Immunity to adult Heligmosomoides polygyrus (Nematospiroides dubius): survival or rejection of adult worms following transplantation to mice refractory to larval challenge

ABSTRACT Experiments were carried out to explore the survival of 14-day adult H. polygyrus following transplantation to mice of four strains, immunized by various protocols. Adult worm establishment and survival was unimpaired in CFLP mice which were totally refractory to larval challenge. Transplanted adult worms were also successful in NIH mice immunized by the 9-day abbreviated infection regime. However, NIH mice exposed to irradiated larvae or subjected to the divided primary infection, expelled transplanted adults. The 9-day abbreviated infection was further examined in SJL and (C57 Bl10 X NIH) F1 mice which expel adult worms during a primary infection and although this regime was unsuccessful in causing NIH mice to reject adult worms, expulsion of adult worms was accelerated in SJL and F1 mice. The survival of adult H. polygyrus was discussed in the context of stage-specific immunity and the delicate balance between the immunogenic stimuli from developing larvae, the immunomodulatory activities of adult stages and the host's genetically determined capacity to respond to these opposing signals.


INTRODUCTION
Heligmosomoides polygyrus is a long-lived parasite in most laboratory strains of mice and is known to survive for 8-10 months following a single oral administration of infective larvae (EHRENFORD, 1954;KEYMER & HIORNS, 1986;BEHNKE et al. 1987). The survival of the parasite is dependent on a delicate balance between the immunogenic stimuli from developing larvae, the immunomodulatory activities of adult worms (BEHNKE, 1987) and the host's genetically determined capacity to respond to these opposing signals (BEHNKE & ROBINSON, 1985). BARTLETT & BALL (1974) concluded that H. polygyrus differed from most other intestinal nematodes in that the fecundity and survival of adult worms seemed to be completely unaffected by acquired resistance, the latter being considered to act only against the larvae, leaving the adult worms totally unimpaired. This remarkable ability of adult H. polygyrus to live in mice totally refractory to larval infections was reinforced by JACOBSON et al. (1982) who demonstrated that immune LAF/J mice, totally resistant to larval challenge, tolerated transplanted adult worms for 2 weeks following implantation.
Despite these studies, evidence is available that adult H. polygyrus are not totally resistant to immune responses in the intestine and that the host's genotype is an important factor in this equation. Some mouse strains have been shown to expel adult worms prematurely (LAF/J-CYPESS et al., 1977;SJL-MITCHELL et al., 1982). 'Present address: Department of Immunology, Mayo Clinic, Rochester, Minnesota 55905, USA. 2 Author to whom all correspondence should be addressed.
Furthermore, experiments have been reported in which effective anti-adult worm responses have been initiated in mice by appropriate laboratory manipulation. Balb/c mice immunized by intraperitoneal injections of adult worms, when challenged orally by larvae, expelled the mature parasites within 20 days of infection (DAY et al. 7 1979). Even the weak responder strain C 57 B1 H ) was induced to reject adult worms by implantation of mature parasites i.p. and concurrent administration of the adjuvant, pertussigen (MITCHELL & MUNOZ, 1983;MITCHELL & CRUISE, 1984).
These studies showed that although adult worms caused chronic primary infections in many widely used laboratory strains of mice and could survive in animals totally resistant to larval infection, the parasites were still susceptible to host effector mechanisms. In the experiments reported in this paper, we began by re-examining the effectiveness if i.p. implanted adult worms in eliciting responses against adult worm challenge. Adult worms were found to survive in CFLP mice, irrespective of the immunizing protocol used even when the mice were resistant to larvae. It was then necessary to compare the efficacy of other immunizing regimes, with well established properties of inducing strong acquired resistance to H. polygyrus in mice and to determine the influence of host genotype on the survival of transplanted adult worms in mice immunized by these procedures.

Animals
Four strains of female mice were used in the present study. Randomly bred CFLP, syngeneic NIH and (C 57 B1 H) X NIH)F, were all bred in the departmental animal house. SJL mice were purchased from Harlan Olac Ltd., Bicester, Oxon. All the animals were maintained under conventional animal house conditions with access to food and water ad libitum.

