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

Véronique Duquesne, Cristina Gastaldi, Aurélie del Cont, Nicolas Cougoule, Andrzej Bober, Marleen Brunain, Gabriela Chioveanu, Noel Demicoli, Petra Deakne Paulus, Pilar Fernandez Somalo, et al.

► To cite this version:

Véronique Duquesne, Cristina Gastaldi, Aurélie del Cont, Nicolas Cougoule, Andrzej Bober, et al. An international inter-laboratory study on Nosema spp. spore detection and quantification through microscopic examination of crushed honey bee abdomens. Journal of Microbiological Methods, 2021, 184, pp.106183. 10.1016/j.mimet.2021.106183. anses-03627158

HAL Id: anses-03627158

https://anses.hal.science/anses-03627158

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1 **An international inter-laboratory study on Nosema spp. spore detection and**

2 **quantification through microscopic examination of crushed honey bee abdomens**

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4 Véronique Duquesneª*, Cristina Gastaldiª, Aurélie Del Contª, Nicolas Cougouleª, Andrzej Boberᵇ¹,

5 Marleen Brunain c¹, Gabriela Chioveanu d¹, Noel Demicoli e¹, Petra Deakne Paulus f¹, Pilar Fernandez

6 Somalo g¹, Miriam Filipova h¹, Eva Forsgreni¹, Anna Granato j¹, Kalinka Gurgulova k¹, Sirpa

7 Heinikainen l¹, Age Kärssinml, Irena Kinduriene n¹, Hemma Köglberger o¹, Konstantinos Oureilidis p¹,

8 Zanda Ozolinaq¹, Martin Pijacek r¹, Metka Pislak Ocepek s¹, Marc Oliver Schäfer t¹, Ivana Tlak

9 Gajgeru¹, Maria José Valeriov¹, Maureen Wakefieldw¹ and Stéphanie Francoª

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11 ªFrench Agency for Food, Environmental and Occupational Health and Safety (ANSES), European

12 Union Reference Laboratory for Bee Health, Honey Bee Pathology Unit, 105 route des Chappes – CS

13 20111, 06902 Sophia Antipolis, France

14 b National Veterinary Research Institute, Department of Honey Bee Diseases, 57 Partyzantow Avenue,
15 24-100, Pulawy, Poland

16 c Laboratory for Molecular Entomology and Bee Pathology, Universiteit Gent, Krijgslaan 281, S2, 2de
17 verdiep, B-9000, Ghent, Belgium

18 d Institute for Diagnosis and Animal Health, NRL for Honey Bee Diseases and Other Useful Insects,
19 Dr. N. Staicovici street No. 63, sector 5, 050557 Bucharest, Romania

20 e National Veterinary Laboratory, Abattoir street, Albert Town, MRS1123 Marsa, Malta

21 f NFCSO Veterinary Diagnostic Directorate Molecular Biology Laboratory, Tábornok utca 2, 1143
22 Budapest, Hungary

23 g Laboratorio Central de Veterinaria, Ctra. M-106 pK M-104, 28110 Algete (Madrid), Spain

24 h State Veterinary and Food Institute, Janoskova 1611/58, 026 01, Dolny Kubin, Slovakia

25 i Swedish University of Agricultural Sciences, Department of Ecology, Box 7044, 750 07 Uppsala,
26 Sweden
27 j Istituto Zooprofilattico Sperimentale delle Venezie, NRL for Beekeeping, Viale dell'Università 10,

28 35020 Legnaro (PD), Italy

29 k NRL Bee Health, NDR VMI, 15 "Pencho Slaveykov" Blvd, 1606 Sofia, Bulgaria

30 l Finnish Food Safety Authority Evira, Veterinary Bacteriology, Research Department,

31 Neulaniementie 4, 70210 Kuopio, Finland

32 m Veterinary and Food Laboratory, Kreutzwaldi 30, 51006 Tartu, Estonia

33 n National Food and Veterinary Risk Assessment Institute, J. Kairiukscio 10, LT-08409 Vilnius,

34 Lithuania

35 o AGES, Department for Apiculture and Bee Protection, Spargelfeldstraß 3 191, 1226, Vienna, Austria

36 q Thessalonica Veterinary Center, Laboratory of Bee Diseases, 26th October str. 80, 54627

37 Thessalonica, Greece

38 p Institute of Food Safety, Animal Health and Environment "BIOR", Lejupes str. 3, 1076 Riga, Latvia

39 r State Veterinary Institute Olomouc, NRL for Honey Bee Health, Jakoubka ze Stribra 1, 77900

40 Olomouc, Czech Republic

41 s Veterinary Faculty, University of Ljubljana, National Veterinary Institute Laboratory for Health Care

42 of Bees, Gerbičeva 60, 1000 Ljubljana, Slovenia

43 t National Reference Laboratory for Bee Diseases, Friedrich-Loeffler-Institut, Südufer 10, 17493

44 Greifswald-Insel Riems, Germany

45 u Laboratory for Honeybee Diseases APISlab, University of Zagreb, Faculty of Veterinary Medicine,

46 Department for Biology and Pathology of Fish and Bees, Heinzelova 55, 10000 Zagreb, Croatia

47 v Instituto Nacional Investigação Agraria e Veterinaria, Rua Genaral Moraes Sarmento, 1500-311,

48 Lisbon, Portugal

49 w National Bee Unit, FERA, Sand Hutton, YO41 ILZ, York, United Kingdom

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51 *Corresponding author: veronique.duquesne@anses.fr, tel.: +33 4 92 94 37 27, fax: +33 4 92 94 37 01

52 l Collaborators contributing equally to this study, with names listed in alphabetical order.

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54 Keywords: diagnosis, interlaboratory comparison, microscopic counting, microsporidia, Nosema spp.
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Abstract

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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.

