Nghiên cứu bệnh do streptococcus iniae gây ra trên cá chẽm (lates calcarifer) và biện pháp phòng bệnh tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY

SUMMARY OF DOCTORAL DISSERTATION
Major: Aquacuture
Code: 9620301

TRUONG THI HOA

STUDY ON Streptococcus iniae INFECTION IN
BARRAMUNDI (Lates calcarifer) AND
PREVETION METHODS

Can Tho, 2019


THIS STUDY WAS ACHIEVED AT CAN THO
UNIVERSITY

Supervior: Assoc Prof. Dr. Dang Thi Hoang Oanh
The dissertation will be defended at the Doctoral Dissertation
Assessment Committee at the University Level
At:……………………………………….……………...
Time & Date:…………………………………………...

Reviewer 1: …………………………………………….
Reviewer 2: …………………………………………….

The dissertation is available at:
Learning Recource Center of Can Tho University
National library Vietnam

PUBLISHED PAPERS OF THE AUTHOR
1. Truong Thi Hoa, Nguyen Ngoc Phuoc, Dang Thi Hoang Oanh,
2018. Isolation and characterization of lactic acid bacteria from
brackish fish species antagonistic to Streptococcus iniae isolated
from haemorrhagic barramundi (Lates calcarifer). Journal of
Agriculture and Rural Development. 338:99-106
2. Truong Thi Hoa, Nguyen Ngoc Phuoc, Dang Thi Hoang Oanh,
2018. Characteristics of Streptococcus iniae infected in barramundi
(Lates calcarifer). Can Tho University Journal of Science.
54(3B):99-106.

i


Chapter 1
OVERVIEW OF THE DISSERTATION
1.1 Introduction
On barramundi, the diseases caused by S. iniae were first
reported in 1999 in Australia (Bromage et al., 1999), after that,
the diseases were also reported in 2006 in Australia (Creeper
and Buller, 2006), in 2010 in Thailand (Suanyuk et al., 2010).
The diseases caused by S. iniae showed signs of haemorrhagic
on skin and fins, popped eyes (Bromage et al., 1999). There are
other signs of diseases such as septicemia on skins,
haemorrhagic on skin, fins, gills and anus, which were reported
in S. iniae infected barramundi and tilapia (Suanyuk et al.,
2010). The accumulative mortality of this diseases can be as
high as 70% in the barramundi fingerlings (Creeper and Buller,

2006).
In Vietnam, the diseases caused by S. iniae were first
reported in 2013 on barramundi raised in Khanh Hoa (Tran Vi
Hich, 2014). In Thua Thien Hue, from 2007, in order to
prevent the diseases caused by intensive culture of white
shrimp, the provincial committee encourage the culture of
marine and brackish water fish species such as grouper, red
snapper, rabbit fish, barramundi…, as alternatives for shrimp in
polutted area. In fact, seabas was used as a popular species by
local farmer and was proven as a high value fish (Ton That
Chat et al., 2010). However, the rapid increase in barramundi
culture lead to environmental poluttion and diseases, including
the haemorrhagic diseases on barramundi.
For treatment, antibiotics are widely used to treat
bacterial pathogens on aquactic animals. The use of antibiotics
had brought some advantages, as well as disadvantages, such
as the antibiotic-resistance problems (Weston, 1996; Tu Thanh
Dung et al., 2008). Therefore, the studies on the bacteria which
have antagonistic ability against bacterial pathogens are widely
popular. In fact, lactic acid bacteria, which have antagonistic
ability against bacterial pathogens and provide advantages to the
aquatic animal health, such as digestive enzymes (Nirunya et al.,
1


2008). Lactic acid bacteria produces antimicrobial peptides and
antifungal substances, bacteriocin which inhibit the development
of pathogens (Ringo et al., 2005; Gatesoupe, 2007).
Lactic acid bacteria can be isolated from different sources
such as fermentative products, intestieve tracts of terrestrial

