Isolation of Lyme disease spirochetes from an ixodid tick in Hokkaido, Japan

Abstract

Several cases of erythema chronicum migrans (ECM) have been reported recently in Japan and these cases diagnosed as Lyme disease serologically (Kawabata et al., 1987; Sasa et al., 1988; Miyamoto et al., 1990). Epidemiologic evaluation of these cases suggested that the ixodid tick, Ixodes persulcatus, was involved in a vector. Lyme disease siprochetes, Borrelia burgdorferi, have been isolated from Ixodes dammini, L pacificus, L scapularis and Dermacentor variabilis in the U.S.A. and from L ricinus and L persulcatus in Europe (Anderson et al., 1985; Schmid, 1985; Korenberg et al., 1986; Piesman, 1989). Recently, the spirochetes were also detected in L persulcatus in the northern part of China (Ai et al., 1988) and in the Khabarovsk territory of the Soviet Union (Korenberg et al., 1989). As no report on the etiological agent of Japanese Lyme disease exists, we attempted to isolate spirochetes from the vector ticks and reservoir hosts. The ixodid ticks were collected from vegetation by dragging a cotton muslin sheet along and through the vegetation during late April to late July, 1989 in the cycling path of Asahikawa and the forest of Furano, Hokkaido, northern Japan. A total of 468 ticks were collected, 345 adult L persulcatus and 123 adult L ovatus. Of these, 152 ticks were used for the isolation of spirochetes. Before dissecting the ticks, they were dipped into 70% ethanol for 30 s, then rinsed with sterile saline. The washed ticks were placed in wells on a glass slide containing a small amount of BSK-I medium (Barbour et al., 1983a), and teased apart under a dissecting microscope. The midgut of each tick was inoculated into 6 ml of BSK medium within a culture tube. The cultures were incubated at 32°C for 6 weeks, and examined by dark-field microscopy at weekly intervals. Feral rodents were captured by Sharman box traps at four