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 pseudomallei :
      1. GenBank Taxonomy No.: 28450
      2. Description: Melioidosis is an infectious disease of humans and animals caused by Burkholderia pseudomallei. It was first described under the name Bacillus pseudomallei by Whitmore and Krishnaswami (1912) following its isolation in Rangoon, Myanmar (Burma) more than 90 years ago. Subsequent reports described the causative agent under a variety of names, including Bacillus whitmori, Pfeifferella whitmori, Pfeifferella pseudomallei, Actinobacillus pseudomallei, Loefferella whitmori, Malleomyces pseudomallei and Pseudomonas pseudomallei. Melioidosis has to be considered an emerging disease with high impact on animal and man. In the past century, it has spread from East Asia to many parts of the world previously not affected. Today it can be imported to regions with inappropriate climate if a subclinical carrier capable of contaminating its surroundings survives. Sporadic acute cases may also be seen in animals imported from enzootic areas(Sprague and Neubauer, 2004).
      3. Variant(s):
         • Burkholderia pseudomallei (Ara- biotype) (Sirisinha et al., 1998):
           • Description: The presence of two distinct biotypes in soil can be reliably distinguished by their ability to assimilate L-arabinose. Whereas some soil isolates could utilize this substrate (Ara+), the remaining soil isolates and all clinical isolates tested so far could not (Ara-)(Sirisinha et al., 1998). The Ara- biotype is virulent(Sonthayanon et al., 2002). The Ara- biotype is found in almost all B. pseudomallei clinical isolates(Thepthai et al., 2001). There was no significant difference between the LD(50)s for clinical and soil Ara- isolates(Smith et al., 1997).
         • Burkholderia pseudomallei (Ara+ biotype) (Website 2):
           • GenBank Taxonomy No.: 95163
           • Description: The Ara+ biotype is capable of assimilating L-arabinose(Sonthayanon et al., 2002). The Ara+ biotype is nonvirulent(Sonthayanon et al., 2002).
         • Burkholderia pseudomallei K96243 (Website 3):
           • GenBank Taxonomy No.: 272560
           • Description: The classical arabinose-negative B. pseudomallei isolate K96243(Songsivilai and Dharakul, 2000). B. pseudomallei strain K96243 was isolated in 1996 from a 34-year-old female diabetic patient in Khon Kaen hospital in Thailand(Holden et al., 2004). The Burkholderia pseudomallei K96243 genome contains multiple type IV pilin-associated loci, including one encoding a putative pilus structural protein (pilA). A pilA deletion mutant has reduced adherence to human epithelial cells and is less virulent in the nematode model of virulence and the murine model of melioidosis, suggesting a role for type IV pili in B. pseudomallei virulence(Essex-Lopresti et al., 2005).
         • Burkholderia pseudomallei 1710a (Website 28):
           • GenBank Taxonomy No.: 320371
         • Burkholderia pseudomallei 1710b (Website 29):
           • GenBank Taxonomy No.: 320372
         • Burkholderia pseudomallei 668 (Website 30):
           • GenBank Taxonomy No.: 320373
         • Burkholderia pseudomallei S13 (Website 31):
           • GenBank Taxonomy No.: 320374

1. B. pseudomalleiLifecycle
   1. Stage Information:
      1. Burkholderia pseudomallei cells:
         • Size: The bacteria are small (0.8 x 1.5 m).
         • Shape: Burkholderia pseudomallei, a causative agent of melioidosis, is a facultative intracellular gram-negative bacillus.
         • Picture(s):
           • TEXT (Website 32)
             
             
             
             Description: TEXT(Website 32).
   2. Description: B. pseudomallei occurs as a soil organism in Southeast Asia and northern Australia, and incidents of melioidosis are generally confined to these endemic areas(Woods et al., 1999).

1. Genome of Burkholderia pseudomallei
   1. Description: The complete genome of B. pseudomallei strain K96243 consists of two circular replicons (European Molecular Biology Laboratory accession nos. BX571965 [GenBank] and BX571966 [GenBank] ) of 4.07 Mb and 3.17 Mb each that have been designated chromosome 1 and chromosome 2. Genomic comparisons with closely and more distantly related bacteria revealed a greater level of gene order conservation and a greater number of orthologous genes on the large chromosome, suggesting that the two replicons have distinct evolutionary origins(Holden et al., 2004).
   2. Burkholderia pseudomallei K96243 chromosome 1
      1. GenBank Accession Number: NC_006350
      2. Size: 4.07 Mb(Holden et al., 2004).
      3. Gene Count: 3,460 coding sequences (CDSs)(Holden et al., 2004).
      4. Description: The large chromosome encodes many of the core functions associated with central metabolism and cell growth(Holden et al., 2004).
   3. Burkholderia pseudomallei K96243 chromosome 2
      1. GenBank Accession Number: NC_006351
      2. Size: 3.17 Mb(Holden et al., 2004).
      3. Gene Count: 2,395 coding sequences (CDSs)(Holden et al., 2004).
      4. Description: The small chromosome carries more accessory functions associated with adaptation and survival in different niches(Holden et al., 2004).

1. Biosafety information for Burkholderia pseudomallei
   1. Level: Biosafety level (BSL) 2 practices, equipment, and containment are recommended for working with known or potentially infectious body fluids, tissue specimens, or cultures. However, a review of work in a clinical laboratory in an area in which melioidosis is endemic indicated low risk to laboratory workers(CDC Report, 2004).

1. Ashdown's selective agar (Ashdown, 1979):
   1. Description: Definitive diagnosis of melioidosis requires a positive culture of B. pseudomallei. Melioidosis must be considered in febrile patients in or returning from endemic regions to enable appropriate samples and laboratory awareness. B.pseudomallei readily grows in commercially available blood culture media but it is not unusual for laboratories in nonendemic locations to misidentify the bacteria. Culture from nonsterile sites can be problematic and the likelihood of successful culture is increased if sputum, throat swabs, ulcer/skin lesion swabs and rectal swabs are placed into Ashdown's medium, a gentamicin-containing liquid transport broth that results in selective growth of B. pseudomallei(Currie, 2003). Ashdown's selective agar (ASA). Ashdown's selective agar (ASA) is the currently favored medium for the isolation and presumptive identification of B. pseudomallei in areas where melioidosis is endemic(Howard and Inglis, 2003).
   2. Medium: A selective medium consisting of trypticase soy agar with 4% glycerol, 5 mg/l crystal violet, 50 mg/l neutral red and 4 mg/l of gentamicin was devised for isolation of Pseudomonas pseudomallei from clinical specimens. Absorption of neutral red was found to be suitable for differentiating this organism from other bacteria, while gentamicin was effective in selecting Ps. pseudomallei from organisms commonly found in clinical material. The medium was more suitable for screening clinical specimens than MacConkey's agar with colistin-S because it was more selective and allowed multiple specimens to be inoculated on a single plate(Ashdown, 1979).
   3. Optimal Temperature: 35C(Howard and Inglis, 2003).
2. Novel Burkholderia pseudomallei selective agar (BPSA) (Howard and Inglis, 2003):
   1. Description: Isolation of Burkholderia pseudomallei currently relies on the use of Ashdown's selective agar (ASA). It was designed a new selective agar (Burkholderia pseudomallei selective agar [BPSA]) to improve recovery of the more easily inhibited strains of B. pseudomallei. B. pseudomallei, Burkholderia cepacia, and Pseudomonas aeruginosa were used to determine the selectivity and sensitivity of BPSA. BPSA was more inhibitory to P. aeruginosa and B. cepacia and should make recognition of Burkholderia species easier due to distinctive colony morphology. BPSA also inhibited Enterococcus, Escherichia, Staphylococcus, and Streptococcus: These results indicate that BPSA is a potential replacement for ASA. BPSA provides large wrinkled colonies faster than ASA. This means that B. pseudomallei colonies are more visible and there is more bacterial growth for supplementary tests. This is particularly important for melioidosis, for public health control strategies that rely on rapid isolation of B. pseudomallei will also benefit from faster detection. This time reduction should also improve the quality of antibiotic treatment for patients. While public health investigations of melioidosis are hampered at present by delays in isolation of B. pseudomallei from nonsterile clinical sites, isolation of B. pseudomallei from environmental specimens such as soil and water takes even longer(Howard and Inglis, 2003).
