8th International Symposium on Tilapia in Aquaculture 2008
1073
EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS
AND DISEASE RESISTANCE OF NILE TILAPIA
(OREOCHROMIS NILOTICUS)
NEVIEN K. M. ABDELKHALEK 1, VIOLA H. ZAKI 2AND M. A. A. YOUSEF
2
1. Present address: Laboratory of Marine Biochemistry, Faculty of Agriculture,
Kyushu University, Japan.
2. Department of Internal Medicine, Infectious and Fish Diseases, Faculty of
Veterinary Medicine, El Mansoura University, Egypt.
Abstract
Different
immunostimulants
were
investigated
to
detect
changes in the activity of innate non-specific immune response,
disease resistance and growth performance of Nile Tilapia
Oreochromis niloticus. In this experiment, fish fed on diets
supplemented with levamisole HCl (225 mg/kg diet), Immunoton ®
(a mixture of vitamins E (150 mg/kg diet) and C (101.2 mg/kg diet)
and control diet for 2 weeks. Total leukocyte count (TLC),
Differential leukocyte count (DLC), phagocytosis have been
measured as indicators for cellular innate immune response.
Lysozyme and protein electrophoresis have been measured as
indicators for humoral innate immune response, in addition to
immunocompetence assay against Aeromonas hydrophila and
growth performance test. The obtained results indicated that,
addition of levamisole HCl to the diet of O. niloticus significantly
enhanced both cellular and humoral innate immune responses and
increased resistance to A. hydrophila infection although they had a
little growth promoting activity. While addition of vitamins C and E
to O. niloticus higher than the requirements needed for growth lead
to maximum growth compared to levamisole but little enhancement
of the immune response as well as resistance to A. hydrophila
infection.
Key
words:
Immunostimulants;
Innate
immune
response;
Oreochromis niloticus.
*
Corresponding author
Nevien Kamel Mohamed Abdelkhalek, PhD
Laboratory of Marine Biochemistry,
Department of Bioscience and Biotechnology,
Graduate School of Bioresource and Bioenvironmental Science,
Kyushu University, Fukuoka 812-8581, Japan.
Tel: +81-92-642-2894 Fax: +81-92-642-2897 mobile: +81- 90- 8404-8906
E-mail. nevien_k@agr.kyushu-u.ac.jp
1074
EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE
OF NILE TILAPIA (OREOCHROMIS NILOTICUS)
INTRODUCTION
With the worldwide fish production and intensive cultivation system, fish are
subjected to many diseases that lead to great losses and decrease in fish production.
The lack of effective disease control has the potential of being the chief limiting
factor of the realization of highly stable fish production (Phillip et al., 2000). Aquatic
animal diseases control in Egypt includes a limited number of Government-approved
antibiotics and chemotherapeutics, beside limited vaccines that can be used to assist
the environmental management (Aly et al., 2008). However, the approach which
concentrates on treatment once disease develops using antibiotic are sometimes of
little value or less successful due to emergence of antibiotic resistant microorganisms which makes this method of control less successful (Anderson, 1992 and
Stoskopf, 1993).
Immunotherapy is an approach that has been actively investigated in recent
years as a method for disease prevention. It does not involve recognition of a
specific antigen or targeting the immune response towards a specific pathogen, but
causes an overall immune response that hastens recognition of foreign proteins
(Campos et al., 1993; Secombes, 1994 and Sordello et al., 1997). So the use of
immunostimulants for prevention of diseases in fish is considered an alternative and
promising area (Sakai, 1999).
Levamisole, originally synthesized as an anti-helminthic, has been widely used as
an immunomodulator in fish either by injection (Siwicki, 1987), immersion (Siwicki
and Korwin-Kosskowski, 1988), oral administration (Siwicki, 1989; Siwicki and
Studnicka, 1994; Mulero et al., 1998b Alvarez et al., 2006) or in vitro
immunostimulation (Siwicki et al., 1992).
Use of vitamins as immunostimulant has been used in many fish including
Atlantic salmon (Waagbo et al., 1992), rainbow trout (VerIhac et al., 1998) and
gilthead sea bream (Mulero et al., 1998a, Ortuno et al., 1999, 2000 and Cuesta et al.,
2001) although not so much data for tilapia. The aim of the present work is to study
the effect of dietary intake of levamisole and Immunoton ® (vitamins C and E) on
innate immune system, resistance to diseases and growth performance of Nile Tilapia
(Oreochromis niloticus).
NEVIEN K. M. ABDELKHALE et al.
1075
MATERIALS AND METHODS
FISH
A total number of 300 apparently healthy Oreochromis niloticus obtained
from a private fish farm in EL Dakahlia Governorate with average body weight of
28.12(±5) g transported alive to the laboratory of Department of Fish Diseases and
Management, Faculty of Veterinary Medicine, El Mansoura University. They were kept
for 2 weeks under observation for acclimatization in glass aquaria (40 × 60 × 100
cm). The water of the aquaria was removed daily, and its temperature was
maintained at 25 ± 1 °C.
IMMUNOSTIMULANTS
Levamisole® HCL
It’s a commercial product available in the market manufactured by Memphis
Pharmaceutical, Cairo, Egypt. It’s used as anti helminthic and immunostimulant for
large animals in Egyptian farms. Each ml contains 0.1g levamisole HCL. The dose
was calculated to be 225mg /kg diet then mixed with the basal diet and pellets were
made. The pellets were prepared biweekly, air dried at room temperature and stored
in a refrigerator (4 °C) for daily use.
Immunoton®
It’s a commercial product available in the market manufactured by El Safa Pharm
for manufacturing, Al Nubaria, Egypt. It’s used as immunostimulant for poultry in
Egyptian farms. Each one kg contains (vitamin E 150 g, vitamin C 101.2 g, sodium
selenite 222 mg, folic acid 521
mg and Lactose up to one 1000 g). It is mixed with
the basal diet at a dose of 1g of the product/kg diet and pellets were made. The
pellets were prepared biweekly, air dried at room temperature and stored in a
refrigerator (4 °C) for daily use.
EXPERIMENTAL DESIGN
Fish were divided into 3 groups (100 fish/group). First group served as a control
group fed on basal diet, 2nd group fed on Levamisole® supplemented diet for 2 weeks
then fed on basal diet till the end of the 4th week. 3rd group fed on Immunoton®
supplemented diet for 2 weeks then fed on basal diet till the end of the 4 th week.
Each group subdivided into 4 subgroups (25 fish/subgroup).
Fish were fed on basal diet of 3000 kcal/kg digestible energy and 30% protein either
control (basal diet only) or immunostimulant supplanted diets at a feeding rate of 30
1076
EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE
OF NILE TILAPIA (OREOCHROMIS NILOTICUS)
g diet/kg biomass/day, divided into two feeding times for 2 weeks then fed on
immunostimulant free diet (basal diet only) till the end of experimental period (the
end of the 4th week) with the same feeding rate and feeding time. The required diets
were prepared biweekly and stored in a refrigerator (4 °C) for daily use.
ASSESSMENT OF GROWTH PERFORMANCE.
Fish samples were collected from each treatment and control groups at 1 st and
28th days of the experiment, then weighted for determining (Average body weight,
Body weight gain, Average daily gain (ADG), condition factor (CF) and specific
growth rate according to Ricker, (1979) using the following equations:
a) Total gain (g / fish) = Wt – Wo
b) Average daily gain (g /fish /day) = Wt – Wo/n
c) Condition factor ( CF) =
Weight (g) x 100
Length (cm) ³
d) Specific growth rate.
SGR= (Lin Wt – Lin Wo) 100 / n
Wo: Is the initial fish weight (gm) at the start of the experiment.
Wt: Is the final fish weight (gm) at the end of the experiment.
n: Is the duration period of the experiment in day.
Lin: Is the natural logarithm.
DETERMINATION OF NON SPECIFIC INNATE IMMUNE RESPONSE
A total number of 20 blood samples were collected from the caudal vein of 20
fish in each group (5 samples from each replicate) on days 1 st, 14th, 21st and 28th of
the experimental period according to Rowley, (1990). Each sample divided into 2
haves (one after adding anticoagulant for examination of total leukocyte count (TLC)
according to Schaperclaus, (1992), differential leukocyte count (DLC) according to
Stoskopf, (1993) using Giemsa stain. Phagocytosis assay (phagocytic activity and
phagocytic index) was determined according to Kawahara et al., (1991) using
Candida albicans culture. While the other half allowed to clot at 4 °C in a refrigerator
for 4hrs then centrifuged at 1500 xg for 10minits. Following centrifugation, the
serum was collected and frozen at - 80 °C until used for Lysozyme (lysoplate) assay
according to Ellis, (1990) using 50 μg/ml Micrococcus lysodictecus and SDSpolyacrylamide serum protein electrophoresis according to Laemmli, (1970).
IMMUNOCOMPETANCE TEST (DISEASE RESISTANCE)
Two weeks after last administration of the medicated diets, 20 fish from each
NEVIEN K. M. ABDELKHALE et al.
1077
of the tested groups (5 fish/replicate) were injected I/P with 0.1 ml/24 hrs broth
culture of A. hydrophila strain containing 3 × 107 viable cells/ml according to (Zaki ,
1991), Clinical signs, moralities were recorded daily for 7 days. The relative level of
protection (RLP) among the challenged fish was determined according to
Ruangroupan et al., (1986).
STATISTICAL ANALYSIS
Data were statistically analyzed using one- way or two- way analysis of
variance (ANOVA) (SAS, 1996). Duncan multiple range test was used to test the
significance among the means (Snedecor and Cochrang, 1989). Differences were
considered significant when P <0.05.
RESULTS
ASSESSMENT OF GROWTH PERFORMANCE
Growth performance (Body weight again, ADG, SGR, CF) of O. niloticus fed on
Immunoton showed a significant increase (at p < 0.05) compared to levamisole or
control groups by the end of the experimental period (28days) (table 1).
Table 1. Growth performance by the end of experiment of O. niloticus fed on
different immunostimulants for 2 weeks
Body weight
(gm/fish)
Parameters
Initial weight
Final weight
Weight gain
ADG
(mg / fish / day)
SGR (% / day)
CF (%)
Control
Levamisole
Immunoton
28.08 ± 0.13
27.94 ± 0.26
28.30±0.27
28.52 ± 1.94 b
1.30 ± 0.12 c
0.047 ±0.01 c
0.126 ± 0.05
b
0.089 ± 0.03
b
28.64 ± 2.08
b
32.36±1.16
a
2.42 ±0.44
ab
3.06±0.56
a
0.086±0.02
ab
0.109±0.02
a
0.326 ± 0.09
a
0.378±0.09
a
0.213 ± 0.08
a
0.306±0.07
a
Means in a column with different superscripts different significantly at P<0.05.
ASSESSMENT OF CELLULAR INNATE IMMUNE RESPONSE
TOTAL LEUKOCYTES COUNT (TLC)
There was a significant increase (at p < 0.05) in TLC through out the experimental
period reached the highest level at end of 4th week of experiment in group
supplemented with levamisole followed by Immunoton supplemented group (fig. 1).
DIFFERENTIAL LEUKOCYTES COUNT (DLC)
Levamisole and Immunoton significantly increased lymphocytes % (at p <
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EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE
OF NILE TILAPIA (OREOCHROMIS NILOTICUS)
0.05) when compared to control that peaked after the 4th week, while induced a nonsignificant effect on the other cell types (fig.2).
PHAGOCYTOSIS
There was a non-significant difference in phagocyte activity (P.A) in all
immunostimulants supplemented groups and control throughout the experimental
period. While phagocyte index (P.I) showed a significant increase during the
experimental period peaked after 4th week for levamisole supplemented group but
did not show a significant increase with Immunoton supplemented group or control
(fig.3).
45
a
40
b
c
35
30
d
25
20
15
1st day
2nd week
3rd week
4th week
10
5
0
Control
Levamisole
Immunoton
Fig. 1. Total leukocytes count (cells x10 3 cell/µl) of O. niloticus fed on different
immunostimulants for 2 weeks.
Columns with the different letter are significantly different at P<0.05.
NEVIEN K. M. ABDELKHALE et al.
100
c
90
bb
1079
bb a
a
c
Control
Levamisole
Immunoton
80
70
60
50
40
1st day
30
2nd week
20
3rd week
4th week
Basophil
Eosinophil
Neutrophil
Monocyt
Lymphocyt
Basophil
Eosinophil
Neutrophil
Monocyt
Lymphocyt
Basophil
Eosinophil
Neutrophil
Monocyt
0
Lymphocyt
10
Fig. 2. Differential leukocyte count of O. niloticus fed on different immunostimulants
for 2 weeks.
