Immune responses and enhanced disease resistance in Chinese drum, Miichthys miiuy (Basilewsky), after oral administration of live or dead cells of Clostridium butyrium CB2

Immune responses and enhanced disease resistance

in Chinese drum, Miichthys miiuy (Basilewsky), after oral

administration of live or dead cells of Clostridium butyrium

CB2

X Pan1,2, T Wu1, Z Song3, H Tang1 and Z Zhao1

1 Department of Chemistry, Zhejiang University, Hangzhou, China

2 Key Laboratory for Molecular Design and Nutrition Engineering, Ningbo Institute of Technology, Zhejiang

University, Ningbo, China

3 College of Life Sciences, Shanghai Fisheries University, Shanghai, China

Abstract

Clostridium butyrium CB2 isolated from chickenswas tested as a potential fish probiotic in the Chi-nese drum, Miichthys miiuy. Fish were fed live(CB), dead CB2 (D-CB) cells (108 cells g)1) orspent culture supernatant (SCS), for 30 days andchallenged with Vibrio anguillarum or Aeromonashydrophila. Survival was higher in both the CB andthe CB-D fed groups, but the SCS group was notsignificantly different from the control. After feed-ing live or dead CB2 cells, there was increase inphagocytic activity of the head kidney macrophages,the lysozyme activity of serum and gut mucosa andimmunoglobulin (Ig) level. The SCS group showedno obvious change in immune parameters. Theresults suggest that live or dead Clostridium butyriumCB2 has an immunomodulatory effect on fish.

Keywords: Clostridium butyrium, innate immunity,Miichthys miiuy, probiotic, specific immunity.

Introduction

Probiotics have been defined by FAO/WHO(2001) as �live microorganisms which when admin-istered in adequate amounts, confer a health benefiton the host�. Because of their beneficial effects, therehas been increasing interest in the use of probiotics

in aquaculture (Kennedy, Tucker, Neidic, Vermeer,Cooper, Jarrell & Sennett 1998; Deschrijver &Ollevier 2000; Robertson, O�Dowd, Burrells, Wil-liams & Austin 2000), particularly the use ofantagonistic probiotic strains to control populationsof potential pathogens through competitive exclu-sion or enhancement of immunity. Previous studieshave demonstrated the potential use of probiotics asa means of reducing mortalities in aquaculture(Gatesoupe 1994; Moriarty 1998; Gram, Melchi-orsen, Spanggaard, Huber & Nielsen 1999; Nik-oskelainen, Salminen, Bylund & Ouwehand 2001;Panigrahi, Kiron, Kobayashi, Puangkaew, Satoh &Sugita 2004; Irene, Alberto, Esteban & Jose 2005).

Probiotics for human and terrestrial animals aredominantly lactic acid bacteria (LAB), but in fishand shellfish many different bacteria, includingAeromonas, Pseudomonas, Bacillus, Carnobacteriumand Lactobacillus, have been evaluated as probiotics(Gatesoupe 1991; Gildberg, Johansen & Bogwald1995; Gildberg & Mikkelsen 1998; Irianto &Austin 2002a,b). However, most of the probioticbacteria used have been derived from fish intestineand, to our knowledge, there are few reportsconcerning the use of exogenous bacteria isolatedfrom terrestrial animals as probiotics foraquaculture.

Clostridium butyricum has been used as a probi-otic for many years in eastern Asian countries suchas Japan, Korea and China (Ito, Hayashi, Iguchi,Endo, Nakao, Kato, Nabeshima & Ogura 1997;Kamiya, Taguchi, Yamaguchi, Osaki, Takahashi

Journal of Fish Diseases 2008, 31, 679–686 doi:10.1111/j.1365-2761.2008.00955.x

Correspondence Dr X Pan, Department of Chemistry, Zhejiang

University, Hangzhou 310027, China

(e-mail: [email protected])

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& Nakamura 1997). The bacteria, which arebutyric acid producing, spore-forming, Gram-positive rods can be found in soil and in intestinesof healthy animals and humans.

This study was aimed to evaluate the mechanismof immune modulation and disease resistanceinduced by a candidate probiotic C. butyriumCB2, isolated from chicken intestine.

Materials and methods

Bacterial strains

Clostridium butyrium CB2, isolated from the intes-tine of healthy chicken, was obtained from theSchool of Biosystems Engineering and Food Sci-ence, Zhejiang University, Hangzhou, China. Thebacteria were identified by sequencing the 16SrRNA gene using the method described by Austin,Robertson & Austin (2003) with the followinguniversal primers: plb16 (5¢-AGAGTTTGATC-CTGGCTCAG-3¢) and mlb16 (5¢-GGCTGCTG-GCACGTAGTTAG-3¢) (Kullen, Sanozky, Crowell& Klaenhammer 2000). The resulting 16 rRNAgene sequences were compared in a BLAST search(Altschul, Gish, Miller, Myers & Lipman 1990).Vibrio anguillarum and Aeromonas hydrophilaoriginally isolated from diseased Nibea albiflorawere obtained from the Aquatic Pathogen Collec-tion Centre of the Ministry of Agriculture(APCCMA) in China. The harvested bacteriawere freeze-dried (REL 206, Kyowa VacuumTech., Tokyo, Japan) by maintaining the bacterialsuspension for 60 h at )20 �C. The freeze-driedform was vacuum packed before storing at )20 �Cuntil use.

Preparation of bacteria

Clostridium butyrium CB2 was cultured in ManRogosa and Sharpe broth (MRS, Sigma ChemicalCo., St. Louis, MI, USA). A pure culture of C.butyrium was inoculated into a conical flask(500 mL) containing MRS broth and incubatedat 26 �C for 24 h on a rotary shaker at 120 gand sub-cultured twice under the same condi-tions. The culture was centrifuged at 10 000 g for20 min at 4 �C, the cell pellet re-suspended inphosphate-buffered saline (PBS; pH 7.2; OxoidLtd, Basingstoke, UK) and inactivated by heatingat 150 �C for 15 min. Inactivation was confirmedby the absence of growth following the seeding of

triplicate plates of MRS agar with 0.1 mLvolumes of the cell suspension and incubationfor 2–3 days at 26 �C. The spent culture super-natant (SCS) was obtained by centrifuging 24 hbacterial cultures at 10 000 g for 30 min at 4 �Cand the protein concentration was tested by themethod of Bradford (1976) using bovine serumalbumin (BSA) as standard. The SCS used foranti-pathogen assays was diluted in PBS (pH 7.2)to a final protein concentration of 2.5 mg mL)1.

Pathogen inhibition assay

The inhibitory activity of viable or dead C. butyriumCB2 and SCS was assessed against V. anguillarumand A. hydrophila by cross-streaking (Austin,Billaud & Stobie 1992) and the spot-on-lawnmethod (Pilet, Dousset, Barre, Novel, Desmazeaud& Piard 1995). For this, 10 mL (109 CFU mL)1)of 10· concentrated freeze dried bacterial suspen-sion in PBS at pH 7.2 was applied to freshlyprepared bacterial lawn on nutrient agar (Oxoid)and zones of inhibition were recorded after over-night incubation at 26 �C.

Fish and experimental conditions

Chinese drums, Miichthys miiuy (Basilewsky),weighing approximately 200–260 g, were pur-chased from net-cages in Xihu Port, ZhejiangProvince, China and transferred to the experi-mental laboratory (Institute of Mingo Tech-BankCo., Ltd, Ningbo, China). The fish were ran-domly divided into six 600 L tanks, each con-taining 40 fish. The flow rate was approximately3 L min)1 and salinity of the water in each tankwas 26&. The water temperature was 25–30 �Cduring the whole trial.

Safety assay of C. butyrium for fish

Bacterial cultures were grown in MRS broth at26 �C for 24 h before centrifuging at 1000 g for10 min at 4 �C, washing twice and re-suspendingin saline. The heat-killed bacteria were preparedby heating the cells in sterile normal saline at150 �C for 15 min. SCS was obtained from a24 h culture by centrifugation for 30 min at10 000 g at 4 �C. 0.1 mL suspensions of SCS(protein concentration, 2.5 mg mL)1), live CB2(108 cells mL)1) and dead CB2 (108 cells mL)1)were injected intraperitoneally (i.p.) and

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Journal of Fish Diseases 2008, 31, 679–686 X Pan et al. Clostridium butyrium as a probiotic in Miichthys miiuy

intramuscularly (i.m.) into separate groups of 10Chinese drum each. Another group of 10 fish wasinjected with saline as controls. In a separatechallenge, 3.0 mL of bacterial suspension, con-taining 108 cells mL)1, was mixed thoroughly in30 g of commercial Chinese drum feed (MingoTech-Bank Co., Ltd, Ningbo, China) to achieve108 bacterial cells g)1 of feed. Groups of 10Chinese drum were then fed three times a dayfor 7 days. Controls were fed with normal dietsupplemented with the same volume of saline.After 7 days, survivors were killed and examinedfor evidence of disease (Austin & Austin 1989).

Detection of viable C. butyrium CB2 in feed andintestine

The basal diet used the commercial productprovided by Mingo Tech-Bank Co., Ltd. Therequired amount of bacterial suspension wassprayed into the feed slowly, mixed in a drummixer and then air dried under sterile conditions for12 h. The viability of bacteria in the feed wasassessed following storage of the diets at 12 �C for2 weeks. Thus, 1.0 g amounts of feed were homog-enized in 9.0 mL volumes of saline and dilutionsprepared to 10)4 in fresh saline. Then, 0.1 mLvolumes were spread over duplicate plates of MRSagar.

Enumeration of live bacteria in the gut wasassessed using intestinal contents and samples ofmucus after 14 days of feeding. For this, 0.05–0.1 gof the posterior intestinal contents was homoge-nized in 0.45–0.9 mL of saline, as appropriate, anddilutions prepared to 10)4. Intestinal mucus (0.1 g)was homogenized in 0.9 mL of saline and 0.1 mLvolumes were spread over the surface of duplicateplates of MRS agar, with incubation at 26 �C.Tentative bacterial identification was achieved as aresult of an examination of colon and micro-morphology, including Gram-stained smears. Purecultures were stored at )70 �C as suspensions in20% (v/v) glycerol.

Feeding experiment and challenge test

Groups of 40 Chinese drum were fed either withbasal diet (control), or diet supplemented withviable C. butyrium (CB), heat-killed C. butyrium(D-CB) or SCS for 30 days. The live and deadbacteria were in freeze-dried form at 108 CFU g)1.SCS was obtained from 24 h cultures by centrifu-

gation for 30 min at 10 000 g at 4 �C. SCS wasadded at 20 mL)1 kg diet (protein concentration,2.5 mg mL)1). Subsequently, the fish were chal-lenged by i.p. injection with a 0.1 mL suspension ofV. anguillarum (1 · 107 cells mL)1) or A. hydro-phila (1 · 107 cells mL)1) as determined by meansof a haemocytometer. Dead and moribund fishwere removed and examined microbiologically andpathologically for up to 14 days post-challenge(Austin & Austin 1989). Swabs were taken from thekidney and inoculated onto tryptone soy agar plates(TSA, CM131; Oxoid) to recover pathogens.

Isolation of head kidney macrophages

Head kidney macrophages of Chinese drum used inthe feeding experiments were isolated by themethod of Secombes (1990) and used for assaysof phagocytic activity. Briefly, the head kidney ofeach fish was pushed through a 100 lm nylon meshwith RPMI 1640 (Sigma) containing 2% (v/v)foetal calf serum (FCS; Gibco, Gaithersberg, MD,USA), 100 U mL)1 of penicillin and streptomycin(P/S; Sigma) and 10 lL mL)1 of heparin (Sigma).The resultant suspensions were layered onto a 34/51% (v/v) Percoll (Sigma) gradient diluted inHank�s balanced salt solution (HBSS; Sigma) beforetubes were centrifuged at 400 g for 25 min at 4 �C.The band of cells lying at the 34–51% interface wascollected and washed twice with HBSS, and using ahaemocytometer slide, the number of cells wasadjusted to 106 cells mL)1 in RPMI 1640 mediumsupplemented with 0.1% (v/v) FCS and100 lL mL)1 P/S. Viability was checked by usingtrypan blue exclusion.

Phagocytic activity

One millilitre of the macrophage-enriched cellsuspension (106 cells mL)1) obtained from eachof 10 individual fish from the three experimentalgroups was allowed to adhere to a methanol-cleanedglass slide for 1 h, after which non-adherent cellswere removed by washing with HBSS. Then,1.0 mL of 0.85 lm diameter latex beads (Sigma)containing 108 particles mL)1 in RPMI mediumwas added to the slides. Phagocytosis was allowed toproceed for 1 h before non-phagocytosed beadswere washed-off with HBSS. Air-dried slides werefixed in absolute methanol for 1 min, removed bytilting and stained by Giemsa�s method (Houwen2000). Approximately 200 cells were counted at

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random and phagocytic activity (PA) was expressedas:

PA ¼ Number of phagocytosing cells

Number of total cells�100

The phagocytic index was determined by thenumber of latex beads phagocytosed per cell.

Serum and intestinal mucus preparation

Blood, collected by tail venepuncture, was allowedto clot at 4 �C for 6 h and serum was obtainedafter centrifugation at 2000 g for 15 min andstored at )70 �C. Intestinal mucus was collectedby scraping with a scalpel from the entiredigestive tract, after the contents had beenremoved by washing with PBS. Four parts ofsodium phosphate buffer (SPB; 2.7 mm Na2H-PO4/1.3 mm NaH2PO4; 0.004 m; pH 7.2) wereadded into each tube containing mucus and thecontents suspended thoroughly by pipetting. Themucus suspensions were centrifuged at 10 000 gfor 20 min at 4 �C and the supernatants werestored at )70 �C.

Lysozyme activity of serum and gut mucus

The lysozyme activity assay followed the previ-ously described method of Lange, Gudmundsdot-tir & Magnadottir (2001) with somemodifications. Thus, 100 mL of serum or mucussupernatant was diluted in four twofold serialdilutions in 0.05 m SPB at pH 6.2 in 96-wellmicrotitre flat bottom plates. Then, each well wasmixed with 100 lL of a 0.4 mg mL)1 suspensionof Micrococcus lysodeikticus (Sigma) in SPB. Themicrotitre plates were incubated at 20 �C and theOD was measured at 570 nm at 0, 15 and30 min using SPB as a blank. As a negativecontrol, SPB replaced serum or mucus. A unit oflysozyme activity was defined as the amount ofserum or mucus causing a decrease in absorbanceof 0.001 min)1.

Total immunoglobulin (Ig) level assay

Total Ig of plasma and gut mucus was analysed bythe method of Siwicki & Anderson (1993). Briefly,samples were diluted 100· with 0.85% NaCl andthe Biuret method was used for determining theprotein content. All reagents were sourced from

Sigma. 0.1 ml of each plasma sample was mixedwith an equal volume of 12% solution of polyeth-ylene glycol (10 000 MW) and incubated for 2 h.Ig was removed by centrifuging at 5000 g at 4 �C.The supernatant was diluted 50· with 0.85% NaCland the protein content was determined asdescribed above. The differences between theprotein values of the untreated and PEG treatedsample correspond to the total Ig content and isexpressed as mg mL)1.

Statistical analysis

The rank-sum test was used to determine thesignificant variation in bacterial growth (P £ 0.05).One-way analysis of variance (ANOVA) was usedto determine significant variation in the humoralimmune responses between treatments (SYSTAT10.0, SPSS, USA).

Results

Characterization of putative probiotic

CB2 isolated from the gastrointestinal tract ofchicken was identified as C. butyrium, with 99%similarity to Clostridium butyrium strain ATCC19398. Suspensions of live or heat-killed bacteriaand SCS had no harmful effects on fish. Whenlive or heat-killed bacteria and SCS were assayedfor their inhibitory activity in vitro, live bacteriahad a higher activity than dead bacteria and SCS(Table 1). Survival of Chinese drum controlgroups after i.p. challenge with V. anguillarumand A. hydrophila were 40% and 56%, respec-tively. After i.p. challenge with V. anguillarumand A. hydrophila, fish fed CB (78% and 70%,respectively) and D-CB (84% and 83%, respec-tively) showed an increased survival. However, the

Table 1 Characterization of the candidate probiotic Clostridiumbutyrium CB2

Suspension

Injection

dose

(CFU mL)1)

Inhibitory activitya

Vibrio

anguillarum

Aeromonas

hydrophila

CB 1 · 108 + +

D-CB 1 · 108 (+) )SCS 0 (+) (+)

+, Strong inhibition; ), no inhibition; (+), delayed growth.aObtained by spot-on-lawn method.

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SCS fed group showed no increased survival(Table 2).

Viability of C. butyrium CB2 in feed andintestine

Feed contained 109 CFU g)1 of CB2 (108

CFU g)1 initial) after 2 weeks storage at 12 �C.After 14 days of feeding, there were 2.1 · 106 and3.5 · 105 CFU g)1 of CB2 in the intestinal con-tent and in the mucus, respectively.

Phagocytic activity

The phagocytic activity of head kidney leucocytesisolated from Chinese drum fed with CB wassignificantly higher (65.6 � 12.8%; P < 0.05)than in the control group (40.5 � 13.3%; Fig. 1a).Phagocytic activity was also significantly higher infish fed with D-CB (60.2 � 11.6%; P < 0.05)than in the control group. However, there was nosignificant difference among groups with respect tothe phagocytic index (control = 10.2 � 1.6,CB = 11.6 � 2.1, D-CB = 9.8 � 1.2 and SCS =8.8 � 1.1) (Fig. 1b).

Lysozyme activity of serum and gut mucus

The lysozyme activity of the serum and the gutmucus of Chinese drum fed CB or D-CB wassignificantly increased compared with the controland SCS groups. There was no significant differencebetween the control and the SCS groups (Fig. 2).

Total Ig level in serum and gut mucus

The total Ig level of serum and gut mucus increasedboth in CB and D-CB treatment groups(P < 0.05). However, the Ig level of the SCSgroup was not significantly different from thecontrol group (P > 0.05) (Fig. 3).

Discussion

The aim of this study was to assay the effects ofClostridium butyrium CB2 isolated from healthychicken intestine on fish immune modulation anddisease resistance by oral administration. Thepotential probiotic strain used in this study had a99% similarity to Clostridium butyrium strainATCC19398 in the PCR-amplified 16S rRNAgene. Although this is the first time that exogenousClostridium butyrium has been demonstrated to be apotential probiotic in marine fish, there have beensome attempts to use Clostridium butyrium inhumans and other animals (Ito, Hayashi, Iguchi,Endo, Nakao, Kato, Nabeshima & Ogura 1997;Kamiya et al. 1997). Other probiotics inludingCarnobacterium divergens, Lactobacillus rhamnosus,Bacillus subtilis, Lactobacillus fructivorans and Car-nobacterium maltaromaticum (Sami, Arthur, Goran,Seppo & Esa 2003; Irene et al. 2005; Kim &Austin 2006; Simona, Massimo, Anna, Raffaella,

Table 2 Survival (%) of Chinese drum fed live C. butyrium CB2

(CB), dead CB2 (D-CB) cells (108 cells g)1) or spent culture

supernatant (SCS) for 30 days after i.p. challenge with Vibrioanguillarum and Aeromonas hydrophila (1 · 106 cells fish)1)

Pathogen

Survival of Chinese drum (%)

Control CB D-CB SCS

V. anguillarum 40 � 2.4 78 � 4.1 70 � 4.5 46 � 4.7

A. hydrophila 56 � 3.2 84 � 5.2 83 � .3.9 62 � 5.3

CB, live C. butyrium; D-CB, heat-killed C. butyrium; SCS, spent culture

supernatant, mean � SD, n = 3.

0

20

40

60

80

100(a) (b)

Control CB D-CB SCS Control CB D-CB SCS

**

0

2

4

6

8

10

12

14

16

Figure 1 The effect of administration of viable Clostridium butyrium (CB), heat-killed C. butyrium (D-CB) and spent culture

supernatant (SCS) on (a) % phagocytosis and (b) phagocytic index in Chinese drum head kidney macrophages harvested at 30 days after

feeding. Data are expressed as mean � SD of 40 fish. *Significant difference, P < 0.05 compared with the values of control group.

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Anna, Victoriano, Oliana, Alberto & Luigi 2007)have been shown to have an immunostimulatoryeffect in fish.

Probiotic bacteria should survive passagethrough the digestive tract and have the capabilityto multiply therein (Havenaar, Brink & HuisIn� 1992). During our feeding experiments,C. butyrium was recovered from the gut contentsafter 7 days, suggesting this strain was able toresist digestive tract fluids. Our results alsosuggest that C. butyrium CB2 live cells, heatkilled cells and SCS are not harmful to fish.Clostridium butyrium inhibited V. anguillarumand A. hydrophila. These results are in agreementwith Motomichi, Haruhiko, Hiroyuki, Takako,Akio & Shigeru (2004) who demonstrated thatC. butyrium had an antagonistic action againstintestinal pathogens in vitro.

An increase in growth rate and decrease inmortalities after challenge tests are commonlyassociated with probiotic activity in fish (Gatesoupe1994; Gram et al. 1999; Irianto & Austin 2002a;Austin et al. 2003). After challenge with V. anguil-larum and A. hydrophila, the fish fed viable or heat-killed C. butyrium CB2 showed low mortalities, but

SCS had no effect. This result is in agreement withSakai, Yoshida, Atsuta & Kobayashi (1995), whodemonstrated resistance to Vibrio spp. in rainbowtrout following oral administration of C. butyrium.Although SCS had an obvious inhibitory effect onV. anguillarum and A. hydrophila in vitro, there wasno significant inhibition effect in fish after oraldelivery, suggesting that the activity of SCS may beaffected by gastrointestinal fluids.

It has been shown that certain probiotic bacteriaare able to stimulate phagocytic activity (Arunacha-lam, Gill & Chandra 2000) and there are reportsthat phagocytic activity of fish head kidney leuco-cytes is increased by feeding both live probioticbacteria-supplemented diets (Nikoskelainen,Ouwehand, Bylund, Salminen & Lilius 2003) andheat killed bacteria-supplemented diets (Irianto &Austin 2003). However, Villamil, Tafalla, Figueras& Novoa (2002) showed that Lactococcus lactissupplementation did not affect the phagocyticactivity of turbot head kidney macrophages after1 week of daily administration. In the presentstudy, the phagocytic activity of head kidneyleucocytes of drum fed live or heat-killedC. butyrium was increased.

0

200

400

600

800

1000

Control CB D-CB SCS

Serum

Mucus

** **

*

* Ly

sozy

me

activ

ity (

U m

L–1)

Figure 2 Lysozyme activity of the serum

(h) and gut mucus ( ) of Chinese drum fed

live Clostridium butyrium (CB), heat-killed

C. butyrium (D-CB) and spent culture

supernatant (SCS). Data are expressed

as mean � SD of 40 fish. Significant

differences *P < 0.05; **P < 0.01

compared with control group.

0

2

4

6

8

10

12

14

Control CB D-CB SCS

SerumMucus*

* *

*

Ig (

mg

mL

–1)

Figure 3 Total Ig levels in serum (h) and

gut mucus ( )of Chinese drum fed live

Clostridium butyrium (CB), heat-killed

C. butyrium (D-CB) and spent culture

supernatant (SCS). Data are expressed

as mean � SD of 40 fish. Significant

differences *P < 0.05; **P < 0.01

compared with control group.

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Lysozyme is a major component of the immunesystem in both invertebrates and vertebrates and candivide peptidoglycan in bacterial cell walls andcause lysis of cells, especially of Gram-positivespecies (Chipman & Sharon 1969). Lysozymeconcentrations in fish have been reported to beincreased after injection of a bacterial product(Chen, Yoshida, Adams, Thompson & Richards1996) and in response to bacterial infection(Møyner, Roed, Sevatdal & Heum 1993). In thisstudy, the lysozyme activity of drum fed liveC. butyrium was significantly increased and amarginal increase was seen with heat-killedC. butyrium. The result is in contrast to the resultsof Panigrahi et al. (2004), who demonstrated thatheat-killed L. acidophilus had no effect on fishlysozyme activity. This difference may reflect thedifferent action of the probiotic strains.

In teleost fish, two types of immunoglobulin(belonging to the IgM and IgD subpopulations) havebeen characterized (Watts, Munday & Burke 2000),with IgM being the main type which resemblesmammalian IgM in both structure and physiologicalcharacteristics. This study showed that administra-tion of live or heat-killed C. butyrium increased thetotal Ig level in serum and gut mucus of drum. Theresults are in line with previous reports, whichsuggested that probiotic bacteria may also inducecell-mediated immunity in fish as well as in mammals(Miettinen, Vuopio & Varkila 1996).

In conclusion, the present study has shown thatexogenous C. butyrium CB2 can affect the immunesystem and enhance disease resistance after oraladministration to Chinese drum.

Acknowledgement

We are grateful for support from the Science andTechnology Bureau of Zhejiang Province, China(Project number: 2006C12098).

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Received: 18 June 2007Revision received: 14 January 2008Accepted: 21 January 2008

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� 2008



Documents

Immune responses and enhanced disease resistance in Chinese drum, Miichthys miiuy (Basilewsky), after oral administration of live or dead cells of Clostridium butyrium CB2