Aquaculture 212 (2002) 21 – 30

www.elsevier.com/locate/aqua-online

Susceptibility of Nile tilapia (Oreochromis niloticus)

to vibriosis due to Vibrio vulnificus
biotype 2 (serovar E)
Belén Fouz a, Elena Alcaide a, Rodolfo Barrera b, Carmen Amaro a,*
a
Departamento de Microbiologı́a y Ecologı́a, Facultad de Biologı́a, Universidad de Valencia,
Burjasot, Valencia 46100 Spain
b
Valenciana de Acuicultura S.A., Puzol, Valencia, Spain
Received 6 September 2001; accepted 19 November 2001

Abstract

The present study documents the susceptibility of Nile tilapia to the experimental vibriosis caused
by Vibrio vulnificus biotype 2 (serovar E) using a reference strain (Spanish Collection of Type
Cultures, CECT 4604) selected for its high degree of virulence for eels. The biotype 1 of this species
is one of the usual organisms involved in epizootics occurred in tilapia. After intraperitoneal
injection, the selected strain developed a haemorrhagic septicaemia similar to eel vibriosis with a
LD50 four log units lower than that exhibited by the type strain of the species, which belongs to the
biotype 1. The results obtained in waterborne and intubation challenges indicated that water and feed
could act as a vehicle for vibriosis transmission to healthy tilapia. Moreover, live cells and the
extracellular products derived from the strain CECT 4604 showed remarkable activity against tilapia
erythrocytes, which correlated with the in vivo production of extensive haemorrhagic areas. Our
results suggest that this bacterium could constitute a serious health hazard for tilapia, especially if it
is cocultured with eels. Thus, vaccination of tilapia with a vaccine against V. vulnificus biotype 2
could be the best strategy to prevent any cross transmission of the disease from eels to tilapia under
intensive rearing conditions.
D 2002 Published by Elsevier Science B.V.

Keywords: Vibrio vulnificus serovar E; Vibrio vulnificus biotype 2; Tilapia; Vibriosis

*
Corresponding author. Tel.: +34-96-3983104; fax: +34-96-3983099.
E-mail address: carmen.amaro@uv.es (C. Amaro).

0044-8486/02/$ - see front matter D 2002 Published by Elsevier Science B.V.

PII: S 0 0 4 4 - 8 4 8 6 ( 0 2 ) 0 0 0 0 2 - 9
22 B. Fouz et al. / Aquaculture 212 (2002) 21–30

1. Introduction

The Nile tilapia Oreochromis niloticus is one of the most important warm-water
cultured fish in the world, both in salt and fresh water. No serious infectious disease seems
to affect this species when it is extensively or moderately cultured (Plumb, 1999).
However, under stress conditions (including poor water quality and high density of fish),
tilapia can easily be infected by several opportunistic pathogens belonging to the genera
Streptococcus, Enterococcus, Aeromonas, Pseudomonas, Vibrio, Flexibacter and Edwar-
siella (Plumb, 1999). Among Vibrio and related genera, Listonella anguillarum, V.
parahaemolyticus and V. vulnificus are the main pathogenic species involved in salt water
culture, and V. mimicus and V. cholerae in fresh water culture (Sakata and Hattori, 1988;
Plumb, 1999). The disease caused by V. vulnificus occurs in the form of sporadic outbreaks
with 10% to 20% of mortality and the main associated symptoms are haemorrhaging
around the bases of fins, prostration in the swimming movements and muscle stiffness
(Sakata and Hattori, 1988).
The species V. vulnificus comprises three biotypes which differ in serological and
biochemical characteristics and host specificity (Tamplin et al., 1982; Tison et al., 1982;
Oliver, 1989; Amaro and Biosca, 1996; Biosca et al., 1997; Bisharat et al., 1999).
Regardless of the biotype, the species comprises strains that can cause in humans either
wound infections after exposure to seawater or handling of fish, or septicaemia after
ingestion of raw shellfish by immunocompromised hosts (Oliver, 1989; Amaro and
Biosca, 1996; Bisharat and Raz, 1996; Bisharat et al., 1999). Biotype 1 has also been
associated to infectious diseases occurred in tilapia and shrimp (Sakata and Hattori, 1988;
Song et al., 1990). Biotype 2 (or serovar E, according to Biosca et al., 1997) causes
severe vibriosis in eels that occurs as epizootics or outbreaks of high mortality responsible
for important economic losses in intensive culture of European eel (Anguilla anguilla)
(Biosca et al., 1991; Austin and Austin, 1993; Biosca, 1994; Dalsgaard et al., 1998; Høi,
1999). The main reservoir for biotype 2 is the eel (Biosca, 1994) from which it is
transmitted to healthy hosts under favourable physico-chemical conditions, using water as
a vehicle of infection (Amaro et al., 1995). In contrast to biotypes 1 and 3, this serovar
has seldom been isolated from water and healthy fish samples (Arias et al., 1999; Høi,
1999).
In Spain, Nile tilapia and European eels have recently started to be reared in a closed
experimental system, since the culture conditions, such as water temperature and salinity,
are similar for both species. We have recently developed and licensed a vaccine (vulni-
vaccine) against V. vulnificus biotype 2, which successfully induces protection in eels during
the main growth period in culture facilities (Fouz et al., 2001). Within this scenario, the
main objective of this study was to determine the susceptibility of tilapia to this pathogen
prior to program tilapia vaccination trials with the vulnivaccine. Since this serovar is phe-
notypically and genotypically homogeneous (Biosca et al., 1997), we selected one strain
which is representative of the highest virulence clone isolated in Spain (Biosca, 1994). We
conducted pathogenicity assays in tilapia with this strain and investigated possible routes of
infection. The type strain of the species, which belongs to biotype 1, was also included in
virulence challenges. In addition, we evaluated the role of toxins produced by the selected
strain in the infectious process as well as the role of serum from tilapia in the defence
 B. Fouz et al. / Aquaculture 212 (2002) 21–30 23

mechanisms. For comparative purposes, experimental assays with eels were conducted in
parallel.

2. Material and methods

2.1. Bacterial strains and growth conditions

The biotype 2 strain E86 (Spanish Collection of Type Cultures, CECT, 4604), originally
isolated from diseased eel kidney and the type strain of the species, CECT 529T, belonging
to biotype 1, were used in this study. The virulence degree as well as the toxicity potential of
strain CECT 4604 are the highest among those of the clones responsible for the epizootics
registered in cultured eels in Spain (Biosca, 1994). Lyophilized stocks were maintained at
room temperature (25 jC) and frozen stocks at 80 jC in marine broth 2216 (Difco
Laboratories) containing 15% (v/v) glycerol. Cells were routinely cultured in Tryptic Soy
Agar or Tryptone Soy Broth (Oxoid) supplemented with 0.5% NaCl (TSA-1 or TSB-1) for
24 h at 28 jC.

2.2. Experimental fish and maintenance conditions

Unvaccinated healthy eels (average weight 8 –10 g) and Nile tilapia (weight ranging
from 5 to 10 g) were used in this study. Fish were obtained from a Spanish fish farm where
both species have recently started to be reared in a closed experimental system with a
common source of water. Prior to challenges, fish were kept separately at 26 F 2 jC for 7
days in the laboratory in glass tanks containing dechlorinated water supplemented with
0.1% (w/v) NaCl supplied with filtration and air systems.

2.3. Challenge experiments

Duplicate experiments were carried out at the temperature routinely used on the farm
(26 F 2 jC) with groups of 8 – 10 fish in each.

2.3.1. Injection challenge

Fish were intraperitoneally (i.p.) injected with 0.1 ml of a cell suspension of strains
CECT 4604 or CECT 529T in phosphate buffered saline solution supplemented with 1%
(w/v) NaCl (PBS-1) (pH 7.0) (Amaro et al., 1992). Injected cell concentrations ranged
from 10 to 108 colony forming units (cfu) per fish. Control groups were injected with
sterile PBS-1 in the same way.

2.3.2. Immersion challenge

Bath challenges were basically performed as described by Amaro et al. (1995). Fish
were exposed separately for 60 min to serial 10-fold dilutions of stationary-phase 18-h
culture in TSB-1 of strains CECT 4604 and CECT 529T in a final volume of 3 l of water at
different salinity (0.1 and 0.5% NaCl). Assayed cell concentrations ranged from 108 to 105
cfu per ml of water. After challenge, each group of fish was placed in a new aquarium, and
24 B. Fouz et al. / Aquaculture 212 (2002) 21–30

the number of culturable cells in the remaining challenge suspension was determined by
plating serial 10-fold dilutions in PBS-1 on duplicate TSA-1 plates. Two control groups of
fish were challenged with sterile TSB-1.

2.3.3. Intubation and feed challenges

A volume of 0.1 ml of 10-fold dilutions in PBS-1 (from 109 to 105 cfu/ml) of strains
CECT 4604 or CECT 529T was inoculated to each fish through the mouth to the stomach
by using a silicone tube (1 mm in diameter) attached to a plastic syringe. In order to
determine the importance of transmission of the pathogen through food, commercial feed
contaminated with the same bacterial suspensions was also intubated to fish in the same
way. To prepare inocula, sterilised feed was aseptically homogenised with PBS-1 (ratio 1/
4, v/v) by incubating the mixture for 24 h at 28 jC. Afterwards, the homogenate was
diluted with a bacterial suspension (v/v) and maintained at room temperature for 30 min to
allow the absorption of the bacterial cells to the particulate material. Non-infected groups
of fish, challenged with sterile PBS-1 or feed homogenate, were also included.
In all cases, fish mortality was recorded daily during an 8 –10-day period and mean
lethal doses (LD50s) were calculated by using the Reed and Muench method (1938).
Anaesthesia was not used for any of the experimental procedures. In all challenges,
signs of moribund and dead fish were recorded, and samples from liver and kidney were
taken aseptically to confirm the cause of death. Mortalities were only considered if the
challenged strain was reisolated as a pure culture from internal organs and serologically or
biochemically confirmed (Amaro et al., 1992).

2.4. Haemolytic activity of whole cells and extracellular products

Extracellular products (ECP) of strains CECT 4604 and CECT 529T were obtained
using the cellophane plate technique of Liu (1957) with slight modifications (Amaro et al.,
1992). ECP protein concentrations were determined following Bradford (1976) using the
Biorad reagent (Biorad Laboratories), and bovine serum albumin (Sigma) as the standard.
Haemolytic activity was evaluated on TSA-1 plates supplemented with 5% (w/v) eel or
tilapia erythrocytes in PBS-1 by the method described by Amaro et al. (1992). Briefly, 5 Al
of a stationary phase cell culture in TSB-1 (109 cfu/ml) or ECP samples were inoculated in
2– 3-mm-diameter wells made on these plates. The ratio between the diameter of the
reaction zone and the well diameter, in duplicate experiments, served as a relative quanti-
fication of the activity.

2.5. Bacterial survival in serum

Bactericidal and bacteriostatic activities in serum from fish were measured as the
percentage survival of the strains in these fluids, according to the procedure defined by
Amaro et al. (1999). Serum samples, pooled from 6 to 8 fish, were obtained as described
previously (Biosca et al., 1993; Collado et al., 2000) and stored at 80 jC until use.
Briefly, stationary phase cell suspensions in PBS-1 were inoculated in duplicate in samples
of serum at a level of approximately 105 cfu/ml and incubated at 26 jC for 4 h. Viable cell
counts were determined by drop plating serial dilutions on TSA-1.
 B. Fouz et al. / Aquaculture 212 (2002) 21–30 25

3. Results

3.1. Injection challenges

The strains of biotypes 1 and 2 used in this study were virulent for tilapia by i.p.
injection (Table 1). Moribund fish showed clear signs of a haemorrhagic septicaemia;
external haemorrhages, mainly located on the head, ventral part of the body and anal fin,
and internal haemorrhages, mainly located in the intestine. Pure cultures of the inoculated
strain were recovered from the internal organs of moribund fish. Although the mean lethal
dose for the biotype 2 strain (4  104 cfu/fish) was lower than that for the biotype 1 strain
(1  108 cfu/fish), in both cases, fish mortality began to occur shortly after injection
(approximately 12-h postchallenge), with subsequent losses in the following 24 –48 h
(Table 1). As expected, the biotype 1 strain was avirulent for eel (LD50 higher than 1  108
cfu/fish) and the biotype 2 strain was highly virulent (LD50 = 1.5  102 cfu/fish) (Table 1).
Moribund eels showed external signs of septicaemia with petechiae and general haemor-
rhages, but without haemorrhages in the intestine. No mortality or visible changes were
observed in any of the control groups.

3.2. Immersion challenges

The biotype 2 strain CECT 4604 infected both fish species in the waterborne chal-
lenges performed at 26 F 2 jC in brackish water (0.5% of salinity). The mean lethal doses
were of 1.0  106 cfu/ml and 1.1  107 cfu/ml for eel and tilapia groups, respectively
(Table 1). When experiments were performed at lower salinities (around 0.1%), a mor-
tality of 100% was registered in both species with doses around 108 cfu/ml. In both cases,
affected fish showed the typical signs of a generalised septicaemia, with extensive hae-
morrhages in the mouth, head and pectoral and anal fins. Intestinal haemorrhages were
also observed in tilapia infected with the biotype 2 strain. The biotype 1 strain was aviru-
lent by bath for tilapia and eels (LD50 higher than 109 cfu/ml), regardless of the water
salinity (Table 1).

Table 1
Results of infectivity of Vibrio vulnificus biotype 2 (serovar E) (strain CECT 4604) and biotype 1 (strain CECT
529T) for Nile tilapia and European eel
Straina Virulence for tilapia/eelb
I.p. injection Immersionc Stomach intubation Feed intubationd
T
CECT 529 +/ / / /
CECT 4604 ++/+ + + +/ + + +/ ++/ +
a
CECT, Spanish Collection of Type Cultures.
b
Degree of virulence expressed as range of mean lethal dose 50% (LD50) after intraperitoneal (i.p.) injection
(cfu/fish), immersion (cfu/ml) or intubation (cfu/g fish) challenges [+++, 101 – 103;++, 104 – 106;+, 107 – 108; ,
> 109].
c
Fish were challenged and maintained in water of salinity 0.5% at 26 F 2 jC.
d
Commercial feed contaminated with bacteria was stomach intubated.
26 B. Fouz et al. / Aquaculture 212 (2002) 21–30

3.3. Intubation and feed challenges

Mortality of 50% occurred at a dose of 1.1  107 cfu/g fish in the group of tilapia chal-
lenged by stomach intubation with bacterial cells of the strain of biotype 2 CECT 4604,
while no mortality was recorded in the group of eels (Table 1). However, use of intubated V.
vulnificus biotype 2-laden feed at the same bacterial dose resulted in the decrease of the
mean lethal doses (LD50 of 2.0  106 cfu/g fish and 2.5  107 cfu/g fish in the group of
tilapia and eels, respectively) (Table 1). Remarkably, moribund fish belonging to both
species displayed internal signs of intestine inflammation. The biotype 1 strain was aviru-
lent by the oral route for tilapia and eels (LD50 higher than 109 cfu/g fish), regardless of the
presence of feed.

3.4. Haemolytic activity of cells and extracellular products

Live cells (at a dose of 106 cfu/Al) and ECPs (at concentrations of z 0.2 Ag of protein/
Al) of the strains of both biotypes were haemolytic against tilapia erythrocytes. The hae-
molytic activities were stronger (average ratio around 3) for tilapia than for eel (average
ratio around 1.5) erythrocytes.

Fig. 1. Survival of the strains CECT 4604 (biotype 2) and CECT 529T (biotype 1) after 4 h of incubation in serum
from Nile tilapia and European eels.
 B. Fouz et al. / Aquaculture 212 (2002) 21–30 27

3.5. Bacterial survival in serum

The biotype 2 strain was able to survive and multiply in samples of serum from both
fish species. The first 2 h after transferring cells to serum, the bacterial survival was around
100 –200%; however, in the following two hours, the cells numbers increased to achieve
survival percentages higher than 1000% at the end of the incubation period (Fig. 1). In
case of the strain of biotype 1, eel serum was strongly bactericidal (survival percentage
around 1%) and tilapia serum was slightly bactericidal (survival percentage around 70%)
(Fig. 1).

4. Discussion

The present study documents the susceptibility of Nile tilapia to the experimental vi-
briosis caused by V. vulnificus biotype 2. This species is one of the usual organisms involved
in epizootics occurred in tilapia (Plumb, 1999). In fact, V. vulnificus biotype 1 has been
reported to cause a mild infectious disease in tilapia (Sakata and Hattori, 1988). In agree-
ment with our previous results, the type strain of the species, which belongs to biotype 1,
was avirulent for eels, regardless of the challenge procedure (Amaro et al., 1992, 1995).
However, this strain was slightly virulent for tilapia after i.p. challenges. Its LD50 was si-
milar to that described by other authors for biotype 1 isolates from diseased tilapia (Sakata
and Hattori, 1988). Moribund tilapia showed symptoms of a haemorrhagic septicaemia
similar to the natural disease (Sakata and Hattori, 1988). In accordance with the secondary
pathogenic character of this biotype, the type strain of V. vulnificus was avirulent by bath and
was sensitive to serum of healthy tilapia.
In contrast, our results with the selected biotype 2 strain suggest that this biotype can
behave as a primary pathogen for tilapia. Firstly, after i.p. challenge, this strain showed a
LD50 more than three log units lower than that of the biotype 1 strains tested by us and
by other authors (Sakata and Hattori, 1988). The biotype 2 strain was less virulent for
tilapia than for eels (LD50 values around two log units higher for tilapia), its main host,
but its virulence degree was similar to those found for other vibrios in their hosts
(Toranzo and Barja, 1993). Other fish species, such as sea bass (Morone saxatilis) and
turbot (Scophthalmus maximus) or sea bream (Sparus aurata) and trout (Oncorhynchus
mykiss), are less susceptible than tilapia (LD50 after i.p. challenge from 105 to 106 cfu/
fish) or resistant (LD50 after i.p. challenge higher than 108 cfu/fish), respectively, to
infection by V. vulnificus biotype 2 (Biosca and Amaro, 1996). Secondly, this strain was
virulent by bath, with a LD50 similar to that exhibited by other biotype 2 isolates for their
natural host (Amaro et al., 1995). The vibriosis could be transmitted to healthy tilapia at
the temperature (26 F 2 jC) and salinity (0.1 –0.5% NaCl) conditions used in culture
facilities. In contrast, well-recognised tilapia pathogens, such as Streptococcus spp., are
unable to infect fish unless skin is scarified prior to bath challenge (Chang and Plumb,
1996). Thirdly, the biotype 2 isolate resisted and successfully multiplied in tilapia serum,
achieving similar cell numbers to eel serum. The resistance to fish serum is essential for
preventing the spreading of septicaemic bacteria to the main body organs (Amaro et al.,
1997). In case of eels, the resistance to serum is conferred by the high molecular weight
28 B. Fouz et al. / Aquaculture 212 (2002) 21–30

