| |

Received: 3 March 2020    Revised: 22 April 2020    Accepted: 30 April 2020

DOI: 10.1111/anu.13096

ORIGINAL ARTICLE

Beneficial effects of Bacillus subtilis on water quality, growth,

immune responses, endotoxemia and protection against
lipopolysaccharide-induced damages in Oreochromis niloticus
under biofloc technology system

Ghasem Mohammadi PhD1  | Taida Juliana Adorian PhD2  |

Gholamreza Rafiee PhD1

1
Department of Fisheries Sciences, Faculty
of Natural Resources, University of Tehran,
Karaj, Iran
2
Department of Animal Science, Federal
University of Santa Maria, Santa Maria,
Brazil
Correspondence
Ghasem Mohammadi, Department of
Fisheries Sciences, Faculty of Natural
Resources, University of Tehran, 331585-
4314 Karaj, Iran.
Email: Ghasem.mohamady@ut.ac.ir
Abstract
The present research explored the effects of Bacillus subtilis on water quality,
growth, immune responses, endotoxemia and protection against lipopolysaccha-
ride (LPS) damages in Nile tilapia Oreochromis niloticus under biofloc system. B. sub-
tilis was added at 0, 1, 2, 3 and 4 grams (1.19 × 108 CFU/g) per kg of basal diet,
named T1 (control), T2, T3, T4 and T5, respectively, and fed to fish (14.82 ± 0.42 g)
for 50 days. The concentrations of TAN, NO2 and NO3 were significantly reduced,
and fish fed probiotics displayed significantly better growth performances versus the
control, concomitantly with significantly enhanced activities of digestive enzymes.
They also showed significantly declined serum glucose and cholesterol vice versa
significantly improved immune responses (total protein, albumin, globulin, lysozyme,
alternative complement, protease, immunoglobulins, alkaline phosphatase and res-
piratory burst), antioxidant capacity (superoxide dismutase, total antioxidant capac-
ity, malondialdehyde) and skin mucus parameters (total protein, lysozyme, alternative
complement, protease, immunoglobulins). Meanwhile, significantly lower endotoxin
(LPS) concentrations were detected in the intestines and serum of fish fed probiotics.
LPS challenge induced profound oxidative stress and impaired immune responses.
Interestingly, probiotic alleviated LPS-induced damages and restored mentioned pa-
rameters. In conclusion, B. subtilis effectively enhanced fish production, immunity
and protection against LPS-induced damages in tilapia under biofloc system.

KEYWORDS

endotoxin, immunostimulant, innate immunity, oxidative stress, probiotic

1 |  I NTRO D U C TI O N waste-free fish (Mohammadi, Rashidian, Hoseinifar, Naserabad, &

Studies conducted over the past five years demonstrate the growing in-
terest in the use of prebiotics, probiotics and other immunostimulants in
aquaculture (Dash et al., 2018; Dawood & Koshio, 2016; Kuebutornye,
Abarike, & Lu, 2019). These substances can, to varying degrees, replace
and/or reduce the use of antibiotics in aquaculture systems, producing
Doan, 2020). What is even more important in intensive production
systems, since animals are subjected to high stocking densities, and,
consequently, there is a higher infection pressure. In this sense, we can
highlight the increased cultivation of tilapia (Oreochromis niloticus) in
the biofloc technology system (BFTS), an engineered system based on
the assimilation of residual nutrients and the conversion to microbial

Aquaculture Nutrition. 2020;00:1–17. wileyonlinelibrary.com/journal/anu© 2020 John Wiley & Sons Ltd     1 |
 |
2       MOHAMMADI et al.
biomass that is used as a natural source of food for fish. This system
allows for increased aquaculture productivity, even with limited water
availability (Avnimelech, 2015). However, it is known that limited water
exchange and/or fish production at high densities deteriorates water
quality and exposes animals to higher stress conditions, resulting in im-
paired growth performance and immune responses, which makes fish
more susceptible to the pathogens commonly found under cultivation
conditions (Nardocci et al., 2014; Plant & LaPatra, 2011; Tort, 2011). In
cases, biofloc technology (BFT) develops opportunistic and pathogenic
species that may compromise the health and survival status of fish
(Avnimelech, 2015; Pacheco-Vega et al., 2018; Prangnell et al., 2016;
Schulze, Alabi, Tattersall-Sheldrake, & Miller, 2006; Schveitzer
et al., 2019). A number of important pathogens inflecting the aquacul-
ture industry are Gram-negative, for example Edwardsiella spp., Vibrio
spp., Aeromonas spp. and Pseudomonas spp. (Jeney, 2017; Pridgeon &
Klesius, 2012). These bacteria cause stress and disease in fish by dif-
ferent mechanisms (Ben Hamed et al., 2018; Jeney, 2017). For example,
they could induce immune response owing to their cell wall component,
known as lipopolysaccharide (LPS) or endotoxin (Jiang et al., 2017). The
gut barrier dysfunction, intestinal bacteria overgrowth and host immu-
nodeficiency increase the risk for endotoxemia, where an excessive
amount of Gram-negative bacteria or their endotoxin translocate/pass
through the intestinal epithelium into the circulatory system, resulting
in inflammation, suppressed immune responses or reduced growth
performance (Berg, 2000; Jiao et al., 2020; Mokkala, Laitinen, & Röytiö,
2016; Swain, Nayak, Nanda, & Dash, 2008; Volkoff & Peter, 2004). The
previous studies show that LPS induced excessive reactive oxygen
species formation, leading to metabolic and immunological alterations,
immune system impairment and disease progress (Galina, 2017; Li
et al., 2020). These conditions demonstrate the need to adopt strate-
gies to improve the health status of cultured fish and also water quality,
thus minimizing these impacts on aquaculture production.
Studies show that Bacillus spp. in diets improve feed efficiency,
immune function, stress responses, disease resistance, gut health,
modulate gut microbiota and also enhance water quality for vari-
ous fish species (Abarike et al., 2018; Cha, Rahimnejad, Yang, Kim,
& Lee, 2013; Gobi et al., 2018; Kuebutornye et al., 2019; Selim &
Reda, 2015), being indicated as a preventive strategy for stress con-
dition and disease outbreaks. However, its efficiency upon dietary
supplementation for fish grown in BFT is not yet studied. Also, there
is a lack of studies on the possible protective effects of probiotic
against LPS-induced damages in fish. Thus, the present study aimed
to evaluate the effects of B. subtilis supplementation in diets of Nile
tilapia (O. niloticus) juveniles raised in the biofloc system and chal-
lenged with bacterial LPS at the end of study.
2 |  M ATE R I A L S A N D M E TH O DS
2.1 | Fish and experimental conditions
Nile tilapia (Oreochromis niloticus) juveniles of mixed sex were pur-
chased from a private fish farm and transferred to a greenhouse
structure at the college of agriculture and natural resources of the
University of Tehran, Karaj, Iran (35°48'13.1"N 51°00'01.3"E). The
fish were acclimated for two weeks to the new experimental condi-
tion and diet, during which time they were fed a commercial feed
(Table 1) at 30 g/kg of estimated biomass three times a day. The gen-
eral health status of fish and behavioural symptoms were monitored.
Before introducing the fish to the experimental treatments, four
cylindrical tanks each were filled with 250L of the water from accli-
matization tank, after which total ammonia nitrogen (TAN) concen-
trations were checked and molasses (0.25 g carbon/ml) was added
to these tanks, assuming that 20 g of carbon is required to assimilate
1 g of TAN by heterotrophic bacteria (Avnimelech, 2015; Ebeling,
Timmons, & Bisogni, 2006). All the tanks were continuously and vig-
orously aerated until total suspended solids (TSS) reached 300 mg/L
and TAN concentrations declined to nearly zero. Finally, the experi-
mental treatments were added with 50 mg/L of microbial flocs.
After inoculation, 300 healthy juveniles (14.82 ± 0.42 g;
9.26 ± 0.09 cm) were randomly allocated into 15 cylindrical fibreglass
tanks (height: 64 cm, diameter: 79 cm, filled depth: 55 cm) containing
270 L (working capacity) of de-chlorinated and aerated water. Each
experimental group had three replicates, and fish were fed experi-
mental diets 3% body weight three times a day (7 a.m., 2 p.m. and 5
p.m.). Each tank was equipped with electric 300 W heating thermo-
stats (SOBO HS-300, China) and submerged diffuser air stones con-
nected to 35W air compressors (70L/min, >0.025 Mpa, Hailea Co.,
China) to control the water temperature and to supply the oxygen,
respectively. A zero-water exchange approach was followed, and
some fresh tap water was added to the tanks periodically to compen-
sate for the water loss due to evaporation. The daily rate of molasses
addition as the carbon source was calculated to obtain a C/N ratio
of 15 based on the available formula (Avnimelech, 2015). The exper-
iment was conducted under natural photoperiod (12:12 light/dark).
2.2 | Probiotic and diet supplementation
2.2.1 | Bacterial strain
The lyophilized powder of probiotic Bacillus subtilis (1.19 × 10 8 CFU/g)
was kindly provided by Dr. Alireza Shenavar Masouleh, manager at
TA B L E 1   The proximate composition of the commercial diet
(Beta Co., Iran)
Proximate analysis
Dry matter (g/kg) 930
Crude proteina  390 ± 20
Crude lipida  150 ± 20
Asha  100 ± 20
Fibre 40>
Gross energy (kcal/kg) 3,800 ± 200
a
g/kg dry matter basis
MOHAMMADI et al.
a probiotic producing company (Zistyar Varna Co, Iran). The bacte-
rial inhibitory activity of this probiotic was checked, as described
below:
2.2.2 | Antimicrobial activity
Aeromonas hydrophila, Escherichia coli and B. subtilis were grown
separately in tryptic soy broth media (Merck, Germany) at 37°C for
24 hr in an incubator shaker at 150 rpm (Pars Azma Co., Iran). Then,
the bacterial suspensions were centrifugated (Centrikon H-401,
Kontron, Italy) at 4,000 g, 4°C for 6 min. The bacterial pellets were
washed with sterile saline (0.9% w/v of NaCl) and resuspended into
the same fresh solution.
Then, A. hydrophila and E. coli suspensions were diluted and
0.1mL (107 cells mL-1; determined using hemocytometer) was sepa-
rately plated on tryptic soy agar plates.
A sterile blank disc (6  mm diameter) inoculated with 5  μl of B.
subtilis culture was placed on the plates and incubated for 24 hr at
37°C. The sterile blank disc in control plate was only added with
sterile saline (0.9% w/v of NaCl). Finally, the radius of inhibition cir-
cles around the disc was measured in millimetres.
2.2.3 | Diet supplementation
1, 2, 3 and 4 grams of the lyophilized probiotic (1.19 × 108 CFU/g)
were separately dissolved in 100 ml sterile physiological serum
(0.9% w/v of NaCl) and sprayed on 1 kg of previously well-grounded
commercial diets (Table 1) named T2, T3, T4 and T5 diet. The diet
used as the control (T1) was only sprayed with an equal amount of
sterile physiological serum. The diets were thoroughly mixed and
pelletized again using a hand pelletizer. All experimental diets were
aseptically dried in an oven at 40°C for 1 hr and kept in sealed plastic
bags at 4°C until use. The concentration of probiotic in lyophilized
powder and its presence in supplemented diets was confirmed using
Bacillus selective media, mannitol-egg-yolk-polymyxin agar (Quelab,
Canada) (Abarike et al., 2018; Allameh et al., 2016; Iranian Standard
and Industrial Researches Institute, 2006). The procedure of supple-
menting the diets was repeated every 17 days using fresh materials
to ensure probiotic viability.
2.3 | Water quality parameters
During the 50-day experimental period, water temperature, pH
(HANNA 9811-5, Romania) and dissolved oxygen (HANNA Hi-
98193, Romania) were measured at a daily interval. Also, water sam-
ples were collected from each tank and passed through filter papers
(Whatman) for inorganic nitrogenous compound measurements. The
total ammonia nitrogen (TAN) concentrations and nitrite (NO2–N)
concentrations were quantified following (Clesceri, Greenbaerg,
& Eaton, 1998; Parson, Maita, & Lalli, 1984), respectively. Nitrate
|
      3
(NO3–N) concentrations and total suspended solids (TSS) were de-
termined according to Ref. (Clesceri et al., 1998).
2.4 | Growth performances
In the early and late experimental period, a biometric assessment
was performed to collect data from the animals, to estimate the
following: weight gain (WG)  = final weight (g) – initial weight (g);
food conversion ratio (FCR) = feed intake/weight gain and con-
sumption (g); specific growth rate (SGR) (%): [(ln (final weight) − ln
(initial weight))/50  days] × 100, where: ln  =  Napierian logarithm;
condition factor (K) = (weight (g)/(length (cm)) 3) × 100, and survival
rates (SR) = final number of fish in tank/ initial number of fish in
the tank × 100.
2.5 | Serum analyses
At the end of the experiment (day 50), five fish were randomly se-
lected from each tank (15 per treatment) and sedated (clove powder,
500 mg/L) to collect their blood. Blood samples were drawn from the
caudal vein and transferred to non-heparinized tubes and instantly
centrifuged at 2,000 g (5 min, 4°C) to separate sera. An amount of
the blood was transferred into heparinized tubes and used for hae-
matological indices assays, as well.
The levels of serum total protein, albumin, glucose, cholesterol
and alkaline phosphatase were quantified using available commer-
cial kits (Pars Azmun Co, Tehran, Iran), and the difference between
serum total protein and serum albumin was considered as serum
globulin concentration (Vali, Mohammadi, Tavabe, Moghadas, &
Naserabad, 2020). Lysozyme activity was determined following the
method of Ref. (Ellis, 1990). The serum protease activities were mea-
sured using the azocasein hydrolysis assay (Palaksha, Shin, Kim, &
Jung, 2008). Alternative complement activities (ACH50) were esti-
mated following Ref. (Yano, 1992). Total immunoglobulin (Ig) con-
tent was quantified according to Ref. (Siwicki & Anderson, 1993).
Superoxide dismutase (SOD) and total malondialdehyde (MDA) ac-
tivities, as well as total antioxidant capacities (TAC), were determined
using commercially available kits (ZellBio GmbH, Germany) (Taheri
Mirghaed, Fayaz, & Hoseini, 2019). Serum endotoxin (lipopolysac-
charide, LPS) contents were determined using an enzyme-linked
immune sorbent assay (ELISA) kit (Hangzhou Eastbiopharm Co. Ltd,
China). The respiratory burst activity (RBA) of blood leucocytes
was determined by assessing the reduction in nitroblue tetrazolium
(Abu-Elala, Marzouk, & Moustafa, 2013). All the tests were repli-
cated two times.
2.6 | Mucus collection and immunological assays
The method of Ref. (Ross, Firth, Wang, Burka, & Johnson, 2000) was
followed to collect fish skin mucus samples at the end of the feeding
 |
4       MOHAMMADI et al.
trial. The six fish were randomly taken from each tank and sedated;
then, their body surface was washed with sterile NaCl solution (50
mM), and individuals were gently shacked for two minutes in poly-
ethylene bags containing 10 ml of NaCl (50 mM), and then, mucus
was collected and transferred to 10-ml sterile tubes and centrifuged
(2,000 g for 10 min at 4°C) to separate wastes. The resultant super-
natants were kept at −20°C until further analysis of mucus protease,
lysozyme and ACH50, as well as total immunoglobulin and total pro-
tein contents, using similar methods described in Section 2.5. All the
tests were run in duplicate.
2.7 | Intestine digestive enzymes and
endotoxin content
The previously sedated fish (four fish per tank) were sacrificed,
and the whole intestine was removed aseptically, washed with cold
sterile saline, pooled and homogenized (Heidolph Instruments,
Germany) in 100 mM Tris-HCl buffer with 0.1 mM EDTA and 0.1%
Triton X-100 at 9:1 ratio (pH 7.8). All these processes were performed
on ice. The homogenate was centrifuged at 20,000 g for 20 min at
4°C, supernatant collected, and then aliquot and keep at −20°C for
further analysis. Lipase activity was determined by a commercial kit
(Bionic Co., Iran). Protease activity was determined using the azoca-
sein hydrolysis assay according to the method of Ross et al., 2000.
Intestine endotoxin contents were determined by using an ELISA kit
(Hangzhou Eastbiopharm Co. Ltd, China). All the tests were repli-
cated two times.
2.8 | Challenge study with bacterial
lipopolysaccharide (LPS)
At the end of experiment, five fish (15 per treatment) were intra-
peritoneally injected with 0.1ml of pure LPS from Escherichia coli
serotype 0127:B8 (Sigma-Aldrich, Germany) (5 mg/kg fish). LPS sus-
pension was prepared in the same day by dissolving the lyophilized
cells in sterile physiological serum (0.9% w/v of NaCl). The admin-
istration dose of LPS was chosen based on previous studies on O.
niloticus (Lazado, Skov, & Pedersen, 2016; Lee et al., 2013). Three
fish in the control group (n  =  9) also received an equal volume of
sterile physiological serum without LPS. Blood was collected from
fish in all treatments (four fish per replicate) 24 hr postinjection and
used for various blood and serum immune parameters assays fol-
lowing protocols described earlier. All the tests were replicated two
times.
2.9 | Statistical analysis
The data normality and homogeneity were checked by applying the
Shapiro-Wilk's test and Levene's test for the equality of variances
(p > .05). The data that were not normal and percent data were log10
and arcsine transformed, respectively (Ray & Lotz, 2017). And then,
one-way analysis of variance (ANOVA) followed by Duncan test
carried out to determine significant differences among treatments,
using probiotic supplementation as factor. A repeated-measures
(RM) ANOVA followed by Tukey's test was used for water quality
parameters analysis, and one-way ANOVA was applied to reveal
the differences among study groups at each sampling day (Ray &
Lotz, 2017; Ren, Li, Dong, Tian, & Xue, 2019). The data for the chal-
lenge test were subjected to paired-samples t test to reveal the sig-
nificant differences in parameters before and after LPS injection. In
all cases, an α-value of 0.05 was used to reveal the significant differ-
ences. All the data are presented as mean ± standard deviation (SD),
and Windows SPSS software (version 24) was used for all statistical
analyses.
3 | R E S U LT S
3.1 | Probiotic antimicrobial activity
The B. subtilis bacteria used in this study displayed antagonistic ac-
tivity against A. hydrophila and E. coli determined by agar diffusion
test. The radius of bacteriostatic circle formed by the B. subtilis was
6 and 7 millimetres for A. hydrophila and E. coli, respectively, showing
a high Gram-negative bacteria inhibiting activity.
3.2 | Water quality parameters
The descriptive statistics of various water quality parameters
(mean, SD, maximum and minimum values) in the study groups dur-
ing 50 days of feeding trial are presented in Table 2. Dissolved oxy-
gen concentrations (D.O.), temperature and pH were similar among
study groups (p > .05; Table 2).
Based on the repeated-measures ANOVA test (Table 3), the ef-
fects of time, probiotic level and their interaction on TAN, NO2, NO3
and TSS concentrations were significant. These mean that a signif-
icant variation existed for tested parameters through time (effect
of time), the probiotic effect varied depending on dosage (effect
of probiotic) and the probiotic effect varied through time (effect of
time × probiotic):
From day 7 until the end of experiment, the TAN concentrations
significantly differed among the study groups (p  < .05). The TAN
concentrations drastically increased and reached to the mean peak
values of 0.51 mg/L (T1), 0.51 mg/L (T2), 0.46 mg/L (T3), 0.39 mg/L
(T4) and 0.34 mg/L (T5) on day 14 of the experiment, after which, the
concentrations of TAN started to decrease, except for the control
where TAN decrease started after day 28. As depicted in Figure 1,
the rate of increase or decrease in TAN concentrations was lower
and higher in probiotic-fed groups, respectively. Besides, mean TAN
concentrations in probiotic-fed groups were significantly lowered
versus the control (p < .05). The highest probiotic supplementation
dose (T5) led to the lowest mean TAN concentrations.
MOHAMMADI et al. |
      5

TA B L E 2   The descriptive statistics of various water quality parameters (mean, SD, maximum and minimum values) in the study groups
during 50 days of feeding trial

Treatments

Parameters T1 T2 T3 T4 T5
a a a a
D.O. (mg/L) 6.71 ± 0.47 6.55 ± 0.12 6.56 ± 0.28 6.49 ± 0.14 6.43 ± 0.79a
(5.44–7.89) (5.40–7.77) (5.46–7.69) (5.58–7.95) (5.65–7.72)
Temperature (oC) 27.44 ± 0.38a 27.37 ± 0.43a 27.45 ± 0.45a 27.51 ± 0.38a 27.42 ± 0.39a
(26.50–28.60) (26.30–28.30) (26.20–28.50) (26.30–28.60) (26.20–28.40)
pH 8.11 ± 0.01a 8.09 ± 0.03a 8.10 ± 0.02a 8.09 ± 0.06a 8.07 ± 0.02a
(8.00–8.29) (8.00–8.29) (8.00–8.29) (8.00–8.29) (7.96–8.29)
TAN (mg/L) 0.32 ± 0.15a 0.28 ± 0.14b 0.21 ± 0.13c 0.20 ± 0.14c 0.15 ± 0.11d
(0.02–0.55) (0.01–0.51) (0.03–0.48) (0.03–0.49) (0.02–0.37)
NO2 (mg/L) 0.19 ± 0.12a 0.16 ± 0.10ab 0.16 ± 0.09abc 0.13 ± 0.07bc 0.12 ± 0.07c
(0.02–0.39) (0.01–0.38) (0.03–0.33) (0.03–0.26) (0.02–0.22)
NO3 (mg/L) 14.25 ± 7.69a 13.97 ± 7.63a 13.10 ± 6.01ab 13.13 ± 7.12ab 12.02 ± 5.50 b
(2.06–26.87) (2.28–24.33) (3.14–20.30) (1.38–21.83) (2.59–18.68)
TSS (mg/L) 283 ± 188c (48–610) 288 ± 193c (45–624) 304 ± 199b (48–650) 313 ± 204ab (57–680) 326 ± 209a (58–690)

Note: Values are expressed as mean ± SD (range). Different superscript letters on each row indicate significant differences determined by repeated-
measures (RM) ANOVA followed by Tukey's test (p < .05).

TA B L E 3   The differences in TAN, NO2, NO3 and TSS among study groups at each sampling time and the effects of sampling time,
probiotic level and their interaction on the presented water parameters

One-way ANOVA (p-value) RM ANOVA (p-value)

Parameters Day 1 Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Day 50 T P T × P

TAN (mg/L) NS ** *** ** *** *** *** *** *** *** ***
NO2 (mg/L) NS NS NS NS *** NS ** *** *** ** ***
NO3 (mg/L) NS NS NS NS *** NS NS *** *** ** ***
TSS (mg/L) NS – *** – ** – ** ** *** *** **

Note: The data were subjected to one-way ANOVA to determine whether differences exist among study groups at each sampling time. A repeated-
measures (RM) ANOVA was also used to determine the significance of time (T), probiotic concentration (P) and their interaction (T × P) effects on
measured parameters.
NS p > .05, *p < .05, **p < .01, ***p < .001.

