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Fish & Shellfish Immunology xxx (2015) 1e7
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Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi
Short communication
Q5 Q4
Dietary supplementation of Avicennia marina extract on immune protection and disease resistance in Amphiprion sebae against Vibrio alginolyticus Nagarajan Balachandran Dhayanithi a, Thipramalai Thankappan Ajithkumar a, Jesu Arockiaraj c, Chellam Balasundaram b, Ramasamy Harikrishnan d, * a
Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai 608 502, Tamil Nadu, India Department of Herbal and Environmental Science, Tamil University, Thanjavur 613 005, Tamil Nadu, India Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattankulathur, 603 203 Chennai, Tamil Nadu, India d Department of Zoology, Pachaiyappa's College for Men, Kanchipuram 631 501, Tamil Nadu, India b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 December 2014 Received in revised form 8 February 2015 Accepted 10 February 2015 Available online xxx
The effect of Avicennia marina aqueous leaf extract on innate immune mechanisms such as total white blood cell counts (WBC), serum lysozyme activity, respiratory burst assay, alternative complement (ACH50) assay, phagocytic activity assay, disease resistance, gut bacteria, and survival rate of clownfish (Amphiprion sebae) against Vibrio alginolyticus is reported. Healthy fish challenged with V. alginolyticus (1 107 cells ml1) were fed with diets supplemented (0, 1, 2, and 4%) with A. marina extract. The survival rate was 85% and 80% in infected fish fed with 4% and 8% supplementation diet; with 1% diet it was 70% while in the infected untreated group it was only 10%. The total gut bacteria flora was high in 8% and 4% supplementation diet groups with 2.8 105 and 4.7 104 cfu/g while it was 8.9 103 cfu/ml in 1% diet group. The immunological parameters significantly increased on weeks 6 and 8 when infected fish were fed with 1% or 4% supplementation diet. This study reports that in clownfish challenged with V. alginolyticus, dietary administration of the 1% or 4% of A. marina extract improved the immune status and survival rate. © 2015 Published by Elsevier Ltd.
Keywords: Avicennia marina Disease resistance Innate immune parameter Mortality Vibrio alginolyticus
1. Introduction Immune system is the defense mechanism against diseases by eliminating the invading pathogens from the host. The classical division of the immune system is categorized into the innate and the adaptive systems. The importance of innate immune system is to defend the fish against the invading pathogens [1] which comprise: (i) the epithelial/mucosal barrier, (ii) the humoral parameters, and (iii) the cellular components. The epithelial and mucosal system of the skin, gills, and alimentary tract act as an extremely important disease barrier since these organs are constantly bathed in media containing potentially harmful agents. Apart from the physical and mechanical defense further protection is afforded since the fish mucus contains several immune defense
* Corresponding author. Tel.: þ91 4362 227937; fax: þ91 4362 227185. E-mail address: [email protected] (R. Harikrishnan).
parameters including antimicrobial peptides, complement factors, and immunoglobulins [2]. Traditional disease control strategies with antibiotics and chemical disinfectants are no longer recommended practices due to the emergence of bacterial resistance and also concerns over environmental impacts and wildlife protection [3e5]. Vaccination, an effective prophylactic method boosts the immune system preventing the incidence of microbial infection. However, there are some methodological issues like being very expensive and stressful to fish. Nowadays a remarkable success has been achieved with herbal immunostimulants as a more environmentally friendly approach in disease management [6e16]. An herbal immunostimulant is defined as a naturally occurring compound that modulates the immune system by increasing the host's resistance against diseases [6e16]. These treatments are primarily aimed at enhancing the innate immune system and therefore of great value as general preventive measures in aquaculture. During the past few years, commercial fish farming has been severely affected due to
http://dx.doi.org/10.1016/j.fsi.2015.02.018 1050-4648/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Dhayanithi NB, et al., Dietary supplementation of Avicennia marina extract on immune protection and disease resistance in Amphiprion sebae against Vibrio alginolyticus, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.02.018
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various diseases especially by bacteria inflicting severe economic losses due to Vibrio alginolyticus, Vibrio carchariae, Pseudomonas sp., and Flexibacter sp. infection. Avicennia marina one of the common tree species of mangrove forest ecosystem belonging to the family Verbenaceae, is a cosmopolitan species widely distributed along tropical and subtropical coastlines. From the bark, leaf, and fruits of A. marina a number of active compounds like alkaloids, phenolics, steroids, terpenoids etc. have been isolated [17e23]. From leaf extract of A. marina by GC-MS we recently identified major phytochemical compounds namely Dehydrovomifolio, Diterpene, Pyrogallol, Levoglucosan, Glycerin, Ketone, Sesquiterpenoids, Phthalates, Dihydro, trimethyl Coumarin, Polysaccharide (unpublished data); these compounds are well-known as antioxidants, enzyme inhibitor, precursors of toxic substances and possess anti-inflammatory, antioxidant, anti-allergic, anticarcinogenic activities [24,25]. The bark, leaves, and fruits of A. marina have reported as antibacterial, antifungal, antiviral agents and also possess anticancer, antiplasmodial, antitumor, and antiulcer properties [26e33]. Recently V. alginolyticus was isolated and reported during breeding time and in the juvenile stages of Amphiprion sebae at the Marine Ornamental Fish Breeding Hatchery in the Centre of Advanced Study in Marine Biology, Annamalai University leading to mass mortality (personal communication). This bacterium also has been isolated from moribund A. sebae collected from the Marine Ornamental Fish Breeding Hatchery [34,35]. Hence to tackle this problem the application of one of the dominant mangrove tree species A. marina known for its medicinal properties [34] was attempted. The demand for ornamental fish like clown fish is rapidly increasing with the consequent increase in the ornamental fish hatcheries. In this back drop since there is a dearth of information with reference to phytotherapeutic effect of A. marina in aquatic animals, the present study was conducted to analyze the immunomodulatory effects and disease resistance for the first time in clownfish, A. sebae against V. alginolyticus infection. 2. Materials and methods 2.1. Collection and extraction of mangrove plants Leaves of A. marina were collected from the Vellar estuary mangroves. The leaves were washed in sterile water and shade dried for one week. The dried leaves were pulverized in a mechanical grinder, sieved, and then stored in an air tight container for further use. One hundred grams of the coarsely powdered sample was successively extracted with 85% ethanol and then filtered. The successive extraction was performed by a cold maceration process for seven days with daily agitation twice. The solvent was evaporated using a rotary vacuum evaporator (Buchi, Flawil, Switzerland) and the residues were stored at 20 C until used for the experiment. 2.2. Vibrio alginolyticus The pathogenic bacterium, V. alginolyticus (AUMOFP2) was previously isolated moribund A. sebae [34] and maintained in the laboratory under standard conditions [34]. Subcultures were maintained on marine zobell agar (Himedia, Mumbai) in slopes at 5 C and routinely tested for pathogenesis by inoculation into marine ornamental fish. Stock culture in marine zobell broth (Himedia, Mumbai) was stored at 70 C in 0.85% NaCl with 20% glycerol (v/v) to provide stable inoculate throughout the experiment. The culture was centrifuged at 1000 g for 10 min at 4 C. The supernatant were discarded and the bacterial pellet was washed
three times and re-suspended with phosphate-buffered saline (PBS) at pH 7.4. The optical density (OD) of the solution was adjusted to 0.5 at 456 nm which corresponded to 1 107 cells ml1. 2.3. Preparation supplementation feed The experimental diets and the proximate composition of the formulated diets were mentioned in Table 1. The ingredients were well mixed and extruded through a pellet extruder. Four experimental pellet diets, 0 (control basal diet without extract), 1, 4, and 8% A. marina extracts sprayed into the basal diet slowly, mixing evenly in a drum mixer, after which it was air dried under sterile conditions for 12 h. The control basal diet was added with the same volume of solvent without the extracts. The pellets were dried in an oven at 30 C for 18 h, packed, and stored in a freezer at 20 C until used. 2.4. Fish Healthy Clownfish, A. sebae (20.2 ± 5.4 g in weight) was collected from the marine ornamental fish hatchery, Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai. The fishes (n ¼ 500) were acclimated in 5000 l cement tank filled with 2500 l of UV treated seawater and was provided continuous aeration. Fish were acclimatized for 2 weeks prior to initiating the experiment and the fish were provided formulated control basal diet (Table 1) twice a day. During the experiment, water temperature was 28.2 C ± 1.4, pH 8.2 ± 0.3, salinity 28 ± 2.2 ppt, dissolved oxygen concentration 5.8 ± 0.6 mg l1, and 14 h light: 10 h dark photoperiod were measured. 2.5. Experimental design and challenge study A total number of 300 fish (A. sebae) were selected and divided into 5 groups of twenty fish each in three replicate (5 20 3) as: 1) uninfected control fish fed basal control diet without extract, 2) infected fish fed basal control diet without extract, 3) infected fish fed 1% extract supplementation feed, 4) infected fish fed 4% extract supplementation feed, and 5) infected fish fed 4% extract supplementation feed. After 2 weeks of feeding, all groups except control group intramuscularly injected with 0.1 ml of V. alginolyticus at a
Table 1 Composition of the formulated fish feed. Ingredients (g/100 g)
0%
1%
2%
4%
Acetes Mytha Ground net oil cake Rice bran Cod liver oil Vitamina & Mineralb complex Herbal extract Proximate composition Crude protein Crude lipid Crude ash Moisture Fibre
50 10 27 10 2 1 0
50 10 27 9 2 1 1
50 10 27 8 2 1 2
50 10 27 6 2 1 4
41.6 7.3 7.7 7.1 3
41.8 7.3 7.8 7.2 3
42.3 7.2 7.8 7.2 3
42.6 7.1 7.9 7.3 2
a Contains (g/100 g premix): DL-calcium pantothenate 0.5, choline bitartrate 10, inositol 0.5, menadione 0.02, niacin 0.5, pyridoxine-HCl 0.05, riboflavin 0.1, thiamine mononitrate 0.05, DL-a-tocopheryl acetate 0.2, retinyl acetate 0.02, biotin 0.005, folic acid 0.015, B12 0.0003, cholecalciferol 0.006, a-cellulose 80.0. b Contains (mg/100 g premix): Al 0.10, Ca 500, Cl 10, Cu 0.5, Co 0.8, Na 0.120, Mg 50, P 5000, K 420, Zn 0.3, Fe 4, I 0.50 Se 0.02, Mn 0.8.
Please cite this article in press as: Dhayanithi NB, et al., Dietary supplementation of Avicennia marina extract on immune protection and disease resistance in Amphiprion sebae against Vibrio alginolyticus, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.02.018
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dose 1 107 cells ml1. The experimental fish was provided the corresponding diet twice a day until the end of experiment. 2.6. Bleeding and sampling Six fish in each tank were sampled for hematology, immunological parameters, and gut micro flora study. The blood was collected from the caudal vasculature following anesthesia with MS-222 (SigmaeAldrich, St. Louis, MO, USA) using a 1 ml heparinized syringe with 24 gauge needles and transferred into a heparinized tube. The plasma was recovered after centrifugation at 2000 rpm for 10 min and immediately stored at e 20 C until used for the experiment. 2.7. Head kidney (HK) leucocytes The head kidney (HK) leukocytes isolates from peripheral blood according to Chung and Secombes [36]. In each fish 0.5 ml of blood was collected and diluted with 2 ml of RPMI 1640 (Gibthai) and then carefully laid onto 3 ml of Histopaque (Sigma) in a 15 ml tube. It was centrifuged at 400 g for 30 min at room temperature, a white buffy coat of leukocytes cells floated on the top of the Histopaque and the opaque interfaces were carefully aspirated with a Pasteur pipette and transferred into a clean 15 ml tube. Make 6 ml with PBS (pH 7.4), gently mixed by aspiration, and centrifugation at 250 g for 10 min. The centrifugation step repeated 3 times to complete remove any residual Histopaque. The isolated HK leukocytes cells were then re-suspended in the PBS and adjusted to the required cell numbers for phagocytosis and respiratory activities. 2.8. Supplemented feed action on gut bacteria Total gut bacteria of the experimental and control group were assessed by the modified protocol of Velmurugan and Citarasu [37] and find out the effects of supplemented feed containing A. marina extracts on the normal gut bacterial flora. The whole gut was removed aseptically in all fish from each group, weighed, and the surface washed with 85% sterile saline and than homogenized. The homogenate was diluted in sterile seawater (32‰) and plated in nutrient agar (Hi-media, India) and TCBS (Hi-media, India). The plates were incubated at 30 C for 48 h and the colonies were counted quantitatively. 2.9. Total white blood cell (WBC) counts The blood was collected from the fishes along with diluting Hayem's fluid and incubated 5 min for complete haemolysis of RBCs. After incubation, the blood mixture was loaded on the improved Neubauer haemocytometer by holding the pipette at 45 angle and the cover slip was placed. The cells were counted and expressed as cells ml1. 2.10. Serum lysozyme activity Serum lysozyme activity was measured according to the modified method described by Ellis [38]. Briefly, 10 ml of individual serum was mixed with 200 ml of a Micrococcus luteus suspension at 0.2 mg ml1 in 0.05 M sodium phosphate buffer (pH 6.2). The mixture was incubated at 27 C and its OD was recorded at 530 nm using an ELISA plate reader. One unit of lysozyme activity was defined as the amount of enzyme produced at a decrease in absorbance of 0.001 min1 ml1 serum. Lysozyme concentrations were calculated from a standard curve of known lysozymes from chicken albumin concentrations.
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2.11. Respiratory burst assay To detect the Nitro Blue Tetrazolium chloride (NBT) activity, 100 ml of blood containing anti-coagulant was placed into a microtitre plate well and then equal amount of 0.2% NBT solution was added. After incubation at room temperature for 30 min, 0.05 ml of NBT blood suspension was transferred to glass tube containing 1 ml of N, N dimethyl formamide and centrifuged at 400 g for 5 min at 4 C. The spectrophotometric reading was measured the OD at 620 nm [39]. 2.12. Alternative complement (ACH50) assay ACH50 was determined adopting the method of Sunyer and Tort [40] with the following small modifications. Sheep red blood cells (SRBCs) were used as target cells in the presence of gelatin veronal buffer. Individual 20 ml aliquots (2-fold dilutions) of a serially diluted serum with EGTA-Mg2þ- GVB buffer (10 mM ethylene glycoltetraacetic acid and 10 mM MgCl2 in GVB) was used as a complement source, which was mixed with 6 ml of SRBC suspension (4 108 cells ml1), and the mixture was incubated at 21 C at pH 7.2 for 2 h. The hemolytic reaction was stopped by adding 200 ml of GVB containing 10 mM EDTA. The mixtures were centrifuged at 1600 g for 10 min at 4 C. The OD of the supernatants was measured at 414 nm using ELISA reader (A). The reactions were supplemented with 6 ml EDTA-GVB, 20 ml EDTA-GVB and 220 ml distilled water to replace the SRBC suspension, the diluted serum and the diluted serum with EDTA-GVB buffer, respectively, as the SRBC blank (B), serum blank (C) and 100% hemolysis sample (D). The degree of hemolysis (Y) was defined as Y ¼ (A (B þ C)) (D C) 1 and calculated and the lysis curve for each specimen was obtained by plotting Y (l Y) 1 against the volume of complements added on a log/log-scaled graph. The volume of serum complement producing 50% hemolysis (ACH50) was determined and the number of ACH50 units ml1 was calculated for each experimental group. 2.13. Phagocytic activity Phagocytic activity was measured from HK leucocytes according to Matsuyama et al. [41]. A suspension of leucocytes (100 ml of 1 107 cells ml1) was placed in a sterile slide and allowed to attach for 30 min at 25 C. Then, 100 ml yeast suspension (Baker's yeast, Type II, Sigma, 1 108 cells ml1) was added to the cell monolayer. The slide was incubated for 45 min at 25 C and then the same was washed three times with PBS to remove the unattached cells and uningested yeasts. After air-drying, the slides were fixed in ethanol, redried and stained with Giemsa. The percentage of phagocytic index was calculated by enumerating phagocytes under a microscope the following formula: number of engulfed bacteria/number of phagocytic cells 100. 2.14. Survival rate Groups of 20 fish each in triplicate were fed with 0, 1, 4, and 8% extract supplemented diets twice a day at 5% of their body weight. After 2 weeks of feeding, the fishes were challenged with V. alginolyticus (1 107 cells ml1) and the survival rate was recorded for 30 days. Our earlier study, the LD50 dose was standardized of the bacterial concentration to give 80% mortality. 2.15. Statistics The data from treated groups were analyzed using one-way analysis of variance (ANOVA) to find out the significant difference to control group at the 5% (P < 0.05) level.
Please cite this article in press as: Dhayanithi NB, et al., Dietary supplementation of Avicennia marina extract on immune protection and disease resistance in Amphiprion sebae against Vibrio alginolyticus, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.02.018
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3. Results
3.6. Action of feed on gut flora
3.1. Total white blood cell (WBC) counts
The gut bacterial flora was analyzed in the experimental and control groups. Results indicate that the gut bacterial microbial flora was altered due to administration of mangrove plant extract. The maximum total viable counts (TVCs) were observed in groups fed with 8% and 4% supplementation diets: 2.8 105 cfu/g (38.32% Bacillus subtilis, 27.75% Bacillus circulans, 15.85% Lactobacillus sp., 11.52% Vibrio fluvialis, and 6.56% M. luteus) and 4.7 104 cfu/g (30.8% B. subtilis, 25.98% B. circulans, 22.1% Lactobacillus sp., 9.58% V. fluvialis, and 11.54% M. luteus). But when fed with 1% supplementation diet was found 8.9 103 cfu/g (29.5% B. subtilis, 25.3% B. circulans, 20.0% Lactobacillus sp., 8.65% V. fluvialis, and 14.55% M. luteus).
The white blood cell (WBCs) of the experimental group compared with control as shown in Fig. 1. The WBC level progressively increased in all supplementation diets as compared to control on weeks 2 and 4. However, it increased to the maximum in all supplementation diets on weeks 6 and 8 when compared to the control. 3.2. Serum lysozyme activity The serum lysozyme activity did not increase statistically (P > 0.05) in all supplementation diets as compared to control on weeks 2 and 4. Its production reached a statistically significant (P < 0.05) with 4% supplementation diet on week 6 and 1% and 4% supplementation diets on week 8 (Fig. 2). 3.3. Respiratory burst activity The respiratory burst activity did not vary in the fish fed with any supplementation diet on weeks 2 and 4 when compared to control (Fig. 3). However, the respiratory burst activity significantly increased in the infected fish fed with 4% or 8% supplementation diets on weeks 6 and 8. 3.4. Alternative complement (ACH50) activity The ACH50 activity did not statistically increase (P > 0.05) with any supplementation diet on weeks 2 and 4 as compared with control (Fig. 4). However, the ACH50 activity showed the maximum on weeks 6 and 8. Interestingly, the activity increased significantly (P < 0.05) in fish fed with 1% and 4% supplementation diets on weeks 6 and 8. 3.5. Phagocytic activity The phagocytic activity did not significantly increased (P > 0.05) when fed with any supplementation diet during the experimental period as compared with control (Fig. 5). However, the phagotytic activity was significantly increased (P < 0.05) when fed with 4% supplementation diet on week 6 and 8 against the pathogens.
Control
0%
1%
4%
3.7. Survival rate The survival rate was 85% and 80% in fish fed with 4% and 8% supplementation diet with A. marina against V. alginolyticus. However, the survival rate declined to 70% when fish fed with 1% supplementation diet while in the infected untreated group it was found 10% (Fig. 6). 4. Discussion Enhancement of the immune system seems to be the most promising method of preventing diseases in fishes. The innate (non-specific) immune system of fish is considered to be the first line of defense against invading pathogens and is more important for fish than mammals [42]. The major components of the innate immune system are macrophages, monocytes, granulocytes, and humoral elements, like lysozyme or complement system [43]. In this context, the immunostimulants and adjuvants used in fish vaccines are of interest, as they offer an alternative to the drugs, chemicals, and antibiotics currently used in fish culture to control the fish diseases. In aquaculture, the use of medicinal herbs as immunostimulants continue to receive more attention since last two decades not only regarding their immune stimulating functions, but also for their growth promoting effects with little or no side effects [6e16]. It has been also shown that herbal based immunostimulants are capable of enhancing immune responses and/or reducing losses from viruses, bacteria and/or parasitic infections in freshwater and marine fishes [6e16,44e50]. A number of compounds such as alkaloids, phenolics, steroids, terpenoids etc. have been isolated from bark, leaf, and fruits of A. marina [17e23] which are exhibited antioxidant, antibacterial,
8%
Control
-3 4
Absorbance at 530 nm
2.5
WBC (10 mm )
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2 1.5 1 0.5 0 2
4
6
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Weeks of feeding Fig. 1. Changes in total white blood cells in fish fed with different dosages (0, 1, 4, and 8%) supplementation diets with A. marina leaf extract against V. alginolyticus infection. The experimental groups statistical significantly different at P < 0.05 level as compared to control.
0%
1%
4%
4.5 4 3.5 3 2.5 2 1.5 1 0.5 0
8%
* *
2
4
6
*
8
Weeks of feeding Fig. 2. Serum lysozyme activity in fish fed with different dosages (0, 1, 8, and 8%) supplementation diets with A. marina leaf extract against V. alginolyticus infection. The experimental groups statistical significantly different at P < 0.05 level as compared to control.
