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Simple Summary Aethina tumida, also called the Small Hive Beetle, is an insect that multiplies primarily in honeybee hives, causing honey losses and weakening colonies. It is native to sub-Saharan Africa and was introduced into different countries and continents over the last 20 years, posing a threat to beekeeping internationally. In case of introduction into a new area, officially approved laboratories (certified by government services) carry out analyses to confirm the outbreak. The reliability of the results is essential in the implementation of management measures. Therefore, a study was organised at the European level to compare the results between official laboratories for two types of methods, used routinely for the identification of A. tumida: morphological examination (form and structure) and DNA testing (genetics). The 22 participants analysed in a blinded way a panel of 12 samples (positive and negative samples). The results were very satisfactory, with the exception of one participant who encountered several anomalies for negative samples and especially for DNA tests, probably related to his inexperience with the method. This study proved the ability of laboratories and analytical methods to identify A. tumida, which is a key element in monitoring and managing this risk. Abstract 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.

L’Anses a été saisie le 05/08/2022 par la Direction générale de l’alimentation (DGAl) pour la réalisation de l’appui scientifique et technique suivant : « Evaluation des mesures de gestion mises en place sur l’île de La Réunion suite à la découverte du petit coléoptère des ruches Aethina tumida et des conditions de leurs adaptations potentielles au regard de l’évolution de la situation épidémiologique ».La DGAL a demandé un appui de l’Anses pour répondre à plusieurs questions. Conformément au contrat établi avec le demandeur (2022-SA-0141), l’objectif de la présente note d’appui scientifique et technique (AST) est d’instruire les deux questions suivantes qui concernent la surveillance : Question 1 relative à la « surveillance » : Evaluer les modalités pratiques de la mise en place d’une surveillance programmée au sein des zones réglementées (zone de surveillance et zone de protection) et sur le reste du territoire de l’Île face aux enjeux d’éradication d’A. tumida et, en particulier, le besoin d’une adaptation des préconisations du document guide du Laboratoire de référence de l’Union Européenne (LRUE) établi en 2016, aux spécificités de l’île de La Réunion. Question 2 relative à l’« endémisation » d’A. tumida : Conduire une réflexion sur les résultats de surveillance et critères qui traduiraient une installation durable.

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, the European Reference Laboratory for Bee Health organised an inter-laboratory comparison on the identification of A. tumida by morphology and real-time PCR, two types of methods used routinely to confirm outbreaks. 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 present comparison is, to our knowledge, the first organised at the international level on the official diagnosis of A. tumida. It 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 in countries where its presence has not yet been detected and where early detection is crucial.

To evaluate the performance of the molecular methods used by national reference laboratories (NRLs) for the identification of Nosema species in bee samples, an inter-laboratory comparison (ILC) was organised in 2015. A total of 20 EU NRLs and 1 non-European NRL participated in this ILC. The specificity of the methods was tested on various Nosema species: Nosema apis, Nosema ceranae and Nosema bombi. The test panel of samples provided to the laboratories contained 17 suspensions of crushed abdomens from naturally and artificially infected honeybees and bumblebees. In addition, data on the routine methods used by the participating laboratories were collected in an online survey, covering all the steps involved in DNA extraction and PCR. Our analysis showed that the 21 NRLs use 21 different protocols, each presenting variations from the DNA extraction step to the PCR step. The results of this ILC indicate that 48% of the participating laboratories returned the expected results. Considering the 21 different methods used, 57% of participating laboratories provided satisfactory results with regard to sensitivity, and 72% with regard to specificity. The results of this ILC clearly highlight the need for improved harmonisation of molecular Nosema identification methods., http://euroreference.mag.anses.fr/en

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Altres ajuts: ANSES/A06-018-2 Border disease virus (BDV) causes high mortality in Pyrenean chamois (Rupicapra pyrenaica) on the French and Spanish sides of the Pyrenees Mountains. We investigated the pathology induced by BDV in pregnant chamois via experimental infection. Three females were inoculated during the second third of pregnancy with a BDV-4 subgroup strain isolated from a wild Pyrenean chamois during an acute epizootic. A fourth pregnant chamois and one nonpregnant ewe were kept as negative controls. Animals were monitored to assess clinical signs, hematology, viremia, and serology. Postmortem examinations included necropsy, histopathology, and quantification of viral RNA in organs. Pregnancy was unsuccessful in all inoculated animals. One died 24 days postinoculation (dpi) without showing any precursory clinical signs. The second animal had profuse diarrhea from 13 dpi to its death at 51 dpi. The third aborted at 46 dpi and was euthanized at 51 dpi. All animals were viremic from 4 dpi until death. Neutralizing antibodies against BDV-4 were detected from 12 dpi. Necropsies showed generalized lymphadenomegaly, associated in one case with disseminated petechial hemorrhages in the digestive tract. Seventy-eight of 79 organs from inoculated adults and their fetuses had detectable viral RNA. The main histologic lesions in adults were mild lymphohistiocytic encephalitis associated with moderate or moderately severe lymphoid depletion. Control animals remained negative for virus (in blood and organs), antibody, and lesions upon postmortem examination. BDV infection during pregnancy in Pyrenean chamois causes severe disease leading to abortion, then death. Despite 100% fetal death following inoculation, viral RNA was recovered from all organs of infected fetuses, suggesting that persistently infected offspring could be born. Our results may help explain the reported decrease in chamois populations in several areas and suggest that great care must be taken when interpreting infection status for wildlife.