Isolation Identification Of Carp Myxobolusand Technology Of Culture In Vitro

Myxosporean,a kind of microscopic multicellularparasites,is affiliated with Metazoan,Myxozoa,and Myxosporea. It is parasitic widely in mostof fish such as silver carps, grass carp,carp,crucian carp and so on.A variety of Myxosporean are parasitic in various tissues and organs of fish, but most species haveunique parasitic sites up to one. Since one Myxozoa wasfirst foundin whitefish muscle of Lake Geneva in 1825. So far,domestic and foreign scholars have been reported about 62 genera and more than 2500 kinds ofMyxosporean.The disease caused by Myxosporean parasitic fishwas called fish myxosporidiasis.The disease is not significantly seasonal and can be found throughout the year.In recent years,with the increasing levels of intensive farming and expansion offarmed species,myxosporidiasis isparticularly outstanding, and its damage is very serious.If the control is not timely,a large number of fish will die, which causes serious economic losses.However, effective and preventive measures are notresearched against the disease.In this study, carp Myxobolus was used as research subjectsto establish its laboratory culture method and optimizeitsculture conditions, and the optimum conditions for culturing Myxobolus in vitro was obtained,which provides a lot of foundationmaterials for the future research of myxosporidiasis.Research contents and results as follows:1.The preliminary investigation of myxosporidiasis at Jilin Province. The incidence of myxosporidiasis in Changchun, Jilin, Baicheng and so on was investigated. Samplesof vital organs were further checked by microscope, and focus tissue was mainly used to make flood sheet and paraffin biopsy.At last, the Myxosporean was identified by PCR and sequencing.Survey results showed that:three kinds of Myxosporean were parasitic on Carassius auratus gibelio species. They are Myxobolus, Thelohanellus and Myxosoma. ForCarassius auratus gibeliobreeding inJilin province,although Thelohanellus cause the disease with high incidence, but not cause fish to death directly.The highest mortality diseases is caused by Myxobolus,and Myxoboluswasidentified as Honghu Myxobolus.2.Effect of different Mediumon Myxobolus proliferation.Muscle and gill tissue of Fresh crucian carp was used respectively to prepare tissue homogenates.Culture conditions was crucian carp tissue homogenates, culture medium(M199,L-15 and DMEM culture medium), serum(FBS,fish serum), antibiotics 1%,at26℃,setting serum concentration gradient, that is 5%,10%,15%,20%. The growth rate wascounted a total of 6 times per 72 hours, and then was calculatedwith formula. The results showed that: optimal conditions in vitro Myxobolus was crucian carp muscle homogenates, DMEM medium, 5% fetal bovine serum;in the test group of crucian carp muscle homogenates, fish serum was used to culture Myxobolus, which did not achieve optimal results, but there are certain growth trends; the test group of crucian carp gill homogenates did not obtain suitable conditions for in vitro Myxobolus.3.Effect of different cellson Myxobolus proliferation.Fin and muscle tissue of fish was applied to culture primary cells.When the cells moved out and grew to the optimal condition, the subculture began. Crucian Carp fin cells, crucian carp muscle cells, FHM, and COwas used as growth vector respectively of Myxobolus.Four bottles of cells were selected and cultured at 26℃, which with 4ml medium(L-15 culture medium or M199 culture medium) 10% FBS, 1% double antibody and 1ml Myxobolus suspension. The growth rate was counted a total of 7 times per 72 hours,and then was calculated with formula. The results showed that: optimal conditions in vitro Myxobolus is FHM, M199 medium, 10% fetal calf serum, antibiotic 1%,at 26 ℃; when grass carp gonad cells was applied as the grow carrier, Myxobolus almost had no proliferation.