We isolated a strain of lymphocystis disease virus(LCDV) from Japanese flounder(Paralichthys olivaceus) cultured in northern China.Based on published sequences of major capsid protein(MCP) gene of LCDV-cn(GenBank:AF126405),we designed two primer sets P1/P2 and P3/P4.We then used one-step or nested PCR and in-situ hybridization(ISH) to detect LCDV and identify the target tissues or cells in infected Japanese flounder.The PCR products were positive in purified viral supernatant,skin nodules,gut,gill,kidney,spleen,stomach,heart,and liver of Japanese flounder.We compared the DNA sequence with 14 MCP nucleotide sequences from GenBank,including Megalocytivirus(OFIV and RSIV),Iridovirus(CzIV and WIV),Ranavirus(TFV and FV3),and Lymphocystivirus(8 LCDV).Based on the alignment,we confirmed the PCR product was from Lymphocystivirus(GenBank accession number DQ279090(LCDV-HD)).Using ISH,we noted the presence of LCDV in the skin nodules,gut,gill,spleen,stomach,and heart of spontaneously infected Japanese flounders.We successfully amplified LCDV fragments from Schlegel's black rockfish(Sebastes schlegeli Higendorf),redwing sea robin(Lepidotrigla microptera Günther) and turbot(Scophthalmus maximus) using the one-step and nested PCR,suggesting the target genes can be widely detected in fish using this method.
A pathogenic bacterium(S636),identified as Streptococcus iniae,was isolated from turbot(Scophthalmus maximus) in 2005.We immunized turbot with formalin-killed S.iniae four times(on days 1,14,21,and 28) by intraperitoneal inoculation.After each vaccination,we obtained serum samples and isolated the lymphocytes from the peripheral blood,spleen,pronephros,and mesonephros.We measured surface Ig-positive(sIg+) lymphocytes and serum antibody levels from these organs using flow cytometry and enzyme-linked immunosorbent assay(ELISA),respectively,using monoclonal antibodies against turbot immunoglobulin.We confirmed that the antibody reacted with both the surface and plasma Ig by confocal laser scanning microscopy and electron microscopy.The percentage of sIg+ in the lymphocytes increased following each successive vaccination.The mean percentage increased from 31.96%(control) to 37.49%,38.36%,42.9%,and 51.63% in the peripheral blood;from 27.09% to 36.63%,36.81%,39.28%,and 46.0% in the spleen;from 22.2% to 28.99%,29.21%,32.83%,and 41.58% in pronephros;and from 18.12% to 22.17%,22.45%,25.69%,and 31.68% in the mesonephros.The ELISA results were consistent with these results.Both the total and specific antibody levels increased with each vaccination.The mean OD value of the specific antibody assay increased from 0.094,to 0.269,0.283,0.333,and 0.421;for total antibody the mean OD value increased from 0.133,to 0.292,0.323,0.413,and 0.527.
