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.

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.

Investigation on the space structure of plague natural foci in the Sanjiangyuan area in Qinghai Province / 中国地方病学杂志

Objective To investigate the space structure of plague natural foci in the area of Lantsang, Yellow and Yangtse River in Qinghai Province to provide references for making decisions to eontrol the occurrence of human plague. Methods Data was collected from the survey on natural foci and surveillance of plague from 1954 to 2006 and descriptive epidemiological method was used to analyze the data. Results Marmata hirnalayana and Microtus fuscua natural foci were known in Sanjiangyuan area. Callopsylla dolabris, Oropsylla silantiewi, Citellophilus sparsilis and Amphipsylla tuta were vectors; Microtus fuscus plague natural foci was in a range of about 9500 km2, distributing in Zhenqin Town, Chengduo County. Marmata himalayana plague natural foci distributed over 13 countries, a range nearly 107 000 km2. By the end of 2006, 450 strains of Yersinia pestis were detected and separated from 6 kinds of rodents, 6 kinds of carnivora, 3 kinds of artiodactyls and 9 insects vectors. Between 1960 and 2006, 238 cases and 134 deaths from plague were reported. Most human plague cases occurred in the months from May to November and usually presented as one of three primary forms-bubonic 17.23%(41/238), septicemic 16.81% (40/238), pneumonic 61.34% (146/238) and other types 4.62% (11/238). However, the first epidemic plague case was mainly the glandular plague. Conclusions Date suggested that plague is still a critical public health problem in Sanjiangyuan area, against which countermaeasure needs to be strengthened in the main epidemic areas. More scientific researches on plague should be carried out. Surveillance networks of reporting suspected plague have been established and reduce the number of human plague cases.

Study on the pathogen of plague in Sanjiangyuan area in Qinghai province / 中华流行病学杂志

Objective To study the biological characteristics of Yersinia pestis and to develop prevention and control program on plague in Sanjiangyuan areas,Qinghai province.Methods To identify the biologic types and molecular biological features of Y.pestis isolated in Sanjiangyuan area from 1954-2007.Results Among the 411 strains ofY.pestis,12 strains belonged to the microtus type Y.pestis with denitrification (-) and donkey-bible gelatin carbohydrate(-) and glycerine(+).399 strains belonged to classic type Y.pestis with denitrification (+) and donkey-hide gelatin carbohydrate (+) and glycerine (+).411 Y.pestis strains had factor F I and Pst I.Among them,VW + strains of Y.pestis accounted for 95.13%(391/411),VW accounted for 4.87%(20/411),Pgm+accounted for 80.78%(332/411 ),Pgm±accounted for 9% (37/411 ) and Pgm-accounted for 10.22% (42/411) respectively.96.82% (213/220) of the Y.pestis strains showed strong virulence to laboratory mice while 3.18% (7/220) of the strains carried medium virulence.90.02% of the tested Y.pestis (370/411) strains had 6×106,45×106,65×106 plasmids.8 types of genome were found among 80 strains of Y.pestis,with 6 of them resembling ZHOU Dongsheng's classification.Two new genome types were found.Conclusion The Y.pestis in the Sanjiangyuan area had the characteristics of plague pathogen,identified in Qinghai-Tibet plateau.It is estimated that human beings are highly susceptible to the disease which spread fast,causing serious signs and symptoms with high death rate.

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>

A retrospective analysis of plague in Three-River Source Region of Qinghai from 1954 to 2007 / 中国地方病学杂志

Objective To analyze plague epidemic tendency in the Three-River Region of Qinghai.Methods Using retrospective study,the Three-River Region during 1954-2006 year pestis epidemic focus were investigated and analyzed.Result Pestis prevailed mainly in Yushu,Chindu,Qumalai,Nangqian,Zhiduo and the Geermu.Tanghla Township.It was first found that the nature plague focus of miefitus existed in Chengduo County.There are 1 5 kinds of 12 branches in 8 trees infected plague animals were founded,336 Yersinia pestis were separated from the driven objects.Among them there were 291 Himalayas marmot body,account for 86.60%of the total,13 of Tibet sheep,accounts for 3.87%.10 of Qinghai field-mouse,accounts for 2.98%,Also there were 114 Yersinia pestis which were separated from each kind of vector insect in vivo.And,46 pestis strains came from the axe shape of flea in vivo account for 40.35%(46/114),38 pestis strains separated from Xie mountain flea,account for 33.33% (38/114).During 1960-2006 years there were 85 human plague cases were founded,238 occurred,134 died,the case fatality rate wero 56.30%(134/238),the popular seasons were started from May to November,the peak season happened in Aug and Sep.After Oct mainly due to Tibet sheep pestis which will cause as the origin of infection.The majority of sickness was pulmonary plague,account for 49.58%(117/238),whereas the first round case caused by the gland bubonic plague,account for 77.12%(91/118).Conclusions There are two pestis strains natural epidemic focus places in Three-River Source Region of Qinghai including the Himalayas marmot pestis strain and the Qinghai field-mouse pestis strain.The case of human pestis strain causes by the marmot strain,the fiehl-mouse mold mushroom spawn causes human pestis strain has not yet discovered,Three-River Source Region of Qinghai is a pestis strain key popular area in Qinghai Province.

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.

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>