In-vivo Test of Chlorella Protein Fragments as Nucleotide Vaccine

Candidates in Grouper Viral Nervous Necrosis (VNN) Infection

against Haematological Response

Uun Yanuhar

, Muhammad Musa, Diana Arfiati, Nico Rahman Caesar and Nur Sakinah Junirahma

Faculty of Fisheries and Marine Science, Brawijaya University, Indonesia

Keywords: Humpback grouper, Chlorella vulgaris, VNN, Haematological.

Abstract: Grouper (Cromileptes altivelis) is a species of fish with important economic values both in the national and

international markets. The disease that has been reported by researchers is Viral Nervous Necrosis (VNN)

which can cause mass death in groupers, especially in larval and juvenile stadia. Based on the problems, a

research is needed on haematological analysis of groupers (Cromileptes altivelis) infected with Viral

Nervous Necrosis by in-vivo testing using protein fragments of C. vulgaris. This research employed an

experiment method using 5 treatments, namely (A) healthy fish, (B) VNN-infected fish, (C) VNN-infected

fish with administration of C.vulgaris crude extracts of 17 µg mL

-1

, (D) VNN-infected fish with

administration of C.vulgaris crude extract of 33 µg mL

-1

, and (E) VNN-infected fish with administration of

C.vulgaris crude extract of 50 µg mL

-1

. Observations of haematological parameters included erythrocytes,

leukocytes, haemoglobin, and haematocrit. The observation results showed an erythrocyte value of 97 x 104

cells/mm

in treatment (C), 107 x 104 cells/mm

in treatment (D), and 94 x 104 cells/mm

in treatment (E).

The observation results of leukocyte values were 150,000 cells/mm

in treatment (C), 133,300 cells/mm

treatment (D), and 139,000 cells/mm

in treatment (E). Furthermore, the observation results of haemoglobin

showed a value of 5 gr/100 ml in treatment (C), 6 gr/100 ml in treatment (D), and 5 gr/100 ml in treatment

(E). As for the haematocrit parameter, the results obtained from the observation were 18% in treatment (C),

22% in treatment (D), and 15% in treatment (E). Based on this research, the haematological status of VNN-

infected groupers was not good. However, the results of the in-vivo testing conducted showed that

administration of C. vulgaris extract gave a positive result on improving the haematological status of

groupers (C. altivelis) infected with VNN with the optimal dose of 33 µg mL

-1

1 INTRODUCTION

One of the potential of sea waters that has been

developed and is starting to show a growth in

international market is grouper. Grouper is widely

distributed in waters that are inhabited by coral in

tropical and subtropical regions. Some types of

grouper that have been targeted in the market are the

duck grouper (Cromileptes altivelis), tiger grouper

(Epinephelus fuscoguttatus), leopard grouper

(Epinephelus leopardus) and mud grouper

(Epinephelus coioides). These types of grouper have

a high selling value. In addition, its cultivation

process only need and use local components.

(Sudaryatma et al., 2012). However, the hybrid of

cantang grouper is experiencing a decrease in

production due to some environmental stresses, for

example, poor water quality, which makes the

cantang grouper is susceptible to viral, bacterial,

stressful infections from time to time resulting in

poor growth and ultimately death. (Noor et al., 2018).

The obstacle of cultivation in the Epinephelus

group (Grouper) in Indonesia is the limited supply of

fish seeds due to pathogenic infections which cause

more than 80% mortality, even up to 100% (Yanuhar

et al., 2012). VNN virus has been reported to infect

cultivated marine fish and has been stipulated in

Ministerial Decree number 26 Year 2013 as Pests

and Diseases of Quarantine Fish (HPIK) Group I.

VNN weakens the nervous system of fish so that the

fish will lose control nerves, will experience

weakness of motion, and eventually death (Yanuhar,

2015).

The development of local natural materials as one

of the countermeasures for controlling the spread of

the VNN virus is very much needed. Natural

Yanuhar, U., Musa, M., Arﬁati, D., Caesar, N. and Junirahma, N.

In-vivo Test of Chlorella Protein Fragments as Nucleotide Vaccine Candidates in Grouper Viral Nervous Necrosis (VNN) Infection against Haematological Response.

DOI: 10.5220/0009588100790083

In Proceedings of the 6th International Conference on Advanced Molecular Bioscience and Biomedical Engineering (ICAMBBE 2019) - Bio-Prospecting Natural Biological Compounds for

Seeds Vaccine and Drug Discovery, pages 79-83

ISBN: 978-989-758-483-1

materials used are derived from natural ingredients

that have not been developed much, one of which is

the use of microalgae. Chlorella vulgaris is a type of

one-celled green microalgae that can grow and be

found in warm climates. C. vulgaris has many

ingredients in it which include protein, vitamins,

minerals, carbohydrates, fats, chlorophyll and beta

carotene (Tang dan Paolo, 2011). The use of

microalgae has been developed, especially in the

field of pharmacology. Microalgae have benefits as

antioxidants for fish because they contain vitamins,

polysaccharides, and bioactive compounds (Yanuhar,

2016).

Blood tests are conducted to establish the

diagnosis of a disease in fish because physiological

disorders in fish will cause changes in blood

components which will then be able to determine the

condition or health status of the fish (Yanuhar et al.,

2019). Based on these problems, research is needed

on the hematological analysis of groupers

(Cromileptes altivelis) infected with Viral Nervous

Necrosis (VNN) by in vivo testing using protein

fragments from C. vulgaris.

2 METHODS

This study utilized crude extracts from C. vulgaris

marine microalgae to be tested on cantang grouper

(Epinephelus sp) infected with Viral Nervous

Necrosis (VNN). C. vulgaris samples were obtained

from the Brackish Aquaculture Fisheries Center

(BPBAP) of Situbondo. The research took place at

the Laboratory of Environment and Biotechnology

Aquatic, Faculty of Fisheries and Marine Sciences,

Brawijaya University and Organic Chemistry

Laboratory, Faculty of Science and Technology,

State Islamic University of Malik Ibrahim Malang.

2.1 Extraction of C. Vulgaris

C. vulgaris was extracted by maceration using

methanol PA solvent in a ratio of 1:5 for 24 hours.

Then it was filtered using filter paper to remove the

pulp so that the extract was obtained with a solvent.

Furthermore, to obtain the extract, the solvent was

removed by using a rotary vacuum evaporator at a

temperature of 40 ºC, with a speed of 60 rpm.

2.2 In-vivo Test of C. vulgaris Extract

in Groupers

In this study, an in-vivo treatment of extracts from

C. vulgaris marine microalgae on Groupers (C.

altivelis) was carried out. The testing process was

carried out orally which refers to Yanuhar (2015),

using 5 treatments, namely (A) healthy fish, (B)

VNN-infected fish, (C) VNN-infected fish with

administration of C.vulgaris crude extracts of 17 µg

-1

, (D) VNN-infected fish with administration of

C.vulgaris crude extract of 33 µg mL

-1

, and (E)

VNN-infected fish with administration of C.vulgaris

crude extract of 50 µg mL

-1

. Oral treatment with the

help of feeding tube was carried out for 3 times,

namely on day 0, 5, and 10. Each rearing tank

contained 12 groupers with a size of 10 cm and the

test treatment was carried out for 24 days.

Hemotological observations were then performed to

determine the effect of in vivo test treatments on the

Groupers.

2.3 Haematological Response

Observations of measured hematologic responses

consisted of erythrocytes, leukocytes, hemoglobin

and hematocrit. Blood samples were taken once at

the end of the study. The method of blood sampling

in fish was carried out according to Svobodova et al.

(2006). This blood sampling was carried out using a

0.5 mL syringe that has previously been added with

Ethylene Diamine Tetra Acetatic Acid (EDTA) at a

dose of 1.50 ± 0.25 mg/mL of blood. The fish was

placed with the head on the left side. Blood samples

were taken using a syringe that pierced the muscles

in the midline of the body behind the anal fin.

2.3.1 Erythrocyte Calculation

The procedure for calculating erythrocytes count

was measured according to Blaxhall and Daisley

(1973), firstly, blood was sucked with a pipette

containing a red stirrer grains to scale 1 (a pipette to

measure red blood cells count), then hayem’s

solution was added to scale 11. The stirring of the

blood in a pipette was done by swinging a hand

holding a pipette like forming a number, specifically

number 8, for 3-5 minutes so that the blood was

mixed evenly. The first two drops of the blood

solution in a pipette were removed, then the drops

were placed on a Neubauer haemocytometer and

were covered with a glass cover. Then red blood

cells count was calculated with the help of a

microscope with 400x magnification. Red blood

cells (erythrocytes) count can be calculated by the

following formula. According to Blaxhall and

Daisley (1973):

Σerythrocytesfoundx10

cells/mm

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

2.3.2 Leukocyte Calculation

The procedure for calculating erythocyte count was

measured according to Blaxhall and Daisley (1973),

blood samples were sucked with a pipette containing

white stirrer grains to a scale of 0.5. Then, the truk’s

solution was added to scale 11. The stirring of the

blood in a pipette was done by swinging a hand

holding a pipette like forming a number, specifically

number 8, for 3-5 minutes so that the blood was

mixed evenly (the same as stirring for the

calculation of red blood cells count). After that, the

first two drops of blood solution from the pipette

were removed, then the solution was dropped to the

haemocytometer, after which it was closed with a

glass cover. The white blood cells (leukocytes)

count can be calculated by the following formula.

According to Blaxhall and Daisley (1973):

Σleukocytesfoundx50cells/mm

3

2.3.3 Hemoglobin Calculation

Measurement of hemoglobin levels was done by

sampling the blood with a sahli pipette up to a scale

of 20 mm

or on a scale of 0.2 ml. Then the tip of

the pipette was cleaned with tissue paper. The blood

in the pipette was transferred into a Hb-meter filled

with 0.1 N HCl to a scale of 10 (red). The blood was

then stirred with a stirring rod for 3-5 minutes. The

distilled water was added to the tube until the color

of the blood was like the color of the standard

solution present in the Hb-meter. The hemoglobin

scale can be seen on the gr % (yellow) pathway

scale, which meant the amount of hemoglobin in

grams per 100 ml of blood.

2.3.4 Hematocrit Calculation

The examination of hematocrit values was

performed using the microhematocrit method.

Microhydematocrit with heparin was inserted into

the collected blood sample, until the blood filled

approximately three quarters (3/4) of the capillary

tube. In addition, one end of the capillary tube was

blocked by sticking it in the wax stopper. Then it

was centrifuged for 5 minutes using a

microhematocrit centrifuge with a speed of 1,500

rpm. In addition, the results were read using a

hematocrit reader and were expressed in % (Vonti,

2008).

3 RESULTS AND DISCUSSION

3.1 Erythrocyte Calculation

Based on the calculation, red blood cells count

(Figure 1.) in treatment (A) was 203 x 104

cells/mm

, in treatment (B) was 48 x 104 cells/mm

in treatment was 97 x 104 cells/mm

, in treatment

(D) was 107 x 104 cells/mm

and in treatment (E)

was 94 x 104 cells/mm

. The highest erythrocytes

count was found in treatment (A), i.e. healthy

grouper without treatment and the lowest was found

in treatment (B) VNN-infected fish.

250

203

200

cells/

mm3

150

97

107

94

100

Cou

nt

50

0

A

B

C

D

E

Treatment

Figure 1. Erythrocyte calculation results. (A) healthy fish,

VNN-infected fish, (C) VNN-infected fish with

administration of C.vulgaris crude extracts of 17 µg mL-1,

VNN-infected fish with administration of C.vulgaris crude

extract of 33 µg mL-1, and (E) VNN-infected fish with

administration of C.vulgaris crude extract of 50 µg mL-1.

Among the three treatments of C. vulgaris

extract tested, treatment (D) at a dose of 33 µg mL

-1

gave the highest increase in the erythrocytes count at

107 x 104 cells/mm

. It showed that administration

of C. vulgaris extract at a dose of 33 μg mL

-1

increased the erythrocytes count of groupers infected

with VNN. The fish blood cells count in teleostei

fish ranged from 1.05 × 106 cells/mm

- 3.0 x 106

cells/mm

. Low erythrocytes count are an indicator

of anemia, while high erythrocytes count indicates

that fish are under stress (Sababalat, 2015).

3.2 Leukocyte Calculation

White blood cells are immune cells that will respond

to the presence of pathogens or foreign objects that

enter the body, the higher the pathogenicity, the

body will produce more white blood cells. In

addition, according to Muiswinkel and Vervoorn

(2006), leukocytes have a variety of functions,

In-vivo Test of Chlorella Protein Fragments as Nucleotide Vaccine Candidates in Grouper Viral Nervous Necrosis (VNN) Infection against

Haematological Response

closely related to the removal of foreign matter

(including pathogenic microorganisms). Based on

the calculation of the leukocytes count (Figure 2.) it

shown the results of leukocyte calculation, as

follows: treatment (A) was 91,350 cells/mm

treatment (B) was 182,050 cells/mm

, treatment (C)

was 150,000 cells/mm

, treatment (D) was 133,300

cells/mm

and treatment (E) of 139,000 cells/mm

3

250,000

133,300

139,000

cells/mm

200,000

182,050

150,000

Leukocytes

150,000

50,000

91,350



100,000

A

B

C

D

Count

Treatment

Figure 2. Leukocyte calculation results. (A) healthy fish,

VNN-infected fish, (C) VNN-infected fish with

administration of C.vulgaris crude extracts of 17 µg mL-1,

VNN-infected fish with administration of C.vulgaris crude

extract of 33 µg mL-1, and (E) VNN-infected fish with

administration of C.vulgaris crude extract of 50 µg mL-1.

Leukocytes or white blood cells are an important

part of the body’s defense system which has the

nature to prey on pathogens that enter the body.

Therefore, leukocytes are very closely related to the

immune system. Leukocytes have a role in cellular

defense and humoral organisms against foreign

substances. Fish have white blood cells called

leukocytes which range from 20,000 - 150,000

cells/mm

(Irianto, 2005). Nearly all treatments

showed leukocyte counts in the normal category

except for VNN-infected fish.

3.3 Hemoglobin Calculation

According to Almanda et al. 2007, low Hb levels

caused the metabolic rate to decrease and the energy

produced to be low. This makes the fish weak and

has no appetite and looks still at the bottom or hangs

under the surface of the water. Normal hemoglobin

levels in fish range from 5.05 to 8.33 grams/100 ml

of blood or grams/%.

6.5

6

5 5

4

A

B C D

E

Treatment

Figure 3. Hemoglobin calculation results. (A) healthy fish,

VNN-infected fish, (C) VNN-infected fish with

administration of C.vulgaris crude extracts of 17 µg mL-1,

VNN-infected fish with administration of C.vulgaris crude

extract of 33 µg mL-1, and (E) VNN-infected fish with

administration of C.vulgaris crude extract of 50 µg mL-1.

The observation results of the highest hemogloblin

levels (Figure 3.) other than healthy fish treated (A)

were found in treated groupers (D) VNN-infected

fish with C.vulgaris crude extract of 33 µg mL-

1with hemoglobin levels of 6.5 gr/100 ml. The

lowest hemoglobin concentration was observed in

treatment of VNN-infected fish with a hemoglobin

level of 4 gr/100 ml.

3.4 Hematocrit Calculation

Hematocrit is a comparison between red blood cells

and blood plasma, and it affects the regulation of red

blood cells. Hematocrit is a means for aquaculture to

find out whether the fish being cultivated has anemia

or not. Hematocrit is the percentage of the volume of

erythrocytes in the blood and its value is related to

red blood cells count. The increase in hematocrit

levels is influenced by two factors, namely changes

in environmental parameters, especially the

temperature and physiological conditions of fish

related to the energy needed (Jawad et al., 2004).

Based on hematocrit observations (Figure 4.) it

obtained results as follow: namely at treatment (A)

by 29%, treatment (B) by 13%, treatment (C) by

18%, treatment (D) by 22% and treatment (E) by

15% . The highest hematocrit value was in the

treated groupers (D), namely the VNN-infected fish

with C.vulgaris crude extract of 33 µg mL-1,which

was around 30%. Whereas the lowest hematocrit

value was in treatment (B), namely VNN-infected

fish, which was around 13%.

ICAMBBE 2019 - 6th ICAMBBE (International Conference on Advance Molecular Bioscience Biomedical Engineering) 2019

(%)

40

29

Hemat

ocrit

30

18

22

10



20

13

Count

0

A

B

C

D

E

Treatment

Figure 4. Hematocrit calculation results. (A) healthy fish,

VNN-infected fish, (C) VNN-infected fish with

administration of C.vulgaris crude extracts of 17 µg mL-1,

VNN-infected fish with administration of C.vulgaris crude

extract of 33 µg mL-1, and (E) VNN-infected fish with

administration of C.vulgaris crude extract of 50 µg mL-1.

4 CONCLUSIONS

The health condition of fish can be seen from the

hematological status which changes the amount

value at the normal range. Based on this study, the

hematological status of groupers infected with VNN

was not good. However, based on the results of in

vivo tests conducted, it was showed that the

administration of C. vulgaris extract gave positive

results on improving the hematological status of

grouper (C. altivelis) infected with VNN with the

most optimal dose of 33 μg mL

-1

ACKNOWLEDGEMENTS

We offer our gratitude to the Ministry of Research,

Technology and Higher Education of Republic of

Indonesia for funding this research through the

Applied Research Excellence for University

(PTUPT) Scheme, 2019.

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In-vivo Test of Chlorella Protein Fragments as Nucleotide Vaccine Candidates in Grouper Viral Nervous Necrosis (VNN) Infection against

Haematological Response