Use of athymic nude mice in a mouse model of Dirofilaria immitis and its utilization in drug screening

Use of athymic nude mice in a mouse model of Dirofilaria immitis and its utilization in drug screening | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Use of athymic nude mice in a mouse model of Dirofilaria immitis and its utilization in drug screening Crystal Fricks, John W McCall, Abdelmoneim Mansour, Utami DiCosty, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8742911/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The heartworm Dirofilaria immitis mouse model has been a recent topic of research and discussion as a tool that is more logistically feasible and economical to study the efficacy of new drugs as opposed to using dogs and/or cats. Often, for the initial screening of novel antifilarial compounds, larval development assay (LDA) is used in vitro. However, a reliable D. immitis mouse model would be beneficial as in vitro benchtop assays cannot fully mimic the in vivo system. Methods Several strains of mice were evaluated for their susceptibility to the infective larval stage of D. immitis while attempting to use a low cost, low maintenance mouse. Experimental infections were performed by injecting third-stage infective larvae subcutaneously and performing necropsies at several time points post-infection (PI). Results By 7–10 days PI, 89–96% of the worms recovered from the subcutaneous tissue were determined to have molted to the fourth stage. Among the mouse strains tested for susceptibility to D. immitis larval infection, the athymic nude mouse strain, J:NU, proved to be the best candidate for developing the heartworm-mouse model. When infected with 50–100 infective third-stage larvae, the recovery rate of fourth stage larvae was consistently 20–30% at 7–10 days PI. This model was then evaluated using the macrocyclic lactones (ML) ivermectin and moxidectin, and emodepside, a cyclic depsipeptide, as a treatment against the third-stage and fourth-stage infective larvae of a ML-susceptible and resistant heartworm isolate. Results show that the MLs had good efficacy and emodepside was nearly 100% effective against the ML susceptible D. immitis isolate. In addition, mice were also necropsied 77 to 96 days post infection of third-stage infective larvae with recovery rates of up to 46% of adult worms found in the heart and lungs as well as other areas of the body. Conclusions Although target animals (dogs and cats) should be used to confirm the efficacy of new drugs to combat D. immitis , this heartworm mouse model will give a more economical and efficient approach to screen novel compounds. Dirofilaria immitis heartworm mouse model macrocyclic lactones Figures Figure 1 Figure 2 Background Heartworm disease, caused by Dirofilaria immitis , is a debilitating malady among canines in warm, humid climates. Heartworm disease can cause severe pulmonary disease and has the potential to be fatal [1]. After the discovery of Ivermectin in the 1970’s [2], macrocyclic lactones (MLs) have been the mainstay of prevention of disease from D. immitis since. In recent history, though, there have been an emergence of cases of heartworm disease from isolates of D. immitis that are resistant to MLs [3]. The necessity to research and develop new heartworm preventatives is therefore an urgent matter as cases of heartworm disease are on the rise [4,5]. However, in keeping with the spirit of the 3Rs - Replacement, Reduction and Refinement [6], there is more of a demand and desire to improve the process of investigating novel therapeutics. In vitro assays, such as the larval migration inhibition assay [7] can be invaluable tools for the initial screening of anthelmintics. Nevertheless, benchtop assays cannot fully mimic the in vivo system. Mice have been used in research and development for both human and other animal diseases for decades. Even as far back as the 1980’s, mice were used for D. immitis research. As one example, ICR mice were injected either subcutaneously or intraperitoneally with 10,000 D. immitis microfilaria (MF) and through a laborious process, MF were recovered up to 6 weeks after inoculation [8]. To study viability of larvae, diffusion chambers were placed in BALB/c mice and third-stage larvae of D. immitis were then introduced into the chambers; live larvae were recovered three weeks later [9]. As with any disease model development process in mice, it is imperative to select the correct strain of mouse to mimic the disease in the normal host species as closely as possible. The athymic nude mouse strain is a staple for human oncology studies [10]. Because they lack a thymus, T cells cannot maturate and that leaves the mice susceptible to and able to accept foreign tissue. It is with that thought in mind that we sought to see if athymic nude mice would be a good candidate for hosting infection with D. immitis . Materials and Methods Animals and Reagents All animal work conducted at TRS was approved by the facility’s IACUC, which follows regulations set forth by AWA and the Guide for Care and Use of Laboratory Animals. Male mice (5–8 weeks of age) were obtained from Jackson Laboratories in Bar Harbor, Maine, USA. Strains purchased were J:Nu (JAX stock #007850), Nu/J (JAX stock #002019), and C57BL/6J (JAX stock #000664). Mice were housed 3 per cage in standard shoebox type IV polycarbonate cages in a dedicated room with a 12 hour light/dark cycle. They were provided with sterile water (by reverse osmosis) and irradiated pelleted food (Teklad Global 18% protein rodent diet 2918) ad lib. Mice were individually identified by applying different colored markings at the base of the tail. Three different drugs -ivermectin (Sigma or Covetrus), moxidectin (Sigma) and emodepside (AchemBlock)- were used for efficacy studies. Hanks Balance Salt Solution (HBSS), purchased from Sigma, was used to facilitate injections of infective larvae and for soaking of carcasses at necropsy. Prednisolone sodium phosphate oral solution 15mg/5ml (Covetrus) and over the counter gel capsules of generic diphenhydramine 25mg were added to the drinking water in some instances. Infections Mice were allowed to acclimate to the facility for at least one week prior to infection. In early studies, diphenhydramine was added to the drinking water 3–5 days prior to infection and was maintained as such until necropsy. For some longer-term studies, prednisolone was also added to the drinking water. On day 0, mice were infected with infective third-stage larvae (L 3 ) of either the macrocyclic lactone (ML) susceptible D. immitis isolate SC-20 or the ML resistant isolate JYD-34. Between 50–100 L 3 in approximately 500 to 700uL of HBSS were injected subcutaneously (SQ) over the caudal dorsum. Larvae were collected from crushed mosquitoes that had fed on blood that contained the infective microfilariae 14 days prior. Treatments Mice were treated SQ on day 1 or day 7 with 100 mg/kg or 50mg/kg of emodepside, ivermectin, or moxidectin or with a vehicle control. Drugs used for SQ injections were dissolved in DMSO (Neogen) containing 0.5% Tween @ 80 (Sigma), with the exception of commercially available ivermectin (Covetrus). In some cases, emodepside was administered via oral gavage. Necropsy Mice were necropsied at various timepoints, depending on the nature of the particular study. After humane euthanasia via gradual carbon dioxide asphyxiation followed by cervical dislocation, larvae were recovered from the subcutaneous tissue. For mice that were necropsied at later timepoints (such as day 77 and beyond), the peritoneal and thoracic cavities and the heart/lungs were examined along with the SQ layer for worm recovery and count. Results In early pilot studies, the J:Nu strain of mouse yielded the most consistent and highest recovery rate of live larvae at necropsy. Larvae were recovered from the C57BL/6J and the NU/J strains, but at a rate of 50–75% less than the J:Nu strain at various timepoints ranging from 7–21 days post infection. We followed out the molting of the larvae in the mice from the third to the fourth stage. A total of 16 mice were infected with 100 L 3 of the SC-20 isolate and 4 mice were necropsied at day 3, 7, 10, or 14 PI. The results are summarized in Fig. 1 . By day 7 PI, the vast majority of larvae (89%) had molted from the third to fourth stage. Molting was determined to have occurred based on the length of the larvae when examined through a dissecting microscope. As a logical next step, we decided to treat infected mice with MLs (ivermectin and moxidectin) and a cyclic depsipeptide (emodepside). Groups of 6 mice each were infected with 100 L 3 of either the SC-20 or JYD-34 isolate and were left untreated, injected with a vehicle control, or injected with ivermectin, moxidectin, or emodepside. Treatment occurred 1 day PI and necropsy was performed 7 days PI. Table 1 summaries the results for mice infected with SC-20 and Table 2 shows the data from the mice infected with the JYD-34 isolate. Both ivermectin and moxidectin showed some efficacy against both isolates of D. immitis (51–75% efficacy) whereas emodepside was 100% effective against the ML-susceptible isolate of SC-20 and 92% effective against the ML-resistant isolate, JYD-34. Table 1 Efficacy