5 Int.J.Curr.Biotechnol. Volume 1; Issue 1; Mar, 2013

Ramesh B., Assessment of Antimicrobial peptides from mucus of fish, Int.J.Curr.Biotechnol., 2013,1(1):5-8.

International Journal of CurrentBiotechnology

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Assessment of Antimicrobial peptides from mucus of fish

Balasubramaniyam Ramesh**Department of Biotechnology, Sri Sankara Arts and Science College, Enathur – 631 561.

A R T I C L E I N F O A B S T R A C T

Article History:Received 02 March 2013Received in revised form 08 March 2013Accepted 19 March 2013Available online 22 March 2013

Key words:Fish mucusSDS PAGEBacterial pathogenProtein

Fish are a diverse group of animals, highly specialized for their aquatic existence andcomprising almost half the number of vertebrate species in existence today. Fish are inintimate contact with their environment, which can contain very high concentrationsof bacteria and viruses. The immune system is composed of numerous organs andcells that act together in a dynamic network in the defense against infection, diseaseand foreign substances. The bacterial pathogens was tested against several organ-isms and maximum inhibition was recorded against Staphylococcus aureus (12 mm),Lactobacillus vulgarius (10mm) and Klebsiella pneumonia (7 mm). The protein estima-tion of fish mucus was observed as (0.865 mg/ml). SDS -PAGE of fish mucus showedmany protein bands with molecular size ranging from 22 to 65 K da.

Introduction

Aquaculture is the cultivation of aquatic animals suchas fish (or) Shellfish (or) of plants such as seaweed ina controlled and sometimes enclosed body of water. InAquaculture, according to the intensive system ofproduction to reduce the cost, fish are kept in highdensities and the possibility for exposure to pathogenswhich can be bacteria, parasites (or) virus, throughoutproduction cycle was becoming high (Laidler et al., 1999).Under such conditions, the problems of the Infectiousfish diseases become serious and have considerableeffects on Aquaculture. In fact, bacterial and viraldiseases of farmed fish have lead to high moralitiesand reduced economical income for the fish farmingindustry (Munro 1933; Pilcher 1980). Though aquacultureis very profitable, it also creates serious economicthreat when diseases outbreaks. Avoiding diseaseepidermis in aquaculture is very important to geteconomic benefit and it is possible only when fishspecies are reared in a good environmental conditionand given priority in fish welfare. Fish welfare or animalright law is related to farmed fish. It means that fishhave right to live a life as good as possible and expressits natural behaviour as much as possible and freefrom negative experiences. Fish are in intimate contactwith their environment through the large surface oftheir gills, skin, and of necessity they defecated into

the medium in which they live, so water quality (interms of dissolved oxygen, CO

2, ammonia and pH) and

the presence of contaminants (organic and inorganicpollutants) are probably the most critical aspects ofthe environment for fish welfare and also the bestdefined (Mellor and Stafford 2001).

Optimal conditions vary markedly between species forexample, catfish do poorly in clear water, whereassalmon do poorly in cloudy water and cyprinid fish arevery tolerant of low dissolved oxygen levels where assalmonid fish are not (Kramer 1987). The mechanismbarrier of the skin impedes entry of the majority ofmicroorganisms into the body (Bressler and Bressler etal., 1989) Mucous membranes lining the alimentary,respiratory, and urogenital tracts are equipped with alayer of mucus which functions to entrap foreignmicroorganisms out of the body. The major componentsof the mucus layer are produced by goblet cells andthese cells start to differentiate in the basal part ofthe epidermis, and then grow in size and move towardsthe surface where they release their content (Pickering,1977). The mucus is a dynamic coat, which passivelyflows over and covers the fish (Powell et al., 1992).

