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.     

Wolbachia and its influence on the pathology and immunology of Dirofilaria immitis infection

Since the definitive identification in 1995 of the bacterial endosymbiont Wolbachia that resides in different tissues of the filarial worm Dirofilaria immitis, there has been increasing interest to understand whether and what role it plays in the pathogenesis of and immune response to heartworm infection. The present study evaluated the effects of treatments on lung pathology in 20 beagle dogs experimentally infected with D. immitis. Dogs in Group 1 were treated with doxycycline (10 mg/kg/day) orally from weeks 0-6, 10-12, 16-18, 22-26, and 28-34. Dogs in Group 2 served as infected, non-treated controls. Dogs in Group 3 were given doxycycline as described for Group 1 combined with weekly oral doses of ivermectin (6 mcg/kg) for 34 weeks and intramuscular (IM) melarsomine (2.5 mg/kg) at week 24, followed by two additional melarsomine injections 24h apart 1 month later. Group 4 received only melarsomine as described for Group 3. Lung lesion criteria, scored by two independent blinded pathologists, included perivascular inflammation and endothelial proliferation. Doxycycline treatment alone had no effect on lesion scores, whereas the combination of doxycycline and ivermectin resulted in less severe perivascular inflammation. All lungs were evaluated for positive immunostaining for the Wolbachia surface protein (WSP). Control dogs showed numerous thrombi, intense perivascular and interstitial inflammation and, occasionally, positive staining for WSP. Interestingly, dogs receiving doxycycline/ivermectin/melarsomine showed significantly less severe arterial lesions and the virtual absence of thrombi.     

Activity of an injectable, sustained-release formulation of moxidectin administered prophylactically to mixed-breed dogs to prevent infection with Dirofilaria immitis.

OBJECTIVE: To test the ability of a single injection of a sustained-release formulation of moxidectin (moxidectin SR) to protect dogs against heartworm infection for 180 days after inoculation with infective third-stage larvae (L3) of Dirofilaria immitis. ANIMALS: 32 adult mixed-breed dogs. PROCEDURE: Dogs were allocated to 4 groups on the basis of weight and sex. Dogs were injected SC with saline (0.9% NaCl) solution or moxidectin SR at the rate of 0.06, 0.17, or 0.5 mg/kg of body weight (day 0). Each dog was inoculated SC with 50 D immitis L3 180 days later. On days 330 and 331, dogs were euthanatized. The heart, lungs, and thoracic cavity were examined, and number and sex of heartworms were determined. RESULTS: A mean of 35.9 heartworms was recovered from untreated control dogs. Fourteen worms were recovered from 1 of 8 dogs given moxidectin SR at the lowest dosage, and none of the dogs in the 2 highest moxidectin treatment groups were infected. Small barely palpable granulomas were detected at injection sites of moxidectin-treated dogs. Frequency and size of granulomas were positively correlated with dose of moxidectin administered. CONCLUSIONS AND CLINICAL RELEVANCE: A single dose of moxidectin SR at a dosage as low as 0.17 mg/kg can safely and reliably confer complete protection against infection after challenge-exposure with D. immitis L3, and protection lasts for at least 180 days. This mode of prophylactic treatment against infection with heartworms effectively eliminates failure of prophylaxis that results from erratic administration of medications designed for monthly administration.     

Prevalence of Dirofilaria immitis in a stray dog population in western New York.

In a survey in 1973 undertaken to determine the prevalence of Dirofilaria immitis in 100 dogs from a public pound in western New York, 2 dogs were found to be positive. Microfilariae of Dipetalonema sp were not present. Hearts and pulmonary blood vessels were dissected for adult worms, and blood samples were examined for microfilariae, using a modified Knott technique. Although adult heartworms and microfilariae have been recovered from wild foxes, coyotes, and wolves in New York, this is the first report of heartworm in domesticated dogs in the state.     

