Sequence analysis of plasmids and application to detection of antibiotics resistance genes in riemerella anatipestifer isolated in Taiwan

• Main Author: 李雁鈴
• Other Authors: 謝快樂
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/22536319275113223438

碩士 === 國立中興大學 === 獸醫學系 === 92 === Riemerella anatipestifer is a Gram-negative rod-shaped bacterium and may cause epizootic infections mostly in water fowls and others. R. anatipestifer infection characterized by infectious serositis, new duck syndrome in duck or exudative septicaemia, infectious influenza in geese. This disease causes acute or chronic contagious septicemic disease and accounts for major economic losses in industrialized duck forms due to weight loss. There has been little work on the molecular basis of the pathogenesis of this organism, and so far no virulence factors or drug resistance genes have been established. This study sixty-six strains of R. anatipestifer were isolated from ducks with infectious serositis and geese in Taiwan. All strains contain seven differently sized plasmids ,and eighty-five percent of the ioslates（56/66）contained plasmids. In this study find the new plasmids, 7-kb and 9-kb plasmids in R. anatipestifer strains. The 7-kb plasmid（designated as pY1）and the 9-kb plasmid（designated as pY2）were completely sequenced to determine if they encoded virulence factors or drug resistance genes. pY1 had 6849 bp and pY2 had 8745 bp. Six ORFs（open reading frames） were identified in the pY1 and eight ORFs were identified in the pY2. There were a new ORF（ORF651）in pY1 and a new ORF（ORF1233）in pY2. The results indicated that ORF651 was a plasmid-associated protein, and it was a water-soluble protein. After blasting ORF651 with other proteins, indicated that ORF651 has disaccharide hexapeptide lipid substrate binding site. There was no drug resistance gene in pY1 and pY2. Then designated PCR primers to amplify the drug resistance gene of R. anatipestifer genome. The results of PCR indicated that R. anatipestifer genome has encoded two types of dihydropeteroate synthase. This is the first report that R. anatipestifer genome encoded drug resistance gene, dihydropeteroate synthase. Compared the PCR results with antibiotic susceptibility test , there were 90.3％ similarity. Thus, using the PCR method could be short the testing time of antibiotic susceptibility.