Isolation And Identification Of Emerging Tick-borne Rickettsia Erhaii And Epidemiological Investigation In Ticks

Research BackgroundAs a diverse population,Ticks were obligate blood-feeding arthropods to be clearly identified as pathogen carriers.Like mosquitoes,ticks were the major vectors of human and livestock diseases around the world.Spotted fever group rickettsia(SFGR),as an important pathogen of tick-borne diseases,causes a large number of zoonotic diseases worldwide.To date,SFGR has been found on all six continents except Antarctica,and more than 15 SFGR species have been confirmed to cause human disease,while more than 20 species of SFGR have been found in mainland China.With the development of molecular techniques,the range of known species within this group of bacteria raised significantly.Due to the limitations of SFGR separation technology,there were few isolates of SFGR,which made SFGR experiments difficult to be performed in the laboratory.the SFGR associated with the arthropods medium,the importance of vertebrate as host,Difference of symptoms between animal infection and human infection,various pathogenic species and host cell interactions were still not clear.At the same time,most of the current studies on ticks focus on the widespread species,and less on the narrowly localized species.H.montgomeryi is mainly distributed in southwest China,but there are few studies on its living habits and the role of vector.However,a variety of spotted fever group rickettsia have been detected and a novel tick-borne rickettsia strain was successfully isolated from H.montgomeryi,suggesting that it has the possibility of spreading multiple SFGRs.Therefore,H.montgomeryi was used for screening,identification and isolation of SFGR.On the basis of obtaining the isolated strain,taking more attention to the studies on genomics and biological characteristics of SFGR will help to understand the infection pathway and mechanism of Rickettsia.Finally,the monitoring of multiple and novel SFGR is an important means to detect and prevent newly human pathogenic rickettsioses.Research ObjectivesFirstly,to explore the application of different methods in the isolation of newly tick-borne rickettsia.Secondly,to find out the biological characteristics of novel spotted fever group rickettsia.Finally,to find out epidemiological characteristics of the novel spotted fever group rickettsia in ticks.Research ContentsFirstly,establish an ecological population of H.montgomeryi under laboratory conditions and obtain a novel SFGR isolated strain,followed by phylogenetic analysis.Secondly,obtain its genome-wide sequence and observe the biological characteristics of the novel SFGR in vitro for identification and its potential pathogenicity analysis.Finally,make an epidemiological investigation in ticks of the novel SFGR in natural environment to find out its distributionResearch MethodsFirstly,an ecological population of H.montgomeryi collected from Yunnan province was established under laboratory conditions and its life cycle was observed.Some ticks were pestled to fragments and inoculated into cells,and then via plaque separation experiment to isolate two kinds of spotted fever group rickettsia mixed in cell.After isolated successfully,DNA was extracted from culture and used for analyzing Rickettsia specific gene fragments for novel SFGR identification and classification.Secondly,the genome-wide sequence of novel SFGR was obtained to analyze its structure and function.The morphology of Rickettsia was observed by Giemsa staining and ultrathin section with electron microscopy.Plotting the growth curve of novel SFGR in various cell lines,observing its cytopathic effect and plaque assays cytotoxicity to describe the biological characteristics of novel SFGR,and find out its invasive ability and virulence on host cells,as well as animal hosts and human potential pathogenicity.Finally,the omp A genes was used to investigate presence of SFGR in tick samples collected from many provinces and cities in the northeast and southwest regions where ticks are plentiful by molecular techniques.including the epidemiological characteristics of novel SFGR to find out its infection level and coinfection with other pathogens in the natural environment.Research ResultsDiscover and isolation of a novel SFGR named R.erhaiiIn this study,an ecological population was established and a life cycle was observed about 110-130 days of H.montgomeryi.Some of them from the same mother tick was inoculated into Vero81 cells after grinded.After a period of culture,Rickettsia was confirmed to exist in the culture and could grow stably in various cells.However,genome-wide sequence about Rickettsia in the culture suggested that the isolated strain might mingled two different SFGRs.According to the plaque purification experiment,the plaques formed were picked out and inoculated into fresh cells for culture,and the two SFGRs were successfully separated.specific gene fragments of SFGR were amplified and phylogenetic analysis showed that one of them was R.yunnanensis isolated in the laboratory earlier.The other strain was found to be different from the existing Rickettsia.According to the identification criteria of the new species of Rickettsia,it was confirmed to be a novel spotted fever group rickettsia named Rickettsia erhaii.Study on genomics and biological characteristics of R.erhaiiThe genome-wide sequence of R.erhaii was obtained,with 32.5% of G+C content similar to other SFGRs.It is most closely related to R.massiliae and R.rhipicephali.The morphology of R.erhaii were observed by Giemsa staining and ultrathin section with electron microscopy,which was long rod-shaped,micro-rod-shaped and spherical.The ultrastructure of R.erhaii was similar to that of other SFGRs,showing typical Rickettsia bilayer structure.Intracellular proliferation of R.erhaii showed that R.erhaii could grow in mammal cells and tick cells,and the load of R.erhaii in Vero81 cells was much higher than that in IDE8 cells,suggesting the possibility of R.erhaii infects host containing animal and human.The toxicity of R.erhaii was studied.By comparing the cytopathic effects of several SFGRs,it was found that R.erhaii was less toxic to cells than other SFGRs.Plaque assays showed that the plaques formed by R.erhaii were relatively concentrated in the form of sheets,with the cells became translucent and when the concentration was higher,cell apoptosis happened.Meanwhile,the plaques formed more slowly and the degree of cell damage was weaker compared with R.yunnanensis.Immune experiments in BAL B/C showed that a 1024-fold increase in titer against R.erhaii,the antiserum of R.erhaii was obtained.Epidemiological investigations on SFGR and R.erhaii infection in ticksA total of 826 ticks were collected from northeast and southwest China,and identified as 16 tick species belonging to 5 genera.The infection rate of spotted fever group rickettsia was 44.44%(356/801),and the infection rate of SFGR was vary greatly among different tick species.Moreover,in sixteen tick species,R.erhaii was only detected in H.montgomeryi collected from Jianchuan,Yunnan,with a positive rate of38.71%(36/93),which was at a high level.R.erhaii also had complex infection with A.phagocytophilum and Babesia sp.BICM002,with infection rates of 26.88% and1.08%,respectively.In addition,the investigation of R.erhaii coinfection revealed that there was a case of R.erhaii and R.yunnanensis combined infection,with a coinfection of 24.73%.Research ConclusionsFirstly,H.montgomeryi is capable of carrying many tick-borne pathogens,including SFGR,Anaplasma,Babesia,suggesting that it may carry multiple pathogens which have not been found yet.At the same time,there is a situation of compound infection in H.montgomeryi.it needs to pay more attention on research.Secondly,among the 16 tick species collected,R.erhaii was only detected in H.montgomeryi,which did not mean that R.erhaii could only be carried by H.montgomeryi.The distribution of R.erhaii could be known more clearly by expanding the collection area,screening different species of ticks and collecting sufficient sample size.Other,inconsistent with traditional knowledge,there are two kinds of spotted fever group rickettsia coinfection in H.montgomeryi in natural,and this may be related to the fact that the two species of spotted fever group rickettsia were not easily detected by using conventional universal primers.Change the primers or adjust the screening strategy to screen more tick species to verify whether the combination of two spotted fever group rickettsia is common or accidental.Finally,plaque formation test can not only be used for bacterial cloning,biological characteristics research and screening of sensitive antimicrobial agents,but also play an important role in Rickettsia isolation.Once various spotted fever group Rickettsia compound infection is common,the plaque purification and isolation test can be a good choice.