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AbstractThe potential of Phaeodactylum tricornutu (a diatom) as a treatment against monogenean and bacterial infections of fish was studied. A monogenean parasite,Gyrodactylus turnbulli, and two bacterial pathogens affecting fish, Streptococcus iniae and Vibrio harveyi were used. The microalgae was cultured atdifferent conditions and extracts were prepared using different solvents, aiming to optimize the conditions for effective therapeutic preparation.
Extracts were screened in vitro against G. turnbulli followed by in vivo immersion treatment against G. turnbulli-infected guppies. In vitro exposure to 5 pptof ethanolic P. tricornutum extract killed 97% of the parasites within 4 hours. Fish were effectively treated by immersion in 2.5 ppt over 24 hours, showingsignificant reduction of both infection prevalence and intensity to almost complete clearance of the parasites.
Anti-bacterial effect against S. iniae and V. harveyi was analysed by the disc diffusion assay. Significant antibacterial effect was confirmed in vitro.Ethanolic extract from 2 weeks-cultured microalgae showed 28 and 14 mm of growth inhibition against S. iniae and V. harveyi, respectively.
The French Associates Institute for Dryland Agriculture and Biotechnology, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Midreshet Ben-Gurion, 8499000, Israel. Email: [email protected]; [email protected]
J. H. Kim, Inna Khozin-Goldberg and Dina Zilberg
Microalgae for the remedy of parasitic and bacterial infections in fish: a substitute to conventional chemical treatments
IntroductionDisease outbreaks cause substantial losses in aquaculture. Traditional chemical and antibiotic treatments pose adverse effects onhuman health and the environment and there is therefore a growing trend and need to develop natural therapeutants foraquaculture. Phaeodactylum tricornutum is a microalga (of the diatoms) with proven anti-bacterial effect.
Our aim was to test the potential of P. tricornutum as a treatment against monogenean and bacterial infections of fish.
ConclusionPhaeodactylum tricornutum-based preparations were confirmed to be affective against a parasite and bacteria affecting fish and can be potentially used todevelop a natural therapeutant for aquaculture. Identification of the active ingredient in the extract is underway.
Further research will be directed to evaluate anti-bacterial treatments in vivo, using infected fish. The application of this preparation against other parasitesof significance to aquaculture should be explored. Finally, a cost-effective production of the extract needs to be developed.
Materials & Methods
2. Antibacterial activity test (disc diffusion assay)
1 week growth
2 weeks growth
Breakage, freeze-drying and extraction with ethanol
S. Iniae on TSA agar V. harveyi on MH agar
Disks with extracts
Measure diameter of growth inhibition
Incubation at 26.5° for 24 h
1. Antiparasitic activity test
Gyrodactylus
turnbulii
Recording death and detachment over time
Breakage by bead beating
Recording no. of parasites on the tail fin before and after treatment
Results
1. Antiparasitic effect of microalgal extracts
Time (mins)
0 60 120 180 240
Mort
alit
y (%
)
0
20
40
60
80
100 S-K1-EtOH
K1-EtOH
S-EtOH
EtOH
Control (S-K1-EtOH)
c
bc
b
b
a
In vitro (tail fin clips)
Treatments
Num
ber
para
site
s pe
r fis
h
0
2
4
6
8
10
12
14
16
0 hour
24 hours
median
mean
100%(1.86)
86%(2.17)
100%(1.93)
93%(2.92)
100%(3.79)
29%(4)
100%(3.71)
0%(0)
b
ab
a
a
*
*
In vivo
Contol (FW) Contol (S-K1-ethanol) S-K1-EtOH 1.25 S-K1-EtOH 2.5
3. Attempting to identify the active compound in the algal extract:
Time (mins)
0 60 120 180 240
Mo
rta
lity
(%)
0
20
40
60
80
100
Cont. EtOH
SCC F34
F34-1a F34-1b
F34-2 F34-3 F34-4 F34-5 F34-6+7 F34-8 F34-9 F34-10
*
*
Time (mins)
0 60 120 180 240
Mort
ality
(%
)
0
20
40
60
80
100
Cont. EtOH
F1
F2
F3
F4
F5
F6 †
*
TLC (thin layer chromatography)Fractionation by SCC (Silica cartridge chromatography)
Minutes
9 10 11 12 13 14 15 16 17 18 19
Volts
-0.10
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
16
:0
16
:1
17
:0
16
:3
18
:01
8:1
w9
18
:1w
7
EP
A
24
:1
TRACE GC-PTV FID
AF34 NL 1b
Name
2. Antibacterial effect of microalgal extracts
V. harveyi (MH agar )
Treatments
Cont. DDW
Cont. DMSO1 week
2 weeks
Inhi
bito
ry d
iam
eter
(m
m)
6
11
16
21
26
31 Streptococcus iniae Vibrio harveyi
*
* †
S. Iniae (TSA agar )
GC analysis of active fraction 1b: