Lipopolysaccharide from Burkholderia thailandensis E264 provides protection in a murine model of melioidosis.

Abstract

Burkholderia thailandensis is a less virulent close relative of Burkholderia pseudomallei, a CDC category B biothreat agent. We have previously shown that lipopolysaccharide (LPS) extracted from B. pseudomallei can provide protection against a lethal challenge of B. pseudomallei in a mouse model of melioidosis. Sugar analysis on LPS from B. thailandensis strain E264 confirmed that this polysaccharide has a similar structure to LPS from B. pseudomallei. Mice were immunised with LPS from B. thailandensis or B. pseudomallei and challenged with a lethal dose of B. pseudomallei strain K96243. Similar protection levels were observed when either LPS was used as the immunogen. This data suggests that B. thailandensis LPS has the potential to be used as part of a subunit based vaccine against pathogenic B. pseudomallei.

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Vaccine 28 (2010) 7551–7555
Contents lists available at ScienceDirect
Vaccine
journal homepage: www.elsevier.com/locate/vaccine
Lipopolysaccharide from Burkholderia thailandensis E264 provides protection in a
murine model of melioidosis
Sarah A. Ngugia, Valeria V. Venturab, Omar Qazic, Sarah V. Hardinga, G. Barrie Kittoc,d,
D. Mark Estese, Anne Dellb, Richard W. Titballf, Timothy P. Atkinsa, Katherine A. Brownb,c,d,
Paul G. Hitchenb,g, Joann L. Priora,∗
aDstl Porton Down, Salisbury, Wiltshire SP4 0JQ, UK
bDepartment of Life Sciences, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK
cInstitute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA
dDepartment of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX 78712, USA
eDepartment of Microbiology and Immunology, Department of Pathology and the Sealy Center for Vaccine Development, University of Texas Medical Branch,
Galveston, TX 77555, USA
fUniversity of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK
gCentre for Integrative Systems Biology, Imperial College London, South Kensington Campus, Exhibition Road, London SW7 2AZ, UK
a r t i c l e i n f o
Article history:
Received 24 May 2010
Received in revised form 10 August 2010
Accepted 11 August 2010
Available online 15 September 2010
Keywords:
Burkholderia thailandensis
Burkholderia pseudomallei
Lipopolysaccharide
a b s t r a c t
Burkholderia thailandensis is a less virulent close relative of Burkholderia pseudomallei, a CDC category B
biothreat agent. We have previously shown that lipopolysaccharide (LPS) extracted from B. pseudomallei
canprovideprotectionagainstalethalchallengeofB.pseudomalleiinamousemodelofmelioidosis.Sugar
analysisonLPSfromB.thailandensisstrainE264confirmedthatthispolysaccharidehasasimilarstructure
to LPS from B. pseudomallei. Mice were immunised with LPS from B. thailandensis or B. pseudomallei and
challenged with a lethal dose of B. pseudomallei strain K96243. Similar protection levels were observed
wheneitherLPSwasusedastheimmunogen.ThisdatasuggeststhatB.thailandensisLPShasthepotential
to be used as part of a subunit based vaccine against pathogenic B. pseudomallei.
Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved.
1. Introduction
Burkholderia pseudomallei is the causative agent of melioido-
sis, a severe infectious disease of humans and animals which is
reported most frequently in SE Asia and Northern Australia [1]. The
disease is difficult to treat with antibiotics because of the intrin-
sic antibiotic resistance of the bacterium [2,3], and there is no
licensed vaccine available for use in humans. Because of the abil-
ity of the bacterium to infect via the respiratory tract, it is also
considered to be a potential bioweapon [4]. Studies in Thailand
have shown that the bacterium can be readily isolated from the
soil in melioidosis endemic areas [5]. In addition, the related bac-
terium Burkholderia thailandensis, can often be isolated from soil
in these areas. The clearest distinction between these two species
is the ability of B. thailandensis to assimilate l-arabinose (Ara+),
whereas B. pseudomallei (Ara−) does not have this capability, and
in fact lacks the entire arabinose assimilation operon [6]. Addition-
ally, B. thailandensis is considered to be avirulent in humans, with
∗Corresponding author. Tel.: +44 01980 614739; fax: +44 01980 614307.
