Isolation And Identification Of Tick Pathogens And Surveillance Study

BackgroundsTick-borne diseases are zoonotic diseases transmitted by ticks.Tick-borne pathogens include Rickettsia,Anaplasma,Ehrlichia,Borrelia,Babesia and viruses.With the development of molecular biology,many pathogens including new species,new strains or new genotypes have been found worldwide,causing widespread concern among public health and medical personnel.The life cycle of ticks includes four stages,containing eggs,larvae,nymphs and adults.Some pathogens can be transmitted by transstadial transmission,while some pathogens,such as rickettsia,can also be transmitted to the next generation by transovarial transmission.Different stages of ticks can feed on different host animals,increasing the transmission efficiency of tick-borne pathogens.Ticks have a long life cycle and each stage of ticks can survive for a long time without sucking blood,which provide pathogens a stable living environment.Therefore,ticks are not only vectors,but also an excellent host.Ticks is to pathogens what water is to fish.In order to prevent the growing threat tick-borne diseases worldwide,surveillance of tick-borne diseases and study on pathogenic mechanisms of pathogens are particularly important.In this study,we classified the tick-borne pathogens into three categories: pathogen which is reported to be pathogenic to humans in other countries and our group has confirmed its existence in China;pathogens which is reported to be pathogenic to humans in other countries,but there is no study on it in China;new emerging pathogens.The pathogenic mechanism of pathogens is the key to the prevention and control of tick-borne diseases.However,there are few researches on pathogen isolation in China,which makes it difficult to study the pathogenic mechanism of pathogens,especially intracellular organism,such as spotted fever group rickettsia and viruses which are difficult to isolate by cell culture in vitro.Due to the complex habitats,rich species,diverse hosts,and public health hazards of infectious diseases,it is of great biological and hygienic significance to carry out the isolation of tick-borne pathogens.At present,most tick-borne diseases researches in China are tick-borne spotted fever,babesiosis,and severe fever with thrombocytopenia syndrome.Among them,studies on spotted fever group rickettsia are relatively thorough.There are four species of spotted fever groups rickettsia infecting people in China.They are Rickettsia.heilongjiangiensis,Candidatus R.tarasevichiae,R.raoultii and R.sibirica.In China,most Borrelia caused disease is Lyme disease whose pathogen is B.burgdorferi sensu lato.Recently,a new type of tick-borne disease has been reported abroad,Borrelia miyamotoi.Clinical symptoms after infection include fever,fatigue,and headaches.However,no cases have been reported in China.As northeastern China is rich in forest,with good ecological environment,diverse species of ticks and host animals,it is an important natural focus of tick-borne diseases.So we conducted a survey in the sentinel hospital on the emerging B.miyamotoi infection to understand its threat to the local population.ObjectivesThe purpose of this study was to isolate tick-borne pathogens from ticks in vitro as to provide the basis for future research of pathogenic mechanisms.At the same time,this study used Mudanjiang Forestry Center Hospital as a sentinel hospital to monitor human infection with B.miyamotoi and investigated the prevalence of B.miyamotoi in ticks to evaluate its harm to public health.MethodsThe study collected questing ticks in northeastern China,Yunnan and Guangxi province by dragging a flag over vegetation,and then isolated tick-borne pathogens by cell culture.For the pathogens successfully isolated,analyzed their evolutionary status and observed their morphology using molecular and morphological methods.For known pathogenic to human pathogens,sentinel surveillance was conducted at the sentinel hospitals from May through July in each year from 2013 to 2015.Human population,who had been bited by ticks in the past two months were enrolled and patients' clinical samples,epidemiological and clinical information were collected.At the same time,ticks were collected using the flag method near the patients' residence.For the positive samples,pathogen-specific gene fragments were amplified to analyze their evolutionary status and summarized their epidemiology information and clinical manifestations.For unknown pathogenicity to human,the study was focued on pathogen correlations with ticks.Pathogen infection in ticks and distribution in tick organs under laboratory conditions was observed and study the role of anti-pathogen key factors in ticks on pathogen infection in vitro.ResultsA strain of R.raoultii was isolated from Dermacentor silvarum collected from northeastern China using Vero cells.The shape of the R.raoultii was observed by Giemsa staining and electronic microscopy,1.0 um-2.6 um in length and 0.2 um-0.4 um in width.The phylogenetic tree revealed that this strain of R.raoultii is consistent with other reported R.raoultii isolate on evolutionary status.Among the 984 patients with tick bite,14 patients infected with B.miyamotoi were diagnosed by PCR and clinical characteristics.Among the 692 questing ticks,19 I.persulcatus