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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
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M. Mariappam/ Life Science Archives (LSA), Volume 1, Issue 2, Page 96 to 111, 2015 97
2015 Published by JPS Scientific Publications Ltd. All right reserved
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
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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
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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