Nematospiroides dubius in mice selected for liability to infection: Modification of parasite biology through host selection

Brindley P. J. and Dobson C. 1982. Nematospiroides dubius in mice selected for liability to infection: modification of parasite biology through host selection. International Journal for Parasitology12: 573–578. Mice selected as liable (L) and refractory (R) over ten generations voided significantly more and less Nematospiroides dubius eggs compared with randomly mated (Rd) mice after primary infections with 100 larvae. There was little difference between the number of parasite eggs voided g^?1 faeces (epg) by individual mice on day 14 compared with day 15 after infection.However there was a significant diurnal variation in the egg values for individual mice but the mean differences observed between the R, Rd and L mice were maintained over a 24 h period. There was a strong correlation between both the total number and the number of female worms, surviving 21 days after infection, and the mean epg 14 and 15 days after infection. Female N. dubius produced more eggs in L mice and fewer eggs in R mice compared with worms in Rd mice. Similarly, worms grew longer in L mice and were shorter in R mice compared with parasites in Rd mice.     

Regulation of toxocariasis in mice selectively reared for high and low immune responses to Nematospiroides dubius

Test mice have been selectively reared for high (H) or low (L) immune responses to Nematospiroides dubius. After secondary infection with N. dubius, the L mice voided ten times as many eggs in their faeces as the H mice, and at necropsy, 71% versus 20% of the inoculum of N. dubius were recovered as adult worms from the L and H mice respectively. Furthermore, N. dubius were more fecund in the L than in H mice. High or low immune responsiveness was not restricted to N. dubius infection in these mice but was also observed during Toxocara canis infection. The migration of T. canis larvae from gut via the liver to skeletal muscle and CNS was inhibited in H versus L mice. Many more larvae were recovered from the livers of H compared with L mice which was indicative of greater immunity in the H mice. The protective immune response in H compared with L mice to both N. dubius and T. canis included pronounced eosinophilia and elevated antiparasite antibody titres.     

The influence of Nematospiroides dubius on subsequent Nippostrongylus brasiliensis infections in mice.

The fecundity and longevity of Nippostrongylus brasiliensis was prolonged in mice previously infected with Nematospiroides dubius only when the former developed from the larval stage in those mice. Such worms appeared to be less immunogenic than worms which developed in mice never exposed to N. dubius. It is proposed that prolonged fecundity and longevity resulted from an adaptation undertaken by the worms in the face of host antibodies which had been developed against the pre-existing N. dubius infection.     

Nematospiroides dubius: genetic control of immunity to infections of mice

Inbred strains of mice differ in their susceptibility and resistance to challenge infections with Nematospiroides dubius. In our studies, F1 hybrid mice from resistant SJL and susceptible CBA parents were resistant to N. dubius challenge infections. Only 22% of backcrosses to SJL were susceptible while backcrosses to CBA had a wide range of susceptibility. Male mice were more susceptible than female mice. In another experiment, inbred strains of mice were compared in their ability to resist N. dubius challenge infection: SJL and A.SW (H-2s) mice became resistant after one immunizing infection, A, A/He (both H-2a), as well as BALB/c and DBA/2 (both H-2d) mice became resistant after two immunizing infections, while C57BL/6 (H-2b), C3H/He, CBA, and AKR (H-2k) mice remained susceptible. The resistance to reinfections was characterized by reduction of worm burdens between Days 6 and 14 postinfection. It was concluded that (1) resistance to N. dubius challenge infections is inherited in a dominant fashion and that multiple genes may influence such response, which in turn might be modulated by the Y chromosome; (2) both MHC and non-MHC genes may influence, in conjunction with the number of exposures to parasite antigens, the resistance to challenge infections.     

Nematospiroides dubius: direct and correlated responses to selection for high and low immune responsiveness in mice

High and low immune responder lines of mice were bred selectively from an allogeneic stock over 10 generations, based on their fecal parasite egg count assayed 3 weeks after reinfection with 100 Nematospiroides dubius larvae. By generation 10, (F10), the low immune response mice voided about 10 times as many fecal N. dubius eggs as the high immune response mice. Realized heritability for the selected trait, fecal egg count after secondary infection (= protective immunity), was 0.35 at F7. F7 was considered the selection limit. Selection for change in fecal egg count did not significantly influence the conformational nor reproductive characteristics of these mice. Significant phenotypic and genetic correlations were evident between the selected character and innate immunity to N. dubius, humoral antibody response to N. dubius infection, and establishment, growth, and reproduction of N. dubius in the selected mice.     

Nematospiroides dubius: multiple infections in mice bred for immune responsiveness

Allogeneic mice were reared over 5 generations in a 2-way selection for high (H) and low (L) immune responsiveness to the intestinal trichostrongylid nematode Nematospiroides dubius. After 5 generations of selective breeding, the H mice passed fewer N. dubius eggs and harbored fewer, smaller and less fecund worms than did the L mice. These trends were observed after each of 4 consecutive infections, which were all treated with levamisole before reinfection. Further, more H than L mice were worm-free, and H mice were sensitized to sterile immunity by fewer infections than were needed by the L mice. These results encourage the view that the selective breeding of host species of agricultural importance is a valuable means of biologic control of nematodiasis.     

Nematospiroides dubius and Nippostrongylus brasiliensis: delayed type hypersensitivity responses to ovalbumin in the infected mouse

Mice (C57BL) infected with the intestinal nematode Nematospiroides dubius showed depressed delayed type hypersensitivity responses to ovalbumin administered subcutaneously in Freund's complete adjuvant. IgG and IgM responses to this inoculum were unaffected. It is unlikely that the depression arose from impairment of the ear test response because responses to an extract of the adult parasite were measurable and ear testing with lipopolysaccharide yielded normal responses in infected mice. Furthermore, mice immunized on the day of infection responded normally, whilst long term infected mice ear challenged with antigen pulsed macrophages gave depressed responses. The in vitro proliferative responses of cells from the spleens and from the lymph nodes draining the site of immunization were enhanced marginally by N. dubius infection. Furthermore, these cells induced normal or elevated adoptive delayed-type hypersensitivity and IgG responses in irradiated recipients. These findings suggest that N. dubius does not compromise the development of ovalbumin specific T cells involved in a delayed type hypersensitivity response. Evidence for the induction of suppressor cells by N. dubius is discussed, and the findings are compared with results obtained with Nippostrongylus brasiliensis, a parasite which is rejected rapidly from the mouse.     

Nippostrongylus brasiliensis infections in mice: the immunological basis of worm expulsion.

Nippostrongylus brasiliensis infections in mice were terminated more rapidly than in rats and immunologically induced damage occurred earlier. Like rats, mice expelled damaged worms more rapidly than normal worms. Recipients of cells from the spleen or mesenteric node of immune mice expelled their worms by day 8 of the infection. Recipients of cells alone or antiserum alone did not expel their worms by day 5 but mice given both cells and antiserum expelled their worms by this stage of the infection. Damaged worms were expelled more rapidly than normal worms from mice given immune cells, This work indicated that antibodies and cells collaborate to expel N. brasiliensis from mice as has been shown to occur in rats.     

Influence of immunizing dose and presence or absence of adult worms on the development of resistance to Nematospiroides dubius challenge infections of mice

H-2 congenic strains expressing resistant (H-2q, H-2f) or susceptible (H-2k) haplotypes were compared for their ability to resist challenge infection with N. dubius following a 6- or 14-day ivermectin-abbreviated immunizing infection. B10.BR mice (H-2k) were considerably more resistant to infection when the priming interval was shortened from 14 to 6 days. B10.Q (H-2q) and B10.M (H-2f) mice resisted challenge regardless of which immunization regimen was used. The influence of parasite numbers on the response to challenge was studied by comparing infections in resistant DBA/1 (H-2q) and susceptible CBA/J (H-2k) mice that differ at both H-2 and non-H-2 genes. DBA/1 mice, immunized with 50 or 150 L3 of N. dubius for 14 days, resisted challenge, whereas mice receiving 300 worms did not. In contrast, CBA/J mice failed to resist challenge at all priming doses tested. When the immunizing infection was shortened from 14 to 6 days, DBA/1 mice resisted challenge regardless of priming dose and CBA/J mice resisted challenge only when the highest dose of 300 worms was used for priming. The data suggest that susceptible strains of mice may be preferentially immunosuppressed, particularly at low infective doses, and that suppression is associated with adult worms present in the lumen of the small intestine.     

