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Hymenolepis nana: Survival in the immunized mouse
EXPERIMENTAL
PARASITOLOGY
49,
Hymenolepis
248-257
nana:
(1980)
Survival
in the Immunized
Mouse
AK~RA ITO’
(Accepted
for publication
3 October
1979)
ITO, A. Hymenolepis nona: Survival in the immunized mouse. Experimenrul Parasifolog> 49, 248-257. Mice initially infected with Hymenolepis nona eggs became completely immune to challenge with mouse-derived cysticercoids (cysts) after more than 10 days. The host possessed at least two separated immune responses, one directed exclusively against reinfection with eggs (early response) and the other against cyst infection (late response). In two different mouse strains the responses showed markedly different duration both for the time lag prior to acquisition of the late response and for the survival of the initially infected worms, but were otherwise similar. The mice became immune to adult tapeworms and expelled the initially infected, destrobilated worms: this third immune response determines the longevity of H. ~UNI in the mouse host. Thus, there is a strong indication that H. many successively changes its immunogenicity during development, each stage stimulating immunity after a time lag. It is possible that the longevity of H. ncrncr in a mouse strain depends on the length of time prior to acquisition of immune responses directed not against the tissue stage (early response), but against the lumen stages (late response and worm expulsion response). INDEX DESCRIPTORS: H?mc~nolrpis ~WW: Cestode: Tapeworm; Mice; Immunity: Expulsion, worm: Survival, worm: lmmunogenicity successive. For experimental study of the immunological features of direct infections of H. nanu in the mouse, infections using mouse-derived cysts (Ito 1977) seem to be more advantageous than those using beetle-derived cysts (Heyneman 1961, 1962a,b; Isaak et al. 1977). Apparently some still unknown factors of the beetle intermediate host may affect antigenicity of beetle-derived cysts and the biological features of the indirect infection in the mouse (Caley 1975; Ghazal and Avery 1974). These effects are completely excluded by the use of mouse-derived cysts (Ito and Yamamoto 1977). A hypothesis has been proposed that, in the relationship between H. nunu and the mouse, two separate immune responses are directed against reinfection with either eggs or mouse-derived cysts (Ito 1978). One is an “early” response directed exclusively against oncospheres and is acquired within 2 to 5 days of an immunizing egg inoculation.
INTRODIJCTION
The relationship between Hymenolepis nc~na and the mouse is one of the most widely studied topics in cestode immunology. Most information has been derived from a comparison of direct egg and indirect beetle-derived cysticercoid (cyst) infections (reviewed by Gemmell 1976; Gemmell and Macnamara 1972; Gemmell and Soulsby 1968; Heyneman 1963; Smyth 1969; Weinmann 1970). Nevertheless, little is known about the immunological features of the direct cycle stages of H. nanu in the immunized mouse host, or the means whereby the highly immunogenic H. nanu can complete its primary infection in the immunized host (Brown 1976; Isaak et ml. 1977; Ito 1978; Ito and Yamamoto 1977; Ito et al. 1978; Weinmann 1966). ’ Present address: University School Gifu 500, Japan.
Department of Medicine,
of Parasitology, Tsukasa-Machi
Gifu 40,
248 0014.4894/80/020248-10$02.00/O Copyright All rights
@ 1980 by Academic Press, Inc. of reproduction in any form reserved.
Hymeno/epis
nancr: SURVIVAL
This is a rapid resistance, which has been described previously (Bailey 1951; Di Conza 1969, 1970; Hearin 1941; Heyneman 1962a; Hunninen 1935; Ito and Yamamoto 1976; Ito er al. 1978; Larsh 1951; Shorb 1933; Weinmann 1966, 1974). The other is a “late” response against the mouse-derived cysts acquired after a time lag of unknown duration (Ito 1978; Ito ef al. 1978). The present study was designed to determine the extent of the time lag prior to acquisition of the late response directed against the mouse-derived cysts, and also to observe the survival of worms in primary infections induced by eggs in two different mouse strains. Also discussed is the mechanism of worm survival in the immunized mouse host and its relation to the immunogenicity of cysts and adult worms. MATERIALS
AND METHODS
Host animals. Two strains of worm-free mice were maintained under worm-free conditions (Ito and Yamamoto 1977). One strain was the random-bred dd strain (dd mice) raised from August 1970 as closed colony in the present laboratory. The other was the inbred BALB/cAnN strain (BALB/c mice) purchased from the Nippon Charles River Company Ltd., Japan as specific pathogen-free mice. By housing a single male with four to six females in estrus on bedding in a plastic cage for 1 week, four to six litters of the same age (+ 3 days) were obtained. These littermates (32 to 48 dd mice, 24 to 36 BALB/c mice), male and female, which were housed separately after weaning, were used as a unit in each experiment. Five- to six-week-old dd mice of both sexes and lo- to 12 week-old BALB/c mice of both sexes were used throughout the experiments, except where otherwise stated. At these ages the body weights of the mice were comparable (20 to 25 g). The maintenance of uninfected mice, and those inoculated with Hymenolepis nana, has been described previously (Ito and Yamamoto 1977). Each unit consisted of two subunits of three infection groups.
IN MICE
249
Thus, mice of each unit were housed in 12 separate cages throughout the experiment. Parasites. Eggs of Hymenolepis nana were collected exclusively from dd mice (Ito and Yamamoto 1977). Preparation or inoculation of shell-free eggs (Berntzen and Voge 1965) or mouse-derived cysticercoids (cysts) (Ito 1977) has been described previously (Ito et al. 1978). Throughout the text “cyst” means mouse-derived cysticercoid, except where otherwise stated. Sevenday-old juvenile or thirty-day-old adult worms were collected from donor(s) that had been given 2 x lo4 or more shell-free eggs, respectively, 7 or 30 days previously. The posterior third of the small intestine harboring a number of juveniles, invisible in situ to the naked eye, or crowded 30-day worms, 5 to 10 mm long, was cut open lengthwise. This was done with care, in order not to damage the worms. Gentle shaking in 0.9% NaCl removed debris. Then the intestinal wall was placed in a glass beaker of fresh 0.9% NaCl and stirred gently for 30 to 60 min at room temperature with a magnetic stirrer. In this way, most of juveniles sank undamaged to the bottom. These worms were repeatedly washed with fresh 0.9% NaCl in a round-bottomed dish, as in the preparation of cysts from the anterior third of the small intestine’s wall (Ito 1977). Since this method seriously damaged 30-day worms, another method was devised: 30-day worms were flushed out with 0.9% NaCl by shaking the intestinal wall vigorously and by repeated washings. This preparation was completed 30 to 60 min before inoculation. Experimental design. In Experiments 1 and 2, 8 units of dd mice and 6 units of BALB/c mice were used. In Experiment 3, 3 units of dd mice only were used. One or two mice of each unit were used as donors and were given 2 x lo4 or more shell-free eggs either 5 days before each cyst inoculation (Experiments 1 and 2), or 30 days before each 30-day worm inoculation (Experiment 3). The mice of each unit, excluding the donors, were randomly di-
250
AKIRA
vided into three groups and infected as follows: In Experiments 1 and 2, group 1 was given eggs only (E), group 2 given eggs and later challenged with cysts (E + C), group 3 was given cysts only (C). In Experiment 3, group 1 was given eggs only (E), group 2 given eggs and later challenged with 30-day worms (E + W), group 3 was given worms only (W). In all experiments, the initial immunization with eggs was achieved by administering 20 or, in a few cases, 40 shell-free eggs in 0.1 to 0.2 ml of 0.9% NaCl on Day 0. Throughout the text, “Day 0” means the day of the immunizing inoculation. Challenge infections consisted of 200 or, in a few cases, 400 cysts in 0.2 ml of 0.9% NaCl (Experiments 1 and 2), or two hundred 30day worms in 0.5 ml of 0.9% NaCl (Experiment 3). Cysts or 30-day worms were introduced into the mouse stomach by a drawn glass pipet while the mice were under light ether anesthesia, and eggs were administered by a glass syringe as previously described (Ito et al. 1978). Details of the experimental design and the number of mice examined are given in the description of each experiment. Assay of egg, cyst, and 30-duy worm infections. In each experimental unit the
groups receiving egg infections were infected from the same batch of eggs. The mice to be given challenge infections (E + C or E + W) were divided into two subgroups and challenged at different times after egg infection. Mice of each subgroup of Experiments 1 and 3 were killed 7 days after each challenge, whereas those of Experiment 2 were killed 10 or 11 days after challenge. In this way it was possible to determine the numbers of egg-derived worms (EdW) recovered on two different days. Adult worms derived from eggs (EdW) and those from cysts (CdW), or EdW and those from 30-day worms (WdW), were easily distinguishable from each other by their number, shape, size, and maturation rates under the experimental design de-
IT0
scribed above (Ito 1978; Ito and Yamamoto 1977). The width of gravid proglottids was also measured with a micrometer. Secondary autoinfection was recorded when massive (approximately 100) worms, 2 or more mm long, were observed by the naked eye (Experiment 3). Autoinfection may be observed when immunologically normal mice are given cysts and examined more than 20 days after cyst inoculation (ACI), but does not occur within 7 to 10 days AC1 (Ito, in preparation). Experiments 1 and 2 were therefore designed to exclude involvement by autoinfection. RESULTS
Immunity qainst Cysts and Survival oj Egg-Derived Worms Experiment I. The time lag before acquisition of immunity to mousederived cysts of Hymenolepis nana was determined in both dd and BALB/c mice by feeding 200 or 400 cysts to test mice at varying intervals following an initial immunizing dose of 20 or 40 shell-free eggs. The egg-derived tapeworms (EdW) and cyst-derived ones (CdW) recovered from E + C group were compared with EdW from E group and CdW from C group, respectively. The results are summarized in Table I. Time lag in dd mice was about 30 days (between 15 and 40 days) after egg inoculation, whereas that in BALB/c mice was about 12 days (between 10 and 15 days). Five- to six-week-old dd mice, as well as those 10 to 12 weeks old, had a lag of more than 15 days. The EdW from the initial infection survived in all the mice which had become immune to the cysts, but the longevity of EdW in BALB/c mice was much shorter than in dd mice. Almost all BALB/c mice lost the initially established EdW between Days 27 and 37, whereas most of dd mice retained them until Day 47, the last day of this experiment (Table I). The maturation rates of surviving EdW were checked by counting the gravid pro-
Hwwnolepis nana: SURVIVAL
IN MICE
251
TABLE 1 Acquisition of Protective Immunity to Mouse-Derived Cysticercoids (Cysts) Induced by Initial Egg Inoculation of Hymenolepis nana into dd, Random-Bred, and BALBlc Inbred Strains of Mice
Days between egg and cyst inoculations
Group dd random-bred Estz (E) E+C
strain
15* 20
25*
30
40 BALBic inbred E E + ct C$ Et Et + C$ Cf Et Et + Cr
strain
” Mice of the E mice of the C and mice used were 5 old. All the mice
No.
of worms
No. of mice infected
Mean
f
SD
recovered Range
No. of mice infected
Cyst-derived
worms
No.
of worms
Mean
+ SD
recovered Range
12/12 14114 o/14 5/5 515 o/5 16116 11117 0117 617 717 017 lo/lo IO/l0 o/10 18120 17120 o/20
7.8 k 3.1 6.7 ? 2.3 0 9 i- 2.8 9.6 k 3.9 0 8.6 k 5.0 8 2 4.0 0 9.1 k 5.9 8.9 r 4.9 0 7 -c 3.1 7.4 k 5.0 0 3.9 + 1.8 3.4 2 2.7 0
5-13 3-15 6-12 6-16 I-17 3-18 I-15 2-17 l-15 l-18 I-11 1-12
0112 14114 14114 o/5 515 515 o/16 15117 17117 o/7 617 717 O/l0 3110 10110 0120 o/20 20120
0 67.9 + 32.3 71.1 f 35.4 0 48.2 k 9.0 44 + 12.6 0 28.1 ‘2 34.1 76.4 k 33.6 0 26.8 -t 31.1 84.3 + 44.2 0 7.7 I? 4.9 78.9 ? 43.9 0 0 73.6 ? 34.6
32- 136 28- 149 39-57 29-63 1-114 12- 140 2-78 35- 167 2-21 31-135
32-
124
mice IO
12
15
20
E+C C E EiC C
worms
mice 10
E+C C E E+C C E E+C
Egg-derived
30
40
6/6 6l6 016 616 717 017 516 6/6 016 5/7 617 o/7 317 017 o/7 016 o/7 017
8.1 8 k 0 15.7 13.3 0 9.2 11.2 0 2.4 1.8 0 1.7 0 0 0 0 0
2 2.9 2.2
4-12 5-11
2 5.2 k 3.4
9-23 8-17
+ 6.6 k 6.6
1-17 2-20
2 0.9 f 1.0
2-4 l-3
2
l-3
1.2
O/6 fY6 616 016 717 717 016 O/6 616 017 017 717 017 o/7 711 016 017 717
0 148.7 155.3 0 27 2 154.6 0 0 143.3 0 0 71.6 0 0 69.6 0 0 52.6
r 28.8 lr 32.1
103- 185 121-194
15.9 k 47.1
10-50 71-201
+ 75.5
73-241
? 34.8
34-
129
k 44.2
20-
138
2 30.0
17-93
and E + C groups were given 20 shell-free eggs (except those marked with t were given 40 shell-free eggs), and E + C groups were given 200 cysts (except those marked with $ were given 400 cysts). dd random-bred strain to 6 weeks old (except those marked with *). BALB/c inbred strain mice (and * of dd mice) were 10 to 12 weeks were killed 7 days after cyst inoculations.
glottids (GP) filled with mature eggs under x 100 magnification (Ito 1978; Ito and Yamamoto 1977). The maturity of EdW, the number of mice examined, and the number of EdW examined are summarized in Table II. In this experiment EdW recovered from dd mice were always much larger than those recovered from BALB/c mice: The largest EdW recovered from dd mice was about 150 mm long, whereas that from BALB/c mice was 80 mm long. In both
mouse strains it was observed, after initially established EdW became mature, that some worms became sterile after release of eggs, and often became smaller by destrobilation. These worms were described as worms of short-term maturity. Others remained mature (these were described as worms of long-term maturity). This characteristic, however, differs markedly between the mouse strains used (Table II). Worms of short-term maturity were found
AKIRA
252
Maturity
IT0
TABLE II Hymenolepis nana Recovered from BALBic Inbred Strains of Mice”
of Egg-Derived
Days
after
dd, Random-Bred,
egg inoculation
and
(AEI)
17
27
37
26126
20120
20120
No. of mature worms (MW)/ No. of worms recovered (B)
1981198
1321132
1171144
No. of MWI No. of L-sized
1981198
1321132
65166
35139
worms
(C)
No. of MWI No. of M-sized
o/o
o/o
52177
19180
worms
(D) o/o
o/o
Oil
o/o
dd random-bred strain mice No. of mice found infected/ No. of mice examined (A)
No. of MWI No. of S-sized
worms
35140 54/I 19
(E) Days
BALBlc strain
47
inbred mice A B C D E
AEI
17
19
22
27
37
47
12112 90197 90193 014 o/o
13113 1101135 1041106 6129 o/o
11112 421101 40155 2135 O/II
11114 6123 6/11 o/7 O/5
3114 115 III oi I 013
0113
” The mice examined include dd, random-bred strain mice as well as all BALBlc, inbred strain mice shown in Table I. Egg-derived worms were divided into three size groups: large (L-sized worms: 70 to 150 mm long, 0.7 to 1.2 mm wide), medium (M-sized worms: 30 to 60 mm long, 0.4 to 0.6 mm wide), and small (S-sized worms: 10 to 20 mm long, less than 0.3 mm wide). Maturation
Rates of Cyst-Derived
TABLE III Hymc~nolrpis ncrncr Recovered BALBic Inbred Strains of Mice”
from
Cyst-derived
Group dd random-bred Egg + Cyst Cyst ((3 E+C C E+C C BALBic E+C C
inbred
strain mice (E + C)
worms
No. of mice infected
IO
414 414 717 717 315 515
51 57.3 26.8 53.7 8 50.8
14.6 15.5 23.5 42.0 4.4 20.3
621204 2081229 621184 3661366 0124 2251254
4.2 10.9 5.1 20.2 0 9.9
414 414
69 t 46.3 88.8 2 68.4
O/276 3261555
0 11.4 k 7.6
30 strain
and
Days between egg and cyst inoculations
20*
No. of worms recovered (mean ? SD)
dd, Random-Bred,
t -t t ? + 2
No. of mature worms
No. of GP of mature worms” (mean ? SD)
2 k -t -c
2.2 6.2 2.6 12.8
i
4.4
mice 10
” Mice of E + C groups were initially given 20 shell-free eggs and challenged with 200 cysts, mice of C groups were initially given 200 cysts. All the mice other than those shown by an asterisk (killed 11 days after cyst inoculation (AU)) were killed IO days ACI. ’ Gravid proglottids.
