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The Oriental rat flea, Xenopsylla cheopis (Rothschild 1903), is a cosmopolitan flea usually found infesting domestic rats. This flea is a well-known major human plague vector in Madagascar. As part of field sampling, fleas and small mammals were collected in the village of South Andranofeno and the natural reserve of Sohisika, two sites of the district of Ankazobe, located in the Central Highlands of Madagascar. Rats inside houses and forest small mammals were trapped using Besancon Technical Services and pitfall traps, respectively. Their fleas were collected and preserved for laboratory works. Collected fleas from the village and forest belonged to five species, which were X. cheopis, Synopsyllus fonquerniei (Wagner and Roubaud 1932) (Siphonaptera: Pulicidae), Echidnophaga gallinacea (Westwood 1875) (Siphonaptera: Pulicidae), Ctenocephalides felisstrongylus (Jordan 1925) (Siphonaptera: Pulicidae), Pulex irritans (Linnaeus 1758) (Siphonaptera: Pulicidae). After sampling in the forest zone, one specimen of X. cheopis was unexpectedly collected while infesting an endemic tenrec Setifer setosus (Schreber 1777) (Afrosoricida: Tenrecidae). Polymerase chain reaction (PCR) diagnosis on all collected fleas allowed detecting plague bacterium Yersinia pestis (Lehmann and Neumann 1896) (Enterobacterales: Yersiniaceae) on nine specimens of the endemic flea S. fonquerniei collected inside forest. The presence of the oriental rat flea in forest highlights the connection between human and wild environments due to animal movements and the fact that the rat flea can infest various hosts. As only one specimen of X. cheopis was collected on S. setosus, we hypothesize that flea was carried from the village to forest. Yersinia pestis infection of forest fleas outlines plague circulation in this sylvatic area.

International audience; Plague is a vector-borne disease caused by Yersinia pestis. Transmitted by fleas from rodent reservoirs, Y. pestis emerged less than 6000 years ago from an enteric bacterial ancestor through events of gene gain and genome reduction. It is a highly remarkable model for the understanding of pathogenic bacteria evolution, and a major concern for public health as highlighted by recent human outbreaks. A complex set of virulence determinants, including the Yersinia outer membrane proteins (Yops), the broad range pro-tease Pla, pathogen-associated molecular patterns (PAMPs) and iron capture systems play critical roles in the molecular strategies that Y. pestis employs to subvert the human immune system, allowing unrestricted bacterial replication in lymph nodes (bubonic plague) and in lungs (pneumonic plague). Some of these immunogenic proteins as well as the capsular antigen F1 are exploited for diagnostic purposes, which are critical in the context of the rapid onset of death in the absence of antibiotic treatment (less than a week for bubonic plague and less than 48 h for pneumonic plague). In here, we review recent research advances on Y. pestis evolution, virulence factors function, bacterial strategies to subvert mammalian innate immune responses, vaccination and problems associated to pneumonic plague diagnosis.

International audience; Immunization with the live-attenuated Yersinia pseudotuberculosis VTnF1 strain producing a Yersinia pestis F1 pseudocapsule efficiently protects mice against bubonic and pneumonic plague. In clinical trials, demonstration of a plague vaccine's efficacy in humans will not be feasible, and correlates of protection will be needed to bridge the immune response of protected animals to that of vaccinated humans. Using serum transfer and vaccination of antibody-deficient µMT mice, we established that both humoral and cellular responses elicited by VTnF1 independently conferred protection against bubonic plague. Thus, correlates were searched for in both responses, using blood only. Mice were vaccinated with increasing doses of VTnF1 to provide a range of immune responses and survival outcomes. The cellular response was evaluated using an in vitro IFNγ release assay, and IFNγ levels were significantly associated with protection, although some survivors were negative for IFNγ, so that IFNγ release is not a fully satisfactory correlate. Abundant serum IgG against the F1 capsule, Yop injectable toxins, and also non-F1 Y.pestis antigens were found, but none against the LcrV antigen. All readouts correlated to survival and to each other, confirming that vaccination triggered multiple protective mechanisms developing in parallel. Anti-F1 IgG was the most stringent correlate of protection, in both inbred BALB/c mice and outbred OF1 mice. This indicates that antibodies (Ab) to F1 play a dominant role for protection even in the presence of Ab to many other targets. Easy to measure, the anti-F1 IgG titer will be useful to evaluate the immune response in other animal species and in clinical trials.

International audience; The emergence of antibiotic-resistant Yersinia pestis strains represents a public health concern. Two antibiotic-resistant Y. pestis strains isolated from Madagascar have been previously identified and characterised. Both strains carried conjugative plasmids that conferred resistance to streptomycin or to multiple antibacterial drugs, respectively. Here we characterised a novel Y. pestis strain (IP2180H) that exhibited resistance to doxycycline. This strain was isolated from a rat in Antananarivo (Madagascar) in 1998. Resistance was carried by a conjugative plasmid (pIP2180H) homologous to pB71 from Salmonella enterica. The plasmid of the previously identified streptomycin-resistant Y. pestis strain was also sequenced and it was found that the three antibiotic resistance Y. pestis plasmids sequenced until now are genetically unrelated and are also unrelated to multidrug resistance plasmids from the phylogenetically close bacterial species Yersinia pseudotuberculosis. The fact that the three antibiotic-resistant Malagasy Y. pestis strains were isolated from different hosts, at different times, from distant locations, and carried unrelated plasmids indicates independent horizontal acquisition of genetic material and further demonstrates the capacity of Y. pestis to acquire antibiotic resistance plasmids under natural conditions. Since these resistance plasmids can frequently carry or easily trap antibiotic resistance cassettes, the emergence of new multidrug-resistant Y. pestis strains may be expected and would represent a major health threat.

Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by homozygous or compound heterozygous gain-of-function mutations in MEFV, encoding pyrin, an inflammasome protein. Heterozygous carrier frequencies for multiple MEFV mutations are high in several Mediterranean populations, suggesting that they confer selective advantage. Among 2,313 Turks, we found extended haplotype homozygosity flanking FMF-associated mutations, indicating evolutionarily recent positive selection of FMF-associated mutations. Two pathogenic pyrin variants independently arose >1,800 years ago. Mutant pyrin interacts less avidly with Yersinia pestis virulence factor YopM than wild type human pyrin, thereby attenuating YopM-induced IL-1β suppression. Relative to healthy controls, leukocytes from FMF patients harboring homozygous or compound heterozygous mutations and from asymptomatic heterozygous carriers released heightened IL-1β specifically in response to Y. pestis. Y. pestis-infected MefvM680I/M680I FMF knock-in mice exhibited IL-1-dependent increased survival relative to wild-type knock-in mice. Thus, FMF mutations that were positively selected in Mediterranean populations confer heightened resistance to Y. pestis.

