MOLECULAR IDENTIFICATION AND CLONAL RELATION OF ATYPICAL ISOLATE Aeromonas salmonicida USING RESTRICTION FRAGMENT LENGTH POLYMORPHISM ( RFLP )

Aeromonas salmonicida is responsible in many cases of furunculosis outbreaks resulting in economic loss of freshwater aquaculture. Four isolates of A. salmonicida have been isolated from fish collected in four different regions in Indonesia and its clonal relation has yet to be determined. In the research, A. salmonicida isolates and ATCC atypical isolate as the control had been checked for their clonal relation using Restriction Fragment Length Polymorphism (RFLP) method in which restriction enzyme of AluI, HaeIII, MboI and EheI were used. PCR test results using the primers 16S rDNA amplicon gave a positive response to the 1300 bp band. The result of RFLP analysis showed that A. salmonicida atypical isolates from Indonesia represent subspecies smithia except isolates from C. macropomum in Yogyakarta in MS and 16S rDNA regions. Isolates from Jambi, Pontianak and Semarang showed a slight variation on enzyme restriction sites. Isolates number 2, 3, 4 and 5 had the same restriction sites using AluI enzyme with MS primer. The restriction enzymes that could give the best result for RFLP method of A. salmonicida were HaeIII, MboI and EheI.


INTRODUCTION
Aeromonas salmonicida is the agent of etiological furunculosis disease which strikes many fish farmings and has caused a significant economic loss in freshwater aquaculture (McCarthy & Roberts, 1980;O'Brien et al., 1994).Typical and atypical terms of A. salmonicida are used to differentiate among strains causing furunculosis on salmon and other strains causing furunculosis on both Salmon and other fishes (Austin & Austin, 1999;Inglis et al., 1993) croses, and hemorrhagic of gill, muscle and intestine (Hiney & Olivier, 1999).This bacterium has also been attributed to disease of ulce r o n carp and flat fish and also erythrodermatitis on carp fish (Austin & Austin, 1999).
The research was aimed to study clonal relationship of all atypical isolates of A. salmonicida using PCR and Restriction Fragment Length Polymorphism (RFLP) from several areas in Indonesia .

Fish
Fish samples used in this research was 470 individuals of goldfish (Carpio cyprinus) sized ± 10 cm in average weight.The fish was collected from several freshwater areas in Indonesia characterized by the occurrences of A. salmonicida.
As the control was atypical isolate of A. salmonicida subsp.smithia (Austin et al.Product of Americans Type Culture Collection (ATCC) Number : 49393 by RFLP).

Characterization
Characterization of bacterium was done through identification of morphology, such as Gram test, motility and ability of fermentation, IMVIC, and biochemical examination using biochemistry test.

Results
The results of 16S rDNA amplification with a primer on all isolates of A. salmonicida gave a positive response to the 1300 bp band.The re sults also showed that iso late s from Yogyakarta, Semarang, Pontianak and Jambi has similarities to isolates of A. salmonicida.This ATCC isolates amplified with the characterization of isolates from Indonesia indicated A. salmonicida (Figure 1).
The results of the test using RFLP on four isolates of atypical A. salmonicida from several regions in Indonesia and isolates from the ATCC with MS primary did not show any band with a restriction enzyme AluI (Figure 2 and 3).Enzymes AluI did not give a positive response to the five isolates because this enzyme has no restriction sites of DNA on all isolates.This can occur because of several factors that affect the action of the enzyme, namely: temperature (0 o C: no activity; 38 o C-40 o C: increased enzyme activity, >38 o C: decreased enzyme activity; 60 o C: enzyme activity is discontinued), water, pH, concentration of enzyme (speed the process of formation or decomposition of the molecule following the concentration of enzyme substrate) and inhibitors (Brown, 2002).HaeIII restriction enzyme showed similarity with isolates no. 1 and 4. Isolates 2, 3, 5 had very different places of restrictions on each other (Figure 4 and 5).Side-cutting enzymes used is shown in Table 1.
HaeIII enzyme was used for PCR-RFLP along with the MS and 16S rDNA primers.With the MS primer, ATCC isolates showed the same restriction sites in isolates 4, while the isolates 2, 3, and 5 respectively show different restriction  M= marker; 1 and 6 : ATCC isolate, 2 and 7 : Yogyakarta isolate, 3 and 8 : Semarang isolate, 4 and 9 : Pontianak isolate, 5 and 10 : Jambi isolate sites.The amplification with 16S rDNA primer showed that 5 isolates of A. salmonicida have mutually different restriction sites.So, it can be concluded that the five isolates had a variety of strains in the area of the 16S rDNA gene (Figure 4 and 5).
In RFLP testing using MboI and EheI enzyme on 5 isolates of atypical A. salmonicida, isolates 3, 4, and 5 showed similar results compared to ATCC isolate (1).There was only one isolate which was isolate number 2 (isolates from Yogyakarta) that had different restriction sites compared to four isolates (Figure 6 and  7).The same result was also found in the use of enzymes EheI.16S rDNA nucleotide sequences at 118-207 bp cut on MboI enzyme and the enzyme EheI 452-1050 bp were consistent with the results of the study by Martinez-Murcia et al. (1992) and Borell et al. (1997) (Table 2).

Characterization
Iso latio n and ide ntificatio n of A. salmonicida from several areas in Indonesia showed the same result.

Discussion
HaeIII enzyme provided a better result on DNA testing of MS gene RFLP and 16S rRNA of A. salmonicida.RFLP testing using enzyme MboI and EheI on 5 isolates of atypical A. salmonicida showed similar results for isolates 3, 4, and 5 with ATCC isolates (1).Isolates from Yogyakarta (2) has a different restriction site compared to the other four isolates.The same result was also found in the use of enzymes EheI.The result of 16S rRNA nucleotide sequence cutting in the enzyme MboI 118-207 bp and 452-1050 bp in EheI enzymes were consistent with the results of the study by Martinez-Murcia et al. (1992) and Borrell et al. (1997).Based on these results, it showed a specific pattern of relationships which are ve ry close in all iso lates of atypical A. salmonicida collection from several regions in Indonesia with atypical isolates of A. salmonicida subsp.smithia from ATCC.Application of RFLP method does not only uncover patterns that can be grouped in a single subspecies (Huys et al., 1997), but also can clarify phenotype pattern of 16S rRNA gene variation within A. salmonicida subsp.smithia (Graf, 1999).
In this regard, dete rminatio n o f endonucleosa enzyme to distinguish genes using the 16S rDNA RFLP method is basically not easy.Enzyme AluI (5'AGCT) is not effective to be used in the primary RFLP testing of MS, but the enzyme HaeIII (5'GGCC) can provide a clear difference between the five iso-lates of A. salmonicida either by using the primers MS or 16S.The RFLP test on isolates of atypical A. salmonicida on MS rDNA showed that the isolate number 2 (from Yogyakarta) is very different from the other 4 isolates based on the use of MboI (5'NGATCN) and EheI (5'GGCGCC) enzymes.
Restriction enzyme that can provide the best result for RFLP method of A.salmonicida is HaeIII, followed in order of accuracy by MboI and EheI enzymes.

MOLECULAR IDENTIFICATION AND CLONAL RELATION OF ATYPICAL ISOLATE Aeromonas salmonicida USING RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP)
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ABSTRACTAeromonas salmonicida is responsible in many cases of furunculosis outbreaks resulting in economic loss of freshwater aquaculture.Four isolates of A. salmonicida have been isolated from fish collected in four different regions in Indonesia and its clonal relation has yet to be determined.In the research, A. salmonicida isolates and ATCC atypical isolate as the control had been checked for their clonal relation using Restriction Fragment Length Polymorphism (RFLP) method in which restriction enzyme of AluI, HaeIII, MboI and EheI were used.PCR test results using the primers 16S rDNA amplicon gave a positive response to the 1300 bp band.The result of RFLP analysis showed that A. salmonicida atypical isolates from Indonesia represent subspecies smithia except isolates from C. macropomum in Yogyakarta in MS and 16S rDNA regions.Isolates from Jambi, Pontianak and Semarang showed a slight variation on enzyme restriction sites.Isolates number 2, 3, 4 and 5 had the same restriction sites using AluI enzyme with MS primer.The restriction enzymes that could give the best result for RFLP method of A. salmonicida were HaeIII, MboI and EheI.KEYWORDS: A. salmonicida, furunculosis, PCR, RFLP, restriction enzyme

Table 1 .
Recognition results of the various restriction enzyme sites of the samples

Table 2 .
Comparison with the pattern of restriction enzyme endonukleus of A. salmonicida isolates

Table 3 .
Result of biochemical characterization of A. salmonicida isolated from goldfish collected from Pontianak, Semarang, Yogyakarta, and Jambi