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Table of Contents:
Taxonomy Information
  1. Species:
    1. Burkholderia mallei. :
      1. Common Name: Burkholderia mallei.
      2. GenBank Taxonomy No.: 13373
      3. Description: Burkholderia mallei was formerly known as Pseudomonas mallei(Website 3). The causative agent of glanders in the genus Burkholderia is Burkholderia mallei. Glanders, one of the oldest diseases known to man, was first described by Aristotle in 330 B.C. Studies by Loeffler and Schuetz in 1882 were the first to identify Burkholderia mallei as the etiologic agent of glanders. Glanders was distributed worldwide until control measures were introduced in the early 20th century(Website 1). Glanders may occur in an acute localized form, as an acute pulmonary infection, or as an acute fulminant, rapidly fatal, sepsis. Combinations of these syndromes may occur in human cases(Website 4).
      4. Variant(s):
        • Burkholderia mallei ATCC 23344. :
          • Common Name: Burkholderia mallei ATCC 23344.
          • GenBank Taxonomy No.: 243160
Lifecycle Information
  1. Burkholderia mallei Lifecycle Information
    1. Stage Information:
      1. Burkholderia mallei lifecycle one stage:
        • Size: Burkholderia mallei cells are 2-5 m long and 0.5 m wide.
        • Shape: Burkholderia mallei cells are fairly straight rods with rounded ends.
Genome Summary
  1. Genome of Burkholderia mallei ATCC 23344.
    1. Description: Burkholderia mallei, sequencing in progress(Website 10).
    2. Burkholderia mallei Chromosome(Website 10)
      1. GenBank Accession Number: NC_002970
      2. Size: 5977371 bp(Website 10).
      3. Description: Contributor: The Institute for Genomic Research (TIGR) http://www.tigr.org(Website 10).
Biosafety Information
  1. General biosafety information
    1. Level: Biosafety level 2 and level 3.
    2. Precautions: Recommended Precautions: Biosafety level 2 practices, containment equipment, and facilities are recommended for activities with clinical materials and cultures known to contain or potentially contain the microorganism. Animal Biosafety level 2 practices, containment equipment, and facilities are recommended for activities with experimentally or naturally infected animals. Additional primary containment and personnel precautions, such as those described for Biosafety level 3, may be indicated for activities with a high potential for aerosol or droplet production, and for activities involving production quantities or concentrations of infectious materials(Website 21).
Culturing Information
  1. Burkholderia mallei Culturing Method :
    1. Description: Burkholderia mallei grows slowly on ordinary nutrient agar, but growth is accelerated with addition of 1-5% glucose and or 5% glycerol. Primary isolation requires 48 hours. Growth is also rapid on most meat infusion nutrient media(Website 20).
    2. Optimal Temperature: Growth optimal temperature is 37.5 degrees celcius(Website 20).
Epidemiology Information:
  1. Outbreak Locations:
    1. The United States has not seen any naturally occurring cases since the 1940s. However, it is still commonly seen among horses in Africa, Asia, the Middle East, and Central and South America(Website 11).
  2. Transmission Information:
    1. From: Horses(Website 11). , To: Humans(Website 11).
      Mechanism: Burkholderia mallei cells are transmitted to humans by inhalation of bacteria in aerosols or dust or contact with infected animals.Risk groups: The sporadic cases have been documented in veterinarians, horse caretakers, and workers in laboratories where the organism is being handled(Website 11).
  3. Environmental Reservoir:
    1. Environmental Reservoir:
      1. Description: Horses, donkeys and mules are the only natural reservoir of glanders(Website 3).
      2. Survival: Burkholderia mallei can survive in room temperature water for as long as 30 days and may be able to survive for a few months in other favorable environments. It is susceptible to heat, light, drying and a variety of chemicals.Disinfection: Burkholderia mallei is susceptible to numerous disinfectants including benzalkonium chloride, iodine, mercuric chloride in alcohol, potassium permanganate, 1% sodium hypochlorite, 70% ethanol and 2% glutaraldehyde. It is less susceptible to phenolic disinfectants. This organism can also be destroyed by heating to 55 degrees celcius for 10 min or by ultraviolet irradiation(Website 3).
  4. Intentional Releases:
    1. Intentional Release Information:
      1. Description: Burkholderia mallei infection(Website 11).
      2. Emergency Contact: If you believe that you have been exposed to a biological or chemical agent, or if you believe an intentional biological threat will occur or is occurring, contact your local health department and/or your local police or other law enforcement agency. CDC Emergency Response Hotline (24 hours) 770-488-7100. Call communicable disease epidemiology 206-361-2914 or the food program 360-586-1249. Call USDA's Meat and Poultry Hotline at 1-800-535-4555, 10 a.m. to 4 p.m., Eastern Time. In the Washington, DC area, call (202) 720-3333. TTY: 1-800-256-7072(Website 13). CDC. Information networks and other information sources. State and Local Health Departments(Website 14).
Diagnostic Tests Information
  1. Organism Detection Test:
    1. Gram Staining :
      1. Time to Perform: minutes-to-1-hour
      2. Description: Burkholderia mallei are fairly straight Gram-negative rods. Gram-staining is a four- part procedure which uses certain dyes to make a bacterial cell stand out against its background. The specimen should be mounted and heat fixed on a slide before you proceed to stain it(Website 22).
      3. False Positive: Not using enough decolorizer may yield a false Gram (+) result(Website 22).
      4. False Negative: Using too much decolorizer could result in a false Gram (-) result(Website 22).
  2. Immunoassay Test:
    1. The Complement-Fixation Test :
      1. Time to Perform: 1-hour-to-1-day
      2. Description: The Complement-fixation (CF) test is an accurate serological test that has been used for glanders diagnosis for many years. It is reported to be 90-95% accurate, serum being positive within 1 week of infection and remaining positive for a long time in chronic cases. The antigen for the CF test is prepared from young cultures by growing the organism on glycerol/agar slopes for 12 hours, and washing off with normal saline. The suspension is then heated for 1 hour at 65 degrees celcius.Serum is diluted 1/5 in veronal (barbiturate) buffered saline containing 0.1% gelatin (VBSG) or CFD (complement fixation diluent - available as tablets) without gelatin, and inactivated for 30 minutes at 56 degrees celcius. Serum of equidae other than horses should be inactivated at 63 degrees celcius for 30 minutes. Two-fold dilutions of the sera are prepared in 96-well round-bottom microtitre plates. Guinea pig complement is diluted in the chosen buffer and 5 (or optionally 4) complement hemolytic units-50% (CH50) are used. Sera, complement and antigen are reacted in the plates and incubated for 1 hour at 37 degrees celcius. (Some laboratories prefer to use overnight incubation at 4 degrees celcius.) A 2% suspension of sensitized washed sheep red blood cells is added, and the plates are incubated for 45 minutes at 37 degrees celcius, then centrifuged for 5 minutes at 600 g.A sample that produces 100% hemolysis at the 1/5 dilution is negative, 25-75% hemolysis is suspicious, and no hemolysis (100% fixation) is positive(Website 8).
      3. False Positive: Unfortunately false-positive results can occur as some strains cross-react with B. pseudomallei, and healthy horses can have a CF titre for a variable period following a mallein test(Website 8).
