Draining lymph node gross pathology.

<p>Macroscopical aspect of lymph nodes two days after inoculation of either saline, ∼5500 <i>Y. pseudotuberculosis</i> cfu or ∼5500 <i>Y. pestis</i> cfu. Lymph node from <i>Y. pseudotuberculosis</i>-infected mice is purulent whereas <i>Y. pestis</i>-infected lymph node is reddish and adherent to neighboring tissues. Bar = 2 mm.</p

Illustrations of some of the criteria used for scoring.

<p>Panels A–E, G, H and J–L display HE stained sections, panels F and I show sections immunostained with <i>Y. pestis</i> specific antibodies. In the text below, the criteria are referred to according to the numbering in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001688#pone-0001688-t001" target="_blank">Table 1</a>. A: Wedge shaped abscess (criterion 4) indicated by arrows. Within the abscess bacterial colonies are visible as pink patches. B: Peripheral layer of PMNs containing bacterial foci (criterion 9), with a blunt demarcation (arrows) from the lymph node tissue (criterion 11). Inset: higher magnification to show the characteristic horseshoe shaped nuclei of the PMNs, and a bacterial focus. C: Layer of PMNs bordering a peripheral band of bacteria, cell debris and PMNs (criterion 10). The inset shows the typical PMN morphology of the cells within the layer. Behind this layer, bacterial aggregates are seen as pink areas (arrowheads) containing purple dots that are, as seen as higher magnification (not shown here) PMNs and cell debris. D: Patch of densely packed bacteria, bordered by PMNs (criterion14). Packed bacteria form a pink 8-shaped area at the centre of the picture (star). E: Atypical bacterial patch (criterion 15). At the center of the picture an aggregate of bacterial rods, not as densely packed as the preceding one, is loosely surrounded by inflammatory cells. F: A large bacterial zone (criterion 17), stained brownish on the preparation. G: Isolated host cells within a bacterial zone (criterion 19). Isolated host cells and cell remnants are seen amid a sea of bacteria which gives a “ground glass” appearance to this part of the LN section. H, <i>left</i>: bacterial infiltration around host cells (criterion 20). A bacterial strand (arrow), that seems to originate from a nearby colony (star), passes between host cells. H, <i>right</i>: Free floating bacterium, indicated by an arrow (criterion 21). I: Zone of reduced tissular density (arrows) outside bacterial areas, which are brownish on this preparation (criterion 26). This image also shows flame-like inward bacterial projections (criterion 18). J: Area of reduced host cell density with a reticular pattern (criterion 27) and containing numerous pycnotic cells (criterion 32). K: Moth eaten appearance (criterion 33) of a lymph node with areas of contrasting tissular densities. L: Vascular congestion (criterion 40), showing bright red on this preparation. <i>Magnifications</i>: Panels A–C, F, I–L : bar = 200 µm. Insets of panels B and C: bar = 50 µm. Panels D, E, G and H: bar = 10 µm.</p

Dendrogram of the draining lymph node histopathological profiles.

<p>Each label indicates (in order): infecting species (P for <i>Y. pestis</i>, T for <i>Y. pseudotuberculosis</i>), delay between mouse infection and lymph node collection (in days), amount of injected cfu. Top scale = % similarity</p

Bacterial loads at injection site and in draining lymph node.

<p>Data are means of log₁₀ of cfu numbers recovered from 77 mice infected over six independent experiments. Bars = Standard Errors. Stars indicate that the data are significantly different between the two <i>Yersinia</i> species (p≤0.0001). The dashed line shows the detection limit (10 cfu). The cross symbol indicates that most <i>Y. pestis</i>-infected mice died between days 2 and 3.</p

Examples of the three histopathological types of the draining lymph nodes.

<p>Lymph nodes were taken 24 h (type 1) and 72 h (type 2 and 3) after inoculation of ∼4500 cfu of <i>Y. pseudotuberculosis</i> (types 1 and 2) or <i>Y. pestis</i> (type 3). Sections were stained by Hematoxylin-Eosin (HE), and by antibodies specific to <i>Y. pseudotuberculosis</i> or to <i>Y. pestis</i> (Bact), to PMNs and to B lymphocytes (BL). Immunostainings give a brownish color. Arrowheads on the HE-stained sections indicate the border of the inflammatory front. Bacteria (arrows) are present as tiny bacterial foci at the periphery (type 1), well delimited patches (type 2) or flame-like formations (type 3). The BL staining shows that the overall structure of the type 1, but not of the type 3, lymph node is preserved, while, in the type 2 lymph node, the inflammatory reaction forced B lymphocytes to the central part of the organ. Bar = 100 µm.</p

Criteria used to score sections of lymph nodes infected with <i>Y. pestis</i> or <i>Y. pseudotuberculosis</i>, and their discriminating indexes.

<p>Discriminating indexes are the test values in the right column. Only significant test values (i.e. >1.96 or <−1.96) are given. Positive and negative test-values are distinctive of, respectively, <i>Y. pestis</i> and <i>Y. pseudotuberculosis</i> infections.</p

Distribution and infiltration pattern of bacteria in dLNs infected with <i>Y</i>. <i>pestis</i> or <i>Y</i>. <i>pseudotuberculosis</i> strains containing or not the pPla plasmid.

<p>Mice were infected id in the ear with 5x10³ cfu of each strain. At 48 h post infection mice were sacrificed and the ipsilateral superficial parotid LN was collected. LN sections were immunostained with an anti-<i>Y</i>. <i>pestis</i> (first two rows) or -<i>Y</i>. <i>pseudotuberculosis</i> (last two rows) antiserum. Bacteria have an orange-brown coloration, and the tissues are counterstained with hematoxylin (blue). (A) and (B) panels represent low- and high-magnification pictures, as indicated by the corresponding scale bars. Strains are denoted as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005222#ppat.1005222.t001" target="_blank">Table 1</a>. The figure shows typical aspects observed in a panel of 68 examined LNs (20 infected with Yp.wt, 26 with Yp(ΔPla), 11 with Yptb* and 11 with Yptb*(Pla)).</p

Impact of pPla on bacterial morphology in the dLN.

<p>LNs were collected 48h after id inoculation of 5x10³ cfu of the indicated strain and sections were immunolabeled with an anti-<i>Y</i>. <i>pestis</i> antiserum. In the left panel, the orange-brown staining delineates abundant and apparently intact bacteria typical of wild-type <i>Y</i>. <i>pestis</i> (Yp.wt) infections, contrasting with the punctate staining pattern observed after infection with the <i>Y</i>. <i>pestis</i> variant devoid of pPla (Yp(∆Pla), right panel).</p

Plasmids and bacterial strains.

<p>Plasmids and bacterial strains.</p

Histology of dLNs infected with <i>Y</i>. <i>pestis</i> or <i>Y</i>. <i>pseudotuberculosis</i> strains containing or not the pPla plasmid.

<p>Mice were infected id in the ear with 5x10³ cfu of each strain and dLNs were collected 48h later. An uninfected LN is shown for comparison. HE: hematoxylin-eosin staining; BL: immunostaining of B lymphocytes (brown), and counterstaining with hematoxylin (blue). In Yp.wt- and Yp(ΔPla)-infected nodes (first two rows): areas of lymphoid tissue depletion and necrosis appear on low-magnification HE pictures (left column) as zones of lower staining intensity or as irregular pink areas; at higher magnification (right column), dissociation of the lymphoid tissue, deposition of eosinophilic fibrillar material, and the presence of necrotic cells and cell debris characterize the necrotic process; typical PMNs harboring horseshoe-shape nuclei are visible (green arrows). In Yptb^* and Yptb^*(Pla)-infected nodes (third and fourth rows): on low-magnification HE pictures, arrowheads indicate the limits of the inflammatory infiltration clearly delineated from the rest of the organ; higher magnifications show that the inflammatory infiltration is essentially made of PMNs (green arrows), which come in close contact with the bacterial foci (stars). In uninfected LNs (last row), the lymphoid tissue density and follicular organization (B cells at the periphery) are normal. Strains are denoted as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005222#ppat.1005222.t001" target="_blank">Table 1</a>. Sections of infected dLNs shown here are from the same dLNs as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005222#ppat.1005222.g001" target="_blank">Fig 1</a>. The position of the fields shown in the right column is marked by grey rectangles in the corresponding low-magnification HE sections.</p