Columns with the different letter are significantly different at P<0.05.
Phagocytes activity
26
24
22
20
18
16
14
12
10
8
6
4
2
0
Phagocytes index
c
b
a
1st day
2nd week
3rd week
4th week
d
Control
Levamisole Immunoton
Control
Levamisole Immunoton
Fig. 3. Phagocytosis of O.niloticus fed on different immunostimulants for 2 weeks.
Columns with the different letter are significantly different at P<0.05.
1080
EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE
OF NILE TILAPIA (OREOCHROMIS NILOTICUS)
ASSESSMENT OF HUMORAL INNATE IMMUNE RESPONSE
LYSOZYME CONCENTRATION
There was a significant increase in Lysozyme concentration started at the end of
3rd week with levamisole supplemented group while Immunoton supplemented group
showed a non significant difference trough out the experimental period (fig. 4).
PROTEIN ELECTROPHORESIS
γ- Globulin concentration significantly increased at the end of the 4 th week with
levamisole (at p < 0.05) in fig.6. While Immunoton was non significantly different
(fig.5). Total albumin, α and β globulin didn’t show a significant difference either in
each treatment through out the experimental period, or between different treatments
including control.
a
500
450
b
400
b
c
350
300
250
200
1st day
2nd week
3rd week
4th week
150
100
50
0
Control
Levamisole
Immunoton
Fig. 4. Lysozyme concentration (μg/ml) of O.niloticus fed on different
immunostimulants for 2 weeks.
Columns with the different letter are significantly different at P<0.05.
NEVIEN K. M. ABDELKHALE et al.
1081
3
2.5
Control
Immunoton
Levamisole
a
2
First day
2nd week
1.5
3rd week
b
4th week
1
d
c
0.5
0
γ-globulin
β- globulin
α – globulin
Albumin
γ-globulin
β- globulin
α – globulin
Albumin
γ-globulin
β- globulin
α – globulin
Albumin
Fig. 5. Protein electrophoresis of O. niloticus fed on different immunostimulants for 2
weeks.
Columns with the different letter are significantly different at P<0.05.
IMMUNOCOMPETANCE TEST (DISEASE RESISTANCE)
Mortality rates have been decreased with levamisole supplemented to 15% and
high relative protection level (83.3) while was 40% with Immunoton supplemented
group with moderate RPL (55.6) compared to 90% mortalities and no protection in
control group table (2).
Clinical pictures of the experimentally infected fish were nearly similar in all
treatments including control but varied in the severity of the developed lesions. They
included poor appetite, loss of equilibrium with erratic movement of some fish,
swimming with head down due to abdominal distension and finally loss of all reflexes
just prior to death. Hemorrhage of all fins, caudal peduncle and ventral abdominal
wall with hemorrhagic and protruded anal opening were seen (fig. 6).
Table 2. Disease resistance of O. niloticus fed on different immunostimulants for 2 weeks
Died fish during 7 days after injection.
No.
Fish groups
of
fish
Mortality
Route of
Type of
injection
Inoculate
Dose
0
1
2
3
4
5
6
7
(%)
RLP
(ml)
Control
20
I/P
A. hydrophila
0.1
_
4
3
3
2
2
3
1
90
0
Levamisole
20
I/P
A. hydrophila
0.1
_
_
1
_
2
_
_
_
15
83.3
Immunoton
20
I/P
A. hydrophila
0.1
_
1
3
_
2
_
2
_
40
55.6
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EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE
OF NILE TILAPIA (OREOCHROMIS NILOTICUS)
b
.a
b
a
Fig. 6. O. niloticus experimentally infected with A. hydrophila with hemorrhage at all fins
(arrow) and caudal peduncle (a) compared to apparently normal healthy fish (b).
DISCUSSION
During the last decade there was an increasing interest in the modulation of
the non specific immune response of fish to elevate the general defense barriers and
hence increase resistance against diseases through use of immunostimulants (Raa,
2000 and Sahoo and Mukherjee, 2002).
