Yersinia pestis

Monograph
Tables
What's New
Reviews
History

Y. pestis is the cause of bubonic, septicemic, and pneumonic plague. Plague is a zoonotic infection with its reservoirs in rodents and other animals. Humans can be considered accidental victims when they are bitten by rodent fleas or handle animal tissues or, rarely, inhale airborne bacteria from coughing patients or from infected animals. For readers interested in reviews that address human plague as well as bacterial genetics and virulence attributes of Y. pestis, they should consult some that were published in the past decade or so (11, 12, 39, 43, 64, 68, 75, 85).

MICROBIOLOGY

Y. pestis is a Gram-negative facultative anaerobic bacterium in the family of Enterobacteriaceae. Its characteristic laboratory features include nonmotility, oxidase-negative, catalase-positive, lactose-negative, indole-negative, phenylalanine deaminase-negative, rhamnose-negative, urease-negative, and optimal growth rate at 28-300C. The genus Yersinia contains two other pathogenic species, Y. enterocolitica and Y. pseudotuberculosis. Molecular clock analysis suggests that Y. pestis emerged as a clone of Y. pseudotuberculosis about 20,000 years ago by acquiring two virulence-associated plasmids that carried genes enabling flea bite transmission and by silencing genes that facilitated enteric transmission (1). Three different biovars, or biotypes, of Y. pestis are found in nature and are believed to have caused pandemics in different centuries and on different continents. The Antiqua biovar originated in the Mediterranean region to initiate the Justinian plague in 541 AD, the Medievalis biovar originated in West Asia before spreading to Europe in 1347 AD, and the Orientalis biovar started in Southern China around 1890 AD before spreading by ships to Africa and Americas to cause our current pandemic. The biovar phenotypes are distinguished by glycerol fermentation and nitrate reduction: Antiqua is glycerol-positive and nitrate-positive, Medievalis is glycerol-positive and nitrate-negative, and Orientalis is glycerol-negative and nitrate-positive.

Virulence Plasmids and Chromosomal Genes

The genome of Y. pestis and its 3 plasmids was published in 2001 (63). All 3 species of pathogenic Yersinia carry the same virulence plasmid with approximately 70 kb of DNA called the low-calcium response plasmid, or pYV or pCD, because it encodes for a type III secretion system (TTSS) and yersinial outer proteins (Yops) and V antigen which are expressed when bacterial growth is restricted by low concentrations of calcium at 37oC. Only Y. pestis has additionally a 9.5 kb plasmid called pPST/pPCP1 that encodes the plasminogen activator (Pla) and the bacteriocin pesticin as well as an approximately 100 kb plasmid called pFra/pMT1 that encodes a capsular protein called fraction 1(F1) antigen and the murine toxin. Chromosomal genes that are instrumental in pathogenicity include a pigmentation locus (pgm) that encodes for iron capture. Within the pgm locus is the hemin storage gene (hms) and a gene for the siderophore yersiniabactin (Ybt) that transports iron to bacteria. A high-pathogenicity island (HPI) within the pgm locus governs Ybt synthesis and transport (15). The anti-phagocytic pH 6 antigen (psa) is likewise chromosomally encoded as are genes for cell wall rough lipopolysaccharide (LPS), which lacks O-antigen side chains of the other Yersinia species. Outer membrane proteins in the Ail family confer resistance to complement-mediated killing and are also chromosomally encoded (3).

Virulence Factors

Several key virulence attributes of Y. pestis have been identified and studied in mutant bacteria lacking them (74). Experimental infections in animals and cell cultures have elucidated roles of virulence factors, some of which have been incorporated into promising vaccines. The factors to be included here are the F1 antigen, V antigen, Yops, Pla, murine toxin, pgm, psa, LPS, outer membrane proteins in Ail family, non-fimbrial adhesins in yadBC operon, and outer membrane lipoprotein.

F1 Antigen

Encoded by the 100 kb Fra/pMT1 plasmid, F1 is a protein that forms a fibrillar capsule around the bacterium (23). It causes resistance to phagocytosis by monocytes, macrophages, and dendritic cells through preventing adhesion-receptor interactions. F1 is produced during growth at 37oC but is absent when bacteria grow at lower environmental temperatures of 23-26oC. Thus, F1 antigen is perceived as relevant for plague ecology and pathogenesis by being absent in the flea at lower temperatures. Without F1 antigen, bacteria after a flea bite are taken up by dendritic cells and macrophages, where they survive and grow while producing F1 antigen that allows organisms in later stages of infection to multiply extracellularly while resisting phagocytosis (88).

V Antigen and Yops

The 70 kb low-calcium response plasmid pYV encodes for V antigen, Yops, and a TTSS. Expression of these proteins occurs when bacteria are grown at 37oC and are growth-restricted by low or absent calcium concentrations. V antigen is anti-inflammatory and mediates immunosuppression by upregulating interleukin-10 (IL-10), which in turn downregulates tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) (12). The TTSS is a needle-like injectisome about 5 nm in diameter that can deliver Yops from the bacterium into host cells. Targeted cells to receive Yops in infected animals are mainly neutrophils, macrophages, and dendritic cells (59). When several Yops have been separately analyzed, Yops B and D are parts of the TTSS needle, and V antigen forms the tip of the needle (61). Other Yops have enzymatic or signaling activities and are believed, along with V antigen, to play roles in inhibiting phagocytosis, inhibition of cytokine production, inhibition of platelet aggregation, apoptosis of macrophages, and immune suppression (67). Six of the Yops have defined actions after insertion into host cells: Yop E is a GTPase-activating protein that inhibits phagocytosis by downregulating multiple Rho GTPases; Yop H is a protein tyrosine phosphatase that inhibits phagocytosis; Yop O/YpkA is a threonine kinase that causes apoptosis of macrophages; Yop J/P is a cysteine protease that inhibits TNF-alpha by acting to deubiquitinate proteins resulting in inhibition of nuclear factor-kB (NF-kB) and also induces apoptosis of macrophages (89); Yop T is a cytotoxin that disrupts actin filaments; and Yop M transits to cell nuclei but has no known function (50). In experimental infections of rats, both a fully virulent strain of Yersinia pestis and a pYV-negative variant induced IL-17 production, but the pYV-negative infections results in more polymorphonuclear leukocytes (PMNs) in buboes with fewer bacteria, indiating that pYV suppressed a strong PMN response (22).

Pla

The small 9.5 kb plasmid encodes the Pla, which is an outer membrane aspartate protease (31). It’s considered a key virulence factor for both bubonic and pneumonic plague, but its several biological activities have resulted in changing concepts about its role in pathogenesis. Originally it was proposed as a modulator of blood coagulation with specific temperature-dependent properties. At environmental temperatures below 26oC it acted as a coagulase to clot blood meals in the flea gut, but the relevance of this function in flea transmission has been doubted (25, 27, 40). At a warmer temperature of 37oC, it exhibits activity to convert plasminogen to plasmin, which is fibrinolytic in rodents or humans to lyse fibrin clots. This lysis of fibrin clots at a flea bite site could release Y. pestis to migrate more readily to lymph nodes. The current view is that Pla is a spreading factor in subcutaneous tissues immediately after a flea bite. Additionally, Pla binds to and cleaves components of the extracellular matrix, such as fibronectin and laminin, furthering the ability of bacteria to spread in animal tissues after their introduction by a flea bite (47). Pla, as well as F1, may be released by bacteria into tissues during growth by outer membrane vesicles, which form on the surface of bacteria in cultures (24). In a mouse model of inhalational pneumonia, Pla was required for death within 4 days, rapid bacterial growth in the lung, extensive inflammation in lung, and higher quantities of pro-inflammatory cytokines (49). Furthermore, Pla inhibits the cationic antimicrobial peptide cathelicidin in pulmonary secretions, and this could enable Y. pestis to be a strong pathogen in the lung (31). As a multi-functional protease, Pla cleaves C3 of the complement system with the possible effect to reduce chemoattraction of leukocytes to sites of infection. Another function of Pla could be adherence to and invasion of nonphagocytic cells, which it was shown to enable in studies of HeLa cells, in which Pla caused rearrangement of actin (5). Interestingly, Pla degrades Yops but not V antigen (12), but it isn’t known whether Yop activity is lost during actual infection because Yops are exported from bacteria by the TTSS.

