Moxidectin: heartworm disease prevention in dogs in the face of emerging macrocyclic lactone resistance.

Heartworm (Dirofilaria immitis) disease continues to increase and spread, remaining one of the most important and pathogenic parasitic diseases of dogs, despite the regular use of macrocyclic lactones (MLs) in preventive products. Dogs harboring strains of D. immitis resistant to MLs, the only drug class available for heartworm prevention in the United States, have been documented and proven. As no new products are available utilizing a novel drug class for the prevention of this disease, the only options for combating ML resistance include increasing the dose and/or changing the dosage regime of current MLs, or by optimizing the formulations of MLs currently available. Moxidectin provides a unique opportunity for optimization of the dose and formulation, which may provide improved efficacy against ML-resistant strains. Currently there are oral, topical, and injectable moxidectin products approved for heartworm prevention in the USA. Two new products (ProHeart® 12 and Simparica Trio®), available in many countries around the world including the USA, take advantage of the unique attributes of moxidectin for providing robust heartworm prevention against the strains of heartworm to which most dogs in the USA will likely be exposed. Both products have demonstrated 100% preventive efficacy in laboratory studies against recently collected field strains of heartworm, and also in large field studies, where the majority of dogs were living in the southern USA in areas where ML resistance has been confirmed to occur, therefore under elevated heartworm challenge. Based on the data summarized here, these products offer important advances in heartworm prevention and provide additional options for veterinarians and pet owners to protect their dogs from developing heartworm disease.

Detection and cross-reaction of Dirofilaria repens using a commercial heartworm antigen test kit.

Antigen enzyme-linked immunosorbent assay tests are widely used for the diagnosis of heartworm infection in dogs. While commercially-available heartworm antigen tests have high sensitivity and specificity, false-negative test results can occur in dogs with low worm burdens, female-only infections, or prior to patency. The use of immune complex dissociation (ICD) methods have demonstrated increased sensitivity in the detection of Dirofilaria immitis antigens and the reversal of false-negative antigen results. However, there are concerns pertaining to false-positive antigen results due to infections of other nematode parasites, especially post-ICD. Therefore, this study evaluated the effect of heat-treatment on serum samples of dogs experimentally-infected with Dirofilaria repens during the course of infection, to assess for potential cross-reactivity on heartworm antigen tests. Archived serum samples from three dogs experimentally-infected with D. repens were utilized. All samples were tested for cross-reactivity pre- and post-heat-treatment using the DiroCHEK® Heartworm Antigen test kit throughout infection (day -9 through 404 days post-infection; dpi). All heat-treated samples tested false-positive starting at 164 dpi and continuing through 404 dpi, thereby testing positive prior to patency. No cross-reactivity was observed for any dog at any time point prior to heat-treatment. Our results suggest that the ICD method decreased the specificity of heartworm antigen tests and caused cross-reactivity of serum from dogs experimentally infected with D. repens. In conclusion, heat-treatment of serum in areas co-endemic for D. repens and D. immitis has limited clinical value, and should be used with caution.

Clinical assessment of heartworm-infected Beagles treated with a combination of imidacloprid/moxidectin and doxycycline, or untreated.

BACKGROUND: Administration of moxidectin topically and doxycycline PO has been utilized experimentally as an alternative treatment for heartworm disease. However, clinical effects of this protocol remain poorly characterized. OBJECTIVE: To evaluate the clinical and postmortem findings associated with administration of doxycycline and monthly 10% imidacloprid + 2.5% moxidectin (IMD + MOX, Advantage Multi/Advocate) to Dirofilaria immitis-experimentally infected as compared to nontreated control dogs. ANIMALS: Sixteen purpose-bred, female, Beagle dogs. METHODS: Prospective, blinded, experimental study. Animals with surgically transplanted adult heartworms were randomized into 2 study groups of equal size: a nontreated control group (n = 8) and an IMD + MOX and doxycycline-treated group (n = 8). Randomization was performed using a complete block design according to circulating microfilarial concentrations, measured before treatment. Serum biochemical profiles, CBCs, thoracic radiographs and echocardiograms were performed prior to and 3 weeks after transplantation, and monthly for 10 months. Postmortem gross and histopathologic evaluations were performed. RESULTS: Compared to control animals, mean ± SD serum alanine aminotransferase (181 ± 203 U/L vs 33 ± 7 U/L; P < .0001) and alkaline phosphatase (246 ± 258 U/L vs 58 ± 19 U/L; P < .0001) activities were significantly higher in the treated group on day 28. Radiographic and echocardiographic evidence of heartworm disease was observed in both groups; however, no significant differences in these variables were noted between groups. Mean ± SD pulmonary arterial thrombus score was significantly higher in the treated vs nontreated group (3.9 ± 0.4 and 1.5 ± 2.1, respectively; P = .01). CONCLUSIONS AND CLINICAL IMPORTANCE: The treatment protocol was well-tolerated with no clinically relevant adverse effects for any variable evaluated during the observational period.

