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.

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.

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.

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.

Preventive efficacy of oral moxidectin at various doses and dosage regimens against macrocyclic lactone-resistant heartworm (Dirofilaria immitis) strains in dogs.

BACKGROUND: Moxidectin has previously shown limited efficacy (≤ 44.4%) against confirmed macrocyclic lactone (ML)-resistant Dirofilaria immitis strains at 3 µg/kg after single and multiple oral dosages. Three studies were conducted to evaluate higher oral moxidectin doses for efficacy against confirmed ML-resistant D. immitis strains. METHODS: Dogs were inoculated with 50 D. immitis L3 and randomly allocated to treatments. Study 1: 6 groups of dogs (n = 8) were inoculated with JYD-34 (Day - 30) and treated as follows: T01, negative control; T02-T05, moxidectin at 3, 6, 12 or 24 µg/kg, respectively, on Day 0 only; T06, moxidectin at 3 µg/kg on Days 0, 30 and 60. Study 2: 10 groups of dogs (n = 5) were inoculated (Day - 30) with either JYD-34 (T01, T03-05) or ZoeLA (T02, T06-T10) and treated as follows: T01 and T02, negative controls; T03-T05, moxidectin at 24, 40 or 60 µg/kg, respectively, on Days 0, 28 and 56; T06 and T09, moxidectin at 3 or 60 µg/kg on Day 0 only; T07, T08 and T10, moxidectin at 24, 40 or 60 µg/kg, respectively, on Days 0, 28 and 56. Study 3: 5 groups of dogs (n = 5) were inoculated with ZoeMO (Day - 28) and treated as follows: T01, negative control; T02, moxidectin at 3 µg/kg moxidectin on Day 0 only; T03-T05, moxidectin at 24, 40 or 60 µg/kg, respectively, on Days 0, 28 and 56. All dogs were necropsied for adult heartworm recovery ~ 4-5 months post-inoculation. RESULTS: All moxidectin-treated dogs showed significantly lower worm counts than controls. The efficacy of moxidectin administered once at 3 µg/kg was 19% (JYD-34), 44.4% (ZoeLA) and 82.1% (ZoeMO). Increasing both the dose and the number of dosages of moxidectin improved efficacy, with 100% protection obtained using three dosages of moxidectin at either 40 µg/kg (JYD-34, ZoeMO) or 60 µg/kg (ZoeLA). Three dosages of 24 µg/kg were also highly effective, providing ≥ 98.8% efficacy for all three strains. CONCLUSIONS: Increasing both the dose and number of consecutive monthly dosages of moxidectin improved the efficacy against ML-resistant heartworms. Based on these data and other technical considerations, the 24 µg/kg dose was considered the optimal dose for further commercial development.

Laboratory and field studies to investigate the efficacy of a novel, orally administered combination product containing moxidectin, sarolaner and pyrantel for the prevention of heartworm disease (Dirofilaria immitis) in dogs.

BACKGROUND: Dirofilaria immitis is a filarial parasite of dogs that can cause serious or fatal cardiopulmonary disease. Three studies were conducted to evaluate the efficacy and safety of monthly treatment with moxidectin in a chewable tablet product in combination with sarolaner and pyrantel to prevent heartworm disease in dogs after experimental challenge and in a clinical field study in the USA. METHODS: In two laboratory studies, dogs (8 per group) that had been inoculated 30 days prior with 50 third-stage D. immitis larvae were randomized to treatment on Day 0 with placebo or combination product, at the minimum dose of 24 µg/kg moxidectin, 2 mg/kg sarolaner and 5 mg/kg pyrantel (as pamoate salt). Study 2 also included groups treated with tablets containing moxidectin-alone (24 µg/kg) or sarolaner-alone (2 mg/kg). Efficacy was evaluated ~ 5 months after inoculation by adult heartworm counts at necropsy. In the field study, 410 dogs ≥ 8 weeks-old from 23 USA veterinary clinics were treated for 11 months with either combination product at 24-48 µg/kg moxidectin, 2-4 mg/kg sarolaner and 5-10 mg/kg pyrantel (n = 272) or Heartgard® Plus (ivermectin/pyrantel) at the label recommended dose rate (n = 138). Efficacy was evaluated on Day 330 using antigen and microfilaria testing to assess adult heartworm infection. RESULTS: In the laboratory studies, there were no heartworms recovered from any dog treated with the combination product or moxidectin alone and all dogs treated with placebo or sarolaner-alone were infected with 20-44 adult heartworms. In the field study, all dogs treated with the combination product tested negative for heartworm infection on Day 330, whereas two dogs treated with Heartgard® Plus tested positive. The Heartgard® Plus-treated dogs that tested heartworm positive were from the lower Mississippi River Valley region, where heartworm resistance has been confirmed to occur. The combination product was well tolerated in all studies. CONCLUSIONS: In laboratory studies, no heartworms were recovered from dogs treated with a single dose of the novel combination product containing moxidectin, sarolaner and pyrantel. Additionally, in the field study no dog tested positive for adult heartworm infection when dosed with the combination product monthly for 11 months, while two dogs treated with Heartgard® Plus tested positive.

Differential ABC transporter gene expression in adult Dirofilaria immitis males and females following in vitro treatment with ivermectin, doxycycline or a combination of both.

BACKGROUND: Combination doxycycline/macrocyclic lactone (ML) protocols have been shown to provide a more rapid adulticidal and microfilaricidal effect than either MLs or doxycycline alone, although female worms were reported to have a higher tolerance to treatments compared to male worms. The present study aimed to evaluate how ABC transporters may be involved in the synergic effect of the combination treatment. Adult worms of D. immitis were treated in vitro for 24 hours with doxycycline (DOXY), ivermectin (IVM) and a combination of both, and changes in the modulation of ABC transporter genes were measured. Levels of doxycycline inside different treatment media, post-treatment, were determined through HPLC analysis. RESULTS: Quantitative RT-PCR analysis showed the presence of changes in the modulation of ABC transporter genes evaluated in this study. In particular, in female worms, the combination treatment induced a substantial increase in gene expressions, especially of Dim-pgp-10 and Dim-haf-4; whereas in male worms, the greatest increase in gene expression was observed for Dim-pgp-10 and Dim-pgp-11 when treated with DMSO + IVM and DMSO + DOXY/IVM. HPLC analysis of the DOXY concentrations in the media after in vitro treatments of male worms showed a slight difference between the DMSO + DOXY samples and the combination (DMSO + DOXY + IVM), while no difference was observed among females. CONCLUSIONS: Further studies are required to explain whether the modulation of cellular efflux plays a role, even partially, in the adulticide effect of doxycycline/macrocyclic lactone combinations in heartworm-infected dogs. To the authors' knowledge, this is the first study to evaluate P-gp expression in adult D. immitis.

ProHeart® 12, a moxidectin extended-release injectable formulation for prevention of heartworm (Dirofilaria immitis) disease in dogs in the USA for 12 months.

BACKGROUND: The efficacy of an extended-release injectable moxidectin (0.5 mg/kg) suspension (ProHeart® 12) (PH 12) in preventing the development of Dirofilaria immitis in dogs for 12 months was investigated in laboratory and field studies in the USA. METHODS: In each of two laboratory studies, 20 dogs ≥ 12 months of age were randomly allocated to receive a subcutaneous injection of saline or PH 12 on Day 0 and were then inoculated with 50 D. immitis third-stage larvae (L3) on Day 365. All dogs were necropsied ~ 5 months post-inoculation for adult worm counts. The field efficacy study included dogs ≥ 10 months of age from 19 veterinary clinics in the USA treated with either 20 monthly doses of Heartgard® Plus (HG Plus) (296 dogs) or two doses of PH 12 (297 dogs) on Days 0 and 365. Efficacy was determined on Days 365, 480 and 605 using adult HW antigen and microfilaria testing to assess adult HW infection. RESULTS: PH 12 was 100% effective in preventing HW disease in all three of these studies. In the laboratory studies, no PH 12-treated dogs had any adult HWs, whereas all control dogs in both studies had adult HWs [geometric mean, 30.2 (range, 22-37) for Study 1 and 32.6 (22-44) for Study 2]. In the field study, all dogs treated with PH 12 tested negative for adult HW infection on all test days (Days, 365, 480 and 605), whereas four dogs receiving HG Plus (positive control) tested positive for HWs during the study (three dogs on Day 365 and one dog on Day 480). All four dogs treated with HG Plus that subsequently tested positive for HWs during the field study were from the lower Mississippi River Valley region, where HW resistance to macrocyclic lactone preventives has been confirmed to occur. PH 12 was significantly better than HG Plus in preventing heartworm disease in the field study (P = 0.0367). PH 12 was well-tolerated in both laboratory and field studies. CONCLUSIONS: A single dose of ProHeart® 12 was 100% effective in preventing heartworm disease in dogs for a full year in both laboratory and field studies.

Efficacy and safety of a new topical formulation containing selamectin and sarolaner in the prevention of heartworm disease and the treatment of roundworm infection in cats presented as veterinary patients in Japan.

The efficacy and safety of a new topical formulation containing selamectin plus sarolaner (Revolution® Plus / Stronghold® Plus, Zoetis) was evaluated for the prevention of heartworm (Dirofilaria immitis) disease and the treatment of roundworm infection in cats enrolled as veterinary patients in two field studies conducted in Japan. In the heartworm field study, 91 cats negative for D. immitis antigen and anti-D. immitis antibody were enrolled and received 9 monthly topical treatments with selamectin plus sarolaner during the period of April to December 2015. Efficacy was assessed by testing post-treatment blood samples collected 8, 12, and 15 months after initiation of treatment for the presence of D. immitis antigen and anti-D. immitis antibody. Eighty-seven cats completed the entire study and were included in the determination of efficacy. No D. immitis antigen or anti-D. immitis antibody were detected in any of the post-treatment samples. In the roundworm field study, completed in the period from April to November 2015, 64 cats with ≥100 roundworm eggs per gram (EPG) of feces were enrolled and allocated randomly in a 1:1 ratio, to receive either selamectin plus sarolaner or emodepside plus praziquantel (Profender®, Bayer). Treatments were administered topically on Days 0 and 30, and efficacy was assessed by fecal EPG counts conducted on Days 14, 30, and 60. All cats completed the entire study. At enrollment, all cats were infected with Toxocara cati. Compared to pre-treatment, geometric mean T. cati EPG counts on Days 14, 30, and 60 were reduced by >99.9% in both treatment groups. There were no treatment-related adverse events in either study. Monthly topical administration of Revolution® Plus / Stronghold® Plus providing a minimum of 6 mg/kg selamectin and 1 mg/kg sarolaner was safe and effective in the prevention of heartworm disease and the treatment of roundworm infection in cats enrolled as veterinary patients in Japan.