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Dietary fucoidan enhance the non-specific immune response and disease resistance in African catfish, Clarias gariepinus, immunosuppressed by cadmium chloride
Accepted Manuscript Title: Dietary fucoidan enhance the non-specific immune response and disease resistance in African catfish, Clarias gariepinus immunosuppressed by cadmium chloride Author: Mohamed El-Boshy Ahmed El-Ashram Engy Risha Fatma Abdelhamid Eman Zahran Ali Gab-Alla PII: DOI: Reference:
S0165-2427(14)00226-8 http://dx.doi.org/doi:10.1016/j.vetimm.2014.10.001 VETIMM 9258
To appear in:
VETIMM
Received date: Revised date: Accepted date:
23-4-2014 28-9-2014 2-10-2014
Please cite this article as: El-Boshy, M., El-Ashram, A., Risha, E., Abdelhamid, F., Zahran, E., Gab-Alla, A.,Dietary fucoidan enhance the non-specific immune response and disease resistance in African catfish, Clarias gariepinus immunosuppressed by cadmium chloride, Veterinary Immunology and Immunopathology (2014), http://dx.doi.org/10.1016/j.vetimm.2014.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Dietary fucoidan enhance the non-specific immune response and disease resistance in African catfish, Clarias gariepinus immunosuppressed by cadmium chloride.
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Eman Zahran4 and Ali Gab-Alla5
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Mohamed El-Boshy1, 3*, Ahmed El-Ashram2, Engy Risha3, Fatma Abdelhamid3,
1. Department of Laboratory Medicine, Faculty of Applied Medical Science, Umm
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Al-Qura University. Makkah, Kingdom of Saudi Arabia. 2. Department of Fish
Diseases, Central Lab for Aquaculture Research (El-
Abbassa), Agriculture Research Center, Egypt.
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3. Department of Clinical Pathology, Faculty of Veterinary Medicine, Mansoura University. Egypt
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4. Department of Internal Medicine, Infections and Fish Diseases, Faculty of Veterinary Medicine, Mansoura University. Egypt
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5. Department of Biological Sciences, Faculty of Applied Sciences, Umm Al- Qura University, Makkah, Kingdom of Saudi Arabia
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* Corresponding author: El-Boshy, M. El-Sayed
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E-mail address: [email protected]
Abstract
Fucoidan is sulfated polysaccharide extracted from seaweed brown algae. This study was designed to evaluate the immunomodulatory effects and disease resistance of dietary fucoidan on catfish, Clarias gariepinus immunosuppressed by cadmium. Three hundred and sixty African catfish, Clarias gariepinus was allocated into six equal groups. The first group served as a control. Groups (F1 & F2) were fed on fucoidan supplemented ration at concentrations of 4 & 6 g/kg diet respectively for 21 days. Groups (Cd, CdF1 & CdF2) were subjected throughout
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the experiment to a sub-lethal concentration of 5 ppm cadmium chloride solution and groups (CdF1 & CdF2) were fed on a ration supplemented with fucoidan. Macrophages oxidative burst, phagocytic activity percentages and lymphocytes
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transformation index were a significant increase in the fucoidan-treated groups, (F1&F2) while serum lysozyme, nitric oxide and bactericidal activity were
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enhanced only in group (F2) when compared with controls. These parameters as
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well as absolute lymphocyte count and survival rate were significantly increased in group (CdF2) when compared with cadmium chloride immunosuppressed group
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(Cd). It could be concluded that the fucoidan can be used as immunostimulant for the farmed African catfish, Clarias gariepinus as it can improve its resistance to
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immunosuppressive stressful conditions.
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Key words: Dietary; Fucoidan; Immunomodulatory; Clarias gariepinus;
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Aeromonas hydrophila; Cadmium chloride.
1. Introduction
Using of natural immunostimulants in fish for the activation of non-specific immune response is promising to increase disease resistance (Traifalgar et al., 2013). The seaweed algae is wealthy with different minerals, vitamins, amino acids, alginic acid and fucoidan. Fucoidan is seaweed algal sulfated polysaccharide with a wide variety of biological activities including; detoxification of heavy metals (Davis et al., 2003), antiviral, antibacterial and antiparasitic action (Chotigeat et al., 2004; Immanuel et al., 2012; Sharma et al., 2014), antioxidant effect (Wang et al.,
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2010). Certainly, the importance of fucoidan in disease resistance and immunomodulatory has been highlighted in other farmed aquatic species such as shrimp (Deachamag et al., 2006; Immanuel et al., 2012;).
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Unfortunately, natural water reservoirs used for aquaculture are polluted with different environmental pollutants and contaminants (Zikic et al., 2001; Tawari-
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Fufeyinet al., 2008). Wastes pollutants, including industrial, agricultural and
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communal wastewater containing high levels of heavy metals like cadmium enter into water reservoirs without their prior treatment (Kumar et al., 2009). It has been
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proved to be toxic to fish at low concentrations in culture systems (Zikic et al., 2001; Kumar et al., 2009). Cadmium water pollution has been reported in different
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lakes in Egypt (El-Shehawi et al., 2007). Immunosuppressive effects of cadmium
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have been reported in Heteropneustes fossilis, common carp, catfish and rainbow
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trout by Radhakrishnan (2010); Basha and Rani (2003); Albergoni and Viola (1995a) and Zelikoff et al. (1995), respectively. The aim of this work was to study
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the immunomodulatory effect of dietary fucoidan in African catfish, Clarias gariepinus, immunosuppressed by cadmium chloride and to evaluate its diseasesresistance after challenge with a virulent strain of A. hydrophila.
2. Materials and Methods 2.1. Experimental Fish A total of 360 apparently healthy African catfish, Clarias gariepinus weighing 100120 g were obtained from the fish farm in Abbassa, Sharkia, Egypt. The fish were maintained in fiberglass tanks supplied with dechlorinated tap water with
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continuous aeration for one week under observation for acclimatization. The pH ranged from 6.9 to 7.5, pH, 7.6-7.9, total hardness, 115-120 mg/L (as CaCO3), and a temperature of 25-27 C was kept throughout the experiment.
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2.2. Rations The standard basal ration was formulated to meet the basic dietary requirements of
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catfish, according to Reis et al. (1989) and it was containing approximately 38% crude protein and crude fat 4%, in brief, 60.0% soybean meal, 26.5% corn grain,
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10.5% fish meal with 0.1 and 0.25 vitamins & mineral mixture respectively.
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Fucoidan, obtained from marine seaweed brown algae, Laminaria japonica (Alibaba, Co., Ltd), which is 95% composed of sulfated esters fucose with
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molecular weight 189 kD. The experimental diets were prepared by thoroughly mixing fucoidan 4 & 6 g/kg of the basal diet in Hobart mixer (D300-T, OH, USA)
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2.3. Pathogen
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and extruded out in 1.5 mm diameter pellets.
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A pure culture of virulent strain of A. hydrophila isolated from naturally infected catfish, Clarias
gariepinus and identified by performing the conventional
biochemical tests (API 20E). It was maintained in nutrient broth (Oxoid) for 24 h at 37 C. The concentration of bacteria was adjusted to 1 X107 CFU/ml by the optical density of the suspension.
2.4. Experimental Design A total number of 360 African catfish were randomly divided into six equal groups (60 fish). Each group consists of equal three replicate, each replicate was 20 in separate tanks. A control group (Cont) was fed on a normal ration. Groups
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(F1&F2) were fed on a diet containing fucoidan at concentrations of 4 & 6 g/kg ration respectively for 21 days. Groups (Cd, CdF1 & CdF2) were subjected throughout the experiment to a sub-lethal concentration of cadmium chloride 5 ppm
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solution (CdCl2 H2O, Sigma Co) according to Kumar et al. (2009) methodology,
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concentrations of 4 & 6 g/kg ration respectively.
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and CdF1 & CdF2 groups were fed on a ration supplemented with fucoidan at
2.5. Sample Collection
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Three replicate from each group (10 fish/ replicate), were randomly selected on the 21st day of the treatment period. Heparinized blood samples (approximately 3.0
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mL) were collected from the caudal vein. One half of each blood sample was
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immediately used for nitroblue tetrazolium (NBT) testing, lymphocyte-
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transformation assay, phagocytic activity, in addition to total and differential leukocyte count. The other half was left to coagulate and serum was separated for
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evaluation of lysozyme, nitric oxide, acid phosphatase and bactericidal activities.
2.6. Culture media
The culture media used in macrophage isolation were prepared according to Miller (1994). Briefly, equal portions of AIM-V (AlbuMAX, Gibco® Life Technologies, USA) and L-15 (Sigma-Chemical, Louis, USA) culture media, 0.05 mM of 2mercaptoethanol, 8% cell culture grade water (Sigma-Chemical)
and 0.09%
Na2HCO3 (Adwia Co. Egypt). Antibiotics, gentamicin 100 mg/mL, streptomycin
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100 mg/mL and penicillin 100 U/mL were added to the culture medium when
2.7. Activities of Macrophages and Lymphocytes 2.7.1. Isolation of head-kidney macrophage
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required.
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The head kidney was isolated according to Secombes (1990). In brief, 10 fish was
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sacrificed by administration of an overdose of anesthetic (3- amino benzoic acid ethyl ester). Head kidneys were removed, pooled, and forced through 100 mm
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nylon mesh with antibiotic free (af) culture media. Macrophage cells were layered onto 34/51% (v/v) Percoll (Sigma-Aldrich) and centrifuged at 400 g for 25 min at 4
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C. The cell layer at the interface was collected and adjusted to 106 cells/mL in af-
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culture media.
2.7.2. Macrophage Respiratory Burst Activity.
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The macrophage oxidative burst was assayed according to Rice et al. (1995). In
summary, 100 μL macrophage suspended cells were dispensed into 96 well plates. The cells were activated with 100 μL of culture media containing 1 mg/mL NBT
and 1 pg/mL Phorbol Myristate Acetate and incubated for 30 min at 27 C. The formazan dissolved by the addition of 140 μL of 2 M KOH and 120 μL diethyl sulphoxide (DMSO) and the optical density was measured at 620 nm using an ELISA reader. Controls were carried out with wells included macrophages and af-culture media alone.
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2.7.3 Macrophage Phagocytic Activity The phagocytic activity of macrophages was determined by the method of Sakai et al. (1995). Briefly, 1.0 mL of macrophages suspension was pipetted into sterile
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glass microscope slides and incubated at room temperature for 1 h. After that, 1.0 mL of a latex bead suspension (0.85 μm, 109 particles/mL, Sigma-Aldrich)
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supplemented with af-culture media was added to the slides and incubated for 2 h at
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room temperature. The slides were fixed with absolute methanol and stained by Giemsa’s method. Around 100 cells were counted microscopically. Phagocytic
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activity (PA) was determined by the following equation:
X100
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PA= Number of phagocytosing cells Number of total cells
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2.7.4. Lymphocytes Proliferation Assay
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This experiment was performed as the method of Guo et al. (2013). In summary, the whole blood samples diluted with an equal volume of culture media,
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then 2 mL were layered over 1 mL lymphocytes separating medium (histopaque1077). The lymphocytes were recovered from the interface after centrifuged at 400 g at -4C for 20 min. Lymphocyte concentration was adjusted to 2 X106 cells /mL after the viability of cells was checked (>95%) by the trypan blue exclusion (0.4%).
50 μL lymphocyte cell suspension and 100 μL culture medium (contain mitogen 50 μg/mL ConA) were added to duplicate wells of microtiter plate. Non- stimulatedcultures were also set without mitogen. The plate was incubated at 27 C with 5% CO2 for 48 hrs. Each culture was pulsed with 20 μL MTT (5 mg /mL) and 2 h later incubation, the culture was centrifuged at 1000g for 10 min. After carefully
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removed the supernatant, 200 μL of DMSO was added to each well. The cultures were read in a plate reader at OD 570 nm. The stimulation index values of
optical density of the same samples without mitogen. 2.8. Activities of Humoral Factors
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2.8.1. Bactericidal Activity.
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lymphocytes were; Mean optical density of mitoge stimulated samples / Mean
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The bactericidal activity of plasma was determined with modifications (Welker et al., 2007). Flat-bottom well was set up in duplicate with 100 μL of serum or Hank's
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balanced salt solution for control and incubated for 120 min with 50 μL suspensions of 24 h live A. hydrophila culture (1 X108 CFU/mL). Fifty μL
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diphenyltetrazolium bromide solution (MTT; 2 mg/mL) was pipetted into each well
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and incubated for 20 min at room temperature to allow the formation of formazan.
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The supernatant discarded and the formazan dissolved by the addition of 200 μL dimethyl sulfoxide (DMSO). The bactericidal activity was read at 560 nm with a
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microtiter plate reader and reporting as absorbance units after subtracting the absorbance of samples from that of controls.
2.8.2. Lysozyme Activity. Serum lysozyme activity was measured according to the method of Ellis (1990) with some modifications. A sample of 0.25 mL plasma was mixed with 0.75 mL Micrococcus lysodeikticus (Sigma Chemical Co) which was suspended in 0.05M
PBS, pH 6.2. The mixture reacted at 25 C for 5 min, and then the OD was measured at 1 min intervals for 5 min at 540 nm (5010, Photometer, BM Co. Germany).
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2.8.3. Nitric Oxide and Acid Phosphatase Activity The serum nitric oxide (NO) and acid phosphatase activity were assayed spectrophotometric using commercial test kits according to the enclosed pamphlet
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(Bio-Chain, Inc. USA).
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2.9. Total and Differential Leukocyte Count
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sample according to Stoskoph (1993).
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Total and differential leukocyte counts were counted in duplicate for each
2.10. Experimental Challenge
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At the end of the experiment, the fish in each experimental group was
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intraperitoneally injected with 200 µL of pathogenic A. hydrophila (0.5 X 108
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CFU/mL). Mortality was observed during the following 14 days post-inoculation. The dead and 20% moribund fish were subjected for bacterial re-isolation to
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confirm A. hydrophila as the cause of death. 2.11. Statistical Analysis The data among treatment groups were analyzed by one way analysis of variance (ANOVA) with post-hoc LSD multiple comparison test using SPSS software (ver. 18.00, USA) to find out the significant differences among treatment groups when P < 0.05. For data of mortalities, data were tested for normal distribution using Kolmogorov-Sminov test. The data were not normally distributed, therefore, Kaplan-Meier survival analyses curve were performed for estimation of survival probabilities with cadmium or death due to cadmium as the primary clinical
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endpoint.
The analyses were performed using GraphPad prism Version
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5.01(GraphPad Software Inc., USA).
