Objective To analyze the phenotype characteristics of Yersinia pestisMicrotus strain 90017 from Lasiopodomys brandtii, so as to provide a theoretical basis for investigating mechanisms of bacterial resistance to phages, the long-term preservation mechanisms of Y. pestis in plague foci, and evolutionary epidemiology of infectious diseases. Methods BALB/c mice were infected subcutaneously with 3 concentrations of Microtus strain of Y. pestis respectively including 2×10⁹,1×10⁶ and 1×10⁵ pieces /ml. Animals are dissected after death to isolated Y. pestis. It were observed on cultural characteristics and plague phage lytic test of Y. pestisMicrotus strain 90017. Result Y. pestis 90017 was cultured in a solid medium at 28°C for 24 hours before and after animal experiment, and it was found that the bacterial growth was slow. The bacterial colony showed typical morphology of Y. pestis after 48-100 hours of incubation. After a 24-hour bacteria lysed at 28°C in plague phage lysis test, the center of phage lysis zone demonstrated the appearance of broken glass and untypical debris of colonies. After 48-100 hours of incubation, in addition to the eroded residual colonies, some relatively complete colonies remained in the test zone where plague phage dripped and flowed through. The areas marked with the control Y. pestis strain 141 showed obvious bacterial lawn, and there was no bacterial growth in the areas where plague phage dripped and flowed through. The resulting phage lysis zone was wider than the trace left by the phage flow. The bacterium 90017 did not recover to its typical state after animal experiment, which was similar to its growth state and the result of plague phage lysis test for plague before animal experiment. Conclusion The coexistence of plague diagnostic bacteriophage-sensitive and -resistant strains in Microtus strain of Y. pestis 90017 from L. brandtii reflects biological complexity of microbial community in natural environment, which may play an important role in maintaining the epidemic and infection process of pestis.

Objective To investigate the characteristics of Yersinia pestis population structure in natural plague foci in the Sanjiangyuan region of Qinghai Province using genomic single nucleotide polymorphisms (SNPs) analysis, so as to provide a theoretical basis for plague prevention and control in this region. Methods A total of 81 representative strains of Y. pestis collected in the Sanjiangyuan region were selected for analysis of variations across 2 298 SNP sites based on whole genome sequencing data. MEGA 6.0 software was used to construct a phylogenetic tree. The structure and distribution characteristics of Y. pestis population in this region were analyzed. Results The 81 strains of were classified into three SNP genotypes, designated as 1.IN, 2.ANT, and 0.PE. The strains in the 1.IN subpopulation were dominant in the natural plague foci of the Sanjiangyuan region, and all of them were isolated from the natural plague focus of Marmotahimalayana. The strains in the 2.ANT and 0.PE subpopulations were distributed across Chengduo County, Nangqian County, and Qumalai County in Yushu Tibetan Autonomous Prefecture. The strains of Y. pestis isolated from the natural plague focus of Neodon fuscus in Qinghai belonged exclusively to the 0.PE4C subpopulation. Conclusion Genomic SNP analysis of Y. pestis subpopulations showed obvious regional distribution characteristics in the Sanjiangyuan region, which provides scientific tips for the epidemiological investigation of plague epidemic in this region.

Objective To investigate the genotype of Yersinia pestis isolated from the natural plague focus of Meriones unguiculatus in Inner Mongolian Plateau using clustered regularly interspaced short palindromic repeats (CRISPR) and the difference in the genotype of strains from the same type of epidemic focus and different areas, to establish the CRISPR gene bank of plague strains from gerbil plague foci, and to lay a foundation for epidemiological trace-back and analysis of epidemic situation. Methods Three pairs of CRISPR primers (YPa, YPb, and YPc) were used for PCR amplification and sequencing of the DNA of experimental strains, and the CRISPR sequence obtained was compared with the latest CRISPR Dictionary reported in literature to obtain CRISPR spacer array and identify genotype. Bionumerics 7.6 software was used to plot clustering charts and analyze the phylogenetic relationship. Results A total of 9 spacers were found in 33 strains of Y. pestis, i.e., 4 types of YPa (a1, a2, a3, and a56), 2 types of YPb (b1 and b2), and 3 types of YPc (c1, c2, and c3). The comparative analysis showed that all strains were classified as 1 CRISPR gene cluster (Cb2) with two genotypes. The strains isolated from Etuoke banner and Hanggin Rear banner of Inner Mongolia were identified as a new genotype, which was named as genotype 2' (a1-a2-a3-a56, b1-b2, c1-c2-c3), and the strains from Urad Front banner of Inner Mongolia, Kangbao county of Hebei province, and Yinchuan city of Ningxia Hui autonomous region were identified as genotype 1 (a1-a2-a3, b1-b2, c1-c2-c3). Conclusion Y. pestis isolated from the plague foci of M. unguiculatus in Inner Mongolian is genetically stable as a whole and is classified as one gene cluster, but a certain degree of microevolution is observed, which reflects that strains from different areas have different genotypes.

Objective To determine the minimum inhibitory concentration (MIC) of 11 antibiotics against 46 strains of Yersinia pestis from the natural plague foci in Gansu province, China and master the sensitivity of these strains to the 11 antibiotics in this region, and to provide a scientific basis for the effective prevention and control of plague. Methods According to American Association of Clinical and Laboratory Standards (CLSI)(2017-M100), the agar dilution method was used to determine the MICs of ofloxacin, ciprofloxacin, sulfamethoxazole/trimethoprim, kanamycin sulfate, streptomycin sulfate, ceftriaxone sodium, ampicillin, chloramphenicol, spectinomycin hydrochloride, cefuroxime sodium, and tetracycline hydrochloride against 46 strains of Y. pestis. Results None of the 46 strains of Y. pestis tested were found to have resistance to one or more of the 11 antibiotics. Among the 11 antibiotics, ceftriaxone sodium had the highest in vitro antibacterial activity (MIC90, 0.03 μg/ml), followed by ciprofloxacin (MIC90, 0.06 μg/ml), sulfamethoxazole/trimethoprim (MIC90, 0.12 μg/ml), ofloxacin (MIC90, 0.25 μg/ml), ampicillin (MIC90, 0.50 μg/ml), cefuroxime sodium (MIC90, 1.00 μg/ml), kanamycin sulfate, streptomycin sulfate, tetracycline hydrochloride, and chloramphenicol (MIC90, 4.00 μg/ml), and spectinomycin hydrochloride had the lowest antibacterial activity (MIC90, 16.00 μg/ml). Conclusion The 46 strains of Y. pestis from the natural plague foci in Gansu province are all sensitive to the 11 antibiotics. Monitoring of drug-resistant strains should be continued in order to detect drug-resistant Y. pestis in the early stage, choose antibiotics rationally, and develop the individualized treatment regimen.

Objective To provide a scientific basis for plague prevention and control in Guoluo Tibetan autonomous prefecture, Qinghai province, China (hereinafter referred to as Guoluo prefecture), by etiological analysis and determination of the antibiotic resistance-related genes of Yersinia pestis in that area. Methods A series of experiments including sugar alcohol fermentation test, toxicity test, virulence factor assay, plasmid analysis, and different region (DFR) genotyping were conducted on 13 Y. pestis strains isolated from Guoluo prefecture from 1978 to 2016. According to the gene sequences (aminoglycosides streptomycin-resistant strA and strB; beta-lactam antibiotics-resistant tem and ctx-m; and sulfonamides-resistant sul1 and sul2) published by the National Center for Biotechnology Information (U.S.), a pair of primers were designed for each gene. Polymerase chain reaction (PCR) amplification of the above 6 antibiotic resistance-related genes was carried out using the DNAs from the above 13 Y. pestis strains as templates, to detect whether they carried antibiotic resistance-related genes. Results According to the biochemical grouping, of the 13 test strains, 9 belonged to Qing-Tibet Plateau ecotype, and 4 belonged to Qilian Mountain ecotype. As shown by the results of the toxicity test, all the 13 strains were velogenic strains and 11 contained four Y. pestis-specific virulence factors. The test strains contained 2 types of plasmids with a molecular weight of 6×10⁶, 45×10⁶, and 65×10⁶ versus 6×10⁶, 45×10⁶, and 52×10⁶, respectively. The DFRs were divided into 4 genomovars, i.e., genomovar 5 (7 strains), genomovar 8 (4 strains), genomovar 36 (1 strain), and genomovar 01b (1 strain). In case of valid positive and negative controls, PCR assay of the antibiotic-resistant genes revealed a negative result for all the above 13 Y. pestis strains, and no strain was found to be resistant to streptomycin, beta-lactam antibiotics, or sulfonamides. Conclusion The strains isolated from Guoluo prefecture show the characteristics of Y. pestis from Qinghai-Tibet Plateau. Despite the fact that no antibiotic-resistant strains were found, which demonstrate high toxicity; therefore, it is necessary to enhance plague surveillance and health promotion to prevent plague spreading from animals to humans.

