Table of Contents:

• Taxonomy Information
• Lifecycle Information
• Genome Information
• Biosafety Information
• Culturing Information
• Epidemiology Information
• Diagnostic Tests Information
• Infected Hosts Information
• Phinet: Pathogen-Host Interaction Network
• Lab Animal Pathobiology & Management
• References
• Data Provenance and Curators

1. Species:
   1. Yersinia pestis (Website 22):
      1. Common Name: Yersinia pestis
      2. GenBank Taxonomy No.: 632
      3. Description: The causative agent of plague was identified by the Swiss microbiologist Alexandre Yersin who was investigating an 1894 outbreak in Hong Kong. Yersinia pestis has undergone several nomenclature changes - Bacterium pestis until 1900, Bacillus pestis until 1923, Pasteurella pestis (after Yersin's mentor), and finally, Yersinia pestis in 1970(Perry and Fetherston, 1997).
      4. Variant(s):
         • Yersinia pestis, biovar Antiqua (Deng et al., 2002):
           • Common Name: Yersinia pestis Antiqua
           • Description: Yersinia pestis strains fall into three subtypes or biovars: Antiqua, Mediaevalis, and Orientalis, each of which is associated with a major pandemic(Deng et al., 2002).
         • Yersinia pestis, biovar Mediaevalis (Deng et al., 2002):
           • Common Name: Yersinia pestis Mediaevalis
           • Description: Yersinia pestis strains fall into three subtypes or biovars: Antiqua, Mediaevalis, and Orientalis, each of which is associated with a major pandemic(Deng et al., 2002).
         • Yersinia pestis, biovar Orientalis (Deng et al., 2002, Radnedge et al., 2001):
           • Description: Yersinia pestis strains fall into three subtypes or biovars: Antiqua, Mediaevalis, and Orientalis, each of which is associated with a major pandemic(Deng et al., 2002). The Orientalis biovar of Yersinia pestis is considered to be the most recently emerged biovar, and it includes all of the strains isolated so far in the United States(Radnedge et al., 2001).
         • Yersinia pestis, KIM strain (Website 23):
           • GenBank Taxonomy No.: 187410
           • Description: The KIM strain belongs to biovar Mediaevalis and its genome has been sequenced(Deng et al., 2002).
         • Yersinia pestis, CO92 strain :
           • GenBank Taxonomy No.: 214092
           • Description: The CO92 strain belongs to biovar Orientalis and its genome has been sequenced(Parkhill et al., 2001).
         • Y. pestis F1+ strain (GB) (Williamson et al., 2000, Eyles et al., 1998):

1. Yersinia pestis information
   1. Stage Information:
      1. Vegetative-cell:
         • Size: 1-3 micrometer X 0.5-0.8 micrometer
         • Shape: Single or short-chained, plump, coccobacillus.
         • Picture(s):
           • Wayson stain of Yersinia pestis (Website 8)
             
             
             
             Description: Wayson stain of Yersinia pestis. Note the characteristic "safety pin" appearance of the bacteria. Copyright: CDC(Cornelis, 2002).

1. Genome of Yersinia pestis, CO92 strain, Yersinia pestis, KIM strain
   1. Description: 7418. The complete genome sequences of Yersinia pestis strains CO92 and KIM are available(Parkhill et al., 2001, Dong et al., 2000, Deng et al., 2002). More than 95% of the sequence is shared by the two genomes. The CO92 genome is approximately 4.65-megabase (Mb), which is about 50 kilobases (kb) larger than the KIM genome(Deng et al., 2002). Both strains have plasmids of 96.2 kb, 70.3 kb, and 9.6 kb(Website 14, Parkhill et al., 2001, Deng et al., 2002). A 5.9 kb plasmid has also been found in some Yersinia pestis isolates(Website 26, Dong et al., 2000).
   2. Strain CO92 genome(Website 14, Parkhill et al., 2001)
      1. GenBank Accession Number: NC_003143
      2. Size: 4653728 bp.
      3. Gene Count: 4008.
      4. Description: Yersinia pestis strain CO92, complete genome.
   3. Strain KIM genome(Website 24, Deng et al., 2002, Website 34)
      1. GenBank Accession Number: NC_004088
      2. Size: 4600755 bp.
      3. Gene Count: 4198 predicted ORFs.
      4. Description: Yersinia pestis strain KIM, complete genome.
   4. Strain CO92 plasmid pCD1(Website 3, Perry and Fetherston, 1997, Parkhill et al., 2001)
      1. GenBank Accession Number: NC_003131
      2. Size: 70305 bp.
      3. Description: Yersinia pestis CO92 plasmid pCD1 (calcium dependence), complete sequence.
   5. Strain CO92 plasmid pPCP1(Website 28, Perry and Fetherston, 1997, Parkhill et al., 2001)
      1. GenBank Accession Number: NC_003132
      2. Size: 9612 bps.
      3. Description: Yersinia pestis CO92 plasmid pPCP1 (pesticin, coagulase, plasminogen activator), complete sequence.
   6. Strain CO92 plasmid pMT1(Website 19, Perry and Fetherston, 1997, Parkhill et al., 2001)
      1. GenBank Accession Number: NC_003134
      2. Size: 96210 bp.
      3. Description: Yersinia pestis CO92 plasmid pMT1 (murine toxin), complete sequence.
   7. Plasmid pYC(Website 26, Dong et al., 2000)
      1. GenBank Accession Number: NC_002144
      2. Size: 5919 bp.
      3. Description: Yersinia pestis plasmid pYC, complete sequence.
   8. Strain KIM plasmid pMT1(Website 29)
      1. GenBank Accession Number: NC_004835
      2. Size: 100984 bp.
      3. Description: Yersinia pestis KIM plasmid pMT1, complete sequence.
   9. Strain KIM plasmid pCD1(Website 30)
      1. GenBank Accession Number: NC_004836
      2. Size: 70504 bp.
      3. Description: Yersinia pestis KIM plasmid pCD1, complete sequence.
   10. Strain KIM plasmid pPCP1(Website 31)
       1. GenBank Accession Number: NC_004837
       2. Size: 9610.
       3. Description: Yersinia pestis KIM plasmid pPCP1, complete sequence.
   11. Strain KIM plasmid pMT-1(Website 32)
       1. GenBank Accession Number: NC_004838
       2. Size: 100990.
       3. Description: Yersinia pestis KIM plasmid pMT-1, complete sequence.
   12. Strain KIM plasmid pCD1(Website 33)
       1. GenBank Accession Number: NC_004839
       2. Size: 70559.
       3. Description: Yersinia pestis KIM plasmid pCD1, complete sequence.

1. General biosafety information
   1. Level: 2(Website 15).
   2. Precautions: Biosafety Level 2 practices, containment equipment and facilities are recommended for all activities involving the handling of potentially infectious clinical materials and cultures. Special care should be taken to avoid the generation of aerosols from infectious materials and during the necropsy of naturally or experimentally infected rodents. Gloves should be worn when handling field-collected or infected laboratory rodents and when there is the likelihood of direct skin contact with infectious materials. Necropsy of rodents is ideally conducted in a biological safety cabinet. Immunization is recommended for personnel working regularly with cultures of Yersinia pestis or infected rodents(Website 15).
2. General biosafety information
   1. Level: 3(Website 15).
   2. Precautions: Biosafety level 3 practices should be used for activities with high potential for droplet or aerosol production, for work with antibiotic-resistant strains, and for activities involving production quantities or concentrations of infectious materials(Website 15).

1. Sheep Blood Agar (SBA) :
   1. Description: Sheep blood agar (SBA) is a general bacteriologic medium used for the isolation and examination of colonial morphology of bacterial organisms. Yersinia pestis organisms are not fastidious and will grow well on any nutrient medium including SBA. Plague bacilli grow slower than most bacteria at 37 degrees celcius, but at 28 degrees celcius they will grow faster than most. Enrichment of medium with 6% sheep red blood cells instead of the standard 5% provides more nutrition and shortens the incubation period. Even though Yersinia pestis may grow faster at 28 degrees celcius, a plate should also be incubated at 37 degrees celcius since diagnostic tests for plague depend primarily on expression of the temperature-regulated F1 antigen(Website 20). Procedure: For cultures, use the sterile loop/stick to inoculate SBA plates. For tissues, the samples are obtained by using the sterile wood stick to punch into the tissue several times, especially in visibly necrotic areas, and then transferring the materials on the wood stick to the agar surface. Inoculate two SBA plates and streak to obtain isolated colonies. For safety, tape the top and bottom of the petri dish together in two places to keep them together; incubate plates, one at 28 degrees celcius (for faster growth) and another at 37 degrees celcius (for F1 antigen expression), for at least 24-48 hours. Examine plates for characteristic colonies(Website 20).
   2. Medium: Sheep blood agar plates (4-6% sheep blood)(Website 20).
   3. Optimal Temperature: 28 degrees celcius(Website 20).
   4. Upper Temperature: 37 degrees celcius(Website 20).
   5. Lower Temperature: 25 degrees celcius(Website 20).
   6. Optimal pH: 7.2 to 7.6(Website 20).
   7. Upper pH: 9.6(Quan, 1987).
   8. Lower pH: 5.0(Quan, 1987).
   9. Doubling Time: 1.25 hours/generation time(Website 20).
   10. Note: Sheep blood agar (SBA) plates are used as the standard solid medium for the isolation and culture of Yersinia pestis. If, however, SBA plates are not available, other general solid medium such as brain heart infusion agar, nutrient agar or trypticase soy agar may be used, though growth of the organism will be slower and colonies will be smaller(Website 20). Atmosphere: Ambient, use of 5% CO2 is acceptable. Hold primary plates for 5 days. Plates should be held for up to 7 days if the patient has been treated with bacteriostatic antibiotic. Yersinia pestis colonies are gray-white, translucent, usually too small to be seen as individual colonies at 24 hours. After incubation for 48 hours, colonies are about 1-2 mm in diameter, gray-white to slightly yellow, and opaque. Under 4X enlargement, after 48-72 hour of incubation, colonies have a raised, irregular "fried egg" appearance, which becomes more prominent as the culture ages. Colonies also can be described as having a "hammered copper," shiny surface. There is little or no hemolysis of the sheep red blood cells(Website 6).
2. Nutrient-rich broths :
   1. Description: Yersinia pestis grows well in nutrient-rich broth such as brain heart infusion (BHI), trypticase soy or nutrient broth. The organisms exhibit a characteristic growth formation in clear broth, whose appearance may be used as an aid to its identification. Because of its slower growth, Yersinia pestis may be quickly overwhelmed by contaminants, but the characteristic clumpedgrowth may still be seen in the broth tube growth. Inoculation in clear, enriched media such as BHI may assist in the recovery of Yersinia pestis, but is not critical to isolation(Website 20). Procedure: Specimens taken from clinical materials or pure cultures should be inoculated into two broth tubes and incubated at 28 degrees celcius (for faster growth) and at 37 degrees celcius (for expression of the diagnostic F1 antigen). Cultures should be incubated for 24-48 hours without agitation. At that time, carefully remove tubes, without agitation, from the incubator, and examine for the characteristic growth pattern. The cultures in broth can be described as suspended flocculent or crumbly clumps ("stalactites"). These clumps are visible at the side and bottom of the tube with the rest of the medium remains clear (an image of Yersinia growth in BHI broth tubes can be viewed at Website 20). Longer incubation will result in the clumps of cells falling to the bottom of the tube and loss of the characteristic formation, but the medium above will still remain clear. The characteristic formation of Yersinia pestis cells can be seen in broth culture even if the culture is contaminated; the broth will be cloudy but the clumps will be visible(Website 20).
   2. Medium: Brain heart infusion broth (BHI). There must be at least 5 cm of BHI in the tube to correctly visualize the characteristic growth of Yersinia pestis(Website 20).