Hym~nolrpis
nancr: SURVIVAL
in dd mice by Day 37 but never by Day 27, whereas in BALB/c mice those were found by Day 17 or 19. In both dd and BALB/c mice, CdW recovered from the mice of the E + C group on Day 17 (challenged with cysts on Day 10) were small (10 to 15 mm long) and immature, whereas those obtained from the control mice of C group were much larger (30 to 60 mm long) and beginning to produce eggs. There was, however, no significant difference between the number of CdW recovered from mice of the E + C and C groups (Table I). Experiment 2. The ability of cysts given in the challenge on Day 10 (in BALB/c mice), or on Day 10, 20, or 30 (in dd mice), to become mature was tested by increasing their development time in the mice from 7 days to 10 or 11 days. Maturation rates of CdW recovered from E + C and C groups were compared. The results are summarized in Table III. EdW recovered from the E and E + C groups are not recorded in this table. It was found that CdW given on Day 10 or even on Day 20 were able to become mature in dd mice, whereas those given on Day 10 in BALB/c mice remained immature.
Infectivity
of Juvenile (7-day Random-Bred,
Age of worms recovered
No. of mice massively infected
dd
‘I-day worms
20120
dd
30-day worms
18120
BALB/c
‘I-day worms
20120
BALBic
30-day worms
2120
” All recipient
Immunity
MICE
253
against Adult Tapeworms
A preliminary experiment, to determine whether lumen-dwelling stages of H. nana, juveniles or adult worms, orally inoculated could establish and develop into mature worms, was done with worms from both dd and BALB/c mice given 2 x lo4 or more shell-free eggs (Table IV). As shown in Table IV, juveniles were recovered by Day 7 from all mice of both strains. However, crowded 30-day worms were recovered from very few BALB/c mice (2/20), but were recovered from almost all dd mice (18/20). Infectivity of these worms was examined 7 days later. The results are summarized in Table IV. These worms were infective via the stomach and became mature. Experiment 3. Based on these findings an experiment was designed to determine whether or not adult tapeworms differed from the cysts in their immunogenicity. For this purpose, dd mice seemed to be much more useful than BALB/c mice because of the longer longevity of H. nana (Tables I, II, and IV). Thus, only dd mice were used as donors and recipients of adult worms. Crowded 30 day-old, adult worms, 5 to 10
TABLE IV Worms) or Adult (30~day Worms) Hymenolcpis or BALBic Inbred Strains of Mice on Oral
Worm recovery from donor mice given a2 x IO4 shell-free eggs
Donor mouse strains
IN
Infectivity
Recipient mouse strains (age) dd (5 weeks) BALBic (IO weeks) dd (IO weeks) dd (5 weeks) dd (IO weeks) dd (30 weeks) dd (I year) dd (5 weeks) BALBlc (5 weeks) Not tested
mice were killed 7 days after worm inoculation.
ncrru Prepared Inoculation
of the worms collected Worm dose administered loo 100 100 100 400 400 100 200 200
from
dd,
from the donors
No. of mice infected 313 414 515 414 717 515 313 313 414
No. of worms recovered Mean k SD 22.7 20.3 23 13.7 63.7 64.8 19 52.3 66.5
-t f t k k i k k t
16.0 9.3 19.8 5.1 56.9 45.3 16.5 10.2 23.6
Range 6-38 14-31 II-57 S-18 19- 172 24- 130 8-38 45-64 47-96
AKIRA
254
mm long, were used as challenges. Maturation rates of 30-day worms just before challenge were variable among populations; some populations consisted of immature worms, but others, in most cases, consisted of mature worms with less than 10 gravid proglottids (GP). Details of this experimental design and the results are summarized in Table V. Mice initially infected with EdW became immune to 30-day worms by Day 40 on the average (between Days 30 and 50), although almost all of these immune mice still retained 40-day-old EdW. However, there seemed to be a tendency for EdW to decrease in numbers between Days 30 and 50 (Tables I and V). The mean number of 30day worm-derived worms (WdW) recovered from the E + W group on Day 47 (challenged on Day 40) was 4.6 ? 2.4, whereas that from the control W group was 54.3 & 15.4. The WdW recovered were of small size, 10 to 20 mm long, but strobilated. Further, some of them were mature worms with not more than 10 GP. By this they were judged to be WdW, but not EdW, since such small-sized EdW, if present, never remained as mature, strobilated
Acquisition
of Protective
HUN,
into
Egg-derived
Days between egg and worm Group
inoculation
515
E&(E) E+W
Worm E E+W W E E+W W E E+W W
IO
(W) 30
40
50
” Mice of E and E + W groups worms. All the mice were killed
a dd
414 014 919 IO/IO O/IO IO/IO 9110 O/IO 5/5 8/R 015
DISCUSSION
These results indicate that mice initially infected with Hymenolepis nana eggs become immune to cyst challenge 10 or more days after egg inoculation. Since an infection, even as great as one with 2000 cysts, does not make the host immune to either cysts or eggs by the seventh day of their prepatent period, whereas a very small number of shell-free eggs such as 5 does make the
Random-Bred
Mean 9.6 10.5 0 7.4 8.3 0 6.3 5.2 0 5.8 5.1 0
Induced Strain 30.day
worms
worms No. of mice infected
forms (see Experiment 1). When 30-day worms were used on Days 10 and 30 to challenge mice previously infected with EdW, the challenging worms became mature, but the maturation rates and sizes of the resulting WdW were somewhat less than those rates and sizes of the WdW from control mice. Autoinfection with more or less than 100 small sized immature worms, 2 to 4 mm long, occurred exclusively in control mice of W groups given mature, 30-day worms (Table V). Clearly a time lag occurred before acquisition of immunity against 30-day worms. This differed from the immunity against cysts, since the mice immunized to cysts retained 30-day worms (Tables I and V).
TABLE V to Adult Tapeworms
Immunity
H~~nr~olrpis
IT0
Range
k 4.0 2 6.5
l- 16 4-17
2 3.7 k 5.4
4-13 lb15
i k
4.3 4.1
I-13 I- I4
i- 4.5 2 4.2
l-12 I- I4
Egg Inoculation
worm-derived
No. of mice infected O/S 4/4 414 019 IO/IO IO/IO O/IO 5/lO IO/IO O/5 018 515
Mean 0 40.8 31.5 0 31.1 37.2 0 4.6 54.3 0 0 46.4
2 SD
Range
f 16.2 5 9.5
21-59 23-42
i k
10.9 10.8
22-44 18-49
i- 2.4 k 15.4
2-8 38-83
2
29-63
were given 20 shell-free eggs, and mice of W and E + W groups 7 days after 30.day worm inoculations.
