VIRULENCE DETERMINATION AMONG VIBRIO HARVEYI HATCHERY ISOLATES THROUGH HAEMOLYSIS AND GROWTH CONSTRAINT

Abstract

Bacterial diseases mainly due to vibriosis in Penaeid shrimp culture implicating several species of Vibrios. Vibrio harveyi is considered as an important causative agent of the systemic vibriosis, which occurs in any bio-fields. The virulence of this bacterium is due to the production and expression of several virulent factors such as hemolysin, cysteine and metalloprotease, phospholipases, exotoxins, luciferase and siderophore. The present study intends the virulence determination among three strains of V. harveyi VSH3, VSH5 and VSH9 isolated from shrimp hatchery along the coastline of Tuticorin, Tamil Nadu during vibriosis outbreaks through their haemolysis and growth performance. Haemolytic activity examined against sheep blood resulted minor or no variations among the organisms in agar plate assay. However, in microtitre assay, Extra Cellular Products (ECP) of all isolates rendered increased activity with increase in dose and time of exposure. The bacterium VSH5 exhibited more haemolytic effect (at 500 µl concentration 72% haemolysis) than that of VSH9 and VSH3 reavled that the bacterium is highly virulent than the others. The bacterium VSH5 showed well growth in the optimum temperature (33°C), NaCl (2%) and pH (7.3). The maximum luminescence was expressed in 37°C, 2.5-3% NaCl and pH 7-9 in 18 to 48 hrs. Diverse colony morphology was observed in VSH5 on solid medium incubated for 3 to 5 days or longer. Growth curve experiment revealed that the bacterim VSH5 is a fast grower, completed its log phase within 7 hrs. Pathogenic strain like V. harveyi VSH5 causes high mortality and affects aquaculture production in hatchery as well as pond level. Hence, control measure against this kind of bacterium is urgently needed for maintain the sustainability of shrimp aquaculture in India and other Asian country.

Full text

G.J.B.B., VOL.3 (1) 2014: 109-114 ISSN 2278 –9103
109
VIRULENCE DETERMINATION AMONG VIBRIO HARVEYI HATCHERY
ISOLATES THROUGH HAEMOLYSIS AND GROWTH CONSTRAINT
K. Ramesh, M. Natarajan, H. Sridhar & S. Umamaheswari*
Microbial Biotechnology Laboratory, Department of Biotechnology
Manonmaniam Sundaranar University, Tirunelveli –627 012, India
Corresponding author * E-mail: umamsu@gmail.com
ABSTRACT
Bacterial diseases mainly due to vibriosis in Penaeid shrimp culture implicating several species of Vibrios. Vibrio harveyi
is considered as an important causative agent of the systemic vibriosis, which occurs in any bio-fields. The virulence of
this bacterium is due to the production and expression of several virulent factors such as hemolysin, cysteine and
metalloprotease, phospholipases, exotoxins, luciferase and siderophore. The present study intends the virulence
determination among three strains of V. harveyi VSH3, VSH5 and VSH9 isolated from shrimp hatchery along the coastline
of Tuticorin, Tamil Nadu during vibriosis outbreaks through their haemolysis and growth performance. Haemolytic
activity examined against sheep blood resulted minor or no variations among the organisms in agar plate assay. However ,
in microtitre assay, Extra Cellular Products (ECP) of all isolates rendered increased activity with increase in dose and time
of exposure. The bacterium VSH5 exhibited more haemolytic effect (at 500 µl concentration 72% haemolysis) than that of
VSH9 and VSH3 reavled that the bacterium is highly virulent than the others. The bacterium VSH5 showed well growth
in the optimum temperature (33°C), NaCl (2%) and pH (7.3). The maximum luminescence was expressed in 37°C, 2.5-3%
NaCl and pH 7-9 in 18 to 48 hrs. Diverse colony morphology was observed in VSH5 on solid medium incubated for 3 to 5
days or longer. Growth curve experiment revealed that the bacterim VSH5 is a fast grower, completed its log phase within
7 hrs. Pathogenic strain like V. harveyi VSH5 causes high mortality and affects aquaculture production in hatchery as well
as pond level. Hence, control measure against this kind of bacterium is urgently needed for maintain the sustainability of
shrimp aquaculture in India and other Asian country.
