Clinic for Poultry

University of Veterinary Medicine Hannover

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

Supervisors: Prof. S. Rautenschlein (Clinic for Poultry, University of

Veterinary Medicine Hannover, Germany)

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

Publications

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)

I

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

II

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

III

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

IV

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

V

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

VI

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

VII

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)

VIII

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

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

2

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

3

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.

4

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).

5

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

6

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

7

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

8

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).

9

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

10

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.

11

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).

12

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).

13

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).

14

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

15

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).

16

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

17

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).

18

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.

19

4. Pathobiology of Heterakis gallinarum mono- and co-infection with

Histomonas meleagridis in layer chicken

20

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

21

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.

22

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).

23

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

24

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

25

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

26

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

27

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-

28

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.

29

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

30

the gut-mucosa two weeks pi was five- to six-fold compared to non-inoculated animals

(Figure 3, P< 0.05). Mono-infected birds (Exp. 2) reacted with mild to moderate T

lymphocyte infiltration in the cecal lamina propria two weeks pi (Figure 2b, c). The

abundance score increase was two- to three- fold in infected birds compared to non-inoculated

animals (Figure 3, P< 0.05). Three to five weeks pi, dually infected birds (Exp. 1) showed

moderate to severe CD4+, CD8α+, TCRαβ (Vβ1)+ and TCRγδ+ T-cells infiltration (Figure

2c, d, Figure 3 , P< 0.05). Five weeks pi, animals in Exp. 2 showed a small increase in the

abundance score that was only significant for CD4+, CD8α+ and TCRγδ+ T-lymphocytes

(Figure 3, P< 0.05).

Following H. g. and H. m co-infection, no IgA+ B lymphocytes were detected in the

destroyed cecal mucosa 2 weeks pi (Exp. 1). Five weeks pi, there were no differences in the

abundance score of IgA+ cells between control and inoculated birds, when the mucosal

structure was almost restored. No changes in IgA+ B cell populations were observed between

mono-infected and parasite-free birds in Exp. 2 (data not shown).

Flow cytometric analysis of splenic lymphocyte populations. Co-infection of H. g. and H.

m. induced a significant decrease in the relative number of CD4+ splenic T lymphocytes at

two weeks pi but not at three and five weeks pi in comparison to non-inoculated layers (Exp.

1), (Figure 4, P< 0.05). In the case of dimetridazol treated birds no changes in the relative

number of splenic CD4+ cell were detected compared to worm free controls (Exp. 2, Figure

4). No differences were seen in the percentages of splenic CD8α+ (Exp.1 & 2) and TCRαβ

(Vβ1)+ and TCRγδ+ (Exp. 2) T lymphocyte populations between infected and parasite-free

birds.

Detection of IFN-γ, IL-4 and IL-13 cytokine mRNA expression levels by real-time

quantitative RT-PCR. Dual infection with H. g. and H. m. (Exp. 1) induced a statistically

significant increase in IFN-γ mRNA expression in the ceca of inoculated birds in comparison

to non-inoculated controls at two weeks pi (Figure 5a, P< 0.05). The changes in IL-4 and IL-

13 cytokine mRNA expression were not significantly different compared to the control group

in this experiment (P>0.05). Mono-infection with H. g. (Exp. 2) resulted in a statistically

significant increase in IL-13 mRNA expression two weeks pi in inoculated birds compared to

31

worm-free controls (Figure 5c, P< 0.05). The changes in IFN-γ and IL-4 mRNA expression in

H. g. mono-infected birds were not significantly different compared to the worm free control

group.

Electro-physiological measurements. The maximal changes in short-circuit currents in ceca

of H. g. and H. m. co-infected birds (Exp. 1) and H. g. mono-infected birds (Exp. 2) in

response to the addition of carbachol, forskolin and NPPB are shown in Figure 6. Mono-

infected birds (Exp. 2) showed responses to carbachol and forskolin, whereas no significant

effect of the infection or the diet was seen in dually infected birds (Exp. 1). Both substances

induced a significantly lower increase in the maximal Isc response in mono-infected birds

compared to non-inoculated ones (Exp. 2), when insoluble NSP were fed (Figure 6a, b, P<

0.05). Forskolin also induced smaller maximal Isc responses in non-inoculated animals fed

with soluble NSP than in non-inoculated animals fed with insoluble NSP. Moreover, forskolin

caused lower short-circuit current responses in inoculated animals fed with soluble NSP than

in inoculated animals receiving control feed (Figure 6b, P< 0.05). NPPB addition resulted in

decreases in Isc in all animals. Dual infection of H. g. and H. m. (Exp. 1) induced a

significantly smaller decrease in the maximal Isc responses in inoculated compared to non-

inoculated animals when fed insoluble NSP. Mono-infection with H. g. (Exp. 2) led to smaller

Isc decreases in tissues of inoculated animals being fed soluble or insoluble NSP instead of

control diet. Maximal Isc response was also less pronounced in non-inoculated birds fed with

soluble NSP compared to non-inoculated animals receiving insoluble NSP (Figure 6c, P<

0.05). DIDS had no effect on Isc response in both experiments (data not shown).

32

Discussion

Mucosal immunity and the secretory response of the intestine play an important role in the

control of parasitic intestinal infections (Khan & Collins, 2004). In this study we investigated

for the first time the gut-associated immune parameters and secretory responses of ceca in

layer chicken in the light of different diets following mono-infection with H. g. and dual

infection with H. g. and H. m.

While NSP showed immunomodulatory effects in the T cell compartment in mammalian

species (Lim et al., 1997; Trushina et al., 2005; Watzl et al., 2005), we did not observe any

NSP-influence on the splenic and cecal T cell numbers in worm-free or in nematode-infected

birds at the investigated points as specified in the experimental setting. In broiler chicken

fructo-oligosaccharide enhanced IgG antibody titers in plasma and reduced the proportion of

B cells but did not affect the percentage of T cells in cecal tonsils (Janardhana et al., 2009). It

may be speculated that splenic and gut T cell populations in layer chicken are not influenced

by NSP, but further studies investigating other immune parameters are needed to support our

findings.

Previous studies on nematode infection in mammals showed that T cells are attracted locally

to the site of infection (Almeria et al., 1997; Bozic et al., 2000; Balic et al., 2002; Perez et al.,

2008). It was suggested that T lymphocytes, especially CD4+ cells, may play a significant

role in resistance to worm infection (Betts et al., 2000; Khan & Collins, 2004; Little et al.,

2005; Rausch et al., 2008; Patel et al., 2009). In this study we demonstrated for the first time

that similar to mammals H. g. infection in chicken also leads to recruitment of T lymphocytes

to the intestinal mucosa. In our experiments the local accumulation of immune cells in the

cecal lamina propria was most pronounced 2 weeks pi for both mono- and dually infected

birds. The observed increase in CD4+, CD8α+, TCRαβ (Vβ1)+ and TCRγδ+ T cell

subpopulation was similar for subsets within each experiment. Co-infection of H. g. and H. m.

resulted in a significantly higher T cell infiltration rate compared to mono-infection with H.

g., which may be due to a higher invasiveness of the protozoan parasite (Brener et al., 2006).

Flow cytometric analysis of splenic T lymphocyte population revealed a significant decrease

in CD4+ T cells in dually infected birds at 2 weeks pi. This effect was not detected for splenic

CD8α+ lymphocytes. This observation may suggest a significant role of CD4+ cells in H. m.

infection, as previously reported for other protozoan parasites (Vervelde et al., 1996; Yun et

33

al., 2000) and provide evidence of an increased intestinal recruitment of CD4+cells to the

cecal lamina propria (Taylor et al., 2009). Mono-infection with H. g. did not cause any

changes in the percentage of splenic T lymphocytes, which suggests that in contrast to high

invasive intestinal nematodes (Dondji et al., 2008; Dondji et al., 2010), the immune response

following H. g. infection is mainly localised in the gut.

Analysis of T helper (Th)1 and Th2 cytokine mRNA expression in cecal tissues demonstrated

an increase in IFN-γ mRNA level in dually infected birds and an increase in IL-13 mRNA

level in mono-infected birds compared to worm-free animals. Our data suggests that mono-

infection with H. g. elicit local Th2 type immune reactions in the cecal lamina propria as was

shown for nematode infections in mammals (Dawson et al., 2005; Patel et al., 2009).

Following H. g. and H. m. co-infection the local immunological reactions are dominated by

H. m. with induction of Th1 type cytokines comparable to the effect reported for other

protozoan infections (Lillehoj, 1998; Yun et al., 2000; Hong et al., 2006).

Previous studies in mammals have demonstrated that nematode infection influences secretory

functions of intestine (Madden et al., 2004; Dawson et al., 2005). To study electrogenic Cl-

secretion in the ceca of chicken we used a modified protocol, which had been established for

the diagnosis of cystic fibrosis in humans and for the characteristics of Cl- secretion in mice

and pigs. Changes in short-circuit current can be attributed to changes in Cl- secretion after

inhibition of the apical Na+ and K+ channels (De Jonge et al., 2004; Bleich et al., 2007;

Leonhard-Marek et al., 2009). Major chloride secretion in intestine epithelium takes place via

the CFTR channel (Barrett & Keely, 2000). This channel can be stimulated in a Ca-dependent

manner by carbachol or in a cAMP-dependent way by forskolin, and is inhibited by NPPB.

No obvious effects of the diet or infection were observed on electrogenic Cl- secretion.

Previous studies in rats showed significant decreases in Cl- ion transport in the proximal

jejunum after dietary supplementation with cellulose or pectin (Schwartz et al., 1982). Our

data did not reproduce this observation in ceca of chicken, where NSP fermentation takes

place. Reduced cecal Cl- secretion of mono-infected birds fed soluble NSP instead of the

control diet suggests that soluble NSP in combination with nematode infection may reduce

intestinal fluid. Reduced Cl- secretion was also observed in H. g. mono-infected animals fed

with the insoluble NSP diet compared to non-inoculated birds. Experiments with pigs have

shown that insoluble NSP benefit nematode infection (Petkevicius et al., 1997; Petkevicius et

34

al., 2001). Reduction of intestinal fluid may be considered as a reaction of the host to a

parasitic developmental stage. Pigs infected with Oesophagostomum dentatum showed higher

Isc responses during the time of nematode penetration into the epithelium (high expulsion

chances) and lower Isc responses at the histiotrophic phase (low expulsion chances)

(Leonhard-Marek & Daugschies, 1997). It may also be suggested that nematodes may

modulate the activation of Cl- channels by their excretory/secretory products to prevent

expulsion.

The changes in electrogenic Cl- secretion showed a similar pattern for mono- and dually

infected birds despite the high grade of destruction of cecal tissue in the course of H. m. co-

infection. We suggest that the secretory response of the intestine was not significantly

affected by the co-infection at five weeks pi, when the intestinal epithelia had already largely

recovered from pathological lesions as shown histologically.

In our experiments we observed clinical signs and pathological changes only in H. g. and H.

m. co-infected chicken. The birds had completely recovered from clinical signs and most of

the macroscopical lesions at 5 weeks pi. This recovery is consistent with previous studies in

chicken (Hess et al., 2006; Powell et al., 2009). Mono-infection with H. g. induced only mild

to moderate local histological reaction in the cecal lamina propria. Bacterial colonies, which

were detected in the damaged mucosa of H. m. infected birds, were absent. The absence of

pathological lesions and detectable bacterial colonies in the cecal tissue may indicate the long-

term adaptation of H. g. in chicken. Possibly, the dimetridazol treatment may also have

affected the bacterial flora of the gut.

Furthermore, in our study H. m. has clearly influenced the establishment of H. g. infection in

the ceca. Previously it had been demonstrated that chicken with pathological evidence of

histomoniasis had lower numbers of H. g. worms compared to those mono-infected with H. g.

(Lund, 1958; Lund, 1967). The low infection rate and the absence of H. g. eggs observed in

our experiments suggest that the severe inflammation induced by H. m. may provide

unfavourable conditions for the establishment of H. g. and decelerate the development of the

nematode.

In summary, local T cell immune reactions in the cecal lamina propria with induction of the

Th2-type cytokine IL-13 dominate the H. g. immunopathogenesis in chicken. NSP did not

influence the gut-associated cellular immune response after H. g. infection. Co-infection of H.

35

g. and H. m. altered the local immune response and induced an increase in mRNA expression

of the Th1-type cytokine IFN-γ, and elicited systemic immune reactions in the spleen.

Furthermore, we did not observe obvious effects of the infection or different feed diets on the

chloride secretion in intestinal epithelia.

With this study we contributed to the understanding of immunological and electro-

physiological responses of the chicken intestine following nematode infection.

36

References

Almeria, S., Canals, A., Zarlenga, D. S. & Gasbarre, L. C. (1997). Isolation and phenotypic

characterization of abomasal mucosal lymphocytes in the course of a primary

Ostertagia ostertagi infection in calves. Veterinary Immunology and

Immunopathology, 57, 87-98.

Balic, A., Bowles, V. M. & Meeusen, E. N. (2002). Mechanisms of immunity to Haemonchus

contortus infection in sheep. Parasite Immunology, 24, 39-46.

Barrett, K. E. & Keely, S. J. (2000). Chloride secretion by the intestinal epithelium: molecular

basis and regulatory aspects. Annual Review of Physiology, 62, 535-572.

Berndt, A., Wilhelm, A., Jugert, C., Pieper, J., Sachse, K. & Methner, U. (2007). Chicken

cecum immune response to Salmonella enterica serovars of different levels of

invasiveness. Infection and Immunity, 75, 5993-6007.

Betts, J., deSchoolmeester, M. L. & Else, K. J. (2000). Trichuris muris: CD4+ T cell-

mediated protection in reconstituted SCID mice. Parasitology, 121(6), 631-637.

Bleich, E. M., Leonhard-Marek, S., Beyerbach, M. & Breves, G. (2007). Characterisation of

chloride currents across the proximal colon in CftrTgH(neoim)1Hgu congenic mice.

Journal of Comparative Physiology B, 177, 61-73.

Bozic, F., Marinculic, A. & Durakovic, E. (2000). Analysis of intestinal intraepithelial

lymphocyte populations in experimental Trichinella spiralis infection of mice. Folia

Parasitologica (Praha), 47, 55-59.

Brener, B., Tortelly, R., Menezes, R. C., Muniz-Pereira, L. C. & Pinto, R. M. (2006).

Prevalence and pathology of the nematode Heterakis gallinarum, the trematode

Paratanaisia bragai, and the protozoan Histomonas meleagridis in the turkey,

Meleagris gallopavo. Memorias do Instituto Oswaldo Cruz, 101, 677-681.

Chan, M. M., Chen, C. L., Ager, L. L. & Cooper, M. D. (1988). Identification of the avian

homologues of mammalian CD4 and CD8 antigens. Journal of Immunology, 140,

2133-2138.

Chen, C. L., Cihak, J., Losch, U. & Cooper, M. D. (1988). Differential expression of two T

cell receptors, TcR1 and TcR2, on chicken lymphocytes. European Journal of

Immunology, 18, 539-543.

37

Dawson, H. D., Beshah, E., Nishi, S., Solano-Aguilar, G., Morimoto, M., Zhao, A., Madden,

K. B., Ledbetter, T. K., Dubey, J. P., Shea-Donohue, T., Lunney, J. K. & Urban, J. F.,

Jr. (2005). Localized multigene expression patterns support an evolving Th1/Th2-like

paradigm in response to infections with Toxoplasma gondii and Ascaris suum.

Infection and Immunity, 73, 1116-1128.

De Jonge, H. R., Ballmann, M., Veeze, H., Bronsveld, I., Stanke, F., Tummler, B. &

Sinaasappel, M. (2004). Ex vivo CF diagnosis by intestinal current measurements

(ICM) in small aperture, circulating Ussing chambers. Journal of Cystic Fibrosis, 3

Suppl 2, 159-163.

Degen, W. G., Daal, N., Rothwell, L., Kaiser, P. & Schijns, V. E. (2005). Th1/Th2

polarization by viral and helminth infection in birds. Veterinary Microbiology, 105,

163-167.

Dondji, B., Bungiro, R. D., Harrison, L. M., Vermeire, J. J., Bifulco, C., McMahon-Pratt, D.

& Cappello, M. (2008). Role for nitric oxide in hookworm-associated immune

suppression. Infection and Immunity, 76, 2560-2567.

Dondji, B., Sun, T., Bungiro, R. D., Vermeire, J. J., Harrison, L. M., Bifulco, C. & Cappello,

M. (2010). CD4 T cells mediate mucosal and systemic immune responses to

experimental hookworm infection. Parasite Immunology, 32, 406-413.

Esquenet, C., De Herdt, P., De Bosschere, H., Ronsmans, S., Ducatelle, R. & Van Erum, J.

(2003). An outbreak of histomoniasis in free-range layer hens. Avian Pathology, 32,

305-308.

Gibbs, B. J. (1962). The occurrence of the protozoan parasite Histomonas meleagridis in the

adults and eggs of the cecal worm Heterakis gallinae. Journal of Protozoology, 9,

288-293.

Hess, M., Grabensteiner, E. & Liebhart, D. (2006). Rapid transmission of the protozoan

parasite Histomonas meleagridis in turkeys and specific pathogen free chickens

following cloacal infection with a mono-eukaryotic culture. Avian Pathology, 35, 280-

285.

Hong, Y. H., Lillehoj, H. S., Lillehoj, E. P. & Lee, S. H. (2006). Changes in immune-related

gene expression and intestinal lymphocyte subpopulations following Eimeria maxima

infection of chickens. Veterinary Immunology and Immunopathology, 114, 259-272.

38

Hu, J., Fuller, L., Armstrong, P. L. & McDougald, L. R. (2006). Histomonas meleagridis in

chickens: attempted transmission in the absence of vectors. Avian Diseases, 50, 277-

279.

Janardhana, V., Broadway, M. M., Bruce, M. P., Lowenthal, J. W., Geier, M. S., Hughes, R.

J. & Bean, A. G. (2009). Prebiotics modulate immune responses in the gut-associated

lymphoid tissue of chickens. Journal of Nutrition, 139, 1404-1409.

Kaiser, P. (2007). The avian immune genome--a glass half-full or half-empty? Cytogenetic

and Genome Research, 117, 221-230.

Kaushik, R. K. & Deorani, V. P. (1969). Studies on tissue responses in primary and

subsequent infections with Heterakis galinae in chickens and on the process of

formation of caecal nodules. Journal of Helminthology, 43, 69-78.

Kelly-Quagliana, N., Buddington (2003). Dietary oligofructose and inulin modulate immune

functions in mice. Nutrition research, 23, 257-267.

Khan, W. I. & Collins, S. M. (2004). Immune-mediated alteration in gut physiology and its

role in host defence in nematode infection. Parasite Immunology, 26, 319-326.

Kurt, M. & Acici, M. (2008). Cross-sectional survey on helminth infections of chickens in the

Samsun region, Turkey. Deutsche Tierarztliche Wochenschrift, 115, 239-242.

Leonhard-Marek, S. & Daugschies, A. (1997). Electro- and transportphysiological changes in

pig proximal colon during parasitic infection. Comparative Biochemistry and

Physiology, Part A Physiology, 118, 345-347.

Leonhard-Marek, S., Hempe, J., Schroeder, B. & Breves, G. (2009). Electrophysiological

characterization of chloride secretion across the jejunum and colon of pigs as affected

by age and weaning. Journal of Comparative Physiology B, 179, 883-896.

Lillehoj, H. S. (1998). Role of T lymphocytes and cytokines in coccidiosis. International

Journal for Parasitology, 28, 1071-1081.

Lim, B. O., Yamada, K., Nonaka, M., Kuramoto, Y., Hung, P. & Sugano, M. (1997). Dietary

fibers modulate indices of intestinal immune function in rats. Journal of Nutrition,

127, 663-667.

Liman, M. & Rautenschlein, S. (2007). Induction of local and systemic immune reactions

following infection of turkeys with avian Metapneumovirus (aMPV) subtypes A and

B. Veterinary Immunology and Immunopathology, 115, 273-285.

39

Little, M. C., Bell, L. V., Cliffe, L. J. & Else, K. J. (2005). The characterization of

intraepithelial lymphocytes, lamina propria leukocytes, and isolated lymphoid follicles

in the large intestine of mice infected with the intestinal nematode parasite Trichuris

muris. Journal of Immunology, 175, 6713-6722.

Lund, E. E. (1958). Growth and development of Heterakis gallinae in turkeys and chickens

infected with Histomonas meleagridis. Journal of Parasitology, 44, 297-301.

Lund, E. E. (1967). Response of four breeds of chickens and one breed of turkeys to

experimental Heterakis and Histomonas infections. Avian Diseases, 11, 491-502.

Lund, E. E., Chute, A. M. & Myers, S. L. (1970). Performance in chickens and turkeys of

chicken-adapted Heterakis gallinarum. Journal of Helminthology, 44, 97-106.

Lund, E. E. & Chute, A. M. (1974). The reproductive potential of Heterakis gallinarum in

various species of galliform birds: implications for survival of H. gallinarum and

Histomonas meleagridis to recent times. International Journal forParasitology, 4,

455-461.

Madden, K. B., Yeung, K. A., Zhao, A., Gause, W. C., Finkelman, F. D., Katona, I. M.,

Urban, J. F., Jr. & Shea-Donohue, T. (2004). Enteric nematodes induce stereotypic

STAT6-dependent alterations in intestinal epithelial cell function. Journal of

Immunology, 172, 5616-5621.

Marcos-Atxutegi, C., Gandolfi, B., Aranguena, T., Sepulveda, R., Arevalo, M. & Simon, F.

(2009). Antibody and inflammatory responses in laying hens with experimental

primary infections of Ascaridia galli. Veterinary Parasitology, 161, 69-75.

Maurer, V., Amsler, Z., Perler, E. & Heckendorn, F. (2009). Poultry litter as a source of

gastrointestinal helminth infections. Veterinary Parasitology, 161, 255-260.

McDougald, L. R. (2005). Blackhead disease (histomoniasis) in poultry: a critical review.

Avian Diseases, 49, 462-476.

Mountzouris, K. C., Balaskas, C., Xanthakos, I., Tzivinikou, A. & Fegeros, K. (2009). Effects

of a multi-species probiotic on biomarkers of competitive exclusion efficacy in

broilers challenged with Salmonella enteritidis. British Poultry Science, 50, 467-478.

Mungube, E. O., Bauni, S. M., Tenhagen, B. A., Wamae, L. W., Nzioka, S. M., Muhammed,

L. & Nginyi, J. M. (2008). Prevalence of parasites of the local scavenging chickens in

40

a selected semi-arid zone of Eastern Kenya. Tropical Animal Health and Production,

40, 101-109.

Owens, B., Tucker, L., Collins, M. A. & McCracken, K. J. (2008). Effects of different feed

additives alone or in combination on broiler performance, gut microflora and ileal

histology. British Poultry Science, 49, 202-212.

Patel, N., Kreider, T., Urban, J. F., Jr. & Gause, W. C. (2009). Characterisation of effector

mechanisms at the host:parasite interface during the immune response to tissue-

dwelling intestinal nematode parasites. International Journal for Parasitology, 39, 13-

21.

Pearce, G. P. (1999). Interactions between dietary fibre, endo-parasites and Lawsonia

intracellularis bacteria in grower-finisher pigs. Veterinary Parasitology, 87, 51-61.

Perez, J., Zafra, R., Buffoni, L., Hernandez, S., Camara, S. & Martinez-Moreno, A. (2008).

Cellular phenotypes in the abomasal mucosa and abomasal lymph nodes of goats

infected with Haemonchus contortus. Journal of Comparative Pathology, 138, 102-

107.

Permin, A., Bisgaard, M., Frandsen, F., Pearman, M., Kold, J. & Nansen, P. (1999).

Prevalence of gastrointestinal helminths in different poultry production systems.

British Poultry Science, 40, 439-443.

Petkevicius, S., Knudsen, K. E., Nansen, P., Roepstorff, A., Skjoth, F. & Jensen, K. (1997).

The impact of diets varying in carbohydrates resistant to endogenous enzymes and

lignin on populations of Ascaris suum and Oesophagostomum dentatum in pigs.

Parasitology, 114 (6), 555-568.

Petkevicius, S., Knudsen, K. E., Nansen, P. & Murrell, K. D. (2001). The effect of dietary

carbohydrates with different digestibility on the populations of Oesophagostomum

dentatum in the intestinal tract of pigs. Parasitology, 123, 315-324.

Petkevicius, S., Bach Knudsen, K. E., Murrell, K. D. & Wachmann, H. (2003). The effect of

inulin and sugar beet fibre on oesophagostomum dentatum infection in pigs.

Parasitology, 127, 61-68.

Petkevicius, S., Thomsen, L. E., Bach Knudsen, K. E., Murrell, K. D., Roepstorff, A. & Boes,

J. (2007). The effect of inulin on new and on patent infections of Trichuris suis in

growing pigs. Parasitology, 134, 121-127.

41

Powell, F. L., Rothwell, L., Clarkson, M. J. & Kaiser, P. (2009). The turkey, compared to the

chicken, fails to mount an effective early immune response to Histomonas meleagridis

in the gut. Parasite Immunology, 31, 312-327.

Rausch, S., Huehn, J., Kirchhoff, D., Rzepecka, J., Schnoeller, C., Pillai, S., Loddenkemper,

C., Scheffold, A., Hamann, A., Lucius, R. & Hartmann, S. (2008). Functional analysis

of effector and regulatory T cells in a parasitic nematode infection. Infection and

Immunity, 76, 1908-1919.

Rautenschlein, S., von Samson-Himmelstjerna, G. & Haase, C. (2007). A comparison of

immune responses to infection with virulent infectious bursal disease virus (IBDV)

between specific-pathogen-free chickens infected at 12 and 28 days of age. Veterinary

Immunology and Immunopathology, 115, 251-260.

Riddell, C. & Gajadhar, A. (1988). Cecal and hepatic granulomas in chickens associated with

Heterakis gallinarum infection. Avian Diseases, 32, 836-838.

Roller, M., Rechkemmer, G. & Watzl, B. (2004). Prebiotic inulin enriched with oligofructose

in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium

lactis modulates intestinal immune functions in rats. Journal of Nutrition, 134, 153-

156.

Ryz, N. R., Meddings, J. B. & Taylor, C. G. (2008). Long-chain inulin increases dendritic

cells in the Peyer's patches and increases ex vivo cytokine secretion in the spleen and

mesenteric lymph nodes of growing female rats, independent of zinc status. British

Journal of Nutrition, 1-11.

Scales, H. E., Ierna, M. X. & Lawrence, C. E. (2007). The role of IL-4, IL-13 and IL-4Ralpha

in the development of protective and pathological responses to Trichinella spiralis.

Parasite Immunology, 29, 81-91.

Schwartz, S. E., Levine, G. D. & Starr, C. M. (1982). Effects of dietary fiber on intestinal ion

fluxes in rats. The American Journal of Clinical Nutrition, 36, 1102-1105.

Seifert, S. & Watzl, B. (2007). Inulin and oligofructose: review of experimental data on

immune modulation. Journal of Nutrition, 137, 2563S-2567S.

Solis de los Santos, F., Donoghue, A. M., Venkitanarayanan, K., Metcalf, J. H., Reyes-

Herrera, I., Dirain, M. L., Aguiar, V. F., Blore, P. J. & Donoghue, D. J. (2009). The

42

natural feed additive caprylic acid decreases Campylobacter jejuni colonization in

market-aged broiler chickens. Poultry Science, 88, 61-64.

Springer, W. T., Johnson, J. & Reid, W. M. (1969). Transmission of histomoniasis with male

heterakis gallinarum (nematoda). Parasitology, 59, 401-405.

Taylor, M. D., van der Werf, N., Harris, A., Graham, A. L., Bain, O., Allen, J. E. & Maizels,

R. M. (2009). Early recruitment of natural CD4+ Foxp3+ Treg cells by infective

larvae determines the outcome of filarial infection. European Journal of Immunology,

39, 192-206.

Trushina, E. N., Martynova, E. A., Nikitiuk, D. B., Mustafina, O. K. & Baigarin, E. K.

(2005). [The influence of dietary inulin and oligofructose on the cell-mediated and

humoral immunity in rats]. Voprosy Pitaniia, 74, 22-27.

Vervelde, L., Vermeulen, A. N. & Jeurissen, S. H. (1996). In situ characterization of

leucocyte subpopulations after infection with Eimeria tenella in chickens. Parasite

Immunology, 18, 247-256.

Watzl, B., Girrbach, S. & Roller, M. (2005). Inulin, oligofructose and immunomodulation.

British Journal of Nutrition, 93 Suppl 1, S49-55.

Yun, C. H., Lillehoj, H. S. & Choi, K. D. (2000). Eimeria tenella infection induces local

gamma interferon production and intestinal lymphocyte subpopulation changes.

Infection and Immunity, 68, 1282-1288.

43

Table 1. Ingredients and nutrient contents of experimental diets.

Item Diet1 C I S Component, % Barley 29.0 26.4 26.4 Wheat 54.0 49.1 49.1 Fishmeal 8.0 7.3 7.3 Casein 4.5 4.1 4.1 Soybean oil 2.0 1.8 1.8 Premix 77046 1.0 0.9 0.9 MCP2 0.9 0.8 0.8 CaCO3 0.6 0.5 0.5 Pea bran meal - 9.1 - Chicory root meal - - 9.1 Analyzed DM, g/kg 898 900 898 Nutrients3, g/kg DM Crude ash 56 53 56 Crude protein 219 203 204 Crude lipids 40 35 37 Sugar 33 30 69 Starch 485 439 415 ADF 32 88 38 NDF 119 173 123 Insoluble NSP 102 173 104 Sol- NSP,g/kg 19 22 24 Inulin,g/kg - - 71 ME4, MJ/kg DM 13.29 12.06 12.25

1C = control diet, I = diet with insoluble NSP, S = diet with soluble NSP 2MCP = mono-calcium phosphate, 3DM = dry matter, ADF = acid detergent fiber, NDF =

neutral detergent fiber, NSP = non-starch polysaccharides 4ME = metabolizable energy

44

Table 2. Real-time quantitative RT-PCR primers and probes.

RNA

taget Probe/primer sequence (5´-3´)

Accession

No.a

28S P HEX-AGGACCGCTACGGACCTCCACCA-BHQ2 X59733

F GGCGAAGCCAGAGGAAACT

R GACGACCGATTTGCACGTC

IL-4 P FAM-AGCAGCACCTCCCTCAAGGCACC-3'TAMRA AJ621735

F AACATGCGTCAGCTCCTGAAT

R TCTGCTAGGAACTTCTCCATTGAA

FC TGCCGCTGATGGAGAGCATCC

RC GTGGAAGAAGGTACGTAGGTCTGCT

IL-13 P FAM-ACACCAGAGTGGCACAAGTGGCTTTCAA-BHQ1 AJ621735

F CAGAGGAGTGCAGGTCCCTTG

R CAGTTCGTCATGCCGTGCAG

FC ACTGAAGGCTGCCCTTGCTC

RC GGTGTAGTTCCCCAGTGCCG

IFN-γ P FAM-AAGCTCCCGATGAACGACTTGA-TAMRA Y07922

F GTGAAAGATATCATGGACCTGG

R TTCTGTAAGATGCTGAAGAGTTC

P: probe, F: forward primer, R: reverse primer, FC: cloning forward primer, RC: cloning

reverse primer. a refers to genomic DNA sequence

45

Table 3. Infection rate and development of macroscopical and microscopical lesions in

chicken inoculated with 200 embryonated eggs of Heterakis gallinarum (H. g.).

% birds per group with cecal lesions at weeks pi

Macroscopical

Microscopical

(severity grade)

Experiment/

/ treatment

with

dimetridazol

H. g.

infection

rate in %

% feces

samples

with H. g.

eggs 2 3 5 2 3 5

1 /no 13 0 93

93

7

93

(4.0)

100

(3.5)

93

(3.0)

2 / yes 93 33 0 0 0 93

(2.0)

86

(1.0)

80

(1.0)

n=14-15 birds per inoculated group. The macroscopically observed lesions were thickening of

cecal wall, hemorrhagic exudate and cheesy core in cecal lumen. Severity grades of

microscopical lesions: 4.0- severe hyperplasy of tunica muscularis, numerous histomonads,

massive lymphocyte, heterophil and macrophage infiltration, coagulation necrosis; 3.0- severe

lymphocyte infiltration in the lamina propria and lymphoid centers in cecal tissue; 2.0-

moderate lymphocyte infiltration in the lamina propria and formation of lymphoid

aggregations; 1.0- mild lymphocyte infiltration in the lamina propria. Non-inoculated birds

were negative for lesions, worms and eggs.

46

Figure 1. 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.

47

Figure 2. 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.

48

***

*

*

0123

4567

2 3 5 2 5

weeks post infection

grou

p av

erag

e ab

unda

nce

scor

e of

CD4

+ ce

lls

non-inoculated inoculated

Exp. 1 Exp. 2

A

**

*

*

*

0

12

34

56

7

2 3 5 2 5

weeks post infection

grou

p av

erag

e ab

unda

nce

scor

e of

CD8

α+ c

ells

non-inoculated inoculated

Exp. 1 Exp. 2

B

**

*

*

01

23

45

67

2 3 5 2 5

weeks post infection

grou

p av

erag

e ab

unda

nce

scor

e of

TCR

αβ (V

β1)+

cel

ls

non-inoculated inoculated

Exp. 1 Exp. 2

C

***

*

*

0

12

34

56

7

2 3 5 2 5

weeks post infection

grou

p av

erag

e ab

unda

nce

scor

e of

TCR

γδ+

cells

non-inoculated inoculated

Exp. 1 Exp. 2D

Figure 3. 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)

49

*

0

10

20

30

40

2 3 5 2 3 5

weeks post infection

% o

f CD

4+ c

ells

in

sple

en ly

mph

ocyt

es

non-inoculated inoculated

Exp. 1 Exp. 2

Figure 4. 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)

50

-15

0

15

30

2 3 5 2 3 5

weeks post infection

x-fo

ld c

hang

e in

IFN-γ

mRN

A ex

pres

sion

leve

ls

*

Exp. 1 Exp. 2

A

-15

0

15

2 3 5 2 3 5

weeks post infection

x-fo

ld c

hang

e in

IL-4

mR

NA

ex

pres

sion

leve

ls

Exp. 1 Exp. 2

B

-15

0

15

2 3 5 2 3 5

weeks post infection

x-fo

ld c

hang

e in

IL-1

3 m

RN

A

expr

essi

on le

vels

*

Exp. 1 Exp. 2

C

Figure 5. 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)

51

*

-0.5

0.0

0.5

1.0

1.5

2.0

2.5

C I S C I S

feed diets

∆ Is

c (µ

Eq*h

-1*c

m2 )

non-inoculated inoculated

Exp. 1 Exp. 2

A

*

0.0

0.5

1.0

1.5

2.0

2.5

C I S C I S

feed diets

∆ Is

c (µ

Eq*h

-1*c

m2 )

non-inoculated inoculated

Exp. 1 Exp. 2

**

B

*

-2.5

-2.0

-1.5

-1.0

-0.5

0.0C I S C I S

feed diets

∆ Is

c (µ

Eq*h

-1*c

m2 )

non-inoculated inoculated

Exp. 1 Exp. 2

**

*

C

52

Figure 6. 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)

53

5. Immunopathogenesis of Ascaridia galli infection in layer chicken

This paper has been published in Developmental and Comparative Immunology. 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

Abstract

Gastro-intestinal nematode infections in mammals are associated with local T lymphocyte

infiltrations, Th2 cytokine induction, and alterations in epithelial cell secretion and

absorption. This study demonstrates that Ascaridia (A.) galli infection in chicken also elicits

local gut-associated immune reactions and changes in the intestinal electrogenic nutrient

transport. In A. galli-infected birds we observed infiltrations of different T cell populations in

the intestinal lamina propria and accumulation of CD4+ lymphocytes in the epithelium. The

Th2 cytokines IL-4 and IL-13 dominated the intestinal immune reactions following A. galli

infection. A. galli-specific systemic IgY antibodies were detected after two weeks post

infection, and did only poorly correlate with detected worm numbers. Electrogenic transport

of alanin and glucose was impaired in A. galli-infected chicken. Our data provide

circumstantial evidence that local immune responses and electro-physiological intestinal

functions may be connected and contribute to the elimination of worm infection.

54

6. General discussion and conclusions

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.

55

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.

56

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 IL-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.

57

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.

58

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.

59

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.

60

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.

61

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

62

(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.

63

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

homologue for mammalian IgE has been described till now. The role of IL-5 in the immune

reactions of birds remains unclear (DAVISON 2008). These components are an important

part of the mammalian immune response following intestinal nematode infections (RAUSCH

et al. 2008; CHIUSO-MINICUCCI et al. 2010). Can it be suggested that the local T cell

infiltrations and the induction of Th2 cytokines IL-4 and IL-13 play a more important role in

the control of avian intestinal nematode infections than in mammalian species? It is not clear,

whether the Th2 immune response and upregulation of IL-13 mRNA expression may protect

avian species against nematode infections. In our experiments the induction of high IL-13 and

IL-4 mRNA levels positively correlated with a higher infection rate and a high average

parasite numbers per bird in A. gall-infected chicken. Also the role of the humoral immune

response and induction of nematode specific serum IgG should be elucidated further. Our

studies did not address the role of innate immune reactions in the control of avian gastro-

intestinal nematode infections, which is also an important component of the immune defense.

Further investigations are required to understand complex interactions between the protective

host immune reactions in avian species and parasite-mediated modulation of the immune

response to ensure its survival and replication.

64

In the future, investigations of resistant and susceptible chicken lines may give an insight in

the protective features of immunological and electro-physiological responses in avian

nematode infections. Re-infection models may provide information on the antigen memory

response in nematode infections in the avian species, and on the influence of re-infection on

the electro-physiological intestinal functions (WANG et al. 1991; O'MALLEY et al. 1993;

BEHNKE et al. 2003; KANOBANA et al. 2003).

65

7. Summary

Anna Schwarz

The influence of non-starch-polysaccharides on experimental infections with Ascaridia

galli and Heterakis gallinarum in layer chicken (Gallus gallus domesticus)

Recent changes in legal requirements for layer-housing in European countries have led to the

substitution of traditional cages with free-range systems and floor husbandry. In these systems

the risk and the prevalence of gastro-intestinal parasitic infections are very high because of

the close contact of the animals to their feces. Ascaridia galli (A. galli) and Heterakis

gallinarum (H. gallinarum) are common poultry nematodes with worldwide distribution,

which may contribute to substantial economic losses in alternative production systems.

Previous studies in mammals demonstrated that local immune reactions and alterations in the

intestinal physiology play an important role in the control of parasitic intestinal infections. It

was shown that feed components such as non-starch polysaccharides (NSP) may modulate

systemic and local gut-associated immune functions. No information is available on the

influence of NSP on the local immune reactions and the course of nematode infections in

chicken.

The goal 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 to characterize the influence of NSP on these parameters. Under

different dietary conditions we investigated local and systemic T cell populations, induction

of local Th1 and Th2 cytokines, the humoral immune response as well as electrophysiological

epithelial functions in the intestine. In addition, we described the influence of H. meleagridis

on the pathology of H. gallinarum infection.

As in mammalian species, mild to moderate T cell infiltrations in the intestinal lamina propria

and induction of the Th2 cytokines IL-4 and IL-13 dominated the local immune reactions

following both nematode mono-infections. In contrast to highly invasive intestinal nematodes,

66

both parasites did not induce any systemic effects on spleen lymphocyte populations. This

observation suggests that the immune response following A. galli and H. gallinarum infection

is primary localized in the gut. A parasite specific systemic IgG antibody response was

observed in A. galli-infected birds. Co-infection with H. gallinarum and H. meleagridis

induced severe destruction of the cecal mucosa in association with strong T cell infiltrations, a

shift from the Th2- to the Th1-type cytokine response, and elicited systemic immune reactions

in the spleen.

As it was previously shown in nematode infections in mammals, electrogenic absorption of

alanin and glucose was impaired in A. galli-infected chicken. The alterations in the intestinal

physiology were only observed in experiment with an increase in IL-4 and IL-13 cytokine

mRNA expression in the intestinal mucosa. This may indicate that an interaction between the

local immunological reactions and electro-physiological intestinal functions may also exist in

avian species.

In contrast to the studies in mammals, NSP did not significantly influence the gut-associated

immune parameters and electro-physiological functions of the intestine in nematode infected

as well as in non-infected chicken.

The present study reveals that both A. galli and H. gallinarum infections in layer chicken

elicit local gut-associated immune reactions and changes in the intestinal electro-

physiological functions which may be comparable to mammalian species.

67

8. Zusammenfassung

Anna Schwarz

Einflüsse von Nicht-Stärke-Polysacchariden (NSP) des Futters auf die Auswirkungen

von experimentellen Ascaridia galli und Heterakis gallinarum Infektionen beim

Haushuhn (Gallus gallus domesticus)

Mit den Änderungen in der EU Gesetzgebung werden alternative Haltungssysteme für

Legehennen wie Freilandhaltung und Bodenhaltungs-Systeme wieder vermehrt eingesetzt. In

diesen Systemen gewinnen Endoparasiten wie Ascaridia galli und Heterakis gallinarum an

Bedeutung. Studien an Säugetieren haben gezeigt, dass lokale Immunreaktionen und die

Anpassung verdauungsphysiologischer Funktionen des Gastrointestinaltrakts eine große Rolle

bei der Abwehr von Nematoden-Infektionen spielen. Futterkomponenten wie Nicht-Stärke-

Polysaccharide (NSP) können die systemischen und lokalen Immunfunktionen sowie die

Darmphysiologie bei Säugern beeinflussen. Diese Aspekte zur Darmimmunität bei

Wurminfektionen unter dem Einfluss von bestimmten Futterkomponenten wurden beim

Geflügel bisher noch nicht untersucht.

Das Ziel der Studie war es, die Wirkungen von NSP auf die Verdauungsphysiologie und

Immunologie in Verbindung mit experimentellen Helminthen-Infektionen beim Haushuhn zu

charakterisieren. Es wurden zellvermittelte Immunparameter, wie lokale und systemische T-

Zellpopulationen und die Induktion von Th1 und Th2-Zytokinen, sowie die elektro- und

transportphysiologischen Eigenschaften des Darmepithels nach experimentellen Ascaridia

(A.) galli und Heterakis (H.) gallinarum Infektionen untersucht. Weiterhin wurde der Einfluss

von Histomonas meleagridis auf die Immunpathogenese von Heterakis gallinarum erfasst.

Es konnte gezeigt werden, dass die beiden Nematoden-Infektionen, ähnlich wie in den

Säugetier-Modellen, milde lokale zellvermittelte Immunreaktionen auslösen. Es kam zu

moderaten T-Zell Infiltrationen in die interstinale Lamina propria und zur Induktion der Th2

Zytokine IL-4 und IL-13. Im Unterschied zu stärker invasiven intestinalen Nematoden hatten

68

Infektionen mit A. galli und H. gallinarum keine systemischen Veränderungen in den

Lymphozytenpopulationen der Milz induziert. Dies weist darauf hin, dass die Immunantwort

bei beiden Nematoden-Infektionen hauptsächlich im Darm lokalisiert ist. Nach A. galli

Infektionen wurde die Bildung von spezifischen Antikörpern im Serum detektiert, deren

Schutzwirkung noch in weiteren Studien geklärt werden muss. Die Koinfektion mit

Histomonas meleagridis dominierte die lokale Immunantwort bei H. gallinarum-infizierten

Tieren. Sie führte zu massiven Zellinfiltrationen in die interstinale Lamina Propria, einer

Verschiebung zu Th1-vermittelten lokalen Immunreaktionen, sowie einer systemischen

Immunantwort in der Milz.

Wie in Säuger-Modellen, hat die A. galli Infektion die transportphysiologischen

Eigenschaften des Darmepithels beeinflusst. Es kann spekuliert werden, dass auch bei Vögeln

ein Zusammenhang zwischen Induktion von Th2 Zytokinen und vermindertem elektrogenen

Nährstofftransport besteht.

Im Gegensatz zu den Studien bei Säugern konnte kein Einfluss von NSP auf die untersuchten

Immunparameter und den elektrogenen Nährstofftransport an den ausgewählten Zeitpunkten

nach der Infektion festgestellt werden.

Zusammenfassend zeigte diese Studie, dass lokale Immunreaktionen und elektro- und

transportphysiologische Eigenschaften des Darmepithels nach Nematoden-Infektionen bei

Hühnern in ähnlicher Weise wie bei Säugern beeinflusst werden.

69

9. References ABDELQADER, A., M. GAULY u. C. B. WOLLNY (2007):

Response of two breeds of chickens to Ascaridia galli infections from two geographic

sources.

Veterinary Parasitology 145, 176-180

ARTIS, D. (2006):

New weapons in the war on worms: identification of putative mechanisms of immune-

mediated expulsion of gastrointestinal nematodes.

International Journal for Parasitology 36, 723-733

AU YEUNG, K. J., A. SMITH, A. ZHAO, K. B. MADDEN, J. ELFREY, C. SULLIVAN, O.

LEVANDER, J. F. URBAN u. T. SHEA-DONOHUE (2005):

Impact of vitamin E or selenium deficiency on nematode-induced alterations in murine

intestinal function.

Experimental Parasitology 109, 201-208

AWAD, W., K. GHAREEB u. J. BOEHM (2008):

Intestinal structure and function of broiler chickens on diets supplemented with a synbiotic

containing Enterococcus faecium and oligosaccharides.

International Journal of Molecular Sciences 9, 2205-2216

AWAD, W. A., K. GHAREEB u. J. BOHM (2010):

Evaluation of the chicory inulin efficacy on ameliorating the intestinal morphology and

modulating the intestinal electrophysiological properties in broiler chickens.

Journal of Animal Physiology and Animal Nutrition

BAKKER, G. C., R. A. DEKKER, R. JONGBLOED u. A. W. JONGBLOED (1998):

Non-starch polysaccharides in pig feeding.

The Veterinary Quarterly 20 Suppl 3, S59-64

70

BANCROFT, A. J., A. N. MCKENZIE u. R. K. GRENCIS (1998):

A critical role for IL-13 in resistance to intestinal nematode infection.

Journal of Immunology 160, 3453-3461

BAR-SHIRA, E., D. SKLAN u. A. FRIEDMAN (2003):

Establishment of immune competence in the avian GALT during the immediate post-hatch

period.

Developmental and Comparative Immunology 27, 147-157

BARRETT, K. E. u. S. J. KEELY (2000):

Chloride secretion by the intestinal epithelium: molecular basis and regulatory aspects.

Annual Review of Physiology 62, 535-572

BEDFORD, M. R. u. H. L. CLASSEN (1992):

Reduction of intestinal viscosity through manipulation of dietary rye and pentosanase

concentration is effected through changes in the carbohydrate composition of the intestinal

aqueous phase and results in improved growth rate and food conversion efficiency of broiler

chicks.

The Journal of Nutrition 122, 560-569

BEFUS, A. D., N. JOHNSTON, G. A. LESLIE u. J. BIENENSTOCK (1980):

Gut-associated lymphoid tissue in the chicken. I. Morphology, ontogeny, and some functional

characteristics of Peyer's patches.

Journal of Immunology 125, 2626-2632

BEHNKE, J. M., A. LOWE, S. CLIFFORD u. D. WAKELIN (2003):

Cellular and serological responses in resistant and susceptible mice exposed to repeated

infection with Heligmosomoides polygyrus bakeri.

Parasite Immunology 25, 333-340

BEN-SMITH, A., D. A. LAMMAS u. J. M. BEHNKE (2003):

71

The relative involvement of Th1 and Th2 associated immune responses in the expulsion of a

primary infection of Heligmosomoides polygyrus in mice of differing response phenotype.

Journal of Helminthology 77, 133-146

BESOLUK, K., E. EKEN, M. BOYDAK u. S. TIPIRDAMAZ (2002):

Morphological studies on Meckel's diverticulum in geese (Anser anser domesticus).

Anatomia, Histologia, Embryologia 31, 290-292

BLEAY, C., C. P. WILKES, S. PATERSON u. M. E. VINEY (2007):

Density-dependent immune responses against the gastrointestinal nematode Strongyloides

ratti.

International Journal for Parasitology 37, 1501-1509

BLEICH, E. M., S. LEONHARD-MAREK, M. BEYERBACH u. G. BREVES (2007):

Characterisation of chloride currents across the proximal colon in CftrTgH(neoim)1Hgu

congenic mice.

Journal of Comparative Physiology 177, 61-73

BLEYEN, N., K. DE GUSSEM, J. DE GUSSEM u. B. M. GODDEERIS (2007):

Specific detection of Histomonas meleagridis in turkeys by a PCR assay with an internal

amplification control.

Veterinary Parasitology 143, 206-213

BLEYEN, N., E. ONS, M. DE GUSSEM u. B. M. GODDEERIS (2009):

Passive immunization against Histomonas meleagridis does not protect turkeys from an

experimental infection.

Avian Pathology 38, 71-76

BODERA, P. (2008):

Influence of prebiotics on the human immune system (GALT).

Recent Patents on Inflammation & Allergy Drug Discovery 2, 149-153

72

BOKKERS, E. A. u. I. J. DE BOER (2009):

Economic, ecological, and social performance of conventional and organic broiler production

in the Netherlands.

British Poultry Science 50, 546-557

BRENER, B., R. TORTELLY, R. C. MENEZES, L. C. MUNIZ-PEREIRA u. R. M. PINTO

(2006):

Prevalence and pathology of the nematode Heterakis gallinarum, the trematode Paratanaisia

bragai, and the protozoan Histomonas meleagridis in the turkey, Meleagris gallopavo.

Memorias do Instituto Oswaldo Cruz 101, 677-681

BRONSVELD, I., F. MEKUS, J. BIJMAN, M. BALLMANN, J. GREIPEL, J.

HUNDRIESER, D. J. HALLEY, U. LAABS, R. BUSCHE, H. R. DE JONGE, B.

TUMMLER u. H. J. VEEZE (2000):

Residual chloride secretion in intestinal tissue of deltaF508 homozygous twins and siblings

with cystic fibrosis. The European CF Twin and Sibling Study Consortium.

Gastroenterology 119, 32-40

BUCY, R. P., C. L. CHEN, J. CIHAK, U. LOSCH u. M. D. COOPER (1988):

Avian T cells expressing gamma delta receptors localize in the splenic sinusoids and the

intestinal epithelium.

Journal of Immunology 141, 2200-2205

BUMSTEAD, J. M., N. BUMSTEAD, L. ROTHWELL u. F. M. TOMLEY (1995):

Comparison of immune responses in inbred lines of chickens to Eimeria maxima and Eimeria

tenella.

Parasitology 111 ( Pt 2), 143-151

BURNS, R. B. (1982):

Histology and immunology of Peyer's patches in the domestic fowl (Gallus domesticus).

73

Research in Veterinary Science 32, 359-367

CAMACHO-ESCOBAR, M. A., J. ARROYO-LEDEZMA u. L. RAMIREZ-CANCINO

(2008):

Diseases of backyard turkeys in the Mexican tropics.

Annals of the New York Academy of Sciences 1149, 368-370

CASTELEYN, C., M. DOOM, E. LAMBRECHTS, W. VAN DEN BROECK, P. SIMOENS

u. P. CORNILLIE (2010):

Locations of gut-associated lymphoid tissue in the 3-month-old chicken: a review.

Avian Pathology 39, 143-150

CHADFIELD, M., A. PERMIN, P. NANSEN u. M. BISGAARD (2001):

Investigation of the parasitic nematode Ascaridia galli (Shrank 1788) as a potential vector for

Salmonella enterica dissemination in poultry.

Parasitology Research 87, 317-325

CHIUSO-MINICUCCI, F., N. M. MARRA, S. F. ZORZELLA-PEZAVENTO, T. G.

FRANCA, L. L. ISHIKAWA, M. R. AMARANTE, A. F. AMARANTE u. A. SARTORI

(2010):

Recovery from Strongyloides venezuelensis infection in Lewis rats is associated with a strong

Th2 response.

Parasite Immunology 32, 74-78

CHOCT, M. u. G. ANNISON (1992):

Anti-nutritive effect of wheat pentosans in broiler chickens: roles of viscosity and gut

microflora.

British Poultry Science 33, 821-834

CHOCT, M., R. J. HUGHES, J. WANG, M. R. BEDFORD, A. J. MORGAN u. G.

ANNISON (1996):

74

Increased small intestinal fermentation is partly responsible for the anti-nutritive activity of

non-starch polysaccharides in chickens.

British Poultry Science 37, 609-621

CLAEREBOUT, E. u. J. VERCRUYSSE (2000):

The immune response and the evaluation of acquired immunity against gastrointestinal

nematodes in cattle: a review.

Parasitology 120 Suppl, S25-42

CLARKSON, M. J. (1963):

Immunological responses to Histomonas meleagridis in the turkey and fowl.

Immunology 6, 156-168

CLIFFE, L. J. u. R. K. GRENCIS (2004):

The Trichuris muris system: a paradigm of resistance and susceptibility to intestinal nematode

infection.

Advances in Parasitology 57, 255-307

COOPER, M. D., C. L. CHEN, R. P. BUCY u. C. B. THOMPSON (1991):

Avian T cell ontogeny.

Advances in Immunology 50, 87-117

CUMMINGS, J. H. u. G. T. MACFARLANE (1997):

Role of intestinal bacteria in nutrient metabolism.

Journal of Parenteral and Enteral Nutrition 21, 357-365

CUMMINGS, J. H. u. A. M. STEPHEN (2007):

Carbohydrate terminology and classification.

European Journal of Clinical Nutrition 61 Suppl 1, S5-18

75

DAHL, C., A. PERMIN, J. P. CHRISTENSEN, M. BISGAARD, A. P. MUHAIRWA, K. M.

PETERSEN, J. S. POULSEN u. A. L. JENSEN (2002):

The effect of concurrent infections with Pasteurella multocida and Ascaridia galli on free

range chickens.

Veterinary Microbiology 86, 313-324

DÄNICKE, S., E. MOORS, A. BEINEKE u. M. GAULY (2009):

Ascaridia galli infection of pullets and intestinal viscosity: consequences for nutrient

retention and gut morphology.

British Poultry Science 50, 512-520

DAŞ, G., F. KAUFMANN, H. ABEL u. M. GAULY (2010):

Effect of extra dietary lysine in Ascaridia galli-infected grower layers.

Veterinary Parasitology 170, 238-243

DAŞ, G., H. ABEL, S. RAUTENSCHLEIN, J. HUMBURG, A. SCHWARZ, G. BREVES u.

M. GAULY (2011a):

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., H. ABEL, S. RAUTENSCHLEIN, J. HUMBURG, A. SCHWARZ, G. BREVES u.

M. GAULY (2011b):

The effects of dietary non-starch polysaccharides on Ascaridia galli infection in grower

layers.

Parasitology (submitted: PAR-2011-0165)

DAVISON, F., KASPERS, B., SCHAT K. (2008):

Avian Immunology.

Elsevier Ltd., London, UK

76

DAWSON, H. D., E. BESHAH, S. NISHI, G. SOLANO-AGUILAR, M. MORIMOTO, A.

ZHAO, K. B. MADDEN, T. K. LEDBETTER, J. P. DUBEY, T. SHEA-DONOHUE, J. K.

LUNNEY u. J. F. URBAN, JR. (2005):

Localized multigene expression patterns support an evolving Th1/Th2-like paradigm in

response to infections with Toxoplasma gondii and Ascaris suum.

Infection and Immunity 73, 1116-1128

D'ELIA, R., J. M. BEHNKE, J. E. BRADLEY u. K. J. ELSE (2009):

Regulatory T cells: a role in the control of helminth-driven intestinal pathology and worm

survival.

Journal of Immunology 182, 2340-2348

DE JONGE, H. R., M. BALLMANN, H. VEEZE, I. BRONSVELD, F. STANKE, B.

TUMMLER u. M. SINAASAPPEL (2004):

Ex vivo CF diagnosis by intestinal current measurements (ICM) in small aperture, circulating

Ussing chambers.

Journal of Cystic Fibrosis 3 Suppl 2, 159-163

DEGEN, W. G., N. DAAL, L. ROTHWELL, P. KAISER u. V. E. SCHIJNS (2005):

Th1/Th2 polarization by viral and helminth infection in birds.

Veterinary Microbiology 105, 163-167

DEL CACHO, E., M. GALLEGO, A. SANZ u. A. ZAPATA (1993):

Characterization of distal lymphoid nodules in the chicken caecum.

The Anatomical Record 237, 512-517

DESSEIN, A. J., W. L. PARKER, S. L. JAMES u. J. R. DAVID (1981):

IgE antibody and resistance to infection. I. Selective suppression of the IgE antibody response

in rats diminishes the resistance and the eosinophil response to Trichinella spiralis infection.

The Journal of Experimental Medicine 153, 423-436

77

DOLIGALSKA, M., J. RZEPECKA, N. DRELA, K. DONSKOW u. M. GERWEL-

WRONKA (2006):

The role of TGF-beta in mice infected with Heligmosomoides polygyrus.

Parasite Immunology 28, 387-395

DONOGHUE, D. J. (2003):

Antibiotic residues in poultry tissues and eggs: human health concerns?

Poultry Science 82, 618-621

DURMIC, Z., D. W. PETHICK, J. R. PLUSKE u. D. J. HAMPSON (1998):

Changes in bacterial populations in the colon of pigs fed different sources of dietary fibre, and

the development of swine dysentery after experimental infection.

Journal of Applied Microbiology 85, 574-582

DUSEL, G., H. KLUGE, K. GLASER, O. SIMON, G. HARTMANN, J. LENGERKEN u. H.

JEROCH (1997):

An investigation into the variability of extract viscosity of wheat-relationship with the content

of non-starch-polysaccharide fractions and metabolisable energy for broiler chickens.

Archiv fur Tierernahrung 50, 121-135

EGERTON, J. R. u. M. F. HANSEN (1955):

Immunity and tolerance of chickens to the roundworm, Ascaridia galli (Schrank).

Experimental Parasitology 4, 335-350

EIGAARD, N. M., T. W. SCHOU, A. PERMIN, J. P. CHRISTENSEN, C. T. EKSTROM, F.

AMBROSINI, D. CIANCI u. M. BISGAARD (2006):

Infection and excretion of Salmonella Enteritidis in two different chicken lines with

concurrent Ascaridia galli infection.

Avian Pathology 35, 487-493

EL-KHOLY, H. u. B. W. KEMPPAINEN (2005):

78

Levamisole residues in chicken tissues and eggs.

Poultry Science 84, 9-13

ENGLYST, K. N., S. LIU u. H. N. ENGLYST (2007):

Nutritional characterization and measurement of dietary carbohydrates.

European Journal of Clinical Nutrition 61 Suppl 1, S19-39

ESQUENET, C., P. DE HERDT, H. DE BOSSCHERE, S. RONSMANS, R. DUCATELLE

u. J. VAN ERUM (2003):

An outbreak of histomoniasis in free-range layer hens.

Avian Pathology 32, 305-308

FINKELMAN, F. D., T. SHEA-DONOHUE, S. C. MORRIS, L. GILDEA, R. STRAIT, K. B.

MADDEN, L. SCHOPF u. J. F. URBAN, JR. (2004):

Interleukin-4- and interleukin-13-mediated host protection against intestinal nematode

parasites.

Immunological Reviews 201, 139-155

GARCIA-AMADO, M. A., J. R. DEL CASTILLO, M. EGLEE PEREZ u. M. G.

DOMINGUEZ-BELLO (2005):

Intestinal D-glucose and L-alanine transport in Japanese quail (Coturnix coturnix).

Poultry Science 84, 947-950

GARRIGA, C., M. MORETO u. J. M. PLANAS (1999):

Hexose transport in the apical and basolateral membranes of enterocytes in chickens adapted

to high and low NaCl intakes.

The Journal of Physiology 514 ( Pt 1), 189-199

GAULY, M., C. DUSS u. G. ERHARDT (2007):

Influence of Ascaridia galli infections and anthelmintic treatments on the behaviour and

social ranks of laying hens (Gallus gallus domesticus).

79

Veterinary Parasitology 146, 271-280

GAULY, M., T. HOMANN u. G. ERHARDT (2005):

Age-related differences of Ascaridia galli egg output and worm burden in chickens following

a single dose infection.

Veterinary Parasitology 128, 141-148

GIBBS, B. J. (1962):

The occurrence of the protozoan parasite Histomonas meleagridis in the adults and eggs of

the cecal worm Heterakis gallinae.

The Journal of Protozoology 9, 288-293

GÖBEL, T. W., B. KASPERS u. M. STANGASSINGER (2001):

NK and T cells constitute two major, functionally distinct intestinal epithelial lymphocyte

subsets in the chicken.

International Immunology 13, 757-762

GRABENSTEINER, E., D. LIEBHART, H. WEISSENBOCK u. M. HESS (2006):

Broad dissemination of Histomonas meleagridis determined by the detection of nucleic acid

in different organs after experimental infection of turkeys and specified pathogen-free

chickens using a mono-eukaryotic culture of the parasite.

Parasitology International 55, 317-322

GRENCIS, R. K. (1997):

Th2-mediated host protective immunity to intestinal nematode infections.

Philosophical Transactions of the Royal Society of London 352, 1377-1384

GRENCIS, R. K. u. A. J. BANCROFT (2004):

Interleukin-13: a key mediator in resistance to gastrointestinal-dwelling nematode parasites.

Clinical Reviews in Allergy & Immunology 26, 51-60

80

GRUBER, A. D., R. GANDHI u. B. U. PAULI (1998):

The murine calcium-sensitive chloride channel (mCaCC) is widely expressed in secretory

epithelia and in other select tissues.

Histochemistry and Cell Biology 110, 43-49

HAFEZ, H. M., R. HAUCK, D. LUSCHOW u. L. MCDOUGALD (2005):

Comparison of the specificity and sensitivity of PCR, nested PCR, and real-time PCR for the

diagnosis of histomoniasis.

Avian Diseases 49, 366-370

HERBERT, D. R., J. Q. YANG, S. P. HOGAN, K. GROSCHWITZ, M. KHODOUN, A.

MUNITZ, T. OREKOV, C. PERKINS, Q. WANG, F. BROMBACHER, J. F. URBAN, JR.,

M. E. ROTHENBERG u. F. D. FINKELMAN (2009):

Intestinal epithelial cell secretion of RELM-beta protects against gastrointestinal worm

infection.

The Journal of Experimental Medicine 206, 2947-2957

HERD, R. P. u. D. J. MCNAUGHT (1975):

Arrested development and the histotropic phase of Ascaridia galli in the chicken.

International Journal for Parasitology 5, 401-406

HESS, M., E. GRABENSTEINER u. D. LIEBHART (2006):

Rapid transmission of the protozoan parasite Histomonas meleagridis in turkeys and specific

pathogen free chickens following cloacal infection with a mono-eukaryotic culture.

Avian Pathology 35, 280-285

HONG, Y. H., H. S. LILLEHOJ, E. P. LILLEHOJ u. S. H. LEE (2006):

Changes in immune-related gene expression and intestinal lymphocyte subpopulations

following Eimeria maxima infection of chickens.

Veterinary Immunology and Immunopathology 114, 259-272

81

HOSONO, A., A. OZAWA, R. KATO, Y. OHNISHI, Y. NAKANISHI, T. KIMURA u. R.

NAKAMURA (2003):

Dietary fructooligosaccharides induce immunoregulation of intestinal IgA secretion by

murine Peyer's patch cells.

Bioscience, Biotechnology, and Biochemistry 67, 758-764

HRYCIW, D. H. u. W. B. GUGGINO (2000):

Cystic fibrosis transmembrane conductance regulator and the outwardly rectifying chloride

channel: a relationship between two chloride channels expressed in epithelial cells.

Clinical and Experimental Pharmacology & Physiology 27, 892-895

HU, J., L. FULLER, P. L. ARMSTRONG u. L. R. MCDOUGALD (2006):

Histomonas meleagridis in chickens: attempted transmission in the absence of vectors.

Avian Diseases 50, 277-279

IKEME, M. M. (1971):

Observations on the pathogenicity and pathology of Ascaridia galli.

Parasitology 63, 169-179

JAMROZ, D., K. JAKOBSEN, K. E. BACH KNUDSEN, A. WILICZKIEWICZ u. J. ORDA

(2002):

Digestibility and energy value of non-starch polysaccharides in young chickens, ducks and

geese, fed diets containing high amounts of barley.

Comparative Biochemistry and Physiology 131, 657-668

JANARDHANA, V., M. M. BROADWAY, M. P. BRUCE, J. W. LOWENTHAL, M. S.

GEIER, R. J. HUGHES u. A. G. BEAN (2009):

Prebiotics modulate immune responses in the gut-associated lymphoid tissue of chickens.

The Journal of Nutrition 139, 1404-1409

JENKINS, D. J., C. W. KENDALL u. V. VUKSAN (1999):

82

Inulin, oligofructose and intestinal function.

The Journal of Nutrition 129, 1431S-1433S

JENTSCH, T. J., V. STEIN, F. WEINREICH u. A. A. ZDEBIK (2002):

Molecular structure and physiological function of chloride channels.

Physiological Reviews 82, 503-568

KAISER, P. (2007):

The avian immune genome--a glass half-full or half-empty?

Cytogenetic and Genome Research 117, 221-230

KAISER, P., T. Y. POH, L. ROTHWELL, S. AVERY, S. BALU, U. S. PATHANIA, S.

HUGHES, M. GOODCHILD, S. MORRELL, M. WATSON, N. BUMSTEAD, J.

KAUFMAN u. J. R. YOUNG (2005):

A genomic analysis of chicken cytokines and chemokines.

Journal of Interferon and Cytokine Research 25, 467-484

KATAKAM, K. K., P. NEJSUM, N. C. KYVSGAARD, C. B. JORGENSEN u. S. M.

THAMSBORG (2010):

Molecular and parasitological tools for the study of Ascaridia galli population dynamics in

chickens.

Avian Pathology 39, 81-85

KAUSHIK, R. K. u. V. P. DEORANI (1969):

Studies on tissue responses in primary and subsequent infections with Heterakis galinae in

chickens and on the process of formation of caecal nodules.

Journal of Helminthology 43, 69-78

KELLY, G. (2009):

Inulin-type prebiotics: a review. (Part 2).

Alternative Medicine Review 14, 36-55

83

KHAN, W. I. u. S. M. COLLINS (2004):

Immune-mediated alteration in gut physiology and its role in host defence in nematode

infection.

Parasite Immunology 26, 319-326

KILPINEN, O., A. ROEPSTORFF, A. PERMIN, G. NORGAARD-NIELSEN, L. G.

LAWSON u. H. B. SIMONSEN (2005):

Influence of Dermanyssus gallinae and Ascaridia galli infections on behaviour and health of

laying hens (Gallus gallus domesticus).

British Poultry Science 46, 26-34

KING, I. L. u. M. MOHRS (2009):

IL-4-producing CD4+ T cells in reactive lymph nodes during helminth infection are T

follicular helper cells.

The Journal of Experimental Medicine 206, 1001-1007

KITAGAWA, H., Y. HIRATSUKA, T. IMAGAWA u. M. UEHARA (1998):

Distribution of lymphoid tissue in the caecal mucosa of chickens.

Journal of Anatomy 192 ( Pt 2), 293-298

KNOTT, M. L., K. I. MATTHAEI, P. S. FOSTER u. L. A. DENT (2009):

The roles of eotaxin and the STAT6 signalling pathway in eosinophil recruitment and host

resistance to the nematodes Nippostrongylus brasiliensis and Heligmosomoides bakeri.

Molecular Immunology 46, 2714-2722

KOSIK-BOGACKA, D. I., I. BARANOWSKA-BOSIACKA u. R. SALAMATIN (2010):

Hymenolepis diminuta: Effect of infection on ion transport in colon and blood picture of rats.

Experimental Parasitology 124, 285-294

KOSIK-BOGACKA, D. I. u. L. KOLODZIEJCZYK (2004):

84

Ion transport in the colon of rats experimentally infected with liver fluke (Fasciola hepatica).

Folia Biologica 52, 243-246

KRAG, L., L. E. THOMSEN u. T. IBURG (2006):

Pathology of Trichuris suis infection in pigs fed an inulin- and a non-inulin-containing diet.

Journal of Veterinary Medicine 53, 405-409

KUDOH, K., J. SHIMIZU, M. WADA, T. TAKITA, Y. KANKE u. S. INNAMI (1998):

Effect of indigestible saccharides on B lymphocyte response of intestinal mucosa and cecal

fermentation in rats.

Journal of Nutritional Science and Vitaminology 44, 103-112

KURT, M. u. M. ACICI (2008):

Cross-sectional survey on helminth infections of chickens in the Samsun region, Turkey.

Deutsche Tierarztliche Wochenschrift 115, 239-242

LEE, D. L. (1969):

Histomonas in the bird and in the nematode Heterakis.

Transactions of the Royal Society of Tropical Medicine and Hygiene 63, 427-428

LEESON, A., J. D. SUMMERS u. L. J. CASTON (1991):

Diet dilution and compensatory growth in broilers.

Poultry Science 70, 867-873

LEONHARD-MAREK, S. u. A. DAUGSCHIES (1997):

Electro- and transportphysiological changes in pig proximal colon during parasitic infection.

Comparative Biochemistry and Physiology 118, 345-347

LEONHARD-MAREK, S., J. HEMPE, B. SCHROEDER u. G. BREVES (2009):

Electrophysiological characterization of chloride secretion across the jejunum and colon of

pigs as affected by age and weaning.

85

Journal of Comparative Physiology 179, 883-896

LILLEHOJ, H. S. (1993):

Avian gut-associated immune system: implication in coccidial vaccine development.

Poultry Science 72, 1306-1311

LILLEHOJ, H. S. u. K. D. CHOI (1998):

Recombinant chicken interferon-gamma-mediated inhibition of Eimeria tenella development

in vitro and reduction of oocyst production and body weight loss following Eimeria

acervulina challenge infection.

Avian Diseases 42, 307-314

LILLEHOJ, H. S. u. K. S. CHUNG (1992):

Postnatal development of T-lymphocyte subpopulations in the intestinal intraepithelium and

lamina propria in chickens.

Veterinary Immunology and Immunopathology 31, 347-360

LILLEHOJ, H. S. u. E. P. LILLEHOJ (2000):

Avian coccidiosis. A review of acquired intestinal immunity and vaccination strategies.

Avian Diseases 44, 408-425

LILLEHOJ, H. S., W. MIN u. R. A. DALLOUL (2004):

Recent progress on the cytokine regulation of intestinal immune responses to Eimeria.

Poultry Science 83, 611-623

LILLEHOJ, H. S. u. M. D. RUFF (1987):

Comparison of disease susceptibility and subclass-specific antibody response in SC and FP

chickens experimentally inoculated with Eimeria tenella, E. acervulina, or E. maxima.

Avian Diseases 31, 112-119

LILLEHOJ, H. S. u. J. M. TROUT (1993):

86

Coccidia: a review of recent advances on immunity and vaccine development.

Avian Pathology 22, 3-31

LILLEHOJ, H. S. u. J. M. TROUT (1996):

Avian gut-associated lymphoid tissues and intestinal immune responses to Eimeria parasites.

Clinical Microbiology Reviews 9, 349-360

LIM, B. O., K. YAMADA, M. NONAKA, Y. KURAMOTO, P. HUNG u. M. SUGANO

(1997):

Dietary fibers modulate indices of intestinal immune function in rats.

The Journal of Nutrition 127, 663-667

LIN, P. H., B. L. SHIH u. J. C. HSU (2010):

Effects of different sources of dietary non-starch polysaccharides on the growth performance,

development of digestive tract and activities of pancreatic enzymes in goslings.

British Poultry Science 51, 270-277

LITTLE, M. C., L. V. BELL, L. J. CLIFFE u. K. J. ELSE (2005):

The characterization of intraepithelial lymphocytes, lamina propria leukocytes, and isolated

lymphoid follicles in the large intestine of mice infected with the intestinal nematode parasite

Trichuris muris.

Journal of Immunology 175, 6713-6722

LUND, E. E. u. A. M. CHUTE (1972):

Reciprocal responses of eight species of galliform birds and three parasites: Heterakis

gallinarum, Histomonas meleagridis, and Parahistomonas wenrichi.

The Journal of Parasitology 58, 940-945

LUND, E. E. u. A. M. CHUTE (1974):

The reproductive potential of Heterakis gallinarum in various species of galliform birds:

implications for survival of H. gallinarum and Histomonas meleagridis to recent times.

87

International Journal for Parasitology 4, 455-461

LUND, E. E., A. M. CHUTE u. S. L. MYERS (1970):

Performance in chickens and turkeys of chicken-adapted Heterakis gallinarum.

Journal of Helminthology 44, 97-106

MADDEN, K. B., L. WHITMAN, C. SULLIVAN, W. C. GAUSE, J. F. URBAN, JR., I. M.

KATONA, F. D. FINKELMAN u. T. SHEA-DONOHUE (2002):

Role of STAT6 and mast cells in IL-4- and IL-13-induced alterations in murine intestinal

epithelial cell function.

Journal of Immunology 169, 4417-4422

MADDEN, K. B., K. A. YEUNG, A. ZHAO, W. C. GAUSE, F. D. FINKELMAN, I. M.

KATONA, J. F. URBAN, JR. u. T. SHEA-DONOHUE (2004):

Enteric nematodes induce stereotypic STAT6-dependent alterations in intestinal epithelial cell

function.

Journal of Immunology 172, 5616-5621

MAGWISHA, H. B., A. A. KASSUKU, N. C. KYVSGAARD u. A. PERMIN (2002):

A comparison of the prevalence and burdens of helminth infections in growers and adult free-

range chickens.

Tropical Animal Health and Production 34, 205-214

MAILLEAU, C., J. CAPEAU u. M. C. BRAHIMI-HORN (1998):

Interrelationship between the Na+/glucose cotransporter and CFTR in Caco-2 cells: relevance

to cystic fibrosis.

Journal of Cellular Physiology 176, 472-481

MAIZELS, R. M., A. BALIC, N. GOMEZ-ESCOBAR, M. NAIR, M. D. TAYLOR u. J. E.

ALLEN (2004):

Helminth parasites--masters of regulation.

88

Immunological Reviews 201, 89-116

MAIZELS, R. M. u. M. YAZDANBAKHSH (2003):

Immune regulation by helminth parasites: cellular and molecular mechanisms.

Nature Reviews 3, 733-744

MANHART, N., A. SPITTLER, H. BERGMEISTER, M. MITTLBOCK u. E. ROTH (2003):

Influence of fructooligosaccharides on Peyer's patch lymphocyte numbers in healthy and

endotoxemic mice.

Nutrition 19(7-8), 657-660

MARCOS-ATXUTEGI, C., B. GANDOLFI, T. ARANGUENA, R. SEPULVEDA, M.

AREVALO u. F. SIMON (2009):

Antibody and inflammatory responses in laying hens with experimental primary infections of

Ascaridia galli.

Veterinary Parasitology 161, 69-75

MARTIN-PACHO, J. R., M. N. MONTOYA, T. ARANGUENA, C. TORO, R. MORCHON,

C. MARCOS-ATXUTEGI u. F. SIMON (2005):

A coprological and serological survey for the prevalence of Ascaridia spp. in laying hens.

Journal of Veterinary Medicine 52, 238-242

MAST, J. u. B. M. GODDEERIS (1999):

Development of immunocompetence of broiler chickens.

Veterinary Immunology and Immunopathology 70, 245-256

MAURER, V., Z. AMSLER, E. PERLER u. F. HECKENDORN (2009):

Poultry litter as a source of gastrointestinal helminth infections.

Veterinary Parasitology 161, 255-260

MCDONALD, D. E., D. W. PETHICK, J. R. PLUSKE u. D. J. HAMPSON (1999):

89

Adverse effects of soluble non-starch polysaccharide (guar gum) on piglet growth and

experimental colibacillosis immediately after weaning.

Research in Veterinary Science 67, 245-250

MCDOUGALD, L. R. (2005):

Blackhead disease (histomoniasis) in poultry: a critical review.

Avian Diseases 49, 462-476

MCKENZIE, G. J., A. BANCROFT, R. K. GRENCIS u. A. N. MCKENZIE (1998):

A distinct role for interleukin-13 in Th2-cell-mediated immune responses.

Current Biology 8, 339-342

MCKENZIE, G. J., P. G. FALLON, C. L. EMSON, R. K. GRENCIS u. A. N. J. MCKENZIE

(1999):

Simultaneous disruption of interleukin (IL)-4 and IL-13 defines individual roles in T helper

cell type 2-mediated responses.

Journal of Experimental Medicine 189, 1565-1572

MEYER, D. (2008):

Prebiotic dietary fibres and the immune system.

Agro Food Industry Hi-Tech 19, 12-15

MUIR, W. I., W. L. BRYDEN u. A. J. HUSBAND (2000):

Immunity, vaccination and the avian intestinal tract.

Developmental and Comparative Immunology 24, 325-342

MUNGUBE, E. O., S. M. BAUNI, B. A. TENHAGEN, L. W. WAMAE, S. M. NZIOKA, L.

MUHAMMED u. J. M. NGINYI (2008):

Prevalence of parasites of the local scavenging chickens in a selected semi-arid zone of

Eastern Kenya.

Tropical Animal Health and Production 40, 101-109

90

O'MALLEY, K. E., T. SLOAN, P. JOYCE u. A. W. BAIRD (1993):

Type I hypersensitivity reactions in intestinal mucosae from rats infected with Fasciola

hepatica.

Parasite Immunology 15, 449-453

OLAH, I. u. B. GLICK (1984):

Meckel's diverticulum. I. Extramedullary myelopoiesis in the yolk sac of hatched chickens

(Gallus domesticus).

The Anatomical Record 208, 243-252

OLAH, I., B. GLICK u. R. L. TAYLOR, JR. (1984):

Meckel's diverticulum. II. A novel lymphoepithelial organ in the chicken.

The Anatomical Record 208, 253-263

PAPPENHEIMER, J. R. (1993):

On the coupling of membrane digestion with intestinal absorption of sugars and amino acids.

The American Journal of Physiology 265, G409-417

PATEL, N., T. KREIDER, J. F. URBAN, JR. u. W. C. GAUSE (2009):

Characterisation of effector mechanisms at the host:parasite interface during the immune

response to tissue-dwelling intestinal nematode parasites.

International Journal for Parasitology 39, 13-21

PEARCE, G. P. (1999):

Interactions between dietary fibre, endo-parasites and Lawsonia intracellularis bacteria in

grower-finisher pigs.

Veterinary Parasitology 87, 51-61

PEREZ, J., R. ZAFRA, L. BUFFONI, S. HERNANDEZ, S. CAMARA u. A. MARTINEZ-

MORENO (2008):

91

Cellular phenotypes in the abomasal mucosa and abomasal lymph nodes of goats infected

with Haemonchus contortus.

Journal of Comparative Pathology 138, 102-107

PERMIN, A., M. BISGAARD, F. FRANDSEN, M. PEARMAN, J. KOLD u. P. NANSEN

(1999):

Prevalence of gastrointestinal helminths in different poultry production systems.

British Poultry Science 40, 439-443

PERMIN, A., H. MAGWISHA, A. A. KASSUKU, P. NANSEN, M. BISGAARD, F.

FRANDSEN u. L. GIBBONS (1997):

A cross-sectional study of helminths in rural scavenging poultry in Tanzania in relation to

season and climate.

Journal of Helminthology 71, 233-240

PERMIN, A. u. H. RANVIG (2001):

Genetic resistance to Ascaridia galli infections in chickens.

Veterinary Parasitology 102, 101-111

PERNTHANER, A., M. STANKIEWICZ, W. CABAJ, A. PFEFFER, R. S. GREEN u. P. G.

DOUCH (1996):

Immune responsiveness of nematode-resistant or susceptible Romney line-bred sheep to

continuous infection with Trichostrongylus axei.

Veterinary Immunology and Immunopathology 51, 137-146

PETKEVICIUS, S., K. E. BACH KNUDSEN, K. D. MURRELL u. H. WACHMANN

(2003):

The effect of inulin and sugar beet fibre on Oesophagostomum dentatum infection in pigs.

Parasitology 127, 61-68

PETKEVICIUS, S., K. E. KNUDSEN, P. NANSEN u. K. D. MURRELL (2001):

92

The effect of dietary carbohydrates with different digestibility on the populations of

Oesophagostomum dentatum in the intestinal tract of pigs.

Parasitology 123, 315-324

PETKEVICIUS, S., K. E. KNUDSEN, P. NANSEN, A. ROEPSTORFF, F. SKJOTH u. K.

JENSEN (1997):

The impact of diets varying in carbohydrates resistant to endogenous enzymes and lignin on

populations of Ascaris suum and Oesophagostomum dentatum in pigs.

Parasitology 114 ( Pt 6), 555-568

PETKEVICIUS, S., K. D. MURRELL, K. E. BACH KNUDSEN, H. JORGENSEN, A.

ROEPSTORFF, A. LAUE u. H. WACHMANN (2004):

Effects of short-chain fatty acids and lactic acids on survival of Oesophagostomum dentatum

in pigs.

Veterinary Parasitology 122, 293-301

PETKEVICIUS, S., P. NANSEN, K. E. BACH KNUDSEN u. F. SKJOTH (1999):

The effect of increasing levels of insoluble dietary fibre on the establishment and persistence

of Oesophagostomum dentatum in pigs.

Parasite (Paris, France) 6, 17-26

PETKEVICIUS, S., L. E. THOMSEN, K. E. BACH KNUDSEN, K. D. MURRELL, A.

ROEPSTORFF u. J. BOES (2007):

The effect of inulin on new and on patent infections of Trichuris suis in growing pigs.

Parasitology 134, 121-127

PLUSKE, J. R., Z. DURMIC, D. W. PETHICK, B. P. MULLAN u. D. J. HAMPSON (1998):

Confirmation of the role of rapidly fermentable carbohydrates in the expression of swine

dysentery in pigs after experimental infection.

The Journal of Nutrition 128, 1737-1744

93

POMPA, G., F. ARIOLI u. M. L. FRACCHIOLLA (2005):

Presence of residues in food of animal origin and related risk.

Veterinary Research Communications 29 Suppl 2, 113-116

POTTS, G. R. (2009):

Long-term changes in the prevalences of caecal nematodes and histomonosis in gamebirds in

the UK and the interaction with poultry.

The Veterinary Record 164, 715-718

POWELL, F. L., L. ROTHWELL, M. J. CLARKSON u. P. KAISER (2009):

The turkey, compared to the chicken, fails to mount an effective early immune response to

Histomonas meleagridis in the gut.

Parasite Immunology 31, 312-327

RAMADAN, H. H. u. N. Y. ABOU ZNADA (1991):

Some pathological and biochemical studies on experimental ascaridiasis in chickens.

Die Nahrung 35, 71-84

RATCLIFFE, M. J. (2006):

Antibodies, immunoglobulin genes and the bursa of Fabricius in chicken B cell development.

Developmental and Comparative Immunology 30, 101-118

RAUSCH, S., J. HUEHN, D. KIRCHHOFF, J. RZEPECKA, C. SCHNOELLER, S. PILLAI,

C. LODDENKEMPER, A. SCHEFFOLD, A. HAMANN, R. LUCIUS u. S. HARTMANN

(2008):

Functional analysis of effector and regulatory T cells in a parasitic nematode infection.

Infection and Immunity 76, 1908-1919

REHMAN, H., C. ROSENKRANZ, J. BOHM u. J. ZENTEK (2007):

Dietary inulin affects the morphology but not the sodium-dependent glucose and glutamine

transport in the jejunum of broilers.

94

Poultry Science 86, 118-122

REID, W. M. u. J. L. CARMON (1958):

Effects of numbers of Ascaridia galli in depressing weight gains in chicks.

The Journal of Parasitology 44, 183-186

REID, W. M., J. L. MABON u. W. C. HARSHBARGER (1973):

Detection of worm parasites in chicken eggs by candling.

Poultry Science 52, 2316-2324

RIDDELL, C. u. A. GAJADHAR (1988):

Cecal and hepatic granulomas in chickens associated with Heterakis gallinarum infection.

Avian Diseases 32, 836-838

ROBERFROID, M. B. (2006):

Introducing inulin-type fructans.

British Journal of Nutrition 93, S13-S25

ROLLER, M., Y. CLUNE, K. COLLINS, G. RECHKEMMER u. B. WATZL (2007):

Consumption of prebiotic inulin enriched with oligofructose in combination with the

probiotics Lactobacillus rhamnosus and Bifidobacterium lactis has minor effects on selected

immune parameters in polypectomised and colon cancer patients.

British Journal of Nutrition 97, 676-684

ROLLER, M., A. PIETRO FEMIA, G. CADERNI, G. RECHKEMMER u. B. WATZL

(2004a):

Intestinal immunity of rats with colon cancer is modulated by oligofructose-enriched inulin

combined with Lactobacillus rhamnosus and Bifidobacterium lactis.

British Journal of Nutrition 92, 931-938

ROLLER, M., G. RECHKEMMER u. B. WATZL (2004b):

95

Prebiotic inulin enriched with oligofructose in combination with the probiotics Lactobacillus

rhamnosus and Bifidobacterium lactis modulates intestinal immune functions in rats.

The Journal of Nutrition 134, 153-156

ROSE, M. E. (1987):

Immunity to Eimeria infections.

Veterinary Immunology and Immunopathology 17, 333-343

ROTHWELL, L., R. A. GRAMZINSKI, M. E. ROSE u. P. KAISER (1995):

Avian coccidiosis: changes in intestinal lymphocyte populations associated with the

development of immunity to Eimeria maxima.

Parasite Immunology 17, 525-533

RUFF, M. D., L. R. MCDOUGALD u. M. F. HANSEN (1970):

Isolation of Histomonas meleagridis from embryonated eggs of Heterakis gallinarum.

The Journal of Protozoology 17, 10-11

RYZ, N. R., J. B. MEDDINGS u. C. G. TAYLOR (2009):

Long-chain inulin increases dendritic cells in the Peyer's patches and increases ex vivo

cytokine secretion in the spleen and mesenteric lymph nodes of growing female rats,

independent of zinc status.

British Journal of Nutrition 101, 1653-1663

SAIF, Y. M. (2008):

Diseases of poultry.

Blackwell Publishing, Ames, Iowa, USA

SCHILTER, H. C., A. T. PEREIRA, P. D. ESCHENAZI, A. FERNANDES, D. SHIM, A. L.

SOUSA, M. M. TEIXEIRA u. D. NEGRAO-CORREA (2010):

Regulation of immune responses to Strongyloides venezuelensis challenge after primary

infection with different larvae doses.

96

Parasite Immunology 32, 184-192

SCHOU, T. W., R. LABOURIAU, A. PERMIN, J. P. CHRISTENSEN, P. SORENSEN, H. P.

CU, V. K. NGUYEN u. H. R. JUUL-MADSEN (2010):

MHC haplotype and susceptibility to experimental infections (Salmonella Enteritidis,

Pasteurella multocida or Ascaridia galli) in a commercial and an indigenous chicken breed.

Veterinary Immunology and Immunopathology 135, 52-63

SCHWARTZ, S. E., G. D. LEVINE u. C. M. STARR (1982):

Effects of dietary fiber on intestinal ion fluxes in rats.

The American Journal of Clinical Nutrition 36, 1102-1105

SEIFERT, S. u. B. WATZL (2007):

Inulin and oligofructose: review of experimental data on immune modulation.

The Journal of Nutrition 137, 2563S-2567S

SHEA-DONOHUE, T., C. SULLIVAN, F. D. FINKELMAN, K. B. MADDEN, S. C.

MORRIS, J. GOLDHILL, V. PINEIRO-CARRERO u. J. F. URBAN, JR. (2001):

The role of IL-4 in Heligmosomoides polygyrus-induced alterations in murine intestinal

epithelial cell function.

Journal of Immunology 167, 2234-2239

SHIMADA, T. u. T. HOSHI (1986):

A comparative study of specificity of the intestinal Na+/sugar cotransport among vertebrates.

Comparative Biochemistry and Physiology, Part A Comp Physiol 84, 365-370

SMITS, C. H. M. u. G. ANNISON (1996):

Non-starch plant polysaccharides in broiler nutrition - Towards a physiologically valid

approach to their determination.

Worlds Poultry Science Journal 52, 203-221

97

SPILLER, R. C. (1994):

Pharmacology of dietary fibre.

Pharmacology & Therapeutics 62, 407-427

SPRINGER, W. T., J. JOHNSON u. W. M. REID (1969):

Transmission of histomoniasis with male Heterakis gallinarum (nematoda).

Parasitology 59, 401-405

SRETER, T., I. VARGA u. L. BEKESI (1996):

Effects of bursectomy and thymectomy on the development of resistance to Cryptosporidium

baileyi in chickens.

Parasitology Research 82, 174-177

SUN, Y., K. G. KOSKI, L. J. WYKES u. M. E. SCOTT (2002):

Dietary pectin, but not cellulose, influences Heligmosomoides polygyrus (Nematoda)

reproduction and intestinal morphology in the mouse.

Parasitology 124, 447-455

TAJIK, H., H. MALEKINEJAD, S. M. RAZAVI-ROUHANI, M. R. PAJOUHI, R.

MAHMOUDI u. A. HAGHNAZARI (2010):

Chloramphenicol residues in chicken liver, kidney and muscle: A comparison among the

antibacterial residues monitoring methods of Four Plate Test, ELISA and HPLC.

Food and Chemical Toxicology 48(8-9), 2464-2468

THOMSEN, L. E., K. E. KNUDSEN, M. S. HEDEMANN u. A. ROEPSTORFF (2006):

The effect of dietary carbohydrates and Trichuris suis infection on pig large intestine tissue

structure, epithelial cell proliferation and mucin characteristics.

Veterinary Parasitology 142, 112-122

THOMSEN, L. E., S. PETKEVICIUS, K. E. BACH KNUDSEN u. A. ROEPSTORFF

(2005):

98

The influence of dietary carbohydrates on experimental infection with Trichuris suis in pigs.

Parasitology 131, 857-865

TIZARD, I. R. (2009):

Veterinary Immunology.

Saunders Elsevier, St. Louis, Missouri, USA

TONGSON, M. S. u. B. M. MCCRAW (1967):

Experimental ascaridiasis: influence of chicken age and infective egg dose on structure of

Ascaridia galli populations.

Experimental Parasitology 21, 160-172

TROUT, J. M. u. H. S. LILLEHOJ (1996):

T lymphocyte roles during Eimeria acervulina and Eimeria tenella infections.

Veterinary Immunology and Immunopathology 53, 163-172

TRUSHINA, E. N., E. A. MARTYNOVA, D. B. NIKITIUK, O. K. MUSTAFINA u. E. K.

BAIGARIN (2005):

[The influence of dietary inulin and oligofructose on the cell-mediated and humoral immunity

in rats].

Voprosy pitaniia 74, 22-27

TSUJI, Y., K. YAMADA, N. HOSOYA u. S. MORIUCHI (1985):

Changes of sugar-evoked transmural potential differences in intestine of rats with

streptozotocin-induced diabetes.

Journal of Nutritional Science and Vitaminology 31, 317-326

TUGWELL, R. L. u. J. E. ACKERT (1952):

On the tissue phase of the life cycle of the fowl nematode Ascaridia galli (Schrank).

The Journal of Parasitology 38, 277-288

99

TVERDOKHLEBOV, P. T. (1966):

[The susceptibility of domestic fowls to Ascaridia galli].

Veterinariia 43, 67-69

URBAN, J., H. FANG, Q. LIU, M. J. EKKENS, S. J. CHEN, D. NGUYEN, V. MITRO, D.

D. DONALDSON, C. BYRD, R. PEACH, S. C. MORRIS, F. D. FINKELMAN, L. SCHOPF

u. W. C. GAUSE (2000):

IL-13-mediated worm expulsion is B7 independent and IFN-gamma sensitive.

Journal of Immunology 164, 4250-4256

USSING, H. H. u. K. ZERAHN (1951):

Active transport of sodium as the source of electric current in the short-circuited isolated frog

skin.

Acta Physiologica Scandinavica 23, 110-127

VAN KRIMPEN, M. M., R. P. KWAKKEL, C. M. VAN DER PEET-SCHWERING, L. A.

DEN HARTOG u. M. W. VERSTEGEN (2009):

Effects of nutrient dilution and nonstarch polysaccharide concentration in rearing and laying

diets on eating behavior and feather damage of rearing and laying hens.

Poultry Science 88, 759-773

VERVELDE, L. u. S. H. JEURISSEN (1993):

Postnatal development of intra-epithelial leukocytes in the chicken digestive tract:

phenotypical characterization in situ.

Cell and Tissue Research 274, 295-301

VON HEIMENDAHL, E., G. BREVES u. H. J. ABEL (2010):

Fiber-related digestive processes in three different breeds of pigs.

Journal of Animal Science 88, 972-981

WANG, Y. Z., J. M. PALMER u. H. J. COOKE (1991):

100

Neuroimmune regulation of colonic secretion in guinea pigs.

The American Journal of Physiology 260, G307-314

WATZL, B., S. GIRRBACH u. M. ROLLER (2005):

Inulin, oligofructose and immunomodulation.

British Journal of Nutrition 93 Suppl 1, S49-55

WESTENDARP, H. (2006):

[Effects of non-starch-polysaccharides in nutrition of monogastric animals].

Deutsche Tierarztliche Wochenschrift 113, 379-384

YUN, C. H., H. S. LILLEHOJ u. K. D. CHOI (2000a):

Eimeria tenella infection induces local gamma interferon production and intestinal

lymphocyte subpopulation changes.

Infection and Immunity 68, 1282-1288

YUN, C. H., H. S. LILLEHOJ u. E. P. LILLEHOJ (2000b):

Intestinal immune responses to coccidiosis.

Developmental and Comparative Immunology 24, 303-324

ZAROS, L. G., P. A. BRICARELLO, A. F. AMARANTE, R. A. ROCHA, F. N.

KOOYMAN, E. DE VRIES u. L. L. COUTINHO (2010):

Cytokine gene expression in response to Haemonchus placei infections in Nelore cattle.

Veterinary Parasitology 171, 68-73

ZHAO, A., J. MCDERMOTT, J. F. URBAN, JR., W. GAUSE, K. B. MADDEN, K. A.

YEUNG, S. C. MORRIS, F. D. FINKELMAN u. T. SHEA-DONOHUE (2003):

Dependence of IL-4, IL-13, and nematode-induced alterations in murine small intestinal

smooth muscle contractility on Stat6 and enteric nerves.

Journal of Immunology 171, 948-954

101

10. Acknowledgements

The studies in this project were financially supported by the Deutsche

Forschungsgemeinschaft (AB 30/8-1, BR 780/14-1).

I would like to express my special thanks to Prof. Silke Rautenschlein, Clinic for Poultry,

University of Veterinary Medicine Hannover and Prof. Gerhard Breves, Institute for

Physiology, University of Veterinary Medicine Hannover for their patient supervision of all

my research work and extensive support of my Ph.D. curriculum.

I also would like to give my deepest thank to our collaborating partners in this project: Prof.

Matthias Gauly, Prof. Hansjorg Abel, Gürbüz Daş, Julia Humburg and Birgit Sohnrey,

Department of Animal Sciences, University of Goettingen for the wonderful team-work in

these four years, their friendly support in various situations and a lot of fun in the sunny Alps.

I also give my special thanks to Alexander Th. A. Weiss, Ph.D., Department of Veterinary

Pathology, Freie Universitaet Berlin for his enormous assistance in all my histo-pathological

examinations and his valuable suggestions in the design of the animal experiments.

I give my deep thanks to Dr. Karl Rohn, Department of Biometry, Epidemiology and

Information Processing, University of Veterinary Medicine Hannover for his patient help in

the statistical evaluation of the data.

I would like to thank Victoria Lebed and Christine Haase from the Clinic for Poultry, and

Marion Burmister from Institute for Physiology, University of Veterinary Medicine Hannover

for their exellent technical support in the lab. I also thank Sonja Bernhard for her assistance at

necropsy.

Furthermore, my deep thanks to all my colleagues from the Clinic for Poultry and Institute for

Physiology for their collaborative help in my research work and pleasant working

atmosphere.