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Plasmodium berghei exoerythrocytic forms develop only in the liver
904
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPIC.~L MEDICINE AND HYGIENE, VOL. 75, No. 6, 1981. CORRESPONDENCE
This study was supported by the Academy of Medicine, Malaysia, through the Tun Razak Research Award to D.S. We are, etc. D. SINNIAH Dept. of Paediatrics, University of Malaya, Kuala Lumpur, Malaysia G. BASKARAN Central Animal House, University of Malaya, Kuala Lumpur, Malaysia B. VITAYALAKSHMI
Dept. of Physiology, Stanley Medical College, Madras, India N. SUNDARAVELLI Institute of Child Health, and Hospital for Children, Madras 8, India References Sinniah D. & Baskaran G. (1981) Margosa oil poisoning as a cause of Reye’s Syndrome. Lancet, i, 487-489.
Accepted for publication
15th July,
1981.
Plasmodium berghei exoerythrocytic forms develop only in the liver MADAM-Since the initial description of the exoerythrocytic (EE) form of Plasmodium cynomolgi in the liver of its simian host (SHORTT & GARNHAM, 1948), all other species of mammalian malaria para&es studied hive been shown to undergo a similar EE develooment within the livers of their respective hosts. The search for EE development at other tissue sites has consistently yielded negative results. Thus, in the case of rodent malaria, extensive histological examinations of tissues other than the liver have failed to produce any evidence of ectopic EE forms even at times when EE forms in the liver were abundant (YOELI & MOST, 1965; YOELI
et al., 1966; BAFORT, 1971).
Nevertheless, these negative morphological findings do not in themselves prove the lack of ectopic EE development. The failure to identify such ectopic EE forms might also be due to a relatively low frequency of their occurrence, or to their small size or abnormal morphology in the non-liver tissues. I therefore decided to investigate the possibility of ectopic development by using a more sensitive assav for the nresence of EE forms than their morphilogical presence, namely, their infectivity. We previously showed that minced liver infected with EE forms of P. berghei was infective when
transferred into the peritoneal cavity of recipient rodents (FOLEY & VANDERBERG, 1977). This established, for the first time, a means of demonstrating the presence of EE forms by their infectivity as well as their morphology. By doing similar studies with tissues other than those of the liver, I have now been able to show that the liver is the only organ in which such infective EE forms can be demonstrated after injection of rats with soorozoites. In view of the fact that the initial invasion site of sporozoites might ‘be lymphoidmacrophage cells, with subsequent early development of the EE form in these cells NERHAVE et al.. 19801, special attention was Daid td tissues rich i; these ceils. The techniques used for these studies were similar to those tlreviouslv described (FOLEY & VANDERBERG, 1977). Female Sprague-Dawley rats (35-85 g) were injected intravenously with l-2 x lo6 sporozoites of the NK-65 strain of P. berghei. At various periods of time between one and 36 hr after inoculation, these rats were killed and several tissues were removed. Liver, spleen, intestine, lungs, kidney and muscle were minced in cold tissue culture medium M 199 into fragments about 0 *5 to 1 *O mm in greatest dimension. Amounts of tissue ranging from loo-250 mg were inoculated intraperitoneally into nembutal-anaesthetized rats. Peritoneal exudate containing macrophages, and bone marrow (from the femur) were also inoculated into rats. The blood of all recipients was then examined periodically for three weeks for evidence of blood forms of P. berghei. The results showed that 92% (45/49) recipients of liver developed parasitaemia, while none of the recipients of other tissues (20-30 rats for each tissue) became patent. These results indicate that, once I’. berghei sporozoites disappear from the circulatory system of a rodent, later stages of EE development of the parasite can be detected only in the liver. This suggests that the entire developmental sequence of EE development is restricted to the liver. This study was supported by the US National Institutes of Health through Research Grant AI 09560. I thank Dr. David A. Foley for his skilled assistance in preliminary studies. I am, etc., J. I?. VANDERBERG Division of Parasitology, Dept. of Microbiology, New York University Medical School, New York, N. Y. 10016, USA References Bafort, J. M. (1971). The biology of rodent malaria with uarticular reference to Plasmodium vinckei vinckli. Annales de la Socie’te’ Belge de Me’decine Tropicale, 51, l-204. Foley, D. A. & Vanderberg, J. (1977). Plasmodium berghei: transmission by intraperitoneal inoculation of immature exoerythrocytic schizonts from rats to rats, mice and hamsters. Experimental Parasitology, 43, 69-81. Shortt, H. E. & Garnham, I?. C. C. (1948). Preerythrocytic stage in mammalian malaria parasites. Nature, 161, 126.
TRANSACTIONS OPTHEROYALSOCIETY OFTROPICALMEDICINE
Verhave, J. P., Meuwissen, J. H. E. T. & Golenser, J. (1980). The dual role of macrophages in the sporozoite-induced malaria infection. A hypothesis. International Journal of Nuclear Medicine and Biology, 7, 149-156. Yoeli, M. & Most, H. (1965). Studies on sporozoiteinduced infections of rodent malaria. I. The preerythrocytic tissue stage of Plasmodium berghei. American Journal of Tropical Medicine and Hygiene, 14, 700-714. Yoeli, M., Upmanis, R. S., Vanderberg, J. & Most, H. (1966). Life cycle and patterns of development of Plasmodium berghei in normal and experimental hosts. Military Medicine, 131 (Suppl.), 900-914.
AND HYGIENE,
VOL.
75, No.
6, 1981.
CORRESPONDENCE
Sickle
cell trait and the immune response to meningococcal vaccines MADAM-During the course of an investigation into the factors influencing the immune response to meningococcal vaccination undertaken during the wet season of 1976 in the northern Nigerian village of Mahuta, we found that subjects with the haemoglobin genotype AS had a higher antibody response to group C meningococcal vaccine than subjects with the genotype AA (GREENWOOD et al., 1980). This effect was nresent in subiects of all ages but was most marked in the young. ‘Over-all, &difference between the two groups was statistically significant. Acute malaria suppresses the immune response to meningococcal vaccines (WILLIAMSON & GREENWOOD, 1978) and we suggested,
Accepted for publication 21st July, 1981.
Association
of cockroaches with an outbreak of dysentery-Comment MAnAIM-Although I recognize that there is epidemiologic evidence to suggest that flies are important in the spread of Shigella (LINDSAY, 1953), and do not wish to condone large numbers of cockroaches in sutlers’ shops, I wish to take issue with the conclusion offered by BURGESS & CHETWYN (1981) that isolation of Shigella dysenteriae from a cockroach even in the midst of an outbreak of shigellosis in humans caused by the same serotype demonstrates a causal relationship between cockroaches and the spread of Shigella. In the course of an investigation of human outbreaks caused by Shigella, this organism may be isolated from a variety of environmental sources that have been contaminated by infected persons; under these circumstances it is not surprising that cockroaches may also be exposed to these pathogens, but it does not suggest that there is a causal relationship. I am, etc., D. N. TAYLOR Enteric Diseases Branch, Bacterial Diseases Division, Center for Infectious Diseases, Centers for Disease Control, AtZanta, GA 30333 USA.
905
therefore,
that the genotype effect might have been mediated through the protective effect of the haemoglobin genotype AS against severe malaria. In the dry season of 1978 we undertook a further meningococcal vaccine study in the town of Malumfashi 30 km from Mahuta village. The main aim of this investigation was to determine if meningococcal vaccination had any effect on meningococcal carriage. The results of this aspect of the study are reported separately (BLAKEBROUGH et al., 1981a). However, the study also provided us with an opportunity to re-examine the effect of haemoglobin genotype on the immune response to meningococcal vaccination. 438 pupils, aged 11 to 20 years, attending the Government Secondarv School, Malumfashi, were vaccinated with 50 pg- of group A meningococcal nolvsaccharide vaccine and with 50 ua of arouv C meningococcal polysaccharide vaccine ?Insiitut McrieGx). Fingerprick blood samples were obtained from 288 nunils before and two weeks after vaccination. Piaima was separated immediately after collection and was stored at -20°C until assayed for group A and group C meningococcal antibody levels by a haemagglutination technique (BLAKEResidual red cells were BROUGH et al., 1981b). stored at -20°C until they were tested for haemoglobin genotype by cellulose acetate electrophoresis. The results of pre- and post-vaccination antibody assays in subjects with normal and abnormal haemoglobin genotypes are shown in Table I. No significant differences between groups were found. Table I-The influence of haemoglobin genotype on theimmune response to meningococcal vaccination. Meningococcal haemagglutinating antibody titres (mean fl standard error of the mean) are expressed as a reciprocal of the log, antibody titre
References Lindsay, D. R., Stewart, W. H. & Watt, J. (1953). Effect of fly control on diarrhea1 disease in an area of moderate morbidity. Public Health Reports, 68, 361-367. Burgess, N. R. H., Chetwyn, K. N. (1981). Association of cockroaches with an outbreak of dysentery. Transactions of the Royal Society of Tropical Medicine and Hygiene, 75, 332-333.
Group A Pre-vaccination Postvaccination
2.9hO.l 4.9hO.l
2.9kO.2 5.0f0.2
2.9&O-4 4.910.5
Accepted
Group C Pre-vaccination Post-vaccination
1.5&0*1 5.7f0.2
1.4f0.2 5.9&O-2
1*3&O-3 6.7&O-7
for
publication
1st August,
1981.
Antibody
Haemoglobin AA AS n.68 n.208
Genotype Other n.12
TRANSACTIONS OF THE ROYAL SOCIETY OF TROPIC.~L MEDICINE AND HYGIENE, VOL. 75, No. 6, 1981. CORRESPONDENCE
This study was supported by the Academy of Medicine, Malaysia, through the Tun Razak Research Award to D.S. We are, etc. D. SINNIAH Dept. of Paediatrics, University of Malaya, Kuala Lumpur, Malaysia G. BASKARAN Central Animal House, University of Malaya, Kuala Lumpur, Malaysia B. VITAYALAKSHMI
Dept. of Physiology, Stanley Medical College, Madras, India N. SUNDARAVELLI Institute of Child Health, and Hospital for Children, Madras 8, India References Sinniah D. & Baskaran G. (1981) Margosa oil poisoning as a cause of Reye’s Syndrome. Lancet, i, 487-489.
Accepted for publication
15th July,
1981.
Plasmodium berghei exoerythrocytic forms develop only in the liver MADAM-Since the initial description of the exoerythrocytic (EE) form of Plasmodium cynomolgi in the liver of its simian host (SHORTT & GARNHAM, 1948), all other species of mammalian malaria para&es studied hive been shown to undergo a similar EE develooment within the livers of their respective hosts. The search for EE development at other tissue sites has consistently yielded negative results. Thus, in the case of rodent malaria, extensive histological examinations of tissues other than the liver have failed to produce any evidence of ectopic EE forms even at times when EE forms in the liver were abundant (YOELI & MOST, 1965; YOELI
et al., 1966; BAFORT, 1971).
Nevertheless, these negative morphological findings do not in themselves prove the lack of ectopic EE development. The failure to identify such ectopic EE forms might also be due to a relatively low frequency of their occurrence, or to their small size or abnormal morphology in the non-liver tissues. I therefore decided to investigate the possibility of ectopic development by using a more sensitive assav for the nresence of EE forms than their morphilogical presence, namely, their infectivity. We previously showed that minced liver infected with EE forms of P. berghei was infective when
transferred into the peritoneal cavity of recipient rodents (FOLEY & VANDERBERG, 1977). This established, for the first time, a means of demonstrating the presence of EE forms by their infectivity as well as their morphology. By doing similar studies with tissues other than those of the liver, I have now been able to show that the liver is the only organ in which such infective EE forms can be demonstrated after injection of rats with soorozoites. In view of the fact that the initial invasion site of sporozoites might ‘be lymphoidmacrophage cells, with subsequent early development of the EE form in these cells NERHAVE et al.. 19801, special attention was Daid td tissues rich i; these ceils. The techniques used for these studies were similar to those tlreviouslv described (FOLEY & VANDERBERG, 1977). Female Sprague-Dawley rats (35-85 g) were injected intravenously with l-2 x lo6 sporozoites of the NK-65 strain of P. berghei. At various periods of time between one and 36 hr after inoculation, these rats were killed and several tissues were removed. Liver, spleen, intestine, lungs, kidney and muscle were minced in cold tissue culture medium M 199 into fragments about 0 *5 to 1 *O mm in greatest dimension. Amounts of tissue ranging from loo-250 mg were inoculated intraperitoneally into nembutal-anaesthetized rats. Peritoneal exudate containing macrophages, and bone marrow (from the femur) were also inoculated into rats. The blood of all recipients was then examined periodically for three weeks for evidence of blood forms of P. berghei. The results showed that 92% (45/49) recipients of liver developed parasitaemia, while none of the recipients of other tissues (20-30 rats for each tissue) became patent. These results indicate that, once I’. berghei sporozoites disappear from the circulatory system of a rodent, later stages of EE development of the parasite can be detected only in the liver. This suggests that the entire developmental sequence of EE development is restricted to the liver. This study was supported by the US National Institutes of Health through Research Grant AI 09560. I thank Dr. David A. Foley for his skilled assistance in preliminary studies. I am, etc., J. I?. VANDERBERG Division of Parasitology, Dept. of Microbiology, New York University Medical School, New York, N. Y. 10016, USA References Bafort, J. M. (1971). The biology of rodent malaria with uarticular reference to Plasmodium vinckei vinckli. Annales de la Socie’te’ Belge de Me’decine Tropicale, 51, l-204. Foley, D. A. & Vanderberg, J. (1977). Plasmodium berghei: transmission by intraperitoneal inoculation of immature exoerythrocytic schizonts from rats to rats, mice and hamsters. Experimental Parasitology, 43, 69-81. Shortt, H. E. & Garnham, I?. C. C. (1948). Preerythrocytic stage in mammalian malaria parasites. Nature, 161, 126.
TRANSACTIONS OPTHEROYALSOCIETY OFTROPICALMEDICINE
Verhave, J. P., Meuwissen, J. H. E. T. & Golenser, J. (1980). The dual role of macrophages in the sporozoite-induced malaria infection. A hypothesis. International Journal of Nuclear Medicine and Biology, 7, 149-156. Yoeli, M. & Most, H. (1965). Studies on sporozoiteinduced infections of rodent malaria. I. The preerythrocytic tissue stage of Plasmodium berghei. American Journal of Tropical Medicine and Hygiene, 14, 700-714. Yoeli, M., Upmanis, R. S., Vanderberg, J. & Most, H. (1966). Life cycle and patterns of development of Plasmodium berghei in normal and experimental hosts. Military Medicine, 131 (Suppl.), 900-914.
AND HYGIENE,
VOL.
75, No.
6, 1981.
CORRESPONDENCE
Sickle
cell trait and the immune response to meningococcal vaccines MADAM-During the course of an investigation into the factors influencing the immune response to meningococcal vaccination undertaken during the wet season of 1976 in the northern Nigerian village of Mahuta, we found that subjects with the haemoglobin genotype AS had a higher antibody response to group C meningococcal vaccine than subjects with the genotype AA (GREENWOOD et al., 1980). This effect was nresent in subiects of all ages but was most marked in the young. ‘Over-all, &difference between the two groups was statistically significant. Acute malaria suppresses the immune response to meningococcal vaccines (WILLIAMSON & GREENWOOD, 1978) and we suggested,
Accepted for publication 21st July, 1981.
Association
of cockroaches with an outbreak of dysentery-Comment MAnAIM-Although I recognize that there is epidemiologic evidence to suggest that flies are important in the spread of Shigella (LINDSAY, 1953), and do not wish to condone large numbers of cockroaches in sutlers’ shops, I wish to take issue with the conclusion offered by BURGESS & CHETWYN (1981) that isolation of Shigella dysenteriae from a cockroach even in the midst of an outbreak of shigellosis in humans caused by the same serotype demonstrates a causal relationship between cockroaches and the spread of Shigella. In the course of an investigation of human outbreaks caused by Shigella, this organism may be isolated from a variety of environmental sources that have been contaminated by infected persons; under these circumstances it is not surprising that cockroaches may also be exposed to these pathogens, but it does not suggest that there is a causal relationship. I am, etc., D. N. TAYLOR Enteric Diseases Branch, Bacterial Diseases Division, Center for Infectious Diseases, Centers for Disease Control, AtZanta, GA 30333 USA.
905
therefore,
that the genotype effect might have been mediated through the protective effect of the haemoglobin genotype AS against severe malaria. In the dry season of 1978 we undertook a further meningococcal vaccine study in the town of Malumfashi 30 km from Mahuta village. The main aim of this investigation was to determine if meningococcal vaccination had any effect on meningococcal carriage. The results of this aspect of the study are reported separately (BLAKEBROUGH et al., 1981a). However, the study also provided us with an opportunity to re-examine the effect of haemoglobin genotype on the immune response to meningococcal vaccination. 438 pupils, aged 11 to 20 years, attending the Government Secondarv School, Malumfashi, were vaccinated with 50 pg- of group A meningococcal nolvsaccharide vaccine and with 50 ua of arouv C meningococcal polysaccharide vaccine ?Insiitut McrieGx). Fingerprick blood samples were obtained from 288 nunils before and two weeks after vaccination. Piaima was separated immediately after collection and was stored at -20°C until assayed for group A and group C meningococcal antibody levels by a haemagglutination technique (BLAKEResidual red cells were BROUGH et al., 1981b). stored at -20°C until they were tested for haemoglobin genotype by cellulose acetate electrophoresis. The results of pre- and post-vaccination antibody assays in subjects with normal and abnormal haemoglobin genotypes are shown in Table I. No significant differences between groups were found. Table I-The influence of haemoglobin genotype on theimmune response to meningococcal vaccination. Meningococcal haemagglutinating antibody titres (mean fl standard error of the mean) are expressed as a reciprocal of the log, antibody titre
References Lindsay, D. R., Stewart, W. H. & Watt, J. (1953). Effect of fly control on diarrhea1 disease in an area of moderate morbidity. Public Health Reports, 68, 361-367. Burgess, N. R. H., Chetwyn, K. N. (1981). Association of cockroaches with an outbreak of dysentery. Transactions of the Royal Society of Tropical Medicine and Hygiene, 75, 332-333.
Group A Pre-vaccination Postvaccination
2.9hO.l 4.9hO.l
2.9kO.2 5.0f0.2
2.9&O-4 4.910.5
Accepted
Group C Pre-vaccination Post-vaccination
1.5&0*1 5.7f0.2
1.4f0.2 5.9&O-2
1*3&O-3 6.7&O-7
for
publication
1st August,
1981.
Antibody
Haemoglobin AA AS n.68 n.208
Genotype Other n.12