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1. Introduction

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Nosemosis is a global disease of adult honey bees. It is caused by a spore-forming unicellular parasite of the Microsporidia group Nosema. However, a recent study based on a molecular comparison of the SSU rRNA gene proposed a new definition of the Nosema clade (Tokarev et al., 2020). The two main species of Nosema causing disorders in honey bees worldwide are Nosemaapis (Zander, 1909) and Nosema ceranae (Fries et al., 1996). Another species, Nosema neumanni, has been found in honey bees in Uganda (Chemurot et al., 2017) but the implications of infections with N. neumanni still have to be studied. Nosemaapis and N. ceranae multiply in the epithelial cells of the posterior ventricle
region. The spores present in the lumen of the digestive tract germinate and release polar filaments that mechanically perforate epithelial cells and through which the sporoplasm enters the cell to multiply. The cell is damaged as a large number of spores is generated. Two types of spores are produced: the primary spores that are capable of transmitting infection to adjacent cells, and the mature environmental spores that may be voided with the faeces or stay in the gut to start a new multiplication cycle (Fries, 1988, Goblirsch, 2018, Higes et al., 2007). *Nosema apis* is a parasite of the European honey bee (*Apis mellifera*) while *N. ceranae*, originally described in the Asian honey bee (*Apis cerana*) but also detected in *A. mellifera* populations in a number of geographically distant regions: Europe (Higes et al., 2006), South and North America (Calderón et al., 2008, Chen et al., 2008), Africa and Asia (Chen et al., 2009). *Nosema* species (spp.) spores have an ovoid morphology. *Nosema ceranae* spores measure approximately 4.7 × 2.7 µm, making them smaller on average than *N.apis* spores, which measure approximately 6 × 3 µm (Fries et al., 2013, Fries et al., 1996, Zander and Böttcher, 1984). However, this slight difference in size is not sufficient for a differential diagnosis in routine microscopy analyses. Molecular methods (PCR) are therefore required to identify *Nosema* spp.

*Nosema* infection between adult bees is spread by the exchange of spores during feeding (trophallaxis) or comb-cleaning. Contaminated beekeeping equipment, honey stores and water also play a role in the transmission of the disease. *Nosema apis* spores expelled with faeces remain viable for over a year. They also remain contagious in honey (MacInnis et al., 2020) and in bee bodies.

The clinical signs of nosemosis are not specific. High infection rates can weaken the colony, leading to varying levels of depopulation in winter or spring. In the case of *N. apis*, dead bees, bees crawling on the ground and traces of diarrhoea may be observed on or around the hive. In contrast, the pathogenic effects of *N. ceranae* on *A. mellifera* colonies are not clearly understood. *N. ceranae* is thought to contribute to colony weakening, particularly in the presence of other sources of stress (Alaux et al., 2010, Doublet et al., 2015, Vidau et al., 2011, Zheng et al., 2015). Climate is also thought to have an effect on the pathogenicity of *N. ceranae*. While it is assumed that in warm areas the chronic stress caused by *N. ceranae* infections might favour colony death (Higes et al., 2008, Martín-Hernández et al., 2018), it was shown on the other hand that *N. ceranae* may be more virulent and better adapted
than *N. apis* in cold climates (Emsen et al., 2016). It should also be noted that bees can sustain high
infection rates of *N. apis/N. ceranae* without apparent symptoms (Meana et al., 2010) (unpublished
data). Given the difficulties of diagnosis, laboratories need to detect and quantify *Nosema* spp. spore
loads in honey bees and to establish a differential diagnosis with other adult honey bee diseases
causing similar disorders (e.g. tracheal acariasis, amoebiasis, chronic paralysis, intoxication, etc.).

Nosemosis is not covered by European Union regulations, nor is it included in the list drawn up by the
World Organisation for Animal Health (OIE). However, the disease is regulated at national level in
some countries. The OIE Manual of Diagnostic tests and Vaccines for Terrestrial Animals describes a
diagnosis method for Nosema disease based on the detection and the quantification of spores by
microscopy (World Organisation for Animal Health (OIE), 2018). A number of official laboratories
have implemented this method.

In response to diagnostic and health issues, and to ensure the quality of the analytical results obtained
within the European Union (EU), the European Union Reference Laboratory (EURL) for Bee Health,
located in the laboratory of the French Agency for Food, Environmental and Occupational Health and
Safety (ANSES) in Sophia-Antipolis (France), organised a InterLaboratory Comparison (ILC) using
microscopy to detect and count Nosema spp. spores in crushed bee samples. This was the first test of
this method organised by the EU. All the EU National Reference Laboratories (NRLs) were invited to
participate in the ILC. The overall objective was to assess the ability of laboratories to establish a
correct result using their routine analysis. Four criteria were evaluated: sensitivity, specificity, trueness
and precision of the results. At the same time, a survey was conducted within the network of EU NRLs
in order to collect information on their analytical methods with the perspective of a possible
harmonisation.

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## 2. Materials and Methods

### 2.1. *Participating laboratories*
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In June 2017, the EURL for Bee Health organised an ILC. A total of 23 NRLs for Bee Health took part in this trial, all from EU member states. In order to ensure the confidentiality of results, each participating laboratory was assigned an individual random code number.

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## 2.2. Reference methods

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Two reference methods were used to characterise and check the homogeneity and stability of the samples used in the ILC: i) a microscopy-based method to detect and count *Nosema* spp. spores, and ii) a PCR-based method to confirm detection of *Nosema* spp. (this method was only used to characterise the samples, i.e. to verify their negative or positive status). The EURL is accredited by the French Accreditation Committee (COFRAC) for these two methods in compliance with the international standard ISO/IEC 17025 on "General requirements for the competence of testing and calibration laboratories" (NF EN ISO/IEC 17025, 2005).

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The microscopic method is based on the procedure developed by Cantwell (Cantwell, 1970) and recommended by the OIE in the Terrestrial Manual (World Organisation for Animal Health (OIE), 2018) intending to detect and evaluate the average infection rate of bees by *Nosema* spp. spores using microscopy. In brief, the procedure involves crushing bee abdomens (60 bees) with a mortar and a pestle in ultrapure water at a rate of one millilitre (1 mL) per bee. The suspension is filtered through two layers of muslin (thin loosely woven cotton fabric) and centrifuged for six minutes at 800 × g to eliminate large debris and to purify the spores. The pellets are then resuspended to a homogeneous suspension in order to restore the initial dilution of 1 mL per bee. Finally, the sample is placed in a calibrated haemocytometer (Malassez counting chamber) and the microscopic examination is performed to detect and count *Nosema* spp. spores. The analytical results are both qualitative (negative versus positive) and quantitative (number *Nosema* spp. spores per mL, i.e. per bee, based on dilution). It should be noted that measurement uncertainty may be high for the visual counting method. It can vary depending on the number of *Nosema* spores and particles (e.g. pollen, yeast) present in the bee's digestive tract, which may interfere with the detection and identification of *Nosema* spores.

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The main steps in the molecular method are as follows. First, 80 µL of the suspension of crushed bee abdomens, prepared for the microscopic examination, is used to extract DNA using High Pure PCR Template Preparation Kit (Roche Diagnostics). The DNA extraction is performed following the "tissue" protocol without any change. The extracted DNA is resuspended in 200 µL of elution buffer according to the manufacturer's recommendations and stored at -20°C ± 5°C until further analysis (used as a template in the PCR). The PCR is performed as follows: 25 µL of the reaction mixture containing 1 U Platinum Taq DNA polymerase (Invitrogen), 0.4 µm of each primer, 0.4 mM dNTPs and adjusted with nuclease-free H₂O to reach a final reaction volume of 20 µL plus 5 µL of extracted DNA. The PCR reactions were run in an Eppendorf Mastercycler® Nexus ThermoCycler under the following cycling conditions: initial denaturation at 94°C for 2 min followed by 35 cycles of 30s at 94°C, 30s at 62°C and 30s at 72°C with a final extension of 7 min at 72°C. The PCR allows the identification of *Nosema* species (*N. apis* and *N. ceranae*) using the species-specific primers described by Martin-Hernandez (Martín-Hernández et al., 2007).

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## 2.3. Inter-comparison samples

The samples from the ANSES collection at Sophia-Antipolis laboratory originated from diagnostic analyses, field studies, experimental infections and collaborations. The panel included two types of samples: crushed *A. mellifera* abdomens, prepared according to the reference method described in the paragraph above, and a filtered suspension of *N. ceranae* spores. The status of each batch of samples (negative or positive for *Nosema* spp., defined spore load) was based on the results obtained with the two independent methods described above. The PCR also demonstrated that all positive samples were infected by *N. ceranae*.

Three negative *Nosema* spp. samples (NEG1, NEG2 and NEG3) were prepared using bees from the ANSES experimental apiary. Three positive *Nosema* spp. samples (POS1, POS2 and POS3) with different infectious loads (3.47E+06, 4.73E+05 and 1.84E+06 spores per mL, i.e. per bee respectively) were included in the panel (Table 1). POS1 and POS2 were prepared using bees naturally infected
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with *N. ceranae* from the EURL sample collection, while POS3 was a suspension of *N. ceranae* spores, prepared from experimentally infected emerging bees and filtered through a 100 µm mesh sieve.

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Each crushed sample was distributed in tubes with a volume of 400 µL per tube and the batches of tubes were stored at -20°C ± 5°C until shipment. The panels sent to participants included ten evaluation samples and a lure sample (Table 1). The lure sample was positive or negative, depending on the participant, was not evaluated and was added to limit the risk of interlaboratory collusion. Two positive *Nosema* spp. samples (POS2 and POS3) were sent in triplicate to assess the accuracy and the trueness of participants' results.

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## 2.4. Sample homogeneity and stability

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The EURL conducted homogeneity and stability tests. Homogeneity tests were performed for all sample batches between February and April 2017, i.e. before shipment. The homogeneity of each batch was tested by means of a duplicate analysis of ten randomly selected samples stored at -20°C. In total, 20 results per batch were obtained. The homogeneity criterion of the negative samples was defined as a number of spores per mL (or bee) not exceeding 2E+04. This corresponds to the detection limit of the microscopic counting method using the Malassez chamber. All the negative samples met this criterion, with no *Nosema* spp. spores detected in any of the selected samples. The homogeneity of the positive samples was evaluated by calculating the standard deviations (SD) between samples in compliance with the formula set out in Annex B of international standard NF ISO 13528 (NF ISO 13528, 2015). The analysis was carried out on the number of spores per mL expressed as decimal logarithm ($\log_{10}$), in order to facilitate data analysis. The homogeneity of the inter-comparison samples was validated against a target standard deviation value ($\sigma_{pt}$) of 0.2. This value was based on the results of an inter-laboratory validation test organised in 2016 within France's official laboratory network. As specified in Annex B (paragraph B.2.2) of international standard NF ISO 13528, it was necessary for inter-sample standard deviation (SD) to fall under the critical value of 0.3 $\sigma_{pt}$. SD values were calculated for the three positive samples. They ranged from 0.02 to 0.05 $\log_{10}$, while the SD/$\sigma_{pt}$ ratios
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were below 0.3 (Table 2). The homogeneity criterion was met and the positive samples were considered homogeneous.

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Stability tests were performed on positive batches. Stability was controlled by a duplicate analysis of three randomly selected samples stored at -20°C ± 5°C. In total, six results were obtained for each sample (Table 3). The tests were carried out one day after the shipment of panels to the participants (D-0) and at the end of the ILC period (deadline for sending in results) (D-20). The stability of the positive samples was evaluated in compliance with the criteria set out in Annex B (paragraph B.5.1) of international standard NF ISO 13528. The results of the stability tests at D-0 and D-20 were compared to those from the homogeneity tests. The difference between the mean of the homogeneity test results (mL) and the mean of the stability test results (m2) was less than the critical value of 0.3 σpt (i.e. |mL-m2| ≤ 0.3 σpt), thus validating the stability of the positive samples during the trial period (Table 3).

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## 2.5. Study design

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The ILC was organised in compliance with the quality requirements described in international standards ISO/IEC 17043 and ISO/IEC 17025 (NF EN ISO/IEC 17025, 2005, NF EN ISO/IEC 17043, 2015). The samples were packed and shipped between the EURL and NRLs in compliance with UN3373 regulations (Biological Substance, Category B).

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Each participating laboratory was anonymously coded with a 1- or 2-digit random number to ensure the confidentiality of results. Each of the samples to be blind-tested was coded with the attribution of a random number between 1 and 11. Participating laboratories received inter-comparison samples with a laboratory code on each tube. After receiving the package, the laboratories were required to store the samples at -20°C ± 5°C until analysis and to send back their results within 15 days. They were asked to report the results: i) qualitatively (detected versus not detected, with samples of ≤ 2 × 10⁴ spores / mL being considered as “not detected”), and ii) quantitatively (number of *Nosema* spp. spores per mL, corresponding to the number of *Nosema* spp. spores per bee). Participating laboratories were required to conduct calculations in compliance with their own analytical methods and, more particularly, according to the type of counting chamber used for analysis.
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Results were evaluated according to four performance criteria:

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1) Specificity, i.e. the ability of the laboratory to give a negative result for a negative sample (NF EN ISO 22117, 2010). The expected specificity rate was 100% of negative results.

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2) Sensitivity, i.e. the ability of the laboratory to give a positive result for a positive sample (NF EN ISO 22117, 2010). The expected sensitivity rate was 100% of positive results.

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3) Trueness, which was evaluated only for positive quantitative results by calculating the z-score in compliance with international standard NF ISO 13528 (NF ISO 13528, 2015).

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4) Precision, which was evaluated only for positive quantitative results, by calculating Mandel's k-value in compliance with international standard NF ISO 5725-2 (NF ISO 5725-2, 1994).

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## 2.6. Technical survey of the analytical methods employed by the ILC participants

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This study was the first step in a process to evaluate the level of harmonisation across the European NRL network for the diagnosis of *Nosema* spp. by microscopy. Participating laboratories were asked to use their own routine methods to analyse the ILC panel of samples. Concurrently with the test, and in order to gather information on these methods, the EURL asked participants to complete an online survey (using Sphinx iQ2 software, version 7.4.0.0, Le Sphinx Développement), detailing each stage of their routine procedure, from grinding the sample to interpreting the results.

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## 2.7. Statistical analysis of results

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In the first instance, a qualitative analysis was conducted in order to evaluate the sensitivity and specificity of results. Conformity was assessed using the reference values obtained during the homogeneity study. Sensitivity and specificity rates were calculated using the formula below, set out in standard NF ISO 22117 (NF EN ISO 22117, 2010):

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- Sensitivity rate: $r_{SE} = n_+ / E_{(n+tot)} \times 100\%$ (where n_+ is the number of positive results found and E_{(n+tot)} is the total number of expected positive samples).

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- Specificity rate: r<sub>SP</sub> = n. / E<sub>(n-tot)</sub> × 100% (where n is the number of negative results found and E<sub>(n-tot)</sub> is the total number of expected negative samples).

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## 3. Results

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### 3.1. Analysis of qualitative results

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Participants identified all the positive samples. Sensitivity was therefore satisfactory for all the participants and complied with the expected rate of 100%. However, the analysis revealed a non-compliance in specificity for the laboratory with code No. 21, which gave a positive result for sample NEG3. Specificity was therefore satisfactory for 95.6% of participants (22 of 23).

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Across the network of the 23 participating EU NRLs, the global sensitivity rate for results was 100% while the global specificity rate was 98.6% (Table 4).

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## 3.2. Analysis of quantitative results

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All the results of the participants were included in the analysis (Supplemental information, Table S1).

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The performance of participants was evaluated by sending three replicates of two different loaded samples (POS2 and POS3), as described in the section "Materials and methods". Figures 1A and 1B show the results of the 23 participants for sample POS2 and sample POS3 respectively. The values assigned to samples POS2 and POS3 are 5.57 and 6.19 $log_{10}$ respectively (with a standard deviation of 0.18 and 0.16 $log_{10}$). The mean and standard deviation were estimated through a robust analysis of all participant data, in compliance with algorithm A described in Appendix C of international standard NF ISO 13528. As the uncertainties relating to the values assigned to the two positive samples could be considered as negligible, they were not included in the interpretation of the results, making it possible to use a z-score for the evaluation.

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## 3.3. Assessment of the trueness of results

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The z-score values are shown in two histograms (Fig. 2). Figures 2A and 2B show the z-score of each participant for the three repetitions of results with POS2 and POS3, respectively.

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As a reminder, if the z-score is 0, the measured value (x) corresponds to the assigned value (x*). The interpretation of z-scores is set out in international standard NF EN ISO / IEC 17043 as: (i) if $|z| \le 2.0$ then the value of z is considered to be acceptable, (ii) if $2.0 < |z| < 3.0$ the value of z is considered to give a warning signal, and (iii) if $|z| \ge 3.0$ the value of z is considered to be unacceptable and generates an action signal.