E-mail address: JLPRIOR@dstl.gov.uk (J.L. Prior).
a median lethal dose (MLD) in hamsters of approximately 1×106
bacteria (48h post-inoculation), when given intra-peritoneally (IP)
[7] and approximately 1×109bacteria (IP) in BALB/c mice [6]. In
contrast, B. pseudomallei is highly virulent in Syrian hamsters hav-
ing an MLD of <10 bacteria (IP), and in BALB/c mice (IP) an MLD of
approximately 100 bacteria [6,7].
Lipopolysaccharide (LPS) is a major component of the outer
membrane of Gram-negative bacteria. It is composed of three main
regions, lipid A, the core and multiple O-antigen units covalently
linked to one another. Lipid A anchors the LPS in the outer mem-
brane, the core is a mixture of sugars and sugar derivatives, whilst
the O-antigen is a polysaccharide of repeating oligosaccharide
units that extends from the cell surface, and typically determines
serotype specificity [8,9]. There are three types of B. pseudomallei
LPS. The predominant type (97%) is known as type A or typical,
whilst the less common LPS (2%) is known as type B or atypical.
Both are smooth types of LPS, whereas the remaining 1% lacking
an O-antigen is termed rough LPS. When LPS separated by sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)
is stained with silver, one of three different profiles is seen. A lad-
der for typical and atypical LPS, whilst no ladder is observed for the
rough strains [10]. The cellular fatty acid profiles and lipid A struc-
0264-410X/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.vaccine.2010.08.058

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tures of B. pseudomallei and B. thailandensis have been described
[11,12]. It was established that these species share the same fatty
acid(FA)profilewiththeexceptionofFAC14:0(2-OH)whichisonly
present in B. pseudomallei [11,12].
LPSextractedfromB.pseudomalleihaspreviouslybeenshownto
provide protection against an otherwise lethal challenge of B. pseu-
domallei in a mouse model of melioidosis. In mice immunised with
LPS from B. pseudomallei strain K96243, 50% survival was seen at
day35,withameantimetodeath(MTTD)of17.6days,incontrastto
100% mortality at day 11 and a MTTD of 2.6 days in naïve mice [13].
However,B.pseudomalleiisaBiosafetylevelthree(BSL-3)pathogen
which makes the purification of LPS a complex and potentially haz-
ardous procedure. It has been reported that B. thailandensis and B.
pseudomalleiproduceasimilarLPSO-antigen.Thisisanunbranched
heteropolymer of disaccharide repeats with the structure, 3-?-d-
glucopyranose-(1→3)-6-deoxy-?-l-talopyranose-(1→), in which
their 6-deoxy-?-l-talopyranosyl residues are variably substituted
with O-acetyl groups at the O-2 or O-4 positions [14–16]. Further-
more, LPS isolated from B. thailandensis and B. pseudomallei show
similar banding patterns on silver-stained SDS-PAGE gels, and in
immuno-blotsprobedwithmelioidosis-positiveserafromhumans,
mice or rabbits [17,18]. The similarity of these LPSs may suggest
that it would be possible to immunise mice with LPS extracted
from B. thailandensis and protect them against a lethal challenge
of B. pseudomallei. The extraction of LPS from the BSL-2 bacterium
B. thailandensis would be less expensive, have reduced safety risks
and be more amenable to scale-up compared to extraction from B.
pseudomallei.
In this work we extracted the LPS from either B. pseudomallei
K96243 or B. thailandensis E264, and analysed them by silver stain-
ingandGC–MStoallowinsightintothestructuralsimilaritiesofthe
LPSexpressedbyeachspecies.Wehavecomparedtheirefficaciesas
protective antigens in a murine melioidosis model of infection, and
determined the antibody isotype of the serum antibody immune
response.
2. Materials and methods
2.1. LPS extraction and gel electrophoresis
LPS was extracted from B. thailandensis strain E264 and B. pseu-
domalleistrainK96243usingacombinationofpreviouslypublished
methods [19,20]. In brief, cell pellets of B. pseudomallei K96243
and B. thailandensis E264 were digested for 16h at 4◦C with
15,000Units of lysozyme (Sigma, UK) per mg of bacteria, prior to
digestion with 20?g/ml of DNase I and RNase A (Sigma, UK) for a
further 16h at room temperature. A modified hot phenol method
followed.LPSwasseparatedbySDS-PAGEona12.5%separatinggel
and visualised by silver staining [21].
2.2. Chemical derivatisation to alditol acetates for GC–MS
analysis
For compositional analysis of the O-antigen by alditol acetate,
the LPS samples were hydrolysed (2M TFA, 121◦C, 2h) and the
monosaccharides reduced (10mg/ml NaBD4room temperature,
2h). After borate removal by repeated evaporation (4×) with 10%
acetic acid in methanol, the sugars were re-acetylated (acetic
anhydride, 100◦C, 1h) and prepared for GC–MS by suspension in
hexanes.
2.3. GC–MS analysis
GC–MS analysis was carried out on a Clarus 500 GC–MS instru-
ment (Perkin-Elmer, USA). Samples were dissolved in hexanes
and injected on a RTX-5MS fused silica column (30mm×0.25mm
Fig. 1. Silver-stained SDS-PAGE of LPS isolated from B. pseudomallei strain K96243
(lane 1) or B. thailandensis E264 (lane 2).
internaldiameter,Restek).Foranalysisofthealditolacetatederiva-
tives, the oven was held at 60◦C for 1min and increased to 190◦C
at 20◦Cmin−1, at which point the temperature was increased to
230◦Cat1◦Cmin−1.Thefinaltemperatureincrementwasto300◦C,
raised at 25◦Cmin−1and held for a total of 5min.
2.4. LPS protective efficacy
Groups of six female BALB/c mice, aged 6–8 weeks (Charles
River Laboratories, Kent, UK) were immunised with purified LPS
extracted from B. thailandensis strain E264 or B. pseudomallei strain
K96243. On days 0, 14 and 28 mice were dosed intra-peritoneally
(IP) with 10?g of LPS in PBS. Mice were tail-bled 4 weeks after
the final immunisation. Blood for each group was pooled and sera
recoveredbycentrifugation.Micewerechallenged35daysafterthe
last immunisation with 55 MLD’s of B. pseudomallei strain K96243
by the IP route, delivered in 0.1ml. All procedures were performed
inaccordancewiththeAnimal(ScientificProcedures)Act1986.Liv-
ers and spleens were harvested from surviving mice for bacterial
counts.
2.5. Serum response to LPS
Levels of IgG1, IgG2a, IgG3 and IgM were measured from sera
collected 4 weeks post-final vaccination by ELISA. Microtitre plates
(Immulon 2HB, USA) were coated using anti-mouse fab-specific

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Fig. 2. GC–MS alditol acetate sugar analysis of hydrolysed and derivatised LPS from (a) B. pseudomallei and (b) B. thailandensis. Non-sugar impurities are labelled X.
antibody (Sigma, UK) at 5?g/ml in PBS and B. pseudomallei K96243
LPS at 5?g/ml, both100?l/well. Plates were washed three times
with 0.05% (v/v) PBS/Tween (Oxoid, UK/Sigma, UK) using an ELx
405 96-well automated plate washer (BioTek Instruments, UK),
and blocked with 200?l/well, 2% (w/v) skimmed milk powder
(Blotto) for 1h at 37◦C. Plates were re-washed as previously
described. A standard curve was prepared and pooled serum sam-
ples diluted 1:100 in Blotto. Diluted naïve sera were included on
each plate as a negative control. Standards and samples were seri-
ally diluted. Plates were incubated for 1h at 37◦C and re-washed.
100?l of HRP-conjugated isotype-specific goat anti-mouse anti-
body, (AbDserotec, UK) was added to each well and incubated at
37◦C for 1h. Plates were washed as previously. 100?l of ABTS
substrate (Sigma, UK) was added to each well; plates were incu-
bated for 15min at 37◦C and read on a Multiskan Ascent plate
reader (Thermo Life sciences, UK) at 414nm. Analysis was car-
ried out using ELISA for Windows version 2.0 (freely available
from http://www.cdc.gov.ncidod/dbmd/bimb/elisa.htm). This uses
a four-parameter logistic fit curve to calculate unknown sample
concentrations of antibody in ?g/ml.
3. Results and discussion
We have shown that LPS extracted from B. thailandensis strain
E264 provides protection against a lethal challenge of B. pseudoma-
llei strain K96243 by the IP route and produces a specific antibody
response.
3.1. LPS profiles
When visualised with silver-staining the LPS extracted from B.
thailandensis shows a characteristic LPS ladder banding pattern. It

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has a profile comparable to the LPS extracted from B. pseudomallei
strain K96243, displaying very similar spacing between O-antigen
units (Fig. 1).
3.2. Sugar composition of B. thailandensis LPS
The purified LPS samples of B. pseudomallei and B. thailandensis
were analysed for sugar composition as alditol acetate-derivatised
monosaccharides to determine their sugar compositions (Fig. 2).
GC–MS analysis revealed close similarities between the B. pseu-
domallei and B. thailandensis O-antigens, although their talose
methylation patterns are not identical.
Both LPSs were found to contain talose and glucose in their
O-antigens which is consistent with previous reports [14–16]. In
addition, the GC–MS spectra contain mannoheptose and rhamnose
which are likely to be derived from the core of the LPS. Differ-
ences in the LPSs were associated with the talose sugar. Thus B.
pseudomallei LPS possessed both methylated and unmethylated
talose, in contrast to the B. thailandensis sample which had only
unmodified talose. The two talose forms in the B. pseudomallei LPS
chromatograms are consistent with the published literature which
has reported that 33% of the talose residues in B. pseudomallei LPS
are 2-O-methylated [15,16]. Our data suggests that the major dif-
ference between the two O-antigens is the absence of methylated
talose in B. thailandensis.
Additional minor components were observed in the B. pseudo-
mallei alditol acetate chromatogram, which are likely associated
with the LPS core and/or contaminating polymers. For exam-
ple a minor deoxymannoheptose peak was observed in the B.
pseudomallei LPS chromatogram, but not in the B. thailandensis
data. This is probably derived from trace amounts of co-purified
capsular polysaccharide which is known to be an unbranched
homopolymer of O-acetylated deoxymannoheptose. The absence
of the deoxymannoheptose sugar in the B. thailandensis LPS anal-
ysis is consistent with the lack of a capsule in this species [22]. In
addition, some galactose is present in B. pseudomallei LPS but not
B. thailandensis LPS. This is likely to have originated from the LPS
coresincetheO-antigenofB.pseudomalleiiswellcharacterisedand
does not contain galactose [14,15].
3.3. Protective efficacy of LPS in a murine model of melioidosis
Despite the difference in the extent of methylation of the talose
monosaccharide between the species, we found, via the experi-
ments described below, that B. thailandensis LPS is able to induce a
protective response against infection with a typical B. pseudomallei
strain.ImmunisationofmicewitheitherLPSfromB.thailandensisor
B. pseudomallei was able to induce protection against a subsequent
challengeof55MLD’sofB.pseudomalleistrainK96243(Fig.3).After
3 days there were no survivors in the naïve group. At the end of
the experiment (day 35 post-challenge) there was 50% survival of
the mice immunised with B. thailandensis LPS compared with 66%
survival of mice immunised with B. pseudomallei LPS. The mean
time to death (MTTD) for naïve mice was 2.2 days, whereas the
MTTD of mice immunised with B. thailandensis or B. pseudomallei
was 32.8 days or 31.5 days, respectively. There was a statistically
significantdifferenceintheMTTDofimmunisedanimalscompared
to the control (p<0.001) using a Log rank test. This was repeated
with a challenge of 24 MLD’s of B. pseudomallei strain K96243 (data
not shown). All naïve mice succumbed by day 18, compared to 50%
survival in those mice immunised with B. thailandensis LPS at the
end of the experiment (day 35 post-challenge). The MTTD for naïve
micewas10.8dayscomparedto32.3daysformiceimmunisedwith
B. thailandensis LPS. The MTTD for naïve mice would be expected
to be higher than observed in the previous experiment due to the
lower challenge dose.
Fig. 3. Survival of BALB/c mice immunised with LPS isolated from B. pseudoma-
llei (O), B. thailandensis (X) or naïve controls (?). Mean time to death in both test
groups was significantly different to the controls (p<0.001) using a Log rank test for
difference.
IthasbeenpostulatedthatnaturalexposuretoB.thailandensisin
melioidosis endemic areas, and the subsequent development of an
antibody response without overt infection may provide protection
against melioidosis [13]. Immunisation with live B. thailandensis
has reportedly provided 50% protection in guinea pigs when chal-
lenged with 200 LD50B. pseudomallei strain 100 [23]. Moreover,
BALB/c mice immunised IP with 1×108cfuml−1of heat killed B.
thailandensis, and subsequently challenged IP with 40 MLD’s B.
pseudomallei K96243 also showed 50% survival. Additionally, all
spleens were free from signs of infection [24]. In this study liv-
ers and spleens were harvested from all surviving mice immunised
with the LPSs to determine bacterial load. Colonies showing the
typicalmorphologicalcharacteristicsofB.pseudomalleiwererecov-
ered from three of the spleens, and all four of the livers from the
survivinganimalsimmunisedwithK96243LPS.B.pseudomalleiwas
also recovered from two of the spleens, and all of the livers of the
three surviving animals immunised with LPS from B. thailandensis,
thus mice vaccinated with LPS do not clear the bacteria and may
ultimately succumb to infection.
3.4. Serum antibody responses to LPS immunisation
LPS is a T-independent antigen, and as expected IgM and IgG3
responses predominated in the serological profile of the host
response to vaccination. Mice immunised with LPS from B. thai-
landensis or B. pseudomallei had measurable antibody response in
IgG3 and IgM for both LPS’.
In conclusion, consistent with previous studies using B. pseudo-
mallei LPS we have demonstrated that B. thailandensis LPS is both
structurally similar, and displays a comparable level of partial pro-
tection against melioidosis in a mouse model of infection [13]. This
offers the possibility of using B. thailandensis LPS or LPS moieties
in vaccines targeted to B. pseudomallei. For example to improve
the efficacy of a vaccine, the LPS could be conjugated to a carrier
protein to generate a T-cell dependent response [25]. Previously
B. pseudomallei LPS has been conjugated to tetanus toxoid or flag-
ellin to produce a glycoconjugate. The passive transfer of antisera
against these glycoconjugates conferred protection to diabetic rats
against B. pseudomallei strain 316c [26,27]. Given the results pre-
sented in this manuscript, it is likely that similar results could be
obtained using B. thailandensis LPS. Furthermore, the reduced costs
and hazards associated with the production of B. thailandensis LPS
suggest that this molecule can be exploited in a wider range of
studies aimed at developing a vaccine against melioidosis infec-
tion.
Acknowledgements
A.D. and P.G.H. are supported by the Biotechnology and
Biological Sciences Research Council (grants BBF0083091 and

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BBC5196701 CISBIC). V.V.V. is supported by a student bursary
from Dstl. This work was also supported by NIH/NIAID grants
1U01AI078008-1 and U54AI057156 (KAB, DME, AGT).
References
[1] Dance DAB. Melioidosis: the tip of the iceberg? Clin Microbiol Rev
1991;4(1):52–60.
[2] Dance DAB, Wuthiekanun V, Chaowagul W, White NJ. The antimicrobial sus-
ceptibility of Pseudomonas pseudomallei—emergence of resistance in vitro and
during treatment. J Antimicrob Chemother 1989;24:295–309.
[3] SamuelM,TiTY.Interventionsfortreatingmelioidosis.CochraneDatabaseSyst
Rev 2001:CD001263.
[4] ChengAC,CurrieBJ.Melioidosis:epidemiology,pathophysiologymanagement.
Clin Microbiol Rev 2005;18(2):383–416.
[5] Wuthiekanun V, Smith MD, Dance DAB, White NJ. Isolation of Pseudomonas
pseudomallei from soil in north-eastern Thailand. Trans R Soc Trop Med Hyg
1995;89:41–3.
[6] Smith MD, Angus BJ, Wuthiekanun V, White NJ. Arabinose assimilation
defines a nonvirulent biotype of Burkholderia pseudomallei. Infect Immun
1997;65(10):4319–21.
[7] Brett PJ, DeShazer D, Woods DE. Characterization of Burkholderia pseudo-
mallei and Burkholderia pseudomallei-like strains. Epidemiol Infect 1997;118:
137–48.
[8] Reeves PR, Hobbs M, Valvano MA, Skurnik M, Whitfeld C, Coplin D, et al., Klena
J, Raetz CRH. Trends Microbiol 1996;4(12):495–503.
[9] Darveau RP, Hancock REW. Procedure for isolation of bacterial lipopolysac-
charides from both smooth and rough Pseudomonas aeruginosa and Salmonella
typhimurium strains. J Bacteriol 1983;155(2):831–8.
[10] Anuntagool N, Wuthiekanun V, White NJ, Currie BJ, Sermswan RW, Won-
gratanacheewin S, et al., Chaiyaroj SC. Lipopolysaccharide heterogeneity
amongBurkholderiapseudomalleifromdifferentgeographicandclinicalorigins.
Am J Trop Med Hyg 2006;74(3):348–52.
[11] Inglis TJJ, Aravena-Roman M, Ching S, Croft K, Wuthiekanun V, Mee BJ. Cel-
lular fatty acid profile distinguishes Burkholderia pseudomallei from avirulent
Burkholderia thailandensis. J Clin Micro 2003;14(10):4812–4.
[12] Novem V, Shui G, Wang D, Bendt AK, Sim SH, Liu Y, et al., Sivalingam SP,
Wenk MR. Structural and biological diversity of lipopolysaccharides from
BurkholderiapseudomalleiandBurkholderiathailandensis.ClinVaccineImmunol
2009;16(10):1420–8.
[13] Nelson M, Prior JL, Lever MS, Jones HE, Atkins TP, Titball RW. Eval-
uationof lipopolysaccharideand
vaccines against experimental melioidosis. J Med Microbiol 2004;53:
1177–82.
capsular polysaccharide assubunit
[14] Knirel YA, Paramonov NA, Shashkov AS, Kochetkov NK, Yarullin RG, Farber
SM, et al. Structure of the polysaccharide chains of Pseudomonas pseudomallei
lipopolysaccharides. Carbohyd Res 1992;233:185–93.
[15] Perry MB, MacLean LL, Schollaardt T, Bryan LE, Ho M. Structural characteriza-
tion of the lipopolysaccharide O-antigens of Burkholderia pseudomallei. Infect
Immun 1995;63(9):3348–52.
[16] Brett PJ, Burtnick MN, Woods DE. The wbiA locus is required for the 2-O-
acetylation of lipopolysaccharide expressed by Burkholderia pseudomallei and
Burkholderia thailandensis. FEMS Microbiol Lett 2003;218:323–8.
[17] Anuntagool N, Intachote P, Wuthiekanun V, White NJ, Sirisinha S. Lipopolysac-
charide from nonvirulent Ara+Burkholderia pseudomallei isolates is immuno-
logically indistinguishable from lipopolysaccharide from virulent Ara−clinical
isolates. Clin Diagn Lab Immun 1998;5(2):225–9.
[18] Qazi O, Prior JL, Judy BM, Whitlock GC, Kitto GB, Torres AG, et al., Brown KA.
Sero-characterisation of lipopolysaccharide from Burkholderia thailandensis. T
Roy Soc Trop Med H 2008;102(Suppl. 1):S58–60.
[19] WeshphalO, JannK.Bacterial
phenol–water and further applications of the procedure. In: Whistler
RL, editor. Methods in carbohydrate chemistry, vol. 5. New York: Academic
Press; 1965. p. 83–91.
[20] Johnson KG, Perry MB. Improved techniques for the preparation of bacterial
lipopolysaccharides. Can J Microbiol 1975;22:29–34.
[21] Chart HLPS. Isolation and characterisation. In: Raton B, Arbor A, editors. Meth-
ods in practical laboratory bacteriology. London, Tokyo: CRC Press; 1994. p.
11–20.
[22] Reckseidler SL, DeShazer D, Sokol PA, Woods DE. Detection of bacte-
rial virulence genes by subtractive hybridization: Identification of capsular
polysaccharide of Burkholderia pseudomallei as a major virulence determinant.
Infect Immun 2001;69(1):34–44.
[23] Iliukhin VI, Senina TV, Plekhanova NG, Antonov VA, Merinova LK, Seimova IK.
Burkholderia thailandensis: biological properties, identification and taxonomy.
Mol Gen Mikrobiol Virusol 2002;1:7–11.
[24] Sarkar-Tyson M, Smither SJ, Harding SV, Atkins TA, Titball RW. Protective effi-
cacy of heat-inactivated B. thailandensis, B. mallei or B. pseudomallei against
experimental melioidosis and glanders. Vaccine 2009;27:4447–51.
[25] Peeters CCAM, Lagerman PR, Weers O, Oomen LA, Hoogerhout P, Beurret M,
et al. Preparation of polysaccharide-conjugate vaccines. In: Robinson, Hudson,
Cranage, editors. Methods in molecular medicine, vol. 87: vaccine protocols.
2nd ed. Totowa, NJ: Humana Press Inc.; 2003. p. 153–73.
[26] Bryan LE, Wong S, Woods DE, Dance DAB, Chaowagul W. Passive protection
of diabetic rats with antisera specific for the polysaccharide portion of the
lipopolysaccharide isolated from Pseudomonas pseudomallei. Can J Infect Dis
Med 1994;5(4):170–8.
[27] Brett PJ, Woods DE. Structural and immunological characterisation of
BurkholderiapseudomalleiO-polysaccharide-flagellinproteinconjugates.Infect
Immun 1996;64(7):2824–8.
lipopolysaccharides,extractionwith