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Neutrophils and eosinophils, isolated from the blood of mice infected with Nematospiroides dubius, were tested for their capacity to damage exsheathed third stage N. dubius larvae in vitro. In the presence of fresh serum from infected mice, both types of granulocyte caused a significant reduction in larval infectivity (up to 40-50%) whereas lymphocytes/monocytes prepared from the same blood samples were inactive. Neutrophils were at least as active as eosinophils, on a cell for cell basis. None of the cells exhibited larvicidal activity in the absence of serum and serum alone had no effect. The reduction in larval infectivity caused by neutrophils in the presence of fresh normal mouse serum (NMS) was only marginally less than that obtained using immune mouse serum (IMS), suggesting that complement, which is activated by the larvae via the alternative pathway and mediates the adherence of both cell types, was able to promote the larvicidal effect of these cells in vitro. In contrast to neutrophils, eosinophils were considerably less effective in NMS than in IMS. Both NMS and IMS were ineffective if they had been heat-inactivated or incubated with methylamine at pH 8.0 to destroy complement activity. The immunoglobulin fraction of IMS was also ineffective in promoting neutrophil or eosinophil-mediated larval damage. These results indicate that in this in vitro system antibodies are incapable of directing the activity of either cell type in the absence of complement. A novel finding of this study was that neutrophils from uninfected mice were unable to reduce larval infectivity in the presence of fresh NMS or IMS. 'Altered' neutrophils possessing larvicidal activity appeared in the blood of mice within 4 days of infection with N. dubius.

Infection by the intestinal nematode parasite Nematospiroides dubius was studied in seven different inbred mouse strains. Although there was some minor variation in the susceptibility of the different strains to a primary infection there were marked differences in their ability to develop resistance to infection following repeated exposure to infective larvae. The strains of mice which developed the best resistance also expelled adult worms arising from the previous infections. The adult worms resulting from a primary infection were slowly eliminated in two inbred strains studied whereas no loss occurred from outbred LACA mice. Although males and females of two strains, C3H/HeJ and CBA/H were equally susceptible to a primary infection, the females developed better resistance than the male mice following two oral administrations of third stage larvae. Infected mice of every strain and both sexes contained high levels of IgG1 in the serum.

The data presented in this paper show that mice may be immunised against re-infection with Nematospiroides dubius by giving live third-stage larvae orally, intravenously, intraperitoneally or subcutaneously. Immunity is directed not against the adult worm but against third-stage larvae following their penetration of the wall of the intestine.

The results given below show that, whilst it is not possible to transfer immunity to infections with Nematospiroides dubius passively with serum from immune mice to normal adult mice, young born to immune females are resistant to this infection. The immunity is dependent on an intake of immunoglobulin via the milk for a period longer than 24 h. The passive transfer of immunity from immune mothers to neonatal mice does not appear to be dependent on a specific class of immunoglobulins.

The role of neutrophils and eosinophils in the acquired resistance of mice to infection with the helminth Nematospiroides dubius has been investigated in vivo by testing infected mice for their resistance to a challenge dose of larvae following treatment with monoclonal rat antibodies (MAbs) specific for mouse granulocytes. Treatment of normal and infected mice with NIMP-R10 MAb reduced the number of neutrophils in the peripheral blood and peritoneal cavity during the ensuing 2-5 days without affecting macrophage or eosinophil levels. The effect of this MAb on immunity depended on the immune status of the mice tested. The partial resistance of mice receiving a primary 'immunising' infection, followed by passive transfer of immune serum and challenge at 4 days, was completely suppressed by NIMP-R10 MAb. The acquired resistance of mice challenged 10 days after being given a single 'immunising' infection was halved by NIMP-R10 treatment, while that of 'twice-immunised' mice (infected on days 0 and 14, challenged on day 24) was unaffected. Treatment of twice-infected mice with the eosinophil-specific MAb NIMP-R6 reduced the number of eosinophils in the blood and peritoneal cavity by 40-60%, but caused no significant loss of immunity. The data indicate that after infection of mice with N. dubius, neutrophils play a predominant role in early resistance to reinfection, but they become progressively less essential as activated macrophages and (following a second infection) eosinophils become prevalent. The lack of effect of NIMP-R6 MAb treatment on the immunity of twice-infected mice may have been due to an insufficient reduction in eosinophil numbers; however, it seems likely that the neutrophils and macrophages present in these mice would have provided adequate protection even in the absence of eosinophils.

The cuticular surface of the infectious third-stage larvae of Nematospiroides dubius activates complement via the alternative pathway. Sensitisation of larvae with complement or with antibodies from the serum of immune mice (resistant to reinfection) promoted the adherence of mouse peritoneal exudate cells to the larval cuticle during incubation in vitro. The infectivity of larvae sensitized with antibody or complement was significantly reduced after incubation with cells from immune mice.

The results of the present study show that peritoneal exudate cells from mice immune to infection with Nematospiroides dubius are able in vitro to damage the third stage infective larvae as measured by a loss in infectivity. The ability of these cells to function in the absence of specific antibody seems to be related to the presence of trypsin labile factors on their surfaces. Lymphocytes from immune mice are also able to damage the larvae. The suggestion is made that 'activated' macrophages may play an important role in immunity to this infection.

The ability of murine neutrophils to confer resistance to mice against infection by the helminth parasite Nematospiroides dubius has been investigated. Mice whose neutrophils had been 'altered' by an immunizing infection with third-stage larvae (L3) of N. dubius exhibited resistance to a challenge dose of L3 given 4 days after the immunizing infection, provided greater than 0.1 ml of immune mouse serum was passively transferred within 0-24 hr of challenge. Normal mouse serum was ineffective, as was immune serum given at the time of challenge to naive (unimmunized) mice. Neutrophils purified from the blood of mice infected 4 days previously were able to reduce the infectivity of exsheathed L3 when incubated with the latter in vitro in the presence of fresh immune serum. In contrast, peritoneal exudate cells collected at the same time were inactive in this test, indicating that activated macrophages capable of damaging L3 were not yet present and that the immunity of 4-day infected mice given immune serum was probably attributable to the presence of 'altered' neutrophils. The capacity of neutrophils to confer resistance to infection in vivo was unambiguously demonstrated by the passive transfer of 98 +/- 4% pure neutrophils, isolated from the blood of 4-day infected (C57Bl X BALB/c)F1 mice, to uninfected F1 mice. Only those mice which received both neutrophils and immune serum exhibited resistance to a challenge infection. In contrast, mice injected with an eosinophil-enriched cell preparation and immune serum were not resistant. These results indicate for the first time that neutrophils have the capacity to damage nematode parasites in vivo and that they are active against N. dubius in mice infected with this parasite.

Eosinophils and neutrophils, purified by density gradient centrifugation from the blood of infected mice resistant to reinfection, were tested for their ability to adhere to the different parasitic larval stages of the murine nematode parasite Nematospiroides dubius. Cells were tested for adherence to larvae which had been sensitised with immune mouse serum (IMS) or normal mouse serum (NMS) in the presence of CA2+ and Mg2+ ions. EDTA, or EGTA. Differences were observed in the degree of cell adherence to the different stages of the parasite. However, the adherence of the two cell types to any given stage of the parasite was similar. Adherence to the sheathed infective third-stage (L3) larvae, 96 h post-infective larvae and to adult worms depended to a large degree on conditions suitable for complement activation (viz. fresh serum and the presence of Ca2+ and Mg2+ ions). Complement was activated both via the alternative pathway by the parasite itself and via the classical pathway by parasite-bound antibodies. In these conditions, cell adherence probably occurred predominantly through the interaction of leucocyte third component of complement (C3) receptors with parasite-bound C3. In contrast, adherence of cells to exsheathed L3 and to the 48 h and 72 h post-infective larval stages appeared to involve antibody/Fc receptor as well as C3/C3 receptor interaction. The data indicate that N. dubius may undergo a series of antigenic changes during its life cycle and that antibodies capable of mediating granulocyte attachment are elicited predominantly against the early tissue developmental forms of the parasite.

The effect of one or more immunising infections on the susceptibility of mice to a subsequent challenge dose of Nematospiroides dubius larvae has been investigated. Following a single infection, animals developed a partial resistance characterized by their capacity to kill no more than half of the challenge dose of larvae. This low degree of immunity prevailed for some weeks after initial infection. A second immunising infection was found to induce a sudden increase in resistance such that at least 95% of a subsequent challenge dose of larvae was killed. This increase occurred 6 to 8 days after the second immunisation. Similar results were observed for mice immunised orally or intravenously. During the course of immunisations, absolute and differential cell levels in the blood and peritoneum as well as serum concentrations of the various immunoglobulin classes were determined. The results are considered in terms of the possible involvement of two distinct mechanisms of immunity to N. dubius, one requiring the participation of macrophages and the other, eosinophils.

BALB/c-nu/nu mice and their intact nu/+ littermates are equally susceptible to infection with third-stage larvae of Nematospiroides dubius. Unlike their heterozygous littermates, however, the nu/nu mice are unable to form ganulomata in the intestinal wall and become only partially resistant to rechallenge. Following two or more infections, nu/nu mice maintain a high burden of adult intestinal worms, whereas worms are lost from immune nu/+ mice. Studies in T cell-injected nu/nu mice suggest that a full complement of T cells is needed to develop maximum resistance against the infective third-stage larvae and to expel adult worms. Measurement of serum immunoglobulin levels indicate that infected nu/+ mice have very high levels of IgG1 whereas the levels of IgG2a are reduced. In infected T cell-injected nu/nu mice, IgG1 levels increase with the number of T cells injected, whereas IgG2a levels are variable but always higher than in infected nu/+ mice.