Standardization of real-time polymerase chain reaction (qPCR) and investigation of Trypanosoma caninum infection in dogs in Brazil.

Trypanosoma caninum was isolated by axenic culture from healthy dog skin in 2009. This parasite has been described in different regions of Brazil, using isolation in culture, molecular detection, and sequencing. However, more sensitive diagnostic methods using specific targets are needed to estimate the real frequency of infection. This study aims to develop a pair of primers based on the 18S region of Trypanosoma caninum rRNA, as well as its investigation and quantification by the qPCR technique in skin, blood, and lymph node samples from dogs in Brazil. The developed primers showed high specificity for T. caninum and without showing cross-reactivity with other trypanosomatids. Furthermore, these primers amplified a 248 bp fragment and were able to detect T. caninum DNA from only 10 DNA copies. Of the 349 dogs analyzed, 54 (15.5 %) were positive. T. caninum infection occurred in all municipalities, with higher frequency in Várzea Grande (37.1 %). Parasitological culture and quantitative PCR were positive in 1.4 % and 14 % of dogs, respectively. Parasite DNA was found in skin, lymph node and blood in 10 %, 3.7 % and 1.7 % of dogs respectively. The mean T. caninum parasite load was highest in skin samples, followed by lymph node and blood, with no significant difference (p = 0.06). T. caninum infection was higher than previously found, pointing out that the frequency of this parasite is underestimated. The parasite load in the biological samples was considered low; however, the skin presented the highest number of DNA copies, reinforcing the predilection for this site and the low occurrence of clinical alterations in infected dogs.