of
wcmns
No. of wcwms recovered
No. of recovered
2 SD
by Initial of Mouse”
16.1
were given
No. of mice autoinfected 015 014 414 019 O/IO IO/IO O/IO Oil0 4110 O/S O/8 5/5
two hundred
30-day
Hymetiolepis
nana: SURVIVAL
host completely immune to egg challenge within 2 to 5 days after egg inoculation (Hearin 1941; Heyneman 1962a; Hunninen 1935; Ito and Yamamoto 1976; Ito et al. 1978; Shorb 1933; Weinmann 1974), it seems highly probable that mice given eggs of H. nana possess at least two acquired immune responses directed exclusively against reinfections with either eggs (early response to inhibit cyst development in the intestinal villi) or cysts (late response to inhibit adult development in the lumen) as previously speculated (Ito 1978). This hypothesis is comparable to that of Campbell (1938a, b, c) in the relationship between Taenia taeniaeformis and the rat. This finding was consistent in the two mouse strains used which showed markedly different durations of both the time lag before onset of the late response and the survival of initially established worms. Egg-induced immunity to mouse-derived cysts seems to be complete, as demonstrated above, but that to beetle-derived cysts has been reported to be incomplete (Heyneman 1962b, 1963; Isaak et al. 1977). It has been speculated that this difference may be due to the difference in antigenicity of cysts recovered from the mammalian host compared with those recovered from the beetle intermediate host (Ito 1978). This speculation still remains to be proven, but now another explanation of the discrepancy may be possible. Egg-induced immunity to beetle-derived cysts may also be complete. If some of the mice given eggs retained sterile, but variable sized EdW as shown in Table II, the EdW might be mistaken for CdW, since littermate controls given eggs, but not challenged with cysts, were not included in the above authors’ works. If so, egg-induced immunity to cysts may be complete not only to mouse-derived but also to beetle-derived cysts. It is recognized that lumenal stages of H. nana seldom infect mice via the stomach (Caley 1975; Coleman and DeSa 1964; Rothman 1959). Caley (1975) reported that
IN
MICE
255
even cysts could not infect mice. However, earlier work (Ito 1977, 1978) and the present results (Table IV) have demonstrated that freshly prepared lumenal stages, cysts, juveniles, and crowded adult worms, can be used to infect mice orally. At present, it seems to be difficult to explain’this discrepancy, but both the preparation of undamaged worms and administering the worms into the mouse stomach with an excess solution such as 0.5 ml of 0.9% NaCl seem to be important factors in the success of the infection in mice. The fact that EdW survived in mice of both dd and BALB/c strains which had become immune to cysts strongly suggests that the immunogenicity may alter during all its developmental stages, from oncosphere to cyst (Heyneman 1963; Ito et al. 1978) and further from cyst to adult tapeworm (Tables I and V; Ito 1978). The experimental evidence supports the hypothesis that because of the time lag before immune responses are acquired, those against H. nana cannot reveal their functions against the initial immunogenic stages of the infection until the established H. nana stops changing its immunogenicity (Ito 1978). This was previously speculated by Brown (1976) and Weinmann (1970). However, in BALB/c mice the longevity of H. nana of primary infection was clearly much shorter than in dd mice (Tables I, II, and IV; Ito 1978) and it has been proven to be affected by a thymus-dependent immunity of worm expulsion (Isaak et al. 1977). The time lag before acquisition of immunity against the lumenal stages in BALB/c mice did not last as long as in dd mice (Table I) and the immunobiological features differed somewhat between the two mouse strains (Tables II and III). Therefore, we may consider that when the time lag is relatively short compared with the speed of H. nana development in the intestinal lumen, the host’s late response somewhat affects the lumenal stage of a primary infection. The lumenal stage might, then, not always com-
256
AKIRA
plete its immunogenic changes due to development or aging. The number of adult worms retained in the host tends to decrease with elapsed time (Tables I, II, and V; Hunninen 1935; Ito 1978; Shorb 1933; Woodland 1924). The surviving adult worms (EdW) were of the two types, short- and long-term maturity. Worms with a short time to maturity had released all eggs produced after maturation and often (but not always) became smaller than those of the same age with a long time to maturity. These smaller worms had destrobilated as observed in H. diminutu (Befus and Threadgold 1975; Heyneman 1962~; Hopkins et al. 1972). This inequality of size and/or fecundity of worms of the same age emerged days earlier in BALB/c mice than in dd mice (Table II). This phenomenon, previously observed by Shorb (1933) and Woodland (1924), seems to be due to different rates of aging influenced by the host’s immune responses. It therefore seems highly probable that the appearance of worms with a short time to maturity is the first step in “worm expulsion.” This was recently proven to be a thymusdependent immunity (Isaak rf al. 1977). It has been found that BALB/c mice become immune to egg challenge by Day 3 after an immunizing inoculation with 20 shell-free eggs. In contrast, dd mice become immune by Day 4 or 5, but never by Day 3 (Ito, unpublished). Thus, the mode of protection to either eggs or cysts, or the manner of worm expulsion, seemsto differ markedly between mouse strains. However, it is more probable that the duration or degree of maturity of EdW is affected by immune response(s) directed against the lumen-dwelling stages themselves rather than by the early response directed agqinst the oncospheres (Ghazal and Avery 1974). A possibility that the early response enhances or accelerates the late response remains, nevertheless, to be tested. The experimental evidence described above suggests that the longevity of H. mna in a
IT0
mouse strain depends on the length of the time lag prior to acquisition of immune responses directed not to the tissue stage (early response), but to the lumenal stages (late response and worm expulsion response). ACKNOWLEDGMENTS I wish and this
to thank
discussions. work during
Professor
K.
Okamoto
Mrs. Mieko Yamamoto its early phases.
for
advice
cooperated
in
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Host-tissue
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and superimposed infections nono var. ,f?trrc,~~o. Journcrl 440-444. BEAUS, A. D., AND THREADGOLD, ble immunological Hymrrwlrpi.t dimirlrttrr ofiy 71, 5255534.
to initial
with of‘
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37,
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A. K., AND VOGt, M. 1965. In ~.itro of oncospheres of four Hymenolepidid Joro-no/ c1.f Ptrrrr.vi~o/og~ 5 1, 235 -242.
BERNT7EN, hatching cestodes.
BROWN. K. N. 1976. Specificity in host-parasite interaction. In “Receptors and Recognition (Series A)” (P. Cuatrecasas and M. F. &eaves, eds.), Vol. CAI
1, pp. 119-175. EY, J. 1975.
Chapman A comparative
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%rit.cc,/rr-ifi
CAMPBEI.L, erty of
Jortmcrl
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ncrncl, the drawf to the process of Prrrrr.~ir~nXl/ndr
D. H. 1938a. The specific serum from rats infected
c~rrr.tsico//i.\. CAMPBELL, protective li.5 and artificially
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London. of the two
c~Jfmmuno/o~:?’
47,
protective propwith C.y.sficrr.crrs 35,
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D. H. 1938b. The specific absorbability of antibodies against C?~ric~c,vccts crcr.s.sic~o/C. /~i.s(fi~rmis in rabbits from infected and immunized animals. Jo~rnrrl <~,~l,nmrrno/-
og.v 35, 205-216. CAMPBELL, D. H. “nonabsorbable” rats infected with
1938~. Further studies on the protective property in serum from C~sric~c,~~r,s c~rrrssic~o//i.r. Joumtrl
of Immrmolo~~y 35, 465 -476. COI~EMAN, R. M., AND DESA, L. M. 1964. Immunological response to implanted adult dwarf tapeworms. Norrrrr (London) 204, 397-398. DI CONZA, J. J. 1969. Protective action of passively transferred immune serum and immunoglobulin fractions against tissue invasive stages of the dwarf tapeworm, Hymc,nolcpi.t none. Expeuimcntul Ptrr~uifology 25, 368-375. DI CONLA, J. J. 1970. Hymenolcpis ncrnrr: Mouse subcutaneous tissue responses to larval stages. E.rperirner~rr~l Porcrsirolo~y 28, 482-492.
nano: SURVIVAL
Hymenolepis
GEMMELI., M. A. 1976. Immunology and regulation of the cestode zoonoses. In “Immunology of Parasitic Infections” (S. Cohen and E. Sadun, eds.), pp. 333-358. Blackwell, Oxford. GEMMELL, M. A., AND MACNAMARA, F. N. 1972. Immune response to tissue parasites. II. Cestodes. In “Immunity to Animal Parasites” (E. I. L. Soulsby, ed.), pp. 236-272. Academic Press, New York. GEMMELL, M. A., AND SOULSBY, E. J. L. 1968. The development of acquired immunity to tapeworms and progress towards active immunization, with special reference to Echinococclrs spp. B~,//etin OJ the
World
Heultk
Orgrrnization
39,
45-55:
GHAZAL, A. M., AND AVERY, R. A. 1974. dynamics of Hymenolepis tmna in mice: and the “crowding effect.” Purositology 403-415. HEARIN, J. T. 1941. Studies on the acquired to the dwarf tapeworm Hymenolepis jicrtrrncr in the mouse host. Amrricun Hygiene
33,
Population Fecundity 69,
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nuntl Journtrl
c1.f
of Immunology
88,
210-220.
