640
Gong, Y.-X. Cao, S.-Q., Shi, X.-Z. & Zhou, S.-C. (1990). Crvotosuoridiosis in Oinedao. China ‘foumal ofParasitic DiseaSeiCo;ltrol,~3,?2.
- -
Guan, J.-S., Cm, S.-L., Yen, X.-L., Zhao, X. H., Zhang, J.Z., Wang, Y.-M. & Tuo, Y. (1990). Preliminary clinical observations of cryptosporidial enteritis. Chinese Jouwtal of Pediatrics, 28,41.
Han, F. (1989). Infant cryptosporidiosis found in China. ChineseJournal of Pediatrics, 27,75-76. Han, F., Tan, W.-X. & Zhou, X.-L. (1987). Human cryptosporidiosis in Naming region-two casereports. Jiangsu Medicine, 12,692. Huang, S.-R., Zhang, H.-J,., Huang, M.-J., Li, S.-Y. & Zhang, L. (1990). Cryptosportdrum infection of calves in the suburbs of Beijing. China ZoonosesJournal, 6,45. Lu, L.-A., Fan, Z.-Z., Li, C.-C. & Liu, B.-Z. (1990). Two casesof infant cryptosporidiosis. HunanMedicine, 7, 106. Luo, M.-Y., Wang, Q.-N. & Zhen,, X.-P. (1988). Cryptosporidial infection and enteritis. Chmese 3ournal of Internal Medicine, 27,686-688. Su, Q.-P., Chen, D.-G., Zhao, Z.-G., Hua, Y., Hua, X.-L., Chen, S., Huang, M. H., Wan, J.-Q., Guo, Y.-H. & Li, S.M. (1989). Cryptosporidiosis from infants in Fuzhou region. China ZoonosesJournal, 5,35-36.
TRANSACTIONS OFTHEROYAL
Su, L.-Y., Chen, X.-C., Li, Y.-H. & Liu, J. H. (1990). Casereport of infant cryptosporidiosis. China Journal of Parasitic : DiseasesControl, 3, 154. Unaar, B. L. I’.. Soave, R., Faver, R. & Nash, T. E. (1986). Enzyme immunoassay detection of immunoglobulin ‘M and G antibodies to Cryptosporidium in immunocompetent and immunocomeromised oersons. 7ouwtal oflnfectious Diseases. lS3,570-578’. ” ’ ’ Yao, F.-B. & Chen, Y.-G. (1989). Preliminary study on the effir cacy of garlicin in treatment of childhood cryptosporidiosis. 3ournal of Xuzhou Medical College, 9,56.
Zhu, F.-C., Hun, Z.-X. & Wang, F.-P. (1990). A case-control studv of childhood crvutosuoridiosis in Wuhu. China Public __ _ Health, 6,503-504.
Zu, S.-X. & Du, M.-W. (1987). Human cryptosporidiosis seen in China.3ouwaal ofAnhuiMedica1 University, 22,276. Zuo, Y.-X., Chen, F.-Q. & Fang, L. (1990). Human and domestic cryptosporidiosis in Yunnan and experimental infection study. China ZoonosesJournal, 6, 34-35. Received 5 February 1992; revised 16 April 1992; accepted for publication 16 April I992
SOCIETY OF TROPICAL MEDICINE ANDHYGIENE (1992) 86, 640
/Short Report 1 Rapid purification and concentration technique for Cryptosporidium parvum oocysts M. Lyagoubi, A. Datry, I. Malet, M. Danis and M. Gentilini Laboratoire, de Parasitologie, Uniti INSERM 313, CHU Piti&Salpet&re, 91 Boulevard de l’H6pita1, 75534 Paris ckdex 13, France Purification of faecal Cyptosporidium oocysts is necessary for infection of cell cultures (in vitro models) and of laboratory animals (in vivo models). For immunological studies purified parasites are essential. C. parzwm oocysts are separated from patients’ stools or from the stools of naturallv or exuerimentallv infected animals. Several techniques are used to isolate the oocysts: PercoB@ gradients (DULSKI & TURNER. 1988). sucrose gradientsu(CURREN? & LONG, 1983) and’saturated NaCl solution (DATRY et al., 1989). In order to obtain a sufficient number of oocysts with these techniques, very long and fastidious manipulations are needed. We propose a technique in which stools are previously filtered through a wide-mesh screen and a Blutter cloth (40 urn pore4 and conserved in potassium bichromate. The stools, diluted 50% in potassium bichromate, are filtered through a moistened hydrophylic cotton column (height 5 cm, diameter 2 cm); 50 ml of this material can be filtered through each column, before it becomes saturated. The filtrate is washed in phosphate-buffered saline (PBS) and centrifuged twice (10 min at 1500 g). The pellet obtained is suspended in 3 ml of PBS and the suspension is placed on a discontinuous angiographine gradient (meglumine sodium amidotrizoate, Radioselectan@, Schering Laboratories). This discon-
tinuous gradient is obtained by pouring 3 ml of a solution containing 3 parts Radioselectan@, 1 part human serum and 3 parts MEM medium on to 3 ml of a solution containing the same ingredients in the proportions 4: 1:2. The tubes are centrifueed at 12 000 P for 10 min in a Beckman horizontal ro’ior centrifuge.“Two dense rings are formed between the 2 liquid-liquid interfaces. We have recovered from the liquid column located above the lower ring, 90% of the oocysts that were introduced; the quality of this purification method is excellent. In the residue, we find bacteria, yeasts and other small debris. The method permits the detection of Cyptosporidium oocysts in a stool-bichromate solution containing as few as 20 oocvstsiml. This technique has been previously used to purify leishmanial amastigotes (MONIOUR et al., 1984). It is useful because of its rapidity and ease compared with the usual techniques and, above all, the quality and yield of the end product. The oocysts obtained are pure, viable and suitable, after excystation and sporozoite liberation, for the infection of cell cultures. References Current, L. W. & Long, I’. L. (1983). Development of human and calf Ctyptosporidium in chicken embryos. 3oumal of Infectious Diseases, 148, 1108-l 113. Datry, A., Danis, M. & Gentilini, M. (1989). Developpement complet de Cryptosporidium en culture cellulaire. Medicine et Sciences, 5, 762-766.
Dulski, I’. & Turner, M. (1988). The purification of sporocysts and sporozoites from Eimena tenella oocysts using PercoIl” density gradients. Avian Diseases, 32, 235-239. Monjour, L., Vouldoukis, I., Brandicourt, 0.) Mazier, D., Alfred, C., Ploton, I. & Gentilini, M. (1984). Rapid large-scale production and isolation of Leishmania amastigotes. Annals of Tropical hfedicine and Parasitology, 78, 423-425. Received 5 December 1990; revised 12 February 1991; accepted for publication 13 February 1992 [subsequently unavoidably delayed by mailing problems]
Gong, Y.-X. Cao, S.-Q., Shi, X.-Z. & Zhou, S.-C. (1990). Crvotosuoridiosis in Oinedao. China ‘foumal ofParasitic DiseaSeiCo;ltrol,~3,?2.
- -
Guan, J.-S., Cm, S.-L., Yen, X.-L., Zhao, X. H., Zhang, J.Z., Wang, Y.-M. & Tuo, Y. (1990). Preliminary clinical observations of cryptosporidial enteritis. Chinese Jouwtal of Pediatrics, 28,41.
Han, F. (1989). Infant cryptosporidiosis found in China. ChineseJournal of Pediatrics, 27,75-76. Han, F., Tan, W.-X. & Zhou, X.-L. (1987). Human cryptosporidiosis in Naming region-two casereports. Jiangsu Medicine, 12,692. Huang, S.-R., Zhang, H.-J,., Huang, M.-J., Li, S.-Y. & Zhang, L. (1990). Cryptosportdrum infection of calves in the suburbs of Beijing. China ZoonosesJournal, 6,45. Lu, L.-A., Fan, Z.-Z., Li, C.-C. & Liu, B.-Z. (1990). Two casesof infant cryptosporidiosis. HunanMedicine, 7, 106. Luo, M.-Y., Wang, Q.-N. & Zhen,, X.-P. (1988). Cryptosporidial infection and enteritis. Chmese 3ournal of Internal Medicine, 27,686-688. Su, Q.-P., Chen, D.-G., Zhao, Z.-G., Hua, Y., Hua, X.-L., Chen, S., Huang, M. H., Wan, J.-Q., Guo, Y.-H. & Li, S.M. (1989). Cryptosporidiosis from infants in Fuzhou region. China ZoonosesJournal, 5,35-36.
TRANSACTIONS OFTHEROYAL
Su, L.-Y., Chen, X.-C., Li, Y.-H. & Liu, J. H. (1990). Casereport of infant cryptosporidiosis. China Journal of Parasitic : DiseasesControl, 3, 154. Unaar, B. L. I’.. Soave, R., Faver, R. & Nash, T. E. (1986). Enzyme immunoassay detection of immunoglobulin ‘M and G antibodies to Cryptosporidium in immunocompetent and immunocomeromised oersons. 7ouwtal oflnfectious Diseases. lS3,570-578’. ” ’ ’ Yao, F.-B. & Chen, Y.-G. (1989). Preliminary study on the effir cacy of garlicin in treatment of childhood cryptosporidiosis. 3ournal of Xuzhou Medical College, 9,56.
Zhu, F.-C., Hun, Z.-X. & Wang, F.-P. (1990). A case-control studv of childhood crvutosuoridiosis in Wuhu. China Public __ _ Health, 6,503-504.
Zu, S.-X. & Du, M.-W. (1987). Human cryptosporidiosis seen in China.3ouwaal ofAnhuiMedica1 University, 22,276. Zuo, Y.-X., Chen, F.-Q. & Fang, L. (1990). Human and domestic cryptosporidiosis in Yunnan and experimental infection study. China ZoonosesJournal, 6, 34-35. Received 5 February 1992; revised 16 April 1992; accepted for publication 16 April I992
SOCIETY OF TROPICAL MEDICINE ANDHYGIENE (1992) 86, 640
/Short Report 1 Rapid purification and concentration technique for Cryptosporidium parvum oocysts M. Lyagoubi, A. Datry, I. Malet, M. Danis and M. Gentilini Laboratoire, de Parasitologie, Uniti INSERM 313, CHU Piti&Salpet&re, 91 Boulevard de l’H6pita1, 75534 Paris ckdex 13, France Purification of faecal Cyptosporidium oocysts is necessary for infection of cell cultures (in vitro models) and of laboratory animals (in vivo models). For immunological studies purified parasites are essential. C. parzwm oocysts are separated from patients’ stools or from the stools of naturallv or exuerimentallv infected animals. Several techniques are used to isolate the oocysts: PercoB@ gradients (DULSKI & TURNER. 1988). sucrose gradientsu(CURREN? & LONG, 1983) and’saturated NaCl solution (DATRY et al., 1989). In order to obtain a sufficient number of oocysts with these techniques, very long and fastidious manipulations are needed. We propose a technique in which stools are previously filtered through a wide-mesh screen and a Blutter cloth (40 urn pore4 and conserved in potassium bichromate. The stools, diluted 50% in potassium bichromate, are filtered through a moistened hydrophylic cotton column (height 5 cm, diameter 2 cm); 50 ml of this material can be filtered through each column, before it becomes saturated. The filtrate is washed in phosphate-buffered saline (PBS) and centrifuged twice (10 min at 1500 g). The pellet obtained is suspended in 3 ml of PBS and the suspension is placed on a discontinuous angiographine gradient (meglumine sodium amidotrizoate, Radioselectan@, Schering Laboratories). This discon-
tinuous gradient is obtained by pouring 3 ml of a solution containing 3 parts Radioselectan@, 1 part human serum and 3 parts MEM medium on to 3 ml of a solution containing the same ingredients in the proportions 4: 1:2. The tubes are centrifueed at 12 000 P for 10 min in a Beckman horizontal ro’ior centrifuge.“Two dense rings are formed between the 2 liquid-liquid interfaces. We have recovered from the liquid column located above the lower ring, 90% of the oocysts that were introduced; the quality of this purification method is excellent. In the residue, we find bacteria, yeasts and other small debris. The method permits the detection of Cyptosporidium oocysts in a stool-bichromate solution containing as few as 20 oocvstsiml. This technique has been previously used to purify leishmanial amastigotes (MONIOUR et al., 1984). It is useful because of its rapidity and ease compared with the usual techniques and, above all, the quality and yield of the end product. The oocysts obtained are pure, viable and suitable, after excystation and sporozoite liberation, for the infection of cell cultures. References Current, L. W. & Long, I’. L. (1983). Development of human and calf Ctyptosporidium in chicken embryos. 3oumal of Infectious Diseases, 148, 1108-l 113. Datry, A., Danis, M. & Gentilini, M. (1989). Developpement complet de Cryptosporidium en culture cellulaire. Medicine et Sciences, 5, 762-766.
Dulski, I’. & Turner, M. (1988). The purification of sporocysts and sporozoites from Eimena tenella oocysts using PercoIl” density gradients. Avian Diseases, 32, 235-239. Monjour, L., Vouldoukis, I., Brandicourt, 0.) Mazier, D., Alfred, C., Ploton, I. & Gentilini, M. (1984). Rapid large-scale production and isolation of Leishmania amastigotes. Annals of Tropical hfedicine and Parasitology, 78, 423-425. Received 5 December 1990; revised 12 February 1991; accepted for publication 13 February 1992 [subsequently unavoidably delayed by mailing problems]
