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Veterinary Parasitology 178 (2011) 121–128

Contents lists available at ScienceDirect

Veterinary Parasitology

journa l homepage: www.e lsev ier .com/ locate /vetpar

ffects of dietary non-starch polysaccharides on establishment andecundity of Heterakis gallinarum in grower layers

ürbüz Dasa,∗, Hansjörg Abela, Silke Rautenschleinb, Julia Humburga, Anna Schwarzb,erhard Brevesc, Matthias Gaulya

University of Göttingen, Department of Animal Sciences, Albrecht-Thaer-Weg 3, 37075 Göttingen, GermanyUniversity of Veterinary Medicine Hannover, Clinic for Poultry, Bünteweg 17, 30559 Hannover, GermanyUniversity of Veterinary Medicine Hannover, Institute for Physiology, Bischofsholer Damm 15, 30173 Hannover, Germany

r t i c l e i n f o

rticle history:eceived 19 August 2010eceived in revised form 8 November 2010ccepted 20 December 2010

eywords:eterakis gallinarumistomonas meleagridisorm fecundity

on-starch polysaccharidesnulinhickenaecaaecal egg counts

a b s t r a c t

It was hypothesized that the establishment and fecundity of Histomonas meleagridis freeHeterakis gallinarum may be affected by dietary non-starch polysaccharides (NSPs). One-day-old female layer chicks (N = 670) were fed ad libitum for 11 wk one of the followingdiets in a three-times repeated experiment: basal diet (CON), basal diet plus pea bran rich ininsoluble NSP (I-NSP), basal diet plus chicory root meal as a source of inulin rich soluble NSP(S-NSP). At the end of wk three, each feeding group was subdivided into an uninfected andan infected group of birds each being inoculated with a placebo or with 200 H. meleagridisfree eggs of H. gallinarum. The birds were slaughtered 8 wk post infection and their wormburdens, the nematode egg excretion, caeca sizes and weights as well as intracaecal pH andvolatile fatty acid (VFA) concentrations were determined.

The NSP supplemented diets and also infection led to reduced body weights (BWs) of birdsand impaired the feed conversion rate (P < 0.001). The NSP supplemented diets increasedaverage length of caecum (P < 0.001) with S-NSP exerting a stronger effect than I-NSP(P < 0.05). Full caeca weight was increased by S-NSP (P < 0.001). Feeding S-NSP loweredintracaecal pH and molar proportion of acetate and increased that of butyrate compared toCON and I-NSP (P < 0.001). Caecal pool of VFA was increased with S-NSP (P < 0.001).

The NSP-diets elevated incidence of infection (P < 0.01), average number of larvae(P < 0.009) and total worm burden (P < 0.001) compared to CON. The daily amount of faecesincreased in NSP-fed birds (P < 0.001). Number of eggs per gram of faeces (EPG), number ofeggs excreted per worm population of a bird within 24 h (EPD) and female worm fecundity(EPD/female worm) were elevated after feeding S-NSP (P ≤ 0.002), whereas I-NSP led tolower EPG/female worm (P < 0.05). The EPD increased in the sequence of CON < I-NSP < S-

NSP (P < 0.001).

It is concluded that the pea bran and chicory root meal used as sources of insoluble andsoluble dietary NSPs, respectively, provided favourable conditions for the establishmentof H. gallinarum in grower layers. Chicory root meal additionally enhanced fecundity ofthe nematode. Therefore, the two natural sources of insoluble and soluble NSPs offer no

ting ag

potential as protec

∗ Corresponding author. Tel.: +49 551 39 19977; fax: +49 551 39 5587.E-mail address: gdas@gwdg.de (G. Das).

304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.vetpar.2010.12.027

ents against H. gallinarum infections in chicken.© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Dietary non-starch polysaccharides (NSPs) have beenshown to influence infections of pigs with common

arasitology 178 (2011) 121–128

Table 1Dry matter (DM), nutrient contents (as g/kg DM) and metabolizableenergy (ME) concentrations of the experimental diets.

Item CONa I-NSPb S-NSPc

DM, g/kg 894 896 895Ash 53 51 53Crude protein 216 199 202Neutral detergent fibre 111 166 110Acid detergent fibre 33 90 40Ether extract 40 36 37Starch 514 476 435Insoluble NSP 103 172 104Soluble NSP 20 23 25Inulin – – 70ME, MJ/kg DMd 13.64 12.58 12.21

a Basal diet (for the components see Das et al., in press).b Insoluble non-starch polysaccharide supplemented diet = 1000 g CON

plus 100 g pea bran. Pea bran contained 86.9% crude fibre (Exafine 500,Socode, Belgium).

c Soluble non-starch polysaccharide supplemented diet = 1000 g CONplus 100 g chicory root meal. Average polymerization degree (DP) of inulin

122 G. Das et al. / Veterinary P

nematodes (Petkevicius et al., 1997, 2001, 2003, 2007)and of chickens with Ascaridia galli (Daenicke et al.,2009). As NSPs are only degradable by the intestinalmicrobiota (Englyst, 1989), their effects on nematodeinfections should mainly be ascribable to alterations ofdigesta characteristics and intestinal microbial fermenta-tion.

In recent years, Heterakis gallinarum has become moreimportant with the increasing number of poultry kept infloor husbandry systems, where the prevalence of thisparasite may reach 84% (Permin et al., 1999; Kaufmannand Gauly, 2009; Maurer et al., 2009). The nematodeis known as the main vector for the transmission ofHistomonas meleagridis, which is brought about by theingestion of embryonated eggs of the nematode by thehost animal (McDougald, 2005). The caeca are not only themain sites of microbial fermentation in poultry (Józefiaket al., 2004), but also the predilection sites of these twoparasites. We have recently shown that dietary NSPsalters interactions between H. gallinarum and H. melea-gridis in birds infected with Histomonas positive nematodeeggs, and either left untreated or treated with dimetri-dazole (Das et al., in press). Although the dimetridazoletreatment eliminates H. meleagridis, it may also affectthe gastrointestinal microbial flora (Fernie et al., 1977;APVMA, 2007), which may then influence the establish-ment of the nematode. Therefore, it must be ensured thatthe nematode is free of H. meleagridis, if the effects ofdietary NSP on a mono H. gallinarum infection are inves-tigated.

We hypothesized that the establishment and fecun-dity of H. gallinarum can be influenced and regulatedby dietary NSP, which are known to alter the intracae-cal environment by affecting intestinal microbial activity.The objective of the present investigation was to exam-ine the effects of low or highly fermentable NSP on theestablishment and fecundity of the nematode, on parame-ters of caecal fermentation and on performance of growerlayers experimentally infected with Histomonas free H. gal-linarum.

2. Materials and methods

2.1. Experimental design and diets

In a three times repeated experiment, conducted inthe years 2008–2009, a total of 670 one-day-old femaleLohmann Selected Leghorn (LSL) chickens were used.Within each repetition, the chicks were divided into threefeeding groups. Each feeding group was fed ad libitum oneof the diets shown in Table 1 from hatch until wk 11 oflife. Nutrient and energy contents of the diets are shown inTable 1, while the composition, methodology for the analy-sis of the experimental diets as well as litter management,temperature and lighting program are given elsewhere in

details (Das et al., in press). Daily feed consumption wasmonitored per group. Drinking water was offered ad libi-tum. At the end of wk three, the birds were marked withwing tags and individual body weights (BWs) were deter-mined for the first time and thereafter at weekly intervals.

in the chicory root meal, DP = 9 (Fibrofos 60, Socode, Belgium).d ME, MJ/kg DM = [(g CP/kg DM × 0.01551) + (g CL/kg DM × 0.03431) + (g

starch/kg DM × 0.01669) + (g sugar/kg DM × 0.01301)]. Sugar contents ofthe diets were estimated based on sugar contents of the components.

2.2. Experimental infection

H. meleagridis free adult H. gallinarum females weregathered from a preliminary animal trial in which adimetridazole treatment was applied to chickens (Das et al.,in press). For the first experimental repetition, femaleworms were incubated at room temperature (20–25 ◦C)for three weeks in media containing 0.1% (w/v) potassiumdichromate (K2Cr2O7). In the second and third experimen-tal repetitions, the eggs of female worms harvested in thepreceding experimental run were used as infection mate-rial and prepared in the same way. Therefore, age of theeggs at infection was around 8 mo, 3 mo and 1 mo in thefirst, second and third repetitions, respectively.

At the end of wk three, each feeding group was subdi-vided into an uninfected control group (40% of birds) and aninfected group (60%). The infected groups were inoculatedwith 200 embryonated eggs of H. gallinarum per bird, whichwere administered orally by a 5 cm esophageal cannula.Uninfected control birds were given 0.2 ml of an aqueousplacebo. Uninfected birds were left in their previous pens,whereas birds of each infected group were placed in newpens within the same experimental stable. The birds did notget any vaccination or anthelmintic treatment throughoutthe experimental period. The pens were thoroughly disin-fected at least 10 d before introducing the birds. Furtherdetails for the preparation of the infection material andinfection procedures are given by Das et al. (in press).

2.3. Faecal sampling and post-mortem examinations

During the last four days of the last two repetitions,birds of the infected groups were placed into individual

cages for a 24 h period of faeces collection (12 birds d−1

group−1). In the cages, the birds had free access to feedand water. Faeces excreted by each bird accumulated inplastic bag-covered boxes underneath the cage. The totalamount of faeces per bird/day was weighed, transferred

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G. Das et al. / Veterinary P

nto a plastic cup and stirred thoroughly for at least 3 mino get a paste-like consistency that guaranteed a homoge-eous distribution of the eggs in the faeces. The numberf eggs per gram of faeces (EPG) was quantified using aodified McMaster counting technique (MAFF, 1986) and

aturated NaCl as the flotation liquid (density = 1.2 g/ml).he minimum detection level was 50 eggs/g faeces.

The birds were slaughtered after electrical stunningwk post-infection (p.i.) at an age of 11 wk. Immediatelyfter slaughter, the gastrointestinal tracts were removednd the visceral organs were separated. Weights of liver+gall bladder), pancreas, full caeca as well as length ofmall intestine and each caecum were measured. Intactaeca from 10 birds per group (60 per repetition) wereeighed, frozen and stored at −18 ◦C until analyzed for

olatile fatty acids (VFAs).The caeca of the infected birds were further processed

or parasitological examinations to determine incidencend number of adult worms, as well as number of larvaes described by Gauly et al. (2008) and Das et al. (in press).he surface moisture of the empty caeca was removedsing paper towel, and the empty caeca weight was deter-ined. Average intact mature worm length was estimated

y measuring 20 female and male worms per bird. In caseshere lower than 20 worms per sex were available, all

he intact worms were measured. Caeca samples of unin-ected control birds (15–20% of each group) were alsorocessed to confirm infection free status of these groups.he caeca of the residual birds of each uninfected groupere pooled and checked for the presence of the nematode.total number of 475 birds and 287 faecal samples were

xamined for determination of worm burdens and EPG,espectively.

.4. pH and volatile fatty acids (VFAs)

The frozen intact caeca were thawed at room tempera-ure. The caecal content was removed from the caeca, andg was weighed and immediately afterwards suspended

n 10 ml of distilled water. The sample was mixed using aortex for around 5 s. The pH was directly measured in thisuspension using a pH electrode (InLab®Easy BNC, Fa. Met-ler Toledo) connected to a pH meter (GC 811, Fa, Schott).hereafter, the suspension was centrifuged at 2000 × g atoom temperature for 20 min. Five millilitres of super-atant was transferred to a glass tube, which contained50 �l international standard (4% methyl-valeric acid inormic acid). The mixture was vortexed and two parallelub-samples of 1.5 ml each were transferred to Eppendorfubes. The parallels were centrifuged at 10,000 × g at roomemperature for 10 min. After centrifugation, the samplesere stored in a refrigerator (+4 ◦C) until gas chromatogra-hy.

For gas chromatography, a combined internal/externaltandard procedure was applied using a packed column10% Carbowax 20 MTPA SP1000 with 1% H3PO4 on Chro-

osorb WAW, 80/100). Temperature for injection port was70 ◦C, for detector 200 ◦C and for column 120 ◦C (isother-al). The gas chromatograph (Shimadzu GC 14B) was

quipped with a flame ionization detector (FID) and hydro-en was used as the carrier gas (Da Costa Gomez, 1999;

ogy 178 (2011) 121–128 123

Abel et al., 2002). The average of the parallels was used forcalculations.

The remaining caecal contents after sampling for VFAswere used to determine dry matter, crude ash and organicmatter of the caecal contents.

2.5. Data management and statistical analyses

2.5.1. Parameter definitions, transformations andrestrictions

Because the data of the infection variables posi-tively skewed (Skewness >0) and showed non-normal(Kolmogorov–Smirnov, P < 0.05) distributions, log-transformations were employed. For this, individualinfection parameters that described worm counts (estab-lishment rate, number of males, females, larvae, and totalworm burden), EPG, total number of eggs excreted perworm population of a bird within 24 h (eggs per day; EPD)and female worm fecundity parameters were transformedby using the natural logarithm (ln) function [ln(y) = Log(y)]to correct for heterogeneity of variance and to producean approximately normally distributed data set. Estab-lishment rate was defined as the number of worms perbird in relation to infection dose. Adult female wormfecundity was defined based both on egg concentrationin gram faeces (EPG per female worm) as well as on totalnumber of the daily excreted eggs (EPD per female worm).Lengths of the male and female worms, sex ratio (numbersof females/males) and the amount of daily faeces of theinfected birds were left untransformed.

In preliminary analyses (with fixed effect of rep-etition), no significant (P > 0.05) interaction effects ofdiet × infection × experimental repetitions on any of theperformance parameters (e.g., BW, ADG) were observed.This partly indicated reproducible effects of diet and infec-tions on the performance parameters over the repetitions.However, because the experimental repetitions were per-formed in different periods of time, the effect of repetitionwas included in the models as a random factor to ensuresafe generalization of the effects of the main factors (dietand infection) and to avoid any possible confounding effectof time, in which the repetitions were performed, with anyof the main factors.

2.5.2. StatisticsStatistical analyses were performed with SAS V9.1.3

(2010). The effect of diet on worm-harboring birds as aproportion of experimentally infected birds (incidence ofinfection) was separately analyzed for each repetition. Thedifferences between incidences of infection among theinfected groups were analyzed with Fisher’s exact test, per-formed for all possible pair-wise combinations of the threeinfected groups.

Establishment rate, worm counts and nematode eggexcretion variables were analyzed with a mixed modelthat included fixed effect of the diets and random effect

of experimental repetitions using the MIXED procedure ofSAS. Data of VFA and visceral organ measurements wereanalyzed with an extended mixed model that includedfixed effects of diets, infection as well as interaction effectof diet and infection. Effect of experimental repetition

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124 G. Das et al. / Veterinary P

was included in the model as random. The model forthe repeatedly measured performance variables (e.g., BW,feed:gain) included fixed effects of diet, infection, exper-imental weeks (as age of birds) as well as all possibleinteractions among these factors. The effect of experimen-tal repetitions was included in the model as a randomfactor. Furthermore, individual random effect of the birdsas the repeated subject within a repetition over the exper-imental weeks, was also included in the model. Therepeatedly (weekly) measured variables were assumed tobe correlated from one measurement date to the next,and thus the covariance structure was set to be compoundsymmetry.

2.5.3. Presentation of the resultsAfter infection at the end of wk three, groups of unin-

fected and infected chickens were kept according to a 3 × 2factorial arrangement of treatments with diet and infec-tion as the main factors. Therefore, unless no significantinteractions between the effects of diet and infection wereencountered, the data are presented as the main effectsof diet and infection. In case of significant interactionsbetween diet and infection, the results are mentioned forthe 6 single treatments in the text. Tukey adjusted posthoc comparisons (˛ = 0.05) were performed to either par-tition effects of the main factors or to determine singlegroup differences when a non interactive significant maineffect or when a significant interaction effect of the mainfactors was encountered, respectively. For the effects ofthe main experimental factors, the results are presentedas least square means (LSMEANS) with common pooledstandard error (PSE).

3. Results

3.1. Feed consumption and performance

During the pre-infection period (1–3 wk), birds receiv-ing I-NSP and S-NSP consumed 6% more and roughly 1% lessfeed, respectively, compared to CON (Table 2). The coef-ficient of variation (CV) of feed consumption, calculated

Table 2Effects of diet and H. gallinarum infection on feed consumption, body weight (BW

Item Dieta

CON I-NSP S-NSP PSEc P-value

Pre-infection period (1–3 wk)Feed consumptiond, g/bird 336 356 332 – –BWe, g 201a 192b 192b 8.778 0.001Feed:gain, g/g 2.10a 2.38b 2.20c 0.094 0.001

Entire period (1–11 wk)Feed consumption4, g/bird 2910 3146 2964 – –BWf, g 989a 960b 954b 16.100 0.001Feed:gain, g/g 3.30a 3.64b 3.50c 0.067 0.001

[(abc) or (AB)]: Different letters within each factor on the same line indicate diffeNo: no infection effect in the pre-infection period.

a CON = basal diet; I-NSP = 1000 g CON + 100 g pea bran; S-NSP = 1000 g CON + 1b Uninfected controls or infected with 200 eggs of H. gallinarum.c Pooled SE.d Calculated from daily group consumptions, therefore no statistical comparisoe Body weight at the end of wk 3 of life.f Estimated from body weight of birds from wk 3 to 11 of life, presented as BW

ogy 178 (2011) 121–128

for repetitions within feeding groups, was smaller than 3%.Regarding the entire experimental period (1–11 wk), birdson the I-NSP and S-NSP diets consumed 8% and almost 2%more than the CON fed group and the CV within feedinggroups was smaller than 5%. Compared to CON, the NSP-diets reduced the BW gain of birds and impaired the feedconversion rate with I-NSP entailing a greater amount offeed per unit BW gain than S-NSP (P < 0.001). Infected birdsconsumed 1.6% less feed and had a lower BW (P < 0.001)accompanied by a higher feed:gain ratio (P < 0.001) thantheir uninfected counterparts.

3.2. Incidence of infection, worm burden and wormfecundity

In repetitions 1 and 3, feeding NSP led to higher inci-dences of infection than CON (P < 0.01), but no differencewas observed in the second repetition. Almost all NSP fedbirds had at least one worm in all three repetitions. In CONfed birds, 88%, 100% and 81% were harboring worms in thefirst, second and third repetitions, respectively. Birds on theNSP-diets had higher establishment rate (P < 0.001), num-ber of larvae (P < 0.009) and total worm burden (P < 0.001)than those receiving CON (Table 3). There was a trend fora lower ratio of female to male worms in birds being fedS-NSP instead of CON (P = 0.094). The worm length was notinfluenced by diet (P > 0.05).

The amount of faeces increased in the NSP-fed birds(P < 0.001) irrespective of the type of NSP (Table 4). The EPG,the EPD and EPD/female worm were elevated after feedingS-NSP (P ≤ 0.002), whereas I-NSP led to lower EPG/femaleworm (P < 0.05). The EPD increased in the sequence ofCON < I-NSP < S-NSP (P < 0.001).

3.3. Visceral organ development

Both types of NSP increased the relative pancreasweight, the caecum length and the empty caeca weight(P < 0.001; Table 5), and, with the exception of relative pan-creas weight, S-NSP exerted a greater effect than I-NSP(P < 0.05). The absolute liver weight was lower in S-NSP

), and feed:gain ratio.

H. gallinarum infectionb Interaction P-value

Uninfected Infected PSEc P-value

No No No No NoNo No No No NoNo No No No NoNo No No No No3030 2983 – – –978A 957B 16.050 0.001 0.5823.43A 3.53B 0.066 0.001 0.430

rences (Tukey, P < 0.05).

00 g chicory root meal.

n could be performed (–).

at the age of 11 wk.

G. Das et al. / Veterinary Parasitology 178 (2011) 121–128 125

Table 3Effects of diet on establishment rate, average number of worms per bird, sex ratio and length of worms in birds infected with H. gallinarum (200 eggs/bird).

Item Dieta PSEb P-value, ≤

CON I-NSP S-NSP

Establishment ratec, % 26.7a 46.9b 49.2b 11.959 0.001Number of female wormsc 26.6a 46.2a 47.6b 12.811 0.001Number of male wormsc 26.6a 45.9a 49.7b 11.481 0.001Number of larvaec 0.15a 1.62b 1.03b 0.470 0.009Total worm burdenc 53.4a 93.7b 98.4b 23.917 0.001Sex ratio, F/M 1.07 1.03 0.95 0.072 0.094Female worm length, mm 11.28 11.47 11.37 0.302 0.111Male worm length, mm 9.59 9.66 9.65 0.217 0.578

(ab): Values with no common letters within rows differ (Tukey, P < 0.05).a CON = basal diet; I-NSP = 1000 g CON + 100 g pea bran; S-NSP = 1000 g CON + 100 g chicory root meal.b Pooled SE.c LSMEANS and PSE represent untransformed data, P-values are based on the transformed data.

Table 4Effects of diet on the amount of faeces, the excretion of nematode eggs and the fecundity estimates of worms in birds infected with H. gallinarum (200eggs/bird).*

Item Dieta PSE P-value, ≤

CON I-NSP S-NSP

Faeces, g bird−1 d−1 28.26a 37.68b 36.43b 4.70 0.001EPGb 449a 581a 780b 180.91 0.002EPG/female wormc 13.2a 10.4b 14.9a 4.85 0.001EPDd 12148a 19138b 26181c 3548.59 0.001EPD/female worme 321.4a 344.0a 490.8b 96.10 0.002

(abc): Values with no common letters within rows differ (Tukey, P < 0.05).a CON = basal diet; I-NSP = 1000 g CON + 100 g pea bran; S-NSP = 1000 g CON + 100 g chicory root meal.b Number of eggs per gram of faeces.

per femulationle wormata, P-v

fantSawwbec

As shown in Table 6, percentage of DM in caecal contents

TE

[

c EPG based female worm fecundity: average number of eggs excretedd Number of eggs per day; total number of eggs excreted per worm pope EPD based female worm fecundity; number of eggs excreted per fema* LSMEANS and pooled standard error (PSE) represent untransformed d

ed birds than the CON fed birds (P = 0.005), however rel-tive weight of liver to BW (hepato-somatic index) wasot influenced by the diets (P = 0.629). The small intes-ine length and the full caeca weight were increased by-NSP (P < 0.05). Infection reduced liver weight (P = 0.005)nd increased the relative pancreas weight (P = 0.003) asell as the full caeca weight (P = 0.027). Empty caeca weight

as also elevated by infection (P < 0.001). An interaction

etween diet and infection (P = 0.039) revealed, that withinach feeding group infected animals had heavier emptyaeca than the uninfected ones, but uninfected S-NSP fed

able 5ffects of diet and H. gallinarum infection on the size of certain visceral organs.

Dieta

CON I-NSP S-NSP PSEc

Liver, g 18.0a 17.7ab 17.3b 0.499HS-Indexd, % (liver/BW) 1.84 1.84 1.85 0.020Pancreas, g 2.35 2.42 2.42 0.044g Pancreas/100 g BW 2.41a 2.53b 2.60b 0.087Small int. length, cm 108.6a 108.5a 112.6b 1.505Caecum length, cm 13.7a 14.1b 15.7c 0.083Full caeca weight, g 6.42a 6.54a 9.52b 0.178Empty caeca weight, g 2.46a 2.61b 3.29c 0.085

(abc) or (AB)]: Different letters within each factor on the same line indicate diffea CON = basal diet; I-NSP = 1000 g CON + 100 g pea bran; S-NSP = 1000 g CON + 1b Uninfected controls (−) or infected with 200 eggs of H. gallinarum (+).c Pooled SE.d HS-index: hepato-somatic index = liver/BW × 100.

ale worm through 1 g of faeces.of a bird within 24 h.

within 24 h.alues and multiple comparisons are based on transformed data.

birds exceeded infected ones receiving CON and did notdiffer from infected I-NSP-fed birds.

3.4. Biochemical characteristics of the caeca

was not influenced by diet (P = 0.246) but was decreasedby infection (P < 0.001). Feeding S-NSP decreased the pro-portion of crude ash and increased organic matter in theDM of caecal contents compared to feeding CON and I-

H. gallinarum infectionb InteractionP-value

P, ≤ − + PSE3 P, ≤0.005 17.9A 17.5B 0.494 0.005 0.8490.629 1.85 1.84 0.020 0.340 0.5870.097 2.39 2.40 0.042 0.559 0.4790.001 2.47A 2.56B 0.086 0.003 0.2440.001 110.2 109.6 1.482 0.362 0.6690.001 14.4 14.5 0.074 0.242 0.0750.001 7.35A 7.64B 0.171 0.027 0.2140.001 2.59A 2.98B 0.083 0.001 0.039

rences (P < 0.05).00 g chicory root meal.

126 G. Das et al. / Veterinary Parasitology 178 (2011) 121–128

Table 6Effects of diet and H. gallinarum infection on biochemical characteristics of the caeca.

Item Dieta H. gallinarum infectionb InteractionP-value

CON I-NSP S-NSP PSEc P, ≤ Inf. (−) Inf. (+) PSEc P, ≤Dry matter (DM), % 17.48 17.63 18.19 0.398 0.246 18.42A 17.11B 0.350 0.001 0.589Crude ash (% of DM) 13.86a 13.15a 11.41b 0.377 0.001 12.65 12.97 0.339 0.317 0.373Organic matter (% of DM) 86.14a 86.85a 88.59b 0.377 0.001 87.35 87.03 0.339 0.317 0.373pH 6.61a 6.59a 6.00b 0.153 0.001 6.32A 6.49B 0.151 0.002 0.210VFA molar ratios, %

Acetate 69a 71a 66b 1.301 0.001 68 69 1.229 0.091 0.785Propionate 15 15 14 2.142 0.581 14 15 2.093 0.389 0.278Butyrate 16a 15a 20b 1.219 0.001 18A 16B 1.183 0.001 0.212

VFA pool, �mold

Acetate 262.6a 247.0a 351.3b 20.769 0.001 312.8A 261.1B 18.945 0.003 0.116Propionate 52.5a 47.5a 69.5b 8.416 0.001 62.0A 51.0B 8.210 0.003 0.014Butyrate 61.0a 52.6a 108.9b 4.705 0.001 86.4A 62.0B 3.799 0.001 0.812Total 376.2a 347.1a 529.7b 30.091 0.001 461.2A 374.2B 27.403 0.001 0.144

[(abc) or (AB)]: Different letters within each factor on the same line indicate significant differences (Tukey, P < 0.05).a ented d

.

t caecal

CON = basal diet; I-NSP = insoluble non-starch polysaccharide supplemb Uninfected controls (−) or infected (+) with 200 eggs of H. gallinarumc Pooled SE.d Calculated as multiplication of VFA concentration by the total amoun

NSP (P < 0.001). Infection did not influence crude ash andorganic matter in the DM of caecal contents (P > 0.05). Feed-ing S-NSP reduced intracaecal pH and molar proportion ofacetate and increased that of butyrate as well as the caecalpools of individual and total VFAs compared to CON andI-NSP (P < 0.001). Infection increased pH (P = 0.002) accom-panied by lower molar proportion of butyrate (P < 0.001),pools of acetate (P = 0.003), butyrate (P < 0.001) as wellas the total VFAs pool (P < 0.001) of the caecal contents.Significant interaction effects of diet and infection wereobserved for the caecal propionate pool (P = 0.014), whichwas smaller (P < 0.05) in infected CON and I-NSP fed birdsand reached a similarly (P > 0.05) high level in all the othergroups.

4. Discussion

The inclusion of pea bran and chicory root meal implieda nutrient dilution in I-NSP and S-NSP diets compared toCON. However, because chickens are able to increase theirfeed intake when a nutritionally diluted diet is offered(Forbes and Shariatmadari, 1994; Halle, 2002; Van Krimpenet al., 2007; Das et al., 2010), the NSP fed birds couldhave consumed similar amounts of basal mixture nutri-ents as the CON fed birds. In fact, this was the case withI-NSP, whereas with S-NSP the increase in feed intake wasnot large enough to reach a similar level of basal mixtureintake compared to CON. In spite of these differences infeed intake and the larger intestines of S-NSP fed birds,the NSP fed birds developed lower BW than those on CON.H. gallinarum is commonly regarded as a non-pathogenicnematode (Taylor et al., 2007). However, in agreement withour previous results (Das et al., in press), growth perfor-mance was impaired in the infected birds independently

of the type of diet.

The increased sizes and weights of empty caeca, pan-creas and small intestine length indicate that NSP feeding,with S-NSP in particular, have caused preferred chan-neling of nutrients to the development of splanchnic

iet; S-NSP = soluble non-starch polysaccharide supplemented diet.

digesta.

tissues and the intestinal tract. Increased relative pan-creas weight in I-NSP and S-NSP fed birds indicates anadaptive response induced either directly by the diet(Iji et al., 2001) or indirectly by the intestinal micro-biota. The longer small intestine in S-NSP fed birdsindicates stimulated fermentation in the pre-caecal intes-tine too. Greater caeca size has repeatedly been observedafter feeding chicken with NSP (Redig, 1989; Clench andMathias, 1995; Jørgensen et al., 1996; Józefiak et al.,2004). Fermentation of inulin in chicken has been reportedto selectively support butyrate producing microorgan-isms and to lower luminal pH (Marounek et al., 1999;Rehman et al., 2008a,b). Volatile fatty acids and butyrate inparticular are known to exert trophic effects on the intesti-nal mucosa (Montagne et al., 2003). In agreement withthese observations, the pools of VFAs and butyrate wereincreased and the pH was lowered in the caeca of birdson the chicory root meal supplemented diet in the presentstudy.

Insoluble and soluble NSP supplemented diets almostdoubled the establishment rate of H. gallinarum. Establish-ment rate of H. gallinarum in a pheasant host system wasshown to be density dependent, i.e., the success rate of lar-vae developing to adult stage decreases as the infectiondose increases (Thompkins and Hudson, 1999). The samepattern has also been observed for A. galli in experimen-tally infected birds with different infection doses (Perminet al., 1997). With regard to the density dependent char-acteristic of the establishment rate, the great differenceobserved in the present study between CON and NSP fedbirds indicates that manifold favourable conditions wereprovided to the nematode by feeding NSP. Although nodifference between worm burdens of I-NSP and S-NSPfed birds was observed, these two diets differed in their

effects on the faecal egg counts and fecundity of the nema-tode.

Egg excretion and fecundity of the nematode havescarcely been quantified, probably due to difficulties insampling appropriate faecal material. Because the nema-

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G. Das et al. / Veterinary P

ode’s eggs are passed to the external environment throughaecal droppings (Fine, 1975), which are periodicallyxcreted within a day (Clarke, 1979), 24 h collection of fae-es originating both from intestines and caeca seems to berucial for a reliable egg quantification of the nematode.part from the periodicity in caecal faeces excretion, theaily total amount of faeces, which was elevated in theSP fed birds in agreement with results of others (Van derlis et al., 1993; Jørgensen et al., 1996), appears to cru-ially influence the EPG counts. Feeding I-NSP led to higherorm burden, but did not increase EPG and a lower EPG

ased fecundity (EPG/female worm) was calculated in com-arison to CON because of the greater amount of faeces.eeding S-NSP also led to greater worm burden and fae-es amount and EPG was in addition increased without anffect on EPG based worm fecundity. Thus, the EPG ren-ered unsatisfactory information about the actual infection

ntensity in the birds, even though the analyzed faecal sam-les were taken from well-stirred daily collections. Basedn EPD, I-NSP and particularly S-NSP increased worm eggxcretion compared to CON and the fecundity (EPD/femaleorm) was also elevated after feeding S-NSP. As shown in

he present study, inclusion of the daily total amount ofaeces for the calculation of EPD eliminates the dilutionffect of faeces and provides more accurate informationbout the actual infection status of the host animal asell as for the actual worm fecundity estimate than EPG

lone.Infection increased caeca size possibly by a histotropic

hase, in which the larvae embed themselves into caecalissue during the larval development as observed in A. gallinfection (Herd and Mcnaught, 1975). It has been shownhat bacteria play an important role for the establishmentf H. gallinarum (Springer et al., 1970). The inulin suppliedith S-NSP in the present study can be regarded as a prebi-

tic similar to pure inulin which has been shown to increaseacteria counts and metabolic activity of the microbesJuskiewicz et al., 2005; Rehman et al., 2008b). It can bessumed that the establishment and fecundity of the bac-eria feeder H. gallinarum (Bilgrami and Gaugler, 2004) wastimulated by NSP fermenting caecal bacteria. An explana-ion for the reduced caecal VFA pool observed in infectedirds can only be found if the rates of VFA production andtilization are investigated in more details. Undoubtedly,he nematode benefited from the altered NSP dependentaecal environment resulting in higher establishment ratend enhanced fecundity. However, when compared to theffects of S-NSP on the gastrointestinal organ developmentnd the parameters describing the intracaecal environmentorganic matter, pH, VFA), the effects of I-NSP were lessrominent than those of S-NSP. Enlarged caecal size andmpty weight induced by feeding both NSP diets mightave offered a larger space, thereby resulting in less com-etition for a higher establishment of larvae, while furtherltered caecal environment due to S-NSP corresponds wello the enhanced fecundity of the nematode in this feeding

roup.

The average establishment rates of the nematode in theON fed birds were similar to those reported for chickens

nfected either with 100 eggs (Gauly et al., 2008) or with–9 eggs/bird (Fine, 1975). However, establishment rates

ogy 178 (2011) 121–128 127

of the nematode in the CON fed birds in the present studywere higher than those of birds on the same diet infectedwith Histomonas contaminant Heterakis and treated withdimetridazole (Das et al., in press). The lower establish-ment rates observed in our preliminary study might haveresulted from an effect of dimetridazole not only againsthistomonads but also against obligately anaerobic bacte-ria species (Fernie et al., 1977; APVMA, 2007), and suchan effect appeared to be more pronounced in dimetrida-zole treated birds on the NSP diets as compared to theestablishment rates in the NSP fed birds in the presentstudy.

A study with the pig whipworm, Trichuris suis, showedthat supplementation of inulin decreased establishment,egg excretion and female worm fecundity (Petkeviciuset al., 2007) when compared with oat hull meal as source ofinsoluble NSP. Similar results have also been reported forthe pig nodule worm, Oesophagostomum dentatum. It hasrepeatedly been shown that lignin rich diets or supplemen-tations of insoluble NSP provided favourable conditionsfor the establishment of the nodule worm, whereas inulinsupplemented diets had a profound deworming effect(Petkevicius et al., 1997, 2001, 2003). The results reportedfor the pig nematodes are in agreement with the favourableeffects of I-NSP to H. gallinarum. However, in contrast tothe pig nematodes, the inulin rich S-NSP fed in the presentstudy also provided most favourable conditions for H. gal-linarum. The differences between responses of differentworm species residing in their specific hosts to the samesubstance (e.g., inulin) is of interest, and should further beinvestigated.

The higher number of excreted H. gallinarum eggs,due to either elevated number of worms in both NSPfeeding groups or additionally due to enhanced wormfecundity in the S-NSP fed birds, would contribute to astrongly contaminated environment and create a higherrisk for new or re-infections of birds under field con-ditions. This is particularly important for organic andfree-range poultry production systems, where energy andnutrient diluted fibre rich, i.e., NSP rich, diets are used(Sundrum et al., 2005; Van de Weerd et al., 2009). In thesesystems, birds are also in close contact with faeces allow-ing nematodes to complete their life cycles. Presumably,high amounts of dietary NSP in the bird’s rations mighthave already contributed to the reported high incidenceand intensity of H. gallinarum infections in such produc-tion systems (Permin et al., 1999; Kaufmann and Gauly,2009).

5. Conclusion

It is concluded that the pea bran and chicory rootmeal used as sources of insoluble and soluble dietary NSP,respectively, provided favourable conditions for the estab-

lishment of H. gallinarum in grower layers. Inulin richchicory root meal additionally enhanced fecundity of thenematode. Therefore, the two natural sources of insolu-ble and soluble NSP offer no potential as protecting agentsagainst H. gallinarum infections in chicken.

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Acknowledgements

Financial supports of the German Research Founda-tion (DFG) and the H. Wilhelm Schaumann Foundation aregratefully acknowledged. The authors thank Dr. Eva Moors,Ms. Birgit Sohnrey, Mr. Rolf Jeromin, Mr. Falko Kaufmann,Mr. Ahmad Idris and Mr. Abdussamad Abdussamad fortheir contributions.

References

Abel, Hj., Immig, I., Harman, E., 2002. Effect of adding caprylic and capricacid to grass on fermentation characteristics during ensiling and in theartificial rumen system RUSITEC. Anim. Feed Sci. Technol. 99, 65–72.

Australian Pesticides and Veterinary Medicine Authority (APVMA),2007. The reconsideration of registrations of products contain-ing Dimetridazole and their associated approved labels. FinalReview Report and Regulatory Decision, p. 3. URL: http://www.apvma.gov.au/products/review/docs/Dimetridazole final report.pdf(accessed 22.10.10).

Bilgrami, A.L., Gaugler, R., 2004. Feeding behaviour. In: Gaugler, R., Bil-grami, A.L. (Eds.), Nematode Behaviour. CABI Publishing, p. 98.

Clarke, P.L., 1979. Coccidial infection with Eimeria tenella and caecal defae-cation in chicks. Br. Poult. Sci. 20, 317–322.

Clench, M.H., Mathias, J.R., 1995. The avian cecum: a review. Wilson Bull.107, 93–121.

Da Costa Gomez, C., 1999. In-vitro-Untersuchungen zur reduktiven Ace-togenese im Pansen. Diss. Agr. Göttingen, 18–25.

Daenicke, S., Moors, E., Beineke, A., Gauly, M., 2009. Ascaridia galli infectionof pullets and intestinal viscosity: consequences for nutrient retentionand gut morphology. Br. Poult. Sci. 50, 512–520.

Das, G., Abel, H., Humburg, J., Schwarz, A., Rautenschlein, S., Breves, G.,Gauly, M. Non-starch polysaccharides alter interactions between Het-erakis gallinarum and Histomonas meleagridis. Vet. Parasitol., in press,doi:10.1016/j.vetpar.2010.11.004.

Das, G., Kaufmann, F., Abel, H., Gauly, M., 2010. Effect of extra dietary lysinein Ascaridia galli-infected grower layers. Vet. Parasitol. 170, 238–243.

Englyst, H., 1989. Classification and measurement of plant polysaccha-rides. Anim. Feed Sci. Technol. 23, 27–42.

Fernie, D.S., Ware, D.A., Park, R.W.A., 1977. The effect of the nitroimida-zole drug dimetridazole on microaerophilic campylobacters. J. Med.Microbiol. 10, 233–240.

Fine, P.E.M., 1975. Quantitative studies on the transmission of Parahis-tomonas wenrichi by ova of Heterakis gallinarum. Parasitology 70,407–417.

Forbes, J.M., Shariatmadari, F., 1994. Diet selection by poultry. World Poult.Sci. J. 50, 7–24.

Gauly, M., Kanan, A., Brandt, H., Weigend, S., Moors, E., Erhardt, G., 2008.Genetic resistance to Heterakis gallinarum in two chicken layer linesfollowing a single dose of infection. Vet. Parasitol. 155, 74–79.

Halle, I., 2002. Einfluss einer gestaffelten Supplementierung von Lysin undMethionin während der Aufzucht auf das Wachstum und auf die Leis-tungsmerkmale der Hennen in der folgenden Legeperiode bei einergestaffelten Protein- und Energieversorgung. Arch. Geflugelkd. 66,66–74.

Herd, R.P., Mcnaught, D.J., 1975. Arrested development and the histotropicphase of Ascaridia galli in the chicken. Int. J. Parasitol. 5, 401–406.

Iji, P.A., Saki, A.A., Tivey, D.R., 2001. Intestinal development and bodygrowth of broiler chicks on diets supplemented with non-starchpolysaccharides. Anim. Feed Sci. Technol. 89, 175–188.

Jørgensen, H., Zhao, X.-Q., Knudsen, K.E.B., Eggum, B.O., 1996. The influ-ence of dietary fibre source and level on the development of thegastrointestinal tract, digestibility and energy metabolism in broilerchickens. Br. J. Nutr. 75, 379–395.

Józefiak, D., Rutkowski, A., Martin, S.A., 2004. Carbohydrate fermentation

in the avian ceca: a review. Anim. Feed Sci. Technol. 113, 1–15.

Juskiewicz, J., Jankowski, J., Zdunczyk, Z., Biedrzycka, E., Koncicki, l.A.,2005. Performance and microbial status of turkeys fed diets containingdifferent levels of inulin. Arch. Geflugelkd. 69, 175–180.

Kaufmann, F., Gauly, M., 2009. Prevalence and burden of helminthes inlocal free range laying hens. In: Book of Abstracts of the 60th Annual

ogy 178 (2011) 121–128

Meeting of the European Federation of Animal Science ,. WageningenAcademic Publishers, p. 553.

MAFF, 1986. Manual Veterinary Parasitological Laboratory Techniques,3rd ed. Ministry of Agriculture, Fisheries and Food, HMSO, London(Reference Book 418).

Marounek, M., Suchorska, O., Savka, O., 1999. Effect of substrate and feedantibiotics on in vitro production of volatile fatty acids and methanein cecal contents of chickens. Anim. Feed Sci. Technol. 80, 223–230.

Maurer, V., Amsler, Z., Perler, E., Heckendorn, F., 2009. Poultry litter asa source of gastrointestinal helminth infections. Vet. Parasitol. 161,255–260.

McDougald, L.R., 2005. Blackhead disease (Histomoniasis) in poultry: acritical review. Avian Dis. 49, 462–476.

Montagne, L., Pluske, J.R., Hampson, D.J., 2003. A review of interactionsbetween dietary fibre and the intestinal mucosa, and their conse-quences on digestive health in young non-ruminant animals. Anim.Feed Sci. Technol. 108, 95–117.

Permin, A., Bojesen, M., Nansen, P., Bisgaard, M., Frandsen, F., Pearman,M., 1997. Ascaridia galli populations in chickens following single infec-tions with different dose levels. Parasitol. Res. 83, 614–617.

Permin, A., Bisgaard, M., Frandsen, F., Pearman, M., Nansen, P., Kold, J.,1999. The prevalence of gastrointestinal helminths in different poultryproduction systems. Br. Poult. Sci. 40, 439–443.

Petkevicius, S., Knudsen, K.E.B., Nansen, P., Roepstorff, A., Skjøth, F.,Jensen, K., 1997. The impact of diets varying in carbohydrates resis-tant to endogenous enzymes and lignin on populations of Ascarissuum and Oesophagostomum dentatum in pigs. Parasitology 114, 555–568.

Petkevicius, S., Knudsen, K.E.B., Nansen, P., Murrel, K.D., 2001. The effectof dietary carbohydrates with different digestibility on the popula-tions of Oesophagostomum dentatum in the intestinal tract of pigs.Parasitology 123, 315–324.

Petkevicius, S., Knudsen, K.E.B., Murrel, K.D., Wachmann, H., 2003. Theeffect of inulin and sugar beet fibre on Oesophagostomum dentatum inpigs. Parasitology 127, 61–68.

Petkevicius, S., Thomsen, L.E., Knudsen, K.E.B., Murrel, K.D., Roepstorff, A.,Boes, J., 2007. The effect of inulin on new and on patent infections ofTrichuris suis in growing pigs. Parasitology 134, 121–127.

Redig, P., 1989. The avian ceca: obligate combustion chambers or facul-tative afterburners? The conditioning influence of diet. J. Exp. Zool.Suppl. 3, 66–69.

Rehman, H., Hellweg, P., Taras, D., Zentek, J., 2008a. Effects of dietaryinulin on the intestinal short chain fatty acids and microbial ecology inbroiler chickens as revealed by denaturing gradient gel electrophore-sis. Poult. Sci. 87, 783–789.

Rehman, H., Böhm, J., Zentek, J., 2008b. Effects of differentially fermentablecarbohydrates on the microbial fermentation profile of the gastroin-testinal tract. J. Anim. Physiol. Anim. Nutr. 92, 471–480.

SAS Institute Inc., 2010. SAS OnlineDoc® Version 9.1.3, Cary, NC, USA.Springer, W.T., Johnson, J., Reid, W.M., 1970. Histomoniasis in gnotobiotic

chickens and turkeys: biological aspects of the role of bacteria in theetiology. Exp. Parasitol. 28, 383–392.

Sundrum, A., Schneider, K., Richter, U., 2005. Possibilities and limitationsof protein supply in organic poultry and pig production. Final ProjectReport EEC 2092/91 (Organic) Revision no. D 4.1 (Part 1). Depart-ment of Animal Nutrition and Animal Health, University of Kassel,Witzenhausen, Germany.

Taylor, M.A., Coop, R.L., Wall, R.L., 2007. Parasites of poultry and game-birds. In: Veterinary Parasitology, 3rd ed. Blackwell Publishing, ISBN978-1-4051-1964-1, p. 496.

Thompkins, D.M., Hudson, P.J., 1999. Regulation of nematode fecundityin the ring-necked pheasant (Phasianus colchicus): not just densitydependence. Parasitology 118, 417–423.

Van de Weerd, H.A., Keatinge, R., Roderick, S., 2009. A review of key health-related welfare issues in organic poultry production. World Poult. Sci.J. 65, 649–684.

Van der Klis, J.D., Van Voorst, A., Van Cruyningen, C., 1993. Effect of asoluble polysaccharide (carboxy methyl cellulose) on the physico-

chemical conditions in the gastrointestinal tract of broilers. Br. Poult.Sci. 34, 971–983.

Van Krimpen, M.M., Kwakkel, R.P., André, G., Van Der Peet-Schwering,C.M.C., Den Hartog, L.A., Verstegen, M.W.A., 2007. Effect of nutrientdilution on feed intake, eating time and performance of hens in earlylay. Br. Poult. Sci. 48, 389–398.