Please cite this article in press as: Dhayanithi NB, et al., Dietary supplementation of Avicennia marina extract on immune protection and disease resistance in Amphiprion sebae against Vibrio alginolyticus, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.02.018
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Control
0%
1%
4%
1%
4%
0.15
*
*
*
0.1 0.05
8%
*
*
25
Precentage (%)
Absorbance at 620 nm
0%
30
*
20 15 10 5
0 2
4
6
8
0
Weeks of feeding
2
Fig. 3. Respiratory burst activity in fish fed with different dosages (0, 1, 4, and 8%) supplementation diets with A. marina leaf extract against V. alginolyticus infection. The experimental groups statistical significantly different at P < 0.05 level as compared to control.
antifungal, anticancer, antiplasmodial, antitumor, antiantiviral, and antiulcer properties [26e33]. Therefore in the present study for the first time we investigated the innate immune response and disease resistance of A. marina extract in the economically important marine ornamental clownfish, A. sebae. The WBCs afford protection in fish against pathogens and chemical factors [51]. In the present study, the WBC level slightly increased in all supplementation diet as compared to control on weeks 2 and 4; its increased to the maximum on weeks 6 and 8. Monitoring these values and gathering information on the profile of leucocytes can reflect on the general immune status in fish. There is a poor correlation between differential composition of leucocytes and increase in the neutrophiles percentage in fish associated with viral or other infections [52]. Lysozyme a mucolytic enzyme produced by leucocytes is an important defense molecule of the innate immune system and plays a vital role in mediating protection against microbial invasion. In the present study, the maximum protection was observed in all supplementation diets on weeks 6 and 8; it was statistically significant (P < 0.05) with 4% supplementation diet on week 6 and 1% and 4% supplementation diets on week 8. A similar activity of lysozyme was observed in Jian carp [53] and in large yellow croaker [54] with traditional Chinese medicine (TCM) formulated from Astragalus radix and Angelica root. Oreochromis niloticus fed with 0.1% or 0.5% supplementation diets A. radix [49] and Labeo rohita fed
Control
0%
1%
4%
*
25
*
*
20 15 10 5 0 2
4
6
8
Weeks of feeding Fig. 4. Alternate complement activity in fish fed with different dosages (0, 1, 4, and 8%) supplementation diets with A. marina leaf extract against V. alginolyticus infection. The experimental groups statistical significantly different at P < 0.05 level as compared to control.
8
with Achyranthes aspera showed significantly enhanced the lysozyme activity [55]. In this study, the respiratory burst activity significantly enhanced with 4% and 8% supplementation diets on weeks 6 and 8. The present results are agreement with the previous report on L. rohita fed with A. aspera diet significantly enhanced superoxide anion production [55]. Jang et al. [56] reported that in vitro treatment with glycyrrhizine isolated from Glycyrrhiza glabra enhanced the respiratory burst activity of macrophages and the proliferative response of lymphocytes in rainbow trout. Phagocytes produce toxic oxygen forms during the process of respiratory burst [57]. Since superoxide anion is the first product to be released from the respiratory burst, the measurement of O2 has been accepted as a precise way of measuring respiratory burst [58]. The respiratory burst activity was observed in large yellow croaker and common carp after feeding with a diet containing a mixture of A. membranaceus and A. sinensis extracts [53,54]. The present study has indicated that the different doses of A. marina extract could increase the alternative complement activity on weeks 6 and 8. Interestingly, the ACH50 activity was found statistically significant with 1% and 4% supplementation diets. This is in contrast to the findings of Cook et al. [59] where oral application of EcoActiva™, a commercial b-glucan preparation, did not change the alternative complement activity. In gilthead seabream also a significant enhancement in serum complement activity was observed after feeding with levamisole
8%
*
6
Fig. 5. Phagocytic activity in fish fed with different dosages (0, 1, 4, and 8%) supplementation diets with A. marina leaf extract against V. alginolyticus infection. The experimental groups statistical significantly different at P < 0.05 level as compared to control.
Survival rate (%)
30
4 Weeks of feeding
35
1
5
Control
8%
0.2
Units ml
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65
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0%
1%
4%
8%
Groups Fig. 6. Survival rate (%) in fish fed with different dosages (0, 1, 4, and 8%) supplementation diets with A. marina leaf extract against V. alginolyticus infection for 30 days.
Please cite this article in press as: Dhayanithi NB, et al., Dietary supplementation of Avicennia marina extract on immune protection and disease resistance in Amphiprion sebae against Vibrio alginolyticus, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.02.018
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containing diet [60]. However, no significant effect was seen on weeks 2 and 4 with any enriched diet with reference to the alternative complement pathway. This is in line with the findings of Cuesta et al. [61] when administration of propolis in gilthead seabream did not result in a significant change in the alternative complement activity. The bactericidal activity of the complement has been well recognized as one of the key killing mechanisms of clearing bacteria in fish [62]. Phagocytic cells are important cellular components of the innate immune system of fish [63] and it is a primitive defense mechanism and an important characteristic of the non-specific immune system [64]. The present results show that infected fish fed with 4% diet significantly enhance the phagocytic activity on weeks 6 and 8. Herbal extracts can also enhance phagocytosis in various fish species [6e16]. On the other hand, no major effect was found with any diet on week 2 and 4. This result suggests that the A. marina extracts containing diet enhanced the phagocytic activity after week 6. These results are agreement with the previous observations Scutellaria extract in O. niloticus [49]. Gut microflora play an important role in the digestive process and disease susceptibility of marine feeders [65]. The wild and cultured aquatic animals harbor a diverse bacterial flora, including Aeromonas, Plesiomonas, Photobacterium, Pseudoalteromonas, Pseudomonas, Vibrio, etc. [66]. So, an understanding of the host intestinal bacterial floral interactions is of much significance for the development of a healthy cultivation environment and also to optimize the growth of benign species. But, the intestinal bacteria, such as Aeromonas and Vibrio often occur as opportunistic pathogens [67]. In this study, the maximum TVCs recorded were 2.8 105 and 4.7 104 (cfu/g) when fish were fed with 8% and 4% supplementation diets, respectively. The present results reveal that the gut microbial flora changed qualitatively and quantitatively which might be due to the action of antibacterial active principles present in the mangrove extracts. Bacterial pathogens could have been retarded by the active extracts, by inhibiting the transcription and arresting the protein synthesis of the bacteria [68]. After being challenged with V. alginolyticus, all the treated groups showed a reduced mortality as compared to the infected untreated group. The maximum survival rate of 85% and 80% were observed in the group fed with 4% and 8% supplementation diet against V. alginolyticus. Enhancement of the non-specific immune parameters by the A. marina extracts could have possibly been an important factor in reducing the percentage mortality, protecting the fish against V. alginolyticus infection. Earlier studies in this direction have also revealed that dietary supplementation of various herbal extracts reduced the mortality and increased the survival rate against bacterial, viral, fungal, and parasitic infection [6e16]. Though, there was no positive correlation between the effect of immunostimulant and dosage, a higher dosage might enhance or inhibit the immune response [6e16]. It is important to estimate the increased protection in the mangrove extract treated fish to determine the efficacy of an immunostimulant. The present study suggests that the mangrove extracts (A. marina leaf) could strengthen the immune system with 4% and 8% in clownfish against V. alginolyticus. Further detailed immunological and molecular studies are needed to extend the application of these results in aquaculture as a prophylactic measure.
