Phenotypic and molecular characterization, and pathogenicity of vibrio harveyi isolated from diseased marine fish in Sabah

Mohammad Tamrin Bin Mohamad Lal, (2012) Phenotypic and molecular characterization, and pathogenicity of vibrio harveyi isolated from diseased marine fish in Sabah. Masters thesis, Universiti Malaysia Sabah.

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Abstract

This study was conducted to characterize Vibrio harveyi isolated from diseased fish specimens and water sample in Sabah. The V. harveyi were Isolated using thiosulphate dtrate bile sucrose (TCSS) agar and detected using PCR amplification of hemolysin gene. The V. harveyi were phenotypically characterized using 67 biochemical tests and subjected to susceptibility test against 11 types of antibiotics. The bacteria were also analyzed using DNA sequencing of two housekeeping genes (16S rRNA and atpA). The virulence of the V. harveyi was determined using challenge test and RFLP-PCR analysis of hemolysin gene. The result of this study revealed that the V. harveyi were detected in four out of seven diseased fish species and water sample. The V. harveyi from the fish specimens and water sample were phylogeneticalty similar to V. harveyi type strains (ATCC 35084, VHJR7 and VHJR4) and other V. harveyi from GenBank. However, they were differed in citrate utilization, urease and f3-galactosidase production tests. Based on these variations, the V. harveyi isolates can be grouped into 5 phenotypic groups. Further analysis on antibiotic susceptibility revealed that all the V. harveyi isolates were susceptible to tetracycline and nalidixic acid; Intermediate sensitive to oxolinic acid and trimetophrim but resistant to vancomycin and ampicillin. However, they responded variably to ciprofloraxin, nitrofurantoin, novobiocin, chloramphenicol and kanamycin. The sequencing analysis of housekeeping genes further confirmed the Identity of the V. harveyi. The phylogenetic tree of 16S rRNA showed that V. harveyi dustered together with the closely related species, V. campbellii and V. rotlferfanus. However, the atpA cleariy separated all the V. harveyiIsolates from Its closely related species. The challenge test showed that all V. harveyi isolates were virulent to Asian seabass (lDtes calcarifet). The RFlP analysis using two restriction enzymes, Ac∧ and Bg∧I, and amino acid sequence analysis showed that all the V. harveyi isolates have similar RFLP-PCR pattern with pathogenic strain of V. harvey; (VHJR7). The present study revealed that the V. harveyi can exist In diseased fish and water samples. The variability of the V. harveyi isolates in the utilization of citrate, urease and β-galactosidase production test can be explained by the horizontal gene transfer of the gene responsible for those phenotypic features in V. harveyi. The various responses to antibiotics suggest that the V. harveyi may contain resistant genes for those antibiotics. The molecular analysis of housekeeping genes revealed that the atpA gene Is more discriminative and can provide accurate species identification for V. harvey; than the 16S rRNA gene. It is also shown in this study that V. harveyi with different phenotypes can cause mortality to Asian seabass. The sequendng and RFLP-PCR analyses of hemolysin gene can be used to differentiate pathogenic strains of V. harveyi from nonpathogenic strains. The data presented In the present study can be useful in the fish vibriosis monitoring program for marine aquaculture in Sabah.

Item Type:Thesis (Masters)
Uncontrolled Keywords:Vibrio harveyi, diseased fish specimen, water sample
Subjects:Q Science > QP Physiology
Divisions:SCHOOL > Borneo Marine Research Institute
ID Code:11825
Deposited By:IR Admin
Deposited On:07 Oct 2015 15:48
Last Modified:07 Oct 2015 15:48

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