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. 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

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.

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).

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).

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).

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).

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:

1. Horses, Human and Animal Models
   1. Taxonomy Information:
      1. Species:
         1. Equidae :
            • Common Name: Equidae
            • GenBank Taxonomy No.: 9788
            • Description: The major hosts are horses (including mules and donkeys). Probably, Burkholderia mallei infections can also occur in dogs, cats, goats and camels. Hamsters and guinea pigs can be infected in the laboratory. Glanders has been only a rare and sporadic disease in humans, and no epidemics have been reported(Website 4).
         2. Homo sapiens :
            • Common Name: Homo sapiens
            • GenBank Taxonomy No.: 9606
            • Description: Human infection has occurred rarely and sporadically among laboratory workers and those in direct and prolonged contact with infected animals(Website 7).
         3. Mesocricetus auratus :
            • Common Name: Mesocricetus auratus
            • GenBank Taxonomy No.: 10036
            • Description: Burkholderia mallei infection hamster model(Website 23).
         4. Mus sp :
            • Common Name: Mus sp
            • GenBank Taxonomy No.: 10095
            • Description: Burkholderia mallei infection mice model(Website 24).
   2. Infection Process:
      1. Infectious Dose: The infectious dose of Burkholderia mallei for humans is not known(Website 18),
      2. Description: Burkholderia mallei enters the human organism through the skin and through mucosal surfaces of the eyes and nose by inhalation of bacteria in aerosols or dust or contact with infected animals(Website 12),
   3. Disease Information:
      1. Glanders(i.e., ) :
         1. Incubation: The incubation period ranges from 10 - 14 days, depending on the inhaled dose(Website 17),
         2. Prognosis:
            Untreated patients with septicemia have fatal outcomes. Before antibiotics, the death rate for septicemic disease was 95%. It is greater than 50% for septicemic disease and 20% for localized disease despite treatment. Overall, mortality is 40%(Website 16),
         3. Diagnosis Summary: Methylene blue stain of exudates may reveal scant small bacilli. Chest X-rays may show miliary lesions, small multiple lung abscesses, or bronchopneumonia. Burkholderia mallei can be cultured from infected secretions using meat nutrients(Website 20),
         4. Symptom Information :
            • Symptom -- Burkholderia mallei infection :
              • Description: Symptoms of glanders: The symptoms of glanders depend upon the route of infection with the organism. The types of infection include localized, pus-forming cutaneous infections, pulmonary infections, bloodstream infections, and chronic suppurative infections of the skin. Generalized symptoms of glanders include fever, muscle aches, chest pain, muscle tightness, and headache. Additional symptoms have included excessive tearing of the eyes, light sensitivity, and diarrhea. Localized infections: If there is a cut or scratch in the skin, a localized infection with ulceration will develop within 1 to 5 days at the site where the bacteria entered the body. Swollen lymph nodes may also be apparent. Infections involving the mucous membranes in the eyes, nose, and respiratory tract will cause increased mucus production from the affected sites.Pulmonary infections: In pulmonary infections, pneumonia, pulmonary abscesses, and pleural effusion can occur. Chest X-rays (CXR) will show localized infection in the lobes of the lungs. Bloodstream infections: Glanders bloodstream infections are usually fatal within 7 to 10 days.Chronic infections: The chronic form of glanders involves multiple abscesses within the muscles of the arms and legs or in the spleen or liver.Symptoms include fever, malaise, pleuritic chest pain, cervical adenopathy, splenomegaly, and generalized papular/pustular eruptions. Mortality rate is over 50% despite antibiotic treatment(Website 19).
         5. Treatment Information:
            • Antibiotic-Amoxicillin-Clavulanate. : Amoxicillin and Clavulanate (Augmentin). Amoxicillin is a semisynthetic antibiotic with a broad spectrum of bactericidal activity against many gram-positive and gram-negative microorganisms. Formulation of amoxicillin and clavulanic acid protects amoxicillin from degradation by beta-lactamase enzymes and effectively extends antibiotic spectrum of amoxicillin to include many bacteria normally resistant to amoxicillin and other beta-lactam antibiotics. Possesses properties of a broad-spectrum antibiotic and a beta-lactamase inhibitor.Adult Dose: 60 mg/kg/d PO divided TID (TID=3 times daily) for local disease or mild toxicity.Pediatric Dose: Administer as in adults(Website 16).
              • Contraindicator: Amoxicillin is contraindicated in pateints with documented hypersensitivity; history of Augmentin-associated cholestatic jaundice and/or hepatic dysfunction. Renal impairment(Website 16).
              • Complication: Probenecid decreases renal tubular secretion of amoxicillin; concurrent use may result in increased and prolonged blood levels of amoxicillin; coadministration of probenecid cannot be recommended; concurrent administration of allopurinol and ampicillin substantially increases incidence of rashes in patients receiving both drugs compared to patients receiving ampicillin alone; unknown whether this potentiation of ampicillin rashes is due to allopurinol or hyperuricemia present in these patients. Pregnancy: Usually safe but benefits must outweigh the risks(Website 16).
              • Drug Resistance: The resistance to amoxicillin and clavulanate of B. mallei is not available.
            • Antibiotic-Tetracycline. : Tetracycline (Minocin) is bacteriostatic antibiotic. Adult Dose: 40 mg/kg/d PO divided tid.Pediatric Dose: Less than 8 years: Not recommended. More than 8 years: Administer as in adults(Website 16).
              • Contraindicator: Tetracycline is contraindicated in pateints with documented hypersensitivity. Pseudotumor cerebri (benign intracranial hypertension) in adults has been associated with use; photosensitivity manifested by exaggerated sunburn reaction has been observed in some individuals; persons with impaired renal function may require a lower dose.Unsafe in pregnancy(Website 16).
              • Complication: Patients receiving anticoagulant therapy may require downward adjustment of anticoagulant dosage; since bacteriostatic drugs may interfere with bactericidal action of penicillin, avoid administering tetracycline-class drugs in conjunction with penicillin; absorption is impaired by antacids containing aluminum, calcium, or magnesium and by iron-containing preparations; concurrent use of methoxyflurane has been reported to result in fatal renal toxicity(Website 16).
              • Drug Resistance: The resistance to tetracycline of B. mallei is not available.
            • Antibiotic-Sulfamethoxazole-Trimethoprim. : Sulfamethoxazole (SMX) and trimethoprim (TMP) (Bactrim, Bactrim DS, Septra, Septra DS). SMX inhibits bacterial synthesis of dihydrofolic acid. TMP blocks production of tetrahydrofolic acid.Adult Dose: Local or mild disease: 4 mg TMP/kg/d and 20 mg SMX/kg/d PO (orally) divided bid.Severe disease: 8 mg TMP/kg/d and 40 mg SMX/kg/d IV (intravenous) divided qid for 2 wk; then PO (orally) for 6 mo.Pediatric Dose: Less than 2 months: Not recommended. More than 2 months: Administer as in adults(Website 16).
              • Contraindicator: Documented hypersensitivity; documented megaloblastic anemia due to folate deficiency, and severe allergies or bronchial asthma. Safety for use during pregnancy has not been established(Website 16).
              • Complication: Local irritation and inflammation due to extravascular infiltration of the infusion have been observed with IV. Increased incidence of thrombocytopenia with purpura has been reported in elderly patients concurrently receiving certain diuretics (primarily thiazides). It may inhibit hepatic metabolism of phenytoin(Website 16).
              • Drug Resistance: The resistance to sulfamethoxazole and trimethoprim of B. mallei is not available.
            • Antibiotic-Ceftazidime. : Ceftazidime (Fortaz) is bactericidal broad spectrum antibiotic (Third generation cephalosporins).Adult Dose: 120 mg/kg/d IV divided tid.Pediatric Dose: Administer as in adults(Website 16).
              • Contraindicator: Ceftazidime (Fortaz)is contraindicated in pateints with documented hypersensitivity. Reduce total daily dosage in patients with renal insufficiency; cephalosporins may be associated with decreased prothrombin activity; those at risk include patients with renal and hepatic impairment, poor nutritional state, and those receiving a protracted course of antimicrobial therapy; caution in individuals with colitis. Pregnancy: Usually safe but benefits must outweigh the risks(Website 16).
              • Complication: Nephrotoxicity has been reported following concomitant administration of cephalosporins with aminoglycoside antibiotics or potent diuretics such as furosemide; chloramphenicol has been demonstrated to be antagonistic to beta-lactam antibiotics, including ceftazidime, based on in vitro studies and time kill curves with enteric gram-negative bacilli; because of possibility of antagonism in vivo, particularly when bactericidal activity is desired, avoid this drug combination(Website 16).
              • Drug Resistance: The resistance to ceftazidime of B. mallei is not available.
            • Antibiotic-Streptomycin : Streptomycin is aminoglycoside antibiotic. Aminoglycoside antibiotic is recommended when less potentially hazardous therapeutic agents are ineffective or contraindicated.Adult Dose: 30 mg/kg/d IM (intramuscular), not to exceed 2 g/d.Pediatric Dose: 25-30 mg/kg/d IM, not to exceed 1-1.5 g/d(Website 16).
              • Contraindicator: Streptomycin is contraindicated in pateints with documented hypersensitivity. Caution in patients with renal failure who are not on dialysis; caution with myasthenia gravis, hypocalcemia, and conditions that depress neuromuscular transmission. Unsafe in pregnancy(Website 16).
              • Complication: Nephrotoxicity may be increased with combination of aminoglycosides and loop diuretics(Website 16).
              • Drug Resistance: The resistance to streptomycin of B. mallei is not available.
   4. Prevention:
      1. Preventing Glanders
         • Description: In countries where glanders is endemic in animals, identification and elimination of the disease in the animal population prevents disease in humans. Burkholderia mallei exists in nature only in infected susceptible hosts and is not found in water, soil, or plants(Website 15), Biosafety Level 3 containment practices are required for laboratory staff when working with these organisms(Website 16), There is no vaccine available for human use(Website 17),
         • Complication: Possible complications include septicemia, osteomyelitis, meningitis, and brain, liver, or splenic abscess(Website 16),
   5. Model System:
      1. Hamsters
         1. Model Host: Mesocricetus auratus.
            Female Syrian hamsters(DeShazer et al., 2001, Fritz et al., 1999),
         2. Model Pathogens:
         3. Description: Female Syrian hamsters, weighing between 70 and 100 g, are infected by the intraperitoneal (IP) route with B. mallei. All animals are observed at least twice daily until death(DeShazer et al., 2001, Fritz et al., 1999),
      1. Mice
         1. Model Host: Mus sp.
            Female BALB/c mice(DeShazer et al., 2001),
         2. Model Pathogens:
         3. Description: Female BALB/c mice, weighing between 20 and 25 g and approximately 68 weeks old, are challenged by aerosol using a whole-body aerosol exposure apparatus in a Class III safety cabinet in a biological safety level 3 containment facility(DeShazer et al., 2001),

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DeShazer et al., 2001: DeShazer D, Waag D, Fritz D, Woods D. Identification of a Burkholderia mallei polysaccharide gene cluster by subtractive hybridization and demonstration that the encoded capsule is an essential virulence determinant. Microbial Pathogenesis. 2001; 30(5); 253-269. [PubMed: 11373120].

Fritz et al., 1999: Fritz D, Vogel P, Brown D, Waag D. The hamster model of intraperitoneal Burkholderia mallei (glanders). Veterinary Pathology. 1999; 36(4); 276-291. [PubMed: 10421094].

Verma et al., 1990: Verma R, Sharma J, Venkateswaran K, Batra H. Development of an avidin-biotin dot enzyme-linked immunosorbent assay and its comparison with other serological tests for diagnosis of glanders in equines. Veterinary Microbiology. 1990; 25(1); 77-85. [PubMed: 2247938].

Website 10: Burkholderia mallei sequence

Website 11: CDC. Glanders. Disease information

Website 12: Mitretec Systems. Bacteria

Website 13: CDC. What to do in an emergency

Website 14: CDC. Information networks

Website 15: USDPI. Glanders

Website 16: Emedicine. Glanders

Website 17: Glanders - Medical Management

Website 18: MSDS. Infectious substances

Website 19: Symptoms of Glanders

Website 20: Medman. Glanders

Website 21: CDC. Agent Summary Statements: Bacterial Agents

Website 22: Gram-staining procedure

Website 23: NCBI. Taxonomy. Syrian hamster

Website 24: NCBI. Taxonomy. Mice

Website 3: Center for Food Security

Website 4: U.S Army Medical Research

Website 7: Clinical bacteria

Website 8: Glanders. Summary

 

PathInfo: George Abramochkin

HazARD: (for the section of Lab Animal Pathobiology & Management)

PHIDIAS: Yongqun "Oliver" He

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