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The present study was conducted to assess the protective efficacy of a trivalent oral vaccine containing chitosan-PLGA encapsulated inactivated viral haemorrhagic septicemia virus (VHSV), Streptococcus parauberis serotype I and Miamiensis avidus antigens, followed by its oral (incorporated in feed) administration to olive flounder (Paralichthys olivaceus) fingerlings for a period of 15-consecutive days. After 35 days of initial vaccination, three separate challenge studies were conducted at the optimal temperature of the targeted pathogens using an intraperitoneal injection route. RPS analysis revealed moderate protection in the immunized group against all the three pathogens viz., VHSV (53.30% RPS), S. parauberis serotype-I (33.30% RPS), and M. avidus (66.75% RPS), as compared to the respective non-vaccinated challenge (NVC) control group. In addition, the immunized fish demonstrated significantly (p 0.05) higher specific antibody titres in serum and significant (p 0.05) upregulation in the transcript levels of immune genes of Igs (IgM, IgT, pIgR), TLRs (TLR 2, TLR 7), cytokines (IL-1β, IL-8) and complement pathway (C3) in the mucosal and systemic tissues than those of NVC control fish, suggesting orchestration of pathogen-specific host immune responses thereby favouring its combativeness against the three pathogens. The expression dynamics of IFN-γ, Mx, caspase 3 genes post VHSV challenge; IFN-γ, TLR 2, caspase 1 genes post S. parauberis serotype I challenge and CD-8α, IL-10, TNF-α genes post M. avidus challenge further substantiates the efficacy of the vaccine in stimulating antiviral, antibacterial and antiparasitic immune responses in the host resulting in their better survival. The findings from the present study reflect that the formulated trivalent oral vaccine incorporating VHSV, S. parauberis serotype I and M. avidus antigens can be a promising prophylactic strategy to prevent the associated disease outbreaks in olive flounder.

Edwardsiella piscicida is a facultative intracellular pathogen that causes hemorrhagic septicemia and haemolytic ascites disease in aquaculture fish. During bacterial infection, macrophages and neutrophils are the first line of host innate immune system. However, the role of neutrophils in response to E. piscicida infection in vivo remains poorly understood. Here, through developing an immersion infection model in the 5 day-post fertilization (dpf) zebrafish larvae, we found that E. piscicida was mainly colonized in intestine, and resulted into significant pathological changes in paraffin sections. Moreover, a dynamic up-regulation of inflammatory cytokines (TNF-α, IL-1β, GCSFb, CXCL8 and MMP9) was detected in zebrafish larvae during E. piscicida infection. Furthermore, a significant recruitment of neutrophils was observed during the E. piscicida infection in Tg(mpx:eGFP) zebrafish larvae. Thus, we utilized the CRISPR/Cas9 system to generate the neutrophil-knockdown (gcsfr-/- crispants) larvae, and found a comparative higher mortality and bacterial colonization in gcsfr-/- crispants, which reveals the critical role of fish neutrophils in bacterial clearance. Taken together, our results developed an effective E. piscicida immersion challenge model in zebrafish larvae to clarify the dynamic of bacterial infection in vivo, which would provide a better understanding of the action about innate immune cells during infection.

Hemorrhagic septicemia of mandarin fish (Siniperca chuatsi) was mainly caused by Aeromonas hydrophila which was an opportunistic pathogen. In recent years, the disease has caused tremendous economic loss with high morbidity and mass mortality in the mandarin fish breeding industry. Histopathological analysis and the immune related gene expression profiles of mandarin fish (S. chuatsi) infected with A. hydrophila were investigated in this study. Transmission electron microscopy (TEM) images showed that the cells of A. hydrophila densely covered with a mass of fimbriae. Histopathological analysis revealed that inflammation, vacuolization and extensive necrosis existed in the gill, liver, spleen and head kidney of the diseased fish. Quantitative real-time PCR was performed to measure mRNA expression levels for six immune related genes in mandarin fish after A. hydrophila infection. The transcriptional analysis of these immune related genes demonstrated that the expression levels of major histocompatibility complex class II (MHC II), T cell receptor α (TCRα), tumor necrosis factor α (TNFα), CC chemokine 3, interleukin 8 (IL-8) and Hepcidin were strongly up-regulated in spleen and head kidney of mandarin fish post-infection. These results will contribute to further study on the pathogenesis and host defensive system in A. hydrophila infection.

Pattern recognition receptor (PRR) toll-like receptors (TLRs), antiviral agent interferon (IFN) and the effector IFN stimulated genes (ISGs) play pivotal role in antiviral innate immunity of a host. The present in-vivo experiment was conducted to investigate the role of these innate immune factors in early phase as well as during recovery of viral haemorrhagic septicaemia virus (VHSV) infection by quantitative real-time reverse transcriptase polymerase chain reaction. A less lethal VHSV infection was generated in olive flounder (Paralichthys olivaceus) and was sampled at 3, 6, and 12h post infection (hpi), and 1, 2, 4, and 7 days post infection (dpi). At 3 hpi, the VHSV N gene was detected in three out of five fish and all five fish showed a relative fold increase of TLR 2, TLR 7, interleukin 8 (IL 8), IFN regulatory factor 3 (IRF 3), IRF 7, and ISG 15. Viral copies rapidly increased at 12 hpi then remained high until 2 dpi. When viral copy numbers were high, a higher expression of immune genes IL 1β, IRF 3, IRF 7, Type I IFN, ISG 15 and Mx was observed. Viral copies were drastically reduced in 4 and 7 dpi fish, and also the immune response was considerably reduced but remained elevated, except for ISG 15 which found equal to control in 7 dpi fish. A high degree of correlation was observed between immune genes and viral copy number in each of the sampled fish at 12 hpi. A fish with ascites sampled at 7 dpi displayed high viral copy but under-expressed immune genes except for Mx. When viral copies were high at 1 and 2 dpi, both TLR 2 and TLR 7 were down-regulated, perhaps indicating immune suppression by the virus. The quick and prolonged elevated expression of the immune genes indicates their crucial role in survival of host against VHSV.

A recombinant viral hemorrhagic septicemia virus (rVHSV-ΔNV-EGFP) that has enhanced green fluorescent protein (EGFP) gene instead of NV gene was previously generated using reverse genetics technology. In this study, potential of the rVHSV-ΔNV-EGFP to be used as a live oral vaccine candidate was assessed. The presence of the recombinant virus in internal organs of orally administered olive flounder (Paralichthys olivaceus) was analyzed by semi-quantitative RT-PCR. Although the recombinant VHSV-specific band was detected only when the number of PCR cycle was increased to 35, the band was detected from internal organs, such as kidney, spleen, and liver of fish that were reared at either 15 °C or 20 °C till even 20 days, suggesting that a few orally administered rVHSV-ΔNV-EGFP might be transported to internal organs, and might keep weak replication ability in the organs. VHSV-neutralizing activity was induced by oral immunization of olive flounder with the NV gene knock-out recombinant VHSV not only in skin and intestinal mucus but also in serum, suggesting that mucosal and systemic adaptive immune responses were elicited by oral immunization. In challenge experiment, groups of fish immunized with 10⁴, 10⁵, and 2 × 10⁵ PFU of rVHSV-ΔNV-EGFP/fish showed 25%, 50%, and 70% of relative percent survival (RPS), respectively. The RPSs were elevated to 60%, 75%, and 90% by a boost immunization in fish boost immunized with 10⁴, 10⁵, and 2 × 10⁵ PFU of rVHSV-ΔNV-EGFP, respectively. The cumulative mortality of fish in the control groups was 100%. Conclusionly, the present results demonstrate that the NV gene knock-out recombinant VHSV administered orally to olive flounder can induce dose- and boosting-dependent VHSV-neutralizing antibody in mucus and serum, and can provide a high protection in olive flounder against a virulent VHSV challenge.

Edwardsiella tarda is a gram-negative pathogen for hemorrhagic septicemia in a broad range of hosts. The type VI secretion system (T6SS) has recently been dissected in E. tarda to secrete EvpC, EvpI and a novel effector protein EvpP. In this study, sequencing and genetic alignments showed that evpP genes from different E. tarda isolates were highly similar and an evpP homolog was also found in Aeromonas hydrophila 0865 isolated from a diseased eel, suggesting the possible lateral gene transfer of evpP or the whole T6SS gene island. With reporter strains carrying gfp gene fused to the evpP promoter region, flow cytometric analysis revealed that transcription of evpP was positively regulated by either the two-component system EsrA-EsrB in E. tarda or the iron concentration in media. Compared with the parental strain, in-frame deletion of evpP in E. tarda EIB202 led to the significantly increased 50% lethal doses in zebrafish (Danio rerio) and Japanese flounder (Paralichthys olivaceus), decreased hemolytic activities, failure to adhere to mucus and reduced serum resistance, and complementation of an intact evpP gene restored these phenotypes in the evpP mutant. Investigation of infection kinetics indicated that the evpP deletion mutant was unable to proliferate in vivo, particularly in immune organs of fish. Moreover, the evpP deletion mutant exhibited incapacity to internalize in EPC cell model in vitro, demonstrating that EvpP in T6SS plays critical roles for invasion mechanism of E. tarda and merits as potential target for attenuated live vaccine construction.

Japanese flounder, Paralichthys olivaceus juveniles were vaccinated against viral hemorrhagic septicemia (VHS) by intramuscular injection of 10 microg of a plasmid DNA vector which encodes the viral hemorrhagic septicemia virus (VHSV) glycoprotein (G) gene under the control of the cytomegalovirus promoter. Experimental challenge of two viral doses (1 x 10(2) TCID50 and 1 x 10(3) TCID50) one month post-vaccination revealed that the G gene was able to induce protective immunity against VHS and this lasted until 21 days after the challenge. The VHSV G-protein gene DNA vaccine had a high protective efficiency, giving relative percentage survival (RPS) values of at least 93%. The defense mechanisms activated by the DNA vaccine were further elucidated by microarray analysis. Non-specific immune response genes such as NK, Kupffer cell receptor, MIP1-alpha and Mx1 protein gene were observed to be up-regulated by the VHSV G-protein DNA vaccine at 1 and 3 days post-immunization. Also, specific immune-related genes including the CD20 receptor, CD8 alpha chain, CD40 and B lymphocyte cell adhesion molecule were also up-regulated during that time. We observed significant up-regulation of some immune-related genes that are necessary for antiviral defense. Significant up- and/or down-regulation of unknown genes was also observed upon DNA vaccination. Our results confirm previous reports that the VHSV G gene elicits strong humoral and cellular immune responses which may play a pivotal role in protecting the fish during virus infections.

Aeromonas hydrophila is a Gram negative bacteria widely distributed in aquatic environments. It is an opportunistic pathogen for fish and terrestrial animals including mammals. In fish it causes motile hemorrhagic septicemia leading to a high mortality and economic losses in aquaculture. In humans it is an emerging pathogen provoking gastroenteritis, septicemia, and skin and soft tissue infections. Some clinical conditions like cancer, hepatic diseases, diabetes and trauma increase the risk to develop a fatal A. hydrophila infection. The pathogenicity of A. hydrophila is multifactorial depending on several virulence factors, including surface polysaccharides (capsule, lipopolysaccharide, and glucan), S-layers, iron-binding systems, exotoxins and extracellular enzymes, secretion systems, fimbriae and other nonfilamentous adhesins, motility and flagella