microalgae for the remedy of parasitic and bacterial ... · the french associates institute for...

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: jihyunkim1129@gmail.com; dzilberg@bgu.ac.il

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: