Mucosal immune responses to Ichthyophthirius multifiliis in the ocular mucosa of rainbow trout (Oncorhynchus mykiss, Walbaum), an ancient teleost fish.

The eye, as a specialized visual organ, is directly exposed to the external environment, and, therefore, it faces constant challenges from external pathogenic organisms and toxins. In the ocular mucosa (OM) of mammals, mucosal-associated lymphoid tissues (MALTs) constitute the primary line of defense. However, the immune defense role of the OM remains unknown in aquatic vertebrates. To gain insights into the immune processes within the OM of teleost fish, we developed an infection model of rainbow trout (Oncorhynchus mykiss) OM using a parasite, Ichthyophthirius multifiliis (Ich). Immunofluorescence, qPCR, and H&E staining revealed that Ich successfully infiltrates the OM of rainbow trout, leading to pathological structural changes, as evidenced by A&B staining. Importantly, the qPCR results indicate an up-regulation of immune-related genes following Ich infection in the OM. Moreover, transcriptome analyses were conducted to detect immune responses and impairments in eye function within the OM of rainbow trout with Ich infection. The results of the transcriptome analysis that Ich infection can cause an extensive immune response in the OM, ultimately affecting ocular function. To the best of our knowledge, our findings represent for the first time that the teleost OM could act as an invasion site for parasites and trigger a strong mucosal immune response to parasitic infection. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00199-6.

Functional characterization of group â ¡ interferon, IFNf in the acipenseriform fish, Chinese sturgeon (Acipenser sinensis).

Teleost fish possess a diversity of type Ⅰ interferons (IFNs) repertoire, which play a crucial role in antiviral and antimicrobial immune responses. In our previous study, IFNe1-3 and IFNb were identified and cloned from Chinese sturgeon (Acipenser sinensis), an acipenseriform fish. However, the absence of Chinese sturgeon genome data has left the question of whether there are other type Ⅰ IFN members in this species unresolved. In this study, we have identified and characterized a novel IFN, IFNf in Chinese sturgeon (AsIFNf). Bioinformatics analysis revealed that the AsIFNf contains a unique disulfide bond (2 cysteines) located in the second exon and fifth exon region, distinguishing it from other reported teleost type I IFNs. Meanwhile, qPCR results showed that AsIFNf mRNA was detectable in all examined tissues and up-regulated in the spleen or kidney in response to poly I: C, Citrobacter freundii, and Spring Viremia of Carp Virus (SVCV), but not by LPS. Furthermore, compared to recombinant AsIFNe2 protein (rAsIFNe2), rAsIFNf exhibited a stronger protective effect on Chinese sturgeon fin cells against SVCV and also induced higher expression of antiviral genes Mx and viperin. Importantly, AsIFNf displayed characteristics similar to antimicrobial peptides (AMPs) with a positive charge and demonstrated a broad spectrum of antimicrobial activity in vitro. These findings provide a theoretical foundation for understanding the primitive structure and function of interferon, as well as deepening our comprehension of the innate immune system and disease defense in the endangered Chinese sturgeon.

Mucosal immune responses of gut IgM in common carp (Cyprinus carpio) following infection with spring viremia of carp virus (SVCV).

Immunoglobulin M (IgM) specifically recognizes various antigens and can activate complement, mediate cytotoxicity, opsonize and agglutinate pathogens to induce phagocytosis, all of which play an important role in immunity. However, the IgM response of common carp (Cyprinus carpio) in the intestinal mucosa after viral infection has not been thoroughly. Therefore, we successfully produced an anti-carp IgM monoclonal antibody and developed a model of viral infection to study the kinetics of immune responses after viral infection. Our results showed that the expression of IL1-ß and Igs were dramatically increased, implying that common carp exhibited a significant innate and adaptive immune response to viral infection. Furthermore, we found that the IgM responses varied between the two infection strategies. At 14 days post-infection (DPI), a significant population of IgM+ B cells were observed in the gut, accompanied by a sharp rise in IgM levels. The immune response to secondary infection started at 7 DPI, suggesting that the IgM response is faster in the gut after re-infection. Importantly, we also explored the variability of different gut compartments to viral infection, and result revealed a stronger immune response in the hindgut than in the foregut and midgut. Overall, our findings indicate that IgM plays an important role in the intestinal immune response following primary and secondary viral infection, in which the hindgut plays a major immune function.

Elucidating the dynamic immune responses within the ocular mucosa of rainbow trout (Oncorhynchus mykiss) after infection with Flavobacterium columnare.

The eye of vertebrates is constantly faced with numerous challenges from aquatic or airborne pathogens. As a crucial first line of defense, the ocular mucosa (OM) protects the visual organ from external threats in vertebrates such as birds and mammals. However, the understanding of ocular mucosal immunity in early vertebrates, such as teleost fish, remains limited, particularly concerning their resistance to bacterial infections. To gain insights into the pivotal role of the OM in antibacterial immunity among teleost fish, we developed a bacterial infection model using Flavobacterium columnare in rainbow trout (Oncorhynchus mykiss). Here the qPCR and immunofluorescence results showed that F. columnare could invade trout OM, suggesting that the OM could be a primary target and barrier for the bacteria. Moreover, immune-related genes (il-6, il-8, il-11, cxcl10, nod1, il1-b, igm, igt, etc.) were upregulated in the OM of trout following F. columnare infection, as confirmed by qPCR, which was further proved through RNA-seq. The results of transcriptome analyses showed that bacterial infection critically triggers a robust immune response, including innate, and adaptive immune-related signaling pathways such as Toll-like, NOD-like, and C-type lectin receptor signaling pathway and immune network for IgA production, which underscores the immune role of the OM in bacterial infection. Interestingly, a substantial reduction in the expression of genes associated with visual function was observed after infection, indicating that bacterial infection could impact ocular function. Overall, our findings have unveiled a robust mucosal immune response to bacterial infection in the teleost OM for the first time, providing valuable insights for future research into the mechanisms and functions of ocular mucosal immunity in early vertebrate species.

Comparative Transcriptomic Analysis Revealed Potential Differential Mechanisms of Grass Carp Reovirus Pathogenicity.

Grass carp reovirus (GCRV), one of the most serious pathogens threatening grass carp (Ctenopharyngodon idella), can lead to grass carp hemorrhagic disease (GCHD). Currently, GCRV can be divided into three genotypes, but the comparison of their pathogenic mechanisms and the host responses remain unclear. In this study, we utilized the Ctenopharyngodon idella kidney (CIK) model infected with GCRV to conduct comparative studies on the three genotypes. We observed a cytopathic effect (CPE) in the GCRV-I and GCRV-III groups, whereas the GCRV-II group did not show any CPE. Moreover, a consistent trend in the mRNA expression levels of antiviral-related genes across all experimental groups of CIK cells was detected via qPCR and further explored through RNA-seq analysis. Importantly, GO/KEGG enrichment analysis showed that GCRV-I, -II, and -III could all activate the immune response in CIK cells, but GCRV-II induced more intense immune responses. Intriguingly, transcriptomic analysis revealed a widespread down-regulation of metabolism processes such as steroid biosynthesis, butanoate metabolism, and N-Glycan biosynthesis in infected CIK cells. Overall, our results reveal the CIK cells showed unique responses in immunity and metabolism in the three genotypes of GCRV infection. These results provide a theoretical basis for understanding the pathogenesis and prevention and control methods of GCRV.

Teleost Eye Is the Portal of IHNV Entry and Contributes to a Robust Mucosal Immune Response.

The ocular mucosa (OM) is an important and unique part of the vertebrate mucosal immune system. The OM plays an important role in maintaining visual function and defending against foreign antigens or microorganisms, while maintaining a balance between the two through complex regulatory mechanisms. However, the function of ocular mucosal defense against foreign pathogens and mucosal immune response in bony fish are still less studied. To acquire deeper understanding into the mucosal immunity of the OM in teleost fish, we established a study of the immune response of rainbow trout (Oncorhynchus mykiss) infected with the infectious hematopoietic necrosis virus (IHNV). Our findings revealed that IHNV could successfully infiltrate the trout's OM, indicating that the OM could be an important portal for the IHNV. Furthermore, qPCR and RNA-Seq analysis results showed that a large number of immune-related genes were significantly upregulated in the OM of trout with IHNV infection. Critically, the results of our RNA-Seq analysis demonstrated that viral infection triggered a robust immune response, as evidenced by the substantial induction of antiviral, innate, and adaptive immune-related genes in the OM of infected fish, which underscored the essential role of the OM in viral infection. Overall, our findings revealed a previously unknown function of teleost OM in antiviral defense, and provided a theoretical basis for the study of the mucosal immunity of fish.

Transcriptome Sequencing Reveals the Antiviral Innate Immunity by IFN-γ in Chinese Sturgeon Macrophages.

To further study the biological function of interferon-gamma (IFN-γ) in the Chinese sturgeon (Acipenser sinensis), we conducted a transcriptome analysis of primary macrophages induced by IFN-γ using Illumina sequencing technology. We obtained 88,879 unigenes, with a total length of 93,919,393 bp, and an average length of 1,057bp. We identified 8,490 differentially expressed genes (DEGs) between the untreated and IFN-γ-treated macrophages, with 4,599 upregulated and 3,891 downregulated. Gene ontology (GO) analysis showed that 4,044 DEGs were enriched in the biological, cellular components, and molecular function categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) identified 278 immunity-related pathways enriched for the DEGs. According to the GO enrichment results, eight key immunity-related genes were screened for verification using qPCR. Results indicate that IFN-γ can activate macrophage Interferon Regulatory Factors (IRFs) and type I interferon (IFN-I), activate RIG-I-like and Toll-like receptor-related pathways, and improve the antiviral ability of macrophages in Chinese sturgeon.