Immunostimulation by phospholipopeptide biosurfactant from Staphylococcus hominis in Oreochromis mossambicus

Fish & Shellfish Immunology 48 (2016) 244e253

Contents lists available at ScienceDirect

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

Full length article

Immunostimulation by phospholipopeptide biosurfactant from Staphylococcus hominis in Oreochromis mossambicus Veluchamy Rajeswari, Sekaran Kalaivani Priyadarshini, Viswanathan Saranya, Ponnusamy Suguna, Rajaiah Shenbagarathai* Postgraduate and Research Department of Zoology and Biotechnology, Lady Doak College, Madurai 625002, Tamil Nadu, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 June 2015 Received in revised form 31 October 2015 Accepted 2 November 2015 Available online 6 November 2015

The immunostimulatory effect of phospholipopeptide biosurfactant from Staphylococcus hominis (GenBank Accession No: KJ564272) was assessed with Oreochromis mossambicus. The non-specific (serum lysozyme activity, serum antiprotease activity, serum peroxidase activity and serum bactericidal activity), specific (bacterial agglutination assay) immune responses and disease resistance activity against Aeromonas hydrophila were examined. Fish were intraperitonially injected with water soluble secondary metabolite (biosurfactant) of S. hominis at a dose of 2 mg, 20 mg and 200 mg kg
1 body weight. Commercial surfactant surfactin (sigma) at 20 mg kg
1 was used as standard and saline as negative control. All the doses of water soluble biosurfactant tested, significantly enhanced the specific, nonspecific immunity and disease resistance from the day of post administration of phospholipopeptide biosurfactant till the tail of the experimental period. These results clearly indicated that the secondary metabolite isolated from S. hominis stimulates the immunity of finfish thereby could enhance aquaculture production. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Secondary metabolite Phospholipopeptide biosurfactant Immunostimulant Specific immune response Non-specific immune response Disease resistance

1. Introduction Aquaculture is a rapidly growing economic area that contributes nearly half (47.3%) of the world's fish food consumption. Nevertheless, one of the main threats to aquaculture is the infectious outbreaks leading to mass mortality in fish [1,2]. A. hydrophila, the motile aeromonads affects a wide variety of freshwater fish species and occasionally marine fish [3]. Improving fish performance in terms of immunity and disease resistance are the major challenges faced by the fish culturists. Though, many measures including routine use of antibiotics have been tried, the excessive and inappropriate use of antibiotics has resulted in cross resistance against pathogens and accumulation of residues in tissues [4]. The use of organic, inorganic and synthetic compounds such as levamisole [5,6], glucan [7,8], vitamin C and E [9e11], chitin [12], FK-565 (Lactoyl tetrapeptide) [13], FCA (Freund's Complete Adjuvant) [14,15] as immunostimulant has been increased in recent years. Generally, many microbial products with low molar mass and the group of

* Corresponding author. E-mail addresses: [email protected], [email protected] (R. Shenbagarathai). http://dx.doi.org/10.1016/j.fsi.2015.11.006 1050-4648/© 2015 Elsevier Ltd. All rights reserved.

naturally occurring polymers are surveyed to have an immunoregulatory activity [16]. The components of bacterial cell wall, such as MDP (Muramyl dipeptide), peptidoglucan and LPS (Lipopolysaccharide), attributed to the immunostimulatory effects of aquatic animals [17e19]. However, their role as immunostimulants is controversial for commercial application due to their adverse health effects [20,21]. Microbial secondary metabolites, a protective subcellular component has received more attention in disease control. Newaj-Fyzul et al. [22], has demonstrated that feeding rainbow trout with cell-free supernatant of Bacillus subtilis AB1 significantly reduced cumulative mortalities after challenge with Aeromonas sp. Arijo et al. [23], also reported that subcellular components of Vibrio harveyi were successful for the stimulation of immunity and the prevention of V. harveyi infections in rainbow trout, Oncorhynchus mykiss (Walbaum). Hence, screening of new immunostimulants from the secondary metabolites (biosurfactants) of microorganisms could be advantageous to strengthen fish immune system and to reduce the quantity of antibiotics required to control infectious diseases. Many Staphylococcus strains have been reported to produce a number of antimicrobial peptides [24e27]. Staphylococcus hominis MBBL 2e9 produced an antimicrobial peptide, called hominicin which exhibited a high bactericidal activity against methicillin-

V. Rajeswari et al. / Fish & Shellfish Immunology 48 (2016) 244e253

resistant Staphylococcus aureus (MRSA) ATCC 11435 and vancomycin-intermediate S. aureus (VISA) CCARM 3501 but also showcased heat-tolerance and pH stability. The main advantages of biosurfactants are their low toxicity, high biodegradability and high stability at extreme conditions, such as high levels of temperatures, salinity and pH [28,29]. In some cases, stability is higher than that of chemical based surfactants [30]. Also, biosurfactant molecules are able to form stable emulsions in solutions with high ionic force, which is especially relevant in aquacultural applications, since most fish farms are located in high salinity environments, making the stability of biosurfactants at high ionic strength is essential. For example, biosurfactants from Cobetia sp. MM1IDA2H-1 [31] exhibited high stability at wide ranges of pH, temperature and strength force (3e20% w/v of sodium chloride). Researchers across the globe have evaluated the use of biosurfactants in a wide variety of potential applications such as environmental bioremediation, biomedical applications, cosmetics, personal care products, perfume and fragrance industry [31]. Similarly, WH1fungin (WF), a lipopeptide surfactin, was verified to have an immunoadjuvant property in mice groups when WF plus Hepatitis B surface antigen (HbsAg) was immunized intranasally or parenterally suggesting its potential for development of more efficient HBV vaccines in the future [32]. Also, a synthetic lipopeptide derived from bacterial lipoprotein P3CSK4, has shown to be an effective immunoadjuvant in parenteral, nasal and oral immunization [33]. Synthetic lipopeptide P3CSK4 constitutes a potent macrophage/monocyte activator resulting in the induction of lymphokine production, phagocytosis, activation for tumour cytotoxicity, tumour necrosis factor- a (TNFa) production, and release of reactive oxygen and nitrogen intermediates [34,35]. However, limited research is available regarding its immunomodulatory activity. Hence the current study focus on the evaluation of immunostimulatory property of extracellular secondary metabolite phospholipopeptide biosurfactant isolated from S. hominis to fish (Oreochromis mossambicus), but the ideas are also applicable to other groups of organisms relevant to aquaculture. 2. Materials and methods 2.1. Fish and their maintenance Wild strain of O. mossambicus (Mossambique tilapia), a cichlid fish was used as the model organism for this study. About 260 male fish weighing 25 ± 5 g were collected from a local fish farm and were acclimatized to laboratory conditions in 21 fibre reinforced plastic tanks (vol. 150 L) consisting of 10 fish each for a period of 4 weeks. They were kept at an ambient temperature of 28 ± 2  C under natural photoperiod (between March and June). Water was changed on alternate days to avoid ammonia accumulation. Fish were fed at a rate of 3% body weight once in a day with a balanced fish diet consisting of 42% dried fish meal, 20% groundnut oil cake, 15% tapioca flour, 15% wheat flour, 5% blood meal and 3% mineralvitamin mixture [36] prepared in the lab. Its nutrient composition consisted of 39% protein, 24% carbohydrate, 11% lipid and 9% ash [37]. A total of 26 tanks with 10 fish per tank were used for the experiment. 2.2. Microbial identification Bacterial isolate were obtained from petrol/diesel contaminated soil and screened for biosurfactant producing ability. The potent strain was subjected to 16 S rDNA based molecular characterization. The genomic DNA of the isolate was amplified using Eu-bacterial primer (50 - AGA GTT TGA TCC TGG CTC AG -30 ; 50 - ACG GCT ACC TTG TTA CGA CTT -30 ) following the method described by Relman

