Effectiveness of photo-ozone therapy against equine Pythium insidiosum

Cutaneous pythiosis is a life-threatening infectious disease. Low-level laser therapy (LLLT) and ozone (O₃) have been used individually in the treatment of infected wounds. The goals of the study were a) to characterize the antimicrobial action of the photo-ozone therapy (LLLT-O₃) against equine Pythium insidiosum, and b) to assess the cytotoxic potential of the LLLT-O₃ in keratinocytes. Specimens of pathogen were isolated from 10 horses. After culturing, 120 hyphae plugs were distributed among four groups (n=30 hyphae plugs/group): LLLT (laser irradiation for 160 sec;), O₃ (exposition to O₃ for 15 min;), LLLT-O₃ (LLLT and O₃ treatments in sequence) and control (untreated plugs). The hyphae growth was measured during the first 14 days post-treatment. Where there was an absence of hyphae growth, the plug was recultured for an additional 7 days. The cytotoxic potential of the treatments against HaCaT keratinocytes was assessed by colorimetric assays. The LLLT-O₃ and O₃ treatments inactivated, respectively, 92.3% (28/30) and 30% (9/30) of the samples. No growth was detected after 7 days reculture of inactivated hyphae plugs on new media. Hyphae growth was visualized in 100% of the control and LLLT hyphae plugs. The viability of HaCaT cells was not affected by the isolated treatments (LLLT and O₃), while the LLLT-O₃ showed slight cytotoxic effect (20%) when compared to the control group (P<0.05). Photo-ozone therapy inactivated equine P. insidiosum hyphae with minimal cytotoxicity in skin cells in vitro.