bacillus subtilis para aeromonas-impreso

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O R I G I N A L A R T I C L E

Bacillus subtilis AB1 controls Aeromonas infection

in rainbow trout (Oncorhynchus mykiss, Walbaum)

A. Newaj-Fyzul1,2, A. A. Adesiyun2, A. Mutani2, A. Ramsubhag3, J. Brunt1 and B. Austin1

1 School of Life Sciences, Heriot-Watt University, Edinburgh, Scotland, UK

2 School of Veterinary Medicine, The University of the West Indies, St Augustine, Trinidad, West Indies

3 Department of Life Sciences, The University of the West Indies, St Augustine, Trinidad, West Indies

Introduction

Recently, there has been an increasing practice of man-

aging bacterial fish diseases by using naturally antagonis-

tic micro-organisms to control populations of potential

pathogens, either by competitive inhibition, enhancement

of fish immunity or by the microbial enhancement of the

environment. Such organisms have been usually referred

to as probiotics (Salminen et al. 1999), and are usually

incorporated into the fish feed. Lactic acid bacteria (LAB)

are among the most common probiotics used in aquacul-

ture, and have been proposed to function as nonspecific

immunostimulants and in environmental enhancement

(Vadstein 1997; Ringø and Gatesoupe 1998; Skjermo and

Vadstein 1999; Robertson et al. 2000). However, a greater

variety of micro-organisms has been considered for use as

probiotics in aquaculture than in other areas of agri-

culture (Irianto and Austin 2002). In this study, Bacillus

subtilis AB1 was isolated and evaluated as a putative pro-

biotic in preventing disease in rainbow trout caused by a

highly virulent strain of Aeromonas sp.

Materials and methods

Fish

Rainbow trout, Oncorhynchus mykiss (Walbaum) (average

weight = 30 g) were obtained from a commercial fish

farm in Scotland. The fish were maintained in continuo-

usly aerated free-flowing dechlorinated fresh water at 17°C

and fed with a commercial pellet diet (Trouw, Wincham,

UK). Representative samples from the fish stock were rou-

tinely examined microbiologically and physically to ensure

the absence of bacterial diseases and parasites following

methods described by Austin and Austin (1989).

Bacterial pathogen

Aeromonas sp. ABE1 originally isolated from diseased til-

apia (Oreochromis sp.) was obtained from the culture

collection of the School of Veterinary Medicine, The

University of the West Indies, St. Augustine, Trinidad.

Pathogenicity of Aeromonas sp. ABE1 against rainbow

Keywords

Aeromonas, Bacillus subtilis AB1, fish disease,

innate immunity, probiotic, specific immunity.

Correspondence

B. Austin, School of Life Sciences, John Muir

Building, Heriot-Watt University, Riccarton,Edinburgh, EH14 4AS, Scotland, UK. E-mail:

[email protected]

2006 ⁄ 1652: received 27 November 2006,

revised 13 March 2007 and accepted 13

March 2007

doi:10.1111/j.1365-2672.2007.03402.x

Abstract

Aim: To develop a probiotic with effectiveness against Aeromonas sp., which

was pathogenic to rainbow trout.

Methods and Results: When Bacillus subtilis AB1, which was obtained from

fish intestine, was administered for 14 days to rainbow trout in feed at a con-

centration of 107 cells per gram either as viable, formalized or sonicated cellsor as cell-free supernatant, the fish survived challenge with the pathogen. AB1

stimulated immune parameters, specifically stimulating respiratory burst, serum

and gut lysozyme, peroxidase, phagocytic killing, total and a1-antiprotease and

lymphocyte populations.

Conclusions: Bacillus subtilis AB1 was effective as a probiotic at controlling

infections by a fish-pathogenic Aeromonas sp. in rainbow trout.

Significance and Impact of the Study: Disease control in fish is possible by

means of the oral application of live and inactivated cells and their subcellular

components with the mode of action reflecting stimulation of the innate

immune response.

Journal of Applied Microbiology ISSN 1364-5072

ª 2007 The Authors

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 1699–1706 1699



trout was determined by challenging the fish intraperiton-

eally (i.p.) and intramuscularly (i.m.) with the pathogen

at different concentrations (102 to 108 cells per millilitre)

and observing for disease development and death over a

7-day period.