    2. Dot ELISA :
      1. Time to Perform: 2-to-7-days
      2. Description: The avidin/biotin dot enzyme-linked immunosorbent assay (dot ELISA) is a promising test for horses. The antigen is prepared from a 5-day growth on glycerol dextrose agar, suspended in sterile distilled water to give a thick suspension, and inactivated by heat at 100 degrees celcius for 1 hour. The sample is centrifuged at 20,000 g for 1 hour, and the volume of water is adjusted to give a 20% packed cell volume. Phenyl methyl sulphonyl fluoride (PMSF), at 2 mM concentration, is added to inhibit protease, and the sample is freeze/thawed six times in liquid nitrogen. It is then centrifuged at 20,000 g for 1 hour, and the supernatant is filtered through a 0.23 m membrane filter. Protein is estimated and the antigen is stored at -20 degrees celcius with merthiolate (1/10,000). The antigen is used at a concentration of 0.6 mg protein/ml.Test procedure.Dot approximately 1 ml of optimally diluted antigen on to the centre of a nitrocellulose dipstick with a micropipette, and allow to dry at 37 degrees celcius for 15 minutes.Unbound nitrocellulose sites are blocked by incubating the dipsticks in 3% skimmed milk in phosphate buffered saline (PBS) at 37 degrees celcius for 30 minutes.After rinsing in PBS, incubate the dipsticks in an appropriate dilution of equine serum for 30 minutes at 37 degrees celcius, and then wash them in three changes of 0.05% PBS/Tween 20 (PBST) with a minimum of five shakings in each wash.Further incubation is carried out in optimally diluted biotinylated anti-horse IgG in PBS (Vector Laboratories, USA. http://www.vectorlabs.com) at 37 degrees celcius for 20 minutes.Wash the dipsticks again as before, and immerse in optimally diluted horseradish peroxidase/avidin D (Vector Laboratories) in 0.5% gelatin/PBS for 8 minutes at room temperature.After washing as before, the enzyme reaction is developed with a substrate solution containing 0.3 mg/ml of diamino-benzidine and 1 ml of H2O2 solution (30%) in PBS.The reaction is developed for 5 minutes and stopped by washing the dipsticks in tap water.A positive reaction is indicated by the appearance of a clearly visible brown dot in the antigen-coated area.Using antigen-dotted, preblocked dipsticks, the test can be completed in approximately 1 hour. Serum or whole blood can be used for the test, and partial hemolysis does not impart any background colour to the antigen-coated area on the nitrocellulose.Low antibody levels, 1/100 or less, can be demonstrated in the normal equine population. Naturally infected and sensitized horses have dot ELISA titres ranging from 1/400 to 1/25,600. With the limited data available from infected horses at this stage, a 1/200 dilution of the serum is recommended as a positive threshold. Positive dot ELISA titres may be seen on day 4 post-infection, but are present from day 6 and persist for 7 weeks, although some animals may still be positive at 11 weeks. Test results are unreliable for up to 6 weeks after a mallein test(Website 8, Verma et al., 1990).
    3. The Mallein Test :
      1. Time to Perform: 1-hour-to-1-day
      2. Description: The mallein test.The mallein purified protein derivative (PPD), which is available commercially, is a solution of water-soluble protein fractions of heat-treated B. mallei. The test depends on infected horses being hypersensitive to mallein. Advanced clinical cases in horses and acute cases in donkeys and mules may give inconclusive results requiring additional methods of diagnosis to be employed.A. The intradermo-palpebral test.This is the most sensitive, reliable and specific test for detecting infected solipeds, and has largely displaced the ophthalmic and subcutaneous tests; 0.1 ml of concentrated mallein PPD is injected intradermally into the lower eyelid and the test is read at 24 and 48 hours. A positive reaction is characterized by marked edematous swelling of the eyelid, and there may be a purulent discharge from the inner canthus or conjunctiva. This is usually accompanied by a rise in temperature. With a negative response, there is usually no reaction or only a little swelling of the lower lid.B. The ophthalmic test.This is less reliable than the intradermo-palpebral test. A few drops of mallein are instilled into the eye at the canthus. In an infected animal, the eyelids, and sometimes the side of the face, become swollen and there may be a little discharge from the eye. The reaction may also occur to a lesser extent in the opposite eye.Mallein PPD for use in performing the intradermo-palpebral and ophthalmic tests is produced commercially by two institutes (Institute for Animal Science and Health (ID-Lelystad), Edelhertweg 15, 8219 PH, Lelystad, The Netherlands, and Min Agr Si Industr. Institut Die Cercertari Si Biopreparate Pasteur, R-7000 - Bucuresti, 77.826 SOS Giulesti Nr 333, Romania)(Website 8).
  3. Nucleic Acid Detection Test: