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>

Epidemiological analysis of plague in Qinghai province between 2000 and 2009 / 中国地方病学杂志

Objective To analyze the endemic features of plague in Qinghai province between 2000 and 2009, discover the law of occurrence and progression, in order to provide a scientific basis for further prevention and treatment of the disease. Methods Descriptive epidemiology was employed to analyze the data from on the spot investigation, monitoring reports and papers published between 2000 and 2009. The indicators included the area, host and media distribution of animal plague and area, time, and population distribution of human plague.Results In Qinghai province between 2000 and 2009, 189 strains of Yersinia pestis were isolated from a variety of animals and insect vectors, including 77 from the marmot, accounting for 40.74%, 40 from Callopaylla dolabris,accounting for 21.16%. Positive serum antibodies against F1 plague were detected in 238 samples, including 90 samples from husbandry dogs, 63 from woodchucks. The areas with Yersinia pestis were consistent with the areas with positive serum antibodies against F1 plague, which distributed mainly along the Qinghai-Tibet railway Wulan county, Delhi and Golmud Multi-county;confirmed that there was natural foci of plague in Qinghai vole. Between 2000 and 2009, 13 events of human plague occurred, with 37 cases and 16 patients died, mortality was 43.24%.Cases were distributed in 11 townships of Tongde, Xinghai, Qilian, Wulan, Tianjun, Nangqian, Qumalai,Chengduo and Zhiduo counties. May to October was the disease season, with September the peak. Pneumonic plague disease type was the main mode of transmission of the plague and patients often contacted with airborne droplets through the air and peeling fresh Marmota. Conclusions Plague in Qinghai province is still grim,strengthening animal plague surveillance, and timely disposal of animal plague, improving the province's agricultural and pastoral areas, especially increase the disease prevention consciousness of the masses are future tasks. Work should be focused on strengthening the prevention and control of plague along Qinghai-Tibet railway,and prevent the occurrence and long-distance transmission of human plague.

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>

Evaluation of immunization protection efficacy of plague subunit vaccine / 中华预防医学杂志

<p><b>OBJECTIVE</b>To evaluate the protective efficacy of plague subunit vaccine, BALB/c mice, guinea pigs and rabbits were used in this study.</p><p><b>METHODS</b>Groups of mice (10 per group), guinea pigs (14 per group) and rabbits (6 per group) were immunized with F1 + rV270 vaccine, EV76 vaccine and alum adjuvant by intramuscular route, respectively. Serum antibody titres of mice, guinea pigs and rabbits were determined by ELISA and the immunized animals were challenged with 10(6) CFU of Y. pestis strain 141 at the 8th week after the primary immunization.</p><p><b>RESULTS</b>The immunized mice, guinea pigs or rabbits with subunit vaccine developed anti-F1 IgG titre of 41 587.3 +/- 2.1, 11 543.7 +/- 2.1 or 522.4 +/- 22.4 and elicited statistical anti-F1 IgG titre difference among them (F = 17.58, P < 0.01). The immunized mice, guinea pigs or rabbits with subunit vaccine had anti-rV270 IgG titre of 15 748.7 +/- 1.6, 12.6 +/- 1.4 or 1648.0 +/- 5.0 and induced statistical anti-rV270 IgG titre difference among them (F value was 16.34, P < 0.01). There was significant anti-F1 IgG titre difference among mice, guinea pigs and rabbits immunized with EV76 vaccine that developed anti-F1 IgG titre of 913.4 +/- 4.5, 937.0 +/- 2.0 or 342.0 +/- 12.0 (F = 23.67, P < 0.01), whereas the immunized mice, guinea pigs and rabbits with EV76 vaccine developed anti-rV270 IgG titre of 12.0 +/- 1.0, 447.0 +/- 10.0, 40.0 +/- 11.0 and there was no anti-rV270 IgG titre difference between them (F = 2.20, P = 0.1314). The immunized mice with subunit vaccine developed significantly higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 30.57 and 19.04, respectively, P < 0.01), and there were no anti-F1 IgG titre differences between the immunized guinea pigs and rabbits (q = 0.04, P = 0.8485). The immunized mice with subunit vaccine developed significantly higher anti-rV270 IgG titres than immunized guinea pigs and rabbits (q value was 27.10 and 19.49, respectively, P < 0.01), and there were no anti-rV270 IgG titre differences between the immunized guinea pigs and rabbits with the subunit vaccine (q = 0.25, P = 0.6187). The immunized mice with EV76 elicited higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 40.67 and 29.10, respectively, P < 0.01), whereas there was no difference of F1 IgG titer between immunized guinea pigs and rabbits (q = 0.06, P = 0.8098). The immunized mice, guinea pigs and rabbits with subunit vaccine provided 100% (10/10), 86% (12/14) and 100% (5/5) protection against 10(6) CFU Y. pestis of challenge, respectively. The immunized mice, guinea pigs and rabbits with EV76 vaccine gave 100% (6/6), 93% (13/14) and 100% (6/6) protection against 10(6) CFU Y. pestis of challenge respectively.</p><p><b>CONCLUSION</b>BALB/c mice is the best small animal model for valuation of protective efficacy of plague subunit vaccine. The guinea pigs showed a high individual variation for this purpose. The rabbits can be used as an alternative model for evaluating plague subunit vaccine.</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.

