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Development of Centrocestus armatus in different final hosts
Veterinary Parasitology 146 (2007) 367–371 www.elsevier.com/locate/vetpar
Short communication
Development of Centrocestus armatus in different final hosts Daisuke Kimura *, Vachel Gay Paller, Shoji Uga Department of Medical Technology, Faculty of Health Sciences, Kobe University School of Medicine, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142, Japan Received 26 July 2005; received in revised form 26 February 2007; accepted 26 February 2007
Abstract In this study, Centrocestus armatus metacercariae were fed orally to hamsters, albino rats, mice, and chicks. Animals were sacrificed and dissected at 1, 3, 5, 7, and 14 days post-infection to determine the development and recovery rate of worms. Results indicated that the average worm recovery rate in hamsters was 25% on the first day post-infection and recovery continued until the 14th day with a gradual decrease in the percentage. Worms were also recovered from mice and albino rats from the first until the third day post-infection, but no worms were recovered thereafter. In chicks, worms were not observed on first day but recovery was positive at 12 h post-infection. Among the four animal hosts, feces from hamsters were the only ones positive for eggs; these were initially observed from the third day and recovery continued until 14 days post-infection. In our study, hamsters are the animal model most suitable for the study of C. armatus when compared to rats, mice, and chicks. # 2007 Elsevier B.V. All rights reserved. Keywords: Centrocestus armatus; Development; Suitable host
1. Introduction Centrocestus armatus was first reported as Stamnosoma armatum by Tanabe in 1922. Chapin (1926) and Price (1932) later clarified that the genus Stamnosoma was synonymous to the genus Centrocestus. This minute intestinal trematode belongs to the family Heterophyidae and lives in the small intestines of fisheating birds and mammals including man. Natural human infections from three species Centrocestus spp. have been reported: C. formosanus var. kurokawai (Kurokawa, 1935) in Japan, C. caninus (Waikagul, 1997) in Thailand, and C. armatus (Hong et al., 1988) in Korea. These species can be distinguished by the
* Corresponding author. Present address: Division of Immunology, Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. Tel.: +81 95 849 7072; fax: +81 95 849 7072. E-mail address: [email protected] (D. Kimura). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.02.032
number of circumoraol spine and/or second intermediate fish. About 20 species of fresh water fish such as Zacco platypus, Pseudorasbora parva, and Carassius carassius are known to act as second intermediate hosts for C. armatus (Tanabe, 1922). Of them, Z. platypus is reported to be heavily infected with C. armatus metacercariae (Hong et al., 1989). The same phenomenon was also observed in our survey area (Kimura and Uga, 2003). The family Heterophyidae, such as Metagonimus spp., are recognized as important human pathogenic parasites. Clinical manifestations of the Metagonimus infection are colicky pain, abdominal tenderness, and mucous. Occasionally, if the intensity of the infection is high, bloody diarrhea can also be evident. Therefore, it is important to conduct studies to obtain information about these parasites. However, it is difficult to use a particular species of Metagonimus spp. for experimental purposes since the taxonomy of this genus is still under discussion (Saito et al., 1997; Lee et al., 2004).
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D. Kimura et al. / Veterinary Parasitology 146 (2007) 367–371
Centrocestus spp. presents some advantages over the Metagonimus species. First, collection of the parasites is easier due to its countrywide distribution (Kagei and Yanohara, 1995; Kimura and Uga, 2003); second, albino rats and hamsters have been reported to be suitable hosts; and third, development in the host body is relatively short (usually 3 days) (Hong et al., 1989). It is considered that these advantages are useful for of its use in an experimental model for these parasites belonging to the family Heterophyidae to observe developmental dynamics and/or morphological changes. The present study was conducted to identify a suitable animal host for the development of C. armatus under laboratory conditions. In addition to rat and hamster that have reported as infected with this fluke, we selected mice as they are the most frequently used laboratory animal and chicks (it is reported that a bird is a final host of this fluke; Yamaguti, 1958). Observations of the development and morphology of C. armatus in four different animal hosts were conducted to better understand host–parasite relationships. 2. Materials and methods 2.1. Recovery of metacercariae Fish caught from the Chikusa River were digested for 1 h using a digest solution (0.1% each of pepsin and HCl in distilled water) at 37 8C, and the solution then centrifuged at 150 g for 5 min. C. armatus metacercariae were collected under a stereoscopic microscope. Only mature metacercariae with an ‘‘X’’-shaped excretory bladder were collected for further study. 2.2. Final hosts Hamsters (Golden hamster, 8–16 weeks old females), mice (ICR strain, 6–8 weeks old females), rats (Wister strain, 4–6 weeks old females), and chicks (40 days old females) were used as experimental final hosts. Hamsters, mice, and rats, three to five animals of each, were purchased from a vendor (Japan SLC, Tokyo, Japan) and used for the experiments. For the recovery of adult worms, about 10–30 C. armatus metacercariae per gram of body weight were fed orally to the four different final hosts. 2.3. Recovery and observation of worms Parasites were recovered from the small intestine of each host at 1, 3, 5, 7, and 14 days post-infection. At the time of recovery, animals were humanely euthanized
and small intestines taken out. Intestines were divided into upper, middle, and lower regions and the number of worms in each region recorded. The parasites were observed using a light microscope (OPTIPHOT, Nikon, Tokyo, Japan) and a scanning electron microscope (SEM) (T-330A, Jeol Ltd., Tokyo, Japan). For the light microscopic observations, parasites were stained with acetocarmine. For the scanning electron microscopic observations, parasites were fixed with 10% neutral buffered formalin, and dehydrated in a graded series of ethyl alcohol. All specimens were dried in a criticalpoint dryer (HCP-2, Hitachi, Tokyo, Japan), and coated with gold (IB-3, Eiko, Tokyo, Japan) according to Uga et al. (1998). 3. Results 3.1. Transition of the numbers of eggs in feces Numbers of eggs appearing in feces in each host are shown in Fig. 1. In hamster, eggs were initially observed in feces on the third day. The number of eggs excreted reached a peak (n = 7) on the seventh day and started to decline thereafter. Contrarily, no eggs were found from the other three hosts. Examinations of feces from these hosts were terminated on day 7 since adult worms of these parasites had disappeared at 1–5 days postinfection. 3.2. Recovery of worms The recovery rates of worms from different hosts after infection are shown in Fig. 2. About 25% (368/1452) of the worms were recovered from hamsters on the first day and then decrease gradually to 8% (109/1400) at the seventh day. Worms were collected from mice and rats until the third day post-infection but no worms were recovered thereafter. In chicks, about 4% (82/2024) worms were recovered at 12 h post-infection but no worm was observed thereafter. On the first day, adult worms were recovered from the upper, middle and lower regions of the small intestines irrespective of suitable or unsuitable hosts. However, on the third day, 81% (362/ 445) of worms were recovered from the upper region. 3.3. Light microscopic observations Fig. 3a shows an adult C. armatus worm collected from a hamster at 7 days post-infection. Measurements of the various vital organs of the adult worms are shown in Table 1. Although body width increased, body length hardly increased with the number of the post-infection
D. Kimura et al. / Veterinary Parasitology 146 (2007) 367–371
369
Fig. 1. Number of eggs recovered from feces in different intervals of post-infection. Data were shown in average of three hamsters, four mice, and each of four rats and chicks.
day. The ovary (OV) was not recognizable on the first day but became conspicuous on the third day. The seminal vesicle (SV) and seminal receptacle (SR) were also initially observed on the third day. Testis (TT) was recognized in all worms, but the left testis grew to be larger than the right. This phenomenon was observed throughout the observation period. Intrauterine eggs (EG) were observed in hamsters and mice from the third day. In hamsters, the number of eggs reached its peak (15) on the seventh day and decreased thereafter until the 14th day (11). This trend corresponds with that in Fig. 1, where the number of eggs recovered from feces peaked on the seventh day and subsequently decreased thereafter. The oral sucker (OS) measured 46 mm on average and always exceeded the size of the ventral sucker (VS), which was 34 mm on average. No size increase in the oral sucker was observed from the seventh day; however, the size of the ventral sucker
gradually increased as the number of post-infection days increased. Therefore, the ratio of the oral to ventral sucker always exceeded a value of one. 3.4. Observations by scanning electron microscopy The tegmental spines of the adult worms (seventh day) appeared as sawtooth-like structures covering the entire dorsal surface of the body (Fig. 3e). The oral sucker (OS) was located at the most anterior edge part of the worm (Fig. 3b) with 44 circumoral spines (CS), measuring 1 mm 2 mm, and aligned alternately in two rows on its circumference (Fig. 3c). A number of sensory papillae (SP) (arrows) were found surrounding the oral sucker. The ventral sucker (VS) was located at the mid-anterior one-third of the body (Fig. 3b). About six sensory papillae were also observed around the circumference of the ventral sucker (Fig. 3d).
Fig. 2. Comparison of recovery rate of Centrocestus armatus from different animal. Vertical bar indicates S.D.
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D. Kimura et al. / Veterinary Parasitology 146 (2007) 367–371
Fig. 3. Light microscopic (a) and scanning electron microscopic (b–e) photographs of C. armatus adult worms. OS: oral sucker, PH: pharynx, IT: intestinal tract, VS: ventral sucker, SV: seminal vesicle, EG: egg, OV: ovary, SR: seminal receptacle, TT: testis, CS: circumoral spine. (a) A 7-day-old worm stained with acetocarmine. (b) Ventral view of the whole body. (c) Oral sucker and sensory papillae (arrows). (d) Ventral sucker and six sensory papillae (arrows) on the rim of acetabulum. (e) Tegmental spines on ventral surface.
4. Discussion In this study, four different species of experimental animals were used to assess a suitable host for C. armatus. Hong et al. (1989) observed the development of C. armatus in albino rat and noted that worms were collected at a ratio of 0–17% between 1 and 28 days of
post-infection. By contrast, in this study we were able to collect adult worms at 15% recovery rate on the first day post-infection but this subsequently decreased until the third day. No worms were recovered on the fifth day in rats. The discrepancy between Hong’s report (1989) and the present study is not clear since the same age and strain of rats was used in both experiments. However, aspects such as different physiological conditions,
Table 1 Measurement of Centrocestus armatus Days postinfection
H
No. of parasites
Body
Ovary
Seminal vesicle
Seminal receptacle
Right test is
Left testis
Intrauterine eggs
La
Wb
L
W
L
W
L
W
L
W
L
W
13 14 16 6 12
420 424 461 397 404
106 109 121 147 171
– 38 47 49 51
– 30 37 39 47
– 84 115 140 161
– 16 22 24 27
– 25 35 40 34
– 24 30 35 31
44 42 50 55 62
26 29 26 36 40
56 54 65 66 71
29 31 32 41 42
– +7 +13 +15 +11
3 3
315 388
85 88
– 24
– 30
– 135
– 35
– 23
– 23
36 30
21 24
46 39
25 19
– +9
c
1 3 5 7 14 Md 1 3 a b c d
Length (mm). Width (mm). Hamster. Mouse.
