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ght and transmission electron microscopy of Vibrio campbellii infection gnotobiotic Artemia franciscana and protection offered by a yeastutant with elevated cell wall glucan

.Y.S.Asanka Gunasekara a, Tom Defoirdt b, Anamaria Rekecki a, Annemie Decostere a,ria Cornelissen c, Patrick Sorgeloos b, Peter Bossier b, Wim Van den Broeck a,*

partment of Morphology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820, Merelbeke, Belgium

oratory of Aquaculture and Artemia Reference Center, Ghent University, Rozier 44, 9000 Ghent, Belgium

partment of Basic Medical Sciences, Faculty of Medicine and Health Sciences, Ghent University, De Pintelaan 185 6B3, 9000 Ghent, Belgium

ntroduction

Until recently, antibiotics were considered to beispensable for controlling high mortalities in aqua-ture. However, this practice is now questioned and

often banned because of environmental and human healthconsequences (Witte, 2000). Therefore, alternative controlstrategies are under consideration (Defoirdt et al., 2007).

The use of preventive and environment-friendlyapproaches such as probiotics, immunostimulants, anti-bacterial peptides and quorum sensing disruption are ofmajor importance in aquaculture facilities (Sakai, 1999;Raa, 2000). Among these approaches, use of specificbiological compounds such as immunostimulants that

T I C L E I N F O

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ived 18 October 2011

ived in revised form 15 February 2012

pted 17 February 2012

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A B S T R A C T

Luminescent vibrios are amongst the most important pathogens in aquaculture, affecting

almost all types of cultured organisms. Vibrio campbellii is one of these most important

pathogens. In this study, the effects of feeding mnn9 yeast cell wall mutant and wild type

yeast strain were investigated in the digestive tract of brine shrimp nauplii, Artemia

franciscana, after experimental infection with V. campbellii (LMG 21363). Gnotobiotic A.

franciscana nauplii were fed daily with dead Aeromonas hydrophila LVS3, and with either

wild type strain of baker’s yeast, Saccharomyces cerevisiae, or mutant strain mnn9, of which

the cell wall contains elevated chitin and glucan and lower mannose levels. After three

days of feeding, some nauplii were challenged with V. campbellii. Mean survival (%),

individual length (mm) and total length (mm) at one day and two days after challenge

were significantly higher in the group fed mnn9 than in the group fed wild type yeast

(81 � 1.50 and 63 � 0.49, 1.56 � 0.07 and 1.13 � 0.02, 38.21 � 3.11 and 21.26 � 0.81

respectively for one day and 50 � 2.37 and 20 � 1.41, 2.33 � 0.01 and 1.24 � 0.04,

34.97 � 5.56 and 7.45 � 1.63 for two days after challenge). Histological examination revealed

that the luminal diameter and enterocyte height of both mid- and hindgut were larger in the

mnn9-fed group. Colonization of the gut lumen by V. campbellii could be observed by

transmission electron microscopy for the group of nauplii fed with wild type yeast.

Furthermore, it was observed that V. campbellii caused damage to the gut epithelium including

shortening and disappearance of the microvilli, destruction of the apical cell membrane and

cell lysis in the nauplii fed wild type yeast. The gut epithelium remained intact in challenged

nauplii fed mnn9 yeast. The morphological findings of the present study further substantiate

previous studies reporting a protective effect of this yeast cell wall mutant.

� 2012 Elsevier B.V. All rights reserved.

Corresponding author. Tel.: +32 92647716; fax: +32 92647790.

E-mail address: wim.vandenbroeck@UGent.be (W. Van den Broeck).

Contents lists available at SciVerse ScienceDirect

Veterinary Microbiology

jo u rn al ho m epag e: ww w.els evier .c o m/lo cat e/vetmic

8-1135/$ – see front matter � 2012 Elsevier B.V. All rights reserved.

10.1016/j.vetmic.2012.02.025

R.A.Y.S.A. Gunasekara et al. / Veterinary Microbiology 158 (2012) 337–343338

boost immune responses and augment disease resistanceof target organisms can be considered as an excellentpreventive tool (Sakai, 1999). Moreover, in cases wheredisease outbreaks are cyclic and predictable, immunosti-mulants may be useful to enhance defense mechanismsand to reduce stress, stress-induced diseases and mor-talities (Raa, 2000). Although the exact mechanism ofimmunostimulation is not yet completely understood,several immunostimulants such as b-glucans (Sung et al.,1996; Sritunyalucksana et al., 1999; Burgents et al., 2004),chitin (Anderson and Siwicki, 1994), lipopolysaccharides(Takahashi et al., 2000) and dead bacteria (Keith et al.,1992) are being applied in vertebrate as well as ininvertebrate cultures, to stimulate the immune responseand to increase protection against a wide range of diseases.

