Objective To analyze the epizootic trend of animal plague in the natural plague foci of Hebei province, China, and to provide evidence for formulating the strategies for the prevention and control of plague in Hebei province. Methods The monitoring data of animal plague in the natural plague foci of Hebei province from 1950 to 2019 were analyzed using Excel 2007 and SPSS 19.0 softwares. The number of Yersinia pestis positive animals was described as constituent ratio. The distribution of different groups was compared by the χ² test. The seasonal distribution of animal plague was analyzed by concentration degree. Results The median interval of plague epizootics in the natural plague foci of Hebei province was 9.5 years. The epizootics had certain seasonality (M=0.387), with a high frequency during October to November and then during April to May. Kangbao pasture (constituent ratio 73.13%) and Zhaoyanghe town (constituent ratio 26.12%) were the main epizootic areas. Meriones unguiculatus (constituent ratio 87.31%) was the main animal involved in the epizootics. The main vectors were Nosopsyllus laeviceps kuzenkovi (constituent ratio 1.49%) and Citellophilus tesquorum mongolicus (constituent ratio 1.49%). Among positive rodents, 92.25% were naturally dead rodents. The number of Y. pestis strains isolated from M. unguiculatus was significantly different between five rounds of plague epizootics (χ²=20.026, P<0.001). The average density of M. unguiculatus was 1.98/hm², the average flea index of body fleas was 1.12, and the average flea index of nest fleas was 3.19. Conclusion Plague epizootics in the natural plague foci of Hebei province show intermittent and relatively stable characteristics. In order to reduce the risk of animal plague, close attention should be paid to the density and parasitic flea index of M. unguiculatus, and measures should be taken timely to kill rodents and fleas in spring and autumn. At the same time, the searching for naturally dead rodents in historical epizootic areas should be strengthened to discover plague epizootics in time and prevent the spread of plague.

Objective To investigate the relationship between rodent fleas and meteorological factors in Meriones unguiculatus plague foci in Hebei province, China. Methods The monitoring data of rodent fleas from 2001 to 2013 and the meteorological data of precipitation, temperature, and humidity during the same period in M. unguiculatus plague foci in Hebei province were collected. According to the interquartile range, the data of precipitation, mean temperature, and mean relative humidity were all divided into four groups, and each four groups were compared in terms of flea infestation rate and flea index. A Spearman rank correlation analysis was used to analyze the correlation between these meteorological factors and flea infestation rate and flea index. Results There were significant differences in the distribution of flea infestation rate between different precipitation and mean temperature groups (H=11.031 and 20.212, both P<0.05). When the precipitation was >60.40 mm and the mean temperature was >18.20℃, the flea infestation rate reached the peak level. There were significant differences in the distribution of flea index between different precipitation, mean temperature, and mean relative humidity groups (H=8.044, 9.254, and 9.082, all P<0.05). When the precipitation was >60.40 mm, the mean temperature was 13.30-18.20℃, and the mean relative humidity was >64.00%, the flea index reached the peak level. The results of correlation analysis showed that flea infestation rate was positively correlated with precipitation and mean temperature (r=0.396 and 0.547, both P<0.05), while flea index was positively correlated with mean temperature (r=0.376, P<0.05). Conclusion Precipitation, mean temperature, and mean relative humidity are the important meteorological factors affecting the flea infestation rate and flea index in M. unguiculatus plague foci in Hebei province. Targeted flea control should be carried out according to the meteorological conditions.

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 master the plague epidemics among animals in Kangbao county, to provide a scientific basis for accurate prediction of plague epidemic risk, and to put forward measures for plague prevention and control. Methods Based on the results of plague surveillance and epidemics among animals in Kangbao county for years, a descriptive epidemiological method was used to analyze the plague surveillance data in Kangbao county from 2014 to 2018. Results From 2014 to 2018, the annual mean densities of Meriones unguiculatus and Citellus dauricus in Kangbao county were 0.14-0.93 inds/hm² and 0.22-0.69 inds/hm², respectively, which showed a decreasing trend in general year by year. The major dominant species of small rodents was Cricetulus barabensis. The mean rate of M. unguiculatus with flea was 22.56%-46.26%; the mean flea index was 0.57-1.25, and the main parasitic flea was Nosopsyllus laeviceps kuzenkovi. The mean rate of C. dauricus with flea was 40.56%-88.46%; the mean flea index was 2.42-6.73, and the main parasitic flea was Citellophilus tesquorum mongolicus. Serological tests showed three positive materials; six strains of Yersinia pestis were isolated. Conclusion Kangbao county has a complex situation of plague surveillance among animals. Even in periods of low density of wild rodents, it is still necessary to continue to strengthen the plague surveillance, continuously do a good job in deratization, and regularly carry out plague risk assessment, so as to prevent the occurrence of plague epidemic in the area.

