Pathomorphological and histological examination of the animals

A complete post-mortem examination was carried out by veterinarians on all wild boar carcasses submitted to the regional diagnostic laboratory in the federal state North Rhine-Westphalia by hunters or local veterinary authorities. African and Classical swine fever were ruled out by means of PCR, Brucella infection and Aujeszky’s disease have been excluded by serological methods. Wild boars examined were found dead or were shot by hunters because of overt clinical disease (abnormal behaviour, lost escape behaviour). Road killed animals were not submitted. Animals weighed between 1.5 and 53 kg. Depending on the state of decomposition samples of brain, lung, heart, liver, spleen, kidney, and intestine were fixed in 10% neutral buffered formalin. From animals with swollen feet, metaphyses of long bones were included. Bones were decalcified using hydrochloric acid (Osteomoll, Merck, Darmstadt, Germany). The formalin fixed tissue was further processed for histology using routine techniques. Sections were cut at 5 μm and stained with hematoxylin and eosin for microscopic examination. Domestic pigs and non-porcine game animals with S. Choleraesuis findings were necropsied in order to determine the cause of illness and death. Processing of samples from these animals for histology was identical as described above.