8th International Symposium on Tilapia in Aquaculture 2008
1329
A TRIAL FOR TREATMENT OF ICHTHYOPHONOSIS IN
CULTURED OREOCHROMIS NILOTICUS USING FUCUS AND
NEEM PLANTS
NADIA A. ABD EL-GHANY1 AND HODA M. L. ABD ALLA2
1. Dept. of Fish Diseases, Animal Health Research Institute, Dokki, Giza, Egypt.
2. Animal Health Research Institute, Zagazig. Lab. Egypt.
Abstract
This is first trial that carried out in Egypt on the treatment of
Ichthyophonosis in Oreochromis niloticus. Clinically naturally
infected O niloticus with Ichthyophonus spp. was observed as
spherical multinucleate spores white and creamy in colour were
very variable in size, found in liver, spleen and kidney and
presence of spores freely with mucus in gills, the germinating
stages, hyphae and endospores were also found.
Different cultures were used in growth of Ichthyophonus hoferi
spores as Eagles minimum essential medium (MEM), thioglycollate
medium and Sabourauds dextrose broth. In addition, solid media
as Sabourauds dextrose agar, all media were supplemented by
different concentration of fetal bovine serum. Healthy O.niloticus
was infected experimentally by spores of Ichthyophonus spp MEM10 pH 3.5 culture as 1ml/ fish. Mortality, clinical signs and
postmortem changes in experimentally infected fish were recorded.
The results of biochemical analysis and hematology showed
increased plasma levels of cortisol, eosinophils and monocytes
while decreased total protein, albumin, total globulin and
lymphocytes in group infected with Ichthyophonosis. In the present
study, we investigated the effect of crude extract of Azadirachta
indica (neem) leaves at dose 5ppm /kg ration and Fucus
vesiculosus extract at dose 2g/kg ration in controlling of such
disease in fish. Approximately, most of these parameters increased
in infected fish treated with neem and fucus .The present data
concluded that neem is more effective than fucus and qualifies as a
safe and efficient in the prevention of Ichthyophonosis in fish.
INTRODUCTION
Ichthyophonus disease is recognized to be of economic significance in both
fish cultured and wild fishes (McVicar, 1999). Its causative agent is Ichthyophonus
hoferi, that has a very broad host spectrum and is known to infect marine and
cultured fish, causing granulomatous systemic disease in vascularized organs such as
heart, spleen, liver and kidneys (Spanggaard et al., 1994).The variability in clinical
signs is undoubtedly related to the site of the fungal invasion, including abnormal
swimming patterns, some fish develop a dark colour. Also, it is the cause of many
unidentified deaths in fishes (Jones and Dawe, 2002).
Diagnosis of Ichthyophonosis depends on external and internal alterations,
isolation of infective fungus which needs special condition and nutrients and
1330
A TRIAL FOR TREATMENT OF ICHTHYOPHONOSIS IN Cultured
OREOCHROMIS NILOTICUS USING FUCUS AND NEEM PLANTS
histopathology for the infected organs. More recently, the application of medicinal
herbs in disease management is gaining momentum because herbal treatment is cost
–effective and has minimal side effect.
Azadirachta indica (neem) is one of the most widely tropical plants and its
leaves contain several chemical and biological active ingredients (Erratum, 2001),
about ten herbs are most commonly used in China to treat fish diseases such as gill
rot, white mouth disease, Aeromonas hydrophilia in Carp and fungal pathogen as
Aphanomyces invadans (Siddiqui et al., 1992 and Harikrishnan et al., 2005).
Fucus vesiculosus is marine algae sulfated polysaccharide with a wide variety of
biological activities, that revealed anti-inflammatory and antiviral (Hayashi et al., 1996)
antibacterial (Sakai, 1999) antifungal (Miles et al., 2001) anticoagulant and
immunomodulating (Kuznetsova et al., 2003) antitumor effects (Skibola et al., 2005)
and antioxidant (Veena et al., 2007).
The present study was carried out to evaluate the effect of Azadirachta
indica (neem) and Fucus vesiculosus in the ration of Nile tilapia (O.niloticus) in treating
Ichthyophonus disease.
MATERIAL AND METHODS
Naturally infected Fish
A total number of 100 Oreochromis niloticus were collected from a private
fish farm Giza Governorate previously suffered from Ichthyophonosis with body weight
range of 50-60 g. for isolation of fungi, and transferred to Fish Diseases Department
at the Animal Health Research Institute. Body weight and length, clinical signs and
postmortem changes were recorded (Innes 1966).
