Saekil Yun

Effect of guava leaves on the growth performance and cytokine gene expression of Labeo rohita and its susceptibility to Aeromonas hydrophila infection

The present study aimed to evaluate the effects of Psidium guajava L. (guava) leaves on the growth and immune response of the fish species Labeo rohita and its susceptibility to Aeromonas hydrophila infection. Diets containing five different concentrations of guava leaves (0% [basal diet], 0.1% [G1], 0.5% [G2], 1% [G3], and 1.5% [G4]) were fed to fish (average weight: 11.1 g) for 60 days. Various growth and immune parameters were examined 60 days post-feeding. Fish were challenged with A. hydrophila at the end of the trial, and mortalities were recorded over 15 days post-infection. We found that growth parameters such as percent weight gain (657.61 ± 9.74) and specific growth rate (3.37 ± 0.021) were significantly higher in G2 group than in the control (P < 0.05). Among the immune parameters examined, lysozyme levels (79.5 ± 5.1 U mL(-1)), leukocyte phagocytic activity (52 ± 4.3%), and alternative complement pathway activity (ACP) (186.1 ± 8.3 U mL(-1)) were significantly high (P < 0.05) in G2 fed group; there was, however, no significant effect of guava leaves at any concentration on plasma IgM level. Of the cytokine-related genes examined, interleukin-1beta (IL-1β) and tumour necrosis factor-alpha (TNF-α) were up-regulated in the head-kidney, intestine, and hepatopancreas of fish fed experimental diets, and expression was significantly higher in G2 and G3 than in the control group. In contrast, gene expression of IL-10, transforming growth factor-beta (TGF-β), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and transcription factor nuclear factor-κB (NF-κB) were down-regulated in the treatment groups. Moreover, fish fed the G2 diet exhibited a significantly higher post-challenge survival rate (66.66%). Collectively, these results suggest that dietary supplementation with guava leaves (at 0.5% concentration) could promote growth performance and strengthen immunity of L. rohita. Guava leaves therefore represent a promising feed additive for carps in aquaculture.

Effect of cellular products of potential probiotic bacteria on the immune response of Labeo rohita and susceptibility to Aeromonas hydrophila infection.

In the present study, the immunological efficacy of cellular components from the potential probiotic bacteria Bacillus subtilis VSG1, Pseudomonas aeruginosa VSG2, and Lactobacillus plantarum VSG3 was evaluated in Labeo rohita fingerlings. Fish were immunized intraperitoneally with 0.1 mL phosphate-buffer solution (PBS) containing 0.1 mg of any of the following cellular components: intercellular products (ICPs) of VSG1 (BS-ICPs), heat-killed whole cell products of VSG2 (PA-HKWCPs), or ICPs of VSG3 (LP-ICPs). Fish injected with 0.1 mL PBS served as the control. Various immunological parameters, including the expression of immune-related genes, were measured 14 and 21 days post-immunization. The fish were challenged with Aeromonas hydrophila and mortality was recorded up to 21 days post-infection. The results revealed that administration of cellular components significantly increased the activity of serum lysozyme and the alternative complement pathway, phagocytosis, and respiratory bursts throughout the experimental period. Total serum protein, albumin, and globulin levels were significantly higher in experimental groups than in the control up to 14 days post-immunization, and decreased thereafter. With respect to immune-related genes, IL-1β, COX-2, iNOS, and IL-10 were highly (P < 0.05) up-regulated in fish immunized with cellular components, compared to the control. The expression of TNF-α and NF-κB was up-regulated in immunized fish up to 14 days post-immunization. Interestingly, fish immunized with LP-ICPs exhibited a significantly higher post-challenge relative percent survival (83.32%), followed by PA-HKWCPs (66.66%), and BS-ICPs (50%). These results indicate that cellular components of probiotic bacteria can influence immune responses, enhance disease protection, and stimulate immune-related gene expression in rohu. Hence, these cellular components may be useful as adjuvants for vaccines in aquaculture.

Chlorophytum borivilianum Polysaccharide Fraction Provokes the Immune Function and Disease Resistance of Labeo rohita against Aeromonas hydrophila

The present study aimed to investigate the effects of Chlorophytum borivilianum polysaccharide (CBP), as a dietary supplement administered at varying concentrations with feed (basal diet), on various cytokine-related responses in Labeo rohita fingerlings. Immune parameters and immune-related gene expressions were measured at 3rd, 4th, and 5th week after feeding. The results revealed that dietary administration of CBP at 0.2% and 0.4% for 4 weeks significantly upregulated serum lysozyme and phagocytic activity. Complement C3 and respiratory burst activity (RBA) were significantly higher after 4 weeks of CBP feeding. The immune related genes IL-8, IL-1β, TNF-α, and iNOS were downregulated (P < 0.05) in groups with 0.2% and 0.4% CBP supplemented diets at week 4. Expression of anti-inflammatory cytokines (IL-10 and TGF-β) was also downregulated (P < 0.5) after 4 weeks of feeding with 0.2% to 0.8% CBP. However, five weeks of CBP administration had no significant effect on immune gene expression, except TNF-α and IL-8. Fish fed with 0.4% CBP for 4 weeks showed maximum resistance against Aeromonas hydrophila (73.3% survival) compared to control. From these results, we recommend that CBP administration at 0.4% for 4 weeks could effectively improve immune response and disease resistance in L. rohita.

Effect of dietary leucine on the growth parameters and expression of antioxidant, immune, and inflammatory genes in the head kidney of Labeo rohita fingerlings

The present investigation evaluated the effects of dietary leucine (Leu) on growth performance, head kidney antioxidant status, and gene expression in Labeo rohita juveniles. Fish were fed with six isonitrogenous diets containing graded levels of Leu at 0.75 (control), 1.7, 3.2, 4.6, 6.3, and 7.6 g Leu kg(-1) of feed for 8 weeks. Compared with the control group, appropriate Leu supplementation significantly enhanced the percent weight gain (PWG), feed intake (FI), and protein efficiency ratio (PER) (P<0.05) but decreased the plasma ammonia content (PAC) (P<0.05). Similarly, optimal Leu supplementation stimulated head kidney glutathione (GSH) content, and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities as compared to the control group; however, a reverse trend was observed in malondialdehyde (MDA) content. Further, relative gene-expression levels of lysozyme, complement C3, β-microglobulin, immunoglobulin-M, SOD, GPx, nuclear factor erythroid 2-related factor 2 (Nrf2), natural killer-cell enhancing factor β (NKEF-β), and toll-like receptor-22 (TLR22) in the head kidney were enhanced (P<0.05) at leucine levels of 4.6 g kg(-1) of feed. Conversely, the mRNA levels of tumour necrosis factor-α (TNF-α), Kelch-like-ECH-associated protein 1 (Keap1), and interleukin 1β (IL-1β) in head kidney were down-regulated by Leu supplementation. Collectively, our results revealed that appropriate Leu supplementation improved fish growth and antioxidant capacity, and regulated the mRNA levels of related signalling molecules in L. rohita juveniles. Based on the quadratic regression analysis of PWG, PER, and PAC, the optimum dietary leucine requirements of L. rohita juveniles were estimated to be 4.7, 4.5, and 4.8 g kg(-1) of feed.

Immunomodulatory Effects of a Bioactive Compound Isolated from Dryopteris crassirhizoma on the Grass Carp Ctenopharyngodon idella

In the present study, we investigated effects of compound kaempferol 3-a-L-(4-O-acetyl)rhamnopyranoside-7-a-L-rhamnopyranoside (SA) isolated from Dryopteris crassirhizoma during immune-related gene expression in Ctenopharyngodon idella head kidney macrophages (CIHKM). The expression of immune-related genes (IL-1β, TNF-α, MyD88, and Mx1) were investigated using real-time PCR at 2 h, 8 h, 12 h, and 24 h after incubation with 1, 10, and 50 μg mL−1 of SA. Furthermore, fish were injected intraperitoneally with 100 μL of SA, and immune parameters such as lysozyme activity, complement C3, SOD, phagocytic activity, and IgM level were examined at 1, 2, and 3 weeks after injection. The differential expression of cytokines was observed after exposure to SA. IL-1β genes displayed significant expression at 2 and 8 h after exposure to 1–10 μg mL−1 of SA. SA also induced gene expression of cytokines such as MyD88, Mx1, and TNF-α. Furthermore, enhanced immune parameters in grass carp confirmed the immunomodulatory activity of SA. Interestingly, this compound has no toxic effect on CIHKM cells as tested by MTT assay. In addition, fish immunised with 10 μg mL−1 of SA exhibited maximum resistance against Aeromonas hydrophila infection. These results suggest that SA has the potential to stimulate immune responses in grass carp.

Efficacy of PLGA microparticle-encapsulated formalin-killed Aeromonas hydrophila cells as a single-shot vaccine against A. hydrophila infection

Control and prevention of disease is a high priority in aquaculture, and vaccination is important to prevent outbreaks. Here, poly(d,l-lactide-co-glycolic acid) (PLGA) microparticles (MPs) approximately 36μm in diameter were used to encapsulate and deliver Aeromonas hydrophila formalin-killed cells (FKC) as an antigen, and the innate and adaptive immune responses of cyprinid loaches and common carp were assessed following vaccination. The antigen was confirmed to be well encapsulated by scanning electron microscopy analysis of PLGA MP sections. Blood and head kidney specimens were collected and analyzed for bacterial agglutination activity and relative mRNA expression of immune-related genes (IL-1β, IL-10, TNF-α, lysozyme C, TGF-β, and IgM) at 2, 4, 6, and 8weeks post vaccination (wpv). For both fish species, the curve of antibody titer over time was shallower in the PLGA group than the FKC group. These titers in loaches and carp were very similar in the two vaccination groups until 8 and 6 wpv, respectively, but differences were subsequently noted in both species until the end of experiment. Loaches and carp were then challenged with A. hydrophila at 12 and 20 wpv, and 10 and 14 wpv, respectively, and relative survival rates were calculated. For both species, the PLGA groups demonstrated higher survival rates at all time points. Relative expression of IL-1β and TNF-α mRNA was significantly upregulated in the PLGA group at 2 and 4 wpv. Moreover, PLGA-MP vaccination increased relative mRNA levels of lysozyme C and IgM, which were significantly higher than those observed with FKC treatment at 2 wpv and 4, 6, and 8 wpv, respectively. In conclusion, PLGA-MP vaccines have the potential to induce longer and more potent immune responses than FKCs alone, and protect both cyprinid loaches and common carp with greater efficiency.

Effects of algal toxin okadaic acid on the non-specific immune and antioxidant response of bay scallop (Argopecten irradians)

&NA; Okadaic acid (OA) is produced by dinoflagellates during harmful algal blooms and is a diarrhetic shellfish‐poisoning (DSP) toxin. This toxin is particularly problematic for bivalves that are cultured for human consumption. This study aimed to reveal the effects of exposure to OA on the non‐specific immune responses of bay scallop, Argopecten irradians. Various immunological parameters (superoxide dismutase (SOD), acid phosphatase (ACP), alkaline phosphatase (ALP), lysozyme activities, and total protein level) were assessed in the hemolymph of bay scallops at 3, 6, 12, 24, and 48 h post‐exposure (hpe) to different concentrations (50, 100, and 500 nM) of OA. Moreover, the expression of immune system‐related genes (MnSOD, PrxV, PGRP, and BD) was also measured. Results showed that SOD and ACP activities were decreased between 12 and 48 hpe. The ALP, lysozyme activities, and total protein levels were also modulated after exposure to different concentrations of OA. The expression of immune‐system‐related genes was also assessed at different time points during the exposure period. Overall, our results suggest that the exposure to OA had negative effects on the antioxidant and non‐specific immune responses, and even disrupted the metabolism of bay scallops, making them more vulnerable to environmental stress‐inducing agents; they provide a better understanding of the response status of bivalves against DSP toxins. HighlightsEffect of okadaic acid (OA) on the bay scallop was investigated.Cytokine responses of bay scallop were altered by OA.Immune and antioxidant responses to OA were time and concentration‐dependent.

Effect of the Algaecide Palmitoleic Acid on the Immune Function of the Bay Scallop Argopecten irradians

Palmitoleic acid (PA), an algicidal compound, is used against the toxin producing dinofagelate Alexandrium tamarense, however, its impact on the edible bay scallop (Argopecten irradians) is still unclear. Therefore, we investigated the impacts of effective algicidal concentrations (20, 40, and 80 mg/L) of PA on immune responses in A. irradians. Various immune parameters including acid phosphatase (ACP) activity, superoxide dismutase (SOD), lysozyme, phagocytic activity, total protein, malondialdehyde (MDA) level, and reactive oxygen species (ROS) production and the expression of immune-related genes (PrxV, CLT-6, MT, and BD) were measured at 3, 6, 12, 24, and 48 h post-exposure (hpe) to PA. Lysozyme activity was lower in scallops at 12–48 hpe to 80 mg/L. SOD, ACP activity, ROS production, the total protein, and MDA level was higher at 12 to 48 hpe with different concentrations of PA. Phagocytic activity increased at 6–12 hpe to 40–80 mg/L of PA, but decreased at 24–48 hpe. The expressions of genes PrxV, CLT-6, MT and BD down-regulated at 3 hpe were observed, while differential expressions from 6–48 hpe with different concentrations of PA. The present study demonstrated that immersing A. irradians in PA at effective concentrations could result in differential effects on non-specific immune responses and expressions of immune-related genes.

Marine Toxin Okadaic Acid Affects the Immune Function of Bay Scallop (Argopecten irradians).

Okadaic acid (OA) is produced by dinoflagellates during harmful algal blooms and is a diarrhetic shellfish poisoning toxin. This toxin is particularly problematic for bivalves that are cultured for human consumption. This study aimed to reveal the effects of exposure to OA on the immune responses of bay scallop, Argopecten irradians. Various immunological parameters were assessed (total hemocyte counts (THC), reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), lactate dehydrogenase (LDH), and nitric oxide (NO) in the hemolymph of scallops at 3, 6, 12, 24, and 48 h post-exposure (hpe) to different concentrations of OA (50, 100, and 500 nM). Moreover, the expression of immune-system-related genes (CLT-6, FREP, HSP90, MT, and Cu/ZnSOD) was also measured. Results showed that ROS, MDA, and NO levels and LDH activity were enhanced after exposure to different concentrations of OA; however, both THC and GSH decreased between 24–48 hpe. The expression of immune-system-related genes was also assessed at different time points during the exposure period. Overall, our results suggest that exposure to OA had negative effects on immune system function, increased oxygenic stress, and disrupted metabolism of bay scallops.

Effects of intracellular products of Bacillus subtilis VSG1 and Lactobacillus plantarum VSG3 on cytokine responses in the head kidney macrophages of Labeo rohita.

The efficiency of intracellular products (ICPs) of the probiotics Bacillus subtilis VSG1 and Lactobacillus plantarum VSG3 in stimulating cytokine responses in the head kidney (HK) macrophages of Labeo rohita was investigated. The HK macrophages were incubated with ICPs and lipopolysaccharide (LPS), and the responses of cytokine genes, namely interleukin-10 (IL-10), IL-1β, IL-12p35, IL-12p40, IL-18, tumour necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB), cyclo-oxygenase-2 (COX-2), interferon-1 (IFN-1), and IFN-γ were assessed by quantitative real-time PCR (qRT-PCR) at 2, 6, 12, 24, and 48 h post-stimulation (hps). Among the proinflammatory cytokines, a strong increase in the gene expression of IL-1β and TNF-α was displayed mainly at 2-6 hps with ICPs, as compared to that of the positive control (LPS) or the negative control (PBS) (P < 0.05). However, COX-2 and NF-κB showed higher expression at 2 and 24 hps, and 6-24 hps with ICPs, respectively. Antiviral cytokines IFN-1 and IFN-γ displayed strong expressions (P < 0.05) at 6-12 hps, and 12-24 hps with ICPs, respectively. Upregulation of the anti-inflammatory cytokine, IL-10, was recorded at 6-24 hps with ICPs, as compared to that controls. Expressions of cell-mediated immune factor genes (IL-12p35, IL-12p40, and IL-18) were also significantly upregulated at different time points, except 48 hps, in HK macrophages stimulated with ICPs. Furthermore, enhanced cellular (phagocytic activity and nitroblue tetrazolium assay) and humoral (lysozyme) immune parameters in stimulated cells confirmed the induction of the inflammatory response. Therefore, the results of this in vitro study indicate that the ICPs of B. subtilis VSG1 or L. plantarum VSG3 have great potential for stimulating the cytokine responses in fish, and are thereby potential immunostimulants to fish. Further studies could be conducted to explore its suitability as an adjuvant vaccine in aquaculture.