Dirofilariasis mouse models for heartworm preclinical research

Abstract

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.

Combinations of the azaquinazoline anti-Wolbachia agent, AWZ1066S, with benzimidazole anthelmintics synergise to mediate sub-seven-day sterilising and curative efficacies in experimental models of filariasis

“…Vehicle and 10 mg/kg ABZ control groups were also included ( Figure 5A ). Using previously established FISH staining with Wolbachia- specific 16S rRNA probes (Walker et al, 2021 ; Marriott et al, 2023 ), we visualised and quantified Wolbachia loads both in hypodermal chords and ovaries in female worms from control and different drug treatment groups. Previous studies have shown that the ovaries have the highest density of germline Wolbachia populations (Bakowski et al, 2019 ) and are also the only tissue site in mature adult worms containing proliferating cells (Foray et al, 2018 ); thus, they serve as an ideal tissue to compare against hypodermal chords.…”

“…In addition, we determined that via depletion of Wolbachia in the mf-stage, AWZ1066S can also block the development of Brugia in the mosquito vector via a deficit in Wolbachia, inhibition of mf chitinase production and failure to exsheath in the insect midgut (Quek et al, 2022). Most recently, we have determined 90% anti-Wolbachia activity of AWZ1066S against developing larvae of the veterinary filaria, Dirofilaria immitis, following 2-day exposures in vivo, extending the potential use-case of this new class of anti-Wolbachia therapeutic from human to veterinary medicine (Turner et al, 2020;Marriott et al, 2023). In this study, compared with a 100 mg per day human equivalent dose exposure of doxycycline (Sharma et al, 2016), which requires 6 weeks in a SCID model of brugian filariasis to mediate >90% anti-Wolbachia activity, we further established the minimum AWZ1066S dose time frame of 5 days which could sustainably deplete Wolbachia to a similar or >99% threshold when sufficient bi-daily dose-exposures were applied against mature B. pahangi in gerbils (Figures 1, 2).…”

Comparative development of human filariae Loa loa, Onchocerca volvulus and Mansonella perstans in immunocompromised mouse strains

“…Therefore, we aim to establish a murine model of M. perstans based on the knowledge of the established animal models of onchocerciasis, lymphatic filariasis and loiasis. Immunocompromised mouse strains that lack adaptive immunity pathways like RAG2 −/− ( 27 , 28 ), IL-4/5 −/− ( 32 ) and NSG strains ( 26 , 36 ) have been proven suitable for human filarial infections and recently, NSG mice have been also proven to be susceptible to infection with the dog heartworm Dirofilaria immits ( 47 , 48 ). Indeed, a 62-66% L3 recovery rate could be obtained upon Loa loa L3 inoculation, whereas M. perstans and O. volvulus L3 were absent in all immunocompromised mouse strains.…”

Development of a novel rodent model for dog heartworm microfilaremia using the severe-combined immunodeficiency mouse

“…Recent studies have established mouse models for L4 development in D. immitis using NOD-, NSG-, or NXG immunode cient mice [23,24]. This study proposes a micro laremia model for D. immitis using SCID mice.…”