Aquaculture 526 (2020) 735392

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Aquaculture
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Gene expression and histopathological changes of Nile tilapia (Oreochromis T

niloticus) infected with Aeromonas hydrophila and Pseudomonas fluorescens
⁎
Ahmed M. Hala, , Manal I. El-Barbaryb
a
Genetics and Genetic Engineering Lab, National Institute of Oceanography and Fisheries, Egypt
b
Fish Diseases Lab, National Institute of Oceanography and Fisheries, Egypt

A R T I C LE I N FO A B S T R A C T

Keywords: Hepcidin, interleukin 1-β (IL-1β), and cytochrome P450 1A (CYP1A) genes play an important role in the
Oreochromis niloticus adaptive immune system in fish. To our knowledge, this is the first observation among the expression level of
Aeromonas hydrophila these genes in response to infection with Aeromonas hydrophila and Pseudomonas fluorescens in Nile tilapia
Pseudomonas fluorescens (Oreochromis niloticus). Also, the effect of these experimentally induced infections was investigated regarding the
Hepcidin
mortality rate, clinical signs, and histopathological changes in the liver and kidney tissues. Three experimental
Interleukin 1-β
Cytochrome P450 1A
fish groups were injected intraperitoneally (IP) by 0.1 ml of phosphate-buffered saline, two of these groups were
Real-time PCR injected by 0.1 ml of 1 × 105 CFU/ml of the suspension of either A. hydrophila or P. fluorescens. The infected
Histopathology groups showed extensive skin hemorrhages, cloudiness of eyes, protrusion of the scales, and severe swelling of
the abdomen with inflammation of the vent and the mortality rate (MR%) was 42.75 and 37.5% in A. hydrophila
and P. fluorescens groups, respectively. The histological changes of the infected O. niloticus with A. hydrophila and
P. fluorescens showed fibrosis, karyolitic necrosis, the disappearance of hepatocytes wall and deposits of he-
mosiderin in the liver, while kidney showed dilation in Bowman's spaces, disconnection of renal tubules and
hemorrhage and necrotic areas between them besides the accumulation of hemosiderin. The relative expression
levels of hepcidin were higher after A. hydrophila than P. fluorescens infection in most studied organs while other
studied genes expression levels were varied between infected fish groups. The relative expression of hepcidin
was increased in the liver, blood, pituitary, kidney, and ovary while IL-1β transcripts were increased in the liver,
kidney, muscle, gill, brain, and blood. CYP1A transcripts were increased in the liver, kidney, gill, and testis. The
magnitude of the increase in IL-1β levels differed from that observed for the hepcidin and CYP1A genes. The
relative expression levels of studied genes were significantly increased in the liver followed by the kidney. No
significant differences were observed in the relative expression of studied genes among the gill, blood, brain, and
pituitary and among the ovary, testis, brain, and pituitary during the bacterial challenge (p < .05). These
findings in response to pathogen challenge could improve our understanding of the defense mechanism of
adaptive immunity genes in O. niloticus organs.

1. Introduction 1999). The pathogenicity of A. hydrophila contributed to the aerolysin-

The adaptive immune response in fish is considered an essential
feature in host defense against pathogenic organisms (Magnadóttir,
2006). Bacterial pathogens as A. hydrophila and many species of Pseu-
domonas have reported causing disease in different fish species and
associated with septicemia. They are secondary invader of wounded
tissue that causes disease in stressed fish (Roberts, 1978; Thune et al.,
1993; Xu et al., 2012). Bacterial pathogens have developed several
strategies to survive inside the host and for counteracting cell defenses.
