Ten raccoon dogs (4 males and 6 femalee, body weight 4.8 ± 0.6 kg) were captured between March 2002 and February 2005. They were collected by trapping from the suburban areas of Gwangju-shi (Gwangju Metropolitan City), Damyang-gun, and Hampyong-gun, Jeollanam-do, Korea. They were brought to the Animal Shelter at the College of Veterinary Medicine, Chonnam National University, and were housed either in dog cages or confined to a room. Upon arrival, the body weight of each animal was measured, and water, commercial dog food, and chicken meat were provided to the animals ad libitum.

Fecal samples from 5 raccoon dogs were parasitologically examined using a modified version of Sheather's sugar centrifugal flotation technique (Sloss, 1994). Fecal samples that were infected with hookworms were mixed with granulated charcoal (Union Carbon Co., Ltd, Naju City, Korea) and cultured in glass jars in darkness at 25℃, in accordance with the methods described by Garcia and Ash (1975). Daily observations of cultures for adequate moisture, and the identification of larvae were conducted with the aid of both compound and dissecting microscopes. Infective larvae present in the mixed cultures were recovered using a Baermann's apparatus (Garcia and Ash, 1975).

The infective 2^nd stage larvae from the charcoal cultures were orally administered to 3 puppies, 3-month-old, helminth-free, mixed-bred, at a dosage of 100 larvae per dog. After 12 days, the dogs reached patency with monospecific hookworm infections. Adult A. miyazakiense worms were collected from the feces of these dogs, following deworming with a commercial anthelmintic (Drontal Plus, Bayer Animal Health, Leverkusen, Germany).

One of the 2 raccoon dogs that discharged hookworm eggs in the feces died soon after it was brought to the Animal Shelter. Numerous adult hookworms were collected in the duodenum of necropsied animal. Some worms collected from a necropsied raccoon dog and some from experimental puppies were fixed with 10% formalin, and then prepared as specimens for microscopic observations. They were identified based on the morphologic characteristics described by Shimada (1979) and Yoshida and Arizono (1976).

The microculture slides used to observe the development of the larvae were prepared using a microscope slide glass (75 × 25 mm) on which a cover glass (22 × 40 mm) was mounted at a height of 0.8 mm, using a couple of polypropylene bars (0.8 × 1.0 × 22 mm) fixed with a silicone-based sealant (Park et al., 2005). The microculture cell space between the slide glass and cover glass held approximately 700 µl of medium. Mature eggs of A. miyazakiense harvested from the feces of infected animals were washed in water and pipetted into the cell space, after which each microculture slide was placed into a disposable petri dish (87 × 15 mm, Green Cross Medical, Inc., Seoul, Korea). Three layers of paper towel wet with deionized distilled water were positioned in the bottoms of the covered Petri dishes in order to provide humidity, and to minimize the evaporation of the culture medium. A couple of wood sticks were placed between the wet paper towel and the microculture slide. Petri dishes with microculture slides were maintained at room temperature (23-25℃). The culture slides were exposed to fluorescent lighting at 400 lux illumination for 12 hr everyday.

Four species of helminth eggs and 1 species of protozoan cyst were detected by the fecal examination of 5 raccoon dogs. Two of the 5 raccoon dogs discharged the eggs of hookworms, Toxocara sp. and Trichuris sp. in their feces. Capillaria sp. eggs and Giardia sp. cysts were observed in the feces of 3 animals (Table 1).

Adult hookworms collected from a necropsied raccoon dog and experimental puppies were whitish in color, rather small and slender, with cuticles striated finely in a transverse direction, and the lengths of the female and male worms were 6.0-8.0 mm and 4.7-5.9 mm, respectively (Fig. 1; Table 2). The mouth part of adult worms had the following features: anterodorsal, buccal capsule composed of 10 articulated plates which included 1 basal, 1 ventral, 2 ventrolateral, 2 lateral, 2 dorsolateral, and 2 mediolateral plates (Fig. 2). The buccal capsule did not harbor cutting plates or teeth, but had a vertebra-shaped basal plate and a ventral pair of conspicuous lancets. Prevulval papillae were observed right-sidedly (Fig. 3). Eggs were 60-65 × 35-40 µm (average, 62.5 × 35 µm) in size, and was indistinguishable morphologically with Ancylostoma caninum (Fig. 4).

Upon incubation of eggs at room temperature, eggs became embryonated within 30 hr and subsequently hatched within 48 hr (Table 3). Only 54% of embryonated eggs were hatched by the 2^nd day of culture, when the eggs were incubated at 25℃. All eggs cultured at 30℃ hatched and became 1st stage larvae within 2 days (data not shown). The average length of the first-stage larvae at the time of hatching was 325 µm (range: 320-330 µm) and had a well-developed digestive system, mouth, oesophagus, and anus.

The 2^nd stage larvae were observed on the 3rd day of culture. The length of larvae was 505-695 µm (av. 638.1 ± 100.4 µm). When we infected to puppies on 14th day of culture, the range of the larval length was 620-720 µm (av. 650 µm) and was ensheathed (Fig. 5). Upon incubation of eggs at room temperature, eggs became embryonated within 30 hr and subsequently hatched within 48 hr (Table 3). The size of larvae grew above 600 µm within 6 days of culture to become an infective stage (Table 4; Fig. 5). Oral inoculation of 2-weeks old infective larvae from the charcoal culture into 3 helminth-free dogs resulted in patency at 12th day after infection, as evidenced by the presence of hookworm eggs in the feces.