Yersinia pestis can infect the Pawlowsky glands of human body lice and be transmitted by louse bite.

Yersinia pestis, the causative agent of plague, is a highly lethal vector-borne pathogen responsible for killing large portions of Europe's population during the Black Death of the Middle Ages. In the wild, Y. pestis cycles between fleas and rodents; occasionally spilling over into humans bitten by infectious fleas. For this reason, fleas and the rats harboring them have been considered the main epidemiological drivers of previous plague pandemics. Human ectoparasites, such as the body louse (Pediculus humanus humanus), have largely been discounted due to their reputation as inefficient vectors of plague bacilli. Using a membrane-feeder adapted strain of body lice, we show that the digestive tract of some body lice become chronically infected with Y. pestis at bacteremia as low as 1 × 105 CFU/ml, and these lice routinely defecate Y. pestis. At higher bacteremia (≥1 × 107 CFU/ml), a subset of the lice develop an infection within the Pawlowsky glands (PGs), a pair of putative accessory salivary glands in the louse head. Lice that developed PG infection transmitted Y. pestis more consistently than those with bacteria only in the digestive tract. These glands are thought to secrete lubricant onto the mouthparts, and we hypothesize that when infected, their secretions contaminate the mouthparts prior to feeding, resulting in bite-based transmission of Y. pestis. The body louse's high level of susceptibility to infection by gram-negative bacteria and their potential to transmit plague bacilli by multiple mechanisms supports the hypothesis that they may have played a role in previous human plague pandemics and local outbreaks.

Acquisition of yersinia murine toxin enabled Yersinia pestis to expand the range of mammalian hosts that sustain flea-borne plague.

Yersinia murine toxin (Ymt) is a phospholipase D encoded on a plasmid acquired by Yersinia pestis after its recent divergence from a Yersinia pseudotuberculosis progenitor. Despite its name, Ymt is not required for virulence but acts to enhance bacterial survival in the flea digestive tract. Certain Y. pestis strains circulating in the Bronze Age lacked Ymt, suggesting that they were not transmitted by fleas. However, we show that the importance of Ymt varies with host blood source. In accordance with the original description, Ymt greatly enhanced Y. pestis survival in fleas infected with bacteremic mouse, human, or black rat blood. In contrast, Ymt was much less important when fleas were infected using brown rat blood. A Y. pestis Ymt- mutant infected fleas nearly as well as the Ymt+ parent strain after feeding on bacteremic brown rat blood, and the mutant was transmitted efficiently by flea bite during the first weeks after infection. The protective function of Ymt correlated with red blood cell digestion kinetics in the flea gut. Thus, early Y. pestis strains that lacked Ymt could have been maintained in flea-brown rat transmission cycles, and perhaps in other hosts with similar blood characteristics. Acquisition of Ymt, however, served to greatly expand the range of hosts that could support flea-borne plague.

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.

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF THE PLAGUE VECTOR XENOPSYLLA BRASILIENSIS.

Members of the flea family Pulicidae have been the focus of many studies due to their significance as diseases vectors of medical and veterinary importance and their cosmopolitan distribution. They often exhibit variation in morphological features that can make correct species identification and management challenging. This may also apply to Xenopsylla brasiliensis (Baker, 1904), an important plague vector. In the current study, we aimed to provide genetic tools for reliable species identification using a DNA barcoding approach. A total of 73 flea specimens was collected from a native host (Namaqua rock mouse, Micaelamys namaquensis) in South Africa and identified morphologically. In addition, we took measurements of 7 morphological characteristics. Subsequently, we successfully generated barcodes of the mitochondrial cytochrome c oxidase subunit I (COI) gene for X. brasiliensis. We validated this approach by comparing our data to COI sequences from Rwandan X. brasiliensis. While sequences from both regions suggested a close relationship between the 2 X. brasiliensis populations, both haplotype and nucleotide diversity were substantially larger for the South African specimens. This may be attributed to human-assisted spread, differences in habitat, and/or host species sampled and merits further study in the future.

Molecular and Genetic Mechanisms That Mediate Transmission of Yersinia pestis by Fleas.

The ability to cause plague in mammals represents only half of the life history of Yersinia pestis. It is also able to colonize and produce a transmissible infection in the digestive tract of the flea, its insect host. Parallel to studies of the molecular mechanisms by which Y. pestis is able to overcome the immune response of its mammalian hosts, disseminate, and produce septicemia, studies of Y. pestis-flea interactions have led to the identification and characterization of important factors that lead to transmission by flea bite. Y. pestis adapts to the unique conditions in the flea gut by altering its metabolic physiology in ways that promote biofilm development, a common strategy by which bacteria cope with a nutrient-limited environment. Biofilm localization to the flea foregut disrupts normal fluid dynamics of blood feeding, resulting in regurgitative transmission. Many of the important genes, regulatory pathways, and molecules required for this process have been identified and are reviewed here.

Ectoparasitic community of the Mahali mole-rat, Cryptomys hottentotus mahali: potential host for vectors of medical importance in South Africa.

BACKGROUND: The endemic rodent family of Bathyergidae in Africa, particularly South Africa, are understudied as reservoirs of diseases of significant medical importance. Considering the diversity and wide distribution of African mole-rats in South Africa, many of these bathyergids could act as carriers of zoonoses. METHODS: The present study assessed the ectoparasite community of the Mahali mole-rat (Cryptomys hottentotus mahali). We aimed to identify possible parasitic arthropods that may infest this mole-rat species and explore host preference, contributions of seasonality, host sex and body mass as well as social class and colony size on ectoparasite assemblage prevalence and abundance. RESULTS: A limited number of ectoparasite species were found on C. h. mahali belonging to two significant taxa: mites (Acari) and fleas, with mites being the most prevalent and abundant. We recorded the presence of X. philoxera, a flea well known as the principal reservoir of plague in the southern African region on the Mahali mole-rats. Only three mite species were collected: Androlaelaps scapularis, Androlaelaps capensis and Laelaps liberiensis. Seasonal peaks in prevalence and abundance of X. philoxera and A. scapularis were observed during summer. Xenopsylla philoxera abundance and A. scapularis loads significantly increased on reproductive mole-rat individuals in comparison to non-reproductive individuals. CONCLUSION: Despite the wide distribution of the subterranean African mole-rats, studies investigating their parasitic fauna remain limited and scarce. This dearth in knowledge raises the concern regarding their potential role as an endemic reservoir for zoonotic diseases. Consequently, additional sampling of their ectoparasitic community throughout their distributional range and research addressing their role as a reservoir for zoonotic diseases in southern Africa are urgently needed.

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.

The role of louse-transmitted diseases in historical plague pandemics.

The rodent-murine ectoparasite-human model of plague transmission does not correspond with historical details around plague pandemics in Europe. New analysis of ancient genomes reveal that Yersinia pestis was unable to be transmitted by rat fleas until around 4000 Before Present, which challenges the rodent-murine ectoparasite-human model of plague transmission and historical details around plague pandemics in Europe. In this Review, we summarise data regarding Y pestis transmission by human lice in the context of genomic evolution and co-transmission of other major epidemic deadly pathogens throughout human history, with the aim of broadening our view of plague transmission. Experimental models support the efficiency of human lice as plague vectors through infected faeces, which suggest that Y pestis could be a louse-borne disease, similar to Borrelia recurrentis, Rickettsia prowazekii, and Bartonella quintana. Studies have shown that louse-borne outbreaks often involve multiple pathogens, and several cases of co-transmission of Y pestis and B quintana have been reported. Furthermore, an exclusive louse-borne bacterium, namely B recurrentis, was found to be circulating in northern Europe during the second plague pandemic (14th-18th century). Current data make it possible to attribute large historical pandemics to multiple bacteria, and suggests that human lice probably played a preponderant role in the interhuman transmission of plague and pathogen co-transmission during previous large epidemics, including plague pandemics.

Comparison of the transmission efficiency and plague progression dynamics associated with two mechanisms by which fleas transmit Yersinia pestis.