Host cell apoptosis by bacteria is the main mechanism to overcome the
host immune defenses (Moss et al., 1999; Weinrauch and Zychlinsky,
related cytotoxic enterotoxin that can lyse red blood cells, destroy cell
wall, with oligomerization and pore creation (Chopra et al., 1993;
Ferguson et al., 1997) causing symptoms including tissues distension,
dropsy, necrosis, hemorrhage and ulcers, exophthalmia, and accumu-
lation of fluid in the scale of the pockets (Karunasagar et al., 1989; Azad
et al., 2001; El-Barbary, 2017). Pseudomonas fluorescens has identified
as a causative agent of Pseudomonas septicemia diseases which caused
some clinical signs as skin hemorrhage, fin rot, detached scales, ascites,
and exophthalmia in different species of fish (Okaeme, 1989; Eissa
et al., 2010; El-Barbary and Hal, 2017).
Fish immune-related genes have a role in immunity and the

⁎
Corresponding author.
E-mail address: ahmedmhal@gmail.com (A.M. Hal).

https://doi.org/10.1016/j.aquaculture.2020.735392
Received 28 August 2019; Received in revised form 27 February 2020
Available online 22 April 2020
0044-8486/ © 2020 Elsevier B.V. All rights reserved.
A.M. Hal and M.I. El-Barbary
evolution of immune response to bacterial infection. Fish hepcidins
have an antibacterial role, and their expression in the liver naturally
induced because of the bacterial infection and affect macrophages and
monocytes activation (Shike et al., 2002; Pinto et al., 2010; Costa et al.,
2011). Early response pro-inflammatory cytokine mediates immune
regulation by interleukin-1 (IL-1) in both adaptive and innate im-
munity. Each of activated macrophages, monocytes, endothelial cells,
lymphocytes, granulocytes, activated T and many other cell types could
cause the secretion of IL-1 (Sigel et al., 1986; Roux-Lombard et al.,
1989). Interleukin-1β has a humoral immune reaction, and it has been
recognized in several teleost fish species, including rainbow trout,
yellowfin sea bream, sea bass, carp, and channel catfish (Zou et al.,
1999; Fujiki et al., 2000; Scapigliati et al., 2001; Wang et al., 2006;
Jiang et al., 2008). Among the cytochrome P450 family, cytochrome
P4501A (CYP1A) contributes to the metabolism of several environ-
mental mutagens and carcinogens (Lee et al., 1998). Also, CYP1A has
used as an indicator of immersion infection with Pseudomonas aerugi-
nosa and Edwardsiella tarda (van Soest et al., 2011). In the other side,
Wang et al. (2009) have compared hepcidin and CYP1A1 genes ex-
pression in response to pollution. The previous studies raise curiosity to
find out the response of CYP1A gene expression compared to hepcidin
and IL-1β against these types of bacterial infections.
The present study aims to 1) investigate the harmful effects of both
A. hydrophila and P. fluorescens on infected O. niloticus liver and kidney
structure, and 2) observe the expression of genes; hepcidin, interleukin-
1β, and CYP1A in blood, liver, gill, muscle, brain, pituitary, kidney,
ovary, and testis to gain a knowledge regarding the effect the bacterial
infection on different fish organs through defensive mechanisms of
studied genes.
2. Material and methods
2.1. Challenged test
Forty-eight of apparently healthy O. niloticus (50 ± 2 g) were di-
vided into three duplicated experimental groups and keeping in six
aquaria that supplied with dechlorinated water with continues aeration,
each aquarium contained eight fish, and the acclimatization period was
14 days. Fish were fed twice daily by commercial diet 30% protein by
feeding rate 3% of body weight. Fish were intraperitoneally (IP) in-
jected by 0.1 ml of 1 × 105 CFU/ml of the suspension of either the
isolates A. hydrophila (A. hydrophila group) or P. fluorescens (P. fluor-
escens group). Fish in the control group were IP injected with 0.1 ml
phosphate-buffered saline. Fish were anesthetized by a clove oil solu-
tion immediately before injection (Hamackova et al., 2006). The clin-
ical signs were observed daily, and samples for histological examination
were collected after 14 days. Blood samples from three fish groups were
collected from the caudal veins. Fish were dissected and the liver, gill,
muscle, kidney, brain, pituitary, blood, ovary, and testis of O. niloticus
were taken after 7 days for gene expression analysis.
