Delayed canine heartworm (Dirofilaria immitis) microfilarial reduction following Advocate™ for dogs (imidacloprid, moxidectin) treatment.

Macrocyclic lactone (ML) anthelmintics are currently the only class of drugs available for canine heartworm prevention. Recent reports of Dirofilaria immitis infection occurring in dogs reportedly receiving 'rigorous' prevention in Queensland, Australia, coupled with the confirmation of ML-resistant isolates in the USA, has led to speculation about the potential emergence of ML-resistance in Australia. In this study, we describe two cases (Dog 1 and 2) of asymptomatic canine heartworm disease in Townsville, Australia, that were reportedly receiving 'rigorous' heartworm prevention according to the owners' claims. We aimed to deploy currently available tools to assess the phenotypic and genotypic ML-resistance status of these two dogs. For phenotypic testing, we performed an in-vivo 7-day microfilariae suppression test using a dose of spot-on moxidectin (Advocate™ for Dogs, 100 g/L imidacloprid + 25 g/L moxidectin). This formulation is marketed as Advantage Multi® for Dogs in the USA, which claims a D. immitis microfilaricidal effect. For genetic testing, an Illumina amplicon metabarcoding approach was used to target single nucleotide polymorphisms (SNPs) previously associated with ML-resistance in D. immitis from the USA. Dog 1 and Dog 2 demonstrated <10 % and <40 % reductions in circulating microfilariae seven days after moxidectin treatment, respectively. These phenotypes were not corroborated by genetic SNP testing, as both dogs were classified as susceptible across all examined markers. To streamline testing of D. immitis SNPs, we developed a rhAmp™ SNP qPCR approach for rapidly genotyping suspect cases of ML-resistant infections at the two major loci (L15709_A and L30575). These findings illustrate a phenomenon shown in some heartworm cases outside the USA, whereby infected dogs are failing to see marked reductions in microfilaraemia after ML treatment but possess an ML-susceptible genotype.

Current status of immunodeficient mouse models as substitutes to reduce cat and dog use in heartworm preclinical research.

Chemoprophylactic prevention of veterinary heartworm disease in companion animals, caused by the vector-borne nematode parasite Dirofilaria immitis, is a multi-billion-dollar global market. Experimental use of cats and dogs in preclinical heartworm drug testing is increasing due to evolving drug-resistance to frontline macrocyclic lactones and renewed investment in alternative preventative drug research. We and others recently published data demonstrating proof-of-concept of utilising lymphopenic severe-combined immunodeficient (SCID) or Recombination Activating Gene (RAG)2 deficient mice with additional knockout of the IL-2/7 receptor gamma chain (γc) as alternative preventative drug screening research models of dirofilariasis. Here we summarise the current knowledge of candidate immunodeficient mouse models tested, including a comparison of susceptibility using different background strains of mice, different D. immitis isolates, following use of anti-inflammatory treatments to further suppress residual innate immunity, and efficacies achieved against different reference anthelmintics. We supplement this precis with new data on treatment response to the veterinary anthelmintic, oxfendazole, and initial evaluation of D. immitis susceptibility in CB.17 SCID and C57BL/6 RAG2 -/-γc -/- mice. We conclude that in addition to NSG and NXG mice, RAG2 -/-γc -/- mice on either a BALB/c or C57BL/6 background offer an alternative screening model option, widening access to academic and commercial laboratories wishing to pursue initial rapid in vivo drug screening whilst avoiding potentially unnecessary cat or dog testing.

Australian Marine and Terrestrial Streptomyces-Derived Surugamides, and Synthetic Analogs, and Their Ability to Inhibit Dirofilaria immitis (Heartworm) Motility.

A bioassay-guided chemical investigation of a bacterium, Streptomyces sp. CMB-MRB032, isolated from sheep feces collected near Bathurst, Victoria, Australia, yielded the known polyketide antimycins A4a (1) and A2a (2) as potent inhibitors of Dirofilaria immitis (heartworm) microfilaria (mf) motility (EC50 0.0013-0.0021 µg/mL), along with the octapeptide surugamide A (3) and the new N-methylated analog surugamide K (4). With biological data suggesting surugamides may also exhibit activity against D. immitis, a GNPS molecular network analysis of a library of microbes sourced from geographically diverse Australian ecosystems identified a further five taxonomically and chemically distinct surugamide producers. Scaled-up cultivation of one such producer, Streptomyces sp. CMB-M0112 isolated from a marine sediment collected at Shorncliff, Qld, Australia, yielded 3 along with the new acyl-surugamides A1-A4 (5-8). Solid-phase peptide synthesis provided additional synthetic analogs, surugamides S1-S3 (9-11), while derivatization of 3 returned the semi-synthetic surugamide S4 (12) and acyl-surugamides AS1-AS3 (13-15). The natural acyl-surugamide A3 (7) and semi-synthetic acyl-surugamide AS3 (15) were shown to selectively inhibit D. immitis mf motility (EC50 3.3-3.4 µg/mL), however, unlike antimycins 1 and 2, were inactive against the gastrointestinal nematode Haemonchus contortus L1-L3 larvae (EC50 > 25 µg/mL) and were not cytotoxic to mammalian cells (human colorectal carcinoma SW620, IC50 > 30 µg/mL). A structure-activity relationship (SAR) study on the surugamides 3-15 revealed that selective acylation of the Lys3-ε-NH2 correlates with anthelmintic activity.

Investigating Dirofilaria immitis isolates infecting domestic canines and their susceptibility/resistance patterns to macrocyclic lactones in the northern region of the Mississippi Delta area (southeast Missouri).

Previous reports of macrocyclic lactone (ML) resistance in Dirofilaria immitis, the parasitic nematode which causes heartworm disease, have mainly been from the southern Mississippi Delta region. Southeast Missouri (SEMO), forming the northern boundary of this region, has not previously been well studied. The area is an ideal propagation region for heartworm infection and possibly for the spread of ML resistance. To assess whether D. immitis isolates infecting domestic canines in SEMO exhibit evidence of resistance to MLs, domestic canines, presented to veterinary facilities testing positive for heartworms through antigen and microfilariae (MF) examination, were utilized in the study. Using a descriptive epidemiological cross-sectional study, from March 2021 through February 2022, blood sample collection from 96 canines living in SEMO testing positive for heartworms were analyzed. MiSeq technology was utilized to sequence specific genetic markers associated with susceptibility/resistance for MLs in D. immitis isolates. Genomic data revealed most D. immitis isolates had genotypic profiles consistent with resistance to MLs. Of the 96 samples tested, 91 (94.8%) had a resistant genotype, 4 (4.2%) had a mixed genotype, and 1 sample (1%) genotyped as susceptible. While detailed and reliable medical histories were not available for most canines, detailed medical history from 2 canines indicated evidence of phenotypic resistance that was consistent with their genotypes. However, in vivo preventive tests are needed to confirm a high frequency of phenotypic ML resistance in D. immitis from this region. Increasing resistance patterns to MLs indicate the approach to heartworm prevention/treatment protocol should be reconsidered. New measures may be required to stop heartworm disease.

First case of macrocyclic lactone-resistant Dirofilaria immitis in Europe - Cause for concern.

Heartworm disease caused by the nematode Dirofilaria immitis is one of the most important parasitoses of dogs. The treatment of the infection is long, complicated, risky and expensive. Conversely, prevention is easy, safe, and effective and it is achieved by the administration of macrocyclic lactones (MLs). In recent years, D. immitis strains resistant to MLs have been described in Southern USA, raising concerns for possible emergence, or spreading in other areas of the world. The present study describes the first case of ML-resistant D. immitis in a dog in Europe. The dog arrived in Rome, Italy, from USA in 2023. Less than 6 months after its arrival in Italy, the dog tested positive for D. immitis circulating antigen and microfilariae, despite it having received monthly the ML milbemycin oxime (plus an isoxazoline) after arrival. The microfilariae suppression test suggested a resistant strain. Microfilariae DNA was examined by droplet digital PCR-based duplex assays targeting four marker positions at single nucleotide polymorphisms (SNP1, SNP2, SNP3, SNP7) which differentiate resistant from susceptible isolates. The genetic analysis showed that microfilariae had a ML-resistant genotype at SNP1 and SNP7 positions, compatible with a resistant strain. It is unlikely that the dog acquired the infection after its arrival in Europe, while it is biologically and epidemiologically plausible that the dog was already infected when imported from USA to Europe. The present report highlights the realistic risk of ML-resistant D. immitis strains being imported and possibly transmitted in Europe and other areas of the world. Monitoring dogs travelling from one area to another, especially if they originate from regions where ML-resistance is well-documented, is imperative. Scientists, practitioners, and pet owners should be aware of the risk and remain vigilant against ML-resistance, in order to monitor and reduce the spreading of resistant D. immitis.

Safety of an oral combination of moxidectin, afoxolaner, and pyrantel pamoate in dogs.

