Veterinary World, EISSN: 2231-0916
Available at www.veterinaryworld.org/Vol.12/November-2019/17.pdf
REVIEW ARTICLE
Open Access
Live vaccines against bacterial fish diseases: A review
Aslizah Mohd-Aris1,2, Mohd Hafiz Ngoo Muhamad-Sofie2, Mohd Zamri-Saad2,3, Hassan Mohd Daud2,4 and
Md. Yasin Ina-Salwany2,5
1. Department of Biology, School of Biology, Faculty of Applied Sciences, Universiti Teknologi MARA, Cawangan Negeri
Sembilan, Kampus Kuala Pilah, Malaysia; 2. Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra
Malaysia, Serdang, Malaysia; 3. Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine,
Universiti Putra Malaysia, Serdang, Malaysia; 4. Department of Veterinary Clinical Studies, Faculty of Veterinary
Medicine, Universiti Putra Malaysia, Serdang, Malaysia; 5. Department of Aquaculture, Faculty of Agriculture, Universiti
Putra Malaysia, Serdang, Malaysia.
Corresponding author: Aslizah Mohd-Aris, e-mail: aslizah@uitm.edu.my
Co-authors: MHNM: sofiehafizngoo@gmail.com, MZ: mzamri@upm.edu.my, HMD: hassanmd@upm.edu.my,
MYI: salwany@upm.edu.my
Received: 29-07-2019, Accepted: 11-10-2019, Published online: 21-11-2019
doi: www.doi.org/10.14202/vetworld.2019.1806-1815 How to cite this article: Mohd-Aris A, Muhamad-Sofie MHN,
Zamri-Saad M, Daud HM, Ina-Salwany MY (2019) Live vaccines against bacterial fish diseases: A review, Veterinary World,
12(11): 1806-1815.
Abstract
Fish diseases are often caused either by bacteria, viruses, fungi, parasites, or a combination of these pathogens. Of these,
bacterial fish diseases are considered to be a major problem in the aquaculture industry. Hence, the prevention of such
diseases by proper vaccination is one of the integral strategies in fish health management, aimed at reducing the fish
mortality rate in the aquaculture farms. Vaccination offers an effective yet low-cost solution to combat the risk of disease in
fish farming. An appropriate vaccination regime to prevent bacterial diseases offers a solution against the harmful effects
of antibiotic applications. This review discusses the role of live-attenuated vaccine in controlling bacterial diseases and
the development of such vaccines and their vaccination strategy. The current achievements and potential applications of
live-attenuated and combined vaccines are also highlighted. Vaccine development is concluded to be a demanding process,
as it must satisfy the requirements of the aquaculture industry.
Keywords: aquaculture, attenuated vaccines, bacterial fish diseases, vaccination.
Introduction
Aquaculture contributes significantly to the
global production sector, particularly in meeting
the increased demand for high-quality food.
Approximately 44% of the total global fish production
is contributed by aquaculture [1]. As reported by Food
and Agriculture Organization [2], the majority of
the fish produced by aquaculture is used for human
consumption. Therefore, to meet the market demands,
several difficulties need to be addressed by the aquaculture sector, including natural variables such as
ecological impacts, poor water quality, and disease
infestations [3-5]. The current strategy to increase
aquaculture productivity is based on intensification and increased commercialization of aquaculture
products [6]. However, efforts for rapid intensification
by aquaculture sectors may have adverse ramifications, such as disease outbreaks [7], which are a major
impediment to the growth of aquaculture [8].
Disease outbreaks have socio-economic impacts
since the cultures of many aquatic species sustain
severe losses. There might be a loss of investment and
Copyright: Mohd-Aris, et al. Open Access. This article is distributed
under the terms of the Creative Commons Attribution 4.0
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License
(http://creativecommons.org/licenses/
by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit
to the original author(s) and the source, provide a link to the
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The Creative Commons Public Domain Dedication waiver (http://
creativecommons.org/publicdomain/zero/1.0/) applies to the data
made available in this article, unless otherwise stated.
Veterinary World, EISSN: 2231-0916
consumer confidence, food shortage due to industry
failure, or cessation of aquaculture operations [6].
Consequently, the production rate, income ability,
power of employment, market access or market shares
can be affected. Several cases of disease outbreaks,
particularly in the Asia-Pacific region, have been
reported. For instance, more than 30% of the total yield
loss was estimated in China, India, and Vietnam, due to
fish diseases [9]. In the Philippines, fish diseases have
resulted in a 75% reduction in household income, and
a 19.4% increase in debt [10]. Moreover, it has been
reported the rainbow trout (Oncorhynchus mykiss)
industry losing its sale for 29.1 million fish during
2018, which 92% loss incurred, due to diseases [11].
Many factors contribute to the susceptibility of
cultured fish to pathogens. In particular, the viability of
pathogens inside the fish and in the water sources often
increases the chances of infection [6]. A study by Albert
and Ransangan [5] revealed that deterioration of water
quality can increase the susceptibility of fish to infection and diseases such as vibriosis. They reported an
increase in fish mortality during periods of high water
temperature, which was consistent with high counts
of Vibrio bacteria in the diseased fish, water column,
and biofilm [5]. Furthermore, Bowater et al. [4] suggested that pollutants, such as heavy metals, present
in the environment may increase the hosts’ susceptibility to disease. According to Shefat [12], bacterial
diseases are most prevalent in farmed fish. Previous
reports on bacterial fish diseases have suggested motile
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aeromonad septicemia, edwardsiellosis, flexibacteriosis,
columnaris, yersiniosis, and bacterial gill disease as the
most common diseases [13]. The bacterial strains capable of predisposing the host to disease are referred to as
primary pathogens, which are not always host-specific.
Some bacterial strains, particularly those infecting
already weakened or damaged hosts, are categorized as
opportunistic pathogens [14].
In cases of vibriosis in Malaysia, Vibrio harveyi
was most frequently isolated, followed by Vibrio
parahaemolyticus, Vibrio alginolyticus, and Vibrio
anguillarum [5]. Vibrio species, including V. alginolyticus, V. harveyi, and V. parahaemolyticus, were also
found to be the causative agents infecting large yellow croakers (Pseudosciaena crocea (Richardson)) in
China [15]. Table-1 [13-19] summarizes some common fish bacterial diseases [13-17], their causative
agents [13-17], the main hosts [13-17], and the commercial vaccines available [18,19]. The table clearly
shows that these bacteria are not host-specific, indicating that cross infections can occur between fish
infected with different pathogens, and that such diseases are induced by several factors.
importantly, vaccines do not have any side effects,
in terms of inducing pathogen resistance, compared to antibiotics [6,22,24]. However, once a disease outbreak occurs, the application of vaccines is
pointless [22].
Vaccines play a significant role in inducing an
immune response and increasing the resistance to
diseases in the host’s system. The immune system of
the host will remain sensitized and ready to respond
to the pathogens encountered by the host [22]. In
fish vaccine development, studies have focused
on vaccine formulation, development of vaccination regimes, and the protective efficacy of these
vaccines. Several types of vaccines, such as killed
whole-cell [25-27], live-attenuated [28-34], DNA
vaccine [35,36], subunits [37-39], anti-idiotypic [40],
and toxoid vaccines [22], have already been developed. To date, most commercially available and
authorized vaccines used in the aquaculture industry
are killed whole-cell vaccines. Other types of vaccines
are being developed, but they are still at the experimental stage or under live animal clinical studies.
Disease Prevention in Aquaculture
The killed whole-cell vaccine, also known as
bacterin, is a common type of bacterial vaccine.
Bacterin and inactivated vaccines are commercially
available and authorized to be used in the aquaculture industry [41]. These vaccines are created using
physical (heat) and chemical mutagenesis, usually
with formalin or chloroform [42]. Adjuvants are often
added to these vaccines, as immune potentiators or
vaccine carriers [43], to increase the vaccine’s efficiency of inducing a potent immune response [27,42].
Firdaus-Nawi et al. [44] demonstrated the increased
effectiveness of killed whole-cell vaccines added with
adjuvants. They found that the addition of incomplete Freund’s adjuvant (20% v/v) to a formalin-killed
Streptococcus agalactiae vaccine resulted in 100%
survival of red tilapia intraperitoneally (IP) challenged
with S. agalactiae, compared to only 50% survival with
non-adjuvanted vaccines [44]. Huang et al. [27] used
two types of adjuvants, the ISA763A – a non-mineral
oil emulsion formulated as a metabolizable adjuvant,
and the AS-F – a mineral oil-based adjuvant which
is not yet commercialized, in the formalin-inactivated
whole-cell vaccine of Streptococcus iniae; intraperitoneal infection in vaccinated Epinephelus coioides
resulted in 100% survival [27].
A preventive approach is the best course of
action to overcome disease outbreaks in aquaculture.
Scientific research on health and environmental constraints of the hosts, the pathogenesis of diseases, and
prevention strategies must be well addressed. To date,
prevention and control of diseases rely on antibiotics
and other chemicals for treatment. However, the use
of antibiotics in the management of fish diseases is
not recommended, due to their negative impacts on
aquatic environments, such as the development of antimicrobial drug resistance in pathogenic strains [3,20].
Instead of chemical disease control strategies, biological strategies can be applied. In addition, biosecurity measures are important in preventing the
occurrence of disease-causing agents in aquaculture.
This includes stringent quarantine measures, egg disinfection, fish traffic control, water treatments, clean
feed, and disposal of carcasses [1]. Biological control
and prevention of infectious diseases in aquaculture
are often achieved with the application of vaccines.
However, the success rate of vaccination depends
on the development of protective vaccines and their
proper application [21].
Bacterial Fish Vaccine Usage in Aquaculture
Vaccines are a powerful tool, proven to provide
an easy, and cost-effective preventive solution to
fish diseases [6,16,22,23]. Vaccines, in addition
to reducing antibiotic dependence and the severity of losses incurred due to diseases, are known
to improve fish health, reduce disease outbreaks,
and provide long-lasting protection against diseases, while leaving no harmful residues in the
product or the environment [6,16,22,23]. More
Veterinary World, EISSN: 2231-0916
Killed whole-cell vaccines
Live-attenuated vaccines
Besides killed vaccines, live-attenuated vaccines
are under strong consideration to be commercialized
as fish vaccines due to their advantages. Scientific
studies are being increasingly focused on live-attenuated vaccines due to several reasons, such as the
virulence factors displayed on the surface, ease of
culturing, cheap production, and clear genetic background [45]. Furthermore, live-attenuated vaccines
can induce cell-mediated and humoral antibodies, in
1807
Diseases1
Pathogen1
Main hosts1
Type of vaccine2
Trade name2
Vaccination route2
BKD
Renibacterium
salmoninarum
Edwardsiella tarda
Salmonids
Arthrobacter vaccine
Renogen
Injection
Salmon, catfish, carps, turbot,
flounder, eel, tilapia
Channel catfish, freshwater
catfish, striped catfish, brown
bullhead, Donio spp.
Cyprinids, salmonids, catfish
carp, trout, perch, tilapia
n.a
n.a
n.a
Edwardsiella ictaluri
vaccine, avirulent live
culture
Flavobacterium columnare
bacterin
Flavobacterium columnare
vaccine, avirulent live
culture
Aeromonas salmonicida
bacterin
IROMP antigens of
Aeromonas salmonicida
Inactivated strain of
Listonella (Vibrio)
anguillarum serovar
O1, Listonella (Vibrio)
anguillarum serovar O2,
Aeromonas salmonicida
subsp salmonicida, Vibrio
salmonicida, and Moritella
viscosa and surface protein
from IPN virus serotype spp.