D. Kimura et al. / Veterinary Parasitology 146 (2007) 367–371
health status and nutrition of the rats along with different laboratory conditions may explain the discrepancy. Mice however showed a relatively high recovery at 50% on the first day but decreased abruptly to about 6% on the third day. On the other hand, hamsters showed a recovery rate of 25% on the third day and gradually decreased thereafter, but persisted until 14 days post-infection. The same trends have been observed in related studies of Metagonimus spp. and Pygidiopsis spp. (Chen, 1942; Chai, 1979). Hong et al. (1989) also mentioned that if susceptibility of a host animal to an intestinal fluke is low, their recovery rate decreased rapidly compared with other animals with relatively higher susceptibilities. Although the recovery rate of worms in hamster ranges only from 8 to 25%, among the different animal hosts tested we considered it a suitable host of C. armatus, whereas the other animals tested were unsuitable hosts. Ovary was initially observed in front of the right testis on the third day. By contrast, Hong et al. (1989) reported, that ovary was observed on the first day postinfection. The reason for this discrepancy is not clear. In our study, the testis was the only genital organ recognized in worms on the first day post-infection. It is interesting to note that there was no increase in size in the oral sucker throughout the observation period. Probably because the oral sucker has already reached its mature size during the metacercaria stage since it is a very important organ for the parasitic mode of living. The recovery of eggs from feces of hamster from the third day post-infection could be considered relatively early compared to other flukes such as Clonorchis sp. and Paragonimus sp., which require 1 month post-infection. On the other hand, feces from mice, albino rat, and chick were examined for the presence of eggs until the seventh day post-infection, but no eggs were recovered. Even though intrauterine eggs were observed in C. armatus on third day of post-infection, no eggs were recovered from mice feces. This was probably due to the very low worm recovery rate at 3 days post-infection, and thus a very limited number or no eggs at all could be found in feces. Under the above conditions the parasites may not be able to continue their life cycle, further confirming that rats and mice should be considered unsuitable hosts for C. armatus development. The study clearly revealed that
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hamsters, compared with rats, mice, and chicks, are the most suitable animal model for the study of C. armatus. References Chai, J.Y., 1979. Study on Metagonimus yokogawai (Katsurada, 1912) in Korea. V. Intestinal pathology in experimentally infected albino rats. Seoul J. Med. 20, 104–117. Chapin, E.A., 1926. Note on the Heterophyidae. J. Parasitol. 12, 180. Chen, H.T., 1942. The metacercaria and adult of Centrocestus formosanus (Nishigori, 1924), with notes on the natural infection of rats and cats with C. armatus (Tanabe, 1922). J. Parasitol. 28, 285– 298. Hong, S.J., Seo, B.S., Lee, S.H., Chai, J.Y., 1988. A human case of Centrocestus armatus infection in Korea. Kor. J. Parasitol. 26, 55– 60. Hong, S.J., Woo, H.C., Chai, J.Y., Chung, S.W., Lee, S.H., Seo, B.S., 1989. Study on Centrocestus armatus in Korea. II. Recovery rate, growth and development of worms in albino rats. Kor. J. Parasitol. 27, 47–56. Kagei, N., Yanohara, Y., 1995. Epidemiological study on Centrocestus formosanus (Nishigori, 1924)—surveys of its infection in Tanegashima, Kagoshima Prefecture, Japan. Jpn. J. Parasitol. 44, 154– 160. Kimura, D., Uga, S., 2003. Epidemiological studies on Centrocestus spp. (Trematoda: Heterophyidae) in Chikusa River basin, Hyogo Prefecture: infection in the 1st intermediate host snail, Semisulcospira libertina. Jpn. J. Environ. Entomol. Zool. 14, 97–103 (in Japanese with English summary). Kurokawa, T., 1935. On a new trematode of genus Stamnosoma proved from a man. Tokyo Iji Shinji 2915, 293–298 (in Japanese). Lee, S.U., Huh, S., Sohn, W.M., Chai, J.Y., 2004. Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai. Kor. J. Parasitol. 42, 129–135. Price, E.W., 1932. On the genera Centrocestus Looss and Stamnosoma Tanabe. J. Parasitol. 18, 309. Saito, S., Chai, J.Y., Kim, K.H., Lee, S.H., Rim, H.J., 1997. Metagonimus miyatai sp. nov (Digenea: Heterophyidae), a new intestinal trematode transmitted by freshwater fishes in Japan and Korea. J. Parasitol. 35, 223–232. Tanabe, H., 1922. Studien uber die trematoden mit Susswasserfischen als Zwischenwirt. I. Stamnosoma armatum n.g., n.sp. Kyoto Igaku Zasshi 19, 239–252 (in Japanese). Uga, S., Morimoto, M., Sato, T., Rai, S.K., 1998. Surface ultrastructure of Heterophyes heterophyes (Trematoda: Heterophyidae) collected from a man. J. Helminthol. Soc. Wash. 65, 119– 122. Waikagul, J., 1997. Human infection of Centrocestus caninus in Thailand. Southeast Asian J. Trop. Med. Public Health. 28, 831–835. Yamaguti, S., 1958. Systema Helminthum, vol. I: Digenetic Trematodes of Vertebrates, pp. 705–707, 872–873.
Short communication
Development of Centrocestus armatus in different final hosts Daisuke Kimura *, Vachel Gay Paller, Shoji Uga Department of Medical Technology, Faculty of Health Sciences, Kobe University School of Medicine, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142, Japan Received 26 July 2005; received in revised form 26 February 2007; accepted 26 February 2007
Abstract In this study, Centrocestus armatus metacercariae were fed orally to hamsters, albino rats, mice, and chicks. Animals were sacrificed and dissected at 1, 3, 5, 7, and 14 days post-infection to determine the development and recovery rate of worms. Results indicated that the average worm recovery rate in hamsters was 25% on the first day post-infection and recovery continued until the 14th day with a gradual decrease in the percentage. Worms were also recovered from mice and albino rats from the first until the third day post-infection, but no worms were recovered thereafter. In chicks, worms were not observed on first day but recovery was positive at 12 h post-infection. Among the four animal hosts, feces from hamsters were the only ones positive for eggs; these were initially observed from the third day and recovery continued until 14 days post-infection. In our study, hamsters are the animal model most suitable for the study of C. armatus when compared to rats, mice, and chicks. # 2007 Elsevier B.V. All rights reserved. Keywords: Centrocestus armatus; Development; Suitable host
1. Introduction Centrocestus armatus was first reported as Stamnosoma armatum by Tanabe in 1922. Chapin (1926) and Price (1932) later clarified that the genus Stamnosoma was synonymous to the genus Centrocestus. This minute intestinal trematode belongs to the family Heterophyidae and lives in the small intestines of fisheating birds and mammals including man. Natural human infections from three species Centrocestus spp. have been reported: C. formosanus var. kurokawai (Kurokawa, 1935) in Japan, C. caninus (Waikagul, 1997) in Thailand, and C. armatus (Hong et al., 1988) in Korea. These species can be distinguished by the
* Corresponding author. Present address: Division of Immunology, Department of Molecular Microbiology and Immunology, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. Tel.: +81 95 849 7072; fax: +81 95 849 7072. E-mail address: [email protected] (D. Kimura). 0304-4017/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2007.02.032
number of circumoraol spine and/or second intermediate fish. About 20 species of fresh water fish such as Zacco platypus, Pseudorasbora parva, and Carassius carassius are known to act as second intermediate hosts for C. armatus (Tanabe, 1922). Of them, Z. platypus is reported to be heavily infected with C. armatus metacercariae (Hong et al., 1989). The same phenomenon was also observed in our survey area (Kimura and Uga, 2003). The family Heterophyidae, such as Metagonimus spp., are recognized as important human pathogenic parasites. Clinical manifestations of the Metagonimus infection are colicky pain, abdominal tenderness, and mucous. Occasionally, if the intensity of the infection is high, bloody diarrhea can also be evident. Therefore, it is important to conduct studies to obtain information about these parasites. However, it is difficult to use a particular species of Metagonimus spp. for experimental purposes since the taxonomy of this genus is still under discussion (Saito et al., 1997; Lee et al., 2004).
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D. Kimura et al. / Veterinary Parasitology 146 (2007) 367–371
Centrocestus spp. presents some advantages over the Metagonimus species. First, collection of the parasites is easier due to its countrywide distribution (Kagei and Yanohara, 1995; Kimura and Uga, 2003); second, albino rats and hamsters have been reported to be suitable hosts; and third, development in the host body is relatively short (usually 3 days) (Hong et al., 1989). It is considered that these advantages are useful for of its use in an experimental model for these parasites belonging to the family Heterophyidae to observe developmental dynamics and/or morphological changes. The present study was conducted to identify a suitable animal host for the development of C. armatus under laboratory conditions. In addition to rat and hamster that have reported as infected with this fluke, we selected mice as they are the most frequently used laboratory animal and chicks (it is reported that a bird is a final host of this fluke; Yamaguti, 1958). Observations of the development and morphology of C. armatus in four different animal hosts were conducted to better understand host–parasite relationships. 2. Materials and methods 2.1. Recovery of metacercariae Fish caught from the Chikusa River were digested for 1 h using a digest solution (0.1% each of pepsin and HCl in distilled water) at 37 8C, and the solution then centrifuged at 150 g for 5 min. C. armatus metacercariae were collected under a stereoscopic microscope. Only mature metacercariae with an ‘‘X’’-shaped excretory bladder were collected for further study. 2.2. Final hosts Hamsters (Golden hamster, 8–16 weeks old females), mice (ICR strain, 6–8 weeks old females), rats (Wister strain, 4–6 weeks old females), and chicks (40 days old females) were used as experimental final hosts. Hamsters, mice, and rats, three to five animals of each, were purchased from a vendor (Japan SLC, Tokyo, Japan) and used for the experiments. For the recovery of adult worms, about 10–30 C. armatus metacercariae per gram of body weight were fed orally to the four different final hosts. 2.3. Recovery and observation of worms Parasites were recovered from the small intestine of each host at 1, 3, 5, 7, and 14 days post-infection. At the time of recovery, animals were humanely euthanized
and small intestines taken out. Intestines were divided into upper, middle, and lower regions and the number of worms in each region recorded. The parasites were observed using a light microscope (OPTIPHOT, Nikon, Tokyo, Japan) and a scanning electron microscope (SEM) (T-330A, Jeol Ltd., Tokyo, Japan). For the light microscopic observations, parasites were stained with acetocarmine. For the scanning electron microscopic observations, parasites were fixed with 10% neutral buffered formalin, and dehydrated in a graded series of ethyl alcohol. All specimens were dried in a criticalpoint dryer (HCP-2, Hitachi, Tokyo, Japan), and coated with gold (IB-3, Eiko, Tokyo, Japan) according to Uga et al. (1998). 3. Results 3.1. Transition of the numbers of eggs in feces Numbers of eggs appearing in feces in each host are shown in Fig. 1. In hamster, eggs were initially observed in feces on the third day. The number of eggs excreted reached a peak (n = 7) on the seventh day and started to decline thereafter. Contrarily, no eggs were found from the other three hosts. Examinations of feces from these hosts were terminated on day 7 since adult worms of these parasites had disappeared at 1–5 days postinfection. 3.2. Recovery of worms The recovery rates of worms from different hosts after infection are shown in Fig. 2. About 25% (368/1452) of the worms were recovered from hamsters on the first day and then decrease gradually to 8% (109/1400) at the seventh day. Worms were collected from mice and rats until the third day post-infection but no worms were recovered thereafter. In chicks, about 4% (82/2024) worms were recovered at 12 h post-infection but no worm was observed thereafter. On the first day, adult worms were recovered from the upper, middle and lower regions of the small intestines irrespective of suitable or unsuitable hosts. However, on the third day, 81% (362/ 445) of worms were recovered from the upper region. 3.3. Light microscopic observations Fig. 3a shows an adult C. armatus worm collected from a hamster at 7 days post-infection. Measurements of the various vital organs of the adult worms are shown in Table 1. Although body width increased, body length hardly increased with the number of the post-infection
D. Kimura et al. / Veterinary Parasitology 146 (2007) 367–371
369
Fig. 1. Number of eggs recovered from feces in different intervals of post-infection. Data were shown in average of three hamsters, four mice, and each of four rats and chicks.
day. The ovary (OV) was not recognizable on the first day but became conspicuous on the third day. The seminal vesicle (SV) and seminal receptacle (SR) were also initially observed on the third day. Testis (TT) was recognized in all worms, but the left testis grew to be larger than the right. This phenomenon was observed throughout the observation period. Intrauterine eggs (EG) were observed in hamsters and mice from the third day. In hamsters, the number of eggs reached its peak (15) on the seventh day and decreased thereafter until the 14th day (11). This trend corresponds with that in Fig. 1, where the number of eggs recovered from feces peaked on the seventh day and subsequently decreased thereafter. The oral sucker (OS) measured 46 mm on average and always exceeded the size of the ventral sucker (VS), which was 34 mm on average. No size increase in the oral sucker was observed from the seventh day; however, the size of the ventral sucker
gradually increased as the number of post-infection days increased. Therefore, the ratio of the oral to ventral sucker always exceeded a value of one. 3.4. Observations by scanning electron microscopy The tegmental spines of the adult worms (seventh day) appeared as sawtooth-like structures covering the entire dorsal surface of the body (Fig. 3e). The oral sucker (OS) was located at the most anterior edge part of the worm (Fig. 3b) with 44 circumoral spines (CS), measuring 1 mm 2 mm, and aligned alternately in two rows on its circumference (Fig. 3c). A number of sensory papillae (SP) (arrows) were found surrounding the oral sucker. The ventral sucker (VS) was located at the mid-anterior one-third of the body (Fig. 3b). About six sensory papillae were also observed around the circumference of the ventral sucker (Fig. 3d).
