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Pathogenicity of Achlya proliferoides and Saprolegnia diclina (Saprolegniaceae)

Associated with Saprolegniosis Outbreaks in Cultured Nile Tilapia (Oreochromis
niloticus)

Article · March 2013

DOI: 10.5829/idosi.wjfms.2013.05.02.7212

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World Journal of Fish and Marine Sciences 5 (2): 188-193, 2013
ISSN 2078-4589
© IDOSI Publications, 2013
DOI: 10.5829/idosi.wjfms.2013.05.02.7212

Pathogenicity of Achlya proliferoides and

Saprolegnia diclina (Saprolegniaceae) Associated with
Saprolegniosis Outbreaks in Cultured Nile Tilapia (Oreochromis niloticus)
1
Mortada M.A. Hussein, 2Walid H. Hassan and 3Maha A. Mahmoud

Department of Fish, Faculty of Veterinary Medicine,

1

Beni-Suef University, Beni-Suef 62512, Egypt

2
Department of Bacteriology, Mycology and Immunology,
Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62512, Egypt
3
Advanced Center for the Diagnosis of Animal and Poultry Diseases, Fayoum 63517, Egypt

Abstract: A study was conducted to evaluate the pathogenicity and pathology of Achlya proliferoides
BSN-M005 and Saprolegnia diclina BSN-M003, isolated from saprolegniosis outbreaks against immature
stages of Nile tilapia, Oreochromis niloticus. The cumulative mortality rates of the tested fish groups that
exposed to high zoospore concentrations of A. proliferoides BSN-M005 and S. diclina BSN-M003 were 60 and
100% respectively. The histopathological changes associated with saprolegniosis lesions induced by S. diclina
BSN-M003, were loss of the epidermis, edema of the hypodermis, different degrees of degenerative changes
in the underling musculature and the fungal elements were observed penetrating the entire musculature. On the
other hand, the histopathological changes associated with saprolegniosis lesions induced by A. proliferoides
BSN-M005 showed mats of hyphae attached the surface of epidermis, necrosis, degenerative changes close
to hyphae and sometimes fungal elements were observed penetrate into dermal layer but never reach the
underling musculature. It is clear from our results that S. diclina BSN-M003 is highly pathogenic than
A. proliferoides BSN-M005 to Nile tilapia.

Key words: Saprolegniosis Achlya proliferoides Saprolegnia diclina Pathogenicity

INTRODUCTION Pathogenic members of the order Saprolegniales,

Saprolegniosis is an infectious fungal disease that is
widespread in all stages of the life cycle of fish. The
disease causes serious losses in fish farms and hatcheries
and considered as one of aquatic disease implicated in
mass mortalities of cultured and wild fish in many
countries [1-3]. The disease appears as cotton wool-like
tufts on the body surface causing destruction of the skin
and/or fins due to cellular necrosis by hyphal
penetration and that is generally restricted to the
epidermis and dermis. Most lesions are caused by
Saprolegnia species that why the disease is called
saprolegniosis, however, other Saprolegniaceae including
Achlya may be primarily cause a clinically identical
saprolegniosis [4-6].
which includes two families, the Saprolegniaceae
(e.g. Achlya, Brevilegnia, Dictyuchus, Saprolegnia and
Thraustotheca) and the Leptolegniaceae (Aphanomyces,
Leptolegnia and Plectospira), totaling 132 species in
about 20 genera [7] are considered the causative agents
that incriminated in such infections. The most important
water moulds in the order Saprolegniales are those
water moulds in the genera Achlya and Saprolegnia that
can be pathogens of many fish species and their eggs
[8-10].
In Egypt, saprolegniosis constitute one of the
most important disease causing troubles in freshwater
cultured fish with several economical losses [11-13].
Recently, mass mortalities associated with an outbreak
of saprolegniosis was recorded among Nile tilapia,

Corresponding Author: Walid H. Hassan, Department of Bacteriology, Mycology and Immunology,

Faculty of Veterinary Medicine, Beni-Suef University, Beni-Suef 62512, Egypt.
Tel: +2 082 2327982.
188
 World J. Fish & Marine Sci., 5 (2): 188-193, 2013
Oreochromis niloticus, fish farm in Beni-Suef
Governorate, Egypt, where as several Saprolegnia and
Achlya isolates were found to be associated with the
disease incidence.
The present study was conducted to evaluate the
pathogenicity and the pathology of two selected isolates
of Saprolegnia and Achlya, isolated during the outbreak
of saprolegniosis, against immature stages of Nile tilapia.
MATERIALS AND METHODS
Fungal Strains: For artificial infection, Achlya
proliferoides BSN-M005 and Saprolegnia diclina
BSN-M003 were used for testing their pathogenicity in
juvenile Nile tilapia. Both isolates were isolated from
outbreak of saprolegniosis in cultured Nile tilapia.
The tested strains were grown in Petri dishes on glucose
yeast-extract (GY) agar and incubated at 20°C. The
zoosporic stages of the tested fungi were obtained by
inoculating agar blocks 1 X 1 cm (3~4 blocks) with
actively growing mycelia of each fungus into Petri dishes
containing 20 ml of GY broth. The cultured plates were
kept at 20°C for 24 hours. The growing mycelia were cut
and washed repeatedly in sterilized tap water (TW) then,
transferred into 20 ml of fresh sterilized TW and kept for
18~24 hours at 20°C [14]. The zoospores were harvested,
counted with a Neubauer chamber (ERMA, R, Tokyo) and
adjusted to two concentrations that were 2 X 103 and 2 X
106 spore/ml. Tufts of the growing mycelia in TW of
both tested fungi were also used for experimental
infection.
Fish: All experiments were performed at Aquatic
Laboratory Unit, Fish Department, Faculty of Veterinary
Medicine, Beni-Suef University, Beni-Suef, Egypt.
Sub-adult Nile tilapia with an average weight of 40±2 g
and 14±2 cm length of both sexes were obtained from a
private tilapia hatchery at Beni-Suef Governorate, Egypt.
The fish were maintained in 500 L fiberglass tank, the
water quality and temperature were monitored daily and
kept within the acceptable range for tilapia. The fish were
fed commercial pellet feed (Joe-Trade Factory, El-Asher
men Ramadan, Cairo, Egypt) twice daily at a ratio of 2% of
their body weight and kept under observation for 2 weeks
prior to the experiment. Feeding was stopped three days
prior to starting the experiment following the various
recommendations for the maintenance of fish bioassay
described by Ellsaesser and Clem [15].
189
All fish experiments were conducted in 10 L aquaria
at 25±2°C. Five tilapia individuals weighting 40±2 g
were placed in each aquarium 24 h prior to the
experiments.
Experimental Design: Two sets of 4 aquaria (10 L) with
5 fish in each were prepared. The first set was designated
for the experimental infection with A. proliferoides
BSN-M005. Two aquaria were used for exposure to the
two concentrations of zoospores of A. proliferoides
BSN-M005, one was used for exposure to mycelial mats,
while the fourth one was used as negative control.
The second set was designated for the experimental
infection with S. diclina BSN-M 003 and treated at the
same manner as mentioned above.
To induce infection, all fish groups including the
negative control were subjected to ami-momi (net-shake)
treatment [16] before being exposed to the zoospores or
mycelial mats of the tested fungi. Briefly, fish were shaken
in the air in a fan-shaped scoop net for 2 min and then
rapidly washed before being returned to their aquaria.
Then, one liter of water was taken from each tested
aquaria and replaced by 1 L of both low or high
concentration of the tested fungal zoospore suspensions
to obtain zoospore concentrations of 2 X 102 (low) and
2 X 105 (high) spore/ml. Control aquaria were treated as
experimental ones with exception. The exception is that
1 L of aquaria water was replaced by 1 L of sterilized
TW. For exposure to fungal mycelia, 2 g wet weight of
mycilial mats of each tested fungi was warped in a piece
of gauze and hanged into their tested aquarium corners.
The aquaria were checked each day after exposure for
10 days, considering the day of exposure as 0-day and
dead and moribund fish were removed for examination.
All fish remaining at the end of the 10-day period were
removed for examination.
Pathological Examination: All fish were examined for
gross pathological changes and those exhibiting lesions
were subjected to routine necropsy. Then, tissue
specimens from moribund and dead fish including all the
body with special intention to head, trunk muscles, gills
and fins were excised and fixed in 10% neutral buffered
formalin solution, embedded in paraffin and sectioned at
4~5 µm. The sections were stained with methanamine
silver nitrate-Grocott’s variation and they counter-stained
with haematoxylin and eosin (Grocott-H and E) and/or
light green (Grocott-Light green).
 World J. Fish & Marine Sci., 5 (2): 188-193, 2013

RESULTS

External Clinical Signs and Mortality Pattern: Artificial

infection of Nile tilapia was done using A. proliferoides
BSN-M005 and S. diclina BSN-M003 and the results
varied considerably with the two fungi used. Certainly,
none of the fish challenged to low zoospore
concentrations and those challenged with mycelial mats
of both tested fungi died or showed clinical signs Fig. 1: Moribund immature Nile tilapia, O. niloticus,
characteristics to saprolegniosis. Furthermore, during shows cotton-like mycelial growth 4 days after
post-mortem examination, none of these fish showed challenging with S. diclina BSN-M 003 Arrows.
external or internal gross lesions. They seemed to be as Scale bar 5cm
apparently healthy as the fish in the negative controls.
On gross observation, fish groups challenged with
high zoospore concentrations of tested S. diclina strains
as well as A. proliferoides became listless, showed erratic
swimming, rose to the water surface or rested on the
bottom of their aquarium. Fish challenged with S. diclina
started to die on the day 2 after challenge showed no
characteristic external signs, while on the days 3-7 both
dead and moribund fishes had mycelial growth on various Fig. 2: Immature Nile tilapia, O. niloticus, died 4 d after
parts of the body surface especially on the head, dorsal being challenged with S. diclina BSN-M 003. Note
and caudal fins (Figs. 1and2). Finally, all fish in that group the saprolegniosis lesion on tail fin and eye
were died within 8 days. On contrary, mortalities among Arrows. Scale bar in cm
fish challenged with A. proliferoides were slightly delayed
and started at the day 6, extended till the end of the
experiment (10 days) and associated with cotton like,
whitish color lesions scattered on the body surface
(Figs. 3and4).
As a matter of fact, the cumulative mortality rates of
the tested fish groups that were exposed to high
zoospore concentrations of A. proliferoides BSN-M005
and S. diclina BSN-M003 were 60 % and 100 %, Fig. 3: Moribund immature Nile tilapia, O. niloticus,
respectively (Table 1). All control negative groups that showing cotton-like mycelial growth scattered on
were exposed only to ami-momi treatment as well as those the head and body surface 6 days after
that exposed to mycelial mats showed survival rates of challenging with A. proliferoides BSN-M005
100% and that was evident till the end of the experiments. Arrows. Scale bar 5cm
In contrast, fish group that exposed to high zoospores of
S. diclina BSN-M003 experienced cumulative mortality
rates of 20, 40, 20 and 20% on the days 2, 4, 6 and 8 after
challenge, respectively. On the other hand, fish group
exposed to high zoospores of A. proliferoides BSN-M005
experienced cumulative mortality rates of 20, 20 and 20%
on the day 6, 8 and 10 after challenge respectively.
Fig. 4: Immature Nile tilapia, O. niloticus, died 8 days after
Histopathological Changes: Generally, the being challenged with A. proliferoides BSN-M005.
histopathological changes associated with experimental Note the saprolegniosis lesion on head, dorsal and
infection with S. diclina BSN-M003 and A. proliferoides tail fin Arrows. Scale bar in cm

190
 World J. Fish & Marine Sci., 5 (2): 188-193, 2013

Table 1: Pathogenicity of A. proliferoides BSN-M005 and Saprolegnia diclina BSN-M 003 to immature Nile tilapia exposed to 2 X 105 spore ml 1

Mortality %
----------------------------------------------------------------------------------------------------------------------
Days After infection A. proliferoides BSN-M005 S. diclina BSN-M 003
2 0.0 20
4 0.0 40
6 20 20
8 20 20
10 20
Total 60 100

Fig. 5: Slight destruction of the epidermal layer of
immature Nile tilapia, O. niloticus, exposed only to
“ami-momi” treatment (arrows). Scale bar = 50µm
Fig. 8: Head of Immature Nile tilapia, O. niloticus, 6 days
after challenge with A. proliferoides BSN-M005.
Note superficial penetration of the fungal elements
to the epidermal layer. Grocott-Light green. Scale
bar = 50µm

Fig. 6: Extensive edema of the hypodermis radiating away

from the invading mycelia of S. diclina BSN-M
003 resulting in marked myofibrillar degeneration
Arrows. Scale bar 50µm Grocott-Light green
Fig. 9: Invading fungal elements of A. proliferoides BSN-
M005 penetrating only epidermal and dermal
layers Arrows. Note the hyperplasic proliferations
and edema around the invading mycelia. Scale bar
30µm. Grocott-HandE)

BSN-M005 were loss of the epidermis, edema of the

Fig. 7: Hyaline degeneration of myofibrillar cells and loss
of nuclei, focal myofibrillar necrosis and edema
(*) together with massive mycelia penetration of
S. diclina BSN-M 003 Arrows. Scale bar 30µm.
Grocott-HandE
hypodermis and different degrees of degenerative
changes in the underling musculature. In contrast,
“ami-momi” treatment frequently caused damage and loss
to the epidermal layer of the treated fishes (Fig. 5) and,
therefore, facilitates the establishment of the infection.
The histological examination of the saprolegniosis
lesion of the tested fish after exposure to high
concentration of S. diclina BSN-M003 zoospores showed
the fungus radiated away from the focus of the infection
and subsequently penetrated the basement membrane,