and 1 H.concinna were positive.According to phylogenetic tree,B.miyamotoi can be divided into three genotypes: Siberian,European,and American.The sequences of B.miyamotoi in this study were similar to Siberian type including those in Russia and Japan,different to the European and American type.Analysis of the clinical symptoms and laboratory parameters of 14 patients found that patients infected with B.miyamotoi had influenza-like symptoms,of which 6(42.9%)patients had fever and their body temperatures ranged from 38.4°C to 39.7°C,6(42.9%)patients had headache,5(35.7%)patients had anorexia,and 3(21.4%)patients had joint pain.One(7.1%)patient each had symptoms of rash,eschar and partial lymphadenopathy.Blood routine and blood biochemical examination revealed that the white blood cell count was elevated in 2 patients(14.3%),platelet count was decreased in 1 patient(7.1%),and both alanine aminotransferase and aspartate aminotransferase were elevated in 2 patients(14.3%).Four of the 14 patients were hospitalized due to high fever(38.4°C-39.7°C),and hospitalization duration varied from 3 to 20 days.Two of the inpatients were treated with doxycycline twice a day for 100 mg.After 3-5 days of treatment with doxycycline,the patient's body temperature returned to normal and other symptoms disappeared.There were no deaths in hospitalized patients,and there was no sequelae in follow-up surveys.Two patients were co-infected with Candidatus R.tarasevichiae and one patient was co-infected with Anaplasma capra.However,compared with other11 patients,the 3 patients with complex infection did not have more complex and serious symptoms,the duration of the disease has not been extended.A novel species of virus,named Guangxi tick virus(GXTV),was isolated from Amblyomma javanense collected from Javanica pangolins in Guangxi province by tick cell line BME/CTVM23.It's a single-stranded positive-sense RNA virus.The whole genome sequence was obtained.GXTV was found to be similar to the previously discovered virus called Jingmen tick virus(JMTV),as a result of their similarity of the whole genome was 93.8%.GXTV was a segmented virus with four gene segments.Both GXTV and JMTV have a special evolutionary status.Two of their segments have the closest evolutionary relationship with the unsegmented Flaviviridae virus.However,the other two segments have previously.There is also a GCXV isolated from mosquitoes that has the same characteristics.However,we have confirmed that GXTV is a virus that replicates all the four segments together in the same cell,but GCXV is a multicomponent virus.In addition,under laboratory conditions,GXTV can infect adult Amblyomma javanense and larval ticks of Rhipicephalus(Boophilus)microplus by,but it is unable to infect adult Haemaphysalis longicornis and nymphal ticks of Rhipicephalus sanguinensis.Through fluorescence in situ hybridization experiments(FISH),we confirmed that GXTV can efficiently replicate in tick salivary glands under laboratory conditions,but it takes about 14 days to transfer from the midguts to the salivary glands.The levels of mRNA of Dicer and Argonatue in the anti-viral RNA interference(RNAi)pathway are significantly inhibited in the process of virus infecting tick cells.ConclusionsA strain of R.raoultii was isolated from Dermacentor silvarum collected from northeastern China using Vero cells,and its evolutionary status was clarified.A series cases of human infection with B.miyamotoi was found in northeastern China.Its clinical features and treatment were described.The main symptoms after onset include fever,fatigue,headache,chills,myalgia,joint pain and nausea.Doxycycline can be used to treat patients.Detection of B.miyamotoi in local ticks confirmed that northeastern China is the natural focus of B.miyamotoi.A novel species of virus was isolated from Amblyomma javanense collected from Javanica pangolins in Guangxi province by tick cell line BME/CTVM23.The virus was named GXTV and its whole genome sequences were obtained,also confirmed its infection in Amblyomma javanense and Rhipicephalus(Boophilus)microplus.The infection in Amblyomma javanense clearly clarified its distribution in the salivary glands and midguts.Compared with GCXV and flavivirus,GXTV have different characteristics in its replication,infection and interaction with ticks.Significance and innovationThe study is the first time to isolate a strain of R.raoultii from ticks by Vero cells in China.Compared with the previously reported isolates,this strain of R.raoultii can be passaged stably,and provides a foundation to the study of the pathogenic mechanism of Rickettsia in the future.For the first time,a series cases of human infection with B.miyamotoi was reported in China.B.miyamotoi was also detected in ticks.The clinical characteristics and treatment methods of characteristics provide an important basis for the diagnosis of B.miyamotoi,which fills the gap in the investigation of B.miyamotoi in China.In this study,a novel tick-borne virus was isolated from the tick cell line BME/CTVM23,which enriched the species of emerging tick-borne virus.The characteristics of the new virus had been preliminarily studied before it was found to infect humans,which provides chances for evalue its pathogenicity and understanding its evolutionary rule and interactions with ticks.