Development of a dual antigen lateral flow immunoassay for detecting Yersinia pestis.

BACKGROUND: Yersinia pestis is the causative agent of plague, a zoonosis associated with small mammals. Plague is a severe disease, especially in the pneumonic and septicemic forms, where fatality rates approach 100% if left untreated. The bacterium is primarily transmitted via flea bite or through direct contact with an infected host. The 2017 plague outbreak in Madagascar resulted in more than 2,400 cases and was highlighted by an increased number of pneumonic infections. Standard diagnostics for plague include laboratory-based assays such as bacterial culture and serology, which are inadequate for administering immediate patient care for pneumonic and septicemic plague. PRINCIPAL FINDINGS: The goal of this study was to develop a sensitive rapid plague prototype that can detect all virulent strains of Y. pestis. Monoclonal antibodies (mAbs) were produced against two Y. pestis antigens, low-calcium response V (LcrV) and capsular fraction-1 (F1), and prototype lateral flow immunoassays (LFI) and enzyme-linked immunosorbent assays (ELISA) were constructed. The LFIs developed for the detection of LcrV and F1 had limits of detection (LOD) of roughly 1-2 ng/mL in surrogate clinical samples (antigens spiked into normal human sera). The optimized antigen-capture ELISAs produced LODs of 74 pg/mL for LcrV and 61 pg/mL for F1 when these antigens were spiked into buffer. A dual antigen LFI prototype comprised of two test lines was evaluated for the detection of both antigens in Y. pestis lysates. The dual format was also evaluated for specificity using a small panel of clinical near-neighbors and other Tier 1 bacterial Select Agents. CONCLUSIONS: LcrV is expressed by all virulent Y. pestis strains, but homologs produced by other Yersinia species can confound assay specificity. F1 is specific to Y. pestis but is not expressed by all virulent strains. Utilizing highly reactive mAbs, a dual-antigen detection (multiplexed) LFI was developed to capitalize on the diagnostic strengths of each target.

The invasive pathogen Yersinia pestis disrupts host blood vasculature to spread and provoke hemorrhages.

Yersinia pestis is a powerful pathogen with a rare invasive capacity. After a flea bite, the plague bacillus can reach the bloodstream in a matter of days giving way to invade the whole organism reaching all organs and provoking disseminated hemorrhages. However, the mechanisms used by this bacterium to cross and disrupt the endothelial vascular barrier remain poorly understood. In this study, an innovative model of in vivo infection was used to focus on the interaction between Y. pestis and its host vascular system. In the draining lymph nodes and in secondary organs, bacteria provoked the porosity and disruption of blood vessels. An in vitro model of endothelial barrier showed a role in this phenotype for the pYV/pCD1 plasmid that carries a Type Three Secretion System. This work supports that the pYV/pCD1 plasmid is responsible for the powerful tissue invasiveness capacity of the plague bacillus and the hemorrhagic features of plague.

Interrelationship of soil moisture and temperature to sylvatic plague cycle among prairie dogs in the Western United States.

Plague, caused by Yersinia pestis, is a flea-borne disease that is endemic in areas throughout the world due to its successful maintenance in a sylvatic cycle, mainly in areas with temperate climates. Burrowing rodents are thought to play a key role in the enzootic maintenance as well as epizootic outbreaks of plague. In the United States, prairie dogs (Cynomys), rodents (Muridae), and ground squirrels (Spermophilus) are susceptible to infection and are parasitized by fleas that transmit plague. In particular, prairie dogs can experience outbreaks that rapidly spread, which can lead to extirpation of colonies. A number of ecological parameters, including climate, are associated with these epizootics. In this study, we asked whether soil parameters, primarily moisture and temperature, are associated with outbreaks of plague in black-tailed prairie dogs and Gunnison's prairie dogs in the Western United States, and at what depth these associations were apparent. We collected publicly available county-level information on the occurrence of population declines or colony extirpation, while historical soil data was collected from SCAN and USCRN stations in counties and states where prairie dogs have been located. The analysis suggests that soil moisture at lower depths correlates with colony die-offs, in addition to temperature near the surface, with key differences within the landscape ecology that impact the occurrence of plague. Overall, the model suggests that the burrow environment may play a significant role in the epizootic spread of disease amongst black-tailed and Gunnison's prairie dogs.

A 5,000-year-old hunter-gatherer already plagued by Yersinia pestis.

A 5,000-year-old Yersinia pestis genome (RV 2039) is reconstructed from a hunter-fisher-gatherer (5300-5050 cal BP) buried at Rinnukalns, Latvia. RV 2039 is the first in a series of ancient strains that evolved shortly after the split of Y. pestis from its antecessor Y. pseudotuberculosis ∼7,000 years ago. The genomic and phylogenetic characteristics of RV 2039 are consistent with the hypothesis that this very early Y. pestis form was most likely less transmissible and maybe even less virulent than later strains. Our data do not support the scenario of a prehistoric pneumonic plague pandemic, as suggested previously for the Neolithic decline. The geographical and temporal distribution of the few prehistoric Y. pestis cases reported so far is more in agreement with single zoonotic events.

Poor vector competence of the human flea, Pulex irritans, to transmit Yersinia pestis.

BACKGROUND: The human flea, Pulex irritans, is widespread globally and has a long association with humans, one of its principal hosts. Its role in plague transmission is still under discussion, although its high prevalence in plague-endemic regions and the presence of infected fleas of this species during plague outbreaks has led to proposals that it has been a significant vector in human-to-human transmission in some historical and present-day epidemiologic situations. However, based on a limited number of studies, P. irritans is considered to be a poor vector and receives very little attention from public health policymakers. In this study we examined the vector competence of P. irritans collected from foxes and owls in the western United States, using a standard protocol and artificial infection system. METHODS: Wild-caught fleas were maintained in the laboratory and infected by allowing them to feed on human or rat blood containing 2 × 108 to 1 × 109 Y. pestis/ml. The fleas were then monitored periodically for infection rate and bacterial load, mortality, feeding rate, bacterial biofilm formation in the foregut (proventricular blockage), and ability to transmit Y. pestis after their single infectious blood meal. RESULTS: P. irritans were susceptible to infection, with more than 30% maintaining high bacterial loads for up to 20 days. Transmission during this time was infrequent and inefficient, however. Consistent with previous studies, a low level of early-phase transmission (3 days after the infectious blood meal) was detected in some trials. Transmission at later time points was also sporadic, and the incidence of proventricular blockage, required for this mode of transmission, was low in fleas infected using rat blood and never occurred in fleas infected using human blood. The highest level of blockage and transmission was seen in fleas infected using rat blood and allowed to feed intermittently rather than daily, indicating that host blood and feeding frequency influence vector competence. CONCLUSIONS: Our results affirm the reputation of P. irritans as a feeble vector compared to rodent flea species examined similarly, and its vector competence may be lower when infected by feeding on bacteremic human blood.

Development of a PCR-lateral flow assay for rapid detection of Yersinia pestis, the causative agent of plague.

Plague is a zoonotic disease caused by Yersinia pestis, a Gram-negative, rod shaped coccobacillus, which is primarily found in rodents and can be transmitted to humans through flea bite. The disease has three major clinical forms bubonic (by flea bite), pneumonic (by respiratory droplets) and septicemic plague. Y. pestis is classified as a category 'A' agent by NIAID, USA due to its high mortality and easy person to person dissemination. The conventional diagnostic methods available for Y. pestis show cross-reactivity with other enteropathogenic bacteria making its detection difficult. There is a need to develop sensitive and specific molecular assay for accurate detection of Y. pestis. PCR is well suited molecular biology tool for rapid diagnosis of plague but after completion of thermal cycling steps, it requires additional time to analyze amplified product using agarose gel electrophoresis. In the present study, PCR assay coupled with lateral flow strips has been developed for rapid detection of Y. pestis. Lateral flow strips give an alternative to gel electrophoresis and permit easy and rapid detection of PCR products. The PCR was performed with 5' 6-FAM and biotin tagged primers specific for Y. pestis, targeting yihN gene located on chromosome. The PCR product was analyzed using lateral flow strips which yielded result within 2-3 minutes. The analytical sensitivity of PCR-lateral flow (PCR-LF) assay was 1 pg genomic DNA of Y. pestis and 500 copies of target DNA sequence harboured in a recombinant plasmid. The assay could detect Y. pestis DNA extracted from spiked human blood samples containing ≥104 CFU per mL of bacteria. The assay was found to be specific and did not cross react with other closely related bacterial species. The developed assay was highly specific, sensitive and also did not require agarose gel electrophoresis for post amplification analysis.

