times blind passage tests to identify the conditions of complete loss of the virus infection capability, the selected inactivation vaccine prepare conditions are Formalin and BPL treatment at 4℃ and 20℃ for 4 days. The vaccination methods used for groupers were injection, immersion, and feeding. For Formalin 1:400 and 1:800 concentration vaccine with 30-days and 40-days immunization periods, TGIV challenge test result shows that the protection effect for all groups are 100%. But further virus re-isolation of the sample groupers shows that both 1:400 concentration groups has detect virus rate 0%; so Formalin treatment with 1:400 concentration has the best protection effect. After 30 days vaccination with BPL- treatment vaccine, the immunization effect was way below expectation, only the 1:800 concentration group has 40% protection rate. Although BPL-treatment vaccine with 40 days vaccination period has worse protection rate than the Formalin groups, but 1:200 and 1:400 concentration groups has better effect than 30 days vaccination period groups, their survival rates are 70% and 50%. For the immersion experiment cases, BPL treatment microcapsule vaccine with 1 hour immunization period has the best survival rate 80%, better than the Formalin treatment cases 50%. For the feeding experiment cases, both microcapsule vaccines with 20-days and 30-days immunization periods have zero protection effect.
目 錄
中文摘要 ………………………………………………………………………… I前言 ………………………………………………………………………...……. 1
文獻整理 ………………………………………………………………………… 3
一、亞洲地區海水魚類之虹彩病毒感染症相關疫情簡介 ………………… 3
二、魚類病毒性疾病防治之相關對策 ……………………………………… 7
三、化學因子不活化病毒之作用機制 ……………………………………… 10
四、魚類病毒性疫苗的簡介 ……………………………………………… 13
材料與方法 …………………………………………………………………… 18
一、臺灣石斑虹彩病毒之不活化實驗 ……………………………………… 18
(一)實驗材料 ………………………………………………………………… 18
(二)研究方法 ………………………………………………………………… 19
1、物理因子對TGIV之不活化實驗 ……………………………………… 19
2、化學因子對TGIV之不活化實驗 ……………………………………… 20
3、TGIV在在蒸餾水、HESS緩衝液及乾燥環境下之活存實驗 ……… 21
二、石斑虹彩病毒不活化疫苗之開發 ………………………………………… 22
(一)實驗材料 ………………………………………………………………….. 22
(二)研究方法 ………………………………………………………………….. 22
1、TGIV不活化疫苗的製作 ……………………………………………… 22
2、TGIV不活化疫苗之施用方法 ………………………………. ………. 23
3、疫苗保護效果之評估 …………………………………………………... 25
4、人工感染實驗魚之病毒再分離 ………………………………. ………. 25
5、檢體內TGIV之PCR檢測 ………………………………. ……………. 25
三、溫度因子對石斑虹彩病毒致病力之影響 ………………. ……………… 27
(一)實驗材料 ………………. ……………………………. ………………… 27
(二)研究方法 ……………. ……………………………. …………………… 27
結果 …………………………………………………………………………… 28
一、臺灣石斑虹彩病毒的不活化實驗 ………………………………………… 28
1、物理因子對病毒的不活化實驗 ………………………………………… 28
2、化學因子對病毒的不活化實驗 ………………………………………… 29
3、TGIV在在蒸餾水、HESS緩衝液及乾燥環境下之活存實驗 ………… 29
二、石斑虹彩病毒不活化疫苗之開發 ………………………………………… 30
1、TGIV不活化疫苗的製備 ……………………………………………… 30
2、TGIV不活化疫苗施用方法的保護效果評估 ………………………… 30
三、溫度因子對於石斑虹彩病毒致病性之影響 ……………………………… 32
討論 …………………………………………………………………………… 33
參考文獻 ……………………………………………………………………… 46
附錄 …………………………………………………………………………… 60
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