MOLECULAR IDENTICATION AND CLONAL RELATION OF ATYPICAL ISOLATE Aeromonas salmonicida
USE METHOD OF RESTRICTION FRAGMENT LENGTH POLYMORPHISM ( RFLP)
PRIYATNA, R. 1.), INDARYULIANTO, S. 1.), KURNIASIH1.), AND
ISNANSETYO, A2.)
1.)
Faculty Of Animal Medicinine University of Gadjah Mada-Yogyakarta
2.)
Faculty Of Agriculture-Fishery Majors of University of Gadjah Mada-Yogyakarta
ABSTRACT
Aeromonas salmonicida represent bacterium cause of furunculosis at fish
resulting loss of economics in freshwater aquaculture. Counted 4 (four) A.
salmonicida isolates have succeeded isolation of fish in 4 (four) region in Indonesia,
but not yet been known its clonal relation. At this research to A. salmonicida isolates and ATCC atypical isolate as control have been checked its clonal relation use method of Restriction Fragment Length Polymorphism ( RFLP) by using restrictation enzyme of AluI, HaeIII, MboI and NarI. Result at examination with RFLP at phase I show the existence of difference of band of DNA at isolate 4 while 1, 2, 3 and 5 isolate showing is same pattern, goodness by using enzyme of AluI and HaeIII. Pattern of RFLP that happened is second of primary (15S and also M5) ranging from 67-421 bp where in all isolates (1, 2, 3, 4 and 5). Location difference of band at isolate 4 more which emerge band in enzyme of AluI on course 160-240 16S rDNA
A. salmonicida gene compared to HaeIII endonucleus which only cutting 3 multiply
in 16S rDNA A. salmonicida gene. At phase test II by using of MboI and NarI enzyme where atypical isolate of A. salmonicida (isolate of 1,2,3,4 and 5) showing result of examination do not show the existence of difference and show strong level is overall of gene sequence of 16S rDNA nucleotide where cutting a lot emerge band in enzyme of AluI on course 160-240 16S rDNA gene A. salmonicida while HaeIII endonucleus only cutting 3 multiply in 16S rDNA gene A. salmonicida
Keyword : A. salmonicida, RFLP
INTRODUCTION
Aeromonas salmonicida represent agent of etiological furunculosis, an disease
which attacking many fish and cause of loss of economics in freshwater aquaculture
(Mccarthy and Roberts; 1980; O'Brien et al, 1994). Typical and atypical term of A.
salmon and of strain causing furunculosis at fish besides fish of salmon (Austin and
Austin 1999; Inglis et al., 1993)
At attacked fish to be marked with septisemia, losing appetite, weak and hurt of
nekrosis and haemorahagik at gill, muscle and intestine (Hiney and Olivier, 1999).
This bacterium had also been attributed to disease of hurt/ulcer at carp fish and flat
fish and also erithrodermatitis at carp fish (Austin and Austin, 1999).
Aeromonas salmonicida represent bacterium of Gram-negatif bipolar, having
the character of optional anaerob, motion less, not have spore, do not have capsule,
in form of short rod, fairish 1,3 - 2,0 µ best growth at temperature 18-25°C(Sakazaki
and of Ballows, 1981; Untergasser, 1989; Ingglis et al., 1993; Austin and Austin,
1999). Isolation A. salmonicida can grow in various standard bacterium media used
occasionally for example Trypticase Soy Agar (TSA) and Brain Heart Infussion Agar
(BHIA) and pregnant nutrient media 0,85 % content salt (Ingglis et al.,., 1993; Austin
et al., 1998)
By using RFLP of 16S rRNA gene, will be obtained by spesific pattern-type for
all strain of species A. salmonicida (Borrel et al., 1997; Figueras et al., 2000). Where
this method can be used to equip gene amplification method 16S rDNA of PCR
through usage 2 endonucleus simultan AluI (AGCT), HaeIII, NarI and MboI (GATC)
(Borrell et al., 1997). Addition 2 enzyme type of NarI (GGCGCC) and of HaeIII
(GGCC) needed to to differentiate gene 16S rDNA A. salmonicida, A. encheleia of
HG 11 (group hybridization 11) Aeromonas spp. (Figueras et al., 2000)
Base of RFLP analysis to pass examination with refer to to identify
biochemical characteristic like : dexarboxylase lysine, dexarboxylase ornithin,
dihydrolase arginin (L/O/A); hydrolysis esculin bile (BAE); product gas from TSI;
Voges-Proskauer test; acid of arabinose, sacrose manitol (A/M/S); acid of
D-Rhamnose (LOUVRE); acid of D-Sorbitol (SOR); acid of salicin (SAL); acid of
lactose; acid of mannose (MAN); and exploiting of citrate (Borrell et al., 1997).
This research is executed by applying molecular method (RFLP) at atypical
atypical of A. salmonicida subsp. smithia Austin et al. product of Americans Type
Culture Collection (ATCC) Number :: 49393.
MATERIALS AND METHOD
1. Fish
Fish Test which utilized in this research is goldfish (Carpio cyprinus) size ± 10
cm counted 500, election of test fish type based of territorial water characteristic
which still in spanning influence of patogenisitas A. salmonicida.
2. Isolate
A. salmonicida isolate the used from Pontianak /isolate 1 (coming from fish of
mas/C. carpio), Semarang/isolate 2 (gurame/O. gouramy), Yogyakarta /isolate 3
(freshwater fish pomfret/ C. macropomum), Jambi/isolate 4 (nila/O. niloticus) and as
control/isolate 5 is atipikal isolate A. salmonicida subsp. smithia Austin et al. from
Americans Type Culture Collection ( ATCC) Number : 49393 .
3. RFLP
Materials for the examination of RFLP method for example : enzyme of AluI
(AGCT), HaeIII (GGCC), MboI and NarI (Boehringer), acetic acid glacial, buffer
loading ( what consist of 0,25 % blue bromphenol, 0,25 % cyanol xylene and 30 %
glyserol), 4 % gel agarose metaphor (PMC Bioproduct) in 0,5 TBE x
(Tris-Borate-Edta) Buffer (Metaphor Agarose Gel in stain by using bromide ethidium 0,5 µg/ml),
dH2O, sol I for 100 ml (AgNO3 0,15 gr + 37%), sol II for 100 ml (Na2CO3 : 3 gr +
Formaldhehyde 151 ml + sodium thiosulfat 5µl/261 mg/ml where is endconcentration
2 mg/ml) and 8 % polycrylamide gel.
4. Characteritation
Characteritationto cause bacterium conducted seen morphology, nature of Gram,
motilitas and nature of other cultural like : ability of ferment, IMVIC, and biochemy
RESULT ANN DISSCUSSION
1.Result
By virtue of result of research show there is inappropriate of used enzyme at
examination with RFLP at phase I where atypical isolate of A. salmonicida from
some region in Indonesia and atypical isolate of ATCC show an difference of band at
isolate 4 while isolate 1, 2, 3 and 5 showing is same pattern, goodness by using of
AluI enzyme (blue colourant)/ 15S primary and HaeIII enzyme (red colourant)/ M5
primary. Pattern of RFLP method that happened is second primary (15S and also M5)
ranging from 67-421 bp where all isolate (1, 2, 3, 4 and 5) emerge band showing do
not happened mistake identify isolate. Location difference of band at isolate 4 more
which emerge band in enzyme of AluI on course 160-240 16S rDNA gene A.
salmonicida while HaeIII endonukleus only cutting 3 multiply in 16S gene rDNA A.
salmonicida (Picture 1). This is matter more caused by technical factor but not of
genetica factor or phenotipic factor from all isolate A. salmonicida which is used in
this research
At phase test II by using of MboI and NarI enzyme where atypical isolate of A.
salmonicida (isolate of 1,2,3,4 and 5) showing result of examination of method of
RFLP do not show the existence of difference and show strong level is overall of
gene sequence of nucleotide 16S rDNA where cutting a lot emerge band in
enzyme of AluI on course 160-240 16S rDNA gene A. salmonicida while HaeIII
endonucleus only cutting 3 (three) multiply in 16S rDNA gene A. salmonicida. This
matter more caused by technical factor but not genetic factor or phenotypic factor
1 2 3 4 5 6 6 5 4 3 2 1
Primer 15S Primer M5
Picture 1. RFLP pattern of 16S rDNA A. salmonicida atypical isolate,
where well : 1. marker; 2. Pontianak isolate 3. Semarang isolate 4. Yogyakarta isolate
5. Jambi isolate and 6. ATCC control isolate.
Picture 2. RFLP pattern of Pontianak atypical isolate of 16S rDNA A. salmonicida.
Picture 3. RFLP pattern of Semarang atypikal isolate of 16S rDNA A. salmonicida.
Picture 5. RFLP pattern of Jambi atypikal isolate of 16S rDNA A. salmonicida
Picture 4. RFLP pattern of ATCC atypikal isolate of 16S rDNA A. salmonicida
subsp. Smithia
Characteritation
Tables 1. Result of characteritation of A. salmonicida isolate
Test Media A. salmonicida Isolat e Atypical
Pontianak Semarang Yogyakarta Jambi ATCC
1 2 3 4 5 6
Broxn Pigmen + + + + +
Blood Agar β
haemolysis
β
haemolysis
β
haemolysis
β
haemolysis
β
haemolysis
Mc. Conkey
Agar
+
(colour)
+
(colour)
+
(colour)
+
(colour)
+
(colour)
Morfology Rod
Gram -
Rod
Gram -
Rod
Gram -
Rod
Gram -
Rod
Voges-Proskauer
- - - - -
Motility - - - - -
Katalase + + + + +
Fermentativ
Metabolisme
+ + + + +
Oxidase + + + + +
TSI Agar :
-Buff -Slant -H2S As Alk - As Alk - As Alk - As Alk - As Alk -
Indol - - - - -
MR - - - - -
Sodium citrat - - - - -
Urea - - - - -
O/F F F F F F
Agar 2%
NaCl
+ + + + +
Agar 4%
NaCl
- - - - -
Gelatin + + + + +
DNA + + + + +
Growth in
37ºC
- - - - -
Glukosa + + + + +
Laktosa - - - - -
Sukrosa + + + + +
Sorbitol - - - - -
Maltosa + + + + +
Arabinosa + + + + +
Dulcitol - - - - -
Ornithin
decarbosilase
- - - - -
3. Discusse
Pursuant to result of research show specific pattern of very close relationship in all A.
salmonicida atypical isolate is collect from some region in Indonesia with atypical
isolate of A. salmonicida subsp. smithia of ATCC. Equation genome A. salmonicida
strain can be conducted by comparing method of RFLP-PCR among A. salmonicida
strain isolate from Indonesian and closely related with isolate of A. salmonicida
subsp. smithia of ATCC, where method application application of RFLP method
besides can yield pattern able to be pattern able to be grouped in one subspesies
able to be grouped in one subspesies (Huys et al., 1997), also by phenotip can clarify
gene variation of 16S rRNA A. salmonicida subsp. smithia (Graph, 1999).
CONCLUSION
Thereby, as an conclusion, A. salmonicida atypical isolate from Indonesian
represent subspecies smithia, for that can be expressed that method of RFLP-PCR
16S rRNA gene can be used in assisting early identifying type of Aeromonas.
Verification identify type with biochemical test remain to be conductedbas according
to diagnosis of clinical to solve differences that happened
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