THE JOURNAL OF INFECTIOUS DISEASES. VOL. 135. :'110. 3 • MARCH 1977 © 1977 by the University of Chicago. All rights reserved.
Toxoplasmosis and the Host-Parasite Relationship in Murine Schistosomiasis Mansoni From the Division of Geographic Medicine, Department of Medicine, Case Western Reserve University and University Hospitals, Cleveland, Ohio; the Division of Immunoparasitology, Naval Medical Research Institute, Bethesda; and the Uniformed University of the Health Sciences, Bethesda, Maryland
Adel A. F. Mahmoud, G. Thomas Strickland, and Kenneth S. Warren
Schistosomiasis is a chronic helminthic infection that afflicts more than 200 million individuals globally. Toxoplasmosis is a ubiquitous protozoan infection with a worldwide prevalence of 34% [1, 2]. Since schistosomiasis has been shown to be largely an immunologic disease [3] and toxoplasmosis has recently been reported to be intensely immunosuppressive [4-6], the interaction between these two infections is of particular interest. The host granulomatous response to Schistosoma mansoni eggs deposited in the portal venules of the liver has been shown to be an immunologic reaction of the cell-mediated type [7]. The granulomas and the subsequent fibrous scarring obstruct portal blood flow, resulting in portal hypertension, congestive splenomegaly, and esophageal varices [8]. Suppression of cellmediated immunological reactivity markedly reReceived for publication May 3, 1976, and in revised form August 6,1976. This work was supported by grant no. AI 08163 from the National Institutes of Health, Bethesda, Maryland. We acknowledge the technical assistance of Pierre A. Peters and Earlene Moss. Please address requests for reprints to Dr. Kenneth S. Warren, Division of Geographic Medicine, Wearn Research Building, University Hospitals, Cleveland, Ohio 44106.
408
duces granulomatous inflammation [9, 10] and has been associated with significant alleviation of disease [11, 12]. Several recent reports have described the immunosuppression induced in animals by toxoplasmosis [4-6]. It has been shown that three weeks after infection there is a depletion of cortical thymocytes and a significant decrease in thymus weight. In addition, thymus-dependent zones of the lymph nodes and spleen were greatly reduced in area [4]. Mice infected with Toxoplasma gondii and subsequently immunized with sheep erythrocytes have depressed spleen plaqueforming cells and diminished serum hemagglutinin and hemolysin responses [5]. Whereas the lymphocyte response to a bone marrow-derived (B-) cell mitogen (lipopolysaccharide) was decreased for two to three weeks after infection with T. gondii, the responses to the thymusderived (T-) cell mitogens phytohemagglutinin and concanavalin A were suppressed significantly for at least 100 days [6]. In the present study we examined the effects of toxoplasma infection on the host granulomatous responses to S. mansoni eggs injected into the pulmonary microvasculature. The effect of toxoplasmosis on the course of natural infection with S. mansoni was also evaluated.
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on July 23, 2015
Infection with Toxoplasma gondii has been shown to suppress markedly the in vitro splenic lymphocyte response to nonspecific mitogens and the in vivo antibody response to sheep red blood cells. The effect of toxoplasmosis on an in vivo cell-mediated response, granuloma formation around Schistosoma mansoni eggs, was examined in the present study. The granulomas were markedly suppressed from two to 20 weeks after infection with T. gondii. In subsequent experiments the effect of toxoplasmosis-induced immunosuppression on the development of hepatosplenic schistosomiasis was evaluated. Mice with the combined infections had markedly smaller hepatic granulomas and lower mean portal pressures than those infected with S. mansoni alone. Although the prevalence of esophageal varices in the mice with schistosomiasis alone was 60%, there was no visible collateral circulation in the animals with both infections.
T. gondii and Response to S. mansoni
Materials and Methods
granuloma area (control) - granuloma area (T. gondij·infected)
~=:..::.:..:::~-=--.:..._--.:.~:::..-_--;-----:-~----X 100, granuloma area (control)
where the granuloma area is the area of inflammation minus the area of the egg.