Objective To investigate the etiological characteristics and epidemiological significance of Yersinia pestis strains in Haibei Tibetan Autonomous Prefecture, Qinghai province, and to provide a scientific basis for the prevention and control of plague in this area. Methods Carbohydrate fermentation test, virulence factor detection, virulence determination, plasmid analysis, and different region (DFR) analysis were performed on a total of 104 Y. pestis strains isolated from Qilian, Menyuan, Haiyan, and Gangcha in Haibei Tibetan Autonomous Prefecture from 1956 to 2011. Results According to the biochemical typing, 73 of the 104 strains tested were classified as the Qinghai-Tibet Plateau ecotype, 25 as the Qilian Mountain ecotype, and 6 as the ecotypes different from those in the natural plague foci of Qinghai province. Plasmids with molecular weights of 6×10⁶, 45×10⁶, and 52×10⁶ were found in all of the strains. Seventy-seven (74.04%) of the 104 strains contained four virulence factors, and 98.51% (66/67) were velogenic strains. According to the DFR analysis, six genomovars were found:genomovar 8 (75 strains), genomovar 44 (20 strains), genomovar 5 (6 strains), genomovar 6 (1 strain), genomovar 7 (1 strain), and genomovar 11 (1 strain). Conclusion The Y. pestis strains isolated in Haibei Tibetan Autonomous Prefecture, Qinghai province have the specific characteristics of Y. pestis strains in Qinghai-Tibet plateau, with complex ecotypes and strong virulence. Monitoring, prevention, and control of plague, as well as dissemination of relative knowledge, should be strengthened in this area.

Objective To analyze the genotypes and genetic characteristics of Yersinia pestis isolated from Hebei plague foci. Methods The DNA of Y. pestis collected in Kangbao county from 1972 to 2017 was extracted by boiling method, the multiple locus variable number tandem repeat (MLVA) and the DFR primers according to a publication were synthesized by a biological company, then amplified by polymerase chain reaction, then genotypes were analyzed by agarose gel electrophoresis. Results All of the strains showed one MLVA genotype, and the main genotypes of Y. pestis from Hebei foci were G17 and G20. The absence of DFRs were 1, 6, 7, 13, 15, 16, 17, 18 in G17, the absence of DFRs were 1, 6, 7, 12, 13, 15, 16, 17, 18 in G20. Conclusion The genetic characteristics of enzootic plague in Hebei province remain stable. Further studies are warranted to confirm whether the epidemics among wildlife in 2017 was caused by spread of neighboring foci or by genetic shifting of the pathogen.

Objective To understand whether there is a drug-or disinfectant-resistant strains in natural plague foci in Tibet, and provide the accurate information for clinical treatment of plague. Methods According to the aminoglycoside resistant gene of streptomycin resistant, StrB, StrA, beta lactam antibiotics TEM, SHV, and CTX-M gene, sulfamilamide resistant Sul1, Sul2, and Sul3, and anti-disinfectant QacEdeltal-sul1 gene sequence the National Center for Biotechnology Information (NCBI) released,a pair of primers in each gene was designed separately. DNA of strains isolated from natural plague foci in Tibet were amplified by PCR using every pair of primers. Results Negative and positive control were established, samples by PCR amplification results were negative, there were no streptomycin, sulfamilamide and beta lactam antimicrobial drug resistance genes and anti-disinfectant genes in strains studied. Conclusion The Tibet autonomous region of natural plague foci did not appear to have drug-or disinfectant-resistant Yersina pestis.

Objective To study the genotype of Yersinia pestis in Hebei plague foci by variable number of tandem repeat (VNTR). Methods Primers were designed according to the confirmed 14＋12 VNTR, to genotype the 116 Y. pestis DNA of Hebei province. Results All of the strains showed one genetype, but they were different from CO92 and EV. Conclusion There is only one genetype of plague, indicating a genetic stability in Y. pestis in Hebei province.

The plague is categorized as a Class A infectious disease in China. Plague foci are widely distributed, and strains have different features and varied virulence in China. It is of great significance for deducing the sudden outbreak of plague and terrorist attack detection, with the strains of different foci for genotyping. The choice of tandem repeats loci is the key of the multiple loci vntr analysis (MLVA) classification results. This assay mainly introduces tandem repeats in Yersinia pestis genotyping in application progress, and provides a reference for other workers in genotyping.

