Sample collection

The project budget allowed for approximately 400 tests in total, corresponding to five tests on each of 80 calves. However, during an outbreak of M. bovis some calves may die or be euthanized because of clinical disease, so we initially enrolled more than 80 calves in the study. During the first visit, the investigating veterinarian and the farmer sampled the calves strategically to ensure that a sufficient number of calves suspected to have M. bovis-associated disease were included, as well as a sample of calves without evidence of clinical disease. The rationale behind this non-random sampling strategy was to maximize the likelihood of including sufficient numbers of animals with and without different clinical signs.

During the herd visits, each calf underwent a clinical examination with a focus on the respiratory and musculoskeletal system, using a standardised clinical protocol, which is available from the corresponding author. One of three veterinarians performed the clinical examinations, and at least one of the authors of the clinical examination protocol was present at each visit.

In addition, a venous blood sample was collected from each calf using a 10 ml plain Vacutainer tube (Kruuse, Denmark). Blood samples were stored in a cool environment and delivered to the Eurofins Steins laboratory (Vejen, Denmark) within 36 h of collection. The serum samples were frozen at − 18 °C.