Development of Pneumocystosis Animal Models: Corticosteriod‐Treated Wistar Rat; SCID Mouse and Nude Rat.

Pneirmocystis carinii pneumonia (PCP) is a major cause of morbidity and mortality in immunocompromised patients suffering from acquired immunodeficiency syndrome, receiving chemotherapy or immunosuppressing drugs for organ transplantation or other pathological conditions. Experimental models of pneumocystosis are used as source of P. carinii because continuous culture systems have not been established. Moreover, laboratory animals are of paramount importance in the study of PCP pathogenesis. The aim of this work was to develop animal models of pneumocystosis in order to evaluate the prophylactic and the therapeutic effect of new anti-Prreunrorystis drugs. Three pneumocystosis animal models have been developed corticosteroid-treated Wistar rats, parasite inoculated severe combined immunodeficiency (SCID) mice and nude rats. MATERIALS AND METHODS. WIFIor ruf nIOdK1: seven-weekold female Wistar rats (Iffa-Credo, France) were treated with dexamethasone at a concentration of 2 mg/l in the drinking water for 12 weeks. Rats were housed in our animal facilities (P2 biosafety level) and were given standard food and water ad libitum. These animals develop spontaneous P. curinii infection after corticosteroid treatment. Each week, 4 to 9 animals were sacrificed and parasite extraction and quantitation was performed. The Stomacher method was used to extract parasites as previously described [2]. The protocol was modified as follows. Lungs were removed aseptically and cut into small pieces in sterile PBS solution. Pieces were homogenised using a Stomacher 400 blender and large particles were removed by filtering the homogenate through sterile gauze. The filtrate was centrifuged at 2900 g for 10 mins and the pellet resuspended in PBS. Quantitation of P. cariirii cystic forms was performed with Toluidine Blue 0 (TE30) stain. SCID mouse model: Ten week old female SCID mice (Iffa-Credo, France) free of latent P. carinii infection were used in the study. Mice were anaesthetised with a drug cockail (ketamindxylacine) and then nasally inoculated with 2.5~10' mouse-derived P. cun'nii organisms [ 11. In order. to obtain viable, pure parasite samples to infect SCID mice, P. carinii extraction was performed as previously described [3]. Briefly, lungs of the infected mice were removed aseptically and cut into small pieces in sterile Dulbecco modified Eagle's medium (DMEM). Parasites were extracted by agitation of the lung pieces, with a magnetic stirrer, and the resulting homogenate was poured through gauze. The pellet was then resuspended in DMEM and the suspension was filtered successively through 250and 63-pm stainless steel mesh and through 8-pm filters. Parasite quantitation, and control for other pathogens, was performed using Giemsa stain. Animals were divided into 2 groups (i) untreated SCID mice, (ii) SCID mice treated with corticosteroids. Dexamethasone was added to the drinking water at 4 mgA, one week prior to parasite inoculation and throughout the experiment. Three, 4, or 5 weeks post-inoculation (p.i.) animals were sacrificed and lungs were removed aseptically. Extraction and quantitation of parasites were performed using the stomacher method as described above. Nude rut model: 10-12 week old female n d n u rats (Harlan, UK), free of latent P. carinii infection were used in the study. Rats were anaesthetised with a drug cochail (ketaminelxylacine) given intraperitoneally. The trachea was sxposed surgically and 100 p1 of the inoculum was injected into the trachea with 0.3 ml of air behind it. All the rats were inoculated with 8.0 x lo7 freshly extracted ratderived P. carinii organisms The wound was closed with Michel clips. Animals were divided into 3 groups (i) untreated rats, (ii) animals treated with dexamethasone (1 rng) starting the day of inoculation and for all the experiment, (iii) animals treated with dexamethasone (1 mg/l) starting 2 weeks before the parasites inoculation and throughout the experiment. Animals were sacrificed at the 40th-day p.i. and the parasites extraction and quantitation were performed using the stomacher method. Morever, the kinetics of infection in nude rats, with 2 weeks previous dexamethasone treatment, were done from week 1 to week 13 pi. SClD mice and nude rats were housed into isolators with positive pressure, and all experiments were done under sterile conditions (vertical laminar flow). RESULTS AND DISCUSSION. WIFm rut model: The results of the kinetics of the infection in corticosteroids-treated Wistar rats are shown in figure 1. Parasites were detected after week 4 of immunosuppression, considering that in these experimental conditions the limit of parasites detection is log 3.0. After week 4 a rapid increase of parasite growth until the week 8 was observed. A wide range of parasite rates was recorded during the increasing phase. Between the weeks 9 to 12, parasites rates reach a plateau becoming more homogenous (log = 8) (Fig. 1). In one satellite group (n = lo), after 21 weeks of immunosuppression, we observed the same level of infection as that of the plateau.