Expulsion of Nematospiroides dubius from the intestine of mice treated with immune serum

Summary This paper describes experiments which demonstrated that the survival of Nematospiroides dubius was severely impaired in mice treated with immune serum. CFLP donor mice were given a series of infections ranging from 25 to 200 infective larvae, at weekly intervals for 6 weeks. The mice were treated with anthelmintic on day 21 and/or day 28 to prevent the accumulation of lethal numbers of parasites in the intestine, and were bled between day 42 and day 49. Female NIH recipient mice were given a total of 2.0–2.5 ml of immune serum i.p., in several separate smaller doses at various times in relation to the day of infection. Between the administration of immune serum begun during the first 4 days of infection and the animals being killed within the next 3 weeks, the mice harboured fewer worms than control animals, the worms were stunted and their fecundity was greatly reduced. Furthermore, these worms were subsequently lost from the intestines of treated mice, during and after the fourth week of infection. These effects on N. dubius were not observed when mice were given normal serum nor when immune serum was administered after day 6 of infection. The delayed rejection of adult worms from mice treated with immune serum is of particular significance and suggests that immune serum contained factors which facilitated the expression of a second component in worm expulsion not normally effective in a primary infection. The possible immunological mechanisms underlying these findings are discussed and related to the immunosuppression which N. dubius is known to induce in the host.


Introduction
The generation of protective immunity to intestinal helminth parasites has been the subject of intensive research in recent years. Attempts have been made to analyse the steps involved in the immune expulsion of parasitic nematodes such as Nippostrongylus brasiliensis, Trichinella spiralis and Trichuris muris, and there is now substantial evidence that two or more components, involving antibody-and thymus-dependant lymphocytes, are required to act in a precise sequence to bring about worm loss (Ogilvie & Parrott 1977, Wakelin 1978. In contrast, there are many host-parasite relationships in which the parasite is not rejected and consequently survives for a long time following infection (Ogilvie & Wilson 1976). These situations must reflect the failure of either the afferent or the efferant arm of the immune system to function normally, but unfortunately, there is little experimental information available to explain the exact mechanisms involved (Ogilvie & Wilson 1976, Wakelin 1978. Following infection with the third stage larvae of Nematospiroides dubius, the mouse develops a chronic infection of many months duration (Bartlett & Ball 1974). Furthermore, most inbred strains of mice do not readily acquire resistance to secondary infections with this parasite, which frequently, therefore, also become patent (Cypess & Zidian 1975, Behnke & Wakelin 1977. In the inbred NIH mouse, however, a divided primary infection is known to stimulate a high level of immunity, which is manifested as an arrested development of larvae in challenge infections and as the destruction of these larvae while still in the intestinal walls , Behnke & Wakelin 1977, Behnke & Parish 1979). The few larvae which complete their histotropic phase of development and return to the gut lumen are rapidly expelled (Behnke & Wakelin 1977).
Whilst several previous workers failed to passively transfer resistance to N . dubius with immune serum (Chaicumpa, Jenkin & Rowley 1976, Cypess 1970, Panter 1969), recent preliminary results have shown that NIH mice could be protected in this way (Behnke & Parish 1978). The purpose of the present investigation, therefore, was to determine the timing of the administration of immune serum for maximum effect against the parasite, and to clarify the time course of worm expulsion to enable mice to be autopsied immediately before and after the process of rejection.

ANIMALS
Immune serum was raised in groups of male and female CFLP mice bred at random and was transferred to inbred female NIH recipients. The mice were bred under conventional animal house conditions in the Zoology Department of Nottingham University.

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 of animals and recovery of worms have already been described (Behnke & Wakelin 1977, Jenkins & Behnke 1977.

IMMUNIZATION OF SERUM DONORS
Approximately equal groups of male and female mice, comprising a total of 60-80 animals were given 125 larvae of N . dubius on days 0, 7, 14, 35 and 42. Anthelmintic was given on day 21 and day 30 to prevent lethal numbers of worms from accumulating in the intestine. The mice were killed and bled between day 42 and day 49. The serum given to recipient mice in Expts 6 and 10 was raised in donor mice according to the following schedule: 25 larvae on day 0,50 larvae on day 7, 100 larvae on day 14, 125 larvae on day 21 and day 35, and 200 larvae on day 42. Anthelmintic was given on day 28 and the mice were killed and bled on day 45. Normal serum was obtained from CFLP mice of a similar age but never having experienced infection with N . dubius. The blood was allowed to clot at room temperature for approximately 1 h and then at 4@C for 4-8 h. Serum was removed after centrifugation at 3000 rpm for 10 min and was stored at -20°C in aliquots corresponding to the total volume of serum to be injected on each occasion.

ANTHELMINTIC
Pyrantel embonate (Strongid-P paste, Pfizer) was used to remove adult N . dubius from the infected mice. A dose of 100 mg/kg body weight 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
One gram of faeces was taken from the pooled faeces of all the mice in each group, deposited over the preceding 24 h, and was dispersed in 8 ml of 50% saturated saline. This suspension was washed through a sieve (aperture size 800 pm) with 35% zinc sulphate solution 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 whole faeces. The relative fecundity (modified from Dineen & Wagland 1966) was calculated as the mean of the egg counts recorded between day 11 and the last day of the experiment .

DETERMINATION OF THE DRY WEIGHT OF N . DUBIUS
Fifteen male and 15 female worms were separated and dried at 100°C for'24 h in a glass vial. The worms were weighed and since female worms are much larger than males, the results were expressed as the dry weight of a pair of worms (one male and one female).

STATISTICAL ANALYSIS OF RESULTS
All the results were analysed for significance by the non-parametric Wilcoxon test (Sokal & Rohlf 1969). A value of P<0.05 was considered to be significant.

THE EFFECT OF IMMUNE SERUM ON THE SURVIVAL OF N . DUBIUS IN RECIPIENT MICE
Several preliminary experiments were carried out in which groups of female NIH mice were treated with immune serum and infected, together with control groups, with 100 larvae of N. dubius.The experimental'details and the results of three such experiments (Expts 1-3) are presented in Table 1. * In Expt. 1, group B was given 1 ml ofimmune serum on days-1,0, + 1, +2, +3, + 5 and +6. In Expt. 2, group D was given 0.25 ml of immune serum on day -1 and 0.5 ml on days 0, + 1 and + 2. In Expt. 3, group F was given 0.5 ml of immune serum on days -1, + 1, + 3, + 5 and + 7.
Group G was given 1 ml of immune serum on day -1. All the groups were challenged with 100 larvae of N. dubius on day 0. t In Expt. 2 the groups were killed on day 37 whereas in Expt. 3 they were killed on day 42. Paired groups were compared and found to have the following statistical significance: The data shows that in Expts 1 and 3, fewer worms were recovered from groups given immune serum (experimental groups) than from the respective control groups killed on day 9; in Expt. 2, the difference between the groups killed on day 9 was not significant. Furthermore, although the difference between the mean worm recovery from the mice in group D (Expt. 2) killed on day 9 and day 37 was not significant, four of the five mice killed on the latter day expelled the majority of the worms. Eighty-two worms were recovered from the fifth mouse in this group. Nevertheless, group D had fewer worms than the control group (C) (P < 0.005), indicating that the immune serum had some effect. Expulsion of worms was clearly demonstrated in Expt. 3 where both groups treated with immune serum (F and G ) lost most of the worm burden by day 42. The control group showed no such loss. .

