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Infectivity and development of Metagonimus yokogawai in experimentally infected domestic ducks (Cairina moschata)
Veterinary Parasitology 168 (2010) 45–50
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Infectivity and development of Metagonimus yokogawai in experimentally infected domestic ducks (Cairina moschata) Ming-Hsien Li a,b, Chien-Wei Liao c, Yueh-Lun Lee d, Hong-Kean Ooi e, Wen-Yuan Du f, Shen-Che Lu g, Hai-I. Huang b, Kua-Eyre Su g, Chia-Kwung Fan c,h,* a
Graduate Institute of Veterinary Microbiology, National Chung Hsing University, Taichung, Taiwan Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan Department of Parasitology, College of Medicine, Taipei Medical University, 250 Wu-Hsin Street, Taipei, Taiwan d Department of Microbiology & Immunology, College of Medicine, Taipei Medical University, 250 Wu-Hsin Street, Taipei, Taiwan e Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan f School of Medicine, Fu Jen Catholic University, Taiwan g Department of Parasitology, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, Section 1, Taipei, Taiwan h Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsin Street, Taipei, Taiwan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 December 2007 Received in revised form 23 September 2009 Accepted 8 October 2009
Information concerning whether fowl such as duck is a suitable reservoir host of Metagonimus yokogawai is largely unclear to date. In the present study, the growth and development of M. yokogawai metacercaria (Mc) in domestic duck (Cairina moschata) was determined by worm recovery rate (WRR) and morphological changes e.g., the size of fluke’s body as well as their internal organs was assessed by using Semichon’s acetocarmine staining. Each duck was orally inoculated with 50 Mcs of M. yokogawai and infected ducks were deeply anesthetized with ether and killed at 1, 2, 3, 4, 5, 6, 7, and 14 days post-infection (DPI). On each date, two infected ducks were killed and the small intestines of each duck were separated into four parts then they were opened longitudinally to harvest the flukes. Results revealed that WRR of M. yokogawai from inoculated ducks increased during early infection with a peak as seen at 4 DPI (28.5 6.9%); thereafter it gradually decreased and a drastic decline was observed in 14 DPI (2.0 1.1%) in the trial. The preference sites for M. yokogawai were low portions of the small intestine; nevertheless the size of fluke’s body and organs developed increasingly with time and they maturated to produce ova from 4 DPI onward in the trial. However, present results indicated that ducks, based on the findings of this study, are not suitable hosts for establishment of M. yokogawai infection because most flukes were expelled from duck’s intestine within 14 days. Nevertheless, it was proposed that ducks might play a certain role in transmitting M. yokogawai when they deposited the ova via feces into marsh where snails and fish were abundant since they could presumably establish transient and possibly patent infections with this parasite. Crown Copyright ß 2009 Published by Elsevier B.V. All rights reserved.
Keywords: Infectivity Development Metagonimus yokogawai Duck
1. Introduction * Corresponding author at: Department of Parasitology, College of Medicine, Taipei Medical University, 250 Wu-Hsin Street, Taipei 110, Taiwan. Tel.: +886 2 27395092; fax: +886 2 27395092. E-mail address: [email protected] (C.-K. Fan).
Metagonimus yokogawai has been one of the most important human intestinal flukes in eastern Asia, especially of China, Japan, Korea, and Taiwan (Chai and Lee, 2002). Human infection with this flukes is usually
0304-4017/$ – see front matter . Crown Copyright ß 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.10.005
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contracted by eating raw and/or undercook fresh-water fish which harbor the metacercaria (Chai and Lee, 2002). Fresh-water fish of many kinds e.g., Plecoglossus altivelis, Zacco pachycephalus, Carassius auratus, and Cyprinus carpio were ever reported to be the second intermediate host of M. yokogawai in those endemic countries (Yokogawa, 1913; Lai, 1982; Akahane et al., 1983). The clinical symptoms due to M. yokogawai infection are generally mild but some of the infected persons may suffer from severe diarrhea and abdominal pain (Chai and Lee, 2002). Several fish-eating mammals including dogs (Cho et al., 1981), rats (Seo et al., 1981), and cats (Huh et al., 1993) reportedly may serve as the natural reservoir host for M. yokogawai and the life span of this fluke in dogs, cats, and rats was estimated with a range of 1–12 months (Koga, 1938). The prepatent period for dogs experimentally infected with M. yokogawai was observed to be about 9 days post-infection (Miyamoto, 1985), and undoubtedly these reservoir hosts play an important role in transmission of M. yokogawai. In Taiwan, there are several natural marshes where snails and fish are abundant (Tsai, 2000) and since various species of migratory wildfowl e.g., Anas crecca, Motacilla flava, and Pluvialis fulva often fly to the warm natural marshes in Taiwan to settle down temporarily during winter (Shiu and Lee, 2003), whether those fowls may play a role in transmission of M. yokogawai is concerned. Although numerous studies concerning infectivity, development and pathology of M. yokogawai in experimental animal have been intensively carried out in various rodent models (Chai et al., 1993, 1994, 1995); regrettably, information concerning whether fowl is a suitable reservoir host of M. yokogawai is largely unclear to date. In the present study, we attempted to evaluate the susceptibility of the muscovy duck (Cairina moschata) to experimental infection with M. yokogawai by designing the monitoring time up to 14 days post-infection (DPI) to assess all the parameters based on the prepatent period of dogs, one natural reservoir host of M. yokogawai, was about 9 days (Miyamoto, 1985). 2. Materials and methods 2.1. Metacercaria collection and the inoculation protocol The method to recover the metacercaria (Mc) of M. yokogawai was adapted from Chai et al. (1995) with slight modifications. Briefly, the scales and minced flesh of the Z. pachycephalus, which has been widely reported to have high infection rate of M. yokogawai Mc in Taiwan (Lai, 1982), were separately digested with artificial digestive fluid (0.1% pepsin solution with 0.7% hydrochloric acid) for about 6–8 h at 37 8C. The sediments were sieved with a nylon mesh with an opening of 80 mm and debris retaining Mc on the mesh was washed out into a 50-ml beaker with saline solution. For each sample, the Mc in the saline were counted and collected under a dissecting microscope (Chai et al., 1995). The structure of the M. yokogawai Mc was examined and confirmed according to the method previously described by Seo and Hong (1969). Briefly, when round to oval-like shaped Mc recovered from scales and minced flesh was
observed to present with oral sucker in the front body and heart-shaped excretory sac with abundant yellow/brown granules in the late body examined under a dissecting microscope that was recognized as M. yokogawai Mc and that they were used in the inoculation experiment. Male muscovy ducks aged 2–3 weeks were obtained from the traditional market in Taipei City. Presence of any parasite e.g., Ascaridia galli in the stools of any duck examined by ether–formalin technique was excluded in the study. Each duck was tested three times for detection of any parasite. Ducks which were negative on fecal examination for parasite were housed in small cage individually in the animal facility of National Taiwan University and maintained on commercial pellet food and water ad libitum. Each duck was orally inoculated with about 50 Mcs of M. yokogawai using a stomach tube (Kino et al., 2006) and a total of 16 ducks were used in the present experiment. All animal experiments were carried out in accordance with institutional Policies and Guidelines for the Care and Use of Laboratory Animals, National Taiwan University and all efforts were made to minimize animal suffering. 2.2. Susceptibility assessed by worm recovery rates and developmental status of M. yokogawai in small intestines of infected ducks Infected ducks were deeply anesthetized with ether and killed by cervical dislocation at 1, 2, 3, 4, 5, 6, 7, and 14 days post-infection (DPI). On each date, two infected ducks were killed and the small intestines of each duck were separated into four equal parts of the same length from anterior to posterior according to a method described by Kino et al. (2006) with slight modifications. After visual observation of intestinal contents and mucosal changes, they were further incubated in saline solution for 1 h at 37 8C and liberated adult worms from each piece of the intestine were counted and identified individually under a microscope according to the relative location of the testes and uterine egg mass (Rim et al., 1996). The position of the worm was scored against the number of the intestinal section in which it was located and the worm recovery rate was calculated, thereafter they were fixed in 10% neutral formalin, stained with Semichon’s acetocarmine to assess various characteristic parameters of the worm recovered, including the morphology, body size, mean diameter of oral and genital-ventral suckers, right, left testis, ovary, or ova was measured using ocular meter at 1000 magnifications under light microscope. At least 3 (14 DPI)–10 (1–7 DPI) recovered worms and 15–25 ova in the uterus per worm were examined per date infection. 2.3. Statistical analysis All data were processed using statistical software system (SPSS, Chicago, IL, USA). For calculation of the significance of differences between times, numbers of recovered worm, size of worm and its internal organ as well as ova, Pearson’s coefficient (r) of correlation and Chi-square test (x2-test) or Student’s t-test was used. For all statistical analysis; a Pvalue of <0.05 was considered statistically significant.
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3. Results 3.1. Selection of ducks used in the experiment Totally, 16 out of 22 ducks purchased from the market showed negative parasitic infections by fecal examinations. The six ducks were all found to be infected with A. galli that were excluded in the present experiments. 3.2. Worm recovery rate (WRR) of M. yokogawai from inoculated ducks The mean (SD) WRR of M. yokogawai began to increase from 1 DPI (16.0 6.7%), and reached a peak at 4 DPI (28.5 6.9%); afterwards it gradually decreased from 26.5 9.9% to 19.5 7.2% during 5–7 DPI. A significantly drastic decrease in WRR was observed at 14 DPI (2.0 1.1%) (Fig. 1). Statistically, a positive correlation between WRR and infection time from 1 to 4 DPI was observed (r = 0.947, P = 0.004), whereas a significantly negative correlation was found between WRR and time from 5 to 14 DPI (r = 0.993, P = 0.003). Overall, a significantly negative correlation was found between WRR and time in the trial (r = 0.700, P = 0.004).
Fig. 1. Chronological changes in the mean worm recovery rate + 1 SD of flukes recovered from sections 1 (Sec 1) to 4 (Sec 4) of the small intestine of ducks experimental infection with Metagonimus yokogawai from 1 to 14 days (D) post-infection.
from 1 to 4 DPI, ranging from 3.5 2.1 to 17.5 10.6, thereafter the numbers of recovered worm reached a peak of 22.5 2.1 at 5 DPI. Afterwards, they fell till to 14.5 7.8 at 7 DPI. None of flukes were found at 14 DPI. Similar findings were also observed in section 4 of small intestine, the highest worm recovery was found at 2 DPI (16.0 22.6), thereafter it fluctuated from 2.0 0.0 to 11.0 4.2 from 3 DPI onwards (Fig. 1).
3.3. The primary sites of M. yokogawai recovered from the ducks’ small intestine
3.4. Development of M. yokogawai in inoculated ducks
None of flukes were recovered from section 1 of small intestine in the trial. In section 2 of small intestine, the flukes were merely recovered during 1–5 DPI, with a range of 0.5 0.4 at 1 DPI to the highest of 10.5 6.4 at 4 DPI; thereafter they dropped to 2.0 1.4 at 5 DPI. The flukes recovery from section 3 of small intestine increased gradually
Overall, the fluke’s length, width, oral sucker, genitalventral sucker, and sexual organs including right, left testis and ovary were observed to be increased in size with time in the trial (Table 1, Fig. 2). Noteworthy, ova did not appear in the fluke’s uterus until 4 DPI and largely the ova size was kept within a range of 24.8 2.2 mm in
Table 1 Morphological features of Metagonimus yokogawai recovered from domestic ducks (Cairina moschata) experimentally inoculated with 50 metacercaria from 1 to 14 days post-infections. Day after infection (no. of worms examined)
1 2 3 4 5 6 7 14
(n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 3)
Day after infection (no. of worms examined)
1 2 3 4 5 6 7 14
(n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 3)
Fluke length (mm)
Fluke width (mm)
Oral sucker (mm)
Genital-ventral (mm)
Length
Width
Length
Width
Length
Width
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
176.0 25.9 231.0 65.2 216.0 31.0 249.0 42.0 283.0 28.3 223.0 22.6 263.0 33.4 274.3 4.4
85.0 9.7 95.0 16.5 102.0 17.5 120.0 27.1 134.0 18.4 107.0 17.0 168.0 39.1 155.5 5.3
22.1 2.9 23.8 2.6 24.4 2.6 23.5 4.1 27.0 2.5 22.8 2.2 27.7 2.9 27.8 1.0
27.5 1.7 34.2 3.7 31.2 2.0 30.0 4.8 35.7 4.6 29.9 3.5 33.8 3.6 35.0 0.8
17.0 2.2 22.1 4.1 24.4 4.5 24.9 4.0 30.8 2.3 22.7 3.6 33.4 4.8 35.3 0.5
20.5 3.5 32.9 5.5 35.7 4.6 32.1 5.5 43.6 5.1 30.5 6.0 47.0 9.0 52.5 0.6
13.7 1.7 24.7 6.6 33.4 10.6 34.9 8.7 46.0 8.2 31.3 6.4 44.8 8.8 46.5 1.3
21.0 2.8 36.7 10.8 46.7 13.1 49.7 14.3 64.9 6.4 44.8 10.3 59.3 8.7 60.3 0.5
Left testis (mm)
Ovary (mm)
sucker
Right testis (mm)
Ova (mm)
Length
Width
Length
Width
Length
Width
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
13.4 1.5 24.0 6.9 31.1 9.0 34.2 9.8 43.1 2.7 31.1 6.1 45.4 11.3 41.5 5.3
19.7 2.9 31.7 9.5 45.5 11.9 48.2 12.3 62.7 4.0 42.0 7.9 55.2 10.9 55.5 1.3
14.3 1.8 23.0 4.6 23.2 3.8 24.6 5.9 31.3 5.2 25.3 3.3 33.0 5.4 38.0 8.2
19.4 1.6 30.8 6.3 31.0 6.6 33.9 8.4 44.2 5.7 31.6 6.2 49.7 10.4 58.3 9.4
0.0 0.0 0.0 0.0 0.0 0.0 26.5 0.7 24.9 1.7 21.0 4.1 25.5 1.1 26.3 0.5
0.0 0.0 0.0 0.0 0.0 0.0 15.0 2.8 16.2 3.2 15.5 2.8 15.5 0.9 16.0 0.0
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Fig. 2. Representative figures indicate (A) 1-day-old juvenile fluke of Metagonimus yokogawai. Internal organs do not develop well and no uterine eggs are seen. Bar = 25 mm. (B) 4-day-old juvenile fluke of Metagonimus yokogawai. Sex organs develop fully to produce several uterine eggs (arrow). Bar = 25 mm. Inset indicating eggs (arrow) in the uterus with high magnifications. (C) 14-day-old adult fluke of Metagonimus yokogawai. Its size and internal organs grow greatly to be the largest and mature fully to produce numerous uterine eggs. Bar = 25 mm.
length 15.2 1.1 mm in width (Table 1, Fig. 2B). All of the detailed figures of various organs as well as ova developed in the flukes were summarized in Table 1. 4. Discussion Although Miyamoto (1985) found wildlife such as fowls of black kite (Miluvs migrans lineatus) captured from Hokkaido, Japan was one natural definitive host of M. yokogawai based on mature M. yokogawai adults with fully developed testis, ovary, and uterine eggs were found in the small intestine by a necropsy study, to date information regarding whether other fowls may play a role in transmission of M. yokogawai is not available. In Taiwan, various species of migratory birds often fly from the cold northern to the warm southern Taiwan to settle down temporarily during winter, thus whether those fowls may play a role in transmission of M. yokogawai is also concerned. The susceptibility of animal hosts to infection with M. yokogawai can be assessed by analyzing certain parameters e.g., chronological worm recovery rate (WRR),
worm growth, and fecundity (Chai et al., 1984). The present results revealed that in infected duck the highest WRR was present at 4 DPI, reaching 28.5% which was much lower than that in inbred strain BALB/c mice (80% at 3 DPI) or outbred strain ICR mice (66% at 1 DPI), interestingly these mice were also considered as not susceptible hosts to M. yokogawai infection (Chai et al., 1984; Guk et al., 2005). In contrast, in cats, dogs, or hamster, the development of worm was good, the recovery rate of worms was high and their maintenance of parasitism was fairly long (Yokogawa and Sano, 1968; Kang et al., 1983). Moreover, the growth of the worm seemed to be strictly limited as evidenced by that the length of the worm recovered from infected ducks at either 7 DPI (263.0 33.4 mm) or 14 DPI (274.3 4.4 mm) was rather smaller than that in BALB/c (535 102 mm at 7 DPI; 667 60 mm at 14 DPI) or C57BL/6J (532 136 mm at 7 DPI; 603 123 mm at 14 DPI) (Guk et al., 2005). However, the statistical significance in the WRR with each subsequent pair of experimental ducks showed apparent variability. The effects may be due to low sample numbers (only two ducks
M.-H. Li et al. / Veterinary Parasitology 168 (2010) 45–50
per inoculation dose), host/parasite biological variability, problems with inoculations, etc. Similarly, Chai et al. (1984) also found that the susceptibility for M. yokogawai was different even among the same mouse host of different strain, indicating genetic background may be involved; nevertheless, complex regulation may exist between parasite and hosts. In addition, the mean size of ova (24.8 mm 15.6 mm) produced from the worm appeared smaller than that in gerbil (30.4 mm 18.0 mm) (Lai, 1982) but its size was similar to that in hamster (27 mm 17 mm) (Rim et al., 1996). Although the present study revealed that metacercaria of M. yokogawai can infect successfully and subsequently matured to produce ova in infected duck from 4 DPI onwards, the present results suggested that duck did not seem to be susceptible to M. yokogawai infection because most worms were expelled within 14 days. This finding was also seen in various small rodent animals experimentally infected with M. yokogawai e.g., inbred strain BALB/c mice and outbred strain