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.

Early migration and development of Dirofilaria immitis in the ferret, Mustela putorius furo.

Twenty-four female ferrets, approximately 4 mo of age, were subcutaneously inoculated with 60 infective larvae of Dirofilaria immitis to determine migration and development of the worms until they became young adults in the heart and associated vessels. Twelve groups of 2 ferrets each were examined at 3, 5, 11, 15, 21, 49, 56, 63, 70, 91, 119, and 140 days postinoculation, respectively, to recover worms. Total worm recovery from each ferret varied from 1.6 to 79.3%. Worms were found mostly in subcutaneous tissue and muscle from the beginning of infection up to and including day 91. Worms were first recovered from the heart at day 70, when 3.8% of the worms had reached this site. By day 119, essentially all of the worms had migrated to the heart, as indicated by similar overall worm recoveries at 119 (mean 59.0%) and 140 days (mean 65.8%). Some worms remained in the tissues even at day 140. The third and fourth molts occurred as early as days 3 and 56, respectively. On day 91, i.e., soon after the worms reached the heart, the mean lengths of males and females were 58.8 mm (range 43.0-75.0 mm) and 63.3 mm (range 55.0-69.0 mm), respectively. At 140 days, male and female worms from the heart measured 118 mm (range 62.0-146.0 mm) and 144 mm (range 105.0-168.0 mm), respectively.