Yersinia pestis can be transmitted by fleas during the first week after an infectious blood meal, termed early-phase or mass transmission, and again after Y. pestis forms a cohesive biofilm in the flea foregut that blocks normal blood feeding. We compared the transmission efficiency and the progression of infection after transmission by Oropsylla montana fleas at both stages. Fleas were allowed to feed on mice three days after an infectious blood meal to evaluate early-phase transmission, or after they had developed complete proventricular blockage. Transmission was variable and rather inefficient by both modes, and the odds of early-phase transmission was positively associated with the number of infected fleas that fed. Disease progression in individual mice bitten by fleas infected with a bioluminescent strain of Y. pestis was tracked. An early prominent focus of infection at the intradermal flea bite site and dissemination to the draining lymph node(s) soon thereafter were common features, but unlike what has been observed in intradermal injection models, this did not invariably lead to further systemic spread and terminal disease. Several of these mice resolved the infection without progression to terminal sepsis and developed an immune response to Y. pestis, particularly those that received an intermediate number of early-phase flea bites. Furthermore, two distinct types of terminal disease were noted: the stereotypical rapid onset terminal disease within four days, or a prolonged onset preceded by an extended, fluctuating infection of the lymph nodes before eventual systemic dissemination. For both modes of transmission, bubonic plague rather than primary septicemic plague was the predominant disease outcome. The results will help to inform mathematical models of flea-borne plague dynamics used to predict the relative contribution of the two transmission modes to epizootic outbreaks that erupt periodically from the normal enzootic background state.

Control measures of a 400-year-old plague epidemic: an example of past efficiency at controlling disease and similarities with current epidemics.

The plague caused by the bacterium Yersinia pestis, provides one of the best historical examples of pandemic infection. It can therefore be considered the first "globalized" disease, thanks also to the crowds that favoured the rebalancing of infectious agents between Europe and the Middle East. In this paper we analyse all the official documents of the time, highlighting the most effective prevention measures implemented in the city of Ferrara during the Italian plague. Historical mortality data for the 1630 Italian plague in northern Italy are first analysed. In contrast to the high rates recorded throughout the area from Milan to Florence, the mortality rate in Ferrara remained normal over the period. From the city's documents it emerged that the authorities, from the 16th century onwards, had already understood that the spread of the contagion could also occur through domestic animals, although rats are never mentioned. The strength of Ferrara's response to the "plague emergency" stems from an efficient and emergency-ready health control system, financed and supported by the "permanent surveillance team of the city and the Pontifical Legation of Ferrara - Azienda Sanitaria Pubblica" even in times of great economic difficulty for the State. Among the various measures that the city of Ferrara adopted to deal with the plague the following should be mentioned: guards at the city gates, lazarettos, safety of doctors, self-isolation and treatment of every suspicious case as if it were a real case of plague, measures to support the poorer classes of the population, veterinary and hygiene standards for the city and for housing, management of Catholic religious functions and the precepts of the Legation of Ferrara, which was under papal control, closure of churches to avoid mass gatherings, and limitations of all kinds of social and economic relations within and outside the population. The broad regimen, laid down in the 16th century, contains extremely modern health rules which are very much in line with those recommended by the WHO and the health authorities of each individual state in the current COVID-19 pandemic, even starting with hand-washing. The fight against epidemics of the past, especially the history of the plague in the 17th century, anticipates very important and valid concepts, and represents a wake-up call for the recent epidemics of emerging pathogens.

Yersinia pestis: the Natural History of Plague.

The Gram-negative bacterium Yersinia pestis is responsible for deadly plague, a zoonotic disease established in stable foci in the Americas, Africa, and Eurasia. Its persistence in the environment relies on the subtle balance between Y. pestis-contaminated soils, burrowing and nonburrowing mammals exhibiting variable degrees of plague susceptibility, and their associated fleas. Transmission from one host to another relies mainly on infected flea bites, inducing typical painful, enlarged lymph nodes referred to as buboes, followed by septicemic dissemination of the pathogen. In contrast, droplet inhalation after close contact with infected mammals induces primary pneumonic plague. Finally, the rarely reported consumption of contaminated raw meat causes pharyngeal and gastrointestinal plague. Point-of-care diagnosis, early antibiotic treatment, and confinement measures contribute to outbreak control despite residual mortality. Mandatory primary prevention relies on the active surveillance of established plague foci and ectoparasite control. Plague is acknowledged to have infected human populations for at least 5,000 years in Eurasia. Y. pestis genomes recovered from affected archaeological sites have suggested clonal evolution from a common ancestor shared with the closely related enteric pathogen Yersinia pseudotuberculosis and have indicated that ymt gene acquisition during the Bronze Age conferred Y. pestis with ectoparasite transmissibility while maintaining its enteric transmissibility. Three historic pandemics, starting in 541 AD and continuing until today, have been described. At present, the third pandemic has become largely quiescent, with hundreds of human cases being reported mainly in a few impoverished African countries, where zoonotic plague is mostly transmitted to people by rodent-associated flea bites.

The changing triad of plague in Uganda: invasive black rats (Rattus rattus), indigenous small mammals, and their fleas.

Rattus rattus was first reported from the West Nile Region of Uganda in 1961, an event that preceded the appearance of the first documented human plague outbreak in 1970. We investigated how invasive R. rattus and native small mammal populations, as well as their fleas, have changed in recent decades. Over an 18-month period, a total of 2,959 small mammals were captured, sampled, and examined for fleas, resulting in the identification of 20 small mammal taxa that were hosts to 5,109 fleas (nine species). Over three-fourths (75.8%) of captured mammals belonged to four taxa: R. rattus, which predominated inside huts, and Arvicanthis niloticus, Mastomys sp., and Crocidura sp., which were more common outside huts. These mammals were hosts for 85.8% of fleas collected, including the efficient plague vectors Xenopsylla cheopis and X. brasiliensis, as well as likely enzootic vectors, Dinopsyllus lypusus and Ctenophthalmus bacopus. Flea loads on small mammals were higher in certain environments in villages with a recent history of plague compared to those that lacked such a history. The significance of these results is discussed in relation to historical data, the initial spread of plague in the WNR and the continuing threat posed by the disease.

Vector-Borne Pathogens in Ectoparasites Collected from High-Elevation Pika Populations.

The American pika, Ochotona princeps, is projected to decline throughout North America as climate change reduces its range, and pikas have already disappeared from several locations. In addition to climate, disease spillover from lower elevation mammalian species might affect pikas. We sampled pika fleas in Colorado and Montana across elevations ranging from 2896 to 3612 m and screened them for the presence of DNA from rodent-associated bacterial pathogens (Bartonella species and Yersinia pestis) to test the hypothesis that flea exchange between pikas and rodents may lead to occurrence of rodent-associated pathogens in pika ectoparasites. We collected 275 fleas from 74 individual pikas at 5 sites in Colorado and one site in Montana. We found that 5.5% of 275 pika fleas in this study tested positive for rodent-associated Bartonella DNA but that variation in Bartonella infection prevalence in fleas among sites was not driven by elevation. Specifically, we detected DNA sequences from two loci (gltA and rpoB) that are most similar to Bartonella grahamii isolates collected from rodents in Canada. We did not detect Y. pestis DNA in our survey. Our results demonstrate evidence of rodent-associated flea-borne bacteria in pika fleas. These findings are also consistent with the hypothesis that rodent-associated pathogens could be acquired by pikas. Flea-borne pathogen spillover from rodents to pikas has the potential to exacerbate the more direct effects of climate that have been suggested to drive pika declines.

Estimating the basic reproduction number for the 2015 bubonic plague outbreak in Nyimba district of Eastern Zambia.

BACKGROUND: Plague is a re-emerging flea-borne infectious disease of global importance and in recent years, Zambia has periodically experienced increased incidence of outbreaks of this disease. However, there are currently no studies in the country that provide a quantitative assessment of the ability of the disease to spread during these outbreaks. This limits our understanding of the epidemiology of the disease especially for planning and implementing quantifiable and cost-effective control measures. To fill this gap, the basic reproduction number, R0, for bubonic plague was estimated in this study, using data from the 2015 Nyimba district outbreak, in the Eastern province of Zambia. R0 is the average number of secondary infections arising from a single infectious individual during their infectious period in an entirely susceptible population. METHODOLOGY/PRINCIPAL FINDINGS: Secondary epidemic data for the most recent 2015 Nyimba district bubonic plague outbreak in Zambia was analyzed. R0 was estimated as a function of the average epidemic doubling time based on the initial exponential growth rate of the outbreak and the average infectious period for bubonic plague. R0 was estimated to range between 1.5599 [95% CI: 1.382-1.7378] and 1.9332 [95% CI: 1.6366-2.2297], with average of 1.7465 [95% CI: 1.5093-1.9838]. Further, an SIR deterministic mathematical model was derived for this infection and this estimated R0 to be between 1.4 to 1.5, which was within the range estimated above. CONCLUSIONS/SIGNIFICANCE: This estimated R0 for bubonic plague is an indication that each bubonic plague case can typically give rise to almost two new cases during these outbreaks. This R0 estimate can now be used to quantitatively analyze and plan measurable interventions against future plague outbreaks in Zambia.

What's eating you? oriental rat flea (Xenopsylla cheopis).

The oriental rat flea (Xenopsylla cheopis) is an ectoparasite of small mammals and a vector of many diseases for which humans are incidental hosts. This species of flea is most widely known for carrying Yersinia pestis and Rickettsia typhi, the causative agents of the plague and murine typhus, respectively. Public health issues related to X cheopis may increase in the future as global warming expands the geographic area in which the fleas can survive. A bioterrorist attack of plague also remains a threat. Extensive research is ongoing regarding X cheopis and its interaction with the bacteria it transmits to find better ways of reducing related morbidity and mortality. Traditional control measures include extermination of small mammal hosts, insecticide use to eliminate the flea itself, and use of antibiotics to control the associated diseases. The future may include targeted insecticide usage to prevent the continued development of resistance as well as new methods of reducing transmission of flea-borne diseases that could eliminate the need for chemical insecticides all together.

New ancient Eastern European Yersinia pestis genomes illuminate the dispersal of plague in Europe.

Yersinia pestis, the causative agent of plague, has been prevalent among humans for at least 5000 years, being accountable for several devastating epidemics in history, including the Black Death. Analyses of the genetic diversity of ancient strains of Y. pestis have shed light on the mechanisms of evolution and the spread of plague in Europe. However, many questions regarding the origins of the pathogen and its long persistence in Europe are still unresolved, especially during the late medieval time period. To address this, we present four newly assembled Y. pestis genomes from Eastern Europe (Poland and Southern Russia), dating from the fifteenth to eighteenth century AD. The analysis of polymorphisms in these genomes and their phylogenetic relationships with other ancient and modern Y. pestis strains may suggest several independent introductions of plague into Eastern Europe or its persistence in different reservoirs. Furthermore, with the reconstruction of a partial Y. pestis genome from rat skeletal remains found in a Polish ossuary, we were able to identify a potential animal reservoir in late medieval Europe. Overall, our results add new information concerning Y. pestis transmission and its evolutionary history in Eastern Europe. This article is part of the theme issue 'Insights into health and disease from ancient biomolecules'.

Acceleration of plague outbreaks in the second pandemic.

Historical records reveal the temporal patterns of a sequence of plague epidemics in London, United Kingdom, from the 14th to 17th centuries. Analysis of these records shows that later epidemics spread significantly faster ("accelerated"). Between the Black Death of 1348 and the later epidemics that culminated with the Great Plague of 1665, we estimate that the epidemic growth rate increased fourfold. Currently available data do not provide enough information to infer the mode of plague transmission in any given epidemic; nevertheless, order-of-magnitude estimates of epidemic parameters suggest that the observed slow growth rates in the 14th century are inconsistent with direct (pneumonic) transmission. We discuss the potential roles of demographic and ecological factors, such as climate change or human or rat population density, in driving the observed acceleration.

Human plague: An old scourge that needs new answers.

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.

Pentaplex real-time PCR for differential detection of Yersinia pestis and Y. pseudotuberculosis and application for testing fleas collected during plague epizootics.

Upon acquiring two unique plasmids (pMT1 and pPCP1) and genome rearrangement during the evolution from Yersinia pseudotuberculosis, the plague causative agent Y. pestis is closely related to Y. pseudotuberculosis genetically but became highly virulent. We developed a pentaplex real-time PCR assay that not only detects both Yersinia species but also differentiates Y. pestis strains regarding their plasmid profiles. The five targets used were Y. pestis-specific ypo2088, caf1, and pst located on the chromosome, plasmids pMT1 and pPCP1, respectively; Y. pseudotuberculosis-specific chromosomal gene opgG; and 18S ribosomal RNA gene as an internal control for flea DNA. All targets showed 100% specificity and high sensitivity with limits of detection ranging from 1 fg to 100 fg, with Y. pestis-specific pst as the most sensitive target. Using the assay, Y. pestis strains were differentiated 100% by their known plasmid profiles. Testing Y. pestis and Y. pseudotuberculosis-spiked flea DNA showed there is no interference from flea DNA on the amplification of targeted genes. Finally, we applied the assay for testing 102 fleas collected from prairie dog burrows where prairie dog die-off was reported months before flea collection. All flea DNA was amplified by 18S rRNA; no Y. pseudotuberculosis was detected; one flea was positive for all Y. pestis-specific targets, confirming local Y. pestis transmission. Our results indicated the assay is sensitive and specific for the detection and differentiation of Y. pestis and Y. pseudotuberculosis. The assay can be used in field investigations for the rapid identification of the plague causative agent.