emergent

Life Sciences Research

Research Article
Aeromonas hydrophila in Nile tilapia (Oreochromis
niloticus) from Brazilian aquaculture: a public health
problem

Marianna Vaz Rodrigues, Maria Fernanda Falcone-Dias, Claire Juliana

Francisco, Gianmarco Silva David, Reinaldo José da Silva, João Pessoa
Araújo Júnior

Abstract
Received: 25 January 2019 Aeromonas hydrophila is a Gram-negative bacterium present in the water,
Accepted: 3 May 2019 which can cause disease in animals, such as fish, frog, and mammals,
Online: 13 May 2019
including humans. In fish, Aeromonosis occurs when it is immunosuppressed
Authors: due to the stress of handling, water quality, parasitism or population density.
M. V. Rodrigues , J. P. A. Júnior
Department of Microbiology and Immunology,
Due the importance of this disease in fish and humans, this study aimed to
Biosciences Institute, Univ. Estadual Paulista detect this bacterium by PCR in Nile tilapia (Oreochromis niloticus) of cage
(UNESP) at Botucatu, Distrito de Rubião Júnior, fish farms localized in hydro-electrical reservoirs of São Paulo state, Brazil
SP CEP 18618-689, Brazil
and describe the lesions found in positive fish by necropsy and histopathology.
M. F. Falcone-Dias, G. S. David Around 360 samples of Oreochromis niloticus specimens were randomly
São Paulo State Agency for Agribusiness sampled at six Brazilian fish farms in November 2014 (n = 180) and in March
Technology (APTA) at Jaú, CP 66 Jaú, SP CEP
17340-000, Brazil 2015 (n = 180). The identification of A. hydrophila by PCR showed the
prevalence since 3.33% to 46.66%. The most common macroscopic lesions
C. J. Francisco, R. J. da Silva
Department of Parasitology, Biosciences
were hemorrhage and splenomegaly, and bacteria colonies, coagulative
Institute, Univ. Estadual Paulista (UNESP) at necrosis, hemorrhage, inflammatory process, melano-macrophages, and
Botucatu, Distrito de Rubião Júnior, SP CEP vacuolar degeneration were microscopic. The pathological and
18618-689, Brazil
histopathological findings showed the presence of an infectious disease, and
[email protected] employing the molecular technique, it was possible to identify that the
analyzed fishes had A. hydrophila. Thus, producers should utilize this
Emer Life Sci Res (2019) 5(1): 48-55 information using histopathology and molecular techniques in tilapia to reduce
economic losses and avoid disease in consumers.
E-ISSN: 2395-6658
P-ISSN: 2395-664X
Keywords aeromonosis, aquaculture, fish diseases, diagnostic, public health
DOI: https://doi.org/10.31783/elsr.2019.514855
Introduction
The aeromonads are Gram-negative, rod-shaped, facultative anaerobic,
nonspore forming bacteria that are autochthonous and widely distributed in
aquatic environments [1]. These bacteria, mainly Aeromonas hydrophila, have
emerged as a foodborne pathogen of extreme importance [1-2]. Aeromonas
spp. have been linked to both food and water-borne diseases in different parts
of the world especially developing countries due to poor hygiene and poor
quality water [3]. Infections in humans with bacteremia [4], respiratory tract
infections [5], gastroenteristrics [6], septicemia [7], urinary tract infection [8],
and traveler’s diarrhea [9] have been associated with Aeromonads. Aeromonas
hydrophila is an important cause of zoonotic diseases (i.e., diseases that can
be spread from animals to humans and vice versa) [10]. In fishes, it is
considered as a significant pathogen causing the motile aeromonad septicemia
(MAS), also known as epizootic ulcerative

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syndrome (EUS) [11]. The symptoms of A. hydrophila infections include swelling of tissues,
dropsy, red sores, necrosis, ulceration, and hemorrhagic septicemia [12]. This bacterium has been found in
several fish species, including Nile Tilapia [13-15]. A. hydrophila has been causing outbreaks in fish farms
with high mortality rates, resulting in severe economic losses to the aquaculture industry worldwide [16-
17].
Aeromonads possess a wide range of virulence factors that enable them to evade the host’s defense
system, spread, and eventually killing the host. Among these factors, there are different toxins and enzymes,
including Lipase (Lip), Serine protease (Ser), Aerolysin (Aer), Cytotoxic enterotoxin (ACT) and
temperature-sensitive protease, Epr (CAI) [13]. These bacteria have attracted the attention of reseachers
because of its ability to grow at cold temperatures [1]. Aeromonas spp. are able to survive and multiply at
low temperatures in a variety of food products and can produce virulence factors even at these low
temperatures [18-19]. Thus, most cases of illness in humans are associated with aquaculture products or
long-term refrigerated ready-to-eat foods. Multiple resistances to some antibiotics has occurred in many
strains of the pathogen, and thus, it has become a problem to cure intestinal disorders in human [1].
Due to the importance of A. hydrophila as a fish pathogen and as an agent of emerging foodborne
diseases, representing a serious public health concern, the aim of this study was to detect this bacterium by
polymerase chain reaction (PCR) in Nile tilapia (Oreochromis niloticus) of cage fish farms localized in
hydroelectrical reservoirs of São Paulo state, Brazil. In addition, we described the lesions found in positive
fish by necropsy and histopathology.

Methodology
Ethics statement
This study was carried out in strict accordance with the recommendations in the following laws: Law
11794/2008 and Decree 6899/2009. The protocol was approved by the Ethics Committee on Animal Use of
the São Paulo State University (UNESP) (Protocol Number: 724-CEUA). The owners of fish farms used in
this study gave their consent for the use of their fish for detecting pathogens and other analysis, which are
not presented in this paper.

Sampling
Oreochromis niloticus specimens were randomly sampled from six fish farms from three different
reservoirs in São Paulo State, Brazil, in November 2014 (n = 180) and in March 2015 (n = 180) (Table 1).
Each fish-farm and sampling was named as A(1): fish farm 1 (first sampling), A(2): fish farm 1 (second
sampling), B(1): fish farm 2 (first sampling), B(2): fish farm 2 (second sampling), C(1): fish farm 3 (first
sampling), C(2): fish farm 3 (second sampling), D(1): fish farm 4 (first sampling), D(2): fish farm 4 (second
sampling), E(1): fish farm 5 (first sampling), E(2): fish farm 5 (second sampling), F(1): fish farm 6 (first
sampling), and F(2): fish farm 6 (second sampling) (Table 1). Fish farm 1 is in the Paranapanema River
Basin, fish farms 2-4 are in the Tiete River Basin, and fish farms 5-6 are in the Grande Paraná River Basin.
Necropsy was performed according to Noga [20]. The organs sampled were the brain, gall bladder, gill, gut,
heart, kidney, liver, muscle, spleen, and stomach for histopathology and molecular analysis. According to
Noga [20], a 1-cm³ portion of each tissue was fixed in 10% neutral buffered formalin followed by the
processing using standard histological techniques and embedded in paraffin. Hematoxylin and eosin were
used for staining.

Bacterial isolation
Six O. niloticus with ulcers in the head and skin were used for bacterial isolation. During necropsy, kidney
was swabbed for culturing in MacConkey agar and incubated at 37°C overnight. Identification was
perfomed by PCR and sequencing.

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Table 1. Number, weight (g), and size (cm) of Oreochromis niloticus sampled at six fish farms in
the first sampling and second sampling
Fish Farm N Weight¹ (g) Weight² (g) Size¹ (cm) Size² (cm)
(𝐗) (𝐗) (𝐗) (𝐗)
A 30 518.67 441.10 19.53 21.64
B 30 434.76 518.99 22.33 22.00
C 30 286.55 537.56 19.49 22.02
D 30 427.47 430.86 21.42 20.73
E 30 234.08 396.82 18.43 22.77
F 30 304.17 424.77 19.71 21.49
Total 180 367.61 458.35 20.15 21.77
¹ First sampling; ² Second sampling.

DNA extraction, PCR (polymerase chain reaction), sequencing, and phylogeny test
The organ tissues collected from each fish were pooled and 20 mg of each sample was used for molecular
analysis. Colorless colonies were isolated and submitted to deoxyribonucleic acid (DNA) extraction. The
DNA extraction for tissues was performed using the Wizard® SV Genomic DNA Purification System kit
(Promega Corporation®) according to the manufacturer’s instructions. For bacterial extraction, DNeasy®
Blood & Tissue Kit (Qiagen®) was used according to manufacturer´s instructions. The DNA was eluted in
elution buffer (nuclease-free water) and kept at -20°C. Purity and quantification of extracted DNA was
measured using a 260/280 absorbance rate in a Nanodrop 2000c (Thermo Fisher Scientific®). Only the
DNA samples with a ratio of >1.7 (260/280 rate) were used in this study.
The colonies extracted were submitted to PCR with universal primers designed by Weisburg et al.,
[21] that recognize 16S rRNA region. For this purpose, the reaction mixture consisted of 10 µL of Gotaq
qPCR Mastermix 2X (Promega), 10 pmol of each primer (Univ16Sf and Univ16r) and 3 µL of DNA was
prepared and finally adjusted to 20 µL by adding nuclease free water. The reaction consisted of an initial
denaturation step of 95°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 54°C for 1 min, and 72°C for
1 min, ended with the finalextension at 72°C for 10 min and a hold at 22°C. The 1507 bp amplicons were
purified with an Ilustra Microspin™ S-400 HR Columns Kit (GE Healthcare®) according to the
manufacturer’s instructions for the identification by Sanger sequencing. For this, the purified amplicon was
sequenced in both directions using BigDye™ Terminator Cycle Sequencing Kit (Applied Biosystems) on
an Applied Biosystems capillary 3500 Genetic Analyzer. The quality of the electropherograms was assessed
in Sequencing Analysis version 5.4 (Applied Biosystems). Further, sequences were identified by similarity
analysis using BLAST (Basic Local Alignment Search Tool) algorithm.
Later, all samples from fish were submitted to a new PCR with specific primers for Aeromonas
hydrophila that recognize ascV gene according to Carvalho-Castro et al., [22]. The reaction mixture
consisted of 10 µL of Gotaq qPCR Mastermix 2X (Promega), 10 pmol of each primer (ascV sense and ascV
antisense), 3 µL of DNA, and nuclease free water to adjust to 20 µL. The reaction consisted of an initial
denaturation step of 95°C for 5 min, followed by 35 cycles of 95°C for 1 min, 58°C for 90 sec, and 72°C for
1 min, finishing with terminal extension at 72 °C for 5 min and a hold at 22 °C. The 890 bp amplicons were
purified with an Ilustra Microspin™ S-400 HR Columns Kit (GE Healthcare®) according to the
manufacturer’s instructions for identification by Sanger sequencing as described above.
The nucleotide sequence of the reaction with universal primers of approximately 1100 bp was used
to query the GenBank library to arrive at the closest type strain and thus, attain a species affiliation and
possible identification to that level. To compare the sequences from different strain found in Genbank
library, the nucleotide sequences were aligned with ClustalW from MEGA software, version 7, and
dendrograms were created by using the neighbor-joining method based on a model by Jukes and Cantor.

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Statistical analysis
The prevalence of A. hydrophila detected by PCR was calculated for each fish farm sampled in the two
sampling times (November 2014 and March 2015). The occurrence of lesions was observed (necropsy and
histopathology) and positive PCR results were also analyzed. All statistical analyses were performed in
Statistic v. 10 (Stat Soft 2011) [23] and visualized in GraphPad Prism version 5.00 for Windows, GraphPad
Software, San Diego California USA, www.graphpad.com.

Results and Discussion

During necropsy, many lesions were observed, which were compatible to bacterial infection, such as ocular
edema, splenomegaly, and ulcer of the skin (Table 2). The fishes that were positive by PCR for A.
hydrophila presented the hemorrhage and splenomegaly as the most common lesions.

Table 2. Prevalence of macroscopic lesions observed in Oreochromis

Niloticus positive for Aeromonas hydrophila by PCR
Lesions A B C F
Hemorrhage 100.00 (8/8) 33.33 (1/3) 66.66 (2/3) 9.09 (1/11)
Ocular edema 12.50 (1/8) 0.00 (0/3) 0.00 (0/8) 0.00 (0/11)
Opacity of the cornea 12.50 (1/8) 0.00 (0/3) 0.00 (0/8) 0.00 (0/11)
Splenomegaly 100.00 (8/8) 0.00 (0/3) 100.00 (3/3) 63.63 (7/11)
Ulcer of the skin 25.00 (2/8) 0.00 (0/3) 33.33 (1/3) 0.00 (0/11)

Oreochromis niloticus with aeromonosis could present hemorrhagic patches, dark discoloration in
skin, and congestion of internal organs [13]. In an experimental infection with A. hydrophila in O. niloticus,
cutaneuous hemorrhage at the base of all fins and in the mouth, ascites with serobloody fluid, and
exophthalmia [22] was observed. However, in this study besides hemorrhage in skin, ulcers of the skin were
also visualized as the most common lesions. According to Noga [20], ulcers appears after the progress of
skin lesions and can lead to exophthalmos, as observed in samples positive for this bacterium.
In histopathology analysis, processes that suggest bacterial infection were also observed. For the
positive animals for A. hydrophila, bacteria colonies, coagulative necrosis, hemorrhage, inflammatory
process, melano-macrophages , and vacuolar degeneration were the most common lesions (Table 3).
Coscelli et al., [24] performed an experiment with turbot (Scophthalmus maximus L.) that was
challenged with A. salmonicida, which presented diffuse infiltrates of inflammatory cells, mainly composed
by monocyte and macrophages on the connective tissue of coelomic cavity, vascular congestion, colonies of
bacteria, hemorrhage and necrosis. As mentioned by these authors, we also detected these lesions (Table 3)
as granulomas, degeneration, and eosinophils cells. As observed in this study, Harikrishnan et al., [25] also
observed granulomatous inflammation. It was an important point because onlt this research describes this
kind of lesion, which could indicate that these granulomas could be a sign that other disease causing
bacteria are also present like Mycobacterium or Francisella, and suggesting the co-infection. The
identification of A. hydrophila by PCR showed prevalence since 3.33% to 46.66%. It can be noted in
Figure 1 that in the second sampling of fish farm F, an increase in the number of infected animals occurred
by this species of bacteria. Jimoh and Jatau [26] detected 47%, while Balaji et al., [27] detected
41.7% of A. hydrophila in Oreochromis, that was similar to our study. However, we observed lower
occurrence that could be due to the water quality and handling of the animals without causing any stress or
lesions in the skin and the place where this bacterium enters in the body of the fish.
Although in this study water quality parameters were not evaluated, the difference in the prevalence
from fish farms (Figure 1) could be due to the immunity of the fishes, since stress causes
immunosuppression and becomes susceptible to infections.
This affirmation was supported by Janda and Abbott [28], which explains that immunosuppressed
fish by spawning or environmental triggers, such as high temperatures or low water levels are more
susceptible to Aeromonas.

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Table 3. Prevalence of microscopic lesions observed in Oreochromis

niloticus positive for Aeromonas hydrophyla by PCR
Lesions A B C F
Bacteria colonies 100.00 (8/8) 66.66 (2/3) 0.00 (0/3) 100.00 (11/11)
Calcification necrosis 0.00 (0/8) 33.33 (1/3) 0.00 (0/3) 9.09 (1/11)
Coagulative necrosis 100.00 (8/8) 100.00 (3/3) 66.66 (2/3) 81.81 (9/11)
Congestion 12.50 (1/8) 0.00 (0/3) 33.33 (1/3) 18.18 (2/11)
Eosinophils cells 87.50 (7/8) 33.33 (1/3) 0.00 (0/3) 18.18 (2/11)
Granulomes 0.00 (0/8) 33.33 (1/3) 66.66 (2/3) 36.36 (4/11)
Hemorrhage 50.00 (4/8) 66.66 (2/3) 33.33 (1/3) 100.00 (11/11)
Inflammatory process 100.00 (8/8) 100.00 (3/3) 100.00 (3/3) 63.63 (7/11)
Melano-macrophages 100.00 (8/8) 100.00 (3/3) 33.33 (1/3) 100.00 (11/11)
Vacuolar degeneration 100.00 (8/8) 100.00 (3/3) 100.00 (3/3) 81.81 (9/11)

Figure 1. Prevalence (%) of Aeromonas hydrophila by PCR in Oreochromis niloticus

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After sequencing and analysis, our sequence was similar (99% of identity) to Aeromonas
hydrophila (genbank: AM992197), confirming that the bacteria isolated in this study was from this species.
In the megablast search (GenBank) more than hundred sequences with identity of the 96% (the closest) with
the sequence of this study were found. Figure 2 has shown the bioinformatics study results with fourteen
sequences found with 96% ID and three sequences of A. hydrophila found in Brazil. According with the
dendrogram, the closest sequence was the A. hydrophila TGDY isolated in China from Betta splendens. For
A. hydrophila found in Brazil have 90 and 91% identityand both were found in Tilapia.

Figure 2. Dendrogram representing the bioinformatics study. The sequence in red was found in this study.
The dendrogram shows respectively for each isolate: the identity with the sequence of this study, the
host and country from which they were isolated and accession number from Genbank.

In the bioinformatics study, we observed that Aeromonas spp. similar to that found in this study
were from different country, organisms and environment, but the majority were found in different fish
species. Among these bacterial sequences, the most important is the A. hydrophila AL09-71 that was
responsible for a MAS disease outbreak in 2009 in West Alabama [29], where it alone led to an estimated
loss of more than 3 million pounds of food size channel catfish. Virulence studies have revealed that AL09-
71, is highly virulent to channel catfish, killing the fish within 24 h post exposure [29-30]. One among
these sequences was isolated from a patient, the A. hydrophila AHNIH1 that carried a 143-kb plasmid
(pASP-135), with a blaKPC-2 gene, TEM β-lactamase, genes encoding resistance to aminoglycosides,
chloramphenicol, fluoroquinolones, macrolides, and mercury [31]. In Brazil, the closest sequence with our

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study were found in Tilapia by Sebastião et al., [32], where several genera related to the pathogenic bacteria
were found and among 178 bacterial isolates, Aeromonas sp. were with higher frequency(31%). Although
in this study, we did not evaluate the presence of Aeromonas in fish fillets, there is an increased risk for
human consumption of raw O. niloticus, since muscle was added to the pool in the samples analyzed for
molecular tests, which had positive results (Figure 1).

Conclusion
The results found in this study by pathological and histopathological techniques showed bacterial infection
in the analyzed fishes, which was also confirmed by the molecular investigation. Since Aeromonas is a
zoonotic bacteria, it is suggested that producers should utilize the histopathological and molecular
techniques to check the presence of this infectious agent to guarantee public health and avoid economic
losses.

Acknowlegments
The authors thank to FAPESP for the financial support (2013/50504-5) and postdoctoral fellowships
(2014/15859-0 to C. J. Francisco, 2015/13025-7 to M. F. Falcone-Dias, and 2014/13718-0 to M. V.
Rodrigues). The authors also thankful to CNPq for the doctor the fellowship to R. J. da Silva of CNPq
(307808/2014-9) and CNPq-PROTAX (440496/2015-2)/FAPESP 2016/50377-1.

Conflict of interest
The authors declare that they have no conflict of interest.

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