EXPERIhfENTAL
PARASITOLOGY
Trichinella
43, 336-341
spiralis:
(1977)
lmmunosuppression of Swiss Mice GAETAN
Institute
in Challenge
M. FAU~ERT
of Parasitology, McGill University, Macdonald College Province of Quebec, HOA lC0, Canada (Accepted
Infections
for publication
Post Office,
29 June 1977)
FAUUERT, G. M. 1977. Trichinella spiralis: 1117munosuppression in challenge infections of Swiss mice. Experimental Para.Gtologrj 43, 336-341. Swiss mice given 200 Trichinella spiralis larvae followed by a further 200 larvae 21 days later were found to be immune to the challenge infection. Immunity caused a significant reduction in adult and muscle larvae. When challenged 56 days after immunization, the intestinal phase was unaffected, but complete protection was expressed against the muscle stage. Immunized mice challenged at Day 21 showed no difference in ‘the plaque-forming cell response of antibody produced against sheep red blood cells (SRBC) at any time during the challenge infection. However, when mice were challenged 56 days after i~nmunizatiun, immunodepression was evident at Days 7 and 14 only, with a return to normal responsiveness by Days 28 and 56. INDEX DESCRIPTOI~S: T~iclainella spimlis; Parasitic nematode; Trichinellosis; Swiss mice; Challenge infection; Plaque-forming Immunosuppression; Sheep erythrocytes; Immunity; cells.
It has been reported that the humoral antibody response to unrelated antigens is lowered in mice previously infected with Trichinella spir&s (Faubert and Tanner 1971, 1974; Cypess et nl. 1973; Lubiniecki and Cypess 1975). The cellular immune response is also affected, since skin allografts and heart transplants survive much longer in T. spiral&infected mice than in noninfected controls ( Svet-Moldavsky et al. 1970; Faubert and Tanner 1975; Ljunstrijm 1976). Capron et al. (1977) have recently reviewed many immunosuppressive phenomena in human parasitic infections, as well as in experimental models. One of the possible defense mechanisms that a parasite might use to survive in the host is the secretion of substances toxic to
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any farm reserved.
the immunological system. Recently, it was reported (Faubert 1976) that the migrating phase of T. spin&s is capable of producing substances which can affect the antibody response of spleen cells to sheep red blood cells (SRBC). A similar phenomenon has also been observed in schistosomiasis by Capron & al. (1977). These authors have demonstrated that Schistosoma ma~oni produces soluble substances that diminish the response of spleen cells to T- and B-cell mitogens. The purpose of this study was twofold. Accounts of the level of immunity have differed widely when mice were challenged a short time after a primary infection (McCoy 1931; Larsh 1963; Rappaport and Wells I951), and wide variations in the expulsion rate of T. spiralis from the mouse intestine may occur in different strains of
ISSN
0014
4894
Trichinella
spiralis:
IMMUNOSUPPRESSION
mice (Wakelin and Lloyd 1976; RiveraOrtiz and Nussenzweig 1976). Therefore, it was necessary to examine the expulsion rate of T. spiralis in Swiss mice during primary and challenge infections in order to determine the time at which immune responsiveness should be assessed. Second, the response of plaque-forming cells (PFC) to SRBC in challenge infections was studied in order to determine whether immunodepression toward SRBC occurs in challenge infections at Day 21 or 56. MATERIALS
AND METHODS
Animals and the Parasite The parasite, Trichinellu spiralis, and the mice used here were described in an earlier paper (Faubert and Tanner 1971). Young female Swiss adult mice, weighing 20 g, were infected with 200 T. spiralis muscle larvae. Recovery of Adult
Parasites
Food was removed from cages on the eve of sacrifice. Mice were killed in ether and the small intestine was removed, slit open along its length, and placed in warm normal saline. The mucosa was scraped gently, and both scrapings and intestine were placed in flasks with 200 ml of saline. During incubation at 37 C, the T. spiralis worms left the intestine. After 2 hr, the contents of each flask were poured through a coarse sieve, allowing the worms to pass through and settle in sedimenting jars. The worms were transferred to a petri dish and the total number was counted under a dissecting microscope. Intestinal debris and mucosal scrapings were examined for further adults. Recovery of Encysked Muscle Larvae T. spiralis larvae were obtained from infected animals as described by Faubert and Tanner (1975), except for the followPrior to digestion, the ing modification: muscles were mascerated in a blender.