Attributes, knowledge, beliefs, and behaviors relating to prevention of heartworm in dogs among members of a national hunting dog club

The Food and Drug Administration Center for Veterinary Medicine (FDA/CVM) has cited an increasing number of reports of failure of heartworm prophylaxis in dogs. Failures may be due to resistance of L(3)-L(4) stage Dirofilaria immitis to the macrocyclic lactone class of compounds used for prophylaxis, lack of compliance with and understanding of administration of prophylactics, individual differences in drug absorption or metabolism, or a combination of these factors. Using the latest scientific information, the American Heartworm Society (AHS) and Companion Animal Parasite Council (CAPC) have developed guidelines for the diagnosis, prevention, and treatment of heartworm infection in dogs. This study summarizes the knowledge, beliefs, and behaviors relating to prevention of heartworm among members of a national hunting dog club, visitors to the club website, and attendees at club-sponsored events. These factors can have a direct effect on the success of heartworm prophylaxis. Results suggest that the dog owners lack confidence in the accuracy of the heartworm test to identify infection, the efficacy of products sold for prevention of heartworm, and the effectiveness of treatment to eliminate adult heartworms from infected dogs. Knowledge about when to begin heartworm preventive medication in a new puppy and the timing of heartworm tests was also lacking among a substantial number of respondents. In order to increase acceptance of prophylaxis and reduce the likelihood of a false conclusion of prophylactic failure, education of dog owners should focus on the need for an appropriately timed annual heartworm test and the importance of administering the last dose of monthly heartworm preventative after the last possible day of potential transmission.     

Comparative efficacy of four commercially available heartworm preventive products against the MP3 laboratory strain of Dirofilaria immitis

A controlled laboratory study was conducted to evaluate the efficacy of four commercial products administered as a single treatment for the prevention of heartworm disease caused by Dirofilaria immitis in dogs. Forty-four commercially sourced Beagle dogs, 6-7 months of age, were received at the test site (Auburn University, Department of Pathobiology) on Study Day (SD) -72 to begin acclimation. On SD -30, each dog was inoculated subcutaneously with 100 infective, third-stage D. immitis larvae (MP3 strain, TRS Laboratories, Inc., Athens, GA). On SD -1, 40 dogs weighing 18.2-25.3 lbs were ranked by decreasing body weight and randomized to five groups of eight dogs each. On SD 0, the dogs assigned to Group 1 were treated orally with ivermectin/pyrantel pamoate chewable tablets, Group 2 dogs were treated orally with milbemycin oxime flavored tablets, Group 3 dogs were treated with selamectin topical solution, and Group 4 dogs were treated with imidacloprid/moxidectin topical solution. Group 5 dogs remained nontreated. Dosages for dogs in Groups 1-4 were based on the individual body weight of each dog and current labeled dose banding for each commercial product. All dogs were fasted overnight prior to treatment. Food was returned four hours after treatment. Animals were observed for abnormal clinical signs involving eyes, feces, respiration, behavioral attitude, locomotion/musculature, or skin conditions at prescribed intervals immediately after treatment and at twice daily intervals thereafter. On SD 90, whole blood was collected and tested for adult heartworm antigen. On SDs 119/120, the dogs were euthanized and subjected to necropsy examination for recovery of adult D. immitis and/or worm fragments. At necropsy, all 8 dogs in the nontreated group were infected with adult D. immitis (34-70 worms/dog, geometric mean (GM)=51.6 worms/dog). One or more adult D. immitis and/or worm fragments were recovered from 7 of 8 of the dogs each in Groups 1-3 (87.5% were heartworm positive). The respective GM worm burdens/dog for Groups 1-3 was 2.3, 2.4, and 2.3 which resulted in 95.6, 95.4 and 95.5% efficacy, respectively. No worms were recovered from any of the 8 dogs in Group 4 resulting in 100% efficacy.     

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.     

Efficacy of an injectable, sustained-release formulation of moxidectin in preventing experimental heartworm infection in mongrel dogs challenged 12 months after administration

The objective of this study was to ascertain the ability of a single subcutaneous injection of a sustained-release (SR) formulation of moxidectin to protect dogs against challenge inoculation with infective Dirofilaria immitis larvae 364 days after administration. Twenty four purpose-bred adult mixed-breed dogs were grouped into three blocks of eight based on weight and sex. Saline solution (0.9% NaCl) or a moxidectin SR formulation at volumes designed to deliver 0.17 or 0.27 mg moxidectin/kg b.w. was injected subcutaneously on day 0. Throughout the post-treatment period, injection sites of all dogs were periodically examined visually and by palpation. Palpable swellings were characterized as to size, consistency and the presence of associated pain or erythema. On day 364, each dog was inoculated subcutaneously with 50 D. immitis L3. On days 510 and 511, dogs were euthanatized, and their hearts, lungs and thoracic cavities were inspected for the presence of adult heartworms. number, sex and viability of recovered heartworms were determined. The mean number of heartworms recovered from dogs that had received the saline control injection was 35.7. No heartworms were recovered from any dog treated with either 0.17 or 0.27 mg moxidectin/kg b.w. For variable periods of time following treatment, small (1-4 mm diameter), firm, subcutaneous swellings could be palpated at the injection sites of dogs treated with 0.17 or 0.27 mg moxidectin/kg b.w. These swellings contracted progressively and eventually disappeared except for the case of one animal treated with 0.27 mg/kg, in which the swelling persisted for the entire study period. At no time during the study was pain or erythema noted at the injection site of any dog, and no dog exhibited any adverse systemic reaction related to treatment. We conclude that under conditions pertaining in this study, a single subcutaneous injection of a moxidectin SR formulation at dosing rates of either 0.17 or 0.27 mg/kg b.w. can safely protect adult dogs against experimental challenge inoculation with infective heartworm larvae for a period of 12 months.     

Evaluation of a single injection of a sustained-release formulation of moxidectin for prevention of experimental heartworm infection after 12 months in dogs

OBJECTIVE: To evaluate the efficacy of a single injection of a sustained-release formulation of moxidectin in preventing heartworm (Dirofilaria immitis) infection for 12 months in dogs. ANIMALS: 14 healthy dogs. PROCEDURE: Group A (nontreated control dogs; n = 6) received sterile vehicle administered SC, and group B (treated dogs; n = 6) received a sustained-release formulation of moxidectin administered SC. All dogs were housed in a heartworm-endemic area for 11.5 months, and heartworm antigen and modified Knott tests were performed monthly. All dogs (including 2 additional control dogs [group C]) were then inoculated with infective-stage larvae (L3) of D. immitis, and 4.5 months later, all dogs were euthanatized and post-mortem examinations were performed. Adult D. immitis were counted and measured, and their age was estimated. RESULTS: All dogs in groups A and C were infected with young (4- to 4.5-month old) adult male and female D. immitis. No dogs in group B were infected with heartworms. CONCLUSIONS AND. CLINICAL RELEVANCE: The age of heartworms recovered suggests that infection was the result of experimental inoculation and not natural exposure to mosquitoes during the 11.5-month period the dogs resided in a heartworm-endemic area. A single SC injection of a sustained-release formulation of moxidectin was effective in providing protection against heartworm infection after 12 months in dogs. This formulation is a convenient method of heartworm prophylaxis that could eliminate the problem of poor owner compliance.     

Prevalence and epidemiological aspects of Dirofilaria immitis in dogs from Kayseri Province, Turkey

This study was conducted to determine the prevalence of Dirofilaria immitis infection and to investigate the risk factors related to heartworm disease in dogs from Kayseri, Turkey. Blood samples were collected from 280 dogs from May 2005 to March 2006 and were examined by membrane filtration-acid phosphatase histochemical staining and antigen Elisa techniques to detect circulating microfilariae and antigens of D. immitis, respectively. Of the total of 280 dogs, 27 were positive for D. immitis with a prevalence value of 9.6%. In addition 29.6% of positive dogs determined to have occult D. immitis infections. D. immitis was the only canine filarial parasite present in the study area. The mean number of microfilariae in infected dogs was 4730+/-5479 per ml of blood. The highest heartworm prevalence were observed in 7-10 age group (28.6%) followed by 4-6 (17.1%) and 0.5-3 (4.8%) age groups. The differences between 0.5-3 and other age groups were found significant, whereas no statistically significant difference was observed between 4-6 and 7-10 age groups. The infection was more prevalent in males, larger breeds and the dogs not on prophylaxis. No statistically significant difference was observed between stray and owned dogs. Our results suggest that heartworm treatment and prophylaxis should be considered in Kayseri Province.     

The occurrence of Dirofilaria immitis in dogs in South Australia

Heart, lung and samples of blood from 230 dogs were examined for infections of filarial parasites. Dirofilaria immitis worms and microfilariae were detected in one dog. Blood samples from a further 1428 dogs were examined for microfilariae and 22 were found to be infected. Eighteen dogs were infected with D immitis microfilariae and four with Dipetolonema reconditum microfilariae. The histories were available for 18 of the dogs infected with heartworm and only seven dogs had not travelled outside South Australia. It was concluded that heartworm infection was endemic in South Australia but the apparent prevalence was only about 1%.     

Dirofilaria immitis infection of a snow leopard (Uncia uncia) in a Japanese zoo with mitochondrial DNA analysis

Three dog heartworms (Dirofilaria immitis) were detected in the lumen of the right cardiac ventriculus and of the pulmonary artery of a captive female snow leopard (Uncia uncia) that died of pancreatic carcinoma at a zoo in Japan. Neither clinical respiratory nor circulatory symptoms caused by the heartworm infection were observed. The filarial worms were identified as D. immitis from the morphologic characteristics of the esophagus, the presence of faint longitudinal ridges on the cuticular surface, the situation of vulva posterior to the esophagus, and the measurements of the body. The heartworms from the snow leopard were identical to that of D. immitis from dogs in the sequence of the cytochrome oxidase I region in the mitochondrial DNA. This host record is the first of D. immitis in U. uncia.     

Prevalence of the Dirofilaria immitis infection in dogs from Merida, Yucatan, Mexico

The aim of the present study was to estimate the prevalence of Dirofilaria immitis infection, and to examine the relationship between host factors (gender, age and breed) and D. immitis infection in dogs. The study was designed as a cross-sectional study. A total of 676 dogs were examined for D. immitis infection. Adult worms (necropsy) and blood samples were collected from all animals for diagnosis of D. immitis. Blood samples were examined using a modified Knott's and thick drop techniques. Fifty-six of 676 dogs were diagnosed infected with D. immitis. The odds of infection were 2.85 times higher in female dogs, compared to male dogs (OR = 2.85, 95% CI = 1.5-5.5, P = 0.01). In addition, the odds of infection were 2.11 times higher in > or =3 years old dogs, compared with younger dogs (OR = 2.11, 95% CI = 1.1-4.2, P = 0.03). This information is important for a better understanding of the epidemiology of D. immitis in dogs in Yucatan, Mexico.     

Can heartworm prevalence in dogs be used as provisional data for assessing the prevalence of the infection in cats?

Cats are considered a susceptible host for Dirofilaria immitis; however, increased host resistance is reflected by relatively low adult worm burdens in natural and experimental infections; the prolonged prepatent period (8 months); the low level and short duration of microfilaremia; and the short life span of adult worms (2-3 years). From April to September 2006, 212 cats and 608 dogs, all exposed for at least one transmission season, were screened for D. immitis infection in a multi-center study in the Po River Valley in northern Italy. Cats were initially evaluated by antibody testing; positive subjects were followed up by antigen testing and echocardiography (and necropsy if death occurred). The prevalence in dogs was 29% by a modified Knott test and antigen testing compared with a prevalence of 4.7% in cats by an antibody test; six of these infections (2.8%) were confirmed by the follow-up evaluations. This field study demonstrated that the prevalence of heartworm infection in cats in this area is within the expected limits of 9-18% of the prevalence in dogs. Antibody testing likely underestimates the real prevalence of D. immitis infection in cats. These results also emphasize the importance of preventive treatment in cats.     

Histopathological comparison of pulmonary artery lesions between raccoon dogs (Nyctereutes procyonoides) and domestic dogs experimentally infected with Dirofilaria immitis

Five raccoon dogs (Nyctereutes procyonoides) and two domestic dogs (Canis familiaris) were subcutaneously infected with 100 infective larvae (L3) of Dirofilaria immitis. Two and five worms, respectively, were collected from two of three raccoon dogs. Villous endarteritis was found in the raccoon dog with five worms and two dogs at 116 days after infection. The number of recovered worms in the raccoon dogs was significantly smaller than that of the domestic dogs having 22 and 29 worms, while histopathological features and the severity of the lesions in the raccoon dogs were similar to those in the domestic dogs. The vascular lesions in two chronically-infected raccoon dogs turned into much severe at 565 and 590 days after inoculation.     

Immunological analysis of agglutination in Dirofilaria immitis microfllariae

The mechanism of agglutination phenomenon of Dirofilaria immitis microfilariae was analyzed. Circulating microfilariae were collected from a D. immitis-infected microfilaremic dog and cultured in the several kinds of sera from dogs and animals. The agglutination of D. immitis microfilariae is a specific phenomenon due to some immune complexes formed with the anti-microfilarial antibody, heat-instable factor(s) and excretory-secretory products of microfilariae. Only live microfilariae were agglutinated and the agglutinated microfilariae remained alive as long as 27 days in culture in vitro.     

Limited efficacy of levamisole against adults of Dirofilaria immitis in a dog

A dog with chronic dirofilariasis (Dirofilaria immitis) was given 15 doses of 12 or 24 mg of levamisole/kg of body weight in 6 treatments within 96 days. Concentration of microfilariae in the blood was determined before and after treatment, and Aedes triseriatus mosquitoes were fed on the dog 1 to 5 days after each treatment and examined for D immitis larvae. Several adult worms were recovered from the dog 160 days after the end of the treatments.     

Nine-month evaluation of dogs after open-heart surgical removal of heartworms

Adult heartworms were surgically removed from 4 infected dogs by use of intracardiac techniques during cardiopulmonary bypass. The number of worms removed ranged from 12 to 14 per dog. Observation for 9 months after surgery gave no clinical evidence of active adult heartworm infection, in spite of the consistent finding of circulating microfilariae during the follow-up period. There were no significant early or late postoperative complications. Results of postoperative hematologic and biochemical studies were unremarkable. At necropsy (following euthanasia, 9 months after surgery) the heart and pulmonary arteries of all dogs were free of adult heartworms; pathologic changes attributable to residual infection were not found.     

Contribution of live heartworms harboring in pulmonary arteries to pulmonary hypertension in dogs with dirofilariasis

To investigate whether adult heartworms harboring in the pulmonary arteries contribute to pulmonary hypertension, we determined the cardio-pulmonary values immediately before and after removal of heartworms from the pulmonary arteries and before and after insertion of live worms in their place. In 10 heartworm-infected dogs, 8 to 46 worms were removed. The mean pulmonary arterial pressure fell significantly from 24.5 +/- 7.9 mmHg to 16.3 +/- 4.9 mmHg (p less than 0.01) immediately after removal. The right cardiac output decreased in 7 of the 10 cases. The total pulmonary resistance and right ventricular stroke work index also decreased. At 24 hours after removal, live heartworms were put back into the pulmonary arteries of their host dog. The mean pulmonary arterial pressure elevated significantly (p less than 0.01) immediately after insertion. The right cardiac output further decreased in 7 of the 10 dogs, and the total pulmonary resistance and right ventricular stroke work index increased. Separate from this, 12 to 42 heartworms were transplanted into the pulmonary arteries of 5 heartworm-free dogs. Immediately after transplantation, the pulmonary arterial pressure did not show any significant change. However, the stroke volume decreased, and the total pulmonary resistance increased. These facts suggest a contribution of live heartworms to the pulmonary hypertension, although there is a complicated interaction among the presence of heartworms, the pulmonary lesions and the pulmonary hypertension.     

Influence of immune treatments against Dirofilaria immitis infection in dogs

Mongrel dogs were inoculated with two kinds of antigenic substances. The first was a phosphate buffered saline extract of whole Dirofilaria immitis mixed with aluminum hydroxide gel (group 1), and the second was an orally administered live Metastrongylus apri infective larvae (L3) (group 2). Both groups were then infected with D. immitis L3. Indirect hemagglutination (IHA) tests showed that the antibody was produced by these inoculations before the infection was introduced, even in dogs inoculated with M. apri. This suggests a cross-reactivity between D. immitis and M. apri. The initial passive cutaneous anaphylaxis (PCA) antibody production was markedly delayed by about 70 days in group 2 compared with the production in the infected control dogs (group 3). The appearance of microfilaremia was also delayed by about one month in group 2 compared with that in the above control group. All dogs were sacrificed after the termination of the observation and worms recovered from the right ventricle and pulmonary arteries were counted and measured. The results indicated that immunization resulting from the homologous worm-somatic antigen might accelerate the growth of the infected larvae, whereas immunization resulting from the heterologous worm antigen, but cross-reactive to D. immitis, might disadvantageously affect the growth.