Dataset Information

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.

SUBMITTER: Kusar D

PROVIDER: S-EPMC8621733 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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

Kušar Darja D Papić Bojan B Zajc Urška U Zdovc Irena I Golob Majda M Žvokelj Lucija L Knific Tanja T Avberšek Jana J Ocepek Matjaž M Pislak Ocepek Metka M

Insects 20211117 11

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 ...[more]

PMID: 34821836

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Project description:Three commercial honey bee operations in Saskatchewan, Canada, with outbreaks of American foulbrood (AFB) and recent or ongoing metaphylactic antibiotic use were intensively sampled to detect spores of Paenibacillus larvae during the summer of 2019. Here, we compared spore concentrations in different sample types within individual hives, assessed the surrogacy potential of honey collected from honey supers in place of brood chamber honey or adult bees within hives, and evaluated the ability of pooled, extracted honey to predict the degree of spore contamination identified through individual hive testing. Samples of honey and bees from hives within apiaries with a recent, confirmed case of AFB in a single hive (index apiaries) and apiaries without clinical evidence of AFB (unaffected apiaries), as well as pooled, apiary-level honey samples from end-of-season extraction, were collected and cultured to detect and enumerate spores. Only a few hives were heavily contaminated by spores in any given apiary. All operations were different from one another with regard to both the overall degree of spore contamination across apiaries and the distribution of spores between index apiaries and unaffected apiaries. Within operations, individual hive spore concentrations in unaffected apiaries were significantly different from index apiaries in the brood chamber (BC) honey, honey super (HS) honey, and BC bees of one of three operations. Across all operations, BC honey was best for discriminating index apiaries from unaffected apiaries (p = 0.001), followed by HS honey (p = 0.06), and BC bees (p = 0.398). HS honey positively correlated with both BC honey (rs = 0.76, p < 0.0001) and bees (rs = 0.50, p < 0.0001) and may be useful as a surrogate for either. Spore concentrations in pooled, extracted honey seem to have predictive potential for overall spore contamination within each operation and may have prognostic value in assessing the risk of future AFB outbreaks at the apiary (or operation) level.

Project description:American foulbrood disease of honey bees is caused by the bacterium Paenibacillus larvae. Infection occurs per os in larvae and systemic infection requires a breaching of the host peritrophic matrix and midgut epithelium. Genetic variation exists for both bacterial virulence and host resistance, and a general immunity is achieved by larvae as they age, the basis of which has not been identified. To quickly identify a pool of candidate genes responsive to P. larvae infection, we sequenced transcripts from larvae inoculated with P. larvae at 12 hours post-emergence and incubated for 72 hours, and compared expression levels to a control cohort. We identified 75 genes with significantly higher expression and six genes with significantly lower expression. In addition to several antimicrobial peptides, two genes encoding peritrophic-matrix domains were also up-regulated. Extracellular matrix proteins, proteases/protease inhibitors, and members of the Osiris gene family were prevalent among differentially regulated genes. However, analysis of Drosophila homologs of differentially expressed genes revealed spatial and temporal patterns consistent with developmental asynchrony as a likely confounder of our results. We therefore used qPCR to measure the consistency of gene expression changes for a subset of differentially expressed genes. A replicate experiment sampled at both 48 and 72 hours post infection allowed further discrimination of genes likely to be involved in host response. The consistently responsive genes in our test set included a hymenopteran-specific protein tyrosine kinase, a hymenopteran specific serine endopeptidase, a cytochrome P450 (CYP9Q1), and a homolog of trynity, a zona pellucida domain protein. Of the known honey bee antimicrobial peptides, apidaecin was responsive at both time-points studied whereas hymenoptaecin was more consistent in its level of change between biological replicates and had the greatest increase in expression by RNA-seq analysis.

Dataset Information

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

Publications

Novel TaqMan PCR Assay for the Quantification of <i>Paenibacillus larvae</i> Spores in Bee-Related Samples.

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