• View in gallery

    Combined survival data from pilot and confirmatory experiments showing partial lethality at the higher inoculating doses of Burkholderia pseudomallei. AD, BALB/c and C57BL/6 mice inoculated with 1 × 103, 1 × 106, 1 × 108, or 7 × 108 colony-forming units (CFU) of B. pseudomallei by gavage and observed for survival. The 1 × 106 and 1 × 108 CFU data represent two independent experiments combined. BALB/c: n = 3, 9, 8, and 4, respectively. C57BL/6: n = 5, 10, 10, and 7, respectively. There was no difference in survival between mouse strains at any dose by the log rank test. E and F, Spleen section photographed with a 4× objective (E) and liver section photographed with a 20× objective (F) stained with hematoxylin and eosin from a BALB/c mouse inoculated with 1 × 106 CFU six weeks earlier. Multiple, large, confluent abscesses with dark blue necrotic cores efface the normal architecture of the spleen (E). In the liver (F), a focal area of hepatocellular necrosis contains clusters of neutrophilic and mononuclear inflammatory cells. The hypercellular appearance of surrounding sinusoids is caused by increased numbers of inflammatory cells. This figure appears in color at www.ajtmh.org.

  • 1.

    White NJ, 2003. Melioidosis. Lancet 361: 17151722.

  • 2.

    Cheng AC, Currie BJ, 2005. Melioidosis: epidemiology, pathophysiology, and management. Clin Microbiol Rev 18: 383416.

  • 3.

    Inglis TJ, Garrow SC, Adams C, Henderson M, Mayo M, 1998. Dry-season outbreak of melioidosis in western Australia. Lancet 352: 1600.

  • 4.

    Inglis TJ, Garrow SC, Henderson M, Clair A, Sampson J, O'Reilly L, Cameron B, 2000. Burkholderia pseudomallei traced to water treatment plant in Australia. Emerg Infect Dis 6: 5659.

    • Search Google Scholar
    • Export Citation
  • 5.

    Currie BJ, Mayo M, Anstey NM, Donohoe P, Haase A, Kemp DJ, 2001. A cluster of melioidosis cases from an endemic region is clonal and is linked to the water supply using molecular typing of Burkholderia pseudomallei isolates. Am J Trop Med Hyg 65: 177179.

    • Search Google Scholar
    • Export Citation
  • 6.

    Titball RW, Russell P, Cuccui J, Easton A, Haque A, Atkins T, Sarkar-Tyson M, Harley V, Wren B, Bancroft GJ, 2008. Burkholderia pseudomallei: animal models of infection. Trans R Soc Trop Med Hyg 102 (Suppl 1): S111S116.

    • Search Google Scholar
    • Export Citation
  • 7.

    Whitmore A, 1913. An account of a glanders-like disease occurring in Rangoon. J Hyg (Lond) 13: 134.

  • 8.

    Stanton AT, Fletcher W, Kanagarayer K, 1924. Two cases of melioidosis. J Hyg (Lond) 23: 268276.

  • 9.

    Stanton AT, Fletcher W, 1925. Melioidosis and its relation to glanders. J Hyg (Lond) 23: 347363.

  • 10.

    Barnes JL, Ketheesan N, 2005. Route of infection in melioidosis. Emerg Infect Dis 11: 638639.

  • 11.

    Ashdown LR, 1979. An improved screening technique for isolation of Pseudomonas pseudomallei from clinical specimens. Pathology 11: 293297.

    • Search Google Scholar
    • Export Citation
  • 12.

    Wuthiekanun V, Anuntagool N, White NJ, Sirisinha S, 2002. Short report: a rapid method for the differentiation of Burkholderia pseudomallei and Burkholderia thailandensis. Am J Trop Med Hyg 66: 759761.

    • Search Google Scholar
    • Export Citation
  • 13.

    Wuthiekanun V, Smith MD, White NJ, 1995. Survival of Burkholderia pseudomallei in the absence of nutrients. Trans R Soc Trop Med Hyg 89: 491.

  • 14.

    Leakey AK, Ulett GC, Hirst RG, 1998. BALB/c and C57Bl/6 mice infected with virulent Burkholderia pseudomallei provide contrasting animal models for the acute and chronic forms of human melioidosis. Microb Pathog 24: 269275.

    • Search Google Scholar
    • Export Citation
  • 15.

    Liu B, Koo GC, Yap EH, Chua KL, Gan YH, 2002. Model of differential susceptibility to mucosal Burkholderia pseudomallei infection. Infect Immun 70: 504511.

    • Search Google Scholar
    • Export Citation
  • 16.

    Tong S, Yang S, Lu Z, He W, 1996. Laboratory investigation of ecological factors influencing the environmental presence of Burkholderia pseudomallei. Microbiol Immunol 40: 451453.

    • Search Google Scholar
    • Export Citation
  • 17.

    Owen SJ, Batzloff M, Chehrehasa F, Meedeniya A, Casart Y, Logue CA, Hirst RG, Peak IR, Mackay-Sim A, Beacham IR, 2009. Nasal-associated lymphoid tissue and olfactory epithelium as portals of entry for Burkholderia pseudomallei in murine melioidosis. J Infect Dis 199: 17611770.

    • Search Google Scholar
    • Export Citation
  • 18.

    Wuthiekanun V, Dance DA, Wattanagoon Y, Supputtamongkol Y, Chaowagul W, White NJ, 1990. The use of selective media for the isolation of Pseudomonas pseudomallei in clinical practice. J Med Microbiol 33: 121126.

    • Search Google Scholar
    • Export Citation
  • 19.

    Kanaphun P, Thirawattanasuk N, Suputtamongkol Y, Naigowit P, Dance DA, Smith MD, White NJ, 1993. Serology and carriage of Pseudomonas pseudomallei: a prospective study in 1000 hospitalized children in northeast Thailand. J Infect Dis 167: 230233.

    • Search Google Scholar
    • Export Citation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Pathogenicity of High-Dose Enteral Inoculation of Burkholderia pseudomallei to Mice

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  • Department of Medicine, and Department of Comparative Medicine, University of Washington School of Medicine, Seattle, Washington; Mahidol-Oxford Tropical Medicine Research Unit, Department of Tropical Hygiene, and Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, United Kingdom

Melioidosis is a frequently lethal tropical infection caused by the environmental saprophyte Burkholderia pseudomallei. Although transcutaneous inoculation and inhalation are considered the primary routes of infection, suggestive clinical evidence implicates ingestion as a possible alternative route. We show that in BALB/c and C57BL/6 mice, direct gastric inoculation of high doses of B. pseudomallei causes systemic infection that may be lethal or cause chronic disseminated infection. Mice may shed bacteria in the stool for weeks after infection, and high titers of B. pseudomallei-specific IgG are detectable. This report of enteric murine melioidosis supports further consideration of this route of infection.

Introduction

Melioidosis is a serious bacterial infection occurring primarily in southeast Asia and northern Australia. The causative agent is Burkholderia pseudomallei, a gram-negative soil saphrophyte and potential biothreat agent.1 Melioidosis occurs after contact with a contaminated environment and therefore is potentially preventable. A major stumbling block to the development of protective strategies is the lack of an evidence base defining routes of acquisition. The prevailing assumption is that most disease occurs as a result of transcutaneous inoculation or inhalation.1 The role of ingestion as a route of infection remains undefined.2 Suggestive but inconclusive evidence is provided by reports of clusters of melioidosis cases in which the strain of B. pseudomallei isolated from a common water source was a genetic match for the strain causing disease.35 Experimental models of melioidosis have focused predominantly on non-enteral routes of infection, all of which have reproducibly resulted in disease.6 Experimental enteric infection has received relatively little consideration since the description of melioidosis in 1913 by Whitmore.710 We developed a model of enteric melioidosis in mice to shed light on ingestion as a route of infection in humans.

Materials and Methods

Bacteria.

Burkholderia pseudomallei 1026b, a clinical isolate obtained from a bacteremic patient from Thailand, was grown in Luria-Bertani broth shaking in air at 37°C, washed twice, resuspended in phosphate-buffered saline (PBS) containing 20% glycerol, and frozen at –80°C. On the day of experiments, the freezer stock was thawed and diluted in PBS to the specific concentration, which was confirmed by quantitative culture on Ashdown agar plates.

Animal model.

Six to ten–week old, female, specific pathogen–free BALB/c and C57BL/6 mice were obtained from Charles River Laboratoriwes, Inc. (Wilmington, MA) and Jackson Laboratories (Bar Harbor, ME), and maintained in a biosafety level 3 animal facility. Food was withheld from the mice for three hours preceding inoculation. Inoculation was performed by direct gastric delivery of 200 μL of bacteria using a 22-gauge, 1.5-inch gavage needle. Mice that had inadvertent nasal or respiratory delivery of bacteria or mice that became moribund or died within two hours of infection, suggesting a complicated procedure, were killed and excluded from the experiments.

Thereafter, mice were monitored daily. Ill animals that had ruffled fur, eye crusting, hunched posture, and lack of resistance to handling were deemed terminal and killed (spontaneous death was not required as an endpoint). In the initial experiment, BALB/c and C57BL/6 mice were inoculated with 1 × 103, 1 × 106, or 1 × 108 colony-forming units (CFU) (n = 3, 5, and 3 and n = 5, 5, and 5, respectively). In the second experiment, BALB/c and C57BL/6 mice were inoculated with 1 × 106, 1 × 108 CFU, or 7 × 108 CFU (n = 4, 5, and 4 and n = 5, 5, and 7, respectively).

For bacterial culture, the left lung, median hepatic lobe, spleen, brain, and mesenteric lymph nodes were removed in a sterile fashion and homogenized in 1 mL of Dulbecco's PBS. Aliquots (100–200 μL) of homogenized tissue were plated in duplicate on Ashdown agar.11 Stool samples were obtained from mice prior to killing and were homogenized and cultured in a similar manner. Burkholderia pseudomallei colonies were counted after 2–4 days of incubation at 37°C. The lower limit of detection was 5–10 CFU/mL. A positive culture was defined as detectable colonies consistent with B. pseudomallei morphotypes on both of the duplicate plates. If colony morphology was atypical or there were mixed morphologies, identification of B. pseudomallei was confirmed by using a monoclonal antibody–based latex agglutination test.12 For histologic analysis, organs were fixed in 4% paraformaldehyde before processing. Sections were stained with hematoxylin and eosin, and with Giemsa or Brown and Brenn stains. Slides were reviewed by a veterinary pathologist (HDL). All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Washington.

Antibody detection.

Heat-killed B. pseudomallei 1026b (1 × 106 CFU in 100 μL of PBS) was added to 96-well plates and stored at 4°C overnight. The plates were washed and blocked with 5% skim milk in PBS at 37°C for two hours. After repeat washing, two-fold serial dilutions of serum in PBS from each infected mouse (in duplicate) and from two uninfected mice were added for two hours. The range of dilutions was 1:32–1:65,536. The plates were washed, goat anti-mouse IgG conjugated to biotin (SouthernBiotech, Birmingham, AL) diluted 1:10,000 in PBS and 1% bovine serum albumin was added, and incubated for two hours. Streptavidin–horseradish peroxidase diluted 1:200 was added for 20 minutes, washing was repeated, and color development was obtained by adding peroxidase substrate solution (Kirkegaard and Perry Laboratories, Gaithersburg, MD). The reaction was quenched with 1 M phosphoric acid and optical densities were determined at an absorbance of 405 nm after subtracting values at 570 nm for correction. A positive antibody titer was defined as the maximal dilution of tested serum that had twice the mean optical density of uninfected serum at the same dilution.

Results

In the initial assessment of pathogenicity from enteral inoculation of B. pseudomallei, all BALB/c and C57BL/6 mice administered 1 × 103 or 1 × 106 CFU survived for six weeks. One C57BL/6 mouse in the 106 CFU group developed paresis of a hind leg after one week. Of the mice inoculated with 1 × 108 CFU, two of three BALB/c mice and three of five C57BL/6 mice died within five days. The experiment was censored at six weeks. The surviving BALB/c mouse appeared hunched and ill; splenomegaly and a large splenic abscess were observed on a limited necropsy sample.

To confirm these findings, the experiment was repeated. In addition to doses of 1 × 106 and 1 × 108 CFU, a higher dose of 7 × 108 CFU was chosen to determine whether greater lethality could be provoked (n = 4, 5, and 4 and n = 5, 5, and 7 for BALB/c and C57BL/6 mice, respectively). One of the BALB/c mice that received 1 × 108 CFU was killed because of an isolated inability to lift its head. The combined survival data from the pilot and confirmatory experiments showing partial lethality at the higher inoculating doses are shown in Figure 1. Survival in the two strains of mice did not differ when analyzed by the log rank test.

Figure 1.
Figure 1.

Combined survival data from pilot and confirmatory experiments showing partial lethality at the higher inoculating doses of Burkholderia pseudomallei. AD, BALB/c and C57BL/6 mice inoculated with 1 × 103, 1 × 106, 1 × 108, or 7 × 108 colony-forming units (CFU) of B. pseudomallei by gavage and observed for survival. The 1 × 106 and 1 × 108 CFU data represent two independent experiments combined. BALB/c: n = 3, 9, 8, and 4, respectively. C57BL/6: n = 5, 10, 10, and 7, respectively. There was no difference in survival between mouse strains at any dose by the log rank test. E and F, Spleen section photographed with a 4× objective (E) and liver section photographed with a 20× objective (F) stained with hematoxylin and eosin from a BALB/c mouse inoculated with 1 × 106 CFU six weeks earlier. Multiple, large, confluent abscesses with dark blue necrotic cores efface the normal architecture of the spleen (E). In the liver (F), a focal area of hepatocellular necrosis contains clusters of neutrophilic and mononuclear inflammatory cells. The hypercellular appearance of surrounding sinusoids is caused by increased numbers of inflammatory cells. This figure appears in color at www.ajtmh.org.

Citation: The American Society of Tropical Medicine and Hygiene 83, 5; 10.4269/ajtmh.2010.10-0306

Lung, liver, spleen, mesenteric lymph node, and, because of the neurologic manifestations observed, brain were harvested and stool samples were obtained from mice in the 1 × 108 and 7 × 108 CFU groups surviving to six weeks. Because abscesses were observed grossly in two BALB/c spleens, particular care was taken to avoid cross-contamination between organs. Ashdown agar and the latex agglutination assay were used to confirm growth of B. pseudomallei. Four of six BALB/c mice but only one of six C57BL/6 mice had detectable bacteria in at least one organ or in stool sample (Table 1). No bacteria were recovered from mesenteric lymph nodes but several mice had positive brain cultures. Recoverable bacteria in any organ were only detected when bacteria were shed in the stool. To examine dissemination in the early phase of infection, BALB/c (n = 4) and C57BL/6 (n = 5) mice were inoculated with 7 × 108 CFU and killed after 24 hours. Lung, liver, spleen, mesenteric lymph node, and stool samples were cultured (Table 1). Burkholderia pseudomallei was recovered from three BALB/c mice and from one C57BL/6 mouse.

Table 1

Distribution of recovered Burkholderia pseudomallei and serum IgG titers after enteral infection of mice*

MicePositive culturesIgG titer
24 hours6 weeks
BALB/c
Inoculum = 7 × 108 CFU
Mouse 1LN, stool
Mouse 2None
Mouse 3Spleen, stool
Mouse 4Lung, spleen, LN, stool
Mouse 5Brain, stool≥ 1:65,536
Mouse 6Stool≥ 1:65,536
Mouse 7None< 1:32
Inoculum = 1 × 108 CFU
Mouse 1None< 1:32
Mouse 2Liver, spleen, stool≥ 1:65,536
Mouse 3Spleen, brain, stool≥ 1:65,536
Inoculum = 1 × 106 CFU
Mouse 11:256
Mouse 21:256
Mouse 3≥ 1:65,536
C57BL/6
Inoculum = 7 × 108 CFU
Mouse 1Lung, stool
Mouse 2None
Mouse 3None
Mouse 4None
Mouse 5None
Mouse 6None< 1:32
Mouse 7None1:16,384
Mouse 8None1:256
Mouse 9None< 1:32
Inoculum = 1 × 108 CFU
Mouse 1None1:2,048
Mouse 2Brain, stool1:32,768
Inoculum = 1 × 106 CFU
Mouse 1< 1:32
Mouse 2< 1:32
Mouse 31:32,768
Mouse 41:4,096
Mouse 51:1,024

Specimens sampled at 24 hours were lung, liver, spleen, mesenteric lymph node (LN), and stool. Specimens sampled at 6 weeks were lung, liver, spleen, mesenteric LN, brain, and stool. B. pseudomallei-specific IgG titers were measured 6 weeks after infection. CFU = colony-forming units; – = not measured; none = negative culture for all specimens sampled.

Histopathologic examination of organs from BALB/c mice six weeks after enteral infection with 1 × 106–7 × 108 CFU infection showed a range of inflammatory lesions. Splenic abscesses were characterized histologically by multiple large coalescing foci of densely necrotic neutrophils and parenchymal cells surrounded by scant histiocytes and a thin fibrous capsule (Figure 1E). Liver sections were characterized by small, widely scattered focal abscesses, prominent Kupffer cells, and an abundance of inflammatory cells within sinusoids (Figure 1F). Extramedullary hematopoietic foci were common within the liver. Mild focal inflammation was present within or near heart valves and focal gliosis was noted within the brain. Lung histopathologic abnormalities were absent. Giemsa and Brown and Brenn stains failed to identify organisms within lesion sites.

To determine whether enteral infection induced an adaptive immune response, serum from surviving mice infected six weeks prior with 1 × 106–7 × 108 CFU were tested for IgG to heat-killed B. pseudomallei (Table 1). All mice with recoverable bacteria on organ sampling had titers of at least 1:32,768 but titers were variable in culture-negative mice.

Discussion

We show in this exploratory study that at high doses B. pseudomallei administered enterally to mice may be rapidly lethal or cause chronic disseminated infection. Infection by this route induces an antibody response. Mice may shed bacteria in the stool for at least six weeks and may demonstrate neurologic involvement. The establishment of this experimental model bolsters efforts to understand the route of infection in human disease.

Melioidosis, which occurs only upon exposure to a contaminated environment, can potentially be prevented if the route of infection is understood. Burkholderia pseudomallei may survive for years in water under experimental conditions,13 and the evidence implicating ingestion as a possible route of infection in humans underscores the need for experimental animal models.35 Although others investigators have reported that oral inoculation may be lethal,7,9,10 our study has defined the clinical and pathologic features of enteric disease. Inoculating doses of 108 CFU may induce lethal disease in BALB/c and C57BL/6 mice. Unlike intravenous and intranasal infection models, we did not observe a survival difference between mouse strains.14,15 The enteral dose required for lethality in both strains is substantially higher than the approximately 5 × 102 CFU/lung that is lethal in an inhalation model of murine melioidosis (West TE, unpublished data). How this translates to infectious doses in humans is presently unclear. There are few data that indicate likely concentrations of B. pseudomallei in environmental water sources in disease-endemic areas. Additionally, chronic ingestion of the pathogen may conceivably result in different clinical manifestations, and impaired host defenses, as are commonly associated with melioidosis, may dramatically increase susceptibility to infection by this route.

Although B. pseudomallei survives poorly in the acidic gastric environment,16 our data show that invasive infection nonetheless may occur after high-dose enteric infection. Given the negative liver cultures at 24 hours, bacterial spread is conceivably initiated by mesenteric lymphatics rather than the portal venous system. The pattern of distant organ infection suggests hematogenous dissemination. Our observations of neurotropism are not unique because bacteria have been cultured from brain in oral, intranasal, intravenous and intraperitoneal models of B. pseudomallei infection.10 Neurologic infection in humans has been well documented, particularly in Australia.1 Although intranasal mouse infection models probably cause direct infection of the brain by the olfactory nerve,17 our results suggest that B. pseudomallei acquired enterally can cross the blood–brain barrier.

Our stool culture data indicate that B. pseudomallei survives for up to six weeks within the lumen of the gut. All mice with detectable infection in any organ had positive stool cultures; thus, recoverable fecal bacteria may be a marker for invasive infection. Likewise, in humans with melioidosis, B. pseudomallei has been isolated from rectal swabs, always in association with bacterial growth from other sites.18 The shedding of bacteria in stool, particularly on a chronic basis, raises important questions about mechanisms of environmental contamination in disease-endemic areas.

Enteric melioidosis induces an adaptive immune response in BALB/c and C57BL/6 mice. Not surprisingly, the B. pseudomallei-specific IgG titer was uniformly higher in the presence of persistent infection. Some mice had no measurable antibodies or detectable bacteria at six weeks, supporting the hypothesis that even at high inoculating doses, infection was not established in these animals. Robust antibody titers were measured in several mice that did not have bacteria recovered from the organs or stool sampled. These mice may have had foci of persistent infection in other organs that were not sampled, or may have developed but then cleared infection prior to sampling. A similar phenomenon after ingestion of B. pseudomallei might explain the seropositivity observed in otherwise healthy humans.19

Potential limitations to our model include the use of a thawed glycerol bacterial stock, which may have altered the interaction of the pathogen with the mucosal immune system. We have used the same stock extensively in an aerosol model of murine melioidosis and found our results to be reproducible and largely consistent with the published literature (West TE, unpublished data). Although we excluded cases of obvious aspiration during inoculation, we cannot exclude microaspiration after gastric delivery of bacteria. However, only two of nine mice had recoverable bacteria in the lung 24 hours after enteral infection, suggesting that this finding was not likely to be a dominant phenomenon.

In summary, this animal model provides supportive evidence that melioidosis may be initiated by ingestion of B. pseudomallei. However, high inoculating doses were required to demonstrate virulence in bolus infection of immunocompentent mice, and the parallels to human melioidosis remain uncertain. Additional investigations of environmental contamination, of experimental enteric disease models, including chronic ingestion and immunocompromised hosts, and of human melioidosis are required.

Acknowledgments:

We thank Loren Kinman, Tony Han, and Anthony Hager for expert technical assistance.

  • 1.

    White NJ, 2003. Melioidosis. Lancet 361: 17151722.

  • 2.

    Cheng AC, Currie BJ, 2005. Melioidosis: epidemiology, pathophysiology, and management. Clin Microbiol Rev 18: 383416.

  • 3.

    Inglis TJ, Garrow SC, Adams C, Henderson M, Mayo M, 1998. Dry-season outbreak of melioidosis in western Australia. Lancet 352: 1600.

  • 4.

    Inglis TJ, Garrow SC, Henderson M, Clair A, Sampson J, O'Reilly L, Cameron B, 2000. Burkholderia pseudomallei traced to water treatment plant in Australia. Emerg Infect Dis 6: 5659.

    • Search Google Scholar
    • Export Citation
  • 5.

    Currie BJ, Mayo M, Anstey NM, Donohoe P, Haase A, Kemp DJ, 2001. A cluster of melioidosis cases from an endemic region is clonal and is linked to the water supply using molecular typing of Burkholderia pseudomallei isolates. Am J Trop Med Hyg 65: 177179.

    • Search Google Scholar
    • Export Citation
  • 6.

    Titball RW, Russell P, Cuccui J, Easton A, Haque A, Atkins T, Sarkar-Tyson M, Harley V, Wren B, Bancroft GJ, 2008. Burkholderia pseudomallei: animal models of infection. Trans R Soc Trop Med Hyg 102 (Suppl 1): S111S116.

    • Search Google Scholar
    • Export Citation
  • 7.

    Whitmore A, 1913. An account of a glanders-like disease occurring in Rangoon. J Hyg (Lond) 13: 134.

  • 8.

    Stanton AT, Fletcher W, Kanagarayer K, 1924. Two cases of melioidosis. J Hyg (Lond) 23: 268276.

  • 9.

    Stanton AT, Fletcher W, 1925. Melioidosis and its relation to glanders. J Hyg (Lond) 23: 347363.

  • 10.

    Barnes JL, Ketheesan N, 2005. Route of infection in melioidosis. Emerg Infect Dis 11: 638639.

  • 11.

    Ashdown LR, 1979. An improved screening technique for isolation of Pseudomonas pseudomallei from clinical specimens. Pathology 11: 293297.

    • Search Google Scholar
    • Export Citation
  • 12.

    Wuthiekanun V, Anuntagool N, White NJ, Sirisinha S, 2002. Short report: a rapid method for the differentiation of Burkholderia pseudomallei and Burkholderia thailandensis. Am J Trop Med Hyg 66: 759761.

    • Search Google Scholar
    • Export Citation
  • 13.

    Wuthiekanun V, Smith MD, White NJ, 1995. Survival of Burkholderia pseudomallei in the absence of nutrients. Trans R Soc Trop Med Hyg 89: 491.

  • 14.

    Leakey AK, Ulett GC, Hirst RG, 1998. BALB/c and C57Bl/6 mice infected with virulent Burkholderia pseudomallei provide contrasting animal models for the acute and chronic forms of human melioidosis. Microb Pathog 24: 269275.

    • Search Google Scholar
    • Export Citation
  • 15.

    Liu B, Koo GC, Yap EH, Chua KL, Gan YH, 2002. Model of differential susceptibility to mucosal Burkholderia pseudomallei infection. Infect Immun 70: 504511.

    • Search Google Scholar
    • Export Citation
  • 16.

    Tong S, Yang S, Lu Z, He W, 1996. Laboratory investigation of ecological factors influencing the environmental presence of Burkholderia pseudomallei. Microbiol Immunol 40: 451453.

    • Search Google Scholar
    • Export Citation
  • 17.

    Owen SJ, Batzloff M, Chehrehasa F, Meedeniya A, Casart Y, Logue CA, Hirst RG, Peak IR, Mackay-Sim A, Beacham IR, 2009. Nasal-associated lymphoid tissue and olfactory epithelium as portals of entry for Burkholderia pseudomallei in murine melioidosis. J Infect Dis 199: 17611770.

    • Search Google Scholar
    • Export Citation
  • 18.

    Wuthiekanun V, Dance DA, Wattanagoon Y, Supputtamongkol Y, Chaowagul W, White NJ, 1990. The use of selective media for the isolation of Pseudomonas pseudomallei in clinical practice. J Med Microbiol 33: 121126.

    • Search Google Scholar
    • Export Citation
  • 19.

    Kanaphun P, Thirawattanasuk N, Suputtamongkol Y, Naigowit P, Dance DA, Smith MD, White NJ, 1993. Serology and carriage of Pseudomonas pseudomallei: a prospective study in 1000 hospitalized children in northeast Thailand. J Infect Dis 167: 230233.

    • Search Google Scholar
    • Export Citation

Author Notes

*Address correspondence to T. Eoin West, Division of Pulmonary and Critical Care Medicine, University of Washington School of Medicine, Harborview Medical Center, Box 359640, 325 9th Avenue Seattle, WA 98104. E-mail: tewest@u.washington.edu

Financial support: T. Eoin West is supported by National Institutes of Health award K08 HL094759 and by a Parker B. Francis Fellowship for Pulmonary Research. Direk Limmathurotsakul, Narisara Chantratita, and Sharon J. Peacock are supported by the Wellcome Trust.

Authors' addresses: T. Eoin West, Nicolle D. Myers, and Shawn J. Skerrett, Division of Pulmonary and Critical Care Medicine, University of Washington School of Medicine, Harborview Medical Center, Seattle, WA, E-mails: tewest@u.washington.edu, ndmyers@u.washington.edu, and shawn@u.washington.edu. Direk Limmathurotsakul, Narisara Chantratita, and Sharon J. Peacock, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, E-mails: direk@tropmedres.ac, narisara@tropmedres.ac, and sharon@tropmedres.ac. H. Denny Liggitt, Department of Comparative Medicine, University of Washington School of Medicine, Seattle, WA, E-mail: dliggitt@u.washington.edu.

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