Adjuvant and immunostimulatory effects of β-glucan administration in combination with lipopolysaccharide enhances survival and some immune parameters in carp challenged with Aeromonas hydrophila

Veterinary Immunology and Immunopathology 114 (2006) 15–24 www.elsevier.com/locate/vetimm

Adjuvant and immunostimulatory effects of b-glucan administration in combination with lipopolysaccharide enhances survival and some immune parameters in carp challenged with Aeromonas hydrophila V. Selvaraj a, K. Sampath b, Vaithilingam Sekar c,* a

Spic Research and Development, Spic Limited, Tuticorin 628005, Tamil Nadu, India b Department of Zoology, V.O.C. College, Tuticorin 628008, Tamil Nadu, India c 2901 Wessex Drive, Ames, IA 50014, USA

Received 22 March 2006; received in revised form 19 June 2006; accepted 23 June 2006

Abstract Combined effects of b-glucan and lipopolysaccharide (LPS) on survival and immune response were studied in Cyprinus carpio that were challenged with the pathogen Aeromonas hydrophila. b-Glucan from Saccharomyces cervisiae and LPS from a virulent strain of A. hydrophila were used in this study. Different concentrations of b-glucan + LPS mixture were administered on days 1, 7, and 14 through different routes (intraperitoneal injection, bathing, and oral administration). Control and test fish were challenged by intraperitoneal injection of LD50 concentration of A. hydrophila on day 16 and subsequently, mortality and relative percent survival (RPS) were recorded. Intraperitoneal injection elicited 100% RPS even at the lowest concentration (100 mg b-glucan + 10 mg LPS); whereas, oral administration improved RPS rate of carps at higher concentration (1% b-glucan + 0.25% LPS). Bathing did not improve the RPS. Test animals injected with even the minimum dose of the immunomodulators (100 mg b-glucan + 10 mg LPS/ fish) had a significant increase in total blood leukocyte counts and an increase in the proportion of neutrophils and monocytes. Superoxide anion production by macrophages was also elevated, which presumably aided the efficient killing of bacterial pathogen. Lower concentration of b-glucan + LPS had an adjuvant effect on antibody production as pretreatment by injection of 100 mg bglucan + 10 mg LPS/fish resulted in higher antibody titer against A. hydrophila following vaccination. RT-PCR analyses showed that the expression of interleukin-1b mRNA did not increase in test fish when compared with the control. Classical and alternative complement pathways were not affected by either the dose or the route of administration of the compounds. It may be concluded that intraperitoneal injection and oral administration, and not the bathing, of b-glucan + LPS mixture in carp could enhance resistance to challenge by A. hydrophila through changes in several non-specific and specific immune responses. # 2006 Elsevier B.V. All rights reserved. Keywords: b-Glucan; LPS; Macrophage; Phagocytosis; Opsonin; NBT assay; Complement; Interleukin-1b mRNA; Carp; Aeromonas hyrophila

1. Introduction Infectious diseases are a major problem in aquaculture causing heavy loss to fish farmers. Chemotherapy, vacci* Corresponding author. E-mail address: [email protected] (V. Sekar). 0165-2427/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2006.06.011

nation, and other prophylactic measures are generally adopted to control the infectious diseases. The use of chemotherapeutic agents (such as antibiotics) leads to development of drug resistance in organisms (Kawakami et al., 1997; Collado et al., 2000). Use of a vaccine triggers the production of specific antibody to only a particular pathogen and is often expensive (Robertsen

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et al., 1990) One of the most promising methods to controlling the disease in aquaculture is the strengthening the defense mechanism of fish through prior administration of immunostimulants. Currently, microbial polymers have been tested as immunomostimulants in various fish species (Robertsen, 1999). Among these, b-glucan and LPS has been studied extensively. For instance, it has been demonstrated that in vivo administration of bglucan and LPS independently can boost protection against several major fish pathogens such as Edwardsiella ictaluri, Vibrio anguillarum, V. salmonicida, Yersinia rukeri, Aeromonas bestiarum and A. hydrophila in different fish species such eels Anguilla japonica, channel catfish Ictalurus punctatus, Atalantic salmon Salmo salar (Robertsen, 1999; Guttvik et al., 2002) and carp Cyprinus carpio (Selvaraj et al., 2004, 2005, 2006). The observed enhanced resistance after the administration of b-glucan and LPS is mediated through the modulation of host defences by increasing total leucocytes, differential count (Kozinska and Guz, 2004; Selvaraj et al., 2004, 2005) and immune parameters such as bacterial killing activity, phagocytosis (Solem et al., 1995; Sakai, 1999; Robertsen, 1999; Dautremepuits et al., 2006), production of antimicrobial mediators including superoxide anion (Sakai, 1999; Robertsen, 1999), interleukin-1b (Secombes et al., 1998, 1999; Fujiki et al., 2000; Engelsma et al., 2001; Selvaraj et al., 2005, 2006), complement activity (Engstad et al., 1992; Verlhac et al., 1996) and also activation of lymphocytes (B cells) which produce specific antibody in brown trout, eels, catfish and carp (Ingram and Alexander, 1980; Salati et al., 1987; Chen and Ainsworth, 1992; Selvaraj et al., 2004, 2005). In addition to independent application, b-glucan has also been used with bacterial vaccine as adjuvant (Figueras et al., 1998) or with LPS as synergients (Cook et al., 2001) so as to increase the immune response and protection of fish against pathogens. In our previous studies, we have reported independent application of bglucan and LPS in carp elicit different immune response (Selvaraj et al., 2005, 2006). Intraperitoneal injection of b-glucan and LPS independently showed enhanced protection against pathogen A. hydrophila. In bath immunostimulation, LPS was found to provide better protection than b-glucan. In contrast to injection and bath immunostimulation, oral administration of bglucan and LPS independently did not show any enhanced protection when compared with control group. Even though injection is the best method, bathing and oral administration are the potentially effective alternative methods for injection as these methods are useful for mass administration of fish of all