In the present study, yeast strains were used as glucansources. Significantly decreased amounts of mannopro-teins (from almost 40% to less than 10%) and b-1,3 glucan(from 45–50% to 35%) and increased amounts of b-1,6glucan (from 10–15% to 30%) and chitin (from less than 2%to 20%) are present in the mnn9 mutant strain ofSaccharomyces cerevisiae (Magnelli et al., 2002). Inprevious studies, better development (Marques et al.,2004) and a higher survival (Soltanian et al., 2007) ofgnotobiotic Artemia nauplii fed with mnn9 yeast werefound when they were either challenged or unchallengedwith Vibrio.

The present study combines a gnotobiotic Artemia testsystem (GART) with sensitive, newly introduced histolo-gical (Gunasekara et al., 2010a) and transmission electronmicroscopic monitoring tools to study the digestive tractmorphology in response to feeding isogenic yeast strains(mnn9 vs. wild type) in an experimental challenge withpathogenic Vibrio campbellii. The digestive tract was ourmain focus because it is a crucial interface between thehost and its environment and represents an importantputative portal of entry for pathogenic micro-organisms(Nehme et al., 2007).

2. Materials and methods

2.1. Culturing and harvesting of yeast

Wild type (WT) strain of baker’s yeast (S. cerevisiae)(BY4741 [genotype, Mat a; his3D1; leu2D0; met15D0;ura3D0]) and its mnn9 isogenic mutant (BY4741; genotypeMat a; his3D1; leu2D0; met15D0; ura3D0; YPL050c::k-anMX4), which harbors a null mutation for the mnn9 generesulting in lower concentration of mannose linked tomannoproteins, compensated by a higher concentration ofchitin and glucans in the cell wall (Magnelli et al., 2002;Marques et al., 2004) were used as feed for Artemia. Thesestrains were chosen because of their protection capacityagainst pathogenic bacteria i.e. protection by mnn9 yeastand no protection by WT yeast (Marques et al., 2005). Bothstrains were kindly provided by the European S. cerevisiae

Archive for Functional Analysis (University of Frankfurt,Frankfurt, Germany).

The yeast cells were streaked on complete yeast extractpeptone dextrose (YEPD) agar plates, containing yeastextract (Sigma, 1% w/v), bacteriological-grade peptone

(Sigma, 1% w/v), D-glucose (Sigma, 2% w/v) and bacter-iological-grade agar (10–20 g/L; ICN), subsequently cul-tured in YEPD medium and harvested according to theprocedure described by Gunasekara et al. (2010a,b). Thedensities were determined by measuring twice the cellconcentration using a Burker hemocytometer. Suspensionswere stored at 4 8C and provided to Artemia nauplii untilthe end of each experiment. All manipulations werecarried out in a laminar flow hood to maintain sterility.

2.2. Culturing and harvesting of bacteria

Aeromonas hydrophila bacterial strain (LVS3) wasselected as the main food because of its positive effecton growth and survival in Artemia (Verschuere et al., 1999,2000). Pure cultures of LVS3 stored at �80 8C, were grownon Marine agar plates, containing Difco Marine broth 2216(37.4 g/L; BD Biosciences) and bacteriological-grade agar,for three days at 28 8C, and subsequently cultured inMarine broth. The cultures were harvested and thedensities were determined according to the proceduredescribed by Marques et al. (2006) and Gunasekara et al.(2010a,b, 2011a). LVS3 cells were killed by autoclaving at120 8C for 20 min.

Vibrio campbellii (LMG 21363) was selected as a virulentpathogenic strain for Artemia nauplii (Verschuere et al.,1999, 2000). It was cultured and harvested using the sameprocedure followed for A. hydrophila.

2.3. Culturing, feeding and challenging of gnotobiotic Artemia

The experiment was carried out using Artemia francis-

cana originating from the Great Salt Lake, Utah. Artemia

cysts were hydrated and decapsulated following themethodology described by Defoirdt et al. (2005) in orderto obtain sterile cysts and subsequently sterile nauplii.After decapsulation, cysts were washed and placed on arotor according to the procedure described by Marqueset al. (2006). After about 20–24 h, 30 nauplii weretransferred to 50 ml sterile culture tubes containing30 ml of filtered (using 0.2 mm filters), autoclaved (at120 8C for 20 min) sea water made with ‘‘Instant Ocean’’(Aquarium systems, Sarrebourg, France). Half of the totaltubes received live WT yeast together with dead LVS3 asdaily food, whereas the other half of the tubes receivedmnn9 yeast together with dead LVS3. All tubes were placedon a rotor turning at 4 rpm. Daily feeding was ad libitum.During the six days of the study period, dead bacteria(LVS3), 90% of the total feed, and either WT or mnn9 yeast,10% of the total feed were administered per culture tubefollowing the feeding schedule described by Marques et al.(2006).

