Real-time quaking-induced conversion (RT-QuIC) is a rapid, specific and highly sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrP Sen ) to detect subinfectious levels of the abnormal isoforms of the prion protein (PrP Sc ). Although RT-QuIC has been successfully used to detect PrP Sc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie have not yet been tested. Therefore, the aims of the present study were to (1) evaluate whether prion seeding activity could be detected in the brain tissues of goats with scrapie using RT-QuIC, (2) optimize reaction conditions to improve scrapie detection in goats, and (3) compare the performance of RT-QuIC for the detection of PrP Sc with the more commonly used ELISA and Western blot assays. We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200 mM sodium chloride, both full-length sheep rPrP Sen substrates (PrP genotypes A 136 R 154 Q 171 and V 136 R 154 Q 171 ) provided good discrimination between scrapie-infected and normal goat brain samples at 10 23 dilution within 15 h. Our findings indicate that RT-QuIC was at least 10 000-fold more sensitive than ELISA and Western blot assays for the detection of scrapie seeding activity in goat brain samples. In addition to PRNP WT samples, positive RT-QuIC reactions were also observed with three PRNP polymorphic goat brain samples (G/S127, I/M142 and H/R143) tested. Taken together, these findings demonstrate that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples.

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Transmission of tick-borne pathogens requires transition between distinct host environments with infection and replication in host-specific cell types. Anaplasma marginale illustrates this transition: in the mammalian host, the bacterium infects and replicates in mature (nonnucleated) erythrocytes, while in the tick vector, replication occurs in nucleated epithelial cells. We hypothesized that proteins containing ankyrin motifs would be expressed by A. marginale only in tick cells and would traffic to the infected host cell nucleus. A. marginale encodes three proteins containing ankyrin motifs, an AnkA orthologue (the AM705 protein), AnkB (the AM926 protein), and AnkC (the AM638 protein). All three A. marginale Anks were confirmed to be expressed during intracellular infection: AnkA is expressed at significantly higher levels in erythrocytes, AnkB is expressed equally by both infected erythrocytes and tick cells, and AnkC is expressed exclusively in tick cells. There was no evidence of any of the Ank proteins trafficking to the nucleus. Thus, the hypothesis that ankyrin-containing motifs were predictive of cell type expression and nuclear localization was rejected. In contrast, AnkA orthologues in the closely related A. phagocytophilum and Ehrlichia chaffeensis have been shown to localize to the host cell nucleus. This difference, together with the lack of a nuclear localization signal in any of the AnkA orthologues, suggests that trafficking may be mediated by a separate transporter rather than by endogenous signals. Selection for divergence in Ank function among Anaplasma and Ehrlichia spp. is supported by both locus and allelic analyses of genes encoding orthologous proteins and their ankyrin motif compositions.

Microcin PDI (MccPDI), a class IIa microcin that is produced by Escherichia coli strains 25 and 284, is known to inhibit foodborne pathogenic enterohemorrhagic E. coli serotypes O157:H7 and O26. Here we demonstrate that MccPDI can inhibit Shigella strains and E. coli isolates that are multidrug resistant, the latter including strains known to cause urinary tract infections in people and companion animals. Two exceptions out of 17 strains were identified. One of the two resistant E. coli isolates (AR0349) has a mutation in a critical amino acid residue that was identified in previous work as a requisite for the MccPDI precursor protein (McpM) to interact with outer membrane porin F (OmpF) on susceptible cells. The second resistant E. coli strain (MAD 96) had no mutations in ompF, but it was PCR positive for two antimicrobial peptides, of which colicin Ia/Ib likely inhibits the MccPDI-producing strain during coculture. Recombinant McpM was still effective against strain MAD 96. In an assessment of how MccPDI affects susceptible strains, results from both an extracellular ATP assay and a nucleic acid staining assay were consistent with membrane damage, while the addition of 200-to 600-Da polyethylene glycol (PEG) to cocultures protected against MccPDI (Ͼ600-Da PEG did not provide protection). Further studies using a paraformaldehyde cross-linking experiment and a bacterial two-hybrid assay demonstrated that MccPDI immunity protein (McpI) forms a multimeric complex with itself and presumably protects the producer strain from within the periplasm through an unknown mechanism. IMPORTANCE Microcins represent potential alternatives to conventional antibiotics for human and veterinary medicine. For them to be applied in this manner, however, we need to better understand their spectrum of activity, how these proteins interact with susceptible cells, and how producer cells are protected against the antimicrobial properties of the microcins. For microcin PDI (MccPDI), we report that the spectrum of activity likely includes most E. coli strains due to a conserved binding motif found on an outer membrane protein. Shigella has this motif as well and is susceptible to MccPDI killing via damage to the bacterial membrane. Receptor specificity suggests that these proteins could be used without causing large-scale disruptions to a microbiota, but this also increases the likelihood that resistance can evolve via random mutations. As with conventional antibiotics, good stewardship will be needed to preserve the efficacy of microcins should they be deployed for clinical use.