The reversibility of intestinal immune expulsion effects on adult Nippostrongylus brasiliensis

An attempt has been made to study the extent and nature of the damage occurring in adult Nippostrongylus brasiliensis undergoing immune expulsion from the rat. It was found that worms are not killed nor irreparably damaged when being rejected. On transfer into naive second recipient rats the rate of re-establishment of worms previously incubated in immune rat recipients for 4-17 hr was high (68-69%) and comparable to that shown by worms from normal recipient rats (48-56%). Similarly, worms taken on days 10, 11, and 12 of a primary infection, already passed to the distal half of the small intestine due to immune expulsion effects, on transfer into naive recipient rats re-established themselves well (rates varying from 62 to 80%) compared to those harvested from their normal habitat in the proximal half of the small intestine (rates varying from 44 to 87%). Worm damage is associated with decreased motility and impaired locomotion capacity. The phenomenon of mucosal trapping occurs during expulsion, but merely to the extent of some 30% of the worm population. It is suggested that in principle, worms subjected to immune expulsion are in a state of acute, transient metabolic crisis. The present results support the enteroallergic indirect mechanism for worm rejection.     

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.     

Immunity against trichostrongylus colubriformis infection in guinea-pigs and sheep: Some comparisons with Nippostrongylus brasiliensis infection in the rat

In Nippostrongylus brasiliensis infection in rats there is evidence that antibodies ‘damage’ the worms rendering them susceptible to other components of the host immune response. Some of the experiments from which this evidence was obtained were repeated in Trichostrongylus colubri-formis infections in guinea-pigs and sheep.Lesions similar to those described in ‘damaged’ N. brasiliensis were present in T. colubriformis, but unlike damaged N. brasiliensis, damaged T. colubriformis were not rapidly expelled from the intestine of either normal or adoptively immunized hosts. Other differences between the two infections included the failure of serum from T. colubriformis-immune donors either to regularly transfer immunity against infection to non-immune recipients or to endow additional immunity on recipients of lymphoid cells from immune donors.It is suggested that the results might reflect differences between the manner in which guinea-pigs and rats achieve immune expulsion of gastro-intestinal nematode parasites.     

Strongyloides ratti: reversibility of immune damage to adult worms

Transplantation experiments were conducted to assess the reversibility or irreversibility of the damage sustained by Strongyloides ratti during infections in the rat host. Worms of different ages from primary and secondary infections were recovered from their original hosts and transplanted surgically into naive rats. The size and fecundity of normal (Days 6–11 postinfection) worms were maintained after transfer. Damaged worms from primary infection (Days 22–26) showed complete recovery of size and fecundity within 10 days of transfer; damaged worms from a secondary infection (Days 6–7) also showed functional recovery but to a lesser extent. The ultrastructural changes observed mainly in the intestine of damaged worms from primary infections, prior to their transfer, were, however, only partially ameliorated following transplantation into new naive hosts; there was no complete return to structural normality. On the other hand, second infection worms did show almost complete ultrastructural recovery. The course of a transplanted infection established with either damaged or normal worms was similar to infections established percutaneously. Increase in the size of transplanted infections from 100 to 250 worms per recipient did not alter the dynamics of the host/parasite relationship. There was no evidence of adaptation in S. ratti and damaged worms, when transplanted into naive rats, were as successful as normal worms in protecting the host against a subcutaneous larval infection. The implications of this work on the present understanding of the phenomenon of autoinfection in experimental rodent strongyloidiasis are discussed.     

Trichinella spiralis: mediation of the intestinal component of protective immunity in the rat by multiple, phase-specific, antiparasitic responses.

Rats infected orally with Trichinella spiralis developed an immunity that was induced by and expressed against separate phases of the parasite's enteral life cycle. Infectious muscle larvae generated an immune response (rapid expulsion) that was directed against the very early intestinal infection and resulted in the expulsion of worms within 24 hr. This response eliminated more than 95% of worms in an oral challenge inoculum. Developing larvae (preadults) also induced an immune response that was expressed against adult worms. The effect on adults was dependent upon continuous exposure of worms to the immune environment throughout their enteral larval development. Immunity induced by preadult T. spiralis was not expressed against adult worms transferred from nonimmune rats. While adult worms were resistant to the immunity engendered by preadults they induced an efficient immunity that was autospecific. Both “preadult” and “adult” immunities were expressed in depression of worm fecundity as well as in the expulsion of adults from the gut. However, the two reactions differed in respect to their kinetics and their efficiency against various worm burdens. Preadult immunity was directed mainly against fecundity whereas adult immunity favored worm expulsion. All responses (rapid expulsion, preadult and adult immunity, and antifecundity) acted synergistically to produce sterile immunity against challenge infections of up to 5000 muscle larvae. These findings indicate that the host protective response to T. spiralis is a complex, multifactorial process that operates sequentially and synergistically to protect the host against reinfection.     

Strongyloides ratti: the role of enteral antigenic stimuli by adult worms in the generation of protective immunity in rats

Immunogenicity of adult Strongyloides ratti was studied in rats. Immunization of rats by intraduodenal implantation of adult worms could completely inhibit the egg production and hasten the expulsion of challenged worms which were developed from subcutaneously inoculated L3 or were implanted intraduodenally as adults. Enteral immunization by intraduodenal implantation of adult worms was, however, not able to affect the esophageal larval output of the challenge infection with L3. In contrast to enteral immunization with adult worms, immunization by full sequence of a primary infection or by a combination of drug-abbreviated infection and adult worm implantation could suppress the esophageal larval output of the challenge infection. The relationship between the host defense mechanism and the life cycle of S. ratti is discussed.     

Transplantation of adult Trichinella spiralis between hosts: worm survival and immunological characteristics of the host--parasite relationship.

A technique for the transplantation of Trichinella spiralis worms directly into the host intestine is described. Infections established by the direct transfer of adult worms were essentially normal both in terms of their survival and reproduction and in their stimulation of, and susceptibility to, host immune responses. Worms transplanted from NIH mouse donors at intervals after infection had an equal ability to survive in the recipient, even when taken from the donor shortly before or during the process of worm expulsion, showing that expulsion does not require worms to be irreversibly damaged. It was noted, however, that after 7 days in the donor the ability of the worm to reproduce in the recipient was temporarily impaired.     

Nippostrongylus brasiliensis: reversibility of reduced-energy status associated with the course of expulsion from the small intestine in rats

Gastrointestinal nematodes require energy for active establishment in the gut against intestinal flow and peristaltic motion. In this study we employed CellTiter-Glo Luminescent Cell Viability Assay to measure the ATP value of individual adult Nippostrongylus brasiliensis during the course of immune-mediated expulsion from the small intestine in rats. The ATP values of adult worms taken from the lumen of the distal small intestine were lower than worms collected from the lumen of the proximal small intestine. Moreover, values from worms in the lumen of the proximal small intestine were lower than those from worms in the mucosa, the preferred site of adult N. brasiliensis. The reduction of ATP values in worms from each region was observed not only at expulsion phase, but also at established phases of the infection suggesting that energy metabolism of the parasites is independent of host immune response. When adult worms with low ATP values on day 12 post-infection were implanted surgically into the small intestine of na?ve rats, the worms re-established in recipients and completely restored the ATP values. Short in vitro culture of adult worms under low oxygen tension resulted in low ATP value in the worms. These results suggested that adult worms were dislodged from their preferred site by intact energy metabolism activity.     

Immunity to intestinal parasites: role of mast cells and goblet cells

Nippostrongylus brasiliensis infection of rats and mice is a model for studying immunity at mucosal surfaces. Adult worms are spontaneously expelled from the intestine at the end of the second week of infection. Expulsion from the jejunum requires the presence of immune T lymphocytes and IgG antibodies. Mucosal mast cells (MMCs) are a prominent part of the jejunal inflammatory response. They are derived from a hematopoietic stem cell, possibly the same precursor as basophils. Their differentiation is not absolutely T dependent but their accumulation at the site of infection is. The possible involvement of IgE antibodies and intestinal MMCs through a "leak lesion" is still uncertain. Increased mucus secretion from epithelial goblet cells is also a prominent feature of the inflammatory reaction at the site of infection. Goblet cell numbers increase two to four times at the onset of worm expulsion; this increase is regulated by T lymphocytes and possibly immune serum. The mechanism of mucus secretion in these infections is not clear; it may be a response to mast cell mediators. Together with antiworm antibodies, intestinal mucus may trap worms and prevent them from surviving in the intervillous spaces of the jejunum. Thus, expulsion of this intestinal parasite may occur through a nonspecific process that is induced by specific immune mechanisms.     

Trichinella spiralis: role of different life cycle phases in induction, maintenance, and expression of rapid expulsion in rats.

The capacity of different phases of the life cycle of Trichinella spiralis to induce rapid expulsion was examined. The phases examined included enteral preadults, enteral adults, and parenteral larvae. All had the ability to induce rapid expulsion although there were significant quantitative differences in their inductive capacity and in the kinetics of expression. Immunization with preadults required only a 48-hr enteral exposure to 2000 worms to induce strong rapid expulsion. In contrast rats required a 14-day exposure to adult worms to elicit a comparable response. After immunization with adults the reaction was demonstrable for only 2 weeks. Parenteral larvae produced only a weak rapid expulsion reaction by themselves and this response did not develop until some 8 weeks after challenge. When immunization with the enteral phases (preadult and adult) was combined with exposure to parenteral larvae a strong and enduring rapid expulsion reaction was observed. Phase specificity was also observed in the susceptibility of worms to the rapid expulsion response. The preadult phases, from infectious larvae to worms of up to 2 days of age were highly susceptible. Older worms, from 3 to 4 days old were not susceptible to rapid expulsion and could invade and establish themselves in the primed intestine for at least a 48-hr period without apparent adverse effects.     

Neutrophil accumulation around Onchocerca worms and chemotaxis of neutrophils are dependent on Wolbachia endobacteria

Unlike in many other helminth infections, neutrophilic granulocytes are major cellular components in the hosts immune response against filarial worms. The pathways that drive the immune response involving neutrophils are unclear. This study shows that Wolbachia endobacteria (detectable by polyclonal antibodies against endobacterial heat shock protein 60 and catalase and by polymerase chain reaction being sensitive to doxycycline treatment) are direct and indirect sources of signals accounting for neutrophil accumulation around adult Onchocerca volvulus filariae. Worm nodules from untreated onchocerciasis patients displayed a strong neutrophil infiltrate adjacent to the live adult worms. In contrast, in patients treated with doxycycline to eliminate the endobacteria from O. volvulus and to render the worms sterile, the neutrophil accumulation around live adult filariae was drastically reduced. Neutrophils were absent in worm nodules from the deer filaria Onchocerca flexuosa, a species which does not contain endobacteria. Extracts of O. volvulus extirpated from untreated patients showed neutrophil chemotactic activity and in addition, induced strong TNF-alpha and IL-8 production in human monocytes, in contrast to filarial extracts obtained after doxycycline treatment. Thus, neutrophil chemotaxis and activation are induced directly by endobacterial products and also indirectly via chemokine induction by monocytes. These results show that the neutrophil response is a characteristic of endobacteria-containing filariae.     

New weapons in the war on worms: identification of putative mechanisms of immune-mediated expulsion of gastrointestinal nematodes

Parasitic nematode infections of humans and livestock continue to impose a significant public health and economic burden worldwide. Murine models of intestinal nematode infection have proved to be relevant and tractable systems to define the cellular and molecular basis of how the host immune system regulates resistance and susceptibility to infection. While susceptibility to chronic infection is propagated by T helper cell type 1 cytokine responses (characterised by production of IL-12, IL-18 and interferon-gamma), immunity to intestinal-dwelling adult nematode worms is critically dependent on a type 2 cytokine response (controlled by CD4+T helper type 2 cells that secrete the cytokines IL-4, IL-5, IL-9 and IL-13). However, the immune effector mechanisms elicited by type 2 cytokines in the gut microenvironment that precipitate worm expulsion have remained elusive. This review focuses on new studies that implicate host intestinal epithelial cells as one of the dominant immune effector cells against this group of pathogens. Specifically, three recently identified type 2 cytokine-dependent pathways that could offer insights into the mechanisms of expulsion of parasitic nematodes will be discussed: (i) the intelectins, a new family of galactose-binding lectins implicated in innate immunity, (ii) the resistin-like molecules, a family of small cysteine-rich proteins expressed by multiple cell types, and (iii) cytokine regulation of intestinal epithelial cell turnover. Identifying how the mammalian immune response fights gastrointestinal nematode infections is providing new insights into host protective immunity. Harnessing these discoveries, coupled with identifying what the targets of these responses are within parasitic nematodes, offers promise in the design of a new generation of anti-parasitic drugs and vaccines.     

Strongyloides venezuelensis: heparin-binding adhesion substances in immunologically damaged adult worms

Immunologically damaged Strongyloides venezuelensis adult worms were examined for their mucosal invasion ability and secretion of heparin-binding adhesion substances. S. venezuelensis was expelled from male Wistar rats 4 to 5 weeks after infection. Four-week-old adult worms were smaller and had fewer eggs than 1-week-old adult worms. One-week-old, 4-week-old, and 5-week-old adult worms equally established in the recipient mouse intestine when surgically implanted. Adult worms of 4 and 5 weeks of age secreted adhesion substances as much as 1-week-old adult worms. There was no difference in the heparin-binding activities and the lectin-binding profile of adhesion substances among adult worms of different ages. The rate of secretion of adhesion substances from the mouth was also identical. Heparin-binding activities were detected in crude adult worm proteins; however, proteins of 5-week-old adult worms had weaker heparin-binding activities than those of 1-week-old adult worms. Western blotting revealed that a number of heparin-binding proteins were lost in 5-week-old adult worms. A heparin-binding protein of 42. 0 kDa, which was consistently expressed in adult worms, was a possible component of heparin-binding adhesion substances which are secreted from the mouth.     

Trichinella spiralis: immunity and inflammation in the expulsion of transplanted adult worms from mice.

The technique of implanting adult Trichinella spiralis into the intestines of mice has been used to assess the contributions of direct, anti-worm immunity and of intestinal inflammation to worm expulsion. The survival after transfer of worms exposed to an effective adoptive immunity in donors was no different from that of worms taken from control donors. Worms taken from donors 8 days after infection, i.e., shortly before the onset of expulsion, showed no increased susceptibility to an immunity adoptively transferred to the recipient mice. When worms were implanted into mice responding to a prior, oral infection they were expelled rapidly. This expulsion was independent of the age of the worms transferred and took place at the same time as the expulsion of the existing infection.     

Co-expulsion of Ascaridia galli and Heterakis gallinarum by chickens

Worm expulsion is known to occur in mammalian hosts exposed to mono-species helminth infections, whilst this phenomenon is poorly described in avian hosts. Mono-species infections, however, are rather rare under natural circumstances. Therefore, we quantified the extent and duration of worm expulsion by chickens experimentally infected with both Ascaridia galli and Heterakis gallinarum, and investigated the accompanying humoral and cell-mediated host immune responses in association with population dynamics of the worms. Results demonstrated the strong co-expulsion of the two ascarid species in three phases. The expulsion patterns were characterized by non-linear alterations separated by species-specific time thresholds. Ascaridia galli burden decreased at a daily expulsion rate (e) of 4.3 worms up to a threshold of 30.5 days p.i., followed by a much lower second expulsion rate (e = 0.46), which resulted in almost, but not entirely, complete expulsion. Heterakis gallinarum was able to induce reinfection within the experimental period (9 weeks). First generation H. gallinarum worms were expelled at a daily rate of e = 0.8 worms until 36.4 days p.i., and thereafter almost no expulsion occurred. Data on both humoral and tissue-specific cellular immune responses collectively indicated that antibody production in chickens with multispecies ascarid infections is triggered by Th2 polarisation. Local Th2 immune responses and mucin-regulating genes are associated with the regulation of worm expulsion. In conclusion, the chicken host is able to eliminate the vast majority of both A. galli and H. gallinarum in three distinct phases. Worm expulsion was strongly associated with the developmental stages of the worms, where the elimination of juvenile stages was specifically targeted. A very small percentage of worms was nevertheless able to survive, reach maturity and induce reinfection if given sufficient time to complete their life cycle. Both humoral and local immune responses were associated with worm expulsion.     

Eosinophil responses of permissive and nonpermissive hosts to the young adult worms of Angiostrongylus cantonensis

Blood and bone marrow eosinophilia was assessed in nonpermissive (guinea pigs) and permissive (rats) hosts following the pulmonary arterial transfers of live or dead young adult worms of Angiostrongylus cantonensis. Guinea pigs showed a marked eosinophilic response to live worms but only a slight response to dead worms. Neither IgE nor haemagglutinating antibodies correlated with the induction of this eosinophilia. In contrast, the rat responded to neither form of the young adult worm. When the guinea pig and the rat were injected with whole worm extract (WWE) of the young adult worms either by an osmotic mini-pump connected to the jugular vein or by intermittent intravenous injections, the former animal showed blood eosinophilia but the latter failed to do so. Guinea pigs also developed blood eosinophilia after continuous exposure to the excretory and secretory products of the young adult worms, administered by the mini-pump. Eosinophil responses to WWE could be induced both in athymic CD-1 (ICR) nude mice and in its heterozygous litter mates, suggesting that T cell-independent mechanism(s) could be involved in the induction of blood eosinophilia in the nonpermissive, mouse host. These data clearly indicate that the eosinophilia-inducing factor(s) and the mechanism of eosinophilia are different in permissive and nonpermissive hosts.     

Some observations on a possible role of lung and fecal IgA antibodies in immunity of rats to Nippostrongylus brasiliensis

Factors influencing lung IgA antibody responses to Nippostrongylus brasiliensis infections and the role of lung and fecal IgA antibodies in immunity to this nematode were studied in rats. Hooded Lister rats were vaccinated subcutaneously with infective larvae radio-attenuated at 80-180 kr or with a single dose of infective larvae somatic proteins administered intravenously or intragastrically, and then challenged 14 days later with normal larvae. It was found that optimal lung IgA antibody responses depended more on the duration of the antigenic stimulation than on the quantity of antigenic material present, although a threshold amount was required. However, comparisons of lung anti-larval IgA antibody levels in rats resistant or susceptible to challenge indicated that these antibodies were not directly involved in specific host protective immunity. Levels of haemagglutinating fecal antibodies reacting with adult nematode metabolites were correlated with the numbers of adult worms recovered from the intestines following vaccination and also with the degree of resistance to reinfection. However, preincubation of adult (day 5) nematodes in media containing the IgA fraction of fecal globulins from primary infected rats did not reduce the ability of these worms to establish and survive in naive rats.     

Trichinella spiralis: acquired immunity in swine

The ability of domestic pigs to develop protective immunity to Trichinella spiralis in response to inoculation with different doses of muscle larvae was assessed. Adult worms developing from the inoculations of 112, 500, and 10,000 larvae were expelled from the intestine about 6 weeks after inoculation. Inoculation with 25,000 larvae, however, resulted in more rapid intestinal worms expulsion, indicating that gut expulsion is dose dependent. Secondary expulsion also tended to be dependent upon primary infection level. Pigs initially inoculated with 500 to 10,000 larvae expelled the challenge infection of adult worms after 22 to 25 days; in contrast, infection by inoculation of only 112 larvae failed to induce significant enchanced gut expulsion of the challenge infection intestinal worms. However, all primary infection levels, including inoculation with 112 larvae, induced nearly absolute resistance to the muscle establishment of larvae from challenge adult worms. The fecundity of female worms recovered from immune pigs was reduced 75% in comparison to controls. These results show that, in contrast to some host species, very rapid gut expulsion does not occur in domestic swine. Yet, immune responses at the gut level are important, perhaps responsible for much of the inhibition reflected as reduction in the establishment of muscle larvae.     

Nippostrongylus brasiliensis: intestinal goblet-cell response in adoptively immunized rats.

Goblet-cell differentiation was studied in the intestinal epithelium of rats infected with the nematode Nippostrongylus brasiliensis. An increase in the proportion of goblet cells occurred at the time of worm expulsion in rats infected with 1000 or 4000 third stage larvae. Adoptive immunization of infected rats with immune-thoracic duct lymphocytes (TDL) induced extensive goblet-cell differentiation whereas the transfer of immune-TDL into normal rats had no effect. The extent of goblet-cell differentiation in adoptively immunized infected rats was proportional to the number of cells transferred. A goblet-cell response also occurred in adoptively immunized rats harboring implanted “normal” and “damaged” worms but recipients of normal worms which were not given cells were unable either to expel their worm burden or to induce a goblet-cell response. Experiments in which the parasites were expelled with an anthelmintic drug suggested that the goblet-cell increase was not simply a repair process associated with the expulsion of the parasites. In all situations where immune expulsion of the parasites occurred, there was a concomitant rise in the proportion of goblet cells. These experiments suggest that thoracic duct lymphocytes either directly or indirectly regulate the differentiation of intestinal goblet cells.     

Echinostoma revolutum: resistance to secondary and superimposed infections in mice

A complete or almost complete resistance (94-100%) to a superimposed Echinostoma revolutum infection existed in mice harboring 20-, 30-, and 40-day-old infections in the range of 2-4 to 30-35 worms, but no resistance was found at challenge Day 10. A similar high level of resistance (85-100%) also existed in mice for at least 6 weeks after natural expulsion of a primary 6 metacercarial infection and for at least 5 weeks after anthelmintic termination of a 30-day-old 20 metacercarial infection. Thymus-deficient nude mice failed to develop resistance to a superimposed infection, and the resistance in normal mice was inhibited by corticosteroid treatment. These findings are all in favor of a host immune response being responsible for the resistance against both a secondary and a superimposed infection. Nearly all the worms of a superimposed infection were, in resistant mice, expelled prior to 24 hr following infection (rapid expulsion), and the few worms circumventing this early expulsion persisted for at least 8 days. Newly excysted juvenile worms implanted intraduodenally into resistant mice were rejected to the same degree as juvenile worms from an oral metacercarial infection indicating that the newly excysted juvenile worms are the target of the host immune response. However, 7-day-old worms implanted intraduodenally into resistant mice survived indicating that adaptation to the host immune response had occurred. In conclusion, this host-parasite model is an example of concomitant immunity because the immunological mechanism responsible for the expulsion of the superimposed infection had no effect on the number of primary worms present.