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.

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.

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.

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.

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.

Atypical case of subcutaneous filariosis in a cat caused by Dirofilaria immitis.

Subcutaneous dirofilariosis is a well-known disease caused mainly by Dirofilaria repens and described in several mammalian species including humans, dogs, and cats. Additionally, early developing stages of the heartworm Dirofilaria immitis are rarely reported in subcutaneous localization from humans and dogs. To our knowledge, confirmed clinical evidence of this condition has not been described in the cats yet, even if the feline hosts can be affected either by the classic adult-related heartworm form or heartworm-associated respiratory disease (HARD) caused by immature stages. A 2 year old, spayed male cat was presented for three subcutaneous nodules on the head and trunk. The cat lived in Northern Italy and was regularly vaccinated and treated monthly with an antiparasitic spot on formulation containing selamectin. One of the three nodules was surgically excised and examined. Histology showed the presence of a nodular lesion in the subcutis characterized by a severe inflammatory infiltrate composed of macrophages, small lymphocytes, with fewer eosinophils, and mast cells, supported by a proliferation of mature fibroblasts (fibrosis). Inflammatory cells were multifocally surrounding sections of parasites identified as adult nematodes. Microscopic features were compatible with D. immitis, which has been molecularly confirmed (98.2% identity to D. immitis isolate OP107739). The cat tested negative for D. immitis antigenemia and the two remaining nodules disappeared spontaneously in a few months. In region where heartworm is prevalent, aberrant localization of D. immitis should be considered in the differential diagnoses of subcutaneous filarial worms in cats and dogs.

Prevention of heartworm infection in dogs using a combination of moxidectin, imidacloprid and praziquantel: evidence from a randomized clinical trial.

The aim of this study was to evaluate the efficacy of a topical combination of moxidectin 3.5%, imidacloprid 10% and praziquantel 10% for the prevention of Dirofilaria immitis (Leidy, 1856) infection in dogs. For this purpose, a randomized and controlled clinical trial was conducted between August 2021 and October 2022, in the municipality of Goiana, state of Pernambuco, north-eastern Brazil, where heartworm is highly prevalent. Of the 213 dogs initially sampled (baseline), 68 (31.9%) were positive for adult antigens (SNAP 4Dx Plus, Idexx) and/or microfilariae (modified Knott's test). On day 0, 140 negative dogs were randomly included in the treatment and control groups, 70 animals each. During the study, 60 dogs (34 treated and 26 untreated) were removed for different reasons. At the end of the study (day 360 ± 2), 36 treated and 44 untreated were sampled and included in the efficacy calculation. The efficacy against the development of adults and microfilariae was 84.7%, with only one treated dog being positive for adult antigens but negative for microfilariae. On the other hand, eight untreated dogs were positive for adult antigens and/or microfilariae, resulting in a significant difference in the number of positives between groups (Chi-square test = 4.706, df = 1, P = 0.0301). Remarkably, the efficacy against the appearance of D. immitis microfilariae was 100% (i.e., all treated dogs negative) and three untreated dogs were positive for microfilariae. The topical combination of moxidectin 3.5%, imidacloprid 10% and praziquantel 10% significantly reduced the risk of D. immitis infection in treated dogs as compared with untreated dogs, in a highly endemic area in north-eastern Brazil.

Efficacy and tolerability of the American Heartworm Society therapeutic protocol in dogs affected by heartworm disease without caval syndrome.

OBJECTIVES: The American Heartworm Society medical protocol represents the current standard of therapy for canine heartworm disease without caval syndrome. However, data on the tolerability of this protocol are limited. This study aimed to describe efficacy and prevalence of possible treatment-related side effects in dogs with heartworm disease treated using the American Heartworm Society protocol. MATERIALS AND METHODS: For this retrospective multi-centre cohort study, dogs diagnosed with classes 1 to 3 heartworm disease that completed the American Heartworm Society medical protocol were searched in four medical databases. Demographic, clinical, diagnostic, therapeutic and outcome data, including the number and type of possible treatment-related side effects, were retrieved. RESULTS: Thirty-five dogs were included. The median age and bodyweight were 6 years (1 to 13 years) and 17.3 kg (4.9 to 50 kg), respectively. Heartworm disease was classified as classes 1, 2 and 3 in 20 of 35, 11 of 35 and four of 35 dogs, respectively. In addition to the therapeutic recommendations of the American Heartworm Society, eight of 35 dogs underwent sedation to favour melarsomine administration, and 30 of 35 received ice at the injection site. After adulticide therapy, all dogs were hospitalised with cage rest [median time 12 hours (6 to 48 hours)]. All dogs survived the treatment. All dogs with long-term follow-up (32/35) became negative. Furthermore, treatment-related side effects were rare, mild and rapidly recovered without the need for supporting therapies; these included depression/lethargy (4/35 dogs), cough (2/35 dogs) and lameness, pain and gastrointestinal signs (1/35 dog each). CLINICAL SIGNIFICANCE: The American Heartworm Society medical protocol is efficient and safe in dogs with classes 1 to 3 heartworm disease.

How Italian practitioners manage dirofilariosis in dogs and cats? Data of a second national survey.

The zoonotic nematodes Dirofilaria immitis and Dirofilaria repens are endemic in many European countries and their distribution is changing. A correct diagnosis is the starting point to reduce their spread and veterinary practitioners represent the first line of defense against these two vector-borne diseases in pets and humans. With the aim to better understand current knowledge by veterinarians and to compare them with the previous survey, an online questionnaire was sent to Italian companion animal veterinary facilities. The overall response rate was 22.3%. Approximately 50% and 5% of facilities have diagnosed at least one case of Dirofilaria spp. in the last year in dogs and cats, respectively. In agreement with international guidelines, of the 665 facilities that responded to the questionnaire, 16.8% used Knott's test in combination with serology (Antigenic test-Ag-) to diagnose heartworm infection in dogs. For the diagnosis of D. repens infection, 13.8% used Knott's test in dogs and cats, and 10.5% used Knott's test only in dogs. Most facilities recommended beginning prevention of canine heartworm disease in April and ending in November. Injectable moxidectin and oral ivermectin were the two most commonly used preventives for D. immitis in dogs, while selamectin was most frequently used in cats. The drug most commonly used for heartworm adulticide treatment in dogs was melarsomine followed by the combined ivermectin-doxycycline treatment. The results of this study suggest that, on the one hand, veterinarians are aware of the recent developments in adulticide therapy, while on the other hand, diagnosis is not always made according to what is recommended by international guidelines and that the start and end times of prevention may not be optimal. Furthermore, the data show an increase in cases of coinfection compared to the previous survey.

New paradigms in research on Dirofilaria immitis.

BACKGROUND: Since the advent of ivermectin (along with melarsomine and doxycycline), heartworm has come to be viewed as a solved problem in veterinary medicine, diminishing investment into non-clinical research on Dirofilaria immitis. However, heartworm infections continue to pose problems for practitioners and their patients and seem to be increasing in frequency and geographic distribution. Resistance to preventative therapies (macrocyclic lactones) complicates the picture. The use of chemotherapy for other kinds of pathogens has benefitted enormously from research into the basic biology of the pathogen and on the host-pathogen interface. A lack of basic information on heartworms as parasites and how they interact with permissive and non-permissive hosts greatly limits the ability to discover new ways to prevent and treat heartworm disease. Recent advances in technical platforms will help overcome the intrinsic barriers that hamper research on D. immitis, most notably, the need for experimentally infected dogs to maintain the life cycle and provide material for experiments. Impressive achievements in the development of laboratory animal models for D. immitis will enhance efforts to discover new drugs for prevention or treatment, to characterize new diagnostic biomarkers and to identify key parasite-derived molecules that are essential for survival in permissive hosts, providing a rational basis for vaccine discovery. A 'genomics toolbox' for D. immitis could enable unprecedented insight into the negotiations between host and parasite that enable survival in a permissive host. The more we know about the pathogen and how it manipulates its host, the better able we will be to protect companion animals far into the future.

