Isolation,Identification And Potential Pathogenicity Of Three Spotted Fever Group Rickettsiae In Southern China

BackgroundTicks are blood-feeding arthropods with a global distribution which parasitize terrestrial vertebrates,including humans.Spotted fever is a tick-borne rickettsiosis caused by spotted fever group rickettsia(SFGR),which is a natural focus disease that seriously threatens human health.China has a high diversity of tick species,125 species have been reported,including 111 hard tick and 14 soft tick species.Novel SFGR species appear constantly and 21 genotypes of SFGR have been identified in the Chinese mainland.Among them,8 genotypes of SFGR have been reported to cause human disease in China.These research areas are mainly distributed in northern China.With the gradual expansion of the research scope of tick-borne rickettsia in China,the DNA of SFGR has been detected from ticks and host animals in South China.However,the research is limited to serological and molecular epidemiological investigation,but the discovery,isolation and identification of novel rickettsia are rarely reported.The ecological environment in southern China was different from those in northern China so that the dominant tick species and the pathogens transmitted by ticks are not the same.We should expand the scope of the investigation,strengthen the isolation and identification of novel pathogens and study on pathogenicity.Research purposeDetecting novel Spotted fever group rickettsia in southern China.Isolation and identification of novel Spotted fever group rickettsia.Studying the potential pathogenicity of novel Spotted fever group rickettsia.MethodIn this study,ticks were collected in Guangdong,Guangxi and Yunnan Province.The omp A and glt A genes were used as PCR targets and to investigate the presence of novel SFGR in each sample.Isolation and culture of the novel rickettsia were performed using cell lines.Morphological features and molecular biological techniques were applied to identify the isolated pathogen.Whole-genome sequencing was performed using Pac Bio sequencing technology.Plaque assays were used to study the differences in the plaque formation process of different rickettsia species.Specific antibodies were produced in host animals by animal immunization experiment.Besides,the novel rickettsia was detected in humans and animals through the sentinel surveillance survey and investigation of natural foci.ResultA total of 938 ticks(7 species)were collected in Guangdong,Guangxi and Yunnan Province.The infection rate of rickettsia was 12.86%(75/583).Two novel SFGR species and one new SFGR record were found.The sequences of Candidatus Rickettsia sp.GD01 were recovered from H.formosensis and were closest to the corresponding sequence of R.tamurae strain AT-1.Rickettsia longicornii strain GD02 was detected in the H.hystricis,D.steini,R.sanguinensis,R.microplus,H.montgomeryi and I.ovatus,which was highly homologous with that of Candidatus Rickettsia longicornii isolated ROK-HL727.The sequences of Rickettsia yunnanensis sp.nov.were recovered from H.montgomeryi and were closest to the corresponding sequence of Rickettsia hoogstraalii isolate Av20.In the present study,R.yunnanensis was successfully isolated and the complete genome sequence of this strain was reported.The morphological study shows that R.yunnanensis is a small short rod or long rod-shaped body(0.8-1.5 × 0.3-0.4 μm).The double membrane structure of rickettsia was detected by transmission electronic microscope.It was found that R.yunnanensis plaque formation rate and cell damage degree were similar to R.heilongjiangensis and R.conorii,which were stronger than R.raoultii.No infection of R.yunnanensis was found in 263 fever patients and 175 host animals.However,17 cases(6.46%)of fever patients were infected R.sibirica(6.08%)and R.raoultii(0.38%).ConclusionThis study is the first discovery of two novel SFGR and one new SFGR record in southern China.One of the novel SFGR was isolated from ticks,and its morphological evidence and whole genome sequence were obtained,which is potentially pathogenic.There are abundant tick species and high diversity of tick-borne pathogens in southern China.The distribution and epidemic of novel tick-borne diseases need further investigation.SignificanceThis study expanded the investigation scope of tick-borne rickettsia in China.two novel SFGR and one new SFGR record were found in southern China where are high biodiversity of tick species and tick-borne pathogens.The possible natural foci of novel SFGR were established in southern China from the serology,etiology and molecular epidemiology,which provided firm foundation for exploration of the pathogenic mechanisms and scientific basis on which to formulate preventive strategies of SFGR.