size with lower handling stress and are safer and easier to administer. As no published information is available on adjuvant and synergistic effects of b-glucan with LPS on the immune response of carp by intraperitoneal, bathing, and oral administration in farmed fish species, we decided to study the synergistic and adjuvant effects of these immunostimulants on the immune response of carp when administered in combination. We also attempted to determine the effective route of administration (i.e., i.p. injection, bathing and oral) and dosage and to assess the impact of b-glucan + LPS administration on relative percent survival (RPS) of C. carpio challenged with the pathogen A. hydrophila. The RPS data obtained was correlated with hematological studies and functional assays performed under in vitro conditions. Bacterial killing, superoxide anion production, complement activity (classical and alternative pathway), specific immune response against an A. hydrophila vaccine, and level of expression of IL-1b mRNA were also studied. 2. Materials and methods 2.1. Experimental animals C. carpio were purchased from Manimutharu dam, Tirunelveli District, Tamil Nadu, India and transported to the laboratory in aerated plastic bags. The animals were allowed to acclimatize to laboratory conditions for 15 days prior to use in experiments. The weight of the animals ranged between 25 and 30 g. The experiments were carried out in glass tanks with 250 l capacity containing tap water at 30
2 8C. The water was changed on alternate days. The fish were fed with a pelleted diet containing 35% crude protein, which was prepared in our laboratory. 2.2. A. hydrophila The pathogen was isolated from infected C. carpi collected from Manimutharu dam as described (Shome and Shome, 1999). Based on the comparative biochemical tests, the isolated bacterium was identified as A. hydrophila. To determine the LD50 concentration of the pathogen, the bacterium was cultured in the laboratory in LB broth at 37 8C for 24 h and the cells were separated by centrifugation at 8000  g and to fulfill the Koch postulate, the pathogenecity test was conducted by intraperitoneal injection of 0.1 ml of live A. hydrophila at a concentration of 2.11  107 cfu ml1 in to the carp (size 25–30 g) at room temperature using PBS as control. The same types of clinical signs were observed after 2

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days of injection. A. hydrophila was then used to find out the LD50 concentration by following the method of (Saeed and Plumb, 1986). The LD50 concentration of pathogen A. hydrophila was 2.94  107 cfu ml1. 2.3. Preparation of b-glucan and LPS LPS was isolated and characterized from a virulent strain of A. hydrophila following published procedures (Tsai and Frasch, 1982; Kido et al., 1990). The characterized LPS were suspended in phosphate buffered saline (PBS), pH 7.2, and heat inactivated at 75 8C for 30 min to reduce its toxicity. b-Glucan was isolated from the yeast, Saccharomyces cerevisiae, and characterized (Northcote and Horne, 1951; Bacon et al., 1969). The characterized b-glucan was suspended in PBS and subjected to sonication to reduce its particle size. 2.4. Experimental design The experimental designs consist of three doses, 3 days, and three routes of administration (i.p. injection, bathing and oral). Thoroughly acclimated carp were taken from the stock and divided into four groups. Among the four groups, three groups were used for compounds administration and fourth group used for control. Each concentration and each mode of administration was tested with 24 fish. Experiments were conducted in duplicate sets, one set was used for challenge study and another for functional assay determination. 1. Fish were i.p. injected with 100 mg b-glucan + 10 mg LPS/fish or 500 mg b-glucan + 50 mg LPS/fish or 1000 mg b-glucan + 100 mg LPS/fish on days 1, 7 and 14. Control group received 0.1 ml of PBS on the same schedule. 2. Fish were bath immunostimulated in 100 l of wellaerated b-glucan + LPS solution-containing tank for about 90 min at the concentrations of 100 mg bglucan + 10 mg LPS ml1 or 500 mg b-glucan + 50 mg LPS ml1 or 1000 mg b-glucan + 100 mg LPS ml1 on days 1, 7 and 14. Control fish were immersed in tap water on the same schedule. 3. Fish received b-glucan + LPS containing pellet feed at the concentration of 0.1% b-glucan + 0.025% LPS or 0.5% b-glucan + 0.125% LPS or 1% b-glucan + 0.25% LPS twice a day at morning and evening on days 1, 7 and 14 during the rest of treatment periods the animals received normal pelleted diet. Control animals were received pellet diet without the test compounds.

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2.5. Challenge study All the 24 test and 24 control fish from one set of each group were challenged with 0.1 ml of LD50 concentration (2.94  107 cfu ml1) of A. hydrophila on day 16 by i.p. injection and mortality was recorded daily for 14 days and RPS was calculated as follows: mortality ð%Þ of untreated controls  mortality ð%Þ of treated  100 RPS ð%Þ ¼ mortality ð%Þ of untreated controls

2.6. In vitro assays Blood and kidney were sampled from b-glucan + LPS treated and control groups as described below: 1. Blood was sampled for hematology and complement assay on day 16 (6 animals from each group). 2. Kidneys were sampled for bacterial killing, NBT assay on day 16 (6 animals from each group). 3. Kidneys were sampled for IL I-b mRNA assay from 3 animals each on day 15 and 16 (6 animals of each group). 4. Blood was sampled for antibody determination on day 33 (6 animals of each group). 2.7. Hematology 2.7.1. Total leucocyte count (TLC) and differential count Total leucocyte count was made in a Neubauer counting chamber. Blood smears were stained with May-Grunwald/Giemsa and 100 leucocytes were counted under the microscope and the percentage of different types of leucocytes was calculated following staining (Schaperclaus et al., 1991). 2.7.2. Isolation of anterior kidney macrophages Macrophages were isolated from kidneys as described by (Braun-Nesje et al., 1981). Briefly, the kidneys of the fish were dissected out and a cell suspension was prepared by pressing the kidney with a glass rod through a stainless steel mesh (diameter 0.3 mm) in a plastic Petri dish on ice. Cells were suspended in L-15 (HI Media, India) supplemented with 0.33 mg ml1 glucose, 100 IU ml1 penicillin–streptomycin and 10% FCS. The medium was adjusted to pH 7.6 and sterilized by syringe filtration and 10 IU ml1 heparin (sterile) was added to the medium.

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Cell suspensions were loaded on to a discontinuous (densities 1.08 and 1.07) Percoll gradient (Sigma) and centrifuged for 40 min at 400  g at 4 8C (Hitachi). The macrophage enriched fraction was collected and the cell number counted in a hemocytometer. The cells were washed in L-15 medium twice, the supernatant discarded and the pellet resuspended in L-15 medium containing 10% FCS at a concentration of 1  106 cells ml1. 2.7.3. Bacterial killing assay Bacterial killing assay was performed as described (Chen and Ainsworth, 1992). Approximately 1  107 cfu ml1 of A. hydrophila was used as a stock. From this, 0.1 ml was taken and mixed with 0.1 ml of macrophage suspension (1  106 cell ml1) and 0.04 ml of pooled fresh carp serum collected from six animals was added, mixed well and incubated for 2 h with occasional shaking in a water bath at 27 8C. After 2 h, 0.1 ml of the bacteria–macrophage mixture was diluted with 9.9 ml of sterile distilled water to release living bacteria from phagocytes. This was serially diluted, plated on LB agar plates, incubated overnight at 37 8C and the number of colonies was counted. 2.7.4. Zymosan A activation Zymosan A activation was performed by the method of (Jorgensen and Robertsen, 1995). Zymosan A (Sigma) 100 mg ml1 was suspended in 0.9% NaCl, boiled in a water bath for 30 min, washed twice in PBS and opsonized by incubation with fresh carp serum (5 mg ml1) for about 1 h at 14 8C. The coated zymosan was washed twice in PBS and diluted to 10 mg ml1 in PBS and stored in small aliquots at 20 8C. 2.7.5. Oxygen burst activity assay Oxygen burst activity assay was performed as described (Chung and Secombes, 1988; Dalmo and Seljelid, 1995). From the macrophage suspension (1  106 cells ml1 in L-15 medium), 100 ml was placed into a 96-well microtiter polystyrene plate and allowed to adhere for 2 h at 18 8C. Non-adhered cells were removed by three washes with 100 ml/well of L-15 culture medium supplemented with 10% FCS. To the macrophage monolayer, 100 ml/well of NBT solution (1 mg ml1 L-15, 10% FCS) containing activated opsonized zymosan A at 500 mg ml1 was added. After 30 min incubation at 12 8C, the medium was removed and the culture was washed twice with isotonic PBS, fixed with 100 ml/well of 100% methanol for 3 min. Subsequently, the cells were washed twice with 70% methanol and air-dried. Formazan was solubilized

in 120 ml of KOH (2 M) and 120 ml of DMSO (100%) and the absorbance was read spectrophotometrically (Hitachi) at 620 nm using KOH/DMSO as blank. 2.7.6. RNA preparation The RNA was prepared from the macrophages of six animals (each day three animals) of each group by gradient centrifugation and washed twice with PBS at 400  g at 4 8C for 5 min; the macrophages pellet containing (1  106 cells ml1 in L-15 medium) was lysed with Trireagent (Sigma) and the RNA pellet was prepared according to the manufacturer’s protocol. Finally, the RNA pellet was dissolved in 30 mL of DEPC-treated water. The concentration of the total RNA was assayed spectrophotometrically at 260 nm (Hitachi). 2.7.7. Preparation of cDNA RNA (5 mg), 1 ml of oligo dT primer (Promega, USA), 1 ml of 10 mM dNTP mix and 10 ml of DEPC water were mixed and incubated at 65 8C for 5 min, placed on ice for 1 min and the pellet was collected by centrifugation at 8000  g. To this pellet, the following components were added: 4 ml of 5 first strand buffer, 1 ml of 0.1 M DTT and 1 ml of RNAsin (40 U/ml, Promega, USA) and mixed gently and incubated at 42 8C for 1 min and 1 ml of (200 U) of MMLV reverse transcriptase (Gibco BRL, USA) was added and incubated at 42 8C for 50 min. The reaction was terminated by incubation at 70 8C for 15 min and by placing the sample on ice for 1 min. The reaction mixture was saved at 20 8C until further use. 2.7.8. PCR assay The synthesized first strand cDNA 2 mg was taken from each sample. PCR reaction was conducted in 20 ml, which contained 1 ml of the first strand cDNA, 0.8 ml of 5 mM of specific forward and reverse primers, 0.4 ml of 10 mM dNTP mixture, 0.5 ml of Taq polymerase, 0.8 ml of 2 mM MgCl2 and 2 ml of 1 buffer and 13.7 ml of sterile Milli-Q water. PCR was performed in a thermocycler (Mini Cycler, MJ Research, USA) under the following conditions— 95 8C, 5 min: 40 cycles at (95 8C for 30 s, 50 8C for 30 s, 72 8C for 1 min) and a final extension at 72 8C for 5 min. Oligonuleotides used as PCR primers were as follows: carp 40S ribosomal protein S11 (433 bp) forward primer 50 -TACAGAACGAGAGGGCTTATC (13–33), reverse primer 50 -TTGGTGACCTTCAGGACATTG (425–445); carp IL-1b (574 bp) forward primer 50 -ACCACTGGATTTGTCAGAAG (76–96), reverse primer 50 -ACAGGGGAAGAACCTGTCATTT-

V. Selvaraj et al. / Veterinary Immunology and Immunopathology 114 (2006) 15–24

CAG (629–649). An aliquot (10 ml) of the PCR product was electrophoresed on a 1.5% agarose gel containing 0.5 mg ml1 of ethidium bromide photographed on a UV transilluminator and intensity of each band was quantified by alpha image documentation and analysis system (Alpha Innotech Corporation, USA). 2.7.9. Preparation and purification of immunoglobulin from carp anti-A. hydrophila serum Carp anti-A. hydrophila serum was prepared and the immunoglobulin was purified from the serum by following method of (Waterstrat et al., 1989; Selvaraj et al., 2005). After purification the purified fraction was tested for agglutination against A. hydrophila at a concentration of 2.4  107 cfu ml1. The agglutinated fraction was tested for purity check by SDS-PAGE by following method of (Laemmli, 1970). 2.7.10. Rabbit anti-carp Ig production Rabbit anti-carp immunoglobulin was prepared according to Murai et al. (1990) and Selvaraj et al. (2005). 2.7.11. Adjuvant effect of b-glucan + LPS on antibody response to A. hydrophila b-Glucan + LPS treated and control fish (six animals from each group) were vaccinated by i.p. with A. hydrophila vaccine in Freund’s incomplete adjuvant (FIA) (Sigma) on day 21 and the booster without FIA was given on day 28. On day 33, blood was drawn; the serum was separated and assayed for antibody titer by ELISA (Waterstrat et al., 1989; Selvaraj et al., 2005).

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2.8.3. Assay of alternative complement pathway (ACP) Alternative complement pathway activity was assayed according to available methods (Matsuyama et al., 1988; Yano et al., 1988). Briefly, 0.5 ml of serially 10-fold diluted carp serum in EGTA-Mg-GVB was placed in a set of test tubes and 0.2 ml of sheep red blood cells suspension (2  106 cells ml1) was added. This mixture was incubated at 15 8C for 90 min. Addition of 2.8 ml of 10 mM EDTA-GVB buffer stopped the hemolytic reaction. After centrifugation, the value y (percent hemolysis/100) was calculated from the OD at 414 nm of the supernatant. The value y/ (1  y) and the reciprocal of the serum dilution were plotted on log–log graph paper and the ACH 50 (units ml1) and the reciprocal dilution giving 50% hemolysis (y(1  y) = 1) were read from the graph. 2.9. Assay of classical complement pathway (CCP) 2.9.1. Carp anti-SRBC serum C. carpio (250–300 g) were intraperitoneally injected with 0.25 ml of sheep red blood cells at a concentration of 2  108 cells ml1 in PBS. One week later, a booster injection was given. After 5 days, blood was collected and the serum separated and carp antisheep red blood cells antibody was determined by heamagglutination using anti serum and sheep red blood cells and incubated the antiserum at 50 8C for 15 min to inactivate the complement (Sakai, 1992) and the CCP was assayed as described (Selvaraj et al., 2005). 2.10. Statistical analysis

2.8. Complements assay 2.8.1. Alternative complement pathway (ACP) activity Blood was collected from the caudal vessels of 6 animals of each group on day 16 and kept at 30 8C for 60 min and subsequently left in a refrigerator for 2 h. The fresh serum was separated by centrifugation at 400  g for 10 min and serum samples pooled, used for alternative complement assay. 2.8.2. Preparation of sheep red blood cells (SRBC) Sheep blood was mixed with an equal volume of Alsevers’s solution and stored at 4 8C. Subsequently, cells were centrifuged at 400  g for 5 min; the pellet of SRBC was washed twice in 10 mM EGTA-Mg-gelatin veronol buffer (GVB) and suspended in the same buffer at a concentration of 2  106 cells/ml for alternative complement pathway assay.

Data were analyzed by Student’s t-test. Results are expressed as mean
standard deviation (S.D.). Differences between compounds treated and control groups were considered statistically significant at p < 0.01 and <0.05. 3. Results 3.1. Relative percent survival (RPS) of carp challenged with A. hydrophila The mortality rates for control groups were 54%, 46% and 50% for i.p., bathing and oral administration, respectively. The RPS was significantly higher in i.p. injected groups (up to 100%) even at the lowest concentration, i.e. 100 mg b-glucan + 10 mg LPS/fish (Table 1). Whereas, in fish that received b-glucan + LPS mixture by oral route (0.5% b-glucan + 0.125% and 1%

1000 b-glucan + 100 LPS

14.2 – – – – – – 250  103
14.977

0.21
0.015

14.2 – – – – – – 240  103
20.132

0.21
0.01

7.1 – – – – – – 190  103
60.277

0.21
0.015 0.22
0.015 0.262
0.006*

b-glucan + 0.25% LPS), the survival rate was higher when compared with bath administration. Bathing route of administration did not increase in the survival rate compared with control group. 3.2. Total leucocyte count (TLC) and differential count b-Glucan + LPS treated fish showed significantly increased TLC, which was directly proportional to the doses of b-glucan + LPS injection. Among the leucocytes, neutrophils were predominant (t = 13.96, n = 6; p < 0.01) followed by monocytes (t = 3.68, n = 6; p < 0.05) (Table 1). Other leucocytes like eosinophils and basophils declined in number. The lymphocyte count did not vary much between the control and experimental groups. 3.3. Bacterial killing assay

0.278
0.009*

– – – – – – – 262  103
20.066

0.255
0.010

A. hydrophila was killed more efficiently by macrophages of fish given intraperitoneal injection and oral administration of b-glucan + LPS than macrophages of fish treated with b-glucan + LPS by bathing (Table 1). Bacterial count was significantly reduced in i.p. injected fish (t = 26.6, n = 6; p < 0.01) even at the lowest concentration (100 mg b-glucan + 10 mg LPS/fish) and in orally administered fish (t = 18.1, n = 6; p < 0.01). Bathing administration did not kill the bacteria significantly even at the highest concentration compared with control animals (t = 1.39, n = 6; p > 0.05).

*

Significant at 5% level;

**

significant at 1% level, n = 24 for RPS, n = 6 for all statistics.

0.21
0.01 0.38
0.015* 0.38
0.005* 0.27
0.005* 0.20
0.015

– – – – – – – 250  103
14.977 100 42.0
0.816** 45.4
0.471** 32.2
1.24* 23.0
1.24 0 0 31  102
4.163** 100 40.0
0.816** 42.3
1.247** 31.3
0.816* 24.0
4.32 0.3
0.46 2.3
0.47 35  102
3.511**

RPS TLC (103 mm1) Neutrophil (%) Monocytes (%) Lymphocyte (%) Basophil (%) Esosinophil (%) Bacterial killing assay (cfu ml1 live bacteria) NBT assay OD 620 nm 1  106 cell ml1

– 24
0.417 26.0
0.816 21.66
0.816 22.66
0.942 18.0
0.816 12.66
0.471 268  103
10.066

100 28.0
0.816 32.0
0.180* 27.0
0.180* 21.0
0.816 10.0
0.816 10.7
0.816 37  102
7.435**

28.5 – – – – – – 69  102
14.29**

50.0 – – – – – – 48  102
17.08**

71.4 – – – – – – 31  102
15.53**

100 b-glucan + 10 LPS Control 1 b-glucan + 0.250 LPS 0.5 b-glucan + 0.125 LPS 0.1b-glucan + 0.025 LPS Control 1000 b-glucan + 100 LPS 500 b-glucan + 50 LPS 100 b-glucan + 10 LPS PBS control

Oral administration of b-glucan + LPS (%) Injection of b-glucan + LPS (mg/fish) Functional assay

Table 1 Effect of b-glucan + LPS administration through injection and oral route on relative percent survival (RPS) and other parameters of carp

500 b-glucan + 50 LPS

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Bath administration of b-glucan + LPS (mg ml1)

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3.4. Superoxide anion production/NBT assay Fish treated with b-glucan + LPS by intraperitoneal injection at all doses showed significant increase (t = 6.085, n = 6; p < 0.05) in superoxide anion production compared with control animals. Oral administration also elicited enhanced superoxide anion production (t = 5.12, n = 6; p < 0.05). Bathing route of administration did not induce any change ( p > 0.05) in the superoxide anion production in all concentrations when compared with control (Table 1). 3.5. RT-PCR analysis RT-PCR analysis assay revealed that the expression of IL-1b mRNA in macrophages of b-glucan + LPS treated fish did not increase at 24 h (day 15) and 48 h (day 16) subsequent to third booster dose application when compared with control group (Fig. 1). The level of

V. Selvaraj et al. / Veterinary Immunology and Immunopathology 114 (2006) 15–24

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Fig. 1. RT-PCR analysis of IL-1b expression in carp kidney macrophages following stimulation with b-glucan + LPS by i.p. injection. Samples were taken for analysis after 24 and 48 h subsequent to third booster dose application. Amplified DNA products separated on agarose gels were visualized after ethidium bromide staining. (A) 24 h samples, IL-1b (lane 1 = non-template control; lane 2 = PBS control; lane 3 = 100 mg + 10 mg; lane 4 = 500 mg + 50 mg; lane 5 = 1000 mg + 100 mg; lane 6 = molecular weight marker). (B) 24 h samples, 40S ribosomal S11 (lane 1 = nontemplate control; lane 2 = PBS control; lane 3 = 100 mg + 10 mg; lane 4 = 500 mg + 50 mg; lane 5 = 1000 mg + 100 mg; lane 6 = molecular weight marker). (C) 48 h samples, IL-1b (lane 1 = non-template control; lane 2 = PBS control; lane 3 = 100 mg + 10 mg; lane 4 = 500 mg + 50 mg; lane 5 = 1000 mg + 100 mg; lane 6 = molecular weight marker). (D) 48 h samples, 40S ribosomal S11 (lane 1 = non-template control; lane 2 = PBS control; lane 3 = 100 mg + 10 mg; lane 4 = 500 mg + 50 mg; lane 5 = 1000 mg + 100 mg; lane 6 = molecular weight marker).

S11 mRNA (used as an internal control to confirm the total RNA concentration) was also almost the same in all samples. 3.6. Adjuvant effect of b-glucan + LPS on antibody production Fish injected (i.p.) with b-glucan + LPS showed higher antibody titer (when compared to the control group) following vaccination with A. hydrophila at

lower concentration rather than higher concentrations. The antibody titer of pooled (n = 6) normal control, PBS control and test animal injected with 100 mg bglucan + 10 mg LPS, 500 mg b-glucan + 50 mg LPS and 1000 mg b-glucan + 100 mg LPS were 13.33, 53.33, 853.33, 106.66 and 53.33, respectively. The antibody titer was almost similar in test and control fish in the bathing method. The antibody titer increased slightly in oral administration compared with bath method (Fig. 2). 3.7. Classical pathway The mean CH 50 unit ml1 of the control group was 40.3
0.40. There was no significant ( p > 0.05) difference in CH 50 unit ml1 between the sera of bglucan + LPS treated and control fish (data not shown). 3.8. Alternative complement pathway b-Glucan + LPS treatment through injection, bathing and oral route did not induce any change ( p > 0.05) in this activity, which for the control group was mean 553
40.8 ACH 50 unit ml1 (data not shown).