   2. Medium: BPSA PREPARATION. BPSA comprised 23.5 g of SMA (BBL, Cockeysville, Md.), 4 g of maltose (Sigma, St. Louis, Mo.), and 100 mg of neutral red (Sigma) in 1 liter of distilled water sterilized at 134C for 10 min (or 121C for 15 min). The agar Is allowed to cool to 40 to 45C, and then 20 mg of gentamicin (Sigma) per liter and 1 ml of 20-g/liter Nile blue (dissolved in 1% dimethyl sulfoxide) are added after filter sterilization via a 0.2-m-pore-size membrane (Pall Corporation, Ann Arbor, Mich.). Ten milliliters of glycerol (equivalent to 1%) (BDH, Merck P/L, Kilsyth, Australia) is added and the medium is placed onto a heated magnetic stirrer at 40C for 5 min before plates are poured (18 ml/plate)(Howard and Inglis, 2003).
   3. Optimal Temperature: 35C(Howard and Inglis, 2003).

1. Outbreak Locations:
   1. Melioidosis occurs in tropical areas between latitudes 20N and 20S, predominantly in Southeast Asia and Northern Australia. Cases in animals have also been reported from Australia, Thailand, the Indian subcontinent, Iran, Saudi Arabia, United Arab Emirates, Chad and South Africa, South Africa, Taiwan, Singapore, Brazil and France. B. pseudomallei has been isolated from horses in Spain. In Western Europe, the outbreak of melioidosis at the Paris Zoo in 1975 was doubtless the most unusual spread of B. pseudomallei in a non-endemic region. This outbreak subsequently spread to other zoos in Paris and Mulhouse and equestrian clubs throughout France, and led to the death or slaughter of an unknown number of animals and at least two human fatalities. In 1992, an outbreak of melioidosis in Britain in primates imported from the Philippines and Indonesia. This outbreak was a further reminder of the potential risks posed by the introduction of environmental foci of B. pseudomallei into non-endemic countries(Sprague and Neubauer, 2004). The highest documented rate being in northeastern Thailand, where melioidosis accounts for 20% of all community-acquired septicaemias(Holden et al., 2004). In Thailand, the first case report appeared in 1955. At present, 20003000 cases of clinical melioidosis are estimated to occur each year in Thailand with a population of 60000000. The incidence rate in highly endemic areas was calculated to be 3.65.5 cases per 100000 population and is seasonal(Leelarasamee, 2000). Melioidosis in Vietnam can be traced back to 1937 when a case of cervical melioidosis was reported in a local journal. Then, 30 years later many American soldiers suffered from fatal pneumonitis during the Vietnam war. Vietnam is also claimed to be an original source of B. pseudomallei afflicting travelers or soldiers who developed melioidosis after their visit. At least ten human isolates and five environmental isolates have been recently studied and their cellular lipid and fatty acid composition investigated. Melioidosis is therefore an emerging disease in Vietnam. With a 60000000 population similar to Thailand, we expect to see in the near future many more reported cases with a variety of clinical manifestations similar to what has been found in Malaysia, Singapore and Thailand(Leelarasamee, 2000). Melioidosis was first described from Australia in an outbreak in sheep in 1949 in Winton, north Queensland. The first human case described was a 32-year-old diabetic from Townsville, north Queensland, who developed fatal septicemic melioidosis. Another case in north Queensland was documented from 1959 and the first reported case in the Northern Territory (NT) was from 1960. Between 1981 and 1983, there were 159 cases of melioidosis in piggeries in the region of the Burnett River. The cases were attributed to a contaminated water supply, possibly associated with preceding heavy rainfall and river flooding and construction of a dam. There have been subsequent sporadic cases of melioidosis of animals in the region up until at least 1996. While this subtropical area is endemic for melioidosis in animals, by far the majority of human cases still occurs above 20S, with disease recognised across northern Australia. The largest numbers of cases are documented at hospital (12S), followed by hospitals in Cairns (17S) and Townsville (19S). Cases are also seen at the smaller hospitals in Mt Isa in western Queensland and Kununurra, Halls Creek, Derby and Broome in the Kimberley region of north Western Australia. The incidence of disease is highest in the north of the NT and possibly also in the small population in the Torres Strait Islands adjacent to Papua New Guinea (PNG) in far north Queensland. It was the situation in the first case diagnosed in Sydney in 1966, recognised at autopsy in a man who had had various septic presentations over a 19-year period. Infection was thought to have possibly been acquired in New Guinea during World War II(Currie et al., 2000 (a)). In addition to the above case, another case was attributed to World War II service in Papua New Guinea (PNG), making a latent period of 24 years from exposure to fatal melioidosis. This patient was from Brisbane and therefore it is also possible that there had been exposure to introduced infection. However, since 1964 at least six cases of melioidosis (four fatal) have been documented from Port Moresby(Currie et al., 2000 (a)). Cases of human melioidosis have been reported from several Indian states in recent years, including Maharashtra, Kerala, Orissa, Tripura, Tamil Nadu, West Bengal, Assam, and possibly Pune, although the evidence for the last is questionable. Of villagers in a rice growing area near Vellore 10.7% were found to be sero-positive(Dance, 2000 (b)). B. pseudomallei, which was previously known to be endemic in Hong Kong, has now repeatedly been isolated from the environment in the island of Hainan and the southern provinces of Guangdong and Guangxi. Furthermore, 16 animal and seven human cases have been reported from these provinces, and a sero-positivity rate of 3.815.2% has been found in the human population. Furthermore, two indigenous cases have recently been reported from Taiwan(Dance, 2000 (b)). There has been very little recent evidence of melioidosis activity in sub-Saharan Africa. An environmental survey in Kenya yielded no isolates of typical B. pseudomallei from 152 soil and water samples. The only case of melioidosis reported since 1991 occurred in a goat in Transvaal, South Africa, although independent confirmation of the identity of this isolate has not been obtained(Dance, 2000 (b)). There have been several isolations of B. pseudomallei reported from the Middle East in the last few years, although none of these has been independently confirmed and so doubt as to the accuracy of the identifications must remain. Sources have included humans (Shibl et al., 1996) and animals in Saudi Arabia, a lake in Egypt, milk in Turkey and a camel in the United Arab Emirates(Dance, 2000 (b)). Three human cases of melioidosis, each confirmed by independent Reference Laboratories, have been reported in the past 4 years. These originated from Martinique, Guadeloupe and Puerto Rico. The Caribbean must thus be regarded as endemic for melioidosis, although further work is needed to determine just how common the disease is there and how widely it is distributed(Dance, 2000 (b)). All recent isolates from other non-endemic areas appear to have been imported from known endemic areas. There has been no repeat of the outbreak described in France in the mid-1970s and known as Laffaire du Jardin des Plantes. Isolates from this latter outbreak have been examined, however, and appear phenotypically indistinguishable from clinical isolates of B. pseudomallei from elsewhere in the world, although belonging to distinct genotypes(Dance, 2000 (b)). Imported melioidosis in the UK acts as a mirror on the rest of the world and provides further evidence that melioidosis is widely distributed in the Indian sub-continent. Of 15 human cases diagnosed in the UK between 1988 and 1998, five originated from Bangladesh and one each from India and Pakistan. Interestingly, cases of melioidosis have also been seen in the UK in monkeys and/or humans from both Indonesia and the Philippines, reflecting endemicity in these two countries, in both of which the disease is seldom if ever diagnosed(Dance, 2000 (b)).
2. Transmission Information:
   1. From: Contaminated soil and surface water(Currie, 2003). , To: Human(Currie, 2003). (Currie, 2003)
      Mechanism: It is now clear that humans and animals are infected by exposure to B. pseudomallei present in soil and surface water in endemic locations(Currie, 2003). There are several modes of acquisition of melioidosis for which there is strong evidence. These include the inoculation of environmental organisms through penetrating wounds or into existing skin lesions, the aspiration of contaminated water during near-drowning episodes and iatrogenic inoculation(Dance, 2000 (a)). A comprehensive review of 252 cases of melioidosis were studied prospectively over a 10 year period described the epidemiological features of the disease in Darwin, northern Australia. Patients ranged from 16 months to 91 years old (mean 47 years, median 49 years), although only 4% were children under 15 years of age. Males outnumbered females 3 : 1. A total of 85% of cases presented during the rainy season; 80% had risk factors that may have predisposed them to infection, particularly diabetes mellitus (37%), alcohol abuse (39%), chronic lung disease (27%), chronic renal disease (10%), and consumption of a local root infusion known as kava (8%). Alcohol abuse and chronic lung disease have not emerged as risk factors for melioidosis in Thailand, and this warrants further study. Cystic fibrosis also appears to be a previously unrecognized risk factor for pulmonary melioidosis, and a total of 10 such cases were identified at the recent World Melioidosis Congress in Australia. Patients with diabetes, chronic renal disease or cystic fibrosis who are travelling to endemic areas should probably be warned of the risk of melioidosis, although this risk is impossible to quantify. A recent case of melioidosis acquired in temperate southern Queensland was a reminder that the disease is not confined to the tropics. The precise mode of infection with B. pseudomallei is often unknown, although most cases are assumed to be acquired by inoculation. However, cases can occur in tourists with no obvious soil or water contact(Dance, 2002). Sporadic cases have resulted from iatrogenic inoculation, laboratory accidents(Dance, 2000 (a)). Serologic follow-up of 60 laboratory workers over 15 years identified three workers with titers suggestive of subclinical infection, consistent with the background seroprevalence in the local community. These data suggest that infection is not easily acquired from routine, open-bench laboratory work with B. pseudomallei(CDC Report, 2004).
   2. From: Human(Dance, 2000 (a)). , To: Human(Dance, 2000 (a)). (Dance, 2000 (a))
      Mechanism: The evidence for other modes of infection, such as person-to-person, animal-to-person, inhalation of environmental organisms, ingestion of environmental organisms, and vector transmission, is relatively weak(Dance, 2000 (a)). Person-to-person transmission of B. pseudomallei is very unusual(Currie, 2003). Venereal transmission was the first report of person-to-person spread of B. pseudomallei infection(McCormick et al., 1975). Two cases of maternal to child transmission of melioidosis are reported from Australia's tropical north. One infant died of overwhelming sepsis. Both lactating mothers had mastitis. In 1 case, Burkholderia pseudomallei isolated from breast milk was identical on pulsed-field gel electrophoresis with that in blood and cerebrospinal fluid isolates from the infant(Ralph et al., 2004).
   3. From: Animals(Sprague and Neubauer, 2004). , To: Human(Sprague and Neubauer, 2004). (Sprague and Neubauer, 2004)
      Mechanism: Zoonotic transmission to humans is extremely unusual, but there are many similar epidemiological and clinical features of melioidosis in animals and humans(Choy et al., 2000). Experimental animals have been infected by inhalation and this mode of acquisition was thought to account for the predominance of helicopter crew with pulmonary melioidosis during the Vietnam War(Dance, 2000 (a)).
3. Environmental Reservoir:
   1. Burkholderia pseudomallei Environmental Reservoir:
      1. Description: The environmental reservoirs for B. pseudomallei are surface water and soil(CDC Report, 2004). Burkholderia pseudomallei is a natural saprophyte that can be isolated from soil and muddy water in endemic areas. It lives in the rhizosphere and is believed to play an important role in denitrification. The agent can be found in soil and clay layers from the surface but more often and regularly in deeper layers from 25 to 120 cm. B. pseudomallei can multiply in soil at a pH from 4 to 8, at a minimal humidity of 10-15% and at temperatures from 4 to 42C but not in estuarine or salt water. It has been noted that the isolation rate was high in places used by animals to rest in the shade. Aerotaxis may account for the bacteria moving actively from deeper layers to the surface soil if it is moistened, e.g. by rain or agricultural practices. The type of soil seems not to be a major factor. The minimum isotherm for a steady establishment in a new geographic area seems to be 11C(Sprague and Neubauer, 2004).
      2. Survival: Burkholderia pseudomallei can survive in water at room temperature for up to 8 weeks, in muddy water for up to 7 months and in soil in the laboratory for up to 30 months. The bacterium is not particularly resistant to UV-irradiation or sunlight. Chlorine has only a bacteriostatic effect on the agent as bacteria were recovered from water containing up to 1000 p.p.m. free chlorine. Lower pH additionally reduces the effect of the disinfectant. However, it can effectively reduce the number of viable bacteria(Sprague and Neubauer, 2004). We still know very little about the climatic, physical, chemical and biological factors which control the proliferation and survival of Burkholderia spp. in the environment(Dance, 2000 (a)).
4. Intentional Releases:
   1. Intentional Release Information:
      1. Description: Melioidosis was an important cause of morbidity and mortality in foreign troops fighting in South East Asia. Concerns of re-activation of latent infection in soldiers returned from Vietnam, with estimates from serology studies of ~225,000 potential cases, resulted in melioidosis being called the "Vietnamese time bomb". However, while occasional cases of re-activation of B. pseudomallei still occur in Vietnam veterans, it is rare in comparison to the numbers exposed. Melioidosis remains a risk for travellers to endemic areas, especially if they have a recognised risk factor as discussed below. Adventure tours resulting in extensive exposure to wet season soils and surface water are a possible scenario for infection. This occurred in early 2002 in a young French tourist who died from fulminant melioidosis septicemic pneumonia after exposure to muddy soils on a camping tour of a tropical Australian wilderness park. Imported melioidosis cases are seen each year in hospitals in southern Australia. Some of these cases are acquired in tourists returning from northern Australia and others are acquired in South East Asia. Occasional imported cases are also reported from Europe and the USA. While the vast majority of these cases are from recent acquisition of B. pseudomallei, as occurs with more than 90% of cases in northern Australia, occasional patients may have chronic melioidosis or have re-activation of disease from a latent focus many years after leaving an endemic area, as noted above in returned soldiers from Vietnam(Currie, 2003). The potential for the bacterium to cause disease after inhalation has also resulted in the inclusion of this pathogen on the Centers for Disease Control list of potential biothreat agents as a Category B agent(Holden et al., 2004).
      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.

1. Organism Detection Test:
   1. Rapid immunofluorescence microscopy (IF) (Wuthiekanun et al., 2005):
      1. Time to Perform: minutes-to-1-hour
      2. Description: The rapid method was a one-step technique in which 1 drop (10 l) of specimen was mixed on a clean glass microscopic slide with an equal volume of conjugate and a coverslip applied. The white blood cells present in pus were lysed prior to examination by the addition of an equal volume of distilled water, and respiratory secretions were mixed with an equal volume of sterile distilled water before examination. Conjugate was used at a 1:200 dilution in blocking buffer. The slides were examined with a fluorescent microscope with a x100 oil-immersion lens. A positive result by either test was recorded when the periphery of the bacilli showed a strong apple-green fluorescence. A slide known to be positive (for a clinical isolate of B. pseudomallei) was prepared and examined in each test run. The results of this rapid method and those of an existing IF method were prospectively compared with the culture results for 776 specimens from patients with suspected melioidosis. The sensitivities of both IF tests were 66%, and the specificities were 99.5 and 99.4%, respectively(Wuthiekanun et al., 2005).
      3. False Positive: Four of the 622 specimens (from 526 patients) that were culture negative for B. pseudomallei were positive by either IF method. Three were false positive by both methods, and an additional sample was false positive only by the standard IF method. Two of these were urine samples that grew Pseudomonas aeruginosa and Acinetobacter spp., respectively, on Ashdown's medium; and two were respiratory secretions that were negative on Ashdown's medium but that grew mixed respiratory flora on blood agar(Wuthiekanun et al., 2005).
2. Immunoassay Test:
   1. ELISA :
      1. Description: An enzyme-linked immunosorbent assay (ELISA) for antibodies to a partly purified culture filtrate antigen. The immunoglobulin G ELISA exhibited a sensitivity of 96% and a specificity of 97%, whereas the immunoglobulin M ELISA had a sensitivity of 74% and a specificity of 99%. The responses were studied in more detail in a smaller group of patients. Immunoglobulin G was detectable in 96-100% of patients with proved or suspected melioidosis, immunoglobulin A was detectable in 86-100%, and immunoglobulin M in 66-85%, whereas immunoglobulin E was not detected. A higher proportion of cases with localized than with septicaemic infections were antibody positive. Immunoglobulin G1 and G2 were the predominant subclasses detected. However, an internationally standardized serodiagnostic test for melioidosis is much needed(Dance, 2002). It can be assumed that all tests described so far cross-react with antibodies raised against B. mallei. Despite all these shortcomings, serology has always been used in veterinary medicine for the identification of anti-Burkholderia antibodies, e.g. in horses, goats and dairy cows. To date, none of the newly developed tests has been or is commercially available and no positive and negative control sera of the various animal species which could be infected with B. pseudomallei are available. Serology is still a technique restricted to a limited number of expert laboratories worldwide. In view of the international transport of animals from endemic to non-endemic areas, the development of a sensitive and specific serological test is unavoidable in order to prevent the spread of B. pseudomallei in the future(Sprague and Neubauer, 2004).
   2. Monoclonal antibody-based latex agglutination :
      1. Description: A monoclonal antibody-based latex agglutination (MAb-LA) test was employed for the rapid identification of Burkholderia pseudomallei in blood culture fluid from patients with community-acquired septicaemia. These patients were admitted to 12 hospitals in the northeastern part of Thailand which is a region known to be endemic for melioidosis. Blood samples were collected and immediately added to the blood culture bottles which were incubated in either automated (five hospitals) or manual (seven hospitals) culture systems. Of a total of 1369 culture-positive specimens, 204 specimens were culture-positive for B. pseudomallei. Of those, 194 (95%) were positive by MAb-LA and the type of blood culture system did not affect positivity rates. The performance of the MAb-LA test on these specimens was highly satisfactory compared with culture detection and confirmation by biochemical test, with 95.1% sensitivity, 99.7% specificity and 98.8% and 99.2% for positive and negative predictive values, respectively. The method described is highly reproducible, simple to perform even by inexperienced laboratory personnel and does not require expensive or elaborate equipment(Anuntagool et al., 2000).
   3. Lipopolysaccharide-specific monoclonal antibody :
      1. Description: The Bps-L1 monoclonal antibody recognized the lipopolysaccharide antigen of 96.8% of B. pseudomallei clinical isolates and was highly specific for B. pseudomallei. The diagnostic value of the latex agglutination test based on Bps-L1 monoclonal antibody was prospectively evaluated in an area endemic for melioidosis. The agglutination test kit was evaluated in 88 blood cultures with gram-negative bacteria identified with Gram staining. The sensitivity and specificity of the test kit were both 100%. These results indicated that the detection of B. pseudomallei lipopolysaccharide by specific monoclonal antibody in a latex agglutination format is clinically useful for the rapid identification of the bacteria in blood cultures in areas endemic for melioidosis(Dharakul et al., 1999).
   4. Monoclonal antibody (MAb) specific to the 30-kDa protein of B. pseudomallei :
      1. Description: The definitive diagnosis of this disease is made by bacterial culture. In this study, it was produced a monoclonal antibody (MAb) specific to the 30-kDa protein of B. pseudomallei by in vivo and in vitro immunization of BALB/c mice with a crude culture filtrate antigen. The MAb could directly agglutinate with all 243 clinical isolates of B. pseudomallei but not with other gram-negative bacteria, except for one strain of Burkholderia mallei. However, the MAb cross-reacted with the gram-positive Bacillus sp. and Streptococcus pyogenes. B. pseudomallei in brain heart infusion broth (BHIB) subcultured from a BacT/Alert automated blood culture system could be identified by simple agglutination with this MAb assay. The sensitivity and specificity of direct agglutination compared to the "gold standard," the culture method, were 94.12 and 98.25%, respectively. However, the MAb adsorbed to polystyrene beads or latex particles directly identified the bacterium in blood culture specimens and in BHIB subcultured from a BacT/Alert automated blood culture system. The sensitivity of the latex agglutination test was 100% for both blood culture and BHIB specimens. The specificity was 85.96 and 96.49% for the blood culture and BHIB specimens, respectively. The specificity could be increased if the nonspecific materials in the blood culture broths were eradicated by centrifugation at low speeds. Thus, a combination of blood culture and the agglutination method could be used for the rapid diagnosis of melioidosis in the routine bacteriological laboratory. This method could speed up detection of the bacterium in blood culture by at least 2 days, compared to the conventional bacterial culture method. In addition, the MAb is stable at room temperature for 2 weeks and at 4, -20, and -70 degrees C for at least 1 year. The latex reagent was stable for at least 6 months at 4 degrees C(Pongsunk et al., 1999).
   5. Monoclonal antibody specific for exopolysaccharide of B. pseudomallei :
      1. Description: In this study it was developed a latex agglutination test based on monoclonal antibody 3015, which is specific for this exopolysaccharide, and evaluated this test for rapid identification of B. pseudomallei grown on agar plates. All 74 environmental and clinical B. pseudomallei strains tested, originating from different areas of Southeast Asia, northern Australia, and Africa, showed a strong and specific agglutination. B. pseudomallei-like organisms and a variety of other bacteria did not react. In conclusion this monoclonal antibody-based test is a simple, rapid, and highly specific method for identifying B. pseudomallei culture isolates from different geographic areas(Steinmetz et al., 1999).
3. Nucleic Acid Detection Test: