ABSTRACT. The antifungal activity of β-thujaplicin was evaluated against 51 Malassezia pachydermatis strains isolated from canine ear canals with or without otitis externa. For comparison, sensitivity tests were performed on M. pachydermatis isolates for nystatin, ketoconazole, and terbinafine HCl, all clinically available antifungal agents. The minimal inhibition concentrations over 50% of the tested isolates (MIC 50 ) were 3.13 µg/ml for β-thujaplicin and nystatin, 0.016 µg/ml for ketoconazole, and 1.56 µg/ml for terbinafine HCl. The antifungal effect for M. pachydermatis of β-thujaplicin compared favorably with commercial antifungal agents. None of the 51 M. pachydermatis isolates showed resistance against any of the tested antibiotics investigated in this study. Ten representative isolates of M. pachydermatis were subcultured for 30 generations at concentrations close to the MIC levels of β-thujaplicin, nystatin, ketoconazole, and terbinafine HCl, and examined to determine whether they had acquired resistance to each drug. As a result, M. pachydermatis was found to achieve resistance more easily for ketoconazole and terbinafine HCl than for β-thujaplicin or nystatin. The MIC 50 of β-thujaplicin did not change during the course of subculture, and it is thought that the potential development of a resistant strain is low, even with continuous infusion for otitis externa therapy. β-Thujaplicin is an inexpensive and safe treatment with anti-inflammatory and deodorant effects that can be recommended as an effective remedy for canine otitis externa.KEY WORDS: β-thujaplicin, canine, Malassezia pachydermatis, otitis externa, sensitivity test.J. Vet. Med. Sci. 67(12): 1243-1247, 2005 Otitis externa is one of the most frequently occurring canine diseases [2,3], and although it is not lethal, it is a chronic disease that troubles both dogs and owners for a prolonged period of time [1]. Malassezia pachydermatis is the most common microorganism isolated from canine otitis externa cases and even from healthy ear canals [12]. Seven species, M. furfur, M. globosa, M. obtusa, M. restricta, M. slooffiae, M. sympodialis, and M. pachydermatis belong to the genus Malassezia, but 6 of the species, all except M. pachydermatis, are characterized by a lipid being required for growth [6]. As M. pachydermatis is a major component of normal canine microbial flora, infections of the canine ear canal must be opportunistic to multiply when the microenvironment changes [1,18]. M. pachydermatis-related otitis externa is very difficult to control, with repeated recurrences, and requires long-term antifungal agent therapy. Recently, the appearance of multidrug-resistant microbes due to long-term pharmaceutical treatment has come a problem for human and veterinary clinical practices, and drug resistance for the genus Candida has been reported [19]. The appearance of resistant strains of M. pachydermatis is a concern. Azoles, such as ketoconazole and itraconazole, polyene derivatives, such as nystatin, and allylamines, such as terbinafine HCl, a...