3. Results and Discussion
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The current study evaluated the effects of cadmium on the non-specific immune
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response of catfish, Clarias gariepinus. Table 1 illustrates the toxicological effects of cadmium on the immune system, which shows evidence of impaired neutrophil
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function, macrophage respiratory burst and phagocytic activity. Phagocytosis and macrophage function is widely used to evaluate aquatic animal health status under
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different environmental stress (Zelikoff et al., 1995; Uribe et al., 2011). Cadmium
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exposure altered macrophage-mediated immune functions, including phagocytosis
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and free radical production in rainbow trout, dab while and common carp (Zelikoff et al., 1995; Hutchinson and Manning 1996; Ghiasi et al., 2012), respectively. It is
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known that the cadmium is a strong stressor to fish and the cortisol secreted during stress reactions, reduces the fish phagocytic activity (Ghiasi et al., 2012). Cortisol effects on immune cells begin with a marked increased expression of interleukin 1β and decreased stimulation of leukocyte function (Fast et al., 2008). Moreover, the bactericidal activity was suppressed with a significantly decreased level of serum lysozyme, nitric oxide and acid phosphatase (Table 2). Lysozyme has a bactericidal effect by destroying cellular walls of bacteria, stimulates phagocytic activity and participates in the regulation of immune cell differentiation and proliferation (Ghiasi et al., 2012). Head kidney lysozyme, liver acid phosphatase
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and serum lysozyme were remarkably reduced in Ictalurus melas, Cyprinus carpio and Clarias garipeinus after cadmium exposure (Albergoni and Viola 1995b; Sovenyi and Szakolczai 1993; El-Shehawi et al., 2007). The reduced bactericidal
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substances and lysozyme after cadmium exposure in fish could be attributed to reducing leukocytes count and function (Ghiasi et al., 2012). NO is an intracellular
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molecule synthesized by a nitric oxide synthases (NOS) and involved in the
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regulation of many cellular functions including mitochondrial metabolism and immune response (Ivanina, et al., 2010). They reported that, Cd exposure resulted
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in a strong reduction NO level as a result of inhibition of NOS activity in oyster gills. The effect of cadmium on total and differential leukocyte count is illustrated
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in Table 3, which shows a significant decrease of the total leukocytes, lymphocyte
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counts besides lymphocyte proliferation. Cadmium may bind to some lymphocyte
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membrane proteins, resulting in impaired of lymphocytic function and suppress the immune response (Albergoni and Viola 1995b). Similar results were observed in
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Heteropneustes fossilis and Cyprinus carpio after exposure to cadmium (Radhakrishnan 2010; Ghiasi et al., 2012). Elevation of the cortisol blood level, as a result of cadmium strong stressor to fish, reduces the life span of lymphocytes, enhances their apoptosis and suppresses their proliferation (Verburg-Van et al., 1999; Radhakrishnan 2010). Meanwhile, lower doses of cadmium enhanced macrophages oxidative burst in Ictalurus melas, total leukocyte count in Clarias gariepinus and leukocyte phagocytosis in Sparus aurata (Albergoni and Viola 1995b; Tawari-Fufeyin et al., 2008; Guardiola et al., 2013), respectively. They
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concluded that lower doses of cadmium enhance certain properties of macrophage function to produce more leukocytes in poisoned fish. Fish macrophages remove bacteria by the production, reactive oxygen species
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during a respiratory burst (Uribe et al., 2011).Table l, illustrates the macrophage oxidative burst and phagocytic activity percentages were enhanced in the fucoidan
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treated groups (F1&F2). Also, our results are in accordance with that carried out on
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shrimp (Chotigeat et al., 2004). Fucoidan enhances macrophage phagocytosis and lymphocyte activation, which is likely to activate a signaling pathway leading to
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enhanced interleukins (IL), IL-12 production from macrophages (Kawashima et al., 2012). Water-soluble polysaccharides extracted from seaweed brown algae, have
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been documented to have a potent mediator of respiratory bursts, phagocytes,
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lysozyme and bactericidal activities by inducing expression of IL-1, IL-6 and
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tumor necrosis factor receptors, which exert a major role in the induction of immune responses in turbot (Peddie et al., 2002), goldfish macrophages (Stafford et
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al., 2003), and shrimp (Kanjana et al., 2011). Moreover, expression of phagocytes peptide proteins was recorded on shrimp macrophages after vaccination with inactivated Vibrio harveyi and fucoidan supplement (Deachamag et al., 2006). The lymphocyte proliferation was significantly enhanced in (F1 &F2) group in comparison with the control group and this is consistent with findings in shrimp (Chotigeat et al., 2004). In-vitro fucoidan, enhanced lymphocyte blastogenesis and the esterase activity of lymphocyte tryptase, which is believed to stimulate lymphocyte proliferation (Hirayasu et al., 2005; Hayashi et al., 2008).
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Fish antimicrobial substances that include NO, reactive oxygen and lysozyme constitute the essential components of the humoral immune defense against invading pathogens (Uribe et al., 2011). The activities of humoral factors are
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summarized in Table2, in which, bactericidal activity, serum lysozyme and nitric oxide were significantly increased in (F2) group in comparison with the control
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group and this agree with the findings in shrimp and sea bass (Bagni et al., 2005;
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Traifalgar et al., 2013). They concluded, enhancement serum bactericidal activity was attributed to the elevated bactericidal substances such as lysozyme, NO and
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phagocyte oxidative burst. Lysozyme is a bacteriolytic enzyme and is a part of the nonspecific defense mechanism in most aquatic animals and the main sources of
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lysozyme are phagocytes (Uribe et al., 2011). Sulfate group in the fucoidan, is
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essential to binding macrophage cell surface receptors and plays a key role in the
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immune-regulating activity of macrophages (Teruya, et al., 2009). Fucoidan has been documented to stimulate NO production, in a macrophage cell line through
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membrane macrophage scavenger receptors and signaling pathway, in addition, enhances the NOS activity (Teruya, et al., 2009; Sharma et al., 2014). Free radicals as reactive oxygen species (ROS) disrupt the normal metabolism, including the immune system and incriminated in the pathogenesis of several toxicities including Cd exposed tilapia (Basha and Rani 2003), catfish (Kumar et al., 2009) and goldfish (Zikic et al., 2001). Cadmium could induce oxidative stress, through inhibiting the mitochondrial electron-transfer chain which enhances superoxide radical production and or interference with cellular antioxidant defense system (Sandrini et al., 2008). The fucoidan at high doses 6 gm/kg diet is effective
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in enhancing the cellular and humoral immunological parameters in cadmium exposed groups, when compared with the lower doses of fucoidan (Table 1,2). Fucoidan exhibited excellent scavenging capacities on ROS and showed a great
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potential for preventing free radical mediated diseases (Chotigeat et al., 2004; Wang et al., 2010). The fucoidan, alleviate the cadmium immunotoxicity in our
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work, could be due to its immune-stimulant action and or its antioxidant properties.
Dietary supplementation of fucoidan led to a reduction in mortalities after challenge
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with A. hydrophila when compared with the control group (Fig. 1). Fish fed fucoidan in F2 group has the greatest survival rate (80%) which significantly differs
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from cadmium exposed group (P= 0.0145). The Cd exposed groups and
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supplemented with two different levels of fucoidan (CdF1 & CdF2) significantly
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increased the survival rate (27% & 37%), respectively in comparison with Cd (7%) exposed group; however, neither of both groups were significantly different from
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each other nor from the control. Together, these data indicate that feed supplementation with fucoidan in the presence of cadmium exposure can enhance the survival rate near the level of the control. These results are consistent with those reported in shrimp challenged with white spot syndrome virus (Chotigeat et al., 2004) and V. harveyi (Traifalgar et al., 2013). Enhanced phagocytic activity, respiratory burst activity, serum lysozyme and nitric oxide were observed in the fucoidan treated groups. Also, the antimicrobial effect of fucoidan could be attributed to the negative charges of the sulfate group of the polysaccharide bind
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with positive charges of amino acids present in microorganism membrane, and thus preventing entry of the microbial agent into the host cell (Immanuel et al., 2012). 4. Conclusion
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In conclusion, feeding fucoidan enhanced the humoral and cellular immunity of African catfish, Clarias gariepinus. Moreover, fucoidan seems to be a promising
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candidate for increasing the resistance against bacterial infections under the
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conditions of heavy metal pollution.
All authors have none to declare.
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Ictalurus punctatus following acute exposure to tributyltin. Archives of
Protein-to-energy ratios in
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production diets and growth, feed conversion and body composition of channel
an
catfish, ictalurus punctatus. Aquaculture, 77, 21-27.
Sakai, M., Kobayashi, M., Yoshida, T., 1995. Activation of rainbow trout,
M
Oncorhynchus mykiss, phagocytic cells by administration of bovine lactoferrin. Comparative Biochemistry and Physiology 110, 755-759.
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Sandrini, J.Z., Juliane, V.L, Francesco, R., Fattorini, D., Nottic, A., Marins, L.F.,
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Jose' Man'a, M., 2008. Antioxidant responses in the nereidid Laeonereis acuta
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(Annelida, polychaeta) after cadmium exposure. Ecotoxicology and Environmental Safety 70, 115-120.
Secombes, C.J., 1990. Isolation of salmonid macrophages and analysis of their killing activity. In: Techniques in Fish Immunology, Vol. 1 (ed. by J.S. Stolen, T.C. Fletcher, D.P. Anderson & B.S. Roberson), pp. 137-163. SOS Publications, Fair Haven, New Jersey.
Sharma, G., Susanta, K., Writoban, B.B., Kuntal, G., Pijush, K.D., 2014. The curative effect of fucoidan on visceral leishmaniasis is mediated by activation of
Page 19 of 27
MAP kinases through specific protein kinase C isoforms. Cellular & Molecular Immunology, doi:10.1038/cmi.68 Sovenyi, J., Szakolczai J., 1993. Studies on the toxic and immunosuppressive
ip t
effects of cadmium on the common carp. Acta Veterinaria Hungarica 41,415426.
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Stafford, J.L., Ellestad, K.K., Maggot, K.E., Belosevic, M. Magor, B.G., 2003. A toll-like receptor (TLR) gene is up-regulated in activated goldfish macrophages.
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Dev Comp Immunol 27, 685-698.
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Stoskoph, M., 1993. Fish Medicine. W.B. Saunders Company, PP128-129. Tawari-Fufeyin, P., Igetei, J., Okoidigun, M.E., 2008. Changes in the catfish
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(Clarias gariepinus) exposed to acute cadmium and lead poisoning. Bioscience Research Communication 20, 271-276.
te
d
Teruya, T., Takemoto, H., Konishi, T., Tako., 2009. Structural characteristics and in vitro macrophage activation of acetyl fucoidan from Cladosiphon okamuranus.
Ac ce p
Glycoconjugate J., 26, 1019-1028. Traifalgar, R.F.M., Corre, V.L., Serrano, A.E., 2013. Efficacy of dietary immunostimulants to enhance the immunological responses and vibriosis resistance of Juvenile Penaeus monodon. Journal of Fisheries and Aquatic Science 8, 340-
354.
Uribe, C., Folch, H., Enriquez, R., and Moran, G, 2011. Innate and adaptive immunity in teleost fish: a review. Veterinarni Medicina, 56, 486-503 Verburg-Van Kemenade, B.M.L., Nowak, B., Eingelsma, M.Y., Wyets, F.A.A., 1999. Differential effects of cortisol on apoptosis and proliferation of Carp B
Page 20 of 27
lymphocytes from head kidney, spleen and blood. Fish and Shellfish Immunology 9, 405-415. Wang, J., Zhang, Q., Zhanga, Z., Song, H., Li, P., 2010. Potential antioxidant and
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anticoagulant capacity of low molecular weight fucoidan fractions extracted from
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Laminaria japonica. International Journal of Biological Macromolecules 46, 6-12. Welker, T.L, Chhorn, L., Mediha, Y.A., Klesius, H.P., 2007. Growth, immune
us
function, and disease and stress resistance of juvenile Nile tilapia (Oreochromis
an
niloticus) fed graded levels of bovine lactoferrin. Aquaculture 262, 156-162. Zelikoff, J.T., Bowser, D., Squibb, K.S., Frenkel K., 1995. Immunotoxicity of low cadmium
exposure
in
fish:
an
alternative
animal
model
for
M
level
235-248.
d
immunotoxicological studies. Journal of Toxicology and Environmental Health 45,
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Zikic, R.V., Stajn, A.S. Pavlovic S.Z., Ognjanovic, B.I., Saicic, Z.S., 2001.
Ac ce p
Activities of superoxide dismutase and catalase in erythrocytes and plasma transaminases of goldfish (Carassius auratus gibelio Bloch.) exposed to cadmium. Physiological Research 50, 105-111.
Table 1. Effect of dietary treatment fucoidan on selected cellular immunological parameters in catfish, Clarias gariepinus after immunosuppressive with
cadmium chloride for 21 days. Table 2. Effect of dietary treatment fucoidan on selected humoral immunological
parameters,
in
catfish,
Clarias
gariepinus
after
immunosuppressive with cadmium chloride for 21 days.
Page 21 of 27
Table 3. Total and differential leukocyte count (mean ± S.E) in catfish, Clarias gariepinus
fed
on a fucoidan
supplement
diet
for 21 days after
immunosuppression induced by cadmium chloride.
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Fig. 1. Cumulative mortality (%) of African catfish, Clarias gariepinus fed fucoidan supplemented diets for 21 days and challenged with pathogenic A.
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te
d
M
an
us
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hydrophila for 14 days.
Page 22 of 27
Table 1. Effect of dietary treatment fucoidan on selected cellular immunological parameters in
Neutrophils NBT assay (OD) unit
0.98
c
±0.08 Macrophage respiratory burst index
7.15c
±0.09
1.01
Cd
c
CdF1
CdF2
ab
0.78b
±0.06
±0.08
±0.07
0.51
a
±0.12
0.62
9.18d
9.86d
3.48a
5.02b
6.84c
±0.26
±0.35
±0.15
±0.12
±0.22
28.2c
38.1d
40.1d
15.1a
21.2b
27.4c
d
±1.85
±2.95
±1.45
±1. 01
±2.12
1.72c
1.75c
2.15c
0.61a
0.98b
1.01b
±0.15
±0. 11
±0.19
±0.05
±0. 06
±0. 09
1.66b
1.95c
2.64d
1.05a
1.25a
1.58b
±0.09
± 0.12
± 0.18 ±0.08
± 0.14
± 0.11
Phagocytic activity %
Ac ce p
te
±1.15
Phagocytic index
0.96
F2 c
M
±0.18
F1
us
Contro
cr
Groups
an
Cellular immune response
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catfish, Clarias gariepinus after immunosuppressive with cadmium chloride for 21 days.
Lymphocyte transformation index
( F1= fucoidan 4 g, F2= fucoidan 6 g , Cd= cadmium). The rows with the same superscript are not statistically significant at P<0.05. (Values are mean ± SE) (n = 10).
Page 23 of 27
Table 2. Effect of dietary treatment fucoidan on selected humoral immunological parameters, in
F1
1.15b
1.32b
1.58 c
±0.09
±0.10
±0.08
9.15c
( U/mL)
±0.31
te 1.12b
Ac ce p
Serum nitric oxide
11.05d 12.31e
d
Serum lysozyme
F2
cr
Cd
CdF1
CdF2
0.72a
0.84a
1.12b
±0.05
±0.08
±0.11
6.15a
7.10b
8.84c
an
Control
M
Bactericidal activity unit
Groups
us
Humoral immune response
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catfish, Clarias gariepinus after immunosuppressive with cadmium chloride for 21 days.
±0.38
±0.45
±0.25
±0.32
±0.48
1.48c
1.51c
0.66a
0.89b
1.08b
(Mmol/mL)
±0.11
±0.10
±0.15
±0.07
±0.09
±0.12
Serum acid phosphatase
1.85c
1.92c
1.95c
0.91a
1.05a
1.38b
U/L
±0.14
±0.11
±0.18
±0.09
±0.10
±0.12
(F1= fucoidan 4 g, F2= fucoidan 6 g , Cd= cadmium). The rows with the same superscript are not statistically significant at P<0.05. (Values are mean ± SE) (n = 10).
Page 24 of 27
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Table 3. Total and differential leukocyte count (mean ± S.E) in catfish, Clarias gariepinus fed on
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a fucoidan supplement diet for 21 days after immunosuppression induced by cadmium
us
chloride. Leukogram
F1
F2
Cd
CdF1
CdF2
Total Leukocytes Count
32.15c
33.21c
34.35c
20.52a
24.54b
26.75 b
(Thousand/ µL)
±2.51
±2.45
±2.62
±1.18
±1.32
±1.95
Neutrophils
10.15a
10.32a
10.12a
8.24a
8.41a
9.14a
±0.92
±1.10
±0.98
±0.78
±0.75
±0.81
Esinophils
0.52b
0.59 b
0.48b
0.24a
0.32a
0.30a
(Thousand/µL)
±0.04
±0.06
±0.05
±0.04
±0.05
±0.03
Basophils
0.022
0
0
0
0.020
0
(Thousand/µL)
±0.02
Lymphocytes
19.95c
20.84c
22.18c
10.41a
14.25b
15.82b
(Thousand/µL)
±1.42
±1.55
±1.64
±1.02
±1.12
±1.18
Monocytes
1.52a
1.45a
1.55a
1.61a
1.56a
1.48a
Ac ce p
(Thousand/ µL)
te
d
M
Control
an
Groups
±0.02
Page 25 of 27
(Thousand/µL)
±0.16
±0.12
±0.17
±0.21
±0.15
±0.10
( F1= fucoidan 4 g, F2= fucoidan 6 g , Cd= cadmium). The rows with the same superscript are not statistically significant at P<0.05. (Values are
Ac ce p
te
d
M
an
us
cr
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mean ± SE) (n = 10).
Page 26 of 27
ip t cr us an M d te Ac ce p Fig. 1. Cumulative mortality (%) of African catfish, Clarias gariepinus fed fucoidan supplements, diet for 21 days and challenged with pathogenic A. hydrophila for 14 days .
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S0165-2427(14)00226-8 http://dx.doi.org/doi:10.1016/j.vetimm.2014.10.001 VETIMM 9258
To appear in:
VETIMM
Received date: Revised date: Accepted date:
23-4-2014 28-9-2014 2-10-2014
Please cite this article as: El-Boshy, M., El-Ashram, A., Risha, E., Abdelhamid, F., Zahran, E., Gab-Alla, A.,Dietary fucoidan enhance the non-specific immune response and disease resistance in African catfish, Clarias gariepinus immunosuppressed by cadmium chloride, Veterinary Immunology and Immunopathology (2014), http://dx.doi.org/10.1016/j.vetimm.2014.10.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Dietary fucoidan enhance the non-specific immune response and disease resistance in African catfish, Clarias gariepinus immunosuppressed by cadmium chloride.
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Eman Zahran4 and Ali Gab-Alla5
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Mohamed El-Boshy1, 3*, Ahmed El-Ashram2, Engy Risha3, Fatma Abdelhamid3,
1. Department of Laboratory Medicine, Faculty of Applied Medical Science, Umm
us
Al-Qura University. Makkah, Kingdom of Saudi Arabia. 2. Department of Fish
Diseases, Central Lab for Aquaculture Research (El-
Abbassa), Agriculture Research Center, Egypt.
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3. Department of Clinical Pathology, Faculty of Veterinary Medicine, Mansoura University. Egypt
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4. Department of Internal Medicine, Infections and Fish Diseases, Faculty of Veterinary Medicine, Mansoura University. Egypt
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5. Department of Biological Sciences, Faculty of Applied Sciences, Umm Al- Qura University, Makkah, Kingdom of Saudi Arabia
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* Corresponding author: El-Boshy, M. El-Sayed
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E-mail address: [email protected]
Abstract
Fucoidan is sulfated polysaccharide extracted from seaweed brown algae. This study was designed to evaluate the immunomodulatory effects and disease resistance of dietary fucoidan on catfish, Clarias gariepinus immunosuppressed by cadmium. Three hundred and sixty African catfish, Clarias gariepinus was allocated into six equal groups. The first group served as a control. Groups (F1 & F2) were fed on fucoidan supplemented ration at concentrations of 4 & 6 g/kg diet respectively for 21 days. Groups (Cd, CdF1 & CdF2) were subjected throughout
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the experiment to a sub-lethal concentration of 5 ppm cadmium chloride solution and groups (CdF1 & CdF2) were fed on a ration supplemented with fucoidan. Macrophages oxidative burst, phagocytic activity percentages and lymphocytes
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transformation index were a significant increase in the fucoidan-treated groups, (F1&F2) while serum lysozyme, nitric oxide and bactericidal activity were
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enhanced only in group (F2) when compared with controls. These parameters as
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well as absolute lymphocyte count and survival rate were significantly increased in group (CdF2) when compared with cadmium chloride immunosuppressed group
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(Cd). It could be concluded that the fucoidan can be used as immunostimulant for the farmed African catfish, Clarias gariepinus as it can improve its resistance to
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immunosuppressive stressful conditions.
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Key words: Dietary; Fucoidan; Immunomodulatory; Clarias gariepinus;
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Aeromonas hydrophila; Cadmium chloride.
1. Introduction
Using of natural immunostimulants in fish for the activation of non-specific immune response is promising to increase disease resistance (Traifalgar et al., 2013). The seaweed algae is wealthy with different minerals, vitamins, amino acids, alginic acid and fucoidan. Fucoidan is seaweed algal sulfated polysaccharide with a wide variety of biological activities including; detoxification of heavy metals (Davis et al., 2003), antiviral, antibacterial and antiparasitic action (Chotigeat et al., 2004; Immanuel et al., 2012; Sharma et al., 2014), antioxidant effect (Wang et al.,
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2010). Certainly, the importance of fucoidan in disease resistance and immunomodulatory has been highlighted in other farmed aquatic species such as shrimp (Deachamag et al., 2006; Immanuel et al., 2012;).
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Unfortunately, natural water reservoirs used for aquaculture are polluted with different environmental pollutants and contaminants (Zikic et al., 2001; Tawari-
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Fufeyinet al., 2008). Wastes pollutants, including industrial, agricultural and
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communal wastewater containing high levels of heavy metals like cadmium enter into water reservoirs without their prior treatment (Kumar et al., 2009). It has been
an
proved to be toxic to fish at low concentrations in culture systems (Zikic et al., 2001; Kumar et al., 2009). Cadmium water pollution has been reported in different
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lakes in Egypt (El-Shehawi et al., 2007). Immunosuppressive effects of cadmium
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have been reported in Heteropneustes fossilis, common carp, catfish and rainbow
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trout by Radhakrishnan (2010); Basha and Rani (2003); Albergoni and Viola (1995a) and Zelikoff et al. (1995), respectively. The aim of this work was to study
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the immunomodulatory effect of dietary fucoidan in African catfish, Clarias gariepinus, immunosuppressed by cadmium chloride and to evaluate its diseasesresistance after challenge with a virulent strain of A. hydrophila.
2. Materials and Methods 2.1. Experimental Fish A total of 360 apparently healthy African catfish, Clarias gariepinus weighing 100120 g were obtained from the fish farm in Abbassa, Sharkia, Egypt. The fish were maintained in fiberglass tanks supplied with dechlorinated tap water with
Page 3 of 27
continuous aeration for one week under observation for acclimatization. The pH ranged from 6.9 to 7.5, pH, 7.6-7.9, total hardness, 115-120 mg/L (as CaCO3), and a temperature of 25-27 C was kept throughout the experiment.
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2.2. Rations The standard basal ration was formulated to meet the basic dietary requirements of
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catfish, according to Reis et al. (1989) and it was containing approximately 38% crude protein and crude fat 4%, in brief, 60.0% soybean meal, 26.5% corn grain,
us
10.5% fish meal with 0.1 and 0.25 vitamins & mineral mixture respectively.
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Fucoidan, obtained from marine seaweed brown algae, Laminaria japonica (Alibaba, Co., Ltd), which is 95% composed of sulfated esters fucose with
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molecular weight 189 kD. The experimental diets were prepared by thoroughly mixing fucoidan 4 & 6 g/kg of the basal diet in Hobart mixer (D300-T, OH, USA)
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2.3. Pathogen
d
and extruded out in 1.5 mm diameter pellets.
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A pure culture of virulent strain of A. hydrophila isolated from naturally infected catfish, Clarias
gariepinus and identified by performing the conventional
biochemical tests (API 20E). It was maintained in nutrient broth (Oxoid) for 24 h at 37 C. The concentration of bacteria was adjusted to 1 X107 CFU/ml by the optical density of the suspension.
2.4. Experimental Design A total number of 360 African catfish were randomly divided into six equal groups (60 fish). Each group consists of equal three replicate, each replicate was 20 in separate tanks. A control group (Cont) was fed on a normal ration. Groups
Page 4 of 27
(F1&F2) were fed on a diet containing fucoidan at concentrations of 4 & 6 g/kg ration respectively for 21 days. Groups (Cd, CdF1 & CdF2) were subjected throughout the experiment to a sub-lethal concentration of cadmium chloride 5 ppm
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solution (CdCl2 H2O, Sigma Co) according to Kumar et al. (2009) methodology,
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concentrations of 4 & 6 g/kg ration respectively.
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and CdF1 & CdF2 groups were fed on a ration supplemented with fucoidan at
2.5. Sample Collection
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Three replicate from each group (10 fish/ replicate), were randomly selected on the 21st day of the treatment period. Heparinized blood samples (approximately 3.0
M
mL) were collected from the caudal vein. One half of each blood sample was
d
immediately used for nitroblue tetrazolium (NBT) testing, lymphocyte-
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transformation assay, phagocytic activity, in addition to total and differential leukocyte count. The other half was left to coagulate and serum was separated for
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evaluation of lysozyme, nitric oxide, acid phosphatase and bactericidal activities.
2.6. Culture media
The culture media used in macrophage isolation were prepared according to Miller (1994). Briefly, equal portions of AIM-V (AlbuMAX, Gibco® Life Technologies, USA) and L-15 (Sigma-Chemical, Louis, USA) culture media, 0.05 mM of 2mercaptoethanol, 8% cell culture grade water (Sigma-Chemical)
and 0.09%
Na2HCO3 (Adwia Co. Egypt). Antibiotics, gentamicin 100 mg/mL, streptomycin
Page 5 of 27
100 mg/mL and penicillin 100 U/mL were added to the culture medium when
2.7. Activities of Macrophages and Lymphocytes 2.7.1. Isolation of head-kidney macrophage
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required.
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The head kidney was isolated according to Secombes (1990). In brief, 10 fish was
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sacrificed by administration of an overdose of anesthetic (3- amino benzoic acid ethyl ester). Head kidneys were removed, pooled, and forced through 100 mm
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nylon mesh with antibiotic free (af) culture media. Macrophage cells were layered onto 34/51% (v/v) Percoll (Sigma-Aldrich) and centrifuged at 400 g for 25 min at 4
M
C. The cell layer at the interface was collected and adjusted to 106 cells/mL in af-
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culture media.
2.7.2. Macrophage Respiratory Burst Activity.
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The macrophage oxidative burst was assayed according to Rice et al. (1995). In
summary, 100 μL macrophage suspended cells were dispensed into 96 well plates. The cells were activated with 100 μL of culture media containing 1 mg/mL NBT
and 1 pg/mL Phorbol Myristate Acetate and incubated for 30 min at 27 C. The formazan dissolved by the addition of 140 μL of 2 M KOH and 120 μL diethyl sulphoxide (DMSO) and the optical density was measured at 620 nm using an ELISA reader. Controls were carried out with wells included macrophages and af-culture media alone.
Page 6 of 27
2.7.3 Macrophage Phagocytic Activity The phagocytic activity of macrophages was determined by the method of Sakai et al. (1995). Briefly, 1.0 mL of macrophages suspension was pipetted into sterile
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glass microscope slides and incubated at room temperature for 1 h. After that, 1.0 mL of a latex bead suspension (0.85 μm, 109 particles/mL, Sigma-Aldrich)
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supplemented with af-culture media was added to the slides and incubated for 2 h at
us
room temperature. The slides were fixed with absolute methanol and stained by Giemsa’s method. Around 100 cells were counted microscopically. Phagocytic
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activity (PA) was determined by the following equation:
X100
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PA= Number of phagocytosing cells Number of total cells
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2.7.4. Lymphocytes Proliferation Assay
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This experiment was performed as the method of Guo et al. (2013). In summary, the whole blood samples diluted with an equal volume of culture media,
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then 2 mL were layered over 1 mL lymphocytes separating medium (histopaque1077). The lymphocytes were recovered from the interface after centrifuged at 400 g at -4C for 20 min. Lymphocyte concentration was adjusted to 2 X106 cells /mL after the viability of cells was checked (>95%) by the trypan blue exclusion (0.4%).
50 μL lymphocyte cell suspension and 100 μL culture medium (contain mitogen 50 μg/mL ConA) were added to duplicate wells of microtiter plate. Non- stimulatedcultures were also set without mitogen. The plate was incubated at 27 C with 5% CO2 for 48 hrs. Each culture was pulsed with 20 μL MTT (5 mg /mL) and 2 h later incubation, the culture was centrifuged at 1000g for 10 min. After carefully
Page 7 of 27
removed the supernatant, 200 μL of DMSO was added to each well. The cultures were read in a plate reader at OD 570 nm. The stimulation index values of
optical density of the same samples without mitogen. 2.8. Activities of Humoral Factors
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2.8.1. Bactericidal Activity.
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lymphocytes were; Mean optical density of mitoge stimulated samples / Mean
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The bactericidal activity of plasma was determined with modifications (Welker et al., 2007). Flat-bottom well was set up in duplicate with 100 μL of serum or Hank's
an
balanced salt solution for control and incubated for 120 min with 50 μL suspensions of 24 h live A. hydrophila culture (1 X108 CFU/mL). Fifty μL
M
diphenyltetrazolium bromide solution (MTT; 2 mg/mL) was pipetted into each well
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and incubated for 20 min at room temperature to allow the formation of formazan.
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The supernatant discarded and the formazan dissolved by the addition of 200 μL dimethyl sulfoxide (DMSO). The bactericidal activity was read at 560 nm with a
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microtiter plate reader and reporting as absorbance units after subtracting the absorbance of samples from that of controls.
2.8.2. Lysozyme Activity. Serum lysozyme activity was measured according to the method of Ellis (1990) with some modifications. A sample of 0.25 mL plasma was mixed with 0.75 mL Micrococcus lysodeikticus (Sigma Chemical Co) which was suspended in 0.05M
PBS, pH 6.2. The mixture reacted at 25 C for 5 min, and then the OD was measured at 1 min intervals for 5 min at 540 nm (5010, Photometer, BM Co. Germany).
Page 8 of 27
2.8.3. Nitric Oxide and Acid Phosphatase Activity The serum nitric oxide (NO) and acid phosphatase activity were assayed spectrophotometric using commercial test kits according to the enclosed pamphlet
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(Bio-Chain, Inc. USA).
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2.9. Total and Differential Leukocyte Count
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sample according to Stoskoph (1993).
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Total and differential leukocyte counts were counted in duplicate for each
2.10. Experimental Challenge
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At the end of the experiment, the fish in each experimental group was
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intraperitoneally injected with 200 µL of pathogenic A. hydrophila (0.5 X 108
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CFU/mL). Mortality was observed during the following 14 days post-inoculation. The dead and 20% moribund fish were subjected for bacterial re-isolation to
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confirm A. hydrophila as the cause of death. 2.11. Statistical Analysis The data among treatment groups were analyzed by one way analysis of variance (ANOVA) with post-hoc LSD multiple comparison test using SPSS software (ver. 18.00, USA) to find out the significant differences among treatment groups when P < 0.05. For data of mortalities, data were tested for normal distribution using Kolmogorov-Sminov test. The data were not normally distributed, therefore, Kaplan-Meier survival analyses curve were performed for estimation of survival probabilities with cadmium or death due to cadmium as the primary clinical
Page 9 of 27
endpoint.
The analyses were performed using GraphPad prism Version
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5.01(GraphPad Software Inc., USA).
3. Results and Discussion
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The current study evaluated the effects of cadmium on the non-specific immune
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response of catfish, Clarias gariepinus. Table 1 illustrates the toxicological effects of cadmium on the immune system, which shows evidence of impaired neutrophil
an
function, macrophage respiratory burst and phagocytic activity. Phagocytosis and macrophage function is widely used to evaluate aquatic animal health status under
M
different environmental stress (Zelikoff et al., 1995; Uribe et al., 2011). Cadmium
d
exposure altered macrophage-mediated immune functions, including phagocytosis
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and free radical production in rainbow trout, dab while and common carp (Zelikoff et al., 1995; Hutchinson and Manning 1996; Ghiasi et al., 2012), respectively. It is
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known that the cadmium is a strong stressor to fish and the cortisol secreted during stress reactions, reduces the fish phagocytic activity (Ghiasi et al., 2012). Cortisol effects on immune cells begin with a marked increased expression of interleukin 1β and decreased stimulation of leukocyte function (Fast et al., 2008). Moreover, the bactericidal activity was suppressed with a significantly decreased level of serum lysozyme, nitric oxide and acid phosphatase (Table 2). Lysozyme has a bactericidal effect by destroying cellular walls of bacteria, stimulates phagocytic activity and participates in the regulation of immune cell differentiation and proliferation (Ghiasi et al., 2012). Head kidney lysozyme, liver acid phosphatase
Page 10 of 27
and serum lysozyme were remarkably reduced in Ictalurus melas, Cyprinus carpio and Clarias garipeinus after cadmium exposure (Albergoni and Viola 1995b; Sovenyi and Szakolczai 1993; El-Shehawi et al., 2007). The reduced bactericidal
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substances and lysozyme after cadmium exposure in fish could be attributed to reducing leukocytes count and function (Ghiasi et al., 2012). NO is an intracellular
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molecule synthesized by a nitric oxide synthases (NOS) and involved in the
us
regulation of many cellular functions including mitochondrial metabolism and immune response (Ivanina, et al., 2010). They reported that, Cd exposure resulted
an
in a strong reduction NO level as a result of inhibition of NOS activity in oyster gills. The effect of cadmium on total and differential leukocyte count is illustrated
M
in Table 3, which shows a significant decrease of the total leukocytes, lymphocyte
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counts besides lymphocyte proliferation. Cadmium may bind to some lymphocyte
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membrane proteins, resulting in impaired of lymphocytic function and suppress the immune response (Albergoni and Viola 1995b). Similar results were observed in
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Heteropneustes fossilis and Cyprinus carpio after exposure to cadmium (Radhakrishnan 2010; Ghiasi et al., 2012). Elevation of the cortisol blood level, as a result of cadmium strong stressor to fish, reduces the life span of lymphocytes, enhances their apoptosis and suppresses their proliferation (Verburg-Van et al., 1999; Radhakrishnan 2010). Meanwhile, lower doses of cadmium enhanced macrophages oxidative burst in Ictalurus melas, total leukocyte count in Clarias gariepinus and leukocyte phagocytosis in Sparus aurata (Albergoni and Viola 1995b; Tawari-Fufeyin et al., 2008; Guardiola et al., 2013), respectively. They
Page 11 of 27
concluded that lower doses of cadmium enhance certain properties of macrophage function to produce more leukocytes in poisoned fish. Fish macrophages remove bacteria by the production, reactive oxygen species
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during a respiratory burst (Uribe et al., 2011).Table l, illustrates the macrophage oxidative burst and phagocytic activity percentages were enhanced in the fucoidan
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treated groups (F1&F2). Also, our results are in accordance with that carried out on
us
shrimp (Chotigeat et al., 2004). Fucoidan enhances macrophage phagocytosis and lymphocyte activation, which is likely to activate a signaling pathway leading to
an
enhanced interleukins (IL), IL-12 production from macrophages (Kawashima et al., 2012). Water-soluble polysaccharides extracted from seaweed brown algae, have
M
been documented to have a potent mediator of respiratory bursts, phagocytes,
d
lysozyme and bactericidal activities by inducing expression of IL-1, IL-6 and
te
tumor necrosis factor receptors, which exert a major role in the induction of immune responses in turbot (Peddie et al., 2002), goldfish macrophages (Stafford et
Ac ce p
al., 2003), and shrimp (Kanjana et al., 2011). Moreover, expression of phagocytes peptide proteins was recorded on shrimp macrophages after vaccination with inactivated Vibrio harveyi and fucoidan supplement (Deachamag et al., 2006). The lymphocyte proliferation was significantly enhanced in (F1 &F2) group in comparison with the control group and this is consistent with findings in shrimp (Chotigeat et al., 2004). In-vitro fucoidan, enhanced lymphocyte blastogenesis and the esterase activity of lymphocyte tryptase, which is believed to stimulate lymphocyte proliferation (Hirayasu et al., 2005; Hayashi et al., 2008).
Page 12 of 27
Fish antimicrobial substances that include NO, reactive oxygen and lysozyme constitute the essential components of the humoral immune defense against invading pathogens (Uribe et al., 2011). The activities of humoral factors are
ip t
summarized in Table2, in which, bactericidal activity, serum lysozyme and nitric oxide were significantly increased in (F2) group in comparison with the control
cr
group and this agree with the findings in shrimp and sea bass (Bagni et al., 2005;
us
Traifalgar et al., 2013). They concluded, enhancement serum bactericidal activity was attributed to the elevated bactericidal substances such as lysozyme, NO and
an
phagocyte oxidative burst. Lysozyme is a bacteriolytic enzyme and is a part of the nonspecific defense mechanism in most aquatic animals and the main sources of
M
lysozyme are phagocytes (Uribe et al., 2011). Sulfate group in the fucoidan, is
d
essential to binding macrophage cell surface receptors and plays a key role in the
te
immune-regulating activity of macrophages (Teruya, et al., 2009). Fucoidan has been documented to stimulate NO production, in a macrophage cell line through
Ac ce p
membrane macrophage scavenger receptors and signaling pathway, in addition, enhances the NOS activity (Teruya, et al., 2009; Sharma et al., 2014). Free radicals as reactive oxygen species (ROS) disrupt the normal metabolism, including the immune system and incriminated in the pathogenesis of several toxicities including Cd exposed tilapia (Basha and Rani 2003), catfish (Kumar et al., 2009) and goldfish (Zikic et al., 2001). Cadmium could induce oxidative stress, through inhibiting the mitochondrial electron-transfer chain which enhances superoxide radical production and or interference with cellular antioxidant defense system (Sandrini et al., 2008). The fucoidan at high doses 6 gm/kg diet is effective
Page 13 of 27
in enhancing the cellular and humoral immunological parameters in cadmium exposed groups, when compared with the lower doses of fucoidan (Table 1,2). Fucoidan exhibited excellent scavenging capacities on ROS and showed a great
ip t
potential for preventing free radical mediated diseases (Chotigeat et al., 2004; Wang et al., 2010). The fucoidan, alleviate the cadmium immunotoxicity in our
us
cr
work, could be due to its immune-stimulant action and or its antioxidant properties.
Dietary supplementation of fucoidan led to a reduction in mortalities after challenge
an
with A. hydrophila when compared with the control group (Fig. 1). Fish fed fucoidan in F2 group has the greatest survival rate (80%) which significantly differs
M
from cadmium exposed group (P= 0.0145). The Cd exposed groups and
d
supplemented with two different levels of fucoidan (CdF1 & CdF2) significantly
te
increased the survival rate (27% & 37%), respectively in comparison with Cd (7%) exposed group; however, neither of both groups were significantly different from
Ac ce p
each other nor from the control. Together, these data indicate that feed supplementation with fucoidan in the presence of cadmium exposure can enhance the survival rate near the level of the control. These results are consistent with those reported in shrimp challenged with white spot syndrome virus (Chotigeat et al., 2004) and V. harveyi (Traifalgar et al., 2013). Enhanced phagocytic activity, respiratory burst activity, serum lysozyme and nitric oxide were observed in the fucoidan treated groups. Also, the antimicrobial effect of fucoidan could be attributed to the negative charges of the sulfate group of the polysaccharide bind
Page 14 of 27
with positive charges of amino acids present in microorganism membrane, and thus preventing entry of the microbial agent into the host cell (Immanuel et al., 2012). 4. Conclusion
ip t
In conclusion, feeding fucoidan enhanced the humoral and cellular immunity of African catfish, Clarias gariepinus. Moreover, fucoidan seems to be a promising
cr
candidate for increasing the resistance against bacterial infections under the
us
conditions of heavy metal pollution.
All authors have none to declare.
M
References
an
Conflict of interest
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Table 1. Effect of dietary treatment fucoidan on selected cellular immunological parameters in catfish, Clarias gariepinus after immunosuppressive with
cadmium chloride for 21 days. Table 2. Effect of dietary treatment fucoidan on selected humoral immunological
parameters,
in
catfish,
Clarias
gariepinus
after
immunosuppressive with cadmium chloride for 21 days.
Page 21 of 27
Table 3. Total and differential leukocyte count (mean ± S.E) in catfish, Clarias gariepinus
fed
on a fucoidan
supplement
diet
for 21 days after
immunosuppression induced by cadmium chloride.
ip t
Fig. 1. Cumulative mortality (%) of African catfish, Clarias gariepinus fed fucoidan supplemented diets for 21 days and challenged with pathogenic A.
Ac ce p
te
d
M
an
us
cr
hydrophila for 14 days.
Page 22 of 27
Table 1. Effect of dietary treatment fucoidan on selected cellular immunological parameters in
Neutrophils NBT assay (OD) unit
0.98
c
±0.08 Macrophage respiratory burst index
7.15c
±0.09
1.01
Cd
c
CdF1
CdF2
ab
0.78b
±0.06
±0.08
±0.07
0.51
a
±0.12
0.62
9.18d
9.86d
3.48a
5.02b
6.84c
±0.26
±0.35
±0.15
±0.12
±0.22
28.2c
38.1d
40.1d
15.1a
21.2b
27.4c
d
±1.85
±2.95
±1.45
±1. 01
±2.12
1.72c
1.75c
2.15c
0.61a
0.98b
1.01b
±0.15
±0. 11
±0.19
±0.05
±0. 06
±0. 09
1.66b
1.95c
2.64d
1.05a
1.25a
1.58b
±0.09
± 0.12
± 0.18 ±0.08
± 0.14
± 0.11
Phagocytic activity %
Ac ce p
te
±1.15
Phagocytic index
0.96
F2 c
M
±0.18
F1
us
Contro
cr
Groups
an
Cellular immune response
ip t
catfish, Clarias gariepinus after immunosuppressive with cadmium chloride for 21 days.
Lymphocyte transformation index
( F1= fucoidan 4 g, F2= fucoidan 6 g , Cd= cadmium). The rows with the same superscript are not statistically significant at P<0.05. (Values are mean ± SE) (n = 10).
Page 23 of 27
Table 2. Effect of dietary treatment fucoidan on selected humoral immunological parameters, in
F1
1.15b
1.32b
1.58 c
±0.09
±0.10
±0.08
9.15c
( U/mL)
±0.31
te 1.12b
Ac ce p
Serum nitric oxide
11.05d 12.31e
d
Serum lysozyme
F2
cr
Cd
CdF1
CdF2
0.72a
0.84a
1.12b
±0.05
±0.08
±0.11
6.15a
7.10b
8.84c
an
Control
M
Bactericidal activity unit
Groups
us
Humoral immune response
ip t
catfish, Clarias gariepinus after immunosuppressive with cadmium chloride for 21 days.
±0.38
±0.45
±0.25
±0.32
±0.48
1.48c
1.51c
0.66a
0.89b
1.08b
(Mmol/mL)
±0.11
±0.10
±0.15
±0.07
±0.09
±0.12
Serum acid phosphatase
1.85c
1.92c
1.95c
0.91a
1.05a
1.38b
U/L
±0.14
±0.11
±0.18
±0.09
±0.10
±0.12
(F1= fucoidan 4 g, F2= fucoidan 6 g , Cd= cadmium). The rows with the same superscript are not statistically significant at P<0.05. (Values are mean ± SE) (n = 10).
Page 24 of 27
ip t
Table 3. Total and differential leukocyte count (mean ± S.E) in catfish, Clarias gariepinus fed on
cr
a fucoidan supplement diet for 21 days after immunosuppression induced by cadmium
us
chloride. Leukogram
F1
F2
Cd
CdF1
CdF2
Total Leukocytes Count
32.15c
33.21c
34.35c
20.52a
24.54b
26.75 b
(Thousand/ µL)
±2.51
±2.45
±2.62
±1.18
±1.32
±1.95
Neutrophils
10.15a
10.32a
10.12a
8.24a
8.41a
9.14a
±0.92
±1.10
±0.98
±0.78
±0.75
±0.81
Esinophils
0.52b
0.59 b
0.48b
0.24a
0.32a
0.30a
(Thousand/µL)
±0.04
±0.06
±0.05
±0.04
±0.05
±0.03
Basophils
0.022
0
0
0
0.020
0
(Thousand/µL)
±0.02
Lymphocytes
19.95c
20.84c
22.18c
10.41a
14.25b
15.82b
(Thousand/µL)
±1.42
±1.55
±1.64
±1.02
±1.12
±1.18
Monocytes
1.52a
1.45a
1.55a
1.61a
1.56a
1.48a
Ac ce p
(Thousand/ µL)
te
d
M
Control
an
Groups
±0.02
Page 25 of 27
(Thousand/µL)
±0.16
±0.12
±0.17
±0.21
±0.15
±0.10
( F1= fucoidan 4 g, F2= fucoidan 6 g , Cd= cadmium). The rows with the same superscript are not statistically significant at P<0.05. (Values are
Ac ce p
te
d
M
an
us
cr
ip t
mean ± SE) (n = 10).
Page 26 of 27
ip t cr us an M d te Ac ce p Fig. 1. Cumulative mortality (%) of African catfish, Clarias gariepinus fed fucoidan supplements, diet for 21 days and challenged with pathogenic A. hydrophila for 14 days .
Page 27 of 27