Molecular Identication And Clonal Relation Of Atypical Isolate Aeromonas Salmonicida Use Method Of Restriction Fragment Length Polymorphism ( Rflp).

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 isolate collected A. salmonicida from some area in Indonesia and as control of isolate

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 Examination of bichemistry conducted at all isolate conventionally .

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 from all A. salmonicida isolate which is used in this research.

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 Gram -

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

REFERENCES

Austin, D. A., Robertson, P.A.W., Wallace, D. K., Daskalov. H., and Austin, B. 1998. Isolation of Aeromonas salmonicida in Association with

Purple-Pigmented Bacteria in Sediment from Scottish Loch. Lethers in Applied Microbiology. Vol. 27 : 349-351.

Austin, B. and Austin, D. A. (Editor). 1999. Bacterial Fish Pathogens: Diseases of

Farmed and Wild Fish, 3rd edn. Chichester: Springer – Praxis.

Borrell, N., S. G. Acinas, M. J. Figueras and A. J. Martínez-Murcia. 1997.

Identification of Aeromonas salmonicida Clinical Isolates by Restrication Fragment Length Polymorphism of PCR-Amplified 16s rRNA Genes. Journal of Clinical Microbiology. Vol. 35(7) : 1671-1674.

Figueras, M. J., Soler, L., Chaćon, M. R., Guarro, J., and Martínez-Murcia, A. J. 2000. Extended Method for Discrimination of Aeromonas spp. By 16S rDNA RFLP Analysis. International Journal of Systematic and Evolutionary

Microbiology. Vol. 50 : 2069-2073.

Graf, J. 1999. Diverse Restriction Fragment Length Polymorphism Patterns of the

PCR-Amplified 16S rRNA Genes in Aeromonas veronii Strains and Possible Misidentification of Aeromonas species. Journal of Clinical Microbiology.

Vol. 37, No. 10 : 3194-3197.

Hiney, M. and Olivier, G. 1999. Furunculosis (Aeromonas salmonicida). In Fish

Diseases and Disorders, vol. 3: Viral, Bacterial and Fungal Infections, pp. 341–425. Edited by P. T. K. Woo & D. W. Bruno. Wallingford: CABI

Publishing.

Huys, G., Kämpfer, P., Altwegg, M., Coopman, R., Janssen, P., Gillis, M. & Kersters, K. 1997. Inclusion of Aeromonas DNA hybridization Group 11 in Aeromonas

encheleia and extended descriptions of the species Aeromonas eucrenophila and A. encheleia. Int J Syst Bacteriol 47, 1157–1164.

Inglis, V., Robert, R. J., and Bromage, N. R. 1993. Bacterial Disease Of Fish. Institute Of Aquaculture. Blackweell Scientific Publication. Oxford, : 122-142.

McCarthy, D. H. and Roberts, R. J. 1980. Furunculosis in Fish – The Present State

Of Our Knowledge In : Advance In Aquatic Microbiology. Academic Press,

London. 293-341.

O’Brien, D., Mooney, J., Ryan, S., Powell, E., Hiney M., Smith, P. R., and Powell., R. 1994. Detection of Aeromonas salmonicids, Causal Agents of

Furunculosis in Salmon Fish, from the Tank Effluent of Hatchery-Reared Atlantik Salmon Smolts. Applied and Enviromental Microbiology. Vol. 60, No.

10 : 3874-3877.

Sakazaki, R. and A. Balows. 1981. The Genera Vibrio, Plesimonas, and Aeromonas, p. 1272-1301. In M. P. Starr, H. G. Triper, A. Balows, and H. G. Schlegel (ed.) The Prokaryotes : a Hanbook of Habitats, Isolation, and Identification of

Bacteria. Springer-Verlag, New York.

Untergasser. 1989. Hand Book Of Fish Diseases.(Translated by Howard, H.H)

T.S.H Publication, Insc. USA, pp : 33.

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 Gram -

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

REFERENCES

Austin, D. A., Robertson, P.A.W., Wallace, D. K., Daskalov. H., and Austin, B. 1998. Isolation of Aeromonas salmonicida in Association with

Purple-Pigmented Bacteria in Sediment from Scottish Loch. Lethers in Applied Microbiology. Vol. 27 : 349-351.

Austin, B. and Austin, D. A. (Editor). 1999. Bacterial Fish Pathogens: Diseases of

Farmed and Wild Fish, 3rd edn. Chichester: Springer – Praxis.

Borrell, N., S. G. Acinas, M. J. Figueras and A. J. Martínez-Murcia. 1997.

Identification of Aeromonas salmonicida Clinical Isolates by Restrication Fragment Length Polymorphism of PCR-Amplified 16s rRNA Genes. Journal of Clinical Microbiology. Vol. 35(7) : 1671-1674.

Figueras, M. J., Soler, L., Chaćon, M. R., Guarro, J., and Martínez-Murcia, A. J.

2000. Extended Method for Discrimination of Aeromonas spp. By 16S rDNA RFLP Analysis. International Journal of Systematic and Evolutionary

Microbiology. Vol. 50 : 2069-2073.

Graf, J. 1999. Diverse Restriction Fragment Length Polymorphism Patterns of the

PCR-Amplified 16S rRNA Genes in Aeromonas veronii Strains and Possible Misidentification of Aeromonas species. Journal of Clinical Microbiology.

Vol. 37, No. 10 : 3194-3197.

Hiney, M. and Olivier, G. 1999. Furunculosis (Aeromonas salmonicida). In Fish

Diseases and Disorders, vol. 3: Viral, Bacterial and Fungal Infections, pp. 341–425. Edited by P. T. K. Woo & D. W. Bruno. Wallingford: CABI

Publishing.

Huys, G., Kämpfer, P., Altwegg, M., Coopman, R., Janssen, P., Gillis, M. & Kersters, K. 1997. Inclusion of Aeromonas DNA hybridization Group 11 in Aeromonas

encheleia and extended descriptions of the species Aeromonas eucrenophila and A. encheleia. Int J Syst Bacteriol 47, 1157–1164.

Inglis, V., Robert, R. J., and Bromage, N. R. 1993. Bacterial Disease Of Fish. Institute Of Aquaculture. Blackweell Scientific Publication. Oxford, : 122-142.

McCarthy, D. H. and Roberts, R. J. 1980. Furunculosis in Fish – The Present State

Of Our Knowledge In : Advance In Aquatic Microbiology. Academic Press,

London. 293-341.

O’Brien, D., Mooney, J., Ryan, S., Powell, E., Hiney M., Smith, P. R., and Powell., R. 1994. Detection of Aeromonas salmonicids, Causal Agents of

Furunculosis in Salmon Fish, from the Tank Effluent of Hatchery-Reared Atlantik Salmon Smolts. Applied and Enviromental Microbiology. Vol. 60, No.

10 : 3874-3877.

Sakazaki, R. and A. Balows. 1981. The Genera Vibrio, Plesimonas, and Aeromonas, p. 1272-1301. In M. P. Starr, H. G. Triper, A. Balows, and H. G. Schlegel (ed.) The Prokaryotes : a Hanbook of Habitats, Isolation, and Identification of

Bacteria. Springer-Verlag, New York.

Untergasser. 1989. Hand Book Of Fish Diseases.(Translated by Howard, H.H)

T.S.H Publication, Insc. USA, pp : 33.