Preliminary Study On Immune Responses Of The Pelophylax Nigromaculata Spleen Macrophages Following Infection With Elizabethkingia Miricola

Elizabethkingia miricola can infect a variety of farmed and wild frogs.It is the leading cause of meningitis-like disease in frog,which is highly contagious and fatal.This disease has been one of the most serious epidemic diseases in farmed frogs in China.However,the pathogenic mechanism of E.miricola and its interaction mechanism with the host are still unclear,thus limiting the prevention and control of related diseases.Macrophages play an important role in frog’s immune system,which can clean invading bacteria by phagocytosis and secreting a serious of inflammatory factors.In order to reveal the interaction between E.miricola and the host,we established in vitro model of the Pelophylax nigromaculata spleen macrophages following infection with E.miricola,which explored the process of E.miricola induced the immune response of macrophages,and resolved the mechanism between E.miricola and P.nigromaculata macrophages by ultrastructural observation,reactive oxygen content measurement and transcriptome analysis.Therefore,it can provide a theoretical basis for the prevention and control of meningitis-like disease in frogs.The main results and conclusions are as follows:1.A method was established for isolation,culture and identification of spleen macrophages in P.nigromaculata.The spleen tissue of P.nigromaculata was filtered through a 100 mesh and the cells were purified by Percoll density gradient centrifugation and cultured at 28°C.After that,macrophages were followed by cytomorphological examination and functional verification.The morphological results showed that the primary cells had typical characteristics of macrophages such as pseudopods and vesicles,besides the cells confluence were over 95% after 1 d in culture;The results of functional validation test showed Lipopolysaccharide(LPS)could significantly induce the generation of superoxide ions in macrophages(P<0.01),after 24 h of LPS-stimulated macrophages,the expression of TNF-α,IL-1β and IL-6was significantly higher than control group(P<0.01).In addition,the phagocytosis of spleen macrophages was 29.33%,21.54%,12.67% for Escherchia coil,Streptococcus agalactiae and E.miricola,respectively.2.In vitro infection experiments showed that E.miricola could induce the production of reactive oxygen species in P.nigromaculata macrophages and activate immune-related signaling pathways.The spleen macrophages of the P.nigromaculata were infected with E.miricola and apoptosis rate and reactive oxygen species were measured.It was found that E.miricola could significantly induce the apoptosis of macrophages(P<0.01).The infected macrophages could produce reactive oxygen species,with the increase of infection time,the content of ROS increased and then decreased.Transcriptome sequencing analysis of macrophages infected with E.miricola for 3 hours reveals that we obtained 2839 different genes,including 1126up-regulated genes and 1713 down-regulated genes.24 immune-related signaling pathways were screened by differential gene enrichment analysis,among which the most significant differences were NOD-like receptor signaling pathway,IL-17 signaling pathway,C-type lectin receptor signaling pathway and RIG-I-like receptor signaling pathway.Transcriptomics data revealed that the related signal pathways played a key role in the immune response of P.nigromaculata macrophages,which provided a reference for studying the interaction mechanism between E.miricola and host frogs.