Development of a loop-mediated isothermal amplification (LAMP) and a direct LAMP for the specific detection of Nosema ceranae, a parasite of honey bees

Lannutti, Lucas; Mira, Anabela; Basualdo, Marina; Rodriguez, Graciela; Erler, Silvio; Silva, Victoria; Gisder, Sebastian; Genersch, Elke; Florin-Christensen, Mónica; Schnittger, Leonhard

doi:10.1007/s00436-020-06915-w

Arthropods and Medical Entomology - Original Paper
Published: 12 October 2020

Development of a loop-mediated isothermal amplification (LAMP) and a direct LAMP for the specific detection of Nosema ceranae, a parasite of honey bees

Parasitology Research volume 119, pages 3947–3956 (2020)Cite this article

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Abstract

Nosema ceranae is a ubiquitous microsporidian pathogen infecting the midgut of honey bees. The infection causes bee nosemosis, a disease associated with malnutrition, dysentery, and lethargic behavior, and results in considerable economic losses in apiculture. The use of a rapid, sensitive, and inexpensive DNA-based molecular detection method assists in the surveillance and eventual control of this pathogen. To this end, a loop-mediated isothermal amplification (LAMP) assay targeting the single-copy gene encoding the polar tube protein 3 (PTP3) has been developed. Genomic DNA of N. ceranae–infected forager bees sampled from distant geographic regions could be reliably amplified using the established LAMP assay. The N. ceranae-LAMP showed higher sensitivity than a classical reference PCR (98.6 vs 95.7%), when both approaches were applied to the detection of N. ceranae. LAMP detected a ten-fold lower infection rate than the reference PCR (1 pg vs 10 pg genomic DNA, respectively). In addition, we show highly specific and sensitive detection of N. ceranae from spore preparations in a direct LAMP format. No cross-reactions with genomic DNA and/or spores from N. apis, often co-infecting A. mellifera, or from N. bombi, infecting bumble bees, were observed. This low-cost and time-saving molecular detection method can be easily applied in simple laboratory settings, facilitating a rapid detection of N. ceranae in honey bees in epidemiological studies, surveillance and control, as well as evaluation of therapeutic measures against nosemosis.

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Funding

This research has been financially supported by grants from the University of Moron, Argentina (PI3/18-09-LS-07, Code: 800201702000023UM), the Argentine Ministry of Science, Technology and Productive Innovation (PICT-2014-1585), and the National Institute of Agricultural Technology, Argentina (2019-PE-E1-I017-001).

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Escuela Superior de Ciencias Exactas y Naturales, Universidad de Morón, Morón, Argentina
Lucas Lannutti, Mónica Florin-Christensen & Leonhard Schnittger
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Buenos Aires, Argentina
Lucas Lannutti, Mónica Florin-Christensen & Leonhard Schnittger
Instituto de Patobiología Veterinaria, CICVyA, INTA-Castelar, Los Reseros y Nicolas Repetto s/n, 1686 Hurlingham, Buenos Aires, Argentina
Anabela Mira, Victoria Silva, Mónica Florin-Christensen & Leonhard Schnittger
Facultad de Ciencias Veterinarias, PROANVET, Universidad Nacional del Centro de la Provincia de Buenos Aires, Tandil, 7000, Buenos Aires, Argentina
Marina Basualdo
Instituto Nacional de Tecnología Agropecuaria EEA Hilario Ascasubi, Hilario Ascasubi, Buenos Aires, Argentina
Graciela Rodriguez
Institut für Biologie, Molekulare Ökologie, Martin-Luther-Universität Halle-Wittenberg, 06099, Halle (Saale), Germany
Silvio Erler
Institute for Bee Protection, Julius Kühn-Institut, Federal Research Centre for Cultivated Plants, 38104, Braunschweig, Germany
Silvio Erler
Department of Molecular Microbiology and Bee Diseases, Institute for Bee Research, Hohen Neuendorf, Germany
Sebastian Gisder & Elke Genersch

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Lucas Lannutti
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Anabela Mira
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Marina Basualdo
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Graciela Rodriguez
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Silvio Erler
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Victoria Silva
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Sebastian Gisder
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Elke Genersch
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Mónica Florin-Christensen
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Leonhard Schnittger
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Contributions

Conceptualization: Leonhard Schnittger. Formal analysis: Lucas Lannutti and Leonhard Schnittger. Investigation: Lucas Lannutti, Anabela Mira, and Victoria Silva. Resources: Marina Basualdo, Graciela Rodriguez, Silvio Erler, Sebastian Gisder, and Elke Genersch. Writing–original draft: Lucas Lannutti, Monica Florin-Christensen, and Leonhard Schnittger. Writing–review and editing: Silvio Erler, Elke Genersch, and Leonhard Schnittger. Visualization: Lucas Lannutti. Supervision: Leonhard Schnittger. Funding acquisition: Leonhard Schnittger, Marina Basualdo, and Monica Florin-Christensen.

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Correspondence to Leonhard Schnittger.

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Lannutti, L., Mira, A., Basualdo, M. et al. Development of a loop-mediated isothermal amplification (LAMP) and a direct LAMP for the specific detection of Nosema ceranae, a parasite of honey bees. Parasitol Res 119, 3947–3956 (2020). https://doi.org/10.1007/s00436-020-06915-w

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Received: 12 August 2020
Accepted: 29 September 2020
Published: 12 October 2020
Issue Date: December 2020
DOI: https://doi.org/10.1007/s00436-020-06915-w

Keywords

Loop-mediated isothermal amplification
LAMP
Microsporidia
Nosema ceranae
Nosema apis
Nosemosis
Apis mellifera
Apis cerana