Aquaculture is the fastest growing industry and is an important economic activity
contributing to the world protein supply. To meet the growing demand for fish
and seafood throughout the world, traditional farming systems have given way
to intensive aquaculture. Intensive culture and adverse environmental conditions
are often attributed to disease due to immune suppression or physiological stress.
High mortality and serious economic losses have been reported due to vibriosis,
a major disease problem in shrimp and prawn (Balasundaram
et al., 2012) aquaculture.
According to Ramalingam and Shyamala (2006), Vibrio
spp. are often considered opportunistic pathogens in shrimp, but primary disease
can also be caused by highly virulent strains. The major species causing vibriosis
in shrimp are Vibrio alginolyticus, V. anguillarum, V. harveyi
and V. parahaemolyticus based on the phenotypic data (Goarant
et al., 1999).
The use of antibiotics is a common practice for the treatment of diseases,
but excessive use of antibiotics has encouraged the evolution of antibiotic-resistant
bacteria (Cabello, 2006). The impact of the intensive
use of antimicrobial agents worldwide for prophylactic and therapeutic purposes
has been associated with the increase of bacterial resistance in the exposed
microbial environment. Currently, multiple antibiotic resistances have been
reported in a wide range of human pathogenic bacteria and also in fish pathogens.
Reservoirs of antibiotic resistance can interact between different ecological
systems and potential transfer of resistant bacteria or resistant genes from
animals to human may occur through the food chain (Sarter
et al., 2007). It has been reported that luminous strains of Vibrio
isolated from shrimp larvae are resistant to antibiotics such as erythromycin,
kanamycin, penicillin and streptomycin (Baticados et
al., 1990). The indiscriminate use of oxytetracycline and chloramphenicol
has led to an increase in the incidence of bacterial resistance in shrimp farms
and hatcheries. According to Immanuel et al. (2004),
Philippines have isolated V. harveyi strains from diseased shrimp that
are resistant to most of the chemotherapeutic agents used in aquaculture systems.
It might be clear from the above that global efforts are needed to promote
more judicious use of antibiotics in aquaculture and that new strategies to
control pathogenic bacteria are needed to make the industry more sustainable.
The limited number of treatment options for aquatic species in certain countries
makes widespread resistance to even one antibiotic class a concern (Uhland
and Higgins, 2006). Therefore, new strategies to control infections are
In general, a good therapeutic antimicrobial agent should have wide spectrum
of activity, must not trigger any adverse reactions or be resistant to their
therapeutic effects. A number of natural products, specifically some herbal
plant, could possess some of these ideal characteristics. The search for antimicrobial
agents has continued to be concentrated on lower plants, fungi and bacteria.
Less research has focused on higher plants although identified plant compounds
such as berberine, emetine, quinine and sanguinarine still find specialized
Neem or the Margosa tree (Azadirachta indica) is abundantly prevalent
in the tropical countries of the world and is well known for its insecticidal
and various types of biomedical properties. Almost every part of neem tree has
been known to possess a wide range of pharmacological properties (Farah
et al., 2006). The medicinal and insecticidal properties of different
parts of neem tree have been well documented. Neem leaves are traditionally
being used as curative against certain fungal and bacterial diseases (Parida
et al., 2002). The present study investigates the inhibitory activity
of the extracts of Azadirachta indica leaves against two most common
shrimp pathogens Vibrio parahaemolyticus and Vibrio alginolyticus
under in vitro conditions.
MATERIALS AND METHODS
Extract preparation: Leaves of neem were collected from trees growing in
the Universiti Putra Malaysia, Malaysia, campus. The collected neem leaves were
thoroughly washed with water to remove dirt. Twenty grams of neem leaves was
then shade dried and ground well by using mixer grinder. The powder was then
put into a filter bag and distilled water was added. Aqueous extract was collected
by squeezing the bag manually. Aqueous extract was prepared in 10, 25, 50, 100
and 200 mg mL-1 concentration. Another portion of the fresh neem
leaves was blended to get the juice of the leaves. The juice was mixed with
distilled water to make into different concentration: 10, 25, 50 and 75%. Hundred
percent concentration was also prepared without mixing with distilled water.
Bacteria: Vibrio parahaemolyticus and Vibrio alginolyticus
were isolated from cultured shrimp and maintained at the Aquatic Animal Health
Unit, Universiti Putra Malaysia and used in this experiment. All cultures subsequently
grown from stored stocks were streaked to obtain single colony prior to use.
The bacteria were cultured on tryptic soy agar (TSA; Merck, Germany) plates
and incubated at 37°C.
Agar plate preparation for sensitivity test: Thirty-eight grams of Mueller-Hinton
agar powder (Merck, Germany) was suspended in 1 L of distilled water together
with 30 g of sodium chloride (3% NaCl) and autoclaved at 121°C for 15 min.
The agar was allowed to cool to 62°C in a water bath before pouring into
petri dish (Ruangpan and Tendencia, 2004).
Agar plate for minimum bactericidal concentration (MBC): Forty grams
of TSA powder was suspended in 1 L of distilled water together with 30 g of
sodium chloride (3% NaCl) and autoclaved at 121°C for 15 min. The agar was
allowed to cool to 62°C in a water bath before pouring into petri dish (Ruangpan
and Tendencia, 2004).
Broth preparation for minimum inhibitory concentration (MIC): Fifteen
grams of Tryptic Soy Broth (TSB; Merck, Germany) was suspended in 500 mL of
distilled water together with 15 g of NaCl and was autoclaved at 121°C for
15 min. The broth was allowed to cool to 62°C in a water bath before pouring
into bijou bottles (Ruangpan and Tendencia, 2004).
Disc diffusion method: Disc diffusion test was done according to the
method of Austin et al. (1995). A single colony
from each of the bacteria species was selected and suspended in sterile saline
until turbidity comparable or adjusted to 0.5 McFarland standard was obtained.
A sterilized cotton swab was dipped into the suspension. The inoculum was then
swabbed over the entire agar surface. Five pieces of 6 mm disc previously impregnated
with different concentrations of herbal extracts were placed onto the agar surface
with sterile forceps. The plates were then incubated at 37°C for 24 h. The
antimicrobial activity of the test materials was observed through zone of inhibition
by measuring the diameter in millimeters (mm) inclusive of the disc. Commercial
antibiotic disc of sulfamethoxazole-trimethoprim (10 μg), erythromycin
(15 μg), doxycycline (30 μg), ampicillin (10 μg), tetracycline
(30 μg) and oxolinic acid (2 μg) was placed on agar plate for comparison
of the zone of inhibition.
Determination of minimal inhibitory concentration (MIC) and minimal bactericidal
concentration (MBC) values: MIC of neem juice was assessed using the broth
microdilution method. An inoculum of the bacteria was prepared and suspension
was adjusted with a turbidity equivalent to 0.5 McFarland standards. Dilutions
of neem juice by two-fold dilution were prepared using sterile TSB so as to
get different range of concentrations. One milliliter of cultured suspension
was added into each tube. Control tubes contained no neem juice. After 24 h
of incubation at 37°C the test tubes were examined for possible growth and
MIC was determined as the lowest concentration that ended with no growth. Tubes
without bacterial growth in the MIC test were streaked onto TSA plates to achieve
MBC against tested bacteria. Bacterial growth was observed after incubation.
The minimum concentration of neem juice that prevents bacterial growth is reported
as the MBC value (Ruangpan and Tendencia, 2004).
Antimicrobial sensitivity test: Aqueous extract of neem leaves did not
produce any zone of inhibition on the culture of V. parahaemolyticus
and V. alginolyticus. Mean diameter of inhibition zone produced by neem
juice of different concentration tested against V. parahaemolyticus
and V. alginolyticus are shown in Table 1.
||Antibacterial activity of Azadirachta indica leaves
juice on Vibrio parahaemolyticus and Vibrio alginolyticus
at different concentrations
|Values are Mean±SE, n = 3
||Antibacterial activity of commercial antibiotic disc on Vibrio
parahaemolyticus and Vibrio alginolyticus
|Values are Mean±SE, n = 3, DO-30: Doxycycline, SXT-25:
Sulfamethoxazole-trimethoprim, E-15: Erythromycin, AMP-10: Ampicillin, TE-30:
Tetracycline, OA-2: Oxolinic acid
Hundred percent neem juice produced 19.6 mm inhibition zone on V. parahaemolyticus
and 14.8 mm on V. alginolyticus. Compared with their sensitivity towards
commercial antibiotic disc, the inhibition zone produced was only larger than
the zone produced by oxolinic acid (2 μg).
Both bacteria tested were sensitive to doxycycline, sulfamethoxazole-trimethoprim,
ampicillin and tetracycline and showed an intermediate susceptibility towards
erythromycin and oxolinic acid (Table 2) (based on the NCCLS
publication M31-A2-Performance Standards for Antimicrobial Disc and Dilution
Susceptibility Test for Bacteria Isolated from Animals).
Minimal inhibitory concentration (MIC): The MIC for the neem juice against
V. parahaemolyticus was 3.13% and against V. alginolyticus was
Minimal bactericidal concentration (MBC): The MBC for the neem juice
against V. parahaemolyticus and V. alginolyticus were 12.50 and
According to Immanuel et al. (2004), treating
microbial infections in fish and shrimp involves dissolving higher quantities
of broad spectrum of chemotherapeutic agents in the culture medium. A disadvantage
of this method is the requirement of large amount of expensive drugs which are
used and discharged in the environment that poses risk to the animals and human
health. An alternative is resorting to herbal compounds having antimicrobial
characteristics instead of synthetic antibiotic drugs.
In the present investigation, two different preparations of neem leaves (aqueous
extract and leaf juice) were used to determine their antimicrobial activity
against V. parahaemolyticus and V. alginolyticus that were isolated
from cultured shrimp. This experiment revealed that aqueous extract that is
prepared from powder of neem leaves did not show any antibacterial activity.
This may be due to deactivating or denaturing of the antimicrobial properties
in the neem leaves during the process of drying (Parida
et al., 2002).
Zone of inhibition produced by neem juice show a linear relationship with the
concentration of the juice. Higher concentration produced bigger zone of inhibition.
One hundred percent concentration of neem juice produced the largest inhibition
zone and the diameter of the zone decreased as the neem juice becomes diluted.
This is sufficient to prove that fresh neem juice contains compound with antibacterial
activities. By comparing the size of inhibition zone, V. parahaemolyticus
showed a higher sensitivity to 100% neem juice.
Compared to the inhibitory zone produced by commercial antibiotics, the inhibitory
zone of neem juice was smaller. This may be because the active compound in neem
juice is not high or concentrated enough to produce the same antibacterial effect
as compared to commercial antibiotics where their bacteriostatic and bactericidal
effect has been determined by a lot of research. Vibrio parahaemolyticus
and V. alginolyticus were moderately sensitive to neem juice at 100%
concentration based on inhibition zone.
Although the result showed lower activities of this neem juice relative to
previous findings (Nkuo-Akenji et al., 2001)
which reported higher potency of this plant against bacteria, they however corroborate
their reports. This variation may be due to the dose of extract in different
studies, the age of the plant and the part of the plant used. The masking effect
by other compounds in the extract may also account for the low activity demonstrated
in this study. However, the weak activity demonstrated by these extracts in
vitro to the bacteria does not necessarily imply that they would demonstrate
weak activities in vivo. Oliver-Bever (1986) and
Garcia et al. (2003) had demonstrated immuno-modulation
of chemical compounds from medicinal plants, many of which had been proven to
be inactive or weakly active in vitro against pathogens. Also, as with
some drugs, some of these plant maybe more potent in vivo due to metabolic
transformation of their components into highly active intermediates (Ngemenya
et al., 2006).
According to Yamamoto (2003), an antimicrobial agent
can be considered as bactericidal agent when the MBC value is no more than four
times of the MIC value. Based on the MBC values, only 12.5% is needed to inhibit
growth of V. parahaemolyticus and 25% to inhibit V. alginolyticus.
Both of these values are less than four times of the MIC value for V. parahaemolyticus
and V. alginolyticus. Based on this, 100% neem juice can be considered
as a bactericidal agent.
Crude preparation of neem juice is an antibacterial agent and is useful for
inhibition of vibrios in shrimp. It is possible that neem may take a role as
an adjuvant to the use of antibiotics or as a replacement of current antibiotics.
The present study showed the effectiveness of neem juice in inhibiting bacteria.
It is recommended that identification of herbs and their medicinal value need
further exploration. Further study also needs to be conducted to determine the
active compound (s) that poses antimicrobial activities and their amount. Evaluation
of neem extract against other important aquatic pathogens also needs to be conducted.
This study was supported by the Ministry of Science, Technology and Innovation,
Malaysia E-Science project No. 04-01-04-SF1016.