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INCOFIMS-2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1036 (2022) 012086 doi:10.1088/1755-1315/1036/1/012086

The effect of Ocimum sanctum L. crude extract on

haematology of Cyprinus carpio infected by Aeromonas
hydrophila
A M Halim1, A B Cahyanurani1,2, and A A Aonullah1
1
Politeknik Kelautan dan Perikanan Sidoarjo, Department of Aquaculture Techniques, Sidoarjo
61253, East Java, Indonesia
2
Corresponding author: [email protected]

Abstract. Cyprinus carpio L. is one of the most important freshwater fish and has been
intensively cultivated. However, microbial infection become restricting component that can
cause economic loss in carp production and the use of commercial antibiotics for therapy
produces adverse side effects. This study was conducted to evaluate the potential of Ocimum
sanctum L. crude extract on haematology of Cyprinus carpio against A. hydrophila infection.
For this purpose, fish that have been infected were immersed in different doses of Ocimum
sanctum L. crude extract (50, 150, 250, 350 ppm) and compared with the control group. After 1
week of immersion, total erythrocytes, total leukocytes, and deferential leukocytes (neutrophils,
monocytes and lymphocytes) were recorded. The result showed that significant increase of total
erythrocytes was obtained at the concentration of 350 ppm Ocimum sanctum L. crude extract.
Furthermore, decreasing of the total leukocytes about 55.06×103 to 25.77×103, also decreasing
deferential leukocytes (monocytes 40%, neutrophils 31% and lymphocytes 49% from the
positive control). These results suggested that 350 ppm of Ocimum sanctum L. crude extract is
beneficial to enhance the haematological status of common carp against A. hydrophila.

1. Introduction
Carp (Cyprinus carpio) is one type of freshwater fish that has important economic value [1]. Nowadays,
to increase the production, carp are intensively cultivated [2,3]. However, in intensive cultivation with
limited environmental conditions, high stocking density, overfeeding problem, as well as inappropriate
water quality management can disturb environmental balance, so that fish become stressed and
susceptible to disease [4,5].
One of the major bacterial pathogens is Aeromonas hydrophila. Aeromonas hydrophila is a
ubiquitous gram-negative rod-shaped bacterium which is commonly isolated from fresh water ponds
and which is a normal inhabitant of the gastrointestinal tract [6]. A. hydrphila is a causative agent of
haemerrhagic septicemia on carp or known as “Motile Aeromonas Septicemia” which now become a
serious problem in fish culture [7,8]. This disease often attacks freshwater fish and can cause outbreaks
disease with a high mortality rate (80 – 100%) within 1 – 2 week [9,10].
Treatment of bacterial attacks is generally done by antibiotics. However, the use of antibiotics can
cause side effects to both pathogen and the fish. Continuous use of antibiotics can cause pathogenic
organisms become resistant, so the use of antibiotics becomes ineffective [11–13]. Whereas in fish it
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INCOFIMS-2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1036 (2022) 012086 doi:10.1088/1755-1315/1036/1/012086

can cause bioaccumulation, and cause carcinogenic effects (cancer-causing) if consumed by humans
[14–16]. Therefore, several alternative strategies to antimicrobial usage have been proposed, including
the use of medicinal plant as biological control agents [17,18].
Ocimum sanctum L. is an aromatic shrub in the basil family Lamiaceae that can be found easily in
Indonesia. It contains many compounds, such as flavonoids, alkanoids, saponins, tannins, phenols,
anthocynins, flavonoids, triterpenoids, tannins which are potential sources for therapeutic importance
[19,20]. Methanolic crude extracts of Ocimum sanctum L. showed significant activity against
Aeromonas hydrophila, Pseudomonas aeruginosa and Edwardsiella tarda [21]. Ocimum sanctum also
acts against Klebsiella spp, Anthrobacter globiformis, Bacillus megaterium, Escherichia coli,
Pseudomonas spp. Staphylococcus aureus, Staphylococcus albus and Vibrio [22,23]. Leaves extract of
O. sanctum affected both specific and non-specific immune responses and disease resistance against
fungal and bacterial infection [24]. Many results proves that Ocimum sanctum L can act as excellent
antimicrobial agent against many microbes. This study was conducted to evaluate the potential of
Ocimum sanctum L. crude extract on haematology of Cyprinus carpio against A. hydrophila infection.

2. Materials and methods

2.1 Fish
The common carp, C. carpio were obtained from Reproduction Laboratory, Faculty of Fisheries dan
Marine Science, University of Brawijaya, Malang, Indonesia. C. carpio with 7-12 cm in length were
used as many as 150 fish. Fish were kept under the same environmental conditions and placed in 150 L
aquarium for a week as an acclimatization period to the laboratory condition and fed with a commercial
diet.

2.2 Plant extract

Ocimum sanctum L. were collected from Vegetable Market, Sengkaling, East Java, Indonesia. All parts
of this plant were washed, dried at room temperature and ground. One kilogram powdered sample was
macerated in 70% ethanol for 3 × 24 hours at room temperature. The resulting extract was concentrated
over a rotary vacuum evaporator and then frieze-dried.

2.3 Aeromonas hydrophila

Aeromonas hydrophila (AT118) was obtained from Control Fish Quarantine Center Quality and Safety
of Fishery Products Class I Surabaya.

2.4 Experimental design

After a weeks of acclimatization period, healthy C. carpio were infected by intraperitoneal injection (IP)
with pathogenic A. hydrophila diluted in distilled water (107 CFU) and were transferred into the same
tanks. The fish were maintained for 1 week and the clinical symptoms after infected with A. hydrophila
bacteria were observed.
After infection with A. hydrophila, blood samples of infected carp were taken and counted for total
erythrocytes, total leukocytes and differential leukocytes. After that, the infected fish were then given
an immersion treatment with different dose of Ocimum sanctum L. extract; of 50 ppm (A), 150 ppm (B),
250 ppm (C) and 350 ppm (D) in 15 L aquarium (10 fish/aquarium) for 24 hours. There were three
replicates for each treatment arranged randomly. In this study, 2 comparison controls were used,
negative control and positive control, negative control as sample treatment without bacterial infection
and without giving basil crude extract, while the positive control as the sample treatment with infection
A. hydrophila bacteria, the negative and positive controls for comparison only. During immersion, each
aquarium were aerated to increase the dissolved oxygen content. After immersion for 24 hours, fish
were transferred to another aquarium and maintained for 1 week. Then observed for total erythrocytes,
total leukocytes and differential leukocytes in fish for 2 days once.

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INCOFIMS-2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1036 (2022) 012086 doi:10.1088/1755-1315/1036/1/012086

2.5 Haematology, biochemistry and serum collection

Blood was collected from the caudal vein using a 1 mL spuit disposable contains Na Sitrat 3,8% as
anticoagulan. Two fish were randomly collected from one of the replicate aquarium for haematological
assays: total erythrocytes, total leukocytes and differential leukocytes. Total erythrocytes were counted
with a haemocytometer using Hayem’s RBC diluting fluid [25]. Total leukocytes were counted with a
haemocytometer using Türk’s solution. Differential leukocytes were counted from one or more blood
slides of each experimental group. The blood smear was fixed with methanol for 5 min by immersing
the entire microscope slide with the blood smear into methanol. After that, the blood smears were
removed from methanol and were allowed to air dry. Then the blood smears were colored by inserting
them into the 10% Giemsa solution for 30 minutes, washed, then allowed to air dry. Blood smears that
have been stained were observed and counted on the under a microscope with 1000x magnification until
it reaches 100 leukocytes. Leukocytes were identified as lymphocytes (LYM), monocytes (MON),
eosinophils (EOS), and neutrophils (NEU) or other leukochytes [26].

2.6 Statistical Analysis

Statistical analysis was performed by one way ANOVA. Analysis of variance (ANOVA) was used to
determine the overall significance of differences among samples and a post hoc Tukey test for multiple
comparisons between groups of samples assembled according to the significant factors and continued
by orthogonal polynomial test. All the statistical analyses were carried out by using SPSS program
version16. Values are expressed as mean standard deviation.

3. Results and discussion

3.1 Red Blood Cell Count
The RBC count was significantly increased in all the treatment groups compared with control group
including post challenge period (Fig. 1). Based on the results of the study, the average of red blood cell
count in the negative control K (-) was 218.4 x 105 cells/ml and the Positive control K (+) was 135.33x
105 cells/ml. While in treatment A (50 ppm) was 176.73 x 10 5 cells/ml, B (150 ppm) was 165.53 x 105
cells/ml, C (250 ppm) was 189.97 x 105 cells/ml and D (350 ppm) was 211.86 x 105 cells/ml. RBC count
in treatment D (350 ppm) included in healthy or normal fish erythrocytes count, because it was still in
the range of total erythrocytes count at negative control K(-). The increase in the number of erythrocytes
in each treatment showed that the crude Ocimum sanctum extract given at different doses was able to
treat fish infected with A. hydrophila bacteria. Similarly, [27] observed that adding Ocimum sanctum
increasing the red blood cell of Labeo rohita and boosting the defence mechanisms and protection
against Aeromonas hydrophila. [28] Amirkhani and Farid observed that Basil (Ocimum basilicum)
supplementation diet containing ethanol extract can prevent infection Aeromonas hydrophila in common
carp (Cyprinus carpio). Ocimum sanctum commonly known as holy basil or tulsi is considered as a
sacred plant in hindu belief and known a queen of herbs. Crude extract of basil in the process of treating
bacterial diseases causes death in A. hydrophila because of its chemical composition, it is suspected that
the active substances that can act as antibacterial is ethanol and flavonoids.

3.2 White Blood Cell Count

Based on the results of the study, the average of white blood cell count (WBC) in common carp in the
negative control K (-) was 8.60 x 103 cells/ml and the positive control K (+) was 15.02 x 10 3 cells/ml.
While in treatment A (50 ppm) was 19.84 x 10 3 cells/ml, B (150 ppm) was 10.65x 10 3 cells/ml, C (250
ppm) was 11.44 x 103 cells/ml and D (350 ppm) was 8.83 x 103 cells/ml (Fig. 2). Based on the results of
the average number of show that the average number of leukocytes of C. carpio in treatment D (350
ppm) is the lowest at 8.83 x 103 cells/ml, this value is in the normal range because it relates to the number
of leukocytes in K (-) which is 8.60 x 103 cells/ml, but it can be seen in treatment C (250 ppm) the
average of leukocyte increased, presumably because leukocytes fight foreign materials (Aeromonas
hydrophila). Total leukocytes in treatment D was the lowest, presumably because the crude leaf extract
of O. sanctum inhibit and kill pathogens because it is suspected that the active substance that acts as an

3
INCOFIMS-2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1036 (2022) 012086 doi:10.1088/1755-1315/1036/1/012086

antibacterial. The results in this study are supported by research conducted by [29] The highest
antibacterial effect of ethanolic E. hirsutum extract may be due to its high content flavonoids, tannins
and steroids. In fact, this compound is known for its strong antimicrobial activity. It was indicated that
the ethanolic extract of E. hirsutum inhibited growth of pathogens on agar plates as well. Effect of
antibacterial activity was observed in Ocimum sanctum. The crude aqueous extract of leaf possesses
some antibacterial and immunomodulatory. The ethanolic extracts from the leaves showed better
activity against the β-lactamase producing methicillin-resistant Staphylococcus aureus and Aeromonas
hydrophila strains [30].

3.3 Differential of Leukocyte

The percentage of neutrophils in treatment A (50 ppm) was 9.44% which showed the highest value
compared to other doses. In those treatment, fish infected with A. hydrophila became stress and got the
loger inflammation. While, the lowest percentage of neutrophils was in treatment D (350 ppm) which
was 4.00% (Fig. 3). This value is to be in the normal range because it corresponds to the percentage of
neutrophils at K (-) which is 4.67%. The number of neutrophils will increase in the event of infection or
attack by bacteria, because neutrophils move by diapedesis through capillary pores and by chemotaxis
to damaged tissue areas. In the fish, the oxygen radicals are at the destruction of bacterial invaders.
Studies on neutrophil activity clearly showed the enhancing effect of O. sanctum leaf extract on
neutrophil respiratory burst (RB) activity [31].
Fish given treatment A (50 ppm) showed the percentage of monocytes and lymphoctes that was close
to the results of the positive control K (+) (Fig. 4 and 5). However, when compared to other treatments,
treatment D (350 ppm) showed the lowest monocyte percentage, which was 9.50%, when compared to
the negative control K (-) of 10.00%. While, the percentage of lymphocyte in treatment D (350 ppm)
was 14% compared to the negative control K (-) 14.3%. In the event of infection by a foreign materials,
monocytes will move quickly from the blood to the infected area to carry out phagocytosis. The ability
of monocytes to penetrate the walls of capillary blood vessels, enter the tissue and differentiate into
macrophage cells. Similarly, the explanation by [32], in whole blood culture, lymphocytes,
thrombocytes and erythrocytes did not show phagocytosis but neutrophil and monocyte like cells
showed active phagocytosis.
The increament doses of O. sanctum leaf extract showed that the percentage of phagocytic activity
in fish infected with A. hydrophila was getting lower. This is presumably because the immune system
in C. carpio more active to kill A. hydroplia bacteria with the addition of basil leaf extract to the highest
dose (350 ppm). The lymphocyte and monocyte plays an important role in primary defense mechanism
because of their engulfing nature against foreign particles. Monocytes kill bacteria by producing reactive
nitrogen intermediates (RNIs) and reactive oxygen intermediates (ROIs) and through the action of
phagolysosomal enzymes [33].
Red Blood Cell 105

250,00 a a
c b
(cell/ml)

200,00 d
e
150,00
100,00
50,00
0,00
K(-) K(+) 50 150 250 350
Dose of Extract (ppm)

Figure 1. Effect of O. sanctum leaf extract on red blood cell count (RBC) during pre and post
challenge. Indicates significant difference (P < 0.05).

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INCOFIMS-2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1036 (2022) 012086 doi:10.1088/1755-1315/1036/1/012086

25,00
a

White Blood Cell (103 cell/ml)

20,00
b
15,00
d c
10,00 e e

5,00

0,00
K (-) K (+) A B C D
Dose of Extract (ppm)

Figure 2. Effect of O. sanctum leaf extract on white blood cell count (WBC) during pre and post
challenge. Indicates significant difference (P < 0.05).

16,00
a
14,00
12,00
Neutrophil (%)

b c
10,00
8,00 d
d
6,00 d
4,00
2,00
0,00
K (-) K (+) A B C D
Dose of Extract (ppm)

Figure 3. Effect of O. sanctum leaf extract on percentage of neutrophil during pre and post challenge.
Indicates significant difference (P < 0.05).

5
INCOFIMS-2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1036 (2022) 012086 doi:10.1088/1755-1315/1036/1/012086

30,00

a
25,00
b
20,00
Monocyte (%)

15,00 c
c
c c
10,00

5,00

0,00
K (-) K (+) A B C D
Dose of Extract (ppm)

Figure 4. Effect of O. sanctum leaf extract on percentage of Monocyte during pre and post challenge.
Indicates significant difference (P < 0.05).

35,00
a
30,00 b

25,00 c
Lymphocyte (%)

20,00
d
d d
15,00

10,00

5,00

0,00
K (-) K (+) A B C D
Dose of Extract (ppm)

Figure 5. Effect of O. sanctum leaf extract on percentage of lymphocyte during pre and post
challenge. Indicates significant difference (P < 0.05).

4. Conclusion
Administration of crude extract of basil leaves (O. sanctum) with different doses gave a significant effect
on the hematological response of C. carpio and gave an effect on total erythrocyte, total leukocytesa and
leukocyte differential after infected with A. hydrophila. The best dose of extract to increase immunity
in C. carpio against bacterial infection A. hydrophila is 350 ppm.

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INCOFIMS-2021 IOP Publishing
IOP Conf. Series: Earth and Environmental Science 1036 (2022) 012086 doi:10.1088/1755-1315/1036/1/012086

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6. Acknowledgments
We thank the editor and reviewers for their insightful comments on the manuscript.