ICR mice in which most worms were expelled within 14 days (Chai et al., 1984; Guk et al., 2005). Interestingly, the time for ova found in the uterus of M. yokogawai and infected ducks present with watery diarrhea both were found accompanying by decreasing total WRR as started from 4 DPI onward in the trial. Although in the present study we did not intend to evaluate the clinical sings of infected ducks, however, one of inoculated ducks seemed likely die of severe watery diarrhea at 14 DPI in the trial due to presence of apparent watery-like feces. It was also evident from 4 DPI onward that worms were frequently recovered from the lower portion (sections 3 and 4) other than the upper or middle portion the preferred site for M. yokogawai to reside in the small intestine as similar results were also seen in murine host experimental infection of M. yokogawai (Chai et al., 1984). Based on these results, we might speculate that when the fecundity of worm began that seemed to be able to produce some antigenic materials capable of eliciting the intestinal immunological response to interfere the invasion and maturation of M. yokogawai and consequently the worm was expelled from the intestine thus leading to decreased WRR and possibly the worm’s relocation to the lower portion of the small intestine of infected ducks from 4 DPI onward in the trial. Previous study has indicated that rats infected with M. yokogawai present with intensive intestinal mucosal response which was proposed to be involved in the expulsion of the worm (Chai et al., 1993) and leukocytes including eosinophil, neutrophil, lymphocytes, and mucosal mast cells were suggested to be involved in the expulsion process of the worm from the small intestine of mice or rats experimentally infected with M. yokogawai (Chai et al., 1984, 1993). However, whether mucosal immunity involved in defending against the M. yokogawai infection in the duck’s intestine remained to be determined. Nevertheless, it might alternatively consider that this phenomenon may simply be a matter of the time required for the host to mount an effective immunological response to the parasite, and is not due to the generation of any maturation-specific fluke antigens. In conclusion, the current work was a pilot study to indicate that fowls such as ducks did not seem to be
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susceptible to M. yokogawai infection because most worms were expelled within 14 days possibly due to intestinal immunological interference thus affecting the growth as well as development of M. yokogawai in duck’s intestine. Therefore, it was proposed a transient role for ducks to transmit M. yokogawai when they deposited the ova via feces into marsh where snails and fish were abundant. Nevertheless, further studies are needed to better elucidate any role ducks might play in the epidemiology of this parasitic infection e.g., assess infectivity and development of ova from ducks in freshwater snail and subsequently whether cercaria shedding from the snails are capable to develop into fully infective metacercaria in the fresh-water fish as assessed by inoculation of fish-eating animals. References Akahane, H., Kusano, F., Hayashi, K., 1983. An epidemiological study on Metagonimiasis in Shiga Prefecture, with special reference to the infection pattern of Metagonimus yokogawai metacercariae of ayu (Plecoglossue altivelis) reared in ponds. Jpn. J. Parasitol. 32, 85–90 (in Japanese with English abstract). Chai, J.Y., Seo, B.S., Lee, S.H., 1984. Study on Metagonimus yokogawai (Katsurada, 1912) in Korea VII Susceptibility of various strains of mice to Metagonimus infection and effect of prednisolone. Kisaengchunghak Chapchi 22, 153–160. Chai, J.Y., Kim, T.H., Kho, W.G., Chung, S.W., Hong, S.T., Lee, S.H., 1993. Mucosal mast cell responses to experimental Metagonimus yokogawai infection in rats. Korean J. Parasitol. 31, 129–134. Chai, J.Y., Yun, T.Y., Kim, J., Huh, S., Choi, M.H., Lee, S.H., 1994. Chronological observation on intestinal histopathology and intraepithelial lymphocytes in the intestine of rats infected with Metagonimus yokogawai. Korean J. Parasitol. 32, 215–221. Chai, J.Y., Kim, J., Lee, S.H., 1995. Invasion of Metagonimus yokogawai into the submucosa layer of the small intestine of immuno-suppressed mice. Korean J. Parasitol. 33, 313–321. Chai, J.Y., Lee, S.H., 2002. Food-borne intestinal trematode infections in the Republic of Korea. Parasitol. Int. 51, 129–154. Cho, S.Y., Kang, S.Y., Ryang, Y.S., 1981. Helminthes infections in the small intestine of stray dogs in Ejungbu City, Kyunggi Do, Korea. Korean J. Parasitol. 19, 55–59. Guk, S.M., Park, J.Y., Seo, M., Han, E.T., Kim, J.L., Chai, J.Y., 2005. Susceptibility of inbred mouse strains to infection with three species of Metagonimus prevalent in the Republic of Korea. J. Parasitol. 91, 12–16. Huh, S., Sohn, W.M., Chai, J.Y., 1993. Intestinal parasites of cats purchased in Seoul. Korean J. Parasitol. 31, 371–373. Kang, S.Y., Cho, S.Y., Chai, J.Y., Lee, J.B., Jang, D.H., 1983. A study on intestinal lesions of experimentally reinfected dogs with Metagonimus yokogawai. Kisaengchunghak Chapchi 21, 58–73 (in Korean with English abstract). Kino, H., Suzuki, T., Oishi, H., Suzuki, S., Yamagiwa, S., Ishiguro, M., 2006. Geographical distribution of Metagonimus yokogawai and M. miyatai in Shizuoka Prefecture, Japan, and their site preferences in the sweetfish, Plecoglossus altivelis, and hamsters. Parasitol. Int. 55, 201–206. Koga, G., 1938. On the condition of worms of Metagonimus and Heterophyes within the final host’s intestinal tract, and effect of worms upon the tract. Nippon Kiseichu Gakkai Kiji 10, 84. Lai, T.S., 1982. Two new fish intermediate host of Metagonimus yokogawai: Acrosscheilus formosanus and Zacco pachycephalus found in Taipei, Taiwan. Med. Res. 4, 1283–1290 (in Chinese with English abstract). Miyamoto, K., 1985. Studies on zoonoses in Hokkaido. 7. Survey of natural definitive hosts of Metagonimus yokogawai. Jpn. J. Parasitol. 34, 371– 376. Rim, H.J., Kim, K.H., Joo, K.H., 1996. Classification and host specificity of Metagonimus spp. from Korean freshwater fish. Korean J. Parasitol. 34, 7–14. Seo, B.S., Hong, N.T., 1969. Study on Metagonimus yokogawai (Katsurada, 1912) in Korea. I. On the metacercaria, its distribution in the second intermediate host and the development in the final host. Kisaengchunghak Chapchi 7, 129–142 (in Korean with English abstract).
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Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Infectivity and development of Metagonimus yokogawai in experimentally infected domestic ducks (Cairina moschata) Ming-Hsien Li a,b, Chien-Wei Liao c, Yueh-Lun Lee d, Hong-Kean Ooi e, Wen-Yuan Du f, Shen-Che Lu g, Hai-I. Huang b, Kua-Eyre Su g, Chia-Kwung Fan c,h,* a
Graduate Institute of Veterinary Microbiology, National Chung Hsing University, Taichung, Taiwan Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan Department of Parasitology, College of Medicine, Taipei Medical University, 250 Wu-Hsin Street, Taipei, Taiwan d Department of Microbiology & Immunology, College of Medicine, Taipei Medical University, 250 Wu-Hsin Street, Taipei, Taiwan e Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan f School of Medicine, Fu Jen Catholic University, Taiwan g Department of Parasitology, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, Section 1, Taipei, Taiwan h Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsin Street, Taipei, Taiwan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 December 2007 Received in revised form 23 September 2009 Accepted 8 October 2009
Information concerning whether fowl such as duck is a suitable reservoir host of Metagonimus yokogawai is largely unclear to date. In the present study, the growth and development of M. yokogawai metacercaria (Mc) in domestic duck (Cairina moschata) was determined by worm recovery rate (WRR) and morphological changes e.g., the size of fluke’s body as well as their internal organs was assessed by using Semichon’s acetocarmine staining. Each duck was orally inoculated with 50 Mcs of M. yokogawai and infected ducks were deeply anesthetized with ether and killed at 1, 2, 3, 4, 5, 6, 7, and 14 days post-infection (DPI). On each date, two infected ducks were killed and the small intestines of each duck were separated into four parts then they were opened longitudinally to harvest the flukes. Results revealed that WRR of M. yokogawai from inoculated ducks increased during early infection with a peak as seen at 4 DPI (28.5 6.9%); thereafter it gradually decreased and a drastic decline was observed in 14 DPI (2.0 1.1%) in the trial. The preference sites for M. yokogawai were low portions of the small intestine; nevertheless the size of fluke’s body and organs developed increasingly with time and they maturated to produce ova from 4 DPI onward in the trial. However, present results indicated that ducks, based on the findings of this study, are not suitable hosts for establishment of M. yokogawai infection because most flukes were expelled from duck’s intestine within 14 days. Nevertheless, it was proposed that ducks might play a certain role in transmitting M. yokogawai when they deposited the ova via feces into marsh where snails and fish were abundant since they could presumably establish transient and possibly patent infections with this parasite. Crown Copyright ß 2009 Published by Elsevier B.V. All rights reserved.
Keywords: Infectivity Development Metagonimus yokogawai Duck
1. Introduction * Corresponding author at: Department of Parasitology, College of Medicine, Taipei Medical University, 250 Wu-Hsin Street, Taipei 110, Taiwan. Tel.: +886 2 27395092; fax: +886 2 27395092. E-mail address: [email protected] (C.-K. Fan).
Metagonimus yokogawai has been one of the most important human intestinal flukes in eastern Asia, especially of China, Japan, Korea, and Taiwan (Chai and Lee, 2002). Human infection with this flukes is usually
0304-4017/$ – see front matter . Crown Copyright ß 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.10.005
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contracted by eating raw and/or undercook fresh-water fish which harbor the metacercaria (Chai and Lee, 2002). Fresh-water fish of many kinds e.g., Plecoglossus altivelis, Zacco pachycephalus, Carassius auratus, and Cyprinus carpio were ever reported to be the second intermediate host of M. yokogawai in those endemic countries (Yokogawa, 1913; Lai, 1982; Akahane et al., 1983). The clinical symptoms due to M. yokogawai infection are generally mild but some of the infected persons may suffer from severe diarrhea and abdominal pain (Chai and Lee, 2002). Several fish-eating mammals including dogs (Cho et al., 1981), rats (Seo et al., 1981), and cats (Huh et al., 1993) reportedly may serve as the natural reservoir host for M. yokogawai and the life span of this fluke in dogs, cats, and rats was estimated with a range of 1–12 months (Koga, 1938). The prepatent period for dogs experimentally infected with M. yokogawai was observed to be about 9 days post-infection (Miyamoto, 1985), and undoubtedly these reservoir hosts play an important role in transmission of M. yokogawai. In Taiwan, there are several natural marshes where snails and fish are abundant (Tsai, 2000) and since various species of migratory wildfowl e.g., Anas crecca, Motacilla flava, and Pluvialis fulva often fly to the warm natural marshes in Taiwan to settle down temporarily during winter (Shiu and Lee, 2003), whether those fowls may play a role in transmission of M. yokogawai is concerned. Although numerous studies concerning infectivity, development and pathology of M. yokogawai in experimental animal have been intensively carried out in various rodent models (Chai et al., 1993, 1994, 1995); regrettably, information concerning whether fowl is a suitable reservoir host of M. yokogawai is largely unclear to date. In the present study, we attempted to evaluate the susceptibility of the muscovy duck (Cairina moschata) to experimental infection with M. yokogawai by designing the monitoring time up to 14 days post-infection (DPI) to assess all the parameters based on the prepatent period of dogs, one natural reservoir host of M. yokogawai, was about 9 days (Miyamoto, 1985). 2. Materials and methods 2.1. Metacercaria collection and the inoculation protocol The method to recover the metacercaria (Mc) of M. yokogawai was adapted from Chai et al. (1995) with slight modifications. Briefly, the scales and minced flesh of the Z. pachycephalus, which has been widely reported to have high infection rate of M. yokogawai Mc in Taiwan (Lai, 1982), were separately digested with artificial digestive fluid (0.1% pepsin solution with 0.7% hydrochloric acid) for about 6–8 h at 37 8C. The sediments were sieved with a nylon mesh with an opening of 80 mm and debris retaining Mc on the mesh was washed out into a 50-ml beaker with saline solution. For each sample, the Mc in the saline were counted and collected under a dissecting microscope (Chai et al., 1995). The structure of the M. yokogawai Mc was examined and confirmed according to the method previously described by Seo and Hong (1969). Briefly, when round to oval-like shaped Mc recovered from scales and minced flesh was
observed to present with oral sucker in the front body and heart-shaped excretory sac with abundant yellow/brown granules in the late body examined under a dissecting microscope that was recognized as M. yokogawai Mc and that they were used in the inoculation experiment. Male muscovy ducks aged 2–3 weeks were obtained from the traditional market in Taipei City. Presence of any parasite e.g., Ascaridia galli in the stools of any duck examined by ether–formalin technique was excluded in the study. Each duck was tested three times for detection of any parasite. Ducks which were negative on fecal examination for parasite were housed in small cage individually in the animal facility of National Taiwan University and maintained on commercial pellet food and water ad libitum. Each duck was orally inoculated with about 50 Mcs of M. yokogawai using a stomach tube (Kino et al., 2006) and a total of 16 ducks were used in the present experiment. All animal experiments were carried out in accordance with institutional Policies and Guidelines for the Care and Use of Laboratory Animals, National Taiwan University and all efforts were made to minimize animal suffering. 2.2. Susceptibility assessed by worm recovery rates and developmental status of M. yokogawai in small intestines of infected ducks Infected ducks were deeply anesthetized with ether and killed by cervical dislocation at 1, 2, 3, 4, 5, 6, 7, and 14 days post-infection (DPI). On each date, two infected ducks were killed and the small intestines of each duck were separated into four equal parts of the same length from anterior to posterior according to a method described by Kino et al. (2006) with slight modifications. After visual observation of intestinal contents and mucosal changes, they were further incubated in saline solution for 1 h at 37 8C and liberated adult worms from each piece of the intestine were counted and identified individually under a microscope according to the relative location of the testes and uterine egg mass (Rim et al., 1996). The position of the worm was scored against the number of the intestinal section in which it was located and the worm recovery rate was calculated, thereafter they were fixed in 10% neutral formalin, stained with Semichon’s acetocarmine to assess various characteristic parameters of the worm recovered, including the morphology, body size, mean diameter of oral and genital-ventral suckers, right, left testis, ovary, or ova was measured using ocular meter at 1000 magnifications under light microscope. At least 3 (14 DPI)–10 (1–7 DPI) recovered worms and 15–25 ova in the uterus per worm were examined per date infection. 2.3. Statistical analysis All data were processed using statistical software system (SPSS, Chicago, IL, USA). For calculation of the significance of differences between times, numbers of recovered worm, size of worm and its internal organ as well as ova, Pearson’s coefficient (r) of correlation and Chi-square test (x2-test) or Student’s t-test was used. For all statistical analysis; a Pvalue of <0.05 was considered statistically significant.
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3. Results 3.1. Selection of ducks used in the experiment Totally, 16 out of 22 ducks purchased from the market showed negative parasitic infections by fecal examinations. The six ducks were all found to be infected with A. galli that were excluded in the present experiments. 3.2. Worm recovery rate (WRR) of M. yokogawai from inoculated ducks The mean (SD) WRR of M. yokogawai began to increase from 1 DPI (16.0 6.7%), and reached a peak at 4 DPI (28.5 6.9%); afterwards it gradually decreased from 26.5 9.9% to 19.5 7.2% during 5–7 DPI. A significantly drastic decrease in WRR was observed at 14 DPI (2.0 1.1%) (Fig. 1). Statistically, a positive correlation between WRR and infection time from 1 to 4 DPI was observed (r = 0.947, P = 0.004), whereas a significantly negative correlation was found between WRR and time from 5 to 14 DPI (r = 0.993, P = 0.003). Overall, a significantly negative correlation was found between WRR and time in the trial (r = 0.700, P = 0.004).
Fig. 1. Chronological changes in the mean worm recovery rate + 1 SD of flukes recovered from sections 1 (Sec 1) to 4 (Sec 4) of the small intestine of ducks experimental infection with Metagonimus yokogawai from 1 to 14 days (D) post-infection.
from 1 to 4 DPI, ranging from 3.5 2.1 to 17.5 10.6, thereafter the numbers of recovered worm reached a peak of 22.5 2.1 at 5 DPI. Afterwards, they fell till to 14.5 7.8 at 7 DPI. None of flukes were found at 14 DPI. Similar findings were also observed in section 4 of small intestine, the highest worm recovery was found at 2 DPI (16.0 22.6), thereafter it fluctuated from 2.0 0.0 to 11.0 4.2 from 3 DPI onwards (Fig. 1).
3.3. The primary sites of M. yokogawai recovered from the ducks’ small intestine
3.4. Development of M. yokogawai in inoculated ducks
None of flukes were recovered from section 1 of small intestine in the trial. In section 2 of small intestine, the flukes were merely recovered during 1–5 DPI, with a range of 0.5 0.4 at 1 DPI to the highest of 10.5 6.4 at 4 DPI; thereafter they dropped to 2.0 1.4 at 5 DPI. The flukes recovery from section 3 of small intestine increased gradually
Overall, the fluke’s length, width, oral sucker, genitalventral sucker, and sexual organs including right, left testis and ovary were observed to be increased in size with time in the trial (Table 1, Fig. 2). Noteworthy, ova did not appear in the fluke’s uterus until 4 DPI and largely the ova size was kept within a range of 24.8 2.2 mm in
Table 1 Morphological features of Metagonimus yokogawai recovered from domestic ducks (Cairina moschata) experimentally inoculated with 50 metacercaria from 1 to 14 days post-infections. Day after infection (no. of worms examined)
1 2 3 4 5 6 7 14
(n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 3)
Day after infection (no. of worms examined)
1 2 3 4 5 6 7 14
(n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 3)
Fluke length (mm)
Fluke width (mm)
Oral sucker (mm)
Genital-ventral (mm)
Length
Width
Length
Width
Length
Width
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
176.0 25.9 231.0 65.2 216.0 31.0 249.0 42.0 283.0 28.3 223.0 22.6 263.0 33.4 274.3 4.4
85.0 9.7 95.0 16.5 102.0 17.5 120.0 27.1 134.0 18.4 107.0 17.0 168.0 39.1 155.5 5.3
22.1 2.9 23.8 2.6 24.4 2.6 23.5 4.1 27.0 2.5 22.8 2.2 27.7 2.9 27.8 1.0
27.5 1.7 34.2 3.7 31.2 2.0 30.0 4.8 35.7 4.6 29.9 3.5 33.8 3.6 35.0 0.8
17.0 2.2 22.1 4.1 24.4 4.5 24.9 4.0 30.8 2.3 22.7 3.6 33.4 4.8 35.3 0.5
20.5 3.5 32.9 5.5 35.7 4.6 32.1 5.5 43.6 5.1 30.5 6.0 47.0 9.0 52.5 0.6
13.7 1.7 24.7 6.6 33.4 10.6 34.9 8.7 46.0 8.2 31.3 6.4 44.8 8.8 46.5 1.3
21.0 2.8 36.7 10.8 46.7 13.1 49.7 14.3 64.9 6.4 44.8 10.3 59.3 8.7 60.3 0.5
Left testis (mm)
Ovary (mm)
sucker
Right testis (mm)
Ova (mm)
Length
Width
Length
Width
Length
Width
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
Mean SD
13.4 1.5 24.0 6.9 31.1 9.0 34.2 9.8 43.1 2.7 31.1 6.1 45.4 11.3 41.5 5.3
19.7 2.9 31.7 9.5 45.5 11.9 48.2 12.3 62.7 4.0 42.0 7.9 55.2 10.9 55.5 1.3
14.3 1.8 23.0 4.6 23.2 3.8 24.6 5.9 31.3 5.2 25.3 3.3 33.0 5.4 38.0 8.2
19.4 1.6 30.8 6.3 31.0 6.6 33.9 8.4 44.2 5.7 31.6 6.2 49.7 10.4 58.3 9.4
0.0 0.0 0.0 0.0 0.0 0.0 26.5 0.7 24.9 1.7 21.0 4.1 25.5 1.1 26.3 0.5
0.0 0.0 0.0 0.0 0.0 0.0 15.0 2.8 16.2 3.2 15.5 2.8 15.5 0.9 16.0 0.0
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Fig. 2. Representative figures indicate (A) 1-day-old juvenile fluke of Metagonimus yokogawai. Internal organs do not develop well and no uterine eggs are seen. Bar = 25 mm. (B) 4-day-old juvenile fluke of Metagonimus yokogawai. Sex organs develop fully to produce several uterine eggs (arrow). Bar = 25 mm. Inset indicating eggs (arrow) in the uterus with high magnifications. (C) 14-day-old adult fluke of Metagonimus yokogawai. Its size and internal organs grow greatly to be the largest and mature fully to produce numerous uterine eggs. Bar = 25 mm.
length 15.2 1.1 mm in width (Table 1, Fig. 2B). All of the detailed figures of various organs as well as ova developed in the flukes were summarized in Table 1. 4. Discussion Although Miyamoto (1985) found wildlife such as fowls of black kite (Miluvs migrans lineatus) captured from Hokkaido, Japan was one natural definitive host of M. yokogawai based on mature M. yokogawai adults with fully developed testis, ovary, and uterine eggs were found in the small intestine by a necropsy study, to date information regarding whether other fowls may play a role in transmission of M. yokogawai is not available. In Taiwan, various species of migratory birds often fly from the cold northern to the warm southern Taiwan to settle down temporarily during winter, thus whether those fowls may play a role in transmission of M. yokogawai is also concerned. The susceptibility of animal hosts to infection with M. yokogawai can be assessed by analyzing certain parameters e.g., chronological worm recovery rate (WRR),
worm growth, and fecundity (Chai et al., 1984). The present results revealed that in infected duck the highest WRR was present at 4 DPI, reaching 28.5% which was much lower than that in inbred strain BALB/c mice (80% at 3 DPI) or outbred strain ICR mice (66% at 1 DPI), interestingly these mice were also considered as not susceptible hosts to M. yokogawai infection (Chai et al., 1984; Guk et al., 2005). In contrast, in cats, dogs, or hamster, the development of worm was good, the recovery rate of worms was high and their maintenance of parasitism was fairly long (Yokogawa and Sano, 1968; Kang et al., 1983). Moreover, the growth of the worm seemed to be strictly limited as evidenced by that the length of the worm recovered from infected ducks at either 7 DPI (263.0 33.4 mm) or 14 DPI (274.3 4.4 mm) was rather smaller than that in BALB/c (535 102 mm at 7 DPI; 667 60 mm at 14 DPI) or C57BL/6J (532 136 mm at 7 DPI; 603 123 mm at 14 DPI) (Guk et al., 2005). However, the statistical significance in the WRR with each subsequent pair of experimental ducks showed apparent variability. The effects may be due to low sample numbers (only two ducks
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per inoculation dose), host/parasite biological variability, problems with inoculations, etc. Similarly, Chai et al. (1984) also found that the susceptibility for M. yokogawai was different even among the same mouse host of different strain, indicating genetic background may be involved; nevertheless, complex regulation may exist between parasite and hosts. In addition, the mean size of ova (24.8 mm 15.6 mm) produced from the worm appeared smaller than that in gerbil (30.4 mm 18.0 mm) (Lai, 1982) but its size was similar to that in hamster (27 mm 17 mm) (Rim et al., 1996). Although the present study revealed that metacercaria of M. yokogawai can infect successfully and subsequently matured to produce ova in infected duck from 4 DPI onwards, the present results suggested that duck did not seem to be susceptible to M. yokogawai infection because most worms were expelled within 14 days. This finding was also seen in various small rodent animals experimentally infected with M. yokogawai e.g., inbred strain BALB/c mice and outbred strain ICR mice in which most worms were expelled within 14 days (Chai et al., 1984; Guk et al., 2005). Interestingly, the time for ova found in the uterus of M. yokogawai and infected ducks present with watery diarrhea both were found accompanying by decreasing total WRR as started from 4 DPI onward in the trial. Although in the present study we did not intend to evaluate the clinical sings of infected ducks, however, one of inoculated ducks seemed likely die of severe watery diarrhea at 14 DPI in the trial due to presence of apparent watery-like feces. It was also evident from 4 DPI onward that worms were frequently recovered from the lower portion (sections 3 and 4) other than the upper or middle portion the preferred site for M. yokogawai to reside in the small intestine as similar results were also seen in murine host experimental infection of M. yokogawai (Chai et al., 1984). Based on these results, we might speculate that when the fecundity of worm began that seemed to be able to produce some antigenic materials capable of eliciting the intestinal immunological response to interfere the invasion and maturation of M. yokogawai and consequently the worm was expelled from the intestine thus leading to decreased WRR and possibly the worm’s relocation to the lower portion of the small intestine of infected ducks from 4 DPI onward in the trial. Previous study has indicated that rats infected with M. yokogawai present with intensive intestinal mucosal response which was proposed to be involved in the expulsion of the worm (Chai et al., 1993) and leukocytes including eosinophil, neutrophil, lymphocytes, and mucosal mast cells were suggested to be involved in the expulsion process of the worm from the small intestine of mice or rats experimentally infected with M. yokogawai (Chai et al., 1984, 1993). However, whether mucosal immunity involved in defending against the M. yokogawai infection in the duck’s intestine remained to be determined. Nevertheless, it might alternatively consider that this phenomenon may simply be a matter of the time required for the host to mount an effective immunological response to the parasite, and is not due to the generation of any maturation-specific fluke antigens. In conclusion, the current work was a pilot study to indicate that fowls such as ducks did not seem to be
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susceptible to M. yokogawai infection because most worms were expelled within 14 days possibly due to intestinal immunological interference thus affecting the growth as well as development of M. yokogawai in duck’s intestine. Therefore, it was proposed a transient role for ducks to transmit M. yokogawai when they deposited the ova via feces into marsh where snails and fish were abundant. Nevertheless, further studies are needed to better elucidate any role ducks might play in the epidemiology of this parasitic infection e.g., assess infectivity and development of ova from ducks in freshwater snail and subsequently whether cercaria shedding from the snails are capable to develop into fully infective metacercaria in the fresh-water fish as assessed by inoculation of fish-eating animals. References Akahane, H., Kusano, F., Hayashi, K., 1983. An epidemiological study on Metagonimiasis in Shiga Prefecture, with special reference to the infection pattern of Metagonimus yokogawai metacercariae of ayu (Plecoglossue altivelis) reared in ponds. Jpn. J. Parasitol. 32, 85–90 (in Japanese with English abstract). Chai, J.Y., Seo, B.S., Lee, S.H., 1984. Study on Metagonimus yokogawai (Katsurada, 1912) in Korea VII Susceptibility of various strains of mice to Metagonimus infection and effect of prednisolone. Kisaengchunghak Chapchi 22, 153–160. Chai, J.Y., Kim, T.H., Kho, W.G., Chung, S.W., Hong, S.T., Lee, S.H., 1993. Mucosal mast cell responses to experimental Metagonimus yokogawai infection in rats. Korean J. Parasitol. 31, 129–134. Chai, J.Y., Yun, T.Y., Kim, J., Huh, S., Choi, M.H., Lee, S.H., 1994. Chronological observation on intestinal histopathology and intraepithelial lymphocytes in the intestine of rats infected with Metagonimus yokogawai. Korean J. Parasitol. 32, 215–221. Chai, J.Y., Kim, J., Lee, S.H., 1995. Invasion of Metagonimus yokogawai into the submucosa layer of the small intestine of immuno-suppressed mice. Korean J. Parasitol. 33, 313–321. Chai, J.Y., Lee, S.H., 2002. Food-borne intestinal trematode infections in the Republic of Korea. Parasitol. Int. 51, 129–154. Cho, S.Y., Kang, S.Y., Ryang, Y.S., 1981. Helminthes infections in the small intestine of stray dogs in Ejungbu City, Kyunggi Do, Korea. Korean J. Parasitol. 19, 55–59. Guk, S.M., Park, J.Y., Seo, M., Han, E.T., Kim, J.L., Chai, J.Y., 2005. Susceptibility of inbred mouse strains to infection with three species of Metagonimus prevalent in the Republic of Korea. J. Parasitol. 91, 12–16. Huh, S., Sohn, W.M., Chai, J.Y., 1993. Intestinal parasites of cats purchased in Seoul. Korean J. Parasitol. 31, 371–373. Kang, S.Y., Cho, S.Y., Chai, J.Y., Lee, J.B., Jang, D.H., 1983. A study on intestinal lesions of experimentally reinfected dogs with Metagonimus yokogawai. Kisaengchunghak Chapchi 21, 58–73 (in Korean with English abstract). Kino, H., Suzuki, T., Oishi, H., Suzuki, S., Yamagiwa, S., Ishiguro, M., 2006. Geographical distribution of Metagonimus yokogawai and M. miyatai in Shizuoka Prefecture, Japan, and their site preferences in the sweetfish, Plecoglossus altivelis, and hamsters. Parasitol. Int. 55, 201–206. Koga, G., 1938. On the condition of worms of Metagonimus and Heterophyes within the final host’s intestinal tract, and effect of worms upon the tract. Nippon Kiseichu Gakkai Kiji 10, 84. Lai, T.S., 1982. Two new fish intermediate host of Metagonimus yokogawai: Acrosscheilus formosanus and Zacco pachycephalus found in Taipei, Taiwan. Med. Res. 4, 1283–1290 (in Chinese with English abstract). Miyamoto, K., 1985. Studies on zoonoses in Hokkaido. 7. Survey of natural definitive hosts of Metagonimus yokogawai. Jpn. J. Parasitol. 34, 371– 376. Rim, H.J., Kim, K.H., Joo, K.H., 1996. Classification and host specificity of Metagonimus spp. from Korean freshwater fish. Korean J. Parasitol. 34, 7–14. Seo, B.S., Hong, N.T., 1969. Study on Metagonimus yokogawai (Katsurada, 1912) in Korea. I. On the metacercaria, its distribution in the second intermediate host and the development in the final host. Kisaengchunghak Chapchi 7, 129–142 (in Korean with English abstract).
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Yokogawa, S., 1913. On a new trematode, of which second intermediate host of Plecoglossus altivelis, with establishment of a new genus for this trematode. Okayama Igakkai Zasshi 266, 337–358 (in Japanese). Yokogawa, M., Sano, M., 1968. Studies on the intestinal flukes IV. On the development of the worm in the experimentally infected animals with metacercariae of Metagonimus yokogawai. Jpn. J. Parasitol. 17, 540–545.