Egyptian Journal of Aquatic Biology & Fisheries

Zoology Department, Faculty of Science,

Ain Shams University, Cairo, Egypt.
ISSN 1110 – 6131
Vol. 28(6): 1425 – 1442 (2024)
www.ejabf.journals.ekb.eg

Pathogenicity Testing of Bacterial Disease in the Barramundi (Lates calcarifer) in

North Aceh

Eva Ayuzar *, Anis Nugrahawati , Mainisa , Salamah , Rachmawati Rusydi

Department of Fisheries and Marine, Faculty of Agribussines, Malikussaleh University, 24355, North
Aceh, Aceh, Indonesia
*
Corresponding Author: [email protected]

ARTICLE INFO ABSTRACT

Article History: The barramundi (Lates calcarifer) is a fish of high economic value, a feature
Received: Aug. 7, 2024 of which has increased its aquaculture. The expansion of aquaculture not only
Accepted: Aug. 24, 2024 leads to higher profits but raises the risks involved in aquaculture as well. One
Online: Dec. 11, 2024 of them is a bacterial disease. Pathogenicity testing is essential to understand
_______________ how these bacteria infect their host, which can inform strategies for managing
bacterial diseases. Preliminary tests identified two bacteria, which were
Keywords:
Bacteria
subjected to molecular DNA identification and sequencing, revealing the
Histopathology presence of Vibrio navarrensis and Acinetobacter lwoffii. The two bacterial
Lates calcarifer strains were then tested on barramundi fingerlings measuring 6- 7cm to
Pathogenicity determine the LD50 for subsequent pathogenicity assessment. The
PCR pathogenicity tests involved seven treatments with three replicates each,
including treatment A, which involved the injection of V. navarrensis 107 CFU
mL-1; treatment B with the injection of V. navarrensis at 106 CFU mL-1;
treatment C with the injection of V. navarrensis 105 CFU mL-1; treatment D
with the injection of A. lwoffii at 107 CFU mL-1; treatment E with the injection
of A. lwoffii at 106 CFU mL-1; treatment F with the injection of A. lwoffii at 105
CFU mL-1; and a control group injected with phosphate buffered saline (PBS).
The LD50 results indicated that V. navarrensis and A. lwoffii bacterial densities
were 1,7 x 105 CFU mL-1 dan 2,1 x 105 CFU mL-1. Significant differences were
noted in mortality rates, average time to death, and blood parameter profiles
across all treatments compared to the control group (P< 0.05).
Histopathological examination of the liver and spleen revealed hepatocyte
damage, cell inflammation, melanomacrophage centers, and cell necrosis. Test
of the intestine showed hemorrhage and hydropic degeneration in the muscular
layer. A series of pathogenicity tests confirmed that these two bacteria are
responsible for causing bacterial disease in Barramundi culture in North Aceh.
INTRODUCTION

North Aceh is a regency consisting of 27 districts. Among them, Dewantara is part

of the coastal region of West Lancang, which includes a village (Gampong) known for its
potential in aquaculture, developed by the local community. The primary commodity
cultivated by aquaculture farmers in this area is barramundi (Lates calcarifer).
Barramundi (L. calcarifer) is a fish of significant economic value, serving as an important
export commodity with extensive markets in America, Europe, Malaysia, and Thailand.
1426 Ayuzar et al. , 2024

Its production has been increasing annually. In 2018, barramundi production reached
30,000 tons, a substantial increase from the previous year's production of 25,051 tons
(Rayes et al., 2013; Ministry of Marine Affairs and Fisheries, 2018). The tall trade
request presents an opportunity for the advancement of barramundi aquaculture, as the
angle is simple to develop and incorporates a tall resilience for changes in saltiness
(Hardianti et al., 2016).
The rising demand for barramundi has led to a significant increase in aquaculture
activities. However, the lack of knowledge about proper farming practices has resulted in
several issues, notably disease outbreaks. Farmers in Dewantara District frequently report
high mortality rates among their farmed barramundi (Lates calcarifer). The observable
symptoms in the deceased fish include surface lesions, darkened body coloration, torn tail
fins, and sloughing gills. Additionally, a decrease in appetite is a concern for farmers in
Dewantara District. These symptoms are suspected to be caused by bacterial infections.
Kurniawan (2012) elucidated that bacterial diseases in fish typically manifest as reddish
lesions on the body surface. Bacterial infection typically results in abnormal changes
(lesions) on the skin or fins, muscle tissues, and internal organs.
One effective and accurate method utilized to measure the pathogenicity of
bacterial diseases is through pathogenicity testing and determining the Lethal Dose 50
(LD50) - the bacterial dose that causes mortality in 50 percent of the infected fish
population. Additional methods include observing clinical symptoms, calculating
mortality rates and average time to death, conducting histopathological examinations of
tissue samples, analyzing the blood profiles of fish affected by bacterial diseases, and
testing antimicrobial sensitivity to assess the effectiveness of antibiotic substances in
inhibiting the growth of bacteria causing these diseases (Izwar et al., 2020). This study
aimed to investigate the bacterial species causing bacterial diseases in barramundi and to
test their pathogenicity.

MATERIALS AND METHODS

Description of study sites

This research was conducted at the Brackish Water Aquaculture Fisheries Center
Ujung Batee, Aceh, Indonesia. Molecular analysis of the bacteria was conducted at PT
Genetica Science Indonesia, Tanggerang City, Banten, Indonesia. Experimental fish was
sent from PT Kembang Tani Farm, North Aceh, Aceh, Indonesia.

Study design
This research comprises preliminary and main studies. The preliminary study
involves molecular analysis of bacteria from Saragih et al. (2024) to definitively identify
the bacterial species. This study involved two bacterial samples, designated as isolate A
and isolate B, which were sent to PT Genetica Science Indonesia for analysis. The PCR
analysis conducted by PT Genetica Science Indonesia included the following steps: (1)
 1427
Pathogenicity Testing of Bacterial Disease in Lates calcarifer in North Aceh

Genomic DNA extraction with Quick-DNA Fungal/Bacterial Miniprep Kit (Zymo

Research, D6005); (2) PCR amplification with (2x) Dream Taq PCR Master Mix (K9021;
Thermo); and (3) Bi-directional sequencing. The sequencing results indicated that isolate
A was Vibrio navarrensis and isolate B was Acinetobacter lwoffii. Subsequently, Koch's
postulates were tested to confirm the virulence of the test bacteria. Koch's postulates were
conducted by injecting each bacterium at a density of 108 CFU mL-1. Observations
regarding clinical symptoms such as changes in behavior, morphology, external
symptoms, internal symptoms, and fish mortality were conducted over a period of 96
hours.
The main study commenced following the completion of the preliminary study. The
pathogenicity test was conducted using a completely randomized design with 6 treatments
and 1 control, each with 3 replicates. The experimental design for the treatments is outlined
in Table (1).

Table 1. Experimental design for the pathogenicity test of bacterial diseases in

barramundi
No Treatment Explanation
1 K Control: fish injected with Phosphate Buffered Saline (PBS)
2 A Fish injected with V. navarrensis 2.3 × 107 CFU mL-1
3 B Fish injected with V. navarrensis 2.2 × 106 CFU mL-1
4 C Fish injected with V. navarrensis 1.7 × 105 CFU mL-1
5 D Fish injected with A. lwoffii 2.1 × 107 CFU mL-1
6 E Fish injected with A. lwoffii 2.5 × 106 CFU mL-1
7 F Fish injected with A. lwoffii 1.7 × 105 CFU mL-1
The research steps included preparing the containers, preparing the test fish,
preparing the bacteria and conducting Koch's postulates, conducting the pathogenicity
test, and making observations. Each bacterial strain was cultured for 24 hours before
being injected into the barramundi fingerlings. The maintenance containers, which had a
volume of 25 liters, were disinfected to prevent contamination. A total of 105 liters of
seawater was used across all containers, with 105 barramundi fingerlings used for the
study.

Preparation of test fish

The fish used in this study were barramundi measuring 7 cm in length. They were
carefully selected to ensure they were in good health and free from any wounds or
physical deformities in order to obtain accurate results.

Preparation of test bacteria and Koch's postulates test

The bacteria were obtained from the study conducted by Saragih et al., (2024).
Both bacterial isolates were purified in TSA media + NaCl 0.9%. Three colonies of each
bacterium were transferred into TSB and homogenized. Each bacterium, with a density of
1428 Ayuzar et al. , 2024

108 CFU, was injected intraperitoneally into 10 barramundis. The injected fish were
observed for changes in behavior and clinical symptoms over a period of 96 hours
(Sarjito, 2010).

LD50 test
The bacterial virulence level was assessed by calculating its LD50 value. LD50
testing was conducted using the Reed-Muench method (1938), with three different
dosage treatments achieved through dilution methods and bacterial density calculations.
The bacterial densities used in the LD50 testing were , , dan cells per 0.1 mL
per fish. The LD50 testing was conducted by injecting healthy barramundi fingerlings
with bacteria at 0.1mL using the intraperitoneal injection method, while the control group
was injected with phosphate buffered saline (PBS). Prior to infection, the test fish were
first coated with wet cloth to minimize stress from handling. After injection, the fish were
returned to the maintenance containers and observed at least every 12 hours. The LD50
testing results will assess bacterial virulence and mortality among the barramundi
fingerlings (Sunarto et al., 2003).

Log negative LD50 = Log concentration in bacteria below 50% + (Proportion Odds
Ratio × Log dilution)
LD50 = Anti Log × Log negative LD50
Pathogenicity test
The fish infected during the LD50 test were monitored to evaluate the pathogenicity
of the test bacteria. Parameters observed included clinical symptoms, mortality rate,
average time to death, and blood profile analysis. Changes observed during the LD50 test
were recorded over a period of 7 days. Throughout the observation period, fish were fed
commercial feed twice daily ad libitum. Fish that died during the study were disposed of
by burial to prevent disease transmission.

Mortality rate
The observation of fish mortality was done every day after pathogen injection. Fish
mortality was calculated by using the formula below (Effendie, 1979):

Meantime of death (MTD)

Fish that was already injected by pathogen was observed detecting the behavior
every day, such as the clinical symptoms, fish mortality, and meant time to death.
Meantime to death was calculated by using the formula below (Hubbert, 1980).
 1429
Pathogenicity Testing of Bacterial Disease in Lates calcarifer in North Aceh

Note:
MTD = Meantime of death (hour)
a = mortality time (hour)
b = number of dead fish

Blood profile parameters

Blood profile parameters analyzed include total erythrocytes, total leukocytes,
haemoglobin, and haematocrit. Test fish injected with bacteria were subjected to blood
profile analysis on days 1, 3, 5, and 7 according to the treatment. Blood sampling from
the fish was conducted using the Caudal Vessel Puncture technique. The syringe needle
was first moistened with an anticoagulant, ethylene diamine tetraacetic acid (EDTA), to
prevent blood clotting during the analysis.

Total Erythrocytes
The erythrocyte count in barramundi was conducted using a hemocytometer. This
device involves drawing a 0.5mL blood sample and mixing it with Hayem's solution up
to the 101 mark. The mixture is then pipetted and gently mixed for 5 minutes.
Subsequently, the blood is dropped into a Neubauer hemocytometer chamber. The
erythrocyte count is observed and calculated using a microscope connected to a computer
screen to facilitate observation (Saparuddin, 2017).

Total leukocytes
The tool used to count white blood cells was a hemocytometer. This device was
employed by drawing a blood sample up to the 0.5mL mark for each treatment, followed
by the addition of Turk's solution up to the 1:11 ratio. The pipette was gently swung to
ensure thorough mixing for 5 minutes. Subsequently, the blood was dropped into a
Neubauer hemocytometer chamber. The number of white blood cells was then observed
and calculated using a microscope connected to a computer screen for easier observation
(Saparuddin, 2017).

Hemoglobin
The procedure for hemoglobin measurement was conducted using the Sahli
method. First, a blood sample was drawn into a pipette up to the 0.2 mL mark. The
pipette tip was then wiped clean with tissue paper. Next, the blood from the pipette was
transferred to an Hb-meter tube filled with 0.1 N HCl up to the 10 mL mark. Next, the
blood was stirred for 3 to 5 minutes, followed by the addition of distilled water (aquades)
into the tube until the blood color matches that of the standard solution in the Hb-meter.
The hemoglobin level was expressed in g 100mL-1 (g dl-1) (Hartika et al., 2014).
1430 Ayuzar et al. , 2024

Haematocrit
Hematocrit levels are determined by filling a capillary tube approximately two-
thirds full with blood from a porcelain dish. The tube's end is tightly sealed with wax and
wrapped in tissue to prevent breakage during centrifugation. The sample is then
centrifuged at 1500rpm for 5 minutes. The red portion of the contents in the tube is
measured as the hematocrit level (Sarkiah et al., 2016).

RESULTS

Molecular analysis of bacteria

Both types of bacteria from the previous study were subjected to molecular testing
and underwent sequencing to accurately identify their types. The DNA concentration of
each bacterial isolate met purity standards with an Absorbance 260/280 1.90 for bacterial
isolate A, and 1.96 for bacterial isolate B (Table 2). A DNA sample is considered pure
when the A260/280 ratio ranges from 1.8 to 2.0. If the ratio falls below 1.8, it indicates
contamination by phenol. Conversely, if the ratio exceeds 2.0, contamination by proteins
or other compounds is inferred (Sambrook & Russell, 2001; Maulida & Lisdiana,
2024).

Table 2. Results of quantitative DNA testing

No Sample Name Conc (ng/µL) A260/280
1 Isolate 1 38.0 1.90
2 Isolate 2 21.5 1.96

Fig. 1. Electrophoresis bands of rDNA gene fragments from bacterial isolate 1 and isolate 2

Observation of clinical symptoms

Prior to conducting the LD50 test, all treatment groups exhibited similar conditions:
responsiveness to feed, normal behavior, active movement, silvery-white body color,
smooth skin surface, intact fins, clear eyes, non-distended abdominal cavity, and well-
preserved anatomical organs. However, following the LD50 test across all treatments,
 1431
Pathogenicity Testing of Bacterial Disease in Lates calcarifer in North Aceh

barramundi showed signs of feed non-responsiveness, abnormal behavior, reduced

activity, darkened body color, rough skin, frayed fins, yellowish-white eyes, distended
abdominal cavity, and hemorrhaging in anatomical organs such as the intestines,
lymphatic system, and kidneys. Detailed clinical symptoms of barramundi are presented
in Table (3).

Table 3. Clinical symptoms of barramundi after LD50 test

Observation of Treatment
morphology and
K A B C D E F
anatomy
Feeding response Responsiv Non Non Non Non Non Responsive
e responsive responsive responsive responsive responsive
Behavior Normal Abnormal Abnormal Abnormal Abnormal Abnormal Abnormal
Movement Active Passive Passive Passive Passive Passive Passive
Body Color White Blackish Blackish Blackish Blackish Yellowing Yellowing
Skin Surface Smooth Rough Rough Rough Rough Rough Rough
Fins condition Intact Chipped Chipped Chipped Chipped Chipped Chipped
Eyes Condition Shiny Yellowing Yellowing Yellowing Yellowing Yellowing Yellowing
White
Abdominal cavity Normal Swollen Swollen Swollen Swollen Swollen Swollen
Condition
Anatomical organ Normal Hemorrhage Hemorrhage Hemorrhage Hemorrhage Hemorrhage Hemorrhage
Notes: K-control; A-Fish injected by V. navarrensis 2.3 × 107 CFU mL-1; B- Fish injected by V. navarrensis 2.2 × 106
CFU mL-1; Fish injected by V. navarrensis 1.7 × 105 CFU mL-1; Fish injected by A. lwoffii 2.1 × 107 CFU mL-1; Fish
injected by A. lwoffii 2.5 × 106 CFU mL-1, Fish injected by A. lwoffii 1.7 × 105 CFU mL-1.

Fish injected with bacteria also exhibited body color changes to a darker shade and
developed lesions on their bodies. This is caused by excessive mucus production, leading
to a rough body texture and reduced pigmentation. Additionally, the bacteria damaged the
fish's body surface and tissue or outer skin layer, resulting in skin decay and ultimately
causing lesions.

LD50
Based on the research results, the LD50 testing on barramundi showed varying
outcomes. The LD50 result for the bacterium V. navarrensis was 1,7 x CFU mL-1.
The LD50 value for the bacterium A. lwoffii was 2,1 x CFU mL-1. The determination
of the LD50 was performed by recording the number of test animal deaths that occurred
within 24 hours after injection. The results of the LD50 test are presented in Table (4).
 1432 Ayuzar et al. , 2024

Table 4. Results of the LD50 test in bacterial pathogenicity testing

Bacterium LD50 (CFU mL-1)
V. navarrensis 1,7 x
A. Lwoffii 2,1 x
a b

c d

Fig. 2. Comparison of clinical symptoms between control fish and diseased fish: a. Control fish
with normal eyes; b. Treated fish with exophthalmia; c. Healthy control fish with intact scales; d.
Treated fish with body lesions and frayed scales

Mortality Rate
Based on the results of the Kruskal-Wallis test analysis, significant differences in
death rates were found across the treatments. The research results show an asymptotic
significance value (asymp. sig.) of 0,003 meaning that the value is < 0,05. This indicates
that the null hypothesis (Ho) is rejected and the alternative hypothesis (H1) is accepted,
meaning there is an effect of the bacterial pathogen on the mortality of barramundi. This
study demonstrates that bacterial pathogens significantly influence the mortality of
barramundi (Fig. 3).
 1433
Pathogenicity Testing of Bacterial Disease in Lates calcarifer in North Aceh

Fig. 3. Mortality of Barramundi (Lates calcarifer) in bacterial pathogenicity testing: K-control: Fish
injected with PBS A: Fish injected with bacteria V. navarrensis 2.3 × 107 CFU mL-1; B- Fish injected with
bacteria V. navarrensis 2.2 × 106 CFU mL-1; Fish injected with bacteria V. navarrensis 1.7 × 105 CFU mL-
1
; Fish injected with bacteria A. lwoffii 2.1 × 107 CFU mL-1; Fish injected with bacteria A. lwoffii 2.5 × 106
CFU mL-1, Fish injected with bacteria A. lwoffii 1.7 × 105 CFU mL-1.
Mean time to death
Based on the results of the Kruskal-Wallis test analysis, significant differences were
found in the mean time to death of barramundi (L. calcarifer) across different treatments.
The study results showed an asymptotic significance value (asymp. Sig) of 0.003,
indicating that this value is less than 0.05. As a result, the null hypothesis (H0) is
rejected, and the alternative hypothesis (H1) is accepted, meaning there is an effect of the
pathogenic bacteria on the average to death of barramundi. The mean times to death for
each treatment are presented in Fig. (4).

Fig. 4. The mean time to death of fish in the pathogenicity test for bacterial disease K-control.; A
- Fish injected with bacteria V. navarrensis 2.3 × 107 CFU mL-1; B - Fish injected with bacteria V.
navarrensis 2.2 × 106 CFU mL-1; Fish injected with bacteria. V. navarrensis 1.7 × 105 CFU mL-1;
Fish injected with bacteria. A. lwoffii 2.1 × 107 CFU mL-1; Fish injected with bacteria. A. lwoffii
2.5 × 106 CFU mL-1, Fish injected with bacteria. A. lwoffii 1.7 × 105 CFU mL-1.
1434 Ayuzar et al. , 2024

Blood Profile
Hemoglobin levels tended to decrease in each treatment, and hematocrit values
were directly proportional to the decreasing erythrocyte and hemoglobin levels over time,
showing a significant difference from the control. The blood analysis can be seen in Figs.
(5, 6, 7, and 8).

Fig. 5. Erythrocyte Cell Graph of Barramundi K-control; A- The fish injected with bacteria V.
navarrensis 2.3 × 107 CFU mL-1; B- The fish injected with bacteria V. navarrensis 2.2 × 106 CFU
mL-1; The fish injected with bacteria V. navarrensis 1.7 × 105 CFU mL-1; The fish injected with
bacteria A. lwoffii 2.1 × 107 CFU mL-1; The fish injected with bacteria A. lwoffii 2.5 × 106 CFU
mL-1; The fish injected with bacteria A. lwoffii 1.7 × 105 CFU mL-1.

Fig. 6. Total leukocyte count in barramundi fish infected with K-control bacteria; A-Fish injected
with bacteria V. navarrensis 2.3 × 107 CFU mL-1; B- Fish injected with bacteria V. navarrensis
2.2 × 106 CFU mL-1; Fish injected with bacteria V. navarrensis 1.7 × 105 CFU mL-1; Fish injected
with bacteria A. lwoffii 2.1 × 107 CFU mL-1; Fish injected with bacteria A. lwoffii 2.5 × 106 CFU
mL-1; Fish injected with bacteria A. lwoffii 1.7 × 105 CFU mL-1.
 1435
Pathogenicity Testing of Bacterial Disease in Lates calcarifer in North Aceh

The hemoglobin (Hb) levels observed in barramundi remain within the normal
range, typically ranging between 5.47 &7.67 g% (Fig. 7). However, a significant
distinction between diseased and healthy fish is evident, as the Hb levels in the treatment
groups progressively decrease over time. This decline is attributable to reduced red blood
cell counts caused by bacterial lysis, leading to diminished blood oxygen levels and
thereby contributing to decreased Hb levels in barramundi.

Fig. 7. Hemoglobin cell morphology in barramundi under K-control; A-Fish injected with
bacteria V. navarrensis 2.3 × 107 CFU mL-1; B- The fish injected with bacteria V.
navarrensis 2.2 × 106 CFU mL-1; The fish injected with bacteria V. navarrensis 1.7 × 105
CFU mL-1; The fish injected with bacteria A. lwoffii 2.1 × 107 CFU mL-1; The fish
injected with bacteria A. lwoffii 2.5 × 106 CFU mL-1, The fish injected with bacteria A.
lwoffii 1.7 × 105 CFU mL-1.

Based on the observations, the hematocrit levels in barramundi infected with

bacteria and those that are not, also exhibit a notable decline (Fig. 8). This is due to
bacterial activity within the bloodstream, which reduces hematocrit levels.
1436 Ayuzar et al. , 2024

Fig. 8. Hematocrit cell morphology in barramundi under K-control; A-Fish injected with
bacteria V. navarrensis 2.3 × 107 CFU mL-1; B- Fish injected with bacteria V. navarrensis
2.2 × 106 CFU mL-1; Fish injected with bacteria V. navarrensis 1.7 × 105 CFU mL-1; Fish
injected with bacteria A. lwoffii 2.1 × 107 CFU mL-1; Fish injected with bacteria A. lwoffii
2.5 × 106 CFU mL-1; Fish injected with bacteria A. lwoffii 1.7 × 105 CFU mL-1.

DISCUSSION

Molecular analysis of bacteria

The results of electrophoresis presented in Fig. (1) show bands at the 1500bp
marker. Each sample exhibits only one band, indicating the presence of a single type of
DNA fragment molecule from bacterial isolates one and two, without any contaminants.
This allows progression to the next stage, sequencing. Based on sequencing results
matched with the NCBI GenBank database, isolate one was identified as Vibrio
navarrensis bacteria with 100% similarity, and isolate two as Acinetobacter lwoffii
bacteria, also with 100% similarity.
V. navarrensis is a gram-negative bacterium first discovered in 1982 from sewage
water in Spain. Gladney & Tarr (2014) characterized V. navarrensis isolates from
various sources, including blood, revealing its potential to become a human pathogen.
Genetically, V. navarrensis shares phenotypic and genotypic similarities with Vibrio
vulnificus and is considered an opportunistic bacterium with a 50% mortality rate
associated with symptoms of septicemia (Thompson et al., 2005; Gladney & Tar, 2014;
Ayuzar et al., 2023) mentioned that vibriosis infections can lead to mortality rates of 80-
90% in barramundi. Gladney et al., (2014) stated that V. navarrensis has the capability to
thrive across a wide range of salinities. This is evidenced by its presence in freshwater,
brackish water, and marine environments. Furthermore, isolations from humans and
 1437
Pathogenicity Testing of Bacterial Disease in Lates calcarifer in North Aceh

household settings indicate that V. navarrensis inhabits a broad ecological niche,

demonstrating extensive genetic diversity within the species.
Similar to V. navarrensis, Acinetobacter bacteria are also Gram-negative bacteria
commonly found in various aquatic environments from freshwater to seawater, soil, and
even in the air, capable of infecting diverse hosts (Wang et al., 2020). Acinetobacter spp.
are not pathogenic; however, they are known to cause nosocomial infections in
immunocompromised ones (Baumann 1968; Berlau et al., 1999; González et al., 2000).
In recent years, there have been several reports of fish infected by Acinetobacter. For
instance, Ictalurus punctatus and Channa striatus have been infected by A. baumannii
(Lu et al., 2008; Rauta et al., 2011), and the rainbow trout (Oncorhynchus mykiss) has
been infected by Acinetobacter johnsonii (Kozińska et al., 2014; Bi et al., 2023).
Recently, Ledesma et al. (2017) postulated that A. lwoffii can cause respiratory infections
in the mammal Lama glama and postencephalitic hydrocephalus with blindness in dogs
(Kim et al., 2017). It has also been linked to esophagitis in the common carp (Cyprinus
carpio) (Kozińska et al., 2014), exophthalmos in the rainbow trout (Oncorhynchus
mykiss) (Dadar et al., 2016), gill disease in the African catfish (Clarias gariepinus)
(Elsayyad et al., 2010), and diseases in Schizothorax (Cao et al., 2018). To our
knowledge, this is the first reported case of V. navarrensis and A. lwoffii infection in
barramundi.

Observation of clinical symptoms

The clinical symptoms observed after the LD50 test across all treatments were
nearly identical. These included unresponsiveness to feed, abnormal movement
behaviors, passivity, darkening of body color, rough body surface, frayed fins, yellowish-
white eyes, bloated abdomen, and bleeding in the organs of the barramundi. These
symptoms are consistent with those of fish affected by bacterial infections or bacterial
diseases. This finding aligns with the research conducted by Napitupulu (2016),
indicating that fish infected by bacteria exhibit clinical symptoms such as lesions on the
body surface, detached scales, abnormal changes (lesions) on the skin or fins, muscle
tissue damage, and damage to internal organs.
Fish injected with bacteria exhibited body color changes to a darker shade and
developed lesions on their bodies. This is caused by excessive mucus production, leading
to a rough body texture and reduced pigmentation. Additionally, the bacteria damage the
fish's body surface and tissue or outer skin layer, resulting in skin decay and ultimately
causing lesions. This observation is consistent with Subachri et al. (2011) research
indicating that the darkening of fish body color is caused by bacteria, viruses, or parasites
disrupting the fish's immune system and affecting mucus production. As a physiological
response, fish infected by bacteria, viruses, or parasites will initially produce excessive
mucus, followed by a drastic reduction in mucus production. This results in a rough skin
texture and the darkening of the body color.
1438 Ayuzar et al. , 2024

The subsequent symptoms exhibited by fish injected with bacteria include a rough
skin surface, frayed fins, and eyes that turn yellowish-white. The rough skin is caused by
excessive mucus production in response to bacterial infection. Frayed fins (Fig. 2a) result
from bacteria attacking and adhering to the tissue or cells in the fin area. The yellowish-
white eyes or exophthalmia (Fig. 2b) are due to cell damage caused by bacterial activity.
Hastari et al., (2014) noted that the clinical symptoms in fish infected with bacteria are
caused by inhibited cell formation, leading fish to respond by producing excess mucus.
Lesions result from bacteria eroding cells and tissues and changes in eye color are due to
cell damage, ultimately causing exophthalmia. This is supported by the study of Cao et
al., (2018), who reported that fish infected with A. lwoffi bacteria exhibit clinical
symptoms such as lesions on the body surface, especially the dorsal region, and
exophthalmia in the eyes.
In contrast to the six treatments, the clinical symptoms in the control group
remained unchanged before and after the LD50 test. This is because the control fish were
only injected with PBS (Phosphate Buffered Saline), a solution with ion conditions
similar to those within the body, thus not causing any effects or clinical symptoms
indicative of bacterial infection. Luo (2020) stated that PBS has good buffering capacity
and can maintain osmotic pressure since it contains salts and amino acids. PBS functions
to regulate pH and osmolarity balance by providing essential water and organic ions.

LD50
Wahjuningrum (2014) stated that the virulence of bacteria is influenced by the
production of enzymes and toxins from the bacterial cells themselves. Differences in
LD50 values are also attributed to variations in bacterial serotypes and biotypes.
Furthermore, the differences in enzyme and toxin production due to the use of different
isolates, may also play a role. A low LD50 value further demonstrates that these bacteria
have the capability to cause infections in host cells, leading to subsequent death. This is
supported by Mangunwardoyo et al., (2016), who stated that death at low LD50 values
is due to high virulence and pathogenicity, as the bacteria possess the ability to transmit,
adhere to host cells, invade host tissues, and ultimately cause infections that lead to death.
Priyadi et al., (2010) further added that, generally, the smaller the LD50 value, the more
toxic the compound or substance. Conversely, the larger the LD50 value, the lower the
toxicity.

Mortality rate
The treatments with the highest mortality rates were Treatment A and D, with an
average of 100%, followed by Treatment B with an average of 75.56%, Treatment E with
an average of 57.77%, Treatment C with an average of 48.89%, and Treatment F with an
average of 42.22%. The control treatment (K) showed a mortality rate of 0%. The
mortality rates are illustrated in Fig. (2). Additionally, treatment C and F showed lower
mortality rates compared to the other 4 treatments. The decrease in bacterial density is
 1439
Pathogenicity Testing of Bacterial Disease in Lates calcarifer in North Aceh

one of the factors contributing to the lower mortality rates in treatments C and F
compared to the others. This indicates that the reduction in the injected bacterial
concentration affects the percentage of deaths. This is consistent with the work of
Susanti (2016), who stated that increasing bacterial concentration by 10 times increases
the percentage of fish deaths, and conversely, decreasing bacterial concentration by 10
times decreases the percentage of fish deaths. The mortality rate in the control treatment
was 0%, indicating that no fish died among those injected with PBS. The absence of
mortality is due to the fact that the test fish were only injected with PBS, without
introduction of any bacteria or foreign pathogens into their bodies. This ensured that there
was no disruption to the fish's immune system or health, maintaining the control fish in a
healthy state throughout the experiment.
Cao et al., (2018) stated that A. lwoffi infection with a density of 107 CFU mL-1
resulted in 100% death in the Schizothorax fish genus. While in this study, A. lwoffi
infection can kill 100% of barramundi with a density of only 106 CFU mL-1. This is
thought to be related to differences in fish species and differences in the environment,
where the bacteria are located.

Mean Time of Death (MTD)

The treatment with the shortest MTD was treatment A, with an average time of
66.40 hours. This was followed by treatment D at 69 hours; treatment B at 69.55 hours;
treatment C at 76.36 hours; treatment E at 79.80 hours; and treatment F, which had the
longest average time to death at 87.31 hours. The control treatment did not exhibit any
death throughout the maintenance period. Statistical analysis showed that V. navarrensis
bacterial infection of 106 CFU mL-1, 105 CFU mL-1, and A. lwoffii bacterial infection with
a density of 107 CFU mL-1 produced the same MTD. These three treatments had the
fastest MTD value among the other treatments, namely 66.40 - 69.55 hours. This is in
accordance with the finding of Sivasankar et al., (2015) postulating that vibriosis
infection can kill fish populations within 1-3 days.

Blood Profile
Blood profile parameters have been used as health indicators in fish (Sayed &
Moneeb, 2015; Nugrahawati et al., 2019). One of the blood parameters used to indicate
the health condition of fish is the total erythrocyte count. The total erythrocyte count in
the six treatments was significantly different (P< 0.05) compared to the control treatment.
At all observation times, the total erythrocyte count in the control treatment was higher
than in the six treatments (Fig. 4). The decrease in erythrocyte count is believed to be
caused by the hemolytic properties of the bacteria and Acinetobacter lwoffii (Kozińska et
al., 2014; Cao et al., 2018) and Vibrio navarrensis (Schwartz et al., 2017) which can
break down the red blood cells.
1440 Ayuzar et al. , 2024

CONCLUSION

The bacteria that attack the white sea bass in North Aceh are V. navarrensis and A.
lwoffi. The bacterial infections by V. navarrensis and A. lwoffi can effectively infect and
induce bacterial diseases in barramundi, as evidenced by clinical symptoms, high
mortality rates, relatively short average time to death, and distinct blood profiles
compared to the control group. A series of pathogenicity tests confirmed that these two
bacteria are responsible for causing bacterial disease in barramundi culture in North
Aceh.

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