Effect of in vitro exposure to Vibrio vulnificuson hydroelectrolytic transport and structural changesof sea bream (Sparus aurata L.) intestine

Fathia Khemiss Æ Salwa Ahmadi Æ Raja Massoudi ÆSonia Ghoul-Mazgar Æ Sihem Safta ÆAli Asghar Moshtaghie Æ Dalila Saıdane

Received: 1 February 2008 / Accepted: 25 August 2008 / Published online: 30 September 2008

� Springer Science+Business Media B.V. 2008

Abstract The everted gut sac technique has been

used to investigate the effect of Vibrio vulnificus on

water and electrolyte (Na?, K?, Cl-, HCO3-)

transport on the intestine of sea bream (Sparus

aurata L.). Both the anterior and the posterior

intestine were incubated in a medium containing

108 V. vulnificus cells ml-1 at 25�C for 2 h. The

presence of V. vulnificus resulted in a significant

reduction (P \ 0.05) of water absorption in the

anterior intestine, while sodium absorption in the

anterior (P \ 0.01) and posterior (P \ 0.05) intestine

was elevated. Chloride absorption was increased, but

the changed was not significant, while potassium

absorption decreased significantly (P \ 0.05), but

only in the posterior intestine. Incubation the sea

bream intestine with V. vulnificus did not affect

carbonate secretion in the anterior segment, whereas

high secretion was stimulated in the posterior

segment (P \ 0.01). Histological evaluations dem-

onstrated damage in the anterior intestine of sea

bream that was characterized by the detachment of

degenerative enterocytes, alterations in the microvilli,

and the presence of a heterogenous cell population,

indicating inflammation. Based on our results, we

conclude that V. vulnificus caused cell damage to the

intestine of sea bream and that the anterior intestine is

more susceptible than the posterior part of the

intestine. Several hypotheses are suggested to explain

our observations, such as the presence of higher

numbers of villosities in the anterior intestine than in

the posterior one and/or the presence of endogenous

bacteria in the posterior intestine which may have a

protector role.

Keywords Electrolyte transport �Everted gut sac � Sparus aurata (L.) �Vibrio vulnificus � Water transport

Introduction

Fish bacteriosis is an infection arising from disruption

of the normal flora and results in fish diseases. These

diseases represent serious economic problems for the

commercial aquaculture of fish and shellfish and also

constitute a health risk to consumers (Lau et al.

F. Khemiss (&)

Laboratory of Physiology, Faculty of Dental Medicine,

Monastir, Tunisia

e-mail: khfathia2002@yahoo.fr

S. Ahmadi � R. Massoudi � D. Saıdane

Laboratory of Physiology, Faculty of Pharmacy,

Monastir, Tunisia

S. Ghoul-Mazgar � S. Safta

Laboratory of Histology–Embryology, Faculty of Dental

Medicine, Monastir, Tunisia

A. A. Moshtaghie

School of Pharmacy Isfahan Medical Sciences, Iran

University, Isfahan, Iran

123

Fish Physiol Biochem (2009) 35:541–549

DOI 10.1007/s10695-008-9265-7

2007). During the last 28 years, numerous studies

have been conducted to identify the gut microbiota of

various fish species (Cahill 1990; Ringø et al. 2004).

Such knowledge is important in order to be able to

evaluate the role of the gastrointestinal (GI) tract and

skin and gill in such infections as these represent

potential routes of infection (Robertson et al. 2000;

Ringø et al. 2003, 2007a).

The GI tract provides an environmental medium

rich with micronutrients and suitable for bacterial

proliferation and growth (Ringø et al. 2001). The

equilibrium of microbiota may be altered by patho-

genic colonization, which may provoke major

intestinal disorders (Gibson-Kueh et al. 2003). During

the last three decades, numerous bacterial species,

such as Aeromonas salmonicida, A. hydrophila,

E. ictaluri, Edwardsiella tarda, Photobacterium

damselae ssp. piscicida, Vibrio anguillarum, and

V. vulnificus, have been recognized to be the most

important pathogenic agents. These bacteria cause

many different fish diseases (Balebona et al. 2001).

Furunculosis is caused by A. salmonicida (Ringø et al.

2004), while septicaemia and ulcer are attributed to

A. hydrophila (Dierckens et al. 1998). Vibrio vulnificus

is a bacteria that may cause local wounds, gastroen-

teritis, and septicaemia (Lin et al. 2006). This bacteria

is virulent to sea bream, as evidenced by experimental

challenge (Li et al. 1999), and investigators have been

reported that fish intestinal damage occurs in the

presence of bacteria invasion (Chopra et al. 2000;

Ringø et al. 2003, 2004, 2007a, b).

Various researchers have explored the functional

responses of the fish intestine to stresses caused by

pathogenic infections (Bakke-Mckellep et al. 2007).

Morphological alterations have been described in

Salmo salar infected by A. salmonicida (Ringø et al.

2004, 2007b).

Sea bream (Sparus aurata L.) is a euryhaline

species common to the Mediterranean Sea and is

considered to be an economically valuable species for

commercial aquaculture (Mojetta and Ghisotto 1995).

We have evaluated the relationship between intestinal

tract infection and water and electrolyte disturbances

based on accepted knowledge that the GI tract plays

an important role in body functioning by ensuring the

nutrient absorption that allows hydroelectrolytic

equilibrium and body growth. We have also deter-

mined the histological alterations of sea bream

intestine in the presence of V. vulnificus, a pathogenic

bacteria (Kobayashi et al. 2004), using the in vitro

everted gut sac (EGS) technique (Osman et al. 1998;

Khemiss et al. 2006).

Materials and methods

Bacteria strain

The Vibrio vulnificus strain used in the present study

was kindly donated by the Laboratory of Analysis

and Control of Chemical and Microbiological Pollu-

tants of Environment (Faculty of Pharmacy Monastir,

Tunisia). This strain was isolated from an infected

liver of a Dicentrarchus labrax specimen. A bacterial

suspension of V. vulnificus was prepared in 40 ml of

Ringer solution (9%) containing (mmol l-1) NaCl

0.154, KCl 0.0034, NaHCO3 0.0024, and CaCl20.0021. The pH was adjusted to 8.5 as described by

Walsh et al. (1991). All chemical products were

obtained from Prolabo (Paris, France).

This suspension was adjusted to a concentration

equal to 108 cells ml-1 (Chabrillon et al. 2005).

Fish

Sea bream Sparus aurata (Linnaeus 1758) was

provided by Hergla Sea and brought (in a specifically

adapted aquarium with ventilation system) to the

National Sea Institute of Sciences and Technology of

Monastir, Tunisia. The fish were held in a tank filled

with sea water for at least 2 weeks prior to the

experiments. The water temperature was maintained

between 18 and 22�C. The fish were fed with a standard

diet (a melange of crushed fish and flours with 43%

protein content and 22% lipid content; Le-Gouessant,

France). Our experiments were made from 8 fish

ranging from 100 to 125 g body mass.

Treatment of animals

Fish were anaesthetized with 0.1% 3-aminobenzoic acid

ethyl-esther. The intestines were removed immediately,

stripped of adhering tissues, and cleaned with Ringer

solution (9%, pH 8.5). The anterior and posterior

intestines were separated into different segments

(medium length of each segment was 7 cm). The EGS

technique was prepared as previously described (Barthel

et al. 1998; Khemiss et al. 2006), maintained at 25�C,

542 Fish Physiol Biochem (2009) 35:541–549

123

and continuously gassed with a mixture of O2/CO2 (95/5).

The total incubation time was 2 h.

Determination of water and electrolytes flux

Water flux was determined in the presence or absence

of V. vulnificus. The results are expressed in milli-

grams of water per gram fresh intestine per hour

(Charpin et al. 1992; Khemiss et al. 2006). The sodium

and potassium fluxes were determined by photometry

(Flame photometer BT634; Biotecnica Instruments,

Rome, Italy), the chloride flux was determined using a

Cobas Integra 400 plus analyzer (Roche Diagnostics,

Indianapolis, IN), and the carbonate flux was deter-

mined by an automatic system (Ultra M Norm

Biomedical, USA). The electrolyte fluxes were

expressed in micromoles per gram of intestine per

hour, as described by Chikh-Isaa et al. (1992).

Histological study

Tissue samples were fixed by immersion in formol

for at least 24 h and then dehydrated and embedded

in paraffin. Serial 5-lm transversal sections were

classically stained with hematoxylin and eosin and

mounted with Canada Baum before being observed

by light microscopy (Martoja and Martoja 1968).

Tissue sections were observed and photographed

under a binocular light microscopy (Axioskop Zeiss).

Statistical study

The statistical analyses were performed using the

statistical Package for Social Sciences (SPSS ver.

10.0; SPSS, Chicago, IL) for Windows. All values are

expressed as mean ± standard error of the mean

(SEM). The statistical significance of the results was

determined using the Student t test, and results were

considered to be significant at P \ 0.05.

Results

Determination of water flux in the presence

or absence of V. vulnificus in the intestine

of sea bream

Control conditions were initially established by mea-

suring the flux in the anterior and posterior intestinal

segments in incubation medium that did not contain

bacteria; these values were 250 ± 38 and 108 ± 11

mg g-1 of fresh intestine h-1, respectively (Fig. 1). The

flux noted in the anterior intestine was significantly

higher than that in the posterior segment (P \ 0.05).

Although the addition of V. vulnificus (108 cells

ml-1) in the incubation medium (Fig. 1) was associ-

ated with a significant decrease of water flux in the

anterior segment (P \ 0.05), this decrease was not

significant in the posterior part of the intestine.

Determination of electrolyte flux (in the presence

or absence of V. vulnificus) in the intestine

of sea bream

The recorded flux of sodium in the two intestinal

segments of sea bream intestine in the absence of

bacteria and after 2 h of incubation is shown in

Table 1. This flux was higher in the anterior segment

than in the posterior one (P \ 0.05). The addition of

bacteria (108 cells ml-1) to the incubation medium

led to a significant elevation in the sodium flux in the

anterior segment (P \ 0.05), but variations in the flux

were larger in the posterior segment (P \ 0.01).

The chloride flux also showed an increased uptake

of 9.21 ± 0.61 and 8.76 ± 0.50 lmol g-1 of fresh

Fig. 1 Effect of Vibrio vulnificus on the variation of water flux

in the intestine of Sparus aurata (anterior and posterior

segments) after 2 h of incubation. The control samples are

everted gut segments (EGS) exposed to only Ringer solution

(9%) for a 2-h incubation. The infected samples EGS

incubated in a suspension of V. vulnificus (108 cells ml-1).

Asterisk indicates that V. vulnificus induced a significant

(P \ 0.05) decrease in water flux in the anterior segment

Fish Physiol Biochem (2009) 35:541–549 543

123

intestine h-1 in the anterior and posterior segments,

respectively (Table 1). The presence of V. vulnificus

(108 cells ml-1) in the incubation medium was not

associated with a significant variation of this flux in

both segments of the intestine.

While the potassium flux recorded in the anterior

segment was 0.50 ± 0.12 lmol g-1 of fresh intestine

h-1, in the posterior segment, it was -0.43 ±

0.01 lmol g-1 of fresh intestine h-1 (Table 1). This

result showed that the potassium flux decreased

significantly in the posterior segment (P \ 0.05) but

that the presence of V. vulnificus did not affect it in

the anterior segment.

In control samples, carbonate flux was higher in

the anterior segment than in the posterior one

(P \ 0.05). Although this flux was not altered in

the anterior segment in the presence of the bacteria

suspension (108cells ml-1), it increased significantly

(P \ 0.01) in the posterior segment.

Histological study

Histological sections of sea bream intestine were

observed by light microscopy. Three layers were

identified: serosa, muscularis, and mucosa. Histolog-

ical studies of the intestine after 2 h of incubation in a

Ringer solution (9%, pH 8.5) at 25�C showed that the

intestine remained intact morphologically with a high

number of villosities (Fig. 2a, b), with more villos-

ities in the anterior segment (Fig. 2a) than in the

posterior one (Fig. 2b). The addition of V. vulnificus

to the incubation medium was associated with a

detachment of the intestinal layer and alterations in

the villosities (Fig. 2c, d).

At higher magnification, the structures of the brush

borders were conserved after 2 h of incubation time

(Fig. 3a, b). The addition of V. vulnificus to the

incubation medium was associated with a detachment

of the intestinal layers, alteration of the villosities

(Figs. 2c, d, 4a), alterations in the brush border

(Fig. 3d), and enterocyte detachment (Fig. 3c, d).

A heterogenous inflammatory cell population and

congestive vessels were noted in the anterior segment

of the intestine (Fig. 4a, b).

Discussion

The everted gut sac technique is very useful in

enabling a simultaneous evaluation of water and Ta

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544 Fish Physiol Biochem (2009) 35:541–549

123

electrolyte transfer. This in vitro method has been

used by a number of researchers (Barthel et al. 1998;

Abatomi et al. 1994; Osman et al. 1998) and

validated by a histological study (Khemiss et al.

2006).

Our initial results showed that the fish intestine

retained its absorption capacity for water and elec-

trolytes during the 2-h incubation period. These

results are in accordance with those of Buddington

et al. (1987).

Under our experimental conditions and in the

absence of bacteria, there was a water transfer from

the mucosal to serosal side, which showed absorp-

tion. Water absorption in the anterior part was

accompanied by Na?, Cl-, and K? absorption and

a secretion of HCO3-. Carbonate secretion in the

teleost fish intestine has also been noted by other

authors (Wilson et al. 2002). In the posterior seg-

ment, water, Na?, and Cl- were absorbed, while K?

and HCO3- were secreted. The addition of the

bacteria to the incubation medium altered water and

sodium absorption in the anterior segment and

carbonate secretion in the posterior one; it did not

affect the transfer of the other electrolytes. This

variability in water and electrolyte movements indi-

cated variable responses in the different segments

studied. Our results show that the movements of

chloride and sodium were comparable to those of

water under the control conditions. We therefore

suggest that the absorption of sodium, chloride, and

water appears to be coupled processes (Skadauge

1974).

Various hypotheses have been put forth to explain

this coupled process between water and NaCl

absorption. One of these has been that water absorp-

tion is attributable to a cotransportor Na? K? 2Cl-

located at the epithelium brush border and acting as a

water pump (Loo et al. 1999). However, Tiruppathi

Fig. 2 Light micrograph of the histological views of the

anterior and the posterior intestine of S. aurata (95). Bar:

45 lm. Stain: Hematoxylin–eosin (HE). a, b Anterior segment

(a) and posterior segment (b) of S. aurata intestine exposed to

Ringer solution (9%) showing serosa (S), muscularis (M) and

highly developed villosities (V). c, d Anterior segment (c) and

posterior segment (d) of S. aurata intestine exposed to bacteria

(108 cells ml-1) showing layer detachment (LD) and altera-

tions in the villosities (VA)

Fish Physiol Biochem (2009) 35:541–549 545

123

suggested that this absorption was due to Na? K?

ATPase located at the basolateral side of the entero-

cyte (Tiruppathi et al. 1983).

Vibrio vulnificus decreased potassium absorption

in the anterior segment and secretion in the posterior

part. The decrease in potassium absorption induces a

decrease in secretion and translates into a change in

the equilibrium of K? balance. The K? secretion may

be related to the activity of 3Na? 2K? pump in

basolateral membrane intestine (Larsen et al. 2002).

For HCO3-, we noted a high secretion in the

anterior intestine compared to the posterior one. Our

results agree with those of Wilson and Grosell

(2003), who suggested that the anterior segment

was the principal site of carbonate secretion in the

fish intestine. These results are also in accordance

with those of a study by Wilson et al. (2002).

Carbonate secretion was noted against an electro-

chemical gradient due to antiport Cl-/HCO3- located

at the basolateral side of the enterocyte (Safsten

1993).

The osmoregulatory function of the intestine is

accomplished via electrolyte transfer by the activa-

tion of the Cl-/HCO3- exchanger and involves the

secretion of carbonate and absorption of chloride.

This mode of transfer may support the secretion

of carbonate and potassium and the absorption of

chloride recorded in our data. The equilibrium of

fluid movements through the intestinal tract results

from these absorption and secretion phenomena.

Aquaporins are channel proteins implied in this fluid

transport (Ma and Verkman 1999), and several

different types of aquaporins and transporters have

been reported (Mayumi et al. 2003) that show

variable distribution, localization, and permeability.

These variations in distribution and localization can

be detected immuno-histochemically (Lignot et al.

2002; Aoki et al. 2003), and they may explain the

difference in water absorption noted in our data for

control samples.

Sea bream has a high capacity for absorbing water,

sodium chloride, and potassium (Evans 1993; Laiz-

Carrion et al. 2005). In the absence of bacteria, we

observed a signficant level of water absorption at the

anterior segment (Fig. 1). This result agrees with

those of previous studies established in other species,

such as Salmo salar (Nordrum et al. 2000). However,

hydro-electrolytic absorption through the intestinal

tract may be different from one species of fish to

another one. In contrast to our data, the flux was

Fig. 3 Light micrograph of

the histological views of the

anterior and the posterior

intestine of S. aurata(9100). Bar: 10 lm. Stain:

HE. a, b Anterior segment

(a) and posterior segment

(b) of S. aurata intestine

exposed to Ringer solution

(9%) showing the border

brush (BB) and enterocytes

(E). (c) Anterior segment of

S. aurata intestine exposed

to bacteria (108 cells ml-1)

showing serious detachment

of the enterocyte (D) and

disappearance of

microvillosities. (d)

Posterior segment of S.aurata intestine exposed to

bacteria (108 cells ml-1)

showing enterocyte

detachment (D) and

discontinued disappearance

of the border brush (BB,

arrows)

546 Fish Physiol Biochem (2009) 35:541–549

123

constantly the same along the intestinal tractus in

Carassius auratus (Smith 1964) and was highly

increased in the posterior intestine of Anguilla

japonica (Aoki et al. 2003), while Ando and Naga-

shima (1996) suggested that water absorption in the

A. japonica intestine may be regulated by the

presence of electrolytes

A number of different experimental approaches

have been used to explore the capacity of water and

electrolyte transfer in the fish intestine. Blanchard

and Grosell (2006) analyzed water transfer in fish

exposed to different environment, while Scott et al.

(2006) used the whole intestine tract ligatured at the

two extremities. In contrast, Grosell et al. (2005)

used only the gut sac of the spiral valve. Bucking and

Wood (2006) used the different compartments of the

GI tract to determine water and ion composition in

each gut sac. We specified two intestinal segments

(anterior or posterior) and also explored the variation

of water and electrolyte flux using an in vitro model

‘‘EGS’’ in the absence or presence of V. vulnificus.

All of these approaches may be complementary to

each other in providing information that will con-

tribute to an elucidation of the mechanism of water

and electrolyte transfer in the fish intestine.

Our histological study of the sea bream intestine in

the absence of V. vulnificus showed that the intestinal

tract remained intact morphologically with a high

number of villosities. This finding confirms that the

sea bream intestinal tract preserves its viability as

well as its functioning after 2 h of incubation.

However, the presence of V. vulnificus provoked

damage in the two intestinal segments. We noted the

presence of a heterogeneous inflammatory cell pop-

ulation and congestive vessels that are indicative of

affected tissues and functions. Similar histological

alterations were recorded in infected fish intestines of

other species (Gibson-Kueh et al. 2003; Ringø et al.

2004, 2007a, b). The alterations noted under our

experimental conditions may be associated to the

inflammation recorded following intestinal exposition

to V. vulnificus. This phenomenon was previously

noted in sea bream (Chaves-Pozo et al. 2004);

however, Shirouzu et al. (1985) and (Morimatsu

et al. (2003) noted the presence of necrosis cells in

human GI tracts infected by V. vulnificus. Based on

our findings, we suggest that V. vulnificus is able to

induce histological alterations in the sea bream

intestine—particularly in the anterior segment.

Analysis of our histological data and the hydro-

electrolytic movement of sea bream intestine leads us

to suggest that the different segments of the fish

intestine present variable sensitivities to pathogenic

factors.

Acknowledgments The authors thank Mr. Boukottaya Samir

for his careful presentation of the manuscript.

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