Aquaculture 516 (2020) 734556

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Aquaculture
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Prevalence, diagnosis and experimental challenge of Dermocystidium sp. T

infection in Nile tilapia (Oreochromis niloticus) in Egypt
Heba H. Mahbouba,∗, Adel Shaheenb
a
Fish Diseases and Management Department, Faculty of Veterinary Medicine, Zagazig University, 44519, Zagazig, Sharkia, Egypt
b
Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Benha University, 13736, Benha, Qalyubia, Egypt

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

Keywords: This study confirms the systemic position of Dermocystidium sp. in Nile tilapia (Oreochromis niloticus) internal
Dermocystidium organs (liver, spleen, kidneys, stomach and intestine). Our work reveals an entire approach for Dermocystidium
Oreochromis niloticus sp. including prevalence, diagnosis, spores multiplication, experimental infection and histopathological ex-
Prevalence amination. A total of 1388 Nile tilapia (766 cultured fish and 622 wild fish) were collected from May 2012 to
Diagnosis
May 2014 and screened for Dermocystidium infection. The total prevalence of infection was 35.5% and was
Experimental challenge
higher in wild (45%) than cultured fish (27.8%). The highest rates of infection were recorded in the largest fish
and in winter. Several different techniques were employed to detect and identify Dermocystidium sp. from the
internal organs of infected hosts including macroscopic examination, microscopic observation of tissue squashes,
histological examination and in vitro culture. Infected fish were sluggish and suffered from severe ulceration and
black discoloration of the skin. The post-mortem lesion included dark-grayish nodules on internal organs, par-
ticularly the liver. Squash preparations from the internal organs revealed spherical spores (signet-ring shape),
which is characteristic for Dermocystidium sp. The spores showed multiplication in the freshly dead host tissue.
Our study demonstrated new successful attempts for in vitro culture of Dermocystidium either on Eagle's
Minimum Essential Medium after adjusting pH of media to 3.5 or on Sabouraud's dextrose agar media enriched
with duck decoction 10%. Experimental infection revealed that intra-gastric route was more pathogenic than the
immersion route. In histopathological sections from infected fish tissue following natural and experimental in-
fection, spherical spores of Dermocystidium were embedded in the affected tissue. To the best of our knowledge,
this study represents the first report of Dermocystidium infection in wild Nile tilapia in Egypt.

1. Introduction salmonid fish by a new species, Dermocystidium macrophagi in rainbow

trout (Oncorhynchus mykiss) from France and characterized by gross
Dermocystidiosis is an important newly recorded highly pathogenic kidney lesions, Landsberg and Paperna (1992) described systemic
systemic disease among cultured and wild Nile tilapia at different lo- granuloma in goldfish caused by a Dermocystidium like aetiological
calities in Egypt (Zagazig, Sharkia Governorate and Benha, Qalyubia agent, Olson and Holt (1995) reported mortalities induced by gill in-
Governorate) that has resulted in higher mortalities. Dermocystidium sp. fection of Dermocystidium salmonis in Oregon salmonids. Shaheen
sometimes affect amphibians (Gonzalez-Hernandez et al., 2010), but (2000) isolated Dermocystidium from the white nodules on the skin and
most commonly affect fish and induce diseases. Previous studies re- fins of Nile tilapia in Egypt, Zhang and Wang (2005) recorded mor-
ported mortality cases associated with Dermocystidium sp. infection in talities caused by Dermocystidium spp. in juvenile catfish Silurus mer-
different fish species and localities, McVicar and Wooten (1980) de- idionalis in China, Dykova and Lom (2007) isolated Dermocystidium koi
scribed mortality events caused by Dermocystidium spp. in Atlantic from the subcutaneous tissue of the host, Cyprinus carpio var. koi, Raffel
salmon (Salmo salar) with occurrence of extensive caseous lesions that et al. (2008) observed high fish mortalities in Notophthalmus viridescens
most prominent in the visceral fat, Wootten and Mcvicar (1982) re- by Amphibiocystidium in the U.S. Pacific Northwest, Langenmayer et al.
ported Dermocystidium infection from cultured eels, Anguilla anguilla (2015) revealed cutaneous infection with Dermocystidium salmonis in
L., in Scotland, Moer et al. (1986) mentioned systemic parasitism of cardinal tetra, Paracheirodon axelrodi, and Kirkbright et al. (2016)

∗
Corresponding author. Department of Fish Diseases and Management, Faculty of Veterinary Medicine, Zagazig University, 44519, Zagazig, Sharkia, Egypt. Tel.:
+7437581260.
E-mail address: [email protected] (H.H. Mahboub).

https://doi.org/10.1016/j.aquaculture.2019.734556
Received 4 April 2019; Received in revised form 12 September 2019; Accepted 30 September 2019
Available online 23 October 2019
0044-8486/ © 2019 Elsevier B.V. All rights reserved.
H.H. Mahboub and A. Shaheen Aquaculture 516 (2020) 734556

linked Dermocystidium-like organism with a mortality cases in yellow meter (ml/L) (WTW, Germany) were used for monitoring water (tem-
perch Perca flavescens (Mitchill) in Ontario, Canada. Dermocystidium perature, pH, salinity, and dissolved oxygen), respectively.
was originally named Perkinsus (Mackin et al., 1950) then it was placed
among the lower fungi (Allen et al., 1968), and later among the pro- 2.3. Clinical and post-mortem examination
tozoans (Levine, 1978; Nash et al., 1989). This taxon has since been
located in a novel clade (provisional name ‘DRIPs’ clade: Dermocysti- All of the collected fish were euthanized using overdose of an-
dium, Rhinosporidium, Ichthyophonus and Psorospermium. The new clade esthesia {5 mg\L of 10% benzocaine (REDA chemicals, Egypt) in 70%
containing Dermocystidium, Rhinosporidium, Ichthyophonus and Psor- ethanol (Al-Nasr pharmaceutical chemicals Co, Egypt)} followed by
ospermium (DRIPs) was named Mesomycetozoa, and includes two or- severing of spinal cord to ensure death (Neiffer and Stamper, 2009)
ders, Dermocystida and Ichthyophonida (Herr et al., 1999). All mem- then clinically and post-mortem examination were performed as de-
bers in the order Dermocystida cause diseases in either fish scribed by Moeller (2019).
(Dermocystidium sp. and the rosette agent) (Rowley et al., 2013;
Glockling et al., 2013; Blazer et al., 2016) or mammals and birds 2.4. Mycological examination
(Rhinosporidium seeberi) (Mendoza et al., 2002).
To our knowledge, infection with Dermocystidium can be superficial Squash preparations were made from the macroscopic grayish no-
that appears as macroscopically visible cutaneous cysts (Zhang and dules on the internal organs (liver, spleen, kidneys, stomach and in-
Wang, 2005; Novotny & Smolova, 2006; Lazăr et al., 2014; Fujimoto testine) of freshly dead infected fish and examined microscopically.
et al., 2018; Plaul et al., 2018; Tooba et al., 2018) or systemic that Pieces from the affected parts were taken using sterile forceps from
induces chronic internal infections (Hedrick et al., 1989; Dykova and the macroscopic grayish nodules on the internal organs and inoculated
Lom, 2007; Landsberg and Paperna, 1992; Bruno, 2001; El-Mansy, under completely aseptic conditions into Tris-buffered Eagle's
2008; Hassan et al., 2014). Minimum Essential Medium (MEM, Sigma M5775, UK), adjusted to pH
Many studies suggest the predisposing factors for occurrence of 3.5, others adjusted to pH 7.0 supplemented with 10% calf serum
Dermocystidiosis; the disease is encouraged by water rich in toxicant (Seradigm,VWR Int, USA), 100 IU/ml penicillin (Adwia, Egypt),
pollutants (Valtonen et al., 2003) and lower temperature (Kasesalu 100 mg/ml streptomycin (Adwia, Egypt), and 100 mg/ml gentamycin
et al., 2000; Höglund et al., 1997; Kirkbright et al., 2016). Dermocys- (Adwia, Egypt). The inoculated tubes of MEM either at pH 3.5 or pH 7.0
tidium infection most commonly occurs in adult and large-sized fish were incubated at 20 ± 2 °C for 5 days and examined daily until the end
(Hassan et al., 2014 and Gjurcevic et al., 2008). of the incubation period.
Diagnosis of the disease is based primarily on fresh mounts for light Subsequently, for further identification according to morphological
microscopy from the affected tissue (Zhang and Wang, 2005; Fujimoto characters, inoculum from positive culture growth in MEM media was
et al., 2018) and on histopathological alterations of the affected tissue; inoculated into Sabouraud Dextrose agar plates (SDA, Difco) with
the spores appear unicellular, containing a large central vacuole occu- chloramphenicol (cidocitene vial/cid Co), other inoculum deposited on
pying most of the cell volume and a peripheral nucleus (Bruno, 2001; SDA plates with chloramphenicol and 10% duck decoction (prepared by
Zhang and Wang, 2005; Lazăr et al., 2014; Hassan et al., 2014). Der- immersing fresh duck excreta in sterilized distilled water for 1 h (hr)
mocystidium species produce zoospores, which are found to be motile, then make filtration for this suspension through sterilized gauze into
infective and highly pathogenic at lower temperature (4 °C) (Olson sterilized flask that sterilized using autoclave before adding this sus-
et al., 1991) After infection with Dermocystidium, the zoospores encyst pension to the media) then incubated at 20 ± 2 °C for 2 weeks.
and enlarge to form large, spherical, multinucleate walled cells inside Multiplication of the spores were monitored though microscopic
the host (Mendoza et al., 2002). examination of infected freshly dead fish tissue that preserved in a re-
The present work aimed to highlight the prevalence and methods of frigerator at 4 °C during the period of examination and examined per-
diagnosis of Dermocystidium infection among wild and cultured Nile iodically for 20 h.
tilapia in Egypt. Moreover, multiplication of spores in dead tissue of
fish, experimental infection to assess its virulence and histopathological 2.5. Experimental infection
alterations in tissue of naturally and experimentally infected fish were
determined. 2.5.1. Preparation of the inoculum (the infective dose)
Dermocystidium colonies were picked up after 5 days of fresh culture
2. Materials and methods growth of the pathogen on SDA at 20 ± 2 °C supplemented with 10%
duck decoction and placed in 1 L of distilled water and stirred by
2.1. Fish specimens vortex, before adding to 9 L of distilled water to make a 10-L
Dermocystidium spore stock suspension. One milliliter of this suspension
A total of 1388 Nile tilapia (766 cultured fish and 622 wild fish was used to determine spore concentration using hemocytometer
weighing 15–30 g and 30–60 g) were collected from private fish farms (Feldman et al., 2000).
in Abbassa and Sahl El-Hosainia in Sharkia Governorate (cultured fish)
and Bahr Moes in Zagazig and Al-Riah El-Tawfiki in Benha, Qalyubia 2.5.2. Design of the experiment
Governorate (wild fish) from May 2012 to May 2014. The fish were A total of 30 apparently healthy Nile tilapia were obtained from
transferred alive from Department of Fish Diseases and Management, Abbassa fish farms, Sharkia Province, Egypt with an average body
Faculty of Veterinary Medicine, Zagazig University, Egypt and ex- weight 40 ± 5 and divided into three equal groups. Fish in the first
amined for any gross and post-mortem abnormalities. Fish were accli- group were firstly anesthetized by 1 ml\ L of 10% benzocaine in 70%
mated by maintaining in glass aquaria with dechlorinated tap water at ethanol (Neiffer and Stamper, 2009) then infected with Dermocystidium
22 ± 2 °C. The water of each glass aquaria was changed daily at a rate through intra-gastric route using a stomach tube with a dose of 1 ml of
of 25%, supplied with air pumps for continuous aeration and fish fed on spore suspension for each fish (each 1 ml contained 1550 spores that
a commercial fish diet 3% of its body weight for 15 days. determined using hemocytometer). The second group was infected
through immersion route by placing fish for 2 h in Dermocystidium spore
2.2. Methods of measurment of water quality suspension (9990 ml, this is the volume of spore suspension after re-
moving 10 ml for inoculating 10 fish through intra-gastric route),
Standard centigrade thermometer (°C) (Orion, USA), pH meter aquarium water partially exchanged daily by percentage of 25% after
(WTW, Germany), Salino-meter (Orion, USA), and dissolved oxygen 2 h of immersion till the end of experiment. While, third group (control

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H.H. Mahboub and A. Shaheen Aquaculture 516 (2020) 734556

group) was divided into 2 equal subgroups, first subgroup received

sterile saline using a stomach tube with a dose of 1 ml/fish, other
subgroup immersed for 2 h in sterile saline suspension. Each group of
fish was placed in a glass aquarium (80 × 30 × 40 cm) and supplied
with dechlorinated tap water (80 L) and continuous aeration at
22 ± 2 °C, pH 7 ± 0.2 and dissolved oxygen 5–6 mg/l−. Fish were
kept under observation for 15 days before starting the experiment.
Clinical symptoms and mortality in the examined fish were recorded
daily for 3 weeks then euthanized as mentioned before (Neiffer and
Stamper, 2009).

