Regeneration of Pulmonary Tissue in a Calf Model of Fibrinonecrotic Bronchopneumonia Induced by Experimental Infection with Chlamydia psittaci

Pneumonia is a cause of high morbidity and mortality in humans. Animal models are indispensable to investigate the complex cellular interactions during lung injury and repair in vivo. The time sequence of lesion development and regeneration is described after endobronchial inoculation of calves with Chlamydia psittaci. Calves were necropsied 2–37 days after inoculation (dpi). Lesions and presence of Chlamydia psittaci were investigated using histology and immunohistochemistry. Calves developed bronchopneumonia at the sites of inoculation. Initially, Chlamydia psittaci replicated in type 1 alveolar epithelial cells followed by an influx of neutrophils, vascular leakage, fibrinous exudation, thrombosis and lobular pulmonary necrosis. Lesions were most extensive at 4 dpi. Beginning at 7 dpi, the number of chlamydial inclusions declined and proliferation of cuboidal alveolar epithelial cells and sprouting of capillaries were seen at the periphery of necrotic tissue. At 14 dpi, most of the necrosis had been replaced with alveoli lined with cuboidal epithelial cells resembling type 2 alveolar epithelial cells and mild fibrosis, and hyperplasia of organized lymphoid tissue were observed. At 37 dpi, regeneration of pulmonary tissue was nearly complete and only small foci of remodeling remained. The well-defined time course of development and regeneration of necrotizing pneumonia allows correlation of morphological findings with clinical data or treatment regimen

A Bovine Model of Respiratory Chlamydia psittaci Infection: Challenge Dose Titration

This study aimed to establish and evaluate a bovine respiratory model of experimentally induced acute C. psittaci infection. Calves are natural hosts and pathogenesis may resemble the situation in humans. Intrabronchial inoculation of C. psittaci strain DC15 was performed in calves aged 2–3 months via bronchoscope at four different challenge doses from 106 to 109 inclusion-forming units (ifu) per animal. Control groups received either UV-inactivated C. psittaci or cell culture medium. While 106 ifu/calf resulted in a mild respiratory infection only, the doses of 107 and 108 induced fever, tachypnea, dry cough, and tachycardia that became apparent 2–3 days post inoculation (dpi) and lasted for about one week. In calves exposed to 109 ifu C. psittaci, the respiratory disease was accompanied by severe systemic illness (apathy, tremor, markedly reduced appetite). At the time point of most pronounced clinical signs (3 dpi) the extent of lung lesions was below 10% of pulmonary tissue in calves inoculated with 106 and 107 ifu, about 15% in calves inoculated with 108 and more than 30% in calves inoculated with 109 ifu C. psittaci. Beside clinical signs and pathologic lesions, the bacterial load of lung tissue and markers of pulmonary inflammation (i.e., cell counts, concentration of proteins and eicosanoids in broncho-alveolar lavage fluid) were positively associated with ifu of viable C. psittaci. While any effect of endotoxin has been ruled out, all effects could be attributed to infection by the replicating bacteria. In conclusion, the calf represents a suitable model of respiratory chlamydial infection. Dose titration revealed that both clinically latent and clinically manifest infection can be reproduced experimentally by either 106 or 108 ifu/calf of C. psittaci DC15 while doses above 108 ifu C. psittaci cannot be recommended for further studies for ethical reasons. This defined model of different clinical expressions of chlamydial infection allows studying host-pathogen interactions

Regeneration of Pulmonary Tissue in a Calf Model of Fibrinonecrotic Bronchopneumonia Induced by Experimental Infection with Chlamydia psittaci

Pneumonia is a cause of high morbidity and mortality in humans. Animal models are indispensable to investigate the complex cellular interactions during lung injury and repair in vivo. The time sequence of lesion development and regeneration is described after endobronchial inoculation of calves with Chlamydia psittaci. Calves were necropsied 2–37 days after inoculation (dpi). Lesions and presence of Chlamydia psittaci were investigated using histology and immunohistochemistry. Calves developed bronchopneumonia at the sites of inoculation. Initially, Chlamydia psittaci replicated in type 1 alveolar epithelial cells followed by an influx of neutrophils, vascular leakage, fibrinous exudation, thrombosis and lobular pulmonary necrosis. Lesions were most extensive at 4 dpi. Beginning at 7 dpi, the number of chlamydial inclusions declined and proliferation of cuboidal alveolar epithelial cells and sprouting of capillaries were seen at the periphery of necrotic tissue. At 14 dpi, most of the necrosis had been replaced with alveoli lined with cuboidal epithelial cells resembling type 2 alveolar epithelial cells and mild fibrosis, and hyperplasia of organized lymphoid tissue were observed. At 37 dpi, regeneration of pulmonary tissue was nearly complete and only small foci of remodeling remained. The well-defined time course of development and regeneration of necrotizing pneumonia allows correlation of morphological findings with clinical data or treatment regimen

Brain Lateralization for Language, Vocabulary Development and Handedness at 18 Months

Is hemisphere lateralization for speech processing linked to handedness? To answer this question, we compared hemisphere lateralization for speech processing and handedness in 18-month-old infants, the age at which infants start to produce words and reach a stable pattern of handedness. To assess hemisphere lateralization for speech perception, we coupled event-related potential (ERP) recordings with a syllable-discrimination paradigm and measured response differences to a change in phoneme or voice (different speaker) in the left and right clusters of electrodes. To assess handedness, we gave a 15-item grasping test to infants. We also evaluated infants' range of vocabulary to assess whether it was associated with direction and degree of handedness and language brain asymmetries. Brain signals in response to a change in phoneme and voice were leftand right-lateralized, respectively, indicating functional brain lateralization for speech processing in infants. Handedness and brain asymmetry for speech processing were not related. In addition, there were no interactions between the range of vocabulary and asymmetry in brain responses, even for a phoneme change. Together, a high degree of right-handedness and greater vocabulary range were associated with an increase in ERP amplitudes in voice condition, irrespective of hemisphere side, suggesting that they influence discrimination during voice processing

Rectal temperature and respiratory rate measured 48–72 hours post inoculation (i.e. peak of clinical signs).

<p>In calves experimentally inoculated with different doses of viable <i>C. psittaci,</i> both rectal temperature (panel A) and respiratory rate (panel B) were significantly increased while no significant changes were seen in control calves. Data are given as Box-and Whisker Plots based on 2 or 3 measurements per calf in controls or infected animals, respectively. Different letters indicate significant differences between groups at given <i>P</i>-level (multiple range test).</p

Markers of pulmonary inflammation assessed in broncho-alveolar lavage fluid (BALF).

<p>Both concentration of total protein (A) and 12-HETE (B) were maximal in calves inoculated with 10⁹ ifu of <i>C. psittaci</i>. Data for control groups are from 2 and 3 dpi combined (Box and Whisker Plots). Data for calves challenged with different doses of viable <i>C. psittaci</i> are given on an individual basis for time points when calves were sacrificed (filled circles: 3 dpi; open circles: 7 dpi and 14 dpi). Kruskal-Wallis test revealed significant differences between groups at given <i>P</i>-level.</p

Development of the total clinical score over time.

<p>Data are given as regression lines and individual data according to the best fitting regression model per group. In control calves, no significant changes of total clinical score occurred after inoculation of medium or inactivated chlamydiae (panel A). In calves experimentally inoculated with different doses of viable <i>C. psittaci</i>, scores of clinical illness increased with challenge doses (panel B). Equations of regression, coefficients of correlation, R-squared values, and probability levels are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030125#pone-0030125-t001" target="_blank">Table 1</a>.</p

Distribution and extent of pulmonary lesions at day 3 pi in a calf inoculated with 10⁹ ifu of <i>C. psittaci</i>.

<p>Dorsal view of the lung and heart (H). Pneumonic lesions present as dark red discolorations (>) in the apical lobes, middle lobe and basal lobes. Note distension of the basal lobes due to severe bronchopneumonia in the inferior segments (hatched lines). Bar = 5 cm.</p

Assessment of regression lines of the clinical scores given in Figure 1.

<p>Assessment of regression lines of the clinical scores given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030125#pone-0030125-g001" target="_blank">Figure 1</a>.</p

Quantity of cells present in broncho-alveolar lavage fluid (BALF) 2–3 days post inoculation in calves experimentally inoculated with different doses of viable <i>C. psittaci</i> (<i>Cp</i>) and in control calves.

<p>Med = median. Kruskal-Wallis test: <i>P</i>≤0.05 indicates significant differences between medians of all groups. <i>W</i> test: <i>P</i>≤0.067 indicates significant differences between two groups. Medians highlighted in bold increased (<b>↑</b>) or decreased (<b>↓</b>) significantly in comparison to controls ‘<i>Cp</i> inactivated’. Data of the two control groups did not differ significantly.</p