Strain-level variations of Dirofilaria immitis microfilariae in two biochemical assays.

BACKGROUND: The increase in reports of resistance to macrocyclic lactones in the canine heartworm, Dirofilaria immitis is alarming. While DNA based tests have been well-validated, they can be expensive. In a previous study, we showed that two biochemical tests adapted to a 96- well plate format and read in a spectrophotometer could detect differences among lab validated D. immitis isolates. The two tests- Resazurin reduction and Hoechst 33342 efflux-detect metabolism and P-glycoprotein activity respectively in microfilariae isolated from infected dog blood. METHODS: Our objective was to optimize the two assays further by testing various assay parameters in D. immitis isolates not tested previously. We tested microfilarial seeding density, incubation time and the effect of in vitro treatment with ivermectin and doxycycline in five other D. immitis isolates-JYD-34, Big Head, Berkeley, Georgia III and LOL. All assays were performed in 3 technical replicates and 2-4 biological replicates. To understand the molecular basis of the assays, we also performed qPCR for selected drug metabolism and elimination associated genes of the ABC transporter and cytochrome P450 gene families. RESULTS: Metabolism and ABC transporter activity as detected by these assays varied between strains. Anthelmintic status (resistant or susceptible) did not correlate with metabolism or P-gp efflux. Basal transcriptional variations were found between strains in ABC transporter and cytochrome P450 genes. CONCLUSIONS: These assays provide a greater understanding of the biochemical variation among isolates of D. immitis, which can be exploited in the future to develop in vitro diagnostic tests capable of differentiating susceptible and resistant isolates.

Dirofilariasis presenting as pleural effusion: a rare case report with unusual manifestations and treatment modalities.

BACKGROUND: We present an extremely rare manifestation of dirofilariasis in the pleural cavity. This is the first human pulmonary dirofilariasis reported in Lithuania; according to our knowledge, only two other patients were documented with this pathology in the world. CASE PRESENTATION: A 72-year-old woman was admitted to the hospital complaining of dyspnea, left-side chest pain, and a dry cough. She was a retiree living alone in the countryside without domestic pets (sometimes stray dogs appear) or a travel history. A complete blood count was within normal limits, with a CRP level of 16.8 mg/l and D-dimer concentration of 900 µg/l, which raised suspicion of pulmonary embolism. In chest computed tomography angiography, pulmonary embolism was excluded, and only left pleural effusion without abnormal lesions was confirmed. Left thoracocentesis was performed, and the pleural fluid was evaluated as an exudate with a predominance of eosinophils (59%), along with the presence of parasites. These parasites exhibited the morphology of Dirofilaria repens. Oral doxycycline (100 mg, twice daily) and albendazole (400 mg, twice daily) were prescribed for a 14-day course. A month later, there were no pathological findings on the chest X-ray, and the patient no longer had respiratory symptoms. However, the patient presented with an emerged, painful palpable right breastmass, where the rash was previously observed. Ultrasound imaging revealed a 1.5 × 2 cm nodule, which was surgically removed. Parasites consistent with Dirofilaria repens were suspected but not definitively identified. Pharmacological treatment for dirofilariasis was not further prescribed. CONCLUSIONS: This case encourages doctors to be more vigilant because the patient, who neither travelled nor kept any pets, contracted dirofilariasis. Diagnostic and treatment guidelines are lacking, necessitating further research. Treatment with doxycycline and albendazole yielded positive outcomes, suggesting potential efficacy for dirofilarial pleuritis.

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.

Whole genome amplification and sequencing of individual Dirofilaria immitis microfilariae.

Dirofilaria immitis is a filarial parasitic nematode of veterinary significance. With the emergence of drug-resistant isolates in the USA, it is imperative to determine the likelihood of resistance occurring in other regions of the world. One approach is to conduct population genetic studies across an extensive geographical range, and to sequence the genomes of individual worms to understand genome-wide genetic variation associated with resistance. The immature life stages of D. immitis found in the host blood are more accessible and less invasive to sample compared to extracting adult stages from the host heart. To assess the use of immature life stages for population genetic analyses, we have performed whole genome amplification and whole-genome sequencing on nine (n = 9) individual D. immitis microfilaria samples isolated from dog blood. On average, less than 1% of mapped reads aligned to each D. immitis genome (nuclear, mitochondrial, and Wolbachia endosymbiont). For the dog genome, an average of over 99% of mapped reads aligned to the nuclear genome and less than 1% aligned to the mitochondrial genome. The average coverage for all D. immitis genomes and the dog nuclear genome was less than 1, while the dog mitochondrial genome had an average coverage of 2.87. The overwhelming proportion of sequencing reads mapping to the dog host genome can be attributed to residual dog blood cells in the microfilariae samples. These results demonstrate the challenges of conducting genome-wide studies on individual immature parasite life stages, particularly in the presence of extraneous host DNA.

Detection and dynamics of tumor necrosis factor alpha in the diagnosis and treatment of canine heartworm disease.

The aim of this study was to determine the concentration of TNF-alpha (TNF-α) in dogs naturally infected with Dirofilaria immitis (D. immitis) and to assess whether there are any changes in TNF-α concentration and their dependence during therapy for heartworm disease (HWD). For this study, 14 client-owned dogs with HWD were selected. Clinical and parasitological examinations (modified Knott test for circulating microfilariae and SNAP Test IDEXX for circulating D. immitis antigen) had been used for diagnosing D. immitis and HWD. All dogs were treated with an alternative therapy for HWD (oral doxycycline 10 mg/kg b.w., once daily for 6 weeks, then alternately 4 weeks without and 2 weeks with the medication, and oral ivermectin 6-14 µg/kg b.w., every 2 weeks). The dogs blood sera at the moment of HWD diagnosis, during and at the end of therapy were frozen for further quantifying of TNF-α (Canine TNF-alpha ELISA kit, Thermo scientific). At the moment of HWD diagnosis TNF-α was detected in 9 dogs (7.21±12.44 pg/ml). Concentration of TNF-α was not significantly change during the therapy, neither related to the level of D. immitis antigen nor to antigen level changes. The alternative therapy for HWD has no influence on TNF-α concentration dynamics.

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

Dirofilaria immitis is a mosquito-borne parasitic nematode that causes fatal heartworm disease in canids. The microfilariae are essential for research, including drug screening and mosquito-parasite interactions. However, no reliable methods for maintaining microfilaria long-term are currently available. Therefore, we used severe combined immunodeficiency (SCID) mice to develop a reliable method for maintaining D. immitis microfilaria. SCID mice were injected intravenously with microfilariae isolated from a D. immitis-infected dog. Microfilariae were detected in blood collected from the tail vein 218 days post-inoculation (dpi) and via cardiac puncture 296 dpi. Microfilariae maintained in and extracted from SCID mice showed infectivity and matured into third-stage larvae (L3s) in the vector mosquito Aedes aegypti. L3s can develop into the fourth stage larvae in vitro. Microfilariae from SCID mice respond normally to ivermectin in vitro. The microfilariae in SCID mice displayed periodicity in the peripheral circulation. The SCID mouse model aided in the separation of microfilariae from cryopreserved specimens. The use of SCID mice enabled the isolation and sustained cultivation of microfilariae from clinical samples. These findings highlight the usefulness of the SCID mouse model for studying D. immitis microfilaremia in canine heartworm research.

Evaluation of diagnostic techniques for early detection of heartworm in experimentally infected dogs: identification of Dirofilaria immitis-derived microRNA in the initial 28 weeks post-inoculation.

BACKGROUND: Dirofilaria immitis, commonly known as heartworm (HW), is a parasitic nematode transmitted by various mosquito species, leading to heartworm disease (HWD) in dogs. Diagnosis of HW typically involves antigen or microfilariae detection, or visualization of adult worms through imaging or post mortem examination. Polymerase chain reaction (PCR) and micro RNA (miRNA) detection have been explored for HW diagnosis. METHODS: Three dogs, previously experimentally infected with HW, underwent blood sampling every 4 weeks for 7 months. Samples were assessed for antigen presence after heat treatment, PCR amplification, and microfilaria examination using Giemsa-stained thick smears. Additionally, whole blood aliquots underwent miRNA deep sequencing and bioinformatic analysis. RESULTS: Heartworm antigen was detectable after heat treatment at 20 weeks post-inoculation and via PCR at 24 weeks, with microfilariae observed in peripheral blood smears at 28 weeks. However, deep miRNA sequencing revealed that the miRNA candidate sequences are not consistently expressed before 28 weeks of infection. CONCLUSIONS: While ancillary molecular methods such as PCR and miRNA sequencing may be less effective than antigen detection for detecting immature larval stages in an early stage of infection, our experimental findings demonstrate that circulating miRNAs can still be detected in 28 weeks post-infection.

Molecular insights and antibody response to Dr20/22 in dogs naturally infected with Dirofilaria repens.

Subcutaneous dirofilariasis, caused by the parasitic nematode Dirofilaria repens, is a growing concern in Europe, affecting both dogs and humans. This study focused on D. repens Dr20/22, a protein encoded by an alt (abundant larval transcript) gene family. While well-documented in L3 larvae of other filariae species, this gene family had not been explored in dirofilariasis. The research involved cloning Dr20/22 cDNA, molecular characterization, and evaluating its potential application in the diagnosis of dirofilariasis. Although Real-Time analysis revealed mRNA expression in both adult worms and microfilariae, the native protein remained undetected in lysates from both developmental stages. This suggests the protein's specificity for L3 larvae and may be related to a process called SLTS (spliced leader trans-splicing), contributing to stage-specific gene expression. The specificity of the antigen for invasive larvae positions it as a promising early marker for dirofilariasis. However, ELISA tests using sera from infected and uninfected dogs indicated limited diagnostic utility. While further research is required, our findings contribute to a deeper understanding of the molecular and immunological aspects of host-parasite interactions and could offer insights into the parasite's strategies for evading the immune system.

Emerging human pulmonary dirofilariasis in Hungary: a single center experience.

BACKGROUND: Human pulmonary dirofilariasis (HPD) is rare in Hungary, and it stems from Dirofilaria immitis, mainly transmitted through mosquito bites, with dogs as primary hosts. Despite its prevalence in veterinary settings, human cases are infrequent. Historically, Mediterranean countries report most HPD cases, but sporadic cases occur in temperate European regions. Radiologically, HPD often manifests in a non-specific manner, resembling pulmonary neoplasms, leading to unnecessary surgery and patient distress. METHODS: This study presents a notable case series from Hungary, encompassing a 12-year period, documenting 5 instances of HPD with the aim to provide baseline estimate of occurrence for future comparison. RESULTS: Among the patients studied, all were of middle age (median: 52 years, range: 37-69) and exhibited tumor-like lesions, primarily localized to the right lung, necessitating lobectomy or wedge resection. Histological examination consistently revealed a necrotizing granulomatous response characterized by remnants of helminths, without the presence of ovules. Furthermore, rigorous diagnostic procedures excluded other potential infectious agents through specialized staining techniques. Polymerase chain reaction analysis definitively confirmed the diagnosis of HPD in each case. CONCLUSIONS: This case series highlights HPD as a seldom zoonosis, with a probable escalation in its occurrence within temperate regions. Therefore, clinicians should maintain a heightened awareness of HPD in the differential diagnosis of pulmonary coin lesions. Early recognition and diagnosis are paramount for appropriate management and prevention of potential complications associated with this increasingly recognized infectious entity.

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.

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.

Subconjunctival Dirofilaria masquerading as nodular scleritis.

A man in his late 50s presented with a gradually enlarging, painless, reddish mass on the white portion of his left eye for 2 weeks. His best-corrected visual acuity was 20/20 in both eyes. Slit-lamp examination showed a congested, nodular, elevated lesion on the temporal bulbar conjunctiva with two pustule-like elevations. Anterior segment optical coherence tomography showed a subconjunctival solid mass rather than an abscess or a cyst. Scleral deroofing was performed and a long thread-like object resembling a dead worm was identified. The worm was removed intact, and its histopathology confirmed the diagnosis of Dirofilaria Peripheral blood smear did not show any microfilariae. No recurrences or new lesions were observed during the follow-up examinations at 1 and 5 months post-surgery. This case highlights the importance of considering a parasitic aetiology in cases of nodular or infectious scleritis.

Evaluation of coagulation and platelet activation state and function in heartworm-infected dogs.

BACKGROUND: Enhanced platelet responses have been demonstrated in heartworm-infected (HWI) dogs; however, the cause and clinical implications of altered platelet function have not been fully elucidated. OBJECTIVE: This study evaluated platelet function in HWI dogs. METHODS: Anticoagulated whole blood collected from eight HWI and eight uninfected dogs was evaluated using turbidometric platelet aggregometry, a platelet function analyzer (PFA-100), a total thrombus analysis system (T-TAS), tissue factor-activated and tissue plasminogen activator modified thromboelastography (TF- and tPA-TEG), CBC, von Willebrand Factor activity, and fibrinogen concentrations. Platelet activation state and the presence of reticulated platelets were assessed via flow cytometric expression of P-selection (CD-62P) and thiazole orange staining. RESULTS: Platelet aggregation responses to adenosine diphosphate (ADP, 10 µM) or collagen (20 µg/mL), PFA-100 closure times, and T-TAS occlusion times did not differ between groups. TEG values TF-R, tPA-R, TF-K, and TF-LY60 were decreased (P = .025, P = .047, P = .038, P = .025) and TF-MA, tPA-MA, TF-G, tPA-G and TF-alpha angle were increased (P < .04) in HWI dogs. HWI dogs had higher fibrinogen concentrations (465.6 ± 161 mg/dL vs 284.5 ± 38 mg/dL, P = .008) and eosinophil counts (0.686 ± 0.27 × 103/µL vs 0.267 ± 0.20 × 103/µL, P = .003). There was no difference in hematocrit, activation state, or percent of reticulated platelets. Non-activated reticulated platelets exhibited higher CD62P expression compared with mature platelets. CONCLUSIONS: Chronic canine heartworm disease was accompanied by hypercoagulability, hyperfibrinogenemia, and decreased fibrinolysis. Enhanced platelet activation was not identified in this group of HWI dogs.

Heartworm-, Flea-, and Tick-associated Diseases in Dogs: A Review of Common Parasites and Drug Classes Prophylactic Against Them.

Throughout recorded history, the canine-human connection has varied by custom, purpose, and intensity. In many cultures worldwide, dogs have long been considered essential workers, protectors and guardians, and, often, an integral part of the family unit. Ensuring the health and quality of life of those companion animals is essential to preserving the bond between dogs and their owners. Fortunately, advances in veterinary science continue to improve treatments and cures for and prophylaxis against a variety of deadly canine diseases, several of which can be sourced to ectoparasites or endoparasites. For many veterinary patients, a customized preparation often proves to be the best therapeutic option, but many compounding-pharmacy stores also include a retail component that offers ready access to manufactured prescription medications, including those prophylactic against canine flea, tick, or heartworm infestation. Because dog owners often need guidance in selecting such products and assistance with obtaining them, this article will be of special interest to ompounders in those pharmacies. To that end, the following content addresses common canine parasites and classes of drugs that prevent the illnesses they cause, with emphasis on heartworm disease.

Prophylaxis Against Heartworm Infection and Flea or Tick Infestation in Dogs: Single Agents and Combination-Drug Products.

From ancient times to the present, parasites and the diseases they transmit have jeopardized the health and wellbeing of working and companion canines worldwide. Many common pests that afflict dogs can be classified as ectoparasites (e.g., fleas, ticks, lice), which serve as vectors of pathogens transmitted as the organism feeds or defecates; or endoparasites (e. g, helminths, protozoa), which can cause slowly progressive subclinical canine diseases as well as acute illnesses associated with high morbidity and mortality rates. Safe, effective antiparasitic prophylaxis in dogs remains a topic of major interest to both veterinarians and their clients, especially with respect to the prevention of canine heartworm infection and flea or tick infestation. Many compounders, especially those whose pharmacy includes a retail component, counsel veterinarians and pet owners about preparations and commercially available medications that prevent or treat parasitic infestations and provide assistance in obtaining those therapies. To support such efforts, this article provides information about single agents and combination-drug products prophylactic against common canine parasites, emerging resistance to those medications, and the toxic effects that such treatments can engender in some canine breeds.

Efficacy of moxidectin, using various dose regimens, against JYD-34, a macrocyclic lactone resistant isolate of Dirofilaria immitis.

BACKGROUND: Macrocyclic lactones (MLs) are the only class of drugs currently commercially available that are effective for preventing heartworm disease. The data presented in this article provide information on the efficacy of oral moxidectin against JYD-34, a known ML-resistant Dirofilaria immitis isolate, when dogs are treated under various dosing regimens. METHODS: Fifty-two purpose-bred Beagle dogs were used in five laboratory studies. All dogs were inoculated with 50 D. immitis third-stage larvae (L3) (JYD-34 isolate) 30 days prior to the first treatment. Dogs were randomized to treatment (four to five animals in each group) with one, three, or five monthly doses of oral moxidectin ranging from 6 to 100 µg/kg body weight. In each study, control dogs were not treated. Five to 6 months after L3 inoculation, dogs were euthanized, and adult worms were counted to evaluate efficacy of the dosing regimens. RESULTS: Adult heartworms were recovered from all control dogs, with an overall geometric mean of 29.7 worms (range 15.2 to 38.0, individual counts ranged from 8 to 51). Five monthly doses of 6 µg/kg provided 83.3% and 90.2%, efficacy, and the same number of monthly doses of 9 µg/kg demonstrated 98.8% and 94.1% efficacy. Three monthly doses of 30 and 50 µg/kg demonstrated 97.9% and 99.0% efficacy, respectively, while a single dose of 100 µg/kg demonstrated 91.1% efficacy. CONCLUSIONS: Five monthly doses of 9 µg/kg provided similar or only marginally lower efficacy against JYD-34, a known ML-resistant isolate, compared to substantially higher doses administered for 3 months. This underscores the importance of duration of exposure to moxidectin when facing ML-resistant isolates. Repeated administration of lower doses of moxidectin are an alternative to higher doses in the prevention of heartworm disease associated with less susceptible or resistant isolates.

Can nematode infection cause internal bleeding in dogs? A case of Dirofilaria immitis infection in cavitary fluids.

A 9-year-old dog was presented with weight loss, respiratory effort, and an enlarged abdomen. Imaging studies and exploratory surgery showed pulmonary and splenic masses and bi-cavitary effusion, later classified as hemorrhage. Cytology of the peritoneal and pleural fluids also revealed several microfilariae. Immunologic and molecular analyses confirmed Dirofilaria immitis infection and histopathology of the spleen indicated a cavernous endothelial proliferation with undefined etiology (hemangiosarcoma vs reaction to parasite infestation). The nematode larvae are speculated to have entered body cavities via erratic migration or via hemorrhage and visceral lesions to be related to parasitism. Nematode infection should be considered as a differential diagnosis for internal bleeding of undetermined origin.

Microfilaruria of Dirofilaria immitis in a dog from Italy.

Dirofilaria immitis is a mosquito-borne nematode-causing canine heartworm disease, with adult worms localized in the pulmonary arteries and right heart. In rare cases, ectopic migration might occur, and adults and blood circulating microfilariae can be found in unusual organs or fluids (e.g., eyes, abdominal cavity, bone marrow, and urine). A 17-year-old mixed-breed female dog was presented in a private veterinary clinic in Italy for hematuria and dysuria. Physical examination showed cardiac mitral murmur with marked respiratory distress and cyanotic mucous membranes after handling. Abdominal ultrasounds revealed a non-specific chronic cystopathy, while the echocardiography showed enlargement of the right heart associated with tricuspid insufficiency and mitral regurgitation, with the presence of an adult filariae in the right ventricular chamber. Circulating microfilariae were observed in the blood smear and molecularly identified as D. immitis. Unusual microfilaruria was detected in the urine sediment. Data presented raise awareness about the occurrence of microfilariae in unusual locations, such as the bladder, suggesting the need of a thorough clinical and laboratory assessment where D. immitis is endemic.

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.