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Optimization of duplex real-time PCR with melting-curve analysis for detecting the microsporidian parasites Nosema apis and Nosema ceranae in Apis mellifera1

  • Karen L. Burgher-MacLellan (a1), Geoffrey R. Williams (a2), Dave Shutler (a3), Kenna MacKenzie (a1) and Richard E.L. Rogers (a4)...

Honey bees, Apis mellifera (L.) (Hymenoptera: Apidae), are parasitized by the microsporidians Nosema apis (Zander) and Nosema ceranae (Fries). Molecular techniques are commonly used to differentiate between these parasites because light microscopy is inadequate. Our objectives were to (i) adapt the previously published duplex polymerase chain reaction (PCR) targeting the 16S rRNA gene of N. apis (321APIS-FOR, 321APIS-REV) and N. ceranae (218MITOC-FOR, 218MITOC-REV) using qualitative real-time PCR assay with SYBR® Green I dye (R-T PCR) and DNA melting-curve analysis, and (ii) determine whether the two Nosema species can be detected simultaneously in honey bees. Total spore counts and purified total genomic DNA were obtained from 37 bee samples (19 individual workers and 18 pooled samples of 15 workers) collected in Nova Scotia, Prince Edward Island, and Newfoundland, Canada. Overall, the prevalence of Nosema species was 86.5% (32/37 samples of bee DNA), based on conventional PCR and the optimized R-T PCR assay. The melting-curve analysis showed three groups of curve profiles that could determine the prevalence of N. apis, N. ceranae, and co-infection (21.9%, 56.2%, and 21.9%, respectively). The duplex R-T PCR assay was efficient, specific, and more sensitive than duplex conventional PCR because co-infection was identified in 5.4% (n = 2) more samples. Sequencing of R-T PCR products confirmed the results of the melting-curve analysis. Duplex R-T PCR with melting-curve analysis is a sensitive and rapid method of detecting N. apis, N. ceranae, and co-infection in honey bees.


Les abeilles domestiques, Apis mellifera (L.) (Hymenoptera: Apidae) sont parasitées par les microsporidies Nosema apis (Zander) et N. ceranae (Fries). Parce que la microscopie optique est inadéquate, on utilise couramment des méthodes moléculaires pour distinguer ces parasites. Nos objectifs sont 1) d'adapter la méthode déjà publiée de la réaction de PCR (amplification en chaîne par polymérase) duplex qui cible le gène 16S de l'ARNr de N. apis (321APIS-FOR et 321APIS-REV) et de N. ceranae (218MITOC-FOR et 218MITOC-REV) à l'aide d'un test qualitatif au vert de SYBR I en temps réel avec une analyse de la courbe de fusion de l'ADN (R-T PCR) et 2) de voir s’il est possible de détecter simultanément les deux espèces de Nosema chez les abeilles. Nous avons obtenu les dénombrements de spores et l'ADN génomique total purifié dans 37 échantillons d'abeilles (19 ouvrières individuelles et 18 échantillons collectifs de 15 ouvrières) récoltés en Nouvelle-Écosse, à l'Île-du-Prince-Édouard et à Terre-Neuve, Canada. La prévalence globale de Nosema est de 86,5 % (32/37 échantillons d'ADN d'abeilles) d'après les analyses de PCR conventionnelle et de R-T PCR optimisée. L’analyse de la courbe de fusion révèle l'existence de trois groupes de profils de courbes qui permettent d'identifier les prévalences de N. apis, de N. ceranae et de co-infections (respectivement 21,9 %, 56,2 % et 21,9 %). Le test de la R-T PCR duplex est efficace, spécifique et plus sensible que la PCR duplex ordinaire parce que la co-infection a pu être décelée dans 5,4 % (n=2) plus d'échantillons. Le séquençage des produits de la R-T PCR confirme les résultats de l'analyse de la courbe de fusion. La PCR duplex au vert SYBR I en temps réel avec une analyse de la courbe de fusion est une méthode sensible et rapide de détection de N. apis, de N. ceranae et des co-infections chez les abeilles.

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Contribution No. 2365 from the Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, Kentville, Nova Scotia.

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O. Berry , and S.D. Sarre 2007. Gel-free species identification using melt-curve analysis. Molecular Ecology Notes, 7: 14. doi:10.1111/j.1471-8286.2006.01541.x.

S.A. Bustin , V. Benes , J.A. Garson , J. Hellemans , J. Huggett , M. Kubista , 2009. The MIQE guidelines: minimum information for the publication of quantitative real-time PCR experiments. Clinical Chemistry, 55: 611622. PMID:19246619 doi:10.1373/clinchem.2008.112797.

T. Cavalier-Smith 1987. Eukaryotes with no mitochondria. Nature (London), 326: 332333. PMID:3561476 doi:10.1038/326332a0.

Y. Chen , J.D. Evans , I.B. Smith , and J.S. Pettis 2008. Nosema ceranae is a long-present and widespread microsporidian infection of the European honey bee (Apis mellifera) in the United States. Journal of Invertebrate Pathology, 97: 186188. PMID:17880997 doi:10.1016/j.jip.2007.07.010.

Y. Chen , Y.D. Evans , L. Zhou , H. Boncristiani , K. Kimura , T. Xiao , A.M. Litkowski , and J.S. Pettis 2009. Asymmetrical coexistence of Nosema ceranae and N. apis in honey bees. Journal of Invertebrate Pathology, 101: 204209. PMID:19467238 doi:10.1016/j.jip.2009.05.012.

I. Fries 1993. Nosema apis: a parasite in the honey bee colony. Bee World, 74: 519.

I. Fries , F. Feng , A. da Silva , S.B. Slemenda , and N.J. Pieniazek 1996. Nosema ceranae n. sp. (Microspora, Nosematidae): morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). European Journal of Protistology, 32: 356365.

P.D.M. Hebert , K. Cywinska , S.L. Ball , and J.R. deWaard 2003. Biological identification through DNA bar codes. Proceedings of the Royal Society of London B, Biological Sciences, 270: 313321. doi:10.1098/rspb.2002.2218.

M. Higes , R. Martín , and A. Meana 2006. Nosema ceranae, a new microsporidian parasite in honey bees in Europe. Journal of Invertebrate Pathology, 92: 9395. PMID:16574143 doi:10.1016/j.jip.2006.02.005.

M. Higes , P. García-Palencia , R. Martín-Hernández , and A. Meana 2007. Experimental infection of Apis mellifera honeybees with Nosema ceranae (Microsporidia). Journal of Invertebrate Pathology, 94: 211217. PMID:17217954 doi:10.1016/j.jip.2006.11.001.

M. Higes , R. Martín-Hernández , C. Botías , E. Garrido Bailón , A.V. González-Porto , L. Barrios , 2008. How natural infection by Nosema ceranae causes honeybee colony collapse. Environmental Microbiology, 10: 26592669. PMID:18647336 doi:10.1111/j.1462-2920.2008.01687.x.

M. Higes , R. Martín-Hernández , E. Garrido Bailón , C. Botías , and A. Meana 2009. The presence of Nosema ceranae (Microsporidia) in North African honey bees (Apis mellifera intermissa). Journal of Apicultural Research, 48: 217219. doi:10.3896/IBRA.

W.F. Huang , J.H. Jiang , and C.H. Wang 2007. A Nosema ceranae isolate from the honey bee Apis mellifera. Apidologie, 38: 3037. doi:10.1051/apido:2006054.

J. Klee , A.M. Besana , E. Genersch , S. Gisder , A. Nanetti , D.Q. Tam , 2007. Widespread dispersal of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera. Journal of Invertebrate Pathology, 96: 110. PMID:17428493 doi:10.1016/j.jip.2007.02.014.

R. Martín-Hernández , A. Meana , L. Prieto , A.M. Salvador , E. Garrido-Bailon , and M. Higes 2007. Outcome of colonization of Apis mellifera by Nosema ceranae. Applied and Environmental Microbiology, 73: 63316338. PMID:17675417 doi:10.1128/AEM.00270-07.

R. Martín-Hernández , A. Meana , P. García-Palenci , P. Marín , C. Botías , E. Garrido-Bailón , 2009. Effect of temperature on the biotic potential of honeybee microsporidia. Applied and Environmental Microbiology. 75: 25542557. PMID: 19233948 doi:10.1128/AEM.02908-08.

R.J. Paxton , J. Klee , S. Korpela , and I. Fries 2007. Nosema ceranae has infected Apis mellifera in Europe since at least 1998 and may be more virulent than Nosema apis. Apidologie, 38: 558565. doi:10.1051/apido:2007037.

K.M. Ririe , R.P. Rasmussen , and C.T. Wittwer 1997. Product differentiation by analysis of DNA melting curves during polymerase chain reaction. Analytical Biochemistry, 245: 154160. PMID:9056205 doi:10.1006/abio.1996.9916.

G.R. Williams , A.B.A. Shafer , R.E.L. Rogers , D. Shutler , and D.T. Stewart 2008 a. First detection of Nosema ceranae, a microsporidian parasite of European honey bees (Apis mellifera), in Canada and central U.S.A. Journal of Invertebrate Pathology, 97: 189192. PMID:17897670 doi:10.1016/j.jip.2007.08.005.

G.R. Williams , M.A. Sampson , D. Shutler , and R.E.L. Rogers 2008 b. Does fumagillin control the recently detected invasive parasite Nosema ceranae in western honey bees (Apis mellifera)? Journal of Invertebrate Pathology, 99: 342344. PMID:18550078 doi:10.1016/j.jip.2008.04.005.

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The Canadian Entomologist
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