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Simultaneous PCR detection of Paenibacillus larvae targeting insertion sequence IS256 and Melissococcus plutonius
Katerina Vlkova1,2, Tomas Erban3, Martin Kamler4
1Biomedical Center, Faculty of Medicine in Pilsen, Charles University, alej Svobody 76, 323 00, Pilsen, Czech Republic.
Abstract:
Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)-American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I-IV (strain DSM 7030-ERIC I, DSM 25430-ERIC II, LMG 16252-ERIC III, DSM 3615-ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.
Insights
A new multiplex PCR method accurately detects American and European foulbrood pathogens, Paenibacillus larvae and Melissococcus plutonius, in honeybees. This rapid detection is crucial for controlling these devastating honeybee diseases.
Area of Science:
- Apiculture
- Veterinary Microbiology
- Molecular Diagnostics
Background:
- American foulbrood (Paenibacillus larvae) and European foulbrood (Melissococcus plutonius) are significant threats to honeybee (Apis mellifera) colonies.
- Rapid and accurate pathogen detection is essential for effective disease management in beekeeping.
Purpose of the Study:
- To develop a novel multiplex PCR assay for simultaneous detection of P. larvae and M. plutonius.
- To optimize a sensitive and specific molecular diagnostic tool for honeybee foulbrood diseases.
Main Methods:
- Multiplex PCR assay design targeting specific genetic markers: IS256 for P. larvae, pMP1 plasmid and ETX/MTX2 toxin genes for M. plutonius.
- Optimization using four P. larvae ERIC genotypes (I-IV) and whole-genome sequencing.
- Validation using 36 clinical samples from infected honeybee colonies in the Czech Republic, including hive debris, workers, and brood.
Main Results:
- The multiplex PCR achieved 93.75% sensitivity and 100% specificity for P. larvae detection in hive debris.
- 100% sensitivity and specificity were obtained for both pathogens in honeybee workers, larval scales, and diseased brood.
- An internal control using a honeybee-specific gene (major royal jelly protein 1) ensured assay reliability.
Conclusions:
- The developed multiplex PCR method provides a reliable, rapid, and accurate tool for simultaneous diagnosis of American and European foulbrood in honeybees.
- This assay facilitates timely intervention and improved disease control strategies, safeguarding honeybee populations.