All fish procedures were approved by the Ethics Committee of
National Institute of Oceanography and Fisheries (NIOF), Egyptian
Ministry of Higher Education and Scientific Research.
2.2. Analysis of gene expression by real-time PCR
The total RNA from each replicate of samples was isolated according
to Chomczynski and Sacchi (1987) and its integrity and concentration
were detected. Total RNA was treated with DNAseI to avert genomic
DNA. The first strand of cDNA was created by using M-MuLV Reverse
transcriptase (SibEnzyme Ltd.) with an oligo-dT12–18 primer (Bio Basic
Inc.). After cDNA synthesis, all first-strand cDNA products were diluted
to 200 ng/μl for the subsequent quantitative real-time PCR amplifica-
tion.
The used primers for relative expressions of hepcidin, IL-1β, CYP1A,
and the internal control beta-actin gene were designed based on
2
Aquaculture 526 (2020) 735392
Table 1
The primers were used to amplify the studied genes.
Primers Accession Sequences 5′-3′ Annealing™
numbers
Hepcidin AY725227 F-GCTGGAGGAGCCAATGAGC 61 °C
R-GTGGTTGTGGGAGCAGGAAT
IL-1β KF747686 F-GATGACGACAAGCCAACCCT 61 °C
R-GCTGATGTACCAGTTGGGGT
CYP1A FJ389918 F-GCAAATGGCTGCTGCTTGTCA 62 °C
R-GTGTATCAAGGGTTCATGCCCT
β-actin EU887951 F-TCAGGGTGTGATGGTGGGTATG 62 °C
R-CTCAGCTCGTTGTAGAAGGTGT
accession numbers as listed in Table 1 and were estimated by real-time
PCR using double-standard curves method. The real-time PCR reactions
for genes expression were in a total volume of 20 μl consisting of 4 μl
HOT FIREPol EvaGreen qPCR Mix Plus (5×), 2 μl cDNA, 0.5 μl each for
Forward/Reverse Primers and 13 μl H2O PCR grade. In a Stratagene
MX3005P (ABI, CA, USA), the thermal profile was performed as 95 °C
for 10 s, followed by 40 cycles involving of 95 °C for 15 s, the annealing
temperature of genes as listed in Table 1 for 15 s and 72 °C for 12 s.
Melting curves of the reaction were produced from 65 °C to 95 °C. The
expression of genes was represented as average gene expression (in
triplicate) ± the standard error of the sample replicates and normal-
ized to β-actin. The relative mRNA levels of genes were calculated by
the ΔΔCT method and values were expressed as log2 ratios.
2.3. Histological examination
Sampling was collected from each group after 14 days of the ex-
periment start of O. niloticus organs (liver and kidney) and fixed in 10%
neutral buffered formalin for 24 h. The histological examination carried
out according to Roberts (2004). Tissue sections were stained with
hematoxylin-eosin.
2.4. Statistical analysis
The data were analyzed using a three-way ANOVA followed by the
Tukey post hoc test to explore the effects of bacterial infections and
gene expression differences among organs. The same letters of a, b, c,
and d indicate no significant difference and different letters indicate
significant differences (p < .05) among organs. All the statistical
analyses were done by the IBM SPSS program version 19.
3. Results
3.1. Mortality rate and clinical observation for experimental infected O.
niloticus
In the control group, no clinical signs or mortality was observed
during the experiment period. In contrast, the mortality rate was 42.75
and 37.5% in groups of A. hydrophila and P. fluorescens, respectively.
Both of A. hydrophila and P. fluorescens groups showed extensive skin
hemorrhages and ulcers, cloudiness of eyes, a detachment of the scales
and severe swelling of the abdomen with inflammation of the vent;
while, internal organs showed severe congestion and enlargement with
fatty change in the liver.
3.2. Histological examination
Liver of the control fish group showed a normal structure of tissue
(Plate 1a), while the liver in A. hydrophila group showed histological
changes as the disappearance of hepatocyte wall, vacuolar degenera-
tion, pyknosis in hepatocytes and dilatation in sinusoids with fibrosis
and hemosiderin accumulation (Plate 1b,c). Hepatocytes in P.
A.M. Hal and M.I. El-Barbary
fluorescens group exhibited pyknosis, vacuolar degeneration, and dis-
appearance of its wall (Plate 1d) and dilatation in sinusoids with severe
lipid vacuoles (Plate 1e).
The kidney in the control group showed a normal structure of renal
tubules and Bowman's space in the glomeruli (Plate 1f), but the kidney
in A. hydrophila group of O. niloticus showed severe tubular degenera-
tion with interstitial mononuclear cell infiltration of the kidney and
hemosiderin deposits (Plate 1g). Additionally, P. fluorescens group
showed dilatation in Bowman's spaces, hemorrhage and areas of ne-
crosis between renal tubules, disconnection of renal tubules, and he-
mosiderin accumulation with inflammatory cells between renal tubules
(Plate 1h).
3.3. Hepcidin, IL-1β and CYP1A genes expression
In O. niloticus organs, hepcidin, IL-1β, and CYP1A genes expression
was measured in response to bacterial infection of A. hydrophila and P.
fluorescens compared to the uninfected group (control). The expression
of genes significantly differed according to the type of bacterial infec-
tion, the studied gene, and among fish organs (p < .05; Fig. 1). At the
level of genes, the relative expression levels of hepcidin were higher in
organs of A. hydrophila group than P. fluorescens group except in
3
Aquaculture 526 (2020) 735392
Plate 1. Histopathological changes of the liver and kidney of
three O. niloticus groups (control, A. hydrophila (Ah), and P.
fluorescens (Ps)) stained with H & E; (a, f) control showed
normal structure tissue of both liver (a ×250) and kidney that
exhibited normal renal tubules, tubular lumen and Bowman's
space in the glomerular (f ×200). (b, c) Ah group showing
disappearance of hepatocyte wall, vacuolar degeneration with
some pyknosis in hepatocytes (bx 400), fibrosis and hemosi-
derin accumulation (c ×300). (d, e) Ps group showed py-
knosis and vacuolar degeneration in the hepatocytes with
disappearance of hepatocyte wall (d ×300), and sever vacu-
lation in hepatocytes (e ×400). Kidney in Ah group showed
severe tubular degeneration with interstitial mononuclear cell
infiltration and hemosiderin accumulation of the kidney (g
×300). (h) Ps group showed disconnection of renal tubules,
and invaded with inflammatory cells between renal tubules
and hemosiderin accumulation (h ×400). v = vacuolar de-
generation, pk = pyknosis, f = fibrosis, g = glomerulus,
bs = Bowman's space, if = infiltration, d = degeneration,
h = hemorrhage, n = necrosis, hs = hemosiderin.
muscles and ovaries. In contrast, the expression level of IL-1β was
higher after infection of P. fluorescens than A. hydrophila in the liver,
gill, muscle, blood, and testis (Fig. 1). The magnitude of the increase in
IL-1β levels differed from that observed for the hepcidin and CYP1A
genes. The relative IL-1β expression levels were varied between the
infected groups and were significantly increased in most infected fish
organs compared to controls (Fig. 1). CYP1A transcripts were sig-
nificantly increased in the liver, gill, kidney, and testis of infected fish
groups (p < .05). The expression of CYP1A gene decreased in the
pituitary of infected groups, and its level decreased in the ovary of A.
hydrophila group (Fig. 1).
At the level of infected fish organs, the relative expression level of
studied genes were significantly increased in the liver (p < .05) fol-
lowed by the kidney. In the liver, kidney, muscle, and brain, the ex-
pression level of IL-1β gene was the highest, while the expression level
of hepcidin gene was the highest in the pituitary. Also, the relative
expression of CYP1A gene was the highest in the testis among studied
genes. The gill, muscle, brain, and testis showed a slight increase of
hepcidin expression level, while the increase of IL-1β expression was a
relatively low level in the pituitary and testis of infected fish groups
(Fig. 1). Differences in the relative expression of studied genes were not
significant among the gill, blood, brain, and pituitary and not
A.M. Hal and M.I. El-Barbary
Fig. 1. Relative expression of hepcidin, IL-1β and CYP1A transcripts in different organs of O. niloticus infected by A. hydrophila (Ah) and P. fluorescens (Ps).
Transcripts levels were normalized against β-actin. * represents a significant difference from control, organs assigned with same letters are not significantly different
using a three-way ANOVA (P < .05) and Tukey's multiple comparison test.
significant among the ovary, testis, brain, and pituitary (p < .05)
during the bacterial challenge.
4. Discussion
The effect of A. hydrophila and P. fluorescens within 14 days on MR%
was 42.75 and 37.5%, respectively. This result agreed with Saad El-
Deen (2014) who recorded that the cumulative mortalities were
53.33% in O. niloticus challenged with Pseudomonas aeraginosa. More-
over, Yambot and Inglis (1994) recorded acute MR among O. niloticus
infected by A. hydrophila. In the present study, some clinical symptoms
observed in A. hydrophila group such as skin hemorrhages and ulcers,
cloudiness of eyes, detachment and protrusion of the scales, a severe
distention of the abdomen with inflammation of the anus. Moreover,
the internal organs (liver and kidney) showed severe congestion and
enlargement. These clinical signs were similar to that described by Laith
and Najiah (2013); El-Barbary (2017) who observed that the O. niloticus
infected by A. hydrophila showed extensive skin ulcers, inflamed vent,
and congestion and enlargement of the fatty liver. Besides, the external
alteration associated with infection by Pseudomonas sp. in most fish
species were hemorrhage and darkness of the skin, fin rot, detached
scales, ascites, exophthalmia and ulceration especially at dorsum part
and at the base of fins with eroded fins (Okaeme, 1989; Eissa et al.,
2010; Saad El-Deen, 2014). Generally, the pathogenicity of Aeromonas
sp. and Pseudomonas sp. might be caused by the extracellular toxins and
enzymes and others have been related to the virulence of them which
cause injury of the body tissues and leads to mortality (Burke et al.,
1982; Brenden and Huizinga, 1986; Stelma et al., 1986). The histolo-
gical changes of the liver of A. hydrophila group showed disappearance
of hepatocytes, vacuolar degeneration, hepatocytes pyknosis, dilatation
in sinusoids with fibrosis and hemosiderin accumulation. These results
are similar to previous observations of El-Barbary (2010, 2017);
Yardimci and Aydin (2011) regarding degenerative changes, lympho-
cytic infiltration with focal necrosis of liver tissues, fibrosis, and accu-
mulation of hemosiderin. In the present study, fish in P. fluorescens
group showed pyknosis, vacuolar degeneration, and disappearance of
hepatocytes wall and dilatation in sinusoids with severe lipid vacuoles
similar to the findings of El-Barbary and Hal (2017). In A. hydrophila
group, the kidney showed tubular degeneration with interstitial
mononuclear cell infiltration and hemosiderin deposits that seems to be
similar to previous studies (Yardimci and Aydin, 2011; Laith and
Najiah, 2013; El-Barbary, 2017). On the other hand, fish in P. fluor-
escens group showed dilatation in Bowman's spaces, hemorrhage and
areas of necrosis in between renal tubules, disconnection of renal tu-
bules and hemosiderin deposits and inflammatory cells between renal
4
Aquaculture 526 (2020) 735392
tubules. These observations are similar to the findings of El-Barbary and
Hal (2017). Hemosiderin accumulation reflects a pathological process
(Wolke et al., 1985), which was observed after hemolytic anemia
(Roberts, 2001). Also, the accumulations of erythrocytes and lympho-
cytes cells in the kidney might cause reduced hemoglobin content that
is usually because of the destruction of erythrocytes and abnormal
movement of erythrocytes through the spleen in fish (Scott and Rogers,
1981; Hibiya, 1982; Zapata and Cooper, 1990). In this study, accu-
mulation of hemosiderin in infected liver and kidney tissues in A. hy-
drophila and P. fluorescens groups agreed with those of Chang and
Plumb (1996) who observed foci of hemosiderin in spleens of common
carp, Cyprinus carpio exposed to A. hydrophila.
