Journal of Aquaculture & Marine Biology

Molecular Detection of Aeromonas hydrophila as the

Main Cause of Outbreak in Tilapia Farms in Egypt

Abstract Research Article

An outbreak recorded in Tilapia farms in Kafrelsheikh governorate with high
Volume 2 Issue 6 - 2015
mortalities ranged between 30-70 % in the summer season of 2014. This study
was conducted for isolation and identification of the causative agent responsible
for mortalities in four fish farms. Twelve Aeromonas isolates were identified by Ibrahim M Aboyadak1*, Nadia G M Ali1 ,
PCR using Aeromonas species primer at the molecular weight of (953 bp) then Ashraf M A S Goda2, Walaa H Aboelgalagel3
all strains were also confirmed by PCR as Aeromonas hydrophila using Aeromonas and Asmaa M E Salam2
hydrophila specific-16S rRNA gene primer at the molecular weight of (103 bp). 1
Fish disease lab, National Institute of Oceanography and
Fishery (NIOF), Egypt
Keywords: Oreochromis niloticus; Outbreak; Fish farm; PCR: Aeromonas hydrophila;
Tilapia; mortality; Kafrelsheikh
2
Aquaculture department, National Institute of Oceanography
and Fishery (NIOF), Egypt
Central Diagnostic & Research Lab, Faculty of Veterinary
3

Medicine Kafrelsheikh University, Egypt

*Corresponding author: Ibrahim M Aboyadak, Fish disease

Introduction lab, National Institute of Oceanography and Fishery (NIOF),
Alexandria, Egypt, Tel: 00201005100472;
The global production of cultured tilapia represent 4.4 million Email:
ton in 2013 [1]. Egypt ranked as third major producer with total Received: August 5, 2015 | Published: September 30, 2015
production of 635843 ton in 2013 [2], approximately, 14.45% of
global cultured tilapia production. Kafrelsheikh is the main tilapia-
culturing center in Egypt as it produces about 320 thousand ton,
which represent 51.2% of Egyptian production and 7.27% of this outbreak in four tilapia farms in Kafrelsheikh by molecular
global production. Bacterial diseases are considered the main and detection using PCR.
most dangerous type of diseases affecting the fish production as
it represents 80 % of fish mortalities [3]. Fish mortalities have Materials and Methods
negative impact on fish farming. Fish farming is not only provide a
source of cheap animal protein and a source of income but also it is Study area
a source of employment. Continuous losses due, to fish mortalities Samples were taken from four fish farms suffered from mass
may make fish farms owners to stop or change the activity that mortalities in Kafrelsheikh governorate north of Egypt. First farm
causes loss of thousands of worker to their jobs. located in Baltim, second farm in Torombat seven and other two
In Kafrelsheikh, the semi intensive tilapia culture is the farms in Elhamol.
only system used in earthen bonds, fish farms supplied with Sample: Sixteen live Nile tilapias (Oreochromis niloticus) showing
agricultural drain water, fish density in studied farms is four fish the clinical signs of septicemia were sampled, as four fish were
per cubic meter. The most common bacterial disease affecting fish taken from each farm. Each fish weight ranged between150-500
is motile Aeromonas septicemia caused by Aeromonas hydrophila. gm. Each fish was packed alive in a separate stile labeled plastic
It is defined as septicemic disease worldwide distributed bag and transported to lab in ice pox.
affecting numerous species of freshwater and marine fishes. It
is also considered as the most significant disease which occurs Clinical examination: The clinical examination was performed
in cultured freshwater fish [4]. Congestion and haemorrhages on according to the method described by Conroy & Hermann [6].
the abdominal wall and at the base of fins with scale erosion at Post mortem examination: The post mortem examination was
different parts of the body were the marked clinical signs observed performed according to the method described by Austin & Austin
[5] who also recorded severe congestion of internal organs with [7].
accumulation of ascetic fluid in abdominal cavity and swollen
kidney and spleen. In Egypt, motile Aeromonas septicemia has Isolation and identification of the causative agent: Under
a potential economic hazard in which it causes severe losses sterile conditions one tryptic soy broth (oxoid) tube was
in cultured freshwater fish including Oreochromis niloticus, inoculated with a platinal lope from heart blood, liver and kidney
common carp, Mugil cephalous and Mugil capeto [6]. Increasing tissues of fish and incubated at 37 ̊C for 18 hrs. Rimler-Shotts
water change, elevation of water column highs and stop feeding media with novobiocin selective supplement (Oxoid) plates were
helped in decreasing the deleterious effect of the outbreak but not streaked with a loopful of cultured broth then incubated at 37 ̊C
solve the problem completely. So the present work aimed for the for 24 hrs.The growing colonies were further identified by PCR.
isolation and identification of the causative agent responsible for

