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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.Viable 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.