THE EFFECT OF TIMING OF ADMINISTRATION OF IMMUNE SERUM O N THE SURVI-VAL OF N . DUBIUS IN RECIPIENT MICE
Since the life cycle of N . dubius involves a histotropic phase of development during which the larvae are deep in the intestinal submucosa and muscularis externu, it was of interest to determine whether this stage of the parasite would be more susceptible to the effects of immune serum than the adult worms which live in the gut lumen. Therefore, in the following three experiments (Expts 4, 5 & 6) groups of mice were infected with N . dubius and treated with immune serum at different times in relation to the day of infection (day 0). The experimental design and the results are shown in Table 2. The pattern of daily egg output from Expt. 4 is illustrated in Fig. 1.
It can clearly be seen from the results ( Table 2, Fig. 1) that in all three experiments normal serum did not affect the survival of N . dubius, nor was there any evidence of a reduction in the fecundity of worms from treated animals. In marked contrast, the survival of N . dubius was severely impaired when treatment with immune serum overlapped with the first 4 days of infection. Furthermore, the relative fecundity of worms from such mice was greatly reduced. The lower worm recoveries from groups given immune serum at the optimum time (day-1 to day+4) reflect not only the loss of some worms during the tissue phase of development, but also the expulsion of worms which had reached the gut lumen. In Expt. 4, a group of 12 mice was given immune serum on day-1 and day+ 1. Six mice were then killed on day 10 when the mean worm recovery was 57.3 k 14.7 and the remainder on day 28 when 6.5 It 4.8 worms were recovered ( Table 2), indicating that expulsion occurred between the second and fourth weeks of infection (P < 0.005).
These results indicate that immune serum only evoked the expulsion of worms when the treatment of recipient mice was initiated within the first 4 days of infection. In Expt. 5, there was a significant reduction in the relative fecundity of worms in the group given immune serum on days +4 and + 6, but the worm t Means significantly lower than the group given normal serum. counts, although lower than in control mice, were not significantly different. In contrast, in Expt. 6 when immune serum was given on days+4,6, 8, 10 and 12, there was both a reduction in worm fecundity and in the number of worms recovered at autopsy. Furthermore, in this experiment there was also a reduction in worm counts from mice treated with immune serum on days+6,8, 10, 12 and 14, whereas in Expt. 5 the group given 1 ml of immune serum on days + 6 and + 8 did not expel worms. When immune serum was administered later, either from day + 7 (Expt. 4), day + 8 (Expt. 5) or day + 1 1 (Expt. 4) onwards, expulsion of worms did not take place and the fecundity of worms was normal, except in Expt. 4 where there was a significant reduction in the fecundity of worms in the group given immune serum on days + 7 and + 9.

THE TIME-COURSE OF THE EXPULSION OF N . DUBIUS FROM MICE TREATED WITH IMMUNE SERUM
When mice infected with N . dubius were given immune serum during the first 4 Expt. 10,0.5 ml of immune serum given on days - t Means significantly lower than control group killed on the same day.
Paired groups were compared and found to have the following statistical significance: days of infection, fewer worms were recovered on day 9 or day 10 ( Table 1). A more substantial reduction in worm numbers took place later and was essentially complete by day 28. In order to clarify the exact timing of worm expulsion, four experiments were carried out (Expts 7,8,9 & 10) in which groups of mice were given immune serum at the optimum time and were killed together with the respective control groups at different times after infection. The experimental details together with the results are summarized in Tables 3 , 4 & 5. * Groups of five to seven mice were given immune serum i.p. and together with control groups of six to eight mice were infected with 100 N. dubills on day 0. The experimental groups were treated with immune serum as follows: Expt. 9, 0.5 ml immune serum given on days -1, + 1, + 3 and +5; Expt. 10. 0.5 ml immune serum given on days -I, + 1, + 3, + 5 and + 7. The worms were weighed as described in the text.
t Means significantly lower than control groups killed on the same day.
There was a significant reduction in the worm counts of mice treated with immune serum on all 15 occasions when control and experimental groups were killed on the same day (Table 3). When treated mice were killed during the first 3 weeks of infection, this reduction was relatively small, ranging from 16% to 41%. A far greater loss, ranging from 52% to 86%, took place only during and after the fourth week of infection. In all four experiments this loss was statistically significant.
The mean dry weight per worm pair was determined for Expts 9 & 10 and the results (Table 4) show that the worms from mice treated with immune serum were significantly lighter. The relative fecundity of the last group to be killed in these experiments was also measured (Table 5). There was a significant reduction in the egg output of the treated groups in all four experiments. * Groups of five to seven mice were given immune serum i.p.
t Means significantly lower than the respective control group.

Discussion
Despite the failure of sqveral groups of workers to transfer immunity to Nematospiroides dubius passively with serum from immune mice (Panter 1969, Cypess 1970, Chaicumpa et al. 1977, the results reported in this paper clearly demonstrated that the survival of N . dubius was severely impaired in mice treated with immune serum. During the period between the administration of immune serum beginning in the first 4 days of infection until they were killed within the next 3 weeks, the mice harboured fewer worms than control mice, the worms were stunted and their fecundity was greatly reduced. It is possible that a proportion of the lower worm recoveries on days 9-10 represented prolonged development of the larvae in the gut mucosa as described by Bartlett & Ball (1974). Certainly, in Expts 7-10 ( Table 3) there was a consistent increase in worm recoveries from treated mice during the third week of infection (days 13-20). However, even on days 15-20 (Expts 7 & 10) when worm recoveries were highest, control mice still had significantly more worms than treated mice and this suggests that not only was the rate of worm development inhibited, but that in addition there was a loss of some larvae during this early phase of the infection. Perhaps the most interesting feature of these experiments was the observation that mice given immune serum expelled most of the worm burden during and after the fourth week of infection, some 2-3 weeks after the last injection of immune serum. The half-life of injected IgG in normal mice is about 4-8 days (Fahey & Sell 1965), whereas in mice infected with N . dubius it can be as short as 2.6-3.5 days (Brown, Crandall & Crandall 1976). Therefore, in agreement with the findings from other experimental systems (Wakelin 1978), it is unlikely that in this host-parasite system, immune serum alone effected worm expulsion. However, it is possible that transferred antibody had some direct effect against N . dubius since the worms recovered from mice treated with immune serum were stunted throughout the experimental period (Table 4). Thus, factors in the transferred serum played a vital initial role in which worms were damaged, thereby making them susceptible to a second component in the host response which resulted in their expulsion. Such a sequence of events is well documented in the case of Nippostrongylus brasiliensis (Ogilvie & Love 1974) and Trichuris muris (Wakelin 1975a).
Recent findings which have established that N . dubius has the capacity to interfere with the immunological activity of the host, are also pertinent in the present context. Thus, the induction and expression of primary and secondary responses to Trichinella spiralis and T. muris was greatly impaired in mice concurrently infected with N. dubius (Jenkins 1977, Jenkins & Behnke 1977, Behnke, Wakelin & Wilson 1979. When mice were challenged with N. dubius 4-6 days after infection with either T. spiralis or T. muris, the primary response to the latter parasites was still markedly affected, suggesting that the larvae of N. dubius were immunosuppressive and that they rapidly interfered with the efferent component of the immune response. On the basis of these results it seems probable that in order to'reject N. dubius, the host must have the capacity to counteract the immunosuppressive factors produced by the larvae in the first few days of infection. It is possible that transferred immune serum in the present study, besides acting directly on the larvae, neutralized parasite toxins, thus facilitating the generation of an effective immune response by the recipients which consequently expelled the damaged worms in the fourth week of infection. The effects of immune Serum on N. dubius were not observed when mice were given normal serum or when immune serum was administered after day 6 of infection. The failure of the immune serum given later in the infection to affect worm fecundity and to cause worm expulsion can conceivably be attributed to an already impaired or blocked host immune system which is incapable of co-operating fully with transferred serum. It is also possible that transferred serum had less access to the worms in the intestinal lumen, although Cypess, Ebersole & Molinari (1977) reported that the concentration of IgG in the gut lumen increased substantially 3-7 days after infection with N . dubius and they suggested that leakage of serum proteins into the lumen occurred in animals infected with this parasite. Protective activity associated with IgG in transferred immune serum has also been described in other gut dwelling helminth parasites (Di Conza 1969, Jones, Edwards & Ogilvie 1970, but it has recently been pointed out that there is no convincing evidence that the severe cytopathological changes seen in N . brasiliensis immediately preceding worm expulsion (Lee 1969, Ogilvie & Hockley 1968) are the direct consequence of antibody activity (Love, Ogilvie & McLaren 1975, Wakelin, 1978. Similar changes can be induced in in vitro culture (Love et al. 1975), and worm expulsion can take place normally in mice incapable of detectable antibody synthesis (Jacobson, Reed & Manning 1977).
The reasons underlying the failure of previous workers to transfer immunity to N . dubius are unknown, but it is possible to identify several important differences between the present work and that already reported in the literature. The donor mice used in the present study were given a series of graded infections which were controlled by treatment with anthelmintic to prevent lethal worm burdens from accumulating in the gut lumen. Bartlett & Ball (1974), who also infected donor mice with a series of overlapping infections, demonstrated a significant delay in the development of N . dubius in recipients, but their experiment was terminated too early to show expulsion. Another important factor is the strain of mice used in the present work. NIH mice are known to respond more rapidly to several intestinal nematode parasites (Wakelin 1975b, Wakelin & Lloyd 1976) than any other strain of inbred mice and it is possible that in the presence of transferred immune serum, the resultant more effective immune response conferred on this strain the capacity to expel worms. Clearly this point will only be resolved when the present study is expanded to involve different strains of mice. Finally, the results reported in this paper have firmly established that resistance to N . dubius can be transferred by immune serum and hence support the hypothesis that protection in resistant mice is immunologically mediated.