Subcellular component of Bacillus subtilis (AN11) induces protective immunity against Aeromonas hydrophila in Labeo rohita (Ham.)

Abstract

Aquatic animals are usually vulnerable to a complex range of life-threatening pathogenic organisms which cause enormous losses in the aquaculture industry. In the present study, the probiotic bacteria, Bacillus subtilis, isolated from the mangrove forest of Bhitarakanika, Odisha, India, was used to determine the efficacy of its subcellular component on the immune system of Labeo rohita and also to evaluate its effectiveness against the fish pathogen, Aeromonas hydrophila. The subcellular component of bacteria was separated and immunized intraperitoneally at a dose of 4 µl, i.e. 40.4 µg protein/µl in healthy L. rohita (average weight 50 ± 5g). A dose of 0.1 ml phosphate buffer saline (PBS) was injected as a replacement of subcellular component in the control. After 1 month of immunization, various haematological parameters including RBC and WBC, non-specific immune factors such as respiratory burst activity, lysozyme, bactericidal activity and serum biochemical activity (i.e. total protein, albumin, globulin, and A/G ratio) were evaluated. The immunized and control fish were infected experimentally with virulent A. hydrophila. Bacterial cell suspension (20 µl) in PBS containing 106 CFU/ml injected intraperitoneally. The mortality rate was observed after the infection. After infection with A. hydrophila, the blood and serum samples were again obtained at 6, 12, 24, 48 and 72 h for assessment of the above parameters to estimate the efficacy of subcellular component. Our result revealed that the WBC, NBT and bactericidal activity were increased significantly (p ≤ 0.05) in the immunized fish as compared to that of the control fish. After being infected with A. hydrophila, the total WBC count and NBT levels of immunized fish were significantly (p ≤ 0.05) risen at 72 h. Likewise, the levels of total protein, albumin, globulin and A/G in the immunized fish were increased than in the control fish. However, albumin and A/G ratio were significantly (p ≤ 0.05) decreased after infected with A. hydrophila, but the highest globulin level was observed at 72 h of post-challenged. Due to its better effect on the non-specific immune function, e.g. respiratory burst (0.44 ± 0.01 ΔOD/min), lysozyme (4.13 ± 0.17 U/min) and bactericidal (88.80% ± 0.18%) activities of fish, this sub-cellular component can be useful as a potential vaccine for bacterial septicaemia caused by Aeromonas hydrophila in aquaculture.