Elimination of Dirofilaria immitis Infection in Dogs, Linosa Island, Italy, 2020-2022.

On Linosa Island, Italy, Dirofilaria immitis infection has been hyperendemic in dogs and seroprevalent among islanders. In 2020, we implemented a heartworm disease elimination program on Linosa Island. Of 54 dogs tested for D. immitis antigen and microfilariae, 28 had positive results and received treatment with oral doxycycline twice daily for 4 weeks plus topical imidacloprid/moxidectin monthly for 12 months. The 26 dogs with negative results received monthly topical imidacloprid/moxidectin as preventive. During month 1, the number of microfilaremic dogs was reduced by 76.5%. From month 2 on, all animals were microfilariae negative, and during months 3 to 9, the number of antigen-positive dogs decreased progressively. Treatment of positive dogs coupled with chemoprophylaxis for noninfected dogs was effective, protecting them from new infections. The elimination program reduced the risk for human infection, representing a One Health paradigm. Monitoring and chemoprophylaxis are advocated to maintain the status of heartworm disease-free area.

Effects of doxycycline dose rate and pre-adulticide wait period on heartworm-associated pathology and adult worm mass.

BACKGROUND: The American Heartworm Society canine guidelines recommend treatment with doxycycline prior to adulticide administration to reduce levels of Wolbachia and its associated metabolites, which are known to be a leading cause of pulmonary pathology. Studies have determined that doxycycline administered at 10 mg/kg BID for 28 days is an effective dose for eliminating Wolbachia, but what has not been determined is the clinical relevance of this elimination. The current guidelines also recommend a 30-day wait period following administration of doxycycline to allow for clearance of metabolites, such as Wolbachia surface protein, and for further reduction in heartworm biomass before administration of adulticide. Reducing the doxycycline dose and eliminating the wait period may carry practical benefits for the animal, client, and practitioner. METHODS: To investigate these treatment practices, Dirofilaria immitis adults were surgically transplanted into each of 45 dogs, which were divided into nine study groups of five dogs each. Seventy-five days after transplantation, two groups each were administered 5, 7.5, or 10 mg/kg BID doxycycline orally for 28 days and 6 µg/kg ivermectin monthly, with three untreated groups serving as controls. Study animals were necropsied and examined prior to treatment as well as 30 and 60 days post-treatment. RESULTS: Mean worm weight was unaffected by dosage but exhibited a significant increase at 30 days and significant decrease at 60 days post-treatment, including in control groups. Histopathology lesion scores did not significantly differ among groups, with the exception of the lung composite score for one untreated group. Liver enzymes, the levels of which are a concern in doxycycline treatment, were also examined, with no abnormalities in alanine aminotransferase or alkaline phosphatase observed. CONCLUSIONS: No consistent worsening of tissue lesions was observed with or without the AHS-recommended 30-day wait period, nor did reduced dosages of doxycycline lead to worsening of pathology or any change in efficacy in depleting worm weight. Mean worm weight did significantly increase prior to, and decrease following, the wait period. Future work that also includes adulticide treatment (i.e. melarsomine) will study treatment recommendations that may improve both animal health and owner compliance.

Efficacy of an oral combination of moxidectin, afoxolaner, and pyrantel pamoate for the prevention of heartworm disease in dogs.

Dirofilaria immitis, the mosquito-borne agent of dirofilariosis, a chronic and sometimes fatal cardiopulmonary canine disease, is endemic in most warm and temperate regions in the world. The efficacy of an oral endectoparasiticide product (test product or TP) combining moxidectin, afoxolaner, and pyrantel pamoate was evaluated for the prevention of heartworm disease in dogs, in two laboratory and one field studies. In each laboratory study, 20 D. immitis-naïve beagle dogs were experimentally infected with D. immitis. Ten control dogs were sham-treated, and ten dogs were administered the TP targeting the minimum effective dose, six times monthly and starting 30 days post infection. At necropsy seven months after inoculations, no heartworms were found in any of the TP treated dog, whereas 19 to 42 live heartworms were found in the control dogs. In each study, treatment efficacy was 100% and the difference between treated and untreated groups was highly significant (p < 0.0001). A field study was conducted through the full transmission season in several heartworm-endemic regions of the United States. One hundred and twenty client-owned dogs that were negative for D. immitis at enrollment were administered twelve monthly oral doses of the TP at label dose. Blood tests for D. immitis antigen and modified Knott's tests for microfilariae remained negative through the full duration of the study, demonstrating that all dogs were protected from heartworm infection during the full transmission season. These studies demonstrated that TP administered monthly for at least six doses is effective at preventing dirofilariosis.

Subcutaneous dirofilariasis due to Dirofilaria immitis in a dog in Brazil: first report.

The aim of this study was to report on the presence of microfilariae of Dirofilaria immitis causing nodular pyogranulomatous dermatitis in a dog in the state of Rio Grande do Norte, northeastern Brazil. A 4-year-old male dachshund dog with lesions in the nostrils and left dorsolateral regions was treated. Tests were requested to aid in making the diagnosis, such as skin cytology, Knott's test, thick smear and histopathology of the lesions. From these, presence of a diffuse pyogranulomatous process was observed and, amidst the cellular material, microfilariae of Dirofilaria spp. A conventional polymerase chain reaction test on tissue samples from the lesions revealed the presence of the species D. immitis. Treatment based on ivermectin (3mg) was administered at a single oral dose of 0.6 mg/kg. In the first seven days there was regression of the lesions, but after 30 days there was recurrence. A new treatment was administered, consisting of 10% imidacloprid + 2.5% moxidectin (4-10 mg/kg), with one application per month for 6 months, and doxycycline (100 mg), 10 mg/kg, 1 tablet, 2 times a day, for 30 days. In conclusion, D. immitis microfilariae caused pyogranulomatous lesions in the subcutaneous tissue of a dog. This had not previously been described in Brazil.

Long-term evaluation of viability of microfilariae and intravenously transplanted adult Dirofilaria immitis in microfilaremic dogs treated with low-dose, short- and long-treatment regimens of doxycycline and ivermectin.

BACKGROUND: Microfilarial (mf) counts were monitored over 21.3 months for any rebound that might occur in counts, and adulticidal efficacy was assessed following administration of low dosage with short- and long-treatment regimens of doxycycline and ivermectin to heartworm-microfilaremic dogs. METHODS: Twelve heartworm-naïve beagles infected with 10 pairs of adult Dirofilaria immitis by intravenous transplantation were randomly allocated to three groups of four dogs. All treatments started on day 0. On day 0, Group 1 (short-treatment regimen) received doxycycline orally at 10 mg/kg once daily for 30 days plus ivermectin orally (minimum, 6 mcg/kg) on days 0 and 30. Group 2 (long-treatment regimen) received doxycycline orally at 10 mg/kg once daily until individual dogs became mf-negative (72-98 days) and ivermectin every other week until individual dogs became mf-negative (6-7 doses). Group 3 was the untreated control. Mf counts and antigen (Ag) tests were conducted. Dogs were necropsied for recovery and enumeration of heartworms on day 647. RESULTS: Day -1 mean mf counts were 15,613, 23,950, and 15,513 mf/ml for groups 1, 2, and 3, respectively. Mean counts for Groups 1 and 2 declined until days 239 and 97, respectively, when all were negative. Group 3 had high mf counts throughout the study. There was not a rebound in mf counts in any of the treated dogs after they became amicrofilaremic. All dogs in group 1 and group 3 were Ag-positive throughout the study and had at least one live female worm at necropsy. All dogs in treated Group 2 were positive for Ag through day 154, but were antigen-negative on days 644 and 647, as all had only male worms. Mean live adult worm recoveries for Groups 1, 2, and 3 were 6.8 (range, 5-8), 3.3 (range, 1-6), and 16.0 (range, 14-17), respectively, with a percent reduction in adult worm counts of 57.5% for Group 1 and 79.3% for Group 2. CONCLUSIONS: These data lend support to the use of the American Heartworm Society Canine Guidelines for adulticide therapy recommending the initiation of doxycycline plus a macrocyclic lactone (ML) at the time of the heartworm-positive diagnosis.