KEYWORDS: Vibrio harveyi VSH5, Haemolysis, Growth Parameters, Colony Variation, Growth Curve.
INTRODUCTION
Shrimp aquaculture is a most important industry in India
and other Asian countries. Shrimp hatcheries along the
coastline involved in shrimp seed production often suffer
enormous economic losses due to luminescent bacterial
disease, commonly called as vibriosis. Vibriosis outbreaks
are being increasingly recognized as a significant
constraint to aquaculture production and trade in
worldwide. The genus Vibrio belongs to the gamma-
proteobacteria and is Gram-negative, usually motile rods
(Thompson et al., 2004). Vibrio disease in aquaculture is
described as vibriosis or bacterial disease, Penaeid
bacterial septicaemia, Penaeid vibriosis, luminescent
vibriosis or red-leg disease (Aguirre-Guzmán et al., 2004).
Among the Vibrios, Vibrio harveyi (luminous Vibrio) is
the main cause of shrimp death infecting larva in the
hatchery as well as in the cultivation pond (Won & Park,
2008). V. harveyi is one of the important etiological agents
of mass mortalities of Penaeus monodon larvae rearing
systems. The virulence of V. harveyi causes 100% losses
at a time in shrimp production (Chythanya et al., 2002;
Musa et al., 2008). Luminescent strains of V. harveyi have
been reported to cause major losses in the shrimp
larviculture in the Phillippine (Lavilla-Pitogo et al., 1990),
Australia (Pizzutto & Hirst, 1995), South America
(Alvarez et al., 1998; Robertson et al., 1998) and Mexico
(Vandenberghe et al., 1999). Although almost all types of
cultured crustaceans can be affected by these bacteria, the
most serious problems have been reported in Penaeid
shrimp culturing (Austin & Zhang, 2006). Adult shrimps
suffering vibriosis may appear hypoxic, shows reddening
of the body with red to brown gills, reduce feeding and
may be observed swimming lethargically at the edges and
surface of ponds (Anderson et al., 1988; Nash et al.,
1992). Vibriosis infected post larvae (PL) exhibits reduced
motility, reduced phototaxis and empty guts (Chen, 1992).
Vibriosis is expressed by a way of number of syndromes
which include oral and enteric vibriosis, appendage and
cuticular vibriosis, localised vibriosis of wounds, shell
disease, systemic vibriosis and septic hepatopancreatitis
(Lightner, 1996). Vibriosis Infected animals shows signs
of lethargy, tissue and appendage necrosis, slow growth,
slow metamorphosis, body malformation,
bioluminescence, muscle opacity and melanisation
(Aguirre-Guzmán et al., 2004). Despite its role as a
serious pathogen of cultured marine animals, the
pathogenic mechanisms of V. harveyi yet have to be fully
elucidated (Austin & Zhang, 2006), although several
different virulent factors have been identified. Some of
the hatcheries along the coastal regions of Tuticorin, South
Tamil Nadu showed the symptoms of luminous vibriosis
during the middle of 2012. The usage of medication and
other treatments could not prevent the disease prevalence
in the hatcheries. In our previous study we isolated several

Vibrio harveyi hatchery isolates through haemolysis and growth constraint
110
vibrio pathogens during Vibriosis outbreak from the above
said regions. Among the isolates, three (VSH3, VSH5 and
VSH9) were phenotypically and genotypically identified
as V. harveyi. Hence, the present study was performed to
select the highly virulent strain among them based on their
hemolytic and growth ability.
