Identification Of Flea Species And Investigation Of Flea-borne Diseases,Northern Of Xinjiang

Object: To identify the fleas and detect the pathogens in the border areas of northern and southern Xinjiang,which laid the foundation for the risk assessment and the prevention of flea-borne diseases in Xinjiang.Additionally,to collect the samples from Wenquan County and classify Yersinia pestis,and provide scientific basis for the biology of main types of host,back the plague vector and the ecological landscape.Methods: 1.From 2015 to 2016,fleas were collected directly from the mammals at seven sites(Buerjin County,Alashankou City,Wenquan County,Jinghe County,Huocheng County,Qapqal Xibe Autonomous County and Xinyuan County)in the northern region of Xinjiang Uygur Autonomous Region,besides,the sheep fleas from Mingfeng County.All of them were identified by morphological examination and molecular analysis of five loci(18S ribosomal DNA,28 S ribosomal DNA,Elongation Factor 1-alpha,Cytochrome Oxidase II and Cytochrome Oxidase I).Phylogenetic analysis was then employed.2.The DNA of five flea species(Echidnophaga oschanini,Xenopsylla gerbilli minax,Coptopsylla Lamellifer ardua,Paradoxopsyllus repandus and Nosopsyllus laeviceps laeviceps)were sequenced by Novogene sequence bacterial V3-V4 variable region for the flea-associated communities,population diversity and richness.3.Combining with the macro bacteria data,a multilocus sequence typing(MLST)was used to detect the Rickettsia spp.in flea samples.Samples were also screened for the presence of Wolbachia spp.with PCR assays targeting wsp and 16 S r DNA.Moreover,three pairs of primers were used to amplify the glt A,ITS and rib C fragment about Bartonella spp..The positive PCR products were sequenced by Sangon Biotech,and the phylogenetic trees were constructed using MEGA 6.0 software.4.We collected serum samples and tissue samples from Wenquan County and flea samples from sample sites.The serum samples were screened with a passive haemagglutination assay to detect immunoglobulin(Ig)G antibodies against the F1 antigen of Y.pestis.All the tissue samples and flea samples were screened for the presence of the caf1 and pla genes and then genotyped by multiple-locus variable-number tandem repeat analysis(MLVA).Results: 1.A total of 5699 fleas belonging to 19 species of 7 families and 16 genera were collected,including E.oschanini,Pulex irritans,X.conformis conformis,X.gerbilli minax,X.cheopis,Ctenocephalides felis felis,Co.Lamellifer ardua,Neopsylla mana,Ctenophthalmus dolichus dolichus,Rhadinopsylla cedestis,Ischnopsyllus octactenus,Pectinoctenus nemorosa,Mesopsylla eucta shikno,Frontopsylla elatoides elatoides,P.repandus,Oropsylla silantiewi,Citellophilus tesquorum dzetysuensis,N.laeviceps laeviceps,Vermipsylla alakurt.Alashankou animal parasitic fleas is changing according to seasonal changes,except for January and February,the fleas numbers is low,the remaining 10 months flea index is high and has certain volatility.2.726626 Tags,5447 OTUs were measured in 5 kinds of fleas of Alashankou area and analysis found that the vast majority of bacteria belonging to four phylum: Proteobacteria,Firmicutes,Actinobacteria and Bacteroidetes,but the abundance showed obvious difference.3.Rickettsia spp.: Candidatus R.barbaria were found from V.alakurt in Minfeng.R.bellii were found in E.oschanin,X.gerbilli minax and P.repandus in Alashankou,and one species older than R.bellii was found in R.cedestis and N.laeviceps laeviceps in same area.Wolbachia spp.: Wolbachia detected in a variety of fleas,including P.irritans,X.conformis conformis,X.gerbilli minax,C.felis felis,F.elatoides elatoides,P.repandus,O.silantiewi,C.tesquorum dzetysuensis,N.laeviceps laeviceps.Molecular analysis showed that these endosymbiont with previous reports there is a big difference in the fleas.Bartonella spp.:1)B.rochalimae were found in X.conformis conformis,X.gerbilli minax;2)B.grahamii were found in X.gerbilli minax;3)B.elizabethae were found in C.Lamellifer ardua,P.repandus and N.laeviceps laeviceps;4)BZ01 were found in F.elatoides elatoides and C.tesquorum dzetysuensis and BZ02 were found in O.silantiewi.All of them belong to the same branch and ogether with the B.Washoensis.4.The serum collected in Wenquan County in 2015 showed that the total positive rate of Marmota baibacina was 2.1%(1/47),shepherd 6.7%(2/30)and Spermophilus undulates 0.7%(1/134).Y.pestis molecular detection found in the O.silantiewi and M.baibacina tissues and the MLVA types were MT86(2-2-2-4-6-8-7-5-2-7-3-3-2-5).Conclusion: 1.All the sequences of 19 species fleas collected were submitted to the Genbank and some sequences is first submit including entire five gene sequences of Co.Lamellifer ardua,Ct.dolichus dolichus,Rh.cedestis,I.octactenus,M.eucta shikno,F.elatoides elatoides,P.repandus,O.silantiewi,C.tesquorum dzetysuensis,V.alakurt and partial sequences of E.oschanini,X.gerbilli minax,N.laeviceps laeviceps.This finding inriched the database information and provide convenience for later study;Alashankou district in vitro animal parasitic fleas seasonal change tip fleas of the number of species have obvious seasonal distribution and diversity.2.Macro bacteria research found there are many pathogenic bacteria and conditional pathogenic bacteria genera in the fleas,showed that we should targeted screening pathogenic agents and do the risk assessment for early warning in this region.3.This finding extends our knowledge of distribution of Rickettsiae,Wolbachia and Bartonella and the profile of carriers.Especially,were found some pathogen human Bartonella in fleas,remind us to pay more attention to the flea spesies of this area.4.Two new plague point were found in the Alataw and Begjintaw mountain,remind local should strengthen surveillance and epidemic of the plague,prevent the emergence of human plague.