Infections with Avian Pathogenic and Fecal Escherichia coli Strains Display Similar Lung Histopathology and Macrophage Apoptosis

The purpose of this study was to compare histopathological changes in the lungs of chickens infected with avian pathogenic (APEC) and avian fecal (Afecal) Escherichia coli strains, and to analyze how the interaction of the bacteria with avian macrophages relates to the outcome of the infection. Chickens were infected intratracheally with three APEC strains, MT78, IMT5155, and UEL17, and one non-pathogenic Afecal strain, IMT5104. The pathogenicity of the strains was assessed by isolating bacteria from lungs, kidneys, and spleens at 24 h post-infection (p.i.). Lungs were examined for histopathological changes at 12, 18, and 24 h p.i. Serial lung sections were stained with hematoxylin and eosin (HE), terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) for detection of apoptotic cells, and an anti-O2 antibody for detection of MT78 and IMT5155. UEL17 and IMT5104 did not cause systemic infections and the extents of lung colonization were two orders of magnitude lower than for the septicemic strains MT78 and IMT5155, yet all four strains caused the same extent of inflammation in the lungs. The inflammation was localized; there were some congested areas next to unaffected areas. Only the inflamed regions became labeled with anti-O2 antibody. TUNEL labeling revealed the presence of apoptotic cells at 12 h p.i in the inflamed regions only, and before any necrotic foci could be seen. The TUNEL-positive cells were very likely dying heterophils, as evidenced by the purulent inflammation. Some of the dying cells observed in avian lungs in situ may also be macrophages, since all four avian E. coli induced caspase 3/7 activation in monolayers of HD11 avian macrophages. In summary, both pathogenic and non-pathogenic fecal strains of avian E. coli produce focal infections in the avian lung, and these are accompanied by inflammation and cell death in the infected areas

Middle East - North Africa and the millennium development goals : implications for German development cooperation

          Closed-loop controlled combustion is a promising technique to improve the overall performance of internal combustion engines and Diesel engines in particular. In order for this technique to be implemented some form of feedback from the combustion process is required. The feedback signal is processed and from it combustionrelated parameters are computed. These parameters are then fed to a control process which drives a series of outputs (e.g. injection timing in Diesel engines) to control their values. This paper’s focus lies on the processing and computation that is needed on the feedback signal before this is ready to be fed to the control process as well as on the electronics necessary to support it. A number of feedback alternatives are briefly discussed and for one of them, the in-cylinder pressure sensor, the CA50 (crank angle in which the integrated heat release curve reaches its 50% value) and the IMEP (Indicated Mean Effective Pressure) are identified as two potential control variables. The hardware architecture of a system capable of calculating both of them on-line is proposed and necessary feasibility size and speed considerations are made by implementing critical blocks in VHDL targeting a flash-based Actel ProASIC3 automotive-grade FPGA

Infections with Avian Pathogenic and Fecal Escherichia coli strains display lung histopathology and macrophage apoptosis

The purpose of this study was to compare histopathological changes in the lungs of chickens infected with avian pathogenic (APEC) and avian fecal (Afecal) Escherichia coli strains, and to analyze how the interaction of the bacteria with avian macrophages relates to the outcome of the infection. Chickens were infected intratracheally with three APEC strains, MT78, IMT5155, and UEL17, and one non-pathogenic Afecal strain, IMT5104. The pathogenicity of the strains was assessed by isolating bacteria from lungs, kidneys, and spleens at 24 h post-infection (p.i.). Lungs were examined for histopathological changes at 12, 18, and 24 h p.i. Serial lung sections were stained with hematoxylin and eosin (HE), terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) for detection of apoptotic cells, and an anti-O2 antibody for detection of MT78 and IMT5155. UEL17 and IMT5104 did not cause systemic infections and the extents of lung colonization were two orders of magnitude lower than for the septicemic strains MT78 and IMT5155, yet all four strains caused the same extent of inflammation in the lungs. The inflammation was localized; there were some congested areas next to unaffected areas. Only the inflamed regions became labeled with anti-O2 antibody. TUNEL labeling revealed the presence of apoptotic cells at 12 h p.i in the inflamed regions only, and before any necrotic foci could be seen. The TUNEL-positive cells were very likely dying heterophils, as evidenced by the purulent inflammation. Some of the dying cells observed in avian lungs in situ may also be macrophages, since all four avian E. coli induced caspase 3/7 activation in monolayers of HD11 avian macrophages. In summary, both pathogenic and non-pathogenic fecal strains of avian E. coli produce focal infections in the avian lung, and these are accompanied by inflammation and cell death in the infected areas

Mean organ lesion scores in chickens infected with APEC MT78 and UEL17.

a<p><i>n</i> = 5 chickens per strain at 24 h p.i.</p>b<p>Organ lesions were scored as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041031#pone.0041031-Anto1" target="_blank">[2]</a>: air sacs 0–3; lungs 0–5; heart 0–3; liver 0–2 and spleen 0–1; maximum additive lesion score: 14.</p

TUNEL-positive cells in the lungs of APEC-infected chickens.

<p>Lungs were fixed at 12 h p.i. (A) Clogged parabronchus of MT78-infected chicken, stained with HE; the boxed region is amplified and was stained with anti-O2 antibody in (D), and by TUNEL in (G). (B) Clogged parabronchus of IMT5155-infected chicken, stained with HE; the boxed region is amplified and was stained with anti-O2 antibody in (E), and by TUNEL in (H). (C) Lung section from PBS-inoculated chicken, showing intact parabronchi, open and aerated atria and air capillaries, and absence of TUNEL-positive cells. (F) Parabronchus of UEL17-infected chicken, stained by TUNEL. (I) Parabronchus of IMT5104-infected chicken, stained by TUNEL. All <i>E. coli</i>-infected chickens had TUNEL-positive cells in inflamed lung areas. Scale bars, A, B and C, 200 µm; D–I, 50 µm. PL, parabronchial lumen; A, atria; AC, air capillary; S, interparabronchial septa; BV, blood vessel.</p

(A) Hydrolysis of caspase 3/7 substrate by cell extracts from <i>E. coli</i>-infected HD11 macrophages.

<p>Macrophages were infected with MT78 (closed squares), UEL17 (open squares), IMT5155 (closed triangles) or IMT5104 (open triangles) at MOI of 150 CFU per cell. At 6 h p.i., cell extracts were prepared and analyzed using the caspase 3/7 substrate as described in Section 2.5. Open circles, uninfected cells; closed circles, cells irradiated with 0.02 J UV (positive control). Data are representative of at least four experiments performed in duplicate; the mean ± standard deviation of <i>V</i>_max for each curve is indicated on the right of the curve. (B) TUNEL analysis of HD11 cells. Macrophages were treated as in (A), and after 8 h, samples were fixed with formaldehyde (3.7% in PBS) and analyzed for TUNEL-positive cells (yellow).</p

Lungs from chickens infected intratracheally with PBS (<i>upper left</i>) or 10⁹ CFU of APEC MT78 (<i>upper centre</i>), UEL17 (<i>upper right</i>), IMT5155 (<i>lower centre</i>), and A_fecal IMT5104 (<i>lower left</i>).

<p>Lungs were fixed at 12 h p.i.. Lungs from UEL17-infected chicken fixed at 24 h p.i. are also shown (<i>lower right</i>). Sections were stained with HE and examined under a light microscope. The lungs of PBS-inoculated chickens had intact parabronchi, and open and aerated atria and air capillaries. The lungs of <i>E. coli</i>-infected chickens had inflamed areas next to intact areas. Scale bars 200 µm. PB, primary bronchus; SB, secondary bronchus; PL, parabronchial lumen; A, atria; AC, air capillary; S, interparabronchial septa; BV, blood vessel; Cat, cartilaginous plates supporting primary bronchus.</p

Numbers of virulence-associated genes and properties of the avian <i>E. coli</i> strains <i>in vivo</i> and in monolayers of HD11 macrophages.

*<p>total no. of genes tested: 46 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041031#pone.0041031-Ewers1" target="_blank">[14]</a>; n.d. = not determined; − = no; + = low; ++ = medium; +++ = high.</p>a<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041031#pone.0041031-Anto1" target="_blank">[2]</a>.</p>b<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041031#pone.0041031-Li1" target="_blank">[5]</a>.</p

Lungs from chickens infected intratracheally with IMT5155 (10⁹ CFU).

<p>Lungs were fixed 24 h p.i.; sections were stained with (A) HE or (B) anti-O2 antibody and counter-stained with hematoxylin, and examined with a light microscope. Inflamed parabronchi coincide with the presence of bacteria, labeled with anti-O2. Scale bars 400 µm. BV, blood vessel.</p