Ability of an oral formulation of afoxolaner to protect dogs from Borrelia burgdorferi infection transmitted by wild Ixodes scapularis ticks.

A randomized, blinded, negative controlled study was conducted to determine whether treatment with afoxolaner (NexGard®, Merial, Inc.) would prevent the transmission of Borrelia burgdorferi to dogs by wild caught Ixodes scapularis ticks. Twenty healthy dogs were randomly assigned to two groups of ten dogs each. Ten dogs were treated orally on Day 0 at a dose near the minimum recommended dose of afoxolaner of 2.5mg/kg (actual doses 2.5-3.1mg/kg) and ten control dogs were not treated. On Day 28, each dog was infested with approximately 50 adult unfed wild caught I. scapularis that had a 67% B. burgdorferi infection rate (determined by polymerase chain reaction). On Day 33, live ticks were counted and removed. No ticks were found on treated dogs while control dogs had an average of 21.4 ticks. To detect infection, the B. burgdorferi-specific C6 antibody SNAP® 4Dx® test (IDEXX) was performed on serum collected before infestation (all dogs seronegative on Days -6 and 27) and on Days 48, 63, 77 and 92. The ten treated dogs remained seronegative through the end of the study (Day 92), while nine out of the ten control dogs were infected, as demonstrated by their seroconversion to being positive for the presence of the B. burgdorferi-specific C6 antibody starting on Day 48. In this study, all dogs treated with NexGard® 28days prior to challenge with wild caught I. scapularis ticks were protected from B. burgdorferi infection, while nine out of the ten untreated control dogs were infected.

Effects of doxycycline on heartworm embryogenesis, transmission, circulating microfilaria, and adult worms in microfilaremic dogs.

Tetracycline treatment of animals or humans infected with filariae that harbor Wolbachia endosymbionts blocks further embryogenesis, and existing microfilariae gradually die. This treatment also kills developing larvae and has a slow-kill effect on adult filariae, all presumably due to elimination of the Wolbachia. Also, Dirofilaria immitis microfilariae in blood collected from dogs up to 25 days after the last dose of doxycycline developed to infective L3 that were normal in appearance and motility in mosquitoes but did not continue to develop or migrate normally after subcutaneous (SC) injection into dogs. The present study was designed to determine whether heartworm microfilariae collected at later times after treatment would regain the ability to continue normal development in a dog. The study also was expected to yield valuable data on the effects of treatment on microfilariae and antigen levels and adult worms. The study was conducted in 16 dogs as two separate replicates at different times. A total of five dogs (two in Replicate A and three in Replicate B) infected either by SC injection of L3 or intravenous transplantation of adult heartworms were given doxycycline orally at 10mg/kg twice daily for 30 days, with three untreated controls. Microfilarial counts in the five treated dogs gradually declined during the 12-13 months after treatment initiation. Two dogs were amicrofilaremic before necropsy and three had 13 or fewer microfilariae/ml. Only one treated dog was negative for heartworm antigen before necropsy. Overall, treated dogs generally had fewer live adult heartworms than controls, and most of their live worms were moribund. All three control dogs remained positive for microfilariae and antigen and had many live worms. L3 from mosquitoes fed on blood collected 73-77 or 161-164 days after initiation of doxycycline treatments were injected SC into five dogs. None of the dogs injected with L3 from mosquitoes fed on blood from doxycycline-treated dogs were ever positive for microfilariae or antigen, and none had worms at necropsy; three control dogs were positive for microfilariae and antigen and had many live worms. These data indicate that doxycycline treatment of microfilaremic dogs gradually reduces numbers of microfilariae and blocks further transmission of heartworms. This latter effect should be highly effective in reducing the rate of selection of heartworms with genes that confer resistance to macrocyclic lactone preventives and microfilaricides. The data also suggest that doxycycline has a slow-kill effect on adult heartworms.

Experimental Dirofilaria immitis infection in dogs: effects of doxycycline and Advantage Multi® administration on immature adult parasites.

To better understand the efficacy of doxycycline and 10% imidacloprid+2.5% moxidectin (Advantage Multi(®); Bayer Animal Health, Shawnee Mission, Kansas) on immature adult Dirofilaria immitis parasites and the results of antigen tests, 12 healthy, randomly selected dogs were experimentally infected with D. immitis and monitored for 407 days. Two dogs in each of three subgroups of four dogs were each infected with six (total of 6 dogs) or 12 (total of 6 dogs) D. immitis infective third-stage larvae (L3) obtained from infected mosquitoes. Doxycycline (10mg/kg per os twice daily×30 days) and 10% imidacloprid+2.5% moxidectin (1ml/kg by topical application every 30 days) treatment was initiated at 105 (Group A) and 149 (Group B) days post infection (PI) in two groups. One subgroup of two dogs given 6 L3 and one subgroup of two dogs given 12 L3 remained as untreated controls (GroupC). Serum obtained regularly throughout the study was evaluated by ELISA (PetChek(®) Heartworm-PF Antigen Test, IDEXX Laboratories, Inc.) for D. immitis adult circulating antigens. Six of the eight dogs in the treated groups had detectable antigenemia starting between 148 and 240 days post infection, but antigen was not detected in any treated dog at the end of the study. In the control subgroups, the dogs that received 6 L3 had no detectable antigen while the two dogs that received 12 L3 had detectable antigen beginning on Day 180 that persisted until the end of the study. None of the infected dogs had evidence of circulating microfilariae. At necropsy, no heartworms were recovered from the treated dogs, but all dogs in the untreated group had viable adult heartworms. These results indicate that early immature adult worms (3.5 and 5 months of age) of D. immitis were susceptible to a combined treatment regimen of doxycycline and 10% imidacloprid+2.5% moxidectin.

Evaluation of lung pathology in Dirofilaria immitis-experimentally infected dogs treated with doxycycline or a combination of doxycycline and ivermectin before administration of melarsomine dihydrochloride.

Adulticide therapy in heartworm (Dirofilaria immitis)-infected dogs can lead to thromboembolism, which can seriously compromise post-treatment health status. Lung pathology following adulticide therapy was evaluated in three groups of experimentally infected dogs. Group 1 was treated with doxycycline at 20 mg/kg per os once daily for 30 days post infection followed by an intramuscular injection of melarsomine dihydrochloride (2.5 mg/kg) at Week 12, followed 1 month later by two injections 24 h apart. Group 2 was treated as described for Group 1, with the addition of ivermectin at 6 mcg/kg given monthly per os for 24 weeks post-infection. Group 3 received melarsomine alone, as described above. All dogs were necropsied at Week 24 and lung pathology was evaluated. Lesion criteria included perivascular inflammation and endothelial proliferation. Lesions were scored by two independent pathologists who were blinded as to treatment. Results indicate that doxycycline treatment alone or combined with ivermectin had lower lesion scores than lungs from dogs who had received melarsomine alone. Dogs that received the combined doxycycline/ivermectin protocol and treated with adulticide showed less severe arterial lesions and the virtual absence of thrombi.

Effects of doxycycline on early infections of Dirofilaria immitis in dogs.

