Prapansak Srisapoome

Efficacy of viable Bacillus pumilus isolated from farmed fish on immune responses and increased disease resistance in Nile tilapia (Oreochromis niloticus): Laboratory and on-farm trials

Abstract Applications of viable Bacillus pumilus AQAHBS01 isolated from Nile tilapia farms as probiotics were studied in both laboratory and farm conditions. In the laboratory, feeding fish (approximately 50 g) with feed containing viable B. pumilus at concentrations of 1 × 107‐109 colony forming units (CFU)/kg elevated fish immune responses, as indicated by their phagocytic activity and superoxide anion levels, and led to more effective disease resistance against Streptococcus agalactiae. However, when these concentrations were applied to Nile tilapia cultures growing in cage culture systems, only B. pumilus AQAHBS01 at concentrations of 1 × 108 and 109 CFU/kg diet could effectively enhance disease resistance against S. agalactiae during the critical period of early to middle April when the temperature reached 33 °C, whereas control fish and fish that consumed B. pumilus AQAHBS01 at concentrations of 1 × 107 CFU/kg showed very rapid streptococcosis‐induced mortality. However, in late April, massive levels of organic matter‐containing water flowed into the culture areas, causing all fish groups to become infected with Flavobacterium columnare. Moreover, the dissolved oxygen levels in the river declined to critical levels of approximately 1.0–1.5 mg/L, causing anorectic effects in fish for long periods of time. This effect may have also gradually killed the cultured fish until the end of the experiment. This information strongly demonstrates the effective application of B. pumilus as a probiotic for streptococcosis resistance in both laboratory and field culture conditions. For on‐farm cage culture practices, however, fluctuations in water quality remain a significant constraint for probiotic application, as they usually induce negative effects on fish health. This decline in health makes fish more fragile and more susceptible to problems from both infectious and non‐infectious diseases, which farmers must consider carefully. HighlightsViable Bacillus pumilus AQAHBS01 isolated from Nile tilapia farms as probiotics were studied in both laboratory and farm conditions.Application B. pumilus AQAHBS01 in feed significantly elevate immune parameters of Nile tilapia in laboratory scale.1×107‐1×109 CFU B. pumilus AQAHBS01/kg diet enhances Streptococcus agalactiae resistance of Nile tilapia in the lab scale.1×108–1×109 CFU B. pumilus AQAHBS01/kg diet increases S. agalactiae resistance of Nile tilapia in on‐farmed cageculture.

Analysis of hematologic alterations, immune responses and metallothionein gene expression in Nile tilapia (Oreochromis niloticus) exposed to silver nanoparticles

Abstract In this study, Nile tilapia (Oreochromis niloticus) fingerlings were used as a model to examine acute and chronic toxicity of silver nanoparticles (AgNP). Expression levels of metallothionein (MT) transcripts in fish exposed to 0, 1 or 100 mg AgNP/kg fish were investigated by quantitative real-time RT-PCR. The results showed MT expression levels were significantly decreased 0.3–0.7-fold in the liver and spleen of fish exposed to 1 or 100 mg AgNP/kg after 6–48 h. In contrast, during this period, MT mRNA expression levels were increased 2–3-fold in the head kidney of the fish exposed to either level of AgNP. Investigations of effects of AgNP on the fish immune responses and hematological parameters revealed that phagocytic activity, the amount of red blood cells (RBC) and the percent hematocrit (%Hct) in fish exposed to AgNP were decreased significantly 1 week after exposure, especially those exposed to 100 mg AgNP/kg. Fish immunized with Streptococcus agalactiae vaccine and simultaneously exposed to 100 mg AgNP/kg presented decreased antibody titers during the early phase. Lastly, a challenge test showed that vaccinated fish exposed to AgNP, regardless of concentration, remained protected against S. agalactiae infection, with a lower mortality (10–20%) compared to 70% in control fish. These findings indicated that expression patterns of the MT gene in the liver, spleen and head kidney at different timepoints could be used to assess acute and chronic exposure of Nile tilapia to AgNP. Additionally, changes in innate immune responses and hematological parameters in fish may prove useful for evaluation of AgNP toxicity. Data obtained in this study strongly support the use of Nile tilapia as an animal model to potentially serve as a bio-indicator of environment contamination caused by AgNP.

Molecular serotyping, virulence gene profiling and pathogenicity of Streptococcus agalactiae isolated from tilapia farms in Thailand by multiplex PCR

This study aimed to biotype Streptococcus agalactiae isolated from tilapia farms in Thailand based on molecular biotyping methods and to determine the correlation between the serotype and virulence of bacteria. In addition to a biotyping (serotyping) technique based on multiplex PCR of cps genes, in this study, we developed multiplex PCR typing of Group B streptococcus (GBS) virulence genes to examine three clusters of virulence genes and their correlation with the pathogenicity of S. agalactiae. The epidemiology of S. agalactiae in Thailand was analysed to provide bacterial genetic information towards a future rational vaccine strategy for tilapia culture systems.

Molecular characterization of Galectin-8 from Nile tilapia (Oreochromis niloticus Linn.) and its response to bacterial infection.

Galectins belong to the family of galactoside-binding proteins and play a major role in the immune and inflammatory responses of vertebrates and invertebrates. The galectin family is divided into three subtypes based on molecular structure; prototypes, chimera types, and tandem-repeated types. We isolated and characterized the cDNA of galectin-8 (OnGal-8) in Nile tilapia (Oreochromis niloticus). OnGal-8 consisted of a 966 bp open reading frame (ORF) that encoded a 321 amino acid protein (43.47 kDa). Homology and phylogenetic tree analysis suggested the protein was clustered with galectin-8s from other animal species and shared at least 56.8% identity with salmon galectin-8. Structurally, the amino acid sequence included two distinct N- and C- terminus carbohydrate recognition domains (CRDs) of 135 and 133 amino acids, respectively, that were connected by a 39 amino acid polypeptide linker. The N- and C-CRDs contained two conserved WG-E-I and WG-E-T motifs, suggesting they have an important role in mediating the specific interactions between OnGal-8 and saccharide moieties such as β-galactoside. The structure of OnGal-8 was characterized by a two-fold symmetric pattern of 10-and 12-stranded antiparallel ß-sheets of both N- and C-CRDs, and the peptide linker presumably formed a random coil similar to the characteristic tandem-repeat type galectin. The expression of OnGal-8 in healthy fish was highest in the skin, intestine, and brain. Experimental challenge of Nile tilapia with S. agalactiae resulted in significant up-regulation of OnGal-8in the spleen after 5 d. Our results suggest that OnGal-8 is involved in the immune response to bacterial infection.

The production of anti-Vibrio harveyi egg yolk immunoglobulin and evaluation of its stability and neutralisation efficacy

In order to replace the antibiotic treatment for control of Vibrio harveyi, a causal agent of luminous disease in Black tiger shrimp, anti-V. harveyi IgY was produced and showed its potential in our preliminary study. However, for further use as feed additive, the IgY stability should be evaluated. The titre of specific IgY was enhanced with an immunostimulant, C-phosphate guanosine oligodeoxynucleotide (CpG-ODN). IgY was stable at natural pH while the activity was decreased below pH 4 or above pH 10. The supplementation of 30% sorbitol significantly enhanced the IgY stability at tested temperatures in excess of 70°C. The activity of IgY remained at 9 and 94% after 4 h incubation in simulated gastric and intestinal fluids, respectively. A storage temperature at −20°C was found to be the best condition. IgY at the concentrations of 1, 5 and 10 mg/ml efficiently inhibited V. harveyi in vitro within 24 h after exposure.

Immunostimulation and yellow head virus (YHV) disease resistance induced by a lignin-based pulping by-product in black tiger shrimp (Penaeus monodon Linn.)

ABSTRACT Yellow head virus (YHV) is classified as one of the most serious pathogens causing a harmful disease in many penaeids, especially black tiger shrimp (Penaeus monodon), with high economic loss. To determine a potent and practical prophylactic strategy for controlling this disease, the toxicity of the by‐product kraft lignin and its ability to control severe YHV infection were investigated in juvenile black tiger shrimp (15.9 ± 1.2 g body weight). The median lethal dosage at 96 h (96‐hrs LD50) of lignin in shrimp was 297 mg/L. Lignin was further added to shrimp diets via top‐dressing to assess its ability to elicit immune stimulation activity. At 14 days after feeding, shrimp fed 1, 3, 5 and 10 g of lignin/kg of diet exhibited significantly higher levels of phagocytic activity (PA) than the control group (P < 0.05). However, differences in total hemocyte count among treatments were not significant during the experimental period (P > 0.05). Additionally, lignin supplementation at 1‐10 g/kg for 14 days failed to protect experimental shrimp against YHV infection. The antiviral activity of lignin against YHV in black tiger shrimp was notable in vitro because compared to control shrimp (96.7 ± 5.8%; P < 0.05), shrimp injected with a pre‐incubated solution of YHV and lignin at 1, 5, 10 and 20 mg/L exhibited significantly lower mortality rates, 23.3 ± 5.8, 16.7 ± 5.8, 23.3 ± 5.8, and 20.0 ± 0.0%, respectively, after a lethal dose of YHV at 14–20 days after injection. These potent effects were clearly supported and confirmed by histopathological and RT‐PCR analyses. Based on these results, the pulping by‐product kraft lignin efficiently inhibits YHV infection in black tiger shrimp. This information will facilitate the development of practical methods to control yellow head disease in the marine shrimp culture industry. HighlightsThe median lethal dosage at 96 h (96‐hrs LD50) of kraft lignin in Penaeus monodon (15.9 ± 1.2 g) was 297 mg/L.Application of kraft lignin 1–10 g/kg diets for 14 days significantly enhanced phagocytic activity in shrimp hemocytes.Lignin supplementation at 1‐10 g/kg diet for 14 days failed to protect experimental shrimp against YHV infection.Immersion 1‐20 mg lignin/L with YHV for 2 h strongly inhibit and effectively protect shrimp from YHV infection.

Molecular and functional analyses of novel anti-lipopolysaccharide factors in giant river prawn (Macrobrachium rosenbergii, De Man) and their expression responses under pathogen and temperature exposure

&NA; Anti‐lipopolysaccharide factor (ALF) is an immune‐related protein that is crucially involved in immune defense mechanisms against invading pathogens in crustaceans. In the current study, three different ALFs of giant river prawn (Mr‐ALF3, Mr‐ALF8 and Mr‐ALF9) were discovered. Based on sequence analysis, Mr‐ALF3 and Mr‐ALF9 were identified as new members of ALFs in crustaceans (groups F and G, respectively). Structurally, each newly identified Mr‐ALF contained three &agr;‐helices packed against a four‐stranded &bgr;‐sheet bearing the LPS‐binding motif, which usually binds to the cell wall components of bacteria. Tissue expression analysis using quantitative real‐time RT‐PCR (qRT‐PCR) demonstrated that Mr‐ALF3 was expressed in most tissues, and the highest expression was in the heart and hemocytes. The Mr‐ALF8 gene was highly expressed in the heart, hemocytes, midgut, hepatopacreas and hindgut, respectively, while the Mr‐ALF9 gene was modestly expressed in the heart and hemocytes, respectively. The transcriptional responses of the Mr‐ALFs to Aeromonas hydrophila and hot/cold temperatures were investigated by qRT‐PCR in the gills, hepatopancreas and hemocytes. We found that all Mr‐ALFs were clearly suppressed in all tested tissues when the experimental prawns were exposed to extreme temperatures (25 and 35 °C). Moreover, the expression levels of these genes were significantly induced in all examined tissues by 2 different concentrations of A. hydrophila (1 × 106 and 1 × 109 CFU/ml), particularly 12 and 96 h after the injection. Finally, binding activity analysis of LPS‐motif peptides of each Mr‐ALF revealed that the LPS peptide of Mr‐ALF3 exhibited the strongest adhesion to two pathogenic Gram‐negative bacteria, A. hydrophila and Vibrio harveyi, and the non‐pathogenic Gram‐positive Bacillus megaterium. The results also showed that the Mr‐ALF8 and Mr‐ALF9 peptides had mild antimicrobial effects against similar tested bacteria. Based on information obtained in this study, novel ALF genes were clearly identified. Analyses of their responses under pathogenic and temperature stresses demonstrated the binding and antimicrobial activities of these ALFs and the consequent physiological effects, indicating their crucial functional roles in the prawn immune system. HighlightsThree novel cDNAs of anti‐lipopolysaccharide factors in giant river prawn (Mr‐ALF3, 8, 9) were cloned and charaterized.Structure of all Mr‐ALF proteins clearly showed &agr;‐helices packed against a four &bgr;‐sheet bearing the LPS binding motif.qRT‐PCR analysis of Mr‐ALFs in response to A. hydrophila in the gills, hepatopancreas and hemocytes was demonstrated.Expression levels of Mr‐ALFs were obviously suppressed in all tested tissues after heat or cold shock induction.LPS‐binding peptides of Mr‐ALFs clearly bound and inhibited both Gram‐positive and negative bacteria.