The Research On Mechanism Of Self-cure In Japanese Flounder(Paralichthys Olivaceus) LCD Infection

The requirement for additional or altemate sources of fish and shellfish has prompted the rapid growth of the aquaculture industry worldwide,which has resulted in the outbreak of aquaculture animal diseases,especially the prevalence of viral infectious diseases. Lymphocystis disease(LCD) is an infectious disease of several seawater fish species.LCD and its causative agent,fish lymphocystis disease virus(LCDV),exist worldwide,and the rate of incidence appears to be increasing.LCDV belongs to the Iridoviridae group of viruses. LCD is the first large scale prevalent disease in seawater fish species.Today,at least there are 42 osections,more than 125 fishses can drive LCDV infect,among them,the sea water fish have 30 sections.The prevalence of LCDV rsults to large loss in seawater cultured industry. Infection disease to the virus appeal to have no special effect medicine still currently,usually is the medicine and antibiotics that makes use of vaccine to carry on the vaccination or the interference virus replication to cure to the disease in the mankind.But the medicament that disturb virus copy usually brings damage to host.The marine products farming mostly uses chemistry to synthesize or antibiotic medicine,result in easily the backlog,the ecological devastation balance of the antibiotic medicine,guide to change anti- medicine germ stub and pollute the water body,also cause our country the exit of the marine products article get difficulty.According to the observation,LCD infection have from the limit,then the disease exist the phenomenon of self-cure.The primary phenomenon of self-cure exist in that the skin have no tumour and the check of virus is negative.The self-cure of the Japanese flounder(paralichthys olivaceus) have close relation to temperature,general at 14 or low in 14℃,the tumor growth compares slowly;But with go up of water temperature,18℃and or so,the tumor growth compares quickly,being taken bad circumstance severity;But water temperature attain 24℃above,the tumor section of body shed off in Japanese flounder,and the disease fish appear from self-cure.Asusual,under the low temperature,the activity of the fish inborn immunity composition compares greatly,under the heat,the fish adaptability immunity,such as the lymphoid cell activity and the antibody activity become effect.Fish displays the season influence of to the sensitivity of the cause body.It is very imporment for the prevalence of pathogeny and the increase of sensitivity to host.As for the researh on mechanism of LCDV self-cure have never be found,but if make pure this problem and may provide way of thinking for the prevention and cure that should get sick.The cytokine,especially the immunity related cytokine,have a ability in the clearance of the virus,by interfering the replication,construction and the secrete of virus in host,So this research,we discuss the immunity defense mechanism of Japanese flounder about disease self-cure by real-time PCR technique and 2D electrophoresis technique,and look for the difference expression serum protein appears in the process of variety.Find out to cure the paroxysm key factor with the period,provide a worthy reference for the disease prevention and cure in the marine products farming.The main results of this thesis are as follows:1 The Japanese flounder different organization were checked by the PCR technique,The result showed that there have no virus in spleen,liver,kidney,gill,heart,bowel,muscle,blood, epidermis of Japanese flounder,So the result indicated that the self-cure Japanese flounder have no virus.2 Organize pathology slice is about liver by PAS dye.It is thus clear that color is deep in self-cure group,showing liver damaged because of the infection of the LCDV-cn and then the synthesize of hepatin reduce.The AB-PAS dyeing of the tumor clear sthat the tumor layer outside the cell present a blue and is an acidity material.The nucleolus in the immature tumor cell that the primary divide flourishing,still appeared nucleolus,the pit became a dumbbell form in some cells.3 The expression of IL-1,TNF,TNFR,IL-8,IL-8R and Mx in different tissue:liver, spleen and kidney,under different temperature:14℃,18℃,22℃and 26℃,using the technique of real-lime PCR.The research indicated the expression of go up IL-1 with the temperature increase,and the expression reached tiptop at 22℃in liver in the expriment and the control groups,but the tiptop is 26℃in spleen and kidney tissue.The expression of Mx indicated that go up with the temperature increase,and the expression reached tiptop at 26℃in liver and spleen tissue,but 22℃in kidney in the expriment groups,and the increase degree isn't obvious in different tissue in control group.The expression of TNF indicated that the expression go up with the temperature increase,and the expression reached tiptop at 22℃in the the liver tissue,but the expression reduced at 26℃in liver tissue in the control groups.The expression of TNFR indicated that the expression go up with the temperature increase,the expression reached tiptop at is 22℃in the liver,spleen and kidney in the expriment groups.The expression of IL-8 indicated that the expression reached tiptop at 22℃in the liver and kidney tissueses,but the expression reached tiptop at 18℃in spleen in the expriment and control groupses.The expression of the IL-8 R indicated that the reached tiptop at 26℃in liver,spleen and kidney,but the expression reached the tiptop at 22 t℃in liver in the expriment groups,the tiptop is 22 t℃in liver and spleen tissue,26℃in kidney tissue respectively,in the control groups.These data indicated that the expression of cell factors increase obviously with the go up of water temperature in the different organization of experiment group,but the increase is not very obvious in the control group,cytokine involve to the non-special immunity of Japanese flounder.The strengthened expression of these cytokineexplains that non-special immunity of Japanese flounder increase in the heat water, also explaining that the ability of clearance virus will strengtheded along with the water temperature,and then accelerate the possibility of self-cure in Japanese flounder..4 The expression level of TCR,MHCⅠand MHCⅡheavy chain of mRNA in different tissues from a group of vaccinated Japanese flounders(Paralichthys olivaceus) and different temperature was investigated using a real-time polymerase chain reaction(real-time PCR) approach.In the liver,the expression tiptop of MHCⅠ,MHCⅡand TCR is at 22℃,26℃,22℃respectively,and the expression multiple is 14.03,16.33,10.6 respectively.In the spleen,the expression tiptop of MHCⅠ,MHCⅡand TCR is at 18℃,26℃,26℃respectively,and the expression multiple is 2.11,16.87,2.89 respectively;In the kidney,the expression tiptop of MHCⅠ,MHCⅡand TCR is at 14℃,26℃,18℃respectively,and the expression multiple is 5.23,9.96,10.9 respectively;The level expression of MHCI was significantly increased in liver,spleen and kidney of the vaccinated group after two days and eight days,and the level of after two days is higher to after eight days,but the expression level of MHCⅡis contrary to MHCⅠ.The level of expression of TCR have no difference in the vaccinated group aider two and eight days.The expression of MHCⅡand TCR depended on temperature and the expression level increase with the temperature。As a whole,the expression level of from tissues in LCDV group The level of expression of MHCⅠ,MHCⅡ,TCR in defferent tissues was different in the LCDV group and control group.The level of expression was significantly increased in LCDV group.These results indicated that the infected Japanese flounders is placed in a kind of stronger immunity response and the activated route of MHCⅠand MHCⅡis different.It is clear that the mutual expression of MHCⅠ,Ⅱgene regulate the immunity activity of the body together.So,the the high expression of the MHC and TCR in the high temperature was advantageous to promote the body to identify to the virus infection cell,strengthen the cell immunity and the body fluid immunity,and thus clearance virus.5.The different protein of blood serum in selt-cure and ailing Japanese flounders was investigated using two-dimensional gel electrophoresis(2-DE).After silver staine,the results indcated that the protein dot is 328±30 in self-cure groups and 315±21 in infected groups. There are 8 protein spots that their expression were increased in selt-cure groups, and added 3 protein spots.After G-250 stain,there are 10 protein spots to be analysed.The two increased spots are identified to C3 and transferrin(TRF) in self-cure groups.Bacause C3 and transferrin are importment nonspecific immune response factors,the high expression of the non- particularity immunity factor is primary factors for the self-cure of Japanese flounders.