Gene enrichment analysis of DEGs specific of the clinical and preclinical stage of the disease.

<p>The most relevant GO terms (y axis) associated to clinical (<b>A</b>) and preclinical (<b>B</b>) phase are listed according to decreasing statistical significance from top to bottom. The threshold for statistical significance is marked by the green lines. The enrichment analysis was performed using DAVID bioinformatics tool 6.7 (NIAID/NIH, USA).</p

List of DEGs characterized by an up-down/down-up pattern of expression.^a

<p>List of DEGs characterized by an up-down/down-up pattern of expression.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153425#t003fn001" target="_blank">^a</a></p

Functional classification of differentially expressed genes in blood of infected cattle versus control group^a.

<p>Functional classification of differentially expressed genes in blood of infected cattle versus control group<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153425#t002fn001" target="_blank">^a</a>.</p

Heat maps representing the DEGs found in clinical and preclinical cattle with atypical BSE.

<p>Two heat maps were generated using the <i>heatmap</i>.<i>2</i> function from the <i>gplots</i> library in <i>R</i> statistical environment. DEGs were hierarchically clustered with complete linkage using the Euclidean metric. The heat maps represent the most significant DEGs (p value ≤.0.05 and fold change ≥ 2) in clinical (A) and preclinical (B) animals compared to the control group. Animals are reported in the x-axis while the differentially expressed probes are in the y-axis.</p

Identification of common DEGs in blood of preclinical and clinical atypical BSE-infected cattle.

<p>(A) Venn diagram showing the number of differentially expressed probe sets in blood of clinical and preclinical cattle. The intersection in grey represents 35 differentially expressed probe sets corresponding to 32 differentially expressed genes (DEGs) that are in common between the two stages of the disease. (B) Expression pattern of the common 32 DEGs. The histograms represent the fold change relative to the control group. PvsCtrl = preclinical versus control, CvsCtrl = clinical versus control.</p

Differential expression of selected genes quantified by microarray and RT-qPCR analysis ^a.

<p>Differential expression of selected genes quantified by microarray and RT-qPCR analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153425#t005fn001" target="_blank">^a</a>.</p

Validation of microarray data by RT-qPCR.

<p>Differential expression of selected genes in blood from preclinical (A) and clinical (B) atypical BSE-infected cattle. Ganulysin (GNLY), X-inactive specific transcript (XIST), pyruvate dehydrogenase kinase 4 (PDK4), CD40 ligand (CD40L), haemoglobin, alpha 2 (HBA2) and Sel-1 Suppressor Of Lin-12-Like 3 protein (SEL1L3). Gene expression (ratio) values are represented as relative to RNA levels in control animals. Ns = not significant; *<i>P</i> value ≤ 0.05.</p

DEGs in common between CvsP, PvsCtrl and CvsCtrl comparisons.

<p>(A) Venn diagram revealed the presence of 22 DEGs in common between CvsP and PvsCtrl comparisons. One DEG (SEL1L3, Sel-1 Suppressor Of Lin-12-Like 3) was common in the three comparisons. (B-C) The normalized expression values of the 22 common DEGs for CvsP and PvsCtrl comparisons are represented by the histograms. As indicated by the schematic figures on the right, these 22 DEGs showed an oscillatory pattern of expression: (B) 9/22 were upregulated in PvsCtrl and then downregulated in CvsP comparisons, while (C) 13/22 were downregulated in PvsCtrl and upregulated in CvsP comparison, respectively. (D) SEL1L3, the only gene found in common among the three comparisons (CvsP, CvsCtrl and PvsCtrl), showed a progressive downregulation during the preclinical and the clinical phase of the infection. P = preclinical, C = clinical and Ctrl = control, <i>vs</i> = <i>versus</i>. DEGs = differentially expressed genes.</p

Haematoxylin and eosin.

<p>A: Frontal cortex of C-BSE (10X); B: Frontal cortex of L-BSE (10X); C: Dorsal nucleus of vagus nerve, brainstem of C-BSE (10X); D: Dorsal nucleus of vagus nerve, brainstem of L-BSE (10X). The single image shows one animal (81556 for C-BSE and 69540 for L-BSE) representative of all animals in the group. Scale bar: 100 μm.</p

Clinical, pathological, and molecular features of classical and L-type atypical-BSE in goats

<div><p>Monitoring of small ruminants for transmissible spongiform encephalopathies (TSEs) has recently become more relevant after two natural scrapie suspected cases of goats were found to be positive for classical BSE (C-BSE). C-BSE probably established itself in this species unrecognized, undermining disease control measures. This opens the possibility that TSEs in goats may remain an animal source for human prion diseases. Currently, there are no data regarding the natural presence of the atypical BSE in caprines. Here we report that C-BSE and L-type atypical BSE (L-BSE) isolates from bovine species are intracerebrally transmissible to goats, with a 100% attack rate and a significantly shorter incubation period and survival time after C-BSE than after L-BSE experimental infection, suggesting a lower species barrier for classical agentin goat. All animals showed nearly the same clinical features of disease characterized by skin lesions, including broken hair and alopecia, and abnormal mental status. Histology and immunohistochemistry showed several differences between C-BSE and L-BSE infection, allowing discrimination between the two different strains. The lymphoreticular involvement we observed in the C-BSE positive goats argues in favour of a peripheral distribution of PrPSc similar to classical scrapie. Western blot and other currently approved screening tests detected both strains in the goats and were able to classify negative control animals. These data demonstrate that active surveillance of small ruminants, as applied to fallen stock and/or healthy slaughter populations in European countries, is able to correctly identify and classify classical and L-BSE and ultimately protect public health.</p></div