STUDIES ON HATCHABILITY OF SCHISTOSOMA JAPONICUM EGGS IN SEVERAL EXTERNAL ENVIRONMENTAL CONDITIONS

Transcription

1 STUDIES ON HATCHABILITY OF SCHISTOSOMA JAPONICUM EGGS IN SEVERAL EXTERNAL ENVIRONMENTAL CONDITIONS JIRO ITO Parasitology Division, National Institute of Health, Tokyo, Japan (Received: March 3rd, 1955) INTRODUCTION Several factors influencing the hatching of Schistosorna japonicum eggs were reported by Miyagawa (1916), Faust and Meleney (1924), Magath and Mathieson (1946), Faust (1947), Maldonado and Matienzo (1948), Ingalls et al (1949) and Sugiura et al (1954), but there were only a few investigators who have dealt with the resistance of the eggs to various external environmental conditions. These problems are essential for the elaboration of ffective measures for their control, but many important points in relation to this aspect had been only superficially studied. As to the effect of high and low temperatures, Miyagawa (1916) and Faust (1947) reported very briefly respectively. Several meagre data concerning the effects of salinity and dryness were found in the reports of Miyagawa (1916), Magath and Mathieson (1946) and Ingalls et al (1947). A series of studies designed to provide the resistance of the eggs necessary for a better understanding of the epidemiology of schistosomiasis japonica was thus undertaken. The present paper reported a series of experiments concerning the influence of temperatures ranging from -20 Ž to 60 Ž, salinity, dryness and exprements of host on the hatchability and resistance of schistosome eggs. GENERAL EXPERIMENTAL PROCEDURE Eggs used were taken from normal-looking stools of an experimentally infected rabbit during 60 `150 days after infection. Half-dryed or bloody mucoid stools were not employed to avoid using the immature or unhatchable eggs. The stools furnished by the rabbit were brought to the laboratory as soon as possible. They were immediately broken up in large quantities of previously boiled spring water (ph ), filtered through a gauze and the menstrum was washed repeatedly with the spring water in a large V-shaped vessel. This cleaned menstrum was gently agitated and each 25 ml of it was introduced into a series of 50 ml Erlenmyer flasks. The time elapsed during the entire procedure of washing the stool and introducing into the flasks was about 30 minutes. The number of eggs in each flask was estimated by counting them contained in 2.5 ml of such menstrum. After treated with each condition the hatched miracidia were accurately counted every 3-6 hours for a period of 3-5 successive days. The procedure was as follows. Almost all water in flask was carefully drawn out by a micropipette without sucking up the egg sediment, 175

2 176 ITO Vol. 8 The miracidia swimming in water were killed with formaline, stained with neutral red, gathered by centrifugation and counted under the low binocular dissecting microscope. Equal amount of spring water was introduced into the flasks and kept at each incubator for the next observation. EFFECT OF TEMPERATURE ON HATCHING In order to determine the optimal temperature on hatching, every three flasks handled as described above were kept at each temperature of 0 Ž, 3 Ž, 8 Ž, 13 Ž, 18 Ž, 23 Ž, 28 Ž, 33 Ž and 38 Ž (Table 1). No hatching of eggs was Table 1. Effect of normal temperatures on hatching observed at 0 Ž and 38 Ž which confirmed the results of Miyagawa (1916), Magath and Mathieson (1946) and Ingalls et al (1949). Eggs were hatched at temperature extremes of 3 Ž and 33 Ž representing the hatching rate of 0.17% and 23.0% respectively. The data showed a wide optimal temperature ranging from 13 Ž to 28 Ž in which no significant difference of hatching rate was existed. The result of this optimal temperature range appeared to favor that established by Sugiura et al (1954). In these optimal temperatures, the hatching of eggs took place over a period of 3 to 5 days, about one-half emerging within 24 hours, almost all doing so withing the.next 24 hours. The hatching curve represented themselves as the sigmoid (Textfig. 1).

3 1955 H ATCHABILITY OF S. JAPONICUM EGGS 177 Fig. 1. Hatching rate in each temperature As to the effects of high and low temperatures on the resistance of eggs, the hatchability of them exposed to temperature ranging from -20 Ž to 60 Ž was tested. The procedure was as follows: a series of flasks containing the egg menstrum was exposed to each temperature for a certain time intervals; then these flasks were removed to the incubator at 28 Ž to make the viable eggs hatch; hatched miracidia were accurately counted every 6 hours for 3 successive days. Increasing and decreasing time intervals at each temperature were always recorded. Total number of hatched miracidia as well as its percentage comparing with that of control was shown in Table 2. The exposing time intervals required for killing all eggs at various temperatures was as follows; 1 second at 60 Ž, 3 minutes at 55 Ž, 30 minutes at -20 Ž, 1 hour at 50 Ž, 4 hours at -10 Ž, 8 hours at 45 Ž and 19 days at 38 Ž. Several eggs were found still alive after the exposure to 0 Ž for 81 days and 3 Ž for 370 days. When employed the total time interval including those of increasing and decreasing of temperatures the results mentioned above were corrected as 4 minutes at 60 Ž, 7 minutes at 55 Ž, 6 hours at -20 Ž and 8 hours at -10 Ž. Current result of 60 Ž appeared to favor that of Miyagawa (1916) who noted that the eggs lost their hatchability at 60 Ž for 5 minutes. As to the low temperature, the current result of 3 Ž showed longer period than that of Faust (1947) who reported that only a few viable eggs were found in 180 days at 4 Ž.

4 178 ITO Vol. S Table 2. Hatchability of eggs exposed to cold and warm temperatures in each time intervals EFFECT OF SALINITY ON HATCHING After preparing a series of flasks in the same manner as mentioned above the water component in the flasks was sufficiently replaced with each 0.2%, 0.5%, 0.8% and 1.0% NaCl-solution. These flasks grouping in three were immediately kept in an incubator at 28 Ž. Then the hatched miracidia were accurately counted every 6 hours for 4 successive days (Table 3). No significant Table 3. Hatchability of eggs in dilute NaCl-solutions at 28 Ž.

5 1955 HATCHABILITY OF S. JAPONICUM EGGS 179 difference was observed between the control and 0.2% NaCl-solution in respect of total hatching rate, whereas the hatching period was somewhat delayed in the latter case. In the cases of 0.5 `1.0% solutions, the higher was the concentration, the lower was the hatching rate and the longer was the hatching period. In 1.0% solution, the hatching rate was only 1.8% compared with that of control. When the salt solution was replaced with spring water at the end of this experiment a large number of miracidia was liberated within a short time interval. Microscopical examination of the sediment revealed the presence of motile embryos within the eggs. These findings proved the fact that the eggs lost no hatchability but were prevented from their hatching in such diluted salt solution. These results agreed with that of Magath and Mathieson (1946) who tested the hatching of eggs in 0.7% NaCl-solution. The next experiments were performed to know the resistance of eggs immersed in a salt solution of high concentration. All procedures were taken place under the temperature of 28 Ž. The water component in a series of flasks was sufficiently replaced with each 1.0%, 2.0%, 3.0% and 4.0% NaCl-solution. These flasks were preserved in an incubator at 28 Ž for 24 hours and 72 hours respectively. Then the eggs in these flasks were washed repeatedly with spring water and were again kept in the same incubator to make the viable eggs hatch. Hatched miracidia were counted every 6 hours for a period of 3 days (Table 4). No apparent effect was observed on the hatchability of eggs immersed Table 4. Effects of salinity and dryness on hatching at 28 Ž. in 1.0% NaCl-solution for 24 hours. This result differed from that of Ingalls et al (1949) who reported that the decreasing effect on hatching rate was observed on the eggs immersed in physiological salt solution for 1.5 hours. In the other cases, the higher was the concentration of solution and the longer was the immersing time interval, the lower was the hatching rate. All eggs lost their hatchability by immersing in 4.0% NaCl-solution for both 24 and 72 hours.

6 ITO 180 Vol. 8 EFFECT OF DRYNESS ON HATCHING Several preliminary experiments revealed the fact that feces of rabbit preserved in an incubator at 28 Ž and relative humidity were almost completely dried up for 24 hours. Current experiments were taken place to determine the hatchability of eggs contained in each feces of 8, 16, 24, 48 and 72 hours drying condition in the same incubator. These dried feces were broken up, washed repeatedly and introduced into a series of flasks with 25 ml of water The flasks were then kept in an incubator at 28 Ž and the hatched miracidia. were counted every 6 hours for 3 successive days. In this experiment, the number of eggs was estimated by that in every milligram feces, because previous test proved the fact that as far as the feces excreted from one rabbit in a short time was concerned, the number of eggs in each milligram of feces was nearly the same. Total number of hatched miracidia and its percentage are shown in Table 4. The data showed that about 90% of eggs lost their hatchability in the feces of 8 hours drying, almost all did so in that of hours drying. Seventy two hours drying time interval was required for killing all eggs in the feces. EFFECT OF EXCREMENTS OF HOST ON HATCHING In the last chapter, it was reported that the eggs contained in dried feces lost their hatchability for 3 days at 28 Ž. In the present experiment, the hatchability of eggs in wet feces and in urine was tested at 8 Ž, 18 Ž and 28 Ž respectively. The feces taken immediately after discharging from an infected rabbit were kept in moist chambers at each temperature for each time interval. After preserving in the moist chamber each feces was broken up, washed and introduced into a series of flasks, and the hatched mira acidia were counted in the same manner as mentioned above. Each test was performed three times and the average data are shown in Table 5. In the case of 28 Ž, all eggs lost Table 5. Hatchability of eggs preserved in the wet feces of rabbit in each time intervals * showed the datum at 20 days interval

7 1955 HATCHABILITY 0F S. JAPONICUM EGGS 181 their hatchability for 20 days preserving time interval, whereas in the cases of 18 Ž and 8 Ž the eggs held their hatchabilities for surprisingly long period. Some eggs were still alive after 113 days at 18 Ž and 180 days at 8 Ž. The next experiments were taken place on the eggs immersed in five urine specimens, namely that of non-infected rabbit, cow and man and that of infected rabbit and cow. The feces from an infected rabbit were broken up in these fresh urine, filtered through a gauze and washed repeatedly with the same urine. Then 25 ml of such menstrum was introduced into each flask. After preserving these flasks in an incubator at 28 Ž, 18 Ž and 8 Ž for several time intervals respectively, the eggs contained in the flasks were sufficiently washed with spring water and kept at 28 Ž to make viable eggs hatch. The data were summarized in Table 6. No significant difference of hatching rate was observed between the eggs immersed in the urine of non-infected animal and that of infected one. All eggs, in general, lost their hatchabilities within a relatively short time interval of immersing in the urine. Moreover, the higher was the temperature the shorter was the immersing time interval for killing them. These time intervals were 1 day in the urine of the rabbit, 2 days in that of cow and 3 days in that of man at 28 Ž. These were prolonged twice as much as that of 28 Ž at the temperature of 18 Ž, and again prolonged four times as much as that of 28 Ž at 8 Ž. At the end of these experiments, microscopical observation of the sediment revealed the fact that all eggs were recognized as a death. SUMMARY AND CONCLUSION The author investigated the effects of temperature, salinity, dryness and excrements of host on the hatchability and resistance of the eggs. Materials were taken from the feces of an experimentally infected rabbit. Obtained results were summarized as follows: 1. No eggs hatch at 0 Ž and 38 Ž. They hatched at temperatures rang ing from 3 Ž to 33 Ž, whereas the optimal temperatures were from 13 Ž to 28 Ž. In these optimal temperatures, the hatching of eggs took place over a period of 3 to 5 days, about one-half emerging within 24 hours, almost all doing so within the next 24 hours. 2. The time intervals required for killing all eggs at various temperatures were as follows ; 1 second at 60 Ž, 3 minutes at 55 Ž, 30 minutes at -20 Ž, 1 hour at 50 Ž, 4 hours at -10 Ž, 8 hours at 45 Ž and 19 days at 38 Ž. Several eggs were found still alive for 81 days at 0 Ž and 370 days at 3 Ž. 3. In the medium of 0.2% NaCl-solution, no apparent effect was observed on the hatchability of eggs, and in that of 1.0%, the hatching rate decreased down to about 2%. Unhatched eggs at that time were recognized as alive. The eggs immersed in 4 % NaCl-solution for 24 hours lost their hatchabilities and were recognized as dead.

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9 1955 HATCHABILITY OF S. JAPONICUM EGGS Eggs in the dried feces lost their hatchability in a relatively short time interval. All eggs did so by preserving in such feces for 3 days at 28 Ž. 5. In the wet feces of the rabbit, all eggs lost their hatchability for 20 days at 28 Ž, but several eggs held that ability for 113 days at 18 Ž and 180 days at 8 Ž. 6. In the urine of the rabbit, cow and man, all eggs lost their hatchability for one or three days at 28 Ž. At the temperature of 18 Ž, these time inter vals were prolonged twice as much as that of 28 Ž, and again prolonged four times as much as that of 28 Ž at the temperature of 8 Ž. 7. Above mentioned results were considered as follows. The schistosome eggs in natural water can easily hatch at the temperatures normally encountered in their natural habitats. Their hatchability may be held for a relatively long period both in low but not freezing water and in wet feces of the rabbit. Their hatchability was on the other hand, easily lost within a short time interval when preserved at the conditions such as freezing and high temperatures, hypertonic medium, dryness and the urine of the host. On publishing this report, author should express my heartfull thanks to the Chief of Parasitology Division, Dr. Yoshitaka Komiya who has done continuously his guidance and revision for this study. Further I wish to express my thanks to Mr. Kazuo Yasuraoka and Miss Koko Yamaguchi for their kindness to help my study. A part of this study was relied on the expenditure of Ministry of Education and Ministry of Health and Welfare. REFERENCES 1. Faust, E: C.: The effects of cold temperatures on the eggs of Schistosoma japo nicum. Jour. Parasitol., 33 (2), , Faust, E. C. and Meleney, H. E.: Studies on schistosomiasis japonica, etc. Amer. J. Hyg. Mono.gr. Ser. 3, 399 pp., Ingalls, J. W., Jr., Hunter, G. W. III, McMullen, D. B. and Bauman, P. M.: The molluscan intermediate host and schistosomiasis japonica 1. Observations on the conditions governing the hatching of the eggs of Schistosoma japonicum. Jour. Parasitol., 35 (2), , Ito, J.: The effects of temperatures on the eggs of Schistosoma japonicum I and II. Nisshin Igaku, 40 (10), , 41 (2), 88-95, 1953 (In Japanese with English summary). 5. Ito, J.: The effects of dryness and salinity on the hatchabilities of eggs of Schistosoma japonicum. Nisshin Igaku, 41 (3), , 1954 (in Japanese with English summary). 6. Ito, J.: The viability of eggs of Schistosoma japonicum in the excrements of several hosts. Nisshin Igaku, 41 (7), , 1954 (in Japanese with English sum mary). 7. Magath, T. B. and Mathieson, D. R.: Factors affecting the hatching of ova of Schistosoma japonicum. Jour. Parasitol., 32 (1), 64-68, Maldonado, J. F. and Acosta-Matienzo, J.: Biological studies on the miracidium of Schistosoma mansoni. Amer. Jour. Trop. Med., 28 (5), , 1948.

10 184 ITO Vol Miyagawa, Y.: Ueber die Verð nderungen der Eier des Schistosoma japonicum unter den Einwirkungen verschiedener physikalischen und chemischen Agenzien u nd ð¹ber einige prophylaktische Massregeln der Schistosomiasis. Mitt. Med. Fak. Univ. Tokyo, 15, , 1916.