Evaluation of renal values during treatment for heartworm disease in 27 client-owned dogs.

BACKGROUND: Canine heartworm disease (CHD) caused by Dirofilaria immitis remains a common preventable disease with increasing incidence in some parts of the USA. The treatment guidelines of the American Heartworm Society (AHS) currently recommend monthly macrocyclic lactone administration, 28 days of doxycycline given orally every 12 h and three injections of melarsomine dihydrochloride (1 injection on day 2 of treatment followed 30 days later by 2 injections 24 h apart). Minocycline has also been utilized when doxycycline is unavailable. The systemic effects of CHD, which particularly impact cardiac and renal function, have been described, with infected dogs often experiencing renal damage characterized by an increase in serum concentrations of renal biomarkers. Although the AHS treatment protocol for CHD has been shown to be safe and effective in most cases, the potential for complications remains. No study as of yet has evaluated changes in symmetric dimethylarginine (SDMA), a sensitive marker of renal function, during treatment for CHD. The purpose of the present study was to evaluate renal function in dogs by measuring serum creatinine and SDMA concentrations during the adulticide treatment period. METHODS: Serum creatinine and SDMA concentrations were measured in 27 client-owned dogs affected by CHD at the following time points: prior to starting doxycycline or minocycline therapy (baseline), during doxycycline or minocycline therapy (interim), at the time of the first dose of melarsomine (first dose), at the time of the second dose of melarsomine (second dose) and at the dog's follow-up visit after treatment, occurring between 1 and 6 months after completion of therapy (post-treatment). Concentrations of creatinine and SDMA were compared between time points using a mixed effects linear model. RESULTS: Mean SDMA concentrations following the second dose of melarsomine were significantly lower (-1.80 ug/dL, t-test, df = 99.067, t = -2.694, P-Value = 0.00829) than baseline concentrations. There were no other statistically significant differences in the concentration of either biomarker between the baseline and the other time points in CHD dogs undergoing treatment. CONCLUSIONS: The results suggest that the current AHS protocol may not have a substantial impact on renal function.

Fluralaner (Bravecto®) treatment kills Aedes aegypti after feeding on Dirofilaria immitis-infected dogs.

BACKGROUND: Transmission of canine heartworm (Dirofilaria immitis) from infected to naïve dogs is dependent on successful mosquito feeding and survival. METHODS: To determine whether treating heartworm-infected dogs with fluralaner (Bravecto®) limits the survival of infected mosquitoes, and potentially the transmission of D. immitis, we allowed female mosquitoes to feed on microfilaremic dogs and evaluated mosquito survival and infection with D. immitis. Eight dogs were experimentally infected with D. immitis. On day 0 (~ 11 months post-infection), four microfilaremic dogs were treated with fluralaner according to label directions while the other four were non-treated controls. Mosquitoes (Aedes aegypti Liverpool) were allowed to feed on each dog on days -7, 2, 30, 56, and 84. Fed mosquitoes were collected, and the number of live mosquitoes determined at 6 h, 24 h, 48 h, and 72 h post-feeding. Surviving mosquitoes held for 2 weeks were dissected to confirm third-stage D. immitis larvae; PCR (12S rRNA gene) was performed post-dissection to identify D. immitis in mosquitoes. RESULTS: Prior to treatment, 98.4%, 85.1%, 60.7%, and 40.3% of mosquitoes fed on microfilaremic dogs were alive at 6 h, 24 h, 48 h, and 72 h post-feeding, respectively. Similarly, mosquitoes fed on microfilaremic, non-treated dogs were alive 6 h post-feeding (98.5-100%) throughout the study. In contrast, mosquitoes fed on fluralaner-treated dogs 2 days after treatment were dead or severely moribund by 6 h post-feeding. At 30 and 56 days post-treatment, > 99% of mosquitoes fed on treated dogs were dead by 24 h. At 84 days post-treatment, 98.4% of mosquitoes fed on treated dogs were dead by 24 h. Before treatment, third-stage larvae of D. immitis were recovered from 15.5% of Ae. aegypti 2 weeks after feeding, and 72.4% were positive for D. immitis by PCR. Similarly, 17.7% of mosquitoes fed on non-treated dogs had D. immitis third-stage larvae 2 weeks after feeding, and 88.2% were positive by PCR. Five mosquitoes fed on fluralaner-treated dogs survived 2 weeks post-feeding, and 4/5 were from day 84. None had third-stage larvae at dissection, and all were PCR-negative. CONCLUSION: The data indicate that fluralaner treatment of dogs kills mosquitoes and thus would be expected to reduce transmission of heartworm in the surrounding community.

