Preparation, <i>M</i>. <i>ulcerans</i> application and swabbing of fresh pigskin from part of one experiment.

(A) The pigskin was divided in 2x2cm squares using a ruler and marker pen; (B) M. ulcerans culture and culture dilutions were applied to each square and allowed to dry before swabbing. Note that N = neat culture, -1,-2, -3, -4 etc are the 10-fold dilutions, B = sterile 7H9 media negative control; (C) Demonstration of swabbing technique for recovery of M. ulcerans from the pigskin surface.</p

All results from human swabs.

Blank cells indicate “not tested” (while all swabs were tested in duplicate at least, some were tested in triplicate). (DOCX)</p

Limit-of-detection for IS<i>2404</i> qPCR from swabbed pigskin.

Plot showing Ct values for two independent M. ulcerans 10-fold dilution series used to inoculate fresh pigskin. The dotted line represents the limit-of-detection (Ct 35). Each dilution was tested by IS2404 qPCR in technical triplicates. Shown are the mean Ct values and standard deviation for the technical replicates. Note that error bars are present but too small to resolve (refer S1 Table).</p

All results from pigskin validation model.

IntroductionMycobacterium ulcerans (MU) causes Buruli ulcer (Buruli), a geographically restricted infection that can result in skin loss, contracture and permanent scarring. Lesion-location maps compiled from more than 640 cases in south eastern Australia suggest biting insects are likely involved in transmission, but it is unclear whether MU is brought by insects to humans or if MU is already on the skin and inoculation is an opportunistic event that need not be insect dependent.MethodsWe validated a PCR swab detection assay and defined its dynamic range using laboratory cultured M. ulcerans and fresh pigskin. We invited volunteers in Buruli-endemic and non-endemic areas to sample their skin surfaces with self-collected skin swabs tested by IS2404 quantitative PCR.ResultsPigskin validation experiments established a limit-of-detection of 0.06 CFU/cm2 at a qPCR cycle threshold (Ct) of 35. Fifty-seven volunteers returned their self-collected kits of 4 swabs (bilateral ankles, calves, wrists, forearms), 10 from control areas and 47 from endemic areas. Collection was timed to coincide with the known peak-transmission period of Buruli. All swabs from human volunteers tested negative (Ct ≥35).ConclusionsM. ulcerans was not detected on the skin of humans from highly Buruli endemic areas.</div

Time-kill assay showing the effect of ivermectin against <i>M</i>. <i>ulcerans</i> over 8-weeks.

<p><i>M</i>. <i>ulcerans</i> was grown in 25cm² culture flasks in the presence of 0, 8 and 20μg/ml ivermectin. Ethanol (solvent for ivermectin) at the concentration in the IVM-20 dose was used in the no-drug control. Weekly, ten-fold dilutions of each culture were plated onto 7H10 agar, using a 3-μl spot-dilution method, with five replicates per dilution. The plates were examined for growth after incubation for 8-weeks at 30°C, and colony forming units per ml calculated. The mean and range for duplicate biological experiments are shown.</p

Antigen-specific IFN-γ production in naïve and vaccinated mice, as tested by ELISPOT.

<p>Number of IFN-γ producing cells in C57BL/6 mice vaccinated against the 9 PkS domains using the pDNA prime/protein boost protocol and stimulated <i>in vitro</i> with the corresponding recombinant protein antigen (5 µg/ml). Results represent mean ± SD of spot forming cells/10⁶ cells of 4–6 mice tested individually as detected in a 48 hr ELISPOT. Data are representative of one of two experiments. White columns: cells without Ag stimulation; black columns: cells stimulated for 48 hr with corresponding antigen.</p

Antigen-specific IL-2 production in naïve and vaccinated mice, as tested by ELISA.

<p>Interleukin-2 levels in 24 hr spleen cell culture supernatants of C57BL/6 mice vaccinated against the 9 PkS domains using the pDNA prime/protein boost protocol and stimulated <i>in vitro</i> with the corresponding recombinant protein antigen (5 µg/ml). Results represent mean ± SD IL-2 values (pg/ml) of 4–6 mice tested individually. Data are representative of one of three experiments.</p

Proteomic Characterization of a Natural Host–Pathogen Interaction: Repertoire of in Vivo Expressed Bacterial and Host Surface-Associated Proteins

Interactions between a host and a bacterial pathogen are mediated by cross-talk between molecules present on, or secreted by, pathogens and host binding-molecules. Identifying proteins involved at this interface would provide substantial insights into this interaction. Although numerous studies have examined in vitro models of infection at the level of transcriptional change and proteomic profiling, there is virtually no information available on naturally occurring host–pathogen interactions in vivo. We employed membrane shaving to identify peptide fragments cleaved from surface-expressed bacterial proteins and also detected proteins originating from the infected host. We optimized this technique for media-cultured Corynebacterium pseudotuberculosis, a sheep pathogen, revealing a set of 247 surface proteins. We then studied a natural host–pathogen interaction by performing membrane shaving on C. pseudotuberculosis harvested directly from naturally infected sheep lymph nodes. Thirty-one bacterial surface proteins were identified, including 13 not identified in culture media, suggesting that a different surface protein repertoire is expressed in this hostile environment. Forty-nine host proteins were identified, including immune mediators and antimicrobial peptides such as cathelicidin. This novel application of proteolytic shaving has documented sets of host and pathogen proteins present at the bacterial surface in an infection of the native host

Primers used for the cloning of nine Pks domains.

<p>Primers used for the cloning of nine Pks domains in plasmid pV1J.ns-tPA. Restriction enzymes used are indicated. Sense primers are listed first and antisense primers second, within primer pairs. All restriction sites are shown in bold.</p

Antigen-specific IFN-γ production in naïve and vaccinated mice, as tested by ELISA.

<p>IFN-γ levels in 72 hr spleen cell culture supernatants of C57BL/6 mice vaccinated against the 9 PkS domains using the pDNA prime/protein boost protocol and stimulated <i>in vitro</i> with the corresponding recombinant protein antigen (5 µg/ml). Results represent mean ± SD IFN-γvalues (pg/ml) of 4–6 mice tested individually. Data are representative of one of three experiments.</p