Study On The Traceability Of Brucella Melitensis From Inner Mongolia And The Characteristics Of Its New Arthropod Host

The unique natural climate and nomadic living environment in Hulun Buir of Inner Mongolia have provided a favorable environment for the breeding and propagation of ticks.With the change of global climate,ticks become more active in grassland regions.As everyone knows,the ticks will not only cause inflammatory reaction to the host's skin through directly biting,but also act as the communication media of many pathogens,and brucellosis might also belong to one of the tick-borne diseases.In recent years,brucellosis occurs frequently among human and animals in various areas of Inner Mongolia,which severely influences the sound development of local livestock industry and even leads to serious social problems.The author collected Dermacentor nuttalli of various developmental phases in Hulun Buir as test materials from 2015 to 2020.The methods of molecular biology were applied to test the specific genes of Brucella melitensis and the products at the gene level and protein level by aiming at the different genders,different developmental phases and different organic tissues of D.nuttalli.It is important that 8 strains of B.melitensis were isolated from the mature tick and tick egg,which strongly proved that D.nuttalli might be one of the natural reservoir hosts for B.melitensis in Inner Mongolia.Later,the tick cell infection test was conducted for isolated strains of B.melitensis in the primary cells of D.nuttalli cultured in vitro.Effective Brucella was detected in the primary cell cytoplasm of D.nuttalli through immunofluorescence and transmission electron microscope methods.This test shows that B.melitensis can adapt to the environment in tick cells.The test results of this study are described as follows:1.From 2015 to 2020,tick samples were collected in 23 pasturing areas of Hulun Buir,Inner Mongolia covering four regions(including Xin Barag Left Banner,Xin Barag Right Banner,Prairie Chenbarhu banner,and Ewenki Autonomous Banner)and Yakeshi,and the tick types were identified.The method of combining traditional morphology and molecular biology was used,and D.nuttalli was determined as the dominant tick species during the period from the last ten days of April to the last ten days of June in Hulun Buir.A genetic tree was drawn according to 16S rRNA genes and CO I genes,and D.nuttalli in this area and D.nuttalli in Russia,Xinjiang,Jilin and Hebei originate from the same ancestor.2.Bcsp31 specific genes of Brucella were detected in 1,911 pieces of D.nuttalli in Inner Mongolia for the first time from 2015 to 2019.According to the results,the highest positive rate of Bcsp31 specific genes in Brucella carried by D.nuttalli can reach 87.80%.The same method successfully detected specific genes of Brucella in ticks of different development stages(including egg,larva,nymph and adult).Such test results indicate that Brucella in D.nuttalli has a long existence cycle,and it might be one of the potential reasons making brucellosis in domestic animal pandemic in pasturing areas.3.The conventional separation method for Brucella was used to successfully isolate 8 strains of Brucella from full-blood D.nuttalli and tick egg.It was verified in the classification test of Brucella that the 6 isolated strains were B.melitensis type 3,named as B.melitensis IMHT1,B.melitensis IMHT2,B.melitensis IMHT3,B.melitensis IMHT4,B.melitensis IMHT5 and B.melitensis IMHT6 separately;the other two were B.melitensis type 2,named as B.melitensis IMHT7 and B.melitensis IMHT8 respectively.The MLST analysis results about 4 isolated strains of Brucella show that these Brucella strains have a proximal evolutionary relationship.The 16S rRNA genes of 4 Brucella strains were registered in GeneBank,and their registration numbers were:MT611102.1,MT611103.1,MT611104.1 and MT611105,1.4.The midgut and salivary gland tissues of ticks are key tissues for the successful mechanical transmission of corresponding reported pathogens of tick-borne diseases.This test conducted successful quantitative determination on the copy number of Brucella Bcsp31 genes in the above two tissues of ticks at gene level through fluorogenic quantitative PCR method.Besides,Brucella Bcsp31 proteins were successfully detected in the above 2 tissues at protein level through immunofluorescence and Western Blot methods.Such results indicate that D.nuttalli has the potential risk of spreading brucellosis among flocks and herds.5.The primary cells of salivary gland and midgut tissues in D.nuttalli were isolated and cultured in vitro.Through optimizing the culture conditions,the optimum culture conditions for tick cells were screened.Later,Brucella was infected with isolated primary cells of salivary gland and midgut.The infection situations about isolated strains of Brucella were detected via immunofluorescence and transmission electron microscope methods.Such test results indicate that the isolated strains of Brucella can adapt to the internal environment in primary cells of ticks cultured in vitro.To sum up,through a series of continuity test,the author systematically traced to the source for epidemic reasons of B.melitensis among flocks and herds in Inner Mongolia(especially Hulun Buir).Besides,it is preliminarily identified that D.nuttalli is the arthropod host carrying Brucella for a long time,and that it might have the potential risk of spreading brucellosis of cattle and sheep.