The antifilarial effects of tetracycline drugs were first demonstrated when they were found to be highly effective against L(3) and L(4) of Brugia pahangi and Litomosoides sigmodontis in rodent models. Tetracyclines are also now known to have activity against microfilariae and adult Dirofilaria immitis, but assessment of their activity against larval and juvenile heartworms has not been reported previously. This study assessed the effects of doxycycline administered orally at 10mg/kg twice daily for 30-day periods at selected times during the early part of the life cycle of D. immitis in dogs with dual infections of D. immitis and B. pahangi. Twenty beagles were randomly allocated by weight to four groups of five dogs each. On Day 0, each dog was given 50 D. immitis L(3) and 200 B. pahangi L(3) by SC injection. Dogs received doxycycline on Days 0-29 (Group 1); Days 40-69 (Group 2); or Days 65-94 (Group 3). Group 4 served as untreated controls. Blood samples were collected for microfilariae counting and antigen testing. Necropsy for collection of adult heartworms and selected tissues were performed Days 218-222. Heartworms recovered were examined by immunohistology, conventional microscopy/transmission electron microscopy, and molecular biology techniques. No live heartworms were recovered from dogs in Group 1; dogs in Group 2 had 0 to 2 live worms (98.4% efficacy), and dogs in Group 3 had 0-36 live worms (69.6% efficacy). All control dogs had live adult heartworms (25-41). The live worms recovered from dogs in Groups 2 and 3 were less developed and smaller that worms from control dogs. Microfilariae were not detected in any dogs in Groups 1 and 2; one dog in Group 3 had 1 microfilariae/ml at necropsy. All control dogs had microfilariae at necropsy. One dog in Group 1 was antigen positive at one sampling (Day 166). One dog in Group 2 was antigen positive Days 196 and 218-222 and three dogs in Group 3 were antigen positive at one or more samplings All five control dogs were antigen positive at all three sampling times. These findings suggest that doxycycline at 10mg/kg orally twice daily for 30 days has efficacy against migrating tissue-phase larvae and juvenile worms and will delay or restrict microfilarial production.

Heartworm and Wolbachia: therapeutic implications.

A safer, more effective adulticidal treatment and a safe method for reducing microfilaremia and breaking transmission of heartworm disease early in the treatment are needed. The present study evaluated efficacy of ivermectin (IVM) and doxycycline (DOXY) alone or together (with or without melarsomine [MEL]) in dogs with induced adult heartworm infection and assessed the ability of microfilariae from DOXY-treated dogs to develop to L3 in Aedes aegypti mosquitoes and subsequently to become reproductive adults in dogs. Thirty beagles were each infected with 16 adult heartworms by intravenous transplantation. Six weeks later, dogs were ranked by microfilarial count and randomly allocated to 6 groups of 5 dogs each. Beginning on Day 0, Group 1 received IVM (6 mcg/kg) weekly for 36 weeks. Group 2 received DOXY (10 mcg/(kgday)) orally Weeks 1-6, 10-11, 16-17, 22-25, and 28-33. Groups 3 and 5 received IVM and DOXY according to doses and schedules used for Groups 1 and 2. At Week 24, Groups 3 and 4 received an intramuscular injection of MEL (2.5 mg/kg), followed 1 month later by two injections 24h apart. Group 6 was not treated. Blood samples were collected for periodic microfilaria counts and antigen (Ag) testing (and later immunologic evaluation and molecular biology procedures). Radiographic and physical examinations, hematology/clinical chemistry testing, and urinalysis were done before infection, before Day 0, and periodically during the treatment period. At 36 weeks, the dogs were euthanized and necropsied for worm recovery, collection of lung, liver, kidney, and spleen samples for examination by immunohistochemistry and conventional histological methods. All dogs treated with IVM + DOXY (with or without MEL) were amicrofilaremic after Week 9. Microfilarial counts gradually decreased in dogs treated with IVM or DOXY, but most had a few microfilariae at necropsy. Microfilarial counts for dogs treated only with MEL were similar to those for controls. Antigen test scores gradually decreased with IVM + DOXY (with or without MEL) and after MEL. Antigen scores for IVM or DOXY alone were similar to controls throughout the study. Reduction of adult worms was 20.3% for IVM, 8.7% for DOXY, 92.8% for IVM + DOXY + MEL, 100% for MEL, and 78.3% for IVM + DOXY. Mosquitoes that fed on blood from DOXY-treated dogs had L3 normal in appearance but were not infective for dogs. Preliminary observations suggest that administration of DOXY+IVM for several months prior to (or without) MEL will eliminate adult HW with less potential for severe thromboembolism than MEL alone.