Clinical and euthanasia observations

Animals were weighed at the same time of day from the day before infection until the end of the experiment, or until death or euthanasia. Animals were examined twice daily for clinical signs of disease and scores were recorded using a ‘clinical observation score’. Monitoring of animals increased to at least four times daily during critical periods in the study (at times when septicaemia was likely to occur or as soon as clinical signs of septicaemia were detected). Clinical signs of disease were assigned a score based upon the following criteria: healthy, 0; ruffled fur, 2; arched back, 3; eyes shut, 3; immobile, 9; euthanasia or death in cage, 10. Clinical scores were not used to trigger euthanasia. In order to meet the requirement of the project license, three criteria for immediate euthanasia were used: (i) immobility defined as a lack of movement even after stimulus such as handling, (ii) neurological signs including repetitive or unusual movement, (iii) weight loss of more than 20% from baseline for more than 24 h. If any animal reached any of these three euthanasia criteria, they were immediately euthanized using a UK Home Office approved Schedule 1 procedure.