Infectious salmon anemia
virus vaccine-Aeromonas
salmonicida‑Vibrio
anguillarum‑ordalii
salmonicida bacterin
Inactivated Lactococcus
garvieae
n.a
AquaVac-ESC™
Immersion
FryVacc1
Immersion
AquaVac-Col™
Immersion
Furogen Dip
Injection
AquaVac® FNM
Injection
Norvax® Minova 6
Injection
Forte V1
Injection
Amalin™ Rensa
Oral
n.a
n.a
Inactivated Photobacterium
damsel
Inactivated strain
Listonella (Vibrio
anguillarum (biotype
I and II) and Photobacterium
damselae spp. piscicida
n.a
AquaVac Photobac Prime™
Immersion/Oral
AquaVac® Vibrio Pasteurella
Injection
n.a
n.a
Edwardsiellosis/Redpest
Edwardsiellosis/Enteric
septicemia
Edwardsiella ictaluri
Flavobacteriosis/Columnaris
Flavobacterium.
columnare,
Flavobacterium
maritimus
Furunculosis
Aeromonas salmonicida
Lactococcosis
Lactococcus garvieae
Motile Aeromonas
Septicaemia
Pasteurellosis
Aeromonas hydrophila,
Aeromonas salmonicida
Photobacterium.
damselae spp. piscicida
Piscirickettsiosis/Rickettsial
septicemia
Piscirickettsia salmonis
Salmons, trout, flounder,
turbot, carp, tilapia, sole
Salmonids, seabream, seabass,
Seriola spp.
Salmonids, bass, carp, trout,
eel, sturgeon, tilapia
Seabream, seabass, ayu,
yellowtail, carp, sturgeon,
hybrid striped bass, tuna, cobia,
snakehead
Salmonids, trout, seabass,
tilapia
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Veterinary World, EISSN: 2231-0916
Table-1: List of fish bacterial diseases, the causative agents and main hosts, and some of the vaccine commercially available in the market.
Diseases1
Pathogen1
Main hosts1
Type of vaccine2
Trade name2
Vaccination route2
Streptococcosis
Streptococcus
agalactiae,
Streptococcus iniae,
Streptococcus
dysgalactiae,
Streptococcus
parauberis,
Streptococcus phocae
Vibrio alginolyticus,
Vibrio
parahaemolyticus,
Vibrio vulnificus, Vibrio
anguillarum
Grouper, salmonids, turbot,
flounder, sturgeon, amberjack,
yellow tail, red porgy,
barramundi, rabbitfish, seabass,
seabream, hybrid striped bass,
catfish, mullet, pomfret, tilapia,
koi, carp
Streptococcus agalactiae
biotype 2 bacterin
1
AquaVac Garvetil/AquaVac
Garvetil Oral; AquaVac®
Strep Sa;
Immersion/Oral
Most marine fish, salmonids,
groupers, cods, red seabream,
gilt-head sea bream, Japanese
flounder, summer flounder,
amberjack, halibut, yellowtail,
seabass, seriolla, milkfish,
horse mackerel, cobria, sole,
eel, tilapia
Streptococcus iniae bacterin
Vibrio anguillarum‑ordalii
bacterin
Norvax® Strep Si
Vibrogen 2
Immersion/Injection
Immersion
Inactivated Vibrio
anguillarum 01 and
02 (Vibrio ordalii)
Inactivated strain
Listonella (Vibrio
anguillarum (biotype I
and II) and Photobacterium
damselae spp. piscicida
Aeromonas salmonicida
Vibrio anguillarum ordalii
salmonicida bacterin
Infectious salmon
anemia virus vaccine
Aeromonas salmonicida
Vibrio anguillarum ordalii
salmonicida bacterin
Yersinia ruckeri bacterin
AquaVac® Vibrio, AquaVac®
Vibrio Oral Boost
Immersion/Oral
AquaVac® Vibrio Pasteurella
Injection
Lipogen Forte
Injection
Forte V1
Injection
Ermogen; AquaVac® ERM;
AquaVac® ERM Oral;
AquaVac® RELERA™
Immersion/Oral
Vibriosis
Yersiniosis/Enteric redmouth
Yersinia ruckeri
Salmonids, trout, eel, minnows,
tilapia
Source: 1[13,14,16,17], 2[18,19]. BKD=Bacterial kidney disease, IROMP=Iron regulated outer membrane protein
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Table-1: (Continued).
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addition to mucosal immunity [46]. Therefore, they
stimulate greater adaptive immune protection in fish,
compared to that induced by inactivated bacterins or
subunit vaccines [47]. Live-attenuated vaccines carry
native antigenic structures that are normally expressed
by pathogens in vivo. This causes a self-limiting infection that mimics, on a smaller scale, the real infection
induced following natural exposure [48]. Immune
responses stimulated by such “mimic” infections
closely resemble those detected in a normal infection.
The antigens produced during a live infection may
respond differently than those administered in the
form of subunit vaccines [49].
Live-attenuated vaccines offer a prolonged and
unaltered antigen presentation, which stimulates
humoral and cell-mediated immune responses [47].
They are incapable of producing clinical disease;
however, they can colonize appropriate sites and stimulate secretory responses [49]. Furthermore, they do
not require adjuvants, and only single or few doses
are needed during vaccination [50]. Attenuation was
traditionally achieved through the induction of random mutation(s) by serial passage of the virulent
strain in specific antibiotics [41,51,52], or on laboratory media [32]. In contrast, the modern attenuation strategy uses genetic modification techniques,
such as random transposon recombination or allelic
exchange replacement [47]. The latter technique has
gained much interest as it offers more stable and definite attenuation, compared to the techniques typically
used for killed whole-cell vaccines.
Potential of Live-attenuated Vaccine in
Aquaculture
Live-attenuated vaccine using traditional attenuation
strategy
A live-attenuated vaccine using selective double
resistance to rifampin-streptomycin was developed
against V. anguillarum strain VAN1000 [51]. It was
tested on juvenile rainbow trout (O. mykiss), where
it was shown to provide good homologous protection against V. anguillarum, but only slight protection
against Aeromonas salmonicida. A live vaccine containing Arthrobacter spp. has also been successfully
demonstrated to cross-protect against Renibacterium
salmoninarum, a pathogen that causes bacterial kidney disease in salmonids [41,52]. It has been licensed
for use on salmonids in North America and Chile [41].
Hu et al. [53] successfully attenuated a mutant,
designated as strain T4DM, using a selection of rifampicin resistance from a virulent V. harveyi strain, T4D.
The mutant strain T4DM was able to induce effective
cross-species protection against both V. harveyi and
V. alginolyticus, when used as a live immersion vaccine.
Live-attenuated strains developed through repeated
in vitro passage have also been shown to provide significant immune protection. Li et al. [32] developed the
attenuated S. agalactiae YM001, through 840 continuous in vitro passages. Tilapia vaccinated with this strain
Veterinary World, EISSN: 2231-0916
(1.0×108 CFU/fish of S. agalactiae YM001) exhibited
96.88% (injection), 67.22% (immersion), and 71.81%
(oral) relative percentage survival (RPS), 15 days
post-vaccination. Furthermore, the hosts challenged
after 30 days showed an RPS of 93.61% (injection),
60.56% (immersion), and 53.16 % (oral) [32].
Live-attenuated vaccine using genetic engineering
strategy
Recent advances in molecular biology, immunology, and genetic engineering have offered
exceptional technological developments in the
fields of pathogenesis and recombinant DNA.
Molecular biology and immunology further reveal
information relating to the identification and characterization of pathogens and their pathogenicity
[54]. Genetic engineering has made the construction of precise attenuated vaccines possible. Sitedirected mutagenesis (SDM) is a reliable strategy
to obtain a well-defined deletion, insertion, or addition in targeted genes [55]. Thus, directed attenuation can be achieved through insertion, deletion, or
disruption in the metabolic pathway(s) or virulence
gene(s) responsible for pathogenicity [46,56,57].
Live-attenuated vaccines developed using this new
approach is remarkably potential and more efficient
than bacterins in eliciting a protective immune
response [31].
Ma et al. [28] successfully developed a two-strain
polyvalent live-attenuated vaccine through genetic
engineering and molecular biology, instead of the traditional serial passage technique. The strains, designated as MVAV6203 (deletion of aromatic amino acid
and folic acid synthesis gene, ΔaroC) and MVAV6204
(deletion of aromatic amino acid and folic acid synthesis gene and siderophore anguibactin, ΔaroCΔangE),
were developed from the V. anguillarum strain
MVM425. The results revealed a 100% protection
in Epinephelus spp. and Paralichthys spp. against V.
anguillarum and V. alginolyticus infections, after being
vaccinated with the two attenuated strains through
intraperitoneal and immersion routes [28]. This indicated that the deletion of the target gene prevented the
synthesis of an aromatic acid, folic acid, and siderophore anguibactin, thus reducing the strains’ ability to
colonize in nature and also in the fish body [28].
