| Scrub Typhus is a zoonotic disease caused by an arthropod-borne Gram-negative specialized intracellular bacterium that poses a serious threat to public health and safety.Its widespread prevalence in the Asia-Pacific region,extending from Afghanistan to China,Korea,the Southwest Pacific islands,and northern Australia,causes disease in 1 million people annually and threatens the health of nearly 1 billion people worldwide.Serologic evidence from African and South American countries suggests that the geographic extent of the disease and the population of risk groups may be even greater.Our country is one of the priority epidemic areas for scrub typhus,with high incidence and mortality rates ranging from 1.4%to 6%seriously threatening the health of our population.Previous studies suggest that scrub typhus is endemic in a wide area south of31°N latitude in China with significant seasonal characteristics,with summer type predominating.Since the first report of scrub typhus epidemic in Shandong in 1986,new epidemic areas have been formed and spread,and the epidemic season in such areas is also different from that in the south,being autumn and winter,leading to a large change in disease characteristics,and the genetic background,pathogenic characteristics,and virulence intensity of the Orientals of scrub typhus may also have mutated and gradually developed toward more pathogenic strains.Rickettsia diseases mainly cause symptoms such as fever,rash and swollen lymph nodes,and clinical features cannot be effectively distinguished from other febrile diseases,which are often misdiagnosed and lead to aggravation,thus causing fatal diseases such as multi-organ damage.In view of the fact that Orientia tsutsugamushi is difficult to be stably cultured and transmitted under conventional laboratory conditions,the requirements for laboratory culture conditions and experimental personnel are high,and the culture of this strain not only requires a large number of normal cells to obtain the isolated strain,but also requires a large number of purified strains for subsequent related research,and the above combined reasons have made fewer domestic researchers,while international research on the pathogenesis of tsutsugamushi is extremely scarce,it is important to establish an economic and rapid diagnostic method.In this study,we attempted to establish a rapid diagnosis method for scrub typhus with relatively high sensitivity and specificity and easy to operate,and to find suitable targets for vaccine development in order to eliminate scrub typhus oriental infection at root.Our study focused on four parts:In the first part,specific primers were designed based on the sequences of TSA and Htr A,cloning and prokaryotic expression vectors of TSA and Htr A were constructed,and the induced expression and conditions of the two proteins were optimized.The results showed that E.coli strains with stable in vitro expression of TSA and Htr A were constructed,while higher protein concentrations were obtained by purification,which laid the foundation for further experiments.In the second part,the study initially establishes the method for the detection of Orientia tsutsugamushi.Colloidal gold rapid detection method:the preserved Escherichia coli strains that can be used to stably express TSA and Htr A were revived,protein induction expression purification was performed,and after obtaining the protein with high purity in vitro,colloidal gold was used for labeling,and the established method was initially evaluated,and the results showed that using clinically confirmed specimens and healthy human.The results showed that the method was highly accurate and significantly reduced the detection time and cost by using confirmed clinical specimens and healthy specimens;Immunofluorescence antigen slice assay:the preserved Orientia tsutsugamushi strains were revived,infected with L929 cells,and antibody incubation was performed after preparation of cell suspension to produce antigen slice for the assay,and the preliminary results showed that the method is low cost and has good accuracy.In the third part,the antigenicity of TSA and Htr A was initially investigated,and HUVECs were stimulated with the two proteins respectively to observe the effects of TSA and Htr A on the phenotype of HUVECs,while the changes of related cytokines were detected using QPCR.The results showed that the levels of relevant cytokines were significantly higher in the experimental group compared with the control group.In the fourth part,Orientia tsutsugamushi infected mouse model and its induced cytokine storm:firstly,the preserved Orientia tsutsugamushi strain was revived and infected with L929 for several times to optimize the culture conditions to enable mass cloning.The cell cultures were collected repeatedly and repeatedly for cell fragmentation followed by differential centrifugation for ultra-purification to obtain a high concentration of Rickettsia suspension,which was finally quantified at a concentration of 1×10~8copies/μl,and infected with type I interferon knockout mice and natural immunodeficient TLR4 mice by the intraperitoneal inoculation route,and basic information such as mouse signs,body temperature and body weight were recorded,while blood and organ specimens were collected.The results showed that compared with the control group,the mice in the type I interferon knockout experimental group showed significant changes in activity and other indicators on the eighth day after infection and death occurred on the tenth day after infection,while the mice in the TLR4 receptor natural deficiency group showed no significant differences in activity and status,and also combined with the HE staining and immunohistochemical results,mice in the experimental group showed significant inflammatory cell infiltration and higher levels of cellular inflammatory factors.In this study,two specific proteins of Orientia tsutsugamushi were successfully expressed in vitro,and a rapid colloidal gold detection method based on TSA of Orientia tsutsugamushi and clinical diagnosis by immunofluorescence antigen slice method were initially established,providing a rapid and effective technical means for clinical laboratory to confirm the diagnosis of Orientia tsutsugamushi and rickettsia diseases.TSA and Htr A were pathogenic to HUVECs,providing a basis for further The establishment of model with lung and spleen damage due to the similar performance of patients after Orientia tsutsugamushi infection can be used as in vivo models for the study of the pathogenicity of Orientia tsutsugamushi,and the preliminary investigation of the pathogenicity of animals infected with rickettsiae in combination with different mouse models. |
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