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Novel TaqMan PCR Assay for the Quantification of Paenibacillus larvae Spores in Bee-Related Samples
Darja Kušar1, Bojan Papić1, Urška Zajc1
1Institute of Microbiology and Parasitology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia.
Abstract:
Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybees. P. larvae spore counts in bee-related samples correlate with the presence of AFB symptoms and may, therefore, be used to identify at-risk colonies. Here, we constructed a TaqMan-based real-time PCR (qPCR) assay targeting a single-copy chromosomal metalloproteinase gene for reliable quantification of P. larvae. The assay was calibrated using digital PCR (dPCR) to allow absolute quantification of P. larvae spores in honey and hive debris samples. The limits of detection and quantification were 8 and 58 spores/g for honey and 188 and 707 spores/mL for hive debris, respectively. To assess the association between AFB clinical symptoms and spore counts, we quantified spores in honey and hive debris samples originating from honeybee colonies with known severity of clinical symptoms. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies but did not differ significantly with regard to the severity of clinical symptoms. For honey, the average spore germination rate was 0.52% (range = 0.04-6.05%), indicating poor and inconsistent in vitro germination. The newly developed qPCR assay allows reliable detection and quantification of P. larvae in honey and hive debris samples but can also be extended to other sample types.
Insights
A new real-time PCR assay accurately quantifies Paenibacillus larvae spores, the cause of American foulbrood (AFB) in honeybees. Higher spore counts in infected colonies indicate risk, but not disease severity.
Area of Science:
- Veterinary Entomology
- Microbiology
- Molecular Diagnostics
Background:
- American foulbrood (AFB), caused by Paenibacillus larvae, is a severe honeybee disease.
- P. larvae spore load in bee samples correlates with AFB presence and colony risk.
- Accurate quantification of P. larvae is crucial for disease management.
Purpose of the Study:
- To develop and validate a real-time PCR (qPCR) assay for reliable quantification of P. larvae spores.
- To determine the association between P. larvae spore counts and AFB clinical symptoms.
- To assess P. larvae spore viability in honey.
Main Methods:
- Construction of a TaqMan-based qPCR assay targeting a single-copy P. larvae gene.
- Calibration of the qPCR assay using digital PCR (dPCR) for absolute quantification.
- Quantification of P. larvae spores in honey and hive debris from colonies with varying AFB severity.
Main Results:
- The qPCR assay demonstrated reliable detection and quantification limits for P. larvae spores in honey and hive debris.
- Significantly higher spore counts were found in AFB-positive colonies compared to asymptomatic ones.
- Spore counts did not significantly correlate with the severity of clinical AFB symptoms.
- In vitro germination rates of P. larvae spores in honey were low and inconsistent.
Conclusions:
- The developed qPCR assay provides a robust tool for detecting and quantifying P. larvae in honey and hive debris.
- Spore quantification can aid in identifying at-risk honeybee colonies.
- Further research may extend the assay's application to other sample types for comprehensive disease surveillance.
