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 a 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.

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

The 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.

Influence of chronic exposure to thiamethoxam and chronic bee paralysis virus on winter honey bees.

Co-exposure to pesticides and viruses is likely to occur in honey bee colonies. Pesticides can be present in pollen, nectar, and persist in stored food (honey and bee bread), and viruses can be highly prevalent in honey bee colonies. Therefore, the present study describes the influence of chronic co-exposure to thiamethoxam and Chronic bee paralysis virus (CBPV) on bee survival, virus loads, expression level of immune and detoxication genes, and pesticide metabolism Experiments were performed on honey bees collected from a winter apiary with reduced viral contaminations. No synergistic effect of co-exposure was observed on bee survival, nor on the ability of bees to metabolise the pesticide into clothianidin. However, we found that co-exposure caused an increase in CBPV loads that reached the viral levels usually found in overt infections. The effect of co-exposure on CBPV replication was associated with down-regulation of vitellogenin and dorsal-1a gene transcription. Nevertheless, the observed effects might be different to those occurring in spring or summer bees, which are more likelyco-exposed to thiamethoxam and CBPV and exhibit a different physiology.

Trueness and precision of the real-time RT-PCR method for quantifying the chronic bee paralysis virus genome in bee homogenates evaluated by a comparative inter-laboratory study.

The Chronic bee paralysis virus (CBPV) is the aetiological agent of chronic bee paralysis, a contagious disease associated with nervous disorders in adult honeybees leading to massive mortalities in front of the hives. Some of the clinical signs frequently reported, such as trembling, may be confused with intoxication syndromes. Therefore, laboratory diagnosis using real-time PCR to quantify CBPV loads is used to confirm disease. Clinical signs of chronic paralysis are usually associated with viral loads higher than 108 copies of CBPV genome copies per bee (8 log10 CBPV/bee). This threshold is used by the European Union Reference Laboratory for Bee Health to diagnose the disease. In 2015, the accuracy of measurements of three CBPV loads (5, 8 and 9 log10 CBPV/bee) was assessed through an inter-laboratory study. Twenty-one participants, including 16 European National Reference Laboratories, received 13 homogenates of CBPV-infected bees adjusted to the three loads. Participants were requested to use the method usually employed for routine diagnosis. The quantitative results (n=270) were analysed according to international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). The standard deviations of measurement reproducibility (SR) were 0.83, 1.06 and 1.16 at viral loads 5, 8 and 9 log10 CBPV/bee, respectively. The inter-laboratory confidence of viral quantification (+/- 1.96SR) at the diagnostic threshold (8 log10 CBPV/bee) was+/- 2.08 log10 CBPV/bee. These results highlight the need to take into account the confidence of measurements in epidemiological studies using results from different laboratories. Considering this confidence, viral loads over 6 log10 CBPV/bee may be considered to indicate probable cases of chronic paralysis.

RNA 1 and RNA 2 Genomic Segments of Chronic Bee Paralysis Virus Are Infectious and Induce Chronic Bee Paralysis Disease.

Chronic bee paralysis virus (CBPV) causes an infectious and contagious disease of adult honeybees. Its segmented genome is composed of two major positive single-stranded RNAs, RNA 1 (3,674 nt) and RNA 2 (2,305 nt). Three minor RNAs (about 1,000 nt each) have been described earlier but they were not detected by sequencing of CBPV genome. In this study, the results of in vivo inoculation of the two purified CBPV major RNAs are presented and demonstrate that RNA 1 and RNA 2 are infectious. Honeybees inoculated with 10(9) RNA copies per bee developed paralysis symptoms within 6 days after inoculation. The number of CBPV RNA copies increased significantly throughout the infection. Moreover, the negative strand of CBPV RNA was detected by RT-PCR, and CBPV particles were visualized by electronic microscopy in inoculated honeybees. Taken together, these results show that CBPV RNA 1 and CBPV RNA 2 segments can induce virus replication and produce CBPV virus particles. Therefore, the three minor RNAs described in early studies are not essential for virus replication. These data are crucial for the development of a reverse genetic system for CBPV.

An assessment of honeybee colony matrices, Apis mellifera (Hymenoptera: Apidae) to monitor pesticide presence in continental France.

The frequency of occurrence and relative concentration of 44 pesticides in apicultural (Apis mellifera) matrices collected from five French locations (24 apiaries) were assessed from 2002 to 2005. The number and nature of the pesticides investigated varied with the matrices examined-living honeybees, pollen loads, honey, and beeswax. Pollen loads and beeswax had the highest frequency of pesticide occurrence among the apiary matrices examined in the present study, whereas honey samples had the lowest. The imidacloprid group and the fipronil group were detected in sufficient amounts in all matrices to allow statistical comparisons. Some seasonal variation was shown when residues were identified in pollen loads. Given the results (highest frequency of presence) and practical aspects (easy to collect; matrix with no turnover, unlike with bees that are naturally renewed), pollen loads were the best matrix for assessing the presence of pesticide residues in the environment in our given conditions.

