动物造模,药效评价,继发肺炎链球菌肺炎 z-bio 2024-07-29 422

　　In the ranking of human disease mortality rates, bacterial pneumonia and influenza rank sixth. Animal models can be used to study the mechanism of interaction between influenza and pneumococcal bacteria.

　　1. Mouse pneumococcal secondary infection model

　　(1) Modeling method: The strains of Streptococcus pneumoniae used were D39 (serotype 2), WU2 (serotype 3), and TIGR4 (serotype 4). The H1N1 mouse adapted strain Influenza A/Puerto Rico 8/34 (PR8) infected with 400 pfu was inhaled into the oral pharynx of mice (dissolved in 50 μ l PBS). After 6 days of viral infection, D39 infected with 5 × 100000 cfu and PspA - infected with 5 × 100000 cfu, Hyl or NanA mutant strain 50 μ l PBS were inhaled through the oropharynx. After 24 hours of pneumococcal infection, euthanized mice were washed with 5ml of lung tissue and homogenized in the lavage solution. The lung tissue was frozen at -80 ℃. Detect the bacterial count in lung homogenate.

　　(2) Result: Prior to inoculation with bacteria, mice exhibited symptoms of influenza virus infection, such as hunchback, vertical hair, and lethargy. After 7 days of influenza virus inoculation, the titer of influenza virus in the lungs of mice ranged from 10 ^ 6 to 10 ^ 7 pfu. The bacterial count in the lung tissue of mice pre infected with influenza virus is much higher than that of uninfected individuals (15000-29000 times). After 6 days of influenza virus infection, reinfection with pneumococcal bacteria can serve as a model for secondary infection of pneumococcal bacteria after influenza, and can be used to study the mechanism of mutual influence and pathogenic effects between influenza and pneumococcal bacteria.

　　2. Ferret model

　　(1) Model making: Influenza virus of 1 × 10 ^ 6 ^ EID50 and pneumococcal of 1 × 10 ^ 6 ^ 6 cfu were diluted with sterile PBS in a volume of 1ml (500 μ l/nostril). Ferrets were anesthetized with 3.5% isoflurane and intranasal infected. Ferrets were infected with influenza virus or PBS control, and after 5 days, they were infected with pneumococcal strain D39 or dropped into PBS. Animals are divided into three groups: influenza virus, pneumococcal infection, or infection with influenza virus followed by pneumococcal infection, with two animals in each group. Observe clinical symptoms and collect blood samples and nasal irrigation fluid at 0, 6, 24, 36, and 48 hours. When collecting nasal flushing solution, 1ml PBS was injected into the ferret nasal cavity (60mg ketamine intramuscular injection anesthesia) and collected. Titrate influenza virus in nasal wash solution with MDCK cells, and culture pneumococcal colonies on soy trypsin agar plates with 3% (vol/vol) sheep red blood cells added. 48 hours after pneumococcal infection, the ferret was euthanized by Anle. Remove the lungs and fix them in 10% neutral formalin. Regular production observation.

　　(2) Result: Ferrets infected with influenza showed typical symptoms of influenza such as runny nose and reduced activity, while ferrets only infected with pneumococcal bacteria did not show runny nose or other respiratory symptoms, nor did they show reduced activity. Ferrets infected with pneumococcal bacteria after influenza virus attack exhibit more severe disease states than those infected alone, manifested as ataxia, refusal to play, and severe coma. Within 36 hours, the white blood cell count of ferrets infected with influenza virus and subsequently infected with pneumococcus increased by 82%, rising from 8.2 × 10 ^ 9 to 14.9 × 10 ^ 9, and showing a left shift in the nucleus. There was no significant change in the white blood cell count of ferrets infected alone, but ferrets infected alone with influenza virus had higher white blood cell counts than those infected alone with pneumococcal bacteria. The titer of pneumococcal bacteria in the nasal lavage fluid of ferrets infected with influenza virus first and then with pneumococcal bacteria is 2-3 log values higher than that of individuals infected with pneumococcal bacteria alone. The titer of influenza virus is similar or slightly higher than that of individuals infected with influenza virus alone. All ferrets showed mild pathological changes in their lungs and negative cultures for pneumococcal bacteria. But there are also some special findings. Ferrets infected only with influenza virus have widened alveolar septa, increased interstitial cells, and increased alveolar epithelial cells. Ferrets infected only with pneumococcal bacteria show infiltration of inflammatory cells mainly composed of neutrophils (Figure 30-1). Ferrets infected with influenza virus and pneumococcal bacteria show proliferation and hypertrophy of bronchial epithelial cells and alveolar epithelial cells, reduced cilia of epithelial cells, exudation in alveolar cavities, and infiltration of monocytes and neutrophils. The clinical symptoms of ferrets infected with influenza virus and subsequently infected with pneumococcal bacteria are more severe than those infected alone, with increased white blood cell count and higher bacterial titers in nasal lavage fluid. However, the pathological changes in the lungs are milder, indicating that ferrets infected with influenza virus first and then pneumococcal bacteria may have first developed upper respiratory tract infections.