Putative probiotics

Bacterial cultures were obtained from the digestive tract of

rainbow trout using the procedures of Brunt and Austin

(2005). Briefly, the digestive tracts of five euthanized fish

were removed in their entirety, and the intestinal contents

including the mucus were emptied into petri dishes before

1Æ0 g quantities were added to 9 ml of 0 Æ9% (w ⁄ v) saline

and vortexed vigorously for 1 min. Then, 10-fold dilu-

tions were prepared in fresh saline to 10)5, and 0Æ1 ml vol-

umes were spread over triplicate plates of tryptone soya

agar (TSA; Oxoid) with incubation at 22°C for up to

7 days. A total of 160 colonies was randomly picked and

purified by streaking onto fresh medium, and assessed for

inhibition against the fish pathogenic aeromonad using

the cross-streak method, spot-on-lawn method and the

overlay method as described by Robertson et al. (2000).

One inhibitory isolate was evaluated at different concen-

trations for possible adverse effects in fish. Briefly, separate

groups of 25 fish were injected i.p. and i.m. with the

organism at concentrations ranging from 104 to 109 cells

per millilitre as determined by means of a haemocytome-

ter slide (Improved Neubauer type; Merck, Whitehouse

Station, NJ, USA) on a Kyowa light microscope at ·400

magnification. The fish were observed for disease signs

daily for up to 14 days (Brunt and Austin 2005). Addi-tionally, the willingness of fish to accept food with the

bacterial culture was tested by feeding separate groups of

25 rainbow trout for 14 days with commercial fish feed

supplemented with putative probiotic at concentrations of

103 to 109 cells per gram. Control fish were also fed with

commercial fish feed but without any added bacterial cul-

ture. The bacterial cultures were examined in the API 20E

and API 50CH rapid identification systems (BioMerieux,

Basingstoke, UK) and by 16S rRNA sequencing. The cul-

tures were stored in tryptone soya broth (TSB; Oxoid)

supplemented with 15% (v ⁄ v) glycerol at )70°C.

Challenge experiments to determine the effectiveness

of putative probiotics

The methods described by Brunt and Austin (2005) were

used to determine the potential usefulness of the cultures

in preventing disease. Probiotic-supplemented fish feed

containing 104, 105, 106, 107, 108 and 109 bacterial cells

per gram were prepared spraying 5 ml volumes of appro-

priate saline suspensions of the organism onto 50 g

batches of feed, with constant mixing. The bacterial

counts in the feed and the survival of the putative probi-

otic on the feed over a 2-month period were determined

by means of the total viable counts on TSA. This was

achieved by homogenizing (VWR disposable homogeniz-

er) 1Æ0 g of feed in 9Æ0 ml of saline, preparing 10-fold

dilutions and spreading 0Æ1 ml amounts over duplicate

TSA plates, which were incubated at 22°C for 7 days.

Additional experiments were conducted to determine the

ability of formalin treated cells or cell extracts to confer

protection against the pathogen. The putative probiotic as

formalized (2Æ0 v ⁄ v formalin for 48 h) cells, sonicated cell

suspensions and cell-free extract were added to fish feed

to a final concentration equivalent to107 cells per gram

following the methods described by Brunt and Austin

(2005).

Separate groups of 25 rainbow trout were fed with the

modified diets, which were refrigerated until use for

14 days before challenging with the pathogenic aeromo-

nad by i.p. injection with 2Æ3 · 106 cells per millilitre,

which was equivalent to 2 · LD50 as determined sepa-

rately (data not shown). Appropriate control groups were

included. Each challenge experiment was repeated three

times.

Determination of the mode of action of probiotics

The modes of action on the putative viable probiotics

were determined using rainbow trout fed with probiotic

dose at 107 cells per gram of fish feed for 14 days. Then,

groups of 25 fish were killed by administration of an

overdose of anaesthetic (3-aminobenzoic acid ethyl ester;Sigma-Aldrich) before collection of blood, tissue or gut

mucus (Brunt and Austin 2005). Each assay was repli-

cated three times.

To estimate the number of probiotic cells in the diges-

tive tract of rainbow trout, the following experiment was

carried out. For the collection of intestinal mucus, the

method of Chabrillon et al. (2005) was followed with

modifications. Thus, the abdomen of each fish was cut

open to expose the gastrointestinal tract. The intestine

from the pylorus to the anus was removed, and its outer

surface was carefully cleaned of its layers of fat. Pressure

was applied to the sides of the intestine so that the mucus

exuded out through the open ends. The mucus and gut

contents were collected separately in pre-weighed sterile

1Æ5-ml Eppendorf tubes and homogenized in sodium

phosphate buffer (SPB; 2Æ7 mmol l)1 Na2HPO4,

1Æ3 mmol l)1 NaH2PO4, 0Æ004 mol l)1; pH 7Æ2), and dilu-

tions prepared to 10)4 before 0Æ1 ml volumes were spread

over duplicate TSA plates with incubation at 30°C for up

to 3 days. Identification and colony counts were done as

described earlier.

Control of Aeromonas infection in rainbow trout A. Newaj-Fyzul et al.

1700 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 1699–1706

ª 2007 The Authors



Sample and cell preparations for immunological assays

After feeding, the fish were euthanized and exsanguinated

by caudal venepuncture using 9 ml capacity Vacuettes

containing a Z Serum Sep Clot Activator (Greiner, Stone-

house, UK). Blood was allowed to clot at 4°C for 2 h,

and the sera were separated by centrifugation(4000 rev min)1 for 25 min at 4°C) and stored at )70°C

for subsequent assays.

Gut mucus was collected as described earlier and cen-

trifuged twice at 13 000 rev min)1 for 25 min at 4°C to

remove particulate and cellular material. The supernatant

was removed and stored at )70°C for lysozyme analysis.

Isolation of head kidney macrophages for evaluation of

phagocytic activity, respiratory burst and bacterial killing

assay, determination of the number of erythrocytes and

leucocytes, the lysozyme and antitrypsin activity, and the

total protein quantity of the serum followed the methods

of Sakai et al. (1995).

Using aseptic techniques, the head kidneys were

removed from rainbow trout and forced through a

100 lm nylon mesh with L-15 medium (Sigma-Aldrich)

containing 2% (v ⁄ v) foetal calf serum (FCS; Gibco,

Paisley, UK), 100 ll ml )1 of penicillin ⁄ streptomycin

(p ⁄ s; 10 000 IU ml )1 ⁄ 10 000 UG; Sigma-Aldrich) and

10 ll ml )1 heparin (Sigma-Aldrich) to isolate the leu-

cocytes. Thus, kidney suspensions were layered carefully

onto a 34–51% (v ⁄ v) Percoll gradient diluted in Hanks

balanced salt solution (HBSS; Sigma-Aldrich). The sam-

ples were centrifuged at 400 g for 25 min at 4°C before

carefully removing the cells lying at the 34–51% inter-

face. The cells were adjusted to 106

cells per millilitrein L-15 medium supplemented with 0Æ1% (v ⁄ v) FCS

and 100 ll ml )1 p ⁄ s. Additional assays were carried

out as follows:

Flow cytometry

Analysis of fish blood using flow cytometry was per-

formed following the methods of Takamasa et al. (2002).

Thus, stock solutions of 3,3¢-Dihexyloxacarbocyanine

[DiOC6(3); Sigma-Aldrich] were prepared in absolute

ethanol to 500 lg ml )1and held in the dark. This stock

solution was diluted 10-fold in HBSS immediately prior

to use. Fresh rainbow trout blood (10 ll) was added to

triplicate test tubes, each containing 1950 ll of HBSS and

40 ll of DiOC6(3) dye solution. This was mixed gently

and incubated at room temperature for 10 min. Follow-

ing staining with DiOC6(3), blood cells were analysed

using a flow cytometer (Cyflow SL). Forward scatter

(FSC), side scatter (SSC) and green fluorescence (FL-1) of

each cell was measured. All data were analysed using the

Flomax (Partec, Munster, Germany) software package.

Bactericidal assay

Macrophage killing activity was assessed according to

Secombes (1990) with modifications. Aeromonas sp. was

grown in TSB for 24 h and adjusted to 107 cells per milli-

litre in saline. Macrophages were adjusted to 106 cells per

millilitre in L-15 medium before adding 0Æ1 ml volumes

to 0Æ1 ml of bacterial suspension. Subsequently, 40 ll of

pooled fresh rainbow trout serum was added, followed by

incubation at 25°C for 2 h with shaking every 15 min.

Volumes (0Æ1 ml) were removed and diluted in 9Æ9 ml of

sterile (121°C 15 min)1) distilled water to release living

bacteria from the phagocytes. This was serially diluted to

10)5, and 100 ll volumes were spread onto triplicate TSA

plates with incubation overnight at 30°C, and the number

of colonies was counted (Selvaraj et al. 2005). Control

assays were carried out in the absence of macrophages to

give 100% survival at all bacterial dilutions.

Total and a1-antiprotease activity of serum

The antitrypsin activity of sera was measured following

the methods described by Magnadottir et al. (2005).

Briefly, 20 ll of serum was incubated with 20 ll of stand-

ard trypsin solution (Sigma-Aldrich, 1000-2000 BAEE,

5 mg ml)1) at room temperature (22°C) for 10 min in

Eppendorf tubes. Two hundred microlitres of 0Æ1 mol l)1

PBS and 250 ll of 2% (w ⁄ v) azocasein solution

(20 mg ml)1 PBS) were added and further incubated for

1 h. Then, 500 ll of 10% trichloro acetic acid (TCA;

Fisher) was added and incubated for another 30 min. The

tubes were centrifuged at 9000 rev min)1

for 5 min before100 ll of the supernatant from each tube was placed into

the wells of a microtitre flat bottom plate (Nalge Nunc,

Hereford, UK) containing 100 ll of 1 N sodium hydrox-

ide. The optical density (OD) was read at 450 nm on a

Smartspec 3000 spectrophotometer (Bio-Rad). Inhibition

of trypsin activity was calculated by comparing with a

100% control sample, which contained the buffer to

replace serum, and a negative control where the buffer

replaced both serum and trypsin.

For a1-antiprotease, the assay was prepared following

the method by Ellis (1999) where 10 ll of serum was incu-

bated with 20 lg trypsin dissolved in 100 ll of Tris–HCl

(50 mmol l)1; p H 8Æ2) (Sigma-Aldrich). All tubes were

made up to 200 ll with Tris–HCl and incubated at room

temperature (22°C) for 1 h. Then, 2 ml of 0 Æ1 mmol l)1

Na-benzoyl-DL-arginine-p-nitroanilide HCl (BAPNA; Sig-

ma-Aldrich) was added and incubated for a further

15 min. The reaction was stopped by adding 500 ll of

30% acetic acid and the OD read at 450 nm. The serum

blank contained 100 ll of Tris instead of trypsin, and the

positive control contained trypsin but no serum.

A. Newaj-Fyzul et al. Control of Aeromonas infection in rainbow trout

ª 2007 The Authors




a-2 macroglobulin in serum

This assay is a modification of a method described by

Ellis (1990), and uses an aniline-arginine dye ester as a

substrate for trypsin, which hydrolyses the aniline dye

resulting in a colour change that can be measured spec-

trophotometrically. Trypsin, at final concentrationsbetween 0 and 35 lg (bovine pancreas Type 1, 100 mg

ml)1 in 0Æ01 mol l)1 Tris–HCl; Sigma-Aldrich; pH 8Æ2),

was incubated with 10 ll of serum at 22°C for 5 min.

Replicates with a range of trypsin concentrations were

used. After the initial incubation, 0Æ5 ml of 2 mmol l)1

BAPNA in distilled water was added and the volume

made up to 1 ml with 0Æ1 mol l)1 Tris–HCl, pH 8Æ2,

followed by incubation at 22°C for 25 min. The reaction

was stopped by the addition of 150 ll of 30% (v ⁄ v) acetic

acid. Each sample was centrifuged at 462 g and then

filtered through a 0Æ22 lm filter (Millipore Millex, Edin-

burgh, UK) into 1 ml cuvettes and read at OD410 in a

spectrophotometer against a blank of BAPNA in buffer

and acetic acid. Controls consisted of the reaction combi-

nation without serum or trypsin. The trypsin hydrolysed

BAPNA under standard reaction conditions at a rate that

produced a change in OD410 of 0Æ112 for each microgram

of trypsin present.

