| Mink hemorrhagic pneumonia is one of the important diseases that seriously endanger the healthy development of mink aquaculture.For a long time in the past,it was thought that the disease was caused by infection with Pseudomonas aeruginosa.However,studies in the past ten years have shown that a single infection of Pseudomonas aeruginosa is not very virulent to mink and cannot cause hemorrhagic pneumonia in mink.There should be other incentives for the disease.Influenza A virus（IAV）of H9N2 subtype is widespread in mink,but it is not very pathogenic to mink,and a single infection only causes mild respiratory diseases in mink.However,H9N2 IAV infection is prone to induce secondary infection of Pseudomonas aeruginosa,resulting in hemorrhagic pneumonia in mink.Klebsiella pneumoniae is a conditional pathogen that is widespread in the environment and often colonizes the upper respiratory tract of mink.This study was conducted to investigate whether Klebsiella pneumoniae infection secondary to H9N2 IAV could induce severe pneumonia.Using Mk/SD/F10/13（H9N2）and Klebsiella pneumoniae-SD-21 strains isolated and preserved in our laboratory,Kunming mice were used as experimental animals to establish a mouse model of Klebsiella pneumoniae infection secondary to H9N2 IAV.176 SPF female Kunming mice（KM mice）were randomly divided into 4 groups with 44 mice in each group.Group 1: single infection with H9N2 IAV（H9 group）,group 2: single infection with Klebsiella pneumoniae（KPN group）,group 3: H9N2 IAV secondary Klebsiella pneumoniae infection（H9 + KPN group）,group 4: inoculated with sterile PBS as a negative control.After inoculation with H9N2 IAV and/or Klebsiella pneumoniae,the clinical symptoms of the mice were observed every day,and the body weight and body temperature of the mice were recorded on the 3rd,5th,7th,9th,and 11 th days,the blood of the mice was collected to prepare serum for anti-H9N2,and the organ tissues（heart,liver,spleen,lung,kidney）of the mice were collected at the same time,the lung index of mice was calculated after weighing the lung tissues;A part of the organ tissues was immersed in 10% neutral buffered formalin to make slices to observe the histopathological changes of mouse organs,and to determine the apoptosis index of lung cells.Another part of the organ tissues was ground for detection of bacterial load,viral load and cytokines.The results showed that H9N2 IAV single-infected mice had no obvious clinical symptoms and no significant weight loss,which indicated that H9N2 IAV was less pathogenic to mice;Compared with H9N2 IAV or Klebsiella pneumoniae single-infected mice,mice infected with H9N2 IAV secondary Klebsiella pneumoniae developed a faster course of disease,exhibited severe respiratory disease,and significantly increased lung index（P < 0.05）.The results of histopathological examination of the lungs of experimental mice showed that the lung tissue of mice single-infected with H9N2 IAV showed moderate pathological changes,the mice single-infected with Klebsiella pneumoniae developed interstitial pneumonia in the lungs,and H9N2 IAV secondary Klebsiella pneumoniae-infected mice had more severe lung histopathological damage than the single-infection groups.The apoptosis of mouse lung cells was detected by TUNEL method.The results showed that compared with H9N2 IAV/Klebsiella pneumoniae single-infected mice,the number of apoptotic cells in the lung tissue of H9N2 IAV secondary Klebsiella pneumoniae-infected mice was significantly increased,and the apoptosis index（AI）was significantly increased（P< 0.05）.The collected mouse organ tissues were ground for detection of H9N2 IAV and Klebsiella pneumoniae.The tissue grinding fluid was inoculated with 10-day-old SPF chicken embryos to detect the H9N2 IAV load in the lungs of mice.The results showed that the H9N2 IAV load in the lungs of mice infected with H9N2 IAV alone was higher than that of mice infected with Klebsiella pneumoniae secondary to H9N2 IAV.The peak of bacterial load in each organ of mice infected with Klebsiella pneumoniae alone appeared at 5dpi,while the peak value of each organ of mice infected with Klebsiella pneumoniae secondary to H9N2 IAV infection appeared at 7dpi.It can be seen that the clearance rate of Klebsiella pneumoniae in each organ（liver,spleen,lung,kidney）of mice infected with Klebsiella pneumoniae secondary to H9N2 IAV was delayed compared to mice infected with Klebsiella pneumoniae alone.In addition,secondary Klebsiella pneumoniae infection affected antiH9N2 antibody titers and cytokine levels,and anti-H9N2 antibody titers in the serum of mice infected with Klebsiella pneumoniae secondary to H9N2 IAV were significantly higher than those of mice single-infected with H9N2 IAV（P < 0.05）.In the early stage of the disease course,the level of IL-1β in the lung homogenate of the mice infected with Klebsiella pneumoniae secondary to H9N2 IAV was significantly higher than that in the H9N2IAV/Klebsiella pneumoniae single infection group（P<0.05）.In the middle stage of the disease course,the level of IL-6 in the lung homogenate of the mice infected with Klebsiella pneumoniae secondary to H9N2 IAV was significantly higher than that of the mice with single infection of H9N2 IAV/Klebsiella pneumoniae（P<0.05）.There were no significant differences in the levels of TNF-αbetween groups of mice.The results showed that H9N2 IAV infection promoted the secondary infection of Klebsiella pneumoniae,resulting in severe respiratory disease in mice,severe lung pathological damage,increased lung apoptosis index,delayed bacterial clearance in the organs,altered levels of cytokines in the lungs,and increased mortality.These findings imply that early prevention of IAV infection is critical to reducing severe diseases caused by secondary bacterial infections in animals.This study enriches the research data on secondary bacterial infection of low pathogenic IAV,provides a new perspective for the prevention and control of mink hemorrhagic pneumonia,and also enriches the research data on IAV-bacterial co-infection pneumonia. |
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