Handling of blood, CSF and autopsy samples obtained in the animal experiments

Before the inoculation of S. suis or PBS (see chapter 4.2.3) into the pig blood from the Vena (V.) cava cranialis was filled into serum and plasma (lithium-heparin) monovettes (SARSTEDT AG & Co. KG, Nümbrecht, Germany). During isoflurane anaesthesia, these blood samples were also collected via indwelling venous catheter (or via the arterial access). The blood samples were taken at 13, 16 and 19 hours after infection. In parallel a sample of 1-2 ml fresh CSF was collected via the CSF catheter if possible. It was ensured that the serum samples stood upright for 30 minutes after time of collection before further working steps. Immediately after the collection the serum, plasma and CSF samples were transferred on ice to the laboratory for the following procedures:

77 4.2.8.1. Processing of the blood samples

First, from the post infection lithium-heparin samples a volume of 200 μl was removed for the determination of colony forming units, for testing for pure S. suis culture and further molecular investigations. The rest was centrifuged at 4°C for 10 minutes at 2100 g (Heraeus™

Multifuge™ X3; ThermoFisher, Waltham, USA). The supernatant was aliquoted and flash frozen in liquid nitrogen. Long-time storage is at -80 ° C.

From the 200 µl aliquot a volume of 20 µl coagulation inhibited blood was pipetted and smeared fragmentally on Columbia agar plate with 6% sheep blood (Oxoid Deutschland GmbH). Therefore, a sterile eyelet was used. After 24 hours of incubation at 37°C the smear was checked for pure S. suis culture by morphology (e.g alpha-hemolyzing colonies).

A further volume of 20 µl was used for the determination of colony forming units (CFU/ml) according to chapter 4.1.2. Dilution series up to 10^-5 were plated for animals suffering from infection symptoms (body temperature > 41°C, CNS symptomatic or turbid CSF), for control animals or animals without infection symptoms dilutions up to 10^-2 were plated. In contrast to the method described in chapter 4.1.2 two dilutions were plated on one plate. The blood agar plates were incubated at 37°C for the next 24 hours.

A volume of 100 µl from the 200 µl aliquot is transferred into 5 ml Todd Hewitt Broth (THB) (Becton, Dickinson and Company, Sparks Glencoe, USA) in a T405-Cultubes™ (Simport®

Scientific Inc., Belœil, Canada) with air exchange. The following incubation was 12-24 hours at 37°C.

Afterwards a fragmented smear (20 µl) with a sterile eyelet was created from this culture on a Columbia agar plate with 6% sheep blood to check again for pure S. suis culture by morphology after 24 hours of incubation at 37°C.

The rest of the sample was centrifuged at 2600 g for 5 minutes. The resulting bacterial pellet was stored at -20 °C for DNA examination.

The serum blood samples were centrifuged at 2100 g for 10 minutes at 4°C after a standing time of 30 minutes. The supernatant was aliquoted and flash frozen in liquid nitrogen. Long-time storage is at -80 ° C.

78 4.2.8.2. Processing of the CSF samples

For the quantification of PMN in the cerebrospinal fluids volumes of 200 µl CSF were first supplemented with paraformaldehyde (PFA) (Science Services GmbH, Munich, Germany) to a final concentration of 4% for fixation in 1.5 ml SafeSeal tubes (SARSTEDT AG & Co. KG, Nümbrecht, Germany). The samples were short-time stored at +4°C and later analyzed together by fluorescence-activated cell sorting (FACS; Attune NxT Flow Cytometer; life technologies Inc, Carlsbad, USA). The analysis was based on Forward Scatter (FSC-A; detection of cell size) 180 and Side Scatter (SSC-A, detection of granularity) 350. The sample volume was 100 μl. The number of neutrophil granulocytes was calculated by gating for specific PMN morphology. In addition, the total number of cells and the number of singlets was determined (using FFS-A versus FFS-H scatter).

For microscopic determination of the cells in the CSF, 50 μl of sample was applied to a standard slide and spread over a small area with a sterile loop. Subsequently, the smear was air-dried and a Diff Quick staining (HAEMA SCHNELLFÄRBUNG Diff-Quick, LT-Sys ®, Berlin, Germany) was performed according to the manufacturer's instructions. The stained samples were then examined microscopically (BX81, Olympus, Tokyo, Japan) to differentiate morphologically different cell types in the CSF. To counteract the altered morphology of the cells by the process of dehydration, the samples were wetted with Roti®-Mount (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) immediately prior to microscopic examination and covered with a coverslip.

