We have identified a globally important clonal complex of M. bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of Mycobacterium bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu,1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than

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Nanopore analysis can be used to study conformational changes in individual peptide or protein molecules. Under an applied voltage there is a change in the event parameters of blockade current or time when a molecule bumps into or translocates through the pore. If a molecule undergoes a conformational change upon binding a ligand or metal ion the event parameters will be altered. The objective of this research was to demonstrate that the conformation of the prion protein (PrP) and prion peptides can be modulated by binding divalent metal ions. Peptides from the octarepeat region (Octa2, (PHGGGWGQ)2 and Octa 4, (PHGGGWGQ)4), residues 106-126 (PrP106-126), and the full-length Bovine recombinant prion (BrecPrP) were studied with an alpha-hemolysin pore. Octa2 readily translocated the pore but significant bumping events occurred on addition of Cu(II) and to a lesser extent Zn(II), demonstrating that complex formation was occurring with concomitant conformational changes. The binding of Cu(II) to Octa4 was more pronounced and at high concentrations only a small proportion of the complex could translocate. Addition of Zn(II) also caused significant changes to the event parameters but Mg(II) and Mn(II) were inert. Addition of Cu(II) to PrP106-126 caused the formation of a very tight complex, which could not translocate the pore. Small changes were observed with Zn(II), but not with Mg(II) or Mn(II). Analysis of BrecPrP showed that about 37% were translocation events, but on addition of Cu(II) or Zn(II) these disappeared and only bumping events were recorded. Suprisingly, addition of Mn(II) caused an increase in translocation events to about 64%. Thus, conformational changes to prions upon binding metal ions are readily observed by nanopore analysis.

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