MATERIALS & METHODS
Bacterial strains
Three strains of V. harveyi VSH3, VSH5 and VSH9 were
isolated and identified from hatchery water during
vibriosis outbreak. The stock cultures maintained in
Microbial Biotechnology Laboratory, Manonmaniam
Sundaranar University, Alwarkurichi were used as target
pathogens throughout the study.
Hemolysin test on agar plates
Hemolysin test was carried out according to the method
described by Austin et al. (2005) with slight modification.
Bacterial strains were grown overnight in marine broth at
25ºC in an incubator shaker. A drop of each of the isolate
was spotted onto freshly prepared blood agar (marine agar
containing 1% de-fibrinated sheep blood) plates. Finally,
the plates were covered with parafilm and kept in an
incubator at 30ºC for 48 hrs. The test was repeated three
times.
Haemolytic activity of ECP
Extra cellular products (ECP) of the Vibrios were obtained
by centrifuging overnight culture in a concentration of
1.5×108cells/ml and tested for haemolytic activity.
Haemolytic titrations were conducted in 96-well microtitre
plates (Tarsons, Kolkata). Five ml of blood was collected
from a healthy universal donor individual (O+ve) and
erythrocytes were collected after repeated washing in
sterile normal saline (0.85% w/v NaCl, pH 7.2) and
resuspended in normal saline to 0.5%. A volume of 0.5ml
of the cell suspension was mixed with various
concentrations of ECP (50, 100, 150, 200……450 and
500µl). Total volume of each well was made up to one ml
with normal saline. The mixtures were incubated for 1 hr
at 37ºC. Haemolytic activity was determined by the
appearance of lytic erythrocytes visibly or
microscopically. Haemolytic activity was also performed
by spectroscopic method (Yang et al., 2005). Following
incubation at 37ºC for 1 hr, the mixture was centrifuged at
1500 rpm for 10 min in a cooling centrifuge. The free
hemoglobin in the supernatants was measured in UV-
Visible spectrophotometer at 540 nm. Drabkin’s solution
(500 µl) and Saline (500 µl) were used as positive
(maximal) and negative (minimal) haemolytic controls.
Each experiment was performed in triplicates for each
concentration.
The percentage haemolysis was calculated according to
the following formula:
% Haemolysis = [At–An/Ac–An] ×100
Where, Atis the absorbance of test sample, Anis
absorbance of the negative control, Acis the absorbance of
the positive control.
Optimal condition for culturing highly virulent strain
of V. harveyi
The bacterium was cultured in both solid and liquid
medium of TSA and TSB. Colony morphology was
studied on TCBS and marine agar. The bacterium was
incubated at various temperatures (from room temperature
to 37°C), in media of various percentages of NaCl
(between 0.5-7%) and various alkalinity (pH 5 -12). Under
these physical conditions growth, colony morphology and
luminescence production were investigated.
Growth curve of virulent strain
About 200 ml of the marine broth was inoculated with the
overnight culture of test organism and kept on Orbital
shaker at room temperature. Observations for growth OD
was taken using a spectrophotometer at 600nm of
absorbance after every 15 min till the log phase was
achieved, after which readings were taken every 30
minutes. Zero reading was calibrated with un-inoculated
broth (control). Growth curve was plotted as OD 600
readings against time and different phases of growth were
hence determined.
RESULTS
Haemolytic activity in plate and microtitre assay
In order to study the haemolytic activity (hemolysin
production), certain volume of bacterial suspension of
each isolate was spot inoculated on the plates
supplemented with sheep blood. Finally opalescence or
clearing zones on plates were noted relative to colony
diameter. Haemolytic activity of each Vibrios were
examined against sheep blood and the result indicated
minor or no such variations occurring between the
organisms (Table 1). Haemolytic activities of the ECP of
isolated Vibrios were screened against normal human
erythrocytes. All the three ECP indicated haemolytic
effect. The results do not clearly report upon the visual
observation of haemolysis in microtitre plate except
negative control (button formation occurrence) but on
microscopic observation it is clear that the VSH5 showed
best haemolysis than the others. However the haemolytic
percentage increased with increase in dose and time of
exposure. Overall, VSH5 exhibited more haemolytic effect
than that of VSH9 and VSH3. The high haemolytic
activity of VSH5 (at 500 µl concentration 72%
haemolysis) suggests that the bacterium is highly virulent
in nature than the others (Fig. 1). Hence it was selected for
further studies.
TABLE 1. Haemolytic activity towards sheep red blood of three isolates of V. harveyi
Strain
Zone of Clearance
(mm)
Colony diameter
(mm)
Ratio
V. harveyi VSH3
20.8±0.2
15.4±0.1
5.3±0.2
V. harveyi VSH5
20.9±0.5
15.1±0.1
5.8±0.5
V. harveyi VSH9
23.1±0.2
17.5±0.2
5.6±0.4
Results are expressed as mean ± standard deviation of three replicates

Vibrio harveyi hatchery isolates through haemolysis and growth constraint
appeared only on solid medium incubated for 3 to 5 days
or longer. The variation was not evident upon daily
subculture. Growth curve experiments revealed that VSH5
is a fast growing bacterial isolate that reached its log phase
after 30 min of incubation at room temperature with
continuous shaking of 150 rpm. The log phase continues
up to 7.0 hrs after which the stationary phase seemed to
start (Fig. 3).
FIGURE 2. Various colony types of VSH5: (A) clouded green small rough colonies on TCBS (B) Smooth colonies with
black pigments on TCBS (C) Small and big margin colonies on marine agar (D) Small dark centered yellow halo around
colonies (E) Clear dark centered green colonies (F) Rough medium sized green unshaped colonies (G) large colonies with
dark green round (H) Large elevated dark green to yellow colonies and (I) Colonies partially digested.
FIGURE 3. Growth curve of V. harveyi VSH5 in Marine broth
DISCUSSION
Nakayama et al. (2006) observed that the ECP of V.
harveyi VP1 showed higher haemolytic activity compared
with VT2 and other strains. A similar trend was also
observed with the strain of V. harveyi VSH5 isolated in the
present study. Several reports have evaluated the
pathogenicity of environmental isolates of V. harveyi to
larva and juvenile penaeid shrimps (Harris & Owen, 1999;
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0
30
60
90
120
150
180
210
240
270
300
330
360
390
420
450
480
510
540
570
Absorbance (OD at 600 nm)
Time in minutes
Absorbance (O.D at 600 nm)
112

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Pizzutto & Hirst, 1995; Ruangpan et al., 1999). One of the
reasons for variation in the virulence levels of Vibrios
reported in this study revealed that infectivity of V.
harveyi is dependent on the virulence factors of the strains
employed (Gomez-Gill et al., 1998). Some studies indicate
that the virulence factors produced by V. harveyi can be
contributed from toxins (either protease or hemolysin)
(Liu & Lee, 1999; Zhang & Austin, 2000; Zhang et al.,
2001). However, other studies represented that the
pathogenicity of V. harveyi is derived from phage in which
genes coding for toxin production are acquired by gene
transduction (Morris & Robert, 1995). The toxin
production in bacteria may be controlled by gene
transduction but some bacteria have been found to express
toxin by a process called quorum sensing (Bernd et al.,
2001; Costi et al., 2002). It is reported that the bacterial
luminescense is produced by different autoinducer in each
genus or species. The major autoinducer of V. harveyi has
been reported to be a long chain aliphatic aldehyde. Lux
gene expression triggers the synthesis and accumulation of
autoinducer during the growth of bacteria. The electron
transport proceeds by the catalytic reaction of luciferase
among the reduced flavin mononucleotide (FMNH2), O2
and a long chain aliphatic aldehyde produces flavin
mononucleotide (FMN) and an aliphatic carboxylic acid
which emits the light (Fisher et al., 1995). The results of
the present study suggested that the temperature may also
influence the expression of luminescence which is in
accordance with the study of Pasharawipas et al., (1998).
The temperature may either stimulate luciferase activity or
the production or function of the autoinducer. The
bacterium VSH5 does not grow at higher temperatures
such as 37°C and above as the temperature may affect the
production and activities of luciferase or autoinducer. The
present study also reports that the luminescence expression
was affected by pH. Optimum pH (7-9) resulted in strong
luminescence which also correlates with the study of
Pasharawipas et al. (1998). The combination of
temperature and alkalinity might find some application in
manipulation of V. harveyi for higher production of poly-
3-hydroxybutyrate (PHB), a raw material in plastic
industry due to its properties like thermoplasticity, water
resistance and biodegradability. It was previously reported
that the production of PHB is related to luminous
expression controlled by the lux autoinducer (Sun et al.,
1994).The variation in colony morphology of VSH5 is of
somewhat interest. Similar variation was obtained by
Pasharawipas et al.(1998) during sub-culturing of V.
harveyi VH1039 on TSA and TCBS. The variability of
VSH5 might involve the fact that it is a lysogenic host of
temperate phages which are rarely found in culture
environment. The variability of other bacteria has also
been reported to be due to transposon like behaviour of
bacteriophages (Reidl & Makalanos, 1995; Belas et al.,
1984). This may be the best explanation for variable
morphology of bacterial colonies since the biochemical
tests did not change for each colony. The growth curve
result of strain VSH5 is in corroboration with the findings
of Aisha & Nuzhat (2011) who recorded the growth curve
of V. harveyi N6. Mortality among the cultured shrimp in
hatcheries is due to the presence of highly virulent strain
like V. harveyi VSH5 with different colony morphology
and growth performance. This proves that there is an
urgent need for a new eco-friendly preventive measure
against the Vibrio pathogens in shrimp aquaculture to
overcome quality seeds (larvae) exports.
Study to determine the virulence levels of the bacterial
pathogen in aquatic animals is a key to prevent vibriosis in
marine aquaculture. The virulence of V. harveyi is
reported dependent on host species (Vera et al., 1992),
doses, time exposure and age of host species (Jun &
Huaishu, 1998) and pathogenic factors of the bacterial
strains (Gomez-Gill et al., 1998). This paper describes the
virulence of three strains of V. harveyi isolated from
shrimp hatchery water based on their haemolytic activity
and growth performance. Highly virulent strain like VSH5
causes mortality on animals and affects aquaculture
production in hatchery level. Hence, control measure
against this kind of bacterial pathogen is urgently needed
for sustainability of shrimp aquaculture in India and other
Asian country.
REFERENCES
Aguirre-Guzmán, G., Meija Ruíz, H. and Ascencio, F.
(2004) A review of extracellular virulence product of Vibrio
sp. important in disease of cultivated shrimp. Aquaculture
Res. 35, 1395-1404.
Aisha, N. and Nuzhat, A. (2011) Isolation and
characterization of indigenous luminescent marine bacteria
from Karachi coast. Acad. Res. Int. 1, 74-83.
Alvarez, J. D., Austin, B., Alvarez, A. M. and Reyes, H.
(1998) Vibrio harveyi: a pathogen of penaeid shrimps and
fish in Venezuela. J. Fish Dis.21, 313-316.
Anderson, I. G., Shamsudin, M. N. and Shariff, M. (1988)
Bacterial septicemia in juvenile tiger shrimp, Penaeus
monodon, cultured in Malaysian brackish water ponds. Asian
Fis. Sci. 2, 93-108.
Austin, B. and Zhang, X. H. (2006) Vibrio harveyi: a
significant pathogen of marine vertebrates and invertebrates.
Lett. Appl. Microbiol. 43, 119-124.
Austin, B., Austin, D., Sutherland, R., Thompson, F. and
Swings, J. (2005) Pathogenicity of vibrios to rainbow trout
(Oncorhynchus mykiss, Walbaum) and Artemia
nauplii. Environ. Microbiol. 7, 1488-1495.
