For many tick-borne organisms, dogs may be utilized as sentinel hosts to estimate the risk of human infection. The detection of antibodies in these animals indicates the circulation of pathogens in each location for a set period of time. The standard protocol for the surveillance of tick-borne diseases established by the Brazilian National Reference Laboratory for Rickettsiosis at the Ezequiel Dias Foundation (FUNED) includes testing the seropositivity of dogs for Rickettsia spp. and Ehrlichia canis. Dog serum samples were collected during FUNED’s surveillance protocol in a preserved area in Brazil. Despite Anaplasma phagocytophilum being endemic in Brazil, this agent is not included in FUNED’s standard protocol. To determine if A. phagocytophilum should be included in the standard testing protocol, a collaboration with Fuller Laboratories was established and the seropositivity for A. phagocytophilum was tested. The estimated prevalence for singular infections: 25% Rickettsia spp.; 52.3% E. canis; and 70.5% A. phagocytophilum. The estimated prevalence for co-infections: 17% Rickettsia spp. and E. canis; 18.2% Rickettsia spp. and A. phagocytophilum; 39.8% E. canis and A. phagocytophilum; and 13.6% Rickettsia spp., E. canis, and A. phagocytophilum. These results confirmed a significant presence of A. phagocytophilum and establishes a potential threat upon the public health.