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period the control group showed percentages of
Lb. delbrueckii
subsp.
delbrueckii
lower than
32%, with the contemporaneous presence of other LAB species. In contrast, the application of
dietary lactobacilli initially isolated from the rumen of goat (
Lb. farciminis
CNCM MA27/6R
and
Lb. rhamnosus
CNCM MA27/6B) did not influence the cultivable heterotrophic bacterial
levels or LAB levels in the intestinal tract of European sea bass (Frouël
et al
. 2008).
Recently, the effects of the
Lb. rhamnosus
on the gut microbiota of a popular ornamental
species, the clownfish, was studied by Avella
et al
. (2010a). Avella and colleagues exposed
clownfish larvae and juveniles to
Lb. rhamnosus
ICM501 at 10
6
CFU ml
-1
by two methods:
via live preys and addition to rearing water (group 2) or via live preys only (group 3). GI
Lactobacillus
levels were estimated by qPCR; the microbiota of clownfish administered with
Lb. rhamnosus
was affected in both larval and juvenile stages in both treatment groups, as
evidenced by the changes of
Lb. rhamnosus
16S rDNA levels. On the contrary, in the clown-
fish control group (group 1; no addition of probiotics) no
Lb. rhamnosus
rDNA was detected.
Although this might not be so surprising, the interesting observation was that
Lb. rhamnosus
levels were significantly higher in treatment group 2 (probiotic provision via feed and rearing
water) than in group 3 (probiotic provision via feed only). As a marine species, clownfish have
an internal osmotic concentration lower than that of the surrounding seawater, and therefore
they drink large quantities of rearing water to replace water loss. The study suggests that addi-
tion of probiotics to the rearing water of marine fish, perhaps more so than that of freshwater
fish, may be a promising way to effectively boost probiotic intake of dietary sources. Although
this is likely difficult or unfeasible on large fish, particularly in sea cages or flow through sys-
tems, this method may be a viable alternative at the larval rearing stage where small scale,
controlled, recirculation facilities are used (refer to
Chapter 16
). Indeed, Avella
et al
. (2010a)
observed that the best probiotic effects on the host were observed in fish provided with
Lb.
rhamnosus
via both live preys and additions to the rearing water.
The zebrafish has become an important model for assessing the gut microbiota of verte-
brates (Rawls
et al
. 2004; 2006; Carnevali
et al
. 2013) and this model has also been used
to assess the efficacy of potential probiotic colonization and GI microbial modulation. In a
recent study, Zhou
et al
. (2012) tested the capabilities of 10
Lactobacillus
strains to adhere
to zebrafish intestine using partial
rpo
B gene DGGE analysis, clone libraries and scanning
electron microscopy. The 10 strains were simultaneously applied via the feed for a period of 4
weeks. The probiotic application led to significantly elevated lactobacilli levels in the GI tract
of probiotic treated fish (1.9 × 10
5
CFU g
-1
of gut) compared to those of the control fish (no
probiotic provision; 1.4 × 10
4
CFU g
-1
). The composition of the lactobacilli in the probiotic
group revealed that the adhesive capabilities of lactobacilli varied greatly;
Lactobacillus bre-
vis
CGMCC 1.2028,
Lactobacillus buchneri
BU 222,
Lb. plantarum
subsp.
plantarum
JCM
1149T,
Lb. rhamnosus
CICC 6141, and
Lb. rhamnosus
JCM 20300 were identified as highly
adhesive gut strains. Two further studies have also verified that
Lb. rhamnosus
(strain IMC
501
®
) has good capacity to populate the GI tract of zebrafish at multiple life stages (Avella
et al
. 2012; Gioacchini
et al
. 2012). Using qPCR Avella
et al
. (2012) verified the presence
of
Lb. rhamnosus
in the GI tract of zebrafish larvae (6 days post fertilization) and juveniles
(9 weeks post fertilization) after administration at 10
6
CFU g
-1
/ml
-1
via live feed and rearing
water. Gioacchini
etal
. (2012) fed adult zebrafish a commercial diet containing
Lb.rhamnosus
(10
6
CFU g
-1
) for 10 days. DGGE revealed the presence of
Lb. rhamnosus
in all replicates of
the probiotic group and subsequently these populations influenced the microbial communities.
DGGE fingerprints from the two treatment groups formed two clusters with approximately
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