Subculture method
N. brasiliensis HUJEG-1 was used for these experiments; it has been utilized in previous assays [5, 9]. Bacterial cultures obtained from mouse lesions were kept frozen at -70°C in 20% skim milk. From these stocks, bacteria were grown on Sabouraud agar at 30°C for 4 to 7 days; then a single colony was placed in a 7 mL sterile Eveljham-Potter device. We added 2.5 mL of sterile saline and the bacterial mass was ground to obtain a homogeneous suspension and turbidity adjusted to McFarland's tube No 1. With this suspension (0.1 mL) we inoculated a 125 ml Erlenmeyer flask containing 33 ml of previously sterilized liquid medium Brain Heart Infusion (BHI). It was incubated with constant agitation at 110 rpm, at 37°C. After 72 h the bacterial mass was harvested by centrifugation at 2,500 rpm for 3 minutes, washed, and ground as above. A new Erlenmeyer flask was inoculated with 0.1 ml of this suspension. These steps were repeated until reaching 130 subcultures (T130). Samples were taken every 10 passages and kept frozen at -70°C, including T0. The entire process took about four years.
Experimental mycetoma in a murine model
Cultures were obtained from the aliquots stored in the deep freezer of passages 40, 80, 100 and 130 (T40, T80, T100 and T130 respectively), as well as T0. The inoculums were prepared using a previously published technique [9], and adjusted to 20 mg (wet weight) of Nocardia brasiliensis in 50 μL of saline solution. Female BALB/c mice, 8-12 weeks-old were injected with 50 μl of the nocardial suspension in the right footpad and the development of lesions was scored from 0 (for no inflammatory changes) to 4+ (extension of the lesions beyond the ankle of the animal with extensive production of inflammation and abscesses) as previously described [9]. The thickness of each lesion was measured with a caliper every week for 12 weeks. The study was approved by the Comité Local de Investigación en Salud No. 1908, Centro de Investigación Biomédica del Noreste, IMSS, and the animal handling was done according to our institutions' guidelines.
Induction of infection resistance in a murine model
In order to study if infection with subcultured N. brasiliensis produced a state of immune resistance we inoculated a group of animals with N. brasiliensis subcultured 130 times in the right foot pad. After 12 weeks the left footpad was inoculated with the non-subcultured bacteria (T0). As a control we inoculated a group of animals of the same age with the non-subcultured isolate in the right footpad. In all cases the development of lesions was scored and measured as described above.
Histopathological analysis comparing T0 and T130strains
For histopathological evaluation of the infection process we obtained biopsies of the inoculated footpad on weeks 1, 3, 5, 7, 9 and 12 post-inoculation in both groups: the T130 re-inoculated group and the control. Biopsies were stored in 4% formalin for further processing and staining with H&E, Kinyoun, and PAS. To identify the subsets of the inflammatory cells, the tissue samples were stained with antibodies against CD4 (helper T cells), CD8 (suppressor/cytotoxic T cells) and CD14 (monocytes). Briefly, sections from affected feet destined for immunohistochemistry were deparaffined, rehydrated and subjected to a sequence of incubation steps starting with sodium citrate (0.01 M) for epitope recuperation. After blocking endogenous peroxidase activity with 1% hydrogen peroxide in methanol, sections were incubated in a humidity chamber during 18 h at 4°C with polyclonal anti-CD4, anti-CD8 and anti-CD14 (Dako Corp., Carpinteria, CA) diluted 1:200 in PBS. Following rinses in PBS, sections were incubated for 20 min at room temperature with biotinylated goat anti-rabbit antibody, and diluted 1:500 in PBS. This was followed by rinses in PBS and 20 min humidity chamber incubation in streptavidin-biotin. Peroxidase activity was visualized by incubating the sections with 3,3',-diaminobenzidine and counterstaining with hematoxilin.
Statistical analysis
The statistical analysis was performed with Statgraphics under the heading of The StatAdvisor and also with SPSS, Sigma Plot and Excel 2007. For comparison between groups an ANOVA analysis was conducted and as an alternative a Kruskal-Wallis test was applied. A P < 0.05 was considered statistically significant.