Effect Evaluation Of Essential Oil Of Oregano On Mice Infected With Pseudorabies Virus

Pseudorabies has been introduced into China for 70 years,which has led to huge economic losses in Chinese pig industry.At present,there are no effective prevention and control drugs for PRV,except for biological safety prevention and control measures such as vaccine immunization.Oregano essential oil is a volatile plant essential oil extracted from oregano.Studies have shown that it has anti-herpes simplex virus activity（HSV-1/2）,but there is no report about the anti-herpesviridae PRV activity of oregano essential oil in vitro and in vivo.In this experiment,SPF Kunming mice（KM mice）were used as experimental objects to evaluate the anti-PRV infection effect of oregano essential oil in vivo,and to provide scientific theoretical basis for the application of oregano essential oil in PRV infection.Experimental design:Through the pre-test,the optimal concentration of oregano essential oil powder was determined to be 250mg/kg BW after diluted and continuously administered to KM mice for 14 days（once a day）.100 male KM mice were randomly divided into five groups（3 drug dose groups,virus control group and solvent control group）.The mice were given oregano essential oil at 250,125 and62.5mg/kg BW for 14 days,and then infected with 10^-4/0.1m L PRV virus solution by intraperitoneal injection on the 8th day（except the solvent control group）.Observe for1 week,and record the clinical symptoms and survival of mice every day.Seven days after the attack,17 mice in each group were randomly selected for blood collection and autopsy,and the gross autopsy changes and organ indexes were observed.Among them,5 mice anticoagulation was used to detect the differentiation of T lymphocyte subsets in peripheral blood by flow cytometry.The organs were frozen with liquid nitrogen and used to measure the viral load of tissues,and formalin solution was fixed for observation of pathological sections of organs.RNA was extracted from duodenum and lung,and then used to measure the mRNA of TLR-like receptor（TLR3/7/8/9）and interferon type I partial genes（IRF-3/7,IFN-I）.Every two mixed samples of serum of 12 mice were used to detect immunoglobulin and cytokine.Through the observation of the above indexes,the anti-PRV effect of oregano essential oil in vivo and its partial mechanism were explored.The results are as follows:（1）Preliminary study on the anti-pseudorabies effect of oregano essential oil in vivo.All concentrations of oregano essential oil can alleviate the clinical symptoms and pathological changes of KM mice infected with PRV,delay the death process,improve the survival rate,significantly and dependently reduce the virus content in various organs（P<0.01）,effectively alleviate the pathological damage of lung and duodenum caused by PRV infection,and help to maintain the normal structure and function of tissues.It shows that oregano essential oil has the effect of anti-PRV in mice in vivo.（2）Effect of oregano essential oil on serum immune indexes of PRV infected mice.Compared with the virus control group,62.5 and 125mg/kg BW oregano essential oil could significantly reduce the spleen index of mice in a dose-dependent manner（P<0.05,0.01）,250 mg/kg BW oregano essential oil could significantly increase the thymus index（P<0.05）,and significantly reduce the spleen and lung index（P<0.05）.250mg/kg BW oregano essential oil can significantly increase the contents of IL-4,IL-10,IFN-γ,Ig G and Ig A in serum（P<0.01）,significantly decreased the contents of TNF-α（P<0.01）,while 125mg/kg BW oregano essential oil can significantly increase IFN-γcontent（P<0.05）.125 and250mg/kg BW oregano essential oil could significantly increase the percentage of blood CD3⁺,CD3⁺CD4⁺,CD3⁺CD8⁺T lymphocytes and the ratio of CD4⁺/CD8⁺（P<0.05 or 0.01）.It shows that oregano essential oil can maintain the immune balance and play the role of anti PRV by improving the ability of humoral and cellular immunity.（3）Effect of oregano essential oil on IFN-Ⅰmediated by TLR like receptors in organs of PRV infected mice.Compared with the virus control group,250mg/kg BW origanum essential oil significantly reduced the expression of TLR3/8/9mRNA in the lung and duodenum（P<0.01）,and 125mg/kg BW origanum essential oil significantly reduced the expression of TLR3/7/9 mRNA in the lung and TLR3/9mRNA in the duodenum（P<0.01）.62.5mg/kg BW origanum essential oil significantly reduced the mRNA expression of TLR7/8/9 in the lung and the mRNA expression of TLR3 in the duodenum（P<0.01）.125 and 250mg/kg BW origanum essential oil could significantly increase the mRNA expression of some genes in the IFN-Ⅰpathway in the lung and duodenum（P<0.01 or P<0.05）,62.5mg/kg BW origanum essential oil significantly increased the mRNA expression of IRF-3,IFN-αin the lung and IFN-αin the duodenum（P<0.01）,and significantly increased the mRNA expression of IRF-7and IFN-βin the duodenum（P<0.05）.These results indicated that origanum essential oil could reduce the over-expression of TLR-like receptor in PRV-infected mice,induce the mRNA expression of IRF3/7 and IFN-I,and relieve the immunosuppressive state of the body,thus exerting the antiviral effect of interferon induced by origanum essential oil.Conclusion:10%oregano essential oil powder at 62.5～250mg/kg BW can reduce the load of PRV in various tissues in a dose-dependent manner,alleviate the pathological changes of tissues,improve the survival rate and have anti-PRV effect.Oregano essential oil can play an anti-PRV role in vivo by improving the humoral and cellular immune function of PRV infected mice,reducing the over-expression of TLR-like receptor after PRV infection,and inducing the expression of IRF-3/7 and IFN-I.The anti-PRV effect of 10%oregano essential oil powder in mice is the most suitable at 250mg/kg BW.