International Journal of Aquaculture, 2016, Vol.6, No.2, 1
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9
2
The use of probiotics is widely applied in order to replace the use of chemotherapeutic agents in animal
production, and to restore the indigenous microbiota. In aquaculture systems, probiotics were described as “a live
microbial adjunct which has a beneficial effect on the host by modifying the host associated or ambient microbial
community, by ensuring improved use of feed or enhancing its nutritional value, by enhancing the host response
toward disease or by improving the quality of its ambient environment” (Verschuere et al., 2000), and later
defined for a wide variety of hosts (ISAPP, 2011). Several beneficial effects were reported for different aquatic
species: inhibitory activity against pathogens, nonspecific immune response stimulation, immunomodulatory
effect, decrease of mortality and increase of growth rate and production (Gullian et al., 2004; Vieira et al., 2010;
Nayak and Mukherjee, 2011; Rahman et al., 2011; Lakshmi et al., 2013).
Different bacterial genus, mainly those classified as lactic acid bacteria (LAB), including lactobacilli, enterococci
and related (Bifidobacteria) have been potentially evaluated as probiotics (Mego et al., 2005; Marcinakova et al.,
2006). Among enterococci, only
Enterococcus faecium
is included in most of the major probiotic products
commercially available for aquatic animal nutrition in the EU (Bogut et al., 2000; Chang and Liu, 2002; Vahjen et
al., 2007; Gatesoupe, 2008; Abumourad et al., 2014).
Pediococcus acidilactici
is a Food Grade Microorganism
(FGM) frequently isolated from dairy products. As they are included in the Qualified Presumption of Safety
(QPS) and found in a variety of ecosystems, are commonly used as probiotic for humans (Balgeri et al., 2013),
terrestrial (Giancamillo et al., 2008; Mikulski et al., 2012), and aquatic animals (Castex et al., 2010; Neissi et al.,
2013 and 2015; Giannenas et al., 2015; Ramos et al., 2015).
In previous studies
E. faecium
strains CRL 1937, CRL 1938, CRL 1940 and CRL 1941 and
P. acidilactici
strain
CRL 1939 were isolated from healthy specimens of
P. mesopotamicus
, selected as potentially probiotic based on
their “in vitro” beneficial properties and grouped into two formulae: LAB containing strains isolated in summer
(
P. acidilactici
CRL 1939 and
E. faecium
CRL 1940 and CRL 1941) and ENT containing enterococci isolated in
winter (
E. faecium
CRL 1937, CRL 1938) (Guidoli et al., 2015). As there are no previous descriptions of
autochthonous strains as probiotics in this fish species, the aim of this work was to evaluate the effect of these two
formulas on the survival, mean weight, biomass and histological parameters of
P. mesopotamicus
larvae. Another
purpose was to select the optimum doses and stages of administration of each formula for the design of a multi-
strain probiotic product.
2 Materials and Methods
2.1 Strains and formulae
Autochthonous
E. faecium
CRL1937, CRL1938, CRL1940 and CRL1941 and
P. acidilactici
CRL1939 strains
were isolated from diverse body areas of healthy
P. mesopotamicus
specimens during different seasons and
selected as potentially probiotic microorganisms based on their ability to express beneficial properties in
in vitro
tests (Table 1) (Guidoli et al., 2015). Strains were grouped into two formulae (LAB and ENT) according to their
characteristics and the season in which were isolated (Table 1). All the microorganisms were included in the
CERELA strain collection and in the Laboratorio de Sanidad Animal of the Estación Experimental Agropecuaria
Rafaela belonging to the Instituto Nacional de Tecnología Agropecuaria (INTA) under Budapest treaty for patent
aims (Conicet and Unne, 2013).
Table 1 Origins and beneficial properties of the potentially beneficial microorganisms assayed
Strain
Season Anatomical part Fish Weight (g) Beneficial properties
Formulae (Selection criteria)
E. faecium
CRL 1937 Winter Medium Intestine 195.25
Inhibition of pathogens and
food borne microorganisms
ENT (Selected enterococci
isolated in winter)
E. faecium
CRL 1938
P. acidilactici
CRL1939
Summer Hole
fish
homogenate
0.379
H
2
O
2
production
and
inhibition of pathogens and
food borne microorganisms
LAB (Selected Lactic Acid
Bacteria isolated in summer)
E. faecium
CRL 1941
0.451
E. faecium
CRL 1940
Gills
1,399
High hydrophobicity and
autoaggregation indexes
