Parasitology, (1979), 78, 67-76 With 2figuresin the text
67
Plasmodium knowlesi in the marmoset (Callithrix jacchus) J. LANGHORNE and S. COHEN Department of Chemical Pathology, Guy's Hospital Medical School, London SEl {Received 20 April 1978) SUMMARY
Common marmosets were shown to be susceptible to Plasmodium knowlesi malaria. The morphology of the parasite was indistinguishable from that observed in the natural host (Macaca fascicularis) and the common laboratory model (Macaca mulatto). A differential susceptibility to P. knowlesi was observed in the 8 marmosets studied. Multiplication rates of parasites were variable over 24 h periods. Five animals died of a fulminating infection within 12—17 days after challenge. Three animals recovered spontaneously from infection and were subsequently resistant to challenge with homologous and heterologous variants and strains of P. knowlesi. This resistance was maintained for intervals up to 100 days between challenge infections. INTRODUCTION
Mechanisms of immunity to Plasmodium knowlesi malaria are poorly understood. Although induction of immunity to P. knowlesi variants and strains by vaccination of rhesus monkeys with merozoites in Freund's complete adjuvant (FCA) has proved successful (Mitchell, Butcher & Cohen, 1975; Richards, Mitchell, Butcher & Cohen, 1977; Mitchell, Butcher, Langhorne & Cohen, 1977) it is not known how the vaccine works and why FCA has proved to be obligatory. Antibodymediated mechanisms are important in vaccinated animals as shown by passive transfer, but previous observations indicate that, in addition, immunity induced by merozoite vaccination may involve cellular mechanisms (Butcher, Mitchell & Cohen, 1978). The outbred nature of rhesus monkeys will not allow investigation of in vivo cellular responses and the transfer of delayed-type hypersensitivity (DTH), since these animals probably differ at many loci within the major histocompatibility complex (MHC). Such differences may result in rejection of transferred cells and inability of the donor and recipient cells to co-operate in a DTH response (Sprent & Miller, 1971). Because of these limitations it was decided to investigate whether another primate model might be more suitable for detailed immunological studies. The common marmoset {Callithrix jacchus) has previously been shown to be susceptible to P. knowlesi (Cruz & de Mello, 1947). A unique feature of marmosets is the high incidence of twinning which occurs in about 80 % of all births (Bernirschke, Anderson & Brownhill, 1962; Gengozian, Batson & Eide, 1964). These twins are haemopoietic chimaerae and are tolerant to co-twin antigens (Porter & Gengozian, 1969, 1973). I t has been shown in mouse bone marrow chimaera models that, 0031-1820/79/0078-0407 $01.00 © 1979 Cambridge University Press
68
J. LANGHORNE AND S. COHEN
although there may be MHC differences between the cells from the two animals, these differences are not recognized. Thus T-cells sensitized in a chimaerie environment are able to operate in thymus-derived cell (T-cell) helper functions and possibly DTH responses with cells from either animal (Von Boehmer, Hudson & Sprent, 1975; Miller, Vadas, Whitelaw & Gamble, 1975). The marmoset, therefore, may be a useful model for the study of cellular mechanisms in the immune response to P. Jcnowlesi. Before immunological studies can be carried out, it is necessary to establish the nature of the P. knowlesi infection in the marmoset. This preliminary report describes the infection of the marmoset C. jacchus with different variants and strains of P. knowlesi, and records observations on the course of parasitaemia in these animals. MATERIALS AND METHODS
Animals Common marmosets (C. jacchus) of either sex, weighing 250-400 g (aged between 2 and 4 years) were caged in a room artificially illuminated between 5.00 a.m. and 5.00 p.m. Two strains of P. knowlesi parasites were used. A Malaysian strain designated W (Butcher & Cohen, 1972) was originally obtained from the Walter-Reed Army Institute of Research, Washington. Serologically distinct variants of the W strain were identified by the scbizont-infected cell agglutination (SICA) test (Brown & Brown, 1965). A Malaysian strain designated Nuri (Brown, Brown & Hills, 1968), maintained at the National Institute for Medical Research, Mill Hill, London, was kindly provided by Dr K. N. Brown. Infection of marmosets with P. knowlesi parasites
Normal marmosets were inoculated intravenously (i.v.) and intramuscularly (i.m.) with a known number of immature schizonts of known strain and variant specificity taken from an infected donor (marmoset or rhesus) monkey. Alternatively, marmosets were inoculated intramuscularly with 1 ml of thawed (whole) infected marmoset or rhesus monkey blood which had been stored at — 80 °C in 15% glycerol/saline. Detection of parasites
First appearance of parasitaemia was assessed on thick bloodfilmshaemolysed and stained in Field's solution. Parasite numbers were estimated by erythrocyte counts. The percentage of red cells parasitized was determined by counting up to 10* erythrocytes on thin blood films stained with Giemsa. A negative count on thick blood film indicates a parasite density of less than 1/106 erythrocytes, as estimated by white cell counts.