Peroxidase content

The total peroxidase content present in serum was meas-

ured according to Dıaz-Rosales et al. (2006). For this,

15 ll of serum was diluted with 35 ll o f C a+2- and

Mg+2

-free HBSS (Sigma) in flat-bottomed 96-well micro-titre plates (Nalge Nunc). Then, 50 ll of 20 mmol l)1

3,3¢,5,5¢-tetramethylbenzidine hydrochloride (TMB; Sig-

ma-Aldrich) and 5 mmol l)1 H2O2 (Sigma-Aldrich) were

added (both substrates of peroxidase). The serum mixture

(150 ll) was transferred from each well to new 96-well

microtitre plates. The colour-change reaction was stopped

after 2 min by adding 50 ll of 2 mol l)1 sulphuric acid

and the OD was read at 540 nm in an ELISA reader

(Dynatech, Guernsey, UK). Standard samples without

serum were also analysed.

Natural haemolytic complement activity

The alternative complement pathway (ACH50) activity

used sheep red blood cells (SRBC; Sigma-Aldrich) as tar-

gets. Equal volumes of SRBC suspension (1Æ7 · 107 cells

per millilitre) in phenol red-free Hank’s buffer (HBSS;

Sigma-Aldrich) containing 0Æ1 mmol l)1 Mg+2 and EGTA

(Sigma-Aldrich) were mixed with serially diluted serum

to give final serum concentrations ranging from 10% to

0Æ078%. After incubation for 90 min at 22°C, the samples

were centrifuged at 400 g for 5 min at 4°C. The relative

haemoglobin content of the supernatants was assessed by

measuring their OD550. The values of maximum (100%)

and minimum (spontaneous) haemolysis were obtained

by adding 100 ll of distilled water or HBSS to 100 ll

samples of SRBC, respectively. The degree of haemolysis

(Y ) was estimated and a lysis curve for each specimenwas obtained by plotting Y ⁄ (1 ) Y ) against the volume of

serum added (ml) on a log ) log scaled graph. The vol-

ume of serum producing 50% haemolysis (ACH50) was

determined and the number of ACH50 units per millilitre

obtained for each experimental group.

Statistics

All fish experiments were repeated three times unless spe-

cified. The data were examined by a range of statistical

methods including Student t -test for comparing immune

responses between probiotic feed and control fish and

anova for comparing probiotic treatments in the chal-

lenge experiments using Instat 2Æ01 statistical software

package (GraphPad Software, San Diego, CA, USA).

Results

The isolate AB1, which was an endospore-forming,

Gram-positive bacterium identified as B. subtilis by

phenotypic traits and 16S rDNA sequencing (with a

sequence homology of 99% when compared with

B. subtilis spizizenii strain PDA), was inhibitory to the

pathogenic Aeromonas sp. Furthermore, AB1 was harmless

to rainbow trout following administration via injection orby feeding. In addition, 14 days after the completion of

the feeding regime, the fish appeared healthy, and the

organism could not be recovered internally or from

around the injection sites.

Feeding to fish for 14 days at 107 cells per gram of feed,

whole, sonicated or formalized cells as well as cell-free

supernatant, led to significantly (P = 0Æ0001) reduced

cumulative mortalities after challenge with Aeromonas sp.

(Table 1). The survival rates after challenge ranged from

65% to 100% for the probiotic-fed as compared to 5% to

15% in the nonprobiotic control-fed fish. Doses of viable

AB1 lower and higher than 107 cells per gram of feed were

less successful at controlling the infection by Aeromonas

(Table 1). Furthermore, AB1 was present in the intestine

at > 104 cells per gram of gut contents and mucus during

the feeding regime, but was absent 4 weeks after switching

to regular feed (Table 2). Moreover, there was only a slow

decline of viability of AB1 in fish feed over 7 weeks at

4°C, but at 22°C there was a steadier decline in the num-

ber of culturable cells from 2Æ1 · 107 g)1 initially to

6Æ1 · 104 g)1 at 56 days (Table 3).



ª 2007 The Authors



Mode of action of the probiotic

Generally, there was stimulation of the immune system

after administering AB1 to rainbow trout. Specifically, the

number of leucocytes increased from 0Æ64 ± 0Æ25 ·

104 ml)1 in the control group to 2Æ8 ± 0Æ2 · 104 ml)1 in

AB1-fed fish (P = 0Æ0002). The erythrocyte counts for the

probiotic treated and control fish were 1Æ3 ± 0Æ5 · 109

and 1Æ5 ± 0Æ3 · 109, respectively, but the difference was

not statistically significant (Table 4). The phagocytic

index of head kidney macrophages of AB1-fed fish

(69 ± 9%) was significantly higher than that of controls

(38 ± 3%) (P = 0Æ018). In addition, the bactericidal

activity of macrophages from AB1-fed fish (2Æ1 · 106 ±

0Æ12 cells per millilitre) was significantly higher than that

of controls (3Æ8 · 10

4

± 0Æ21 cells per millilitre) (P =

0Æ0002) after incubating the pathogen at a dose of

4Æ4 · 107 cells per millilitre with macrophages obtained

from head kidney (Table 4). Moreover, there were statis-

tically significant differences in the respiratory burst activ-

ity of blood macrophages from fish which received

probiotics (0Æ12 ± 0Æ02 units of activity) as compared

with the controls (0Æ06 ± 0Æ005) (P = 0Æ0013). The serum

lysozyme activity was recorded as 1269 ± 134 and

438 ± 75 U ml)1 after 60 min for AB1 treated and con-

trol fish, respectively. The results for gut mucus lysozyme

revealed a significantly higher activity for fish fed with

probiotic as compared with controls (1033 ± 181 and

510 ± 45 at 60 min, respectively) (P = 0Æ001). Total anti-

protease activity as measured by the mean antitrypsin

activity of sera was 86% (±4) and 64% (±3) for AB1 and

controls, respectively. These differences were statistically

significant (P = 0Æ0001). In particular, fish fed with AB1

showed a higher activity for a1-antiprotease (93 ± 2) as

compared with the controls (84 ± 3), with the differences

being significant (Table 4). Although a2-macroglobulin

activity was higher in AB1-fed fish (1Æ42 ± 0Æ12) than the

Table 1 Effect of feeding varying concentration of AB1 on the survi-

val of rainbow trout after challenging with Aeromonas sp.

No. of bacterial cells (CFU g)1)

% survival

AB1 Control

104 65* 15

105

72* 10106 86* 5

107 100* 15

108 68* 15

109 65* 10

Sonicated cells (107) 100* 5

Formalized cells (107) 100* 10

Cell-free supernatant (107) 100* 10

means of three replicates of 25 fish used in treatment.

*Significant at < 5% level.

Table 2 Survival of probiotic in the intestines of rainbow trout after

feeding for 14 days

Sample

Mean number of cells g)1(CFU) in gut

Treatment Probiotic cells Total viable count

Gut contents Control None detected 4Æ6 ± 0Æ8 · 107

AB1 5Æ35 ±1Æ2 · 104 2Æ4 ± 1Æ1 · 104

Gut mucus Control None detected 1Æ3 ± 0Æ6 · 106

AB1 8Æ1 ± 2Æ8 · 104 3Æ6 ± 1Æ4 · 104

n = 10.

Table 3 Survival of probiotic (AB1) in feed maintained at different

temperatures

Day

Number of cells on feed (CFU g)1)

4°C 22°C

0 2Æ1 · 108* 2Æ1 · 108*

1 2Æ0 · 108 2Æ0 · 108

3 2Æ0 · 108 5Æ7 · 107

7 6Æ9 · 108 7Æ4 · 106

14 5Æ3 · 107 6Æ8 · 106

21 5Æ2 · 107 3Æ4 · 106

28 4Æ7 · 107 1Æ2 · 106

35 3Æ8 · 107 8Æ3 · 105

42 3Æ0 · 107 1Æ4 · 105

49 2Æ4 · 107 5Æ2 · 104

56 2Æ3 · 10

7

6Æ1 · 10

4

*Initial count.

Table 4 Immunological response of rainbow trout after feeding AB1

for 14 days

Immunological parameter Probiotic AB1 Control

Erythrocytes (·109 ml)1) 1Æ3 ± 0Æ5 1Æ5 ± 0Æ3

Leucocytes (·104 ml)1) 2Æ8 ± 0Æ2* 0Æ64 ± 0Æ25

Phagocytic activity (%) 69 ± 9* 38 ± 3

Bactericidal activity (CFU ml)1) 2Æ1 · 106 ± 0

Æ12* 3

Æ8 · 104 ± 0

Æ21

Respiratory burst (OD630) 0Æ12 ± 0Æ02* 0Æ06 ± 0Æ01

Gut mucus lysozyme (U ml)1) 1033 ± 181* 510 ± 45

Serum lysozyme (U ml)1) 1269 ± 134* 438 ± 75

Total antiprotease activity

(% trypsin inhibition)

86 ± 4* 64 ± 3

a1-antiprotease 93 ± 2* 84 ± 3

a2-macroglobulin 1Æ42 ± 0Æ12 1Æ39 ± 0Æ11

Peroxidase assay (OD 540) 1Æ25 ± 0Æ09* 0Æ77 ± 0Æ06

Complement activity

(ACH50 U ml)1)

68 ± 8 64 ± 6

Total proteins (mg ml)1) 42 ± 3* 33 ± 4

*Significant at < 5% level.

Data represent the average ± standard deviation from a triplicate setof 25 fish.


ª 2007 The Authors




controls (1Æ39 ± 0Æ11), these values were not significantly

different (P > 0Æ05). Again, the serum peroxidase content

from fish fed with probiotic was higher (1Æ25 ± 0Æ09) than

the controls (0Æ77 ± 0Æ06) (P < 0Æ01). Flow cytometry of

the whole blood revealed a significantly higher count of

leucocytes in AB1 treated (2Æ8 · 104 ± 0Æ20) as compared

with control fish (0Æ64 · 104 ± 0Æ25) (P = 0Æ001) (Fig. 1).

There was no significant difference (P > 0Æ05) in natural

haemolytic complement activity levels of serum between

AB1 fed (68 ± 8) and control fish (64 ± 6). In contrast,

total protein was significantly higher (P < 0Æ01) in AB1-

fed groups (42 ± 3 g dl)1) as compared with the controls

(33 ± 4 g dl)1

) (Table 4).

Discussion

Bacillus subtilis AB1 was able to effectively protect rain-

bow trout against virulent Aeromonas sp., and thus can

be classified as a probiotic agent. The fact that B. subtilis

AB1 was isolated from the gut of apparently healthy rain-

bow trout confirms the potential role of gut micro-organ-

isms in exerting an important role in the wellbeing of the

host fish (Cunningham-Rundles and Lin 1998). There is

increasing evidence that Bacillus spp. is beneficial in pro-

tecting against bacterial pathogens. Moreover, B. subtilis

has demonstrated antibiosis against pathogenic Vibrio

spp., and has also been used to improve pond water

quality, leading to increased survival of black tiger prawns

(Vaseeharan et al. 2004).

The results of the present study indicate that B. subtilis

AB1 stimulated both cellular and humoral immune

responses, which may have provided the rainbow trout

with adequate protection to survive the challenge by the

highly virulent Aeromonas sp. The role of probiotics in

influencing immune responses in fish has been previously

reported as having important regulatory effects on the

innate and adaptive immune responses of the host (Aus-

tin et al. 1995).

The immune responses of rainbow trout to B. subtilis

AB1 included a significant increase in the number of leu-

cocytes (Fig. 1) as well as enhanced respiratory burst and

phagocytic activity. Similar results have been reported for

LAB in turbot (Villamil et al. 2002), Lactobacillus

rhamnosus in rainbow trout (Nikoskelainen et al. 2003),

Bacillus toyoi in European eel (Chang and Liu 2002), Car-

nobacterium sp. in rainbow trout (Kim and Austin 2006)

and Vibrio sp. in gilthead sea bream (Sparus aurata L.)(Dıaz-Rosales et al. 2006).

The increased respiratory burst activity, which is a

measure of the superoxide anion (O2

)) and its deriva-

tives, may have contributed to the ability of the probiotic

to protect against the pathogen insofar as reactive oxida-

tive species (ROS) compounds are known to contribute

extracellular killing of pathogens (Hardie et al. 1996; Itou

et al. 1997). The corresponding increase in peroxidase

activity was not surprising because these enzymes will be

required to remove reactive-free radicals that may be

harmful to the fish. However, Dıaz-Rosales et al. (2006)

did not find any significant increase in respiratory burst

activity or peroxidase activity in gilthead sea bream fed

with heat-inactivated probiotic. The role of peroxide

activity in protecting fish following exposure to probiotics

is, therefore, unclear. However, these cellular responses

could provide a mechanism to account for the probiotic

properties of select bacteria.

