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was subsequently quantified by Makridis et al. (2000b) using immunocolony blot (ICB) and
enzyme-linked immunosorbent assay (ELISA). The addition of 10 7 bacteria ml -1 in the rotifer
rearing tanks for a short period (20-30 min) led to a total dominance (100% of total CFU) of
the added bacteria and a load of 10 3 -10 4 bacteria rotifer −1 . Once transferred to seawater,
the probiotic persisted in the prey for 4 to 24 h. This study demonstrated that the bioencap-
sulation is highly dependent on several variables such as bacteria concentration, grazing rate
and presence of microalgae. Particle size is however the main factor for the initial grazing by
the rotifers, so the average cell size and the degree to which bacteria aggregate are the most
important factors (Makridis et al. 2000a).
In a first feeding trial with turbot larvae, Makridis et al. (2000b) reported that the bioen-
capsulation of strain 4:44 in rotifers resulted in probiotic population of the GI tract (up to 10 4
bacteria larva -1 ), indicating that the bioencapsulation is an effective way to deliver the pro-
biotic into larvae. However, it was also shown that the bacteria bioencapsulated into rotifers
were quickly released (in 2-4 h) from rotifers to the culture water and that the bacteria were
present in the water of the larval rearing tanks. This finding suggests that larvae may ingest
bacteria also by drinking. The presence of the probiotic did not modify total CFU levels in
larvae or improve larval survival and growth, but it could potentially improve resistance of
larvae against pathogens due to antagonistic activities (Makridis et al. 2000a).
The bioencapsulation of the probiotic Phaeobacter 27-4 in the rotifer Brachionus plicatilis
by three different protocols was monitored by Pintado et al. (2010) using culture-dependent
methods and DGGE. The results demonstrated a rapid (3 h) incorporation of the probiotic
by rotifers (10 2 CFU rotifer -1 ) and subsequent maintenance for 48 h, allowing the larvae to
graze on the rotifers and to incorporate the probiotic. DGGE fingerprints showed a shift of
bacterial communities of the rotifers due to temporal colonization by the probiotic. Planas
etal. (2006) fed turbot larvae on rotifers enriched with Phaeobacter 27-4 in parallel to induced
infections with the fish pathogen V.anguillarum , following model challenge trials (Planas et al .
2005). These authors demonstrated a clear in vivo probiotic effect, reducing the accumulated
mortality of the larvae to the level of the uninfected control larvae. The probiotic bacteria was
detected by immunohistochemistry in the intestine of turbot larva fed rotifers enriched with
Phaeobacter 27-4 (Figure 16.1). However, Phaeobacter 27-4 did not colonize turbot larvae
intestinal epithelia and continuous additions of the probiont were necessary to maintain an
effective concentration of the probiotic in seawater and rotifers.
Encouraging results in the reduction of mortality were also achieved in sea bream larvae
when Lactobacillus fructivorans (isolated from the sea bream gut) and Lb. plantarum (from
human faeces) were bioencapsulated in rotifers and Artemia (Carnevali et al. 2004). Both
strains were administered concomitantly (4:1 ratio) and specifically identified in larvae by
culture in selective medium (MRS) and biochemical tests (API 50 CHL). It was observed that
the non-autochthonous Lb.plantarum was prevalent during early stages (35 days post hatching
(dph)), indicating that the establishment of the gut microbiota occurs in a non-selective way.
The autochthonous Lb.fructivorans became predominant at the end of the experiment (90 dph),
probably due to the more favourable condition of the GI tract for colonization by that strain
at that developmental stage. These results were related to an immunostimulatory effect and
the development of gut-associated lymphoid tissue (GALT) (Picchietti et al. 2007). At 90 dph
probiotic- fed larvae showed a higher density of B lymphocytes and Ig + cells and acidophilic
granulocytes in the intestinal epithelium than the control fish. These results showed the impor-
tance of live feed supplementation with probiotics in early stages of larvae development. On
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