Murine Toxin

The large 100 kb plasmid encodes the murine toxin, so-called because it was originally described as lethal to mice and rats with toxicity that antagonized beta-adrenergic activity. More recently, this toxin , shown to be a phospholipase D, has been recast as instrumental in flea transmission by allowing bacteria after ingestion to survive and multiply in the flea midgut (37, 40). The phospholipase D may neutralize an antibacterial substance produced from rodent plasma by digestion in the flea midgut.

Pgm Locus Including hms and Ybt

Virulent Y. pestis needs iron for growth and uses the pgm locus to adsorb hemin, which contains ferric iron in the form of ferriprotoporphyrin chloride. It is called pigmentation locus because it is responsible for binding Congo red dye in culture media as well as hemin. Storage of hemin is governed by the hms locus, which has recently been shown necessary for efficient flea transmission because the hms gene directs development of a biofilm produced by Y. pestis in the flea’s proventriculus (40). The biofilm forms aggregates of bacteria that block flea swallowing, leading to regurgitation of Y. pestis into bite wounds when fleas attempt feeding on their next host. Also encoded by the pgm locus is the Ybt siderophore-dependent iron transport system (32). This siderophore is essential for removing iron from host proteins that chelate iron or heme. Another gene in the high-pathogenicity island of the pgm locus is RipA, which permits Y. pestis to survive in activated macrophages (69).

Psa

This anti-phagocytic antigen is a fimbrial structure that is expressed at 37oC in acidic medium. Encoded by chromosomal genes it has a surface structure and function similar to that of F1 antigen but acts independently of F1 to reduce uptake of bacteria by mouse macrophages (38). In a mouse model of bubonic plague, psa was upregulated by a transcriptional regulator gene RovA and was instrumental in early bacterial proliferation in regional lymph nodes after subcutaneous inoculation (16).

LPS

The Gram-negative cell wall structural molecule of LPS in Y. pestis is encoded by chromosomal genes. It differs from LPS of other enteric Gram-negative bacteria by not having an O side chain. Thus, colonies of Y. pestis on agar growth media appear rough rather than smooth. The shortened molecule of LPS contains fully toxic lipid A comprised of a disaccharide backbone to which are attached short-chain fatty acids and phosphate groups, which can produce fever and septic shock, and an oligosaccharide core that includes keto-deoxyoctanoic acid (KDO) and heptoses (42). This shortened LPS, sometimes called lipooligosaccharide (LOS), attaches to a receptor called dendritic cell specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN) that is on dendritic cells and macrophages. These cells transport Y. pestis to lymph nodes and, thereby, both present its antigens to the immune system and set the stage for an inflamed bubo (88). Several biological functions of Y. pestis have been correlated with LPS structure. Resistance to complement-mediated killing by serum has been correlated with increased content of N-acetyl-glucoseamine in the oligosaccharide core of bacterial strains with varying virulence (2, 66). Resistance to the cationic antibiotic polymyxin B is a general feature of virulent Y. pestis, and this resistance is greater when bacteria are grown at 25oC than at 37oC, resulting in lipid A with more interspersed molecules of 4-amino-4-deoxyarabinose (2). Bacteria grown at the lower temperature also produced lipid A with more acylation of the disaccharide backbone by fatty acids, and this lipid A stimulated macrophages to elaborate more TNF-alpha than lipid A from bacteria grown at 37oC (41). In experiments with bacteria other than Y. pestis, Pla activity was shown to require rough LPS, whereas O antigen from smooth LPS inhibited Pla activity (46).

Outer Membrane Proteins in Ail Family

Chromosomally encoded Ail proteins confer resistance to complement-mediated serum killing, allowing Y. pestis to survive in mammalian tissues such as blood and to be transmitted by fleas that ingest blood meals containing Y. pestis. These protective proteins are essential for virulence because rough colonies of Gram-negative bacilli lacking O side chains are usually susceptible to complement-mediated lysis and thus unable to cause sepsis (3).

Non-fimbrial Adhesins in yadBC operon

Surface proteins that allow Y. pestis to adhere to epithelial cells and to macrophages are believed to be important in early events in pathogenesis. Mutant bacteria defective in adhesins showed less invasion of epithelial cells and reduced lethality for mice when injected subcutaneously (27).

Outer Membrane Braun lipoprotein (Lpp)

A lipoprotein in the outer membrane of Y. pestis may play a pathogenic role in plague because Lpp acts synergistically with LPS to cause septic shock. Mutants of Y. pestis defective in Lpp showed reduced virulence in mice infected by both parenteral and intranasal routes (80).

EPIDEMIOLOGY

Occurrence

Plague is enzootic in rodents, with infection sometimes reaching humans, on the continents of Africa, Asia, South America, and North America. Only the continents of Europe, Australia, and Antarctica are spared. The majority of human cases reported to the World Health Organization (WHO) in recent years have been in African countries, followed in frequency by Asian countries, with the least numbers reported from the Americas (13, 86).

Considering the decade 2000-2009, the WHO reports of confirmed and suspected human cases in all countries was 21,725 with 1,612 deaths, for a case-fatality rate of 7.4%%. Countries that reported more than 100 cases were, from greatest to fewest: Congo (10,581 cases), Madaagscar (7,182 cases), Zambia (1,309 cases), Uganda (972 cases), Mozambique (600 cases), Tanzania (230 cases), China (227 cases), Peru (185 cases), Malawi (170 cases) and Indonesia (100 cases). The USA reported 57 cases. In Algeria plague re-emerged in 2003 with 18 reported cases and one death after 50 years of being free of the disease (6). An outbreak of 8 cases with 3 deaths was reported in 2004 in the Indian state of Uttaranchal (60). Also in 2004, there were 4 cases of pneumonic plague reported from Uganda with 3 deaths. Two of these were secondary pneumonias following bubonic and septicemic disease and 2 cases were primary inhalational pneumonia in caregivers of the secondary cases (4). In 2006 in USA, 13 cases with 2 deaths were reported in the states of New Mexico, Colorado, California, and Texas (17).

Transmission and Animal Reservoirs

The majority of the world’s cases of human plague are bubonic cases that are caused by rodent flea bites. Humans are considered accidental victims of this zoonotic infection when animal fleas aberrantly bite persons, sometimes prompted by an animal’s death from plague after which the flea seeks a new source of blood meals. The incubation period from flea bite to symptomatic disease is 2-10 days (29). Humans can be viewed as playing no role in the maintenance of plague in nature because rodent populations and their fleas suffice and because humans are poor transmitters of short-lived outbreaks of pneumonic plague. Most infected fleas come from the domestic black rat Rattus rattus or the brown sewer ratRattus norvegicus. The most common and efficient flea vector is Xenopsylla cheopis, but many other flea species can transmit plague. The oriental rat flea X. cheopis is more susceptible than other fleas to having the proventriculus of its digestive tract blocked by a blood meal containing Y. pestis because bacterial growth enables formation of an aggregative biofilm on the spicules of the proventriculus (40). Blocked fleas are unable to clear their midguts of infected blood, leading them to bite repeatedly and regurgitate bacteria into the skin of its next host. Unblocked fleas, under experimental conditions, were more effective in transmission than blocked fleas (25). Furthermore, the human flea Pulex irritans and the body louse Pediculus humanus humanus have been shown to contain Y. pestis (71, 72) with the implication that these alternative vectors might transmit plague under certain conditions or transmitted it in previous pandemics. In Madagascar flea transmission occurs mainly from R. rattus in the highlands but in the coastal city of Mahajanga house shrews also harbor infection (8). In addition to flea transmission, some cases are caused by direct handling of animal tissues when bacteria are inoculated through skin lacerations or when aerosolized bacteria are inhaled. Two of the cases in USA in 2006 were hunters who had skinned rabbits (17). Animals associated with human cases found to be infected or to harbor infected fleas in USA were white-tailed antelope squirrels, ground squirrels, rock squirrels, cottontail rabbits, jack rabbits, prairie dogs, deer mice, Colorado chipmunks, and wood rats (56, 58). One case in India in 2002 was a hunter who had killed and skinned a sick wildcat (34). In Mongolia in 2002, there were 6 cases of plague for which the most common mode of acquisition was hunters skinning sick marmots (86). In Qinghai Province of China in 2004, there were 19 human cases with 8 deaths most were hunters who butchered marmots (51). Domestic cats in USA were the source of 23 cases of human plague between 1977 and 1998 due to bites and scratches in most but, in 5 cases of primary pneumonia, due to inhaling cats’ respiratory secretions (29). Cats and other carnivores get their infection by eating rodents, but these carnivores play a minor role in plague epidemiology probably because their fleas are inefficient transmitters of infection. The first outbreak of pneumonic plague originating from a sick domestic dog occurred in China in 2009. A herdsman was the index case, who spread it to 11 others, resulting in 3 deaths (84). A sick camel in Saudi Arabia in 1994 was the source of 5 cases with 2 deaths; four persons who ate raw camel liver developed pharyngitis and one who had butchered the camel developed an axillary bubo (7). Camel meat also was the source of an outbreak of pharyngeal plague in Jordan in 1997 and an outbreak of gastroenteritis in Afghanistan in 2007 (13).

Primary inhalational lung infection is a rare form of transmission but can propagate person-to-person outbreaks. The index patient usually starts with bubonic or septicemic illness from a flea bite which develops into secondary pneumonia from bacteremic spread. Coughing produces airborne droplets that are inhaled by family or other up-close personal contacts. In Madagascar in 1997, an outbreak occurred that affected 18 persons with 8 deaths (73). The index case, who had bubonic plague with secondary pneumonia, spread infection to a traditional healer, who sucked bacteremic blood from the patient’s skin acquiring lung contagion that he passed to family and another patient before dying. At a funeral for the healer more cases occurred from airborne exposure. In Himachal State of northern India in 2002, an outbreak of pneumonic plague resulted in 16 cases and 4 deaths (34). It was started by a hunter who killed and skinned a sick wildcat, developing fever 5 days later followed by cough and hemoptysis suggestive of pneumonia. Before dying he infected family and other patients in a hospital. Incubation periods for primary pneumonia are about 3 days after contact with a coughing patient, and death usually follows in another 3 days unless antibiotic is administered on the first day of symptoms or prophylactically. Outbreaks of pneumonic plague have been restricted in recent years to a small number of cases over a few weeks because spread is inefficient by large droplets that require close contact with coughing patients in the last hours of terminal illness (44).

Susceptible Persons

All ages and both genders are susceptible to disease. Distributions by age and gender were not given in the WHO report (86), but most cases in recent decades were children with a slight preponderance of males. In Madagascar in 1996-1998, 61% of confirmed and presumptive cases were children and adolescents (0-19 years) and 57% were male (20). In the USA in 2006, the age range was 13-79 years, and 8 of the 13 cases were female (17). Exposure of persons to infected fleas where local rodents are transmitting infection is most determinative. Occurrence of an epizootic with a visible die-off of rodents that harbored plentiful fleas increases the chance of human cases. High incidences of cases are associated with poverty that results in substandard housing that isn’t rat-proof. In the U.S., however, increasing numbers of plague cases have occurred in affluent suburbs of Sante Fe and Albuquerque, where dwellings have wood piles that attract rodents (78). Warm climates in developing countries give rise to exposure of skin to flea bites due to persons’ uncovered legs and feet. Unsettled conditions of war and relocations of refugees with lack of public health services favor plague because rodents will feed on garbage in greater proximity to people’s dwellings. In Uganda, living conditions in plague areas were characterized by storage of garbage beside people's huts, in which they slept on mats of reeds or straw and allowed dogs inside (26).

Seasonality

Human plague in all endemic countries shows seasonal variation. The peak season corresponds to timing of epizootics with die-offs of susceptible rodents. These seasons can often be correlated with increases in fertility of rodent fleas, increases in rodent populations, and greater proximity of humans to infected animals. In USA the plague season is February-August (17). In Vietnam cases occur mainly in January-April. The highlands of Madagascar have a peak season in October-February, but the coastal city of Maharanja shows disease mainly in August-October (8). In Tanzania, the months of December- March are the peak season.

CLINICAL MANIFESTATIONS

Bubonic Plague

Bubonic plague is the most common clinical form of disease, present in about 80% of reported cases. After a flea bite or cutaneous inoculation of bacteria from handling animal tissues, organisms migrate to the nearest lymph node, where they proliferate and elicit inflammation that leads to fever and a swollen, painful, and tender lymph node, which is the bubo. Around the lymph node there is edema due to obstruction of lymphatic flow. Overlying skin is often warm and erythematous. Locations of buboes are, from most to least common, femoral, inguinal, axillary, cervical, and supraclavicular. Other symptoms that sometimes accompany bubonic plague are chills, headache, weakness, myalgias, anorexia, nausea, vomiting, and diarrhea. Cutaneous manifestations are present in about a fourth of cases as a pustule or eschar at the site of the presumed flea bite. Some patients develop petechial rashes and purpura due to vasculitis and disseminated intravascular coagulation. These skin changes rarely progress to cutaneous necrosis and gangrene that has necessitated amputation of digits or limbs several weeks after patients have recovered from their acute infection. Some patients develop secondary pneumonia a few days after the onset of the bubo with cough, dyspnea, pleuritic pain, and hemoptysis. Less often some patients develop meningitis a few days after the onset of the bubo with headache, stiff neck, and mental confusion. The bubo, in a minority of cases, may suppurate and spontaneously drain pus through the skin several days after the onset of disease. Untreated infection causes death in about 50% of cases in about 3-5 days after onset of symptoms with clinical features of septic shock, but antibiotic therapy reduces mortality to about 10%. Vital signs typically reveal elevated temperature, elevated pulse and respiratory rates, and decreases in blood pressure. Blood tests frequently show elevated white blood cell counts with increased band forms, reduced platelet counts, elevated creatinine concentrations, and elevated liver function tests.

Septicemic Plague

When plague bacteria inoculated by flea bite or handling of animal tissues fail to localize in a lymph node but proliferate in other tissues to circulate in the blood, the septicemic form evolves. This clinical form without a bubo goes often unrecognized until a blood culture is processed because it is a nonspecific febrile illness. Accompanying symptoms are similar to bubonic plague, as is the development of secondary pneumonia and meningitis. In USA about 25% of plague is septicemic with a mortality of 30-50%, which is higher than for bubonic because of delays in diagnosis and giving patients appropriate antibiotics. Recent patients in USA presented with abdominal pain, bloody stools, hematemesis, pulmonary infiltrates, hypoxemia, acidosis, thrombocytopenia, and renal failure (17, 58).

Pneumonic Plague

Primary pneumonia from inhalation of infected droplets is the rarest of the clinical forms. No cases of person-to-person transmission of pneumonic plague have been reported in USA since 1924, but 5 cases occurred in persons who inhaled aerosolized secretions from sick domestic cats (29). The incubation period averages 3 days. All patients die in about 3-5 days after onset of symptoms unless they are promptly treated within 24 hours after onset of symptoms. Typical symptoms are fever, chills, cough, hemoptysis, and pleuritic chest pain. Chest x-rays show infiltrates or lobar consolidation. Death follows cyanosis and septic shock. Secondary pneumonia in a patient with bubonic plague is more common than primary pneumonia and presents as cough and pulmonary infiltrate a few days after a bubo has developed.

LABORATORY DIAGNOSIS

Microscopic Examination of Clinical Specimens

The mainstay of rapid bedside diagnosis of bubonic plague is examination of the bubo aspirate. A sterile needle on a syringe containing one ml of sterile saline is inserted through skin into the center of the bubo. Saline is injected and immediately aspirated by vigorous withdrawal of the plunger until blood-tinged liquid appears in the syringe. A drop is placed onto a microscope slide for Gram stain or Wayson’s stain, which contains methylene blue. A diagnostic specimen contains many Gram-negative bacilli or blue bacilli with Wayson’s stain. The bacilli usually display darker staining at their ends giving characteristic bipolar accentuation, or the classical “safety pin” appearance. Other specimens, such as skin pus, sputum, and spinal fluid may also reveal bacilli on microscopic examination.

Culture of Y. pestis

Ever since the discovery of the causative bacterium by Alexandre Yersin in Hong Kong in 1894, isolation of the organism by culture is the traditional diagnostic method of choice. It is also the “gold standard” for a diagnosis of plague. Specimens of bubo aspirate, blood or sputum should be collected and appropriately plated onto blood and MacConkey agars and inoculated into nutrient broth for incubation at 37oC. Gram stain will reveal small Gram-negative bacilli. Characteristic colonies are small after 24 hours of growth and may require 48-72 hours to display lactose-negative colonies with irregular edges. Biochemical tests reveal subcultures to be catalase-positive, oxidase-negative, urease-negative, indole-negative, phenylalanine deaminase-negative, rhamnose-negative, and ONPG (O-nitrophenyl-beta-D-galactopyranoside)-positive (64, 68). On triple-sugar-iron slants it gives alkaline/acid reaction without gas or H2S. It is nonmotile. Reference laboratories confirm Y. pestis by specific bacteriophage lysis (21). Automated commercial methods don’t includeY. pestis in their data base and are likely to misidentify it (75).

Immunodiagnosis

A direct immunofluorescence assay using an antibody against F1 antigen can be applied to smears of bubo aspirates or sputum for rapid diagnosis or can be applied to bacteria from cultures. An enzyme-linked immunosorbent assay (ELISA) using antibody against F1 antigen for capture is in use at reference laboratories to detect F1 in clinical specimens. Patients with F1 antigen in blood showed a mortality rate of 17% in Madagascar, and most of those who died had concentrations of antigen greater than one µg/ml (18). A rapid dipstick test utilizing two monoclonal antibodies against F1 antigen – one as a capture antibody on a nitrocellulose strip, the other attached to colloidal gold particles on a polyester release pad - has been developed for field use (19). The Pasteur Institute workers applied the dipstick to bubo aspirates and other specimens in Madagascar finding it highly specific and potentially more sensitive than both culture and F1 ELISA. In an effort to modernize plague diagnosis, they propose using the dipstick at the bedside, omitting the cumbersome microscopic examination that is subject to vagaries of human judgment, and confirming diagnosis by culture. Hemagglutinating antibodies against F1 antigen rise in serum during the first week of illness and can be measured for evidence of plague infection. Paired sera, in acute and convalescent phases of illness, should demonstrate a 4-fold or greater rise in antibody titer, or a single serum specimen with a titer of 1:10 or greater is diagnostic. An ELISA is available to measure antibodies against F1 antigen in both classes of IgM, signifying recent or current infection, and of IgG, signifying infection at a more remote time.

Polymerase Chain Reaction (PCR)

PCR tests that use structural genes for F1 antigen, Pla, and murine toxin have been developed and may prove useful as specific and rapid tests for plague diagnosis (70). A PCR assay that amplified a portion of the F1 gene, when applied to bubo aspirates in Madagascar, showed high specificity but disappointing sensitivities of 35% to 89% when compared to culture or F1 ELISA (70). In sputum specimens to which Y. pestis was added, real-time PCR probe for F1 antigen in extracted DNA detected 102-104 cfu/ml in less than 5 hours with sensitivity dependent on inhibitors of the reaction in sputum (53). Another real-time PCR with probes for 16S rRNA and the 3 plague virulence factors was highly sensitive and specific with results in 3 hours (83). A need for these rapid genetic tests has been driven by preparations for bioterrorism defense because they can be adapted for use in the field by persons without microbiological training.

PATHOGENESIS

Flea Bite

Bubonic plague is initiated by inoculation of Y. pestis from an infected flea. The infective dose can be as low as one organism in experimental injections of cultured bacteria into animals, but actual counts of transmitted bacteria by fleas under experimental observation averaged over 600 per flea bite (54). Efficiency of transmission in nature appears to be low because fleas must feed on rodents with terminal high-grade bacteremia to ingest infective meals that average only 0. 12 µL. Bacteria inoculated by a flea that had its proventriculus blocked by a biofilm elaborated by Y. pestis are more resistant to phagocytosis by neutrophils than bacteria not associated with a biofilm and thus are more capable of surviving in the human (40). Furthermore, Y. pestis multiplies faster at 25oC than at 37oC and would thus be expected to grow in the flea midgut, where its murine toxin protects it from antibacterial substances elaborated by the flea. At 25oC its LPS additionally has greater resistance to phagocytic killing by cationic antimicrobial peptides present in host phagocytes (2). On the other hand, Y. pestis from fleas at usual ambient temperatures around 25oC do not have the anti-phagocytic F1 antigen nor do they produce anti-phagocytic Yops or V antigen, which require a higher temperature of 37oC.

Adherence and Spread

After inoculation into subcutaneous tissues, Y. pestis gets a foothold by adhesion to proteins in the extracellular matrix (ECM). This ability to adhere to mammalian tissues requires Pla, which specifically degrades a component of the ECM called laminin. Degradation of laminin probably allows bacteria to spread and aids their migration to lymph node (47). Pla also has the effect to generate plasmin for lysis of fibrin clots. Spread of bacteria is potentially assisted by fibrinolysis because fibrin clots would be formed at the flea bite wound. Migration to lymph nodes may also be facilitated by rough LPS of Y. pestis attaching to the DC-SIGN receptor on dendritic cells and macrophages, which in turn carry organisms via lymphatic vessels (88). Some of the inoculum that remains at the site of the bite multiply to elicit local inflammation as demonstrated by the red papule that forms in experimental rat infection (79) and the occurrence in some patients of pustules or eschars containing Y. pestis in skin areas drained by lymph nodes that became buboes. Early growth of bacteria in subcutaneous tissues at 37oC would result in generations of bacteria that possess the F1 antigen as well as V antigen and Yops.

Uptake by Macrophages and Neutrophils

A portion of inoculated bacteria is promptly phagocytosed by macrophages and neutrophils. Most bacteria ingested by neutrophils are killed, followed by apoptosis of the neutrophils, but most bacteria ingested by macrophages survive and multiply to acquire the anti-phagocytic F1 antigen, V antigen, and Yops. Essential steps for replication of Y. pestisin macrophages are uptake into phagosomes, enlargement of phagosomes, fusion of phagosomes with lysosomes, and resistance to killing by antimicrobial peptides like cathepsin D as well as resistance to unfavorable low concentrations of magnesium. A bacterial gene PhoP regulates this survival in macrophages by encoding activation of DNA transcription (33). Additionally, the RipA gene allows Y. pestis to survive in activated macrophages exposed to interferon-gamma (IFN-gamma) by reducing nitric oxide in the cells (69). Eventually bacteria are released from macrophages to multiply extracellularly up to massive loads that ultimately kill patients (57). Released bacteria resist further phagocytosis by using Yops and the TTSS to induce apoptosis of macrophages and neutrophils as well as keeping pro-inflammatory cytokines like TNF-alpha and IFN-gamma in check, with the result that bacteria proliferate unhindered. Experimental activation of macrophages by adding pro-inflammatory cytokines could, however, result in bacterial death (57).

Bubo Formation and Extracellular Growth

Y. pestis reaches a regional lymph node from the flea bite site by lymphatic flow. In experimental rat infection, extracellular bacteria appear in the marginal sinus of lymph nodes, and bacteria in macrophages and neutrophils arrive into germinal centers (79). Acute lymphadenitis develops with infiltration of the node by neutrophils. Additionally, the bubo contains thrombosed blood vessels, fibrin, hemorrhage, necrosis, and sheets of extracellular bacteria. Lymph node architecture is destroyed. Periglandular tissue also becomes involved with a sero-sanguinous exudate comprised of bacteria, blood cells, and fibrin. In experimental rat infection, increasing numbers of apoptotic cells were observed in lymph nodes between 36 and 72 hours after infection while extracellular bacteria were increasing and macrophages were declining (79). This rapid growth of bacteria is due, in part, to the ability ofY. pestis to acquire iron from host tissues through its chromosomal hms and Ybt genes (32).

Pathogen Recognition by Toll-like Receptors (TLR)

Relevant to uptake of Y. pestis by phagocytic cells, these host cells carry TLRs that recognize bacterial molecules and send signals to their nuclei for synthesis of cytokines. In particular, TLR2 recognizes bacterial cell wall peptidoglycan or lipoprotein and TLR4 recognizes LPS (11, 81). These TLRs activate the transcription factor nuclear factor-kB (NF-kB) to start production of pro-inflammatory cytokines including TNF-alpha, interleukin-6 (IL-6), and IFN-gamma, which mediate symptomatic disease and help control infection by activating phagocytes. At the same time, anti-inflammatory events are initiated by antigen V, which, acting through TLR2, induces IL-10, leading to inhibition of TLR synthesis and inhibition of NF-kB activation (12). The balancing of cytokine expression determines largely how disease progresses. Further, TLRs in plague serve as a bridge between the initial innate immune system encounter of bacteria with phagocytes and the adaptive immune response by T lymphocytes because mononuclear phagocytes differentiate into dendritic cells that present antigens to T lymphocytes (77).