HEYNEMAN, D. 1962b. Studies on helminth immunity. I. Comparison between lumenal and tissue phases of infection in the white mouse by Hymeno/epi.s ncrncr (Cestoda: Hymenolepididae). Americrrn Jorrrnnl of Tropkrrl Medicine and Hygiene 11, 46-63. HEYNEMAN, D. 1962~. Studies on helminth immunity. II. Influence of Hymenolepis nuna (Cestoda: Hymenolepididae) in dual infections with H. diminrlrrr in white mice and rats. Expcrimenfal Porrrsitology 12, 7- 18. HEYNEMAN, D. 1963. Host-parasite resistance patterns-Some implications from experimental studies with helminths. Annrrls of the Nell, York Awdemy of Sc,iences 113, 114- 129. HOPKINS, C. A., SUBRAMANIAN, G., AND STALLARD, H. 1972. The development of Hymrnolepis diminrrtcr in primary and secondary infections in mice. Porrrsitology 64, 401-412. HUNNINEN, A. V. 1935. Studies on the life history and host-parasite relations of H~mc,,2o/epis.~trtc,rncr (H.
MICE
var. fruternu
Jorwnal
of Hygiene
257
Stiles) 22,
in white
mice.
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414-443.
ISAAK,
D. D., JACOBSON, R. H., AND REED, N. D. The course of Hymenolepis nuns infections in thymus-deficient mice. Internnrionol Archi\,es oj 1977.
Allergy
und
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55,
504-513.
ITO, A. 1977. A simple method for collecting infective cysticercoids of Hymrnolepis ncrncr from the mouse intestine. Jorrrnul oj’Prrrusito/ogy 63, 167- 168. ITO, A. 1978. Hymenolepis ncrncr: Protective immunity against mouse-derived cysticercoids induced by initial inoculation with eggs. Experimentcrl Purrrsifology 46, 12-19. ITO, A., AND YAMAMOTO, M. 1976. The mode of active protection against Hymenolepis nrrncr reinfection in mice inoculated with different doses of shell-free eggs. Jnpcrnese Jorrrnul of’ Porcrsitology 25, 247-253. ITO, A., AND YAMAMOTO, M. 1977. Maturation of initially infected Hymenolrpis ncrna under rapid protective immunity against reinfection with the eggs. Jopcrnese
71-87.
HEYNEMAN, D. 1961. Studies on helminth immunity. III. Experimental verification of autoreinfection from cysticercoids of Hymrnolepis ntrncr in the white mouse. Jorrrnrrl of In&tiotr.s Dise~~srs 109, lo- 18. HEYNEMAN, D. 1962a. Studies on helminth immunity. IV. Rapid onset of resistance by the white mouse against a challenging infection with eggs of Hvmenolepis nuns (Cestoda: Hymenolepididae). Journal
nuns
IN
Jorrrnul
of Pumsitology
26,
30 I-306.
ITO, A., YAMAMOTO, M., AND OKAMOTO, K. 1978. Primary infection with mouse-derived cysticercoids of Hymenolepis ncrntr prepared from baby or adult mice and secondary infections with eggs or cysticercoids. Internrrtionrrl Jorrrncrl Jbr Purasitology 8, 149- 153. LARSH, J. E. 1951. Host-parasite relationships in cestode infections, with emphasis on host resistance. Jorrrncrl of Parusitology 37, 343-352. ROTHMAN, A. H. 1959. Studies on the excystment of tapeworms. Expcrimentrrl Pamsitology 8, 336-364. SHORB, D. A. 1933. Host-parasite relations of Hymenolepisjkaterna in the rat and mouse. Americon Jorrrnrrl of Hygiene 18, 74- 113. SMYTH, J. D. 1969. “The Physiology of Cestodes.” Oliver & Boyd, Edinburgh. WEINMANN, C. J. 1966. Immunity mechanisms in cestode infection. In “Biology of Parasites” (E. J. L. Soulsby, ed.), pp. 301-320. Academic Press, New York. WEINMANN, C. J. 1970. Immunity in mammalian hosts. In “Immunity to Parasitic Animals” (G. J. Jackson, R. Herman, and I. Singer, eds.), Vol. II, pp. 1021- 1059. Appleton, New York. WEINMANN, C. J. 1974. Hymenolepis nono in mice: A potential model for the study of intestinal immunity. Procerdings
o,f‘ the
Purc:.sitology
Munich,
WOODLAND,
Third
W. N. F.
Hymenolepis
jktprncr
of the white
mouse.
Inrerncrtionctl
Congress
of’
life-cycle
of stiles)
589.
1924.
On
(H. ncrncr Portrsitology
the
var..frcrternu
16, 69-83.
PARASITOLOGY
49,
Hymenolepis
248-257
nana:
(1980)
Survival
in the Immunized
Mouse
AK~RA ITO’
(Accepted
for publication
3 October
1979)
ITO, A. Hymenolepis nona: Survival in the immunized mouse. Experimenrul Parasifolog> 49, 248-257. Mice initially infected with Hymenolepis nona eggs became completely immune to challenge with mouse-derived cysticercoids (cysts) after more than 10 days. The host possessed at least two separated immune responses, one directed exclusively against reinfection with eggs (early response) and the other against cyst infection (late response). In two different mouse strains the responses showed markedly different duration both for the time lag prior to acquisition of the late response and for the survival of the initially infected worms, but were otherwise similar. The mice became immune to adult tapeworms and expelled the initially infected, destrobilated worms: this third immune response determines the longevity of H. ~UNI in the mouse host. Thus, there is a strong indication that H. many successively changes its immunogenicity during development, each stage stimulating immunity after a time lag. It is possible that the longevity of H. ncrncr in a mouse strain depends on the length of time prior to acquisition of immune responses directed not against the tissue stage (early response), but against the lumen stages (late response and worm expulsion response). INDEX DESCRIPTORS: H?mc~nolrpis ~WW: Cestode: Tapeworm; Mice; Immunity: Expulsion, worm: Survival, worm: lmmunogenicity successive. For experimental study of the immunological features of direct infections of H. nanu in the mouse, infections using mouse-derived cysts (Ito 1977) seem to be more advantageous than those using beetle-derived cysts (Heyneman 1961, 1962a,b; Isaak et al. 1977). Apparently some still unknown factors of the beetle intermediate host may affect antigenicity of beetle-derived cysts and the biological features of the indirect infection in the mouse (Caley 1975; Ghazal and Avery 1974). These effects are completely excluded by the use of mouse-derived cysts (Ito and Yamamoto 1977). A hypothesis has been proposed that, in the relationship between H. nunu and the mouse, two separate immune responses are directed against reinfection with either eggs or mouse-derived cysts (Ito 1978). One is an “early” response directed exclusively against oncospheres and is acquired within 2 to 5 days of an immunizing egg inoculation.
INTRODIJCTION
The relationship between Hymenolepis nc~na and the mouse is one of the most widely studied topics in cestode immunology. Most information has been derived from a comparison of direct egg and indirect beetle-derived cysticercoid (cyst) infections (reviewed by Gemmell 1976; Gemmell and Macnamara 1972; Gemmell and Soulsby 1968; Heyneman 1963; Smyth 1969; Weinmann 1970). Nevertheless, little is known about the immunological features of the direct cycle stages of H. nanu in the immunized mouse host, or the means whereby the highly immunogenic H. nanu can complete its primary infection in the immunized host (Brown 1976; Isaak et ml. 1977; Ito 1978; Ito and Yamamoto 1977; Ito et al. 1978; Weinmann 1966). ’ Present address: University School Gifu 500, Japan.
Department of Medicine,
of Parasitology, Tsukasa-Machi
Gifu 40,
248 0014.4894/80/020248-10$02.00/O Copyright All rights
@ 1980 by Academic Press, Inc. of reproduction in any form reserved.
Hymeno/epis
nancr: SURVIVAL
This is a rapid resistance, which has been described previously (Bailey 1951; Di Conza 1969, 1970; Hearin 1941; Heyneman 1962a; Hunninen 1935; Ito and Yamamoto 1976; Ito er al. 1978; Larsh 1951; Shorb 1933; Weinmann 1966, 1974). The other is a “late” response against the mouse-derived cysts acquired after a time lag of unknown duration (Ito 1978; Ito ef al. 1978). The present study was designed to determine the extent of the time lag prior to acquisition of the late response directed against the mouse-derived cysts, and also to observe the survival of worms in primary infections induced by eggs in two different mouse strains. Also discussed is the mechanism of worm survival in the immunized mouse host and its relation to the immunogenicity of cysts and adult worms. MATERIALS
AND METHODS
Host animals. Two strains of worm-free mice were maintained under worm-free conditions (Ito and Yamamoto 1977). One strain was the random-bred dd strain (dd mice) raised from August 1970 as closed colony in the present laboratory. The other was the inbred BALB/cAnN strain (BALB/c mice) purchased from the Nippon Charles River Company Ltd., Japan as specific pathogen-free mice. By housing a single male with four to six females in estrus on bedding in a plastic cage for 1 week, four to six litters of the same age (+ 3 days) were obtained. These littermates (32 to 48 dd mice, 24 to 36 BALB/c mice), male and female, which were housed separately after weaning, were used as a unit in each experiment. Five- to six-week-old dd mice of both sexes and lo- to 12 week-old BALB/c mice of both sexes were used throughout the experiments, except where otherwise stated. At these ages the body weights of the mice were comparable (20 to 25 g). The maintenance of uninfected mice, and those inoculated with Hymenolepis nana, has been described previously (Ito and Yamamoto 1977). Each unit consisted of two subunits of three infection groups.
IN MICE
249
Thus, mice of each unit were housed in 12 separate cages throughout the experiment. Parasites. Eggs of Hymenolepis nana were collected exclusively from dd mice (Ito and Yamamoto 1977). Preparation or inoculation of shell-free eggs (Berntzen and Voge 1965) or mouse-derived cysticercoids (cysts) (Ito 1977) has been described previously (Ito et al. 1978). Throughout the text “cyst” means mouse-derived cysticercoid, except where otherwise stated. Sevenday-old juvenile or thirty-day-old adult worms were collected from donor(s) that had been given 2 x lo4 or more shell-free eggs, respectively, 7 or 30 days previously. The posterior third of the small intestine harboring a number of juveniles, invisible in situ to the naked eye, or crowded 30-day worms, 5 to 10 mm long, was cut open lengthwise. This was done with care, in order not to damage the worms. Gentle shaking in 0.9% NaCl removed debris. Then the intestinal wall was placed in a glass beaker of fresh 0.9% NaCl and stirred gently for 30 to 60 min at room temperature with a magnetic stirrer. In this way, most of juveniles sank undamaged to the bottom. These worms were repeatedly washed with fresh 0.9% NaCl in a round-bottomed dish, as in the preparation of cysts from the anterior third of the small intestine’s wall (Ito 1977). Since this method seriously damaged 30-day worms, another method was devised: 30-day worms were flushed out with 0.9% NaCl by shaking the intestinal wall vigorously and by repeated washings. This preparation was completed 30 to 60 min before inoculation. Experimental design. In Experiments 1 and 2, 8 units of dd mice and 6 units of BALB/c mice were used. In Experiment 3, 3 units of dd mice only were used. One or two mice of each unit were used as donors and were given 2 x lo4 or more shell-free eggs either 5 days before each cyst inoculation (Experiments 1 and 2), or 30 days before each 30-day worm inoculation (Experiment 3). The mice of each unit, excluding the donors, were randomly di-
250
AKIRA
vided into three groups and infected as follows: In Experiments 1 and 2, group 1 was given eggs only (E), group 2 given eggs and later challenged with cysts (E + C), group 3 was given cysts only (C). In Experiment 3, group 1 was given eggs only (E), group 2 given eggs and later challenged with 30-day worms (E + W), group 3 was given worms only (W). In all experiments, the initial immunization with eggs was achieved by administering 20 or, in a few cases, 40 shell-free eggs in 0.1 to 0.2 ml of 0.9% NaCl on Day 0. Throughout the text, “Day 0” means the day of the immunizing inoculation. Challenge infections consisted of 200 or, in a few cases, 400 cysts in 0.2 ml of 0.9% NaCl (Experiments 1 and 2), or two hundred 30day worms in 0.5 ml of 0.9% NaCl (Experiment 3). Cysts or 30-day worms were introduced into the mouse stomach by a drawn glass pipet while the mice were under light ether anesthesia, and eggs were administered by a glass syringe as previously described (Ito et al. 1978). Details of the experimental design and the number of mice examined are given in the description of each experiment. Assay of egg, cyst, and 30-duy worm infections. In each experimental unit the
groups receiving egg infections were infected from the same batch of eggs. The mice to be given challenge infections (E + C or E + W) were divided into two subgroups and challenged at different times after egg infection. Mice of each subgroup of Experiments 1 and 3 were killed 7 days after each challenge, whereas those of Experiment 2 were killed 10 or 11 days after challenge. In this way it was possible to determine the numbers of egg-derived worms (EdW) recovered on two different days. Adult worms derived from eggs (EdW) and those from cysts (CdW), or EdW and those from 30-day worms (WdW), were easily distinguishable from each other by their number, shape, size, and maturation rates under the experimental design de-
IT0
scribed above (Ito 1978; Ito and Yamamoto 1977). The width of gravid proglottids was also measured with a micrometer. Secondary autoinfection was recorded when massive (approximately 100) worms, 2 or more mm long, were observed by the naked eye (Experiment 3). Autoinfection may be observed when immunologically normal mice are given cysts and examined more than 20 days after cyst inoculation (ACI), but does not occur within 7 to 10 days AC1 (Ito, in preparation). Experiments 1 and 2 were therefore designed to exclude involvement by autoinfection. RESULTS
Immunity qainst Cysts and Survival oj Egg-Derived Worms Experiment I. The time lag before acquisition of immunity to mousederived cysts of Hymenolepis nana was determined in both dd and BALB/c mice by feeding 200 or 400 cysts to test mice at varying intervals following an initial immunizing dose of 20 or 40 shell-free eggs. The egg-derived tapeworms (EdW) and cyst-derived ones (CdW) recovered from E + C group were compared with EdW from E group and CdW from C group, respectively. The results are summarized in Table I. Time lag in dd mice was about 30 days (between 15 and 40 days) after egg inoculation, whereas that in BALB/c mice was about 12 days (between 10 and 15 days). Five- to six-week-old dd mice, as well as those 10 to 12 weeks old, had a lag of more than 15 days. The EdW from the initial infection survived in all the mice which had become immune to the cysts, but the longevity of EdW in BALB/c mice was much shorter than in dd mice. Almost all BALB/c mice lost the initially established EdW between Days 27 and 37, whereas most of dd mice retained them until Day 47, the last day of this experiment (Table I). The maturation rates of surviving EdW were checked by counting the gravid pro-
Hwwnolepis nana: SURVIVAL
IN MICE
251
TABLE 1 Acquisition of Protective Immunity to Mouse-Derived Cysticercoids (Cysts) Induced by Initial Egg Inoculation of Hymenolepis nana into dd, Random-Bred, and BALBlc Inbred Strains of Mice
Days between egg and cyst inoculations
Group dd random-bred Estz (E) E+C
strain
15* 20
25*
30
40 BALBic inbred E E + ct C$ Et Et + C$ Cf Et Et + Cr
strain
” Mice of the E mice of the C and mice used were 5 old. All the mice
No.
of worms
No. of mice infected
Mean
f
SD
recovered Range
No. of mice infected
Cyst-derived
worms
No.
of worms
Mean
+ SD
recovered Range
12/12 14114 o/14 5/5 515 o/5 16116 11117 0117 617 717 017 lo/lo IO/l0 o/10 18120 17120 o/20
7.8 k 3.1 6.7 ? 2.3 0 9 i- 2.8 9.6 k 3.9 0 8.6 k 5.0 8 2 4.0 0 9.1 k 5.9 8.9 r 4.9 0 7 -c 3.1 7.4 k 5.0 0 3.9 + 1.8 3.4 2 2.7 0
5-13 3-15 6-12 6-16 I-17 3-18 I-15 2-17 l-15 l-18 I-11 1-12
0112 14114 14114 o/5 515 515 o/16 15117 17117 o/7 617 717 O/l0 3110 10110 0120 o/20 20120
0 67.9 + 32.3 71.1 f 35.4 0 48.2 k 9.0 44 + 12.6 0 28.1 ‘2 34.1 76.4 k 33.6 0 26.8 -t 31.1 84.3 + 44.2 0 7.7 I? 4.9 78.9 ? 43.9 0 0 73.6 ? 34.6
32- 136 28- 149 39-57 29-63 1-114 12- 140 2-78 35- 167 2-21 31-135
32-
124
mice IO
12
15
20
E+C C E EiC C
worms
mice 10
E+C C E E+C C E E+C
Egg-derived
30
40
6/6 6l6 016 616 717 017 516 6/6 016 5/7 617 o/7 317 017 o/7 016 o/7 017
8.1 8 k 0 15.7 13.3 0 9.2 11.2 0 2.4 1.8 0 1.7 0 0 0 0 0
2 2.9 2.2
4-12 5-11
2 5.2 k 3.4
9-23 8-17
+ 6.6 k 6.6
1-17 2-20
2 0.9 f 1.0
2-4 l-3
2
l-3
1.2
O/6 fY6 616 016 717 717 016 O/6 616 017 017 717 017 o/7 711 016 017 717
0 148.7 155.3 0 27 2 154.6 0 0 143.3 0 0 71.6 0 0 69.6 0 0 52.6
r 28.8 lr 32.1
103- 185 121-194
15.9 k 47.1
10-50 71-201
+ 75.5
73-241
? 34.8
34-
129
k 44.2
20-
138
2 30.0
17-93
and E + C groups were given 20 shell-free eggs (except those marked with t were given 40 shell-free eggs), and E + C groups were given 200 cysts (except those marked with $ were given 400 cysts). dd random-bred strain to 6 weeks old (except those marked with *). BALB/c inbred strain mice (and * of dd mice) were 10 to 12 weeks were killed 7 days after cyst inoculations.
glottids (GP) filled with mature eggs under x 100 magnification (Ito 1978; Ito and Yamamoto 1977). The maturity of EdW, the number of mice examined, and the number of EdW examined are summarized in Table II. In this experiment EdW recovered from dd mice were always much larger than those recovered from BALB/c mice: The largest EdW recovered from dd mice was about 150 mm long, whereas that from BALB/c mice was 80 mm long. In both
mouse strains it was observed, after initially established EdW became mature, that some worms became sterile after release of eggs, and often became smaller by destrobilation. These worms were described as worms of short-term maturity. Others remained mature (these were described as worms of long-term maturity). This characteristic, however, differs markedly between the mouse strains used (Table II). Worms of short-term maturity were found
AKIRA
252
Maturity
IT0
TABLE II Hymenolepis nana Recovered from BALBic Inbred Strains of Mice”
of Egg-Derived
Days
after
dd, Random-Bred,
egg inoculation
and
(AEI)
17
27
37
26126
20120
20120
No. of mature worms (MW)/ No. of worms recovered (B)
1981198
1321132
1171144
No. of MWI No. of L-sized
1981198
1321132
65166
35139
worms
(C)
No. of MWI No. of M-sized
o/o
o/o
52177
19180
worms
(D) o/o
o/o
Oil
o/o
dd random-bred strain mice No. of mice found infected/ No. of mice examined (A)
No. of MWI No. of S-sized
worms
35140 54/I 19
(E) Days
BALBlc strain
47
inbred mice A B C D E
AEI
17
19
22
27
37
47
12112 90197 90193 014 o/o
13113 1101135 1041106 6129 o/o
11112 421101 40155 2135 O/II
11114 6123 6/11 o/7 O/5
3114 115 III oi I 013
0113
” The mice examined include dd, random-bred strain mice as well as all BALBlc, inbred strain mice shown in Table I. Egg-derived worms were divided into three size groups: large (L-sized worms: 70 to 150 mm long, 0.7 to 1.2 mm wide), medium (M-sized worms: 30 to 60 mm long, 0.4 to 0.6 mm wide), and small (S-sized worms: 10 to 20 mm long, less than 0.3 mm wide). Maturation
Rates of Cyst-Derived
TABLE III Hymc~nolrpis ncrncr Recovered BALBic Inbred Strains of Mice”
from
Cyst-derived
Group dd random-bred Egg + Cyst Cyst ((3 E+C C E+C C BALBic E+C C
inbred
strain mice (E + C)
worms
No. of mice infected
IO
414 414 717 717 315 515
51 57.3 26.8 53.7 8 50.8
14.6 15.5 23.5 42.0 4.4 20.3
621204 2081229 621184 3661366 0124 2251254
4.2 10.9 5.1 20.2 0 9.9
414 414
69 t 46.3 88.8 2 68.4
O/276 3261555
0 11.4 k 7.6
30 strain
and
Days between egg and cyst inoculations
20*
No. of worms recovered (mean ? SD)
dd, Random-Bred,
t -t t ? + 2
No. of mature worms
No. of GP of mature worms” (mean ? SD)
2 k -t -c
2.2 6.2 2.6 12.8
i
4.4
mice 10
” Mice of E + C groups were initially given 20 shell-free eggs and challenged with 200 cysts, mice of C groups were initially given 200 cysts. All the mice other than those shown by an asterisk (killed 11 days after cyst inoculation (AU)) were killed IO days ACI. ’ Gravid proglottids.
Hym~nolrpis
nancr: SURVIVAL
in dd mice by Day 37 but never by Day 27, whereas in BALB/c mice those were found by Day 17 or 19. In both dd and BALB/c mice, CdW recovered from the mice of the E + C group on Day 17 (challenged with cysts on Day 10) were small (10 to 15 mm long) and immature, whereas those obtained from the control mice of C group were much larger (30 to 60 mm long) and beginning to produce eggs. There was, however, no significant difference between the number of CdW recovered from mice of the E + C and C groups (Table I). Experiment 2. The ability of cysts given in the challenge on Day 10 (in BALB/c mice), or on Day 10, 20, or 30 (in dd mice), to become mature was tested by increasing their development time in the mice from 7 days to 10 or 11 days. Maturation rates of CdW recovered from E + C and C groups were compared. The results are summarized in Table III. EdW recovered from the E and E + C groups are not recorded in this table. It was found that CdW given on Day 10 or even on Day 20 were able to become mature in dd mice, whereas those given on Day 10 in BALB/c mice remained immature.
Infectivity
of Juvenile (7-day Random-Bred,
Age of worms recovered
No. of mice massively infected
dd
‘I-day worms
20120
dd
30-day worms
18120
BALB/c
‘I-day worms
20120
BALBic
30-day worms
2120
” All recipient
Immunity
MICE
253
against Adult Tapeworms
A preliminary experiment, to determine whether lumen-dwelling stages of H. nana, juveniles or adult worms, orally inoculated could establish and develop into mature worms, was done with worms from both dd and BALB/c mice given 2 x lo4 or more shell-free eggs (Table IV). As shown in Table IV, juveniles were recovered by Day 7 from all mice of both strains. However, crowded 30-day worms were recovered from very few BALB/c mice (2/20), but were recovered from almost all dd mice (18/20). Infectivity of these worms was examined 7 days later. The results are summarized in Table IV. These worms were infective via the stomach and became mature. Experiment 3. Based on these findings an experiment was designed to determine whether or not adult tapeworms differed from the cysts in their immunogenicity. For this purpose, dd mice seemed to be much more useful than BALB/c mice because of the longer longevity of H. nana (Tables I, II, and IV). Thus, only dd mice were used as donors and recipients of adult worms. Crowded 30 day-old, adult worms, 5 to 10
TABLE IV Worms) or Adult (30~day Worms) Hymenolcpis or BALBic Inbred Strains of Mice on Oral
Worm recovery from donor mice given a2 x IO4 shell-free eggs
Donor mouse strains
IN
Infectivity
Recipient mouse strains (age) dd (5 weeks) BALBic (IO weeks) dd (IO weeks) dd (5 weeks) dd (IO weeks) dd (30 weeks) dd (I year) dd (5 weeks) BALBlc (5 weeks) Not tested
mice were killed 7 days after worm inoculation.
ncrru Prepared Inoculation
of the worms collected Worm dose administered loo 100 100 100 400 400 100 200 200
from
dd,
from the donors
No. of mice infected 313 414 515 414 717 515 313 313 414
No. of worms recovered Mean k SD 22.7 20.3 23 13.7 63.7 64.8 19 52.3 66.5
-t f t k k i k k t
16.0 9.3 19.8 5.1 56.9 45.3 16.5 10.2 23.6
Range 6-38 14-31 II-57 S-18 19- 172 24- 130 8-38 45-64 47-96
AKIRA
254
mm long, were used as challenges. Maturation rates of 30-day worms just before challenge were variable among populations; some populations consisted of immature worms, but others, in most cases, consisted of mature worms with less than 10 gravid proglottids (GP). Details of this experimental design and the results are summarized in Table V. Mice initially infected with EdW became immune to 30-day worms by Day 40 on the average (between Days 30 and 50), although almost all of these immune mice still retained 40-day-old EdW. However, there seemed to be a tendency for EdW to decrease in numbers between Days 30 and 50 (Tables I and V). The mean number of 30day worm-derived worms (WdW) recovered from the E + W group on Day 47 (challenged on Day 40) was 4.6 ? 2.4, whereas that from the control W group was 54.3 & 15.4. The WdW recovered were of small size, 10 to 20 mm long, but strobilated. Further, some of them were mature worms with not more than 10 GP. By this they were judged to be WdW, but not EdW, since such small-sized EdW, if present, never remained as mature, strobilated
Acquisition
of Protective
HUN,
into
Egg-derived
Days between egg and worm Group
inoculation
515
E&(E) E+W
Worm E E+W W E E+W W E E+W W
IO
(W) 30
40
50
” Mice of E and E + W groups worms. All the mice were killed
a dd
414 014 919 IO/IO O/IO IO/IO 9110 O/IO 5/5 8/R 015
DISCUSSION
These results indicate that mice initially infected with Hymenolepis nana eggs become immune to cyst challenge 10 or more days after egg inoculation. Since an infection, even as great as one with 2000 cysts, does not make the host immune to either cysts or eggs by the seventh day of their prepatent period, whereas a very small number of shell-free eggs such as 5 does make the
Random-Bred
Mean 9.6 10.5 0 7.4 8.3 0 6.3 5.2 0 5.8 5.1 0
Induced Strain 30.day
worms
worms No. of mice infected
forms (see Experiment 1). When 30-day worms were used on Days 10 and 30 to challenge mice previously infected with EdW, the challenging worms became mature, but the maturation rates and sizes of the resulting WdW were somewhat less than those rates and sizes of the WdW from control mice. Autoinfection with more or less than 100 small sized immature worms, 2 to 4 mm long, occurred exclusively in control mice of W groups given mature, 30-day worms (Table V). Clearly a time lag occurred before acquisition of immunity against 30-day worms. This differed from the immunity against cysts, since the mice immunized to cysts retained 30-day worms (Tables I and V).
TABLE V to Adult Tapeworms
Immunity
H~~nr~olrpis
IT0
Range
k 4.0 2 6.5
l- 16 4-17
2 3.7 k 5.4
4-13 lb15
i k
4.3 4.1
I-13 I- I4
i- 4.5 2 4.2
l-12 I- I4
Egg Inoculation
worm-derived
No. of mice infected O/S 4/4 414 019 IO/IO IO/IO O/IO 5/lO IO/IO O/5 018 515
Mean 0 40.8 31.5 0 31.1 37.2 0 4.6 54.3 0 0 46.4
2 SD
Range
f 16.2 5 9.5
21-59 23-42
i k
10.9 10.8
22-44 18-49
i- 2.4 k 15.4
2-8 38-83
2
29-63
were given 20 shell-free eggs, and mice of W and E + W groups 7 days after 30.day worm inoculations.
of
wcmns
No. of wcwms recovered
No. of recovered
2 SD
by Initial of Mouse”
16.1
were given
No. of mice autoinfected 015 014 414 019 O/IO IO/IO O/IO Oil0 4110 O/S O/8 5/5
two hundred
30-day
Hymetiolepis
nana: SURVIVAL
host completely immune to egg challenge within 2 to 5 days after egg inoculation (Hearin 1941; Heyneman 1962a; Hunninen 1935; Ito and Yamamoto 1976; Ito et al. 1978; Shorb 1933; Weinmann 1974), it seems highly probable that mice given eggs of H. nana possess at least two acquired immune responses directed exclusively against reinfections with either eggs (early response to inhibit cyst development in the intestinal villi) or cysts (late response to inhibit adult development in the lumen) as previously speculated (Ito 1978). This hypothesis is comparable to that of Campbell (1938a, b, c) in the relationship between Taenia taeniaeformis and the rat. This finding was consistent in the two mouse strains used which showed markedly different durations of both the time lag before onset of the late response and the survival of initially established worms. Egg-induced immunity to mouse-derived cysts seems to be complete, as demonstrated above, but that to beetle-derived cysts has been reported to be incomplete (Heyneman 1962b, 1963; Isaak et al. 1977). It has been speculated that this difference may be due to the difference in antigenicity of cysts recovered from the mammalian host compared with those recovered from the beetle intermediate host (Ito 1978). This speculation still remains to be proven, but now another explanation of the discrepancy may be possible. Egg-induced immunity to beetle-derived cysts may also be complete. If some of the mice given eggs retained sterile, but variable sized EdW as shown in Table II, the EdW might be mistaken for CdW, since littermate controls given eggs, but not challenged with cysts, were not included in the above authors’ works. If so, egg-induced immunity to cysts may be complete not only to mouse-derived but also to beetle-derived cysts. It is recognized that lumenal stages of H. nana seldom infect mice via the stomach (Caley 1975; Coleman and DeSa 1964; Rothman 1959). Caley (1975) reported that
IN
MICE
255
even cysts could not infect mice. However, earlier work (Ito 1977, 1978) and the present results (Table IV) have demonstrated that freshly prepared lumenal stages, cysts, juveniles, and crowded adult worms, can be used to infect mice orally. At present, it seems to be difficult to explain’this discrepancy, but both the preparation of undamaged worms and administering the worms into the mouse stomach with an excess solution such as 0.5 ml of 0.9% NaCl seem to be important factors in the success of the infection in mice. The fact that EdW survived in mice of both dd and BALB/c strains which had become immune to cysts strongly suggests that the immunogenicity may alter during all its developmental stages, from oncosphere to cyst (Heyneman 1963; Ito et al. 1978) and further from cyst to adult tapeworm (Tables I and V; Ito 1978). The experimental evidence supports the hypothesis that because of the time lag before immune responses are acquired, those against H. nana cannot reveal their functions against the initial immunogenic stages of the infection until the established H. nana stops changing its immunogenicity (Ito 1978). This was previously speculated by Brown (1976) and Weinmann (1970). However, in BALB/c mice the longevity of H. nana of primary infection was clearly much shorter than in dd mice (Tables I, II, and IV; Ito 1978) and it has been proven to be affected by a thymus-dependent immunity of worm expulsion (Isaak et al. 1977). The time lag before acquisition of immunity against the lumenal stages in BALB/c mice did not last as long as in dd mice (Table I) and the immunobiological features differed somewhat between the two mouse strains (Tables II and III). Therefore, we may consider that when the time lag is relatively short compared with the speed of H. nana development in the intestinal lumen, the host’s late response somewhat affects the lumenal stage of a primary infection. The lumenal stage might, then, not always com-
256
AKIRA
plete its immunogenic changes due to development or aging. The number of adult worms retained in the host tends to decrease with elapsed time (Tables I, II, and V; Hunninen 1935; Ito 1978; Shorb 1933; Woodland 1924). The surviving adult worms (EdW) were of the two types, short- and long-term maturity. Worms with a short time to maturity had released all eggs produced after maturation and often (but not always) became smaller than those of the same age with a long time to maturity. These smaller worms had destrobilated as observed in H. diminutu (Befus and Threadgold 1975; Heyneman 1962~; Hopkins et al. 1972). This inequality of size and/or fecundity of worms of the same age emerged days earlier in BALB/c mice than in dd mice (Table II). This phenomenon, previously observed by Shorb (1933) and Woodland (1924), seems to be due to different rates of aging influenced by the host’s immune responses. It therefore seems highly probable that the appearance of worms with a short time to maturity is the first step in “worm expulsion.” This was recently proven to be a thymusdependent immunity (Isaak rf al. 1977). It has been found that BALB/c mice become immune to egg challenge by Day 3 after an immunizing inoculation with 20 shell-free eggs. In contrast, dd mice become immune by Day 4 or 5, but never by Day 3 (Ito, unpublished). Thus, the mode of protection to either eggs or cysts, or the manner of worm expulsion, seemsto differ markedly between mouse strains. However, it is more probable that the duration or degree of maturity of EdW is affected by immune response(s) directed against the lumen-dwelling stages themselves rather than by the early response directed agqinst the oncospheres (Ghazal and Avery 1974). A possibility that the early response enhances or accelerates the late response remains, nevertheless, to be tested. The experimental evidence described above suggests that the longevity of H. mna in a
IT0
mouse strain depends on the length of the time lag prior to acquisition of immune responses directed not to the tissue stage (early response), but to the lumenal stages (late response and worm expulsion response). ACKNOWLEDGMENTS I wish and this
to thank
discussions. work during
Professor
K.
Okamoto
Mrs. Mieko Yamamoto its early phases.
for
advice
cooperated
in
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