Protective Effect Of Lactobacillus Reuteri On Mice Infected With H9N2 Subtype Avian Influenza Virus

The effects of probiotics,which colonize the host gut,have significant implications for human and animal health.Due to their application in regulating the balance of intestinal flora,stimulating the activity of endogenous beneficial microorganisms and activating the host immune system and other aspects of intestinal microecological environment,they have been paid close attention to and received extensive attention and recognition in the medical field.Recent studies have shown that oral probiotics can effectively prevent or treat lactose intolerance and other intestinal related diseases,while lactic acid bacteria have been proven to adhere to toxic and harmful substances,stimulate intestinal mucosal immune tissue,reduce morbidity,etc.Lactobacillus reuteri can improve the resistance of the host to exogenous microorganisms and enhance the metabolism and growth performance of animals by maintaining and regulating the balance of intestinal flora.It can be seen that Lactobacillus reuteri,as a common intestinal probiotic,has a crucial effect on intestinal microecological mucosal immunity.Therefore,it is of great significance to study intestinal probiotics and their immune-related mechanisms for the treatment of H9N2 subtype avian influenza virus.In 1966,H9N2 subtype Avian Influenza Virus（AIV）was first isolated from turkeys somewhere in the United States.In the early 1990s,it was first isolated from chickens in China.In the following decades,Related viruses and their subtypes have become endemic in farmed poultry in most parts of Asia,the Middle East,West Africa and North Africa.As a zoonotic disease,the virus can infect not only poultry but also humans,causing great damage to the breeding industry.This study explored the protective effect of Lactobacillus reuteri against H9N2 subtype AIV.The specific research contents and experimental results are as follows:（1）C57BL/6J mice were used to screen the probiotics with the best protective effect against H9N2 subtype AIV infection,and the mice were randomly divided into 5 groups:Experimental group（Ef1+H9N2）,（Ef2+H9N2）,（Lr+H9N2）,normal control group（PBS+PBS）and infected control group（PBS+H9N2）.Mice in the experimental group were fed Enterococcus faecalis and Lactobacillus reuteri respectively for one week,and then challenged with H9N2 subtype AIV.The survival status of mice was monitored daily after challenge and the changes of body weight were recorded.The protection of probiotics against H9N2 subtype AIV was evaluated by weight loss rate and survival,and the best probiotics were selected.The results showed that L.reuteri had the best protective effect against H9N2 subtype avian influenza virus infection by reducing the rate of weight loss and mortality in mice.（2）Two challenge groups（Lr+H9N2）,（PBS+H9N2）and two control groups（Lr,PBS）were set up and fed with Lactobacillus reuteri and PBS,respectively.After one week of feeding,H9N2 subtype AIV was challenged.After challenge,the survival status of mice was monitored daily and the change of body weight was recorded.The lung tissues of mice were collected and fixed 7 days after the challenge for pathological section analysis.Serum and alveolar lavage solution were collected,and IFN-αand IFN-βcontents were analyzed by ELISA kit.Part of lung tissue was collected and stored at-80℃for virus titer determination.The challenge protection of Lactobacillus reuteri against H9N2 subtype AIV was evaluated by weight loss rate and survival of mice,contents of IFN-αand IFN-βin alveolar lavage fluid and serum,lung section observation and pathological damage score,and virus titer of lung detected by TCID50.The results showed that L.reuteri had a good effect on improving the immunity of mice.It significantly increased the contents of IFN-αand IFN-βin serum and alveolar lavage fluid,enhanced the intensity of innate immune response,reduced the lung tissue damage caused by infection with H9N2 subtype AIV,and significantly reduced the virus titer in lung tissue.（3）The effects of Lactobacillus reuteri on type I interferon and anti-H9N2 subtype AIV infection were tested by IFNAR1^-/-mice and wild mice.C57BL/6J mice and IFNAR1^-/-mice were divided into two groups,which were fed Lactobacillus reuteri screened,and fed for one week after challenge.The potential role of type I interferon in providing protection against H9N2 subtype AIV infection by probiotics was proved by pathological section and immunofluorescence analysis.The results showed that after Lactobacillus reuteri treatment,the weight loss rate and mortality of mice in the gene deletion group were higher,the degree of lung pathological injury and the content of viruses in the lung were significantly increased,while the wild group mice could improve the anti-influenza virus ability by increasing the level of type I interferon,which was consistent with the results of the previous animal model.It lays a foundation for the development of anti-influenza microecological agents and anti-influenza drugs in the future.In this study,a mouse protective model was established,and the dominant probiotics with the most protective effect were selected from the three kinds of probiotics.The effects of probiotics on intestinal microecological environment and host innate immune response of mice were verified through in vitro and in vivo experiments,and the interaction between probiotics and type I interferon was further clarified,which laid the foundation for the development of anti-H9N2 subtype AIV microecological preparations and related drugs in the future.