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Resistance to superinfection with Plasmodium berghei in rats fed a protein-free diet
382
Resistance
to superinfection
with Plasmodium protein-free diet
J. S. EDIRISINGHE ‘*, E. B. FERN’ Departments of Human Nutrition’
AND
berghei
in rats fed a
G. A. T. TARGE-I-?
and Medical Protomology2, London School of Hygiene and Tropical Medicine, Keppel Street, London WClE 7HT
S-
The development of resistance to reinfection with Plasmodium berghei was studied in rats in which the primary infection had been almost totally suppressed by feeding a protein-free diet (peak parasitaemia 05%; patent for only the first four days after inoculation). On Days 5, 9, IS, 23 and 28 after primary inocuhN.ion groups of animals were challenged with the same strain of parasite. At the same time the diet was changed to &at of a 17% casein formula. The development of resistance as judged by the level of tmrasitaemiafollowinn challenee reached a significant ievel nine days after he prim-&y inoculation and almost complete protection by Day 23 of the study. The protective activity was immunological since it could be transferred to other animals by a single intravenous injection of a suspension of spleen cells from infected donors. The study illustrates that infected animals experiencing severe protein malnutrition are still capable of mounting a substantial immune response to malaria. Introduction The ability to suppressa malarial infection through the quality of a diet was first demonstrated by MAEGRAITH et al. in 1952. By maintaining rats exclusively on milk diets they were able to reduce the level of parasitaemia in animals infected with Plasm+ dim berg&i. The mechanism for this attentuation was subsequently shown by HAWKING (1954), amongst others, to be associated with the requirement for 4-amino benzoic acid (PABA) by the parasite and the relative deficiency of this vitamin in milk. In 1966 KRETSCHMARshowed that mice in which the development of the primary infection had been suppressed with a milk diet were able to acquire significant resistance to reinfection with the same s&n of parasite. Similar findings were reported earlier bv ADLER (19581 usina a meat diet (also KRETXHMAR deficient *in PABA)‘ and’ P. v&&i. (196fi) concluded that this immunity, obtained through dietary control of the primary infection, depended on the duration of contact with the antigen and not on the density of ,the parasite in the blood. GILBERTSON et al. (1970), however, using a similar experimental model, concluded that the acquired immunity was dependent on both the level of the original infection and its duration. We have studied immunity resulting from dietary suppression in rats where the primary infection with P. berghei has been almost totally controlled by an absence of protein in the diet (EDIRISINGHE et al., 1981a, b; see also Table I). When dietary protein is available in excess of metabolic requirements of the host the effect of infection with P. berg&i (or P. VincketJis severe, producing high peak parasitaemias
and considerably mortality. In contrast when a protein-free diet is given peak parasitaemias are always well below 1% and no mortalities occur. Materials and Methods Male hooded rats, with an initial body weight of between 60 and 70 g, were stabilized on a hieh motein diet (17% case&& below) for at least &e days before inoculation. Infection was achieved by intravenous injection of red cells parasitized with P. bmghei (Anka strain) obtained directly from donor rats. Two powdered diets identical in all respect other than their protein content were used in the study. The first was a protein-free diet and the second contained 17% casein, by weight: both formulations contained identical levels of PABA, iron and free fatty acids. Details of the two diets &e given by EDIRNNGHE et al. (1981a, b). Parasitaemiaswere estimated from thin fib& of&l blood stained with 10% (v/v) Giemsa stain in 15 mM phosphate buffer (pH 7.2) and reticulocyte numbers were determined from thin films of blood stained with Brilliant Cresyl Blue. Parasitaemiasand growth rates of all animals were monitored on alternative days up to 24 days after the last inoculation. Resistance to Superinfection
Six groups of rats (A-F) were used. At the beginning of the study all animals were changed from the high protein to the protein-free diet and those in Groups B-F were infected with 1 x IO6 parasitized red blood cells. These groups were challenged with the samestrain of P. bmhei (4 X lo6 oarasitized red cells) on Days 5 (Group-B), 9 (Group-C), 15 (Group D), 23 (Group E) and 28 (Group F) after the initial infection. At the time of challenge the diet of rats in groups B, C and D was changed back to the high protein formula. Those challenged on Day 23 (Group E) and Day 28 (Group F) had their diets changed on Day 21. Group A control animals received only a primary infection. This w-as given after 15 days on the protein-free diet (1 x lo6 parasitized red cells), thereafter the rats were fed the high protein diet. In addition, small samplesof blood were taken 5,9, 15 and 23 days after primary infection from all rats of Group E and injected into fresh rats, each recipient receiving 12.5 ~1of the pooled blood. The purpose of this was to detect latent infection in animals on the protein-free diet. Microscopically parasites could not be detected in the blood of these rats after Dav 4. The recipient animals were maintained throughoit on the high protein formula.
*Present address: Dept. of Medical Parasitology, Faculty of Medicine, University of Sri Lanka, Peradeniya Campus, Sri Lanka.
J. S. EDlRlSINGHE
Table I-Effect of protein free and high protein diets on the course of a primary infection with P. berghei Rats were fed either a 0% casein or a 17% casein diet from the day of infection. Results are means + SEM, the figures in parentheses indicating the number of animals surviving. Zero (0) in the table denotes the microscopic absence of parasites in the entire blood film Percentage Parasitaemia + SEM Protein-free Diet
High Protein (17%) Diet -
0*16~0*01 0*52to-01 O-27&0*01 0+09z!Y0-01 0 ii x 8 0
2.lfo.2 7.5To.7 10.8:0-7 28*1+1*4 24*41t1.6 54.4+4*6 so*o+ 1.0 64-l -
et
383
d.
suspension (FORD, 1978). The technique involved carefully fragmenting the spleens in cold ‘IX 199 medium (Wellcome Laboratories) containing 2% foetal calf serum. The fragments were slowly passed through a stainless steel mesh into a glass tube and resulting particles allowed to sediment for 10 min. The supernatant was centrifuged at 300 g for 10 min to separate subcellular debris and the pellet was reconstituted in 2 ml of fresh medium. Cell counts were made in an improved Neubauer haemocytometer and viability was assessedby the Trypan Blue exclusion test. Between 88 and 90% of the total cell population was found to exclude the dye. Blood for serum was also taken immediately after the donor animals were killed. It was left at 20°C for 30 min and the clots removed by centrifugation. Serum and spleen cells from the infected rats were injected intravenously into three groups of four rats. The first group received 5 X 10’ spleen cells in 250 pl volume, the second group received O-5 ml serum 100 g body-weight and the third group were injected with both cells and serum at the same respective doses. Spleen cells and serum from the uninfected donors were injected into a further three groups of rats by exactly the same protocol. There were three animals in each of these control groups. Results The effect of dietary protein on the course of P. berghei infection in rats is shown in Table I. Except
Serum and Stdeen Cell Transfer
Serum and-spleencells were transferred together or separately, from infected and uninfected donors maintained on the protein-free diet into fresh rats. The recipients were fed the high protein diet throughout and were infected with P. bwhea’within 45 min of the transfer of serum and spleen cells. The details are as follows: 12 rats were infected and immediately transferred to the protein-free diet. They were maintained on this diet for either 5 or 15 days (identical to Groups B and D in Table II). A second group of six uninfected animals, the control group, was maintained on the protein-free diet for a period of 15 days alone. At the end of each period, the animals were killed, and the spleens removed and processed to obtam a cell
for the protein content both diets were identical: Table KI shows the results of the challenge experiment. The control animals (Group A) which received, their primary infections after 15 days on a protein-free diet showed very little resistance to infection. Peak parasitaemiaswere high and all animals had died by the 18th day following inoculation. Bats challenged after five days of infection on the protein-free dtet (Group B) showed a decreased mortality and reduced peak parasitaemias but the reduction in parasitaemia was not significantly different from the control values. However, Group C rats, challenged after nine days of infection on the proteinfree diet, did show a si~i~c~t decrease in peak parasitaemia.The development of resistancewas most apparent in rats of Groups D, E and F, challenged on Days 15, 23 and 28. Here mortality was absent and
Table ILDevelopmeot of resistance to reinfection with P. berghei Details of the study are given in the text. The results shown are those for a 24-day period following the challeuge infection, or in the case of the control group, are those following the primary infection. Statistical significance of differences between the challenged groupsand the control animals was assessedby Student’s t-teat for samples with unequal variance, in the case of the Day 5 group, and for samples with equal variances for the Day 9, 15, 23 and 28 groups (ARMITAGE 1971); l p = ~0.05, **p E t0.001 Group A f! D z
Day of challenge
Peak Parasitaemia (%f SEM)
Day of Peak Parasitaemia
Controls 5
45*31t 4.2 22*5+ 10.4 21*9& 7*7* 7*6+ 7-O** O-41 0*3** @6+ O-3**
16
1; ti
ii f 4
No. of Animals Showing Parasites in the Blood z: 616 4/6 2t5 416
No. of Deaths 6 3 1 t! 0
384
RESISTANCE
4
12 *a Days after Infection
TO
16
SUPERINFECTION
WITH
MALARIA
20
Fig. 1. The transfer of spleen cells fmm uninfected and infected donors and the subsequent development of infection (F. bmgket) in recipient animals Details are given in the .text. Recipients of spleen cells from uninkcted rats on a protein-free diet (m) and from infected animals on the same diet after either 5 days (0) or 15 days of infection (0). Mortalities in the recipient groups were 66%, 25% and O%, respectively. Relative infection, in the recipients receiving spleen cells from the three groups of donors were 1.00 (tminfected mntmls), 050 (5 day it&ted) and 0.11 (IS day infected).
Fig. 2. The transfer of serum fmm uninfected and infected donors and the subsequent development of infection (P. bmghei) in recipient animals. Details are given in the text. Recipients of serum fmm uninfected controls (m), or from infected animals after either 5 days (0) or 15 days (0) on a protein-free diet. Mortalities in these three groups of recipients were 66%, 50% and 0% and the relative infections were 1.00, 1.10 and 0.93 respectively.
Days after Infection Fig. 3. The transfer of both spleen cells and serum from uninfected and infected donors and the subsequent development of infection (P. bergha] in recipient animals. Details are exactly as those for Figs. 1 and 2. Mortalities were 100% (uninfected controls), 50% (5 day infected) and 0% (15 day infected) and the relative infections were 1.00, W52 and 0.03, respectively.
the peak parasitaemias were considerably reduced. Some animals in each group were apparently aparasitaemic. In all experimental groups (B-F) the peaks in parasitaemia after challenge occurred much earlier than during the primary infection of the controls. Tests for latency of the disease during the primary infection of rats maintained on the protein-free diet indicated that circulating parasites, although undetectable by microscopy after Day 4, were still present, and remained virulent, up to 23 days after the initial infection (last day tested). Subinoculation on days 5, 9, 15 or 23 produced a normal course of the disease in the recipients and all but one of them died as a consequence. The results are not shown. The results of the study on serum and spleen cell transfer are shown in Figs. 1, 2 and 3. Fig. 1 illustrates the effect of spleen cell transfer from infected animals, after either 5 or 15 days of primary infection on a protein-free diet, and from uninfected control animals after 15 days on the same diet. Transfer of spleen cells conveyed a considerable protection to the recipients, particularly those from donor animals infected for 15 days on the protein-free diet. The relative infection in the groups receiving spleen cells from 5-day and 15-day infected donors was 50% and 11% respectively of the level in the animals receiving spleen cells from uninfected donors. The relative infection has been based on numeric integration of the level of parasitaemia against time. Mortality was seen in recipients of spleen cells from uninfected and 5-day infected animals but not in the group receiving cells from 15-day infected animals.
J. S. EDIRISINGHE
Apart from a slight apparent suppression of the infection during the first few days, serum transfer from the infected rats did not lower the level of parasitaemia in the recipients (Fig. 2). In relative terms the infection in the groups receiving serum from 5-day and 15day infected donors were 110% and 93% of the level seen in the control group receiving serum from uninfected animals. Mortalities occurred in both the control group and in recipients of serum from S-day infected animals but, as with the spleen cell transfer, there were no deaths in the group receiving serum from 15day infected animals. When both spleen cells and serum were transferred (Fig. 3) the protection conveyed was similar to that seen after transfer of spleen cells alone (Fig. 1). Animals receiving cells from both S-day and 15day infected donors produced lower peak parasitaemia than the controls (relative infections were 52% and 3% respectively). ’ Discussion
The results of the challenge study (Table II) and the transfer study (Figs. 1, 2 and 3) illustrate two important features. First, Table II shows that, for 21 days at least, severe protein depletion in rats did not interfere with the capacity to develop adequate resistanceagainst reinfection with P. berg/L. During this period the animals were in substantial negative nitrogen balance which resulted in a total loss of body weight of 33%. The ability to mount an effective immune response after chronic protein insufficiency has also been reported by COOPERet al. (1974). Secondly, despite very low levels of circulating parasites (Table I), resistance to reinfection by these animals was apparent within a relatively short spaceof time. Table II illustrates that by the 9th day after infection on the protein-free diet the rats were able to reduce significantly the extent of the challenge infection. Even after five days some resistance was evident from the protective effect of spleen cell transfer (Figs. 1 and 3). As parasite density in the blood remained very low for the first four days and submicroscopic from then on, it would seemthat the level of parasitaemiais of little or no importance to the development of immunity. In this respectour findings agree with those of JACOBI & KRET~CHMAR (1962), ADLER & GUNDERS (1965) and KRETSCHMAR (1966) but differ from those of GILBERTSONet al. (1970). However, the distribution of P. berghei in the body is known to be heterogeneous(ALGAR1963; DESOWITZ & BARNWELL,1976) and the development of resistance could as a consequence have resulted from localized concentrations of the parasite in compartments other than the peripheral circulation. Using crush and impression smearswe examined the parasite density in liver, lung, heart, kidney, bone marrow and spleen of infected rats after 21 days on the protein-free diet. The spleen was the only tissue in which parasitized red cells were observed. Although by Day 21 the level of infection was low (0.5% of the erythrocyte population) it was equal to the maximum parasitaemiaseen in the peripheral blood (on Day 2). As we did not measurethe tissue parasitaemiasat any time other than Day 21, we cannot comment on whether the parasite density is ever important to the development of resistance. Further investigations are necessary.
et al.
385
However, the duration of contact with the parasite did appear to be important for the development of the immune response because resistance increased over the first 23 days of the study (Table II). During this period parasiteswere known to be present in the body of the animal, albeit in extremely low numbers. These results support the conclusions of both KRETSCHMAR (1966) and GILBERTSONet al. (1970). The observed resistanceto reinfection seenin Table II could be transferred by intravenous passageof a suspension of spleen cells from infected donors to fresh animals (Fig. 1). Parasitaemiasresulting from infection of the recipients were appreciably lower than in control animals receiving cells from uninfected donors. The exact way in which the spleen cells conveyed protection is not known becausethe suspension used in the transfer was not fractionated. Several mechanisms could have operated. These include transferred B cells acting as a source for immunoglobulin production, or, transferred T cells stimulating antibody synthesis in the B cells of the recipients (BROWNet al.. 1976; GRAVELY& KREIER. 1976).The spleen cells ‘may also have conferred’ prot’ection through cytotoxic (COLEMANet al., 1975) and/or through phagocytic activity (ZUCKERMANet al., 1973; HAMBURGER& KREIER, 1976). The transfer of serum from infected animals fed the protein-free diet, in contrast, did not control subsequent infection in the recipients, although some suppression of parasitaemia was seenduringthe early stages (Fig. 2). The abilitv to transfer resistance through se‘;um’hasbeen shown by BROWN(1969) and others, but the development of suppressive activity in serum is relatively slow. DIGGS& OSLER(1969) found the maximum level of protective antibodies occurred 74 days after infection and detectable levels were first apparent after 12 days in one study (STECHSHULTE er aL, 1969) and 20 days in another (PHILLIPS& JONES, 1972). We might have seen a more pronounced response in the present study if serum from more chronically infected donors had been used of if the amount transferred (0.5 ml) had been increased. Because of the severe malnourished state of the donors and the small blood volume of the recipients, neither was really possible in practice. The results in Fig. 2, nevertheless, reflect the conclusion of PHILLIPS & JONES(1972) who reported the protective activity of serum to be short lived. In addition in the group receiving serum from donor animals infected for 15 days (Group C) the absenceof mortality despite the high parasitaemiasuggeststhat serum factors may possess antitoxic rather than antiparasitic activity (JERUSALEM, 1968). ._ Transferring both serum and spleen cells to recipients (Fia. 3) oroduced a similar level of orotection to that seen’frdm transfer of spleen cells alone (Fig. 1). The purpose of the transfer study (Figs. 1,2 and 3) was to show that the acquired resistanceto reinfection (Table II) was not the result of factors other than those associatedwith immunity. Reticulocytosis, for example, is decreasedin rats fed the protein-free diet (EDI~SI~GHE~Z al., 198la, b). As these immature red cells are oreferentiallv invaded bv P. bewhei (SINGER. 1954; G~RNHAM,
1566),
low
&iculo&te
Lumbers
could have impeded the development of infection. The transfer study shows that this was not the reason for the suppression of parasitaemia after reinfection.
RESISTANCE
386
TO
SUPERINFWX-ION
In addition-in the chalienge study animals of Groups E and F were fed the high protein diet two and seven days before reinoculation in order to increase the reticulocyte numbers. At the time of challenge reticulocytes accounted for at least 10% of the red cell population. Despite this, subsequent parasitaemias remained very low. The acquired resistance shown in Table II is also not related to the metabolic (nutritional) status of the host as all animals were fed the high protein diet at or before the time of challenee. The hieh orotein diet ensured optimum metabolic conditions in’the host for the development of infection, as seenwith the control group (Table II). The results of the present study indicate that, in rats, immunity to malaria can develop both in the presenceof very low numbers of circulating parasites and during severe protein malnutrition. The technique of suppressing a malarial infection with a protein-free diet is a very simple and reliable method for obtaining large numbers of animals which exhibit a high degree of resistance to this disease. Acknowledgements
J. S. E. and E. B. F. wish to thank respectively the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseasesand the Wellcome Trust for financial support. References
Adler, S. (1958). The effect of meat diets on the course of infections with PIormodiumvi&k
Research Council of Israel,
in mice. Bulletin of the
7E, 9-14.
Adler, S. & Gunders, A. E. (1965). Immunisation of mice against a virulent strain of P/asmodium Yin&i by artificial mild infections on a diet deficient in paraaminobenzoic acid. Israel Jotanal of Medical Science, 1, 441. Algar, N. E. (1963). Distribution of Plasm&urn berghei in tissues of rats, mice and hamsters.Joumui of Prorozoology, 10, 6-10. Armitage,I’. (1971). Statistical Methoa!s in Medical Research. Oxford and Edinburnh: Blackwell Scientific Publications, pp. 118-126. Brown, I. N. (1969). Immunological aspects of malaria infections. Advances in Immunology, 11, 267-349. Brown, K. N., Jarra, W. & Hills, L. A. (1976). T-cells and protective immunity to Pbxmodium bqhei in rats. Infection and Immunify, 14, 858-871. Coleman, R. M., Rencricca, N. J., Stout, J. P., Brissette, W. H. & Smith, D. M. (1975). Splenic mediated erythrocyte cytotoxicity in malaria. Immwwlogy, 29, 49-54. Cooper, W. C., Good, R. A. & Mariani, T. (1974). Effect of protein insufficiency on immune respnsiveness. American Journal of Clinical
Dcsowitz, R. S. & kwell,
Nutrition,
27, 647464.
J. W. (1976). Plamtodium
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MALARIA
Edrisinehe. 1. S.. Fern; E. B. & Tireett. 6. A. T. f1981a). Dier&y’&ppiession’of rodent m&ria: Transactions of the f$&%ctety
of Troptcal Medtcme and Hygtene, 75,
Edirisinghe; J. S., Fern, E. B. & Targett, G. A. T. (1981b). The influence of dietary protein on rhe development of malaria. Annals of T$&l Paedia&cs, 1, 87-91. Ford, W. L. (1978). Handbook of Experimental Immunology. Volume II. Cellulm Immunology. (Third edit.). Oxford and Edinburgh. Blackwell Scientific Publications, pp. 23.1-23.22. Garnham, P. C.C. (1966). Malaria Parasites and Ocher Haemosporidia. Oxford: Blackwell Publications, p. 443. Gilbertson, M., Maegraith, B. J. & Fletcher, K. A. (1970). Resistanceto superinfection with Plasmodium berghei in mice in which the original infection was suppressedby a milk diet. Annals of Tropical Medicine and Parasitology, 64. -.> 497-512. .-. ---.
Gravely, S. M. & Kreier, J. P. (1976). Adoptive transfer of immunitv lo Plasmodium be&ei with immuneT and B lympho&tes. Infection and &muniry, 14, 184-190. Hamburger, J. & Kreier, J. P. (1976). Plasmodium berghei: use of free blood stage parasites to demonstrate protective humoral activity in the serum of recovered rats. Experimental Parasitology, 40, 158-169. Hawking, F. (1954). Milk, p-amino benzoate and malaria of rats and monkeys. British Medical Journal, i, 425429. Jacobi, K. & Kretschmar, W. (1962). Die milchtherapie der malaria-infektion (Pkasmodiumberghet] bei der maus. fehrift fiir Tropenmedizin und Parasitologic, 13, 286Jerusalem, C. (1%8). Active immunization against malaria (Plasmodium berghei) I: Definition of antimalarial immunity. Zeitschrift ftir Tropenmedizin und Parasiwlogie, 19, 171-181. Kretschmar, W. (1%6). Die bedeurung der paminobenzoesaiire fiir den krankheitsverlauf und die immunitat bei der malaria im tier (Pfasmodium berghei) und in meuschen(Plasmodium fakipamm) I. Versuche en NMRI-mausen. Zeitschrift fir Tropenmedizin und Parasitologie, 17, 301-320. Maegraith, B. G., Deegan, T. & Jones, E. S. (1952). Suppression of malaria (Plasmodium berghei) by milk. British Medical Journal, ii, 1382-1384. Phillips, R. S. & Jones, V. E. (1972). Immunity to Plasmodium berghei in rats: maximum level of protective antibody activity are associated with eradication of infection. Parasiwfogy, 64, 117-127. Singer, I. (1954). The effect of splenectomy or phenylhydrazine on infections with Plasmodium berghei in the white mouse. 3oum~l of Infectious Diseases, 94, 159-163. Stechshulte, D. J., Briggs, T. & Welldq, B. T. (1969). Characterization of protective antibodies produced in Plasmodiumberghei infected rats. Military Medicine, 134, 1140-1146. Zuckerman, A., Spira, D. & Row, N. (1973). A quantitative study of phagocytes in the, spleen of rats infected with Plasmodium berghei. In: Dynamic Aspects of HostParasite Relationships. Zuckerman, A. and Weiss, D. W. $E$i.s). Volume 1. New York: Academic Press, pp.
berghei: deep vascular sequestration of young forms m the heart and kidney of the white rat. Annals of Tropical Medicine and Parasitolo~, 70, 475-476.
Diggs, C. L. & Osler, A. G. (1969). Humoral immunity in rodent malaria II. Inhibition of parasitaemia by serum antibody. Journal of Immunology, 102. 298-305.
Accepted for publication 9th October, 1981.
Resistance
to superinfection
with Plasmodium protein-free diet
J. S. EDIRISINGHE ‘*, E. B. FERN’ Departments of Human Nutrition’
AND
berghei
in rats fed a
G. A. T. TARGE-I-?
and Medical Protomology2, London School of Hygiene and Tropical Medicine, Keppel Street, London WClE 7HT
S-
The development of resistance to reinfection with Plasmodium berghei was studied in rats in which the primary infection had been almost totally suppressed by feeding a protein-free diet (peak parasitaemia 05%; patent for only the first four days after inoculation). On Days 5, 9, IS, 23 and 28 after primary inocuhN.ion groups of animals were challenged with the same strain of parasite. At the same time the diet was changed to &at of a 17% casein formula. The development of resistance as judged by the level of tmrasitaemiafollowinn challenee reached a significant ievel nine days after he prim-&y inoculation and almost complete protection by Day 23 of the study. The protective activity was immunological since it could be transferred to other animals by a single intravenous injection of a suspension of spleen cells from infected donors. The study illustrates that infected animals experiencing severe protein malnutrition are still capable of mounting a substantial immune response to malaria. Introduction The ability to suppressa malarial infection through the quality of a diet was first demonstrated by MAEGRAITH et al. in 1952. By maintaining rats exclusively on milk diets they were able to reduce the level of parasitaemia in animals infected with Plasm+ dim berg&i. The mechanism for this attentuation was subsequently shown by HAWKING (1954), amongst others, to be associated with the requirement for 4-amino benzoic acid (PABA) by the parasite and the relative deficiency of this vitamin in milk. In 1966 KRETSCHMARshowed that mice in which the development of the primary infection had been suppressed with a milk diet were able to acquire significant resistance to reinfection with the same s&n of parasite. Similar findings were reported earlier bv ADLER (19581 usina a meat diet (also KRETXHMAR deficient *in PABA)‘ and’ P. v&&i. (196fi) concluded that this immunity, obtained through dietary control of the primary infection, depended on the duration of contact with the antigen and not on the density of ,the parasite in the blood. GILBERTSON et al. (1970), however, using a similar experimental model, concluded that the acquired immunity was dependent on both the level of the original infection and its duration. We have studied immunity resulting from dietary suppression in rats where the primary infection with P. berghei has been almost totally controlled by an absence of protein in the diet (EDIRISINGHE et al., 1981a, b; see also Table I). When dietary protein is available in excess of metabolic requirements of the host the effect of infection with P. berg&i (or P. VincketJis severe, producing high peak parasitaemias
and considerably mortality. In contrast when a protein-free diet is given peak parasitaemias are always well below 1% and no mortalities occur. Materials and Methods Male hooded rats, with an initial body weight of between 60 and 70 g, were stabilized on a hieh motein diet (17% case&& below) for at least &e days before inoculation. Infection was achieved by intravenous injection of red cells parasitized with P. bmghei (Anka strain) obtained directly from donor rats. Two powdered diets identical in all respect other than their protein content were used in the study. The first was a protein-free diet and the second contained 17% casein, by weight: both formulations contained identical levels of PABA, iron and free fatty acids. Details of the two diets &e given by EDIRNNGHE et al. (1981a, b). Parasitaemiaswere estimated from thin fib& of&l blood stained with 10% (v/v) Giemsa stain in 15 mM phosphate buffer (pH 7.2) and reticulocyte numbers were determined from thin films of blood stained with Brilliant Cresyl Blue. Parasitaemiasand growth rates of all animals were monitored on alternative days up to 24 days after the last inoculation. Resistance to Superinfection
Six groups of rats (A-F) were used. At the beginning of the study all animals were changed from the high protein to the protein-free diet and those in Groups B-F were infected with 1 x IO6 parasitized red blood cells. These groups were challenged with the samestrain of P. bmhei (4 X lo6 oarasitized red cells) on Days 5 (Group-B), 9 (Group-C), 15 (Group D), 23 (Group E) and 28 (Group F) after the initial infection. At the time of challenge the diet of rats in groups B, C and D was changed back to the high protein formula. Those challenged on Day 23 (Group E) and Day 28 (Group F) had their diets changed on Day 21. Group A control animals received only a primary infection. This w-as given after 15 days on the protein-free diet (1 x lo6 parasitized red cells), thereafter the rats were fed the high protein diet. In addition, small samplesof blood were taken 5,9, 15 and 23 days after primary infection from all rats of Group E and injected into fresh rats, each recipient receiving 12.5 ~1of the pooled blood. The purpose of this was to detect latent infection in animals on the protein-free diet. Microscopically parasites could not be detected in the blood of these rats after Dav 4. The recipient animals were maintained throughoit on the high protein formula.
*Present address: Dept. of Medical Parasitology, Faculty of Medicine, University of Sri Lanka, Peradeniya Campus, Sri Lanka.
