Transmission Of Babesia Microti By Ticks In Tengchong

Ticks are vectors of many pathogens including viruses,bacteria and parasites,and are considered to be the second important vector caused multi-diseases threating to human health after mosquitoes worldwide.Babesiosis,caused by the intraerythrocytic parasite in the genus of Babesia,is one of the most important tick-borne diseases for wild and domestic animals.In China,human babesiosis has been increasingly reported in recent years.It is believed that babesiosis is an emerging infectious disease in the country.Three babesia species have been documented to infect humans in China,i.e.B.microti,B.divergens and B.venatorum.However,in southern areas of China such as Guangxi Province and Yunnan Province,most of the human infections with Babesia spp.have been caused by B.microti.However,competent vectors of B.microti in these areas of the country are not yet well understood,and more investigations are needed.Bacterial endosymbionts have been widely used in the fields of control of vectors and vector-borne diseases in recent years.However,only few studies have been performed on bacterial endosymbionts of ticks in China,most of which focus on diversity of microbial community or presence of endosymbionts and pathogens in some tick species.It is believed that more studies should be focused on functions of endosymbionts in ticks because tick-borne diseases are getting more and more intention and endosymbionts have good prospects in controls of vectors and vector-borne diseases.Tengchong,Yunnan Province,located in the southwest border of the country,is considered to be one of an epidemic focus of tick-borne diseases because several human cases of B.microti infection have been reported there.To understand the distribution of tick species,the infections of B.microti and other pathogens in ticks,and the risk of tick-biting in local population,an investigation was performed in Tengchong County from July to October,2014.Sixteen villages in Tengchong Country were selected arbitrarily according to ownership of the domestic animals and following the suggestions of local experts.Infesting and questing ticks were collected from the villages.The ticks were then identified and detected for pathogen infection.As a result of the investigation,a total of 900 infesting and 558 host-seeking ticks from six species were collected.Three tick species were identified,i.e.Rhipicephalus microplus,R.haemaphysaloides,and Haemaphysalis longicornis.The three others were phylogenetically closest to Ixodes ovatus,H.cornigera and H.kitaokai(or H.inermis),separately.The infection rate of I.ovatus with B.microti was 6.5%(12/186).However,B.microti infections were not detected from other tick species.Some other pathogens were also reported in ticks from Tengchong County,including B.bigemina,B.divergens,B.orientalis,Theileria orientalis,T.luwenshuni,an unnamed Theileria species and Piroplasmida species,an Anaplasma species,a Borrelia species,an Ehrlichia species,and a Rickettsiella species.In the second part of the thesis,several engorged ticks of different species were taken back and maintained in our laboratory.Two tick species,i.e.R.haemaphysaloides and parthenogenetic H.longicornis,were finally successfully maintained and passaged in our laboratory.And then the transmission competence for B.microti of the two tick species was examined with artificial infection experiments.Our experiments revealed that transstadial transmission of R.haemaphysaloides with B.microti was proved in both the larvae to nymph transstadial and the nymph to adult routes.However,transstadial transmission of parthenogenetic H.longicornis was only proved in the nymph to adult routes.In the third part,16S rRNA gene based next generation sequencing was firstly performed to investigate the bacterial diversity of R.haemaphysaloides and parthenogenetic H.longicornis.And then the influence of endosymbionts on the reproductive fitness of the two tick species was explored by treating the engorged females with antibiotics.The result showed that both a Coxiella sp.bacterial endosymbiont and a Rickettsia sp.were demonstrated inside of R.haemaphysaloides,while only a Coxiella sp.bacterial endosymbiont presented in parthenogenetic H.longicornis.Tetracycline and kanamycin treatment not only reduced the relative copies in eggs of the two tick species,but also decreased the hatching rate of the eggs.And the decreasing hatching rates were associated with the relative copies of the Coxiella sp.bacterial endosymbiont in both tick species.Finally,kanamycin,ciprofloxacin or PBS treated offspring larvae were used in artificial transmission experiments to explore the impacts of bacterial endosymbionts on susceptibility of R.haemaphysaloides to B.microti.Our study revealed that both kanamycin and ciprofloxacin treatment reduced the relative copies of the Coxiella sp.and the Rickettsia sp.However,kanamycin had more impacts on endosymbionts than ciprofloxacin.The infection rates of nymphs in kanamycin treated groups were higher than that in ciprofloxacin or PBS treated groups(P<0.001).And the increased infection rates in kanamycin treated groups were only associated with the reduced relative copies of the Coxiella sp.In conclusion,our study demonstrate that B.microti can be transmitted artificially by both R.haemaphysaloides and parthenogenetic H.longicornis.Three tick species,i.e.I.ovatus,R.haemaphysaloides and parthenogenetic H.longicornis,are considered to be potential vector of B.microti in Tengchong County..The Coxiella sp.might be the primary bacterial endosymbiont associated with the reproductive function of both R.haemaphysaloides and parthenogenetic H.longicornis.And the Coxiella sp.might play a role in the anti-infection effect to B.microti for R.haemaphysaloides.