IntroductionDirofilariasis, including heartworm disease, is a major emergent veterinary parasitic infection and a human zoonosis. Currently, experimental infections of cats and dogs are used in veterinary heartworm preclinical drug research.MethodsAs a refined alternativein vivoheartworm preventative drug screen, we assessed lymphopenic mouse strains with ablation of the interleukin-2/7 common gamma chain (γc) as susceptible to the larval development phase ofDirofilaria immitis.ResultsNon-obese diabetic (NOD) severe combined immunodeficiency (SCID)γc−/−(NSG and NXG) and recombination-activating gene (RAG)2−/−γc−/−mouse strains yielded viableD. immitislarvae at 2–4 weeks post-infection, including the use of different batches ofD. immitisinfectious larvae, differentD. immitisisolates, and at different laboratories. Mice did not display any clinical signs associated with infection for up to 4 weeks. Developing larvae were found in subcutaneous and muscle fascia tissues, which is the natural site of this stage of heartworm in dogs. Compared within vitro-propagated larvae at day 14,in vivo-derived larvae had completed the L4 molt, were significantly larger, and contained expandedWolbachiaendobacteria titres. We established anex vivoL4 paralytic screening system whereby assays with moxidectin or levamisole highlighted discrepancies in relative drug sensitivities in comparison within vitro-reared L4D. immitis. We demonstrated effective depletion ofWolbachiaby 70%−90% inD. immitisL4 following 2- to 7-day oralin vivoexposures of NSG- or NXG-infected mice with doxycycline or the rapid-acting investigational drug, AWZ1066S. We validated NSG and NXGD. immitismouse models as a filaricide screen byin vivotreatments with single injections of moxidectin, which mediated a 60%−88% reduction in L4 larvae at 14–28 days.DiscussionFuture adoption of these mouse models will benefit end-user laboratories conducting research and development of novel heartworm preventatives via increased access, rapid turnaround, and reduced costs and may simultaneously decrease the need for experimental cat or dog use.