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ISOLATION, CHARACTERISATION AND MOLECULAR IDENTIFICATION OF

VIBRIO HARVEYI FROM MORIBUND FARM SHRIMPS IN PUDUCHERRY, INDIA

Article · January 2019

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UGC Approved Journal No: 63495

ISOLATION, CHARACTERISATION AND MOLECULAR IDENTIFICATION OF VIBRIO HARVEYI

FROM MORIBUND FARM SHRIMPS IN PUDUCHERRY, INDIA

G Shanmugavel*1, G Krishnamoorthy2

1. Department of Zoology, KM Centre for PG Studies,

Government of Puducherry, Puducherry 605008, India
2. Department of Zoology, Tagore Government Arts and Science College,
Government of Puducherry, Puducherry 605008, India
*(Correspondence author: Email: idshanvel@gmail.com)

ABSTRACT
Vibrio harveyi is the most common bacterial species associated with crustacean vibriosis often causing sizeable
economic losses. Shrimp samples were collected from culture ponds located near Kaliveli Lake are subjected to
investigation. Bacterial strain was isolated from the hepatopancreas of Litopenaeus vannamei and was cultured in
Vibrio harveyi agar (VHA). Isolates were evaluated for characterization and identification using biochemical and
physiological test. The result obtained from biochemical and physiological test confirms that the bacterial isolates
belong to the species V.harveyi. The isolated V.harveyi strain was identified by 16S rDNA sequencing (Molecular-
based detection technique) and these data were confirmed with NCBI BLAST data match. The present study
indicates that V. harveyi is more prevalent bacterial stain cause mortality in farm shrimp.

KEYWORDS: Kaliveli Lake, 16s rDNA sequence, Vibrio harveyi, PCR.

INTRODUCTION
Vibriosis is one of the major bacterial diseases responsible for the mass mortality of cultured shrimp worldwide (Chen
et al., 2000). Outbreaks of vibriosis may occur when the environmental factors trigger the rapid multiplication of
bacteria already tolerated at low levels within shrimp blood (Sizemore and Davis, 1985). V. harveyi is an opportunistic
pathogen which is considered as the most frequently implicated in vibriosis, in penaeid livestock (Austin and Zhang,
2006).

Isolation and identification of the causative agent is the initial step towards understanding the nature of a disease in any
environment. Accurate identification is indeed for developing appropriate prophylactic measures in an aquaculture
setting. Conventional methods of bacterial identification have major drawbacks. Thus, an array of molecular techniques
is gaining popularity for the identification of different aquaculture-related bacterial pathogens. PCR based molecular
techniques, such as 16S rDNA sequence identification, Colony hybridization by species-specific probes, Fluorescent In
Situ Hybridization (FISH), Ribotyping, Restriction Fragment Length Polymorphism (RFLP), Amplified Fragment
Length Polymorphism (AFLP), Random Amplified Polymorphic DNA (RAPD) etc., have yielded the most valuable
information and new insights into the identification of closely related marine bacteria. Among DNA sequence-based
identification, analysis of 16S rDNA and other housekeeping gene sequences are the most popular and precise methods
currently used to identify closely related Vibrio (Chatterjee and Haldar, 2012). PCR technique is a valuable tool for a
rapid and exact identification of V.harveyi using 16S rDNA sequences than phenotypic characterization (Leslie et al.,
2013). The objective of the present research is to isolate, characterize and identification Vibrio harveyi bacteria from
the hepatopancreas of moribund Litopenaeus vannamei.

MATERIALS AND METHODS

Animal collection:
A total of 20 adult moribund L.vannamei were collected randomly during disease outbreak season from culture pond
near Kaliveli Lake (12o 7’ 11.02’ N, 79o 51’ 27.66’ E) located 25 km north of Puducherry, southeast coast of India.
Collected samples were transported to a laboratory in an aseptic plastic bag.

Isolation and Characterization of Vibrio harveyi:

The hepatopancreas of infected shrimps was excised and washed extensively with 0.9% saline water. Hepatopancreas
was weighed and the known amount of tissue sample was homogenized with Tris buffer (pH.7.0) for a minute. The
homogenized tissue sample was then centrifuged at 7000 rpm for 5 minutes and the clear supernatant was aspirated in

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to clean centrifuge tube. The clear supernatant was made into serial dilution (10-1 from 10-9) using sterile water in the
aseptic condition. From the diluted sample, 0.1 ml of sample was taken from 10-1 to 10-9 and spread over on specialized
Vibrio harveyi selective agar medium separately and one plate maintained as a control without a sample. This
specialized agar inhibits the growth of other Vibrio species. The plates were incubated at 37 oC for 24 to 48 hours to
observe the bacterial colonies growth. In such term the grow out clones were isolated and subjected for phenotype
characterization using various physiological and biochemical tests based on the methods outlined in the Bergey’s
Manual of Determinative Bacteriology (Vishniac, 1974).

Molecular identification:
The isolates identified based on phenotypic characterization, it was further confirmed of their identity based on
sequence analyses of 16S rDNA.

Extraction of genomic DNA:

The genomic DNA was extracted from bacterial cell suspension culture in Luria-Bertani broth. A sample of 2 ml of
bacterial cell suspension was centrifuged at 15,000 g for 10 min at 4 oC. The pellet was collected and re-suspended in
500μL of TNE buffer (10mM Tris-Cl, 0.15 mM NaCl, 1 mM EDTA, pH 8.0) and centrifuged again at 15,000 g for 10
min at 4°C. Subsequently, the pellets were re-suspended in 500 μL buffer for cell lysis (0.05 mM Tris-HCl, pH 8.0,
0.05 mM EDTA, 0.1 mM NaCl, 2%, SDS, 0.2 % PVP and 0.1% mercaptoethanol) (Lee et al., 2003) and 10μL of
proteinase K (20mg/ml) was added and incubated initially for 1 hr at 37°C and then for 2 hr at 55°C. The extraction
was further carried out by phenol-chloroform method (Sambrook and Russell, 2001). The sample was deproteinated by
adding an equal volume of phenol (Tris- equilibrated, pH 8.0), chloroform and isoamyl alcohol (25:24:1) mixture. The
aqueous layers and phenol were separated by centrifugation at 15,000g for 15 min at 4°C. The aqueous phase was
pipetted out carefully into a fresh tube and the process was repeated once again. Following this, an equal volume of
chloroform and isoamyl alcohol mixture (24:1) was added, mixed by gentle inversion and centrifuged at 15,000 g for
15 min at 4°C. The aqueous phase was separated and transferred to a fresh tube. Then, the DNA was precipitated by
incubation at -20°C overnight after adding an equal volume of ice-cold absolute ethanol. The precipitated DNA was
collected by centrifugation at 15,000 g for 15 min at 4°C and the pellet washed with 70% chilled ethanol.
Centrifugation was repeated once again then the supernatant decanted and the tubes left open until the pellet got dried.
The DNA pellet was dissolved in 100µL MilliQ (Millipore) grade water. Isolation of DNA was carried out using
electrophoresis in 1% agarose gel. The isolated DNA was quantified spectrophotometrically (Abs 260) and the purity
assessed by calculating the ratio of absorbance at 260 nm and 280 nm (Abs 260/Abs280).
Concentration of DNA (µg µL-1) = Abs260×50×dilution factor

PCR amplification, cloning, and sequencing:

Amplification of 16S rRNA gene was performed according to Reddy et al. (2000) using universal primers 16 S1 (GAG
TTT GAT CCT GGC TCA) and 16 S2 (ACG GCT ACC TTG TTA CGA CTT) in DNA thermal cycler (Eppendorf,
Germany). The reaction mixture (final volume 25 µL) contained 2.5 µL 10 X buffer, 1 µL 10 pmol each of
oligonucleotide primer, 1.5 µL DNA template, 2.5 µL 2.5 mM each deoxynucleoside triphosphate, 1 µL Taq
polymerase, and the remaining volume make up with sterile Milli Q water. The amplification profile consisted of an
initial denaturation for 5 min at 95°C followed by 34 cycles of denaturation for 20 sec at 94°C, annealing for 30 sec at
58°C and extension for 2 min at 68°C followed by a final extension for 10 min at 68°C. The PCR product was
separated on 1 % agarose gel prepared in 1x TAE buffer and stained with ethidium bromide.

Gel purification was done using GenEluteTM Gel Extraction kit (Sigma, USA). For purifying the PCR products, the
agarose gel that contained DNA fragments of appropriate size was excised and taken in a 1.5 mL tube, weighed and
added 3 gel volumes (~450 µL ) of gel solubilization solution and incubated for 10 min at 60 °C with repeated
vortexing in every 2 min. After incubation, added 1 gel volume (~150 µL) of 100% isopropanol, mixed gently until it
became homogenous. This solubilized gel solution was loaded into the binding column that was pretreated with column
preparation solution, centrifuged at 12,000 x g for 1 min. Added 700 µL wash solution and centrifuged at 12,000 x g
for 1 min, repeat the centrifugation, and residual wash solution was removed. The binding column was transferred to a
fresh collection tube (2mL MCT) and added 50 µL of preheated (at 65°C) 10 mM Tris-HCl (pH 9.0), centrifuged at
12,000 x g for 1 min, stored at -20°C. The concentration of DNA was measured spectrophotometrically at 260/280 nm
in a UV-VIS spectrometer (1800, Shimadzu, Japan). The purified PCR product of 16S rRNA gene was sequenced using
the dideoxynucleotide chain termination method (Musa et al., 2008) on a DNA sequencer (Model 373A, Applied Bios

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2008). The obtained nucleotide sequence was matched with the Genbank database by submitting the sequence in NCBI
BLAST to find out the percentage of identity.

RESULTS
Vibrio harveyi is gram-negative, nonspore-forming, straight rod bacterium and classified as chemoorganotroph. Vibrio
harveyi agar (VHA) was used for isolation and enumeration of V. harveyi. This medium is able to differentiate V.
harveyi from 15 other Vibrio species and has been shown to inhibit the growth of other Pseudomonas spp. and
Flavobacterium spp. The colonies of V. harveyi on VHA were green in color with a dark center (Fig. 1).

Fig. 1: Vibrio harveyi colony in Vibrio Harveyi agar

Table 1: Biochemical and physiological characteristics of

Vibrio harveyi isolates from farm shrimp
Bacterial Colonies
1 2 3 4 5 6 7 8 9 10
Characteristics

Gram stain -/S -/S -/S -/S -/S -/S -/S -/S -/S -/S
Glucose fermentation + + + + + + + + + +
Oxidase + + + + + + + + + +
Catalase + + + + + + + + + +
Motility + + + + + + + + + +
Formation of Indole + + + + + + + + + +
Formation of H2S - - - - - - - - - -
Luminescence + + + + + + + + + +
Gelatin liquefaction - - - - - - - - - -
Arginine dihydrolase - - - - - - - - - -
Amylase + + + + + + + + + +
Colony colour on TCBS G G G G G G G G G G
β Blood hemolysis + + + + + + + + + +
Sensitivity to vibriostat + + + + + + + + + +
0/129
Oxidase and fermentation +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+
Phenylalanine deaminase - - - - - - - - - -
Identification VH VH VH VH VH VH VH VH VH VH

VH = Vibrio harveyi, S = Short rod, G = Green, + denotes = Positive, - denotes = Negative

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Biochemical and physiological characteristics of the V. harveyi was done systematically (Table 1). All isolates were
gram-negative under light microscopic (Labomed 3100 microscopy, USA) observation after gram stain. All isolates
were able to ferment the glucose and they are positive to glucose fermentation. All bacterial isolates were showed the
result for oxidase, catalase and motility test but unable to produce hydrogen sulfide. The production of amylase was
also observed by the formation of a clear zone around the colony. The isolates were able to degrade tryptophan and
produce indole as the final product, sensitive to vibriostat 0/129 (150 μg/disk) and produced green colonies on
thiosulfate citrate bile salt agar (TCBS agar). The isolates were observed to be luminous in the darkroom after 24 hours
of growth. Isolated colonies were failed to utilize the L-arginine, phenylalanine, and gelatinase with a negative result. A
positive result for both oxidative and fermentative test of isolates because they utilize starch and lipid. Colonies were
able to haemolyse horse’s blood which resulted in the breakdown of horse blood agar plate around the bacterial colony
known as β-hemolysis.

Molecular identification of the isolated Vibrio harveyi strains was carried out based on 16S rDNA sequence analysis.
The sequence of the 1380 bp 16S rDNA PCR amplicon from isolate was determined and deposited at GenBank with
accession numbers MK578896.1. The nucleotide sequence analysis of the 16S rDNA fragments from this bacteria
revealed a high percentage of identity (>99) to 16S rDNA nucleotide sequences belonging to V. harveyi, they were also
showed high similarity (94-98%) to other Vibrio species. Therefore, the isolated were identified as V. harveyi based on
their morphological, cultural, physiological, biochemical characteristics and 16S rDNA sequence analyses.

DISCUSSION
Vibrio harveyi has been implicated as the causative agent for vibriosis in shrimps (Tendencia et al., 2004). Isolation of
Vibrio harveyi from infected L. vannamei hepatopancreas was done using Vibrio harveyi agar (VHA) medium. The
VHA media contain cellobiose and ornithine as carbon sources. The organisms that decarboxylate ornithine produce a
basic pH change and appear blue in the medium. The organisms that utilize cellobiose produce acidic pH change and
the resulting colonies appear green. Vibrio spp. can utilize either ornithine or cellobiose but Vibrio harveyi can utilize
them both, and thus appear as light green with dark center or yellow halo, and hence are unique in colony morphology
in the medium. Isolates with pathogenic potential were isolated according to their ability to lyse starch (Zamora-
Rodriguez, 2003), positive hydrophobicity (Khuntia et al., 2008), and production of extracellular enzymes such as
proteases and lipases (Farzanfar, 2006).

Isolated colonies show luminous, Gram-negative and possess short rod cell morphology; these morphological
appearances were similar to isolated V. harveyi from P. monodon in the Philippines described by Leano et al. (1998).
Baumann and Schubert, (1984) explained that most of the V. harveyi strains were positive to both oxidase and
fermentative tests. Bacteria were positive for oxidase, catalase, and motility tests and sensitive to vibriostat indicating
confirmed grouping to the Vibrionaceae. V. harveyi isolated from semi-intensive penaeid shrimp hatcheries were also
positive for motility, utilization of glucose, starch and tryptophan (Abraham and Palaniappan, 2004). The study clearly
evidenced that those isolates were able to obtain energy sources from glucose, starch, and tryptophan. The isolated
clones were unable to obtain energy through arginine hydrolysis based on the negative result for the arginine hydrolysis
test. It could not utilize citrate and unable to produce hydrogen sulfide which shows a negative result. Clones were
positive to gelatin liquefaction test. Isolated bacteria unable to utilize phenylalanine thus it shows a negative result. β
haemolytic activity refers to the ability to lyse the whole cell of erythrocytes (Pollack et al., 2002). Hemolytic bacteria
are capable to synthesize exotoxins that cause partial or total lysis of blood erythrocytes of various animals, which was
a desirable feature in our isolation.

The 16S and the 23S ribosomal RNAs are essential to the viability of bacterial cells, hence the genes coding for them
are highly conserved. However, these genes also contain short variable sequences useful for characterization and
discrimination of microbial populations at the level of family and, in many cases, at the level of genus and species. This
combination of conserved and variable sites makes these molecules ideal taxonomic markers to identify vibrio spp. by
PCR amplification and gene sequencing (Cano-Gomez et al., 2009). The 16S rRNA genes are considered the standard
marker for Vibrio phylogeny though since the gene evolves slowly, the differences between species are limited and
therefore often unable to resolve closely related bacterial strains (Sawabe et al., 2007, Nagpal et al., 1998).
Khamesipour et al., (2014) suggested that molecular-based detection techniques including PCR can be useful for a fast
and accurate determination of the pathogens at the early phases of infection. PCR technique was employed using 16S
rRNA gene sequence information is widely used for molecular identification of bacteria species (Dewhirst et al., 2015).

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In the present study V. harveyi was isolate identified from collected hepatopancreas of Litopenaeus vannamei from
culture pond, indicates its host infection biology and hepatopancreas the primary target tissue of V.harveyi infection
(Soto-Rodriguez et al., 2003). Its phenotypic characterization through different biochemical tests indicates that the
isolates were V.harveyi. Apart from this the clear cut genotypic identification through 16S rDNA gene sequencing
revealed that the isolated bacterial colony is V.harveyi strain with more than 99% similarity with V.harveyi strain.
These finding add new evidences to the wide distribution of this species, which has been frequently reported as one of
the most abundant Vibrio species in marine environments, especially in south east coast of Tamil Nadu, India
(Abraham and Palaniappan, 2004). Therefore, the use of PCR method to identified the V. harveyi in shrimp will
facilitate disease identification and improve the cultivation process for successful harvest.

CONCLUSION
The pathogenic strain of V. harveyi causes mortality and affects aquaculture production of L.vannamei. Conventional
biochemical and physiological tests were revealed the V.harveyi infection is a causative pathogen in L.vannamei shrimp
taken for this study. Molecular identification by 16S rDNA sequencing using PCR is indeed tooled to reveal the exact
genetic identity. Based on the present research, 16S rDNA sequencing using PCR is a convenient method to identified
pathogenic bacteria responsible for vibriosis. There is also a need to develop the shrimp culture practice and control the
pathogenic microbes is very essential. Hence, drug development against the bacterial pathogen is urgently needed for
the sake of sustainability of L.vannamei aquaculture.

ACKNOWLEDGMENT
We are very grateful to the Director, K.M. Centre for PG Studies, Puducherry, India, for providing us the facilities to
carry out this research work.

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