| BackgroundRodents play an important role in the transmission of zoonoses,including bacterial,viral and parasitic rodent-borne diseases.Rodent-borne diseases have seriously affected peoples’ helath and social economic development.It is an important public health problem.We used molecular biology techniques and immunological methods to detect the pathogens in rodents and humans from animal tissue samples and human serum samples collected in Qingdao,Shandong Province.We also expressed the recombinant protein of pseudorabies virus(PRV)and analyzed the protective immunity of the target protein in mice to provide the basis for vaccine development.Objective1.Epidemiological survey of zoonosis in Qingdao,Shandong a)To investigate the species and distribution of rodents,and determine whether they were infected with Orientia tsutsugamushi,Brucella,Babesia,and PRV.b)To determine the prevalence of Orientia tsutsugamushi and Brucella in healthy people in Qingdao City.2.Analysis of the protectivity of PRV recombinant proteina)Construction,expression,and purification of the recombinant PRV gB proteins.b)Determine the neutralizing antigenic epitope of PRV gB proteins and determine the protecitiy of of the recombinant protein.Methods1.From 2012 to 2016,small animals were collected from Qingdao,Shandong Province.All small animals were classified and recorded according to the morphology,aseptically dissected to collect animal tissues.These tissues were frozen at-80℃.2.The animal spleen DNA were extracted.The PCR were conducted based on the Orientia tsutsugamushi 56-kDa TSA gene,the Brucella 16S rRNA/bcsp31/omp2 gene,the Babesia 18S rDNA and the PRV gB/gC/gD gene.The positive PCR products were cloned and sequenced,the sequencing results were compared by blast in NCBI,the homology analysis was carried out by MegAlign software and the phylogenetic analysis was analyzed by Mega 7.0 software.3.Orientia tsutsugamushi antibody and Brucella antibody in human sera were detected with ELISA,and the 56-kDa TSA gene of Orientia tsutsugamushi,the 16S rRNA,bcsp31 and omp2 gene of Brucella were amplified with PCR with each gene specific primers.4.PRV gB gene was divided into five segments based on its domains.The fragments were cloned into pGEX-KG plasmids and named as pGEX-KG-gB-A,pGEX-KG-gB-B,pGEX-KG-gB-C,pGEX-KG-gB-D,pGEX-KG-gB-E,respectively.The fusion proteins Pr-A,Pr-B,Pr-C,Pr-D,and Pr-E were purified with AKTA system.5.Female BALB/c mice were immunized seperately with the proteins Pr-A,Pr-B,Pr-C,Pr-D,or Pr-E mixed with the same volume of adjuvant.The mouse serum was collected,and neutralizing antibody titers were assayed by plaque reduction neutralization test(PRNT).6.The lymphocyte proliferation assay was carried out with CCK-8.The secretion level of IFN-y in splenocytes was determined with ELISpot kit and qPCR.The secretion level of IL-4 in splenocytes was determined with ELISA and qPCR.7.The behavioral and survival status of mice were observed for 21 days after virus challenge.To confirm the mice died of the PRV,PCR was performed to amplify the gB and gE in the dead moue tissues.Results1.From 2012 to 2016,7 animal species belonging to 5 genera,3 families and 2 orders were captured in Qingdao,Shandong,including Apodemus agrarius,Cricetulus barabensis,Tscherskia Triton,Mus musculus,Rattus norvegicus,niviventer configurianus and Crocidura lasiura.The dominant species in this study was Apodemus agrarius,followed by Mus musculus.The dominant species of indoor captured rodents were Mus musculus(41.9%,18/43)and Rattus norvegicus(51.2%,22/43).The dominant species of outdoor captured rodents were Apodemus agrarius(58%,69/119).All Tscherskia triton(18),Niviventer confucianus(8),Cricetulus barabensis(7)and most Apodemus agrarius(98.6%,69/70)were caught in the field;Most Rattus norvegicus(91.7%,22/24)and most Mus musculus(66.7%,18/27)were obtained indoors.2.The Orientia tsutsugamushi 56-kDa TSA gene was successfully amplified from 162 animal spleens,but the 18S rRNA gene of Babesia,the gB,gC,and gE gene of pseudorabies and the 16S rRNA,bcsp31,omp2 gene of Brucella were not positive with PCR.In this study,the overall prevalence of Orientia tsutsugamushi was 4.3%(7/162),including Apodemus agrarius(4.3%,3/70),Mus musculus(7.4%,2/27),Niviventer confucianus(12.5%,1/8)and Cricetulus barabensis(14.3%,1/7),which were all captured in autumn.Positive Apodemus agrarius(4.3%,3/69),positive Niviventer confucianu(12.5%,1/8)and positive Cricetulus barabensis(14.3%,1/7)were captured in the field.Positive Mus musculus(11.1%,2/18)were captured indoors.The positive rates of Orientia tsutsugamushi in outdoor and indoor animals were 4.2%and 4.7%,respectively.3.In this study,seven sequences of Orientia tsutsugamushi based on 56-kDa TSA gene were obtained.Phylogenetic analysis showed that 6 strains were Kawasaki type and 1 strain was STA-07 type.Homology analysis showed that the nucleotide identity ranged from 91.7%to 100%among these 6 strains and they were identified with Kawasaki(GenBank Number:M63383)from 91.4%to 95.7%.4.A total of 229 human sera were collected in Pingdu City,Qingdao,Shandong Province in 2019.The population ages were among 26 to 78 years old,with a median age of 53 years old.Men accounted for 66.4%and women 33.6%.There was no significant difference in gender distribution among different age groups(P>0.05).5.By ELISA,the positive rates of Orientia tsutsugamushi total antibodies and IgM antibody in human serum were 10.0%(23/229)and 3.1%(7/229),respectively.The positive rates of Brucella IgG antibody and IgM antibody in human serum were 3.1%(7/229)and 2.6%(6/229),respectively.Among 7 Brucella IgG positive persons,6 of them were also IgM positive at the same time.The target band of Orientia tsutsugamushi DNA or Brucella DNA was not amplified by PCR in their antibody positive samples.6.The segments of PRV gB was divided as sections:gB-a(59aa-255aa),gB-b(163aa-376aa),gB-C(357aa-600aa),gB-d(473aa-685aa)and gB-e(600aa-797aa).The recombinant plasmid pGEX-KG-gB-A,pGEX-KG-gB-B,pGEX-KG-gB-C,pGEX-KG-gB-D and pGEX-KG-gB-E were successfully constructed.7.The fusion proteins were purified with GST affinity chromatography and Sephadex sieve chromatography with AKTA chromatography system.The purified protein Pr-A,Pr-B,Pr-C,Pr-D and Pr-E were collected.8.Plaque reduction neutralization test(PRNT)showed the PRNT50 of the PA,Pb,PC,PD,PE,p-ctrl and Ctrl groups was 16,64,16,64,256,>256,<4 respectively.It was considered that the group PE mice were produced higher neutralizing antibodies.9.CCK-8 assay showed that the proliferation degree of splenocytes in group PE was significantly higher than that in control mice(P<0.01).10.ELISpot indicated that spot forming cells(SFCs)were significantly higher in group PE(P<0.01),qPCR showed that the relative content of IFN-y in splenocytes of group PE was increased significantly higher than that in control mice(P<0.05).ELISA and qPCR showed that there was no significant difference in the secretion of IL-4 between PE group and the control group.11.Two weeks after the last immunization,the mice were injected intraperitoneally,and the behavior and health status of the mice were observed for 21 days.After challenge,the mice in the control group began to have itching,hyperactivity and mania on the 6th day,the first mouse died on the 7th day,and the last mouse died on the 11th day.The mice in PE group behaved normally during the observation period,and the survival rate was 100%.12.DNA was extracted from the brain,heart,liver,spleen,kidney and lung of all the dead mice.gB and gE gene were amplified from all dead mice.Conclusion1.People were at risk of contracting Orientia tsutsugamushi at home even if they were not engaged in fields.2.Orientia tsutsugamushi and Brucella was endemic in Qingdao.3.Pr-e(600aa-797aa),the segment protein of PRV gB,could induce mice a higher level of neutralizing antibody,and protect mice from the PRV lethal challenge. |
PDF Full Text Request