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Yersinia pestis, the causative agent of plague, is a highly virulent bacterium that poses a significant threat to human health. Preserving this bacterium in a viable state is crucial for research and diagnostic purposes. This paper presents and evaluates a simple lyophilization protocol for the long-term storage of Y. pestis strains from Fiocruz-CYP, aiming to explore its impact on viability and long-term stability, while replacing the currently used methodologies. The lyophilization tests were conducted using the non-virulent Y. pestis strain EV76, subjected to the lyophilization process under vacuum conditions. Viability assessment was performed to evaluate the effects of lyophilization and storage conditions on Y. pestis under multiple temperature conditions (- 80 °C, - 20 °C, 4-8 °C and room temperature). The lyophilization protocol employed in this study consistently demonstrated its efficacy in maintaining high viability rates for Y. pestis samples in a up to one year follow-up. The storage temperature that consistently exhibited the highest recovery rates was - 80 °C, followed by - 20 °C and 4-8 °C. Microscopic analysis of the post-lyophilized cultures revealed preserved morphological features, consistent with viable bacteria. The high viability rates observed in the preserved samples indicate the successful preservation of Y. pestis using this protocol. Overall, the presented lyophilization protocol provides a valuable tool for the long-term storage of Y. pestis, offering stability, viability, and functionality. By refining the currently used methods of lyophilization, this protocol can improve long-term preservation for Y. pestis strains collections, facilitating research efforts, diagnostic procedures, and the development of preventive and therapeutic strategies against plague.

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Background: Yersinia pestis, a Gram-negative bacterium, is the causative agent of plague. Y. pestis is a zoonotic pathogen that occasionally infects humans and became endemic in the western United States after spreading from California in 1899. Methods: To better understand evolutionary patterns in Y. pestis from the southwestern United States, we sequenced and analyzed 22 novel genomes from New Mexico. Analytical methods included, assembly, multiple sequences alignment, phylogenetic tree reconstruction, genotype-phenotype correlation, and selection pressure. Results: We identified four genes, including Yscp and locus tag YPO3944, which contained codons undergoing negative selection. We also observed 42 nucleotide sites displaying a statistically significant skew in the observed residue distribution based on the year of isolation. Overall, the three genes with the most statistically significant variations that associated with metadata for these isolates were sapA, fliC, and argD. Phylogenetic analyses point to a single introduction of Y. pestis into the United States with two subsequent, independent movements into New Mexico. Taken together, these analyses shed light on the evolutionary history of this pathogen in the southwestern US over a focused time range and confirm a single origin and introduction into North America.

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Yersinia pestis, the etiological agent of the plague, is considered a genetically homogeneous species. Brazil is currently in a period of epidemiological silence but plague antibodies are still detected in sentinel animals, suggesting disease activity in the sylvatic cycle. The present study deployed an in silico approach to analyze virulence factors among 407 Brazilian genomes of Y. pestis belonging to the Fiocruz Collection (1966-1997). The pangenome analysis associated several known virulence factors of Y. pestis in clades according to the presence or absence of genes. Four main strain clades (C, E, G, and H) exhibited the absence of various virulence genes. Notably, clade G displayed the highest number of absent genes, while clade E showed a significant absence of genes related to the T6SS secretion system and clade H predominantly demonstrated the absence of plasmid-related genes. These results suggest attenuation of virulence in these strains over time. The cgMLST analysis associated genomic and epidemiological data highlighting evolutionary patterns related to the isolation years and outbreaks of Y. pestis in Brazil. Thus, the results contribute to the understanding of the genetic diversity and virulence within Y. pestis and the potential for utilizing genomic data in epidemiological investigations.

