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.

Objective To determine the optimal distance between ribosomal binding site (RBS) and translation initiation site (TIS) by statistical analysis of RBS-TIS distances in Yersinia pestis CO92. Methods All copies of 3′-terminal 16S rRNA and 177 annotated RBS sequences in CO92 were analyzed to identify the characteristics of Y. pestis RBS sequences. The positions and number of all RBS sequences in CO92 and their distances from the TIS ATG (GTG, TTG, CTG) were determined by statistical analysis. The characteristics of the 20 upstream bases in the annotated coding DNA sequences (CDSs) in CO92 were observed. Results A total of 5081 potential RBS sequences were found in CO92 genome. Of the 5081 RBS sequences, 2909 had downstream open reading frames (ORFs); of the 2909 RBS sequences, 1541 had the ORFs identical to the annotated CDSs, and 535 had the same termination sites as the annotated CDSs but had different initiation sites. In the 20 upstream bases of 3887 annotated CO92 CDSs, 57.7% contained RBS sequences. Conclusion The most frequent distances between RBS and TIS were 7 and 8 bases in CO92 genome. RBS can be an important gene index for identification.

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.

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.

【Abstract】 Objective To deeply know the role of the reporting of dead rodents in the monitering and control of commensal rodent plague and its cooperation with linking surveillance method. Methods  （1）To compare and analyze the function of dead rodents reporting and three plague?risk indicators reporting on the timely finding of the plague in rodents. （2）To analyze the relationship between dead rodents reporting and linking surveillance method and study the function of the latter. Results （1） The reporting of dead rodents contributes to, and other two plague?risk indicators reporting has no relation to the detection of commensal rodent plague among rodents.（2）The two HD techniques in monitoring and control system of commensal rodent plague are dead rodents reporting and linking surveillance method, of which, the former is mainly in charge of the detection of the plague among rodents in time, and the latter has two roles, namely both decreasing and founding the epidemic foci among rodents, especially in decreasing them. Conclusion The emphasis should be put on the popularization of 2HD technique in order to detect and control rodent plague in time in the monitoring and control of commensal rodent plague, and at the same time,  the reporting of three plague?risk indicator should be understood and treated correctly.

Objective To know deeply the incidence mechanism of commensal rodent plague and scientifically work out or choose appropriate surveillance and control measures.Methods The transmission route from rodents to humans of commensal rodent plague was expressed by mathematical language to show up the inherent relations among the decisive factors,and its relevant monitoring methods were analyzed and compared.Results(1)An incidence mode function of the plague was derived.(2) The linking surveillance method was more effective than the parallel surveillance method,and the former was more accordant with the epidemiology principles,the statistics requirements and the "benefit-cost ratio" requirement because of its smaller workload.Conclusion(1)The incidence mode function on commensal rodent plague disclosed the inherent relations among the decisive factors of the disease,and had the important guide function and wide application prospects to its prevention and control.(2)The linking surveillance method should be suited to the surveillance of plague compared to the parallel surveillance method.

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 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 Most known Bartonella.species are arthropod borne and cat fleas can carry and transmit this organism from cats to cats. The study intended to make clear whether some Bartonellaspecies,which are emerging pathogens,could be carried or transmitted by fleas in natural enviroments in China. Methods In this study,fleas were collected from domestic cats,dogs and rats hosts from June to July 2003 in Yunlong county,Yunnan province. PCR assay with genus. Bartonella specific primers were applied to Bartonelladetection in every group of fleas which were same species and came from one captured animal. Results Ctenocephalides felis,C. orientis, Pulex irritans, Xenopsylla cheopis, Leptopsylla segnis, Ctenophthalmus quadratus, Neopsylla specialis specialiscollected from the host animals were 251.One group of Ct. felis including 8 tested fleas were PCR positive with three pairs of primers for Bartonella species.Five Leptopsylla segnisfrom a Rattus tanezumi flavipectus were also amplified DNA fragments of Bartonella species. Conclusion These findings indicate that fleas associated with cats and rats can carry Bartonella species and may play an important role in this pathogen transmission among animals and humans in China.

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.