of various drugs when administered to mice 1 day after infection with the ML-susceptible SC-20 isolate of D. immitis and necropsied 7 days PI Treatment Group (SQ injection) N = 6 per group Isolate No. of Infective L 3 Injected Average Total Larvae Recovered Average Normalized Efficacy Based on Controls Untreated Control SC-20 100 24.20 N/A Vehicle Control SC-20 100 25.33 N/A Ivermectin 50mg/kg SC-20 100 8.67 65.05% Moxidectin 50mg/kg SC-20 100 6.00 75.81% Emodepside 100mg/kg SC-20 100 0 100% Emodepside 50mg/kg SC-20 100 0 100% Table 2 Efficacy of various drugs when administered to mice 1 day after infection with the ML-resistant JYD-34 isolate of D. immitis and necropsied 7 days PI Treatment Group (SQ injection) N = 6 per group Isolate No. of Infective L 3 Injected Average Total Larvae Recovered Average Normalized Efficacy Based on Controls Vehicle Control JYD-34 100 24.67 N/A Ivermectin 50mg/kg JYD-34 100 12.00 51.36% Moxidectin 50mg/kg JYD-34 100 6.50 73.65% Emodepside 100mg/kg JYD-34 100 2.00 92% Up until this next set of studies, diphenhydramine had been added to the drinking water a few days before infection and was maintained in the water until necropsy. For the following studies, the drinking water was not supplemented with diphenhydramine. In addition, we wanted to see the recovery rate of larvae if only 50 L 3 were injected and to examine the effects of emodepside if administered orally (PO) via oral gavage. We also strove to see the efficacy of emodepside if given 7 days PI as to target the fourth-stage larvae (L 4 ). Four groups of 3 mice were infected with 50 L 3 (2 groups infected with the SC-20 isolate and 2 groups with the JYD-34 isolate) and necropsied at 7 days PI with half being treated with emodepside PO 1 day PI. Six groups of 3 mice were infected 100 L 3 (3 groups infected with the SC-20 isolate and 3 groups with the JYD-34 isolate) and necropsied 10 days PI with some groups being treated with emodepside PO or SQ 7 days PI. Table 3 summarizes the study design and the results. Table 3 Results of infecting mice (n = 3 per group) with 50 L 3 of D. immitis and necropsying 7 days PI with or without treatment with emodepside at 1 day PI and results of infecting mice with 100 L 3 of D. immitis and necropsying 10 days PI with or without treatment with emodepside at 7 days PI Isolate No. of Infective L 3 Injected Treatment (50mg/kg) Route of treatment Days Post Infection at Treatment Days Post Infection at Necropsy Average % recovery of larvae Average Normalized Efficacy Based on Controls SC20 50 Emodepside PO 1 7 3.3% 91.3% SC20 50 None N/A N/A 7 38.0% N/A JYD-34 50 Emodepside PO 1 7 12.7% 72.0% JYD-34 50 None N/A N/A 7 45.3% N/A SC20 100 Emodepside PO 7 10 0.0% 100% SC20 100 Emodepside SQ N/A 10 0.3% 98.4% SC20 100 None N/A N/A 10 19.3% N/A JYD-34 100 Emodepside PO 7 10 1.0% 97.4% JYD-34 100 Emodepside SQ 7 10 1.0% 97.4% JYD-34 100 None N/A N/A 10 38.0% N/A In this group of studies, we noted that, infecting mice with only 50 L 3 larvae, recovery rates were just as good or better (up to 38% for SC-20 and up to 45% for JYD) than when we infected with 100 larvae. In addition, emodepside was nearly 100% effective at killing the larvae of both isolates when mice were treated 7 days PI, regardless of route of treatment. Efficacy rates for the PO route of treatment were lower for both D. immitis isolates when mice were treated 1 day PI as compared to treatment 7 days PI. It is also evident by the rate of larval recovery from untreated mice that diphenhydramine is not required to maintain D. immitis infection in the mice. We also conducted studies in which we allowed the infective L 3 of the SC-20 isolate to molt and grow into adult worms within the mice. Mice were subcutaneously infected with either 100 or 50 infective L 3 (Table 4 ) and most were necropsied on day 96 PI. Prednisolone was added to the mice’s drinking water for the duration of these studies. Results in Table 4 show a recovery rate of at least 26% of adult worms in mice infected with 100 larvae and a recovery rate of at least 40% from mice infected with only 50 larvae. Worms were found in various areas of the body with most adult worms recovered from the heart/lungs/thoracic cavity by Day 96 (Fig. 2 ) and ranged in length from 3cm to 8cm. A subset of mice were treated with 50mg/kg emodepside SQ on day 1 PI. No adult worms were recovered from those mice. Table 4 Recovery rate of adult D. immitis worms from mice after being necropsied 77 to 96 days post infection with SC-20 infective L 3 and the anatomical location of the worms Average No. of Worms Recovered Isolate No. of mice Days Post Infection at Necropsy No. of Infective L 3 Injected Subcutaneous Tissue Peritoneal Cavity Thoracic Cavity Heart Lungs Average Total No. of Worms Recovered SC-20 3 77 50 6.3 2.3 4.0 3.7 2.3 18.7 SC-20 6 79 50 6.0 1.0 1.8 2.7 0.7 12.0 SC-20 5 93–96 50 5.0 3.8 2.4 7.0 3.0 20.8 SC-20 8 96 100 4.5 5.6 3.9 8.5 4.0 26.6 Discussion We have shown that the athymic J:Nu strain of mouse is a susceptible host for infection with D. immitis , with an average recovery rate of 24% and even up to 45% at 7–10 days PI. For decades, a variety of tissues from a variety of species have successfully been transplanted into nude mice [11] leading to a wealth of new information aiding in the treatment of various maladies, especially oncological diseases [12]. Other strains of mice have also been shown to harbor D. immitis infection weeks after injection with third-stage infective larvae, including the NSG mouse [13, 14] and C57BL/6 Rag2/Il-2rγ −/− mice [15]. Having a functional immune system may be important for some anti parasiticides to work properly, though. Ivermectin disrupts protein release from the excretory-secretory apparatus of microfilaria [16] which then allows the parasite to become vulnerable to the host’s immune system. Neutrophils and PMBCs adhere to microfilaria in the presence of ivermectin in vitro [17] further solidifying the importance of functional innate immune cells for MLs to properly operate. As shown in Table 5 , adapted from the Jackson Laboratories website [18], the J:Nu strain of mice have a robust innate immune system when compared to wild type C57BL/6J mice and the severely immunocompromised NSG mice. Only the adaptive immunity of T cells is lacking in the J:Nu mice compared to the wild type mice. This semi-functional immune system is not only lucrative for drug testing, but it also allows for increased vigor of the mice. Table 5 Mouse Hematology Physiological Data Summary a Parameter Units Male J:Nu b (JAX Strain 007850) Male C57BL/6J c (JAX Strain 0006664) Male NSG c (JAX Strain 005557) White Blood Cell count 10 3 cells/uL 9.1 6.4 0.71 Red Blood Cell count 10 6 cells/uL 9.6 8.2 6.96 Neutrophil count 10 3 cells/uL 1.4 1.5 0.35 Lymphocyte count 10 3 cells/uL 7.1 4.41 0.2 Monocyte count 10 3 cells/uL 0.23 0.34 0.12 Eosinophil Count 10 3 cells/uL 0.25 0.08 0.04 Basophil Count 10 3 cells/uL 0.03 0.01 0 Splenic B Cells % B220+ 48.9 56.1 0.114 Splenic T Cells % CD3e+ 0.02 21.3 0.016 a data adapted from the JAX® Physiological Data Summary b homozygous for Foxn1 nu /Foxn1 nu (nude) and based on 10 mice 4 weeks of age c based on 20–30 mice 8 weeks of age As shown in Fig. 1 , about 90% of the D. immitis larvae have molted from the L 3 to the L 4 stage in the mouse by 7 days PI. This allows for quick turnaround time of evaluation of the molting of larvae in this in vivo model. Tables 1 and 2 show that emodepside is quite effective at killing both the ML-susceptible isolate of SC-20 and the ML-resistant isolate of JYD-34 when administered to the mice 1 day PI. As there are no standardized published doses for MLs and emodepside in mice, we used literature as a guide and focused on studies that treated with only one dose. One example shows the efficacy of emodepside against Trichuris muris was 100% when administered to mice at 75mg/kg PO [19]. It is not unreasonable to suspect that emodepside will be effective at lower doses. It was also encouraging to see that emodepside is quite effective at killing the larvae when given to mice 7 days PI and necropsying just 3 days later (Table 3 ). This is important as the current heartworm preventatives on the market do heavily rely on targeting the early fourth-stage larvae of D. immitis in the dog. The J:Nu mice also served as a successful host to allow third-stage infective larvae of D. immitis to mature into young adult worms (Table 4 ). Early pilot studies (unpublished data) did suggest that additional immunosuppression was required for this to take place. The need for immunosuppression in order to recover adult worms has been documented before in a rat model of D. immitis [20]. Therefore, these mice were given prednisolone in their drinking water for the duration of the studies. The majority of adult worms were recovered from the heart and lungs and thoracic cavity. As some mice were starting to show signs of dyspnea, we did not allow the infection to continue longer than 96 days and elected for humane euthanasia and necropsy before the timepoint that would have allowed for a patent infection. A small subset of mice began to display signs of dyspnea between days 77–79 PI. Once any level of distress was noted in the mice, they were immediately