Modulation of innate immunity, expression of cytokine genes and disease resistance against Aeromonas hydrophila infection in goldfish (Carassius auratus) by dietary supplementation with Exiguobacterium acetylicum S01.

Probiotic strains play an increasing role in the production of healthy animals used as a food source. Elucidating the mechanisms of action that allow probiotic-driven immunomodulation may facilitate different applications such as the prevention of infectious diseases in food organisms. This study elucidates the probiotic effects of Exiguobacterium acetylicum S01 on the growth, haematological profile, innate immune capacity, expression of cytokine genes, and resistance to diseases of Carassius auratus caused by Aeromonas hydrophila infection. Three fish groups were fed with the following diets containing different doses of E. acetylicum S01 (CFU g-1): basal diet 0 (BD, without probiotic), 2.5 × 107 (DI) and 2.7 × 109 (DII)-CFU g-1 for 4 weeks. After 4 weeks, the fish were injected intraperitoneally with A. hydrophila and the percentage of survival was recorded over 21 days of post-challenge. Results revealed that dietary supplementation of E. acetylicum S01 significantly (P < 0.05) enhanced the growth, haematological profile and cellular immune responses including respiratory burst, phagocytic activities and antimicrobial enzymes (myeloperoxidase and lysozyme) and total immunoglobulin levels were improved by probiotic feeding at both occasions. Comparatively, expression of c- and g-type lysozyme followed by pro- and anti-inflammatory cytokines (IL-1ß, IL-10 and TGFß) was up-regulated in kidney, head-kidney and spleen. Moreover, fish fed with diet DII had a significantly higher (P < 0.05) survival rate (73.2%) after challenging. The survival rate was only 33.2% of the BD group against A. hydrophila infection. Our results revealed that E. acetylicum S01 delivered probiotic in feed exerts its influence on growth performance and provides disease resistance by stimulating the immune system at the cellular and molecular levels in C. auratus.

Antibiotic resistance and virulence traits of bacterial pathogens from infected freshwater fish, Labeo rohita.

Bacterial infectious diseases are a main dangerous problem in Aquaculture farming. It causes multiple diseases in fish as well as in human being and it has considerable virulence potential. In this connection, the moot of study focus to discriminate bacterial isolates recovered from naturally diseased Labeo rohita fish and their virulent characteristics. Based on the ß-haemolysis factor, four isolates (KADR11, KADR12, KADR13 and KADR14) were selected for further delineation. These bacterial isolates showed high similarity with Providencia rettgeri, Aeromonas sp., Aeromonas sp. and Aeromonas enteropelogenes respectively, using partial 16S r-RNA gene amplification and biochemical characterizations were also supported. The further study investigates the virulence characteristics of isolates showed separation of outer membrane proteins (OMPs) and lipopolysaccharides (LPS) which appeared between 19_80 kDa and 20_100 kDa in SDS_PAGE analysis respectively. All the four strains were complete resistant (100%) to ß-lactam antibiotics. L. rohita were injected intraperitoneally with 0 (control), 2.0 × 104, 2.0 × 105, 2.0 × 106, 2.0 × 107 and 2.0 × 108 cells/fish of Providencia rettgeri KADR11, Aeromonas sp. KADR12, Aeromonas sp. KADR13 and Aeromonas enteropelogenes KADR14 for the determination of lethal dose 50 (LD50) values, which were 2.4 × 107, 4.1 × 105, 2.7 × 107 and 7.4 × 105 cells/fish respectively. The results indicated that isolated strains were possessed the high pathogenic potential for L. rohita.

Effects of the potential probiotics Bacillus aerophilus KADR3 in inducing immunity and disease resistance in Labeo rohita.

