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Honeybees originating from 10 different countries (Austria, Poland, Germany, Hungary, Slovenia, Nepal, Sri Lanka, the United Arab Emirates, Canada, and New Zealand) located on four continents were analyzed for the presence of deformed wing virus (DWV) nucleic acid by reverse transcription-PCR. Two target regions within the DWV genome were selected for PCR amplification and subsequent sequencing, i.e., a region within the putative VP2 and VP4 structural-protein genes and a region within the RNA helicase enzyme gene. DWV nucleic acid was amplified from 34 honeybee samples representing all the above-mentioned countries with the notable exception of New Zealand. The amplification products were sequenced, and phylogenetic analyses of both genomic regions were performed independently. The phylogenetic analyses included all sequences determined in this study as well as previously published DWV sequences and the sequences of two closely related viruses, Kakugo virus (KGV) and Varroa destructor virus 1 (VDV-1). In the sequenced regions, the DWV genome turned out to be highly conserved, independent of the geographic origins of the honeybee samples: the partial sequences exhibited 98 to 99% nucleotide sequence identity. Substitutions were most frequently observed at the same positions in the various DWV sequences. Due to the high level of sequence conservation, no significant clustering of the samples in the phylogenetic trees could be identified. On the other hand, the phylogenetic analyses support a genetic segregation of KGV and VDV-1 from DWV.

The occurrence, prevalence, and distribution patterns of acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV) were investigated in 90 Austrian honeybee colonies suffering from symptoms of depopulation, sudden collapse, paralysis, or dark coloring by employing reverse transcription-PCR. Infestation with parasites was also recorded. The samples originated from all parts of Austria. The most prevalent virus was DWV, present in 91% of samples, followed by ABPV, SBV, and BQCV (68%, 49%, and 30%, respectively). CBPV was detected in 10% of colonies, while KBV was not present in any sample. In most samples, more than one virus was identified. The distribution pattern of ABPV, BQCV, CBPV, and SBV varied considerably in the different geographic regions investigated, while DWV was widespread in all Austrian federal states. In bees that showed dark coloring and disorientation, CBPV was always detected. Simultaneous infections of DWV and ABPV were most frequently observed in colonies suffering from weakness, depopulation, and sudden collapse. Bees obtained from apparently healthy colonies within the same apiaries showed a similar distribution pattern of viruses; however, the relative virus load was 10 to 126 times lower than in bees from diseased colonies. A limited number of bee samples from surrounding central European countries (Germany, Poland, Hungary, and Slovenia) were also tested for the presence of the above viruses. Variances were found in the distribution of BQCV and SBV.

PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P. larvae spores. The most sensitive primer pair detected P. larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not P. larvae, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs.