Dietary levamisole modulates the immune response

Aquaculture Research, 2006, 37, 500^509

doi:10.1111/j.1365-2109.2006.01456.x

Dietary levamisole modulates the immune response and disease resistance of Asian catfish Clarias

batrachus (Linnaeus) Jaya Kumari & Pramoda K Sahoo Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar, India Correspondence: P K Sahoo, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751002, India. E-mail: [email protected], [email protected]

Abstract In order to determine the immunomodulatory e¡ect of dietary levamisole in Asian cat¢sh (Clarias batrachus), ¢sh were fed four di¡erent diets for 10 days: a formulated diet as control and the same diet supplemented with 50, 150 or 450 mg levamisole kg 1 feed. The serum bacterial agglutination titre against Aeromonas hydrophila as a measure of speci¢c immunity, serum haemagglutination titre, natural haemolytic complement activity (ACH50), myeloperoxidase and lysozyme activities, total protein level and oxidative radical production by neutrophils as a measure of non-speci¢c immunity as well as disease resistance against A. hydrophila challenge to separate vaccinated and non-vaccinated groups were evaluated at 0, 1, 2 and 3 weeks after last administration of levamisole. Levamisole supplement at the lowest level (50 mg kg 1) signi¢cantly enhanced oxidative radical production and serum myeloperoxidase (MPO) content immediately after 10 days of feeding, which reached peak values after 3 and 2 weeks of feeding respectively. Haemolytic complement and haemagglutination titre were signi¢cantly enhanced after 3 and 1 weeks respectively. Haemolytic complement activity and MPO activities were signi¢cantly raised to 150 mg kg 1 after 3 and 2 weeks, respectively. At the highest level of levamisole feeding (450 mg kg 1) signi¢cant decreases in superoxide production and complement activity were measured immediately after levamisole feeding, which returned to the normal level after 1 week postfeeding. Fish were challenged with a virulent strain of A. hydrophila at 0, 1, 2 and 3 weeks after levamisole feeding, and the cumulative per cent survival was recorded over 10 days. Feeding levamisole at 50, 150 or 450 mg kg 1

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increased per cent survival in vaccinated ¢sh immediately after levamisole feeding, and survival was signi¢cantly higher at 450 mg kg 1. There was no di¡erence in mortality patterns in non-vaccinated ¢sh. The results support the use of levamisole at 50 mg kg 1 feed for 10 days as an immunostimulant in Asian cat¢sh farming.

Keywords: levamisole, immunomodulation, disease resistance, Asian cat¢sh (Clarias batrachus)

Introduction The growth of intensive aquaculture production has led to a growing interest in treating or preventing ¢sh diseases. Traditionally, antibiotics have been used in aquaculture for the prevention and treatment of bacterial diseases. However, the use of antibiotics in aquaculture poses threats such as development of bacterial strains that are resistant to antibiotic treatment, or the occurrence of antibiotic residues in ¢sh farmed for human consumers’ (FAO 2002). Based on these two considerations, the potential uses of existing antibiotics and approval of new ones for aquaculture are limited. Alternatively, vaccines against speci¢c pathogens have been developed with varying degree of success. In India, no commercial vaccine or recommended immunotherapy is currently available for cat¢sh culture. The wide range of pathogens in ¢sh farming also limits vaccine e¡ectiveness. Nonspeci¢c immunity in ¢sh is important for resistance against a range of diseases, whereas vaccination protects against a speci¢c pathogen. Thus, immunostimulants hold promise as alternative strategies against infectious aquatic diseases and have received