IX MICE
337
PFC Assay The method for assaying IgM PFC was described earlier (Faubert 1976). Statistical
Analysis
In all cases, a statistical analysis of mean values was done using Student’s t test. P values less than 0.05 were considered significant. Experimental
Protocol
Experiment 1. The pattern of the primary and challenge infections in Swiss mice was determined by two different techniques: counting the number of adults in the intestine and counting the encysted larvae. To count the adults in the intestine, three groups of 36 mice were used. Group 1A received a single infection at Day 0, whereas Groups 1B and 1C received second infections at Days 21 and 56, respectively. The mice in each group were killed in Groups of six at Days 4, 6, 8, 10, 12, and 14 postinfection. Experiment 2. To count the encysted T. spiralis larvae, three, groups of 10 mice, 2A, 2B, and 2C, were infected as above and killed 28 days after the last infection. Two other groups of 10 mice were also infected at the same time as the challenge infections at Days 21 and 56, as a control for groups 2B and 2C, respectively. Experiment 3. To assay the PFC response to SRBC, each of 120 mice was infected with 200 T. spiralis muscle larvae. They were then divided into three groups: 3A, 3B, and 3C. Groups 3B and 3C were challenged with 200 muscle larvae at Days 21 and 56, respectively. Each animal was injected intraperitoneally with 0.2 ml of a 5% suspension of SRBC 4 days before the mouse spleen cells were assayed for PFC. With each group, an additional 16 mice were injected with the same amount of SRBC only, and served as controls. Since the pattern of the infection is shorter in
GAETAN hf. FAUBEKT
I
:I.: :::. . .. . ..: .: :. .._. ._. :.:.,: :::.. .::: .I.!:; >y;.;. :.:. :::: :(:I :;,:;. :.,.. ::: ‘.. .:: :: -:.:. .::: _._. .:.. : ....‘. ..:...;. :: : ;.,:I
~
4
q q
‘... .::i: :;i :‘.I: ,t::. :.:: .:-. ::: .:-. . $1 ..::: :.: ... .: ..: ::.: .:‘; ‘;>,. f:>,j h .::. 6
6
days
post-infecti
Primary
infection
Day
21
challenge
Day
56
challenge
10
12
14
on
FIG. 1. Mean numbers of adult Trichinellu spiralis worms counted in the intestine of Swiss mice in a primary infection (solid bars), challenge infection at Day 21 (open bars), or challenge infection at Day 56 .( dotted bars).
the group of mice challenged at Day 21 (Fig. l), the SRBC were given earlier. RESULTS
Expulsion of Adult Worms from the Intestine The expulsion of adult Trichinda spiralis worms from the intestine of Swiss mice is presented in Fig. 1 and is expressed as the mean number of worms recovered. In Group 1A (solid bars), approximately 45% of the inoculum was normally recovered at Day 4, which agrees with Rappaport and Wells (1951). The numbers decreased gradually until Day 14, by which time most of the worms had left the small intestine. In Group 1B (open bars), only 16% of the inoculum was recovered at Day 4, and the females were found to be stunted. The population was virtually eliminated by Day 6 (P < 0.001) . This result confirms a recent report by Love et al. (1976), in which they have shown that reinfected rats are immune to T. spiralis. The immunological response in the rat system is faster than in the mouse. These results suggest that the mice in this group possess a strong immunity to the challenge
infection. In Group 1C (dotted bars), 36% of the original inoculum was counted at Day 4. This number is not significantly different from the number of worms in Group 1A (P > 0.05). The expulsion rate from Days 4 to 14 in Group 1C was also quite similar to that observed in Group 1A. Also, the female worms in Group 1C were not at all stunted, as in the Day-21 challenge (Group lB), but were identical in size to those in the primary infections (Group 1A). Enajsted
Muscle Laruae
Due to these peculiar results, the apparent lack of immunity was investigated further. The number of encysted larvae in the muscle was used as an assay for the immunity to T. spidis (Fig. 2). The mean numbers of encysted larvae per gram of muscle in Groups 2A and 2B were the same. This assay suggests that all of the mice were completely immune to the parasite, including the group of mice challenged at Day 56. The infectivity of the larvae used in the challenge infection was determined by infecting normal mice. The dashed lines represent the number
Trichinella
spiralis:
IMMUNOSUPPRESSION IN MICE
of encysted larvae counted in the controls of Groups 2B and 2C to illustrate the infectivity of the larvae. They also represent the number of encysted larvae expected if no immunity is shown in Groups 2B and 2c. PFC in Primuy
and Challenge Infection
The total number of PFC per spleen from mice receiving a single infection is presented in Fig. 3. These results confirm a previous report (Faubert 1976). A much lower mean number of PFC is obtained if the SRBC are given during the migrating phase (Day 14), compared to a control group (Fig. 3A). A statistically different number of PFC was also observed at Day 28 (P < 0.01). However, at Days 7 and 56 after the T. spiralis infection, there was no difference in the mean number of PFC between those two groups and their controls (P > 0.05). It is important to note that the immunodepression observed in the primary infection when the infected mice are challenged with SRBC at Day 14 or 28 does not appear in the secondary infection ( Fig. 3B ) . The phenomenon of immunodepression reappears in the group of mice receiving the challenge infection at Day 56 (Fig. 3C). A much lower number of PFC was counted per spleen if the mice were injected with SRBC at Days 3 and 11 after the challenge infection (P < 0.01). After a challenge infection at Day 56, the kinetics of immunodepression are different than after a primary infection, In the latter case, the phenomenon is shown earlier (Day 7) and disappears much faster (Day 28).
DISCUSSION By counting the number of Trichinella s@ralis adults in the intestine and measuring the expulsion rate, it appears that there is no protection in Swiss mice challenged at Day 56. On the other hand, by counting the number of encysted larvae in the mus-
LIO00 FIG. 2. Mean numbers of encysted larvae per gram of muscle in mice receiving a single infection of 200 T~ichinellu spiralis larvae (A) and a second infection at Day 21 (B) or Day 56 (C). Dashed lines represent the numbers of encysted larvae expected if no immunity is shown.
cle, the conclusion might be that the mice are well protected. Therefore, counting the number of adults or measuring the expulsion rate is not sufficient for the evaluation of immunity in trichinellosis. The in u&o bioassay used by Despommier et al. (1977) to test the fecundity of the adult worm is a much better test for immunity against the adult worms at the intestinal level. Recently it has been demonstrated that two kinds of immunity exist against T. spiralis. Grove et al. (1977) have shown that the eosinophils play a role in resistance to the systemic phase of a primary T. spiralis infection in mice, but not against the adult phase. The data obtained here from the 56-day challenge group support their results, since a similar number of adult T. spiralis was counted by comparison with the primary infection. Furthermore, the number of encysted muscle larvae is much lower in this group, showing a complete protection. Immunodepression toward SRBC was observed in the group with challenge infection at Day 56, but not in the Day-21 challenge group (3B). The absence of immunodepression in Group 3B may be explained by the lack of the migrating phase
GAETAN hi. FAUBERT
340
300 250 200 150 100 50 0 5
11
24
54 DAYS
DAYS
3. Mean numbers of PFC per spleen at different times postinfection in a primary Trichinellu spiralis infection (A) and challenge infection at Day 21 (B) or Day 56 (C). FIG.
of T. spiralis. However, Ljungstriim and Huldt (1977) have observed immunodepression toward SRHC in a challenge infection at Day 21. The much larger number of infective larvae used in their esperiment might explain the discontinuity between my results and theirs! The data presented here show that the expulsion rate of the adult worms in Group 1C is quite similar to that of the primary infection (Fig. 1A). It appears that the adults remain in the intestine long enough to release newborn larvae which will depress the immunological system (Fig. 3C). Unfortunately, this hypothesis is not supported, since no increase in the number of encysted larvae was counted in the group of mice challenged at Day 56. There are, therefore, two possibilities left to explain the immunodepression in this group. First, newborn larvae could be released from the adult worms when the mice are challenged at Day 56 and rapidly destroyed by the eosinophils (Grove et al. 1977). Second, the observed immunodepression could be the result of an over-reaction of the immunological system to the secondary antigenie stimulus which is T. spiralis; this will
result in a more rapid appearance of antibody against the parasite, since we are dealing with a typical secondary immune response, but might also facilitate antigenie competition to an unrelated antigen like SRRC. These two possible explanations are now under active investigation in this laboratory. ACKNOWLEIKMENTS The author gratefully acknowledges the assistance of Catherine Rowe in the preparation of the manuscript, and also the assistance of Brigitte hlullner. The valuable scientific comments of Dr. Steven Ackerman were also appreciated. The author wishes to acknowledge a Formation de Chercheurs et d’Action ConccrtSe Programme grant to G.4l.F. Research at the Institute of Parasitology is supported by the National Research Council of Canada and the Formation de Chercheurs et d’Action Concert&e du hlinist&e de l’Education du Qubbec.
REFERENCES CAPHON, A., CAMUS, D.,
J. P., AXD E. 1977. Alterations de la rcponse immune au tours des infections parasitaires. Anna.? d’lmmunologie (in press). CYPESS, R. I-I., LUIXNECKI, A. S., AND HAMMON, W. 1973. Immunosuppression and increased susceptibility to Japanese B encephalitis virus in TrichincZZu s~Jir&-infected mice. Proceedings LE
BOUUENNER-FISCHER,
DESSAIST,
Trichinella
spiralis:
IMMZ JNOSUPPRESSION
of t/lc Society of Expcrimentd Riology (New York) 143, 469473. DESPOMMIER, D. D., CAMPBELL, W. C., AND BLAIR, L. S. 1977. The in vitro and in vivo analysis of immunity to Trichinella spiralis in mice and rats. Parusitology 74, 109-119. FAUBERT, G., AND TANNER, C. E. 1971. T. spiralis: Inhibition of sheep hemagglutinin in mice. Experimental Parasitology 30, 120-123. FAUBERT, G., AND TANNER, C. E. 1974. The suppression of sheep rosette-forming cells and the inability of mouse bone-marrow cells to reconstitute competence after infection with the nematode Trichinella spirahs. Immunology 27, 201-505. FAUBERT, G., AND TANNER, C. E. 1975. Leucoagglutination and cytotoxicity of the serum of infected mice and of extracts of T. spiralis larvae and the capacity of infected mouse serum to prolong skin allografts. Immunology 28, 1041-1050. FAUBERT, G. 1976. Depression of the plaqueforming cells to sheep red blood cells by the new-born larvae of Trichinella spiralis. Immunology 30, 485489. GROVE, D. I., MAH~LIOUD, M. A., AND WARREN, K. S. 1977. Eosinophils and resistance to Trichinella spiralis. The Journal of Experimental Medicine 145, 755-759. LARSH, J. E. 1963. Experimental trichiniasis. In “Advances in Parasitology” ( B. Dawes, ed. ), Vol. 1, pp. 213-286. Academic Press, London. LOVE, R. J., OGILVIE, B. M., AI\TD MCLAREN, D. J. 1976. The immune mechanism which expels the intestinal stage of TrichineZZa spiralis from rats. Immunology 30, 7-15.
IN MICE
341
LJUNGSTH&, I. 1976. The effect of experimental trichinellosis on immune response to unrelated antigens. Proceedings of the Fourth International Conference on Trichinellosis, Poznan, Poland (in press). LJUNGSTRGM, I., AND HULDT, G. 1977. Effect of experimental trichinosis on unrelated humoral and cell mediated immunity. Acta Pathologica Microbiology Scandinavia Section C 85, 131141. LUBINIECKI, A. S., AND CYPESS, R. H. 1975. Immunological sequelae of T. spiralis infection in mice. Effect on the antibody response to SRBC and Japanese B encephalitis virus. Infection ImmunoZogy 11, 1306-1311. McCoy, 0. R. 1931. Immunity of rats to reinfection with Trichinella spiralis. American Journal of Hygiene 14, 484494. RAPPAPORT, I., AND WELLS, H. S. 1951. Studies on trichinosis. I. Immunity to reinfection in mice following a single light infection. ]ournaZ of Infectious Diseases 88, 248-253. RIVERA-ORTIZ, C. I., AND NUSSENZWEIG, R. 1976. Trichinella spiralis: Anaphylactic antibody formation and susceptibility in strains of inbred mice. Experimental Parasitology 39, 7-17. SVET-MOLDAVSKY, G. J., SHAGHIYAN, G. S., CHERNYAKHOVSKAYA, I. Y., MKHEIDZE, D. M., LITOVCHENKO, T. A., OZERETSKOVSKAYA, N. N., AND KADAGHIDZE, Z. G. 1970. Inhibition of skin allograft rejection in Trichinellu infected mice. Transplantation 9, 69-70. WA~ELIN, D., AND LLOYD, M. 1976. Immunity to primary and challenge infections of Trichinella spiralis in mice: A re-examination of conventional parameters. Parasitology 72, 173-182.
PARASITOLOGY
Trichinella
43, 336-341
spiralis:
(1977)
lmmunosuppression of Swiss Mice GAETAN
Institute
in Challenge
M. FAU~ERT
of Parasitology, McGill University, Macdonald College Province of Quebec, HOA lC0, Canada (Accepted
Infections
for publication
Post Office,
29 June 1977)
FAUUERT, G. M. 1977. Trichinella spiralis: 1117munosuppression in challenge infections of Swiss mice. Experimental Para.Gtologrj 43, 336-341. Swiss mice given 200 Trichinella spiralis larvae followed by a further 200 larvae 21 days later were found to be immune to the challenge infection. Immunity caused a significant reduction in adult and muscle larvae. When challenged 56 days after immunization, the intestinal phase was unaffected, but complete protection was expressed against the muscle stage. Immunized mice challenged at Day 21 showed no difference in ‘the plaque-forming cell response of antibody produced against sheep red blood cells (SRBC) at any time during the challenge infection. However, when mice were challenged 56 days after i~nmunizatiun, immunodepression was evident at Days 7 and 14 only, with a return to normal responsiveness by Days 28 and 56. INDEX DESCRIPTOI~S: T~iclainella spimlis; Parasitic nematode; Trichinellosis; Swiss mice; Challenge infection; Plaque-forming Immunosuppression; Sheep erythrocytes; Immunity; cells.
It has been reported that the humoral antibody response to unrelated antigens is lowered in mice previously infected with Trichinella spir&s (Faubert and Tanner 1971, 1974; Cypess et nl. 1973; Lubiniecki and Cypess 1975). The cellular immune response is also affected, since skin allografts and heart transplants survive much longer in T. spiral&infected mice than in noninfected controls ( Svet-Moldavsky et al. 1970; Faubert and Tanner 1975; Ljunstrijm 1976). Capron et al. (1977) have recently reviewed many immunosuppressive phenomena in human parasitic infections, as well as in experimental models. One of the possible defense mechanisms that a parasite might use to survive in the host is the secretion of substances toxic to
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any farm reserved.
the immunological system. Recently, it was reported (Faubert 1976) that the migrating phase of T. spin&s is capable of producing substances which can affect the antibody response of spleen cells to sheep red blood cells (SRBC). A similar phenomenon has also been observed in schistosomiasis by Capron & al. (1977). These authors have demonstrated that Schistosoma ma~oni produces soluble substances that diminish the response of spleen cells to T- and B-cell mitogens. The purpose of this study was twofold. Accounts of the level of immunity have differed widely when mice were challenged a short time after a primary infection (McCoy 1931; Larsh 1963; Rappaport and Wells I951), and wide variations in the expulsion rate of T. spiralis from the mouse intestine may occur in different strains of
ISSN
0014
4894
Trichinella
spiralis:
IMMUNOSUPPRESSION
mice (Wakelin and Lloyd 1976; RiveraOrtiz and Nussenzweig 1976). Therefore, it was necessary to examine the expulsion rate of T. spiralis in Swiss mice during primary and challenge infections in order to determine the time at which immune responsiveness should be assessed. Second, the response of plaque-forming cells (PFC) to SRBC in challenge infections was studied in order to determine whether immunodepression toward SRBC occurs in challenge infections at Day 21 or 56. MATERIALS
AND METHODS
Animals and the Parasite The parasite, Trichinellu spiralis, and the mice used here were described in an earlier paper (Faubert and Tanner 1971). Young female Swiss adult mice, weighing 20 g, were infected with 200 T. spiralis muscle larvae. Recovery of Adult
Parasites
Food was removed from cages on the eve of sacrifice. Mice were killed in ether and the small intestine was removed, slit open along its length, and placed in warm normal saline. The mucosa was scraped gently, and both scrapings and intestine were placed in flasks with 200 ml of saline. During incubation at 37 C, the T. spiralis worms left the intestine. After 2 hr, the contents of each flask were poured through a coarse sieve, allowing the worms to pass through and settle in sedimenting jars. The worms were transferred to a petri dish and the total number was counted under a dissecting microscope. Intestinal debris and mucosal scrapings were examined for further adults. Recovery of Encysked Muscle Larvae T. spiralis larvae were obtained from infected animals as described by Faubert and Tanner (1975), except for the followPrior to digestion, the ing modification: muscles were mascerated in a blender.
IX MICE
337
PFC Assay The method for assaying IgM PFC was described earlier (Faubert 1976). Statistical
Analysis
In all cases, a statistical analysis of mean values was done using Student’s t test. P values less than 0.05 were considered significant. Experimental
Protocol
Experiment 1. The pattern of the primary and challenge infections in Swiss mice was determined by two different techniques: counting the number of adults in the intestine and counting the encysted larvae. To count the adults in the intestine, three groups of 36 mice were used. Group 1A received a single infection at Day 0, whereas Groups 1B and 1C received second infections at Days 21 and 56, respectively. The mice in each group were killed in Groups of six at Days 4, 6, 8, 10, 12, and 14 postinfection. Experiment 2. To count the encysted T. spiralis larvae, three, groups of 10 mice, 2A, 2B, and 2C, were infected as above and killed 28 days after the last infection. Two other groups of 10 mice were also infected at the same time as the challenge infections at Days 21 and 56, as a control for groups 2B and 2C, respectively. Experiment 3. To assay the PFC response to SRBC, each of 120 mice was infected with 200 T. spiralis muscle larvae. They were then divided into three groups: 3A, 3B, and 3C. Groups 3B and 3C were challenged with 200 muscle larvae at Days 21 and 56, respectively. Each animal was injected intraperitoneally with 0.2 ml of a 5% suspension of SRBC 4 days before the mouse spleen cells were assayed for PFC. With each group, an additional 16 mice were injected with the same amount of SRBC only, and served as controls. Since the pattern of the infection is shorter in
GAETAN hf. FAUBEKT
I
:I.: :::. . .. . ..: .: :. .._. ._. :.:.,: :::.. .::: .I.!:; >y;.;. :.:. :::: :(:I :;,:;. :.,.. ::: ‘.. .:: :: -:.:. .::: _._. .:.. : ....‘. ..:...;. :: : ;.,:I
~
4
q q
‘... .::i: :;i :‘.I: ,t::. :.:: .:-. ::: .:-. . $1 ..::: :.: ... .: ..: ::.: .:‘; ‘;>,. f:>,j h .::. 6
6
days
post-infecti
Primary
infection
Day
21
challenge
Day
56
challenge
10
12
14
on
FIG. 1. Mean numbers of adult Trichinellu spiralis worms counted in the intestine of Swiss mice in a primary infection (solid bars), challenge infection at Day 21 (open bars), or challenge infection at Day 56 .( dotted bars).
the group of mice challenged at Day 21 (Fig. l), the SRBC were given earlier. RESULTS
Expulsion of Adult Worms from the Intestine The expulsion of adult Trichinda spiralis worms from the intestine of Swiss mice is presented in Fig. 1 and is expressed as the mean number of worms recovered. In Group 1A (solid bars), approximately 45% of the inoculum was normally recovered at Day 4, which agrees with Rappaport and Wells (1951). The numbers decreased gradually until Day 14, by which time most of the worms had left the small intestine. In Group 1B (open bars), only 16% of the inoculum was recovered at Day 4, and the females were found to be stunted. The population was virtually eliminated by Day 6 (P < 0.001) . This result confirms a recent report by Love et al. (1976), in which they have shown that reinfected rats are immune to T. spiralis. The immunological response in the rat system is faster than in the mouse. These results suggest that the mice in this group possess a strong immunity to the challenge
infection. In Group 1C (dotted bars), 36% of the original inoculum was counted at Day 4. This number is not significantly different from the number of worms in Group 1A (P > 0.05). The expulsion rate from Days 4 to 14 in Group 1C was also quite similar to that observed in Group 1A. Also, the female worms in Group 1C were not at all stunted, as in the Day-21 challenge (Group lB), but were identical in size to those in the primary infections (Group 1A). Enajsted
Muscle Laruae
Due to these peculiar results, the apparent lack of immunity was investigated further. The number of encysted larvae in the muscle was used as an assay for the immunity to T. spidis (Fig. 2). The mean numbers of encysted larvae per gram of muscle in Groups 2A and 2B were the same. This assay suggests that all of the mice were completely immune to the parasite, including the group of mice challenged at Day 56. The infectivity of the larvae used in the challenge infection was determined by infecting normal mice. The dashed lines represent the number
Trichinella
spiralis:
IMMUNOSUPPRESSION IN MICE
of encysted larvae counted in the controls of Groups 2B and 2C to illustrate the infectivity of the larvae. They also represent the number of encysted larvae expected if no immunity is shown in Groups 2B and 2c. PFC in Primuy
and Challenge Infection
The total number of PFC per spleen from mice receiving a single infection is presented in Fig. 3. These results confirm a previous report (Faubert 1976). A much lower mean number of PFC is obtained if the SRBC are given during the migrating phase (Day 14), compared to a control group (Fig. 3A). A statistically different number of PFC was also observed at Day 28 (P < 0.01). However, at Days 7 and 56 after the T. spiralis infection, there was no difference in the mean number of PFC between those two groups and their controls (P > 0.05). It is important to note that the immunodepression observed in the primary infection when the infected mice are challenged with SRBC at Day 14 or 28 does not appear in the secondary infection ( Fig. 3B ) . The phenomenon of immunodepression reappears in the group of mice receiving the challenge infection at Day 56 (Fig. 3C). A much lower number of PFC was counted per spleen if the mice were injected with SRBC at Days 3 and 11 after the challenge infection (P < 0.01). After a challenge infection at Day 56, the kinetics of immunodepression are different than after a primary infection, In the latter case, the phenomenon is shown earlier (Day 7) and disappears much faster (Day 28).
DISCUSSION By counting the number of Trichinella s@ralis adults in the intestine and measuring the expulsion rate, it appears that there is no protection in Swiss mice challenged at Day 56. On the other hand, by counting the number of encysted larvae in the mus-
LIO00 FIG. 2. Mean numbers of encysted larvae per gram of muscle in mice receiving a single infection of 200 T~ichinellu spiralis larvae (A) and a second infection at Day 21 (B) or Day 56 (C). Dashed lines represent the numbers of encysted larvae expected if no immunity is shown.
cle, the conclusion might be that the mice are well protected. Therefore, counting the number of adults or measuring the expulsion rate is not sufficient for the evaluation of immunity in trichinellosis. The in u&o bioassay used by Despommier et al. (1977) to test the fecundity of the adult worm is a much better test for immunity against the adult worms at the intestinal level. Recently it has been demonstrated that two kinds of immunity exist against T. spiralis. Grove et al. (1977) have shown that the eosinophils play a role in resistance to the systemic phase of a primary T. spiralis infection in mice, but not against the adult phase. The data obtained here from the 56-day challenge group support their results, since a similar number of adult T. spiralis was counted by comparison with the primary infection. Furthermore, the number of encysted muscle larvae is much lower in this group, showing a complete protection. Immunodepression toward SRBC was observed in the group with challenge infection at Day 56, but not in the Day-21 challenge group (3B). The absence of immunodepression in Group 3B may be explained by the lack of the migrating phase
GAETAN hi. FAUBERT
340
300 250 200 150 100 50 0 5
11
24
54 DAYS
DAYS
3. Mean numbers of PFC per spleen at different times postinfection in a primary Trichinellu spiralis infection (A) and challenge infection at Day 21 (B) or Day 56 (C). FIG.
of T. spiralis. However, Ljungstriim and Huldt (1977) have observed immunodepression toward SRHC in a challenge infection at Day 21. The much larger number of infective larvae used in their esperiment might explain the discontinuity between my results and theirs! The data presented here show that the expulsion rate of the adult worms in Group 1C is quite similar to that of the primary infection (Fig. 1A). It appears that the adults remain in the intestine long enough to release newborn larvae which will depress the immunological system (Fig. 3C). Unfortunately, this hypothesis is not supported, since no increase in the number of encysted larvae was counted in the group of mice challenged at Day 56. There are, therefore, two possibilities left to explain the immunodepression in this group. First, newborn larvae could be released from the adult worms when the mice are challenged at Day 56 and rapidly destroyed by the eosinophils (Grove et al. 1977). Second, the observed immunodepression could be the result of an over-reaction of the immunological system to the secondary antigenie stimulus which is T. spiralis; this will
result in a more rapid appearance of antibody against the parasite, since we are dealing with a typical secondary immune response, but might also facilitate antigenie competition to an unrelated antigen like SRRC. These two possible explanations are now under active investigation in this laboratory. ACKNOWLEIKMENTS The author gratefully acknowledges the assistance of Catherine Rowe in the preparation of the manuscript, and also the assistance of Brigitte hlullner. The valuable scientific comments of Dr. Steven Ackerman were also appreciated. The author wishes to acknowledge a Formation de Chercheurs et d’Action ConccrtSe Programme grant to G.4l.F. Research at the Institute of Parasitology is supported by the National Research Council of Canada and the Formation de Chercheurs et d’Action Concert&e du hlinist&e de l’Education du Qubbec.
REFERENCES CAPHON, A., CAMUS, D.,
J. P., AXD E. 1977. Alterations de la rcponse immune au tours des infections parasitaires. Anna.? d’lmmunologie (in press). CYPESS, R. I-I., LUIXNECKI, A. S., AND HAMMON, W. 1973. Immunosuppression and increased susceptibility to Japanese B encephalitis virus in TrichincZZu s~Jir&-infected mice. Proceedings LE
BOUUENNER-FISCHER,
DESSAIST,
Trichinella
spiralis:
IMMZ JNOSUPPRESSION
of t/lc Society of Expcrimentd Riology (New York) 143, 469473. DESPOMMIER, D. D., CAMPBELL, W. C., AND BLAIR, L. S. 1977. The in vitro and in vivo analysis of immunity to Trichinella spiralis in mice and rats. Parusitology 74, 109-119. FAUBERT, G., AND TANNER, C. E. 1971. T. spiralis: Inhibition of sheep hemagglutinin in mice. Experimental Parasitology 30, 120-123. FAUBERT, G., AND TANNER, C. E. 1974. The suppression of sheep rosette-forming cells and the inability of mouse bone-marrow cells to reconstitute competence after infection with the nematode Trichinella spirahs. Immunology 27, 201-505. FAUBERT, G., AND TANNER, C. E. 1975. Leucoagglutination and cytotoxicity of the serum of infected mice and of extracts of T. spiralis larvae and the capacity of infected mouse serum to prolong skin allografts. Immunology 28, 1041-1050. FAUBERT, G. 1976. Depression of the plaqueforming cells to sheep red blood cells by the new-born larvae of Trichinella spiralis. Immunology 30, 485489. GROVE, D. I., MAH~LIOUD, M. A., AND WARREN, K. S. 1977. Eosinophils and resistance to Trichinella spiralis. The Journal of Experimental Medicine 145, 755-759. LARSH, J. E. 1963. Experimental trichiniasis. In “Advances in Parasitology” ( B. Dawes, ed. ), Vol. 1, pp. 213-286. Academic Press, London. LOVE, R. J., OGILVIE, B. M., AI\TD MCLAREN, D. J. 1976. The immune mechanism which expels the intestinal stage of TrichineZZa spiralis from rats. Immunology 30, 7-15.
IN MICE
341
LJUNGSTH&, I. 1976. The effect of experimental trichinellosis on immune response to unrelated antigens. Proceedings of the Fourth International Conference on Trichinellosis, Poznan, Poland (in press). LJUNGSTRGM, I., AND HULDT, G. 1977. Effect of experimental trichinosis on unrelated humoral and cell mediated immunity. Acta Pathologica Microbiology Scandinavia Section C 85, 131141. LUBINIECKI, A. S., AND CYPESS, R. H. 1975. Immunological sequelae of T. spiralis infection in mice. Effect on the antibody response to SRBC and Japanese B encephalitis virus. Infection ImmunoZogy 11, 1306-1311. McCoy, 0. R. 1931. Immunity of rats to reinfection with Trichinella spiralis. American Journal of Hygiene 14, 484494. RAPPAPORT, I., AND WELLS, H. S. 1951. Studies on trichinosis. I. Immunity to reinfection in mice following a single light infection. ]ournaZ of Infectious Diseases 88, 248-253. RIVERA-ORTIZ, C. I., AND NUSSENZWEIG, R. 1976. Trichinella spiralis: Anaphylactic antibody formation and susceptibility in strains of inbred mice. Experimental Parasitology 39, 7-17. SVET-MOLDAVSKY, G. J., SHAGHIYAN, G. S., CHERNYAKHOVSKAYA, I. Y., MKHEIDZE, D. M., LITOVCHENKO, T. A., OZERETSKOVSKAYA, N. N., AND KADAGHIDZE, Z. G. 1970. Inhibition of skin allograft rejection in Trichinellu infected mice. Transplantation 9, 69-70. WA~ELIN, D., AND LLOYD, M. 1976. Immunity to primary and challenge infections of Trichinella spiralis in mice: A re-examination of conventional parameters. Parasitology 72, 173-182.