Analysis the epidemiology and clinical feature of plague cases from 1981 to 2006 year in China / 中国地方病学杂志

Objective To summarize the epidemiological feature of plague cases oceuwed in China.Methods The epidemiological and clinical data from 1981 to 2006 year in China were analyzed with descriptive study method.Result Nine hundred and seveneteen human plague cases were diagnosed in 9 provinces(regions) from 1981 to 2006 years,105 cases died,the mortality rate being 11.45%,and they distributed in 69 counties (cities or banners).In Qinghai Province 108 cases were diagnosed,the mortality rate was 46.30%(50/108),the cases distributed in 17 counties(cities);137 cans in Guizhou,distributing in 2 counties(cities);517 cases in Yunnan,distributing in 26 counties(cities).Plague cases peaked separately in 1983,1990,1996 and 2000 years,they were 25,75,98 and 254 separately.The principal spreading ways were breathing flying particles,touching,skinning and eating marmot in Qinghai;750 cases were of bubonic plague,among whom 4 cases in Tibet died,the fatality rate was 0.53(4/750);121 cases were of pneumonic prague,among whom 65 cases died,was accounting for 53.72%(65/121);31 cases were of septieaemic plague,and 30 cases died(one cases was cured in Inner Mongolia),accounting for 96.77%(30/31).Others were brain plague,intestinal plague,tonsil plague and plague cellulites,which were cured.Conclusion From 1990,human plague epidemical scope and intensity is enlarging continuously compared with 1980-1990 and there is a trend of going up gradually in China.

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.

Epidemiologic analysis on human plague of Sanjiangyun Area in Qinghai Province, China / 中国地方病学杂志

Objective To study on epidemiologic characteristics of human plague cases in Sanjiangyuan Area,and provide theoretical basis to work out the preventive measures.Methods Based upon the epidemiology information from the human plague case data bank of Qinghai Province,human plague data were analyzed retrospectively in Sanjiangyun Area by sorting,verifying and summing up of these data,including some of case file and monitoring data.Results Except for 12 years in the period of 1960 to 2006,there were human plague cases happened every year.The morbidity occurred mainly in 12 counties of 4 states,including Yushu,Guolou,Huangnan and Hainan,and Tanggula Town of Geermu City,a total of 85 human plague episodes were occurred,resulting 238 onsets,134 deaths,and a matality rate of 56.30%.The sources of infection were respectively Himalayan mormot 27.31%(65/238),artiodactyls 14.71%(32/238),carnivora 2.10%(5/238),Lagomorpha 0.42%(1/238),the pneumonic plague patient 49.16%(117/238),and biting of flea 6.30%(15/238).The prevalent season was from May all the way to November,the peak-months were August and September.After October,the sheep as the source of infection initiating human being plague accounted for 23.53%.Among the clinical types,the most prevalent type was pneumonic type(61.34%),and the rest,glandular type(17.23%),septic type(16.81%)and other types(4.62%),but the first plague case in each epidemic was mainly the glandular plague.Conclusions In recent years,the tendency of human plague prevalence increases in Sanjiangyuan Area,it is urgent to improve and adjust the prevention and treatment measures in time.