Assessing the potential cross-reactivity using a commercial heartworm ELISA kits of serum from dogs naturally infected with Onchocerca lupi.

Onchocerca lupi is an emerging zoonotic parasite of dogs, endemic to the southwestern USA and areas of the Old World. Currently, there are no specific serological diagnostic tests able to detect O. lupi infection. Recent literature has demonstrated that commercially available heartworm antigen tests, despite being highly sensitive, may cross-react with infections by other filarid nematodes. There is no information on potential cross-reactivity of such tests in serum of dogs infected with O. lupi. Our objective was to assess serum samples of dogs naturally-infected with O. lupi for potential cross-reactivity before and after heat-treatment using a commercial heartworm ELISA kit. We obtained serum from 23 dogs naturally-infected with O. lupi. These dogs presented with ocular disease, and were consulted to schedule either surgical removal of ocular nodules due to infection or enucleation. Samples were tested in triplicate using the DiroCHEK® Heartworm Antigen Test kit (Synbiotics Corporation, Zoetis, Kalamazoo, MI, USA) following the manufacturers' protocol pre- and post-heat-treatment. Samples were heat-treated using a dry heat block at 103 °C for 10 min and then centrifuged at 1818×g for 20 min. Out of a total of 23 dogs, 19 (82.6 %) had no antigen detected regardless of heat-treatment, three dogs tested positive before and after heat-treatment, and a single dog turned positive after heat-treatment. These three dogs that were positive before and after heat-treatment were confirmedly co-infected with Dirofilaria immitis by the veterinarians responsible for these cases, and we were unable to get the history or follow up with the dog that turned positive post-heat-treatment only. Our data suggest that O. lupi infections should not result in false-positives when using the DiroCHEK® in dog serum, before or after heat-treatment. Dogs with clinical ocular onchocercosis that test antigen-positive in DiroCHEK® are likely co-infected with D. immitis, and should be further tested, including evaluation of microfilariae in blood and diagnostic imaging. If heartworm infection is confirmed, the animals should be enrolled in the recommended treatment protocol in accordance to the guidelines of the American Heartworm Society or other local organizations.

Canine gastrointestinal nematode transmission potential in municipal dog parks in the southeast United States.

Monthly canine parasite prophylactic products prevent not only adult heartworm infection, but also patent infections with specific gastrointestinal parasites. While most monthly products control and treat certain hookworm and roundworm infections, fewer are labeled for whipworm (Trichuris vulpis). Therefore, we hypothesized that fecal samples collected from municipal dog parks will have a greater prevalence of whipworm eggs compared to hookworm and roundworm eggs. In this study, canine fecal samples were collected from municipal dog parks in three southeastern states, with up to 20 fecal samples were collected from each park. A total of 200 fecal samples were obtained from dog parks in Georgia, North Carolina, and South Carolina. All fecal samples were examined for the presence of gastrointestinal helminths by a simple centrifugal flotation using sheather's sugar flotation solution. Of the 200 samples collected, 27% were positive for gastrointestinal helminths by fecal flotation. Of these infected fecal samples, 8.5%, 17%, and 1.5% contained whipworm, hookworm, and roundworm, respectively. However, the majority of hookworm-positive samples were collected from one park, whereas whipworm and roundworm samples were collected from multiple parks. These results could indicate that dogs are at risk of infection by all three parasites at dog parks, and that preventive strategies may need to be tailored not only to the specific region, but to specific infected dog parks.

Acetic acid as an alternative reagent in the modified Knott test.

The suitability of acetic acid as a safer alternative to formalin in the modified Knott test was evaluated for the diagnosis of canine heartworm (Dirofilaria immitis). Microfilaria concentration was measured by both methods and found to agree within reasonable limits (-5.84 % bias; -88.1-76.4 % limits of agreement). The level of agreement was lower when samples were prepared with a 24 h delay, but this was due to the formalin method tending to yield lower counts (-20.1 % bias; -90.5-50.2 % limits of agreement). Clearing the sample of hemoglobin improves readability and is a key feature of the modified Knott test. Hemolysis was significantly lower in the acetic acid method than the formalin method as measured by red blood cell count (6.83 × 106 and 8.79 × 106 cells/ml, respectively; p = 0.015) and absorbance at 415 nm (33.20 and 34.75, respectively; p < 0.001). Visual assessment, however, revealed little practical difference in readability. Finally, lengths of microfilariae were measured to ensure the validity of species identification by the acetic acid method; mean length was significantly shorter after acetic acid treatment (273 µm) than formalin treatment (316 µm; p < 0.001). Length reduction was also observed in acetic acid-treated Acanthocheilonema reconditum (254 µm versus 262 µm; p = 0.035), though these samples were stored prior to testing and are not directly comparable. We conclude that, while the readability of samples is similar for both methods, species differentiation must still be accomplished by other means. For most clinical purposes in determining the presence or absence of blood circulating microfilariae, however, acetic acid appears to be a suitable alternative to formalin in the modified Knott test.

Efficacy and side effects of doxycycline versus minocycline in the three-dose melarsomine canine adulticidal heartworm treatment protocol.

BACKGROUND: The American Heartworm Society currently recommends the use of a monthly macrocyclic lactone, a 28-day course of 10 mg/kg doxycycline BID, and the 3-dose protocol of melarsomine dihydrochloride for the treatment of canine heartworm disease. Doxycycline is necessary for the reduction of the bacterium Wolbachia, found in all heartworm life-stages. Previous price increases and decreasing availability prompted us to evaluate alternative tetracycline antibiotics, i.e. minocycline, for the reduction of Wolbachia during canine heartworm treatment. METHODS: Thirty-two heartworm-positive dogs were randomized to receive 10 mg/kg or 5 mg/kg of either doxycycline or minocycline for 28 days BID, for a total of 8 dogs per experimental group. All dogs received 6 months of Heartgard Plus® (ivermectin/pyrantel) and the 3-dose protocol of 2.5 mg/kg melarsomine dihydrochloride. Blood samples were collected prior to the initiation of treatment, every 7 days throughout tetracycline treatment, and then monthly thereafter until the dog tested negative for the presence of heartworm antigen. DNA was isolated from circulating microfilarial samples and qPCR was performed on each sample. RESULTS: A greater number of dogs in the 10 mg/kg doxycycline and minocycline treated groups experienced gastrointestinal side effects as compared to the 5 mg/kg doxycycline and minocycline treated groups. All eight dogs in the 10 mg/kg doxycycline-treated group tested negative for the presence of Wolbachia DNA by 28 days post-tetracycline treatment. A total of two dogs in both the 5 mg/kg doxycycline- and 10 mg/kg minocycline-treated groups and three dogs in the 5 mg/kg minocycline-treated group remained positive for the presence of Wolbachia DNA by the end of tetracycline treatment. CONCLUSIONS: No lung pathology was assessed in this clinical trial, therefore the clinical effect of the remaining Wolbachia DNA in the 10 mg/kg minocycline-, 5 mg/kg doxycycline- and 5 mg/kg minocycline-treated groups cannot be determined. Owner compliance in the proper administration of these tetracyclines may be impacted by the increased severe gastrointestinal side effects reported for the 10 mg/kg doxycycline- and minocycline-treated groups. We recommend that veterinarians prescribe the recommended 10 mg/kg doxycycline for canine heartworm treatment and reduce the dosage to 5 mg/kg in cases of severe gastrointestinal side effects in order to improve owner compliance in administration of medications.

Evaluation of heat-treating heartworm-positive canine serum samples during treatment with Advantage Multi® for Dogs and doxycycline.

