Fish & Shellfish Immunology 36 (2014) 485e493

Contents lists available at ScienceDirect

Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi

Full length article

Protection of ornamental gold fish Carassius auratus against Aeromonas hydrophila by treating Ixora coccinea active principles Paulraj Anusha, Vijayaragavan Thangaviji, Subramanian Velmurugan, Mariavincent Michaelbabu, Thavasimuthu Citarasu* Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam, Kanyakumari, 629502 Tamilnadu, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 August 2013 Received in revised form 18 November 2013 Accepted 16 December 2013 Available online 28 December 2013

Herbals such as Ixora coccinea, Daemia extensa and Tridax procumbens were selected to screen in vitro antibacterial and immunostimulant activity against the freshwater fish pathogen Aeromonas hydrophila using different organic polar and non-polar solvents. Initial screening results revealed that, ethyl acetate extracts and its purified fraction of I. coccinea was able to suppress the A. hydrophila strains at more than 15 mm of zone of inhibition and positive immunostimulant activity. The purified active fraction, which eluted from H40: EA60 mobile phase was structurally characterized by GCeMS analysis. Two compounds such as Diethyl Phthalate (1,2-Benzene dicarboxylic acid, monobutyl ester) and Dibutyl Phthalate were characterized using NIST database search. In order to study the in vivo immunostimulant influence of the compounds, the crude extracts (ICE) and purified fractions (ICF) were incorporated to the artificial diets at the concentration of 400 mg kg
1 and fed to the ornamental gold fish Carassius auratus for 30 days. After termination of feeding experiment, they were challenged with highly virulent A. hydrophila AHV-1 which was isolated from infected gold fish and studied the survival, specific bacterial load reduction, serum biochemistry, haematology, immunology and histological parameters. The control diet fed fishes succumbed to death within five days at 100% mortality whereas ICE and ICF fed groups survived 60 and 80% respectively after 10 days. The diets also helped to decrease the Aeromonas load after challenge and significantly (P  0.01) improved the serum albumin, globulin and protein. The diets also helped to increase the RBC and haemoglobin level significantly (P  0.05) from the control group. Surprisingly the immunological parameters like phagocytic activity, serum bactericidal activity and lysozyme activity were significantly increased (P  0.001) in the experimental diets. Macrophages and erythrocytes were abundantly expressed in the treated groups and the present work concluded that, the Phthalate derivatives from I. coccinea helps to stimulate the immune system against A. hydrophila challenge in C. auratus. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Aeromonas hydrophila Antibacterial Carassius auratus Ixora coccinea Immunostimulants

1. Introduction Ornamental fishes are nowadays rapidly gaining importance because of their aesthetic value and also due to their immense commercial value in the export trade world over [1]. The art of keeping aquarium is ancient and it started around 800 B.C. in China with the gold fish Carassius auratus, which is in demand and a popular ornamental fish throughout the world even today. These fishes are often called as “living jewels” due to their varied colour, shape and behaviour [2]. In the ornamental aquaculture sector, ornamental fish breeding, culture and trade provide excellent

* Corresponding author. Tel./fax: þ91 4652 253078. E-mail addresses: [email protected], [email protected] (T. Citarasu). 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.12.006

opportunities as a non-food fishery activity for employment and income generation. It is environment friendly, socially acceptable and involves low investment for adopting as a small scale enterprise with high return. The attractive colouration and quiet disposition of ornamental fish provide a source of joy and peace for people irrespective of age group [3]. Nowadays, ornamental aquaculture industry is facing bacterial disease outbreaks resulting high mortality and loss of economy. Gold fish C. auratus has high susceptibility to aeromonads and are commonly valuable for experimental animals [4]. Aeromonas hydrophila, gram negative facultative anaerobic short bacillus, causes red fin disease, haemorrhagic septicaemia, motile aeromonad septicaemia and other infections in C. auratus [5]. Potential virulence factors of A. hydrophila, which contribute to their pathogenicity, include the production of endotoxins, extracellular

486

P. Anusha et al. / Fish & Shellfish Immunology 36 (2014) 485e493

enterotoxins, haemolysin, cytotoxins and protease, the ability to adhere the cells, and the possession of certain surface proteins [6]. Outbreaks of motile aeromonad septicaemia usually occur whether fish are immune compromised due to unpleasant environment or predisposing factors leading to stresses such as temperature, overcrowding, organic pollution and hypoxia. The current bacterial disease treatment protocols are rather difficult, non effective, un-safety, costly and create so many environmental hazards such as residual effects, biomagnifications and resistant strain development etc [7]. Humoural and cellular immune responses of common carp have been shown to be suppressed during treatment with oxytetracycline, and an increased number of granulocytes were observed in the spleen of treated fish [8]. Increasingly, antibiotic resistance of bacterial pathogens is reported from all areas of aquaculture, ranging from warm water to cold water. The use of antibiotics in the fish/shrimp hatcheries has led to biomagnifications that in turn leads to rejection of the total consignment during export [9]. Marine product development export authority (MPEDA) of India has also banned more than 20 antibiotics due to their bad effects. In order to achieve optimal fish production, better prophylactic, diagnostic and therapeutic measures are warranted during fish farming operations. To develop the alternative practices for disease management in aquaculture, attention should be diverted to find novel drugs, especially from plant sources. Plant-derived compounds act as a better antibacterial, antiviral, immunostimulant and antistress effect in fish and shellfish aquaculture [7,10e12]. Plants are the storehouses and rich sources of safe and cheap chemicals. Many plant-derived compounds have been found to have non-specific immunostimulating properties in animals, of which more than a dozen have been evaluated in fin and shellfishes [13,14]. Sivaram et al. [15] successfully controlled the Vibrio pathogen, and improved the immune system of grouper larviculture using herbal methanolic extracts. These findings suggest that phytochemicals could be an alternative to the chemotherapeutic molecules and safe to use in aquaculture. Also, the herbal immunostimulants Emblica officinalis, Cynodon dactylon and Adathoda vasica improved the immune system and reduced microbial infection in the gold fish C. auratus [16], and similar work was carried out by Magdelin [17] on the ornamental fish Poecilia sphenops using herbal immunostimulants. The extracts of the flowers and leaves of Ixora coccinea have been reported to show cytotoxic, antitumour [18]; antimicrobial, anti-inflammatory [19] and antioxidant activities [20]. The present study focus on the antibacterial and immunostimulant effect of I. coccinea extracts on ornamental gold fish C. auratus against A. hydrophila challenge. 2. Materials and methods 2.1. Source of A. hydrophila A highly virulent strain of A. hydrophila (AHV-1; GenBank: HQ331525.1) and medium virulent strain CMST-1 (GenBank: JX575135.1) used for this study were isolated from infected ornamental gold fish C. auratus during massive outbreak of disease in the ornamental fish hatchery at Nagercoil, Kanyakumari District of India [21,22]. 2.2. Herbal extracts for in vitro antibacterial activity against virulent A. hydrophila Three herbal extracts including I. coccinea, Daemia extensa and Tridax procumbens having antibacterial and immunostimulant characteristics were selected following Nadkarni [23] and Citarasu [9]. These were extracted serially with hexane, ethyl acetate and

methanol. Aqueous extracts were concentrated using lyophilizer and stored at 4  C for further use. 2.2.1. In vitro antibacterial activity Known quantity of different plant organic extracts condensate were impregnated in 5-mm diameter sterile paper discs (Himedia, India) and screened against virulent A. hydrophila (AHV-1 & CMST1) with three replicates of the disc diffusion test as described by Bauer et al. [24]. After drying, the impregnated discs with herbal extracts were carefully dispensed with uniform distances over Muller Hinton agar surface and correct implantation was assured by applying gentle pressure over the disc. Control tests were carried out using sterile discs without impregnation of the herbal extracts. All plates were kept at 35  C for 24 h incubation. After the incubation, plates were studied for inhibitory zone formation of antibacterial extracts on microbial lawns in agar surface. 2.2.2. In vitro immunostimulant activity In vitro immunostimulation was carried out following the method of Sritunyalucksana et al. [25] with slight modification. Blood was bled from uninfected C. auratus and left to clot at 25  2  C for 1 h and stored in
80  C deep freezer for 5 min and thawing to induce lysis. Repeated freeze and thaw cycle resulted in complete haemocyte lysis. The liquid fraction was then collected and designated as the Cell lysate fraction (CLF). One gram of plant extract condensate was dissolved in 100 ml of distilled water (W/v) as stock solutions. Five microlitres of each extracts was incubated with 100 ml of CLF at 25  2  C for 1 h. The immunostimulantincubated CLF was used for testing the antibacterial assay with A. hydrophila [12]. The 100 ml of immunostimulated CLF was incubated again with 100 ml A. hydrophila bacterial culture 1  103 cfu ml
1 for 30 min at 25  2  C. Control experiments were performed for A. hydrophila incubated with CLF without incubation of immunostimulants herbal extracts. Triplicate samples of 20 ml each were drop-transferred to Tryptic Soy Agar (TSA) (Hi media, India) to obtain bacterial counts (CFU) after incubation at 30  C for 12 h. Based on the positive immunostimulation results, the extracts were selected for further analysis. 2.3. Purification of the ethyl acetate extract of I. coccinea Based on the better antibacterial and immunostimulant activities among the herbal extracts, ethyl acetate extract was selected for further works including purification, characterization and in vivo delivery. For purification, preparative silica column chromatography (50e80 mm particle size; 30 cm column length; 0.5 ml elution flow rate and three bed volume elution) was used. The column was equilibrated with 100% hexane and one gram of the condensate of the ethyl acetate extract of I. coccinea was loaded on the top of the column. Different proportions of the mobile phases such as hexane/ ethyl acetate and ethyl acetate/methanol were used for eluting the compounds. The different fractions were collected, concentrated in a rotary evaporator and stored at 4  C. The fractions were spotted on silica gel plates GF254 (Merck), 20  20 cm, 1 mm thick and the chromatogram was developed using, hexane: ethyl acetate (8:2) as mobile phase. The plates were visualized under short UV wavelength. Secondary antibacterial and immunostimulant screening was also performed against the virulent A. hydrophila strains using different fractions following the method mentioned the Section 2.2. 2.4. Structural characterization of I. coccinea fraction by GCeMS analysis The fraction which eluted by H 40: EA 60 mobile phase was selected for GCeMS analysis based on the highest zone of inhibition

P. Anusha et al. / Fish & Shellfish Immunology 36 (2014) 485e493 Table 1 Composition of basal diet supplemented with I. coccinea active principles. Ingredients

Brown fish meal Shrimp head meal Squid meal Squid liver powder Wheat gluten Wheat flour Soy bean meal Broken rice Fish oil Vitamin premix Mineral mix I. coccinea crude extracts I. coccinea purified fraction Others

Diet composition (g kg
1) ICE

ICF

280 100 20 30 60 200 100 102 20 7.8 40 0.4 e 39.8

280 100 20 30 60 200 100 102 20 7.8 40 e 0.4 39.8

487

healthy fishes (50 fish tank
1) were stocked in each treatment group. Water quality of the control and experimental tanks was monitored daily for dissolved oxygen 6.2e7.5 mg
l, pH 7.2e8.1. The tanks had a capacity of 500 l with a flow-through system having a water flow rate of 1 ml min
1. The fishes were fed ad libitum thrice daily with the experimental formulated diet at a ratio of 10% of their body weight per day throughout the period of study. Partial water exchange was done daily to remove waste feed and faecal materials and the experimental period prolonged for 30 days. 2.7. Bacterial challenge and specific bacterial reduction

after secondary screening. The active fraction of I. coccinea was analysed using Gas Chromatography Mass Spectrometer (GC Clarus 500 Perkin Elmer). The column was Elite-5 MS (5% Diphenyl/95% Dimethyl poly siloxane), 30  0.25 mm  0.25 mm df. Column condition was programmed as column oven temperature 110  C. Up to 200  C at the rate of 10  C/min-No hold, Up to 280  C at the rate of 5  C/min-9 min hold. Injector temperature port was 280  C and total GC running time was 36 min. Helium was used as the carrier gas at a flow rate of 1 ml/min. Mass detector Turbo mass goldPerkin Elmer was used. Mass spectra were recorded in the scan mode at 70 eV (45e450 amu). The data obtained was compared with the mass spectral compounds available in the NIST-05 library.

After the termination of feeding experiment (i.e. the 31st day morning), 10 fishes from each tank were intraperitoneally injected with 100 ml of live AHV-1 at a LC50 dose concentration of 1  107 cfu fish
1. Survival and pathological signs were monitored in every 3 h intervals for 10 days. Blood was collected from the caudal vein of the challenged and un-challenged fish with a 1 ml plastic syringe and rinsed with anticoagulant (10% trisodium citrate), and 100 ml of blood was plated in Aeromonas agar for Aeromonas count. The fishes were then weighed aseptically and immersed in 50 ppm Formalin solution for 5 min (to remove external bacteria). Then they were washed thoroughly under sterilized water for 30 s to remove the remaining surface bacteria and disinfectant. The muscle was dissected out using sterile scissors and homogenized with 5 ml of 85% sterile saline and diluted up to 10-fold. Using a sterilized pipette, a 100 ml sample was taken and poured into Aeromonas isolation medium. Triplicates were maintained for each sample and incubated at 37  C for 48 h.

2.5. Diet preparation

2.8. Haematological parameters

Diets were prepared following the basal ratio of 45.1% protein; 7.2% lipids, 14.6% ash, 7.1% moisture and 3% fibre (Table 1). Two test diets were prepared by incorporating the ethyl acetate extract of I. coccinea (ICE) and the fraction eluted from H 40: EA 60 mobile phase (ICF) and mixed to the feeds in the concentration of 400 mg kg
1 diets based on the optimal level of herbal extracts to fish and shellfishes [26]. A control diet, devoid of herbal immunostimulant was also prepared. To prepare the diets, ingredients were mixed thoroughly and 4% gelatin solution containing the active principles at the appropriate concentration was added along with the oil ingredients. Sufficient water was added and the pH was adjusted to 7  0.1. The mixture was then cold extruded, cut into pellets, air dried and stored at room temperature.

Twenty five of randomly chosen fish from each tank were taken and anaesthetized with 50 mg MS-222 dm
3 of water. Blood was collected from the caudal vein using a 1 ml plastic syringe rinsed with anticoagulant. Part of the blood was transferred immediately and added to an equal volume of 10% trisodium citrate and then stored at 4  C. The remaining blood was kept at room temperature for 1 h, without anticoagulant (to collect the serum), and stored at
40  C. Live unfixed blood sample was quickly drawn into a RBC dilution pipette up to 0.5 marks and diluted up to 10
1 marks using RBC diluting fluid (Mercuric chloride 2.5 g, Sodium chloride 0.5 g, sodium sulphate 2.5 g, distilled water 100 ml with the addition of 0.01% gentian violet). After filling the pipette was shaken several minutes to prevent coagulation and uniform staining. The first few drops of blood were discarded and the Neubuer’s Haemocytometer was loaded immediately with the blood, which was allowed to fill the chamber by capillary motion. Cells found in the entire centre 1 mm squares were counted and the following formula of Jones (1962) was adopted for THC calculation.

2.6. Experimental setup and feeding Healthy gold fish C. auratus had the mean weight of 19.2  1 g were purchased from local ornamental fish hatchery and accli-

  Haemocytes in 1 mm square  Dilution  Depth of chamber Total Count cells=mm3 ¼ Number of 1 mm square counted

matized in fibreglass-reinforced plastic aquaria (1000 l capacity) for 10 days to meet established laboratory conditions and they were fed with control feeds which prepared for the experimental works. After acclimatization, triplicate tanks containing 150

Haemoglobin level was determined by the method of cyanomethaemoglobin method described by Van Kampen and Zijlstra [27]. Total erythrocyte count was performed following the method of Hendricks [28] using a haemocynometer.

488

P. Anusha et al. / Fish & Shellfish Immunology 36 (2014) 485e493

2.9. Blood serum biochemistry Saturated ammonium sulphate (NH4)2SO4, (40%) was added to 100 ml of serum in microcentrifuge tubes, mixed well and left to stand for 1 h at room temperature. The tubes were centrifuged at 10,000  g (10 min), and the supernatant (albumin fraction) from each tube was collected into separate tubes. The precipitate (globulin fraction) was then dissolved in 500 ml of distilled water. The protein content of both albumin and globulin fractions was determined by the method of Lowry et al. [29]. 2.10. Immunological parameters To study the serum bactericidal activity, five fishes from each group were injected with 100 ml of live virulent A. hydrophila suspension at the rate of 105 cells ml
1 and blood samples were collected 10 and 90 min after injection. One hundred micro litres of blood was serially diluted and plated in the Aeromonas agar (Hi media, India). For the phagocytic assay, 107 cells of formalin killed A. hydrophila were added to 100 ml of pooled blood samples in a sterile microplate and incubated for 30 min at 25  C after thorough mixing in the well. Following incubation, the bloodebacteria suspension was mixed gently, and 50 ml of this suspension was smeared on 3 glass slides. After air drying, the smears were fixed in 95% ethanol, redried and stained with May-Grunwald’s Giemsa. The phagocytic cells and phagocytosed bacteria were counted [30]. Intra-agar lysozyme activity was performed against Micrococcus luteus culture by diluting the blood cells and delivering a drop onto the agar wells. Once the drops were allowed time to absorb into the agar, the wells were incubated upside down for 24e48 h at 37  C. The wells were then scored for the highest dilution of blood cells capable of lysing the test microbes. 2.11. Histology studies After 4th day of A. hydrophila challenge, muscle tissues were dissected out from the control and experimental fishes and fixed in 10% buffered formalin for 24 h. After 24 h, the fixed muscle tissues were processed in graded levels of alcohol, cleared in xylene, impregnated and embedded in paraffin wax. Trimmed blocks of the embedded tissues were treated, overnight, with 0.5% trichloroacetic acid (TCA) as a decalcification process. Decalcified tissue was thoroughly washed in running water and sectioned (5e6 mm). Deparafinized sections were then rehydrated in graded alcohol, stained with H & E, dehydrated, cleared in xylene and mounted using DPX. Stained sections were observed under a compound microscope (Cos Lab, India) and the observations were recorded and photographed [31]. 2.12. Data analysis One-way and two-way ANOVAs were carried out using the SPSS statistics data package and Ky plot, respectively. Means were compared at 0.05 to 0.001% levels. 3. Results 3.1. In vitro antibacterial and immunostimulant screening of herbal extracts In vitro antibacterial and immunostimulant activities for the herbal organic solvent extracts were given in Table 2. Among the three different herbals, the herbal extracts of I. coccinea were able to suppress the growth of both virulent A. hydrophila AHV-1 and

Table 2 Antibacterial and immunostimulant screening of herbal organic solvent extracts against selected highly virulent A. hydrophila isolates. Herbal plant

Extraction

Antibacterial activity by zone of inhibition (mm)

Immunostimulant activity

AHV-1

AHV-1

CMST-1

Hexane 12.23  0.20 13.4  0.21 e Ethyl acetate 17.13  0.11 18.23  0.20 e Methanol 12.16  0.15 10.13  0.11 _ Tridax Hexane 9.13  0.11 5.13  0.12 þ procumbens Ethyl acetate 8.16  0.15 9.1  0.1 þ Methanol 7.23  0.20 8.06  0.05 þ Daemia extensa Hexane 7.1  0.11 5.2  0.20 þ Ethyl acetate 5.13  0.12 9.13  0.11 þ Methanol 9.1  0.1 7.2  0.26 e Ixora coccinea

CMST-1 e e e þþ þ þ þþ e þ

e: no growth, þ: minimum growth; þþ: maximum growth.

CMST-1 at more than 10 mm zone of inhibition. The maximum antibacterial activity of 17.13 and 18.23 mm zone of inhibition were found in the ethyl acetate extract of I. coccinea against AHV-1 and CMST-1 A. hydrophila strains respectively. The in vitro immunostimulant activity also reflected the same way of antibacterial activity in I. coccinea extracts. There was no Aeromonas growth observed in the I. coccinea extract incubated CLF and there was a high Aeromonas growth observed in the other extracts, T. procumbens and D. extensa. 3.2. In vitro antibacterial and immunostimulant screening of I. coccinea fractions The screening of column eluted fractions against A. hydrophila revealed that, the fractions which eluted from H40: EA60 and H50: EA50 had the highest antibacterial activity. The highest antibacterial activity of 19.2 and 17.12 mm zone of inhibition against AHV-1 and CMST-1 by treating H40: EA60 fraction and 14.4 and 10.32 mm of zone of inhibition against AHV-1 and CMST-1 by treating H50: EA50 respectively. The same fractions had also responsible for highest immunostimulant activity among the tested fractions. There is no Aeromonas growth observed in the fractions which were eluted from H40: EA60, H50: EA50 and H60: EA40 respectively (Table 3). 3.3. Structural characterization of I. coccinea fraction by GCeMS analysis The ethyl acetate extract of I. coccinea fraction eluted from H40: EA60 through silica column chromatography had two higher Table 3 Antibacterial and immunostimulant screening of I. coccinea ethyl acetate fractions eluted by column chromatography against selected highly virulent A. hydrophila isolates. Fractions eluted by hexane and ethyl acetate (%)*

Antibacterial activity by zone of inhibition (mm)

H H H H H H H H

5.4 9.04 19.2 14.4 11.02 4.07d e e

20: 30: 40: 50: 60: 70: 80: 90:

EA80 EA 70 EA 60 EA 50 EA 40 EA 30 EA 20 EA 10

AHV-1      

Immunostimulant activity

CMST-1 0.10a 0.03b 0.26c 0.08d 0.15e 0.11f

4.2 7.04 17.12 10.32 13.8c 3.8c e e

     

0.2a 0.03b 0.24c 0.02d 0.20e 0.03a

AHV-1

CMST-1

þþ þ _ _ _ þþ þþ þþ

þþ þ _ _ _ þþ þþ þþ

*Fractions eluted by the mobile phases of hexane (H) and ethyl acetate (EA). e: no growth, þ: minimum growth; þþ: maximum growth. Means with the same superscript do not significantly (P  0.001) e One Way ANOVA.

P. Anusha et al. / Fish & Shellfish Immunology 36 (2014) 485e493

489

Fig. 1. Structural characterization of I. coccinea active fraction by GCeMS analysis.

intensity spectral peaks at 8.72 and 12.72% (Fig. 1). Based on the NIST search database, the spectral peak of 8.72 was confirmed as Diethyl Phthalate (1,2-Benzene dicarboxylic acid, monobutyl ester). The molecular formula is C12H14O4 and the molecular weight is 222. The other spectral peak of 12.72 was confirmed as Dibutyl Phthalate with the molecular formula and weight of C16H22O4 and 278 respectively (Fig. 2a and b). 3.4. Survival of I. coccinea treated C. auratus after A. hydrophila challenge The control, ICE and ICF treated C. auratus fishes were challenged with highly virulent A. hydrophila AHV-1 after completion of experiment. The control fishes were succumbed to cent percentage death within five days. Surprisingly, the ICE treatment helped to increase the survival to 60% and ICF treatment helped to 80% survival due to the antibacterial/immunostimulant active principles in the I. coccinea extracts and fraction. Two Way ANOVA revealed that the survival was significantly (F ¼ 41.12; P  0.001) increased from control group to herbal extracts treated groups (Fig. 3). 3.5. Specific bacterial reduction of I. coccinea treated C. auratus after A. hydrophila challenge The specific Aeromonas count in blood and muscle after I. coccinea extracts and fraction treatment in C. auratus are given in Table 4 up to 8 days. In control, C. auratus the initial Aeromonas count observed was 5.7  104 cfu ml
1 after 2nd days in blood and this was increased after 8 days when the bacterial count was 4.5  106. In muscles, the initial count of 3.7  102 was observed after 2 days and the count increased to the maximum of 3.7  105 cfu g
1 after 8th days. Fortunately, the I. coccinea extracts and fraction helped to drastically decrease the Aeromonas count in blood as well as muscle. After 8th days, the Aeromonas count observed in the blood were 2.5  102 cfu ml
1 and 1.7  102 cfu g
1 in muscle respectively in ICE treated C. auratus. The lowest Aeromonas counts were also observed in blood (0.5  101 cfu ml
1) and muscle (0.3  101 cfu g
1) of ICF treated C. auratus after 8 days.

Fig. 2. a&b. Phthalate compounds detected from the peak of GCeMS analysis. Diethyl Phthalate (1,2-Benzene dicarboxylic acid, monobutyl ester) detected from the peak at the retention time of 8.72 (2a) and Dibutyl Phthalate detected from the peak at the retention time of 12.72 (2b).

3.6. Serum biochemistry of I. coccinea treated C. auratus after A. hydrophila challenge The serum albumin observed was 153.5 mg/ml in control C. auratus after A. hydrophila challenge. The albumin level was significantly (P  0.01) increased to 164.3 and 168.3 mg/ml in ICE and ICF treated C. auratus respectively after A. hydrophila challenge. The lowest serum globulin (147.1 mg ml
1) was observed in control group due to pathogenic interruption. The ICE and ICF treatment helped to increase the globulin level to 160.1 and 163.5 mg ml
1 respectively and which suppressed the bacterial count. Before treatment, the serum protein observed was 298.2 mg/ml in blank control group. The level drastically came down to 278.5 mg ml
1 due to immunosuppression. The serum protein level significantly (P  0.01) improved of 309.3 and 315.3 mg ml
1 in ICE and ICF treated C. auratus respectively and it reflects immunostimulation (Table 5). 3.7. Haematological studies of I. coccinea treated C. auratus after A. hydrophila challenge The haematological parameters of I. coccinea treated C. auratus after A. hydrophila challenge was given in Fig. 4. The RBC count observed was 0.854  106 mm3
1 in control group whereas the I. coccinea extracts had significantly (P  0.05) increased results. The count increased to 1.32 and 1.42  106 mm3
1 in ICE and ICF treated C. auratus respectively. The lowest haemoglobin level (6.72 mg dl
1) was observed in control group and this was significantly (P  0.05) increased to 8.6 and 8.82 mg dl
1 in ICE and ICF treated C. auratus respectively.

Fig. 3. Survival of I. coccinea treated C. auratus after A. hydrophila AHV-1 challenge. Values are significantly differed each other’s (F ¼ 41.12; P  0.001) e Two Way ANOVA.

490

P. Anusha et al. / Fish & Shellfish Immunology 36 (2014) 485e493

Table 4 Aeromonas count in blood and muscle samples of I. coccinea treated C. auratus after A. hydrophila AHV-1 challenge. Treatments

Aeromonas count Blood (cfu ml
1)

Blank control Control ICE ICF

Muscle (cfu g
1)

2 Days

4 Days

8 Days

2 Days

4 Days

8 Days

e 5.7  104  0.32  101 2.6  104  0.3  101 2.3  104  0.5  101

e 7.3  105  0.7  101 6.0  103  0. 5  101 1.2  102  0.3  101

e 4.5  106  1.1  101 2.5  102  0.4  101 0.5  101  0.0

e 3.7  102  0.5  101 3.7  103  0.2  101 9.3  102  0. 02  101

e 2.1  103  0. 6  101 2.9  103  0.1  101 0.5  102  0.01  101

e 3.7  105  0.31  101 1.7  102  0.1  101 0.3  101  0.0

Table 5 Blood serum biochemistry of I. coccinea treated C. auratus after A. hydrophila AHV-1 challenge. Treatment

Serum biochemistry (mg ml
1) Serum albumin

Blank control Control ICE ICF

160.2 153.5 164.3 168.3

   

0.03a 0.15b 0.26c 0.06d

Serum globulin 153.4 147.1 160.1 163.5

   

0.2a 0.1b 0.1c 0.2d

Serum protein 298.2 278.5 309.3 315.3

   

0.15a 0.08b 0.05c 0.15d

Means with the same superscript do not significantly (P  0.01) e One Way ANOVA.

3.8. Immunological studies of I. coccinea treated C. auratus after A. hydrophila challenge The immunological parameters of serum bactericidal activity (%), phagocytic activity (%) and serum lysozyme activity (IU/ml) are given in Table 6. The serum bactericidal activity observed was 3.5% in control group fishes. This was significantly (P  0.001) increased to 11.10, 15.17% in ICE and ICF treated groups respectively. The active compounds (ICF) helped to increase the activity to maximum 3 times from the control group. The phagocytic activity also reflected the same way as in the serum bactericidal activity. The maximum phagocytic activity observed was 47.4% in ICF treated C. auratus and the activity increased more than one times from the control group. The lowest serum lysozyme activity of 23.1 IU/ml was observed in control group and this significantly (P  0.001) increased to 40.7 and 47.05 IU/ml in ICE and ICF treated groups respectively due to

immune enhancement of the active compounds in the extract and fraction. 3.9. Histology of I. coccinea treated C. auratus after A. hydrophila challenge The photomicrograph of the histological data is given in the plates by H&E staining (Fig. 5aec). Fig. 5b and c, macrophages and erythrocytes are abundantly expressed among the fragmented skeletal muscle fibres due to the immune enhancement by ICE and ICF treatment. In control C. auratus there is no expression observed and the active compounds such as diethyl and dibutyl phthalate from I. coccinea helped to induce the macrophages expression against the A. hydrophila interruption. 4. Discussion The aqueous and organic solvent extracts of I. coccinea leaf, flower and roots showed highly significant antibacterial activity against various bacterial pathogens including Staphylococcus aureus, Bacillus pumilius, Enterococcus faecalis, Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa [32e34]. The ethyl acetate extracts and its purified fractions are effectively suppressed the A. hydrophila strains (AHV-1 and CMST-1) at more than 15 mm of zone of inhibition. The phthalate type of active compounds was present in the extracts help to inhibit the A. hydrophila at maximum level. Generally herbal extracts are powerful antibacterial agents against various aquatic important bacterial pathogens. Vibrio harveyi was successfully controlled by the extract of Acalypha indica, Hygrophila spinosa, Picrorhiza kurooa, Tinospora cordifolia and Zingiber officinale in vitro and in vivo level on Epinephalus tauvina larviculture [26]. Also the herbal extracts including Adhatoda vasika, Murraya koenigii, Ocimum basilicum, Psoralea corylifolia and Quercus infectoria highly influenced to suppress the pathogenic vibrios and A. hydrophila isolated from shrimps and freshwater ornamental fishes respectively [35]. The I. coccinea ethyl acetate extract and its few fractions which eluted from H 40: EA 60, H 50: EA 50 and H 60: EA 40 had immunostimulant activity by suppressing the A. hydrophila growth during CLF incubation. The active compound of the extract/fractions was responsible for

Table 6 Immunological parameters of I. coccinea treated C. auratus after A. hydrophila AHV-1 challenge. Treatment

Immunological parameters Serum bactericidal activity (%)

Fig. 4. Haematological parameters of I. coccinea treated C. auratus after A. hydrophila AHV-1 challenge. Means with the same superscript do not significantly (P  0.05) e One Way ANOVA.

Blank control Control ICE ICF

6.17 3.52 11.10 15.7

   

0.03a 0.35b 0.08c 1.32d

Phagocytic activity (%) 35. 4 17.8 43.2 47.4

   

1.05a 1.21b 2.73c 2.56d

Serum lysozyme activity (IU ml
1) 31.7 23.1 40.7 47.05

   

1.34a 1.53b 2.33c 2.76d

Means with the same superscript do not significantly (P  0.001) e One Way ANOVA.

P. Anusha et al. / Fish & Shellfish Immunology 36 (2014) 485e493

Fig 5. aec. Histochemistry of the muscle section of (200) control (5a), ICE (5b) and ICF (5c) of I. coccinea treated C. auratus after A. hydrophila AHV-1 challenge. Macrophages and erythrocytes are abundantly expressed among the fragmented skeletal muscle fibres in b and c.

immunostimulation. The immunostimulant activity was proved by Raja Rajeswari et al. [12] in Fenneropenaeus indicus by treating the methanolic extracts of Acalypha indica, Hygrophila spinosa, Picrorhiza kurooa, Tinospora cordifolia and Zingiber officinale against V. harveyi infection. Ocimum sanctum and Withania somnifera were reported for their immunostimulant properties in experimental animals [36,11]. Diethyl Phthalate (1,2-Benzene dicarboxylic acid, monobutyl ester) and Dibutyl Phthalate was confirmed in the active fraction eluted from H 40: EA 60 by GCeMS analysis. Due to the enrichment of those active compounds in the fractions, it was responsible for high antibacterial and immunostimulant activities which induce the immune system. The same compound has been already reported from the methanol extract of the aerial part of Hypericum hyssopifolium [37]. Gaikwad et al. [38] identified the compound diethyl phthalate from Cassia auriculata by the spectral analysis of MS, IR, 1H NMR, 13C NMR, etc. Phyllanthus muellerianus showed presence of bis (2-ethyloctyl) phthalate and bis (2-ethylicosyl) phthalate (VI) [39]. The compound bis (2-methylheptyl) phthalate

491

identified from the plant Pongamia pinnata had antiviral activity against WSSV [40]. P. pinnata has also been shown to exhibit antibactericidal activity against Bacillus anthracis, Bacillus mycoides, Bacillus putiles, E. coli, Pseudomonas mangiferae, Salmonella typhimurium, Sarcina lutea, S. aureus, Staphylococcus albus and Xanthomonas [41]. The presence of dibutyl phthalate from Mimusops elengi [42], Leea indica [43], Alstonia scholaris, Torreya grandis, Achyrathes bidentata, Rheum glabricaule [44] has been also reported. The herbals having the characteristics of antimicrobial and immunostimulants were able to increase the survival and reduce the pathogenic load against pathogenic challenge by improving immune system in fishes and shrimps [12,26]. In the present study, 60% of the survival was increased by ICE fed C. auratus and 80% increase by ICF fed C. autratus from the control group. The ICF helps to increases the survival to 20% from the crude ICE. The methanolic extracts of Cynodon dactylon, Aegle marmelos, Tinospora cordifolia, Picrorhiza kurooa and Eclipta alba incorporated diets were well influenced by better survival, reduction in the viral load [10]. The I. coccinea extracts, ICE and ICF were also highly influenced to reduce the Aeromonas load in blood and muscle significantly after 8th days from the challenge when compared to the control. The extracts helped to boost the immune system against the A. hydrophila challenge and it reflects the increased survival and reduced bacterial load to 2.5  102 and 0.5  101 cfu ml
1 in ICE and ICF after 8th days respectively. Emblica officinale, Cynodon dactylon and Adathoda vasica were reported as herbal immunostimulants that improved the immune system and reduced the microbial infection in the gold fish C. auratus [16]. The saponin immunoadjuvant from Asparagus racemosus along with the OMP and BF vaccine delivered to C. auratus significantly decreased the Aeromonas load after A. hydrophila challenge [45]. Manju and Nair [46] have reported an increase in serum protein content as well as albumin content in Anabas testudineus fed with aqueous leaf, stem and root extracts of Aegle marmelos. The increased serum albumin, globulin and protein in the ICE and ICF treated fishes after A. hydrophila challenge reflected the stimulation of immune factors. Generally the immunostimulated or vaccine treated animals have higher serum biochemical parameters. The active principles of the phthalate derivatives of the extracts influenced to increase the serum biochemical parameters. In C. auratus, haemoglobin and haematocrit values improved by feeding herbal extracts compared to the controls [47]. Herbal immunostimulants such as Lonicera japonica and Ganoderma lucidum also help to increase the immunoglobulin level in Oreochromis niloticus against A. hydrophila challenge. Dina Rairakhwada et al. [48] found that the globulin content was significantly enhanced in levan-fed common carp fingerlings. The haematological parameters including RBC and haemoglobin also significantly increased from the control group to ICE and ICF treated C. auratus after A. hydrophila challenge. The RBC count was drastically came down from the blank control to control group due to the absence of immunostimulation given through feed. Sahu et al. [49] reported that WBC and RBC counts were higher in Labeo rohita finger- lings fed Mangifera indica kernel when compared to controls. Di (2-ethylhexyl) phthalate (DEHP) mediates the immune system mainly by triggering the production of reactive oxygen species (ROS) and nitric oxide (NO) in higher animals [50]. Recent research in higher animals also revealed that DEHP had the potential to interact with the immune system through its enhancement of IL-4 production [51]. The diethyl and dibutyl phthalate compounds presence in the fraction (ICF) helps to increase the serum bactericidal activity of around three times against A. hydrophila. The phagocytic activity and lysozyme activity was increased more than one times from control group to ICF group. The herbal immunostimulant extracts were able to induce the specific and non-specific

492

P. Anusha et al. / Fish & Shellfish Immunology 36 (2014) 485e493

immune factors in fish and shellfish species reported by several authors. The extracts of Chinese herbs, Rheum officinale, Andrographis paniculata, Isatis indigotica and L. japonica helps to increase phagocytosis of white blood cells of crucian carp [52]. Oncorhynchus mykiss fed with the diets containing Viscum album, Urtica dioica and Zingiber officinale improved the non-specific defense mechanisms, including extracellular and intracellular respiratory burst activities, phagocytosis in blood leucocytes and total plasma protein level etc [53]. Supplementing the OMP and BF vaccines with the immunoadjuvant A. racemosus helped to improve phagocytosis, serum bactericidal activity, albumin:globulin ratio in C. auratus [45]. The level of serum lysozyme was also enhanced in L. rohita after feeding the fish with Achyranthes asperea seed [54]. Elevated lysozyme was also observed in Japanese eel (Anguilla japonica) after feeding with Korean mistletoe extract (KM-110; Viscum album Coloratum) [55]. The histochemistry data revealed that, the diethyl and dibutyl phthalate compounds helps to highly influence the expression of macrophages and erythrocytes abundantly expressed among the fragmented skeletal muscle fibres due to the immunostimulations. The macrophages are the immune related cells that only expressed in the ICE and ICF treated fishes against A. hydrophila challenges. Phthalates have been shown to enhance the effect of immunogens, which means they have an adjuvant effect [56,57]. Monoethylhexyl phthalate detected by GCeMS analysis in the extracts of Eupatorium odoratum possesses antibacterial activity against nine bacterial sp and antioxidant activity [58]. Based on the results, the phthalates compounds are highly influential to inhibit the growth of A. hydrophila and stimulate the immune system of the ornamental gold fish C. auratus. Acknowledgements The authors gratefully acknowledge the University Grants Commission (UGC), New Delhi, Government of India, for its financial support, in the form Special Assistance Programme (SAP) [UGC NO. F.3-24/2012 (SAP-II) dated October 2012]. References [1] Ahilan B, Jegan K, Felix N, Ravaneswaran K. Influence of botanical additives on the growth and colouration of adult gold fish Corrassicus auratus (Linn). J Vet Anim Sci 2008;4(4):129e34. [2] Nasser AKV, Rajkumar U. Ornamental fish e prospects for culture. Souvenir issued on the occasion of the inauguration of Visakhapatnam R C of CMFRI; 17 October 2001. pp. 24e6. [3] Swain SK, Mallik D, Mishra S, Sarkar B, Routray P. Ornamental fish as model animals for biotechnological research. In: Mishra, Juwarkar, editors. Environmental biotechnology. Delhi: APH Publ. Corp.; 2007. pp. 293e328. [4] Austin B, Austin DA. Bacterial fish pathogens. In: Schuster Chichester S, editor. Diseases of farmed and wild fish. 2nd ed. Springer Publication; 1993. pp. 111e7. [5] Biradar SS, Goud NR, Neogi U, Saumya R. In vitro and in vivo antibacterial studies of medicinal plant on motile aeromonad septicemia in fish caused by Aeromonas hydrophila. J Fish Aquat Sci 2007;2(6):417e21. [6] Howard SP, Buckley JT. Activation of the hole forming toxin aerolysin by extracellular processing. J Bacteriol 1985;163:336e40. [7] Citarasu T, Babu MM, Sekar RJR, Marian PM. Developing artemia enriched herbal diet for producing quality larvae in Penaeus monodon fabricius. Asian Fish Sci 2002;15:21e32. [8] Rijkers GT, Teunissen AG, Van Oosterom R, Van Muiswinkel WB. The immune system of cyprinid fish: the immunosuppressive effect of the antibiotic oxytetracycline in carp (Cyprinus carpio L). Aquaculture 1980;19:177e89. [9] Citarasu T. Herbal biomedicines: a new opportunity for aquaculture industry. Aquac Int 2010;18:403e14. [10] Citarasu T, Sivaram V, Immanuel G, Rout N, Murugan V. Influence of selected Indian immunostimulant herbs against white spot syndrome virus (WSSV) infection in black tiger shrimp, Penaeus monodon with reference to haematological, biochemical and immunological changes. Fish Shellfish Immunol 2006;21:372e84. [11] Yogeeswaran A, Velmurugan S, Punitha SMJ, Babu MM, Selvaraj T, Kumaran T, et al. Protection of Penaeus monodon against white spot syndrome virus by inactivated vaccine with herbal immunostimulants. Fish Shellfish Immunol 2012;32(6):1058e67.

[12] Raja Rajeswari P, Velmurugan S, Michael Babu M, Kesavan K, Citarasu T. A study on the influence of selected Indian herbal active principles on enhancing the immune system in Fenneropenaeus indicus against Vibrio harveyi infection. Aquac Int 2012:1e14. [13] Su MS, Liu KF, Chang CF, Liao IC. Enhancement of grass prawn Penaeus monodon postlarvae viability by b-1,3-glucan from Schizophyllum commune. J Taiwan Fish Res 1995;3:125e32. [14] Liao IC, Su MS, Chang CF, Her BY, Kojima T. Enhancement of the resistance of grass prawn Penaeus monodon against Vibrio damsela infection by beta-1,3glucan. J Fish Soc Taiwan 1996;23:109e16. [15] Sivaram V, Babu MM, Immanuel G, Murugadass S, Citarasu T, Marian MP. Growth and immune response of juvenile greasy groupers (Epinephelus tauvina) fed with herbal antibacterial active principle supplemented diets against V. harveyi infections. Aquaculture 2004;237:9e20. [16] Minomol M. Culture of Gold fish Carassius auratus using medicinal plants having immunostimulant characteristics [M. Phil dissertation]. India: M. S. University; 2005. [17] Magdelin SM. Culture of ornamental fish, Black molly (Poecilia sphenops) using medicinal plants having immunostimulant characteristics [M. Phil dissertation]. India: Manonmaniam Sundaranar University; 2005. [18] Latha PG, Panikkar KR. Cytotoxic and antitumour principles from Ixora coccinea flowers. Cancer Lett 1998;130(1e2):197e202. [19] Ratnasooriya WD, Deraniyagala SA, Galhena G, Liyanage SSP, Bathige SDNK, Jayakody JRAC. Anti-inflammatory activity of the aqueous leaf extract of Ixora coccinea. Pharm Biol 2005;43:147e52. [20] Saha MR, Alam MA, Akter R, Jahangir R. In-vitro free radical scavenging activity of Ixora coccinea L. Bangladesh J Pharmacol 2008;3(2):90e6. [21] Thanga Viji V, Michael Babu M, Velmurugan S, Kumaran T, Anand SB, Gunasekaran P, et al. Virulence factors and molecular cloning of outer membrane protein (OMP) gene from virulent Aeromonas hydrophila isolated from infected gold fish Carassius auratus. Bangladesh J Microbiol 2011;28:70e5. [22] Thanga Viji V, Michaelbabu M, Anand SB, Gunasekaran P, Citarasu T. Immunization with the Aeromonas OMP provides protection against Aeromonas hydrophila in goldfish (Carassius auratus). J Microb Biochem Technol 2012;4:45e9. [23] Nadkarni KM. Indian materia medica with ayurvedic, unani, tibbi, sidda, allopathic, homeopathi, naturopathi and home remedies appendices and indexes, vols. I and II. New Delhi, India: Ram Pintograph; 1995. [24] Bauer AW, Kirby WMM, Sherris JC, Truck M. Antibiotic susceptibility testing by a standardized single disc method. Am J Clin Pathol 1966;45:493e6. [25] Sritunyalucksana K, Sithisarn P, Withayachumnarnkul B, Flegel TW. Activation of prophenoloxidase, agglutinin and antibacterial activity in haemolymph of the black tiger prawn, Penaeus monodon, by immunostimulants. Fish Shellfish Immunol 1999;9:21e30. [26] Punitha SMJ, Babu MM, Sivaram V, Shankar VS, Dhas SA, Mahesh TC, et al. Immunostimulating influence of herbal biomedicines on non-specific immunity in grouper Epinephelus tauvina juvenile against Vibrio harveyi infection. Aquac Int 2008;16:511e23. [27] Van Kampen EJ, Zijlstra WG. Standardization of hemoglobinometry. 11. The hemiglobincyanide method. Clin Chim Acta 1961;6:538e44. [28] Hendricks LJ. Erythrocyte counts and haemoglobin determinations for two species of suckers, genus Catostomus from Colorado. Copeia 1952;4:2655266. [29] Lowry OH, Rosenberough NJ, Farr AL, Randal RJ. Protein measurement with folin phenol reagent. J Biochem 1951;193:265e75. [30] Park KH, Jeong HD. Enhanced resistance against Edwardsiella tarda infection in tilapia (Oreochromis niloticus) by administration of protein-bound polysaccharide. Aquaculture 1996;143:135e43. [31] Bernet D, Schmidt H, Meier W, Burkhardt-Holm P, Wahli T. Histopathology in fish: proposal for a protocol to assess aquatic pollution. J Fish Dis 1999;22:25e34. [32] Latha PG, Abraham TK, Panikkar KR. Antimicrobial properties of Ixora coccinea L. (Rubiaceae). Anc Sci Life 4 April 1995;XIV:286e91. [33] Sharma MC, Sharma S. Preliminary phytochemical and antimicrobial investigations of the aqueous extract of Ixora coccinea Linn and Commelina benghalensis L. on Gram-positive and Gram-negative microorganisms. MiddleEast J Sci Res 2010;6(5):436e9. [34] Selvaraj N, Lakshmanan B, Mazumder PM, Karuppasamy M, Jena SS, Pattnaik AK. Evaluation of wound healing and antimicrobial potentials of Ixora coccinea root extract. Asian Pac J Trop Med 2011:959e63. [35] Velmurugan S, Punitha SMJ, Michael Babu M, Selvaraj T, Citarasu T. Select of Indian antibacterial medicine characteristics herbal to replace antibiotics for shrimp Penaeus monodon post larvae. J Appl Aquac 2010;22:230e9. [36] Davis L, Kuttan G. Immunomodulatory activity of Withania somnifera. J Ethnopharmacol 2000;71:193e200. [37] Cakir A, Mavi A, Yyldyrym A, Emin Duru M, Harmandar M, Kazaz C. Isolation and characterization of antioxidant phenolic compounds from the aerial parts of Hypericum hyssopifolium L. by activity-guided fractionation. J Ethnopharmacol 2003;87:73e83. [38] Gaikwad S, Devare S, Kale A, Deshpande Nirmala R, Salvekar Jyoti P. Isolation and characterisation of a diethyl pthalate, an bioactive compound from Cassia auriculata L. Int J Pharm Sci Rev Res 2013;19(1):56e7. [39] Roy RN, Laskar S, Sen SK. Dibutyl phthalate, the bioactive compound produced by Streptomyces albidoflavus 321.2. Microbiol Res 2006;161:121e6. [40] Rameshthangam P, Ramasamy P. Antiviral activity of bis(2-methylheptyl) phthalate isolated from Pongamia pinnata leaves against white spot syndrome virus of Penaeus monodon fabricius. Virus Res 2007;126:38e44.

P. Anusha et al. / Fish & Shellfish Immunology 36 (2014) 485e493 [41] Baswa M, Rath C, Dash SK, Mishra RK. Anti bacterial activity of Karanj (Pongamia pinnata) and neem (Azardirachta indica) seed oil: a preliminary report. Microbios 2001;105:183e9. [42] Namikoshi M, Fujiwara T, Nishikawa TK. Mar Drugs 2006;4:290e7. [43] Ruikar AD, Gadkari TV, Phalgune UD, Puranik VG, Deshpande NR. Studies on aerial part of Artimisia pallens for the phenol flavonoid content. Chem Nat Comp 2010;213:6. [44] Srinivasan GV, Sharanappa P, Leela NK, Sadashiva CT, Vijayan KK. Anti-microbial activity and biochemical constituents of two edible and medicinal mushrooms of mid-Western, Uganda. Nat Prod Rad 2009;8:488e93. [45] Thanga Viji V, Deepa K, Velmurugan S, Birdilla Selva Donio M, Adlin Jenifer J, Michael Babu M, et al. Vaccination strategies to protect goldfish Carassius auratus against Aeromonas hydrophila infection. Dis Aquat Org 2013;104:45e 57. [46] Manju K, Nair GRJ. Effect of Indian beal tree extract on protein metabolism in the fish Anabas testudineus. J Ecotoxicol Environ Monit 2004;14:221e6. [47] Harikrishnan R, Balasundaram C. In vitro and in vivo studies of the use of some medicinal herbals against the pathogen Aeromonas hydrophila in goldfish. J Aquat Anim Health 2008;20:165e76. [48] Rairakhwada D, Pal AK, Bhathena ZP, Sahu NP, Jha A, Mukherjee SC. Dietary microbial levan enhances cellular non-specific immunity and survival of common carp (Cyprinus carpio) juveniles. Fish Shellfish Immunol 2007;22(5): 477e86. [49] Sahu S, Das BK, Pradhan J, Mohapatra BC, Mishra BK, Sarangi NN. Effect of Magnifera indica kernel as a feed additive on immunity and resistance to Aeromonas hydrophila in Labeo rohita fingerlings. Fish Shellfish Immunol 2007;23:109e18.

493

[50] Lu Y, Zhang P, Li C, Su X, Jin C, Li Y, et al. Characterisation of immune-related gene expression in clam (Venerupis philippinarum) under exposure to di(2ethylhexyl) phthalate. Fish Shellfish Immunol 2013;34:142e6. [51] Lee MH, Park J, Chung SW, Kang BY, Kim SH, Kim TS. Enhancement of interleukin-4 production in activated CD4 þ T cells by diphthalate plasticizers via increased NFAT binding activity. Int Arch Allergy Immunol 2004;134:213e 22. [52] Chen CF, Kusuda RA. Preliminary study on the adjuvant activity of henbane in oral vaccination of grass carp (Ctenopharyngodon idellus). J Huazhong Agric Univ 1996;15:157e61. [53] Dügenci S, Arda KN, Candan K. Some medicinal plants as immunostimulant for fish. J Ethnopharmacol 2003;88:99e106. [54] Rao YV, Das BK, Pradhan J, Chakrabarti R. Effect of Achyranthes aspera on the immunity and survival of L. rohita infected with Aeromonas hydrophila. Fish Shellfish Immunol 2006;20:263e73. [55] Choi SH, Park KH, Yoon TJ, Kim JB, Jang Y, Choe CH. Dietary Korean mistletoe enhances cellular non specific immune responses and survival of Japanese eel (Anguilla japonica). Fish Shellfish Immunol 2008;24:67e73. [56] Hansen JS, Larsen ST, Poulsen LK, Nielsen GD. Does lipophilicity per se induce adjuvant effects? Methyl palmitate as model substance does not affect ovalbumin sensitization. J Toxicol Environ Health A 2007;70:128e37. [57] Larsen ST, Nielsen GD. The adjuvant effect of di-(2-ethylhexyl) phthalate is mediated through a PPARa-independent mechanism. Toxicol Lett 2007;170: 223e8. [58] Venkata Raman B, Samuel LA, Pardha Saradhi M, Narashimha Rao B, Naga Vamsi Krishna A, Sudhakar M, et al. Antibacterial, antioxidant activity and GCMS analysis of Eupatorium odoratum. Asian J Pharm Clin Res 2012;5(2).