Aquaculture 535 (2021) 736345

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Feed supplementation with quorum quenching probiotics with

anti-virulence potential improved innate immune responses, antioxidant
capacity and disease resistance in Asian seabass (Lates calcarifer)
Reza Ghanei-Motlagh a, Darioush Gharibi b, c, *, Takavar Mohammadian a, c, **,
Mohammad Khosravi b, Esmaeil Mahmoudi d, Mojtaba Zarea a, Simon Menanteau-Ledouble e, f,
Mansour El-Matbouli e
a
Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, 61357-831351, Ahvaz, Iran
b
Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, 61357-831351, Ahvaz, Iran
c
Member of Excellence Center of Warm Water Fish Health, Shahid Chamran University of Ahvaz, 61357-831351, Ahvaz, Iran
d
Department of Plant Protection, Faculty of Agriculture, Isfahan (Khorasgan) Branch, Islamic Azad University, 158-81595, Isfahan, Iran
e
Clinical Division of Fish Medicine, University of Veterinary Medicine, 1210 Vienna, Austria
f
Department of Chemistry and Bioscience, Aalborg University, DK-9220 Aalborg East, Denmark

A R T I C L E I N F O A B S T R A C T

Keywords: Application of multifunctional probiotics is considered to be a practical strategy for the control of infections caused
Quorum quenching probiotics by Gram-negative bacteria. Consequently, the present study was designed to investigate the possible inhibitory
Vibrio infection effects of quorum quenching (QQ) probiotics on several N-acyl-homoserine lactone (AHL)-associated virulence
Virulence factor
factors in Vibrio alginolyticus and V. harveyi in vitro, and to evaluate the impacts of their administration in the diet on
Innate immune response
non-specific immune responses, antioxidant capacity and resistance to V. alginolyticus in Asian seabass. For the in
Antioxidant capacity
Asian seabass vitro assay, the effect of exogenous AHL was assayed on the regulation of several virulence factors including lytic
enzymes (amylase, gelatinase, hemolysin, lecithinase, lipase and protease productions), swarming and swimming
motilities, and biofilm formation in V. alginolyticus and V. harveyi. Exogenous 3-oxo-C10-HSL positively regulated the
production of amylase, gelatinase, protease and biofilm in V. alginolyticus. Exogenous 3-oh-C4-HSL positively
regulated swarming, swimming and biofilm formation in V. harveyi. To determine the virulence-inhibitory effects of
QQ probiotics on the AHL-dependent virulence factors, cell-free lysates were obtained from Bacillus thuringiensis QQ1
and B. cereus QQ2. Both probionts could reduce the production of amylase, gelatinase and protease in V. alginolyticus.
Bacillus thuringiensis QQ1 was also able to decrease the biofilm formed by V. alginolyticus. Bacillus thuringiensis QQ1
and B. cereus QQ2 decreased swarming and swimming motilities, and biofilm formation in V. harveyi. In order to
assay the effectiveness of the QQ probiotics in vivo, Asian seabass were fed with basal diet (control) and basal diet
supplemented with 109 CFU/g feed of B. thuringiensis QQ1 or B. cereus QQ2 for 8 weeks. At the end of pre-infection
trial (day 42), catalase (CAT), superoxide dismutase (SOD), lysozyme, antiprotease, myeloperoxidase (MPO), res­
piratory burst (RB) and bactericidal activities were significantly (p < 0.05) higher in the probiotic-treated fish than
un-treated fish. After challenge with V. alginolyticus, significantly (p < 0.05) higher levels of glutathione (GSH) in the
QQ2 treatment (day 49) and lower levels of malondialdehyde (MDA) in both probiotic groups were observed
compared to the control group. Moreover, CAT, MPO, RB, alternative pathway of complement (APC), lysozyme,
antiprotease and bactericidal activities were significantly (p < 0.05) higher in the probiotic treatments compared to
the control group after 7 and 14 days post-infection. Both probionts significantly (p < 0.05) improved the cumulative
survival in Asian seabass against V. alginolyticus. Our results indicate that QQ probiotics can modulate immune and
antioxidant responses, and provide high protection against Vibrio infection in Asian seabass.

* Corresponding authors at: Darioush Gharibi, Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, 61357-831351,
Ahvaz, Iran.
** Correspondence to: Takavar Mohammadian, Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, 61357-
831351, Ahvaz, Iran.
E-mail addresses: d.gharibi@scu.ac.ir (D. Gharibi), t.mohammadian@scu.ac.ir (T. Mohammadian).