C4 C6 C7 C17 C14 C3 Cll C12 W3 W3 W3 Wl W3 Wl W3
W3
Variant Marmoset Rhesus Marmoset Rhesus Rhesus Rhesus Marmoset Rhesus
Source
)
oute i.v. i.m. i.v. i.m. i.v. i.m. i.m. i.m. 5 7 5 6 7 10 5 6
Pre-patent period (days) 69 44 75 5-3 82 35 48 18
(%)
Maximum parasitaemia 7 10 7 7 10 15 15 15
Duration of patency (days)
Not determined since marmosets wero infected with 1 ml of infected, frozen whole blood.
N.D. N.D.
2xlO4 105
N.D.
2x10*
N.D.*
108
Marmoset No. of parasites
P. knowlesi infection
Table 1. Infection of marmosets vrith Plasmodium knowlesi
Survived Survived Survived
12 17 12 13 16
Died (day)
O5
to
70
J. LANGHOENE AND S. COHEN 100
r
100
•S
10
0-1
001
5
10 Days after challenge
15
Fig. 1. Course of parasitaemia in 5 marmosets which died, of Plasmodium hnowlesi infection. (O—O), C4; ( • — • ) , C6; (Q—D), C7; ( • — • ) , C14; (A—A), C17. The animals were challenged with Wl or W3 variants of P. knowlesi as described in Table 1. Day of death of each animal is indicated in Table 1. RESULTS
Course of P. knowlesi infection in marmosets
Eight marmosets were infected with W3 or Wl variant of P. knowlesi by intravenous or intramuscular injection of organisms obtained from infected donor marmosets or rhesus monkeys or fromfrozen,infected blood. The parasite variants, routes of infection and resultant parasitaemias are summarized in Table 1. Parasites were first observed in peripheral blood within 5-10 days. In those animals injected with a known number of parasitized erythrocytes the pre-patent period was longer than that predicted from a logarithmic growth rate of the parasite.
P. knowlesi in the marmoset
71
100
10
2
10
0-1
001
10
15
20
25
30
Days after challenge
Fig. 2. Course of parasitaemia in 3 marmosets that survived an initial challenge with Plasmodium knowlesi. ( • — • ) , C3; (O—O), C l l ; O—3),C12. The animals were challenged as described in Table 1.
Maximum parasitaemias of 5-3-82% were recorded within 9-21 days of blood challenge. The course of P. knowlesi infections in these animals is shown in Figs 1 and 2. Five of the 8 animals showed pre-patent periods of 5-7 days and died of P. knowlesi infection within 13—17 days after challenge by intravenous or intramuscular routes (Fig. 1). Three animals infected by intramuscular inoculation survived blood challenge (Fig. 2). Parasites were apparent in peripheral blood after 5-10 days and persisted for 15 days, after which time thick blood films became negative suggesting a concentration of less than 1 parasite/10 6 erythrocytes. In all these animals, parasites were rapidly cleared from the blood; parasitaemias of 18-48% were reduced to less than 1 parasite/104 erythrocytes in 3-5 days.
126
78 217 316 43 182 210
W3
Wl
W3
Marmoset
C3
Cll
C12
106 105 104
10 106 10s
s
10 s
No. of parasites
W3 Wl Nuri
W3 Wl Nuri
W3
' Strain or variant
Challenge infection
.V. .V. .V.
.V. .V. •V.
i.v
Route
—
-t
7 9
4
5 1
1
—
4 1
2
PreMaximum Duration of patent parasitaemia period (parasites/10* patency (days) erythrocytes) (days)
ft
w
O
Q
GO
E AND
* Days after initial infection with P. knowlesi. t Indicates that no parasites were observed by thick or thin film analysis.
Day*
Parasite variant of initial infection
Table 2. Results of homologous and heterologous challenge of 3 marmosets which had recovered from a primarg infection with Plasmodium knowlesi LANGHO
P. knowlesi in the marmoset
73
The morphology of the erythrocyte forms of P . knowlesi was indistinguishable from parasites infecting rhesus monkey erythrocytes. The erythrocyte cycle in marmoset blood occupied 24 h and was synchronous. Mature schizonts containing 8-16 merozoites were present in the peripheral circulation. Multiplication rates over 24 h periods were variable; logarithmic parasite multiplication was recorded in only 2 animals (C4 and C14) which died of P. hnowlesi infection. Multiplication rates of 2- to 8-fold were observed over 24 h periods in the remaining 6 animals. Challenge of marmosets with homologous and heterologous variants and a heterologous strain of P. knowlesi The 3 marmosets surviving initial blood challenge with W3 or Wl variants of P . knowlesi were subsequently challenged with W3 and Wl variants and Nuri strain at various times after initial infection (Table 2). All three animals survived challenge with an homologous or heterologous variant or heterologous strain. Two of the 3 animals (C3 and CH) developed low parasitaemias of brief duration. The pre-patent period in these animals was 4-9 days. The maximum parasitaemia recorded was 5 parasites/104 erythrocytes, and parasites were observed in peripheral blood for a period of 1-4 days. Two of the animals (Oil and C12) when challenged with a heterologous strain of P. knowlesi (Nuri strain) did not develop patent parasitaemias. These preliminary results suggest that marmosets surviving an initial infection with P . knowlesi are subsequently resistant to infection with heterologous variants or strains of this parasite. Resistance is retained during intervals of at least 100 days between challenge infections.