NexGard®PLUS (moxidectin, afoxolaner, and pyrantel pamoate), is an oral combination product for dogs indicated for the prevention of heartworm disease, the treatment and prevention of flea and tick infestations, and the treatment of gastro-intestinal nematode infections. The safety of this product in dogs was evaluated in three studies. Study #1 was a margin-of-safety study conducted in puppies, dosed six times at 28-day intervals at 1X, 3X, or 5X multiples of the maximum exposure dose (equivalent to 24 µg/kg moxidectin, 5 mg/kg afoxolaner, and 10 mg/kg pyrantel). In Study #2, the product was administered to ABCB1-deficient collie dogs at a 1X dose twice at a 28-day interval, and at a 3X or 5X dose once. Study #3 evaluated the safety of the product at 1X and 3X doses administered three times at 4-week intervals, to dogs harboring adult Dirofilaria immitis. In the three studies, the safety was evaluated on the basis of multiple clinical observations and physical examinations, including a complete assessment of toxicity to macrocyclic lactones, and on comprehensive clinical and anatomical pathology evaluations in Study #1. No clinically significant combination product-related effects were observed in any of the three studies. No signs of macrocyclic lactone toxicity were observed in the ABCB1-deficient collie dogs. Some mild and self-resolving instances of emesis or diarrhea were occasionally observed in the 3X and 5X dosed dogs. NexGard® PLUS was demonstrated to be safe following multiple administrations in puppies, in ABCB1-deficient collie dogs, and in microfilaremic dogs infected with adult D. immitis.

Genotyping USA laboratory-maintained isolates and European clinical isolates of Dirofilaria immitis to assess macrocyclic lactone susceptibility or resistance at predictive SNP sites using droplet digital PCR.

Dirofilaria immitis is a parasitic nematode that causes cardiovascular dirofilariosis ("heartworm disease") primarily in canids. The principal approach for mitigating heartworm infection involves the use of macrocyclic lactone (ML) for prophylaxis. Recent research has substantiated the emergence of D. immitis displaying resistance to MLs in the USA. Numerous factors, such as the mobility of companion animals and competent vectors could impact the spread of drug resistance. Genomic analysis has unveiled that isolates resistant to ML exhibit unique genetic profiles when compared to their wild-type (susceptible) counterparts. Out of the ten single nucleotide polymorphism (SNP) markers validated in clinical samples of D. immitis from the USA, four have demonstrated their effectiveness in distinguishing between isolates with varying ML efficacy phenotypes. This study explores the potential of these confirmed SNPs for conducting surveillance studies. Genotypic analysis using SNP markers emerges as a valuable tool for carrying out surveys and evaluating individual clinical isolates. Two USA laboratory-maintained isolates (Berkeley, WildCat) and twenty-five random European clinical samples of either adult worms or microfilariae (mf) pools isolated from domestic dogs, were tested by droplet digital PCR (ddPCR)-based duplex assay. This approach elucidates genetic evidence pertaining to the development of drug resistance and provides baseline data on resistance related genotypes in Europe. The data on these clinical samples suggests genotypes consistent with the continued efficacy of ML treatment regimens in Europe. In addition, this assay can be significant in discriminating cases of drug-resistance from those possibly due to non-compliance to the recommended preventive protocols.

Efficacy of afoxolaner (NexGard®) in preventing the transmission of Leishmania infantum and Dirofilaria immitis to sheltered dogs in a highly endemic area.

BACKGROUND: Leishmania infantum and Dirofilaria immitis are among the most important canine vector-borne pathogens (CVBPs) of zoonotic concern in Europe. In endemic areas for both of these CVBPs, the use of systemic ectoparasiticides, such as afoxolaner (NexGard®; Boehringer Ingelheim Animal Health), may have the potential for controlling these infections. The aim of this study was to assess, for the first time, the insecticidal efficacy of NexGard® in decreasing the transmission of D. immitis and L. infantum to sheltered dogs living in a hyperendemic area, compared to the year before treatment, as well as its impact on the abundance of mosquito and sand fly populations. METHODS: All dogs (n = 179) enrolled in the study were divided into two groups based on their infection status at enrollment: a non-infected group (G1) and an infected group (G2; infected with D. immitis, L. infantum or both). The study was conducted from March 2020 to March 2021. In order to exclude all animals infected with L. infantum and D. immitis before March 2020 (sampling time: T0), dogs in G1 were sampled in June (T1; i.e. T0 + 90 days) and in October 2020 (T2; i.e. T0 + 210 days). From March to September 2020, all animals (G1 and G2) were weighed and treated monthly with NexGard®. Animals in G1 were tested for the last time in March 2021 (T3; i.e. T0 + 330 days) for assessing post-treatment incidence rate of infection and prevention efficacy. RESULTS: The post-treatment incidence of D. immitis was 3.7% (1/27; 95% confidence interval [CI]: 0.2-18.1) and that of L. infantum was 3.6% (3/83; 95% CI: 1.0-10.1). Considering the annual incidence in 2019 and 2020, the protective efficacy against D. immitis and L. infantum infections was 94.2 and 64%, respectively. Of the female mosquitoes collected (n = 146), only one pool out of 50 tested positive for D. immitis DNA, whereas out of 1252 female Sergentomya minuta specimens collected, only four tested positive for L. infantum (0.3%). CONCLUSIONS: Afoxolaner is efficacious in decreasing the rate of transmission of both D. immitis and L. infantum; however, comparison of the pre- and post-treatment period demonstrated that there was a significant difference only in the seasonal incidences of D. immitis infection. Preventive measures are recommended throughout the year in endemic areas to reduce the risk of pathogen transmission to animals and humans.

Comparative preventive efficacy of ProHeart® 12, Heartgard® Plus and Interceptor® Plus against a macrocyclic lactone-resistant strain (JYD-34) of heartworm (Dirofilaria immitis) in dogs.

BACKGROUND: The current studies compared ProHeart® 12, Heartgard® Plus and Interceptor® Plus for preventive efficacy against JYD-34, a macrocyclic lactone (ML)-resistant strain of Dirofilaria immitis in dogs. METHODS: In two studies, each using 24 adult beagles, dogs were allocated to four treatment groups (n = 6): placebo-treated control; ProHeart 12 as per label (0.5 mg/kg moxidectin); Heartgard Plus (HGP) as per label (minimum 6 µg/kg ivermectin); and Interceptor Plus (INP) as per label (minimum 0.5 mg/kg milbemycin oxime). In both studies, ProHeart 12 was administered as a single subcutaneous dose on day 0, and HGP and INP were administered orally on days 0, 30, 60, 90, 120 and 150. In Studies 1 and 2, dogs were inoculated with 50 third-stage heartworm larvae (JYD-34 strain) on days -30 and 165, respectively. In Study 2, treatment for both HGP and INP was continued on days 180, 210, 240, 270, 300 and 330. Adult heartworm recoveries were performed on day 185 in Study 1 and on day 360 in Study 2. RESULTS: In Studies 1 and 2, all placebo-treated dogs developed adult heartworm infections (geometric mean, 29.9 and 34.9 worms/dog, respectively). A single dose of ProHeart 12 was 100% effective in preventing the development of adult JYD-34 heartworms when treatment was initiated 30 days after heartworm inoculation, while six consecutive monthly doses of HGP and INP were only 10.5% and 14.6% effective, respectively. The mean worm count for the ProHeart 12-treated group was significantly lower (P < 0.0001) than that for the placebo control, HGP- and INP-treated groups. In Study 2, the dogs treated with ProHeart 12 had an efficacy of 98.3%. All dogs treated with HGP and INP for 12 consecutive months had adult heartworms with efficacies of 37.7% and 34.9%, respectively. The mean worm count for the ProHeart 12-treated dogs was significantly lower (P < 0.0001) than those for the control group, HGP- and INP-treated groups. CONCLUSIONS: A single administration of ProHeart 12 was 98-100% effective in preventing the development of the ML-resistant JYD-34 heartworm strain and was significantly better than multiple consecutive monthly doses of either Heartgard Plus or Interceptor Plus in both studies.

Developmental regulation of Dirofilaria immitis microfilariae and evaluation of ecdysone signaling pathway transcript level using droplet digital PCR.

BACKGROUND: Current measures for the prevention of dirofilariasis, caused by the dog heartworm, Dirofilaria immitis, rely on macrocyclic lactones, but evidence of drug-resistant isolates has called for alternative approaches to disease intervention. As microfilariae are known to be in a state of developmental arrest in their mammalian host and then undergo two molts once inside the arthropod, the aim of this study was to look at the developmental regulation of D. immitis microfilariae that occurs in their arthropod host using in vitro approaches and to investigate the role of the ecdysone signaling system in this development regulation. METHODS: Dirofilaria immitis microfilariae extracted from dog blood were incubated under various culture conditions to identify those most suitable for in vitro culture and development of the microfilariae, and to determine the effects of fetal bovine serum (FBS), mosquito cells, and ecdysteroid on the development of the microfilariae. Transcript levels of the ecdysone signaling pathway components were measured with droplet digital PCR (ddPCR). RESULTS: In vitro conditions that best promote early development of D. immitis microfilariae to the "late sausage stage" have been identified, although shedding of the cuticle was not observed. FBS had inhibitory effects on the development and motility of the microfilariae, but media conditioned with Anopheles gambiae cells were favorable to microfilarial growth. The transcript level study using ddPCR also showed that ecdysone signaling system components were upregulated in developing microfilariae and that 20-hydroxyecdysone increased the proportion of larvae developing to the sausage and late sausage stages in vitro. CONCLUSIONS: The arthropod host environment provides cues required for the rapid development of D. immitis microfilariae, and the ecdysone signaling system may play an important role in filarial nematode developmental transitions. This study contributes to a better understanding of the developmental process of D. immitis microfilariae.

Efficacy of semi-annual therapy of an extended-release injectable moxidectin suspension and oral doxycycline in Dirofilaria immitis naturally infected dogs.

BACKGROUND: Dirofilaria immitis is a life-threatening nematode spreading globally. Arsenical treatment is currently recommended for removal of adult worms. However, arsenical treatment is not available in some countries, and there are dogs that cannot tolerate the rapid kill of adult worms; therefore, alternative adulticide slow-kill treatments are needed. Criticisms against the use of these alternative protocols include the potential for allowing disease to progress and for the development of ML-resistant worms. METHODS: The efficacy of a protocol that includes semi-annual doses (i.e. every 6 months) of commercially available extended-release injectable moxidectin suspension (ProHeart® SR-12) with 30-day oral administration of doxycycline was studied in 20 dogs with naturally occurring D. immitis infections. Each dog received treatment with ProHeart® SR-12 (0.5 mg moxidectin/kg) by subcutaneous injection and oral doxycycline (10 mg/kg/bid × 30 days) every 6 months until two consecutive negative antigen test results were obtained. Pulmonary and cardiac evaluations were performed by radiographic and echocardiographic parameters. Physical examinations, complete blood counts, clinical chemistry profiles, microfilariae and antigen tests were performed periodically. RESULTS: At enrollment, all dogs were positive for D. immitis antigen and 18 were microfilaremic. On day 30, microfilaremia counts decreased, and all dogs became amicrofilaremic by day 150. On day 180, 11 dogs were antigen-negative, and 7 more became negative by day 360. The two remaining antigen-positive dogs converted to negative by day 540 or 810. All antigen tests performed 180 days after the first negative test were negative. There was no decline in cardiac performance of the dogs throughout the study. Overall, pulmonary clinical conditions, presence of worms by echocardiography, and enlargement of caudal and main pulmonary arteries improved after treatment. Physical examinations, complete blood count results, and clinical chemistry profiles were within normal reference values. Respiratory conditions were improved, no damage to the heart was observed, and the treatment protocol was well tolerated by the animals. CONCLUSIONS: This alternative adulticide treatment was efficacious and well tolerated in naturally infected dogs. The injectable formulation provides the advantage of having veterinarians able to administer, monitor, and assess the efficacy and condition of the dog throughout the treatment and post-treatment periods.

Comparative sequences of the Wolbachia genomes of drug-sensitive and resistant isolates of Dirofilaria immitis.

The recent identification of isolates of D. immitis with confirmed resistance to the macrocyclic lactone preventatives presents an opportunity for comparative genomic studies using these isolates, and examining the genetic diversity within and between them. We studied the genomes of Wolbachia endosymbionts of five isolates of D. immitis maintained at the University of Georgia. Missouri and Georgia-2 are maintained as drug susceptible isolates, and JYD-27, Yazoo-2013 and Metairie-2014 are resistant to the macrocyclic lactone preventatives. We used whole genome amplification followed by Illumina-based sequencing from 8 to 12 individual microfilariae from each of the five isolates, obtaining a depth of coverage of approximately 40-75 fold for each. The Illumina sequences were used to create new genome assemblies for all the Wolbachia isolates studied. Comparisons of the Wolbachia sequences revealed more than 3000 sequence variations in each isolate. We identified 67 loci specific in resistant isolates but not in susceptible isolates, including 18 genes affected.Phylogenetic analysis suggested that the endosymbionts of the drug-susceptible isolates are more closely related to each other than to those from any of the resistant parasites. This level of variation in the Wolbachia endosymbionts of D. immitis isolates suggests a potential for selection for resistance against drugs targeting them.

Laboratory assays reveal diverse phenotypes among microfilariae of Dirofilaria immitis isolates with known macrocyclic lactone susceptibility status.

Prevention of canine heartworm disease caused by Dirofilaria immitis relies on chemoprophylaxis with macrocyclic lactone anthelmintics. Alarmingly, there are increased reports of D. immitis isolates with resistance to macrocyclic lactones and the ability to break through prophylaxis. Yet, there is not a well-established laboratory assay that can utilize biochemical phenotypes of microfilariae to predict drug resistance status. In this study we evaluated laboratory assays measuring cell permeability, metabolism, and P-glycoprotein-mediated efflux. Our assays revealed that trypan blue, propidium iodide staining, and resazurin metabolism could detect differences among D. immitis isolates but none of these approaches could accurately predict drug susceptibility status for all resistant isolates tested. P-glycoprotein assays suggested that the repertoire of P-gp expression is likely to vary among isolates, and investigation of pharmacological differences among different P-gp genes is warranted. Further research is needed to investigate and optimize laboratory assays for D. immitis microfilariae, and caution should be applied when adapting cell death assays to drug screening studies for nematode parasites.

The development of the dog heartworm is highly sensitive to sterols which activate the orthologue of the nuclear receptor DAF-12.

Prevention therapy against Dirofilaria immitis in companion animals is currently threatened by the emergence of isolates resistant to macrocyclic lactone anthelmintics. Understanding the control over developmental processes in D. immitis is important for elucidating new approaches to heartworm control. The nuclear receptor DAF-12 plays a role in the entry and exit of dauer stage in Caenorhabditis elegans and in the development of free-living infective third-stage larvae (iL3) of some Clade IV and V parasitic nematodes. We identified a DAF-12 ortholog in the clade III nematode D. immitis and found that it exhibited a much higher affinity for dafachronic acids than described with other nematode DAF-12 investigated so far. We also modelled the DimDAF-12 structure and characterized the residues involved with DA binding. Moreover, we showed that cholesterol derivatives impacted the molting process from the iL3 to the fourth-stage larvae. Since D. immitis is unable to synthesize cholesterol and only completes its development upon host infection, we hypothesize that host environment contributes to its further molting inside the host vertebrate. Our discovery contributes to a better understanding of the developmental checkpoints of D. immitis and offers new perspectives for the development of novel therapies against filarial infections.

Preventive efficacy of four or six monthly oral doses of 24 µg/kg moxidectin compared to six monthly doses of Heartgard® Plus or Interceptor® Plus against macrocyclic lactone-resistant heartworm (Dirofilaria immitis) strains in dogs.

BACKGROUND: Recent reports indicated that increasing the monthly oral dosage and the number of consecutive monthly doses of moxidectin improved the efficacy against macrocyclic lactone (ML)-resistant Dirofilaria immitis. The two laboratory studies reported here evaluated the efficacy of four or six monthly oral doses of 24 µg/kg moxidectin compared to six monthly doses of either Heartgard® Plus (ivermectin/pyrantel) or Interceptor® Plus (milbemycin oxime/praziquantel) against ML-resistant D. immitis strains. METHODS: Dogs were inoculated 30 days prior to first treatment with 50 third-stage (L3) larvae of a ML-resistant strain of D. immitis, ZoeLA or JYD-34. In each study, dogs (six per group) were randomized to treatment with six monthly doses of placebo, four or six monthly doses of 24 µg/kg moxidectin, or six monthly doses of Heartgard® Plus or Interceptor® Plus at their label dose rates. Efficacy was evaluated by adult heartworm counts approximately nine months after L3 inoculation. RESULTS: All negative-control dogs were infected with adult heartworms (geometric mean, 35.6; range, 24-41) for ZoeLA and (geometric mean, 32.9; range, 30-37) for JYD-34. Efficacies against ZoeLA for moxidectin, Heartgard® Plus and Interceptor® Plus were ≥ 96.1%, 18.7% and 21.2%, respectively. Adult counts for both moxidectin-treated groups were significantly lower than negative control (P < 0.0001), significantly lower than Heartgard® Plus and Interceptor® Plus (P < 0.0001), but not significantly different from each other (P = 0.5876). Counts for Heartgard® Plus and Interceptor® Plus were not significantly different than negative control (P ≥ 0.2471). Efficacies against JYD-34 were ≥ 95.9%, 63.9% and 54.6% for moxidectin, Heartgard® Plus and Interceptor® Plus, respectively. Counts for all groups were significantly lower than negative control (P ≤ 0.0001). Counts for six monthly doses of moxidectin were significantly lower than those for four monthly doses (P = 0.0470), and the counts for both moxidectin-treated groups were significantly lower than Heartgard® Plus and Interceptor® Plus (P ≤ 0.0002). CONCLUSIONS: Moxidectin administered orally at 24 µg/kg to dogs for four or six consecutive months was ≥ 95.9% effective in preventing the development of two ML-resistant heartworm strains and resulted in significantly fewer adult D. immitis than in dogs treated with Heartgard® Plus or Interceptor® Plus when administered for six consecutive months at their approved label dosages in two laboratory efficacy studies.

Using population genetics to examine relationships of Dirofilaria immitis based on both macrocyclic lactone-resistance status and geography.

Prevention of infection with canine heartworm (Dirofilaria immitis) is based on the compliant administration of macrocyclic lactone (ML) drugs. Resistance to ML drugs is well documented in D. immitis; however, there remains a paucity of information on the spatial distribution and prevalence of resistant isolates. This project aims to improve understanding of ML-resistance by using a population genetic approach. We developed a large panel of microsatellite loci and identified 12 novel highly polymorphic markers. These 12, and five previously published markers were used to screen pools of microfilariae from 16 confirmed drug-susceptible, 25 confirmed drug-resistant, and from 10 suspected drug-resistant field isolates. In isolates where microfilarial suppression testing indicated resistance, Spatial Principal Component Analysis (sPCoA), Neighbor Joining Trees and Bayesian clustering all revealed high genetic similarity between pre- and post-treatment samples. Somewhat surprisingly, the Neighbor Joining tree and sPCoA generated using pairwise Nei's distances did not reveal clustering for resistant isolates, nor did it reveal state-level geographic clustering from samples collected in Georgia, Louisiana or Mississippi. In contrast, Discriminant Analysis of Principle Components was able to discriminate between susceptible, suspected-resistant and resistant samples. However, no resistance-associated markers were detected, and this clustering was driven by the combined effects of multiple alleles across multiple loci. Additionally, we measured unexpectedly large genetic distances between different passages of laboratory strains that originated from the same source infection. This finding strongly suggests that the genetic makeup of laboratory isolates can change substantially with each passage, likely due to genetic bottlenecking. Taken together, these data suggest greater than expected genetic variability in the resistant isolates, and in D. immitis overall. Our results also suggest that microsatellite genotyping lacks the sensitivity to detect a specific genetic signature for resistance. Future investigations using genomic analyses will be required to elucidate the genetic relationships of ML-resistant isolates.

Non-Arsenical heartworm adulticidal therapy using topical moxidectin-imidacloprid and doxycycline: A prospective case series.

This prospective case series evaluated the adulticidal efficacy of topical 10 % moxidectin/2.5 % imidacloprid (M/I; Advantage Multi®, Bayer, Shawnee Mission, KS, USA) and doxycycline in dogs with naturally occurring heartworm infection (HWI). Twenty-two dogs with HWI whose owners declined melarsomine were treated with M/I at the preventive dosage twice monthly for 90 days then monthly thereafter and doxycycline (median [interquartile range; IQR] dosage 12.6 [12.0-16.1] mg/kg/day) for the first 15 days. Although strict activity restriction was not imposed, owners were asked to prevent their dogs from exercising strenuously. This protocol was referred to as the MOXY protocol. Antigen testing was performed every 30-60 days, until dogs had 'no antigen detected' (NAD). Twenty-one of the 22 dogs ultimately converted to NAD by 434 days (median [IQR]), 234 (179-303). One dog remained positive 701 days after MOXY initiation and was considered a treatment failure. All sera which converted to NAD on HW antigen testing were retested after heat-treatment. Twelve dogs had NAD on the heat-treated test on the same day as having their first NAD on the conventional test. Six of 9 dogs testing positive after heat-treatment were retested and all 6 had NAD on a heat-treated test within 2-3 months. Microfilaremia was cleared in all 8 dogs re-tested. Four dogs required treatment for cough, thought due to heartworm (HW) death, an average of 89 days after initiation of MOXY. This cough was most likely due to pneumonitis with heartworm-pulmonary thromboembolism. One dog required hospitalization for 24 -h and recovered fully with corticosteroid therapy and supportive care and 2 dogs were treated in an outpatient fashion with steroids. The MOXY protocol was tolerated and 96 % (21/22) of dogs converted to NAD, though 2 dogs required greater than 1 year to achieve this result. Nonaresenical-adulticide therapy may result in pneumonitis and heartworm-pulmonary thromboembolism at unpredictable times, potentially months after initiation of macrocyclic lactone therapy and exercise restriction should be considered when using a nonarsenical protocol. Although not currently recommended by the American Heartworm Society (AHS), non-arsenical strategies are in use and the goal of this study was to evaluate the efficacy, duration of therapy, and safety of an accelerated dosing protocol of M/I with doxycycline.

Activation of the Toll pathway in Aedes aegypti blocks the development of emerging third-stage larvae of drug-resistant Dirofilaria immitis.

Dirofilaria immitis is the globally distributed agent of heartworm disease. Infection in canines causes debilitating disease that can be fatal if left untreated. Macrocyclic lactones can prevent heartworm disease in dogs, cats and ferrets by killing larvae before they develop into adult worms in the pulmonary artery. However, administration of prophylactic drugs to wild canids to prevent D. immitis infection is not feasible. Furthermore, a vaccine against heartworm is currently unavailable and drug resistant D. immitis have been identified, highlighting the need for new strategies to prevent parasite transmission. We recently established a method to block development of emerging third-stage larvae (eL3) from the mosquito Aedes aegypti by over-activating the Toll pathway, one of the major innate immune signaling pathways in mosquitoes. Our previous study used a drug-sensitive strain of D. immitis and it remains unknown if the strategy is effective against different D. immitis genotypes and, more importantly, if it would work against drug-resistant genotypes. The purpose of this study was to determine whether Toll pathway activation is capable of blocking eL3 development of D. immitis strains that are resistant to macrocyclic lactones. We infected mosquitoes with two independent strains of D. immitis previously confirmed as being resistant to macrocyclic lactones, and then activated Toll signaling by RNAi-mediated silencing of the pathway inhibitor, IκB/Cactus, and quantitatively measured eL3 development. Similar to the drug-sensitive strain, eL3 were strongly reduced by Toll activation in both drug-resistant strains. Furthermore, similar to the drug-sensitive strain, the reduction of eL3 in both drug-resistant strains suggests a defect in larval invasion of, or development in, the Malpighian tubules - the organ in the mosquito to which microfilariae migrate after ingestion and where the larvae undergo several developmental molts. In summary, Toll pathway activation blocks the development of three distinct D. immitis genotypes, including two different drug-resistant genotypes. If this strategy can be applied to heartworm vectors in the field, it may help reduce the spread of disease and is not predicted to favor the spread of drug resistance.

The efficacy of a topical formulation of selamectin plus sarolaner in preventing the development of a macrocyclic lactone-resistant strain of Dirofilaria immitis in cats.

Revolution®/Stronghold® Plus, a topical endectocide incorporating 6 mg/kg selamectin plus 1 mg/kg sarolaner, is approved for use in cats to prevent heartworm disease. The efficacy of selamectin has not previously been evaluated against any macrocyclic lactone (ML)-resistant heartworm strains in cats for prevention of heartworm disease. In this study, an experimental combination formulation of selamectin (6 mg/kg) plus sarolaner (2 mg/kg) was assessed for preventing the development of a ML-resistant strain of Dirofilaria immitis in cats. Forty purpose-bred domestic shorted-haired cats (20 males; 20 females) from 7-9 months of age and negative for heartworm antigen prior to study inclusion were used. On Day -30, cats were inoculated with 100 D. immitis L3 (ZoeMO strain) subcutaneously in the inguinal area. Cats were randomly allocated to one of the following four treatments with associated dosing regimens: T01 (vehicle-treated control on Days 0, 28, and 56), T02 (single dose of selamectin plus sarolaner combination on Day 0 only), T03 (selamectin plus sarolaner combination on Days 0, 28, and 56) or T04 (single dose of selamectin on Day 0 only). All treatments were administered topically in an isopropyl alcohol-based formulation. Selamectin was administered at 6 mg/kg in both standalone and combination formulations. Sarolaner was administered at 2 mg/kg. Cats were necropsied on Day ∼145 (∼175 days post infection) and adult worms were counted. Nine of ten cats in the control group (T01) were infected with adult worms (range, 1-23; geometric mean, 3.5). In contrast, all cats in T03 had zero heartworms. Only two cats in T02 (0-3; 0.2) and a single cat in the T04 (0-1; 0.1) had heartworms. Compared to T01 (control cats), all treated cats had significantly (p < 0.0001) reduced worm burdens, with treatment efficacies of 100% (T03), 93.5% (T02) and 98% (T04). A topical combination of selamectin (6 mg/kg) plus sarolaner (2 mg/kg) was 100% efficacious in preventing the development of an ML-resistant strain of D. immitis (ZoeMO) in cats when administered as three consecutive monthly treatments. A single dose was highly (93.5%) but incompletely effective.

Novel anti-Wolbachia drugs, a new approach in the treatment and prevention of veterinary filariasis?

Filarial nematodes are tissue-dwelling parasitic worms that can cause a range of disfiguring pathologies in humans and potentially lethal infections of companion animals. The bacterial endosymbiont, Wolbachia, is present within most human and veterinary filarial pathogens, including the causative agent of heartworm disease, Dirofilaria immitis. Doxycycline-mediated drug targeting of Wolbachia leads to sterility, clearance of microfilariae and gradual death of adult filariae. This mode of action is attractive in the treatment of filariasis because it avoids severe host inflammatory adverse reactions invoked by rapid-killing anthelmintic agents. However, doxycycline needs to be taken for four weeks to exert curative activity. In this review, we discuss the evidence that Wolbachia drug targeting is efficacious in blocking filarial larval development as well as in the treatment of chronic filarial disease. We present the current portfolio of next-generation anti-Wolbachia candidates discovered through phenotypic screening of chemical libraries and validated in a range of in vitro and in vivo filarial infection models. Several novel chemotypes have been identified with selected narrow-spectrum anti-Wolbachia specificity and superior time-to-kill kinetics compared with doxycycline. We discuss the opportunities of developing these novel anti-Wolbachia agents as either cures, adjunct therapies or new preventatives for the treatment of veterinary filariasis.