Objective To understand the mutations of Yersinia pestis, all of strains isolated in Tibet during 2009 to 2011 were selected to identify the biochemical features of the pathogen. Methods Traditional methods like glycerol fermentation, glucose metabolism and nitrate reduction were carried out. Tested strains isolated from Tibet during the year of 2009 and 2011, were incubated in biochemical action tubes made recently in 28℃ for 14 d, and the color of every tubes was observed and recorded each day. Results All of 111 tested strains were able to reduce nitrate and ferment glycerol, but unable to catabolize rhamnose; Nine of tested strains were capable of catabolizing arabinose, six of them isolated from Naqu and the other three from southern valley of the Qinghai-Tibet Plateau; The results of arabinose metabolism of the other 102 strains were all negative. Conclusion It was suggested that all tested strains belong to Biovar Antiqua. Those Y. pestis strains isolated from Naqu located in the northern Tibet were Qinghai-Tibet Plateau Ecotype, and most strains isolated from southern valley of the Qinghai-Tibet Plateau were Gangdise Mountain Ecotype except only three strains, which had the same biochemical features as the strains of Qinghai-Tibet Plateau Ecotype. It needs to utilize molecular methods through analyzing genetic markers to type the three special Y. pestis strains.

Objective To study the features of gene composition and distribution of three common plasmids among different Yersinia pestis strains. Methods The global structure of three plasmids, pMT1, pCD1, and pPCP1, in the nine Y. pestis strains with completed whole genome sequencing was comparatively analyzed by homologous alignment of coding sequences and automatic analysis using the Mauve software. Results The pMT1 of Y. pestis showed rearrangement of large gene fragments, and it was divided into four major gene blocks. The plasmid structures of pMT1 in Orientalis Biovar strain CO92, Medievalis Biovar strain KIM, and Microtus Biovar strain 91001 were different from each other. The plasmid structures of pMT1 in all the other six Antiqua Biovar strains were almost identical, yet different from those of CO92, KIM, and 91001. Plasmid pCD1 was divided into three major gene blocks;however, the global structure of the plasmid was the same among all strains except a different insertion position of IS100. The order and orientation of all coding sequences in plasmid pPCP1 were consistent in all strains. Conclusion Plasmids pCD1 and pPCP1 have conservative gene structure among different Y. pestis strains. A certain degree of variation in the plasmid structure of pMT1 has occurred during the long-term evolution of Y. pestis.

Objective To develop a simple genotyping method for rapid differentiation of Yersinia pestis strains between Microtus and non-Microtus biotypes. Methods Specific primers were designed and applied to amplify the aspartase gene (aspA) through polymerase chain reaction (PCR) among 93 Y. pestis strains of diverse origins and biotypes. The PCR products were genotyped by restriction fragment length polymorphism (RFLP) analysis using the restriction enzyme Hpy CH4Ⅳ. Genetic polymorphisms of aspA were determined by direct DNA sequencing. Results Among the 93 test strains, the amplified products of aspA digested with Hpy CH4Ⅳ showed two genotypes: Microtus biotype strains (38) all had two RFLP bands in sizes of 101 and 126 bp, respectively; and non-Microtus biotype strains (58) consistently had one single RFLP band in size of 227 bp. Direct sequencing data confirmed that mutation at the recognition site for restriction enzyme led to differences in the RFLP banding patterns. Conclusion A new genotyping method (aspA-PCR-RFLP) was established for rapid differentiation of Y. pestis strains between Microtus biotype with low virulence and non-Microtus biotype with high virulence according to different RFLP banding patterns. The proposed method can be used for epidemiologic surveillance in plague foci.

Objective To develop a simple genotyping method for rapid differentiation of Yersinia pestis strains between Microtus and non-Microtus biotypes. Methods Specific primers were designed and applied to amplify the aspartase gene (aspA) through polymerase chain reaction (PCR) among 93 Y. pestis strains of diverse origins and biotypes. The PCR products were genotyped by restriction fragment length polymorphism (RFLP) analysis using the restriction enzyme Hpy CH4Ⅳ. Genetic polymorphisms of aspA were determined by direct DNA sequencing. Results Among the 93 test strains, the amplified products of aspA digested with Hpy CH4Ⅳ showed two genotypes: Microtus biotype strains (38) all had two RFLP bands in sizes of 101 and 126 bp, respectively; and non-Microtus biotype strains (58) consistently had one single RFLP band in size of 227 bp. Direct sequencing data confirmed that mutation at the recognition site for restriction enzyme led to differences in the RFLP banding patterns. Conclusion A new genotyping method (aspA-PCR-RFLP) was established for rapid differentiation of Y. pestis strains between Microtus biotype with low virulence and non-Microtus biotype with high virulence according to different RFLP banding patterns. The proposed method can be used for epidemiologic surveillance in plague foci.

Objective To detect pesticin (Pst) antibody in the sera of plague host animals and to investigate the feasibility of using Pst as the diagnostic reagent in plague antibody detection. Methods A total of 351 serum samples of plague host animals from different sources were subjected to indirect enzyme?linked immunosorbent assay (ELISA) for detection of recombinant Pst antibody and F1 antibody. Results Pst antibody was found in the serum samples of plague host animals, and the serum level of Pst antibody increased significantly as the serum level of F1 antibody rose. The ELISA absorbance value was 0.438 in plague patients 20 months later, and the Pst antibody in serum remained at a detectable level. Conclusion Pst antibody detection can be used along with F1 antibody detection, so as to make plague antibody detection more reliable.

Yersinia pestis bacteriophage, a strongly specific bacterial virus, is commonly used for identification of the plague pathogen. Early research on this bacteriophage has focused mainly on the natural isolation, biological characteristics, and diagnostic or therapeutic applications. In recent years, advances in microbial genomics and proteomics have shed light on the role of this bacteriophage in the evolution of Y. pestis. This paper summarizes recent progress in Y. pestis bacteriophage research regarding genome structure and related pathogenicity and immunology, for the development of new molecular-based diagnostic modalities and medicines.

Single nucleotide polymorphisms (SNPs) mainly refer to the polymorphism of DNA sequence caused by a single nucleotide mutation, including the synonymous SNPs and non-synonymous SNPs. With the rapid development of sequencing technology, a large number of bacterial genome sequences are available. So, it’s possible to identify potential SNPs sites by sequencing technology and bioinformatics methods. Also, SNPs, because of their own characteristics, have been widely used as a new molecular marker in bacterial genotyping, evolution and epidemiology research. In this paper, advances in the research on the genome-wide search of SNPs sites and analysis of the Yersinia pestis microevolution based on SNPs data are reviewed.

Objective To study the subspecies of Francisella tularensis in China and the genetic relationships among the various strains of them. Methods Ten strains of F. tularensis from North China were subject to PCR using two specific primers C1C4 and RD1. Their subspecies were identified based on the length of the amplification products. At the same time, PCR on three specific genes was performed using fopA, tul4 and 16S rRNA primers, followed by sequencing. Based on the three specific genes, phylogenetic analysis was conducted using MEGA 4 software to involve the 10 strains of F. tularensis and the three strains of F. tularensis type B and one strain of subsp. novicida published on the NCBI website. Results The 10 strains of F. tularensis were identified as type B based on the PCR results using two specific primers C1C4 and RD1. According to the phylogenetic tree structured by MEGA 4, the 10 strains of F. tularensis from China can be classified into two types: B1 type, including 410108, 410109 and 410111, and B2 type including the other seven strains. In contrast, the three foreign strains were of type B3. Conclusion The F. tularensis isolated in North China may be predominated by type B. As for the origin of F. tularensis type B, the F. tularensis in China has probably emerged earlier than those in Europe and America. Phylogenetic analysis based on the three specific genes can be used as a reliable genotyping tool for F. tularensis.

The natural focus of Marmota himalayana plague on Qinghai-Tibet plateau is one of the most active plague foci in China. In particular, since the beginning of the 1990s, epizootic epidemics have continually emerged year after year in conjunction with regional outbreaks, occasionally affecting human beings. This paper systematically analyzes the geographical characteristics of the focus of M. himalayana plague on Qinghai-Tibet plateau, prevalence patterns of animal and human plagues, etiologic characteristics of plague and current status of plague surveillance, providing the basis for further development and adjustment of the prevention and control strategies.

Objective To evaluate the sensitivity of the four test methods, hemagglutination test, gold immunochromatographic assay (GICA),  enzyme-linked  immune  sorbent assay (ELISA)  and  polymerase  chain  reaction (PCR).  Methods The  minimal detectable dilution of antiserum of a bacteria?immunity rabbit via serological approaches for F1 antibody detection, the minimal detectable concentration in F1 antigen measurement, and the minimal concentration of template with a detectable gene pair, fra and pla, were evaluated. Results For the F1 antibody test, the minimal dilution detected was 1∶64 through IHA, 1∶1000 through GICA, and 1∶204 800 through ELISA. For F1 antigen test, the minimal detectable concentration was 2 ng/ml by RIHA and 50 ng/ml by GICA. The minimal concentration of template was 0.21 ng/μl when fra and pla were both detected. Conclusion In the serum consisting mainly of antibody IgG, the ELISA method has a higher sensitivity detecting F1 antibodies. The RIHA has higher sensitivity detecting F1 antigens. To minimize misdiagnosis, a minimal concentration of template of 0.21 ng/μl is required to diagnose plague when using PCR.

Objective To know about the genetic characteristics of a new kind of Yersinia pestis, isolated at Yulong, Yunnan province of China, and to analyze the relationship between these strains and the other types of Y.pestis of China. Methods The primers were designed according to six IS285 sites of DNA genome of CO92 strain. The differences between Yulong strains and other strains of Y.pestis were analyzed by PCR and cluster analysis. Results Compared to CO92 strain, Chinese strains had some variation at some IS285 sites. 4 of the 5 Yulong strains had the same profile of IS285, while the other one presented variation at one site. On the bases of PCR, the types of Chinese Y.pestis could be clustered as 8 groups. The 5 Yulong strains were divided into two groups. Four strains of them, plus Dianxi zonggu and Dianmin jumin, most of strains isolating from Marmota baibacina, and all of strains isolating from Marmota himalayana belongs to one group. Conclusion The genetic characteristic of Y.pestis from Yulong was known primarily, suggesting the importance of Yulong strains during the prevalence and evolution of Chinese Y.pestis strains.

Objective Study on the control ratios of rodents' parasitic flea and ground free flea after resort spraying on the ground in the focus of house mice plague.Methods After diluted the liquid with water(1∶160) 5% Fendona was resort sprayed on the indoor and the outdoor(80 ml/m ²) according to per house 100 g the powder,especially on the foot of a wall,the kennel,the holes and traces of rodents(150-180 ml/m ²).Through counted control ratios with the indices of rodents' parasitic flea and the ground free flea before the application and after the application.Results The control ratios of rodents parasitic flea and ground free flea are 33.54% and 71.39% respectively.Conclusion On-the-spot the control ratios of rodents' parasitic flea is very limited,but the rural sanitation is key to the question.

Objective To evaluate application of the F1 antigen(antibody) ELISA test Kit in plague surveillence.Methods The samples from plague surveillance were detected by the routine method,ELISA and gold immuno chromatographic assay(GICA),and statistical analysis their results.Results 1798 tissue samples of human and animals were detected with bacterial culture,ELISA and RIHA.ELISA detecting rate(7.01%) were significantly higher than that of bacterial culture method( P<0.01),and their coincidence were 98.23%.4401 serum samples of human and animals were detected with IHA,ELISA and GICA.ELISA detecting rate were no discrepancy with IHA( P>0.05),but the coincidence were low(61.04%).When ELISA and GICA were used together,the coincidence can be improved(91.56%).Conclusion The F1 antigen(antibody) ELISA test kit for the diagnosis of plague is suit for the early detection of plague particularly in plague surveillence.

