ANIMAL MODEL OF
Pneumocystis Pneumonia
HUMAN DISEASE Animal Model: Pneumocystis carinii Pneumonia in the Immunosuppressed Rat Contributed by: F. W. Chandler, Jr., DVM, PhD, Pathology Division, Bureau of Laboratories, Center for Disease Control, Public Health Service, US Department of Health, Education, and Welfare, Atlanta, GA 30333; J. K. Frenkel, MD, PhD, Department of Pathology and Oncology, University of Kansas Medical Center, Kansas City, KS 66103; and W. G. Campbell, Jr., MD, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322. Biologic Features
Rats in most colonies have latent Pneumocystis carinii infection, and pneumonia can be produced experimentally by giving the rats any of several immunosuppressive agents. Recrudescence can be initiated by the subcutaneous (s.c.) injection twice weekly of 25 mg cortisone acetate, alternating with cortisol, into 200-gm rats for 6 to 8 weeks.1 The doses are increased if the rats gain weight and are decreased if the weight falls below 160 g. Dexamethasone, 0.01 mg/ml, in the drinking water can also be used 2,3 in 250-gm rats on a low (8%) protein diet (ICN Pharmaceuticals, Inc., Cleveland), which alone sometimes gives rise to a low-grade pneumocystosis.4 Cyclophosphamide alone, 20 to 40 mg/kg body weight p.o. three times weekly, is immunosuppressive, whereas chlorambucil, 8 to 16 mg/kg body weight p.o. three times weekly, and aminopterin, 0.4 to 0.8 mg/kg body weight p.o. three times weekly, give rise to Pneumocystis pneumonia only when subeffective doses of cortisone are added.' To forestall death from other concomitant infections before pneumocystosis develops, tetracycline (1: 1000) is added to the drinking water, especially to inhibit Corynebacterium kutscheri, and amphotericin B (1 mg) is injected s.c. three times weekly to inhibit Aspergillus and Phycomycetes. After 5 to 8 weeks, almost all the rats will die because of diffuse filling of alveoli with numerous Pneumocystis organisms. Pneumocystis pneumonia in rats is characterized by intact alveoli filled with foamy, eosinophilic material containing indistinct coccus-like nuclei Publication sponsored by the Registry of Comparative Pathology of the Armed Forces Institute of Pathology and supported by Public Health Service Grant RR 00301 from the Division of Research Resources, US Department of Health, Education and Welfare, under the auspices of Universities Associated for Research and Education in Pathology, Inc. 0002-9440/79/0510-0571$01 .00 571 © 1979 American Association of Pathologists
and occasional macrophages (Figure 1). The tinctorial characteristics of Pneumocystis by light microscopy (Figures 1 and 2) and the ultrastructural features of the organism (Figures 3 and 4) are well known."15-7 In brief, a diagnosis can be made from the cysts that usually contain eight nucleated organisms on impression films stained with Giemsa (Figure 2). Lung sections stained with hematoxylin-eosin demonstrate only the Pneulmocystis nuclei; the periodic acid-Schiff stain identifies, in addition, the cysts, but the toluidine blue and methenamine silver stains demonstrate mainly the cyst wall and are therefore invaluable for low-power searches (Figure 1). Pnetumocystis cysts need to be carefully differentiated from yeasts, however, by demonstrating the nuclei of the eight organisms (Figures 2 and 4). Comparison With Human Disease
Pneumocystosis in the hypercorticoid or otherwise immunosuppressed rat is similar to pneumocystosis in patients treated with immunosuppressive agents to maintain a transplant or with cytostatic agents to inhibit leukemia or lymphoreticular neoplasia.8 Pneumocystic disease in humans and animals is prima facie evidence of immunodeficiency. Pneumocystosis is also seen in children with hypogammaglobulinemia, dysgammaglobulinemia, cell-mediated disease, or combined immunodeficiency disease. In malnourished premature infants, a form of interstitial plasma cell pneumonia results (and the disease is still so cited in the Index Medicuis). No satisfactory animal models are available for these pathogenetic mechanisms. Pneumocystosis has been produced, however, by injecting Pneumocystis organisms into athymic mice.3 Pneulmocystis has also been found in cortisone-treated rabbits and in dogs, swine, horses, goats, owl monkeys, and chimpanzees.9 The human and rat Pneuimocystis has apparently been cultivated in the presence of mammalian and avian cells.'0", Because the literature is replete with references to P carinii from humans, it should be emphasized that this species
Figure 1-Variable intra-alveolar filling with Pneumocystis organisms and macrophages in a Inset- Detail Sprague-Dawley rat imm unosuppressed for 45 days. Only cysts are stained. of the deeply stained ovoid to crescent-shaped cysts. (Gomori methenamine silver with H&E counterstain, x300; Inset, Gomori methenamine silver, X600) Figure 2-Impression smear from a Pneumocystis-infected rat lung. Both nucleated cysts (NC) and trophozoites (T) are seen. (Giemsa stain, X1500)
4. S.
Figure 3-Electron micrograph of five pleomorphic trophozoites of Pneumocystis carinii along alveolar wall of rat lung. N, nucleoplasm. (Lead citrate and uranyl acetate, X21,000)
designates the form in rats. Because there is serologic and biologic evidence that rats and humans are infected with distinct species, the human form has been designated P jiroveci in honor of Otto Jirovec who, with J. Vanek, linked pneumocystis to illness in humans.7 The literature on Pneumocystis infection in humans and animals has been extensively reviewed and annotated.12 Usefulness of the Model
The immunosuppressed rat with Pneumocystis pneumonia offers an excellent opportunity for studying the pathogenesis, immunity, and chemotherapy of the disease. Stopping cortisone treatment leads to spontaneous phagocytosis of Pneumocystis by macrophages.1 Phagocytosis is also observed, although hypercorticism is maintained, after chemotherapy with sulfadiazine and pyrimethamine, or pentamidine,' or with trimethoprim and sulfamethoxazole.3 Transmission, as affected by different degrees of contact, has been studied in isolators.2 The organisms can be freed from lung tissue by digestion in 0.1% collagenase 2 or by bronchial lavage, permitting in vitro studies of adherence to cells, opsonization by antibody, and phagocytosis.3 Availability
P carinii pneumonia has been found after cortisone treatment in rats of the following strains: Sprague-Dawley, Fisher, and Long-Evans. For noninfected rats, caesarian-derived, barrier-sustained stocks should be tried.2 They must be tested by iminunosuppression for freedom from Pneumocystis infection, because monitoring of pathogen free rats does not usually include this organisin.
Figure 4-Electron micrograph of cystic organism with intracystic bodies. Note residual cytoplasmic organelles. M, mitochondria. (Lead citrate and uranyl acetate, X26,000)
References Frenkel JK, Good JT, Shultz JA: Latent Pneuimocy~stis infection of rats, relapse, and chemotherapy. Lab Invest 15:1559-1.577, 1966 2. Hendley JO, Weller TH: Activation and transmission in rats of infection with Pneuimocystis. Proc Soc Exp Biol Med 137:1401-1404, 1971 3. Masur IH, Jones TC: The interaction in vitro of Pneuimocystis carinii with macrophages and L-cells. J Exp Med 147:157-170, 1978 4. Hughes WT, Price RA, Sisko F, Havron WS, Kafatos AG, Schonland M, Smythe 1.
5. 6.
7. 8.
9.
10. 11.
12. 13.
PM: Protein-calorie malnutrition: A host determinant for Pneumocystis carinii infection. Am J Dis Child 128:44-52, 1974 Barton EG Jr, Campbell WG Jr: Pnentmocystis carinii in lungs of rats treated with cortisone acetate. Am J Pathol 54:209-236, 1969 Campbell WG Jr: Ultrastructure of Pneiinmocystis in human lung: Life cycle in human pneumocystosis. Arch Pathol 93:312-324, 1972 Frenkel JKK: PneGTmocystis jiroveci n. sp. from man: Morphology, physiology, and immunology in relation to pathology. Nat] Cancer Inst Monogr 43:13-27, 1976 Robbins JB, DeVita VT Jr, Dutz W (editors): Symposium on Pnetnmocystis carinii Infection. National Cancer Institute Monograph 43. Washington, DC, US Government Printing Office, 1976 Chandler FW Jr, McClure HM, Campbell WG Jr, Watts JC: Pulmonary pneumocystosis in nonhuman primates. Arch Pathol Lab Med 100:163-167, 1976 Pifer LL, Hughes WT, Murphy MJ: Propogation ofPneeu mocystis carinii in vitro. Ped Res 11:305-316, 1977 Latorre CR, Sulzer AJ, Norman LG: Serial propagation of Pneumocystis carinii in cell line cultures. Appl Envir Microbiol 33:1204-1206, 1977 Gajdutsek DC: Pneumocystis carinii as the cause of human disease: Historical perspective and magnitude of the problem. Introductory remarks. Natl Cancer Inst Monogr 43:1-10, 1976 Hughes WT, McNabb PC, Makres TD, Feldman 5: Efficacy of trimethoprim and sulfamethoxazole in the prevention and treatment of Pnenmocystis carinii pneumonitis. Antimicrob Agents Chemother 5:289-293, 1974
Pneumocystis Pneumonia
HUMAN DISEASE Animal Model: Pneumocystis carinii Pneumonia in the Immunosuppressed Rat Contributed by: F. W. Chandler, Jr., DVM, PhD, Pathology Division, Bureau of Laboratories, Center for Disease Control, Public Health Service, US Department of Health, Education, and Welfare, Atlanta, GA 30333; J. K. Frenkel, MD, PhD, Department of Pathology and Oncology, University of Kansas Medical Center, Kansas City, KS 66103; and W. G. Campbell, Jr., MD, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322. Biologic Features
Rats in most colonies have latent Pneumocystis carinii infection, and pneumonia can be produced experimentally by giving the rats any of several immunosuppressive agents. Recrudescence can be initiated by the subcutaneous (s.c.) injection twice weekly of 25 mg cortisone acetate, alternating with cortisol, into 200-gm rats for 6 to 8 weeks.1 The doses are increased if the rats gain weight and are decreased if the weight falls below 160 g. Dexamethasone, 0.01 mg/ml, in the drinking water can also be used 2,3 in 250-gm rats on a low (8%) protein diet (ICN Pharmaceuticals, Inc., Cleveland), which alone sometimes gives rise to a low-grade pneumocystosis.4 Cyclophosphamide alone, 20 to 40 mg/kg body weight p.o. three times weekly, is immunosuppressive, whereas chlorambucil, 8 to 16 mg/kg body weight p.o. three times weekly, and aminopterin, 0.4 to 0.8 mg/kg body weight p.o. three times weekly, give rise to Pneumocystis pneumonia only when subeffective doses of cortisone are added.' To forestall death from other concomitant infections before pneumocystosis develops, tetracycline (1: 1000) is added to the drinking water, especially to inhibit Corynebacterium kutscheri, and amphotericin B (1 mg) is injected s.c. three times weekly to inhibit Aspergillus and Phycomycetes. After 5 to 8 weeks, almost all the rats will die because of diffuse filling of alveoli with numerous Pneumocystis organisms. Pneumocystis pneumonia in rats is characterized by intact alveoli filled with foamy, eosinophilic material containing indistinct coccus-like nuclei Publication sponsored by the Registry of Comparative Pathology of the Armed Forces Institute of Pathology and supported by Public Health Service Grant RR 00301 from the Division of Research Resources, US Department of Health, Education and Welfare, under the auspices of Universities Associated for Research and Education in Pathology, Inc. 0002-9440/79/0510-0571$01 .00 571 © 1979 American Association of Pathologists
and occasional macrophages (Figure 1). The tinctorial characteristics of Pneumocystis by light microscopy (Figures 1 and 2) and the ultrastructural features of the organism (Figures 3 and 4) are well known."15-7 In brief, a diagnosis can be made from the cysts that usually contain eight nucleated organisms on impression films stained with Giemsa (Figure 2). Lung sections stained with hematoxylin-eosin demonstrate only the Pneulmocystis nuclei; the periodic acid-Schiff stain identifies, in addition, the cysts, but the toluidine blue and methenamine silver stains demonstrate mainly the cyst wall and are therefore invaluable for low-power searches (Figure 1). Pnetumocystis cysts need to be carefully differentiated from yeasts, however, by demonstrating the nuclei of the eight organisms (Figures 2 and 4). Comparison With Human Disease
Pneumocystosis in the hypercorticoid or otherwise immunosuppressed rat is similar to pneumocystosis in patients treated with immunosuppressive agents to maintain a transplant or with cytostatic agents to inhibit leukemia or lymphoreticular neoplasia.8 Pneumocystic disease in humans and animals is prima facie evidence of immunodeficiency. Pneumocystosis is also seen in children with hypogammaglobulinemia, dysgammaglobulinemia, cell-mediated disease, or combined immunodeficiency disease. In malnourished premature infants, a form of interstitial plasma cell pneumonia results (and the disease is still so cited in the Index Medicuis). No satisfactory animal models are available for these pathogenetic mechanisms. Pneumocystosis has been produced, however, by injecting Pneumocystis organisms into athymic mice.3 Pneulmocystis has also been found in cortisone-treated rabbits and in dogs, swine, horses, goats, owl monkeys, and chimpanzees.9 The human and rat Pneuimocystis has apparently been cultivated in the presence of mammalian and avian cells.'0", Because the literature is replete with references to P carinii from humans, it should be emphasized that this species
Figure 1-Variable intra-alveolar filling with Pneumocystis organisms and macrophages in a Inset- Detail Sprague-Dawley rat imm unosuppressed for 45 days. Only cysts are stained. of the deeply stained ovoid to crescent-shaped cysts. (Gomori methenamine silver with H&E counterstain, x300; Inset, Gomori methenamine silver, X600) Figure 2-Impression smear from a Pneumocystis-infected rat lung. Both nucleated cysts (NC) and trophozoites (T) are seen. (Giemsa stain, X1500)
4. S.
Figure 3-Electron micrograph of five pleomorphic trophozoites of Pneumocystis carinii along alveolar wall of rat lung. N, nucleoplasm. (Lead citrate and uranyl acetate, X21,000)
designates the form in rats. Because there is serologic and biologic evidence that rats and humans are infected with distinct species, the human form has been designated P jiroveci in honor of Otto Jirovec who, with J. Vanek, linked pneumocystis to illness in humans.7 The literature on Pneumocystis infection in humans and animals has been extensively reviewed and annotated.12 Usefulness of the Model
The immunosuppressed rat with Pneumocystis pneumonia offers an excellent opportunity for studying the pathogenesis, immunity, and chemotherapy of the disease. Stopping cortisone treatment leads to spontaneous phagocytosis of Pneumocystis by macrophages.1 Phagocytosis is also observed, although hypercorticism is maintained, after chemotherapy with sulfadiazine and pyrimethamine, or pentamidine,' or with trimethoprim and sulfamethoxazole.3 Transmission, as affected by different degrees of contact, has been studied in isolators.2 The organisms can be freed from lung tissue by digestion in 0.1% collagenase 2 or by bronchial lavage, permitting in vitro studies of adherence to cells, opsonization by antibody, and phagocytosis.3 Availability
P carinii pneumonia has been found after cortisone treatment in rats of the following strains: Sprague-Dawley, Fisher, and Long-Evans. For noninfected rats, caesarian-derived, barrier-sustained stocks should be tried.2 They must be tested by iminunosuppression for freedom from Pneumocystis infection, because monitoring of pathogen free rats does not usually include this organisin.
Figure 4-Electron micrograph of cystic organism with intracystic bodies. Note residual cytoplasmic organelles. M, mitochondria. (Lead citrate and uranyl acetate, X26,000)
References Frenkel JK, Good JT, Shultz JA: Latent Pneuimocy~stis infection of rats, relapse, and chemotherapy. Lab Invest 15:1559-1.577, 1966 2. Hendley JO, Weller TH: Activation and transmission in rats of infection with Pneuimocystis. Proc Soc Exp Biol Med 137:1401-1404, 1971 3. Masur IH, Jones TC: The interaction in vitro of Pneuimocystis carinii with macrophages and L-cells. J Exp Med 147:157-170, 1978 4. Hughes WT, Price RA, Sisko F, Havron WS, Kafatos AG, Schonland M, Smythe 1.
5. 6.
7. 8.
9.
10. 11.
12. 13.
PM: Protein-calorie malnutrition: A host determinant for Pneumocystis carinii infection. Am J Dis Child 128:44-52, 1974 Barton EG Jr, Campbell WG Jr: Pnentmocystis carinii in lungs of rats treated with cortisone acetate. Am J Pathol 54:209-236, 1969 Campbell WG Jr: Ultrastructure of Pneiinmocystis in human lung: Life cycle in human pneumocystosis. Arch Pathol 93:312-324, 1972 Frenkel JKK: PneGTmocystis jiroveci n. sp. from man: Morphology, physiology, and immunology in relation to pathology. Nat] Cancer Inst Monogr 43:13-27, 1976 Robbins JB, DeVita VT Jr, Dutz W (editors): Symposium on Pnetnmocystis carinii Infection. National Cancer Institute Monograph 43. Washington, DC, US Government Printing Office, 1976 Chandler FW Jr, McClure HM, Campbell WG Jr, Watts JC: Pulmonary pneumocystosis in nonhuman primates. Arch Pathol Lab Med 100:163-167, 1976 Pifer LL, Hughes WT, Murphy MJ: Propogation ofPneeu mocystis carinii in vitro. Ped Res 11:305-316, 1977 Latorre CR, Sulzer AJ, Norman LG: Serial propagation of Pneumocystis carinii in cell line cultures. Appl Envir Microbiol 33:1204-1206, 1977 Gajdutsek DC: Pneumocystis carinii as the cause of human disease: Historical perspective and magnitude of the problem. Introductory remarks. Natl Cancer Inst Monogr 43:1-10, 1976 Hughes WT, McNabb PC, Makres TD, Feldman 5: Efficacy of trimethoprim and sulfamethoxazole in the prevention and treatment of Pnenmocystis carinii pneumonitis. Antimicrob Agents Chemother 5:289-293, 1974
