A wheat-based diet enhances colonization with the intestinal spirochaete Brachyspira intermedia in experimentally infected laying hens

Abstract

This study investigated the influence of feeding diets based on cereals with different non-starch polysaccharide content on colonization with the intestinal spirochaete Brachyspira intermedia in experimentally infected laying hens. The diets were based on wheat, barley, or barley and sorghum, all with or without supplementation with exogenous dietary enzymes. Six groups of 12 individually caged laying hens were fed the respective diets for 2 weeks, and then challenged at 20 weeks of age by crop tube with 10⁸ to 10⁹ colony forming units of active motile spirochaetes on five successive days. Eggs were collected daily. The birds were weighed weekly, and caecal faeces collected for assessment of water content. Every 3 to 4 days cloacal swabs were collected and subjected to culture and subsequent polymerase chain reaction for B. intermedia. The birds were killed at 27 weeks of age. Caecal contents were cultured for spirochaetes, and the viscosity of the ileal and rectal contents assessed. Dietary soluble non-starch polysaccharide content was not a consistent predictor of viscosity in the ileum, and the addition of enzymes did not significantly reduce the viscosity. A dietary influence on colonization was observed, with birds fed wheat having significantly more colonization than the birds fed the other diets. No significant association was found between either dietary soluble non-starch polysaccharide content or ileal viscosity and colonization with B. intermedia, as assessed by faecal excretion of the spirochaete. Dietary enzymes did not consistently or significantly reduce colonization. It was not established how wheat enhances colonization by B. intermedia, but the results suggest that wheat-based diets should be avoided in flocks infected with B. intermedia.

1 Introduction

An increase in water content of the faeces of laying hens can cause problems for individual flocks. Problems include difficulties with mechanical cleaning, faecal staining of eggshells, increased odour from sheds, and attraction of flies. It is generally accepted that diet has a strong influence on faecal consistency and water content. Particularly in broilers, it has been shown that an increased non-starch polysaccharide (NSP) content of the diet, especially the soluble NSP (sNSP) content, can result in wet, sticky faeces together with an increased viscosity of the intestinal contents (Choct & Hughes, 1997). Exogenous dietary enzymes designed to hydrolyse these sNSPs are now routinely used in broiler diets to overcome such problems (Bedford & Schulze, 1998). Addition of enzymes to layer diets that contain sNSP from wheat, barley or rye also has been reported to reduce both the viscosity of the intestinal contents and the incidence of dirty eggs (Lazaro et al., 2003).

Besides dietary effects, increased faecal water content in chickens can result from infection with a number of different pathogenic microorganisms. Among these, the anaerobic intestinal spirochaete species Brachyspira intermedia, Brachyspira pilosicoli and Brachyspira alvinipulli can all infect the caeca and rectum of adult birds (McLaren et al., 1997 ). Infection with the first two species is widespread in commercial layer and broiler breeder flocks (McLaren et al., 1996; Stephens & Hampson, 1999). Experimentally, infection of laying hens with B. intermedia has been shown to cause both increased faecal water content and reduced egg production (Hampson & McLaren, 1999).

Recently, the authors found that the duration of colonization with B. intermedia in laying hens that were fed a wheat-based diet could be reduced by the dietary inclusion of a commercial enzyme supplement (Avizyme® l302; Danisco, Marlborough, UK), designed to hydrolyse the sNSP in wheat (Hampson et al., 2002). At the same time, work in pigs showed that increasing the digesta viscosity by addition of high-viscosity carboxymethylcellulose to the diet predisposed to infection with the intestinal spirochaete B. pilosicoli (Hopwood et al., 2002). Hence, the protection observed in the laying hens might have resulted from the enzyme acting to reduce the viscosity of the intestinal digesta, which in turn led to less colonization by the spirochaete. The mechanism(s) by which viscosity and spirochaete colonization might be linked are not clear, but, for example, viscous intestinal contents may decrease the feed transit time, leading to the presence of additional substrate for the spirochaetes in the caeca. Alternatively, the viscous contents might alter the thickness or composition of the mucus overlying the caecal epithelium, such that the highly motile spirochaetes obtain a selective advantage.

The purpose of the current experiment was to investigate the association between dietary sNSP, viscosity of the intestinal contents and the colonization of laying hens with B. intermedia following experimental infection. The hypothesis being tested was that increased digesta viscosity arising from the presence of sNSP in the diet would enhance colonization with B. intermedia, and that hydrolysing the sNSP with specific exogenous enzymes would result in a reduced digesta viscosity and less spirochaetal colonization. In order to manipulate the sNSP content of the experimental diets, each was based on a different cereal grain or combination of grains with high sNSP contents, with or without the addition of appropriate dietary enzymes designed to hydrolyse the sNSP in the diet.

The long-term aim of the work was to identify means to reduce or modify infections with intestinal spirochaetes by the use of specific diets or dietary treatments, without having to resort to antibiotic therapy.

2 Materials and Methods

This experiment was conducted with the approval of the Murdoch University Animal Ethics Committee.

2.1 Experimental birds

Seventy-two ISA-Brown laying hens were purchased from a commercial breeder at 18 weeks of age, and were housed in individual adjacent cages with mesh floors located in an environmentally controlled (25°C) facility. The hens were subjected to 12 h artificial light each day. Experimental infection commenced at 20 weeks of age, once the hens were in lay, and they were euthanased at 27 weeks of age.

2.2 Diets and feeding

Three diets were fed, with each dietary group being divided into the diet with or without an appropriate commercial dietary exogenous enzyme being added to the dry diets (i.e. six groups in total). The specifications of the diets were typical for laying hens of this age. The cereal sources used in the three diets were wheat, barley, or barley+sorghum, respectively (Table 1). The NSP contribution from all the non-cereal sources in the diet were balanced across diets, so that any difference in NSP content between diets was due to the cereal of interest. The NSP contents of the dry diets were measured in the laboratory of Professor Mingan Choct in the School of Rural Science and Agriculture at the University of New England, using the standard methodology described by Spiller (1993); they are summarized in Table 2 . Diets were fed as a mash, and the mash and fresh drinking water were available ad libitum.

Composition of the diets used in the experiment

Diet	Wheat diet	Barley diet	Barley+sorghum diet
Ingredient (%)
Wheat	49.24	–	–
Barley	–	50.58	24.65
Sorghum	–	–	25.76
Choline chloride, 75%	0.01	0.01	0.01
Groats	15.00	15.000	15.00
Lupin kernel	7.00	7.00	7.00
Soya-bean meal	11.20	10.00	10.00
Meat and bone meal	2.00	2.35	3.50
Fishmeal (Peruvian)	1.00	1.00	1.00
Canola oil	2.00	2.15	1.00
Limestone fine	10.40	10.00	10.45
Dicalcium phosphate	1.20	1.05	0.75
Salt	0.31	0.23	0.24
DL-Methionine	0.13	0.13	0.14
L-Lysine HCl	0.01	–	–
Enzymes premix	0.25	0.25	0.25
Household layer premix^a	0.25	0.25	0.25
Calculated analysis
AME (kcal/kg)^b	2738	2739	2735
Protein (%)	17.28	17.14	17.00
Calcium (%)	4.45	4.29	4.50
Total phosphate (%)	0.63	0.61	0.63
Available phosphate (%)	0.42	0.40	0.40
Methionine (%)	0.37	0.37	0.37
Total sulphur amino acids (%)	0.68	0.68	0.66
Lysine (%)	0.79	0.80	0.78
Threonine (%)	0.60	0.61	0.60
Tryptophane (%)	0.19	0.18	0.18
Leucine (%)	1.22	1.20	1.33
Fat (%)	4.76	5.10	4.33
Linoleic acid (%)	2.11	2.29	1.73
Crude fibre (%)	3.03	3.87	3.13
Sodium (%)	0.16	0.14	0.16
^aContaining minerals and vitamins to meet the nutritional requirements of birds of this age and weight (National Research Council, 1994).^bApparent metabolizable energy.

Non-starch polysaccharide (NSP) content of the six experimental diets (g/kg)

	Cereal base used in the experimental diets
NSP and free sugar content	Wheat	Wheat+enzyme	Barley	Barley+enzyme	Barley+sorghum	Barley+sorghum+enzyme
Insoluble NSP	75.93	67.72	93.90	85.78	65.61	70.12
Soluble NSP	15.66	17.79	30.65	30.41	20.44	20.98
Total NSP	91.59	85.51	124.55	116.19	86.05	91.10
Free sugars^a	22.82	24.87	26.23	26.48	20.44	24.68
^aIncluding rhamnose, fucose, ribose, arabinose, xylose, mannose, galactose and glucose.

Dietary enzymes from DSM Nutritional Products (French's Forest, NSW, Australia) were added to duplicates of each diet. Ronozyme WX at 200 g/tonne was added to the wheat-based diet: this contained endo-1-4-β-xylanase (IUB No, 3.2.1.8) with a minimal activity of 1000 units of xylanase activity/g. Ronozyme A at 200 g/tonne was added to the barley-based diet: this contained α-amylase (IUB 3.2.1.1) with a minimum activity of 200 units of α-amylase activity/g, and endo-1,3-1,4-β-glucanase (IUB 3.2.1.6) with a minimum activity of 350 units of β-gluconase activity/g. For the barley+sorghum diet, both Ronozyme WX and Ronozyme A were added, both at 100 g/tonne. The enzyme replaced the equivalent amount of cereal in the respective diet.

2.3 Experimental infection

B. intermedia strain HB60 was obtained as a frozen stock culture from the collection held by the Reference Centre for Intestinal Spirochaetes at Murdoch University. The strain was originally isolated from a Western Australian layer flock with a wet litter problem (McLaren et al., 1996), and had previously been used to experimentally infect laying hens (Hampson & McLaren, 1999; Hampson et al., 2002). The strain was thawed and grown in Kunkle's anaerobic broth medium (Kunkle et al., 1986) at 37°C on a rocking platform until early log-phase growth was achieved, when the spirochaetes were actively motile. The growth and absence of contamination was monitored by examining aliquots taken at daily intervals under a phase contrast microscope. At 20 weeks of age, cloacal swabs were taken and cultured to ensure that the hens were not colonized by intestinal spirochaetes. All the hens then were orally inoculated via a crop tube with 4 ml actively growing culture, on five consecutive days. The broths contained approximately 10⁸ to 10⁹ colony forming units/ml.

2.4 Experimental monitoring

The hens were weighed on entry to the experiment at 18 weeks of age and at weekly intervals thereafter. Eggs were collected daily, counted and weighed. At weekly intervals, starting at 21 weeks, aluminium foil was placed under the cage of each hen, and after 1 h individual caecal faeces samples were identified and collected. Approximately 1-g portions of the samples were weighed, then dried to constant weight at 120°C in a hot air oven to determine the moisture content. Immediately before the experimental infection and then every 2–4 days, starting 2 days after the last experimental inoculation, cloacal swabs were taken from each hen. A total of 17 sets of these post-infection swabs was collected (i.e. 204 samples in total). Excreta samples were plated to selective agar for isolation of intestinal spirochaetes, consisting of Trypticase Soy agar (BBL, Cockeysville, MD, USA) containing 400 μg/ml spectinomycin and 25 μg/ml each of colistin and vancomycin (Jenkinson & Wingar, 1981). Plates were incubated at 37°C in an anaerobic jar in an atmosphere of 94% H₂ and 6% CO₂ for 7 days, and then were scored visually for the presence or absence of intestinal spirochaete growth.

Bacterial growth on the primary isolation plates was subjected to specific polymerase chain reaction (PCR) amplification for the detection and identification of B. intermedia. A cell-pick method was used to ohtain spirochaetal DNA from the plates (Atyeo et al., 1998). The tip of a sterile wooden toothpick was used to stab through the area of spirochaete growth, and the material adhering to the tip was resuspended in 50 μl ultra-pure water before being boiled for 30 sec. A 25 μl volume was added to the B. intermedia PCR reaction, which was designed to amplify a 567 base pair portion of the NADH oxidase gene (nox). The primer pair used was Intl (5′-AGAGTTTGATGAYAATTATGAC-3′) and Int2 (5′-ATAAACATCAGGATCTTTGC-3′). DNA was amplified by hot-start PCR in a 25 μl total volume using HotStarTaq DNA polymerase (QIAGEN GmbH, Hilden, Germany), according to the manufacturer's instructions. Briefly, amplification mixtures consisted of 1× PCR buffer (containing 1.5 mM MgCl₂), 0.5 u HotStarTaq DNA polymerase, 0.2 mM each dNTP (Amersham Pharmacia Biotech AB, Uppsala, Sweden), 0.5 μM primer pair and 2.5 μl chromosomal template DNA. Cycling conditions involved an initial 15 min HotStarTaq DNA polymerase activation step at 95°C, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec, and a primer extension at 72°C for 1 min. The PCR products were subjected to electrophoresis in 1.5% (w/v) agarose gels in 1× TAE buffer (40 mM Tris-acetate, 1 mM ethylenediaminetetra-acetic acid), stained with ethidium bromide and viewed over ultrariolet light.

2.5 Postmortem examination

Six weeks after the last experimental inoculation, at 27 weeks of age, the birds were killed by cervical dislocation and were subjected to postmortem examination. The caeca and rectum were opened and examined for evidence of gross changes. Ileal and rectal contents were collected for viscosity measurements from each of the birds at postmortem, and immediately frozen at −80°C. A bacteriology swab was rubbed on the wall of one caecum, and this was cultured for spirochaetes as for faecal samples. A section of the other caecum was placed in 10% neutral buffered formalin as a fixative for subsequent histological examination. This tissue was processed through to paraffin blocks, sectioned at 4 μm and stained with haematoxylin and eosin before being examined.

2.6 Viscosity of intestinal contents

Ileal and rectal contents were thawed and measurements of the viscosity (mPa sec) performed using a Brookfield LVDV-II+ cone plate (CP40) rotational viscometer (Brookfield Engineering Laboratories, Inc., Stoughton, MA, USA). The samples were diluted 1:1 (v/v) with distilled water, mixed and centrifuged at 12 000g for 8 min. The viscosity of 0.5 ml supernatant fractions was measured at 25°C, applying a shear rate of 60/sec (McDonald et al., 2001).

2.7 Statistical analysis

Comparisons were made between the groups of birds, using InStat version 3 (Graphpad Software; San Diego, CA, USA). The number of birds that were colonized with B. intermedia in each group, and the number of faecal samples that were positive for B. intermedia relative to the total number of samples that were taken over the course of the experiment, were compared using chi-squared tests. Weekly group bird weights, faecal moisture content, egg numbers, egg weights and intestinal viscosity values were compared using one-way analysis of variance. Means were compared using the Tukey–Kramer multiple comparisons test, and significance was accepted at the 5% level. Regression analyses were made between each of the dietary sNSP, insoluble NSP and total NSP contents and the viscosity of the ileal and rectal contents in the birds on these diets. Regression analyses also were made between the dietary sNSP, insoluble NSP and total NSP content and colonization with B. intermedia (number ot sample days culture positive).

3 Results

3.1 Colonization with B. intermedia

The results for faecal excretion of B. intermedia during the weekly samplings are presented in Table 3. Not all birds became colonized, and there was no significant group effect on the number of birds that shed the spirochaete. The faeces of several birds were positive at all or most of the samplings taken over periods of up to 4 weeks. There was a significant overall effect of diet on number of days when faecal samples were positive χ²=19.831, P=0.001). Subsequent pair-wise comparison of colonization rates between dietary groups indicated the birds on the wheat-based diet without enzyme had significantly more colonization than birds in all other groups, except those receiving wheat with enzyme. In addition, the group receiving barley with enzyme demonstrated significantly less colonization than both the other cereal groups receiving enzyme. No other differences were significant. For the wheat and the barley diets, addition of enzyme to the diets was associated with a trend for less colonization, but the reverse occurred with the barley+sorghum diet. Regression analysis of sNSP, insoluble NSP and total NSP content of the diets all gave slopes of the lines that were not significantly different from zero (r ² values of 0.526, 0.046 and 0.175, respectively; P values of 0.103, 0.684 and 0.409, respectively). Regression analysis of ileal and rectal viscosity values against colonization also failed to reveal significant relationships. The r ² value for the regressions were 0.212 and 0.163 and the P values were 0.358 and 0.428, respectively.

Colonization of birds with B. intermedia, showing number of colonized hens and of faecal-positive samples of a possible 204 taken at 17 samplings between 21 and 26 weeks of age

	Cereal base of diet
Colonization	Wheat	Wheat+enzyme	Barley	Barley+enzyme	Barley+sorghum	Barley+sorghum+enzyme	P value
Number of hens colonized	8	8	7	5	7	8	Not significant
Number of samples positive	42^A	30^AB	25^BC	14^C	20^BC	27^B	0.001
Note: Group values having different superscripts differ at the 5% level of significance (paired chi-squared analysis).

3.2 Faecal water content

No significant group effects on faecal water content were identified at any sampling time. Group mean faecal water contents ranged from 71.2% to 79.9%.

3.3 Body weights and egg production parameters

The only significant group differences in body weight occurred towards the end of the experiment. There was an overall effect (P=0.042) when the birds were 26 weeks of age, with birds fed barley+sorghum+enzyme tending to be heavier than the other groups of birds. At 27 weeks of age the significance increased (P=0.026), with the birds fed barley+sorghum+enzyme now being significantly heavier than those fed barley without enzyme (2.07±0.12 kg compared with 1.92±0.12 kg).

The only significant group effect on egg production occurred when the birds were 23 weeks of age (P=0.027). The birds receiving wheat without enzyme produced significantly fewer eggs than the group receiving barley without enzyme (4.3±2.4 eggs per week compared with 6.8±0.4 eggs per week). No other group differences in egg numbers or weights were significant at any age.

3.4 Postmortem findings

At postmortem, all birds were in good condition. No pathological changes were found, and no histological evidence of typhlitis was observed. B. intermedia was isolated from the caeca of six birds: two fed wheat, one fed barley, two fed barley+sorgum with enzyme and one fed the latter diet without enzyme.

3.5 Viscosity of the digestu

Group results for digesta viscosity are presented in Table 4. There was a significant dietary effect at the ileum (P=<0.001), with birds fed barley + enzyme having significantly more viscous digesta than birds in the other groups, except for those receiving barley without enzyme. There were no other significant differences between groups. Addition of enzyme was associated with a numerically lower group mean ileal digesta viscosity in birds fed wheat and barley + sorghum, but with a higher viscosity in birds fed barley. Regression analyses of sNSP, insoluble NSP and total NSP content of the six diets against mean ileal viscosity gave r ² values of 0.454, 0.475 and 0.51, respectively, and the slopes were not significantly different from zero (P=0.142, 0.130 and 0.111, respectively).

Mean and standard deviation of viscosity of intestinal contents (mPa sec) at postmortem

	Cereal base of diet
Site	Wheat	Wheat+enzyme	Barley	Barley+enzyme	Barley+sorghum	Barley+sorghum+enzyme	P value
Ileum	195^A±0.22	1.69^A±0.28	2.01^AB±0.13	2.41^B±0.70	1.84^A±0.40	1.61^A±0.20	<0.001
Rectum	1.77±0.45	1.48±0.21	1.72±0.24	1.62±0.41	1.47±0.25	1.48±0.36	0.150
Note: Group means having different superscripts differ at the 5% level of significance (Tukey–Kramer multiple comparisons test).

Viscosity values in the rectum were lower than in the ileum, and no significant group differences were detected at this site. Addition of enzyme was associated with a numerically lower mean rectal viscosity in birds fed wheat, and a slightly lower value in those fed barley, but there were no differences for the birds fed barley + sorghum with or without enzyme. Regression analyses of sNSP, insoluble NSP and total NSP content of the diets against mean rectal viscosity gave r ² values of 0.055, 0.527 and 0.327, respectively. As in the ileum, none of the slopes were significantly different from zero (P=0.655, 0.102 and 0.236, respectively).

4 Discussion

This experiment utilized diets differing in their NSP content. The sNSP content of the barley-based diet was about twice that of the wheat-based diet, while the sNSP content of the barley+sorghum diet was a little higher than that of the wheat diets (Table 2). In comparison with the sNSP contents, the insoluble NSP and the total NSP contents of the barley-based diet were only about 20% higher than those of the other two diets. Hence, in relation to NSP, the difference in the diets mainly resided in their sNSP content. It was not surprising that addition of the enzymes to the dry diets had little or no effect on their measured NSP contents, as these enzymes require moisture for activation. In retrospect, it was realized that it would have been more informative to have moistened the diets and allowed the enzymes time to act before measuring the NSP content.

The different diets resulted in different viscosities of the ileal digesta, but this effect was not observed in the rectum, presumably as a result of bacterial degradation of remaining dietary substrate in the caeca. As expected, the barley-based diets, with the greatest sNSP content, resulted in the greatest viscosity of the ileal digesta. Across all six diets, however, the relationship between sNSP content and viscosity of the ileal digesta was not linear. For example, the wheat-based diet without enzyme had about one-half of the sNSP content of the barley diet without enzyme, but the mean viscosity of the ileal contents in birds on the two diets was similar. One explanation for this difference could be that specific subfractions of the sNSP, or other factors or components of the wheat besides sNSP, could act independently to increase digesta viscosity values in the ileum of laying birds.