Macrophage mediated resistance to Babesia microti in Nematospiroides dubius--infected mice

Infection with the gastrointestinal nematode, Nematospiroides dubius, induced resistance to a concurrent infection with Babesia microti in mice. This enhanced resistance to B. microti occurred during infection with the larval, rather than the adult stages of N. dubius. Splenectomy or the injection of carrageenan or silica abrogated the protective effect against B. microti induced by infection with N. dubius. Peritoneal macrophages harvested from infected mice produced inhibition of the development of B. microti in vitro. This inhibition was greatest using macrophages harvested from mice recovered from infection with B. microti and from mice infected with the larval stages of N. dubius. Also, supernatants harvested from cultures of macrophages from N. dubius-infected mice suppressed B. microti development in vitro.     

Responses of inbred mouse strains to infection with intestinal nematodes

Comparisons were made of the immune and inflammatory responses of four strains of inbred mice to infection with the intestinal nematodes Trichinella spiralis and Nippostrongylus brasiliensis to determine whether genetically determined 'high responsiveness' to infection, seen most clearly in intestinal responses, is independent of the parasite concerned and necessarily correlated with protection. The time course of infection was followed by counting adult worms at intervals after infection. Mucosal mast cells and Paneth cell numbers were determined as indices of the intestinal inflammatory response. Levels of IgG2a and IgG1 antibodies and of the cytokines IFN-gamma and IL-5 released from in vitro-stimulated mesenteric node lymphocytes were measured to assess type 1 and type 2 responses. NIH and CBA mice were the most resistant to T. spiralis and N. brasiliensis respectively, resistance in each case being correlated with the most intense intestinal inflammatory responses. C57BL/10 (B10) and B10.BR were the least resistant to T. spiralis, but were as resistant as CBA to N. brasiliensis, despite their intestinal inflammatory responses to both parasites being much lower than the other two strains. Mice infected with T. spiralis made the expected switch from a type 1 (IFN-gamma) to a type 2 (IL-5) response between days 2 and 8, and there were no significant differences in levels of these cytokines between the strains. In contrast, when infected with N. brasiliensis, CBA showed an IFN-gamma response at day 4, all strains switching to IL-5 by day 8 and NIH mice releasing the greatest amount of IL-5. The results indicate that the "high responder" phenotype to intestinal nematode infection is in part determined by host characteristics, but is also determined by the parasite concerned--seen most clearly by the differences between NIH and CBA when infected with T. spiralis and N. brasiliensis. The fact that "low responder" B10 background mice were more resistant to N. brasiliensis than "high responder" NIH implies that each parasite elicits a particular pattern of protective host responses, rather than parasites being differentially susceptible to the same response profile.     

Genetic variation and host-parasite relations: Nematospiroides dubius in mice.

Our work deals with aspects of the genetics and immunology of host-parasite relationships as they influence the development of protective immunity and the phenomenon of coevolution. The aim is to understand parasitism through analyses of host specificity. In earlier studies we examined the inheritance of resistances in mice to infections with Nematospiroides dubius (=Heligmosomoides polygyrus) and established the predominant role played by antibodies in protective immunity. Here we report information concerning the selection of lines of N. dubius that differ in their ability to survive antagonistic immunological reactions from mice. Challenge infections with groups of these mice, immunized and protected by previous repeated infections, show that worms selected to survive the immunity that kills other worms do so by inhibiting the effectiveness of the cellular rather than humoral elements of protective immunity.     

Concurrent infections with Trypanosoma brucei and Nippostrongylus brasiliensis in mice deficient in inducible nitric oxide

Concurrent infection with Trypanosoma brucei (Tb) delays the normal protective responses of mice to the gastrointestinal parasite Nippostrongylus brasiliensis (Nb). The course of such infections was followed in mice genetically deficient in inducible nitric oxide synthase (INOS) to assess the role of nitric oxide (NO) in this effect. The time course of trypanosome infection in INOS deficient (INOS-/-) mice was similar to that in wild type (WT) and heterozygote (INOS+/-) mice but did not result in NO production. Although concurrent infection with Tb increased initial susceptibility to Nb in INOS-/- mice, the immune-mediated loss of N. brasiliensis and the associated decline in faecal egg output occurred more rapidly then in WT and INOS+/- littermates. Concurrent infection with trypanosomes markedly suppressed Concanavalin A (ConA)-induced in vitro proliferation of splenic lymphocytes in all groups, but had little effect on the responses of mesenteric node lymphocytes. Trypanosome infection was also associated with increased early release of interferon-gamma and reduced IL-5 from lymphocytes stimulated in vitro with ConA, but did not affect later release of IL-5. The overall similarity of proliferative and cytokine responses in WT, INOS+/- and INOS-/- mice suggest that the suppressive effects of T. brucei on N. brasiliensis infection do not simply reflect depressed lymphocyte responsiveness or altered cytokine profiles. NO appears to be involved in suppression only of the later phases of the host responses to Nb.     

Physiological responses of rats to primary infection with Nippostrongylus brasiliensis

The physiological responses of well-nourished rats to primary infection with the intestinal nematode Nippostrongylus brasiliensis were examined. Infected rats fed ad libitum were compared with uninfected control rats fed ad lib. and with uninfected rats which were pair-fed to the infected rats. Following infection with N. brasiliensis rat food intake was reduced from day 2 post infection (pi) and there were two periods of minimal food intake (days 2 to 3 and 8 to 9 pi). The water intake of infected rats was only reduced on days 2, 3 and 9 pi and not to the same extent as food intake. Muscle catabolism in infected rats was more severe than could be explained on the basis of their food intake reduction. The rectal temperature and rate of oxygen consumption per g body-weight of rats was not significantly altered by the infection. Host responses to N. brasiliensis are compared with those seen in microbial infections and some of them are found to be considerably different.     

Impairment of primary expulsion of Trichuris muris in mice concurrently infected with Nematospiroides dubius

Primary immune expulsion of Trichuris muris was markedly delayed by concurrent infection with Nematospiroides dubius. Maximum delay of expulsion was dependent on size and timing of N. dubius infection relative to T. muris infection. In NIH mice infection with 400 N. dubius larvae immediately before or after T. muris infection was found to be most effective in suppressing expulsion. Infection on day 8 of T. muris infection, when mice are sensitized to T. muris, also impaired expulsion. From this evidence it is suggested that the larvae of N. dubius are immunosuppressive and that the efferent role of the immune response to T. muris is inhibited. The results are discussed in terms of non-specific immunosuppression and their relevance to the tropical disease situation is emphasized.     

Host influences on reproduction and establishment of mouse-adapted Nippostrongylus brasiliensis (Nematoda)

Various influences of the host altered the establishment and reproduction of mouse-adapted Nippostrongylus brasiliensis. Establishment and egg production were greater in mature male than in female mice. Worm fecundity increased similarly in both sexes of mice prior to 41 days of host age. Male mice at 62 days of age or older harbored more N. brasiliensis than did females, but the number of nematodes was similar in younger hosts of both sexes. Crowding of male mice significantly increased the establishment of helminths, but not the production of eggs by N. brasiliensis. Pregnancy of the host significantly increased both establishment of and reproduction by N. brasiliensis. Ligation of the bile duct of the mouse reduced both establishment and fecundity of the nematode after subcutaneous or surgical infection with larvae and adults, respectively.     

Nematospiroides dubius: influence of adjuvants on immunity in mice vaccinated with antigens isolated by affinity chromatography from adult worms

Five adjuvants were examined for their ability to potentiate the immune response of mice to soluble antigens from adult Nematospiroides dubius prepared by affinity chromatography against antibodies from repeatedly infected mice as ligands (IMIgAg). Immunized mice were better protected against N. dubius by IMIgAg injected intraperitoneally with either pertussigen (75%) or aluminium hydroxide (Alum) (67%) as adjuvants than with Freud's complete (54%) or incomplete adjuvants (31%). Protection was correlated with elevated specific antibody values and with cellular responses. Quil A was toxic to recipient mice at the concentration used. Alum may be a more practical adjuvant than pertussigen, which may activate protective immunity only in specific recipient genotypes and oil-based adjuvants which appear to be less efficient, to vaccinate mice with soluble parasite antigens.     

Nematospiroides dubius: passive transfer of protective immunity to mice with monoclonal antibodies

Nine hybridoma cell lines secreting monoclonal antibodies specific for Nematospiroides dubius were produced by fusion of the mouse myeloma cell line NS-1 to either spleen cells or mesenteric lymph node cells from mice repeatedly infected with N. dubius. Seven of the antibodies were identified as IgM and two as IgG1. Each monoclonal antibody bound to polypeptide epitopes on both infective larvae (L3) and adult worms. However, five antibodies bound preferentially to L3 and three to adult worms. All nine antibodies reacted with high molecular weight protein antigens. Passive protective immunity in Balb/c mice was demonstrated with monoclonal antibodies Nd2 and Nd3 in ascites fluid which stunted both male and female worms and reduced parasite fecundity.     

Hymenolepis citelli (Cestoda) and Nematospiroides dubius (Nematoda): interspecific interaction in mice

In mice concurrently infected with Hymenolepis citelli and Nematospiroides dubius, survival of the tapeworm was prolonged, and there was an impairment of the efferent arm of the response to the cestode. The immunological rejection of a six cysticercoid primary H. citelli infection was delayed by the N. dubius infection. The growth of the cestode was poorer in concurrently infected mice, and this effect was rapid, being evident within 4 days of the N. dubius infection. Maximum biomass in the controls was reached on Day 20, whereas in the concurrently infected mice it was reached on Day 25. The induction of acquired immunity to homologous H. citelli infection was suppressed, although the expression of a secondary response against homologous challenge was not abrogated in doubly infected mice. The results are discussed with reference to the immunodepressive effects the nematode is known to have on heterologous antigenic stimulation.     

IgE formation and Fc receptor-positive lymphocytes in normal, immuno-deficient, and auto-immune mice infected with Nippostrongylus brasiliensis

The IgE serum levels and IgE FcR-positive lymphocytes (Fc epsilon R) in the spleen and mesenteric lymph nodes (MLN) of normal and immunologically mutant strains of mice were determined before and 14 days after infection with Nippostrongylus brasiliensis (Nbr) parasites. By IgE rosetting of cells immunofluorescently stained for sIg. Thy-1.2, Lyt-2, and L3T4, only sIg+ IgE rosetting lymphocytes were detected in both normal and Nbr-infected mice. IgE high responder mice had the same percentage of Fc epsilon R+ spleen and MLN lymphocytes as low responder mice. After Nbr infection, the percentages of splenic and MLN Fc epsilon R+ cells increased in parallel to a similar increase of sIg+ B cells. Athymic C57BL/6J-nu mice had 62% Fc epsilon R+ spleen and 85% Fc epsilon R+ MLN cells before and after Nbr infection, but IgE serum levels were less than 5 ng IgE/ml. C57BL/6J mice with the viable moth-eaten mutation mev which have almost exclusively Ly-1+ B cells, had less than 1% Fc epsilon R+ lymphocytes and formed only small amounts of IgE. C57BL/6J mice with the lymphoproliferation (lpr) or generalized lymphoproliferative disease (gld) mutations had low numbers of Fc epsilon R+ cells but formed 15 to 30 times more IgE after Nbr infection than control C57BL/6J mice. The IgE response of mice with the beige mutation (bg) did not differ from control mice. Mice with the xid mutation had few Fc epsilon R+ and sIg+ cells but showed high IgE responses. These data demonstrate that Fc epsilon R are typical cell surface markers for approximately 90% of murine Ly-1-, sIg+ B cells and that the number of Fc epsilon R+ cells does not correlate with the capacity of the mice to form IgE. The IgE response to Nbr infection is normal in mice homozygous for the bg mutation, elevated in mice homozygous for the xid, lpr, and gld mutations, and decreased in mice homozygous for the mev and nu mutations.