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Intramuscular (i.m.) injection is one of the common delivery methods of vaccination in aquaculture, which could induce an ideal immune protection to fish. In the present study, the olive flounders ( Paralichthys olivaceus ) were injected intramuscularly with 200 μl of three concentrations of formalin-inactivated Edwardsiella tarda bacterin (10 7 , 10 8 , 10 9 CFU ml −1 ) to investigate the transportation and dynamic distribution of antigen uptake in tissues by absolute real-time quantitative PCR (qPCR). The amount of uptaken antigen increased firstly, and then decreased. The peak occurred first in the blood at 6–9 h after i.m. injection, and in the spleen and head kidney at 9–15 h, then in the liver, gill and muscle at 15–24 h, finally in the skin and intestine at 36 h. The amount of uptaken antigen was highest in the head kidney, followed by in the spleen, blood, gill, and liver, and lowest in the muscle, skin and intestine. Among the three dose groups, the amount of uptaken antigen in all tested tissues became higher with the increasing dose of injected bacterin. Moreover, the tissue distribution of antigen uptake was investigated by indirect immunofluorescence assay (IIFA) at 15 h after i.m. injection with 200 μl of 10 8 CFU ml −1 E. tarda bacterin. The distribution of antigen was mainly observed in the head kidney, then in the spleen, blood, liver, gill and muscle, and least in the skin and intestine, which correlated with the results of absolute qPCR detection. Furthermore, the expression levels of MHC Iα, MHC IIα, CD4-1 and CD8α were detected by RT-qPCR. The expression of these four genes peaked highest in the head kidney, followed by in the spleen, liver, blood and gill, and lowest in the muscle, skin and intestine, and the levels increased in parallel with the increasing dose of injected vaccine. All these results provided an important insight into the dynamic transportation of antigen uptake, and also deepened the understanding of immune response to the i.m. injection. [ABSTRACT FROM AUTHOR]

Abstract: Flounder gill (FG) cell line derived from the gill tissue of Japanese flounder (Paralichthys olivaceus) was employed as a target cell for investigating the molecules involved in lymphocystis disease virus (LCDV) binding and infection. Indirect immunofluorescence assay showed that LCDV could attach to the FG cells membrane susceptibly. By virus overlay protein binding assay (VOPBA), a 37.6kDa LCDV-binding protein was detected in FG cells'' membrane. Two dimensional gel electrophoresis analysis revealed that the 37.6kDa protein was a single polypeptide with PI of 6.0. Mass spectrometric analysis showed that the 37.6kDa protein had a strong association with a coiled-coil domain-containing protein. While treated with trypsin or sodium periodate, the binding ability of the 37.6kDa protein to LCDV was inhibited, which suggested that sugar chains of the 37.6kDa glycosylated protein were involved in virus binding. Polyclonal antibodies were obtained by immunizing the rabbit with electroeluted 37.6kDa protein, which showed a dose-dependent blocking effect to the binding between LCDV and 37.6kDa protein in modified VOPBA, and also inhibited LCDV infection to FG cells in virus infection assay. These results demonstrated that the 37.6kDa trypsin-sensitive glycoprotein might be a potential receptor for LCDV infection to FG cells. [Copyright &y& Elsevier]

In our previous work, two monoclonal antibodies (Mabs) against granulocytes of shrimp ( Fenneropenaeus chinensis ) had been produced, in this paper, haemocyte subpopulations were analyzed by flow cytometry (FCM) using the Mabs. Then immunomagnetic bead (IMB) method was applied for separation hyalinocytes and granulocytes using the Mabs. The separated hyalinocytes and granulocytes were analyzed by FCM, indirect immunofluorescence assay, Giemsa staining and transmission electron microscopy, respectively. The results showed the proportion of hyalinocytes in haemolymph of F . chinensis was 15.14 ± 1.22%, and that of granulocytes was 75.43 ± 2.31%. After two times separation by IMB, the purity rate of hyalinocytes and granulocytes was 96.27 ± 1.06% and 98.13 ± 0.86%, respectively. The hyalinocytes possessed 0.60–0.85 in nucleus/cytoplasm (N/C) ratio and had few granule in cytoplasm, whereas the separated granulocytes with N/C ratio of 0.12–0.36 and high electronic density of double membrane granules. The results reported the separation of haemocyte subpopulations using Mabs in shrimp for the first time, and the hyalinocytes and granulocytes isolated by IMB could be used for their differential protein analysis. [ABSTRACT FROM AUTHOR]

Abstract: To investigate the interaction between white spot syndrome virus (WSSV)-VP37 and gill membrane proteins (GMPs) of Pacific white shrimp (Litopenaeus vannamei), the VP37 protein was expressed and purified, and a distinct 53 kDa VP37-binding protein band was identified in GMPs by virus overlay protein binding assay and GST pull-down assay. By electroelution, the VP37 binding protein was purified and identified as F1ATP synthase β (F1ATPase β) subunit by Mass Spectrometry. The purified F1ATPase β subunit was used to immunize BALB/C mice to produce monoclonal antibodies (Mabs). After cell fusion, sixteen hybridomas secreting Mabs against F1ATPase β subunit of L. vannamei were screened by enzyme-linked immunosorbant assay (ELISA), three of which designated as 1D5, 1E8 and 2H4 were cloned by limiting dilution and further characterized by indirect immunofluorescence assay (IIFA) and western blotting. The results of IIFA showed that specific fluorescence signals located at the peripheral zone of the gills of L. vannamei. Western blotting demonstrated that three Mabs reacted specifically with the 53 kDa protein band in GMPs of L. vannamei. By IIFA, the Mabs could also cross-react with the gill cells of three other WSSV-susceptible shrimps Fenneropenaeus chinensis, Penaeus monodon and Marsupenaeus japonicus. Furthermore, the three anti-F1ATPase β subunit Mabs could partially block the binding of WSSV to GMPs by ELISA in vitro, and also exhibited direct anti-WSSV activity in shrimp by neutralization assay in vivo. These findings suggested that F1ATPase β subunit involved in WSSV infection in L. vannamei. [Copyright &y& Elsevier]

Abstract: A monoclonal antibody (MAb 6H7) specific to granulocytes of scallop Chlamys farreri was produced by immunising mice with separated granulocytes as an antigen. Characterised using a flow cytometric immunofluorescence assay, MAb 6H7 reacted to granulocytes by 87.1% of total positive haemocytes. At the ultrastructural level, MAb 6H7 demonstrated epitope in cytoplasmic granules of granulocytes. Western blotting analysis indicated that a peptide of 155 kDa was recognised by MAb 6H7. It was therefore used to investigate granulocyte variation in C. farreri after acute viral necrobiotic virus (AVNV) infection using an enzyme-linked immunosorbent assay. The result illustrated that granulocytes varied greatly by AVNV infection, and their amount significantly increased on day 1 post-injection, then decreased on days 2, 3 and 4, thereafter, rebounded and approached to a second peak on day 6, finally went down gradually to the control level on day 8. [Copyright &y& Elsevier]