   3. Optimal Temperature: 28 degrees celcius(Website 20).
   4. Upper Temperature: 37 degrees celcius(Website 20).
   5. Lower Temperature: 25 degrees celcius(Website 20).
   6. Optimal pH: 7.2 to 7.6(Website 20).
   7. Doubling Time: 1.25 hours/generation time(Website 20).
   8. Note: Sample preparation for culturing: Lower respiratory tract (pneumonic): Bronchial wash or transtracheal aspirate (1 ml). Sputum may be examined but this is not advised because of contamination by normal throat flora. Blood (septicemic): Collect appropriate blood volume and number of sets per established laboratory protocol. Note: In suspected cases of plague, an additional blood or broth culture (general nutrient broth) should be incubated at room temperature (22-28 degrees celcius), the temperature at which Yersinia pestis grows faster. Do not shake or rock the additional broth culture so that the characteristic growth formation of Yersinia pestis can be clearly visualized. Aspirate of involved tissue (bubonic) or biopsied specimen: Liver, spleen, bone marrow, lung. Note: Aspirates may yield little material; therefore, a sterile saline flush may be needed to obtain an adequate amount of specimen. Syringe and needle of aspirated sample should be capped, secured by tape, and sent to the laboratory. Respiratory/sputum: Transport specimens in sterile, screw-capped containers at room temperature. If it is known that material will be transported from 2-24 hours after collection, then store container and transport at 2-8 degrees celcius. Blood: Transport samples directly to the laboratory at ambient temperature. Hold them at ambient temperature until they are placed onto the blood culture instrument or incubator. Do not refrigerate. Follow established laboratory protocol for processing blood cultures. Tissue aspirate/biopsy specimen: Submit tissue or aspirate in a sterile container. For small samples, add 1-2 drops of sterile normal saline to keep the tissue moist. Transport the sample at room temperature for immediate processing. Keep the specimen chilled if processing of the specimen will be delayed(Website 6).
3. BIN selective agar :
   1. Description: Growth of Yersinia pestis on BIN selective agar: Existing media designed for selective isolation of clinically important members of the genus Yersinia were found to be unsatisfactory for the growth and isolation of Yersinia pestis. Ber et al., (2003) reported the development of a new selective agar medium (termed BIN) that supports the growth of Y. pestis. The development of the formulation of this medium was based on a fluorescence screening system designed for monitoring bacterial growth on semisolid media, using a green fluorescent protein-expressing strain. High-throughput combinatorial experiments can be conducted for the quantitative evaluation of the effect of different medium components on growth. Generation of fluorescence plots in this system, using microplates, allowed the quantitative evaluation of the growth rate of Y. pestis EV76 cultures in different agar compositions(Ber et al., 2003). The final BIN formulation is based on brain heart infusion (BHI) agar, to which the selective agents irgasan, cholate salts (sodium cholate and sodium deoxycholate), crystal violet, and nystatin were introduced. It was found that BIN agar is more efficient in supporting colony formation and recovery of Y. pestis than are the conventional semisolid media MacConkey agar and Yersinia-selective agar (cefsulodin-irgasan-novobiocin agar)(Ber et al., 2003). In summary, the BIN medium is superior to the WHO-recommended selective medium, MacConkey medium, as well as to the commercial CIN medium for the isolation and recovery of Y. pestis from pure and fresh samples as well as from background environments where the bacterium is expected to be under stress(Ber et al., 2003).
   2. Medium: The BIN formulation is based on brain heart infusion (BHI) agar, to which the selective agents irgasan (0.0008 g/liter), cholate salts (sodium cholate (0.5 g/liter) and sodium deoxycholate (0.5 g/liter)), crystal violet (0.001 g/liter), and nystatin (0.025 g/liter) were introduced(Ber et al., 2003).

1. Outbreak Locations:
   1. Reservoirs for Yersinia pestis are present on nearly every major continent(Perry and Fetherston, 1997).
   2. Most human cases of plague are reported from developing countries in Asia and Africa. During 1990-1995, 12,988 cases of plague, with 1009 deaths (8%) were reported to the World Health Organization. The countries that reported more than 100 cases (from greatest to least) were Tanzania, Madagascar, Democratic Republic of Congo, Vietnam, Peru, India, Myanmar, Zimbabwe, Mozambique, Uganda, and China(Butler, 2000).
   3. In the United States, about 10 cases occur each year; mostly in the southwestern states(Butler, 2000).
2. Transmission Information:
   1. From: Rat flea , To: Homo sapiens (Perry and Fetherston, 1997)
      Mechanism: Transmission of Yersinia pestis from fleas to humans occurs primarily via the bites of infected fleas(Perry and Fetherston, 1997). Fleas acquire Yersinia pestis from an infected blood meal(Perry and Fetherston, 1997).
