EXPERIMENTAL
40, 411-420
PARASITOLOGY
Trypanosoma
(1976)
cruzi: Lactate Dehydrogenase and Infections in Mice
lsoenzymes
TERESA I. MERCADO Laboratoy of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Department of Health, Education, and Welfare, Bethesda, Maryland 20014, U.S.A. (Accepted
for publication
19 January
1976)
T. I. 1976. Trypanosoma
cruzi: Lactate dehydrogenase isoenzymes Parasitology 40, 411420. Total plasma LDH
and infecisoenzyme (EC 1.1.1.27) levels increased significantly over the normal level in mice infected with strains of Trypanosoma cmzi from three different geographic locations, but some strain differences were observed. The most rapid increase was exhibited by the blood-induced Tulahuen strain, but this strain, unlike the House 510 or House 11, did not elicit an increase during the early period of infection. Overall increases in LDH-1 and LDH-2, heart isoenzymes, were most marked in vector-derived House 510 infections, but, as in the Tulahuen strain, a considerable increase was also observed in blood-induced infections. The House 510 strain also elicited significant increases in LDH-4; these were particularly high during the early period of the blood-induced infection. By contrast, the vector-derived Tulahuen strain elicited a higher increase in LDH-4 during the early period than the House 510 or House 11 strains. Comparable similarites and differences were also observed in regard to LDH-3, 5, and “X.” The most marked isoenzyme increases were those of “LDH-X” exhibited by the blood-induced House 510 and vector-derived Tulahuen strains. Parallel histopathologic studies of liver, heart, and skeletal muscle disclosed s&&cant pathology in all the infections. Animals with blood-induced Tulahuen strain infections characteristically showed extensive necrosis with marked multiplication of parasites throughout the liver, .but little or no evident damage to the heart and skeleal muscle. Animals infected with House 510 and House 11 strains exhibited minimal pathology in the liver but severe damage to the heart and skeletal muscle. Increases in LDH-4 and LDH-5, isoenzymes which represent both liver and skeletal muscle, in blood-induced Tulahuen infections were attributed largely to liver damage, but in the House 510 and House 11 infeotions were related more to skeletal muscle. INDEX DESCRIPTORS: Chagas’ disease; Trypanosomu cTuzi; Parasite strains; Blood-induced infections; Rho&us< prolixus; Vector-derived infections; Lactate dehydrogenase isoenzymes (EC 1.1.1.27); Mice; Plasma; Liver; Heart; Skeletal muscle; Disc electrophoresis; Densitometry; Planimetry; Histochemical staining; Histopathology. MERCAW,
tions in mice.
Experimental
tions in regard to lactate dehydrogenase (LDH) (EC 1.1.1.27) isoenzymes in medical diagnosis. It is well known that following myocardial infarction an elevation of LDH isoenzymes designated 1 and 2 is observed in the plasma and that an increase of LDH-isoenzyme 5 usually accompanies liver or skeletal muscle disease. It has been
The study of isoenzymes has been the subject of extensive research in recent years (Vessel1 and Bear-n 1957, Wroblewski et al. 1966, Wroblewski and Gregory 1961, Wieme and van Maercke 1961, Vessell 1961, Allen 1961, Kaplan 1968, Lubrano et al 1971, Garbus 1971). Of particular importance have been the contribu411
1976by AcademicPress,Inc. reproductionin any form resved.
412
TERESA I. MERCADO
further demonstrated that the particular isoenzymes which are increased correspond to those contributed most prominently by the tissue which is pathologically involved. Studies in this laboratory on host pathology produced by Trypanosoma cruzi have indicated that certain strains of this parasite are more pathogenic for muscle whereas others cause greater damage in liver. These observations and knowledge of the role of isoenzymes in disease led us to examine whether in T. cruzi infections, as in some of the studies mentioned above, tissue injury could be assessed on the basis of the LDH isoenzyme level of the plasma. The results of such a study are described in the present report. MATERIALS
AND METHODS
Strains of Trypanosoma cruzi from three different geographic locations were studied in blood-induced and vector-derived in( 1) Tulahuen fections. These were: (Chile), (2) H ouse 510 (Costa Rica), and (3) House 11 (Nicaragua). Our earlier observations, that vector-derived infections preferentially produced parasitization of heart and skeletal muscle, but not of liver, suggested the study of this type of infection in addition to the blood-induced type. The blood-induced House 510 and House 11 strains had been maintained by consecutive blood passages (every 10 to 15 TABLE l’arasitemia
of Mice Infected
days) during a period of 2 years; the blood-induced Tulahuen strain was passed every 6 to 8 days for about 10 years. Fourto six-week-old NIH male mice were used. Inocula of 5 to 10 million trypanosomes were administered intraperitoneally. The vector-derived House 510 and House 11 strains had been maintained by alternate vector (Rhodnius prolixus) mouse passages for 5 years; the vector-passed Tulahuen strain had undergone five alternate passages over a period of 10 months. Inocula of 6000 to 94,000 parasites derived from Rho&Gus were administered intraperitoneally. Parasitemia was estimated by hemocytometer counting of the blood. The infections were classified as “early,” “intermediate,” and “acute” (Table 1). Blood, obtained by cardiac puncture under ether anesthesia, was centrifuged at 1000 rpm for 10 min at 4 C. Discontinuous polyacrylamide gel electrophoresis of the separated plasma was performed (Dietz and Lubrano 1967) employing a Canalco model 1200 bath assembly (electrode polarity at normal) and a Buchler power source. Gel tubes, 76 x 7 mm, were used and the gel solution allowed to polymerize to a height of 48 mm. The samples were electrophoresed in Tris-glycine buffer at pH 8.3 and the bath assembly was maintained in ice throughout the period of migration. An electric current of 5 mA/ tube was used for 30 min. I with
cruz+
Strains
Infection/periods of pittency Tulahuen Early Blood-induced Days Number of animals
40, 411-420
PARASITOLOGY
Trypanosoma
(1976)
cruzi: Lactate Dehydrogenase and Infections in Mice
lsoenzymes
TERESA I. MERCADO Laboratoy of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Department of Health, Education, and Welfare, Bethesda, Maryland 20014, U.S.A. (Accepted
for publication
19 January
1976)
T. I. 1976. Trypanosoma
cruzi: Lactate dehydrogenase isoenzymes Parasitology 40, 411420. Total plasma LDH
and infecisoenzyme (EC 1.1.1.27) levels increased significantly over the normal level in mice infected with strains of Trypanosoma cmzi from three different geographic locations, but some strain differences were observed. The most rapid increase was exhibited by the blood-induced Tulahuen strain, but this strain, unlike the House 510 or House 11, did not elicit an increase during the early period of infection. Overall increases in LDH-1 and LDH-2, heart isoenzymes, were most marked in vector-derived House 510 infections, but, as in the Tulahuen strain, a considerable increase was also observed in blood-induced infections. The House 510 strain also elicited significant increases in LDH-4; these were particularly high during the early period of the blood-induced infection. By contrast, the vector-derived Tulahuen strain elicited a higher increase in LDH-4 during the early period than the House 510 or House 11 strains. Comparable similarites and differences were also observed in regard to LDH-3, 5, and “X.” The most marked isoenzyme increases were those of “LDH-X” exhibited by the blood-induced House 510 and vector-derived Tulahuen strains. Parallel histopathologic studies of liver, heart, and skeletal muscle disclosed s&&cant pathology in all the infections. Animals with blood-induced Tulahuen strain infections characteristically showed extensive necrosis with marked multiplication of parasites throughout the liver, .but little or no evident damage to the heart and skeleal muscle. Animals infected with House 510 and House 11 strains exhibited minimal pathology in the liver but severe damage to the heart and skeletal muscle. Increases in LDH-4 and LDH-5, isoenzymes which represent both liver and skeletal muscle, in blood-induced Tulahuen infections were attributed largely to liver damage, but in the House 510 and House 11 infeotions were related more to skeletal muscle. INDEX DESCRIPTORS: Chagas’ disease; Trypanosomu cTuzi; Parasite strains; Blood-induced infections; Rho&us< prolixus; Vector-derived infections; Lactate dehydrogenase isoenzymes (EC 1.1.1.27); Mice; Plasma; Liver; Heart; Skeletal muscle; Disc electrophoresis; Densitometry; Planimetry; Histochemical staining; Histopathology. MERCAW,
tions in mice.
Experimental
tions in regard to lactate dehydrogenase (LDH) (EC 1.1.1.27) isoenzymes in medical diagnosis. It is well known that following myocardial infarction an elevation of LDH isoenzymes designated 1 and 2 is observed in the plasma and that an increase of LDH-isoenzyme 5 usually accompanies liver or skeletal muscle disease. It has been
The study of isoenzymes has been the subject of extensive research in recent years (Vessel1 and Bear-n 1957, Wroblewski et al. 1966, Wroblewski and Gregory 1961, Wieme and van Maercke 1961, Vessell 1961, Allen 1961, Kaplan 1968, Lubrano et al 1971, Garbus 1971). Of particular importance have been the contribu411
1976by AcademicPress,Inc. reproductionin any form resved.
412
TERESA I. MERCADO
further demonstrated that the particular isoenzymes which are increased correspond to those contributed most prominently by the tissue which is pathologically involved. Studies in this laboratory on host pathology produced by Trypanosoma cruzi have indicated that certain strains of this parasite are more pathogenic for muscle whereas others cause greater damage in liver. These observations and knowledge of the role of isoenzymes in disease led us to examine whether in T. cruzi infections, as in some of the studies mentioned above, tissue injury could be assessed on the basis of the LDH isoenzyme level of the plasma. The results of such a study are described in the present report. MATERIALS
AND METHODS
Strains of Trypanosoma cruzi from three different geographic locations were studied in blood-induced and vector-derived in( 1) Tulahuen fections. These were: (Chile), (2) H ouse 510 (Costa Rica), and (3) House 11 (Nicaragua). Our earlier observations, that vector-derived infections preferentially produced parasitization of heart and skeletal muscle, but not of liver, suggested the study of this type of infection in addition to the blood-induced type. The blood-induced House 510 and House 11 strains had been maintained by consecutive blood passages (every 10 to 15 TABLE l’arasitemia
of Mice Infected
days) during a period of 2 years; the blood-induced Tulahuen strain was passed every 6 to 8 days for about 10 years. Fourto six-week-old NIH male mice were used. Inocula of 5 to 10 million trypanosomes were administered intraperitoneally. The vector-derived House 510 and House 11 strains had been maintained by alternate vector (Rhodnius prolixus) mouse passages for 5 years; the vector-passed Tulahuen strain had undergone five alternate passages over a period of 10 months. Inocula of 6000 to 94,000 parasites derived from Rho&Gus were administered intraperitoneally. Parasitemia was estimated by hemocytometer counting of the blood. The infections were classified as “early,” “intermediate,” and “acute” (Table 1). Blood, obtained by cardiac puncture under ether anesthesia, was centrifuged at 1000 rpm for 10 min at 4 C. Discontinuous polyacrylamide gel electrophoresis of the separated plasma was performed (Dietz and Lubrano 1967) employing a Canalco model 1200 bath assembly (electrode polarity at normal) and a Buchler power source. Gel tubes, 76 x 7 mm, were used and the gel solution allowed to polymerize to a height of 48 mm. The samples were electrophoresed in Tris-glycine buffer at pH 8.3 and the bath assembly was maintained in ice throughout the period of migration. An electric current of 5 mA/ tube was used for 30 min. I with
cruz+
Strains
Infection/periods of pittency Tulahuen Early Blood-induced Days Number of animals