In the present study, the relative expression of hepcidin, IL-1β, and
CYP1A was compared in the liver, gill, muscle, brain, pituitary, kidney,
blood, ovary, and testis of Nile tilapia in response to the bacterial in-
fection by A. hydrophila or P. fluorescens. Previous results showed that
these genes expression has been induced because of the infection in
different fish organs such as hepcidin expression in organs of blunt
snout bream (Liang et al., 2013), IL-1β expression in organs of large-
mouth bass (Ho et al., 2016), and in channel catfish organs (Wang et al.,
2006) and CYP1A expression levels in zebrafish larvae (Guo et al.,
2019). The results of this study revealed that hepcidin expression level
was more affected by A. hydrophila than P. fluorescens infection in most
studied fish organs. This finding could be returned to the secretion of
catecholate-type siderophore amonabactin by A. hydrophila, which is
related to both higher infectivity and iron restriction states, as noted by
Stintzi and Raymond (2000). Moreover, Jiang et al. (2017) observed
that the transcription of hepcidin gene was upregulated in the liver of
zebrafish by A. hydrophila DNA stimulation, which accompanied with
an increase of hepcidin protein; thus, an enhanced bactericidal activity
against A. hydrophila. Also, A. hydrophila produces a different extra-
cellular toxins and enzymes have been associated with its pathogenicity
(Allan and Stevenson, 1981; Rodriguez et al., 1993; Gogal et al., 1999;
Garcia-Abiado et al., 2004; El-Barbary, 2010), this could be contributed
to a high MR% in A. hydrophila group in our study. The harmful effect of
the pathogenic bacteria in this study observed in the liver and kidney
tissues, and the response of these tissues to this infection was demon-
strated by the presence of melano-macrophage centers (MMCs). Hence,
MMCs in the liver and kidney tissues in response to the infection were
related to an increase of hepcidin gene expression levels in our study.
Our observation agreed with Johnson et al. (1999) who reported that
MMCs described as aggressive phagocytic activity against bacteria and
fungi infections. Moreover, MMCs quickly remove the injected foreign
materials from the circulation (Herraez and Zapata, 1986; Brattgjerd
and Evensen, 1996). In our study, low levels of hepcidin expression
A.M. Hal and M.I. El-Barbary
were detected in the gill and brain compared to the liver after 7 days of
the bacterial injection. In agreement, Liang et al. (2013) observed that
the upregulation of hepcidin expression level decreased in the gill after
6 h post-injection and in the brain after 3 days post-injection compared
to the hepcidin expression levels in the liver raised until 6 days post-
injection of A. hydrophila in blunt snout bream.
The relative IL-1β expression levels were varied in fish organs of the
infected groups in the present study. Our findings agreed with Wang
et al. (2006) who observed that gene expression depend on organ type
more than infection type. In this study, the relative IL-1β expression
levels were significantly increased in most infected fish organs com-
pared to controls. This observation could be returned to the role of IL-1
in the first line of the immune defense, as explained by Schmitz et al.
(2005). Additionally, IL-1β gene showed more strong role in humoral
immune response and recognized in various teleost fish species (Zou
et al., 1999; Fujiki et al., 2000; Scapigliati et al., 2001; Wang et al.,
2006; Jiang et al., 2008). Unlike hepcidin expression in this study, IL-1β
expression levels were increased in fish gill, muscle, and brain. In the
gill, our findings were in line with Wu et al. (2015) who observed the
upregulation of IL-1β transcription in the gill of large yellow croaker
against Vibrio alginolyticus. In the muscle, Taechavasonyoo et al. (2013)
found an increase of IL-1β expression in the muscle injected with a
plasmid encoding IL-1β of Japanese flounder. Moreover, our findings of
IL-1β expression in the brain could result from its important role in the
host antibacterial immune response in central nervous system (CNS)
infectious diseases, as observed previously (Zwijnenburg et al., 2003a,
2003b; Kielian et al., 2004) and it could be considered a key blood-
brain barrier in tissue injury (Kielian, 2009). The blood-brain barrier in
the CNS has a unique feature to protect and isolate the brain from the
systemic infection and breakage or injury to this barrier allows the
brain to be susceptible to infection, as such, the ability to rapidly pro-
duce antimicrobial peptides could complement the existing defensive
mechanisms (Neves et al., 2015). In our study, the slight increase of IL-
1β expression level was observed in infected fish testis. Our observation
could be due to the inhibition effect of testosterone by peripheral blood
mononuclear cells, as noted previously (Jara et al., 2006).
In the present study, the relative expression levels of CYP1A were
increased in most fish organs against the bacterial infection. In agree-
ment, Guo et al. (2019) observed that CYP1A was upregulated in zeb-
rafish larvae by immersion and caudal vein microinjection of Vibrio
parahaemolyticus. In this study, the expression of CYP1A was decreased
only in fish ovary in the case of A. hydrophila infection and was showed
negligible induction in fish brain under P. fluorescens infection. Similar
findings were observed by van Soest et al. (2011) who found little to no
induction of CYP1A expression because of intravenous E. tarda injec-
tion, but early induced in E. tarda-immersed zebrafish embryos. These
differences raise the curiosity to find out the immunological role of
CYP1A gene expression in response to the type and exposure mode of
the bacterial infection.
In conclusion, A. hydrophila and P. fluorescens infections caused
clinical signs in infected Nile tilapia, O. niloticus, and their mortality
rate was 37.5% and 42.75%, respectively. This was associated with
severe histopathological changes in the liver and kidney tissues. The
relative expression levels of hepcidin were higher in most studied or-
gans of A. hydrophila group than P. fluorescens group, while other stu-
died genes expression levels were varied between infected groups. The
magnitude of the increase in IL-1β levels differed from that observed for
the hepcidin and CYP1A genes in infected fish groups. Unlike hepcidin
and CYP1A expressions, the relative IL-1β expression levels were sig-
nificantly increased in most fish organs of infected groups. CYP1A
transcripts were significantly increased in the liver, gill, kidney, and
testis of infected fish groups. The expression of CYP1A gene decreased
in the pituitary of infected groups, and its level decreased in the ovary
of A. hydrophila group. The relative expression levels of studied genes
were significantly increased in the liver followed by the kidney.
Differences in relative genes expression were not significant among the
5
Aquaculture 526 (2020) 735392
gill, blood, brain, and pituitary and not significant among the ovary,
testis, brain, and pituitary during the bacterial challenge. These find-
ings may help to understand the defense mechanism of adaptive im-
munity genes in O. niloticus organs.
Ethics statement
The experiment complied with the regulations of the Egyptian
Ministry of Higher Education and Scientific Research and National
Institute of Oceanography and Fisheries (NIOF) regarding the use of fish
in scientific experiments and had the approval of NIOF Ethics
Committee.
Declaration of Competing Interest
The authors declare that there is no conflict of interest.
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