humanely euthanized and necropsied. Young adult worms were recovered from the mice at that earlier timepoint. Many of the worms were in the thoracic musculature as they were still continuing their trek into the thoracic cavity. However, in our lab, we were able to successfully harvest these worms without damaging them. We did have 2 mice that remained very healthy until 102 days PI. During worm recovery, it was noted that the worms had grown and matured enough that the sex of the worms distinguishable. This appears to be the exception, though, as most mice begin to experience caval syndrome starting around 90 days PI. Being able to recover adult D. immitis worms from mice less than three months after infection allows for many other types of research to be conducted, both in vitro and in vivo . In our lab, we are currently utilizing these worms in benchtop assays to evaluate how novel compounds affect the motility and viability of the worms. In addition, we have successfully taken young adult D. immitis worms from a mouse 92 days PI and subcutaneously injected them into a dog. The infection became patent and full grown adult worms up to 30cm long were harvested from the dog. The use of this technology has the ability to significantly decrease the number of dogs used in heartworm research. It is necessary to deduce the effects of compounds on adult heartworms and microfilaria in vivo as MLs are very effective at decreasing MF in dogs with patent infections [21, 22], which could lead to severe reactions as the MF die. A method to decrease the amount of time dogs are kept during the prepatent period has been established by hyper-infecting a few donor dogs and then surgically transplanting those harvested adult worms into the jugular vein of recipient dogs [23]. The use of young adult worms harvested from mice and then inserted subcutaneously could prove to be an alternative method to hyper-infecting donor dogs. As new isolates of D. immitis become available to our lab, we have begun to test the efficacy of established compounds within this mouse model. Our Georgia-IV isolate was validated in July 2023 and early mouse models show that ivermectin is up to 96% effective against this isolate (unpublished data). Once a new isolate is validated, there is a limited time in which pivotal studies may be conducted with that isolate. The Food and Drug Administration and Center for Veterinary Medicine require that laboratory dose confirmation studies should be conducted from current circulating D. immitis isolates obtained from the United States and established for experimental use within 5 years of the start of the study [24]. It is important for researchers to know if the isolate is susceptible or resistant to MLs. Single nucleotide polymorphisms are now being used to genotypically detect ML resistant, susceptible, or mixed isolates of D. immitis [25]. Our mouse model is another tool to determine the potential susceptibility or resistance of new D. immitis isolates in a timely manner. In addition, by taking advantage of the quick turnaround in evaluating the efficacy of novel compounds in this mouse model, researchers can narrow down their candidate pool before beginning the lengthier process of investigating the products in the laboratory dog model. Conclusions Our studies show that the athymic nude J:Nu mouse is a susceptible host for infection with D. immitis and can maintain the infection through the development of the third-stage infective larvae into adult worms. This strain of mouse does not require extra immunosuppression to harbor the infection of the larvae for up to two weeks. Having a mostly intact immune system allows for the animals to remain more thrifty in a non-barrier rodent vivarium, though good husbandry practices are still strictly observed. The presence of neutrophils and lymphocytes also allow for the variable of having the interaction of investigational drugs and immune cells in vivo. As the landscape of heartworm disease is changing with the advent of ML-resistant isolates gaining traction, the way we approach the research is shifting, as well [26]. The use of the J:Nu mouse proves to be an invaluable tool to not only perform quick initial evaluations of novel investigational compounds, but this mouse can also lead the way in helping to further understand the biology of a parasite that can undeniably lead to debilitating and deadly disease in pets around the world. Abbreviations AWA Animal Welfare Act HBSS Hanks balanced salt solution IACUC Institutional Animal Care and Use Committee L 3 Third-stage larvae L 4 Fourth-stage larvae LDA Larval development assay MF Microfilaria ML Macrocyclic lactones No. Number PI Post-infection PO orally SC subcutaneous Declarations Acknowledgements The authors would like to thank all the study site personnel for their support in the conduct of these studies. Funding TRS Labs, Inc. Availability of data and materials Not applicable Authors’ Contributions CF: Conceptualization, methodology, writing—original draft, writing—review and editing, veterinary care and procedures. JWM: Conceptualization, methodology, writing—review and editing. AM: parasitology and laboratory assistance, methodology, writing—review and editing. UD: parasitology and laboratory assistance, methodology, writing—review and editing. SM: project administration, research coordinator Ethics approval and consent to participate The TRS Labs’ Institutional Animal Care and Use Committee approved the study prior to implementation. Consent for publication All of the authors approved the manuscript before submission. Competing interests None of the authors have any competing interests in this study. References Noack S, Harrington J, Carithers DS, Kaminsky R, Selzer PM. Heartworm disease - Overview, intervention, and industry perspective. Int J Parasitol Drugs Drug Resist. 2021 Aug;16:65–89. 10.1016/j.ijpddr.2021.03.004. Burg RW, Miller BM, Baker EE, Birnbaum J, Currie SA, Hartman R, et al. Avermectins, new family of potent anthelmintic agents: producing organism and fermentation. Antimicrob Agents Chemother. 1979 Mar;15(3):361-7. 10.1128/AAC.15.3.361. Gyles C. Heartworm resistance. Can Vet J. 2011 Dec;52(12):1279-80. Drake J, Wiseman S. Increasing incidence of Dirofilaria immitis in dogs in USA with focus on the southeast region 2013–2016. Parasit Vectors. 2018 Jan 17;11(1):39. 10.1186/s13071-018-2631-0. American Heartworm Society - Heartworm incidence maps. https://heartwormsociety.org/veterinary-resources/incidence-maps. Accessed 19 Jan 2026. Hubrecht RC, Carter E. The 3Rs and Humane Experimental Technique: Implementing Change. Animals (Basel). 2019 Sep 30;9(10):754. 10.3390/ani9100754. Evans CC, Moorhead AR, Storey BE, Wolstenholme AJ, Kaplan RM. Development of an in vitro bioassay for measuring susceptibility to macrocyclic lactone anthelmintics in Dirofilaria immitis . Int J Parasitol Drugs Drug Resist. 2013 Jun 6;3:102-8. 10.1016/j.ijpddr.2013.05.001. Sakamoto M, Kimura E, Aoki Y, Jakajima Y. Subcutaneous and intraperitoneal inoculation of Dirofilaria immitis microfilariae into mice. Jpn. J. Parasitol. 1984 Oct; 33(5):415–420 Abraham D, Grieve RB, Mika-Grieve M, Seibert BP. Active and passive immunization of mice against larval Dirofilaria immitis . J Parasitol. 1988;74(2):275–282. Szadvari I, Krizanova O, Babula P. Athymic nude mice as an experimental model for cancer treatment. Physiol Res. 2016;65(Suppl 4):S441-S453. 10.33549/physiolres.933526 Leclerc M, Panijel J, Toyama K. Heterotransplantation of invertebrate organs to congenitally athymic (nude) mice. Transplantation. 1975 Jan;19(1):74–76. 10.1097/00007890-197501000-00014 Giovanella BC, Fogh J. The nude mouse in cancer research. Adv. Cancer Res. 1985, 44, 69–120. Hess JA, Eberhard ML, Segura-Lepe M, Grundner-Culemann K, Kracher B, Shryock J, et al. A rodent model for Dirofilaria immitis , canine heartworm: parasite growth, development, and drug sensitivity in NSG mice. Sci Rep. 2023;13(1):976. 10.1038/s41598-023-27537-z Marriott AE, Dagley JL, Hegde S, Steven A, Fricks C., DiCosty U, et al. Dirofilariasis mouse models for heartworm preclinical research. Frontiers in Microbiology. 2023(14). 10.3389/fmicb.2023.1208301 Risch F, Ludwig-Erdmann C, Hoerauf A, Sager H, Hübner MP. Development of adult Dirofilaria immitis worms in the Rag2/Il-2rγ-/- mouse model. Int J Parasitol. Published online January 19, 2024. doi:10.1016/j.ijpara.2024.01.004 Moreno Y, Nabhan JF, Solomon J, Mackenzie CD, Geary TG. Ivermectin disrupts the function of the excretory-secretory apparatus in microfilariae of Brugia malayi . Proc Natl Acad Sci U S A. 2010 Nov 16;107(46):20120-5 Vatta AF, Dzimianski M, Storey BE, Camus MS, Moorhead AR, Kaplan RM, et al. Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro. Vet Parasitol. 2014 Nov 15;206(1–2):38–42. 10.1016/j.vetpar.2014.02.004. Karpstein T, Pasche V, Häberli C, Scandale I, Neodo A, Keiser J. Evaluation of emodepside in laboratory models of human intestinal nematode and schistosome infections. Parasit Vectors. 2019;12(1):226. 10.1186/s13071-019-3476-x Mills BJ, McTier Tl, Knauer CS, Woods DJ. Anthelmintic laboratory animal model for heartworm. WO 2020/146338 A1, 16 July 2020. Riggs KL, Haney D, Wiseman S. Safety of Credelio Quattro™ (lotilaner, moxidectin, praziquantel, and pyrantel chewable tablets) in dogs infected with adult heartworms ( Dirofilaria immitis ). Parasit Vectors. 