Parasite
The methods used to maintain H. polygyrus, infect mice and recover worms at autopsy have all been described previously (JENKINS & BEHNKE, 1977). Faecal egg counts were monitored as reported by BEHNKE & PARISH (1979). A detailed description of the methods used to transplant adult worms was given by BEHNKE et al. (1983). Worms for transplantation were obtained from CFLP mice infected with 250-400 L 3 (third-stage larvae), 14 days earlier.

Immunization of mice
Several different methods were used to immunize mice to larval or adult stages of H. polygyrus. Most of these have been described previously but briefly the following procedures were used: Experiments 1-3. Adult worms were isolated, washed three times in warm sterile saline and injected into the peritoneal cavity of mice in 0-5 ml of sterile saline using a sharp 18 gauge hypodermic needle. Larvae were cleaned by repeated washing in saline and were injected in 0-5 ml of sterile saline through a 21-gauge needle. Both larvae and adult worms were killed prior to injection by freezing to -40°C and thawing, twice in rapid succession. Experiment 4. The divided primary infection protocol adopted was described by BEHNKE & WAKELIN (1977). Mice were given 100 L 3 larvae on days 0, 2, 4, 7, 9, 11 and were treated with pyrantel embonate (Strongid-P paste -Pfizer) on days 15, 22,29, 35 and 42. The animals were challenged on day 51. This protocol allows the overlapping development of larval stages, prolonging larval exposure in the intestine and limits adult worm populations by the anthelmintic treatments administered from day 15 onwards. It is extremely effective at inducing acquired resistance to larval challenge.
Experiments 5, 8 and 9. The 9-day abbreviated infection regime used was described by BEHNKE & ROBINSON (1985). Mice were given a single dose of L 3 on day 0 and were treated with pyrantel on days 9, 10, 14 and 21. Challenge infections were administered a week later. This protocol allows the synchronized normal development of L 3 and L 4 but the anthelmintic treatment from day 9 onwards removes the adult worms from the gut lumen as they emerge from the submucosa without affecting parasites which are still developing in the tissues. This regime has been found to be very effective in discriminating between weak and strong responder mouse strains.
Experiments 6, 7 and 9. Immunization by irradiated larvae was carried out as described by BEHNKE et al. (1980) and HAGAN et al. (1981). Infective larvae were exposed to 25 krad of gamma radiation from a Cobalt 60 source. Mice were given 250 L 3 on day 0 and were challenged on day 21 (Experiment 6), day 24 (Experiment 7) or day 28 (Experiment 9). This protocol does not permit the development of adult parasites but the L 3 and L 4 stages are believed to persist as arrested forms in the intestinal tissues (ALI & BEHNKE, 1985).

Statistical analysis of results
Faecal egg counts are presented as the mean value of 4 counts on each 1 sample of faeces. Worm counts are given as the mean worm burden ±S.E.M. When applicable the non-parametric Mann-Whitney U test was used to compare groups for significant difference (SOKAL & ROHLF, 1969) and a value of P<0-05 was considered to represent a significant difference.