A volume of 20 µl CSF was smeared fragmentally on a Columbia agar plate with 6% sheep (Oxoid Deutschland GmbH). Therefore, a sterile eyelet was used. After 24 hours of incubation at 37°C the smear was checked for pure S. suis culture by morphology (e.g alpha-hemolyzing colonies).

A further volume of 20 µl was used for the determination of colony forming units (CFU/ml) according to chapter 4.1.2. Dilution series up to 10^-5 were plated for animals suffering from infection symptoms (body temperature > 41°C, CNS symptomatic or turbid CSF), for control animals or animals without infection symptoms dilutions up to 10^-2 were plated. In contrast to

79

the method described in chapter 4.1.2 two dilutions were plated on one plate, except for the 10^-5 dilution. The blood agar plates were incubated at 37°C for the next 24 hours.

A volume of 100 µl CSF was transferred into 5 ml Todd Hewitt Broth (THB) (Becton, Dickinson and Company, Sparks Glencoe, USA) in a T405-Cultubes™ (Simport® Scientific Inc., Belœil, Canada) with air exchange. The following incubation was 12-24 hours at 37°C. Afterwards a fragmented smear (20 µl) with a sterile eyelet was created from this culture on a Columbia agar plate with 6% sheep blood to check for pure S. suis culture after 24 hours of further incubation.

In case of turbidity of the THB medium, the suspension was centrifuged at 2600 g for 5 minutes. The resulting bacterial pellet was stored at -20 °C for DNA examination.

All leftover CSF on the day of the experiment was aliquoted and flash frozen in liquid nitrogen.

The temperature for long-time storage is at -80 ° C.

4.2.8.3. Processing of the autopsy samples

After completion of the respective measurements on the living animal, the pig was euthanized for animal welfare reasons in state of general anaesthesia by applicating 3-4 ml of T 61 ® (Intervet Deutschland GmbH, Unterschleißheim, Germany) intravenously. After death was determined by means of electrical monitoring and auscultation, necropsy was performed to examine certain organs macroscopically for signs of infection. Different samples were taken for the microbiological follow-up examination or histopathological examination. For the latter, the samples were embedded directly in 10 % formalin. Table 17 provides information on the organ specific samples.

Organ Microbiological sample Histopathological sample (formalin)

Liver (Lobus quadratus) 1.5 x 1.5 cm 2.0 x 3.0 cm

Spleen 1.5 x 1.5 cm 2.0 x 3.0 cm

Lung (Lobus cranialis sinister) 2.0 x 3.0 cm 2.0 x 3.0 cm

Tonsil sterile swab¹ (pre-infection)

1.0 x 1.0 cm

1.0 x 1.0 cm

Peritoneum sterile swab¹ 2.0 x 3.0 cm

Pleura costalis sterile swab¹ 2.0 x 3.0 cm

Pericardium sterile swab¹ -

Heart (Valva atrioventricularis sinister) sterile swab¹ Ventriculus cordis sinister

80

Brain sterile swab¹

(Fissura longitudinalis cerebri)

complete

Carpal + Tarsal joint (left, right) 0.1-1.5 ml aspirate Synovial membrane

1 Amies medium, SARSTEDT AG & Co. KG, Nümbrecht, Germany

Table 17 Overview of organ samplings for S. suis detection

For the microbiological examination, the organ pieces were placed in plastic dishes and were undergoing the following procedure:

The organ pieces were dipped in 100% alcohol (except lung) and were briefly flamed. With flamed, sterile scissors and tweezers a fresh cut was generated and pressed 2-3 times for 1-2 seconds on a Columbia agar plate with 6% sheep blood. About 1/3 of the plate was stamped.

From this, a fractionated smear was made with a sterile loop.

From punctures (e.g. joint fluids), one drop each from the syringe was dropped on the agar plate and streaked fragmentally. Swab samples were spun down to 1/3 of the agar plate and again a fragmented smear was made. For tonsil samples this was additionally performed on agar plates for the differentiation of streptococci and staphylococci (Oxoid Deutschland GmbH, Columbia-CNA).

The agar plates were then incubated at 37 ° C for 48 hours. Afterwards, S. suis suspicious colonies (alpha-hemolyzing colonies) were picked up with a loop. The loop was then smeared fragmentally on a new plate for further incubating the next 24 hours (subcultivation).

S. suis suspect colonies were inoculated in 10 ml of THB and incubated for 12-24 hours in a T405-Cultubes™ (Simport® Scientific Inc., Belœil, Canada) with air exchange. In case of turbidity of the THB medium, the suspension was centrifuged at 2600 g for 5 minutes. The resulting bacterial pellet was stored at -20 °C for DNA.