Interactive effects of pesticide exposure and pathogen infection on bee health - a critical analysis.

Bees are fundamentally important for pollination services and declines in populations could have significant economic and environmental implications. Pesticide exposure and pathogen infection are recognised as potential stressors impacting upon bee populations and recently there has been a surge in research on pesticide-disease interactions to reflect environmentally realistic scenarios better. We critically analyse the findings on pesticide-disease interactions, including effects on the survival, pathogen loads and immunity of bees, and assess the suitability of various endpoints to inform our mechanistic understanding of these interactions. We show that pesticide exposure and pathogen infection have not yet been found to interact to affect worker survival under field-realistic scenarios. Colony-level implications of pesticide effects on Nosema infections, viral loads and honey bee immunity remain unclear as these effects have been observed in a laboratory setting only using a small range of pesticide exposures, generally exceeding those likely to occur in the natural environment, and assessing a highly selected series of immune-related endpoints. Future research priorities include the need for a better understanding of pesticide effects on the antimicrobial peptide (AMP) component of an individual's immune response and on social defence behaviours. Interactions between pesticide exposure and bacterial and fungal infections have yet to be addressed. The paucity of studies in non-Apis bee species is a further major knowledge gap.

Detection of an activating c-kit mutation by real-time PCR in patients with anaphylaxis.

A well-characterised gain-of-function point mutation within exon 17 of the c-kit proto-oncogene known as Asp816Val is present in patients with mastocytosis. Activation of mast cells through this receptor primes them for IgE-dependent activation, and patients with mastocytosis are at increased risk of anaphylaxis. We hypothesised that the Asp816Val mutation is associated with a history of anaphylaxis in the general population. A mismatch amplification real-time PCR assay was developed and validated to test for the Asp816Val mutation. Subjects were recruited to four subject groups: normal non-atopics, atopics without anaphylaxis, food-induced anaphylactics and non-food anaphylactics. Blood samples collected from forty subjects were tested for the presence of Asp816Val. Thirteen subjects were found to carry the mutation; normals (2/9), atopics (2/10), food anaphylactics (5/11) and non-food anaphylactics (4/10). Statistical analysis of the data determined that there was no significant difference between the numbers of subjects found to carry the Asp816Val mutation in each of the groups although a trend towards an increased occurrence in anaphylactics was observed. In summary, the hypothesis that the presence of the Asp816Val mutation is linked to the occurrence of anaphylaxis was not supported, but interestingly, we have shown for the first time Asp816Val may occur more frequently than previously reported within the general population.

Determination of percentage of RoundUp Ready soya in soya flour using real-time polymerase chain reaction: interlaboratory study.

An interlaboratory study was conducted to evaluate a method for determination of the percentage of RoundUp Ready (RR) soya in soya flour using Taqman technology. The method included DNA extraction from the test portion with cetyltrimethylammonium bromide buffer followed by chloroform extraction and Wizard resin cleanup steps. The DNA was then assayed with primer and probe sets specific for lectin as the endogenous control and the RR insert as the target. The percentage of RR soya in the soya fraction of the sample was calculated by using a matrix-matched standard curve. Ten samples of split-level blind duplicates were sent to 22 laboratories in 12 countries worldwide. Test portions contained 0, 0.5, 0.7,1.6, 2, and 3.9% (w/w) RR soya prepared gravimetrically from commercially available RR standard reference materials. Based on the results for test materials, the relative standard deviation for repeatability (RSDr) for the method ranged from 9.3 to 19.3% and, for reproducibility (RSDR), ranged from 20.3 to 33.7%.

Development of a microsatellite-based method for the differentiation of European wild boar (Sus scrofa scrofa) from domestic pig breeds (Sus scrofa domestica) in food.

Twenty microsatellites (simple sequence repeats, SSR) were used to discriminate wild boar from domestic pig and to identify mixtures of the two. Reference groups of wild boar and pig samples were collected from the UK and Europe for genetic assignment tests. Bayesian Analysis of Populations software (BAPs) gave 100% correct assignment for blind wild boar and pig samples and correctly identified mixed samples. DNA was extracted from 12 commercial food samples (11 labeled as containing wild boar) including patés, salamis, and sausage, and good SSR profiles were obtained. Eleven samples were correctly assigned as pig, and two as mixed meats. One sample sold as wild boar meat was clearly assigned as pig. A further 10 blind samples of meat cuts were analyzed, eight wild boar and two pig, and all were correctly assigned.

Identification of Agrobacterium spp. present within Brassica napus seed by TaqMan PCR--implications for GM screening procedures.

A fluorogenic probe (fliG-P), designed within a chromosomal DNA sequence, was used in a TaqMan PCR assay to identify Agrobacterium spp. The TaqMan assay detected 58 of 59 Agrobacterium strains tested, but did not detect 13 other Rhizobiaceae strains. Seedlings were grown from seven lots of surface-sterilised Brassica napus seed. Seedlings from these samples were placed in phosphate buffer and the resulting suspensions used to inoculate broth media selective for Agrobacterium biovars 1 and 2. Lysed broths (after 48 h incubation) were used as template in the fliG TaqMan PCR to detect Agrobacterium sp. in one of the seed samples. Individual Agrobacterium strains were isolated from this sample and tested by three Ti-plasmid conventional PCR assays. None of the strains possessed a plasmid. This is the first report of Agrobacterium sp. present within the seed of B. napus, a crop routinely screened for genetically modified DNA contamination using PCR assays with Agrobacterium sequences as targets.