Effects of hyperoxia during oxygen nanobubble treatment on innate immunity, growth performance, gill histology, and gut microbiome in Nile tilapia, Oreochromis niloticus.

Oxygen nanobubble (NB-O2) technology has been introduced to the aquaculture industry in recent years. This treatment usually results in a tremendously high level of dissolved oxygen (DO) in the water. However, little is known about the possible negative effects of hyperoxia due to NB-O2 treatment (hyper-NB-O2) on farmed fish. Here, we investigated i) the effect of short-term hyper-NB-O2 exposure (single treatment) on the innate immunity in Nile tilapia, Oreochromis niloticus, and ii) the effect of long-term hyper-NB-O2 exposure (26-day treatments) on survival, growth performance, gill histology, and gut microbiome in Nile tilapia. A single treatment with NB-O2 for 10 min in 50 L of water resulted in 24.2 ± 0.04 mg/L DO (approximately 2-3 × 107 nanoscale oxygen bubbles/mL). This treatment did not result in differences in expression of several immune-related genes (e.g., TNF-α, LYZ and HPS70) in various tissues (e.g., gill, head kidney, and spleen) compared to the non-treated control. Over a 26-day period of exposure, no significant differences were observed in survival and growth performance of the fish, but minor histological changes were occasionally noted on the gills. Analysis of the gut microbiome revealed a significant increase in the genera Bosea, Exiguobacterium, Hyphomicrobium, and Singulisphaera in the group receiving NB-O2. Moreover, no signs of "gas bubble disease" were observed in the fish throughout the duration of the experiment. Overall, these results suggest that both short- and long-term hyper-NB-O2 exposure appears to be benign and has no obvious adverse effects on fish.

Usefulness of the pancreas as a prime target for histopathological diagnosis of Tilapia parvovirus (TiPV) infection in Nile tilapia, Oreochromis niloticus.

Tilapia parvovirus (TiPV) is an emerging virus reportedly associated with disease and mortality in farmed tilapia. Although previous descriptions of histopathological changes are available, the lesions reported in these are not pathognomonic. Here, we report Cowdry type A inclusion bodies (CAIB) in the pancreas as a diagnostic histopathological feature found in adult Nile tilapia naturally infected with TiPV. This type of inclusion body has been well-known as a histopathological landmark for the diagnosis of other parvoviral infections in shrimp and terrestrial species. Interestingly, this lesion could be exclusively observed in pancreatic acinar cells, both in the hepatopancreas and pancreatic tissue along the intestine. In situ hybridization (ISH) using a TiPV-specific probe revealed the intranuclear presence of TiPV DNA in multiple tissues, including the liver, pancreas, kidney, spleen, gills and the membrane of oocytes in the ovary. These findings suggest that although TiPV can replicate in several tissue types, CAIB manifest exclusively in pancreatic tissues. In addition to TiPV, most diseased fish were co-infected with Streptococcus agalactiae, and presented with multifocal granulomas secondary to this bacterial infection. Partial genome amplification of TiPV was successful and revealed high nucleotide identity (>99%) to previously reported isolates. In summary, this study highlights the usefulness of pancreatic tissue as a prime target for histopathological diagnosis of TiPV in diseased Nile tilapia. This pattern may be critical when determining the presence of TiPV infection in new geographic areas, where ancillary testing may not be available. TiPV pathogenesis in this landmark organ warrants further investigation.

Pre-treatment of Nile tilapia (Oreochromis niloticus) with ozone nanobubbles improve efficacy of heat-killed Streptococcus agalactiae immersion vaccine.

Nanobubble technology has shown appealing technical benefits and potential applications in aquaculture. We recently found that treatment with ozone nanobubbles (NB-O3) activated expression of several immune-related genes leading to effective response to subsequent exposure to fish pathogens. In this study, we investigated whether pre-treatment of Nile tilapia (Oreochromis niloticus) with NB-O3 can enhance specific immune responses and improve efficacy of immersion vaccination against Streptococcus agalactiae. Spleen and head kidney of fish in the vaccinated groups showed a substantial upregulation in expression levels of pro-inflammatory cytokine genes (IL-1ß, TNF-α, IL-6) and immunoglobulin classes (IgM, IgD, IgT) compared with the unvaccinated control groups. The mRNA transcript of pro-inflammatory cytokine genes was greatest (approx. 2.8-3.3 folds) on day 7 post-vaccination, whereas the relative expression of immunoglobulin genes was greatest (approx. 3.2-4.1 folds) on day 21 post-immunization. Both systemic and mucosal IgM antibodies were elicited in vaccinated groups. As the result, the cumulative survival rate of the vaccinated groups was found to be higher than that of the unvaccinated groups, with a relative percent survival (RPS) ranging from 52.9 to 70.5%. However, fish in the vaccinated groups that received pre-treatment with NB-O3, bacterial antigen uptakes, expression levels of IL-1ß, TNF-α, IL-6,IgM, IgD, and IgT, as well as the specific-IgM antibody levels and percent survival, were all slightly or significantly higher than that of the vaccinated group without pre-treatment with NB-O3. Taken together, our findings suggest that utilizing pre-treatment with NB-O3 may improve the immune response and efficacy of immersion vaccination in Nile tilapia.

Characterization and protective effects of lytic bacteriophage pAh6.2TG against a pathogenic multidrug-resistant Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus).

Bacteriophage (phage) is considered as one of the alternatives to antibiotics and an environmentally friendly approach to tackle antimicrobial resistance (AMR) in aquaculture. Here, we reported isolation, morphology and genomic characterizations of a newly isolated lytic phage, designated pAh6.2TG. Host range and stability of pAh6.2TG in different environmental conditions, and protective efficacy against a pathogenic multidrug-resistant (MDR) Aeromonas hydrophila in Nile tilapia were subsequently evaluated. The results showed that pAh6.2TG is a member of the new family Chaseviridae which has genome size of 51,780 bp, encoding 65 putative open reading frames (ORFs) and is most closely related to Aeromonas phage PVN02 (99.33% nucleotide identity). The pAh6.2TG was highly specific to A. hydrophila and infected 83.3% tested strains of MDR A. hydrophila (10 out of 12) with relative stability at pH 7-9, temperature 0-40°C and salinity 0-40 ppt. In experimental challenge, pAh6.2TG treatments significantly improved survivability of Nile tilapia exposed to a lethal dose of the pathogenic MDR A. hydrophila, with relative per cent survival (RPS) of 73.3% and 50% for phage multiplicity of infection (MOI) 1.0 and 0.1, respectively. Phage treatment significantly reduced the concentration of A. hydrophila in both water and fish body. Interestingly, the surviving fish from A. hydrophila challenged groups provoked specific antibody (IgM) against this bacterium. In summary, the findings suggested that the lytic phage pAh6.2TG is an effective alternative to antibiotics to control MDR A. hydrophila in tilapia and possibly other freshwater fish.

Ozone nanobubble modulates the innate defense system of Nile tilapia (Oreochromis niloticus) against Streptococcus agalactiae.

Ozone nanobubble (NB-O3) is a promising technology for improving dissolved oxygen and reducing bacterial concentration in aquaculture systems. Here, we investigated the effects of NB-O3 on the innate immunity of fish by monitoring the expression levels of nonspecific immune-related genes (IL-1ß, IL-2ß, TNF-α), heat-shock protein genes (HSP70, HSP90-α), and a bacteriolytic enzyme, C-type lysozyme, gene (LYZ) post-treatment with this technology. Following exposure to NB-O3, the different tissues of Nile tilapia (Oreochromis niloticus) were collected over time for quantitative real-time PCR (qPCR) analysis. The expression of all the genes evaluated in the gills, the head kidney, and the spleen of the NB-O3 treated group was significantly up-regulated compared to that in the untreated control group. The expression levels were the highest (approx. 2 to 4-fold) at 15 min and 3 h post-exposure and then decreased from 6 to 24 h. These findings suggested that NB-O3 could switch on the innate immunity genes of Nile tilapia. Thus, we hypothesized that the NB-O3-immune-activated fish would respond more effectively to subsequent bacterial infections, thereby improving survivability compared to that of untreated fish. To test this hypothesis, 3 h post NB-O3 exposed fish and unexposed fish were challenged with a lethal dose of Streptococcus agalactiae. Interestingly, the survival rate of the NB-O3 group was significantly higher than that of the non-treated controls, with a relative percent survival (RPS) of 60-70%. Together, these findings indicate, for the first time, that NB-O3 may trigger the nonspecific defense system of the fish, thereby improving fish survivability during subsequent bacterial infections. This research identified another potential benefit of NB-O3 in aquaculture for preventing infectious bacterial diseases.