Objective To investigate the genotypes and genetic characteristics of Yersinia pestis strains isolated from Kangbao county in Hebei province, China in 2017. Methods The DNA of three Y. pestis strains isolated from Kangbao county in Hebei province in 2017 was extracted by boiling method and kit extraction method. According to the literature, the primers of 22 different regions (DFRs) and pMT1 were synthesized and the DFR sites were amplified by PCR. The genotype was analyzed after agarose gel electrophoresis. Results The DFRs in the three strains were DFR02-05, DFR08-11, DFR14, DFR19-22, and pMT1, but not DFR01, 06, 07, 12, 13, and 15-18. The genotype was G20 after alignment. Conclusion The genotype of Y. pestis isolated from the plague foci in Hebei province in 2017 is G20 and different from G17 isolated previously. The emergence of new genotype provides a basis for further study of the genetic characteristics and epidemiological characteristics of the strain.

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 identify Yersinia pestis from the Mongolian gerbils and EV76 bacteria of plague by different region (DFR) fragment genotyping. Methods The DNA of 6 strains of Y. pestis and EV76 isolates was extracted by boiling method from Hangjinqi of Inner Mongolia in 2015, Yinchuan city of Ningxia Hui Autonomous Region in 2013, and Kangbao county of Hebei province in 2003. The DFR primers according to a report were synthesized by biological company, then amplified by PCR, analyzed genotype through agarose gel electrophoresis. Results The DFR genotypes of Y. pestis from Mongolian gerbils were G11, G17, and G20. The absent points of G11 were DFR01, 06, 07, 13, 15, 16, and 17; DFR18 was also absent for G17 in addition to the above; The absent point of G20 also included DFR12 in addition to G17. The absence DFRs of EV76 were 01, 02, 04, and 10. Conclusion Using the differential fragment genotyping method can quickly distinguish Y. pestis isolates from Mongolian gerbils and EV76. At the same time, it is possible to trace the source of the epidemic rapidly by identifying the genotype of wild strains.

Objective To explore the epidemic factors of animal plague in Hebei province, China, and provide scientific basis for the prevention and control of plague. Methods The materials were collected and rodents were tested by "four step test" of plague, and the fleas were tested by group method or single stomach pulling method. Results A total of 240 358 rodents were tested, and 123 positive plague materials were found. Five of 14 856 vectors tested positive for Yersinia pestis. There were 7 animal plague prevalent years in Hebei province. Conclusion The prevalence of animal plague epidemic occurred intermittently in plague natural foci of Hebei province, the main change of hosts was a primary cause of animal plague, especially the gerbil density in animal plague epidemic years.

At the initial stage of epidemics, the bacteria of plague natural foci were more virulent, nitrogen removal was stable, and the plasmid species were relatively complete and stable. But at the end of the epidemic, the virulence of the plague was weaker, nitrogen removal was not stable, and plasmids tended to be absent. There were great differences in the morphology of plague bacteria in nature. Biological, biochemical, plasmid and virulence of Yersinia pestis were investigated in Hebei province, China. The mechanism of the existence of plague was revealed in some way.

Objective To study the genotype and epidemiological characteristics of Yersinia pestis in Hebei foci. Methods Primers were designed according to the confirmed 22 different segments (DFR), to genotype the 116 Y. pestis DNA of Hebei province. Results All of the strains lack these DFR of 01, 06, 07, 13, 15-18, to compare the genetic typing system of DFR, Y. pestis from Hebei province belong to the 17 genotype, distributed in the northern Kangbao county neighboring Huade, Baiqi, and Taipusiqi in the Inner Mongolia Autonomous Region. Conclusion The Y. pestis is stable genetically in Hebei plague foci, and have only one genotype, so the epidemic tends to stabilize.

Plague is an enzootic infectious disease. The rodent animals are primary reservoir hosts of plague. There are 223 kinds of animal which have been found to be infected with plague in nature. Plague-tolerant animals which can be naturally infected with plague without symptoms serve as plague-indicators. These animals also play a very important role in the spread of the plague. The research of plague-indicating animals was reviewed in this paper which provides scientific basis for plague prevention and control in China.

Objective To evaluate enzyme immunostaining technique for detection of F1 antigen of Yersinia pestis in rodents. Methods Visceral organ specimens of 266 mice infected with virulent Y. pestis and 207 control rodent specimens were detected by horseradish peroxidase labeled plague F1 monoclonal antibody (HRP-F1McAb) enzyme immunostaining technique, and in comparison with the RIHA and RGICA methods．Results Coincidence was 98.52% between HRP-F1McAb enzyme immunostaining technique and RIHA, Kappa=0.970, and the difference was statistically significant in the positive detection rates (χ²=5.140, P=0.016); Coincidence was 98.50% between HRP-F1McAb enzyme immunostaining technique and RGICA, Kappa=0.901, with statistically insignificant difference in the positive detection rates (χ²=0.250, P=0.625). Sensitivity of HRP-F1McAb enzyme immunostaining technique was 100%, specificity was 97.02%, positive predictive value was 97.14%, negative predictive value was 100%, and Youden index was 0.970． Conclusion The enzyme immunostaining technique is sensitive and specific, fast and simple in detection of plague F1 antigen. It is a valuable detection technique in early and rapid diagnosis of plague in rodents．

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 characterize the genosome of 116 Yersinia pestis strains isolated from plague foci in Hebei province in China. Methods All the strains were detected by polymerase chain reaction (PCR) with 15 pairs variable number tandem repeat (VNTR) provided by China CDC, then the length of results were analyzed. Results The numbers and lengths of the repeat sequences from all strains are same with the single primer, and different with the different primers, such as the length of all stains with the prime M52 is 153 bp, and the number of the repeat sequences is 3, then with the prime M59, the length and the number is 250 bp and 3 respectively. Conclusion Multiple loci VNTR analysis (MLVA) genetic typing is reliable, and it is stable genetic mark of Y. pestis from Hebei province. To build the database of the plague with MLVA is useful for the investigation of the plague variation and source.

Objective To get an insight into the characteristics of temporal clustering and distribution of patients with hemorrhagic fever with renal syndrome (HFRS) from 2005 to 2010 in Henan province, with the concentration ratio and circular distribution methods evaluated. Methods The incidence data on HFRS from 2005 to 2010 in Henan province were analyzed by concentration ratio combined with circular distribution. Results The M value of the concentration ratio of HFRS in Henan province from 2005 to 2010 by year was 0.356, 0.243, 0.370, 0.341, 0.489 and 0.746,respectively, with the mean angle of the circular distribution from 2005 to 2010 being 338.21°, 12.47°, 296.03°, 341.19°, 319.87°and 314.42° respectively. Except for the years 2006 and 2007,incidence of HFRS in Henan province took on obvious seasonal characteristics (P＜0.05), with the morbidity peak not at the same time (P＜0.05). Conclusion Obvious seasonal characteristics can be seen in the morbidity of HFRS in Henan province with the peak incidence seen in November and December. There has been a trend of the peak incidence occurring more earlier in recent years．

The main hosts in plague natural foci play a key role in the spread of plague and the preservation of Yersinia pestis. However, the plague hosts in natural foci have been showing diversity in recent years. Especially the non-primary hosts in plague natural foci have become a classic mode of plague transmission, posing a serious threat to human health. The relationship between hosts and plague transmission is presented in this paper to provide guidance for the development of measures for plague monitoring, prevention and control.

To further study the structure and nature of the foci of plague in Hebei province, this article summarizes the biological characteristics of Yersinia pestis isolates in the regions by analyzing the large amounts of data collected. By introducing the biochemical characteristics, nutritional needs, plasmid characteristics, virulence factors, virulence and toxins and other aspects, the study has concluded that Y. pestis isolated in Hebei province belongs to Group B of the Ordos Plateau, of which the nutritional needs are characterized by glycine semi-dependence and tryptophan dependence. These strains contain 4 kinds of plasmids, of which 13×106 plasmid is specifically carried by Meriones unguiculatus in the Inner Mongolian. Some strains lack the 45×106 plasmid, though all of isolates were F1 antigen and streptozotocin (PstⅠ)-positive. However, only a certain portion of the strains were positive for pigmentation (Pgm) and VW antigen, indicating genetic instability and moderate toxicity.

　　【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 value application effects on plague surveillance using different methods.Methods Rodent serums were detected by indirect hemagglutination assay(IHA),gold-immunochromatography assay(GICA-Ag),enzymelinked immunosorbent assay(ELISA-Ag).Rodent viscera were detected by gold-immunochromatography assay(GICA-Ab),enzymelinked immunosorbent assay(ELISA-Ab),polymerase chain reaction(PCR),bacterial isolation,reverse indirect hemagglutination assay(RIHA).Results Antibody detection rates in serums using IHA,GICA-Ag,ELISA-Ag were 0.32%,0.32%,1.26%,respectively.Positive detection rates of GICA-Ab,ELISA-Ab,PCR,RIHA,bacterial isolation in rodent viscera were 3.15%,0.70%,4.90%,0.70%,0.70%,respectively.Conclusion GICA,ELISA and PCR are valueable to extend and apply.They are suitable to use in basic level and fast diagnosis.

Objective Plague animal material is tested by 4-pair-primer-PCR which is compaired with traditional “4 piece test” to find a quick diagnostic way for plague. Methods Narcotics test and 4-pair-primer-PCR are both done in traditional way. Results 52 positive specimen of 4-pair-primer-PCR are found from 150,and 62 of traditional way are found. Both the ways have no discrepancy(χ²=1.42,P> 0.05 ). Conclusion 4-pair-primer-PCR is a quick,peculiar,sensitive method for the diagnosis and surveillance of plague.