Fungal examination
Fresh smears of liver, heart, spleen, kidneys and gills were examined by light
microscope immediately or few hours after death for the presence of spores or any
other stages of Ichthyophonus. The inoculum used consisted of fungal material
removed aseptically from suspected organs to culture medium (Faisal et al., 1985).
According to Rand and Cone (1990), the inoculum was cultured aseptically in
different
broths,
Eagles
minimum
essential
medium
(MEM,
Sigma
M5775),
thioglycollate medium (TGC, Oxoide) and Sabourauds dextrose broth medium (SDB,
Difco) using sterile tubes with 5ml medium/tubes. The fungus was also inoculated into
solid media of Sabourauds dextrose agar (SDA, Difco). All media were supplemented
by different concentration of fetal bovine serum (FBS, Sigma F4010), the percentages
of FBS and the resulting pH of each
medium are listed in Table 1. All media were
NADIA A. ABD EL-GHANY AND HODA M. L. ABD ALLA
1331
supplemented by penicillin at 100UI/ml and with streptomycin at 100ug/ml.The culture
in tubes and plates were incubated at 14 0C for 15days.
Identification of the fungal growth was performed by microscopically
examination of wet mount preparation and stained by lactophenol cotton blue (LPCB)
as described by (Spanggaard et al., 1995).The isolates were subcultured in MEM-10
pH 3.5 for experimental infection.
Table 1. List of the media used for in vitro culture
Medium
FBS%
pH
MEM-10
10
3.5
MEM-10
10
7
TGC-10
10
6.6
SDB-10
10
3.5
SDA-1
1
5.5
Trial for treatment in experimental infection
Rations
Standard commercial ration formulated to contain 32%crude protein (8.0% menhaden
fish meal, 45.0% soybean meal, 25.0 cornmeal, 14.0% wheat middling) and 5.6%
crude lipid. The ration was supplemented with 5ppm of Azadirachta indica (neem)
extract/kg ration and 2 g Fucus vesiculosus extract /kg ration. Dietary ingredients
were thoroughly mixed in a Hobart mixer and extruded through a 2.5-mm diameter
diet in a Hobart meat grinder. The pellets were air-dried at room temperature, broken
into small pieces, sieved to obtain appropriate size and stored in a freezer at -8°C until
used.
Experimental fish
A total number of 150 apparently healthy Nile tilapia fingerlings were transported from
a private commercial fish farm to fish diseases department at the Animal Health
Research Institute and acclimated to the laboratory conditions in glass aquaria
supplied with an air and dechlorinated tap water at 21 ±1°C for a month before the
start of the experiment. At the end of acclimation period, 30 fish were randomly
collected and subjected to mycological examination as previously mentioned to
confirm that these fish were free from infection.
Experimental Design
One hundred and twenty O. niloticus were divided into two groups. The first group (20
fish) was designated as a negative control. The second one (100 fish) was infected by
using stomach tube containing I. hoferi MEM-10 pH 3.5 culture as 1ml/fish for one
time according to
Dorier and Degrang, (1961). The mortality and clinical
1332
A TRIAL FOR TREATMENT OF ICHTHYOPHONOSIS IN Cultured
OREOCHROMIS NILOTICUS USING FUCUS AND NEEM PLANTS
abnormalities were recorded during one month of infection. At the end of 30 days of
infection, some of the infected fish (15) were scarified for mycological examination to
verify the presence of infection and the survived fish (60) was subdivided into three
equal sub-groups. Group A was represented as infected positive control. Group B was
fed on ration containing 5ppm of Azadirachta indica (neem) extract/kg ration
(Harikrishnan et al, 2005). Group C was fed on ration containing 2 g Fucus vesiculosus
extract /kg ration (Farias et al., 2004). The treatment was continued for three months.
At the end of experiment, all survivors were examined for infection as previously
mentioned.
Blood samples
Five blood samples were obtained from each group by cutting tail in hepranized
tube then were separated and kept at 20±1 °C until used for biochemical analysis.
Estimation of cortisol was carried out after Pickering, (1981), Triiodothyronine (T3)
and Tetraiodothyronine (T4) according to (Abroham 1981) using Radioimmunoassay
kits.
Serum total protein albumin and total globulins were performed using 7150
Automatic blood chemistry analyzer (Ciba- Corning Diagnostic Crop). Total leucocytic
count (TLC) was estimated according to (Stoskoph, 1993) and differential leucocytic
count was carried out after (Hibiya, 1982).
Histopathological Examination
Tissue specimens from suspected examined organs were fixed in 10%buffered
formalin, embedded in paraffin, sectioned and stained with Haematoxylin and Eosine
(H&E) as well as periodic Acid Schiff methods (PAS) according to (Roberts 1978).
Statistical analysis
The obtained data were analyzed statistically by analysis of variance according to
(Snedecor and Cochran 1982).
RESULTS
Clinical picture
The general clinical signs in infected fish were represented as sluggish movements,
darkening of the colour, ulceration of skin, depressions in the bones of the head above
and under the eyes (Fig. 1). Post-mortem examination of the infected fish revealed
presence of white, well defined, macroscopic nodules of the size of a pin head in
kidneys, spleen and liver (Fig. 2).
Mycological examination
Squash preparation of gills and internal organs of such fish revealed the presence
of various life stages of Ichthyophonus hoferi in liver, kidneys and spleen. The
NADIA A. ABD EL-GHANY AND HODA M. L. ABD ALLA
1333
recognized stages were distension of the spore wall was observed with the formation
of a hyphae or new budding yeast like spores (Fig, 3&4). Development of uni or
binucleated endospores and multinucleate spore (resting spore) which vary greatly in
diameter and germinating flask shaped cyst or uninucleated endospores scattered
between tissues of the organs (Fig 5).
The first isolation of Ichthyophonus from infected organs was achieved in all the
media assayed, though optimal growths of the organism were observed in MEM-10
and SD broth-10 (Fig 6 and 7). Results of cultivation of infected materials on SDA-1
revealed hyphae growth both on the surface and into the substance from 4-6 days
(Fig 8), the hyphae growth increased to full fill plate within 10-14 days post
inoculation formation of hyphae and spores was observed and large elongated
chlamydospores from the apex or middle of the hyphae appeared (Fig 9 and 10).
Germination of spores was observed in TGC-10 and SD-10 after 2-4 days of
incubation. Long septate hyphae were formed and the cytoplasm was released from
the spore to the newly formed hyphae (Fig 11). The growth of hyphae was higher on
SD broth-10 PH 3.5 are a septate, characterized by oblong apices and extensive
branching after one day of inoculation, hyphae developed bulbous tips, which rounded
up to form large, thick-walled spores (Fig 12). Endogenous division of spores
occurred, and spherical uni or bi-nucleate bodies were formed after cleavage.
The endospores were released by short and thick hyphae, after their release they
grew, the nucleus divided, and multinucleate spores were formed. Ichthyophonus
growths were optimal in MEM-10 and SD-10 with pH 7 and 3.5 respectively. The
fungus was sub cultured in this medium for more than 1 year with monthly or
bimonthly passages.
The examination of 100 cultured O. niloticus was classified as: 20 infected
when the organism was isolated by invitro culture, 15 clinically infected or diseased
when visible white lesions were observed on at least one organ and confirmed to be
Ichthyophonus by culture, 45 sub-clinically infected when visible lesions were not
apparent but Ichthyophonus was detected microscopically and 20 negative when
Ichthyophonus couldn't be identified by any of the above techniques.
Experimental infection
O. niloticus were susceptible to Ichthyophonus hoferi where the cumulative mortality
rate reached 25% in one month (Table, 2). Most of moribund O. niloticus showed
emaciation, skin ulceration with abdominal distension in some cases. Post-mortem
examination of moribund fish revealed presence of one to a few focal, creamy white
patches in liver, spleen and kidney. Microscopic examination of infected organs
revealed the presence of characteristic forms of Ichthyophonus hoferi similar of
1334
A TRIAL FOR TREATMENT OF ICHTHYOPHONOSIS IN Cultured
OREOCHROMIS NILOTICUS USING FUCUS AND NEEM PLANTS
naturally infected fish. Also, fungal reisolation from infected organs on MEM-10 and
SD-10 with pH 7 and 3.5 respectively were positive for Ichthyophonus hoferi.
Treatment effects
Treatment trials exerted zero mortality in Azadirachta indica (neem) extract while
it was 25% in the treated group by Fucus vesiculosus extract
No fungus was reisolated from organs of O. niloticus after treatment with
Azadirachta indica and isolated from organs after treatment with Fucus vesiculosus
(Table, 3).
Haematological and biochemical analysis
The present data exhibited that O. niloticus infected with Ichthyophonus hoferi
revealed a significant elevation in the levels of plasma cortisol, while plasma levels of
T3 and T4 did not change than the control ones throughout all periods of the trial
(Table, 4). At the same time the treated groups exhibited a significant increase in
plasma levels of cortisol, T3 and T4 with both Azadirachta indica (neem) and Fucus
vesiculosus throughout all the treatment periods except at the first month of the
treatment with neem, T3 did not change significantly than the control group.
Table , (5) showed a significant reduction in the concentration of total protein,
albumin and total globulin in fish infected with Ichthyophonus hoferi throughout all the
experimental periods, except albumin at first month of infection did not appear any
changes in comparison with control.
In addition, the to treated groups showed a significant increase in the
concentration of total protein, total globulin in group treated with neem extract and
albumin in group treated with fucus extract while albumin of neem extract treated
group and globulin of fucus extract treated group did not change significantly at all
periods treatment in comparison with the control groups.
Data in (Table, 6) illustrates the total and differential leucocytic counts, there was
leucocytosis in Ichthyophonus hoferi infected fish at first month of infection while
leucopenia was detected at second and third months of infection. Also this group
appeared lymphocytopenia, neutropenia, monocytosis and eosinophila at all periods of
the trial.
However treatment with Azadirachta indica (neem) and fucus vesiculosus
exhibited elevation of the total leucocytic count and lymphocytes with decrease
monocytes and eosinophils throughout all treated periods except at first and third
month of neem extract treated group and first month of fucus extract treated group,
eosinophils did not differs than the control .
The histopathological sections from infected organs with Ichthyophonus hoferi
revealed that Kidney there were diffuse extraverted erythrocytes all over the renal
NADIA A. ABD EL-GHANY AND HODA M. L. ABD ALLA
1335
tissue in between the tubule, the epithelium cells lining the renal tubules showed
swelling with vacuolar cytoplasm Fig (13).
Liver there were severe dilatation of central veins and sinusoid. The hepatocytes
showed different stages of necrobiotic changes with complete rupture of the cells
associated with appearance of some fungus spores strongly positive PAS and
inflammatory cells infiltration Fig (14-15).
Gills there were oedema with mononuclear leucocytic inflammatory cells as well as
dilated blood vessels with presence of spores were noticed as strongly positive PAS Fig
(16-17).
DISCUSSION
Ichthyophonosis, having a wide host and is defined as one of the economic
significant affections in fish culture and wild fisheries. In this study, majority of
infected fish missed external lesions except for darkening of the colour, ulceration of
skin, depressions in the bones of the head above and under the eyes as those nearly
noticed by Kent et al., (2001). Sindermann and Scattergood (1954) described
behavioral changes in salmonids, curvature of spine in herring and salmonids with
darkening of the skin were all seen in few cases indicating that the fungus invade the
hosts nervous system. In herring and trout roughening of skin were noted by
Reichenback-klinke
(1973).
Internal
examination
of
examined
O.niloticus
demonstrated light gray raised nodules in liver, spleen and kidneys, the organs richly
supplied with blood, which indicate the systemic nature of Ichthyophonus. Squash
preparation from these nodules demonstrated that the parasite seen were refractile
thick double walled spores. There were developmental stages of Ichthyophonus hoferi
observed as latent resting stage, yeast like stage and hyphae stage. Difference in the
developmental pattern of Ichthyophonus hoferi were observed according to the
conditions surrounding the fungus in tissue of fish host. According to McVicar (1982),
such differences probably reflect the nutrients available and the conditions
surrounding the developing fungus.
Therefore, the endospores formed in fish could be responsible for septicemia and
subsequent infection of organs. The formation of new spores by budding like (yeast
like) germination could be a means of proliferation with the granuloma and not an
occasional developmental form as reported by Dorier and Degrang (1961). This
dimorphism has been demonstrated in Mucor rouxii (Bartnicki-Garcia 1973),
Histoplasma and Candida (Alexopoulos and Mims 1985).
Ichthyophonus growth and development was achieved in all the media assayed and
was generally similar to that reported by other authors for Ichthyophonus hoferi or
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A TRIAL FOR TREATMENT OF ICHTHYOPHONOSIS IN Cultured
OREOCHROMIS NILOTICUS USING FUCUS AND NEEM PLANTS
other forms of Ichthyophonus (Chien et al., 1979; McVicar 1982; and Okamoto et al,.
1985). Different development patterns were observed, according to the medium used,
in spite of this polymorphic development, characteristics of fungi and related
organisms (Bruns et al,. 1991).
The development in media with acid pH (SD, TGC and MEM-10) associated with
germination of multinucleate spores, branching of the formed a septate hyphae and
formation of terminal spores in the hyphal tips, was similar described by Okamoto et
al. (1985), Ana and Pilar (1999) for Ichthyophonus hoferi grown in MEM-10 at the
same pH value. In MEM-10 at the pH 7 we observed internal cleavage and release of
endospores through short hyphae that was rather similar to pattern one described by
the above mentioned authors in MEM-10 at the pH 7.The growth of fungus was
achieved on Sabouraudes dextrose agar (SDA-1) grow into the substrate within 3-7
days. The growth become heavy and filled the plate within 2 weeks post inoculation.
However Sindermann and Scattergood (1954) described germination of the inoculum
from herring after 7-10 days on SDA-1within 3-20°C with optimum temperature 10°C.
Resting spore germination may produce slender non-septated microhyphae and
tubular club shaped macrohyphae. This may produced large spherical thick walled
spores or cleaved to numerous hyphal (endospores), this agree with the finding of
Chine et al., (1979) and Ana and Pilar (1999) .In our cultures we never observed
motile forms such as those described by McVicar (1982) and Spanggaard et al.,
(1995), though some ovoid forms, apparently similar to those described by these
authors, were sometimes present.
Experimentally infected O. niloticus showed nearly similar clinical signs,
postmortem and histopathological alterations to those observed in naturally infected
ones. The mortality percentages was 25% in the infected groups with Ichthyophonus
cultured on MEM-10 adjusted at pH 3.5 for 30 days. On the other hand, the control
group showed neither clinical signs and postmortem lesion nor mortality. Such findings
were met by Ana and Pilar (1999), Jones and Dawe (2002) and Kocan and
Hershberger (2006) who induced infections in different fish species by exposure to a
pure culture of Ichthyophonus hoferi.
Treatment trials exerted zero mortality in Azadirachta indica (neem) extract and
No fungus was reisolated from organs of O. niloticus after treatment.
These results were in agreement with those of Harikrishnan et al.,( 2005) who
reported the inhibitory action of Azadirachta indica (neem) leaves and seeds extract
on a wide spectrum of microorganisms including fungi as Aphanomyces invadans and
also agreed with those of Tandan et al .,(1998) who reported the wound healing effect
of neem because of increased cutaneous capillary permeability at the site of the
NADIA A. ABD EL-GHANY AND HODA M. L. ABD ALLA
1337
wound. In group treated by Fucus vesiculosus extract mortality rate was 18.75% and
fungus isolated from organs after treatment with Fucus vesiculosus. These results
were in disagreement with Kubitza (2000) who explained that Fucus vesiculosus
enhanced non specific immunity and diseases resistance.
Farias et al., (2004) recorded that Fucus increase diseases resistance in O. niloticus,
Cyprinus carpio and shrimp Penaeus monodon respectively.
It has been presumed that disease constitutes an environmental stressor in
fish
Wedemeyer&Mcleady(1981).The obtained results revealed a significant increase
in the levels of plasma cortisol in Ichthyophoniasis infected fish, while there were no
changes in the levels of plasma T3 and T4 throughout all periods of the trial.
The increase in the cortisol levels in this study may be related to that cortisol
production is an important factor underlying the course of infectious diseases in fish
(Pickering, 1981).Also; Dunn (1989) indicated that infections activate the same type of
neural response as physical stressors.
In contrast to, Rand and Cone (1991) found that cortisol did not change in fish
infected with Ichthyophonus hoferi, whereas the same authors supported the present
study with thyroid hormone where they recorded that T4 did not differ in
Ichthyophonasis infected fish than control. In addition, the present data exhibited
increased levels of cortisol, T3 and T4 in both treated groups throughout all
experimental periods but T3 did not exhibit any change at the first month in group
treated with neem. Concerning, elevation of these hormones after treatment with
neem and fucus may be these herbals induce the production of cytokines in a variety
of cells (Talwar et al., 1997 and Hirayasu et al., 2005) and these cytokines modulate
the neuroendocrine functions of the hypothalamus as well as acting on the pituitary,
thyroid, pancreas, adrenal glands and gonads to modulate hormone release (Kennedy
& Jones, 1991). It is worthy to mention that corticosteroids has biphasic effect on
immune system, they produce an initial inhibition of macrophage and T cells
development following a stressor stimulus but prolonged release of corticosteroids(by
lower concentration) may stimulate the immune response (Brown, 1994).
Also thyroid hormones (T3 & T4) stimulate the release of thymic hormones and
maturation of T lymphocytes (Berczi, 1986). The present data demonstrated
decreased concentration of total protein, albumin and total globulin in fish infected
with Ichthyophonus hoferi.
'
In accordance with this result Rand and Cone (1990) found depressed protein in
rainbow trout infected with Ichthyophonus hoferi and this may be attributed to
pathology associated with high numbers of resting spores in kidney, spleen, heart and
liver, as supported by histopathological results in this study.
1338
A TRIAL FOR TREATMENT OF ICHTHYOPHONOSIS IN Cultured
OREOCHROMIS NILOTICUS USING FUCUS AND NEEM PLANTS
Interestingly, these parameters increased significantly with both treated groups except
albumin in group treated with neem and total globulin in group treated with fucus
throughout all the treatment periods. This may be referred to herbal medicines help to
heal the lesion (Harikrishnan et al. 2003) and neem may induce the state of nonspecific increased resistance in fish.
In this data, infected group showed
significantly increase in the numbers of
circulating leucocytes after first month with monocytosis at all experimental periods,
while there were leucopenia after second and third month with lymphocytopenia and
neutropenia throughout all periods of the trial, Similarly Harikrishnan etal.,(2005)
found that increased the total leucocytic count in the common carp infected with
fungal pathogen, Aphanomyces Invadans suggesting that the production of white
blood cells proportionate to any stress of infection, where pathogen may induce
endotoxin and tissue injury which is a potent stimulant to initiate the proliferation,
differentiation, and activation of B lymphocyte and macrophage (Xiang etal.,2008).
Leucopenia after long period of infection in the present data is consistent with Rand
and Cone (1990) and Qureshi et al., (2001) who recorded leucopenia in fish having
fungal infection, suggesting that leucopenia is a generalized host response and
supports that there is cell mediated response to wall of the resting spore ( Mc Vicar,
1982).
Interestingly, the fish treated with neem or fucus exhibited a significant increase
in the total leucocytic count and lymphocytes., these results were supported by
Upadhyayet , etal.,(1992)who reported that treatment with neem ( Azadirachta
indica)resulted in the maximum number of leucocytes and macrophages exhibiting
enhanced phagocytic activity .Furthermore Kuznetsova et al.,(2003) who found that
fucoidan from brown algae Fucus evanesces stimulated phagocytic and bactericide
activity at leucocytes of mice peritoneal exudates.
The histopathological changes induced by Ichthyophonus hoferi in fish were nearly
agreed with those mentioned by Shaheen and Easa (1996) in wild and cultured tilapia.
Based
on
aforementioned
results,
the
following
conclusions
could
be
recommended as the effective role of neem (Azadirachta indica) in the treatment of
Ichthyophonosis in O. niloticus fish is more better than fucus since neem stimulated
both humoral and cell mediate immunity and succeeded for the first time to eradicate
all the Ichthyophonus spores in fish after three months of treatments, while fucus
stimulated cell mediate immunity only and succeeded to eradicate about 75% of
Ichthyophonosis spores.
1339
NADIA A. ABD EL-GHANY AND HODA M. L. ABD ALLA
Acknowledgements
"We extend our sincere thanks to Prof. Dr/ Nahla R.El-Khatib, Chief Research, Fish
Diseases Department, Animal Health Research Institute, for her
scientific advice
and help in this study."
Table 2. The mortality rate during 30 days of infection.
Group
No. of fish
Mortality
No.
%
Non-infected
20
0
0
Infected
100
25
25
Table 3. The mortality rate among the experimentally infected fish after the
application of treatment.
Fish group
Control(group I)
A (I. hoferi)
Infected group
B (I. hoferi + neem)
C (I. hoferi + fucus)
No. of fish
No. of
Mortality
dead
rate
20
0
0
20
15
75%
20
0
0
20
5
25%
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A TRIAL FOR TREATMENT OF ICHTHYOPHONOSIS IN Cultured
OREOCHROMIS NILOTICUS USING FUCUS AND NEEM PLANTS
Table 4. Blood plasma levels of cortisol, T3, T4 in tilapia fish experimentally infected with Ichthyophonus hoferi with daily treatment of neem and
fucus extract for three months
Cortisol ( µg/dl )
T3 ( ng / ml )
T4 ( µg/dl )
1st month
2nd month
3rd month
1st month
2nd month
3rd month
1st month
2nd month
3rd month
2.18 ± 0.07
2.80 ± 0.22
2.50 ± 0.23
2.40 ± 0.14
2.42 ± 0.14
2.59 ± 0.12
1.53 ± 0.13
1.48 ± 0.14
1.54 ± 0.15
d
d
d
b
c
b
c
b
b
5.27 ± 0.35
11.70 ± 0.38
5.10 ± 0.43
2.55 ± 0.17
2.51 ± 0.13
2.68 ± 0.19
1.61 ± 0.13
1.44 ± 0.13
1.48 ± 0.15
a
a
a
b
c
b
c
b
b
4.12 ± 0.44
3.70 ± 0.22
3.50 ± 0.31
2.93 ± 0.09
3.19 ± 0.21
3.20 ± 0.41
2.10 ± 0.12
2.54 ± 0.16
2.70± 0.13
group
c
c
c
b
b
a
b
a
a
I. hoferi
4.85 ± 0.11
4.50 ± 0.34
3.90 ± 0.33
4.45 ± 0.19
4.62 ± 0.14
3.56 ± 0.19
2.81 ± 0.21
2.71 ± 0.21
2.90 ± 0.26
b
b
b
a
a
a
a
a
a
control
group
I. hoferi
group
I. hoferi
+ neem
+ fucus
group
Mean ± SE Means in the same column followed by different letters are significantly different ( P< 0.05 )
1341
NADIA A. ABD EL-GHANY AND HODA M. L. ABD ALLA
Table 5. Plasma total protein ,albumin total globulin in tilapia fish experimentally infected with Ichthyophonus hoferi with daily treatment of neem and
fucus extract for three months
Protein ( g / dl )
Albumin ( g / dl )
Total globulin ( g / dl )
1st month
2nd month
3rd month
1st month
2nd month
3rd month
1st month
2nd month
3rd month
3.00 ± 0.15
3.20 ± 0.23
3.30 ± 0.23
1.21 ± 0.03
1.11 ± 0.03
1.15 ± 0.02
1.79 ± 0.04
2.09 ± 0.09
2.15 ± 0.07
c
c
c
b
b
b
b
b
b
2.18 ± 0.20
2.10 ±0.14
2.30 ± 0.18
1.29 ± 0.03
0.86 ± 0.01
0.98 ± 0.02
0.89 ± 0.04
1.34 ± 0.06
1.32 ± 0.11
d
c
d
b
c
c
c
c
c
4.42 ± 0.16
5.20 ± 0.43
5.60 ± 0.24
1.25 ± 0.03
1.15 ± 0.06
1.17 ± 0.04
3.17 ± 0.06
4.05 ± 0.06
4.43 ± 0.12
group
a
a
a
b
b
b
a
a
a
I. hoferi
4.06 ± 0.07
4.00 ± 0.32
4.16 ± 0.24
2.23 ± 0.06
1.84 ± 0.17
2.02 ± 0.03
1.83 ± 0.13
2.16 ± 0.08
2.14 ±0.07
b
b
b
a
a
a
b
b
b
control
group
I. hoferi
group
I. hoferi
+ neem
+ fucus
group
Mean ± SE Means in the same column followed by different letters are significantly different ( P< 0.05 )
1342
A TRIAL FOR TREATMENT OF ICHTHYOPHONOSIS IN Cultured
OREOCHROMIS NILOTICUS USING FUCUS AND NEEM PLANTS
Table 6. Total and differential leucocytic counts of tilapia fish experimentally infected with Ichthyophonus hoferi with daily treatment of neem and
fucus extract for three months
TLC 10³/ µl
Lymphocytes 10³/ µl
1st
month
2nd
month
3rd
month
34.1 ±
1.6
35.6 ±
1.4
34.9
1.7
b
b
b
48.8 ±
1.8
28.1 ±
1.1
27.4
0.57
a
c
c
48.4 ±
1.4
46.5 ±
1.2
47.1
1.07
group
a
a
a
I. hoferi
47.0 ±
1.6
44.9 ±
1.4
47.3
1.3
a
a
a
control
1st month
2nd
month
69.4 ± 1.43
68.6
2.24
b
b
49.6 ± 1.53
48.0
1.41
c
c
74.4 ± 7.11
74.2
1.24
a
a
75.0 ± 1.58
75.2
1.24
a
a
±
Eosinophils
10³/ µl
10³/ µl
3rd month
1st month
2nd month
27.8 ± 3.08
1.2 ± 0.37
1.4 ± 0.24
a
b
b
43.6 ± 1.02
3.6 ± 0.40
3.0 ± 0.44
b
a
a
25.2 ± 1.98
0.60 ± 0.24
0.6 ± 0.24
a
b
c
24.4 ± 2.01
1.0 ± 0.31
0.20
0.20
a
b
c
Neutrophils10³/ µl
3rd
month
±
69.2
1.01
1st month
2nd
month
27.0 ± 1.51
27.0
1.92
a
a
41.8 ± 1.82
43.0
1.22
b
b
24.0 ± 1.58
23.8
0.86
a
a
23.0 ± 1.87
23.6
2.06
a
a
±
±
group
I. hoferi
±
b
±
46.6
1.50
±
±
group
I. hoferi
±
c
±
73.8
1.06
±
±
+ neem
±
a
±
74.0
1.58
±
±
+ fucus
group
a
Mean ± SE Means in the same column followed by different letters are significantly different ( P< 0.05 )
±
NADIA A. ABD EL-GHANY AND HODA M. L. ABD ALLA
1343
1344
A TRIAL FOR TREATMENT OF ICHTHYOPHONOSIS IN Cultured
OREOCHROMIS NILOTICUS USING FUCUS AND NEEM PLANTS
1345
NADIA A. ABD EL-GHANY AND HODA M. L. ABD ALLA
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OREOCHROMIS NILOTICUS USING FUCUS AND NEEM PLANTS
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‫‪1349‬‬
‫‪NADIA A. ABD EL-GHANY AND HODA M. L. ABD ALLA‬‬
‫محاولة عالج مرض االكثيوفونس بنبات الفيكيس و نبات النيم‬
‫فى أسماك البلطى النيلى المستزرعة‬
‫ناديه عبد الغني ‪ ،‬هدي عبد هللا‬
‫أجريت هذه الدراسه ألول مرة كمحاولة لعالج مرض االكثيوفونس فى أسماك البلطى المستزرعة‬
‫وكانت العالمات األكلينكية لألسماك المصابة بالمرض طبيعيا متمثلة فى دكانة وتقرحات فى الجلد مع‬
‫وجود درنات بيضاء اللون صغيرة فى الحجم فى األحشاء الداخلية (الكبد‪ -‬الطحال –الكلى ) والخياشيم ‪.‬‬
‫وينمو الفطر داخل السمكة فى ثالث مراحل (مرحلة السكون – مرحلة ذات التبرعم ومرحلة تكوين الغزل‬
‫الفطرى وفيها تظهر الخيوط الغير المقسمة ويخرج من هذه الخيوط نتوأت داخلية تعرف بأجسام الخيوط‬
‫التى تكون فيما بعد الهيفات الجديدة‪ .‬أستخدمت منبتات عديدة لكى تساعد فى نمو الفطر مثال (المم‪-‬‬
‫الثيوجليكوالت السائل –شوربة السابارود والسابارود دكستروز اجار مضافا اليهم نسب مختلفة من السيرم‬
‫البقرى) ‪.‬‬
‫تم اجراء عدوى صناعية على أسماك بلطى سليمة وخالية من الفطر عن طريق تأكيل الحبيبات‬
‫الجرثومية للفطر فى المم مع ‪ %01‬سيرم بقرى عند أس هيدروجينى ‪ 5.3‬بجرعة ‪0‬مل‪/‬سمكة ‪.‬‬
‫تم تسجيل نسب النفوق و العالمات الظاهرية والداخلية لألسماك المصابة صناعيا ‪.‬‬
‫ومن التحليل الكيميائى وقياسات الدم أظهرت النتائج الى زيادة مستوى هرمون الكورتيزول و‬
‫الجلوبين الكلى فى بالزما الدم كما زادت أعداد الخاليا المتعادلة والخاليا الكلوية بينما قلت تركيزات كل‬
‫من البروتين الكلى والالبومين باألضافة الى نقصان أعداد خاليا الدم البيضاء والليمفاوية فى أسماك‬
‫المجموعة المصابة باألكثيوفونس ولقد أوضحت النتائج أن معظم هذه القياسات زادت فى أسماك‬
‫المجموعة المعالجة بمستخلص النيم بجرعة‬
‫‪3‬جزء فى المليون لكل كجم من العليقةوأسماك المجموعة‬
‫المعالجة بمستخلص الفيكس بجرعة ‪2‬مليجرام لكل كجم من العليقة ‪.‬‬
‫وبينت النتائج أن مستخلص نبات النيم أكثر تأثي ار من مستخلص الفيكس(الطحالب البنية) على فطر‬
‫األكثيوفونس وأن هذا النبات أمن وفعال فى معالجة األسماك المصابة بهذا الفطر‪.‬‬