Multiplex PCR identification and culture-independent quantification of Bacillus licheniformis by qPCR using specific DNA markers.

Probiotics benefits in fish farming have been usually inferred appraising the effects observed on the host and not through the direct assessment of probiotic dynamics in the host gut microbiota. To overcome this gap, quantitative PCR (qPCR) can be a powerful approach to study the bacterial dynamics in fish gut microbiota. The presented work proposes four B. licheniformis-specific DNA markers and details a qPCR method to track putative probiotics B. licheniformis on fish gut. The four B. licheniformis-specific DNA markers - BL5B (hypothetical protein BL00303), BL8A (serA2), BL13C (rfaB) and BL18A (ligD) - were selected and validated by PCR and multiplex-PCR with 20 B. licheniformis isolates and a broad range of non-target bacteria. To assess the dynamics of B. licheniformis in the digesta of farmed fish, a qPCR was validated using markers BL8A and BL18A and calibration curves obtained for both markers with digesta samples spiked with B. licheniformis cells showed a high correlation (R2 > 0.99) over 6 log units (CFU/reaction), and a limit of detection (LOD) as low as 247 CFUs/reaction. Furthermore, the consistent qPCR repeatability and reproducibility underline the specificity and reliability of the qPCR proposed. Ultimately, the possibility to monitor the dynamics of B. licheniformis probiotics in the gut microbiota of farmed fish might be instrumental to optimize best practices in aquaculture.

Nutritional history does not modulate hepatic oxidative status of European sea bass (Dicentrarchus labrax) submitted to handling stress.

The aim of the present study was to assess the impact of an acute handling stress on hepatic oxidative status of European sea bass (Dicentrarchus labrax) juveniles fed diets differing in lipid so urce and carbohydrate content. For that purpose, four diets were formulated with fish oil (FO) and vegetable oils (VO) as lipid source and with 20 or 0% gelatinized starch as carbohydrate source. Triplicate groups of fish with 74 g were fed each diet during 13 weeks and then subjected to an acute handling stress. Stress exposure decreased hematocrit (Ht) and hemoglobin (Hb) levels. Independent of dietary treatment, stress exposure increased hepatic lipid peroxidation (LPO). Stressed fish exhibited lower glucose 6-phosphate dehydrogenase (G6PD), catalase (CAT), and superoxide dismutase (SOD) activities, independent of previous nutritional history. In the VO groups, stress exposure increased glutathione peroxidase (GPX) activity. Diet composition had no effect on Ht and Hb levels. In contrast, dietary carbohydrate decreased hepatic LPO and CAT activity and increased glutathione reductase (GR) and G6PD activities. Dietary lipids had no effect on LPO. Fish fed the VO diets exhibited higher G6PD activity than fish fed the FO diets. In conclusion, dietary carbohydrates contributed to the reduction of oxidative stress in fish. However, under the imposed handling stress conditions, liver enzymatic antioxidant mechanisms were not enhanced, which may explain the overall increased oxidative stress.