Bubonic is the most prevalent plague form in Madagascar. Indoor ground application of insecticide dust is the conventional method used to control potentially infected rodent fleas that transmit the plague bacterium from rodents to humans. The use of bait stations is an alternative approach for vector control during plague epidemics, as well as a preventive control method during non-epidemic seasons. Bait stations have many advantages, principally by reducing the amount of insecticide used, lowering the cost of the treatment and minimizing insecticide exposure in the environment. A previous study reported promising results on controlling simultaneously the reservoir and vectors, when slow-acting rodenticide was incorporated in bait stations called “Boîtes de Kartman”. However, little evidence of an effective control of the fleas prior to the elimination of rodents was found. In this study, we evaluated bait stations containing insecticide powder and non-toxic attractive rodent bait for their potential to control rat fleas. Its efficacy was compared to the standard method. The impact of both methods on indoor and outdoor rodent fleas, as well as the human household flea Pulex irritans were analyzed at different time points after treatments. Bait stations did not cause any significant immediate or delayed reduction of rat fleas and increasing the number of operational bait stations per household did not significantly improve their efficacy. Insecticide ground dusting appeared to be the most efficient method to control indoor rat fleas. Both methods appeared to have little impact on the density of outdoor rat fleas and human fleas. These results demonstrate limited effectiveness for bait stations and encourage the maintenance of insecticide dusting as a first-line control strategy in case of epidemic emergence of plague, when immediate effect on rodent fleas is needed. Recommendations are given to improve the efficacy of the bait station method., Author summary Insecticide ground dusting inside houses is the recommended measure to control rat fleas responsible for bubonic plague transmission. The main inconvenience of this method is the direct contact of houseowners to the toxic insecticide dust and spillage in environment. A bait station approach, where the insecticide is confined in a box or tunnel containing rodent attractant, seems to be a valuable complementary or alternative vector control tool. However currently, little is known about its real efficacy on reducing or eliminating fleas harbored by rats. Guidelines regarding its implementation (density and duration of use) as vector control tool are lacking. Those questions were addressed during a field trial study, where bait stations were deployed at different densities per household and followed up at different time points. The efficacy of bait station was compared to the standard method. The present study allowed to demonstrate that bait station approach requires more improvements to be efficient. Meanwhile, insecticide ground dusting is still recommended for to control rat fleas during epidemics.

Yersinia pestis, the bacterial causative agent of plague, remains an important threat to human health. Plague is a rodent-borne disease that has historically shown an outstanding ability to colonize and persist across different species, habitats, and environments while provoking sporadic cases, outbreaks, and deadly global epidemics among humans. Between September and November 2017, an outbreak of urban pneumonic plague was declared in Madagascar, which refocused the attention of the scientific community on this ancient human scourge. Given recent trends and plague’s resilience to control in the wild, its high fatality rate in humans without early treatment, and its capacity to disrupt social and healthcare systems, human plague should be considered as a neglected threat. A workshop was held in Paris in July 2018 to review current knowledge about plague and to identify the scientific research priorities to eradicate plague as a human threat. It was concluded that an urgent commitment is needed to develop and fund a strong research agenda aiming to fill the current knowledge gaps structured around 4 main axes: (i) an improved understanding of the ecological interactions among the reservoir, vector, pathogen, and environment; (ii) human and societal responses; (iii) improved diagnostic tools and case management; and (iv) vaccine development. These axes should be cross-cutting, translational, and focused on delivering context-specific strategies. Results of this research should feed a global control and prevention strategy within a “One Health” approach., Author summary The historical aspect of plague makes for fascinating reading, due to its capacity to disrupt human society and its socioeconomic and cultural impacts throughout human history. We argue that the Madagascar outbreak in 2017 is a tipping point in human plague epidemiology and a call to elevate research priorities on plague as a matter of some urgency. In contrast with what occurred with the Ebola virus disease crisis in West Africa between 2013 and 2015 and the new coronaviruses (the emergence of severe acute respiratory syndrome coronavirus [SARS-CoV] and Middle East respiratory syndrome coronavirus [MERS-CoV] as early warnings of the current severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] pandemic), we have an opportunity to act preventively and enable evidence-based measures to avoid major health crises due to plague outbreaks in the near future.

Identifying key reservoirs for zoonoses is crucial for understanding variation in incidence. Plague re-emerged in Mahajanga, Madagascar in the 1990s but there has been no confirmed case since 1999. Here we combine ecological and genetic data, from during and after the epidemics, with experimental infections to examine the role of the shrew Suncus murinus in the plague epidemiological cycle. The predominance of S. murinus captures during the epidemics, their carriage of the flea vector and their infection with Yersinia pestis suggest they played an important role in the maintenance and transmission of plague. S. murinus exhibit a high but variable resistance to experimental Y. pestis infections, providing evidence of its ability to act as a maintenance host. Genetic analyses of the strains isolated from various hosts were consistent with two partially-linked transmission cycles, with plague persisting within the S. murinus population, occasionally spilling over into the rat and human populations. The recent isolation from a rat in Mahajanga of a Y. pestis strain genetically close to shrew strains obtained during the epidemics reinforces this hypothesis and suggests circulation of plague continues. The observed decline in S. murinus and Xenopsylla cheopis since the epidemics appears to have decreased the frequency of spillover events to the more susceptible rats, which act as a source of infection for humans. Although this may explain the lack of confirmed human cases in recent years, the current circulation of plague within the city highlights the continuing health threat., Author summary The reemergence of plague is related to the persistence and dynamics of its reservoirs and vectors. During the human plague outbreaks that re-occurred in Mahajanga harbor, Madagascar in the 1990s, the shrew Suncus murinus was found infected with Yersinia pestis. Combining field surveys, experimental infections and genetic analysis, we examined the role of Asian shrew Suncus murinus in plague transmission and maintenance comparatively with others potential hosts in Mahajanga. The genomes of nineteen Y. pestis isolates recovered from humans, shrews, rats and fleas in this focus were sequenced and compared. We observed the predominance of S. murinus captured during the epidemics and their carriage of the flea vector. Shrews exhibited high resistance to Y. pestis experimental infections. Genetic analyses of the strains isolated from various hosts were consistent with two partially-linked transmission cycles, with plague persisting within the S. murinus population, occasionally spilling over into the rat and human populations. The isolation of a Y. pestis strain from a rat in Mahajanga in 2014 genetically close to shrew strains reinforces this hypothesis. The current circulation of plague within the city highlights the continuing health threat.

International audience; Background. Susceptibility to infection is in part genetically driven, and C57BL/6 mice resist various pathogens through the proinflammatory response of their M1 macrophages (MPs). However, they are susceptible to plague. It has been reported elsewhere that Mus spretus SEG mice resist plague and develop an immune response characterized by a strong recruitment of MPs. Methods. The responses of C57BL/6 and SEG MPs exposed to Yersinia pestis in vitro were examined. Results. SEG MPs exhibit a stronger bactericidal activity with higher nitric oxide production, a more proinflammatory polarized cytokine response, and a higher resistance to Y. pestis-induced apoptosis. This response was not specific to Y. pestis and involved a reduced sensitivity to M2 polarization/signal transducer and activator of transcription 6 activation and inhibition of caspase 8. The enhanced M1 profile was inducible in C57BL/6 MPs in vitro, and when transferred to susceptible C57BL/6 mice, these MPs significantly increased survival of bubonic plague. Conclusions. MPs can develop an enhanced functional profile beyond the prototypic M1, characterized by an even more potent proinflammatory response coordinated with resistance to killing. This programming plays a key role in the plague-resistance phenotype and may be similarly significant in other highly lethal infections, suggesting that orienting the MP response may represent a new therapeutic approach.

International audience

International audience; Yersinia pestis, the agent of plague, is among the deadliest bacterial pathogens affecting humans, and is a potential biological weapon. Because antibiotic resistant strains of Yersinia pestis have been observed or could be engineered for evil use, vaccination against plague might become the only means to reduce mortality. Although plague is re-emerging in many countries, a vaccine with worldwide license is currently lacking. The vaccine strategy described here is based on an oral vaccination with an attenuated strain of Yersinia pseudotuberculosis. Indeed, this species is genetically almost identical to Y. pestis, but has a much lower pathogenicity and a higher genomic stability. Gradual modifications of the wild-type Yersinia pseudotuberculosis strain IP32953 were performed to generate a safe and immunogenic vaccine. Genes coding for three essential virulence factors were deleted from this strain. To increase cross-species immunogenicity, an F1-encapsulated Y. pseudotuberculosis strain was then generated. For this, the Y. pestis caf operon, which encodes F1, was inserted first on a plasmid, and subsequently into the chromosome. The successive steps achieved to reach maximal vaccine potential are described, and how each step affected bacterial virulence and the development of a protective immune response is discussed. The final version of the vaccine, named VTnF1, provides a highly efficient and long-lasting protection against both bubonic and pneumonic plague after a single oral vaccine dose. Since a Y. pestis strain deprived of F1 exist or could be engineered, we also analyzed the protection conferred by the vaccine against such strain and found that it also confers full protection against the two forms of plague. Thus, the properties of VTnF1 makes it one of the most efficient candidate vaccine for mass vaccination in tropical endemic areas as well as for populations exposed to bioterrorism.

Background Genetic analysis of pathogenic organisms is a useful tool for linking human cases together and/or to potential environmental sources. The resulting data can also provide information on evolutionary patterns within a targeted species and phenotypic traits. However, the instruments often used to generate genotyping data, such as single nucleotide polymorphisms (SNPs), can be expensive and sometimes require advanced technologies to implement. This places many genotyping tools out of reach for laboratories that do not specialize in genetic studies and/or lack the requisite financial and technological resources. To address this issue, we developed a low cost and low tech genotyping system, termed agarose-MAMA, which combines traditional PCR and agarose gel electrophoresis to target phylogenetically informative SNPs. Methodology/Principal findings To demonstrate the utility of this approach for generating genotype data in a resource-constrained area (Madagascar), we designed an agarose-MAMA system targeting previously characterized SNPs within Yersinia pestis, the causative agent of plague. We then used this system to genetically type pathogenic strains of Y. pestis in a Malagasy laboratory not specialized in genetic studies, the Institut Pasteur de Madagascar (IPM). We conducted rigorous assay performance validations to assess potential variation introduced by differing research facilities, reagents, and personnel and found no difference in SNP genotyping results. These agarose-MAMA PCR assays are currently employed as an investigative tool at IPM, providing Malagasy researchers a means to improve the value of their plague epidemiological investigations by linking outbreaks to potential sources through genetic characterization of isolates and to improve understanding of disease ecology that may contribute to a long-term control effort. Conclusions The success of our study demonstrates that the SNP-based genotyping capacity of laboratories in developing countries can be expanded with manageable financial cost for resource constraint laboratories. This is a practical formula that reduces resource-driven limitations to genetic research and promises to advance global collective knowledge of infectious diseases emanating from resource limited regions of the world., Author summary Although genetic characterization of pathogenic organisms is a powerful tool for investigating outbreak origins and transmission, associated high upfront costs and demanding technological maintenance exclude this tool for many under-resourced laboratories. Paradoxically, resource constrained regions commonly suffer from high rates of infectious diseases and could benefit most from genetic tracking tools. One such country is Madagascar, which lacks resources to acquire high tech genetic typing equipment, yet suffers from seasonal human plague outbreaks. A serious disease, plague is caused by the clonal bacterium, Yersinia pestis, and is capable of causing human outbreaks. Using plague as a model organism, we developed a genetic typing method that requires only basic, widely used molecular machinery. Our tools target unique single mutations in the Y. pestis genome to assign isolates to distinct phylogenetic groups with known geographical distributions. Transfer of this technology to Madagascar permits genetic characterization of strains from current outbreaks. This eliminates the need for external genetic analysis and expands the research capacity of this resource-constrained laboratory by allowing rapid, in-house strain typing. Ultimately, our goal is to help improve the ability of local institutes to genetically characterize circulating strains, link outbreaks to originating sources, and improve our understanding of the ecology of tropical diseases in resource-limited regions of the world.

Yersinia pestisis the etiologic agent of bubonic and pneumonic plagues. It is speculated thatY. pestishijacks antigen-presenting cells (APCs), such as dendritic cells (DCs) and alveolar macrophages, in order to be delivered to lymph nodes. However, how APCs initially capture the bacterium remains uncharacterized. It is well known that HIV-1 uses human DC-specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN) (CD209) receptor, expressed by APCs, to be captured and delivered to target cell, such as CD4+lymphocytes. Several gram-negative bacteria utilize their core lipopolysaccharides (LPS) as ligands to interact with the human DC-SIGN. Therefore, it is possible thatY. pestis, whose core LPS is naturally exposed, might exploit DC-SIGN to invade APCs. We demonstrate in this study thatY. pestisdirectly interacts with DC-SIGN and invades both DCs and alveolar macrophages. In contrast, when engineered to cover the core LPS,Y. pestisloses its ability to invade DCs, alveolar macrophages, and DC-SIGN-expressing transfectants. The interaction betweenY. pestisand human DCs can be reduced by a combination treatment with anti-CD209 and anti-CD207 antibodies. This study shows that human DC-SIGN is a receptor forY. pestisthat promotes phagocytosis by DCs in vitro.