2.6. Histopathological examination

Tissue specimens were taken from suspected lesions on liver, spleen,

stomach and intestine from naturally and experimentally infected fish
tissue and fixed in 10% phosphate buffered formalin, embedded in
paraffin, sectioned and stained with Hematoxylin and Eosin stain (H&E)
(Bancroft and Layton, 2013) and Periodic acid–Schiff (PAS) stain
(Totty, 2002).

3. Results

3.1. Prevalence of dermocystidium infection among the examined O.

niloticus

As depicted in Table (1) out of 1388 examined Nile tilapia only 213/
766 (27.8%) cultured fish and 280/622 (45%) wild fish were infected
Fig. 1. O. niloticus infected with Dermocystidium sp. showing scale loss, severe
with Dermocystidium. The highest prevalence of infection in cultured ulceration of the skin and caudal fin rot (Fig. 1A). Post-mortem lesion of O.
fish was recorded in Sahl El-Hosainia followed by Abbassa, with rates of niloticus infected with Dermocystidium sp. showing dark-grayish nodules on the
31.2% and 24.4, while the prevalence of infection in wild fish was liver with enlargement and congestion (Fig. 1B).
highest in Al-Riah El-Tawfiki (Nile branch in Benha, Qalyubia Gover-
norate) followed by Bahr Moes (Nile branch in Zagazig, Sharkia Gov-
3.2. Water quality
ernorate) with rates of 49% and 40.9%. The highest rate of infection
recorded in winter (63.6%) followed by spring (38.6%), autumn
Determination of water quality revealed that, water temperature of
(28.8%) and finally summer (11.8%).
the ponds varied with the season, 35 ± 5 °C; 19 ± 2 °C; 14 ± 5 °C
Higher infections were recorded in adults (49.9%) than juveniles
and 21 ± 2 °C for summer, autumn, winter and spring seasons re-
(21%). Lesions were observed mostly in liver (58.6%), spleen (23.3%),
spectively. The pH of water was 6.5–8.5, 5.8–6.9, 5.9–7.2 and 6.6–8.1
kidneys (10%), stomach (3.4%) and finally the intestine (2%).

Table 1
Prevalence of infection in wild and cultured O. niloticus in relation to different seasons and localities.
No. of infected fish No. of examined fish Locality Type and number of fish Season

% No Cultured Wild Total

6% 6 100 Abbassa 205 363 Summer

11.4% 12 105 Sahl El-Hosainia
13.3% 10 75 Bahr Moes 158
18% 15 83 Al-Riah El-Tawfiki
11.8% 43 Total
21.7% 20 92 Abbassa 178 344 Autumn
29% 25 86 Sahl El-Hosainia
27.4% 23 84 Bahr Moes 166
37.8% 31 82 Al-Riah El-Tawfiki
28.8% 99 Total
41.7% 40 96 Abbassa 195 352 Winter
51.5% 51 99 Sahl El-Hosainia
80.8% 63 78 Bahr Moes 157
88.6% 70 79 Al-Riah El-Tawfiki
63.6% 224 Total
29% 27 93 Abbassa 188 329 Spring
33.7% 32 95 Sahl El-Hosainia
42.2% 30 71 Bahr Moes 141
54.3% 38 70 Al-Riah El-Tawfiki
38.6% 127 Total
27.8% 213 766 1388 Total
45% 280 622
35.5% 493 1388

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H.H. Mahboub and A. Shaheen Aquaculture 516 (2020) 734556

Fig. 2. Squash preparation from infected liver (Fig. 2A), spleen (Fig. 2B), stomach (Fig. 2C) and intestine (Fig. 2D) revealing unicellular spores, each spore with a
large central vacuole occupying most of the cell volume and a peripheral nucleus.

Fig. 4. Growth of Dermocystidium after 3 days on SDA media with 10% duck
decoction; the colony appears to be on the surface of the media, with neither
Fig. 3. Growth of Dermocystidium spores after 5 days on Eagle's Minimum submerged nor aerial growth, like a flower, its center resembling pollen grains
Essential Medium adjusted to pH 3.5 and supplemented with 10% calf serum, that are creamy-colored and pasty in consistency (Fig. 4A). Wet preparation of
100 IU/ml penicillin, 100 mg/ml streptomycin, and 100 mg/ml gentamycin the culture is showing cysts containing numerous spores of Dermocystidium sp.
indicated by change in the color of the media into light rosy color (Fig. 3A). Wet (Fig. 4B).
preparation of the culture is revealing aggregation of a large number of equal-
sized spores of Dermocystidium (Fig. 3B). (For interpretation of the references to
content varied from 4 to 6 mg/L in all localities.
color in this figure legend, the reader is referred to the Web version of this
article.)
3.3. Signs of infection on diseased fish
in Abbassa farms, Sahl El-Hosainia private fish farm, Al-Riah El-Tawfiki
Infected fish showed sluggish movements, black discoloration, scale
and Bahr Moes respectively. While salinity of the water was 0.5–0.8 g/
loss, severe ulceration of the skin and fin rot (Fig. 1A). The post-mortem
L, 1.8–2.1 g/L, 1.2–1.9 g/L and 0.7–1.1 g/L in Abbassa, Sahl El-
lesion involved congestion and enlargement in all internal organs
Hosainia, Al-Riah El-Tawfiki and Bahr Moes respectively. Oxygen
especially the liver, with the presence of dark-grayish patches or

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H.H. Mahboub and A. Shaheen Aquaculture 516 (2020) 734556

Fig. 5. Growth of Dermocystidium after 10 days on

SDA media with 10% duck decoction; the colony is
adhered to the surface of media, radiating on the
media like sun-rays that are creamy-colored and
lighter in color than the boundaries (Fig. 5A). Wet
preparation of the culture is showing dense produc-
tion of enlarged spores of Dermocystidium sp.
(Fig. 5B). (For interpretation of the references to
color in this figure legend, the reader is referred to
the Web version of this article.)

Fig. 6. Multiplication of Dermocystidium spores

through squash preparations from freshly dead host
tissues revealing primarily spherical, thick-walled
spores (Fig. 6A); after 8 h, appearance of mother cell
(mature trophozoite) containing four developing
Dermocystidium spores (Fig. 6B); after 15 h, start of
excystation of spores through an elongated tubular
extension of the mother cell (arrowhead) (Fig. 6C);
and finally after 20 h, a large number of spores are
scattered within the liver tissue (arrowhead)
(Fig. 6D).

nodules (Fig. 1B). Microscopic identification of these nodules or parts aggregation of a large number of equal-sized spores of Dermocystidium
from the internal organs that did not have any apparent lesions re- (Fig. 3B).
vealed cysts with numerous spores or unicellular spores (signet-ring Further identification was performed only from positive culture
shape) containing a large central vacuole occupying most of the cell growth after 3 days on SDA media plates supplemented with chlor-
volume and a peripheral nucleus in different organs, liver (Fig. 2A), amphenicol and 10% duck decoction. Colonies appeared on the surface
spleen (Fig. 2B), stomach (Fig. 2C), intestine (Fig. 2D). of the media, with neither submerged nor aerial growth, like flowers,
the center of each colony resembling pollen grains that were creamy-
3.4. Identification of Dermocystidium sp. colored and pasty in consistency (Fig. 4A). Microscopically, wet pre-
paration of the culture revealed the presence of cysts containing nu-
Dermocystidium spores revealed only positive culture growth after 5 merous spores of Dermocystidium sp. (Fig. 4B). After 7 days, the shape of
days on Eagle's Minimum Essential Medium (MEM) that adjusted to pH the colony changed, and wet preparation of the culture revealed the
3.5, supplemented with 10% calf serum, 100 IU/ml penicillin, 100 mg/ presence of large numbers of enlarged spores of Dermocystidium sp.
ml streptomycin, and 100 mg/ml gentamycin. Its growth indicated a After 10 days of incubation, the colony adhered to the surface of the
change in the color of the media into light rosy color (Fig. 3A). Mi- media and radiated across the media like sun-rays, which were creamy-
croscopic examination for wet preparation from MEM revealed colored and lighter in color than the edges of the colony (Fig. 5A). Wet

5
H.H. Mahboub and A. Shaheen Aquaculture 516 (2020) 734556

Fig. 7. O. niloticus experimentally infected

with Dermocystidium through the immersion
route showing blackish nodule on the liver
(arrowhead) (Fig. 7A). Squash preparation
from the experimentally infected liver re-
vealed spherical cysts (signet ring stage)
containing a large central vacuole occu-
pying most of the cell volume and a per-
ipheral nucleus (arrowhead) (Fig. 7B). O.
niloticus experimentally infected with Der-
mocystidium through the intra-gastric route
showing hyperinflation of the stomach (ar-
rowhead) (Fig. 7C). Microscopic examina-
tion of the stomach revealed the aggregation
of a large number of Dermocystidium spores
(Fig. 7D). The intestine is black-colored and
corrugated with dark greenish contents
(Fig. 7E). Microscopic examination of the
intestine revealing a cyst with discharge
tubes containing a large number of Dermo-
cystidium spores and the release of some
spores through the discharge tubes (Fig. 7F).

preparation of the culture revealed dense production of enlarged spores of appetite, severe emaciation, sluggish movements, aggregation near
of Dermocystidium sp. (Fig. 5B). the side of the aquarium, loss of escape reflex, gasping air from the
surface and dark skin coloration. In the case of Nile tilapia experi-
3.5. Spores multiplication in the freshly dead host tissue mentally infected through the immersion route, clinical signs were
observed after 6 days and the most prominent post-mortem lesion was
Dermocystidium spores had the ability for multiplication in the the presence of black nodule on the liver of most fish (Fig. 7A). Squash
freshly dead host tissues, firstly; squash preparations were done directly preparation from the experimentally infected liver revealed spherical
from the internal organs (liver, spleen and kidneys) of freshly dead host cysts (signet ring stage) containing a large central vacuole occupying
tissue, especially from the grayish nodules found in the liver that re- most of the cell volume and a peripheral nucleus (Fig. 7B). In the case of
vealed presence of spherical, thick-walled spores, each with a large fish experimentally infected through intra-gastric route (using a sto-
central vacuole occupying most of the cell volume and with a periph- mach tube), clinical signs were observed after 3 days (emaciation and
eral nucleus (Fig. 6A); after 8 h, other squash preparations were per- dark skin coloration) and the postmortem lesions were congestion and
formed from the internal organs of refrigerated freshly dead host tissue, enlargement of all internal organs especially liver, with dark nodules on
mother cells (mature trophozoites) containing four developing Dermo- it, as well as hyperinflation of the stomach (Fig. 7C). Microscopically, a
cystidium spores (Fig. 6B) appeared; after 15 h, excystation of the spores large number of Dermocystidium spores were found aggregated in the
began through an elongated tubular extension of the mother cell stomach (Fig. 7D). The intestine was black in color and corrugated with
(Fig. 6C); and after 20 h, a large number of spores were observed dark greenish contents in all positive mortality cases (Fig. 7E). Micro-
scattered in the liver tissue (Fig. 6D). scopically, the intestine showed a cyst with discharge tubes containing
a large number of Dermocystidium spores, some of which were released
3.6. Clinical signs on experimentally infected Nile tilapia through the discharge tubes (Fig. 7F).
Dermocystidium sp. was pathogenic for Nile tilapia with a varying
Fish showed similar signs as in naturally infected fish including lack degree of virulence according to the route of infection. In the first group

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H.H. Mahboub and A. Shaheen Aquaculture 516 (2020) 734556

spleen; characterized by central area of eosinophilic granular necrosis

surrounded by numerous epitheliod cells and lymphocytes (Fig. 9C).