2025 Apr 14;18(1):138. 10.1186/s13071-025-06732-z. Grant T, Wiseman S, Snyder DE. Effects of milbemycin oxime, combined with spinosad, when administered orally to microfilaremic dogs infected with adult heartworms ( Dirofilaria immitis ). J Am Vet Med Assoc. 2020;252:1084–9. Rawlings CA, McCall JW. Surgical transplantation of adult Dirofilaria immitis to study heartworm infection and disease in dogs. Am J Vet Res, 1985; 46(1):221–224 CVM GFI #276 Effectiveness of Anthelmintics: Specific Recommendations for Products Proposed for the Prevention of Heartworm Disease in Dogs. https://www.fda.gov/media/163319/download. Accessed 19 Jan 2026. Curry, E, Tack D, Rodgriguez J, Brehm-Lowe D, Letherer J, Lineberry M, et al. Surveillance of single nucleotide polymorphisms correlated to macrocyclic lactone resistance in Dirofilaria immitis from client-owned dogs across the United States. International Journal for Parasitology: Drugs and Drug Resistance 2025. 10.1016/j.ijpddr.2025.100604. Geary T. New paradigms in research on Dirofilaria immitis . Parasit Vectors. 2023, 16:247. 10.1186/s13071-023-05762-9 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8742911","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":587669664,"identity":"96beff84-8671-4ee9-a5b1-49852896dec8","order_by":0,"name":"Crystal Fricks","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA10lEQVRIie2PsQrCMBRFXwkkS2pXQdRfeOIg/o3SwUV/IThlivg73UwItEs+oJstzoKjDoJRnETaujnkvOUO93B5AIHAP0I0PG/kY6SjbReFLl7KlPv8Vki74ruwVJ2VRMa1vjqxOgyKSt8yATO2141KP2doVGk3qrdGs3MW5so2r+BYgoWL3ijOQcdSA5Zpi0LpUxErzlll7lIAHk9dlJIs/AjaWBK/0vJ+P6dglLMT5TjaobMcXYqNSiIpuVxzMWaK1fU5EyMsTNU88wn/rR4IBAKBrzwAAGJKTwxDkKYAAAAASUVORK5CYII=","orcid":"","institution":"TRS Labs, Inc","correspondingAuthor":true,"prefix":"","firstName":"Crystal","middleName":"","lastName":"Fricks","suffix":""},{"id":587669665,"identity":"087d69a8-0db1-4458-b7e8-2a040e2b926d","order_by":1,"name":"John W McCall","email":"","orcid":"","institution":"TRS Labs, Inc","correspondingAuthor":false,"prefix":"","firstName":"John","middleName":"W","lastName":"McCall","suffix":""},{"id":587669666,"identity":"3d6d17e8-e4b7-4487-a119-192274a46b4a","order_by":2,"name":"Abdelmoneim Mansour","email":"","orcid":"","institution":"TRS Labs, Inc","correspondingAuthor":false,"prefix":"","firstName":"Abdelmoneim","middleName":"","lastName":"Mansour","suffix":""},{"id":587669667,"identity":"54ac9858-1ba8-4b70-96ae-8721206e927a","order_by":3,"name":"Utami DiCosty","email":"","orcid":"","institution":"TRS Labs, Inc","correspondingAuthor":false,"prefix":"","firstName":"Utami","middleName":"","lastName":"DiCosty","suffix":""},{"id":587669668,"identity":"6a3889bb-d3a3-4c69-92a4-999c6e148e7b","order_by":4,"name":"Scott McCall","email":"","orcid":"","institution":"TRS Labs, Inc","correspondingAuthor":false,"prefix":"","firstName":"Scott","middleName":"","lastName":"McCall","suffix":""}],"badges":[],"createdAt":"2026-01-30 15:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8742911/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8742911/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102415396,"identity":"cb5ed298-0a08-49d5-b6e6-6ee6b0476111","added_by":"auto","created_at":"2026-02-11 12:43:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":204841,"visible":true,"origin":"","legend":"\u003cp\u003eAverage percentage of third or fourth stage larvae recovered from mice (n = 4 per timepoint) infected with 100 L\u003csub\u003e3\u003c/sub\u003e of the SC-20 isolate at different time points post infection\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8742911/v1/b5e38faddc41bc7dc3371bf4.jpg"},{"id":102415406,"identity":"c38fb2b4-3975-4233-a90c-bdd8e33299e4","added_by":"auto","created_at":"2026-02-11 12:43:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":140369,"visible":true,"origin":"","legend":"\u003cp\u003eAnatomic location of adult worms recovered from mice 93-96 days post-infection with \u003cem\u003eD. immitis \u003c/em\u003eL\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8742911/v1/8afe0488844df5b610560e47.jpg"},{"id":106401684,"identity":"a57f11cb-62d3-4579-b65c-9baaf2019373","added_by":"auto","created_at":"2026-04-08 09:09:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1188819,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8742911/v1/a32a0a61-15c7-472d-8b0f-6330c137d11b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Use of athymic nude mice in a mouse model of Dirofilaria immitis and its utilization in drug screening","fulltext":[{"header":"Background","content":"\u003cp\u003eHeartworm disease, caused by \u003cem\u003eDirofilaria immitis\u003c/em\u003e, is a debilitating malady among canines in warm, humid climates. Heartworm disease can cause severe pulmonary disease and has the potential to be fatal [1]. After the discovery of Ivermectin in the 1970\u0026rsquo;s [2], macrocyclic lactones (MLs) have been the mainstay of prevention of disease from \u003cem\u003eD. immitis\u003c/em\u003e since. In recent history, though, there have been an emergence of cases of heartworm disease from isolates of \u003cem\u003eD. immitis\u003c/em\u003e that are resistant to MLs [3]. The necessity to research and develop new heartworm preventatives is therefore an urgent matter as cases of heartworm disease are on the rise [4,5]. However, in keeping with the spirit of the 3Rs - Replacement, Reduction and Refinement [6], there is more of a demand and desire to improve the process of investigating novel therapeutics. \u003cem\u003eIn vitro\u003c/em\u003e assays, such as the larval migration inhibition assay [7] can be invaluable tools for the initial screening of anthelmintics. Nevertheless, benchtop assays cannot fully mimic the \u003cem\u003ein vivo\u003c/em\u003e system.\u003c/p\u003e \u003cp\u003eMice have been used in research and development for both human and other animal diseases for decades. Even as far back as the 1980\u0026rsquo;s, mice were used for \u003cem\u003eD. immitis\u003c/em\u003e research. As one example, ICR mice were injected either subcutaneously or intraperitoneally with 10,000 \u003cem\u003eD. immitis\u003c/em\u003e microfilaria (MF) and through a laborious process, MF were recovered up to 6 weeks after inoculation [8]. To study viability of larvae, diffusion chambers were placed in BALB/c mice and third-stage larvae of \u003cem\u003eD. immitis\u003c/em\u003e were then introduced into the chambers; live larvae were recovered three weeks later [9]. As with any disease model development process in mice, it is imperative to select the correct strain of mouse to mimic the disease in the normal host species as closely as possible. The athymic nude mouse strain is a staple for human oncology studies [10]. Because they lack a thymus, T cells cannot maturate and that leaves the mice susceptible to and able to accept foreign tissue. It is with that thought in mind that we sought to see if athymic nude mice would be a good candidate for hosting infection with \u003cem\u003eD. immitis\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and Reagents\u003c/h2\u003e \u003cp\u003e All animal work conducted at TRS was approved by the facility\u0026rsquo;s IACUC, which follows regulations set forth by AWA and the Guide for Care and Use of Laboratory Animals. Male mice (5\u0026ndash;8 weeks of age) were obtained from Jackson Laboratories in Bar Harbor, Maine, USA. Strains purchased were J:Nu (JAX stock #007850), Nu/J (JAX stock #002019), and C57BL/6J (JAX stock #000664). Mice were housed 3 per cage in standard shoebox type IV polycarbonate cages in a dedicated room with a 12 hour light/dark cycle. They were provided with sterile water (by reverse osmosis) and irradiated pelleted food (Teklad Global 18% protein rodent diet 2918) ad lib. Mice were individually identified by applying different colored markings at the base of the tail. Three different drugs -ivermectin (Sigma or Covetrus), moxidectin (Sigma) and emodepside (AchemBlock)- were used for efficacy studies. Hanks Balance Salt Solution (HBSS), purchased from Sigma, was used to facilitate injections of infective larvae and for soaking of carcasses at necropsy. Prednisolone sodium phosphate oral solution 15mg/5ml (Covetrus) and over the counter gel capsules of generic diphenhydramine 25mg were added to the drinking water in some instances.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInfections\u003c/h3\u003e\n\u003cp\u003eMice were allowed to acclimate to the facility for at least one week prior to infection. In early studies, diphenhydramine was added to the drinking water 3\u0026ndash;5 days prior to infection and was maintained as such until necropsy. For some longer-term studies, prednisolone was also added to the drinking water. On day 0, mice were infected with infective third-stage larvae (L\u003csub\u003e3\u003c/sub\u003e) of either the macrocyclic lactone (ML) susceptible \u003cem\u003eD. immitis\u003c/em\u003e isolate SC-20 or the ML resistant isolate JYD-34. Between 50\u0026ndash;100 L\u003csub\u003e3\u003c/sub\u003e in approximately 500 to 700uL of HBSS were injected subcutaneously (SQ) over the caudal dorsum. Larvae were collected from crushed mosquitoes that had fed on blood that contained the infective microfilariae 14 days prior.\u003c/p\u003e\n\u003ch3\u003eTreatments\u003c/h3\u003e\n\u003cp\u003eMice were treated SQ on day 1 or day 7 with 100 mg/kg or 50mg/kg of emodepside, ivermectin, or moxidectin or with a vehicle control. Drugs used for SQ injections were dissolved in DMSO (Neogen) containing 0.5% Tween\u003csup\u003e@\u003c/sup\u003e80 (Sigma), with the exception of commercially available ivermectin (Covetrus). In some cases, emodepside was administered via oral gavage.\u003c/p\u003e\n\u003ch3\u003eNecropsy\u003c/h3\u003e\n\u003cp\u003eMice were necropsied at various timepoints, depending on the nature of the particular study. After humane euthanasia via gradual carbon dioxide asphyxiation followed by cervical dislocation, larvae were recovered from the subcutaneous tissue. For mice that were necropsied at later timepoints (such as day 77 and beyond), the peritoneal and thoracic cavities and the heart/lungs were examined along with the SQ layer for worm recovery and count.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eIn early pilot studies, the J:Nu strain of mouse yielded the most consistent and highest recovery rate of live larvae at necropsy. Larvae were recovered from the C57BL/6J and the NU/J strains, but at a rate of 50\u0026ndash;75% less than the J:Nu strain at various timepoints ranging from 7\u0026ndash;21 days post infection.\u003c/p\u003e \u003cp\u003eWe followed out the molting of the larvae in the mice from the third to the fourth stage. A total of 16 mice were infected with 100 L\u003csub\u003e3\u003c/sub\u003e of the SC-20 isolate and 4 mice were necropsied at day 3, 7, 10, or 14 PI. The results are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. By day 7 PI, the vast majority of larvae (89%) had molted from the third to fourth stage. Molting was determined to have occurred based on the length of the larvae when examined through a dissecting microscope.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs a logical next step, we decided to treat infected mice with MLs (ivermectin and moxidectin) and a cyclic depsipeptide (emodepside). Groups of 6 mice each were infected with 100 L\u003csub\u003e3\u003c/sub\u003e of either the SC-20 or JYD-34 isolate and were left untreated, injected with a vehicle control, or injected with ivermectin, moxidectin, or emodepside. Treatment occurred 1 day PI and necropsy was performed 7 days PI. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e summaries the results for mice infected with SC-20 and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the data from the mice infected with the JYD-34 isolate. Both ivermectin and moxidectin showed some efficacy against both isolates of \u003cem\u003eD. immitis\u003c/em\u003e (51\u0026ndash;75% efficacy) whereas emodepside was 100% effective against the ML-susceptible isolate of SC-20 and 92% effective against the ML-resistant isolate, JYD-34.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEfficacy of various drugs when administered to mice 1 day after infection with the ML-susceptible SC-20 isolate of \u003cem\u003eD. immitis\u003c/em\u003e and necropsied 7 days PI\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment Group (SQ injection)\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;6 per group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of Infective L\u003csub\u003e3\u003c/sub\u003e Injected\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAverage Total Larvae Recovered\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAverage Normalized Efficacy Based on Controls\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUntreated Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVehicle Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e25.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIvermectin 50mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e65.05%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoxidectin 50mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e75.81%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmodepside 100mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmodepside 50mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSC-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEfficacy of various drugs when administered to mice 1 day after infection with the ML-resistant JYD-34 isolate of \u003cem\u003eD. immitis\u003c/em\u003e and necropsied 7 days PI\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment Group (SQ injection)\u003c/p\u003e \u003cp\u003eN\u0026thinsp;=\u0026thinsp;6 per group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of Infective L\u003csub\u003e3\u003c/sub\u003e Injected\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAverage Total Larvae Recovered\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAverage Normalized Efficacy Based on Controls\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVehicle Control\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJYD-34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIvermectin 50mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJYD-34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51.36%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoxidectin 50mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJYD-34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e73.65%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEmodepside 100mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJYD-34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e92%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eUp until this next set of studies, diphenhydramine had been added to the drinking water a few days before infection and was maintained in the water until necropsy. For the following studies, the drinking water was not supplemented with diphenhydramine. In addition, we wanted to see the recovery rate of larvae if only 50 L\u003csub\u003e3\u003c/sub\u003e were injected and to examine the effects of emodepside if administered orally (PO) via oral gavage. We also strove to see the efficacy of emodepside if given 7 days PI as to target the fourth-stage larvae (L\u003csub\u003e4\u003c/sub\u003e). Four groups of 3 mice were infected with 50 L\u003csub\u003e3\u003c/sub\u003e (2 groups infected with the SC-20 isolate and 2 groups with the JYD-34 isolate) and necropsied at 7 days PI with half being treated with emodepside PO 1 day PI. Six groups of 3 mice were infected 100 L\u003csub\u003e3\u003c/sub\u003e (3 groups infected with the SC-20 isolate and 3 groups with the JYD-34 isolate) and necropsied 10 days PI with some groups being treated with emodepside PO or SQ 7 days PI. Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e summarizes the study design and the results.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eResults of infecting mice (n\u0026thinsp;=\u0026thinsp;3 per group) with 50 L\u003csub\u003e3\u003c/sub\u003e of \u003cem\u003eD. immitis\u003c/em\u003e and necropsying 7 days PI with or without treatment with emodepside at 1 day PI and results of infecting mice with 100 L\u003csub\u003e3\u003c/sub\u003e of \u003cem\u003eD. immitis\u003c/em\u003e and necropsying 10 days PI with or without treatment with emodepside at 7 days PI\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of Infective L\u003csub\u003e3\u003c/sub\u003e Injected\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003cp\u003e(50mg/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRoute of treatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDays Post Infection at Treatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDays Post Infection at Necropsy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAverage % recovery of larvae\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eAverage Normalized Efficacy Based on Controls\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSC20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEmodepside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e91.3%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSC20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e38.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJYD-34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEmodepside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e12.7%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e72.0%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJYD-34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e45.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSC20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEmodepside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSC20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEmodepside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e98.4%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSC20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e19.3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJYD-34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEmodepside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e97.4%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJYD-34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEmodepside\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSQ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e97.4%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJYD-34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e38.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn this group of studies, we noted that, infecting mice with only 50 L\u003csub\u003e3\u003c/sub\u003e larvae, recovery rates were just as good or better (up to 38% for SC-20 and up to 45% for JYD) than when we infected with 100 larvae. In addition, emodepside was nearly 100% effective at killing the larvae of both isolates when mice were treated 7 days PI, regardless of route of treatment. Efficacy rates for the PO route of treatment were lower for both \u003cem\u003eD. immitis\u003c/em\u003e isolates when mice were treated 1 day PI as compared to treatment 7 days PI. It is also evident by the rate of larval recovery from untreated mice that diphenhydramine is not required to maintain \u003cem\u003eD. immitis\u003c/em\u003e infection in the mice.\u003c/p\u003e \u003cp\u003eWe also conducted studies in which we allowed the infective L\u003csub\u003e3\u003c/sub\u003e of the SC-20 isolate to molt and grow into adult worms within the mice. Mice were subcutaneously infected with either 100 or 50 infective L\u003csub\u003e3\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and most were necropsied on day 96 PI. Prednisolone was added to the mice\u0026rsquo;s drinking water for the duration of these studies. Results in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e show a recovery rate of at least 26% of adult worms in mice infected with 100 larvae and a recovery rate of at least 40% from mice infected with only 50 larvae. Worms were found in various areas of the body with most adult worms recovered from the heart/lungs/thoracic cavity by Day 96 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and ranged in length from 3cm to 8cm. A subset of mice were treated with 50mg/kg emodepside SQ on day 1 PI. No adult worms were recovered from those mice.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRecovery rate of adult \u003cem\u003eD. immitis\u003c/em\u003e worms from mice after being necropsied 77 to 96 days post infection with SC-20 infective L\u003csub\u003e3\u003c/sub\u003e and the anatomical location of the worms\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c9\" namest=\"c5\"\u003e \u003cp\u003eAverage No. of Worms Recovered\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of mice\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDays Post Infection at Necropsy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo. of Infective L\u003csub\u003e3\u003c/sub\u003e Injected\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSubcutaneous Tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePeritoneal Cavity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThoracic Cavity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eHeart\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eLungs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003cp\u003eTotal No. of Worms Recovered\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSC-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e18.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSC-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSC-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e93\u0026ndash;96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e20.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSC-20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e8.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e26.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have shown that the athymic J:Nu strain of mouse is a susceptible host for infection with \u003cem\u003eD. immitis\u003c/em\u003e, with an average recovery rate of 24% and even up to 45% at 7\u0026ndash;10 days PI. For decades, a variety of tissues from a variety of species have successfully been transplanted into nude mice [11] leading to a wealth of new information aiding in the treatment of various maladies, especially oncological diseases [12]. Other strains of mice have also been shown to harbor \u003cem\u003eD. immitis\u003c/em\u003e infection weeks after injection with third-stage infective larvae, including the NSG mouse [13, 14] and C57BL/6 \u003cem\u003eRag2/Il-2rγ\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice [15]. Having a functional immune system may be important for some anti parasiticides to work properly, though. Ivermectin disrupts protein release from the excretory-secretory apparatus of microfilaria [16] which then allows the parasite to become vulnerable to the host\u0026rsquo;s immune system. Neutrophils and PMBCs adhere to microfilaria in the presence of ivermectin \u003cem\u003ein vitro\u003c/em\u003e [17] further solidifying the importance of functional innate immune cells for MLs to properly operate. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, adapted from the Jackson Laboratories website [18], the J:Nu strain of mice have a robust innate immune system when compared to wild type C57BL/6J mice and the severely immunocompromised NSG mice. Only the adaptive immunity of T cells is lacking in the J:Nu mice compared to the wild type mice. This semi-functional immune system is not only lucrative for drug testing, but it also allows for increased vigor of the mice.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMouse Hematology Physiological Data Summary \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUnits\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMale J:Nu \u003csup\u003eb\u003c/sup\u003e (JAX Strain 007850)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMale C57BL/6J \u003csup\u003ec\u003c/sup\u003e (JAX Strain 0006664)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMale NSG \u003csup\u003ec\u003c/sup\u003e (JAX Strain 005557)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWhite Blood Cell count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e3\u003c/sup\u003e cells/uL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRed Blood Cell count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e6\u003c/sup\u003e cells/uL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNeutrophil count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e3\u003c/sup\u003e cells/uL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphocyte count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e3\u003c/sup\u003e cells/uL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMonocyte count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e3\u003c/sup\u003e cells/uL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEosinophil Count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e3\u003c/sup\u003e cells/uL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasophil Count\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10\u003csup\u003e3\u003c/sup\u003e cells/uL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSplenic B Cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% B220+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e48.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e56.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.114\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSplenic T Cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e% CD3e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e21.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.016\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e data adapted from the JAX\u0026reg;\u0026nbsp;Physiological Data Summary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c5\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003eb\u003c/sup\u003e homozygous for \u003cem\u003eFoxn1\u003c/em\u003e\u003csup\u003e\u003cem\u003enu\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/Foxn1\u003c/em\u003e\u003csup\u003e\u003cem\u003enu\u003c/em\u003e\u003c/sup\u003e (nude) and based on 10 mice 4 weeks of age\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003ec\u003c/sup\u003e based on 20\u0026ndash;30 mice 8 weeks of age\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, about 90% of the \u003cem\u003eD. immitis\u003c/em\u003e larvae have molted from the L\u003csub\u003e3\u003c/sub\u003e to the L\u003csub\u003e4\u003c/sub\u003e stage in the mouse by 7 days PI. This allows for quick turnaround time of evaluation of the molting of larvae in this \u003cem\u003ein vivo\u003c/em\u003e model. Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e show that emodepside is quite effective at killing both the ML-susceptible isolate of SC-20 and the ML-resistant isolate of JYD-34 when administered to the mice 1 day PI. As there are no standardized published doses for MLs and emodepside in mice, we used literature as a guide and focused on studies that treated with only one dose. One example shows the efficacy of emodepside against \u003cem\u003eTrichuris muris\u003c/em\u003e was 100% when administered to mice at 75mg/kg PO [19]. It is not unreasonable to suspect that emodepside will be effective at lower doses. It was also encouraging to see that emodepside is quite effective at killing the larvae when given to mice 7 days PI and necropsying just 3 days later (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This is important as the current heartworm preventatives on the market do heavily rely on targeting the early fourth-stage larvae of \u003cem\u003eD. immitis\u003c/em\u003e in the dog.