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 alternative in vivo heartworm 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 of Dirofilaria immitis.ResultsNon-obese diabetic (NOD) severe combined immunodeficiency (SCID)γc−/− (NSG and NXG) and recombination-activating gene (RAG)2−/−γc−/− mouse strains yielded viable D. immitis larvae at 2–4 weeks post-infection, including the use of different batches of D. immitis infectious larvae, different D. immitis isolates, 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 with in vitro-propagated larvae at day 14, in vivo-derived larvae had completed the L4 molt, were significantly larger, and contained expanded Wolbachia endobacteria titres. We established an ex vivo L4 paralytic screening system whereby assays with moxidectin or levamisole highlighted discrepancies in relative drug sensitivities in comparison with in vitro-reared L4 D. immitis. We demonstrated effective depletion of Wolbachia by 70%−90% in D. immitis L4 following 2- to 7-day oral in vivo exposures of NSG- or NXG-infected mice with doxycycline or the rapid-acting investigational drug, AWZ1066S. We validated NSG and NXG D. immitis mouse models as a filaricide screen by in vivo treatments 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.
Use of experimental cats and dogs in veterinary heartworm preclinical drug research is increasing. As a potential alternative primary in vivo heartworm preventative drug screen, we assessed lymphopenic mice with ablation of the interleukin-2/7 common gamma chain (gc) as susceptible to the larval development phase of D. immitis. Non-obese diabetic (NOD) Severe Combined ImmunoDeficient (SCID)gc-/-(NSG / NXG) mice consistently yielded viable D. immitis larvae at 2-4 weeks post-infection across multiple experiments, different batches of infectious larvae inoculates, different isolates of D. immitis and at independent laboratories. Mice did not display any overt clinical signs associated with infection up to 4 weeks. Developing larvae were found in subcutaneous and muscle fascia tissues, the natural site of this stage of heartworm in dogs. Larvae retrieved from NSG / NXG mice were mid-L4 stage of development. Compared with 14-day in vitro propagated larvae, in vivo derived L4 were significantly larger and contained expanded Wolbachia endobacteria titres, determined by QPCR and Fluorescent in situ Hybridisation (FISH). We established an ex vivo 6-day L4 paralytic screening system against nematodicidal agents (moxidectin, levamisole) which highlighted discrepancies in relative drug sensitivities in comparison with in vitro reared L4 D. immitis. We demonstrated effective depletion of Wolbachia by 70-90% in D. immitis L4 following 2-7 day oral in vivo exposures of NSG / NXG infected mice with doxycycline or the rapid-acting investigational anti-Wolbachia drug, AWZ1066S. We validated the NSG / NXG mouse model as a filaricide drug screen by in vivo treatments with single injections of moxidectin, which mediated 60-88% reduction in L4 larvae at 14-28 days. Future adoption of the mouse model as a first-line efficacy screen will benefit end-user laboratories conducting research and development of novel heartworm preventatives via increased access, rapid turnaround and reduced costs whilst simultaneously decreasing need for experimental cat or dog use.
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