Belas, R., Mileham, A., Simon, M. and Silverman, M. (1984)
Transposon mutagenesis of marine Vibrio sp. J.
Bacteriol. 158, 890-896.
Bernd, K., Michael, D. P. B., Bettina, A. B., Markus, H. and
Andreas, P. (2001) Group A Streptococcal growth phase-
associated virulence factor regulation by a novel operon
(Fas) with homologies to two-component-type regulators
requires a small RNA molecule. Mol. Microbiol. 39, 392-
406.
Chen, D. (1992) An overview of the disease situation,
diagnostic techniques, treatments and preventatives used on
shrimp farms in China. In: Fuls, W. and K. L. Main (Ed.):
Diseases of cultured penaeid shrimp in Asia and the Unites
States. Hawaii, pp. 47-55.

Vibrio harveyi hatchery isolates through haemolysis and growth constraint
114
Chythanya, R., Karunasagar, I. and Karunasagar, I. (2002)
Inhibition of shrimp pathogenic vibrios by a marine
Pseudomonas I-2 strain. Aquaculture 208, 1-10.
Costi, D. S., Eleftherios, M., Kavindra, V. S., Xiang, Q.,
Danielle, A. G., Murray, B. E., Ausubel, F. M. and
Calderwood, S. B. (2002) Virulence effect of Enterococcus
faecalis protease genes and the quorum-sensing locus fsr in
Caenorhabditis elegans and mice. Infect. Immunol. 70, 5647-
5650.
Fisher, A. J., Raushel, F. M., Baldwin, T. O. and Rayment, I.
(1995)Three dimensional structure of bacterial luciferase
from Vibrio harveyi at 2.4 A° resolution. Biochem. 34, 6581-
6586.
Gomez-Gill, B., Herrera-Vega, M. A., Abreu-Gobois, F. A.
and Roque, A. (1998) Bioencapsulation of two diferent Vibrio
species in nauplii of the brine shrimp (Artemia franciscana).
Appl. Environ. Microbiol. 64, 2318-2322.
Harris, L. J. and Owens, L. (1999) Production of exotoxins by
two luminous Vibrio harveyi strains known to be primary
pathogens of Penaeus monodon larvae. Dis. Aquat. Org.38,
11-22.
Jun, L. I. and Huai-Shu, X. (1998) Isolation and biological
characteristics of Vibrio harveyi affecting hatchery-reared
Penaeus chinensis larvae. Chin. J. Oceanol.Limnol. 29, 353–
361.
Lavilla-Pitogo, C. R. Baticados, M., C. L., Cruz Lacierda, E.
R. and de La Peña L. D. (1990) Occurrence of luminous
bacterial disease of Penaeus monodon larvae in the
Philippines. Aquaculture 91, 1-13.
Lightner, D. V. (1996) A Handbook of pathology and
diagnostic procedures for diseases of Penaeid shrimp. World
Aquaculture Society, Baton Rouge, Louisiana, USA.
Liu, P.C. and Lee, K. K. (1999) Cysteine protease is a major
exotoxin of pathogenic luminous Vibrio harveyi in the tiger
prawn, Penaeus monodon.Lett. Appl .Microbiol.28, 428-
430.
Morris, P. and Robert, G. H. (1995) Classification of isolates
of Vibrio harveyi virulent to Penaeus monodon larvae by
protein profile analysis and M13 DNA fingerprinting. Dis.
Aquat. Org. 21, 61-68.
Musa, N., Wei, L. S. and Wee, W. (2008) Phenotypic and
genotypic characteristics of Vibrio harveyi isolated from
black tiger shimp (Penaeus monodon). World J. Appl. Sci. 3,
885-902.
Nakayama, T., Nomura, N. and Matsumura, M. (2006) Study
on the relationship of protease production and luminescence
in Vibrio harveyi. J. Appl. Microbiol. 101, 200-205.
Nash, G., Nithimathachoke, C., Tungmandi, C.,
Arkarjarmorin, A., Prathampipat, P. and Ruamthaveesub P.
(1992) Vibriosis and its control in pond-reared Penaeus
monodon in Thailand. In: Shariff, I.M., R. P. Subasinghe and
J. R. Arthur (Ed.): Diseases in Asian Aquaculture. The
Phillippines, pp. 143-155.
Pasharawipas, T., Sriurairatana, S., Direkbusarakom, S.,
Donayadol, Y., Thaikua, S., Ruangpan, L. and Flegel, T. W.
(1998) Luminous Vibrio harveyi associated with tea brown
gill syndrome in black tiger shrimp. In: Flegel, T. W. (Ed.):
Advances in shrimp biotechnology, National Center for
Genetic Engineering and Biotechnology, Bangkok.
Pizzutto, M. and Hirst, R. G. (1995) Classification of isolates
of Vibrio harveyi virulent to Penaeus monodon larvae by
protein profile analysis and M13 DNA fingerprinting. Dis.
Aquat. Org. 21, 61-68.
Reidl, J. and Mekalanos, J. (1995) Characterization of Vibrio
cholera bacteriophage K139 and use of a novel mini-
transposon to identify a phage-encoded virulence factor. Mol.
Microbiol. 18, 685-701.
Robertson, P. A. W., Calderon, J., Carrera, L., Stark, J. R.,
Zherdmant, M. and Austin, B. (1998) Experimental Vibrio
harveyi infections in Penaeus vannamei larvae. Dis. Aquat.
Org. 32, 151-155.
Ruangpan, L., Danayadol, Y., Direkbusarakom, S.,
Siurairatana, S. and Flegel, T. W. (1999) Lethal toxicity of
Vibrio harveyi to cultivated Penaeus monodon induced by a
bacteriophage. Dis. Aquat. Org. 35, 195–201.
Sun, W., Cao, J. G., Teng, K. and Meighen, E. A. (1994)
Biosynthesis of poly 3-hydroxybutyrate in the luminescent
bacterium, Vibrio harveyi and regulation by the lux
autoinducer, N-(3-hydroxybutanoyl) homoserine lactone. J.
Biol. Chem. 269, 20785-20790.
Thompson, F. L., Iida, T. and Swings, J. (2004) Biodiversity
of vibrios. Microbiol. Mol. Biol. Rev. 68, 403-431.
Vandenberghe, J., Verdonck, L., Robles Arozarena, R.,
Rivera, G., Bolland, A., Balladares, M., Gomez-Gil, B.,
Calderon, J., Sorgeloos, P. and Swings, J. (1999) Vibrios
associated with Litopenaeus vannamei larvae, postlarvae,
broodstock, and hatchery probionts. Appl. Environ.
Microbiol. 65, 2592-2597.
Vera, P., Navas, J. I. and Quintero, M. C. (1992)
Experimental study of the virulence of three species of
Vibrio bacteria in Penaeus japonicus (Bate 1881) juveniles.
Aquaculture 107, 119-123.
Won, K. M., Park, S. (2008) Pathogenicity of Vibrio harveyi
to cultured marine fishes in Korea. Aquaculture 285, 8-13.
Yang, Z. G., Sun, H. X. and Fang, W. H. (2005) Haemolytic
activities and adjuvant effect of Astragalus membranaceus
saponins (AMS) on the immune responses to ovalbumin in
mice. Vaccine 23, 5196-5203.
Zhang, X. H, and Austin, B. (2000) Pathogenicity of Vibrio
harveyi to Salmonids. J. Fish Dis. 23, 93-102.
Zhang, X. H., Meaden, P. G. and Austin, B. (2001)
Duplication of hemolysin genes in a virulent isolate of Vibrio
harveyi.Appl. Environ. Microbiol. 67, 3161-3167.