191
 World J. Fish & Marine Sci., 5 (2): 188-193, 2013
extended into the dermis and, in most cases, continued to
grow into the hypodermis and musculature. Extensive
edema of hypodermis was present, radiating away from
the mycelial mass into non-affected areas. The edema
extended into the muscle mass, resulting in marked
myofibrillar degenerative changes. The initial change was
hyaline degeneration of myofibrillar cells, followed by loss
of nuclei together with focal myofibrillar necrosis
(Figs. 6 and 7). Frequently, changes in the underling
musculature ranged from complete sarcoplasmic loss to
mild degeneration and fungal elements were observed
penetrating the entire musculature. One the other hand,
for the tested fish that exposed to zoospores of
A. proliferoides BSN-M005, only mats of hyphae attached
the surface of epidermis were observed (Figs. 8 and 9)
together with necrosis and degenerative changes close to
hyphae. In other sites, fungal elements were observed
penetrate into dermal layer but never reach the underling
musculature.
DISCUSSION
In earlier studies conducted by Hussein and Hatai
[16], saprolegniosis was successfully induced in 5 species
of salmonids by using “ami-momi” treatment and
confirmed that the method was extremely useful as the
method of artificial infection. In this study, the
“ami-momi” treatment was applied to induce artificial
infection with A. proliferoides BSN-M005 and S. diclina
BSN-M003 in sub-adult Nile tilapia. Interestingly, data
generated with the presented study showed that none of
the fish groups challenged with low zoospores or mycelial
mats of either tested fungi died, nor did those in the
control groups, although they were exposed to
“ami-momi” treatment at the same level of the tested
groups. Similar results were obtained by Hatai and
Hoshiai [2] who used the same technique for examining
the pathogenicity of S. parasitica and S. diclina to
immature Coho salmon and they also recorded that no
mortalities occurred among fish groups exposed only to
“ami-momi” treatment. On contrary, the results obtained
by other authors in relation to the use of “ami-momi”
treatment to induce saprolegniosis are contradictory.
Thus, Fregeneda et al. [17] elucidated that the “ami-momi”
treatment predisposes the experimental infection of
immature rainbow trout with Saprolegnia; however, it
could kill small fish.
The results in (Table 1) show that A. proliferoides
BSN-M005 and S. diclina BSN-M003 could be pathogenic
to Nile tilapia. All fish groups exposed to high
192
concentrations of zoospores of both tested fungi showed
different levels mortalities. However fish group exposed
to S. diclina BSN-M003 showed high cumulative
mortalities (100 %) than those exposed to A. proliferoides
BSN-M005 (60 %) indicating that S. diclina BSN-M003
infection can cause severe destruction of the superficial
tissue and the direct cause of death is probably related to
the massive osmoregulatory problems [6, 18].
The histopathological demonstrations of the
saprolegniosis lesions of tested fish that exposed to high
zoospores of S. diclina BSN-M003 showed the radiating
fungal elements first destroy the epidermis followed by
penetration of basement membrane and subsequent
invasion into the dermis, hypodermis and subcutaneous
musculature (Figs. 6 and 7). The epidermal cells and
collagen fibers were necrosed and degenerated close to
the hyphae. These pathological findings were consistent
with those previously described [2, 16, 18].
As far as we know, these pathological findings
probably referred to enzymatic activities (chymotrypsin-
like, serine proteases) of such opportunistic fungi
including Saprolegnia as well as Achlya were also likely
to play a role in their pathogenicity, their virulence and
invasiveness against their hosts [19-22]. On the other
hand, the hitopathological picture related to fish that
exposed to high zoospores of A. proliferoides BSN-M005,
revealed superficial invasion of the fungal elements only
to the epidermis and to a little extend to hypodermal layer
(Figs. 8 and 9). The possible explanation is may be related
to the lake of enzymatic activities of A. proliferoides
BSN-M005. This is may be supported by what is reported
by Kales [23] who found that S. ferax and Saprolegnia
spp. had a proteolytic activity more than A. ambisexualis.
It is clear from the present study that S. diclina
BSN-M003 is highly pathogenic than A. proliferoides
BSN-M005 to Nile tilapia. Even though the virulence of
the two species was different, further studies on the factor
(s) involved in virulence of fish-pathogenic strains of
Saprolegniales are required.
ACKNOWLEDGEMENTS
The authors would like to thank Fish department,
faculty of Veterinary Medicine, Beni Suef University, for
supporting this research work.
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