Abstract

Background:
The increasing need for sustainable aquaculture practices has directed attention toward probiotics as viable, eco-friendly alternatives to antibiotics. Bacillus subtilis, a resilient, spore-forming bacterium, is recognized for its immunomodulatory and growth-promoting potential in aquaculture species.

Aim:
This study aimed to assess the histopathological effects of dietary inclusion of an indigenous B. subtilis, isolated from the intestinal tract of common carp (Cyprinus carpio), administered at graded levels to determine dose-dependent tissue responses.

Methods:
A controlled 8-week feeding trial was conducted on 72 common carp randomly assigned to four dietary treatments: a basal control diet and three diets supplemented with B. subtilis at 1, 10, and 100 mg/kg feed. The bacterial strain was taxonomically validated through molecular identification of the isolate by 16S rRNA and endoglucanase gene amplification. Post-trial, hepatic and renal tissues were processed for histopathological analysis using hematoxylin and eosin staining to assess structural integrity and pathological changes.

Results:
Histological examination revealed a clear dose-dependent pattern of tissue response. The 1 mg/kg group exhibited minor histological alterations, including mild glomerular swelling and slight renal hemorrhage, with largely preserved tissue architecture. At 10 mg/kg, moderate renal tubule degeneration, epithelial desquamation, and the formation of hyaline casts were observed, suggesting early nephrotoxic effects. The highest supplementation level (100 mg/kg) induced pronounced hepatic degeneration characterized by cytoplasmic vacuolation, sinusoidal dilatation, Kupffer cell hyperplasia, vascular congestion, and mild periportal inflammation. Similarly, renal tissues exhibited tubular necrosis, glomerular atrophy, and interstitial hemorrhage. No pathological changes were observed in control fish, which displayed intact hepatic and renal architecture.

Conclusion:
Low-dose dietary supplementation (1 mg/kg) of indigenous B. subtilis appears histologically safe, showing only mild alterations without functional impairment. However, higher doses may induce moderate tissue damage. Dose optimization is essential for the safe and effective use of native probiotics in aquaculture.

Key words: Bacillus subtilis; Histological biomarkers; Dose-dependent effects; Fish tissue integrity; Aquaculture probiotics.