Submit Manuscript | http://medcraveonline.com J Aquac Mar Biol 2015, 2(6): 00045

Molecular Detection of Aeromonas hydrophila as the Main Cause of Outbreak in Tilapia Copyright:
©2015Aboyadak et al. 2/4
Farms in Egypt

The isolates not grown on: Rimler-Shotts media were cultured and Aboyadak [12]. The clinical signs observed on infected
on tryptic soy agar (Oxoid), the recovered colonies were stained fish including hemorrhagic patches can be attributed to both
with Gram stain. bacterial invasion and colonization also, to toxins produced by
invading microorganism. Postmortem examination revealed
Identification of collected strains using PCR: presence of severe enteritis with sloughing of intestinal mucosa.
DNA extraction: Extraction of bacterial DNA was performed by Yellow ascetic fluid in the abdominal cavity of some samples
thermolysis according to the method described by Ahmed et al. was observed. Liver was congested and in some cases was pale
[8]. 100 µl of overnight cultured broth was mixed with 400 µl of enlarged with distended gall bladder. Spleen was enlarged and
sterile distilled water in eppendorf tubes that was transferred to congested (Figure 3 & 4). The described Postmortem lesions
the heat block for 5 mins at 95 ̊C followed by centrifugation at were in agreement with the results described by Asaad [5],
15000 rpm for 2 min at 4 ̊C, the supernatant was used as DNA Aboyadak [12], Huizinga et al. [13] and AOAD [14]. The internal
template and stored at – 20 ̊C for PCR study (Table 1). lesions appeared on infected fish were attributed to both bacterial
invasion and colonization also, to its produced toxins described
PCR amplification of targeted DNA: by Boulanger et al. [15]. Twelve from sixteen isolate were grown
a) PCR mixture for both reactions (detection of Aeromo- on R-S media giving small yellow colonies which are characteristic
nas species and Aeromonas hydrophila): Reactions were for Aeromonas hydrophila as described by Cipriano [16] and for
performed in a total volume of 25 μl. Each volume containing, confirmation PCR identification was performed.
5 μl of 5X master mix (taql/high yield- Jena Bioscience, Jena, The PCR amplification with Aeromonas spp. specific primer
Germany, consists of DNA polymerase, dNTPs mixture, (NH4) identified twelve Aeromonas spp. isolate as specific band
SO4, MgCl2, Tween 20, Nonidet P-40, stabilizers+1.25 μl of each appeared by electrophoresis at a molecular weight of 953 bp
primer (forward and transverse) (20 pmol/μl), + 5 μl of geno- which is specific for Aeromonas spp. as showed in Figure 5 that
mic DNA + 12.5 μl double deionized distilled water. was typically described by Lee et al. [9] and Aboelgalagel [17].
b) Thermal cycle adjustment for: The PCR amplification using Aeromonas hydrophila specific
primer (16S rRNA) identified twelve Aeromonas hydrophila
i. Amplification of Aeromonas species targeted DNA as strain as the PCR assay results in the amplification of 103 bp
described by Lee et al. [9]: Amplification was carried out band specific Aeromonas hydrophila, as showed in Figure 6. The
in thermal cycler (Peltier thermal cycler Model: MG 96+ en- same results were observed by Tarakhan et al. [10], Aboelgalagel
zyme® USA), with an initial denaturation at 94°C for 4 mins [17] and Zakaria [18]. Uncontrolled mortalities were recorded
followed by 35 cycles of denaturation each at 94°C for 1 min, in several farms with different rearing facilities associated with
annealing at 68 °C for 30 sec and an extension step at 72°C for high temperature occurred in July and August. Not only high
45 sec. After the end of cycle’s one final extension step at 72°C temperature was recorded in this period, but also, high ammonia
for 10 mins was performed. levels triggered the stability of rearing conditions of tilapia ponds.
According to Irgeui et al. [19] any change in water parameters will
ii. Amplification of Aeromonas hydrophila targeted DNA lead for immune stress conditions allowing bacterial infections to
(specific-16S rRNA gene as described by Trankhan et al. take place. The rest four isolates that not grow on Rimler-Shotts
[10]: Amplification was carried out in thermal cycler, with an media were recultured on tryptic soy agar giving yellow colonies.
initial denaturation at 94°C for 2 mins, followed by 35 cycles Gram staining revealed filamentous long pleomorphic gram-
of denaturation, each at 94°C for 30 sec, annealing at 55.5 °C negative rods that indicating the presence of Flavobacterium
for 30 sec and an extension step at 72°C for 30 sec, After the columnare. This bacterium isolated from cutaneous lesions
end of cycles one final extension step at 72°C for 10 mins was (depigmented area) of affected fish (Figure 7) which was similar
performed. to that described by Cain & Lafrentz [20]. The culture characters
DNA assay of the causative agent and the PCR identification indicated that
Aeromonas hydrophila was the major cause of the outbreak
DNA was assayed by agarose gel-electrophoresis, 5µl of affecting tilapia farms in Kafrelsheikh governorate causing high
PCR products were electrophoresed on 1.5% agarose gels for mortalities as the causative agent was isolated from three farms
Aeromonas spp. & 2% agarose gels for Aeromonas hydrophila. out of four examined farms in a total prevalence of 75 %.
The agarose gels supplied with 0.6 μg/ml ethidium bromide in 1X
Table 1: Primers used for detection of Aeromonas species and Aeromonas
tris acetate EDTA buffer in gel electrophoresis apparatus (SCIE- hydrophila.
PLAS, UK). DNA bands were visualized over a UV transilluminator
(Winpact Scientific, USA). Target Primer Name Oligonucleotide Size
Sequence (5´-3´) (Bp)
Results and Discussion
Aeromonas AER-F CTA CTT TTG CCG 953 bp
Clinical examination of diseased fish samples revealed species AER-R GCG AGC GG
presence of general signs of septicemia including hemorrhagic TGA TTC CCG AAG
patches on the skin, base of pectoral fin and around anal opening. GCA CTC CC
Scale desquamation and skin ulcerations observed with fin Aeromonas 16SrRNA-F GGCCTTGCGCGATTG- 103 bp
erosions. Uni and bilateral exophthalmia with abdominal ascitis hydrophila 16SrRNA-R TATAT GTGGCGGAT-
in some samples are present (Figure 1 & 2). The observed clinical CATCTTCTCAGA
signs were similar to that described by Asaad [5], Lewbart [11]

Citation: Aboyadak IM, Ali NGM, Goda AMAS, Aboelgalagel WH, Alnokrashy AME (2015) Molecular Detection of Aeromonas hydrophila as the Main Cause
of Outbreak in Tilapia Farms in Egypt. J Aquac Mar Biol 2(6): 00045. DOI: 10.15406/jamb.2015.02.00045
Molecular Detection of Aeromonas hydrophila as the Main Cause of Outbreak in Tilapia Copyright:
©2015Aboyadak et al. 3/4
Farms in Egypt

Figure 1: Oreochromis niloticus naturally infected with Aeromonas

hydrophila showing skin hemorrhage. Figure 4: Oreochromis niloticus naturally infected with Aeromonas
hydrophila showing enteritis, intestine filled with purulent exudates
(a), pale enlarged liver (b) with enlarged gall bladder (c).

Figure 2: Oreochromis niloticus naturally infected with Aeromonas hy- Figure 5: 1.5% Agarose gel electrophoresis of PCR product showing
drophila showing skin hemorrhage at the base of pectoral fin with hem- specific Aeromonas spp. bands at 953 bp.
orrhagic skin ulcer under the dorsal fin. M: 100 bp DNA size marker; ctN: control negative

Figure 6: 2 % Agarose gel electrophoresis of PCR product showing

specific Aeromonas hydrophila bands at 103 bp.
M: 100 bp DNA size marker; ctN: control negative
Figure 3: Oreochromis niloticus naturally infected with Aeromonas
hydrophila showing severe enteritis, congestion and hemorrhages (a).

Citation: Aboyadak IM, Ali NGM, Goda AMAS, Aboelgalagel WH, Alnokrashy AME (2015) Molecular Detection of Aeromonas hydrophila as the Main Cause
of Outbreak in Tilapia Farms in Egypt. J Aquac Mar Biol 2(6): 00045. DOI: 10.15406/jamb.2015.02.00045
Molecular Detection of Aeromonas hydrophila as the Main Cause of Outbreak in Tilapia Copyright:
©2015Aboyadak et al. 4/4
Farms in Egypt

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Citation: Aboyadak IM, Ali NGM, Goda AMAS, Aboelgalagel WH, Alnokrashy AME (2015) Molecular Detection of Aeromonas hydrophila as the Main Cause
of Outbreak in Tilapia Farms in Egypt. J Aquac Mar Biol 2(6): 00045. DOI: 10.15406/jamb.2015.02.00045