Survival ofH. polygyrus in CFLP mice immunized by 3 intraperitoneal injections of adult worms or 3 doses of L 3 stages
Initially, several exploratory experiments were carried out in CFLP mice in order to determine whether i.p. injections of adult worms would sensitize mice and enable them to reject a challenge infection with transplanted adult worms. These preliminary studies confirmed that adult H. polygyrus could survive in mice resistant to larval challenge. Table I summarizes the results from three such experiments in which the treatments used to induce resistance were 50 adult worms injected i.p. on days -25, -23 and -21, 500 L 3 injected on the same days or 200 L 3 given orally, again on days -25, -23 and -21. All the mice, together with a naive control group, were treated with pyrantel 14 and 7 days before challenge to eliminate worms which had established in the intestine and the animals were challenged either with 50 adult worms by laparotomy or 200 L 3 orally.
Adult worms survived without loss for 56 days in mice which showed varying levels of resistance to larval challenge (Table I). When 3 doses of 50 adult worms injected i.p. were used, up to 28-5% protection from larval challenge was observed (Experiment 3, groups BvD). Mice immunized by 3 doses of 500 L 3 given i.p. or  200 L 3 given orally showed respectively 47-7% (Experiment 3, groups BvF) and 62-5% (Experiment 3, groups BvH) reduction in parasite survival to day 56. Curiously, whilst these treatments failed to cause the rejection of transplanted adult worms, adult stages of H. polygyrus developing from the challenge inoculum were rejected more rapidly than in naive control mice. Thus in Experiment 3, mice immunized by 50 adult worms, 500 L 3 i.p. and 200 L 3 orally sustained respectively a further 45%, 44% and 46% reduction in worm burden between day 14 and 56. In contrast, naive control mice lost only 19-9% of their worm burden over the same period (group AvB).

Survival and rejection of H. polygyrus in NIH and CFLP mice immunized by 3 different immunizing protocols
The preceding experiments established that transplanted adult worms could indeed survive in CFLP mice immunized by treatments which elicited varying degrees of resistance to larval challenge. However, adult worms which survived the tissue phase of infection in immunized mice were less successful at avoiding expulsion since some were rejected within 8 weeks of infection. Thus there appeared to be an intrinsic difference in the capacity of these distinct adult worm populations to overcome the host's intestinal immune response. In an effort to explore further the resistance of transplanted adult worms to host effector mechanisms, a series of experiments was carried out using three further immunizing protocols, all known to stimulate strong acquired resistance to a challenge infection with H. polygyrus. Three of these experiments were carried out using NIH mice, a strain considered a strong responder to this parasite. In each experiment naive mice and mice immunized by the relevant immunizing protocol were challenged either by transplanted adult worms or by infective larvae given orally. The course of infection was then followed by faecal egg counts and parasite survival was assessed by worm recovery. The results are summarized in Table II. As can be seen, all three procedures were highly immunogenic in NIH mice and produced respectively 98-1, 93-8, and 98-9% protection against larval challenge. The protocols differed, however, with respect to their efficacy in mediating resistance to challenge by transplanted adult worms. Two protocols, irradiated larvae (Experiment 7) and the divided primary infection (Experiment 4) caused 80-2 and 97-4% loss of adult worms respectively. Faecal egg counts (Fig. 1) showed that in Experiment 7, adult worms established in mice immunized by irradiated larvae, initially producing high egg counts, but egg production dropped promptly and stayed depressed suggesting that many of the adult worms had been expelled within 2 weeks of transplantation. In control mice (Experiment 7, group A) faecal egg counts were more steady and the parasites survived for 6 weeks post transplantation. Faecal egg count data is not shown for Experiment 4, but no eggs were detected in the faeces of group B mice (immunized by the divided primary infection) on days 13, 20 and 27, whereas the naive mice receiving adult worms by laparotomy (group A) had counts of 800, 1200 and 1100 on these days.
In contrast to NIH mice, CFLP mice were unable to reject transplanted adult worms when immunized by irradiated larvae (Experiment 6) despite the fact that the same protocol rendered CFLP mice almost totally refractory to larval challenge (95-9% protection). Furthermore, NIH mice were unable to reject adult worms when immunized by the 9-day abbreviated infection regime (Experiment 5). Again the latter protocol was shown to be highly immunogenic and elicited 93-8% protection against larval challenge. Indeed, in order to allow for a possible delayed response to transplanted adult worms, this experiment was continued for 10 weeks,  but at autopsy worm counts were not significantly different in groups A and B. Nevertheless faecal egg counts in group B were depressed relative to group A throughout the period of observation and were very low in the 6-10 weeks of infection suggesting that despite the hosts failure to reject the worms, an anti-parasite response was initiated.

Rejection of adult H. polygyrus from (C 57 Bl 10 X NIH) Fj hybrid and SJL mice.
Recent studies have shown that three strains of mice expel adult H. polygyrus developing from orally administered L 3 within 10 weeks of primary infection (ROBINSON et al., in press). In (C 57 Bl 10 X NIH)Fi hybrids this is a dose-dependent phenomenon, low intensity infections being expelled earlier than high intensity infections. SJL mice, however, expel H. polygyrus in a dose-independent fashion usually within 7 weeks of larval administration. In the final section of this paper, we report the results of experiments carried out to determine whether the immunizing protocol, which failed to elicit resistance to transplanted adult worms in NIH, would accelerate worm rejection by these two strains of mice.
Experiment 8 was designed along the lines of Experiment 5. Thus 27 Fj mice were arranged into 4 groups A(n=4), B(n=6), C(n=9) and D(n=8) which were treated identically to the mice in Experiment 5, except that groups C and D were challenged by 150 L3 larvae and not 50. This experiment was monitored by faecal egg counts as shown in Fig. 2 and the mice were finally killed for worm counts on day 56 post challenge. Naive Fi mice receiving transplanted adult worms (Group A), produced eggs in their faeces throughout the period of observation, but the egg counts declined towards week 5 and at autopsy 14-8±7-4 worms were recovered. Two of the mice still had high parasite numbers, the remainder had lost the majority of worms. In contrast immunized mice receiving transplanted adult worms (Group B) had rejected all the parasites by day 56 but the worms had established following transplantation as eggs were detected in the faeces from day 4 to 39. Thus ¥i mice immunized by the 9-day abbreviated infection protocol showed enhanced resistance to adult parasites which were rejected prematurely in relation to naive controls. No eggs were detected at any time in the faeces of immunized F, mice challenged with L 3 (group D) and no parasites were recovered at autopsy. Group C which comprised naive F, mice given L 3 orally produced eggs throughout the experiment and at autopsy 28-2±15-5 worms were recovered but the worm burdens were variable and 4 of the 9 mice had expelled the parasites before day 56. Thus in this group also, expulsion of adult worms (developing from L 3 larvae) had been initiated, some mice shedding their worm burden before the termination of the experiment.
In the final experiment SJL mice were used. One group was designated the naive control group (A) and the other two groups were immunized, one by the abbreviated infection protocol (B) and the other by irradiated larvae (C). All three groups received transplanted adult worms and the course of infection was monitored by faecal egg counts. Mice in group C ceased producing eggs on day 29, those in group B on day 44, whilst eggs were detected in group A until day 57. At autopsy, the majority of mice were totally without worms.

DISCUSSION
H. polygyrus is clearly a remarkable parasite whose adult stages survive in situations where larvae cannot form patent infections. Our results have drawn attention again to this property of adult H. polygyrus and we have extended previous studies by exploring the survival of adult worms in mouse strains which have the capacity to expel primary infections with this organism.
One interpretation of the results from our experiments could be that they reflect stage-specific resistance e.g. resistance induced by larvae only acting on larval and not adult parasites. Indeed, H. polygyrus expresses stage-specific antigens (PRITCHARD et al., 1984) and in Experiment 3, mice immunized by adult worms did not express anti-larval resistance, but subsequently rejected adult worms developing from the challenge inoculum. However, the same protocol did not accelerate the rejection of transplanted adult worms and although this may seem paradoxical, there are reasons for believing that the two adult worm populations may be phenotypically distinct. Although no biochemical, enzymatic or antigenic differences have been reported, parasites developing in partially resistant hosts are stunted and less fecund than normal (BARTLETT & BALL, 1974;DOBSON, 1982;SITEPU & DOBSON, 1982) and by analogy with Nippostrongylus brasiliensis might be expected to show other differences (OGILIVIE & JONES, 1973). Such worms may already be partially damaged as a consequence of developing in immune animals and hence may be more susceptible to host effector mechanisms.
However, stage-specific immunity is not an explanation which is entirely compatible with our data. Immunization with irradiated larvae induced strong resistance to larval challenge in both CFLP and NIH mice (Experiments 6 & 7, Table II) but allowed transplanted parasites to establish and survive in the former strain but not in the latter. Larvae subjected to 25 krad of irradiation do not develop into mature worms (BEHNKE et al., 1980) and in consequence the mice would have been exposed only to prolonged stimulation by L 3 and L 4 antigens. Both strains, therefore, had comparable experience of larval antigens, but only one acquired an anti-adult worm response.
It is also pertinent that NIH mice were unable to expel transplanted adult worms when immunized by the 9-day abbreviated infection. Again, one explanation may be that the 9-day abbreviated protocol elicited anti-larval stage-specific immunity and for this reason we explored the effects of this immunizing protocol in two strains of mice which rank as stronger responders than NIH mice ((C 57 Bl 10 X NIH)F, and SJL strains; BEHNKE & ROBINSON, 1985). In both strains, the protocol accelerated the rejection of transplanted adult worms and we conclude that stage-specific immunity was unlikely to have been involved, unless there were genetically determined qualitative differences between the strains in recognizing and responding to the inducing signals from the inoculum. Mice exposed to the 9-day abbreviated infection would have experienced only larval stages of H. polygyrus, but would not have been subjected to such prolonged stimulation as mice exposed to the irradiated larvae (ALI & BEHNKE, 1985). Irradiated larvae stimulate strong resistance to challenge in both NIH and C 57 B 10 mice (the latter is a weak responder strain to H. polygyrus) but the 9-day abbreviated infection is only effective in the former (BEHNKE & ROBINSON, 1985). Therefore, the explanation for the different efficacies of the two immunizing protocols in inducing anti-adult worm responses in NIH mice may reside in the varying intensity of stimulation which they provide.
An alternate explanation for our results may lie in considering the host's response to the immunomodulatory mechanisms used by adult worms for evading immunity (reviewed BEHNKE, 1987). CFLP mice may have been more severely compromised by the immunodulatory effects of adult parasites than NIH mice and hence worms were able to survive only in immunized individuals of the former strain. There is further evidence in support of this suggestion. Thus adult worms can readily be shown to depress homologous immunity in CFLP mice immunized by irradiated larvae (BEHNKE et al., 1983) and similar results have been found in C 57 B1 H1 mice, but we have been unable to repeat these observations in NIH mice. In one experiment, 104 adult H. polygyrus resulted in 12% reduction of immunity elicited by the irradiated vaccine in NIH mice, whereas in C 57 Bl ln only 13 adult worms caused 84% depression (Crawford, Behnke & Pritchard, unpublished observations).
It is quite clear from our results and those of others (JACOBSON et al., 1982) that under a variety of circumstances adult H. polygyrus can evade host protective responses, but the parasites are not always successful. An important additional factor, not investigated previously in this context, appears to be the host genotype. Mouse strains such as SJL, (C 57 B1 U ) X NIH)F, and LAF,/J have the capacity to expel adult worms and if immunomodulation is an important component of the parasite's evasive strategies, it follows that these strains must benefit from a genetically determined insusceptibility to parasite mediated immunomodulation. The latter is an attractive, testable but as yet unproven hypothesis.
It is interesting to note here that, the expulsion of primary infection adult worms from SJL mice is not accompanied by an intestinal mast cell response, such as that mounted by mice of this strain to Trichinella spiralis or secondary infections with H. polygyrus (DEHLAWI et al., 1987). SJL mice can therefore respond with a mastocytosis but fail to do so during a primary infection, presumably because the response is depressed by adult worms. In other words, H. polygyrus are partially successful in immunomodulating in SJL mice, in so far as the mast cell response is incapacitated, but the fact that the worms are lost implies that other component processes of the overall response in the intestine are less severely affected. These observations have not yet been extended to encompass other mouse strains which expel adult worms but such experiments are currently in progress. The key to understanding the survival strategy of H. polygyrus and hopefully other long-lived nematode parasites, will be to identify qualitative differences in response phenotype associated with survival or loss of adult parasites and to link these to genetic differences between the mouse strains.