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SCVMJ, XXI (1) 2016
185
Characteristics of Infectious Dropsy from an Epizootic of Cultured
Common Carp (Cyprinus carpio L.) With Special Investigation to Swim-
Bladder Lesions
Salah M. Aly1 and Mona M. Ismail2,
1Dept of Pathology, Fac. Vet. Medicine
Fish Farming and Technology
Institute, Suez Canal University, Ismailia, Egypt.
2Dept of Fish Diseases and Management, Fac. Vet. Medicine, Suez Canal
University, Ismailia, Egypt.
Abstract:
Infectious dropsy was recorded in 2015 among a group of Common
carp (Cyprinus carpio L.) collected from several private fish farms
at Sharkiya Province, Egypt. Out of 200 clinical cases, 180 (90%)
Common carp were found infected by bacteria. The bacterial isolates
revealed a mixed infection with Aeromonas hydrophila and
Pseudomonas fluorescens.The affected fish presented typical signs
of infectious dropsy including hemorrhagic lesions in the skin, fin,
tail, and eye. Loss of scale with sluggish movement and imbalance
were also observed in some fish. Grossly, the diseased fish exhibited
symptoms of septicemia such as congestion and hemorrhages in the
skin and internal organs with distended anus, exophthalmia and
ascites. The antimicrobial resistance of the isolated bacteria was
higher with Oxytetracycline (OX) and lower with Ciprofloxacin
(CIP). The other used antimicrobials showed variable resistance to
both bacterial isolates. Experimental infection was done on 100
Common carp and revealed same clinical findings and gross lesions
of the field study with 76 % mortality. Microscopically, the internal
organs showed degenerative changes, focal necrosis, circulatory
disturbances and inflammatory reactions. The swim bladder mucosa
of infected carp exhibited necrosis, epithelial sloughing with
congestion and lymphocytic infiltration. This study describes
Infectious dropsy among cultured carp in Egypt and highlighted the
importance of implementing preventive measures to control this
infection.
Key words: Common Carp, Infectious dropsy, bacteria, antibiotics,
swim bladder.
Introduction:
Aquaculture, in Egypt, express a
fast development during the last
decades with over 99 percent
produced by private farms (FAO,
2012, GAFRD 2014). While this
186 Salah Aly and Mona Ismail
growth is much appreciated in terms
of food security, outbreaks due to
disease hinder the development of
aquaculture and have a negative
impact on the economy not only in
Egypt but also in many countries.
Bacterial infections in fish are one
of the challenges that influence
sustainability of aquaculture
production in Egypt and elsewhere
(Aly 1994, Parvez and Mudarris,
2014).
Infectious dropsy (ID) in common
carp is one of the bacterial
hemorrhagic septicemic disease that
has been described by many
investigators (Kumar and Dey
(1991); Bohai et al., 1993).
Definitive diagnosis of ID based on
clinical findings and the detection
of the etiology. Bacterial
hemorrhagic septicemia is caused
by P. fluorescens
Wakabayashi
and Egusa, 1972 and Shiose et al.,
1974) which act as a primary
pathogen of freshwater fish and an
opportunistic bacteria for variable
marine and brackish waters fishes
(Hadi et al., 2002; Alicia et al.,
2004 and Foysal et al., 2011).
Other studies mentioned that A.
hydrophila may act as a primary
pathogen for fish or secondary
invaders for cases of hemorrhagic
septicemia (Candan et al., 1995;
Kozinska, 2002 and Güvener &
Timur, 2005). Recently, A.
hydrophila and P. fluorescens were
recorded as the cause of Bacterial
Hemorrhagic Septicemia (BHS) in
Cyprinus carpio and Channa
striatus (Parvez and Mudarris,
2014). The present work was,
therefore, undertaken to identify the
etiological agents of ID in Common
carp (Cyprinus carpio L.) and to
describe the symptoms and
pathological lesions of the disease
with special focus on the swim
bladder.
Materials and methods
1. Fish and Sample collection:
In the Early spring of 2015 complex
non-recognized disease occurred
suddenly during the production
stage of Common carp (Cyprinus
carpio L.) in several fish farms at
Sharkia Province. A total number of
200 clinically diseased common
carp, weighed 50 – 150 g, of both
sexes were collected as a random
samples, transferred alive to the
laboratory and kept in well-aerated
glass aquaria at 25 °C and examined
clinically using the methods
described by Lucky (1977) with
special attention to abnormal
coloration, swimming behavior,
respiratory manifestation, escape
reflex and its appetite.
2. Bacteriological examination:
A total of 200 clinically diseased
Common carp were collected and
sacrificed by decapitation and
disinfected with 70% ethanol.
Bacteriological swab samples were
taken from the cleaned surface of
liver, spleen, kidneys and swim
bladder after gentle removal of the
superficial layer and sterilization of
the exposed surface of the organ.
The bacteriological swabs were
inoculated in Trypticase soya broth
(TSB) and nutrient broth. The broth
SCVMJ, XXI (1) 2016
187
cultures were aerobically incubated
at 20-25 ºC for 18-24 hrs. A loopful
of broth was cultured on selective
media (Rimler-shotts agar, Ordal’s
Pseudomonas F. agar, Mac-
Conkey’s agar, trypticase soya
agar). The inoculated plates were
then incubated at 25-30 ºC for 24-
48 hrs and colonies were picked up
to nutrient slope agar and incubated
at 25-30 ºC for 24-48 hrs. The
isolated bacteria were identified via
morphological as well as
biochemical examinations as
reported by Frerichs and Hendrie
(1985) and Scheperclaus et al.
(1992).
3. Determination of antimicrobial
resistance:
The resistance of A. hydrophila and
P. fluorescens, that isolated in the
present study, was tested through
disk diffusion using The Mueller–
Hinton agar (Difco.). Six
antimicrobial agents were used
[chloramphenicol C, (30 µg, < 13
mm); oxytetracycline, OX (30 µg, <
15 mm); ciprofloxacin, CIP (5 µg, <
16 mm); Kanamycin K, (30 µg, <
13 mm);
Sulphamethoxazole/Trimethoprim,
SXT (25 µg, < 11 mm), and
Nalidixic acid, NA.(30 µg, < 14
mm)]. The sensitive isolates were
differentiated from the resistant one
through the use of break point
values. All the used disks were
purchased from Oxoid. The assays
using disk diffusion were prepared
based on the CLSI
recommendations (CLSI, 2005 a &
b).
4. Experimental infections:
One hundred apparently healthy
Common carp (150 ± 50 gm) were
divided into 2 equal groups. Each
group was reared in two glass
aquaria each of 250 liters capacity.
All Common carp were fed a
balanced diet suitable for the given
fish species and kept 2 weeks
before the experiment for
acclimatization and observation.
The food ingredients were
purchased from private suppliers
and prepared in the form of pellets.
Fish of first group injected I/P with
0.5 ml (108 cells / ml) of equal
mixture of isolated A. hydrophila
and P. fluorescens from clinically
infected Common carp. Second
group act as a control injected I/P
with 0.5 ml sterile broth. The
injections were done according to
Lucky, (1977) and Scheperclaus et
al. (1992). All experimentally
infected Common carp were noticed
for clinical findings or mortalities
throughout the experiment. The
clinically diseased fishes were
subjected to postmortem and
histopathological examinations after
two weeks post-infection. In
addition, bacterial re-isolation was
done.
5. Histopathological examination:
Specimens from the internal organs
and swim bladder of experimentally
infected and control Common carp
were fixed in 10% neutral buffered
formalin. Paraffin sections (5mu
thick) were obtained and stained
with hematoxylin and eosin (H&E)
(Bancroft et al., 1996).
188 Salah Aly and Mona Ismail
Results
1. Field study:
A. Clinical findings:
The adult infected Common carp
revealed exophthalmia and
abdominal distension where the
anal orifice was frequently
protruded in addition to loss of
response to the external stimuli in
young infected Common carp.
Hemorrhagic eyeballs together with
sluggish movement and imbalance
were also observed in some fishes.
B. Identification of the Isolates:
Out 200 clinically diseased
Common carp, 180 fish (90%) were
found infected by bacteria. The
isolates were assessed for their
morphological and biochemical
characteristics. Among these, two
isolates were pathogenic bacteria,
the first isolate was Gram negative,
rod shaped, motile with polar
flagella, catalase positive, oxidative
bacteria, able to ferment glucose
identified as A. hydrophila (85.7%).
The second isolate was Gram
negative, rod shaped, motile with
polar flagella, catalase positive,
oxidative bacteria, produced acid
from glucose in paraffin free media
but unable to ferment glucose
identified as P. fluorescens (84.3%).
C. Antimicrobial resistance:
The antimicrobial resistance of the
isolated A. hydrophila and P.
fluorescens was higher with
Oxytetracycline (OX) and lower
with Ciprofloxacin (CIP). The other
used antimicrobials
[Chloramphenicol (C),
Sulphamethoxazole/Trimethoprim
(SXT), Nalidixic acid (NA) &
Kanamycine (K)] showed variable
resistance to both bacterial isolates
(Table, 1).
2. Experimental study:
The clinical signs, appeared in 96%
of experimented common carp,
were similar to the natural infection,
it was observed within 10 – 24 hrs
post-infection with A. hydrophila
and P. fluorescens. The infected
fish exhibited loss of appetite, loss
of balance, upward position,
intermittent hyperexcitation and
finally loss of reflex prior to death.
Mortality reached 76 % by the end
of the observation period (2 weeks).
Grossly, the experimentally infected
Common carp revealed loss of
scales and petechial hemorrhages
on the body surface and underlying
muscles with distended abdomen
(Fig. 1). The liver was enlarged
with pale white necrotic foci on its
surface, bloody fluid was seen in
the pericardium and peritoneum
together with congested kidneys
and intestine (Fig. 2). The posterior
chamber of the swim bladder found
congested while the anterior
chamber appeared whitish gray with
petechial hemorrhages (Fig. 3).
Microscopically, the internal organs
showed signs of septicemia where
degenerative changes, focal
necrosis, circulatory disturbances
and inflammatory reactions in heart
and liver were evident (Figs. 4 & 5).
The swim bladder in some cases
showed extensive necrosis in the
epithelial lining with focal
aggregation of melanomacrophage
SCVMJ, XXI (1) 2016
189
cells in the subepithelial tissue. In
other cases, the swim bladder
showed mild congestion, edema and
leukocytic infiltration in the
submucosa and focal hyalinization
in the muscle layer (Fig. 6). In the
majority of cases, the swim bladder
showed marked congestion with
hemorrhage, diffuse necrosis and
mononuclear cells infiltration (Fig.
7). The mucosa, submucosa and
muscularis were focally replaced by
marked fibrous connective tissue
proliferation (Fig. 8).
The Common carp of the control
group did not show any clinical
signs of diseases with no gross or
microscopic lesions.
Fig. 1: Common carp post-infection with A. hydrophila and P. fluorescens
showing loss of scales, petechial hemorrhages on the body surface and
distended abdomen.
Fig. 2: Common carp post-infection with A. hydrophila and P. fluorescens
showing enlarged pale liver white necrotic foci on its surface. Bloody fluid
was seen in the body cavities together with congested kidneys and intestine.
Fig. 3: Swim bladder of common carp post-infection with A. hydrophila and
P. fluorescens showing congested posterior chamber and whitish gray
hemorrhagic anterior chamber.
Fig. 4: Heart of Common carp post-infection with A. hydrophila and P.
fluorescens showing focal necrosis and massive inflammatory reactions in
the myocardium. H & E stain, x 250.
3
4
1
2
190 Salah Aly and Mona Ismail
Fig. 5: Liver of Common carp post-infection with A. hydrophila and P.
fluorescens showing vacuolar degeneration, focal necrosis and focal
inflammatory reactions in the hepatic parenchyma. H & E stain, x 250.
Fig. 6: Swim bladder of Common carp post-infection with A. hydrophila and
P. fluorescens showing extensive necrosis in the epithelial lining with
congestion, edema and leukocytic infiltration in the submucosa. H & E stain,
x 250.
Fig. 7: Swim bladder of Common carp post-infection with A. hydrophila and P.
fluorescens showing marked congestion and hemorrhage. H & E stain, x 250.
Fig. 8: Swim bladder of Common carp post-infection with A. hydrophila and
P. fluorescens showing marked fibrous connective tissue proliferation in the
submucosa and muscularis. Van Geisons stain, X 250.
Table (1): Resistance of A. hydrophila and P. fluorescens that isolated from
the infected common carp to the tested antimicrobials.
Bacteria
Antimicrobial resistance (%)
C
SXT
CIP
NA
OX
K
A. hydrophila
61.00
57.67
00.00
46.67
73.33
53.22
P. fluorescens
66.00
75.33
21.67
85.33
100.00
97.00
C = Chloramphenicol, SXT = Sulphamethoxazole/Trimethoprim, CIP =
Ciprofloxacin, NA = Nalidixic acid, OX = Oxytetracycline, K = Kanamycine.
5
6
7
8
SCVMJ, XXI (1) 2016
191
Discussion
In the present study, the diseased
fish was collected with expression
of hemorrhagic lesions in the eye
and skin together with sluggish
movement, loss of balance and
distended abdomen. These results
attributed to the mixed bacterial
infection and similar symptoms
have also been reported by others
(Kumar and Dey, 1991; Snieszko
and Bullock, 1976; Moln´ar and
Csaba, 2005 and Foysal et al.,
2011).
Bacteriological isolation from
diseased carp revealed two
pathogenic bacteriare exhibited the
morphological and biochemical
properties resembled of A.
hydrophila and P. fluorescens.
These isolates supported by several
studies (Miyashita, 1984; Joseph
and Carnahan,1994; Candan et
al., 1995; Kozinska, 2002; Güvener
and Timur, 2005; Shiose et al.,
1974; Hadi et al., 2002; Alicia et
al., 2004; Foysal et al., 2011 and
Parvez and Mudarris, 2014).
Richards and Roberts (1978)
pointed out that, A. hydrophila and
P. fluorescens are ubiquitious in the
aquatic environment and frequently
implicated in the aetiology of
bacterial hemorrhagic septicemia.
Kozi´nska and Pe´kala (2012)
reported that, all strains of A.
hydrophila caused skin ulcers as
well as septicaemia in carp where
carp skin showed great
susceptibility to infection of all
Aeromonas strains. Recent study
indicated that A.hydrophila and
P.fluorescens were the etiological
agents for bacterial hemorrhagic
septicemic disease in Cyprinus
carpio (Parvez and Mudarris,
2014). Additionally, it is reported
that, Aeromonas spp. constitute
very often the component of mixed
bacterial flora isolated from
asymptomatic carriers as well as
from fish with various disease
conditions caused sometimes by
bacteria belonging to completely
different taxa (Kozi´nska and
Pe´kala, 2012).
A high degree of resistance towards
tetracyclines has been displayed by
P. fluorescens (100%) and A.
hydrophila (73%). the percentage of
tetracycline resistance in our study
was consistent with several studies
in aquatic culture (Penders and
Stobberingh (2008); Schmidt et al.,
(2001); Petersen and Dalsgaard
(2003) and Akinbowale et al.,
2007). Although A. hydrophila and
P. fluorescens displayed decreased
susceptibility to the 1st generation
quinolones nalidixic acid they were
highly susceptible to the newer
generation ciprofloxacin which
could be due to the recent use of
ciprofloxacin in aquaculture. This
result is consistent with that of
Sreedharan et al., 2012. In
addition, our finding revealed the
high resistance of both isolates to
chloramphenicol which is
consistent with that of Nguyen et
al., (2014) and Chang et al.,
(2007).
The isolation of bacteria from
mixed bacterial flora does not
192 Salah Aly and Mona Ismail
always indicate that they are
primary factor of a disease, so
experimental infection was carried
out to reproduce the recorded field
finding. The experimental study
confirm that both bacterial isolates
are able to produce the disease.
The gross pathological examination
of the affected organs indicated
necrotic foci in the liver, distended
gall bladder and shrinkage kidney
together with hemopericardium and
hemoperitoneum. These
pathological findings are in
accordance with that reported by
Kumar et al, (1986); Kumar and
Dey (1986) and Rober et al., 2000.
In relation to swim bladder, it
appears that our paper is the first to
describe the histopathology of the
swim bladder in relation to
challenge with mixed A. hydrophila
and P. fluorescens.
Conclusion:
For understanding the disease
process in mixed infection, external
clinical symptoms together with
histopathological changes are
important. The findings presented in
this study may be helpful in
facilitating the diagnosis of
bacterial hemorrhagic septicemia
caused by A. hydrophila and P.
fluorescens in Common carp but
complementary studies are needed
in order to better understand the
pathogenesis of the disease and to
set and implement preventive
measures to control this disease in
Egyptian aquaculture.
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