Trial I

4.2.2.1. Experimental design and diets

Fifty-four, day-old male and female broiler chickens (Ross 308) purchased from a commercial hatchery (Geflügelhof Schulz, Graz, Austria) were randomly divided into three groups.

Chickens were kept in floor pens using wood shavings bedding and were fed ad libitum (Fig.

6A). Three diets – a maize based (M), a maize-wheat based (M+W) and a M+W diet supplemented with 135 mg kg^-1 NSP-degrading enzyme (M+WE) - were supplied by Georgikon Faculty, University of Pannonia. The enzyme used in the M+WE diet was a Grindazym GP15000 product containing a combination of xylanase and glucanase. Diets were

isocaloric and isonitrogenous and were prepared to meet the nutrient requirements of Ross 308 broilers (Aviagen, 2014a). Composition and nutrient contents of diets are shown in Table 1.

Starter, grower and finisher diets were fed between days 1-10, days 11-24 and days 25-35, respectively. Each diet was fed one of the groups contained 18 chickens at the beginning of the experiment. Chickens were monitored daily for any adverse effects and clinical signs. Body weight of all chickens was measured on days 10, 24 and prior to euthanasia.

On days 1 and 14 of age, Campylobacter presence in chickens were tested by taking cloacal swabs which were direct-plated on Campylobacter Blood-Free Agar (CBFA; CM0739, OXOID, Hampshire, UK) for Campylobacter determination (42 °C, 48 hrs).

On day 14, the chickens were infected orally with 10⁸ CFU C. jejuni using crop gavages.

Chickens were killed 7, 14 and 21 days post infection (DPI) and bacteriological, histological and digesta samples were taken. At each time point 6 chickens per group were euthanized for sampling. The gut section ileum is referred as a part of the small intestine starting from the Meckel’s diverticuli to the ileocecal junction.

Fig. 6. Broiler chickens kept on wood shavings and on straw litter in Trial I (A) and II (B).

A B

Table 1. Composition of experimental diets in Trial I (g/kg)

Abbreviations: M – maize based diet; M+W – maize-wheat based diet;

*Premix was supplied by Visonka Kft. (Páhi, Hungary). The active ingredients contained in the vitamin-mineral premix were as follows (per kg of diet):

Starter and grower premix - Vitamin A - 2,4x10⁶ IU, Vitamin D3 - 8x10⁵ IU, Vitamin E – 1x10⁴ IU, Vitamin K3

Ingredient Starter Grower Finisher

M M+W M M+W M M+W

4.2.2.2. Challenge organisms and Campylobacter enumeration

Reference strain Campylobacter jejuni NCTC 12744 was cultured in LB medium (Lennox L broth base, Invitrogen by Life Technologies Corporation, California, USA) at 42^oC for 48 h under microaerobic conditions using GENbox microaer bags (BioMerieux, Vienna, Austria).

The C. jejuni bacteria were enumerated by preparing 10-fold dilutions in PBS (Gibco Life Technologies Corporation, California, USA) and plated on Campylosel agar (BioMerieux, Vienna, Austria), followed by microaerobic incubation at 42^oC for 48 h.

One gram of ileum (5 cm below to Meckel’s diverticuli) and cecum including content and a piece of intestinal tissue were aseptically taken, homogenized and a 10-fold dilution series was made in PBS. One hundred µl of each dilution were inoculated onto Campylosel agars (BioMerieux, Vienna, Austria). Plates were incubated at 42^oC under microaerobic condition.

Greyish, gleaming and bulging colonies were counted after 48 h of incubation and the presence of Campylobacter was confirmed by examining colonial morphology, motility and shape of the bacteria.

4.2.2.3. Analytical methods

Fresh ileal and cecal contents were diluted immediately after collection with distilled water (1:5) and vortexed manually by shaking for 1 minute. Measurement of the pH values were carried out with a SNEX electrode (pH200A Portable pH meter equipped with CS1068 SNEX pH Sensor, CLEAN Instruments, Sanghai).

To measure the ileal digesta viscosity, 2 g of digesta were frozen and stored at -80^oC. After thawing samples were centrifuged (12,000 G for 10 min) and the viscosity of the supernatant (0.5 ml) was measured using a Brookfield DV II+ viscometer (Brookfield Engineering Laboratories, Stoughton, MA, USA) at 25^oC with a CP40 cone and shear rate of 60-600s^-1 (Fig.

7).

Fig. 7. Computer controlled viscosity measurement with a Brookfield DV II+ viscometer attached to a temperature controlled circulating water bath (A) and pipetting of 0.5 ml ileal supernatant into the cone of the viscosimeter(B).

For SCFA analyses 1 g of cecal content samples were frozen, and were stored at -80 ^oC and the analyses were prepared as described by Atteh et al. (2008). A standard SCFA mixture (20 mmol l^-1) of acetic, propionic, isobutyric, butyric, isovaleric, valeric acid was used for calibration as external standard.

One microliter of the ether phase extract was injected into a Gas Chromatograph (TRACE 2000, Thermo Scientific, USA). The instrument was equipped with a Nukol Fused Silica Capillary Column (30 m x 0.25 mm with a film thickness of 0.25 µm; Supelco, USA). The carrier gas was helium with a pressure of 83 kPa. The detector type was FID with a split injector (1:50).

Injector and detector temperatures were 220 and 250 ^oC, respectively.

Tissue samples were taken from ileum close to the junction of Meckel’s diverticulum for histomorphological examination. Samples were fixed in 5% buffered formalin. The processing consisted of serial dehydration, clearing and impregnation with wax. Tissue sections, 5 m thick (three cross-sections) from each of 6 chickens per treatments, were cut by a microtome and were fixed on slides.

A routine staining procedure was carried out using hematoxylin and eosin. The slides were examined on an Olympus BX43F light microscope (Olympus Corporation, Tokyo, Japan) fitted

A B

with digital video camera (Olympus DP-26) using Olympus Stream 1.7 software. The images were analyzed with ImageJ software (Version 1.47) developed by National Institutes of Health (Maryland, USA). A total of 10 intact, well-oriented crypt-villus units were selected in triplicate for each intestinal cross-section for all samples (Fig. 8). Apparent villus surface area were calculated as: (villus height * (apical transverse + basal transverse)/2)/10⁶.

Fig. 8. Hematoxillin-eosin stained ileal cross section. Numbers (1-5) indicate measurements of histomorphology (1_villus height, 2_crypt depth, 3_basal transverse, 4_apical transverse, 5_muscle layer thickness)

4.2.2.4. Statistical analyses

All data were analysed by using SPSS 16.0 software. The arrangement of the results for viscosity, SCFA, pH and histomorphology data was regarded as a 3 x 3 general linear model, with dietary treatments and sampling time points as independent variables. Differences were considered significant at a level of P ≤ 0.05.

Campylobacter counts were analyzed for diet and time effect separately by Kruskal-Wallis tests. Prior to statistical evaluation Campylobacter counts were scored on a scale from 1-6, respectively, ranging them as follows: (1) to <10^1,5, (2)10^1,5-10³, (3)10³-10^4,5, (4)10^4,5-10⁶, (5)10⁶-10^7,5, (6)>10^7,5.