The influence of non-starch-polysaccharides on experimental infections with Ascaridia galli and Heterakis gallinarum in layer
chicken (Gallus gallus domesticus)
THESIS
Submitted in partial fulfilment of the requirements for the degree
DOCTOR OF PHILOSOPHY (PhD)
at the University of Veterinary Medicine Hannover
by Anna Schwarz (Sankt-Petersburg)
Hannover, Germany 2011
Prof. G. Breves (Institute for Physiology, University of Veterinary Medicine Hannover, Germany)
Advisory committee: Prof. Th. Schnieder (Institute for Parasitology, University of Veterinary Medicine Hannover, Germany)
Prof. M. Hess (Clinic for Avian, Reptile and Fish medicine, University of Veterinary Medicine Vienna, Austria)
Prof. S. Rautenschlein Prof. G. Breves
1st Evaluation: Prof. S. Rautenschlein
Prof. G. Breves
Prof. Th. Schnieder
2nd Evaluation: Prof. Th. W. Göbel (Institute for Animal Physiology, Department of Veterinary Sciences, University of Munich, Germany)
Date of oral exam: 26.05.2011
This study was funded by the Deutsche Forschungsgemeinschaft (AB 30/8-1, BR 780/14-1)
Meiner Familie, Dirk und Linda
Schwarz, A., Gauly M., Abel H.J., Daş G., Humburg J., Weiss A.Th.A., Breves G., Rautenschlein S. (2011):
Pathobiology of Heterakis gallinarum mono- and co-infection with Histomonas meleagridis in layer chicken.
Avian Pathology (in press: DOI: 10.1080/03079457.2011.561280)
Schwarz, A., Gauly M., Abel H.J., Daş G., Humburg J., Rohn K., Breves G., Rautenschlein S. (2011):
Immunopathogenesis of Ascaridia galli infection in layer chicken.
Developmental and Comparative Immunology, 35(7), 774-784
Daş, G., Abel, H.J., Humburg, J., Schwarz, A., Rautenschlein, S., Breves, G., Gauly, M.
(2011):
Non-starch polysaccharides alter interaction between Heterakis gallinarum and Histomonas meleagridis.
Veterinary Parasitology , 176(2-3), 208-216
Daş, G., Abel, H.J., Humburg, J., Schwarz, A., Rautenschlein, S., Breves, G., Gauly, M.
(2011):
Effects of dietary non-starch polysaccharides on establishment and fecundity of Heterakis gallinarum in grower layers.
Veterinary Parasitology, 178(1-2), 121-128
Daş, G., Abel, H.J., Humburg, J., Schwarz, A., Rautenschlein, S., Breves, G., Gauly, M.
(2011):
The effects of dietary non-starch polysaccharides on Ascaridia galli infection in grower layers.
Parasitology (submitted: PAR-2011-0165)
Table of contents
Table of contents ...I List of abbreviations... III List of figures ... V List of tables ... VIII
1. Introduction ... 1
2. Literature review ... 4
2.1. New trends in poultry production... 4
2.2. A. galli and H. gallinarum infections... 4
2.2.1. A. galli... 4
2.2.2. H. gallinarum... 5
2.3. H. meleagridis... 6
2.4. General aspects of the avian enteric immune system... 7
2.4.1. Immunity to enteric parasitic infections in birds... 9
2.5. Immunity to enteric nematode infections in mammals ... 11
2.6. Non-starch polysaccharides... 12
2.6.1. Local and systemic effect of NSP on the immune system ... 13
2.6.2. Effect of NSP on nematode infections ... 14
2.7. Chloride secretion and nutrient transport in the intestine ... 15
2.7.1. Effect of NSP on electrogenic chloride secretion and nutrient transport... 16
2.7.2. Influence of helminthic infection on electrogenic chloride secretion and nutrient transport in the intestine ... 16
3. Goals and objectives... 18
4. Pathobiology of Heterakis gallinarum mono- and co-infection with Histomonas meleagridis in layer chicken ... 19
5. Immunopathogenesis of Ascaridia galli infection in layer chicken... 53
6. General discussion and conclusions... 54
6.1. Local T cell-mediated immune reactions to intestinal nematode infection ... 54
6.2. Induction of local Th1/Th2 cytokines ... 56
6.3. Systemic immune reactions in the spleen to intestinal nematode infections ... 57
6.4. Development of systemic worm-specific IgG in serum... 58
6.5. Influence of the infection on electrogenic chloride secretion and nutrient
transport... 58
6.6. Effect of NSP on the immune response and elelecro-physiological intestinal functions in nematode infections... 60
6.7. Consideration concerning the A. galli and H. gallinarum infection models... 62
6.8. Conclusions, open questions and further perspectives... 63
7. Summary ... 65
8. Zusammenfassung... 67
9. References ... 69
10. Acknowledgements ... 101
List of abbreviations
ANOVA analysis of variance
A. galli Ascaridia galli
cAMP cyclic adenosine monophosphate CD3, 4 or 8 (+) cluster of differentiation 3, 4 or 8 (positive)
CFTR cystic fibrosis transmembrane conductance regulator
Cl chloride
Ct cycle threshold
DAB 3.3´-diaminobenzidine
DIDS 4,4´-diisothiocyanostilbene-2,2´-disulfonic acid ELISA enzyme-linked immunosorbent assay
EU European Union
Exp. experiment
FACS fluorescence-activated cell sorting
FITC fluorescein
GADPH glyceraldehyde-3-phosphate dehydrogenase GALT gut associated lymphoid tissue Gt transepithelial tissue conductances H. gallinarum Heterakis gallinarum
H. g. Heterakis gallinarum
H. meleagridis Histomonas meleagridis H&E Haematoxilin & Eosin H. meleagridis Histomonas meleagridis
H. m. Histomonas meleagridis
IEL intraepithelial lymphocytes
IFN interferon
Ig immunoglobulin
IL Interleukin
Isc short-circuit current
LP lamina propria
LPL lamina propria lymphocytes
mRNA messenger ribonucleic acid
MALT mucosa-associated lymphoid tissue
MLN mesenteric lymph node
NK natural killer (cells)
NPPB 5-nitro-2-(3-phenylpropylamino) benzoate
NSP non-starch polysaccharides
O.D. optical density
pi post infection
R-PE phycoerythrin
rRNA ribosomal ribonucleic acid
RT-PCR reverse transcription-polymerase chain reaction
SCFA short-chain fatty acids
SD standard deviation
S/P (ratio) sample/positive (ratio)
spp. Species
SPRD SpectralRed
TCRαβ or γδ (+) T cell receptor αβ or γδ (positive)
TEA tetraethylammonium
Th T helper (cells)
w/v weight per volume
List of figures Chapter 3
Fig. 3. 1. 46
Patho-histological lesions of the cecal wall of chicken inoculated with 200 embryonated eggs of Heterakis gallinarum (H. g.). 1a: non-inoculated control animal. 1b-c-d-e: H. g. and Histomonas meleagridis co-infection (Exp. 1). 1b:
severe interstitial lymphocyte infiltration 2 weeks pi. Mucosal architecture is destroyed. Arrows show histomonads. 1c: mucosal structure is partly restored 3 weeks pi, severe lymphocyte and heterophil infiltration in the lamina propria.
1d: re-epithelisation process 3 weeks pi. 1e: moderate lymphocyte infiltration in the lamina propria and formation of lymphoid centres in cecal mucosa 5 weeks pi. 1f: Exp. 2: mild to moderate lymphocyte infiltration in the lamina propria following H. g. mono-infection. Bars = 300µm in 1a, 1b, 1c, 1e, 1f and =80 µm in 1d.
Fig. 3. 2. 47
Abundance score of cecal T lymphocytes in the lamina propria of birds orally infected with 200 Heterakis gallinarum (H. g.) eggs. 2a: non-inoculated animal.
2b: H. g. mono-infection two or five weeks pi (Exp. 2). 2c: H. g. mono- infection two weeks pi or H. g. and Histomonas meleagridis (H. m.) co- infection five weeks pi (Exp. 1). 2d, e, f: H. g. and H. m. co-infection two and three weeks pi. Black cells indicate positive lymphocytes. 2a: score 1-some scattered positive cells. 2b, c, d: score 2- mild-, score 3- moderate-, score 4- severe lymphocyte infiltration, respectively, tissue architecture not affected. 2e, f: score 5- moderate-, score 6-severe lymphocyte infiltration, respectively, tissue architecture affected. Bars =300µm.
Fig. 3. 3. 48
Changes in local T lymphocyte populations in the cecal lamina propria following Heterakis gallinarum (H. g.) and Histomonas meleagridis co- infection (Exp. 1) and H. g. mono-infection (Exp. 2). Data represent the group
mean of the abundance score ± standard deviation. 3a: CD4+ cells. 3b: CD8α+
cells. 3c: TCRαβ (Vβ1)+ cells. 3d: TCRγδ+ cells. *Significantly different to the non-inoculated group of the same experiment (Exp. 1: n=15, Exp. 2: n=14-15, Wilcoxon Rank Sum Test, P<0.05)
Fig. 3. 4. 49
Flow cytometric analysis of splenic CD4+ lymphocytes. In both experiments birds of one group were inoculated with 200 embryonated Heterakis gallinarum (H. g.) eggs. Exp. 1: dual infection with H. g. and Histomonas meleagridis. Exp. 2: mono-infection with H. g. *Significantly different to the non-inoculated group of the same experiment (Exp. 1: n=15, Exp. 2: n=14-15, t-test, P<0.05)
Fig. 3. 5. 50
Quantification of cytokine mRNA expression levels in cecal tissue of chicken dually infected with Heterakis gallinarum (H. g.) and Histomonas meleagridis (Exp. 1) and mono-infected with H. g. (Exp. 2). The data are corrected for variation in input RNA by 28S mRNA levels and are presented as x-fold change in mRNA expression levels in the ceca of inoculated birds in comparison to non-inoculated controls. 5a: IFN-γ, 5b: IL-4, 5c: IL-13.
*Significantly different to the non-inoculated birds of the same experiment (n=3, t-test, P<0.05)
Fig. 3. 6. 51
Maximal changes in short-circuit currents (ΔIsc) as a mass for the changes in chloride secretion in cecal epithelium of layer chicken inoculated with 200 embrionated Heterakis gallinarum (H. g.) eggs. Birds were allotted to three different diets. Exp. 1: dual infection with H. g. and Histomonas meleagridis.
Exp. 2: mono-infection with H. g. 6a: addition of carbachol- stimulator of Ca- dependent chloride secretion. 6b: addition of forskolin- stimulator of cAMP- dependent chloride secretion. 6c: addition of NPPB- inhibitor of the CFTR
channel, via major chloride secretion occurs. C: control diet, I: diet with 9.1%
of insoluble NSP, S: diet with 9.1% soluble NSP. *Significant effect of the diet or infection between the bird groups of the same experiment (Exp. 1: n=4-5, Exp 2: n=5, P<0.05)
List of tables Chapter 3
Table 3. 1. 43
Ingredients and nutrient contents of experimental diets.
Table 3. 2. 44
Real-time quantitative RT-PCR primers and probes.
Table 3. 3. 45
Infection rate and development of macroscopical and microscopical lesions in chicken inoculated with 200 embryonated eggs of Heterakis gallinarum (H. g.).
1. Introduction
Recent changes in legal requirements for layer-housing and in consumer demands in European countries have led to the substitution of traditional cages with free-range systems and floor husbandry. These systems benefit the spread of parasitic infections because of the close contact of the animals to their feces and lead to an increase in the prevalence of helmintic infections in modern poultry production. Ascaridia galli (A. galli) and Heterakis gallinarum (H. gallinarum) are worldwide distributed nematodes. They are very common in alternative production systems and in case of multifactorial diseases may contribute to substantial economic losses. Infections with A. galli are associated with higher feed conversion rates and decrease in body weight gain and egg production. The main economic importance of H. gallinarum is due to its role as a carrier of Histomonas meleagridis (H.
meleagridis), a protozoan parasite which induces blackhead disease.
In the past, synthetic anthelmintics have been used to control parasitic infections. This has led to the development and spread of resistances among parasites, contaminated the environment and may lead to residues in food products. At present, the use of many effective anthelmintic drugs is prohibited or restricted due to consumer safety reasons, and new ways to influence chicken health are under investigation. Understanding of immunological and physiological processes in the intestine of chicken in the course of helmintic infections is essential to finding alternative control strategies.
Extensive studies in mammals have shown that nematode infections are associated with local cell-infiltrations in the intestinal mucosa and induction of a highly polarized T helper (Th) 2 cytokine response. The studies in mice resistant to Trichuris muris reveal that at the time of worm expulsion the inflammation in the intestine has been dominated by infiltrating CD4+
cells in epithelium and CD4+, CD8α+ cells in the lamina propria. So far, not much information is available on specific immune reactions following parasitic infections in birds.
In comparison to mammals, the avian immune system includes some additional structures to the gut associated lymphoid tissue (GALT), such as the bursa cloacalis, cecal tonsils and Meckels diverticulum, but birds do not possess lymph nodes. This suggests the important role
of GALT as a secondary lymphoid structure in the course of avian intestinal infections.
Recently, it was demonstrated that Th2-polarisation of the immune response and induction of systemic circulating specific IgG antibodies in the course of nematode infection also exists in avian species. However, no studies have been conducted so far to identify the local cell- mediated immune parameters following nematode infections in birds.
Health, general condition and productivity of animals are highly dependent on proper physiological functions of the intestine. In mammalian models, it has been demonstrated that intestinal electrogenic nutrient transport and epithelial cell secretion are affected in the course of nematode infections. Pigs infected with Ascaris suum showed an increase in intestinal chloride secretion during a period of self-curing, which correlates with the net luminal rise of fluid and reduction in electrogenic glucose transport. In birds, no investigations have been done so far concerning the role of nematode infections on the intestinal electro-physiological parameters.
At present, different feed additives are tested as alternatives to the use of chemotherapeutic substances in poultry production. It has been shown in mammalian models, that non-starch polysaccharides (NSP) may have a beneficial effect on the general condition of the animal and may have an influence on systemic and local immune functions. It has also been observed that intestinal nematode infections in mammals are affected by NSP. Diets with inclusion of inulin as a source of soluble NSP reduce nematode worm burden and egg excretion. In contrast, non-soluble NSP diets benefit the establishment and survival rate of nematodes. No information is available on the influence of NSP on the local immune reactions and the course of nematode infections in chicken.
The aim of the project was to investigate immunological and electro-physiological parameters in the intestine following experimental infection with A. galli and H. gallinarum in chicken, as well as the influence of NSP on these parameters. We hypothesized that the local and systemic immune response, as well as the electrogenic nutrient transport and secretory functions of the intestine in chicken might be affected by the nematode infection similarly as in mammalian species. As in mammalian models, we also expected to observe NSP influence
on these parameters. Under different dietary conditions we investigated local and systemic T cell populations, induction of local Th1 and Th2 cytokines, humoral immune response, as well as electrogenic alanin and glucose transport and chloride secretion following experimental A. galli and H. gallinarum infections. In addition, we characterised the influence H. meleagridis on H. gallinarum infection.
2. Literature review
2.1. New trends in poultry production
According to directive no. 1999/74/EC, new regulations for the protection and welfare of laying hens will be implemented in the EU from 01.01.2012. It requires the substitution of traditional battery cages with enriched cage systems, floor husbandry and free-range systems (ESQUENET et al. 2003). In these production systems animals stay in close contact to their feces. This may lead to re-emergence and high prevalence of parasitic infections (PERMIN et al. 1999; MARTIN-PACHO et al. 2005; GAULY et al. 2007; MAURER et al. 2009). In addition, changes in consumer demand into the direction of biological products, which are not burdened with chemotherapeutic residues, have occurred in recent years (DONOGHUE 2003;
EL-KHOLY u. KEMPPAINEN 2005; POMPA et al. 2005; BOKKERS u. DE BOER 2009;
TAJIK et al. 2010). As a result, increased numbers of laying hens will be kept in alternative housing systems (MARCOS-ATXUTEGI et al. 2009; DAŞ et al. 2010; KATAKAM et al.
2010), and the relevance of gastro-intestinal parasitic infections for layer chicken will grow.
2.2. A. galli and H. gallinarum infections
A. galli and H. gallinarum are the most common poultry helminths. They are distributed worldwide and play an important economic role in litter and free-range production systems (RAMADAN u. ABOU ZNADA 1991; PERMIN et al. 1999; PERMIN u. RANVIG 2001;
MAGWISHA et al. 2002; MARTIN-PACHO et al. 2005; ABDELQADER et al. 2007; KURT u. ACICI 2008; MUNGUBE et al. 2008; MAURER et al. 2009).
2.2.1. A. galli
A. galli is a nematode, which was first described by Schrank in 1788. It parasites in the small intestine of domestic and wild birds, and has been reported in chicken, turkey, dove, duck, and goose (TVERDOKHLEBOV 1966; PERMIN et al. 1997; PERMIN et al. 1999;
CAMACHO-ESCOBAR et al. 2008; SAIF 2008; KATAKAM et al. 2010). A. galli worms are large and white yellowish. Male worms (50-70 mm) are smaller than female worms (60-116 mm). Normally, A. galli is found in the lumen of the small intestine, but at high infestation rates it can occasionally migrate into the oesophagus, crop, gizzard, body cavity, oviduct and eggs (REID et al. 1973; SAIF 2008).
A. galli has a direct life cycle. Shed eggs first need to embryonate in the litter or soil to become infective. Under optimum temperature and moisture conditions the process takes about 10-12 days (SAIF 2008). Ingested embryonated eggs, which bear infective third larvae, hatch within 24 hours in either the proventriculus or duodenum of the susceptible host. The larvae live in the lumen of the duodenum for the first 8-9 days and then penetrate the mucosa during the tissue phase (TUGWELL u. ACKERT 1952; HERD u. MCNAUGHT 1975). The length of the tissue phase is dependent on the ingested infectious dose of embryonated worm eggs. At a high dose it may be prolonged (HERD u. MCNAUGHT 1975; KATAKAM et al.
2010). The young worms return to the lumen by day 17 or 18, where they mature at 28-30 days of age. Grasshoppers or earthworms may serve as paratenic hosts for A. galli eggs, without further development of the infectious larvae in the invertebrates (SAIF 2008).
Independent from the previous infection status, chicken older than 3 months show considerable resistance to the infection with A. galli (TONGSON u. MCCRAW 1967).
Infection with A. galli may contribute to substantial economic losses (PERMIN u. RANVIG 2001). It is associated with higher feed conversion rates and decrease in body weight gain and egg production. The weight depression of the host correlates with the A. galli worm burden (REID u. CARMON 1958). Severe infections with A. galli may also result in an increased mortality rate (IKEME 1971; RAMADAN u. ABOU ZNADA 1991; GAULY et al. 2005;
KILPINEN et al. 2005; DAŞ et al. 2010) and occasionally in the migration of the parasite into the eggs of laying hens (REID et al. 1973). In addition, A. galli plays a role in the dissemination of Salmonella enterica (CHADFIELD et al. 2001; EIGAARD et al. 2006) and enhances infections with Pasteurella multocida (DAHL et al. 2002) or coccidia species (SAIF 2008). A. galli worms are also able to transmit avian reoviruses (SAIF 2008; KATAKAM et al. 2010).
2.2.2. H. gallinarum
H. gallinarum was described by Schrank in 1788. The parasite is the one of the most frequently diagnosed nematode in the digestive tract of galliform birds (LUND et al. 1970;
PERMIN et al. 1999; MAURER et al. 2009). Larval stages and adults of H. gallinarum
colonize ceca of chicken, turkeys, ducks, geese, grouse, guinea fowl, partridges, pheasants, and quail (LUND u. CHUTE 1972, 1974; SAIF 2008; POTTS 2009). The ring-necked pheasant is most susceptible to the infection, followed by the guinea fowl and chicken (LUND u. CHUTE 1972). Adult worms of H. gallinarum are white, and male worms are 7-13 mm long, while females are 10-15 mm long. The eggs of H. gallinarum are not embryonated at the time of deposition (SAIF 2008).
Similar to A. galli, H. gallinarum has a direct life cycle. The eggs reach an infective stage in approximately two weeks, depending on the environmental conditions. The larvae hatch in the upper intestine of susceptible hosts, and migrate to the ceca within 24 hours. Until 12 days post-exposure the larvae of H. gallinarum are closely associated with the cecal mucosa, but they do not undergo a true tissue phase (SAIF 2008). H. gallinarum eggs may be ingested by earthworms, where they can survive for months.
Infections with H. gallinarum are generally subclinical. Infected birds show inflammation and thickening of cecal walls. The severity of the lesions depends on the parasite burden. In cases of heavy infection, the formation of nodules in the cecal mucosa and hepatic granulomas have been observed (KAUSHIK u. DEORANI 1969; RIDDELL u. GAJADHAR 1988).
The main economic importance of H. gallinarum is due to its role as a vector for Histomonas meleagridis, a protozoan parasite, which induces the blackhead disease (GIBBS 1962; LEE 1969; SPRINGER et al. 1969; LUND u. CHUTE 1974; ESQUENET et al. 2003). Direct transmission of H. meleagridis was achieved using larvae, eggs and also male worms of H.
gallinarum (SPRINGER et al. 1969; RUFF et al. 1970).
2.3. H. meleagridis
H. meleagridis is an ameboid protozoan, which frequently affects gallinaceous birds (HAFEZ et al. 2005; GRABENSTEINER et al. 2006; BLEYEN et al. 2007; BLEYEN et al. 2009). It induces typhlohepatitis with severe pathological lesions in ceca and liver, and mortality in susceptible hosts (ESQUENET et al. 2003; MCDOUGALD 2005; POWELL et al. 2009). The disease was first described in turkeys in 1895. Birds usually die due to the damages of the
liver (SAIF 2008). Both, chicken and turkey are susceptible to the disease, but usually the turkey is more severely affected than chicken. Whereas the cecal lesions in chicken heal rapidly, turkey develop progressively severe cecal lesions and later liver lesions, which may result in 80-100% mortality rate in turkey flocks (GRABENSTEINER et al. 2006; HESS et al.
2006; SAIF 2008; POWELL et al. 2009). Histomoniasis in chicken results in high morbidity, loss of flock uniformity but usually only in low mortality (MCDOUGALD 2005;
GRABENSTEINER et al. 2006; POWELL et al. 2009). Main clinical signs of histomoniasis in chicken are non-specific (HAFEZ et al. 2005; GRABENSTEINER et al. 2006). Infected birds show depression, ruffled feathers and closed eyes; occasionally the feces may contain blood and caseous cores. During the acute phase of the disease the cecal wall of infected birds become thickened and hyperemic and the lumen is filled with fibrinous to fibrino- hemorrhagic exudates. Recovered chicken often remain carriers (CLARKSON 1963; SAIF 2008). At present, no registered drug is available in the European Union for the prevention and treatment of histomoniasis in commercial poultry (HESS et al. 2006).
Survival and transmission of H. meleagridis is directly associated with the cecal nematode H.
gallinarum (GIBBS 1962; LEE 1969; RUFF et al. 1970). Direct transmission of H.
meleagridis in chicken occurs at much lower rates than in turkey (HESS et al. 2006) or in some studies it could not be demonstrated at all (HU et al. 2006). This emphasises the importance of H. gallinarum as a vector for H. meleagridis in chicken.
2.4. General aspects of the avian enteric immune system
As in mammalian species, the avian immune system includes well developed mucosa- associated lymphoid tissue (MALT), which is the first line of defence on mucosal surfaces (LILLEHOJ u. LILLEHOJ 2000; YUN et al. 2000b; BAR-SHIRA et al. 2003). MALT represents the largest lymphoid organ in the body and consists of antigen-presenting cells, immunoregulatory cells and effector cells, which are mainly located in the lamina propria (LP) mucosae and tela submucosa. The lymphoid tissue of avian MALT is organized in lymphoid follicles, as well as scattered or aggregated lymphoid cells (LILLEHOJ u. TROUT 1996; DAVISON 2008; CASTELEYN et al. 2010). A major component of MALT, which is located in the intestinal tract, is called gut-associated lymphoid tissue (GALT). It contains
more than half of the total lymphocyte pool of the MALT (YUN et al. 2000b) and mounts immune responses against various parasitic, viral and bacterial enteral pathogens (ROTHWELL et al. 1995; MAST u. GODDEERIS 1999; MUIR et al. 2000).
Morphologically, GALT consists of two layers, which are separated by a basal membrane. In the outer layer are located intraepithelial lymphocytes (IEL), which are scattered between epithelial cells, and beneath the basal membrane are the lamina propria, which is rich in lymphocytes and submucosa (LILLEHOJ u. LILLEHOJ 2000; DAVISON 2008).
In comparison to mammals, the avian immune system does not possess structured peripheral lymph nodes (BAR-SHIRA et al. 2003; DAVISON 2008; CASTELEYN et al. 2010). This emphasises the role of the avian GALT as the major secondary lymphoid organ for the defence against avian intestinal infections (OLAH et al. 1984; LILLEHOJ u. TROUT 1996;
MUIR et al. 2000).
Avian GALT contains unique lymphoid structures along the gut (YUN et al. 2000b;
CASTELEYN et al. 2010), such as the cecal tonsils (DEL CACHO et al. 1993; KITAGAWA et al. 1998; JANARDHANA et al. 2009), Meckels diverticulum (OLAH u. GLICK 1984;
BESOLUK et al. 2002) and the bursa cloacalis (RATCLIFFE 2006; CASTELEYN et al.
2010), which have not been described for mammalian species. In addition, birds possess, analogue to mammals, Payer Patches (BEFUS et al. 1980; BURNS 1982), lymphoid follicles within the lamina propria, with varying degrees of organisation, and single lymphoid cells scattered throughout the epithelium and lamina propria of the GALT (YUN et al. 2000b).
Antigen stimulation in the gut of chicken usually leads to the development of diffuse lymphoid tissue in the GALT (DAVISON 2008).
Avian GALT consists of a diverse set of lymphoid cell subsets. Heterophils, eosinophils, macrophages, natural killer cells, dendritic cells and T and B lymphocytes are present in different proportions along the gut, dependent on age, location and antigen stimulation (LILLEHOJ u. CHUNG 1992; LILLEHOJ 1993; GÖBEL et al. 2001; BAR-SHIRA et al.
2003).
IEL are a special cell population of the GALT. Avian IEL mainly consist of TCRαβ + and TCRγδ+ T cells and natural killer cells (NK) (GÖBEL et al. 2001; DAVISON 2008). Most of the avian IEL T cells express a CD8α co-receptor, whereas TCRγδ+CD8α+ IEL are more dominant than TCRαβ+CD8α+ IEL (BUCY et al. 1988; COOPER et al. 1991; LILLEHOJ et al. 2004). The population of IEL CD4+ T cells is very small and B cells are almost absent among those (LILLEHOJ 1993; VERVELDE u. JEURISSEN 1993). IEL have been shown to release several cytokines, such as different interleukins and IFN-γ and influence the activities of intestinal epithelial cells (YUN et al. 2000b).
In the lamina propria various leukocytes, such as granulocytes, macrophages, dendritic cells and B- and T lymphocytes are present. B and T cells compound about 90% of the LP lymphocyte pool, the rest are NK cells (DAVISON 2008). In contrast to IEL, CD4+ T cells are more numerous among the LP lymphocytes than CD8α+ T cell subsets, and TCRαβ+ T cells are more dominant than TCRγδ+ lymphocytes (ROTHWELL et al. 1995). Most of the B lymphocytes in the LP express the secretory IgA isotype (YUN et al. 2000b).
In comparison to mammals, chicken lack some components of the anthelmintic worm responses that are controlled by the Th2 cytokines and are important in the immune reactions following parasitic infections in mammalian species. Chicken have a reduced repertoire of polymorphonuclear cells, neutrophils, eosinophils, and basophils. They are replaced by heterophils, which are predominant cell type in the innate inflammatory reactions. Recently it has been shown that the chicken orthologue of the gene for the Th2 cytokine IL-5, which is important in the mobilization of the bone marrow eosinophil pool in mammals is a pseudogene (KAISER et al. 2005). IgE, which is produced by B cells and play an essential role in nematode resistance in mammals, has not been described for birds. It is suggested that avian IgG partly fulfils the functions of mammalian IgE (DAVISON 2008).
2.4.1. Immunity to enteric parasitic infections in birds
At present, the knowledge about immunity to enteric parasites in birds is mainly based on studies with protozoan parasites in chicken, such as Eimeria. It has been shown that mechanisms of resistance can vary between different Eimeria species (spp.), and the level of
immunity to Eimeria is highly influenced by the genetics of the host (LILLEHOJ u. RUFF 1987; ROSE 1987; BUMSTEAD et al. 1995; TROUT u. LILLEHOJ 1996).
T lymphocytes have been shown to play a crucial role in immunity to coccidia in chicken (LILLEHOJ u. TROUT 1993; TROUT u. LILLEHOJ 1996). The protective immunity to Eimeria has been shown to be TCRαβ+ T cell dependent, in which both CD4+ and CD8+
cells are involved (LILLEHOJ u. TROUT 1996; TROUT u. LILLEHOJ 1996; DAVISON 2008). Partial depletion of CD4+ cells generated by intra-peritoneal injections of anti-CD4 monoclonal antibodies resulted in an increased oocyst shedding rate following primary Eimeria tenella infection in chicken (TROUT u. LILLEHOJ 1996). The mRNA expression of numerous cytokines in the intestinal tissue was upregulated due to Eimeria infections in chicken, but only the T helper (Th) 1 type cytokine IFN-γ induced a protective effect (LILLEHOJ u. CHOI 1998; YUN et al. 2000a; HONG et al. 2006).
The role of cell-mediated immunity in intestinal protozoan infections has also been demonstrated in other parasite models. Studies on thymectomized and bursectomized chicken, which were infected with Cryptosporidium baileyi, indicated a primary role of T cells in the resistance to the infection. Thymectomized chicken showed an increase in the total parasite oocyst shedding rate and failed to resist challenge infection (SRETER et al. 1996).
Not much work has been published so far on the specific immune reactions following helmintic infections in birds. Recently, it has been demonstrated, that Th2 polarisation of the immune response and induction of systemic circulating specific IgG antibodies in the course of nematode infection also exists in avian species. The studies, which were performed on Ascaridia galli-infected chicken, demonstrated systemic and local increase in IL-4 and IL-13 mRNA expression in splenic and ileal tissues (DEGEN et al. 2005; KAISER 2007). It has also been shown that chicken develop circulating IgG antibodies against A. galli soluble somatic antigen and embryonated egg extract starting two to three weeks after infection (MARCOS-ATXUTEGI et al. 2009). However, there is a lack of information on the local cell-mediated immunity in nematode infection in chicken.
2.5. Immunity to enteric nematode infections in mammals
Nematodes are fully adapted obligate parasites, which can notably modulate host immune response to ensure their survival and replication (MAIZELS et al. 2004; RAUSCH et al.
2008). In general, infections with parasitic nematodes cause only mild or subclinical disease (TIZARD 2009). The extend of the parasite burden is controlled by genetic factors, by the host immune response to the parasite and by the initial infection doses (PERNTHANER et al.
1996; LITTLE et al. 2005; BLEAY et al. 2007; SCHILTER et al. 2010).
The Th2-driven immune response has been shown to be protective in gastrointestinal helminth infections in mammals (MAIZELS u. YAZDANBAKHSH 2003; CLIFFE u.
GRENCIS 2004; PATEL et al. 2009). It is associated with antigen-specific local T cell infiltrations (LITTLE et al. 2005; PEREZ et al. 2008) and production of type-2 cytokines such as IL-4, IL-5, IL-10 and IL-13 (GRENCIS 1997; SHEA-DONOHUE et al. 2001; BEHNKE et al. 2003; FINKELMAN et al. 2004; CHIUSO-MINICUCCI et al. 2010). This leads to the high-level tissue eosinophilia, intestinal mastocytosis, goblet cell hyperplasia and production of parasite specific IgG1 and IgE antibodies (CLAEREBOUT u. VERCRUYSSE 2000; BEN- SMITH et al. 2003; ARTIS 2006).
The cytokines IL-4 and IL-13 have an essential role in the immune response to intestinal nematode infections (BANCROFT et al. 1998; MCKENZIE et al. 1998; GRENCIS u.
BANCROFT 2004; HERBERT et al. 2009). IL-4 stimulates development of Th2-type cells, as well as B cells and promotes an IgE response. IgE causes mast cell degranulation and release of vasoactive molecules and cytokines, which stimulates intestinal smooth muscle contraction, increases vascular permeability and results in the expulsion of the worms (DESSEIN et al. 1981; URBAN et al. 2000; KING u. MOHRS 2009; PATEL et al. 2009). IL- 13 stimulates epithelial cell proliferation and, as IL-4 also promotes intestinal muscle contractility (ZHAO et al. 2003; KHAN u. COLLINS 2004).
Secretion of IL-5 by Th2 cells, which is considered to be an important part of the mammalian Th2 immune response in nematode infections, leads to the mobilisation of the bone marrow eosinophil pool (MCKENZIE et al. 1999; DOLIGALSKA et al. 2006; KNOTT et al. 2009).
Eosinophils bind to IgE-coated parasites, degranulate and damage the worm cuticula by their enzymes. This IgE-dependent eosinophil-mediated response is most effective against larval tissue stages.
Local activation of the mucosal immune system and secretion of inflammatory mediators in nematode infections are considered to affect the functions of ion channels in the intestinal epithelium (MADDEN et al. 2004; KOSIK-BOGACKA et al. 2010) and directly control some of the physiological intestinal functions, such as motility and mucus production (KHAN u.
COLLINS 2004).
2.6. Non-starch polysaccharides
Non-starch polysaccharides (NSP) belong to the group of dietary carbohydrates. They are non-starch macromolecular polymers of monosaccharides linked by glycosidic bonds with a degree of polymerization of ten and more (CUMMINGS u. STEPHEN 2007; ENGLYST et al. 2007). In mammals and birds NSP cannot be degraded by endogenous enzymes of the animal and are considered as prebiotics. The fermentation of NSP occurs mainly in cecum and colon by intestinal bacteria (CUMMINGS u. MACFARLANE 1997; BAKKER et al.
1998; WATZL et al. 2005; ROBERFROID 2006; WESTENDARP 2006). According to their physical properties, they can be divided into water-soluble and water-insoluble fractions, which is of relevance for their nutritional value (SPILLER 1994).
Soluble NSP include pectins, pentosans, fructans, beta-glucans and carboxymethylcellulose.
Inulin is a naturally occurring polysaccharide, which is often used as a source of soluble NSP in animal nutrition. Soluble NSP are known to possess anti-nutritional properties by encapsulating nutrients and preventing access of digesting enzymes, or by changing the microbial composition and activity in the intestine (BEDFORD u. CLASSEN 1992; CHOCT et al. 1996; PLUSKE et al. 1998; JENKINS et al. 1999; MCDONALD et al. 1999; JAMROZ et al. 2002). Furthermore, they increase viscosity of the digesta and slow down the passage rate of nutrients (CHOCT u. ANNISON 1992; DUSEL et al. 1997; LIN et al. 2010).
Cellulose and arabinoxylans belong to the insoluble NSP. Feed components such as rice shells and straw powder are especially rich in insoluble NSP. They increase the intestinal passage of nutrients and the volume of digesta. Insoluble NSP are considered to reduce microbial activity and pathogenicity of bacterial populations in the intestine due to their laxative effect (LEESON et al. 1991; SMITS u. ANNISON 1996; DURMIC et al. 1998; VAN KRIMPEN et al. 2009).
2.6.1. Local and systemic effect of NSP on the immune system
Extensive studies in mammals have demonstrated that NSP may modulate systemic and especially local gut-associated immune functions (ROLLER et al. 2004a; ROLLER et al.
2007; BODERA 2008; MEYER 2008; KELLY 2009). Most of these studies have focused on the role of soluble NSP (inulin) on the immune system.
NSP showed various immunomodulatory effects in the T- and B lymphocyte compartment in mammalian species (MANHART et al. 2003; WATZL et al. 2005; KRAG et al. 2006).
Addition of dietary inulin and oligofructose to the diet of rats has led to an increase in T lymphocytes and major histocompatibility complex II molecules in splenic, thymus and mesenteric lymph node (MLN) cells (TRUSHINA et al. 2005). Oral administration of NSP induced proliferation of IgA-producing B lymphocytes in the intestinal mucosa of rats (KUDOH et al. 1998). Pectin in the diet of rats significantly increased the CD4+/CD8+ ratio in MLN lymphocytes (LIM et al. 1997).
Also, local and systemic cytokine production levels, as well as concentration of secretory IgA in ileum and cecum may be influenced by NSP (SEIFERT u. WATZL 2007). Inulin enriched with oligofructose enhanced the production of IL-10 in Peyer's patches as well as the concentration of secretory IgA in the cecum of rats (ROLLER et al. 2004b). Addition of fructooligosaccharides to the diet of mice induced increased production of IFN-γ, IL-10, IL-5 and IL-6 by CD4+ cells in Peyer's patches (HOSONO et al. 2003). Inulin-fed rats showed a higher ex vivo secretion of IL-2, IL-10 and IFN-γ in spleen and mesenteric lymph node cell cultures, as well as a higher proportion of dendritic cells in the Peyer’s patches (RYZ et al.
2008).
Not much information is available how NSP may modulate the immune system of birds.
Recently, it has been shown, that fructo-oligosaccharide reduced the proportion of B cells but did not affect the percentage of T cells in cecal tonsils, and enhanced IgG antibody titers in plasma of broiler chicken (JANARDHANA et al. 2009).
The investigations in mammalian models demonstrated that NSP primarily modulate immune parameters on the GALT level, but it may also come to a systemic activation of leukocytes in the spleen. The production of short-chain fatty acids (SCFA) which bind to SCFA-receptors on leucocytes, or direct influence of lactic acid-producing microorganisms on immune cells are considered to be responsible for the immuno-modulating effects (WATZL et al. 2005).
2.6.2. Effect of NSP on nematode infections
Until now most of the studies investigating the effects of NSP on nematode infections were performed in mammalian models. It was shown that dietary fibre has an influence on parasite establishment and survival in the host (PEARCE 1999; THOMSEN et al. 2006). This influence was shown to be associated with water-soluble and water-insoluble properties of NSP. Inclusion of soluble NSP such as inulin in the diet of pigs infected with Trichuris suis or Oesophagostomum dentatum led to a significant reduction in worm establishment, egg excretion and female worm fecundity (PETKEVICIUS et al. 2003; THOMSEN et al. 2005;
KRAG et al. 2006; PETKEVICIUS et al. 2007). In contrast, diets enriched with insoluble NSP provide favourable conditions for the establishment and survival of Oesophagostomum dentatum in the large intestine of pigs (PETKEVICIUS et al. 1997; PETKEVICIUS et al.
1999; PETKEVICIUS et al. 2001). Opposing results to those collected throughout experiments in pigs were demonstrated in a study in mice infected with Heligmosomoides polygyrus. The parasite establishment was elevated, when the animals were fed a pectin- enriched diet, whereas cellulose did not affect establishment, reproduction and survival of the parasite (SUN et al. 2002).
A study with A. galli-infected chicken demonstrated a reduction in the number of worms and fecal egg shedding rate, when the birds were fed a soluble NSP enriched diet, which
additionally was supplemented with NSP-hydrolyzing enzyme (DÄNICKE et al. 2009). The actual influence of soluble and insoluble NSP in nematode infections in birds has not been yet investigated.
The exact mechanism of the influence of dietary fibre on the course of nematode infections is still unknown. It is suggested that microbial degradation of NSP induce changes in bacterial populations of intestinal microflora and their metabolic products, such as concentrations of short-chain fatty acids and lactic acids, which may have an impact on helminth survival (PETKEVICIUS et al. 2004).
2.7. Chloride secretion and nutrient transport in the intestine
Originally, the Ussing chamber technique had been developed to study electrolyte transport across the frog skin (USSING u. ZERAHN 1951). At present, the USSING method is often used to measure changes in the short-circuit currents, which are associated with the changes in electrogenic ion transport as well as to calculate transcellular nutrient transport processes across intestinal epithelia (TSUJI et al. 1985; SHIMADA u. HOSHI 1986; GARRIGA et al.
1999; DE JONGE et al. 2004; BLEICH et al. 2007).
The volume of intestinal fluid and the water content of the ingesta are regulated by the transport of chloride ions across intestinal epithelia (LEONHARD-MAREK et al. 2009).
Major chloride secretion in intestinal epithelium occurs via the cystic fibrosis transmembrane regulator (CFTR) channel (BARRETT u. KEELY 2000). The existence of alternative chloride channels and the outwardly rectifying chloride channels have been described in mammalian models, but their exact physiological relevance has not yet been identified (GRUBER et al.
1998; BRONSVELD et al. 2000; HRYCIW u. GUGGINO 2000; JENTSCH et al. 2002). The CFTR channel can be stimulated by elevation of intracellular Ca2+ by carbachol or in a cAMP-dependent way by forskolin, and is inhibited by 5-nitro-2-(3-phenylpropylamino) benzoate (NPPB) (LEONHARD-MAREK et al. 2009).
Active transcellular transport of glucose and amino acids in the intestine is coupled with sodium (MAILLEAU et al. 1998; GARCIA-AMADO et al. 2005; AWAD et al. 2008).
Absorption of glucose is mediated by the Na-glucose cotransporter-1. Amino acids, for example, alanin are transported via carrier proteins located in the apical and basolateral membranes (PAPPENHEIMER 1993). In addition to active transcellular mechanisms, paracellular transport of glucose and amino acids is under discussion (GARCIA-AMADO et al. 2005; REHMAN et al. 2007).
2.7.1. Effect of NSP on electrogenic chloride secretion and nutrient transport The influence of dietary fibre on electro-physiological functions of the intestine has been investigated in different mammalian and avian models. In some studies, the supplementation of inulin or dried sugar beet pulp as a source of soluble NSP did not influence epithelial glucose transport in the small intestine of pigs (VON HEIMENDAHL et al. 2010) and jejunal glutamine and glucose transport in broilers (REHMAN et al. 2007). In the study of Awad et al. 2010, an increase of active transcellular glucose transport was observed in jejunal tissues of broilers, fed with an inulin supplemented diet. However, the level of the increase did not reach significance.
Dietary fibre was shown to have an effect on intestinal chloride secretion. The investigation in rats showed significant decreases in Cl- ion transport in the proximal jejunum after dietary supplementation with cellulose or pectin (SCHWARTZ et al. 1982).
2.7.2. Influence of helminthic infection on electrogenic chloride secretion and nutrient transport in the intestine
Investigations in mammalian models demonstrated that infections with gastrointestinal helminths may affect secretory responses (KOSIK-BOGACKA u. KOLODZIEJCZYK 2004;
KOSIK-BOGACKA et al. 2010) and eletrogenic nutrient transport of the intestine (SHEA- DONOHUE et al. 2001).
The changes in chloride secretion, which correlate with a water influx in the intestinal lumen, seem to depend on the parasite stage and the previous sensitization of the host to the parasite antigen (O'MALLEY et al. 1993). The addition of Trichinella spiralis antigen to the colon segments of guinea pigs in the USSING chambers induced an increase in the short-circuit
currents in immune animals as a response to the antigen. The changes in non-immune animals were not observed (WANG et al. 1991). An increase in Cl- secretion in response to histamine was observed in Ascaris suum infected pigs during the period of self-curing (DAWSON et al.
2005). Also pigs infected with Oesophagostomum dentatum showed alterations in chloride secretion which were dependent on the parasite stage (LEONHARD-MAREK u.
DAUGSCHIES 1997).
Helminth-induced reduction in sodium-linked glucose absorption was observed in pigs infected with Ascaris suum (DAWSON et al. 2005) and in mice inoculated with Heligmosomoides polygyrus, Nippostrongylus brasiliensis and Trichinella spiralis (SHEA- DONOHUE et al. 2001; MADDEN et al. 2004; AU YEUNG et al. 2005).
The alterations in intestinal ion transport are connected to the activation of the immune system. It has been shown that induction of Th2-cytokines IL-4 and IL-13 in response to nematode infections influenced absorption, secretion and permeability of epithelial cells. The changes in intestinal electro-physiological functions were dependent on the activation of the STAT6 signaling pathway (MADDEN et al. 2002; MADDEN et al. 2004).
3. Goals and objectives
The aim of the project was to investigate immunological and electro-physiological parameters in the intestine following experimental infection with Ascaridia galli and Heterakis gallinarum in layer chicken and to characterise the influence of non-starch polysaccharides on these parameters.
We hypothesized that the local and systemic immune response, as well as the electrogenic nutrient transport and secretory functions of the intestine in chicken might be affected by the nematode infection similarly as in mammalian species. As in mammalian models, we also expected to observe NSP influence on these parameters.
Under different dietary conditions we investigated:
1) local and systemic T cell populations 2) induction of local Th1 and Th2 cytokines 3) specific IgG antibody development in serum
4) electro-physiological epithelial functions in the intestine, such as chloride secretion and electrogenic alanin and glucose transport.
In addition, we characterized the influence of H. meleagridis on H. gallinarum infection.
4. Pathobiology of Heterakis gallinarum mono- and co-infection with Histomonas meleagridis in layer chicken
Pathobiology of Heterakis gallinarum mono- and co-infection with Histomonas meleagridis in layer chicken
Short title to use as a running head:
H. gallinarum infection in chicken
Corresponding autor:
Prof. Silke Rautenschlein Phone: ++49 511 9538763 Fax: ++49 511 9538580
E-mail: silke.rautenschlein@tiho-hannover.de
Anna Schwarz1, Matthias Gauly2, Hansjörg Abel3, Gürbüz Daş2, Julia Humburg3, Alexander Th. A. Weiss5, Gerhard Breves4, Silke Rautenschlein1*
1University of Veterinary Medicine Hannover, Clinic for Poultry, Bünteweg 17, 30559 Hannover, Germany, 2University of Goettingen, Department of Animal Sciences, Albrecht Thaer Weg 3, 37075 Goettingen, Germany, 3Department of Animal Sciences, University of Göttingen, Kellnerweg 6- 37077 Göttingen, Germany, 4University of Veterinary Medicine Hannover, Institute for Physiology, Bischofsholer Damm 15, 30173 Hannover, Germany,
5Freie Universitaet Berlin, Department of Veterinary Pathology, Robert-von-Ostertag-Str. 15, 14163 Germany
Abstract
Not much is known about the induction and modulation of gut-associated immune reactions after nematode infection in chicken. The objective of this study was to compare the pathogenesis, induction of immune reactions and electrophysiological changes of the gut after mono-infection with Heterakis gallinarum (H. g.) and after dual infection with H. g. and Histomonas meleagridis (H. m.) in layer chicken. In two experiments three-week old chicken were inoculated with embryonated H. g. eggs, which were positive for H. m. While birds of the first experiment were left untreated, those of the second were treated with dimetridazol to prevent H. m. co-infection. Mild to moderate histological lesions and local immune reactions with a significant increase in CD4+, CD8α+, TCRαβ+ and TCRδγ+ cells in the lamina propria and induction of the Th2- cytokine IL-13 dominated the H. g. immune response at two weeks post infection (pi). Co-infection with H. g. and H. m. induced an increase in mRNA expression of the Th1 cytokine IFN-γ, furthermore a decrease in splenic CD4+ cells and severe destruction of the cecal mucosa in association with strong T cell infiltration in the cecal lamina propria. No obvious effects on the chloride secretion of the cecal epithelium, which was investigated once the mucosa had almost recovered from the infection, could be observed in either of the two experiments. These results suggest that the local T cell reactions to nematode infections in chicken may be comparable to mammals and may be shifted from a Th2 to a Th1 dominated response when accompanied by a protozoan infection.
Introduction
Due to changes in the legal requirements for layer-housing a shift from cage to alternative production systems is occurring in European countries. This change to more housing on litter and free-range production has led to the re-emergence of parasitic infections, such as the infection with Heterakis gallinarum (H. g.) (Permin et al., 1999; Maurer et al., 2009). H. g. is one of the most frequently diagnosed nematode within the digestive tract of galliform birds (Lund et al., 1970). Infection with H. g. is generally subclinical, but H. g. may also function as a vector for Histomonas meleagridis (H. m.), which is known to induce severe pathological lesions in gut and liver and leads to high mortality rates in susceptible hosts (Gibbs, 1962;
Springer et al., 1969; Lund & Chute, 1974; Esquenet et al., 2003). In contrast to turkey, histomoniasis in chicken is known to show a high morbidity but low mortality (McDougald, 2005). Direct transmission of H. m. has not been demonstrated for chicken in some studies (Hu et al., 2006) or may occur at lower rates than observed in turkey (Hess et al., 2006). This emphasises the importance of H. g. as a vector for H. m. in chicken.
Although many studies have investigated the prevalence of H. g. in chicken (Kurt & Acici, 2008; Mungube et al., 2008; Maurer et al., 2009) and the induction of pathological lesions after H. g. infection (Kaushik & Deorani, 1969; Riddell & Gajadhar, 1988), no information is available on specific gut-associated immune reactions following the infection. A variety of studies in mammalian species demonstrated the importance of cell-mediated immune reactions in the clearance of nematode infection. Especially the Th2 immune response dominates following a nematode infection (Dawson et al., 2005; Little et al., 2005; Scales et al., 2007). In mice it has been demonstrated that local activation of T cells may play a role in the expulsion of the cecal nematode Trichuris muris. In resistant mice, which develop a Th2 response, the number of infiltrating CD4+ and CD8+ cells in the epithelium and lamina propria of the gut was highest at the time of worm expulsion (Little et al., 2005). Increased numbers of T cells, particularly CD4+ and TCR γδ+ lymphocytes were observed in the abomasal mucosa of goats following primary infection with Haemonchus contortus (Perez et al., 2008). The few studies on Ascaridia galli infection in chicken demonstrated the systemic and local increase in IL-4 and IL-13 mRNA expression in splenic and ileal tissues (Degen et al., 2005; Kaiser, 2007) and induction of circulating IgG antibodies starting two to three weeks pi (Marcos-Atxutegi et al., 2009).
With regard to the ceca as the major site of H. g. and H. m. infection it may be hypothesized that the secretory response might be affected by the infection as well. Pigs infected with Oesophagostomum dentatum showed depending on the parasite stage, alterations in chloride secretion (Leonhard-Marek & Daugschies, 1997), which in turn is correlated to the water content of digesta (Leonhard-Marek et al., 2009). Significant increase in Cl- secretion in response to histamine during the period of self-healing was also observed in Ascaris suum infected pigs suggesting a net rise in fluid in the intestinal lumen at this stage (Dawson et al., 2005).
At present, the use of many effective antihelmintic drugs is prohibited or restricted due to consumer safety reasons. New ways to influence chicken health by feed additives are under investigation (Owens et al., 2008; Mountzouris et al., 2009; Solis de los Santos et al., 2009).
It was shown in different mammalian models that non-starch polysaccharides (NSP) may have a beneficial effect on systemic and local immune functions and general performance of the animal (Kelly-Quagliana, 2003; Roller et al., 2004; Seifert & Watzl, 2007). Inulin-fed rats showed a higher proportion of dendritic cells in the Peyer’s patches and higher ex vivo secretion of IL-2, IL-10 and IFN-γ in spleen and mesenteric lymph node cell cultures (Ryz et al., 2008). Furthermore, NSP influenced the course of intestinal nematode infection in mammalian species (Petkevicius et al., 1997; Pearce, 1999; Petkevicius et al., 2001;
Petkevicius et al., 2003; Petkevicius et al., 2007). There is no information available if NSP may influence gut immunity, nematode infection and electro-physiological parameters in chicken.
The objective of this study was to investigate immunological and electrophysiological parameters of the intestine in response to experimental infection with the nematode H. g. In addition, we characterised the influence of a co-infection of H. g. and H. m. on these parameters, as well as the impact of NSP on the outcome of the infections.
Materials and Methods
Animals. One day old female Lohmann Selected Leghorn (LSL) chicken were obtained from Lohmann Animal Breeding GmbH, Cuxhaven, Germany. The chicken were housed in two isolation rooms. Infected and non-infected groups were kept separately. They were randomly split to three separate groups within each room and kept according to the regulations set for
Animal Welfare. Water and feed was offered ad libitum. No vaccination program was applied.
Heterakis gallinarum. The adult female worms were collected from infected chicken, which had been obtained from different farms. The collected worm eggs were positive for Histomonas meleagridis. After incubation of the eggs in 0.5% (w/v) formalin for 3 weeks at room temperature the embryonated eggs were stored at 4°C for one to eight months until inoculation.
Diets. Both, the animals of the non-infected and infected groups were allotted to three different diets: control feed; control feed containing 9.1% of pea bran meal as a source of insoluble non-starch polysaccharides (NSP); control feed containing 9.1% of chicory root meal as a source of soluble NSP. The diets were offered in pelleted form. The percentages of the ingredients and the analysis of the diets are given in Table 1.
Haematoxilin & Eosin (H&E) staining and patho-histology. Samples of distal cecum were fixed for 24 hours in 4% phosphate-buffered formalin and then processed for patho- histological examination by standard methods after H&E staining. The H&E-stained tissue sections were examined by light microscopy for lesions such as epithelial erosion and ulceration, lymphocyte and heterophil infiltration and cell aggregation as well as accumulation of fibrin exudate in the lumen.
Flow cytometric analysis. Single cell suspensions of spleen leukocytes were prepared using a slightly modified method, to one previously described (Liman & Rautenschlein, 2007).
The combination of the following antibodies was used to detect splenic T cells: mouse-anti- chicken-CD4 and -CD8α antibodies (Exp. 1 & 2) (Chan et al., 1988) and mouse-anti-chicken- TCRαβ (Vβ1) and -TCRγδ antibodies (Exp. 2) (Chen et al., 1988), conjugated to phycoerythrin (R-PE) and to fluorescein (FITC), respectively (Southern Biotech, provided by Biozol, Eching, Germany). The antibodies were diluted in FACS buffer to a final concentration of 0.5 µg (anti-CD4), 0.8 µg (anti-CD8α), 2 µg (anti-TCRαβ (Vβ1)) and 5 µg (anti-TCRγδ) per ml. The percentage of stained cells was determined using the Beckman
Coulter Epics XL© flow cytometer and EXPO 32 ADC software program (Beckman Coulter Company, Miami, Florida). The lymphocyte population was gated according to size and granularity, and 10.000 events per sample were analysed based on positive staining with FITC and R-PE.
Immunohistochemistry. Cryostat sections of distal ceca (8-µm thick) were processed as described previously (Vervelde et al., 1996; Berndt et al., 2007). Sections were stained with the following mouse-anti-chicken unlabeled monoclonal antibodies: anti-CD4, anti-CD8α, anti-TCRαβ (Vβ1), anti-TCRγδ (at 0.5 µg/ml each) and anti-IgA (0.05 µg/ml) (Southern Biotech, provided by Biozol). The secondary anti-mouse IgG biotinylated antibody, ABC reagent (Vectastain® Elite® ABC Kit,Vector Laboratories Inc.) and the 3,3´-diaminobenzidine (DAB) peroxidase substrate Kit (Vector Laboratories Inc.) were used according to the manufacturer’s instructions. The different lymphocyte populations in the cecal lamina propria were evaluated semi-quantitatively using the abundance score (Figure 3).
Real-time quantitative RT-PCR. Total RNA was isolated from distal cecum with 1000 µl TrifastGOLD (Peqlab, Erlangen, Germany) per sample according to the manufacturer’s instructions.
Cytokine mRNA expression levels were quantified using TaqMan quantitative RT-PCR.
Specific primers, cloning primers and probes are provided in Table 2 (Rautenschlein et al., 2007; Powell et al., 2009). Real-time quantitative RT-PCR was performed using the Brilliant® II QRT-PCR one-step master mix kit (STRATAGENE, Agilent Technologies Company, USA). Amplification and quantification of specific products was done using the Mx3005PTM thermal cycle system and Mx3005PTM Q PCR Software (STRATAGENE, Agilent Technologies Company). The following cycle profile was applied: one cycle at 50°C for 30 min and 95°C for 10 min, and 40 cycles at 95°C for 20 s and 60°C for one min. Results are expressed as x-fold change in mRNA expression levels in the tissues of inoculated birds compared to non-inoculated controls. The differences in template RNA levels of individual transcripts were normalised to 28S rRNA as previously described (Powell et al., 2009).
Expression levels of 28S rRNA stayed constant in the tissues of inoculated and non- inoculated animals showing the same threshold cycle values (Ct) throughout both
experiments. GADPH was tested as an additional house-keeping gene (Rautenschlein et al., 2007) but its expression levels in the cecal samples were not as stable as the expression level of 28S rRNA. To generate standard curves the target gene segment of chicken IFN-γ was cloned into the pCR3.1. vector (Invitrogen, Germany) (Rautenschlein et al., 2007) and target gene segments of chicken IL-4 and IL-13 – into the pCR®4-TOPO® vector (Invitrogen) following standard procedures.
Electrophysiological measurements. After removing the tunica serosa the segments of the distal cecum were mounted in Ussing chambers with the exposed area of 1 cm2. The standard buffer solutions contained (mmol/l): NaCl 113.6, KCl 5.4, CaCl2*2H2O 1.2, MgCl2*6H2O 1.2, Na2HPO4*2H2O 1.2, NaH2PO4*H2O 0.3, NaHCO3 21.0, glucose 10.0, HCl 0.4 and mannitol 23.0 at the serosal side, and NaCl 113.6, KCl 5.4, CaCl2*2H2O 1.2, MgCl2*6H2O 1.2, Na2HPO4*2H2O 1.2, NaH2PO4*H2O 0.3, NaHCO3 21.0, HCl 0.4 and mannitol 31.96 at the mucosal side of the tissues. All chemicals were obtained from Merck KG, Darmstadt, Germany. The buffer solutions had an osmolarity of 300 mosmol/l and pH of 7.45, when aerated with carbogen at 37 °C. To reduce endogenous production of prostaglandin the buffers were supplemented with Indomethacin (10 µmol/l). Short-circuit currents (Isc) and transepithelial tissue conductances (Gt) were measured using a computer controlled voltage- clamp device (Mußler Ingenieurbüro für Mess- und Datentechnik, Aachen, Germany). Gt were determined by applying a current pulse of 100 µA for 200 ms every 6 s. The following chemicals were added to the chambers with recovery intervals of 20-30 mins: amiloride (0.1 mmol/l) in combination with tetraethylammonium (TEA) (5 mmol/l) and Ba2+ (1mmol/l) mucosal to inhibit apical Na+ and K+ channels; Carbachol (0.1 mmol/l) and Forskolin (0.01 mmol/l) serosal as stimulators of the chloride secretion, 4,4´-diisothiocyanostilbene-2,2´- disulfonic acid (DIDS) (0.2 mmol/l) mucosal to inhibit alternative chloride channels and 5- nitro-2-(3-phenylpropylamino) benzoate (NPPB) (0.5 mmol/l) serosal as an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR) channel. The substances with an exception of Ba2+ (Merck KG, Darmstadt, Germany) were obtained from Sigma-Aldrich Chemicals, St. Louis, MO, USA. Three cecal segments from each bird in Exp. 1 and two segments from each bird in Exp. 2 were used. The electrical responses were measured as a
difference of an average of two to three basal values before and two to three values after reaching the maximal response to the application of the respective substance.
Experimental protocol. Experiment 1. One hundred twenty animals were randomly divided into three groups of n=40. The groups were kept on control diets or diets containing 9.1% of soluble or insoluble NSP. At 3 weeks of age 20 birds per group were randomly selected and orally inoculated with 200 embryonated H. g. eggs that were positive for H. m. Throughout the experiments clinical examinations were carried out daily and a measure of body weight gain weekly. Necropsy was carried out two, three and five weeks post infection (pi). Four to five birds chosen at random from each subgroup were necropsied and examined for pathological lesions. Samples of distal cecum were taken for histo-pathological and immunohistochemical examination and spleen samples for flow cytometric analysis. Distal cecal tissue of three birds per inoculated and non-inoculated group allotted to control diet was taken for quantification of cytokine mRNA expression levels by quantitative RT-PCR. On the last necropsy day parasite numbers from one cecum per animal were counted and the excrements of the birds were examined for the presence of H. g. eggs. For electrophysiological measurements four to five birds chosen at random per diet and infection group were necropsied at five weeks pi, and samples of distal ceca were analysed in the Ussing chamber.
Experiment 2. The birds were preventively treated via drinking water with dimetridazole (0.05% w/v) (Chevi-col, Chevita GmbH, Germany) against an infection with H. m. from two days before inoculation until day 7 post inoculation with H. g. embryonated eggs. The experimental setup was identical to Exp. 1 with the two following exceptions. Cecal samples for immunohistochemical examination were only taken two and five weeks pi. At nine weeks pi, a total of 30 animals (five birds chosen at random from each subgroup) were necropsied for electrophysiological measurements.
Statistical methods. All data are expressed as mean per group ± standard deviation (SD).
The differences between groups were determined by paired t-test and Wilcoxon Rank Sum Test. The effects of diets and infection as two independent factors were investigated by two-
way ANOVA. P values of <0.05 were considered as significant. The statistical analyses were performed using the SAS® 9.1 programme.
Results
Influence of diet on performance, lesion development and immune parameters. No difference was seen between the feeding groups with regard to clinical signs, pathological and histo-pathological lesions and local as well as systemic immune reactions at the investigated points under the experimental conditions of the present study. Therefore, for the evaluation of these parameters the three different feeding groups were treated as replicates and combined to one inoculated and non-inoculated animal group.
Clinical and post-mortem observations. In Exp. 1 birds of the dually infected group showed mild clinical signs such as depression and ruffled feathers beginning at one week pi. The mortality throughout this experiment was 0.8% - one bird died on day 13 pi. In Exp. 2 animals preventively treated with dimetridazol (0.05% w/v) against H. m. co-infection did not show any clinical signs or mortality. Dually infected birds in Exp. 1 showed a significant reduction in body weight beginning at two weeks pi until the end of the trial (P <0.05), while in Exp. 2 a significant drop in body weight was only observed at two weeks pi (P <0.05) (data not shown).
Pathological examination of dually infected birds in Exp. 1 showed formation of fibrinous to fibrino-hemorrhagic exudates in cecal lumen of 14 out of 15 inoculated chicken at two and three weeks pi (Table 3). One to three focal hepatic necrotic areas were found in six out of 15 birds two weeks pi. At five weeks pi only one bird out of 15 showed macroscopic lesions in the ceca. No pathological lesions were observed in H. g. mono-infected birds in Exp. 2 (Table 3).
The infection rate for H. g. (% of worm-positive animals out of inoculated animals) was 13%
in the dually infected group in Exp. 1 and 93% in the mono-infected group in Exp. 2. No feces sample of inoculated birds in Exp. 1 and 33% of the feces samples of inoculated birds in Exp. 2 were positive for H. g. eggs at five weeks pi (Table 3). The average number of detected H. g. in ceca five weeks pi was 0.33 worms per bird (min 0 and max 3 worms) in Exp. 1 and 12.1 worms per bird (min 0 and max 37 worms) in Exp. 2.
Non-inoculated birds did not show any clinical signs or pathological lesions and were worm- negative in both experiments.
Histo-pathology. The development and incidence of histo-pathological lesions after inoculation of the chicken with embryonated H. g. eggs are shown in the Table 3. In the case of H. m. co-infection we observed severe interstitial lymphocyte, heterophil and macrophage infiltration, complete ulceration of intestinal epithelium and accumulation of fibrin exudates and detritus in the lumen of ceca in 93% (14 out of 15) of inoculated birds two weeks pi (Figure 1b, Exp. 1). Numerous histomonads and moderate numbers of bacterial colonies were found in the severely hyperplasic tunica muscularis. Three weeks pi, all inoculated birds still showed histo-pathological lesions. The structure of the mucosa showed reorganisation and re- epithelisation and still contained severe lymphocyte and heterophil infiltration in the lamina propria (Figure 1c, d). Five weeks pi, 93% (14 out of 15) of inoculated birds showed moderate lymphocyte infiltration in the lamina propria and formation of lymphoid centers in cecal mucosa (Figure 1e).
Mono-infection with H. g. (Exp. 2) resulted in mild to moderate lymphocyte infiltration of the lamina propria and formation of lymphoid aggregations. Bacterial colonies were not observed. Two, three and five weeks pi 93%, 86%, 80% of inoculated chicken showed the indicated microscopical lesions, respectively (Figure 1f), while the remaining birds were free of detectable histo-pathological changes.
No microscopical lesions were observed in the non-inoculated groups of either experiment (Figure 1a).
Changes in local lymphocyte populations. Different T cell subsets were investigated immunohistochemically in the lamina propria of the ceca of control and inoculated birds (Figure 2, 3). For the semi-quantitative evaluation we used an abundance score based on the incidence of positive stained cells in the cecal mucosa and on the changes in the tissue structure due to the infection (Figure 2). The changes in local T lymphocyte populations were comparable for the different T cell subtypes in each experiment (Figure 3). Dually infected birds (Exp. 1) showed severe T lymphocyte infiltration in the lamina propria (Figure 2e, f).
The increase in the abundance score of CD4+, CD8α+, TCRαβ (Vβ1)+ and TCRγδ+ T cells in