Background No efficient vaccine against plague is currently available. We previously showed that a genetically attenuated Yersinia pseudotuberculosis producing the Yersinia pestis F1 antigen was an efficient live oral vaccine against pneumonic plague. This candidate vaccine however failed to confer full protection against bubonic plague and did not produce F1 stably. Methodology/Principal Findings The caf operon encoding F1 was inserted into the chromosome of a genetically attenuated Y. pseudotuberculosis, yielding the VTnF1 strain, which stably produced the F1 capsule. Given orally to mice, VTnF1 persisted two weeks in the mouse gut and induced a high humoral response targeting both F1 and other Y. pestis antigens. The strong cellular response elicited was directed mostly against targets other than F1, but also against F1. It involved cells with a Th1—Th17 effector profile, producing IFNγ, IL-17, and IL-10. A single oral dose (108 CFU) of VTnF1 conferred 100% protection against pneumonic plague using a high-dose challenge (3,300 LD50) caused by the fully virulent Y. pestis CO92. Moreover, vaccination protected 100% of mice from bubonic plague caused by a challenge with 100 LD50 Y. pestis and 93% against a high-dose infection (10,000 LD50). Protection involved fast-acting mechanisms controlling Y. pestis spread out of the injection site, and the protection provided was long-lasting, with 93% and 50% of mice surviving bubonic and pneumonic plague respectively, six months after vaccination. Vaccinated mice also survived bubonic and pneumonic plague caused by a high-dose of non-encapsulated (F1-) Y. pestis. Significance VTnF1 is an easy-to-produce, genetically stable plague vaccine candidate, providing a highly efficient and long-lasting protection against both bubonic and pneumonic plague caused by wild type or un-encapsulated (F1-negative) Y. pestis. To our knowledge, VTnF1 is the only plague vaccine ever reported that could provide high and durable protection against the two forms of plague after a single oral administration., Author Summary Yersinia pestis, the agent of plague, is among the deadliest infectious agents affecting humans. Injected in the skin by infected fleas, Y. pestis causes bubonic plague, which occasionally evolves into the very lethal and contagious pneumonic plague. Y. pestis is also a dangerous potential bioweapon but no plague vaccine is available. The current study describes the development of a vaccine highly efficient against plague in both its bubonic and pneumonic forms. The strategy consists of a live, avirulent, genetically modified Yersinia pseudotuberculosis that produces the capsule antigen of Y. pestis, named F1. The goal was to propose a vaccine that would be both easy to produce rapidly in large amounts with high quality, and easy to administer to individuals via a single oral dose. The VTnF1 strain described fulfills these demands. The immune response generated is long-lasting, involving both antibodies and memory cells directed against F1 and other antigens. We conclude that VTnF1 is a very promising candidate vaccine against plague.

Activation and/or recruitment of the host plasmin, a fibrinolytic enzyme also active on extracellular matrix components, is a common invasive strategy of bacterial pathogens. Yersinia pestis, the bubonic plague agent, expresses the multifunctional surface protease Pla, which activates plasmin and inactivates fibrinolysis inhibitors. Pla is encoded by the pPla plasmid. Following intradermal inoculation, Y. pestis has the capacity to multiply in and cause destruction of the lymph node (LN) draining the entry site. The closely related, pPla-negative, Y. pseudotuberculosis species lacks this capacity. We hypothesized that tissue damage and bacterial multiplication occurring in the LN during bubonic plague were linked and both driven by pPla. Using a set of pPla-positive and pPla-negative Y. pestis and Y. pseudotuberculosis strains in a mouse model of intradermal injection, we found that pPla is not required for bacterial translocation to the LN. We also observed that a pPla-cured Y. pestis caused the same extensive histological lesions as the wild type strain. Furthermore, the Y. pseudotuberculosis histological pattern, characterized by infectious foci limited by inflammatory cell infiltrates with normal tissue density and follicular organization, was unchanged after introduction of pPla. However, the presence of pPla enabled Y. pseudotuberculosis to increase its bacterial load up to that of Y. pestis. Similarly, lack of pPla strongly reduced Y. pestis titers in LNs of infected mice. This pPla-mediated enhancing effect on bacterial load was directly dependent on the proteolytic activity of Pla. Immunohistochemistry of Pla-negative Y. pestis-infected LNs revealed extensive bacterial lysis, unlike the numerous, apparently intact, microorganisms seen in wild type Y. pestis-infected preparations. Therefore, our study demonstrates that tissue destruction and bacterial survival/multiplication are dissociated in the bubo and that the primary action of Pla is to protect bacteria from destruction rather than to alter the tissue environment to favor Y. pestis propagation in the host., Author Summary The hallmark of bubonic plague, a disease that ravaged Medieval Europe and is still prevalent in several countries, is the bubo, a highly inflammatory and painful lymph node, which is characterized by high concentrations of bacteria within a severely damaged organ. Yersinia pestis, the causative agent, expresses a surface protease, Pla, critical to the development of bubonic plague. This multitarget protease has the potential to activate the fibrinolytic pathway and to promote destruction of extracellular protein networks within tissues. Hence, it was expected that Pla was responsible for the tissue destructions of the bubo, and consequently, for bacterial propagation and virulence. However, we found, using various engineered Yersinia strains in a mouse model of bubonic plague, that Pla proteolytic activity was dispensable for lymph node alteration, but was required to achieve high bacterial loads in the organ. Further analysis showed that Pla is essential for preventing the bacteria from being destroyed in the host. Therefore, the role of Pla as a virulence factor is to protect Y. pestis survival and integrity in the host, rather than to assist its spread through tissue destruction.

International audience; Fipronil was evaluated as a systemic control agent for the rat flea Xenopsylla cheopis (Rothschild), the main vector of Yersinia pestis (Yersin), the causative agent of plague, in Madagascar. The effectiveness of fipronil as a systemic control agent against X. cheopis was assessed by determining the toxicity values of the " Lethal Dose 50 " (LD 50). Two techniques were used to evaluate the systemic action of the insecticide on the vector: 1) an artificial feeding device filled with blood–fipronil mixture from which X. cheopis was fed and 2) rodent hosts, Rattus norvegicus (Berkenhout) and Rattus rattus (L.), which fed on fipronil-treated bait. As a standardized control method, the susceptibility of X. cheopis to fipronil was evaluated by exposure to impregnated paper within World Health Organization (WHO) insecticide test protocol to compare its effect to the systemic activity of the studied insecticide. Results showed that when administered in a systemic way, fipronil appears to be more effective: the toxicity level was evaluated to be ninefold higher compared with the WHO test. Compared with other methods, which require indiscriminate dusting of rodent burrows and human dwellings, fipronil applied in a systemic way enables the direct targeting of the plague vector. Thus, this method appears to be a superior alternative to fipronil-dusting for the control of the main plague vector in Madagascar. However, subsequent tests in the field are necessary to confirm the suitability of fipronil administration in a systemic way on large scales.

IL-10 is an anti-inflammatory mediator, important in limiting immunopathology. Its impact is influenced both by the timing and localization of its release. Here we show that NK cells rapidly express IL-10 during acute infection with the rapidly disseminating pathogens Toxoplasma gondii, Listeria monocytogenes or Yersinia pestis. Direct IL-12 signals proved necessary and sufficient for NK induction of IL-10. NK cells from T. gondii-infected mice inhibited dendritic cell release of IL-12 in an IL-10-dependent manner and NK cell depletion resulted in elevated serum IL-12. Together these data suggest an innate, negative feedback loop, in which IL-12 limits its own production by eliciting IL-10 from NK cells. In contrast to the systemic pathogens, NK cell IL-10 was not elicited by locally restricted infection with influenza virus or with a Y. pestis strain attenuated to prevent dissemination. Thus, systemic infections uniquely engage NK cells in an IL-10-mediated immunoregulatory circuit that functions to alleviate inflammation during sepsis.

International audience; Yersinia pestis, the plague bacillus, has a rodent-flea-rodent life cycle but can also persist in the environment for various periods of time. There is now a convenient and effective test (F1-dipstick) for the rapid identification of Y. pestis from human patient or rodent samples, but this test cannot be applied to environmental or flea materials because the F1 capsule is mostly produced at 37uC. The plasminogen activator (PLA), a key virulence factor encoded by a Y. pestis-specific plasmid, is synthesized both at 20uC and 37uC, making it a good candidate antigen for environmental detection of Y. pestis by immunological methods. A recombinant PLA protein from Y. pestis synthesized by an Escherichia coli strain was used to produce monoclonal antibodies (mAbs). PLA-specific mAbs devoid of cross-reactions with other homologous proteins were further cloned. A pair of mAbs was selected based on its specificity, sensitivity, comprehensiveness, and ability to react with Y. pestis strains grown at different temperatures. These antibodies were used to develop a highly sensitive one-step PLA-enzyme immunoassay (PLA-EIA) and an immunostrip (PLA-dipstick), usable as a rapid test under field conditions. These two PLA-immunometric tests could be valuable, in addition to the F1-disptick, to confirm human plague diagnosis in non-endemic areas (WHO standard case definition). They have the supplementary advantage of allowing a rapid and easy detection of Y. pestis in environmental and flea samples, and would therefore be of great value for surveillance and epidemiological investigations of plague foci. Finally, they will be able to detect natural or genetically engineered F1-negative Y. pestis strains in human patients and environmental samples. Citation: Simon S, Demeure C, Lamourette P, Filali S, Plaisance M, et al. (2013) Fast and Simple Detection of Yersinia pestis Applicable to Field Investigation of Plague Foci.

International audience; We have previously described SEG/Pas as the first mouse inbred strain able to survive subcutaneous injection of virulent Yersinia pestis, the agent of plague, and we identified Yprl1, Yprl2 and Yprl3 as three quantitative trait loci (QTLs) controlling this exceptional phenotype in females from a backcross between SEG/Pas and C57BL/6 strains. We have now developed congenic strains to further characterize the extent and effect of these genomic regions. In this study, we confirm the importance of two of these regions, both in males and females, while the third one may well be a spurious association. We show that no genomic region alone is able to increase the survival of C57BL/6 mice, but that C57BL/6 mice carrying both Yprl2 and Yprl3 exhibit intermediate resistance. Each of these two QTLs contains at least two subregions, which are required to increase survival. Finally, through the analysis of congenic strains in an F1 background, we establish the mode of inheritance of the SEG-derived resistance alleles. Altogether, this study has clarified and enhanced our understanding of the genetic architecture of resistance to plague in SEG/Pas mice.

International audience; Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI) to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba), followed by a colonization of the draining inguinal lymph node(s), and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response.

International audience; Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI) to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba), followed by a colonization of the draining inguinal lymph node(s), and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response.

Background Plague was introduced to Madagascar in 1898 and continues to be a significant human health problem. It exists mainly in the central highlands, but in the 1990s was reintroduced to the port city of Mahajanga, where it caused extensive human outbreaks. Despite its prevalence, the phylogeography and molecular epidemiology of Y. pestis in Madagascar has been difficult to study due to the great genetic similarity among isolates. We examine island-wide geographic-genetic patterns based upon whole-genome discovery of SNPs, SNP genotyping and hypervariable variable-number tandem repeat (VNTR) loci to gain insight into the maintenance and spread of Y. pestis in Madagascar. Methodology/Principal Findings We analyzed a set of 262 Malagasy isolates using a set of 56 SNPs and a 43-locus multi-locus VNTR analysis (MLVA) system. We then analyzed the geographic distribution of the subclades and identified patterns related to the maintenance and spread of plague in Madagascar. We find relatively high levels of VNTR diversity in addition to several SNP differences. We identify two major groups, Groups I and II, which are subsequently divided into 11 and 4 subclades, respectively. Y. pestis appears to be maintained in several geographically separate subpopulations. There is also evidence for multiple long distance transfers of Y. pestis, likely human mediated. Such transfers have resulted in the reintroduction and establishment of plague in the port city of Mahajanga, where there is evidence for multiple transfers both from and to the central highlands. Conclusions/Significance The maintenance and spread of Y. pestis in Madagascar is a dynamic and highly active process that relies on the natural cycle between the primary host, the black rat, and its flea vectors as well as human activity., Author Summary Plague, caused by the bacterium Yersinia pestis, has been a problem in Madagascar since it was introduced in 1898. It mainly affects the central highlands, but also has caused several large outbreaks in the port city of Mahajanga, after it was reintroduced there in the 1990s. Despite its prevalence, the genetic diversity and related geographic distribution of different genetic groups of Y. pestis in Madagascar has been difficult to study due to the great genetic similarity among isolates. We subtyped a set of Malagasy isolates and identified two major genetic groups that were subsequently divided into 11 and 4 subgroups, respectively. Y. pestis appears to be maintained in several geographically separate subpopulations. There is also evidence for multiple long distance transfers of Y. pestis, likely human mediated. Such transfers have resulted in the reintroduction and establishment of plague in the port city of Mahajanga where there is evidence for multiple transfers both from and to the central highlands. The maintenance and spread of Y. pestis in Madagascar is a dynamic and highly active process that relies on the natural cycle between the primary host, the black rat, and its flea vectors as well as human activity.

International audience; Laboratory mice are well known to be highly susceptible to virulent strains of Yersinia pestis in experimental models of bubonic plague. We have found that Mus spretus-derived SEG/Pas (SEG) mice are exceptionally resistant to virulent CO92 and 6/69 wild type strains. Upon subcutaneous injection of 10(2) colony-forming units (CFU), 90% of females and 68% of males survived, compared with only an 8% survival rate for both male and female C57BL/6 mice. Furthermore, half of the SEG mice survived a challenge of up to 10(7) CFU. The time required for mortality was similar between B6 and SEG, suggesting that survival is dependent on early rather than late processes. The analysis of 322 backcross mice identified three significant quantitative trait loci (QTLs) on chromosomes 3, 4 and 6, with dominant SEG protective alleles. Each QTL increased the survival rate by approximately 20%. The three QTLs function additively, thereby accounting for 67% of the difference between the parental phenotypes. Mice heterozygous for the three QTLs were just as resistant as SEG mice to Y. pestis challenge. The SEG strain therefore offers an invaluable opportunity to unravel mechanisms and underlying genetic factors of resistance against Y. pestis infection.

