Short communication

Use of dead probiotic cells to control furunculosis

in rainbow trout, Oncorhynchus mykiss (Walbaum)
A Irianto
1
and B Austin
2
1 Faculty of Biology, Jenderal Soedirman University, Purwokerto, Indonesia
2 School of Life Sciences, Heriot-Watt University, Edinburgh, UK
Keywords: control, furunculosis, probiotic cells,
rainbow trout.
There has been increasing interest in the use of
probiotics in aquaculture (e.g. Douillet & Langdon
1994; Gildberg, Mikkelsen, Sandaker & Ringo
1997; Kennedy, Tucker, Neidic, Vermeer, Cooper,
Jarrell & Sennett 1998; DeSchrijver & Ollevier
2000; Robertson, ODowd, Burrells, Williams &
Austin 2000), and a diverse range of Gram-positive
(such as Carnobacterium inhibens) (Robertson et al.
2000) and Gram-negative bacteria, including Vibrio
alginolyticus (Garriques & Arevalo 1995), have been
evaluated at various times. Following previous suc-
cess with live probiotics (Irianto & Austin 2002a),
research has focused on the usefulness of inactivated
preparations for the control of furunculosis in
rainbow trout, Oncorhynchus mykiss (Walbaum).
Rainbow trout (average weight 1 and 15 g)
were obtained from commercial sh farms in
Scotland and maintained in aerated free owing
de-chlorinated fresh water at 12 C. The health
status was examined immediately upon arrival in
the aquaria and at 12 week periods thereafter
(after Austin & Austin 1989).
The source of the probiotics (unidentied Gram-
positive coccus A1-6, V. uvialis A3-47S, Aero-
monas hydrophila A3-51 and Carnobacterium
BA211) has been published previously (Irianto &
Austin 2002a). Stock cultures were freeze-dried and
stored at 70 C as suspensions in 20% (v/v)
glycerol. The organisms were grown in tryptone
soya broth (Oxoid, Basingstoke, UK), harvested by
centrifugation at 1000 g for 10 min at 4 C, and
suspended in 0.9% (w/v) saline to 10
10
cells mL
)1
followed by the addition of 40% (v/v) formalde-
hyde to 0.2% (v/v) with incubation at 20 C for
4 days. Inactivation was conrmed by the absence
of growth following the seeding of triplicate plates
of tryptone soya agar (Oxoid) with 0.1 mL volumes
of the cell suspension and incubation for 23 days
at room temperature. Volumes were mixed thor-
oughly in 100 g amounts of commercial dry
rainbow trout feed (Trouw, Glasgow, UK) to
achieve a dose equivalent to 10
7
bacterial cells per
gram of feed. Groups of 50 rainbow trout were fed
to satiation four times daily, for 14 days. Then,
rainbow trout fry were immersed for 5 min in a
suspension of 10
7
cells mL
)1
of a virulent culture
of A. salmonicida. Fingerlings were challenged by
cohabitation, in which rainbow trout infected
intraperitoneally with 10
6
cells of A. salmonicida
were added to groups of rainbow trout which had
been administered with the putative probiotic at a
rate of 10 infected rainbow trout per group of 50
sh. The rainbow trout were maintained for a
further 14 days and all sh were examined micro-
biologically for the presence of A. salmonicida.
Duplicate groups of sh were used to determine
the mode of action of the probiotics. The methods
for determining the presence of serum antibodies
and the number of erythrocytes and leucocytes in
blood have been described previously (Irianto &
Austin 2002a). Thus, blood was collected from
Journal of Fish Diseases 2003, 26, 5962
Correspondence Prof. B. Austin, School of Life Sciences,
John Muir Building, Heriot-Watt University, Riccarton,
Edinburgh EH14 4AS, UK
(e-mail: b.austin@hw.ac.uk)
59
2003
Blackwell Publishing Ltd
groups of 10 freshly killed sh by venepuncture
using 9 mL capacity Vacuettes containing heparin
(Greiner, Gloucester, UK). The kidneys were
removed from the same groups of 10 sh and used
to determine the number and phagocytic activity of
the macrophages (after Sakai, Kobayashi & Yoshida
1995). Micrococcus luteus (formerly M. lysodeikticus)
[NCIMB (National Collection of Industrial and
Marine Bacteria, Aberdeen, UK) 9278] was used for
the determination of lysozyme activity (Sarder,
Thompson, Penman & McAndrew 2001; Irianto &
Austin2002a). Freshly killed rainbowtrout fromeach
group were used to collect cutaneous and intestinal
mucus, which were used in a previously described
enzyme-linked immunosorbent assay (ELISA) to
determine the presence of antibodies to A. salmonicida
(Durbin 1997; Irianto & Austin 2002a).
The quantitative data were used to calculate
standard deviations, analysis of variance, and the
honestly signicance test (HST) (Steele & Torrie
1996), contained on the Minitab 13 Program
Package (Minitab, Coventry, UK).
With rainbow trout fry and ngerlings, there was
benet in using the formalin-inactivated bacterial
preparations for 14-day feeding regimes. Thus,
following challenge with A. salmonicida, 92% of the
fry died within 14 days compared with 18, 16, 16
and 70% of the sh administered with A1-6,
A3-47S, A3-51 and BA211, respectively (Table 1).
Using rainbow trout ngerlings, 60% of the
controls died after challenge with A. salmonicida
compared with 4, 4, 8 and 0% of the groups fed
with A1-6, A3-47S, A3-51 and BA211, respectively
(Table 1). With the exception of the group fed with
formalin-inactivated cells of A3-51, there was a
slight weight gain among the ngerlings compared
with the controls, although the situation regarding
the fry was less clear (Table 1).
The data did not reveal any serum or mucus
antibodies to A. salmonicida in ngerlings which
had received the formalin-inactivated bacterial
preparations, although more time may be necessary
for detectable antibody production. Thus, the
ELISA gave consistently negative values with all
the serum samples. However, compared with the
controls, there were increased numbers of erythro-
cytes especially after 7 and 21 days (Table 2),
although the analysis of variance indicated that
there was no signicant difference between the data
(P < 0.05). For example, the number of erythro-
cytes in the controls were 4.3 0.2 10
8
and
14.2 0.8 10
8
mL
)1
at 7 and 21 days, respect-
ively, whereas the corresponding counts for the
sh which had received inactivated cells of A1-6
were 9.1 0.6 10
8
and 23.6 7.5 10
8
mL
)1
(Table 2). A higher number of leucocytes was
recorded in sh fed with inactivated probiotics after
7 and 14 days, but less so at 21 days, i.e. 7 days
after the cessation of feeding with the dead bacterial
cells, compared with the controls (Table 2). Thus,
after feeding with dead probiotic cells for 7 days, the
number of leucocytes was 1.4 0.3 10
7
mL
)1
Table 1 Effectiveness of dead probiotic cells to control furun-
culosis in groups of 50 rainbow trout
Probiotic ref. no. Mortality (%)
Average weight
of survivors (g)
Rainbow trout fry
(initial average weight 1 g)
A1-6 18 1.74
A3-47S 16 1.92
A3-51 16 1.79
BA211 70 2.01
Control 92 1.86
Rainbow trout ngerlings
(initial average weight 15 g)
A1-6 4 27.6
A3-47S 4 27.8
A3-51 8 25.6
BA211 0 27.7
Control 60 25.9
Table 2 Average numbers of erythrocytes and leucocytes, and lysozyme activity in rainbow trout (average weight 15 g) after
administering dead probiotics for 14 days
Erythrocytes
( 10
8
mL
)1
)
Leucocytes
( 10
7
mL
)1
)
Lysozyme activity
( 10
3
units min
)1
)
Probiotic Day 7 Day 14 Day 21 Day 7 Day 14 Day 21 Day 7 Day 14 Day 21
A1-6 9.1 0.6 8.7 0.4 23.6 7.5 4.3 0.6 5.4 0.8 5.3 1.9 0.4 0.1 0.5 0.6 0.1
A3-47S 8.6 0.4 10.2 0.9 22.5 3.3 4.4 0.3 5.1 1.3 8.0 0.1 0.2 0.1 0.3 0.1 0.4 0.1
A3-51 10.3 2.3 11.5 0.7 26.9 1.5 4.2 0.6 5.4 0.1 6.7 0.2 0.4 0.1 0.5 0.1 0.5 0.1
BA211 10.9 1.4 11.9 1.5 27.1 2.0 3.7 0.7 7.7 2.1 8.0 0.4 0.5 0.1 0.5 0.1 0.5 0.1
Control 4.3 0.2 10.3 2.8 14.2 0.8 1.4 0.3 3.3 0.1 6.6 0.3 0.1 0.1 0.3 0.1 0.3 0.2
60
2003
Blackwell Publishing Ltd
Journal of Fish Diseases 2003, 26, 5962 A Irianto and B Austin Dead probiotic cells to control furunculosis
for the controls, compared with 4.3 0.6 10
7
,
4.4 0.3 10
7
, 4.2 0.6 10
7
and 3.7
0.7 10
7
mL
)1
for A1-6, A3-47S, A3-51 and
BA211, respectively (Table 2). Analysis of variance
(P < 0.01) and HST (P < 0.01) revealed sig-
nicant differences between the data. Although
there were increased levels of lysozyme activity after
7 days of feeding with dead probiotic cells (Table
2), the data were not statistically signicant by
analysis of variance (P < 0.05). In contrast, the
data for the number of macrophages was signicant
by analysis of variance (P < 0.01) and HST
(P < 0.01). Thus, compared with the controls,
with A1-6, A3-51 and BA211 there were enhanced
number of macrophages in the kidney (Table 3).
