海水魚寄生性白點病原蟲(Cryptocaryon irritans)之研究及免疫控制

Abstract

中文摘要 海水白點蟲 (Cryptocaryon irritans)，是造成海水魚類白點病的原生動物寄生蟲。本研究描述海水白點蟲發育學及分子生物學的特徵，以及其體外培養技術。此外亦評估點帶石斑魚 (Epinephelus coioides) 對抗海水白點蟲產生的體液性和細胞性免疫反應；以及測定經免疫過的魚，是否被賦予保護作用。 在台灣的海水白點蟲各分離株中，分析其發育學特徵，以及包含部分 18 S、完整的第一內轉錄間隔區 (internal transcribed spacer) 和部分 5.8 S 的核醣體 DNA 序列。發現台灣海水白點蟲分離株在聚集、附著和感染部位等發育學特徵上並不一致。澎湖和美國的海水白點蟲分離株核酸序列相同；馬來西亞和以色列分離株核酸序列也相同。成偶比對中，台灣海水白點蟲分離株的變異百分率，比基因資料庫中所列出的序列，顯示出有更高度的變異。親緣關係樹可區分出海水變異株和低鹽度變異株兩類群。在水源有限的半淡鹹水池，或者海水箱網養殖上，採用操作鹽度來控制海水白點蟲病有施行上的困難，因此發展新的防治策略有其需要。 進行了三次體外 (in vitro) 培養海水白點蟲的實驗。纖毛幼蟲 (theront) 能附著在 tryptic soy agar (TSA, 3% NaCl) 固體培養基上，之後長大成為營養體 (trophont)；纖毛幼蟲亦可在加強養份的液體培養基中轉型成營養體。所有離體試驗中所培養出的營養體體形大小，都落在記載中魚體 (in vivo) 發育寄生蟲的大小範圍內。這些結果顯示體外培養具有潛在的可行性。然而從營養體轉型成孢囊體 (tomonts)，仍需找到啟動轉型的必要因子。 將福馬林固定的纖毛幼蟲腹腔注射石斑幼魚進行疫苗效果測試，二次試驗分別在免疫後25 天和 17 天進行，將活的孢囊投入魚缸進行感染。第一次試驗中接受高劑量疫苗 (100 μg/fish) 的魚在感染後 22 天內沒有死亡，接受低劑量疫苗 (10 μg/fish) 的魚累積死亡率為 40%，而對照組(注射 PBS) 則 90% 死亡。第二次試驗中，感染 5 天或 7 天後，疫苗免疫過的石斑魚產生的營養體和孢囊體的數量，分別都顯著地比注射 PBS者少；感染後 9 天內，疫苗(高劑量)免疫過的魚累積死亡率為 37.5%，對照組則 100% 死亡。之前曝露接觸海水白點蟲過的石斑幼魚和成魚，再感染後 3 週，用 ELISA 測定其黏液抗體力價，比之前沒曝露過海水白點蟲的魚來得高。而且第三次曝露感染比起第一次曝露感染，所產生的孢囊體顯著較小。這些結果指出免疫過的石斑魚被賦予了保護性，對海水白點蟲纖毛幼蟲的附著、入侵和發育，石斑魚的皮膚可能扮演防止及限制的主要角色。 先天 (innate) 和後天 (adaptive) 免疫中的細胞性免疫在寄生蟲的排除上，扮演不可或缺的角色。周邊血液中嗜酸性球族群減少、白血球滲入感染區域、表皮層黏液細胞增生，以及分泌抗體的 B 細胞出現在表皮層，都指出協同體液性免疫，細胞性免疫作用的活化；並進一步地解釋，賦予在免疫過的石斑魚對抗原生動物海水白點蟲的保護作用的機制。 海水白點蟲的多變異性，使得收集不同分離株成為免疫研究的必要工作。海水白點蟲的體外培養可能有希望提供長期、穩定的產量，以供應疫苗發展。魚體防禦體表寄生蟲感染，黏液中專一性抗體顯示極其重要，細胞性免疫亦是必要的。

Abstract This study described the developmental and molecular characteristics of Cryptocaryon irritans, a protozoan parasite causing the white spot disease in marine fishes, and its in vitro culture. In addition, the paper also assessed the humoral and cellular immune responses of the grouper Epinephelus coioides immunized against C. irritans and determined whether protection is conferred on immunized fish. Developmental characteristics and sequences of the ribosomal DNA regions such as part of 18 S, entire first internal transcribed spacer, and part of 5.8 S of various Taiwan isolates of C. irritans were determined. The parasite showed variation in its developmental characteristics such as aggregation, adherence, and site of infection. Isolates from Pingtung and the USA had identical nucleotide sequences while the isolate from Malaysia was identical to Israel. Percentage variation among Taiwan isolates, when the sequences were compared pairwise, showed a higher degree of variation than those whose sequences were listed in the GenBank. The phylogenetic tree distinguished the seawater species of C. irritans from the low salinity variant. Salinity manipulation to check cryptocaryoniasis in brackishwater ponds with limited water source and marine cage sites is not feasible hence there is a need to develop new strategies for its control and prevention. Three experiments were carried out for the in vitro culture of C. irritans. Attachment of theront parasites and subsequent enlargement into trophonts were realized in tryptic soy agar (TSA, 3% NaCl). Transformation of theronts into trophonts in an enriched liquid was also realized. Results showed that the in vitro culture of C. irritans is potentially feasible as evidenced by the enlargement of the trophonts within the recorded in vivo size range using either a solid media as attachment substrate or a liquid media without attachment. There is a need, however, to determine the essential factors that will trigger the transformation of the trophonts into viable tomonts. Vaccine-immunization was carried out by intraperitoneal injection of formalin-killed theronts into the grouper fingerling. At 25-day and 17-day post-immunization, live tomonts were seeded into the tanks to challenge the fish in the first replicate and second replicate, respectively. In the first replicate, no mortality was monitored on fish that received high dose vaccine (100 μg/fish) while 40% and 90% cumulative mortalities were recorded in low dose group (10 μg/fish) and control group (PBS-injected), respectively, at 22-day post challenge. In the second replicate, significantly fewer trophonts and tomonts in the vaccine-immunized group than the PBS-injected fish were observed at 5-day post-challenge and 7-day post challenge, respectively. Cumulative mortalities of 37.5% and 100% were observed in the vaccine-immunized group (high dose) and the control group at 9-day post challenge. Antibody titers in the mucus detected by ELISA were significantly higher in C. irritans–exposed grouper fingerlings and large grouper at 3-wk post infection compared to fish that had no previous exposure. In addition, significantly smaller tomonts were produced after three successive exposures of the same fish than those produced in fish after single exposure. Results suggest that protective immunity was conferred on the immunized grouper. The skin of immunized grouper may have played a major role in preventing or limiting the adhesion, invasion, and development of C. irritans theronts. The cellular responses in the innate and adaptive immunity play a vital role in the elimination of the parasite. The down-modulation of eosinophil population in the peripheral blood, infiltration of leukocytes in the infected site, proliferation of mucus cells in the epidermal layer, and presence of antibody-secreting B cells in the epidermal layer all point to the activation of the cellular immunity in coperation with humoral immunity. All of this further explains the mechanisms of protection conferred in immunized grouper against the protozoan parasite C. irritans. The existence of diverse C. irritans necessitates the collection of different isolates for immune studies. The possible in vitro culture showed promise and may provide a long term and a stable supply of C. irritans for vaccine development. Cellular immunity is necessary in the defense of fish and the specific antibodies in the mucus are extremely important against a parasitic infection.