Phage Therapy, A Novel Approach To Control Bacterial Fish
Pathogens In Aquaculture Sector
Kumar1, Yogendra Prasad1,
Prabjeet Singh2 and Vinod Kumar Verma2
Biotechnology and Fish Pathology Laboratory,Department of Animal
Science, M.J.P. Rohilkhand University, Bareilly-243006 (U.P.)
2College of fisheries, G.B.Pant University of Agriculture
and Technology, Pantnagar, Uttrakhand, India.
Our freshwater aquaculture is mainly dominated by Indian major carp
(IMC) comprising of Labeo rohita, Catla catla and Cirrhinus
mrigala and accounts to about 80% of total inland fish
production. They are main candidate fish species of freshwater
aquaculture, extensively cultured and have proved to be boon in
upliftment rural economy. Due to various stressful conditions in
aquaculture like overcrowding, poor water quality, competition for
feed etc the occurrence of infectious diseaseshas now become a common
Among the infectious diseases, bacterial fish diseases are reported
to infest to most of the cultivable as well as wild fish species.
There are 40 - 60 bacterial fish pathogens found to be involved in
fish diseases. Occurrence of Epizootic Ulcerative Syndrome (EUS)
which has devastated the fish industries during nineties badly and
ensured heavy economic loss is a self evident proof. Generally,
fishes are prone to various microbial diseases because they live in
potentially hostile world filled with bewilder array of infectious
microbes which would very happily use fish as aliment and render them
to a variety of ailments.
Diseases like Columnaris, Bacterial Haemorrhagic Septicaemia (BHS),
Infectious Abdominal Dropsy, Furunculosis and Coldwater disease etc.
are very common in fish hatchery and culture systems. Among the
bacterial pathogens Flavobacterium, Pseudomonas and
Aeromonas spp. are known to be great impeder of Indian
aquaculture by being involved in various diseases.
To alleviate the incidences of bacterial diseases in aquaculture,
different anti-microbial chemicals KMnO4, CuSO4,
H2O2, Formalin, Benzolkonium
chloride, strains (Crystal violet, Methylene blue and Malachite
green), lime, common salt and finally antibiotics (Ofloxacin,
Tetracycline, Erythromycin, Neomycin etc.) and vaccine have been
used. Now, there has been sporadic application of probiotics and
adjuvant to cope up with such problems.
But in general, chemicals are toxic to fish and aquatic ecosystem
and some of them (like Malachite green) gets accumulated in different
organs of fish. Thus it seems that chemicals are toxic to fish and
the young ones can not tolerate their high concentration. Moreover,
use of antibiotics for antimicrobial property has resulted in
antibiotic resistance in pathogenic bacteria and posed serious
problems in the treatment of infectious disease. Similarly,
antibiotics not only destroy the targeted bacterium but also destroy
the general micro flora in intestine of fish and also disturb the
ecological balance of water body. The emerging
crisis of resistance to antibiotics (fig. 1) has led to renewed
interest in other methods of controlling bacterial infections. The
applications of probiotics (which are beneficial microorganisms or
their products) have been made in aquaculture in order to develop
immunocompetance in fish to combat with bacterial diseases and also
inhibit the colonization of potential pathogens in the digestive
tract through competition exclusion principle. But generally
probiotics are low — immunogenic in nature, temperature and
salinity sensitive and cumbersome in application.
As far as vaccination is concerned, it has proved to be an excellent
method in disease combating in poultry, animal and humans due to
trouble-free application in them. While in aquaculture, fishes are
under water, large scale cultured crop and it is just impossible to
vaccinate each and every fish. Therefore, vaccination becomes a
tedious job for large-scale aquaculture systems.
Therefore, the usual methods employed to control the disease caused
by this pathogen are not proving effective and there is increasing
trend of multiple drug resistance (MDR) development in pathogenic
bacteria. Due to such reasons they have not been proved as an
appropriate and suitable strategy to combat with potential pathogens
In the non availability of appropriate strategy to eradicate
bacterial pathogens, bacteriophages appear to be the most plausible
and appropriate candidate to overcome the above problems.
Bacteriophage may prove to be a good candidate to mitigate such
problem due to various reasons. Bacteriophages (phages) are viruses
that infect bacteria. Like all viruses, phages are obligate
intracellular parasites, which have no intrinsic metabolism and
require the metabolic machinery of the host cell to support their
reproduction. They subsist on the bacterial cells and lead lytic and
lysogenic life cycle and make the survival of host extremely
difficult. They are species specific, self perpetuating and
genetically flexible in nature. Bacteriophages are highly abundant in
the aquatic environment ranging from 104 ml-1
to in excess of 108 ml-1. Numbers are typically
3-10 times greater than the bacterial counts, although there is
substantial variation between ecosystems.