For example, 1 week after feeding sh with
dead cells of A3-51, i.e. at day 21, the number of
kidney macrophages was estimated as 14.7
1.6 10
8
g
)1
compared with only 2.6
0.8 10
8
g
)1
in the controls (HST, P < 0.01).
However, feeding with dead cells of A3-47S
led to reduced numbers of macrophages, i.e.
1.7 0.1 10
8
g
)1
, at day 21. Whereas it
appeared that use of A3-47S led to the greatest
proportion of dead macrophages (8.8 1.5%
compared with 6.2 2.7% in the controls), the
data were not signicant by analysis of variance
(P < 0.05). Nevertheless, statistical signicance by
analysis of variance (P < 0.01) and HST
(P < 0.05) was recorded for phagocytic activity
of A3-47S. With this organism there was least
phagocytic activity (73.3 2.7%) compared with
the controls (70.2 3.8%) (Table 3).
It is generally accepted that probiotics are live
microbial feed supplements that somehow have
benet for the host (Fuller 1987), although there
has been a virement in the actual denition (Irianto
& Austin 2002b). Yet, the current study has
revealed that formalin-inactivated cells, applied as
feed additives, have a benecial effect at controlling
furunculosis in rainbow trout. This raises an
interesting paradox concerning whether or not
inactivated cells should be regarded as probiotics
or even as oral vaccines. Notwithstanding, there is
clearly benet with using dead cells.
In comparison with the previous work with live
cells (Irianto & Austin 2002a), there was no
evidence for the development of serum or mucus
antibodies, but this is not so surprising in view of
the comparatively short time elapsing from uptake
on feed to challenge, i.e. 14 days. Nevertheless,
there was evidence of other cellular immunity
factors resulting from use of the dead bacterial cells.
Compared with the use of live cells for 14 days
(Irianto & Austin 2002a), there were less erythro-
cytes and kidney macrophages, smaller proportions
of dead macrophages and reduced phagocytic and
lysozyme activities following use of the formalized
preparations. However, the levels of these factors
were still greater than those of the controls. In
contrast, the number of leucocytes was much
greater than with live cells (Irianto & Austin
2002a). Certainly, the data suggest that cellular
rather than humoral immunity is a factor in
explaining the benet of these inactivated bacterial
cell preparations.
In conclusion, the four isolates were clearly
benecial for rainbow trout when administered
inactivated as feed additives. The comparatively
poor performance of BA211 in rainbow trout fry is
curious, and merits further examination. It remains
for large-scale trials to determine the usefulness of
these probiotics in eld conditions.
Acknowledgement
This study was supported by the Indonesian
Government through the Development of
Undergraduate Education Project to Jenderal Soe-
dirman University, Purwokerto.
Table 3 Average numbers of kidney macrophages in rainbow trout (average weight 15 g) after administering dead probiotics for 7
and 14 days
Macrophages in kidney ( 10
8
g
)1
) Percentage of dead macrophages Phagocytic activity (%)
Probiotic Day 7 Day 14 Day 21 Day 7 Day 14 Day 21 Day 7 Day 14 Day 21
A1-6 2.1 0.7 2.7 0.4 3.5 0.4 6.5 2.7 5.5 0.9 4.2 1.1 86.5 2.4 77.7 2.8 76.0 3.0
A3-47S 1.6 0.2 1.6 0.1 1.7 0.1 5.9 0.4 5.4 1.6 8.8 1.5 81.6 4.3 79.0 1.2 73.3 2.7
A3-51 7.1 0.1 11.4 0.4 14.7 1.6 2.4 0.9 5.3 0.8 5.2 0.5 80.6 2.7 84.8 2.4 75.0 2.4
BA211 2.2 0.1 2.6 0.1 2.9 0.3 5.9 2.6 6.9 2.0 4.5 2.3 73.0 1.1 75.4 0.8 73.8 0.9
Control 2.3 0.5 2.5 0.7 2.6 0.8 9.2 2.5 7.3 0.9 6.2 2.7 72.5 2.0 60.7 1.0 70.2 3.8
Journal of Fish Diseases 2003, 26, 5962 A Irianto and B Austin Dead probiotic cells to control furunculosis
61
2003
Blackwell Publishing Ltd
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Received: 27 March 2002
Accepted: 1 August 2002
Journal of Fish Diseases 2003, 26, 5962 A Irianto and B Austin Dead probiotic cells to control furunculosis
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2003
Blackwell Publishing Ltd