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

The purpose of this study was to improve and standardise restriction endonu clease analysis (REA) for discriminating isolates of serogroup B Pasteurell a multocida associated with haemorrhagic septicaemia in wild and domestic a nimals and to create a reference database that can be used for epidemiologi cal studies. Two techniques for extraction and isolation of chromosomal DNA were compared, a DNAzol (R) method and an enzymic lysis followed by a two- phase partition method. No differences were observed between DNA fingerprin t profiles with either technique; however, the former technique was faster and easier to perform. P. multocida isolated from different animals in diff erent countries representing serotypes B:2, B:3, B:3,4 and B:4 were subject ed to REA with HhaI and HpaII endonucleases. Forty-eight fingerprint profil es were distinguished among 222 isolates when only HhaI was used. By combin ing the data from REA with HhaI and HpaII used separately, 88 different gro ups could be distinguished among the same isolates, Following digestion wit h HhaI and electrophoresis, the DNA of all serotype B:2 isolates produced f ingerprint profiles characterised by two trailing bands at similar to 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 pro file 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 end emic.