Historically, they were discovered by Twort (1915) and D’ Herelle
(1917) in the pre-antibiotic era. Now it has been proved that phage
therapy decline the bacterial population below threshold number to a
level where the host defense can take care of remaining bacteria.
There are two types of bacteriophage
reproduction: (1) Lytic - The virus attaches to a host cell and
injects its nucleic acid into the cell, directing the host to produce
numerous progeny. These are then released by a fatal bursting of the
cell, allowing the cycle to begin again. (2) Lysogenic - The nucleic
acid of the virus becomes part of the host genome and reproduces
genetic material (prophage) in the host cell. An induction event,
such as a physiological stressor, can trigger this reproduction to
switch to lytic. In general, the replication of phage in the
bacterial cell occurs in five steps: adsorption, penetration of
genetic material, replication, maturation and lysis. During
adsorption, the phage gets attached to the cell in order to infect
its genetic material in to the host cell. Penetration involves the
actual infection of the genetic material. In replication, the viral
genetic material takes over the host metabolic machinery for its own
replication. While the phage becomes mature and goes into it's
infectious state (maturation) and releases its progeny through lysis.
Lysis occurs when the phage particle releases lytic enzyme (Lysin)
that causes the cell wall to loosen, leaving it weak enough for the
break through of the matured phage.
Lysogenic bacteriophages may incorporate into
the genome of the bacterium rather than being lytic. These phages are
poor candidates for therapy as they do not provide the rapid growth
in phage numbers. Therefore, lytic phages are good candidate for
therapeutic as well as prophylactic use against pathogenic bacterial
fish diseases. This is evidently confirmed in in
vitro test, in which lytic phages
clears the bacterial lawn in Petri plate and forms clear plaques
(fig. 2). During the lytic life cycle the number of phages
released (burst size) after lysis varies from 50-409 new phage
particles (Yang et al., 2010). One of the advantages of phage
therapy over antibiotics is that they are species specific.
Therefore, they can destroy only the harmful bacteria without
affecting the regular microflora of the environment. Antibiotics
generally target both pathogenic and non-pathogenic microflora.
Therefore, phage therapy is safer and there is no need of repeated
administration as phages can replicate as long as the host cells are
available. On the contrary, antibiotics undergo metabolic destruction
and if at stable, they need in numerous molecules to act on bacteria.
1: Showing drug resistance activity against various antibiotics by
2: In vitro
lytic activity of phage, showing
with plaque formation in
the bacterial lawn of Pseudomonas
In aquaculture the first isolation of phages against Aeromonas
salmonicida, an etiological agent of Furunculosis in Coho salmon
(Oncorhynchus kisutch) was made by Paterson et al.
(1969) and Popoff, (1971). Some studies showed that viral particles
in the marine waters are generally found at concentrations ranging
from 104-107 particles per ml. Treatment with
bacteriophage was shown to improve survival of shrimp larvae (Penaeus
monodon) and it was suggested that bacteriophage have a potential
for biocontrol of Vibiro harveyi of shrimp (Penaeus
monodon). Good numbers of phages have been isolated against
important bacterial fish pathogens, such as Edwardsiella tarda,
causative agent of Edwardsiellosis; Yarsinia ruckeri, causative
agent of red mouth disease in Salmo gairdnerii; Aeromonas
hydrophila causative agent of abdominal dropsy; Lactococcous
garvieae, causative agent of serious disease in Yellow tail
(Seriola quinqueradiate), Pseudomonas plecoglossicida
infect ayu (Plecoglossus altivelis), Aeromonas
salmonicida in brook trout (Oncorhynchus fontinalis),
Pseudomonas fluorescens in Rohu (Labeo rohita),
Flavobacterium psychrophilum in coldwater fishes and
Flavobacterium columnare in Clarias batrachus and Labeo
Bacteriophages can be administered through any rout viz.
im(intra muscular), ip(intra peritoneal), bath and oral. Various
researchers found that no phage neutralizing antibodies were found in
phage treated fish. All of these studies have demonstrated the
potential of specific phages to significantly reduce the impacts of
their bacterial hosts, with a resulting positive effect on fish
survival. The studies emphasize the need for further investigations
of the possibilities in using phages as an alternative to antibiotic
treatment of other fish diseases in aquaculture and it can also be
use in fish processing industry against spoilage causing bacteria.
Therefore, phage therapy may be a realistic alternative approach for
controlling pathogenic bacteria in aquaculture owing to its several
advantages over the conventional antibiotics and other methods
against pathogenic multiple drug resistant (MDR) bacteria.
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Seafood — Fish — Crustacea
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