Three days after hatching, half of the culture tubes ofboth groups of nauplii were challenged with V. campbellii

at a final concentration of 5 � 106 cells ml�1. The non-challenged culture tubes also underwent the samemanipulation as the challenged groups. Each treatmenthad six replicates. Sampling for survival, length measure-ments, histology and transmission electron microscopyoccurred at one day after challenge and two days afterchallenge.

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R.A.Y.S.A. Gunasekara et al. / Veterinary Microbiology 158 (2012) 337–343 339

Verification of axenity

The axenity of the decapsulated cysts, the Artemia

ture water and the dead LVS3 suspension (afteroclaving) was verified using plating. Absence orsence of bacterial growth was monitored after fives of incubation at 28 8C of 100 ml of decapsulated cysts,

ture medium or feed plated on Marine agar (n = 2).

Survival, individual length and total length

For estimating the survival, the number of swimmingae was counted followed by calculating the survival

centage. Individual length (IL) of Artemia nauplii wasasured by fixing them in Lugol’s solution (5 g iodine

and 10 g potassium iodide (KI) mixed with 85 mltilled water) and using a dissecting microscopeipped with a drawing mirror, a digital plan measure, the software Artemia 1.0 (courtesy of Marnix Vanme). The total length was calculated using theber of live nauplii per culture tube and the mean IL,

ording to the formula:Total length (millimeters per culture tube) = number ofvivors � mean IL

Histological analysis

Nauplii sampled for histological analysis were fixed andher processed following the methodology described byasekara et al. (2010b). Per group and per day, sixplii were used for histological analysis. The histological

tions were examined under a motorized light micro-pe (Olympus BX 61, Olympus Belgium, Aartselaar,gium) linked to a digital camera (Olympus DP 50,mpus Belgium).

Transmission electron microscopy (TEM)

Samples for TEM were fixed in Karnovsky’s fixativeowed by post fixation in 1% osmium tetroxide ascribed by Gunasekara et al., 2011b. Subsequently, theples were dehydrated using a graded series of alcohol

–100%) and finally embedded in SPURR’s resin. Aftermination of semithin sections stained with toluidinee to localize the regions of interest, ultrathin sections ofnm thickness were made using a Leica EM UC 6amicrotome (Leica Mircosystems GmbH), andunted on formvar coated single slot copper gridsborimpex N.V., Brussels, Belgium). The sections weret-stained with uranyl acetate and lead citrate (Leica EMin, Leica Microsystems GmbH) and viewed on a Jeol0 EXII TEM (JEOL Ltd., Tokyo, Japan) at 80 kV

elerating voltage.

Statistical analysis

The mean number of survival, individual length, totalgth of Artemia nauplii subjected to different treatmentsre compared separately using independent samplest, STATISTICA 7 (Tulsa, Oklahoma, USA). Each statis- le

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R.A.Y.S.A. Gunasekara et al. / Veterinary Microbiology 158 (2012) 337–343340

3. Results

3.1. Percentage survival, individual length and total length

Survival and individual length were determined in bothmnn9 and wild type yeast-fed groups four and fivedays after hatching. The survival, individual length andtotal length of the challenged nauplii were significantlyhigher, at a 0.05 level of probability, in the mnn9-fedgroup when compared to the wild type yeast-fed groupon both days (81 � 1.50 and 63 � 0.49, 1.56 � 0.07 and1.13 � 0.02, 38.21 � 3.11 and 21.26 � 0.81 respectively forone day and 50 � 2.37 and 20 � 1.41, 2.33 � 0.01 and1.24 � 0.04, 34.97 � 5.56 and 7.45 � 1.63 respectively fortwo days after challenge). Furthermore, in unchallengednauplii values for all the above-mentioned factors were alsosignificantly higher, at a 0.05 level of probability, in the mnn9yeast-fed group as compared to the wild type yeast-fed group

of nauplii both on four and five days after hatching (98 � 0.48and 82 � 1.29, 1.80 � 0.16 and 1.23 � 0.03, 52.79 � 5.11 and30.15 � 1.43 respectively for four days and 88 � 0.64 and63 � 4.08, 2.37 � 0.09 and 1.31 � 0.08, 62.38 � 2.67 and25.85 � 6.17 respectively for five days) (Table 1).