Fig. 2. Antibody titer of carp pretreated with different modes of administration of b-glucan + LPS. Injection (no. 1 = normal control without vaccination; no. 2 = control with vaccination; no. 3 = 100 mg b-glucan + 10 mg LPS; no. 4 = 500 mg b-glucan + 50 mg LPS; no. 5 = 1000 mg b-glucan + 100 mg LPS/fish), bathing (no. 1 = normal control without vaccination; no. 2 = control with vaccination; no. 3 = 100 mg b-glucan + 10 mg LPS; no. 4 = 500 mg b-glucan + 50 mg LPS; no. 5 = 1000 mg b-glucan + 100 mg LPS ml1), oral route (no. 1 = normal control without vaccination; no. 2 = control with vaccination; no. 3 = 0.1% b-glucan + 0.025% LPS; no. 4 = 0.5% b-glucan + 0.125% LPS; no. 5 = 1% b-glucan + 0.25 LPS%) following vaccination with A. hydrophila.

4. Discussion The present study revealed that intraperitoneal injection of b-glucan + LPS mixture even at the lowest concentration (100 mg b-glucan + 10 mg LPS/fish) gave 100% protection of C. carpio against an i.p. challenge with the pathogen A. hydrophila. Many previous workers have reported the increased protection of various fish against several pathogens when b-glucan and LPS was given alone by intraperitoneal injection

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(Saeed and Plumb, 1986; Robertsen et al., 1990; AlHarbi and Austin, 1992; Chen and Ainsworth, 1992; Selvaraj et al., 2004, 2005, 2006). However, oral administration of the mixture improved the survival rate only at higher concentrations (0.5% b-glucan + 0.125% LPS, and 1% b-glucan + 0.25% LPS). A similar result was also observed by Baulny et al. (1996) with oral administration of b-glucan along with the vaccine. Animals received the mixture through bathing did not show any significant change in the survival rate even at the highest concentration (1000 mg b-glucan + 100 mg LPS/ml). We observed that the protective effect of bglucan + LPS mixture was correlated with several functional assays under in vitro conditions. The total leucocytes number was increased in b-glucan + LPS injected fish. The highest leucocyte count was found at a concentration of 1000 mg b-glucan + 100 mg LPS/fish compared to control group. Among the leucocytes, neutrophils and monocytes had increased significantly. The present results are in accordance with the findings of others (Jorgensen and Robertsen, 1995; Kozinska and Guz, 2004; Selvaraj et al., 2004, 2005). The ability of macrophage to kill pathogenic microbes is probably one of the most important mechanisms of protection against disease among fish. The present study was performed with macrophages obtained from fish treated with bglucan + LPS mixture by different modes of administration. Intraperitoneal injection and oral administration of b-glucan + LPS mixture enhanced the superoxide anion production that may be involved in the destruction of A. hydrophila. This was correlated with bactericidal assay where the survival of A. hydrophila exposed to macrophages from fish pretreated with b-glucan + LPS mixture was dramatically reduced compared with the survival of the pathogen exposed to macrophages obtained from control fish. Cook et al. (2001) have tested to find out the synergistic effect of commercial glucan + LPS by incubating the macrophages with EcoActiva glucan with LPS in vitro and reported similar increased respiratory burst activity over LPS alone. Enhanced bacterial killing activity of b-glucan and LPS in independently treated fish has also been reported (Chen and Ainsworth, 1992; Jorgensen and Robertsen, 1995; Solem et al., 1995; Baulny et al., 1996; Selvaraj et al., 2005). It has been suggested that the observed enhanced resistance after the administration of b-glucan and LPS could be mediated through the modulation of host defences by increasing interleukin-1b (Secombes et al., 1998, 1999; Fujiki et al., 2000; Engelsma et al., 2001; Selvaraj et al., 2005, 2006). Hence, RT-PCR analysis