NO2 concentrations gradually increased from the beginning of
the study until day 28. For the first 21 days, the NO2 concentra-
tions were statistically comparable among the study groups at each
sampling point (p  > .05). The highest values for T5 treatment were
recorded on day 21 (0.21 mg/L). In other groups, however, NO2 con-
centrations peaked on day 28 as follows: 0.32 mg/L (T1), 0.32 mg/L
(T2), 0.31 mg/L (T3) and 0.24 mg/L (T4). Afterwards, NO2 started
to decrease and significant differences were detected among study
groups (Table 3). The mean NO2 concentrations in T4 and T5 were
significantly lower compared to the control.
Meanwhile, NO3 concentrations gradually increased during the
initial 21 days of experiment, with no significant differences among
study groups at each sampling day (p  > .05). On day 28, however,
NO3 concentrations significantly differed among study groups. NO3
concentrations in different treatments remained rather constant
after day 28 until the end of experiment. Besides, only the T5 group
demonstrated significantly lower mean nitrate (NO3) concentrations
relative to the control (p < .05).
TSS concentrations gradually increased during the study, and
except for the first sampling day, there were significant differences
among study treatments on other days. The mean TSS concentra-
tions in T3, T4 and T5 groups were significantly higher in comparison
with those of the T2 group or the control.
3.3 | Growth performances
The effects of B. subtilis supplementation in the diet at different
doses for 50 days on the growth performances of O. niloticus are
presented in Table 4. In groups fed T4 and T5 diets, the final weight,
weight gain, FCR and SGR were significantly higher than the other
groups (p < .05). Also, the T5 diet provided the highest weight gain
and SGR. However, the highest condition factor was recorded for
fish in the T4 group, following T5 and T3, which significantly dif-
fered from T2 or the control. No fish mortality occurred during the
experimental period.
 |
6       MOHAMMADI et al.

F I G U R E 1   The fluctuations
(mean ± SD) of TAN, NO2, NO3 and TSS
concentrations from each study group
during the 50-day experimental period
MOHAMMADI et al.       7 |
TA B L E 4   Growth performances of O. niloticus fed different concentrations of probiotic B. subtilis at the end of experiment

Treatments

Parameters T1 T2 T3 T4 T5
a a a a
Initial weight (g) 14.94 ± 0.16 14.83 ± 0.77 14.81 ± 0.24 15.01 ± 0.34 14.52 ± 0.51a
Final weight (g) 52.13 ± 0.27d 56.79 ± 0.37c 60.09 ± 1.52b 65.99 ± 0.83a 67.27 ± 1.52a
e d c b
Weight gain (g) 37.18 ± 0.20 41.96 ± 0.51 45.28 ± 1.61 50.98 ± 0.73 52.74 ± 0.80a
FCR 1.55 ± 0.009a 1.54 ± 0.008a 1.52 ± 0.009b 1.48 ± 0.01c 1.48 ± 0.008c
−1 d c c b
SGR (% day ) 2.49 ± 0.01 2.68 ± 0.09 2.80 ± 0.06 2.96 ± 0.04 3.10 ± 0.07a
Condition factor (%) 1.78 ± 0.02b 1.76 ± 0.09b 1.82 ± 0.03ab 1.88 ± 0.01a 1.86 ± 0.05ab
Survival (%) 100 100 100 100 100

Note: Values are expressed as mean ± standard deviation (SD). Different letters on the rows indicate significant differences by the Duncan test
(p < .05).
Abbreviations: FCR: food conversion ratio, SGR: specific growth rate.

TA B L E 5   Serological parameters of O. niloticus fed different concentrations of probiotic B. subtilis at the end of experiment

Treatments

Parameters T1 T2 T3 T4 T5
a a a b
Glucose (mg/dl) 52.36 ± 1.88 51.91 ± 2.61 50.69 ± 2.23 44.75 ± 2.26 46.42 ± 2.78b
Cholesterol (mg/dl) 221.27 ± 4.07a 209.75 ± 11.60ab 189.77 ± 6.16cd 201.38 ± 4.85bc 185.89 ± 18.20 d
c c b a
Total protein (g/dl) 5.76 ± 0.29 6.02 ± 0.24 6.41 ± 0.21 7.05 ± 0.43 6.61 ± 0.33b
Albumin (g/dl) 2.08 ± 0.05c 2.17 ± 0.17c 2.40 ± 0.12b 2.70 ± 0.12a 2.60 ± 0.09a
c c b a
Globulin (g/dl) 2.00 ± 0.08 2.08 ± 1.09 2.45 ± 0.13 2.76 ± 0.07 2.67 ± 0.12a
Albumin:Globulin 1.04 ± 0.03a 1.04 ± 0.08a 0.98 ± 0.04b 0.97 ± 0.02b 0.97 ± 0.03b
b b ab ab
Total antioxidant capacity (μM/L) 1.23 ± 0.08 1.23 ± 0.09 1.32 ± 0.07 1.33 ± 0.09 1.37 ± 0.07a
Superoxide dismutase activity (units/ml) 105.64 ± 1.74d 107.04 ± 2.47d 115.32 ± 1.96c 128.48 ± 1.50a 123.72 ± 3.88b
a ab b c
Malondialdehyde activity (μM/L) 16.98 ± 0.21 16.23 ± 0.69 15.91 ± 0.80 13.08 ± 1.24 13.37 ± 0.75c
Lysozyme activity (units/ml min−1) 33.88 ± 1.14d 36.40 ± 1.51d 43.78 ± 1.03c 55.12 ± 3.31a 48.23 ± 4.41b
d c b a
Alternative complement activity (units/ml) 126.09 ± 2.94 130.80 ± 3.00 137.22 ± 4.72 150.43 ± 4.58 148.13 ± 1.80a
Total Immunoglobulins (g/dl) 26.71 ± 0.40 c 27.73 ± 1.05c 29.53 ± 1.39ab 30.17 ± 1.07a 28.11 ± 2.40 bc
c b b a
Protease activity (units/ml) 26.82 ± 4.01 31.19 ± 2.54 31.44 ± 0.34 37.70 ± 2.72 35.51 ± 5.14a
Alkaline phosphatase activity (units/L) 48.77 ± 3.98e 54.35 ± 4.55d 62.23 ± 2.46c 74.95 ± 1.89a 67.30 ± 2.08b
d c b a
Respiratory burst activity (OD 650 nm) 0.18 ± 0.02 0.22 ± 0.002 0.27 ± 0.007 0.34 ± 0.02 0.34 ± 0.02a
Endotoxin (EU/ml) 16.22 ± 0.80a 15.89 ± 0.95a 15.90 ± 1.52a 13.66 ± 0.76b 13.90 ± 1.48b

Note: Values are expressed as mean ± standard deviation (SD). Different superscripts in each row indicate significant differences by the Duncan test
(p < .05).

3.4 | Serum parameters resulted in significantly higher superoxide dismutase (SOD) activities

The effects of B. subtilis dietary supplementation for 50 days on
serum biochemical and immunological parameters of O. niloticus are
presented in Table 5. The serum glucose was significantly reduced in
T4 and T5 treatments compared to other groups (p < .05).
The lowest cholesterol levels were recorded for T5 and then
T3, which significantly differed to the control. Total protein values
were significantly higher in T4 relative to other groups. The albu-
min and globulin values were significantly highest in T4 and T5. The
serum total antioxidant capacity (TAC) was significantly enhanced
by the T5 diet compared to the control. Also, T4 and T3 or T5 diets
and the lowest malondialdehyde (MDA), respectively, compared to
other treatments. The fish given T4, T5 and T3 diets had significantly
higher lysozyme (LYZ) activities, respectively, compared to remain-
ing groups. Fish in T4 and T5 displayed significantly higher alterna-
tive complement (ACH50) activities compared to other treatments.
Total immunoglobulin (Ig) of fish in T3 or T4 was significantly higher
compared to other groups. The serum protease activities in T4 or
T5 were significantly higher versus the control. The alkaline phos-
phatase (ALP) activity significantly differed among study groups as
follows: T4 > T5> T3 > T2 > T1. The trend of changes in respiratory
burst activity (RBA) for study groups was similar to ALP but without
 |
8       MOHAMMADI et al.

any significant differences between T4 and T5 groups. Significantly
higher serum endotoxin (LPS) levels were detected in T4 or T5 com-
pared to other groups.
3.5 | Skin mucus parameters
The results of skin mucus parameter analysis of the O. niloticus after
50 days of feeding trial with diets containing different levels of B.
subtilis are presented in Table 6. Fish in T4 displayed significantly
higher mucus total protein and protease activity compared to other
groups (p < .05). Probiotic administration in T3, T4 and T5 resulted in
significantly higher skin mucus LYZ and total Ig relative to the con-
trol. Meanwhile, the highest mucus ACH50 activities were similarly
from T3, T4 and T5, which statistically differed from the other two
treatments.
3.6 | Digestive enzymes and intestinal endotoxin
The results of digestive enzymes and endotoxin analysis in intes-
tines of the O. niloticus after 50 days of feeding trial with incre-
mental doses of B. subtilis are presented in Table 7. Lipase activities
significantly increased in a probiotic dose-dependent manner when
compared to the control (p < .05). Meanwhile, fish in T4 and T5 diets
presented the highest protease activity vice versa the lowest intes-
tinal endotoxin levels, which statistically differed from other groups.
3.7 | Challenge with bacterial lipopolysaccharide
The results of the challenge study of the O. niloticus with lipopoly-
saccharide (LPS) are presented in Figure 2.
The levels of MDA and RBA in the Ctrl/LPS group significantly
increased relative to the Ctrl/NaCl group (p < .05). MDA levels also
significantly increased in all probiotic supplemented groups com-
pared to before challenge, yet they still were significantly lower than
the Ctrl/LPS group (p < .05). Meanwhile, however, fish fed T2 and T3
diets displayed significantly reduced RBA when comparing to before
LPS injection. RBA in fish fed T4 or T5 diets remained unchanged
comparing to before injection (p > .05).
Fish in Ctrl/LPS group showed significantly decreased SOD, ALP,
ACH50 and LYZ activities as well as total Ig concentrations versus
fish in all other groups (p < .05). SOD, ALP and ACH50 activities also
significantly dropped in probiotic-fed groups when comparing to
before LPS challenge, but the levels of these parameters still were
significantly higher than the Ctrl/LPS group. Except for the T5 group,
the total Ig concentrations in other probiotic-fed groups significantly
declined compared to before challenge; however, Ig concentrations
remained statistically higher than the Ctrl/LPS group (p < .05).
4 | D I S CU S S I O N
In the present study, the mean values of water physicochemi-
cal properties or corresponding range values in different treat-
ments were favourable for the production of tilapia in the BFTS
(Avnimelech, 2015). At the beginning of study, the variations of
toxic inorganic nitrogenous compounds, as depicted in Figure 1, fol-
lowed the reported classic sequence of nitrogen species in biofloc
systems which are linked to the growth and development of che-
moautotrophic bacterial populations (Avnimelech, 2015; Ebeling
et al., 2006; Samocha, Prangnell, Samocha, & Staresinic, 2019).
TAN concentrations peaked during the first two weeks due to in-
sufficient ammonia-oxidizing bacteria populations, after which NO2
concentrations accumulated during the initial four weeks as a result
of TAN to NO2 conversion. By the establishment of nitrite-oxidizing
bacteria populations, NO2 concentrations declined, starting from
the 5th week to the end of experiment. This was similar to the re-
sults of Ref. (Luo, Chen, Tan, Abakari, & Tan, 2020). In the current
study, NO3 concentrations gradually increased from the beginning
of the study until the 5th week. The accumulation of NO3 is indica-
tive of the nitrification process in BFTS (Luo et al., 2020). However,
the continued decrease in TAN and NO2 while rather constant NO3
concentrations from 5th week to the end of study suggests that het-
erotrophic bacteria dominated over chemoautotrophs, and nitrogen
removal was mainly controlled by immobilization. Meanwhile, B.
subtilis administration beneficially enhanced water quality in terms

