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Observations on human isospora
556 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL ~/[EDICINE AND HYGIENE.
Vol. 62.
No. 4.
1968.
O B S E R V A T I O N S ON H U M A N ISOSPORA
V. ZAMAN
Department of Parasitology, Faculty of Medicine, University of Singapore Introduction
It is generally believed that ~zo different species of Isospora are found in man-I. belli (WE~o~, 1923) and I. h~?ninis (Rivolta, 1878) DOBV.LL, 1919. ELSDON-DEW and F~EDtC~N (1953) have presented photographic evidence to illustrate these differences. Contrary to this belief are the views presented by HENDERSONet al. (1963) who feel that the species should be regarded as one. 6 cases of Isospora infection were recently studied by the author in Singapore. These were followed by weekly stool examination and careful observation of the morphology of the o6cyst before and after sporulation. The results of these investigations lend support to the suggestion of HENDERSON et al. (1963) that I. belli and I. hominis are synonymous. Mature o6cysts from these patients were introduced into the stomachs of 3 gibbons, 2 of which became infected (ZAMAN, 1967). Material and m e t h o d s
Freshly passed stools from patients with Isospora infection were examined immediately, by direct smear, followed by zinc sulphate and sugar flotation (each with a specific gravity of approximately 1.180). The oScysts were photographed in black and white with a Leitz Orthomat, on Panatomic X (Kodak, ASA 32) with a green filter. To induce sporulation, stools were first washed in distilled water by centrifugation and then transferred to conical flasks containing 2% potassium bichromate solution. The contents of these flasks were stirred by a magnetic stirrer for up to 24 hours. They were kept at room temperature, 25-30°C. Some of the stools were similarly stirred in distilled water only, without potassium bichromate. Stool samples were taken out at 12 hours and 24 hours to study the extent of sporulation. The samples showing a high percentage of sporulation were then washed free of potassium bichromate and stored at 5°C. in distilled water for other experimental studies. Results
The freshly passed o6cysts measured 22-33 × ll-16Vt. The sporulated o6cysts in which the o6cyst wall had collapsed measured 17-22 × 10-16 ~. In the freshly passed stools most of the o6cysts were found in the one sporoblast stage (Fig. 1), some in the two sporoblast stage (Fig. 2). Occasionally, even earlier stages were seen in which the sporoblast had not formed (Fig. 3). The shape of the o6cysts varied from spindle-shape to more elongated forms with a slight constriction at one pole. Occasionally, o6cysts were seen filled with granular contents, which did not sporulate (Fig. 4). I am grateful to Dr. Wong Yip Chong of the Woodbridge Hospital, Singapore, whose co-operation made this study possible.
FIG. 3. An early oiicyst in which the sporoFIG. 2. An &cyst showing two sporoblasts. FIG. 1. An oijcyst showing a single sporoblast. FIG. 4. Two okysts, one in which the sporocysts have The clear area in the centre probably represents the nucleus. blast has not formed. FIG. 5. An oiicyst with a single sporocyst containing formed and the other which is filled with coarse granules and has failed to develop. FIG. 6. A mature &cyst showing sporozoites inside the sporocysts. FIG. I. A mature ticyst sporozoites and a large residual mass. collapsed &cyst wall. FIG. 8. A mature oacyst showing a completely showing a partially collapsed oikyst wall. (To Face Page 556)
v. ZAM~
557
These were presumed to be either unfertilized or dead. Sometimes abnormal forms containing a single sporocyst were observed (Fig. 5). WFrcyor~and O'Com~OR (1926) had also noticed such abnormal forms. Sporulated otcysts and spor0cysts were also seen, twice, in the freshly passed stools of one patient. The otcysts suspended in potassium bichromate and distilled water began to sporulate within 12 hours; in 24 hours 80-90% had sporulated. A typical sporulated ot~cyst showed crescent-shaped sporozoites in each sporocyst (Fig. 6). More sporulation was obtained in specimens kept in 2% potassium bichromate solution than in distilled water. In 10 to 15% of the sporulated oiScysts the otcyst wall showed varying degrees of disintegration. In some it was partially collapsed (Fig. 7) and in others it was completely collapsed, leaving just 2 sporocysts attached to each other (Fig. 8). These sporocysts resisted separation even after considerable pressure on the coverglass, probably owing to the binding effect of the collapsed o~cyst wall. These otcysts appeared to be identical with the forms generally described as L hominis.
Discussion The resuks show that there is a considerable variation in the size and shape of lsospora belli otcysts, some of which, such as the presence of monosporic otcysts, could
be attributed to abnormal development. One of the criteria used for differentiating L hominis from 1. belli is the belief that the former are passed as fully mature otcysts. However, in I. belli sporulation is almost complete within 25 hours, and it is, therefore, easy to imagine situations when sporulated forms of 1. belli are passed in the stools. In this series there was one case in which sporulated forms and sporocysts were observed in the stools. The other criterion used for differentiating L hominis from L belli is the absence of the otcyst wall in the former. This difference is also unreliable as a number of oScysts showing disintegrated otcyst walls were observed in specimens kept for sporulation. The damage to the otcyst wall cannot be due to potassium bichromate, as similar forms were observed even when the stools were kept in distilled water. It is therefore suggested that L hominis be regarded as synonymous with I. belli, according to the view put forward by HENnERSONet al. (1963).
Summary The morphology of Isospora otcysts was studied from 6 cases of human infection. There was a considerable variation in the size, shape and the state of development of the otcysts in freshly passed stools. 80-90% of the otcysts completed sporulation in 24 hours in potassium bichromate solution. The sporulated otcysts showed varying degrees of disintegration of the otcyst wall. In some cases the wall was partially collapsed; in others it was completely collapsed leaving just 2 sporocysts attached to each other. In view of these findings I suggest that L hominis be regarded as synonymous with I. belli. REFERENCES DOBELL, C. (1919). Parasitology, 11, 147. ELDSON-DEw, R. & FREEDMAN,L. (1953). Trans. R. Soc. trop. Med. Hyg., 47, 209. HENDEnSON, H. E., GILLEPSlE,G. W., KAPLAN,P. & STEBER,M. (1963). Am. ft. Hyg., 78, 302. WENVON, C. M. (1923). Ann. trop. Med. Parasit., 17, 23. Z.~a~, V. (1967). Trans. R. Soc. trop. 214ed. Hyg., 61, 857.
Vol. 62.
No. 4.
1968.
O B S E R V A T I O N S ON H U M A N ISOSPORA
V. ZAMAN
Department of Parasitology, Faculty of Medicine, University of Singapore Introduction
It is generally believed that ~zo different species of Isospora are found in man-I. belli (WE~o~, 1923) and I. h~?ninis (Rivolta, 1878) DOBV.LL, 1919. ELSDON-DEW and F~EDtC~N (1953) have presented photographic evidence to illustrate these differences. Contrary to this belief are the views presented by HENDERSONet al. (1963) who feel that the species should be regarded as one. 6 cases of Isospora infection were recently studied by the author in Singapore. These were followed by weekly stool examination and careful observation of the morphology of the o6cyst before and after sporulation. The results of these investigations lend support to the suggestion of HENDERSON et al. (1963) that I. belli and I. hominis are synonymous. Mature o6cysts from these patients were introduced into the stomachs of 3 gibbons, 2 of which became infected (ZAMAN, 1967). Material and m e t h o d s
Freshly passed stools from patients with Isospora infection were examined immediately, by direct smear, followed by zinc sulphate and sugar flotation (each with a specific gravity of approximately 1.180). The oScysts were photographed in black and white with a Leitz Orthomat, on Panatomic X (Kodak, ASA 32) with a green filter. To induce sporulation, stools were first washed in distilled water by centrifugation and then transferred to conical flasks containing 2% potassium bichromate solution. The contents of these flasks were stirred by a magnetic stirrer for up to 24 hours. They were kept at room temperature, 25-30°C. Some of the stools were similarly stirred in distilled water only, without potassium bichromate. Stool samples were taken out at 12 hours and 24 hours to study the extent of sporulation. The samples showing a high percentage of sporulation were then washed free of potassium bichromate and stored at 5°C. in distilled water for other experimental studies. Results
The freshly passed o6cysts measured 22-33 × ll-16Vt. The sporulated o6cysts in which the o6cyst wall had collapsed measured 17-22 × 10-16 ~. In the freshly passed stools most of the o6cysts were found in the one sporoblast stage (Fig. 1), some in the two sporoblast stage (Fig. 2). Occasionally, even earlier stages were seen in which the sporoblast had not formed (Fig. 3). The shape of the o6cysts varied from spindle-shape to more elongated forms with a slight constriction at one pole. Occasionally, o6cysts were seen filled with granular contents, which did not sporulate (Fig. 4). I am grateful to Dr. Wong Yip Chong of the Woodbridge Hospital, Singapore, whose co-operation made this study possible.