portion of the O-side chain of the serovar E LPS (Amaro et al., 1997). Biotype 1 strains
that lack this molecule are sensitive to the alternative pathway of complement and are
destroyed in the first stages of infection (Amaro et al., 1997). In the case of tilapia, the
effect of serum on the biotype 1 strain was more bacteriostatic than bactericidal. This fact
could explain why biotype 1 strains behave as a secondary pathogen for tilapia but not
for eels.
In all i.p. and bath challenges, moribund tilapia showed the main symptoms of natural
vibriosis in eels (Biosca et al., 1991), which are extensive haemorrhagic zones on the
entire body surface, especially on the mouth, gills and bases of fins. The strong in vitro
haemolytic activity showed by live cells and ECPs of both strains correlated with the in
vivo production of these extensive haemorrhagic areas observed in the experimental
challenges. Nevertheless, the virulence degree does not seem to be directly correlated with
the intensity of haemolytic activity since the strain of biotype 1 was as haemolytic as the
biotype 2 strain. Interestingly, moribund tilapia showed haemorrhages in the intestine that
were not observed in moribund eels. Thus, we tested whether tilapia vibriosis could be
transmitted through the oral route. The results obtained in stomach intubation and feed
challenges indicated that only the biotype 2 strain could infect healthy tilapia through the
oral route with or without feed. When the same dose of bacteria were given to eels, they
were infected only if bacteria of this serovar were protected with feed, probably because
cells alone were destroyed in the eel stomach. Consequently, tilapia seems to be more
susceptible than eel to infection by V. vulnificus biotype 2 through the oral route. The
ability to establish systemic infections by intragastric inoculation has been also reported
for other fish pathogens such as Enterococcus spp. in turbot (Romalde et al., 1996).
In summary, Nile tilapia could be considered as a susceptible V. vulnificus biotype 2
host, with water and feed being involved in its horizontal transmission. The absence of
reports on the isolation of biotype 2 from diseased tilapia could be due to the fact that its
occurrence is scarce in the aquatic environment (Arias et al., 1999; Høi, 1999). Within this
scenario, vaccination of tilapia with a vaccine, such as vulnivaccine (Fouz et al., 2001),
once brought into coculture facilities, could be the best strategy to prevent V. vulnificus
biotype 2 disease and improve fish survival under adverse environmental conditions. Such
practices would be necessary in farms where there have been previous vibriosis outbreaks
or epizootics and the bacterium could be present in water, either associated with particulate
material or with fish in a carrier state. Moreover, since the pathogen has been described as
an opportunistic human pathogen (Amaro and Biosca, 1996) and has recently been
involved in a septicaemic infection affecting a man injured by the fin of a live tilapia (Chan
et al., 1999), fish farmers should take appropriate measures during vibriosis episodes.

Acknowledgements

This work has been financed by the Spanish projects CICYT IFD97-0800 and ACU01-
009-C2-1 from the Ministerio de Educación y Ciencia and from the Ministerio de Ciencia
y Tecnologı́a, respectively. We thank Rafael Ruano and José Tornero from the Generalitat
Valenciana for supplying eels for assays. We also thank Barraclough-Donnellan for their
help with the English text.
 B. Fouz et al. / Aquaculture 212 (2002) 21–30 29

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