J. Gen. Appl. Microbiol.

, 52, 273280 (2006)

Full Paper
Differential detection of vibrios pathogenic to shrimp by multiplex PCR
Christian Danve M. Castroverde,1 Boris B. San Luis,1 Rosario G. Monsalud,2 and Cynthia T. Hedreyda1,*
1

Molecular Microbiology Laboratory, National Institute of Molecular Biology and Biotechnology, University of the Philippines, Diliman,
Quezon City 1101, Philippines
2
National Institute of Molecular Biology and Biotechnology, University of the Philippines Los Baos College, Laguna, Philippines
(Received March 24, 2006; Accepted September 8, 2006)

The research was focused on the multiplex polymerase chain reaction (PCR) differential detection of shrimp pathogens Vibrio harveyi, Vibrio campbellii and isolates from a variant strain of
Vibrio (referred to as Philippine Vibrio isolates in this study) exhibiting characteristics distinct
from these two species. Sequence alignment of the hemolysin gene from type strains Vibrio harveyi (NBRC 15634) and Vibrio campbellii (NBRC 15631), as well as 10 variant Philippine Vibrio
isolates, was performed in order to design a set of hemolysin-targeted primers for the specific
detection of the Philippine Vibrio isolates. Primer PNhemo amplified a 320-bp hemolysin gene
fragment of the Philippine Vibrio isolates in PCR using 65C annealing temperature, but did not
amplify the target gene fragment in type strains V. harveyi and V. campbellii. Another new primer
(VcatoxR) targeting the toxR gene was designed for the specific detection of type strain V. campbellii under stringent 65C annealing temperature. PCR using VcatoxR primer resulted in the specific amplification of a 245-bp V. campbellii toxR fragment. The simultaneous use of three primer
sets in PCR, including PNhemo and VcatoxR (the two new primers designed in this study), and a
primer VhtoxR (previously reported for the specific detection of V. harveyi), resulted in differential profiles with 390-bp, 245-bp, and 320-bp amplicons for V. harveyi, V. campbellii, and variant
Philippine Vibrio isolates, respectively. Presence of all three types of Vibrio shrimp pathogens in
the sample could be detected with a multiplex PCR profile containing all the expected size amplicons.
Key Wordshemolysin; toxR; Vibrio campbellii; Vibrio harveyi; vibriosis

Introduction

Vibrio infestations in the mid-1990s led to the nearcollapse of the black tiger shrimp Penaeus monodon
industry in the Philippines (Lavilla-Pitogo et al., 1998)
with V. harveyi (Karunasagar et al., 1994; LavillaPitogo et al., 1990) and V. campbellii (De la Pea et
al., 2001) as main causative agents in shrimp disease
* Address reprint requests to: Dr. Cynthia T. Hedreyda, Molecular Microbiology Laboratory, National Institute of Molecular Biology and Biotechnology, University of the Philippines, Diliman,
Quezon City 1101, Philippines.
E-mail: hedreyda@laguna.net

or vibriosis. In addition to these two species, Vibrio isolates from infected shrimps in different parts of the
Philippines, were observed to cause high mortalities of
shrimp postlarvae (De la Pea et al., 2001). These isolates were identified on the basis of biochemical tests
to belong to either V. harveyi or V. campbellii but were
observed to exhibit characteristics distinct from type
strains of both species (Cortado et al., 2004; Raoa
and Hedreyda, 2005). All ten isolates possess luciferase and ornithine decarboxylase gene homologues characteristic of V. harveyi (Raoa and
Hedreyda, 2006) but their toxR and hemolysin gene
sequences were exhibiting 9293% similarity to V.
campbellii and only 7475% to V. harveyi (Raoa and

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CASTROVERDE et al.

Hedreyda, 2005; San Luis and Hedreyda, 2005).