animals and aquatic animals. Several studies on instestial tract
bacteria of fish from different living environment showed that
lactic acid bacteria exist in the instestial tract of fish but do not
dominate other bacteria in the tract (Ringo et al., 2010). Hence,
the addition of lactic acid bacteria to the diet of fish can improve
the ability against bacterial pathogens and improve the natural
immune defense of fish (Lauzon and Ringo, 2011). However,
there was no report about the effect of the addition of lactic
acid bacteria to the fish diet to the inspecific immune factors of
barramundi.
From these perspectives, I carried out the thesis entittled:
“Study on the diseases caused by Streptococcus iniae on
barramundi (Lates calcarifer) and potential prevention
solutions” to supply novel information about diseases caused by
Streptococcus iniae on barramundi in order to support the
sustainable development of barramundi culture.
1.2 Objective of the study
General aims: to supply novel information about diseases
caused by Streptococcus iniae on barramundi in order to
support the sustainable development of barramundi culture.
Specific aims: to determine the pathogenic characteristics
of Streptococcus iniae on barramundi and provide potential
diagnosis and prevention solutions for this diseases.
1.3 Scope of the study
This study focus on the haemorrhagic diseases caused by
Streptococcus iniae on barramundi in Thua Thien Hue
province, to isolate the lactic acid bacteria isolates from the
intestinal tracts of rabbit fish, barramundi, tilapia which
demonstrated the antagonistic activities to S. iniae, as well as
determinating the haematology profile and the antagonistic

2


activities of barramundi serum to the pathogen S. iniae on
barramundi fingerlings.
1.4 Contents of the study
To determine the pathogenic characteristics of
Streptococcus iniae on barramundi
To isolate the lactic acid bacteria isolates from the intestinal
tracts of rabbit fish, barramundi and tilapia.
To determine the haematology profile and the antagonistic
activities of barramundi serum to the pathogen S. iniae on
barramundi fingerlings.

1.5 Scientific significance of the dissertation
This study will supply the information of the pathogenesis
characteristics of haemorrhagic diseases caused by Streptococcus
iniae on barramundi. To assess the effect of the addition of lactic
acid bacteria to the fish diet to the inspecific immune factors of
barramundi. The results of this study can be used to make
probiotics for prevention of haemorrhagic diseases caused by
Streptococcus iniae on barramundi. Besides the applied meaning,
this study will help the researcher to improve the research ability
and provide a valuable reference for research institutes,
universities and students.

1.6 New findings of the dissertation
The harmorrhagic diseases caused by S. iniae was studied
and the histology of this diseases in barramundi in Thua Thien
Hue province was described.

Three isolates of Lactobacillus fermentum from the
intestinal tracts of barramundi and rabbit fish were recovered,
and identified which showed strong antagonistic acitivity to the
S. iniae.
L. fermentum isolate (C21) was used as supplement to the
commercial diet to improve some innative immune system
against the S. iniae pathogens on barramundi.

3


Chapter 3
METHODOLOGY
3.1 Research objects
Streptococcus iniae isolates from barramundi (Lates
calcarifer) have signs of haemorrhagic diseases
Lactic acid bacteria isolates from the intestinal tracts of
rabbit fish (Siganus guttatus), barramundi (Lates calcarifer)
and tilapia (Oreochromis niloticus)
Barramundi (Lates calcarifer)
3.2 Time and date of study
This study was carried out from 12/2014 to 8/2018 at the
wet lab and microbilogy lab of Department of Fish Pathology,
University of Agriculture and Forestry, Hue University.
3.4 Methodology
3.4.1 Methodology of determination of pathogenic
characteristics of S. iniae on barramundi
3.4.1.1 Isolate and identify the S. iniae on barramundi
Sampling method
A total of 87 haemorrhagic fish were sampled at 10

barramundi monoculture farms in Thua Thien Hue province
from 01/2016 to 12/2016. Fish sampled showed clinical signs
of skin haemorrhagic, popped eyes and septicemia. Fish were
stored in styryofoam carton and shipped to the Lab for
analysis.
Method of culture, identification and biochemistry tests
of bacteria
Bacteria was cultured and identified based on the method
of Frerichs and Millar, (1993).
16S rRNA sequencing
The S. iniae identification was also done by 16S rRNA
sequencing
with
primers
Sin
1:
4


CTAGAGTACACATGTAGCT(AGCT)AAG and Sin 2:
GGATTTTCCACTCCCATTAC according to Zlotkin et al.,
(1998). The PCR protocol was followed the protocol of Zlotkin
et al. (1998) with 1X buffer 10X; 1,5mM MgCl2; 200μM
dNTPs; 2,5 U Taq DNA polymerase; 0,5μL forward primer
(Sin1); 0,5μL reversed primer (Sin 2) and 500 ng DNA
sample. Thermal cycles PCR is 94°C in 3’; 94°C in 60’’; 55°C
in 60’’; 72°C in 90’’; with 35 cycles; 72°C in 10’. The PCR
product length is approximately 300 bp.
The PCR product was then purified by kit KIT Isolate II
PCR and Gel (BioLine, Germany) and send to the Macrogen