BACKGROUND: The use of heat-treatment in canine and feline serum has been hypothesized to break the formation of antigen-antibody complexes, thereby freeing the heartworm antigen allowing for detection by commercially available heartworm antigen kits. While studies have analyzed the effect of heat-treating serum and plasma samples in the detection of heartworm antigen, these studies have not utilized necropsy verified results for validation. This study evaluated the use of heat-treating serum samples in experimentally infected dogs during adulticidal treatment in comparison with necropsy adult heartworm recovery. METHODS: As part of a primary study, a total of 16 dogs were experimentally infected with 16 sexually mature adult heartworms using surgical transplantation, allocating 8 dogs in both the control and treated group. Treated dogs received 10 months of topical administration of Advantage Multi® for Dogs (10% Imidacloprid + 2.5% Moxidectin) every 4 weeks and 30 days of 10 mg/kg doxycycline BID. Blood samples were collected from all study animals prior to surgical transplantation of adult heartworms, on study days 0, 1, 3, 7, 14, 21, 28, and every 4 weeks thereafter for the duration of this study. Concentration of heartworm antigen was tested using the DiroCHEK® heartworm antigen test kit using serum samples both pre- and post-heat-treatment. Serum samples were heat-treated at 103 °C in a dry heat block for 10 min and centrifuging at 1818× g for 20 min. RESULTS: There were a total of 4 instances (days 56, 140, 224 and 252) in 3 treated dogs in which a serum sample converted from negative for the detection of heartworm antigen prior to heat-treatment to positive for the detection of heartworm antigen post-heat-treatment. At necropsy, these dogs had no adult heartworms recovered and were all negative on antigen testing prior to and after heat treatment. There was 100% accuracy in the detection of either no infection, or 1-2 adult heartworm infections using the DiroCHEK in serum samples with and without heat-treatment at the time of necropsy. CONCLUSIONS: The DiroCHEK accurately diagnosed all dogs with live adults recovered at necropsy as heartworm antigen positive and all those dogs with no live adults recovered at necropsy as heartworm antigen negative without the use of heat-treatment for samples taken on the day of necropsy. Therefore, these results indicate that the use of heat-treating serum samples did not provide data of any additional value in the diagnosis of heartworm-positive dogs receiving treatment in this study. Additionally, these results may indicate that the conversion of serum samples from negative to positive for the presence of heartworm antigen with heat-treatment may not always accurately diagnose live adult heartworm infections since no adult heartworms were recovered at necropsy for those dogs in which a conversion event occurred. These conversion events may be detecting residual antigen leftover after all adult worms have died or may even be detecting off- target antigens, which have been denatured during heat-treatment. While a necropsy was not performed at the time of the conversion events, no live adult worms were recovered from any of the dogs in which a conversion event occurred earlier in treatment.

Does evaluation of in vitro microfilarial motility reflect the resistance status of Dirofilaria immitis isolates to macrocyclic lactones?

BACKGROUND: Several reports have confirmed that macrocyclic lactone-resistant isolates of Dirofilaria immitis are circulating in the United States; however, the prevalence and potential impact of drug resistance is unknown. We wished to assess computer-aided measurements of motility as a method for rapidly assessing the resistance status of parasite isolates. METHODS: Blood containing microfilariae (MF) from two clinical cases with a high suspicion of resistance was fed to mosquitoes and the resultant L3 injected into dogs that were then treated with six doses of Heartgard® Plus (ivermectin + pyrantel; Merial Limited) at 30-day intervals. In both cases patent heartworm infections resulted despite the preventive treatment. Microfilariae isolated from these dogs and other isolates of known resistance status were exposed to varying concentrations of ivermectin in vitro and their motility assessed 24 h later using computer-processed high-definition video imaging. RESULTS: We produced two isolates, Yazoo-2013 and Metairie-2014, which established patent infections despite Heartgard® Plus treatments. Measurements of the motility of MF of these and other isolates (Missouri, MP3 and JYD-27) following exposure to varying concentrations of ivermectin did not distinguish between susceptible and resistant heartworm populations. There was some evidence that the method of MF isolation had an influence on the motility and drug susceptibility of the MF. CONCLUSIONS: We confirmed that drug-resistant heartworms are circulating in the southern United States, but that motility measurements in the presence of ivermectin are not a reliable method for their detection. This implies that the drug does not kill the microfilariae via paralysis.