https://doi.org/10.1016/j.aquaculture.2021.736345
Received 1 September 2020; Received in revised form 26 November 2020; Accepted 1 January 2021
Available online 5 January 2021
0044-8486/© 2021 Elsevier B.V. All rights reserved.
R. Ghanei-Motlagh et al.
1. Introduction
Outbreaks of diseases caused by bacteria belonging to the family
Vibrionaceae have resulted in serious economic losses in marine aqua­
culture (Wu et al., 2020). During the past decades, the occurrence of
infections caused by Vibrio spp. and V. alginolyticus and V. harveyi in
particular has dramatically increased in aquaculture, in association with
the intensification of rearing systems, global warming and climate
change (Vezzulli et al., 2013). In addition, metabolic versatility of these
bacteria has contributed to their abundance, invasion and pathogenicity
(Montánchez et al., 2019). Disease and mortalities attributed to
V. alginolyticus and V. harveyi have been reported in marine cultured fish
including Asian seabass (Lates calcarifer), European seabass (Dicen­
trarchus labrax), gilt-head seabream (Sparus aurata), turbot (Scoph­
thalmus maximus), Senegal sole (Solea senegalensis), groupers
(Epinephelus spp.), yellow croaker (Pseudosciaena crocea) and common
seahorse (Hippocampus kuda) (Zorrilla et al., 2003; Krupesha Sharma
et al., 2012; Liu et al., 2016; Mohamad et al., 2019b; Zuo et al., 2019; Xie
et al., 2020; Wu et al., 2020; Zhang et al., 2020).
The major virulence factors involved in the pathogenicity of Vibrio
spp. belonging to the Harveyi clade include lytic enzymes, extracellular
polysaccharides, biofilm formation, secretion systems, adhesion factors,
siderophores and several bacteriophage-encoded factors (Aguirre-
Guzmán et al., 2004; Darshanee Ruwandeepika et al., 2012; Gu et al.,
2016; Zhang et al., 2019). However, previous studies have revealed that
the presence of virulence genes is not necessarily correlated with the
pathogenicity of V. alginolyticus and V. harveyi (Kahla-Nakbi et al., 2009;
Ruwandeepika et al., 2010). On the other hand, it has been demon­
strated that quorum sensing (QS) is conserved in Vibrio spp. within the
Harveyi clade (Ruwandeepika et al., 2010; Mohamad et al., 2019a). In
Gram-negative bacteria, QS is mostly mediated by small molecules
called Autoinducer-1 (or AHL) and Autoinducer-2 (AI-2). N-acyl-
homoserine lactones (AHLs) are a major class of QS signals and play key
roles in colonization, survival, invasion and pathogenesis of Vibrio spp.
(Liu et al., 2018). Despite many studies conducted on the regulation of
several virulence factors by QS in Vibrio spp. belonging to the Harveyi
clade, the direct effects of AHLs on control of QS-associated virulence
factors has not yet been well investigated in V. harveyi and
V. alginolyticus. Detection of autoinducers in V. harveyi is performed by
cognate membrane-bound receptors at the cell surface (Ng and Bassler,
2009). At a threshold concentration, autoinducers bind to their re­
ceptors which activates the production of the LuxR protein which acti­
vates or represses the transcription of several QS-related genes such as
those associated with virulence (Rutherford et al., 2011). In
V. alginolyticus, the QS signaling system has been reported to be similar
to that of V. harveyi and the master regulator LuxR play crucial roles in
regulation of various QS-associated genes at high cell density (Wang
et al., 2007; Rui et al., 2008; Tian et al., 2008b; Ye et al., 2008). LuxT (a
QS regulator different from that of V. harveyi) and VqsA (Vibrio quorum
sensing activator) have also been identified as the other pivotal QS
regulators in V. alginolyticus (Liu et al., 2012; Gao et al., 2018; Zhang
et al., 2019).
Generally, adoption of strict biosecurity protocols and vaccination is
the most efficient method to prevent bacterial diseases in aquaculture
(Ina-Salwany et al., 2019). However, implementing many of the bio­
security measures routinely applied in land-based aquaculture systems is
not practically possible in sea-based rearing systems. Additionally,
production of cross-protective vaccines against marine vibriosis that
could be effectively used in local fish farms has been limited (Li et al.,
2016). In recent years, use of dietary immunostimulants has also
emerged as an effective and eco-friendly method for prophylaxis and
treatment of bacterial diseases such as vibriosis in aquaculture farms
(Wang et al., 2017a). On the contrary, antibiotics remain the most
common compounds used in the treatment of bacterial infections.
Nevertheless, the resistance of Vibrio pathogens to a wide spectrum of
antibiotics and the adverse effects of antibiotics on host, environment
2
Aquaculture 535 (2021) 736345
and humans have hindered the application of antibiotics for the control
of vibriosis in aquaculture and reduce the sustainability of this practice
(Defoirdt et al., 2004; Nhan et al., 2010). Regarding the involvement of
AHL signals in the regulation of various virulence-associated pheno­
types, the intervention measures associated with inhibition of AHL
synthesis, degradation of AHL and prevention of AHL-receptor interac­
tion have been investigated in previous studies (Liu et al., 2018). The QQ
strategy, among others, has been demonstrated as a practically anti-
virulence method for control of fish pathogens (Chu et al., 2014; Chen
et al., 2020a, 2020b). Moreover, in contrast with the harsh selective
pressure imposed by antibiotics during treatment of bacterial infections,
QQ bacteria are believed to exert less selective pressure and attenuate
the virulence of pathogens without negative effect on their growth
(Defoirdt, 2018).
Over the last two decades, application of probiotic strains belonging
to the genus Bacillus and in particular spore-forming ones has been
increasingly adopted in aquaculture (Soltani et al., 2019). In our pre­
vious studies, Bacillus thuringiensis QQ1 and B. cereus QQ2 showed high
degrading activities against AHLs associated with several pathogenic
Vibrio spp. in fish. The probiotic potential of these strains was also
demonstrated using in vitro and in vivo assays (Ghanei-Motlagh et al.,
2020a; Ghanei-Motlagh et al., 2020b). In the present study, we inves­
tigated the potential of these autochthonous QQ probiotics against
several virulence-associated phenotypes regulated by AHL-related QS in
V. alginolyticus and V. harveyi in vitro, and evaluate their efficacy as
modulators of immune and antioxidant responses in Asian seabass
before and after experimental infection with V. alginolyticus. The find­
ings presented here suggest that QQ probiotics have high potential to
control marine vibriosis in aquaculture. Indeed, Bacillus strains with QQ
activity not only can attenuate the virulence of Gram-negative patho­
gens but also have the potential to stimulate the host defense systems,
and therefore their application can confer high levels of protection.
2. Materials and methods
2.1. Effect of exogenous AHL on several virulence-associated phenotypes
of V. alginolyticus and V. harveyi
2.1.1. Virulent pathogens, synthetic AHLs and bacterial culture
The pathogens used in this study, V. alginolyticus RT45GH and
V. harveyi SB9612N4, were isolated from diseased Asian seabass reared
in floating cages, Iran. Vibrio alginolyticus RT45GH was identified as
described in Section 2.5 and used for both in vitro and in vivo experiments
carried out in this study. Identification of V. harveyi SB9612N4 was done
using genus- and species-specifc genes in a previous study (Ghanei-
Motlagh et al., 2020a), and the pathogen was used for in vitro tests
conducted in the present research. In our previous study (Ghanei-Mot­
lagh et al., 2020b), other Vibrio strains had been used, but in the present
study, it was important to use clinical isolates with known virulence
potential. Because these isolates had not previously been tested in regard
of their QS apparatus, a preliminary test was performed to explore and
confirm the AHL molecules produced by V. alginolyticus RT45GH and
V. harveyi SB9612N4. The thin layer chromatography method was
applied alongside screening using Agrobacterium tumefaciens NTL4
(pZLR4) as a biosensor to detect AHLs extracted from the pathogens,
according to a protocol that we had previously applied for other Vibrio
isolates (Ghanei-Motlagh et al., 2020b). Regarding the involvement of
AHL molecules in the regulation of certain virulence factors and biofilm
formation in Vibrio spp., the effect of exogenous AHL was investigated
on the activity of lytic enzymes (amylase, gelatinase, hemolysin, leci­
thinase, lipase and protease), swarming and swimming motilities as well
as biofilm formation in the pathogens. In this study, N-(3-Oxodeca­
noyl)-L-homoserine lactone (3-oxo-C10-HSL) and N-[(RS)-3-Hydrox­
ybutyryl]-L-homoserine lactone (3-oh-C4-HSL) (Sigma-Aldrich,
Germany) were used with respect to the AHL generated by the strains
V. alginolyticus RT45GH and V. harveyi SB9612N4, respectively. Each of
R. Ghanei-Motlagh et al.
the synthetic AHLs was dissolved in 0.3 mL of absolute ethanol (Merck,
Germany), and then diluted in 4.7 mL of distilled water (pH 6.6) to
prepare a stock solution containing 1 mg/mL (Defoirdt et al., 2011).
Vibrio alginolyticus and V. harveyi were separately grown in marine
broth (MB 2216, BD Difco, USA) medium at 28 ◦ C for 12 h at 150 rpm.
The cultures were diluted in sterile MB and adjusted to an optical density
of 0.25 at 600 nm (OD600) using a Biophotometer (No 6131, Eppendorf,
Germany). Subsequently, a total of 5 μL of the diluted cultures were
gently spotted in the center of different agar plates (Natrah et al., 2011).
The plates were incubated at 28 ◦ C according to the respective incuba­
tion periods for each assay. The diameter of the colonies and the zones
around the colonies was measured, and the ratio was calculated (Latorre
et al., 2016). All assays were repeated at least in triplicate.
2.1.2. Effect of exogenous AHLs on production of lytic enzymes
Standard plates (80 mm × 15 mm) were used for lytic enzymes assay.
Amylase activity was detected on starch medium consisting 10 g soluble
starch, 4 g casein peptone, 20 g NaCl and 15 g agar per liter (final pH
6.6). Following the incubation of the inoculated plates for 4 days, they
were covered with 5 mL of Gram’s iodine solution and the clear halo
around the colonies were developed and measured (Reda et al., 2018b).
Gelatin hydrolysis was observed in Luria-Bertani (LB, Merck, Darmstadt,
Germany) medium containing 10 g tryptone, 5 g yeast extract, 30 g
gelatin (Sigma-Aldrich), 20 g NaCl and 15 g agar per liter (Khan et al.,
2019). After inoculation, the plates were incubated for 3 days, and then
were cooled at 4 ◦ C for 5 h. The turbid halos around the colonies were
observed and the diameter of the halos was measured. Marine agar (MA
2216, BD Difco, USA) supplemented with 5% defibrinated sheep blood
(SR0051D, Oxoid, UK) was used for determination of hemolytic activity
(Bunpa et al., 2016). The inoculated blood agar plates were incubated
for up to 4 days and the diameters of the clear zones were measured.
Lecithinase (phospholipase) activity was examined using MA supple­
mented with 2% raw egg yolk (v/v) (Hörmansdorfer et al., 2000). After
inoculation, the lecithinase assay plates were incubated for 5 days and
the opalescent zones surrounding the colonies were measured. Lipase
activity was assessed using T medium composed of 1% tryptone, 1%
Tween 80 (Sigma-Aldrich), 1 mM CaCl2.2H2O, 2% NaCl and 1.5% agar
(Ghanei-Motlagh et al., 2020b). The inoculated plates were incubated
up to 7 days and opaque halos around the bacterial colonies were
recorded. Casein hydrolysis (protease activity) was observed in skim
milk medium containing 100 g skim milk powder (Merck, Germany), 20
g NaCl and 15 g agar per liter (Reda et al., 2018b). The inoculated petri
dishes were subsequently incubated up to 5 days and the transparent
halos around the colonies were measured. The media used for lytic
enzyme activities were supplemented with 5 mg/L of 3-oxo-C10-HSL or
3-oh-C4-HSL dissolved in the solvent (Defoirdt et al., 2011). Control
plates were supplemented with the same volume (125 μL) of the solvent
without AHLs. The final pH of all media (supplemented with or without
AHL) were adjusted to 6.6 using PIPES buffer (Sigma-Aldrich). Supple­
mentation with AHL was carried out when the temperature of auto­
claved media reached about 40 ◦ C.
2.1.3. Effect of synthetic AHLs on swarming and swimming motilities
Swarming motility assay was carried out on tryptic soy broth (TSB,
Merck, Darmstadt, Germany) medium supplemented with 0.75% agar
and 2% NaCl (Daniels et al., 2004). TSB medium containing 0.3% agar
and 2% NaCl was used for measurement of swimming motility (Rui
et al., 2008). The components of swarming and swimming media were
autoclaved at 121 ◦ C for 15 min, cooled and poured in large Petri plates
(145 mm × 20 mm). After inoculation, the swarming and swimming
plates were incubated in upright position up to 24 h and the diameter of
bacterial migration surrounding the inoculation site was recorded. The
media used to assess flagellar motility were supplemented with 5 mg/L
of 3-oxo-C10-HSL or 3-oh-C4-HSL dissolved in the solvent (Defoirdt et al.,
2011). Control plates were supplemented with the same volume (200
μL) of the solvent without AHLs. The final pH of swarming and
3
Aquaculture 535 (2021) 736345
swimming media (supplemented with or without AHL) were adjusted to
6.6. Synthetic AHLs were added to the media when the temperature
reached about 40 ◦ C.
2.1.4. Effect of AHL treatment on biofilm formation
The effect of exogenous AHL on biofilm formation of V. alginolyticus
and V. harveyi was assessed in 96-well polystyrene microtiter plate as
previously described with modification (Mizan et al., 2016). The path­
ogens were separately grown overnight at 28 ◦ C in sterile TSB supple­
mented with 2% NaCl. The bacterial cultures were diluted and adjusted
to an OD600 of 1 in fresh TSB. Subsequently, a total of 10 μL of the
adjusted suspensions were inoculated into the wells pre-filled with 200
μL sterile TSB (buffered) and 200 μL sterile TSB containing 3-oxo-C10-
HSL (for V. alginolyticus) or 3-oh-C4-HSL (for V. harveyi) at final con­
centration of 10 μmol/L in six replications and three independent assays
(Liu et al., 2017). The blank wells were filled with 200 μL of sterile TSB.
The plates were statically incubated in a wet chamber at 28 ◦ C for 36 h,
and the planktonic cells were transferred to a new flat-bottomed
microtitre plate. Afterwards, the OD600 was measured using a micro­
plate reader (ELx800, BioTek, USA). The wells were rinsed twice with
250 μL of phosphate buffered saline (PBS) to remove the non-adherent
bacteria. The attached cells were fixed with 250 μL per well of abso­
lute methanol for 30 min and the fixator was discarded. The plates were
dried at 60 ◦ C for 45 min, stained with 220 μL of 0.5% filtered crystal
violet for 15 min and submerged in a tub of water to remove the excess
dye. Thereafter, the plates were air-dried and the stain attached to the
adherent cells was solubilized with 200 μL per well of absolute ethanol
at room temperature for 10 min. The stain extracted with ethanol (200
μL) was pipetted in a new flat-bottomed microtitre plate and the OD590
was measured. Biofilm formation index (BFI) was calculated using the
formula, BFI = (A - B) / (C - D), where A is the OD590 of the stained test
wells, B is the OD590 of the stained blank wells, C is the OD600 of the
wells containing the planktonic cells in TSB and D is the OD600 of the
blank wells containing TSB.
2.2. The impact of AHL degradation on AHL-associated virulence
phenotypes of V. alginolyticus and V. harveyi
2.2.1. QQ activity and preparation of cell-free lysates from QQ probiotics
In our previous study, B. thuringiensis QQ1 and B. cereus QQ2 showed
potent AHL-degrading activity by production of cell-bound QQ lacto­
nase. The QQ activity of both strains against synthetic 3-oxo-C10-HSL
and 3-oh-C4-HSL, and crude AHL extracted from V. harveyi SB9612N4
was confirmed previously (Ghanei-Motlagh et al., 2020a; Ghanei-Mot­
lagh et al., 2020b). In this study, the potential of both probionts against
the crude AHL extract obtained from V. alginolyticus RT45GH was
investigated using the agar well diffusion method, as described else­
where (Ghanei-Motlagh et al., 2020b). In order to prepare the cell-free
lysate from QQ strains, 500 mL of 24 h cultures of B. thuringiensis QQ1
and B. cereus QQ2 in TSB supplemented with 2% NaCl were separately
dispensed in 50 mL conical centrifuge tubes and centrifuged at 4000 g
for 20 min (Eppendorf Centrifuge 5810 R, Hamburg, Germany). The
supernatants were discarded and the obtained pellets were washed twice
with sterile PBS (1×, pH 7) and resuspended in 30 mL of the same buffer.
The bacterial suspensions were intermittently sonicated in a cold water
bath at a frequency of 20 kHz for 10 min (Bandelin Sonopuls HD 2070,
Berlin, Germany) and centrifuged at 4000 g for 30 min at 4 ◦ C. The
supernatants were filter-sterilized using 0.2 μm syringe filters and the
protein concentration of cell-free lysates was quantified and adjusted to
50 mg/mL by the Pierce protein assay kit (Thermo Scientific, USA),
using bovine serum albumin (BSA) as standard (Torres et al., 2016).
2.2.2. Effects of cell-free lysates on production of lytic enzymes, flagellar
motility and biofilm formation
The effect of cell-free lysates containing QQ lactonase was investi­
gated on the inhibition of virulence phenotypes that were positively
R. Ghanei-Motlagh et al.
regulated by exogenous AHL. In order to determine the effect of cell-free
lysates on lytic enzymes (amylase, protease and gelatinase), swarming
motility and swimming motility, the respective media were sterilized at
121 ◦ C for 15 min and cooled in a water bath (45 ◦ C). The plates used for
lytic enzymes assays were prepared using 25 mL of the respective media
(as control) or 20 mL of the corresponding media overlaid with 5 mL of
double strength media and cell-free lysates (from B. thuringiensis QQ1
and B. cereus QQ2) in similar ratio (v/v). The plates applied for
swarming and swimming assays were prepared using 40 mL of the
respective media (as control) or 30 mL of the appertaining media
overlaid with 10 mL of double strength media and cell-free lysates in
equal proportion (v/v). Subsequently, a total of 5 μL of the adjusted
cultures of V. alginolyticus or V. harveyi (OD600 of 0.25) were inoculated
into the middle of the test plates. The incubation conditions were similar
to those described in Sections 2.1.2 and 2.1.3.
The impact of cell-free lysates of QQ probiotics on biofilm formation
of tested V. alginolyticus and V. harveyi was investigated in microtiter
plates (Raafat et al., 2019). Biofilm assays were performed in three in­
dependent experiments. The wells were initially filled with 100 μL of
double strength TSB (pH 7). Subsequently, a total of 100 μL of lysate
B. thuringiensis QQ1, lysate B. cereus QQ2 or sterile PBS (as blank) were
added to the wells in six replications. After inoculation with 10 μL of
diluted suspensions of V. alginolyticus or V. harveyi (OD600 of 1), the
plates were incubated without agitation at 28 ◦ C for 36 h. Biofilm for­
mation was quantified using crystal violet assay and biofilm formation
index (BFI) was calculated as described in Section 2.1.4.
2.3. Preparation of experimental diets and fish maintenance
A commercial feed formulated for marine fish (EX-MF1, Beyza Feed
Mill, Shiraz, Iran) was used as basal diet (crude protein, 47%; crude
lipid, 18%; crude fiber, 2%; ash, 14%; total phosphorus, 1.1%; digestible
energy, 4200 kcal/kg feed; moisture, < 12%; pellet diameter, 3.5–4
mm). Before preparation of the experimental diets, a preliminary test
was performed to estimate the actual numbers of the viable probiotic
cells inoculated to feed. Bacillus thuringiensis QQ1 and B. cereus QQ2
were individually inoculated to TSB supplemented with 2% NaCl and
the media were incubated at 28 ◦ C for 36 h at 150 rpm. The bacterial
cultures were pelleted down by centrifugation at 3500 g for 20 min and
the supernatants were discarded. The harvested cells were washed twice
with sterile PBS, resuspended in the same buffer and thoroughly ho­
mogenized by vortexing. The optical density of the bacterial suspensions
was measured and adjusted to an OD600 of 2.5 and serial dilutions were
prepared from the suspensions to calculate the number of viable bacteria
for each dilution using the spread plate method. The appropriate volume
(3 mL per 30 g feed) of the desired dilution (1 × 1011 CFU/mL) was
gently sprayed on the diets. The diets were subsequently coated using a
solution containing 1% gelatin in sterile PBS (2 mL per 30 g feed) to
stabilize the viable probiont cells in feed (Kiran et al., 2020). Prior to and
after coating with gelatin, sprayed diets (5 g) were dried and then ho­
mogenized in sterile PBS (45 mL). The obtained suspensions were seri­
ally diluted in the same buffer to calculate the CFUs on tryptic soy agar
(TSA) medium supplemented with 2% NaCl. This allowed to adjust the
final concentration of probiotic suspensions at 109 CFU/g feed. Subse­
quently, three experimental diets were prepared (twice per week to
maintain optimal freshness of the feed): basal diet containing
B. thuringiensis QQ1 at 109 CFU/g feed (QQ1 group), basal diet con­
taining B. cereus QQ2 at 109 CFU/g feed (QQ2 group) and basal diet
sprayed with sterile PBS (control group). After spraying with gelatin, the
experimental diets were dried at room temperature and stored at 4 ◦ C in
ziplock plastic bags until used (Mohammadian et al., 2018).
Asian seabass (50 ± 0.5 g) were obtained from a local farm (Bushehr
province, Iran). A total of 10 fish were randomly selected and eutha­
nized using 400 mg/L of tricaine methanesulfonate (MS-222, Sigma-
Aldrich, Germany) (Priborsky and Velisek, 2018). After a preliminary
physical inspection showing them to be healthy on appearance and
4
Aquaculture 535 (2021) 736345
presenting no external signs of infection, randomly selected fish were
examined using wet mount microscopy before sampling of their
anterior-kidney and brain for plating on TSA media supplemented with
2% NaCl. These microbiological samples were found negative following
incubation of the media at 29 ◦ C for 48 h. The healthy fish were accli­
matized to the experimental conditions for two weeks prior to the
commencement of experiment. After adaptation, a total of 375 fish were
arbitrarily divided into fifteen 300-L round fiberglass tanks (5 replica­
tions per treatment and 25 fish per tank) and fed ad libitum with assigned
diets thrice per day under a regular photoperiod regimen (16 L,8D) for 6
weeks. Feeding with the experimental diets was done during the trial
period (before and after experimantal infection). Fish tanks were pro­
vided with filtered and UV-treated running seawater, and water quality
parameters were maintained within a standard range as follows: tem­
perature- 27-30 ◦ C; dissolved oxygen- 9-10 mg/L; pH- 7.4-7.6; NO−2 -N-
< 0.1 mg/L; NH3-N- < 0.05 mg/L (Ghanei-Motlagh et al., 2020a).
2.4. Bacterial challenge and sample collection
Vibrio alginolyticus cultivated overnight in TSB supplemented with
2% NaCl was centrifuged at 4000 g for 10 min and the supernatant was
discarded. The bacterial pellet was washed using sterile PBS and
resuspended in the same buffer. The bacterial suspension was adjusted
to an OD600 of 1, and then 10-fold serial dilutions were prepared from
the adjusted suspension, followed by calculation of viable count of
V. alginolyticus (CFU/mL) by plating on TSA media supplemented with
2% NaCl. Subsequently, 0.1 mL of the dilutions containing 106 to 1010
CFU/mL of V. alginolyticus were injected intraperitoneally to Asian
seabass weighing 50 ± 7 g (15 fish per dilution in two replicates). After
injection, the mortalities were recorded daily over a period of 14 days
(Supplementary Table 1) and then the median lethal dose (LD50) of the
pathogen was determined by Probit analysis in the SPSS program
(version 22) (Mohammadian et al., 2019b). After feeding with the
experimental diets for 6 weeks, the fish from all replicates of each group
were intraperitoneally injected with 0.1 mL per fish of a bacterial sus­
pension pre-adjusted to 6 × 108 CFU/mL of viable V. alginolyticus (ac­
cording to the 14-days LD50).
Subsequently, three replicates were sampled for assessment of
immunological parameters and antioxidant enzymes. The remaining
two replicates were monitored twice daily for 2 weeks and mortalities
were recorded and removed. Blood and liver were sampled at days 42
(immediately before infection), 49 (7 days after infection) and 56 (14
days after infection) from the beginning of the experiment. Prior to
sampling, fish were starved for 24 h and then anesthetized with 2-phe­
noxyethanol (0.3 mL/L) (Ashouri et al., 2020). Blood samples were
obtained from the caudal vein (3 fish per replicate, 9 fish per treatment)
using a 3 mL syringe. One portion of the blood collected was transferred
into heparinized microtube in order to measure respiratory burst ac­
tivity (RBA). The other part was placed in sterile Eppendorf tube,
refrigerated at 4 ◦ C for 1 h and centrifuged at 10,000 g for 5 min at 4 ◦ C
(Mikro 220R, Hettich, Germany). The sera (supernatants) were subse­
quently separated and were kept at − 80 ◦ C for further analysis. At the
same time, three fish per replicate (9 fish per treatments) were dissected
under aseptic conditions and liver samples were removed, divided into
two portions and immediately stored at − 80 ◦ C until assayed.
2.5. Identification of V. alginolyticus isolated from head kidney
After the experimental challenge, dead fish were subjected to
microbiological examination and the cause of mortalities was confirmed
by re-isolation and identification of the bacteria from anterior kidney.
The total genomic DNA was extracted from the recovered bacteria as
previously described (Ghanei-Motlagh et al., 2020a). A duplex PCR
assay was used to amplify the genes 16S rRNA (Vibrio genus-specific
gene) and collagenase (V. alginolyticus species-specific gene) using the
primer sets V⋅16S-700F (5′ -CGGTGAAATGCGTAGAGAT-3′ ) and V⋅16S-
R. Ghanei-Motlagh et al.
1325R (5′ -TTACTAGCGATTCCGAGTTC-3′ ), and VA-F (5′ -CGAGTA­
CAGTCACTTGAAAGCC-3′ ) and VA-R (5′ -CACAACA­
GAACTCGCGTTACC-3′ ), respectively (Di Pinto et al., 2005; Tarr et al.,
2007). The reaction mixture and PCR conditions were similar to those
previously described (Ghanei-Motlagh et al., 2020a). The non-
parametric method Kaplan-Meier was employed to estimate the sur­
vival rates followed by the log-rank test for their pairwise comparisons
at 0.05 level of significance in the SPSS program. The present study was
approved by the institutional ethics committee of the Shahid Chamran
University of Ahvaz, Iran, under approval number EE / 98.24.3.71483 /
scu.ac.ir. All animal experiments in this study were followed according
to the guide for the care and use of laboratory animals by the National
Academy of Sciences (NIH publications No. 8023, revised 1978).
2.6. Antioxidant activity and lipid peroxidation assays
The first portion of liver specimens was individually homogenized in
a pre-cooled homogenization buffer (final pH 7.5, 5:1 v/w) composed of
potassium phosphate buffer (KH2PO4, 100 mM), phenyl­
methylsulphonyl fluoride (PMSF, 0.1 mM) and NaCl (1.8%) using a
tissue homogenizer (Heidolph instruments, Germany) (Regoli et al.,
2011). The homogenates were centrifuged at 10,000 g for 10 min at 4 ◦ C
(Mikro 220R, Hettich, Germany) and the supernatants were used for
measurement of antioxidant markers. The second portion of liver sam­
ples were separately homogenized in ice-cold phosphate buffer (0.1 M,
final pH 7.4, 10,1 v/w) and the resultant supernatants were utilized for
determination of malondialdehyde (MDA) concentration (Peixoto et al.,
2016). The protein content of the sera and hepatic extracts was
measured by the biuret method using a commercial kit (Pars Azmoon
Co., Tehran, Iran) and used to normalize the parameters related to
oxidative stress.
Catalase (CAT) activity was estimated according to the method
described by Koroliuk et al. (1988). Each of the samples was combined
with Tric-HCl buffer (0.05 mM, pH 7.8) containing hydrogen peroxide
(H2O2, 10 mM) and the mixture was incubated in a dark place at room
temperature for precisely 10 min. Thereafter, ammonium molybdate
(4%) was added to react with the remaining hydrogen peroxide (pro­
duction of a yellow complex) and terminate the reaction. The optical
density at 410 nm was measured against the blank and CAT activity in
the specimens of serum and liver was expressed in nmol/mg pro/min.
The activity of superoxide dismutase (SOD) was assayed as suggested
by Kono (1978). The reaction mixture was composed of the solution A
(50 mM sodium carbonate and 0.1 mM EDTA in distilled water, pH
10.2), Triton X-100 (0.6% in solution A), nitroblue tetrazolium (NBT, 60
μM in solution A), hydroxylamine hydrochloride (20 mM, pH 6) and the
sample (or solution A for the blank). The wells of a microtiter plate were
initially filled with the solution A (150 μL in blank wells, 130 μL in wells
used for liver extract and 140 μL in wells used for serum), NBT (30 μL),
Triton (20 μL) and the samples (20 μL of liver extract or 10 μL of serum).
Subsequently, hydroxylamine hydrochloride (50 μL) was added to the
wells to initiate the reaction (total volume of the reaction: 250 μL). The
optical density at 560 nm was measured immediately and after incu­
bation at 37 ◦ C for 5 min using a microplate reader (ELx800, BioTek,
USA). One unit of SOD is defined as the amount of enzyme that results in
50% inhibition of NBT reduction.
The concentration of reduced glutathione (GSH) was measured
following the method of Ellman (1959). Assay reaction buffer was
comprised of 0.1 M sodium phosphate and 1 mM EDTA (final pH 8).
Ellman’s reagent solution was prepared by dissolving 4 mg of 5,5′ -
dithio-bis-(2-nitrobenzoic acid) (DTNB, Sigma-Aldrich, Germany) in 1
mL of the reaction buffer and used for detection of sulfhydryl (thiol)
group present in GSH. Prior to determination of GSH content in the
samples, a stock solution (15 mM) containing cysteine (as a thiol-
containing compound) was prepared by dissolving 26.34 mg of
cysteine hydrochloride monohydrate (Sigma-Aldrich, Germany) in 10
mL of the reaction buffer. The solution was then diluted (tenfold) to
5
Aquaculture 535 (2021) 736345
prepare a standard stock solution (1.5 mM). The standard (St) and
sample tubes were initially filled with the required volumes of the re­
action buffer (2700, 2650, 2600, 2550, 2500 μL in St1, St2, St3, St4, St5
tubes and 2600 μL in sample tubes). Ellman’s reagent solution (50 μL)
was subsequently added to all tubes. In order to initiate the reaction, the
required volumes of standard stock solution (50, 100, 150, 200, 250 μL
in St1, St2, St3, St4, St5 tubes) and the samples (150 μL) were added to the
respective tubes. The blank tubes were filled with the reaction buffer
(2750 μL) and Ellman’s reagent solution (50 μL) without standard or
sample. The tubes were incubated at room temperature for 15 min to
develop a yellow colored product, and then the optical density at 412
nm was measured against the blank. A standard curve was constructed
by plotting the OD values obtained at 412 nm for the standards versus the
cysteine concentrations and used for the quantification of GSH content
in liver and serum samples.
The concentration of MDA in serum and liver specimens was
measured by the thiobarbituric acid reactive substances (TBARS) assay
(Ghanei-Motlagh et al., 2017). Briefly, each sample was treated with a
reagent containing the similar ratio of (1,1,1) thiobarbituric acid (TBA,
0.37%), hydrochloric acid (HCl, 0.25 N) and trichloroacetic acid (TCA,
15%). The reaction mixtures were placed in a water bath at 90 ◦ C for 15
min, cooled and centrifuged at 3000 g for 10 min at room temperature.
The optical density of the supernatants was measured at 535 nm against
the blank, and the concentration of MDA was calculated using the molar
extinction coefficient of MDA (156,000 M− 1 cm− 1) and expressed as
μmol/mg pro.
2.7. Immunological assays
The APC activity was determined using the spontaneous potential of
serum for lysis of rabbit red blood cells (RaRBCs). Briefly, blood
collected in Alsever’s solution was combined with veronal-buffered sa­
line (VBS, pH 7.4) solution in an equal ratio and centrifuged at 3000 g for
10 min at 4 ◦ C. The buffy coat was discarded, and the RBCs were washed
three times and diluted in VBS to prepare a 1.4% suspension (5 × 108
cells/mL). Fifty microliter of appropriate dilutions of serum, either
active or heat-inactivated (30 min at 56 ◦ C), was mixed with 350 μL of
VBS in Eppendorf tubes, and then 100 μL of the adjusted RaRBCs were
added and the reaction mixture was incubated at 24 ◦ C for 90 min with
gentle shaking. The ruptured and unlysed RBCs were pelleted down by
centrifugation at 10,000 for 5 min at 4 ◦ C and 200 μL of the supernatant
were pipetted into a microtiter plate. The optical density was measured
at 412 nm using a microtiter plate reader (M965/965+, Metertech Inc.,
Taiwan) and the difference between the absorbance of active and heat-
inactivated serum was reported as the APC activity in each sample
(Morgan, 2000).
Serum bactericidal activity against V. alginolyticus was measured in
flat-bottomed microtiter plate as previously described with modification
(Subramanian et al., 2008; Khosravi et al., 2018). Briefly, the sera were
diluted in VBS (1,2, v:v) and 50 μL of either the active or heat-
inactivated serum (from the same sample) were separately pipetted
into each well. Subsequently, 50 μL of the bacterial suspension (pre-
adjusted to 1 × 106 CFU/mL in VBS) were inoculated to the wells and the
plate was incubated at 24 ◦ C for 100 min. Thereafter, 100 μL of sterile
TSB containing 2% NaCl were added to the wells and the plate was
incubated at 28 ◦ C for 18 h. The optical density was measured at 600 nm
after 0 and 18 h incubation and the difference between the activity of
active and heat-inactivated serum was calculated and reported as the
bactericidal activity of each sample. The higher growth of the pathogen
in the wells (higher OD) was associated with the lower bactericidal ac­
tivity in the sample.
Serum lysozyme activity was quantified by the turbidimetric assay as
previously described (Mohammadian et al., 2019a). Briefly, Micrococcus
lysodeikticus (0.2 mg/mL, Sigma-Aldrich, Germany) was suspended in
sodium phosphate buffer (20 mM, pH 5.8), and 135 μL of the homoge­
neous suspension was placed in the wells of a 96-well microtiter plate
R. Ghanei-Motlagh et al.
and 15 μL of the serum samples were added. The absorbance was read at
450 nm every 2 min at room temperature for a total of 6 min. The results
were expressed as unit per mL and one unit of lysozyme activity was
corresponded to the amount of enzyme that results in a reduction of
0.001 units of absorbance at 450 nm per min.
In order to assess myeloperoxidase (MPO) activity, 15 μL of serum
was diluted in 135 μL of Hanks’ balanced salt solution (HBSS, modified
without Ca2+ and Mg2+) in 96-well plate. Subsequently, 25 μL of
3,3′ ,5,5′ -tetramethylbenzidine (TMB, 20 mM, Sigma-Aldrich) and 25 μL
of diluted hydrogen peroxide (H2O2, 5 mM, Sigma-Aldrich) were added
to each well. The reaction was stopped precisely after 2 min by adding
25 μL of diulted sulfuric acid (H2SO4, 4 M, Sigma-Aldrich) and the op­
tical density was measured at 450 nm in a microplate reader (Mohapatra
et al., 2014).
To determine antiprotease activity, 15 μL of a solution containing
trypsin at final concentration of 125 μg/mL (prepared from a 0.25%
standard trypsin solution, Kalazist Co., Iran; diluted in 50 mM Tris HCl,
pH 8.2) was incubated with 15 μL of serum in Eppendorf tube at room
temperature for 10 min. The tubes (in triplicate) containing Tris HCl (15
μL) + trypsin solution (15 μL) were used as negative control (absence of
antitrypsin activity). Subsequently, 500 μL of the substrate Nα-Benzoyl-
DL-arginine p-nitroanilide hydrochloride (BAPNA, 2 mM, homoge­
neously dissolved in 50 mM Tris HCl, pH 8.2) was added and the total
volume in the tubes was made up to 1 mL. After incubation of the tubes
for 30 min at room temperature, 200 μL of acetic acid (30%) was added
to terminate the reaction and the mixture was centrifuged at 3500 g for
10 min. Finally, 200 μL of the supernatants were pipetted into the wells
of a flat-bottomed microplate and the optical density was measured at
410 nm (Bowden et al., 1997). The blank was individually prepared by
addition of acetic acid to the mixture of trypsin + serum before adding
the substrate BAPNA (inhibition of yellow color development). The
percentage of trypsin inhibition was calculated using the following
equation:
Trypsin inhibition (%) = [(ODnegative control – ODsample)/(ODnegative
control)] × 100.
RBA in the heparinized blood samples was measured by NBT
reduction assay. Briefly, 0.1 mL of blood sample was combined with 0.1
mL of 0.2% NBT solution and the mixture was incubated for 30 min at
room temperature. Thereafter, 0.1 mL of the mixture was pipetted into a
glass tube containing 2 mL of N,N-Dimethylformamide (DMF), followed
by centrifugation at 3000 g for 5 min at room temperature. Finally, the
optimal density of the purple-blue supernatant was measured against
the blank (DMF) at 540 nm (Cha et al., 2013).
2.8. Statistical analysis
All data were analyzed using IBM SPSS Statistics 22 and expressed as
mean ± standard deviation (SD). The normality of the data was inves­
tigated by the Shapiro-Wilk test. One-way analysis of variance (ANOVA)
followed by Tukey’s post hoc test (for data obtained from the measure­
ment of immunological parameters and oxidative stress markers, and
those from the assay of each virulence factor in media with or without
QQ lysates) and independent student’s t-test (for data from the assay of
each virulence factor in the presence or absence of exogenous AHL) were
used to determine significant differences between the mean values. The
level of significance was considered at p < 0.05.
3. Results
3.1. AHL-producing activity and effect of exogenous AHL on tested
virulence factors of V. alginolyticus and V. harveyi
Fig. 1 represents the AHL produced by the pathogens on TLC plates
overlaid with A. tumefaciens NTL4. The production of 3-oxo-C10-HSL and
3-oh-C4-HSL was respectively detected in tested V. alginolyticus and
V. harveyi. As represented in Table 1 and Fig. 2, supplementation with
6
Aquaculture 535 (2021) 736345
synthetic 3-oxo-C10-HSL significantly (p < 0.05) increased lytic enzymes
production (amylase, gelatinase, protease) and biofilm formation in
tested V. alginolyticus. AHL treatment had no significant (p > 0.05) effect
on hemolysis activity of V. alginolyticus. Lecithinase and lipase pro­
ductions as well as swarming and swimming motilities were signifi­
cantly (p < 0.05) reduced in tested V. alginolyticus when cultured in
media supplemented with exogenous AHL. The impact of treatment with
synthetic 3-oh-C4-HSL on tested phenotypes related to V. harveyi is re­
ported in Table 1 and Fig. 2. Swarming motility, swimming motility and
biofilm formation were significantly (p < 0.05) increased after supple­
mentation with 3-oh-C4-HSL. Contrarily, lecithinase and lipase pro­
ductions were significantly (p < 0.05) decreased upon AHL treatment.
Moreover, no significant (p > 0.05) differences were observed for
gelatinase, protease, amylase and hemolysis activities in media supple­
mented with or without 3-oh-C4-HSL.
3.2. QQ activity and anti-virulence potential of the tested probiotics
Bacillus thuringiensis QQ1 and B. cereus QQ2 were able to degrade the
crude AHL extract obtained from V. alginolyticus RT45GH (Fig. 3). As
presented in Table 2 and Fig. 3, the productions of amylase, gelatinase
and protease by V. alginolyticus were significantly (p < 0.05) reduced in
media supplemented with lysates QQ1 and QQ2, although no significant
differences (p > 0.05) were observed between the lysates tested for this
potential. Biofilm formation by V. alginolyticus was significantly (p <
0.05) decreased in the presence of lysate QQ1 (Table 2). As shown in
Table 3 and Fig. 3, swarming motility, swimming motility and biofilm
formation were significantly (p < 0.05) decreased when V. harveyi was
cultured in media supplemented with lysates from QQ probiotics.
However, no significant (p > 0.05) differences were observed between
the lysates QQ1 and QQ2 for inhibition of the mentioned phenotypes
(Table 3 and Fig. 3).
3.3. Serum and liver oxidative stress indicators
The data in Table 4 describe the serum and liver antioxidant capacity
of Asian seabass fed with QQ probiotics prior to and after infection with
V. alginolyticus. After 6 weeks of feeding, serum and liver CAT activities
were enhanced (p < 0.05) in the probiotic groups compared to the
control group, while no significant (p > 0.05) difference was observed in
their levels between the probiotic treatments before infection. An
Fig. 1. AHL-producing activity of the pathogens. A-B: Visualization of AHL
extracted from V. alginolyticus RT45GH (A) and V. harveyi SB9612N4 (B) on TLC
plates overlaid with a thin layer agar containing the biosensor A. tumefaciens
NTL4. Comparison of the patterns of AHL extract from V. alginolyticus RT45GH
(lane 1) and V. harveyi SB9612N4 (lane 3) with those of synthetic standards 3-
oxo-C10-HSL (lane 2) and 3-oh-C4-HSL, respectively (lane 4).
R. Ghanei-Motlagh et al. Aquaculture 535 (2021) 736345

