Comparison of results for alternative detection methods and culture (MGIT broth or Lowenstein-Jensen slope).

a<p>Determined by McNemar's Chi-Square test using GenStat Release 14.2.</p><p>Results for all 280 lymph node samples are not broken down by lymph node type.</p

Distribution of <i>M. bovis</i> spoligotypes isolated by culture and IMS-MGIT culture.

<p>Spoligotypes 269 (140*), 142* and 263* are thought to be spoligotypes 140, 142 and 263 with spacer 15 signal absent which have arisen due to lesser amounts of <i>M. bovis</i> DNA being present in IMS-MGIT cultures.</p

Interrelationships between Culture, IMS-PCR and IMS-MGIT PCR positive results.

<p>(A) visibly lesioned lymph nodes from reactor cattle, (B) non-visibly lesioned lymph nodes from reactor cattle, (C) visibly lesioned lymph nodes from non-reactor cattle detected at routine slaughter, and (D) all lymph node samples, irrespective of type.</p

Comparison of IMS-PCR and Direct PCR results for 280 lymph node samples –74 non-visibly lesioned (NVL) and 206 visibly lesioned (VL).

a<p>Determined by McNemar's Chi-Square test using Genstat version 14.2.</p>b<p>Includes 166 VL and 74 NVL samples from TB skin test reactor animals plus 40 VL samples from non-reactor animals detected at routine slaughter.</p><p>Data represent number of samples with each combination of test results.</p

Interrelationships between Culture, Direct PCR and IMS-PCR positive results for all 280 lymph node samples, irrespective of type.

<p>Interrelationships between Culture, Direct PCR and IMS-PCR positive results for all 280 lymph node samples, irrespective of type.</p

CD172a cells infiltrate rESAT-6:CFP-10 injection sites.

<p><i>Mycobacterium bovis</i>-infected cattle (n = 5) received 400 µg rESAT-6:CFP-10 intradermally and reactions were characterized after 6 days. Injection sites were collected at necropsy, snap frozen, and evaluated by immunohistochemistry for expression of (A) CD172a, (B) CD11c, (C) CD3, and (D) CD14 on cellular surfaces. Few B cells or γδ T cells were detected (data not shown).</p

Expansion of CD172a⁺ cells in response to ESAT-6/CFP-10 stimulation.

<p>Isolated PBMC were stained with PKH67 and cultured for 6d with media only, rMPB83, a pool of overlapping ESAT-6 and CFP-10 peptides, or rESAT-6:CFP-10. Data are depicted as: (A) dot plots (CD172a-PE (y-axis) versus PKH67 (x-axis, green fluorescence), (B) histograms generated by Modfit Proliferation Wizard analysis of PKH67 staining intensity (gated on CD172a-PE⁺ cells within the live gate) and (C) mean (±SEM) percent CD172a⁺ cells within PBMC cultures (stimulation indicated in the lower margin) from non-infected (open bars, n = 9, includes non- (n = 3), BCG- (n = 3), and ΔRD1- (n = 3) vaccinates) or <i>M. bovis</i>-infected (closed bars, n = 20) cattle. Responses did not differ between controls, BCG- and ΔRD1- vaccinates; thus, these groups were combined. Gate R2 in panel A highlights the CD172a⁺, PKH67^lo proliferative fraction. For panels A and B, data from a single <i>M. bovis</i>-infected animal are provided that are indicative of a representative response.</p

rESAT-6:CFP-10 induces granulomatous inflammation with multi-nucleated giant cells.

<p><i>Mycobacterium bovis</i>-infected cattle (n = 5) received 400 µg rESAT-6:CFP-10 intradermally and reactions were characterized after 6 days. Injection sites were collected at necropsy, fixed in formalin, and stained with hematoxylin and eosin. Injection sites consisted of predominately mononuclear cell infiltrates with intermittent, yet consistently detected, multi-nucleated giant cells (arrows). Prior studies have demonstrated that the inflammatory response to rESAT-6 is specific to <i>M. bovis</i> infection <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006414#pone.0006414-Pollock1" target="_blank">[48]</a>. Also, intradermal injection of a BCG-vaccinated calf with 400 µg rESAT-6:CFP-10 did not elicit a DTH response and multi-nucleated giant cells were not detected within the injection site biopsy from this negative control animal.</p

In vitro expansion of CD3⁻, CD172a⁺ cells within PBMC cultures from <i>M. bovis</i> infected cattle stimulated with ESAT-6/CFP-10.

a<p>Data are presented as mean (±standard error) percent of CD3⁻, CD172a⁺, PKH67^lo cells (R2 gate in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006414#pone-0006414-g001" target="_blank">Fig. 1A</a>). Isolated mononuclear cells were stained with PKH67 (a green fluorescent dye used for cell proliferation analysis), cultured for 6 days with or without stimulation as indicated in the upper margin, and analyzed by flow cytometry for phenotype and PKH67 staining intensity. Both BCG and ΔRD-1 attenuated <i>M. bovis</i> vaccine strains lack ESAT-6, CFP-10, and select ESX-1 secretion apparatus genes; thus, these strains do not produce ESAT-6 or CFP-10. In contrast to ESAT-6/CFP10, stimulation with MPB83, another immunodominant antigen of <i>M. bovis</i>, does not result in expansion of CD172a⁺ cells.</p

Multinucleate giant cells elicited by rESAT-6:CFP10 are composed of a ring of CD172a⁺ cells.

<p><i>Mycobacterium bovis</i>-infected cattle (n = 5) received 400 µg rESAT-6:CFP-10 intradermally and reactions were characterized after 6 days. Injection sites were collected at necropsy, snap frozen, and evaluated by immunohistochemistry for expression of CD172a. Arrows depict a CD172a⁺ multi-nucleate giant cell consisting of a ring of peripherally located nuclei and abundant cytoplasm located centrally.</p