Objective To apply a rapid and special method for the detection of Yersinia pestis by multiplex polymerase chain reaction(PCR).Methods The F1 antigen, pla gene, inv gene and a specific segment on the chromosome were co-amplified by PCR.An internal control(IC)was required in order to prevent false negative results that might be caused by PCR inhibitors.Other enteric pathogenic bacteria DNA were amplified as control.Results The amplicons of Yersinia pestis DNA were the same with the anticipative products.Only internal control amplicon was gotten while amplifying other enteric pathogenic bacteria DNA.Conclusion This method is specific and rapid and can identify Yersinia pestis.It is useful for detection of Yersinia pestis.

Objective To develop a polymerase chain reaction(PCR) method to detect Yersina pestis by multiplex primers(M-PCR).Methods Four pairs of primers,originated from the genes F1(specific capsular antigen fraction 1), pla(palsminogen activator), Hms and Inv encoded on the two kinds of 65×10 ⁶ plasmids and two chromosomal DNA,were designed and 164 strains ofc Y.pestis were amplified with multiplex primers.Results One hundred and fifty-two of 164 strains of(Y.pestis) showed positive in M-PCR.Only 12 strains of them isolated from Yunnan were negative with amplification of the Hms gene.Conclusion M-PCR method showed satisfactory sensitivity,specificity and stability for detecting and identifying Y.pestis DNA and could be used in surveillance and rapid diagnosis for plague.

Objective To establish and assess different methods for detection of Yersinia pestis from artificially contaminated soil and infected mice tissues by realtime fluorescence polymerase chain reaction. Methods Different methods for isolation and purification of nucleic acids from soil and mice tissues contaminated by Y.pestis vaccine strain were established. Three combinations of primers and probes designed according to caf1 and pla and hms genes of Y.pestis were used to assessed these methods. The optimal systems were confirmed by comparing the amplification results. Results The method of NaIGlassmilk is the most effective,and ten thousand of bacteria in one gram soil could be directly detected by this system. Conclusion The NaIGlassmilk method and the Realtime fluorescence polymerase chain reaction could directly and rapidly detect Y.pestis in contaminated issues or soil.

Objective:To develop a kind of method for detection of Bacillus Anthracis in the environment.Methods:Plasmid extraction and PCR amplification.Result:2 pairs of primers were used,and 2 different fragments were obtained from the positive samples.Conclusion:The PCR amplification can be as a method for detection of the Bacillus anthracis in the environment,moveover,this method can evaluate pathogenicity of the Bacillus anthracis.

Objective:Analyzing genetic characters of Yersinia pestis strains isolated from Shiqu County,Sichuan Province,China,and comparing with those of other types of Y.pestis in China.Methods:PCR of specific genes,Random Amplifying Polymorphism DNA,Pulse-field gel electrophoresis,and rRNA gene fingerprinting.Results:The results showed that Shiqu strains were typical Y.pestis strains with all the tested virulence and other specific genes.The strains from Microtus fuscus,however,had unique characters,significantly different from the strains isolated from other parts of China.Conclusion:The results suggested that a new type of plague natural focus existed in that area;The human outbreak of 1999 was infected outside of those natural foci;The focus should be put under close surveillance,and its risk to human beings has to be determined.

Objective:To determine the genetic relationships between different strains of Yersinia pestis.Method:Transfer the characters between different Y.pestis strains into standardized resemblity values and then perform cluster analysis.Results:The strains studied could be devided into two subspecies and four types.Conclusion:The resemble degree are high due to a same epidemic resulting types,the differences of types from foci are more outstanding.