The aim of the present study was to evaluate the dietary administration of Bacillus aerophilus KADR3 on enhancing the immunity and disease resistance against Aeromonas hydrophila infection in Labeo rohita. B. aerophilus culture supernatant exhibited antagonistic activity against fish pathogenic bacteria in an agar well diffusion assay. Four fish groups were fed with either of following diets containing different concentrations of B. aerophilus KADR3 (cfu g-1): 0 (control), 1 × 107 (DI), 1 × 108 (DII), and 1 × 109 (DIII) -cfu g-1. Various innate immune parameters were measured at - 3rd and 6th - week post-feeding. At the end of 6th week, fish were challenged intraperitoneally with A. hydrophila and survival percentage was recorded over 10 days post-challenge. Studied immunological parameters viz. serum lysozyme, phagocytosis, serum total protein, respiratory bursts, serum IgM levels, superoxide dismutase and alternative complement pathway activities were significantly enhanced (P < 0.05) in fish groups fed with B. aerophilus KADR3 supplemented diets, with the highest values were observed in DII (108 cfu g-1) fed group. Further, B. aerophilus supplementation at 108 cfu g-1 exhibited highest post-challenge survivability i.e. 72.83%, followed by DIII (64.19%) and DI (41.97%). Our results collectively suggest that B. aerophilus can potentially be used as probiotic strain in aquaculture to enhance the immunity and disease resistance with an optimal dietary supplementation of 108 cfu g-1.

Isolation of potential probiotic Bacillus spp. and assessment of their subcellular components to induce immune responses in Labeo rohita against Aeromonas hydrophila.

Bacillus species isolated from the gut of healthy Labeo rohita (Hamilton) were screened for antibacterial activity against selected fish pathogens. Among the isolates, KADR5 and KADR6 showed antibacterial activity, tolerated low pH and high bile concentrations and were susceptibility to various antibiotics. Based on morphological and biochemical tests and 16S rRNA gene analysis the probiotic strains KADR5 and KADR6 were identified as Bacillus licheniformis and Bacillus pumilus, respectively. The immune stimulatory effect of subcellular components of probiotic Bacillus licheniformis KADR5 and Bacillus pumilus KADR6 in L. rohita against Aeromonas hydrophila infection was studied. Fish were immunized intraperitoneally in case of subcellular components [cell wall proteins (CWPs), extracellular proteins (ECPs), whole cell proteins (WCPs)] and orally in case of live cells (10(8) CFU/g of feed). After 14th day of administration, fishes from each group were challenged intraperitoneally with 0.1 ml of A. hydrophila cell suspension in PBS (10(5) cells ml(-1)). Groups immunized with subcellular components and live cells had significantly lower mortalities of 20-40% and 23-33%, respectively in comparison to control (80% mortality). The non specific immune factors in the cellular components and viable cells of the probiotics increased the expression of lysozyme and respiratory burst. Use of WCPs and CWPs resulted in better protection against A. hydrophila in L. rohita. Our results clearly reflect the potential of cellular components of the probiotics Bacillus species for the protection of fish against A. hydrophila infection by enhancing the immune response.

Characterization of Bacillus spp. from the gastrointestinal tract of Labeo rohita--towards to identify novel probiotics against fish pathogens.

The aim of the present study is to screen and characterize endogenous microbiota Bacillus spp. from the gastrointestinal (GI) tract of Labeo rohita in order to evaluate their probiotic attributes. A total of 74 isolates from the GI of L. rohita were evaluated for their antimicrobial properties by agar well-diffusion method against fish pathogens. Based on the better antibacterial features, three isolates (KADR1, KADR3, and KADR4) were selected for further delineation. The three selected isolates exhibited higher tolerance to bile salt, moderate tolerance to low pH, high surface hydrophobicity to solvents, and capable to autoaggregate. All three isolates demonstrated notable proteolytic, catalase activity and susceptibility to various antibiotics. Partial 16S rRNA sequencing revealed that the isolates exhibited 99 % sequence homology with Bacillus subtilis, Bacillus aerophilus, and Bacillus firmus of the database substantiating morphological and physiological characterization. Survivability in low pH and bile salt ensures their adaptability in the fish intestinal microenvironment. The ability to autoaggregate reveals colonization potential in the GI of the fish. Absence of hemolytic activity, antibiotic susceptibility to certain antibiotics, presence of protease and catalase activity, and non-pathogenic caliber of the above-mentioned isolates could be feasible characteristics when considering them as probiotics in the aquaculture industry.