Further variation of the adulticide protocol for the treatment of canine heartworm infection: can it be even shorter and cost less?

BACKGROUND: This retrospective study evaluated modified three-dose melarsomine treatment protocols in a shelter setting and compared them to the American Heartworm Society (AHS)-recommended protocol. METHODS: As compared with the AHS protocol, the shelter protocols utilized doxycycline 10 mg/kg once daily (SID) or twice daily (BID), and varied the time from initiation of doxycycline (day 1) to the first melarsomine injection (M1). Dogs were retrospectively grouped based on the shelter's current protocol (M1 on day 14; Group A) and the AHS protocol (M1 on day 60; Group C), allowing a week on either side of the target M1 day. Treatments that fell outside these ranges formed two additional treatment groups (Groups B and D). Respiratory complications were defined as respiratory signs requiring additional treatment, and were statistically compared for Groups A and C. New respiratory signs and gastrointestinal (GI) signs were compared between dogs receiving SID or BID doxycycline. RESULTS: One hundred fifty-seven dogs with asymptomatic or mild heartworm disease at presentation were included. All dogs survived to discharge. There was no statistically significant difference between Groups A (n = 79) and C (n = 27) for new respiratory signs post-melarsomine (P = 0.73). The time to M1 for 14 dogs that developed new respiratory signs was a median of 19 days, compared with 22 days for 143 dogs without new respiratory signs (P = 0.2). Respiratory complications post-melarsomine were uncommon. New respiratory signs post-melarsomine occurred in 10/109 (9.2%) dogs receiving SID doxycycline and 4/48 (8.3%) dogs receiving BID doxycycline (P > 0.999). GI signs prior to M1 were recorded for 40/109 (36.7%) dogs receiving SID doxycycline and 25/48 (52.1%) receiving BID doxycycline (P = 0.08). Forty-four follow-up antigen test results were available; all tests performed > 3 months after the third melarsomine injection were negative. CONCLUSIONS: This study provided support for initiating melarsomine after 14 days of doxycycline and for a lower doxycycline dose. Shorter and less expensive treatment protocols can increase lifesaving capacity and improve quality of life for shelter dogs by reducing the duration of exercise restriction and length of stay.

Heartworm adulticide treatment: a tropical perspective.

Dirofilaria immitis (the canine heartworm) is widespread in the tropics, with prevalence surpassing 30% in high-risk areas. In addition to the suitable climatic conditions that favour mosquito abundance and filarial larva development, there is low compliance with the recommended year-round use of preventives in these transmission hotspots. This represents a major concern, considering that melarsomine (first-line heartworm adulticide) is unavailable in several tropical countries, resulting in the so-called slow-kill protocol being the only available adulticide treatment option. In this article, the members of TroCCAP (Tropical Council for Companion Animal Parasites) review the current distribution of heartworm in the tropics and the availability of melarsomine, and discuss alternatives for the management of heartworm infections in dogs.