245

[38] and sequenced (Eurofins India Ltd.,). The resulted nucleotide sequence was analysed using NCBI BLAST and were submitted to NCBI and accession number was obtained. 2.3. Biosurfactant screening, production, extraction and characterization Different biosurfactant screening methods were performed to select the potential biosurfactant producing microorganism. The methods adopted were (a) Hemolytic activity in 5% blood agar plate [39]; (b) Oil spreading technique [39]; (c) Parafilm M test [40] and (d) Emulsification activity by adding kerosene and equal volume of cell free supernatant [41]. Bacterial strain was grown in a minimal salt (MS) medium containing (g/l): yeast extract e 1; KH2PO4 e 1.4; K2HPO4 e 2.2; MgSO4 7. H2O e 0.6; was used throughout the study. The basal minimal medium was supplemented with 2 ml of trace element solution and palmolein oil 2% (v/v) as the sole carbon source. The composition of trace element solution involved (g/l): Ortho phosphoric acid e 0.6; Cobaltous chloride e 0.4; Zinc sulphate e 0.2; Magnesium chloride e 0.06; Sodium molybdate e 0.06; Copper sulphate e 0.02. The trace element solution was added after the production media were autoclaved, prior to inoculation by filtering it through 0.2 mm membrane filters, sterilized by filtration (Millipore Corp., Bedford, MA, USA). The cultures were centrifuged after incubation at 30  C for 3 days, and the cell-free filtrates were used ~ a et al., [42]). in the experiments (Modified method of Gudin Biosurfactant was precipitated by adjusting the pH to 2.0 in the cell-free filtrate using 6 N HCl and kept at 4  C overnight. Pellet thus precipitated was collected by centrifugation (8000 rpm for 15 min at 20  C) and dissolved in distilled water. Then the pH was adjusted to 8.0 with 1 N NaOH, and the extract was lyophilized [43]. Partial purification of water soluble biosurfactant was performed by dialysis. Preliminary identification of partially purified phospholipopeptide biosurfactant was detected using biuret test [44], phosphate test [45] FTIR (PerkineElmer Spectrum RX1, Shelton, Connecticut), spectrum in the range of 450e4000 cm
1 at a resolution of 4 cm
1 (Madurai Kamaraj University, Madurai) [46], GC e MS spectra coupled to mass detector turbo mass gold e perkin Elmer (Indian Institute of Crop Processing Technology, Thanjavur) [47] and Agilent 1100 HP e HPLC (Sankara Nethralaya, Chennai) were used to confirm the phospholipopeptide nature of the extracted biosurfactant [48]. 2.4. Assessment of nonspecific immunity To analyze the effect of phospholipopeptide biosurfactant on nonspecific immune mechanisms, five groups/tanks each containing 10 fish each were administered, ip, with 0.2 ml water soluble fraction of biosurfactant at doses of 2, 20 and 200 mg/kg body weight and 20 mg/kg concentration of surfactin. A preliminary test was performed to determine the LC50 dose of the biosurfactant [49] and the sub-lethal concentrations were derived for test substance. Then fish were bled at regular intervals of 5 days till day 20 postimmunization. To detect pre-treatment levels of immune mechanisms, fish were also bled 2 days prior to the start of the experiment. The blood was collected in serological tubes and stored overnight at 4  C in a refrigerator. The clot was then spun down at 400  g for 10 min to separate the serum and stored at
20  C until further use. 2.4.1. Lysozyme activity Lysozyme activity was measured by the method of Parry et al. [50], in combination with the microplate adaptation of Hutchinson

246

V. Rajeswari et al. / Fish & Shellfish Immunology 48 (2016) 244e253

and Manning [51]. In this turbidimetric assay, 0.03% lyophilized Micrococcus lysodeikticus in 0.05 mM sodium phosphate buffer (pH 6.2) was used as substrate. Ten microlitres of fish serum was added to 250 ml of bacterial suspension in a “U” bottom microtitre plate and the reduction in absorbance at 490 nm was determined after 0.5 and 4.5 min incubation at 25  C using a microplate reader (VersaMax, Molecular Devices, CA, USA). One unit of lysozyme activity was defined as a reduction in absorbance of 0.001 per min. 2.4.2. Antiprotease assay For detecting the level of antiprotease in serum, 10 ml of serum was added to 20 ml of trypsin (1 mg/ml in 0.01 M Tris HCl). The tubes were incubated for 5 min at room temperature. About 500 ml of 2 mM BAPNA (N-a-benzoyl-DL-arginine-p-nitroanilide) was added and the volume was made up to 1 ml using 0.1 M TriseHCl. After 25 min incubation at room temperature, 150 ml of 30% acetic acid was added to stop the reaction [52]. The OD was read at 410 nm and percentage trypsin inhibition was calculated using the formula,

Percentage trypsin inhibition ¼

Trypsin blank OD
Sample OD Trypsin blank OD

2.4.3. Total serum peroxidase activity To measure total peroxidase (PO) content in serum, about 10 ml of serum was diluted with 90 ml of Hanks balanced salt solution (HBSS) containing Mg2þ and EGTA in 96-well plates. Then 35 ml of 20 mM 3, 30 , 5, 50 -tetramethyl benzidine hydrochloride (TMB-HCl) was added and incubated for 2 min at room temperature. The colour change reaction was stopped after 2 min by adding 50 ml of 2 N sulphuric acid (H2SO4) and OD was read at 450 nm [53]. 2.4.4. Serum antibacterial activity To measure serum bactericidal assay, about 20 ml of test serum, control serum and 20 ml of HBSS (without serum) respectively were added in a ‘U’ bottom 96 well microtiter plate. To this, 20 ml of 24 h old Aeromonas hydrophila culture was added. This mixture was incubated for 150 min at room temperature. MTT solution of 25 ml was added and incubated for 10 min to allow the formation of formazan precipitate. The plates were centrifuged at 1500 rpm for 10 min and the supernatant was discarded. 200 ml of DMSO was added per well to dissolve the precipitate. Purple colour was measured colorimetrically at 560 nm. Results were interpreted as % control [54].

Percent control ¼

Sample O:D  100 Blank O:D

regular intervals of 7 days till day 28 post-immunization. About 200 ml of blood was drawn and the whole bleeding procedure was completed within 1 min to minimize the stress to fish [57]. The blood was collected in serological tubes and stored overnight at 4  C in a refrigerator. The clot was then spun down at 400  g for 10 min to separate the serum. The sera were de-complemented by incubating at 47  C for 30 min in a water bath [58] before storing at
20  C until further use. Antibody titrations were performed in 96 well “V” bottom microtiter plate using the bacterial agglutination assay. Briefly, 50 ml of serum was added to the first well and two fold serial dilutions were made with physiological saline. A volume of 50 ml of A. hydrophila suspension (1  108 cells ml
1) was added to each well. The microtiter plate was incubated at room temperature for 2 h. The highest dilution of serum sample that showed detectable macroscopic agglutination was recorded and expressed as log2 antibody titre of the serum. 2.6. Disease resistance test For performing disease resistance test, 5 groups/tanks, each in triplicates (n ¼ 150) of fish were intraperitoneally administered, with 0.2 ml of water soluble fraction of phospholipopeptide biosurfactant at doses of 2, 20 and 200 mg/kg body weight. Standard and negative control groups were injected with 20 mg/kg concentration of surfactin (Sigma) and saline respectively on Day 0. Fish were challenged with live virulent A. hydrophila. Before the start of the experiment, a separate group of fish administered, ip, with 0.2 ml of PBS (control) was challenged with different doses of live virulent A. hydrophila (LD50 dose of 1  109 cells ml
1) on day 8. A cumulative mortality was recorded for 15 days. The mortality symptoms observed include, haemorrhagic septicemia, distended abdomen and lesions on the ventral surface of the body. The cause of death was confirmed by re-isolating the organisms from liver of 10% of dead fish using Aeromonas isolation medium for A. hydrophila (Hi media, Mumbai, India). Mortality was observed for 96 h and results were expressed as percent mortality [59]. 2.7. Statistical analysis The data were expressed as arithmetic mean ± standard error (SE). Statistical analysis of data involved one-way analysis of variance (ANOVA) followed by Tukey's posthoc multiple comparison tests. The level of significance were expressed as P-value less or greater than 0.05. All statistical calculations were performed using the software, Sigma stat 2.0 (Jandel Corporation, USA). 3. Results

2.5. Assessment of specific immune response by bacterial agglutination assay

3.1. Production and characterization of phospholipopeptide biosurfactant

To analyze the effect of phospholipopeptide biosurfactant on specific immune response, five tanks/groups containing 10 fish each were administered, i. p, with 0.2 ml water soluble fraction of biosurfactant at doses of 2, 20 and 200 mg/kg body weight. Correspondingly, control fish received 0.2 ml saline and 20 mg/kg concentration of standard surfactin (Sigma) was administered for a group on Day 0. After 2 days of administration, fish were primed i. p. with 0.1 ml of heat killed A. hydrophila (1.2  108 cells ml
1), and later given an i. p. booster dose of 0.1 ml of A. hydrophila (6  108 cells ml
1) after 3 days [55]. An unimmunized control group of 10 fish were also maintained to rule out earlier exposure of the antigen injected. The fish were bled from common cardinal vein using 1 ml tuberculin syringe fitted with 24-gauge needle [56] at

Attempts were made to isolate bacterial strains exhibiting excellent emulsification activity and surface properties. Among the hundreds of bacterial strains obtained from hydrocarbon contaminated soil, a gram positive, catalase positive coccus was selected as a powerful biologically significant biosurfactant producer. The 16 s rDNA sequence analysis (GenBank Accession No: KJ564272) exhibited 96% similarity with S. hominis. Based on the different biosurfactant screening tests such as haemolytic activity, oil spreading test, Parafilm M test and emulsification activity, the S. hominis was confirmed for the production of biosurfactants. Biochemical tests were also carried out in order to determine the type of biosurfactant produced in this study. The violet ring that developed when the crude extracellular biosurfactant was added

V. Rajeswari et al. / Fish & Shellfish Immunology 48 (2016) 244e253

with biuret reagent indicated that the biosurfactant was lipoprotein or short-chain polypeptides in nature. Moreover, phosphate assay confirmed that biosurfactant was a phospholipid type. These results demonstrated that the extracellular biosurfactant of S. hominis is a phospo-lipo-peptide. FTIR spectral analysis also confirmed the phospholipopeptide nature of the extracted biosurfactant (Fig. 1). GCeMS analysis revealed that the lipid moiety contains a mixture of 17 linear and branched b-hydroxy fatty acids ranging in size from C8 to C27 (Fig. 2; Table 1). Based on HPLC analysis, the peptide moiety is composed of the following amino acids: Glycine, valine, alanine, isoleucine, leucine, serine, threonine, phenylalanine, tyrosine, asparagine, glutamine, methionine, lysine, arginine and histidine (Fig. 3; Table 2).

247

significantly enhanced (P < 0.05) serum peroxidase activity on different days of analysis (Fig. 6). The value of serum myeloperoxidase activity in fish administered with 200 mg kg
1 (79.29 ± 1.85) of phospholipopeptide biosurfactant was significantly higher compared with standard surfactin at 20 mg kg
1 (52.70 ± 2.45) (P < 0.05). Furthermore on the days 15 and 20, there was decrease in the stimulation activity. 3.2.4. Serum bactericidal activity In this study, all the doses of phospholipopeptide biosurfactant administered through intraperitoneal injection enhanced serum bactericidal activity against live A. hydrophila on almost all the days tested as compared with the controls, with the differences being significant on day 5, 10 and 15 (P < 0.05) as depicted in Fig. 7.

3.2. Nonspecific immune response in O. mossambicus 3.3. Antibody response against A. hydrophila 3.2.1. Lysozyme activity All the doses of extract of phospholipopeptide biosurfactant, administered intraperitoneally exhibited significant enhancement (P < 0.05) of serum lysozyme activity with highest magnitude (Fig. 4) from Day 5. The level of stimulation sustained till day 20 when compared with control as well as with pre-treatment activity levels. The maximal lysozyme activity (1949.16 ± 72.9 units/ml) was observed on day 15 in the group treated with the highest dose (200 mg kg
1) of biosurfactant (Fig. 4). Fish administered intraperitoneally with either 2 mg kg
1 or 20 mg kg
1 biosurfactant also exhibited significant enhancement (P < 0.05) of serum lysozyme activity from day 5 and it sustained till day 15. 3.2.2. Serum antiprotease activity The phospholipopeptide biosurfactant administered intraperitoneally elevated the serum antiprotease activity (79.29 ± 1.85% trypsin activity) on day 10 in the group treated with the highest dose (200 mg kg
1). Fish administered with 20 mg phospholipopeptide biosurfactant followed a similar trend as with 200 mg phospholipopeptide biosurfactant administered fish and sustained in the same level of activity till day 15 (Fig. 5). 3.2.3. Myeloperoxidase activity Intraperitoneal administration of all concentrations of phospholipopeptide biosurfactant 2 mg kg
1, 20 mg kg
1, 200 mg kg
1

The results of bacterial agglutination assay showed that fish administered, intraperitoneally with 20 and 200 mg kg
1 of water soluble fractions of biosurfactant showed significantly (P < 0.05) higher magnitude of antibody response to heat killed A. hydrophila (Fig. 8) on the days 21 and 28 post-immunization when compared with standard surfactin (sigma). 3.4. Disease resistance against A. hydrophila In the present study, analysis of percent mortality following challenge with live A. hydrophila showed significant protection in treated O. mossambicus in a dose dependent manner (Fig. 9). It is evident that among the doses tested, 200 mg kg
1 concentration administered significantly (P < 0.05) exhibited lowest percent mortality of 30% and gave protection against A. hydrophila when compared with the corresponding control groups. 4. Discussion Several molecules of bacterial origin, such as lipopolysaccharides, lipoproteins and glycoproteins, as well as by enzymes produced by immune cells, such as cytokines, transferrin, lysozyme and interleukins [60e63] are involved in the activation of innate immune system in fish. The present study demonstrated that the

Fig. 1. FT-IR spectral analysis of phospholipopeptide biosurfactant extracted from S. hominis.

248

V. Rajeswari et al. / Fish & Shellfish Immunology 48 (2016) 244e253

Fig. 2. GCeMS analysis of the biosurfactant extracted from S. hominis.

Table 1 Major compounds identified from the biosurfactants from S. hominis by GCeMS analysis. SI. No

Retention time

Name of the compounds

Molecular formula

Molecular weight

Peak area %

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

6.88 7.41 8.22 9.34 9.69 10.41 11.54 12.05 14.25 14.39 15.88 16.42 16.77 17.69 19.07 19.46 20.45

Dodecane, 2,6,10-trimethyl Dodecanoic acid, methyl ester Hexadecane Dodecyl acrylate Methyl tetradecanoate Octadecane Octadecane, 2-methylHexadecanoic acid, methyl ester 9-Octadecenoic acid (Z)-, methyl ester Octadecanoic acid, methyl ester 9-Hexadecenoic acid 7-Hexadecenoic acid, methyl ester, (Z)Eicosanoic acid, methyl ester Heptacosane Heneicosane Octadecanoic acid, 9, 10-dichloro-, methyl ester Tert-Hexadecanethiol

C15H32 C13H22O C16H34 C15H28O2 C15H30O2 C18H38 C19H40 C17H34O2 C16H36O2 C19H38O2 C16H30O2 C17H32O2 C21H42O2 C27H56 C21H44 C19H36Cl2O2 C16H34S

212 214 226 240 242 254 268 270 296 298 254 268 326 380 296 366 258

0.37 0.60 0.27 0.59 1.39 0.37 0.38 25.91 53.84 5.47 1.13 0.39 0.95 1.64 1.97 0.53 1.80

phospholipopeptide biosurfactant produced by S. hominis strain was able to modulate some specific, non-specific immune functions in O. mossambicus. The primary screening of biosurfactant extracted from S. hominis was performed by standard methods. The results of hemolytic method was in accordance with Youssef et al. [64]; as biosurfactant lyses the blood cells and exhibited the colorless, transparent ring around the well. In the oil displacement method, displacement of oil over the water surface was observed. The drop lacking biosurfactant remains beaded due to the hydrophobicity of the oil surface that causes aggregation of droplets [65]. The parafilm M test showed positive result with 0.5 cm diameter. Emulsification index (% EI24) measurement was found to be a more reliable method for quantification of the soluble biosurfactant in the medium. In S. hominis, higher emulsifying index was observed (50%) that is higher than that of P. aeruginosa UKMP14T which showed

percentage of emulsification (25.49 ± 0.00%) at 24hr [66]. For analyzing the type of the biosurfactant present in the sample, Okpokwasili and Ibiene [45] used phosphate test to determine phospholipid biosurfactant of crude extract from a culture of Pseudomonas sp. grown on kerosene-supplemented mineral salts medium. In this study, colorless phosphate assay solution changed to yellow indicated positive result for phospholipid. Biuret reagent turned to violet or pink ring due to reaction of peptide bond proteins or short-chain polypeptides. This test was applied in order to detect lipopeptide biosurfactant in the sample [44]. In this study, positive result obtained (color change to violet) when crude biosurfactant extract was dissolved in biuret reagent. These results of preliminary identification revealed that the biosurfactant produced by S. hominis was phospho-lipo-peptide. The basic functional groups present in the phospholipopeptide

V. Rajeswari et al. / Fish & Shellfish Immunology 48 (2016) 244e253

249

Fig. 3. Amino acid analysis the biosurfactant extracted from S. hominis by HPLC.

Table 2 HPLC analysis of amino acids in biosurfactants from S. hominis. SI.No.

Amino acids

Level in nmoles/ml

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Aspartic acid Glutamic acid Serine Histidine Glycine Threonine Arginine Alanine Tyrosine Methionine Valine Phenylalanine Isoleucine Leucine Lysine

748 656 423 231 981 561 443 546 155 226 264 330 346 608 398

biosurfactant were identified based on the FTIR analysis. The FTIR spectrum of biosurfactant contained the absorbance peak centered around 3439 cm
1, ranging from 3100 cm
1 to 3600 cm
1 (Fig. 1), which is the typical feature of compounds containing carbon and amino groups and was caused due to stretching vibrations of CeH and NeH bonds. The absorbance peak at 2926 cm
1 indicated the presence of alkyl chains (CH2 and CH3). The absorbance peak at 1647 cm
1 implied that peptide groups are present in the sample. A absorbance signal at 1458 cm
1 was due to bending vibrations of CeH bonds associated with alkyl chains [46]. The phosphate group asymmetric stretching vibrations appeared in the region 1234 cm
1 [67]. GCeMS spectrometry was used to elucidate the composition of fatty acids in the biosurfactant. Predominance of methyl ester of octanoic and 3-hydroxydecanoic acid indicated that C8 and C10 were the main fatty acid components of the purified biosurfactant. It was similar with previously reports of Saikia et al. [68]. GCeMS results showed that the biosurfactant was a mixture of 17 different b-hydroxy fatty acids (C8 to C27) (Fig. 2; Table 1). The results

Fig. 4. Effect of phospholipopeptide biosurfactant on serum lysozyme activity in O. mossambicus. Each point represents the mean ± SE of 10 fish. Different alphabets represent statistical significance at P < 0.05 between the groups tested by a posteriori Tukey test.

obtained in this study were in accordance with Yakimov et al. [69] wherein lipopeptide surfactant from Bacillus licheniformis BAS50 had a mixture of 14 - b-hydroxy fatty acids ranging in size from C12 to C17. Based on HPLC analysis, the amino acids detected in the biosurfactant contained aliphatic side chains amino acids (Glycine, valine, alanine, isoleucine and leucine); amino acid with hydroxylic group (Serine and threonine); amino acids with aromatic group

250

V. Rajeswari et al. / Fish & Shellfish Immunology 48 (2016) 244e253

Fig. 5. Effect of phospholipopeptide biosurfactant on serum antiprotease in O. mossambicus. Each point represents the mean ± SE of 10 fish. Different alphabets represent statistical significance at P < 0.05 between the groups tested by a posteriori Tukey test.

Fig. 6. Effect of phospholipopeptide biosurfactant on total serum peroxidases in O. mossambicus. Each point represents the mean ± SE of 10 fish. Different alphabets represent statistical significance at P < 0.05 between the groups tested by a posteriori Tukey test.

Fig. 7. Effect of phospholipopeptide biosurfactant on serum bactericidal activity in O. mossambicus. Each point represents the mean ± SE of 10 fish. Different alphabets represent statistical significance at P < 0.05 between the groups tested by a posteriori Tukey test.

Fig. 8. Effect of phospholipopeptide biosurfactant on the antibody response to bacterial cells in O. mossambicus. Each point represents the mean ± SE of 10 fish. Different alphabets represent statistical significance at P < 0.05 between the groups tested by a posteriori Tukey test.

V. Rajeswari et al. / Fish & Shellfish Immunology 48 (2016) 244e253

Fig. 9. Effect of phospholipopeptide biosurfactant on disease resistance against live virulent Aeromonas hydrophila in O. mossambicus in terms of percentage mortality. Each bar represents the mean ± SE of 10 fish. Different alphabets represent statistical significance at P < 0.05 between the groups tested by a posteriori Tukey test.

(Phenylalanine, Tyrosine), amino acids with acidic group (Asparagine, and glutamine), amino acid with sulfur group (Methionine) and amino acid with basic group (Lysine, arginine, histidine) (Fig. 3; Table 2). Similarly, iturins are known to contain a fatty acid chain length of C14 to C16 [70] along with a cyclic peptide of seven amino acids. Lysozyme, known to be an important nonspecific immune mediator against bacterial infections which breaks b-1,4 glycosidic acids and N-acetyl glucosamine in the peptidoglucan of bacteria cell walls [71]. Phospholipopeptide biosurfactant supplementation at all the three different concentration was found to be effective in stimulating serum lysozyme activity at varying degrees (Fig. 4). Similar effect was also seen in fish injected with a range of immunostimulants for various periods. For example, stimulation of lysozyme activity has been recognized after three days and four weeks of injection with from day 5 to the tail of the experiment in a cyclic tetrapeptide from bacillus species like 4-trans-hydroxy-Lproline and cyclo-(L-Pro-Gly)2 [72]. Antiprotease activity is an important innate defense parameter of fish [73e76]. Several studies have been investigated regarding antiprotease levels in fish species; particularly a2-macroglobulin activity [77]. Extracellular products (ECP) of Aeromonas sp. typically contain a variety of proteases, which is neutralised by factors possessing antiprotease activity present in fish serum [78]. The results of the present study indicated that water soluble extract of biosurfactant enhanced the activity of natural antiproteases in the serum, which provided effective defence against invading bacterial pathogens (Fig. 5). Similarly, enhancement of antiprotease activity has been reported in Kelp grouper, Epinephelus bruneus when 1% chitin and chitosan was fed in diet for 2 and 4 weeks [79]. Fish neutrophils have phagocytic, chemotactic and bactericidal functions, an intense respiratory burst and a peroxidase (myeloperoxidase, MPO) activity [80,81]. The process of degranulation is essential for the release of MPO and activation of the halide production pathway, as well as release of a diverse mixture of antimicrobial enzymes. All the concentrations of phospholipopeptide biosurfactant significantly enhanced serum peroxidase activity on different days of analysis (Fig. 6). Similarly, stimulatory effects of glucans on neutrophils, as well as other components of the immune system, have also been recognized [82,83]. Live bacterial cells contain the enzyme succinate-dehydro genase (SDH) which oxidized the 3-(4,5-dimethylthiazol-3-yl)-5diphenyltetrazolium bromide (MTT) dye to formazon crystals

251

while the dead cells did not [84]. The increased serum bactericidal activity indicated that various humoral factors involved in innate and/or adaptive immunities are elevated in the serum to protect the host effectively from infection. The higher bactericidal activities are possibly due to a higher production of O2
. In this study, higher levels of bactericidal activity was observed (Fig. 7) in accordance with cyclic tetrapeptides Cyclo-(L-Pro-Gly)2, a secondary metabolite produced from bacillus genus [71]. Some lipopeptide biosurfactants are also reported to exhibit antifungal properties, moderate antibacterial, hemolytic properties and exhibit antitumor and anti-viral activities [85]. EPS-1 polysaccharide from thermo tolerant Bacillus licheniformis strain was reported to possess antiviral and immunomodulatory effect [86e88]. In the present investigation, phospholipopeptide biosurfactant administered intraperitoneally enhanced antibody response to A. hydrophila (Fig. 8). Lipopeptides were reported to exhibit lytic and growtheinhibitory activities against a broad range of microorganisms [89]. The results were similar to the activity of synthetic lipopeptides (Murine influenza viral epitope NP 147e155 covalently attached to two palmitic acids) which safely induced specific memory immune response in mice and humans of therapeutic importance [90]. The overall consequence of an immunostimulant administration is reflected in the host's ability to resist infection. The results obtained confirmed the fact that the biosurfactant was effective enough in fighting off the bacterial challenge (Fig. 9). Earlier studies also revealed that secondary metabolite produced from Bacillus genus namely, 4-trans-hydroxy-L-proline and cyclo-(L-Pro-Gly)2 treatment increased the survival rate of the fish infected with A. hydrophila [91]. Also a peptide FK-565 (heptanoyl-g-D-glutamyl(L)-meso-diaminopimelyl-(D)-alanine) isolated from the culture supernatant of Streptomyces olivaceogriseus into rainbow trout (Salmo gairdneri) increased their resistance to Aeromonas salmonicida, following the activation of phagocytic cells [91]. The characteristic structural element in lipopeptides is a specific fatty acid, which was combined with an amino acid moiety. As a consequence of this amphiphilic structure, lipopeptides have various interesting biological properties. The present study showed that phospholipopeptide biosurfactant, a secondary metabolite derived from S. hominis strain from petrol/diesel contaminated soil have significantly enhanced the specific, non-specific immunity and disease resistance of O. mossambicus following intraperitoneal administration through improvement of various immune parameters such as bacterial agglutination assay, serum lysozyme activity, serum antiprotease activity, serum peroxidase activity, serum bactericidal activity and disease resistant to A. hydrophila. Therefore, phospholipopeptide biosurfactant of S. hominis is a promising immunostimulant which can be utilized in aquaculture to prevent diseases and disease outbreaks. Acknowledgement The authors thank the DST-INSPIRE and DBT-BIF vide reference BT/BI/25/001/2006 (Vol.-II) for financial support. References [1] M.A. Dinamarca, C.J. Ibacache-Quiroga, J.R. Ojeda, J.M. Troncoso, Marine microbial biosurfactants: biological functions and physical properties as the basis for innovations to prevent and treat infectious diseases in aquaculture, in: ndez-Vilas (Ed.), Microbial Pathogens and Strategies for Combating A. Me Them: Science, Technology and Education, FORMATEX, 2013. [2] B. Gomez-Gil, A. Roque, J.F. Turnbull, The use and selection of probiotic bacteria for use in the culture of larval aquatic organisms, Aquaculture 191 (2000) 259e270. [3] W.H. Chu, C.P. Lu, Multiplex PCR assay for the detection of pathogenic Aeromonas hydrophila, J. Fish Dis. 28 (2005) 437e441.

252

V. Rajeswari et al. / Fish & Shellfish Immunology 48 (2016) 244e253

[4] K. Nomoto, Prevention of infections by probiotics, J. Biosci. Bioeng. 100 (2005) 583e592. [5] V.L. Findlay, B.L. Munday, The immunomodulatory effects of levamisole on the nonspecific immune system of Atlantic salmon, Salmo salar L, J. Fish Dis. 23 (2000) 369e378. [6] A. Cuesta, J. Meseguer, M.A. Esteban, Total serum immunoglobulin M levels are affected by immunomodulators in seabream (Sparus aurata L.), Vet. Immunol. Immunopathol. 101 (2004) 203e210. [7] G. Jeney, D.P. Anderson, Glucan injection of bath exposure given alone or in combination with a bacterin enhances the non-specific defence mechanisms in rainbow trout (Oncorhynchus mykiss), Aquaculture 116 (1993) 315e329. [8] R.A. Dalmo, R. Seljelid, The immunomodulatory effect of LPS, laminaran and sulphated laminaran (beta (1, 3)-D-glucan) on Atlantic salmon, Salmo salar L., macrophages in vitro, J. Fish Dis. 18 (1995) 175e185. [9] V.S. Blazer, R.E. Wocke, The effects of alpha-tocopherol on the immune response and non-specific resistance factors of rainbow trout (Salmo gairdneri Richardson), Aquaculture 37 (1984) 1e9. [10] L.J. Hardie, T.C. Fletcher, C.J. Secombes, The effect of vitamin E on the immune response of the Atlantic salmon (Salmo salar L.), Aquaculture 87 (1990) 1e13. [11] J. Ortuno, A. Cuesta, M. Angeles Esteban, J. Meseguer, Effect of oral administration of high vitamin C and E dosages on the gilthead seabream (Sparus aurata L.) innate immune system, Vet. Immunol. Immunopathol. 79 (2001) 167e180. [12] M. Sakai, H. Kamiya, S. Atsuta, S. Ishii, M. Kobayashi, The immunostimulating effects of chitin in rainbow trout, Oncorhynchus mykiss, Dis. Asian Aquac. I (1992) 413e417. [13] T. Kitao, T. Yoshida, D.P. Anderson, O.W. Dixon, A. Blanch, Immunostimulation of antibody- producing cells and humoral antibody to fish bacterins by a biological response modifier, J. Fish Biol. 31 (1987) 87e91. [14] G. Olivier, T.P. Evelyn, R. Lallier, Immunity to Aeromonas salmonicida in coho salmon (Oncorhynchus kisutch) induced by modified Freund's complete adjuvant: its non-specific nature and the probable role of macrophages in the phenomenon, Dev. Comp. Immunol. 9 (1985) 419e432. [15] Y. Kajita, M. Sakai, S. Atsuta, M. Kobayashi, Immunpotentiation activity of Freund's complete adjuvant in rainbow trout Oncorhynchus mykiss, Nippon Suisan Gakkaishi Bull. 58 (1992) 433e437. , Immunomodulators isolated from microor[16] Z. Vanek, J. Mateju, E. Curdova ganisms, Folia Microbiol. 36 (1991) 99e111. [17] H. Kodama, Y. Hirota, N. Mukamoto, T. Baba, I. Azumn, Activation of rainbow trout (Oncorhynchus mykiss) phagocytes by muramyl dipeptide, Dev. Comp. Immunol. 17 (1993) 129e140. [18] S.T. Solem, J.B. Jørgensen, B. Robertsen, Stimulation of respiratory burst and phagocytic activity in Atlantic salmon (Salmo salar L.) macrophages by lipopolysaccharide, Fish Shellfish Immunol. 5 (1995) 475e491. [19] K. Matsuo, I. Miyazano, The influence of long-term administration of peptidoglucan on disease resistance and growth of juvenile rainbow trout, Nippon Suisan Gakkaishi 59 (1993) 1377e1379. [20] J. Raa, The use of immune-stimulants in fish and shellfish feeds, in: Advances  n Acuícola V. Memorias del V Simposium Internacional de en Nutricio  n Acuícola, 2000, pp. 19e22. Nutricio [21] J. Raa, The use of immunostimulatory substances in fish and shellfish farming, Rev. Fish. Sci. 4 (1996) 229e288. [22] A. Newaj-Fyzul, A.A. Adesiyun, A. Mutani, A. Ramsubhag, J. Brunt, B. Austin, Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum), J. Appl. Microbiol. 103 (2007) 1699e1706.  n, P. Díaz-Rosales, B. Austin, Subcellular com[23] S. Arijo, J. Brunt, M. Chabrillo ponents of Vibrio harveyi and probiotics induce immune responses in rainbow trout, Oncorhynchus mykiss (Walbaum), against V. harveyi, J. Fish Dis. 31 (2008) 579e590. [24] A.S. Dajani, Z. Taube, Plasmid-mediated production of Staphylococci in bacteriophage type 71 Staphylococcus aureus, Antimicrob. Agents Chemother. 5 (1974) 594e598. [25] M. Hille, S. Kies, F. Gotz, A. Peschel, Dual role of GdmH in producer immunity and secretion of the staphylococcal lantibiotics gallidermin and epidermin, Appl. Environ. Microbiol. 67 (2001) 1380e1383. [26] D.J. Netz, R. Pohl, A.G. Beck-Sickinger, T. Selmer, A.J. Pierik, C. Bastos Mdo, H.G. Sahl, Biochemical characterisation and genetic analysis of aureocin A53, a new, a typical bacteriocin from Staphylococcus aureus, J. Mol. Biol. 319 (2002) 745e756. [27] Y. Aso, H. Koga, T. Sashihara, J. Nagao, Y. Kanemasa, J. Nakayama, K. Sonomoto, Description of complete DNA sequence of two plasmids from the nukacin ISK1 producer, Staphylococcus warneri ISK-1, Plasmid 53 (2005) 164e178. [28] J.D. Van Hamme, A. Singh, O. Ward, Physiological aspects. Part 1 in a series of papers devoted to surfactants in microbiology and biotechnology, Biotechnol. Adv. 24 (2006) 604. [29] A. Singh, J.D. Van Hamme, O. Ward, Surfactants in microbiology and biotechnology: Part 2. Application aspects, Biotechnol. Adv. 25 (2007) 99. ~ a, E. Alameda, Performance of a biosurfactant produce by [30] D. Altmajer, E. Gudin Bacillus subtilis strain isolated from crude oil samples as compared to commercial chemical surfactants, Colloid Surf. B Biointerfaces 89 (2012) 167e174. [31] Le TT. Nguyen, Biosurfactants: Characterization, Microemulsion Formation and Potential Applications, Thesis (Ph.D.), Dissertation Abstracts International, 2009, p. 71. [32] Z. Gao, X. Zhao, S. Lee, J. Li, H. Liao, X. Zhou, J. Wu, Q. Gaofu, WH1 fungin a surfactin cyclic lipopeptide is a novel oral immunoadjuvant, Vaccine 31

(2013) 2796e2803. [33] W. Baier, M. Loleit, B. Fischer, Generation of antibodies directed against the low-immunogenic peptide-toxins microcystin-LR/RR and nodularin, Int. J. Immunopharmacol. 22 (2000) 339e353. [34] P. Hoffmann, M. Jimenez-Dias, M. Loleit, Preparation of human and murine monoclonal antibodies: antigens combined with or conjugated to lipopeptides constitute potent immunogens for in vitro and in vivo immunizations, Hum. Antib. Hybrid. 1 (1990) 137e144. [35] M. Kreutz, U. Ackermann, S. Hauschildt, S.W. Krause, D. Riedel, W.G. Bessler, R. Andreesen, A comparative analysis of cytokine production and tolerance induction by bacterial lipopeptides, lipopolysaccharides, and Staphyloccocus aureus in human monocytes, Immunology 92 (1997) 396e401. [36] C. Ibacache-Quiroga, J. Ojed, G. Espinoza-Vergara, P. Olivero, M. Cuellar, M.A. Dinamarca, The hydrocarbon-degrading marine bacterium Cobetia sp. strain MM1IDA2H-1 produces a biosurfactant that interferes with quorum sensing of fish pathogens by signal hijacking, Microb. Biotechnol. 6 (4) (July 2013) 394e405. [37] D. Christybapita, M. Divyagnaneswari, R. Dinakaran Michael, Oral administration of Eclipta alba leaf aqueous extract enhances the non-specific immune responses and disease resistance of Oreochromis mossambicus, Fish Shellfish Immunol. 4 (2007) 840e852. [38] D.A. Relman, Universal bacterial 16S rDNA amplification and sequencing, in: Diagnostic Molecular Microbiology, 1993, pp. 489e495. [39] H.B. Ayed, M. Jridi, H. Maalej, M. Nasri, N. Hmidet, Characterization and stability of biosurfactant produced by Bacillus mojavensis A21 and its application in enhancing solubility of hydrocarbon, J. Chem. Technol. Biotechnol. 14 (2013) 23e29. [40] S. Techaoei, P. Leelapornpisid, D. Santiarwarn, S. Lumyong, Preliminary screening of biosurfactant producing microorganisms isolated from hot spring and garages in northern Thailand, KMITL Sci. Tech. J. 7 (2007) 1e38. [41] A. Hamzah, N. Sabturani, S. Radiman, Screening and optimization of biosurfactant production by the hydrocarbon-degrading bacteria, Sains Malays. 42 (2013) 615e623. ~ a, J.F.B. Pereira, L.R. Rodrigues, J.A.P. Coutinho, J.A. Teixeira, Isolation [42] E.J. Gudin and study of microorganisms from oil samples for application in microbial enhanced oil recovery, Int. Biodeterior. Biodegrad. 68 (2012) 56e64. [43] M. Abouseoud, R. Maachi, A. Amrane, S. Boudergua, A. Nabi, Evaluation of different carbon and nitrogen sources in production of biosurfactant by Pseudomonas fluorescens, Desalination 223 (2008) 143e151. [44] C. Feigner, F. Besson, G. Michel, Studies on lipopeptide biosynthesis by Bacillus subtilis: isolation and characterization of iturin, surfactin mutants, FEMS Microbiol. Lett. 127 (1995) 11e15. [45] G.C. Okpokwasili, A.A. Ibiene, Enhancement of recovery of residual oil using a biosurfactant slug, Afr. J. Biotechnol. 5 (2006) 453e456. [46] T. Varadavenkatesan, V. RamachandraMurty, Production of a lipopeptide biosurfactant by a novel Bacillus sp. and its applicability to enhanced oil recovery, ISRN Microbiol. (2013), 8 pages. [47] S.Y. Lee, Bacterial polyhydroxyalkanoates, Biotechnol. Bioeng. 49 (1996) 1e14. [48] K. Sobha, T.A. Susan, Evaluation of amino acid composition of Phaseolus mungo (black Gram) and Labeo rohita (Rohu) in the perspective of human dietary requirements, Res. J. Pharm. Biol. Chem. Sci. 5 (2014) 542e549. [49] Jha, Toxicology impact of house hold detergent, surf on digestion in tissue of fresh water fish Clarias batrachus (Linn), Fish Res. (2008) 107e114. [50] R.M. Parry, R.C. Chandan, R.M. Shahani, A rapid sensitive assay of muramidase, Proc. Soc. Exp. Biol. Med. 119 (1965) 384e386. [51] T.H. Hutchinson, M.J. Manning, Seasonal trends in serum lysozyme activity and total protein concentration in dab (Limanda limanda L) sampled from Lyme Bay, UK, Fish Shellfish Immunol. 6 (1996) 473e482. [52] T.J. Bowden, R. Butler, I.R. Bricknell, A.E. Ellis, Serum trypsin inhibitory activity in five species of farmed fish, Fish Shellfish Immunol. 7 (1997) 377e385. [53] M.J. Quade, J.A. Roth, A rapid, direct assay to measure degranulation of bovine neutrophil primary granules, Vet. Immunol. Immunopathol. 58 (1997) 239e248. [54] T.L. Welker, L. Chhorn, Y.A. Mediha, H.K. Klesius, Growth, immune function, and disease and stress resistance of juvenile Nile tilapia (Oreochromis niloticus) fed graded levels of bovine lactoferrin, Aquaculture 262 (2007) 156e162. [55] C. Binuramesh, M. Prabakaran, D. Steinhagen, R.D. Michael, Effect of chronic confinement stress on the immune responses in different sex ratio groups of Oreochromis mossambicus (Peters), Aquaculture 250 (2005) 47e59. [56] R.D. Michael, S.D. Srinivas, K. Sailendri, V.R. Muthukkaruppan, A rapid method for repetitive bleeding in fish, Indian J. Exp. Biol. 32 (1994) 838e839. [57] D.K. Sakai, Heat inactivation of complements and immune hemolysis reactions in rainbow trout, masu salmon, coho salmon, goldfish and tilapia, Bull. Jpn. Soc. Sci. Fish. 47 (1981) 565e571. [58] I. Karunasagar, A. Ali, S.K. Otta, Immunization with bacterial antigens, Infections with motile aeromonads, Dev. Biol. Stand. 90 (1997) 135e141. [59] D.F. Amend, Potency testing of fish vaccines, Dev. Biol. Stand. 49 (1981) 447e454. [60] B. Magnadottir, Innate immunity of fish (overview), Fish Shellfish Immunol. 20 (2006) 137e151. [61] B. Magnadottir, S. Lange, S. Gudmundsdottir, J. Bogwald, R. Dalmo, Ontogeny of humoral immune parameters in fish, Fish Shellfish Immunol. 19 (2005) 429e439. [62] S.A. Arancibia, C.J. Beltran, I.M. Aguirre, P. Silva, A.L. Peralta, M.A. Hermoso, Toll-like receptors are key participants in innate immune responses, Biol. Res.

V. Rajeswari et al. / Fish & Shellfish Immunology 48 (2016) 244e253 40 (2007) 97e112. [63] Y. Palti, Toll like receptors in bony fish: from genomics to function, Dev. Comp. Immunol. 35 (2011) 1263e1272. [64] N.H. Youssef, K.E. Duncan, D.P. Nagle, K.N. Savage, R.M. Knapp, M.J. McInerney, Comparison of methods to detect biosurfactant production by diverse microorganisms, J. Microbiol. Methods 56 (2006) 339e347. [65] S. Nasr, M.R. Soudi, M.R. Mehrnia, M.H. Sarrafzadeh, Characterization of novel biosurfactant producing strains of Bacillus sp. isolated from petroleum contaminated soil, Iran. J. Microbiol. 1 (2009) 54e61. [66] A. Hamzah, N. Sabturani, S. Radiman, Screening and optimization of biosurfactant production by the hydrocarbon-degrading bacteria, Sains Malays. 42 (2013) 615e623. lix, F.M. Gon, Infrared studies of protein-induced [67] J.L.R. Arrondo, M. Fe perturbation of lipids in lipoproteins and membranes, Chem. Phys. Lipids (1998) 53e68. [68] R.R. Saikia, S. Deka, M. Deka, H. Sarma, Optimization of environmental factors for improved production of rhamnolipid biosurfactant by Pseudomonas aeruginosa RS29 on glycerol, J. Basic Microbiol. 52 (2012) 446e457. [69] M.M. Yakimov, K.N. Timmis, V. Wray, H.L. Fredrickson, Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface Bacillus licheniformis BAS50, Appl. Environ. Microbiol. 61 (1995) 1706e1713. [70] M.L. Hourdou, F. Besson, I. Tenoux, G. Michel, Fatty acids and b-amino acid syntheses in strains of Bacillus subtilis producing iturinic antibiotics, Lipids 24 (1989) 940e944. [71] T.C. Fletcher, Modulation of non-specific host defences in fish, Vet. Immunol. Immunopathol. 12 (1986) 59e67. [72] G.X. Wang, Y.T. Liu, F.Y. Li, H.T. Gao, Y. Lei, X.L. Liu, Immunostimulatory activities of Bacillus simplex DR-834 to carp (Cyprinus carpio), Fish Shellfish Immunol. 29 (2010) 378e387. [73] T.J. Bowden, R. Butler, I.R. Bricknell, A.E. Ellis, Serum trypsin inhibitory activity in five species of farmed fish, Fish Shellfish Immunol. 7 (1997) 377e385. [74] D.K. Sakai, Repertoire of complement in immunological defence mechanisms of fish, Annu. Rev. Fish Dis. (1992) 223e247. ttir, H. Jo nsdo ttir, S. Helgason, B. Bjo € rnsson, T. Jørgensen, [75] B. Magnado €m, Humoral immune parameters in Atlantic cod (Gadus morhua L.) I: L. Pilstro the effects of environmental temperature, Comp. Biochem. Physiol. 122B (1999a) 173e180. ttir, H. Jo nsdo ttir, S. Helgason, B. Bjo € rnsson, T. Jørgensen, [76] B. Magnado € m, Humoral immune parameters in Atlantic cod (Gadus morhua L.) II: L. Pilstro the effects of size and gender under different environmental conditions, Comp. Biochem. Physiol. 122B (1999b) 181e188. [77] R. Salte, H.M. Gjøen, K. Norberg, T. Gjedrem, Plasma protein levels as potential marker traits for resistance to furunculosis, J. Fish Dis. 16 (1993) 561e568. [78] S.J. Freedman, The role of a2-macroglobulin in furunculosis: a comparison of rainbow trout and brook trout, Comp. Biochem. Physiol. B 98 (1991) 549e553.

253

[79] R. Harikrishnan, J.S. Kim, C. Balasundaram, M.S. Heo, Dietary supplementation with chitin and chitosan on haematology and innate immune response in Epinephelus bruneus against Philasterides dicentrarchi, Exp. Parasitol. 131 (2012) 116e124. [80] A. Rodrigues, M.A. Esteban, J. Meseguer, Phagocytosis and peroxide release by seabream (Sparus aurata L.) leucocytes in response to yeast cells, Anat. Rec. Part A 272A (2003) 415e423. [81] D. Pali c, C.B. Andreasen, E.D. Frank, B.W. Menzel, J.A. Roth, Gradient separation and cytochemical characterisation of neutrophils from kidney of fathead minnow (Pimephales promelas Rafinesque, 1820), Fish Shellfish Immunol. 18 (2005a) 263e267. [82] D. Pali c, C.B. Andreasen, B.W. Menzel, J.A. Roth, A rapid, direct assay to measure degranulation of primary granules in neutrophils from kidney of fathead minnow (Pimephales promelas Rafinesque, 1820), Fish Shellfish Immunol. 19 (2005b) 217e227. [83] C.S. Engstad, R.E. Engstad, J.O. Olsen, B. Osterud, The effect of soluble beta-1,3glucan and lipopolysaccharide on cytokine production and coagulation activation in whole blood, Int. Immunopharmacol. 2 (2002) 1585e1597. [84] T.L. Welker, C. Lim, Shelby R. Yildirim-Aksoy, P.H. Klesius, Immune response and resistance to stress and Edwardsiella ictaluri challenge in channel catfish, Ictalurus punctatus, fed diets containing commercial whole-cell yeast or yeast subcomponents, J. World Aquac. Soc. 38 (2007) 24e35. [85] D. Kishore, K.M. Ashis, Assessment of mosquito larvicidal potency of cyclic lipopeptides produced by Bacillus subtilis strains, Acta Trop. 97 (2006) 168e173. [86] G. Seydlov, J. Svobodov, Review of surfactin chemical properties and the potential biomedical applications, Cent. Eur. J. Med. 3 (2008) 123e133. [87] M.B.S. Donio, S.F.A. Ronica, V. Thanga Viji, S. Velmurugan, J.A. Jenifer, M. Michaelbabu, P. Dhar, T. Citarasu, Halomonas sp. BS4, A biosurfactant producing halophilic bacterium isolated from solar salt works in India and their biomedical importance, Springer Plus 2 (2013) 149. [88] M.B.S. Donio, S.F.A. Ronica, V. Thanga Viji, S. Velmurugan, J.A. Jenifer, M. Michaelbabu, T. Citarasu, Isolation and characterization of halophilic Bacillus sp BS3 able to producing pharmacologically important biosurfactants, Asian J. Trop. Med. 6 (2013) 876e883. [89] J.M. Raaijmakers, I.D. Bruijn, O. Nybroe, M. Ongena, Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics, FEMS Microbiol. Rev. 34 (2010) 1037e1062. [90] A. Vitiello, G. Ishioka, H.M. Grey, R. Rose, P. Farness, R. Lafond, et al., Development of a lipopeptide-based therapeutic vaccine to treat chronic HBV infection.I. Induction of a primary cytotoxic T lymphocyte response in humans, J. Clin. Investig. 95 (1995) 341e349. [91] H. Tada, S. Aiba, K. Shibata, T.Z. Ohteki, H. Takada, Synergistic effect of Nod1 and Nod2 agonists with toll-like receptor agonists on human dendritic cells to generate interleukin-12 and T helper type 1 Cell, Infect. Immun. 73 (2005 D) 7967e7976.