Infection with S. mansoni. Three experiments were performed in which 10 T. gondii cysts were injected ip into groups of mice four weeks before, one day before, and four weeks after the onset of schistosomiasis. Infection with S. mansoni was induced by sc injection of 20 cercariae of a Puerto Rican strain of the parasite. For each experiment, animals were subdivided into four groups of 20 mice each: uninfected controls, T. gondii-infected mice, S. mansoniinfected mice, and mice wi th combined infections. Animals were followed closely during the experimental period, and all fatalities were recorded. Eight weeks after the schistosome infection, 10 mice were chosen randomly from each group and examined for body weight, liver and spleen weights, portal vein pressure, esophageal varices, hematocrit, and histopathology of the liver [14]. In addition, the diameters of the granulomas around S. mansoni eggs in the livers were measured in stained sections with a Cooke-A.EJ. image-spli tting measuring eyepiece (Vickers Instruments, Woburn, Mass.) [14]. In the two groups of animals infected with Toxoplasma one day before infection with S. mansoni, a high mortality rate was observed between six and eight weeks. At eight weeks, when the pathophysiological studies were performed, it was noted that these animals had multiple necrotic liver lesions. The experiment was repeated to confirm these findings, and when these animals were killed, their livers, which also contained multiple necrotic areas, were removed aseptically. Samples from the cut surface of these livers were cultured aerobically and anaerobically [15] by Dr. K. V. Gopalakrishna of the Veterans Administration Hospital of Cleveland, Ohio. Sections of liver tissue were stained with giemsa solution and examined for Actinobacillus piliformis [16]. In addition, the effect on these animals of the administration of specific therapy for toxoplasmosis, beginning at five weeks (when egg deposition by the schistosomes begins) a~d continuing for three weeks, was evaluated In terms of the mortali ty of the animals and the development of the necrotic areas in their livers. Sulphadimidine and pyrimethamine were administered daily by a blunt-tip cannula in doses of 3.2 mg and 0.01 mg per mouse, respectively [17].
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on July 23, 2015
Mice. Swiss albino female mice weighing 1820 g were purchased from Carworth Farms, New City, N. Y. T. gondii infection. To obtain T. gondii cysts, we removed the brains of mice infected with the Gleadle strain for five to six weeks and emulsified them in Hanks' balanced salt solution (Flow Laboratories, Rockville, Md.) in a tissue grinder. Cysts were counted in three separate 20pJ aliquots of brain suspensionpla~ed on gla~s slides with cover slips on top. Expenmental ammals were given ip injections of 10 cysts in 0.2 ml of brain emulsion to which penicillin (100 unitsj ml) and streptomycin (10 IJ-g/ml) were added. Similar volumes of brain emulsion from uninfected mice with added antibiotics were injected ip into uninfected control animals. . Granuloma formation around S. mansoru eggs. For the study of the effect of toxoplasmosis on granuloma formation, S. mansoni eggs were.isolated from the livers of mice infected for eight weeks [13] and were injected iv into the pulmonary microvasculature of T. gondii-infected and control mice. Each animal received 1,500 eggs suspended in 0.5 ml of 0.85% NaCl. Th~ gran~ Iomatous responses elicited were examined III separate groups of mice that had been infected with T. gondii for two to 30 weeks. One group of mice was sensitized with an ip 'injection of 1,500 eggs eight days before iv injection of eggs, and the response was examined four, eight, 12, 20, and 30 weeks after the iv injection. On day 8 after iv injection of eggs, the lungs were removed, sections were prepared, and the size of the granulomatous reaction was determined ('1T MC Particle Measurement Computer; Millipore Corp., Bedford, Mass.) as previously described [10]. Approximately 50 granulomas were measured within each experimental group of five to six animals, and the mean lesion area was calculated. The percentage of change in the area of inflammation due to toxoplasmosis was calculated according to the formula