TA B L E 6   Skin mucus parameters of O. niloticus fed different concentrations of probiotic B. subtilis at the end of experiment

Treatments

Parameters T1 T2 T3 T4 T5
c b b a
Total protein (g/L) 172.44 ± 3.33 187.97 ± 3.32 190.42 ± 1.63 206.46.54 ± 1.10 191.93 ± 2.02b
Lysozyme activity (units/ml min−1) 23.16 ± 1.85c 25.13 ± 1.63c 28.68 ± 0.27b 32.28 ± 1.84a 30.85 ± 1.32ab
d c bc a
Protease (units/ml) 8.60 ± 1.34 13.75 ± 1.58 14.89 ± 1.34 19.45 ± 0.87 16.27 ± 0.31b
Alternative complement activity (units/ 83.85 ± 1.65c 87.68 ± 0.87b 91.70 ± 2.70a 93.97 ± 1.27a 92.57 ± 1.17a
ml)
Total Immunoglobulins (g/L) 18.43 ± 0.33c 19.72 ± 1.36c 22.97 ± 1.68b 26.26 ± 1.35a 24.57 ± 1.13ab

Note: Values are expressed as mean ± standard deviation (SD). Different superscripts in each row indicate significant differences by the Duncan test
(p < .05).
MOHAMMADI et al. |
      9

TA B L E 7   Intestines digestive enzymes and endotoxin levels of O. niloticus fed different concentrations of probiotic B. subtilis at the end of
experiment

Treatments

Parameters T1 T2 T3 T4 T5
c bc b a
Protease activity (units/kg) 158.44 ± 3.54 157.66 ± 4.28 163.65 ± 6.82 178.54 ± 4.34 174.94 ± 3.09a
Lipase activity (units/kg) 43.88 ± 3.27d 54.70 ± 6.01c 59.69 ± 1.96b 65.40 ± 3.62a 61.43 ± 4.43ab
a ab b c
Endotoxin (ng/ml extract) 3.67 ± 0.12 3.49 ± 0.19 3.32 ± 0.35 2.97 ± 0.15 3.01 ± 2.29c

Note: Values are expressed as mean ± standard deviation (SD). Different superscripts in each row indicate significant differences by the Duncan test
(p < .05).

of inorganic nitrogen species concentrations. The removal rate of
TAN, NO2 and NO3 was reported 1.17 times higher by the addi-
tion of B. subtilis during aquaculture wastewater treatment (Lu,
Tan, Luo, & Liang, 2012). Also, Bacillus spp. in diet or culture water
significantly reduced ammonia concentrations in the culture of ti-
lapia and common carp in a conventional system (CS) and BFTS,
respectively (Dash et al., 2018; Elsabagh et al., 2018). This can be
attributed to the higher protein digestion/assimilation efficacy by
fish, thus lower faecal nitrogen or ammonia entering the water
(Green, Rawles, Schrader, Gaylord, & McEntire, 2019), supported
by the higher activities of digestive enzymes and fish production in
probiotic groups (Table 7). TSS changes over time reflect the bacte-
rial biomass production in the BFTS (Kamilya et al., 2017). Hence,
the higher nitrogen (TAN, NO2, NO3) uptake by probiotic bacteria
entering into culture water through fish excreta or alteration of the
microbial community by probiotics might resulted in water quality
improvement (Elsabagh et al., 2018; Green et al., 2019; Wang, Li, &
Lin, 2008). Higher levels of TSS are reported by probiotics addition
to the culture water of carp in BFTS (Dash et al., 2018), as was in
the current study. Similarly, Bacillus spp. in the diet of tilapia signifi-
cantly increased total dissolved solids, a parameter closely related
to TSS, in a CS (Elsabagh et al., 2018).
The application of B. subtilis increased the production of tila-
pia (Table 4). Significantly improved growth performances are re-
ported by the application of this probiotic in fish and broilers (Sayed
Hassani, Jourdehi, Zelti, Masouleh, & Lakani, 2020; Sayed Hassani
et al., 2016; Vase-Khavari et al., 2019). Similar beneficial effects
are reported for Bacillus and Lactobacillus probiotics on the growth
performances of tilapia (Elsabagh et al., 2018; Mohammadi, Rafiee,
& Abdelrahman, 2020; Van Doan et al., 2019). Bacillus bacteria col-
onize the digestive tract, produce organic acids and detoxify the
harmful constituents of the feed, thus improving the food digestibil-
ity and absorption (Di et al., 2019; Elsabagh et al., 2018). This is sup-
ported by the increased activity of protease and lipases in digestive
tract of probiotic-fed groups (Table 7). The capabilities of B. subtilis
in the production of extracellular enzymes are well documented (Di
et al., 2019; Guo et al., 2016). For example, tilapia and African cat-
fish fed Bacillus displayed markedly increased protease, lipase and
amylase activities (Liu et al., 2017; Reda, El-Hady, Selim, & El-Sayed,
2018). Besides, the probiotic cells may be directly consumed as a
source of nutrients, supplying the host with vitamins, exogenous nu-
trients, fatty acids (Dhanasiri et al., 2011; Promthale, Pongtippatee,
Withyachumnarnkul, & Wongprasert, 2019; Sugita, Miyajima, &
Deguchi, 1991; Wang, Ran, Ringø, & Zhou, 2018).
B. subtilis positively elevated the concentrations of total pro-
teins, albumin, globulin and total immunoglobulins in plasma
(Table 5). The nutritional and health status of fish as well as its liver
conditions are reflected in these indices. Hence, the promoted lev-
els are redolent of improved nutrition and health status of fish fed
probiotic (Harikrishnan, Kim, Kim, Balasundaram, & Heo, 2011b).
Significantly improved haematology and immunology are reported
by the application of this probiotic in fish and broilers (Sayed
Hassani et al., 2020; Sayed Hassani et al., 2016; Vase-Khavari
et al., 2019). Similar results are reported with the administration of
Bacillus spp. for tilapia and other fish species (Elsabagh et al., 2018;
Harikrishnan et al., 2011b; Kumar, Mukherjee, Prasad, & Pal, 2006;
Reda et al., 2018) in CS.
Meanwhile, fish fed probiotics presented the higher serum
LYZ, ACH50, RBA and protease activities (Table 5). Tilapia fed
Bacillus spp. in CS (Selim & Reda, 2015; Telli et al., 2014) or
Lactobacillus spp. in BFTS (Mohammadi, Rashidian, et al., 2020;
Van Doan et al., 2019) exhibited similar responses. LYZ hydroly-
ses Gram-positive cytoderm and in conjunction with complement
proteins even some Gram-negative bacteria (GNB). The increased
activity of LYZ is attributed to the increased number or activity
of leucocytes (Paulsen, Lunde, Engstad, & Robertsen, 2003). In
the present study, probiotic-fed fish had higher RBA, supporting
the former statement (Table 5). RBA is a critical innate defence
mechanism, leading to the production of oxygen radicals against
pathogens by leucocytes (Harikrishnan et al., 2011b). Besides,
the serum proteases destruct pathogens and activate other im-
mune components such as complement proteins (Hajirezaee,
Mohammadi, & Naserabad, 2020; Modanloo, Soltanian, Akhlaghi,
& Hoseinifar, 2017). The probiotics or their cell components inter-
act with the immune system through microbe-associated molecu-
lar patterns (MAMPs), which are recognized by specific receptors
such as Toll-like receptors. These bindings at gut-associated lym-
phoid tissue stimulate immune cells, leading to the release of cyto-
kines and improved immune responses (Kuebutornye et al., 2020).
Endotoxin is the major component of the cell envelope of GNB
and known as a potent microbial initiator of inflammation (Fujihara
et al., 2003; Swain et al., 2008). Endotoxin can beneficially enhance,
at low levels, or adversely suppress, at high levels, the immune re-
sponses of the host (Guttvik et al., 2002; Nayak et al., 2008). The
 |
10       MOHAMMADI et al.

/
/ l
/

F I G U R E 2   (Continued)
MOHAMMADI et al. |
      11

// l

F I G U R E 2   Results of the challenge study with bacterial lipopolysaccharide (LPS). Values are expressed as mean ± standard deviation (SD).
Different superscripts denote significant differences in parameters after challenge by the Duncan test (p < .05). Arrows on each bar show a
significant increase (↑) or decrease (↓) in parameters compared to before challenge determined by the t test (p < .05)

elevated serum LPS content may be a sign of increased GNB trans- intestinal GNB over-growth or fish immunodeficiency increases
location or infections (Bates, Akerlund, Mittge, & Guillemin, 2007; the risk for endotoxemia, resulting in over-stimulation of immune
Fickert, Pollheimer, Österreicher, & Trauner, 2013). Therefore, cells, inflammation, immunosuppression, and thus reduced fish
any gut barrier dysfunction/damage, gut increased permeability, growth (Berg, 2000; Fickert et al., 2013; Swain et al., 2008; Volkoff
 |
12       MOHAMMADI et al.
& Peter, 2004). For instance, the Gram-negative bacteria Vibrio par-
ahaemolyticus infection impaired intestinal barrier and increased
haemolymph endotoxin in shrimp (Jiao et al., 2020). In the current
study, B. subtilis at high levels effectively lowered the content of
endotoxin in plasma and intestines of tilapia. There are not similar
studies on fish to testify our results. However, probiotics were found
beneficial in improving gut mucosal barrier function and reducing
intestinal and serum endotoxin levels in mammals (He & Shi, 2017;
Sabico et  al.,  2017; Szulińska, Łoniewski, van Hemert, Sobieska, &
Bogdański, 2018; Urao, Fujimoto, Lane, Seo, & Miyano, 1999). These
could be due to the competitive act for adhesion sites and nutri-
ents, production of pathogen inhibiting substances, and improved
gut and/or serum immune responses (Farzanfar, 2006). Significantly
lower Proteobacteria were recorded in the intestines of fish fed pro-
biotic in a CS (Yang et al., 2019) or giant river prawn when probiotics
were added to the water in a BFTS (Miao et al., 2017). The supple-
mentation of tilapia diet with a mixture of antimicrobial peptides
produced by Bacillus significantly inhibited E. coli and lowered serum
endotoxin levels (Chen et al., 2016). This is similar to our study where
B. subtilis showed antagonistic activity against A. hydrophyla and E.
coli (see Section 3.1).
ALP has antimicrobial actions and plays a role in the dephos-
phorylation of various substrates, including LPS. The removal of a
phosphate group from LPS by ALP attenuates the biological effects
of the whole molecule (Bates et al., 2007). Therefore, the higher ac-
tivity of serum ALP in fish fed probiotics (Table 5) may be due to
the low substrate (LPS) concentrations (Chen et al., 2011; Poelstra
et al., 1997), thereby sparing ALP enzyme. This finding is further sup-
ported by studies showing that ALP is located in specific granules in
neutrophils, where also LPS accumulates (Dalmo & Bøgwald, 1996;
Poelstra et al., 1997). Besides, the higher ALP activity of fish fed pro-
biotics has also been attributed to the interactions between probi-
otics and immune cells, leading to stimulated and enhanced immune
responses (Kuebutornye et al., 2020), as discussed earlier.
B. subtilis promoted a protective effect against oxidative stress by
significantly increasing TAC and SOD activities vice versa decreas-
ing MDA activity (Table 5). The antioxidant enzymes nullify reactive
oxygen species (ROS) and their negative effects on cell proteins
and lipids, lowering MDA values (Abarike et al., 2018; Mohammadi,
Rashidian, et al., 2020). Similarly, tilapia fed Bacillus spp. was more
tolerant to oxidative stress in serum, skin mucus and intestines,
indicating better health and immune status (Abarike et al., 2018;
Kuebutornye et al., 2020). Bacillus spp. can act as antigen-present-
ing cells, thereby stimulating the secretion of antioxidant enzymes
by immune cells. They also produce antioxidant enzymes or ROS
scavenging exopolysaccharides, thereby sparing the antioxidant en-
zymes of the host (Kuebutornye et al., 2020). These reinforce the
idea that B. subtilis dietary supplementation at selected concentra-
tions effectively protects tilapia from oxidative stress when reared
in BFTS.
B. subtilis effectively reduced the serum glucose, implying
that fish were experiencing lower stress conditions (Hajirezaee
et al., 2020). The probiotics can lower the effects of stressors,
decrease oxidative stress and reduce intestinal endotoxin, inflam-
mation, insulin resistance and hyperglycaemia (Neissi, Rafiee,
Nematollahi, Razavi, & Maniei, 2015; Ruan et al., 2015). Similar
results were observed in other fish species fed different probiot-
ics in CS (Al-Dohail, Hashim, & Aliyu-Paiko, 2009; Chen, Wooster,
& Bowser, 2004; Neissi et al., 2015). The reduced serum choles-
terol indicates better health conditions and flesh quality (Al-Dohail
et al., 2009). The decreased serum cholesterol herein is similar to
the findings of (Dawood, Koshio, Ishikawa, & Yokoyama, 2015; Ngugi
et al., 2015). Such beneficial effects are attributed to the modulatory
role of probiotics on cholesterol and lipid metabolism in the liver, or
they can directly assimilate cholesterol and interfere with choles-
terol absorption by the host (Dehkohneh, Jafari, & Fahimi, 2019; Kim
et al., 2017; Rawls, Samuel, & Gordon, 2004).
The fish skin mucosal layer (SML) is the first line of defence
against pathogens. The protein fraction of mucus plays a cen-
tral role in maintaining mucosal health (Lazado & Caipang, 2014;
Mohammadi, Rashidian, et al., 2020). The SML proteins were en-
hanced by B. subtilis (Table 6). This is similar to the results of previ-
ous studies (Kuebutornye et al., 2020; Mohammadi, Rashidian, et al.,
2020; Van Doan et al., 2019). It might be due to promoted activity or
count of immune cells in mucosal-associated lymphoid tissue. These
cells are recruited/directed to different tissues upon receiving sig-
nals, such as cytokines and chemokines (Lazado & Caipang, 2014).
This study affirms that B. subtilis positively stimulates the mucosal
immune system of tilapia in BFTS.
There is a lack of studies on the protective effects of probiotic
against LPS-induced damages in fish. Therefore, the efficacy of B.
subtilis in promoting the immune function and protection of tilapia
was further studied by conducting a challenge experiment with
bacterial LPS. LPS can cause oxidative stress through excessive
ROS formation, leading to metabolic and immunological alterations,
immune system impairment and thus disease progress (Jiang et al.,
2017; Li et al., 2020). LPS injection herein significantly reduced SOD
vice versa increased MDA and RBA in the serum of Ctrl/LPS group
(Figure 2). Similarly, different fish species injected with LPS at 4 mg/
kg or Chinese mitten crab at 400 μg/kg showed a profound reduc-
tion in SOD and elevation in MDA, RBA and ROS (Jiang et al., 2017;
Li et al., 2020; Liu et al., 2019; Ren, Xu, et al., 2019). However, SOD,
MDA and RBA levels in probiotic groups were significantly improved
after challenge compared to Ctrl/LPS group. In fact, probiotic sup-
plementation markedly inhibited the increase in MDA or RBA in
serum upon LPS challenge, which was concomitant with higher SOD
activity. It suggests that B. subtilis attenuated LPS-induced oxida-
tive stress in fish by its antioxidative capacities, as discussed ear-
lier. Such beneficial effects against LPS are documented in rats fed
Lactobacillus spp. and Bifidobacterium infantis (Osman, Adawi, Ahrné,
Jeppsson, & Molin, 2007; Y. Wang et al., 2013).
LPS challenge significantly reduced serum ALP, ACH50, LYZ and
total Ig in Ctrl/LPS group (Figure 2). This is similar to other studies
either injecting fish with LPS or giving them diets containing LPS
(Lazado et al., 2016; Li et al., 2020; Li, et al., 2019; Nayak et al., 2008;
Nya & Austin, 2010). ACH50 is the main pathway to complement
MOHAMMADI et al.
activation when encountering LPS (Nya & Austin, 2010). LYZ, ACH50
and Ig are involved in elimination, opsonization and detoxification
of pathogens and their toxins (Hajirezaee et al., 2020). Therefore,
LPS as a MAMP might decline these parameters through over-stim-
ulation and exhaustion of immune cells. LPS exposure profoundly
elevated intracellular ROS production and depleted ATP production
in fish lymphocytes (Xiang, Peng, Dong, Yang, & Shao, 2008). These
statements are further supported by previous studies showing in-
creased LYZ, ACH50 and Ig at low, but not high LPS concentrations
(Bich Hang, Milla, Gillardin, Phuong, & Kestemont, 2013; Nayak
et al., 2008; Nya & Austin, 2010). The reduced ALP after the chal-
lenge is related to the detoxification of bacterial LPS by altering its
chemical structure (Ran et al., 2016). The biological effects of LPS
such as toxicity and macrophage or complement activation are lost
following such modifications (Bates et al., 2007). Hence, the reduced
ALP after the challenge is suggestive of the protection of fish against
LPS.
On the other hand, fish fed probiotic diets displayed significantly
reduced ACH50, LYZ, ALP and total Ig compared to before challenge
(Figure 2). However, these reductions were significantly attenuated
by probiotic supplementation. These data show that probiotic could
help fish to cope with LPS challenge. Such beneficial effects of immu-
nostimulants other than probiotics against LPS in fish are reported
by previous studies (Li et al., 2020; Li, et al., 2019). In addition, feed-
ing diets containing probiotics remarkedly restored immune parame-
ters in fish subjected to pathogenic bacteria (Harikrishnan, Kim, Kim,
Balasundaram, & Heo, 2011a, 2011c; Kumar et al., 2006; Pirarat,
Kobayashi, Katagiri, Maita, & Endo, 2006).
5 | CO N C LU S I O N
In conclusion, the present study demonstrated that B. subtilis sup-
plementation in the diet of O. niloticus cultured using BFTS effec-
tively enhanced water quality, fish production and fish health status,
as well as decreased endotoxemia. The results also showed that LPS
injection induced oxidative stress and impaired immune responses.
However, B. subtilis supplementation successfully relieved fish from
LPS-induced damages. The protective effects of the probiotic B. sub-
tilis on reducing the severity of LPS-induced damages may be related
to promoting the levels of immune factors and antioxidant enzymes.
AC K N OW L E D G E M E N T S
The authors express their sincere thanks to Mr. Reza Ashouri, Mr.
Farhad Konyeh, Mr. Abdolmajid Abdi and Mr. Bahman Pouryounes
at the University of Tehran, Iran, for their kind assistance in differ-
ent steps of this study. We also appreciate anonymous reviewers for
their valuable remarks which contributed to the quality of this paper.
DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets generated during and/or analysed during the current
study are available from the corresponding author on request.
|
      13
ORCID
Ghasem Mohammadi  https://orcid.org/0000-0001-9711-7827
Taida Juliana Adorian  https://orcid.org/0000-0002-8217-0067
Gholamreza Rafiee  https://orcid.org/0000-0002-0621-8890
REFERENCES
Abarike, E. D., Cai, J., Lu, Y., Yu, H., Chen, L., Jian, J., … Kuebutornye,
F. K. A. (2018). Effects of a commercial probiotic BS containing
Bacillus subtilis and Bacillus licheniformis on growth, immune re-
sponse and disease resistance in Nile tilapia, Oreochromis niloticus.
Fish & Shellfish Immunology, 82, 229–238. https://doi.org/10.1016/j.
fsi.2018.08.037
Abu-Elala, N., Marzouk, M., & Moustafa, M. (2013). Use of differ-
ent Saccharomyces cerevisiae biotic forms as immune-modula-
tor and growth promoter for Oreochromis niloticus challenged
with some fish pathogens. International Journal of Veterinary
Science and Medicine, 1(1), 21–29. http://dx.doi.org/10.1016/j.
ijvsm.2013.05.001
Al-Dohail, M. A., Hashim, R., & Aliyu-Paiko, M. (2009). Effects of
the probiotic, Lactobacillus acidophilus, on the growth perfor-
mance, haematology parameters and immunoglobulin concen-
tration in African Catfish (Clarias gariepinus, Burchell 1822) fin-
gerling. Aquaculture Research, 40(14), 1642–1652. http://dx.doi.
org/10.1111/j.1365-2109.2009.02265.x
Allameh, S. K., Yusoff, F. M., Ringø, E., Daud, H. M., Saad, C. R., & Ideris,
A. (2016). Effects of dietary mono- and multiprobiotic strains on
growth performance, gut bacteria and body composition of Javanese
carp (Puntius gonionotus, Bleeker 1850). Aquaculture Nutrition, 22(2),
367–373. http://dx.doi.org/10.1111/anu.12265
Avnimelech, Y. (2015). Biofloc technology-A practical guide book (3rd edn.,
pp. 1–258). Baton Rouge, LA: The World Aquaculture Society.
Bates, J. M., Akerlund, J., Mittge, E., & Guillemin, K. (2007). Intestinal Alkaline
Phosphatase Detoxifies Lipopolysaccharide and Prevents Inflammation
in Zebrafish in Response to the Gut Microbiota. Cell Host & Microbe,
2(6), 371–382. https://doi.org/10.1016/j.chom.2007.10.010
Ben Hamed, S., Tavares Ranzani-Paiva, M. J., Tachibana, L., de Carla Dias,
D., Ishikawa, C. M., & Esteban, M. A. (2018). Fish pathogen bacte-
ria: Adhesion, parameters influencing virulence and interaction with
host cells. Fish & Shellfish Immunology, 80, 550–562. https://doi.
org/10.1016/j.fsi.2018.06.053
Berg, R. D. (2000). Bacterial translocation from the gastrointestinal tract.
Advances in Experimental Medicine and Biology, 437, 11–30. https://
doi.org/10.1007/978-1-4615-4143-1_2
Bich Hang, B. T., Milla, S., Gillardin, V., Phuong, N. T., & Kestemont, P.
(2013). In vivo effects of Escherichia coli lipopolysaccharide on reg-
ulation of immune response and protein expression in striped catfish
(Pangasianodon hypophthalmus). Fish & Shellfish Immunology, 34(1),
339–347. https://doi.org/10.1016/J.FSI.2012.11.025
Cha, J. H., Rahimnejad, S., Yang, S. Y., Kim, K. W., & Lee, K. J. (2013).
Evaluations of Bacillus spp. as dietary additives on growth performance,
innate immunity and disease resistance of olive flounder (Paralichthys ol-
ivaceus) against Streptococcus iniae and as water additives. Aquaculture,
402-403, 50–57. https://doi.org/10.1016/j.aquac​ulture.2013.03.030
Chen, C. Y., Wooster, G. A., & Bowser, P. R. (2004). Comparative blood
chemistry and histopathology of tilapia infected with Vibrio vulnifi-
cus or Streptococcus iniae or exposed to carbon tetrachloride, genta-
micin, or copper sulfate. Aquaculture, 239(1-4), 421–443. https://doi.
org/10.1016/j.aquac​ulture.2004.05.033
Chen, K. T., Malo, M. S., Beasley-Topliffe, L. K., Poelstra, K., Millan, J.
L., Mostafa, G., … Hodin, R. A. (2011). A role for intestinal alkaline
phosphatase in the maintenance of local gut immunity. Digestive
Diseases and Sciences, 56(4), 1020–1027. https://doi.org/10.1007/
s1062​0 -010-1396-x
 |
14       MOHAMMADI et al.
Chen, Y. B., Hu, J., Lyu, Q. J., Liu, L. J., Wen, L. F., Yang, X. K., & Zhao,
H. H. (2016). The effects of Natucin C-Natucin P mixture on blood
biochemical parameters, antioxidant activity and non-specific im-
mune responses in tilapia (Oreochromis niloticus). Fish & Shellfish
Immunology, 55, 367–373. https://doi.org/10.1016/j.fsi.2016.06.016
Clesceri, L. S., Greenbaerg, A. E., & Eaton, A. D. (1998). Standard methods
for examination of water and wastewater. Washington, DC: American
Public Health Association (APHA).
Dalmo, R. A., & Bøgwald, J. (1996). Distribution of intravenously and
perorally administeredAeromonas salmonicidalipopolysaccharide in
Atlantic salmon,Salmo salarL. Fish & Shellfish Immunology, 6(6), 427–
441. https://doi.org/10.1006/fsim.1996.0041
Dash, P., Tandel, R. S., Bhat, R. A. H., Mallik, S., Pandey, N. N., Singh,
A. K., & Sarma, D. (2018). The addition of probiotic bacteria to mi-
crobial floc: Water quality, growth, non-specific immune response
and disease resistance of Cyprinus carpio in mid-Himalayan alti-
tude. Aquaculture, 495, 961–969. https://doi.org/10.1016/j.aquac​
ulture.2018.06.056
Dawood, M. A. O., & Koshio, S. (2016). Recent advances in the role of
probiotics and prebiotics in carp aquaculture: A review. Aquaculture,
454, 243–251. https://doi.org/10.1016/j.aquac​ulture.2015.12.033
Dawood, M. A. O., Koshio, S., Ishikawa, M., & Yokoyama, S. (2015). Effects
of heat killed lactobacillus plantarum (LP20) supplemental diets on
growth performance, stress resistance and immune response of
red sea bream, pagrus major. Aquaculture, 442, 29–36. https://doi.
org/10.1016/j.aquac​ulture.2015.02.005
Dehkohneh, A., Jafari, P., & Fahimi, H. (2019). Effects of probiotic
Lactobacillus paracasei TD3 on moderation of cholesterol biosyn-
thesis pathway in rats. Iranian Journal of Basic Medical Sciences, 22(9),
1004–1009. https://doi.org/10.22038​/ijbms.2019.33933.8073
Dhanasiri, A. K. S., Brunvold, L., Brinchmann, M. F., Korsnes, K., Bergh,
Ø., & Kiron, V. (2011). Changes in the Intestinal Microbiota of Wild
Atlantic cod Gadus morhua L. Upon Captive Rearing. Microbial
Ecology, 61(1), 20–30. https://doi.org/10.1007/s0024​8-010-9673-y
Di, J., Chu, Z., Zhang, S., Huang, J., Du, H., & Wei, Q. (2019). Evaluation
of the potential probiotic Bacillus subtilis isolated from two ancient
sturgeons on growth performance, serum immunity and disease
resistance of Acipenser dabryanus. Fish & Shellfish Immunology, 93,
711–719. https://doi.org/10.1016/j.fsi.2019.08.020
Ebeling, J. M., Timmons, M. B., & Bisogni, J. J. (2006). Engineering anal-
ysis of the stoichiometry of photoautotrophic, autotrophic, and het-
erotrophic removal of ammonia-nitrogen in aquaculture systems.
Aquaculture, 257(1-4), 346–358. https://doi.org/10.1016/j.aquac​
ulture.2006.03.019
Ellis, A. E. (1990). Lysozyme assays. Techniques in Fish Immunology, 1,
101–103.
Elsabagh, M., Mohamed, R., Moustafa, E. M., Hamza, A., Farrag, F.,
Decamp, O., … Eltholth, M. (2018). Assessing the impact of Bacillus
strains mixture probiotic on water quality, growth performance,
blood profile and intestinal morphology of Nile tilapia, Oreochromis
niloticus. Aquaculture Nutrition, 24(6), 1613–1622. http://dx.doi.
org/10.1111/anu.12797
Farzanfar, A. (2006). The use of probiotics in shrimp aquaculture: Table
1. FEMS Immunology & Medical Microbiology, 48(2), 149–158. http://
dx.doi.org/10.1111/j.1574-695x.2006.00116.x
Fickert, P., Pollheimer, M. J., Österreicher, C. H., & Trauner, M. (2013).
Animal models of cholestasis. In P. M. Conn (Ed.), Animal Models for
the Study of Human Disease, (Vol. 1, 1st edn., pp. 331–410). San Diego,
CA: Academic Press. https://doi.org/10.1016/B978-0-12-41589​
4-8.00015​- 4
Fujihara, M., Muroi, M., Tanamoto, K. I., Suzuki, T., Azuma, H., & Ikeda,
H. (2003). Molecular mechanisms of macrophage activation and
deactivation by lipopolysaccharide: Roles of the receptor com-
plex. Pharmacology & Therapeutics, 100(2), 171–194. https://doi.
org/10.1016/j.pharm​thera.2003.08.003
Galina, J. (2017). Fish Diseases: Prevention and Control Strategies (Vol. 1, 1,
1–278). San Diego, CA: Academic Press.
Gobi, N., Vaseeharan, B., Chen, J. C., Rekha, R., Vijayakumar, S., Anjugam,
M., & Iswarya, A. (2018). Dietary supplementation of probiotic
Bacillus licheniformis Dahb1 improves growth performance, mucus
and serum immune parameters, antioxidant enzyme activity as well
as resistance against Aeromonas hydrophila in tilapia Oreochromis
mossambicus. Fish & Shellfish Immunology, 74, 501–508. https://doi.
org/10.1016/j.fsi.2017.12.066
Green, B. W., Rawles, S. D., Schrader, K. K., Gaylord, T. G., & McEntire,
M. E. (2019). Effects of dietary protein content on hybrid tilapia
(Oreochromis aureus × O. niloticus) performance, common micro-
bial off-flavor compounds, and water quality dynamics in an outdoor
biofloc technology production system. Aquaculture, 503, 571–582.
https://doi.org/10.1016/j.aquac​ulture.2019.01.034
Guo, X., Chen, D. D., Peng, K. S., Cui, Z. W., Zhang, X. J., Li, S., & Zhang, Y.
A. (2016). Identification and characterization of Bacillus subtilis from
grass carp (Ctenopharynodon idellus) for use as probiotic additives
in aquatic feed. Fish & Shellfish Immunology, 52, 74–84. https://doi.
org/10.1016/j.fsi.2016.03.017
Guttvik, A., Paulsen, B., Dalmo, R. A., Espelid, S., Lund, V., & Bøgwald,
J. (2002). Oral administration of lipopolysaccharide to Atlantic
salmon (Salmo salar L.) fry. Uptake, distribution, influence on growth
and immune stimulation. Aquaculture, 214(1-4), 35–53. https://doi.
org/10.1016/S0044​-8486(02)00358​-7.
Hajirezaee, S., Mohammadi, G., & Naserabad, S. S. (2020). The protective
effects of vitamin C on common carp (Cyprinus carpio) exposed to
titanium oxide nanoparticles (TiO2-NPs). Aquaculture, 518, 734734.
https://doi.org/10.1016/j.aquac​ulture.2019.734734
Harikrishnan, R., Kim, M. C., Kim, J. S., Balasundaram, C., & Heo, M. S.
(2011a). Immunomodulatory effect of probiotics enriched diets on
Uronema marinum infected olive flounder. Fish & Shellfish Immunology,
30(3), 964–971. https://doi.org/10.1016/j.fsi.2011.01.030
Harikrishnan, R., Kim, M. C., Kim, J. S., Balasundaram, C., & Heo, M. S.
(2011b). Probiotics and herbal mixtures enhance the growth, blood
constituents, and nonspecific immune response in Paralichthys oli-
vaceus against Streptococcus parauberis. Fish & Shellfish Immunology,
31(2), 310–317. https://doi.org/10.1016/j.fsi.2011.05.020
Harikrishnan, R., Kim, M.-C., Kim, J.-S., Balasundaram, C., & Heo, M.-S.
(2011). Probiotics and herbal mixtures enhance the growth, blood
constituents, and nonspecific immune response in Paralichthys oli-
vaceus against Streptococcus parauberis. Fish & Shellfish Immunology,
31(2), 310–317. http://dx.doi.org/10.1016/j.fsi.2011.05.020
He, M., & Shi, B. (2017). Gut microbiota as a potential target of metabolic
syndrome: The role of probiotics and prebiotics. Cell & Bioscience,
7(1). http://dx.doi.org/10.1186/s1357​8-017-0183-1
Iranian Standard and Industrial Researches Institute (2006). Horizontal
method for the enumeration of presumptive Bacillus cereus. Microbiology
of food and animal feeding stuffs, Method 2324, (Vol. 1, 1st edn., pp. 1–
29). Tehran, Iran: Institute of Standard and Industrial Research of Iran.
Jiang, J., Yin, L., Li, J.-Y., Li, Q., Shi, D., Feng, L., … Zhou, X.-Q. (2017).
Glutamate attenuates lipopolysaccharide-induced oxidative dam-
age and mRNA expression changes of tight junction and defensin
proteins, inflammatory and apoptosis response signaling molecules
in the intestine of fish. Fish and Shellfish Immunology, 70, 473–484.
https://doi.org/10.1016/j.fsi.2017.09.035
Jiao, L. F., Dai, T. M., Zhong, S. Q., Jin, M., Sun, P., & Zhou, Q. C. (2020).
Vibrio parahaemolyticus infection impaired intestinal barrier func-
tion and nutrient absorption in Litopenaeus vannamei. Fish & Shellfish
Immunology, 99, 184–189. https://doi.org/10.1016/j.fsi.2020.02.009
Kamilya, D., Debbarma, M., Pal, P., Kheti, B., Sarkar, S., & Singh, S. T.
(2017). Biofloc technology application in indoor culture of Labeo ro-
hita (Hamilton, 1822) fingerlings: The effects on inorganic nitrogen
control, growth and immunity. Chemosphere, 182, 8–14. https://doi.
org/10.1016/j.chemo​sphere.2017.05.021
MOHAMMADI et al.
Kim, S.-J., Park, S., Sin, H.-S., Jang, S.-H., Lee, S.-W., Kim, S.-Y., … Yang,
D. (2017). Hypocholesterolemic effects of probiotic mixture on di-
et-induced hypercholesterolemic rats. Nutrients, 9(3), 293. https://
doi.org/10.3390/nu903​0293
Kuebutornye, F. K. A., Abarike, E. D., & Lu, Y. (2019). A review on the
application of Bacillus as probiotics in aquaculture. Fish & Shellfish
Immunology, 87, 820–828. https://doi.org/10.1016/j.fsi.2019.02.010
Kuebutornye, F. K. A., Wang, Z., Lu, Y., Abarike, E. D., Sakyi, M. E., Li, Y.,
… Hlordzi, V. (2020). Effects of three host-associated Bacillus spe-
cies on mucosal immunity and gut health of Nile tilapia, Oreochromis
niloticus and its resistance against Aeromonas hydrophila infection.
Fish & Shellfish Immunology, 97, 83–95. https://doi.org/10.1016/J.
FSI.2019.12.046
Kumar, R., Mukherjee, S. C., Prasad, K. P., & Pal, A. K. (2006). Evaluation
of Bacillus subtilis as a probiotic to Indian major carp Labeo ro-
hita (Ham.). Aquaculture Research, 37(12), 1215–1221. https://doi.
org/10.1111/j.1365-2109.2006.01551.x
Lazado, C. C., & Caipang, C. M. A. (2014). Mucosal immunity and pro-
biotics in fish. Fish & Shellfish Immunology, 39(1), 78–89. https://doi.
org/10.1016/j.fsi.2014.04.015
Lazado, C. C., Skov, P. V., & Pedersen, P. B. (2016). Innate immune de-
fenses exhibit circadian rhythmicity and differential temporal sen-
sitivity to a bacterial endotoxin in Nile tilapia (Oreochromis niloticus).
Fish & Shellfish Immunology, 55, 613–622. https://doi.org/10.1016/j.
fsi.2016.06.040
Lee, S.-H., Peng, K.-C., Lee, L.-H., Pan, C.-Y., Hour, A.-L., Her, G. M.,
… Chen, J.-Y. (2013). Characterization of tilapia (Oreochromis ni-
loticus) viperin expression, and inhibition of bacterial growth and
modulation of immune-related gene expression by electrotransfer
of viperin DNA into zebrafish muscle. Veterinary Immunology and
Immunopathology, 151(3-4), 217–228. https://doi.org/10.1016/j.
vetimm.2012.11.010
Li, M.-Y., Guo, W.-Q., Guo, G.-L., Zhu, X.-M., Niu, X.-T., Shan, X.-F.,
… Zhang, D.-M. (2020). Effects of dietary astaxanthin on lipo-
polysaccharide-induced oxidative stress, immune responses and
glucocorticoid receptor (GR)-related gene expression in Channa
argus. Aquaculture, 517, 734816. https://doi.org/10.1016/J.AQUAC​
ULTURE.2019.734816
Li, M.-Y., Zhu, X.-M., Niu, X.-T., Chen, X.-M., Tian, J.-X., Kong, Y.-D., …
Wang, G.-Q. (2019). Effects of dietary Allium mongolicum Regel
polysaccharide on growth, lipopolysaccharide-induced antioxidant
responses and immune responses in Channa argus. Molecular Biology
Reports, 46(2), 2221–2230. https://doi.org/10.1007/s1103​ 3-019-
04677​-y
Liu, H., Wang, S., Cai, Y., Guo, X., Cao, Z., Zhang, Y., … Zhou, Y. (2017).
Dietary administration of Bacillus subtilis HAINUP40 enhances
growth, digestive enzyme activities, innate immune responses and
disease resistance of tilapia, Oreochromis niloticus. Fish & Shellfish
Immunology, 60, 326–333. https://doi.org/10.1016/j.fsi.2016.12.003
Liu, J.-D., Liu, W.-B., Zhang, C.-Y., Xu, C.-Y., Zheng, X.-C., Zhang, D.-D.,
& Chi, C. (2020). Dietary glutathione supplementation enhances an-
tioxidant activity and protects against lipopolysaccharide-induced
acute hepatopancreatic injury and cell apoptosis in Chinese mitten
crab, Eriocheir sinensis. Fish & Shellfish Immunology, 97, 440–454.
https://doi.org/10.1016/j.fsi.2019.12.049
Lu, L., Tan, H., Luo, G., & Liang, W. (2012). The effects of Bacillus sub-
tilis on nitrogen recycling from aquaculture solid waste using het-
erotrophic nitrogen assimilation in sequencing batch reactors.
Bioresource Technology, 124, 180–185. https://doi.org/10.1016/j.
biort​ech.2012.07.084
Luo, G., Chen, X., Tan, J., Abakari, G., & Tan, H. (2020). Effects of carbo-
hydrate addition strategy and biofloc levels on the establishment of
nitrification in biofloc technology aquaculture systems. Aquaculture,
514, 734441. https://doi.org/10.1016/j.aquac​ulture.2019.734441
|
      15
Miao, S., Zhu, J., Zhao, C., Sun, L., Zhang, X., & Chen, G. (2017). Effects
of C/N ratio control combined with probiotics on the immune re-
sponse, disease resistance, intestinal microbiota and morphology
of giant freshwater prawn (Macrobrachium rosenbergii). Aquaculture,
476, 125–133. https://doi.org/10.1016/j.aquac​ulture.2017.04.027
Modanloo, M., Soltanian, S., Akhlaghi, M., & Hoseinifar, S. H. (2017).
The effects of single or combined administration of galactooligo-
saccharide and Pediococcus acidilactici on cutaneous mucus im-
mune parameters, humoral immune responses and immune related
genes expression in common carp (Cyprinus carpio) fingerlings. Fish
& Shellfish Immunology, 70, 391–397. https://doi.org/10.1016/j.
fsi.2017.09.032
Mohammadi, G., Rafiee, G., & Abdelrahman, H. A. (2020). Effects of di-
etary Lactobacillus plantarum (KC426951) in biofloc and stagnant-re-
newal culture systems on growth performance, mucosal parameters,
and serum innate responses of Nile tilapia Oreochromis niloticus.
Fish Physiology and Biochemistry, 4, 1–15. http://dx.doi.org/10.1007/
s1069​5-020-00777​-w
Mohammadi, G., Rashidian, G., Hoseinifar, S. H., Naserabad, S. S., &
Doan, H. V. (2020). Ginger (Zingiber officinale) extract affects
growth performance, body composition, haematology, serum and
mucosal immune parameters in common carp (Cyprinus carpio). Fish
& Shellfish Immunology, 99, 267–273. https://doi.org/10.1016/j.
fsi.2020.01.032
Mokkala, K., Laitinen, K., & Röytiö, H. (2016). Bifidobacterium lactis
420 and fish oil enhance intestinal epithelial integrity in Caco-2
cells. Nutrition Research, 36(3), 246–252. https://doi.org/10.1016/j.
nutres.2015.11.014
Nardocci, G., Navarro, C., Cortés, P. P., Imarai, M., Montoya, M.,
Valenzuela, B., … Fernández, R. (2014). Neuroendocrine mecha-
nisms for immune system regulation during stress in fish. Fish &
Shellfish Immunology, 40(2), 531–538. https://doi.org/10.1016/j.
fsi.2014.08.001
Nayak, S. K., Swain, P., Nanda, P. K., Dash, S., Shukla, S., Meher, P. K., &
Maiti, N. K. (2008). Effect of endotoxin on the immunity of Indian
major carp, Labeo rohita. Fish & Shellfish Immunology, 24(4), 394–399.
https://doi.org/10.1016/j.fsi.2007.09.005
Neissi, A., Rafiee, G., Nematollahi, M., Razavi, S. H., & Maniei, F.
(2015). Influence of supplemented diet with Pediococcus acidilac-
tici on non-specific immunity and stress indicators in green terror
(Aequidens rivulatus) during hypoxia. Fish & Shellfish Immunology,
45(1), 13–18. https://doi.org/10.1016/j.fsi.2015.04.008
Ngugi, C. C., Oyoo-Okoth, E., Mugo-Bundi, J., Orina, P. S., Chemoiwa,
E. J., & Aloo, P. A. (2015). Effects of dietary administration of sting-
ing nettle (Urtica dioica) on the growth performance, biochemical,
hematological and immunological parameters in juvenile and adult
Victoria Labeo (Labeo victorianus) challenged with Aeromonas hy-
drophila. Fish & Shellfish Immunology, 44(2), 533–541. https://doi.
org/10.1016/j.fsi.2015.03.025
Nya E. J., & Austin B. (2010). Use of bacterial lipopolysaccharide (LPS)
as an immunostimulant for the control ofAeromonas hydrophilain-
fections in rainbow troutOncorhynchus mykiss(Walbaum).
Journal of Applied Microbiology, 108(2), 686–694. http://dx.doi.
org/10.1111/j.1365-2672.2009.04464.x
Osman, N., Adawi, D., Ahrné, S., Jeppsson, B., & Molin, G. (2007).
Endotoxin- and d-galactosamine-induced liver injury improved by
the administration of Lactobacillus, Bifidobacterium and blueberry.
Digestive and Liver Disease, 39(9), 849–856. https://doi.org/10.1016/J.
DLD.2007.06.001
Pacheco-Vega, J. M., Cadena-Roa, M. A., Leyva-Flores, J. A., Zavala-Leal,
O. I., Pérez-Bravo, E., & Ruiz-Velazco, J. M. J. (2018). Effect of isolated
bacteria and microalgae on the biofloc characteristics in the Pacific
white shrimp culture. Aquaculture Reports, 11, 24–30. https://doi.
org/10.1016/j.aqrep.2018.05.003
 |
16       MOHAMMADI et al.
Palaksha, K. J., Shin, G. W., Kim, Y. R., & Jung, T. S. (2008). Evaluation
of non-specific immune components from the skin mucus of olive
flounder (Paralichthys olivaceus). Fish & Shellfish Immunology, 24(4),
479–488. https://doi.org/10.1016/j.fsi.2008.01.005
Parson, T. R., Maita, Y., & Lalli, C. M. (1984). A Manual of chemical and
biological methods for seawater analysis (Vol. 1, 1 edn., pp. 1–173).
Oxford, UK. Pergamon.
Paulsen, S. M., Lunde, H., Engstad, R. E., & Robertsen, B. (2003). In vivo
effects of β-glucan and LPS on regulation of lysozyme activity and
mRNA expression in Atlantic salmon (Salmo salar L.). Fish & Shellfish
Immunology, 14(1), 39–54. https://doi.org/10.1006/fsim.2002.0416
Pirarat, N., Kobayashi, T., Katagiri, T., Maita, M., & Endo, M. (2006). Protective
effects and mechanisms of a probiotic bacterium Lactobacillus rham-
nosus against experimental Edwardsiella tarda infection in tilapia
(Oreochromis niloticus). Veterinary Immunology and Immunopathology,
113(3-4), 339–347. https://doi.org/10.1016/j.vetimm.2006.06.003
Plant, K. P., & LaPatra, S. E. (2011). Advances in fish vaccine delivery.
Developmental & Comparative Immunology, 35(12), 1256–1262.
https://doi.org/10.1016/j.dci.2011.03.007
Poelstra, K., Bakker, W. W., Klok, P. A., Kamps, J. A. A. M., Hardonk,
M. J., & Meijer, D. K. F. (1997). Dephosphorylation of endotoxin by
alkaline phosphatase in vivo. American Journal of Pathology, 151(4),
1163–1169.
Prangnell, D. I., Castro, L. F., Ali, A. S., Browdy, C. L., Zimba, P. V., Laramore,
S. E., & Samocha T. M. (2016). Some Limiting Factors in Superintensive
Production of Juvenile Pacific White Shrimp,Litopenaeus vanna-
mei, in No-water-exchange, Biofloc-dominated Systems. Journal
of the World Aquaculture Society, 47(3), 396–413. http://dx.doi.
org/10.1111/jwas.12275
Prangnell, D. I., Samocha, T. M., & Staresinic, N. (2019). Water. In T. M.
Samocha (Ed.), Sustainable Biofloc Systems for Marine Shrimp (Vol. 1,
1 st edn., pp. 37–58). San Diego, CA: Academic Press. https://doi.
org/10.1016/B978-0-12-81804​0 -2.00004​-6
Pridgeon J. W., & Klesius, P. H. (2012). Major bacterial diseases in aqua-
culture and their vaccine development. CAB Reviews: Perspectives in
Agriculture, Veterinary Science, Nutrition and Natural Resources, 7(48),
1–16. http://dx.doi.org/10.1079/pavsn​nr201​27048
Promthale, P., Pongtippatee, P., Withyachumnarnkul, B., & Wongprasert,
K. (2019). Bioflocs substituted fishmeal feed stimulates immune
response and protects shrimp from Vibrio parahaemolyticus in-
fection. Fish & Shellfish Immunology, 93, 1067–1075. https://doi.
org/10.1016/j.fsi.2019.07.084
Ran, C., Huang, L. U., Hu, J., Tacon, P., He, S., Li, Z., … Zhou, Z. (2016).
Effects of dietary live and heat-inactive baker’s yeast on growth,
gut health, and disease resistance of Nile tilapia under high rear-
ing density. Fish & Shellfish Immunology, 56, 263–271. https://doi.
org/10.1016/j.fsi.2016.07.001
Rawls, J. F., Samuel, B. S., & Gordon, J. I. (2004). From The Cover:
Gnotobiotic zebrafish reveal evolutionarily conserved responses to
the gut microbiota. Proceedings of the National Academy of Sciences,
101(13), 4596–4601. http://dx.doi.org/10.1073/pnas.04007​06101
Ray, A. J., & Lotz, J. M. (2017). Comparing salinities of 10, 20, and 30‰
in intensive, commercial-scale biofloc shrimp (Litopenaeus vann-
amei) production systems. Aquaculture, 476, 29–36. https://doi.
org/10.1016/j.aquac​ulture.2017.03.047
Reda, R. M., El-Hady, M. A., Selim, K. M., & El-Sayed, H. M. (2018).
Comparative study of three predominant gut Bacillus strains and a
commercial B. amyloliquefaciens as probiotics on the performance of
Clarias gariepinus. Fish & Shellfish Immunology, 80, 416–425. https://
doi.org/10.1016/j.fsi.2018.06.031
Ren, G., Xu, L., Lu, T., Zhang, Y., Wang, Y., & Yin, J. (2019). Protective
effects of lentinan on lipopolysaccharide induced inflammatory
response in intestine of juvenile taimen (Hucho taimen, Pallas).
International Journal of Biological Macromolecules, 121, 317–325.
https://doi.org/10.1016/j.ijbio​mac.2018.09.121
Ren, W., Li, L., Dong, S., Tian, X., & Xue, Y. (2019). Effects of C/N ratio and
light on ammonia nitrogen uptake in Litopenaeus vannamei culture
tanks. Aquaculture, 498, 123–131. https://doi.org/10.1016/j.aquac​
ulture.2018.08.043
Ross, N. W., Firth, K. J., Wang, A., Burka, J. F., & Johnson S. C. (2000).
Changes in hydrolytic enzyme activities of naïve Atlantic salmon
Salmo salar skin mucus due to infection with the salmon louse
Lepeophtheirus salmonis and cortisol implantation. Diseases of
Aquatic Organisms, 41, 43–51. http://dx.doi.org/10.3354/dao04​1043
Ruan, Y., Sun, J., He, J., Chen, F., Chen, R., & Chen, H. (2015). Effect of
probiotics on glycemic control: A systematic review and meta-anal-
ysis of randomized, controlled trials. PLoS ONE, 10(7), e0132121–
https://doi.org/10.1371/journ​al.pone.0132121
Sabico, S., Al-Mashharawi, A., Al-Daghri, N. M., Yakout, S., Alnaami, A.
M., Alokail, M. S., & McTernan, P. G. (2017). Effects of a multi-strain
probiotic supplement for 12 weeks in circulating endotoxin levels
and cardiometabolic profiles of medication naïve T2DM patients: a
randomized clinical trial. Journal of Translational Medicine, 15(1), 1–9.
http://dx.doi.org/10.1186/s1296​7-017-1354-x
Sayed Hassani, M. H., Jourdehi, A. Y., Zelti, A. H., Masouleh, A. S., &
Lakani, F. B. (2020). Effects of commercial superzist probiotic on
growth performance and hematological and immune indices in fin-
gerlings Acipenser baerii. Aquaculture International, 28(1), 377–387.
https://doi.org/10.1007/s1049​9-019-00468​-1
Sayed Hassani, M., Pourelti Hossien, A., Shenavar, A., Bagerzadeh Lakani,
F., Yazdani Sadati, M. A., Shakourian, M., & Kamranjo, G. (2016).
Effects of commercial aquaculture probiotic on growth performance
and immune system of Acipenser baerii juveniles. Future of Sturgeon
Aquaculture, 1, 78–79.
Schulze, A. D., Alabi, A. O., Tattersall-Sheldrake, A. R., & Miller, K.
M. (2006). Bacterial diversity in a marine hatchery: Balance be-
tween pathogenic and potentially probiotic bacterial strains.
Aquaculture, 256(1-4), 50–73. https://doi.org/10.1016/j.aquac​
ulture.2006.02.008
Schveitzer, R., Fonseca, G., Orteney, N., Menezes, F. C. T., Thompson, F.
L., Thompson, C. C., & Gregoracci, G. B. (2019). The role of sedimen-
tation in the structuring of microbial communities in biofloc-dom-
inated aquaculture tanks. Aquaculture, 514, 734493. https://doi.
org/10.1016/j.aquac​ulture.2019.734493
Selim, K. M., & Reda, R. M. (2015). Improvement of immunity and disease
resistance in the Nile tilapia, Oreochromis niloticus, by dietary sup-
plementation with Bacillus amyloliquefaciens. Shellfish Immunology,
44(2), 496–503. https://doi.org/10.1016/j.fsi.2015.03.004
Siwicki, A. K., & Anderson, D. P. (1993). An easy spectrophotometric
assay for determining total protein and immunoglobulin levels in
fish sera: Correlation to fish health. Technical Fish Immunology, 3,
23–30.
Sugita, H., Miyajima, C., & Deguchi, Y. (1991). The vitamin B12-producing
ability of the intestinal microflora of freshwater fish. Aquaculture,
8486(91), 267–276. https://doi.org/10.1016/0044-8486(91)90028​-6
Swain, P., Nayak, S. K., Nanda, P. K., & Dash, S. (2008). Biological effects
of bacterial lipopolysaccharide (endotoxin) in fish: A review. Fish &
Shellfish Immunology, 25(3), 191–201. https://doi.org/10.1016/j.
fsi.2008.04.009
Szulińska, M., Łoniewski, I., van Hemert, S., Sobieska, M., & Bogdański,
P. (2018). Dose-dependent effects of multispecies probiotic supple-
mentation on the lipopolysaccharide (LPS) level and cardiometabolic
profile in obese postmenopausal women: A 12-week randomized
clinical trial. Nutrients, 10(6), 773–https://doi.org/10.3390/nu100​
60773
Taheri Mirghaed, A., Fayaz, S., & Hoseini, S. M. (2019). Effects of dietary
1,8-cineole supplementation on serum stress and antioxidant mark-
ers of common carp (Cyprinus carpio) acutely exposed to ambient
ammonia. Aquaculture, 509, 8–15. https://doi.org/10.1016/j.aquac​
ulture.2019.04.071
MOHAMMADI et al.
Telli, G. S., Ranzani-Paiva, M. J. T., Dias, D. D. C., Sussel, F. R., Ishikawa,
C. M., & Tachibana, L. (2014). Dietary administration of Bacillus
subtilis on hematology and non-specific immunity of Nile tilapia
Oreochromis niloticus raised at different stocking densities. Fish
& Shellfish Immunology, 39(2), 305–311. https://doi.org/10.1016/j.
fsi.2014.05.025
Tort, L. (2011). Stress and immune modulation in fish. Developmental
& Comparative Immunology, 35(12), 1366–1375. https://doi.
org/10.1016/j.dci.2011.07.002
Urao, M., Fujimoto, T., Lane, G. J., Seo, G. I., & Miyano, T. (1999). Does
probiotics administration decrease serum endotoxin levels in infants?
Journal of Pediatric Surgery, 34(2), 273–276. https://doi.org/10.1016/
S0022​-3468(99)90189​-6
Vali, S., Mohammadi, G., Tavabe, K. R., Moghadas, F., & Naserabad, S.
S. (2020). The effects of silver nanoparticles (Ag-NPs) sublethal
concentrations on common carp (Cyprinus carpio): Bioaccumulation,
hematology, serum biochemistry and immunology, antioxi-
dant enzymes, and skin mucosal responses. Ecotoxicology and
Environmental Safety, 194, 110353. https://doi.org/10.1016/j.
ecoenv.2020.110353
Van Doan, H., Hoseinifar, S. H., Naraballobh, W., Jaturasitha, S.,
Tongsiri, S., Chitmanat, C., & Ringø, E. (2019). Dietary inclusion of
Orange peels derived pectin and Lactobacillus plantarum for Nile
tilapia (Oreochromis niloticus) cultured under indoor biofloc sys-
tems. Aquaculture, 508, 98–105. https://doi.org/10.1016/j.aquac​
ulture.2019.03.067
Vase-Khavari, K., Mortezavi, S. H., Rasouli, B., Khusro, A., Salem, A. Z.
M., & Seidavi, A. (2019). The effect of three tropical medicinal plants
and superzist probiotic on growth performance, carcass character-
istics, blood constitutes, immune response, and gut microflora of
broiler. Tropical Animal Health and Production, 51(1), 33–42. https://
doi.org/10.1007/s1125​0 -018-1656-x
Volkoff, H., & Peter, R. E. (2004). Effects of lipopolysaccharide treat-
ment on feeding of goldfish: Role of appetite-regulating peptides.
Brain Research, 998(2), 139–147. https://doi.org/10.1016/j.brain​
res.2003.11.011
|
      17
Wang, A. R., Ran, C., Ringø, E., & Zhou, Z. G. (2018). Progress in fish
gastrointestinal microbiota research. Reviews in Aquaculture, 10(3),
626–640. https://doi.org/10.1111/raq.12191
Wang, Y. B., Li, J. R., & Lin, J. (2008). Probiotics in aquaculture: Challenges
and outlook. Aquaculture, 281(1-4), 1–4. https://doi.org/10.1016/j.
aquac​ulture.2008.06.002
Wang, Y., Li, Y., Xie, J., Zhang, Y., Wang, J., Sun, X., & Zhang, H. (2013).
Protective effects of probiotic Lactobacillus casei Zhang against endo-
toxin- and d-galactosamine-induced liver injury in rats via anti-oxidative
and anti-inflammatory capacities. International Immunopharmacology,
15(1), 30–37. https://doi.org/10.1016/J.INTIMP.2012.10.026
Xiang, L. X., Peng, B., Dong, W. R., Yang, Z. F., & Shao, J. Z. (2008).
Lipopolysaccharide induces apoptosis in Carassius auratus lympho-
cytes, a possible role in pathogenesis of bacterial infection in fish.
Developmental & Comparative Immunology, 32(8), 992–1001. https://
doi.org/10.1016/j.dci.2008.01.009
Yang, H.-L., Sun, Y.-Z., Hu, X. I., Ye, J.-D., Lu, K.-L., Hu, L.-H., & Zhang, J.-J.
(2019). Bacillus pumilus SE5 originated PG and LTA tuned the intes-
tinal TLRs/MyD88 signaling and microbiota in grouper (Epinephelus
coioides). Fish & Shellfish Immunology, 88, 266–271. https://doi.
org/10.1016/j.fsi.2019.03.005
Yano, T. (1992). Assays of hemolytic complement activity. In J. S. Stolen,
T. C. Fletcher, D. P. Anderson, B. S. Roberson, & W. B. van Muiswinkel
(Eds.), Techniques in fish immunology (pp. 131–141). Fair Haven, NJ:
SOS Publications. http://pubs.er.usgs.gov/publi​c atio​n/70006954
How to cite this article: Mohammadi G, Adorian TJ, Rafiee G.
Beneficial effects of Bacillus subtilis on water quality, growth,
immune responses, endotoxemia and protection against
lipopolysaccharide-induced damages in Oreochromis niloticus
under biofloc technology system. Aquacult Nutr.
2020;00:1–17. https://doi.org/10.1111/anu.13096