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The Infection Experiment of Paragonimus Westermani in Its First

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The Infection Experiment of Paragonimus Westermani in Its First Jap. J. M. Sc. & Biol., 14, 39-43, 1961 INFECTION EXPERIMENT OF PARAGONIMUS WESTERMANI IN ITS FIRST INTERMEDIATE HOST, SEMISULCOSPIRA LIBERTINA YOSHITAKA KOMIYA, NORIJI SUZUKI AND YOICHI ITO Department of Parasitology, National Institute of Health , Tokyo (Received: February 28th, 1961) The infection experiment of Paragonimus westermani in its first intermediate host, Semisulcospira libertina, was made by Nakagawa (1915), Kobayashi (1918), Yokogawa (1919) and Miyairi (1919, 1934). But due to the difficulty in rearing this snail for a sufficient time after infection, no cercaria thereof was obtained in these experiments . The present authors succeeded in maintaining this snail for a long time after exposure to miracidia, rendering possible the experimental infection of it with the miracidia of Paragonimus westermani, and were able to obtain the cercariae of this worm . The following is the summary of the results. MATERIALS AND METHODS Miracidia of P. westermani : Metacercariae were obtained from the second intermediate host Eriocheir japonicus collected from Kannami Area of Shizuoka Prefecture . They were fed to dogs, which discharged Paragonimus eggs in feces after a few months. These eggs were washed several times with tap water, collected, put into a Petri dish with water and maintained for 12 to 20 days at the temperature of 30•Ž up to the time when the miracidia were fully developed in their shells. The miracidia hatching was made by adding a sudden change of temperature to the eggs . Snail : Young specimens of Semisulcospira libertina Gould , 5 to 10 mm in shell length, were collected from Kannami Area of Shizuoka Prefecture and were maintained in a water tank in the laboratory for the experimental purpose . The reason why the young specimens of snails were applied was that they possessed less resistance to the infection with the miracidia and that they could have less chance of natural infection, because the area in which they were collected was one of the endemic areas of Paragonimiasis . Those snails were deliberately collected from the endemic area because, if there were strains of different resistances to the infection with Paragoni - mus, those from the endemic area were considered to have a comparatively lower resistance to it . Snails after being exposed to miracidia were put in cages of vinyl net and were maintained in the water tank at the temperature of about 25•Ž. As their feed materials , artificial solid feeds and some kinds of water weed were applied . The water tank was continuously aerated. Methods of infection: About 200 cc of water containing numerous miracidia were put into a Petri dish, into which about 50 to 75 specimens of the snail were put . The number of miracidia per snail was about 140-2 ,900. The number of snails used for each series of experiments is shown in Table 1. After the snails were exposed to miracidia for about 20 hours at the room tempera- ture, they were removed from the dish to the water tank and maintained . The same number of snails as that used in each series of experiment was maintained as control, and they were also dissected and confirmed to be negative for Paragonimus infection. The examination of Paragonimus larvae in snails : On every week following exposure to miracidia, 3 snails were dissected at a time and examined for the presence of larvae of Paragonimus . The sporocyst could be discriminated from the snail tissue particularly by the presence of their This study was supported by a grant from the Ministry of Education. 小 宮 義 孝 ・鈴 木 了 司 ・伊 藤 洋 一(国 立 予 防衛 生 研 究 所 寄 生 虫部) 39 40 KOMIYA et al. Vol. 14 Table 1. Average number of exposed miracidia per snail in each experiment proper flame cells. The rediae of Paragonimus were very difficult to make difference from those of other species already naturally infected. Snails used in these experiments were, however, maintained in the laboratory for a considerable time prior to exposure. Therefore, if they had been already naturally infected with Paragonimus or other trematodes, the rediae of the latters would have attained almost to the level of their maturity even at the time of the initial dissection. Thus rediae contained in the experimental snails could be differentiated from others by their size and structure besides their characteristic features. RESULTS Eight experimental series were carried out during the period of approximately 2 years and 248 specimens of snails were dissected after exposure to miracidia of Paragonimus. The results are shown in Fig. 1. In this figure are presented characters of Paragonimus found in the snails, of which character S indicates sporocyst, R redia and C cercaria and the time of appearance of these characters in the snails which habored them. Fig. 1. Appearance of sporocyst, redia and cercaria in eight experimental series. 1961 INFECTION OF P. WESTERMANI IN S. LIBERTINA 41 24 hours after exposure (•~320) 3 weeks after exposure (•~500) Fig. 2. Sporocyst. Fig. 3. Daughter redia. Ten weeks Fig. 4. Cercaria. Eleven weeks after exposure. after exposure. (Experimental series No. 8). (Experimnetal series No. 13). 42 KOMIYA et al. Vol. 14 Three specimens being dissected every week, only sporocysts were found in snail tissues up to 3 weeks after exposure. Numerous sporocysts, usually hundreds in number , were seen directly after exposure, but they became fewer in number as the time elapsed . Mother rediae were seen, hundreds in number, in one series as early as five weeks after exposure, and among snails of series No. 8, 9 and 13 characteristic cercariae of Parago- nimus westermani were recognized 9 or more weeks after exposure . The number of snails haboring cercariae were 6 out of totally 14 specimens. DISCUSSION As stated above in 6 specimens of snails dissected 9 or more weeks after exposure were found cercariae of Paragonimus westermani. But there remains the possibility that these snails might already be naturally infected before experimental exposure to miracidia, because the snails used in the experiments were collected from an endemic area of Paragonimiasis. But such a possibility can be denied for the following reasons. Namely, (1) all snails of all control groups set on every experimental series were proved to be negative for Paragonimus larvae ; (2) all the snails used in the experiments had been maintained, as already mentioned, for a considerable time in the laboratory at the temperature of about 18-23•Ž before the experimental exposure to miracidia. Hence, if natural infection of Paragonimus had occurred in those snails in fields, more developed rediae or cercariae would have been recovered during the earliest weeks after exposure ; (3) as shown in Fig. 1, in the experimental series No. 13 in which cercariae were found during suc- cessive 3 weeks from 9 to 11 weeks after exposure, totally 3 positive cases were obtained among 7 specimens, representing the probability of about 42.9 per cent, whereas the rate of the natural infection of Paragonimus westermani in the endemic area from where the snails were collected was only 5 per cent. It is noticed here, however, that the infectivity of Paragonimus westermani to snails appeared rather too low as compared with that of P. kellicotti and P. ohirai in spite of the exposure to massive miracidia. In case of experimental infections of the latter 2 species cercariae were easily obtained from the experimentally infected snails exposured to miracidia (Ameel, 1934; Yokogawa et al., 1958). Such differences would make one suspect whether other mode of infection would be operative in fields. The results of the experimental infection above mentioned showed that the number of sporocysts per snail decreased so rapidly with time after exposure. This fact appears to support the above mentioned suspicion. To make clear this problem, further study is to be undertaken. SUMMARY Since no cercaria of Paragonimus westermani has yet been obtained from an experi- mental infection of Semisulcospira libertine, its natural first intermediate host, with its miracidia, young specimens of Semisulcospira libertina, were collected from an endemic area of P. westerman i, then infected with its miracidia en masse and maintained in the laboratory. For about 10 to 20 weeks after exposure to miracidia, cercariae of this worm were first obtained experimentally. But the rate of the infection appeared rather too low. A discussion was made concerning this point. REFERENCE AMEEL, D. J. (1934) : Paragonimus, its life history and distribution in North America and its taxonomy (Trematoda : Troglotrematidae), Am. J. Hyg., 19, 279-317. 43 1961 INFECTION OF P. WESTERMANI IN S. LIBERTINA AMEEL, D. J., CORT, W. W. & VAN DER WOUDE, ANNE (1951) : Development of the mother sporocyst and rediae of Paragonimus kellicotti Ward 1908. J. Parasotol., 37, 396-404. ANDO, R. (1915 a) : Studies on Paragonimus westermani (II). •gChugai-Iji-Sinpo•h, 847, 827- 840 (text in Japanese). ANDO, R. (1915 b) : Studies on Paragonimus westermani (III). •gChugai-Iji-Sinpo•h, 851, 1109-1127 (text in Japanese). ANDO, R. (1920a) : Studies on Melania libertina as a first intermediate host of Paragonimus westermani (I). •gTokyo-Iji-Shinshi•h, 2175, 861-865 ; 2178, 987-991 (text in Japanese). ANDO, R. (1920b) : Morphological and biological studies on cercaria of Paragonimus wester- mani. •gChugai-Iji-Shinpo•h, 983, 286-297 (text in Japanese). ITO, J., MOCHIZUKI, H. & NOGUCHI, M. (1959) : Studies on the cercariae parasitic in Semisu- lcospira libertina in Shizuoka Prefecture. Jap. J. Parasitol., 8, 918-922 (text in Japanese with English summary). KOBAYASHI, H. (1918) : Studies on first intermediate host of Paragonimus westermani. •gCho- sen Igakkai Zasshi•h, 21, 1-8 (text in Japanese). KOBAYASHI, H. (1924) : The supplemental report on the development of Paragonimus wester- mani. •gChosen Igakkai Zasshi•h, 50, 80 (text in Japanese). MIYAIRI, K.
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