Community Research and Development Information Service - CORDIS

Tool-kit for detection of defined Microsporidia

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 7 down to 10 spores diluted in 100 of either (i) water or (ii) host bumble bee homogenate to simulate natural N.bombi infection (equivalent to the DNA from 10 6 spores down to 1 spore per

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-speci W c 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 and the tools described, reduce the detection level of Microsporidia in bumble bees. This will aid in keeping breeding facilities free from Microsporidia infections.

The method has been described in detail in scientific media and a lab manual for using the tool kit made available on the project web site and in a public technical report from the project presented at the project End User meeting.

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Queen's University of Belfast
University Road
BT7 1NN Belfast
United Kingdom
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