Etiological analysis and epidemiological significance of plague in Qinghai, 1980-2011 / 中国热带医学

@#Abstract: Objective　To analyze the pathogenic characteristics and epidemiological significance of human plague related strains in Qinghai Province in recent 30 years, so as to provide scientific basis for on-the-spot disposal and prevention and control measures of plague outbreak in Qinghai Province. Methods　A total of 35 strains of Yersinia pestis isolated from 29 typical human plague outbreaks in Qinghai Province from 1980 to 2011 were selected and studied by biochemical fermentation experiments. Virulence factors detection of Fraction 1 antigen (Fra1), virulence antigen (VW), pigmentation (Pgm) and Yersinia pestis Ⅰ (PstⅠ), determinants and genotyping of differential regions (DFRs) were used to study the pathogenic characteristics. At the same time, according to the epidemic situation of human and animal plague in Qinghai Province in recent years, the current situation of plague prevention and control and epidemic characteristics were analyzed. Results　The biotypes of 35 strains of Yersinia pestis were classical, and the biotypes of 29 strains (82.86%) were of Qinghai-Tibet Plateau type, mainly distributed in southern Qinghai and around lake areas, 2 strains (5.71%) belonged to Qilian Mountains type, mainly distributed in Qilian mountains, and 6 genotypes were identified by DFR. Among them, 16 were type 5, 12 were type 8, 2 were type 10, 1 was type 36, 3 were type 30 and 1 was type 1b, the strains of type 5 and 1b were mainly distributed around the lake and the southern foot of Qilian Mountains, while the strains of type 8, 10, 36 and 30 were mainly distributed in the southern part of Qinghai. Conclusions　The pathogen of Yersinia pestis in Qinghai Plateau has complex biochemical types, the epidemic situation among animals is continuous year after year, the situation of prevention and control is serious, the occurrence and prevalence of plague seriously endanger people's health and social development, so it is necessary to do a solid job in the prevention and control of plague to ensure the safety of people's lives.

The pathogenic ecology research on plague in Qinghai plateau / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the pathogenic ecology characteristics of plague in Qinghai plateau.</p><p><b>METHODS</b>Applied molecular biology techniques, conventional technologies and geographic information system (GIS) to study phenotypic traits, plasmid spectrum, genotype, infected host and media spectrum etc.of 952 Yersinia pestis strains in Qinghai plateau plague foci, which were separated from different host and media in different regions during 1954 to 2012.</p><p><b>RESULTS</b>The ecotypes of these strains were Qingzang plateau (91.49%, 871/952),Qilian mountain (6.41%, 61/952) and Microtus fuscus (1.26%, 12/952).83.6% (796/952) of these strains contained all the 4 virulence factors (Fr1, Pesticin1,Virulence antigen, and Pigmentation), 93.26% (367/392) were velogenic strains confirmed by virulence test.725 Yersinia pestis strains were separated from Qinghai plateau plague foci carried 9 kinds of plasmid, among which 713 strains from Marmot himalayan plague foci carried 9 kinds of plasmid, the Mr were 6×10(6), 7×10(6), 23×10(6), 27×10(6), 30×10(6), 45×10(6), 52×10(6), 65×10(6) and 92×10(6) respectively. 12 Yersinia pestis strains were separated from Microtus fuscus plague foci carried only 3 kinds of plasmid, the Mr were 6×10(6), 45×10(6), 65×10(6). Meanwhile, the strains carrying large plasmid (52×10(6), 65×10(6) and 92×10(6)) were only distributed in particular geographical location, which had the category property. The research also confirmed that 841 Yersinia pestis strains from two kinds of plague foci in Qinghai plateau had 11 genomovars. The strains of Marmot himalayan plague foci were given priority to genomovar 5 and 8, amounted to 611 strains, genomovar 8 accounted for 56.00% (471/841), genomovar 5 accounted for 23.07% (194/841). Besides, 3 new genomovars, including new 1(62 strains), new 2(52 strains), new 3(48 strains) were newly founded, and 12 strains of Microtus fuscus plague foci were genomovar 14.</p><p><b>CONCLUSION</b>The main host and media of Qinghai plateau plague foci directly affected the spatial distribution regularities of plague epidemic and the pathogens characteristics, meanwhile the polymorphism of plague ecological geographic landscape leds to the complexity of Yersinia pestis' genotype.</p>

Observation of protective effect on mice after immunization with Yersinia pestis capsular antigen F1 and recombinant rV270 / 中国地方病学杂志

Objective To evaluate the protective effect of Yersinia pestis capsular antigen F1 and recombinant rV270 on mice after immunization with them.Methods According to body weight,40 female Balb/c mice aged 6 to 8 weeks were randomly divided into four experimental groups(Fl-10 μg + aluminum adjuvant,F1-20 μg + aluminum adjuvant,rV-10 μg + aluminum adjuvant,and rV-20 μg + aluminum adjuvant) and a control group,8 in each group.Mice in experimental groups were immunized with the natural antigen F1 and recombinant antigen rV270 adsorbed to 25％ aluminum adjuvant and the control group was immunized with the same amount of aluminum adjuvant.Each mouse was immunized at the hind leg muscle with 100 ml immunizing agent,then a booster immunization was done once on the 21st day after the first immunization.The blood of all mice was collected on the 8th week after the first immunization,serum antibody titers were detected by ELISA and the data of antibody titers were analyzed by t test for comparison between groups.At the same time the mice were injected subcutaneously with 2000-fold LD50 of Yersinia pestis virulent strain 141,after 14 days,the protective effect of immunization was analyzed.Results The control group did not produce antibody.Antibody geometric mean titers (GMT) of the F1-10 mg + aluminum adjuvant and F1-20 mg + aluminum adjuvant groups were 1 ∶ 30443.9,and 1 ∶21527.8,respectively,and compared between the two groups,the difference was not statistically significant (t =1.1282,P ＞ 0.05).The GMTs of the rV-10 μg + aluminum adjuvant and rV-20 μg + aluminum adjuvant groups were 1 ∶ 13957.3 and 1 ∶18100.9,respectively,and compared between the two groups,the difference was not statistically significant(t =0.9408,P ＞ 0.05 ).After subcutaneous injection with Yersinia pestis virulent strain 141,all mice died in the control group but all survived in the experimental group.Conclusion The immune activity of natural antigen F1 and recombinant rV270 is high,which can be used as the main component of subunit vaccine in the plague subunit vaccine study.

Epidemiologicai analysis of plague in Qinghai province from 2001 to 2010 / 中国地方病学杂志

Objective To explore the plague epidemical trend of nearly a 10 years data in Qinghai province to provide basis for making the prevention and control measures. Method The regional distribution and time distribution of animal and human plague, monitoring and plague foci of survey data in Qinghai from 2001 to 2010 were analyzed with Excel software 2003. Results In Qinghai province, a total of 167 strains of Yersinia pestis were isolated from infected animals and insects in 10 years. Yersinia pestis was mainly distributed in Wulan,Delinha, Geermu, and Tianjun, along the Qinghai-Xizang railway. Human plague was occurred every year from 2001 to 2010 except 2002, 2007, 2008, and 2010. In the 10 years, there were 37 plague cases and 16 of these cases died, the mortality was 43.24％. The plague cases were mainly distributed in Nangqian, Qumalai, Chenduo,Zhiduo, Xinghai, Tongde, Tianjun, Wulan and Qilian. And these cases were found mostly in the period from May to October, especially in the period from August to October. Major clinical type of the plague cases was lung-type (62.16％,23/37). Conclusions The plague epidemic situation in Qinghai province is still severe, animal plague occurred year after year, and human plague outbreaks occasionally. Monitoring and early warning in the key areas should be strengthened, and the comprehensive measures of plague prevention and control should be carried out to reduce the incidence and prevalence of plague.

Different strategies for preparation of non-tagged rV270 protein and its efficacy against Yersinia pestis challenge / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study.</p><p><b>METHODS</b>A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co(2+) affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography.</p><p><b>RESULTS</b>Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)₃ adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 10⁶ CFU of Y. pestis virulent strain 141.</p><p><b>CONCLUSION</b>The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.</p>

Identification of 2 strains of suspected Yersinia pestis isolated from Marmot,,himalayana in Dege County,Sichuan Province / 中国地方病学杂志

Objective Throush identify biochemical characteristics and virulence factors of 2 strains suspected Yersinia pestis(Y.pestis)isolated from the dead Marmota himalayana(M.himalayana)to confirm the nature epidemic focus in Dege County,Sichuan Province.Methods Y.pestis was analyzed by specific staining and shape,culturing characteristics,splitting-test by bacteriophage,test of biochemical characteristics and glycolysis ability,virulence factors,virulence,nutritional requirement,plasmid,genetic test and genetic type. Results The tested strains were Gram staining bacilus.The main biochemical characteristics were Arabinose(+)、 Rhamnose(-),Maltose(+),Melibiose(-),Glycerol(+),Denitrification(+).The virulence factors with FI+.VW+, Pgm+,Pst I+;and with the common 6.0×106,45.0×106,65.0×106 plasmids,also with the virulence-relative plasmid gene.Both their absolutely lethal dose(LD100)in mice were 50 bacteria.The nutritional requirement appeared which were depended on Phenylalanine and Methionine.With the Genomovar 5 genotype characteristics of M.himalayana plague foci of Qinghai-Tibet plateau.The difference between tested strains and Yersinia pseudotubercuosis on the 3 different culture medium was obvious.The tested strains had a Y.pestis' specific 3a fragment,Pst I and FI-Ag,at 22 ℃,the strains could be split by bacteriophage completely.Conclusions According to the diagnostic criteria of plague in China,the 2 suspected strains isolated from Dege County,Sichuan Province ale confirmed as Y.pestis.both with powerful virulenceand with the characteristics of the Y.pestis of M.himahtyana in Qinghai-Tibet plateau plague natural focus.

Study on plague pathogeny in areas along Qinghai-Tibet Railroad in Qinghai from 2001-2006 / 中国地方病学杂志

Objective To study the features of Yersinia pestis(Y.pestis)in areas along Qinghai-Tibet Railroad in Qinghai Province.Methods To identify the biologic types and the molecular biological feathers of Y.pestis isolated from areas along Qinghai-Tibet Railroad in Qinghai from 2001-2006.Results All the tested Y.pestis was biologically of classical type and ecologically of Qinghai-Tibet plateau type.The Y.pestis had high virulence.The Y.pestis of 65×106 plasmids was distributed in the Tanggula area,the Y.pestis of 52×106plasmids,in Tianjun and Delingha areas.The Y.pestis srains carried 52 × 106 plasmids.except the two containing 65 X 106 plasmids in Wulan County.The genetic type of Y.pestis in Tanggula was type 5 and that in Zongwulong of Delingha,Saishike,Keke,Tongpu of Wulan was type 8 except 2 strains of Y.pestis isolated from woodchuck and the patients in Dananwan of Tongpu,Wulan County were type 15.Conclusion The Y.pestis in the area along Qinghai-Tibet Railroad in Qinghai belongs to Qinghai-Tibet plateau type with high virulence.

A molecular epidemiological study on human plague fulminant epidemic in Qinghai, 2004 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the epidemiology of genotyping Yersinia pestis isolated in the fulminant epidemics of human plague in Qinghai province in 2004.</p><p><b>METHODS</b>Primer pairs targeting the twenty-three different identified regions (DFRs) were designed to detect the presence or deletion of each DFR in 13 strains of Yersinia pestis isolated from the fulminant epidemic of human plague in Qinghai province in 2004.</p><p><b>RESULTS</b>There were 4 genomovars, i.e. Genomovar 8, 10, 15 and 16 in the 13 strains of Yersinia pestis identified. The genomovar of all the strains of Yersinia pestis isolated from Nangqian county was Genomovar 10. Among the two strains of Yersinia pestis isolated from Wulan county, the genomovar of one strain was Genomovar 8 and the other was Genomovar 10. The genomovars of all the strains of Yersinia pestis isolated from Qilian, Qumalai and Chengduo county belonged to Genomovar 16.</p><p><b>CONCLUSION</b>It was demonstrated that the genotyping of Yersinia pestis appeared to be a powerful tool for investigating human plague epidemics.</p>

Study on the genotyping and microevolution of Yersinia pestis in the Qinghai-Tibet Plateau / 中华流行病学杂志

<p><b>OBJECTIVE</b>To study the distribution of genomovars and microevolution of Yersinia pestis in the Qinghai-Tibet Plateau.</p><p><b>METHODS</b>Primer pairs targeting the twenty-two different regions(DFRs) were designed for detecting the presence or deletion of each DFR in 297 strains isolated from the Qinghai-Tibet Plateau.</p><p><b>RESULTS</b>9 genomovars, i. e. Genomovar 1, 5, 6, 7, 8, 10, 11, new type and Ype-ancestor were identified in the Marmota himalayana plague focus of the Qinghai-Tibet Plateau. Among these genomovars, genomovar 5,8 and 10 were dominant types. The total rate of the three genomovars was 80.6% (204/253) and the genomovars in different regions were different. All of 44 strains of Y. pestis in the Microtus fuscus plague focus of the Qinghai-Tibet Plateau belonged to genomovar 14.</p><p><b>CONCLUSION</b>The distribution of genomovars of Y. pestis in the Qinghai-Tibet plateau had remarkable characteristics geographically. Based on the distribution of genomovars of Y. pestis, the routes of transmission and microevolution of Y. pestis were proposed.</p>