Acknowledgment Authors thank the authorities of Annamalai University for providing facilities. The first and second authors are thankful to the University Grants Commission (UGC), New Delhi for providing financial assistance. Reference No. U.G.C. No. 33-384/2007 (SR).
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Dietary supplementation of Avicennia marina extract on immune protection and disease resistance in Amphiprion sebae against Vibrio alginolyticus Nagarajan Balachandran Dhayanithi a, Thipramalai Thankappan Ajithkumar a, Jesu Arockiaraj c, Chellam Balasundaram b, Ramasamy Harikrishnan d, * a
Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai 608 502, Tamil Nadu, India Department of Herbal and Environmental Science, Tamil University, Thanjavur 613 005, Tamil Nadu, India Division of Fisheries Biotechnology & Molecular Biology, Department of Biotechnology, Faculty of Science and Humanities, SRM University, Kattankulathur, 603 203 Chennai, Tamil Nadu, India d Department of Zoology, Pachaiyappa's College for Men, Kanchipuram 631 501, Tamil Nadu, India b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 December 2014 Received in revised form 8 February 2015 Accepted 10 February 2015 Available online xxx
The effect of Avicennia marina aqueous leaf extract on innate immune mechanisms such as total white blood cell counts (WBC), serum lysozyme activity, respiratory burst assay, alternative complement (ACH50) assay, phagocytic activity assay, disease resistance, gut bacteria, and survival rate of clownfish (Amphiprion sebae) against Vibrio alginolyticus is reported. Healthy fish challenged with V. alginolyticus (1 107 cells ml1) were fed with diets supplemented (0, 1, 2, and 4%) with A. marina extract. The survival rate was 85% and 80% in infected fish fed with 4% and 8% supplementation diet; with 1% diet it was 70% while in the infected untreated group it was only 10%. The total gut bacteria flora was high in 8% and 4% supplementation diet groups with 2.8 105 and 4.7 104 cfu/g while it was 8.9 103 cfu/ml in 1% diet group. The immunological parameters significantly increased on weeks 6 and 8 when infected fish were fed with 1% or 4% supplementation diet. This study reports that in clownfish challenged with V. alginolyticus, dietary administration of the 1% or 4% of A. marina extract improved the immune status and survival rate. © 2015 Published by Elsevier Ltd.
Keywords: Avicennia marina Disease resistance Innate immune parameter Mortality Vibrio alginolyticus
1. Introduction Immune system is the defense mechanism against diseases by eliminating the invading pathogens from the host. The classical division of the immune system is categorized into the innate and the adaptive systems. The importance of innate immune system is to defend the fish against the invading pathogens [1] which comprise: (i) the epithelial/mucosal barrier, (ii) the humoral parameters, and (iii) the cellular components. The epithelial and mucosal system of the skin, gills, and alimentary tract act as an extremely important disease barrier since these organs are constantly bathed in media containing potentially harmful agents. Apart from the physical and mechanical defense further protection is afforded since the fish mucus contains several immune defense
* Corresponding author. Tel.: þ91 4362 227937; fax: þ91 4362 227185. E-mail address: [email protected] (R. Harikrishnan).
parameters including antimicrobial peptides, complement factors, and immunoglobulins [2]. Traditional disease control strategies with antibiotics and chemical disinfectants are no longer recommended practices due to the emergence of bacterial resistance and also concerns over environmental impacts and wildlife protection [3e5]. Vaccination, an effective prophylactic method boosts the immune system preventing the incidence of microbial infection. However, there are some methodological issues like being very expensive and stressful to fish. Nowadays a remarkable success has been achieved with herbal immunostimulants as a more environmentally friendly approach in disease management [6e16]. An herbal immunostimulant is defined as a naturally occurring compound that modulates the immune system by increasing the host's resistance against diseases [6e16]. These treatments are primarily aimed at enhancing the innate immune system and therefore of great value as general preventive measures in aquaculture. During the past few years, commercial fish farming has been severely affected due to
http://dx.doi.org/10.1016/j.fsi.2015.02.018 1050-4648/© 2015 Published by Elsevier Ltd.
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various diseases especially by bacteria inflicting severe economic losses due to Vibrio alginolyticus, Vibrio carchariae, Pseudomonas sp., and Flexibacter sp. infection. Avicennia marina one of the common tree species of mangrove forest ecosystem belonging to the family Verbenaceae, is a cosmopolitan species widely distributed along tropical and subtropical coastlines. From the bark, leaf, and fruits of A. marina a number of active compounds like alkaloids, phenolics, steroids, terpenoids etc. have been isolated [17e23]. From leaf extract of A. marina by GC-MS we recently identified major phytochemical compounds namely Dehydrovomifolio, Diterpene, Pyrogallol, Levoglucosan, Glycerin, Ketone, Sesquiterpenoids, Phthalates, Dihydro, trimethyl Coumarin, Polysaccharide (unpublished data); these compounds are well-known as antioxidants, enzyme inhibitor, precursors of toxic substances and possess anti-inflammatory, antioxidant, anti-allergic, anticarcinogenic activities [24,25]. The bark, leaves, and fruits of A. marina have reported as antibacterial, antifungal, antiviral agents and also possess anticancer, antiplasmodial, antitumor, and antiulcer properties [26e33]. Recently V. alginolyticus was isolated and reported during breeding time and in the juvenile stages of Amphiprion sebae at the Marine Ornamental Fish Breeding Hatchery in the Centre of Advanced Study in Marine Biology, Annamalai University leading to mass mortality (personal communication). This bacterium also has been isolated from moribund A. sebae collected from the Marine Ornamental Fish Breeding Hatchery [34,35]. Hence to tackle this problem the application of one of the dominant mangrove tree species A. marina known for its medicinal properties [34] was attempted. The demand for ornamental fish like clown fish is rapidly increasing with the consequent increase in the ornamental fish hatcheries. In this back drop since there is a dearth of information with reference to phytotherapeutic effect of A. marina in aquatic animals, the present study was conducted to analyze the immunomodulatory effects and disease resistance for the first time in clownfish, A. sebae against V. alginolyticus infection. 2. Materials and methods 2.1. Collection and extraction of mangrove plants Leaves of A. marina were collected from the Vellar estuary mangroves. The leaves were washed in sterile water and shade dried for one week. The dried leaves were pulverized in a mechanical grinder, sieved, and then stored in an air tight container for further use. One hundred grams of the coarsely powdered sample was successively extracted with 85% ethanol and then filtered. The successive extraction was performed by a cold maceration process for seven days with daily agitation twice. The solvent was evaporated using a rotary vacuum evaporator (Buchi, Flawil, Switzerland) and the residues were stored at 20 C until used for the experiment. 2.2. Vibrio alginolyticus The pathogenic bacterium, V. alginolyticus (AUMOFP2) was previously isolated moribund A. sebae [34] and maintained in the laboratory under standard conditions [34]. Subcultures were maintained on marine zobell agar (Himedia, Mumbai) in slopes at 5 C and routinely tested for pathogenesis by inoculation into marine ornamental fish. Stock culture in marine zobell broth (Himedia, Mumbai) was stored at 70 C in 0.85% NaCl with 20% glycerol (v/v) to provide stable inoculate throughout the experiment. The culture was centrifuged at 1000 g for 10 min at 4 C. The supernatant were discarded and the bacterial pellet was washed
three times and re-suspended with phosphate-buffered saline (PBS) at pH 7.4. The optical density (OD) of the solution was adjusted to 0.5 at 456 nm which corresponded to 1 107 cells ml1. 2.3. Preparation supplementation feed The experimental diets and the proximate composition of the formulated diets were mentioned in Table 1. The ingredients were well mixed and extruded through a pellet extruder. Four experimental pellet diets, 0 (control basal diet without extract), 1, 4, and 8% A. marina extracts sprayed into the basal diet slowly, mixing evenly in a drum mixer, after which it was air dried under sterile conditions for 12 h. The control basal diet was added with the same volume of solvent without the extracts. The pellets were dried in an oven at 30 C for 18 h, packed, and stored in a freezer at 20 C until used. 2.4. Fish Healthy Clownfish, A. sebae (20.2 ± 5.4 g in weight) was collected from the marine ornamental fish hatchery, Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai. The fishes (n ¼ 500) were acclimated in 5000 l cement tank filled with 2500 l of UV treated seawater and was provided continuous aeration. Fish were acclimatized for 2 weeks prior to initiating the experiment and the fish were provided formulated control basal diet (Table 1) twice a day. During the experiment, water temperature was 28.2 C ± 1.4, pH 8.2 ± 0.3, salinity 28 ± 2.2 ppt, dissolved oxygen concentration 5.8 ± 0.6 mg l1, and 14 h light: 10 h dark photoperiod were measured. 2.5. Experimental design and challenge study A total number of 300 fish (A. sebae) were selected and divided into 5 groups of twenty fish each in three replicate (5 20 3) as: 1) uninfected control fish fed basal control diet without extract, 2) infected fish fed basal control diet without extract, 3) infected fish fed 1% extract supplementation feed, 4) infected fish fed 4% extract supplementation feed, and 5) infected fish fed 4% extract supplementation feed. After 2 weeks of feeding, all groups except control group intramuscularly injected with 0.1 ml of V. alginolyticus at a
Table 1 Composition of the formulated fish feed. Ingredients (g/100 g)
0%
1%
2%
4%
Acetes Mytha Ground net oil cake Rice bran Cod liver oil Vitamina & Mineralb complex Herbal extract Proximate composition Crude protein Crude lipid Crude ash Moisture Fibre
50 10 27 10 2 1 0
50 10 27 9 2 1 1
50 10 27 8 2 1 2
50 10 27 6 2 1 4
41.6 7.3 7.7 7.1 3
41.8 7.3 7.8 7.2 3
42.3 7.2 7.8 7.2 3
42.6 7.1 7.9 7.3 2
a Contains (g/100 g premix): DL-calcium pantothenate 0.5, choline bitartrate 10, inositol 0.5, menadione 0.02, niacin 0.5, pyridoxine-HCl 0.05, riboflavin 0.1, thiamine mononitrate 0.05, DL-a-tocopheryl acetate 0.2, retinyl acetate 0.02, biotin 0.005, folic acid 0.015, B12 0.0003, cholecalciferol 0.006, a-cellulose 80.0. b Contains (mg/100 g premix): Al 0.10, Ca 500, Cl 10, Cu 0.5, Co 0.8, Na 0.120, Mg 50, P 5000, K 420, Zn 0.3, Fe 4, I 0.50 Se 0.02, Mn 0.8.
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dose 1 107 cells ml1. The experimental fish was provided the corresponding diet twice a day until the end of experiment. 2.6. Bleeding and sampling Six fish in each tank were sampled for hematology, immunological parameters, and gut micro flora study. The blood was collected from the caudal vasculature following anesthesia with MS-222 (SigmaeAldrich, St. Louis, MO, USA) using a 1 ml heparinized syringe with 24 gauge needles and transferred into a heparinized tube. The plasma was recovered after centrifugation at 2000 rpm for 10 min and immediately stored at e 20 C until used for the experiment. 2.7. Head kidney (HK) leucocytes The head kidney (HK) leukocytes isolates from peripheral blood according to Chung and Secombes [36]. In each fish 0.5 ml of blood was collected and diluted with 2 ml of RPMI 1640 (Gibthai) and then carefully laid onto 3 ml of Histopaque (Sigma) in a 15 ml tube. It was centrifuged at 400 g for 30 min at room temperature, a white buffy coat of leukocytes cells floated on the top of the Histopaque and the opaque interfaces were carefully aspirated with a Pasteur pipette and transferred into a clean 15 ml tube. Make 6 ml with PBS (pH 7.4), gently mixed by aspiration, and centrifugation at 250 g for 10 min. The centrifugation step repeated 3 times to complete remove any residual Histopaque. The isolated HK leukocytes cells were then re-suspended in the PBS and adjusted to the required cell numbers for phagocytosis and respiratory activities. 2.8. Supplemented feed action on gut bacteria Total gut bacteria of the experimental and control group were assessed by the modified protocol of Velmurugan and Citarasu [37] and find out the effects of supplemented feed containing A. marina extracts on the normal gut bacterial flora. The whole gut was removed aseptically in all fish from each group, weighed, and the surface washed with 85% sterile saline and than homogenized. The homogenate was diluted in sterile seawater (32‰) and plated in nutrient agar (Hi-media, India) and TCBS (Hi-media, India). The plates were incubated at 30 C for 48 h and the colonies were counted quantitatively. 2.9. Total white blood cell (WBC) counts The blood was collected from the fishes along with diluting Hayem's fluid and incubated 5 min for complete haemolysis of RBCs. After incubation, the blood mixture was loaded on the improved Neubauer haemocytometer by holding the pipette at 45 angle and the cover slip was placed. The cells were counted and expressed as cells ml1. 2.10. Serum lysozyme activity Serum lysozyme activity was measured according to the modified method described by Ellis [38]. Briefly, 10 ml of individual serum was mixed with 200 ml of a Micrococcus luteus suspension at 0.2 mg ml1 in 0.05 M sodium phosphate buffer (pH 6.2). The mixture was incubated at 27 C and its OD was recorded at 530 nm using an ELISA plate reader. One unit of lysozyme activity was defined as the amount of enzyme produced at a decrease in absorbance of 0.001 min1 ml1 serum. Lysozyme concentrations were calculated from a standard curve of known lysozymes from chicken albumin concentrations.
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2.11. Respiratory burst assay To detect the Nitro Blue Tetrazolium chloride (NBT) activity, 100 ml of blood containing anti-coagulant was placed into a microtitre plate well and then equal amount of 0.2% NBT solution was added. After incubation at room temperature for 30 min, 0.05 ml of NBT blood suspension was transferred to glass tube containing 1 ml of N, N dimethyl formamide and centrifuged at 400 g for 5 min at 4 C. The spectrophotometric reading was measured the OD at 620 nm [39]. 2.12. Alternative complement (ACH50) assay ACH50 was determined adopting the method of Sunyer and Tort [40] with the following small modifications. Sheep red blood cells (SRBCs) were used as target cells in the presence of gelatin veronal buffer. Individual 20 ml aliquots (2-fold dilutions) of a serially diluted serum with EGTA-Mg2þ- GVB buffer (10 mM ethylene glycoltetraacetic acid and 10 mM MgCl2 in GVB) was used as a complement source, which was mixed with 6 ml of SRBC suspension (4 108 cells ml1), and the mixture was incubated at 21 C at pH 7.2 for 2 h. The hemolytic reaction was stopped by adding 200 ml of GVB containing 10 mM EDTA. The mixtures were centrifuged at 1600 g for 10 min at 4 C. The OD of the supernatants was measured at 414 nm using ELISA reader (A). The reactions were supplemented with 6 ml EDTA-GVB, 20 ml EDTA-GVB and 220 ml distilled water to replace the SRBC suspension, the diluted serum and the diluted serum with EDTA-GVB buffer, respectively, as the SRBC blank (B), serum blank (C) and 100% hemolysis sample (D). The degree of hemolysis (Y) was defined as Y ¼ (A (B þ C)) (D C) 1 and calculated and the lysis curve for each specimen was obtained by plotting Y (l Y) 1 against the volume of complements added on a log/log-scaled graph. The volume of serum complement producing 50% hemolysis (ACH50) was determined and the number of ACH50 units ml1 was calculated for each experimental group. 2.13. Phagocytic activity Phagocytic activity was measured from HK leucocytes according to Matsuyama et al. [41]. A suspension of leucocytes (100 ml of 1 107 cells ml1) was placed in a sterile slide and allowed to attach for 30 min at 25 C. Then, 100 ml yeast suspension (Baker's yeast, Type II, Sigma, 1 108 cells ml1) was added to the cell monolayer. The slide was incubated for 45 min at 25 C and then the same was washed three times with PBS to remove the unattached cells and uningested yeasts. After air-drying, the slides were fixed in ethanol, redried and stained with Giemsa. The percentage of phagocytic index was calculated by enumerating phagocytes under a microscope the following formula: number of engulfed bacteria/number of phagocytic cells 100. 2.14. Survival rate Groups of 20 fish each in triplicate were fed with 0, 1, 4, and 8% extract supplemented diets twice a day at 5% of their body weight. After 2 weeks of feeding, the fishes were challenged with V. alginolyticus (1 107 cells ml1) and the survival rate was recorded for 30 days. Our earlier study, the LD50 dose was standardized of the bacterial concentration to give 80% mortality. 2.15. Statistics The data from treated groups were analyzed using one-way analysis of variance (ANOVA) to find out the significant difference to control group at the 5% (P < 0.05) level.
Please cite this article in press as: Dhayanithi NB, et al., Dietary supplementation of Avicennia marina extract on immune protection and disease resistance in Amphiprion sebae against Vibrio alginolyticus, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.02.018
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3. Results
3.6. Action of feed on gut flora
3.1. Total white blood cell (WBC) counts
The gut bacterial flora was analyzed in the experimental and control groups. Results indicate that the gut bacterial microbial flora was altered due to administration of mangrove plant extract. The maximum total viable counts (TVCs) were observed in groups fed with 8% and 4% supplementation diets: 2.8 105 cfu/g (38.32% Bacillus subtilis, 27.75% Bacillus circulans, 15.85% Lactobacillus sp., 11.52% Vibrio fluvialis, and 6.56% M. luteus) and 4.7 104 cfu/g (30.8% B. subtilis, 25.98% B. circulans, 22.1% Lactobacillus sp., 9.58% V. fluvialis, and 11.54% M. luteus). But when fed with 1% supplementation diet was found 8.9 103 cfu/g (29.5% B. subtilis, 25.3% B. circulans, 20.0% Lactobacillus sp., 8.65% V. fluvialis, and 14.55% M. luteus).
The white blood cell (WBCs) of the experimental group compared with control as shown in Fig. 1. The WBC level progressively increased in all supplementation diets as compared to control on weeks 2 and 4. However, it increased to the maximum in all supplementation diets on weeks 6 and 8 when compared to the control. 3.2. Serum lysozyme activity The serum lysozyme activity did not increase statistically (P > 0.05) in all supplementation diets as compared to control on weeks 2 and 4. Its production reached a statistically significant (P < 0.05) with 4% supplementation diet on week 6 and 1% and 4% supplementation diets on week 8 (Fig. 2). 3.3. Respiratory burst activity The respiratory burst activity did not vary in the fish fed with any supplementation diet on weeks 2 and 4 when compared to control (Fig. 3). However, the respiratory burst activity significantly increased in the infected fish fed with 4% or 8% supplementation diets on weeks 6 and 8. 3.4. Alternative complement (ACH50) activity The ACH50 activity did not statistically increase (P > 0.05) with any supplementation diet on weeks 2 and 4 as compared with control (Fig. 4). However, the ACH50 activity showed the maximum on weeks 6 and 8. Interestingly, the activity increased significantly (P < 0.05) in fish fed with 1% and 4% supplementation diets on weeks 6 and 8. 3.5. Phagocytic activity The phagocytic activity did not significantly increased (P > 0.05) when fed with any supplementation diet during the experimental period as compared with control (Fig. 5). However, the phagotytic activity was significantly increased (P < 0.05) when fed with 4% supplementation diet on week 6 and 8 against the pathogens.
Control
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3.7. Survival rate The survival rate was 85% and 80% in fish fed with 4% and 8% supplementation diet with A. marina against V. alginolyticus. However, the survival rate declined to 70% when fish fed with 1% supplementation diet while in the infected untreated group it was found 10% (Fig. 6). 4. Discussion Enhancement of the immune system seems to be the most promising method of preventing diseases in fishes. The innate (non-specific) immune system of fish is considered to be the first line of defense against invading pathogens and is more important for fish than mammals [42]. The major components of the innate immune system are macrophages, monocytes, granulocytes, and humoral elements, like lysozyme or complement system [43]. In this context, the immunostimulants and adjuvants used in fish vaccines are of interest, as they offer an alternative to the drugs, chemicals, and antibiotics currently used in fish culture to control the fish diseases. In aquaculture, the use of medicinal herbs as immunostimulants continue to receive more attention since last two decades not only regarding their immune stimulating functions, but also for their growth promoting effects with little or no side effects [6e16]. It has been also shown that herbal based immunostimulants are capable of enhancing immune responses and/or reducing losses from viruses, bacteria and/or parasitic infections in freshwater and marine fishes [6e16,44e50]. A number of compounds such as alkaloids, phenolics, steroids, terpenoids etc. have been isolated from bark, leaf, and fruits of A. marina [17e23] which are exhibited antioxidant, antibacterial,
8%
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Weeks of feeding Fig. 1. Changes in total white blood cells in fish fed with different dosages (0, 1, 4, and 8%) supplementation diets with A. marina leaf extract against V. alginolyticus infection. The experimental groups statistical significantly different at P < 0.05 level as compared to control.
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Weeks of feeding Fig. 2. Serum lysozyme activity in fish fed with different dosages (0, 1, 8, and 8%) supplementation diets with A. marina leaf extract against V. alginolyticus infection. The experimental groups statistical significantly different at P < 0.05 level as compared to control.
Please cite this article in press as: Dhayanithi NB, et al., Dietary supplementation of Avicennia marina extract on immune protection and disease resistance in Amphiprion sebae against Vibrio alginolyticus, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.02.018
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Fig. 3. Respiratory burst activity in fish fed with different dosages (0, 1, 4, and 8%) supplementation diets with A. marina leaf extract against V. alginolyticus infection. The experimental groups statistical significantly different at P < 0.05 level as compared to control.
antifungal, anticancer, antiplasmodial, antitumor, antiantiviral, and antiulcer properties [26e33]. Therefore in the present study for the first time we investigated the innate immune response and disease resistance of A. marina extract in the economically important marine ornamental clownfish, A. sebae. The WBCs afford protection in fish against pathogens and chemical factors [51]. In the present study, the WBC level slightly increased in all supplementation diet as compared to control on weeks 2 and 4; its increased to the maximum on weeks 6 and 8. Monitoring these values and gathering information on the profile of leucocytes can reflect on the general immune status in fish. There is a poor correlation between differential composition of leucocytes and increase in the neutrophiles percentage in fish associated with viral or other infections [52]. Lysozyme a mucolytic enzyme produced by leucocytes is an important defense molecule of the innate immune system and plays a vital role in mediating protection against microbial invasion. In the present study, the maximum protection was observed in all supplementation diets on weeks 6 and 8; it was statistically significant (P < 0.05) with 4% supplementation diet on week 6 and 1% and 4% supplementation diets on week 8. A similar activity of lysozyme was observed in Jian carp [53] and in large yellow croaker [54] with traditional Chinese medicine (TCM) formulated from Astragalus radix and Angelica root. Oreochromis niloticus fed with 0.1% or 0.5% supplementation diets A. radix [49] and Labeo rohita fed
Control
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Weeks of feeding Fig. 4. Alternate complement activity in fish fed with different dosages (0, 1, 4, and 8%) supplementation diets with A. marina leaf extract against V. alginolyticus infection. The experimental groups statistical significantly different at P < 0.05 level as compared to control.
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with Achyranthes aspera showed significantly enhanced the lysozyme activity [55]. In this study, the respiratory burst activity significantly enhanced with 4% and 8% supplementation diets on weeks 6 and 8. The present results are agreement with the previous report on L. rohita fed with A. aspera diet significantly enhanced superoxide anion production [55]. Jang et al. [56] reported that in vitro treatment with glycyrrhizine isolated from Glycyrrhiza glabra enhanced the respiratory burst activity of macrophages and the proliferative response of lymphocytes in rainbow trout. Phagocytes produce toxic oxygen forms during the process of respiratory burst [57]. Since superoxide anion is the first product to be released from the respiratory burst, the measurement of O2 has been accepted as a precise way of measuring respiratory burst [58]. The respiratory burst activity was observed in large yellow croaker and common carp after feeding with a diet containing a mixture of A. membranaceus and A. sinensis extracts [53,54]. The present study has indicated that the different doses of A. marina extract could increase the alternative complement activity on weeks 6 and 8. Interestingly, the ACH50 activity was found statistically significant with 1% and 4% supplementation diets. This is in contrast to the findings of Cook et al. [59] where oral application of EcoActiva™, a commercial b-glucan preparation, did not change the alternative complement activity. In gilthead seabream also a significant enhancement in serum complement activity was observed after feeding with levamisole
8%
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Fig. 5. Phagocytic activity in fish fed with different dosages (0, 1, 4, and 8%) supplementation diets with A. marina leaf extract against V. alginolyticus infection. The experimental groups statistical significantly different at P < 0.05 level as compared to control.
Survival rate (%)
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Groups Fig. 6. Survival rate (%) in fish fed with different dosages (0, 1, 4, and 8%) supplementation diets with A. marina leaf extract against V. alginolyticus infection for 30 days.
Please cite this article in press as: Dhayanithi NB, et al., Dietary supplementation of Avicennia marina extract on immune protection and disease resistance in Amphiprion sebae against Vibrio alginolyticus, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.02.018
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containing diet [60]. However, no significant effect was seen on weeks 2 and 4 with any enriched diet with reference to the alternative complement pathway. This is in line with the findings of Cuesta et al. [61] when administration of propolis in gilthead seabream did not result in a significant change in the alternative complement activity. The bactericidal activity of the complement has been well recognized as one of the key killing mechanisms of clearing bacteria in fish [62]. Phagocytic cells are important cellular components of the innate immune system of fish [63] and it is a primitive defense mechanism and an important characteristic of the non-specific immune system [64]. The present results show that infected fish fed with 4% diet significantly enhance the phagocytic activity on weeks 6 and 8. Herbal extracts can also enhance phagocytosis in various fish species [6e16]. On the other hand, no major effect was found with any diet on week 2 and 4. This result suggests that the A. marina extracts containing diet enhanced the phagocytic activity after week 6. These results are agreement with the previous observations Scutellaria extract in O. niloticus [49]. Gut microflora play an important role in the digestive process and disease susceptibility of marine feeders [65]. The wild and cultured aquatic animals harbor a diverse bacterial flora, including Aeromonas, Plesiomonas, Photobacterium, Pseudoalteromonas, Pseudomonas, Vibrio, etc. [66]. So, an understanding of the host intestinal bacterial floral interactions is of much significance for the development of a healthy cultivation environment and also to optimize the growth of benign species. But, the intestinal bacteria, such as Aeromonas and Vibrio often occur as opportunistic pathogens [67]. In this study, the maximum TVCs recorded were 2.8 105 and 4.7 104 (cfu/g) when fish were fed with 8% and 4% supplementation diets, respectively. The present results reveal that the gut microbial flora changed qualitatively and quantitatively which might be due to the action of antibacterial active principles present in the mangrove extracts. Bacterial pathogens could have been retarded by the active extracts, by inhibiting the transcription and arresting the protein synthesis of the bacteria [68]. After being challenged with V. alginolyticus, all the treated groups showed a reduced mortality as compared to the infected untreated group. The maximum survival rate of 85% and 80% were observed in the group fed with 4% and 8% supplementation diet against V. alginolyticus. Enhancement of the non-specific immune parameters by the A. marina extracts could have possibly been an important factor in reducing the percentage mortality, protecting the fish against V. alginolyticus infection. Earlier studies in this direction have also revealed that dietary supplementation of various herbal extracts reduced the mortality and increased the survival rate against bacterial, viral, fungal, and parasitic infection [6e16]. Though, there was no positive correlation between the effect of immunostimulant and dosage, a higher dosage might enhance or inhibit the immune response [6e16]. It is important to estimate the increased protection in the mangrove extract treated fish to determine the efficacy of an immunostimulant. The present study suggests that the mangrove extracts (A. marina leaf) could strengthen the immune system with 4% and 8% in clownfish against V. alginolyticus. Further detailed immunological and molecular studies are needed to extend the application of these results in aquaculture as a prophylactic measure.
Acknowledgment Authors thank the authorities of Annamalai University for providing facilities. The first and second authors are thankful to the University Grants Commission (UGC), New Delhi for providing financial assistance. Reference No. U.G.C. No. 33-384/2007 (SR).
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Please cite this article in press as: Dhayanithi NB, et al., Dietary supplementation of Avicennia marina extract on immune protection and disease resistance in Amphiprion sebae against Vibrio alginolyticus, Fish & Shellfish Immunology (2015), http://dx.doi.org/10.1016/j.fsi.2015.02.018
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