DISCUSSION
These studies confirm early observations of Cruz & de Mello (1947) that the common marmoset, C. jacchus, is susceptible to P . knowlesi. The morphology of the erythrocytic forms of P . knowlesi was indistinguishable from that found in the natural host, Macacafascicularis, and the common laboratory host, M. mulatta, the rhesus monkey. The parasite cycle was of 24 h duration and was synchronous. The course of parasitaemia in the marmoset differed, however, from that seen in the rhesus monkey. In the latter, the infection usually has a rapidly fatal outcome with logarithmic multiplication of the parasite (Garnham, 1966). P . knowlesi infection of marmosets did not produce a uniformly fatal infection; 3 of the 8 animals survived an initial challenge infection. The animals that survived were all infected by intramuscular inoculation while those that died were inoculated either intravenously or intramuscularly. Cruz & de Mello (1947) observed that, occasionally, marmosets infected by intramuscular routes spontaneously recovered from infection. In addition to animals surviving challenge infections, it was observed in this study that the pre-patent periods were longer than expected of a logarithmically multiplicating parasite. This was also apparent in the early patent parasitaemias of the majority of the animals. Although the mature schizonts contained 8-16 merozoites, multiplication rates were less than 10-fold over a 24 h period.
74
J. LANGHORNE AND S. COHEN
The observed variations in survival and parasite multiplication rates suggest a differential susceptibility of individual animals to P. knowlesi, but the underlying mechanisms have not been established. There may be components in the serum which affect the viability of released merozoites, or certain erythrocytes may lack the receptors necessary for merozoite invasion. The extended pre-patent periods and the initial low multiplication rates of parasites may reflect the selection of those merozoites able to survive in the plasma and invade other erythrocytes. In vitro studies of the invasion of P. knowlesi merozoites with erythrocytes and sera of various animals demonstrated that the New World douroucouli monkey, Aotus trivirgatus, was less susceptible to P. knowlesi than the Old World rhesus monkey a difference apparently due to some property of the erythrocyte rather than to serum factors (Butcher, Mitchell & Cohen, 1973). Similar studies with a range of marmoset erythrocytes and sera may help to elucidate the mechanisms underlying susceptibility or relative resistance of individual animals to P. knowlesi. The ability of marmosets to recover from P. knowlesi infection may be due to a combination of mechanisms. Non-immunological factors which determine lower initial parasite multiplication rates may allow sufficient time for the development of acquired immunity. Since these infections were initiated by intramuscular inoculation, local inflammatory responses, particularly in those animals infected with parasites in rhesus monkey erythrocytes, may have reduced the number of viable parasites entering the peripheral circulation. The mechanism of parasite clearance in these animals is not yet understood, but it is likely to be predominantly immunological, as subsequent challenge with the same parasite variant produced a negligible or low grade parasitaemia of short duration. These animals were also resistant to challenge with a heterologous variant of the same strain, and to a heterologous strain of P. knowlesi, suggesting that the protective immune response may be directed against common antigens of P. knowlesi rather than variantspecific antigens. There are no comparable studies of the rhesus monkey since P. knowlesi infection is almost invariably fatal if left untreated. A degree of immunity can, however, be induced in the rhesus by infection and sub-curative drug therapy resulting in periodic relapses and low-grade infection. Each relapse is attributable to a serologically distinct variant. Chronic infection produces an immunity with broad variant specificity (Brown & Brown, 1965; Brown, Brown & Hills, 1968; Voller & Rossan, 1969) which may be due in part to T-cell sensitization to common malarial antigens (Brown, 1971) or B-cell activation leading to production of low titres of antibody directed against several serological variants (Butcher & Cohen, 1972). Merozoite vaccination protects rhesus monkeys against homologous or heterologous challenge with erythrocytic stages or the blood infections following mosquito-transmitted (sporozoite) stages of P. knowlesi (Mitchell, Butcher & Cohen, 1975; Mitchell, Butcher, Langhorne & Cohen, 1977; Richards, Mitchell, Butcher & Cohen, 1977). The immunity induced in the rhesus monkey by such vaccination is similar in terms of specificity and degree to that obtained in marmosets which survive an initial challenge infection. The merozoite vaccine may stimulate a specific response in rhesus monkeys to antigens which are present on a wide
P. knowlesi in the marmoset
75
range of P. knowlesi parasites, but are poorly immunogenic in a natural infection. In certain marmosets these antigens may be significantly immunogenic in the natural infection, thus enabling them to mount an effective immune response. The nature of the immune responses induced in marmosets recovered from P. knowlesi infection and in marmosets vaccinated with merozoites in Freund's complete adjuvant are currently being investigated. The marmoset provides an interesting and convenient model for such studies since in vivo immune responses can readily be investigated in chimaeric marmoset twins. This work was supported by the Medical Research Council and the World Health Organization. We would like to thank Drs G. H. Mitchell and G. A. Butcher for helpful discussion, and Miss Marilyn Burnikell and Miss Deborah Cutter for skilled technical assistance.
REFERENCES K., ANDEBSON, J. M. & BROWNHILL, L. E. (1962). Marrow chimerism in the marmosets. Science 138, 513-15. BBOWN, K. N. (1971). Protective immunity to malaria provides a model for the survival of cells in an unmunologically hostile environment. Nature, London 230, 163—7. BROWN, K. N. & BROWN, I. N. (1965). Immunity to malaria: antigenic variation in chronic infections of Plasmodium knowlesi. Nature, London 208, 1286-8. BERNTRSCHKE,
BROWN, K. N., BROWN, I. N. & HELLS, L. A. (1968). Immunity to malaria: the antibody
response to antigenic variation by Plasmodium knowlesi. Immunology 14, 127—38. G. A. & COHEN, S. (1972). Antigenic variation and protective immunity in Plasmodium knowlesi malaria. Immunology 23, 503-21. BUTCHER, G. A., MITCHELL, G. H. & COHEN, S. (1973). Mechanism of host specificity in malarial infection. Nature, London 244, 40-2. BUTCHER, G. A., MITCHELL, G. H. & COHEN, S. (1978). Antibody mediated mechanisms of immunity to malaria induced by vaccination with Plasmodium knowlesi merozoites. Immunology 34, 77-86. CRUZ, W. O. & DE MELLO, R. P. (1947). Infeccao de Macaco sul americano 'sagul' (Callithrix jacchus, Linnaeus 1758) com o Plasmodium knowlesi. Memoriae do Instituto Oswaldo Cruz 45, 119-21. GARNKAM, P. C. C. (1966). Malaria Parasites and other Haemosporidia. Oxford: Blackwell Scientific Press. GENGOZIAN, N., BATSON, J. S. & EIDE, P. (1964). Haematologic and cytogenic evidence for haemopoietic chimerism in the marmoset, Tamerinus nigricollis. Cytogenetics (Basel) 3, 384-93. MILLER, J. F. A. P., VADAS, M. A., WHITELAW, A. & GAMBLE, J. (1975). H—2 gene complex restricts transfer of delayed-type hypersensitivity in mice. Proceedings oj the National Academy of Sciences (USA) 72, 5095-8. MITCHELL, G. H., BUTCHER, G. A. & COHEN, S. (1975). Merozoite vaccination against Plasmodium knowlesi malaria. Immunology 29, 397-407. MITCHELL, G. H., BUTCHER, G. A., LANGHOBNE, J. & COHEN, S. (1977). A freeze-dxied merozoite vaccine effective against Plasmodium knowlesi malaria. Clinical and Experimental Immunology 28, 276-9. PORTER, R. P. & GENGOZIAN, N. (1969). Immunological tolerance and rejection of skin allografts in the marmoset. Transplantation 8, 653-65. PORTER, R. P. & GENGOZIAN, N. (1973). Immunological responsiveness and tolerance of marmoset lymphoid tissue in vitro. Transplantation 16, 221-30. RICHAKDS, W. H. G., MITCHELL, G. EL, BUTCHER, G. A. & COHEN, S. (1977). Merozoite vaccination of rhesus monkeys against Plasmodium knowlesi malaria; immunity to sporozoite (mosquito-transmitted) challenge. Parasitology 74, 191-8. SPRENT, J. & MILLER, J. F. A. P. (1971). Activation of thymus cells by histocompatibility antigens. Nature, New Biology 234, 195-8. BUTCHER,
76
J. LANQBOBNE AND S. COHEN
A. & ROSSAN, R. N. (1969). Immiinological studies on simian malaria iii. Immunity to challenge and antigenic variation in Plasmodium hnovilesi. Transactions of the Royal Society of Tropical Medicine and Hygiene 63, 507-23. VON BoEffMEK, H., HUDSON, L. & SPKENT, J. (1975). Collaboration of histoincompatible T and B lymphocytes using cells from tetraparental bone marrow chimeras. Journal of Experimental Medicine 142, 989-97. VOIXEB,
Printed in Great Britain
67
Plasmodium knowlesi in the marmoset (Callithrix jacchus) J. LANGHORNE and S. COHEN Department of Chemical Pathology, Guy's Hospital Medical School, London SEl {Received 20 April 1978) SUMMARY
Common marmosets were shown to be susceptible to Plasmodium knowlesi malaria. The morphology of the parasite was indistinguishable from that observed in the natural host (Macaca fascicularis) and the common laboratory model (Macaca mulatto). A differential susceptibility to P. knowlesi was observed in the 8 marmosets studied. Multiplication rates of parasites were variable over 24 h periods. Five animals died of a fulminating infection within 12—17 days after challenge. Three animals recovered spontaneously from infection and were subsequently resistant to challenge with homologous and heterologous variants and strains of P. knowlesi. This resistance was maintained for intervals up to 100 days between challenge infections. INTRODUCTION
Mechanisms of immunity to Plasmodium knowlesi malaria are poorly understood. Although induction of immunity to P. knowlesi variants and strains by vaccination of rhesus monkeys with merozoites in Freund's complete adjuvant (FCA) has proved successful (Mitchell, Butcher & Cohen, 1975; Richards, Mitchell, Butcher & Cohen, 1977; Mitchell, Butcher, Langhorne & Cohen, 1977) it is not known how the vaccine works and why FCA has proved to be obligatory. Antibodymediated mechanisms are important in vaccinated animals as shown by passive transfer, but previous observations indicate that, in addition, immunity induced by merozoite vaccination may involve cellular mechanisms (Butcher, Mitchell & Cohen, 1978). The outbred nature of rhesus monkeys will not allow investigation of in vivo cellular responses and the transfer of delayed-type hypersensitivity (DTH), since these animals probably differ at many loci within the major histocompatibility complex (MHC). Such differences may result in rejection of transferred cells and inability of the donor and recipient cells to co-operate in a DTH response (Sprent & Miller, 1971). Because of these limitations it was decided to investigate whether another primate model might be more suitable for detailed immunological studies. The common marmoset {Callithrix jacchus) has previously been shown to be susceptible to P. knowlesi (Cruz & de Mello, 1947). A unique feature of marmosets is the high incidence of twinning which occurs in about 80 % of all births (Bernirschke, Anderson & Brownhill, 1962; Gengozian, Batson & Eide, 1964). These twins are haemopoietic chimaerae and are tolerant to co-twin antigens (Porter & Gengozian, 1969, 1973). I t has been shown in mouse bone marrow chimaera models that, 0031-1820/79/0078-0407 $01.00 © 1979 Cambridge University Press
68
J. LANGHORNE AND S. COHEN
although there may be MHC differences between the cells from the two animals, these differences are not recognized. Thus T-cells sensitized in a chimaerie environment are able to operate in thymus-derived cell (T-cell) helper functions and possibly DTH responses with cells from either animal (Von Boehmer, Hudson & Sprent, 1975; Miller, Vadas, Whitelaw & Gamble, 1975). The marmoset, therefore, may be a useful model for the study of cellular mechanisms in the immune response to P. Jcnowlesi. Before immunological studies can be carried out, it is necessary to establish the nature of the P. knowlesi infection in the marmoset. This preliminary report describes the infection of the marmoset C. jacchus with different variants and strains of P. knowlesi, and records observations on the course of parasitaemia in these animals. MATERIALS AND METHODS
Animals Common marmosets (C. jacchus) of either sex, weighing 250-400 g (aged between 2 and 4 years) were caged in a room artificially illuminated between 5.00 a.m. and 5.00 p.m. Two strains of P. knowlesi parasites were used. A Malaysian strain designated W (Butcher & Cohen, 1972) was originally obtained from the Walter-Reed Army Institute of Research, Washington. Serologically distinct variants of the W strain were identified by the scbizont-infected cell agglutination (SICA) test (Brown & Brown, 1965). A Malaysian strain designated Nuri (Brown, Brown & Hills, 1968), maintained at the National Institute for Medical Research, Mill Hill, London, was kindly provided by Dr K. N. Brown. Infection of marmosets with P. knowlesi parasites
Normal marmosets were inoculated intravenously (i.v.) and intramuscularly (i.m.) with a known number of immature schizonts of known strain and variant specificity taken from an infected donor (marmoset or rhesus) monkey. Alternatively, marmosets were inoculated intramuscularly with 1 ml of thawed (whole) infected marmoset or rhesus monkey blood which had been stored at — 80 °C in 15% glycerol/saline. Detection of parasites
First appearance of parasitaemia was assessed on thick bloodfilmshaemolysed and stained in Field's solution. Parasite numbers were estimated by erythrocyte counts. The percentage of red cells parasitized was determined by counting up to 10* erythrocytes on thin blood films stained with Giemsa. A negative count on thick blood film indicates a parasite density of less than 1/106 erythrocytes, as estimated by white cell counts.
C4 C6 C7 C17 C14 C3 Cll C12 W3 W3 W3 Wl W3 Wl W3
W3
Variant Marmoset Rhesus Marmoset Rhesus Rhesus Rhesus Marmoset Rhesus
Source
)
oute i.v. i.m. i.v. i.m. i.v. i.m. i.m. i.m. 5 7 5 6 7 10 5 6
Pre-patent period (days) 69 44 75 5-3 82 35 48 18
(%)
Maximum parasitaemia 7 10 7 7 10 15 15 15
Duration of patency (days)
Not determined since marmosets wero infected with 1 ml of infected, frozen whole blood.
N.D. N.D.
2xlO4 105
N.D.
2x10*
N.D.*
108
Marmoset No. of parasites
P. knowlesi infection
Table 1. Infection of marmosets vrith Plasmodium knowlesi
Survived Survived Survived
12 17 12 13 16
Died (day)
O5
to
70
J. LANGHOENE AND S. COHEN 100
r
100
•S
10
0-1
001
5
10 Days after challenge
15
Fig. 1. Course of parasitaemia in 5 marmosets which died, of Plasmodium hnowlesi infection. (O—O), C4; ( • — • ) , C6; (Q—D), C7; ( • — • ) , C14; (A—A), C17. The animals were challenged with Wl or W3 variants of P. knowlesi as described in Table 1. Day of death of each animal is indicated in Table 1. RESULTS
Course of P. knowlesi infection in marmosets
Eight marmosets were infected with W3 or Wl variant of P. knowlesi by intravenous or intramuscular injection of organisms obtained from infected donor marmosets or rhesus monkeys or fromfrozen,infected blood. The parasite variants, routes of infection and resultant parasitaemias are summarized in Table 1. Parasites were first observed in peripheral blood within 5-10 days. In those animals injected with a known number of parasitized erythrocytes the pre-patent period was longer than that predicted from a logarithmic growth rate of the parasite.
P. knowlesi in the marmoset
71
100
10
2
10
0-1
001
10
15
20
25
30
Days after challenge
Fig. 2. Course of parasitaemia in 3 marmosets that survived an initial challenge with Plasmodium knowlesi. ( • — • ) , C3; (O—O), C l l ; O—3),C12. The animals were challenged as described in Table 1.
Maximum parasitaemias of 5-3-82% were recorded within 9-21 days of blood challenge. The course of P. knowlesi infections in these animals is shown in Figs 1 and 2. Five of the 8 animals showed pre-patent periods of 5-7 days and died of P. knowlesi infection within 13—17 days after challenge by intravenous or intramuscular routes (Fig. 1). Three animals infected by intramuscular inoculation survived blood challenge (Fig. 2). Parasites were apparent in peripheral blood after 5-10 days and persisted for 15 days, after which time thick blood films became negative suggesting a concentration of less than 1 parasite/10 6 erythrocytes. In all these animals, parasites were rapidly cleared from the blood; parasitaemias of 18-48% were reduced to less than 1 parasite/104 erythrocytes in 3-5 days.
126
78 217 316 43 182 210
W3
Wl
W3
Marmoset
C3
Cll
C12
106 105 104
10 106 10s
s
10 s
No. of parasites
W3 Wl Nuri
W3 Wl Nuri
W3
' Strain or variant
Challenge infection
.V. .V. .V.
.V. .V. •V.
i.v
Route
—
-t
7 9
4
5 1
1
—
4 1
2
PreMaximum Duration of patent parasitaemia period (parasites/10* patency (days) erythrocytes) (days)
ft
w
O
Q
GO
E AND
* Days after initial infection with P. knowlesi. t Indicates that no parasites were observed by thick or thin film analysis.
Day*
Parasite variant of initial infection
Table 2. Results of homologous and heterologous challenge of 3 marmosets which had recovered from a primarg infection with Plasmodium knowlesi LANGHO
P. knowlesi in the marmoset
73
The morphology of the erythrocyte forms of P . knowlesi was indistinguishable from parasites infecting rhesus monkey erythrocytes. The erythrocyte cycle in marmoset blood occupied 24 h and was synchronous. Mature schizonts containing 8-16 merozoites were present in the peripheral circulation. Multiplication rates over 24 h periods were variable; logarithmic parasite multiplication was recorded in only 2 animals (C4 and C14) which died of P. hnowlesi infection. Multiplication rates of 2- to 8-fold were observed over 24 h periods in the remaining 6 animals. Challenge of marmosets with homologous and heterologous variants and a heterologous strain of P. knowlesi The 3 marmosets surviving initial blood challenge with W3 or Wl variants of P . knowlesi were subsequently challenged with W3 and Wl variants and Nuri strain at various times after initial infection (Table 2). All three animals survived challenge with an homologous or heterologous variant or heterologous strain. Two of the 3 animals (C3 and CH) developed low parasitaemias of brief duration. The pre-patent period in these animals was 4-9 days. The maximum parasitaemia recorded was 5 parasites/104 erythrocytes, and parasites were observed in peripheral blood for a period of 1-4 days. Two of the animals (Oil and C12) when challenged with a heterologous strain of P. knowlesi (Nuri strain) did not develop patent parasitaemias. These preliminary results suggest that marmosets surviving an initial infection with P . knowlesi are subsequently resistant to infection with heterologous variants or strains of this parasite. Resistance is retained during intervals of at least 100 days between challenge infections.
DISCUSSION
These studies confirm early observations of Cruz & de Mello (1947) that the common marmoset, C. jacchus, is susceptible to P . knowlesi. The morphology of the erythrocytic forms of P . knowlesi was indistinguishable from that found in the natural host, Macacafascicularis, and the common laboratory host, M. mulatta, the rhesus monkey. The parasite cycle was of 24 h duration and was synchronous. The course of parasitaemia in the marmoset differed, however, from that seen in the rhesus monkey. In the latter, the infection usually has a rapidly fatal outcome with logarithmic multiplication of the parasite (Garnham, 1966). P . knowlesi infection of marmosets did not produce a uniformly fatal infection; 3 of the 8 animals survived an initial challenge infection. The animals that survived were all infected by intramuscular inoculation while those that died were inoculated either intravenously or intramuscularly. Cruz & de Mello (1947) observed that, occasionally, marmosets infected by intramuscular routes spontaneously recovered from infection. In addition to animals surviving challenge infections, it was observed in this study that the pre-patent periods were longer than expected of a logarithmically multiplicating parasite. This was also apparent in the early patent parasitaemias of the majority of the animals. Although the mature schizonts contained 8-16 merozoites, multiplication rates were less than 10-fold over a 24 h period.
74
J. LANGHORNE AND S. COHEN
The observed variations in survival and parasite multiplication rates suggest a differential susceptibility of individual animals to P. knowlesi, but the underlying mechanisms have not been established. There may be components in the serum which affect the viability of released merozoites, or certain erythrocytes may lack the receptors necessary for merozoite invasion. The extended pre-patent periods and the initial low multiplication rates of parasites may reflect the selection of those merozoites able to survive in the plasma and invade other erythrocytes. In vitro studies of the invasion of P. knowlesi merozoites with erythrocytes and sera of various animals demonstrated that the New World douroucouli monkey, Aotus trivirgatus, was less susceptible to P. knowlesi than the Old World rhesus monkey a difference apparently due to some property of the erythrocyte rather than to serum factors (Butcher, Mitchell & Cohen, 1973). Similar studies with a range of marmoset erythrocytes and sera may help to elucidate the mechanisms underlying susceptibility or relative resistance of individual animals to P. knowlesi. The ability of marmosets to recover from P. knowlesi infection may be due to a combination of mechanisms. Non-immunological factors which determine lower initial parasite multiplication rates may allow sufficient time for the development of acquired immunity. Since these infections were initiated by intramuscular inoculation, local inflammatory responses, particularly in those animals infected with parasites in rhesus monkey erythrocytes, may have reduced the number of viable parasites entering the peripheral circulation. The mechanism of parasite clearance in these animals is not yet understood, but it is likely to be predominantly immunological, as subsequent challenge with the same parasite variant produced a negligible or low grade parasitaemia of short duration. These animals were also resistant to challenge with a heterologous variant of the same strain, and to a heterologous strain of P. knowlesi, suggesting that the protective immune response may be directed against common antigens of P. knowlesi rather than variantspecific antigens. There are no comparable studies of the rhesus monkey since P. knowlesi infection is almost invariably fatal if left untreated. A degree of immunity can, however, be induced in the rhesus by infection and sub-curative drug therapy resulting in periodic relapses and low-grade infection. Each relapse is attributable to a serologically distinct variant. Chronic infection produces an immunity with broad variant specificity (Brown & Brown, 1965; Brown, Brown & Hills, 1968; Voller & Rossan, 1969) which may be due in part to T-cell sensitization to common malarial antigens (Brown, 1971) or B-cell activation leading to production of low titres of antibody directed against several serological variants (Butcher & Cohen, 1972). Merozoite vaccination protects rhesus monkeys against homologous or heterologous challenge with erythrocytic stages or the blood infections following mosquito-transmitted (sporozoite) stages of P. knowlesi (Mitchell, Butcher & Cohen, 1975; Mitchell, Butcher, Langhorne & Cohen, 1977; Richards, Mitchell, Butcher & Cohen, 1977). The immunity induced in the rhesus monkey by such vaccination is similar in terms of specificity and degree to that obtained in marmosets which survive an initial challenge infection. The merozoite vaccine may stimulate a specific response in rhesus monkeys to antigens which are present on a wide
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range of P. knowlesi parasites, but are poorly immunogenic in a natural infection. In certain marmosets these antigens may be significantly immunogenic in the natural infection, thus enabling them to mount an effective immune response. The nature of the immune responses induced in marmosets recovered from P. knowlesi infection and in marmosets vaccinated with merozoites in Freund's complete adjuvant are currently being investigated. The marmoset provides an interesting and convenient model for such studies since in vivo immune responses can readily be investigated in chimaeric marmoset twins. This work was supported by the Medical Research Council and the World Health Organization. We would like to thank Drs G. H. Mitchell and G. A. Butcher for helpful discussion, and Miss Marilyn Burnikell and Miss Deborah Cutter for skilled technical assistance.
REFERENCES K., ANDEBSON, J. M. & BROWNHILL, L. E. (1962). Marrow chimerism in the marmosets. Science 138, 513-15. BBOWN, K. N. (1971). Protective immunity to malaria provides a model for the survival of cells in an unmunologically hostile environment. Nature, London 230, 163—7. BROWN, K. N. & BROWN, I. N. (1965). Immunity to malaria: antigenic variation in chronic infections of Plasmodium knowlesi. Nature, London 208, 1286-8. BERNTRSCHKE,
BROWN, K. N., BROWN, I. N. & HELLS, L. A. (1968). Immunity to malaria: the antibody
response to antigenic variation by Plasmodium knowlesi. Immunology 14, 127—38. G. A. & COHEN, S. (1972). Antigenic variation and protective immunity in Plasmodium knowlesi malaria. Immunology 23, 503-21. BUTCHER, G. A., MITCHELL, G. H. & COHEN, S. (1973). Mechanism of host specificity in malarial infection. Nature, London 244, 40-2. BUTCHER, G. A., MITCHELL, G. H. & COHEN, S. (1978). Antibody mediated mechanisms of immunity to malaria induced by vaccination with Plasmodium knowlesi merozoites. Immunology 34, 77-86. CRUZ, W. O. & DE MELLO, R. P. (1947). Infeccao de Macaco sul americano 'sagul' (Callithrix jacchus, Linnaeus 1758) com o Plasmodium knowlesi. Memoriae do Instituto Oswaldo Cruz 45, 119-21. GARNKAM, P. C. C. (1966). Malaria Parasites and other Haemosporidia. Oxford: Blackwell Scientific Press. GENGOZIAN, N., BATSON, J. S. & EIDE, P. (1964). Haematologic and cytogenic evidence for haemopoietic chimerism in the marmoset, Tamerinus nigricollis. Cytogenetics (Basel) 3, 384-93. MILLER, J. F. A. P., VADAS, M. A., WHITELAW, A. & GAMBLE, J. (1975). H—2 gene complex restricts transfer of delayed-type hypersensitivity in mice. Proceedings oj the National Academy of Sciences (USA) 72, 5095-8. MITCHELL, G. H., BUTCHER, G. A. & COHEN, S. (1975). Merozoite vaccination against Plasmodium knowlesi malaria. Immunology 29, 397-407. MITCHELL, G. H., BUTCHER, G. A., LANGHOBNE, J. & COHEN, S. (1977). A freeze-dxied merozoite vaccine effective against Plasmodium knowlesi malaria. Clinical and Experimental Immunology 28, 276-9. PORTER, R. P. & GENGOZIAN, N. (1969). Immunological tolerance and rejection of skin allografts in the marmoset. Transplantation 8, 653-65. PORTER, R. P. & GENGOZIAN, N. (1973). Immunological responsiveness and tolerance of marmoset lymphoid tissue in vitro. Transplantation 16, 221-30. RICHAKDS, W. H. G., MITCHELL, G. EL, BUTCHER, G. A. & COHEN, S. (1977). Merozoite vaccination of rhesus monkeys against Plasmodium knowlesi malaria; immunity to sporozoite (mosquito-transmitted) challenge. Parasitology 74, 191-8. SPRENT, J. & MILLER, J. F. A. P. (1971). Activation of thymus cells by histocompatibility antigens. Nature, New Biology 234, 195-8. BUTCHER,
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A. & ROSSAN, R. N. (1969). Immiinological studies on simian malaria iii. Immunity to challenge and antigenic variation in Plasmodium hnovilesi. Transactions of the Royal Society of Tropical Medicine and Hygiene 63, 507-23. VON BoEffMEK, H., HUDSON, L. & SPKENT, J. (1975). Collaboration of histoincompatible T and B lymphocytes using cells from tetraparental bone marrow chimeras. Journal of Experimental Medicine 142, 989-97. VOIXEB,
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