Experimental evolution of parasite life-history traits in Strongyloides ratti (Nematoda)

Evolutionary ecology predicts that parasite life-history traits, including a parasite's survivorship and fecundity within a host, will evolve in response to selection and that their evolution will be constrained by trade-offs between traits. Here, we test these predictions using a nematode parasite of rats, Strongyloides ratti, as a model. We performed a selection experiment by passage of parasite progeny from either early in an infection ('fast' lines) or late in an infection ('slow' lines). We found that parasite fecundity responded to selection but that parasite survivorship did not. We found a trade-off mediated via conspecific density-dependent constraints; namely, that fast lines exhibit higher density-independent fecundity than slow lines, but fast lines suffered greater reduction in fecundity in the presence of density-dependent constraints than slow lines. We also found that slow lines both stimulate a higher level of IgG1, which is a marker for a Th2-type immune response, and show less of a reduction in fecundity in response to IgG1 levels than for fast lines. Our results confirm the general prediction that parasite life-history traits can evolve in response to selection and indicate that such evolutionary responses may have significant implications for the epidemiology of infectious disease.     

Immunological responses elicited by different infection regimes with Strongyloides ratti

Nematode infections are a ubiquitous feature of vertebrate life. In nature, such nematode infections are acquired by continued exposure to infective stages over a prolonged period of time. By contrast, experimental laboratory infections are typically induced by the administration of a single (and often large) dose of infective stages. Previous work has shown that the size of an infection dose can have significant effects on anti-nematode immune responses. Here we investigated the effect of different infection regimes of Strongyloides ratti, comparing single and repeated dose infections, on the host immune response that was elicited. We considered and compared infections of the same size, but administered in different ways. We considered infection size in two ways: the maximum dose of worms administered and the cumulative worm exposure time. We found that both infection regimes resulted in Th2-type immune response, characterised by IL4 and IL13 produced by S. ratti stimulated mesenteric lymph node cells, anti-S. ratti IgG(1) and intestinal rat mast cell protease II (RMCPII) production. We observed some small quantitative immunological differences between different infection regimes, in which the concentration of IL4, IL13, anti-S. ratti IgG(1) and IgG(2a) and RMCPII were affected. However, these differences were quantitatively relatively modest compared with the temporal dynamics of the anti-S. ratti immune response as a whole.     

Antibodies and coinfection drive variation in nematode burdens in wild mice

Coinfections with parasitic helminths and microparasites are highly common in nature and can lead to complex within-host interactions between parasite species which can cause negative health outcomes for humans, and domestic and wild animals. Many of these negative health effects worsen with increasing parasite burdens. However, even though many studies have identified several key factors that determine worm burdens across various host systems, less is known about how the immune response interacts with these factors and what the consequences are for the outcome of within-host parasite interactions. We investigated two interacting gastrointestinal parasites of wild wood mice, Heligmosomoides polygyrus (nematode) and Eimeria spp. (coccidia), in order to investigate how host demographic factors, coinfection and the host’s immune response affected parasite burdens and infection probability, and to determine what factors predict parasite-specific and total antibody levels. We found that antibody levels were the only factors that significantly influenced variation in both H. polygyrus burden and infection probability, and Eimeria spp. infection probability. Total faecal IgA was negatively associated with H. polygyrus burden and Eimeria spp. infection, whereas H. polygyrus-specific IgG1 was positively associated with H. polygyrus infection. We further found that the presence of Eimeria spp. had a negative effect on both faecal IgA and H. polygyrus-specific IgG1. Our results show that even in the context of natural demographic and immunological variation amongst individuals, we were able to decipher a role for the host humoral immune response in shaping the within-host interaction between H. polygyrus and Eimeria spp.     

Cysteine protease B of Leishmania mexicana inhibits host Th1 responses and protective immunity

C3H mice infected with Leishmania mexicana fail to develop a protective Th1 response, and are unable to cure. In this study, we show that L. mexicana cysteine proteases suppress the antileishmanial immune response. Previous studies demonstrated that deletion of the entire multicopy cysteine protease B (CPB) gene array in L. mexicana is associated with decreased parasite virulence, potentially attributable to factors related to parasite fitness rather than to direct effects on the host immune response. We now show that C3H mice infected with the L. mexicana deletion mutant (Deltacpb) initially develop lesions that grow at rates comparable to those of wild-type L. mexicana-infected mice. However, in contrast to controls, Deltacpb-induced lesions heal with an accompanying Th1 immune response. Lesion resolution was Th1 dependent, as Deltacpb-infected IL-12p40(-/-) and STAT4(-/-) mice developed high parasite burdens and progressive disease. Moreover, when L. major was transfected with a cosmid expressing multiple L. mexicana CPB genes, this parasite induced a significantly lower IFN-gamma response compared with wild-type L. major. These data indicate that cysteine proteases of L. mexicana are critical in suppressing protective immune responses and that inhibition of CPB may prove to be a valuable immunomodulatory strategy for chronic forms of leishmaniasis.     

Altered T helper responses in CD40 and interleukin-12 deficient mice reveal a critical role for Th1 responses in eliminating the helminth parasite Taenia crassiceps

A key feature of helminth infections is the induction of strong Th2-biased immune responses in their hosts. We have previously found that Th2-like responses mediate susceptibility to the helminth parasite Taenia crassiceps, probably by inhibiting Th1 responses required for the development of protective immunity against this parasite. Here we show that mice lacking interleukin-12p35 (IL-12p35-/-) following T. crassiceps infection, failed to mount a Th1 response, but developed a strong Th2-type response, produced higher levels of IgG1, IgE, interleukin-4, interleukin-5 as well as interleukin-13 than wild-type mice, and became highly susceptible to the larval stage of this cestode. In contrast, similarly-infected CD40 deficient BALB/c mice (CD40-/-) displayed impairment of both Th1 and Th2-type responses associated with low levels of interferon-gamma as well as IgE, interleukin-4, interleukin-5 and interleukin-13, but efficiently controlled T. crassiceps infection. Together, these findings suggest a detrimental role for Th2-biased responses during the larval stage of T. crassiceps infection. Furthermore, they also suggest a pivotal role for CD40 in developing Th2-type responses.     

Schistosoma mansoni and alpha-galactosylceramide: prophylactic effect of Th1 Immune suppression in a mouse model of Graves' hyperthyroidism

Graves' hyperthyroidism, an organ-specific autoimmune disease mediated by stimulatory thyrotropin receptor (TSHR) autoantibodies, has been considered a Th2-dominant disease. However, recent data with mouse Graves' models are conflicting. For example, we recently demonstrated that injection of BALB/c mice with adenovirus coding the TSHR induced Graves' hyperthyroidism characterized by mixed Th1 and Th2 immune responses against the TSHR, and that transient coexpression of the Th2 cytokine IL-4 by adenovirus skewed Ag-specific immune response toward Th2 and suppressed disease induction. To gain further insight into the relationship between immune polarization and Graves' disease, we evaluated the effect of Th2 immune polarization by helminth Schistosoma mansoni infection and alpha-galactosylceramide (alpha-GalCer), both known to bias the systemic immune response to Th2, on Graves' disease. S. mansoni infection first induced mixed Th1 and Th2 immune responses to soluble worm Ags, followed by a Th2 response to soluble egg Ags. Prior infection with S. mansoni suppressed the Th1-type anti-TSHR immune response, as demonstrated by impaired Ag-specific IFN-gamma secretion of splenocytes and decreased titers of IgG2a subclass anti-TSHR Abs, and also prevented disease development. Similarly, alpha-GalCer suppressed Ag-specific splenocyte secretion of IFN-gamma and prevented disease induction. However, once the anti-TSHR immune response was fully induced, S. mansoni or alpha-GalCer was ineffective in curing disease. These data support the Th1 theory in Graves' disease and indicate that suppression of the Th1-type immune response at the time of Ag priming may be crucial for inhibiting the pathogenic anti-TSHR immune response.     

TLR9 is required for the gut-associated lymphoid tissue response following oral infection of Toxoplasma gondii

TLRs expressed by a variety of cells, including epithelial cells, B cells, and dendritic cells, are important initiators of the immune response following stimulation with various microbial products. Several of the TLRs require the adaptor protein, MyD88, which is an important mediator for the immune response following Toxoplasma gondii infection. Previously, TLR9-mediated innate immune responses were predominantly associated with ligation of unmethylated bacterial CpG DNA. In this study, we show that TLR9 is required for the Th1-type inflammatory response that ensues following oral infection with T. gondii. After oral infection with T. gondii, susceptible wild-type (WT; C57BL/6) but not TLR9(-/-) (B6 background) mice develop a Th1-dependent acute lethal ileitis; TLR9(-/-) mice have higher parasite burdens than control WT mice, consistent with depressed IFN-gamma-dependent parasite killing. A reduction in the total T cell and IFN-gamma-producing T cell frequencies was observed in the lamina propria of the TLR9(-/-) parasite-infected mice. TLR9 and type I IFN production was observed by cells from infected intestines in WT mice. TLR9 expression by dendritic cell populations is essential for their expansion in the mesenteric lymph nodes of infected mice. Infection of chimeric mice deleted of TLR9 in either the hemopoietic or nonhemopoietic compartments demonstrated that TLR9 expression by cells from both compartments is important for efficient T cell responses to oral infection. These observations demonstrate that TLR9 mediates the innate response to oral parasite infection and is involved in the development of an effective Th1-type immune response.     

Strongyloides ratti infection modulates B and T cell responses to third party antigens

It is estimated that over one third of the world population is infected with helminths, Strongyloides ssp. accounting for approximately 30-100 million cases. As helminth infections often result in a modulation of the host's immune system, infected people may display impaired responses to concurrent infections and to third party antigens. Here, we employ the experimental system of murine Strongyloides ratti infection to investigate the impact of helminth infections on experimental vaccinations. We demonstrate that concurrent infection with S. ratti strongly affected the humoral response to a thymus dependent model antigen, whereby predominantly Th1 associated IgG2b production was suppressed. We provide evidence that this suppression was due to modulation of T helper cell and not B cell function as the responses to a thymus independent model antigen remained unchanged in S. ratti infected mice. Moreover, using an adoptive transfer system, we show that infection with S. ratti directly interfered with antigen-specific proliferation of T cell receptor transgenic CD4(+) T helper cells in vivo. Finally, using IL-10 deficient mice and mice that selectively lack T helper cell derived IL-10 we rule out a role for host-derived IL-10 in mediating the suppression of thymus dependent model antigen response in S. ratti infected mice.     

Expression of genes in gastrointestinal and lymphatic tissues during parasite infection in sheep genetically resistant or susceptible to Trichostrongylus colubriformis and Haemonchus contortus

Resistance to an acute gastrointestinal nematode (GIN) infection is dependent on the ability of the host to recognise the parasite and mount a protective Th2 response. It is hypothesised that lambs which are genetically susceptible to GIN will differentially up-regulate Th1-type genes and therefore remain susceptible to chronic parasitism compared with genetically resistant lambs which will differentially up-regulate Th2-type genes and clear the parasite infection. Two selection flocks, in which lines of Merino sheep produced lambs genetically resistant or susceptible to GIN, were acutely challenged once or thrice with either Haemonchus contortus or Trichostrongylus colubriformis. Faecal-egg counts (FECs), and plasma and tissue anti-parasite (H. contortus or T. colubriformis) antibody isotype responses showed that resistant animals challenged three times with T. colubriformis established a protective Th2 response (negligible FEC, IgG1 and IgE) whereas susceptible animals required multiple challenges to establish a significant IgG1 response despite FECs remaining high. Trichostrongylus colubriformis elicited a more pronounced host response than H. contortus. RNA extracted from tissues at the site of each parasite infection and associated lymph nodes were interrogated by microarray and quantitative PCR analyses to correlate host gene expression to FECs and antibody responses. The IFN-γ inducible gene cxcl10 was up-regulated in the susceptible line of the Trichostrongylus selection flock sheep after a tertiary challenge with the parasites H. contortus and T. colubriformis. However, a uniform pattern of genes was not up-regulated in resistant animals from both selection flocks during both parasite infections, suggesting that the mode of host resistance to these parasites is different, although some similarities in host susceptibility were apparent.     

Immune expulsion of the trichostrongylid Cooperia oncophora is associated with increased eosinophilia and mucosal IgA

Previous experiments have shown that a primary infection with 100000 infective larvae of the trichostrongylid Cooperia oncophora allows discrimination between different type of responder animals based on the speed by which the parasite is expelled from the host. In most of the animals (intermediate responders) the expulsion occurs 35-42 days after infection. This experiment was carried out to investigate which mechanisms contribute to the clearance of the parasite from the intestine. Sequential necropsy of the animals 14, 28 and 42 days after infection together with a segmental division of the small intestine, allowed us to characterise essential components associated with development of immunity and expulsion of the parasite from its niche. The results show that during the patent phase of the infection the parasite preferentially resides in the proximal gut. Forty-two days after infection ongoing expulsion is characterised by a migration of the worms to the more distal part of the intestine. Expulsion of the adult worm population appears to be mast-cell independent and is associated with a significant increase in parasite-specific mucous IgA and IgG1 as well as with an influx of eosinophils in the intestinal lamina propria. Although we did not observe a specific lymphocyte recruitment into the intestinal mucosa, the accumulation of eosinophils seems to be mediated by CD4+ cells. We measured significant negative correlations between the number of eosinophils and the expulsion rate of the parasite expressed by sex ratio and ratio eggs per gram faeces. Parasite-specific mucosal IgA levels were negatively correlated to the fecundity of the worms, expressed as number of eggs per female worm. Our results describe the involvement of both eosinophils and mucosal IgA in the regulation of C. oncophora expulsion and suggest the development of a Th2 effector immune response.     

Effect of immunostimulatory oligodeoxynucleotides on host responses and the establishment of Brugia pahangi in Mongolian gerbils (Meriones unguiculatus)

Infection of humans with filarial parasites has long been associated with the maintenance of a dominant Th2-type host immune response. This is reflected by increases in interleukin (IL)-4- and IL-5-producing T cells, elevated immunoglobulin (Ig)E and IgG4 levels, and a pronounced eosinophilia. The Mongolian gerbil (Meriones unguiculatus) is permissive for the filarial nematodes Brugia malayi and B. pahangi. As in humans, persistent microfilaremic infections of gerbils with Brugia spp. results in increases in Th2 cytokines such as IL-4 and IL-5. The association of dominant Th2 cytokine profiles with the maintenance of infection suggests that the introduction of Brugia spp. into a strongly Th1-biased environment may adversely affect parasite establishment. Indeed, studies conducted in mice with B. malayi suggest that depleting Th1 effectors such as interferon (IFN)-gamma and nitric oxide results in increased worm recoveries. In the present studies, the Mongolian gerbil was used as a model to investigate the effect of a dominant Th1 cytokine environment on the establishment of B. pahangi. Intraperitoneal (i.p.) administration of immunostimulatory oligodeoxynucleotide (IS ODN) induced the production of IFN-gamma in the peritoneal exudate cells and spleen of gerbils. The presence of IFN-gamma at the time of B. pahangi infection did result in an altered host immune response to B. pahangi. Gerbils that received IS ODN before i.p. B. pahangi infections showed lower levels of the Th2-type cytokines IL-4 and IL-5, compared with animals that received B. pahangi alone (0 + Bp). This alteration in cytokine profile, however, did not alter the establishment or development of B. pahangi in the peritoneal cavity. Furthermore, there was no difference in the granulomatous response of gerbils to soluble adult B. pahangi antigen bound to beads embolized in their lungs, regardless of treatment group, suggesting that IL-4 and IL-5 are not essential contributors to the systemic host inflammatory response to B. pahangi in this model.     

Spectrum of gut immunologic reactions: selective induction of distinct responses to Vibrio cholerae WO7 and its toxin

Past studies with Vibrio cholerae have shown that cholera toxin (CT) is mainly responsible for inducing T helper type 2 (Th2) responses with systemic IgG1, IgE and mucosal secretory IgA (sIgA) antibodies. In this study, V. cholerae WO7, which produces novel toxin unrelated to CT, was given orally to mice in order to determine whether the strain V. cholerae WO7 differs from V. cholerae 569B, which produces CT, in the nature of responses generated at the gut and splenic level. The analysis of immune responses evoked by V. cholerae WO7 in the gut of mice revealed striking differences as compared to those elicited by V. cholerae 569B infection. To assess the T helper cell type responses, lymphocytes from Peyer's patches and the spleen were stimulated in vitro for studying the cytokine patterns. PP and SP lymphoid cells from V. cholerae WO7 infected animals elaborated significant amounts of IL-2, IFN-gamma and IL-12 by 7 days p.i., suggesting a Th1 type of response. However by 15 days p.i., the PP and SP lymphoid cells secreted only IL-6 and IL-10 with traces of IFN-gamma. On the other hand, infection with V. cholerae 569B yielded mainly Th2 type responses at Peyer's patches as well as the splenic level. Infection with both V. cholerae WO7 and 569B induced toxin-specific IgA secreting cells at the gut and splenic level along with IgG1 secreting cells, indicating that both V. cholerae WO7 and 569B evoke an antigen-specific Th2 type of response in the gut as well as spleen. The persistence of IgA along with Th1-type cytokines indicates an alternate induction mechanism since mucosal IgA responses are usually associated with Th2-type responses. These observations are suggestive of a common mechanism employed by the host to clear different strains of V. cholerae infection (569B and WO7 in this case), while the nature of toxins elaborated failed to modulate the net outcome of the infection caused by V. cholerae.     

Who is in control of the stickleback immune system: interactions between Schistocephalus solidus and its specific vertebrate host

The cestode Schistocephalus solidus is a frequent parasite of three-spined sticklebacks and has a large impact on its host's fitness. Selection pressure should therefore be high on stickleback defence mechanisms, like an efficient immune system, and also on parasite strategies to overcome these. Even though there are indications for manipulation of the immune system of its specific second intermediate host by the cestode, nothing is yet known about the chronology of specific interactions of S. solidus with the stickleback immune system. We here expected sticklebacks to first mount an innate immune response directly post-exposure to the parasite to clear the infection at an early stage and after an initial lag phase to upregulate adaptive immunity. Most interestingly, we did not find any upregulation of the specific lymphocyte-mediated immune response. Also, the pattern of activation of the innate immune system did not match our expectations: the proliferation of monocytes followed fluctuating kinetics suggesting that the parasite repeatedly installs a new surface coat not immunogenic to the host. Furthermore, the respiratory burst activity, which has the potential to clear an early S. solidus infection, was upregulated very late during infection, when the parasite was too big to be cleared but ready for transmission to its final host. We here suggest that the late activation of the innate immune system interferes with the neuroendocrine system, which mediates reduced predation avoidance behaviour and so facilitates the transmission to the final host.     

Toll-like receptor (TLR)2 and TLR3 sensing is required for dendritic cell activation, but dispensable to control Schistosoma mansoni infection and pathology

Toll-like receptors (TLRs) play an important role in the innate recognition of pathogens by dendritic cells (DCs) and in the induction of immune responses. However, relatively little is known about their functions in innate/acquired responses to complex eukaryotic microorganisms, including helminth parasites. That Schistosoma mansoni eggs activate myeloid DCs through TLR2 and TLR3 has been shown by us and others, but the consequences of this combined activation are still unknown. We show that the engagement of both TLR2 and TLR3 by schistosome eggs is important for the production of inflammatory cytokines and interferon-stimulated genes, such as some chemokines, by DCs. Strikingly, DCs sensitized with ovalbumin in the presence of parasite eggs dramatically reduce the release of Th2-type cytokines by ovalbumin-specific T lymphocytes, an effect that fully depends on TLR3. Finally, although TLR2 and TLR3 have no role in host resistance and in egg-induced granuloma formation in S. mansoni-infected mice, they individually and additionally increase the Th1/Th2 balance of the immune response. Thus, TLR2 and TLR3 sensing is required to shape the immune response during murine schistosomiasis, but is dispensable to control infection and pathology.     

Bigger groups have fewer parasites and similar cortisol levels: a multi-group analysis in red colobus monkeys

If stress and disease impose fitness costs, and if those costs vary as a function of group size, then stress and disease should exert selection pressures on group size. We assessed the relationships between group size, stress, and parasite infections across nine groups of red colobus monkeys (Procolobus rufomitratus) in Kibale National Park, Uganda. We used fecal cortisol as a measure of physiological stress and examined fecal samples to assess the prevalence and intensity of gastrointestinal helminth infections. We also examined the effect of behaviors that could potentially reduce parasite transmission (e.g., increasing group spread and reducing social interactions). We found that cortisol was not significantly related to group size, but parasite prevalence was negatively related to group size and group spread. The observed increase in group spread could have reduced the rate of parasite transmission in larger groups; however, it is not clear whether this was a density-dependent behavioral counter-strategy to infection or a response to food competition that also reduced parasite transmission. The results do not support the suggestion that gastrointestinal parasitism or stress directly imposed group-size-related fitness costs, and we cannot conclude that they are among the mechanisms limiting group size in red colobus monkeys.     

Use of repeated measure linear modeling to analyze longitudinal data from experimental parasite infections.

Here the use of repeated measure linear modeling to analyze experimental infections is demonstrated. This method of analysis makes full use of serial measurements on the same host throughout the course of an infection while taking into account correlations between measures within the same host. As an example, repeated measure linear modeling is used to analyze worm output from an experimental infection of Strongyloides ratti, a nematode parasite of rats. This analysis allows differences in the temporal dynamics of infection of two lines of S. ratti to be dissected.     

Immunocompetence and helminth community of the white-toothed shrew, <Emphasis Type="Italic">Crocidura russula</Emphasis> from the Montseny Natural Park,Spain

Host immunocompetence assessed by spleen size and response to phytohaemagglutinin (PHA) injection may give some indications on the control of parasite infection and on host mediation effect, through immunity, on parasite community structure. We investigated the helminth community and immunocompetence of the white-toothed shrew in a small area to test the relationship between immunocompetence and intensity of helminth infection. At the proximate level and if spleen mass and PHA response reflect the level of immunocompetence, we expected that individuals with a large spleen or a high PHA response should harbour a lower parasite load than individuals with relatively small spleen or low PHA response. In addition, we predicted that the structure of the helminth community should be mediated by the host’s immune defence. Spleen mass was linked to helminth infection. Nematodes and cestodes were negatively associated within hosts. PHA response was not related to total helminth intensity of infection but was negatively related to cestode intensity and positively to nematode intensity. This result suggests either a differential modulating effect on immunity by the two groups of worms or the existence of an antagonistic association between nematodes and cestodes mediated by the immune response of the host.     

Echinococcus granulosus: different cytokine profiles are induced by single versus multiple experimental infections in dogs

Modulation of host responses is an important strategy by which parasites ensure successful establishment and persistence. Host counteraction against this modulation may be required for the host to develop resistance to infection. In this pilot study, experimental infection of dogs with Echinococcus granulosus induced a strong polarization of the cytokine response towards a Th2 phenotype. Consecutive rounds of infection and cure induced resistance to infection resulting in a dramatically lower parasite burden. Repeatedly-infected resistant dogs also lost immune polarization and developed a balanced Th1/Th2 response. No major differences were observed in the production of regulatory cytokines (IL-10, TGF-β) between dogs with high parasite load and dogs with only few intestinal parasites. These results suggest that E. granulosus-driven immunomodulation contributes to successful infection in the definitive host. This information might be relevant for the development of more effective vaccines against this stage of the parasite.     

Immunoglobulin subclass responses of wild brown rats to Sarcocystis singaporensis

Immunoglobulin subclass responses of wild brown rats (Rattus norvegicus) from southeastern Asia to the endemic cyst-forming coccidian Sarcocystis singaporensis were characterised. The antibody response of brown rats to wild-type parasites (high reproductive capacity) showed a Th1 profile during acute infection, namely elevated concentrations of parasite-specific IgG2b and IgG2c and absence of IgG1. Chronic infection (bradyzoite development) resulted in a mixed Th1/Th2 pattern whereby significant concentrations of IgG1 appeared. A primary infection with 1000 sporocysts eight days before challenge induced protection, accompanied by significant concentrations of IgA and IgG2, particularly IgG2a. Western blot analysis of rat sera, using sporozoite and bradyzoite-extracts as antigen, revealed that IgG1, IgG2a, and IgG2b predominantly recognised molecules between 70-80 kDa in one or the other stage. Some of the antibodies were possibly directed against a 79 kDa heat shock protein of sporozoites. An apparent unresponsiveness to molecules in the low molecular weight range, particularly of bradyzoite antigens, was observed. This was abrogated by infection of rats with an avirulent strain of S. singaporensis (low reproductive capacity) indicating that a parasite that was less adapted to its host provoked a stronger immune response. These results suggest the existence of an immune evasion strategy used by Sarcocystis and show that wild rodents may be suitable as immunological research objects, reflecting the natural situation.