In vivo assessment of antimicrobial agents against Toxoplasma gondii by quantification of parasites in the blood, lungs, and brain of infected mice

Abstract

The in vivo effects of antimicrobial agents against Toxoplasma gondii were evaluated in mice that were infected intraperitoneally with 104 tachyzoites of the RH strain by determination of survival rates and study of the kinetics of growth of T. gondi in infected mice. At various intervals after infection, subcultures of serial dilutions of blood, lung, and brain homogenates were performed in fibroblast tissue cultures for determination of parasitic loads. Pyrimethamine (18.5 mg/kg per day), sulfadiazine (375 mg/kg per day), and clindamycin (300 mg/kg per day) were administered for 10 days from day 1 or day 4 after infection. Untreated control mice died within 9 days and showed early and predominant lung involvement. All mice treated with sulfadiazine administered from day 1 survived and were apparently healthy; parasitic loads decreased early after treatment, but a relapse was observed 5 days after the cessation of therapy. When pyrimethamine was administered from day 1, 7 of 11 mice died within 25 days; by determination of parasitic loads, the effect of pyrimethamine was only demonstrable from day 6, and a relapse was constantly observed after the cessation of therapy. When pyrimethamine and sulfadiazine were administered in combination, 100% of mice survived; when therapy was started at day 1, parasites remained undetectable; in mice treated from day 4, parasites were eradicated by day 8 but infection relapsed 8 days after the cessation of therapy. All mice treated with clindamycin from day 1 or day 4 died within 10 days, but parasitemia was always undetectable. These results indicate that study of the kinetics of parasitic loads in blood and organs may provide additional information on the effect of antimicrobial agents against T. gondii in regard to the evolution of the infection and may represent a reliable basis for the determination of therapeutic regimens in humans.In vivo experimental studies of Toxoplasma gondii are usually performed in mice that are either acutely infected by intraperitoneal injection of tachyzoites or chronically infected by peroral ingestion or intraperitoneal injection of cysts. Another alternative, which better fits with the pathophysiology of toxoplasmosis in immunocompromised human patients, is to induce a reactivation in chronically infected mice by depleting the CD4+ lymphocyte subpopulation (19), administering anti-gamma interferon (18), or creating a model of intracerebral infection by local injection of tachyzoites (11,12). Assessment of the efficacy of antimicrobial agents is based on the comparative study of mean survival times of untreated and treated mice, enumeration of brain cysts, and in some cases, histologic examination of organs and subinoculation to mice of dilutions of organ homogenates. The survival rate is an important parameter which can be determined easily but which is poorly informative regarding the mode of drug action. Since additional histological studies and mice subinoculations are time-consuming and may not be applied for extensive studies, we propos...

Synergistic activity of azithromycin and pyrimethamine or sulfadiazine in acute experimental toxoplasmosis

“…All the control mice died within 7 (range, 5 to 7) days. As in a previous study (11), parasite burdens were constantly higher in lung than in brain or blood; at day 4, the mean values of four experiments (20 mice) were 6.31 ± 0.7 log units in lung, 3.8 ± 0.4 in brain, and 2.3 ± 2 in blood.…”

“…In contrast, parasites were not detectable in blood and were cleared from lungs. This specific efficacy was probably the main reason for the prolonged survival of treated mice, since pulmonary toxoplasmosis is the principal cause of death in this model of acute infection (11). This special effect on lung infection may be explained in part by the pharmacokinetics of azithromycin.…”

Innate Refractoriness of the Lewis Rat to Toxoplasmosis Is a Dominant Trait That Is Intrinsic to Bone Marrow-Derived Cells

“…Following 2 h of incubation at 37°C, the suspension was distributed into two six-well culture plates, diluted in PBS, and scanned by light microscopy (magnification, ϫ20) to count the blue-stained cysts. Determination of parasite burden in the organs was performed by a tissue culture method adapted from that of Piketty et al (25): (i) blood (200 l) of infected rats was collected from the retro-orbital sinus into Vacutainer Systems tubes (HEMOGARD SST; Becton Dickinson), diluted into 4 ml of PBSA (0.01 M PBS, pH 7.4, supplemented with 100 U/ml penicillin, 50 l/ml streptomycin, 0.04 l/ml gentamicin, and 2.5 g/ml amphotericin B), and (ii) the organs were removed, washed in PBSA, weighed, and homogenized, using a Potter homogenizer, into various volumes of PBSA (4 ml for the spleen and 2 ml for mesenteric lymphatic nodes). Serial fourfold dilution of homogenates were prepared in Dulbecco's modified Eagle's medium supplemented with antibiotics.…”

Synergistic activity of clarithromycin and minocycline in an animal model of acute experimental toxoplasmosis

“…Briefly, serial fourfold dilutions of each blood or organ suspension were prepared in the culture medium, and then 40 ,ul of each dilution was inoculated into duplicate wells of tissue culture plates. Previous experiments using the same experimental procedure had shown that antimicrobial agents in ground tissue had no effect on parasitic growth in the cultures (12). After the parasite burden for five mice (+ 1 standard error) was calculated for each time point.…”

“…Monitoring of infection involved the estimation of survival rates by using the Kaplan Meier product limit method and sequential examination of parasite burden in blood, brains, and lungs on days 4, 7, 10, 14, 22, and 30 after infection; on day 30, liver and spleen tissues were also examined. At each time point, five mice from each group were sacrificed and blood and organ homogenates were cultured as described previously (12). Briefly, serial fourfold dilutions of each blood or organ suspension were prepared in the culture medium, and then 40 ,ul of each dilution was inoculated into duplicate wells of tissue culture plates.…”