<i>MAP</i> morphotypes survive 70°C and are positive for <i>MAP 16SrRNA</i> and <i>IS900</i>.

<p>(A) <i>MAP K-10</i> log phase and spores, <i>B. subtilis</i> and <i>C. perfringens</i> were heat treated at 70°C for 30 min and subsequently treated with 2% lysozyme, PK, or kanamycin. Heat treated samples were plated on MB7H9 or blood agar and incubated at 37°C under aerobic or anaerobic conditions. (B) Heat treated cultures were plated on blood agar to determine growth of any contaminates. (C) <i>16SrRNA</i> sequences of germinated heat treated <i>MAP</i> spores compared to reference sequences from <i>MAP</i>, <i>Bacillus</i> spp., <i>Streptomyces</i> spp. <i>and Clostridium</i> spp.. Ten colonies from each plate were selected for sequences. Sequences shown are a consensus from the ten colonies. (D) <i>IS900</i> duplex PCR of germinated heat treated <i>MAP</i> spores.</p

<i>MAP</i> morphotype induction is dependent upon temperature.

<p>One year old MB7H9 <i>MAP</i> broth cultures were inoculated on A) A–K agar and C) BHI agar for 72 h at 37°C and 39°C. <i>MAP</i> showed growth only at 39°C compared with <i>B. subtilis</i> and <i>E. coli K-12</i> controls, which had substantial growth at both temperatures (A). Spore enrichment was determined by malachite green staining (B). In order to confirm purity of <i>MAP</i> culture, the year old <i>MAP</i> culture was grown on BHI agar and determined to be free of any contaminating organisms (C).</p

Ultrastructural characterization of <i>MAP</i> morphologies.

<p>Fine <i>MAP</i> morphotype structure was determined by TEM (A). TEM images were taken of log-phase, dormant and A–K <i>MAP</i> cultures. While the dormant <i>MAP</i> culture showed a mix of vegetative cells and spores, A–K <i>MAP</i> cultures displayed typical spore characteristics, including a cortex, plasma membrane and coat layers. (B) All <i>MAP cultures</i> were assessed for contamination of duplex and normal PCR of <i>IS900</i>, <i>spoIVA</i> and <i>Clostridium 16SrDNA.</i> Only <i>MAP</i> samples contained the <i>IS900</i> element and did not amplify <i>Bacillus</i> and <i>Clostridium</i> related genes. (C) Spore formation was confirmed by the detection of dipicolnic acid (DPA) using a colorimetric assay. DPA is a chemical found within the spore core of endospores. Intact and autoclaved mycobactin J (250.0 µg/mL) were used as controls. Each sample was conducted in triplicate.</p

Sporulation occurs in multiple <i>MAP</i> strains.

<p><i>MAP</i> strains <i>7565</i>, <i>Ben</i> and <i>Linda</i> were inoculated on A–K agar. Biomasses were collected and processed for TEM. All strains show characteristic spore structures.</p

Primers used in this study.

±<p>Motiwala, A.S., et al. <i>Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis: evidence for limited strain diversity, strain sharing, and identification of unique targets for diagnosis.</i> J Clin Micobiol, 2003. <b>41</b>(5):2015–26.</p>ŧ<p>Bull, T.J., et al. <i>Characterization of IS900 loci in Mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typing.</i> Microbiology, 2000. <b>146</b>:3285.</p>Ψ<p>Wang, R.F., et al. <i>A 16S rDNA-based PCR method for rapid and specific detection of Clostridium perferingens in food.</i> Mol Cell Probes,m1994. <b>8</b>(2): 131–7.</p

Dormant <i>MAP</i> cultures upregulate spore-related transcripts.

<p>(A) A BLAST comparison and reciprocal BLAST searches were conducted between known sporulation genes from <i>Bacillus spp.^†</i> and <i>Streptomyces spp.^‡</i> against <i>MAP</i>. Percent similarity was determined by protein alignment. (B) Quantitative real-time PCR was performed on dormant <i>MAP</i> cultures to determine the presence of <i>carD (MAP0475</i> and <i>MAP0987</i>) and <i>spo0A</i> (<i>MAP1002c</i>). All three genes are upregulated in comparison to log-phase <i>MAP K-10</i> culture. All samples were conducted in triplicate. C) Multiple sequence alignment of selected CarD proteins. CarD has recently been shown by Stallings et al. to be necessary component of stringency regulation in mycobacteria. Other studies indicate that the stringency response is also necessary for the initiation of sporulation. A multiple sequence alignment of CarD amino acid sequences from mycobacteria and sporulating bacteria was conducted using CLUSTALW.</p

<i>MAP</i> spores retain infectivity and germinate into acid-fast bacilli in a bovine MDM model.

<p><i>MAP</i> spores, <i>MAP</i> log-phase and <i>Bacillus subtilis</i> spores were allowed to infect MDMs for 0.5, 2, 6, 24, and 48 h p.i.. <i>MAP</i> spores readily infected MDMs and germinated by 24 h p.i.. Upon 48 h p.i., MDMs were lysed and <i>MAP</i> spores successfully germinated into acid fast bacilli.</p

DNA aptamers against <i>F. tularensis</i> specifically capture <i>F. tularensis</i> in mixed bacteria samples.

<p>A biotinylated aptamer cocktail composed of 10 aptamers (0.4 pmol each) was bound to M-280 streptavidin Dynabeads. <i>F. tularensis</i> (1–10⁶ cells/mL) was separately mixed with <b>A)</b> 10⁹CFU/mL of soil bacteria or <b>B)</b> lettuce bacteria and aptamer cocktail beads or control beads (beads without aptamers) and incubated for 1 h at room temperature. Samples were denatured and the supernatant was subjected to real-time PCR analysis. Bead controls showed Cp values greater than 34 and were considered negative for <i>F. tularensis</i>. Legend: Aptamers = black bar, Aptamers and Soil Bacteria = dark grey bar.</p

<i>F. tularensis</i> spent medium enhances growth and is specific for <i>F. tularensis</i>.

<p><b>A)</b><i>F. tularensis</i> was cultured for 24 h and diluted on TSA containing 0.1% L-cysteine (<i>left</i>) and TSA containing 0.1% L-cysteine and 10% spent medium (<i>right</i>). A ten-fold dilution series of <i>F. tularensis</i> was created (10⁻¹–10⁻⁶, left to right). <b>B)</b> Lettuce bacteria were obtained from stomacher processing of 10 g of lettuce in 50 mL of TSB containing 0.1% L-cysteine. Soil bacteria were obtained from culturing of 1 g of soil, hay and dust in TSB containing 0.1% L-cysteine overnight at 37°C with shaking at 120 rpm. Bacteria from lettuce and soil sources were diluted to 10⁻⁶ and plated on TSA supplemented with 0.1% L-cysteine (control) and control agar supplemented with 10% <i>F. tularensis</i> spent filtrate. Samples were incubated overnight at 37°C and the CFU/mL was calculated. Legend: Control = white bar, Spent filtrate = medium grey bar.</p

Carnosine increases <i>F. tularensis</i> growth.

<p><i>F. tularensis</i> (10² cells/mL) were incubated overnight at 37°C with agitation in control broth alone or supplemented with 10% spent culture filtrate or various concentrations of carnosine (0.625 mg/mL–10 mg/mL). DNA was extracted from all samples and analyzed by <i>fopA</i> real-time PCR. All samples were conducted in triplicate with technical triplicates. Legend: Control = white bar, Spent filtrate = dark grey bar, [carnosine = 10 mg/mL (light grey bar), 5.0 mg/mL (medium grey bar), 2.5 mg/mL (checkered pattern bar), 1.25 mg/mL (diagonal pattern bar) and 0.625 mg/mL (diamond pattern bar)].</p