Effects Of Aeromonas Hydrophila On The Hemocyte And The Interleukin IL-1β And IL-10 Expression Of Rana Dybowskii

In recent decades,the populations of amphibians has dropped sharply all over the word,presumably due to climate change,habitat loss and fragmentation,ultraviolet radiation increases,environmental pollution and infectious diseases.This phenomenon has been drawing broad attention in scientific community.And it has been suggested that the pandemic has aggravated the decrease of amphibians.Aeromonas hydrophila is a common pathogenic microorganism which often infect amphibians.The "red leg disease" which caused by infection with Ah is a kind of frequent disease severeil imperils amphibians’lives.Blood and immune factors are important components of the amphibians immune system.The research on amphibian blood are usually concentrated on evolutionary standpoint,and the attention to amphibian innate immune responses often focus on antimicrobial peptides.There is scarce study on the change of the blood physiological index and expression of cytokines in amphibians.In this study,we selected Aeromonas hydrophila as infection source,and Rana dybowskii,a dominant amphibian in Northeast of China,as the research object,to research the change of the blood physiological index under the influence of Ah by microscopy.And the dynamics changes of the expression of cytokines IL-1β and IL-10 in main visceral organs of R.dybowskii under stress of different concentration of bacteria was explored by Fluorescence quantitative PCR and Western Blot.We hope to study the immune response mechanism of amphibians to pathogenic organisms initially from cytokine balance perspective,and provide the foundation for further understanding of the amphibian immune defense mechanism.The results of this study are as follows:The peripheral blood cell count and microscopic analysis showed that the indexes of R.dybowskii blood cell such as hemocyte concentration and the proportion of various hemocytes had altered after infected Ah.The quantity of peripheral red-cell count decreased per unit volume but had no statistical changes.The red-cell size increased obviously.The total of leukocyte had increase significantly and the proportion of differential leukocyte had changed.The percentage of monocytes and neutrophils increased significantly,the percentage of small lymphocytes dropped significantly.The proportion of macrolymphocytes,eosinophils and basophils had no evident change.The results of real-time PCR analysis revealed that the expression of IL-1β and IL-10 mRNA increased at 4-72h after Ah infection in the liver,spleen,kidney and lung of R.dybowskii.The expression levels and trends of IL-1β and IL-10 in four organs are different for tissue specificity.Overall,the expression of IL-1β in liver and spleen was higher than kidney and lung,and it reached the peak at 12h after infection in most cases.The expression of IL-10 in liver,spleen and kidney was higher than lung,and it reached the peak at 24h in most cases which was behind IL-1β.So we selected the liver to perform Western Blot to test the expression level of Pro-IL-1β protein infected by different concentrations of Ah.The result of Western Blot basically accords with the result of Real-time PCR.The expression of Pro-IL-1β protein at the peak also declined with the increasing of the concentration of Ah.We speculated that the body had triggered the negative feedback control to reduce the inflammatory reaction to protect itself.