MOLECULAR DIAGNOSIS OF VIBRIO HARVEYI

ISOLATED FROM SALT PAN AND MARINE

PRAWN PENAEUS MONODON
 INTRODUCTION
 Shrimp farming is fast developing in India, especially in the
Southern coastal states at a tremendous space, aiming at
economic up lift.

 The prawn culture is wide spread through out the tropical world.
Penaeus monodon, the black tiger shrimp is the most widely
cultured species

 Penaeus monodon is one of the most widely cultured species

involved in this industry (Shishehchian et al., 1999).

 The technique for the commercial larval culture system of

penaeus monodon was first developed in Taiwan in 1968
(Laio et al., 1995).
 Common names of Penaeus monodon are Giant Tiger prawn, Jumbo tiger
prawn and Black tiger shrimp.
 Shrimp require energy with primary on protein (~ 40%) fat, (~ 7 ~10%) and
carbohydrate (~ 30%).
 Shrimp or Prawn aquaculture production in much of the world is depressed
by disease, particularly caused by luminous Vibrio and or viruses (Moriarty).
 Vibrio species, especially the luminous Vibrio harveyi, have been implicated
as the main bacterial pathogen of prawns.
 It is a gram negative bacterium which can be seen in a darkened
environment.
 Vibrio harveyi is a bioluminescent (light generating) marine (Saltwater)
bacterium.
 Vibrio species have been reported to cause serious diseases of larval and Post larval
stages of Penaeus monodon.
 Vibrio harveyi is the causative agent of luminous bacterial disease in shrimp.
 A Polymerase chain reaction techniques used for rapid detection of Vibrio harveyi in
Penaeus monodon brood stock, eggs, larvae, water etc., because bio chemical
identification can involve many tests and seeks to perform.
 SCOPE OF THE WORK
 Isolation of the Vibrio harveyi from water, soil samples collected

from saltpan and prawn samples.

 Biochemical Characterization of Vibrio harveyi.

 Molecular diagnosis of Vibrio harveyi from infected larvae using

PCR.

 Determining the effect of probiotic on Vibrio harveyi.

 Determining the antibiotic susceptibility of Vibrio harveyi using

selected antibiotics.
MATERIALS AND METHODS
1. ISOLATION OF THE PATHOGEN
2. SELECTION OF PATHOGEN

i) Morphological identification
ii) Biochemical identification
iii) Molecular identification

3. PROBIOTIC EFFECT ON VIBRIO HARVEYI

BACTERIAL STRAIN
4. ANTAGONISM ASSAY
5. ANTIMICROBIAL ACTIVITY
 RESULTS
TABLE 1-ENUMERATION OF VIBRIO HARVEYI

Number of colonies per gram and ml

Name of he samples

x 10-3

Soil 13,3000

Water 32,000

Penaeus monodon 23,000

BIOCHEMICAL CHARACTERIZATION IDENTIFICATION

S.No Biochemical characteristics Results

1) Gram’s staining Gram negative
2) Shape curved rods
3) Motility Motile
4) Oxidase Positive
5) MR Positive
6) VP Negative
7) Indole Positive
8) Bioluminiscence Positive
9) TCBS Green coloured colonies.
10) Amylase Clear zone around the colony
11) Lipase Absence of Zone
12) H2S production Absent
13) Gelatinase Gelatin liquefied
CULTURAL CHARACTERISTICS
MOLECULAR IDENTIFICATION OF VIBRIO HARVEYI
 Diameter of inhibition
Zone (mm )
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0
5
10
15
20
25
30
35

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Ba
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Probiotics
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Probiotics effects against Vibrio harveyi
 Com mercial Antibiotics effects against
Vibrio harveyi

30

25
Diameter of inhibition

20
zone (mm)

15

10

in ol in ci
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cill ic yc x
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A am Va C
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Antibiotics
 DISCUSSION

 Vibrio harveyi is a gram negative luminescent marine bacterium

isolated both in a free living state (Lavilla – pitogo et al., 1990) as well
as a commensal organism in the enteric contents of marine animals
(Hoyt and sizemore, 1982).

 In our findings Vibrio harveyi normally present in free living form but
prawn Penaeus monodon containing very lower in amount.

 Use of antibiotic leads to development of drug resistant bacteria and

reduced efficacy of anitibiotic treatment for human animal diseases
(Moriarty, 1997).

 The antagonestic studies of our work bacteria such as Lactobacillus,

Pseudomonas, Bacillus, Saccharomyces cerevisiae.
 In our study, an infected larva was collected and its DNA was

extracted and subjected to PCR analysis. After 35 cycles, the DNA

was taken and electrophoresis was carried out and the DNA bands

were observed. In prawn larvae have lesser amount of Vibrio

harveyi.

 The probiotic study against vibrio harveyi was studied the probiotic

such as lactobacillus, Bacillus, Pseudomonas, Saccharomyces

cervisiae. The results were compared with commercial antibiotics.

 SUMMARY

 In this study mainly focusing three aspects. They are isolation of

vibrio harveyi from water, soil and prawn samples from marine

area. Second identification of pathogen through biochemical &

molecular characterization. Finally determined the effect of

probiotics on Vibrio harveyi & compared with standard antibiotics.

 Normally the pathogen Vibrio harveyi present in the entire

sample. In ponds occurrence of Vibrio harveyi in prawn larvae is

diagnosed by PCR.
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