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SUMMARY
Glanders is a contagious and fatal disease of horses, donkeys, and mules, and is caused by infection with the bacterium Burkholderia mallei (the name recently changed from Pseudomonas mallei and was previously classified variously as Pfeifferella, Loefflerella, Malleomyces or Actinobacillus). The disease causes nodules and ulcerations in the upper respiratory tract and lungs. A skin form also occurs, known as 'farcy'. Control of glanders requires testing of suspect clinical cases, screening of apparently normal equids, and elimination of positive reactors. It is transmitted to humans and all infected or potentially infected material must be handled a laboratory that meets the requirements for Containment Group 3 pathogens.
Identification of the agent: Smears from fresh material may reveal Gram-negative nonsporulating, nonencapsulated rods. The presence of a capsule-like cover has been established by electron microscopy. The bacteria grow aerobically and prefer media that contains glycerol. Unlike the Pseudomonas species, Burkholderia mallei is nonmotile. Guinea-pigs are highly susceptible, and males are used for testing potentially infected material. Intraperitoneal injections are given to attempt to elicit the Strauss reaction (orchitis).
Mallein and serological tests: The mallein test is a sensitive and specific clinical test for glanders. Mallein, a protein fraction of the organism, is injected intradermo-palpebrally or given by eyedrop. In infected animals, the eyelid swells markedly in 1-2 days. A complement fixation test, and an enzyme-linked immunosorbent assay are the most accurate and reliable serological tests for diagnostic use. A rose bengal plate agglutination test has recently been developed in Russia; it has been validated in Russia only.
Requirements for vaccines and diagnostic biologicals: There are no vaccines. Mallein purified protein derivative is currently available commercially from institutes in The Netherlands and Romania (see footnote 1).
A. INTRODUCTION
Glanders is a bacterial disease of perissodactyls or odd-toed ungulates with zoonotic potential that has been known since ancient times, and that is caused by infection with the bacterium Burkholderia mallei (the name recently changed from Pseudomonas mallei (25) and was previously classified variously as Pfeifferella, Loefflerella, Malleomyces or Actinobacillus). Outbreaks of the disease may occur in members of the cat family living in the wild or in zoological gardens. Susceptibility to glanders has been proved in camels, bears, wolfs and dogs. Carnivores may become infected by eating infected meat, but cattle, sheep and pigs are resistant (16). Glanders in the acute form occurs most frequently in donkeys and mules, which run a high fever and exhibit respiratory signs (swollen nostrils, dyspnoea, and pneumonia); death occurs within a few days. In horses, glanders generally runs a more chronic course and they may survive for several years. Chronic and subclinical 'occult' cases are dangerous sources of infection.
In horses, inflammatory nodules and ulcers develop in the nasal passages and give rise to a sticky yellow discharge, accompanied by enlarged firm submaxillary lymph nodes. Stellate scarring follows healing of the ulcers. The formation of nodular abscesses in the lungs is accompanied by progressive debility, febrile episodes, coughing and dyspnoea. Diarrhoea and polyuria can also occur. In the skin form ('farcy'), the lymphatics are enlarged and nodular abscesses ('buds') of 0.5-2.5 cm develop, which ulcerate and discharge yellow oily pus. Discharges from the respiratory tract and skin are infective, and transmission between animals, which is facilitated by close contact, is by inhalation, ingestion of contaminated material (e.g. from infected feed and water troughs), or by inoculation (e.g. via a harness). The incubation period is from a few days to many months (17).
Glanders is transmissible to humans by direct contact with sick animals or infected materials. In the untreated acute disease, there can be 95% mortality within 3 weeks. However, survival is possible if the infected person is treated early and aggressively with multiple systemic antibiotic therapies (11, 19). A chronic form with abscessation also occurs. When handling suspect or known infected animals or fomites, stringent precautions should be taken to prevent self-infection or transmission of the bacterium to other equids. Laboratory samples should be securely packaged, kept cool and shipped as outlined in Chapter I.1.1. of this Terrestrial Manual. All manipulations with potentially infected material must be performed in a laboratory that meets the requirements for Containment Group 3 pathogens as outlined in Appendix I.1.6.1 of Chapter I.1.6 of this Terrestrial Manual.
Glanders has been eradicated from many countries by statutory testing, elimination of infected animals, and import restrictions. It persists in some Asian, African and South American countries.
