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

Abstract

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.