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Information on equine infectious anaemia (EIA) in mules, including those with an equivocal reaction in agar gel immunodiffusion test (AGIDT), is scarce. For this, a study was conducted to evaluate the clinical, viral loads and pathological findings of two groups of naturally infected asymptomatic mules, respectively with a negative/equivocal and positive AGIDT reactivity, which were subjected to pharmacological immune suppression (IS). A non-infected control was included in the study that remained negative during the observation period. Throughout the whole study, even repeated episodes of recrudescence of EIA were observed in 9 infected mules, independently from their AGIDT reactivity. These events were generally characterised by mild, transient alterations, typical of the EIA acute form represented by hyperthermia and thrombocytopenia, in concomitance with viral RNA (vRNA) peaks that were higher in the Post-IS period, reaching values similar to those of horses during the clinical acute phase of EIA. Total tissue viral nucleic acid loads were greatest in animals with the major vRNA activity and in particular in those with negative/equivocal AGIDT reactivity. vRNA replication levels were around 10-1000 times lower than those reported in horses, with the animals still presenting typical alterations of EIA reactivation. Macroscopic lesions were absent in all the infected animals while histological alterations were characterised by lymphomonocyte infiltrates and moderate hemosiderosis in the cytoplasm of macrophages. On the basis of the above results, even mules with an equivocal/negative AGIDT reaction may act as EIAV reservoirs. Moreover, such animals could escape detection due to the low AGIDT sensitivity and therefore contribute to the maintenance and spread of the infection., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)