<I> Cryptosporidium Parvum</I>

Rat Model for Dual Opportunistic Pathogen Prophylaxis: Cryptosporidium parvum and Pneumocystis carinii

Helieh S. Oz, Walter T. Hughes,* and Jerold E. Rehg

Cryptosporidium parvum and Pneumocystis carinii are basis of results of fecal flotation and direct examination of causes of common opportunistic infections in patients with ac- fecal smears. Rats were then immunosuppressed by admin- quired immune deficiency syndrome (AIDS) and cancer and in istration of dexamethasone, USP (oral solution; Roxane organ transplant recipients (1). Cryptosporidiosis, a self-limit- Laboratory, Inc., Columbus, Ohio; 2.5 mg/L) in the drinking ing enteric disease in clinically normal individuals, is often a water for 3 weeks. Tetracycline, which we usually use in our serious chronic enteric disease in immunocompromised pa- PCP rat model to prevent bacterial infection, was not added tients (2). Although P. carinii pneumonitis (PCP) rarely devel- to the drinking water to avoid alterations of the intestinal ops in the immunocompetent host, it may develop as a flora that could interfere with C. parvum infection. Medi- life-threatening disease in up to 70% of patients with AIDS (3). cated or nonmedicated feed (Laboratory Rodent Diet 5001; Immunocompromised patients have been reported to be Purina Mills, Inc., St. Louis, Mo.) and water were provided infected concomitantly with C. parvum and P. carinii (4), ad libitum. Toxoplasma gondii and Giardia lamblia (5), and P. carinii To establish C. parvum infection, rats were inoculated and T. gondii (6). In rats, several compounds have proven orally with 3 to 5.5 x 106 oocysts of bovine origin (continuous effective against infection with P. carinii, including passage in rats) on day 10 of immunosuppression, and dex- trimethoprim-sulfamethoxazole (7), atovaquone (8), amethasone was continued for an additional 11 days. All clindamycin-primaquine (9), trimetrexate (10), pentami- rats were infected with C. parvum by 11 days after oocyst dine (11), and lasalocid (12); or with C. parvum, including inoculation. Our previous studies indicated that rats of the lasalocid (13), diethyldithiocarbamate (14), azithromycin, weight and sex used in this study consume 30 to 50 ml of dex- and paromomycin (15). Because of adverse reactions to amethasone-medicated water per day (16) and are heavily in- some compounds and relapse of infection in some AIDS fected with P. carinii 3 weeks after immunosuppression, using patients, more effective drugs are needed. An approach concentrations of 2.0 or 2.5 mg of dexamethasone/L of water under investigation to prevent infections in AIDS patients (17). Animal care and use procedures were in compliance is referred to as the multiple opportunistic pathogen pro- with the National Institutes of Health Guide for the Care phylaxis strategy (MOPPS). The goal of this strategy is to and Use of Laboratory Animals and were approved by the identify a single drug that is effective against two or more Institutional Animal Care and Usage Committee of St. Jude opportunistic organisms. In the investigation reported Children’s Research Hospital. here, concomitant infections with C. parvum and P. carinii Study drugs were started on day 10 of immunosuppres- in dexamethasone-immunosuppressed rats were estab- sion and continued daily until day 21. Rats alive on day 21 lished to study the efficacy of 17C91 (a prodrug of were euthanized in a CO2 chamber; ileum and lung speci- atovaquone), maduramicin, azithromycin, trimethoprim- mens were removed for histologic examination, and lung sulfamethoxazole, and other compounds. imprints were prepared on slides. A portion of each right Three- to four-month-old, 200- to 225-g female rats lung was fixed in buffered 10% formalin. The lung sections (Harlan Sprague Dawley, Indianapolis, Ind.), free of antibod- and imprint smears were stained by use of the Gomori ies to adventitious murine viruses and mycoplasmal anti- methenamine silver method (7, 8). Lung sections were bodies, were studied in groups of 13 to 22, housed five per scored for severity of PCP lesions as described (17). The en- open-bottom cage. The rats were maintained in a cubicle tire lung section was viewed. Briefly, lesions were scored as with 48 air exchanges/h, humidity of 60 Ϯ 10%, temperature follows: 0 = no cysts; 1+ = cysts sparsely distributed with <1 of 24 Ϯ 2ЊC, and a 12/12-h light/dark cycle in a biosafety organism/25 high-power fields; 2+ = focal areas of P. carinii level-2 facility that has animal care and use programs ac- inflammation surrounded by 10 to 25 high-power fields of credited by AAALAC, International. Rats were determined normal lung parenchyma; and 3+ = diffuse desquamative P. to be free of C. parvum and other enteric protozoans on the carinii pneumonitis with extensive infiltration by cysts in >90% of high-power fields. Animals dying during the first 10 Department of Infectious Diseases and Comparative Medicine Division, days of immunosuppression were not evaluated for P. carinii St. Jude Children’s Research Hospital, Memphis, Tennessee because our previous studies indicated that the infection *Address correspondence to: Dr. Walter T. Hughes, St. Jude Children’s does not become discernible until later than 10 days of dex- Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105.

331 Vol 49, No 3 Laboratory Animal Science June 1999 amethasone administration (1). For assessment of C. acutely acquired from the environment early in life. In ei- parvum infection, a 2-cm linear section of the terminal por- ther event, immunosuppression impairs the immunologic tion of the ileum, the site of heaviest cryptosporidial infec- defenses, and P. carinii proliferates in the lungs, causing tion (18), was formalin fixed, sectioned longitudinally, pneumonitis. Pulmonary cryptosporidiosis rarely develops stained with hematoxylin and eosin (H&E), and examined in animals (19). In this study, C. parvum developmental for C. parvum developmental forms. All epithelial cells in the forms were not detected in the lungs by use of differential 2-cm section were examined for C. parvum developmental staining techniques. forms in the brush border of the ileal epithelium. A rat was When the established MOPPS model was used, 81 of 86 considered infected if one developmental form was observed. (94%) evaluable control rats developed PCP, and 100% were The infection load was quantitated by counting the number infected with C. parvum (Table 1). The effect of antimicrobial of C. parvum developmental forms in the epithelia of 10 ileal compounds varied between the two opportunistic infections. villi (14). Lung sections from C. parvum-infected rats were The 17C91 (atovaquone; 200 mg/kg/day) protected 100% (12 of also stained with H&E, Diffquik, and Kinyoun acid-fast 12) of the rats against PCP, whereas the ileum of all rats was methods (Becton Dickinson, BBL Laboratories, Cockeysville, infected with C. parvum. Azithromycin (400 mg/kg/day) was Md.) and were examined for C. parvum infection. Statistical 100% effective against C. parvum infection in the ileum, but analysis was performed, using Fisher’s exact test. Moribund PCP was prevented in only 44% (8 of 18) of rats. The combina- and dead rats were not evaluated. tion of azithromycin and trimethoprim-sulfamethoxazole Maduramicin (Cygro; American Cyanamid, Wayne, N.J.); (Bactrim) was highly effective against ileal C. parvum infection 17C91 (a prodrug of atovaquone; Glaxo-Wellcome, Research and PCP. Seventy-seven percent (10 of 13) of rats treated Triangle Park, N.C.); azithromycin (Azithromax; Pfizer, New with this combination regimen (azithromycin-trimethoprim- York, N.Y.); trimethoprim-sulfamethoxazole (Bactrim; Roche sulfamethoxazole) were protected against P. carinii, whereas Laboratory, Nutley, N.J.); recombinant mouse interferon- 23% (3 of 13) of the rats had mild PCP (1+). None of the rats gamma (Genentech Inc., San Francisco, Calif.); thalidomide had C. parvum infection in the ileum. Eighty-one of the eighty- (Pharmaceutical Inc., Westfield, N.J.); ivermectin (Agri Vet, six (94%) evaluable untreated control rats had PCP; scores Rahway, N.J.); and amphotericin B, bithionol, diloxanide were 0 in 5%, 1+ in 24%, 2+ in 53%, and 3+ in 18% of the rats. furoate, and diethyldithiocarbamate (Sigma Chemical Co., Brun-Pascaud et al. (20) described a rat model for combined St. Louis, Mo.) were used at various dosages (Table 1). These infection with P. carinii and T. gondii requiring 7 weeks of im- compounds were administered in the feed except for daily munosuppression, low-protein diet, and T. gondii inoculation. doses of amphotericin B (20 mg/kg of body weight, subcuta- Atovaquone was partially effective; however, trimethoprim- neously) and interferon-gamma (250,000 units, intraperito- sulfamethoxazole eradicated T. gondii and prevented P. carinii neally). Drug combinations were evaluated for azithromycin infection. In our study, 17C91 (atovaquone) was 100% effective plus trimethoprim-sulfamethoxazole and azithromycin plus against PCP but had no effect on C. parvum infection in the il- diethyldithiocarbamate. Medicated feed pellets were pre- eum. The effect of 17C91 (atovaquone) on PCP in this study pared by mixing the calculated dosage of drugs in the calcu- was similar to our previous findings with 566C80, the original lated daily requirement of pulverized feed, wetting to a thick formulation of atovaquone (8). The greater effectiveness of paste, and pressing into pellets, which remained solidly atovaquone against PCP in this study compared with that of formed with drying (16–18). Our previous experience (un- the report of Brun-Pascaud et al. (20) may be related to the published) indicates that dexamethasone-treated rats con- method of oral administration. We reported (8) that atovaquone sume feed containing various drugs at the same rate as their (566C80) is more effective in rats when given in the feed rather regular diet. The body weight of each group of rats, the than by gavage, as was done by Brun-Pascaud et al. (20). weight of feed, and the volume of water consumed were mea- Interferon-gamma had a partial effect on PCP. Sixty-two per- sured every other day to ensure that rats received the ex- cent of the rats failed to develop PCP, and 23% were test pected dose of the drugs. Also, the amount of food consumed negative for C. parvum infection in the ileum. Moreover, in- during the preceding 2 days was used as the measure for the terferon-gamma decreased the ileal parasite load from a con- next 2 days. All test drugs were consumed at the expected trol median of 25 organisms/villus to 0.5 organisms/villus. rates (16, 17). Drug concentrations did not require adjust- These data agree with those reported for PCP alone (21). ment because as weight decreased, the quantity of feed con- Diethyldithiocarbamate (900 mg/kg/day) prevented ileal C. sumed decreased proportionally. parvum infection in 70% of rats, but had no effect on P. carinii Immunocompromised patients are often infected with pneumonitis. Maduramicin (0.5 mg/kg/day) was found to be multiple opportunistic pathogens (3–6). We established a 100% effective against C. parvum, but was only partially effec- MOPPS model in rats to study two common pathogens, C. tive (47%) against P. carinii infection. Other compounds, such parvum and P. carinii, of immunocompromised and AIDS as amphotericin B, thalidomide, bithionol, ivermectin, and patients. We previously reported that a high dosage of dex- diloxanide furoate, had minimal or no effect against PCP and amethasone induced severe PCP in female rats after 3 C. parvum infection. weeks of immunosuppression, whereas onset in male rats To the authors’ knowledge, drug efficacy against coinfections began after 5 to 6 weeks (17). In the study reported here, fe- with C. parvum and P. carinii in an animal model has not been male rats also were inoculated with C. parvum oocysts, and reported. We documented that concurrent infection with C. concurrent Cryptosporidium infection developed in the il- parvum and P. carinii did not markedly modify the course of eum. Pneumocystis carinii is presumed to be latent in rats or infection of either pathogen, because the results we obtained

332 Note

Table 1. Effectiveness of various drugs on combined Pneumocystis carinii and Cryptosporidium parvum infections in rats Dosage P. carinii C. parvum administered Mg of drug No. of infectedb infectedc Drug (mg/kg/day) (20 g of feed) ratsa No. % No. % 17C91 200 40 12 (20) 0 0d 12 100 Bithionol 2 0.4 12 (20) 9 75 12 100 Ivermectin 0.4 0.08 12 (20) 9 75 12 100 Diloxanide furoate 160 32 12 (20) 10 83 12 100 Control 0 0 14 (20) 14 100 14 100 Maduramicin 0.5 0.1 17 (19) 9 53d 0 0d Maduramicin 0.25 0.05 14 (19) 10 71 13 93 Control 0 0 18 (20) 18 100 18 100 AZM 400 80 18 (20) 10 56 0 0d AZM 200 40 11 (20) 8 73 0 0d Amphotericin Be 20 4 12 (20) 12 100 12 100 Control 0 0 22 (22) 20 91 22 100 DTC 900 180 9 (13) 9 100 3 30d DTC 300 60 11 (13) 11 100 7 64 DTC 37.5 7.5 10 (13) 10 100 9 90 Control 0 0 17 (19) 16 94 17 100 AZM + DTC 50 + 600 10 + 120 16 (17) 15 94 0 0d AZM + TMP-SMZ ff13 (17) 3 23d 0 0d ␥INFg 250,000 u 50,000 u 13 (17) 5 38d 10 77 Thalidomide 150 30 15 (17) 13 87 15 100 Control 0 0 15 (17) 13 87 15 100 aNumber of rats evaluated (no. of rats initially in group; only survivors at day 21 were evaluated). Nonsurvivor deaths occurred during the first 10 days of immunosuppression before drugs were started. bRats with detectable P. carinii cysts in lung imprint. cRats with detectable C. parvum in the ileum. dSignificantly (P = < 0.01) fewer rats infected in comparison with controls by use of Fisher’s exact test. eGiven subcutaneously. fAzithromycin (AZM, 200 mg/kg/day) and trimethoprim (TMP, 14 mg/kg/day)-sulfamethoxazole (SMZ, 70 mg/kg/day). Drug concentrations per 20 g of feed = 4.0 mg of AZM, 2.8 mg of TMP, and 14 mg of SMZ. gGiven intraperitoneally. DTC = diethyldithiocarbamate; ␥INF = interferon gamma. were similar to those obtained using our dexamethasone-rat 5. Angarano, G., P. Maggi, M. A. Bari, et al. 1997. Giardiasis in models of P. carinii pneumonitis and C. parvum infection (17, HIV: a possible role in patients with severe immune deficiency. 18). Our data are also consistent with those of studies in which Eur. Epidemiol. 13:485–487. 6. Yinnon, A. M., and R. F. Betts. 1994. Pneumocystis carinii some of these antimicrobials were evaluated against the two pneumonia associated with concurrent cryptococcosis or toxo- pathogens separately (7, 8, 14, 15). Therefore, the proposed plasmosis in patients with AIDS. Clin. Infect. Dis. 18: model allows effectual screening of drugs for the two opportu- 113–114. nistic infections simultaneously and minimizes the time and 7. Hughes, W. T., P. C. McNabb, T. D. Markres, et al. 1974. Effi- cacy of trimethoprim and sulfamethoxazole in the prevention number of animals required. and treatment of Pneumocystis carinii pneumonitis. Antimicrob. Agents Chemother. 5:289–293. 8. Hughes, W. T., V. L. Gray, W. E. Gutteridge, et al. 1990. Effi- Acknowledgements cacy of a hydroxynaphthoquinone, 566C80, in experimental Pneumocystis carinii pneumonitis. Antimicrob. Agents Our special thanks to David Barber and Christy Nagy for tech- Chemother. 34:225–228. nical support. 9. Queener, S. F., M. S. Bartlett, J. D. Richardson, et al. 1988. This study was supported in part by NIH grant nos. P30 Activity of clindamycin with primaquine against Pneumocystis CA21765 and AI33288 and the American Lebanese Syrian Asso- carinii in vitro and in vivo. Antimicrob. Agents Chemother. ciated Charities. 32:807–813. This work was partially presented at the 4th Conference on 10. Allegro, C. J., J. A. Kovacs, J. C. Drake, et al. 1987. Activity Retroviruses and Opportunistic Infections, Washington D.C., 1997, of antifolates against Pneumocystis carinii dihydrofolate reduc- abstract no. 287. None of the authors has commercial or other tase and identification of a potent new agent. J. Exp. Med. associations that might pose a conflict of interest. 165:926–931. 11. Waldman, R. H., D. E. Pearce, and R. A. Martin. 1973. Pen- tamidine isethionate levels in lungs, livers and kidneys of rats References after aerosol or intramuscular administration. Am. Rev. Respir. 1. Hughes, W. T. 1987. Pneumocystis carinii pneumonitis, vol. 1, Dis. 108:1004–1006. p. 1–131. CRC Press, Boca Raton, Fla. 12. Oz, H. S., W. T. Hughes, and J. E. Rehg. 1997. Efficacy of 2. Soave, R., and W. D. Johnson. 1988. Cryptosporidium and lasalocid against Pneumocystis carinii pneumonitis. Antimicrob. Isospora belli infections. J. Infect. Dis. 157:225–229. Agents Chemother. 41:191–192. 3. Kovacs, J. A., J. W. Hiemenz, A. M. Macher, et al. 1984. 13. Rehg, J. E. 1993. Anticryptosporidial activity of lasalocid and Pneumocystis carinii: a comparison between patients with ac- other ionophorous antibiotics in immunosuppressed rats. quired immunodeficiency syndrome and patients with other J. Infect. Dis. 168:1566–1569. immunodeficiencies. Ann. Intern. Med. 100:663–671. 14. Rehg, J. E. 1996. Effect of diethyldithiocarbamate on 4. Scaglia, M., C. Atzori, G. Marchetti, et al. 1994. Effective- Cryptosporidium parvum infection in immunosuppressed rats. ness of aminosidine (Paromomycin) sulfate in chronic J. Parasitol. 82:158–162. Cryptosporidium diarrhea in AIDS patients: an open, uncon- 15. Rehg, J. E. 1994. A comparison of anticryptosporidial activity trolled, prospective clinical trial. J. Infect. Dis. 170: of paromomycin with that of other aminoglycosides and 1349–1350. azithromycin in immunosuppressed rats. J. Infect. Dis. 170:934–938.

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16. Oz, H. S., and W. T. Hughes. 1996. Effect of sex and dexa- 20. Brun-Pascaud, M., F. Chau, A. M. Simonpoli, et al. 1994. methasone dose on the experimental host for Pneumocystis Experimental evaluation of combined prophylaxis against carinii. Lab. Anim. Sci. 46:109–110. murine Pneumocystis carinii and toxoplasmosis. J. Infect. Dis. 17. Rehg, J. E., M. L. Hancock, and D. B. Woodmansee. 1988. 170:653–658. Characterization of a dexamethasone-treated rat model of 21. Shear, H. L., G. Valladares, and M. A. Narachi. 1990. En- cryptosporidial infection. J. Infect. Dis. 158:1406–1407. hanced treatment of Pneumocystis carinii pneumonia in rats 18. Hughes, W. T., and B. L. Smith. 1984. Efficacy of with interferon-␥ and reduced doses of trimethoprim/ diaminodiphenylsulfone and other drugs in murine Pneumocystis sulfamethoxazole. J. Acquir. Immune Defic. Syndr. 3:943–948. carinii pneumonitis. Antimicrob. Agents Chemother. 26:436–440. 19. Pavlasek, I. 1984. First record of developmental stages of Cryptosporidium sp. in various organs of experimentally infected mice and spontaneously infected calves. Folia Parasitol. 31:191–192.

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