Clin. exp. Immunol. (1978) 32, 25-40.
Susceptibility of inbred strains of mice to Trypanosoma congolense: correlation with changes in spleen lymphocyte populations W. I. MORRISON, G. E. ROELANTS, KATHLEEN S. MAYOR-WITHEY & M. MURRAY The International Laboratory for Research on Animal Diseases, Nairobi, Kenya (Received 27 September 1977) SUMMARY
A comparison was made of the susceptibility of eight inbred strains of mice to infection with Trypanosoma congolense. Marked differences in susceptibility as judged by survival were found between the different strains. The capacity of certain strains to survive longer than others appeared to be related to their ability to limit the numbers of trypanosomes in the circulation. There was no difference in the infectivity of T. congolense for mice of high and low susceptibility. Furthermore, the findings of similar prepatent periods suggested that the initial replication rate was similar in the different strains. These findings suggested that the level of parasitaemia in different strains of may reflect differences in the nature or quality of the immune response to the trypanosome. In all of the strains of mice a marked increase in splenic B and null lymphocytes was found. This, allied to the finding of an increase in the background plaque-forming cells to sheep erythrocytes, indicated, as suggested by other workers, that trypanosome infection results in a non-specific polyclonal activation of lymphocytes, and that this affects primarily B lymphocytes. In strains of mice which survived longest, i.e. C57B1/6J and AKR/A, the increase in splenic B and null cells was less marked. Whether this is associated with a decreased susceptibility of these strains to polyclonal activation induced by trypanosome infection, or whether it is merely the result of lower levels of parasitaemia, remains to be determined. By comparing T. congolense infection in three strains of mice congenic at the H-2 locus, representing H-2a, H-2b and H-2k haplotypes, it was found that the susceptibility was not associated with the H-2 haplotype. The finding that (A/J x C57Bl/6J)F1 hybrids were of similar susceptibility as the C57B1/6J parents indicated that the relative resistance of this strain is inherited as a dominant trait, although in the early stages of infection the F1 hybrids consistently showed somewhat higher levels of parasitaemia than the C57B1/6J mice. Athymic nude mice and surgically splenectomized mice were found to be more susceptible to T. congolense infection than intact mice of the same strain. However, the effect of splenectomy was much less pronounced in C57B1/6J mice than in the relatively more susceptible BALB/c/A mice.
INTRODUCTION Trypanosomiasis is a major disease in Africa, where it involves man and domestic animals (Ormerod, 1970; Fiennes, 1970). The disease is caused by several species of extracellular flagellated protozoa of the genus Trypanosoma, which are transmitted by tsetse flies. Infection is characterized by the appearance of successive waves of parasitaemia, each containing one or more populations of organisms with distinct surface antigenicity (Doyle, 1977). Persistence of infection is associated with a general depression Correspondence: Dr W. I. Morrison, The International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya. 0099-9104/78/0400-0025$02.00 © 1978 Blackwell Scientific Publications
25
26
W. I. Morrison
et al.
