An indirect ELISA based on a soluble extract of Besnoitia besnoiti tachyzoites was developed and standardised. A set of positive and negative reference bovine sera were characterised using an immunofluorescence antibody test and Western blot. A cut-off with a relative index per cent of 8.1 was determined for equal sensitivity and specificity (100 per cent) by twograph receiver operating characteristic analysis. Cross-reactions with other closely related Apicomplexan parasites were discarded. The standardised ELISA was then used during an outbreak of bovine besnoitiosis in a mountainous area of central Spain. The outbreak occurred in nine herds, and 358 animals that shared grazing lands during the summer season were affected. Clinical examination and blood sampling were carried out for all animals, and skin biopsies were obtained from animals with skin lesions. The confirmatory diagnosis was carried out by means of the indirect ELISA, together with the identification of tissue cysts by microscopy. Most of the animals were seropositive (90·5 per cent), but only 43 per cent of seropositive cattle developed clinical signs compatible with besnoitiosis. Additionally, a significant increase in seroprevalence and clinical signs was found to be associated with the increasing age of the animals, suggesting rapid horizontal transmission of the disease.
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