Lysozyme is an important humoral innate defence

parameter, and is widely distributed in invertebrates and

vertebrates (Magnadottir et al. 2005). Lysozyme has an

Control bloodAB1 fed blood

R2

R1

R3

FL1

100 101 102

- -

103 104100 101 102 103 104

SCC

4 0 9 5

0

4 0 9 5

0

FL1

SCC

Figure 1 Analysis of fish blood using flow

cytometry. Flow cytometry analysis of

DiOC6(3) stained blood cells obtained from

control fish and AB1 fed fish. The graph illus-

trates the percentage cell populations within

whole blood samples. R1 are erythrocytes and

R2 are leucocytes. R3 is the total blood cells.



ª 2007 The Authors



antibacterial activity by attacking peptidoglycan in the cell

wall of bacteria, predominantly Gram-positive bacteria,

thereby causing lysis and stimulation of phagocytosis of

bacteria by phagocytic cells (Ellis 1990). An increase in

the lysozyme concentration in fish blood can be caused

by infections or invasion by foreign material (Siwicki

et al. 1998). Certainly, B. subtilis AB1 influenced the pro-duction of higher levels of lysozyme activity in fish serum

and gut mucus. This increase may have also contributed

to the survival of fish challenged with the pathogen.

Panigrahi et al. (2005) and Kim and Austin (2006) have

similarly demonstrated increases in gut and serum lyso-

zyme in fish fed with L. rhamnosus and Carnobacterium

maltaromaticum B26 and Carnobacterium divergens B33,

respectively. It can be suggested, however, that fish fed

with AB1 resulted in increased serum and gut lysozyme,

which enhanced the immune efficiency of fish to with-

stand challenge with the pathogen.

Bacterial pathogens produce proteolytic enzymes to aid

in the breakdown of host tissues, but protease inhibitors

may be present in sera and other body fluids (Bowden

et al. 1997). These inhibitors also serve in the homeosta-

sis of body fluids, and are involved in acute phase reac-

tions and in defence against pathogens that secrete

proteolytic enzymes (Magnadottir 2006). Fish plasma

contains a number of protease inhibitors, principally

a1-antiprotease, a2-antiplasmin and a2-macroglobulin,

which have been demonstrated to have a role in restrict-

ing the ability of bacteria to survive in vivo (Ellis 2001).

Although several studies have investigated antiprotease

levels in fish species, particularly a2-macroglobulin

activity (Bowden et al. 1997), there is negligible informa-tion concerning modulation in fish other than by infec-

tion. It was also significant that AB1 induced higher

levels of total, a1-antiprotease and a2-antiprotease inhibi-

tors in the probiotic-fed fish as compared with the con-

trols. Similarly, Vasudeva Rao and Chakrabarti (2005)

reported significantly higher total and a1-protease levels

in carp (Catla catla) administered with feeds supplemen-

ted with herbs. These authors noted that the best func-

tion of the a2-macroglobulin antiproteases family

concerns with the clearance of active proteases from tis-

sue fluids. The results of this study indicate that feeding

rainbow trout with AB1 enhanced nonspecific factors

of the immune system by enhancing the level of natural

antiproteases in the serum. Possibly, these may have

provided some defence against infection by the pathogen.

Previously, Mihal et al. (1990), Nikoskelainen et al.

(2003) and Panigrahi et al. (2007) demonstrated that pro-

biotics induced significant and positive effects on comple-

ment levels. In the present study, differences in

complement levels were not statistically significant when

compared with the controls.

Overall, the present study reinforces the view that bac-

terial cultures may well contribute to disease control

strategies in aquaculture. Certainly, AB1 was effective in

preventing disease caused by highly virulent Aeromonas

sp. in rainbow trout.

Acknowledgements

This study was supported by the Commonwealth Split

Site studentship, the Postgraduate Research Fund of The

University of the West Indies and Scalar Scientific and

Technical Supplies of Trinidad and Tobago.

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