Molecular Identification Of Rickettsia In Hard Ticks In Some Regions Of China

Hard ticks are a group of obligate blood sucking arthropod,which are distributed world widely.It can parasitize almost all terrestrial vertebrates including human beings.A wide variety of pathogens may be carried and transmitted by hard ticks,including Rickettsia spp.,Ehrlichia spp.,Anaplasma spp.,Coxiella spp.,Babesia spp.,virus,etc..Therefore,hard ticks are usually considered to be the most important vectors of pathogens.Rickettsiae are a kind of obligate intracellular bacteria parasitizing exclusively inside eukaryotic cells.Rickettsial diseases including spotted fever,scrub typhus,ehrlichiosis,and anaplasmosis,which caused by spotted fever group rickettsia(SFGR),Orientia tsutsugamushi,Ehrlichia spp,and Anaplasma spp.,respectively.Although C.burnetii has been classified into Legionellales,Q fever caused by C.burnetii still belongs to a kind of rickettsial diseases.Most of the rickettsial pathogens may be carried and transmitted by hard ticks.From 2019 to 2020,1,252 hard ticks were collected from Raohe of Heilongjiang,Yanbian of Jilin,Hulunbeier of Inner Mongolia,Jimo of Shandong,Jinzhai of Anhui,Sui and Luotian of Hubei,Chaya of Tibet,and Weishan and Lincang of Yunnan.These tick were identified as Dermacentor silvarum,D.nuttalli,Ixodes persuleatus,I.pavlovskyi,Haemaphysalis concinna,H.Iongicornis,Hyalomma Isaaci,Rhipicephalus sanguineus or Rh.microplus based on morphological and gene characterizations.A whole DNA sample of each tick was prepared and the pooled DNA sample were analyzied by metagenomic next generation sequencing(m NGS)in order to understand what kinds of bacteria carried by these ticks.In addition,each tick DNA sample was detected by rickettsial genus/group-specific nested polymerase chain reaction(PCR)and the positive DNA fragments amplified were sequenced in order to identify the rickettsial species.In m NGS analysis,Rickettsia,Anaplasma,and Coxiella in the pooled tick samples were found by the taxonomic profiles at genus level.The DNA fragment numbers of Rickettsia in different tick species and in the same tick species from different regions were significantly different.The DNA fragment number of Anaplasma in Hy.Isaaci ticks from Chaya of Tibet was over 12000 and the DNA fragment number of Coxiella,Rickettsia,Ehrlichia or Wolbachia was over 2000.The abundance of Coxiella was the highest in Hy.Isaaci from Chaya of Tibet,and the abundance of Rickettsia was the highest in D.nuttalli from Hulunbeier of Inner Mongolia.The abundance of Anaplasma was the highest in H.Iongicornis of Jinzhai of Anhui.In PCR amplification,Coxiella endosymbiont of H.longicornis type was detected in 32.46% of the ticks(112/345),Coxiella endosymbiont of D.silvarum type was detected in 53.36% of the ticks(143/268),Coxiella endosymbiont of Rh.microplus type was detected in 25.28% of the ticks(43/178),Coxiella endosymbiont of RFE03 type was detected in 1.22% of the ticks(3/245).C.burnetii was detected in 4.13% of the ticks(18/436),R.raoultii was detected in 30.21% of the ticks(100/331),Candidatus R.tarasevichiae was detected in 9.60% of the ticks(34/354),R.aeschlimannii was detected in 92.86% of the ticks(13/14),R.japonica was detected in 24.81% of the ticks(128/516),Anaplasma sp.Mongolia was detected in 25.71% of the ticks(63/245),and A.marginale was detected in 13.19% of the ticks(68/516).A total of 138 hard ticks(11.02%,138/1252)were found to carry more than one rickettsial species.Finally,Rh.microplus in Lincang of Yunnan was homogenated and inoculated to yolk sac of egg,and C.burnetii was successfully detected using PCR and classfied into the type of 9-27-4-6-9-5 type using multiple locus variable number tandem repeat analysis(MLVA).In conclusion,hard ticks collected from a varied of regions of China were identified carrying various rickettsial pathogens.The knowledge of rickettsial pathogens inside the hard ticks in these regions provide a basis for the local prevention and control of the rickettsial diseases.