was performed to determine the effect, if any, on the expression of IL-1b in response to b-glucan + LPS mixture administration by injection. Our present findings indicate that IL-1b gene transcription was not increased by intraperitoneal injection of bglucan + LPS mixture. Previously, it has been reported that application of b-glucan and LPS independently increased the expression of IL-1b mRNA. Fujiki et al. (2000) and Selvaraj et al. (2005) have reported that IL1b expression in carp is induced in head kidney macrophages with the application of sodium alginate, scleroglucan, and yeast glucan. The trout and carp leucocytes and macrophages had enhanced expression of IL-1b subsequent to in vivo or in vitro administration of gram-negative bacteria or LPS (Secombes et al., 1998, 1999; Selvaraj et al., 2006). Our present findings suggest that the induction may be transient and hence, it is possible that the expression of the gene had peaked prior to the present sampling period. The present investigation shows that intraperitoneal injection of b-glucan + LPS mixture evoked higher antibody titers following vaccination. Similarly, Aakre et al. (1994) have reported that injection of A. salmonicida vaccine with yeast glucan into Atlantic salmon enhanced antibody response. This enhancement was seen only with the injection of minimum amount of b-glucan + LPS mixture; at higher concentrations the antibody response was almost like the control group. This may be because the LPS derived from any earlier A. hydrophila infections could have induced some antibody response prior to the vaccination. Hence, the vaccine might have complexed with the antibody. The complement, another component of the non-specific humoral immune response, was also studied in the present work. b-Glucan + LPS treatment through injection, bathing, and oral route did not induce any change in the classical as well as alternative pathways when compared with control animals. Baulny et al. (1996) and Robertsen (1999) have also noted that oral administration of b-glucan did not induce any change in complement pathway. However, Engstad et al. (1992) have reported a two- to five-fold enhanced complement activity in Atlantic salmon when b-glucan was administered by i.p. injection. Verlhac et al. (1996) have also observed an enhanced complement activity when b-glucan + Vitamin C was administered orally to trout. In the present study, intraperitoneal injection was found to be the most effective method as even the minimum dose of b-glucan + LPS mixture was sufficient to get 100% protection against the pathogen. This enhanced protection observed might be due to the

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adjuvant effect of available b-glucan with LPS, which could continuously induce the macrophage to elicit nonspecific cellular immune response. Acknowledgments The first author is grateful to SPIC Research and Development Department for permitting him to carryout this research program and he thanks Dr. R. Palaniappan, Joint General Manager of SPIC R&D for continuous encouragement. References Aakre, R., Wergeland, H.I., Aasjord, P.M., Endresen, C., 1994. Enhanced antibody response in Atlantic salmon (Salmo salar L.) to Aeromonas salmonicida cell wall antigens using a bacterin containing b-1, 3-M-Glucan as adjuvant. Fish Shellfish Immunol. 4, 47–61. Al-Harbi, A.H., Austin, B., 1992. Immune response of turbot, Scophthalmus maximus (L.), to lipopolysaccharide from a – pathogenic cytophaga – like bacterium. J. Fish Dis. 15, 449–452. Bacon, J.S.D., Farmer, V.C., Jones, D., Taylor, F.I., 1969. The glucan components of the cell wall of baker’s yeast (Saccharomyces cerevisiae) considered in relation to its ultrastructure. J. Biochem. 114, 557–569. Baulny, M.O.D., Quentel, C., Fournier, V., Lamour, F., Gouvello, R.L., 1996. Effect of long-term oral administration of b-glucan as an immunostimulant or an adjuvant on some non-specific parameters of the immune response of turbot Scophthalmus maximus. Dis. Aquat. Org. 26, 139–147. Braun-Nesje, R., Bertheussen, K., Kaplan, G., Seljelid, R., 1981. Salmonid macrophages: separation, in vitro culture and characterization. J. Fish Dis. 4, 141–151. Collado, R., Fouz, B., Sanjuan, E., Amaro, C., 2000. Effectiveness of different vaccine formulations against vibriosis caused by Vibrio vulnificus serovar E (biotype 2) in European eels Anguilla anguilla. Disease Aquatic Org. 91, 91–101. Chen, D., Ainsworth, A.J., 1992. Glucan administration potentiates immune defence mechanisms of channel catfish. Ictalurus punctalus Rafinesque. J. Fish Dis. 15, 295–304. Chung, S., Secombes, C.J., 1988. Analysis of events occurring within teleost marcophages during the respiratory burst. Comp. Biochem. Physiol. 34, 383–390. Cook, M.T., Hayball, P.J., Hutchinson, W., Nowak, B., Hayball, J., 2001. The efficacy of a commercial b-glucan preparation, EcoActiva, on stimulating respiratory burst activity of head-kidney macrophages from pink snapper (Pagrus auratuw), Sparidae. Fish Shellfish immunol. 11, 661–672. Dalmo, R.A., Seljelid, R., 1995. The immunomodulatory effect of LPS, laminaran and sulphated laminaran b-(1,3)-D-glucan on Atlantic salmon, Salmo salar L., macrophages in vitro. J. Fish Dis. 18, 175–185. Dautremepuits, C., Fortier, M., Croisetiere, S., Belhumeur, P., Fournier, M., 2006. Modulation of juvenile brook trout (Salvelinus fontinalis) cellular immune system after Aeromonas salmonicida challenge. Vet. Immunol. Immunopathol. 110, 27–36. Engelsma, M.Y., Stet, R.J.M., Schipper, H., Verburg-van Kemende, B.M.L., 2001. Regulation of interleukin 1 beta RNA expression in

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