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Bioassay of Plasmodium berghei
LABORATORY
MEETING
5
I n addition to these direct comparisons, other interesting features were demonstrated: (1) P. b. berghei; 50½ hours; pseudocytomere formation. (2) P. b. berghei; 50½ hours; vacuolation. (3) P. b. berghei; 36 hours; nuclear structure as seen after acid hydrolysis. (4) P. berghei subspecies; 50 hours; pattern of nuclei in large but immature schizonts as seen after acid hydrolysis. (5) P. b. berghei; 50~ hours; a mature schizont in a smear from one of several very heavily infected livers. REFERENCES LANDAU, I. & CHABAUD, A. G. (1965). C.R. Acad. Sci., Paris, 260, 230. YOELI, M. & MOST, H. (1965). Amer. J. trop. Med. Hyg., 13, 700.
Dr. Irene Landau
Laboratoire de Zoologie (Vers), Museum National d'Histoire Naturelle, Paris E x o - e r y t h r o c y t i c schizonts o f a rodent p l a s m o d i u m in the fiver o f a wild Thamnomys rutilans 4 schizonts were found in sections of the liver of a Thamnomys rutilans captured at the Station de la Mabok6 du Mus6um National d'Histoire Naturelle, 150 km. south-west of Bangui, in the R6publique Centrafricalne. Blood smears from the rodent showed a mixed infection of Plasmodium chabaudi and a new subspecies of P. berghei. It was killed 11 days after capture. The examination of blood smears from all adult Thamnomys captured in the same area in March 1965 revealed an infection rate of 100% for P. chabaudi, and over 50% for the subspecies of P. berghei (LANDAU and CHABAtrD, 1965). Parasitaemia in naturally infected rodents is always low, and persists for a long time (at least 1½ years, and probably longer). The liver schizonts (3 of which were exhibited) are believed to represent secondary exo-erythrocytic forms of one of the two plasmodia of T. rutilans. Their main characteristics are a distinct limiting membrane, a small number of nuclei, a vacuolated cytoplasm, and an enlargement of the host cell nucleus. REFERENCE
LANDAU, I. & CHABAVD, A, G. (1965).
C.R. Acad. Sci., Paris, 260, 230.
Professor Meir Yoeli, Dr. R a s m a S. U p m a n i s , Dr. J e r o m e Vanderberg and Dr. Harry Most
N e w York University School of Medicine The p r e - e r y t h r o c y t i c d e v e l o p m e n t o f Plasmodium berghei The growth of the pre-erythrocytic tissue schizonts of Plasmodium berghei in the parenchyma cells of the liver, in experimentally infected tree rats (Thamnomys surdaster), young albino rats, golden hamster and white mice, was demonstrated in photomicrographs and histological sections. Some of the tissue schizonts shown derived from animals inoculated successively with massive doses of sporozoites of P. berghei obtained from experimentally infected Anopheles stephensi. Other pre-erythrocytic growth stages Shown were obtained from liver biopsies of animals inoculated intravenously and intraperitoneally with 500,000 to 750,000 sporozoites. It is not difficult to perform 3 liver biopsies within a period of 48 hours after sporozoite inoculation, and the animal may survive to show a blood infection. T h e liver biopsy technique has made possible a more detailed and exact study of the age and growth forms of the pre-erythrocytic tissue stages of P. berghei. From the study of this experimental material, including biopsies at 5½, 18, 24 and 29-48 hours after sporo~
6
LABORATORY MEETING
zoite inoculation, it has been shown that P. berghei undergoes a primary tissue schizogony in the parenchyma cells of the liver. The development of a schizont to maturity takes 48-50 hours. At this time the peripheral blood is invaded by merozoites from bursting schizonts and the infectivity of the blood may be ascertained by subinoculation. T h e number of the merozoites in the mature schizont varies from 1500 in the small schizonts, to 5000 in the medium-sized schizonts, and to approximately 10,000 in the few large schizonts found. T h e growth and pattern of development of the pre-erythrocytic schizogony of P. berghei greatly resembles that of other mammalian plasmodia. There are, no doubt, a number of morphological and structural differences between different species which may be used for diagnosis. It is in the speed and in the innate growth rhythm of the parasite that P. berghei so strikingly differs from the pre-erythrocytic growth of the primate plasmodia. T h e primary pre-erythrocytic development of rodent malaria is 3 times faster than the swift growth of P. falciparum and P. knowlesi, and its maturation requires only one fourth of the time required for P. vivax and P. cynomologi. REFERENCES
(sent separately by Prof. Yoeli; not mentioned in text) YOELI, M. & MOST, H. (1965). Nature, Lond., 205, 715. (1965). Trans. R. Soc. trop. Med. Hyg., 59, 255. & MOST, H. (1965). Amer. ft. trop. Med. Hyg., 14, 700. ~ , VANDERBERG, J., UPMANIS, R. & MOST, H. Nature, Lond., in press. ~ , UPMANIS, R. & MOST, H. Bull. Soc. Path. exot., in press.
Dr. D. C. Warhurst
National Institute for Medical Research, Mill Hill, London, N.W.7 Bioassay of Plasmodlum berghei In blood-passaged P. berghei in mice, there is a linear relationship between log inoculum size and "pre-2% period" (the period between intravenous inoculation of parasites and infection of 2% of the red cells). To find the pre-2% period, only one blood film need be taken daily from each mouse. Two consecutive parasitaemia values, one below and one above 2%, give an interpolated estimate of pre-2% period in days to 2 or 3 decimal places. T h e technique was used to measure percentage survival of 2 strains of P. berghei on freezing to - 7 8 ° C . in 7% dimethylsulphoxide (DMSO) (B.D.H. Lab. Reagent). During freezing the temperature was lowered at 2°C. per minute down to --20°C. T h e 1 ml. ampoules were then rapidly thawed at 37°C. Two serial 10-fold dilutions of control parasitized red cells, parasitized red cells after suspension in D M S O and parasitized red ceils after suspension in D M S O , freezing and thawing, were inoculated into groups of 5 mice, and pre-2% periods were measured. Analysis of variance for the 2-point assays showed that the apparent growth rate of the parasites was unaffected by any of the treatments, so that valid comparisons could be made. In strain A (chloroquine-resistant) 41.5% of parasites survived D M S O alone (95% confidence-limits, 131.2"to 9 . 3 % ) , while 3"4yo survived after D M S O and freezing (limits, 13.8 to 0.2%). I n strain C 2 (a normal strain), 45.1°/0 survived after D M S O (limits, 70.9 to 27.5%) and 0.9°/0 after freezing (limits 2 . 0 to 0.3°/0). Application of the pre-2~/o period technique to drug tests in vivo was demonstrated. T h e advantages of the technique include: (1) applicability to most in vivo or in vitro treatments; (2) data produced (parasite inoculum versus pre-2% period) are easily analysed by standard bio-assay methods; (3) comparisons of treatments are made at the same low level of parasitaemia, when the risk of interference from acquired immunity of the test animals is low.
Acknowledgements Analysis of variance was kindly carried out by Miss M. V. Mussett and Miss P. Woodward of the Division of Biological Standards. I am grateful to Dr. P. ]. Walker and Dr. F. Hawking for advice.
MEETING
5
I n addition to these direct comparisons, other interesting features were demonstrated: (1) P. b. berghei; 50½ hours; pseudocytomere formation. (2) P. b. berghei; 50½ hours; vacuolation. (3) P. b. berghei; 36 hours; nuclear structure as seen after acid hydrolysis. (4) P. berghei subspecies; 50 hours; pattern of nuclei in large but immature schizonts as seen after acid hydrolysis. (5) P. b. berghei; 50~ hours; a mature schizont in a smear from one of several very heavily infected livers. REFERENCES LANDAU, I. & CHABAUD, A. G. (1965). C.R. Acad. Sci., Paris, 260, 230. YOELI, M. & MOST, H. (1965). Amer. J. trop. Med. Hyg., 13, 700.
Dr. Irene Landau
Laboratoire de Zoologie (Vers), Museum National d'Histoire Naturelle, Paris E x o - e r y t h r o c y t i c schizonts o f a rodent p l a s m o d i u m in the fiver o f a wild Thamnomys rutilans 4 schizonts were found in sections of the liver of a Thamnomys rutilans captured at the Station de la Mabok6 du Mus6um National d'Histoire Naturelle, 150 km. south-west of Bangui, in the R6publique Centrafricalne. Blood smears from the rodent showed a mixed infection of Plasmodium chabaudi and a new subspecies of P. berghei. It was killed 11 days after capture. The examination of blood smears from all adult Thamnomys captured in the same area in March 1965 revealed an infection rate of 100% for P. chabaudi, and over 50% for the subspecies of P. berghei (LANDAU and CHABAtrD, 1965). Parasitaemia in naturally infected rodents is always low, and persists for a long time (at least 1½ years, and probably longer). The liver schizonts (3 of which were exhibited) are believed to represent secondary exo-erythrocytic forms of one of the two plasmodia of T. rutilans. Their main characteristics are a distinct limiting membrane, a small number of nuclei, a vacuolated cytoplasm, and an enlargement of the host cell nucleus. REFERENCE
LANDAU, I. & CHABAVD, A, G. (1965).
C.R. Acad. Sci., Paris, 260, 230.
Professor Meir Yoeli, Dr. R a s m a S. U p m a n i s , Dr. J e r o m e Vanderberg and Dr. Harry Most
N e w York University School of Medicine The p r e - e r y t h r o c y t i c d e v e l o p m e n t o f Plasmodium berghei The growth of the pre-erythrocytic tissue schizonts of Plasmodium berghei in the parenchyma cells of the liver, in experimentally infected tree rats (Thamnomys surdaster), young albino rats, golden hamster and white mice, was demonstrated in photomicrographs and histological sections. Some of the tissue schizonts shown derived from animals inoculated successively with massive doses of sporozoites of P. berghei obtained from experimentally infected Anopheles stephensi. Other pre-erythrocytic growth stages Shown were obtained from liver biopsies of animals inoculated intravenously and intraperitoneally with 500,000 to 750,000 sporozoites. It is not difficult to perform 3 liver biopsies within a period of 48 hours after sporozoite inoculation, and the animal may survive to show a blood infection. T h e liver biopsy technique has made possible a more detailed and exact study of the age and growth forms of the pre-erythrocytic tissue stages of P. berghei. From the study of this experimental material, including biopsies at 5½, 18, 24 and 29-48 hours after sporo~
6
LABORATORY MEETING
zoite inoculation, it has been shown that P. berghei undergoes a primary tissue schizogony in the parenchyma cells of the liver. The development of a schizont to maturity takes 48-50 hours. At this time the peripheral blood is invaded by merozoites from bursting schizonts and the infectivity of the blood may be ascertained by subinoculation. T h e number of the merozoites in the mature schizont varies from 1500 in the small schizonts, to 5000 in the medium-sized schizonts, and to approximately 10,000 in the few large schizonts found. T h e growth and pattern of development of the pre-erythrocytic schizogony of P. berghei greatly resembles that of other mammalian plasmodia. There are, no doubt, a number of morphological and structural differences between different species which may be used for diagnosis. It is in the speed and in the innate growth rhythm of the parasite that P. berghei so strikingly differs from the pre-erythrocytic growth of the primate plasmodia. T h e primary pre-erythrocytic development of rodent malaria is 3 times faster than the swift growth of P. falciparum and P. knowlesi, and its maturation requires only one fourth of the time required for P. vivax and P. cynomologi. REFERENCES
(sent separately by Prof. Yoeli; not mentioned in text) YOELI, M. & MOST, H. (1965). Nature, Lond., 205, 715. (1965). Trans. R. Soc. trop. Med. Hyg., 59, 255. & MOST, H. (1965). Amer. ft. trop. Med. Hyg., 14, 700. ~ , VANDERBERG, J., UPMANIS, R. & MOST, H. Nature, Lond., in press. ~ , UPMANIS, R. & MOST, H. Bull. Soc. Path. exot., in press.
Dr. D. C. Warhurst
National Institute for Medical Research, Mill Hill, London, N.W.7 Bioassay of Plasmodlum berghei In blood-passaged P. berghei in mice, there is a linear relationship between log inoculum size and "pre-2% period" (the period between intravenous inoculation of parasites and infection of 2% of the red cells). To find the pre-2% period, only one blood film need be taken daily from each mouse. Two consecutive parasitaemia values, one below and one above 2%, give an interpolated estimate of pre-2% period in days to 2 or 3 decimal places. T h e technique was used to measure percentage survival of 2 strains of P. berghei on freezing to - 7 8 ° C . in 7% dimethylsulphoxide (DMSO) (B.D.H. Lab. Reagent). During freezing the temperature was lowered at 2°C. per minute down to --20°C. T h e 1 ml. ampoules were then rapidly thawed at 37°C. Two serial 10-fold dilutions of control parasitized red cells, parasitized red cells after suspension in D M S O and parasitized red ceils after suspension in D M S O , freezing and thawing, were inoculated into groups of 5 mice, and pre-2% periods were measured. Analysis of variance for the 2-point assays showed that the apparent growth rate of the parasites was unaffected by any of the treatments, so that valid comparisons could be made. In strain A (chloroquine-resistant) 41.5% of parasites survived D M S O alone (95% confidence-limits, 131.2"to 9 . 3 % ) , while 3"4yo survived after D M S O and freezing (limits, 13.8 to 0.2%). I n strain C 2 (a normal strain), 45.1°/0 survived after D M S O (limits, 70.9 to 27.5%) and 0.9°/0 after freezing (limits 2 . 0 to 0.3°/0). Application of the pre-2~/o period technique to drug tests in vivo was demonstrated. T h e advantages of the technique include: (1) applicability to most in vivo or in vitro treatments; (2) data produced (parasite inoculum versus pre-2% period) are easily analysed by standard bio-assay methods; (3) comparisons of treatments are made at the same low level of parasitaemia, when the risk of interference from acquired immunity of the test animals is low.
Acknowledgements Analysis of variance was kindly carried out by Miss M. V. Mussett and Miss P. Woodward of the Division of Biological Standards. I am grateful to Dr. P. ]. Walker and Dr. F. Hawking for advice.