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Restriction endonuclease analysis using Hhal and Hpall to discriminate among group B Pasteurella multocida associated
1Avian and Swine Respiratory Diseases Research Unit, USDA/Agricultural Research Service/National Animal Disease Center, PO Box 70, Ames, IA 50010, USA.
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 DNAzol 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 approximately 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.
Insights
Restriction endonuclease analysis (REA) effectively differentiates Pasteurella multocida serogroup B isolates. Combining enzymes HhaI and HpaII significantly enhances discrimination for epidemiological studies of haemorrhagic septicaemia.
Area of Science:
- Veterinary Microbiology
- Molecular Epidemiology
Background:
- Haemorrhagic septicaemia, caused by Pasteurella multocida serogroup B, affects both wild and domestic animals.
- Accurate strain discrimination is crucial for epidemiological investigations and disease control.
Purpose of the Study:
- To standardize and improve restriction endonuclease analysis (REA) for Pasteurella multocida serogroup B.
- To develop a reference database for epidemiological studies of haemorrhagic septicaemia.
Main Methods:
- Comparison of two DNA extraction methods: DNAzol versus enzymic lysis with two-phase partition.
- Application of REA using HhaI and HpaII endonucleases on 222 Pasteurella multocida isolates.
- Analysis of DNA fingerprint profiles generated by REA.
Main Results:
- DNAzol method identified as faster and easier for DNA extraction without compromising fingerprint profiles.
- Using HhaI alone distinguished 48 profiles; combining HhaI and HpaII identified 88 distinct groups.
- Serotype B:2 isolates showed unique DNA fingerprint profiles with HhaI, distinct from other serotypes.
- REA profiles remained stable after laboratory passage or mouse inoculation.
Conclusions:
- Standardized REA with HhaI and HpaII provides high discriminatory power for Pasteurella multocida serogroup B.
- The developed method and database are valuable tools for studying the genetic diversity and epidemiology of haemorrhagic septicaemia.