FIG. 3. An early oiicyst in which the sporoFIG. 2. An &cyst showing two sporoblasts. FIG. 1. An oijcyst showing a single sporoblast. FIG. 4. Two okysts, one in which the sporocysts have The clear area in the centre probably represents the nucleus. blast has not formed. FIG. 5. An oiicyst with a single sporocyst containing formed and the other which is filled with coarse granules and has failed to develop. FIG. 6. A mature &cyst showing sporozoites inside the sporocysts. FIG. I. A mature ticyst sporozoites and a large residual mass. collapsed &cyst wall. FIG. 8. A mature oacyst showing a completely showing a partially collapsed oikyst wall. (To Face Page 556)
v. ZAM~
557
These were presumed to be either unfertilized or dead. Sometimes abnormal forms containing a single sporocyst were observed (Fig. 5). WFrcyor~and O'Com~OR (1926) had also noticed such abnormal forms. Sporulated otcysts and spor0cysts were also seen, twice, in the freshly passed stools of one patient. The otcysts suspended in potassium bichromate and distilled water began to sporulate within 12 hours; in 24 hours 80-90% had sporulated. A typical sporulated ot~cyst showed crescent-shaped sporozoites in each sporocyst (Fig. 6). More sporulation was obtained in specimens kept in 2% potassium bichromate solution than in distilled water. In 10 to 15% of the sporulated oiScysts the otcyst wall showed varying degrees of disintegration. In some it was partially collapsed (Fig. 7) and in others it was completely collapsed, leaving just 2 sporocysts attached to each other (Fig. 8). These sporocysts resisted separation even after considerable pressure on the coverglass, probably owing to the binding effect of the collapsed o~cyst wall. These otcysts appeared to be identical with the forms generally described as L hominis.
Discussion The resuks show that there is a considerable variation in the size and shape of lsospora belli otcysts, some of which, such as the presence of monosporic otcysts, could
be attributed to abnormal development. One of the criteria used for differentiating L hominis from 1. belli is the belief that the former are passed as fully mature otcysts. However, in I. belli sporulation is almost complete within 25 hours, and it is, therefore, easy to imagine situations when sporulated forms of 1. belli are passed in the stools. In this series there was one case in which sporulated forms and sporocysts were observed in the stools. The other criterion used for differentiating L hominis from L belli is the absence of the otcyst wall in the former. This difference is also unreliable as a number of oScysts showing disintegrated otcyst walls were observed in specimens kept for sporulation. The damage to the otcyst wall cannot be due to potassium bichromate, as similar forms were observed even when the stools were kept in distilled water. It is therefore suggested that L hominis be regarded as synonymous with I. belli, according to the view put forward by HENnERSONet al. (1963).
Summary The morphology of Isospora otcysts was studied from 6 cases of human infection. There was a considerable variation in the size, shape and the state of development of the otcysts in freshly passed stools. 80-90% of the otcysts completed sporulation in 24 hours in potassium bichromate solution. The sporulated otcysts showed varying degrees of disintegration of the otcyst wall. In some cases the wall was partially collapsed; in others it was completely collapsed leaving just 2 sporocysts attached to each other. In view of these findings I suggest that L hominis be regarded as synonymous with I. belli. REFERENCES DOBELL, C. (1919). Parasitology, 11, 147. ELDSON-DEw, R. & FREEDMAN,L. (1953). Trans. R. Soc. trop. Med. Hyg., 47, 209. HENDEnSON, H. E., GILLEPSlE,G. W., KAPLAN,P. & STEBER,M. (1963). Am. ft. Hyg., 78, 302. WENVON, C. M. (1923). Ann. trop. Med. Parasit., 17, 23. Z.~a~, V. (1967). Trans. R. Soc. trop. 214ed. Hyg., 61, 857.