Mucus slime is the material that makes fish slippery.Its ‘slipperiness’ is the result of its high water contentand the presence of high-molecular weight, gel-forming, macromolecules. The skin of fish is a dynamictissue whose cellular makeup known to be influencedby factors such as season, stress, diseases,

*Corresponding author. Tel: 91-9787088802 (M);

Email address: brbioinfo@gmail.com

ISSN: 2321 - 8371

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development stage and environmental conditions(Blackstock and Pickering, 1982). A great deal of researchsupports the notion that layers of mucus accumulateon the skin and gills of fish that are stressed by disease,adverse environmental conditions and handling (Handyand Eddy, 1991). The skin surfaces support considerableconcentration of gradients, particularly for sodium andchloride in fresh water or seawater. Fish biologistssuspected that mucus might be involved in ionregulation.

The present study demonstrated the Antibacterialactivity and protein content in mucus of several fishspecies (Austin and Mcintosh, 1988) yet this activityseems to vary from fish species to fish species and canbe specific towards certain bacteria (Noya et al., 1995).

Materials and Methods

Fish Collection and MaintenanceMucus sample was collected from Oncorhynchus mykiss,Channa striatus were obtained from a local fish marketin Kanchipuram. Then the fish were stocked into the500 L capacity circular plastic tanks. The fishacclimatized to laboratory conditions in a tap waterand they were maintained for one week. During thisperiod the fish were fed with commercial feed once aday at ad libitum. Every day 50 % of the water waschanged. After one week of acclimatization the fishwere used for mucus collection. Only healthy fish werechosen for mucus collection. Dead fish or fish with skinlesions were removed from the tanks.

Mucus CollectionMucus was collected by a modified method of(Subramanian et al 2008). Fish was starved for one dayprior to mucus collection. On the day of mucus collectionfish was washed and transferred into a sterilepolyethylene bag for 10 to 20 minutes and moved frontand back to slough off the fish mucus. Then, the fishwas returned to recovery tanks. Fish mucus samplesobtained from five fish was then pooled and stored inrefrigerator at 4°C until further use.

Mucus ExtractionThe aqueous extract of fish mucus was prepared usinga method as described by (Hellio et al 2002). Fifty ml offish mucus was mixed with 50 ml of distilled water and

homogenized using a polytron homogenizer. Themixture was then centrifuged at 30,000 g for 30 minutesat 4°C. Supernatant was then collected and filteredwith Whatman no.1 filter paper. The filtrate was thencollected and stored in refrigerator at 4°C.

Protein EstimationThe protein concentration of the samples wasdetermined by the method of (Lowry et al., 1951) withbovine serum albumin as standard. To 5ml of Lowryreagent, add 1ml of suitably diluted sample and themixture was kept at room temperature for 10min. Tothis add 0.5ml of Folin’s reagents and kept at darkcondition for 30mins. The absorbance was taken at640nm.

Protein separation and QuantificationMost commonly the strong anionic detergent SDS isused combination with a reducing agent and heated todisassociate the proteins before they are loaded onthe gel. The amount of SDS bound is alwaysproportional to molecular weight of the poly peptidesand is independent of their sequence. The protein wereseparated by SDS-PAGE electrophoresis and size ofpolypeptide chains of given protein can be determinedby comparing its electrophoretic mobility in SDS-PAGEgel with mobility marker proteins of known molecularweight (Laemmli, 1973).

Antimicrobial assaysStaphylococcus aureus, Salmonella typhi, Salmonellaparatyphi, Klebsiella oxytoca, Pseudomonas aeruginosa, E.coli,Proteus mirabilis, Lactobacillus vulgaris, Vibrio cholera andKlebsiella pneumonia. All the bacteria mentioned abovewere incubated at 35±0.10C for 24h by inoculation intoNutrient Broth while the fungal cultures were incubatedin Potato Dextrose Broth at 25±0.10C for 48h. Thesecultures were spread-plate on Mueller Hinton Agarusing a sterile cotton swab and wells were made inthe plates with the help of a cork borer (0.85cm). Thetest compound (0.1ml) was introduced into the welland the plates were incubated (Rusell et al., 1977).

Results

Protein contentThe fish mucus was estimated for its protein level andthe results shows that the fish mucus contains 0.865mg/ml of protein content

Protein separation and QuantificationSodium Dodecyl Sulphate Polyacrylamide GelElectrophoresis SDS-PAGE is an excellent tool to identifyand monitor proteins during purification and to accessthe homogeneity. SDS Polyacrylamide GelElectrophoresis (SDS-PAGE) of fish mucus showed many

Fig 1: Antimicrobial activity of fish mucus against bacte-rial pathogens

7 Int.J.Curr.Biotechnol. Volume 1; Issue 1; Mar, 2013

protein bands with molecular size ranging from 22KDaas shown in Fig 1.

Antibacterial AssaysAntibacterial activity was determined and the bacterialpathogens (Staphylococcus aureus, Salmonella typhi,Salmonella paratyphi, Klebsiella oxytoca, Pseudomonasaeruginosa, E.coli, Proteus mirabilis, Lactobacillus vulgaris,Vibrio cholera and Klebsiella pneumonia) were susceptibleto fish mucus. Maximum inhibition was recordedagainst Staphylococcus aureus (12mm), Lactobacillusvulgaris (10mm), Proteus mirabilis (9mm), Salmonella andVibrio cholera (8mm) whereas Klebsiella pneumonia were

and antibacterial activities in the fish mucus. Thispeptide may play a role in protection againstintracellular or extracellular pathogens (Fernandes andSmith, 2002). It was demonstrated that there weresignificant histological and biochemical differencesbetween the skin and mucus of rainbow trout, Cohoand Atlantic salmon, which may change as a result ofdifferent environments. Variation in these innateimmune factors is likely to have differing influence oneach species response to disease processes (Fast etal., 2002)

ReferencesAustin, B., and D. Mcintosh. 1988. Natural Antibacterial

Compounds on the Surface of Rainbow-Trout,Salmo-Gairdneri Richardson. Journal of FishDiseases 11: 275-277.

Blackstock, N., and A. D. Pickering. 1982. Changes in theConcentration and Histochemistry ofEpidermal Mucous Cells During the Alevin andFry Stages of the Brown Trout Salmo-Trutta.Journal of Zoology 197: 463-471.

Bressler, R. S., and C. H. Bressler. 1989. Functionalanatomy of the skin. Clin Podiatr Med Surg 6:229-46.

Dalmo, R.A., Ingebrigtsen, K., Bogwald, J., 1997. Non-speciûc defence mechanisms in ûsh, withparticular reference to the reticuloendothelialsystem (RES). J. Fish Dis. 20, 241–273

Fast, M.D (2002) Skin morphology and humoral non-specific parameter of mucus and plasma inrainbow trout, coho and atlantic salmon.CompBiochem Physiol. Vol-132, 645-657

Fevolden SE, Roed KH. Cortisol and immunecharacteristics in rainbow trout (Oncorhynchusmykiss) selected for high or low tolerance tostress. J Fish Biol 1993; 43: 919-30

Handy, R. D., and F. B. Eddy. 1991. The Absence of Mucuson the Secondary Lamellae of UnstressedRainbow-Trout, Oncorhynchus-Mykiss(Walbaum). Journal of Fish Biology 38: 153-155.

Hellio C, Pons AM, Beaupoil C, Bourgougnon N, Legal Y.Antibacterial, antifungal and cytotoxicactivities of extract from fish epidermis andepidermal mucus. Int J Antimicrob Agents 2002;20: 214-219.

Kramer, D. L. 1987. Dissolved-Oxygen and Fish Behavior.Environmental Biology of Fishes 18: 81-92.

Laemmli, U. K. and M. Favre (1973). “Maturation of thehead of bacteriophage T4. I. DNA packagingevents.” Journal of Molecular Biology. 80(4):575-99.

Laidler, L. A., J. W. Treasurer, A. N. Grant, and D. I. Cox.1999. Atypical Aeromonas salmonicidainfection in wrasse (Labridae) used as cleanerfish of farmed Atlantic salmon, Salmo salarL., in Scotland. Journal of Fish Diseases 22:209-213.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Proteinmeasurement with the Folin phenol reagent. JBiol Chem. 1951 Nov;193(1):265–275.

Mellor, D. J., and K. J. Stafford. 2001. Integrating practical,regulatory and ethical strategies forenhancing farm animal welfare. AustralianVeterinary Journal 79: 762-768.

Munro, A. J. 1993. Publishing the Findings of ClinicalResearch. British Medical Journal 307: 1340-1341.

Noya, M., B. Magarinos, A. E. Toranzo, and J. Lamas. 1995.Sequential Pathology of ExperimentalPasteurellosis in Gilthead Seabream Sparus-Aurata - a L ightMicroscopic and Electron-Microscopic Study. Diseases of AquaticOrganisms 21: 177-186.

Pickering, A. D., and D. J . Macey. 1977. Structure,Histochemistry and Effect of Handling on

showed slightly lower inhibition (8mm) as shown inthe Table 1 and Fig 2.

DiscussionOver the past years, it has also been shown that mucusplays a role in the prevention of colonization byparasites, bacteria and fungi and the antibacterial roleof mucus has been known for many years (Austin andMcintosh, 1988). Fish mucus was found as a source ofantimicrobial products (Hellio et al., 2002).The skin mucous layer and epidermis are important infish defense because they are the first sites ofinteraction between the host and potential pathogens.Within these layers are many enzymes andantimicrobial proteins, which are thought to beinvolved in innate immunity of the fish (Dalmo et al.,1997). Differences in activities of ntimicrobial enzymes,such as lysozyme and proteases, and how they relateto the structure and composition of mucus andepidermal layers, may also relate to the differencesobserved in disease resistance. Lysozyme, anantimicrobial, hydrophobic protein found in fish bloodand tissues has been studied most extensively inassociation with disease resistance (Lie et al., 1989)and cortisol stress response (Fevolden and Roed, 1993).The antibacterial activity in mucus samples of fivesalmon and five cod was tested in different proteinconcentrations, against four different bacterial strains(E.coli, Lactobacillus anguillarum, Clastridium glutamicumand Staphylococcus aureus). Growth curves of the bacteriaand water were considered as negative controls, andwere compared against the curves of bacterial culturesadded in the different samples. Antibacterial activitywas conducted in the serum and mucus of rainbow trout(Oncorhynchus mykiss) and found a number ofantibacterial factors increase in concentrationfollowing immunization and that these probably playeda role in protection against microbial disease (Raingerand Rowley, 1993).Elutes from the solid phase extraction were tested forantibacterial activity against E.coli, Lactobacillusanguillarum, Clastridium glutamicum and Staphylococcusaureus was detected in extracts from several tissues inall species tested, but mainly in the haemolymph andhaemocyte extracts. L.anguillarum and C.glutamicumwere found the most sensitive micro-organisms.However, Hellio et al., 2002) studied antibacterial,antifungal and cytotoxic activities of extracts from fishepidermis and epidermal mucus and found antifungal

Table 1: Antibacterial activity of Fish muscus

Strains Zone of Clearance (mm)

Staphylococcus aureas 12Salmonella typhi 8Salmonella paratyphi 8Klebsiella oxytoca 7Pseudomonas aeruginosa 10E. coli 10Proteus mirabilis 9Lactobacillus vulgaris 10Vibrio cholera 8Klebsiella pneumonia 7

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Mucous Cells of Epidermis of Char Salvelinus-Alpinus (L). Journal of Fish Biology 10: 505-&.

Pilcher, K. S., and J. L. Fryer. 1980. The Viral Diseases ofFish - a Review through 1978 Diseases of ProvenViral Etiology. Crc Critical Reviews inMicrobiology 7: 287-363.

Powell, M. D., D. J. Speare, and J. F. Burka. 1992. Fixationof Mucus on Rainbow-Trout (Oncorhynchus-Mykiss Walbaum) Gills for Light and Electron-Microscopy. Journal of Fish Biology 41: 813-824.

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