In another study, flounders (Paralichthys
olivaceus) vaccinated with 107 CFU/ml attenuated
strain ΔaroAΔesrB exhibited 100% RPS against
107 CFU/ml Edwardsiella tarda [32]. This implies
that live-attenuated vaccines can stimulate a cell-mediated immune response, while non-living vaccines
cannot. Mou et al. demonstrated that isocitrate dehydrogenase mutation in V. anguillarum resulted in virulence attenuation and subsequent protection in rainbow trout (O. mykiss) [58]. Insertional mutagenesis in
isocitrate dehydrogenase (icd) gene of V. anguillarum
M93Sm successfully inhibited the synthesis of α-ketoglutarate in V. anguillarum (XM420) icd mutant. After
2 weeks of immersion with icd mutant in 1.5% salt
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solutions at a dose of 4×106 CFU/ml, 90% survival
was recorded in O. mykiss, compared to 30% survival
of fish immersed in its parental strain. It was found
the icd mutant showed strong attenuation in virulence,
resulting in a decrease in growth yield, when comparing to the wild type, due to its inability to synthesize
α-ketoglutarate, an important component for central
metabolism of the pathogen [58].
Mohd-Aris et al. [34] successfully developed
a V. harveyi mutant by protease deletion, as a candidate live-attenuated vaccine against vibriosis in
Epinephelus fuscoguttatus. They employed SDM and
allelic exchange replacement techniques to genetically attenuate the V. harveyi strain MVh-vhs. The
MVh-vhs strain was shown to be safe when tested
in the host, suggesting that the attenuation of virulence-associated protease MVh-vhs decreases the virulence properties. However, further IP vaccination
of E. fuscoguttatus with a single dose of the attenuated strain at 105 CFU/fish showed 52% RPS after
being challenged with 108 CFU/fish of the parental
strain [34]. This suggests that the administration dosage during vaccination, may improve the protective
efficacy of the MVh-vhs strain. Higher survival was
observed in Artemia salina larvae incubated with
107 CFU/mL of the live-attenuated strain MVh-vhs,
6 h post-incubation. Furthermore, A. salina larvae
incubated with MVh-vhs (109 CFU/mL) showed a
higher survival rate when challenged with pathogenic
V. harveyi (Vh1), V. alginolyticus (VA2), and V. parahaemolyticus (FORC_008), 24 h after incubation [59].
Combined live-attenuated vaccines
A combined live-attenuated vaccine utilizes a
“ghost” or vector to harbor foreign materials obtained
from the pathogen, to express and evoke the host’s
immune system [60]. The primary advantage of
live-attenuated vectors is their ability to deliver
multiple antigens, of different species, in a single
dose. Other advantages include the mimicry of natural infection, intrinsic adjuvant characteristics, and the
possibility of being administered through the mucosal route, rather than the more laborious intraperitoneal route [56]. In addition, combined vaccines can
achieve high expression of antigens, due to the plasmid-mediated expression system. Table-2 [31,61-64]
summarizes recent studies related to combined
live-attenuation [61-64]. Goa et al. [31] described
the capability of a combined vaccine, consisting of
live-attenuated E. tarda WED and V. anguillarum
MVAV6203, to evoke better immune-mediated
protection in turbot (Scophthalmus maximus) and
zebrafish (Danio rerio) against E. tarda EIB202
and V. anguillarum MVM425, with the activation
of toll-like receptors and Class I and Class II major
histocompatibility complexes.
Constraints that Limit the Potential of Live
Vaccines
Risks in the protective efficacy of live vaccines
Despite the remarkable advantages of live
vaccines, a few disadvantages have been discerned.
Although attenuation strategies produce attenuated
isolates, the isolates only persist for a short duration,
between 24 and 72 h, and fail to stimulate adequate
immunity in young fish [46]. Live vaccines also have
the risk of producing low-grade infections when the
vaccine agents replicate in the hosts. They may even
result in systemic symptoms, displaying some features of the original infection [65].
Table-2: Combined live-attenuated vaccine of Vibrio spp.
Bacterial vector
Method of
combined vaccine
Protection
against
Research findings
Avirulent Vibrio
anguillarum (MVAV6203)
Inoculation of
Pseudomonas
syringae (ICMP3023)
inaV gene
Inoculation of
Edwardsiella tarda
pUTatgap plasmid
Vibrio anguillarum
and Pseudomonas
syringae
The expression of foreign antigen
in vector was expressed both in
cytoplasms an OMP of vector
[61]
Vibrio anguillarum
and Edwardsiella
tarda
Survival of 80% and 67%
when challenged with Vibrio
anguillarum and Edwardsiella
tarda, respectively, in
turbot (Scophthalmus maximus)
Survival of 90% and 70%
when challenged with Vibrio
anguillarum and Edwardsiella
tarda, respectively, in
zebrafish (Danio rerio)
Survival of 87% and 67%
when challenged with Vibrio
anguillarum and Aeromonas
hydrophila, respectively, in
turbot (Scophthalmus maximus)
Survival of 100% and 0%
when challenged with Vibrio
anguillarum and Edwardsiella
tarda, respectively, in
zebrafish (Danio rerio)
[62]
Avirulent Vibrio
anguillarum (MVAV6203)
Avirulent Vibrio
anguillarum (MVAV6203)
and Edwardsiella
tarda (WED)
Polyvalent live
attenuated vaccine
Vibrio anguillarum
and Edwardsiella
tarda
Avirulent Vibrio
anguillarum (MVAV6203)
Inoculation of
Aeromonas
hydrophila (LSA34)
GAPDH strain AV/
pN-gapA
Inoculation of
Edwardsiella
tarda (EIB202) EseB
OMP
Vibrio anguillarum
and Aeromonas
hydrophila
Avirulent Vibrio
anguillarum (MVAV6203)
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Vibrio anguillarum
and Edwardsiella
tarda
References
[31]
[63]
[64]
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Stability and maintenance of live vaccines
Compared to killed vaccines, live vaccines are
less stable and have a shorter shelf life. This may be
due to the nature of live cells, which are easily affected
by environmental factors, for example, susceptibility
to damages or destructions by high temperatures due to
heat-labile characteristics [50]. It is important to provide a cold or refrigerated environment (2-8°C) during
handling, storage, and distribution of live vaccines,
to ensure stability throughout their designated shelf
life [66]. This leads to higher operational and handling
costs, thus adding to the total expenses for vaccination.
Commercialization process and legislation hurdles
In addition to operational and handling costs,
extensive research, such as risk assessments and clinical testing of live-attenuated bacteria, requires huge
investments before vaccine registration [67]. All
costs incurred during the development of live vaccines greatly influence their market price [50]. As a
result, most live vaccines are still at the research stage.
Another issue with live vaccines is the regulatory hurdles in vaccine registration [57]. The procedure from
research to obtaining a valid license for this type of
vaccine is rather long and often exorbitant. Moreover,
legislation on the control and administration of vaccines varies greatly from country to country [68]. For
example, in the EU, the USA, and Japan, a licensed
vaccine is required to be included while importing
aquaculture products [69]. Therefore, tedious regulatory hurdles cannot be neglected. Concerns related to
costing, budgeting, stable formulation, fill-finish step,
and economical production are some of the limitations
in the application of live vaccines, especially if the
vaccine is targeted for use by aquaculture practitioners
or farmers in developed countries [66,70].
Stability in virulence attenuation properties
Another limitation of using a live vaccine is the
possibility of back-mutation and reversion to its virulent phenotype [8,67,68] which may occur due to
changes in the bacteria, or compromising conditions
in the host. The attenuated strains might be well tolerated by healthy individuals, but some may induce
auto-immune responses, causing local inflammation
and other adverse reactions [71]. Thus, a strategy to
reduce virulence reversion during live vaccine development is to attenuate multiple genes instead of a
single gene [57]. Furthermore, there is a risk of introducing pathogenic strains from live-attenuated agents
into the aquatic environment, which might become a
pathogenic source for other species [72]. Immersion
vaccination has been preferred by most fish farmers to
date [73], as the processing and vaccine application is
easier. However, developments in vaccine production
and processing technologies, storage, and delivery
methods are required.
Negative public perception
All vaccines, including live vaccines, carry some
risk, even when they present an excellent track record
Veterinary World, EISSN: 2231-0916
in terms of safety in human and veterinary use [57].
The long-term challenge for live vaccines is to infuse
understanding and shape public perception [65]. Live
vaccines are often the subject of unsubstantiated accusations by anti-vaccine movements; they are faced
with public resistance and voiced against strongly as
they are genetically modified [57,65]. Therefore, it is
necessary to properly design, in addition to conducting efficacy and other related tests to gather essential
data to refute false claims raised by the public. Safety
aspects must be prioritized to diminish the undesirable
impacts of live vaccines. This approach will greatly
help to rectify and fortify public trust toward vaccination, which is important for the aquaculture sector, to
exploit the benefits of live vaccines [65].
Safety issues and environmental release
There is also a risk for this type of vaccine to
spread from a vaccinated to an unvaccinated individual, due to the release of the pathogen in the environment, or exposure to non-target animals [50,57].
For example, a worst-case scenario would be where
water samples treated with attenuated strains are accidentally released into the open environment during
disposal. Under these circumstances, safety of the
environment and the residing population is jeopardized, as the attenuated strain can cause infections
in the human population [46]. The potential risk of
admission and transmission needs to be scrutinized,
especially by the person in charge of the vaccination
process. Thus, the evaluation of the potential impact of
environmental release and the risk of horizontal gene
transfer is highly critical [50]. It is crucial to monitor the biosafety aspects of the attenuated vaccines
applied in aquaculture. As proposed by Ma et al. [28],
the genetic background of the mutation must be clear,
a double deletion should be considered to eliminate
the reversion of virulence characteristics, and the
attenuation should be definite, so that the environmental safety and controllability of the vaccine are feasible, and the possibility of exposing the pathogen to the
environment can be minimized.
Conclusion and Future Prospects
Vaccination strategy is an integral part of comprehensive fish health management. It is the best preventive strategy to combat the spread of fish diseases
by inducing defense mechanisms against the risk
of bacterial disease outbreaks. Hence, fundamental
knowledge of diseases and pathogen profiles, in addition to the basic economic background of operational
costs, is an essential requirement in the design of
suitable vaccination strategies. There are promising
indicators that live vaccines have great potential to
be further exploited as alternative vaccines. However,
each presumable benefit and implication must be
carefully assessed when designing a new candidate
live vaccine. In spite of the potential problems and
undesired ramifications, the holistic advantages still
1812
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outweigh the disadvantages, thus, endeavoring to
develop new live vaccines is a worthy investment. It
is strongly suggested that all possible limitations must
be critically addressed before employing live-attenuated vaccines in aquaculture sectors. Overall market
demand, integration of suitable vaccination regimes,
and good disease management unequivocally facilitate improvement in fish survival rates, further boosting the production of the aquaculture industry.
7.
8.
Authors’ Contributions
AM conceived and framed the main idea of this
manuscript. AM and MHNM prepared the first draft.
The first draft was read, criticized, and corrected by
MYI, MZ, and HMD. AM proofread the second draft
and finalized the manuscript. All authors have read
and approved the final manuscript.
Acknowledgments
The authors are thankful to the Institute of
Bioscience, Universiti Putra Malaysia and School
of Biology, Faculty of Applied Sciences, Universiti
Teknologi MARA, Negeri Sembilan Branch, Kuala
Pilah Campus, for providing the facilities required
for this review and also thank to the Asian Fisheries
Society, for awarding the AFS-Kanazawa Research
Fellowship Grant to Aslizah Mohd Aris.
9.
10.
11.
12.
Competing Interests
The authors declare that they have no competing
interests.
13.
Publisher’s Note
14.
Veterinary World remains neutral with regard
to jurisdictional claims in published institutional
affiliation.
15.
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