Nontawith Areechon

Effects of the dietary supplementation of mixed probiotic spores of Bacillus amyloliquefaciens 54A, and Bacillus pumilus 47B on growth, innate immunity and stress responses of striped catfish (Pangasianodon hypophthalmus).

ABSTRACT The study used the mixed probiotics of Bacillus amyloliquefaciens 54A and B. pumilus 47B isolated from striped catfish (Pangasianodon hypophthalmus) intestine aiming to stimulate growth performance, innate immunity, stress tolerance of striped catfish. The average weight gain (AWG), specific growth rate (SGR), and feed conversion ratio (FCR) were analyzed after fish were fed the mixture of probiotics (B. amyloliquefaciens 54A and B. pumilus 47B) at concentrations of 1 × 108, 3 × 108, and 5 × 108 CFU g−1 feed for 90 days. Immunity parameters, survival rate of fish challenged with Edwardsiella ictaluri and ammonia tolerance were also investigated. The amounts of B. amyloliquefaciens and B. pumilus were counted and identified by specific primer pairs of Ba1‐F/Ba1‐R, and 16‐F/Bpu‐R to confirm the presence of probiotics in fish intestine. The AWG (476.6 ± 7.81 g fish−1) of fish fed probiotics at 5 × 108 CFU g−1 was significant higher than the control (390 ± 25.7 g fish−1) after 90 days of feeding, but there was no significant (P > 0.05) effect of probiotics on FCR and SGR. Fish fed diet containing probiotics at 5 × 108 CFU g−1 also expressed resistance to E. ictaluri infection and higher immune parameters such as phagocytic activity, respiratory bursts, and lysozyme activity than the control. Stress response with ammonia showed significantly lower mortality rate (25%, 20% and 27%) of fish fed probiotics at all three levels of 1, 3 and 5 × 108 CFU g−1 than the fish fed control diet (75%). The study also demonstrated that the probiotics survived in the intestine of striped catfish after 90 days of feeding. Therefore, the dietary supplementation of a mixture of B. amyloliquefaciens and B. pumilus at 5 × 108 CFU g−1 can be used to improve the health and growth rate of striped catfish. HIGHLIGHTSThe spores of B. amyloliquefaciens 54A and B. pumilus 47B were first applied as probiotics in striped catfish.Dietary supplementation with B. amyloliquefaciens 54A and B. pumilus 47B for 90 days enhanced fish growth.Innate immunity of striped catfish were stimulated after feeding B. amyloliquefaciens 54A and B. pumilus 47B for 90 days.The mixture of B. amyloliquefaciens 54A and B. pumilus 47B in the diet enhanced disease resistance and stress tolerance.Colonization of B. amyloliquefaciens 54A and B. pumilus 47B in the intestine of striped catfish was observed.

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification - by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose - was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0-11.0 and an optimal temperature of approximately 55-60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis-Menten constant (Km) and catalytic constant (Kcat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s(-1), respectively. The catalytic efficiency (Kcat/Km) was 238 s(-1) mM(-1).

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.

Draft Genome Sequences of Streptococcus agalactiae Serotype Ia and III Isolates from Tilapia Farms in Thailand.

ABSTRACT Streptococcus agalactiae serotypes Ia and III were isolated from infected tilapia in cage and pond culture farms in Thailand during 2012 to 2014, in which pathogenicity analysis demonstrated that serotype III showed higher virulence than serotype Ia. Here, we report the draft genome sequencing of piscine S. agalactiae serotypes Ia and III.

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.