To the Editor: Yersinia pestis (family Enterobacteriaceae) is a bacterium that can cause high rates of death in susceptible mammals and can provoke septicemic, pneumonic, and bubonic plague in humans (1). This zoonotic pathogen can be transmitted directly by infectious droplets or by contact with contaminated fluid or tissue or indirectly through flea bites (1). Plague was introduced into Madagascar in 1898 from rat-infested steamships that had sailed from affected areas (2). Now, Madagascar is 1 of 2 countries in Africa that have reported cases of human plague every year since 1991 (3). During January 2008–January 2013, the number of human plague cases reported in Madagascar ranged from 312 to 648 per year. Of these, 61.8%–75.5% were laboratory confirmed (National Plague Laboratory of the Ministry of Health, pers. comm.). Most (>83%) confirmed cases were bubonic plague, which most commonly results from flea bites, suggesting that these bites were the most common mode of Y. pestis transmission. In Madagascar, Xenopsylla cheopis fleas have been known as the primary plague vector in urban areas, whereas Synopsyllus fonquerniei fleas have been usually involved in plague transmission in rural areas (2). In January 2013, a total of 9 suspected bubonic plague cases, 3 confirmed, were reported in Soavina, a rural area in the district of Ambatofinandrana, Madagascar. Domestic fleas were collected with candle traps inside 5 houses during 3 nights (Table). Fleas were also caught on small mammals trapped inside houses and outside in the sisal fences and rice fields (Table). A total of 319 fleas belonging to 5 species in 5 genera were collected inside and outside the houses, an average of 44 per house (maximum 71): Pulex irritans, Echidnophaga gallinacea, and Ctenocephalides canis fleas were collected inside the houses (244, 76.5%), and S. fonquerniei and X. cheopis fleas were collected outside (75, 23.5%). The human flea, P. irritans, was the most collected flea species (233, 73.3%), followed by S. fonquerniei (62, 19.4%), X. cheopis (13, 4.1%), E. gallinacea (10, 3.1%), and C. canis (1, 0.3%). Table Fleas collected inside and outside houses in Soavina, Madagascar, January 2013 Bacterial DNA was extracted from 277 fleas of 5 species: 233 P. irritans, 24 S. fonquerniei, 9 X. cheopis, 10 E. gallinacea, and 1 C. canis. PCR to detect Y. pestis was performed by using primers YP1 (5′-ATC TTA CTT TCC GTG AGA AG-3′) and YP2 (5′-CTT GGA TGT TGA GCT TCC TA-3′) to amplify a 478-bp fragment (4). Y. pestis DNA was then amplified and genotyped by Beckman Coulter Genomics Inc. (Takeley, United Kingdom). The positive control was Y. pestis reference strain (strain 6/69, 3 × 108 bacteria/mL; Institut Pasteur de Madagascar). Detection of Y. pestis was carried out on 274 fleas belonging to 5 flea species: 230 P. irritans (181 unfed and 49 engorged), 24 S. fonquerniei (15 unfed and 9 engorged), 9 X. cheopis (8 unfed and 1 engorged), 10 E. gallinacea (blood-feeding status not identified), and 1 unfed C. canis. Y. pestis was detected in 9 P. irritans fleas (7 male [6 unfed and 1 engorged] and 2 [engorged] female) from 3 houses, including the house where a confirmed human case of plague had occurred (Technical Appendix). Eight sequences (GenBank accession nos. {"type":"entrez-nucleotide-range","attrs":{"text":"KJ361938-KJ361945","start_term":"KJ361938","end_term":"KJ361945","start_term_id":"608789158","end_term_id":"608789172"}}KJ361938-KJ361945) were obtained and share 99% nucleotide homology with plasminogen activator genes of Y. pestis published in GenBank (accession nos. {"type":"entrez-nucleotide","attrs":{"text":"AF528537","term_id":"22212811","term_text":"AF528537"}}AF528537, {"type":"entrez-nucleotide","attrs":{"text":"AY305870","term_id":"32250732","term_text":"AY305870"}}AY305870). No Y. pestis was detected in the 24 S. fonquerniei, 9 X. cheopis, 10 E. gallinacea, or 1 C. canis fleas collected. Although only X. cheopis and S. fonqueniei fleas had previously been described as plague vectors in Madagascar, P. irritans fleas were most commonly collected during this field study; engorged and unfed male and female P. irritans fleas carried Y. pestis. Other studies have found P. irritans fleas in the plague risk area in other countries in Africa (5,6); one study found that P. irritans fleas may play a role in plague epidemiology in Tanzania (5). Data on P. irritans fleas in rats make it unlikely that these fleas are involved in rat-to-human transmission of Y. pestis in Madagascar. During 1922–1995, a total of 118,608 rats were caught and examined in Madagascar, but only 148 P. irritans fleas were identified, and none have been found on rats since 1996 (http://www.pasteur.mg/spip.php?rubrique124). The high density of P. irritans fleas we observed in villages where plague outbreaks occurred in late 2012 and early 2013 (http://www.pasteur.mg/spip.php?rubrique124) supports the possibility that P. irritans fleas played a role in domestic human-to-human transmission of Y. pestis during these outbreaks. Technical Appendix: Agarose gel electrophoresis showing positive PCR results for Yersinia pestis in DNA of 9 Pulex irritans fleas collected from 3 houses in an area of Madagascar where plague outbreaks occurred in late 2012 and early 2013. Click here to view.(47K, pdf)

From AD 1347 to AD 1353, the Black Death killed tens of millions of people in Europe, leaving misery and devastation in its wake, with successive epidemics ravaging the continent until the 18th century. The etiology of this disease has remained highly controversial, ranging from claims based on genetics and the historical descriptions of symptoms that it was caused by Yersinia pestis to conclusions that it must have been caused by other pathogens. It has also been disputed whether plague had the same etiology in northern and southern Europe. Here we identified DNA and protein signatures specific for Y. pestis in human skeletons from mass graves in northern, central and southern Europe that were associated archaeologically with the Black Death and subsequent resurgences. We confirm that Y. pestis caused the Black Death and later epidemics on the entire European continent over the course of four centuries. Furthermore, on the basis of 17 single nucleotide polymorphisms plus the absence of a deletion in glpD gene, our aDNA results identified two previously unknown but related clades of Y. pestis associated with distinct medieval mass graves. These findings suggest that plague was imported to Europe on two or more occasions, each following a distinct route. These two clades are ancestral to modern isolates of Y. pestis biovars Orientalis and Medievalis. Our results clarify the etiology of the Black Death and provide a paradigm for a detailed historical reconstruction of the infection routes followed by this disease., Author Summary Several historical epidemic waves of plague have been attributed to Yersinia pestis, the etiologic agent of modern plague. The most famous of these was the second pandemic which was active in Europe from AD 1347 until 1750, and began with the ‘Black Death’. The most informative method to establish the etiological nature of these ancient infections should be the analysis of ancient DNA, but the results of this method have been controversial. Here, by combining ancient DNA analyses and protein-specific detection, we demonstrate unambiguously that Y. pestis caused the Black Death. Furthermore, we show that at least two variants of Y. pestis spread over Europe during the second pandemic. The analysis of up to 20 diagnostic markers reveals that the two variants evolved near the time that phylogenetic branches 1 and 2 separated and may no longer exist. Our results thus resolve a long-standing debate about the etiology of the Black Death and provide key information about the evolution of the plague bacillus and the spread of the disease during the Middle Ages.

Yersinia pestis , the causative agent of plague, has recently diverged from the less virulent enteropathogen Yersinia pseudotuberculosis . Its emergence has been characterized by massive genetic loss and inactivation and limited gene acquisition. The acquired genes include two plasmids, a filamentous phage, and a few chromosomal loci. The aim of this study was to characterize the chromosomal regions acquired by Y. pestis . Following in silico comparative analysis and PCR screening of 98 strains of Y. pseudotuberculosis and Y. pestis , we found that eight chromosomal loci (six regions [R1 pe to R6 pe ] and two coding sequences [CDS1 pe and CDS2 pe ]) specified Y. pestis . Signatures of integration by site specific or homologous recombination were identified for most of them. These acquisitions and the loss of ancestral DNA sequences were concentrated in a chromosomal region opposite to the origin of replication. The specific regions were acquired very early during Y. pestis evolution and were retained during its microevolution, suggesting that they might bring some selective advantages. Only one region (R3 pe ), predicted to carry a lambdoid prophage, is most likely no longer functional because of mutations. With the exception of R1 pe and R2 pe , which have the potential to encode a restriction/modification and a sugar transport system, respectively, no functions could be predicted for the other Y. pestis -specific loci. To determine the role of the eight chromosomal loci in the physiology and pathogenicity of the plague bacillus, each of them was individually deleted from the bacterial chromosome. None of the deletants exhibited defects during growth in vitro . Using the Xenopsylla cheopis flea model, all deletants retained the capacity to produce a stable and persistent infection and to block fleas. Similarly, none of the deletants caused any acute flea toxicity. In the mouse model of infection, all deletants were fully virulent upon subcutaneous or aerosol infections. Therefore, our results suggest that acquisition of new chromosomal materials has not been of major importance in the dramatic change of life cycle that has accompanied the emergence of Y. pestis .

International audience; Yersinia pestis, the causative agent of bubonic plague, employs its type III secretion system to inject toxins into target cells, a crucial step in infection establishment. LcrV is an essential component of the T3SS of Yersinia spp, and is able to associate at the tip of the secretion needle and take part in the translocation of anti-host effector proteins into the eukaryotic cell cytoplasm. Upon cell contact, LcrV is also released into the surrounding medium where it has been shown to block the normal inflammatory response, although details of this mechanism have remained elusive. In this work, we reveal a key aspect of the immunomodulatory function of LcrV by showing that it interacts directly and with nanomolar affinity with the inflammatory cytokine IFNγ. In addition, we generate specific IFNγ mutants that show decreased interaction capabilities towards LcrV, enabling us to map the interaction region to two basic C-terminal clusters of IFNγ. Lastly, we show that the LcrV-IFNγ interaction can be disrupted by a number of inhibitors, some of which display nanomolar affinity. This study thus not only identifies novel potential inhibitors that could be developed for the control of Yersinia-induced infection, but also highlights the diversity of the strategies used by Y. pestis to evade the immune system, with the hijacking of pleiotropic cytokines being a long-range mechanism that potentially plays a key role in the severity of plague.

International audience; In Madagascar, the black rat, Rattus rattus, is the main reservoir of plague (Yersinia pestis infection), a disease still responsible for hundreds of cases each year in this country. This study used experimental plague challenge to assess susceptibility in wild-caught rats to better understand how R. rattus can act as a plague reservoir. An important difference in plague resistance between rat populations from the plague focus (central highlands) and those from the plague-free zone (low altitude area) was confirmed to be a widespread phenomenon. In rats from the plague focus, we observed that sex influenced plague susceptibility, with males slightly more resistant than females. Other individual factors investigated (weight and habitat of sampling) did not affect plague resistance. When infected at high bacterial dose (more than 10⁵ bacteria injected), rats from the plague focus died mainly within 3-5 days and produced specific antibodies, whereas after low-dose infection (< 5,000 bacteria), delayed mortality was observed and surviving seronegative rats were not uncommon. These results concerning plague resistance level and the course of infection in the black rat would contribute to a better understanding of plague circulation in Madagascar.

International audience; Plague is a pandemic human invasive disease caused by the bacterial agent Yersinia pestis. We here report a comparison of 17 whole genomes of Y. pestis isolates from global sources. We also screened a global collection of 286 Y. pestis isolates for 933 SNPs using Sequenom MassArray SNP typing. We conducted phylogenetic analyses on this sequence variation dataset, assigned isolates to populations based on maximum parsimony and, from these results, made inferences regarding historical transmission routes. Our phylogenetic analysis suggests that Y. pestis evolved in or near China and spread through multiple radiations to Europe, South America, Africa and Southeast Asia, leading to country-specific lineages that can be traced by lineage-specific SNPs. All 626 current isolates from the United States reflect one radiation, and 82 isolates from Madagascar represent a second radiation. Subsequent local microevolution of Y. pestis is marked by sequential, geographically specific SNPs.

International audience; Yersinia pestis isolates were genotyped analyzing the polymorphic DNA regions named variable number tandem repeats (VNTR). Allele variants were studied by high-resolution melting analysis (HRMA) of polymerase chain reaction fragments obtained for 25 VNTR loci. After comparison with previous results, 14 loci gave distinguishable normalized melting curves and allowed to correctly assign alleles. This HRMA typing technique permits to differentiate Y. pestis isolates and turned out to be robust, reproducible, and cheap.

International audience; Yersinia pestis is the aetiologic agent of plague. Without appropriate treatment, the pathogen rapidly causes septicaemia, the terminal and fatal phase of the disease. In order to identify bacterial genes which are essential during septicaemic plague in humans, we performed a transcriptome analysis on the fully virulent Y. pestis CO92 strain grown in either decomplemented human plasma or Luria-Bertani medium, incubated at either 28 or 37 degrees C and harvested at either the mid-exponential or the stationary growth phase. Y. pestis genes involved in 12 iron-acquisition systems and one iron-storage system (bfr, bfd) were specifically induced in human plasma. Of these, the ybt and tonB genes (encoding the yersiniabactin siderophore virulence factor and the siderophore transporter, respectively) were induced at 37 degrees C, i.e. under conditions mimicking the mammalian environment. Growth in human plasma also upregulated genes involved in the synthesis of five fimbrial-like structures (including the Psa virulence factor), and in purine/pyrimidine metabolism (the nrd genes). Genes known to play a role in the virulence of several bacterial pathogens (such as those encoding the Lpp lipoprotein and non-iron metal-uptake proteins) were induced in human plasma, during either the exponential or the stationary phase. Finally, 120 genes encoding proteins of unknown function were upregulated in human plasma. Eleven of these genes were specifically transcribed at 37 degrees C and may thus represent new virulence factors that are important during the septicaemic phase of human plague.

Two historical plague pandemics were likely caused by Orientalis-like strains of Yersinia pestis., Three pandemics have been attributed to plague in the last 1,500 years. Yersinia pestis caused the third, and its DNA was found in human remains from the second. The Antiqua biovar of Y. pestis may have caused the first pandemic; the other two biovars, Medievalis and Orientalis, may have caused the second and third pandemics, respectively. To test this hypothesis, we designed an original genotyping system based on intergenic spacer sequencing called multiple spacer typing (MST). We found that MST differentiated every biovar in a collection of 36 Y. pestis isolates representative of the three biovars. When MST was applied to dental pulp collected from remains of eight persons who likely died in the first and second pandemics, this system identified original sequences that matched those of Y. pestis Orientalis. These data indicate that Y. pestis caused cases of Justinian plague. The two historical plague pandemics were likely caused by Orientalis-like strains.

International audience; Nucleoside monophosphate kinases (NMPKs) are essential catalysts for bacterial growth and multiplication. These enzymes display high primary sequence identities among members of the family Enterobacteriaceae. Yersinia pestis, the causative agent of plague, belongs to this family. However, it was previously shown that its thymidylate kinase (TMPKyp) exhibits biochemical properties significantly different from those of its Escherichia coli counterpart [Chenal-Francisque, Tourneux, Carniel, Christova, Li de la Sierra, Barzu and Gilles (1999) Eur. J. Biochem. 265, 112-119]. In this work, the adenylate kinase (AK) of Y. pestis (AKyp) was characterized. As with TMPKyp, AKyp displayed a lower thermodynamic stability than other studied AKs. Two mutations in AK (Ser129Phe and Pro87Ser), previously shown to induce a thermosensitive growth defect in E. coli, were introduced into AKyp. The recombinant variants had a lower stability than wild-type AKyp and a higher susceptibility to proteolytic digestion. When the Pro87Ser substitution was introduced into the chromosomal adk gene of Y. pestis, growth of the mutant strain was altered at the non-permissive temperature of 37 degree C. In virulence testings, less than 50 colony forming units (CFU) of wild-type Y. pestis killed 100% of the mice upon subcutaneous infection, whereas bacterial loads as high as 1.5 x 10(4) CFU of the adk mutant were unable to kill any animals.

International audience; We report the complete 119,443-bp sequence of the pgm locus from Yersinia pestis and its flanking regions. Sequence analysis confirms that the 102-kb unstable pgm locus is composed of two distinct parts: the pigmentation segment and a high-pathogenicity island (HPI) which carries virulence genes involved in iron acquisition (yersiniabactin biosynthetic gene cluster). Within the HPI, three genes coding for proteins related to phage proteins were uncovered. They are located at both extremities indicating that the entire HPI was acquired en bloc by phage-mediated horizontal transfer. We identified, within the pigmentation segment, two novel loci that may be involved in virulence: a fimbriae gene cluster and a locus probably encoding a two component regulatory system similar to the BvgAS regulatory system of Bordetella pertussis . Three genes containing frameshift mutations and two genes interrupted by insertion element insertion were found within this region. To investigate diversity among different Y. pestis and Yersinia pseudotuberculosis strains, the sequence of selected regions of the pgm locus and flanking regions were compared from 20 different Y. pestis and 10 Y. pseudotuberculosis strains. The results showed that the genes interrupted in Y. pestis are intact in Y. pseudotuberculosis . However, one of these mutations, in the bvgS homologue, is only present in Y. pestis strains of biovar Orientalis and not in those of the biovars Antiqua and Medievalis. The results obtained by analysis of variable positions in the sequence are in accordance with historical records, confirming that biovar Orientalis is the most recent lineage. Furthermore, sequence comparisons among 29 Yersinia strains suggest that Y. pestis is a recently emerged pathogen that is probably entering the initial phase of reductive evolution.

The precise nature of the pathogen having caused early plague pandemics is uncertain. Although Yersinia pestis is a likely candidate for all three plague pandemics, the very rare direct evidence that can be deduced from ancient DNA (aDNA) analysis is controversial. Moreover, which of the three biovars, Antiqua, Medievalis or Orientalis, was associated with these pandemics is still debated. There is a need for phylogenetic analysis performed on Y. pestis strains isolated from countries from which plague probably arose and is still endemic. In addition there exist technical difficulties inherent to aDNA investigations and a lack of appropriate genetic targets. The recently described CRISPRs (clustered regularly interspaced short palindromic repeats) may represent such a target. CRISPR loci consist of a succession of highly conserved regions separated by specific "spacers" usually of viral origin. To be of use, data describing the mechanisms of evolution and diversity of CRISPRs in Y. pestis, its closest neighbors, and other species which might contaminate ancient DNA, are necessary. The investigation of closely related Y. pestis isolates has revealed recent mutation events in which elements constituting CRISPRs were acquired or lost, providing essential insight on their evolution. Rules deduced represent the basis for subsequent interpretation. In the present study, the CRISPR loci from representative Y. pestis and Yersinia pseudotuberculosis strains were investigated by PCR amplification and sequence analysis. The investigation of this wider panel of strains, including other subspecies or ecotypes within Y. pestis and also Y. pseudotuberculosis strains provides a database of the existing CRISPR spacers and helps predict the expected CRISPR structure of the Y. pestis ancestor. This knowledge will open the way to the development of a spoligotyping assay, in which spacers can be amplified even from highly degraded DNA samples. The data obtained show that CRISPR analysis can provide a very powerful typing tool, adapted to the systematic, large-scale genotyping of Y. pestis isolates, and the creation of international typing databases. In addition, CRISPRs do constitute a very promising new tool and genetic target to investigate ancient DNA. The corresponding genetic targets are small (

Fleas are wingless insects that parasitize mammals and more rarely birds. They are able to jump and may bite people. Adult fleas are hematophagous. The impact of fleas on public health is related to their ability to act as vectors for transmission of infectious agents during blood meals. The purpose of this article is to describe fleas and the diseases that they transmit to humans. Special focus is placed on epidemiological aspects., Les puces sont des insectes sans ailes, ectoparasites de mammifères, plus rarement d'oiseaux, et dont certaines espèces peuvent piquer l'homme. Les puces adultes ont la faculté de sauter et sont hématophages. L'importance des puces en santé publique humaine est surtout liée à leur capacité de transmission d'agents de maladies infectieuses au cours du repas sanguin. Dans cette revue, nous présentons les puces et les maladies humaines auxquelles elles sont liées, en nous concentrant sur les aspects épidémiologiques.