| Coccidiosis is the most serious intestinal parasitosis in chicken industry caused by one or more Eimeria.It can cause significant weight loss,malnutrition,blood loss,dehydration and increased susceptibility to other diseases,resulting in worldwide economic losses of up to $3 billion per year.Innate immune system is the first line of defense against microbial invasion,in which macrophages play an important role in innate and adaptive immunity.Macrophages can be activated by a variety of cytokines and pathogen related molecular models,such as lipopolysaccharide,CpG DNA signals,thus initiating innate immune response.In this study,chicken macrophages(HD11)were used as host cells for E.tenella sporozoites,and the development of E.tenella sporozoites in chicken macrophages(HD11)and macrophages in vitro were studied Anti-infective immunity produced by cells(HD11).The details are as follows:1.In vitro culture of HD11 cells from the sporozoites of Eimeria tenella.HD11 macrophages were inoculated with E.tenella sporozoites,and cell slides were prepared and observed with Giemsa staining microscope.It was found that E.tenella sporozoites successfully invaded HD11 cells but showed no signs of development.Macrophages were cultured in vitro to 50 h The cells are lysed,the sporozoites are free in the medium and have good vigor.2.The experimental group,HD11 cells infected with live sporozoites group,blank control group,the sporozoite suspension was replaced with the same volume of medium,positive control group,the sporozoite suspension was replaced with the positive control reagent in the kit.The levels of nitric oxide(NO)and reactive oxygen species(ROS)in the cells were detected by fluorescent probe method.The results showed that compared with the blank control group,the NO content in HD11 cells after the live sporozoite infection was significantly increased at 4 hpi(P <0.05),8 hpi reached the highest and no longer changed with time,while ROS content increased significantly at 2 hpi(P <0.05),6 hpi reached the highest and gradually decreased with time.3.HD11 cells were infected by viable sporozoites,HD11 cells by heat inactivated sporozoites and blank cells(with equal amount of cell culture medium).The mRNA levels of 8 host cytokines(IL-1β,IL-6,IL-10,IL-12p40,IFN-γ,TNF-α,iNOS and IRF7)related to E.tenella infection were detected by qPCR The immune response of phagocytes in coccidial infections is preliminarily described.The results showed that: Compared with the blank control group,the expression of IL-1 βand iNOS mRNA in HD11 cells infected with live sporozoites increased significantly at 6 HPI(P < 0.05),while IL-6,IL-10,IL-12p40,IFN-γ,TNF-α,iNOS and IRF7 increased significantly at 10 HPI(P < 0.05).Compared with the blank control group,the expression of IL-6 and IFN-γ mRNA in HD11 cells increased significantly at 4HPI(P < 0.05),IL-1,TNF-α and iNOS increased significantly at 6 HPI(P < 0.05),while IL-10,IL-12 and IRF7 increased significantly at 10 HPI(P < 0.05).It is suggested that the significant up regulation of cytokines in the immune response of macrophages to coccidia may be related to the inhibition of coccidia development and proliferation or elimination of coccidia infection.In conclusion,this study found that the sporozoites of Eimeria tenella can invade macrophages,but can not develop in cells,and macrophages have no digestion or other adverse effects on the sporozoites,but macrophages produce a large number of cytokines,which provides a theoretical basis for the role of macrophages in anti coccidial immune response. |
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