OBJECTIVETo apply and evaluate new methods regarding specific gene and antigen detection in plague surveillance program.

METHODS1798 samples from natural foci of plague were tested, using internal quality control multiple-polymerase chain reaction, F1 antigen marked by immuno chromatographic assay and enzyme linked immunosorbent assay. Culture of Yersinia pestis and reverse indirect hemagglutination assay were used as reference diagnostic methods.

RESULTSThe overall positive rate of culture on Yersinia pestis together with gene and antigen detection was 7.34%, showing an 16.81% increase when comparing to 6.28% using Yersinia pestis culture method alone. The rate of coincidence was 97.13%.

CONCLUSIONThe new standard being used for specific gene and antigen detection could increase the positive rate of diagnosis on plague.

Animals ; Bacterial Proteins ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Mice ; Plague ; microbiology ; Polymerase Chain Reaction ; Yersinia pestis ; genetics ; immunology ; pathogenicity

OBJECTIVETo understand the distribution, fauna, population structure of host animals and their parasitic fleas as well as popular dynamic of animal plague of natural plague foci in Junggar Basin.

METHODSSample materials and data of animals and vector insects were collected using ecological methods and the population structures were analyzed statistically. F1 antibody of Yersinia pestis in rodents' serum and organ suspension was detected by means of IHA while the pathogen of Y. pestis in rodents and vector insects was detected by means of aetiological detections and the isolated Y. pestis was detected using biochemical methods.

RESULTSThe small mammals which were found in Junggar Basin belonged to 17 species of 11 genera 7 families. Of them, 13 species of rodents were included whose parasitic fleas belonged to 19 species of 10 genera 8 families. The average coverage of Rhombomys opimus hole-community was 22.5% in Junggar Basin with the average density of R. opimus hole-community was 15.9/hm2 and the average rate of habitat of the hole-community was 70.2%. In the R. opimus community, the average density of rodents was 3.1/hole-community, and 34.4/hm2 in the nature plague foci. In the population structure of the hole-community of R. opimus, R. opimus accounted for 72.9% in the total captured rodents, Meriones meridianus was 24.5% while the others were 2.6%. In the nocturnal community of rodents, M. meridianus accounted for 64.0% in total captured rodents, Dipus sagitta was 15.1%, M. erythrourns was 7.5% and the others were 13.4%. In the rodents community of Junggar Basin, the rate of R. opimus with fleas was 84.9%, which was the highest, followed by M. tamariscinus, Euchoreutes naso and M. erythrourns, with the rates as 71.4%, 66.7% and 62.7% respectively. The rate of M. meridianus with fleas was 38.3%. There were 16 species of parasitic fleas in R. opimus, with the total flea index as 8.58 and the dominant species was Xenopsylla skrjabini. There were 17 and 16 kinds of fleas in M. erythrourns and M. meridianus respectively with the total flea index were 1.59 and 1.15, with dominant fleas were Nosopsyllus laeviceps and X. skrjabini. The serum and organ suspension of 3179 rodents which belonged to 12 species were detected by means of IHA, of them 174 samples were positive and the positive rate was 5.5%. There were 1356 samples of R. opimus in these materials, and 164 were positive, accounted for 12.1%. The samples of M. meridianus were 1255, with 9 positive, accounted for 0.7%. The samples of D. sagitta were 116 with 1 positive and the rate was 0.9%. The samples of other rodents were 452 but were all negative. There were in total 2975 organs collected from rodents, when detected by methods of isolated of Y. pestis. 15 strains of Y. pestis were isolated from 1243 R. opimus, and 2 strains isolated from 1230 M. meridianus. A total number of 11 647 fleas from rodents were detected by methods of isolated of Y. pestis in which 1 strain of Y. pestis was isolated from 4713 X. skrjabini, and 6 were isolated from 2101 Xenopsylla minax, 1 from 328 Xenopsylla conformis conformis and 1 from 250 Echidnophaga oschanini. Among the other 4255 fleas, none was isolated. The biochemical properties of these Y. pestis which isolated from Junggar Basin were positive of Maltose, Ejiao sugar and Glycerol, and negative of Rhamnose and Nitrogen, which were all strongly poisonous to mouse.

CONCLUSIONThe natural plague foci in Junggar Basin spread all over the whole Junggar Basin. There were animal plague cases found in 12 counties (cites) while Karamy, Bole, Jimusaer and Qitai were confirmed as plague foci counties (cities). Animals and vector insects of the foci were complicated but the ecological system was stable. R. opimus was recognized as the dominant host animal and its biochemical type belonged to the Middle Ages, suggesting that the foci was a new type of natural plague foci.

Animals ; China ; epidemiology ; Gerbillinae ; microbiology ; Mice ; Plague ; epidemiology ; microbiology ; Rodent Diseases ; epidemiology ; microbiology ; Yersinia pestis ; immunology ; pathogenicity

OBJECTIVETo investigate the functional relations between the putative proteins YpCD1.08, YpCD1.09, YpCD1.16 encoded in pCD1 plasmid of Yersinia pestis and its type III secretion system (T3SS).

METHODSMutants of YpCD1.08, YpCD1.09, YpCD1.16 were constructed using λ-Red recombinant system. The growth curves of the mutant strains cultivated in TMH medium with or without calcium at 26 °C and 37 °C were determined to analyze the low calcium response phenotype. The transcription levels of ΔYpCD1.08, ΔYpCD1.09, ΔYpCD1.16 in Yersinia pestis and the dependence to temperature were determined using real time RT-PCR after cultivation at 26 °C and 37 °C and extraction of RNA. A β-lactamases reporter system was adopted to study the influence of these genes on the translocation of effector YopE of T3SS.

