International Journal of Aquaculture, 2017, Vol.7, No.23, 143-158
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in the laboratory condition for 2 weeks and divided into three equal groups (two experimental, one control) with
three replicates in each case. The candidate isolates were grown in TSB (30°C, 24 h), centrifuged (2800 g, 15 min,
at 4°C) and cell pellets were suspended in sterile 0.9% saline. Each experimental fish received intra-peritoneal (IP)
injection (1.0 mL) of microbial suspension (10
9
cells mL
-1
). The fish in control group were injected with sterile
0.9% saline (Mesalhy et al., 2008). Fish were fed
ad libitum
with a diet containing approximately 35% crude
protein having fish meal as the chief protein source. All groups were kept under observation for 21 days and
health status was checked every day for development of any disease symptom.
2.12 Identification of isolates by 16S rRNA and 18S rRNA gene sequence analysis
The most promising two microbial isolates were identified through either 16S rRNA (bacteria) or 18S rRNA
(yeast) partial gene sequence analyses after isolation and PCR amplification following the methods described in
Dutta et al. (2015) and Gadanho et al. (2003), respectively, with minor modifications. The gene encoding 16S
rRNA
was
amplified
using
27f
(5’-AGAGTTTGATCCTGGCTCAG-3’)
and
1492r
(5’-GGTTACCTTGTTACGACTT-3’) universal primers by polymerase chain reaction (PCR).
For 18S rRNA sequence analysis, an InstaGene Matrix (BIO-RAD) was used for extraction of genomic DNA
samples. The ‘Internal Transcribed Spacer’ (ITS) region in the 18S rDNA fragment was amplified using ITS1
(5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-30’) as the forward and
reverse primers. The amplification products were purified with a multiscreen filter plate (Millipore Corp, Bedford,
MA) and sent to a commercial house for Sanger sequencing (ABI Prism 3730XL DNA analyzer; Applied
Biosystems, Foster City, CA).
Sequenced data were edited (BioEdit Sequence Alignment Editor, Version 7.2.0), aligned and analyzed to find out
the closest homolog using National Centre for Biotechnology Information (NCBI) GenBank database, and
deposited to the NCBI GenBank. Phylogenetic trees were prepared using MEGA5.1 Beta 4 software following the
Minimum Evolution Method.
2.13 Statistical analysis
Statistical analysis of the quantitative enzyme activity data was performed by the one-way analysis of variance
(ANOVA), followed by Tukey’s test according to Zar (1999) using SPSS Version 10 software (Kinnear and Gray,
2000).
3 Results
Analysis of microbiological community in the GI tract revealed that considerable number of culturable aerobic or
facultative anaerobic heterotrophic bacterial and yeast population were present on both PI and DI regions of
O. niloticus
(Table 1). Heterotrophic and diverse extracellular enzyme-producing microbial populations were
found to be highest in the DI region than that of the PI region. While considering extracellular enzyme-producing
bacteria, proteolytic population was dominated in the DI region (LVC = 5.36), which was followed by amylolytic
bacteria (LVC = 4.87) and cellulolytic bacteria (LVC = 4.11). Phytate-degrading population was noticed to be the
lowest (LVC = 3.25).
Altogether 97 bacteria were isolated, out of which 10 extracellular enzyme producing strains (4 from PI and 6
from DI) were primarily selected through qualitative enzyme assay. The intensity of transparent zone (halo)
produced by the isolates are presented as score. Maximum and minimum scores of bacterial isolates were 27 and
17, respectively (Table 2). In case of yeast, out of the 32 yeast isolates, 5 extracellular enzyme-producing strains
were selected primarily through qualitative enzyme assay, with maximum and minimum scores being 16 and 11,
respectively which were further evaluated by quantitative enzyme assay to select the most promising isolates
(Table 2). The results of quantitative estimation of extracellular amylase, protease, lipase, cellulase, phytase and
xylanase production by the microbial strains are depicted in Table 3. The results revealed significant differences in
the enzyme activities between different microbial isolates.