Reciprocal Regulation between Fur and Two RyhB Homologs in Yersinia pestis, and Roles of RyhBs in Biofilm Formation.

OBJECTIVE: To investigate reciprocal regulation between Fur and two RyhB homologs in Yersinia pestis( Y. pestis), as well as the roles of RyhBs in biofilm formation. METHODS: Regulatory relationships were assessed by a combination of colony morphology assay, primer extension, electrophoretic mobility shift assay and DNase I footprinting. RESULTS: Fur bound to the promoter-proximal DNA regions of ryhB1 and ryhB2 to repress their transcription, while both RyhB1 and RyhB2 repressed the expression of Fur at the post-transcriptional level. In addition, both RyhB1 and RyhB2 positively regulated Y. pestis biofilm exopolysaccharide (EPS) production and the expression of hmsHFRS and hmsT. CONCLUSION: Fur and the two RyhB homologs exert negative reciprocal regulation, and RyhB homologs have a positive regulatory effect on biofilm formation in Y. pestis.

Rodent hosts and flea vectors in Brazilian plague foci: a review.

Plague, caused by the Yersinia pestis bacterium, has several foci scattered throughout a large area from the Brazilian territory that ranges from the Northeastern State of Ceará to the Southeastern State of Minas Gerais and another separated area at the State of Rio de Janeiro. This review gathers data from plague control and surveillance programs on the occurrence and geographic distribution of rodent hosts and flea vectors in the Brazilian plague areas during the period of from 1952 to 2019. Furthermore, we discuss how the interaction between Y. pestis and some rodent host species may play a role in the disease dynamics. The absence of human cases nowadays in Brazil does not mean that it was eradicated. The dynamics of plague in Brazil and in other countries where it was introduced during the 3rd pandemic are quite alike, alternating epidemics with decades of quiescence. Hence, it remains an important epidemic disease of global concern. The existence of a large animal reservoir and competent vectors demonstrate a need for continuous surveillance to prevent new outbreaks of this disease in humans.

Reciprocal Regulation between Fur and Two RyhB Homologs in / 生物医学与环境科学（英文）

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Bacteria use structural imperfect mimicry to hijack the host interactome.

Bacteria use protein-protein interactions to infect their hosts and hijack fundamental pathways, which ensures their survival and proliferation. Hence, the infectious capacity of the pathogen is closely related to its ability to interact with host proteins. Here, we show that hubs in the host-pathogen interactome are isolated in the pathogen network by adapting the geometry of the interacting interfaces. An imperfect mimicry of the eukaryotic interfaces allows pathogen proteins to actively bind to the host's target while preventing deleterious effects on the pathogen interactome. Understanding how bacteria recognize eukaryotic proteins may pave the way for the rational design of new antibiotic molecules.

Influence of Temperature on Development of Yersinia pestis Foregut Blockage in Xenopsylla cheopis (Siphonaptera: Pulicidae) and Oropsylla montana (Siphonaptera: Ceratophyllidae).

Plague, caused by the flea-transmitted bacterial pathogen Yersinia pestis, is primarily a disease of wild rodents distributed in temperate and tropical zones worldwide. The ability of Y. pestis to develop a biofilm blockage that obstructs the flea foregut proventriculus facilitates its efficient transmission through regurgitation into the host bite site during flea blood sucking. While it is known that temperature influences transmission, it is not well-known if blockage dynamics are similarly in accord with temperature. Here, we determine the influence of the biologically relevant temperatures, 10 and 21°C, on blockage development in flea species, Xenopsylla cheopis (Rothschild) and Oropsylla montana (Baker), respectively, characterized by geographical distribution as cosmopolitan, tropical or endemic, temperate. We find that both species exhibit delayed development of blockage at 10°C. In Y. pestis infected X. cheopis, this is accompanied by significantly lower survival rates and slightly decreased blockage rates, even though these fleas maintain similar rates of persistent infection as at 21°C. Conversely, irrespective of infection status, O. montana withstand 21 and 10°C similarly well and show significant infection rate increases and slightly greater blocking rates at 10 versus 21°C, emphasizing that cooler temperatures are favorable for Y. pestis transmission from this species. These findings assert that temperature is a relevant parameter to consider in assessing flea transmission efficiency in distinct flea species residing in diverse geographical regions that host endemic plague foci. This is important to predict behavioral dynamics of plague regarding epizootic outbreaks and enzootic maintenance and improve timeous implementation of flea control programs.