From the presented results, use of levamisole as feed additive at a dose of
(225 mg / kg of diet for 2 weeks has shown to be a powerful activator of non specific
innate immune response. This increase in total leukocyte count, lymphocyte count
and phagocytosis after 2 weeks from the last administration of levamisole could be
attributed to stimulation of leukiopoiesis and lymphocyte proliferation by levamisole
(Courntney and Robertsen, 1995) although in our study levamisole did not
significantly increased monocytes and neutrophils numbers but increasing their
activity to phagocytose candida albicans. Similarly Siwicki, (1989) reported an
increase in TLC and lymphocyte count with dietary intake of levamisole to common
carp at a dose of 5 mg/kg body weight for 15 days. Furthermore, increased
phagocytic activity (P.A) and phagocytic index (P.I) were prominent after 2 weeks
from last administration of levamisole in fish kept at 22 oC and lasted for 3 months.
Also Mulero et al., (1998 b) observed increased (P.A) and (P.I) in gilt head sea bream
fed on levamisole supplemented diet for 10 days especially at low doses (125, 250
mg/kg diet) while a dose of 500 mg/kg diet had a suppressive effect.
From our presented data, dietary intake of levamisole also increased serum
lysozyme concentrations of O. niloticus. Similar results in carp obtained by Siwicki,
(1989) who observed an increase in serum lysozyme concentrations in both fish
groups kept at 12 oC and 22 oC but the highest serum lysozyme concentrations was
observed in fish kept at 22oC. Another study in rohu fed on levamisole in a dose of 5
mg/kg but in 5 consecutive occasions for 60 days recorded a significant increase in
NEVIEN K. M. ABDELKHALE et al.
1083
lysozyme concentrations (Sahoo and Mukherjee, 2001). As it is clear that lysozyme is
mainly produced by activated macrophages (Gordon et al., 1974), so the increase in
lysozyme concentration may be attributed to the increase in the activity phagocytic
cells.
Protein electrophoresis is available tool in assessing humoral immunity (kaneko
et al., 1997). From our result, γ-globulin (the immunity related protein fraction) was
the only to be increased after levamisole stimulation while total protein and total
albumin levels remain unchanged, which shows the important role of γ-globulin in
immune response.
Supporting this findings, results obtained by Sahoo and Mukherjee, (2001) in
which total protein levels remain unchanged irrespective to aflatoxin/ levamisole
exposure, although the increased albumin / globulin ratio noted due to aflatoxin
treatment was restored by levamisole feeding. Another study by EL-Boushy and ELAshram, (2002) in African catfish reported that levamisole found to restore the total
protein level in immunocompromised fish and increasing γ-globulin levels in healthy
fish.
It is well known that the ideal conditions are always prevailing in commercial
farms that make
fish continuously stressed. Under these
conditions,
the
requirements for certain nutrients will almost certainly increase due to an increased
activity of the immune system. From the present study the combination of vitamins C
and E could cause little enhancement of the cellular innate immune response while
had a non significant effect on humoral one. This may be attributed to the low doses
of vit. C (101.2 mg\kg diet) and E (150mg\kg diet) present in the supplemented diet
and so it could not induce maximum immunity but induce enhanced immune
response through leukopoiesis and enhanced lymphocytes proliferation. Similar
results on rainbow trout obtained by Wahli et al., (1997) who reported that,
combination of vit.C and E significantly increase lymphocyte proliferation. In contrast,
Mulero et al., (1998a) found that in vitro addition of either vitamin C or E individually
had no effect on the phagocytic activity of gilt head sea bream leukocytes and even
combination of both vitamins failed to further increase such activity at any of the
tested concentrations (1-100 µg / ml) vitamin C or (0.01- 10 µg / ml) vitamin E for
48 hrs.
Regarding to resistance to infection, both of the tested immunostimulants had
been found to increase resistance of O. niloticus against A. hydrophila infection
although it was little with Immunoton. This resistance may be due to the increased
activity of phagocytic cells with subsequent increase in lysozyme activity and
bactericidal activity of fish phagocytes/macrophages (Robertsen et al., 1994). Many
1084
EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE
OF NILE TILAPIA (OREOCHROMIS NILOTICUS)
studies similarly, recorded increased resistance against A.hydrophila challenge after
stimulation with levamisole (Oliver et al., 1985; Baba et al., 1993; Jeney and
Anderson, 1993 and Sahoo and Mukherjee, 2001), while the lower resistance in case
of Immunoton could be attributed to the low doses of Vit. C (101.2 mg \ kg diet) and
vit. E (150 mg \ kg diet) presents in the supplemented diet and so it could not induce
maximum immunity. Although these doses were above the minimal levels needed for
growth in addition to presence of lactose in the product that could help in growth
promoting activity with maximum feed utilization. This explains why it showed the
highest growth stimulator compared to levamisole. Results obtained by Ortuno et al.,
(1999 and 2000) showed that, a moderately high dietary dose of vit. E together with
adequate vitamin E / C ratio are needed for maximum growth and enhanced immune
response.
So in conclusion, dietary intake of levamisole to O. niloticus enhances non
specific innate immunity rather than growth performance. While addition of a
combination of vitamins C and E could significantly enhance growth performance but
higher doses are needed for maximum immunity.
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‫‪1088‬‬
‫‪EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE‬‬
‫)‪OF NILE TILAPIA (OREOCHROMIS NILOTICUS‬‬
‫تأثير بعض منشطات المناعة على الحالة الصحية ومقاومة األمراض ألسماك البلطى النيلى‬
‫نفين كامل محمد عبد الخالق‪ , 1‬فيوال حسن ذكى‪ , 2‬محمد أحمد على‬
‫يوسف‪2‬‬
‫‪ .1‬قسم العلوم والتكنولوجيا الحيوية‪ -‬كلية الزراعة‪ -‬جامعة كيوشو‪ -‬اليابان‪.‬‬
‫‪ .2‬قسم األمراض الباطنة والمعدية وأمراض األسماك‪-‬كلية الطب البيطرى‪-‬جامعة المنصورة‪-‬مصر‪.‬‬
‫أجريت هذه الدراسة بهدف دراسة تأثير بعض منشطات المناعة المختلفة على األستجابة المناعية‬
‫غير المتخصصة ومقاومة العدوى بميكروب األيروموناس هيدروفيال وكفاءة النمو ألسماك البلطى‬
‫النيلى‪.‬‬
‫فى هذه التجربة تم تغذية أسماك البلطى النيلى (‪ )5±28‬جرام وزن متوسط على عالئق مختلفة‬
‫مزودة بالمنشط المناعى الليفاميزول بجرعة ‪ 225‬مللى جرام لكل كيلو جرام عليقة واألميونوتون و هو‬
‫مخلوط من ( فيتامين " ج" و "هـ " مضاف اليهم الصوديوم سيلينيت و حمض الفـوليك وسكر الالكتوز)‬
‫بجرعة واحد جرام لكل كيلو جرام عليقة ‪ .‬تم تغذية االسماك لمدة أسبوعين بمعدل ‪ 30‬جرام من‬
‫العليقة لكل كيلو جرام من الوزن الحي للسمكة يوميا‪ .‬وقد وجد أن اضافة الليفاميزول أدت الى زيادة‬
‫االستجابة المناعـية الخلوية من حيث زيادة في العدد الكلي لكرات الدم البيضاء وعـدد الخاليا الليمفاوية‬
‫باالضافة إلى زيادة قدرت الخاليا االكولة النشطة على التهام فطر الكانديدا البيكانس والتى وصلت‬
‫ألعلى معدالتها عند نهاية االسبوع الرابع باألضافة الى زيادة االستجابة المناعية غير الخلوية حيث‬
‫أدت إلى زيادة تركيز وكفاءة االنزيمات المحللة في المصل والتي وصلت أعلى مستوى لها عند نهاية‬
‫االسبوع الثالث بجانب ارتفاع معنوي ملحوظ في الجاماجلوبيلين والذي وصل الى اعلى مستوى له عند‬
‫نهاية االسبوع الرابع مع انخفاض معدل النفوق الناتج عن العدوى البكتيرية ببكتيريا االيروموناس‬
‫إلى‪ %15‬مقارنة بالمجموعة الضابطة الموجبة والذى وصل الى ‪ %90‬ولكن لم تكن معنوية التاثير‬
‫على كفاءة النمو بينما أضافة األميونوتون ادت الى زيادة معنوية وملحوظة في كفاءة النمو سجلت‬
‫اعلى معدالتها بالمقارنة بالليفاميزول أو المجموعة الضابطةو انخفاض معدل النفوق‬
‫إلى‪%40‬‬
‫مقارنة بالمجموعة الضابطة الموجبة وبالرغم من أنة لم يكن معنوى التاثير على األستجابة المناعية‬
‫مقارنة بالليفاميزول‪.‬‬