15 lsa - mariyappan.pdf

7/24/2019 15 LSA - Mariyappan.pdf

1/15

Available online at www.jpsscientificpublications.com

Life Science Archives

LSA)

ISSN: 2454-1354

Volume 1; Issue - 2; Year 2015; Page: 96 - 111

2015 Published by JPS Scientific Publications Ltd. All right reserved

Research ArticleEFFICACY OF PLANT EXTRACTS ADDITIVES ON IMMUNITY AND

RESISTANCE AGAINST Aeromonas hydrophi laIN COMMON CARP,

Cyprinus carpio

M. Mariappan*, J. Prakash Sahaya Leon and K. Balakrishnan

Department of Zoology DDE, Annamalai University, Chidambaram 608 002, Tamil Nadu, India

Abstract

Fish farmers used vaccines to control diseases can result in the development of drug resistant

pathogens, environmental pollution and accumulation of residues in fish. In recent years, application ofmedicinal valuable plants in aquaculture is considered as a promising alternative to vaccines. The presentinvestigation was designed to study the eight plant extracts and screened their antimicrobial activities against

Aeromonashydrophila, a bacterial pathogen in common carp, Cyprinuscarpio. Using disc diffusion assays

five extracts among these ten (Azadirachtaindica, Andrographispaniculata, Allium sativum

SolanumtricobatumandOcimum sanctum) were selected and equal proportions of them were mixedthoroughly with the artificial feeds at concentrations of 0.0 (A), 100 (B), 200 (C), 400 (D) and 800 (D) mgkg

-1of dry diet. The experiment was conducted with the adult fishes for a period of 60 days. The prepared

diets were fed the common carp during experiment and then challenged withA. hydrophilawhich was given

by intraperitoneal injection. To evaluate the immune response and resistance againstA. hydrophilainfection

of fish, hematological, biochemical and immunological parameters were investigated at 15, 30, 45 and 60

days of feeding and also 15th

day of post-challenge. Results shows that RBC, WBC, Hb, Ht, MCV, MCHMCHC, serum total protein, glucose, cholesterol, serum bactericidal activity and lysozyme activity were

increased in fish fed with plant extract mixed diets compared to the control group. At the end of post-

challenge test, the survival rates were 42.43% in control group (A) and 62.75, 79.35, 84.65 and 89.57% ingroup B, C and D respectively. These results indicate that addition of herbal extract can promote immunity

and can prevent disease in common carp.

Article HistoryReceived: 20.03.2015

Revised : 30.03.2015

Accepted: 04.04.2015

Key words: Aeromonas hydrophila, Cyprinus

carpioand Plant extracts.

1.

IntroductionVaccination is one of the most promising

methods for to enhance the immune system in fish

to prevent diseases. Vaccines, enhance the

acquired immune response of the fish and a singlevaccine is effective only one type of pathogens

* Corresponding author: M. Mariappan

Tel.: +91-8428223898

E-mail: [email protected]

(Laszlo Ardo et al., 2008), but Aeromonas

hydrophila is a heterogenous species, having

variable antigens, that vaccines development isextremely complex (Ramasamy et al., 2010)

However, the use of vaccine in aquaculture has

received considerable attention because their usecan lead to the development of drug resistant

bacteria and the accumulation in fish cause risk to


2/15

M. Mariappam/ Life Science Archives (LSA), Volume 1, Issue 2, Page 96 to 111, 2015 97


consumers and the environment (Alderman and

Hastings, 1998; Adithepchaikarn et al., 2008).

To overcome the problems of vaccines,

immunostimulants were used to enhance theinnate immune response (Galeotti, 1998; Sakai,

1999; Laszlo et al., 2008). An immunostimulantsis naturally occurring compound that modulates

the immune system by increasing the hostsresistance against diseases that in most

circumstances are caused by pathogens (Peddieet

al., 2002). The use of immunostimulants for theprevention of disease in fishes is considered as an

attractive and promising area in the field of

aquaculture (Chinnasamy et al., 2013). The

modulation of immune response by varioussubstances has been reported, including synthetic,

bacterial, animal and plant products (Vasudeva

Rao et al., 2006). Among this plant extracts have

beneficial effects without any side effects. Someplants are rich sources of compounds like volatile

oils, saponins, phenolic compounds, tannins,

alkaloids, polypeptides and polysaccharides.These natural products have various activities like

antistress, appetizer, antimicrobials and

immunostimulant (Citarasu et al., 2002; Citarasu

et al., 2003).

Various studies revealed that plant extracts

have enormous therapeutic potential. Moreover,they are cheaper, safer, non-toxic, biodegradable

and biocompatible (Subeena and Navaraj, 2013).Several plant materials or extracts such as

Astragalus membranaceus (Shao et al., 2004),

Azadirachta indica, Oscimum sanctum and

Curcuma longa (Ramasamyet al., 2010), Viscumalbum, Urticadioica and Zingiber officinale

(Dugenci et al., 2003; Chinnasamy et al., 2013),

Ocimum tenuiflorum, Zingiber officinale and

Allium cepa (Iruthayam et al., 2014), Eucalyptus

spp. and Plelargoni amroseum. Mehrak et al.(2013) have been reported to enhance the

immunity and elevated resistance against thedisease causing agents. However, there was no

report in different concentration of plant extracts

in dietary supplementation feed on hematological

and innate immune response of common carpagainst A. hydrophila. Pastly, studies have used

hematological indices and immunological assay to

determine the health status of fishes

(Harikrishnan, 2003; Harikrishnan, 2009)

Therefore, the aim of the present study was to

investigate the effects of dietary mixed plantextract supplementation feed on hematological

changes and innate immunity of Cyprinus carpio

againstA. hydrophila.

2. Methodology

2.1. Fish

Common carp, C. carpio obtained from alocal fish farm in Cholatharam, Tamilnadu, India

were transported to the laboratory in plastic bags

filled with oxygenated water. The fishes were

acclimatized in cement tank containing 500 l ofwater for 2 week under laboratory conditions

(12/12 hrs light/dark cycle), feeding with basal

experimental diet without supplementation of the

plant extracts (Table - 1) at a rate of 2% of theirbody weight/day. The observed water quality

parameters were dissolved oxygen concentration

7.0 7.5 mg l-1

, pH 7.2 7.6 and temperature at

26 2C during the whole trial. The 50% of waterwas renewed daily to remove the unfed and fecal

materials.

2.2. Growth ofA. hydrophil a

A. hydrophila was obtained from theDepartment of Microbiology, Annamalai

University, Chidambaram, Tamilnadu andmaintained in the laboratory under standard

conditions. Sub-cultures were maintained onTryptone soya agar (Hi-media, Mumbai) in slopes

at 5C. Stock culture in tryptic soy broth (Hi-

media, Mumbai) was stored at -70C in 0.85%NaCl with 20% glycerol to provide stable

inoculate throughout the experiment (Chabot and

Thune, 1991). The broth was incubated overnight

in a shaker for 24 hrs at 20C, and then centrifugedat 10,000 rpm for 20 min at 4C. The supernatant

was discarded and the bacterial pellet was washedthree times with phosphate-buffered saline (PBS)at pH 7.2.

2.3. Preparation of Plant extracts

The parts of eight plant species used in this

study are listed in Table - 2. Fresh parts of these

plant species were collected around AnnamalaUniversity campus during October 2012. The

plants were washed in sterile distilled water and


3/15



separately shade dried for 10 days till weight

constancy was achieved. Each plant parts were

finely powdered in an electric blender. Extracts ofthe powdered plant parts were prepared using

water, 95% ethanol, as solvents at ratios of 1:10

(w/v). The extractions were carried out at room

temperature. The extracts obtained werecentrifuged at 5,000 rpm for 15 min. The filtrate

obtained from ethanol was evaporated to dryness

at 40C in a rotary evaporator and the water

extract was then freeze-dried by using a freezedrier system. Finally, the extracts were stored at

4C until use (Arabshah Delouee and Urooj,

2007).

2.4. Screening of the Plant extract againstA.

hydrophila

A disc diffusion method (Bauer et al.,1996) with some modifications was used to detect

antimicrobial activity of plant extracts against A.hydrophila. Each 500 g of extract was

impregnated in 5mm diameter sterile paper discs

and allowed to dry. Dried antimicrobial discs with

impregnated herbal extracts were carefullydispensed with uniform distances over soyabean

casein digest agar surface and correct implantation

was assured by applying gentle pressure over thedisc. The plates were incubated at 30C for 24

hrs, after which the inhibitory zone formation ofantimicrobial extracts around the paper discs were

measured and recorded (Table - 2).

2.5. Fish diet preparation

The formulated fish feed was prepared inthe laboratory using soyaflour and fish meals as

the protein sources (Table - 1). Based on the zone

of inhibition, Azadirachta indica, Andrographis

paniculata, Allium sativum, Solanum tricobatumand Ocimum sanctumwere selected for the present

study. To enrich the normal diet with in 100, 200,400 and 800 mg kg-1

(w/w) of the five plantsextract was added into the feed slowly, mixing

part in a drum mixer, after which it was air dried

under sterile conditions for 12 hrs. Control dietwas prepared using the same composition of

ingredients except the extract mixture. The pellets

were dried in an oven at 30C for 18 hrs, packed

and stored at -2C in air tight containers until fed.

2.6. Fish feeding experiment

Fishes were randomly divided into five

groups in triplicate each with 50 fishes (n = 50

fishes/group). They were fed with 0, 100, 200400 and 800 mg kg

-1extract mixture (later it will

be called as group A (control), group B, group Cgroup D and group E, respectively). The fish were

fed twice a day at 8:00 and 14:00 hrs at 2% of thebody weight until the end of the experiment.

2.7. Blood sampling

Blood samples of five fish/group were

selected randomly from each group and collect

blood from caudal vein using 1 ml tuberculinsyringe fitted with 24 gauge needle (Michael eal., 1994) on 15, 30, 45 and 60 days after the

feeding. Fishes were anesthetized with 100 mg of

tricaine methane sulphonate (MS-222). Individuafish were sampled only once to avoid the influence

on the assays due to multiple bleeding and

handling stress on the fish. One half of each bloodsample was immediately used for hematological

examination, while the other half was mixed with

heparin anticoagulant then frozen at 4C using

EDTA serum tubes. The serum tubes were placedat room temperature and allowed to clot for 2 hrs

The supernatant serum was separated and

collected from the remaining blood after

centrifugation (1500 rpm for 20 min) and stored at-70 C for biochemical and immunological

analyses.

2.8. Challenges with virulent pathogen

The susceptibility of the fish fed plantextracts to a bacterial challenge was examine invivo A. hydrophila was used as the challenge

strain. Groups of 25 fishes fed with 0, 100, 200

400 and 800 mg kg-1

extracts were challenged 60days after the start of treatments. Carp were

injected intraperitoneally with 100 L of live Ahydrophila (1 10

8cfu mL

-1). The fish were

observed regularly at 12 hrs intervals for mortalityupto 15 days. The surviving fish were than

sampled for serum and blood parameters as per the

method described earlier.

2.9. Hematology

Red blood cell (RBC: 106mm

-3) and white

blood cell (WBC: 103mm

-3) were counted


4/15



manually by haemocytometry (Samuel, 1986)

using Neubauer haemocytometer after diluting

blood samples by adding of Hayem solution forRBC and Turk solution for WBC, respectively.

Haemoglobin (Hb: g dL-1

) concentration of the

blood was estimated by the method of using

Shalis haemoglobinometer (Samuel, 1986).Haematocrit (Ht: %) was measured with

microcentrifuge method, using standard

heparinized micro haematocrit capillary tubes (75

mm at 7000 rpm for 10 min). The derivederythrocytes of mean corpuscular volume (MCV:

m3), mean corpuscular haemoglobin (MCH: pg)

and mean corpuscular haemoglobin concentration(MCHC: g dL

-1) were calculated according to

Dacie and Lewis (1991). The total protein (TP:

mg dL-1

), glucose (GLU: mg dL-1

) and cholesterol

(CHO: mmol-1) were determined by Hawk et al.(1954).

2.10. Respiratory burst activity

Respiratory burst activity was measured by

the Nitro blue tetrazolium assay (NBT),

Secombesis (1990) method with the modificationdescribed by Satasiak and Baumann (1996). The

heparinized blood was collected in silica-coated

Eppendorf tubes and the buffy coat was separatedby centrifuging at 10,000 rpm for 10 min. Fifty

microlitres of the blood coat was placed in each ofthe 96 wells of U bottom microtitre plates and

incubated at 37 C for 1 hour, to facilitateadhesion of cells. Then, the supernatant was

removed and 50 ml of 0.3% NBT was added.

After incubation, NBT was removed. The cellswere then fixed with 100% methanol and washed

thrice with 70% methanol. The plates were air-

dried. Sixty microlitres of 2N KOH and 70

microlitres dimethyl sulphoxide were added toeach well to dissolve the formazan blue

precipitate. The turquoise-blue coloured solutionwas then read in an ELISA reader at 655 nm.

2.11. Bactericidal activity

Serum bactericidal activity was estimatedby following the procedure of Kajita (1990). An

equal volume (100 L) of serum and bacterial

suspension was mixed and incubated for 1 hour at

25 C. A blank was prepared by replacing serumwith sterile PBS buffer. The mixture was then

diluted with sterile PBS at a ratio of 1:10. The

serum-bacterial mixture (100 L) was pour-plated

in nutrient agar and the plates were incubated for24 hrs at 37 C. The number of viable bacteria was

determined by counting the colonies grown on

nutrient agar plates. Data on bactericidal activity

were calculated as follows

Positive control CFU Sample CFU /(Positive control CFU/100)

2.12. Lysozyme assay

Plasma lysozyme activity was measured

spectrophotometrically by the method of Sankaran

and Gurnani (1972). The lysozyme substrate was a0.02% (w/v) suspension of Micrococcuslysodeikticus made up in phosphate buffer (0.05

M, pH 6.2). Lyophilized hen egg white lysozyme

was used as a standard. A new standard curve wasprepared for each assay. Standard solutions as

well as samples were added to the substrate at

25C. The results were expressed as mg mL-

equivalent of hen egg white enzyme activity.

2.13. Statistics

Results are presented as the average (standard error) for five fish, and were compared at

each time point using one-way ANOVA andDunns multiple range tests (SAS package)

Significant differences between experimentalgroups were expressed at a significant level of P

15 lsa - mariyappan.pdf

Documents