Assessment of parasitological findings in heartworm-infected beagles treated with Advantage Multi® for dogs (10% imidacloprid + 2.5% moxidectin) and doxycycline.

BACKGROUND: Anecdotal reports support the position that the adulticidal heartworm treatment utilizing doxycycline and Advantage Multi®/Advocate® for Dogs (10% imidacloprid + 2.5% moxidectin) has successfully converted antigen-positive dogs to antigen-negative. To date, no controlled experimental studies have demonstrated the adulticidal efficacy of this treatment regimen. The aim of this study was to evaluate the parasitological and clinical efficacy of Advantage Multi® for Dogs (IMD + MOX) and doxycycline in heartworm-infected beagles. METHODS: This study utilized 16 dogs, 8 dogs in each of non-treated control and treated groups. A total of 16 adult Dirofilaria immitis (Missouri strain) were surgically transplanted into the jugular vein of each study dog. The treatment regimen of monthly IMD + MOX topically (per labeled dosage and administration) for 10 months and 10 mg/kg doxycycline BID orally for 30 days was initiated 30 days post-surgical transplant. Echocardiograms, radiographs, complete blood counts, clinical chemistry profiles, heartworm antigenemia and microfilaremia were evaluated every 4 weeks. Serum samples were assayed for heartworm antigen using the DiroCHEK® heartworm antigen test. The DiroCHEK® was performed according to the manufacturer's recommendations and read using a spectrophotometer at 490 nm. RESULTS: All dogs tested positive for the presence of heartworm antigen post-surgical transplant and prior to treatment. Heartworm antigen levels began declining in treated dogs 3 months post-treatment. Non-treated control dogs remained antigen-positive. No microfilariae were detected in treated dogs after 21 days post-treatment. At necropsy, adult heartworms were recovered from all non-treated control dogs with a range of 10-12 adult worms/dog for an average recovery of 10.6 adult heartworms/dog. In the IMD + MOX- and doxycycline-treated dogs, the range of adult heartworms recovered was 0-2 adult worms/dog, with five dogs having no adult heartworms present. The average adult heartworm recovery was 0.6/dog in the treated group. This treatment regimen demonstrated a 95.9% efficacy in eliminating adult heartworms (P < 0.0001). CONCLUSIONS: This study demonstrated that this treatment regimen successfully eliminated D. immitis microfilariae by 21 days post-treatment, reduced heartworm antigen concentration over time, and had a 95.9% efficacy in the elimination of mature adult heartworms. Based on this study, we conclude that this treatment regimen is a relatively quick, reliable and safe option to treat canine heartworm infection as compared to other treatment regimens involving macrocyclic lactones, when the approved drug melarsomine dihydrochloride is unavailable, contraindicated or declined by an owner unable to afford the more costly treatment or concerned about the potential side effects.

Assessment of Blood Collection from the Lateral Saphenous Vein for Microfilaria Counts in Mongolian Gerbils (Meriones unguiculatus) Infected with Brugia pahangi.

The NIH guidelines for survival bleeding of mice and rats note that using the retroorbital plexus has a greater potential for complications than do other methods of blood collection and that this procedure should be performed on anesthetized animals. Lateral saphenous vein puncture has a low potential for complications and can be performed without anesthesia. Mongolian gerbils (Meriones unguiculatus) are the preferred rodent model for filarial parasite research. To monitor microfilaria counts in the blood, blood sampling from the orbital plexus has been the standard. Our goal was to refine the blood collection technique. To determine whether blood collection from the lateral saphenous vein was a feasible alternative to retroorbital sampling, we compared microfilaria counts in blood samples collected by both methods from 21 gerbils infected with the filarial parasitic worm Brugia pahangi. Lateral saphenous vein counts were equivalent to retroorbital counts at relatively high counts (greater than 50 microfilariae per 20 µL) but were significantly lower than retroorbital counts when microfilarial concentrations were lower. Our results indicate that although retroorbital collection may be preferable when low concentrations of microfilariae need to be enumerated, the lateral saphenous vein is a suitable alternative site for blood sampling to determine microfilaremia and is a feasible refinement that can benefit the wellbeing of gerbils.