Table 1
Effect of exogenous AHLs on virulence factors of V. alginolyticus and V. harveyi.
Phenotype Vibrio alginolyticus Vibrio harveyi

Control Exogenous AHL AHL-QS regulation Control Exogenous AHLs AHL-QS regulation

Amylase 0.60 ± 0.10 2.58 ± 0.26* Positive 1.07 ± 0.07 1.02 ± 0.21 Unaffected
Gelatinase 1.45 ± 0.23 2.35 ± 0.05* Positive 4.39 ± 0.60 4.58 ± 0.36 Unaffected
Hemolysis 0.27 ± 0.03 0.28 ± 0.02 Unaffected 0.47 ± 0.04 0.48 ± 0.06 Unaffected
Lecithinase 0.48 ± 0.07 0.27 ± 0.01* Negative 0.48 ± 0.03 0.20 ± 0.01* Negative
Lipase 0.68 ± 0.07 0.23 ± 0.02* Negative 1.38 ± 0.35 0.57 ± 0.90* Negative
Protease 0.59 ± 0.03 1.30 ± 0.11* Positive 1.68 ± 0.15 1.79 ± 0.10 Unaffected
Swarming 11.57 ± 0.55 8.04 ± 0.35* Negative 3.92 ± 0.25 14.48 ± 0.23* Positive
Swimming 9.92 ± 0.15 6.65 ± 0.21* Negative 3.26 ± 0.26 14.37 ± 0.29* Positive
BFI 1.02 ± 0.10 1.90 ± 0.16* Positive 3.32 ± 0.26 11.98 ± 1.59* Positive

Asterisks show the significant (p < 0.05) differences between columns for each of the virulence factors of V. alginolyticus or V. harveyi (independent sample t-test). The
data were expressed as mean ± SD of three independent experiments. The values related to lytic enzymes assay were calculated by dividing the diameters of the zones
around the colonies by the diameters of the colonies in millimeter. The results of swarming and swimming motility assays were expressed in centimeter. BFI: biofilm
formation index.

Fig. 2. Effect of synthetic AHLs on virulence phenotypes. A: Effect of exogenous 3-oxo-C10-HSL on virulence factors of V. alginolyticus. B: Effect of exogenous 3-oh-C4-
HSL on virulence determinants of V. harveyi.

7
R. Ghanei-Motlagh et al. Aquaculture 535 (2021) 736345

Table 2
Effects of cell-free lysates of B. thuringiensis QQ1 and B. cereus QQ2 on the
virulence phenotypes (positively regulated by AHL) of V. alginolyticus.
Phenotype Vibrio alginolyticus

Control Lysate QQ1 Lysate QQ2

Amylase 0.61 ± 0.01a 0.29 ± 0.03b 0.24 ± 0.08b

Gelatinase 1.39 ± 0.19a 0.37 ± 0.15b 0.38 ± 0.08b
Protease 0.62 ± 0.09a 0.31 ± 0.10b 0.27 ± 0.03b
BFI 1.02 ± 0.10a 0.42 ± 0.06b 0.96 ± 0.21a

The values with different letters in each row denote significant differences for
each assay (Tukey’s multiple range test, p < 0.05). The data are mean ± SD of
three independent experiments. The values related to lytic enzymes assay were
calculated by dividing the diameters of the zones around the colonies by the
diameters of the colonies in millimeter. BFI: biofilm formation index.

Table 3
Effects of cell-free lysates of B. thuringiensis QQ1 and B. cereus QQ2 on the
virulence phenotypes (positively regulated by AHL) of V. harveyi.
Phenotype Vibrio harveyi

Control Lysate QQ1 Lysate QQ2

a b
Swarming 3.78 ± 0.47 1.12 ± 0.11 1.08 ± 0.06b
Swimming 3.11 ± 0.21a 1.48 ± 0.32b 1.56 ± 0.21b
BFI 3.32 ± 0.26a 2.32 ± 0.50b 1.84 ± 0.33b

The values with different letters in each row indicate significant differences for
each assay (Tukey’s multiple range test, p < 0.05). The data are mean ± SD of
three independent experiments. The results of swarming and swimming motility
assays were expressed in centimeter. BFI: biofilm formation index.