r 2006 The Authors. Journal Compilation r 2006 Blackwell Publishing Ltd


Immunomodulation by dietary levamisole in Clarias batrachus J Kumari & P K Sahoo

heightened attention in recent years (Anderson1992; Sakai 1999; Sahoo & Mukherjee 1999, 2001; Kumari, Swain & Sahoo 2003). One such potential immunostimulant is levamisole, a levo-isomer of tetramisole that has been widely used as an antihelminthic drug in terrestrial livestock. It has also been shown with several aquatic species to be a potent immunostimulant in the modulation of T-cell function (Renoux 1980), cytotoxic activity of leucocytes (Cuesta, Esteban & Meseguer 2002), phagocytosis, respiratory burst (Siwicki 1989; Mulero, Esteban, Munoz & Meseguer 1998; Findlay & Munday 2000) and macrophage-activating factor (Mulero, Esteban, Munoz et al.1998). In some ¢sh species, levamisole has been demonstrated to enhance non-speci¢c or speci¢c immune responses when given alone or as an adjuvant with a vaccine (Sakai 1999). Special attention must be paid to the doses and the timing for sustainable enhancement, as the e¡ects of levamisole are dose dependent and time dependent. High doses of levamisole may suppress the immune responses and low doses may not be e¡ective (Anderson, Siwicki, Dixon & Lizzi 1989; Siwicki, Anderson & Dixon 1990). However, no information exists concerning the e¡ects of levamisole in Asian cat¢sh, Clarias batrachus, a candidate culture species in diversi¢ed Indian aquaculture systems. Only lactoferrin has been evaluated for its e⁄cacy in this ¢sh species as an immunomodulatory substance (Kumari et al. 2003). The present study was undertaken to examine the e¡ect of di¡erent doses of levamisole, supplemented in the diet, on the non-speci¢c and speci¢c immune responses as well as disease resistance against a common pathogenic bacteria Aeromonas hydrophila in Asian cat¢sh.

Materials and methods Fish Asian cat¢sh, average weight 52.4 3.07g and 9.22 0.68 g (utilized for challenge study only), were obtained from the Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, Bhubaneswar, India. Five ¢sh were maintained in 48 numbers of 500 L circular cement tanks, and acclimatized for 15 days before each of the experiment. The ¢sh were fed a formulated pelleted diet (hand pelletized in the laboratory) at 3% of body weight once daily (Kumari & Sahoo 2005b). About 50% of water was exchanged daily along with the uneaten

feed and faecal material. Water parameters were measured at 5-day intervals to maintain optimal levels (dissolved oxygen: 5.65 0.72 mg L 1; pH: 8.2 0.82; nitrites: 0.015 0.009 mg L 1; ammonia: 0.109 0.024 mg L 1) throughout the experiment. The water temperature during the experiment was 29^31 1C.

Experimental design Four such experiments were carried out to measure non-speci¢c and speci¢c immune parameters, challenges to non-vaccinated and vaccinated ¢sh. Two hundred and forty ¢sh utilized in each experiment were divided into four groups (A, B, C and D) with 60 ¢sh in each group kept in 12 tanks. Each group was divided into four subgroups, according to four time periods of 0, 1, 2 and 3 weeks after the last administration of levamisole (15 ¢sh per three tanks for each time period). Each group was fed either a pelleted diet (control group A) or the same diet supplemented with levamisole hydrochloride (Sigma, St Louis, MO, USA) using carboxy methyl cellulose (CMC) as binder at a rate of 50, 150 or 450 mg kg 1 feed (groups B, C and D respectively) for 10 days. The pellet size ranged from 0.5 to 0.8 cm2. Fish were habituated to eat the given diet immediately after dropping to avoid leaching loss of levamisole. After 10 days, all ¢sh were fed the control diet. The non-speci¢c and speci¢c immune parameters and challenge study were carried out immediately (0) and 1, 2 and 3 weeks after the last administration of levamisole with ¢ve ¢sh for each dietary subgroup at each time period. Fish were anaesthetized with 2-phenoxyethanol (Sigma) (100 mL phenoxyethanol L 1 of water) before any injection or blood collection from the caudal vein (Kumari et al. 2003; Kumari & Sahoo 2005a). The experiments were run in triplicate. Because of the large number of ¢sh and tank requirements for a single experiment, four experiments were carried out one after the other within a period of 10 weeks utilizing the same stock of cat¢sh.

Sample collection Blood samples were collected 0,1, 2 and 3 weeks after the last administration of levamisole from the caudal vein with a 24-gauge needle and 2 mL syringe. Blood samples were divided into two aliquots; one was heparinized (50 IU mL 1 of blood) and the other was allowed to clot at room temperature for 30 min and

r 2006 The Authors. Journal Compilation r 2006 Blackwell Publishing Ltd, Aquaculture Research, 37, 500^509

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kept at 4 1C for 3 h. The serum samples collected were frozen at 70 1C until further analysis.

Non-speci¢c immune response Nitroblue tetrazolium (NBT) assay and myeloperoxidase (MPO) activity The oxidative radical production by neutrophils during respiratory burst was measured by the NBT assay (Anderson & Siwicki 1995). Brie£y, heparinized blood and 0.2% NBT solution were mixed in equal proportion (1:1), incubated for 30 min at 25 1C, and then 50 mL of the mixture was dispensed into glass tubes. For solubilization of reduced formazan product, 1mL of dimethyl formamide (SRL, Mumbai, India) was added and centrifuged at 2000 g for 5 min. Finally, the supernatant was taken and the extent of NBT reduction was measured at an optical density of 540 nm. Dimethyl formamide was used as the blank. The total peroxide content present in serum was measured according to Quade and Roth (1997) using a partially modi¢ed technique (Sahoo, Kumari & Mishra 2005).


vating the lytic reaction (Sunyer & Tort 1995). The results are expressed as ACH50 (U mL 1), the reciprocal serum dilution giving 50% haemolysis. Haemagglutination assays were performed as in Kumari and Sahoo (2005a). The total protein content in serum was measured following Bradford (1976) method, using bovine serum albumin as a standard protein.

Speci¢c immune response For this experiment, ¢sh from all groups were injected intraperitoneally with 0.1mL of 1% formalin-killed A. hydrophila bacterin (2.7 108 cells harvested in 0.1mL phosphate-bu¡ered saline (PBS) and mixed with an equal volume of Freund’s complete adjuvant). Fish were fed the control diet until day 20. From days 21to 30, the experimental groups B, C and D were fed with their respective doses of levamisole-supplemented diet. The ¢sh were bled at weekly intervals after levamisole feeding and collected sera were utilized to determine antibody titres using microtitre plates following Plumb and Areechon (1990).

Lysozyme activity A turbidometric assay utilizing lyophilized Micrococcus lysodeikticus cells (Sigma) was used to determine lysozyme activity in serum. A slight modi¢cation of the previously described method (Sankaran & Shanto 1972; Studnicka, Siwicki & Ryka 1986) was used to measure lysozyme activity. A suspension of 150 mL of M. lysodeikticus (0.2 mg mL 1 in 0.02 M sodium acetate bu¡er, pH 5.5) was added to previously dispense 15 mL of serum samples in a 96 well U-bottom microtitre plate (Tarson, Kolkata, India). Initial OD was taken at 450 nm immediately after adding the substrate and ¢nal OD was taken after1h incubation at 25 1C. Lyophilized hen egg white lysozyme, HEWL (Sigma) was used to develop a standard curve. Serum lysozyme values were expressed as microgram per millilitre equivalent of hen egg white lysozyme activity. Alternative complement activity (ACH50) Alternative complement activity was assayed following a previously described technique (Matsuyama,Tanaka, Nakao & Yano 1988; Yano 1992) with partial modi¢cations (Kumari & Sahoo 2005a) by using rabbit red blood cells (RaRBC), previously demonstrated as one of the better mammalian blood cells for acti-

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Disease resistance Asian cat¢sh (average weight 9.22 0.68 g) (juveniles from single parental stock) were utilized for studying the disease resistance (in both non-vaccinated and vaccinated ones) against a common freshwater ¢sh pathogen A. hydrophila, as described in our previous study (Kumari et al. 2003). Fifteen ¢sh kept in three tanks (¢ve ¢sh per replicate) for each group at a particular time period were vaccinated with A. hydrophila bacterin and fed the respective levamisolesupplemented diet for 10 days. Subsequently, at 0, 1, 2 and 3 weeks post-levamisole feeding, each ¢sh was injected intraperitoneally with 0.1mL PBS containing106 live cells of a 24 h culture in tryptone soya broth. Per cent survival was measured for 10 days based on our earlier observations that mortality reached its plateau after 1 week (Sahoo, Mukherjee & Sahoo 1998). The cause of mortality was con¢rmed by reisolating the organism from the kidney of 10% of dead ¢sh as described earlier (Kumari et al. 2003).

Statistical analysis All assays on individual ¢sh were performed in triplicate and data were represented as mean SE for




three sets of each experimental sub-groups. Cumulative per cent survival data were arcsine transformed before using analysis of variance (ANOVA). Two-way ANOVA was used to determine the interaction between dose and time period. Di¡erences between means were analysed by using one-way analysis followed by Duncan’s multiple range tests to determine signi¢cant di¡erences using Po0.05.

signi¢cantly (Po0.05) higher myeloperoxide content at 0,1and 2 weeks after levamisole feeding compared with control ¢sh, but returned back to normal at week 3 (Fig. 2). However, the ¢sh from groups C and D revealed increased peroxidase content after1and 2 weeks of levamisole feeding followed by a signi¢cant decline at week 3. The interaction between dose and time of levamisole feeding signi¢cantly a¡ected NBT and MPO activities (Table 1).

Results Serum lysozyme activity

Non-Speci¢c immune response NBT assay and MPO activity The e¡ect of dietary levamisole on the activity of neutrophilic leucocytes was measured by NBT and MPO assays. The ability of neutrophils to produce reactive oxygen radicals during respiratory burst activity, measured by the NBT assay, was found to be signi¢cantly (Po0.05) higher in group B ¢sh supplied with 50 mg levamisole kg 1 diet compared with control ¢sh. This activity reached its highest level at 3 weeks after levamisole feeding. On the other hand, there was a decrease in NBT activity in the groups of two higher doses of levamisole at di¡erent time periods in comparison with the control (Fig. 1). The release of MPO enzyme mostly by the azurophilic granules of neutrophils during oxidative respiratory burst activity, as measured by serum peroxidase content, was also signi¢cantly (Po0.05) a¡ected by the levamisole-supplemented diets (Fig. 2). Group B ¢sh showed

Serum lysozyme activity was not in£uenced by levamisole feeding at any measurement point. The lysozyme ranged between 13.71 and 18.24 mg mL 1. However, a signi¢cant reduction in lysozyme activity but not di¡erent from the control was noted after 3 weeks in ¢sh fed the highest dose of levamisole (group D) (13.71 mg mL 1) compared with the lowest dose (group B) ¢sh (17.47 mg mL 1). Alternative complement activity (ACH50) Serum complement activity measured as the mean number of ACH50 units per millilitre serum was low at week 0 in all the levamisole-fed groups compared with the control ¢sh, but showed signi¢cant increase in its activity at weeks 1 and 3, irrespective of dose levels of levamisole (Fig. 3). However, groups B and C had lower ACH50 activities at 2 weeks compared with the other two groups. The interaction between time 2.5

0.6 0.5 0.4

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0.3 0.2 0.1

Optical density at 450 nm

Optical density at 540 nm

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0 1 3 0 2 Week(s) after levamisole feeding

Figure 1 In£uence of di¡erent dietary doses of levamisole on nitroblue tetrazolium activity in Clarias batrachus at 0, 1, 2 and 3 weeks after 10 days of levamisole feeding. Data represent the mean SE (n 5 5). Statistical di¡erences (Po0.05) between control and levamisole-fed groups are indicated by letters (a, b, c) over the bars. (group A 5 0 mg, group B 5 50 mg, group C 5150 mg and group D 5 450 mg kg 1 feed).

0

1 2 3 Week(s) after levamisole feeding

Figure 2 In£uence of di¡erent dietary doses of levamisole on total myeloperoxidase content of Clarias batrachus at 0, 1, 2 and 3 weeks after levamisole feeding. Data represent the mean SE (n 5 5). Statistical di¡erences (Po0.05) between control and levamisole-fed groups are indicated by letters (a, b, c) over the bars (group A 5 0 mg, group B 5 50 mg, group C 5150 mg and group D 5 450 mg kg 1 feed).


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450

Parameter

Dose

Time interval

Dose Time interval

NBT activity Myeloperoxidase activity Lysozyme activity Alternative complement activity (ACH50) Haemagglutination titre Total protein Bacterial agglutination titre Vaccinated challenge Non-vaccinated challenge

NS

NS

NS

NS NS NS NS

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ACH50 (units/ml)

60

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40 30

b a bc c

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a

300 250

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200 150 100 50

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b bc

a c

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d

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However, feeding of graded levels of levamisole did not in£uence the total serum protein level (ranging between 4.46 and 5.92 g dL 1) compared with control (ranging between 4.59 and 5.1g dL 1).

c

20

Speci¢c immune response

10 0 0

1

2

3

Week(s) after levamisole feeding

Figure 3 In£uence of di¡erent dietary doses of levamisole on alternative complement activity (ACH50) of Clarias batrachus at 0, 1, 2 and 3 weeks after levamisole feeding. Data represent the mean SE (n 5 5). Statistical di¡erences (Po0.05) between control and levamisole-fed groups are indicated by letters (a, b, c, d) over the bars (group A 5 0 mg, group B 5 50 mg, group C 5150 mg and group D 5 450 mg kg 1 feed).

elapsed and dose of feeding levamisole signi¢cantly a¡ected serum complement activity (Table 1). Natural haemagglutination titre and total serum protein Dietary intake of levamisole also resulted in enhanced haemagglutination titre at all doses measured at any of the week intervals. Group B ¢sh showed the highest titre, 1 or 2 weeks after levamisole feeding (Fig. 4). The other two groups, C and D, showed a signi¢cant increase in HA titre at week 3.

504

B

Figure 4 E¡ect of di¡erent dietary doses of levamisole on haemagglutination titre of Clarias batrachus at 0, 1, 2 and 3 weeks after levamisole feeding. Data represent the mean SE (n 5 5). Statistical di¡erences (Po0.05) between control and levamisole-fed groups are indicated by letters (a, b, c, d) over the bars. (group A 5 0 mg, group B 5 50 mg, group C 5150 mg and group D 5 450 mg kg 1 feed). b

a

A

0 2 1 3 Weeks(s) after levamisole feeding

aa

50

400 350

0

Signi¢cance levels are Po0.05, Po0.01 and NS P40.05. NBT, nitroblue tetrazolium.

70

Haemagglutination titre

Table 1 Results of two-way analysis of variance (ANOVA) Two-way ANOVA1


Levamisole did not enhance the antibody production at any point of time compared with control (data ranging between 400.0 and 608.0), although a numerical rise in antibody titre was measured in all levamisole-fed groups (ranging between 597.33 and 1173.33). Disease resistance The cumulative per cent survival after challenge with A. hydrophila in both vaccinated and non-vaccinated ¢sh after10 days of levamisole feeding is shown in Figs. 5 and 6. Signi¢cantly lower survival was recorded in the control ¢sh (group A) than any other levamisole-fed ¢sh 1 week onwards. Higher survival (%) was recorded in vaccinated ¢sh immediately after 10 days of levamisole feeding compared with the control ¢sh. Subsequently, a signi¢cant increase in cumulative per cent survival was noticed in group B ¢sh after 1 week, whereas the highest dose group D ¢sh showed a signi¢cant decrease in per cent survival 2 weeks after levamisole feeding. After 3 weeks no signi¢cant change in per cent survival was recorded. The per cent survival in vaccinated ¢sh was



Survival (%)

90 80

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Figure 5 Survival (%) at 0,1, 2 and 3 weeks after 10 days of feeding diet with di¡erent levels of levamisole in nonvaccinated cat¢sh challenged intraperitoneally with Aeromonas hydrophila. Data represent the mean SE (n 5 5). Statistical di¡erences (Po0.05) between control and levamisole fed groups are indicated by letters (a,b,c) over the bars (group A 5 0 mg, group B 5 50 mg, group C 5150 mg and group D 5 450 mg kg 1 feed).

A

100

B D

C

80 Survival (%)

a a

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60

b

ab ab

a ab

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40

a a

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20 0

0


Figure 6 Survival (%) in vaccinated cat¢sh challenged intraperitoneally with Aeromonas hydrophila at 0, 1, 2 and 3 weeks after feeding with graded levels of levamisole for 10 days. Data represent the mean SE (n 5 5). Statistical di¡erences (Po0.05) between control and levamisole-fed groups are indicated by letters (a, b) over the bars (group A 5 0 mg, group B 5 50 mg, group C 5150 mg and group D 5 450 mg kg 1 feed).

quite high in comparison with non-vaccinated ¢sh, irrespective of levamisole feeding. Discussion Levamisole is a synthetic compound that is used as an antihelminthic agent in animals. A variety of immunomodulatory e¡ects of levamisole has been established in higher vertebrates. Among the most

widespread e¡ects seems to be enhancement of serum lysozyme activity, serum antibody titres after immunization, the number of leucocytes, phagocyte activities, the expression of cytokines by macrophages, lymphocyte proliferation and antitumour responses (Woods, Siegel & Chirigos 1974; Symoens & Rosenthal 1977; Moertal, Fleming, MacDonald, Haller, Laurie, Goodman, Ungerleider, Emerson, Tormey, Glick, Veeder & Mailliard 1990; Kimball, Schneider, Fisher & Clark 1992; Tempero, Haga, Sivinski, Birt, Klassen & Thiele 1995). Levamisole enhances the non-speci¢c/innate immune response as it does with the acquired response (acting as an adjuvant). Cells treated with levamisole exhibited enhanced cGMP levels, which in turn also increased microtubular assembly and cell mobility (Anderson, Glover, Koornhof & Rabson 1976). However, the mechanism by which levamisole stimulates the immune system cells is not well known. Fish culture is an important industry throughout the world, and antibiotics, vaccines and immunostimulants have been widely used as prophylactic measures to control disease in farmed ¢sh (Anderson 1992). Immunostimulants have received most of the attention during the last two decades because of little or no side e¡ects. Among these immunostimulants, levamisole has been shown to stimulate carp, Cyprinus carpio (Siwicki 1987, 1989; Baba, Watase & Yoshinaga 1993), rohu (Sahoo & Mukherjee 1999, 2001, 2002), rainbow trout, Oncorhynchus mykiss (Kajita, Sakai, Atsuta & Kobayashi 1990; Siwicki, Anderson & Rumsey 1994), gilthead seabream, Sparus aurata (Mulero, Esteban, Munoz et al.1998), Atlantic salmon, Salmo salar (Findlay & Munday 2000), hybrid striped bass (Li,Wang & Gatlin III 2004) and cobia, Rachycentron canadum (Leano, Guo, Chang & Liao 2004) immune responses and disease resistance. In the above-mentioned studies, both the innate and speci¢c humoral (complement and lysozyme activities as well as the speci¢c antibody titres) and cellular (phagocytic cell activities, non-speci¢c cytotoxic cell (NCC) activity and plaque forming cells) immune responses were enhanced after in vitro or in vivo levamisole treatments. Levamisole has also been indicated as a growth-promoting factor for sheep (Cabaj, Stankiewicz, Jomas & Moore 1995) and ¢sh (Siwicki & Korwing-Kossakowski 1988; Meseguer, Esteban & Mulero 1997; Mulero, Esteban & Meseguer 1998). Although positive e¡ects on the immune system have been demonstrated (depending on administration time and dosage), some authors have reported negative or immunosuppressive e¡ects (Anderson


505


et al. 1989; Siwicki et al. 1990; Mulero et al. 1998; Li et al. 2004). To our knowledge, no studies focus on the optimal dose, sustainable time period and e⁄cacy of levamisole in the commercially important Asian cat¢sh, C. batrachus. Therefore, the present work attempted to address this lack of information by describing the general action of levamisole on the innate and acquired immune system of Asian cat¢sh. Based on a previous study carried out in seabream (Mulero, Esteban, Munoz et al. 1998) and also taking the above points into consideration, we used levels of 50, 150 and 450 mg levamisole kg 1 feed and measured the immune response and disease resistance of cat¢sh 0, 1, 2 and 3 weeks after 10 days of levamisole feeding. Dietary levamisole caused a signi¢cant increase in superoxide production at the low dose (50 mg levamisole kg 1 diet) immediately after 10 days of levamisole feeding and it reached its maximum activity after 3 weeks. On the other hand, subsequent higher dose (i.e., 150 and 450 mg levamisole kg 1 diet) suppressed superoxide anion production immediately after the last levamisole feeding, but regained its normal value after 1 week. This result is in agreement with a previous in vitro study by Siwicki et al. (1990). The case regarding serum peroxidase content as measured through MPO activity was similar. At the low-dose level the MPO activity was signi¢cantly enhanced, which lasted up to 2 weeks post-levamisole feeding, but a moderate-to-high dose levamisole enhanced MPO activity up to a certain time period of 2 weeks, after which a clear reduction occurred at 3 weeks. The highest stimulatory index of MPO activity after 2 weeks post-levamisole feeding was found in group B ¢sh. The above results of the elevated respiratory burst and MPO activity are in agreement with previous studies carried out in Atlantic salmon (Findlay & Munday 2000), rohu (Sahoo & Mukherjee 2001) and Cyprinus carpio (Siwicki 1989). Lysozyme, known to be an important non-speci¢c immune mediator against parasitic, bacterial and viral infections, exhibits increased activity in ¢sh blood in response to infection. However, Fevolden, Roed and Gjerde (1994) discussed a negative correlation between elevated lysozyme and resistance of Atlantic salmon to two bacterial pathogens. In contrast to the previous studies, none of the di¡erent doses of levamisole a¡ected the lysozyme activity of Asian cat¢sh in the present study. On the other hand, the highest dose (group D) ¢sh showed a marked reduction in lysozyme activity after 3 weeks with respect to only the lowest dose (group B) ¢sh, but not di¡erent from

506


control. The alternative complement pathway considered as one of the main non-speci¢c immune responses was elevated at all the dose levels after 1 and 3 weeks post-levamisole feeding only, which is in agreement with the previous study done by Mulero, Esteban, Munoz et al. (1998). However, ACH50 level remained either low or at par to that of control ¢sh after 2 or 4 weeks post-levamisole feeding. Although many of the immunostimulants augment ACH50 activity (Kajita et al. 1990; Engstad, Robertsen & Frivold 1992, Matsuyama, Mangindaan & Yano 1992), each possess di¡erent temporal kinetics (Mulero, Esteban, Munoz et al. 1998; Peddie, Zou & Secombes 2002). Similarly, haemagglutination activity was also enhanced at all the doses of levamisole and its maximum activity was observed after 2 weeks postfeeding of 50 mg levamisole kg 1 feed for 10 days. Similar to the previous study carried out by Sahoo and Mukherjee (1999, 2001), total protein level remain unchanged irrespective of levamisole exposure, which might be indicative of the lower sensitivity of this test. Levamisole in a previous study was shown to be a stimulant of speci¢c immune response if given in appropriate doses when administered with or after the antigen (Siwicki 1989). The serum antibody titre showed an increase in antibody titre after levamisole feeding, although the di¡erences were not statistically signi¢cant from the control ¢sh. This may be due to high individual variation marked in this study and also observed in this species earlier (Kumari et al. 2003). The activation of the above-mentioned non-speci¢c immunological functions is associated with extracellular microbiocidal action, thus providing increased protection against virulent pathogens and infectious diseases, which was correlated in this study. Non-vaccinated levamisole-fed Asian cat¢sh showed higher resistance after experimental infection with A. hydrophila with cumulative % survival being the highest (70) in group C,1week after the last administration of levamisole, followed by group B (57) at the same time period. The same pattern was obtained after 2 weeks post-levamisole feeding. Survival in the control group was low (16^30%), and the di¡erence in survival was statistically signi¢cant after 1 and 2 weeks post-levamisole feeding. Similarly, in rohu, feeding of levamisole to healthy ¢sh signi¢cantly increased per cent survival to 75% as compared with 45% survival in control ¢sh after experimental challenge with A. hydrophila (Sahoo & Mukherjee 2001). Increased resistance was also observed after administration of immunostimulants in




gilthead sea bream challenged with Vibrio anguillarum (Mulero, Esteban, Munoz et al. 1998), common carp challenged with A. hydrophila (Baba et al. 1993) and brook trout challenged with A. salmonicida (Anderson, Siwicki & Rumsey 1995). On the other hand, in case of vaccinated and challenged cat¢sh, there was a signi¢cant increase in per cent survival (55.5^ 77.5%) at all three levels of levamisole-fed groups immediately after the last administration of levamisole and in group B after 1 week, compared with control groups (45^55%), thus providing additional protection to vaccinated ¢sh against A. hydrophila challenge. This increased protection against virulent pathogens was often correlated with the enhancement of the above immune parameters, as reported in other ¢sh species (Kajita et al. 1990; Nikl, Albright & Evelyn 1991; Anderson & Jeney 1992; Baba et al. 1993; Jeney & Anderson 1993; Anderson et al. 1995; Mulero, Esteban, Munoz et al. 1998). The above result con¢rms the immunomodulatory role of dietary levamisole at a low dose of 50 mg kg 1 feed, and subsequent higher doses show null or negative e¡ects on a few of the immune parameters. Several previous experiments have used much higher doses of dietary levamisole (250, 500 and 1000 mg levamisole kg 1 feed) for stimulating nonspeci¢c immune response (Mulero, Esteban, Munoz et al. 1998; Leano et al. 2004). On the other hand, Li et al. (2004) showed that 1000 mg levamisole kg 1 feed administered for 3 weeks suppressed growth, and 100 mg and 1000 mg mL 1 levamisole solution in vitro suppressed superoxide anion production after 24 h incubation. Similarly, in seabream (Anderson et al.1995), a level of 5 mg mL 1 levamisole in vitro enhanced NBT reduction, phagocytosis and adherence of spleen phagocytes, whereas higher concentrations (25 and 50 mg mL 1) for 48 h inhibited phagocytic functions. These con£icting results concerning the doses of levamisole required to enhance ¢sh immune functions and disease resistance may be explained by di¡erences in the method of levamisole administration, ¢sh age and immunological status or interspeci¢c variations (Mulero, Esteban, Munoz et al. 1998). To conclude, the above ¢ndings provide evidence that antihelminthic drug levamisole, when added to a ¢sh diet, activates several humoral and cellular innate immune responses of Asian cat¢sh, particularly at 50 mg kg 1 feed for10 days at a feeding rate of 3%. The stimulation e¡ect of the treatment was noticed 0^2 weeks post-administration of levamisole. Optimal doses, administration times and duration of induced protection have been established in an

attempt to provide a useful approach for protecting cultured cat¢sh against infectious/pathogenic agents and/or stressors. Furthermore, the prophylactic use of levamisole may be of value in situations known to result in stress and exposure to disease.

Acknowledgments The authors wish to thank the Director, Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, Bhubaneswar, India, for providing the requisite facilities and the Cat¢sh Unit of the institute for providing the ¢sh during the study.

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