While the epizootic cycle of plague has not been completely understood, infection with Y. pestis is fundamentally a disease of rodents, and plague is found in various endemic foci around the world. Humans are incidental, “dead-end” hosts that become infected when the plague bacillus is transmit ted via flea bite or by exposure to fluids and tissues from an infected animal. As a result of such exposure, a serious infection will develop, often with a high mortality (40–100%). Plague has caused at least three major pandemics in previous centuries. The first pandemic occurred during the time of the Byzantine Empire in the 6th century, and the second pandemic, often called the “Black Death,” began in Central Asia and subsequently spread along ancient trade routes and reached Europe in 1346, where it spread quickly between the years 1347 and 1354, killing an estimated one-third of the population. During the 300 years following the “Black Death,” plague caused numerous smaller epidemics in various European countries. The third pandemic began during the 1850s in China, from where it spread via trade routes and steam ships to many countries, worldwide. While Y. pestis is an organism of considerable historic importance and interest, its potential use as an agent of Biowarfare has also been well recognized and documented. The ability of this organism to be easily transmitted by aerosol and the severity and high mortality associated with pneumonic plague make Y. pestis quite suitable for being a potential agent of biowarfare.

Morphology and Identification

Y. pestis is a Gram-negative rod that exhibits striking bipolar staining with special stains such as Wright, Giemsa, Wayson, or methylene blue (Figure 1). It is nonmotile. It grows as a facultative anaerobe on many bacteriologic media and can be readily isolated when sterile specimens such as blood or lymph node aspirates are plated onto sheep blood agar. Growth is more rapid when agar plates are incubated at 28°C. In cultures on sheep blood agar incubated at 37°C, colonies may be smaller when compared to colonies from agar plates incubated at 28°C. To enhance the recovery of Y. pestis from a nonsterile site specimen (eg, sputum), it is recommended to inoculate the specimen onto cefsulodin-irgasan-novobiocin (CIN) agar and incubate agar plates at 25–28°C. Colonies of Y. pestis are typically gray to white, sometimes opaque, and are 1–1.5 mm in diameter with irregular edges; the organism does not produce hemolysis.

Fig1. Y. pestis (arrows) in blood, Wright-Giemsa stain. Some of the Y. pestis have bipolar staining, which gives them a hairpin-like appearance. Original magnification ×1000. (Courtesy of K Gage, Plague Section, Centers for Disease Control and Prevention, Ft. Collins, CO.)

 Antigenic Structure

 All yersiniae possess lipopolysaccharides that have endotoxic activity when released. Y. pestis and Y. enterocolitica also produce antigens and toxins that act as virulence factors. They have type III secretion systems that consist of a membrane spanning complex that allows the bacteria to inject proteins directly into cytoplasm of the host cells. The virulent yersiniae produce V and W antigens, which are encoded by genes on a plasmid of approximately 70 kb. This is essential for virulence; the V and W antigens yield the requirement for calcium for growth at 37°C. Compared with the other pathogenic yersiniae, Y. pestis has gained additional plasmids. pPCP1 is a 9.5-kb plasmid that contains genes that yield plasminogen-activating protease that has temperature-dependent coagulase activity (20–28°C, the temperature of the flea) and fibrinolytic activity (35–37°C, the temperature of the host). This factor is involved in dissemination of the organism from the flea bite injection site. The pFra/pMT plasmid (80–101 kb) encodes the capsular protein (fraction F1) that is produced mainly at 37°C and confers antiphagocytic properties. In addition, this plasmid contains genes that encode phospholipase D, which is required for organism survival in the flea midgut.

Y. pestis and Y. enterocolitica have a pathogenicity island (PAI) that encodes for an iron-scavenging siderophore , yersiniabactin.

 Pathogenesis and Pathology

When a flea feeds on a rodent infected with Y. pestis, the ingested organisms multiply in the gut of the flea and, helped by the coagulase, block its proventriculus so that no food can pass through. Subsequently, the “blocked” and hungry flea bites ferociously, and the aspirated blood, contaminated with Y. pestis from the flea, is regurgitated into the bite wound. The inoculated organisms may be phagocytosed by polymorphonuclear cells and macrophages. The Y. pestis organisms are killed by the polymorphonuclear cells but multiply in the macrophages; because the bacteria are multiplying at 37°C, they produce the antiphagocytic protein and subsequently are able to resist phagocytosis. The pathogens rapidly reach the lymphatics, and an intense hemorrhagic inflammation develops in the enlarged lymph nodes, which may undergo necrosis and become fluctuant. Although the invasion may stop there, Y. pestis organisms often reach the bloodstream and become widely disseminated. Hemorrhagic and necrotic lesions may develop in all organs; meningitis, pneumonia, and serosan guineous pleuropericarditis are prominent features.

Primary pneumonic plague results from inhalation of infective droplets (usually from a coughing patient), and it is characterized by hemorrhagic consolidation, sepsis, and death.

Clinical Findings

 The clinical manifestations of plague depend on the route of exposure, and three forms of the disease have been described: bubonic plague, pneumonic plague, septicemic plague. Bubonic plague is by far the most common clinical presentation of infection with Y. pestis. After an incubation period of 2–7 days, there is a sudden onset of high fever and development of painful lymphadenopathy, commonly with greatly enlarged, tender lymph nodes (buboes) in the neck, groin, or axillae. Septicemic plague can occur spontaneously or as a complication of untreated bubonic plague. In this form of the disease, Y. pestis multiplies intravascularly and can be seen in blood smears. Patients typically present with a sudden onset of high fever, chills, and weakness, progressing rapidly to septic shock with associated disseminated intravascular coagulation, hypotension (septic shock), altered mental status, and renal and cardiac failure. Bleeding into skin and organs can also occur. Vomiting and diarrhea may develop during the early stages of septicemic plague. Terminally, signs of pneumonia and meningitis can appear. Primary pneumonic plague results from direct inhalation of organisms into the lung. This form of the disease typically occurs through close and direct contact with another patient who has pneumonic plague, and symptoms begin within 1–4 days after the exposure. Patients often have a fulminant course with chest pain, cough, hemoptysis, and severe respiratory distress. Secondary pneumonic plague is a complication in approximately 10% of patients with bubonic plague through hematogenous spread of the organisms from the buboes and often in the setting of delayed or inappropriate antibiotic treatment.

Diagnostic Laboratory Tests

Plague should be suspected in febrile patients who have been exposed to rodents in known endemic areas. Rapid recognition and laboratory confirmation of the disease are essential to institute lifesaving therapy.

 A. Specimens

 Blood is taken for culture and aspirates of enlarged lymph nodes for smear and culture. Acute and convalescent sera may be examined for antibody levels. In pneumonia, sputum is cultured; in possible meningitis, cerebrospinal fluid is taken for smear and culture.

B. Smears

 Y. pestis are small Gram-negative bacilli that appear as single cells or as pairs or short chains in clinical material. Wright, Giemsa, or Wayson stains may be more useful when staining material from a suspected buboes or a positive blood culture result because of the striking bipolar appearance (safety pin shape) of the organism using these stains that is not evident on a direct Gram-stain. More specific direct staining methods (possibly available through reference laboratories) include the use of fluorescent antibody stains targeting the capsular F1 antigen.

 C. Culture

All materials are cultured on blood agar, chocolate, and Mac Conkey agar plates and in brain–heart infusion broth. Growth on solid media may be slow, requiring more than 48 hours, but blood culture results are often positive in 24 hours. Cultures can be tentatively identified by biochemical reactions. Y. pestis produces nonlactose-fermenting colonies on MacConkey agar, and it grows better at 25°C than at 37°C. The organism is catalase positive; indole, oxidase, and urease negative; and nonmotile. The last two reactions are useful in differentiating Y. pestis from other pathogenic yersiniae. The use of automated (commercial) identification systems using various biochemical reactions is not recommended for the identification of Y. pestis, and an organ ism with the abovementioned characteristics should be referred to a public health laboratory for more confirmatory testing. Definite identification of cultures is best done by immunofluorescence or by lysis by a specific Y. pestis bacteriophage (confir mation available through state health department laboratories and by consultation with the Centers for Disease Control and Prevention [CDC], Plague Branch, Fort Collins, CO).

All cultures are highly infectious and must be handled with extreme caution inside a biological safety cabinet.

 D. Serology

In patients who have not been previously vaccinated, a convalescent serum antibody titer of 1:16 or greater is presumptive evidence of Y. pestis infection. A titer rise in two sequential specimens confirms the serologic diagnosis.

Treatment

 Unless promptly treated, plague may have a mortality rate of nearly 50%; pneumonic plague has a mortality rate of nearly 100%. The drug of choice is streptomycin, but the more readily available aminoglycoside gentamicin has been shown to be as effective. Streptomycin is nephrotoxic and ototoxic and should therefore be cautiously used in elderly patients, pregnant women, and in children. For these patient groups as well as other patients with contraindications to the use of aminoglycosides, doxycycline, or ciprofloxacin (and other fluoroquinolones) are considered alternative drugs for the treatment of plague. These agents can also be given in combination with streptomycin or gentamicin. Antimicrobial drug resistance in Y. pestis has never been documented in the United States and has rarely been noted in Y. pestis isolates in other parts of the world.

Epidemiology and Control

Plague is an infection of wild rodents (field mice, gerbils, moles, skunks, and other animals) that occurs in many parts of the world. The primary enzootic areas are India, Southeast Asia (especially Vietnam), Africa, and North and South America. The western states of the United States and Mexico also contain reservoirs of infection. Epizootics with high mortality rates occur intermittently; at such times, the infection can spread to domestic rodents (eg, rats) and other animals (eg, cats), and humans can be infected by flea bites or by contact. The commonest vector of plague is the rat flea (Xenopsylla cheopis), but other fleas may also transmit the infection. Y. pestis does not form spores and is extremely susceptible to UV radiation (eg, sunlight) and desiccation. There fore, it is reasonable to expect that the majority of organisms become nonviable within 1 hour following the release into the environment. However, studies have also demonstrated the plague bacilli are capable of surviving in soil for prolonged periods of time. The control of plague requires surveys of infected animals, vectors, and human contacts; in the United States, this is done by county and state agencies with support from the Plague Branch of the CDC and by destruction of plague-infected animals. If a human case is diagnosed, health authorities must be notified promptly. All patients with suspected plague should be isolated, particularly if pulmonary involvement has not been ruled out. All specimens must be treated with extreme caution. Postexposure prophylaxis with either doxycycline or ciprofloxacin is indicated for people who have a known or documented exposure to plague; typical exposures include close contact with a pneumonic plague patient or direct contact with contaminated/infected fluids and/or tissues. Postexposure prophylaxis is usually given for 7 days following the exposure. Mass chemoprophylaxis is considered as a likely public health response following an intentional release of Y. pestis, and the CDC and State Public Health Departments have formulated detailed guidelines for the response to such an event. Aside from chemoprophylaxis, disease preventative measures for the risk of an occupational exposure (eg, emergency first responder personnel) include the use of biosafety level 3 personal protective equipment (ie, Tyvek suits, booties, gloves, or HEPA-filtered respirators).

 Killed whole-cell vaccines are no longer available in the United States. Because of concern for Y. pestis being a potential agent of biowarfare and bioterrorism, numerous vaccines are currently under development.