Deciphering the genome and secondary metabolome of the plant pathogen Fusarium culmorum.

Fusarium culmorum is one of the most important fungal plant pathogens that causes diseases on a wide diversity of cereal and non-cereal crops. We report herein for the first time the genome sequence of F. culmorum strain PV and its associated secondary metabolome that plays a role in the interaction with other microorganisms and contributes to its pathogenicity on plants. The genome revealed the presence of two terpene synthases, trichodiene and longiborneol synthase, which generate an array of volatile terpenes. Furthermore, we identified two gene clusters, deoxynivalenol and zearalenone, which encode for the production of mycotoxins. Linking the production of mycotoxins with in vitro bioassays, we found high virulence of F. culmorum PV on maize, barley and wheat. By using ultra-performance liquid chromatography-mass spectrometry, we confirmed several compounds important for the behaviour and lifestyle of F. culmorum. This research sets the basis for future studies in microbe-plant interactions.

Collaborative study on the effect of grinding on the detection of bones from processed animal proteins in feed by light microscopy.

Bone fragments are essential structures for the detection of processed animal proteins (PAPs) in feed by light microscopy for official controls according to Annex VI of European Union Regulation EC/152/2009. The preparation of samples submitted for analysis requires a grinding step to make them suitable for microscopic slide preparation and observation. However, there are no technical guidelines set down for this step despite the fact that it can lead to an increase in bone numbers due to fragmentation. This was demonstrated by an in-house study carried out by the Irish National Reference Laboratory (NRL) for animal protein detection. The present collaborative study investigated the possible effects of three different grinding conditions on the final result for a feed adulterated with 0.05 and 0.01% (w/w) of PAP. The microscopic analysis either combined or not with an Alizarin Red staining was carried out by 10 different laboratories. The results demonstrated that although a large variation in the numbers of bone fragments was noted, five of the six different grinding/staining combinations applied at two levels of PAP adulteration did not significantly (at p = 0.05) differ from one another. The only exception occurred when grinding the feed containing 0.05% of PAP with a rotor mill equipped with a 0.5-mm sieve and combined with a staining which resulted in a greater number of bone fragments by forced fragmentation. Overall, the impact of the grinding/staining combinations on the final results was shown to be negligible when considering the regulatory limit of detection (LOD) requirement for the method and the current rules of implementation of the light microscopic method. From a total of 180 analyses carried out on the feed matrix containing 0.05% of PAP no false-negative result was observed, and at a level of 0.01% PAP only 10 false-negative results occurred.

Distribution of chloramphenicol to tissues, plasma and urine in pigs after oral intake of low doses.

Toxic effects of chloramphenicol in humans caused the ban for its use in food-producing animals in the EU. A minimum required performance level (MRPL) was specified for chloramphenicol at 0.3 µg kg(-1) for various matrices, including urine. In 2012, residues of chloramphenicol were found in pig urine and muscle without signs of illegal use. Regarding its natural occurrence in straw, it was hypothesised that this might be the source, straw being compulsory for use as bedding material for pigs in Sweden. Therefore, we investigated if low daily doses of chloramphenicol (4, 40 and 400 µg/pig) given orally during 14 days could result in residues in pig tissues and urine. A dose-related increase of residues was found in muscle, plasma, kidney and urine (showing the highest levels), but no chloramphenicol was found in the liver. At the lowest dose, residues were below the MRPL in all tissues except in the urine. However, in the middle dose, residues were above the MRPL in all tissues except muscle, and at the highest dose in all matrices. This study proves that exposure of pigs to chloramphenicol in doses occurring naturally in straw could result in residues above the MRPL in plasma, kidney and especially urine.

Occurrence of chloramphenicol in cereal straw in north-western Europe.

Two surveys are presented of straw analysed for naturally occurring chloramphenicol (CAP), a drug banned for use in food-producing animals. In the first study, CAP was analysed by LC-MS/MS and detected in 37 out of 105 straw samples originating from the Netherlands, France, the UK, Germany and Denmark. The highest level found was 6.3 µg kg(-1), the average 0.6 µg kg(-1) and the median 0.2 µg kg(-1). The second study included a method comparison between ELISA and LC-MS/MS and a survey of CAP in cereal straw sampled at farms in all areas of Sweden. A total of 215 samples were screened by ELISA and a subset of 26 samples was also analysed by LC-MS/MS. Fifty-four of the samples contained more than 1 µg kg(-1) CAP and the highest level found was 32 µg kg(-1) (confirmed by LC-MS/MS). The highest contents of CAP in this study were allocated to the Baltic sea coast in the south-eastern part of Sweden (the county of Skåne and the Baltic Sea isle of Gotland). These results indicate a high incidence of CAP in straw in north-west Europe and have a severe impact on the enforcement of European Union legislation.

Uncertainty from sampling in measurements of aflatoxins in animal feedingstuffs: application of the Eurachem/CITAC guidelines.

The duplicate method for estimating uncertainty from measurement including sampling is presented in the Eurachem/CITAC guide. The applicability of this method as a tool for verifying sampling plans for mycotoxins was assessed in three case studies with aflatoxin B(1) in animal feedingstuffs. Aspects considered included strategies for obtaining samples from contaminated lots, assumptions about distributions, approaches for statistical analysis, log(10)-transformation of test data and applicability of uncertainty estimates. The results showed that when duplicate aggregate samples are formed by interpenetrating sampling, repeated measurements from a lot can be assumed to approximately follow a normal or lognormal distribution. Due to the large variation in toxin concentration between sampling targets and sometimes very large uncertainty arising from sampling and sample preparation (U(rel) ≥ 50%), estimation of uncertainty from log(10)-transformed data was found to be a more generally applicable approach than application of robust ANOVA.