Pathogens In Ticks And Experimental Study On Maintenance And Transmission Of SFTSV By Haemaphysalis Longicornis

Ticks are efficient vectors of multiple pathogens due to their potential interactionswith a wide spectrum of vertebrate hosts during their life cycle. Ticks areclimate-adaptable with a wide geographical distribution. Ticks are vectors of a varietyof pathogens including viruses(Tick-borne encephalitis virus, Crimean-Congohemorrhagic fever virus, Severe fever with thrombocytopenia syndrome bunyavirus, etal.), bacteria(Francisella tularensis, et al.), Rickettsiales(SFG Rickettsia, Anaplasma, etal.), Borrelia burgdorferi, protozoa (Babesia), mycoplasma and chlamydia et al.Tick-borne diseases have been increasing in recent years endangering the health of theresidents and domestic animals that aroused wide concern. The investigation ofpathogens carried by ticks can help us to understand the spectrum of tick-bornepathogens, and has a profound significance to our knowledge of zoonotic diseases.Firstly,16S RNA gene library method was used to investigate the bacterialdiversity of ticks (Dermacentor niveus Neumann) collected from Tacheng, XinjiangUygur Autonomous Region, China. A total of452clones were successfullysequenced and assigned to four phyla. The dominant phylum was Proteobacteria,accounting for62.8%of all the clones of16S rRNA gene at the confidence level of80%. The other sequences were assigned to the phyla Bacteroidetes, Firmicutes,Actinobacteria and account for13.5%,12.4%, and11.3%respectively. Particularly,our result indicated Rickettsia raoultii and R. slovaca in D. niveus ticks of this area.The results provided a view of bacteria diversity associated with D. niveus ticks in thenatural environment of Tacheng and pinpointed its potential capabilities oftransmitting TIBOLA/DEBONEL (tick-borne lymphadenopathy/Dermacentor-bornenecrosis erythema and lymphadenopathy).However, the16S RNA gene library method can only screen out prokaryoticpathogens. Due to the genomic diversity of different pathogens, the currentmetagenomic approaches for microbial analysis require specific protocols to detectDNA viruses, RNA viruses, and other cellular pathogens. Because of this, sampleprocessing is often labor intensive and costly. Since small RNA fractions couldcontain RNA metabolites derived from all RNA species, such as rRNAs, tRNAs, mRNA, snRNA, snoRNA, we developed a strategy using the deep sequencing ofsRNA to identify multiple types of microorganisms in Heamaphysalis longicornisticks collected from Beijing and Shanghai. The mechanism of RNA silencing, orinterference, as a form of viral immunity, begins with the recognition of a viraldouble-stranded or structured RNA by the Dicer nuclease family, which results inshort interfering RNA (21-26nt). What’s more, since small RNA fractions couldcontain RNA metabolites derived from all RNA species, such as rRNAs, tRNAs,mRNA, snRNA, snoRNA, we hypothesize that it would be possible to use deepsequencing of sRNA as a universal strategy to identify multiple types ofmicroorganisms other than viruses, including prokaryotic and eukaryotic pathogens.Rickettsia spp. and Coxiella spp. were indicated to exist in H. longicornis tickscollected in Beijing and Shanghai and then were confirmed by experiments. Our studydemonstrated the reuse of the sRNA deep sequencing data would have the potential totrace the origin of pathogens or discover novel agents of emerging/re-emerginginfectious diseases.Sporadic cases of severe acute febrile illness with unidentified cause have beennoted after the surveillance of infectious diseases in China has been significantlyenhanced since2004. The major clinical manifestations were flu-like symptoms,thrombocytopenia and leukocytopenia. Most patients are sporadic and a few clustersof cases have also been found. In2011, a novel virus (Severe fever withthrombocytopenia syndrome virus， SFTSV), which belonged to the family ofBunyaviridae and the genus of Phlebovirus, was convincingly demonstrated to be thecause of SFTS, and Koch’s postulates for establishing the causal link have largelybeen satisfied.However, the clinical presentation of SFTS is not specific, but consistent withmany infectious causes including Anaplasma phagocytophilum and Ehrlichia specieswhich were also tick-borne pathogens. Consequently, we conducted an investigationon the diversity of pathogens carried by H. longicornis collected form SFTS epidemicarea Xinyang, Henan province using a improved method combined with the principleof the previous two approaches. A. bovis (9.03%,13/144), A. centrale (1.39%,2/144),R. heilongjiangensis (2.08%,3/144), R. sp. LON-13(0.69%,1/144), R. raoultii(3.47%,5/144), Babesia sp. MA#361-1(0.69%,1/144) and SFTSV(18.75%,27/144) werefound in H. longicornis ticks. Dual infection with A. bovis and SFTSV was discoveredin the collected ticks (2.08%,3/144). Meanwhile, the phylogenetic analysis of SFTSV complete sequences showed the homology of SFTSV from H. longicornis and localpatients.At present, the ecological cycle of the SFTSV is not yet known.Human-to-human transmission of SFTS through blood contact has been reported.Secretions and body fluid were also suspected to be the routes of transmission. Untilnow, there were only field investigation of domestic animals and insects fromepidemic areas. These previous studies indicated that ticks, especially H. longicornisticks were the major potential insect vector to transmit SFTSV. However, the vectorstatus of the tick can not be determined from such field data. Additional vectorcompetence studies must be carried out in the laboratory before a particular tick canbe concluded as a vector. Three criteria must be confirmed to designate a tick as avector of a given pathogen:(1) Ability of ticks to become infected by feeding on theinfected hosts.(2) The ability of fed ticks to trans-stadially pass the pathogen to thenext generation, or through the following molt to the next stage of the life cycle.(3)The ability of molted stages to transmit the pathogen to na ve hosts during bloodfeeding. We conducted the experiments for further understand of the ability ofmaintaining and transmission of SFTSV by H. longicornis ticks.SFTSV-free-H. longicornis raised in laboratory and SFTSV strain “PhlebovirusWCH/97/HN/China/2011” were used for the study. The ticks were microinjected withSFTSV. RT-PCR and IFA were used for the detection of SFTSV in ticks and hostmice.40female adults were microinjected through the anal with the suspension ofSFTSV and PBS respectively. The ticks and subsequent generation ticks wereexamined to evaluate the ability of maintaining and transmission of SFTSV by H.longicornis ticks.Our results showed the phenomenon that SFTSV transmitted from midgut tosalivarygland. The multiplication of virus was presented during the reproductiveprocess of ticks. The filial eggs, larvae, nymphs and adults were SFTSV positive. TheSFTSV were shown to be located at salivary gland, ovary, midgut by the IFA assay.The positive result of hemolymph could explain the migration of virus betweendifferent tissues. The positive result of saliva indicated the possibility that the H.longicornis could transmit SFTSV to the host. Nucleic acid of SFTSV and IgG wereshowed positive in Balb/c mice used for feeding ticks. Our study confirmed thetransstadial and transovarial transmission of SFTSV in H. longicornis ticks and theability of transmitting SFTSV to the hosts by H. longicornis ticks. Our study provided more evidences to reveal the role of H. longicornis ticks in the SFTSV ecologicalcycle.In summary, firstly we explored metagenomic strategies for screeningbroad-spectrum pathogens carried by ticks. An efficient strategy of screening forpathogens in wild-caught ticks and mosquitoes by reusing small RNA deepsequencing data was established, and the feasibility of this method was confirmed bythe attempt at H.longicornis ticks from which SFG Rickettsia and Coxiella weredetected. Moreover, an improved method combined with the principle of the previoustwo approaches using the Ion PGM system for high-throughput sequencing wasdeveloped. An investigation on the diversity of pathogens carried by H. longicorniscollected form SFTS epidemic area using this method and Anaplasma, SFG Rickettsia,Babesia and SFTSV were detected. Afterward, on the other hand, the phylogeneticanalysis of SFTSV complete sequences demonstrated the homology of SFTSV fromH. longicornis and local patients. In the following study, we found that SFTSVlocated at the salivary gland, midgut and ovary. The transstadial and transovarialtransmission of SFTSV in H. longicornis ticks and the ability of transmitting SFTSVto the hosts by H. longicornis ticks were also confirmed by our study.