Latobacillus Spp Case Study

Abstract
The antagonistic effect of Lactobacillus spp. against Vibrio harveyi and its probiotic effect on growth, digestibility and immune response for fresh water prawn, Macrobrachium rosenbergii were determined. Juvenile prawns (1.0±0.2g in weight, 0.45±0.02cm in length) were examined following feeding with Lactobacillus spp.-incorporated feed for 60 days. Lactobacillus spp. was isolated from prawn body. Larvae were fed twice per day with probiotic mixed feed for treatment group. As compared to the control group, significant reduction of V. harveyi growth was observed at 8 hr and at 50 days in in-vitro and in-vivo challenge test, respectively. Treatment group showed better performance than the control one in terms of growth, digestibility

was performed using a pair of primer Vibr-f: 5’- CGG TGA AAT GCG TAG AGA T-3’ and Vibr-r: 5’-TTA CTA GCG ATT CCG AGT TC-3’ (Tarr et al. 2007). A standard PCR was executed with a 50 µl reaction mixture containing 50 ng of template DNA, Top DNA Polymerase (Bioneer Corporation, Daejeon, Korea) at 0.5U/µl, 2.0 mMMgCl, 0.4 µM primers, 200 µM dNTP and 1X buffer as recommended by the manufacturer. PCR parameters were carried out in a thermal cycler (C1000TM, BIO-RAD, USA) as follows: pre-denaturation at 94 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 1 min, and extension at 72 °C for 1 min. The whole PCR parameters were terminated by a final extension step at 72 °C for 10 min. Amplification of 16S/23S intergenic spacer regions specific for Lactobacillus spp. performed with the set of primers; LbLMA1-rev: 5’-CTC AAA ACT AAA CAA AGT TTC-3’ and R16-1: 5’-CTT GTA CAC ACC GCC CGT CA-3’ (Dubernet et al., 2002). PCRs were performed following similar procedure as for virA gene of V. harveyi with editing annealing temperature at 58 °C. The PCR products were analyzed by gel electrophoresis in 2% agarose (Bioneer Corporation) gel. DNA bands were visualized by High Performance UV Transilluminator (UVP, CA, USA) and photographed using the DigiDoc-It 120 gel documentation system (UVP). To compare the size of double stranded DNA from 100 to 2,000 base pairs, 100 bp designed DNA markers were used. The DNA marker consists of 13 double stranded DNA fragments ranging in sizes from 100 to 1,000 (Bioneer,