ISOLATION OF STAPHYLOCOCCUS SP. IN CAGE–CULTURED

ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–
CULTURED TILAPIA OF DIFFERENT WATER BODIES
By
Siti-Zahrah A., Shahidan H., Wan Norazlan G., M-Noor Amal A.,Nur-Nazifah M.,& Misri S1.
National Fish Health Research Centre(NaFisH),
Fisheries Research Institute(FRI),
11960 Batu Maung, Penang, Malaysia
PPBUK, Pulau Sayak, Kuala Muda, Kedah
NaFIS 2008
Simposium Perikanan Kebangsaan
14-16 Julai 2008
Wisma Darul Iman, Kuala Terengganu
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
INTRODUCTION



Staphylococcus sp. infection in fish has been
occasionally reported but not too common
In Japan, reported in red sea bream and yellowtail
(Kusuda & Sugiyama,1981) causing exophthalmus
and skin ulcers by Staph. epidermidis
In India,reported in silver carp (Shah & Tyagi,
1986)causing corneal damage progressing to
phthisis bulbi by Staph. aureus
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
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ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
INTRODUCTION




In Greece, sea bream and sea bass (Dicentrarchus
labrax) were affected causing external skin and fin
lesions by genera of Staphylococcus and
Micrococcus.
In Malaysia, no reported cases so far and this could
be the first report of fish infected by Staphylococcus
sp.
This is found in red tilapia that was sampled from 5
different water bodies that is, reservoirs, river, exmining pool, canal and pond.
Staphylococcus is persistently isolated during the
course of tilapia sampling for pathogen,
Streptococcus sp.
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
2
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
OBJECTIVES
As such, the study thus also aims to:



To determine the species of Staphylococcus and
most dominant infecting red tilapia cage culture
To determine the infection rate of Staphylococcus in
study sites from different water bodies and
To determine its possible role and significance in
red tilapia cage-culture
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
3
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
MATERIALS & METHOD
Tilapia sampling(25-30) at study sites
from Oct. 2006 to Dec.2007
Post-mortem / Necropsy ‘in-situ’
Primary isolations from organs (brain, eye, kidney)
Subculture to obtain pure colony in BA
Morphology & Color
Presumptive test (Gram staining & catalase)
Bacterial ID using API STAPH
Data recording & analysis
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
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ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
SAMPLING SITES : MADA CANAL
Sg. Kodiang
Sg. Arau
Sg. Tunjang
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
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ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
SAMPLING SITES : POND & EX-MINING POOL
PPA JITRA, KEDAH
PANTAI KAMLOON, PENANG
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
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ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
SAMPLING SITES : RIVER – SG TERENGGANU
B. SELAT, B. KEPONG, P. ALI AND K. KEJIR
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
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ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
SAMPLING SITES : RESERVOIRS
PEDU LAKE, KEDAH
KENYIR LAKE,TERENGGANU
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
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ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
RESULTS & DISCUSSION
Table 1. The percentage of most common isolated Staphylococcus
spp. from red tilapia including species already reported in fish
Type of Staph spp.
Percentage %
Micrococcus spp.
15
Kocuria varians
12.6
S. xylosus
12.1
S. sciuri
12.1
S. aureus
7
S. warneri
3.4
S. epidermidis
2.4
S. haemolitycus
1.5
S. chromogenes
0.6
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
9
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
RESULTS & DISCUSSION
Most common isolated species from the study sites
Percentage of most common isolated Staphyloccoccus spp. and
pathogenic species
١٦
Percentage %
١٤
١٢
١٠
٨
٦
٤
٢
og
en
es
hr
om
us
S.
c
ol
ity
c
id
is
S.
ha
em
pi
de
r
m
i
S.
e
ar
ne
r
S.
w
us
S.
au
re
ci
ur
i
S.
s
S.
xy
lo
su
s
va
ria
ns
Ko
cu
ria
M
ic
r
oc
oc
c
us
s
pp
.
٠
Type of species
• More than 10 species of Staphylococcus isolated from all the sites
• Micrococcus spp, Koc. varians, Staph.xylosus and Staph. sciuri are the most dorminant
showing 15%, 13%, 12% and 12% respectively
• Staph. aureus, Staph. warneri, Staph. epidermidis, Staph. haemolyticus and Staph.
chromogens are the species already reported to infect fish
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
10
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
RESULTS & DISCUSSION
Morphological characteristics of pure Staphylococcus colony on BA
•Whitish
opaque
(offwhite) in
colour
•Round &
convex
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
11
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
RESULTS & DISCUSSION
BACTERIAL ID-API STAPH
Good ID for the dominant
isolates of Staphylococcus sp.
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
12
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
RESULTS & DISCUSSION
BACTERIAL ID-API STAPH
Good ID for the dominant
isolates of Staphylococcus sp.
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
13
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
RESULTS & DISCUSSION
Mean Infection rate(%) of Staphylococcus spp in tilapia from 2006 to 2007
from the study sites
Percentage mean of Staphylococcus spp. at study sites (٢٠٠٦٢٠٠٧)
١٤
percentage mean %
١٢
١٠
٨
٦
٤
٢
JA
NG
TU
N
JIT
RA
KE
NY
IR
TA
SI
K
K.
KE
JIR
KO
DI
AN
G
B.
KE
PO
NG
TA
SI
K
PE
DU
LO
N
AM
P.
K
P.
AL
I
B.
SE
LA
T
AR
AU
٠
Sampling site
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
14
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
RESULTS & DISCUSSION
Mean infection rate (%) of Staphylococcus spp. in tilapia from different water bodies(2006 & 2007)
Percentage mean infection of Staph spp. at reservoirs in ٢٠٠٦ to
٢٠٠٧
percentage mean %
١٤
١٢
١٠
٨
T. Kenyir
٦
T. Pedu
٤
٢
٠
٢٠٠٦
٢٠٠٧
percentage mean %
Percentage mean infection of Staph spp. at rivers in ٢٠٠٦ to
٢٠٠٧
١٨
١٦
١٤
١٢
١٠
٨
٦
٤
٢
٠
B.SELAT
Mean infection rate
between reservoir and
river ranges from
10-12 %
B. KEPONG
P. ALI
K. KEJIR
٢٠٠٦
٢٠٠٧
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
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ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
RESULTS & DISCUSSION
Percentage mean infection of Staph spp. at canals in ٢٠٠٦ to
٢٠٠٧
Mean infection rate in Arau is
consistent in both years
٣٠
percentage mean %
٢٥
٢٠
ARAU
١٥
KODIANG
١٠
TUNJANG
Percentage mean infection of Staph spp. at mining pool in ٢٠٠٦
to ٢٠٠٧
٥
٠
١٤
٢٠٠٧
١٢
percentage mean %
٢٠٠٦
Percentage mean infection of Staph spp. at pond in ٢٠٠٦ to
٢٠٠٧
١٠
٨
P. KAMLOON
٦
٤
٢
٠
٢٠٠٦
١٢
٢٠٠٧
percentage mean %
١٠
٨
٦
JITRA
٤
٢
٠
٢٠٠٦
٢٠٠٧
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
Mean infection rate from exmining pool is below 12%,while
pond is <10%
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ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
RESULTS & DISCUSSION
Gross morphological observations of sampled tilapia (sub-clinical)
External observation
Tilapia mostly looks
healthy without any
clinical symptoms
Internal organs
showing no obvious
abnormalities
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
Internal observation
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ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
RESULTS & DISCUSSION
Comparison of correlation analysis with or without Staphylococcus presence in relation
to Streptococcal infection and mortality in tilapia & water quality parameters in Tasik Pedu
Statistics 9.0
Correlations (Pearson)
DEAD
TEMP SULFIDE
SA
TEMP
0.5058
SULFIDE 0.4896 0.3551
SA
0.7140 0.5445 0.3994
PH
0.5533 0.8230 0.5789 0.5864
Cases Included 12 Missing Cases 0
Table.3 Correlations
analysis of Strep.
agalactiae infection &
mortality in tilapia
including water quality
parameters with
Staphylococcus infection
as weighting variable
Table.2 Correlations analysis of
Strep. agalactiae infection &
mortality in tilapia including water
quality parameters without
Staphylococcus.
Statistics 9.0
Correlations (Pearson)
Weighting Variable STAPH_PED
DEAD_PEDU TEMP_PEDU SULFIDE SA_PEDU
TEMP_PEDU 0.8325
SULFIDE
0.8502
0.7588
SA_PEDU
0.9312
0.7336
0.8239
PH
0.8985
0.8001
0.8970
0.8842
Cases Included 8 Missing Cases 112
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
18
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
SUMMARY & CONCLUSION
 More than 10 Staphylococcus species were identified
from sampled red tilapia in all the study sites
 Four common species isolated were Micrococcus sp,
Kocuria varians, S. xylosus and S. scuiri which
ranged from 15%, 12.6% and 12% for the last two
species.
 Except for Kocuria varians and S. scuiri, most of the
Staphylococcus sp. has been reported to infect fish
and causing disease problem
 Example S. aureus, has been reported in silver carp
in India (Shah & Tyagi, 1986)causing corneal
damage
 S. epidermidis is both reported in marine(sea
bream,Japan & Greece, yellowtail,Japan, Kusuda &
Sugiyama,1981) as well as fresh water fish(tilapia,
Taiwan,Shih-Ling Huang et al.,1999)
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
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ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
SUMMARY & CONCLUSION



S. haemolyticus(in scallop) causing severe disease
outbreak in China, (Peilin Zheng,et al.,2008)
S. chromogens, S. warneri,S. xylosus, S. capitis, S.
cohnii are some of the species that infect seabream
and sea bass in Greece causing dark body, fin and
skin necrosis to shallow ulcers. Exophthalmia,
progressing inflammation and necrosis of the eye are
not uncommon, including erratic swimming.
Similarly, in Iran cultured rainbow trout was affected
by the above S. chromogens, S. warneri,S.
epidermidis, and S. cohnii causing lethargy,
darkening, exophthalmia, hemorrhages around the
eye and base of skin. These Staphylococcus spp
were isolated especially in spring and summer
(Seyedeh,S.M., et al.,2008).
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
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ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
SUMMARY & CONCLUSION

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In Malaysia however, the sampled tilapia showed
mainly sub-clinical infection
Hence, tilapia look seemingly healthy showing no
clinical symptoms whatsoever, particularly those
cultured in ponds, and ex-mining pool.
This was surprising, even though the mean
Staphylococcus infection rate in the above water
bodies range between 8-12%
However, red tilapia in Arau were occasionally
observed to be pale & anemic, showing sunken eyes
at times and skinny, where highest mean infection
rate of Staphylococcus infection was recorded.
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
21
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
SUMMARY & CONCLUSION


Though grossly sampled tilapia did not show little or
no clinical signs especially in the pond and ex-mining
pool, Staphylococcus infection could play certain role
in causing Streptococcal infection in tilapia at other
sites, example reservoir
In Pedu for example, using Staphylococcus infection
rate as weighting variable in Pearson Correlation
analysis (Stats. Ver.9), it was shown that, there is
further improved strong correlation of Streptococcus
agalactiae infection to temperature, pH and sulphide
including mortality of tilapia compared to one without
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
22
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
SUMMARY & CONCLUSION

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This is possibly due to the presence of exotoxins
such as luekocidin (LukE/LukD) that enhance
spreading of infection and colonisation of pathogens
within the host, example in S. aureus (Adam Bownik,
2006).
His study showed that leukocidin inhibit Tand Blymphocyte proliferation as a result of cell lysis or
due to cytotoxic effect on the macrophages.
Others have shown that it also has
immunosuppressive influence in higher vertebrates
which is obviously quite similar effect in fish.
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
23
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
SUMMARY & CONCLUSION



Hence, Staphylococcus presence in red tilapia, made
it easily succumbed to colonization by Streptococcus
sp infection
Therefore, its presence is significant in contributing
serious Streptococcal infection in tilapia
This can be seen in reservoirs especially T. Pedu
and T. Kenyir where, besides other environmental
factors, Staphylococcus infection could possibly be
the physiological factor causing serious problem of
Streptococcal infection in red tilapia
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
24
ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
ACKNOWLEDGEMENT



Thank to all the support staffs of Bacteriology lab as
well as the RAs that has been working hard to ensure
that this project is successful.
Also sincere thanks to all the cage-cultured tilapia
farmers that has willingly cooperate with NaFisH to
help us in our research project.
Lastly, to all those that help us one way or another in
this project.
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
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ISOLATION OF STAPHYLOCOCCUS SPP. IN CAGE–CULTURED TILAPIA OF
DIFFERENT WATER BODIES
ACKNOWLEDGEMENT
THANK YOU
© 2008 National Fish Health Research Centre (NaFisH). All Rights Reserved.
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