Zeitschrift für
Z. Parasitenk. 54, 209-216 (1977)
Parasitenkunde Parasitologic Research
@ by Springer-Verlag 1977
Immunization of NMRI Mice Against Virulent Toxoplasma gondii. Differing Efficacy of Eleven Cyst-Forming Toxoplasma Strains K.N. Masihi and H. Werner Robert Koch-Institute,Departmentof Medical Parasitology,Nordufer20, D-1000 Berlin 65
Summary. Mice were infected with eleven cyst-forming Toxoplasma strains of varying virulence and challenged 1 month later with highly virulent BK strain parasites. The early cellular reaction in vivo was estimated by collecting the peritoneal exudates 24 h after challenge. This consisted of 54.34-77.61% lymphocytes and 0-8.88% infected macrophages in the eleven immunized groups in contrast to 27.17% lymphocytes and 18.64% infected macrophages in the control group. The peritoneal exudate 72 h after chaUenge comprised of 55.88-73.86% lymphocytes and 0-6.97% infected macrophages compared to 25.88% lymphocytes and 92% infected macrophages in the control group. Following the virulent challenge, deaths occurred in seven of the eleven groups immunized with live strains and ranged 5-42% at the end of a 6-week observation period. The most virulent of the Toxoplasma strains used - Alt and Gail - gave higher mortality while the least virulent o n e s - 558, 1070, K8 and KSU - were solidly resistant. The significantly different mortality obtained with strains Alt, Gail and Witting in a similar experiment performed at an interval of 7 months is discussed. Acquisition of immunity to Toxoplasma usually follows recovery from a natural infection and is ascribed to a premunition phenomenon. Cysts containing slow proliferating parasites have been found in various tissues of infected hosts and may persist even when antibody is no longer measurable (Werner and Pichl, 1969). Epidemiological studies based on detection of humoral antibodies by conventional serology or cell-mediated immunity by skin tests have shown human infections to be widespread. However, the incidence of positivity has been found to vary according to geographical location. A signißcant proportion of women in child-bearing age may be negative and thus carry higher risks of congenital transmission. Moreover, reactivated Toxoplasma-infectäon is being encountered more frequently in immunosuppressed patients (Ruskin and Remington, 1976). In view of the problems associated with early detection and chemotherapy, prophylactic measures against Toxoplasmosis assume increased importance. Experimental infections of laboratory animals have shown that the intrauterine transmission of Toxoplasma to foetus is likely to be dependent on various factors inc!uding the host species and the strain of parasite. Diaplacental transmission to the foetus was observed with six of the eleven Toxoplasma strains in latent infected mice
210
K.N. Masihi and H.Werner
( W e r n e r et al., 1977) but similar infection o f rabbits b e f o r e m a t i n g was successful in p r e v e n t i n g c o n g e n i t a l infection o f the foetus ( J a n i t s c h k e and Jörren, 1970; W e r n e r et al., 1977). T h e latter has clinical correlates in p r o s p e c t i v e studies of p r e g n a n t w o m e n ( D e s m o n t s and C o u v r e u r , 1974; Berger and Piekarski, 1975). N o cases o f affected infants w e r e o b s e r v e d w h e n m a t e r n a l infection h a d o c c u r r e d prior to c o n c e p t i o n . This a p p r o a c h has been used to p r e v e n t o v i n e a b o r t i o n s w h i c h are a cause o f c o n s i d e r a b l e e c o n o m i c loss in sheep rearing a r e a s ( W a t s o n and Beverley, 1971). A l t h o u g h at p r e s e n t it is h a z a r d o u s to a t t e m p t v a c c i n a t i o n , any future feasibility of applying specific i m m u n o p r o p h y l a x i s to h u m a n s or cats w o u l d depend, a m o n g s t o t h e r i m p o r t a n t c o n s i d e r a t i o n s , on suitable Toxoplasma o r g a n i s m s . M u c h requires to be k n o w n a b o u t parasite strain variability and its effect o n p r o t e c t i o n against s u b s e q u e n t challenge. T h e present p a p e r deals with a c o m p a r i s o n o f eleven different strains in mice,
Materials and Methods Animals. Six-week-old female NMRI mice weighing 20-25 g were obtained from the SPF-colony maintained by the Bundesgesundheitsamt. Ten animals each were kept in Makrolon Type II cages and housed in an air-conditioned room at 22 ° C and 40% humidity. They were red with Herilan Hann ® standard pellets and water ad libitum. Toxoplasma Strains. Eleven strains isolated originally from different sources and maintained by regular passage were used. Basic data on these is given in Table 1. Strain 1070 had been isolated from a human case just 1 month before the present experiments were started.
Table 1. Basic data on strains used for immunization Toxoplasma strain 248 558 1070 K8 KSU Mo S162 $930 Alt Gail Witting a
Isolated from
Maintained since a
Human
6 years
Human Human Rabbit Cat Human Pig Pig Human Human Human
5 years 1 month 12 years 4 years 1 year 8 years 8 years 16 years 4 years 14 years
Maintained in Mastomys natalensis M. natalensis NMRI mice M. natalensis M. nataIensis M. natalensis M. natalensis M. natalensis NMRI mice NMRI mice NMRI mice
Last passage before (months)
Cyst index/ML
9
1,200
6 1 6 10 9 10 10 6 6 6
280 80 160 360 160 200 240 200 320 240
In this laboratory
Preparation of Cyst Suspensions and Primary Infection. Brains from chronically infected Mastomys natalensis or NMRI mice were triturated with glass beads in phosphate buffered saline, pH 7.2. Hornogenate from two or three brains was adjusted to contain 10 cysts/0.3 ml. Animals in groups of 25 were injected with this dose by intraperitoneal route. To increase the total number of survivors in groups injected with more virulent strains, 1 week after infection 20 animals from each group received i.p. a single dose of 2 mg Sulfamethoxypyrazine + 0.02 mg Pyrimethamine per mouse (Werner et al., 1972). Initial mortality after primary infection was observed in remaining untreated mice.
