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The LD50 of Asian Catfish (Pangasius

bocourti, Sauvage 1870) challenge to
pathogen Aeromonas hydrophila
FW52 strain
Hien Van Doan, Amnuaysilpa Suksri

La Pensée

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Pensee Journal Vol 75, No. 10;Oct 2013

The LD50 of Asian Catfish (Pangasius bocourti, Sauvage 1870)

challenge to pathogen Aeromonas hydrophila FW52 strain
Hien Van Doan
Department of Aquaculture, Faculty of Agriculture
Quangbinh University, Quangbinh 45000, Vietnam
Tel: +66-90-029-9995 Email: hienqbuni@gmail.com

Sompong DOOLGINDACHABAPORN (Corresponding author)

Department of Fisheries, Faculty of Agriculture
Khon Kaen University, Khon Kaen 40002, Thailand
Tel: +66 -85-004-5090 Email: sompng_d@kku.ac.th

Amnuaysilpa SUKSRI
Department of Plant sciences and Natural Resources
Faculty of Agriculture, Khon Kaen University
Khon Kaen 40002, Thailand
Tel: +66-81-047-7514 Email: amnuay_s@kku.ac.th

The research is financed by Khon Kaen University under a KKU Scholarship for ASEAN and GMS
Countries

Abstract
This study aims to determine the lethal dose (96-h LD50) of the bacteria Aeromonas hydrophila of Asian
Catfish (Pangasius bocourti), which will be used in challenge tests. One hundred and sixty two fish
(weight of 8.65 ± 0.11g fish-1) were divided into nine treatments, with the use of different bacterial
concentrations: T0 - control (0.85% saline solution); T1 (1x105 cfu ml-1); T2 (1x106 cfu ml-1); T3 (5x106 cfu
ml-1); T4 (1x107 cfu ml-1); T5 (5x107 cfu ml-1); T6 (1x108 cfu ml-1); T7 (5x108 cfu ml-1), and T8 (1x109 cfu
ml-1). Fish were intraperitoneally injected with 0.1 ml of bacterial suspensions and distributed in 27-
aquarium with 100 litres of water in each. The water variables were monitored and fish mortality was
observed. A Completely Randomized Design (CRD) with three replications was applied and the 96-h
LD50 was estimated according to the trimmed Spearman-Karber method. Fish mortality rate increased
with the increase of bacterial concentrations of A. hydrophila (T0 = 0%; T1 = 0%; T2= 0%; T3 = 11.1%, T4
= 33.3%; T5 = 66.7% T6 = 88.9%; and T7, T8 = 100%) after 96h of injection and the first mortalities were
observed after 24 hours (h), although the signs of the bacterial infection were already observed 12h after
the injection. The results indicated that the 96-h LD50 value of A. hydrophila was 2.24 x 108 cfu ml-1.
Keywords: Aeromonas hydrophila, 96-h LD50, Pangasius bocourti
1. Introduction
Asian catfish (Pangasius bocourti) is one species of the Pangasiidae family (Orban et al., 2008). This
fish has been recently interested in culturing along the Mekong river region as a new economic species.
The frozen fillets of its not only supply for local consumption but also exporting to international market
(Thammapat et al., 2010). The demand of fish products have been rapidly increasing (Anonymous, 2005)
because of its desirable qualities such as white flesh and low-fat content (Orban et al., 2008) as well as
easily digestible protein (Økland et al., 2005). It was found that this species gave the fastest growth being
recorded in many aquaculture sectors (Phuong and Oanh, 2009). This fish is well adapted species, but
very sensitive to handle and transport stress condition. As a result of utmost activity, scare and mucus

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losses are observed, facilitating the occurrence of diseases which can cause major losses to the fish
culture.
The study of reducing stress effect has been conducting on some fish species. The application of this,
however, can only minimize the stress responses during the stressor agent and consequently the mortality
(Oliveira et al., 2011). Therefore, more researches in order to solve the adverse effects of the stress
responses after stressor condition is necessary. Handling technique is one of the alternatives techniques
have been recently developed to prevent fish diseases and improve immunological resistance in intensive
culture conditions (Chakrabarti and Rao, 2006). Challenge tests have been developed to evaluate the
efficiency and security of new aquaculture products with the use of pathogenically bacteria as a stressor
agent, because of the simple techniques and high quality results. These tests were carried out for
controlling the organism responses to an infectious agent or even in preventing fish mortality (Sahu et al.,
2007).
Aeromonas hydrophila is known as dominant pathogens which cause ulcers on fish and is often
associated with tail and fin decay, haemorrhagic septicaemia, and epizootic ulcerative syndrome. This
pathogen also causes significant economic losses to the important fish farming industry Tellez-Bañuelos
et al. (2010). High water temperature and elevated organic matter, in association with other stressor
factors viz. high stocking densities, traumas due to inappropriate handling, hypoxia, nutritional
deficiencies and infections can cause out-break of disease. Using these bacteria as an efficiency indicator
for new products that can minimize stress in intensive fish culture systems can contribute to the
improvement its aquaculture in the Mekong region. Therefore, the objective of the present work is to
determine the tolerance limit (96-h LD50) of the bacteria Aeromonas hydrophila to Pangasius bocourti,
to be applied in challenge tests.
2. Materials and methods
2.1 Bacterial strain and culture conditions
Aeromonas hydrophila strain FW52 was obtained from Department of Microbiology, Faculty of Science,
Khon Kaen University. They were isolated from diseased tilapia and β–haemolytic. Then it was grown in
BHI broth and incubation at 35oC for 18 hours. The cells were harvested by centrifugation at 5000 rpm
for 15 min at 4oC. The bacterial pellets was washed and resuspended in a normal saline solution (NSS)
0.85% NaCl.
2.2 Bacterial standard curve
In order to prepare standard cure, the cells suspension were diluted by normal saline at different
concentrations (0.01, 0.03, 0.05, 0.08, 0.1, 0.3, 0.5 and 0.8) total volume was adjusted to 5 ml. Then
Optical Density (OD600) was measured with the use of spectrophotometer. The numbers of cells were
identified with the use of spread plate count technique. Each concentration (range from 0.01 to 0.8) was
serially diluted with NSS into four concentrations (10-4, 10-5, 10-6, and 10-7). Then it was spread in double
petri dishes for bacterial count.
2.3 Experiment design
The experiment was carried out at the Department of Fisheries, Faculty of Agriculture, Khon Kaen
University during a period of 4 days (from January 1st to 4th 2013). The catfish fingerlings were obtained
from Payao Freshwater Fisheries Centre, Department of Fisheries, Ministry of Agriculture and
Cooperatives, Thailand. Fish were acclimatized to laboratory conditions for 1 month in a 1,000 litres
fibre-tank upon arrival. They were fed with a commercial catfish diet (inland production) where the
formula consisted of crude protein (40%), lipid (3%), fibre (3%), with moisture content of 12%. Prior to
experiment one hundred and sixty two juvenile fish (weighing of 8.65 ± 0.11g fish-1) were divided into
nine treatments. Each treatment was cultured of 18 fish in three replications. The fish were
intraperitoneally injected (0.1 ml) with the use of different A. hydrophila concentrations viz. T0= Control
(0.85% saline solution); T1 = 1x 105; T2 = 1 x 106; T3 = 5 x 106; T4 = 1 x 107; T5 = 5x107; T6= 1x108;
T7=5x108 and T8=1x109 cfu ml-1. Fish were fasted for 24h before the beginning of the experiment. For
inoculation, fish were previously fasted and anesthetized with the use of ice, inoculated in the
intraperitoneally cavity with 0.1 ml the different bacterial suspensions and then distributed in twenty
seven 100-litre test aquarium where aerator was used in a semi-static system. The experiment was carried

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out for 96 hours, in which changing in fish behaviour and swimming, physical attribute, and mortality
rates were recorded.
2.4 Water quality measurement
The water quality of each aquarium was daily measured at 9:00 AM to prevent any alteration that would
affect the results. Dissolved oxygen (DO) and temperature were measured with use of DO meter. The pH
was measured with the use of a pH-meter.
2.5 Statistical analysis
All data were subjected to one-way analysis of variance (ANOVA) and when the differences among
groups were identified, multiple comparisons in means were made with Duncan’s Multiple-Range Test
through the computer programme (SAS, 2003). The 96-h LD50 (lethal dose to 50% of a population) was
estimated according to the formulation described by the trimmed Spearman-Karber method (Hamilton et
al. (1977)
3. Results
3.1 Water quality
Water quality parameters in each aquarium during the experimental period are presented in the Tables 1,
2 and 3. The DO, pH and temperature did not show any statistical significantly differences throughout the
experimental period.
3.2 Fish mortality and lethal dose (LD50)
The result revealed that first mortality was observed after injected 36 hours. However, the symptoms of
bacterial infection, such as fin erosion and scales losses, were observed at 12 hours after the inoculation.
The result also indicated that no mortalities were observed during 96-hour experiment for the treatments
T0 (control) and fish that injected with low concentrations (1x105 and 1x106 cfu ml-1) of A. hydrophila
temporarily changed of its fins appearance (los of scales and body ulcerations), behaviour and swimming,
but fish quickly recovered after 24 hours. The fish that injected with intermediate concentrations (5x106,
1x107, 5x107, 1x108 cfu ml-1) showed notable erratic swimming and several changes in physical
appearances such as haemorrages, fins erosion, superficial stains, ulcerative lesions, exophthalmia,
abdominal distension and mucus excess were observed. Fish signs of infection and death rate increased as
the increase of bacterial concentration. All injected fish died was observed in higher concentrations of
bacteria (5x108 and 1x109 cfu ml-1). The correlation between the various doses of A. hydrophila and the
mortality rate of juvenile Asian catfish exposed for 96 hours is shown in Table 4 and 5.
Using fish mortality data during the 96-h experiment, the LD50 was determined by the trimmed
Spearman-Karber method. The result showed that lethal dose of A. hydrophila to 50% of a population of
P. bocourti was 2.24 x 108 cfu ml-1l of saline solution.
4. Discussion
Water quality plays a very important role in aquaculture not only in the ponds system but also in
laboratory tanks systems. It is very necessary to observe and record water quality parameters in fish
aquarium during experimental period, because if the variation of any parameter can affect the results
(Andrade et al., 2006; Affonso et al., 2007). This is extremely important when determining the tolerance
of given species to stressor agents in order to assure reliable results, which dictate the animal tolerance
limits (Avilez et al., 2004). Rahman et al. (2001) carried the work to identify the effect of temperature on
Aeromonas hydrophila infection in goldfish, Carassius auratus. The result indicated that temperature
affects both the cell membrane structure of A. hydrophila and phagocytic activity of goldfish
macrophages, resulting in varying fish mortality when infected at different temperatures. Indifferent
experiment, studies involving Piaractus mesopotamicus indicate that water temperature seems to have an
influence on the mortality rates caused by A. hydrophila and septicemia outbreaks are associated with
highwater temperatures (Garcia et al., 2009). In the present work, most of water parameters were in
appropriate range, which considered being adequate for the development and health of Pangasius
bocourti (Jiwyam, 2010).
Aeromonas hydrophila is one pre-dominant pathogens affecting worldwide fish culture Tellez-Bañuelos
et al. (2010). It has been reported to causes mass mortalities in several species. (Pridgeon and Klesius,
2011) have reported that A. hydrophila appears to have emerged as a primary pathogen, which causes the

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loss of more than 3 million pounds of channel catfish (Ictalurus punctatus) in 2009 alone. The outbreak
of disease also happened to carp (Cyprinus carpio L., Labeo rohita Ham.), snakehead gourami (Channa
striatus Bloch or Channa punctatus Bloch) and catfish (Clarias gariepinus Burchell or Clarias batrachus
L.), and is the aetiological agent of several diseases including haemorrhagic septicaemia, asymptomatic
septicaemia, ulcerative infections, tail rot, and fin rot (Rahman et al., 2001). In present study, not only the
ulcerative lesions on the body but also mucus excess were observed.
The symptoms causes by infection of A. hydrophila beside clinical signs were described in this study; the
studies with different symptoms also have been reported in different fish species. The study of Angka et
al. (1995) with the use of walking catfish has indicated that the infection of A. hydrophila can also cause
necrotic hemorrhaging in internal organs, mainly kidneys and liver and fluid deposition in the abdominal
cavity. Garcia et al. (2009) with the use of pacu when challenged with this bacterium have indicated that
distended abdominal cavity with clear ascites content.
The effects of A. hydrophila on fish seems likely vary between species due to the different of its
resistance to the infection, and immune system. Furthermore, it could possibly depend on bacterial
virulence, the size of animal, water quality, way of infection and many others experimental conditions. In
present study the results indicated that first mortality was observed after injected 36 hours and the LD50
of A. hydrophila to 50% of a population of P. bocourti was 2.24 x 108 cfu ml-1 of saline solution. The
results were different when compare to the study carried out by Andrade et al. (2006) with the use of 55 g
matrinxa (Brycon amazonicus). The results have indicated that the LD50 of A. hydrophila to fish was a
4.6 x 1011 cfu ml-1. These inferior values, when compared to the results found in these experiments, can
suggest that the lethal dose can also vary according to the animal size. In contrast, Oliveira et al. (2011)
carried out experiment with the use of the fish matrinxa (Brycon amazonicus) initial weight 63.23 g
indicated that the lethal concentration for 50% of the population, in 96 hours, of 6.66 x 1011 cells ml-1 of
saline solution. The results different between two experiments could possibly be the fish size. The bigger
fish may have the higher tolerance to pathogens. Bharadwaj et al. (2013) carried out experiment with the
use of Labeo rohita when challenged to difference strains of A. hydrophila. The results indicated that the
A. hydrophila –C36 strain was found to be virulent as the challenged fish at 2.2x109, 2.2x108 and 2.2x107
cells fish-1 recorded 100% mortality within 24h of challenge and first mortality recorded after 3h of
challenge at 2.2x109 cells fish-1. There was no mortality at 2.2x106 cells fish-1 up to 7 days of post-
challenge. The LD50 value for A. hydrophila-C36 strain was calculated to be 5.71×106 cells fish-1. The
work carried out by Pridgeon et al. (2013) in which the channel catfish were exposed to different strains
of A. hydrophila viz. 2010 isolates and AL98-CB1. The result indicated that the LD50 of 2010 isolates
(1.3 x 105 cfu fish-1) to channel catfish was about 200 times more virulent compare to AL98-C1B
(2.8x107 cfu fish-1).
5. Conclusion
According to the analysed result from present study, it is suggested that Pangasius bocourti presents high
tolerance to A. hydrophila being the lethal concentration for 50% of the population in 96 hours was 2.24 x
108 cfu ml-1 of saline solution.
Acknowledgments
The author would like to express his deepest and sincere gratitude to his advisor Associated Professor Dr.
Sompong Doolgindachbaporn for his kindness in supervising research experiments. Without whom this
work would not have been possible. Professor Dr. Amnuaysilpa Suksri, Department of Plant sciences and
Natural Resources, Faculty of Agriculture and Assoct. Prof. Saowanit Toncpim, Depatment of
Micobiology, Faculty of Science, Khon Kaen University for their kindness, advices, and moral support
during this study was carried out. This work would not have been possible without help from Ratchanu
Meidong, Ph.D student, Microbiology Department, Faculty of Science, Khon Kaen University.
6. References
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Table 1. pH during 96h of intraperitoneally inoculation of different A. hydrophila concentrations1

pH
Treatments
0h 24h 48h 72h 96h
T0 (control) 7.47 ± 0.03a 7.63 ± 0.09 a 7.47 ± 0.05 a 7.83 ± 0.05 a 7.77 ± 0.04 a
T1 (1x105) 7.52 ± 0.04 a 7.54 ± 0.07 a 7.44 ± 0.06 a 7.78 ± 0.10 a 7.77 ± 0.04 a
6 a
T2 (1x10 ) 7.56 ± 0.05 7.59 ± 0.03 a 7.56 ± 0.02 a 7.80 ± 0.05 a
7.81 ± 0.02 a
6 a
T3 (5x10 ) 7.53 ± 0.03 7.65 ± 0.03 a 7.55 ± 0.05 a 7.71 ± 0.09 a
7.76 ± 0.03 a
7 a
T4 (1x10 ) 7.49 ± 0.02 7.59 ± 0.01 a 7.48 ± 0.03 a 7.81 ± 0.03 a
7.76 ± 0.01 a
7 a
T5 (5x10 ) 7.49 ± 0.04 7.67 ± 0.02 a 7.54 ± 0.03 a 7.89 ± 0.02 a
7.86 ± 0.01 a
8 a
T6 (1x10 ) 7.55 ± 0.03 7.61 ± 0.08 a 7.53 ± 0.03 a 7.81 ± 0.10 a
7.75 ± 0.06 a
8 a
T7 (5x10 ) 7.46 ± 0.00 7.65 ± 0.14 a 7.48 ± 0.03 a 7.91 ± 0.08 a
7.85 ± 0.01 a
9 a
T8 (1x10 ) 7.49± 0.02 7.58 ± 0.10 a 7.53 ± 0.04 a 7.90 ± 0.03 a
7.84 ± 0.03 a
1
Values are mean ± standard error of triplicate groups. Means in the same column with different
superscripts are significantly different (P < 0.05).

Table 2. DO during 96h of intraperitoneally inoculation of different A. hydrophila concentrations2

DO (Dissolved Oxygen)
Treatments
0h 24h 48h 72h 96h
T0 (control) 6.30 ± 0.10 a 6.26 ± 0.14 a 6.02 ± 0.36 a 6.16 ± 0.25 a 5.97 ± 0.24 a
T1 (1x105) 6.15 ± 0.13 a 6.01 ± 0.04 a 5.79 ± 0.26 a 6.05 ± 0.19 a 6.15 ± 0.18 a
6 a a a a
T2 (1x10 ) 6.46 ± 0.12 6.52 ± 0.10 6.21 ± 0.08 6.31 ± 0.12 6.23 ± 0.10 a
6 a a a a
T3 (5x10 ) 6.49 ± 0.14 6.59 ± 0.26 6.42 ± 0.16 6.26 ± 0.29 5.82 ± 0.41 a
7 a a a a
T4 (1x10 ) 6.53 ± 0.07 6.25 ± 0.30 6.13 ± 0.23 6.13 ± 0.21 6.14 ± 0.25 a
7 a a a a
T5 (5x10 ) 6.47 ± 0.05 6.41 ± 0.23 5.85 ± 0.26 6.19 ± 0.36 6.23 ± 0.27 a
8 a a a a
T6 (1x10 ) 6.52 ± 0.20 6.24 ± 0.41 6.24 ± 0.25 6.16 ± 0.37 5.87 ± 0.29 a
8 a a a a
T7 (5x10 ) 6.26 ± 0.19 6.12 ± 0.04 6.26 ± 0.12 6.02 ± 0.14 5.60 ± 0.05 a
9 a a a a
T8 (1x10 ) 6.27 ± 0.36 6.15 ± 0.04 5.98 ± 0.48 5.68 ± 0.24 6.03 ± 0.06 a
2
Values are mean ± standard error of triplicate groups. Means in the same column with different
superscripts are significantly different (P < 0.05).

Table 3. Temperature during 96h of intraperitoneally inoculation of different A. hydrophila

concentrations3
Temperature
Treatments
0h 24h 48h 72h 96h
T0 (control) 26.73 ± 0.09 a 26.87 ± 0.03 a 27.26 ± 0.09 a 27.13 ± 0.03 a 27.33 ± 0.03 a
T1 (1x105) 26.63 ± 0.09 a 26.83 ± 0.07 a 27.13 ± 0.03 a 27.13 ± 0.03 a 27.23 ± 0.09 a
6 a
T2 (1x10 ) 26.57 ± 0.07 26.73 ± 0.03 a 27.13 ± 0.03 a 27.13 ± 0.03 a
27.23 ± 0.07 a
6 a
T3 (5x10 ) 26.60 ± 0.06 26.83 ± 0.03 a 27.07 ± 0.09 a 27.20 ± 0.06 a
27.17 ± 0.03 a
7 a
T4 (1x10 ) 26.67 ± 0.19 26.83 ± 0.03 a 27.23 ± 0.12 a 27.13 ± 0.03 a
27.17 ± 0.03 a
7 a
T5 (5x10 ) 26.60 ± 0.06 26.83 ± 0.07 a 27.27 ± 0.07 a 27.13 ± 0.03 a
27.16 ± 0.03 a
8 a
T6 (1x10 ) 26.63 ± 0.09 26.83 ± 0.03 a 27.27 ± 0.03 a 27.13 ± 0.03 a
27.20 ± 0.06 a
8 a
T7 (5x10 ) 26.76 ± 0.03 26.80 ± 0.06 a 27.20 ± 0.06 a 27.13 ± 0.03 a
27.30 ± 0.06 a
9 a
T8 (1x10 ) 26.73 ± 0.12 26.80 ± 0.06 a 27.13 ± 0.03 a 27.12 ± 0.03 a
27.20 ± 0.06 a
3
Values are mean ± standard error of triplicate groups. Means in the same column with different
superscripts are significantly different (P < 0.05).
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Table 4. Lethal dose 50 (LD50) of A. hydrophila to juvenile Asian catfish and correlation between dose
of A. hydrophila (cfu ml-1) and mortality rate during 96h
Treatments Percent of dead Pangasius bocourti
24h 36h 48h 72h 96h
T0 (Saline 0.85%) 0 0 0 0 0
T1 (1x105) 0 0 0 0 0
T2 (1x106) 0 0 0 0 0
T3 (5x106) 0 0 11.11 11.11 11.11
T4 (1x107) 0 0 22.22 33.33 33.33
T5 (5x107) 0 55.55 61.11 66.67 66.67
T6 (1x108) 0 61.11 77.77 83.33 88.88
T7 (5x108) 0 55.55 100 100 100
T8 (1x109) 0 83.33 94.44 100 100

Table 5. Lethal dose 50 (LD50) of A. hydrophila to juvenile Asian catfish and correlation between dose
of A. hydrophila (cfu ml-1) and mortality rate during 96h
Treatments No. of dead Pangasius bocourti
24h 36h 48h 72h 96h
T0 (Saline 0.85%) 0 0 0 0 0
T1 (1x105) 0 0 0 0 0
T2 (1x106) 0 0 0 0 0
T3 (5x106) 0 0 2 2 2
T4 (1x107) 0 0 4 6 6
T5 (5x107) 0 10 11 12 12
T6 (1x108) 0 11 14 15 16
T7 (5x108) 0 10 18 18 18
T8 (1x109) 0 15 17 18 18

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