A refined model of how Yersinia pestis produces a transmissible infection in its flea vector.

In flea-borne plague, blockage of the flea's foregut by Yersinia pestis hastens transmission to the mammalian host. Based on microscopy observations, we first suggest that flea blockage results from primary infection of the foregut and not from midgut colonization. In this model, flea infection is characterized by the recurrent production of a mass that fills the lumen of the proventriculus and encompasses a large number of Y. pestis. This recurrence phase ends when the proventricular cast is hard enough to block blood ingestion. We further showed that ymt (known to be essential for flea infection) is crucial for cast production, whereas the hmsHFRS operon (known to be essential for the formation of the biofilm that blocks the gut) is needed for cast consolidation. By screening a library of mutants (each lacking a locus previously known to be upregulated in the flea gut) for biofilm formation, we found that rpiA is important for flea blockage but not for colonization of the midgut. This locus may initially be required to resist toxic compounds within the proventricular cast. However, once the bacterium has adapted to the flea, rpiA helps to form the biofilm that consolidates the proventricular cast. Lastly, we used genetic techniques to demonstrate that ribose-5-phosphate isomerase activity (due to the recent gain of a second copy of rpiA (y2892)) accentuated blockage but not midgut colonization. It is noteworthy that rpiA is an ancestral gene, hmsHFRS and rpiA2 were acquired by the recent ancestor of Y. pestis, and ymt was acquired by Y. pestis itself. Our present results (i) highlight the physiopathological and molecular mechanisms leading to flea blockage, (ii) show that the role of a gene like rpiA changes in space and in time during an infection, and (iii) emphasize that evolution is a gradual process punctuated by sudden jumps.

First Genome Sequence of the Gunnison's Prairie Dog (Cynomys gunnisoni), a Keystone Species and Player in the Transmission of Sylvatic Plague.

Prairie dogs (genus Cynomys) are a charismatic symbol of the American West. Their large social aggregations and complex vocalizations have been the subject of scientific and popular interest for decades. A large body of literature has documented their role as keystone species of western North America's grasslands: They generate habitat for other vertebrates, increase nutrient availability for plants, and act as a food source for mammalian, squamate, and avian predators. An additional keystone role lies in their extreme susceptibility to sylvatic plague (caused by Yersinia pestis), which results in periodic population extinctions, thereby generating spatiotemporal heterogeneity in both biotic communities and ecological processes. Here, we report the first Cynomys genome for a Gunnison's prairie dog (C. gunnisoni gunnisoni) from Telluride, Colorado (USA). The genome was constructed using a hybrid assembly of PacBio and Illumina reads and assembled with MaSuRCA and PBJelly, which resulted in a scaffold N50 of 824 kb. Total genome size was 2.67 Gb, with 32.46% of the bases occurring in repeat regions. We recovered 94.9% (91% complete) of the single copy orthologs using the mammalian Benchmarking Universal Single-Copy Orthologs database and detected 49,377 gene models (332,141 coding regions). Pairwise Sequentially Markovian Coalescent showed support for long-term stable population size followed by a steady decline beginning near the end of the Pleistocene, as well as a recent population reduction. The genome will aid in studies of mammalian evolution, disease resistance, and the genomic basis of life history traits in ground squirrels.

Evolutionary selection of biofilm-mediated extended phenotypes in Yersinia pestis in response to a fluctuating environment.

Yersinia pestis is transmitted from fleas to rodents when the bacterium develops an extensive biofilm in the foregut of a flea, starving it into a feeding frenzy, or, alternatively, during a brief period directly after feeding on a bacteremic host. These two transmission modes are in a trade-off regulated by the amount of biofilm produced by the bacterium. Here by investigating 446 global isolated Y. pestis genomes, including 78 newly sequenced isolates sampled over 40 years from a plague focus in China, we provide evidence for strong selection pressures on the RNA polymerase ω-subunit encoding gene rpoZ. We demonstrate that rpoZ variants have an increased rate of biofilm production in vitro, and that they evolve in the ecosystem during colder and drier periods. Our results support the notion that the bacterium is constantly adapting-through extended phenotype changes in the fleas-in response to climate-driven changes in the niche.

The Cyclic AMP Receptor Protein Regulates Quorum Sensing and Global Gene Expression in Yersinia pestis during Planktonic Growth and Growth in Biofilms.

Cyclic AMP (cAMP) receptor protein (Crp) is an important transcriptional regulator of Yersinia pestis Expression of crp increases during pneumonic plague as the pathogen depletes glucose and forms large biofilms within lungs. To better understand control of Y. pestis Crp, we determined a 1.8-Å crystal structure of the protein-cAMP complex. We found that compared to Escherichia coli Crp, C helix amino acid substitutions in Y. pestis Crp did not impact the cAMP dependency of Crp to bind DNA promoters. To investigate Y. pestis Crp-regulated genes during plague pneumonia, we performed RNA sequencing on both wild-type and Δcrp mutant bacteria growing in planktonic and biofilm states in minimal media with glucose or glycerol. Y. pestis Crp was found to dramatically alter expression of hundreds of genes in a manner dependent upon carbon source and growth state. Gel shift assays confirmed direct regulation of the malT and ptsG promoters, and Crp was then linked to Y. pestis growth on maltose as a sole carbon source. Iron regulation genes ybtA and fyuA were found to be indirectly regulated by Crp. A new connection between carbon source and quorum sensing was revealed as Crp was found to regulate production of acyl-homoserine lactones (AHLs) through direct and indirect regulation of genes for AHL synthetases and receptors. AHLs were subsequently identified in the lungs of Y. pestis-infected mice when crp expression was highest in Y. pestis biofilms. Thus, in addition to the well-studied pla gene, other Crp-regulated genes likely have important functions during plague infection.IMPORTANCE Bacterial pathogens have evolved extensive signaling pathways to translate environmental signals into changes in gene expression. While Crp has long been appreciated for its role in regulating metabolism of carbon sources in many bacterial species, transcriptional profiling has revealed that this protein regulates many other aspects of bacterial physiology. The plague pathogen Y. pestis requires this global regulator to survive in blood, skin, and lungs. During disease progression, this organism adapts to changes within these niches. In addition to regulating genes for metabolism of nonglucose sugars, we found that Crp regulates genes for virulence, metal acquisition, and quorum sensing by direct or indirect mechanisms. Thus, this single transcriptional regulator, which responds to changes in available carbon sources, can regulate multiple critical behaviors for causing disease.

Plague in Zimbabwe from 1974 to 2018: A review article.

Plague is a zoonotic disease caused by the bacterium Yersinia pestis and is transmitted through the bites of infected rodent fleas. Plague is well known for causing 3 major human pandemics that have killed millions of people since 541 A.D. The aim of this Review is to provide an overview of the epidemiology and ecology of plague in Zimbabwe with special emphasis on its introduction, its potential reservoirs and vectors, and possible causes of its persistence and cyclic outbreaks. To achieve this, we carried out a search and document reported plague outbreaks in Zimbabwe. In the country, human plague cases have been reported in Hwange, Nkayi, and Lupane since 1974. The highest number of cases occurred in 1994 in the Nkayi district of Matabeleland North Province with a total of 329 confirmed human cases and 28 deaths. Plague is encountered in 2 different foci in the country, sylvatic and rural. Risk factors for contracting plague in the country include man-to-rodent contact, cultivation, hunting, cattle herding, handling of infected materials, camping in forests, and anthropic invasion of new areas. Plague is now enzootic in Zimbabwe, and the most recent case was reported in 2012, hence its effective control requires up-to-date information on the epidemiology and ecology of the disease. This can be achieved through continuous monitoring and awareness programs in plague-prone areas.

Nutrient depletion may trigger the Yersinia pestis OmpR-EnvZ regulatory system to promote flea-borne plague transmission.

The flea's lumen gut is a poorly documented environment where the agent of flea-borne plague, Yersinia pestis, must replicate to produce a transmissible infection. Here, we report that both the acidic pH and osmolarity of the lumen's contents display simple harmonic oscillations with different periods. Since an acidic pH and osmolarity are two of three known stimuli of the OmpR-EnvZ two-component system in bacteria, we investigated the role and function of this Y. pestis system in fleas. By monitoring the in vivo expression pattern of three OmpR-EnvZ-regulated genes, we concluded that the flea gut environment triggers OmpR-EnvZ. This activation was not, however, correlated with changes in pH and osmolarity but matched the pattern of nutrient depletion (the third known stimulus for OmpR-EnvZ). Lastly, we found that the OmpR-EnvZ and the OmpF porin are needed to produce the biofilm that ultimately obstructs the flea's gut and thus hastens the flea-borne transmission of plague. Taken as a whole, our data suggest that the flea gut is a complex, fluctuating environment in which Y. pestis senses nutrient depletion via OmpR-EnvZ. Once activated, the latter triggers a molecular program (including at least OmpF) that produces the biofilm required for efficient plague transmission.

An Intradermal Model for Yersinia pestis Inoculation.

The dermis and the subcutaneous space vary in many fundamental characteristics, which include composition of lymphatic vessels, density of blood vasculature, and cells of the immune response. Traditional approaches employ the subcutaneous space as the preferred layer of the skin to inoculate Yersinia pestis for bubonic plague studies. Because fleas transmit Y. pestis in nature, and because these insects target the dermal layer of the skin, an intradermal model of infection is more biologically relevant than a subcutaneous model. Among many features, the use of an intradermal model results in robust and reproducible colonization of lymph nodes, blood, and deeper tissues. Remarkably, intradermal inoculation in the murine ear pinna also allows for the study of cutaneous infection without severely disrupting the architecture and physiology of the skin.

Intranasal Inoculation of Mice with Yersinia pestis and Processing of Pulmonary Tissue for Analysis.

Pneumonic plague is a rapidly progressing and highly lethal pneumonia caused by pulmonary infection with Yersinia pestis. Disease is marked by the rapid replication of bacteria in the lungs in the absence of symptoms, followed by the abrupt onset of a highly lethal inflammatory response. A murine intranasal infection model has been key to characterizing the progression of disease. Mice are a natural Y. pestis host, and murine disease closely mirrors what is seen during human infection. Intranasal inoculation of mice with fully virulent Y. pestis strains allows for the detailed analysis of key bacterial and host factors that define disease progression. In this chapter I describe a method for intranasal inoculation of mice with Y. pestis, as well as techniques for processing lung tissue for analysis. These include protocols for isolating whole lungs and lavage fluid for measure of bacterial burden, transcriptomics, cytokine/chemokine expression, and flow cytometry. These techniques can be used to evaluate disease parameters of interest during typical infection, infection with bacterial mutants, or infection in the presence of pharmacological agents aimed at targeting specific host or bacterial factors. Combining a highly relevant murine infection model with these techniques provides a powerful platform for fully evaluating the progression of pneumonic plague.

Standardized Method for Aerosol Challenge of Rodents with Yersinia pestis for Modeling Primary Pneumonic Plague.

Primary pneumonic plague occurs when Yersinia pestis is inhaled into the lower respiratory tract where it invades the alveoli and grows. Rapid bacterial growth eventually elicits a neutrophilic inflammatory response that is ineffective and damaging, leading to accelerated progression of disease. In the laboratory, modeling of primary pneumonic plague can be accomplished by instillation of bacterial culture in the nares of anesthetized mice and rats. Although primary pneumonic plague can develop from this method, variability in dosing and side effects of anesthesia can complicate data interpretation. In contrast, aerosol challenge models allow for well-controlled studies of pneumonic plague with minimal experimental bias and unwanted side effects. For these reasons, antibiotic testing and the licensing of new treatments depend on efficacy data generated from aerosol delivery of Y. pestis in order to more accurately model transmission and the early stages of human pneumonic plague. In order to meet this need, we have extensively characterized pneumonic plague in mice and rats challenged by nose-only exposure to Yersinia pestis. With this approach, simultaneous challenge of large cohorts of animals, gently restrained and not anesthetized, assures safe, well-controlled, unbiased, and uniform infection. In this chapter, we present a standardized method for reproducible aerosol delivery of wild-type Y. pestis to rodents for experimental models of primary pneumonic plague.