MEDIDAS A TOMAR

During both experiments, the caecal contents and spleen were taken

aseptically at each post mortem. Spleen and caecal contents were added to 1 x PBS in a 1:10 dilution. Spleen samples were homogenised using a MicroStomacher 80 (Seward, UK) and the caecal contents were vortexed to form a suspension. Spleen and caecal content samples were serially diluted in 1 x PBS to 10-5 and 10-11 respectively and plated onto Brilliant Green agar (BGA) (Oxoid, UK). The plates were incubated at 37oC for 18 hours and then the bacteria were enumerated.

During post mortem analysis for experiment 2, additional samples were collected and stored for histology and for the immunological experiments outlined in Chapter 5. Ileum and spleen samples were collected and stored in 4% paraformaldehyde at room temperature for histology. Ileum, spleen and caecal tonsil samples were embedded onto cork in O.C.T. compound (tissue- tek), snap frozen in liquid nitrogen and stored at -80°C for

immunohistochemical analysis. Ileum, spleen and caecal tonsil samples were also stored in RNAlater (Sigma-Aldrich, UK) at -20°C for RT-PCR. Serum samples were collected from the heart using 21 mm needles. The serum was centrifuged at 13000 x g for 5 minutes. The supernatant was removed and stored at -20°C for ELISA and western blot.

86 4.2.5 Histological analysis

Ileum and spleen tissue samples from experiment 2 were stored in 4% paraformaldehyde for histological analysis. Tissue samples were placed in plastic cassettes and put on a tissue processor overnight, to dehydrate the tissue and embed it in paraffin wax. A microtome was used to cut 4 µm sections of the tissue samples, which were collected onto slides.

The sections were stained using haematoxylin and eosin. Samples were dewaxed in xylene for 5 minutes and then transferred to containers of

descending grades of alcohol (100%, 96%, 86% and 70%) to distilled water. Sections were stained for 5 minutes using Mayer’s Haemalum and then placed under running water for 6 minutes. Following this, sections were stained with Eosin for 2 minutes. Sections were dehydrated in 3 containers of 95% alcohol for 1 minute per container (repeated 3 times), 3 containers of absolute ethanol and 3 containers of xylene and then mounted in D.P.X.

4.3 Results

4.3.1 Experiment 1 – Pilot poultry infection experiment

Colony counts of Salmonella were taken at 3 DPI. All of the S. Virchow isolates colonised the gut to high levels, with caecal content counts ranging between log10 5.5-7.4 cfu/g (Figure 4.1). No significant differences were found between caecal content colony counts for the S. Virchow infected groups (P = >0.233). The S. Virchow isolates had similar caecal content colony counts to S. Typhimurium F98 and S. Typhimurium 238, which had a counts of log10 6.40 cfu/g and log10 6.00 cfu/g respectively. No significant differences were found between the S. Virchow colony counts and the S. Typhimurium colony counts (P = >0.107).

S. Virchow isolates 55, 59 and 60 showed the ability to systemically colonise the chickens by 3 DPI, with spleen counts ranging between log10 1.5-2.1 cfu/g (Figure 4.1). The 3 S. Virchow isolates showed similar levels of colonisation

87

in the spleen compared to S. Typhimurium F98 and S. Typhimurium 238. The spleen colony counts for S. Typhimurium F98 and S. Typhimurium 238 were log10 2.00 cfu/g and log10 1.80 cfu/g respectively. No significant differences were found between colony counts of the S. Virchow infected groups and the S. Typhimurium infected groups in the spleen (P = >0.148).

Figure 4.1: Experiment 1: Log10 colony counts for the caecal contents and spleen at 3 DPI. S. Virchow = 55 HU, 56 HU, 59 CH and 60 CH; ST F98 = S. Typhimurium F98; ST 238 = S. Typhimurium 238. Error bars represent the standard error of the mean, which was calculated from 5 birds per time point.

4.3.2 Experiment 2 – Poultry infection experiment

Colony counts of Salmonella were taken at 5, 11 and 26 DPI from the caecal contents and spleen. S. Virchow 60 was present in the caecal contents by 5 DPI at a concentration of log10 8.0 cfu/g, which increased to log10 9.0 cfu/g by 11 DPI and declined to log10 7.0 cfu/g by 26 DPI (Figure 4.2). S. Virchow 60 showed a similar pattern of infection to S. Typhimurium F98, which was at a concentration of log10 9.0 cfu/g at 5 DPI, log10 12.0 cfu/g at 11 DPI and log10 7.0 cfu/g at 26 DPI. The caecal content counts for S. Virchow showed no

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 55 HU 56 HU 59 CH 60 CH ST F98 ST 238 M ea n log 10 n u mb er o f cfu /g Isolate Number caecal contents spleen

88

significant differences to counts of S. Typhimurium F98 at each time point (P = >0.068).

S. Virchow 60 was isolated from the spleen at a concentration of log10 3.0 cfu/g at 5 DPI and at 11 DPI; however, it was cleared from the spleen by 26 DPI (Figure 4.3). S. Typhimurium F98 increased from log10 2.0 cfu/g at 5 DPI to log10 4.0 cfu/g at 11 DPI; however it was also cleared from the spleen by 26 DPI and was not significantly higher in the spleen than S. Virchow at any time point (P = >0.136).

Figure 4.2: Experiment 2: Log10 caecal contentcounts at 5, 11 and 26 DPI. Error bars represent the standard error of the mean, which was calculated from 5 birds per time point.

1.00 3.00 5.00 7.00 9.00 11.00 13.00 5 11 26 M ea n log 10 n u mb er o f cfu /g

Number of days post infection

S. Virchow 60 S. Typhimurium F98

89

Figure 4.3: Experiment 2: Log10 spleen counts at 5, 11 and 26 DPI. Error bars represent the standard error of the mean, which was calculated from 5 birds per time point.