   2. From: Homo sapiens , To: Homo sapiens (Perry and Fetherston, 1997)
      Mechanism: Pneumonic plague epidemics can occur via the spread of respiratory droplets between humans, however this type of epidemic is currently uncommon due to the advent of effective antibiotics and modern public health measures(Perry and Fetherston, 1997).
   3. From: Other Mammals , To: Homo sapiens (Website 10)
      Mechanism: Inhaling droplets expelled by the coughing of a plague-infected animal (especially house cats) can result in plague of the lungs (plague pneumonia)(Website 10).
3. Environmental Reservoir:
   1. Rodents(Perry and Fetherston, 1997, Website 12, Website 16, Inglesby et al., 2000, Butler, 2000):
      1. Description: Plaque is primarily a zoonotic infection, occurring in urban or wild rodent populations(Perry and Fetherston, 1997, Butler, 2000, Website 12). Rodents that could be characterized as enzootic hosts (i.e., in what rodent populations Yersinia pestis is found naturally) have not been conclusively identified, but certain species of rat, vole, mouse, and gerbil are suspected(Perry and Fetherston, 1997).
      2. Survival: Data from a study by Rose et al. (2003), suggest that Yersinia pestis maintains viability for extended periods under controlled conditions. Small numbers of cells suspended in phosphate buffer survived 2 to 4 hours after visible drying on stainless steel, polyethylene, or glass and beyond 48 hours on paper. Cells suspended in brain heart infusion broth (BHI) persisted more than 72 hours on stainless steel, polyethylene, and glass. Small numbers of cells suspended in BHI were still viable at 120 hours on paper(Rose et al., 2003). Yersinia pestis may remain viable for months to years at freezing temperatures and may furthermore be viable in dry sputum, flea feces and buried bodies(Website 16). Although some reports suggest that the bacterium may survive in the soil for some time, there is no evidence to suggest environmental risk to humans in this setting and thus no need for environmental decontamination of an area exposed to an aerosol of plague. Yersinia pestis is very sensitive to the action of sunlight and heating and does not survive long outside the host. In a World Health Organization (WHO) analysis, in a worst case scenario, a plague aerosol was estimated to be effective and infectious for as long as 1 hour. In the setting of a clandestine release of plague bacilli, the aerosol would have dissipated long before the first case of pneumonic plague occurred(Inglesby et al., 2000).
   2. Rat flea(Perry and Fetherston, 1997):
      1. Description: 31 species (of the 1500 identified flea species) have been proven to be plaque vectors. Xenopsylla cheopis, the Oriental rat flea, is the classic vector for plaque and is the standard against which all other fleas are measured(Perry and Fetherston, 1997).
   3. Mouse(Guiyole et al., 1994):
      1. Description: Mouse can be infected with Yersinia pestis(Guiyole et al., 1994).
   4. Other Mammals(Website 11, Perry and Fetherston, 1997):
      1. Description: Over 200 species of mammals in 73 genera have been reported to be naturally infected with Yersinia pestis, however rodents are the most important hosts(Perry and Fetherston, 1997). Identified hosts include rats, prairie dogs, squirrels, dogs, cats, and rabbits(Website 11).
4. Intentional Releases:
   1. Intentional Release Information(Website 27):
      1. Description: The Japanese used the plague during their invasion of China before the outbreak of World War II. The agent was delivered by four different methods (see "delivery mechanism"). Several hundred Chinese civilians died as a result of these attacks and a Japanese unit that entered an infected area also suffered heavy casualties(Website 27).
      2. Delivery Mechanism: Methods used by the Japanese to disseminate plaque in China:1. Dropping contaminated rice for rats to feed on 2. Dropping contaminated materials such as lint 3. Dropping infected fleas over the target in a cluster bomb-like weapon called Uji that scattered porcelain bomblets containing infected fleas. The fleas were released when the bomblet shattered.4. An attempt to use bacteria encapsulated in a water-soluble matrix(Website 27).
   2. Intentional Release Information(Website 27, Website 20):
      1. Description: Yersinia pestis can be manufactured by fermentation at relatively low temperatures without affecting its properties as a biological warfare agent. Yersinia pestis can be stored relatively easily because it can survive at low or freezing temperatures for extended periods as long as there is water available. It can survive for 30 days in water. It can also be freeze-dried and stored for up to ten years without loss of viability, (i.e. can be revived and cultured). A 1970 study by the World Health Organization found that the the organism could remain viable for up to an hour after dispersal as an aerosol. Under poor conditions for dispersal, there is a rapid loss of viability (up to 70% per minute) depending on the temperature and humidity of the atmosphere(Website 27).
      2. Emergency Contact: Contact the local FBI, state public health laboratory, and the state public health department. Local FBI agents will forward isolates to a state health department laboratory as is necessary. Consultation with a state health department laboratory is strongly encouraged as soon as Yersinia pestis is suspected as a bioterrorist threat agent(Website 20).
      3. Delivery Mechanism: An aerosol made up of bacteria-containing droplets of the size best suited for absorption in the lungs (1-5 micrometers), dispensed over an unprotected population, could kill a very large percentage (90-100%) of those exposed(Website 27).
      4. Containment: The unique experience of plague control in its natural foci was accumulated in the Soviet Union in the 1930s-1970s. At that period the main measure taken for conditioning these foci was the extermination of rodents, the main carriers of plague, and their fleas as vectors with the aim of breaking, as it was then believed, the continuous process of the transmission of Yersinia pestis among rodents. These measures, carried out in many natural foci for several decades, did not bring desired results; in none of these areas natural foci of plague could not be liquidated, completely or in part. The experience of the anthropogenic transformation of the landscapes of the natural foci of plague revealed that the best antiplague effect was obtained after vast territories were completely plowed up and used afterwards annually for monoculture(Diatlov, 2003).

1. Organism Detection Test:
   1. Gram stain (Website 6, Website 9, Perry and Fetherston, 1997, Website 20, Butler, 2000):
      1. Description: The Gram stain can be used as supportive, but not confirmatory evidence of Yersinia pestis infection(Website 9, Perry and Fetherston, 1997, Website 20). Procedure: Gram stain per standard laboratory protocol. Smears for staining may be prepared in order of likely positive results (i.e., cultures, bubo aspirates, tissue, blood, and sputum specimens)(Website 6). Because a bubo does not contain liquid pus, it may be necessary to inject saline into the bubo and immediately reaspirate it(Butler, 2000). Interpretation: Stained specimens containing Yersinia pestis often reveal plump, gram-negative rods, 1-2 micrometer X 0.5 micrometer, that are seen mostly as single cells or pairs and short chains in liquid media (see Website 6 or Website 20 for an image of Yersinia pestis Gram stain). Note: Patients with pneumonic plague may be secondarily infected with Streptococcus pneumoniae. Both of these organisms may be visualized in the sputum smears. It is imperative to evaluate such smears for the presence of gram-negative rods around the leukocytes (not necessarily intracellularly)(Website 20, Website 6).
   2. Differential stains (Website 6, Website 9, Perry and Fetherston, 1997, Website 20, Butler, 2000):
      1. Time to Perform: minutes-to-1-hour
      2. Description: The Wright-Giemsa or Wayson differential stains can be used as supportive, but not confirmatory evidence of Yersinia pestis infection(Website 9, Perry and Fetherston, 1997, Website 20). The Wright-Giemsa stains are the most reliable for accurately highlighting the bipolar staining characteristics of Yersinia pestis, whereas the Gram stain may not(Website 6). The most suitable materials for differential staining include a bubo aspirate, sputum, blood smears and tissues (lung, spleen, liver)(Website 20). Procedure: A detailed protocol for Wright-Giemsa/Wayson staining is available at Website 20. Interpretation: Consistent, striking bipolar safety-pin morphology of plump bacilli is characteristic of Yersinia, Pasteurella species, and other organisms (see Website 20 or Website 6 for image). All Yersinia pestis may stain as bipolar cells, but all bipolar-staining cells are not Yersinia pestis. Therefore, specimens taken from areas with a wide variety of normal flora (nasal, pharyngeal, and throat) may lead to mistaken interpretation. Bacterial cells picked from freshly passaged agar/broth growth tend to exhibit very little bipolarity, because the cells are too small; however, upon prolonged incubation, the cells would be more likely to exhibit the characteristic bipolar safety-pin shapes. When stained smear reveals the cells with the characteristic safety-pin morphology, the specimen should be forwarded to the state health department for isolation, identification and evaluation(Website 20).