0.05) activity of serum and liver SOD after infection. In all groups, the
highest (p < 0.05) activities of serum and liver SOD were recorded 14
days post-infection. After infection (days 49 and 56), the level of GSH in
serum and liver showed a reduction in all groups relative to pre-
infection. Nevertheless, serum GSH at day 49 and liver GSH at day 56
were significantly higher in QQ2 than the other groups. Moreover, MDA
content in serum and liver was significantly (p < 0.05) lower in the
probiotic groups compared to the control group after challenge.

3.4. Immune responses

Fig. 3. AHL-degrading activity and anti-virulence potential of the tested pro­
biotics. A: Agar well diffusion assay for detection of AHL-degrading activity of
B. thuringiensis QQ1 (left plate) and B. cereus QQ2 (middle plate) against AHL
extracted from V. alginolyticus RT45GH on Luria-Bertani (LB) agar with the
lawn of A. tumefaciens NTL4. AHL re-extracted from LB broth supplemented
with crude extract obtained from V. alginolyticus RT45GH was used as negative
control (right plate). The presence and absence of blue color in A. tumefaciens
NTL4 indicate negative and positive QQ activity, respectively. B-C: Effects of
cell-free lysates of B. thuringiensis QQ1 and B. cereus QQ2 on amylasSV0151e,
gelatinase and protease activities of V. alginolyticus (B), and swarming and
swimming motilites of V. harveyi (C). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of
this article.)
enhancement in serum and liver SOD activity was observed in the
treated groups compared to the control group at day 42, however the
highest activity (p < 0.05) was noticed in fish received B. cereus QQ2. No
significant (p > 0.05) alteration in serum and hepatic levels of GSH and
MDA was found between the experimental and control groups at day 42.
Experimental infection with V. alginolyticus resulted in increased serum
and liver CAT activities in all groups compared to pre-infection. How­
ever, the probiont groups displayed higher CAT activities (p < 0.05)
relative to the control group. Moreover, no significant (p > 0.05) dif­
ference in serum or liver CAT activity was observed between the pro­
biont groups. Feeding with B. cereus QQ2 resulted in the highest (p <
Fig. 4 represents the effect of QQ probiotics on immunological pa­
rameters of Asian seabass before or after challenge with V. alginolyticus.
After 6 weeks feeding with QQ probiotics, serum lysozyme activity was
significantly (p < 0.05) increased in the probiont groups compared to
the control group, and the highest activity was observed in the QQ1
treatment. After infection (days 49 and 56), lysozyme activity was
significantly (p < 0.05) decreased in all treatments relative to pre-
infection, although it was significantly (p < 0.05) higher in the QQ
treatments compared to the control group. Moreover, the highest lyso­
zyme activity (p < 0.05) was recorded in QQ1 treatment post-infection.
Serum APC activity was significantly (p < 0.05) enhanced in fish
receiving B. cereus QQ2 for 6 weeks. After challenge, APC activity was
significantly (p < 0.05) higher in the probiotic groups than the control
group. In this study, a significant (p < 0.05) increase in serum anti­
protease activity was recorded in fish fed with QQ probionts before and
after infection. However, no significant (p > 0.05) difference was
observed between the probiotic groups for this activity. According to the
results, serum MPO activity was significantly (p < 0.05) elevated in
treated groups compared to the control group and the highest activity
was detected in the treatment QQ1. After challenge with V. alginolyticus,
MPO was initially increased (day 49) and subsequently decreased (day
56) in all groups. However, it was significantly (p < 0.05) higher in the
treated groups relative to the control group at both intervals. Blood RBA
was significantly (p < 0.05) increased in the probiotic treatments, as
compared to the control group. A significant (p < 0.05) increase in RBA

8
R. Ghanei-Motlagh et al. Aquaculture 535 (2021) 736345

Table 4 3.5. Cumulative survival after infection with V. alginolyticus

Effect of feeding with quorum quenching probiotics on antioxidant activity and
lipid peroxidation in serum and liver of Asian seabass before and after infection The earliest and the latest mortalities were recorded in the control
with V. alginolyticus. group at days 1 and 11 after infection, respectively. Administration of
Parameters Experimental Pre-infection Post-infection QQ probionts significantly (p < 0.05) decreased the cumulative mor­
groups
Day 42 Day 49 Day 56 tality in Asian seabass infected with V. alginolyticus (Fig. 5). The cu­
mulative survival percentage was significantly (p < 0.05) higher in fish
Serum
CAT Control 8.12 ± 1.36c,B 11.95 ± 13.87 ±
fed with B. cereus QQ2 (97.1%) and B. thuringiensis QQ1 (86.8%)
1.48b,B 1.08a,B compared to the control group (36%), whereas no significant (p > 0.05)
QQ1 11.56 ± 1.20c, 18.52 ± 21.43 ± difference was observed for cumulative survival percentage between the
A
3.02b,A 2.43a,A probiotic groups. Bacteriological examination confirmed the infection
QQ2 10.63 ± 19.90 ± 19.59 ±
caused by of V. alginolyticus in the mortalities occurred (Fig. 5). Fewer
1.52b,A 1.63a,A 2.43a,A
SOD Control 45.39 ± 61.89 ± 202.48 ± and less severe gross external lesions were recorded in fish that had
6.12b,B 5.54b,B 25.36a,C received the probiotic diets, whereas petechial hemorrhage and skin
QQ1 63.75 ± 70.86 ± 296.23 ± ulcers were observed in the fish fed with regular diet at the end of
5.10b,A 5.99b,AB 11.63a,B experiment (Fig. 5).
QQ2 65.65 ± 80.82 ± 335.68 ±
2.76b,A 7.07b,A 20.43a,A
GSH Control 211.59 ± 141.27 ± 129.31 ± 4. Discussion
37.99a,A 4.15b,B 14.13b,A
QQ1 249.03 ± 165.07 ± 155.45 ± It has been previously reported that various strains of V. alginolyticus
40.13a,A 28.08b,AB 23.30b,A
can produce a wide range of AHL molecules mainly dominated by 3-oh-
QQ2 225.08 ± 176.31 ± 162.91 ±
37.99a,A 18.14ab,A 22.38b,A C4-HSL, 3-oxo-C10-HSL and 3-oxo-C14-HSL (Liu et al., 2017). In this
MDA Control 11.92 ± 1.78c, 19.41 ± 25.03 ± study, the regulatory effect of exogenous 3-oxo-C10-HSL was investi­
A
1.88b,A 2.75a,A gated in tested V. alginolyticus with respect to the AHL produced by the
QQ1 9.99 ± 2.35b,A 14.40 ± 16.27 ±
pathogen. According to the results, treatment with exogenous 3-oxo-C10-
1.54a,B 1.77a,B
QQ2 10.38 ± 13.78 ± 14.95 ±
HSL could regulate lytic enzymes production, flagellar motility and
2.02b,A 2.26a,B 1.02a,B biofilm formation in tested V. alginolyticus. To our knowledge, the pre­
Liver sent study is the first one reporting that exogenous AHL is correlated to
CAT Control 14.34 ± 1.92c, 17.35 ± 21.06 ± the regulation of several virulence factors in V. alginolyticus. Regarding
B
1.62b,B 1.74a,B
c, the fact that the signals from all autoinducers (including AHLs) ulti­
QQ1 23.06 ± 2.85 28.17 ± 36.30 ±
A
2.25b,A 2.62a,A mately involve LuxR, treatment with exogenous AHL was probably
QQ2 21.03 ± 2.61c, 29.87 ± 35.09 ± associated with the QS regulation of putative virulence factors through
A
2.28b,A 1.81a,A the protein LuxR in V. alginolyticus (luxRval). In this regard, it has been
SOD Control 86.58 ± 111.65 ± 431.94 ±
shown that luxRval plays a key role in the positive regulation of extra­
10.31b,B 12.83b,B 35.77a,B
QQ1 93.03 ± 122.61 ± 460.20 ±
cellular proteases (ECP), extracellular polysaccharides (EPS) and nega­
6.91b,B 10.37b,B 57.11a,B tive regulation of flagellar motility (Rui et al., 2008). However, LuxO
QQ2 116.11 ± 169.09 ± 704.63 ± protein (Wang et al., 2007; Rui et al., 2009), LuxS protein (Tian et al.,
9.90b,A 34.14b,A 52.58a,A 2008b; Ye et al., 2008), LuxT protein (Liu et al., 2012) and other QS-
GSH Control 1240.87 ± 895.93 ± 356.64 ±
associated regulators (Cao et al., 2011; Liu et al., 2011; Sheng et al.,
145.24a,A 115.87b,A 37.32c,B
QQ1 1253.35 ± 1077.63 ± 399.02 ± 2013; Gu et al., 2016; Gao et al., 2018; Liu et al., 2020) have also been
99.92a,A 119.88b,A 15.38c,B demonstrated to play pivotal roles in regulation of ECP, EPS, flagellar
QQ2 1411.63 ± 1037.24 ± 785.72 ± motility and biofilm formation in V. alginolyticus. Therefore, further
233.87a,A 152.37b,A 48.80c,A studies are required to unravel the mechanisms by which exogenous
MDA Control 20.42 ± 3.83c, 39.99 ± 45.79 ±
A
3.48b,A 4.11a,A
AHLs can regulate virulence factors in V. alginolyticus. Based on the re­
QQ1 22.41 ± 28.10 ± 32.20 ± sults, there was an inverse relation between flagellar motility and bio­
3.18b,A 4.52a,B 2.45a,B film formation in tested V. alginolyticus. This may suggest that exogenous
QQ2 20.17 ± 23.66 ± 26.83 ± 3-oxo-C10-HSL has a regulatory function in the alteration of biofilm
3.07a,A 2.09ab,B 2.16a,C
structure instead the initiation of biofilm formation (induced by the
The values were expressed as mean ± SD (n = 9). For each parameter, small and presence of the flagellum). Consistently with our findings, Liu et al.
capital letters indicate different comparisons. Different small letters denote (2017) indicated that exogenous 3-oxo-C10-HSL could promote biofilm
significant (p < 0.05) differences between the values in each row. Different formation and alter biofilm structure in several strains of V. alginolyticus
capital letters represent significant (p < 0.05) differences between the values in in a dose- and temperature-dependent manner.
each column. CAT units were reported as nmol/mg pro/min. SOD units were
Vibrio harveyi produces and detects three autoinducers including
reported as mU/mg pro/min. GSH units were expressed as nmol/mg pro. MDA
Harveyi autoinducer 1 (HAI-1), autoinducer-2 (AI-2) and cholera
units were expressed as μmol/mg pro.
autoinducer-1 (CAI-1) to regulate a large numbers of QS-related genes
(Chen et al., 2020a, 2020b), however, only the first of these molecules is
was also recorded in all groups after infection with V. alginolyticus
an AHL. In this study, the effect of the AHL molecule produced by
compared to pre-infection. However, RBA was higher in the probiotic
V. harveyi (HAI-1, 3-oh-C4-HSL) was investigated on the putative viru­
groups relative to the control group and the highest RBA (p < 0.05) was
lence factors of the pathogen. Our findings demonstrated the positive
observed in the treatment QQ1 after challenge. In the present research,
regulatory function of exogenous 3-oh-C4-HSL on control of swarming
bactericidal activity was significantly (p < 0.05) higher in the probiont-
motility, swimming motiliy and biofilm formation in tested V. harveyi,
treated fish than the control group before and after infection. The
suggesting that HAI-1 can be involved in initial invasion and coloniza­
highest bactericidal activity (p < 0.05) was also recorded in fish treated
tion of this pathogen. To our knowledge, this is the first study showing
with B. cereus QQ2 after experimental challenge.
that exogenous 3-oh-C4-HSL could increase the swarming motility and
biofilm formation in V. harveyi. In this sense, Anetzberger et al. (2009)
revealed that QS positively regulate biofilm formation by V. harveyi and

9
R. Ghanei-Motlagh et al. Aquaculture 535 (2021) 736345

Fig. 4. Non-specific immune responses in Asian seabass fed or un-fed with quorum quenching probiotics before and after infection with V. alginolyticus. The values
were expressed as mean ± SD (n = 9). Small and capital letters indicate different comparisons. Different lowercase letters on the bars denote significant (p < 0.05)
differences between the experimental groups at a specified interval. Different uppercase letters on the bars represent significant (p < 0.05) differences for each of the
experimental groups at various sampling days.

heterogeneity of the population apparently contributed to enhancement
of biofilm formation in the pathogen. Moreover, our results are in
accordance with Yang and Defoirdt (2015) who demonstrated that HAI-
1 is a crucial signal molecule associated with swimming motility in
V. harveyi. On the other hand, treatment with exogenous 3-oh-C4-HSL
had no significant impact on gelatinase, protease, amylase and hemo­
lysis activities in tested V. harveyi. Our results are in line with a previous
study that indicated that AI-2 and CAI-1 but not HAI-1 are the pivotal
signal molecules involved in the caseinase and hemolysin productions in
V. harveyi, implying that all autoinducers of the pathogen are necessary
for full virulence towards various hosts (Hong et al., 2016). Moreover,
the reduced productions of lipase and lecithinase (phospholipase) by
V. harveyi in medium supplemented with 3-oh-C4-HSL suggest that HAI-
1 can be one of the QS signals involved in the regulation of these en­
zymes in V. harveyi. In this sense, Natrah et al. (2011) reported that
phospholipase activity is negatively regulated by QS, while lipase ac­
tivity was found to be independent of QS in V. harveyi.
Previous studies demonstrated that pathogenic strains of
V. alginolyticus and V. harveyi can produce a wide range of extracellular
products enabling the pathogens to damage and infect their hosts
(Aguirre-Guzmán et al., 2004). In this regard, it has been reported that
lytic exoenzymes including hemolysin, proteases (caseinase and gelati­
nase), lecithinase, lipase and amylase are associated with the pathoge­
nicity of V. alginolyticus (Hörmansdorfer et al., 2000; Tian et al., 2008a)
and V. harveyi (Zhang et al., 2020). Lytic enzymes are regarded as
virulence factors involved in nutrient acquisition through the degrada­
tion of host tissues. They also enable pathogens to penetrate into host
tissues and spread throughout body (Aguirre-Guzmán et al., 2004).
Besides, flagellar motility and biofilm formation allow pathogens to
adhere to host tissues and colonize the host surfaces (Liu et al., 2011;
Echazarreta and Klose, 2019). Biofilm formation also contributes to drug
resistance, persistence and evasion of a pathogen from the host’s im­
mune system (Liu et al., 2018). The results of the present study showed
the possible anti-virulence potential of both QQ strains against
V. alginolyticus and V. harveyi in vitro. QQ probionts decreased the pro­
duction of lytic enzymes (protease, gelatinase and amylase) and biofilm
formation in tested V. alginolyticus, showing their potential for inhibition
of entrance, penetration and colonization of the pathogen. Moreover,

10
R. Ghanei-Motlagh et al.
Fig. 5. Fish survival, pathogen detection and external lesions after experi­
mental challenge. A: Kaplan-Meier survival curves for Asia seabass fed with or
without dietary QQ probiotics during 14 days infection with V. alginolyticus. B:
Agarose gel electrophoresis illustrating the results of duplex PCR of the 16S
rRNA (product size 663 bp) and the V. alginolyticus collagenase (product size
737 bp) genes from V. alginolyticus isolated from anterior kidney of infected fish
(lane 1), V. alginolyticus obtained from pure culture (lane 2) and V. alginolyticus
IBRC-M 10727 as the positive control (PC). Negative control (NC); 100-bp DNA
Marker (M). C: The representative fish from each group after experimental
infection with V. alginolyticus (day 56). Petechial hemorrhage and skin ulcers
(yellow arrows) in Asian seabass fed with basal diet (control group) versus
restricted external lesions in Asian seabass fed with diets containing
B. thuringiensis QQ1 (QQ1 group) and B. cereus QQ2 (QQ2 group). (For inter­
pretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
11
Aquaculture 535 (2021) 736345
flagellar motility and biofilm formation were significantly decreased
when V. harveyi was cultured in media supplemented with lysates from
QQ probiotics, suggesting that degradation of HAI-1 can be effective in
control of V. harveyi through reduction of adherence, colonization and
initial invasion of the pathogen. In accordance with our results, the
purified protein AHL-Lactonase (AiiA) from host-associated
B. licheniformis DAHB1 could inhibit the biofilm formed by
V. parahaemolyticus DAHP1 (Vinoj et al., 2014). Moreover,
B. thuringiensis and B. cereus isolated from rainbow trout (Oncorhynchus
mykiss) could decrease the biofilm formation and swimming motility in
Yersinia ruckeri (Torabi Delshad et al., 2018). Bacillus cereus QSI-1 and
B. firmus sw40, isolated from gibel carp (Carassius auratus gibelio), also
suppressed protease production, hemolytic activity and biofilm forma­
tion in Aeromonas hydrophila (Chu et al., 2014; Li et al., 2019).
The modulatory effects of probiotics on antioxidant defense system
of fish have been demonstrated in previous studies (Thy et al., 2017). In
the present research, higher serum and hepatic activities of CAT and
SOD were recorded in Asian seabass fed with dietary QQ probiotics for 6
weeks, while the levels of GSH and MDA showed no significant alter­
ation among the groups. In this regard, feeding of gibel carp with an
AHL-degrading probiotic (B. firmus sw40) led to an increased level of
serum SOD but reduced contents of serum MDA and GSH (Li et al.,
2019). The elevated activities of serum SOD, CAT and GSH as well as
decreased level of MDA were observed in Pengze crucian carp (Carassius
auratus var. Pengze) fed with B. cereus (Yang et al., 2020). Moreover,
dietary supplementation with B. cereus 39HN resulted in the highest
levels of serum SOD in African catfish (Clarias gariepinus) (Reda et al.,
2018a). These protective effects of dietary probiotics on prevention of
oxidative stress may be attributed to their potential for direct scavenging
active oxidants, production of enzymes and metabolites with antioxi­
dant activity, positive modulation of intestinal microbiota composition,
biosynthesis of essential micronutrients with antioxidant activity such as
vitamins B12 and B6, increased bioavailability of dietary antioxidants
from the gut and regulation of antioxidant-associated signaling path­
ways in host (Mogna et al., 2012; Taş et al., 2014; Wang et al., 2017b).
SOD, CAT, GSH and MDA are among the most reliable markers
associated with oxidative stress response. SOD and CAT are two essential
components of the antioxidant enzyme system catalyzing the dis­
mutation of the superoxide anion radical to hydrogen peroxide and
water, and the decomposition of hydrogen peroxide into oxygen and
water, respectively (Peixoto et al., 2013). GSH is a non-enzymatic
antioxidant with high abundance of sulfhydryl (thiol) groups that
plays a key role in maintaining of intracellular oxidoreductive balance
through scavenging reactive oxygen species (such as hydrogen peroxide
in conjunction with glutathione peroxidase, GPx) and detoxification of
organic hydroperoxides (Zengin and Yilmaz, 2016). The reduction in
hepatic GSH is associated with depletion during oxidative stress,
decreased synthesis and increased utilization (Lu, 2020). MDA is one of
the end products of lipid peroxidation and is frequently used as a
biomarker of cell membrane injury (Naderi et al., 2019). In this study,
elevated activities of CAT and SOD, reduced contents of GSH, and
increased levels of MDA were observed after infection with
V. alginolyticus in both probiotic and control groups compared to pre-
infection, indicating the induced oxidative stress in fish IP-infected
with the pathogen. However, higher activities of CAT, SOD and GSH,
and lower levels of MDA, were recorded in the probiotic groups than the
control group after infection, which may reflect the protective effects of
the tested probiotics against infection with V. alginolyticus. The induc­
tion of oxidative stress after Vibrio infection has previously been re­
ported in aquatic animals. For example, elevation of RB and SOD
activities was observed in haemocytes of white shrimp (Litopenaeus
vannamei) infected with V. alginolyticus in a manner similarly to our
present findings (Liu et al., 2007). A reduction in GSH level and an
elevation in MDA content were also reported in blue shrimp
(L. stylirostris) challenged with V. nigripulchritudo compared to un-
challenged group, whereas probiotic-fed shrimp showed a reverse
R. Ghanei-Motlagh et al.
trend after infection (Castex et al., 2010).
Probiotics can modulate the host immune responses through inter­
action with immune components within the gut or mucus, triggering
immune-related signaling pathways and their effects on the production
of cytokines (Nayak, 2010; Thy et al., 2017). According to our results,
lysozyme activity was significantly higher in the probiotic-fed fish, and
the highest activity was observed in fish fed with B. thuringiensis QQ1.
Lysozyme is a bacteriolytic enzyme that directly hydrolyse the pepti­
doglycan layer of Gram-positive bacteria. It has also a lytic activity
against Gram-negative bacteria after damage by complement system
which exposes the peptidoglycan cell wall (Nakanishi et al., 2018).
Furthermore, as an opsonin, lysozyme activate phagocytes and the
complement system (Magnadóttir, 2006). Our results are in line with
those of Jiang et al. (2019) who reported that administration of dietary
B. cereus QSI-1 at 5 × 108 CFU/g feed for 15 days could enhance lyso­
zyme and APC activities in skin mucus of crucian carp. In this study, a
reduction in lysozyme activity was observed in fish infected with
V. alginolyticus, as compared to un-challenged fish. Similarly, Nile tilapia
infected with V. parahaemolyticus showed a lower serum lysozyme ac­
tivity but a higher RBA compared to pre-infection (Balfry et al., 1997).
Moreover, dietary Lactobacillus acidophilus increased serum lysozyme
activity in striped snakehead (Channa striata) after infection with
A. hydrophila (Talpur et al., 2014).
Serum APC is an important humoral component of the non-specific
immune system with bactericidal, chemotactic and opsonic activities
(Budiño et al., 2006). Lipopolysaccharides from Gram-negative bacteria
and foreign red blood cells are among the known activators of this
pathway (Biller-Takahashi et al., 2013; Nakanishi et al., 2018). In this
study, serum APC activity was significantly increased in fish fed with
dietary B. cereus QQ2 for 6 weeks. It has been previously reported that
dietary B. aerius B81e significantly increased serum APC, lysozyme,
bactericidal activities and RB in Pla-Mong (Pangasius bocourti), in a
manner consistent with our present results (Meidong et al., 2018). After
challenge, APC activity was higher in the probiotic groups compared to
the control group. In accordance with our results, a higher APC activity
was reported in Nile tilapia fed with L. rhamnosus after challenge with
Edwardsiella tarda (Pirarat et al., 2006), and olive flounder (Paralichthys
olivaceus) fed with diets containing probiotic and herbal extract after
infection with Streptococcus parauberis (Harikrishnan et al., 2011a).
Serum protease inhibitors are involved in acute phase reactions,
homeostasis of the body fluids and defense against protease-producing
bacteria (Magnadóttir, 2006). Our findings revealed that serum anti­
protease activity was higher in fish fed with QQ probionts prior to and
after infection. In agreement with our results, Nile tilapia fed with diets
containing B. subtilis and B. licheniformis had significantly enhanced
levels of serum antiprotease in comparison with un-treated fish (Abarike
et al., 2018). Moreover, feeding with dietary L. sakei BK19 promoted
several immune parameters including serum antiprotease activity in fish
infected with S. iniae, S. parauberis and E. tarda (Harikrishnan et al.,
2010; Harikrishnan et al., 2011b).
MPO is a major enzyme present in azurophilic (primary) granules of
granulocytic cells (mostly neutrophils) and modulates the immune
response and inflammatory processes. MPO is involved in both oxidative
(chlorination and peroxidase activities) and antioxidant (superoxidase,
SOD and CAT activities) activities (Winterbourn and Kettle, 2013).
Under normal conditions, MPO and its derivatives provide an effective
microbicidal system against pathogens. However, excessive production
of MPO-derived oxidants has been associated with several pathological
circumstances in higher developed organisms (Van der Veen et al.,
2009). According to the results, a higher serum MPO activity was
recorded in probiont-fed groups compared to the control group before
and after infection. Similarly to our results, serum MPO, liver SOD and
liver CAT were significantly enhanced in Japanese eel (Anguilla japonica)
supplemented with dietary L. pentosus PL11 compared to those fed with
basal diet before and after infection caused by E. tarda (Lee et al., 2013).
Leukocyte RB is known as one of the most important mechanisms
12
Aquaculture 535 (2021) 736345
responsible for potent microbicidal activity against a wide range of
pathogens through the production of reactive superoxide intermediates
(Biller and Takahashi, 2018). In the present research, RBA was higher in
the probiotic treatments than the control group before and after
experimental infection with V. alginolyticus. In this regard, a significantly
higher blood RBA was observed in olive flounder fed with Bacillus pro­
biotics before and after infection with S. iniae (Cha et al., 2013).
Moreover, dietary supplementation with B. subtilis significantly
increased RBA in rohu (Labeo rohita) challenged with A. hydrophila
(Kumar et al., 2008; Devi et al., 2019).
Serum bactericidal activity is associated with the activities of leu­
kocytes, complement system, lysozyme and other humoral factors
(Biller-Takahashi et al., 2013). Based upon the present results, bacteri­
cidal activities were higher in the probiotic groups relative to the control
group before and after infection. Moreover, fish fed with dietary
B. cereus QQ2 had the highest bactericidal activity after challenge,
significantly higher than the QQ1 and control groups (day 56). Similarly
to our findings, a significantly higher serum bactericidal activity was
recorded in African catfish fed with B. thuringiensis for 15 days
(Reneshwary et al., 2011). Moreover, the highest serum bactericidal and
lysozyme activities were observed in African catfish fed with diets
containing 1010 CFU/kg feed of autochthonous B. cereus 39HN for 30
days (Reda et al., 2018a). Besides, Catla (Catla catla) treated by dietary
B. subtilis showed higher bactericidal activities before and after infection
with A. hydrophila (Kumar et al., 2015).
In the present study, administration of both QQ probiotics signifi­
cantly increased the cumulative survival in Asian seabass infected with
V. alginolyticus. Moreover, fish fed with B. cereus QQ2 had a higher
survival percentage (albeit not significantly) compared to those fed with
B. thuringiensis QQ1. Similar differences had previously been reported in
rainbow trout and Asian seabass intraperitoneally infected with
Y. ruckeri and V. harveyi, respectively (Torabi Delshad et al., 2018;
Ghanei-Motlagh et al., 2020a). However, no clear mechanism has been
suggested for this protection and for the difference in protectiveness
between QQ bacteria. A possible explanation may be differences in the
probiotic’s capabilities to modulate physiological responses in fish. For
example, fish fed with B. cereus QQ2 had significantly higher SOD, GSH
and bactericidal activities compared to those fed with B. thuringiensis
QQ1 after infection, facilitating the elimination of the pathogen. How­
ever, further research is needed to confirm this hypothesis with respect
to positive modulation of the other parameters in fish fed with
B. thuringiensis QQ1. The improvement of survival after experimental
infection with V. alginolyticus can be explained by the anti-virulence
potential of B. thuringiensis QQ1 and B. cereus QQ2 combined with the
positive impacts of QQ probiotics on modulation of immune and anti­
oxidant responses in Asian seabass. However, the direct involvement of
QQ potential of the tested Bacillus strains in protection of fish against IP
infection with the pathogen needs further studies. Notably regarding the
fact that the strains used were not in direct contact with the pathogen,
they could not actively eliminate the AHL produced by the pathogen
from their surroundings under these circumstances. This suggests that
other protective mechanisms beside QQ had taken place. These results
are consistent with several previous studies that have shown the pro­
tective effects of QQ bacteria in fish models after exposure with different
routes of infection (IP injection and immersion) with A. hydrophila and
Y. ruckeri (Chu et al., 2011; Torabi Delshad et al., 2018; Chen et al.,
2020a, 2020b). Our findings illustrate that the probiotic effects of both
tested QQ strains were highly effective in protection of Asian seabass
from infection of V. alginolyticus.
The results obtained in this study suggest that, after full validation of
their efficacy in the field and safety considerations, application of QQ
bacteria with high probiotic potential could be developed commercially
as novel dietary supplements for control of infections caused by Vibrio
pathogens in the aquaculture industry. Moreover, the characteristic of
spore formation by certain QQ probionts (Bacillus spp.) protects these
kinds of strains from pressure and heat during the manufacturing
R. Ghanei-Motlagh et al.
processes, which simplifies their incorporation into commercial pellets
(Merrifield et al., 2010; Song et al., 2012).
5. Conclusion
In conclusion, our findings showed the anti-virulence potential of
B. thuringiensis QQ1 and B. cereus QQ2 against several virulence phe­
notypes associated with AHL-based QS in V. alginolyticus and V. harveyi
in vitro. Dietary supplementation with these QQ probiotics improved
innate immune responses and antioxidant capacity in Asian seabass
prior to and after challenge with V. alginolyticus and was associated with
a reduced severity of the infection. Therefore, we suggest that AHL-
degrading bacteria with probiotic activities have high potential to effi­
ciently protect Asian seabass from Vibrio infection. However, further
investigations are required to determine the exact mode of actions
related to QQ potential in protection of fish IP-infected by Vibrio
pathogens.
CRediT authorship contribution statement
Reza Ghanei-Motlagh: Conceptualization, Methodology, Formal
analysis, Writing - original draft, Writing - review & editing. Darioush
Gharibi: Supervision, Methodology, Investigation. Takavar Moham­
madian: Supervision, Methodology, Formal analysis, Investigation.
Mohammad Khosravi: Methodology. Esmaeil Mahmoudi: Methodol­
ogy. Mojtaba Zarea: Methodology. Simon Menanteau-Ledouble:
Methodology, Writing - review & editing. Mansour El-Matbouli: Su­
pervision, Visualization, Investigation.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This study was supported by a grant from Shahid Chamran Univer­
sity of Ahvaz Research Council (Grant No. vC98.299). The funding body
played no role in the design of the study or analysis of the data. We
gratefully thank Prof. Dr. Masoud Ghorbanpoor for providing the spe­
cific primers. The authors appreciate Dr. Sakineh Mashjoor for her
assistance in assessment of the antioxidant parameters, Mojtaba Emam
and Javad Seyedi for their assistance during in vivo trials, and Dr. M
Shokouhmand and Mag. M. Srivastava for the valuable help in bacteri­
ological experiments.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.aquaculture.2021.736345.
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