Dissemination and Sepsis

If disease were confined to lymph nodes, bubonic plague would not be so often fatal. Bacteria break out of the bubo into blood to seed other organs that contain macrophages, mainly other lymph nodes, spleen, and liver. There, the same progression follows as occurred in the bubo from intracellular multiplication to extracellular explosive growth with extensive inflammatory injury. In experimental infections of rats and mice, the spleen is a crucial organ for propagation of bacteria toward high-grade lethal septicemia. In mice, splenic neutrophils initially limited growth of Y. pestis before the neutrophils underwent apoptosis. Bacteria next grew in macrophages followed by extracellular growth as well as apoptosis of the macrophages (79). In rats, 72 hours after inoculation, spleens showed marked splenitis, disruption of architecture with loss of periarterial lymphatic sheaths, lymphocytolysis in white pulp, and infiltration by bacteria mixed with fibrin, rare neutrophils, and macrophages (79). At this time, blood culture showed large numbers of Y. pestisbetween log10 cfu/ml of 3.4 and 9.0. For high-grade bacteremia to develop in plague, Y. pestis requires resistance to lysis by serum complement that is provided by LPS, outer membrane Ail proteins, and, possibly, activity of Pla to inactivate complement component C3. Overwhelming sepsis that leads to shock, disseminated intravascular coagulation, renal failure, and coma can be attributed to sheer bacterial mass that mounts in the spleen, blood, and lymph nodes, containing a sufficient quantity of LPS to be lethal.

Pneumonia

By bacteremic seeding, lung tissue sometimes becomes infected and develops its own inflammatory pathology with an added danger that Y. pestis will be aerosolized into droplets by coughing to spread to other persons. Primary plague pneumonia is the most lethal form of Y. pestis infection in nature. All untreated patients are destined to die after an incubation period of about 3 days after contact with a coughing patient plus another 3 days after the onset of symptoms. Inhaled bacteria initiate lung infection by adhering to respiratory tract epithelial cells, which was attributable to fimbrial psa in experiments with mutant organisms (52). Lungs of experimental animals show alveolar hemorrhage, extracellular bacteria in alveoli, neutrophilic infiltration, and destruction of lung architecture. Infection is lethal because bacteria spread from lung to blood and spleen to result in septicemia. In mouse lung, 2-3 days after inoculation, pro-inflammatory cytokines TNF, IFN-gamma, IL-6, and monocyte chemoattractant protein-1 were increased (48). Pla was essential for rapid replication of Y. pestis in airways because mutant bacteria without Pla proved nonlethal in mice. Its role could be that plasminogen activation to plasmin results in freeing bacteria from fibrin strands, thus allowing dissemination through the lung (49).

SUSCEPTIBILITY IN VITRO AND IN VIVO

In Vitro

Y. pestis isolates from human cases have generally shown consistent susceptibility to beta-lactams, tetracyclines, aminoglycosides, chloramphenicol, and fluoroquinolones (28,36, 85). On the other hand, most isolates were resistant to colistin, polymyxin B, and macrolides, including clarithromycin (28). Only 2 human isolates, both from Madagascar in 1995, have been reported as highly resistant to drugs in use for plague. The first case of drug resistance was in a 16-year-old male with bubonic plague. His organism was resistant to streptomycin, chloramphenicol, tetracycline, sulfonamide, ampicillin, kanamycin, and spectinomycin. It was susceptible to trimethoprim, which was the drug credited with saving his life because he had received streptomycin plus the combination of trimethoprim and sulfamethoxazole. This multidrug resistance was carried by a 150 kb plasmid, which was transferable in vitro to E. coli and back to Y. pestis. The second case was a 14-year-old patient who was infected with a strain that was resistant only to streptomycin. He also recovered while receiving trimethoprim-sulfamethoxozole. This resistance was encoded by a 40 kb plasmid that was transferable to other isolates of Y. pestis and was demonstrated to be transferable to other species in the flea gut (30).

In Vivo

When mice have been challenged with virulent Y. pestis by the subcutaneous, intravenous, or intranasal route, fatal systemic disease results that simulates human bubonic and pneumonic plague. In intravenous or subcutaneous infection, treatment with streptomycin, ceftriaxone, doxycycline, ciprofloxacin, and ofloxacin were effective in preventing death (9,76). In intranasal infections, treatment with streptomycin, ciprofloxacin, and ofloxacin were most effective (14). Cephalosporins, including ceftriaxone, were effective when started 24 hours after challenge but were inferior to aminoglycosides and fluoroquinolones when started 42 hours after challenge (14). The third generation fluoroquinolones, gatifloxacin and moxifloxacin, in both systemic and pneumonic models of infection were effective (82). In mice infected intranasally and made neutropenic by cyclophosphamide, both levofloxacin and gentamicin, which are bactericidal drugs, were highly effective. Doxycycline was effective in non-neutropenic mice, but this bacteriostatic antibiotic allowed regrowth of residual bacteria in neutropenic mice (35). A further advantage of fluoroquinolones was that, when drug-resistant mutants of Y. pestis were tested for pathogenicity in a neutropenic mouse thigh model, levofloxacin-resistant mutants were less pathogenic than streptomycin-resistant mutants (55). Human infections have been successfully treated for 60 years with the following drugs alone or in combination with others: streptomycin, tetracycline, chloramphenicol, doxycycline, gentamicin, kanamycin, and trimethoprim-sulfamethoxazole. Treatment with fluoroquinolons has been limited to one success with ciprofloxacin in the U.S. (45) and six surviving patients given ciprofloxacin in Algeria (71).

ANTIMICROBIAL THERAPY

The drug of choice for plague was streptomycin for several decades after its introduction in 1948. Streptomycin is credited with saving thousands of lives in Vietnam in the 1960-1975 war years. In Madagascar, the treatment of choice remains streptomycin, which is given together with trimethoprim-sulfamethoxazole. This country produces its own streptomycin, which is exclusively used for plague. However, manufacturers in other countries discontinued its production more than a decade ago due to nephrotoxicity and ototoxicity, which included deafness in infants whose mothers received it during pregnancy. G entamicin, also an aminoglycoside with some of the same toxicity potential, has largely replaced streptomycin, having the advantage of being measurable in serum assays in order for physicians to adjust dosage in order to avoid toxicity. Patients with plague have been successfully treated with gentamicin both in USA (10) and Tanzania (62). Gentamicin is recommended for intravenous or intramuscular dosing of 5mg/kg body weight a day in 3 divided doses or once daily for 7-10 days. The dose must be reduced when the creatinine clearance is below 50 ml per minute. Peak concentrations should be 4-10 µg per ml and trough concentrations 1-2 µg per ml. Equally effective and available for oral dosing are tetracycline and doxycycline. Tetracycline can be administered in a daily dose to adults of 2-4 g in 4 divided doses for 7-10 days. Doxycycline is preferred for its convenient dosing of 100 mg twice daily for adults or 2.2 mg per kg body weight twice daily for children. Doxycycline doesn’t need adjustment for diminished creatinine clearance, but tetracycline shouldn’t be used in renal failure. To be life-saving, antibiotic treatment must be started early. In an antibiotic trial in Tanzania, most patients sought care after only one day of symptoms, resulting in fatalities in only 5% of the cases (62). Fatal cases in USA, for which days of illness before treatment were recorded, were sick for 2-5 days (17, 29).

ADJUNCTIVE THERAPY

In most cases, antibiotic therapy suffices to cure patients. Defervescence occurs within a few days and buboes subside in size and pain in 1-2 weeks. A small percentage of patients will have buboes that suppurate and may benefit from incision and drainage. Some patients with advanced disease require hospitalization, even intensive care, for sepsis or pneumonia. Adjunctive therapy is sometimes appropriate with intravenous fluids, oxygen, mechanical ventilation, vasopressors, hemodialysis, and blood transfusion. In rare cases of gangrene affecting digits or extremities, amputations and skin grafting have been carried out.

ENDPOINTS FOR MONITORING THERAPY

Defervescence and stabilization of vital signs should be accomplished through antibiotic treatment and supportive care. Reductions in bubo size and tenderness should be monitored during therapy. Cultures of bubo aspirate and blood should be obtained for diagnosis. Antimicrobial susceptibilities of isolates should be determined to ensure that patients don’t have an unexpected antimicrobial-resistant infection. Follow-up cultures are not advised because neither relapse nor emergence of antimicrobial resistance during therapy is likely. Patients with cough should have a chest x-ray carried out as well as a sputum culture for Y. pestis. Toxicities of drug therapy should be monitored by symptomatic systems review and measurement of serum creatinine when aminoglycoside antibiotic is employed.

VACCINES

Vaccines to prevent plague have been used for more than a half-century for persons in endemic areas of the world, but the formalin-killed plague vaccine was discontinued by its U.S. manufacturer in 1998. A live attenuated vaccine that lacks the pgm genes has long been used in Europe and other countries but is not commercially available. New subunit vaccines that contain F1 antigen and V antigen of Y. pestis are currently being tested for safety and immunogenicity.

PREVENTION

In plague endemic areas, people should protect themselves by avoiding close contact with rodents, other animals likely to carry infection, and animal fleas (87). Anti-plague measures include making houses rodent-proof, removing harborage and food for rodents near homes, such as open garbage containers and old tires, dusting homes with insecticide powder, and wearing protective shoes, socks, and long pants. Hunters should not skin dead animals. To prevent person-to-person spread, patients with plague who have cough should be evaluated for pneumonia with a chest x-ray. If pneumonia is present, patients should be placed on respiratory isolation to protect against droplet spread to nearby contact persons for 4 days after antibiotic treatment is started.

REFERENCES

1. Achtman M, Morelli G, Zhu P, Wirth T, Diehl I, Kusecek B, Vogler AJ, Wagner DM, Allender CJ, Easterday WR, Chenal-Francisquel V, Worsham P, Thomson NR, Parkhill J, Lindler LE, Carniel E, Keim P. Microevolution and history of the plague bacillus, Yersinia pestis. PNAS 2004;101:17837-17842. [PubMed]

2. Anisimov AP, Dentovskaya SV, Titareva GM, Bakhteeva IV, Shaikhutdinova RZ, Balakhonov SV, Lindner B, Kocharova NA, Senchenkova SN, Holst O, Pier GB, Knirel YA. Intraspecies and temperature-dependent variations in susceptibility of Yersinia pestis to the bactericidal action of serum and to polymyxin B. Infect Immun 2005;73:7324-7331. [PubMed]

3. Bartra SS, Styer KL, O’Bryant DM, Nilles ML, Hinnebusch BJ, Aballay A, Plano GV. Resistance of Yersinia pestis to complement-dependent killing is mediated by the Ail outer membrane protein. Infect Immun 2008;76:612-622. [PubMed]

4. Begier EM, Asiki G, Anywaine Z, Yockey B, Schriefer ME, Aleti P, Ogen-Odoi A, Staples JE, Sexton C, Bearden SW, Kool JL. Pneumonic plague cluster, Uganda, 2004. Emerg Infect Dis 2006;12:460-467. [PubMed]

5. Benedek O, Nagy G, Emody L. Intracellular signalling and cytoskeletal rearrangement involved in Yersinia pestis plasminogen activator(Pla) mediated HeLa cell invasion. Microbiol Pathogen 2004;37:47-54. [PubMed]

6. Betherat E, Bekhoucha S, Chougrani S, Razik F, Duchemin JB, Houti L, Deharib L, Fayolle C, Makrerougrass B, Dali-Yahia R, Bellal R, Belhabri L, Chaieb A, Tikhomirov E, Carniel E. Plague reappearance in Algeria after 50 years, 2003. Emerg Infect Dis 2007;13:1459-1462. [PubMed]

7. Bin Saeed AA, Al-Hamadan NA, Fontaine RE. Plague from eating raw camel liver. Emerg Infect Dis 2005; 11:1456-1457. [PubMed]

8. Boisier P, Rahalison L, Rasolomaharo M, Ratsitorahina M, Mahafaly M, Razafimahefa M, Duplantier J-M, Ratsifasoamanana L, Chanteau S. Epidemiologic features of four successive annual outbreaks of bubonic plague in Mahajanga, Madagascar. Emerg Infect Dis 2002;8: 311-316. [PubMed]

9. Bonacorsi SP, Scavizzi MR, Guiyoule A, Amouroux JH, Carniel E. Assessment of a fluoroquinolone, three β-lactams, two aminoglycosides, and a cycline in the treatment of murine Yersinia pestis infection. Antimicrob Agents Chemother 1994; 38:481-486. [PubMed]

10. Boulanger LL, Ettestad P, Fogarty JD, Dennis DT, Romig D, Mertz G. Gentamicin and tetracycline for the treatment human plague: review of 75 cases in New Mexico, 1985-1999. Clin Infect Dis 2004: 38: 663-669. [PubMed]

11. Brubaker RR. How the structural gene products of Yersinia pestis relate to virulence. Future Microbiol 2007; 2;377-385. [PubMed]

12. Brubaker RR. Interleukin-10 and inhibition of innate immunity to Yersiniae: roles of Yops and LcrV(V antigen). Infect Immun 2003; 71: 3673-3681. [PubMed]

13. Butler T. Plague gives surprises in the first decade of the 21st century in the United States and worldwide. Am J Trop Med Hyg 2013; 89:788-793. [PubMed]

14. Byrne WR, Welkos SL, Pitt ML, Davis KJ, Brueckner RP, Ezzell JW, Nelson GO, Vaccaro JR, Battersby LC, Friedlander AM. Antimicrobial treatment of experimental pneumonic plague in mice. Antimicrob Agents Chemother 1998; 42:675-681. [PubMed]

15. Carniel E. The Yersinia high-pathogenicity island. Internatl Microbiol 1999; 2:161-167. [PubMed]

16. Cathelyn JS, Crosby SD, Lathem WW, Goldman WE, Miller VL. RovA, a global regulator of Yersinia pestis, specifically required for bubonic plague. PNAS 2006; 103: 13514-13519.[PubMed]

17. Centers for Disease Control. Human plague – four states, 2006. Morb Mort Wkly Rep 2006; 55: 940-944. [PubMed]

18. Chanteau S, Rabarijaona L, O’Brien T, Rahalison L, Hager J, Boisier P, Burans J, Rasolomaharo M. F1 antigenaemia in bubonic plague patients, a marker of gravity and efficacy of therapy. Trans R Soc Trop Med Hyg 1998; 92: 572-573. [PubMed]

19. Chanteau S, Rahalison L, Ralafiarisoa L, Foulon J, Ratsitorahina M, Ratsifasoamanana L, Carniel E, Nato F. Development and testing of a rapid diagnostic test for bubonic and pneumonic plague. Lancet 2003; 361:211-216. [PubMed]

20. Chanteau S, Ratsitorahina M, Rahalison L, Rasoamanana B, Chan F, Boisier P, Rabeson D, Roux J. Current epidemiology of human plague in Madagascar. Microb Infect 2000; 2:25-31.[PubMed]

21. Chu MC. Laboratory manual for plague diagnostic tests. Geneva, Switzerland: Centers for Disease Control and Prevention and World Health Organization, 2000. [PubMed]

22. Comer JE, Sturdevant DE, Carmody AB, Virtaneva K, Gardner D, Long D, Rosenke R, Porcella SF, Hinnebusch BJ. Transcriptomic and innate immune responses to Yersinia pestis in the lymph node during bubonic plague. Infect Immun 2010;78:5086-5098. [PubMed]

23. Du Y, Rosqvist R, Forsberg A. Role of fraction 1 antigen of Yersinia pestis in inhibition of phagocytosis. Infect Immun 2002; 70:1453-1460. [PubMed]

24. Eddy JL, Gielda LM, Caulfield AJ, Rangel SM, Lathem WW. Production of outer membrane vesicles by the plague pathogen Yersinia pestis. PLoS One 2014; 9:e107002.[PubMed]

25. Eisen RJ, Bearden SW, Wilder AP, Montenieri JA, Antolin MF, Gage KL. Early-phase transmission of Yersinia pestis by unblocked fleas as a mechanism explaining rapidly spreading plague epizootics. Proc Natl Acad Sci USA. 2006;103:15380-15385. [PubMed]

26. Eisen RJ, MacMillan K, Atiku LA, Mpanga JT, Zielinski-Gutierrez E, Graham CB, Boegler KA, Enscore RE, Gage KL. Identification of risk factors for plague in the West Nile region of Uganda. Am J Trop Med Hyg 2014; 90:1047-1058. [PubMed]

27. Forman S, Wulff CR, Myers-Morales T, Cowan C, Perry RD, Straley SC. yadBC of Yersinia pestis, a new virulence determinant for bubonic plague. Infect Immun 2008; 76:578-587.[PubMed]

28. Frean J, Klugman KP, Arntzen L, Bukofzer S. Susceptibility of Yersinia pestis to novel and conventional antimicrobial agents. J Antimicrob Chemother 2003; 52: 294-296. [PubMed]

29. Gage KL, Dennis DT, Orloski KA, Ettestad P, Brown TL, Reynolds PJ, Pape WJ, Fritz CL, Carter LG, Stein JD. Cases of cat-associated human plague in western USA, 1977-1998. Clin Infect Dis 2000; 30:893-900. [PubMed]

30. Galimand M, Carniel E, Courvalin P. Resistance of Yersinia pestis to antimicrobial agents. Antimicrob Agents Chemother 2006; 50:3233-3236. [PubMed]

31. Galvan EM, Lasaro MAS, Schifferli DM. Capsular antigen fraction 1 and Pla modulate the susceptibility of Yersinia pestis to pulmonary antimicrobial peptides such as cathelicidin. Infect Immun 2008; 76:1456-1464. [PubMed]

32. Geoffroy VA, Fetherston JD, Perry RD. Yersinia pestis YbtU and YbtT are involved in synthesis of the siderophore Yersiniabactin but have different effects on regulation. Infect Immun 2000; 68:4452-4461. [PubMed]

33. Grabenstein JP, Fukuto HS, Palmer LE, Bliska JB. Characterization of phagosome trafficking and identification of PhoP –regulated genes important for survival of Yersinia pestis in macrophages. Infect Immun 2006; 74: 3727-3741. [PubMed]

34. Gupta ML, Sharma A. Pneumonic plague, Northern India, 2002. Emerg Infect Dis 2007; 13:664- 666. [PubMed]

35. Heine HS, Louie A, Sorgel F, Bassett J, Miller L, Sullivan LJ, Kinzig-Schippers M, Drusano GL. Comparison of 2 antibiotics that inhibit protein synthesis for the treatment of infection by Yersinia pestis delivered by aerosol in a mouse model of pneumonic plague. J Infect Dis 2007; 196:782-787. [PubMed]

36. Hernandez E, Girardet M, Ramisse F, Vidal D, Cavallo J-D. Antibiotic susceptibilities of 94 isolates of Yersinia pestis to 24 antimicrobial agents. J Antimicrob Chemother 2003; 52: 1029-1031. [PubMed]

37. Hinnebusch BJ, Rudolf AE, Cherepanov P, Dixon JE, Schwan TG, Forsberg A. Role of Yersinia murine toxin in survival of Yersinia pestis in the midgut of the flea vector. Science 2002;296:733-735.[PubMed]

38. Huang X-Z, Lindler LE. The pH 6 antigen is an antiphagocytic factor produced by Yersinia pestis independent of Yersinia outer proteins and capsule antigen. Infect Immun 2004; 72:7212-7219. [PubMed]

39. Huang X-Z, Nikolich MP, Lindler LE. Current trends in plague research: from genomics to virulence. Clin Med Res 2006; 4:189- 199. [PubMed]

40. Jarrett CO, Deak E, Isherwood KE, Oyston PC, Fischer ER, Whitney AR, Kobayashi SD, DeLeo FR, Hinnebusch BJ. Transmission of Yersinia pestis from an infectious biofilm in the flea vector. J Infect Dis 2004; 190:783-792. [PubMed]

41. Kawahara K, Tsukano H, Watanabe H, Lindner B, Matsuura M. Modification of the structure and activity of lipidA in Yersinia pestis lipopolysaccharide by growth temperature. Infect Immun 2002; 70:4092-4098. [PubMed]

42. Knirel YA, Dentovskaya SV, Senchenkova SN, Shaikhutdinova RZ, Kocharova NA, Anisimov AP. Structural features and structural variability of the lipopolysaccharide of Yersinia pestis, the cause of plague. J Endotox Res 2006; 12:3-9. [PubMed]

43. Koirala J. Plague: disease, management, and recognition of act of terrorism. Infect Dis Clin NA 2006; 20:273-287. [PubMed]

44. Kool J. Risk of person-to-person transmission of pneumonic plague. Clin Infect Dis 2005; 40: 1166-1172. [PubMed]

45. Kuberski T, Robinson L, Schurgin A. A case of plague successfully treated with ciprofloxacin and sympathetic blockade for treatment of gangrene. Clin Infect Dis 2003; 36: 521-523. [PubMed]

46. Kukkonen M, Suomalainen M, Kylonen P, Lahteenmaki K, Lang H, Virkola R, Helander IM, Holst O, Korhonen TK. Lack of O-antigen is essential for plasminogen activation by Yersinia pestis and Salmonella enterica. Mol Microbiol 2004; 51:215-225. [PubMed]

47. Lahteenmaki K, Virkola R, Saren A, Emody L, Korhonen TK. Expression of plasminogen activator Pla of Yersinia pestis enhances bacterial attachment to the mammalian extracellular matrix. Infect Immun 1998; 66: 5755- 5762. [PubMed]

48. Lathem WW, Crosby SD, Miller VL, Goldman WE. Progression of primary pneumonic plague: a mouse model of infection, pathology, and bacterial transcriptional activity. PNAS 2005; 102: 17786-17791. [PubMed]

49. Lathem WW, Price PA, Miller VL, Goldman WE. A plasminogen-activating protease specifically controls the development of primary pneumonic plague. Science 2007; 315:509-513. [PubMed]

50. Lemaitre N, Sebbane F, Long D, Hinnebusch BJ. Yersinia pestis YopJ suppresses tumor necrosis factor alpha induction and contributes to apoptosis of immune cells in the lymph node but is not required for virulence in a rat model of bubonic plague.Infect Immun 2006; 74:5126-5131. [PubMed]

51. Li M, Song Y, Li B, Wang Z, Yang R, Jiang L, Yang R. Asymptomatic Yersinia pestis infection, China. Emerg Infect Dis 2005; 11: 1494-1496. [PubMed]

52. Liu F, Chen H, Galvan EM, Lasaro MA, Schifferli DM. Effects of Psa and F1 on the adhesive and invasive interactions of Yersinia pestis with human respiratory tract epithelial cells. Infect Immun 2006; 74; 5636-5644. [PubMed]

53. Loiez C, Herwegh S, Wallet F, Armand S, Guinet F, Courcol RJ. Detection of Yersinia pestis in sputum by real-time PCR. J Clin Microbiol 2003; 41:4873-4875. [PubMed]

54. Lorange EA, Race BL, Sebbane F, Hinnebusch BJ. Poor vector competence of fleas and the evolution of hypervirulence in Yersinia pestis. J Infect Dis 2005; 191:1907-1912.[PubMed]

55. Louie A, Deziel MR, Liu W, Drusano GL. Impact of resistance selection and mutant growth fitness on the relative efficacies of streptomycin and levofloxacin for plague therapy. Antimicrob Agents Chemother 2007: 51: 2661-2667. [PubMed]

56. Lowell JL, Magner DM, Atshaber B, Antolin MF, Vogler AJ, Keim P, Chu MC, Gage KL. Identifying sources of human exposure to plague. J Clin Microbiol 2005; 43: 650-656.[PubMed]

57. Lukaszewski RA, Kenny DJ, Taylor R, Rees DGC, Hartley MG, Oyston PCF. Pathogenesis of Yersinia pestis infection in BALB/c mice: effects on host macrophages and neutrophils. Infect Immun 2005; 73:7142-7150. [PubMed]

58. Margolis DA, Burns J, Reed SL, Ginsberg MM, O’Grady TC, Vinetz JM. Case report: septicemic plague in a community hospital in California. Am J Trop Med Hyg 2008; 78: 868-871. [PubMed]

59. Marketon MM, DePaolo RW, DeBord KL, Jabri B, Schneewind O. Plague bacteria target immune cells during infection. Science 2005; 309: 1739-1741. [PubMed]

60. Mittal V, Rana UVS, Jain SK, Kumar K, Pal IS, Arya RC, Ichhpujani RL, Lal S, Agarwal SP. Quick control of bubonic plague outbreak in Uttar Kashi, India. J Commun Dis 2004; 36:233-239. [PubMed]

61. Mueller CA, Broz P, Muller SA, Ringler P, Erne-Brand F, Sorg I, Kuhn M, Engel A, Cornelis GR. The V-antigen of Yersinia forms a distinct structure at the tip of injectisome needles. Science 2005; 310; 674-676. [PubMed]

62. Mwengee W, Butler T, Mgema S, Mhina G, Almasi Y, Bradley C, Formanik JB, Rochester CG. Treatment of plague with gentamicin or doxycycline in a randomized clinical trial in Tanzania. Clin Infect Dis 2006; 42: 614-621. [PubMed]

63. Parkhill J, Wren BW, Thomson NR, Titball RW, Holden MTG, Prentice MB, Sebalhia M, James KD, Churcher C, Mungall KL, Baker S, Basham D, Bentley SD, Brooks K, Cerdeno-Terrago AM, Chillingworth T, Cronin A, Davies RM, Davis P, Dougan G, Feltwell T, Hamlin N, Holroyd S, Jagels K, Karlyshev AV, Leather S, Moule S, Oyston PCF, Quail M, Rutherford K, Simmonds M, Skelton J, Stevens K, Whitehead S, Barrell BG. Genome sequence of Yersinia pestis, the causative agent of plague. Nature 2001; 413:523-527.[PubMed]

64. Perry RD, Fetherston JD. Yersinia pestis – etiologic agent of plague. Clin Microbiol Rev 1997; 10:35-66. [PubMed]

65. Piarroux R, Abedi AA, Shako JC, Kebela B, Karhemere S, Diatta G, Davoust B, Raoult D, Drancourt M. Plague epidemics and lice, Democratic Republic of the Congo. Emerg Infect Dis 2013; 19:505-506. [PubMed]

66. Porat R, McCabe WR, Brubaker RR. Lipopolysaccharide-associated resistance to killing of yersiniae by complement. J Endotox Res 1995; 2: 91-97. [PubMed]

67. Pouliot K, Pan N, Wang S, Lu S, Lien E, Goguen JD. Evaluation of the role of LcrV-Toll-like receptor 2-mediated immunomodulation in the virulence of Yersinia pestis. Infect Immun 2007; 75: 3571-3580. [PubMed]

68. Prentice MB, Rahalison L. Plague. Lancet 2007; 369:1196-1207. [PubMed]

69. Pujol C, Grabenstein JP, Perry RD, Bliska JB. Replication of Yersinia pestis in interferon γ-activated macrophages requires ripA, a gene encoded in the pigmentation locus. PNAS 2005;102:12909-12914. [PubMed]

70. Rahalison L, Vololonirina E, Ratsitorahina M, Chanteau S. Diagnosis of bubonic plague by PCR in Madagascar under field conditions. J Clin Microbiol 2000: 38:260-263.[PubMed]

71. Raoult D, Mouffok N, Bitam I, Piarroux R, Drancourt M. Plague: history and contemporary analysis. J Infect 2013; 66:18-26. [PubMed]

72. Ratovonjato J, Rajerison M, Rahelinirina S, Boyer S. Yersinia pestis in Pulex irritans fleas during plague outbreak, Madagascar. Emerg Infect Dis 2014; 20:1414-1415. [PubMed]

73. Ratsitorahina M, Chanteau S, Rahalison L, Ratsifasoamanana L, Boisier P. Epidemiological and diagnostic aspects of the outbreak of pneumonic plague in Madagascar. Lancet 2000;355: 111-113. [PubMed]

74. Reboul A, Lemaitre N, Titecat M, Merchez M, Deloison G, Ricard I, Pradel E, Marceau M, Sebbane F. Yersinia pestis requires the 2-component regulatory system OmpR-EnvZ to resist innate immunity during early and late stages of plague. J Infect Dis 2014; 210:1367-1375. [PubMed]

75. Rollins SE, Rollins SM, Ryan ET. Yersinia pestis and the plague. Am J Clin Pathol 2003; 119(Suppl 1):S78-S85. [PubMed]

76. Russell P, Eley SM, Bell DL, Manchee RJ, Titball RW. Doxycycline or ciprofloxacin prophylaxis and therapy against experimental Yersinia pestis infection in mice. J Antimicrob Chemother 1996; 37:769-774. [PubMed]

77. Saikh KU, Kissner TL, Sultana A, Ruthel G, Ulrich RG. Human monocytes infected with Yersinia pestis express cell surface TLR9 and differentiate into dendritic cells. J. Immunol 2004; 173: 7426-7434. [PubMed]

78. Schotthoefer AM, Eisen RJ, Kugeler KJ, Ettestad P, Reynolds PJ, Brown T, Enscore RE, Cheek J, Bueno R, Targhetta J, Montenieri JA, Gage KL. Changing socioeconomic indicators of human plague, New Mexico, USA. Emerg Infect Dis J 2012; 18:1151-1154. [PubMed]

79. Sebbane F, Gardner D, Long D, Gowen BB, Hinnebusch BJ. Kinetics of disease progression and host response in a rat model of bubonic plague. Am J Pathol 2005; 166:1427-1439. [PubMed]

80. Sha J, Agar SL, Baze WB, Olano JP, Fadl AA, Erova TE, Wang S, Foltz SM, Suarez G, Motin VL, Chaudhan S, Klimpel GR, Peterson JW, Chopra AK. Braun lipoprotein(Lpp) contributes to virulence of Yersiniae: potential role of Lpp in inducing bubonic and pneumonic plague. Infect Immun 2008; 76: 1390-1409. [PubMed]

81. Sing A, Reithmeier-Rost D, Granfors K, Hill J, Roggenkamp A, Heesemann J. A hypervariable N-terminal region of Yersinia LcrV determines Toll-like receptor 2-mediated IL-10 induction and mouse virulence. PNAS 2005; 102: 16049-16054. [PubMed]

82. Steward J, Lever MS, Russell P, Beedham RJ, Stagg AJ, Taylor RR, Brooks TJG. Efficacy of the latest fluoroquinolones against experimental Yersinia pestis. Int J Antimicrob Agents 2004; 24: 609-612. [PubMed]

83. Tomaso H, Reisinger EC, Al Dahouk S, Frangoulidis D, Rakin A, Landt O, Neubauer H. Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridization probes. FEMS Immunol Med Microbiol 2003; 38: 116-126. [PubMed]

84. Wang H, Cui Y, Wang Z, Wang X, Guo Z, Yan Y, Li C, Cui B, Xiao X, Yang Y, Qi Z, Wang G, Wei B, Yu S, He D, Chen H, Chen G, Song Y, Yang R. A dog-associated primary pneumonic plague in Qinhai Province, China. Clin Infect Dis 2011; 52:185-190. [PubMed]

85. Wong JD, Barash JR, Sandfort RF, Janda JM. Susceptibilities of Yersinia pestis strains to 12 antimicrobial agents. Antimicrob Agents Chemother 2000; 44: 1995-1996. [PubMed]

86. World Health Organization. Human plague in 2002 and 2003. Wkly Epidemiol Rec 2004; 79:301-308. [PubMed]

87. World Health Organization. Plague overview. Wkly Epidemiol Rec 2005; 80:138-140. [PubMed]

88. Zhang P, Skurnik M, Zhang S-S, Schwartz O, Kalyanasundaram R, Bulgheresi S, He JJ, Klena JD, Hinnebusch BJ, Chen T. Human dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin(CD209) is a receptor for Yersinia pestis that promotes phagocytosis by dendritic cells. Infect Immun 2008; 76:2070-2079. [PubMed]

89. Zhou H, Monack DM, Kayagaki N, Wertz I, Yin J, Wolf B, Dixit VM. Yersinia virulence factor YopJ acts as a deubiquitinase to inhibit NF-κB activation. J Exp Med 2005; 202: 1327-1332 [PubMed]

None