May 24, 1990 - London School ofHygiene and Tropical Medicine, Keppel Street, London ... St Pancras Way, London NW] OPE,2 MoredunResearch Institute, ...
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1990, p. 1498-1500 0066-4804/90/081498-03$02.00/0 Copyright C 1990, American Society for Microbiology
Vol. 34, No. 8
In Vitro Cultivation of Cryptosporidium parvum and Screening for Anticryptosporidial Drugs V. McDONALD,l 2* R. STABLES,"2 D. C. WARHURST,12 M. R. BARER,"2 D. A. BLEWETT,3 H. D. CHAPMAN,4 G. M. CONNOLLY,5 P. L. CHIODINI,"2 AND K. P. W. J. McADAM"2 London School of Hygiene and Tropical Medicine, Keppel Street, London WCJE 7HT,' Hospital for Tropical Diseases, St Pancras Way, London NW] OPE,2 Moredun Research Institute, Edinburgh EH17 7JH,3 Houghton Laboratory, Institute for Animal Health, Huntingdon PE17 2DA,4 and St. Stephens Hospital, London SWIO 9TH,5 United Kingdom Received 16 August 1989/Accepted 24 May 1990
Sporozoites of Cryptosporidium parvum which were excysted in vitro from oocysts isolated from calves or patients with acquired immune deficiency syndrome underwent development in monolayers of the mouse fibroblast cell line L929. Asexual multiplication occurred, with the maximum numbers of parasites usually being observed between 24 and 48 h after infection. Gametocytes were also found, but their numbers were relatively small compared with those of the asexual stages. A study was made of the effect on parasite development of 20 antimicrobial agents, most of which were anticoccidial or antimalarial agents. The majority of the drugs had a limited inhibitory effect on parasite development, but usually only at high concentrations. The two most active drugs were monensin and halofuginone, which reduced parasite multiplication by more than 90% at high concentrations. In the case of monensin, however, inhibition of parasite development at higher concentrations was due, at least in part, to a toxic effect of the drug on the host cells.
Cryptosporidium parvum, an obligate intracellular parasitic protozoan of the suborder Eucoccidiida, is now recognized as a common cause of diarrhea in a variety of mammals, including humans. The parasite has no vector, and infection normally occurs by the fecal-oral route. The infective stage is the oocyst which contains four naked sporozoites, and following ingestion of the oocyst by a susceptible host, the sporozoites excyst and initiate endogenous development in epithelial cells. The development consists of asexual multiplication followed by gametogony and oocyst formation. Oocysts of the new generation sporulate endogenously, and while a degree of autoinfection probably occurs (3), the majority of oocysts are excreted. Cryptosporidiosis caused by C. parvum is usually a selflimiting disease, and the infection tends to be located mainly in the intestine of the immunocompetent host. In humans, the symptoms which are commonly reported, in addition to diarrhea, are vomiting, abdominal pain, and headache. With immunocompromised individuals, including those with acquired immune deficiency syndrome (AIDS), there can be extraintestinal spread of infection, particularly to the respiratory tract. Additionally, the diarrhea often persists and becomes life threatening. Many antimicrobial drugs have been tested in animals or humans infected with Cryptosporidium sp., but none has been consistently effective against this parasite (4). There is a clear requirement, therefore, to intensify the search for anticryptosporidial drugs. The screening of drugs is hindered by the lack of a suitable small-animal model system for cryptosporidiosis caused by C. parvum. Adult mice, for example, are resistant to infection and produce no clinical signs of disease (10). Infections have been observed in drug-immunosuppressed rats (9), but it is not clear whether the levels were sufficiently high for easy measurement by standard parasitological methods. Neonatal animals can be infected, however; and the newborn mouse has been used for in vivo drug studies (14). *
The demonstration of the complete development of C. parvum in tissue culture by Current and Haynes (2) presented the alternative possibility of an in vitro system for drug screening. We report here details of the adaptation of an in vitro culture system for C. parvum to investigate the anticryptosporidial activity of drugs and the results obtained with a number of antimicrobial agents. MATERIALS AND METHODS Cell line. The mouse fibroblast cell line L929 was used. The cells were maintained at 37°C in 25-cm2 culture flasks in medium containing 1 or 10% fetal bovine serum (GIBCO Laboratories), which was heat inactivated for 30 min at 60°C, in RPMI 1640 medium with 25 mM HEPES (N2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 102 U of penicillin per ml, and 102 ,g of streptomycin per ml. Monolayers of fibroblasts were prepared on 13-mm-diameter round cover slips (Gallenkamp) in 24-well plastic tissue culture plates or 8-well plastic chamber slides (Miles Laboratories, Inc.). The numbers of cells added to each well were 5 x 104 (in 0.5 ml) and 3 x 104 (in 0.3 ml), respectively, in plates and chamber slides. The cells were maintained at 37°C in a candle jar (12). Parasites. Oocysts of C. parvum were isolated from stools of infected calves or patients with AIDS. The human stools were homogenized in an equal volume of water and filtered through a metal sieve with a mesh size of 425 ,im. Excess fatty material was removed by ether sedimentation. Ether (1 ml) was mixed with 9 ml of each homogenate and centrifuged (200 x g for 10 min), and the supernatant with the fatty plug was discarded. This preliminary treatment was not required for the calf stools. The subsequent procedures for the two types of stools were similar. The fecal homogenate was allowed to stand until flocculent material had settled. The upper layer, which contained most of the oocysts, was removed and centrifuged at 200 x g for 10 min. The pellet was suspended in a saturated sodium chloride or Sheather sucrose solution, and after centrifugation (200 x g for 10 min) the oocysts were isolated from the top of the superna-
Corresponding author. 1498
Drug Amphotericin Bb Amproliumb Arprinocidc Chloroquineb Cycloguanilb
Diclazurilb Glycarbylamidec Halofantrineb Halofuginoneb Methyl benzoquateb Monensinb Nigericinb Oxytetracyclineb Robenidinec Proguanilb
Pyrimethamineb Spiramycinb Sulfaquinoxilinec Venturicidinc
Zidovudineb
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VOL. 34, 1990
TABLE 1. Inhibitory effects of antimicrobial agents on development of C. parvum in L929 cells Mean no. of parasites/50 fields at drug concn (pLg/ml) oft: 0.0064
0
13.5 15.3 69.7 182.6 23.6 13.3 87.5 17.3 19.0 10.0 31.3 44.3 74.3 79.6 54.0 47.7 37.6 27.3 31.3 27.3
± ± ± ± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
3.5 11.0 3.5 29.1 9.6 3.2 4.9 2.5 1.7 2.0 4.9 14.2 18.9 14.8 15.3 4.0 10.0 14.9 3.5 6.6
13.3 ± 2.9 51.0 ± 19.0
10.7 ± 3.2 37.3 ± 10.0
39.6 ± 12.5
0.032
17.6 12.0 56.0 144.0 19.6 12.3
± ± ± ± ±
100.6 17.6 6.7 11.0 20.3 }2.0 76.0 80.6 57.3 41.0 27.6 16.3 29.6 32.6
± ± ± ± ± ±
0.16
4.5 2.0 15.6 29.8 4.0 ± 3.5
14.3 8.7 50.3 143.3 16.6
± ± ± ± ±
7.6 0.6 16.8 40.0 4.7
30.0 6.5 5.6 4.4 10.8 14.8 18.3 28.8 5.6 4.4 8.5 4.6 4.5 2.5
69.0 16.0 2.7 12.6 13.3 33.7 64.3 58.6 59.0 25.0 23.0 13.6 30.6 28.6
± ± ± ± ± ± ± ± ± ± ± ± ± ±
12.7 3.8 1.5 4.7 5.7 10.9 8.6 11.3 2.6 10.0 2.5 6.6 6.5 5.5
± ±
± ± ± ± ± ±
16.0 9.7 33.6 117.3 17.0 12.0 51.0 11.3
1.0 2.5 15.8 5.1 7.0 ± 3.0 ± 5.2 ± 3.5 2.0 5.6 ± 4.2 1.6 ± 0.6d 15.0 ± 7.5d 59.3 ± 14.3 71.6 ± 7.5 52.6 ± 4.9 23.0 ± 7.0 26.3 ± 5.8 5.6 ± 1.2 23.0 ± 4.6 31.3 ± 5.5 ± ± ± ± ±
20
4
0.8
13.0 9.3 62.3 125.6 18.3 11.3 46.0 6.3 1.3 7.0
41.3 80.6 51.3 12.3 22.7 4.6 17.0 28.0
± 2.6 ± 3.8 ± 14.8
18.0 ± 4.4
± ± ± ± ± ± ±
12.8 5.5 6.6 10.0 2.8 1.1 5.3
107.0 ± 15.1 18.0 ± 11.3 10.0 ± 3.0
± ± ± ± ± ± ± ±
13.6 24.6 14.9
52.0 ± 4.6 73.6 ± 28.3 24.3 ± 11.9
5.3 ± 0.5 6.3 ±3.8
5.7d 10.0 4.0 3.5 5.2
4.6 ± 1.2 8.7 ± 3.2 10.6 ± 7.2d
a Each value (mean + standard deviation) is based on the mean of three samples. Approximately 1% of cells in the monolayer were infected when 15 parasites per 50 fields were counted (there were about 30 fibroblasts in each field). b An isolate of C. parvum from a calf was used. c An isolate of C. parvum from a human was used. d Higher concentrations disrupted monolayers.
tant. The oocysts were then washed in water three times and stored in 2.5% potassium dichromate at 4°C. They were washed four times in Hanks balanced salt solution before use. Excystation of sporozoites was achieved by incubating oocysts at 37°C with 0.5% trypsin in Hanks balanced salt solution for 30 min, washing once in RPMI 1640 medium, and incubating in 0.4% bovine bile salts (Sigma Chemical Co.) at 37°C for 45 min. The excystation mixture was washed with medium (centrifugation at 200 x g for 10 min), and the parasites were immediately applied to monolayers. Measurement of drug activity in C. parvum-infected monolayers. Twenty-four hours after preparing the monolayers, the growth medium was changed to one containing drug at one of five concentrations between 0.0064 and 4 ,ug/ml or 0.032 and 20 ,xg/ml. Sporozoites, 105 in a volume of 25 to 30 1.l of medium, were then added. Controls in which the growth medium contained no drug were also prepared. The monolayers were incubated for 24 h at 37°C in the candle jar, and then the medium was removed and, following three washes with Hanks medium, they were fixed with 70%o methanol and stained with Giemsa stain. Parasite development was examined by microscopic observation of about 1,500 cells by using a x 100 objective and oil immersion. The number of parasites per 50 fields was calculated, ensuring that as wide an area of the monolayer as possible was scanned. Undeveloped sporozoites were sometimes observed, but these could be clearly differentiated from developing parasites, usually appearing to be rounded and bloated and staining less intensely than the endogenous stages. Most unexcysted oocysts were unstained, but a few were stained blue. A mean count of parasite endogenous stages was obtained from three monolayers given the same concentration of drug. Antimicrobial agents. The drugs tested were used at an initial concentration of 1 mg/ml in distilled water (amphotericin B, amprolium, oxytetracycline, spiramycin, and sul-
faquinoxiline), ethanol (chloroquine, halofantrine, monensin, and nigericin), methanol (arprinocid, cycloguanil, diclazuril, glycarbylamide, halofuginone, methyl benzoquate, robenidine, proguanil, and zidovudine), dimethyl sulfoxide (venturicidin), or lactic acid (pyrimethamine). Most of the drugs were selected for their known anticoccidial and/or antimalarial properties, but antifungal agents (amphotericin B and venturicidin) and an antiviral agent (zidovudine) were also included. Preliminary experiments demonstrated that the organic solvents in growth medium, in the absence of drug and at the highest concentration used in the experiments (2%), had no measurable effect on parasite development. RESULTS In vitro endogenous development of C. parvum. Similar patterns of parasite development were observed with the isolates from humans and calves. Developing parasites were found within 90 min of the addition of the sporozoites, and a few meronts were present at 4 h. Increasing numbers of asexual stages could be seen in the following hours, usually reaching a peak (a mean of 10 to 182 parasites per 50 fields, with about 15 parasites per 50 fields being equivalent to 1% of the fibroblasts being infected) at 24 to 48 h. Subsequently, the number of parasites leveled off or gradually decreased. Macrogametocytes were occasionally observed at about 24 h, but were more numerous at about 72 h or later. Relatively few sexual stages were seen overall (less than 10o of the parasite population at 72 h), and there was no evidence that a second cycle of development took place. From the description of in vitro development presented above, it is clear that quantitation of the effect of drugs on sexual development would have been difficult. It was decided, therefore, at least for the initial studies, to concentrate on examination of asexual development. Accordingly, for rapid evaluation of the results, drug experiments were terminated after 24 h.
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ANTIMICROB. AGENTS CHEMOTHER.
Effect of drugs on parasite development. The results of the activities of 20 drugs on the development of C. parvum in L929 cells are presented in Table 1. None of the drugs was able to inhibit parasite growth completely, even at the highest concentrations used (usually 4 or 20 jig/ml, depending on the cytopathic effect of the drug). The majority of drugs, including the anticoccidial agents, produced no more than a 50 to 75% reduction in growth. There was no evidence that the two parasite isolates differed significantly in their susceptibilities to the same drug. Only two drugs, monensin and halofuginone, were found to inhibit parasite development by more than 90%. In the case of monensin, however, concentrations of 4 ,ug/ml or greater had a cytopathic effect on the monolayer.
been due to some recovery of the ability to develop appropriate immune effector mechanisms. Many of the drugs used in this culture system were previously tested in animals infected with C. parvum (for a review, see reference 4), and in general, the results of our in
DISCUSSION An in vitro culture system for C. parvum was used to measure the anticryptosporidial activity of a number of drugs, most of which were anticoccidial agents. Technically, it is a relatively simple system to use, and from the time of monolayer preparation, a drug test could be completed in about 48 h. One disadvantage was that gametocyte development was comparatively rare, and hence, only the effect on asexual development could be readily measured. Most of the 20 drugs studied had some inhibitory effect on parasite growth, but usually only at high concentrations, and so are unlikely to be of use for chemotherapy of cryptosporidiosis. The most effective drugs were monensin and halofuginone, which reduced parasite development by more than 90% at the highest concentrations used (0.8 and 4 ,g/ml, respectively). However, these drugs completely inhibited the development of Eimeria tenella in vitro at substantially lower concentrations (5). Monensin was clearly cytopathic at 4 ,ug/ml, suggesting that its antiparasitic activity is not entirely direct. Interestingly, nigericin, which is structurally related to monensin, was also cytopathic but appeared to be less effective against parasite development. In experimental studies with animals infected with C. parvum, monensin had no therapeutic effect (6, 14); halofuginone treatment was also ineffective in one study (14), but in another study (7), it resulted in a reduction in the output of oocysts from infected lambs. There has been controversy concerning whether spiramycin is useful for the treatment of cryptosporidiosis, with some reports indicating that it is beneficial (8) and others concluding that it has no effect (11, 13, 14). In the present study, spiramycin was not strongly active against the parasite, which would support the view that this drug has little therapeutic value. The poor activity of zidovudine, an anti-human immunodeficiency virus drug, against the parasite indicates, as suggested by Connolly et al. (1), that the clinical and parasitological improvement in AIDS patients with cryptosporidiosis who were given this drug may have
LITERATURE CITED 1. Connolly, G. M., M. S. Dryden, D. C. Shannon, and B. G. Gazzard. 1988. Cryptosporidial diarrhoea in AIDS and its treatment. Gut 29:593-597. 2. Current, W. L., and T. B. Haynes. 1984. Complete development of Cryptosporidium in cell cultures. Science 224:603-605. 3. Current, W. L., and N. C. Reese. 1986. A comparison of endogenous development of three isolates of Cryptosporidium in suckling mice. J. Protozool. 33:98-108. 4. Fayer, R., and B. L. Ungar. 1986. Cryptosporidium spp. and cryptosporidiosis. Microbiol. Rev. 50:458-483. 5. Folz, S. D., B. L. Lee, L. H. Nowakowski, and G. A. Conder. 1988. Anticoccidial evaluation of halofuginone, lasalocid, maduramicin, monensin and salinomycin. Vet. Parasitol. 28:1-9. 6. Moon, H. W., G. N. Woode, and F. A. Ahrens. 1982. Attempted chemoprophylaxis of cryptosporidiosis in calves. Vet. Rec. 110:181. 7. Naciri, M., and D. Yvore. 1988. Treatment of cryptosporidiosis in lambs using halofuginone lactate, p. 120-121. In K. W. Angus and D. A. Blewett (ed.), Cryptosporidiosis: Proceedings of the First International Workshop. The Animal Diseases Research Association, Edinburgh. 8. Portnoy, D., M. L. Whiteside, E. Budeley, and C. L. Macleod. 1984. Treatment of intestinal cryptosporidiosis with spiramycin. Ann. Intern. Med. 101:202-204. 9. Rehg, J. E., M. L. Hancock, and D. B. Woodmansee. 1987. Characterization of cyclophosphamide-rat model of cryptosporidiosis. Infect. Immun. 55:2669-2674. 10. Sherwood, D., K. W. Angus, D. R. Snodgrass, and S. Tzipori. 1982. Experimental cryptosporidiosis in laboratory mice. Infect. Immun. 38:471-475. 11. Soave, R. 1988. Clinical cryptosporidiosis in man, p. 19-26. In K. W. Angus and D. A. Blewett (ed.), Cryptosporidiosis:
vitro and the earlier in vivo experiments are in agreement. An in vitro culture system such as the one described here should, therefore, be of value as a rapid and reliable means of screening potential anticryptosporidial agents prior to testing in animals. ACKNOWLEDGMENT
This work was funded by a grant from the Wellcome Trust.
Proceedings of the First International Workshop. The Animal Research Association, Edinburgh. 12. Trager, W., and J. B. Jensen. 1976. Human malaria parasites in continuous culture. Science 193:673-675. 13. Tzipori, S. 1988. Cryptosporidiosis in perspective. Adv. Parasitol. 27:63-129. 14. Tzipori, S., I. Campbell, and K. W. Angus. 1982. The therapeutic effect of 16 antimicrobial agents on Cryptosporidium infection in mice. Aust. J. Exp. Biol. Med. Sci. 60:187-190.