Laboratory – South Korea for sequencing.
3.4.1.2 Barramundi challenged with S. iniae
Fish preparation
Barramundi fingerlings, average length of 6.6±0.4
cm/fish, average weight of 7.2±0.5 g/fish. The healthy fish
were acclimated for 14 days prior to challenge and were fed
Nanolis C food (Ocialis, Vietnam) 2 times/day at 8 and 17
hours, at 8% bodyweight each.
Experimental design
Experiment 1: Determination of toxicity of bacteria
The experiment on the determination of toxicity of
bacteria based on the method of Dilok (2012). The experiment
consisted of 11 treatments, including 10 challenged treatments
at 109 CFU/mL to the cavity of fish. The S. iniae bacteria
which caused mortality within 3 days of experiment to be
considered as highly toxic strains of bacteria for experiment 2.
Experiment 2: Determination of 50% lethal dose (LD50 Lethal dose 50)
The experiment on the determination of 50% lethal dose
based on the method of Reed and Muench (1938). The
experiment was conducted in 14 days, fish was checked 4
times/week, fish was checked 4 times a week for cumulative
mortality. The 50% lethal dose was determined based on the
5


cumulative mortality in each treatment after experiment.
Experiment 3: The determination of challenge dose of
bacteria
The challenged experiment of bacteria was determined
based on the method of Dilok (2012), (Table 3.4).

Table 3.4: The determination of challenge dose of bacteria
isolated HTA1 and HTA3 on barramundi.
Isolates Experiments

HTA1
HTA3

1
2
Control

Fish
(fish/tank)
10
10
10

Bacterial
dentity
(CFU/mL)
LD50
LD50
Saline solution

Replicates

3
3
3

During 14 days of experiment, fish was checked 4
times/week for fish health and the fish with signs of diseases to
isolate the bacterial strains and histopathology study.
3.4.1.3 The determination of histopathology characteristics of
barramundi infected S. iniae
The barramundi showed signs of diseases and uninfected
fish from the experiment 3 for histopathology study. The tissue
of liver, spleen, kidney, brain of infected diseases and healthy
fish was fixed in formalin solution 4%. The fixation tissue was
then analysed based on the method of Mohamed (2009). The
samples were then observed under microscopes.
3.4.2 Identification and selections of lactic acid bacteria from
gut of marine fishes which have antagonistic activities
against S. iniae
3.4.2.1 Identification of lactic acid bacteria from gut of seabsss,
rabbit fish and tilapia
Sampling
Barramundi, tilapia and rabbit fish were caught from
nature, with adult size. At the point of sampling, fish was
healthy and show no signs of diseases. Fish was then carried to
the Laboratory and dissected. Bacterial samples from gut was
6


then used for identification.
Lactic acid bacteria identification method
Lactic acid bacteria identification followed the method of
Nirunya et al. (2008).
Lactic acid bacteria identification based on physical and
biochemical characteristics

Biochemical tests were conducts: Gram test, catalase,
oxidase, mobility, spore production, CaCO3 degration and
gelatin liquidation. Bacteria was then identified based on the
methods of Cowan and Steels (Barrow and Feltham, 1993).
3.4.2.2 The determination of antagonistic activities of lactic
acid bacteria against S. iniae
The determination of antagonistic activities of lactic acid
bacteria against S. iniae was followed the method of Hamza et
al. (2012). The wells-agar distribution method was used to
determine the antagonistic activities of lactic acid bacteria
against S. iniae The antagonistic activities of lactic acid
bacteria against S. iniae was followed the method of Galindo
(2004).
3.4.2.3 The identification lactic acid bacteria which had
antagonistic activities of against S. iniae
The lactic acid bacteria identification was also done by
16S
rRNA
sequencing
with
primers
LacF:
AGCAGTAGGGAATCTTCCA
and
LacR:
ATTCCACCGCTACACATG according to Nikolaou et al.
(2011). The PCR protocol was followed the protocol of
Nikolaou et al. (2011). The master mix included: 50 ng DNA,
10 pmol forward primer, 10 pmol reverse primer, 5 µL PCR
buffer (10X), 10 pmol dNTP, 5U Enzyme pfu and PCR water

for 50 µL. Thermal cycles PCR is 95°C in 5’; 95°C in 45’’;
48°C in 30’’; 72°C in 60’’; with 30 cycles; 72°C in 7’. The
PCR product length is approximately 345 bp.
PCR product was then purified by KIT Isolate II PCR and
7


Gel (BioLine, Germany) purification test and was sent to the
Macrogen Laboratory – South Korea for the acid nucleic
sequencing.
3.4.3. Study on the effect of the addition of lactic acid bacteria
to the fish diet to the the haematology profile and the
antagonistic activities against S. iniae of barramundi
3.4.3.1 Fish preparation
The number of fish for experiment is 240 individuals, with
average length is 8.3±0.4 cm/fish and average weight is
11.4±2.3 g/fish. The fish was fed Nanolis C (Ocialis, Vietnam)
twice a day at 8 and 17h, at 8% bodyweight.
3.4.3.2 Bacterial preparation
The S. iniae isolate HTA1 was used for challenge, with
the concentration of 1.9x105 CFU/mL and L. fermentum isolate
C21, with the concentration of 109 CFU/g feed.
3.4.3.3 Experimental design
The experiment was designed according to Allameh et al.
(2013). The experiment to determine the effect of L. fermentum
supplement to the barramundi diet to the haematology profile
and the antagonistic ability of barramundi serum to S. iniae
were designed in 4 treatments with triplicates. The negative
control (NT 1): no L. fermentum supplement to the barramundi
diet and no S. iniae i.p injection. The positive control (NT 2):

no L. fermentum supplement to the barramundi diet and S. iniae
i.p injection to the barramundi cavity after 14 days with the
dose of 1.9x105 CFU/mL/fish. The treatment 1 (NT 3): L.
fermentum supplement to the barramundi diet at 109 CFU/g in
feed and no S. iniae i.p injection. The treatment 2 (NT 4): L.
fermentum supplement to the barramundi diet at 109 CFU/g in
feed for 14 days before i.p injection to the barramundi with the
dose of 1.9x105 CFU S. iniae /mL/fish.
Table 3.6: The experiment to determine the effect of L.
fermentum supplement to the barramundi diet to the
8


haematology profile and the antagonistic ability to S. iniae

NT 1

Number of
fish
(individual)
20

NT 2

20

Treatments

L. fermentum
C21

S. iniae
HTA1

-

1.9x105
CFU/mL/fish
1.9x105
CFU/mL/fish

9

NT 3

20

10 CFU/g feed

NT 4

20

109 CFU/g feed

Replicates
3
3
3
3

Identification: S. iniae was injected to fish cavity after 14 days of experiment

The experiment run for 28 days, the number of red blood
cells, total white blood cells, lymphocytes, monocytes,
leukocytes, thrombocytes of fish on day 1, 14, 21 and 28 to
determine the haematology profile and the antagonistic ability
to S. iniae.
3.4.3.4 Method for the determination of the haematology
profile
Red blood cells count: according to the method of Natt
và Herrick (1952).
Total white blood cells count: Sample was prepared on
lame and was dyed based on Humason, 1979 (cited by Rowley,
1990).
Total white blood cells count and other haemolymphs
were determined based on Chinabut et al. (1991) and Hrubec et
al. (2000).
3.4.3.5 The antagonistic ability of barramundi serum to S. iniae

The antagonistic ability of barramundi serum to S. iniae
was determined based on method of Phuong et al. (2007). The
S. iniae inhibited (Suc (%)) by barramundi serum was calculated
as followed:
Suc (%) =

OD sample - OD blank
OD control - OD bank

x

100

With: Suc (%): Ratio (%) S. iniae inhibited by barramundi
serum; OD- Optical Density
3.4.3.6 Determining lysozyme activity in serum
9


The lysozyme activity in serum was determined based
on the method of Kumar et al. (2007). The lysozyme activity in
serum was determined by unit/min/mg protein of serum. One
unit of lysozyme (U) was determined by the amount of
lysozyme absorbed at 450 nm with 0.001 absorb unit/min/mg
(U/min/mg).
3.4.3.7 Survivals determiniation
Fish survivals was conuted at 14 days post challenge of
S. iniae, based on the method of (Kumar et al., 2007):
Survival (%) =

Survival fish after challenge with S. iniae
Total fish challenged with S. iniae

x 100

The effective level of L. fermentum in diet was
calculated based on the protection level (relative percentage of
survival - RPS) (%) with method from (Ellis, 1988).
3.5 Data analysis
Crude data was input and analysed on Microsoft Excel
2016, and the analysed by ANOVA based on General Linear

Model on SPSS version 20.

Chapter 4
RESULTS AND DISCUSSION
4.1 Ppathogenic characteristics of Streptococcus iniae on
barramundi
4.1.1 Isolate and identify the S. iniae on barramundi
4.1.1.1 Result of isolate the S. iniae on barramundi
A total of 87 haemorrhagic barramundi were collected to
isolate bacteria. The clinical signs showed that of haemorrhagic
barramundi showed include erratic swimming, intra-ocular,
corneal opacity and hemorrhage in the eye, at the base of the
fins and in the opercula, abdominal sinuses contain mucus,
hemorrhagic viscera, kidney swelling, hemorrhagic brain
(Figure 4.1).
Bacterial isolation from 87 moribund barramundi
isolated 42 isolates of Streptococcus spp. from the brain,
kidney, spleen and liver of fish (20 isolates from the brain
10


(63%), 9 isolates in the kidney (21.4%), 7 isolates in the spleen
(16.7%) and 6 isolates in the liver (14.3%).

Figure 4.1. Infected fish used for bacterial isolate
(A- cornea opacity and hemorrhage in the eye; B- intra-ocular and
corneal opacity in the eye, abdominal sinuses contain mucus)

4.1.1.2 Morphological, physiological and biochemical
characteristics of the bacterial isolates on barramundi

The result of sampling, isolation and culture of bacteria
showed that on TSA + plate, after 24 to 48 hours of culture at
28oC, bacteria developed into small colonies with diameters of
0.7 - 1 mm, opaque white, even edges, no pigmentation. The
result of Gram staining determined Gram (+) bacteria and
cocci-shaped (Figure 4.2).

Figure 4.2: Colonies morphology and Gram staining of S. iniae (AColonies morphology of S. iniae on TSA + plate; B- Gram staining
of S. iniae isolated from barramundi (100X))

The bacterial isolates were non-motile, catalase and
oxidase negative, lysin decarboxylase negative, positive for
hydrolysis of starch, no growth on the TSB plate containing
11


6.5% NaCl and the TSA plate did not supplement NaCl.
Results of slide agglutination test by using Slidex Strepto Plus
revealed no reaction with A, B, C, D, F, G serotypes of
Lancefield group (Table 4.2). On BA + plates, all isolates
showed β-haemolys.
Using the API 20 Strep system test, the isolates tested
were positive with hydrolysis of esculin and negative for
hydrolysis of hippurate, positive for pyrrolidonyl arylamidase,
β-Glucuronidase, alkaline phosphatase, arginine dihydrolase và
leucine arylamidase, negative for Voges-Proskauer, αGalactosidase, β-Galactosidase.
Results of determined biochemical characteristics and
compared with S. iniae isolate by Bromage et al. (1999) and S.
iniae isolate of Rahmatullah et al. (2017), 42 isolates of
Streptococcus spp. isolated from barramundi were identified as

S. iniae.
4.1.2 Challenge of barramundi with Streptococcus iniae
4.1.2.1 Results of determine of bacterial virulence
Results of determine of bacterial virulence showed that
S. iniae HTA1 and HTA3 isolates were fish 100% mortality in
3 days of experiment. Therefore, S. iniae HTA1 and HTA3
isolates were chosen for from the isolates obtained from
moribund barramundi for determine the LD50.
4.1.2.2 Results of determine of the lethal dose 50% (Lethal
dose 50 - LD50)
HTA1 and HTA3 in intraperitoneal (i.p.) injection
healthy barramundi were 1.9x105 CFU/mL and 1.5x105
CFU/mL, respectively.
4.1.2.3 Results of the determination of challenge dose of
bacteria
The determination of challenge dose of bacteria isolated
HTA1 and HTA3 on barramundi showed that After 48h
challenged with the HTA1 or HTA3 isolate by intraperitoneal
12


(i.p.) injection, fish showed the typical clinical signs of
diseases such as anorexia, haemorrhagic on body, and fins,
exophthalmia eyes and viremia. After 72h i.p injection
challenged, the mortality was observed. The cumulative
mortality was the highest at 8 days post challenged, accounted
for 76.7% (HTA1 isolate) and 80% (HTA3 isolate), (Figure
4.6). Whereas, in the control group, uninfected fish showed no
clinical sign, no death, and no S. iniae were found.

Cumulative mortalities (%)

100.0
90.0
80.0
70.0
60.0
50.0
40.0
30.0
20.0
10.0
0.0

HTA1

1

2

3

4

5

6

7

8

HTA3

ĐC

9 10 11 12 13 14

Days post challenge
Figure 4.6: The cumulative mortalities in barramundi in 14 days
exposed to S. iniae HTA1 and HTA3 isolates by intraperitoneal (i.p.)
injection

4.1.2.4 Histopathological characteristics of barramundi
infected with S. iniae
Histopathological analysis of S. iniae infected
barramundi revealed structural changed in liver, kidney, spleen
and brain. Necrosis and haemorhagic were observed in liver,
kidney, spleen and brain in S. iniae infected barramundi under
experimental condition.

13


Figure 4.7: Liver tissue of barramundi infected with S. iniae (H&E
staining), (100X); (S. iniae in liver tissue of barramundi after 3 days
of challenge (arrows))

Figure 4.8: Kidney tissue of barramundi infected with S. iniae (H&E
staining), (100X); (a- melanomacrophage in the kidney tissue

(arrows);b- structure of renal tubules changed in kidney tissue
(arrows))

Figure 4.9: Spleen tissue of barramundi infected with S. iniae (H&E
staining), (100X); (melanomacrophage in the spleen tissue (arrows))

14


Figure 4.10: Brain tissue of barramundi infected with S. iniae (H&E
staining), (100X); (S. iniae in brain tissue of barramundi after 3 days
of challenge (arrows))

4.1.2.5 The results of identification HTA1 and HTA3 isolates
by polymerase chain reaction and the acid nucleic sequencing
The 2 isolated of HTA1 and HTA3 were identified via
specific 16S rRNA amplification and sequencing. The results
showed that they are S. iniae.
4.2 Identification and selections of lactic acid bacteria from
gut of brackish fishes which have antagonistic activities
against S. iniae
4.2.1 Result of isolation of lactic acid bacteria isolates
The results showed that 61 lactic acid bacterial isolates
were isolated from the gut of adult tilapia, barramundi and
rabbit fish. All isolates were Gram-positive, rod or coccishaped, non spore formation, non-mobile, negative for catalase
and oxidase, CaCO3 degration and no gelatin liquidation (Table
4.7)
Table 4.7: Result of isolation of lactic acid bacteria from
tilapia, barramundi and rabbit fish
Fish

Barramundi
Tilapia
Rabbit fish

Quantity
(individual)
50
50
50

Isolated tissue
Ruột
Ruột
Ruột

15

Lactic acid
bacteria isolates
27
19
15


Figure 4.13. Lactic acid bacteria shape after Gram staining
(100X), (A - Rod-shaped bacteria; B - Cocci-shaped bacteria)
Table 4.8: Morphological, physiological and biochemical
characteristics of the lactic acid bacterial isolated from tilapia,
barramundi and rabbit fish
Test

Colony color
Cell morphology
Rods
Cocci
Gram-staining
Mobile
Spore formation
Catalase
Oxidase
CaCO3 degration
Gelatin liquidation
Growth in MRS agar 1% NaCl
Growth in MRS agar 1,5% NaCl
Growth in MRS agar 2% NaCl
Growth in TSA 1,5% NaCl
Growth in TSA 2% NaCl
Growth in TSA 1,5% NaCl
Temperature, 4oC
Temperature, 28oC
Temperature, 37oC

Test results of lactic acid bacteria
Barramundi
Tilapia
Rabbit fish
(n = 27)
(n = 19)
(n = 15)
Opalescent Opalescent Opalescent

white
white
white
18
9
+
+
+
+
+
+
+

14
5
+
+
+
+
+
+
+

13
2
+
+
+
+
+

+
+

+
+
+
+
+
+
Identification: “+”: positive; “-”: negative

16


4.2.2 The results of determine of antagonistic activities of
lactic acid bacteria against S. iniae
The results of determine of antagonistic activities of
lactic acid bacteria against S. iniae showed that within the
collection of these 61 lactic acid bacterial isolates, 28 isolates
antagonistic to S. iniae, especially, the 3 isolated of C21, D1
and D7 showed strong antagonistic activity to S. iniae
(inhibition zone larger than or equal to 20 mm in diameter).
(Table 4.9).
Table 4.9: The results of determine of antagonistic activities of
lactic acid bacteria against S. iniae
Isolates Antagonistic Isolates Antagonistic Isolates Antagonistic
activity
activity
activity
D1

D2
D3
D4
D5

+++
++
+
-

C7
C8
C9
C10
C11

++
-

D6
D7
D8
D9
D10
D11
D12
D13
D14
D15
C1

C2
C3
C4
C5
C6

R1
R2
R3
R4
R5

+
++
++
-

C12
+
R6
+++
C13
R7
C14
R8
C15
R9
+
C16
R10

+
++
C17
+
R11
C18
R12
C19
+
R13
+
C20
R14
+
C21
+++
R15
+
C22
R16
+
C23
+
R17
+
+
C24
R18
++
+

C25
R19
+
+
C26
+
++
C27
Identification: (-): no inhibition zone recorded or inhibition zone less
than or equal to 1 mm in diameter; (+): inhibition zone from 1 to 5 mm in
diameter; (++): inhibition zone from 6 to 20 mm in diameter; (+++):
inhibition zone larger than or equal to 20 mm in diameter

17


4.2.3 The results of identify lactic acid bacteria which had
antagonistic activities of against S. iniae
The results of electrophoresis PCR of C21, D1 and D7
isolates showed successful amplification of the gene with the
size of 345bp. The 3 isolated of C21, D1 and D7 were
identified via specific 16S rRNA amplification, sequencing and
the phylogenetic tree. The results showed that they are
Lactobacillus fermentum
4.3 The effect of the addition of lactic acid bacteria to the
fish diet to the the haematology profile and the antagonistic
activities against S. iniae of barramundi
4.3.1 The results of determined of the haematology profile
Blood samples were colleted on day 1, 14, 21 and 28
for hamatological analysis and the ability of fish serum

collected to identify the against of S. iniae. The results showed
that, on day 21, the number of reb blood cells and total white
blood cells of fish in NT 3 (L. fermentum supplement to the
barramundi diet at 109 CFU/g in feed and no S. iniae i.p
injection) and NT 4 (L. fermentum supplement to the
barramundi diet at 109 CFU/g in feed for 14 days before i.p
injection to the barramundi with the dose of 1.9x105 CFU S.
iniae /mL/fish) were significantly higher than those from NT 1
(no L. fermentum supplement to the barramundi diet and no S.
iniae i.p injection) and NT 2 (no L. fermentum supplement to
the barramundi diet and S. iniae i.p injection to the barramundi
cavity after 14 days with the dose of 1.9x105 CFU/mL/fish),
(p<0.05). On day 28, the number of total white blood cells of
fish in NT 2, NT 3 and NT 4 were significantly higher than
those from NT 1, (p<0.05).

18


Red bloods cells (x106
cells /mm3)

6

NT 1

NT 2

NT 3

NT 4
c
ac

5
b b

4
3

a

a a

a a
a a

c
a

d
b

b

2
1
0
1

14

21

28

Days post challenge
Figure 4.18: Red blood cells density in barramundi blood

White blood cells (x105
cells/mm3)

NT 1

NT 2

NT 3

5.00

b

b b

4.00
3.00

NT 4
b b

a a a

a

a

a

a

a

b b

a

2.00
1.00
0.00
1

14

21

28

Days post challenge

Figure 4.19. White blood cells density in barramundi blood

4.3.2 The antagonistic ability of barramundi serum to S. iniae
The results of the antagonistic ability of barramundi
serum to S. iniae showed that the antagonistic ability of
barramundi serum to S. iniae from NT 4 (L. fermentum
supplement to the barramundi diet at 109 CFU/g in feed for 14
days before i.p injection to the barramundi with the dose of
1.9x105 CFU S. iniae /mL/fish) was significantly higher than
those from NT 1 (no L. fermentum supplement to the
barramundi diet and no S. iniae i.p injection) and NT 2 (no L.
19


fermentum supplement to the barramundi diet and S. iniae i.p
injection to the barramundi cavity after 14 days with the dose
of 1.9x105 CFU/mL/fish), (p<0.05). (Table 4.10)
Table 4.10: The antagonistic ability of barramundi serum to S. iniae
Treatments
The antagonistic ability of barramundi serum to
S. iniae (%); (Aveg±SD)
Day 1
Day 14
Day 21
Day 28
NT 1
21.3±3.2a 26.7±1.5a
31.0±4.6a 35.0±5.0a
NT 2
24.7±5.0a 30.0±5.6a 43.0±3.0b 46.3±3.5b
NT 3
26.0±4.0a 32.3±8.0a

55.0±5.0c 60.7±1.2c
NT 4
24.7±2.9a 34.3±4.7a 74.0±2.6d 64.3±2.1c
The values in the same column have different letters (a, b, c, d), the
difference is statistically significant (p <0.05)

4.3.3 Lysozyme activity in serum
The results of the lysozyme activity in serum of fish
showwed that the lysozyme activity in NT 3 (L. fermentum
supplement to the barramundi diet at 109 CFU/g in feed and no
S. iniae i.p injection) and NT 4 (L. fermentum supplement to
the barramundi diet at 109 CFU/g in feed for 14 days before i.p
injection to the barramundi with the dose of 1.9x105 CFU S.
iniae /mL/fish) treatments were significantly higher than those
in NT 1 (no L. fermentum supplement to the barramundi diet
and no S. iniae i.p injection) and NT 2 (no L. fermentum
supplement to the barramundi diet and S. iniae i.p injection to
the barramundi cavity after 14 days with the dose of 1.9x105
CFU/mL/fish) treatments (p<0.05). The lysozyme activity of
fish in NT 4 (L. fermentum supplement to the barramundi diet
at 109 CFU/g in feed for 14 days before i.p injection to the
barramundi with the dose of 1.9x105 CFU S. iniae /mL/fish)
treatment was not significantly higher than those in NT 2 (no L.
fermentum supplement to the barramundi diet and S. iniae i.p

20


injection to the barramundi cavity after 14 days with the dose
of 1.9x105 CFU/mL/fish) treatment, (p>0.05). (Figure 4.28)

NT 1

Lysozyme activity serum
(U/min/mg)

1500.0

NT 2

NT 3

NT 4
b

bc c

1000.0

a a a a

a

ab

b b

b b

b

a

a

500.0

0.0
1

14

21

28

Days post challenge

Figure 4.28: Lysozyme activity in barramundi serum
4.3.4 Barramundi survival rate
The survival rate of barramundi in NT 2 (no L.
fermentum supplement to the barramundi diet and S. iniae i.p
injection to the barramundi cavity after 14 days with the dose
of 1.9x105 CFU/mL/fish) and NT 4 (L. fermentum supplement
to the barramundi diet at 109 CFU/g in feed for 14 days before
i.p injection to the barramundi with the dose of 1.9x105 CFU S.
iniae /mL/fish) were 23.7% and 52.3%, respectively (Figure
4.29). The relative percentage of survival with L. fermentum
supplement to the barramundi diet was 37.5%.
Fish survival rate (%)

NT 1

NT 2

NT 3

NT 4

150.0
100.0
50.0
0.0
1

2

3

4

5

6

7

8

9

10 11 12 13 14

Day post challenge with S. iniae

Figure 4.29. Fish survival rate after infection with S. iniae
21


Figure 4.30. The result of S. iniae infection in barramundi
(A- healthy barramundi before experiment; B- healthy
barramundi after experiment; C- hemorrhagic on the skin; Dcorneal opacity in the eye and abdominal sinuses contain
mucus; E- healthy barramundi brain, no hemorrhage; Fhemorrhage in the brain)
The result of S. iniae infection in barramundi showed
that in NT 2 and NT 4, barramundi were hemorrhaged on the
skin, corneal opacity in the eye, abdominal sinuses contain
mucus and hemorrhage in the brain (Figure 4.30). Whereas, in
the NT 1 and NT 2, uninfected fish showed no clinical sign, no
death, and no S. iniae were found.

Chapter 5
CONCLUSION AND RECOMMENDATION
5.1 Conclusion
Streptococcus iniae is the main cause of hemorrhagic
disease in barramundi cultured in Thua Thien Hue. The results
of challenged tests of 2 isolates S. iniae HTA1 and HTA3 on
barramundi indicated the LD50 of 2 isolates HTA1 and HTA3
were 1.9x105 CFU/mL and 1.5x105CFU/mL, respectively. The
22