Genotyping of Yersinia pestis by different regions and its epidemiological characteristics in Yunnan Province / 中国地方病学杂志

Objective To genotype Yersinia pestis and explore intrinsic relationship among different ecotypes of Yersinia pestis in Yunnan foci.Methods A total of 171 strains from three types of Yersinia pestis,house mouse,wild-type mouse and Yulong Yersinia pestis,were tested.Twenty-three different regions (DFR) were used to genotype and cluster analysis was performed using BioNumerics 5.0.Results A total of 171 Yersinia pestis were divided into 7 genotypes by 23 DFRs,which were Genomovar5,Genomovar7,Genomovar9 and 4 newly discovered genotypes.The genotypes of all Yulong plague were Genomovar5.The genotypes of the 16 strains of wild-type mouse plague (the highland of northwestern Yunnan Province type) were divided to 3 genotypes,13 of them were Genomovar 7,2 of them were Genomovar9,and 1 of them was newly discovered genotype Genomovaryn1.The genotypes of the 148 strains of house mouse plague(the residential area of Yunnan and Fujian Provinces type) were divided into 4 genotypes,145 of them were Genomovar9,and 3 of them were newly discovered including Genomovar-yn2,-yn3 and-yn4.The ecological typing results of clustering showed genotype of Yulong plague was similar to the highland of northwestern Yunnan Province type(wild-type mouse plague),and the percentage of similarity was up to 87.20％,but only up to 73.75％ to the residential area of Yunnan and Fujian Provinces type (house mouse plague).The genotypes of 2 wild-type strains of the highland of northwestern Yunnan Province type(wild-type mouse) and main genotypes of the residential area of Yunnan and Fujian Provinces type(house mouse)were Genomovar 9.The genotype of Genomovar-yn 1 of the highland of northwestern Yunnan Province type was similar to Genomovar 7,but lack of DFR 11.The genotypes of Genomovar-yn2,-yn3 and-yn4 of the residential area of Yunnan and Fujian Provinces type were similar to Genomovar 9,but lack of DFR 10,DFR 9 and DFR 11,respectively.Conclusions One newly genotype strain is found in wild-type mouse plague and 3 newly genotype strains are founded in house mouse plague.Wild-type mouse strains are founded in the house mouse strains.The similarity of genotype between Yulong plague and the highland of northwestern Yunnan Province type (wild-type mouse plague) is high while the similarity between Yulong plague and the residential area of Yunnan and Fujian Provinces type(house mouse plague) is low.

Comparative genomic analysis of gene variations of two Chinese Yersinia pestis isolates from vaccine strain EV76 / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate genomic variations of two Chinese Yersinia pestis isolates that were isolated from different plague foci obtained from vaccine strain EV76 from the Yunnan province of China.</p><p><b>METHODS</b>A microarray containing 12 000 probes covering the entire genome of seven Yersinia pestis and two Yersinia pseudotuberculosis strains, was used. PCR assays were performed to confirm microarray results.</p><p><b>RESULTS</b>The gene variations detected included the absence of five genes related to the synthesis of betaine in both EV76 and another sequenced attenuated strain, KIM D27. Several genes related to phage-related membrane proteins were found to be absent in the Antiqua biovar Yunnan strain, 485, which was isolated from a rodent plague foci.</p><p><b>CONCLUSION</b>These findings provide initial insight into the distinct strains isolated from natural foci, within their genomic context, including Yunnan Y. pestis strains. This information will be used therefore to establish subsequent comparisons of these sequences with published complete genomes of other strains.</p>

Screening of marker gene in Yersinia pestis plasmid / 中国地方病学杂志

Objective To screen the conservative,stable and specific DNA signature sequence in the plasmid of Yersinia pestis.Methods Specific validation trials and stability of the qualification test were carried out to 40 strains of Yersinia pestis,47 strains of non-Yersinia pestis of home and wild types of rodent in Yunnan,by using 32 DNA sequences derived from Yersinia pestis in the plasmid and conventional PCR technology,and Yersinia pestis vaccine strain EV76 as a positive control.Results Four pairs of relatively conservative,stable and specific DNA marker genes were screened:YPMT1.05c,YPMT1.03c,YPMT1.42 and YPMT1.04c.Conclusions The 4 pairs of Yersinia pestis DNA signature sequences can be used for rapid diagnosis of plague.

Prokaryotic expression of plasminogen activator factor and its specific fragment of Yersinia pestis / 中国地方病学杂志

Objective To express the plasminogen activator(Pla) of Yersinia pestis and one of its gene fragments,and to detect their immunological reactivity.Methods The pla gene and its specific gene fragment pla-c were amplified by PCR using the EV76 strain as a template.PCR products were then ligated with the plasmid pET32a (+).The recombinant plasmids pET32a (+)-pla and pET32a (+)-pla-c were subsequently trausformed into E.coli BL21 (DE3).The expressed products were purified by HIS affinity chromatography,and their immunological reactivity was detected by Western blotting.Results The recombinant Pla(52.8 × 103) was expressed as inclusion bodies,and the recombinant Pla-c protein (24.0 × 103) was expressed in the soluble form.These two recombinant proteins reacted with anti-Yersinia pestis EV76 rabbit sera.Conclusions The recombinant Pla and its specific fragments have displayed immunological reactivity,and can be served as an alternative diagnosis method for Yersinia pestis.

Comparison of Yersinia pestis genomes from a new natural foci in China / 中国地方病学杂志

Objective Measurement and analysis of the complete genome sequences of Yersinia Pestis from a new plague natural foci and adjacent foci in China, to know the genetic relationship among the epidemic strain isolated in Yulong (D 106004) and Jianchuan strains (D 182038) and the Tibetan strain ( Z 176003 ). Methods Three complete genome sequences were sequenced using the whole-genome shotgun and Solexa method and comparative genomics analysis was done among the three sequences. Genome comparative analysis among the coding sequences was done by BLAST software, SNPs finding was done by the program, genome rearrangements were analyzed using MAUVE software. Results All of the genomes of Yersinia pestis strains D182038, D106004 and Z176003 consist of a single circular chromosome and three virulence plasmids, pMT1, pCD1 and pPCP1. They had similar characteristics in chromosome and plasmid features, and there were no significant difference in coming sequence (CDS) of the cluster of orthologous groups of proteins (COG) functional classification and the number of insertion sequence in the three strains (x2 =3.03, 0.257, all P ＞ 0.05). The comparative genomics results showed that the three bacteria had 2882 genes with 100％ homology, of 3636 genes predicted in D106004, 2994 were identical with D182038's and 3113 with Z176003's, and of which 240 had 90％ homology with D182038's and 200 with Z176003 's. Synonymous single nucleotide polymorphisms(sSNPs) were 59 and 68, and non-synonymous SNPs(nsSNPs) were 104 and 203 between strains D106004 and Z176003/D182038. There were 11 segments rearrangements between D106004 and Z176003, which was less than 16 segments rearrangements between D106004 and D182038. ConclusionsThe three strains are highly homologous, the Yulong strain has more similarity with Tibet strain than with Jianchuan strain, the strain from Yulong foci may be evolved from Tibet foci.

Discussion on transmission mechanism of wild rodent plague to human in the northwestern area of Yunnan province / 中国地方病学杂志

Objective To explore the relationship between wild rodent plague and human in wild rodent plague foci of the northwestern area in Yunnan to probe the possible transmission mechanism of wild rodent plague to human. Methods Data of component ratio of rodents and fleas was collected in different areas from 1985 - 1995. Activities and habits of residents regarding the way they keep cats and dogs and parasitic fleas and free fleas indoor were investigated, the dog serum was collected for detecting F1 antibody. Results Eothenomys miletus were main rodents in farmland and shrub, accounting for 48.00% (4753/9902) and 54.50% (4282/7857), Apodemus chevrieri were main rodents in garden, being 50.47% (1332/2639). The component ratio of Neopsylla specialis specialis was 13.31%(229/1720), 12.31%(1678/13 739) and 10.87%(957/8802) respectively in garden, farmland and shrub, higher than in indoor. The component ratio of Frantcpsylla spodix was 39.88% (686/1720), the highest in garden. Thirty-two per cent (32/100) of residents kept cats,in which 63% (20/32) with cat fleas, 68% (68/100) of villages kept dogs, in which 76%(52/68) with fleas. Eighteen parasitic fleas were caught from 43 dogs with a flea index of 0.119 and a rate for fleas of 11.63%, 7 pulex were collected from 17 indoor. Forty-three blood serum samples were obtained from dogs, among which 3 were positive blood serum. Conclusions Residents touch affected animals or media in different situations. The possibility of transmission for wild rodent plague to human exists in loci in a chain of wild rodent plague → fleas or predation → homebred animal plague (cats or dogs) →touching or respiratory → human.

Comparative study on biochemical characteristic of Yersinia pestis from some natural focuses of plague in Yunnan Province / 中国地方病学杂志

Objective To compare the difference of biochemical characteristics and virulent Pst Ⅰ of Yersinia pestis strains in traditional focuses of plague in Yunna Province and in the new focuses of plague in Yulong County. Methods The identification data of biochemical characteristics(Rhamnose, Glycerol, Maltose, L-Arabina and Melibiose fermentation) and virulence factor(Pst Ⅰ) from different focuses of plague in Yunna Province were Retrospectively collected by tube test followed by the analysis using statistics software SAS 8.0 by Fisher exact probability of disordered two-way R × C table χ2 test. Results Among 48 strains of Yersinia pestis from hantaan type plague focus, 1 strain fermented L-maltose, 48 strains fermented Glycerol. Among 165 strains of Yersinia pestis from the Soul type plague focus, 1 strain did not ferment L-maltose, only one of them fermented Glycerol. 1 strain from the Soul type plague focus was confirmed to have mutation, for the test of nitrate reduction reaction was negative. All 5 strains of Yersinia pestis from the new focuses of plague in Yulong County fermented L-maltose and Glycerol. The statistical result showed that the differences in L-maltose and Glycerol fermentation of Yersinia pestis from different natural focuses of plague in Yunnan Province were statistically siguificant (P < 0.01). The differences of other biochemical characteristics and Pst Ⅰ were not statistically significant (P > 0.01). Conclusions Biochemical characteristics of Yersinia pestis from the hantaan type plague focus and the Soul type plague focus in Yunnan province are overlapping. Biochemical characteristics of Yersinia pestis from the new focuses of plague in Yulong County are different from those tradition focuses of plague in Yunna Province but share similarities to those from Unquiculatus focuses in North Tibet.

Determination of the serum antibody in pneumonic plague patients / 中国地方病学杂志

Objective To analyze the species of the antibody and immune responsibility in pneumonic plague patients in order to pave the way to screen the new sub-unit of the vaccine to provide the experimental basis. Methods Using the virulence-related protein microarray containing 149 proteins of Yersinia pestis (Y.pestis), the species of the antibody and immune responsibility were analyzed in serum of two pneumonic plague patients in six months after onset. Results Eighty-eight gene coded proteins were detected out the related antibodies except YPMT1.23c, YPMT1.86, YPO0406 and YPO1071 in patient 1. Forty-three antibodies from gene coded protein were analyzed, other forty-nine had not been identified in patient 2. Thirty-nine antibodies were detected in both patients. The proteins YPMT1.81c, YPMT1.84, YPCD1.31c, rw10, YPCD1.28, YPCD1.58, YPMT1.62c, YPO3247-related antibodies increased significantly by 109.96,176.4 ;20.64,17.73 ;16.50,7.16 ;23.51,7.65 ;46.00,25.61 ;4.50,8.24 ;5.98,5.08 ;23.98,4.76 folds, respectively. Conclusions The study on the antibody in pneumonic plague patients helps us to select the potential vaccine candidates, which reveals that eight proteins are the immunity diagnosis targets and the research key of sub-unit vaccine.

