Mammalian microfold cells (MCs) are specialized epithelial cells (ECs) that initiate mucosal immune responses through the uptake and transcytosis of luminal antigens. However, such type of antigen-sampling cells remains largely unknown in teleost fish. In this study, the lectins Ulex europaeus agglutinin-1 (UEA-1) and wheat germ agglutinin (WGA), as well as the monoclonal antibody (mAb) NKM-16-2-4 raised against mouse MCs, were used to identify the MCs in the intestines of olive flounder (Paralichthys olivaceus). These cells exhibit binding characteristics with UEA-1 and mAb NKM-16-2-4, similar to mammalian MCs, and were negative for acid and alkaline phosphatase staining. In addition, unique NKM-16-2-4⁺/WGA⁺ or UEA-1⁺/WGA⁺ cells were also identified. The UEA-1⁺ (including UEA-1⁺/WGA⁺) cells of the anterior, mid, and posterior intestines could take up inactivated Vibrio anguillarum and fluorescent microspheres. These were transported across the epithelium to the underlying lamina propria. Consequently, we renamed these UEA-1⁺ cells as MCs. The MCs of flounder showed short and irregular microvilli, without a mammalian-type basal pocket-like structure, but macrophages and lymphocytes at their basal areas. Co-staining confirmed that the flounder MCs lacked MHC II expression, and that MHC II⁺, IgM⁺ B, and CD83⁺ dendritic cells were underneath or adjacent to the MCs. After oral administration, inactivated V. anguillarum were detected in the MHC II⁺ cells at 6 h. These results indicated that the MCs in teleost intestines act as entry points within the epithelial barrier for particulate antigens. They also provide data valuable for developing novel mucosal vaccines targeting the MCs in teleosts.

To compare the fixation effect of four fixation methods on fish mucosal tissues, and screen the fixation methods suitable for the preservation of mucous cells and their secreted mucus, in this study, the mucosa-associated lymphoid tissues (MALTs) of flounder (Paralichthys olivaceus) were fixed by using different protocols including fixation method (Methacarn and Carnoy fixative solution) and fixed time (2 h and 24 h), and paraffin sections were prepared. After hematoxylin-eosin (HE) staining, Alcian blue-periodate Schiff reaction (AB-PAS), and combined lectin- and immuno-histochemical staining (CLIH) were performed, the fixation and staining effects were evaluated based on histological characteristics and three parameters including goblet cell number, mucin staining area and mucus layer integrity. The results showed that AB-PAS staining clearly distinguished neutral mucin, acidic mucin and mixed mucin, and identified 4 types of mucous cells, and exhibited a better staining effect on mucous cells and mucus layer than HE staining. By using different fixation methods and fixed times, tissue sections with relatively complete structure and clear mucous cells were obtained although a certain degree of difference existed in the fixation and staining effect. In Methacarn fixation groups, the tissue morphological integrity, staining properties and mucous layer preservation effect were obviously better than those of Carnoy's fixation treatment. Of the third, Methacarn fixation solution for 2 h was found to be the best fixation protocol. In this fixation group, the mucosal tissue morphology was more complete and clearer, with less mucus shedding and higher mucin content, and the mucus secretion process can be clearly observed. These results provided a fixation method for paraffin section to facilitate the observation of fish mucous cells and mucus secretion, and a technical support for the subsequent basic research on the regulation of mucin secretion and the mucosal defense role of the mucous cells in fish.

To understand the function of fish pyloric caeca and the response characteristics of mucous cells, in this study, the histological structure of the pyloric caeca, and types of mucous cells and their immune responses after vaccination were studied in flounder (Paralichthys olivaceus) by using histology, histochemistry, indirect immunofluorescence, qRT-PCR and ELISA techniques. The research showed that there were four blind tubes in the pyloric caeca, which consisted of mucosal, submucosal, muscular and serous layers from inside to outside, respectively. The mucosal epithelium contained four types of mucous cells, of which, the number of type Ⅱ mucous cells was the largest, type Ⅰ mucous cells took the second place, while the amount of type Ⅲ and Ⅳ mucous cells was very small. After injection of inactivated Edwardsiella tarda, the types and numbers of mucous cells changed significantly. The number of type Ⅰ mucous cells was reduced while type Ⅱ, Ⅲ and Ⅳ mucous cells were increased. Also, the total number of mucous cells was increased, peaked on the 3^rd day after immunization, and the proliferation of mucous cells could be observed on the 2^nd to 3^rd day. The mucous cells of the pyloric caeca of flounder could express mucin Muc2 gene, but not Muc5ac gene. Post immunization, mucin Muc2 and its gene transcript in the pyloric caeca first increased and then decreased, and the muc2 transcripts peaked at 48 h while the Muc2 protein peaked at 24 h. These results revealed that vaccine immunization induced changes in the types of the mucous cells in the pyloric caeca of flounder, and increases of the number of mucous cells and the expression of mucin Muc2 gene, indicating that the mucus barrier was strengthened. These findings provided a theoretical reference for further understanding the immune defense function of fish pyloric caeca and the crucial role of mucous cells in fish mucosal immunity.