Objective To update the information on the species of ectoparasitic fleas on small mammals in Gonghe county, Qinghai province, China. Methods Small mammals were captured by night trapping method, and ectoparasitic fleas were collected from them. The slide specimens of fleas were made, and these fleas were classified and identified based on morphological characteristics. Results A total of 785 ectoparasitic fleas (13 species, 8 genera, 3 families) were collected from small mammals in Gonghe county. Conclusion Neopsylla bidentatiformis (Wagner, 1883) is a new record in Qinghai province.

　　【Abstract】　Objective　To study  the  practicability  of  double  antigens  sandwich  enzyme  linked  immunosorbent  assay（DAgS?ELISA）on the detection of Yersinia pestis F1 antibodies. Methods　A total of 558 samples antibodies of anti?F1 antigen were detected by DAgS?ELISA and trace indirect hemagglutination assay (trace?IHA). Results　Thirty three samples were positive tested by IHA, 31 positive by DAgS?ELISA, the positive accordance rate was 90.91%, 99.81% for negative accordance rate, 99.28% for the total accordance rate. The positive rate detected by IHA and DAgS?ELISA were 5.91% and 5.56% respectively, and no statistics difference was found (χ2＝0.25，P＝0.625). About 27 the immuno?serum were positive detected by IHA and DAgS?ELISA methods, and the sensitivity of IHA test were all higher than that of DAgS?ELISA (t＝3.023, P＝0.006). Conclusion　Sensitivity of DAgS?ELISA is lower than that of trace?IHA, but its specificity is better and no primary inhibitory phenomena, and could exempt from leak detection in the preliminary screening.

Objective To study the sensitivity of colloidal gold immunobinding and analyze the distribution of crowd bubonic plague F1 immune body in Hebei province.Methods 196 serums and 162 serums of healthy people were sampled from history areas affected by plague and plague foci among animals(Zhangjiakou city) in Hebei natural plague foci and fur processing workers in Baoding.100 serums were collected from non-plague foci as control.Colloedal gold immunobinding and indirect hemagglutination test(IHA) were used to detect,at the same time,the minimum limit of detection of these two methods was compared.Results There were 3 positive serums in 196 serum samples,and the positive rate was 1.53%.None positive samples was found in 162 serum samples.The control were all negative.Conclusion The minimum detection of colloedal gold immunobinding was less than that of IHA.There were positive serum samples in plague natural foci,and the people age whose serum presented positive exceeded 40 years old.The samples collected from Baoding fur processing workers and non-plague foci were all negative.2 samples were positive after vaccination by investigation,and others needed be further explored.

Objective To study the three-dimensional distribution and maintenance mechanisms of plague F1 antibody of healthy population in the plague natural foci of Hebei province. Methods To sample 196 blood samples from three administrative villages belonging to plague natural foci formerly, and collect 100 blood samples from non-infected areas as the control. Indirect hemagglutination test(IHA) and colloedal gold immunobinding were used in this test. Results There were 4 positive among 196 blood samples, and the positive rate was 2.04%. There were 4 positive samples in IHA and 3 positive samples in colloedal gold immunobinding. The human serums collected from non-infected areas were all negative after tested by theses two methods. There was significant difference between the positive rate of F1 antibody in epidemic areas and the positive rate of F1 antibody in non-epidemic areas (χ₂=0.822,P<0.05). Conclusion At present, there are still a number of F1 antibody positive population in the natural epidemic focus of Hebei, and it needs further investigation and analysis to clarify.

Objective:Investigate the epidemic of leptospirosis in north-west of Hubei province in 1990'S.Methods:The serum of leptospirosis cases were measured using MAT and Dot-ELISA methods,health population immune level with MAT method.The investigation of the source adopt mouse-clip in the evening,cultivatet rat and pig kidney cortex,separate leptospiroal and identify leptospiroal serum groups.Results:The cases of leptospirosis were few 1990s ago and later the continued outbreaks of leptospirosis were in Zhuxi,Xiangyang and Yicheng and so on in north-west of the Hubei province.All patients(964) accounted for 88.93% in cases of 40 years.The time of outbreak mostly centred on September to October,which prolonged for about one month than that in brief precent areas of all Hubei province.The patient's clinical categories were grippotyphosa type and lung bleed type,which accounted for 70.47% and 21.12% respectively in all patients.The density of mice was 3.62% and mice carrier rate 14.37%.The positive rate of antibody was 41.50% in healthy groups.Conclusions:The leptospiral groups among leptospiral patients were mainly icterohaemorrhagiae.They carried the same groups of source of infection such as pigs and mice.