Serum epidemiological investigation of F1 antibody of population having healed from plague in Yunnan Province / 中国地方病学杂志

Objective To provide theoretical and scientific evidences for plague control,through understanding the F1 antibody level distribution and affected factor of population having healed from plague from plague natural focus of Rat.flavipectus in Yunnan Province.Methods The places and population investigated were chosen according to plague surveillant data in Yunnan Province from 1986 to 2005,using caso-control study and quesfionary. All samples were detected by indirect hemagghtination(IHA),including 248 serum samples from population having heaIed from phsue in 23 counties as case group.295 senlm samples from healthy population inoculated with EV vaccine in 7 counties as artificial immunization group, and 235 serum samples from healthy population not inoculated it in a non-plagued foei county as negative comparison group,with the diagnosing standard for positive titor being not less than 1:20.Results(①The difference WSS statistically significant(X2=44.80,P<0.05)between plague and non-plagued foci with F1 antibody positive rates being 22.10%(120/543)and 0(0/235),respectively.② The F1 antibodv positive rate of case group,35.89%(89/248)and geometric mean titer(GMT)1:84,was higher than that of artiIicial immunization group,which was 10.51%(31/295)and with GMT 1:34,respectively,the difference being statistically significant(X2=50.41,P<0.0125);the positive rate of case group wgs hisher than the neganve comparison group,the difference being statistically significant(X2=103.39,P<0.0125):the posifive mte of artificial immunization group was higher than the negative comparison group,the difference being statisticallv significant(X2=26.23,P<0.0125).③The differences were not statistically significant in the F1 antibedy positive rates of case group for age,sex,nation and occupation(X2=1.88,2.01,5.46,0.04,P>0.05).④The difference was not statistically significant in 89 plague patients with positive F1 antibody at the time of onset and rehabilitation(t= 1.23,P>0.05).Conclusions ①Plague FI antibody in people distributes the sanle a8 the plague natural focus of Rat.flavipectus does in Yunnan Province.②For naturally infected plague patients,only 1/3 popuhtion get long- term immunity,and still 2/3 can be infected again.The protecting rate and effect of naturally acquired immunity due to infection of plague are better than amfieially acquired immunity from inoculation of EV vaccine.③For the population having healed from plague,the positive rotes of FI antibody are not affected by age,sex,nation and occupation,however for those whose plague F1 antibody is still positive after some time,the titer will remain or even increase.

Seroepidemiological survey of F1 antibody of indicator animals in wild rodents loci of plague in YunnanProvince / 中国地方病学杂志

Objective To study on the effect of indicator animals in plague surveillance throngh detecting F1 antibody against Yersiniapestis(Y.pestis)in indicator animals in wild rat plague foci,provide scientific evidence for plague control and determining the range of epidemic area.Methods According to investigation scheme of wild rodents plagne foci in Yunnan Province,indicator animals Canis familiarils and Felis catu(C.familiarils and F.catus)related to the plague were investigated in 75 villages,14 township and 10 counties around Yulong County,and living rodents were captured by cage,sera of indicator animals and rodents relevant to plague were simultaneously collected and detected for F1 antibody against Y.pestis using indirect hemagglutination(IHA).Results Seropositivity rate of indicator animals were 6.76%(202/2987),being 24.69% in C.familiaris and 24.69% in F.catus,there were statistical significance(X2＝87.32,P＜0.01)between C familiaris and F catus,the latter beingmore than the former.But F1 antibody of rodents sera were not detected,its seropositivity rate was zero.there was a statistical significance(P＜0.01)between indicator animals and rodents.Conclusions Through serocpidemiological survey of indicator animals,new wild rat plague natural focus has been confirmed in YuLong County and Gucheng District in LiJiang City,therefore,serocpidemiological surveillance of indicator animals is very important for plague control and prevention.