Stimulation of immunity to Nematospiroides dubius in mice using larvae attenuated by cobalt 60 irradiation

Infective larvae of Nematospiroides dubius attenuated by cobalt 60 irradiation are extremely effective at stimulating immunity. Previously, such levels of protection could only be obtained with multiple immunizations of normal larvae. The critical factor underlying this protective response appears to be the dose of irradiation given to the immunizing infection. Various doses of irradiation have been tested and the most effective of these range between 10 and 30 krad. The experiments show that provided this level of irradiation is used, the number of immunizing infections is relatively unimportant. Such use of irradiated larvae will be of value in attempting to analyse the immune mechanisms which operate against N. dubius. The possible mechanisms of immunity to N. dubius are discussed.


Introduction
The nematode, Nematospiroides dubius (Heligmosomoides polygyrus) in mice provides a convenient laboratory model of a chronic parasitic infection. After an 8-day tissue phase in the muscular wall of the gut, the parasites emerge to the lumen of the small intestine, where, as adults, they can survive for up to 8 months (Ehrenford 1954).
Coupled with this long survival is the failure of a primary infection to stimulate protective immunity to a subsequent challenge. Most attempts to stimulate immunity have used multiple immunization schedules involving a series of infections and drug treatments (Behnke & Parish 1979a). As shown by Prowse, Ey & Jenkin (1978), two infections would appear to be the minimum effective immunizing dose. They found one dose to be ineffective, but obtained greater than 95% protection with two doses.
The use of irradiation-attenuated parasites to stimulate immunity has never been attempted in this system, although the effects of cobalt 60 irradiation on the survival of the parasite have been reported (Behnke, Parish & Hagan 1980). If irradiation-attenuated parasites proved to be effective they would provide an extremely useful tool with which to evaluate the immune mechanisms that operate, often ineffectively, against N . dubius.
This paper describes the results of experiments designed to determine, firstly, whether irradiated parasites do stimulate immunity and, secondly, the combination of dose of irradiation and number of immunizing doses that is most effective in stimulating this immunity.

Materials and methods
Experiments were carried out in both Nottingham and Glasgow Universities.

Animals
Inbred male NIH mice were used in all experiments. These were purchased from Hacking and Churchill Limited (Huntingdon) or bred under conventional animal house conditions in the Zoology Department of Nottingham University. Mice, 6 8 weeks old at the start of each experiment, were killed in groups of six or seven.

Nematospiroides dubius
The strain of N . dubius used in the present study was obtained in 1975 from the Wellcome Research Laboratories, Beckenham, and has since been maintained in outbred CFLP mice. The maintenance of the parasite, and the methods used for infection and recovery of worms have already been described (Behnke & Wakelin 1977, Jenkins & Behnke 1977.

Irradiation of infective larvae
Larvae were irradiated using a cobalt 60 source as described by Behnke et al. (1980).

Anthelmintic
Pyrantel embonate (strongid-P paste, Pfizer) was used to remove adult N . dubius from infected mice. A dose of 100 mg/kg was administered orally as an aqueous suspension. This dose level is known to be adequate for the removal of all adult worms from the intestinal lumen (Behnke & Wakelin 1977).

Faecal egg counts
Half a gram to one gram of fresh faeces collected each morning from the pooled faeces of all the mice in each group was dispersed in 30 ml of 50% saturated saline. This suspension was washed through a sieve (aperture size 300 pm) with 100% saturated saline and the eggs were counted after flotation in standard McMaster counting slides as described by Gordon & Whitlock (1939).
The counts were expressed as the number of eggs per gram of faeces.

Statistical analysis of results
The results were analysed by the non-parametric Wilcoxon test. A value of P < 0.05 was considered to be significant.

EFFECT OF IMMUNIZATION WITH NORMAL OR IRRADIATED LARVAE
In the first experiment, two groups of mice were infected with 200 normal or 200,lO krad irradiated larvae on day 0 and both these groups, plus a group of age-matched controls, were treated with pyrantel on days 34 and 35. All the groups were infected with 100 normal larvae on day 42 and mice were killed on 14 and 35 days post-challenge. The results are shown in Figure 1. A second experiment using a shorter immunizing period and 25 krad irradiated larvae was carried out. Two groups of mice were infected with 100 normal or 100 irradiated larvae on day 0 followed by pyrantel treatment on days 14 and 15. Challenge infection was given on day 28 and all groups, including challenge controls, were killed on day 14 post challenge. The results are shown in Table 1.
In experiment 1, larvae irradiated at 10 krad clearly conferred greater protection against a subsequent challenge than did normal larvae, as assessed by worm recoveries on both day 14 and day 35 post-challenge. Although, in terms of numbers of worms recovered, there was no significant difference in immunizing capacity between normal and 25 krad irradiated larvae in experiment 2, unlike experiment 1, the challenge worms recovered were smaller and less mature than those recovered from mice immunized with normal larvae. It would seem, therefore, that mice immunized with irradiated larvae are more capable of arresting the development (see Behnke & Parish 1979b) of a subsequent challenge than are mice immunized with normal larvae.

LARVAE
The above results showed that irradiated larvae were more effective at stimulating immunity than normal larvae. An investigation of the effect of varying the dose of irradiation on the immunogenicity of larvae was undertaken. A total of five experiments was carried out. In two of these, a protocol similar to that of experiment 1 was used, but with mice killed on day 35 post-challenge only and mice were immunized with 200 normal larvae or with larvae irradiated at 5, 10, 15,20,30 and 40 krad. The combined results are shown in Figure 2 (expt 3). In a further experiment groups of mice were infected with 300 normal, 7.5, 15 or 25 krad irradiated larvae, treated with pyrantel on days 14 and 15, and challenged with 100 normal larvae on day 18. All the mice were killed 14 days later. The results are shown in Table 2 (expt 4). C 1 x 300 (7.5 krad) larvae 77 & 9.9 A vs C, A vs D, A vs E, B vs D, B vs E, P < 0.05 all combinations.
In both these experiments, the protective effect of immunizing with irradiated larvae increased with increasing dose of irradiation. Although this is evident from the mean worm recoveries shown in Figure 2, the variation within the groups was large. However, as the figures shown above the barlines indicate, the numbers of mice in each group which were > 90% protected against a challenge infection increased with increasing dose of irradiation.

CHALLENGE INFECTION
In this experiment (expt 5) all immunizing doses were of 300 normal or 300, 25 krad irradiated larvae. Four groups of mice were immunized at 16-day intervals with 4 x , 3 x , 2 x and 1 x 300,25 krad irradiated larvae and two other groups were immunized with 4 x and 1 x normal larvae. Each immunizing dose was terminated using pyrantel after 14 days. All groups, including age matched controls were challenged with 100 normal larvae and killed on day 35 post-challenge. Faecal egg counts from these groups are shown in    showed a clear difference between the effectiveness of normal and irradiated larvae. As assessed by day 35 worm recoveries, immunization with 1 x 300,25 krad larvae provided more than 95% protection against challenge, whereas 1 x 300 normal conferred no protection. However, immunization with the latter did delay the onset of patency but the egg counts reached control levels at day 16 whereas egg counts from mice immunized with I x 300,25 krad larvae, even at maximum levels, reached only one tenth ofcontrol output. On no occasion were eggs recorded from mice immunized with 4 x normal larvae or mice immunized with 2 x , 3 x and 4 x , 25 krad irradiated larvae.

Discussion
As Mulligan et a f . (1961) have shown, successful use of irradiated larvae as immunizing agents against nematode infections depends upon the selection of the most effective components from a large number of variables. In the mouse-N. dubius system we have shown quite conclusively that irradiated N . dubius larvae are effective at stimulating immunity against a subsequent challenge and that the most important variable is the level of irradiation in attenuation. The number of immunizing doses given is relatively unimportant provided the correct dose of irradiation has been used. We have further evidence (unpublished) to suggest that the size of the immunizing inoculum is not an important variable.
Although the exact details of survival and development of irradiated larvae are not known, the levels of irradiation used in our experiments prevent normal maturation and emergence of the larvae. As a result, the host is exposed for a prolonged period to larval antigens and it is this we believe that stimulates high levels of protective immunity. This view is supported by the fact that single infections of normal larvae fail to stimulate protective immunity, whereas multiple infections which extend the duration of host exposure to larval stages, can produce strong immunity (see expt 5). Further evidence can be drawn from the fact that the least effective stimulation of immunity by irradiated larvae was observed with those irradiated at 5 krad; these are known to develop and emerge into the lumen at the same time as normal larvae (Behnke et a!. 1980).
Prowse et af. (1979) have shown that different mouse strains subjected to similar immunizing schedules develop different levels of resistance to a subsequent challenge infection. The high level of protection, > 95% obtained here using one immunization with 300 (25 krad) cobalt 60 irradiated larvae, is no doubt due in part to the fact that these experiments have been conducted in NIH mice which are known to respond rapidly to nematode infections (Wakelin 1980). Whether or not all mouse strains respond as well as do NIH to immunization with irradiated larvae is the subject of a further investigation. The steady rise in the number of responding mice in the groups of experiment 3, a phenomenon which is more characteristic of outbred rather than inbred strain of mice, suggests that even for NIH mice there is a threshold level of antigenic stimulation which must be exceeded before an effective response can be mounted.
The host mechanisms which act against N . dubius are not well understood. Jones & Rubin (1974) have shown the presence of macrophages and eosinophils in the granulomatous lesions which develop around larval stages in the intestinal wall; eosinophils were associated particularly with larval dissolution. Chaicumpa & Jenkin (1978) and Prowse et al. (1978) have suggested that macrophages are responsible for the partial immunity resulting from a single immunizing infection; macrophages from these mice have been shown to adhere to infective third stage larvae in uiuo. However, only after two immunizing infections with normal larvae did they obtain more than 95% protection. Coinciding with this protection was a blood eosinophilia and the appearance of eosinophils in peritoneal exudates. They concluded that eosinophils may be involved in a second immune mechanism which gives rise to this high level ofprotection. As yet we have not determined whether eosinophils appear after a single immunization with cobalt 60 irradiated larvae.
By attenuating larvae with cobalt 60 irradiation, the characteristics of infection with N . duhius have been dramatically altered. Single infections with normal larvae produce only poor immunity which is manifested by a delay in the onset of patency, possibly due to a slight delay in larval development. Irradiated larvae, on the other hand, produce a highly protective immunity which greatly reduces the number of parasites reaching maturity.
The results of this work indicate that once activated, the immune mechanisms of NIH mice, which normally fail to work against a single N . dubius infection, are extremely effective. Irradiated larvae will provide a means by which the underlying mechanisms of immune responsiveness and non-responsiveness in various strains of mice can be studied.