of immune responsiveness (Goodwin et al., 1972; Freeman et al., 1973; Murray et al., 1974; Hudson et
al., 1976), splenomegaly, lymph node enlargement
and elevated levels of circulating immunoglobulins,
particularly of the IgM class (Murray, 1974; Mattern et al., 1961; Houba, Brown & Allison, 1969; Luckins, 1976). However, it would appear that much of this immunoglobulin is not directed against trypanosome antigens, and it has been postulated that trypanosomes induce a polyclonal lymphocyte activation (Urquhart et al., 1973; Greenwood, 1974). Polyclonal B- and T-lymphocyte activators have been widely studied in vitro, where they lead to both proliferation and differentiation (Janossy & Greaves, 1975; Kearney & Lawton, 1975; Askonas et al., 1976). In vivo, one might postulate that such an activation could lead to a general exhaustion of antigen-reactive cell clones, and to an ineffective immune response upon antigenic challenge. Recent experimental studies of bovine trypanosomiasis have shown that certain breeds of cattle are less susceptible to trypanosome infection than others (Murray et al., 1978); the reason for this is not known, although it is thought to have a genetic basis. The genetic control of immune responsiveness to a variety of antigens has been amply documented in the mouse and the guinea-pig, and is most often controlled by immune response (Ir) genes located in the main histocompatibility region (reviewed by Bluestein, 1977). Obviously the genetic heterogeneity of bovine populations precludes a critical analysis of the factors responsible for differences in susceptibility to trypanosomiasis. Thus the present study was initiated in order to compare the susceptibility of different inbred strains of mice to infection with T. congolense, a major pathogen of the bovine population. The main parameters chosen were duration of survival and levels of parasitaemia. They were correlated to changes in splenic B-, T- and null cell populations to assess the target and relevance of the postulated polyclonal activation. Moreover, the role of the T compartment of the immune system and of the spleen as the major reactive lymphoid organ in this disease were investigated by infecting congenitally athymic nude mice and surgically splenectomized mice. MATERIALS AND METHODS Mice. Eight inbred strains of mice were selected for this study. These were A/J, SWR/J, C3H/HeJ, DBA/1J and 129/J from the Jackson Laboratory, Bar Harbor, Maine, U.S.A., and AKR/A, C57BI/6J and BALB/c/A from GI Bomholtgard Ltd, Denmark. Congenitally athymic BALB/c nude mice were also from GI Bomholtgard Ltd. In addition, (C57B1/6 x A/J)F1 hybrids, C57Bl/lOSn and the congenic resistant strains B1O.A/SgSn and B10.BR/SgSn were obtained from the Jackson Laboratory. Only female mice were used. All mice were 10-12 weeks of age at the onset of the experiments. The characteristics of the mice selected are shown in Table 1. Groups of BALB/c/A and C57B1/6J mice were splenectomized 2 weeks prior to starting the experiments. Trypanosomes. The strain of T. congolense used in the experiments reported here in detail was kindly provided by Dr A. Njogu of the East African Trypanosomiasis Research Organization. This strain was known to produce long-standing infections in outbred mice. It was originally isolated in the Busoga region of Uganda, from a naturally infected bovine source, by sub-inoculation of blood into mice. The isolate was then passaged a further sixteen times in mice and stabilate material from infected mouse blood (EATRO 209) was prepared and stored in liquid nitrogen (Cunningham, Lumsden & Webber, 1963). Subsequently, the isolate underwent three rapid passages in outbred mice before a further stabilate was prepared, from which the mice in the present series of experiments were infected. This stabilate, T. congolence 5E-12, contained approximately 16 ID50 units per 1000 motile organisms, as determined by a serial infectivity titration carried out in mice. A further two stabilates of T. congolense were used. One of these, 5E-13, was also a derivative of EATRO 209, having undergone eight rapid passages in outbred mice. The other, ILRAD C-24, was prepared directly from the blood of a naturally infected dog which was presented with clinical illness at the University of Nairobi, Veterinary Faculty Small Animal Clinic. Measurement of parasitaemia. The onset of detectable parasitaemia was determined by daily examination of unstained wet blood films prepared from tail blood. These were examined by phase-contrast microscopy using a combination of Phaco 2 40/0 65 objective and Periplan GWx 10 eye-pieces (E. Leitz Wetzlar, Giessen, Germany). Approximately 100 fields were examined before a sample was considered negative. In order to quantify the levels of parasitaemia once organisms were detectable, trypanosome counts were performed daily on samples of tail blood. 5 of blood from each mouse was mixed with 45 I1 of a staining solution of Ziehl Neelsen-Carbol Fuchsin and the trypanosomes were counted at an appropriate dilution in a haemocytometer. Immunofluorescence staining of spleen cells. Procedures to characterize the various mouse spleen lymphocytes by virtue of their surface markers have been described in detail (Loor & Roelants, 1975; Roelants et al., 1975). B-lineage cells were defined as 0- Ig, T-lineage cells as 0 g and null cells as -1g. 0 The small population of0+ cells present in the mouse spleen was not taken into consideration in these experiments (Roelants et al., 1975).
,u
Ige
Susceptibility to Trypanosoma congolense
27
TABLE 1. The genetic characteristics of inbred strains of mice used in a study of susceptibility to infection with T. congolense*
Ig allotype Mouse strain A/J SWR/J
129/J BALB/c/A DBA/1J C3H/HeJ AKR/A C57BI/6J C57B1/lOSn B10.A/SgSn B10.BR/SgSn
H-2
Ss
a q b d q k k b b a k
h h h h h 1 I h h h I
Slp
Tlat
Mls
Igl
Ig2
a
a a c c
c
e c
Not a
o o a
a
b b b b b a
o
a
o o o o
b b
a? a?
a
c a
c a
b b b b
d b b b b
c
a? c a Not aorc Not a or c Not a or c Not a or c Not a or c
Ig3
Ig4
9 9
a a a? a
9 9? 9? 9? 9?
a a b b b? b?
* Compiled from: E. L. Green (1965) Handbook on Genetically Standardized JAX mice (ed. E. L. Green). Bar Harbor Times Publishing Co.; and Klein, J. (1975) Biology of the Mouse Histocompatibility-2 Complex. Springer-Verlag, Berlin. t Tla' expresses antigens 1, 2 and 3; Tlab, negative; Tlac expresses antigen 2.
Spleen cells were suspended by teasing with tissue combs (Dr I. Lefkovits, Basel Institute for Immunology, Switzerland) into RPMI 1640 medium (Gibco Bio-Cult, Glasgow, Scotland) supplemented with heat-inactivated 10% foetal calf serum (Gibco) 10 mMNaN3 and 0 5% albumin (Sigma, St Louis, Missouri, U.S.A.) and buffered to pH 7-2-7-4 with 0 03 M N-2hydroxyethyl-piperazine-N-2-ethane sulphonic acid (HEPES) (Flow Laboratories Ltd, Irvine, Scotland) and NaOH. Cell clumps were permitted to settle for 2 min and the suspension was passed through three layers of cotton gauze. Cells were then washed three times before treatment with the fluorescent reagents. All procedures were carried out at around 0°C. A double-labelling technique was used for fluorescence staining. In a first step, 3 x 106 cells in 0-1 ml were exposed for 30 min to 0-1 ml AKR antiserum directed against the Thy 1-2 (OC3H) alloantigen, diluted 1:20, or to normal AKR serum as a control, and subsequently washed three times. In a second step, all cells in 0-1 ml, were exposed for 30 min to 0 1 ml (50 pg) of a IgG fraction of a sheep antiserum directed against mouse Ig (polyvalent) and labelled with tetramethyl rhodamine isothiocyanate (TRITC) (BBL, Cockeysville, Maryland, U.S.A.) (Loor & Roelants, 1975). The cells were again washed three times, smeared and fixed for 5 min in absolute ethanol. The cell preparations were mounted in glycerol-barbital buffer (0.1 M; pH 8-6; 9:1, v/v), and the coverslip margins sealed with nail polish. The cells were examined using a Leitz Orthoplan microscope equipped with a HBO 100 mercury lamp and a Ploemopak 2 vertical illuminator (E. Leitz Wetzlar, Giessen, Germany). Leitz 63 or 40/1-30 Oel Phaco 3 fluoresz objectives were combined with Periplan GW x 4 eye-pieces. The frequency of 9 + Ig- cells was computed by subtracting the frequency of TRITC-positive cells found iri the control (i.e. 0 - Ig + cells) from the total frequency of TRITC-positive cells. Cells which were dead before fixation were easily recognizable by their morphology, using phase-contrast microscopy, and by the diffuse uptake of fluorescent reagents. Only cells which were viable before fixation were taken into account. The total number of cells per spleen and the population of viable cells were estimated by eosin exclusion on cells in suspension before treatment with the fluorescent reagents. The results were expressed as total number of viable cells of each type per spleen. The mean value from three individual mice was used for each point. Main experimental design. To compare susceptibility of mice to T. congolense infection, sixty mice ofeach of the eight strains were inoculated intraperitoneally with 5 x 103 motile trypanosomes (T. congolense 5E-12), equivalent to 80 ID50 units. A further ten mice of each strain were maintained as uninfected controls. Of the sixty infected mice, thirty were killed at intervals for collection of serum and spleen cells. To determine the duration of survival, the remaining thirty of each strain were allowed to run the natural course of infection until death occurred. Twelve of these animals were monitored daily for changes in parasitaemia until day 26 of the experiment, by which time all surviving mice had undergone three recurrent waves of parasitaemia. On the basis of the above experiment, F1 hybrids derived from the most resistant (C57BI/6J) and the most susceptible (A/J) strains were infected and their susceptibility compared with the parental strains. Fifty splenectomized BALB/c/A and fifty splenectomized C57B1/6J mice, along with fifty athymic BALB/c nude mice, were treated in the same way and compared with intact mice of the same strain. In order to assess the influence of H-2 and Tia haplotypes on the susceptibility, groups of thirty-five C57Bl/lOSn (H-2b, Tlab) and congenic strains B1O.A/SgSn (H-2a, Tlaa) and B10.BR/SgSn (H-2k, Tlaa) were infected with T. congolense and
W. I. Morrison et af.
28
monitored for parasitaemia and survival. Preliminary studies had indicated that C57Bl/10Sn were of similar susceptibility as C57B1/6J mice. To determine if the relative susceptibility of the different strains of mice was similar using other isolates of T. congolense, mice of each of the eight inbred strains were infected with two other T. congolense isolates (5E-13 and ILRAD C-24), using similar infective doses, and survival and parasitaemia followed as above. In addition, a group of BALB/c nude mice was infected with T. congolense 5E-13.
RESULTS Susceptibility of inbred strains of Mice The mean survival time for each strain and the percentage of deaths at intervals throughout the experiment are shown in Table 2. Marked differences in survival time following inoculation with T. congolense were found between the different strains of mice. In certain strains, such as A/J and SWR/J, most animals died within 30 days following inoculation while in others, such as AKR/A and C57BI/6J, the majority of animals were still alive by day 60. The remaining strains showed various intermediate patterns of survival. TABLE 2. Duration of survival in inbred strains of mice infected with T. congolenlse
Percentage deaths by days after infection: Mouse Strain
15
20
67 8 85 7 42 0 88 0 36-6 58-5 129/J 15-5 20-0 BALB/c/A 0 0 DBA/1J 0 0 C3H/HeJ 0 0 AKR/A 0 0 C57B1/6J 0 0 (B6A)F1 BALB/c (nu/nu) 33 3 76 2 BALB/c/A (splenectomized) 44 4 96-3
A/J SWR/J
C57B1/6J (splenectomized)
0
0
40
100 100 90-2 28 8 62-2 12 5 79 28 0 100
60
100 64 4 100 50 0 13 6 55 28
80
120
140
100
100 454 977 13 9 611 8-3 30 6
100 944 63-9
15-8 16-9 22-6 49 5 36-3 59 0 81 7 1102 133-2 18 0 16 0
100 11 1
Mean survix al time (days)
17 8
35 7 75-0
89 3
93-4
In all strains except A/J and C3H/HeJ the mean prepatent period, i.e. the time from inoculation until first detectable parasitaemia, was between 6-5 and 7 1 days. In A/J mice the prepatent period was 5-8 days, which was significantly shorter (P
Susceptibility of inbred strains of mice to Trypanosoma congolense: correlation with changes in spleen lymphocyte populations W. I. MORRISON, G. E. ROELANTS, KATHLEEN S. MAYOR-WITHEY & M. MURRAY The International Laboratory for Research on Animal Diseases, Nairobi, Kenya (Received 27 September 1977) SUMMARY
A comparison was made of the susceptibility of eight inbred strains of mice to infection with Trypanosoma congolense. Marked differences in susceptibility as judged by survival were found between the different strains. The capacity of certain strains to survive longer than others appeared to be related to their ability to limit the numbers of trypanosomes in the circulation. There was no difference in the infectivity of T. congolense for mice of high and low susceptibility. Furthermore, the findings of similar prepatent periods suggested that the initial replication rate was similar in the different strains. These findings suggested that the level of parasitaemia in different strains of may reflect differences in the nature or quality of the immune response to the trypanosome. In all of the strains of mice a marked increase in splenic B and null lymphocytes was found. This, allied to the finding of an increase in the background plaque-forming cells to sheep erythrocytes, indicated, as suggested by other workers, that trypanosome infection results in a non-specific polyclonal activation of lymphocytes, and that this affects primarily B lymphocytes. In strains of mice which survived longest, i.e. C57B1/6J and AKR/A, the increase in splenic B and null cells was less marked. Whether this is associated with a decreased susceptibility of these strains to polyclonal activation induced by trypanosome infection, or whether it is merely the result of lower levels of parasitaemia, remains to be determined. By comparing T. congolense infection in three strains of mice congenic at the H-2 locus, representing H-2a, H-2b and H-2k haplotypes, it was found that the susceptibility was not associated with the H-2 haplotype. The finding that (A/J x C57Bl/6J)F1 hybrids were of similar susceptibility as the C57B1/6J parents indicated that the relative resistance of this strain is inherited as a dominant trait, although in the early stages of infection the F1 hybrids consistently showed somewhat higher levels of parasitaemia than the C57B1/6J mice. Athymic nude mice and surgically splenectomized mice were found to be more susceptible to T. congolense infection than intact mice of the same strain. However, the effect of splenectomy was much less pronounced in C57B1/6J mice than in the relatively more susceptible BALB/c/A mice.
INTRODUCTION Trypanosomiasis is a major disease in Africa, where it involves man and domestic animals (Ormerod, 1970; Fiennes, 1970). The disease is caused by several species of extracellular flagellated protozoa of the genus Trypanosoma, which are transmitted by tsetse flies. Infection is characterized by the appearance of successive waves of parasitaemia, each containing one or more populations of organisms with distinct surface antigenicity (Doyle, 1977). Persistence of infection is associated with a general depression Correspondence: Dr W. I. Morrison, The International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya. 0099-9104/78/0400-0025$02.00 © 1978 Blackwell Scientific Publications
25
26
W. I. Morrison
et al.
of immune responsiveness (Goodwin et al., 1972; Freeman et al., 1973; Murray et al., 1974; Hudson et
al., 1976), splenomegaly, lymph node enlargement
and elevated levels of circulating immunoglobulins,
particularly of the IgM class (Murray, 1974; Mattern et al., 1961; Houba, Brown & Allison, 1969; Luckins, 1976). However, it would appear that much of this immunoglobulin is not directed against trypanosome antigens, and it has been postulated that trypanosomes induce a polyclonal lymphocyte activation (Urquhart et al., 1973; Greenwood, 1974). Polyclonal B- and T-lymphocyte activators have been widely studied in vitro, where they lead to both proliferation and differentiation (Janossy & Greaves, 1975; Kearney & Lawton, 1975; Askonas et al., 1976). In vivo, one might postulate that such an activation could lead to a general exhaustion of antigen-reactive cell clones, and to an ineffective immune response upon antigenic challenge. Recent experimental studies of bovine trypanosomiasis have shown that certain breeds of cattle are less susceptible to trypanosome infection than others (Murray et al., 1978); the reason for this is not known, although it is thought to have a genetic basis. The genetic control of immune responsiveness to a variety of antigens has been amply documented in the mouse and the guinea-pig, and is most often controlled by immune response (Ir) genes located in the main histocompatibility region (reviewed by Bluestein, 1977). Obviously the genetic heterogeneity of bovine populations precludes a critical analysis of the factors responsible for differences in susceptibility to trypanosomiasis. Thus the present study was initiated in order to compare the susceptibility of different inbred strains of mice to infection with T. congolense, a major pathogen of the bovine population. The main parameters chosen were duration of survival and levels of parasitaemia. They were correlated to changes in splenic B-, T- and null cell populations to assess the target and relevance of the postulated polyclonal activation. Moreover, the role of the T compartment of the immune system and of the spleen as the major reactive lymphoid organ in this disease were investigated by infecting congenitally athymic nude mice and surgically splenectomized mice. MATERIALS AND METHODS Mice. Eight inbred strains of mice were selected for this study. These were A/J, SWR/J, C3H/HeJ, DBA/1J and 129/J from the Jackson Laboratory, Bar Harbor, Maine, U.S.A., and AKR/A, C57BI/6J and BALB/c/A from GI Bomholtgard Ltd, Denmark. Congenitally athymic BALB/c nude mice were also from GI Bomholtgard Ltd. In addition, (C57B1/6 x A/J)F1 hybrids, C57Bl/lOSn and the congenic resistant strains B1O.A/SgSn and B10.BR/SgSn were obtained from the Jackson Laboratory. Only female mice were used. All mice were 10-12 weeks of age at the onset of the experiments. The characteristics of the mice selected are shown in Table 1. Groups of BALB/c/A and C57B1/6J mice were splenectomized 2 weeks prior to starting the experiments. Trypanosomes. The strain of T. congolense used in the experiments reported here in detail was kindly provided by Dr A. Njogu of the East African Trypanosomiasis Research Organization. This strain was known to produce long-standing infections in outbred mice. It was originally isolated in the Busoga region of Uganda, from a naturally infected bovine source, by sub-inoculation of blood into mice. The isolate was then passaged a further sixteen times in mice and stabilate material from infected mouse blood (EATRO 209) was prepared and stored in liquid nitrogen (Cunningham, Lumsden & Webber, 1963). Subsequently, the isolate underwent three rapid passages in outbred mice before a further stabilate was prepared, from which the mice in the present series of experiments were infected. This stabilate, T. congolence 5E-12, contained approximately 16 ID50 units per 1000 motile organisms, as determined by a serial infectivity titration carried out in mice. A further two stabilates of T. congolense were used. One of these, 5E-13, was also a derivative of EATRO 209, having undergone eight rapid passages in outbred mice. The other, ILRAD C-24, was prepared directly from the blood of a naturally infected dog which was presented with clinical illness at the University of Nairobi, Veterinary Faculty Small Animal Clinic. Measurement of parasitaemia. The onset of detectable parasitaemia was determined by daily examination of unstained wet blood films prepared from tail blood. These were examined by phase-contrast microscopy using a combination of Phaco 2 40/0 65 objective and Periplan GWx 10 eye-pieces (E. Leitz Wetzlar, Giessen, Germany). Approximately 100 fields were examined before a sample was considered negative. In order to quantify the levels of parasitaemia once organisms were detectable, trypanosome counts were performed daily on samples of tail blood. 5 of blood from each mouse was mixed with 45 I1 of a staining solution of Ziehl Neelsen-Carbol Fuchsin and the trypanosomes were counted at an appropriate dilution in a haemocytometer. Immunofluorescence staining of spleen cells. Procedures to characterize the various mouse spleen lymphocytes by virtue of their surface markers have been described in detail (Loor & Roelants, 1975; Roelants et al., 1975). B-lineage cells were defined as 0- Ig, T-lineage cells as 0 g and null cells as -1g. 0 The small population of0+ cells present in the mouse spleen was not taken into consideration in these experiments (Roelants et al., 1975).
,u
Ige
Susceptibility to Trypanosoma congolense
27
TABLE 1. The genetic characteristics of inbred strains of mice used in a study of susceptibility to infection with T. congolense*
Ig allotype Mouse strain A/J SWR/J
129/J BALB/c/A DBA/1J C3H/HeJ AKR/A C57BI/6J C57B1/lOSn B10.A/SgSn B10.BR/SgSn
H-2
Ss
a q b d q k k b b a k
h h h h h 1 I h h h I
Slp
Tlat
Mls
Igl
Ig2
a
a a c c
c
e c
Not a
o o a
a
b b b b b a
o
a
o o o o
b b
a? a?
a
c a
c a
b b b b
d b b b b
c
a? c a Not aorc Not a or c Not a or c Not a or c Not a or c
Ig3
Ig4
9 9
a a a? a
9 9? 9? 9? 9?
a a b b b? b?
* Compiled from: E. L. Green (1965) Handbook on Genetically Standardized JAX mice (ed. E. L. Green). Bar Harbor Times Publishing Co.; and Klein, J. (1975) Biology of the Mouse Histocompatibility-2 Complex. Springer-Verlag, Berlin. t Tla' expresses antigens 1, 2 and 3; Tlab, negative; Tlac expresses antigen 2.
Spleen cells were suspended by teasing with tissue combs (Dr I. Lefkovits, Basel Institute for Immunology, Switzerland) into RPMI 1640 medium (Gibco Bio-Cult, Glasgow, Scotland) supplemented with heat-inactivated 10% foetal calf serum (Gibco) 10 mMNaN3 and 0 5% albumin (Sigma, St Louis, Missouri, U.S.A.) and buffered to pH 7-2-7-4 with 0 03 M N-2hydroxyethyl-piperazine-N-2-ethane sulphonic acid (HEPES) (Flow Laboratories Ltd, Irvine, Scotland) and NaOH. Cell clumps were permitted to settle for 2 min and the suspension was passed through three layers of cotton gauze. Cells were then washed three times before treatment with the fluorescent reagents. All procedures were carried out at around 0°C. A double-labelling technique was used for fluorescence staining. In a first step, 3 x 106 cells in 0-1 ml were exposed for 30 min to 0-1 ml AKR antiserum directed against the Thy 1-2 (OC3H) alloantigen, diluted 1:20, or to normal AKR serum as a control, and subsequently washed three times. In a second step, all cells in 0-1 ml, were exposed for 30 min to 0 1 ml (50 pg) of a IgG fraction of a sheep antiserum directed against mouse Ig (polyvalent) and labelled with tetramethyl rhodamine isothiocyanate (TRITC) (BBL, Cockeysville, Maryland, U.S.A.) (Loor & Roelants, 1975). The cells were again washed three times, smeared and fixed for 5 min in absolute ethanol. The cell preparations were mounted in glycerol-barbital buffer (0.1 M; pH 8-6; 9:1, v/v), and the coverslip margins sealed with nail polish. The cells were examined using a Leitz Orthoplan microscope equipped with a HBO 100 mercury lamp and a Ploemopak 2 vertical illuminator (E. Leitz Wetzlar, Giessen, Germany). Leitz 63 or 40/1-30 Oel Phaco 3 fluoresz objectives were combined with Periplan GW x 4 eye-pieces. The frequency of 9 + Ig- cells was computed by subtracting the frequency of TRITC-positive cells found iri the control (i.e. 0 - Ig + cells) from the total frequency of TRITC-positive cells. Cells which were dead before fixation were easily recognizable by their morphology, using phase-contrast microscopy, and by the diffuse uptake of fluorescent reagents. Only cells which were viable before fixation were taken into account. The total number of cells per spleen and the population of viable cells were estimated by eosin exclusion on cells in suspension before treatment with the fluorescent reagents. The results were expressed as total number of viable cells of each type per spleen. The mean value from three individual mice was used for each point. Main experimental design. To compare susceptibility of mice to T. congolense infection, sixty mice ofeach of the eight strains were inoculated intraperitoneally with 5 x 103 motile trypanosomes (T. congolense 5E-12), equivalent to 80 ID50 units. A further ten mice of each strain were maintained as uninfected controls. Of the sixty infected mice, thirty were killed at intervals for collection of serum and spleen cells. To determine the duration of survival, the remaining thirty of each strain were allowed to run the natural course of infection until death occurred. Twelve of these animals were monitored daily for changes in parasitaemia until day 26 of the experiment, by which time all surviving mice had undergone three recurrent waves of parasitaemia. On the basis of the above experiment, F1 hybrids derived from the most resistant (C57BI/6J) and the most susceptible (A/J) strains were infected and their susceptibility compared with the parental strains. Fifty splenectomized BALB/c/A and fifty splenectomized C57B1/6J mice, along with fifty athymic BALB/c nude mice, were treated in the same way and compared with intact mice of the same strain. In order to assess the influence of H-2 and Tia haplotypes on the susceptibility, groups of thirty-five C57Bl/lOSn (H-2b, Tlab) and congenic strains B1O.A/SgSn (H-2a, Tlaa) and B10.BR/SgSn (H-2k, Tlaa) were infected with T. congolense and
W. I. Morrison et af.
28
monitored for parasitaemia and survival. Preliminary studies had indicated that C57Bl/10Sn were of similar susceptibility as C57B1/6J mice. To determine if the relative susceptibility of the different strains of mice was similar using other isolates of T. congolense, mice of each of the eight inbred strains were infected with two other T. congolense isolates (5E-13 and ILRAD C-24), using similar infective doses, and survival and parasitaemia followed as above. In addition, a group of BALB/c nude mice was infected with T. congolense 5E-13.
RESULTS Susceptibility of inbred strains of Mice The mean survival time for each strain and the percentage of deaths at intervals throughout the experiment are shown in Table 2. Marked differences in survival time following inoculation with T. congolense were found between the different strains of mice. In certain strains, such as A/J and SWR/J, most animals died within 30 days following inoculation while in others, such as AKR/A and C57BI/6J, the majority of animals were still alive by day 60. The remaining strains showed various intermediate patterns of survival. TABLE 2. Duration of survival in inbred strains of mice infected with T. congolenlse
Percentage deaths by days after infection: Mouse Strain
15
20
67 8 85 7 42 0 88 0 36-6 58-5 129/J 15-5 20-0 BALB/c/A 0 0 DBA/1J 0 0 C3H/HeJ 0 0 AKR/A 0 0 C57B1/6J 0 0 (B6A)F1 BALB/c (nu/nu) 33 3 76 2 BALB/c/A (splenectomized) 44 4 96-3
A/J SWR/J
C57B1/6J (splenectomized)
0
0
40
100 100 90-2 28 8 62-2 12 5 79 28 0 100
60
100 64 4 100 50 0 13 6 55 28
80
120
140
100
100 454 977 13 9 611 8-3 30 6
100 944 63-9
15-8 16-9 22-6 49 5 36-3 59 0 81 7 1102 133-2 18 0 16 0
100 11 1
Mean survix al time (days)
17 8
35 7 75-0
89 3
93-4
In all strains except A/J and C3H/HeJ the mean prepatent period, i.e. the time from inoculation until first detectable parasitaemia, was between 6-5 and 7 1 days. In A/J mice the prepatent period was 5-8 days, which was significantly shorter (P
