INTRODUCTION

Mesocestoides spp. are members of the tapeworm order Cyclophyllidea. However, they have some different characters from other groups of cyclophyllideans. In their life cycle, 3 hosts including 2 intermediate and 1 definitive hosts, are required. Adult worms live in the small intestines of carnivorous mammals, i.e., the dog, cat, fox, wolf, coyote, jackal, raccoon, badger, lynx, and some species of wild felines [1-11], and rarely birds and humans [12-17]. The tetrathyridium, the metacestode of Mesocestoides spp., is found in the abdominal cavity of a great variety of intermediate hosts, such as amphibia, reptiles, birds, and mammals [18,19]. Meanwhile, the role of coprophagic arthropods as its first intermediate host is presumed as Padgett and Boyce [20] detected Mesocestoides sp. DNA from ants. There were experimental trials to infect insects with eggs; however, enough evidence has not yet been obtained [21]. Morphologically, the median ventral position of the genital atrium and bipartite vitelline gland in these tapeworms are unique and differ from other groups of cyclophyllideans [22].

In the Republic of Korea (=Korea), Kobayashi [23] reported adult Mesocestoides infection from dogs in Seoul in 1928. Thereafter, Cho et al. [24] in 1982 detected tetrathyridia in a snake, Elaphe rufodorsata, from Gangwon-do [24]. Two cases of human infections with adult Mesocestoides lineatus were reported by Choi et al. [25] and Eom et al. [16]. However, there have been few studies on the life cycle of Mesocestoides tapeworms in Korea. Moreover, there is no information on biological relationships between the tetrathyridia in intermediate hosts and the adults in definitive hosts. In the present study, we succesfully obtained adult tapeworms of M. lineatus from 2 hamsters and a dog which were experimentally infected with tetrathyridia collected from Chinese snakes. The morphological characteristics of the tetrathyridia and adult tapeworms are described herein.

MATERIALS AND METHODS

A substantial number of snakes obtained from the Incheon customs officer were transferred to the Parasitology Laboratory of Gyeongsang National University School of Medicine in November, 2003. Among them, 421 snakes which included Agkistrodon saxatilis (n=60), Elaphe schrenckii (n=80), Dinodon rufozonatum (n=120), Agkistrodon brevicaudus (n=111), and Elaphe davidi (n=50) were examined to detect parasitic helminths including tetrathyridial larvae (Table 1). The muscle and viscera of each snake were isolated after skinned off and artificially digested with pepsin-HCl solution in an incubator at 36℃ for 2-5 hr. The digested material was washed with 0.85% saline until the supernatant became clear, and the sediments were examined under a stereomicroscope. Some collected tetrathyridia were fixed in 10% formalin under a cover glass pressure, and 5-10 larvae per animal were orally infected to 3 rats, 5 hamsters, 2 cats, and 2 dogs to obtain the adult Mesocestoides tapeworm. About 1 month later, experimental animals were killed after anesthesia, and their intestines were isolated and longitudinally opened with a pair of scissors in a beaker with 0.85% saline. The intestinal contents of each animal were washed with 0.85% saline until the supernatant cleared, and the sediments were examined with naked eyes and under a stereomicroscope. The adult tapeworms were fixed in 10% formalin under a slide glass pressure. The tetrathyridia and adults fixed with 10% formalin were stained with Semichon's acetocarmine and observed using a light microscope with a micrometer.

In order to observe the surface ultrastructure of the tetrathyridium, some worms were washed several times in 0.2 M cacodylate buffer (pH 7.2) and fixed in 2.5% glutaraldehyde at 4℃. After washing 3 times with the same buffer, they were dehydrated through a graded alcohol series (50%, 70%, 80%, 90%, 95%, and absolute alcohol), dried in a critical point dryer, coated with gold in the JFC-1100E ion sputtering device (JEOL, Tokyo, Japan), and observed using a scanning electron microscope (Philips XL-30S, Amsterdam, Netherlands) with an accelerating voltage of 20 kV.

RESULTS

DISCUSSION

In the present study, tetrathyridia of M. lineatus were detected in 2 species of snakes, A. saxatilis and E. schrenckii, from China. No tetrathyridia were found in the other 3 snake species, D. rufozonatum, A. brevicaudus, and E. davidi. However, these 3 snake species have been reported as the intermediate hosts for Gnathostoma hispidum [26,27]. Generally, foodborne parasite infections are related to the food-chain in the natural ecosystem. Snakes, as the predator or a transport host, are infected with gnathostome larvae or tetrathyridia by preying upon the other intermediate or transport hosts, such as amphibians, reptiles, avians, and small mammals. How are the real intermediate hosts infected with the tetrathyridia of Mesocestoides spp. then? It is difficult to answer because of the link between the eggs and tetrathyridia (the first intermediate host necessary?) is obscure in the life cycle of Mesocestoides spp. However, tetrathyridia have been detected in various kinds of animals, i.e., 22 reptilian spp., 15 avian spp., and 20 mammalian spp. [18,19]. Therefore, snakes as an upper-level predator may be easily infected by feeding their prey, i.e., reptiles, birds, and small mammals. As the snake intermediate hosts, Agkistrodon halys and E. rufodorsata were reported in Japan and Korea [24,28].

So far, about 30 human infections with Mesocestoides spp. adults have been documented in Japan, China, Korea, USA, Ruanda-Urundi, and Greenland [14-17]. Human infections are acquired by ingesting raw wild animals containing tetrathyridia. Several species of snakes, such as A. brevicaudus, A. halys, Elaphe quadrivirgata and E. rufodorsata, and chickens were reported as the source of human infections in Japan and Korea [14,16,24,25,28].

Adult Mesocestoides tapeworms have been found in surveys of carnivorous mammals, such as, dogs, cats, foxes, wolves, coyotes, jackals, raccoons, badgers, lynxes, and some species of wild felines [1-11]. Among the carnivorous mammals, foxes were the most frequently examined at various localities of the world and their infection rate with Mesocestoides spp. was relatively high [9,29-31]. Recently, Hrčkova et al. [9] detected Mesocestoides tapeworms from 41.9% out of 3,157 red foxes in central Europe. Magi et al. [29] found M. lineatus in 45.4% of 129 red fox from central Italy. Saeed et al. [30] also detected Mesocestoides sp. tapeworms from 35.6% of 1,040 red foxes in Denmark. In these endemic areas, foxes play a crucial role as the definitive host in maintaining the life cycle. What kinds of wild animals are the natural definitive hosts for M. lineatus in Korea? Adult Mesocestoides tapeworms were found in dogs and 2 human cases in Korea [16,23,25]; however, no reports are available on wild carnivorous mammals. Hence, keen observations are needed for detection of Mesocestoides tapeworms in surveys of wild carnivorous mammals in Korea.

To obtain adult worms, we orally infected 5-10 tetrathyridial larvae to 3 rats, 5 hamsters, 2 cats and 2 dogs. However, no worms were recovered in 3 rats, 3 hamsters, 2 cats, and 1 dog, and the fate of uninfected larvae is uncertain. Most of them may have died and passed out of the digestive tract with feces. However, some survived larvae might have migrated into the other organs and provoked the tetrathyridiasis like the canine peritoneal larval cestodosis (CPLC) [19]. Unfortunately, however, we examined only the small intestine of experimental animals to detect adult worms. It is a new finding in this study that hamsters are an experimental definitive host for M. lineatus.

Mesocestoides sp. tapeworms have morphological characteristics, such as the median ventral position of the genital atrium and bipartite vitelline gland in the mature proglottid and a paruterine organ in the gravid proglottid, which differ from those of other cyclophyllidean tapeworms. Especially, the paruterine organ in gravid proglottids is unique and regarded as a diagnostic key in the subfamily Mesocestoidinae [22]. On the other hand, morphological characteristics of adult worms observed in the present study were identical with those of M. lineatus, which were previously reported by Kamegai et al. in 1967 [32] and Eom et al. in 1992 [16] (Table 3). Morphological characteristics of the metacestode and adult of M. lineatus observed in the present study will be helpful in taxonomic studies of Mesocestoides tapeworms.