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Melioidosis is a disease of the tropics caused by the facultative intracellular bacterium Burkholderia pseudomallei. In human infection, increased levels of IFN‐γ in addition to the chemokines interferon‐γ‐inducible protein 10 (IP‐10) and monocyte interferon‐γ‐inducible protein (Mig) have been demonstrated. However, the role of these and other chemokines in the pathogenesis of melioidosis remains unknown. Using BALB/c and C57BL/6 mice as models of the acute and chronic forms of human melioidosis, the induction of mRNA was assessed for various chemokines and CSF (G‐CSF, M‐CSF, GM‐CSF, IP‐10, Mig, RANTES, MCP‐1, KC and MIP‐2) in spleen and liver following B. pseudomallei infection. Patterns of chemokine and CSF induction were similar in liver and spleen; however, responses were typically greater in spleen, which reflected higher tissue bacterial loads. In BALB/c mice, high‐level expression of mRNA for all chemokines and CSF investigated was demonstrated at day 3 postinfection, correlating with peak bacterial load and extensive infiltration of leucocytes. In contrast, increased mRNA expression and bacterial numbers in C57BL/6 mice were greatest between 4 and 14 days following infection. This paralleled increases in the size and number of abscesses in liver and spleen of C57BL/6 mice at days 3 and 14 postinfection. Earlier induction of cytokine‐induced neutrophil chemoattractant (KC), macrophage inflammatory protein‐2 (MIP‐2), monocyte chemoattractant protein‐1 (MCP‐1), granulocyte‐macrophage CSF (GM‐CSF) and macrophage CSF (M‐CSF) mRNA was demonstrated in spleen, while MIP‐2, MCP‐1, IP‐10 and Mig were demonstrated in liver of BALB/c mice when compared to spleen and liver of C57BL/6. The magnitude of cellular responses observed in the tissue correlated with increased levels of the chemokines and CSF investigated, as well as bacterial load. Compared with C57BL/6 mice, greater infiltration of neutrophils was observed in liver and spleen of BALB/c mice at day 3. In contrast, early lesions in C57BL/6 mice predominantly comprised macrophages. These results suggest that the inability of BALB/c mice to contain the infection at sites of inflammation may underlie the susceptible phenotype of this mouse strain towards B. pseudomallei infection.

Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS‐3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS‐3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti‐BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme‐linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei.

Melioidosis is a bacterial infection caused by Burkholderia pseudomallei. The aim of this study was to determine whether a cell-mediated adaptive immune response against B. pseudomallei developed in patients who had recovered from melioidosis. Lymphocyte proliferation assays were done on peripheral blood mononuclear cells from patients (n=13) and control subjects (n=10) to determine the lymphocyte response to B. pseudomallei antigens. Production of interferon-gamma and interleukin-10 was also determined. Activation of T cell subsets was assessed by fluorescence-activated cell sorter analysis, using antibodies to CD4, CD8, and CD69 antigens. Lymphocyte proliferation and interferon-gamma production in response to B. pseudomallei antigens were significantly higher (P<.001 for both) in patients than in control subjects. There was also an increase in the percentage of activated CD4+ (P<.004) and activated CD8+ T cells (P<.035) in cell cultures from patients. The development of such a cell-mediated immune response in patients may be essential for their survival.

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Melioidosis is caused by the facultative intracellular bacterium, Burkholderia pseudomallei. Using C57BL/6 mice, we investigated the role of macrophages, TNF-a, TNF receptor-1 (TNFR1) and TNF receptor-2 (TNFR2) in host defense against B. pseudomallei using an experimental model of melioidosis. This study has demonstrated that in vivo depletion of macrophages renders C57BL/6 mice highly susceptible to intranasal infection with B. pseudomallei, with significant mortality occurring within 5 days of infection. Using knockout mice, we have also shown that TNF-a and both TNFR1 and TNFR2 are required for optimal control of B. pseudomallei infection. Compared with control mice, increased bacterial loads were demonstrated in spleen and liver of knockout mice at day 2 postinfection, correlating with increased inflammatory infiltrates comprised predominantly of neutrophils and widespread necrosis. Following infection with B. pseudomallei, mortality rates of 85.7%, 70% and 91.7% were observed for mice deficient in TNF-a, TNFR1 and TNFR2, respectively. Comparison of survival, bacterial loads and histology indicate that macrophages, TNF-a, TNFR1 or TNFR2 play a role in controlling rapid dissemination of B. pseudomallei.

Melioidosis is a potentially fatal disease caused by the bacterium, Burkholderia pseudomallei. The current study was carried out to determine the mechanisms involved in the development of protective immunity in a murine model of melioidosis. Following intravenous infection with B. pseudomallei, both C57BL/6 and BALB/c mice demonstrated delayed-type hypersensitivity responses and lymphocyte proliferation towards B. pseudomallei antigens, indicating the generation of B. pseudomalleispecific lymphocytes. Adoptive transfer of these lymphocytes to naïve C57BL/6 mice was demonstrated by a delayed-type hypersensitivity response. Mice were not protected from a subsequent lethal challenge with a highly virulent strain of B. pseudomallei, suggesting that a single intravenous dose of the bacterium is insufficient to induce a protective adaptive immune response. Attempts to induce resistance in susceptible BALB/c mice used repetitive low-dose exposure to live B. pseudomallei. Immune responses and resistance following subcutaneous immunization with live B. pseudomallei were compared with exposure to heat-killed, culture filtrate and sonicated B. pseudomallei antigens. Compared to heat-killed B. pseudomallei, significant protection was generated in BALB/c mice following immunization with live bacteria. Our studies also demonstrate that the type of immune response generated in vivo is influenced by the antigenic preparation of B. pseudomallei used for immunization.