Fig. 2. Comparison of recovery rate of Centrocestus armatus from different animal. Vertical bar indicates S.D.
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D. Kimura et al. / Veterinary Parasitology 146 (2007) 367–371
Fig. 3. Light microscopic (a) and scanning electron microscopic (b–e) photographs of C. armatus adult worms. OS: oral sucker, PH: pharynx, IT: intestinal tract, VS: ventral sucker, SV: seminal vesicle, EG: egg, OV: ovary, SR: seminal receptacle, TT: testis, CS: circumoral spine. (a) A 7-day-old worm stained with acetocarmine. (b) Ventral view of the whole body. (c) Oral sucker and sensory papillae (arrows). (d) Ventral sucker and six sensory papillae (arrows) on the rim of acetabulum. (e) Tegmental spines on ventral surface.
4. Discussion In this study, four different species of experimental animals were used to assess a suitable host for C. armatus. Hong et al. (1989) observed the development of C. armatus in albino rat and noted that worms were collected at a ratio of 0–17% between 1 and 28 days of
post-infection. By contrast, in this study we were able to collect adult worms at 15% recovery rate on the first day post-infection but this subsequently decreased until the third day. No worms were recovered on the fifth day in rats. The discrepancy between Hong’s report (1989) and the present study is not clear since the same age and strain of rats was used in both experiments. However, aspects such as different physiological conditions,
Table 1 Measurement of Centrocestus armatus Days postinfection
H
No. of parasites
Body
Ovary
Seminal vesicle
Seminal receptacle
Right test is
Left testis
Intrauterine eggs
La
Wb
L
W
L
W
L
W
L
W
L
W
13 14 16 6 12
420 424 461 397 404
106 109 121 147 171
– 38 47 49 51
– 30 37 39 47
– 84 115 140 161
– 16 22 24 27
– 25 35 40 34
– 24 30 35 31
44 42 50 55 62
26 29 26 36 40
56 54 65 66 71
29 31 32 41 42
– +7 +13 +15 +11
3 3
315 388
85 88
– 24
– 30
– 135
– 35
– 23
– 23
36 30
21 24
46 39
25 19
– +9
c
1 3 5 7 14 Md 1 3 a b c d
Length (mm). Width (mm). Hamster. Mouse.