Addition of the recommended dose rates of dietary enzymes, each designed for diets containing the particular cereal types used, had no significant effect on the viscosity of the digesta. Interestingly, a similar lack of effect of enzymes on digesta viscosity in laying hens has recently been reported (Roberts, 2003). In part, this lack of effect may have been due to the low intestinal viscosity values found in all hens, in both studies. Generally, enzymes are believed to be more effective and useful in broilers than in adult birds, probably because the intestinal viscosity is greater and more limiting in younger birds (Almirall et al., 1995; Petersen et al., 1999). In the study by Lazaro et al. (2003), where enzymes successfully reduced digesta viscosity in laying hens, viscosity values were considerably greater than those found here.

In the current study there was a tendency for the different enzymes to have different effects. Hens fed wheat tended to have lower viscosities in the ileum and rectum if their diets were supplemented with xylanase, but in the case of the barley diet the viscosities were actually higher in the ileal contents in hens receiving α-amylase and β-glucanase than in those not receiving enzymes. As NSPs in barley and wheat have different branching structures (Bacic & Stone, 1981), the effectiveness of any particular enzyme will be more related to its appropriateness and efficacy for the substrate than to the total quantity of NSP in the diet. Hence, when evaluating the effectiveness of dietary enzymes, it is necessary to consider these in the context of their use with the specific cereal sources for which they are designed.

A highly significant dietary effect on colonization with B. intermedia was found, with hens fed wheat shedding spirochaetes in their faeces more frequently than birds on the other diets. This result was somewhat unexpected, as the hens fed the diet with the lowest sNSP (wheat) had the greatest colonization, and those with the highest sNSP (barley) had the least colonization. The sNSP content of the diet therefore did not predict colonization rates. Similarly, the insoluble NSP, total NSP or Free sugar contents of the diets were not related to colonization rates. The wheat diet did result in ileal and rectal viscosities similar to those with the barley diets, but overall regressions of viscosity values against colonization did not reveal a significant relationship between the two at either intestinal site. Hence this experiment also failed to show a clear relationship between viscosity of the ileal and rectal contents and colonization with B. intermedia. The specific properties of the wheat-based diets that have this effect of encouraging colonization remain unknown, and require more detailed analysis. It is possible that wheat has some other non-sNSP-related effects on the physio-chemical properties of the digesta, or on components of the intestinal microflora, which in turn predispose to colonization by intestinal spirochaetes. A better understanding of these interactions could greatly assist the development of control strategies for these bacteria, and possibly even for other enteric pathogens.

The hens given the enzyme preparation with the wheat diet in the current experiment tended to have less colonization than those receiving wheat without enzyme, but this difference was not significant. In our previous experimental work a significant enzyme effect occurred (Hampson et al., 2002), although a different enzyme preparation was used (Avizyme® 1302, which included a protease with the xylanase). Besides the action of the protease, the two xylanases also might have different sites of catalytic action, and different efficiencies against different NSP components. Further work is required to determine to what extent and by what mechanisms different dietary enzyme preparations might influence colonization with B. intermedia.

In this experiment neither infection with B. intermedia nor feeding the various dietary combinations had obvious influences on production parameters or on health. This lack of effect may reflect the small group sizes used, the relatively stress-free and uncrowded environment in which the birds were housed, and the relatively short experimental period. Had the experiments been conducted under commercial conditions, greater differences may have been seen.

In conclusion, this experiment demonstrated that there are dietary influences on colonization of laying hens with B. intermedia, with wheat predisposing to colonization. The sNSP content of the diet was not the main influence, and the viscosities of the ileal and rectal contents also were not good predictors of whether or not birds were susceptible to infection. Similarly, in this experiment, the sNSP content of the diet was not a good predictor of the viscosity of the ileal or rectal digesta in laying hens. Addition of dietary enzymes did not significantly change the viscosity of the ileal or rectal contents under the experimental conditions used, and depending on diet, addition of dietary enzyme had inconsistent and non-significant effects on altering the susceptibility of the hens to colonization by B. intermedia.

Translations of the abstract in French, German and Spanish are available on the Avian Pathology website.

Acknowledgments

This study was funded through a grant from the Australian Chicken Meat Council and the Egg Industry Council (the latter now the Australian Egg Corporation), through the Rural Industries Research and Development Corporation. The authors thank Carol Stephens from the Toowoomba Veterinary Laboratory for assistance with planning the experiment, Jeff Ross of DSM Products for donating the enzymes used in the study, Daniel Goussac of WesFeeds Pty Ltd for formulation of the diets, Professor Mingam Choct of the University of New England, Armidale for the NSP analysis of the diets, and Fiona Cavaney for undertaking the viscosity measurements.