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Restriction endonuclease analysis using HhaI and HpaII to discriminate among group B Pasteurella multocida associated with haemorrhagic septicaemia

Avian and Swine Respiratory Diseases Research Unit, USDA/Agricultural Research Service/National Animal Disease Center, PO Box 70, Ames, IA 50010, USA

Corresponding author: Dr R. B. Rimler (e-mail: rrimler{at}nadc.ars.usda.gov).

Received 16 March 1999; revised manuscript received 12 June 1999; accepted 30 June 1999.

Abstract

The purpose of this study was to improve and standardise restriction endonuclease analysis (REA) for discriminating isolates of serogroup B Pasteurella multocida associated with haemorrhagic septicaemia in wild and domestic animals and to create a reference database that can be used for epidemiological studies. Two techniques for extraction and isolation of chromosomal DNA were compared, a DNAzol1 method and an enzymic lysis followed by a two-phase partition method. No differences were observed between DNA fingerprint profiles with either technique; however, the former technique was faster and easier to perform. P. multocida isolated from different animals in different countries representing serotypes B:2, B:3, B:3,4 and B:4 were subjected to REA with HhaI and HpaII endonucleases. Forty-eight fingerprint profiles were distinguished among 222 isolates when only HhaI was used. By combining the data from REA with HhaI and HpaII used separately, 88 different groups could be distinguished among the same isolates. Following digestion with HhaI and electrophoresis, the DNA of all serotype B:2 isolates produced fingerprint profiles characterised by two trailing bands at 8.4–7.1 kb which have not been observed in any other serotypes of P. multocida. Passage of three serotype B:2 isolates on laboratory media or two serotype B:2 isolates through mice did not result in a change of DNA fingerprint profile detectable by REA. The findings with 59 isolates from Sri Lanka showed that REA was highly discriminative in determining the genetic diversity of serotype B:2 P. multocida in an area where haemorrhagic septicaemia is endemic.

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