Primer

3.1 The isolation of Aeromonas from several pond waters, healthy fish, and infected fish The isolate of A. hydrophila grown for 24 hours at 37C on Rimler-Shoots+novobiocin medium should show bright yellow color with white edge. Figure 1 shows the control of isolate A. hydrophila ATCC 7699 grown on RS+novobiocin medium. Isolate selection on RS medium resulted in 95 isolates, presumed to be A. hydrophila, which would run the phenospecies test (morphology and biochemistry), based on the protocol of SNI 7303 (2009), plus one control isolate the A. hydrophila ATCC 7699 obtained from Microbiologic Co.

Figure 1. The Isolate of A. hydrophila ATCC 7699 Grown on the Rimler-Shoots Agar Medium + Antibiotic Novobiocin at 37C for 24 Hours

3.2 The

hydrophila as the control, i.e. around 700 bp (685 bp). While the amplicon resulted from the primer 760 showed various results and sizes. The amplification results using the two pairs of primers showed different results from the same isolate. The Figure 2 shows the examples of various amplification results for the tested two pairs of primers. The seven isolates tested, i.e. A. hydrophila ATCC 7699, SfB, SfN, SfL, SfM, SfP, and PfKT9 had 685 bp amplicon, however when were amplified using the second primers, only the control isolate, SfB, and PfKT9 had 760 bp amplicon. The isolate SfN had two amplicons, which were different from the control isolate; isolate SfL and SfP had single amplicon, but was different from the control, while the isolate SfM did not have

and four nonaeromonas species as the outgroup. Those comparing sequences were from genBank and had been verified in EzTaxon. The 38 16S rRNA sequences were aligned and compared. The multialignment result of those 38 isolates showed the samples strongly predicted to be A. hydrophila were not always in one group with the control isolate A. hydrophila ATCC 7699, also not in one group with the reference of A. hydrophila sequence from genBank (A. hydrophila ATCC 7699 and A. hydrophila ATCC 49140). The isolates which were in a group with A. hydrophila were SfB, HfMlp, HfNp, PfKT9, PfKBl, while the isolate HfT was in a group with A. jandaei, and PfKC with A. enteropelogenes and A. taiwanensis. Isolates divided into six groups, the grouping by region of sequence 16SrRNA 980-1010. The isolates belonged to the first group were in group with A. hydrophila, the second group with A. media and A. salmonicida, the third group contained A. aquariorum, A. punctata, A. caviae and A. taiwanensis. The fourth group was in group with A. enteropelogenes, dan A. jandaei. The fifth group with A. veronii and the sixth group is outgroup. The second group had the characteristic on region 1216, while the fourth and fifth group had the characteristic on region 1234-1239 and