The Study Of Cultivating Ichthyophthirius Multifiliis In Vitro And The Effect Of Artesunate On Ichthyophthirius Multifiliis

Ichthyophthirius multifiliis is a globly distributed parasite which obligately parasites on freshwater·fishes, and caused ichthyophthiriasis. The disease causes high mortality under the conditions of intensive aquaculture, and a great economic loses annually.Four culture systems were applied to cultivate the parasite which were the EMEM medium included 5% foetal calf serum,EMEM included 5% eel serum, the RPMI1640 medium included 5% foetal calf serum, the RPMI1640 included 5% eel serum respectively. The four mediums were diluted with 1:1, 1:2, 1:3, 1:4, 1:5, 1:6 ratio, which were used to cultivate the parasite in vitro. The survival and the size of the theront of the parasite were observed in this trial. The results show that the EMEM included 5% eel serum was the most optimum culture systems, in which the longest suvival time of the parasite was 22d. Especially, the ratio of dilution was 1:1 of the EMEM included 5% eel serum, in which the Size(μm±SD) development of I. reached 30.17±2.05μm was significantly bigger than other test group(P<0.05).I. multifiliis incubateded in vitro were used to screen medicines. The blood cell counting assay, MTT assay and CBB assay had been used estensively counting the number of cell. The purpose of thise study were to screen the suitable culture medium for I. multifiliis in vitro and determine the accurate counting to evalute growth of I. multifiliis in vitro.This study showed the MTT assay was suitable to count the number of I.multifiliis in the culture medium EMEM included 5% eel serum. The number of I. multifiliis mutiplied by 7.6 times at 8th day after inoculation in the culture medium.The suitable culture medium for the growth of I. multifiliis was used to basal Culture mediun for screening drug. The I. multifiliis treated with the different dilution of Artesunate (AS) and chloroquine phosphate (CQP).The results showed that: the two drug had the effects of growth suppression of I. multifiliis in vitro, The I. multifiliis treated by AS at different dilution at 24h,48h,96h,the Lethal concentration (LC₅₀) was 1.7mg/ml, 1.4mg/ml, 1.08mg/ml. The I. multifiliis treated by CQP at different dilution at 24h, 48h, 96h, the LC₅₀ was 0.0051mg/ml, 0.0035mg/ml, 0.0024mg/ml.The effects of growth suppression of I. multifiliis of CQP were better than AS. The enzymes activities of Na⁺K⁺ATP, SOD, GSH-PX, CAT were decreased and the activities of MDA was increased after treated by AS. The enzymes activities of Na⁺K⁺ ATP, GSH-PX were decreased and the activities of MDA, SOD, CAT were increased after the treated by CQP.The methods of DNA extraction and 18SrRNA sequence amplification were studied in I. multifiliis. The results indicated that the concentrated of I. multifiliis incubateded in vitro were lysed by cell lysis solution and their DNA were extracted by phenol-chorine technique, using this DNA templat; the 18SrRNA flagments of I. multifiliis were amplified. They were 292bp,500bp,577bp,366bp neuleotides respectively.