409
Mahmoud, Strickland, and Warren
410
Results
Toxoplasmosis and the Host-Parasite Relationship in Murine Schistosomiasis Mansoni From the Division of Geographic Medicine, Department of Medicine, Case Western Reserve University and University Hospitals, Cleveland, Ohio; the Division of Immunoparasitology, Naval Medical Research Institute, Bethesda; and the Uniformed University of the Health Sciences, Bethesda, Maryland
Adel A. F. Mahmoud, G. Thomas Strickland, and Kenneth S. Warren
Schistosomiasis is a chronic helminthic infection that afflicts more than 200 million individuals globally. Toxoplasmosis is a ubiquitous protozoan infection with a worldwide prevalence of 34% [1, 2]. Since schistosomiasis has been shown to be largely an immunologic disease [3] and toxoplasmosis has recently been reported to be intensely immunosuppressive [4-6], the interaction between these two infections is of particular interest. The host granulomatous response to Schistosoma mansoni eggs deposited in the portal venules of the liver has been shown to be an immunologic reaction of the cell-mediated type [7]. The granulomas and the subsequent fibrous scarring obstruct portal blood flow, resulting in portal hypertension, congestive splenomegaly, and esophageal varices [8]. Suppression of cellmediated immunological reactivity markedly reReceived for publication May 3, 1976, and in revised form August 6,1976. This work was supported by grant no. AI 08163 from the National Institutes of Health, Bethesda, Maryland. We acknowledge the technical assistance of Pierre A. Peters and Earlene Moss. Please address requests for reprints to Dr. Kenneth S. Warren, Division of Geographic Medicine, Wearn Research Building, University Hospitals, Cleveland, Ohio 44106.
408
duces granulomatous inflammation [9, 10] and has been associated with significant alleviation of disease [11, 12]. Several recent reports have described the immunosuppression induced in animals by toxoplasmosis [4-6]. It has been shown that three weeks after infection there is a depletion of cortical thymocytes and a significant decrease in thymus weight. In addition, thymus-dependent zones of the lymph nodes and spleen were greatly reduced in area [4]. Mice infected with Toxoplasma gondii and subsequently immunized with sheep erythrocytes have depressed spleen plaqueforming cells and diminished serum hemagglutinin and hemolysin responses [5]. Whereas the lymphocyte response to a bone marrow-derived (B-) cell mitogen (lipopolysaccharide) was decreased for two to three weeks after infection with T. gondii, the responses to the thymusderived (T-) cell mitogens phytohemagglutinin and concanavalin A were suppressed significantly for at least 100 days [6]. In the present study we examined the effects of toxoplasma infection on the host granulomatous responses to S. mansoni eggs injected into the pulmonary microvasculature. The effect of toxoplasmosis on the course of natural infection with S. mansoni was also evaluated.
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on July 23, 2015
Infection with Toxoplasma gondii has been shown to suppress markedly the in vitro splenic lymphocyte response to nonspecific mitogens and the in vivo antibody response to sheep red blood cells. The effect of toxoplasmosis on an in vivo cell-mediated response, granuloma formation around Schistosoma mansoni eggs, was examined in the present study. The granulomas were markedly suppressed from two to 20 weeks after infection with T. gondii. In subsequent experiments the effect of toxoplasmosis-induced immunosuppression on the development of hepatosplenic schistosomiasis was evaluated. Mice with the combined infections had markedly smaller hepatic granulomas and lower mean portal pressures than those infected with S. mansoni alone. Although the prevalence of esophageal varices in the mice with schistosomiasis alone was 60%, there was no visible collateral circulation in the animals with both infections.
T. gondii and Response to S. mansoni
Materials and Methods
granuloma area (control) - granuloma area (T. gondij·infected)
~=:..::.:..:::~-=--.:..._--.:.~:::..-_--;-----:-~----X 100, granuloma area (control)
where the granuloma area is the area of inflammation minus the area of the egg.
Infection with S. mansoni. Three experiments were performed in which 10 T. gondii cysts were injected ip into groups of mice four weeks before, one day before, and four weeks after the onset of schistosomiasis. Infection with S. mansoni was induced by sc injection of 20 cercariae of a Puerto Rican strain of the parasite. For each experiment, animals were subdivided into four groups of 20 mice each: uninfected controls, T. gondii-infected mice, S. mansoniinfected mice, and mice wi th combined infections. Animals were followed closely during the experimental period, and all fatalities were recorded. Eight weeks after the schistosome infection, 10 mice were chosen randomly from each group and examined for body weight, liver and spleen weights, portal vein pressure, esophageal varices, hematocrit, and histopathology of the liver [14]. In addition, the diameters of the granulomas around S. mansoni eggs in the livers were measured in stained sections with a Cooke-A.EJ. image-spli tting measuring eyepiece (Vickers Instruments, Woburn, Mass.) [14]. In the two groups of animals infected with Toxoplasma one day before infection with S. mansoni, a high mortality rate was observed between six and eight weeks. At eight weeks, when the pathophysiological studies were performed, it was noted that these animals had multiple necrotic liver lesions. The experiment was repeated to confirm these findings, and when these animals were killed, their livers, which also contained multiple necrotic areas, were removed aseptically. Samples from the cut surface of these livers were cultured aerobically and anaerobically [15] by Dr. K. V. Gopalakrishna of the Veterans Administration Hospital of Cleveland, Ohio. Sections of liver tissue were stained with giemsa solution and examined for Actinobacillus piliformis [16]. In addition, the effect on these animals of the administration of specific therapy for toxoplasmosis, beginning at five weeks (when egg deposition by the schistosomes begins) a~d continuing for three weeks, was evaluated In terms of the mortali ty of the animals and the development of the necrotic areas in their livers. Sulphadimidine and pyrimethamine were administered daily by a blunt-tip cannula in doses of 3.2 mg and 0.01 mg per mouse, respectively [17].
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on July 23, 2015
Mice. Swiss albino female mice weighing 1820 g were purchased from Carworth Farms, New City, N. Y. T. gondii infection. To obtain T. gondii cysts, we removed the brains of mice infected with the Gleadle strain for five to six weeks and emulsified them in Hanks' balanced salt solution (Flow Laboratories, Rockville, Md.) in a tissue grinder. Cysts were counted in three separate 20pJ aliquots of brain suspensionpla~ed on gla~s slides with cover slips on top. Expenmental ammals were given ip injections of 10 cysts in 0.2 ml of brain emulsion to which penicillin (100 unitsj ml) and streptomycin (10 IJ-g/ml) were added. Similar volumes of brain emulsion from uninfected mice with added antibiotics were injected ip into uninfected control animals. . Granuloma formation around S. mansoru eggs. For the study of the effect of toxoplasmosis on granuloma formation, S. mansoni eggs were.isolated from the livers of mice infected for eight weeks [13] and were injected iv into the pulmonary microvasculature of T. gondii-infected and control mice. Each animal received 1,500 eggs suspended in 0.5 ml of 0.85% NaCl. Th~ gran~ Iomatous responses elicited were examined III separate groups of mice that had been infected with T. gondii for two to 30 weeks. One group of mice was sensitized with an ip 'injection of 1,500 eggs eight days before iv injection of eggs, and the response was examined four, eight, 12, 20, and 30 weeks after the iv injection. On day 8 after iv injection of eggs, the lungs were removed, sections were prepared, and the size of the granulomatous reaction was determined ('1T MC Particle Measurement Computer; Millipore Corp., Bedford, Mass.) as previously described [10]. Approximately 50 granulomas were measured within each experimental group of five to six animals, and the mean lesion area was calculated. The percentage of change in the area of inflammation due to toxoplasmosis was calculated according to the formula
409
Mahmoud, Strickland, and Warren
410
Results