D. Kimura et al. / Veterinary Parasitology 146 (2007) 367–371
health status and nutrition of the rats along with different laboratory conditions may explain the discrepancy. Mice however showed a relatively high recovery at 50% on the first day but decreased abruptly to about 6% on the third day. On the other hand, hamsters showed a recovery rate of 25% on the third day and gradually decreased thereafter, but persisted until 14 days post-infection. The same trends have been observed in related studies of Metagonimus spp. and Pygidiopsis spp. (Chen, 1942; Chai, 1979). Hong et al. (1989) also mentioned that if susceptibility of a host animal to an intestinal fluke is low, their recovery rate decreased rapidly compared with other animals with relatively higher susceptibilities. Although the recovery rate of worms in hamster ranges only from 8 to 25%, among the different animal hosts tested we considered it a suitable host of C. armatus, whereas the other animals tested were unsuitable hosts. Ovary was initially observed in front of the right testis on the third day. By contrast, Hong et al. (1989) reported, that ovary was observed on the first day postinfection. The reason for this discrepancy is not clear. In our study, the testis was the only genital organ recognized in worms on the first day post-infection. It is interesting to note that there was no increase in size in the oral sucker throughout the observation period. Probably because the oral sucker has already reached its mature size during the metacercaria stage since it is a very important organ for the parasitic mode of living. The recovery of eggs from feces of hamster from the third day post-infection could be considered relatively early compared to other flukes such as Clonorchis sp. and Paragonimus sp., which require 1 month post-infection. On the other hand, feces from mice, albino rat, and chick were examined for the presence of eggs until the seventh day post-infection, but no eggs were recovered. Even though intrauterine eggs were observed in C. armatus on third day of post-infection, no eggs were recovered from mice feces. This was probably due to the very low worm recovery rate at 3 days post-infection, and thus a very limited number or no eggs at all could be found in feces. Under the above conditions the parasites may not be able to continue their life cycle, further confirming that rats and mice should be considered unsuitable hosts for C. armatus development. The study clearly revealed that
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hamsters, compared with rats, mice, and chicks, are the most suitable animal model for the study of C. armatus. References Chai, J.Y., 1979. Study on Metagonimus yokogawai (Katsurada, 1912) in Korea. V. Intestinal pathology in experimentally infected albino rats. Seoul J. Med. 20, 104–117. Chapin, E.A., 1926. Note on the Heterophyidae. J. Parasitol. 12, 180. Chen, H.T., 1942. The metacercaria and adult of Centrocestus formosanus (Nishigori, 1924), with notes on the natural infection of rats and cats with C. armatus (Tanabe, 1922). J. Parasitol. 28, 285– 298. Hong, S.J., Seo, B.S., Lee, S.H., Chai, J.Y., 1988. A human case of Centrocestus armatus infection in Korea. Kor. J. Parasitol. 26, 55– 60. Hong, S.J., Woo, H.C., Chai, J.Y., Chung, S.W., Lee, S.H., Seo, B.S., 1989. Study on Centrocestus armatus in Korea. II. Recovery rate, growth and development of worms in albino rats. Kor. J. Parasitol. 27, 47–56. Kagei, N., Yanohara, Y., 1995. Epidemiological study on Centrocestus formosanus (Nishigori, 1924)—surveys of its infection in Tanegashima, Kagoshima Prefecture, Japan. Jpn. J. Parasitol. 44, 154– 160. Kimura, D., Uga, S., 2003. Epidemiological studies on Centrocestus spp. (Trematoda: Heterophyidae) in Chikusa River basin, Hyogo Prefecture: infection in the 1st intermediate host snail, Semisulcospira libertina. Jpn. J. Environ. Entomol. Zool. 14, 97–103 (in Japanese with English summary). Kurokawa, T., 1935. On a new trematode of genus Stamnosoma proved from a man. Tokyo Iji Shinji 2915, 293–298 (in Japanese). Lee, S.U., Huh, S., Sohn, W.M., Chai, J.Y., 2004. Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai. Kor. J. Parasitol. 42, 129–135. Price, E.W., 1932. On the genera Centrocestus Looss and Stamnosoma Tanabe. J. Parasitol. 18, 309. Saito, S., Chai, J.Y., Kim, K.H., Lee, S.H., Rim, H.J., 1997. Metagonimus miyatai sp. nov (Digenea: Heterophyidae), a new intestinal trematode transmitted by freshwater fishes in Japan and Korea. J. Parasitol. 35, 223–232. Tanabe, H., 1922. Studien uber die trematoden mit Susswasserfischen als Zwischenwirt. I. Stamnosoma armatum n.g., n.sp. Kyoto Igaku Zasshi 19, 239–252 (in Japanese). Uga, S., Morimoto, M., Sato, T., Rai, S.K., 1998. Surface ultrastructure of Heterophyes heterophyes (Trematoda: Heterophyidae) collected from a man. J. Helminthol. Soc. Wash. 65, 119– 122. Waikagul, J., 1997. Human infection of Centrocestus caninus in Thailand. Southeast Asian J. Trop. Med. Public Health. 28, 831–835. Yamaguti, S., 1958. Systema Helminthum, vol. I: Digenetic Trematodes of Vertebrates, pp. 705–707, 872–873.