Local immune reactions and alterations in the intestinal physiology play an important role in the control of parasitic intestinal infections (SHEA-DONOHUE et al. 2001; KHAN u.
COLLINS 2004; MADDEN et al. 2004). Previous studies in mammals demonstrated that feed components such as NSP may modulate systemic and local gut-associated immune functions (SEIFERT u. WATZL 2007; BODERA 2008; MEYER 2008), and influence intestinal parasitic infection (PETKEVICIUS et al. 2001; PETKEVICIUS et al. 2003; THOMSEN et al.
2005). In this project we investigated local and systemic immune responses and electro-physiological epithelial functions in the intestine of layer chicken following nematode infection with A. galli and H. gallinarum under different dietary conditions.
In our experiments gut-associated immune parameters and electrogenic nutrient transport in layer chicken were affected by nematode infection as observed in mammalian species. NSP did not significantly influence gut-associated cellular immune responses and electro-physiological functions of the intestine neither in nematode infected nor in non-infected chicken.
6.1. Local T cell-mediated immune reactions to intestinal nematode infection
Both nematodes have led to the recruitment of CD4+, CD8α+, TCRαβ+ and TCRγδ+ T lymphocytes to the intestinal mucosa, as it was previously shown for nematode infections in mammals (MCCLURE et al. 1992; BALIC et al. 2002; LITTLE et al. 2005; PEREZ et al.
2008). The local T cell infiltrations in the intestinal lamina propria were most significant at two weeks post infection and declined later. At this period the establishment of A. galli takes place in the jejunum, and the adult stage of H. gallinarum starts developing in the cecal lumen. We suggest that this time may be critical in the immunological control of A. galli and H. gallinarum infection in chicken. Mice resistant to Trichuris muris showed the highest number of infiltrating CD4+ and CD8+ lymphocytes in the epithelium and the lamina propria around the time of the parasite expulsion (LITTLE et al. 2005). We suggest that T cell infiltrations in the intestinal lamina propria in A. galli and H. gallinarum infected chicken may be most distinct, when the chances of probable expulsion of the nematodes are high.
Further studies with resistant and succeptible chicken lines might support this observation.
Also earlier necropsy times might be taken into consideration to assess the immune reactions during the larval stages.
Interestingly, the lymphocyte infiltrations in the intestinal lamina propria were similar for CD4+, CD8α+, TCRαβ+ and TCRγδ+T cell subsets, and were comparable between jejunum for A. galli- and cecum for H. gallinarum-infected chicken. Both, small intestines and cecum developed diffuse lymphoid infiltrations as a reaction to the local stimulation with nematode antigen (DAVISON 2008).
Flow cytometric analysis of intestinal IEL showed a two-fold increase in CD4+ cells in the duodenal mucosa at the beginning of the tissue phase in A. galli-infected chicken. The investigation of the intestinal IEL was only carried out in the small intestines of A. galli-inoculated birds, because we were not able to isolate enough intra-epithelial lymphocytes from the cecal tissue of H. gallinarum-infected chicken. The increase in CD4+ cells in the duodenal mucosa correlated with low worm numbers and the absence of parasite eggs in the feces of A. galli-inoculated birds. Previous studies in mammals suggested that CD4+ cells may play an important role in resistance to intestinal nematode infections (KHAN u.
COLLINS 2004; LITTLE et al. 2005; RAUSCH et al. 2008). Primed as well as naïve CD4+
cells mediated protective immunity to Trichuris muris at the gut level in the absence of antibody in mice (BETTS et al. 2000). From our results it may be speculated that the significant increase in CD4+ duodenal IEL may have caused the elimination of the nematode during the larval tissue stage. Further experiments need to be done to confirm this speculation.
Co-infection of H. gallinarum and H. meleagridis significantly increased the T cell infiltration rate in the cecal lamina propria compared to mono-infection with H. gallinarum. This may be explained by a higher invasiveness and pathogenicity of this protozoan parasite (ESQUENET et al. 2003; BRENER et al. 2006) leading to massive tissue destruction and even mortality.
6.2. Induction of local Th1/Th2 cytokines
Beside antigen-specific local T lymphocyte infiltrations, gastro-intestinal helmintic infections in mammalian species are associated with an induction of a highly polarised type 2 cytokine response (PATEL et al. 2009; DAWSON et al. 2005). It was demonstrated that development of the dominating Th2 immune response often leads to elimination of the nematode infection whereas Th1 type immune responses support chronic infection (CLIFFE u. GRENCIS 2004).
Especially IL-4 and IL-13 were shown to play an important role in the resistance to nematode infections (FINKELMAN et al. 2004; RAUSCH et al. 2008; HERBERT et al. 2009).
Previous investigations demonstrated that Th1 and Th2 polarisation of the immune response may also be observed in avian species. Increased mRNA levels of Th2 cytokines 4 and IL-13 were detected in the intestines of chicken infected with A. galli (DEGEN et al. 2005;
KAISER 2007), whereas viral infection of chicken with Newcastle disease virus has induced an increase of the Th1 cytokine IFN-γ (DEGEN et al. 2005). The results of our study show that, as in mammals, chicken develop a Th2 immune response following an intestinal nematode infection. Coinciding with T lymphocyte infiltrations, we observed a local increase in the Th2 cytokines in the intestine of A. galli and H. gallinarum-infected birds. In contrast to the A. galli model, where we detected a significant increase in both IL-4 and IL-13 cytokine levels in the jejunal tissue, H. gallinarum-infected birds reacted with an upregulation of only IL-13 mRNA expression in cecum. It might be concluded that A. galli in comparison with H. gallinarum may elicit more distinct local immune reactions in the gut mucosa.
Although we observed a two-fold increase in the relative percentage of CD4+ intraepithelial lymphocytes during the duodenal tissue phase in A. galli-infected chicken, no changes were seen in the duodenum in Th2 cytokine mRNA expression. CD4+ lymphocytes represent only a very small population among the IEL (VERVELDE u. JEURISSEN), and for our investigations we used the entire duodenal tissue. Due to a relatively high dilution of the sample mRNA to prevent inhibition reactions during the qRT-PCR-reaction, possible changes in the relatively Th2 cytokine levels might have been below the detection limit.
Further infestigations are needed to conceive the exact role of the induced Th2 immune response in the control of nematode infections in chicken. In the A. galli infection model the high levels of the Th2 cytokines correlated positively with the high infection rate and the high average number of the parasites per bird. Higher upregulation of the Th1 cytokine IFN-γ has led to a lower infection rate and lower worm burden in infected chicken. The studies in mammals (ZAROS et al. 2010; CLIFFE u. GRENCIS 2004) indicated that the Th2 immune response is often sufficiently protective in intestinal parasitic infections. This does not exactly coincide with our observations on nematode infection in chicken.
In this study, we demonstrated for the first time that the local immune response in nematode-infected chicken may be shifted from a Th2 to a Th1 dominated response if accompanied by a protozoan infection. These findings confirm the dichotomy model in avian species (DEGEN et al. 2005). Dual infection of H. gallinarum and H. meleagridis elicited a significant increase in the mRNA expression of the Th1 cytokine IFN-γ, but not of the Th2 cytokine IL-13.
Increase of IFN-γ was previously reported to have a protective effect on Eimeria infections in chicken (YUN et al. 2000; HONG et al. 2006).
6.3. Systemic immune reactions in the spleen to intestinal nematode infections
Both, A. galli and H. gallinarum infections did not cause any significant changes in the splenic lymphocyte population. It was demonstrated in mammals that high invasive intestinal nematodes may reduce the percentage of splenic CD4+ cells (DONDJI et al. 2008). The absence of a systemic immune reaction in spleen in A. galli- and H. gallinarum-infected birds suggests that in contrast to high invasive intestinal nematodes (DONDJI et al. 2010) the immune response following A. galli and H. gallinarum infection is primary localised in the gut.
In H. gallinarum and H. meleagridis dual infected chicken we observed together with severe T cell infiltrations in the cecal lamina propria a reduction in the relative percentage of CD4+
splenic T lymphocytes. This shows that co-infection with H. meleagridis, a highly invasive intestinal pathogen, (BRENER et al. 2006) dominates not only local gut-associated immune responses but also systemic immune reactions in spleen.
6.4. Development of systemic worm-specific IgG in serum
Induction of circulating worm-specific IgG antibodies following nematode infection was investigated by ELISA. For the development of the ELISA-systems the worm soluble somatic antigen was prepared from adult A. galli and H. gallinarum worms, which were collected from the intestines of naturally infected chicken. In our experiments, H. gallinarum worms were always contaminated with H. meleagridis. This may have contributed to the non-specific background in the H. gallinarum ELISA, which made serologic differentiation between worm-infected and non-infected birds not possible under our experimental conditions.
Consistent with previous studies (MARTIN-PACHO et al. 2005; MARCOS-ATXUTEGI et al. 2009), we observed development of worm-specific serum IgG antibodies in A. galli-infected birds starting two to three weeks after the infection. It is not known whether the systemic IgG antibodies play a protective role in A. galli infection in chicken (EGERTON u.
HANSEN 1955). In our experiments there was only a very low correlation between detected worms per bird, infection rate and systemic IgG levels. However, the highest increase in the group average S/P ratio in inoculated birds was observed in the experiment with the highest infection rate. Interestingly, ELISA S/P ratios at the last necropsy in the first two experiments were comparable between infected worm-negative birds and infected worm-positive birds.
6.5. Influence of the infection on electrogenic chloride secretion and nutrient transport
Studies in mammals have shown that gastro-intestinal nematodes may affect chloride secretion and sodium-linked glucose absorption of the intestinal epithelia (LEONHARD-MAREK u. DAUGSCHIES 1997; AU YEUNG et al. 2005; DAWSON et al. 2005).
Moreover, it was demonstrated that secretory functions of the intestinal epithelial cells are kept under direct immunological control during nematode infections (SHEA-DONOHUE et al. 2001; MADDEN et al. 2004). In this study we investigated electrogenic alanin and glucose absorption in ileal tissues of A. galli-infected birds and electrogenic chloride secretion in cecal tissues of H. gallinarum-infected birds.
Coinciding with the previous observations in mammals (SHEA-DONOHUE et al. 2001;
DAWSON et al. 2005), A. galli-infected chicken demonstrated a significant reduction in electrogenic alanin and glucose absorption. These alterations in the intestinal physiology were only seen in correlation with the increase in local Th2 cytokines IL-4 and IL-13 mRNA expression in the intestinal mucosa. It was previously demonstrated in mammals that upregulation of the Th2-cytokines IL-4 and IL-13 in response to nematode infections induced changes in intestinal cell electrogenic secretion and impaired glucose absorption (MADDEN et al. 2002). These alterations in the intestinal physiology were shown to be dependent on activation of the STAT6 signaling pathway (MADDEN et al. 2004). The activation of the STAT6 signaling pathway also promotes intestinal muscle contractility and contributes to the expulsion of the intestinal parasites (ZHAO et al. 2003; KHAN u. COLLINS 2004).
Our findings indicate that the connection between the local immune reactions and electro-physiological intestinal functions also exists in avian species. Induction of the Th2 immune response following nematode infection may affect intestinal epithelial cell functions and influence electrogenic nutrient transport in chicken. The exact mechanisms of these alterations have to be identified in further studies in avian species.
In contrast to mammals (LEONHARD-MAREK u. DAUGSCHIES 1997; MADDEN et al.
2002; DAWSON et al. 2005), H. gallinarum infection did not significantly influence chloride secretion in ceca of chicken. Although the increase in local IL-13 mRNA expression was seen in H. gallinarum-infected birds two weeks post infection, it was not sufficient to induce significant changes in the electro-physiological functions of the intestinal epithelial cells, which were measured 9 weeks post infection. This might have been due to weaker local immune reactions in response to H. gallinarum infection in comparison with A. galli. It might also be concluded that there is no direct correlation between local immune response and electro-physiological response of the intestines, and not all nematodes are able to influence electrogenic chloride secretion in birds. It should also be considered that the electro-physiological measurements in H. gallinarum-infected birds were performed 9 weeks post infection, when the main cecal immune responses had already declined.
Interestingly, H. meleagridis co-infection also did not significantly influence cecal chloride secretion. Despite substantial destruction of cecal tissues during the early phase of the protozoan infection, the alteration patterns in the short-circuit currents were similar to those of H. gallinarum-infected birds. In contrast to turkey, chicken mainly recover from H.
meleagridis infection (HESS et al. 2006; POWELL et al. 2009). Also in our experiments, we observed only minor histological lesions of cecal epithelia at the time of the electro-physiological measurements 5 weeks post infection. At this time the gut epithelia were mostly recovered from H. meleagridis infection, the protozoan parasites were eliminated, and we suggest that the changes in the short-circuit currents were dominated by H. gallinarum worms.
6.6. Effect of NSP on the immune response and elelecro-physiological intestinal functions in nematode infections
No systemic and local effects of NSP were observed on the cell-mediated immune response in non-infected chicken and in A. galli- and H. gallinarum-infected birds. These findings do not coincide with the observation in mammals, where NSP demonstrated various immunomodulatory effects in the T and B lymphocyte compartment (LIM et al. 1997;
WATZL et al. 2005). Studies in rats showed that insoluble NSP increased the number of T cells in spleen, thymus and mesenteric lymph nodes (TRUSHINA et al. 2005). Also proliferation of IgA+ B cells was observed in the mucosa in the small intestines and cecum of rats receiving NSP supplemented diet (KUDOH et al. 1998). In our experiments, NSP had no significant effect on T lymphocyte populations in spleen and in the intestinal mucosa, as well as on intestinal IgA+ B lymphocytes. This might indicate that NSP may not influence the local and systemic cell-mediated immunity in layer chicken similarly as in mammals. Other B cell subsets, macrophage and dendritic cell populations may be affected by NSP in layer chicken and should be investigated in the future. In a recent study, fructo-oligosaccharides reduced the proportion of B cells in cecal tonsils and enhanced IgG antibody titers in plasma of broiler chicken (JANARDHANA et al. 2009). It may also be taken into consideration that longer NSP feeding periods are needed to detect changes in the immune response of layer chicken.
In previous studies NSP have significantly influenced the course of intestinal nematode infections in mammals (PEARCE 1999; THOMSEN et al. 2005, PETKEVICIUS et al. 1999).
Inclusion of soluble NSP such as inulin reduced the worm burdens (PETKEVICIUS et al.
2003; THOMSEN et al. 2005), whereas insoluble NSP favoured the establishement of the parasites (PETKEVICIUS et al. 1997; PETKEVICIUS et al. 2001). In layer chicken, both soluble and insoluble NSP elevated the incidence of A. galli and H. gallinarum infection and worm burdens per bird (DAŞ et al. 2011a; DAŞ et al. 2011b). In our experiments, no significant changes in the cellular immune reaction or circulating parasit specific IgG were observed in infected groups receiving NSP diet compared with infected birds receiving control diet. We suggest that NSP may modulate the immune response in the course of nematode infection in layer chicken in a different way or not at all compared with mammals.
Further investigations are needed to understand the mechanisms how NSP influence nematode infections in layers.
Epithelial electrogenic alanin and glucose transport in ileal tissues, which was assessed in A.
galli-infected birds, was not influenced by dietary fibre in our experiments. These findings coincide with observations obtained in mammalian studies (VON HEIMENDAHL et al.
2010) and in broilers (REHMAN et al. 2007).
Although the cecum is considered to be the main site of dietary fibre fermentation, NSP did not significantly influence the secretory responses in cecal epithelia in non-infected chicken compared to the controls. The findings in chicken do not coincide with the investigations in mammals, where soluble and insoluble NSP induced a significant reduction in Cl- ion transport in the proximal jejunum in rats (SCHWARTZ et al. 1982).
An influence of infection on chloride secretion was observed in H. gallinarum mono-infected chicken receiving NSP diet. This might indicate that NSP in combination with nematode infection may affect intestinal chloride secretion. We also observed reduced Cl- secretion in H. gallinarum mono-infected birds fed with the insoluble NSP diet compared to non-inoculated controls. Previously, it shown that insoluble NSP facilitate nematode infection
(PETKEVICIUS et al. 1997; PETKEVICIUS et al. 2001). Reduction in Cl- secretion will lead to a reduced intestinal fluid, which might benefit the survival of the worms.
No significant interractions between diet and infection could be detected for H. gallinarum and H. meleagridis dual infection.
6.7. Consideration concerning the A. galli and H. gallinarum infection models
In the A. galli model, infection rate and worm burden per bird varied substantially between experiments. We also observed variations in immunological and electro-physiological responses of the infected birds between individual experiments. For each experiment, we used separate A. galli egg preparations, and the worm eggs had been obtained from field-infected chicken. Although we have no information on the genetic diversity of the nematodes in the sampled region, it may be suggested that variations between A. galli genotypes might have influenced the immune reactions. Previously it was shown that different isolates of parasite were able to elicit different immune responses (D'ELIA et al. 2009). For future experiments it may be advantageous to use laboratory isolates to obtain more reproducible results. On the other hand, one problem using of laboratory strains may be the adaptation of the nematode and the loss of virulence.
Also genetic variations between birds may influence immune responses to intestinal parasites (SCHOU et al. 2010). It is possible that the genotype of the chicken in our experiments may have changed slightly throughout the 3 year trial period, because offsprings of different parents of the same lines were used.
One critical point of the H. gallinarum infection model was the contamination of the worm eggs with the protozoan H. meleagridis. As for the A. galli model, we also used eggs from field worm isolates for H. gallinarum infection. To study the immune response and electro-physiological response of the intestine to sole nematode infection we preventively treated the birds with dimetridazole. It is possible that dimetridazol treatment may also have affected the bacterial flora of the gut, which would influence local immune reactions and intestinal electro-physiological parameters.
6.8. Conclusions, open questions and further perspectives
As it was previously shown in mammalian species, local immune reactions including the upregulation of Th2 cytokines and T cell infiltrations dominate A. galli and H. gallinarum infections in layer chicken. The immune response in the intestinal mucosa may also affect the electrogenic nutrient transport in the intestinal epithelia of birds. Co-infection with H.
meleagridis altered the local immune response by H. gallinarum-infected chicken from the Th2 type to a Th1 type and elicited systemic immune reactions in the spleen. NSP did not influence the gut-associated cellular immune responses and electro-physiological responses in layer chicken as it was previously observed in mammals.
Our experiments provided the first comprehensive results on the induction of local and systemic immune responses following nematode infections in birds. There are still many open questions. In comparison to mammals, birds have only very few eosinophils, and no avian
Our experiments provided the first comprehensive results on the induction of local and systemic immune responses following nematode infections in birds. There are still many open questions. In comparison to mammals, birds have only very few eosinophils, and no avian