| Background: Yersinia pestis (Y. pestis) is the causative agent of bubonic and pneumonic plague, which has taken off numerous lives in human history. The worldwide plague cases have been going up since the last decade. Great concern has been attracted because of possibility of abusing Y. pestis as a bioterrorism agent. Therefore, plague still poses significant threats on the public health in the 21st century. However, little is known about the pathological changes of infected organs in inhalation plague, nor the biological changes of Y. pestis during the development and progression of plague. Recently, researches on this pathogen were mostly focused on the ability to resist the innate host defense mechanisms, such as phagocytosis, and inflammatory responses induced by macrophages and neutrophils. Although anumber of virulence-related genes of Y pestis have been identified in the past years, the exact functions of these genes and their encoded proteins in plague progressing are still needed to be clarified.Objective: To investigate the disease progression in the experimental inhaled plague model in mice, and the expression profiling of some virulence related genes of Y. pestis during the infection.Methods: The model of inhalation plague was reconstructed by intranasal delivering Y. pestis into BALB/c mice. The lung, liver, spleen, heart and thymus of the infected mice were collected and examined by light microscopy and transmission electron microscopy to characterize the lesions at different time after the exposure. The polymorphonuclear leucocytes (PMNs) in the lung sections were stained with the Naphthol AS-D chloroacetate esterase kit, and the ratio of PMNs area to the total area of the visual fields was measured by imaging analysis system and the computer software. The degree of acute inflammation in pneumonic plague was quantified by statistically evaluating the data. Apoptotic cells in the infected tissues were detected by immunohistochemistry. Expression profiles of five virulence-related genes in Y. pestis {cafl, icrV, icrG, pla andpsaA) were determined by real-time PCR, and immunohistochemistry was utilized to visualize the corresponding proteins (Fl, LcrV, LcrG, Pla and pH6) in the infected organs.The main results:1. Most mice infected by intranasal delivery died in 2-3 days, The mice with pneumonic plague died in less than 3 days, and mortality of the miceafter the exposure to Y. pestis by inhalation was 100%.2. Two types of plague were developed after intranasal challenge, i.e. the typical primary pneumonic plague and non-pneumonic plague, with obviously different histopathological changes in the lung.3. According to the PMN ratio, the primary pneumonic plague can be divided into three stages, prophase, metaphase and anaphase.4. The basic pathologic changes in infected lung include acutely suppurative inflammation, hemorrhage, necrosis and interstitial aggregates of Y. pestis bacilli. By transmission electron microscopy, it was observed that pulmonary type I and II epithelial cells underwent degeneration and necrosis, and multilamellar bodys in pulmonary type II epithelial cells reduced dramatically or even disappeared. Moreover, the PMNs and macrophages of infected lung were markedly changed. Their mitochondria were obviously swollen and endoplasmic reticula were dilated. Pyknosis, karyorrhexis, karyolysis and phagolysosome were observed. A lot of organelles including lysosomes were released into the lung interstitial spaces. The fraction I envelope (Fl) antigen and fimbria-like structure were found on the cell surface of Y. pestis in alveolar lumina by transmission electron microscopy. Y. pestis bacilli in interstitial spaces were enwrapped with thick capsule instead of fimbria-like structure.5. In the infected liver at the terminal stage of the disease, significant changes were found, such as scattered focal necrotic areas containing degenerate and necrosis with neutrophil infiltration. Y. pestis was not be seenin the necrotic area.6. At the early stage of the disease, the red and white pulp of spleen revealed reactive hyperplasia. At the terminal stage of the disease, there were hemorrhage with infiltration, necrosis and apoptosis of neutrophils and macrophage in red pulp of spleen. The white pulp showed multifocal lymphocytolysis, resulting in moderate to complete loss of the white pulp.7. At anaphase of the disease, multifocal lymphocytolysis and apoptosis also appeared in the thymus of infected mice.8. Acidophilic degeneration even necrosis in cardiac muscle cell at anaphase was found.9. In infected mice, the expression of five virulence-related proteins (Fl, LcrV, LcrG, Pla, PsaA) on the surface of Y. pestis were demonstrated by immunohistochemistry staining with the corresponding antibodies.10. Detailed real-time PCR analysis revealed that five virulent-related genes {cafl, icrV, icrG, pla andpsaA) were transcribed differently at different time and in different organs after the exposure to Y. pestis. Cafl maintained a high level at prophase without tissue difference. Both IcrV and IcrG had phase difference and tissue difference. Pla played a role mainly at anaphase. PsaA expression was staggered, which up-regulated rapidly and down-regulated slowly or rapidly subsequently.The main conclusion:1. The inhalation plague model in mice was successfully reconstructed by intranasal delivering Y. pestis into mice. Two types of plague were induced inthe mice, i.e. the typical primary pneumonic plague and the non-pneumonic plague. The mice developing to primary pneumonic plague died in less than three days after the challenge. The mortality of mice plague by intranasal infection was 100%.2. The basic histopathological changes of primary pneumonic plague include acute suppurative inflammation, hemorrhage, necrosis and interstitial multiplication of Y. pestis, and multi-organs pathological changes in the septicemic plague dead-ends.3. By PMN staining and computer image analysis, the degree of acute inflammation of primary pneumonic plague can be quantified. The three selected time points were just the periods that showed the distinct pathological changes.4. The morphology of Y. pestis underwent remarkable changes to adapt the various living environment encountered in the different organs of the host.5. The innate immune cells of the host were severely destroyed, which demonstrated that Y. pestis mainly targets the immune cells during infection.6. All five virulence-related proteins of Y. pestis, (Fl, LcrV, LcrG, Pla, and pH6) were expressed on the surface of bacteria in infected tissues.7. The in vivo transcription profiling of these five genes suggested that, the differential transcription was closely related with their functions during infection The results imply the interaction between Y. pestis and the host during the plague progression.The main creative results:1. The present study systematically expatiated the changes of histopathology and ultrastructure in infected tissues of experimental inhalation plague models.2. By transmission electron microscopy observation, the fimbrial-like structure, Fl antigen and capsule of Y.pestis were firstly visualized in infected host tissues, which elucidated the morphological changes of Y. pestis in various living environments encountered in the host.3. By means of PMN staining and computer image analysis, acute inflammation of primary pneumonic plague was firstly quantified, and the progression of pneumonic plague was divided into three stages.4. By immunohistochemistry with special antibodies, five important virulence-related proteins of Y. pestis (Fl, LcrV, LcrG, Pla and pH6) were detected in the organs of the infected mice. The detection of LcrG, Pla and pH6 was firstly reported.5. Real-time PCR revealed the time-course in vivo transcription profiles during the plague developing. The changing transcription levels of these genes were closely related with their functions in the pathogenesis, which might reflect the underlying interaction between Y. pestis and the host in the course of plague.
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