Colectores solares, reciclaje de aguas grises y paneles solares:

0 Clean interdigital space with no dermatitis lesion or fetid smell 1 Slight interdigital dermatitis, irritation of the skin but dry

2 Slight interdigital dermatitis with a fetid smell < 5% skin affected 3 Moderate interdigital dermatitis with a fetid smell, 5-25% skin affected 4 Severe interdigital dermatitis with a fetid smell, >25% skin affected B: Classification of SFR lesions

0 A clean digit with no lesions

1 An active or healing footrot lesion with a degree of separation of the sole of the digit 2 An active footrot lesion with a marked degree of separation of the sole of the digit 3 An active footrot lesion with extensive under-running of the wall hoof horn 4 An active footrot lesion with complete under-running of the wall hoof horn

Figure 3.1: Interdigital dermatitis (ID) lesion chart. Images correspond to the description given in Table 3.3 (Source: Research group footrot archives, University of Warwick).

ID 1 ID 2

ID 3 ID 4

Figure 3.2: Severe footrot (SFR) lesion chart. Images correspond to the description given in Table 3.3 (Source: Research group footrot archives, University of Warwick).

One observer carried out all disease assessments to avoid inter-observer bias (Foddai et al., 2012). All data were recorded using an Electronic Identification (EID) Reader. Swabs (Eurotubo® _{Collection swab with transport media, Deltalab, Rubi, Spain) were taken by}

trained researchers from the interdigital skin of the foot and from the gingival crevice of the mouth by passing the sterile wooden end of cotton swab 5 successive times over the skin while rotating it (Figure 3.3). Swabs were transferred into cryogenic vials with 300 µlPBS pH 7.4 and stored at -20 °C.

SFR 1 SFR 2

Figure 3.3: Sheep sampling procedure. Lesions were assessed (A); swabs were taken from the interdigital space of the foot (B) and the gingival cavity (C).

3.3.2.2 Sampling procedures unique to study 1

For study 1 only, an additional swab for culture was taken from each foot (Eurotubo®

Collection swab with transport media, Deltalab, Rubi, Spain). Swabs were taken as above with the sterile wooden end of the cotton swab. Swabs for culture were transferred into transport media (BBL™ CultureSwab™ Plus, BD, New Jersey) and immediately streaked on HA plates and incubated (Chapter 2, section 2.2) to avoid sample deterioration (Catagay and Hickford, 2005).

Faecal samples were randomly collected from the pasture with sterile spatulas and transferred into sterile universal containers. Five samples were obtained from faecal parts that had become in contact with the environment and 5 samples were taken from the faecal sample core. All environmental samples were stored at -20 °C. On the day or processing samples were defrosted and weighed out (0.5 g of soil, 0.2g of grass and 0.1g of faeces).

3.3.2.3 Sampling procedures unique to study 2

For study 2 only (faecal samples were randomly collected from the field in study 1) a faecal sample weighing no more than 5g was collected by manual extraction with a gloved hand from the rectum of each animal and placed into a sterile universal container (procedure carried out under Home Office license – PPL 70/8392). If no faeces were

B

present in the rectum a swab sample was taken and placed into a cryotube containing 0.3 ml of PBS pH 7.4. If a sheep defecated prior to this procedure, efforts were made to collect the faecal sample before contact with the ground, removing the requirement for a rectal sample. Faecal samples from the pasture were not collected.

3.3.2.4 Sampling procedures for collection of soil and grass samples (Studies 1 and 2)

Soil and grass samples were collected from pre-designated sampling sites from the field were sheep were kept. Samples were taken from 2 high and 1 low-traffic area (HTA, LTA). Areas were identified by observation of sheep movement and farmer knowledge. LTA’s were sampled using a quadrant approach with points on the quadrant for sampling chosen using a random number generator (http://www.randomizer.org/form.htm) each week One sample was taken from a chosen HTA and an additional 2 samples were collected 1m and 2m from the HTA respectively (Figures 3.4 and 3.5).

Soil samples at 0-1 and 4-5 cm depths were taken using a soil corer and transferred into sterile universal containers using sterile spatulas. When grass was present at a location, a sample was collected, and transferred to sterile universal containers. Fresh gloves were used between sample collections. Both soil corer and single use gloves were cleaned with DNA remover wipes between samples (PCR Clean™Wipes, Minerva Biolabs, Berlin, Germany). Two additional soil samples (0-1cm, 4-5 cm) were taken from the 3 traffic areas for the determination of soil moisture content.

Figure 3.4: High-traffic sampling areas for study 1 (A) and 2 (B). A gate and a tree were animals congregate were chosen as high traffic areas for study 1. For study 2, the area in front of a feeding trough and a water trough were chosen (image from www.google.co.uk/maps).

Food trough

Water trough Tree

Gate

Figure 3.5: Sampling strategy for low traffic areas (LTA’s) (A) and high traffic areas (HTA’s) (B). A: A 20m2 _{quadrant was chosen and soil and grass samples were taken from 5 random}

locations. Nodes on the quadrant were numbered 1-25 and sampling points (x) were chosen using a random number generator. B: Low traffic areas where sampled (x) in the centre of the low traffic areas and at 1m and 2m distance.

3.3.2.5 Collection of climate data (Studies 1 and 2)

In order to relate climate features to FR lesions and D. nodosus bacterial loads, local climate data (mean, minimum and maximum ambient temperatures [°C] and total rainfall [mm]) was collected by consulting climate data from the Warwick weather station (http://warwick-weather.com).

During every visit soil temperature was recorded in each area using a general-purpose thermometer (Fisher®Brand, Loughborough, UK).

A TROUGH B 1m

3.3.3 Analysis of samples in the laboratory (Studies 1 and 2)

All samples from study 1 were analyzed in the laboratory. For study 2, all 640 soil and grass samples were analyzed. A selection of samples collected from sheep (feet, gingival cavity and faeces) was analyzed based on ID and SFR scores of sheep which were assessed after completion of the trial. The selection of the samples is therefore described in section 3.5.1 (Results).

3.3.3.1 DNA extractions

Samples were selected in random order and thawed before processing. Swabs were removed from cryotubes along with any liquid accumulated and placed in Eppendorf tubes containing 0.5 g micro-beads in preparation for DNA extraction. Samples from the pasture as well as faecal samples were weighted and 0.5g of soil, 0.2g of grass and 0.1g of faecal matter were also placed in Eppendorf tubes with micro-beads. DNA was extracted from all samples according to Purdy (2005) with the addition of a polyethylene glycol (PEG) precipitation step (Ogram et al., 1988) for soil grass and faecal samples, according the optimized protocols described in chapter 2, section 2.3. Every batch of DNA extraction included one blank as experimental control, using PBS pH 7.4 as a sample substitute.

3.3.3.2 Quantification of Dichelobacter nodosus using real-time PCR (Studies 1 and 2)

In order to assess detection and quantification of D. nodosus in all sample types, samples were submitted through the D. nodosus rpoD assay using the Applied Biosystems 7500 Fast Real-time PCR System (Calvo-Bado et al., 2011, Chapter 2, Section 2.9), for quantification. All samples were processed without technical replicates initially to determine detection of D. nodosus. Quantification of positive samples was subsequently carried out in triplicate. All protocols including cycling conditions and the preparation of standard curves are described in chapter 2 (Chapter 2, Section 2.9). Every PCR run included a non-template control (sterile water) and all DNA EB’s were also tested.

3.3.3.3 Determination of soil moisture content (Study 2)

Two additional soil samples (0-1 cm and 4-5 cm) were collected per area for the analysis of soil moisture during each sampling visit. Soil moisture samples were collected from the pasture as described in section 3.4.2.4. Samples were weighed a maximum of 1 hour after collection and dried in the oven at 110 °C for 24 hours. Samples were re-weighed and soil moisture (%) was calculated using the following formula: