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FP5

POLLINATOR PARASITES Résumé de rapport

Project ID: QLK5-CT-2002-00741
Financé au titre de: FP5-LIFE QUALITY
Pays: United Kingdom

Molecular genetic tools for the analysis of Microsporidia in Bombus spp

A polymerase chain reaction (PCR) based method was developed for the specific and sensitive diagnosis of the microsporidian parasite Nosema bombi in bumble bees (Bombus spp.). Four primer pairs, amplifying ribosomal RNA (rRNA) gene fragments, were tested on N. bombi and the related microsporidia Nosema apis and Nosema ceranae, both of which infect honey bees.

Only primer pair Nbombi-SSU-Jf1/Jr1 could distinguish N.bombi (323 bp amplicon) from these other bee parasites. Primer pairs Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2 were then tested for their sensitivity with N. bombi spore concentrations from 10 000 000 down to 10 spores diluted in 100 microliters of either (i) water or (ii) host bumble bee homogenate to simulate natural N. bombi infection (equivalent to the DNA from 1 000 000 spores down to 1 spore per PCR).

Though the N.bombi -speci W c primer pair Nbombi-SSU-Jf1/Jr1 was relatively insensitive, as few as 10 spores per extract (equivalent to 1 spore per PCR) were detectable using the N. bombi -non-specific primer pair ITS-f2/r2, which amplifies a short fragment of È120 bp. Testing 99 bumble bees for N. bombi infection by light microscopy versus PCR diagnosis with the highly sensitive primer pair ITS-f2/r2 showed the latter to be more accurate. PCR diagnosis of N. bombi using a combination of two primer pairs (Nbombi-SSU-Jf1/Jr1 and ITS-f2/r2) provides increased specificity, sensitivity, and detection of all developmental stages compared with light microscopy.

The results have been published in scientific media.

Development of sensitive and specific tools for Microsporidia detection allows for the further development of protocols that can be used in any laboratory equipped for routine PCR analysis. This should have interest for molecular biologists as well as for those interested in bumble bee pathology.

Contact

Robert PAXTON, (Head of Unit)
Tél.: +44-2890972127
Fax: +44-2890975877
E-mail