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Geographically diverse Australian isolates of Melissococcus pluton exhibit minimal genotypic diversity by restriction
S P Djordjevic1, L A Smith, W A Forbes
1NSW Agriculture, Microbiology and Immunology Section, Elizabeth Macarthur Agricultural Institute, Camden, Australia. djordjs@agric.nsw.gov.au
Abstract:
Melissococcus pluton, the causative agent of European foulbrood is an economically significant disease of honey bees (Apis mellifera) across most regions of the world and is prevalent throughout most states of Australia. 49 Isolates of M. pluton recovered from diseased colonies or honey samples in New South Wales, Queensland, South Australia, Tasmania and Victoria were compared using SDS-PAGE, Western immunoblotting and restriction endonuclease analyses. DNA profiles of all 49 geographically diverse isolates showed remarkably similar AluI profiles although four isolates (one each from Queensland, South Australia, New South Wales and Victoria) displayed minor profile variations compared to AluI patterns of all other isolates. DNA from a subset of the 49 Australian and three isolates from the United Kingdom were digested separately with the restriction endonucleases CfoI, RsaI and DraI. Restriction endonuclease fragment patterns generated using these enzymes were also similar although minor variations were noted. SDS-PAGE of whole cell proteins from 13 of the 49 isolates from different states of Australia, including the four isolates which displayed minor profile variations (AluI) produced indistinguishable patterns. Major immunoreactive proteins of approximate molecular masses of 21, 24, 28, 30, 36, 40, 44, 56, 60, 71, 79 and 95 kDa were observed in immunoblots of whole cell lysates of 22 of the 49 isolates and reacted with rabbit hyperimmune antibodies raised against M. pluton whole cells. Neither SDS-PAGE or immunoblotting was capable of distinguishing differences between geographically diverse isolates of M. pluton. Collectively these data confirm that Australian isolates of M. pluton are genetically homogeneous and that this species may be clonal. Plasmid DNA was not detected in whole cell DNA profiles of any isolate resolved using agarose gel electrophoresis.
Insights
Melissococcus pluton causes European foulbrood in honey bees. Genetic analysis of Australian isolates revealed remarkable homogeneity, suggesting this bee pathogen may be clonal.
Area of Science:
- Apiculture
- Microbiology
- Genetics
Background:
- European foulbrood, caused by Melissococcus pluton, is a significant honey bee disease globally and in Australia.
- Understanding the genetic diversity of M. pluton is crucial for disease management strategies.
Purpose of the Study:
- To genetically characterize 49 Australian isolates of Melissococcus pluton.
- To compare Australian M. pluton isolates with those from the United Kingdom.
- To determine the genetic homogeneity and potential clonal nature of M. pluton in Australia.
Main Methods:
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell proteins.
- Western immunoblotting using rabbit hyperimmune antibodies against M. pluton.
- Restriction endonuclease analyses (AluI, CfoI, RsaI, DraI) of DNA profiles.
- Agarose gel electrophoresis for plasmid DNA detection.
Main Results:
- SDS-PAGE and immunoblotting showed indistinguishable protein profiles across geographically diverse Australian isolates.
- Restriction endonuclease analyses revealed highly similar DNA profiles, with only minor variations in four isolates.
- No plasmid DNA was detected in any of the analyzed M. pluton isolates.
- Geographically widespread Australian isolates demonstrated significant genetic homogeneity.
Conclusions:
- Australian isolates of Melissococcus pluton are genetically homogeneous.
- The genetic uniformity suggests that M. pluton may exist as a clonal population in Australia.
- Current molecular methods are insufficient to differentiate between geographically diverse M. pluton isolates.