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United States Department of Agriculture

Agricultural Research Service

Title: Restriction Endonuclease Analysis Using Hhai and Hpaii to Discriminate among Group B Pasteurella Multocida Associated with Haemorrhagic Septicaemia

Author
item Rimler, Richard

Submitted to: Journal of Medical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 30, 1999
Publication Date: N/A

Interpretive Summary: Hemorrhagic septicemia is a disease which occurs in cattle and buffalo, but other domesticated and wild animal species can be affected. It is caused by specific kinds of bacteria (group B Pasteurella multocida) and mainly occurs in Asia and Africa. However, outbreaks are sometimes seen in North America and Europe. Laboratory and computer methods were developed to distinguish strains of these bacteria, and a reference database was create for use in epidemiological studies of outbreaks. Using these methods, a fingerprint can be made of a bacteria isolated from an outbreak. An image of the fingerprint can be sent by e-mail to the National Animal Disease Center where comparisons can be made with fingerprints archived in a database. This methodology and database will be useful for veterinarians and diagnostic laboratories who encounter this disease and are concerned with its epidemiology in domestic and wild animals. Comparisons of bacteria from outbreaks can be made without having to acquire cultures fro different laboratories and countries.

Technical Abstract: The purpose of this study was to improve and standardize 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 DNAzol method and an enzymatic lysis followed by two-phase partition method. No differences were observed between DNA fingerprint profiles using either techniques. 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 using HhaI and HpaII endo- nucleases. Forty-eight fingerprint profiles were distinguished among 222 isolates when only Hhal was used. By combining the data from REA using HhaI and HpaII separately, 88 different groups could be distinguished among the same isolates. Following digestion with Hhal and electrophoresis, the DNA of all serotype B:2 isolates produced finger- print profiles characterised by two trailing bands at ~8.4 to 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 to 2 serotype B:2 isolates through mice did not result in a change of DNA fingerprint profile detectable by REA. The findings with fifty-nine isolates from Sri Lanka showed that REA was highly discriminative in determining the genetic diversity of serotyp B:2 P. multocida in an area where haemorrhagic septicaemia is endemic.

Last Modified: 4/19/2014
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