Longitudinal assessment of Ehrlichia canis bacterial load in naturally infected dogs using droplet digital PCR

Persistence of detectable Ehrlichia canis DNA after a full course of doxycycline treatment remains poorly described. Highly sensitive molecular tools are essential for monitoring this phenomenon. This study aimed to monitor E. canis DNAemia in naturally infected dogs over a 60-day follow-up period using droplet digital PCR (ddPCR) and to compare its performance with TaqMan real-time PCR (qPCR). Forty-one seropositive dogs (identified by the IDEXX SNAP 4Dx Plus test) exhibiting clinical signs consistent with canine monocytic ehrlichiosis (CME) were enrolled as the Day 0 group. Follow-up samples were collected during recovery from partially overlapping cohorts at Days 30 (n = 21) and 60 (n = 14). At Day 0, ddPCR detected E. canis in 33 of 41 dogs (82.92 %), outperforming qPCR (25/41; 60.97 %). At Days 30 and 60, ddPCR detected E. canis DNA in 12 of 21 (57.14 %) and 5 of 14 (35.71 %) dogs, respectively, while qPCR detected only 2 of 21 (9.52 %) at Day 30 and none at Day 60. Quantification of bacterial loads showed a significant decline from a median of 2.28 DNA copies/µL at Day 0-0.067 copies/µL at Day 30 (P < 0.05), and 0.0614 copies/µL by Day 60 (P < 0.05). Dogs with hematocrit (Hct) levels below 25 % on Day 0 were significantly more likely to have ddPCR-positive results at Day 30 (P = 0.035), suggesting a potential association between severe anemia at diagnosis and persistent DNAemia. The high sensitivity of ddPCR revealed persistence of detectable E. canis DNA with very low bacterial loads. These prolonged bacterial DNAemia require further studies incorporating viability assays to clarify whether residual DNAemia reflects persistent viable infection or non-viable bacterial DNA.