\u003c/p\u003e \u003cp\u003eThe J:Nu mice also served as a successful host to allow third-stage infective larvae of \u003cem\u003eD. immitis\u003c/em\u003e to mature into young adult worms (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Early pilot studies (unpublished data) did suggest that additional immunosuppression was required for this to take place. The need for immunosuppression in order to recover adult worms has been documented before in a rat model of \u003cem\u003eD. immitis\u003c/em\u003e [20]. Therefore, these mice were given prednisolone in their drinking water for the duration of the studies. The majority of adult worms were recovered from the heart and lungs and thoracic cavity. As some mice were starting to show signs of dyspnea, we did not allow the infection to continue longer than 96 days and elected for humane euthanasia and necropsy before the timepoint that would have allowed for a patent infection. A small subset of mice began to display signs of dyspnea between days 77\u0026ndash;79 PI. Once any level of distress was noted in the mice, they were immediately humanely euthanized and necropsied. Young adult worms were recovered from the mice at that earlier timepoint. Many of the worms were in the thoracic musculature as they were still continuing their trek into the thoracic cavity. However, in our lab, we were able to successfully harvest these worms without damaging them. We did have 2 mice that remained very healthy until 102 days PI. During worm recovery, it was noted that the worms had grown and matured enough that the sex of the worms distinguishable. This appears to be the exception, though, as most mice begin to experience caval syndrome starting around 90 days PI.\u003c/p\u003e \u003cp\u003eBeing able to recover adult \u003cem\u003eD. immitis\u003c/em\u003e worms from mice less than three months after infection allows for many other types of research to be conducted, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. In our lab, we are currently utilizing these worms in benchtop assays to evaluate how novel compounds affect the motility and viability of the worms. In addition, we have successfully taken young adult \u003cem\u003eD. immitis\u003c/em\u003e worms from a mouse 92 days PI and subcutaneously injected them into a dog. The infection became patent and full grown adult worms up to 30cm long were harvested from the dog. The use of this technology has the ability to significantly decrease the number of dogs used in heartworm research. It is necessary to deduce the effects of compounds on adult heartworms and microfilaria \u003cem\u003ein vivo\u003c/em\u003e as MLs are very effective at decreasing MF in dogs with patent infections [21, 22], which could lead to severe reactions as the MF die. A method to decrease the amount of time dogs are kept during the prepatent period has been established by hyper-infecting a few donor dogs and then surgically transplanting those harvested adult worms into the jugular vein of recipient dogs [23]. The use of young adult worms harvested from mice and then inserted subcutaneously could prove to be an alternative method to hyper-infecting donor dogs.\u003c/p\u003e \u003cp\u003eAs new isolates of \u003cem\u003eD. immitis\u003c/em\u003e become available to our lab, we have begun to test the efficacy of established compounds within this mouse model. Our Georgia-IV isolate was validated in July 2023 and early mouse models show that ivermectin is up to 96% effective against this isolate (unpublished data). Once a new isolate is validated, there is a limited time in which pivotal studies may be conducted with that isolate. The Food and Drug Administration and Center for Veterinary Medicine require that laboratory dose confirmation studies should be conducted from current circulating \u003cem\u003eD. immitis\u003c/em\u003e isolates obtained from the United States and established for experimental use within 5 years of the start of the study [24]. It is important for researchers to know if the isolate is susceptible or resistant to MLs. Single nucleotide polymorphisms are now being used to genotypically detect ML resistant, susceptible, or mixed isolates of \u003cem\u003eD. immitis\u003c/em\u003e [25]. Our mouse model is another tool to determine the potential susceptibility or resistance of new \u003cem\u003eD. immitis\u003c/em\u003e isolates in a timely manner. In addition, by taking advantage of the quick turnaround in evaluating the efficacy of novel compounds in this mouse model, researchers can narrow down their candidate pool before beginning the lengthier process of investigating the products in the laboratory dog model.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur studies show that the athymic nude J:Nu mouse is a susceptible host for infection with \u003cem\u003eD. immitis\u003c/em\u003e and can maintain the infection through the development of the third-stage infective larvae into adult worms. This strain of mouse does not require extra immunosuppression to harbor the infection of the larvae for up to two weeks. Having a mostly intact immune system allows for the animals to remain more thrifty in a non-barrier rodent vivarium, though good husbandry practices are still strictly observed. The presence of neutrophils and lymphocytes also allow for the variable of having the interaction of investigational drugs and immune cells \u003cem\u003ein vivo.\u003c/em\u003e As the landscape of heartworm disease is changing with the advent of ML-resistant isolates gaining traction, the way we approach the research is shifting, as well [26]. The use of the J:Nu mouse proves to be an invaluable tool to not only perform quick initial evaluations of novel investigational compounds, but this mouse can also lead the way in helping to further understand the biology of a parasite that can undeniably lead to debilitating and deadly disease in pets around the world.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAWA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnimal Welfare Act\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHBSS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHanks balanced salt solution\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIACUC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInstitutional Animal Care and Use Committee\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eL\u003csub\u003e3\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThird-stage larvae\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eL\u003csub\u003e4\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFourth-stage larvae\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLDA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLarval development assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMicrofilaria\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eML\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMacrocyclic lactones\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNo.\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePost-infection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eorally\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esubcutaneous\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cu\u003eAcknowledgements\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank all the study site personnel for their support in the conduct of these studies.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eFunding\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eTRS Labs, Inc.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAvailability of data and materials\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eAuthors’ Contributions\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eCF: Conceptualization, methodology, writing—original draft, writing—review and editing, veterinary care and procedures. JWM: Conceptualization, methodology, writing—review and editing. AM: parasitology and laboratory assistance, methodology, writing—review and editing. UD: parasitology and laboratory assistance, methodology, writing—review and editing. SM: project administration, research coordinator\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eEthics approval and consent to participate\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eThe TRS Labs’ Institutional Animal Care and Use Committee approved the study prior to implementation.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eConsent for publication\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eAll of the authors approved the manuscript before submission.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eCompeting interests\u003c/u\u003e\u003c/p\u003e\n\u003cp\u003eNone of the authors have any competing interests in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNoack S, Harrington J, Carithers DS, Kaminsky R, Selzer PM. Heartworm disease - Overview, intervention, and industry perspective. Int J Parasitol Drugs Drug Resist. 2021 Aug;16:65–89. 10.1016/j.ijpddr.2021.03.004.\u003c/li\u003e\n\u003cli\u003eBurg RW, Miller BM, Baker EE, Birnbaum J, Currie SA, Hartman R, et al. Avermectins, new family of potent anthelmintic agents: producing organism and fermentation. Antimicrob Agents Chemother. 1979 Mar;15(3):361-7. 10.1128/AAC.15.3.361.\u003c/li\u003e\n\u003cli\u003eGyles C. Heartworm resistance. Can Vet J. 2011 Dec;52(12):1279-80.\u003c/li\u003e\n\u003cli\u003eDrake J, Wiseman S. Increasing incidence of \u003cem\u003eDirofilaria immitis\u003c/em\u003e in dogs in USA with focus on the southeast region 2013–2016. Parasit Vectors. 2018 Jan 17;11(1):39. 10.1186/s13071-018-2631-0.\u003c/li\u003e\n\u003cli\u003eAmerican Heartworm Society - Heartworm incidence maps. https://heartwormsociety.org/veterinary-resources/incidence-maps. Accessed 19 Jan 2026.\u003c/li\u003e\n\u003cli\u003eHubrecht RC, Carter E. The 3Rs and Humane Experimental Technique: Implementing Change. Animals (Basel). 2019 Sep 30;9(10):754. 10.3390/ani9100754.\u003c/li\u003e\n\u003cli\u003eEvans CC, Moorhead AR, Storey BE, Wolstenholme AJ, Kaplan RM. Development of an in vitro bioassay for measuring susceptibility to macrocyclic lactone anthelmintics in \u003cem\u003eDirofilaria immitis\u003c/em\u003e. Int J Parasitol Drugs Drug Resist. 2013 Jun 6;3:102-8. 10.1016/j.ijpddr.2013.05.001.\u003c/li\u003e\n\u003cli\u003eSakamoto M, Kimura E, Aoki Y, Jakajima Y. Subcutaneous and intraperitoneal inoculation of \u003cem\u003eDirofilaria immitis\u003c/em\u003e microfilariae into mice. Jpn. J. Parasitol. 1984 Oct; 33(5):415–420\u003c/li\u003e\n\u003cli\u003eAbraham D, Grieve RB, Mika-Grieve M, Seibert BP. Active and passive immunization of mice against larval \u003cem\u003eDirofilaria immitis\u003c/em\u003e. J Parasitol. 1988;74(2):275–282.\u003c/li\u003e\n\u003cli\u003eSzadvari I, Krizanova O, Babula P. Athymic nude mice as an experimental model for cancer treatment. Physiol Res. 2016;65(Suppl 4):S441-S453. 10.33549/physiolres.933526\u003c/li\u003e\n\u003cli\u003eLeclerc M, Panijel J, Toyama K. Heterotransplantation of invertebrate organs to congenitally athymic (nude) mice. Transplantation. 1975 Jan;19(1):74–76. 10.1097/00007890-197501000-00014\u003c/li\u003e\n\u003cli\u003eGiovanella BC, Fogh J. The nude mouse in cancer research. Adv. Cancer Res. 1985, 44, 69–120.\u003c/li\u003e\n\u003cli\u003eHess JA, Eberhard ML, Segura-Lepe M, Grundner-Culemann K, Kracher B, Shryock J, et al. A rodent model for \u003cem\u003eDirofilaria immitis\u003c/em\u003e, canine heartworm: parasite growth, development, and drug sensitivity in NSG mice. Sci Rep. 2023;13(1):976. 10.1038/s41598-023-27537-z\u003c/li\u003e\n\u003cli\u003eMarriott AE, Dagley JL, Hegde S, Steven A, Fricks C., DiCosty U, et al. \u003cem\u003eDirofilariasis\u003c/em\u003e mouse models for heartworm preclinical research. Frontiers in Microbiology. 2023(14). 10.3389/fmicb.2023.1208301\u003c/li\u003e\n\u003cli\u003eRisch F, Ludwig-Erdmann C, Hoerauf A, Sager H, Hübner MP. Development of adult \u003cem\u003eDirofilaria immitis\u003c/em\u003e worms in the Rag2/Il-2rγ-/- mouse model. Int J Parasitol. Published online January 19, 2024. doi:10.1016/j.ijpara.2024.01.004\u003c/li\u003e\n\u003cli\u003eMoreno Y, Nabhan JF, Solomon J, Mackenzie CD, Geary TG. Ivermectin disrupts the function of the excretory-secretory apparatus in microfilariae of \u003cem\u003eBrugia malayi\u003c/em\u003e. Proc Natl Acad Sci U S A. 2010 Nov 16;107(46):20120-5\u003c/li\u003e\n\u003cli\u003eVatta AF, Dzimianski M, Storey BE, Camus MS, Moorhead AR, Kaplan RM, et al. Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to \u003cem\u003eDirofilaria immitis\u003c/em\u003e microfilariae in vitro. Vet Parasitol. 2014 Nov 15;206(1–2):38–42. 10.1016/j.vetpar.2014.02.004.\u003c/li\u003e\n\u003cli\u003eKarpstein T, Pasche V, Häberli C, Scandale I, Neodo A, Keiser J. Evaluation of emodepside in laboratory models of human intestinal nematode and schistosome infections. Parasit Vectors. 2019;12(1):226. 10.1186/s13071-019-3476-x\u003c/li\u003e\n\u003cli\u003eMills BJ, McTier Tl, Knauer CS, Woods DJ. Anthelmintic laboratory animal model for heartworm. WO 2020/146338 A1, 16 July 2020.\u003c/li\u003e\n\u003cli\u003eRiggs KL, Haney D, Wiseman S. Safety of Credelio Quattro™ (lotilaner, moxidectin, praziquantel, and pyrantel chewable tablets) in dogs infected with adult heartworms (\u003cem\u003eDirofilaria immitis\u003c/em\u003e). Parasit Vectors. 2025 Apr 14;18(1):138. 10.1186/s13071-025-06732-z.\u003c/li\u003e\n\u003cli\u003eGrant T, Wiseman S, Snyder DE. Effects of milbemycin oxime, combined with spinosad, when administered orally to microfilaremic dogs infected with adult heartworms (\u003cem\u003eDirofilaria immitis\u003c/em\u003e). J Am Vet Med Assoc. 2020;252:1084–9.\u003c/li\u003e\n\u003cli\u003eRawlings CA, McCall JW. Surgical transplantation of adult Dirofilaria immitis to study heartworm infection and disease in dogs. Am J Vet Res, 1985; 46(1):221–224\u003c/li\u003e\n\u003cli\u003eCVM GFI #276 Effectiveness of Anthelmintics: Specific Recommendations for Products Proposed for the Prevention of Heartworm Disease in Dogs. https://www.fda.gov/media/163319/download. Accessed 19 Jan 2026.\u003c/li\u003e\n\u003cli\u003eCurry, E, Tack D, Rodgriguez J, Brehm-Lowe D, Letherer J, Lineberry M, et al. Surveillance of single nucleotide polymorphisms correlated to macrocyclic lactone resistance in \u003cem\u003eDirofilaria immitis\u003c/em\u003e from client-owned dogs across the United States. International Journal for Parasitology: Drugs and Drug Resistance 2025. 10.1016/j.ijpddr.2025.100604.\u003c/li\u003e\n\u003cli\u003eGeary T. New paradigms in research on \u003cem\u003eDirofilaria immitis\u003c/em\u003e. Parasit Vectors. 2023, 16:247. 10.1186/s13071-023-05762-9\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Dirofilaria immitis, heartworm mouse model, macrocyclic lactones","lastPublishedDoi":"10.21203/rs.3.rs-8742911/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8742911/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe heartworm \u003cem\u003eDirofilaria immitis\u003c/em\u003e mouse model has been a recent topic of research and discussion as a tool that is more logistically feasible and economical to study the efficacy of new drugs as opposed to using dogs and/or cats. Often, for the initial screening of novel antifilarial compounds, larval development assay (LDA) is used \u003cem\u003ein vitro.\u003c/em\u003e However, a reliable \u003cem\u003eD. immitis\u003c/em\u003e mouse model would be beneficial as \u003cem\u003ein vitro\u003c/em\u003e benchtop assays cannot fully mimic the \u003cem\u003ein vivo\u003c/em\u003e system.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSeveral strains of mice were evaluated for their susceptibility to the infective larval stage of \u003cem\u003eD. immitis\u003c/em\u003e while attempting to use a low cost, low maintenance mouse. Experimental infections were performed by injecting third-stage infective larvae subcutaneously and performing necropsies at several time points post-infection (PI).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBy 7\u0026ndash;10 days PI, 89\u0026ndash;96% of the worms recovered from the subcutaneous tissue were determined to have molted to the fourth stage. Among the mouse strains tested for susceptibility to \u003cem\u003eD. immitis\u003c/em\u003e larval infection, the athymic nude mouse strain, J:NU, proved to be the best candidate for developing the heartworm-mouse model. When infected with 50\u0026ndash;100 infective third-stage larvae, the recovery rate of fourth stage larvae was consistently 20\u0026ndash;30% at 7\u0026ndash;10 days PI. This model was then evaluated using the macrocyclic lactones (ML) ivermectin and moxidectin, and emodepside, a cyclic depsipeptide, as a treatment against the third-stage and fourth-stage infective larvae of a ML-susceptible and resistant heartworm isolate. Results show that the MLs had good efficacy and emodepside was nearly 100% effective against the ML susceptible \u003cem\u003eD. immitis\u003c/em\u003e isolate. In addition, mice were also necropsied 77 to 96 days post infection of third-stage infective larvae with recovery rates of up to 46% of adult worms found in the heart and lungs as well as other areas of the body.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eAlthough target animals (dogs and cats) should be used to confirm the efficacy of new drugs to combat \u003cem\u003eD. immitis\u003c/em\u003e, this heartworm mouse model will give a more economical and efficient approach to screen novel compounds.\u003c/p\u003e","manuscriptTitle":"Use of athymic nude mice in a mouse model of Dirofilaria immitis and its utilization in drug screening","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 12:38:20","doi":"10.21203/rs.3.rs-8742911/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"42142e27-8ce1-4552-a704-b72f93094de8","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-01T10:58:40+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 12:38:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8742911","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8742911","identity":"rs-8742911","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}