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Journal of Ocean University of China (Oceanic and Coastal Sea Research)

ISSN 1672-5182, January 30, 2007, Vol.6, No.1, pp.76-79


http://www.ouc.edu.cn/xbywb/
E-mail:xbywb@ouc.edu.cn

Review

Probiotics as Control Agents in Aquaculture


Gmez R. Geovanny D1),*, Balczar Jos Luis2), and MA Shen1)
1) Mariculture Research Laboratory, Ocean University of China, Qingdao 266003, P. R.China
2) Laboratory of Fish Pathology, Faculty of Veterinary Medicine, University of Zaragoza,
C /. Miguel Servet 177, Zaragoza 50013, Spain
(Received December 12, 2005; accepted October 8, 2006)
Abstract Infectious diseases constitute a limiting factor in the development of the aquaculture production, and control has solely
concentrated on the use of antibiotics. However, the massive use of antibiotics for the control of diseases has been questioned by
acquisition of antibiotic resistance and the need of alternative is of prime importance. Probiotics, live microorganisms administered
in adequate amounts that confer a healthy effect on the host, are emerging as significant microbial food supplements in the field of
prophylaxis.
Key words

aquaculture; diseases; prevention; probiotics

DOI 10.1007/s11802- 007- 0076-8

1 Introduction
Intensive aquaculture system produces substantially
higher yields than do semi-intensive systems. However,
intensification results in stressful environmental changes
which can produce various kinds of problems in aquatic
organisms, principally diseases of diverse aetiology
(Angulo, 2000). The most common control of the diseases was concentrated on the use of antibiotics. However,
the utility of antibiotics as a preventive measure has been
questioned by acquisition of genes that result in antibiotics resistance and horizontal spread of plasmids from fish
pathogenic to humans. Plasmids carrying resistance de-

terminants have been transferred in vitro from fish


pathogens to human pathogens, such as Vibrio cholerae
and V. parahemolyticus and to potential human pathogens,
including Escherichia coli (Angulo, 2000).
Several alternative strategies for the prevention and
control of diseases have been proposed and have already
been applied successfully in aquaculture, such as the use
of vaccines, the use of immunostimulants for the enhancement of the non-specific defense mechanisms of the
host and the use of probiotics.
Probiotics, live microorganisms administered in adequate amounts that confer a health effect on the host, are
emerging as significant microbial food supplements in the
field of prophylaxis (Table 1).

Table 1 Probiotics considered for use in aquaculture


Probiotic
Lactobacillus plantarum
Micrococcus luteos
Bacillus sp. S11
G-probiotic Pseudomonas fluorescens
Aeromonas Hydrophila,
Vibrio fluvialis, Carnobacterium sp.,
Micrococcus luteus
Enterococcus faesium
SF68

Source
Turbot larvae

Used on

Method of administration

Reference

Scophthalmus maximus

Indirectly via rotifers

Oncorhynchus mykiss

Premix with feed

Penaeus monodon
Oreochromis niloticus
Rainbow trout (Oncorhynchus mykiss)

Premix with feed


Addition to diet
Addition to culture water
Addition to diet

Digestive tract of rainbow trout

Rainbow trout

Addition to diet

Irianto and Austin,


2002

Commercial products
(Cernivet)

Anguilla anguilla

Addition to diet

Chang and Liu, 2002

Digestive tract (Oncorhynchus mykiss)


Penaeus monodon
Commercial product
Iced freshwater fish
(Lates niloticus)

* Corresponding author. E-mail: geovanny78ec@yahoo.com

Gatesoupe, 1994
Irianto and Austin,
2002
Rengipat et al., 1998
Naik et al., 1999
Gram et al., 1999

Gmez R. G. D. et al.: Probiotics as Control Agents in Aquaculture

2 Modes of Action
The mode of action of probiotic strains is likely to be
multifactorial and, from existing evidence, appears to be
strain specific (Touhy et al., 2003). Enhancement of
colonization resistance and/or direct inhibitory effects
against pathogens is likely to be important in situations in
which probiotics have reduced the incidence and duration
of diseases. Probiotic strains have inhibited pathogenic
bacteria both in vitro and in vivo through several different
mechanisms. This includes protection creating a hostile
environment for pathogens by the production of inhibitory compounds such as bacteriocins, siderophores, lysozymes, proteases, hydrogen peroxide, formation of
ammonia and diacetyl, alteration of pH values by the
production of organic acid (Verschuere et al., 2000),
competition for essential nutrients and adhesion sites
(Vine et al., 2004), supply of essential nutrients and enzymes resulting in enhanced nutrition of the cultured
animal (Wang et al., 2000), direct uptake of dissolved
organic material mediated by the bacteria (Dalmin et al.,
2001), modulating interactions with the environment and
the development of beneficial immune responses (Gullian
et al., 2004).
Competitive exclusion is a phenomenon whereby an
established microbiota prevents or reduces the colonization of bacterial pathogen by competition for the attachment sites on the mucosa, competition for nutrients, and
production of inhibitory substances by the microbiota
which prevents replication and destroys the challenging
bacteria. Several mechanisms are involved in the adhesion of microorganisms to intestinal epithelial cells such
as passive forces, electrostatic interactions, hydrophobic,
steric forces, lipoteichoic acids and specific structures.
These properties could confer a competitive advantage
important for bacterial maintenance in the animals gastrointestinal tract. Since mucosal surfaces are colonized
by a large, complex and dynamic collection of microorganisms, it is extremely important for intestinal epithelial
cells and the intestinal mucosal immune system to discriminate between pathogen and non-pathogen bacteria
and to generate an appropriate response, maintaining a
state of homeostasis with microbiota or initiating inflammatory responses against pathogens (Shi and Walker,
2004).
Several studies on the beneficial effects of probiotics in
the digestive process of the fish have been reported.
Ring et al. (1995) suggested that species of Agrobacterium, Pseudomonas, Brevibacterium, Microbacterium and
Staphylococcus isolated from the intestinal tract of alpine
trout (Salvelinus alpinus L.) contributed to the nutritional
process by the production of lipases that increase the absorption of lipids. Sugita et al. (1997) reported bacteria
that constitute the intestinal microbiota (especially Aeromonas sp. and Pseudomonas sp.) from freshwater fish in
Japan (carp, channel catfish, tilapia and japanese ell),

77

which produce amilasas that helps in the digestion of the


starch. In addition, a study in oscar (Astronotus ocellatus),
fish angel (Pterophyllum scalare), and southern flounder
(Paralichthys lethostigma) suggested that the intestinal
anaerobic bacteria can play a role in the digestive process
of the fish by providing a variety of enzymes such as
cabohydrases, phosphatases, esterases, lipases and peptidases that help in the absorption of nutrients (Ramirez
and Dixon, 2003).
The non-specific immune system can be stimulated by
probiotics. It has been demonstrated that oral administration of Clostridium butyricum bacteria to rainbow trout
enhanced the resistance of fish to vibriosis by increasing
the phagocytic activity of leucocytes (Sakai et al., 1995).
Rengpipat et al. (2000) mentioned that the use of Bacillus
sp. (strain S11) provided disease protection by activating
both cellular and humoral immune defenses in tiger
shrimp (P. monodon). Balczar (2003) demonstrated that
the administration of a mixture of bacterial strains (Bacillus and Vibrios sp.) positively influenced the growth
and survival of juveniles of white shrimp and presented a
protective effect against the pathogens Vibrio harveyi and
white spot syndrome virus. This protection was due to a
stimulation of the immune system by increasing phagocytosis and antibacterial activity. In addition, Nikoskelainen et al. (2003) showed that administration of a lactic
acid bacterium Lactobacillus rhamnosus (strain ATCC
53103) at a level of 105 cfu g-1 feed, stimulated the
respiratory burst in rainbow trout (Oncorhynchus mykiss).

3 Selection of Probiotics
A common way to screen the candidate probiotics is to
perform in vitro antagonism test, since bacterial antagonism is a common phenomenon in nature. Therefore, several investigators have used this phenomenon to select
candidates by in vitro tests, in which pathogens are exposed to the candidate probiotics or their extracellular
products in liquid and/or solid medium. Nevertheless, it
has been reported that in vitro activity using liquid and/or
solid medium can not be used to predict a possible effect
in vivo. For example, in vitro antagonism of Pseudomo
nas fluorescents (strain AH2) against A. salmonicida did
not confer protection in Atlantic salmon against furunculosis, but it conferred protection against vibriosis (Gram
et al., 2001). Therefore, it is necessary to know the origin
of the stock (preferably to use strains isolated from the
host in study) and its security (non-pathogenic).
Some bacteria used as candidate probiotics have antiviral effects. Although the exact mechanism by which
these bacteria do this is not known, laboratory tests indicate that the inactivation of viruses can occur by chemical
and biological substances, such as extracts from marine
algae and extracellular agents of bacteria. It has been reported that strains of Pseudomonas sp., Vibrios sp.,
Aeromonas sp., and groups of coryneforms isolated from
salmonid hatcheries, showed antiviral activity against

78

Journal of Ocean University of China

infectious hematopoietic necrosis virus (IHNV) with


more than 50 % plaque reduction (Kamei et al., 1988).
Girones et al. (1989) reported that a marine bacterium,
tentatively classified in the genus Moraxella, showed
antiviral capacity, with high specificity for poliovirus.
Direkbusarakom et al. (1998) isolated two strains of Vibrio spp. NICA 1030 and NICA 1031 from a black tiger
shrimp hatchery. These isolates displayed antiviral activities against IHNV and Oncorhynchus masou virus
(OMV), with percentages of plaque reduction between
62% and 99%, respectively.
Strain viability and maintenance of desirable characteristics during product manufacture and storage is also a
necessary for probiotics strain. Strain survival will depend on such factors as the final product pH, the presence
of other microorganisms, the storage temperature and the
presence or absence of microbial inhibitor in the substrate.
Finally, it is necessary to make an economic analysis
(cost/benefit) before practical application (Gomez-Gil
et al., 2000).

4 Mode of Application of the Probiotics


The probiotics can be added to the host or its ambient
environment in several ways: 1) addition to the artificial
diet, 2) addition to the culture water, 3) bathing and addition via live food (Verschuere et al., 2000; Gomez-Gil
et al., 2000). The concept of inoculating in the culture
system and adding to live food is advisable when we use
small volumes. The inclusion in the artificial diet is more
adapted when using greater volumes; however, this requires of sufficient number of probiotics that last when
they reach the gastrointestinal tract and present capacity
to adhere to the epithelial mucosa. A study carried out by
Gatesoupe (1994) demonstrated a significant increase
with respect to the control in the weight of turbots
(Scophthalmus maximus) fed with rotifers enriched with
acid-lactic bacteria for 20 d.

5 Pioneering Studies
The first report seems to be attributed to Yasuda and
Taga (1980), who suggested that bacteria would be found
to be useful not only as food but also as biological controllers of fish diseases and activators of nutrient regeneration. Later, Nogami and Maeda (1992) isolated a strain
PM-4 from the rearing water of larval Penaeus monodon,
with good survival and molting rates. The bacterium was
identified as Thalassobacter utilis (Nogami et al., 1997).
This biocontrol treatment increased the survival of the
larvae and repressed the growth of Vibrio anguillarium
and the fungus Haliphthoros sp. (Nogami et al., 1997).
Since 1995, there have been increasing successes with
the use of probiotics in the Ecuadorian shrimp industry,
especially in controlling the high incidence of larval diseases. A V. alginolyticus strain isolated from seawater on

Vol.6, No.1, 2007

plates of thiosulphate citrate bile salt sucrose agar, was


inoculated daily into 25 and 60 t larval rearing tanks containing Litopenaeus vannamei postlarvae at a bacterial
concentration of 2103 cell mL-1. The use of Vibrio alginolyticus strains as a probiotics has been recommended to
increase survival and growth of white shrimp (Litopenaeus vannamei). Competitive exclusion of potential
pathogenic bacteria effectively reduces or eliminates the
need for antibiotic prophylaxis in intensive larviculture
system (Garriques and Arevalo, 1995). In addition, it has
been reported that by use of probiotics in Ecuadorian
hatcheries in 1993, production volumes increased by 35 %,
and overall antimicrobial use decreased by 94% (Griffith,
1995).

6 Conclusions
Relevant information shows that probiotics can reduce
the incidence and duration of diseases. The use of probiotics can increase the survival rate and control the high
incidence of larval diseases. It is essential to understand
the mechanisms of action in order to define selection criteria for potential probiotics. Using probiotics in aquaculture has been in large part a result of historical and
empirical use and not based on scientific criteria.

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