Immunization of Mice Against T. gondii
211
Preparation of Toxoplasma Suspension for Challenge. Virulent BK strain was employed. Parasites were harvestd by washing out the peritoneal cavity with 2 ml of phosphate buffered saline on the 3rd day ofinfection. The suspension was adjusted to contain 3 x 103 Toxoplasma per 0.3 rel.
Cellular Reaction in vivo. Peritoneal cavities of 2-3 mice were rinsed with 1 ml of phosphate buffered saline each and the exudates pooled. The cell deposit after centrifugation was stained with Giemsa. Serology. Individual tail blood from 3 mice in each group was collected in the 4th week after infection before the challenge. Antibody titers were measured by Sabin-Feldman dye test. Enhanced Virulenee Due to Infectious Agents. To rule out the observed change (increase) in virulence of strains Alt, Gail and Witting (Table 2) by association with infectious agents (Berger and Schoop, 1967) these strains were injected into quails. Four weeks after infection, re isolation was achieved by inoculating quail brains into mice. The virulenee had not changed. Table 2. Initial mortality of mice infected with various Toxoplasma strainsa
Toxoplasma strain
Cumulative percent mortality Untreated group No. weeks 1 2
248 558 1070 K8 KSU Mo S162 $930 Alt Gail Witting
0 0 0 0 0 0 0 0 20 (0) c 20 (0) 0 (0)
40 20 0 0 0 0 0 60 100 (70) 80 (28) 60 (36)
Treated group b No. weeks 1 2 3
3
4
40 20 0 0 0 0 0 60
40 20 0 0 0 0 0 60
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 5 70
5 0 0 0 0 0 0 10 70
10 0 0 0 0 10 5 10 70
100
0
40
40
40
60
0
15
15
15
(78) 80 (28) 60 (42)
a Each mouse was injected with 10 brain cysts h Chemotherapy adrninistered i.p. on day 7 post-infection (2 mg Sulfamethoxypyrazine + 0.02 mg Pyrimethamine per mouse) « Figures in parenthesis show percent mortality before 7 months The SFT titers are given in Table 4 To test further for possible contamination with pathogenic viruses, the cyst-containing brain suspension was frozen and then immediately thawed again 10 times to kill the cystozoites. No infectious agents could be shown after injecting this suspension in white mice. Finally, the brain suspension was centrifuged for 3 h at 10,000 g in a refrigerated centrifuge and the supernatant injected in mice. The animals remained healthy. These foregoing experiments demonstrated that out Toxoplasma strains were free from infectious agents whieh could not thus be responsible for increased mortafity of laboratory animals simulating an enhanced virulence.
Results
T h e relative p a t h o g e n i c i t y o f eleven Toxoplasma s t r a i n s as d e t e r m i n e d b y initial m o r t a l i t y for m i c e are p r e s e n t e d in T a b l e 2. Alt a n d Gail w e r e the m o s t virulent while
212
K.N. Masihi and H.Werner
1070, K8 and KSU were the least virulent of the strains used. Four weeks after infection, animals were challenged intraperitoneally with 3 × 103 virulent BK Toxoplasma. This dose gives a mean survival time of 7.75 + 0.50 days in normal mice.
Early Cellular Reaction in vivo After Challenge. Exudates coUected at 24 and 72 h after challenge showed a marked difference between the cellular constitutions of the immunized and not immunized animals as is shown in Table 3. In the former, eight groups showed a decrease and two showed slight increases (0.51 and 0.56) in percent of infected macrophages which ranged from 0--8.88% at 24 h and 0-6.97% at 72 h.
Table 3. Early cellular reaction in vivo after challenge (%)
Toxoplasma
Time after challenge
strain 24 h Lymphocytes present
Macrophages infected
72 h L y m p h o c y t e s Macrophages present infected
248 558 1070 K8 KSU Mo S162 $930 Alt Gail Witting
54.34 61.31 70.22 77.61 75.10 68.44 70.48 76.52 60.98 57.64 64.12
3.33 1.78 4.40 5.80 0.00 6.25 8.88 4.50 3.44 7.14 1.60
63.20 63.05 65.50 69.50 71.05 73.86 62.73 68.12 68.20 55.88 67.63
3.84 0.00 1.01 3.22 0.00 4.25 6.97 2.12 4.00 4.44 0.72
Controls
27.17
18.64
25.86
92.00
Values for 24 and 72 h each were obtained from pooled exudates of 2 to 3 mice
In contrast, the control group showed an increase from 18.64% infected macrophages at 24 h to 92% at 72 h. In this group the challenge inoculum had increased in peritoneal cavity to a mean of 1.2 x 106 free parasites per mouse on the 3rd day. No free Toxoplasma were observed in the others except strain 1070 which had a mean of 4.3 x 104 organisms. The number of lymphocytes ranged from 5476% in the immunized group while in controls it was only 25-27%.
Resistance oflmmunized Animals to Challenge. Daily deaths were recorded for a period of 6 weeks after virulent BK challenge. The data on these and the SFT titer ranges a few days before challenge are presented in Table 4. Strains Alt, Gail and Witting had been previously used 7 months before in a similar experiment. Results of that and the present experiment are shown graphically in Figures 1 and 2. Of the eleven Toxoplasma strains used, deaths were registered in seven groups and ranged from 5-42% at 6 weeks post-challenge. All strains showing initial mortality following drug administration 1 week after infection also gave incomplete protection. The most virulent strains used, Alt and Gail, had higher mortality rates, the least virulent ones gave solid resistance on challenge.
Immunization of Mice Against T. gondii
213
Table 4. Results of virulent BK strain challenge of mice immunized with eleven cyst-forming Toxoplasma strains
Toxoplasma strain
SFT range
Total challenged
Cumulative percent dead weeks post-challenge 1
248 558 1070 K8 KSU Mo $162 $930 Alt Gail Witting
1,000-4,000 1,000-4,000 1,000-4,000 4,000 4,000 4,000 1,000-16,000 1,000 1,000--4,000 (4,000)" 1,000-4,000 (1,000-4,000) 256-1,000 (16,000
l7 20 18 20 20 19 20 17 14 (9) 19 (15) 15 (12)
Controls
20
2
3
4
5
6
5.88 5.88 5.88 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10.52 10.52 10.52 0 0 5.26 5.00 5.00 5.00 0 0 0 5.88 5.88 5.88 0 5.88 5.88 35.71 42.85 7.14 7.14 35.71 35.71 (o) (11.11) (11.11) (0) (0) (0) 0 10.52 31.57 31.57 31.57 31.57 (13.33) (26.66) (26.66) (33.33) (0) (0) 0 0 0 6.66 0 0 (50) (50) (8.33) (33.33) (41.66) (50) 90 (95)
100 b (100) b
a Figures in parenthesis show results before 7 months u On day 8 post-challenge
o ALT
100
o
80"
Z
»
"
~ GAI L
> rr
60. taJ
Z UJ
[3WITTING 40"
n-" Lid
n
20"
L~IICON TROL !
!
NUMBER 0E DAYS AFTER CHALLANGE
Fig. 1. Animals were infected with strains Alt (O), Gail (Q), Witting ([3) or saline (mm)and chaUenged 30 days later with virulent BK Toxoplasma. This experiment was performed 7 months before
214
K.N. Masihi and H.Werner
100
aWlTTING
0
z 80" » »
a GAIL
nr"
Sb
oALT
60-
LLI
40LLI r,d, LId
o_ 20-
~ICONTROL I
0
5
I
I
l
I
10 15 20 25 N!JNBER OF DAYS AFTER CHALLANGE
.
30
Fig. 2. Animals were infectedwith strains Alt (O), Gail (O), Witting (tq) or saline (1) and challenged 30 days later with virulentBK Toxoplasma
Discussion
The present study shows that prior immunization of the host with a live Toxoplasma strain does not always result in complete protection against subsequent challenge with a high virulent strain. Different strains vary vastly in their behaviour and relative pathogenicity for a particular host. Immunization with more virulent strains proved to be less efficient while ones with similar initial mortality for mice gave differing levels of protection. The observations with the strains Alt, Gail and Witting are of particular interest as they had been utilized 7 months before without a chemotherapy in a preliminary experiment. At that time strain Alt gave none, Gail 28% and Witting 50% mortality 4 weeks after 3 × 103 BK challenge. In this previous study, Gail strain was comparable to Beverley strain in that the initial mortality by both was up to 30% 2 weeks after inoculation of 10-15 brain cysts (Reikvam et al., 1975) but unlike former, the latter one was resistant to virulent challenge. Between then and the time when present experiments were performed, the initial virulence of these three strains had undergone an increase and completely different results were obtained. Alt gave 35.71%, Gail 31.57%, and Witting 0% mortality 1 month after similar BK challenge. Six-week-old NMRI mice maintained under same conditions were used for both experiments. These results suggest that factors other than virulence may be operative in immunization with live strains and at present remain to be defined. The respective roles of both humoral and cell-mediated immunity in protection against this parasite has received much attention in recent years. Specific antibody has been found to be effective against extracellular organisms which are lysed in the presence of an "accessory factor" by activating the properdin-dependent pathway of complement system and in rendering the parasites susceptible to intracellular destruction by macrophages (Anderson et al., 1976; Jones et al., 1975; Sethi et al.,
Immunization of Mice Against T. gondii
215
1975; Stadtsbaeder et al., 1975). Multiple transfer of immune serum 48 hours prior to challenge with LDT0 of cyst-forming Alt strain conferred significant resistance to normal recipient mice as assessed by a 55% reduction in the number of brain cysts and almost 90% survival rate (Masihi and Werner, in press). In addition, alleviation of established infection could be attained by an immunotherapy with a high fiter immune serum (Werner et al., 1977). However, Toxoplasma is an obligate intracellular parasite and studies with naturally and experimentally infected rabbits suggested an important role for cell-mediated immunity (Huldt, 1966). This was further emphasized by the demonstration of adoptive transfer of immunity with spleen and lymph node cells (Frenkel, 1967). Prior treatment of mice with either antilymphocyte (Nakayama and Aoki, 1970) or antithymocyte sera (Strannegard and Lycke, 1972) also greatly reduced the survival times following challenge. The evidence from these studies suggests that both humoral and cell-mediated immune responses are important in controlling Toxoplasma infections. Cell-mediated immunity to an intracellular infection involves interaction between specifically sensitized lymphocytes and macrophages (Mackaness, 1969). In the present study, early peritoneal exudates from immunized groups after challenge comprised of 54-77% lymphocytes, in comparison to 25-27% in controls. It is probable that these large numbers of lymphocytes in immunized mice included sensitized cells capable of specifically activating the macrophages to destroy intracellular parasites. The bulk of the lymphocytes in these animals were of the small type and are in concurrence with previous observations (Pelster, 1975). Vastly reduced numbers of infected macrophages in immunized animals was in marked contrast to controls which had 92% of such cells. This confirms and extends the earlier results (Pelster, 1975; Stadtsbaeder et al., 1975). Toxoplasma immune mice possess macrophages with enhanced microbicidal capacity (Remington et al., 1972) and infected cells in these animals may show a prolongation of average generation time of parasites (Jones et al., 1975). High titers of circulating antibody present at the time of challenge may have also helped in intracellular killing of antibody-coated parasites by normal macrophages. The host may show different levels of protection after in vivo challenge in spite of the presence of macrophages capable of being activated to kill Toxoplasma in vitro (Swartzberg et al., 1975). Differences in numbers of parasitized macrophages and mortality suggest that some variation in the degree of macrophage activation by different strains may exist. Conflicting reported results of attempts to confer specific anti-Toxoplasma activity on normal macrophages, by immune lymphocytes and antigen or products of such an interaction (Jones et al., 1975; Sethi et al., 1975; Shirahata et al., 1976), may be reflective of differences in strains used for respective immunizations.
References
Anderson, S.E., Bautista, S.C., Remington, J.S.: Specific antibody-dependentkilling of Toxoplasma gondiiby normal macrophages.Clin. exp. Immunol.26, 375-380 ( 1976) Berger, J., Piekarski, G.: Die Bedeutung der Toxoplasma-Infektion für Schwangerschaftsverlaufund Kindesentwicklung. Ergebnisse einer prospektiven Studie. Geburtsh. u. Frauenheilk. 35, 89-97 (1975) Berger, J., Schoop, G.: Über ein an den Toxoplasma-StammBK (Winser van Thiel 1948) gekoppeltes, infektiöses Agens. Z. Med. Mikrobiol. Immunol. 153,269-283 (1967) Desmonts, G., Couvreur0J.: Congenital toxoplasmosis. A prospective study of 378 pregnancies. N. Eng. J. Med. 290, 1110-1116 (1974)
216
K.N. Masihi and H.Werner
Frenkel, J.K.: Adoptive immunity to intracellular infection. J. Immunol. 98, 1309-1319 (1967) Huldt, G.: Experimental toxoplasmosis. Acta path. microbiol, stand. 68, 605-621 (1966) Janitschke, K., Jörren, H.R.: Untersuchungen über die Bedeutung der intrauterinen Übertragung von Toxoplasmen für die Verbreitung der Infektion bei Hauskaninchen. Z. Tropenmed. Parasit. 21, 246-251 (1970) Jones, T.C., Len, L., Hirsch, J.A.: Assessment in vitro of immunity against Toxoplasma gondii. J. Exp. Med. 141, 466-482 (1975) Mackaness, G.B.: The influence of immunologically committed lymphoid cells on macrophage activity in vivo. J. Exp. Med. 129, 973-992 (1969) Masihi, K.N., Werner, H.: The effect of passively transferred heterologous serum on Toxoplasma gondii in NMRI mice. Inftuence of the treatment on course of infection and cyst formation. Zbl. Bakt. Hyg., I. Abt. Orig. A (in press) Nakayama, I., Aoki, T.: The influence of antilymphocyte serum on the resistance of mice to Toxoplasma infection. Jap. J. Parasit. 19, 573-582 (1970) Pelster, B.: Zelluläre Immunreaktionen bei der weißen Maus nach Superinfektion mit Toxoplasma gondii. Z. Parasitenk. 48, 95-110 (1975) Reikvam, A., Grammeltvedt, R., Hoiby, E.A.: Activated mouse macrophages: Morphology, lysosomal biochemistry, and microbicidal properties of in vivo and in vitro activated cells. Acta path. microbiol, scand, Sect. C 83, 129-138 (1975) Remington, J.S., Krahenbuhl, J.L., Medenhall, J.W.: A role for activated macrophages in resistance to infection with Toxoplasma. Infect. Immun. 6, 829-834 (1972) Ruskin, J., Remington, J.S.: Toxoplasmosis in the compromised host. Ann. Intern. Med. 84, 193-199 (1976) Sethi, K.K., Pelster, B., Suzuki, N., Piekarski, G., Brandis, H.: Immunity to 1bxoplasmagondii induced in vitro in non-immune mouse macrophages with specifically immune lymphocytes. J. Immunol. 115, 1151-1158 (1975) Shirahata, T., Shimizu, K., Suzuki, N.: Effects of immune lymphocyte products and serum antibody on multiplication of Toxoplasma in murine peritoneal macrophages. Z. Parasitenk. 49, 11-23 (1976) Stadtsbaeder, S., Nguyen, B.T., Calvin-Preval, M.C.: Respective role of antibodies and immune macrophages during acquired immunity against toxoplasmosis in mice. Ann. Immunol. (Inst. Pasteur) 126 C, 461-474 (1975) Strannegard, O., Lycke, E.: Effect of antilymphocyte serum on experimental toxoplasmosis in mice. Infect. Immun. 5, 769-774 (1972) Swartzberg, J.E., Krahenbuhl, J.L., Remington, J.S.: Dichotomy between macrophage activation and degree of protection against Listeria monocytogenes and Toxoplasma gondii in mice stimulated witb Corynebacteriumparvum. Infect. Immun. 12, 1037-1043 (1975) Watson, W.A., Beverley, J.K.A.: Epizootics of toxoplasmosis causing ovine abortion. Vet. Rec. 88, 120-123 (1971) Werner, H., Janitschke, K., Masihi, K.N., Adusu, E,: Über die Wirkung von Toxoplasma-Antikörpern auf T. gondii nach Reinfektion. Zbl. Bakt. Hyg., I. Abt. Orig. A238, 128-142 (1977) Werner, H., Masihi, K.N., Tischer, I., Adusu, E.: The effect of chemoimmunotherapy with SDDS, pyrimethamine and anti-Toxoplasma serum on Toxoplasma gondii cysts in latent infected NMRI mice. Z. Tropenmed, Parasit. (in press) Werner, H., Pichl. H.: Vergleichende Untersuchungen an zystenbildenden Toxoplasma-Stämmen. II. Zystenentwicklung und humorale Antikörperbildung. Zbl. Bakt. Hyg. I. Abt. Orig. A 210, 402-416 (1969)
Received September3, 1976
Z. Parasitenk. 54, 209-216 (1977)
Parasitenkunde Parasitologic Research
@ by Springer-Verlag 1977
Immunization of NMRI Mice Against Virulent Toxoplasma gondii. Differing Efficacy of Eleven Cyst-Forming Toxoplasma Strains K.N. Masihi and H. Werner Robert Koch-Institute,Departmentof Medical Parasitology,Nordufer20, D-1000 Berlin 65
Summary. Mice were infected with eleven cyst-forming Toxoplasma strains of varying virulence and challenged 1 month later with highly virulent BK strain parasites. The early cellular reaction in vivo was estimated by collecting the peritoneal exudates 24 h after challenge. This consisted of 54.34-77.61% lymphocytes and 0-8.88% infected macrophages in the eleven immunized groups in contrast to 27.17% lymphocytes and 18.64% infected macrophages in the control group. The peritoneal exudate 72 h after chaUenge comprised of 55.88-73.86% lymphocytes and 0-6.97% infected macrophages compared to 25.88% lymphocytes and 92% infected macrophages in the control group. Following the virulent challenge, deaths occurred in seven of the eleven groups immunized with live strains and ranged 5-42% at the end of a 6-week observation period. The most virulent of the Toxoplasma strains used - Alt and Gail - gave higher mortality while the least virulent o n e s - 558, 1070, K8 and KSU - were solidly resistant. The significantly different mortality obtained with strains Alt, Gail and Witting in a similar experiment performed at an interval of 7 months is discussed. Acquisition of immunity to Toxoplasma usually follows recovery from a natural infection and is ascribed to a premunition phenomenon. Cysts containing slow proliferating parasites have been found in various tissues of infected hosts and may persist even when antibody is no longer measurable (Werner and Pichl, 1969). Epidemiological studies based on detection of humoral antibodies by conventional serology or cell-mediated immunity by skin tests have shown human infections to be widespread. However, the incidence of positivity has been found to vary according to geographical location. A signißcant proportion of women in child-bearing age may be negative and thus carry higher risks of congenital transmission. Moreover, reactivated Toxoplasma-infectäon is being encountered more frequently in immunosuppressed patients (Ruskin and Remington, 1976). In view of the problems associated with early detection and chemotherapy, prophylactic measures against Toxoplasmosis assume increased importance. Experimental infections of laboratory animals have shown that the intrauterine transmission of Toxoplasma to foetus is likely to be dependent on various factors inc!uding the host species and the strain of parasite. Diaplacental transmission to the foetus was observed with six of the eleven Toxoplasma strains in latent infected mice
210
K.N. Masihi and H.Werner
( W e r n e r et al., 1977) but similar infection o f rabbits b e f o r e m a t i n g was successful in p r e v e n t i n g c o n g e n i t a l infection o f the foetus ( J a n i t s c h k e and Jörren, 1970; W e r n e r et al., 1977). T h e latter has clinical correlates in p r o s p e c t i v e studies of p r e g n a n t w o m e n ( D e s m o n t s and C o u v r e u r , 1974; Berger and Piekarski, 1975). N o cases o f affected infants w e r e o b s e r v e d w h e n m a t e r n a l infection h a d o c c u r r e d prior to c o n c e p t i o n . This a p p r o a c h has been used to p r e v e n t o v i n e a b o r t i o n s w h i c h are a cause o f c o n s i d e r a b l e e c o n o m i c loss in sheep rearing a r e a s ( W a t s o n and Beverley, 1971). A l t h o u g h at p r e s e n t it is h a z a r d o u s to a t t e m p t v a c c i n a t i o n , any future feasibility of applying specific i m m u n o p r o p h y l a x i s to h u m a n s or cats w o u l d depend, a m o n g s t o t h e r i m p o r t a n t c o n s i d e r a t i o n s , on suitable Toxoplasma o r g a n i s m s . M u c h requires to be k n o w n a b o u t parasite strain variability and its effect o n p r o t e c t i o n against s u b s e q u e n t challenge. T h e present p a p e r deals with a c o m p a r i s o n o f eleven different strains in mice,
Materials and Methods Animals. Six-week-old female NMRI mice weighing 20-25 g were obtained from the SPF-colony maintained by the Bundesgesundheitsamt. Ten animals each were kept in Makrolon Type II cages and housed in an air-conditioned room at 22 ° C and 40% humidity. They were red with Herilan Hann ® standard pellets and water ad libitum. Toxoplasma Strains. Eleven strains isolated originally from different sources and maintained by regular passage were used. Basic data on these is given in Table 1. Strain 1070 had been isolated from a human case just 1 month before the present experiments were started.
Table 1. Basic data on strains used for immunization Toxoplasma strain 248 558 1070 K8 KSU Mo S162 $930 Alt Gail Witting a
Isolated from
Maintained since a
Human
6 years
Human Human Rabbit Cat Human Pig Pig Human Human Human
5 years 1 month 12 years 4 years 1 year 8 years 8 years 16 years 4 years 14 years
Maintained in Mastomys natalensis M. natalensis NMRI mice M. natalensis M. nataIensis M. natalensis M. natalensis M. natalensis NMRI mice NMRI mice NMRI mice
Last passage before (months)
Cyst index/ML
9
1,200
6 1 6 10 9 10 10 6 6 6
280 80 160 360 160 200 240 200 320 240
In this laboratory
Preparation of Cyst Suspensions and Primary Infection. Brains from chronically infected Mastomys natalensis or NMRI mice were triturated with glass beads in phosphate buffered saline, pH 7.2. Hornogenate from two or three brains was adjusted to contain 10 cysts/0.3 ml. Animals in groups of 25 were injected with this dose by intraperitoneal route. To increase the total number of survivors in groups injected with more virulent strains, 1 week after infection 20 animals from each group received i.p. a single dose of 2 mg Sulfamethoxypyrazine + 0.02 mg Pyrimethamine per mouse (Werner et al., 1972). Initial mortality after primary infection was observed in remaining untreated mice.
Immunization of Mice Against T. gondii
211
Preparation of Toxoplasma Suspension for Challenge. Virulent BK strain was employed. Parasites were harvestd by washing out the peritoneal cavity with 2 ml of phosphate buffered saline on the 3rd day ofinfection. The suspension was adjusted to contain 3 x 103 Toxoplasma per 0.3 rel.
Cellular Reaction in vivo. Peritoneal cavities of 2-3 mice were rinsed with 1 ml of phosphate buffered saline each and the exudates pooled. The cell deposit after centrifugation was stained with Giemsa. Serology. Individual tail blood from 3 mice in each group was collected in the 4th week after infection before the challenge. Antibody titers were measured by Sabin-Feldman dye test. Enhanced Virulenee Due to Infectious Agents. To rule out the observed change (increase) in virulence of strains Alt, Gail and Witting (Table 2) by association with infectious agents (Berger and Schoop, 1967) these strains were injected into quails. Four weeks after infection, re isolation was achieved by inoculating quail brains into mice. The virulenee had not changed. Table 2. Initial mortality of mice infected with various Toxoplasma strainsa
Toxoplasma strain
Cumulative percent mortality Untreated group No. weeks 1 2
248 558 1070 K8 KSU Mo S162 $930 Alt Gail Witting
0 0 0 0 0 0 0 0 20 (0) c 20 (0) 0 (0)
40 20 0 0 0 0 0 60 100 (70) 80 (28) 60 (36)
Treated group b No. weeks 1 2 3
3
4
40 20 0 0 0 0 0 60
40 20 0 0 0 0 0 60
0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 5 70
5 0 0 0 0 0 0 10 70
10 0 0 0 0 10 5 10 70
100
0
40
40
40
60
0
15
15
15
(78) 80 (28) 60 (42)
a Each mouse was injected with 10 brain cysts h Chemotherapy adrninistered i.p. on day 7 post-infection (2 mg Sulfamethoxypyrazine + 0.02 mg Pyrimethamine per mouse) « Figures in parenthesis show percent mortality before 7 months The SFT titers are given in Table 4 To test further for possible contamination with pathogenic viruses, the cyst-containing brain suspension was frozen and then immediately thawed again 10 times to kill the cystozoites. No infectious agents could be shown after injecting this suspension in white mice. Finally, the brain suspension was centrifuged for 3 h at 10,000 g in a refrigerated centrifuge and the supernatant injected in mice. The animals remained healthy. These foregoing experiments demonstrated that out Toxoplasma strains were free from infectious agents whieh could not thus be responsible for increased mortafity of laboratory animals simulating an enhanced virulence.
Results
T h e relative p a t h o g e n i c i t y o f eleven Toxoplasma s t r a i n s as d e t e r m i n e d b y initial m o r t a l i t y for m i c e are p r e s e n t e d in T a b l e 2. Alt a n d Gail w e r e the m o s t virulent while
212
K.N. Masihi and H.Werner
1070, K8 and KSU were the least virulent of the strains used. Four weeks after infection, animals were challenged intraperitoneally with 3 × 103 virulent BK Toxoplasma. This dose gives a mean survival time of 7.75 + 0.50 days in normal mice.
Early Cellular Reaction in vivo After Challenge. Exudates coUected at 24 and 72 h after challenge showed a marked difference between the cellular constitutions of the immunized and not immunized animals as is shown in Table 3. In the former, eight groups showed a decrease and two showed slight increases (0.51 and 0.56) in percent of infected macrophages which ranged from 0--8.88% at 24 h and 0-6.97% at 72 h.
Table 3. Early cellular reaction in vivo after challenge (%)
Toxoplasma
Time after challenge
strain 24 h Lymphocytes present
Macrophages infected
72 h L y m p h o c y t e s Macrophages present infected
248 558 1070 K8 KSU Mo S162 $930 Alt Gail Witting
54.34 61.31 70.22 77.61 75.10 68.44 70.48 76.52 60.98 57.64 64.12
3.33 1.78 4.40 5.80 0.00 6.25 8.88 4.50 3.44 7.14 1.60
63.20 63.05 65.50 69.50 71.05 73.86 62.73 68.12 68.20 55.88 67.63
3.84 0.00 1.01 3.22 0.00 4.25 6.97 2.12 4.00 4.44 0.72
Controls
27.17
18.64
25.86
92.00
Values for 24 and 72 h each were obtained from pooled exudates of 2 to 3 mice
In contrast, the control group showed an increase from 18.64% infected macrophages at 24 h to 92% at 72 h. In this group the challenge inoculum had increased in peritoneal cavity to a mean of 1.2 x 106 free parasites per mouse on the 3rd day. No free Toxoplasma were observed in the others except strain 1070 which had a mean of 4.3 x 104 organisms. The number of lymphocytes ranged from 5476% in the immunized group while in controls it was only 25-27%.
Resistance oflmmunized Animals to Challenge. Daily deaths were recorded for a period of 6 weeks after virulent BK challenge. The data on these and the SFT titer ranges a few days before challenge are presented in Table 4. Strains Alt, Gail and Witting had been previously used 7 months before in a similar experiment. Results of that and the present experiment are shown graphically in Figures 1 and 2. Of the eleven Toxoplasma strains used, deaths were registered in seven groups and ranged from 5-42% at 6 weeks post-challenge. All strains showing initial mortality following drug administration 1 week after infection also gave incomplete protection. The most virulent strains used, Alt and Gail, had higher mortality rates, the least virulent ones gave solid resistance on challenge.
Immunization of Mice Against T. gondii
213
Table 4. Results of virulent BK strain challenge of mice immunized with eleven cyst-forming Toxoplasma strains
Toxoplasma strain
SFT range
Total challenged
Cumulative percent dead weeks post-challenge 1
248 558 1070 K8 KSU Mo $162 $930 Alt Gail Witting
1,000-4,000 1,000-4,000 1,000-4,000 4,000 4,000 4,000 1,000-16,000 1,000 1,000--4,000 (4,000)" 1,000-4,000 (1,000-4,000) 256-1,000 (16,000
l7 20 18 20 20 19 20 17 14 (9) 19 (15) 15 (12)
Controls
20
2
3
4
5
6
5.88 5.88 5.88 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10.52 10.52 10.52 0 0 5.26 5.00 5.00 5.00 0 0 0 5.88 5.88 5.88 0 5.88 5.88 35.71 42.85 7.14 7.14 35.71 35.71 (o) (11.11) (11.11) (0) (0) (0) 0 10.52 31.57 31.57 31.57 31.57 (13.33) (26.66) (26.66) (33.33) (0) (0) 0 0 0 6.66 0 0 (50) (50) (8.33) (33.33) (41.66) (50) 90 (95)
100 b (100) b
a Figures in parenthesis show results before 7 months u On day 8 post-challenge
o ALT
100
o
80"
Z
»
"
~ GAI L
> rr
60. taJ
Z UJ
[3WITTING 40"
n-" Lid
n
20"
L~IICON TROL !
!
NUMBER 0E DAYS AFTER CHALLANGE
Fig. 1. Animals were infected with strains Alt (O), Gail (Q), Witting ([3) or saline (mm)and chaUenged 30 days later with virulent BK Toxoplasma. This experiment was performed 7 months before
214
K.N. Masihi and H.Werner
100
aWlTTING
0
z 80" » »
a GAIL
nr"
Sb
oALT
60-
LLI
40LLI r,d, LId
o_ 20-
~ICONTROL I
0
5
I
I
l
I
10 15 20 25 N!JNBER OF DAYS AFTER CHALLANGE
.
30
Fig. 2. Animals were infectedwith strains Alt (O), Gail (O), Witting (tq) or saline (1) and challenged 30 days later with virulentBK Toxoplasma
Discussion
The present study shows that prior immunization of the host with a live Toxoplasma strain does not always result in complete protection against subsequent challenge with a high virulent strain. Different strains vary vastly in their behaviour and relative pathogenicity for a particular host. Immunization with more virulent strains proved to be less efficient while ones with similar initial mortality for mice gave differing levels of protection. The observations with the strains Alt, Gail and Witting are of particular interest as they had been utilized 7 months before without a chemotherapy in a preliminary experiment. At that time strain Alt gave none, Gail 28% and Witting 50% mortality 4 weeks after 3 × 103 BK challenge. In this previous study, Gail strain was comparable to Beverley strain in that the initial mortality by both was up to 30% 2 weeks after inoculation of 10-15 brain cysts (Reikvam et al., 1975) but unlike former, the latter one was resistant to virulent challenge. Between then and the time when present experiments were performed, the initial virulence of these three strains had undergone an increase and completely different results were obtained. Alt gave 35.71%, Gail 31.57%, and Witting 0% mortality 1 month after similar BK challenge. Six-week-old NMRI mice maintained under same conditions were used for both experiments. These results suggest that factors other than virulence may be operative in immunization with live strains and at present remain to be defined. The respective roles of both humoral and cell-mediated immunity in protection against this parasite has received much attention in recent years. Specific antibody has been found to be effective against extracellular organisms which are lysed in the presence of an "accessory factor" by activating the properdin-dependent pathway of complement system and in rendering the parasites susceptible to intracellular destruction by macrophages (Anderson et al., 1976; Jones et al., 1975; Sethi et al.,
Immunization of Mice Against T. gondii
215
1975; Stadtsbaeder et al., 1975). Multiple transfer of immune serum 48 hours prior to challenge with LDT0 of cyst-forming Alt strain conferred significant resistance to normal recipient mice as assessed by a 55% reduction in the number of brain cysts and almost 90% survival rate (Masihi and Werner, in press). In addition, alleviation of established infection could be attained by an immunotherapy with a high fiter immune serum (Werner et al., 1977). However, Toxoplasma is an obligate intracellular parasite and studies with naturally and experimentally infected rabbits suggested an important role for cell-mediated immunity (Huldt, 1966). This was further emphasized by the demonstration of adoptive transfer of immunity with spleen and lymph node cells (Frenkel, 1967). Prior treatment of mice with either antilymphocyte (Nakayama and Aoki, 1970) or antithymocyte sera (Strannegard and Lycke, 1972) also greatly reduced the survival times following challenge. The evidence from these studies suggests that both humoral and cell-mediated immune responses are important in controlling Toxoplasma infections. Cell-mediated immunity to an intracellular infection involves interaction between specifically sensitized lymphocytes and macrophages (Mackaness, 1969). In the present study, early peritoneal exudates from immunized groups after challenge comprised of 54-77% lymphocytes, in comparison to 25-27% in controls. It is probable that these large numbers of lymphocytes in immunized mice included sensitized cells capable of specifically activating the macrophages to destroy intracellular parasites. The bulk of the lymphocytes in these animals were of the small type and are in concurrence with previous observations (Pelster, 1975). Vastly reduced numbers of infected macrophages in immunized animals was in marked contrast to controls which had 92% of such cells. This confirms and extends the earlier results (Pelster, 1975; Stadtsbaeder et al., 1975). Toxoplasma immune mice possess macrophages with enhanced microbicidal capacity (Remington et al., 1972) and infected cells in these animals may show a prolongation of average generation time of parasites (Jones et al., 1975). High titers of circulating antibody present at the time of challenge may have also helped in intracellular killing of antibody-coated parasites by normal macrophages. The host may show different levels of protection after in vivo challenge in spite of the presence of macrophages capable of being activated to kill Toxoplasma in vitro (Swartzberg et al., 1975). Differences in numbers of parasitized macrophages and mortality suggest that some variation in the degree of macrophage activation by different strains may exist. Conflicting reported results of attempts to confer specific anti-Toxoplasma activity on normal macrophages, by immune lymphocytes and antigen or products of such an interaction (Jones et al., 1975; Sethi et al., 1975; Shirahata et al., 1976), may be reflective of differences in strains used for respective immunizations.
References
Anderson, S.E., Bautista, S.C., Remington, J.S.: Specific antibody-dependentkilling of Toxoplasma gondiiby normal macrophages.Clin. exp. Immunol.26, 375-380 ( 1976) Berger, J., Piekarski, G.: Die Bedeutung der Toxoplasma-Infektion für Schwangerschaftsverlaufund Kindesentwicklung. Ergebnisse einer prospektiven Studie. Geburtsh. u. Frauenheilk. 35, 89-97 (1975) Berger, J., Schoop, G.: Über ein an den Toxoplasma-StammBK (Winser van Thiel 1948) gekoppeltes, infektiöses Agens. Z. Med. Mikrobiol. Immunol. 153,269-283 (1967) Desmonts, G., Couvreur0J.: Congenital toxoplasmosis. A prospective study of 378 pregnancies. N. Eng. J. Med. 290, 1110-1116 (1974)
216
K.N. Masihi and H.Werner
Frenkel, J.K.: Adoptive immunity to intracellular infection. J. Immunol. 98, 1309-1319 (1967) Huldt, G.: Experimental toxoplasmosis. Acta path. microbiol, stand. 68, 605-621 (1966) Janitschke, K., Jörren, H.R.: Untersuchungen über die Bedeutung der intrauterinen Übertragung von Toxoplasmen für die Verbreitung der Infektion bei Hauskaninchen. Z. Tropenmed. Parasit. 21, 246-251 (1970) Jones, T.C., Len, L., Hirsch, J.A.: Assessment in vitro of immunity against Toxoplasma gondii. J. Exp. Med. 141, 466-482 (1975) Mackaness, G.B.: The influence of immunologically committed lymphoid cells on macrophage activity in vivo. J. Exp. Med. 129, 973-992 (1969) Masihi, K.N., Werner, H.: The effect of passively transferred heterologous serum on Toxoplasma gondii in NMRI mice. Inftuence of the treatment on course of infection and cyst formation. Zbl. Bakt. Hyg., I. Abt. Orig. A (in press) Nakayama, I., Aoki, T.: The influence of antilymphocyte serum on the resistance of mice to Toxoplasma infection. Jap. J. Parasit. 19, 573-582 (1970) Pelster, B.: Zelluläre Immunreaktionen bei der weißen Maus nach Superinfektion mit Toxoplasma gondii. Z. Parasitenk. 48, 95-110 (1975) Reikvam, A., Grammeltvedt, R., Hoiby, E.A.: Activated mouse macrophages: Morphology, lysosomal biochemistry, and microbicidal properties of in vivo and in vitro activated cells. Acta path. microbiol, scand, Sect. C 83, 129-138 (1975) Remington, J.S., Krahenbuhl, J.L., Medenhall, J.W.: A role for activated macrophages in resistance to infection with Toxoplasma. Infect. Immun. 6, 829-834 (1972) Ruskin, J., Remington, J.S.: Toxoplasmosis in the compromised host. Ann. Intern. Med. 84, 193-199 (1976) Sethi, K.K., Pelster, B., Suzuki, N., Piekarski, G., Brandis, H.: Immunity to 1bxoplasmagondii induced in vitro in non-immune mouse macrophages with specifically immune lymphocytes. J. Immunol. 115, 1151-1158 (1975) Shirahata, T., Shimizu, K., Suzuki, N.: Effects of immune lymphocyte products and serum antibody on multiplication of Toxoplasma in murine peritoneal macrophages. Z. Parasitenk. 49, 11-23 (1976) Stadtsbaeder, S., Nguyen, B.T., Calvin-Preval, M.C.: Respective role of antibodies and immune macrophages during acquired immunity against toxoplasmosis in mice. Ann. Immunol. (Inst. Pasteur) 126 C, 461-474 (1975) Strannegard, O., Lycke, E.: Effect of antilymphocyte serum on experimental toxoplasmosis in mice. Infect. Immun. 5, 769-774 (1972) Swartzberg, J.E., Krahenbuhl, J.L., Remington, J.S.: Dichotomy between macrophage activation and degree of protection against Listeria monocytogenes and Toxoplasma gondii in mice stimulated witb Corynebacteriumparvum. Infect. Immun. 12, 1037-1043 (1975) Watson, W.A., Beverley, J.K.A.: Epizootics of toxoplasmosis causing ovine abortion. Vet. Rec. 88, 120-123 (1971) Werner, H., Janitschke, K., Masihi, K.N., Adusu, E,: Über die Wirkung von Toxoplasma-Antikörpern auf T. gondii nach Reinfektion. Zbl. Bakt. Hyg., I. Abt. Orig. A238, 128-142 (1977) Werner, H., Masihi, K.N., Tischer, I., Adusu, E.: The effect of chemoimmunotherapy with SDDS, pyrimethamine and anti-Toxoplasma serum on Toxoplasma gondii cysts in latent infected NMRI mice. Z. Tropenmed, Parasit. (in press) Werner, H., Pichl. H.: Vergleichende Untersuchungen an zystenbildenden Toxoplasma-Stämmen. II. Zystenentwicklung und humorale Antikörperbildung. Zbl. Bakt. Hyg. I. Abt. Orig. A 210, 402-416 (1969)
Received September3, 1976
