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This report describes the stege I/II development of a new direct agglutination test (DAT) for the diagnosis of canine visceral leishmaniasis (CVL) using freeze-dried antigen produced Coomassie blue-stained Leishmania (Leishmania) infantum promastigotes. In stage I, 16 canine serum samples, collected from eight dogs carrying CVL and eight healthy dogs, were assessed with the DAT using 2-mercaptoethanol (2-ME), N-acetyl-cysteine (NAC), kaolin or NAC plus urea (NAC+U) to improve the assay conditions. Stage II assessed the diagnostic accuracy with 100 serum samples collected from dogs with symptomatic CVL and clinically healthy dogs, comparing the four different sample diluents. The CVL-DAT prototype kit showed equivalent performances when 2-ME, NAC or NAC+U were used: 97.1% sensitivity (CI: 83-99.8%), 97% specificity (CI: 88.5-99.5%) and a 97% diagnostic accuracy (CI: 90.8-99.2). With kaolin, a 94.1% sensitivity (CI: 79-99%), 97% specificity (CI: 88.5-99.5%) and 96% diagnostic accuracy were observed (CI: 89.5-98.7), with no statistically significant differences among the four reagents (p=1.0). The NAC plus urea in sample diluent decreased non-specific agglutination, promoted a better defined sharp-edged blue spot and was thus chosen as a component for the new DAT prototype to diagnose canine VL, designated DAT-Canis.