Novel TaqMan PCR Assay for the Quantification of Paenibacillus larvae Spores in Bee-Related Samples

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.

Molecular Detection and Differentiation of Arthropod, Fungal, Protozoan, Bacterial and Viral Pathogens of Honeybees

“…However, their implementation can be complicated due to the higher risk of amplicon contamination in the first and the high cost of equipment and reagents in the latter. Recently, a novel TaqMan ® qPCR reaction was published, allowing for a very sensitive quantification of P. larvae spores from honey and hive debris, based on a highly conserved single-copy gene encoding for a metalloproteinase [ 268 ].…”

American Foulbrood—Old and Always New Challenge

“…In order to prevent the spread of the disease from the infected apiary, samples of honey, comb, and bees can be used to detect AFB in bee colonies that show no clinical signs of the disease. Nowadays, routine sampling of bees and honey is increasingly used in regional control programs (monitoring) to detect endospores of the causative agent of AFB worldwide [ 8 , 14 , 15 , 16 , 30 , 45 , 47 , 48 , 49 ].…”

“…In the identification of the causative agent of AFB, molecular techniques are applied. Polymerase chain reaction (PCR), real-time polymerase chain reaction (real-time PCR), and pulsed-field gel electrophoresis (PFGE) are the most commonly used in laboratories [ 2 , 4 , 16 , 22 , 25 , 30 , 46 , 47 , 49 , 57 , 58 ]. Real-time PCR analysis of the 16S rDNA gene of P. larvae represents an alternative, rapid diagnostic tool.…”

“…The microscopic method of detecting P. larvae endospores is definitely less sensitive than microbiological isolation, but both methods are less sensitive than PCR methods [ 2 , 47 , 48 , 61 , 62 ]. The presence of P. larvae can be determined by microbiological isolation and PCR methods from bee colonies in which the disease has not occurred [ 16 , 25 , 46 , 47 , 49 ].…”

“…It is also a duty of all responsible people in the beekeeping production to regularly monitoring the health condition of bee colonies, to conduct inspections, to launch anti-AFB initiatives, to implement necessary legal measures, and to share any suspicions of AFB with the competent veterinary services [ 6 , 8 , 15 , 44 , 49 , 65 ]. When working in the apiary and with bee colonies, special attention should be paid to bee and colony health.…”