3.2. Histological observations

Longitudinal and transverse histological sections wereused to evaluate the development and growth of thedigestive tract of brine shrimp nauplii. The diameters ofthe mid- and hindguts were clearly larger in the mnn9-fedgroup both on the first and second day after challenge, andthe nauplii were larger in size compared to the WT yeast-fed group (Fig. 1a and b). The study further revealed thatthe cell thickness of the midgut in the mnn9 yeast-fedgroup was higher compared to the WT yeast-fed group ofnauplii (Fig. 2a and b).

Fig. 1. Representative sagittal histological sections of Artemia nauplii two days after challenge with Vibrio campbellii fed with dead Aeromonas hydrophila and

wild type yeast (a), and mnn9 yeast (b). The nauplii are larger in the group fed with dead A. hydrophila and mnn9 yeast. Legend: (1) midgut epithelial cells;

(2) hindgut epithelial cells.

Fig. 2. Representative longitudinal histological sections of the midgut-hindgut transition of Artemia nauplii two days after challenge with V. campbellii fed

with dead A. hydrophila and wild type yeast (a), and mnn9 yeast (b). The diameter of the gut is larger and the enterocytes are thicker in the group fed with

dead A. hydrophila and mnn9 yeast. Legend: (1) midgut epithelial cells; (2) midgut-hindgut transition; (3) hindgut epithelial cells; (4) gut content.

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R.A.Y.S.A. Gunasekara et al. / Veterinary Microbiology 158 (2012) 337–343 341

Transmission electron microscopy observations

Colonization of the digestive tract of Artemia nauplii byampbellii was investigated using transmission electronroscopy (Fig. 3).

In the nauplii fed with wild type yeast, at two dayspost challenge, the midgut cells were more cuboidal.Bacterial accumulation in the gut lumen could beobserved. Bacteria were more often localized close tothe apical parts of the epithelial cells when compared to

3. Transmission electron micrographs of midgut cells of Artemia nauplii two days after challenge with V. campbellii fed with dead A. hydrophila and wild

yeast (a–f) and mnn9 yeast (g–h). (a) Adhering bacteria; (b) enlarged micrograph of adhering bacteria; (c–d) invaginated enterocyte cell membrane; e.

upted cell membrane (indicated with arrows), (f) putative adhesion; (g–h) bacterial accumulation in the midgut lumen (arrow-heads). Legend: (1)

gut enterocytes; (2) junctional complex; (3) nucleus; (4) microvilli; (5) bacterium; (6) midgut lumen.

R.A.Y.S.A. Gunasekara et al. / Veterinary Microbiology 158 (2012) 337–343342

the nauplii fed with mnn9 yeast. The membrane of theapical part of some of the cells was disrupted (Fig. 3b ande). Putative adhesion and invagination into epithelialcells were obvious (Fig. 3b–e). The enterocyte microvilliwere becoming short or absent. Several bacteria wereapproaching the cellular junctions (Fig. 3f). However, thecellular junctional complexes were still intact (Fig. 3a).Cellular effacement and cell lysis could also be observed,in particular more caudally in the midgut. Expulsion ofepithelial cells in the caudal region of the midgut wasobserved.

In mnn9-fed nauplii at two days after challenge, themidgut enterocytes were columnar. Bacteria were mainlyconfined to the lumen and approach of bacteria towardsthe epithelial cells could not be seen. In contrast to themidgut epithelial cells of the nauplii fed with wild typeyeast, the cell membranes including the apical surfacebordering the gut lumen were intact. Microvilli were intactand clear as well. Hence, in conclusion the gut integritywas well preserved in nauplii fed with mnn9 yeast (Fig. 3gand h). In both groups bacterial accumulation in themidgut-hindgut transition zone was not observed.

4. Discussion

The present study indicates that V. campbellii cancolonize the brine shrimp larval gut, which is considered tobe one of the most important sites of interaction with theexternal environment and provides one of the majorportals of entry for pathogens. A yeast mutant withelevated cell wall glucan levels protects Artemia naupliipreventing that damage caused by the pathogen. Theseobservations coincided with an increased survival, a betterdevelopment and growth in either challenged or unchal-lenged nauplii when fed with glucan rich mnn9 yeast asalso observed by Marques et al. (2004) and Soltanian et al.(2007).

As far as we know, this is the first report showing that V.

campbellii colonizes the digestive tract of Artemia nauplii asverified with transmission electron microscopy which is anappropriate tool to elucidate the mechanisms involved inthe entry of pathogens (Ringø et al., 2007). Accumulation ofbacterial cells in the gut lumen after 48 h of challenge wasvisible. It is assumed that V. campbellii has the capability ofstrong growth in the gut of Artemia (Haldar et al., 2010). Inthe group fed wild type yeast, bacteria could be detectedcloser to the apical parts of the enterocytes, which was notthe case in the group fed mnn9. Furthermore, somedamage to the gut epithelial cells including invagination ofhost cell membrane, which is considered to be anattachment site (Neutra, 1984) and membrane disruptioncould be observed in the nauplii fed wild type yeast.Microvilli were short or absent, which is in agreement withVerschuere et al. (2000) who experimentally infected thebrine shrimp host with virulent pathogen Vibrio proteo-

lyticus. Although approach of bacteria towards the junc-tions were visible in the group fed wild type yeast, cellularjunctional complexes were still intact in both groups ofnauplii, in agreement with Nehme et al. (2007) who failedto detect bacteria approaching the junctions at early stages

Histological appraisal of digestive tract specimensrevealed that the nauplii fed with mnn9 yeast containedhigher enterocytes and showed a better development, thelatter being in agreement with Marques et al. (2004). Yeastrepresented only 10% of the total diet provided, yet underthese conditions the yeast phenotype/genotype has astrong effect on Artemia performances. As reported byCoutteau et al. (1990), b-glucanase activity is present inthe digestive tract of Artemia. However, mannase activity isabsent making the external mannoprotein layer of the wildtype yeast cell wall probably the major barrier for Artemia

to digest yeast cells. The altered cell wall of mnn9, withdecreased levels of mannoproteins, probably rendered thediet more nutritious.

Better nutrition and defense capacity of Artemia to resistagainst pathogenic infection seemed to be correlated (Rojas-Garcıa et al., 2009). Although the Artemia innate defensesystem is poorly documented (Verschuere et al., 2000),protection against the presence of pathogens is suggestivefor the existence of efficient antimicrobial barriers at the gutepithelial level (Rojas-Garcıa et al., 2009). These protectivemechanisms might include production of mucus by gobletcells, the acidic micro-environment at the luminal surface ofthe gut epithelium, cell turnover, peristalsis and gastricacidity some of which are present in fish (Ringø et al., 2007).Gastrointestinal mucus secreted by epithelial goblet cells isone of the protective mechanisms against adherence ofpathogenic bacteria to the underlying epithelium in bothfish (Westerdahl et al., 1991) and endothermic animals(Smith and Butler, 1974; Maxson et al., 1994) although thereis no information available for fish larvae or fry (Ringø et al.,2007). Epithelial goblet cells could not be detected using PAS(periodic-acid-Schiff) stain in both groups of nauplii at thisstage (unpublished data), and therefore mucus protection inthe digestive tract of Artemia nauplii against pathogens isdoubtful. Further investigations are therefore warranted tofind which mechanisms may be involved in digestive tractprotection of Artemia nauplii against pathogenic invasion.

In conclusion, the results obtained in this studyrevealed that V. campbellii damages the gut epitheliumof Artemia nauplii and that the destructive effect of V.

campbellii could be prevented by administration of yeastcell wall mutant mnn9, containing higher glucan, chitinand lower mannose content than in wild type yeast. Thisprotection could be attributed to an immunomodulatoryeffect of mnn9 yeast cells and/or the improved nutritivevalue of mnn9 yeast, resulting in stronger animals. Furtherresearch including immunological studies together withbiochemical assays is needed to unravel the mechanismswhich mediate the observed protection of the host. Thepresented knowledge can be of importance for introducingpreventive strategies for V. campbellii infections in brineshrimp hosts.

Acknowledgments

This study was supported by Special Research Grant(Bijzonder Onderzoeksfonds, BOF, grant numbers B/07289/02; 05B01906) of Ghent University, Belgium awarded tothe first author and by the Foundation for Scientific

Research-Flanders (FWO) (project number G.0491.08). of the infection of Drosophila.

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R.A.Y.S.A. Gunasekara et al. / Veterinary Microbiology 158 (2012) 337–343 343

We thank Prof. Paul Simoens and Dr. Christopheteleyn for the constructive comments. Lobke de Bels,t de Pauw, Brigitte Van Moffaert, Tom Baelemans andn Pieters are acknowledged for their excellent technicalistance.

erences

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