Influence of pesticide residues on honey bee (Hymenoptera: Apidae) colony health in France.

A 3-yr field survey was carried out in France, from 2002 to 2005, to study honey bee (Apis mellifera L.) colony health in relation to pesticide residues found in the colonies. This study was motivated by recent massive losses of honey bee colonies, and our objective was to examine the possible relationship between low levels of pesticide residues in apicultural matrices (honey, pollen collected by honey bees, beeswax) and colony health as measured by colony mortality and adult and brood population abundance. When all apicultural matrices were pooled together, the number of pesticide residue detected per sampling period (four sampling periods per year) and per apiary ranged from 0 to 9, with the most frequent being two (29.6%). No pesticide residues were detected during 12.7% of the sampling periods. Residues of imidacloprid and 6- chloronicotinic acid were the most frequently detected in pollen loads, honey, and honey bee matrices. Several pairs of active ingredients were present concurrently within honey bees and in pollen loads but not in beeswax and honey samples. No statistical relationship was found between colony mortality and pesticide residues. When pesticide residues from all matrices were pooled together, a mixed model analysis did not show a significant relationship between the presence of pesticide residues and the abundance of brood and adults, and no statistical relationship was found between colony mortality and pesticide residues. Thus, although certain pesticide residues were detected in apicultural matrices and occasionally with another pesticide residual, more work is needed to determine the role these residues play in affecting colony health.

First detection of Israeli acute paralysis virus (IAPV) in France, a dicistrovirus affecting honeybees (Apis mellifera).

Bee samples were collected in French apiaries that displayed severe losses and mortality during the winter (from November 2007 to March 2008). They were screened for the presence of Israeli acute paralysis virus (IAPV) by using RT-PCR. Five out of 35 surveyed apiaries, located in two different geographical areas, were found positive. This represents the first reported detection of IAPV in France. The specificity of the PCR products was checked by sequencing. The phylogenetic analysis showed that French isolates of IAPV were closely related to a cluster including American and Australian isolates. Nevertheless, most of American isolates previously reported to be associated to Colony Collapse Disorder (CCD) and an Israeli isolate first isolated in 2004 from dead bees were included in another cluster. Since IAPV was detected in only 14% of the affected apiaries, it was not possible to establish a causal link between IAPV and the severe winter losses that occurred.

Detection of Chronic bee paralysis virus (CBPV) genome and its replicative RNA form in various hosts and possible ways of spread.

Detection of Chronic bee paralysis virus (CBPV) is reported for the first time in two species of ants (Camponotus vagus and Formica rufa) and in Varroa destructor. A quantitative real-time PCR (qPCR) method was used to detect and quantify CBPV in infected bees, ants and mites. A minus-strand-specific RT-PCR was used to assess viral replication. These results suggest a new way by which the infection may be spread and other sites of viral persistence in the close apiary environment.

A survey of pesticide residues in pollen loads collected by honey bees in France.

In 2002, a field survey was initiated on French apiaries to monitor weakness of honey bee, Apis mellifera L., colonies. Apiaries were evenly distributed in five sites located on continental France. Five colonies were randomly selected in each apiary, leading to a total of 125 studied honey bee colonies. For 3 yr (starting in autumn 2002), colonies were visited four times per year: after winter, before summer, during summer, and before winter. Pollen loads from traps were collected at each visit. Multiresidue analyses were performed in pollen to search residues of 36 different molecules. Specific analyses were conducted to search fipronil and metabolites and also imidacloprid and metabolites. Residues of 19 searched compounds were found in samples. Contamination by pesticides ranged from 50 to 0%. Coumaphos and tau-fluvalinate residues were the most concentrated of all residues (mean concentrations were 925.0 and 487.2 microg/kg, respectively). Fipronil and metabolite contents were superior to the limit of detection in 16 samples. Residues of fipronil were found in 10 samples. Nine samples contained the sulfone compound, and three samples contained the desulfinyl compound. Residues of imidacloprid and 6-chloronicotinic acid were found in 69% of samples. Imidacloprid contents were quantified in 11 samples with values ranging from 1.1 to 5.7 microg/kg. 6-Chloronicotinic acid content was superior to the limit of quantification in 28 samples with values ranging from 0.6 to 9.3 microg/kg. Statistical tests showed no difference between places of sampling with the exception of fipronil. Possible origins of these contaminations, concentration and toxicity of pesticides, and the possible consequences for bees are discussed.