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Inhibition of growth of a tapeworm Hymenolepis diminuta in its normal host (rat)
lnternarional JournalforParosirology Printed in Great Briroin
Vol. 2 I . No. 1, pp. 47-55, I99 I 0
002&7519/91 $3.00 + 0.00 Pergamon Press p/c 1991 Ausmdian Societyfoor Parasirology
INHIBITION OF GROWTH OF A TAPEWORM HYMENOLEPIS DIMINUTA IN ITS NORMAL HOST (RAT) C. A. HOPKINS*
and J. ANDREAssENt$
* Wellcome Laboratories for Experimental Parasitology, University of Glasgow, Glasgow, Scotland, t Department of Parasitology, The Institute of Population Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark
U.K.
(Received 24 April 1990; accepted 6 August 1990) Abstract-HoPKmS C. A. and ANDREASSENJ. 1991. Inhibition of growth of a tapeworm Hymenolepis diminuta in its normal host (rat). International Journalfor Parasitology 21: 41-55. The biomass of I-day-old worms of Hymenolepis diminuta in secondary infections, administered to rats 3-10 days after chemotherapeutically expelling a primary infection, was 70-90% less, and the worms were more posteriorly distributed, than in naive controls. The strong depressive effect on growth waned rapidly over 2-5 weeks, but
even in rats not challenged until 17 months later, worm growth was weakly depressed by 30%. The extent to which growth was depressed in a secondary infection was independent of the number of worms in the challenge but increased with number of worms in the immunizing infection up to four to eight worms. Further increase up to 64 worms had little effect. This suggests, as it is known that the biomass of worms in a rat reaches a maximum with infections of between five and 10 worms, that the change in the intestine is proportional to biomass, not number, of worms. It is argued that partially suppressed immunoinflammatory changes in the intestine, which will affect secondary worms so strongly, will also have depressed growth and fecundity effects on the primary worms, that a dynamic equilibrium is reached between the strength of the intestinal response and the biomass of the tapeworm, and that it is reaching this equilibrium, not a ‘crowding effect’, which limits H. diminuta to a level compatible with the survival of the rat. INDEX
KEY WORDS:
Hymenolepis
diminuta; cestodes;
rat; intestine; immunity; growth.
infections as it is in the mouse? The fact that rejection from heavy worm infections is “completely inhibited by cortisone treatment” (Hindsbo, Andreassen & Hesselberg, 1975) and delayed by treating the rat with anti-thymocyte-serum (Hindsbo, Andreassen & Ruitenberg, 1982) suggests an immunological component. Furthermore, Andreassen & Hopkins (1980) observed that a heavy, 50-worm, immunizing infection had a profound effect on the growth of worms in a secondary infection given 1 week after expelling the primary infection, but rats challenged 6 weeks after expulsion of the primary worms were only weakly ‘protected’. Following chemotherapeutic expulsion of a light, five-worm infection, growth of secondary worms administered 1 week later was statistically depressed, but not if administration was delayed for 6 weeks. Resolution of the problem of whether there is a long lasting, presumably immunologically mediated, memory following a primary infection, which depresses the growth of worms in a secondary infection, was made more difficult at the time as, under Scottish laboratory conditions, the Wistar rats gave a much weaker secondary response that they had in Denmark. A very similar problem was encountered by Chappell & Pike (1977) using Hooded Lister rats from two sources, one set rejected worms, the other set did not.
INTRODUCTION THE
rat tapeworm Hymenolepis diminuta is recognized immunologically by a mouse and rejected (Hopkins, Subramanian & Stallard, 1972a,b). A primary infection induces very long, probably life-long, immunological memory (Hopkins, 1982), which leads to growth of worms in a challenge infection stopping within 48 h (Hopkins & Zajac, 1976) and, following pathological changes in the tegument (Befus & Threadgold, 1975; McCaigue, Halton & Hopkins, 1986; McCaigue & Halton, 1987), destrobilation and, later, the expulsion of the scolex. In a rat, no worm loss occurs from a light (up to about 10) worm infection (Harris & Turton, 1973; Hesselberg & Andreassen, 1975; Chappell & Pike, 1976; Pike & Chappell, 1981) but all these workers observed worm loss from heavy infections, often preceded by destrobilation. The questions that arose were as follows: is the rejection of worms from heavy infections brought about by physiological changes in the intestine, often referred to as a ‘crowding effect’, or is it immunologically mediated and, if the latter, why is the process not effective against even single worm
$ To whom all correspondence should be addressed. 47
48
C. A. HOPKINS and J. ANDREASSEN
In this paper, we report on experiments using improved design and methods which have permitted us to measure more precisely the difference in growth of worms in previously infected and uninfected rats. In particular, our objectives have been to (i) verify the previous conclusion that for l-2 weeks following the expulsion of an infection the intestine is strongly inimical to growth of a challenge infection, (ii) determine whether this effect gradually fades completely or whether it fades at first but then remains detectable indefinitely, (iii) determine the relationship between the number of worms in the challenge infection and the strength of the response elicited (in the process, a more precise understanding has emerged of how growth of worms in a primary infection is affected by the number present), and (iv) determine the effect of number of worms in a primary infection on the gro’wth of a challenge infection. MATERIALS AND METHODS Specific pathogen-free (SPF) 0 Wistar rats were purchased at 67 weeks of age from the University of Nottingham Animal Unit and kept under conventional conditions. Rats were caged in sixes and, unless stated to the contrary, three rats in each cage were given a primary immunizing infection of Hymenolepis diminuta when 8 weeks rt 2 days old and the other three used as controls. All rats received an anthelmintic, on day 2 1 (or 28) of the immunizing infection, which was repeated on day 22 (or 29). In most experiments, oxyclosanide (Zanil, I.C.I.) was used, given by stomach tube at a dose level of 170 mg kg-’ (Hopkins, Grant & Stallard, 1973). In later experiments, the safer drug, praziquantel (Droncit, Bayer) was used at 50 mg kg-‘. (A tablet of ‘Droncit’ containing 50 mg of praziquantel was crushed, dissolved in 0.3 ml of Cremophor EL and diluted with water to 5 ml; the required dose, 1 ml for a 200 g rat, was administered by stomach tube.) H. diminuta has been maintained by passage through rats or hamsters and the beetle Triboiium confusum since it was obtained in 1963 from Rice University, Houston. Cysticercoids recovered by dissecting beetles, usually 3-6-week-old infections, in modified Hanks’ balanced salt solution (HBSS) (Hopkins & Barr, 1982), were administered by stomach tube within 1h. Worms were recovered by flushing out the intestine with HBSS. If the recovery was < lOO%, the intestine was slit longitudinally and examined under warm HBSS. On the few occasions where worm recovery was c 80%, the intestine was cut into l&15 cm sections and incubated in HBSS at 37-38-C for 1-3 h. At intervals, the incubating dish was examined for worms which had detached from the intestine. This latter procedure reveals very small, 0.5-5 mm worms but is not quantitative; however, such small worms have virtually no effect on the total biomass. Worm biomass (total mass of worms in a rat), determined after drying the worms at 95°C for l-3 days, was transformed to a log base before calculating the mean and S.D. For ease of interpretation, the antilog of this mean and S.D.is used in the figures and text. The justification for using log transformed data is that H. diminuta is growing almost at an exponential rate on day 8, the age at which worms were recovered. Statistical analysis was by Wilcoxon rank test, except where stated, and differences were considered statistically significant when P I 0.05. Note on drug eficacy. The double treatment with oxyclosanide at I70 mg kg-’ is very effective but, in heavy
infections, a worm very occasionally survives. Presumably this is because in heavy infections some worms may be attached much further back in the intestine. In the present work, two of the 168 rats treated with oxyclosanide harboured a primary worm at autopsy and were eliminated from the experiment. Because the chance of survival is so low, killing of a drug control group is of no predictive value and hence unjustifiable. However, primary worms which have survived, even if drug induced destrobilization has occurred, are immediately identifiable. Tests showed that no inhibition of growth occurred in worms administered 2 or more days after ‘Zanil’ treatment and as the challenge infection, with two exceptions, was never given until at least 6 days after the second drug treatment, any surviving primary worm had grown 4 or more days before the secondary infection was given. At autopsy 8 days later, a surviving primary worm would be at least the equivalent of a 12-day-old worm in size which, as worms between 8 and 12 days of age are approximately doubling their mass daily, is instantly distinguishable from an S-day-old-worm. We have had no survival of H. diminuta following praziquantel at 50 mg kg _’ in tests or in any experiment during extensive use over 4 years.
RESULTS Duration of immunological memory Five-worm primary, #-worm secondary. Immunized and control rats were kept separately at six per cage. The five-worm immunizing infection was removed by treatment with ‘Zanil’ days 21 and 22 postinfection (pi.). Three days (day 24) to 7 weeks later, one cage of control and one of immunized rats were challenged with 40 cysticercoids. Worms were recovered when 8 days old by pinning the intestine on a black wax-tray, opening longitudinally and lightly covering with HBSS. This is not as reliable a method as that used in the other experiments (Materials and Methods) for finding small (C 1 cm long) worms, especially if they 130 c
3d
I
2
3
4
5
6
7
Weeks after primary worms removed
FIG. I. Effect in rats of a five-Hymenolepis diminuta primary infection, removed 21 days pi., on the biomass of 8-day-old worms in 40-worm challenge infections. Mean worm biomass & s. D. in controls (0, primary infected) and immunized (0, secondary infected) rats. n = 6, except for five in immunized group day 3 and control group week 5.
Inhibition
of H. diminuta in the rat
of growth
.
49
A
.
P
I
20
510 Day
rat
35
was ctwllenged
115
70
after
primary
infection
150
terminated
FIG. 2. Effect in rats of a 16-worm Hymenolepis diminuta primary infection on the growth of worms in a secondary infection of 20 worms, given S-150 days after expulsion of the primary, immunizing infection on day 28. Lower plot: mean worm biomass f S.D. (n = 9) per rat of 8-dayold worms from immunized (0) and control (0) rats. Upper plot: A, mean worm biomass per rat in immunized rats as a % of that in control rats.
are in the posterior third of the small intestine, with food debris f mucus, but was used in order to determine the position of the worms. Mean worm recovery was 84% (range 82-87) in the six groups of control rats and 85% (84-85) in the immunized groups challenged after 3-7 weeks, but only 71% (67-73) in the 3 days-2 weeks challenged groups (Fig. 1). Growth of worms in rats challenged 3 days-2 weeks after the primary worms had been expelled was strongly depressed. This effect weakened but worm growth was still depressed by 30% after 5-7 weeks. Differences were statistically significant (‘P test), except in week 5, where t = 2.113, P < 0.1. Mean worm position, expressed as distance from the pylorus , was as follows: in control rats infected on day 3,21 cm, S.D. 12 cm; week 1,23 f 13 cm; week 2,23 f 14 cm; in the immune rats,
38 f 22 cm, 43 f 20 cm and 38 f 22 cm, respectively. In rats challenged 3, 5 and 7 weeks after the primary worms had been expelled, the mean position of the worms in the immune rats (39,47 and 43 cm) did not differ significantly from those in the controls (37, 34 and 46 cm). Mean length of the small intestines was approximately 100 cm in control groups but 3-9 cm longer in immunized groups. Fifly-worm primary, 40-worm secondary. To determine whether a heavier primary infection would modify the response, an experiment was set up using the same protocol, except that rats were immunized with 50 cysticercoids and worm recovery was by flushing and incubating (see Materials and Methods). The biomasses of the 40-worm challenge infections in immunized rats, infected 1, 2, 3 and 15 weeks after
50
C. A. HOPKINSand J. ANDREASSEN
removal of the primary infection, were 11, 54, 59 and 68%, respectively of that of the worms in the control groups. The difference between the worms from immunized rats (mean 45.2 mg per rat) and controls (mean 66.2 mg per rat) challenged 15 weeks after expulsion of the immunizing infection was highly significant (P < 0.01). Expressed as a % of the mean, inter-rat variance in worm biomass was much less 15 weeks after the immunizing infection had been expelled (log mean biomass 1.655, SD. 0.036 mg) than shortly after, e.g. 1 week (log mean 1.012, S.D. 0.395 mg). Sixteen-worm primary, 20-worm secondary. This experiment (Fig. 2) differed from the previous ones in that: rats on removal from the SPF breeding colony were kept in an isolation room under standard light and temperature conditions throughout the experiment. Cages were arranged in a specific pattern on the racks, to minimize spatially distributed variables. Empty cages were substituted when full ones were removed. The immunizing infection, 16 worms, was for 28 days and removed using praziquantel. Mean worm biomass was calculated by recovering worms from nine immune or control rats. Worm recovery was 98.8% (1245 worms from 1260 cysticercoids) in the controls and 95.5% (1203 worms) from the immunized rats. Secondary worms in all groups were significantly lighter that the controls, P < 0.01, except on day 115, when P = 0.05. The initial resistance, which depressed growth by over 80% (day 5, 82.3%; day 10, 82.9%) fell to 44% by day 35 and then probably reached a plateau around 30% (Fig. 2, A). Long-term memory: 30-worm primary, 20-worm secondary. Twelve of 24 rats were immunized with 30 worms and treated with ‘Zanil’ after 21 days. Nine control and nine immunized rats were challenged 36 weeks later and worms recovered on day 8. The mean number and biomass of worms in the control rats were 19.8 and 67 mg, in the immune rats 19.4 and 46 mg, i.e. growth was suppressed by 3 1%. The difference was highly significant, P < 0.01. The other six rats were challenged after 17 months (52 1 days); mean recovery was 19.3 worms per rat in both groups and mean worm biomass was 100 mg in the controls and 60 mg in the immune rats. Taken in conjunction with the above results, this small experiment suggests that the longterm component of memory persists as long as the rat lives. @hence
of number of worms in the secondary infection on the degree ofprotection expressed by immunized rats The design of the next two experiments was similar, except that in Experiment 1 the immunizing infection was 50 worms and in Experiment 2 it was 30 worms. (This difference in the immunizing infection will have had little if any effect-see next section.) The immunizing infection was terminated by ‘Zanil’ on days 21 and 22 pi. Control (previously uninfected) and immunized rats were infected on day 28 with l-50 cysticercoids. Rats were killed on day 36, when the
FIG. 3. Effect of the number of Hymenolepis diminuta in an infection on the biomass of S-day-old worms recovered from primary (0, a) and secondary (0, A) infections in rats. Solid regression lines calculated from 0 and 0 data. Broken lines are extrapolations. A, A Replicate experiment. Points are means of six or nine rats, see Results. infections were 8 days old. In Experiment 1, each point (Fig. 3, 0, 0) is the mean worm biomass from six rats, three in each of two cages. The result of the two-worm challenge to the immunized rats is not plotted, as in one cage the mean burden per rat was 5.6 mg, in the other cage, 10.6 mg, which is similar to the controls (10.1 mg, Fig. 3, 0). This suggests that the immunizing infection had been missed in the rats in the second cage but, as it could indicate a threshold at which a response may or may not be evoked, the number of rats in Experiment 2 (Fig. 3, A, A) was increased to nine in the one- and two-worm control and immunized groups. The mean number of worms recovered per group in Experiment 1 was: rats infected with two cysticercoids, controls loo%, immunized 100%; with five cysticercoids, 100 and 97%, respectively; with 10, 98 and 97%; with 20,93 and 96%; with 35,97 and 96%; and with 50 cysticercoids, 93 and 90%. As the difference between the number of cysticercoids administered and worms recovered never exceeded lo%, and the difference between the number of worms recovered from an immunized group and its control did not exceed 3%, no correction for these minor differences has been made in plotting the results and calculating the regression lines (Fig. 3). Plotting the results of Experiment 1 showed that the best fit to a straight line was obtained when both the dependent variable (total biomass of worms in a rat, 8
51
Inhibition of growth of H. diminuta in the rat days p.i.) and the independent variable (number of worms in the infection) were plotted on logarithmic scales (Fig. 3, 0, 0). Worm biomass in relation to number of worms in the control rats, i.e. in a primary infection Examination of the data showing worm biomass per rat in relation to number of worms in the control rats (Fig. 3, 0) reveals a close fit (r = 0.997) to the calculated regression line: logy
= 0.757 + 0.802 log x.
Thus the regression line predicts that a single worm infection should have on day 8 a biomass of 5.7 mg (log 0.757) and that doubling the number of worms leads to a 74% increase in biomass. Experiment 2 was carried out, inter alia, to test these predictions. The mean mass of one-worm infections in the control rats was 6.3 mg (predicted 5.7), of twoworm infections, 9.5 mg (predicted 10.0) and of loworm, 42.5 mg (predicted 36.5). It can be seen that these results (Fig. 3, A) fit closely the regression line calculated from the data of Experiment 1.
Znjruence of number of worms in the immunizing (primary) infection on the strength of protection induced against a secondary infection Rats were immunized with two-, five-, IO- or 20cysticercoid infections. ‘Zanil’ was administered on days 28 and 29. All rats were challenged with 20 cysticercoids on day 35 and killed 8 days later. Six immunized and six control rats were necropsied at each level of infection to determine the protection induced. Excluding two immunized rats from which only 11 and 12 worms were recovered, no rat had less than 17 worms and the mean recovery was 19.4 worms per control rat and 18.7 per immunized rat.
.
.
.
Worm biomass in relation to number of worms in a secondary infection The logarithm of worm biomass in the immune rats in Experiment 1 (Fig. 3, 0) also showed a linear correlation with the logarithm of the number of worms, r = 0.990. The calculated regression line is: logy
= 0.422 + 0.707 log x.
The predicted value of y when x = 1 is 2.6 mg and &hen x = 2, 4.3 mg; the observed values (Fig. 3, A) were 3.1 and 4.5 mg, respectively. The predicted biomass of a IO-worm challenge was 13.5 mg, observed 16.5 mg. The results of Experiment 2 showed that extrapolation of the regression line back to a oneworm challenge was justified, i.e. no threshold exists below which the growth of secondary worms is not affected. The strength of the immune response in relation to number of worms in the challenge Visual inspection of the slope of the two regression lines in Fig. 3 suggests that they are nearly parallel. Covariance analysis reveals no significant difference between the regression coefficients (F = 2.695, giving P > 0.1 < 0.25). This implies that the strength of the response expressed against a secondary infection is either not, or only slightly, affected by the number of worms in the secondary infection. The difference in elevation between the two lines is highly significant (F = 258.5; P < < 0.01) and indicates that the growth of the worms in the rats, challenged 7 days after a 50-worm immunizing infection had been removed, was only 40% of that in the controls, i.e. growth had been 60% suppressed.
FIG.4. Effect in rats ofthe number OfHymenolepis diminuta in the immunizing infection on the growth of a 20-worm challenge given on day 35, 7 days after expelling the immunizing infection. Lower plot: mean worm biomass f SD (n = 6) of 8-day-old worms per rat in immunized (0) and in control (0) rats. Upper plot: A, mean worm biomass per rat in immunized rats as a % of that in control rats. The S.D. of the biomass of worms (lower plot, Fig. 4) reveals that within control groups there was considerable uniformity but that within immunized groups, the rats were much more variable in their ability to support worm growth. The upper plot (Fig. 4) indicates the ambiguity of the results. The points fit a regression line log y = 1.8803 - 0.2545 log x quite well, r = - 0.9537, implying that the intestine is altered in proportion to the log of the number of worms in the infection; but the plot could also signify
52
C. A.
HOPKINS and J. ANDREASSEN
TABLE I-EFFECXOFVARYINGTHENUMBEROF Hymenolepisdiminuta INTHEIMMUN~UNGINFECTIONTERMINATEDONDAY GROWTHOVER 8 DAYSOFA 20-WORMSECONDARYINFEflIONINRATSGlVENONDAY 35
Number of worms in immunizing infection 1 2 4 8 16 32 64
Worms in control* rats? Number Biomass per recovered, rat, mean f s.o. mean (mg) 18.8 19.3 18.8 19.3 18.0 19.3 19.0
54f 12 61f 8 46f 11 52zk 9 54f 13 61 f 20 54f 8
Number recovered, mean 19.3 19.4 19.0 17.8 17.4 13.4 17.1
Worms in immunized rats? Rats arranged according to worm biomass Rat 1 Rat 2 Rats 3-6, Rats 7-8, mean mean (mg) (mg) (mg) (mg) 65 62 27 28 24 41 20
51 56 11 8 10 9 9
48 41 8 2.8 3.2 3.8 3.8
31 26 1.1 1.2 0.5 0.2 0.3
28 ONTHE
Overall depression in growth$ (%I 12 32 81 88 89 87 90
* Twenty-worm primary, no immunizing infection. t n = 8. Rat 1 was the rat with the heaviest biomass of worms, rat 2 the second heaviest, rats 3-6 the next four heaviest and rats 7-8 the lightest worm biomass, arithmetic means. $ % decrease in mean biomass of worms from the immunized rats compared with the controls.
that above five to 10 worms, there was little additional effect. To determine which was the correct interpretation, we carried out a second experiment. The same protocol was used, except that the immunizing infection was one to 64 worms and the number of rats at each level of infection was nine immunized and nine controls. In four of the control groups, one rat had very few and/or very light worms. Inclusion of such rats would have distorted the mean number and mass ofworms per rat (Table 1, columns 2 and 3). To avoid possible bias, we excluded the rat with the lightest worm biomass from all groups. To better illustrate the variation in response within the groups of immunized rats, the results are set out in detail (Table 1). Immunizing with one to four worms showed a progressive effect. A one-worm immunizing infection depressed growth by 12%. Doubling the immunizing dose led to an approximately 150% increase in growth depression (column 9). Worm recovery, 96% (column 4), in these weakly immunized rats was similar to that of the controls. In the groups of rats immunized with eight to 64 worms, the response pattern was similar and can be divided into four categories: Rat 1: A low responder rat (Table 1, column 5) in which worms grew well, reaching 4&60% of the mean mass of the control worms (column 3). Rat 2: A rat in which appreciable worm growth occurred (column 6). Rats 3-6: Four rats in which growth was strongly depressed (column 7). Rats 7-8: Two (or three, if the ninth rat is included) high responder rats in which, with one exception, growth was suppressed by over 99% (column 8). Only one of the 32 rats immunized with eight to 64 worms had a worm biomass as great as the minimum biomass in a control rat. In particular, it should be noted how approximately 50% of the total biomass of worms from the group of eight rats depended on the
chance occurrence of one low responding rat. Worm recovery was lower than in the control groups (cf. columns 4 and 2) but this was probably due to failure to find some of the very small worms, which would have had a negligible effect on the biomasses recorded. Two general points emerge from the table. One, the maximum immunizing effect is reached by an infection of four to eight worms (column 9). Two, growth of primary worms in the control rats was low compared with that in the other experiments described here and worm growth in secondary infections was depressed even more markedly, i.e. the rats were more responsive to an H. diminuta infection. DISCUSSION The response by a rat to a primary infection with HymenoIepis diminuta can be divided into two phases. For 2-4 weeks following the chemotherapeutic expulsion of a primary infection, growth of secondary worms is strongly depressed. This ‘strong response’ phase wanes rapidly but is followed, after about 5 weeks, by a second phase, which persists with little change (examined up to 17 months), during which growth of worms is weakly (by about 30%) but consistently depressed (Figs. 1,2 and text). This pattern of host response is similar to, though weaker than, that shown by a number of hosts following rejection of intestinal nematodes (Miller, 1984). A challenge infection shortly after spontaneous cure has occurred is marked by an acute ‘rapid expulsion’ response but later stimulates only an anamnestic response. ‘Rapid expulsion’ was shown by Bell & McGregor (1980) to involve two components, one immunologically specific, the other non-specific. It seems probable that the non-specific component is linked to the persistence of the hyperplasia of inflammatory cells (reviewed by Miller, 1987a,b). There is little direct evidence for the involvement of inflammatory cells against H. diminuta, though
Inhibition of growth of H. diminutu in the rat Hindsbo et al. (1982) observed small increases in mast cells and eosinophils in the mucosa of infected rats. However, indirect evidence of the importance of a non-specific inflammatory response modulating the growth of H. diminuta is strong. When an inflammatory response was induced by administering Trichinella spiralis, the growth of a concurrent H. dimi~uta infection, 650% of the controis, was inversely proportional to the log of the number of T. spiralis and hence possibly to the strength of the inflammatory response (calculated from Christie, Wakelin & Wilson, 1979). Likewise, the growth of H. diminuto in concurrent infections with Nippostrongylus brasifiensis, over the period when the latter are rejected, was depressed by over 98% (Hopkins, unpublished). Clearly, growth of H. diminuta in the rat is depressed by a non-specific heterologous inflammato~ response and to an extent similar to that observed in a challenge H. diminuta infection given shortly after expelling a primary infection (Table 1). The high level of anti-worm reactivity expressed by the intestinal wall shortly after expelling an infection will, presumably, have existed and have been at least as strong just prior to the expulsion of the primary worms. This implies either that the growth and fecundity of the primary worms was also depressed, or that worms ‘adapt’ during development (cf. Howard, 1977). Assuming this antiworm effect is modulated by immune and/or inflammatory cells, it would be interesting to know whether the biomass and fecundity of a mature worm infation increase when an agent such as cortisone is administered. The strength of the response induced in rats depended upon worm number up to between four and eight (Table I, Fig. 4) but two lines of work suggest that the critical parameter was biomass, not number of worms. Firstly, Hopkins & Barr (1982) who infected rats with 100 irradiated cysticercoids (65% of which survived the 21 days immunizing period but had negligible biomass) found, on challenging with normal cysticercoids 7 days later, that growth of worms was not significantly different from that in the controls, whereas in mice, eight irradiated cysticercoids induced strong protection. Secondly, a number of investigators (Read, 1951; Roberts, 1961; Chappell & Pike, 1976, 1977; Boddington & Mettrick, 1981) have shown that the biomass of H. diminuta, determined 17-28 days p.i., usually increases with numbers of up to five to 10 worms but thereafter there is little increase. [The linear relationship, when expressed on a log basis, between number of worms, one to 50, and biomass, in a primary infection (Fig. 3) is not a contradiction to the foregoing, in fact it is what would be expected if the depressive response on growth is proportional to biomass. We measured growth only over 8 days p.i., during much of which time the biomass is very small and hence stimulation of the intestine would be small and therefore evoke little counter-effect on growth of even a 50-worm infection.] It appears therefore that in a rat, unlike in a mouse,
53
the response engendered by H. diminuta is related to worm biomass. [See also the greater strength (Table 1) and duration (Fig. 2) of the strong response in experiments where the immunization period was increased from 21 to 28 days.] This would be a tolerable compromise between a ‘good’ parasite and a ‘good’ host; as worm mass increases, the intestinal (inflammatory?) response increases, which depresses growth to an equilibrium. The situation is certainly more complicated than this because, when rats are infected with large numbers, e.g. 40-100 worms, the equilibrium is overshot and an intense response leads to loss of worms in the soft, even diarrhoeal, faeces for 2-3 days, In other cases, worms become widely dispersed in the intestine and partially or completely destrobilated (Hindsbo et al., 1982). Whether the strong intestinal response is induced by direct physicochemical stimulus from the large mass of worm, which judging by the extensive villous fusion and atrophy induced (Martin & Holland, 1984) is considerable, or whether it is mediated through a weakened antigenspecific response is unknown, though the existence of long memory strongly suggests that the ‘weak response’ at least is lymphocyte mediated. The question also arises as to what causes the very poor growth of secondary worms during the strong response phase. The evidence is clear that the intestinal milieu has been altered, the depression in worm growth is almost, if not completely, independent of the number of worms in the challenge (Fig. 3), and the position of the worms in the intestine is quite different from that in control rats. Harris & Turton (1973), Choromanski (1980) and Machnicka & Choromanski (1983) have reported the formation of specific antibodies and sensitized macrophages and it is known that in vitro complement damages the tegument and induces destrobilation (Christensen, Bogh & Andreassen, 1986), but whether any of these are important in vivo has yet to be demonstrated. One point that may be relevant is that worm recovery from rats in the strong response phase often involved untangling a mass of ‘sticky’ worms bound in long tubes of mucus, which supports Bell, Adams & Ogden’s (1984) observations and Miller’s (1987b) conclusion that “mucus serves an important function in protection . . . “. Group size and method of caging and housing rats were changed in later experiments (see text accompanying Fig. 2). This was necessary because a depression of about 30% in growth of secondary worms, which commonly occurred during the weak-response phase, was often too small to be statistically significant, because of the large inter-rat variance in worm biomass. Equally worrying was an observation made in some other experiments that considerable inter-cage variance in mean worm biomass could occur between groups of rats supposedly treated identically. Without knowing the causes of either inter-rat or inter-cage variance, an empirical approach was adopted. To avoid the possibility that any difference between worm
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C. A. HOPKINS and J. ANDREASSEN
growth in control and immunized rats arose from inter-cage variation, control and immunized rats were caged three plus three together. However, by adding inter-cage variance to groups of control and immune rats, variance in mean worm biomass was increased. This was countered by increasing the number of rats to nine per group. The immunizing period was increased to 28 days, because this had been very effective (Table 1) but the influence of the immunizing period requires further investigation (vi& supra). Finally, the number of worms in the immunizing dose was greatly reduced, with much saving of time, as an experiment (Table 1) had shown that at least the short-term response is as strongly induced by eight as by 64 worms, and another experiment (Fig. 1) suggested that the weak long-term response was also induced by a low immunizing infection. We conclude that H. diminuta survives in a rat because of a partial defect in, or suppression of, the antigen-specific response but that the non-specific (inflammatory?) defence cells are stimulated, directly or indirectly, in proportion to the mass of tapeworm, thereby increasingly inhibiting growth and leading to the establishment of a dynamic equilibrium. Acknowledgements-We wish to thank Miss Iona Barr for technical assistance, the M.R.C., London, for financial support (Grant G 8009375T) and the Danish Natural Science Research Council for a travel grant to Glasgow for J. A. REFERENCES ANDREASSEN
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&
HOPKINS
C. A. 1980. Immunologically mediated rejection of Hymenolepis diminuta by its normal host, the rat. Journal of Parasitology 66: 898-903. BEFUS A. D. & THREADC~LD L. T. 1975. Possible immunological damage to the tegument of Hymenolepis diminuta in mice and rats. Parasitology 71: 525-534. BELL R. G. & MCGREGOR D. D. 1980. Requirement for two discrete stimuli for induction of the intestinal rapid expulsion response against Trichinella spiralis in rats. Infection and Immunity 29: 186-193. BELL R. G., ADAMS L. S. & OGDEN R. W. 1984. Intestinal mucus trapping in the rapid expulsion of Trichinella spiralis by rats: induction and expression analyzed by quantitative worm recovery. Infection and Immunity 45: 267-272. BODDINGTONM. J. & METTRICK D. F. 1981. Production and reproduction in Hymenolepis diminuta (Platyhelminthes: Cestoda). Canadian Journal of Zoology 59: 1962-1972. CHAPPELL L. H. & PIKE A. W. 1976. Loss of Hymenolepis diminuta from the rat. International Journal for Parasitology 6: 333-339. CHAPPELL L. H. & PIKE A. W. 1977. Interactions between Hymenolepis diminuta and the rat. In: Biochemistry of Parasites and Host-Parasite Relationships (Edited by VAN DEN BOSSCHEH.), pp. 379-384. Elsevier/North Holland, Amsterdam. CHOROMANSKIL. 1980. Humoral and cellular responses in mice and rats infected with Hymenolepis diminuta. Bulletin de I’AcadPmie Polonaise des Sciences. Serie des Sciences Biologiques 28: 707-7 13. CHRISTENSEN J. P. B., BBGH H. 0. & ANDREASSENJ. 1986. Hymenolepis diminuta: the effect of serum on different ages
of worm in vitro. International Journalfor Parasitology 16: 447453. CHRISTIEP. R., WAKELIND. &WILSON M. M. 1979. The effect of the expulsion phase of Trichinella spiralis on Hymenolepis diminuta infection in rats. Parasitology 78: 323330. HARRIS W. G. & TURTON J. A. 1973. Antibody response to tapeworm (Hymenolepis diminuta) in the rat. Nature (London) 246 521-522. HESSELBERGC. A. & ANDREASSENJ. 1975. Some influences of population density on Hymenolepis diminuta in rats. Parasitology 71: 517-523. HINDSBO O., ANDREASSEN J. & HESSELBERG C. A. 1975. Immunity to Hymenolepis diminuta in the rat. Norwegian Journal of Zoology 23: 197. HINDSBO O., ANDREASSENJ. & RUITENBERG J. 1982. Immunological and histopathological reactions of the rat against the tapeworm Hymenolepis diminuta and the effects of antithymocyte serum. Parasite Immunology 4: 5976. HOPKINS C. A., SUBRAMANIANG. & STALLARD H. 1972a. The development of Hymenolepis diminuta in primary and secondary infections in mice. Parasitology 64: 401412. HOPKINS C. A., SUBRAMANIANG. & STALLARDH. 1972b. The effect of immunosuppressants on the development of Hymenolepis diminuta in mice. Parasitology 65: 11 l120. HOPKINS C. A., GRANT P. M. & STALLARD H. 1973. The effect of oxyclozanide on Hymenolepis microstoma and H. diminuta. Parasitology 66: 355-365. HOPKINS C. A. & ZAJAC A. 1976. Transplantation of Hymenolepis diminuta into naive, immune and irradiated mice. Parasitology 73: 73-8 1. HOPKINSC. A. 1982. Immunological memory in mice to adult Hymenolepis diminuta (Cestoda). Journal of Parasitology 68: 32-38. HOPKINSC. A. & BARR I. F. 1982. The source of antigen in an adult tapeworm. International Journalfor Parasitology 12: 327-333. HOWARD R. J. 1977. Hymenolepis microstoma: a change in susceptibility to resistance with increasing age of the parasite. Parasitology 75: 241-249. MACHNICKA B. & CHOROMANSKIL. 1983. Immune response and immunodepression in Hymenolepis diminuta infection in rats. Zeitschrtftfiir Parasitenkunde 69: 239-245. MARTIN J. & HOLLAND C. 1984. Scanning electron microscope studies of the mucosa of rats infected with H. diminuta (Cestoda). Journal of Helminthology 58: 93-99. MCCAIGUE M. D., HALTON D. W. & HOPKINS C. A. 1986. Hymenolepis diminuta: ultrastructural abnormalities in worms from C57 mice. Experimental Parasitology 62: 51L 60. MCCAIGUE M. D. & HALTON D. W. 1987. Immunological damage to Hymenolepis diminuta following a challenge infection in C57 mice. International Journal for Parasitology 17: 795-803. MILLER H. R. P. 1984. The protective mucosal response against gastrointestinal nematodes in ruminants and laboratory animals. Veterinary Immunology and Immunopathology 6: 167-259. MILLER H. R. P. 1987a. Immunopathology of nematode infestation and expulsion. In: Immunopathology of the Small Intestine (Edited by MARSH M. N.), pp. 177-208. John Wiley & Sons, Chichester. MILLER H. R. P. 1987b. Gastrointestinal mucus, a medium
Inhibition
of growth
for survival and for elimination of parasitic nematodes and protozoa. Parasitology 94: S77-100. PIKE A. W. & CHAPPELL L. H. 1981. Hymenolepis diminuta: worm loss and worm weight loss in- long-term infections of the rat. Experimental Parasitology 51: 3541.
of H. diminuta in the rat
55
READ C. P. 1951. The ‘crowding effect’ in tapeworm infections. Journal of Parasitology 37: 174178. ROEIERT~L. S. 1961. The influence of population density on patterns and physiology of growth in Hymenojepis diminuta (Cestoda: Cyclophyllidea) in the definitive host. Experimental Parasitology 11: 332-37 1.
Vol. 2 I . No. 1, pp. 47-55, I99 I 0
002&7519/91 $3.00 + 0.00 Pergamon Press p/c 1991 Ausmdian Societyfoor Parasirology
INHIBITION OF GROWTH OF A TAPEWORM HYMENOLEPIS DIMINUTA IN ITS NORMAL HOST (RAT) C. A. HOPKINS*
and J. ANDREAssENt$
* Wellcome Laboratories for Experimental Parasitology, University of Glasgow, Glasgow, Scotland, t Department of Parasitology, The Institute of Population Biology, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark
U.K.
(Received 24 April 1990; accepted 6 August 1990) Abstract-HoPKmS C. A. and ANDREASSENJ. 1991. Inhibition of growth of a tapeworm Hymenolepis diminuta in its normal host (rat). International Journalfor Parasitology 21: 41-55. The biomass of I-day-old worms of Hymenolepis diminuta in secondary infections, administered to rats 3-10 days after chemotherapeutically expelling a primary infection, was 70-90% less, and the worms were more posteriorly distributed, than in naive controls. The strong depressive effect on growth waned rapidly over 2-5 weeks, but
even in rats not challenged until 17 months later, worm growth was weakly depressed by 30%. The extent to which growth was depressed in a secondary infection was independent of the number of worms in the challenge but increased with number of worms in the immunizing infection up to four to eight worms. Further increase up to 64 worms had little effect. This suggests, as it is known that the biomass of worms in a rat reaches a maximum with infections of between five and 10 worms, that the change in the intestine is proportional to biomass, not number, of worms. It is argued that partially suppressed immunoinflammatory changes in the intestine, which will affect secondary worms so strongly, will also have depressed growth and fecundity effects on the primary worms, that a dynamic equilibrium is reached between the strength of the intestinal response and the biomass of the tapeworm, and that it is reaching this equilibrium, not a ‘crowding effect’, which limits H. diminuta to a level compatible with the survival of the rat. INDEX
KEY WORDS:
Hymenolepis
diminuta; cestodes;
rat; intestine; immunity; growth.
infections as it is in the mouse? The fact that rejection from heavy worm infections is “completely inhibited by cortisone treatment” (Hindsbo, Andreassen & Hesselberg, 1975) and delayed by treating the rat with anti-thymocyte-serum (Hindsbo, Andreassen & Ruitenberg, 1982) suggests an immunological component. Furthermore, Andreassen & Hopkins (1980) observed that a heavy, 50-worm, immunizing infection had a profound effect on the growth of worms in a secondary infection given 1 week after expelling the primary infection, but rats challenged 6 weeks after expulsion of the primary worms were only weakly ‘protected’. Following chemotherapeutic expulsion of a light, five-worm infection, growth of secondary worms administered 1 week later was statistically depressed, but not if administration was delayed for 6 weeks. Resolution of the problem of whether there is a long lasting, presumably immunologically mediated, memory following a primary infection, which depresses the growth of worms in a secondary infection, was made more difficult at the time as, under Scottish laboratory conditions, the Wistar rats gave a much weaker secondary response that they had in Denmark. A very similar problem was encountered by Chappell & Pike (1977) using Hooded Lister rats from two sources, one set rejected worms, the other set did not.
INTRODUCTION THE
rat tapeworm Hymenolepis diminuta is recognized immunologically by a mouse and rejected (Hopkins, Subramanian & Stallard, 1972a,b). A primary infection induces very long, probably life-long, immunological memory (Hopkins, 1982), which leads to growth of worms in a challenge infection stopping within 48 h (Hopkins & Zajac, 1976) and, following pathological changes in the tegument (Befus & Threadgold, 1975; McCaigue, Halton & Hopkins, 1986; McCaigue & Halton, 1987), destrobilation and, later, the expulsion of the scolex. In a rat, no worm loss occurs from a light (up to about 10) worm infection (Harris & Turton, 1973; Hesselberg & Andreassen, 1975; Chappell & Pike, 1976; Pike & Chappell, 1981) but all these workers observed worm loss from heavy infections, often preceded by destrobilation. The questions that arose were as follows: is the rejection of worms from heavy infections brought about by physiological changes in the intestine, often referred to as a ‘crowding effect’, or is it immunologically mediated and, if the latter, why is the process not effective against even single worm
$ To whom all correspondence should be addressed. 47
48
C. A. HOPKINS and J. ANDREASSEN
In this paper, we report on experiments using improved design and methods which have permitted us to measure more precisely the difference in growth of worms in previously infected and uninfected rats. In particular, our objectives have been to (i) verify the previous conclusion that for l-2 weeks following the expulsion of an infection the intestine is strongly inimical to growth of a challenge infection, (ii) determine whether this effect gradually fades completely or whether it fades at first but then remains detectable indefinitely, (iii) determine the relationship between the number of worms in the challenge infection and the strength of the response elicited (in the process, a more precise understanding has emerged of how growth of worms in a primary infection is affected by the number present), and (iv) determine the effect of number of worms in a primary infection on the gro’wth of a challenge infection. MATERIALS AND METHODS Specific pathogen-free (SPF) 0 Wistar rats were purchased at 67 weeks of age from the University of Nottingham Animal Unit and kept under conventional conditions. Rats were caged in sixes and, unless stated to the contrary, three rats in each cage were given a primary immunizing infection of Hymenolepis diminuta when 8 weeks rt 2 days old and the other three used as controls. All rats received an anthelmintic, on day 2 1 (or 28) of the immunizing infection, which was repeated on day 22 (or 29). In most experiments, oxyclosanide (Zanil, I.C.I.) was used, given by stomach tube at a dose level of 170 mg kg-’ (Hopkins, Grant & Stallard, 1973). In later experiments, the safer drug, praziquantel (Droncit, Bayer) was used at 50 mg kg-‘. (A tablet of ‘Droncit’ containing 50 mg of praziquantel was crushed, dissolved in 0.3 ml of Cremophor EL and diluted with water to 5 ml; the required dose, 1 ml for a 200 g rat, was administered by stomach tube.) H. diminuta has been maintained by passage through rats or hamsters and the beetle Triboiium confusum since it was obtained in 1963 from Rice University, Houston. Cysticercoids recovered by dissecting beetles, usually 3-6-week-old infections, in modified Hanks’ balanced salt solution (HBSS) (Hopkins & Barr, 1982), were administered by stomach tube within 1h. Worms were recovered by flushing out the intestine with HBSS. If the recovery was < lOO%, the intestine was slit longitudinally and examined under warm HBSS. On the few occasions where worm recovery was c 80%, the intestine was cut into l&15 cm sections and incubated in HBSS at 37-38-C for 1-3 h. At intervals, the incubating dish was examined for worms which had detached from the intestine. This latter procedure reveals very small, 0.5-5 mm worms but is not quantitative; however, such small worms have virtually no effect on the total biomass. Worm biomass (total mass of worms in a rat), determined after drying the worms at 95°C for l-3 days, was transformed to a log base before calculating the mean and S.D. For ease of interpretation, the antilog of this mean and S.D.is used in the figures and text. The justification for using log transformed data is that H. diminuta is growing almost at an exponential rate on day 8, the age at which worms were recovered. Statistical analysis was by Wilcoxon rank test, except where stated, and differences were considered statistically significant when P I 0.05. Note on drug eficacy. The double treatment with oxyclosanide at I70 mg kg-’ is very effective but, in heavy
infections, a worm very occasionally survives. Presumably this is because in heavy infections some worms may be attached much further back in the intestine. In the present work, two of the 168 rats treated with oxyclosanide harboured a primary worm at autopsy and were eliminated from the experiment. Because the chance of survival is so low, killing of a drug control group is of no predictive value and hence unjustifiable. However, primary worms which have survived, even if drug induced destrobilization has occurred, are immediately identifiable. Tests showed that no inhibition of growth occurred in worms administered 2 or more days after ‘Zanil’ treatment and as the challenge infection, with two exceptions, was never given until at least 6 days after the second drug treatment, any surviving primary worm had grown 4 or more days before the secondary infection was given. At autopsy 8 days later, a surviving primary worm would be at least the equivalent of a 12-day-old worm in size which, as worms between 8 and 12 days of age are approximately doubling their mass daily, is instantly distinguishable from an S-day-old-worm. We have had no survival of H. diminuta following praziquantel at 50 mg kg _’ in tests or in any experiment during extensive use over 4 years.
RESULTS Duration of immunological memory Five-worm primary, #-worm secondary. Immunized and control rats were kept separately at six per cage. The five-worm immunizing infection was removed by treatment with ‘Zanil’ days 21 and 22 postinfection (pi.). Three days (day 24) to 7 weeks later, one cage of control and one of immunized rats were challenged with 40 cysticercoids. Worms were recovered when 8 days old by pinning the intestine on a black wax-tray, opening longitudinally and lightly covering with HBSS. This is not as reliable a method as that used in the other experiments (Materials and Methods) for finding small (C 1 cm long) worms, especially if they 130 c
3d
I
2
3
4
5
6
7
Weeks after primary worms removed
FIG. I. Effect in rats of a five-Hymenolepis diminuta primary infection, removed 21 days pi., on the biomass of 8-day-old worms in 40-worm challenge infections. Mean worm biomass & s. D. in controls (0, primary infected) and immunized (0, secondary infected) rats. n = 6, except for five in immunized group day 3 and control group week 5.
Inhibition
of H. diminuta in the rat
of growth
.
49
A
.
P
I
20
510 Day
rat
35
was ctwllenged
115
70
after
primary
infection
150
terminated
FIG. 2. Effect in rats of a 16-worm Hymenolepis diminuta primary infection on the growth of worms in a secondary infection of 20 worms, given S-150 days after expulsion of the primary, immunizing infection on day 28. Lower plot: mean worm biomass f S.D. (n = 9) per rat of 8-dayold worms from immunized (0) and control (0) rats. Upper plot: A, mean worm biomass per rat in immunized rats as a % of that in control rats.
are in the posterior third of the small intestine, with food debris f mucus, but was used in order to determine the position of the worms. Mean worm recovery was 84% (range 82-87) in the six groups of control rats and 85% (84-85) in the immunized groups challenged after 3-7 weeks, but only 71% (67-73) in the 3 days-2 weeks challenged groups (Fig. 1). Growth of worms in rats challenged 3 days-2 weeks after the primary worms had been expelled was strongly depressed. This effect weakened but worm growth was still depressed by 30% after 5-7 weeks. Differences were statistically significant (‘P test), except in week 5, where t = 2.113, P < 0.1. Mean worm position, expressed as distance from the pylorus , was as follows: in control rats infected on day 3,21 cm, S.D. 12 cm; week 1,23 f 13 cm; week 2,23 f 14 cm; in the immune rats,
38 f 22 cm, 43 f 20 cm and 38 f 22 cm, respectively. In rats challenged 3, 5 and 7 weeks after the primary worms had been expelled, the mean position of the worms in the immune rats (39,47 and 43 cm) did not differ significantly from those in the controls (37, 34 and 46 cm). Mean length of the small intestines was approximately 100 cm in control groups but 3-9 cm longer in immunized groups. Fifly-worm primary, 40-worm secondary. To determine whether a heavier primary infection would modify the response, an experiment was set up using the same protocol, except that rats were immunized with 50 cysticercoids and worm recovery was by flushing and incubating (see Materials and Methods). The biomasses of the 40-worm challenge infections in immunized rats, infected 1, 2, 3 and 15 weeks after
50
C. A. HOPKINSand J. ANDREASSEN
removal of the primary infection, were 11, 54, 59 and 68%, respectively of that of the worms in the control groups. The difference between the worms from immunized rats (mean 45.2 mg per rat) and controls (mean 66.2 mg per rat) challenged 15 weeks after expulsion of the immunizing infection was highly significant (P < 0.01). Expressed as a % of the mean, inter-rat variance in worm biomass was much less 15 weeks after the immunizing infection had been expelled (log mean biomass 1.655, SD. 0.036 mg) than shortly after, e.g. 1 week (log mean 1.012, S.D. 0.395 mg). Sixteen-worm primary, 20-worm secondary. This experiment (Fig. 2) differed from the previous ones in that: rats on removal from the SPF breeding colony were kept in an isolation room under standard light and temperature conditions throughout the experiment. Cages were arranged in a specific pattern on the racks, to minimize spatially distributed variables. Empty cages were substituted when full ones were removed. The immunizing infection, 16 worms, was for 28 days and removed using praziquantel. Mean worm biomass was calculated by recovering worms from nine immune or control rats. Worm recovery was 98.8% (1245 worms from 1260 cysticercoids) in the controls and 95.5% (1203 worms) from the immunized rats. Secondary worms in all groups were significantly lighter that the controls, P < 0.01, except on day 115, when P = 0.05. The initial resistance, which depressed growth by over 80% (day 5, 82.3%; day 10, 82.9%) fell to 44% by day 35 and then probably reached a plateau around 30% (Fig. 2, A). Long-term memory: 30-worm primary, 20-worm secondary. Twelve of 24 rats were immunized with 30 worms and treated with ‘Zanil’ after 21 days. Nine control and nine immunized rats were challenged 36 weeks later and worms recovered on day 8. The mean number and biomass of worms in the control rats were 19.8 and 67 mg, in the immune rats 19.4 and 46 mg, i.e. growth was suppressed by 3 1%. The difference was highly significant, P < 0.01. The other six rats were challenged after 17 months (52 1 days); mean recovery was 19.3 worms per rat in both groups and mean worm biomass was 100 mg in the controls and 60 mg in the immune rats. Taken in conjunction with the above results, this small experiment suggests that the longterm component of memory persists as long as the rat lives. @hence
of number of worms in the secondary infection on the degree ofprotection expressed by immunized rats The design of the next two experiments was similar, except that in Experiment 1 the immunizing infection was 50 worms and in Experiment 2 it was 30 worms. (This difference in the immunizing infection will have had little if any effect-see next section.) The immunizing infection was terminated by ‘Zanil’ on days 21 and 22 pi. Control (previously uninfected) and immunized rats were infected on day 28 with l-50 cysticercoids. Rats were killed on day 36, when the
FIG. 3. Effect of the number of Hymenolepis diminuta in an infection on the biomass of S-day-old worms recovered from primary (0, a) and secondary (0, A) infections in rats. Solid regression lines calculated from 0 and 0 data. Broken lines are extrapolations. A, A Replicate experiment. Points are means of six or nine rats, see Results. infections were 8 days old. In Experiment 1, each point (Fig. 3, 0, 0) is the mean worm biomass from six rats, three in each of two cages. The result of the two-worm challenge to the immunized rats is not plotted, as in one cage the mean burden per rat was 5.6 mg, in the other cage, 10.6 mg, which is similar to the controls (10.1 mg, Fig. 3, 0). This suggests that the immunizing infection had been missed in the rats in the second cage but, as it could indicate a threshold at which a response may or may not be evoked, the number of rats in Experiment 2 (Fig. 3, A, A) was increased to nine in the one- and two-worm control and immunized groups. The mean number of worms recovered per group in Experiment 1 was: rats infected with two cysticercoids, controls loo%, immunized 100%; with five cysticercoids, 100 and 97%, respectively; with 10, 98 and 97%; with 20,93 and 96%; with 35,97 and 96%; and with 50 cysticercoids, 93 and 90%. As the difference between the number of cysticercoids administered and worms recovered never exceeded lo%, and the difference between the number of worms recovered from an immunized group and its control did not exceed 3%, no correction for these minor differences has been made in plotting the results and calculating the regression lines (Fig. 3). Plotting the results of Experiment 1 showed that the best fit to a straight line was obtained when both the dependent variable (total biomass of worms in a rat, 8
51
Inhibition of growth of H. diminuta in the rat days p.i.) and the independent variable (number of worms in the infection) were plotted on logarithmic scales (Fig. 3, 0, 0). Worm biomass in relation to number of worms in the control rats, i.e. in a primary infection Examination of the data showing worm biomass per rat in relation to number of worms in the control rats (Fig. 3, 0) reveals a close fit (r = 0.997) to the calculated regression line: logy
= 0.757 + 0.802 log x.
Thus the regression line predicts that a single worm infection should have on day 8 a biomass of 5.7 mg (log 0.757) and that doubling the number of worms leads to a 74% increase in biomass. Experiment 2 was carried out, inter alia, to test these predictions. The mean mass of one-worm infections in the control rats was 6.3 mg (predicted 5.7), of twoworm infections, 9.5 mg (predicted 10.0) and of loworm, 42.5 mg (predicted 36.5). It can be seen that these results (Fig. 3, A) fit closely the regression line calculated from the data of Experiment 1.
Znjruence of number of worms in the immunizing (primary) infection on the strength of protection induced against a secondary infection Rats were immunized with two-, five-, IO- or 20cysticercoid infections. ‘Zanil’ was administered on days 28 and 29. All rats were challenged with 20 cysticercoids on day 35 and killed 8 days later. Six immunized and six control rats were necropsied at each level of infection to determine the protection induced. Excluding two immunized rats from which only 11 and 12 worms were recovered, no rat had less than 17 worms and the mean recovery was 19.4 worms per control rat and 18.7 per immunized rat.
.
.
.
Worm biomass in relation to number of worms in a secondary infection The logarithm of worm biomass in the immune rats in Experiment 1 (Fig. 3, 0) also showed a linear correlation with the logarithm of the number of worms, r = 0.990. The calculated regression line is: logy
= 0.422 + 0.707 log x.
The predicted value of y when x = 1 is 2.6 mg and &hen x = 2, 4.3 mg; the observed values (Fig. 3, A) were 3.1 and 4.5 mg, respectively. The predicted biomass of a IO-worm challenge was 13.5 mg, observed 16.5 mg. The results of Experiment 2 showed that extrapolation of the regression line back to a oneworm challenge was justified, i.e. no threshold exists below which the growth of secondary worms is not affected. The strength of the immune response in relation to number of worms in the challenge Visual inspection of the slope of the two regression lines in Fig. 3 suggests that they are nearly parallel. Covariance analysis reveals no significant difference between the regression coefficients (F = 2.695, giving P > 0.1 < 0.25). This implies that the strength of the response expressed against a secondary infection is either not, or only slightly, affected by the number of worms in the secondary infection. The difference in elevation between the two lines is highly significant (F = 258.5; P < < 0.01) and indicates that the growth of the worms in the rats, challenged 7 days after a 50-worm immunizing infection had been removed, was only 40% of that in the controls, i.e. growth had been 60% suppressed.
FIG.4. Effect in rats ofthe number OfHymenolepis diminuta in the immunizing infection on the growth of a 20-worm challenge given on day 35, 7 days after expelling the immunizing infection. Lower plot: mean worm biomass f SD (n = 6) of 8-day-old worms per rat in immunized (0) and in control (0) rats. Upper plot: A, mean worm biomass per rat in immunized rats as a % of that in control rats. The S.D. of the biomass of worms (lower plot, Fig. 4) reveals that within control groups there was considerable uniformity but that within immunized groups, the rats were much more variable in their ability to support worm growth. The upper plot (Fig. 4) indicates the ambiguity of the results. The points fit a regression line log y = 1.8803 - 0.2545 log x quite well, r = - 0.9537, implying that the intestine is altered in proportion to the log of the number of worms in the infection; but the plot could also signify
52
C. A.
HOPKINS and J. ANDREASSEN
TABLE I-EFFECXOFVARYINGTHENUMBEROF Hymenolepisdiminuta INTHEIMMUN~UNGINFECTIONTERMINATEDONDAY GROWTHOVER 8 DAYSOFA 20-WORMSECONDARYINFEflIONINRATSGlVENONDAY 35
Number of worms in immunizing infection 1 2 4 8 16 32 64
Worms in control* rats? Number Biomass per recovered, rat, mean f s.o. mean (mg) 18.8 19.3 18.8 19.3 18.0 19.3 19.0
54f 12 61f 8 46f 11 52zk 9 54f 13 61 f 20 54f 8
Number recovered, mean 19.3 19.4 19.0 17.8 17.4 13.4 17.1
Worms in immunized rats? Rats arranged according to worm biomass Rat 1 Rat 2 Rats 3-6, Rats 7-8, mean mean (mg) (mg) (mg) (mg) 65 62 27 28 24 41 20
51 56 11 8 10 9 9
48 41 8 2.8 3.2 3.8 3.8
31 26 1.1 1.2 0.5 0.2 0.3
28 ONTHE
Overall depression in growth$ (%I 12 32 81 88 89 87 90
* Twenty-worm primary, no immunizing infection. t n = 8. Rat 1 was the rat with the heaviest biomass of worms, rat 2 the second heaviest, rats 3-6 the next four heaviest and rats 7-8 the lightest worm biomass, arithmetic means. $ % decrease in mean biomass of worms from the immunized rats compared with the controls.
that above five to 10 worms, there was little additional effect. To determine which was the correct interpretation, we carried out a second experiment. The same protocol was used, except that the immunizing infection was one to 64 worms and the number of rats at each level of infection was nine immunized and nine controls. In four of the control groups, one rat had very few and/or very light worms. Inclusion of such rats would have distorted the mean number and mass ofworms per rat (Table 1, columns 2 and 3). To avoid possible bias, we excluded the rat with the lightest worm biomass from all groups. To better illustrate the variation in response within the groups of immunized rats, the results are set out in detail (Table 1). Immunizing with one to four worms showed a progressive effect. A one-worm immunizing infection depressed growth by 12%. Doubling the immunizing dose led to an approximately 150% increase in growth depression (column 9). Worm recovery, 96% (column 4), in these weakly immunized rats was similar to that of the controls. In the groups of rats immunized with eight to 64 worms, the response pattern was similar and can be divided into four categories: Rat 1: A low responder rat (Table 1, column 5) in which worms grew well, reaching 4&60% of the mean mass of the control worms (column 3). Rat 2: A rat in which appreciable worm growth occurred (column 6). Rats 3-6: Four rats in which growth was strongly depressed (column 7). Rats 7-8: Two (or three, if the ninth rat is included) high responder rats in which, with one exception, growth was suppressed by over 99% (column 8). Only one of the 32 rats immunized with eight to 64 worms had a worm biomass as great as the minimum biomass in a control rat. In particular, it should be noted how approximately 50% of the total biomass of worms from the group of eight rats depended on the
chance occurrence of one low responding rat. Worm recovery was lower than in the control groups (cf. columns 4 and 2) but this was probably due to failure to find some of the very small worms, which would have had a negligible effect on the biomasses recorded. Two general points emerge from the table. One, the maximum immunizing effect is reached by an infection of four to eight worms (column 9). Two, growth of primary worms in the control rats was low compared with that in the other experiments described here and worm growth in secondary infections was depressed even more markedly, i.e. the rats were more responsive to an H. diminuta infection. DISCUSSION The response by a rat to a primary infection with HymenoIepis diminuta can be divided into two phases. For 2-4 weeks following the chemotherapeutic expulsion of a primary infection, growth of secondary worms is strongly depressed. This ‘strong response’ phase wanes rapidly but is followed, after about 5 weeks, by a second phase, which persists with little change (examined up to 17 months), during which growth of worms is weakly (by about 30%) but consistently depressed (Figs. 1,2 and text). This pattern of host response is similar to, though weaker than, that shown by a number of hosts following rejection of intestinal nematodes (Miller, 1984). A challenge infection shortly after spontaneous cure has occurred is marked by an acute ‘rapid expulsion’ response but later stimulates only an anamnestic response. ‘Rapid expulsion’ was shown by Bell & McGregor (1980) to involve two components, one immunologically specific, the other non-specific. It seems probable that the non-specific component is linked to the persistence of the hyperplasia of inflammatory cells (reviewed by Miller, 1987a,b). There is little direct evidence for the involvement of inflammatory cells against H. diminuta, though
Inhibition of growth of H. diminutu in the rat Hindsbo et al. (1982) observed small increases in mast cells and eosinophils in the mucosa of infected rats. However, indirect evidence of the importance of a non-specific inflammatory response modulating the growth of H. diminuta is strong. When an inflammatory response was induced by administering Trichinella spiralis, the growth of a concurrent H. dimi~uta infection, 650% of the controis, was inversely proportional to the log of the number of T. spiralis and hence possibly to the strength of the inflammatory response (calculated from Christie, Wakelin & Wilson, 1979). Likewise, the growth of H. diminuto in concurrent infections with Nippostrongylus brasifiensis, over the period when the latter are rejected, was depressed by over 98% (Hopkins, unpublished). Clearly, growth of H. diminuta in the rat is depressed by a non-specific heterologous inflammato~ response and to an extent similar to that observed in a challenge H. diminuta infection given shortly after expelling a primary infection (Table 1). The high level of anti-worm reactivity expressed by the intestinal wall shortly after expelling an infection will, presumably, have existed and have been at least as strong just prior to the expulsion of the primary worms. This implies either that the growth and fecundity of the primary worms was also depressed, or that worms ‘adapt’ during development (cf. Howard, 1977). Assuming this antiworm effect is modulated by immune and/or inflammatory cells, it would be interesting to know whether the biomass and fecundity of a mature worm infation increase when an agent such as cortisone is administered. The strength of the response induced in rats depended upon worm number up to between four and eight (Table I, Fig. 4) but two lines of work suggest that the critical parameter was biomass, not number of worms. Firstly, Hopkins & Barr (1982) who infected rats with 100 irradiated cysticercoids (65% of which survived the 21 days immunizing period but had negligible biomass) found, on challenging with normal cysticercoids 7 days later, that growth of worms was not significantly different from that in the controls, whereas in mice, eight irradiated cysticercoids induced strong protection. Secondly, a number of investigators (Read, 1951; Roberts, 1961; Chappell & Pike, 1976, 1977; Boddington & Mettrick, 1981) have shown that the biomass of H. diminuta, determined 17-28 days p.i., usually increases with numbers of up to five to 10 worms but thereafter there is little increase. [The linear relationship, when expressed on a log basis, between number of worms, one to 50, and biomass, in a primary infection (Fig. 3) is not a contradiction to the foregoing, in fact it is what would be expected if the depressive response on growth is proportional to biomass. We measured growth only over 8 days p.i., during much of which time the biomass is very small and hence stimulation of the intestine would be small and therefore evoke little counter-effect on growth of even a 50-worm infection.] It appears therefore that in a rat, unlike in a mouse,
53
the response engendered by H. diminuta is related to worm biomass. [See also the greater strength (Table 1) and duration (Fig. 2) of the strong response in experiments where the immunization period was increased from 21 to 28 days.] This would be a tolerable compromise between a ‘good’ parasite and a ‘good’ host; as worm mass increases, the intestinal (inflammatory?) response increases, which depresses growth to an equilibrium. The situation is certainly more complicated than this because, when rats are infected with large numbers, e.g. 40-100 worms, the equilibrium is overshot and an intense response leads to loss of worms in the soft, even diarrhoeal, faeces for 2-3 days, In other cases, worms become widely dispersed in the intestine and partially or completely destrobilated (Hindsbo et al., 1982). Whether the strong intestinal response is induced by direct physicochemical stimulus from the large mass of worm, which judging by the extensive villous fusion and atrophy induced (Martin & Holland, 1984) is considerable, or whether it is mediated through a weakened antigenspecific response is unknown, though the existence of long memory strongly suggests that the ‘weak response’ at least is lymphocyte mediated. The question also arises as to what causes the very poor growth of secondary worms during the strong response phase. The evidence is clear that the intestinal milieu has been altered, the depression in worm growth is almost, if not completely, independent of the number of worms in the challenge (Fig. 3), and the position of the worms in the intestine is quite different from that in control rats. Harris & Turton (1973), Choromanski (1980) and Machnicka & Choromanski (1983) have reported the formation of specific antibodies and sensitized macrophages and it is known that in vitro complement damages the tegument and induces destrobilation (Christensen, Bogh & Andreassen, 1986), but whether any of these are important in vivo has yet to be demonstrated. One point that may be relevant is that worm recovery from rats in the strong response phase often involved untangling a mass of ‘sticky’ worms bound in long tubes of mucus, which supports Bell, Adams & Ogden’s (1984) observations and Miller’s (1987b) conclusion that “mucus serves an important function in protection . . . “. Group size and method of caging and housing rats were changed in later experiments (see text accompanying Fig. 2). This was necessary because a depression of about 30% in growth of secondary worms, which commonly occurred during the weak-response phase, was often too small to be statistically significant, because of the large inter-rat variance in worm biomass. Equally worrying was an observation made in some other experiments that considerable inter-cage variance in mean worm biomass could occur between groups of rats supposedly treated identically. Without knowing the causes of either inter-rat or inter-cage variance, an empirical approach was adopted. To avoid the possibility that any difference between worm
54
C. A. HOPKINS and J. ANDREASSEN
growth in control and immunized rats arose from inter-cage variation, control and immunized rats were caged three plus three together. However, by adding inter-cage variance to groups of control and immune rats, variance in mean worm biomass was increased. This was countered by increasing the number of rats to nine per group. The immunizing period was increased to 28 days, because this had been very effective (Table 1) but the influence of the immunizing period requires further investigation (vi& supra). Finally, the number of worms in the immunizing dose was greatly reduced, with much saving of time, as an experiment (Table 1) had shown that at least the short-term response is as strongly induced by eight as by 64 worms, and another experiment (Fig. 1) suggested that the weak long-term response was also induced by a low immunizing infection. We conclude that H. diminuta survives in a rat because of a partial defect in, or suppression of, the antigen-specific response but that the non-specific (inflammatory?) defence cells are stimulated, directly or indirectly, in proportion to the mass of tapeworm, thereby increasingly inhibiting growth and leading to the establishment of a dynamic equilibrium. Acknowledgements-We wish to thank Miss Iona Barr for technical assistance, the M.R.C., London, for financial support (Grant G 8009375T) and the Danish Natural Science Research Council for a travel grant to Glasgow for J. A. REFERENCES ANDREASSEN
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