Effect of doxycycline on Wolbachia levels in Dirofilaria immitis worms from five canine hearthworms cases: Immunehistochemical staining analysis

Abstract

Doxycycline, an essential component of adulticidal treatment for canine heartworm, targets the elimination of Wolbachia pipientis, an endosymbiotic bacterium whose release triggers a proinflammatory innate immune response in the host. Therefore, effective eradication of Wolbachia is crucial before proceeding with adulticidal therapy. This study aimed to evaluate the effects of doxycycline administered at two different doses (10 mg/kg BID and 5 mg/kg BID) on Wolbachia levels in Dirofilaria immitis worms harvested from dogs undergoing surgical parasite removal.

Five infected dogs with high Wolbachia and its associated metabolites were studied. Two dogs received no antibiotic treatment before surgery, two received doxycycline at 5 mg/kg BID, and one dog was treated with doxycycline at 10 mg/kg BID for 30 days. Parasite removal was performed under fluoroscopic guidance using Flexible Alligator Forceps through a right jugular incision, with 10-20 worms (males and females) extracted per animal. These worms were preserved in formalin and sent to a laboratory for processing, followed by immunohistochemical staining using anti-WSP (Wolbachia surface protein) antibodies to detect Wolbachia.

Staining results demonstrated a significant reduction in Wolbachia levels in worms treated with both doxycycline doses compared to the control group. However, some sections still showed staining compatible with the presence of the bacterium in some points of the lateral cords. Additionally, treated female worms exhibited destruction of larvae and damage to morulae. These findings suggest that both doxycycline doses are effective in reducing Wolbachia levels in Dirofilaria immitis worms. Staining analysis doses are equally effective in reducing Wolbachia levels, which may impact the management and treatment of canine heartworm.