Thirty deoxynivalenol-producing F. culmorum strains, isolated from wheat grains, were incubated in vitro and analyzed for trichothecene production. Seventeen strains produced more than 1 ppm of deoxynivalenol and acetyldeoxynivalenol and were considered high-deoxynivalenol-producing strains, whereas 13 F. culmorum strains produced less than 0.07 ppm of trichothecenes and were considered low-deoxynivalenol-producing strains. For all strains, a 550-base portion of the trichodiene synthase gene (tri5) was amplified and sequenced. According to the tri5 data, the F. culmorum strains tested clustered into two groups that correlated with in vitro deoxynivalenol production. For three high-producing and three low-producing F. culmorum strains, the tri5-tri6 intergenic region was then sequenced, which confirmed the two separate clusters within the F. culmorum strains. According to the tri5-tri6 sequence data, specific PCR primers were designed to allow differentiation of high-producing from low-producing F. culmorum strains.Trichothecenes, including deoxynivalenol, acetyldeoxynivalenol, nivalenol, and fusarenone X, are sesquiterpene toxins produced by Fusarium species, including Fusarium culmorum, which are common fungal contaminants of cereals. Trichothecenes can be found naturally worldwide on cereals (1,9,18,27,38,45,48,54,55,59), and the consumption of these toxins is a potential problem for humans and farm animals (14, 47).It has been established that some Fusarium species, including F. graminearum and F culmorum, are able to produce B trichothecenes, such as deoxynivalenol and acetyldeoxynivalenol, while other species are not (31). These two types of Fusarium strains (producers and nonproducers) can be distinguished on the basis of DNA polymorphism in the ␤-tubulin gene (43) as well as in the large ribosomal subunit or the internal transcribed spacer (21,37,43).According to their trichothecene production, some Fusarium species, such as F graminearum, have been divided into two chemotypes: (i) the nivalenol chemotype, which includes isolates producing nivalenol and fusarenone X, and (ii) the deoxynivalenol chemotype, which includes isolates producing deoxynivalenol and acetyldeoxynivalenol (26,53). Similar observations have been made for F. culmorum strains (28, 39).In addition, it has been demonstrated that, within the same species and in the same culture conditions, toxin production by Fusarium strain may vary sharply; some strains produce large amounts of trichothecenes, whereas others produce small or undetectable amounts of trichothecenes (3,5,19,28,30,35,36,39,53,57). Until now, no method except in vitro culture has been available to distinguish high-producing from low-producing Fusarium strains.Several genes involved in the biosynthesis of trichothecenes have been described, most of them localized in a gene cluster. The tri5 gene encodes the trichodiene synthase, which catalyzes the first step in the biosynthesis of trichothecenes. The nucleotide sequence of the tri5 gene has been characterized in several Fusa...

We investigated the diagnostic reliability of pregnancy detection using changes in interferon stimulated gene (ISG) messenger RNA (mRNA) levels in circulating immune cells in ewes. Two different groups of ewes (an experimental group, experiment 1 and a farm group, experiment 2) were oestrus-synchronized and blood sampled on day 14 (D0 = day of insemination in control animals, experiment 1) and day 15 (experiment 2). Real-time PCR were performed to evaluate the abundance of different ISG mRNAs. In the experimental group, peripheral blood mononuclear cells of 29 ewes born and bred in experimental facilities were isolated using a Percoll gradient method. Gene expression for Chemokine (C-X-C motif) ligand 10 ( CXCL10), Myxovirus (influenza virus) resistance 1 ( MX1) and Signal transducer and activator of transcription 1 (STAT1) mRNA were, respectively, 8.3-fold, 6.1-fold and 2.7-fold higher ( P < 0.001) in pregnant compared with non-pregnant ewes. The receiver operating characteristic (ROC) curves generated from the real-time PCR data demonstrated that a reliable cut-off could be established for CXCL10, MX1 and STAT1. In the farm group of animals, peripheral blood leucocytes of 37 cross-bred multiparous ewes bought from several herds were isolated using the PAXgene ® procedure. This blood sampling procedure is achievable in farms, whereas the Percoll method is not. No significant differences ( P > 0.10) in CXCL10, STAT1, MX1, Myxovirus (influenza virus) resistance 2 ( MX2) and ISG15 ubiquitin-like modifier ( ISG15) mRNA expression were found between pregnant and non-pregnant ewes. The ROC curves and the hierarchical classification generated from the real-time PCR data failed to discriminate between pregnant and non-pregnant animals. In this group of animals, our results show a strong variability in ISG expression patterns: 17% of animals identified as non-pregnant by the five tests were in fact pregnant, only 52% of pregnant animals had at least two positive results (two genes above threshold), whereas up to five positive results (five genes above threshold) were needed to avoid misclassification. In conclusion, this study illustrates the high variability in ISG expression levels in immune circulating cells during early pregnancy and, therefore, highlights the limits of using ISG expression levels in blood samples, collected on PAXgene ® tubes on farms, for early pregnancy detection in sheep.Keywords: interferon stimulated genes, pregnancy diagnosis, early pregnancy, implantation, sheep ImplicationsThis study aimed to investigate the accuracy of gene transcript quantification in blood (interferon stimulated genes (ISG)) as a method of early pregnancy diagnosis in sheep. This method of pregnancy detection is based on a single blood sample, whereas routine pregnancy diagnosis, namely ultrasonography, requires specific equipment and a skilled operator. Our results show a higher variability in ISG expression patterns in animals from commercial herds than from a controlled research station. Consequently, this prevents th...

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