EXPERIMENTAL
PARASITOLOGY
Schistosoma
41,74-82
mansoni:
(1977)
Resistance
PAUL M. KNOPF, THOMAS
to Reinfection
B. NUTMAN,
in the Rat
AND JOHN A. REASONER
Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island 02912, U.S.A. (Accepted for publication
24 February 1976)
KNOPF, PAUL M., NUTMAN, THOMAS B., AND REASONER, JOHN A, 1977. Schistosoma mansoni: Resistance to reinfection in the rat. Experimental Parasitology 41, 74-82. Resistance to reinfection by Schistosomu mansoni cercariae in Sprague-Dawley rats was analyzed, using the liver perfusion assay. The effects of varying experimental parameters, e.g. dose of cercariae in primary or challenge infections, time interval between infections, and time of worm recovery by liver perfusion, on the level of resistance were determined. Primary infections with doses ranging from 50 to 1000 cercariae/rat induce a significant resistance to reinfection, from 40% to 80%. A primary infection with 10 cercariae/rat was without effect. Resistance is established at a time interval of 4 weeks between primary and challenge infections, and its persists for at least 12 weeks. Increasing the challengeinfection dose to 5000 cercariae/rat abolishes resistance to reinfection. It is concluded that resistance is not due to a nutritional limitation on worm survival (“athrepsia”). Therefore, once-infected rats produce toxic factors which limit challenge worm survival. Worm burdens in the liver were measured during a &-month period in once- and twice-infected rats. We demonstrate that the time course of appearance of challenge worms in the liver is not significantly altered by the presence of primary-infection worms. Similarly, arrival of challenge womrs does not influence the expulsion of primary-infection worms. Thus, resistance is the result of an absolute decrease of challenge-infection worms in twice-infected rats. INDEX DESCRIPTORS: Schistosomiasis; Bilharziasis; Snail fever; Schistosoma mansoni; Trematode; Blood fluke; Cercariae; Sprague-Dawley rat; Snail; Biamphalaria gkzbrata; Resistance to reinfection; Liver perfusion; Athrepsia.
INTRODUCTION
In several laboratory animal model systems of human schistosomiasis,it has been demonstrated that previously infected animals are partially resistant to reinfection by cercariae (Ritchie et al. 1963; Sadun and Bruce, 1964; Smithers and Terry, 1965b, c; Maddison et al. 1970; Damian et al. 1974; Webbe et al. 1974; Sher et al. 1974; Phillips et al. 1975). If the resistance is a consequence of lethal immune reactions against worms of the challenge infection, it would be interesting to study the immunogens inducing these responses,since they would be
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
candidates for a protective vaccine against schistosomiasis. The reduction in yield of worms in twiceinfected animals has been attributed to a decrease in the number of challenge-infection worms recovered. It is theoretically possible that reduced yields in twice-infected animals could be due also to elimination of residual primary-infection worms (Damian et al. 1974) or to changes in the transit time of challenge-infection worms to the liver (Sher et al. 1974) in these animals. Experiments performed in our studies examine these possibilities. Other factors may also influence survival
ISSN 0014-4894
Schistosoma
mansoni:
RESISTANCE TO REINFECTION
of challenge-infection worms. In general, there are two categories of changes which might occur in once-infected animals that would interfere with survival of worms from a subsequent infection. One type of change is termed “athrepsia” (Lichtenbcrg 1962)) which translates: Once-infected animals are deficient in factors (e.g., nutrients) required for the development or survival of worms. The other type of change may be described as follows: Once-infected animals produce toxic factors which block development of or are lethal to worms. Immune responscsare included in the latter category. To distinguish between these two categories, we have examined the effects of varying certain parameters in the experimental protocol on resistance to reinfection in the rat, e.g., dose of cercariae, time interval between infections, etc. The conclusions drawn from the results of these investigations are presented. MATERIALS
AND METHODS
Life cycle maintenance. The life cycle of Schistosoma mansoni (Puerto Rican strain) was maintained using CD-l, outbred, albino mice (Charles River Laboratory) as the primary host and Biomphaluria glabrata snails (albino strain) as the intermediate host. Mice (20-25 g, female) were exposed to 150-250 cercariae for 30 min, by the method of Lee and Lewert ( 1956). Young snails, 5-8 mm in diameter, were exposed to about 15 miracidia/snail for 3 hr, in groups of 60-70 snails in a crystallizing dish at 26 C. Cercariae were shed from snails infected 5-12 weeks previously. Miracidia were prepared from eggs in the liver of mice infected 7-9 weeks, by the method of Lee and Lewert (1956), modified by using 1.8% NaCI, buffered with 0.01 M phosphate, pH 7.4, instead of 0.9% NaCl. Rat infection procedure. Sprague-Dawley rats (CD-l, Charles River Laboratory), female, 80-100 g, were infected by the ring method of Smithers and Terry (1965a). Rats were infected by adding an appro-
IN THE RAT
$65
priatc volume of cercarial suspension. After 20 min, the liquid was removed and checked for free cercariae. We routinely found lessthan 5% of the cercariae remaining in this liquid. The ccrcarial suspensions wcrc diluted to about 500 organisms/ml or less, except for infections of greater than 1000 cercariae/rat. A suspension of about 500 cercariae/ml, when checked for reproducibility of sampling, was shown to contain 458 + 13 (mean * SD, 10 samples). Cercarial doses quoted in the text are rounded off to the nearest hundred. To infect rats with 10 cercariae, 50-~1 droplets of a suitably diluted suspension were distributed into wells of a depression slide. Droplets containing a total of 10 cercariae were transferred to infection rings, and the wells were rinsed and then checked for residual cercariae after transferring the rinse liquid. For reinfection, we followed the same procedure. Age-matched rats were used for the challenge-infection controls. Figure 1 depicts the infection scheme. Worm burden determination. Liver worm burdens were determined by the perfusion method of Radke et al. (1961)) with modification. Perfusing fluid consisted of 0.15 M NaCI, 10 units/ml of heparin, 0.001 M carbachol. Perfusate from the portal vein was suctioned into a flask. All fluid in the collecting flask was then poured through a 45-pm nylon screen filter and the flask was rinsed. Worms collected on the screen were transferred to a petri dish for counting. The liver was removed and the lobes pressed flat to survey for worms still trapped. Worms were counted by examining the contents of the dish and the pressed liver under a dissecting microscope. Statistical treatment of data. The number of rats/group (n) was generally 5-7. Perfusions were performed daily during a 2- to 4-day period with the midpoint at a weekly interval, e.g., Days 27-29 for a Week 4 estimate of worm burden. Animals from every group (see Fig. 1) were perfused each day to minimize sampling bias.
76
KNOPF, NLJTMAN
Mean (X) and standard error of the mean (SEM) were calculated. To calculate the second-infection worm burden in group B (twice-infected) rats, the mean worm burden in A (WA) was subtracted from the mean worm burden in B ( Wu) and the SEM of the difference calculated. This value ( W, - Wa) was compared to Wc and the Student’s t test was performed for determining significance. The degrees of freedom were (nB + nc - 2). Percentage of resistance was calculated by the following equation:
=
WC
-
(FB
-
WA)
x
lo0
WC
RESULTS
1. Resistance to Reinfection: Dependence on Primary Infection Cercariue (N,)
Experiments were performed to determine the minimum primary-infection dose of cercariae which induced resistance to reinfection. The protocol used for this study is given in Methods (Fig. 1). By minimizing Ni, the contribution of average residual worm burden (WA) in the calculation to A
C
)
WC
d2 FIG. 1. Experimental protocol for determination of resistance to Schistosomu munsoni reinfection. Three groups of age-matched rats are subjected to the following infection scheme. Group A is exposed to N, cercariae (primary-infection control). Group B is exposed to:Nl cercariae (primary infection); several weeks later (At), the same rats are exposed to N, cercariae (challenge infection). Group C is exposed to Na cercariae (challenge-infection control), At weeks after the primary-infection date. All rats are perfused at a specified time after the challenge-infection date and worm burdens are determined (WA, WB, WC).
AND REASONER
determine the average number of new worm ( Wn - WA) is reduced, thereby increasing the certainty of this determination. The doses of cercariae used in the primary infection (N,) were 10, 50, 200, 500, and 1000, respectively. The two lowest doses extend downward the range used by previous investigators. The rats were challenged with 500 cercariae ( Nz), 4 weeks after the primary infection (At) and perfused 4 weeks after challenge. The results of these studies are presented in Table IA. Rats infected the first time with 50 to 1000 ccrcariae were resistant to reinfection: ( WB - WA) < ( Wc). The percentage of resistance was 40 to 80% and statistically significant. Total worm burdens in rats infected with Nr = 50 cercariae was sufficiently less than that of the challenge-infection control to be significantly different (P < 0.05) before subtraction of residual worm burden (WA). Rats infected with 10 cercariae did not develop significant resistance. 2. Resistance to Reinfection: Dependence on the Time Interval between Infections
The persistence of resistance induced by 50 or 500 cercariae ( N1) was determined. The time interval between primary and challenge infections was extended to 12 weeks, and the rats challenged with 500 cercariae (N,). The results of this experiment are given in Table IB. The experiment shows that resistance persists for at least 12 weeks. The percentage of resistance for Ni = 500 cercariae dropped from 82% (At = 4 weeks) to 53% (At = 12 weeks); it was not a statistically significant difference ( P> 0.10). In both primary-infection control groups, there were worms remaining in the rat livers for 16 weeks. 3. Resistance to Reinfection: Dependence on Dose of Challenge-Infection Cercariae (Nz)
An experiment was performed to determine the dependence of resistance on chal-
Schistosoma
munsoni:
RESISTANCE
TO
TABLE
REINFECTION
IN
THE
77
RAT
I
Percentage of Rat Resistance to Schistosoma nlansoni Reinfection as a Funct,ion of Primary-Infect,ion of Cerceriae (NJ and of Time Interval between Infection (At)u
17” _---
__.._~~
.~ -~~~
1’:trl A 10 50
NI) 3.0 f
0.8 (7)"
G.8 f 1.1 (4) f 1.7 (3) 18.X f 3.9 (-5)
200
ii00 1000
IT-” - TV:,
35.7
6!).7 * 43.4 f 38.2 f 48.7 f 48.2 f
7..i
(3)
(69.7 40.4 31.4 13.0 29.4
4..i (11) 10.2 3.0 8.2
(.i) (f) (vi) 17,
l’M1, B 50
4.0 f 14.7 f
500
1 .r, (::) 2.7 (3)
41.7f 46.1 f
2.4 10.3
(6) (7)
f f
T..i) 4.6
f 10.3 f f
= 72.6
3.4 9.t f
::7.7 f 31.4 f
Dose
IZh (“iI
I’
4 44 57 82 GO
N.S.
PARASITOLOGY
Schistosoma
41,74-82
mansoni:
(1977)
Resistance
PAUL M. KNOPF, THOMAS
to Reinfection
B. NUTMAN,
in the Rat
AND JOHN A. REASONER
Division of Biological and Medical Sciences, Brown University, Providence, Rhode Island 02912, U.S.A. (Accepted for publication
24 February 1976)
KNOPF, PAUL M., NUTMAN, THOMAS B., AND REASONER, JOHN A, 1977. Schistosoma mansoni: Resistance to reinfection in the rat. Experimental Parasitology 41, 74-82. Resistance to reinfection by Schistosomu mansoni cercariae in Sprague-Dawley rats was analyzed, using the liver perfusion assay. The effects of varying experimental parameters, e.g. dose of cercariae in primary or challenge infections, time interval between infections, and time of worm recovery by liver perfusion, on the level of resistance were determined. Primary infections with doses ranging from 50 to 1000 cercariae/rat induce a significant resistance to reinfection, from 40% to 80%. A primary infection with 10 cercariae/rat was without effect. Resistance is established at a time interval of 4 weeks between primary and challenge infections, and its persists for at least 12 weeks. Increasing the challengeinfection dose to 5000 cercariae/rat abolishes resistance to reinfection. It is concluded that resistance is not due to a nutritional limitation on worm survival (“athrepsia”). Therefore, once-infected rats produce toxic factors which limit challenge worm survival. Worm burdens in the liver were measured during a &-month period in once- and twice-infected rats. We demonstrate that the time course of appearance of challenge worms in the liver is not significantly altered by the presence of primary-infection worms. Similarly, arrival of challenge womrs does not influence the expulsion of primary-infection worms. Thus, resistance is the result of an absolute decrease of challenge-infection worms in twice-infected rats. INDEX DESCRIPTORS: Schistosomiasis; Bilharziasis; Snail fever; Schistosoma mansoni; Trematode; Blood fluke; Cercariae; Sprague-Dawley rat; Snail; Biamphalaria gkzbrata; Resistance to reinfection; Liver perfusion; Athrepsia.
INTRODUCTION
In several laboratory animal model systems of human schistosomiasis,it has been demonstrated that previously infected animals are partially resistant to reinfection by cercariae (Ritchie et al. 1963; Sadun and Bruce, 1964; Smithers and Terry, 1965b, c; Maddison et al. 1970; Damian et al. 1974; Webbe et al. 1974; Sher et al. 1974; Phillips et al. 1975). If the resistance is a consequence of lethal immune reactions against worms of the challenge infection, it would be interesting to study the immunogens inducing these responses,since they would be
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
candidates for a protective vaccine against schistosomiasis. The reduction in yield of worms in twiceinfected animals has been attributed to a decrease in the number of challenge-infection worms recovered. It is theoretically possible that reduced yields in twice-infected animals could be due also to elimination of residual primary-infection worms (Damian et al. 1974) or to changes in the transit time of challenge-infection worms to the liver (Sher et al. 1974) in these animals. Experiments performed in our studies examine these possibilities. Other factors may also influence survival
ISSN 0014-4894
Schistosoma
mansoni:
RESISTANCE TO REINFECTION
of challenge-infection worms. In general, there are two categories of changes which might occur in once-infected animals that would interfere with survival of worms from a subsequent infection. One type of change is termed “athrepsia” (Lichtenbcrg 1962)) which translates: Once-infected animals are deficient in factors (e.g., nutrients) required for the development or survival of worms. The other type of change may be described as follows: Once-infected animals produce toxic factors which block development of or are lethal to worms. Immune responscsare included in the latter category. To distinguish between these two categories, we have examined the effects of varying certain parameters in the experimental protocol on resistance to reinfection in the rat, e.g., dose of cercariae, time interval between infections, etc. The conclusions drawn from the results of these investigations are presented. MATERIALS
AND METHODS
Life cycle maintenance. The life cycle of Schistosoma mansoni (Puerto Rican strain) was maintained using CD-l, outbred, albino mice (Charles River Laboratory) as the primary host and Biomphaluria glabrata snails (albino strain) as the intermediate host. Mice (20-25 g, female) were exposed to 150-250 cercariae for 30 min, by the method of Lee and Lewert ( 1956). Young snails, 5-8 mm in diameter, were exposed to about 15 miracidia/snail for 3 hr, in groups of 60-70 snails in a crystallizing dish at 26 C. Cercariae were shed from snails infected 5-12 weeks previously. Miracidia were prepared from eggs in the liver of mice infected 7-9 weeks, by the method of Lee and Lewert (1956), modified by using 1.8% NaCI, buffered with 0.01 M phosphate, pH 7.4, instead of 0.9% NaCl. Rat infection procedure. Sprague-Dawley rats (CD-l, Charles River Laboratory), female, 80-100 g, were infected by the ring method of Smithers and Terry (1965a). Rats were infected by adding an appro-
IN THE RAT
$65
priatc volume of cercarial suspension. After 20 min, the liquid was removed and checked for free cercariae. We routinely found lessthan 5% of the cercariae remaining in this liquid. The ccrcarial suspensions wcrc diluted to about 500 organisms/ml or less, except for infections of greater than 1000 cercariae/rat. A suspension of about 500 cercariae/ml, when checked for reproducibility of sampling, was shown to contain 458 + 13 (mean * SD, 10 samples). Cercarial doses quoted in the text are rounded off to the nearest hundred. To infect rats with 10 cercariae, 50-~1 droplets of a suitably diluted suspension were distributed into wells of a depression slide. Droplets containing a total of 10 cercariae were transferred to infection rings, and the wells were rinsed and then checked for residual cercariae after transferring the rinse liquid. For reinfection, we followed the same procedure. Age-matched rats were used for the challenge-infection controls. Figure 1 depicts the infection scheme. Worm burden determination. Liver worm burdens were determined by the perfusion method of Radke et al. (1961)) with modification. Perfusing fluid consisted of 0.15 M NaCI, 10 units/ml of heparin, 0.001 M carbachol. Perfusate from the portal vein was suctioned into a flask. All fluid in the collecting flask was then poured through a 45-pm nylon screen filter and the flask was rinsed. Worms collected on the screen were transferred to a petri dish for counting. The liver was removed and the lobes pressed flat to survey for worms still trapped. Worms were counted by examining the contents of the dish and the pressed liver under a dissecting microscope. Statistical treatment of data. The number of rats/group (n) was generally 5-7. Perfusions were performed daily during a 2- to 4-day period with the midpoint at a weekly interval, e.g., Days 27-29 for a Week 4 estimate of worm burden. Animals from every group (see Fig. 1) were perfused each day to minimize sampling bias.
76
KNOPF, NLJTMAN
Mean (X) and standard error of the mean (SEM) were calculated. To calculate the second-infection worm burden in group B (twice-infected) rats, the mean worm burden in A (WA) was subtracted from the mean worm burden in B ( Wu) and the SEM of the difference calculated. This value ( W, - Wa) was compared to Wc and the Student’s t test was performed for determining significance. The degrees of freedom were (nB + nc - 2). Percentage of resistance was calculated by the following equation:
=
WC
-
(FB
-
WA)
x
lo0
WC
RESULTS
1. Resistance to Reinfection: Dependence on Primary Infection Cercariue (N,)
Experiments were performed to determine the minimum primary-infection dose of cercariae which induced resistance to reinfection. The protocol used for this study is given in Methods (Fig. 1). By minimizing Ni, the contribution of average residual worm burden (WA) in the calculation to A
C
)
WC
d2 FIG. 1. Experimental protocol for determination of resistance to Schistosomu munsoni reinfection. Three groups of age-matched rats are subjected to the following infection scheme. Group A is exposed to N, cercariae (primary-infection control). Group B is exposed to:Nl cercariae (primary infection); several weeks later (At), the same rats are exposed to N, cercariae (challenge infection). Group C is exposed to Na cercariae (challenge-infection control), At weeks after the primary-infection date. All rats are perfused at a specified time after the challenge-infection date and worm burdens are determined (WA, WB, WC).
AND REASONER
determine the average number of new worm ( Wn - WA) is reduced, thereby increasing the certainty of this determination. The doses of cercariae used in the primary infection (N,) were 10, 50, 200, 500, and 1000, respectively. The two lowest doses extend downward the range used by previous investigators. The rats were challenged with 500 cercariae ( Nz), 4 weeks after the primary infection (At) and perfused 4 weeks after challenge. The results of these studies are presented in Table IA. Rats infected the first time with 50 to 1000 ccrcariae were resistant to reinfection: ( WB - WA) < ( Wc). The percentage of resistance was 40 to 80% and statistically significant. Total worm burdens in rats infected with Nr = 50 cercariae was sufficiently less than that of the challenge-infection control to be significantly different (P < 0.05) before subtraction of residual worm burden (WA). Rats infected with 10 cercariae did not develop significant resistance. 2. Resistance to Reinfection: Dependence on the Time Interval between Infections
The persistence of resistance induced by 50 or 500 cercariae ( N1) was determined. The time interval between primary and challenge infections was extended to 12 weeks, and the rats challenged with 500 cercariae (N,). The results of this experiment are given in Table IB. The experiment shows that resistance persists for at least 12 weeks. The percentage of resistance for Ni = 500 cercariae dropped from 82% (At = 4 weeks) to 53% (At = 12 weeks); it was not a statistically significant difference ( P> 0.10). In both primary-infection control groups, there were worms remaining in the rat livers for 16 weeks. 3. Resistance to Reinfection: Dependence on Dose of Challenge-Infection Cercariae (Nz)
An experiment was performed to determine the dependence of resistance on chal-
Schistosoma
munsoni:
RESISTANCE
TO
TABLE
REINFECTION
IN
THE
77
RAT
I
Percentage of Rat Resistance to Schistosoma nlansoni Reinfection as a Funct,ion of Primary-Infect,ion of Cerceriae (NJ and of Time Interval between Infection (At)u
17” _---
__.._~~
.~ -~~~
1’:trl A 10 50
NI) 3.0 f
0.8 (7)"
G.8 f 1.1 (4) f 1.7 (3) 18.X f 3.9 (-5)
200
ii00 1000
IT-” - TV:,
35.7
6!).7 * 43.4 f 38.2 f 48.7 f 48.2 f
7..i
(3)
(69.7 40.4 31.4 13.0 29.4
4..i (11) 10.2 3.0 8.2
(.i) (f) (vi) 17,
l’M1, B 50
4.0 f 14.7 f
500
1 .r, (::) 2.7 (3)
41.7f 46.1 f
2.4 10.3
(6) (7)
f f
T..i) 4.6
f 10.3 f f
= 72.6
3.4 9.t f
::7.7 f 31.4 f
Dose
IZh (“iI
I’
4 44 57 82 GO
N.S.
