The History of Discovery
Plague is a highly devastating epidemic disease still known from the times of antiquity. It followed human civilization from the deep past.
Three global pandemics of plague were registered in the written history – the Justinian Plague of 541 AD that affected the all ancient world, lasted for two centuries and caused about 25 mln deaths; the Great Plague or “Black Death” of Middle Ages that killed 60% of European population, and the last plague pandemic started in China in the 1860s and continued for several decades with about 10 mln victims.
In XX century the total number of plague cases gradually declined. Nowadays between 1,000 and 2,000 plague reports are delivered annually to WHO, but this statistics is regarded as seriously underestimated.
The causative agent of plague, Yersinia pestis, was discovered by the French microbiologist A. Yersin in Hong Kong in 1894 during the last global pandemic of the disease.
Classification of Yersinia pestis
Yersinia genus belongs to the family Enterobacteriaceae.
Yersinia pestis, a causative agent of plague is the most virulent yersinia representative.
Structure and Properties of Yersinia pestis
All yersiniae are similar with other enterobacteria – small polymorphic gram-negative rods that have characteristic bipolar stain. Stained microbial cells look like “closed safety pins” on microscopy. Y. pestis are nonmotile enterobacteria, in contrast to other yersiniae, which express flagella. In smears from tissues and cultures Y. pestis is found to have a delicate capsule.
Yersiniae can grow on ordinary nutrient media. The optimal temperature for cultivation is 25-30°C. The growth on blood agar after 48 h of incubation at 35°C yields gray-white opaque colonies 1-2 mm in diameter usually without hemolysis. The colonies resemble “fried-egg” or “crumpled lace handkerchief” at appearance.
Various lactose-containing media (McConkey, or EMB agar) and several special media are used for yersiniae cultivation. Small lactose-negative colonies arise after 24 h of incubation at 35°C. In meat broth the cultures form a pellicle on the surface with threadlike growth resembling stalactites. Y. pestis is virulent in R form.
Yersiniae are facultatively anaerobic bacteria. As all enterobacteria, they are catalase-positive and oxidase negative. Y. pestis has rather weak and variable biochemical activity. The bacterium ferments glucose, maltose, galactose, mannitol and some other carbohydrates with acid end products, but can’t metabolize sucrose, and in most cases lactose. It reduces nitrates to nitrites. Y. pestis neither liquefy gelatin, nor produce indole. They are urease negative.
Y. pestis express many antigens and toxins that act as virulence factors. O-antigen contains lipopolysaccharides that have endotoxic activity, when released. Capsular K- (or F1) antigen of glycoprotein nature is shown to protect bacteria against phagocytosis.
Protein V-Ag and lipoprotein W-Ag can also develop anti-phagocytic activity.
All virulence factors of Y. pestis are under tight genetic control.
A 72-kb virulence plasmid (pYV – plasmid of Yersinia virulence) is responsible for microbial adherence, invasiveness, intercellular spread, and ability to survive and propagate within-host lymphoid tissues. It is essential for mirobial virulence; avirulent strains lack this plasmid. The plasmid contains yersinia pathogenicity island or Yop virulon.
Yop virulon encodes invasive yersinia outer proteins (Yop proteins) and structures of type III bacterial secretion system (injectisome, or needle complex), composed of about 30 protein units (so-called Ysc proteins). Needle complex promotes microbial adherence to epithelial or immune cells and delivers invasive Yop proteins into cytoplasm of the host cells. Injected Yops impair the dynamics of the cytoskeleton, allow microbial penetration and intracellular spread, thereby promoting further bacterial invasion.
Yop proteins sharply diminish the production of proinflammatory cytokines by macrophages and other immune cells, thus maintaining bacterial survival within immune cells and tissues. Also they stimulate macrophage apoptosis.
Among secreted exotoxins, one is lethal for mice in amounts of 1 ng. This deleterious protein substance is extremely cardiotoxic for experimental animals. Its role in human infection is not clearly elucidated.
K- (or F1), V- and W-antigens protect bacteria against phagocytosis.
Y. pestis expresses plasminogen activator – a potent agressive enzyme complex. At 28°C (the normal temperature of the flea body) it shows coagulase activity supporting bacterial dwelling within insect vector, whereas at 35-37°C (human body temperature) it affords fibrinolysin activity, thereby breaking down the tissue matrix and promoting microbial invasion. The latter is additionally stimulated by microbial hyaluronidase.
Also the bacteria produce bacteriocins (or pesticins) that antagonize normal microbiota.
Bacterial lipopolysaccharide has potent endotoxic activity. In human body conditions at 37oC the structure of lipid part of endotoxin alters, and modified LPS loses its capacity to stimulate macrophages thus maintaining microbial survival.
The plague agents can withstand low temperatures. At 0°С they live for 6 months. Y. pestis survives in water for 30 days; in milk for 90 days; in bubonic pus for 20-30 days; in sputum for 10 days.
Y. pestis is very sensitive to drying and high temperatures. Boiling kills the microbials within 1 minute, when heated to 60°C they are inactivated in 1 hour. Standard disinfectants in ordinary concentrations (e.g., 5% phenol) readily destroy them in 5-10 minutes.
Epidemiology, Pathogenesis and Clinical Findings in Plague
Plague is a zoonotic disease of rodents and other animals that is usually transmitted to humans via fleabites.
Rodents, among them black rats, grey rats, mice, gophers, marmots (tarbagans) and many others are susceptible to plague. More than 300 rodent species as primary sources of infection may spontaneously contract the disease.
When an insect vector (flea) feeds on a rodent infected with Y. pestis, the ingested bacteria multiply in the gut of the flea. Microbial cells block vector’s digestive tract owing to coagulase action. Then the flea attacks and bites the mammal host, and flea gut contents contaminated with Y. pestis become expelled into the bite wound.
At the temperature of the flea body 28°C Y. pestis neither secrete virulence proteins, nor alter the structure of LPS and express capsular F1-Ag.
In human body at 37°C the inversion of microbial metabolism occurs. By activation of injectisome Y. pestis delivers virulence effector proteins into macrophages and other immune cells. They paralyze the activity of innate immune response with profound inhibition of cytokine secretion. Alteration of LPS structure and expression of bacterial F1-, V- and W-Ags protects the bacteria against fagocytosis.
Fibrinolysin activity of bacterial plasminogen activator and hyaluronidase facilitate microbial dissemination.
Thus, the powerful mechanisms of suppression of innate immunity essential for Y. pestis create the conditions for generalized systemic devastating infection.
Depending on the location of the pathogen, virulence of the microbe, and host immunity human plague is manifested in three major forms: bubonic, pneumonic, and septicemic. More seldom are cutaneous and intestinal forms.
Fleabite results in the bubonic form of plague, characterized by the sudden rise of fever and an extremely painful lymphadenitis known as bubo. It usually appears in the groin or axillae.
Skin dark-purple lesions may develop during the systemic stage of the infection. They rapidly become necrotic and likely account for the plague name “black death”.
The term septicemic plague describes fulminant disseminative infection without bubonic lymphadenitis.
Pneumonic plague arises after hematogenous spread of yersiniae from a bubo to the lungs (secondary pneumonic) or via the direct inhalation of pathogens.
Direct inhalation of Y. pestis sharply accelerates the disease transmission between humans (primary pneumonic plague). The pneumonic disease is highly contagious and easily spread among individuals via airborne route.
As the result of systemic infection, hemorrhagic and necrotic lesions emerge in all organs and tissues. They lead to meningitis, pneumonia and other inflammatory disorders followed by kidney, liver and cardiovascular failure. Disseminated intravascular coagulation (DIC) entails hypotension and collapse.
Administration of antimicrobial agents (e.g., gentamycin or tetracycline) early in the course of the disease can reduce mortality from approximately 50% in untreated plague cases to about 5-10%. After patient’s recovery a stable immunity of long duration is acquired. Due to its prominent virulence, rapid air-droplet spread of pneumonic disease and high fatality of infection if not treated Y. pestis is ascertained as the potential agent of bioterrorism and biological warfare.
According to United States regulations, the list of “Biological Select Agents or Toxins” comprises microorganisms and their toxins that possess “…the potential to pose a severe threat to public health and safety”. Y. pestis stays in Tier 1 of US Select Agents list (the highest rank of public threat). Criteria for Tier 1 are summarized as follows: “(1) ability to produce a mass casualty event or devastating effects to the economy; (2) communicability; (3) low infectious dose; (4) history of or current interest in weaponization based on threat reporting”.
Laboratory Diagnosis of Plague
For isolation of Y. pestis the specimens from blood, sputum, or lymph node aspirates are taken. The bacteria are also recovered from autopsy material (organs, blood, lungs, lymph node samples), rodent corpses, fleas, foodstuffs, water, etc. At the first stage of examination Y. pestis are often detected by microscopy in smears stained by Gram method or methylene blue.
Immunofluorescence test is used for rapid detection of bacteria in clinical samples. Microbial nucleic acids are detected by PCR. Cultures of Y. pestis should be operated in special biosafety facilities (BSL-2 – biosafety level 2) with minimization of procedures that may create aerosols.
Microbial isolation is performed in ordinary media supplemented with antiseptics, e.g., gentian violet that inhibits concomitant microflora. Growth on media incubated at 35-37°C is slower than growth at 28°C or room temperature. Any suspected Y. pestis isolate should be delivered to the state reference laboratory for identification.
For biological tests (animal experimental infection) isolated pure cultures or specimens are inoculated into guinea pigs. If plague agents are present, the animals die in 5-7 days. Y. pestis is further identified according to their biochemical and antigenic properties and by phage typing. It should be differentiated from other yersiniae, e.g. the causative agent of pseudotuberculosis. Serological testing of patients detects arisen antibody levels to Y. pestis by agglutination or ELISA test.
Treatment and Prophylaxis of Plague
Aminoglycoside antibiotics (streptomycin or gentamycin) are used for the treatment of the plague. The drugs are effective even in pneumonic plague. Good results were obtained from a combination of streptomycin with tetracyclines and passive treatment with anti-plague immune globulin. The latter can be used for urgent post-exposure prophylaxis of the disease.
Specific prophylaxis is afforded with live EV vaccine and formaldehyde-inactivated vaccine. The live vaccine is administered intra- or subcutaneously, orally (in tablets), or by inhalation of vaccine aerosol. The persons from the groups of risk working in the areas of infection are vaccinated (e.g., medical personnel, veterinary workers, hunters, herdsmen, etc.). Immunity lasts for about 1 year. Booster injections of inactivated vaccine are possible in 6-12 months. The efficacy of vaccination is generally moderate.