EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE OF NILE TILAPIA (OREOCHROMIS NILOTICUS

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. [email protected]

mailto:[email protected]

EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE OF NILE TILAPIA (OREOCHROMIS NILOTICUS)

1074

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 micro-

organisms 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 4th 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

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 1st 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) =

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 1st, 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 SDS-

polyacrylamide serum protein electrophoresis according to Laemmli, (1970).

IMMUNOCOMPETANCE TEST (DISEASE RESISTANCE)

Two weeks after last administration of the medicated diets, 20 fish from each

Weight (g) x 100

Length (cm) ³


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

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 <

Parameters Control Levamisole Immunoton

Bo

dy w

eig

ht

(gm

/fi

sh

)

Initial weight 28.08 ± 0.13 27.94 ± 0.26 28.30±0.27

Final weight

28.52 ± 1.94 b

28.64 ± 2.08 b

32.36±1.16 a

Weight gain

1.30 ± 0.12 c

2.42 ±0.44 ab

3.06±0.56 a

ADG (mg / fish / day)

0.047 ±0.01 c

0.086±0.02 ab 0.109±0.02 a

SGR (% / day)

0.126 ± 0.05 b

0.326 ± 0.09 a 0.378±0.09 a

CF (%)

0.089 ± 0.03 b 0.213 ± 0.08 a 0.306±0.07 a


1078

0.05) when compared to control that peaked after the 4th week, while induced a non-

significant 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).

0

5

10

15

20

25

30

35

40

45

Control Levamisole Immunoton

1st day

2nd week

3rd week

4th week

c

b

a

d

Fig. 1. Total leukocytes count (cells x103 cell/µl) of O. niloticus fed on different

immunostimulants for 2 weeks. Columns with the different letter are significantly different at P<0.05.


0

10

20

30

40

50

60

70

80

90

100

Lym

phocyt

Monocyt

Neutr

ophil

Eosin

ophil

Basophil

Lym

phocyt

Monocyt

Neutr

ophil

Eosin

ophil

Basophil

Lym

phocyt

Monocyt

Neutr

ophil

Eosin

ophil

Basophil

1st day

2nd week

3rd week

4th week

abb

cbb

a

c


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.

0

2

4

6

8

10

12

14

16

18

20

22

24

26

Control Levamisole Immunoton Control Levamisole Immunoton

1st day

2nd week

3rd week

4th weekab

c

d

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.

Phagocytes activity

Phagocytes index


1080

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 4th 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.

0

50

100

150

200

250

300

350

400

450

500


1st day

2nd week

3rd week

4th week

a

bb

c

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.


0

0.5

1

1.5

2

2.5

3

Alb

um

in

α –

glo

bulin

β- g

lobulin

γ-g

lobulin

Alb

um

in

α –

glo

bulin

β- g

lobulin

γ-g

lobulin

Alb

um

in

α –

glo

bulin

β- g

lobulin

γ-g

lobulin

First day

2nd week

3rd week

4th week

d

Control LevamisoleImmunoton

a

b

c

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

Fish groups

No.

of

fish

Route of

injection

Type of

Inoculate

Dose

(ml)

Died fish during 7 days after injection.

Mortality

(%)

RLP

0 1 2 3 4 5 6 7

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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.

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

a b

b

a


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 EL-

Ashram, (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

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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تأثير بعض منشطات المناعة على الحالة الصحية ومقاومة األمراض ألسماك البلطى النيلى

2, محمد أحمد على يوسف 2, فيوال حسن ذكى 1نفين كامل محمد عبد الخالق

اليابان. -جامعة كيوشو -كلية الزراعة -قسم العلوم والتكنولوجيا الحيوية .1 مصر.-جامعة المنصورة-كلية الطب البيطرى-ماكقسم األمراض الباطنة والمعدية وأمراض األس .2

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.( جرام وزن متوسط على عالئق مختلفة 5±22ى النيلى )فى هذه التجربة تم تغذية أسماك البلط

مللى جرام لكل كيلو جرام عليقة واألميونوتون و هو 225مزودة بالمنشط المناعى الليفاميزول بجرعة مخلوط من ) فيتامين " ج" و "هـ " مضاف اليهم الصوديوم سيلينيت و حمض الفـوليك وسكر الالكتوز(

جرام من 03بمعدل لمدة أسبوعين االسماك يةتغذ رام عليقة . تم بجرعة واحد جرام لكل كيلو جزيادة أدت الى اضافة الليفاميزول وقد وجد أن العليقة لكل كيلو جرام من الوزن الحي للسمكة يوميا.

دد الخاليا الليمفاوية ـية الخلوية من حيث زيادة في العدد الكلي لكرات الدم البيضاء وعـاالستجابة المناعوصلت والتىباالضافة إلى زيادة قدرت الخاليا االكولة النشطة على التهام فطر الكانديدا البيكانس

االسبوع الرابع باألضافة الى زيادة االستجابة المناعية غير الخلوية حيث نهاية ألعلى معدالتها عند نهاية أدت إلى زيادة تركيز وكفاءة االنزيمات المحللة في المصل والتي وصلت أعلى مستوى لها عند

ارتفاع معنوي ملحوظ في الجاماجلوبيلين والذي وصل الى اعلى مستوى له عند بجانب االسبوع الثالثالناتج عن العدوى البكتيرية ببكتيريا االيروموناس نفوقمعدل الانخفاض االسبوع الرابع مع نهايةولكن لم تكن معنوية التاثير %03الموجبة والذى وصل الى مجموعة الضابطةبالمقارنة %15إلى

و سجلت زيادة معنوية وملحوظة في كفاءة النمبينما أضافة األميونوتون ادت الى على كفاءة النمو %03إلى نفوقمعدل الانخفاض اعلى معدالتها بالمقارنة بالليفاميزول أو المجموعة الضابطةو

وبالرغم من أنة لم يكن معنوى التاثير على األستجابة المناعية الموجبة مجموعة الضابطةبالمقارنة مقارنة بالليفاميزول.

EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE OF NILE TILAPIA (OREOCHROMIS NILOTICUS

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