Background: Babesia rossi (B. rossi) causes inflammatory disease in dogs that models malaria, triggering hemolytic anemia and multiple organ failure. Neutrophil extracellular trap (NET) formation drives inflammation in malaria, but its role in babesiosis pathogenesis is unexplored. Hypotheses:B. rossi will stimulate NETosis in vivo, evidenced by increased plasma cell-free DNA (cfDNA) in infected dogs. Serum from B. rossi-infected dogs and B.rossi parasite lysate will stimulate NETosis ex vivo in healthy canine neutrophils.
Methods: Plasma was collected from dogs pre and post B. rossi infection. Plasma cfDNA was quantified with picogreen. Using an ex vivo model, isolated donor neutrophils were incubated with pre or post-infection serum and phorbol-12-myristate-13-acetate (PMA; NET inducer) or media (unstimulated), stained with dyes labeling extracellular DNA/NETs (SYTOX orange) and intracellular DNA (SYTO green). Percent NET-forming neutrophils was determined using fluorescence confocal microscopy; a two-way ANOVA compared volume/treatment effects. Isolated neutrophils were incubated with B. rossi lysate or RBC lysate control, fixed, and stained with citrullinated histone antibody and DNA dye for NET identification.
Results: Plasma cfDNA was higher post-infection than pre-infection (p=0.0312; Wilcoxon signed rank test) (Figure 1). Pre and post-infection serum did not impact NETosis in unstimulated neutrophils, but unexpectedly inhibited PMA-induced NETosis (treatment effect; p< 0.0001). B. rossi parasite lysate induced NETosis. Conclusions/Clinical Importance:B. rossi parasite-induced NETosis and increased plasma cfDNA in infected dogs suggest NETosis contributes to B. rossi pathogenesis. Therapeutic regulation of NETosis may improve babesiosis outcomes.
