Binder 1

Reviews in Fish Biology and Fisheries 13: 111140, 2003.
2003 Kluwer Academic Publishers. Printed in the Netherlands.
111
Applications and needs of fish and shellfish cell culture for disease control
in aquaculture
Alberto J. Villena
Departamento de Biologa Celular y Anatoma, Facultad de Ciencias Biologicas y Ambientales, Universidad de
Leon, 24071-Leon, Spain (Phone and Fax: +34 987 291487; E-mail: dbcavc@unileon.es)
Accepted 15 July 2003
Contents
Abstract
Scope
Diseases constrain the development of aquaculture
Aetiology of aquatic animal diseases
Application of cell culture methods to protect aquatic animal health
Nutritional diseases and the relationship between feeding and immunocompetence
Toxicological diseases
Infectious diseases
Cell culture methods in diagnosis of infectious diseases
Safe use of drugs for disease control
Study of pathogenicity mechanisms
Characterization of the host defense mechanisms
Vaccination and immunostimulation
Disease resistant strains of aquatic animals
Needs for the further development of fish and shellfish cell culture
Availability of cell lines and of cell culture methods
Standardization of cell and tissue culture methods
Validation of in vitro assays
Conclusion
Acknowledgements
References
page 111
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Key words: aquaculture, cell culture, disease, fish, shellfish

Abstract
This review focuses on the application of cell culture and in vitro methods to address some of the key elements
identified as strategies for integrated health management in aquaculture, including the standardization and validation of diagnostic methods, the development of new safe therapeutants, and the implementation of effective disease
control methodologies. It is expected that any long-term rise in seafood production will depend on the future
progress of aquaculture. However, the development of aquaculture faces a number of problems, of which diseases
particularly the emergence of new pathogens represent serious risks for the production of aquatic animals,
but also for the health of fish farmers and of the consumers of aquaculture products. The complex interactions
underlying disease outbreak and progression may be better studied using in vitro models that use cell/tissue culture
methods and experimental systems, in which the interactions between aggressors and the host can be dissected.
Researchers have developed in vitro assays for fish toxicological, pathological, and immunological studies, as the
in vitro assays allow higher control of the conditions of the experiments. The combination of cell/tissue cultures
and in vitro assays reduces also the variability of the in vivo responses, which are due to the unavoidable effects
of stress and of environmental influences, and to the disparate genetic backgrounds of farmed fish and shellfish
species.
112
Scope
This review focuses on the applications of cell culture
methods to develop and implement strategies for
disease control in aquaculture. Fish and shellfish
farming constitute an important resource for healthy
food, and are a very important source of prosperity
in many countries, and it is expected that any longterm rise in seafood production will depend on the
future progress of aquaculture (FAO Fisheries Department, 2000). However, the development of aquaculture faces a number of problems, of which diseases
of diverse aetiologies, as the emergence of pathogens, are of particular importance. Disease outbreaks
result from interrelated environmental and pathogenic
factors that interact with genetic and physiological
traits of the farmed animals. An understanding of such
relationships is important for the prevention, control,
and treatment of disease in aquaculture (Subasinghe,
1997). In vitro models based upon the use of cell/tissue
culture methods may help to analyze such interactions, and to acquire knowledge for health protection
and disease management in aquaculture. The purpose
of this review is to show how cell culture methods,
and the associated in vitro assays, have contributed,
and may contribute in the future, to the understanding
of the fish and shellfish pathologies, and to implement strategies for health protection of aquacultured
animals. Several applications of cell culture, and of
the pertinent in vitro assays, are reviewed to illustrate
their uses on the study of nutritional, toxicological,
and infectious diseases of farmed fish and shellfish.
Moreover, a description of the needs for the further
development of fish and shellfish cell culture resources
is presented.
For specific details on fish and shellfish cell
culture methods, the reader should consult any of
the books and reviews that in recent years have
covered such subjects (Bayne, 1998; Rinkevich, 1999;
Toullec, 1999; Freshney, 2000; Mothersill and Austin,
2000). Also, several laboratory manuals have collected
specific in vitro techniques for fish (Stolen et al.,
1990, 1992, 1997; Ganassin et al., 2000) and shellfish
(Stolen et al., 1995) immunology and pathology, many
of which are applicable in cell culture assays.
Diseases constrain the development of aquaculture

According to the FAO Conference on Aquaculture
in the Third Millennium (Aquaculture Development
Beyond 2000), aquaculture is an economic activity of

increasing importance that contributes to the global
food supply, to food security in terms of sustainable
resources, and especially for low income, developing
countries to economic development and rural livelihoods. Fisheries have played such a role in many
cases, but supply of food fish from capture fisheries
is likely to decline with population increase (Rana,
1997; Watson and Pauly, 2001). In contrast, aquacultured production had grown around 56% every five
years (Rana, 1997). In 2000, the SOFIA report (FAO
Fisheries Department, 2000) stated that the potential of aquaculture to contribute to food production
has not yet been realized, and that this could not be
achieved without consistent, responsible policies and
goals that encourage sustainable development. Among
these, the Bangkok Declaration on Aquaculture Development Beyond 2000 (NACA/FAO, 2000) included
the rational management of aquatic animal health.
Addressing aquatic animal health issues has
become an urgent requirement for the sustaining
growth of aquaculture (Subasinghe, 1997). The emergence of new pathogens, particularly intracellular
bacteria and viruses, represents serious risks for the
health of aquatic animals, but also of fish farmers
and of the consumers of aquaculture products. Since
disease outbreak and progression depend on the
relationships between the host traits (susceptibility,
genetic background, stress . . .), on the nature of the
aggressor (pathogens, toxicants, toxins . . .), and on
several environmental factors (Snieszko, 1974; Bly et
al., 1997; Hedrick, 1998), an understanding of the
relationships between host, aggressor, and environment is important for the prevention, control and
treatment of diseases (Subasinghe, 1997). Rational
strategies for disease management in aquaculture
should target two main objectives: (a) the protection of aquatic animal health, with the aim to reduce
animal losses due to diseases and to lessen economics
costs to maintain healthy cultures (Subasinghe, 1997);
and (b) the protection of human health, for which
aquacultured products may represent different kinds
of risks (Howgate et al., 1997; Durborow, 1999; Joint
FAO/NACA/WHO Study Group, 1999). The most
important hazards for human health related to aquaculture are transmissible zoonosis, as parasitic helmints,
bacteria, and viruses (Durborow, 1999; Jehane and
Rawlin, 2000), and food-borne hazards represented
by biological microorganisms and biotoxins (Howgate
et al., 1997; Joint FAO/NACA/WHO Study Group,
1999). The World Health Organization (WHO) esti-
113
mated that in 1995 about 39 million people worldwide were infested with parasites transmitted by the
ingestion of raw or improperly cooked freshwater fish
and crustaceans (cited in FAO Fisheries Department,
2000). This review focuses on the first objective,
but it is worthwhile to indicate that the strategies
implemented for the rational management of aquatic
animal health will also contribute to making seafood
consumption safer (Howgate et al., 1997).
Aetiology of aquatic animal diseases

Diseases affecting aquacultured animals can be of
various aetiologies, including: (a) anatomical and/or
functional traumas, including tumors and external
injuries; (b) nutritional or unfit feeding (Bell et al.,
1991, 1993; Blazer, 1992; Sealey and Gatlin, 1999);
(c) toxicological diseases, which in aquaculture are
frequently associated with water quality (Dunier and
Siwicki, 1993; Zelikoff, 1993; Bols et al., 2001),
but also with food residues, particularly fungal toxins
(Sinnhuber et al., 1978), and with inappropriate drug
(disinfectants, antibiotics . . .) dosages (Michel and
Alderman, 1992); and (d) infectious agents, as viruses,
bacteria, and parasites, which are by far those of
major incidence in aquatic animal health (Subasinghe,
1997). It is also important to note that in many
cases susceptibility to disease in aquaculture involves
mutual interactions between several of such aetiological factors.
Application of cell culture methods to protect

aquatic animal health
Several cell culture methods and associated in vitro
assays, which dissect the pertinent biological mechanisms (i.e., immune responses), are applicable for
implementing preventive and disease control measures
in aquaculture. In vitro models are experimental
systems that allow higher control of the conditions of
the assays, reducing also the variability of responses
due to unavoidable stress responses, in which the
interactions between aggressors and the host can
be analyzed. Such methods are being applied to
standardize and validate diagnostic methods, and to
develop new safe, therapeutants and effective disease
control methodologies. The application of similar in
vitro methods to shellfish farming is lagging behind,
and there is an urgent need for the development of
validated long-term cell cultures and cell lines from

molluscs and crustaceans. A considerable number of
fish cell lines have been developed (Fryer and Lannan,
1994), but only a very few established cell lines are
available from shellfish species (Tapay et al., 1995;
Neumann et al., 2000b; Chen and Wang, 1999a).
The cell culture methodologies of major importance
for aquatic animal health are summarised in Tables 1
and 2.
The development of such methodologies was due
in part to the increasing importance of aquaculture.
Notwithstanding, some fish and shellfish species have
become important research models that are playing
important roles in the development of modern biology.
Thus, the zebrafish (Danio rerio) is a fundamental
animal model for developmental biology (Haffter and
Nusslein-Volhard, 1996; Trede et al., 2001), and it
is expected that this species, as well as the pufferfish (Fugu rubripes), will be of great importance in
the development of eukaryotic genomics (Talbot and
Hopkins, 2000; Taylor and Semple, 2002). Advanced
biological research in such animal models should
also provide scientific knowledge and technical backgrounds for the development and application of new
methods to control disease in aquacultured fish and
shellfish species. For instance, studies on the zebrafish
haematopoiesis and lymphopoiesis (Trede et al., 2001;
Hansen and Zapata, 1998; Lieschke, 2001; Barreda
and Belosevic, 2001) may provide tools to enhance the
responses to vaccination in aquacultured fish.
Nutritional diseases and the relationship between

feeding and immunocompetence
The application of cell culture techniques to study
the relationships between nutrition requirements and
disease in aquaculture would be exemplified by
studies on the nutritional requirements for polyunsaturated fatty acids (PUFA) between different farmed
fish species (Table 3). Lipids are an indispensable component of aquacultured fish feeding (Tacon,
1996), and dietary deficiencies in essential fatty acids
have been related to lordosis and muscular dystrophy,
or to no alterations depending on the fish species
(Takeuchi, 1996). Several in vitro methods, which
included the use of several cell lines, and of hepatocyte and astrocyte primary cell cultures from different
fish species, were used to study in vitro the elongation and desaturation of different PUFA. These in
vitro models were also useful to unveiling the pro-
114
Table 1. Major available cell culture methods used to study fish and shellfish pathology
Culture systems
References
From fish
Many permanent epithelioid, fibroblastic and leukocyte cell lines
Primary cultures and subcultures of dispersed cells from blood and organs
Primary tissue cultures of explants from different organs
Some histotypic and organotypic cultures
From shellfish
Primary cell and tissue cultures from several organs
Short-term histotypic and organotypic cultures
Two cell lines from the lymphoid organ of the shrimp Penaeus stylirostris#
A cell line for nervous tissue of the crayfish Orconectes limosus
A long-term cell line from heart tissue of the oyster Crassostrea gigas
A long-term cell line from heart tissue of the clam Meretrix lusoria
Fryer and Lannan, 1994; this review
Bayne, 1998; Rinkevich, 1999;

Toullec, 1999; Freshney, 2000
Tapay et al., 1995
Neumann et al., 2000
Chen and Wang, 1999a
The term permanent is used, as it is not known if most of such fish cell lines are immortalised; # Other name: Litopenaeus stylirostris.
Table 2. Major available in vitro assays used to study fish and shellfish pathology
Target
Assays for
Cell biology
Cell phenotype
Genotoxicity
Cell integrity and growth
Cell behavior
Modulation of cell metabolism
Specific cell function(s)
Cell morphology, cell polarisation, expression of genes and/or cytoplasmic/membrane molecules

Mutagenesis, chromosome aberrations, carcinogenesis
Cell viability and proliferation (blastogenesis, mitogenesis)
Cell-cell communication, adhesion, motility, secretion, endocytosis . . .
Activation/inhibition of specific metabolic pathways (enzyme activity, Ca++ influx . . .)
Assays for the specific capacities of a cell type (production of antibodies, phagocytosis . . .)
Tissue and organ physiology

Modulation of metabolism
Cell differentiation/turnover
Functions that depend of the preservation of the tissue/organ microstructure

Tissue regeneration, haematopoiesis, angiogenesis . . .
Major references in: Rinkevich, 1999; Stolen et al., 1990, 1992, 1995, 1997; Toullec, 1999; Ganassin et al., 2000; Mothersill and Austin,
2000.
inflammatory mechanisms underlying the relationship

between dietary PUFA and cardiac lesions in Atlantic
salmon (Bell et al., 1993). Similar studies combining
leukocyte (Ashton et al., 1994; Bell et al., 1996) and
astrocyte (Tocher et al., 1996) cultures assessed the
effect of fatty acid diet on fish inflammatory responses,
as modifications of dietary lipids affected the in vitro
production of the leukotrienes LTB4 and LTB5 by
isolated salmon kidney macrophages.
A similar in vitro approach using cell cultures
of T- and B-cells from peripheral blood leukocytes
(Bly et al., 1990), and of long-term monocyte cell
lines (Vallejo et al., 1992b) from the channel catfish
(Ictalurus punctatus) was used to demonstrate the relationship between the lipid composition of leukocyte
cell membranes, especially PUFA and cholesterol, and
the thermal adaptation of fish leukocytes through the
modification of membrane fluidity (Bly and Clem,

1992).
Toxicological diseases
Aquatic animals have provided models for many
toxicological studies, and it is well known that
many aquatic invertebrate species, including farmed
molluscs such as mussels, are good bioindicators
of environmental pollution (DAdamo et al., 1977;
Sheehan, 2000). As social and scientific constrains
are pushing forward the development of the In vitro
Toxicology discipline, several cell culture methods
and in vitro assays have been developed and used
for such purposes; particularly short-term primary
cultures and cell lines from fish tissues have provided
115
Table 3. Cell cultures used to study the selectivity and metabolism of fatty acids in fish and their effects on leukocyte reactivity
Cell culture systems
In vitro assays
References
Ghioni et al., 1999, 2001

Tocher et al., 1995
Tocher and Dick, 2001
SAF-1 (Gilthead sea bream, Sparus aurata, fin)
Cell viability and growth

Metabolic transformation of
Polyunsaturated fatty acids
(PUFA)
PLHC-1 (Topminnow, Poeciliopsis lucida, hepatocarcinoma)

TF (Turbot, Scophthalmus maximus fin)
Channel catfish, Ictalurus punctatus, monocyte-like
Antigen processing and

presentation at different
temperatures
Permanent cell lines

AS (Atlantic salmon, Salmo salar, heart, liver, kidney and spleen tissues)
EPC (Carp epithelial papilloma, Epithelioma papulosum cyprini)
Primary cultures
Astroglial brain cells from
Rainbow trout (Oncorhynchus mykiss)

Turbot (Scophthalmus maximus)
Isolated hepatocytes from S. salar

Macrophage-enriched and leukocyte cultures from S. salar pronephros
successful cell culture systems for environmental

toxicology (Babich and Borenfreund, 1991; Castao
and Tarazona, 1995; Castao et al., 1996; Segner,
1998; Segner and Cravedi, 2001).
Concerning fish health, there are numerous studies
that have demonstrated the influence of different
aquatic inorganic and organic pollutants on disease
susceptibility in farmed fish (Zeeman and Brindley,
1981; Dunier and Siwicki, 1993; Zelikoff, 1993;
Anderson and Zeeman, 1995; Bols et al., 2001), and
the effects of aquatic toxicants in the fishs innate
immune system have been reviewed recently by Bols
et al. (2001). Researchers have applied cell culture
methods to dissect the underlying mechanisms of the
immunomodulation effects of heavy metals and fungal
toxins, and their relationships with fish health. Some
of the cell cultures methods and in vitro assays that
have applied in such studies are shown in Table 4.
As an example, Aflatoxin B1 is a mycotoxin that
has carcinogenic and immunomodulatory effects in
many vertebrates, and the rainbow trout is particularly
susceptible to the carcinogenic effects of aflatoxin B1 ,
either after exposure in feed (Sinnhuber et al., 1978)
or in water (Croy et al., 1980). The mechanisms
Metabolic transformation
of PUFA
Production of prostaglandins
and leukotrienes
Metabolic transformation
of PUFA
Production of prostaglandins
and leukotrienes
Leukocyte motility
(chemotaxis)
Tocher and Ghioni, 1999

Ghioni et al., 2001
Tocher et al., 1995
Tocher et al., 1995
Ghioni et al., 1999, 2001
Tocher et al., 1996
Tocher and Wilson, 1990;

Tocher and Sargent, 1990
Tocher et al., 1996
Tocher et al., 1997
Ashton et al., 1994;
Bell et al., 1996
underlying aflatoxin B1-induced hepatic carcinogenesis in trout, which involves the biotransformation of
aflatoxin B1 by hepatic enzymes of the cytochrome
P450 (CYP) family, have been investigated in vitro
using cultures of isolated hepatocytes, in which the
expression of hepatic CYP and the occurrence of DNA
mutations were analyzed. Also, the immunomodulatory effects of aflatoxin B1 in rainbow trout have
been investigated using in vitro assays with leukocytes from salmonids exposed in vivo in feed or
in water as embryos (Ottinger and Kaattari, 2000),
and in leukocytes exposed to the mycotoxin in vitro
(Table 4).
On the other hand, the toxicity of certain compounds, as polycyclic aromatic hydrocarbons (PAH)
and aflatoxins, is due to the metabolites resulting
of the biotransformation of the initial product, a
phenomenon that can be studied in vitro using fish
cell cultures (Loveland et al., 1987; Behrens et al.,
2001). However, such biotransformation is mediated
by specific enzymes, which in many cases may not be
well expressed in simple cultures of isolated cells, as
demonstrated by the use of cultures of isolated hepatocytes versus hepatocyte aggregate cultures to study the
116
Table 4. Examples of cell culture methods and assays applied to study the effects of the in vitro exposure to toxicants in fish
a) Immunomodulation by metals
Primary cultures
Leukocytes from
Toxicant
In vitro assays
References
Zebrafish (Danio rerio) pronephros
Copper
Zinc
Rougier et al., 1994
O. mykiss peritoneal macrophages
Nickel
O. mykiss pronephros
Mercury
Phagocytosis
Natural cytotoxic
activity (NCC)
Cell motility
Phagocytosis
Leukocyte viability
Phagocytosis
Production of ROS#
Production of antibodyforming cells
Bowser et al., 1994

Voccia et al., 1994
Spleen sections from O. mykiss
Copper
b) Effects of polycyclic aromatic hydrocarbons (PAHs)
Toxicant
In vitro assays
References

RTL-W1 (Rainbow trout, O. mykiss, liver)
Nine PAHs
Induction of
cytochrome P4501A
Behrens et al., 2001
Primary cultures
Isolated hepatocytes from S. salar
Several PAHs
Induction of
biotransforming
enzymes
(CYP450 family)
Mutagenesis DNA
covalent binding
Bailey et al., 1984;

Behrens et al., 2001
Aflatoxin B1
Coculture of isolated hepatocytes from rainbow trout

and the cell line RTG-2 (rainbow trout gonad)
Isolated PBLs from O. mykiss
3-Methylcholanthrene
Benzo[a]pyrene
Benzo[a]anthracene
Beta-naphthoflavone
Aflatoxin B1
Induction of
cytochrome P4501A
Leukocyte viability
B-cell mitogenesis
to LPS
Secretion of polyclonal
antibodies after LPS
stimulation
Anderson et al., 1989
Croy et al., 1980;

Bailey et al., 1984;
Loveland et al., 1987;
Scholz and Segner,
1999
Arkoosh and Kaattari,

1987
Ottinger and Kaattari,
1998
# ROS: Reactive oxygen species; LPS: bacterial lipopolysaccharide.
estrogenic potency of different xenobiotics (Flouriot

et al., 1993; Petit et al., 1997). Also, it has been
described that a rainbow trout liver cell line (RTL-W1)
was more sensitive to polycyclic aromatic hydrocarbons, as determined by the expression of cytochrome
P4501A, than isolated hepatocytes in primary culture
(Behrens et al., 2001). As an alternative to organotypic
cultures, co-cultures of rainbow trout isolated hepatocytes with the RTG-2 cell line have been used for such
studies (Scholz and Segner, 1999).
Concerning the application of invertebrate cell
cultures in toxicology, short-term primary cell/tissue
cultures have been used (Table 5), due to the lack of
validated aquatic invertebrate cell lines. The expres-
sion of detoxifying enzymes, the detection of DNA

breakage, the study of cell viability, and of some other
cell responses were some of the parameters analyzed.
Infectious diseases
Research on the prevention of infectious diseases is
the field in which cell cultures and in vitro assays
have been mostly applied in aquaculture. The implementation of preventive and control strategies requires
obtaining sufficient information on: a) the pathogenicity mechanisms; b) the interactions between the host
and the pathogens; and c) the defense mechanisms
117
Table 5. Some shellfish cell culture methods used for in vitro toxicology studies
Primary cultures
Toxicant
In vitro assays
References
Isolated cells from the digestive gland of mussel (Mytilus edulis)
Several
contaminants,
including
PAHs
Fenitrothion#
Cell viability
Expression of
detoxifying enzymes
DNA strand-breakage
Expression of
detoxifying enzymes
Cell viability
Birmelin et al., 1998a;

Mitchelmore et al.,
1998
Isolated cells from the hepatopancreas of crayfish

(Procambarus clarkii)
Tissue cultures from
Heart of oyster (Crassostrea gigas)
Gill of clam (Ruditapes decussatus)
Isolated haemocytes from M. edulis
Isolated haemocytes from oyster (Crassostrea virginica)
Mexel-432
By-products of
chlorination
PAHs
Tributyltin
Triforine
Birmelin et al., 1998b

Domart-Coulon et al.,
2000
Structural alterations
of lysosomes
Phagocytosis
Production of ROS
Cell viability
Phagocytosis
Grundy et al., 1996a, b
Anderson et al., 1997

Alvarez and Friedl,
1990
# Organophosphate pesticide; Organic molluscicide; ROS: Reactive oxygen species; Fungicide.
of the host. Also, improving disease resistance by

selecting appropriated genetic traits is becoming a
new, interesting approach. As indicated below, cell
culture methods and allied in vitro assays are being
applied to achieve each of such strategies.
Cell culture methods in diagnosis of infectious
diseases
Cell culture methodologies are essential tools for the
diagnosis of viral diseases in farmed fish (Wolff,
1988; Hetrick and Hedrick, 1993). Such techniques
are mainly based on the use of fish cell lines sensitive to different viruses (Ahne, 1985; Hetrick and
Hedrick, 1993; O.I.E., 2000), in which viral infection
is primarily demonstrated by the cytopathic (due to
cell lysis) or syncytial (produced by cell-cell fusion)
effects (Table 6). Furthermore, viral in vitro infection of susceptible cell lines facilitates several serological techniques for surveillance and diagnosis of viral
diseases (O.I.E., 2000), including the detection of fish
antibodies against the pathogenic viruses by means of
the viral neutralization test (LaPatra, 1996). Interestingly, the susceptibility of the cell lines to a particular
virus might not be related to the in vivo susceptibility
to the viral disease, and several non-salmonid cell lines
are routinely used for the diagnosis of salmonid viral
diseases (Lorenzen et al., 1999a; Hostnik and Jencic,
2000).
As the occurrence of cytopathic/syncytial effects
may take several days, and in some cases no such
effects occur under some culture conditions, in the

last ten years serological methods that use monoclonal
antibodies (Nicholson, 1993; Lorenzo et al., 1995) and
molecular biology techniques, as the polymerase chain
reaction (PCR; Miller et al., 1998a), or combination
of the two (Estepa et al., 1995), have been applied to
speed up the diagnosis of fish viruses (Table 6).
Cell culture has been used also for the detection
of some intracellular bacterial fish pathogens, as in
Rickettsiae spp. (Fryer and Lannan, 1996) and Renibacterium salmoninarum (McIntosh et al., 1997). The
shared characteristic between such microorganisms
is that they are fastidious to grow in bacteriological
media. For instance, it may take longer than a month
to obtain colonies of R. salmoninarum in bacteriological cultures, but this bacteria can be grown on one
week in the EPC cell line from carp, a fish that is not
susceptible to the pathogen (McIntosh et al., 1997).
The pathogen grows intracellularly in the EPC cells,
and such culture method also allowed for the expression of specific virulence factors of R. salmoninarum
and for the maintenance of the in vivo virulence of the
isolates (McIntosh et al., 1997).
Concerning viral diseases in farmed shellfish,
some primary cell cultures from crustaceans and
molluscs (Table 7) have been used to grow and
to titrate viral pathogens, as there are not validated and available permanent cell lines from farmed
molluscans (Mulcahy, 2000) and crustaceans (Mulford
and Villena, 2000). For crustaceans, monolayers
from the lymphoid organs, circulating haemocytes,
118
Table 6. Synopsis of cell culture methods used for diagnosis of intracellular pathogens in farmed fish
Y
Monolayers of various cell lines=
Viruses
In vitro assays
References
Many fish DNA

and RNA viruses
Cytopathic or syncytial effects
Wolff, 1988;
Hetrick and Hedrick, 1993;
Ahne, 1985
Immunodetection of viral antigens

(FAT, IPAP, ELISA . . .)
O.I.E. 2000; LaPatra, 1996
Viral neutralisation test
LaPatra, 1996;
Lorenzo et al., 1996;
Nicholson, 1993
PCR methods
Lorenzo et al., 1996;

Miller et al., 1998b
Immunocapture and PCR methods
Estepa et al., 1995
IHN-V#
Viral titration
Hostnik and Jencic, 2000
Bacteria
In vitro assays
References
CHSE-214
P. salmonis
Cytopathic effect and FAT
Fryer and Lannan, 1996
EPC
R. salmoninarum
Immunodetection (APAAP$ )
McIntosh et al., 1997
Cell suspensions of freshly

trypsinised EPC cells
of specific virulence factors

=
Y Most frequently used cell lines for these studies were: CHSE-214 (Chinook salmon embryo) and RTG-2 from salmonids; EPC and FHM
(Fathead minnow, Pimephales promelas) from cyprinids; BB (Brown bullhead, Ictalurus nebulosus) and CCO (Channel catfish, Ictalurus
punctatus, ovary) from catfishes; BF-2 (Bluegill, Lepomis macrochirus, fry) from sunfish.
ELISA: enzyme-linked immunosorbent assay; FAT: Immunofluorescent antibody test; IPAP: indirect immunoperoxidase antibody assay;
# IHN-V: Infectious haematopoietic necrosis rhabdovirus; VHS-V: Viral haemorrhagic septicaemia rhabdovirus; Piscirickettsia salmonis;
Renibacterium salmoninarum.
ovary, and heart of penaeid shrimps have been used.

Concerning molluscs, primary cultures of haemocytes
were used to isolate an unidentified virus causing
amyotrophia in the Japanese black abalone (Nakatsugawa et al., 1999). Also, diagnostic techniques for
some viral shellfish diseases have been performed
using permanent fish cell lines, in which certain
viruses isolated from crustaceans replicate (Loh et al.,
1990; Lu and Loh, 1992). The same approach also has
been tested with relative success for some molluscan
viruses. Although such methods have not been validated for shellfish health surveillance and certification
(O.I.E., 2000), their further development may be of
great help to identifying new emerging viral pathogens
(Austin, 2000; Elston, 2000).
sents an increasing risk for human health (Midtvedt

and Lingaas, 1992; Tenover and Hughes, 1996) and
for the sustainable use of the aquatic environment
(Subasinghe, 1997). In addition, some drugs have
long-term toxic and immunomodulatory effects on
fish (Van der Heijden et al., 1992; Stoffregen et al.,
1996). The immunomodulating effects of several antibiotics and of organophosphorus pesticides have been
investigated using leukocyte cell cultures from fish,
in which different immunological parameters were
assayed (Table 8). Results from such studies allowed
establishing correlation between in vitro immunosuppression, and the interference of various antimicrobial
drugs (Lunden et al., 1998, 1999) and of pesticides
(Dunier and Siwicki, 1993) with immune responses in
vivo.
Safe use of drugs for disease control

The use of pesticides, disinfectants, and antimicrobial drugs has been growing in parallel with the
development of aquaculture (Grave et al., 1997),
and their rational use is a need for the growth of
aquaculture (Barg and Lavilla-Pitogo, 1996). It is
becoming evident that the use of such products repre-
Study of pathogenicity mechanisms

The use of cell cultures is a must to study the
biology and pathogenic mechanisms of culturable
viruses affecting aquaculture (Wolff, 1988; Hetrick
and Hedrick, 1993; Bernard and Bremont, 1995;
119
Table 7. Cell culture methods used for isolation of viral pathogens in shellfish
Crustacean
viruses
Permanent fish cell lines
Epithelioma papulosum cyprini (EPC)
Y
IHHNV=
YHV
In vitro assays
References
Cytopathic effect effects

and quantal titration assays
Loh et al., 1990;

Lu and Loh, 1992
Primary cultures from shellfish tissues

Monolayers of circulating haemocytes and tissue
cultures of lymphoid organ, ovary, and heart from
shrimps Penaeus monodon, P. japonicum and
P. penicillatus
Lymphoid organ and ovary of P. monodon
PmSNPV#
WSV
YHV
Cytopathic effect and

electron microscopy
Chen et al., 1995;

Chen and Wang, 1999b
WSV
Lymphoid organ from P. vannamei and P. stylirostris
YHV
Subcultures of lymphoid (Oka) organ from

P. stylirostris
WSV
Cytopathic effect and

quantal titration assays
Nadala et al., 1997;

Kasornchandra et al., 1999
Lu et al., 1995

EK-1 (Eel, Anguilla japonica, kidney) and EPC
BF-2, AS, FHM and GF
(Blue striped Grunt, Haemulon sciurus, fin)
BF-2
Primary cultures from shellfish

Primary cultures of haemocytes from Japanese
black abalone (Nordotis discus discus)
Tapay et al., 1997
Molluscan
viruses
In vitro assays
References
Akoya-virus
(IPN)-like
reovirusc/
Reo-like virus
13P2
Cytopathic effect
Cytopathic effect
Cytopathic effect
Miyazaki et al., 1999

Hill, 1976;
Hill and Alderman, 1977
Meyers, 1979
Viral agent of
amyotrophia
Cytopathic effect
Nakatsugawa et al., 1999
=
Y IHHNV: Infectious hypodermal and haematopoietic necrosis virus; # PmSNPV: singly enveloped nuclear polyhedrosis virus from P. monodon
(formerly MBV for monodon-type baculovirus); YHW: yellowhead virus; WSV: white spot virus (also called CBV for Chinese baculovirus);
From Japanese pearl oyster, Pinctada fucata martensii; c/ From oyster Ostrea edulis and clam Tellina tenuis; From Eastern oyster, Crassostrea
virginica.
Essbauer and Ahne, 2001). Such studies does not

include only diagnostic procedures (see Tables 6 and
7), but also the analysis of the interactions between
fish viruses and their host cells to identifying some
of the mechanisms involved in the different steps of
viral infection. The literature on this subject is so
extensive (i.e., see Woo and Bruno, 1999) that a
comprehensive description of the different cell culture
methods and of in vitro assays used for the study of
fish viral pathogenesis is well beyond the scope of
this review. However, the analysis of the pathogenesis of the rhabdovirus causing haemorrhagic viral
septicaemia (VHS) in salmonids is a paradigmatic
example of the use of such methods, which were
utilized to analyze the susceptibility of different cells
and tissues to infection (Estepa et al., 1992; Diago
et al., 1993a; Tafalla et al., 1998), to characterize
proteins involved in viral entry in cells (Coll, 1997;

Bearzotti et al., 1999; Estepa et al., 2001), and to study
the modulation of the viral replication by endogenous
nitric oxide production (Tafalla et al., 1999).
As indicated for the diagnosis of viruses, the
lack of well-developed cell culture methods from
molluscans and crustaceans has prevented the use
of permanent invertebrate cell lines to analyze the
pathogenesis of major viral diseases affecting aquacultured shellfish (McGladdery, 1999), so that a few in
vitro studies have been done using primary fish tissue
cultures or fish cell lines (see Table 7).
However, cell culture methods also have been
developed to study the virulence factors of bacterial
(Toranzo and Barja, 1993; Austin and Cross, 1998;
Austin and Austin, 1999) and parasitic (Nielsen and
Buchmann, 2000; Buchmann et al., 2000; Nolan and
120
Table 8. Cell culture methods used in studies on the safe use of drugs in fish farming
Primary cultures from fish tissues
Antibiotics
In vitro assays
References
PBLs and leukocyte cell

suspensions from spleen and
kidney of carp (Cyprinus carpio)
Tetracyclines, sulfonamides,
sulfonamides-trimethoprim,
ampicillin, macrolides,
lincomycin, gentamicin and
chloramphenicol
Oxolinic acid, oxytetracycline
florfenicol and sulfadiazinetrimethoprim
Mitogenic response to PHA#

after in vitro exposure
Grondel et al., 1985;

Grondel and Bo Sten,
1982
Mitogenic response to PHA

and to LPS after in vitro
exposure, or after oral
administration of antibiotics
Lunden and Bylund,

2000
Pesticides
In vitro assays
References
Macrophage-enriched cultures from

pronephros and spleen of C. carpio
Lindane, trichlorfon,
dichlorvos and atrazinec/
Summarized in Dunier
and Siwicki, 1993
Lymphocyte cell suspensions from

pronephros and spleen of C. carpio
Lindane, trichlorfon and

dichlorvos
PBLs and macrophage-enriched

cultures from pronephros of rainbow
trout (O. mykiss)
Lindane
Phagocytosis and production

Y after in vitro
of ROS=
exposure
Mitogenic proliferation to
PHA or ConA after in vitro
exposure
Calcium influx and
production of ROS after
in vitro exposure
PBLs and leukocyte cell suspensions

from pronephros of rainbow trout
(O. mykiss)
# PHA: phytohaemagglutinin (T-cell mitogen); LPS: bacterial lipopolysaccharide (B-cell mitogen);

Organophosphorous insecticides; c/ Chlorinated triazine herbicide; =
Y ROS: Reactive oxygen species.
Johnson, 2000) fish pathogens, as well as for some

bacteria (Montero and Austin, 1999) and parasites
(Mourton et al., 1992) affecting aquatic invertebrates.
Many different in vitro methods have been applied for
such purposes (Table 9), but a large part of the studies
dealing with bacterial pathogenicity have focused on
the cytotoxic effects of extracellular products on fish
cells to identify virulence factors.
Moreover, cell culture methodologies have provided a deeper knowledge on the mechanisms of the
host-pathogen interactions for some bacterial pathogens. In vitro assays have been used to analyze the
pathogenicity mechanisms involved in the different
steps of the diseases, as bacterial attachment and entry
into the host, avoidance of defense mechanisms, and
persistence in chronic, asymptomatic phases of the
infection (Evenden et al., 1993; Austin and Austin,
1999). This is the case for bacteria that are obligate
or facultative intracellular pathogens, as Mycoplasma
sp., Mycobacterium sp., some Aeromonas sp., or R.
salmoninarum (Table 9). Moreover, the combination
of cell culture and of molecular biology methodologies provides new and useful tools for the investigation
of pathogens and host, as has been demonstrated for E.
tarda (Ling et al., 2000). In this study, virulent and
avirulent strains of pathogen were transformed with
plasmids encoding the green (GFP) and blue (BFP)
Betoulle et al., 2000a, b
Organochlorine insecticide;
fluorescent proteins, and the tagged bacteria were

used to examine the molecular mechanisms underlying the in vitro adherence and invasion of EPC
cells.
Concerning the pathogenesis of parasites, the fish
EPC cell line has been used to grow some fish parasites in vitro, and to study the interactions between the
pathogen and the target and defense tissues of the host
(Table 9). It is interesting that cultures of pronephros
macrophages have allowed investigation of the mechanisms that underlay the resistance or susceptibility
to the microsporidian Loma salmonae, which infects
Pacific salmons (Oncorhynchus spp.), but not Atlantic
salmon (Shaw et al., 2001).
Concerning shellfish, the pathogenicity mechanisms of some Vibrio species, and the cytotoxic effects
of their extracellular products on crustaceans, and in
vitro defense reactions of cultured molluscan haemocytes to bacterial and parasitic intracellular pathogens have been studied (Table 9). For crustaceans,
infectivity studies in vitro have allowed establishment of good correlation between the in vivo and in
vitro virulence of various Vibrio isolates of prawn
(Montero, 1998; Mulford 2001). It is important to
note that no cell culture methods are available to
study the pathogenesis of molluscan viruses (Chen and
Wang, 1999b), but several primary long-term cultures
121
Table 9. Some cell culture methods used in studies on pathogenicity of fish and shellfish bacterial and parasitic pathogens
Bacterial fish pathogens
In vitro assays
References
Renibacterium salmoninarum
Bacterial growth Expression

of virulence factors
McIntosh et al., 1997
SAF-1: Gilt-head seabream

(Sparus aurata) fin
Listonella anguillarum,
Vibrio alginolyticus and
V. harveyii
Cytopathic effects of
extracellular products
Bejar et al., 1997
EPC, RTG-2, CHSE-214, RTH-149:

Rainbow trout (O. mykiss) hepatoma
cells
Aeromonas hydrophila and

A. salmonicida
Bacterial adherence and
invasion of fish cells
Survival of intracellular
bacteria
Austin and Austin, 1999;

Garduo et al., 2000
EPC and GF
L.anguillarum and
Photobacterium damselae
subsp. Damselae
Microscopy study of
cell-pathogen interactions
of signal
Analysis of transduction
pathways that precede
bacterial internalization
Wang et al., 1998
EPC
P. damselae subsp. piscicida
Bacterial adherence and

invasion of fish cells
Magarinos et al., 1996;

Lopez-Doriga et al., 2000.

the kidney of blue gourami
(Trichogaster trichopterus)
Edwardsiella tarda
Cell division and survival

of intracellular bacteria
Production of ROS#
Ling et al., 2000;

Rao et al., 2001

pronephros of rainbow trout
(O. mykiss)
L. anguillarum
Phagocytosis
Bactericidal activity
Production of ROS
Boesen et al., 2001
Macrophage-enriched cultures
from pronephros of O. mykiss
Mycobacterium spp.
Electron microscopy
Chen et al., 1998
Gill explants from rainbow trout
Mycoplasma mobile
Histopathology and
electron microscopy
Stadtnder and Kirchhoff,

1995;
Stadtlnder et al., 1995

pronephros of O. mykiss
R. salmoninarum
Bacterial growth
Production of ROS
Hardie et al., 1996
Electron microscopy
Flao et al., 1996
Fish parasites
In vitro assays
References
Ichthyophthirius multifiliis
(ciliate)
Stimulation of parasite development: attachment, survival

and growth of trophonts
Nielsen and Buchmann,

2000
Gyrodactylus derjavini
(ectoparasitic monogenean)
Encapsulation and degradation of the ectoparasites by the

epithelial cells
Buchmann et al., 2000
Loma salmonae
Phagocytosis
Shaw et al., 2001

EPC
Explants from gills of O. mykiss

EPC

the pronephros of Atlantic salmon
(S. salar) and of Chinook salmon
(O. tshawytscha)
122
Table 9. Continued

EPC
Shrimp primary cell cultures from
haemocyte and ovary of shrimp
Penaeus stylirostris
Monolayers of circulating haemocytes
and explants from ovary and
haematopoietic organs of Dublin Bay
prawn (Nephrops norvegicus)
Haemocytes from Eastern oyster,
Crassostrea virginica
Haemocytes from oysters Ostrea edulis
and C. gigas
Haemocytes from C. virginica and
C. gigas
Bacterial shellfish pathogens
In vitro assays
References
V. penaeicida, V. alginolyticus
and V. nigripulchritudo
Goarant et al., 2000
V. penaeicida, V. alginolyticus
and V. nigripulchritudo
Goarant et al., 2000
V. harveyi
Cytopathic effects
Cytotoxicity of extracellular
products
Montero, 1998;
Mulford, 2001
L. anguillarum
Production of ROS
Bonamia ostrea
Electron microscopy of
parasite engulfment
Production of ROS
Phagocytosis
Bramble and Anderson,

1998
Boulo et al., 1991;
Mourton et al., 1992
Haplosporidium nelsoni
Ford et al., 1993
# ROS: Reactive oxygen species.
of lymphoid and haematopoietic organs, and of circulating haemocytes have been used for replication of
crustacean viruses (see above). In a recent study, Wang
et al. (2000) were able to demonstrate the morphogenesis of the virus causing the white spot syndrome of
Penaeus monodon using primary cell cultures from the
lymphoid organ of this species.
Characterization of the host defense mechanisms

This is the field in which cell culture methods have
provided a great wealth of information useful for
disease protection of farmed fish and shellfish. A
deep understanding of the defense responses, and of
their cell mediators, is needed to develop preventive
strategies based upon vaccination and immunostimulation (Evelyn, 1997). The functional capacities of the
different cell populations and humoral factors involved
in fish and shellfish defense have been investigated
using a variety of in vitro assays (Table 10). Such
methods have been essential tools to understanding
the complexity of the defense mechanisms, and
to exploiting them for the health protection of the
aquacultured species.
Concerning immunological studies in fish, the
combination of in vitro assays, cell separation, and
cell cultures techniques, and particularly the devel-
opment of clonal leukocyte cell lines from channel

catfish, Ictalurus punctatus, have provided the body
of knowledge necessary to establish the existence of T,
B, natural killer, and immune accessory cell in teleosts
(Warr, 1997; Miller et al., 1998b). Before the development of monoclonal antibodies against specific phenotypic markers for fish T-cells (Scapigliati et al., 1999),
the functional characterization of the T-cell (immunoglobulin negative, Ig ) and B-cell (Ig+ ) populations
in fish had to rely first on immunological in vitro
assays, which were performed in cultures of leukocytes isolated from peripheral blood and lymphoid
organs (spleen and kidney). The in vitro assays used
to delineate the specific T-cell competence included
mitogenic responses (Etlinger et al., 1976; Caspi et
al., 1984; Sizemore et al., 1984), specific alloantigendriven cell proliferation (Caspi and Avtalion, 1984b;
Miller et al., 1986), and cytotoxicity against allogenic
(Verlhac et al., 1990; Yoshida et al., 1995) and virusinfected cells (Hogan et al., 1996). B-cell function
was assayed by means of the in vitro formation of
antibody-forming cells and secretion of Igs to culture
supernatants (Miller et al., 1985; Kaattari et al., 1986).
When they became available, the in vitro immunological experiments were performed in morphologically and functionally distinct catfish cell lines resembling B-cells, T-cells, and monocytes (Vallejo et al.,
1991; Miller et al., 1994; Warr, 1997; Stuge et al.,
123
Table 10. Some cell culture methods applied to study innate- and specific-immune defence mechanisms of fish
Description
Permanent non-leukocyte cell lines
RTG-2, EPC
Interferon (INF)-producing
cell lines
In vitro assays
References
Inhibition of viral infectivity
de Sena and Rio, 1975
Permanent lymphoid cell lines

T-cell lines from channel
catfish, I. punctatus
Lymphoid cell lines from

PBLs
Proliferative responses to mitogens,

antigens and growth factors
Mixed leukocyte reaction (MLR)
T-cell B-cell cooperation
Cytotoxicity assays: antigen-specific,
Y activities
NCC and NK=
Production and effects on cytokines
Molecular biology of T-cell activation
Vallejo et al., 1992a;

Miller et al., 1998b;
Stuge et al., 2000
T-cell line from rainbow trout,

O. mykiss
A lymphoid cell line that

express TCR
Proliferative response induced by

the specific antigen (the G protein of the
VSH-virus)
Estepa and Coll, 1997;

Estepa et al., 1999
B-cell lines from channel

catfish, I. punctatus
Lymphoid cell lines from

PBLs
Proliferative responses induced by

mitogens, antigens and growth factors
Antibody-forming cell assays and
secretion of antibodies
Molecular biology of B-cell activation
Miller et al., 1994, 1998b
Antigen trapping, processing and

presentation
Production and effects of cytokines
Vallejo et al., 1991;

Miller et al., 1998b
Carp (CLC)

Phagocytosis
Production of ROS#
Faisal and Ahne, 1990;

Weyts et al., 1997
Goldfish (GMCL)
Chemotaxis
Phagocytosis
Production of ROS
Production of reactive nitrogen
intermediates
Wang et al., 1995
Rainbow trout (RTS11)
Bactericidal activity (lysozyme-like

activity)
Phagocytosis of latex beads and yeast
Uptake of acetylated low density
lipoprotein and of acridine orange
Production of ROS
Ganassin and Bols, 1998;

Hong et al., 2001
Clonogenic assays for

haemopoietic cell clones
in semi-solid matrixes
Production of myeloid colonies

Mitogenic responses
Finegan and Mulcahy, 1987;

Estepa and Coll, 1992;
Ganassin and Bols, 2000
Cell cultures of mixtures

of lymphoid cells,
monocytes, granulocytes
and thrombocytes
Production of myeloid colonies

Cell migration (chemotaxis and
chemokinesis) assays
Production and effects of IL-2 like
cytokines
Moritomo et al., 1993

Caspi and Avtalion, 1984a;
Grondel and Harmsen, 1984
Permanent macrophage cell lines

Channel catfish, I. punctatus
Mononuclear or dendriticlike cell lines from various
origins
Primary leukocyte cultures

Haematopoietic cells from:
Rainbow trout
From carp
Unfractionated leukocyte cell
suspensions from blood and
lymphoid organs of carp
124
Table 10. Continued
Fractionated leukocyte
populations from blood
and lymphoid organs
Primary tissue cultures

Tissue explants from
pronephros and spleen
from rainbow trout
Description
In vitro assays
References
Macrophages from
goldfish, C. auratus and
in vitro derived kidney
macrophages (IVDKM)
Monocyte macrophage cell

differentiation and functional
characterisation of macrophage
subpopulations
Production of ROS
Production of reactive nitrogen
intermediates
Neumann et al., 2000a, b
Neutrophils and
macrophages from carp
Phagocytic activity
Production of ROS
Kemenade et al., 1994
Neutrophils from carp
Cytotoxic activity against mammalian

tumour cell lines
Kurata et al., 1995
Neutrophils from rainbow

tout
Migration assays
Production of ROS
Hamdani et al., 1998
EGCsc/ from O. mykiss
Endocytosis and intracellular

degradation of proteins
Dorin et al.,1993
EGCs from sea bass,

D. labrax
Cytotoxic assays for EGCs
Cammarata et al., 2000
Histotypic and
organotypic tissue
cultures formed by a
heterogeneous stromal
and haemopoietic cell
populations
Defence responses (mitogenic and

antigen cell proliferation, antibodyforming cell and cytotoxic assays)
Haemopoiesis and production and
effects of haemopoietic growth factors
Anderson et al., 1986, 1989

Diago et al., 1998;
Flao et al., 1998
Identification of dendritic cells
Angiogenesis in vitro
Diago et al., 2000
Long-term cultures from the

stroma of pronephros and
spleen from rainbow trout
Cell cultures of reticular

and endothelial cells
forming multilayers
Matrix and feeder layers for the support

of haemopoiesis
Production of haemopoietic growth
factors
Establishment of stromal cell lines
Moritomo et al., 1990;

Diago et al., 1993b;
Explants from gut, skin and

gills from rainbow trout
Tissue cultures of organ

explants including
epithelial and connective
tissues
Responses of leukocytes, EGCs,

fibroblastic and epithelial cells
Production of cytokines and defence
substances (lysozyme)
Mothersill et al., 1995,

Flao, 1996;
Flao et al., 1996, 1997;
Holland and Rowley, 1998
# ROS: Reactive oxygen species; NCC: Natural cytotoxic cell; =

Y NK: Natural killer cell; /c EGCs: Eosinophilic granular cells.
2000), which in some cases have been cloned. Such

cloned cell lines have been considered indispensable tools for dissecting the physiology, biochemistry
and molecular biology of teleost immune responses
(Clem et al., 1996). Also, some monocyte/macrophage
cell lines have been developed in carp (Faisal and
Ahne, 1990; Weyts et al., 1997), goldfish (Wang et
al., 1995), and rainbow trout (Ganassin and Bols,
1998), but no other leukocyte cell lines are available
for non-catfish fishes (Table 10). Interestingly, a longterm culture of rainbow trout lymphoid cells showing
specific viral antigen-dependent cell proliferation and
T-cell-like features, as the expression of the alpha- and
beta-chains of the T cell receptor, has been reported

(Estepa and Coll, 1997; Estepa et al., 1999).
The functions of fish monocyte/macrophage cells
include migration on response to chemoattractants,
phagocytosis, microbicidal activities, secretion of
cytokines and interleukins, and antigen processing
and presentation (Secombes and Fletcher, 1992;
Vallejo et al., 1992a). Each one of these functions
has been investigated (Table 10), and now can be
routinely assayed in vitro. The existence of different
macrophage subpopulations in fish has been also
analyzed using leukocyte cultures from lymphoid
organs, in which the existence of different stages
125
of monocyte/macrophage differentiation (Neumann et
al., 2000a) and of dendritic-like cells (Ganassin and
Bols, 1996) were demonstrated.
In vitro methods have been applied also to study
other leukocyte populations (Table 10). The existence
of non-specific cytotoxic cells (NCC), resembling
mammalian natural killer (NK) cells, has been proven,
and their killing mechanisms characterized, in vitro
(Greenlee et al., 1991; Evans and Jaso-Friedmann,
1992). There are few studies on isolated populations
of granulocytes, mainly neutrophils, due to the lack
of effective methods to separate them from other nonlymphoid cell populations. Notwithstanding, differential centrifugation on Percoll gradients (Bayne, 1986)
or monoclonal antibodies against granulocytes (Slierendrecht et al., 1995; Hamdani et al., 1998) provided
methods to obtain granulocyte-enriched cultures from
carp and rainbow trout, which were used to study
their microbicidal, cytotoxic, migratory activities, and
activation of respiratory burst (production of reactive
oxygen species; ROS). Eosinophilic granulocytes and
eosinophilic granular cells (EGCs) are usually very
scarce in healthy fish, and thus there are very few in
vitro studies on these cell types. The in vitro endocytic, proteolytic, and cytotoxic activities of the EGCs,
considered to be counterparts of mammalian mast
cells (Reite, 1998), were studied after their isolation from the intestine of rainbow trout (Dorin et
al., 1993) and the peritoneal cavity of Dicentrarchus
labrax (Cammarata et al., 2000).
Likewise, cell culture has been an indispensable tool to study presence and functions of fish
cytokines. Until recently, biological assays have been
the only method to demonstrate the activity of putative
cytokines in fish (Secombes, 1991). Lately, there
have been substantial progresses in the biochemical
characterization and gene sequencing of a number of
fish cytokines, and even in their production by DNA
recombinant technologies (Secombes et al., 1999).
Interferon activity was the first cytokine demonstrated
in fish using viral-infected cultures of the RTG-2 cell
line and assaying the ability of the supernatants from
such cell cultures to reduce the cytopathic effect in
cultures that were pre-treated with the supernatants
before the viral infection (de Sena and Rio, 1975).
Mammalian macrophage cell lines have been used
to test the putative presence of fish IL-1, but recently
the biological activity of a trout recombinant IL-1beta has been successfully and more appropriately
tested using a trout macrophage cell line (Hong
et al., 2001). Detection of IL-2 activity was done
in in vitro assays involving leukocyte proliferation in

response to alloantigens or to lymphocyte mitogens
(Caspi and Avtalion, 1984a; Grondel and Harmsen,
1984). Cytokines that modulate the migratory activity
(MIF) or that activate the metabolism and defense
activities (MAF) of macrophages were also identified
using exposure of macrophage cultures to the supernatants of leukocytes stimulated with T-cell mitogens
(Secombes, 1991).
Another approach to dissecting the complexity of
the lymphoid system in fish consists of the use of
primary tissue cultures from the different lymphoid/
haematopoietic organs. Such studies intended either to
analyze some immune mechanisms, such as production of antibody-forming cells, or the in vitro differentiation of blood cells, either in cultures of isolated
leukocytes in soft matrixes (like agarose, fibrin clots,
or methylcellulose) or in cultures from whole haematopoietic organs (Table 10). Subcultures of haematopoietic and lymphoid organs have been also utilized
as a source of stromal cell lines. The only available
description of in vitro angiogenesis in fish was done
on long-term cultures of a trout pronephric stromal cell
line (Diago et al., 2000).
Some researchers have developed methods to
establishing tissue cultures from the skin, gills, and
gut of various fish species, in which the local defense
responses can be studied (Table 10), as these external
organs represent the first defense barriers that prevent
the entry of pathogens. In this sense, a promising
approach to study the physiology of the EGCs consists
in the use of cultured explants of EGC-rich organs,
as the gills, to observe the differentiation, maturation, and degranulation processes in such cells after
exposure to pathogens, bacterial products (Flao et
al., 1996, 1997), or cytokines (Holland and Rowley,
1998).
The in vitro characterization of the different shellfish haemocyte populations was performed after the
development of techniques for their isolation by differential centrifugation on Percoll/Ficoll density gradients (Sderhll and Smith, 1983; Cheng and Downs,
1988). However, due to the lack of appropriate cell
culture methods, most studies dealing with defense
cells, as haemocytes, have used very short-term
(hours) cultures. Notwithstanding, Walton and Smith
(1999) have developed a technique for the culture
of hyaline haemocytes from decapods that allow the
cells to remain viable and to retain the phagocytic
capacity for 14 days. Several in vitro assays (Table 11)
have been applied on short-term cultures of shellfish
126
haemocytes to assess their antimicrobial abilities, as
the degranulation of haemocytes, and activation of the
prophenoloxidase system, phagocytosis, and cytotoxic
capacities. Also, the production of oxidative metabolites (Adema et al., 1991; Ottaviani et al., 1993;
Arumugam et al., 2000), cell adhesion (Johansson and
Sderhll, 1988; Huang et al., 2000), and chemotactic
responses (Yip et al., 2001) have been studied in vitro
using haemocyte cultures from shellfish.
Vaccination and immunostimulation

These methods, which exploit the enhancement of the
self-defense mechanism of the animals, constitute the
most promising tools for health protection in aquaculture (Anderson 1997; Evelyn, 1997; Sakai, 1999).
The in vivo testing of the protection conferred by
the vaccination, in immunization-challenge trials, has
been the only effective procedure to determine vaccine
potency in fish (Nordmo, 1997). It has been difficult to standardize the in vivo immunization-challenge
protocols in fish, and to obtain reproducible results
from them, probably due to the interferences between
immunocompetence, stress, and environmental factors
(Bly et al., 1997), and the disparate genetic background of farmed fish (Nordmo, 1997). Therefore,
in a limited number of studies some researchers
have tried to establish a correlation between in vitro
immune responses to immunogens (Table 12), and
the in vivo efficacy of the vaccine. Most of such
studies have focused on bacterial vaccines (Reitan and
Secombes, 1997), and have included assays for antibody forming cells and in vitro secretion of specific
antibodies, lymphocyte proliferation, and secretion of
cytokines (Yui and Kaattari, 1987; Midtlyng et al.,
1996; Marsden et al., 1996). While a clear relationship between in vitro cellular immune responses could
be demonstrated for protective antigens (Reitan and
Secombes, 1997; Marsden et al., 1996), the major
problem for in vitro antibody production assays is
that antigen preparations from gram negative bacteria
contains lipopolysaccharide (LPS), which elicits mitogenic and polyclonal activation of B-cells (Yui and
Kaattari, 1987).
Concerning viral vaccines, only a few studies
have used in vitro assays for cell immune responses
to identify protective antigens, and mainly when
recombinant viral proteins were available. For the
rhabdovirus causing viral haemorrhagic septicaemia
(VHS) in salmonids, the in vitro proliferative
responses of pronephros leukocytes to native, recombinant proteins, and synthetic peptides of the viral
nucleocapsid and of the membrane glycoprotein were
assayed (Estepa et al., 1994; Lorenzo et al., 1995).
Higher proliferative responses of pronephros leukocytes from rainbow trout survivors of VHS infection
were obtained for the native membrane glycoprotein,
and the assay allowed identification of some peptides
containing putative protective epitopes (Lorenzo et al.,
1995).
An alternative and promising method for fish
vaccination consists of the application of recombinant
DNA methods to develop DNA-vaccines (Anderson et
al., 1996a, b). In DNA vaccination animals are transfected with DNA vectors so that the host cells express
the appropriate pathogen epitope(s) (Anderson et al.,
1996a; Leong et al., 1997). The antigen may be then
recognized by T-cells, in the context of the appropriate host molecules of the major complex of histocompatibility (MHC), thus resulting in an effective
immune response, which ideally should be similar to
that deployed in response to the pathogen infection
(Kanellos et al., 1999a; Corbeil et al., 1999; Lorenzen
et al., 1999b; Fernndez-Alonso et al., 2001). Several
fish cell lines (Table 12) have been used for the selection of appropriate DNA vectors and reporter genes,
and of reagents and conditions to facilitate the transfection of the fish cells, that would allow for the
correct expression (in intensity and duration) of the
epitopes, and to confirm the expression of the antigens in transfected fish cells (Anderson et al., 1996b).
Such studies are necessary steps for the development
of DNA-vaccine technology (Kanellos et al., 1999b).
Immunostimulants primarily activate non-specific
cellular mechanisms (reviewed by Sakai, 1999),
and therefore most in vitro studies have focused
on the activation of macrophages and granulocytes
(Table 12). Some studies analyzed antigen-specific
humoral responses after in vitro exposure of cultures
of leukocyte suspensions or of spleen explants to
immunostimulants. However, most of the research in
this field has been done using fish leukocyte cultures,
and the assays comprised study of the proliferation of
lymphocytes, and of the phagocytic and microbicidal
capacities of macrophages and granulocytes. Such
studies investigated the effects and cell mechanisms of
immunostimulants used now in aquaculture (as yeast
-glucan). Jensen and Robertsen (2002) have used two
pronephric cell lines (SKK-1 and TO) from Atlantic
salmon to study the effects and cell mechanisms
of synthetic double-stranded RNA polyinosinic poly-
127
Table 11. Some short-term cell culture methods applied to study cell defence mechanisms of shellfish
Description
In vitro assays
References
In vitro degranulation in the presence

of the bacteria Moraxella sp.
Smith and Sderhll, 1983
Monolayers of separated
populations of the different
haemocyte types
Cytotoxic capacity against

mammalian tumoral and non-tumoral
cell lines
Sderhll et al., 1985
Granular haemocytes from

shore crab, Carcinus maenas
granular haemocytes
Haemocyte adhesion and degranulation assays mediated by cell adhesion

proteins and -1,3-glucan binding
proteins
Sderhll et al., 1986;

Thornqvist et al., 1994
Hyaline cells from shore crab,

Carcinus maenas
hyaline haemocytes
Phagocytosis: opsonic activity of cell

adhesion proteins
Monolayers of isolated
haemocyte populations
from mussel, Mytilus
galloprovincialis
Cell suspensions of unfractionated haemocyte
Production of ROS mediated by

PMA# , laminarin or phagocytosis
Phagocytosis of yeast
Arumugam et al., 2000
Granular haemocytes from

crayfish, Pacifastacus
leniusculus
Cell suspensions of separated

granular haemocytes
Haemocyte adhesion mediated by cell

adhesion proteins
Johansson and Sderhll,

1988;
Huang et al., 2000
Short-term cultures of haemocytes from shrimp Penaeus

penicillatus
Cell suspensions of unfractionated haemocytes
Chemotaxis towards bacterial formyl

peptide
Yip et al., 2001

Monolayers of mixed or
Haemocytes from the
separated (hyaline, granular
freshwater crayfish,
and semigranular) haemocytes
Astacus astacus
on glass coverslips
# PMA: Phorbol 12-myristate 13-acetate; ROS: Reactive oxygen species.
cytidylic acid (poly I:C) in antiviral resistance against

infectious pancreatic necrosis (IPNV) and infectious
salmon anaemia (ISAV) viruses.
Since invertebrates lack antigen-specific immune
responses, immunostimulants may represent useful
tools to enhance disease resistance in farmed shellfish
(Song et al., 1997; Roch, 1999), but very few studies
involving in vitro assays with shellfish defense cells
have been done (Table 12).
Disease resistant strains of aquatic animals

An economical and environmentally friendly method
to control infectious diseases is the selection or
development of animal strains resistant to specific
pathogens (Mialhe et al., 1995; Roch, 1999; Van
Muiswinkel et al., 1999). This approach can be
obtained either through the genetic selection of appropriated traits, as higher immune responses (Gjedrem
and Gjoen, 1995; Wiegertjes et al., 1996; Gaffney
and Bushek, 1996; Naciri-Graven et al., 1998) or
lower susceptibility to stress (Fevolden et al., 1994), or
by genetic engineering creating transgenic animals in
which appropriate genes are introduced (Roch, 1999;

Mialhe et al., 1995). As indicated above for the development of DNA vaccines, cell culture methods can be
helpful to select the appropriate genes and methods
to obtain stable transgenic animals (Iyengar et al.,
1996). Thus, in vitro assays on fish cell lines have
been used to search for vectors that may be used to
obtain transgenic farmed fish (Du et al., 1992). Also,
cell transfection in primary cultures of dissociated
embryo cells, and from heart, is being used to test
the suitability of transfection systems and selection of
appropriate promoters in molluscs (Boulo et al., 1996,
2000).
Needs for the further development of fish and

shellfish cell culture
The potential of cell culture techniques and associated methodologies to provide tools and strategies
for disease control in aquaculture has not been yet
achieved, mainly due to the poor standardization of
fish cell culture methods, and to the lack of suitable techniques to develop cell lines from shellfish.
128
Table 12. Examples of cell culture and in vitro assays applied for the development and assessment of vaccines and immunostimulants for
aquaculture
Vaccine development
In vitro assays
References
EPC, RTG-2, rainbow trout hepatoma

cells (RTH149) and tilapia TO2 cells
Search for appropriate vectors

and transfection conditions to
develop fish DNA-vaccines
and transgenic fish
Expression of reporter genes

(luciferase, -galactosidase)
EPC cell line
FHM and EPC cell lines
Confirmation of the
expression of pathogen genes
from candidate DNA vaccines
Transfection assays with plasmid

containing the genes encoding for
the G or N proteins of IHN-V
Transfection assays with plasmid
containing the genes encoding for
the G or N proteins of VHS-Vc/
Liu et al., 1990; Bearzotti et al., 1992; Moav

et al., 1992; Winkler
et al., 1992; Betancourt
et al., 1993; FernandezAlonso et al., 1999
Anderson et al., 1996b
Assays for immunostimulants
In vitro assays
References
Cell lines from pronephros (SHK-1)

and TO from S. salar
Effect of the in vitro exposure

to synthetic double-stranded
RNA (poly I:C)
Expression of interferon-induced
proteins (Mx proteins)
Antiviral resistance against in
vitro infection with IPN-V# and
ISA-V
Jensen and Robertsen,

2002
Vaccine development
In vitro assays
References
Leukocyte suspensions from blood

and lymphoid organs
Evaluation of native,
recombinant proteins and
synthetic peptides as vaccines
Leukocyte proliferation
Yui and Kaattari, 1987;

Estepa et al., 1994;
Lorenzo et al., 1995
In vitro assays
References
Spleen explants from O. mykiss
Levamisole
Siwicki et al., 1990
Neutrophiles from the kidney and

peritoneal cavity of the plaice,
Pleuronectes platessa
FMLP
Numbers of antibody producing

cells against Y. ruckeri O-antigen
or DNP-Ficoll
Cell migration (chemokinesis)
Leukocyte cultures from blood and

spleen of O. mykiss
Leukocyte cultures from the
pronephros of O. mykiss
Peptide FK65
Zymosan
Vitamin C

pronephros of S. salar and L. limanda
-Glucans
Production of ROS
Spreading of adherent cells
Y or antigen-induced
PHA=
proliferation of lymphocytes
Secretion of MAF
Production of ROS
Phagocytosis
Production of ROS
Expression of lysozyme gene

and secretion of lysozyme to
the culture medium
Lorenzen et al., 1998;

Heppell et al., 1998
Nash et al., 1986
Kitao et al., 1987;

Kitao and Yoshida, 1991
Hardie et al., 1993
Robertsen et al., 1994;

Sveinbjrnsson and
Seljelid, 1994; Tahir
and Secombes, 1996
Paulsen et al., 2001
In vitro assays
References
Pecten maximus haemocytes

Short-term cultures of monolayers
of unfractionated haemocytes from
Tiger shrimp, Penaeus monodon
Zymosan
Zymosan
-Glucans
Production of ROS
Production of ROS
Le Gall et al., 1991

Song and Hsieh, 1994
IHN-V: Infectious haematopoietic necrosis virus; c/ VHS-V: Viral haemorrhagic septicaemia virus; # IPN-V: Infectious pancreatic necrosis
Y PHA: Phytohaemaglutinin; MAF: Macrophage activating
virus; ISA-V: Infectious salmon anaemia virus; ROS: Reactive oxygen species; =
factor; FMLP: N-formylmethionyl-leucyl-phenylalanine.
129
On the other hand, some of the current uses of cell
culture methods for aquaculture, as diagnosis, are
being replaced by faster and less-laborious molecular
biology methods. Also, future studies on the characterization of fish and shellfish defence responses will
depend less on cell cultures and more on the development of antibodies and molecular probes to identify
and to quantify cellular and humoral mediators of the
immune mechanisms, as leukocyte subpopulations,
growth factors and cytokines (Reitan and Secombes,
1997).
However, there is still a large spectrum of applications in which cell cultures would be indispensable. Of particular interest are those needed to study
the pathogenic mechanisms of intracellular pathogens,
and to isolate and characterize new viral pathogens.
Moreover, several in vitro assays are not available for
fish/shellfish species because they need cell culture
methods that have not been developed, as cell lines or
well-characterized long-term cultures from shellfish,
or histotypic/organotypic fish cultures. The progress
of cell culture methods for aquaculture will depend on
the adoption of strategies that would facilitate: 1) the
unrestrained availability of current cell lines, and the
dissemination of knowledge and cell culture methods
for fish and shellfish cell culture; 2) the development
of standardized and repetitive cell culture techniques;
and 3) the validation of in vitro assays on cell cultures.
These points are further discussed below.
Availability of cell lines and of cell culture methods
Fryer and Lannan (1994) reported the existence
of about 160 well-characterized cell lines from 34
families of bony fishes, However, a very few number
of them are readily available from international cell
culture repositories (ATCC or ECACC). Also, the
few cell lines that have been described from crustaceans, two from SV-40 transformed primary cultures
of the lymphoid organ of the shrimp Penaeus stylirostris (Tapay et al., 1995), and one developed very
recently from a crayfish (Neumann et al., 2000b), are
not available in any cell culture repository.
Standardization of cell and tissue culture methods
There are two major points relating to the standardization of cell cultures: a) the study of requirements
for the appropriate maintenance of cells and tissue
in culture (Freshney, 2000; Mulford and Villena,
2000), and b) the characterization and authentication
of cell lines and tissue cultures (Kaplan and Hukku,

1998; Hay, 1998; Lyons-Alcantara, 2000). A major
problem in fish and shellfish cell culture is that there
is a large number of fish and shellfish species that
are being used as research models. Many of such
species live in different aquatic environments and
show disparate physiological requirements, and there
is a lack of knowledge of the nutritional and physiological requirements of fish and shellfish cells grown
in vitro. Many investigators in fish cell culture have
borrowed mammalian cell culture techniques (Bols
and Lee, 1991), and little research has been done on
the specific requisites of fish/shellfish cells in culture.
For instance, the response of several cell lines from
marine and freshwater fish to different culture media,
variations of saline strength and osmotic pressure, and
to the temperature of incubation have been analyzed
(Fernandez et al., 1993a, b, c). Also, Bols and
collaborators have investigated the influence of several
nutritional supplements, such as glutamine (Bols et
al., 1994; Ganassin et al., 1998) and nucleosides
(Ganassin and Bols, 1992; Ganassin et al., 1994), on
the growth of fish cell lines and leukocytes. However,
such systematized studies are scarce in fish, and even
fewer investigations have been done for shellfish cell
culture (Domart-Coulon et al., 1994; Buchanan et al.,
1999; Mulcahy, 2000; Mulford and Villena, 2000;
Mulford et al., 2000).
Another important point to be standardized is
the requirement and source of autologous, allogenic
and/or xenogenic serum supplements or replacements
in culture media for fish (Barlian and Bols, 1991;
Ganassin et al., 1994) and shellfish (Mulford and
Villena, 2000) cells. A valid approach to solve this
problem is to develop serum-free media. Such standardized media are available for some fish tissue
cultures and cell lines used in immunology (Luft et
al., 1991, 1994) and toxicology (Segner and Cravedi,
2001; Kohlpoth and Rusche, 1997; Dowling and
Mothersill, 2001). Some attempts to use a defined
culture medium have been done in shellfish (Ghosh et
al., 1995; Mulford and Villena, 2000; Shimizu et al.,
2001), but with little success for the long-term growth
of the cells.
The other need for the standardization of cell lines,
as well as for obtaining reproducible tissue cultures
from aquatic animals, is the authentication and study
of provenance of the cell lines and tissue cultures. This
requires the characterization of the cultured cells, but
in some cases it has been difficult to confirm the identity of the cultured cells. For instance, the morphology
130
of some fish cell lines depends on the culture conditions and of the culture medium (Diago et al., 1998;
Pagniello et al., 2002). Moreover, in shellfish there is
a great diversity of cell types, many of which show
a high plasticity in shape and structure, depending on
the age or biological cycles of the animals. Moreover,
many cell types are ill-characterized in vivo, so that
it may be difficult to recognize them in vitro (LyonsAlcantara, 2000).
It is difficult to ascertain whether fish cell lines
are immortalized or not, and if immortalization is
due to transformation. For instance, T- and B-cell
lines from channel catfish have been described as
unique from their putative mammalian counterparts
in that they are immortalised without the need for
exogenous factors or overt attempts at transformation (Clem et al., 1996). Such long-term cell lines
express high levels of telomerase activity indefinitely
(Barker et al., 2000), but this enzyme, as well as the
heat shock protein 70 (Hsp70) gene, were expressed
constitutively only in immortal cell lines (Barker et al.,
2002). Also, telomerase activity was detectable in cell
lines established from the eye of pufferfish fry Fugu
(Takifugu) niphobles and Fugu (Takifugu) rubripes
(Bradford et al., 1997), and these authors interpreted
this fact as evidence that such cell lines were capable
of indefinite proliferation. On the other hand, the presence of retroviruses have been reported in some cell
lines from tumor cells, such as leukaemia cells of the
Chinook salmon, O. tshawytscha (Eaton et al., 1993),
and neurofibroma of damselfish, Pomacentrus partitus
(Schmale et al., 1996), but also in cell lines from
normal tissues of several species (Frerichs et al., 1991;
Petry et al., 1992; Iwamoto et al., 2000). However,
it remains unchecked whether other permanent cell
lines, including those available from cell repositories,
are transformed by retroviral infection.
Another point of major concern is the presence of
eucaryotic endosymbionts in most aquatic invertebrates (Rinkevich, 1999), which are able to contaminate and overgrow cell cultures (Pomponi et al.,
1997; Lyons-Alcantara, 2000). Contamination by
thraustochytrids, unicellular heterotrophs considered
members of the stramenopiles, has been described
in cell cultures from different marine invertebrates,
including molluscs (Mulcahy, 2000). Pomponi et
al. (1997) reported that eucaryotic endosymbionts
forming outgrowths in invertebrate cell cultures have
been frequently misinterpreted as true invertebrate cell
cultures or cell lines. Therefore specific phenotypic
markers or genetic probes should be used to authen-
ticate the nature of the cultured cells. For instance,

gene probes were used to authenticate primary cell
cultures from crustaceans (West et al., 1999). Also,
DNA fingerprinting, a technique that allows comparison of the genetic structure of the cultured cells
with that of the specie from which the cells originated
(Webb and Debenham, 1992), allowed confirmation
of the origin of the RTG-2 cell line from rainbow trout
(Becerril et al., 2001).
Validation of in vitro assays
Ideally, cell and tissue cultures are substitutes of
whole-animal models, in which in vitro assays can be
applied to address specific biological issues. However,
to obtain meaningful information from them, the in
vitro test should be repetitive and reproducible, that is,
they should be validated according to standard procedures (Anderson, 1990; Ball et al., 1995). Moreover,
the information obtained from the in vitro assays
should be compared with data from in vivo studies on
the particular biological issue in order to establish in
vitro/in vivo correlation (Quinn and Mothersill, 2000).
Due to the limited amount of information still available on the in vivo biological responses of farmed fish
and shellfish or to the complexity of their physiological responses, validated assays have not been fully
established for such animal models. However, in vitro
test for aquatic toxicology and carcinogenesis are
promising tools in fish cell cultures (Castao et al.,
1996; Segner, 1998).
Concerning the defense responses, no validated
assays are available for the in vitro assessment of
vaccines, even in mammals (Lucken, 1999; Redhead
et al., 1999). However, the in vitro assays developed
for mammalian vaccinology also allow dissection and
scrutiny of different components of the cellular and
humoral immune mechanisms (Kourilsky et al., 1998;
Clay et al., 2001). A similar approach would produce
significant knowledge for the advancement of fish
vaccinology, but this requires that the appropriate
reagents, as for instance monoclonal antibodies to
T- and B-cell subsets, and to cytokines, should be
available, and used to implement relevant in vitro
immunological tests (Reitan and Secombes, 1997).
Conclusion
Cell culture and related in vitro assays have provided
useful knowledge for disease control in aquaculture.
131
Moreover, in vitro assays may provide the necessary tools to examine the defense responses of fish
and shellfish, avoiding the disturbance of the environmental and stress effects on their in vivo physiological responses, to which the ectothermic animals are
particularly sensitive. New techniques from molecular
biology, as well as the availability of new reagents
(such as monoclonal antibodies), are replacing some
of the traditional uses of cell culture for disease control
in aquaculture. Nevertheless, cell culture will be an
indispensable technique for the isolation and characterization of new pathogenic viruses and intracellular
bacteria, and for the study of pathogenicity mechanisms. Also, cell culture will facilitate in vitro studies
to dissect complex physiological responses.
It is expected that the increasing economic importance of disease in aquaculture will impel the progress
of cell culture methods for rational health management in aquaculture. Such advances should include
the establishment of new fish and shellfish cell lines,
and the development of methods for long-term maintenance of primary cell, histotypic and organotypic
cultures. Such methodological advances will come
from the standardization of cell culture methods, the
development of new reagents, and the validation of in
vitro assays.
Acknowledgements
The author express thanks to Dr. Julio Coll (INIA,
Madrid) for the critical reading of the manuscript,
and to the anonymous reviewers and editor who
contributed to improve the quality of this review.
The financial support of the EU (contract No. EGCVAC- QLK2-CT-1999-00799), and of the EU FEDER
Programme and the Spanish Plan Nacional de I+D
(project No. 1FD97-0467) is acknowledged.
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Reviews in Fish Biology and Fisheries 13: 111140, 2003.

2003 Kluwer Academic Publishers. Printed in the Netherlands.

Key words: aquaculture, cell culture, disease, fish, shellfish

Diseases constrain the development of aquaculture

Beyond 2000), aquaculture is an economic activity of

Aetiology of aquatic animal diseases

Application of cell culture methods to protect

validated long-term cell cultures and cell lines from

Nutritional diseases and the relationship between

Fryer and Lannan, 1994; this review

Bayne, 1998; Rinkevich, 1999;

Cell morphology, cell polarisation, expression of genes and/or cytoplasmic/membrane molecules

Tissue and organ physiology

Functions that depend of the preservation of the tissue/organ microstructure

inflammatory mechanisms underlying the relationship

modification of membrane fluidity (Bly and Clem,

Ghioni et al., 1999, 2001

SAF-1 (Gilthead sea bream, Sparus aurata, fin)

Cell viability and growth

PLHC-1 (Topminnow, Poeciliopsis lucida, hepatocarcinoma)

Channel catfish, Ictalurus punctatus, monocyte-like

Antigen processing and

Permanent cell lines

Rainbow trout (Oncorhynchus mykiss)

Isolated hepatocytes from S. salar

successful cell culture systems for environmental

Tocher and Ghioni, 1999

Tocher and Wilson, 1990;

Zebrafish (Danio rerio) pronephros

Rougier et al., 1994

O. mykiss peritoneal macrophages

Bowser et al., 1994

Spleen sections from O. mykiss

b) Effects of polycyclic aromatic hydrocarbons (PAHs)

Permanent cell lines

Behrens et al., 2001

Bailey et al., 1984;

Coculture of isolated hepatocytes from rainbow trout

Isolated PBLs from O. mykiss

Anderson et al., 1989

Croy et al., 1980;

Arkoosh and Kaattari,

# ROS: Reactive oxygen species; LPS: bacterial lipopolysaccharide.

estrogenic potency of different xenobiotics (Flouriot

sion of detoxifying enzymes, the detection of DNA

Isolated cells from the digestive gland of mussel (Mytilus edulis)

Birmelin et al., 1998a;

Isolated cells from the hepatopancreas of crayfish

Isolated haemocytes from oyster (Crassostrea virginica)

Birmelin et al., 1998b

Grundy et al., 1996a, b

Anderson et al., 1997

# Organophosphate pesticide; Organic molluscicide; ROS: Reactive oxygen species; Fungicide.

of the host. Also, improving disease resistance by

effects occur under some culture conditions, in the

Many fish DNA

Cytopathic or syncytial effects

Immunodetection of viral antigens

O.I.E. 2000; LaPatra, 1996

Viral neutralisation test

Lorenzo et al., 1996;

Immunocapture and PCR methods

Estepa et al., 1995