B. DIAGNOSTIC TECHNIQUES
| 1. | Identification of the agent
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| | Cases for specific glanders investigation should be differentiated on clinical grounds from other chronic infections of the nasal mucosae or sinuses, and from strangles (Streptococcus equi infection), ulcerative lymphangitis (Corynebacterium pseudotuberculosis), pseudotuberculosis (Yersinia pseudotuberculosis) and sporotrichosis (Sporotrichium spp.). Glanders should be excluded positively from suspected cases of epizootic lymphangitis (caused by Histoplasma farciminosum), with which it has many clinical similarities. In humans in particular, glanders should be distinguished from melioidosis (B. pseudomallei infection), which is caused by an organism with close similarities to B. mallei (16).
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| | a) | Morphology of Burkholderia mallei
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| | | The organisms are fairly numerous in smears from fresh lesions, but in older lesions they are scanty (22). They should be stained by methylene blue or Gram stain. The organisms are mainly extracellular, fairly straight Gram-negative rods with rounded ends, 2-5 µm long and 0.3-0.8 µm wide with granular inclusions of various size, often stain irregularly and do not have capsules or form spores. The presence of a capsule-like cover has been established by electron microscopy. This capsule is composed of neutral carbohydrates and serves to protect the cell from unfavourable environmental factors. Unlike other organisms in the Pseudomonas group, Burkholderia mallei have no flagellae and are therefore nonmotile (13). The organisms are difficult to demonstrate in tissue sections, where they may have a beaded appearance (15). In culture media, they vary in appearance depending on the age of the culture and type of medium. In older cultures, there is much pleomorphism. Branching filaments form on the surface of broth cultures.
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| | b) | Cultural characteristics
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| | | It is preferable to attempt isolation from unopened uncontaminated lesions (15). The organism is aerobic and facultatively anaerobic only in the presence of nitrate (6, 13), growing optimally at 37°C (14). It grows well, but slowly, on ordinary culture media, 48-hour incubation of cultures is recommended; glycerol enrichment is particularly useful. After a few days on glycerol agar, there is a confluent, slightly cream-coloured growth that is smooth, moist, and viscid. With continued incubation, the growth thickens and becomes dark brown and tough. It also grows well on glycerol potato agar and in glycerol broth, on which a slimy pellicle forms. On plain nutrient agar, the growth is much less luxuriant, and growth is poor on gelatin (20).
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| | | Alterations to characteristics may occur in vitro, so fresh isolates should be used for identification reactions. Litmus milk is slightly acidified by B. mallei, and coagulation may occur after long incubation. The organism reduces nitrates. Although some workers have claimed that glucose is the only carbohydrate that is fermented (slowly and inconstantly), other workers have shown that if an appropriate medium and indicator are used, glucose and other carbohydrates, such as arabinose, fructose, galactose and mannose, are consistently fermented by B. mallei (5). Indole is not produced, horse blood is not haemolysed and no diffusible pigments are produced in cultures (13). A commercial laboratory test kit (e.g. API [Analytical Profile Index] system: Analytab Products, BioMerieux or Biolog [Hayward, California]) can be used for easy confirmation that an organism belongs to the Pseudomonas group. Lack of motility is then of special relevance. A bacteriophage specific for B. mallei is available (23).
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| | | All culture media prepared should be subjected to quality control and must support growth of the suspect organism from a small inoculum. The reference strain should be cultured in parallel with the suspicious samples to ensure that the tests are working correctly.
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| | | In contaminated samples, supplementation of media with substances that inhibit the growth of Gram-positive organisms (e.g. crystal violet, proflavine) has proved useful, as has pretreatment with penicillin (1000 units/ml for 3 hours at 37°C) (22). A selective medium has been developed (24) composed of polymyxin E (1000 units), bacitracin (250 units), and actidione (0.25 mg) incorporated into nutrient agar (100 ml) containing glycerine (4%), donkey or horse serum (10%), and ovine haemoglobin or trypton agar (0.1%).
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| | | Outside the body, the organism has little resistance to drying, heat, light or chemicals, so that survival beyond 2 weeks is unlikely. Under favourable conditions, however, it can probably survive a few months. Burkholderia mallei can remain viable in tap water for at least 1 month (20). For disinfection, benzalkonium chloride or 'roccal' (1/2000), sodium hypochlorite (500 ppm available chlorine), iodine, mercuric chloride in alcohol, and potassium permanganate have been shown to be highly effective against B. mallei (14). Phenolic disinfectants are less effective.
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| | c) | Laboratory animal inoculation
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| | | Guinea-pigs, hamsters and cats have been used for diagnosis when necessary. If isolation in a laboratory animal is considered unavoidable, suspected material is inoculated intraperitoneally into a male guinea-pig. Positive material will cause a severe localised peritonitis and orchitis (the Strauss reaction). The number of organisms and their virulence determines the severity of the lesions. Additional steps are used when the test material is heavily contaminated (7). The Strauss reaction is not specific for glanders, and other organisms can elicit it. Bacteriological examination of infected testes should confirm the specificity of the response obtained.
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| | d) | Other methods
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| | | There have been recent advances in the application of molecular biological techniques to the detection of glanders. A polymerase chain reaction (PCR) for the specific detection of B. mallei DNA has been developed (2) that allows differentiation between B. mallei and B. pseudomallei. The technique has not yet been fully validated or gained wide acceptance. However, PCR has the potential to be a safe, fast method to confirm infection. Methods to differentiate between B.mallei and B. pseudomallei are important as this cannot be reliably accomplished by the current serological tests.
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| 2. | Mallein and serological tests
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The mallein test (a prescribed test for international trade)
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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 (1).
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The intradermo-palpebral test
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This is the most sensitive, reliable and specific test for detecting infected perissodactyls or odd-toed ungulates, and has largely displaced the ophthalmic and subcutaneous tests (3): 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 characterised by marked oedematous 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.
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The ophthalmic test
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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.
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The subcutaneous test
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This test interferes with subsequent serological diagnosis and so the other two mallein tests are preferred. Also, the test may not be acceptable in some countries. The horse's temperature has to be under 102°F (38.8°C) on the day before the test, at the time of the injection, and at 9, 12 and 15 hours after the injection. A 10 cm square skin patch in the middle of the neck is clipped and disinfected; 2.5 ml of dilute mallein are injected subcutaneously into the centre of the patch. With a positive test, the horse develops a pyrexia of 104°F (40.0°C) or over during the first 15 hours, and a firm painful swelling with raised edges develops within 24 hours at the injection site. In nonglandered horses, there is no, or minimal, transient local swelling. Doubtful reactors may be retested after 14 days using a double dose of mallein.
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b) |
Complement fixation test (a prescribed test for international trade)
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Although not as sensitive as the mallein test, the CF test is an accurate serological test that has been used for glanders diagnosis for many years (3). It is reported to be 90-95% accurate, serum being positive within 1 week of infection and remaining positive in the case of exacerbation of the chronic process (20).
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Antigen preparation (10)
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Flasks of beef infusion broth with 3% glycerol are inoculated with log-phase growth B. mallei and incubated at 37°C for 8-12 weeks.
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The cultures are inactivated by exposing the flasks to flowing steam (100°C) for 60 minutes.
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The clear supernatant is decanted and filtered. The filtrate is heated again by exposure to live steam for 75 minutes on 3 consecutive days, and clarified by centrifugation.
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The clarified product is concentrated to one-tenth the original volume by evaporation on a steam or hot water bath.
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Concentrated antigen is bottled in brown-glass bottles to protect from light and stored at 4°C. Antigen has been shown to be stable for at least 10 years in this concentrated state.
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Lots of antigen are prepared by diluting the concentrated antigen 1/20 with sterile physiological saline with 0.5% phenol. The diluted antigen is dispensed into brown-glass vials and store at
4°C. The final working dilution is determined by a block titration. The final working dilution for CF test use is made at the time the CF test is performed.
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The resulting antigen is primarily lipopolysaccharide. An alternative procedure is to use young cultures by growing the organism on glycerol-agar slopes for 12 hours and washing off with normal saline. A suspension of the culture is heated for 1 hour at 70°C and the heat-treated bacterial suspension is used as antigen. The disadvantage of this antigen preparation method is that the antigen contains all the bacterial cell components. The antigen should be safety tested by inoculating blood agar plates.
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Test procedure (18)
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Serum is diluted 1/5 in veronal (barbiturate) buffered saline containing 0.1% gelatin (VBSG) or CFD (complement fixation diluent - available as tablets) without gelatine.
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Diluted serum is inactivated for 30 minutes at 56°C. (Serum of equidae other than horses should be inactivated at 63°C for 30 minutes.)
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Twofold dilutions of the sera are prepared in 96-well round-bottom microtitre plates.
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Guinea-pig complement is diluted in the chosen buffer and 5 (or optionally 4) complement haemolytic units-50% (CH50) are used.
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Sera, complement and antigen are reacted in the plates and incubated for 1 hour at 37°C. (An alternate acceptable procedure is overnight incubation at 4°C.)
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A 2% suspension of sensitised washed sheep red blood cells is added.
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Plates are incubated for 45 minutes at 37°C, and then centrifuged for 5 minutes at 600 g.