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We developed a simple new selective LB-based medium, named CYP broth, suitable for recovering long-term stored Y. pestis subcultures and for isolation of Y. pestis strains from field-caught samples for the Plague surveillance. It aimed to inhibit the growth contaminating microorganisms and enrich Y. pestis growth through iron supplementation. The performance of CYP broth on microbial growth from different gram-negative and gram-positive strains from American Type Culture Collection (ATCC®) and other clinical isolates, field-caught rodent samples, and more importantly, on several vials of ancient Y. pestis subcultures was evaluated. Additionally, other pathogenic Yersinia species such as Y. pseudotuberculosis and Y. enterocolitica were also successfully isolated with CYP broth. Selectivity tests and bacterial growth performance on CYP broth (LB broth supplemented with Cefsulodine, Irgasan, Novobiocin, nystatin and ferrioxamine E) were evaluated in comparison with LB broth without additive; LB broth/CIN, LB broth/nystatin and with traditional agar media including LB agar without additive, and LB agar and Cefsulodin-Irgasan-Novobiocin Agar (CIN agar) supplemented with 50 µg/mL of nystatin. Of note, the CYP broth had a recovery twofold higher than those of the CIN supplemented media or other regular media. Additionally, selectivity tests and bacterial growth performance were also evaluated on CYP broth in the absence of ferrioxamine E. The cultures were incubated at 28 °C and visually inspected for microbiological growth analysis and O.D.625 nm measurement between 0 and 120 h. The presence and purity of Y. pestis growth were confirmed by bacteriophage and multiplex PCR tests. Altogether, CYP broth provides an enhanced growth of Y. pestis at 28 °C, while inhibiting contaminant microorganisms. The media is a simple, but powerful tool to improve the reactivation and decontamination of ancient Y. pestis culture collections and for the isolation of Y. pestis strains for the Plague surveillance from various backgrounds. KEY POINTS: • The newly described CYP broth improves the recuperation of ancient/contaminated Yersinia pestis culture collections • CYP broth was also efficient in reducing environmental contamination in field-capture samples, improving Y. pestis isolation • CYP broth can also be used for the isolation of Y. enterocolitica and Y. pseudotuberculosis.

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Plague meningitis is a serious and often fatal manifestation of Yersinia pestis infection. In the aftermath of a bioweapon attack with Y pestis, this typically rare manifestation may develop in a substantial number of patients, particularly if treatment delays occur. Risk factors, clinical evolution, and optimal treatment strategies for plague meningitis are not well understood. We searched PubMed Central and other databases for reports of plague meningitis in any language. Articles containing descriptions of patients with plague meningitis and their treatment and outcomes were included. Among 1,496 articles identified in our search, 56 articles describing 84 cases from 1898 to 2015 met inclusion criteria. The median age of patients was 16 years (range 6 weeks to 64 years); 68% were male. Most patients (n = 50, 60%) developed meningitis following primary bubonic plague. Common signs and symptoms included fever (n = 56, 66%), nuchal rigidity (n = 38, 45%), and headache (n = 33, 36%); 29% (n = 24) of patients had focal neurologic deficits such as cranial nerve abnormalities. Almost all (n = 23, 96%) of the 24 patients who did not receive antimicrobials died, and 42% (n = 25) of the 59 patients treated with antimicrobials died. The case fatality rate of patients grouped by antimicrobial received was 50% (1 out of 2) for fluoroquinolones, 19% (4 out of 21) for aminoglycosides, 14% (2 out of 14) for sulfonamides, 11% (2 out of 18) for chloramphenicol, and 0% (0 out of 13) for tetracyclines. Plague meningitis most often occurs as a complication of bubonic plague and can cause focal neurologic deficits. Survival is more likely in patients who receive antimicrobials; tetracyclines, aminoglycosides, and chloramphenicol had the lowest associated case fatality rates.

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The plague caused by the Yersinia pestis bacterium is primarily a flea-transmitted zoonosis of rodents that can also be conveyed to humans and other mammals. In this work, we analyzed the spatial and temporal distribution of rodent populations during epizootic and enzootic periods of the plague in the municipality of Exu, northeastern Brazil. The geospatial analyses showed that all the rodent species appeared through the whole territory of the municipality, with different occurrence hotspots for the different species. Important fluctuations in the rodent populations were observed, with a reduction in the wild rodent fauna following the end of a plague epizootic period, mostly represented by Necromys lasiurus and an increase in the commensal species Rattus rattus. A higher abundance of rats might lead to an increased exposure of human populations, favoring spillovers of plague and other rodent-borne diseases. Our analysis highlights the role of wild rodent species as amplifier hosts and of commensal rats (R. rattus) as preserver hosts in the enzootic period of a specific transmission infection area.

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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.

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The Yersinia pestis capsular antigen F1 is widely used in plague laboratory diagnosis. Here, we describe the production of an F1 recombinant protein within reduced time and biosafety requirements. Its evaluation in hemagglutination tests indicated that the recombinant F1 can replace the conventional F1 protein for plague diagnosis.

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Plague is a low incidence flea-borne zoonosis that is often fatal if treatment is delayed or inadequate. Outbreaks occur sporadically and human cases are often preceded by epizootics among rodents. Early recognition of epizootics coupled with appropriate prevention measures should reduce plague morbidity and mortality. For nearly a century, the flea index (a measure of fleas per host) has been used as a measure of risk for epizootic spread and human plague case occurrence, yet the practicality and effectiveness of its use in surveillance programs has not been evaluated rigorously. We sought to determine whether long-term monitoring of the Xenopsylla flea index on hut-dwelling rats in sentinel villages in the plague-endemic West Nile region of Uganda accurately predicted plague occurrence in the surrounding parish. Based on observations spanning ~6 yr, we showed that on average, the Xenopsylla flea index increased prior to the start of the annual plague season and tended to be higher in years when plague activity was reported in humans or rodents compared with years when it was not. However, this labor-intensive effort had limited spatial coverage and was a poor predictor of plague activity within sentinel parishes.

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Objetivo: caracterizar aspectos epidemiológicos e clínicos fundamentais, discutir a metodologia do diagnóstico e tecer recomendações sobre as condutas perante a suspeição de casos de peste. Métodos: revisão bibliográfica e levantamento das internações e mortes por peste registradas no Sistema de Informações Hospitalares do Sistema Único de Saúde. Resultados e conclusões: a existência de diagnósticos equivocados de uma doença potencialmente fatal, além dos registros hipotéticos de internações e mortes, constitui um desafio a ser superado, pois espelha uma situação inaceitável, em que um possível e insólito diagnóstico não é investigado e acumula-se nos sistemas de informação.

Objective: to characterize ground epidemiological and clinical aspects of, discuss the methodology of diagnosis and draw recommendations about the management of suspect cases of plague. Methods: literature review and data collection of hospitalizations and deaths due to plague recorded in the Hospital Information System of the Unified Health System. Results and conclusions: the existence of mistaken diagnoses of a potentially fatal disease, as well as hypothetical records of hospitalization and deaths, is a challenge to be overcome, because it reflects an unacceptable panorama in which a possible and unusual diagnosis is not investigated and accumulates in the information systems.

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RESUMEN La peste es una enfermedad reemergente causada por Yersinia pestis. Los humanos generalmente adquieren la enfermedad por picaduras de pulgas. La peste es una enfermedad sistémica fulminante, siendo la peste neumónica la forma más letal. El diagnóstico tardío es una de las principales causas de mortalidad y diseminación de la enfermedad, dado que limita la efectividad de las medidas de control. Presentamos el caso de un varón de 42 años, que previamente había viajado a una zona endémica de peste, y luego presentó hiperpirexia, hipotensión, y adenopatía inguinal inflamatoria. A pesar del cuadro clínico muy característico, nadie (antes del ingreso a nuestro hospital) sospechó peste. Se inició una combinación antibiótica efectiva y tratamiento intensivo recién al quinto día de enfermedad. El paciente evolucionó con shock séptico, falla respiratoria, y muerte. Se confirmó peste por reacción en cadena de polimerasa (PCR). Este caso enfatiza la importancia de tener un alto índice de sospecha para peste.

ABSTRACT Plague is a re-emerging disease caused by the bacteria Yersinia pestis. Humans usually get the disease through the bite of an infected flea. Plague is a fulminant systemic disease, with pneumonic plague being the most lethal form. Late diagnosis is one of the main causes of mortality and spread of the disease, as it limits the effectiveness of control measures. We present the case of a 42-year-old male, who had previously traveled to an endemic plague area and then presented hyperpyrexia, hypotension, and inflammatory inguinal adenopathy. Despite the very characteristic clinical picture, nobody (before admission to our hospital) suspected plague. An effective combination of antibiotics and intensive treatment was initiated only on the fifth day of illness. The patient went into septic shock, respiratory failure, and death. Plague was confirmed by polymerase chain reaction (PCR). This case emphasizes the importance of having a high suspicion rate for plague.

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We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23 CFU for pure culture, whereas 2.3 × 104 or 2.3 × 106 CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3 × 106 CFU, but PCR was negative at the level of 2.3 × 107 CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3 × 103 or 2.3 × 106 CFU, whereas 2.3 × 105 or 2.3 × 107 CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.(AU)

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ABSTRACT We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23 CFU for pure culture, whereas 2.3 × 104 or 2.3 × 106 CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3 × 106 CFU, but PCR was negative at the level of 2.3 × 107 CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3 × 103 or 2.3 × 106 CFU, whereas 2.3 × 105 or 2.3 × 107 CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.

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: The presence of keystone species can influence disease dynamics through changes in species diversity and composition of vector and host communities. In this study, we compared 1) the diversity of small mammals; 2) the prevalence, abundance, and intensity of arthropod vectors; and 3) the prevalence of Yersinia pestis, Francisella tularensis, and Bartonella spp. in vectors, between two grassland communities of northern Sonora, Mexico, one with (La Mesa [LM]) and one without (Los Fresnos [LF]) black-tailed prairie dogs ( Cynomys ludovicianus). The mammal community in LF exhibited higher species richness and diversity than LM, and species composition was different between the two communities. Flea species richness, prevalence, abundance, and intensity, were higher in LM than in LF. The most abundant fleas were Oropsylla hirsuta and Pulex simulans, and C. ludovicianus was the host with the highest flea intensity and richness. There was no serologic evidence for the presence of Y. pestis and F. tularensis in any community, but Bartonella spp. was present in 18% of the total samples. Some specificity was observed between Bartonella species, flea species, and mammal species. Although prairie dogs can indirectly affect the diversity and abundance of hosts and vectors, dynamics of vector-borne diseases at these spatial and temporal scales may be more dependent on the vector and pathogen specificity.

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We developed a loop-mediated isothermal amplification (LAMP) assay for the detection of Y. pestis by targeting the 3a sequence on chromosome. All 11 species of the genus Yersinia were used to evaluate the specificity of LAMP and PCR, demonstrating that the primers had a high level of specificity. The sensitivity of LAMP or PCR was 2.3 or 23CFU for pure culture, whereas 2.3×104 or 2.3×106CFU for simulated spleen and lung samples. For simulated liver samples, the sensitivity of LAMP was 2.3×106CFU, but PCR was negative at the level of 2.3×107CFU. After simulated spleen and lung samples were treated with magnetic beads, the sensitivity of LAMP or PCR was 2.3×103 or 2.3×106CFU, whereas 2.3×105 or 2.3×107CFU for magnetic bead-treated liver samples. These results indicated that some components in the tissues could inhibit LAMP and PCR, and liver tissue samples had a stronger inhibition to LAMP and PCR than spleen and lung tissue samples. LAMP has a higher sensitivity than PCR, and magnetic bead capture of DNAs could remarkably increase the sensitivity of LAMP. LAMP is a simple, rapid and sensitive assay suitable for application in the field or poverty areas.

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ABSTRACT Objectives: The plague, which is an infectious disease caused by Yersinia pestis, still threatens many populations in several countries. The worldwide increase in human plague cases and the potential use of the bacteria as a biological weapon reinforce the need to study the immunity that is induced by potential vaccine candidates. To determine the immunogenicity of antigenic preparations based on the F1 protein and the total extract from Y. pestis, we assessed the role of these antigens in inducing an immune response. Methods: The immunogenicity of antigenic preparations based on the Y. pestis (YP) total extract and the Y. pestis fraction 1 capsular antigen protein (F1) was determined in Swiss-Webster mice immunized with 40 µg or 20 µg for each preparation. Immunophenotyping was performed by flow cytometry. Results: Animals immunized with the YP total extract did not elicit detectable anti-F1 antibodies (Ab) in the hemaglutination/inhibition (HA/HI) test. Animals immunized with 40 µg or 20 µg of the F1 protein produced anti-F1 Abs, with titres ranging from 1/16 to 1/8132. The average of CD3+-CD4+ and CD3+-CD8+ T cells did not differ significantly between the groups. Neither YP total extract nor F1 protein induced a significant expression of IFN-γ and IL-10 in CD4+ T lymphocytes. In addition, F1 failed to induce IFN-γ expression in CD8+ T cells, unlike the YP total extract. Conclusion: The results showed that F1 protein is not an immunogenic T cell antigen, although the YP total extract (40 µg dose) favoured CD8+ T cell-mediated cellular immunity.

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Abstract INTRODUCTION: In Brazil, the plague is established in several foci located mainly in the northeastern part of the country, where it alternates between active and quiescent periods. These foci in the State of Ceará have high epidemiological importance. In addition to other plague detection activities, plague areas can be monitored through serological surveys of dogs and cats (domestic carnivores), which, following feeding on plague-infected rodents, can develop mild to severe forms of the disease and produce long-lasting antibodies. This study aimed to characterize the circulation dynamics and spatial distribution of Yersinia pestis antibodies in dogs and cats in plague foci areas of Ceará. METHODS: An ecological study was conducted to analyze the temporal series and spatial distribution of secondary data obtained from domestic carnivore serum surveillance in Ceará's plague areas from 1990 to 2014. RESULTS: Joinpoint analysis revealed that the overall trend was a reduction in antibody-positive animals. The mean proportion of antibody-positivity during the whole study period was 1.5% (3,023/203,311) for dogs, and 0.7% (426/61,135) for cats, with more than 4% antibody-positivity in dogs in 1997 and 2002. Antibody titers ranging from 1/16 to 1/64 were frequent. Despite fluctuations and a significant reduction, in recent years, there were antibody-positive animals annually throughout the study period, and the localities containing antibody-positive animals increased in number. CONCLUSION: Yersinia pestis is actively circulating in the study areas, posing a danger to the human population.

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OBJECTIVES: The plague, which is an infectious disease caused by Yersinia pestis, still threatens many populations in several countries. The worldwide increase in human plague cases and the potential use of the bacteria as a biological weapon reinforce the need to study the immunity that is induced by potential vaccine candidates. To determine the immunogenicity of antigenic preparations based on the F1 protein and the total extract from Y. pestis, we assessed the role of these antigens in inducing an immune response. METHODS: The immunogenicity of antigenic preparations based on the Y. pestis (YP) total extract and the Y. pestis fraction 1 capsular antigen protein (F1) was determined in Swiss-Webster mice immunized with 40µg or 20µg for each preparation. Immunophenotyping was performed by flow cytometry. RESULTS: Animals immunized with the YP total extract did not elicit detectable anti-F1 antibodies (Ab) in the hemaglutination/inhibition (HA/HI) test. Animals immunized with 40µg or 20µg of the F1 protein produced anti-F1 Abs, with titres ranging from 1/16 to 1/8132. The average of CD3+-CD4+ and CD3+-CD8+ T cells did not differ significantly between the groups. Neither YP total extract nor F1 protein induced a significant expression of IFN-γ and IL-10 in CD4+ T lymphocytes. In addition, F1 failed to induce IFN-γ expression in CD8+ T cells, unlike the YP total extract. CONCLUSION: The results showed that F1 protein is not an immunogenic T cell antigen, although the YP total extract (40µg dose) favoured CD8+ T cell-mediated cellular immunity.

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INTRODUCTION: Plague is an acute, infectious zoonotic disease, primarily of wild rodents and their fleas, that affects humans and other mammals. In Brazil, several plague foci are located in the northeast region. Plague surveillance based on monitoring of rodents was discontinued in 2007, and the current information on rodent populations is unsatisfactory. Our purpose was to update the information on rodents and other small mammals in plague foci in northeastern Brazil. METHODOLOGY: Nine surveys in the historically most important northeastern plague areas were conducted in 2013-2015. RESULTS: In this study, 393 animals (13 rodent and four marsupial species) were entrapped. The plague bacterium Yersinia pestis was not detected in tissue sample cultures from the 225 animals that were analyzed. Eighty sera samples were analyzed for anti-F1 antibodies by hemagglutination (HA) and protein A ELISA tests, and all were negative, except for one marsupial, Monodelphis domestica, which was HA positive. CONCLUSIONS: Qualitative and quantitative differences in the animal populations were observed in the areas surveyed, and the antibody positive marsupial indicated that plague continues to circulate in the wild.

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