RESULTSWhen grown in TMH medium without calcium at 26 °C and 37 °C, the growth curve of the YpCD1.08, YpCD1.09, YpCD1.16 mutants were similar to that of the wild-type strain, indicating that the low calcium response of all the mutants were normal. The ratios of YpCD1.08, YpCD1.09, YpCD1.16 gene transcriptional level at 37 °C and 26 °C were 2.3 ± 0.3, 2.3 ± 0.5 and 3.2 ± 0.7, respectively, indicating that these genes were transcribed in Yersinia pestis and their transcription regulations showed a temperature-dependence that was consistent with the well established temperature-dependent expression of Yersinia T3SS genes. The β-lactamases reporter assays demonstrated that ΔYpCD1.08 could translocate much higher level of YopE into HeLa cells, since that the light intensity ratio of 477/520 nm at 140 min was 2.5, whereas it was 1.8 for the wild-type strain, and the values in ΔYpCD1.09 and ΔYpCD1.16 were similar to the wild-type strain.

CONCLUSIONYpCD1.08, YpCD1.09, YpCD1.16 gene are likely to be the new members of T3SS, and the putative protein YpCD1.08 could play some roles in YopE secretion and translocation.

Bacterial Outer Membrane Proteins ; secretion ; Bacterial Secretion Systems ; genetics ; Genes, Bacterial ; Plasmids ; Protein Interaction Mapping ; Yersinia pestis ; genetics ; metabolism ; pathogenicity

Objective: To understand the genotype of the Yersinia (Y.) pestis strains isolated from Heqing county, Yunnan province in 2017 and provide evidence for the prevention and control of plague in this area. Methods: Ten Y. pestis strains isolated from Heqing were typed by the detections of different region (DFR) and clustered regularly interspaced short palindromic repeats (CRISPRs) as well as multiple-locus variable-number tandem repeat analysis (MLVA). And the results were compared with those of the 93 Y. pestis strains from the adjacent plague foci of Heqing obtained from the established database for clustering analysis. Results: The results showed that Heqing strains had the same type of DFR (Genomovar 05) and CRISPRs (Cluster Ca7, Type 22) with isolates from the plague focus in Lijiang. Heqing strains and Lijiang strains were in the same cluster in MST and only VNTR loci N2117 and M23 of Heqing strains were different from that of Lijiang strains. Conclusion: The Y. pestis strains isolated from Heqing in 2017 were highly homogenous with the strains isolated from wild rodents in plague focus in Lijiang, and Heqing plague might be the result of further southward spread of Lijiang plague.
Animals ; China/epidemiology* ; Epidemiological Monitoring ; Genotype ; Minisatellite Repeats ; Molecular Typing ; Plague/microbiology* ; Rodentia/microbiology* ; Yersinia pestis/pathogenicity*

OBJECTIVETo study the pathogenic ecology characteristics of plague in Qinghai plateau.

METHODSApplied 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.

RESULTSThe 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.

CONCLUSIONThe 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.

Animals ; Arvicolinae ; microbiology ; China ; epidemiology ; Disease Reservoirs ; microbiology ; Ecology ; Genotype ; Marmota ; microbiology ; Plague ; epidemiology ; microbiology ; Virulence ; genetics ; Yersinia pestis ; genetics ; pathogenicity

Animals ; Bacterial Proteins ; genetics ; metabolism ; Biofilms ; growth & development ; Gene Expression Regulation, Bacterial ; Lipopolysaccharides ; chemistry ; metabolism ; Operon ; Promoter Regions, Genetic ; Protein Binding ; Siphonaptera ; microbiology ; Species Specificity ; Transcription, Genetic ; Transferases ; genetics ; metabolism ; Virulence ; Yersinia pestis ; genetics ; metabolism ; pathogenicity ; Yersinia pseudotuberculosis ; genetics ; metabolism

OBJECTIVETo find out the differences between 102 kb pgm locus of Yersinia pestis isolated from Microtus brandti with of other types, and the characters of Yersinia pestis isolated from Microtus brandti caused by their makeup of the 102 kb pgm locus.

METHODS102 kb pgm locus of Yersinia pestis isolated from Microtus brandti and Yersinia pestis isolated from Marmota himalayana were amplified by polymerase chain reation (PCR) with 25 pair of nested primers. The PCR products of one pair of primer were obviously different, and then cloned and sequenced. Sequences were searched against current protein and nucleotide databases, using BLAST.

RESULTSThe 102 kb pgm locus of Yersinia pestis isolated from Microtus brandti was devoid one IS100. In addition, it had more copies than other types in the similar variable-number tandem repeat sequences.

CONCLUSIONThe 102 kb pgm locus of Yersinia pestis was different from that of other types. It had only one IS100 flanked it, which corresponded to the character that its pgm(+) phenotype was stable. Further study was needed to confirm the relationship between the diminution virulence of Yersinia pestis isolated from Microtus brandti and the loss of IS100 and other changes.

Animals ; Arvicolinae ; microbiology ; Bacterial Proteins ; genetics ; metabolism ; China ; epidemiology ; Cloning, Molecular ; DNA, Bacterial ; Humans ; Pigments, Biological ; genetics ; Plague ; epidemiology ; microbiology ; Polymerase Chain Reaction ; Repetitive Sequences, Nucleic Acid ; Sequence Analysis, DNA ; Virulence ; genetics ; Yersinia pestis ; genetics ; pathogenicity