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A sample that produces 100% haemolysis at the 1/5 dilution is negative, 25-75% haemolysis is suspicious, and no haemolysis (100% fixation) is positive. Unfortunately, false-positive results can occur, and B. pseudomallei and B. mallei cross react and cannot be differentiated by serology (3). Also healthy horses can have a false positive CF reaction for a variable period following a mallein intradermal test.
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| | c) | Enzyme-linked immunosorbent assays
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| | | Both plate and membrane (blot) enzyme-linked immunosorbent assays (ELISAs) have been reported for the serodiagnosis of glanders, but none of these procedures has been shown to differentiate serologically between B. mallei and B. pseudomallei. Blotting approaches have involved both dipstick dot-blot and electrophoretically separated and transferred western blot methods (8, 21). A competitive ELISA that uses an anti-lipopolysaccharide monoclonal antibody has also been developed and found to be similar to the CF test in performance (9). Continuing development of monoclonal antibody reagents specific for B. mallei antigenic components offers the potential for more specific ELISAs in the foreseeable future that will help resolve questionable test results of quarantined imported horses (4, 12). At this time, none of these tests has been validated.
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| | d) | Other serological tests
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| | | The avidin-biotin dot ELISA has been described (21), but has not yet been widely used or validated. The antigen is heat-inactivated bacterial culture that has been concentrated and purified. A dot of this antigen is placed on a nitrocellulose dipstick that is then used to test for antibody against B. mallei in equine serum. 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 haemolysis does not impart any background colour to the antigen-coated area on the nitrocellulose.
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| | | The rose bengal plate agglutination test (RBT) has been described for the diagnosis of glanders in horses and other susceptible animals; the test has been validated in Russia only. The antigen is a heat-inactivated bacterial suspension coloured with rose bengal, which is used in a plate agglutination test.
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| | | The accuracy of other agglutination tests and precipitin is unsatisfactory for use in control programmes. Horses with chronic glanders and those in a debilitated condition give negative or inconclusive results.
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C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
No vaccines are available.
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).
The ID-Lelystad has provided the following information on requirements for mallein PPD.
| 1. | Seed management
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| | Three strains of Burkholderia mallei are employed in the production of mallein PPD, namely Bogor strain (originating from Indonesia), Mukteswar strain (India) and Zagreb strain (Yugoslavia). The seed material is kept as a stock of freeze-dried cultures. The strains are subcultured on to glycerol agar at 37°C for 1-2 days. For maintaining virulence and antigenicity, the strains may be passaged through guinea-pigs.
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| 2. | Method of manufacture
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| | Dorset(Henley medium, enriched by the addition of trace elements, is used for production of mallein PPD. The liquid medium is inoculated with a thick saline suspension of B. mallei, grown on glycerol agar. The production medium is incubated at 37°C for about 10 weeks. The bacteria are then killed by steaming for 3 hours in a Koch's steriliser. The fluid is then passed through a layer of cotton wool to remove coarse bacterial clumps. The resulting turbid fluid is cleared by membrane filtration, and one part trichloroacetic acid 40% is immediately added to nine parts culture filtrate. The mixture is allowed to stand overnight and a light brownish to greyish precipitate settles.
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| | The supernatant fluid is pipetted off and discarded. The precipitate is centrifuged for 15 minutes at 2500 g and the layer of precipitate is washed three or more times in a solution of 5% NaCl, pH 3, until the pH is 2.7. The washed precipitate is dissolved by stirring with a minimum of an alkaline solvent. The fluid is dark brown and a pH of 6.7 will be obtained. This mallein concentrate has to be centrifuged thoroughly and the supernatant is diluted with an equal amount of a glucose buffer solution. The protein content of this product is estimated by the Kjeldahl method and freeze-dried after it has been dispensed into ampoules.
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| 3. | In-process control
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| | During the period of incubation, the flasks are inspected frequently for any signs of contamination, and suspect flasks are discarded. A typical growth of the B. mallei cultures comprises turbidity, sedimentation, some surface growth with a tendency towards sinking, and the formation of a conspicuous slightly orange-coloured ring along the margin of the surface of the medium.
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| 4. | Batch control
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| | Each batch of mallein PPD is tested for sterility, safety, preservatives, protein content and potency.
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| | Sterility testing is performed according to the European Pharmacopoeia guidelines.
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| | The examination for safety is conducted on from five to ten normal healthy horses by carrying out the intradermo-palpebral test. The resulting swelling should be, at most, barely detectable and transient, without any signs of conjunctival discharge.
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| | Preparations containing phenol as a preservative should not contain more than 0.5% (w/v) phenol. The protein content should be not less than 0.95 mg/ml and not more than 1.05 mg/ml.
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| | Potency testing is performed in guinea-pigs and horses. The animals are sensitised by subcutaneous inoculation with a concentrated suspension of heat-killed B. mallei in paraffin oil or incomplete Freund's adjuvant. Cattle can also be used instead of horses. The production batch is bioassayed against a standard mallein PPD by intradermal injection in 0.1 ml doses in such a way that complete randomisation is obtained.
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| | In guinea-pigs, the different areas of erythema are measured after 24 hours, and in horses the increase in skin thickness is measured by calipers. The results are statistically evaluated, using standard statistical methods for parallel-line assays.
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| 2. | Bauernfeind A., Roller C., Meyer D., Jungwirth R. & Schneider I. (1998). Molecular procedure for rapid detection of Burkholderia mallei and Burkholderia pseudomallei. J. Clin. Microbiol., 36, 2737-2741.
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| 5. | Evans D. (1966). Utilization of carbohydrates by Actinobacillus mallei. Can. J. Microbiol., 12, 625-639.
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| 9. | Katz J., Dewald R. & Nicholson J. (2000). Procedurally similar competitive immunoassay systems for the serodiagnosis of Babesia equi, Babesia caballi, Trypanosoma equiperdum, and Burkholderia mallei infection in horses. J. Vet. Diagn. Invest., 12, 46-50.
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| 10. | Kelser R.A. & Reynolds F.H.K. (1935). Special veterinary laboratory methods, complement fixation test for glanders. In: Laboratory Methods for the United States Army, Simons J.S. & Gantzkow C.J., eds. Lea and Febiger, Philadelphia, USA, 973-987.
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| 11. | Khan A.S. & Ashford D.A. (2001). Ready or not - Preparedness for bioterrorism. New Engl. J. Med., 345, 287-289.
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| 12. | Khrapova N.P., Tikhonov N.G., Prokhvatilova E.V. & Kulakov M.I. (1995). The outlook for improving the immunoglobulin preparations for the detection and identification of the causative agents of glanders and melioidosis. Med. Parazitol., 4, 49-53.
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| 15. | Miller W., Pannel L., Cravitz L., Tanner A. & Ingalls M. (1948). Studies on certain biological characteristics of Malleomyces mallei and Malleomyces pseudomallei I. Morphology, cultivation, viability, and isolation from contaminated specimens. J. Bacteriol., 55, 115-126.
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| 16. | Minett F.C. (1959). Glanders (and melioidosis). In: Infectious Diseases of Animals. Vol. 1. Diseases due to Bacteria, Stableforth A.W. & Galloway I.A., eds. Butterworths Scientific Publications, London, UK, 296-309.
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| 17. | Monlux W.S. (1984). Diseases affecting food and working animals. In: Foreign Animal Diseases. United States Department of Agriculture, US Government Printing Office, Washington DC, USA, 178-185.
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| 19. | Srinivasan A., Kraus C.N., DeShazer D., Becker P.M., Dick J.D., Spacek L., Bartlett J.G., Byrne W.R. & Thomas D.L. (2001). Glanders in a military research microbiologist. New Engl. J. Med., 345, 256-258.
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| 20. | Steele J.H. (1980). Bacterial, rickettsial and mycotic diseases. In: CRC Handbook Series in Zoonoses, Section A, Vol. I, Steele J.H., ed. CRC Press, Boca Raton, USA, 339-351.
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| 21. | Verma R.D., Sharma J.K., Venkateswaran K.S. & Batra H.V. (1990). Development of an avidin-biotin dot enzyme-linked immunosorbent assay and its comparison with other serological tests for diagnosis of glanders in equines. Vet. Microbiol., 25, 77-85.
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| 22. | Wilson G.S. & Miles A.A. (1964). Glanders and melioidosis. In: Topley and Wilson's Principles of Bacteriology and Immunity. E. Arnold, London, UK, 1714-1717.
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| 23. | Woods D.E., Jeddeloh J.A., Fritz D.L. & DeShazer D. (2002). Burkholderia thailandensis E125 harbors a template bacteriophage specific for Burkholderia mallei. J. Bacteriol., 184, 4003-4017.
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| 24. | Xie X., Xu F., Xu B., Duan X. & Gong R. (1980). A New Selective Medium for Isolation of Glanders Bacilli. Collected papers of veterinary research. Control Institute of Veterinary Biologics, Ministry of Agriculture, Peking, China (People's Rep. of), 6, 83-90.
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| 25. | Yabuuchi E., Kosako Y., Oyaizu H., Yano I., Hotta H., Hashimoto Y., Ezaki T. & Arakawa M. (1992). Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol. Immunol., 36, 1251-1275.
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