At present, the shrimp pathogenic Vibrio isolates
from the Philippines appear to be variants of V. harveyi
but classification and identification of these variant
shrimp pathogens are still the subject of investigation
because sequences for the toxR and hemolysin genes
are significantly different from sequences of type strain
V. harveyi. In this paper, therefore, these 10 shrimp
pathogens are designated as shrimp pathogenic variant Philippine Vibrio isolates. Because these variant
Vibrios were the predominant pathogens obtained
from infected shrimps in different parts of the Philippines (De la Pea et al., 2001), they appear to belong
to a significant strain of shrimp pathogen in the country. In assessing shrimp and shrimp ponds for the
presence of Vibrios pathogenic to shrimp, it is therefore necessary to include PCR detection primers for
these variant Philippine Vibrio isolates.
In this study, the PCR primers were designed to target regions of the hemolysin and toxR genes of the
shrimp pathogens. Hemolysin is a potent exotoxin that
is responsible for red blood cell (erythrocyte) membrane disruption, cell lysis, and other infection
processes (Zhang and Austin, 2005). The toxR gene
product is a 32.5-kDa transmembrane protein that regulates virulence gene expression (Miller et al., 1987).
Sequences from both hemolysin and toxR genes have
been used to develop single locus PCR-based protocols for the detection of Vibrio species (Bej et al.,
1999; Kim et al., 1999; Lee et al., 1998; Vuddhakul et
al., 2000). Single locus toxR and hemolysin gene-targeted PCR were developed for the specific detection
of type strain V. harveyi (Conejero and Hedreyda,
2003, 2004) and a toxR-targeted PCR was reported
for the detection of shrimp pathogenic Philippine Vibrio
isolates (Raoa and Hedreyda, 2005). In this study,
however, hemolysin and toxR-targeted multiplex PCR
was used for the differential detection of three strains
of Vibrio shrimp pathogens.
This paper reports the design of two new primers, a
set of primers for the specific amplification of a fragment of the hemolysin gene from variant Philippine
Vibrio isolates and another new set of primers for the
specific amplification of a region of the toxR gene from
type strain Vibrio campbellii NBRC 15631. These new
sets of primers were used in multiplex PCR together
with a primer pair previously reported for the specific
detection of type strain V. harveyi NBRC 15634. The
simultaneous use of three sets of primers resulted in

Vol. 52

differential detection of shrimp pathogenic type strain

V. harveyi, type strain V. campbellii, and the variant
Philippine Vibrio isolates, in a single PCR run. The resulting agarose gel electrophoresis profile of the multiplex PCR could reveal the type or types of Vibrio
shrimp pathogens present in the sample.
Materials and Methods

Bacterial isolates. Ten pathogenic Vibrios isolated

from various parts in the Philippines (De la Pea et al.,
2001) were used (CRW-9807, PIZ-9824, NRW-9805,
BR-9807, BRW-9804, CRWP-9805, BS-9904, PN9801, SW-9702 and PIZ-9806). Type strains V. harveyi
(NBRC 15634) and V. campbellii (NBRC 15631) were
obtained from the NITE Biological Resource Center
(NBRC), Japan.
Maintenance of cultures. Vibrio bacteria were cultured in Nutrient agar (Pronadisa, Laboratorios Conda)
with 1.5% sodium chloride at 30C overnight or Nutrient broth with 1.5% sodium chloride at 30C overnight
with shaking.
Genomic DNA extraction. Genomic DNA extracts
were obtained for each bacterium used in the study
following the NucleoSpin Tissue Kits User Manual
2002 (BD Biosciences, Clontech, CA, USA).
Design of hemolysin-targeted primers specific to
Philippine Vibrio isolates. The hemolysin gene sequences of type strain V. harveyi NBRC 15634
(DQ269128), V. campbellii NBRC 15631 (DQ356918)
and ten Philippine Vibrio isolates were aligned using
ClustalW (www.ebi.ac.uk/clustalw/). The region exhibiting greatest deviation between the sequences of the
two type strains and the ten Philippine Vibrio isolates
were chosen as the annealing sites. In addition to this,
the primer annealing sites were chosen to be within
conserved regions within the hemolyisn gene of the10
variant Philippine Vibrio isolates. The newly designed
primer pair was referred to as PNhemo with PNhemoF (forward primer) and PNhemo-R (reverse primer)
and synthesized by Genset Oligos Corp. (Singapore).
Amplification of the target hemolysin gene fragment
from the Philippine Vibrio isolates. The PCR reaction
mixtures were prepared by mixing genomic DNA, 1

PCR buffer, 0.2 mM nucleotides, 0.5 mM of each primer
(PNhemo-F and PNhemo-R) and 0.025 U/ml Taq DNA
polymerase (TaKaRa). The reactions were then
loaded into the thermocycler (Eppendorf) with the following conditions: 30 cycles of denaturation (94C for

2006

Differential detection of Vibrios pathogenic to shrimp

275

Fig. 1. Aligned hemolysin gene sequences of ten Philippine Vibrio isolates, type strain V. harveyi and type strain V.
campbellii, showing the annealing sites for the PNhemo primer pair as shaded.
Base deviations within the annealing sites are unshaded. The expected fragment size is 320 bp. Numbers above the
alignment are relative to the hemolysin gene sequence of the Philippine Vibrio isolates.

30 s), annealing (65C for 30 s) and extension (72C

for 30 s).
Design of toxR-targeted primers specific to V. campbellii NBRC 15631. A new primer set VcatoxR that
works with more stringent annealing temperatures was
manually designed resulting in primer pairs referred to
as VcatoxR-F (forward primer) and VcatoxR-R (reverse primer). The primers were synthesized by
Genset Oligos Corp.
Amplification of the toxR fragment from V. campbellii. The PCR reaction mixtures were prepared by mixing genomic DNA, 1
PCR buffer, 0.2 mM nucleotides,
0.5 mM of each primer (VcatoxR-F and VcatoxR-R)
and 0.025 U/ml Taq DNA polymerase (TaKaRa). The
reactions were then loaded into the thermocycler (Eppendorf) with the following conditions: 30 cycles of
denaturation (94C for 30 s), annealing (62 to 65C
for 30 s) and extension (72C for 30 s).
Hemolysin and toxR-targeted multiplex PCR. The
PCR reaction mixtures were prepared by mixing genomic DNA, 1
PCR buffer, 0.5 mM of MgCl2, 0.2 mM
nucleotides, 0.5 mM of all three primer sets (PNhemo,
VcatoxR primers and VhtoxR, Table 3) and 0.025 U/ml
Taq DNA polymerase (TaKaRa). The reactions were
then loaded into the thermocycler (Eppendorf) with
the following conditions: 30 cycles of denaturation
(94C for 30 s), annealing (65C for 30 s) and extension (72C for 30 s).
Purification and direct sequencing of amplicons.
The amplified 320-bp product from Vibrio sp. NRW9805 and 245-bp product from V. campbellii and 390bp product from V. harveyi were purified using the NucleoTrap PCR Purification Kit (Clontech Laboratories). After Big-Dye labeling and ethanol/EDTA
cleanup, the gene fragments were submitted to the

Medical Biotechnology and Genome Research Laboratory (NIMBB, UP Diliman, Philippines) for sequencing. Sequences were then compared to database sequences using BLAST (http://www.ncbi.nlm.nih.gov/
BLAST/).
Results

The initial step towards developing a multiplex PCR

protocol for differential detection of three types of Vibrio shrimp pathogens is to select or design PCR
primers that will result in specific amplification of target
genes from each type of pathogen.
Design of hemolysin-targeted detection primers for
variant Philippine Vibrio isolates
Sequence alignment (Fig. 1 and Table1) revealed
the percentage similarity between type strain V. campbellii and the ten Philippine Vibrio isolates which
ranged from 9397% compared to lower percentage
similarity of about 8285% between V. harveyi and the
variant Philippine isolates. Design of the PNhemo
primers (Table 2) for the specific detection of variant
Philippine Vibrio isolates was performed based on regions that exhibit conserved sequences among the ten
Philippine Vibrio isolates but exhibit deviating sequences between the Philippine isolates and the type
strains V. harveyi and V. campbellii. The deviations in
the terminal base of the primer 3 end clamping sites
in type strain V. harveyi and V. campbellii, were expected to affect annealing of the primer to genes of the
non-target Vibrios. The annealing sites for the primer
pair PNhemo are shown in Fig. 1 while the features of
the newly-designed primer set are shown in Table 2.

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CASTROVERDE et al.
Table 1.

Vol. 52

Percent hemolysin gene sequence homology among Philippine Vibrio isolates and type strains
V. harveyi and V. campbellii using ClustalW.
HOMOLOGY (%)

CRW9807

CRW-9807
PIZ-9824
NRW-9805
BR-9807
BRW-9804
CRWP-9805
BS-9904
PN-9801
SW-9702
PIZ-9806
V. campbellii
NBRC 15631
V. harveyi
NBRC 15634

PIZ9824

NRW9805

BR9807

BRW9804

CRWP9805

BS9904

PN9801

SW9702

96

99
96

99
95
99

98
94
98
98

100
96
99
99
98

97
93
96
97
97
97

98
95
98
98
99
98
97

97
94
97
98
97
97
99
97

V.
V.
PIZ- campbellii harveyi
9806
NBRC
NBRC
15631
15634
99
95
98
98
99
99
97
99
97

96
99
99
98
100
97
98
97
99

96
95
94
96
93
95
94
95

99
98
99
96
98
97
98

98
99
97
98
98
98

98
97
99
97
99

97
98
97
99

97
99
97

97
99

97

96

93

96

96

97

96

96

97

97

96

85

82

85

85

84

85

85

85

85

85

Table 2.

Sequence
Number of bases
GC content
Tm
Annealing site

96
93
96
96
97
96
96
97
97
96

85
82
85
85
84
85
85
85
85
85
79

79

Properties of the new hemolysin gene-targeted PNhemo primers.

Forward primer (5 primer)

Reverse primer (3 primer)

5-GCATTGGGTGACAGCTTGTCG-3
21
57%
66C
104124

5-CGGTTGTAGTTCATGAAGTCATTC-3
24
42%
68C
400423

Amplification of the 320-bp hemolysin fragment from

Philippine Vibrio isolates
Specific PCR detection of the 10 Philippine Vibrio
isolates using the newly designed PNhemo-F and PNhemo-R primers was optimized at 65C annealing
temperature (Fig. 2), producing a 320-bp hemolysin
gene amplicon that was not produced using type
strains V. harveyi and V. campbellii DNA templates.
Sequence analysis revealed that the 320-bp amplicon
is a partial hemolysin gene of Vibrio sp. (data not
shown).
Design of toxR-targeted PCR detection primers specific for type strain V. campbellii
A new primer set VcatoxR was designed for the specific detection of V. campbellii using a stringent anneal-

ing temperature of 65C. The primer design was focused on increasing the number of G and C bases that
was expected to produce primers with a high melting
temperature. The sequence and features of the newlydesigned primer set are shown in Table 3.
Amplification of the 245-bp toxR fragment from V.
campbellii
PCR using the newly designed V. campbellii-specific
VcatoxR primers produced the expected 245-bp toxR
amplicon (Fig. 3) when using a type strain V. campbellii DNA template and 65C annealing temperature. No
amplification was observed in PCR using primer VcatoxR with DNA templates from V. harveyi and variant
Philippine Vibrio isolates and 65C annealing temperature (data not shown). This new primer set was later

2006

Differential detection of Vibrios pathogenic to shrimp

277

Fig. 2. Optimized single PCR detection of Philippine Vibrio isolates using PNhemo primers and 65C annealing
temperature. Lanes 112 contain templates of V. campbellii, V. harveyi and the ten Philippine Vibrio isolates CRW9807, PIZ-9824, NRW-9805, BR-9807, BRW-9804, CRWP-9805, BS-9904, PN-9801, SW-9702 and PIZ-9806, respectively. Lane M contains 100-bp ladder molecular weight markers.

Table 3.

Sequences and parameters of the new V. campbellii toxR gene-targted VcatoxR primers.

Sequence
Number of bases
GC content
Tm
Annealing site

Forward primer (5 primer)

Reverse primer (3 primer)

5-CCGCTTTCTGCTGACTCTACC-3
21
57%
66C
151 171

5-GGCTTAGTCAACATCAGTACACAG-3
24
46%
70C
372 395

used together with other primers for the differential

multiplex PCR detection of Vibrio shrimp pathogens
using a stringent 65C annealing temperature.

Fig. 3. Single PCR detection of V. campbellii using VcatoxR primer pair and different annealing temperatures.
Lanes 14, PCR using templates of V. campbellii at progressively higher annealing temperatures 62C, 63C, 64C, 65C,
respectively. Lane M contains 100-bp ladder molecular weight
markers.

Differential detection by multiplex PCR

Three primer pairs (Table 4) were used in the differential detection of three kinds of Vibrio shrimp
pathogens by multiplex PCR using an annealing temperature of 65C. Distinguishable PCR profiles (Fig. 4)
were generated depending on what DNA template was
added (DNA template from type strain V. harveyi, type
strain V. campbellii, or variant Philippine Vibrio isolates). In multiplex PCR, the PNhemo, VcatoxR, and
VhtoxR (Conejero and Hedreyda, 2003) primers
specifically amplified a 320-bp Philippine Vibrio isolate
hemolysin gene fragment (Fig. 4, lanes 313), a 245bp toxR fragment from type strain V. campbellii (Fig. 4,
lanes 1 and 13), and the 390-bp toxR fragment from
type strain V. harveyi (Fig. 4, lanes 2 and 13) , respectively. When all three kinds of Vibrio shrimp pathogens
were present in the sample, three bands were ob-

278

CASTROVERDE et al.
Table 4.

Vol. 52

Summary of hemolysin and toxR -targeted primers used in multiplex PCR.

Primer
name

Primer sequence

Annealing
site

Expected
product size

Primer
design

PNhemo-F
PNhemo-R

5-GCATTGGGTGACAGCTTGTCG-3
5-CGGTTGTAGTTCATGAAGTCATTC-3

104 124a
400 423a

320 bp

In this study

VhtoxR-F

5-TTCTGAAGCAGCACTCAC-3

159 176c

390 bp

Conejero and
Hedreyda, 2003

VhtoxR-R
VcatoxR-F
VcatoxR-R

5-TCGACTGGTGAAGACTCA-3
5-CCGCTTTCTGCTGACTCTACC-3
5-GGCTTAGTCAACATCAGTACACAG-3

534 551c
151 171b
372 395b

245 bp

In this study

Nucleotide positions of the primers are relative to the first nucleotide of partial hemolysin gene sequence obtained in Philippine
Vibrio isolates.
b
Nucleotide positions of the primers are relative to the first nucleotide of partial toxR gene sequence obtained in V. campbellii
NBRC 15631 (AY946037).
c
Nucleotide positions of the primers are relative to the first nucleotide of partial toxR gene sequence obtained in V. harveyi NBRC
15634 (AY247418).

Fig. 4. Multiplex PCR using three sets of primers (PNhemo, VcatoxR, VhtoxR) and 65C annealing temperature.
DNA templates from lanes 1 and 2, V. campbellii and V. harveyi, lanes 312, ten Philippine Vibrio isolates; lane 13, V.
campbellii, V. harveyi and Philippine isolate BS-9904), and lane 14, no template control. M is a 100-bp ladder.

served in the PCR profile (Fig. 4, lane 13).

Discussion

An oligonucleotide primer pair specific to Philippine

Vibrio isolates (PNhemo primers) and targeting the hemolysin gene, was designed after sequence data
analysis and alignment of the hemolysin gene of type
strains V. harveyi and V. campbellii with ten variant
Philippine Vibrio isolates. Design of the PNhemo
primers for the specific detection of variant Philippine
Vibrio isolates was based on regions that exhibit conserved sequences among the ten Philippine Vibrio isolates but exhibit deviating sequences between the
Philippine isolates and the type strains V. harveyi and

V. campbellii. The newly designed PNhemo forward

primer exhibited one and four base sequence variations with type strains V. harveyi and V. campbellii annealing sites, respectively. The reverse primer had
only a single base deviation at the terminal base of the
primer 3 end in type strain V. harveyi and V. campbellii. The single base deviations located in the 3 end
primer clamping site of the forward primer in V. harveyi
and the reverse primers for both V. harveyi and V.
campbellii, were predicted to be crucial in preventing
annealing (at 65C) of the primers to the hemolysin
genes of non-target type strains V. harveyi and V.
campbellii. Primer annealing sites in the Philippine Vibrio isolates NRW-9805 and BS-9904, exhibited four
and one base sequence deviation from the designed

2006

Differential detection of Vibrios pathogenic to shrimp

primer, respectively. The base deviations, however,

were located in the internal regions of the primer annealing sites and are expected to be not as crucial in
preventing annealing.
Absence of amplification using the PNhemo primers
with DNA templates from both type strains V. harveyi
and V. campbellii (Fig. 2, lanes 1 and 2), suggests that
the single base pair differences at the primer 3 end
clamping site were adequate to prevent annealing of
the primers to the hemolysin genes in type strains of
both species. The PNhemo primers, on the other
hand, amplified the expected 320-bp amplicon only
from the Philippine Vibrio isolates using a stringent annealing temperature of 65C (Fig. 2, lanes 312). In
two Philippine Vibrio isolates, NRW-9805 and BS9904, the base deviations within the internal regions of
the primer annealing site did not affect annealing and
amplification of the target gene in Philippine Vibrio isolates (Fig. 2, lanes 5 and 9). As predicted, these deviations within internal regions of the annealing sites were
not as crucial in preventing annealing and amplification
as the single terminal base deviations in the primer 3
end clamping sites.
A toxR-targeted PCR primer set (VctoxR, 5-TTTCTGCTGACTCTACC-3 forward primer and 5-CAACATCAGTACACAGAC-3 reverse primer) was designed in an earlier study for the detection of type
strain Vibrio campbellii. This primer pair did not amplify
the target gene at 65C and did amplify the target
gene at 6063C but was not specific for V. campbelli
at these lower annealing temperatures (data not
shown). A new primer pair VcatoxR, with forward
primer 5-CCGCTTTCTGCTGACTCTACC-3 is a
modification of the original VctoxR forward primer and
anneals 4 bases upstream of the original annealing
site. Four bases (three cytosines and one guanine in
bold letters) were added to the 5-end of the forward
primer. The new VcatoxR reverse primer (5GGCTTAGTCAACATCAGTACACAG-3)
was
designed to anneal eight bases upstream of the original
annealing site with eight bases (including one cytosine
and three guanines in bold letters) added to the primer
5-end and two bases removed from the 3-end. The
new primers have additional cytosines and guanines
(C and G) which are expected to increase their melting
temperature and allow annealing even at the higher
stringent temperature of 65C, to the V. campbellii target toxR gene fragment but not to genes of non-target
V. harveyi or Philippine Vibrio isolates.

279

As predicted, the use of the new toxR-targeted VcatoxR primers in PCR using V. campbellii template
DNA, resulted in the production of the 245-bp amplicon at an annealing temperature of as high as 65C
(Fig. 3). No amplicons were observed in PCR using
VcatoxR primers, and the DNA template from type
strain V. harveyi or any of the Philippine Vibrio isolates. Sequence analysis (data not shown) confirmed
that the amplicon is the target toxR gene fragment.
This is an improvement in molecular diagnostics since
this new primer pair is now useful in multiplex PCR
using 65C annealing temperature.
The use of the newly designed primers (hemolysintargeted primers for detecting Philippine Vibrio isolates
and toxR-targeted PCR primers for specific detection
of type strain V. campbellii NBRC 15631) in this study,
in combination with the toxR-targeted primer specific
for the detection of V. harveyi (Conejero and
Hedreyda, 2003), will be useful in differential detection
of type strain-like V. harveyi, type strain-like V. campbellii, and strains exhibiting characteristics similar to
the 10 variant Philippine Vibrio isolates used in this
study. PCR profiles obtained in this study reveal (Fig.
4) that the presence of any of these Vibrio strains in
environmental samples, shrimp ponds, or diseased
shrimps, could be determined in a single multiplex
PCR run using the 3 sets of multiplex PCR primers
used in this study.
Acknowledgments
The authors would like to thank the NIMBB, University of the
Philippines Diliman for the research funding, University of the
Philippines System for the Creative Research Grant, Dr.
Leobert de la Pea for the Philippine Vibrio isolates, and NITE
Biological Resource Center (NBRC), Japan for the type strains
Vibrio harveyi and Vibrio campbellii.

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