We have evaluated the plague epidemic situation in the Three Rivers Source Region,Qinghai Province in recent 20 years to provide the basis for revising the plague prevention countermeasures.We have analyzed the time distribution and the plague epidemic situation between animals and human beings during twenty years in the Three Rivers Source Region,Qinghai Province by describing epidemiology.The animal plague in the natural source of Marmot plague was extremely serious in the Three Rivers Source Region during the past 20 years.It mainly distributes in Yushu State and Tanggula County,Germu City and the serious season ran through the whole period of marmot camp ground activities and the peak was between June and August.Human plague epidemic took place between May and October each year and reached its peak between July and September.The peak covered about 41.67％ at most.It mainly endemic distributes in Nangqian and Xinghai Country.During the past 20 years,we have totally found 14 human plague epidemics in the Three Rivers Source Region,among which 48 cases took place and there were deaths of 17 cases.The fatality rate was 35.42％.The lung type was the majority among 48 cases,which startde with the glandular type.During the past 20 years the plague epidemic has been active and the animal plague epidemic broke out continuously in the Three Rivers Source Region,Qinghai Province.The variety of animals and insects infected the plague epidemic was increasing.The human plague epidemics is most serious with high fatality rate,but it is on the decline as a whole.

To understand pathogen spectrum of nontuberculosis Mycobacteria (NTM) and the dominant NTM in Gansu Province and provide the scientific basis for the effective prevention and treatment of NTM diseases,875 Mycobacteria isolates were collected from 2012 to 2014 in Lanzhou Pulmonary Hospital,NTM species were identified by means of PNB/TCH differentiate medium and 16S rRNA gene sequence analysis respectively.Forty-six isolats of NTM were identied from 875 PNB/TCH.Then with 16S rRNA gene sequence analysis,the NTM strains were identified to 3 strains of Nocadia and 43 strains of NTM,including M.intracellulare,M.kansasii,M.avium,M.senegalense,M.gordonae,M.szulgai,M.peregrinumand M.fortuitum.Among them,there were 31 strains of M.intracellulare,which accounted for 72.09％ of the total number of NTM strains.The dominant nontuberculosis Mycobacteria in Gansu Province were mainly M.intracellulare.The application of molecular biology can rapidly and accurately identify the species of nontuberculosis Mycobacteria,and can provide relevant evidence for clinical diagnosis and therapy.

At present,corresponding cell-free parasite DNA molecules (CFPD) has been detected in serum,plasma,urine,saliva and other bodily fluids of a variety of the patients with parasitic diseases.Due to its high specificity and sensitivity,the CFPD shows a strong advantage of noninvasive diagnosis and continuous monitoring,etc.in parasitic diseases.This article namely reviews the current research of CFPD in the patients with parasitic disease at home and abroad in recent years,so as to provide new ideas for the development direction of parasitic disease diagnosis in the future.The current related problems are discussed in the mean time.

Cryptosporidium spp.are protozoan parasites that infect the epithelial cells of the gstrointestinal tract of hosts.In humans,cryptosporidiosis is usually a self-limiting infection in immunocompetent individuals,but severe diarrhea and dissemination to extra-intestinal sites can occur in high-risk individuals,such as the very young,the elderly,immunedeficiency individuals,particularly in HIV-positive patients.So far,molecular epidemiological data have confirmed the presence of 30 species and over 40 genotypes with genus Cryptosporidium,with 21 species and genotypes being found in humans.The majority of human cryptosporidiosis cases are responsible for C.hominis and C.parvum.Human cases caused by C.meleagridis,C.ubiquitum,C.felis and C.canis have been increasing.Besides that,with data accumulation of molecular epidemiology of human cryptosporidiosis,some more Cryptosporidium species and genotypes were newly identified in humans.This paper mainly reviews epidemiology status of these new emerging Cryptosporidium species and genotypes in humans.

We investigate the different expression of toxin gene mazF3,6,9 and antitoxin gene mazE3,6,9 in the drug-resistance Mycobacterium tuberculosis,we used quantitative real-time polymerase chin reaction method to detect the expression level of toxin gene mazF3,6,9 and antitoxin gene mazE3,6,9 in M.tuberculosis (20 mono-resistance strains,20 multidrug resistance strains and standard strain H37Rv).The differences of gene expression levels between groups were analyzed by oneway ANOVA.Contrasting with control group,toxin genes mazF6,9 were up-regulated expression levels both in mono-resistance (11.1519±22.31721;8.4306±17.97897) and multidrug resistance (4.6016±1.29018;6.9627±6.92948),had statistical significance (P＜0.01),mazF3 expression levels had statistical significance neither in mono-resistance nor in multidrug resistance (P＞0.05);antitoxin genes mazE3 was in down-expression level,and had statistical significance both in mono-resistance (0.3606±0.12527) and multidrug resistance (0.2016±0.16542) (P＜0.01),mazE6 had no statistical significance (P＞0.05)either in mono-resistance or multi drug resistance,mazE9 only in multidrug resistance(0.3989±0.37679) was in downexpression level,and has statistical significance (P＜0.001).The toxin gene mazF6,9 and antitoxin gene mazE3,9 may participate in the drug-resistance formation of M.tuberculosis.

For investigating the epidemiology and genetic characteristics of enterovirus 71 (EV71) in Fujian from 2010 to 2015,we analyzed the surveillance data of EV71 and sequenced VP1 genes of 72 EV71 strains randomly picked from the past 6 years.The overall infection rate was gradually down and one incidence peak (from May to July) was observed each year.Major infectious population were focused on Xiamen,Fuzhou,Nanping and Quanzhou,the ages ranged from one to three years old.Scattered children were the most infected ones.The proportion of EV71 in the severe case was higher than in the HMFD(χ2 =732.064 5,P＜0.000 1).EV71 circulated from 2010 to 2015 in Fujian Province was belonged to subgenotype C4a in consistent with vaccine strain (H07).Compared with the VP1 of vaccine strains,the divergence of complete VP1 nucleotide sequence was gradually expanding as time distance increased,but the sequence of amino acid was not found obvious difference.Variations in 4 key immune epitopes of amino acid had not appeared a regular pattern in year and not consistent with the trend of proportion of EV71 in HMFD.As a result,we considered the epidemiology characteristics of EV71 in Fujian was obvious,72 strains still belonged to C4a subgenotype and had no outstanding antigenic drift or mutation.Extensive epidemiology surveillance and genetic characteristic are needed for the application of EV71 vaccine.

Currently,the BCG is used to prevent tuberculosis,but the immune effect is not ideal due to varied reasons.The existence of drug-resistant strains of tuberculosis and the increased prevalence and incidence of AIDS have leaded to the increased incidence of TB year by year.Therefore,the development of new tuberculosis vaccine is imminent In this paper,the latest research results in recent years for tuberculosis live vector vaccines were summarized,which provide a theoretical reference for further research and development of new TB vaccines.

We evaluated clinical application effect of gene chip for detection of rifampin (RFP) and isoniazid (INH) resistance in Mycobacterium tuberculosis (MTB).Rifampin and isoniazid drug-resistance gene loci were detected by gene chip with sputum specimens from smear-positive tuberculosis patients and clinical strains,comparing the results of detection.BACTEC MGIT 960 drug susceptibility test results were used as control to evaluate the detection performance of gene chip.The sequences of the polymerase chain reaction products of the rpoB,katG and inhA genes from 999 strains identified as Mycobacterium tuberculosis were determined to confirm the mutations by DNA sequencing.Results showed that 100 cases were identified as nontuberculous mycobacteria by gene chip in the 1 108 cases of smear-positive samples.Among the rest 1 008 samples,there were only 9 cases of microarray results different from BACTEC MGIT960 culture-positive strains,achieving the coincidences of 99.1％.Compared with BACTEC MGIT 960 drug susceptibility test results,the gene chip method displayed a concordance of 98.1 ％ and 94.5 ％ for RFP and INH respectively in the 999 strains.Compared with the DNA sequencing method,the accuracy of gene chip method was 99.6％ for rifampin resistance and 99.8％ for isoniazid resistance.It's concluded that the gene chip technology can quickly and accurately detect rifampin and isoniazid resistance in MTB and can be used directly for the detection of sputum samples.

We investigated the correlation between toxin gene exoS,exoU and fluoroquinolone resistance in lower respiratory tract infection with P.aeruginosa so as to provide guidance for reasonable treatment of clinical infections.We collected P.aeruginosa of sputum samples in hospitalized patients from October 2015 to March 2016.The antimicrobial susceptibility was tested by liquid dilution method.The exoS and exoU genes were detected by PCR technique.Results showed that forty-six P.aeruginosa strains were identified from sputum.The exoS and exoU gene positive rate were 86.96 ％ (40/46) and 69.57 ％ (32/ 46) respectively,and the highest proportion of genotype was exoS+/exoU+ (60.87％,28/46).Among them,36.96％ (17/ 46) were multiple drug-resistant bacteria(MDR).Fluoroquinolone non-sensitive (FQ-NS) strain were 78.95％ (15/19) for MDR and 89.47 ％ (17/19) exoU gene were positive,which was significantly higher than the fluoroquinolone sensitive strains (FQ-S).Compared with the FQ-S strain,FQ-NS strains were serious drug resistance.The drug resistant rate of eefepime and aztreonam were more than 70％,and then meropenem and imipenem were more than 50％.The drugs of lower resistance rate in FQ-NS strain had polymyxin B(10.53％,2/19),amikacin(10.53％,2/19),ceftazidime (15.79％,3/19) and gentamicin (21.05％,4/19).P.aeruginosa of lower respiratory infection carried toxin genes exoS and exoU were higher,the main genetpy was exoS+/exoU+.FQ-NS strains were higher drug resistance rate and a higher proportion of exoU+ strains than FQ-S strains.We should strengthen virulence genes test and drug resistance monitoring in clinical practice.

We expressed B cell epitopes of dengue virus envelope protein and NS1 protein in prokaryotic cells,and purified and evaluated for its serological activities.A recombinant multi-epitope chimeric gene named rE including eight B cell epitopes was connected by linker peptide (EAAAK)2 and cloned into prokaryotic expression vector pET-28a(+),and transformed into E.coli BL21(DE3) cells for expression under induction of IPTG.The expressed recombinant protein was purified with 6× His purification media,and identified by SDS-PAGE and Western blot,and its antigenicity was analyzed by using an indirect ELISA assay.The recombinant expression vector pET28a-rE was constructed and expressed in BL21 (DE3) successfully,but the recombinant proteins mainly appeared as inclusion bodies.The target protein was obtained with high purity through the purification of affinity chromatography.SDS-PAGE and Western blot analysis showed that the molecular weight of fusion protein was in the expected line.The established indirect ELISA has high accuracy.This recombinant peptide antigen expressed in E.coli has good potential for serum testing.