To better understandthe immune response mechanism of fish gill-associated lymphoid tissue to pathogens, the transcriptome sequencing was performed for the gill tissue from healthy and Vibrio anguillarum infected flounder (Paralichthys olivaceus) in this study with the differentially expressed genes (DEGs) analyzed with GO, KEGG, GSEA and other bioinformatics methods. In total, 620 DEGs were obtained, which included 334 up-regulated genes and 286 down-regulated genes. Eight DEGs were randomly selected for validation of the accuracy and reliability of RNA-seq data by real-time qPCR. The results showed that the expression trend of these eight genes was consistent with the transcriptome sequencing results. The enrichment analysis indicated that the DEGs were enriched in the terms related to autophagy and immune response activation and regulation. The immune-related pathways such as complement and coagulation cascades, leukocyte transendothelial migration, NOD-like receptor signaling pathway, JAK-STAT signaling pathway were activated, and the pathways related to disease and cell adhesion were also significantly enriched. It was found that the lectin genes related to complement activation and antibacterial immunity were up-regulated. The C-type lectin represented by CD209 and MBL changed significantly and was the hub gene in the gene interaction network. As the hub genes of the interaction network, pattern recognition receptors tlr13 and nlrc3 were also significantly up-regulated, and played a key regulatory role in antibacterial mucosal immunity. In addition, the expressions of apoptosis related genes (boka and bcl2l15), phagocytosis related genes (lyg and pla2g4c), mucin gene muc5ac, muc5b and muc19, mucosal defense barrier related genes (cldn3d and esama), and T and B lymphocyte related genes also changed significantly, which were involved in the mucosal immune responses. These results provided a theoretical basis for better understanding the mucosal immune response mechanism of teleost to pathogen invasion.

In order to enrich the epidemiological data of largemouth bass virus (LMBV) and further understand the characteristics and pathogenic mechanism of the virus, in this study, the diseased largemouth bass (Micropterus salmoides) with signs of redness, swelling and hemorrhages on body surface was collected from a fish farm in Yantai, Shandong Province, China. The virus isolation and identification were performed by cell culture, histopathological analysis, PCR detection and phylogenetic analysis. No parasitic or bacterial infection was found in the diseased fish. Epithelioma papulosum cyprini (EPC) cells were inoculated with the homogenate of diseased fish tissues, and the cytopathic effect including rounding up and detachment of cultured cells appeared. The enveloped virions with a diameter of about 150 nm were observed in the cytoplasm of EPC cells under transmission electron microscope (TEM), which were hexagonal in shape like iridovirus. According to the main capsid protein (MCP) gene sequence of ranavirus retrieved from NCBI, the specific primers were designed and a gene sequence with a length of 1392 bp was obtained after PCR amplification. By phylogenetic analysis, this virus strain clustered together with other 13 ranaviruses. Among them, the similarity of the isolated virus with 7 virus strains including Santee-Cooper ranavirus (SCRV), Mandarinfish ranavirus (MRV) and largemouth bass ulcerative syndrome virus (LBUSV) was up to 100%. In vivo infection of healthy largemouth bass demonstrated that the mortality rate reached to 100% on day 7 after intraperitoneal injection of 200 μL 10⁵ TCID₅₀/mL virus. These results collectively revealed that the isolated virus belonged to the genus Ranavirus of family Iridoviridae, and accordingly we named it largemouth bass ranavirus LMBV-CN2022 strain. The histopathological observation showed that a large amount of hemosiderin was deposited in liver, spleen and kidney tissues. In addition, we found the virus particles in spleen and kidney under TEM.