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The effect of mitomycin c treatment of infected erythrocytes on infection with Babesia microti and Babesia rodhaini in mice
THE EFFECT OF MITOMYCIN C TREATMENT OF INFECTED ERYTHROCYTES ON INFECTION WITH BABESIA MZCROTZAND BABESZA RODHAZNZ IN MICE E. MEEUSEN*, S. LLOYDand E. J. L. SOULSBY Department of Clinical Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge, U.K. (Received 13 December 1983; in revisedform 8 June 1984) Abstract-Mmusm E., LLOYDS. and SOULSBY E. J. L. 1985. The effect of mitomycin C treatment of infected erythrocytes on infection with Bubesia microti and Bubesia rodhuini in mice. International Journal for Parasitology 15: 65-69. In vitro treatment of Bubesiu microti infected erythrocytes with mitomycin C before their injection into mice prolonged the prepatent period of infection, reduced the levels of the infection in the ‘breakthrough’ parasitaemia and induced protection against reinfection. Treatment of B. microti with mitomycin C at a concentration of 25 pg ml-f resulted in a mean peak parasitaemia of 6.2% in the infected mice compared with 46.5% in control mice injected with untreated B. microti parasites. In addition, mice survived a normally fatal B. rodhaini infection if injected with 6.2 x 107 infected erythrocytes treated with 25 pg ml-l mitomycin C and four of five mice survived infection with 6.2 x lo5 similarly treated infected erythrocytes. However, the degree of protection against B. rodhuini was dependent on the concentration of mitomycin C used to treat the parasites and treatment of 5 x 107 infected erythrocytes with 50 vg ml-r resulted in survival of only four of the five infected mice. In addition, when 100 pg ml-t of mitomycin C was used to treat B. rodhaini parasites, the course of infection, although delayed, was indistinguishable from that seen in the control mice and all the mice died. The latter results and the lack of efficacy of comparable numbers of heat killed parasites suggested the necessity for sufficient, non-replicating, mitomycin C treated parasites to metabolize and produce and/or present protective antigens to the host.
INDEX KEY WORDS: Bubesiu spp.; mice; immunization; mitomycin C treated infected erythrocytes.
INTRODUCTION
PREVIOUS studies on vaccination against haemoprotozoan parasites have demonstrated that much greater quantities of dead antigen are required to induce levels of protection against infection similar to those seen using smaller quantities of living parasite antigen (Jerusalem & Eling, 1969). In addition, Phillips (1971) observed that injection of irradiated Babesia parasites into rats produced higher levels of protection than did the injection of equivalent numbers of killed parasites. Similarly, the injection of irradiated Babesia infected erythrocytes has been shown to protect both splenectomized and intact calves against a challenge infection provided that the irradiation doses used were such as to still permit an infection to develop. Irradiation doses which completely inhibited infection abrogated the protective effect (Mahoney, Wright & Ketterer, 1973;
*Address for correspondence: Dr. E. Meeusen, Department of Microbiology, University of Melbourne, Victoria 3052, Australia.
Purnell, Lewis & Brocklesby, 1979). In contrast, rodents were protected against a challenge infection whether or not the irradiation doses completely prevented infection (Phillips, 1971; Bishop & Kuttler, 1974). The effects of radiation on haemoprotozoan parasites are not fully understood but it is generally accepted that radiation can prevent the replication of cells while leaving some functions of the cell intact (Trigg, Phillips & Gutteridge, 1972). Therefore, these latter functions may be important in the induction of a protective immune response while irradiation prevented or retarded the fatal effects of an overwhelming parasitaemia. Mitomycin C, used at optimal concentrations, has been shown to prevent cell replication without preventing protein synthesis although both these functions are affected adversely at high concentrations of mitomycin C (Szybalski & Iyer, 1964). The present study describes the effect of mitomycin C on the course of infection with Babesia microti and Babesia rodhaini in mice injected with such treated parasites and the development of protection against infection in the animals. 65
E. MEEUSEN, S. LLOYD and E. J. L. SOULSBY
66
MATERIALS AND METHODS
IO0
I.J.P. VOL. 15. 1985
1
Parasites and experimental host. The Kings strain of B. microti was kindly provided by Professor F. E. G. Cox (London University) and B. rodhaini by the Molten0 Institute (University of Cambridge). Mice normally overcome an infection with B. microti whereas infection with B. rodhaini in mice generally is fatal (Cox & Young, 1969). Infections were always initiated by the intravenous injection of infected erythrocytes (I.E.). Inbred female NIH mice (Hacking and Churchill, Huntingdon) were used in all experiments. Parasitaemias and reticulocytosis. Parasitaemias were calculated as the percent erythrocytes infected (I.E.) on Giemsa stained blood smears. Immature red blood cells of the mouse stain blue with Giemsa and it has been shown that there is good correlation between the numbers of immature red blood cells stained blue with Giemsa and the numbers of reticulocytes observed with brilliant cresyl blue staining such that reticulocytosis could be assessed on Giemsa stained blood smears (Meeusen E.N.T. unpublished, Ph.D. thesis, University of Cambridge, 1982).
Mitomycin
C
treatment
of
infected
erythrocytes.
Infected blood was collected, after the mice had been killed with ether, by open heart punture into syringes containing heparin. The cells were washed twice by centrifugation in Hanks balanced salt solution and resuspended at a concentration of 2 x IO7 I.E. ml-1 in RPM1 1640 containing 10% foetal calf serum (FCS). Mitomycin C (Sigma Chemical Co., Poole) was added at a final concentration of 25, SO or 100 pg ml-l red blood cells and the mixture incubated for 30 min at 37°C (Swain, 1980). The treated cells were washed three times with cold RPM1 1640/10%FCS, readjusted to the required concentration and injected intravenously into mice.
Heat inacfivation of infected erythrocytes. B. rodhaini infected at 45°C
erythrocytes
were heat inactivated
in a water bath
for 45 min (D’Antonio, 1972). RESULTS
Babesia microti
groups, each of five mice, were injected with either 2 x 106 untreated I.E. or 3 x 107 I.E. treated with 25 pg ml-1 mitomycin C. The course of the subsequent parasitaemias is depicted in Fig. 1. Parasitaemias in the control mice injected with untreated I.E. increased as expected to a mean peak parasitaemia of 46.5% on day 11 of infection. In contrast, in mice injected with approx. 10 times the number of mitomycin C treated I.E. there was an initial decline in the number of infected cells immediately after infection. This was followed by a considerable delay of at least 7 days in the appearance of a rising parasitaemia and a mean peak parasitaemia of only 6.2% was seen. However, this low peak of parasitaemia in mice infected with mitomycin C treated I.E. was maintained for 4 days before the infection fell to levels below those seen in the control mice. This experiment was repeated once with similar results. B. microti causes a uniform logarithmetically increasing parasitaemia (Cox, 1975) and therefore the number of parasites initiating an infection can be Two
FIG. 1. Mean B. microti parasitaemia f standard (s.D.) in mice injected with (a) 2 x 106 untreated
deviation
B. microti infected erythrocytes . . . . . . ; or (b) 3 x lo7 Z?.microti I.E. treated with 25 vg ml-l mitomycin C ~.
estimated by extrapolation of the straight semilogarithmetic line of parasitaemia. Extrapolation of the parasitaemia of the mice injected with mitomycin C treated erythrocytes corresponded to an infection initiated with 3-4x 104 I.E. This suggested that approx. 0.1-@2% of the parasites were able to replicate after mitomycin C treatment. The parasitaemia arising from these remaining replicating parasites in the mitomycin C treated cell inoculum will hereafter be referred to as the ‘breakthrough’ infection. Babesia rodhaini
The results obtained
with the above study with
B. microti suggested that similar treatment of B. rodhaini I.E. might lead to protection and survival of mice to a normally fatal B. rodhaini infection. In
an attempt to eliminate or reduce the number of proliferating parasites remaining in the inoculum after treatment with mitomycin C, the concentration of mitomycin C used for the in vitro treatment of the infected cells was increased. Thus, mitomycin C was used to treat B. rodhaini I.E. at a concentration of 25.50 and 100 pg ml-l. The parasitaemias resulting from the injection of such treated cells are depicted in Fig. 2. Although the prepatent periods of infection were progressively delayed with increasing mitomycin C concentration used to treat the cells, a ‘breakthrough’ infection still appeared in all the animals. Extrapolation of the parasitaemia suggested that approx. 0.16, 0.05 and 0.005% of the parasites were not prevented from replicating after treatment with 25, 50 or 100 pg ml-l mitomycin C ml-l, respectively. On the other hand, increasing the mitomycin C concentration used for the in vitro of the cells resulted
I.J.P.VOL. 15. 1985
67
Immunization against Babesiu spp. in mice
D1VS
AfTtR
INfICTION
FIG. 2. Mean B. rodhoini parasitaemia -r- S.D. in mice injected with (a) 5.7 x 10’ untreated B. rodhaini1.E.. ... . ; (b) 6.2 x 10’ I.E. treated with 25 c(g ml-1 mitdmycin C ----_; (c) 5x IO7 I.E. treated with 50 pg ml-’ mitomycin C _ _ _ _ --; or (d) 5 x lo7 I.E. treated with 100 c(g ml-’ mitomycin C_._._._._. + =time of death of an animal.
a decrease in the protection conferred against this ‘breakthrough’ infection. Thus, while all the animals survived and showed reduced peak parasitaemias when injected with infected cells treated with 25 pg ml-1 mitomycin C, treatment of the I.E. with 100 pg ml-1 mitomycin C resulted in a course of infection which, although delayed, was indistinguishable from that seen in the control mice and none of the mice survived the infection. Treatment of the I.E. with 50 pg ml-1 mitomycin C resulted in an intermediate course of infection and four of the five mice survived infection. In a second experiment eight control mice were infected with 5.5~ 107 untreated B. rodhaini I.E. and 10 mice were injected with 5.7~ 107 B. rodhaini I.E. treated with 25 pg ml-1 mitomycin C. All the control mice infected with untreated B. rodhaini parasites died within 80 h of infection whereas all the 10 mice infected with the mitomycin C treated parasites survived. The maximum peak parasitaemia seen in these latter animals was 35.6% and the parasitaemia had reached undetectable levels 12 days after infection. When the number of injected erythrocytes treated with 25 pg ml-l mitomycin C was reduced lOO-fold, from 6.2 x 107 to 6.2 x 10s I.E., the prepatent period of the ‘breakthrough’ infection was delayed and, despite the small number of mitomycin C treated parasites injected, four of the five animals still survived the infection (Fig. 3). In this experiment, the group of mice which received the higher number of mitomycin C treated infected cells showed a more prolonged reticulocytosis (Fig. 4) even though the parasitaemia was less marked. The slope of each parasitaemia on a semilogarithmetic graph should be representative of the rate of multiplication of the parasites in each group of mice.
The calculated value of the slope for the control group of mice injected with untreated erythrocytes was b = 0.03 1. Values for the experimental groups of mice were close to that of the control with the exception of the two groups of mice which were injected with protective, high inocula of I.E. treated with 25 and 50 pg ml-1 mitomycin C. These two groups both showed a slope of b = 0.026. All the surviving mice were given a challenge infection with untreated B. rodhaini parasites 28 days or 3-5 months after the experimental infection and all the mice survived this challenge infection compared with previously uninfected mice which died 3-4 days after infection. The importance of metabolism by the parasites in the above study was examined by comparing B. rodhaini infection in mice injected with mitomycin C treated I.E. or heat killed I.E. Five mice each received 5 x 107 heat inactivated infected cells. No parasitaemia was observed by day 8 of the experiment. At this time the mice were infected by the intravenous injection of 2x 106 B. rodhaini I.E. obtained from the rising ‘breakthrough’ parasitaemia of mice injected with 6.2x 10’ mitomycin C (25 lg ml-l) treated I.E. The subsequent parasitaemia was high and comparable to that seen in previously uninfected, naive mice. Four of the five mice died and one survived after suffering a severe infection (Fig. 3). In another experiment mice were injected with 8 x 107 heat inactivated B. rodhaini and 12 days later infected with 5.3 x 107 normal B. rodhaini I.E. from the same liquid nitrogen stabilate. All the mice died within 4 days of the challenge infection.
DISCUSSION
vitro mitomycin C treatment at a concentration of 25 pg ml-l is known to affect cell replication without affecting protein synthesis of lymphocytes (Szybalski & Iyer, 1964; Swain, 1980). However, some replication of Babesia parasites occurred since ‘breakthrough’ infections, originating from the mitomycin C treated inocula, were always apparent. Nevertheless, significant protection against these ‘breakthrough’ infections and against subsequent challenge infection with normal parasites had been induced in the mice when mitomycin C was used at a concentration of 25 or 50 yg ml-1 to treat the B. rodhaini infected red blood cells in the inocula. The observed protection could be the result of host sensitization by the whole mitomycin C treated inoculum comprising mainly non-replicating parasites or it is possible that mitomycin C treatment selected a population of attentuated Babesia parasites with a slower multiplication rate which permitted the host time to mount a protective immune response. Two observations argue against the latter explanation. The few replicating parasites remaining after treatment of B. rodhaini with 100 pg ml-1 mitomycin C multiplied at a rate similar to that of untreated paraIn
E. MEEUSEN. S. LLOYD and
68
E. J. L.
to prevent
FIG. 3. Mean B. rodhaini parasitaemia * S.D. in mice injected on day 0 with (a) 6.2 x 10’ B. rodhaini I.E. treated with 25 c(g ml-’ mitomycin C_ ; (b) 6.2x 16 I.E. treated with 25 c(g ml-l mitomycin C________; or (c) 4x 10’ heat inactivated I?. rodhnini I.E. followed by 2~ 106 untreated I.E. given on day 8 . . . . . . . . . . + = time of death of an animal and _ _ _ _ parasitaemia in one animal after day 10.
I
II
II
DAYSLFTER
I4
I6
I8
INFECTION
FIG. 4. Mean reticulocytosis + SD. in mice injected with or (b) 6.2x105_______8. (a) 6.2 x 10’ rodhaini I.E. treated with 25 c(gml-1 mitomycin C. + = time of death of an animal whose reticulocytosis is included in the mean until this time.
sites and were equally pathogenic. Also, the parasitaemia induced in the first group of mice which had been injected with heat inactivated parasites was initiated with infected cells from the rising ‘breakthrough’ infection in mice injected with parasites treated with mitomycin C at 25 ,ug ml-t. These latter mice controlled the infection yet the same parasites when transferred to new hosts multiplied as if untreated and caused fatal infections. Thus, it seems likely that the protection against Babesia observed in these experiments was associated with the presence of the whole inoculum of mitomycin C treated parasites injected. That protein synthesis by these mitomycin C treated parasites was required is suggested by the fact that injection of a similar number of heat inactivated parasites did not induce protection against infection. Further, when a similar number of B. rodhaini were treated with mitomycin C at a concentration of 100 pg ml-t, a concentration likely
SOULSBY
1.3.~.VOL. 15. 1985
protein metabolism (Szybalski & lyer, 1964) and likely to kill the cells affected by the treatment, the protection induced by mitomycin C treatment was abolished. Therefore, it is suggested that mitomycin C, when used at optimal concentrations, permits the parasite to produce and/or present protective antigens to the host while, since replication of the majority of the parasites is prevented, the host has time to mount a protective immune response. An additional aspect is that mice injected with a large number, 6.2~ 107, mitomycin C treated B. rodhaim’ showed a prolonged reticulocytosis despite their reduced parasitaemia in comparison to mice injected with fewer, 6.2 x lo*, similarly treated parasites. It is possible that a pathological mechanism acting on the red cells is involved also. The results obtained with mitomycin C treated B. microti I.E. suggest that two mechanisms might be involved in the observed protection. In normal infections, parasitaemias due to B. microti rise rapidly to peak at around 45% erythrocytes infected followed by a rapid decline in parasitaemia. In contrast, the first phase of protection in mice injected with mitomycin C treated cells was manifest by a stable peak parasitaemia which plateaued at a low level (6Oro)for at least 4 days. This might be associated either with prevention of replication or destruction of cells at the same rate as the generation of new parasites. The second phase of protection was characterized by a sudden fall in the numbers of parasitized cells similar to that seen during a normal primary infection and often referred to as the ‘crisis’ period. Quinn & Wyler (1979) working with Plasmodium berghei in rats thought that the accelerated clearance of infected cells could be due to a sudden alteration in the microcirculation in the enlarged spleen resulting in an increased flow through the ‘open’ pathways thus allowing more contact between infected cells and macrophages. It is possible that this mechanism also applies after a primary B. microti infection and that a certain amount of parasite proliferation is required for its activation. In addition, specific or non-specific activation of macrophages and their synergistic action with antibodies in killing Bubesia parasites (Bautista & Kreier, 1980) could also accelerate the reduction of parasitaemia during the second phase of protection. The first stage of protection, represented by a stable peak parasitaemia, was not observed in infections initiated with mitomycin C treated B. rodhaini infected cells. This could be due to the more prolific growth of B. rodhaini which may result in an earlier start of the second phase of protection thus obscuring the first phase. In general, the results obtained in the mitomycin C experiments were comparable to those obtained using irradiated Babesia parasites (Phillips, 1971; Mahoney et al., 1973; Purnell et al., 1979). The in vitro mitomycin C treatment of infected erythrocytes would appear to provide a simpler and more efficient method for experimental immunization against
Immunization against Eabesia spp. in mice
I.J.P. VOL. 15. 1985
parasites. Thus, while only 50% survival of mice could be obtained by the injection of a single dose of irradiated B. rodhaini parasites, the present experiments achieved 100% survival using infected erythrocytes treated with 25 c(g ml-1 mitomycin C. The technique warrants further investigation for its use in experimental immunization against haemoprotozoan parasites.
Bubesia
work was supported by funds from the Wellcome Trust. E. Meeusen was the recipient of a British Council Fellowship. We would like to thank
Acknowledgements-This
Mr. M. Frattasi
for assistance
with the animals.
REFERENCES BAUTISTAC. R. & KREIER J. phages and immune serum in short-term cultures. 313-324. BISHOP J. P. & KUTTLER
P. 1980. The action of macroon growth of Babe& microti Tropenmed. Parasitol. 31:
K. L. 1974. Infectivity and immunogenicity of irradiated Babesia rodhaini. Journal
of Protozoology 21: 758-760. Cox F. E. G. 1975. Factors affecting infections of mammals with intraerythrocytic protozoa. Symposia of the Society for Experimental Biology 24: 429-45 1. Cox F. E. G. & YOUNGA. S. 1969. Acquired immunity to Babesia microti and Babesia rodhaini on mice. ParasitoIogy 59: 257-268.
69
D’ANTONIO L. E. 1972. Plasmodium berghei: vaccination of mice against malaria with heat inactivated parasitized blood. ExperimentalParasitology 31: 82-87. JERUSALEM C. & ELING W. 1969. Active immunization against Plusmodium berghei malaria in mice using
different preparations of plasmodial antigen and different pathways of administration. BuIIetin of the World Health Organization 40: 807-818. MAHONEY D. F., WRIGHT I. C. & KETTERER E. J. 1973. Bubesiu urgentina: the infectivity and immunogenicity of irradiated blood parasites for splenectomized calves. International Journalfor Parasitology 3: 209-217. PHILLIPS R. S. 1971. Immunity of rats following injection of WCo irradiated Bubesiu rodhaini infected red cells. Parasitology 60: 22 1-23 1. PURNELL R. E., LEWIS D. & BROCKLESBY D. W. 1979. Babesiu major: protection of intact calves against homologous challenge by the injection of irradiated piroplasms. International Journal for Parasitology 9: 69-71. QUINN T. C. & WYLER D. J. 1979. Intravascular clearance of parasitized erythrocytes in rodent malaria. Journal of Clinical Investigation 63: 1187-l 194. SWAIN S. L. 1980. Mitomycin C. In: Selected Methods in CeIIuIar Immunology (Edited by MISHELL B. B. & SHIIGI S. M.), pp. 240-241. Freeman, San Francisco. SZYBALSKIW. & IYER V. N. 1964. Crosslinkinp, of DNA bv enzymatically or chemically activated mitomycins or porfiromycins, bifunctionally “alkylating” antibiotics. Federation Proceedings 23: 946-955. TRIGG P. I., PHILLIPS R. S. & GUTTERIDG~ W. E. 1972. The effects of y-radiation on Plasmodium knowlesi. International Journalfor Parasitology 2: 13 l-138.
INDEX KEY WORDS: Bubesiu spp.; mice; immunization; mitomycin C treated infected erythrocytes.
INTRODUCTION
PREVIOUS studies on vaccination against haemoprotozoan parasites have demonstrated that much greater quantities of dead antigen are required to induce levels of protection against infection similar to those seen using smaller quantities of living parasite antigen (Jerusalem & Eling, 1969). In addition, Phillips (1971) observed that injection of irradiated Babesia parasites into rats produced higher levels of protection than did the injection of equivalent numbers of killed parasites. Similarly, the injection of irradiated Babesia infected erythrocytes has been shown to protect both splenectomized and intact calves against a challenge infection provided that the irradiation doses used were such as to still permit an infection to develop. Irradiation doses which completely inhibited infection abrogated the protective effect (Mahoney, Wright & Ketterer, 1973;
*Address for correspondence: Dr. E. Meeusen, Department of Microbiology, University of Melbourne, Victoria 3052, Australia.
Purnell, Lewis & Brocklesby, 1979). In contrast, rodents were protected against a challenge infection whether or not the irradiation doses completely prevented infection (Phillips, 1971; Bishop & Kuttler, 1974). The effects of radiation on haemoprotozoan parasites are not fully understood but it is generally accepted that radiation can prevent the replication of cells while leaving some functions of the cell intact (Trigg, Phillips & Gutteridge, 1972). Therefore, these latter functions may be important in the induction of a protective immune response while irradiation prevented or retarded the fatal effects of an overwhelming parasitaemia. Mitomycin C, used at optimal concentrations, has been shown to prevent cell replication without preventing protein synthesis although both these functions are affected adversely at high concentrations of mitomycin C (Szybalski & Iyer, 1964). The present study describes the effect of mitomycin C on the course of infection with Babesia microti and Babesia rodhaini in mice injected with such treated parasites and the development of protection against infection in the animals. 65
E. MEEUSEN, S. LLOYD and E. J. L. SOULSBY
66
MATERIALS AND METHODS
IO0
I.J.P. VOL. 15. 1985
1
Parasites and experimental host. The Kings strain of B. microti was kindly provided by Professor F. E. G. Cox (London University) and B. rodhaini by the Molten0 Institute (University of Cambridge). Mice normally overcome an infection with B. microti whereas infection with B. rodhaini in mice generally is fatal (Cox & Young, 1969). Infections were always initiated by the intravenous injection of infected erythrocytes (I.E.). Inbred female NIH mice (Hacking and Churchill, Huntingdon) were used in all experiments. Parasitaemias and reticulocytosis. Parasitaemias were calculated as the percent erythrocytes infected (I.E.) on Giemsa stained blood smears. Immature red blood cells of the mouse stain blue with Giemsa and it has been shown that there is good correlation between the numbers of immature red blood cells stained blue with Giemsa and the numbers of reticulocytes observed with brilliant cresyl blue staining such that reticulocytosis could be assessed on Giemsa stained blood smears (Meeusen E.N.T. unpublished, Ph.D. thesis, University of Cambridge, 1982).
Mitomycin
C
treatment
of
infected
erythrocytes.
Infected blood was collected, after the mice had been killed with ether, by open heart punture into syringes containing heparin. The cells were washed twice by centrifugation in Hanks balanced salt solution and resuspended at a concentration of 2 x IO7 I.E. ml-1 in RPM1 1640 containing 10% foetal calf serum (FCS). Mitomycin C (Sigma Chemical Co., Poole) was added at a final concentration of 25, SO or 100 pg ml-l red blood cells and the mixture incubated for 30 min at 37°C (Swain, 1980). The treated cells were washed three times with cold RPM1 1640/10%FCS, readjusted to the required concentration and injected intravenously into mice.
Heat inacfivation of infected erythrocytes. B. rodhaini infected at 45°C
erythrocytes
were heat inactivated
in a water bath
for 45 min (D’Antonio, 1972). RESULTS
Babesia microti
groups, each of five mice, were injected with either 2 x 106 untreated I.E. or 3 x 107 I.E. treated with 25 pg ml-1 mitomycin C. The course of the subsequent parasitaemias is depicted in Fig. 1. Parasitaemias in the control mice injected with untreated I.E. increased as expected to a mean peak parasitaemia of 46.5% on day 11 of infection. In contrast, in mice injected with approx. 10 times the number of mitomycin C treated I.E. there was an initial decline in the number of infected cells immediately after infection. This was followed by a considerable delay of at least 7 days in the appearance of a rising parasitaemia and a mean peak parasitaemia of only 6.2% was seen. However, this low peak of parasitaemia in mice infected with mitomycin C treated I.E. was maintained for 4 days before the infection fell to levels below those seen in the control mice. This experiment was repeated once with similar results. B. microti causes a uniform logarithmetically increasing parasitaemia (Cox, 1975) and therefore the number of parasites initiating an infection can be Two
FIG. 1. Mean B. microti parasitaemia f standard (s.D.) in mice injected with (a) 2 x 106 untreated
deviation
B. microti infected erythrocytes . . . . . . ; or (b) 3 x lo7 Z?.microti I.E. treated with 25 vg ml-l mitomycin C ~.
estimated by extrapolation of the straight semilogarithmetic line of parasitaemia. Extrapolation of the parasitaemia of the mice injected with mitomycin C treated erythrocytes corresponded to an infection initiated with 3-4x 104 I.E. This suggested that approx. 0.1-@2% of the parasites were able to replicate after mitomycin C treatment. The parasitaemia arising from these remaining replicating parasites in the mitomycin C treated cell inoculum will hereafter be referred to as the ‘breakthrough’ infection. Babesia rodhaini
The results obtained
with the above study with
B. microti suggested that similar treatment of B. rodhaini I.E. might lead to protection and survival of mice to a normally fatal B. rodhaini infection. In
an attempt to eliminate or reduce the number of proliferating parasites remaining in the inoculum after treatment with mitomycin C, the concentration of mitomycin C used for the in vitro treatment of the infected cells was increased. Thus, mitomycin C was used to treat B. rodhaini I.E. at a concentration of 25.50 and 100 pg ml-l. The parasitaemias resulting from the injection of such treated cells are depicted in Fig. 2. Although the prepatent periods of infection were progressively delayed with increasing mitomycin C concentration used to treat the cells, a ‘breakthrough’ infection still appeared in all the animals. Extrapolation of the parasitaemia suggested that approx. 0.16, 0.05 and 0.005% of the parasites were not prevented from replicating after treatment with 25, 50 or 100 pg ml-l mitomycin C ml-l, respectively. On the other hand, increasing the mitomycin C concentration used for the in vitro of the cells resulted
I.J.P.VOL. 15. 1985
67
Immunization against Babesiu spp. in mice
D1VS
AfTtR
INfICTION
FIG. 2. Mean B. rodhoini parasitaemia -r- S.D. in mice injected with (a) 5.7 x 10’ untreated B. rodhaini1.E.. ... . ; (b) 6.2 x 10’ I.E. treated with 25 c(g ml-1 mitdmycin C ----_; (c) 5x IO7 I.E. treated with 50 pg ml-’ mitomycin C _ _ _ _ --; or (d) 5 x lo7 I.E. treated with 100 c(g ml-’ mitomycin C_._._._._. + =time of death of an animal.
a decrease in the protection conferred against this ‘breakthrough’ infection. Thus, while all the animals survived and showed reduced peak parasitaemias when injected with infected cells treated with 25 pg ml-1 mitomycin C, treatment of the I.E. with 100 pg ml-1 mitomycin C resulted in a course of infection which, although delayed, was indistinguishable from that seen in the control mice and none of the mice survived the infection. Treatment of the I.E. with 50 pg ml-1 mitomycin C resulted in an intermediate course of infection and four of the five mice survived infection. In a second experiment eight control mice were infected with 5.5~ 107 untreated B. rodhaini I.E. and 10 mice were injected with 5.7~ 107 B. rodhaini I.E. treated with 25 pg ml-1 mitomycin C. All the control mice infected with untreated B. rodhaini parasites died within 80 h of infection whereas all the 10 mice infected with the mitomycin C treated parasites survived. The maximum peak parasitaemia seen in these latter animals was 35.6% and the parasitaemia had reached undetectable levels 12 days after infection. When the number of injected erythrocytes treated with 25 pg ml-l mitomycin C was reduced lOO-fold, from 6.2 x 107 to 6.2 x 10s I.E., the prepatent period of the ‘breakthrough’ infection was delayed and, despite the small number of mitomycin C treated parasites injected, four of the five animals still survived the infection (Fig. 3). In this experiment, the group of mice which received the higher number of mitomycin C treated infected cells showed a more prolonged reticulocytosis (Fig. 4) even though the parasitaemia was less marked. The slope of each parasitaemia on a semilogarithmetic graph should be representative of the rate of multiplication of the parasites in each group of mice.
The calculated value of the slope for the control group of mice injected with untreated erythrocytes was b = 0.03 1. Values for the experimental groups of mice were close to that of the control with the exception of the two groups of mice which were injected with protective, high inocula of I.E. treated with 25 and 50 pg ml-1 mitomycin C. These two groups both showed a slope of b = 0.026. All the surviving mice were given a challenge infection with untreated B. rodhaini parasites 28 days or 3-5 months after the experimental infection and all the mice survived this challenge infection compared with previously uninfected mice which died 3-4 days after infection. The importance of metabolism by the parasites in the above study was examined by comparing B. rodhaini infection in mice injected with mitomycin C treated I.E. or heat killed I.E. Five mice each received 5 x 107 heat inactivated infected cells. No parasitaemia was observed by day 8 of the experiment. At this time the mice were infected by the intravenous injection of 2x 106 B. rodhaini I.E. obtained from the rising ‘breakthrough’ parasitaemia of mice injected with 6.2x 10’ mitomycin C (25 lg ml-l) treated I.E. The subsequent parasitaemia was high and comparable to that seen in previously uninfected, naive mice. Four of the five mice died and one survived after suffering a severe infection (Fig. 3). In another experiment mice were injected with 8 x 107 heat inactivated B. rodhaini and 12 days later infected with 5.3 x 107 normal B. rodhaini I.E. from the same liquid nitrogen stabilate. All the mice died within 4 days of the challenge infection.
DISCUSSION
vitro mitomycin C treatment at a concentration of 25 pg ml-l is known to affect cell replication without affecting protein synthesis of lymphocytes (Szybalski & Iyer, 1964; Swain, 1980). However, some replication of Babesia parasites occurred since ‘breakthrough’ infections, originating from the mitomycin C treated inocula, were always apparent. Nevertheless, significant protection against these ‘breakthrough’ infections and against subsequent challenge infection with normal parasites had been induced in the mice when mitomycin C was used at a concentration of 25 or 50 yg ml-1 to treat the B. rodhaini infected red blood cells in the inocula. The observed protection could be the result of host sensitization by the whole mitomycin C treated inoculum comprising mainly non-replicating parasites or it is possible that mitomycin C treatment selected a population of attentuated Babesia parasites with a slower multiplication rate which permitted the host time to mount a protective immune response. Two observations argue against the latter explanation. The few replicating parasites remaining after treatment of B. rodhaini with 100 pg ml-1 mitomycin C multiplied at a rate similar to that of untreated paraIn
E. MEEUSEN. S. LLOYD and
68
E. J. L.
to prevent
FIG. 3. Mean B. rodhaini parasitaemia * S.D. in mice injected on day 0 with (a) 6.2 x 10’ B. rodhaini I.E. treated with 25 c(g ml-’ mitomycin C_ ; (b) 6.2x 16 I.E. treated with 25 c(g ml-l mitomycin C________; or (c) 4x 10’ heat inactivated I?. rodhnini I.E. followed by 2~ 106 untreated I.E. given on day 8 . . . . . . . . . . + = time of death of an animal and _ _ _ _ parasitaemia in one animal after day 10.
I
II
II
DAYSLFTER
I4
I6
I8
INFECTION
FIG. 4. Mean reticulocytosis + SD. in mice injected with or (b) 6.2x105_______8. (a) 6.2 x 10’ rodhaini I.E. treated with 25 c(gml-1 mitomycin C. + = time of death of an animal whose reticulocytosis is included in the mean until this time.
sites and were equally pathogenic. Also, the parasitaemia induced in the first group of mice which had been injected with heat inactivated parasites was initiated with infected cells from the rising ‘breakthrough’ infection in mice injected with parasites treated with mitomycin C at 25 ,ug ml-t. These latter mice controlled the infection yet the same parasites when transferred to new hosts multiplied as if untreated and caused fatal infections. Thus, it seems likely that the protection against Babesia observed in these experiments was associated with the presence of the whole inoculum of mitomycin C treated parasites injected. That protein synthesis by these mitomycin C treated parasites was required is suggested by the fact that injection of a similar number of heat inactivated parasites did not induce protection against infection. Further, when a similar number of B. rodhaini were treated with mitomycin C at a concentration of 100 pg ml-t, a concentration likely
SOULSBY
1.3.~.VOL. 15. 1985
protein metabolism (Szybalski & lyer, 1964) and likely to kill the cells affected by the treatment, the protection induced by mitomycin C treatment was abolished. Therefore, it is suggested that mitomycin C, when used at optimal concentrations, permits the parasite to produce and/or present protective antigens to the host while, since replication of the majority of the parasites is prevented, the host has time to mount a protective immune response. An additional aspect is that mice injected with a large number, 6.2~ 107, mitomycin C treated B. rodhaim’ showed a prolonged reticulocytosis despite their reduced parasitaemia in comparison to mice injected with fewer, 6.2 x lo*, similarly treated parasites. It is possible that a pathological mechanism acting on the red cells is involved also. The results obtained with mitomycin C treated B. microti I.E. suggest that two mechanisms might be involved in the observed protection. In normal infections, parasitaemias due to B. microti rise rapidly to peak at around 45% erythrocytes infected followed by a rapid decline in parasitaemia. In contrast, the first phase of protection in mice injected with mitomycin C treated cells was manifest by a stable peak parasitaemia which plateaued at a low level (6Oro)for at least 4 days. This might be associated either with prevention of replication or destruction of cells at the same rate as the generation of new parasites. The second phase of protection was characterized by a sudden fall in the numbers of parasitized cells similar to that seen during a normal primary infection and often referred to as the ‘crisis’ period. Quinn & Wyler (1979) working with Plasmodium berghei in rats thought that the accelerated clearance of infected cells could be due to a sudden alteration in the microcirculation in the enlarged spleen resulting in an increased flow through the ‘open’ pathways thus allowing more contact between infected cells and macrophages. It is possible that this mechanism also applies after a primary B. microti infection and that a certain amount of parasite proliferation is required for its activation. In addition, specific or non-specific activation of macrophages and their synergistic action with antibodies in killing Bubesia parasites (Bautista & Kreier, 1980) could also accelerate the reduction of parasitaemia during the second phase of protection. The first stage of protection, represented by a stable peak parasitaemia, was not observed in infections initiated with mitomycin C treated B. rodhaini infected cells. This could be due to the more prolific growth of B. rodhaini which may result in an earlier start of the second phase of protection thus obscuring the first phase. In general, the results obtained in the mitomycin C experiments were comparable to those obtained using irradiated Babesia parasites (Phillips, 1971; Mahoney et al., 1973; Purnell et al., 1979). The in vitro mitomycin C treatment of infected erythrocytes would appear to provide a simpler and more efficient method for experimental immunization against
Immunization against Eabesia spp. in mice
I.J.P. VOL. 15. 1985
parasites. Thus, while only 50% survival of mice could be obtained by the injection of a single dose of irradiated B. rodhaini parasites, the present experiments achieved 100% survival using infected erythrocytes treated with 25 c(g ml-1 mitomycin C. The technique warrants further investigation for its use in experimental immunization against haemoprotozoan parasites.
Bubesia
work was supported by funds from the Wellcome Trust. E. Meeusen was the recipient of a British Council Fellowship. We would like to thank
Acknowledgements-This
Mr. M. Frattasi
for assistance
with the animals.
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D’ANTONIO L. E. 1972. Plasmodium berghei: vaccination of mice against malaria with heat inactivated parasitized blood. ExperimentalParasitology 31: 82-87. JERUSALEM C. & ELING W. 1969. Active immunization against Plusmodium berghei malaria in mice using
different preparations of plasmodial antigen and different pathways of administration. BuIIetin of the World Health Organization 40: 807-818. MAHONEY D. F., WRIGHT I. C. & KETTERER E. J. 1973. Bubesiu urgentina: the infectivity and immunogenicity of irradiated blood parasites for splenectomized calves. International Journalfor Parasitology 3: 209-217. PHILLIPS R. S. 1971. Immunity of rats following injection of WCo irradiated Bubesiu rodhaini infected red cells. Parasitology 60: 22 1-23 1. PURNELL R. E., LEWIS D. & BROCKLESBY D. W. 1979. Babesiu major: protection of intact calves against homologous challenge by the injection of irradiated piroplasms. International Journal for Parasitology 9: 69-71. QUINN T. C. & WYLER D. J. 1979. Intravascular clearance of parasitized erythrocytes in rodent malaria. Journal of Clinical Investigation 63: 1187-l 194. SWAIN S. L. 1980. Mitomycin C. In: Selected Methods in CeIIuIar Immunology (Edited by MISHELL B. B. & SHIIGI S. M.), pp. 240-241. Freeman, San Francisco. SZYBALSKIW. & IYER V. N. 1964. Crosslinkinp, of DNA bv enzymatically or chemically activated mitomycins or porfiromycins, bifunctionally “alkylating” antibiotics. Federation Proceedings 23: 946-955. TRIGG P. I., PHILLIPS R. S. & GUTTERIDG~ W. E. 1972. The effects of y-radiation on Plasmodium knowlesi. International Journalfor Parasitology 2: 13 l-138.