This paper describes a new bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon. The affected shrimp showed white spots similar to those caused by white spot syndrome virus (WSSV), but the shrimp remained active and grew normally without significant mortalities. The study revealed no evidence of WSSV infection using electron microscopy, histopathology and nested polymerase chain reaction. Electron microscopy indicated bacteria associated with white spot formation, and with degeneration and discoloration of the cuticle as a result of erosion of the epicuticle and underlying cuticular layers. Grossly the white spots in BWSS and WSS look similar but showed different profiles under wet mount microscopy. The bacterial white spots were lichen-like, having perforated centers unlike the melanized dots in WSSV-induced white spots. Bacteriological examination showed that the dominant isolate in the lesions was Bacillus subtilis. The occurrence of BWSS may be associated with the regular use of probiotics containing B. subtilis in shrimp ponds. The externally induced white spot lesions were localized at the integumental tissues, i.e., cuticle and epidermis, and connective tissues. Damage to the deeper tissues was limited. The BWS lesions are non-fatal in the absence of other complications and are usually shed through molting.
In this study, potential probiotic strains were isolated from fermented pickles based on antagonistic activity against two shrimp pathogens (Vibrio harveyi and Vibrio parahaemolyticus). Two strains L10 and G1 were identified by biochemical tests, followed by16S ribosomal RNA gene sequence analysis as Bacillus subtilis, and characterized by PCR amplification of repetitive bacterial DNA elements (Rep-PCR). Subsequently, B. subtilis L10 and G1 strains were tested for antibacterial activity under different physical conditions, including culture medium, salinity, pH and temperature using the agar well diffusion assay. Among the different culture media, LB broth was the most suitable medium for antibacterial production. Both strains showed the highest level of antibacterial activity against two pathogens at 30 °C and 1.0% NaCl. Under the pH conditions, strain G1 showed the greatest activity against V. harveyi at pH 7.3-8.0 and against V. parahaemolyticus at pH 6.0-8.0, whereas strain L10 showed the greatest activity against two pathogens at pH 7.3. The cell-free supernatants of both strains were treated with four different enzymes in order to characterize the antibacterial substances against V. harveyi. The result showed considerable reduction of antibacterial activity for both strains, indicating the proteinaceous nature of the antibacterial substances. A wide range of tolerance to NaCl, pH and temperature was also recorded for both strains. In addition, both strains showed no virulence effect in juvenile shrimp Litopenaeus vannamei. On the basis of these results and safety of strains to L. vannamei, they may be considered for future challenge experiments in shrimp as a very promising alternative to the use of antibiotics.
Bacillus strain NS 8, a lipase-producing bacterium isolated from a Malaysian hot spring, is able to tolerate a broad range of temperature and pH, which makes it beneficial for this study. It generated PCR products with molecular weight of 1,532 bp, and the 16S rRNA sequence analysis identified it as Bacillus subtilis with accession number AB110598. It showed a 71% similarity index with B. subtilis using Biolog Microstation System. Its lipase production was optimized using a shake flask system by changing the physical (agitation speed, pH and temperature) and nutritional (nitrogen, carbon and minerals) factors. The most suitable combination of the basal medium for lipase production was 2.5% olive oil (carbon), 1.5% peptone (nitrogen), 0.1% MgSO(4) (mineral) at an optimum temperature of 50°C, pH 7.5 and 150 rpm agitation, giving an enzyme yield of 4.23 U/ml. Statistical optimization using response surface methodology was carried out. An optimum lipase production of 5.67 U/ml was achieved when olive oil concentration of 3%, peptone 2%, MgSO(4)·7H(2)O 0.2% and an agitation rate of 200 rpm were combined. Lipase production was further carried out inside a 2-liter bioreactor, which yielded an enzyme activity of 14.5 U/ml after 15 h of incubation.