   3. Fluorescent Antibody Test (Website 9, Perry and Fetherston, 1997):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: A positive fluorescent antibody test for the F1 antigen can be used as presumptive evidence of a Yersinia pestis infection(Website 9, Perry and Fetherston, 1997). The antibody is available at many western United States hospitals and from the Centers for Disease Control. The F1 antigen is predominantly expressed by Yersinia pestis at 37 degrees celcius. Samples that have been refrigerated for more than 30 hours, from cultures that were incubated at room temperatures less than 35 degrees celcius, or from fleas, will be negative(Perry and Fetherston, 1997).
   4. Yersinia pestis broth culture appearance :
      1. Time to Perform: 1-to-2-days
      2. Description: After 24-48 hours of incubation, a broth culture of Yersinia pestis can be described as suspended flocculent or crumbly clumps ("stalactites"). These clumps are visible at the side and bottom of the tube with the rest of the medium remaining clear (an image of Yersinia growth in broth tubes can be viewed at Website 20). Longer incubation will result in the clumps of cells falling to the bottom of the tube and loss of the characteristic formation, but the medium above will still remain clear. The characteristic formation of Yersinia pestis cells can be seen in broth culture even if the culture is contaminated; the broth will be cloudy but the clumps will be visible(Website 20).
   5. Yersinia pestis colony appearance :
      1. Time to Perform: 2-to-7-days
      2. Description: Yersinia pestis grows as gray-white, translucent colonies, usually too small to be seen as individual colonies at 24 hours. After incubation for 48 hours, colonies are about 1-2 mm in diameter, gray-white to slightly yellow color and opaque. Highly passaged and laboratory adapted strains grow faster and colonies are larger. Under 4X enlargement, after 48-72 hours of incubation, colonies have a raised, irregular "fried egg" morphology, which becomes more prominent as the culture ages (see image at Website 20). Colonies can also be described as having a "hammered copper," shiny surface (see image at Website 20). There is little or no hemolysis of the sheep red blood cells(Perry and Fetherston, 1997, Website 20).
   6. Lysis by specific bacteriophage :
      1. Description: Lysis by a specific bacteriophage is used by the Centers for Disease Control to conclusively identify Yersinia pestis(Website 9, Perry and Fetherston, 1997). Bacteriophage preparations and protocols for their use are available from plague reference laboratories(Quan, 1987).
2. Immunoassay Test:
   1. Antibody detection (Website 9, Perry and Fetherston, 1997):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: Serologic data can be used to assess Yersinia pestis infection, however it is not considered a rapid diagnostic technique and is therefore, often used retrospectively to confirm cases of plague(Perry and Fetherston, 1997). Samples are analyzed for anti-F1 antibodies by passive hemagglutination testing(Website 9, Perry and Fetherston, 1997). Plague is presumed if a single serum sample has a >1:10 anti-F1 antibody titer. Plague is considered confirmed when one of the following two agglutination conditions are met: (1) if two serum specimens demonstrate a four-fold anti-F1 antigen titer difference, (2) if a single serum sample has a titer of >1:128, and the patient has no known previous plague exposure or vaccination history(Website 9, Perry and Fetherston, 1997).
   2. Antigen capture ELISA (Chanteau et al., 2000):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: In 2000, Chanteau et. al. reported results from using an F1 antigen capture ELISA (developed and provided by the Naval Medical Research Institute, Bethesda, Maryland, USA) on patient samples in Madagascar. Using bacteriology as the gold standard reference assay, the sensitivity of the F1 ELISA was 100% in bubo aspirates, 52% in serum, and 58% in urine specimens. In culture-negative patients, the F1 ELISA was positive in 10% of bubo aspirates, 5% of serum, and 7% of urine specimens for whom a seroconversion for anti-F1 antibodies was also observed(Chanteau et al., 2000).
      3. False Negative: 0% in bubo aspirate, 48% in serum, 42% in urine.
   3. Immunochromatography - dipstick assay (Website 25, Chanteau et al., 2000):
      1. Time to Perform: minutes-to-1-hour
      2. Description: In 2000, Chanteau et. al. reported results from using an F1 dipstick assay (developed and provided by the Naval Medical Research Institute, Bethesda, Maryland, USA) on patient samples in Madagascar. The sensitivity of the assay was 98% on bubo aspirates (Chanteau et al., 2000). Subsequently, Dr. Chanteau reported that they had developed a dipstick assay that could be performed directly on clinical samples including sputum. The assay had a cut-off of 0.5 ng F1/ml and was used to confirm 600 cases of suspected plague in Madagascar(Website 25).
   4. Immunochromatography - dipstick assay (Chanteau et al., 2003):
      1. Time to Perform: minutes-to-1-hour
      2. Description: In 2003, Chanteau et. al. reported a rapid diagnostic test (RDT) for bubonic and pneumonic plague that used monoclonal antibodies to the F1 antigen of Yersinia pestis. The RDT is a specific, sensitive, and reliable test that can easily be done by health workers at the patient's bedside, for the rapid diagnosis of pneumonic and bubonic plague. This test will be of key importance for the control of plague in endemic countries. The RDT was derived from a prototype developed by the Naval Medical Research Institute (Bethesda, Maryland, USA) and assessed in Madagascar. For this new test, a combination of hybridomas B181 and G618, produced at the Institut Pasteur was used. The original RDT combined F104-A-G1 Mab with a polyclonal rabbit antiserum, and was based on one-step, vertical-flow immunochromatography. The RDT is as specific as, and at least as sensitive as, the two available standard methods. The excellent specificity of the RDT, its low detection threshold, and the higher number of positive specimens detected among samples from patients with suspected plague, suggest a greater sensitivity than bacteriology and ELISA(Chanteau et al., 2003).
   5. Immunomagnetic Separation - Flow Cytometry Detection Method (Splettstoesser et al., 2003):
      1. Time to Perform: minutes-to-1-hour
      2. Description: Splettstoesser et al., (2003) introduced and evaluated a combination of immunomagnetic separation and flow cytometry for the serodiagnosis of human plague. The preparation of paramagnetic beads indirectly coated with F1 capture antigen (F1 CA) took approximately 90 min. The time for preparing and analyzing 20 serum samples was 110 min for the flow cytometric assay. Compared with a recently published combination of an anti-F1 CA enzyme-linked immunosorbent assay (ELISA) and immunoblot, the new assay showed the same sensitivity as the ELISA and almost the same specificity (99.0 versus 100%) as the immunoblot(Splettstoesser et al., 2003).
   6. Bioluminescence - AB cell-based sensor (Rider et al., 2003):
      1. Time to Perform: minutes-to-1-hour
      2. Description: A pathogen sensor that achieves an optimal combination of speed and sensitivity through the use of B lymphocytes: members of the adaptive immune system that have evolved to identify pathogens very efficiently. B cell lines were engineered to express cytosolic aequorin, a calcium-sensitive bioluminescent protein from the Aequoria victoria jellyfish, as well as membrane-bound antibodies specific for pathogens of interest. Cross-linking of the antibodies by even low levels of the appropriate pathogen elevated intracellular calcium concentrations within seconds, causing the aequorin to emit light. Rider et al., (2003) named the sensor CANARY (Cellular Analysis and Notification of Antigen Risks and Yields). Cells specific for Yersinia pestis, the bacterium that causes plague, could detect as few as 50 colony-forming units (CFU) in a total assay time of less than 3 min, which included a concentration step. The probability of detection for Y. pestis ranged from 62% for 20 CFU to 99% for 200 CFU, whereas the false-positive rate for the CANARY assay was 0.4%. These cells did not respond to large numbers of unrelated bacteria (Francisella tularensis), nor did excess F. tularensis block the response to very low levels of Y. pestis(Rider et al., 2003).
3. Nucleic Acid Detection Test: