Veterinary Parasitology, 42 ( 1992 ) 27-31 Elsevier Science Publishers B.V., Amsterdam
27
In vitro multiplication of Cryptosporidium parvum in mouse peritoneal macrophages F. Martinez, C. Mascaro l, M.J. Rosales, J. Diaz, J. Cifuentes and A. Osuna Department of Molecular Parasitology, Institute of Biotechnology, School of Science, University of Granada, E-18071, Spain (Accepted 10 October 1991 )
ABSTRACT Martinez, F., Mascaro, C., Rosales, M.J., Diaz, J., Cifuentes, J. and Osuna, A., 1992. In vitro multiplication of Cryptosporidium parvum in mouse peritoneal macrophages. Vet. Parasitol., 42:27-31.
Cryptosporidium parvum of bovine origin was developed in vitro in unsensitized mouse peritoneal macroplaages. Macrophages growing in RPMI medium were infected with sporozoites or with oocysts, and after staining infections were studied by light microscopy. A high parasitic index was obtained with multiple infections occurring commonly. This is a simple method for the study of Cryptosporidium biology, and for in vitro assays of pharmacological activity.
INTRODUCTION
The development of Cryptosporidium in intestinal epithelial cells (Current and Reese, 1986), chick embryo chorioallantoic membrane cells (Current and Long, 1983), and in vitro cultured cells (Current and Haynes, 1984; Wagner and Das, 1986; Datry et al., 1989; Flanigan et al., 1991 ) has been described. Although Cryptosporidium is mainly an intestinal parasite, in immunodepressed individuals it spreads to extraintestinal locations, and has been detected in the bile duct and the respiratory tract (Forgacs et al., 1983; Pitlik et al., 1983 ). Villanueva et al. (1984) found Cryptosporidium oocysts in alveolar macrophages of AIDS patients. For that reason, it is interesting to study the parasite's ability to multiply in macrophages which may serve as vehicles to facilitate the invasion of other organs and tissues, as previously reported by Marcial and Madara (1986) in the cytoplasm of intestinal M cells. ~Author to whom correspondence should be addressed.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
28
F. MARTINEZET AL.
MATERIALS AND METHODS
Oocysts of Cryptosporidium parvum were obtained from the feces of spontaneously parasitized young calves from a dairy farm (Rosales et al., 1990). The oocysts were concentrated and purified by filtration through nylon nets, and sterilized in an antibiotic solution. Digestion to release the sporozoites was performed according to Current and Haynes (1984). Macrophages were obtained from albino mice 4-8 weeks old (Swiss OF-1 ) by washing the peritoneal cavity with Hank's solution. The cells were concentrated by centrifugation at 2 5 0 0 × g for 5 min, and the sediment was resuspended in RPMI m e d i u m (Gibco, USA) supplemented with 10% inactivated fetal calf serum (IFCS). The macrophages were then distributed in flat-bottomed microtiter plates (Nunclon, Denmark) in which the bottom of each well had been fitted with a glass disc, and a drop of IFCS had been added to facilitate adhesion. After 24 h the plates were examined by light microscope to confirm adhesion of the macrophages to the substrate. Infection was then induced with the sporozoites or with undigested oocysts resuspended in RPMI m e d i u m with 10% IFCS, and inoculated in each case at a parasite:cell ratio of 1: 1. After 12 h the m e d i u m was replaced, in order to remove parasites' outside cells and the incubation continued at 37°C in an atmosphere of 5% CO2. At intervals of 24, 48 and 72 h, glass discs containing infected macrophages were removed from the microwells and placed in methanol for fixation, and the cultures were stained with alcian-blue and Giemsa for observation by light microscope. TABLE1 Percentage of parasitized mouse peritoneal macrophages (P) in cultures inoculated wilh sporozoites (E) or with oocysts (O), and of different C~vptosporidium development stages Post-inoculation time
(h)
24
P Mu MI M2 Und c; 9 O
48
72
E
O
E
O
E
O
64 24.6 3.7 49.3 12.1 2.4 . 7.4
42 3.7 11.2 48 17.5 3.7
61 16.2 8.1 30.6 38.7 2 . 4
57 18.9 26.1 21.4 16.5 7.1
55 6.8 4.2 56.9 27.7 1.3
9.5
2.7
66 15 6.5 56.5 6.5 6.5 6.5 2.1
.
. 3.7
.
Mu, undifferentiated meronts, mostly with two chromatinic spots; M l, type I meronts, with six or eight well-formed merozoites; M2, type 1I meronts with four well-formed merozoites; Und, undifferentiated forms without chromatinic spots; ~ , well-formed microgamonts; 9, macrogamonts; C), maturc oocysts with four sporozoites.
CR YPTOSPORIDIUM GROWTH IN MOUSE MACROPHAGES
29
RESULTS Both oocysts and sporozoites o f Cryptosporidium were able to infect mouse peritoneal macrophages. For each culture period the n u m b e r of each form were counted per 100 macrophages; the m e a n values per four wells are given in Table l, which also shows the different morphological forms observed (see Fig. 1 ). Multiple infections were evident, with m a n y cells simultaneously having more than one form of the parasite. The parasite appears within a parasitophorus vacuole in the macrophage cytoplasm, mostly located near the
Fig. 1. Representative developmental stages of Cryptosporidiumin mouse peritoneal macrophages. Alcian blue and Giemsa stain ( × 2000): A, undifferentiated meront; B, type I meront with developingmerozoites; C and D, type II meronts with four merozoites; E, undifferentiated forms without chromatinic spots; F, microgamont; G, macrogamont; H, immature oocyst; I, mature oocyst.
30
F. MARTINEZ ET AL.
host cell nucleus. The successive life cycle stages are similar to those which we have observed in intestinal mouse mucus and in chicken embryos inoculated with C. parvum. DISCUSSION
In this work we have tried to verify the multiplication of C. parvum in macrophages. The count of parasites was carried out 24 h post-inoculation to avoid including parasite forms (sporozoites and oocysts) that could have been phagocytized by macrophages and were in the first steps of digestion. The results clearly demonstrate the survival of C. parvum in unsensitized mouse peritoneal macrophages. Most development stages were type 11 meronts (Table 1 ). Sexual forms and mature oocysts were scarce. Even after 72 h of culture the parasite/macrophage ratio remained elevated, evidencing the resistance of the parasite to lysosomal digestion. It is this resistance that permits C. parvum to multiply in the cytoplasm of phagocytic cells. Confirmation of the hypothesis proposed by other authors (Marcial and Madara, 1986 ) that C. parvum is able to multiply in non-epithelial cells is an important step toward explaining the extraintestinal spread of this parasite and the difficulties which have been reported in the eradication of infection in immunodepressed patients. At the same time, our findings open new avenues of investigation in vitro and in vivo of the factors involved in recognition, adhesion and phagocytosis of the protozoan. ACKNOWLEDGMENTS
This work was supported by the Comisi6n Interministerial de Ciencia y Tecnologia through Project no. 87/0867, and by the Fondo Investigaciones Sanitarias de la Seguridad Social through Project 90/0910. We thank Karen Shashok for translating the original manuscript into English.
REFERENCES Current, W.L. and Haynes, T.B., 1984. Complete development of Cryptosporidium in cell culture. Science, 224: 603-605. Current, W.L. and Long, P.L., 1983. Development of human and calfCryptosporidium in chicken embryos. J. Infect. Dis., 148:1108-1113. Current, W.L. and Reese, N.C., 1986. A comparison of endogenous development of three isolates of Cryptosporidium in suckling mice. J. Protozool., 33: 98-108. Datry, A., Danis, M. and Gentilini, M., 1989. Developpement complet de Cryptosporidium en culture cellulaire, applications. Med. Sci., 5: 762-766. Flanigan, P.T., Toshiba, A., Marshall, R., Soave, R., Aikawa, M. and Kaetzel, C., 1991. Asexual development of Cryptosporidiurn parvum within a differentiated human enterocyte cell line. Infect. Immun., 59: 234-239.
CR YPTOSPORIDIUM GROWTH IN MOUSE MACROPHAGES
31
Forgacs, P.A., Tarshis, M., Federman, L., Mele, L., Silverman, M.L. and Shea, J.A., 1983. Intestinal and bronchial cryptosporidiosis in an immunodeficient homosexual man. Ann. Intern. Med., 99: 793-794. Marcial, M.A. and Madara, J.L., 1986. Cryptosporidium: cellular localization, structural analysis of absorptive cell-parasite membrane-membrane interactions in guinea pigs, suggestion of protozoan transport by M cells. Gastroenterology, 90: 583-584. Pitlik, L.S., Fainstein, V., Rios, A., Guarda, L., Mansell, P.W. and Hersh, E.M., 1983. Cryptosporidial cholecystitis. N. Engl. J. Med., 308: 976. Rosales, M.J., Cifuentes, J., Diaz, J., Martlnez, F. and Mascar6, C., 1990. Epidemiological study on bovine cryptosporidiosis. Bull. Soc. Frang. Parasitol., 8: 880. Villanueva, T.G., Kaufman, D. and Gillooley, J.F., 1984. Respiratory cryptosporidiosis in the acquired immune deficiency syndrome. Use of modified cold kinyoun and hemocolor stains for rapid diagnoses. J. Am. Med. Assoc., 252: 1298-1301. Wagner, E.O. and Das, M.P., 1986. Cryptosporidium in cell culture. Jpn. J. Parasitol., 35: 253255.
27
In vitro multiplication of Cryptosporidium parvum in mouse peritoneal macrophages F. Martinez, C. Mascaro l, M.J. Rosales, J. Diaz, J. Cifuentes and A. Osuna Department of Molecular Parasitology, Institute of Biotechnology, School of Science, University of Granada, E-18071, Spain (Accepted 10 October 1991 )
ABSTRACT Martinez, F., Mascaro, C., Rosales, M.J., Diaz, J., Cifuentes, J. and Osuna, A., 1992. In vitro multiplication of Cryptosporidium parvum in mouse peritoneal macrophages. Vet. Parasitol., 42:27-31.
Cryptosporidium parvum of bovine origin was developed in vitro in unsensitized mouse peritoneal macroplaages. Macrophages growing in RPMI medium were infected with sporozoites or with oocysts, and after staining infections were studied by light microscopy. A high parasitic index was obtained with multiple infections occurring commonly. This is a simple method for the study of Cryptosporidium biology, and for in vitro assays of pharmacological activity.
INTRODUCTION
The development of Cryptosporidium in intestinal epithelial cells (Current and Reese, 1986), chick embryo chorioallantoic membrane cells (Current and Long, 1983), and in vitro cultured cells (Current and Haynes, 1984; Wagner and Das, 1986; Datry et al., 1989; Flanigan et al., 1991 ) has been described. Although Cryptosporidium is mainly an intestinal parasite, in immunodepressed individuals it spreads to extraintestinal locations, and has been detected in the bile duct and the respiratory tract (Forgacs et al., 1983; Pitlik et al., 1983 ). Villanueva et al. (1984) found Cryptosporidium oocysts in alveolar macrophages of AIDS patients. For that reason, it is interesting to study the parasite's ability to multiply in macrophages which may serve as vehicles to facilitate the invasion of other organs and tissues, as previously reported by Marcial and Madara (1986) in the cytoplasm of intestinal M cells. ~Author to whom correspondence should be addressed.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
28
F. MARTINEZET AL.
MATERIALS AND METHODS
Oocysts of Cryptosporidium parvum were obtained from the feces of spontaneously parasitized young calves from a dairy farm (Rosales et al., 1990). The oocysts were concentrated and purified by filtration through nylon nets, and sterilized in an antibiotic solution. Digestion to release the sporozoites was performed according to Current and Haynes (1984). Macrophages were obtained from albino mice 4-8 weeks old (Swiss OF-1 ) by washing the peritoneal cavity with Hank's solution. The cells were concentrated by centrifugation at 2 5 0 0 × g for 5 min, and the sediment was resuspended in RPMI m e d i u m (Gibco, USA) supplemented with 10% inactivated fetal calf serum (IFCS). The macrophages were then distributed in flat-bottomed microtiter plates (Nunclon, Denmark) in which the bottom of each well had been fitted with a glass disc, and a drop of IFCS had been added to facilitate adhesion. After 24 h the plates were examined by light microscope to confirm adhesion of the macrophages to the substrate. Infection was then induced with the sporozoites or with undigested oocysts resuspended in RPMI m e d i u m with 10% IFCS, and inoculated in each case at a parasite:cell ratio of 1: 1. After 12 h the m e d i u m was replaced, in order to remove parasites' outside cells and the incubation continued at 37°C in an atmosphere of 5% CO2. At intervals of 24, 48 and 72 h, glass discs containing infected macrophages were removed from the microwells and placed in methanol for fixation, and the cultures were stained with alcian-blue and Giemsa for observation by light microscope. TABLE1 Percentage of parasitized mouse peritoneal macrophages (P) in cultures inoculated wilh sporozoites (E) or with oocysts (O), and of different C~vptosporidium development stages Post-inoculation time
(h)
24
P Mu MI M2 Und c; 9 O
48
72
E
O
E
O
E
O
64 24.6 3.7 49.3 12.1 2.4 . 7.4
42 3.7 11.2 48 17.5 3.7
61 16.2 8.1 30.6 38.7 2 . 4
57 18.9 26.1 21.4 16.5 7.1
55 6.8 4.2 56.9 27.7 1.3
9.5
2.7
66 15 6.5 56.5 6.5 6.5 6.5 2.1
.
. 3.7
.
Mu, undifferentiated meronts, mostly with two chromatinic spots; M l, type I meronts, with six or eight well-formed merozoites; M2, type 1I meronts with four well-formed merozoites; Und, undifferentiated forms without chromatinic spots; ~ , well-formed microgamonts; 9, macrogamonts; C), maturc oocysts with four sporozoites.
CR YPTOSPORIDIUM GROWTH IN MOUSE MACROPHAGES
29
RESULTS Both oocysts and sporozoites o f Cryptosporidium were able to infect mouse peritoneal macrophages. For each culture period the n u m b e r of each form were counted per 100 macrophages; the m e a n values per four wells are given in Table l, which also shows the different morphological forms observed (see Fig. 1 ). Multiple infections were evident, with m a n y cells simultaneously having more than one form of the parasite. The parasite appears within a parasitophorus vacuole in the macrophage cytoplasm, mostly located near the
Fig. 1. Representative developmental stages of Cryptosporidiumin mouse peritoneal macrophages. Alcian blue and Giemsa stain ( × 2000): A, undifferentiated meront; B, type I meront with developingmerozoites; C and D, type II meronts with four merozoites; E, undifferentiated forms without chromatinic spots; F, microgamont; G, macrogamont; H, immature oocyst; I, mature oocyst.
30
F. MARTINEZ ET AL.
host cell nucleus. The successive life cycle stages are similar to those which we have observed in intestinal mouse mucus and in chicken embryos inoculated with C. parvum. DISCUSSION
In this work we have tried to verify the multiplication of C. parvum in macrophages. The count of parasites was carried out 24 h post-inoculation to avoid including parasite forms (sporozoites and oocysts) that could have been phagocytized by macrophages and were in the first steps of digestion. The results clearly demonstrate the survival of C. parvum in unsensitized mouse peritoneal macrophages. Most development stages were type 11 meronts (Table 1 ). Sexual forms and mature oocysts were scarce. Even after 72 h of culture the parasite/macrophage ratio remained elevated, evidencing the resistance of the parasite to lysosomal digestion. It is this resistance that permits C. parvum to multiply in the cytoplasm of phagocytic cells. Confirmation of the hypothesis proposed by other authors (Marcial and Madara, 1986 ) that C. parvum is able to multiply in non-epithelial cells is an important step toward explaining the extraintestinal spread of this parasite and the difficulties which have been reported in the eradication of infection in immunodepressed patients. At the same time, our findings open new avenues of investigation in vitro and in vivo of the factors involved in recognition, adhesion and phagocytosis of the protozoan. ACKNOWLEDGMENTS
This work was supported by the Comisi6n Interministerial de Ciencia y Tecnologia through Project no. 87/0867, and by the Fondo Investigaciones Sanitarias de la Seguridad Social through Project 90/0910. We thank Karen Shashok for translating the original manuscript into English.
REFERENCES Current, W.L. and Haynes, T.B., 1984. Complete development of Cryptosporidium in cell culture. Science, 224: 603-605. Current, W.L. and Long, P.L., 1983. Development of human and calfCryptosporidium in chicken embryos. J. Infect. Dis., 148:1108-1113. Current, W.L. and Reese, N.C., 1986. A comparison of endogenous development of three isolates of Cryptosporidium in suckling mice. J. Protozool., 33: 98-108. Datry, A., Danis, M. and Gentilini, M., 1989. Developpement complet de Cryptosporidium en culture cellulaire, applications. Med. Sci., 5: 762-766. Flanigan, P.T., Toshiba, A., Marshall, R., Soave, R., Aikawa, M. and Kaetzel, C., 1991. Asexual development of Cryptosporidiurn parvum within a differentiated human enterocyte cell line. Infect. Immun., 59: 234-239.
CR YPTOSPORIDIUM GROWTH IN MOUSE MACROPHAGES
31
Forgacs, P.A., Tarshis, M., Federman, L., Mele, L., Silverman, M.L. and Shea, J.A., 1983. Intestinal and bronchial cryptosporidiosis in an immunodeficient homosexual man. Ann. Intern. Med., 99: 793-794. Marcial, M.A. and Madara, J.L., 1986. Cryptosporidium: cellular localization, structural analysis of absorptive cell-parasite membrane-membrane interactions in guinea pigs, suggestion of protozoan transport by M cells. Gastroenterology, 90: 583-584. Pitlik, L.S., Fainstein, V., Rios, A., Guarda, L., Mansell, P.W. and Hersh, E.M., 1983. Cryptosporidial cholecystitis. N. Engl. J. Med., 308: 976. Rosales, M.J., Cifuentes, J., Diaz, J., Martlnez, F. and Mascar6, C., 1990. Epidemiological study on bovine cryptosporidiosis. Bull. Soc. Frang. Parasitol., 8: 880. Villanueva, T.G., Kaufman, D. and Gillooley, J.F., 1984. Respiratory cryptosporidiosis in the acquired immune deficiency syndrome. Use of modified cold kinyoun and hemocolor stains for rapid diagnoses. J. Am. Med. Assoc., 252: 1298-1301. Wagner, E.O. and Das, M.P., 1986. Cryptosporidium in cell culture. Jpn. J. Parasitol., 35: 253255.
