Sib Sankar Giri

Potential probiotic Lactobacillus plantarum VSG3 improves the growth, immunity, and disease resistance of tropical freshwater fish, Labeo rohita

The study evaluated the effects of dietary doses of Lactobacillus plantarum VSG3 on the growth performance, immunity, and disease resistance of Labeo rohita juveniles against Aeromonas hydrophila infection. Fish (mean body weight 60 g) were fed with diet containing 0 (control), 10(6), 10(8), 10(10) cfu g(-1)L. plantarum VSG3 for 60 days. Various growth and immune parameters were examined at 30 and 60 days of post-feeding. Fish were challenged with A. hydrophila 60 days post-feeding and mortalities were recorded over 10 days post-infection. Results showed that administration of VSG3 for 60 days had significant effects (P < 0.05) on the specific growth rate (SGR) and feed utilization efficiency of L. rohita. Dietary administration of L. plantarum VSG3 significantly increased the serum lysozyme and alternative complement pathway (ACP) activities, phagocytosis and respiratory burst activity in L. rohita throughout the experimental period. The highest superoxide dismutase (SOD) activity (P < 0.05) was observed in the fish group fed diet containing VSG3 at 10(8) cfu g(-1). The serum IgM levels were significantly higher in the experimental groups compared to the control group after 30 days of feeding; but, the result was opposite after 60 days of feeding. Further, fish fed the diet containing 10(8) cfu g(-1)L. plantarum VSG3 had significantly higher (P < 0.05) post-challenge survival rate (77.7%). These results collectively suggest that dietary supplementation of L. plantarum VSG3 at 10(8) cfu g(-1) to L. rohita is optimal for enhancing the growth, immunity, and disease resistance against A. hydrophila infection.

Effects of dietary supplementation of potential probiotic Pseudomonas aeruginosa VSG-2 on the innate immunity and disease resistance of tropical freshwater fish, Labeo rohita

The effects of dietary Pseudomonas aeruginosa VSG-2 supplementation on innate immunity and protection against Aeromonas hydrophila infection were evaluated in Labeo rohita. Fish were fed for 60 days with control diet or 3 experimental diets containing P. aeruginosa VSG-2 at 10(5), 10(7), and 10(9) cfu g(-l), respectively. Various innate immune parameters were examined at 30 and 60 days post-feeding. Fish were challenged with A. hydrophila 60 days post-feeding and mortalities were recorded over 10 days post-infection. Dietary supplementation of P. aeruginosa VSG-2 significantly increased serum lysozyme and alternative complement pathway (ACP) activities, phagocytosis, and respiratory burst activity in head kidney macrophages of L. rohita throughout the experimental period. Superoxide dismutase (SOD) activity significantly increased after 60 days in the groups fed diets containing 10(7) and 10(9) cfu g(-1) P aeruginosa. Serum IgM levels were significantly higher in the treatment groups than in the control group after 30 days of feeding; however, the opposite result was observed at 60 days. Moreover, fish fed diets containing 10(7) and 10(9) cfu g(-1)P. aeruginosa had significantly higher post-challenge survival rates against A. hydrophila infection. Further, P. aeruginosa VSG-2 was found to be safe for mammals. These results indicate that dietary P. aeruginosa VSG-2 supplementation at 10(7) cfu g(-1) can effectively improve innate immunity and disease resistance in L. rohita.

Purification and partial characterization of a detergent and oxidizing agent stable alkaline protease from a newly isolated Bacillus subtilis VSG-4 of tropical soil

An extracellular detergent tolerant protease producing strain VSG-4 was isolated from tropical soil sample and identified as Bacillus subtilis based on morphological, biochemical characteristics as well as 16S-rRNA gene sequencing. The VSG-4 protease was purified to homogeneity using ammonium sulphate precipitation, dialysis and sephadex G-200 gel permeation chromatography with a 17.4 purification fold. The purified enzyme was active and stable over a broad range of pH (8.0–11.0, optimum at 9.0) and temperature (40°C to 60°C, optimum at 50°C). The thermostability of the enzyme was significantly increased by the addition CaCl2. This enzyme was strongly inhibited by PMSF and DFP, suggesting that it belongs to the serine protease superfamily. The purified VSG-4 alkaline protease showed remarkable stability in anionic (5 mM SDS) and ionic (1% Trion X-100 and 1% Tween 80) detergents. It retained 97±2% and 83.6±1.1% of its initial activity after 1 h preincubation in the presence of 1 % H2O2 and 1 % sodium perborate, respectively. Furthermore, the purified enzyme showed excellent stability and compatibility with some commercial laundry detergents besides its stain removal capacity. Considering these promising properties, VSG-4 protease may find tremendous application in laundry detergent formulations.

Isolation and characterization of probiotic properties of Lactobacilli isolated from rat fecal microbiota.

The objective of the present study was to characterize lactobacilli isolates from the feces of male Wistar rats. Various physiological features of the candidate probiotic isolates were preliminarily investigated, including tolerance to simulated gastric juice and bile salts, antimicrobial activity, antibiotic susceptibility and in vitro aggregation. Based on their morphological and biochemical characteristics, four potential probiotic isolates (CS2, CS3, CS4, and CS7) were screened. The isolates showed good tolerance to stimulated gastric juice and bile salts. CS4 and CS7 exhibited strong antibacterial activities against the pathogens tested as assessed in neutral pH culture supernatants. All lactobacilli isolates were susceptible to all the tested antibiotics, except vancomycin. Moreover, the isolate CS4 and CS7 were found to possess stronger cell surface traits such as hydrophobicity, auto‐aggregation and co‐aggregation capacity. In addition, CS4 and CS7 had greater β‐galactosidase activities than the others. Biochemical tests and 16S rRNA gene sequencing confirmed that CS2, CS3, CS4 and CS7 are Lactobacillus intestinalis PJ2, L. sakei PJ3, L. helveticus PJ4, and L. plantarum PJ7, respectively. Based on the obtained results, L. helveticus PJ4 and L. plantarum PJ7 are ideal in vitro probiotic candidates and require further in vivo evaluation.

Bacteriophage application to control the contaminated water with Shigella

Shigella is one of the most important waterborne and foodborne pathogens around the world. Emergence of antibiotic-resistant Shigella has made the development of alternatives to conventional antibiotics necessary. In this study, a virulent Myoviridae bacteriophage, pSs-1 was isolated from environmental water in South Korea and showed infectivity to S. flexneri as well as S. sonnei strains. One-step growth analysis showed that pSs-1 has a short latent period (25 min) and a large burst size (97 PFU/cell). According to the genomic analysis, pSs-1 contains 164,999 bp of genome with a G + C content of 35.54% and it is considered as a member of the T4-like bacteriophage group. These results showed that pSs-1 may have potential as a biocontrol agent instead of conventional antibiotics for shigellosis.

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.

Immobilization of carbonic anhydrase enzyme purified from Bacillus subtilis VSG-4 and its application as CO(2) sequesterer.

The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan–alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co2+, Cu2+, and Fe3+, increased the enzyme activity, whereas CA activity was inhibited by Pb2+, Hg2+, ethylenediamine tetraacetic acid (EDTA), 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO2 to CaCO3. The maximum CO2 sequestration potential was achieved with immobilized CA (480 mg CaCO3/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO2 sequestration.

Dietary Administration of Banana (Musa acuminata) Peel Flour Affects the Growth, Antioxidant Status, Cytokine Responses, and Disease Susceptibility of Rohu, Labeo rohita

To explore the feasibility of Musa acuminata (banana) peels as a feed additive, effects of banana peel flour (BPF) on the growth and immune functions of Labeo rohita were evaluated. Diets containing five different concentrations of BPF (0% [basal diet], 1% [B1], 3% [B3], 5% [B5], and 7% [B7]) were fed to the fish (average weight: 15.3 g) for 60 days. The final weight gain and specific growth rate were higher (P < 0.05) in the B5 group. The most significant improvements in immune parameters such as lysozyme, alternative complement pathway, leukocyte phagocytic, superoxide dismutase, and catalase activities were observed in the B5 group. However, the B5 group exhibited the lowest malondialdehyde activity. IgM and glutathione peroxidise activities were significantly elevated in the treatment groups, except in B1, after only 30 days of feeding. Of the examined cytokine-related genes, IL-1β, TNF-α, and HSP70 were upregulated in the head kidney and hepatopancreas, and expressions were generally higher in the B3 and B5 groups. Moreover, B5 group challenged with Aeromonas hydrophila 60 days after feeding exhibited the highest survival rate (70%; P < 0.05). These results suggest that dietary BPF at 5% could promote growth performance and strengthen immunity in L. rohita.

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.

Role of HSP70 in cytoplasm protection against thermal stress in rohu, Labeo rohita.

To understand the function of HSP70 of Labeo rohita (LrHSP70) in cellular protection, LrHSP70 ORF cDNA was inserted into the plasmid of pET-32a(+) or pEGFP-L1. Then, the recombinant plasmids were transformed or transfected into Escherichia coli cells, mouse myeloma cells (MPC-11) or fish hepatoma cells (PLHC-1). Western blot results revealed that LrHSP70 was expressed in E. coli cells and molecular weight was estimated to be 70 kDa. In cells, LrHSP70 was over-expressed following thermal or cold stress. Results revealed that LrHSP70 protected prokaryotic cells against thermal or cold extremes as well as played the same role in MPC-11 and PLHC-1 cells. After heat treatment at 42 °C for 1 h, the viability of the cell was declined considerably. PLHC-1 cells with pEGFP-L1/LrHSP70 exhibited a higher survival rate (50%) than wild-type cells (18%) or cells with only pEGFP-L1 (21.2%). When the time lag extended to 2 h, the survival rates were 30%, 3.4% and 5.3% respectively. The present study revealed that LrHSP70 plays an important role in response to thermal and cold stress in fish.