J. S. EDlRlSINGHE
Table I-Effect of protein free and high protein diets on the course of a primary infection with P. berghei Rats were fed either a 0% casein or a 17% casein diet from the day of infection. Results are means + SEM, the figures in parentheses indicating the number of animals surviving. Zero (0) in the table denotes the microscopic absence of parasites in the entire blood film Percentage Parasitaemia + SEM Protein-free Diet
High Protein (17%) Diet -
0*16~0*01 0*52to-01 O-27&0*01 0+09z!Y0-01 0 ii x 8 0
2.lfo.2 7.5To.7 10.8:0-7 28*1+1*4 24*41t1.6 54.4+4*6 so*o+ 1.0 64-l -
et
383
d.
suspension (FORD, 1978). The technique involved carefully fragmenting the spleens in cold ‘IX 199 medium (Wellcome Laboratories) containing 2% foetal calf serum. The fragments were slowly passed through a stainless steel mesh into a glass tube and resulting particles allowed to sediment for 10 min. The supernatant was centrifuged at 300 g for 10 min to separate subcellular debris and the pellet was reconstituted in 2 ml of fresh medium. Cell counts were made in an improved Neubauer haemocytometer and viability was assessedby the Trypan Blue exclusion test. Between 88 and 90% of the total cell population was found to exclude the dye. Blood for serum was also taken immediately after the donor animals were killed. It was left at 20°C for 30 min and the clots removed by centrifugation. Serum and spleen cells from the infected rats were injected intravenously into three groups of four rats. The first group received 5 X 10’ spleen cells in 250 pl volume, the second group received O-5 ml serum 100 g body-weight and the third group were injected with both cells and serum at the same respective doses. Spleen cells and serum from the uninfected donors were injected into a further three groups of rats by exactly the same protocol. There were three animals in each of these control groups. Results The effect of dietary protein on the course of P. berghei infection in rats is shown in Table I. Except
Serum and Stdeen Cell Transfer
Serum and-spleencells were transferred together or separately, from infected and uninfected donors maintained on the protein-free diet into fresh rats. The recipients were fed the high protein diet throughout and were infected with P. bwhea’within 45 min of the transfer of serum and spleen cells. The details are as follows: 12 rats were infected and immediately transferred to the protein-free diet. They were maintained on this diet for either 5 or 15 days (identical to Groups B and D in Table II). A second group of six uninfected animals, the control group, was maintained on the protein-free diet for a period of 15 days alone. At the end of each period, the animals were killed, and the spleens removed and processed to obtam a cell
for the protein content both diets were identical: Table KI shows the results of the challenge experiment. The control animals (Group A) which received, their primary infections after 15 days on a protein-free diet showed very little resistance to infection. Peak parasitaemiaswere high and all animals had died by the 18th day following inoculation. Bats challenged after five days of infection on the protein-free dtet (Group B) showed a decreased mortality and reduced peak parasitaemias but the reduction in parasitaemia was not significantly different from the control values. However, Group C rats, challenged after nine days of infection on the proteinfree diet, did show a si~i~c~t decrease in peak parasitaemia.The development of resistancewas most apparent in rats of Groups D, E and F, challenged on Days 15, 23 and 28. Here mortality was absent and
Table ILDevelopmeot of resistance to reinfection with P. berghei Details of the study are given in the text. The results shown are those for a 24-day period following the challeuge infection, or in the case of the control group, are those following the primary infection. Statistical significance of differences between the challenged groupsand the control animals was assessedby Student’s t-teat for samples with unequal variance, in the case of the Day 5 group, and for samples with equal variances for the Day 9, 15, 23 and 28 groups (ARMITAGE 1971); l p = ~0.05, **p E t0.001 Group A f! D z
Day of challenge
Peak Parasitaemia (%f SEM)
Day of Peak Parasitaemia
Controls 5
45*31t 4.2 22*5+ 10.4 21*9& 7*7* 7*6+ 7-O** O-41 0*3** @6+ O-3**
16
1; ti
ii f 4
No. of Animals Showing Parasites in the Blood z: 616 4/6 2t5 416
No. of Deaths 6 3 1 t! 0
384
RESISTANCE
4
12 *a Days after Infection
TO
16
SUPERINFECTION
WITH
MALARIA
20
Fig. 1. The transfer of spleen cells fmm uninfected and infected donors and the subsequent development of infection (F. bmgket) in recipient animals Details are given in the .text. Recipients of spleen cells from uninkcted rats on a protein-free diet (m) and from infected animals on the same diet after either 5 days (0) or 15 days of infection (0). Mortalities in the recipient groups were 66%, 25% and O%, respectively. Relative infection, in the recipients receiving spleen cells from the three groups of donors were 1.00 (tminfected mntmls), 050 (5 day it&ted) and 0.11 (IS day infected).
Fig. 2. The transfer of serum fmm uninfected and infected donors and the subsequent development of infection (P. bmghei) in recipient animals. Details are given in the text. Recipients of serum fmm uninfected controls (m), or from infected animals after either 5 days (0) or 15 days (0) on a protein-free diet. Mortalities in these three groups of recipients were 66%, 50% and 0% and the relative infections were 1.00, 1.10 and 0.93 respectively.
Days after Infection Fig. 3. The transfer of both spleen cells and serum from uninfected and infected donors and the subsequent development of infection (P. bergha] in recipient animals. Details are exactly as those for Figs. 1 and 2. Mortalities were 100% (uninfected controls), 50% (5 day infected) and 0% (15 day infected) and the relative infections were 1.00, W52 and 0.03, respectively.
the peak parasitaemias were considerably reduced. Some animals in each group were apparently aparasitaemic. In all experimental groups (B-F) the peaks in parasitaemia after challenge occurred much earlier than during the primary infection of the controls. Tests for latency of the disease during the primary infection of rats maintained on the protein-free diet indicated that circulating parasites, although undetectable by microscopy after Day 4, were still present, and remained virulent, up to 23 days after the initial infection (last day tested). Subinoculation on days 5, 9, 15 or 23 produced a normal course of the disease in the recipients and all but one of them died as a consequence. The results are not shown. The results of the study on serum and spleen cell transfer are shown in Figs. 1, 2 and 3. Fig. 1 illustrates the effect of spleen cell transfer from infected animals, after either 5 or 15 days of primary infection on a protein-free diet, and from uninfected control animals after 15 days on the same diet. Transfer of spleen cells conveyed a considerable protection to the recipients, particularly those from donor animals infected for 15 days on the protein-free diet. The relative infection in the groups receiving spleen cells from 5-day and 15-day infected donors was 50% and 11% respectively of the level in the animals receiving spleen cells from uninfected donors. The relative infection has been based on numeric integration of the level of parasitaemia against time. Mortality was seen in recipients of spleen cells from uninfected and 5-day infected animals but not in the group receiving cells from 15-day infected animals.
J. S. EDIRISINGHE
Apart from a slight apparent suppression of the infection during the first few days, serum transfer from the infected rats did not lower the level of parasitaemia in the recipients (Fig. 2). In relative terms the infection in the groups receiving serum from 5-day and 15day infected donors were 110% and 93% of the level seen in the control group receiving serum from uninfected animals. Mortalities occurred in both the control group and in recipients of serum from S-day infected animals but, as with the spleen cell transfer, there were no deaths in the group receiving serum from 15day infected animals. When both spleen cells and serum were transferred (Fig. 3) the protection conveyed was similar to that seen after transfer of spleen cells alone (Fig. 1). Animals receiving cells from both S-day and 15day infected donors produced lower peak parasitaemia than the controls (relative infections were 52% and 3% respectively). ’ Discussion
The results of the challenge study (Table II) and the transfer study (Figs. 1, 2 and 3) illustrate two important features. First, Table II shows that, for 21 days at least, severe protein depletion in rats did not interfere with the capacity to develop adequate resistanceagainst reinfection with P. berg/L. During this period the animals were in substantial negative nitrogen balance which resulted in a total loss of body weight of 33%. The ability to mount an effective immune response after chronic protein insufficiency has also been reported by COOPERet al. (1974). Secondly, despite very low levels of circulating parasites (Table I), resistance to reinfection by these animals was apparent within a relatively short spaceof time. Table II illustrates that by the 9th day after infection on the protein-free diet the rats were able to reduce significantly the extent of the challenge infection. Even after five days some resistance was evident from the protective effect of spleen cell transfer (Figs. 1 and 3). As parasite density in the blood remained very low for the first four days and submicroscopic from then on, it would seemthat the level of parasitaemiais of little or no importance to the development of immunity. In this respectour findings agree with those of JACOBI & KRET~CHMAR (1962), ADLER & GUNDERS (1965) and KRETSCHMAR (1966) but differ from those of GILBERTSONet al. (1970). However, the distribution of P. berghei in the body is known to be heterogeneous(ALGAR1963; DESOWITZ & BARNWELL,1976) and the development of resistance could as a consequence have resulted from localized concentrations of the parasite in compartments other than the peripheral circulation. Using crush and impression smearswe examined the parasite density in liver, lung, heart, kidney, bone marrow and spleen of infected rats after 21 days on the protein-free diet. The spleen was the only tissue in which parasitized red cells were observed. Although by Day 21 the level of infection was low (0.5% of the erythrocyte population) it was equal to the maximum parasitaemiaseen in the peripheral blood (on Day 2). As we did not measurethe tissue parasitaemiasat any time other than Day 21, we cannot comment on whether the parasite density is ever important to the development of resistance. Further investigations are necessary.
et al.
385
However, the duration of contact with the parasite did appear to be important for the development of the immune response because resistance increased over the first 23 days of the study (Table II). During this period parasiteswere known to be present in the body of the animal, albeit in extremely low numbers. These results support the conclusions of both KRETSCHMAR (1966) and GILBERTSONet al. (1970). The observed resistanceto reinfection seenin Table II could be transferred by intravenous passageof a suspension of spleen cells from infected donors to fresh animals (Fig. 1). Parasitaemiasresulting from infection of the recipients were appreciably lower than in control animals receiving cells from uninfected donors. The exact way in which the spleen cells conveyed protection is not known becausethe suspension used in the transfer was not fractionated. Several mechanisms could have operated. These include transferred B cells acting as a source for immunoglobulin production, or, transferred T cells stimulating antibody synthesis in the B cells of the recipients (BROWNet al.. 1976; GRAVELY& KREIER. 1976).The spleen cells ‘may also have conferred’ prot’ection through cytotoxic (COLEMANet al., 1975) and/or through phagocytic activity (ZUCKERMANet al., 1973; HAMBURGER& KREIER, 1976). The transfer of serum from infected animals fed the protein-free diet, in contrast, did not control subsequent infection in the recipients, although some suppression of parasitaemia was seenduringthe early stages (Fig. 2). The abilitv to transfer resistance through se‘;um’hasbeen shown by BROWN(1969) and others, but the development of suppressive activity in serum is relatively slow. DIGGS& OSLER(1969) found the maximum level of protective antibodies occurred 74 days after infection and detectable levels were first apparent after 12 days in one study (STECHSHULTE er aL, 1969) and 20 days in another (PHILLIPS& JONES, 1972). We might have seen a more pronounced response in the present study if serum from more chronically infected donors had been used of if the amount transferred (0.5 ml) had been increased. Because of the severe malnourished state of the donors and the small blood volume of the recipients, neither was really possible in practice. The results in Fig. 2, nevertheless, reflect the conclusion of PHILLIPS & JONES(1972) who reported the protective activity of serum to be short lived. In addition in the group receiving serum from donor animals infected for 15 days (Group C) the absenceof mortality despite the high parasitaemiasuggeststhat serum factors may possess antitoxic rather than antiparasitic activity (JERUSALEM, 1968). ._ Transferring both serum and spleen cells to recipients (Fia. 3) oroduced a similar level of orotection to that seen’frdm transfer of spleen cells alone (Fig. 1). The purpose of the transfer study (Figs. 1,2 and 3) was to show that the acquired resistanceto reinfection (Table II) was not the result of factors other than those associatedwith immunity. Reticulocytosis, for example, is decreasedin rats fed the protein-free diet (EDI~SI~GHE~Z al., 198la, b). As these immature red cells are oreferentiallv invaded bv P. bewhei (SINGER. 1954; G~RNHAM,
1566),
low
&iculo&te
Lumbers
could have impeded the development of infection. The transfer study shows that this was not the reason for the suppression of parasitaemia after reinfection.
RESISTANCE
386
TO
SUPERINFWX-ION
In addition-in the chalienge study animals of Groups E and F were fed the high protein diet two and seven days before reinoculation in order to increase the reticulocyte numbers. At the time of challenge reticulocytes accounted for at least 10% of the red cell population. Despite this, subsequent parasitaemias remained very low. The acquired resistance shown in Table II is also not related to the metabolic (nutritional) status of the host as all animals were fed the high protein diet at or before the time of challenee. The hieh orotein diet ensured optimum metabolic conditions in’the host for the development of infection, as seenwith the control group (Table II). The results of the present study indicate that, in rats, immunity to malaria can develop both in the presenceof very low numbers of circulating parasites and during severe protein malnutrition. The technique of suppressing a malarial infection with a protein-free diet is a very simple and reliable method for obtaining large numbers of animals which exhibit a high degree of resistance to this disease. Acknowledgements
J. S. E. and E. B. F. wish to thank respectively the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseasesand the Wellcome Trust for financial support. References
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Adler, S. & Gunders, A. E. (1965). Immunisation of mice against a virulent strain of P/asmodium Yin&i by artificial mild infections on a diet deficient in paraaminobenzoic acid. Israel Jotanal of Medical Science, 1, 441. Algar, N. E. (1963). Distribution of Plasm&urn berghei in tissues of rats, mice and hamsters.Joumui of Prorozoology, 10, 6-10. Armitage,I’. (1971). Statistical Methoa!s in Medical Research. Oxford and Edinburnh: Blackwell Scientific Publications, pp. 118-126. Brown, I. N. (1969). Immunological aspects of malaria infections. Advances in Immunology, 11, 267-349. Brown, K. N., Jarra, W. & Hills, L. A. (1976). T-cells and protective immunity to Pbxmodium bqhei in rats. Infection and Immunify, 14, 858-871. Coleman, R. M., Rencricca, N. J., Stout, J. P., Brissette, W. H. & Smith, D. M. (1975). Splenic mediated erythrocyte cytotoxicity in malaria. Immwwlogy, 29, 49-54. Cooper, W. C., Good, R. A. & Mariani, T. (1974). Effect of protein insufficiency on immune respnsiveness. American Journal of Clinical
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Edirisinghe; J. S., Fern, E. B. & Targett, G. A. T. (1981b). The influence of dietary protein on rhe development of malaria. Annals of T$&l Paedia&cs, 1, 87-91. Ford, W. L. (1978). Handbook of Experimental Immunology. Volume II. Cellulm Immunology. (Third edit.). Oxford and Edinburgh. Blackwell Scientific Publications, pp. 23.1-23.22. Garnham, P. C.C. (1966). Malaria Parasites and Ocher Haemosporidia. Oxford: Blackwell Publications, p. 443. Gilbertson, M., Maegraith, B. J. & Fletcher, K. A. (1970). Resistanceto superinfection with Plasmodium berghei in mice in which the original infection was suppressedby a milk diet. Annals of Tropical Medicine and Parasitology, 64. -.> 497-512. .-. ---.
Gravely, S. M. & Kreier, J. P. (1976). Adoptive transfer of immunitv lo Plasmodium be&ei with immuneT and B lympho&tes. Infection and &muniry, 14, 184-190. Hamburger, J. & Kreier, J. P. (1976). Plasmodium berghei: use of free blood stage parasites to demonstrate protective humoral activity in the serum of recovered rats. Experimental Parasitology, 40, 158-169. Hawking, F. (1954). Milk, p-amino benzoate and malaria of rats and monkeys. British Medical Journal, i, 425429. Jacobi, K. & Kretschmar, W. (1962). Die milchtherapie der malaria-infektion (Pkasmodiumberghet] bei der maus. fehrift fiir Tropenmedizin und Parasitologic, 13, 286Jerusalem, C. (1%8). Active immunization against malaria (Plasmodium berghei) I: Definition of antimalarial immunity. Zeitschrift ftir Tropenmedizin und Parasiwlogie, 19, 171-181. Kretschmar, W. (1%6). Die bedeurung der paminobenzoesaiire fiir den krankheitsverlauf und die immunitat bei der malaria im tier (Pfasmodium berghei) und in meuschen(Plasmodium fakipamm) I. Versuche en NMRI-mausen. Zeitschrift fir Tropenmedizin und Parasitologie, 17, 301-320. Maegraith, B. G., Deegan, T. & Jones, E. S. (1952). Suppression of malaria (Plasmodium berghei) by milk. British Medical Journal, ii, 1382-1384. Phillips, R. S. & Jones, V. E. (1972). Immunity to Plasmodium berghei in rats: maximum level of protective antibody activity are associated with eradication of infection. Parasiwfogy, 64, 117-127. Singer, I. (1954). The effect of splenectomy or phenylhydrazine on infections with Plasmodium berghei in the white mouse. 3oum~l of Infectious Diseases, 94, 159-163. Stechshulte, D. J., Briggs, T. & Welldq, B. T. (1969). Characterization of protective antibodies produced in Plasmodiumberghei infected rats. Military Medicine, 134, 1140-1146. Zuckerman, A., Spira, D. & Row, N. (1973). A quantitative study of phagocytes in the, spleen of rats infected with Plasmodium berghei. In: Dynamic Aspects of HostParasite Relationships. Zuckerman, A. and Weiss, D. W. $E$i.s). Volume 1. New York: Academic Press, pp.
berghei: deep vascular sequestration of young forms m the heart and kidney of the white rat. Annals of Tropical Medicine and Parasitolo~, 70, 475-476.
Diggs, C. L. & Osler, A. G. (1969). Humoral immunity in rodent malaria II. Inhibition of parasitaemia by serum antibody. Journal of Immunology, 102. 298-305.
Accepted for publication 9th October, 1981.