4. Discussion

In this study, we report Dermocystidium sp. infection for the first

time in wild Nile tilapia. The total rate of Dermocystidium infection was
59.5% among the examined fish and was higher in wild than cultured
Nile tilapia, which may be attributed to the pollution, human activities
and high organic matter that are common in wild populations that
causing a source of stress for fish. Consequently, may impact on im-
mune system, lower resistance against infection and induce mortalities.
Following Valtonen et al. (2003) who compared parasite communities
in fish taken from a polluted lake and found that D. percae infection in
Perca fluviatilis was correlated with toxicant polluted water, while, de-
crease of D. percae on perch fins increased immune responses in the fish
that reflecting better water quality. Also, Chauhan (2012) added that,
occurrence and parasitic activity of aquatic fungi are expected more in
specific regions rich in organic pollutants. In addition, Budria (2017)
revealed that water pollution by organic (e.g. urea introduced with
waste water), and inorganic (e.g. fertilizers) compounds causing water
turbidity, oxygen depletion and appearance of toxic algal blooms that
increase host susceptibility for infection with inducing mortalities.
Olson & Holt (1995) isolated D. salmonis from wild Oregon salmonids,
also, Kasesalu et al. (2000) isolated D. vejdovskyi on pike and D. percae
on perch from natural water bodies. Moreover, Plaul et al. (2018) re-
vealed superficial Dermocystidium infections in new wild ornamental
fish (Hemigrammus sp.), Regarding the localities, infection in cultured
population was higher at Sahl -El-Hosainia private fish farm than at
Abbassa fish farm, while, in wild population, it was higher in Al-Riah
Fig. 8. Liver of naturally infected fish showing ruptured cyst wall with dis- El-Tawfiki than Bahr Moes. This may be attributed to the higher water
semination of Dermocystidium spores (arrowhead) in adjacent tissues (Fig. 8A, H salinity and lower pH of water (slightly acidic) at Sahl El-Hosainia than
&E stain, 20 μm Scale bar). Liver of experimentally infected fish showing dif- Abbassa farm and in Riah El-Tawfiki than Bahr Moes that aid in pro-
ferent size and developmental stages of Dermocystidium spores (arrowhead) liferation of Dermocystidium spores. Our results were also confirmed
(Fig. 8B, H&E stain, 20 μm bar). when the organism was inoculated on MEM media and the growth
appeared at only acidic pH. Moreover, Stewart (1966) found that de-
(intra-gastric route through a stomach tube), mortalities started on the gree of infection with Dermocystidium marinum rises sharply when water
fourth day and continued 2 weeks post-infection, reaching 90%, so salinity increases, in addition, La Peyre et al. (2006) mentioned that
Dermocystidium can be considered highly virulent via intra-gastric perkinsus olseni in oysters that originating from high salinity areas had
route. In the second group (immersion route), mortalities began on the higher viability and proliferation at the higher salinities. Manley et al.
seventh day and reached 70% 2 weeks post-infection, so Dermocystidium (2009), described that Dermocystidium sp. was less prevalent when
can also be considered a high risk route for infection via immersion salinity levels were lower than normal. The highest infection rate was
route. Both control subgroups showed 0% mortality. Re-isolation of the seen in the winter season, which was consistent with Höglund et al.
fungus on MEM media revealed a positive result for Dermocystidium sp. (1997) who stated that infection of salmon with Dermocystidium sp.
usually commences when water conditions are cold, Kasesalu et al.
(2000) detected highest prevalence of infection by D. cyprinid in cy-
3.7. Histopathological examination prinus carpio during the 4 winter periods. In addition to Kirkbright et al.
(2016) who isolated Dermocystidium-like organism from perch perca in
Histopathological examination of naturally and experimentally in- winter. While, Hassan et al. (2014) revealed that the lowest infection
fected fish revealed multifocal Dermocystidium cysts of different sizes in rate in Lethrinus nebulosus with Dermocystidium arabica was in winter.
the liver, spleen, stomach and intestines. The cysts were filled with Dermocystidium infection was higher in adults than juveniles, in our
numerous spherical to oval spores surrounded by thin connective tissue point of view, it is possible to be a chronic long lasting disease that
wall with minimal inflammatory reaction composed of few mono- infection takes long time starting from acquiring the infection till af-
nuclear cells. Occasionally, the wall of the cyst ruptured with dis- fecting the internal organs and inducing the clinical signs, perhaps ju-
semination of spores in adjacent tissues (Fig. 8A), accompanied by in- venile fish can acquire the infection but it takes long time to induce the
filtrates of moderate numbers of macrophages and lymphocytes. The disease and clinical signs appear in adult stage. In addition, adult fish
spores were not uniform with a large central vacuole and the nucleus consume more food or prey than juveniles, so it is more vulnerable to
restricted to a narrow peripheral layer that exhibited positive reaction infection. Out results were consistent with Hedrick et al. (1989) who
by PAS stain. Several types of spores of different size and develop- described systemic Dermocystidium infections as slowly progressing
mental stages were observed in the liver (Fig. 8B). Occasionally, the chronic infections of the visceral organs. Also, Wildgoose (1995),
spores were small in diameter, and contained a prominent nucleus, Gjurcevic et al. (2008) and Hassan et al. (2014) stated Dermocystidium
while others were large with abundant basophilic or eosinophilic cy- infection in older adult and larger fish species. While, Olson and Holt
toplasm. There were aggregates of spores in stomach (Fig. 9A) and (1995) and Fujimoto et al (2018) demonstrated the highest infection in
intestinal lumen admixed with exfoliated epithelial cells; rarely seen in juvenile and fingerling. Dermocystidium spores were observed in the
lamina propria accompanied by hyperplasia of lining epithelium internal organs with a higher prevalence of infection in liver followed
(Fig. 9B). Moreover, systemic granuloma was rarely observed in the by the spleen, then kidneys, stomach and finally intestine, our results

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H.H. Mahboub and A. Shaheen Aquaculture 516 (2020) 734556

Fig. 9. Stomach of naturally infected fish showing aggregates of spores (arrowhead) in lumen admixed with exfoliated epithelial cells (Fig. 9A, PAS reaction,
20 μm bar). Intestine of experimentally infected fish showing few Dermocystidium spores (arrowhead) in lamina propria accompanied by hyperplasia of lining
epithelium and desquamated epithelium inside lumen (head) (Fig. 9B, H&E stain, 100 μm bar). Spleen of experimentally infected fish showing granuloma composed
of central area of eosinophilic granular necrosis surrounded by numerous epitheliod cells and lymphocytes (Fig. 9C, H&E stain, 20 μm bar).

were matched to El-Mansy (2008) who isolated Dermocystidium ae- in localities with high load of organic matter. Culture of Dermocystidium
gyptiacus from the intestine of cultured Nile tilapia and Hassan et al. on both MEM at pH 3.5 and SDA with 10% duck decoction appears to
(2014) who reported Dermocystidium arabica in liver, spleen and kid- be a sensitive and effective method for isolating the organism in re-
neys of Spangled emperor, Lethrinus nebulosus. In this study, the ma- cently dead individuals and should be considered the diagnostic stan-
jority of infected fish suffered from sluggish movement, black dis- dard.
coloration, scale loss, severe ulceration of the skin and fin rot. Post- Dermocystidium has a negative impact on fisheries due to mortalities
mortem lesions were grayish patches or nodules on the internal organs, caused by the pathogen especially in wild populations; this may be
especially the liver. Hassan et al. (2014) described similar findings for attributed to rapid multiplication of its spores in the freshly dead host
Dermocystidium arabica, including a dull opaque body color, detached tissue and existing in large numbers after 20hrs. This ensures acquiring
scales and emaciation with a sunken belly in addition to grossly visible the infection if fish fed on infected fish viscera carrying Dermocystidium
yellow blotches or spots within the musculature of Lethrinus nebulosus. spores. Fernández Robledo et al. (2011) described the typical multi-
Identification of Dermocystidium through a tissue squash revealed plication pattern of Perkinsus marinus in oyster tissue, in which tro-
spherical cysts (mature trophozoites or signet ring stage) containing a phozoites proliferate (schizogony) giving rise to 4–32 trophozoites,
large central vacuole occupying most of the cell volume and a periph- which are released upon rupture of the schizont cell wall.
eral nucleus, similar to previous descriptions by El-Mansy (2008); Lazăr Experimental infection by Dermocystidium revealed that fish suffered
et al. (2014); Hassan et al. (2014); Fujimoto et al (2018). similar clinical signs and post-mortem lesions as in natural infection;
In vitro culture of Dermocystidium on MEM at only acidic pH 3.5 also, this pathogen is likelihood of being a parasite rather than fungus
establishes a basic explanation that Dermocystidium spores prefer acidic due to occurrence of cystic lesions in various tissues and absence of
environment to proliferate; it may explain a part of pathogen life cycle: hyphae. Following Lotman et al. (2000) who also described visible cysts
after acquiring infection through ingestion, Dermocystidium spores in the genus Dermocystidium.While Dykova and Lom (2007) mentioned
could prefer acidic pH of stomach to proliferate. While, in vitro culture hyphal forms in some Dermocystidium. Moreover, fish can acquire the
of Dermocystidium on SDA supplemented with chloramphenicol and infection through both intra-gastric and immersion routes, but intra-
10% duck decoction revealed that Dermocystidium needs a source of gastric route is likelihood of being a higher-risk route for acquisition of
organic matter to grow and eventually induces the infection, this in- infection through inducing higher mortality than immersion route.
formation was also confirmed when occurrence of infection was higher Furthermore, such experimental infection clarified the life cycle of

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H.H. Mahboub and A. Shaheen Aquaculture 516 (2020) 734556

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