An international inter-laboratory study on Nosema spp. spore detection and quantification through microscopic examination of crushed honey bee abdomens

Nosemosis is a microsporidian disease causing mortality and weakening of honey bee colonies, especially in the event of co-exposure to other sources of stress. As a result, the disease is regulated in some countries. Reliable and harmonised diagnosis is crucial to ensure the quality of surveillance and research results. For this reason, the first European Interlaboratory Comparison (ILC) was organised in 2017 in order to assess both the methods and the results obtained by National Reference Laboratories (NRLs) in counting Nosema spp. spores by microscopy. Implementing their own routine conditions of analysis, the 23 participants were asked to perform an assay on & nbsp;a & nbsp;panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study concluded that using microscopy to detect and quantify spores of Nosema spp. was reliable and valid.panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study conclude

The Pathogens Spillover and Incidence Correlation in Bumblebees and Honeybees in Slovenia

Slovenia has a long tradition of beekeeping and a high density of honeybee colonies, but less is known about bumblebees and their pathogens. Therefore, a study was conducted to define the incidence and prevalence of pathogens in bumblebees and to determine whether there are links between infections in bumblebees and honeybees. In 2017 and 2018, clinically healthy workers of bumblebees (Bombus spp.) and honeybees (Apis mellifera) were collected on flowers at four different locations in Slovenia. In addition, bumblebee queens were also collected in 2018. Several pathogens were detected in the bumblebee workers using PCR and RT-PCR methods: 8.8% on acute bee paralysis virus (ABPV), 58.5% on black queen cell virus (BQCV), 6.8% on deformed wing virus (DWV), 24.5% on sacbrood bee virus (SBV), 15.6% on Lake Sinai virus (LSV), 16.3% on Nosema bombi, 8.2% on Nosema ceranae, 15.0% on Apicystis bombi and 17.0% on Crithidia bombi. In bumblebee queens, only the presence of BQCV, A. bombi and C. bombi was detected with 73.3, 26.3 and 33.3% positive samples, respectively. This study confirmed that several pathogens are regularly detected in both bumblebees and honeybees. Further studies on the pathogen transmission routes are required

Genetic diversity of deformed wing virus from Apis mellifera carnica (Hymenoptera:Apidae) and varroa mite (Mesostigmata: Varroidae)

Deformed wing virus (DWV) is one of the most widespread viruses that infect honey bee colonies. The route of infection is directly through contaminated food, feces, and air, or indirectly through the varroa mite, which acts as a vector. Positive DWV samples were obtained from Carniolan honey bee (Apis mellifera carnica) colonies and of varroa mites from the whole territory of Slovenia during a survey between 2007 and 2014. Nucleotide sequences of 471 nucleotides for the L protein gene and 573 nucleotides for the helicase gene were compared. High genetic diversity was observed among these Slovenian Carniolan honey bee DWV field samples, as well as with almost all the strains previously found in other countries in Europe. Phylogenetic analyses in two regions of the viral genome show that several of the DWV strains obtained from honey bees and varroa are genetically very closely related, confirming the important role of varroa in the transmission of DWV. Identification of closely related sequences also confirmed that the same strains of DWV have been successfully transmitted between various honey bee colonies and apiaries. It has also been established that simultaneous infection, in one apiary, of honey bees with two or more different strains of DWV is quite frequent. This is phylogenetic study that compares honey bee and varroa DWV strains from Carniolan honeybees

Determination of genetically identical strains of four honeybee viruses in bumblebee positive samples

In recent years, there has been growing evidence that certain types of honeybee viruses could be transmitted between different pollinators. Within a voluntary monitoring programme, 180 honeybee samples (Apis mellifera carnica) were collected from affected apiaries between 2007 and 2018. Also from August 2017 to August 2018, a total 148 samples of healthy bumblebees (Bombus lapidarius, B. pascuorum, B. terrestris, B. lucorum, B. hortorum, B. sylvarum, B. humilis) were collected at four different locations in Slovenia, and all samples were tested by using RT-PCR methods for six honeybee viruses. Direct sequencing of a total 158 positive samples (acute bee paralysis virus (ABPV n = 33), black queen cell virus (BQCV n = 75), sacbrood bee virus (SBV n = 25) and Lake Sinai virus (LSV n = 25)) was performed from obtained RT-PCR products. The genetic comparison of identified positive samples of bumblebees and detected honeybee field strains of ABPV, BQCV, SBV, and LSV demonstrated 98.74% to 100% nucleotide identity between both species. This study not only provides evidence that honeybees and bumblebees are infected with genetically identical or closely related viral strains of four endemically present honeybee viruses but also detected a high diversity of circulating strains in bumblebees, similar as was observed among honeybees. Important new genetic data for endemic strains circulating in honeybees and bumblebees in Slovenia are presented

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

Reliability of Morphological and PCR Methods for the Official Diagnosis of Aethina tumida (Coleoptera: Nitidulidae): A European Inter-Laboratory Comparison

International audienceThe Small Hive Beetle (Aethina tumida Murray, 1867) is an invasive scavenger of honeybees. Originally endemic in sub-Saharan Africa, it is regulated internationally in order to preserve the areas still free from this species. To ensure the reliability of official diagnoses in case of introduction, an inter-laboratory comparison was organised on the identification of A. tumida by morphology and real-time PCR. Twenty-two National Reference Laboratories in Europe participated in the study and analysed 12 samples with adult coleopterans and insect larvae. The performance of the laboratories was evaluated in terms of sensitivity and specificity. Sensitivity was satisfactory for all the participants and both types of methods, thus fully meeting the diagnostic challenge of confirming all truly positive cases as positive. Two participants encountered specificity problems. For one, the anomaly was minor whereas, for the other, the issues concerned a larger number of results, especially real-time PCR, which probably were related to inexperience with this technique. The comparison demonstrated the reliability of official diagnosis, including the entire analytical process of A. tumida identification: from the first step of the analysis to the expression of opinions. The performed diagnostic tools, in parallel with field surveillance, are essential to managing A. tumida introduction

Development and evaluation of a core genome multilocus sequence typing scheme for Paenibacillus larvae, the deadly American foulbrood pathogen of honeybees

Paenibacillus larvae is the causative agent of the fatal American foulbrood disease in honeybees (Apis mellifera). Strain identification is vital for preventing the spread of the disease. To date, the most accessible and robust scheme to identify strains is the multilocus sequence typing (MLST) method. However, this approach has limited resolution, especially for epidemiological studies. As the cost of whole-genome sequencing has decreased and as it becomes increasingly available to most laboratories, an extended MLST based on the core genome (cgMLST) presents a valuable tool for high-resolution investigations. In this study, we present a standardized, robust cgMLST scheme for P. larvae typing using whole-genome sequencing. A total of 333 genomes were used to identify, validate and evaluate 2419 core genes. The cgMLST allowed fine-scale differentiation between samples that had the same profile using traditional MLST and allowed for the characterization of strains impossible by MLST. The scheme was successfully used to trace a localized Swedish outbreak, where a cluster of 38 isolates was linked to a country-wide beekeeping operation. cgMLST greatly enhances the power of a traditional typing scheme, while preserving the same stability and standardization for sharing results and methods across different laboratories