Myeloperoxidase (MPO) is a cationic leukocyte haloperoxidase and together with other proteins, they possess activities against various microorganisms and are involved in extracellular trap (ET) formation. The present work describes the gene and deduced protein sequences, and functions of MPO in flounder (PoMPO). The PoMPO possesses a 2313 bp open reading frame (ORF) that encodes a protein of 770 amino acids. The highest PoMPO mRNA expression levels were found in the head kidney, followed by peritoneal cells, gill, spleen, skin, muscle, and liver. PoMPO was expressed in MHCII⁺ and GCSFR⁺ cells which indicated that PoMPO mainly is expressed in flounder macrophages and granulocytes. Bacterial lipopolysaccharide-stimulated peritoneal leukocytes showed an increased protein level of PoMPO while it seemed that LPS also promoted the migration of MPO⁺ cells from the head kidney into the peripheral blood and peritoneal cavity. After phorbol 12-myristate 13-acetate (PMA) or bacterial stimulation, flounder leukocytes produced typical ET structures containing DNA with decoration by MPO. The ETs containing DNA and PoMPO effectively inhibited the proliferation of ET-trapped bacteria. Blocking PoMPO with antibodies decreased the enzymatic activity, which attenuated the antibacterial activity of ETs. This study pinpoints the involvement of ETs in flounder innate responses to pathogens.

Streptococcus iniae is a severe Gram-positive pathogen that can infect a wide range of freshwater and marine fish species. In continuation of our earlier studies on the development of S. iniae vaccine candidates, pyruvate dehydrogenase E1 subunit alpha (PDHA1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were highly efficacious in protecting flounder (Paralichthys olivaceus) against S. iniae. In the present study, to investigate the potential of multi-epitope vaccination strategy to prevent flounder against S. iniae infection, the liner B-cell epitopes of PDHA1 and GAPDH proteins were predicted using a bioinformatics approach and were identified by immunoassay, and recombinant B-cell multi-epitopes of PDHA1 and GAPDH (rMEPIP and rMEPIG) containing immunodominant epitope-concentrated domains were expressed in Escherichia coli BL21 (DE3) and were used as a subunit vaccine to immunize healthy flounder, while recombinant PDHA1 (rPDHA1), GAPDH (rGAPDH) and formalin-inactivated S. iniae (FKC) served as controls. Then, the immunoprotection efficacy of rMEPIP and rMEPIG was evaluated by determining the percentages of CD4-1⁺, CD4-2⁺, CD8β⁺ T lymphocytes and surface-IgM-positive (sIgM⁺) lymphocytes in peripheral blood leucocytes (PBLs), spleen leucocytes (SPLs) and head kidney leucocytes (HKLs), as well as total IgM, specific IgM, and relative percentage survival (RPS) post immunization, respectively. It was found that fish immunized with rPDHA1, rGAPDH, rMEPIP, rMEPIG and FKC showed significant increases in sIgM⁺, CD4-1⁺, CD4-2⁺, and CD8β⁺ lymphocytes and production of total IgM and specific IgM against S. iniae or recombinant proteins rPDHA1 and rGAPDH, which indicated the activation of humoral and cellular immune responses after vaccination. Moreover, RPS rate of the multi-epitope vaccine rMEPIP and rMEPIG groups reached 74.07% and 77.78%, higher than that of rPDHA1 and rGAPDH (62.96% and 66.67%) and KFC (48.15%). These results demonstrated that B-cell multi-epitope protein vaccination, rMEPIP and rMEPIG, could give a better protective effect against S. iniae infection, which provided a promising strategy to design the efficient vaccine in teleost fish.

The peritoneal cavity plays an important role in the immune response, and intraperitoneal administration is an ideal vaccination route in fish. However, immune responses in the peritoneal cavity of teleost fish are still not completely characterized. This study characterized the morphology of peritoneal cavity cells (PerC cells) and their composition in flounder (Paralichthys olivaceus). Flow cytometric analysis of the resident PerC cells revealed two populations varying in granularity and size. One population, approximately 15.43% ± 1.8%, was smaller with a lower granularity, designated as lymphocytes. The other population of the cells, about 78.17% ± 3.52%, was larger with higher granularity and was designated as myeloid cells. The results of cytochemical staining and transmission electron microscopy indicated that peritoneal cavity in flounder normally contains a resident population of leukocytes dominated by granulocytes, macrophages, dendritic cells, and lymphocytes. The percentages of IgM⁺, CD4⁺, G-CSFR⁺, MHCII⁺, and CD83⁺ leukocytes among PerC cells determined by flow cytometry were 3.13% ± 0.4%, 2.83% ± 0.53%, 21.12% ± 1.44%, 27.11% ± 3.30%, and 19.64% ± 0.31%, respectively. Further, the changes in IgM⁺, CD4⁺, G-CSFR⁺, MHCII⁺, and CD83⁺ leukocytes in flounder after Vibrio anguillarum infection and immunization were compared. The composition changed rapidly after the infection or vaccination treatment and included two stages, a non-specific stage dominated by phagocytes and a specific immune stage dominated by lymphocytes. Due to the virulence effectors of bacteria, the infected group exhibited a more intense and complicated PerC cells immune response than that of the immunization group. Following our previous study, this is the first report on the morphology and composition of PerC cells and the early activation of PerC cells in flounder response to V. anguillarum infection and vaccination.