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Five participants delivered unacceptable results, with a $|z| \ge 3.0$: the participants with code No. 20 and No. 27 for sample POS2, the participants with code No. 5 and No. 21 for sample POS3, and the participant with code No. 40 for both samples. These results were assessed as non-compliant.

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At ILC level, trueness was satisfactory (i.e. $|z| < 3.0$) for 92.8% of the results provided by participants.

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## 3.4. Assessment of the precision of results
For 23 participants and three repeats, the critical Mandel's k-value at the 1% significance level was 2.08. Given that less than 25% of k-values were below the critical value, the data of all participants could be used for analysis. The Mandel's k-values (Fig. 3) show that 87% of participants (20 laboratories) achieved good repeatability with both samples. Three participants encountered problems with this criterion: the participant with code No. 27 for sample POS2, and the participants with code No. 5 and No. 21 for sample POS3.

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324 3.5. *Technical survey of analytical methods employed*

The online survey was sent out in June 2017. Of the 23 ILC participants, seven laboratories (30%) were accredited for the diagnosis of nosemosis by microscopy. Most methods (75%) were recommended by or adapted from the OIE Manual. The other methods were internal or based on methods published in literature. One laboratory (No. 21) was using a method for the first time with the ILC.

A wide diversity of counting chambers was used for counting Nosema spp. spores (Fig. 4). The Bürker and Neubauer chambers were the most frequent choices (25% and 30% respectively). It should be noted that two participants did not perform routine counting, preferring to make a semi-quantitative evaluation of the Nosema spp. spore load (giving rise to analytical results such as “sporadic occurrence”, “weak infestation”, “moderate infestation”, “strong infestation”, depending on the number of spores observed per microscopic field).

About 60% of the laboratories used a single microscopic preparation (i.e. one microscopic slide prepared from the abdomen suspension), while around 40% used several slides (i.e. several microscopic slides prepared from the suspension) in order to calculate the mean of the different counts as their final result. Moreover, some laboratories carried out several counts on the same microscopic slide. One laboratory used two preparations, each slide being counted twice by different analysts.

To finish, it should be also noted that some laboratories adjusted the spore-counting protocol depending on the sample (e.g. spore load). In the event of a high number of spores, for instance, 60% of laboratories said that they diluted the suspension.
## 4. Discussion

The ILC was carried out with 23 NRLs from the EU. Its purpose was to evaluate their level of competence in using microscopy to detect and quantify *Nosema* spp. spores in crushed honey bee abdomens. Of the 23 participants, 18 laboratories (78%) obtained compliant results for all evaluation criteria (specificity, sensitivity, trueness and precision) (Table 5). Nine results, attributed to five participants, failed to meet the defined criteria: i) one for a lack of specificity, ii) five for a lack of trueness, iii) three for a lack of precision. A lack of trueness was observed for laboratory No. 40, which globally over-estimated the number of spores in the two positive samples included in triplicate as part of the panel (POS2 and POS3). This laboratory was accredited for the method used; its procedure was based on a single count with a Malassez chamber. The results of participant No. 20 showed a lack of trueness for sample POS2, tending - in contrast - towards under-estimation. The method employed by this laboratory for the ILC relied on a single microscopic preparation counted once with a Neubauer chamber. The results of participant No. 21 showed a lack of trueness, precision and specificity. It should be noted that this laboratory did not apply a routine method, which could explain these non-compliances. Likewise, participant No. 27 encountered problems of trueness and precision. This participant did not make routine use of a counting chamber, but gave semi-quantitative results expressed by crosses in the current analysis (the number of crosses depending on the number of *Nosema* spp. spores observed in the microscopic field). A lack of trueness and precision was also observed for laboratory No. 5, although this participant was using an accredited method, based on the preparation of two microscopic slides counted twice by two different analysts using a Bürker-Türk chamber.

Following the ILC, the laboratories undertook an investigation in collaboration with the EURL to identify and resolve the causes of the non-conformities observed. In one case, the anomaly of trueness was directly linked to an error in the formula used for converting the number of spores counted with the haemocytometer in "spores per mL". The problems of trueness and precision were also explained by a possible incomplete defrosting of the tubes and/or a lack of vortexing before analysis, resulting in
insufficient homogenisation of the samples. Moreover, one laboratory indicated that diluting the most heavily loaded samples would have improved the precision of its results and should have been carried out. However, it should be noted that some participants obtained satisfactory results in the ILC without diluting the samples. Although no reason was specifically determined, a confusion between *Nosema* spp. spores and refractive artefacts could explain the anomaly in specificity, given that the laboratory had little experience in using this method. In 2018, to check the efficacy of the corrective actions carried out, the EURL sent a second panel of samples to the two laboratories interested in taking part in a new assay. The results were satisfactory, proving that the causes of the non-conformities had been resolved.

The ILC on *Nosema* spp diagnosis by microscopy was the first assay involving the official laboratories of EU member states. Concerning the network of the 23 participating NRLs, test results showed global specificity of 98.6%, and global sensitivity of 100%. From a quantitative standpoint, trueness and precision were satisfactory for 92.8% and 93.5% of results respectively. These data demonstrated the performance of the network to provide the reliable analytical results that are essential to ensuring the quality of surveillance and study data. Although the analytical methods used by the NRLs were based on the same principle set out in the OIE Terrestrial Manual, they implemented different technical procedures for preparing microscopic slides and counting. The data collected did not reveal any apparent link between the diversity of procedures and the conformity of results.

The trial used crushed abdomen samples. It would not have been feasible to use whole bees as *Nosema* spp. infection in honeybees is heterogeneous between individuals and not experimentally controllable. However, using whole bees as inter-comparison samples would have made it possible to evaluate the analytical methods in their entirety, i.e. including the first stages of the method: sampling for analysis, bee preparation, buffer type, grinding process and filtration procedures. The data collected by the questionnaire revealed a wide diversity in practices that could also influence results. For instance, some laboratories do not use only bee abdomens but whole bees or digestive tracts only in their routine analyses (16% and 8% of the participants respectively). Moreover, around 40% of the laboratories do not filter the suspension before microscopic examination. The presence of a significant quantity of particles and artefacts (such as pollen or yeast present in the digestive tract) could have an
399 impact on the visual detection and counting of spores. A harmonised approach is certainly necessary
400 for these early stages of the method.

401 Finally, the survey found that most NRLs did not have a diagnosis threshold for the clinical disease of
402 nosemosis (i.e. a Nosema spp. spore load suggestive of an overt infection). Even when this threshold
403 did exist, it varied from one million to several million or nine million spores per bee for N. apis (20%
404 of laboratories having a threshold for this species). In the case of N. ceranae, two laboratories had a
405 diagnosis threshold of one million of spores per bee. A recent study conducted in North America
406 (Canada, Ontario) found that high levels of N. ceranae infections were significantly associated with
407 reduced bee populations and food stores in colonies, and indeed suggested an intervention threshold of
408 one million of spores per bee (Emsen et al., 2020). Several laboratories also said that the clinical signs
409 observed in apiaries were considered in the interpretation of results. Harmonising the way in which
410 results are interpreted, taking account not only of the spore load, but also the clinical signs and
411 associated field information (e.g. presence of other sources of stress), would consolidate diagnostic
412 modalities for nosemosis in the long term.

413 To conclude, the results of the ILC on Nosema spp. spore detection and counting were satisfactory
414 overall. However, it should be mentioned that the panel, consisting of ten samples (three negatives and
415 three positives, including two in triplicate) was relatively small. Further investigations with larger
416 sample sizes should be conducted to consolidate the results and the conclusions of this study. In
417 addition, the small number of anomalies identified must be relativised in view of the significant
418 measurement uncertainties of the visual counting method and the absence of a scientifically
419 established threshold for the diagnosis of N. apis and N. ceranae.

420

421

**Declaration of Competing interest**

422 The authors confirm that there are no known conflicts of interest associated with this publication.

423

424

**Funding**
425 This project was supported by the European Commission, Directorate General for Health and Food

426 Safety, Brussels.
<table><caption>427 Table 1. Composition of the test panel of samples sent to participating laboratories.</caption><thead><tr><th rowspan="2">Samples</th><th rowspan="2">Detection</th><th colspan="2">Microscopic examination</th><th rowspan="2">Nosema species</th><th rowspan="2">Criterion evaluated</th></tr><tr><th>Counting** (spores/ml)</th><th>SD*** (spores/ml)</th></tr></thead><tbody><tr><td>POS1</td><td>Positive</td><td>3.47E+06</td><td>2.83E+05</td><td>N. ceranae</td><td>Sensitivity</td></tr><tr><td>POS2*</td><td>Positive</td><td>4.73E+05</td><td>8.39E+04</td><td>N. ceranae</td><td>Sensitivity</td></tr><tr><td>POS3*</td><td>Positive</td><td>1.84E+06</td><td>2.01E+05</td><td>N. ceranae</td><td>Sensitivity</td></tr><tr><td>NEG1</td><td>Negative</td><td>-</td><td>-</td><td>-</td><td>Specificity</td></tr><tr><td>NEG2</td><td>Negative</td><td>-</td><td>-</td><td>-</td><td>Specificity</td></tr><tr><td>NEG3</td><td>Negative</td><td>-</td><td>-</td><td>-</td><td>Specificity</td></tr><tr><td>Lure</td><td>Positive or negative</td><td>7.04E+05</td><td>1.96E+05</td><td>N. ceranae</td><td>not evaluated</td></tr></tbody></table>

428 * Samples tested in triplicate

429 ** Mean of the homogeneity study results

430 *** Standard deviation evaluated within the homogeneity study

431
<table><caption>Table 2. Homogeneity of positive samples</caption><thead><tr><th>Sample code</th><th>m1</th><th>SD</th><th>SD/σpt</th><th>Criteria SD/σpt ≤ 0.3</th></tr></thead><tbody><tr><td>POS1</td><td>6.54</td><td>0.02</td><td>0.11</td><td>OK</td></tr><tr><td>POS2</td><td>5.67</td><td>0.05</td><td>0.22</td><td>OK</td></tr><tr><td>POS3</td><td>6.26</td><td>0.03</td><td>0.19</td><td>OK</td></tr></tbody></table>

m1: log₁₀ of the mean of the results obtained by the homogeneity study

SD: inter-sample standard deviation

SD/σpt≤ 0.3: homogeneity criterion according to Annex B of international standard NF ISO 13528
<table><caption>Table 3. Stability of positive samples</caption><thead><tr><th></th><th>Sample code</th><th>m1</th><th>m2</th><th>m1-m2</th><th>(m1-m2)/σpt</th><th>Criteria (m1-m2)/σpt ≤ 0.3</th></tr></thead><tbody><tr><td rowspan="3">D0</td><td>POS1</td><td>6.54</td><td>6.50</td><td>0.04</td><td>0.17</td><td>OK</td></tr><tr><td>POS2</td><td>5.67</td><td>5.63</td><td>0.04</td><td>0.17</td><td>OK</td></tr><tr><td>POS3</td><td>6.26</td><td>6.22</td><td>0.04</td><td>0.23</td><td>OK</td></tr><tr><td rowspan="3">D20</td><td>POS1</td><td>6.54</td><td>6.50</td><td>0.04</td><td>0.18</td><td>OK</td></tr><tr><td>POS2</td><td>5.67</td><td>5.63</td><td>0.04</td><td>0.18</td><td>OK</td></tr><tr><td>POS3</td><td>6.26</td><td>6.21</td><td>0.05</td><td>0.28</td><td>OK</td></tr></tbody></table>

442

m1: log₁₀ of the mean of the results obtained by the homogeneity study

m2: log₁₀ of the mean of the results obtained by the stability study

(m1-m2)/σpt ≤ 0.3: stability criterion according to Annex B of international standard NF ISO 13528

446
<table><caption>447 Table 4. Sensitivity and specificity rates attained by each participating laboratory.</caption><thead><tr><th>Laboratory Code</th><th>Sensitivity<sup>a</sup> (%)</th><th>95% confidence interval (%)</th><th>Specificity<sup>b</sup> (%)</th><th>95% confidence interval (%)</th></tr></thead><tbody><tr><td>lab1</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab2</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab3</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab4</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab5</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab6</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab7</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab8</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab15</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab16</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab17</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab18</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab20</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab21</td><td>100</td><td>64.6 to 100</td><td>66.7</td><td>20.8 to 93.8</td></tr><tr><td>lab22</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab23</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab24</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab25</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab26</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab27</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab38</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab40</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr><tr><td>lab41</td><td>100</td><td>64.6 to 100</td><td>100</td><td>43.9 to 100</td></tr></tbody><tfoot><tr><td>Overall</td><td>100 (161/161)</td><td>97.7 to 100</td><td>98.6 (68/69)</td><td>92.2 to 99.7</td></tr></tfoot></table>

448

a Calculation of the sensitivity rate was based on 7 samples (see Table 1).

449

b Calculation of the specificity rate was based on 3 samples (see Table 1).

450
<table><caption>Table 5. Participant fulfilment of proficiency test criteria.</caption><thead><tr><th rowspan="2">Criteria</th><th colspan="20">Participant code</th></tr><tr><th>1</th><th>2</th><th>3</th><th>4</th><th>5</th><th>6</th><th>7</th><th>8</th><th>15</th><th>16</th><th>17</th><th>18</th><th>20</th><th>21</th><th>22</th><th>23</th><th>24</th><th>25</th><th>26</th><th>27</th><th>38</th><th>40</th><th>41</th></tr></thead><tbody><tr><th>Specificity</th><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td></tr><tr><th>Sensitivity</th><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td></tr><tr><th>Trueness</th><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>No</td><td>No</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>No</td><td>Yes</td><td>No</td></tr><tr><th>Precision</th><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>Yes</td><td>No</td><td>Yes</td><td>Yes</td></tr></tbody></table>

452

453
454 Supplemental information

455 Table S1. Raw data, mean and standard deviation (expressed in log₁₀) obtained by each participating
456 laboratory.

<table><thead><tr><th rowspan="2">Laboratory Code</th><th colspan="10">Sample code</th></tr><tr><th>POS1<sup>a</sup></th><th>POS2<sup>b</sup></th><th>POS2 Mean (SD)</th><th>POS3<sup>b</sup></th><th>POS3 Mean (SD)</th></tr></thead><tbody><tr><td>lab1</td><td>6.25</td><td>5.46</td><td>5.35</td><td>5.37</td><td>5.40 (0.06)</td><td>6.06</td><td>5.98</td><td>6.01</td><td>6.02 (0.04)</td></tr><tr><td>lab2</td><td>6.46</td><td>5.64</td><td>5.38</td><td>5.60</td><td>5.54 (0.14)</td><td>6.17</td><td>6.26</td><td>6.24</td><td>6.22 (0.04)</td></tr><tr><td>lab3</td><td>6.28</td><td>5.35</td><td>5.11</td><td>5.20</td><td>5.22 (0.12)</td><td>5.92</td><td>6.08</td><td>5.89</td><td>5.96 (0.11)</td></tr><tr><td>lab4</td><td>6.44</td><td>5.54</td><td>5.48</td><td>5.60</td><td>5.54 (0.06)</td><td>6.16</td><td>6.27</td><td>6.30</td><td>6.24 (0.07)</td></tr><tr><td>lab5</td><td>6.52</td><td>5.82</td><td>5.92</td><td>5.93</td><td>5.89 (0.06)</td><td>6.28</td><td>6.71</td><td>6.68</td><td>6.56 (0.24)</td></tr><tr><td>lab6</td><td>6.40</td><td>5.62</td><td>5.61</td><td>5.67</td><td>5.63 (0.03)</td><td>6.30</td><td>6.23</td><td>6.27</td><td>6.26 (0.04)</td></tr><tr><td>lab7</td><td>6.66</td><td>5.64</td><td>5.63</td><td>5.70</td><td>5.66 (0.04)</td><td>6.40</td><td>6.20</td><td>6.38</td><td>6.33 (0.11)</td></tr><tr><td>lab8</td><td>5.89</td><td>5.61</td><td>5.63</td><td>5.64</td><td>5.63 (0.02)</td><td>6.22</td><td>6.21</td><td>6.16</td><td>6.20 (0.04)</td></tr><tr><td>lab15</td><td>6.42</td><td>5.67</td><td>5.62</td><td>5.68</td><td>5.65 (0.03)</td><td>6.49</td><td>6.26</td><td>6.30</td><td>6.35 (0.12)</td></tr><tr><td>lab16</td><td>6.42</td><td>5.51</td><td>5.61</td><td>5.52</td><td>5.55 (0.05)</td><td>6.13</td><td>6.13</td><td>6.10</td><td>6.12 (0.02)</td></tr><tr><td>lab17</td><td>6.40</td><td>5.61</td><td>5.62</td><td>5.59</td><td>5.61 (0.01)</td><td>6.23</td><td>6.28</td><td>6.25</td><td>6.25 (0.03)</td></tr><tr><td>lab18</td><td>6.36</td><td>5.48</td><td>5.85</td><td>5.54</td><td>5.62 (0.20)</td><td>6.19</td><td>6.26</td><td>6.24</td><td>6.23 (0.03)</td></tr><tr><td>lab20</td><td>5.98</td><td>5.35</td><td>5.18</td><td>5.00</td><td>5.18 (0.18)</td><td>5.75</td><td>5.92</td><td>5.72</td><td>5.80 (0.11)</td></tr><tr><td>lab21</td><td>5.96</td><td>5.23</td><td>5.64</td><td>5.65</td><td>5.50 (0.24)</td><td>6.07</td><td>5.48</td><td>5.78</td><td>5.78 (0.29)</td></tr><tr><td>lab22</td><td>6.29</td><td>5.56</td><td>5.58</td><td>5.56</td><td>5.56 (0.01)</td><td>6.18</td><td>6.20</td><td>6.22</td><td>6.20 (0.02)</td></tr><tr><td>lab23</td><td>6.35</td><td>5.66</td><td>5.76</td><td>5.78</td><td>5.73 (0.06)</td><td>6.20</td><td>6.29</td><td>6.23</td><td>6.24 (0.05)</td></tr><tr><td>lab24</td><td>6.46</td><td>5.74</td><td>5.85</td><td>5.81</td><td>5.80 (0.05)</td><td>6.24</td><td>6.26</td><td>6.28</td><td>6.26 (0.02)</td></tr><tr><td>lab25</td><td>6.08</td><td>5.35</td><td>5.38</td><td>5.34</td><td>5.36 (0.02)</td><td>5.87</td><td>6.17</td><td>6.16</td><td>6.07 (0.17)</td></tr><tr><td>lab26</td><td>6.40</td><td>5.81</td><td>5.74</td><td>5.51</td><td>5.69 (0.16)</td><td>6.16</td><td>6.27</td><td>6.31</td><td>6.25 (0.08)</td></tr><tr><td>lab27</td><td>6.29</td><td>5.00</td><td>5.66</td><td>5.38</td><td>5.35 (0.33)</td><td>6.13</td><td>6.03</td><td>5.88</td><td>6.01 (0.12)</td></tr><tr><td>lab38</td><td>6.40</td><td>5.51</td><td>5.44</td><td>5.46</td><td>5.47 (0.04)</td><td>6.17</td><td>6.10</td><td>6.22</td><td>6.16 (0.06)</td></tr><tr><td>lab40</td><td>6.86</td><td>6.15</td><td>6.15</td><td>5.78</td><td>6.02 (0.21)</td><td>6.81</td><td>6.79</td><td>6.95</td><td>6.85 (0.09)</td></tr><tr><td>lab41</td><td>6.40</td><td>5.51</td><td>5.60</td><td>5.56</td><td>5.56 (0.05)</td><td>6.25</td><td>6.25</td><td>6.23</td><td>6.24 (0.01)</td></tr></tbody></table>

457

458 a log₁₀ of the POS1 sample: one result per participant.

459 b log₁₀ of the POS2 and POS3: three results per participant.

460

461
Figure 1. Experimental results of participants when quantifying *Nosema* spp. spores in the ILC samples. Each participant tested three replicates of the POS2 sample (Fig 1A) and three replicates of the POS3 sample (Fig 1B). The bullet points indicate the number of *Nosema* spp. spores per bee (expressed in log₁₀/bee) quantified by microscopy. The empty red box is the mean value found by each participant. The red lines indicate the robust mean (X).

<figure><img src="image_2.png" /></figure>

<figure><img src="image_3.png" /></figure>
Figure 2. Z-scores calculated for each participant quantifying *Nosema* spores in the inter-laboratory comparison samples. The boxes indicate the individual z-score for the three replicates of the POS2 sample (Fig 2A) and POS3 sample (Fig 2B) tested by each participant. The yellow and red lines indicate the limits of ± 2 and ± 3 respectively.

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

474

475

476
Figure 3. Mandel’s k-value calculated for each participant quantifying *Nosema* spores in the inter-laboratory comparison samples. The boxes indicate the individual z-score for the three replicates of the POS2 sample (Fig 3A) and POS3 sample (Fig 3B) tested by each participant. The 1% significance level is indicated by the red line (k = 2.08).

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

481

482

483

484
485 Figure 4. Type of counting chambers used by the 23 participants.

<figure><img src="image_8.png" /></figure>

486
487
488 References

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492 Calderón, R.A., Sanchez, L.A., Yañez, O., Fallas, N., 2008. Presence of *Nosema ceranae* in 493 Africanized honey bee colonies in Costa Rica. *J. Apic. Research.* 47, 328-329.

494 Cantwell, G.E., 1970. Standard methods for counting Nosema spores. Am. Bee Journal. 110, 222-223.

495 Chemurot, M., De Smet, L., Brunain, M., De Rycke, R., de Graaf, D.C., 2017. Nosema neumanni n. 496 sp. (Microsporidia, Nosematidae), a new microsporidian parasite of honeybees, Apis mellifera in 497 Uganda. Eur. J. Protistology. 61, 13-19.

498 Chen, Y., Evans, J.D., Smith, I.B., Pettis, J.S., 2008. Nosema ceranae is a long-present and wide- 499 spread microsporidian infection of the European honey bee (Apis mellifera) in the United States. J. 500 Invertebr. Pathol. 97, 186-188.

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