Aquaculture

Health & Disease

Management
Issue 1 - June 2010

Asian vaccine protects grouper

juveniles against vibriosis

Published by

Disease control & management in the global aquaculture sector

 Aquaculture Health & Disease Management | Vibriosis
Asian vaccine protects grouper
juveniles against vibriosis
This report from Thailand describes a trial in which an injectable vaccine successfully protected orange-
spotted grouper from an experimental challenge with Vibrio pathogens
by John S Clark, Jirasak Tangtrongpiros and Nantarika Chansue
V
accinating juvenile fish by
injection has given a high
level of protection against
bacteria causing vibriosis, in
a study involving cultured
orange-spotted grouper in Thailand. The
bivalent Vibrio vaccine was administered
intraperitoneally to each fish. Although
this method of administration is difficult
to apply in routine farm management,
the evidence from the study strongly
suggests it should be a good prophylactic
measure against mortalities due to Vibrio
pathogens.
The orange-spotted grouper
Epinephelus coioides has formed part of
a diversification of culture in Thailand over
recent years (de Silva and Phillips, 2007)
that illustrates the expanding diversity of
Septicaemia of hepatic tissue
marine fish species being farmed in Asian
aquaculture. Across the region, however,
vibriosis has caused catastrophic eco-
nomic losses in higher value species such
Vibriosis | Aquaculture Health & Disease Management
as grouper as well as the snapper and
pompano.
A most common disease
Vibriosis is the most common disease
affecting the culture of marine finfish in
the Asian region and many epizootics have
been attributed
in particular
Table 1: Means of water quality parameters.
to Vibrio
Parameters Mean level and S.D.
cholerae.
Conventional
treatments Temperature
regionally use
antibiotics.
Unfortunately,
pH
Salinity
these are often
applied incor-
Hardness
rectly. Therefore they fail to control the
disease and they can lead to the spread of
bacterial resistance.
Many other problems are
associated with the use of
antibiotics. For example, the
efficiency of oral delivery is
questionable because sick fish
often do not eat. Antibiotics
that are water soluble may
leach from feed. The efficacy
of using some antibiotics such
as oxytetracycline is highly
reduced where they form
complexes with calcium and
magnesium ions in seawater
or hard water. Technically, of
course, it should always be remembered
that antibiotics cannot work unless they
are delivered to the correct target organ
or tissue at the appropriate concentration.
Also, by their very nature, they act mainly
on bacterial pathogens; they have no direct
effect on viruses and other pathogens.
Vaccines, by contrast, can act against
both bacteria and viruses and there is
even experimental evidence that they can
be active against parasitic infections. But
Parameters Mean level and S.D.
27.5±0.5°C Alkalinity 60±19.27 ppm
7.66±0.23
34ppt
Ammonia
Nitrite
0.88±0.77 ppm
1.56±1.55 ppm
>1000 ppm 23.15±18.61bc 77.59
it is also important to remember another
difference in the fact that antibiotics cure
whereas vaccines prevent. So while the
application of an antibiotic is a curative
measure or therapy against an existing
infection, a vaccine is applied preventively
to prepare the immune system before a
specific challenge.What is more, the protec-
tion secured by vaccinating normally lasts
longer than that from applying antibiotics.
Vaccination reduces
antibiotic useage
Data from Norway show that vaccina-
tion of fish has greatly reduced the use of
antibiotics in Norwegian salmon produc-
tion. Over a period of two decades while
salmonid production increased by a factor
of 20 times, the weight of antibiotics used
each year dropped from a high of over
50 tonnes in 1984 to less than 2 tonnes
Aquaculture Health & Disease Management | Vibriosis
in 2004. Additionally, the situation where fish were then held for 21 days with daily from moribund sea bass and cultured at the
the only fish vaccines available were for monitoring for mortality. Veterinary Medical Aquatic Animal Research
salmonids is now changing. New vaccines Centre of the Faculty of Veterinary Science
are being registered in Asia for Asian spe- Monitoring water quality at Chulalongkorn University, Thailand.
cies, such as yellowtail in Japan, grass carp in As with all 840 fish involved in the Bacteria were identified using API® 20E
China and a recently launched commercial study, these grouper
vaccine for use in Asian sea bass, tilapia and had been purchased
other species in some south-east Asian from a private farm
countries (Komar et al., 2005). in Thailand when they
Various studies have been carried out
into the efficacy of various Vibrio vaccine
types on the survival of different grouper
were approximately
5.75g in weight and
approximately 4.31 cm
species. In the present study, juvenile
grouper were injected with a bivalent Vibrio
vaccine prepared (NovaQsol Holdings Inc.,
in length. There were
kept in plastic aquaria
for an acclimatization
Philippines) from two virulent strains of period of 14 days and
Figure 1. Determination of LD50 and LD100 for V. chol-
V. harveyi and V. parahaemolyticus. These the sacrifice of two
erae in juvenile spotted grouper.
strains had been isolated from diseased, fish to verify that they
moribund fish located in a farm near Phuket, were free of Vibrio.
Thailand. Throughout the study, half of the water in (BioMerieux, Madrid, Spain) and the API
The vaccine’s safety was established each tank was exchanged every alternate profiles compared with the API database
by testing on a total of 240 juvenile day. A commercial test kit (AQUA-VBC, (Apilab Plus, version 3.3.3.; BioMerieux).
orange-spotted grouper which had been Thailand) was used to monitor water qual- To establish virulence, doses ranging
separated into four groups of 60 fish ity parameters of pH, hardness, ammonia, from 103 to 1010 CFU of bacteria per fish
each. Sub-groups of 20 fish within each nitrite and alkalinity. Salinity was measured were injected intra-peritoneally into 540
group either were injected with 0.05 ml each week with a refractometer. orange-spotted juveniles. Each dose had
or 0.10 ml of vaccine, or given a 0.10 ml The experimental bacterial challenge 3 replicates with 20 fish in each replicate.
saline injection as a positive control, or for the vaccine assessment consisted of Control groups were challenged with sterile
left uninjected. Immunised and control Vibrio cholerae that had been isolated
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Aquaculture Health & Disease Management | Vibriosis
 Aquaculture Health & Disease Management | Vibriosis

0.9 % (v/v) saline. The aim was to find
pathogen doses that would kill 50% and
100% of injected fish within three days, so
that the mean lethal dose could be calcu-
Measures to sustain a
successful aquaculture
(Tan et al., 2006)
Recommended
1. Use healthy (not necessarily
cheap) fry
2. Quarantine incoming animals
3. Use formulated pelleted feed
4. Grade fish periodically
5. Monitor water quality
were significantly lower than for all infected
groups. Although not significantly different
from the 0.10 ml vaccinated fish for per-
centage mortality, the 0.05 ml vaccine group
registered a lower RPS of 45.76%.
Higher survival and
lower mortality
Clearly, therefore, injecting 0.10 ml of
the bivalent vaccine gave a higher rela-
tive survival and the lowest mortality rate
of any group in the test. The test also
demonstrated that the European model of
using vaccination as a primary prophylactic
tool could be applied successfully in Asia to
reduce losses in spotted grouper juveniles
Acute peritonitis from a Vibrio infection.
This points to a new direction in Asian
grouper culture for health management. So
lated by the Reed and Muench procedure as far, the development of effective strategies in
the basis for deciding the optimal challenge Asia for disease prevention and control has
dose to be used during testing of vaccine been hampered by a lack of data on the wide
efficacy. variety of disease-causing agents present and
Figure 1 illustrates the clear dose the immune systems of Asian fish species.
response curve found from the virulence Technical support in such matters as disease
assays. The LD100 dose (killing 100% of diagnosis is often lacking at farm level.
fish within three days) for this particular There is also a stark contrast between
isolate of Vibrio cholerae was 1x1010 CFU/ the Asian situation and modern salmonid
ml, with the LD50 established as 1x108 aquaculture in countries such as Norway.
CFU/ml. Intensified salmon production has brought
At 21 days after immunization, the vacci- a separation between the hatchery sites
nated and control fish were challenged with producing fry and those for grow-out,
the LD100 as a 0.10 ml injection adminis- together with optimized feed strategies and
tered intraperitoneally while the positive virtually disease-free fingerlings. However,
controls received 0.10 ml of sterile saline. the segregation in year classes that is
Monitored for three days post-challenge, obligatory for salmon in Europe does not
Authors
Dr John S Clark obtained his Ph.D.
in micro-encapsulation at Heriot-Watt
University in Edinburgh, Scotland in 1983.
He currently works on formulation of
anti-parasitics, vaccines, nutritional/
health products for a variety of clients.
Email: aquavax2003@yahoo.co.uk
Dr Nantarika Chansue is the
Associate Professor and Director
of the Veterinary Medicine Aquatic
Animal Research Centre (VMAARC) at
Chulalongkorn University in Bangkok.
She specializes in disease identifica-
tion and control methods for shrimp,
marine and freshwater fish, turtles and
aquatic mammals. Aquatic immunoto-
xicology has been her major research
interest for some 20 years.
Professor Jirasak Tangtrongpiros
is the head of the Veterinary Medicine
Aquatic Animal Research Centre
(VMAARC) at Chulalongkorn
University in Bangkok, Thailand. He
specialises in aquatic animal disease
and his primary interest is in methods
for disease prevention and control.
production, development and sustainable
expansion of marine fish aquaculture in
the Asian region. Most Asian aquaculture
is operated on a small scale. Added to the
other factors mentioned and the diversity
of organisms in tropical waters, this contrib-
utes to a truly challenging disease situation
in which pathogens have various opportuni-
ties for entry points. Only now are moves
being made to correct the failure to collect,
report and analyse epidemiological data in
the event of a disease outbreak.
On a more positive note, more attention
has been given in past few years to the iden-
tification of aetiological agents involved in
fish disease epidemics. The following studies
give a solid indication of the diversity of
major pathogens affecting the fish farming
industry in Asia (Bondad-Reantaso et al.,
2005; Komar et al., 2005; Labrie et al., 2005;
Leong et al., 2005, 2006 and Tan et al., 2003).
Meanwhile, significant progress has been
made in the field of vaccine research and
development (Grisez and Tan, 2005).
But as Asian aquaculture continues to
grow, inevitably its problems with disease
will worsen unless key steps are taken. New
disease control concepts must be imple-
mented. This includes the use of healthy
fry, quarantine measures, optimized feeding
and good husbandry techniques. Monitoring
diseases by surveillance and reporting is
essential, as is good sanitation and an
emphasis on being responsible in the use
of chemicals and antibiotics when diseases
occur.
There will certainly be a role for vac-
cination as the overall emphasis changes
to preventing diseases rather than treating
or curing them. Vaccination on its own is
not sufficient on its own to guarantee high
survival and profitability (see box), but it is
one of the tools to employ in good health
management strategies.
Acknowledgements
The authors would like to convey their
thanks to all the staff of VMAARC in
Chulalongkorn University, Bangkok
Thailand, and to the supporting staff of
the Research Institute at Rayong, Rayong
Province, Thailand, and finally to Drs. Gee,
Na, Lin and Jim for their endless patience.
References are available on request.

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6. Record diseases and feeding
7. Observe withdrawal period of
drugs
8. Remove dead fish at least once a
day
9. Clean and disinfect equipment
10. Vaccination if available
NOT recommended
1. Place your farm too close to
others
2. Have several species in one farm
3. Use fingerlings from unknown
sources
4. Overstock (to overcome low
survival)
5. Use trash fish
6. Overfeed
7. Use drugs without diagnosis
8. Leave or throw dead fish in the
water
9. Restock fish without cleaning the
cages
10. Ignore diseases until heavy mor-
tality occurs
there was a 100% mortality rate for the fish yet exist in Asia.
in the positive control group. All dead fish in Asian farms also commonly produce
the challenge experiment were confirmed different species of fish at the same site,
by re-culture and re-identification using API
20E as positive for Vibrio cholerae infection.
they make widespread use of trash fish as
feed and their fry are often wild-caught or audience of industry
professionals
Results of the efficacy test in Table 1 derived from wild-caught brood stock. With
confirmed that giving 0.10 ml of the vaccine farm licensing and zoning policies virtually
provided the highest rate of protection non-existent in most Asian countries, the
against Vibrio cholerae.This vaccination gave spread of diseases is promoted by having
relative percentage survival (RPS) values of too many fish and too many farms in a • You can advertise within this space for £1139
up to 77.59% compared with the relevant concentrated area. or see the back page of this document for other prices & sizes
controls. Its percentage mortality rates It explains why the salmon farms of
Norway or Chile consider a • The advert size can be changed to suit your existing artwork
survival rate of lower than • Advertising space within this feature is limited and sold on a first
Table 2: Percentage mortality of juvenile orange-spotted grouper,
non-vaccinated and vaccinated by two intraperitoneally injected 95% as a sign of a disease come first served basis, so hurry to ensure you get the space you
doses of a Vibrio vaccine and relative percentage survival (RPS) outbreak, whereas a survival Display advertising:
require
after challenge with Vibrio cholerae. rate of 50% at fish farms in
Asia is often considered to Ad size price • Deadline for advertising is 18/06/10
Group Mortality* RPS
be acceptable. Contact Caroline Wearn today to book your space:
Full page £2036
Tel: +44 1242 267706
NC Control, no infection 2.63±3.72c 97.44
The biggest Mini page £1504
Email: carolinew@aquafeed.co.uk
PC Control, normal saline IP 100.00±0.00a 0.00 constraint to Half page £1139
T1 0.05 ml. vaccine IP 54.04±31.73b 45.76 procution Third page £772
Disease undoubtedly
T2 0.10 ml. vaccine IP 23.15±18.61bc 77.59
represents one of the big- Quarter page £625
Data was subjected to two way analysis of variance using an SPSS
program. Significant differences were determined at P<0.05. gest constraints to the Eighth page £457
 Aquaculture
Health & Disease
Management
Vibriosis

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Disease control & management in the global aquaculture sector