Effect of Some Immunostimulants on Health Status and Disease Resistance of Nile Tilapia

8th International Symposium on Tilapia in Aquaculture 2008 49

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

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]

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). NEVIEN K. M. ABDELKHALE et al. 51

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).

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 52 EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE OF NILE TILAPIA (OREOCHROMIS NILOTICUS) 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 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 b 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 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) NEVIEN K. M. ABDELKHALE et al. 53 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 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 54 EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE OF NILE TILAPIA (OREOCHROMIS NILOTICUS)

Parameters Control Levamisole Immunoton t

h ) Initial weight 28.08 ± 0.13 27.94 ± 0.26 28.30±0.27 g h i s e i f w / b b a y

m Final weight 28.52 ± 1.94 28.64 ± 2.08 32.36±1.16 d g o ( B

Weight gain 1.30 ± 0.12 c 2.42 ±0.44 3.06±0.56 a ab ADG 0.047 ±0.01 c 0.086±0.02 ab 0.109±0.02 a (mg / fish / day) 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

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 < 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). NEVIEN K. M. ABDELKHALE et al. 55

45 a 40 b 35 c 30 d 25 20

15 1st day 10 2nd week 3rd week 5 4th week 0 Control Levamisole Immunoton

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.

100 a bb a bb c 90 c Control Levamisole Immunoton 80 70 60 50 40

1 s t d a y 30 2 n d w e e k 20 3 r d w e e k 4 t h w e e k 10 0 l l l l l l i i i t l t l t l i i i t t t i i i h h h y y y h h h y y y h h h c c c p p p c c c p p p p p p o o o o o o o o o o o o o o o s s s n n n h h h r r r n n n i a i a i a o t o t o t p p p s s s u u u B B B M M M o o o m m m e e e E E E y y y N N N L L L

Fig. 2. Differential leukocyte count of O. niloticus fed on different immunostimulants 56 EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE OF NILE TILAPIA (OREOCHROMIS NILOTICUS)

for 2 weeks.

Columns with the different letter are significantly different at P<0.05. Phagocytes activity

26 24 22 20 18 Phagocytes index 16 14 12 1st day 10 2nd week 8 3rd week a 4th week b 6 c 4 d 2 0 Control Levamisole Immunoton Control Levamisole Immunoton

Fig. 3. Phagocytosis of O.niloticus fed on different immunostimulants for 2 weeks.

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. NEVIEN K. M. ABDELKHALE et al. 57

500 a 450 b b 400 c 350 300 250 200 150 1st day 100 2nd week 3rd week 50 4th week 0 Control Levamisole Immunoton

Fig. 4. Lysozyme concentration (μg/ml) of O.niloticus fed on different immunostimulants for 2 weeks.

Immunoton 2.5 Control Levamisole

2 a F i r s t d a y 2 n d w e e k 1.5 3 r d w e e k b 4 t h w e e k 1 c d 0.5

0 A α β γ A α β γ A α β γ ------l l l – – –

b g b g b g g g g

u l u l u l g g g l o l o l o m o m o m o l l l b b b o o o b b b i u i u i u b b b n u n u n u l l l u u u l l l i i i i i i n n n l l l n n n i i i n n n

Fig. 5. Protein electrophoresis of O. niloticus fed on different immunostimulants for 2 weeks.

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

Fish groups No. Route of Type of (%) 0 1 2 3 4 5 6 7 of injection Inoculate Dose fish (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

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). NEVIEN K. M. ABDELKHALE et al. 59

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

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 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 NEVIEN K. M. ABDELKHALE et al. 61 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 المراض لسماك البلطى النيلى

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

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

أجريت هذه الدراسة بهدف دراسة تأثير بعض منشطات المناعة المختلفة على الستجابة المناعية غير المتخصصة ومقاومة العدوى بميكروب اليروموناس هيدروفيل وكفاءة النمو لسماك البلطى النيلى. فى هذه التجربة تم تغذية أسماك البلطى النيلى (28±5) جرام وزن متوسط على علئق مختلفة مزودة بالمنشط المناعى الليفاميزول بجرعة 225 مللى جرام لكل كيلو جرام عليقة والميونوتون و هو مخلوط من ( فيتامين " ج" و "ه " مضاف اليهم الصوديوم سيلينيت و حمض الفوليك وسكر اللكتوز) بجرعة واحد جرام لكل كيلو جرام عليقة . تم تغذية السماك لمدة أسبوعين بمعدل 30 جرام من العليقة لكل كيلو جرام من الوزن الحي للسمكة يوميا. وقد وجد أن اضافة الليفاميزول أدت الى زيادة الستجابة المناعية الخلوية من حيث زيادة في العدد الكلي لكرات الدم البيضاء وعدد الخليا الليمفاوية بالضافة إلى زيادة قدرت الخليا الكولة النشطة على التهام فطر الكانديدا البيكانس والتى وصلت لعلى معدلتها عند نهاية السبوع الرابع بالضافة الى زيادة الستجابة المناعية غير الخلوية حيث أدت إلى زيادة تركيز وكفاءة النزيمات المحللة في المصل والتي وصلت أعلى مستوى لها عند نهاية السبوع الثالث بجانب ارتفاع معنوي ملحوظ في الجاماجلوبيلين والذي وصل الى اعلى مستوى له عند نهاية السبوع الرابع مع انخفاض معدل النفوق الناتج عن العدوى البكتيرية ببكتيريا اليروموناس إلى 15% مقارنة بالمجموعة الضابطة الموجبة والذى وصل الى 90% ولكن لم تكن معنوية التاثير على كفاءة النمو بينما أضافة الميونوتون ادت الى زيادة معنوية وملحوظة في كفاءة النمو سجلت اعلى معدلتها بالمقارنة بالليفاميزول أو المجموعة الضابطةو انخفاض معدل النفوق إلى 40% مقارنة بالمجموعة الضابطة الموجبة وبالرغم من أنة لم يكن معنوى التاثير على الستجابة المناعية مقارنة بالليفاميزول. 66 EFFECT OF SOME IMMUNOSTIMULANTS ON HEALTH STATUS AND DISEASE RESISTANCE OF NILE TILAPIA (OREOCHROMIS NILOTICUS)