Yersinia Pestis DNA Research Articles

Improving the extraction of ancient Yersinia pestis genomes from the dental pulp

Ancient DNA preserved in the dental pulp offers the opportunity to characterize the genome of some of the deadliest pathogens in human history. However, while DNA capture technologies help, focus sequencing efforts, and therefore, reduce experimental costs, the recovery of ancient pathogen DNA remains challenging. Here, we tracked the kinetics of ancient Yersinia pestis DNA release in solution during a pre-digestion of the dental pulp. We found that most of the ancient Y.pestis DNA is released within 60min at 37°C in our experimental conditions. We recommend a simple pre-digestion as an economical procedure to obtain extracts enriched in ancient pathogen DNA, as longer digestion times release other types of templates, including host DNA. Combining this procedure with DNA capture, we characterized the genome sequences of 12 ancient Y.pestis bacteria from France dating to the second pandemic outbreaks of the 17th and 18th centuries Common Era.

The plague’s impact paleodemographic and genetic measures in 15th to 16th century Gdańsk

Yersinia pestis caused plagues and haunted Gdańsk several times during the 15th and 16th centuries. This study focuses on the following demographic effects: 1/ distributions of deceased by age in a plagued city, 2/ parameters of the life tables, 3/ estimation of the natural increase. To assess genetic effects of the plague, measures of the opportunity for natural selection were considered. Skeletal remains of 283 people from the 15th – 16th century ossuary 3009 from the Dominican Monastery in Gdańsk provided research material. Yersinia pestis DNA in this skeletal material has already been found (Morozowa et al. 2017, 2020). Distributions of the deceased by age in the study sample were compared with those for Gdańsk before the plague and with those for the mass burial of plague victims in the 14th century Lübeck. Neither catastrophic mortality was found in the material studied, nor selective nature of the plague with regard to sex and age had been demonstrated. Using the Weiss method, the rate of natural increase r=–0.005 was reconstructed. With the wide dating range of the ossuary and the fact that it contains results of both the epidemic and “normal” mortality, the natural increase value at this level seems justified. There was a deterioration in the values of life tables parameters, especially life expectancy. Newborn life expectancy dropped to 19.5–22.6 years and for a 20-year-old to 17.7 years. The measures of opportunity for natural selection also deteriorated primarily due to child mortality: the biological state index Ibs values were low (within the 0.3–0.4 range) and values of the Im Crow’s index about 1.0. Natural selection also acted on adults as evidenced by values of the gross potential reproduction rate Rpot below 0.7. Demographically the study sample was at the level of the early Middle Ages rather than the Rennaisance.

New Epizootic Territory in Gorno-Altai High-Mountain Natural Plague Focus on the Ukok Plateau

The aim of the work – analysis of peculiarities of the plague epizooty found in the southern steppe part of the Ukok Plateau in 2020.Materials and methods. Epizootiological survey was conducted over the area of 1573 km2 in July and August, 2020. 141 mammals, 157 ectoparasites (including 152 fleas), 17 bone remains samples, and 50 regurgitates of predatory birds have been tested for plague.Results and discussion. The background species of mammals – natural hosts of Yersinia pestis in the southern steppe part of the Ukok plateau are gray marmot, long-tailed souslik, and Daurian pika. Abundance of the gray marmot was high and amounted to 1.7±0.18 inhabited burrows per 1 ha (n=30) with 90 % occupancy. Three strains of Yersinia pestis ssp. pestis have been isolated: from Oropsylla alaskensis fleas collected from long-tailed souslik, gray marmot carcass, and regurgitates of predatory birds. Yersinia pestis DNA have been detected in 14 objects. Eight positive results of serological tests have been obtained. The epizooty area that was confirmed by Yersinia pestis isolation totaled 252 km2 . As a result of the survey the circulation of Yersinia pestis ssp. pestis have been for the first time established on the Ukok Plateau. The target species – the gray marmot, as well as the long-tailed souslik, are actively involved into the epizooty.

Yersinia pestis DNA from Skeletal Remains from the 6th Century AD Reveals Insights into Justinianic Plague

Yersinia pestis, the etiologic agent of the disease plague, has been implicated in three historical pandemics. These include the third pandemic of the 19th and 20th centuries, during which plague was spread around the world, and the second pandemic of the 14th–17th centuries, which included the infamous epidemic known as the Black Death. Previous studies have confirmed that Y. pestis caused these two more recent pandemics. However, a highly spirited debate still continues as to whether Y. pestis caused the so-called Justinianic Plague of the 6th–8th centuries AD. By analyzing ancient DNA in two independent ancient DNA laboratories, we confirmed unambiguously the presence of Y. pestis DNA in human skeletal remains from an Early Medieval cemetery. In addition, we narrowed the phylogenetic position of the responsible strain down to major branch 0 on the Y. pestis phylogeny, specifically between nodes N03 and N05. Our findings confirm that Y. pestis was responsible for the Justinianic Plague, which should end the controversy regarding the etiology of this pandemic. The first genotype of a Y. pestis strain that caused the Late Antique plague provides important information about the history of the plague bacillus and suggests that the first pandemic also originated in Asia, similar to the other two plague pandemics.

Analysis on genotype distributions and epidemiological characteristics of Yersinia pestis plague foci in Gansu province

To study the genotype distributions and epidemiological characteristics of Yersinia pestis in Gansu province. Primers were designed according to the confirmed 23 differential sections, to genotype the 202 Yersinia pestis DNA of Gansu province by PCR, and to analyze its distribution and epidemiological characteristics. Yersinia pestis in Gansu province could be divided into eight genotypes: 1b, 5, 7, 8, 13, 26, new genotype 1 (GS1) and new genotype 2 (GS2). They were distributed in various regions. 1b, 8 and GS1 genotypes of Yersinia pestis had been identified since 1960s but the 7, 13 and 26 genotypes had not been isolated for more than 40 years while GS2 and 5 genotypes had been isolated since 1990s. 1b, 8 and GS1 genotypes of Yersinia pestis continued to be violently prevalent since 1960s but 7, 13 and 26 genotypes had not been isolated for more than 40 years while GS2 and 5 genotypes had started to be popular since 1990s.

Plague: History and contemporary analysis

Plague has caused ravaging outbreaks, including the Justinian plague and the "black death" in the Middle Ages. The causative agents of these outbreaks have been confirmed using modern molecular tests. The vector of plague during pandemics remains the subject of controversy. Nowadays, plague must be suspected in all areas where plague is endemic in rodents when patients present with adenitis or with pneumonia with a bloody expectorate. Diagnosis is more difficult in the situation of the reemergence of plague, as in Algeria for example, told by the first physician involved in that outbreak (NM). When in doubt, it is preferable to prescribe treatment with doxycycline while waiting for the test results because of the risk of fatality in individuals with plague. The typical bubo is a type of adenitis that is painful, red and nonfluctuating. The diagnosis is simple when microbiological analysis is conducted. Plague is a likely diagnosis when one sees gram-negative bacilli in lymph node aspirate or biopsy samples. Yersinia pestis grows very easily in blood cultures and is easy to identify by biochemical tests and MALDI-TOF mass spectrometry. Pneumonic plague and septicemic plague without adenitis are difficult to diagnose, and these diagnoses are often made by chance or retrospectively when cases are not part of an epidemic or related to another specific epidemiologic context. The treatment of plague must be based on gentamicin or doxycycline. Treatment with one of these antibiotics must be started as soon as plague is suspected. Analysis of past plague epidemics by using modern laboratory tools illustrated the value of epidemic buboes for the clinical diagnosis of plague; and brought new concepts regarding its transmission by human ectoparasites.

Brief communication: Co‐detection of Bartonella quintana and Yersinia pestis in an 11th–15th burial site in Bondy, France

Historical and anthropological data suggest that skeletons excavated from an 11th to 15th century mass grave in Bondy, France, may be those of victims of the Great Plague. Using high-throughput real-time PCR investigation of the dental pulp collected from 14 teeth from five such skeletons, we detected Bartonella quintana DNA in three individuals and Yersinia pestis DNA in two individuals. DNA from five other deadly pathogens was not found. Suicide PCR genotyping confirmed Y. pestis DNA belonging to the Orientalis biotype. One individual had co-infection. These data suggest a plague epidemic in a population already infected by the body louse-transmitted B. quintana or a body louse-driven transmission of the plague that drove a medieval epidemic in inland Europe.

High Throughput, Multiplexed Pathogen Detection Authenticates Plague Waves in Medieval Venice, Italy

BackgroundHistorical records suggest that multiple burial sites from the 14th–16th centuries in Venice, Italy, were used during the Black Death and subsequent plague epidemics.Methodology/Principal FindingsHigh throughput, multiplexed real-time PCR detected DNA of seven highly transmissible pathogens in 173 dental pulp specimens collected from 46 graves. Bartonella quintana DNA was identified in five (2.9%) samples, including three from the 16th century and two from the 15th century, and Yersinia pestis DNA was detected in three (1.7%) samples, including two from the 14th century and one from the 16th century. Partial glpD gene sequencing indicated that the detected Y. pestis was the Orientalis biotype.ConclusionsThese data document for the first time successive plague epidemics in the medieval European city where quarantine was first instituted in the 14th century.

Detection of Yersinia pestis DNA in Prairie Dog–Associated Fleas by Polymerase Chain Reaction Assay of Purified DNA

We evaluated, refined, and applied well-established polymerase chain reaction (PCR) techniques for detecting Yersinia pestis DNA in fleas (mainly Oropsylla spp.) collected from prairie dog (Cynomys spp.) burrows. Based on results from PCR of avirulent Y. pestis strain A1122 DNA, we used DNA purification and primers for the plasminogen activator gene to screen field-collected fleas. We detected Y. pestis DNA in flea pools from two black-tailed prairie dog (Cynomys ludovicianus) colonies with evidence of recent plague epizootics, and from one of four white-tailed prairie dog (Cynomys leucurus) colony complexes (Wolf Creek) where evidence of epizootic plague was lacking. Relative flea abundance and occurrence of Y. pestis DNA among flea pools appeared to vary over time at Wolf Creek. Both DNA purification and primer sequences appeared to influence the likelihood of detecting Y. pestis DNA by PCR in fleas collected from prairie dog burrows in the absence of observed epizootic plague. Presence of Y. pestis plasmid DNA in fleas collected from prairie dog burrows at Wolf Creek may represent evidence that infected fleas were somehow being maintained in that system between epizootics, consistent with the hypothesized enzootic maintenance of plague in prairie dog colony complexes elsewhere.

Antigen engineering can play a critical role in the protective immunity elicited by Yersinia pestis DNA vaccines

The use of a DNA immunization approach to deliver protective antigens against Yersinia pestis ( Y. pestis) has been successful in previously reported studies. In the current study, the gene designs for V and F1, two well-studied virulent factors serving as main targets for vaccine development, were altered to explore additional options in hopes of improving the protective immunity of DNA vaccines expressing these two antigens. Compared to the wild type V gene DNA vaccines, the use of codon optimized V gene sequences was effective in improving the antigen expression, titers of anti-V antibody responses, and survival against a mucosal lethal challenge. For the F1 DNA vaccine, removal of the N-terminal hydrophobic region was able to improve protective immunity. However, adding a mammalian signal peptide sequence to F1 actually led to reduced protection despite it inducing slightly higher anti-F1 antibody responses. The F1 gene can be fused with a gene coding for YscF, a newly confirmed partial protective antigen for Y. pestis, to produce DNA vaccines that express fused F1 and YscF antigens. One design, in particular, that had YscF fused to the downstream sequence of F1, produced better protection than separate F1 or YscF DNA vaccines, suggesting a potential synergistic effect between these two antigens. Findings from the above studies indicated that there are multiple approaches to optimize the protective immunity for plague DNA vaccines. Most importantly, proper antigen engineering to produce optimal antigen gene inserts in DNA vaccines can clearly play a major role in the future designs of a wide range of DNA vaccines.

New multispecific array as a tool for electrochemical impedance spectroscopy-based biosensing

Using electrochemical impedance spectroscopy (EIS) for biosensing applications typically requires repetitive experiments. To address this need, we have designed a multispecific electrochemical array with eight individually addressable 2 mm-diameter gold working electrodes for rapid biosensing data accumulation by EIS in the presence of redox agent. The array allows to incorporate multiple negative controls in the course of a single binding experiment, as well as to perform parallel identical experiments to improve reliability of detection. The array is fit with attached electrochemical cell with Ag/AgCl mini reference electrode and can be used to process macro samples of 0.5–1 ml or micro samples of 5 μl in a drop-wise fashion. Eight individual EIS measurements are completed in 15 min. The reported array is disposable, economical and is easy to use. Examples of array use for label-free genetic sensing of 2.7 kb-long target Yersinia pestis DNA and for protein sensing of Ricin Toxin Chain A (RTA) are presented. We suggest the reported array design as a tool for researchers in the area of EIS sensing.

Yersinia pestisOrientalis in Remains of Ancient Plague Patients

Yersinia pestis DNA was recently detected in human remains from 2 ancient plague pandemics in France and Germany. We have now sequenced Y. pestis glpD gene in such remains, showing a 93-bp deletion specific for biotype Orientalis. These data show that only Orientalis type caused the 3 plague pandemics.

Homology with a repeated Yersinia pestis DNA sequence IS100 correlates with pesticin sensitivity in Yersinia pseudotuberculosis.

We have identified IS100 sequences in a specific subset of Yersinia pseudotuberculosis isolates that were also sensitive to the Y. pestis-produced bacteriocin, pesticin. In contrast, Y. pseudotuberculosis strains which did not contain IS100 sequences were not sensitive to pesticin. We propose that IS100 serves as a molecular marker that identifies a subset of Y. pseudotuberculosis isolates that have a particularly close evolutionary and/or ecological relationship with Y. pestis.

Development of a diagnostic test for Yersinia pestis by the polymerase chain reaction.

A 501 bp caf1 gene fragment and a 443 bp of pla gene fragment carried by 100 kb (pFra) and 10 kb (pPst) species-specific extrachromosomal replicons, respectively, were used as targets to study the conditions under which DNA amplification by polymerase chain reaction (PCR) may be applied to detect and identify Yersinia pestis DNA in cell lysates of pure cultures and biological samples. The sensitivity limit of PCR with the crude cell lysates of Y. pestis EV was estimated as 10-50 cfu in reaction mixture. When target Y. pestis EV cells were mixed with fresh blood of white mice, which contained 0.4% potassium citrate, the PCR detection level varied from 400 to 100 cfu ml-1 of blood depending on the method used for preparing the sample. In our tests PCR was effective for the detection of yersinia in the blood of white laboratory mice experimentally infected with virulent Y. pestis KM638 strain. This method can be considered convenient for routine detection and identification of Y. pestis.