Detection of Nosema ceranae in honey bees from Croatia

Abstract

honey bee, distribution, Nosema apis, Nosema ceranae, Croatia Journal of Apicultural Research and Bee World 49(4): 340-341 (2010) © IBRA 2010 DOI: 10.3896/IBRA.1.49.4.08 Previously, Nosema infections in European honey bees have been attributed solely to N. apis (Ellis and Munn, 2005), but it now appears that N. ceranae is an emerging pathogen that has increased its distribution (Klee et al., 2007). Nosema ceranae has not been detected in Croatia to date, but we have suspected its presence in pure or mixed infections with N. apis, because of a high percentage of Nosema infections being found during summer, and, because N. ceranae has been diagnosed in some neighbouring countries. The aim of this research was to determine the presence of N. ceranae, its prevalence and distribution in all 21 districts of Croatia, using a multiplex PCR. The diversity of Nosema species was studied using 204 honey bee samples originating mainly from bee colonies with different pathological problems like depopulation, weakness or high colony mortality. Samples of dead bees were collected during the first spring inspection of colonies, in February and March 2009. Each common sample represented one location or one apiary. Samples of honey bees were analyzed according to the OIE Manual (Anon, 2008). After light microscopy, a total of 150 spore samples from different localities were selected and investigated by multiplex PCR. Extraction of genomic DNA and further molecular analysis was performed as follows: for each of selected suspensions of isolated Nosema spores, an aliquot of 50 μl was transferred to a fresh tube, boiled at 100°C for 30 mins and centrifuged at 14,000 g for 10 mins. Thirty μl of supernatant was removed and supplemented with 10x TE buffer to a final concentration of 10mM Tris and 5mM EDTA, pH8. This supernatant served as source of template DNA and was stored at -20°C, or, used immediately for multiplex PCR (Higes et al., 2006). Primers were selected, taking into account that primer sequences were specific to each of the two species, and that both amplicons could be simultaneously amplified (Anon, 2008) and separated using agarose gel electrophoresis for visualization of results. The molecular size of PCR products were determined by electrophoresis in a 2% agarose TAE (Tris-acetate-ethylene diamine tetra-acetic acid) gel in standard TAE buffer, stained with SYBR green, and visualized using UviTec gel documentation system. Results of microscopic examinations about presence of Nosema spores in samples are presented in Table 1. PCR and electrophoresis results showed that N. ceranae was the only Nosema species found to infect honey bees from our widespread geographic collection from Croatia. N. ceranae infected bees were found in positive samples collected from randomly selected areas of all epizootiological units and in all three climatic areas, i.e. Mediterranean, mountain, and continental parts. Nosema apis alone or mixed infections of N. apis and N. ceranae were not detected at all.