Isolation And Identification Of Novel Tick-borne Rickettsia And The Study On The Tetracycline Intervention Of It In Trans-phase Transmission Of Vector Ticks

BackgroundSpotted fever is a common tick-borne disease caused by Spotted fever group rickettsia(SFGR)which can infect human by ticks bites.In recent years,Chinese scholars have identified novel Rickettsia in ticks,such as Rickettsia massiliae was found in Rhipicephalus turanicus in Xinjiang,and Candidatus Rickettsia longicornii was detected in Haemaphysalis longicornis in Yanbian,Jilin.But there have been no isolates of two Rickettsia in China,and no cases have been reported in our country.The transmission of Rickettsia is related to vector ticks,and studies have confirmed that Rickettsia can transmit and maintain pathogenicity at different stages of tick development.Beside Rickettsia,there are other microbes in ticks,for example,Coxiella forms a symbiotic relationship with most ticks.Studies have found that reducing the load of Coxiella by antibiotic intervention can influence the reproductive capacity of Amblyomma americanum and Haemaphysalis longicornis,and it can also affect the growth of larval ticks.However,there have been no report as to whether antibiotic intervention may affect the endophytic bacteria(such as Coxiella)or other microbes,thereby affecting the transmission of Rickettsia in ticks.Purposes1.We conduct detection of novel Rickettsia in ticks from some regions in our country to found and isolate novel Rickettsia,which provides a basis for the follow-up investigation of pathogens pathogenicity and human infections,and it provides background data.2.Taking vector ticks as the research object,and tetracycline is used to intervene ticks.Evaluating the impact of intervention on Rickettsia and Coxiella,and understanding the relationship of two bacteria to provide a new ideas for the prevention and control of tick-borne diseases and vectors.MethodsWe collect ticks from some areas in China and breed them in the laboratory.Nested PCR is used to detect spotted fever group Rickettsia in nucleic acid molecules of free ticks,female ticks and eggs.The infection in ticks is obtained through sequencing and analysis of sequences,in addition,novel Rickettsia is isolated.Based on the established Rickettsia positive and negative Dermacentor silvarum populations,starting from larval ticks biting Bal B/C mice,and ticks are divided into 3 groups such as normal group,control group and antibiotic treatment group.Fluorescence quantitative PCR is used to detect the load of Coxiella and Rickettsia in ticks from different groups,the foldchange between different groups is calculated by 2^-??Ct method,statistical method is use to test whether differences are statistically significant,and we describe the trend of trans-phase transmission and the effect of antibiotic on two bacteria.Through16S r RNA V3-V4 hypervariable region amplicon sequencing,then,describe the composition of the microbiome in different groups of vector ticks,and explain the effect of antibiotic intervention on the vector tick.Results1.Results of the first partRhipicephalus microplus and Haemaphysalis montgomeryi were respectively infected with Candidatus Rickettsia longicornii,Rickettsia massiliae,and their eggs were as Rickettsia positive with 60%(12/20),50%(5/10)detection rate.Haemaphysalis qinghaiensis,Haemaphysalis campanulata,Dermacentor silvarum were respectively infected with Uncultured Rickettsia sp.,Rickettsia japonica,Rickettsia raoultii,and the detection rate were 20.8%(5/24),60%(3/5),81%(13/16).Candidatus R.longicornii,R.massiliae were respectively isolated from eggs of R.microplus and nymph of H.montgomeryi.2.Results of the second partThe relative load of R.raoultii and Coxiella of four states in tick(egg,larval,nymph,adult)in the non-intervention group were obtained.Among the Rickettsia-positive populations,taking eggs as reference,the average fold change of Rickettsia in larval,nymph,female tick and male tick decreased firstly and then increased.In both two populations,the average fold change of Coxiella increased in Trans-phase period of ticks,and it was highest in nymph period.The average fold change of Coxiella in adult of positive population and larval and nymph of negative population increased,and the difference was statistically significant.In the positive population,the change of R.raoultii load was not significant in the intervention group relative to the control group;However,relative to their respective control group,the average change of Coxiella load in intervention groups which both the larvae and nymph were intervened with antibiotic was increased when ticks molting into adult ticks one week(4.22(female)(P<0.05),15.18(male)),but when ticks molting into adult ticks one month,the average change of Coxiella load of continuous intervention groups was decreased(0.49(female),0.11(male)).In the negative population,compared with the control group,the average fold change of Coxiella load increased in continuous intervention group when nymph molting into female adult tick for one week(2.85)(P<0.05),and it decreased after one month(0.83).Among two populations,both larvae and nymph were in intervention group or control group,compared ticks molting into adult ticks one month with one week in these two groups.The average change of R.raoultii was both decreased in two groups,the average fold change of R.raoultii in adult ticks of continuous intervention and male ticks of control group reduced,and the difference was statistically significant;Coxiella decreased in continuous intervention group(0.91(female)?0.06(male)(P<0.05)),and it increased in continuous control group(7.94(female)(P<0.05)?8.75(male)(P<0.05))in positive population;in negative population,the average fold change of Coxiella load was 1.37(female)in continuous intervention group and 4.94(female)(P<0.05)in continuous control group.Through species annotations,we found that species abundance and diversity in male adult was higher than in female ticks in non-intervention groups in both populations.Among the positive population,compared with continuous control group,the species abundance was higher in continuous intervention group when nymph molting into adult ticks(female and male)one week,but it was lower after one month;compared with adult ticks which nymph molting into one week,the species abundance decreased when nymph molting into adult ticks one month in continuous intervention group,but it increased in continuous control group.From analysis of the genus level,it can be seen that the abundance of Coxiella genus of ticks(molting into female and male adult ticks for one week)in continuous intervention group was higher than in control group,while the abundance of Rickettsia genus was lower,but molting into adult ticks(male and female)after one month,the abundance of Coxiella genus in continuous intervention group was lower than in control group,while the abundance of Rickettsia genus was higher than control group in the positive population.Conclusions1.Novel Rickettsia sp.that detected and isolated from R.microplus in Sichuan and H.montgomeryi in Yunnan were Candidatus Rickettsia longicornii and R.massiliae,respectively.Two pathogens can be transmitted through eggs,making them long-lived in ticks.It makes threat to human health.2.In the Rickettsia-positive population,tetracycline may play a role in stimulating the growth of symbiotic bacteria like Coxiella or other strains in a certain period when ticks are complete molting to the next state,but it may inhibit the growth of Coxiella in a later period,and tetracycline may have no significance effect on R.raoultii in trans-phase transmission.3.Coxiella and Rickettsia genus may have a antagonism relationship in adult ticks.