Heartworm Infection Research Articles

Detection of heartworm infection in dogs via PCR amplification and electrospray ionization mass spectrometry of nucleic acid extracts from whole blood samples

Detection of heartworm infection in dogs via PCR amplification and electrospray ionization mass spectrometry of nucleic acid extracts from whole blood samples

Detection of heartworm infection in dogs via PCR amplification and electrospray ionization mass spectrometry of nucleic acid extracts from whole blood samples

To develop and evaluate a rapid and accurate assay involving PCR amplification and electrospray ionization mass spectrometry of nucleic acid extracts from whole blood samples for the detection of Dirofilaria immitis infection in dogs. Whole blood nucleic acid extracts from 29 dogs experimentally infected with D immitis (and in which circulating D immitis antigen was detected) and 10 uninfected dogs. 16 of the 29 whole blood samples from infected dogs were examined at the time of collection for circulating microfilaria. Nucleic acids were extracted from all whole blood specimens and underwent PCR amplification with 12 PCR primer pairs designed to detect a wide range of pathogens (including the Wolbachia endosymbiont of D immitis) and electrospray ionization mass spectrometry. On the basis of assay results, heartworm infection was detected in 13 of 13 antigen-positive dogs of unknown microfilaria status, 11 of 11 antigen-positive dogs with circulating microfilaria, 0 of 3 antigen-positive dogs tested at 3 months after larval infection, 0 of 2 antigen-positive dogs with occult infections, and 0 of 10 uninfected dogs. With the assay under investigation, it was possible to identify D immitis infection in dogs with circulating microfilaria via detection of the obligate Wolbachia endosymbiont of D immitis. It was not possible to identify dogs with occult infections, which suggested that circulating microfilaria must be present to detect infection with this assay, although further studies would be required to verify that finding.

Macrocyclic Lactones in the Treatment and Control of Parasitism in Small Companion Animals

Macrocyclic lactones (MLs) have many anti-parasitic applications in small companion animal medicine. They were first developed as chemoprophylactics against heartworm (Dirofilaria immitis) infection to be applied monthly for retroactive killing of third- and fourth-stage larvae. ML-containing products formulated for oral (ivermectin, milbemycin oxime), topical (selamectin, moxidectin) or injectable sustained release (moxidectin, ivermectin) are approved for heartworm prevention in dogs or cats. Clearance of microfilariae and gradual or "soft" killing of adult heartworms constitute increasingly prevalent extra-label uses of MLs against D. immitis. Some commercial ML formulations contain sufficient levels of active ingredient (milbemycin oxime, selamectin, moxidectin) to support additional label claims against gastrointestinal nematode parasites such as hookworms (Ancylostoma spp.) and ascarid round worms (Toxocara spp. and Toxascaris leonina). Beyond these approved applications, safe, extra-label uses of MLs against nematodes parasitizing the urinary tract, such as Capillaria spp., and parasites of the tissues, such as Dipetalonema reconditum, Dirofilaria repens, Thelazia spp. and Spirocerca lupi, in dogs and cats as well as exotic pets have been reported. MLs as a group have intrinsic insecticidal and acaricidal activity, and topical or otic formulations of certain compounds (selamectin, moxidectin, milbemycin oxime or ivermectin) are approved for treatment and control of fleas, certain ixodid ticks, sarcoptiform and demodectic mange mites and psoroptiform ear mites. Extra-label applications of MLs against ectoparasites include notoedric mange mites, dermanyssids such as Ornythonussus bacoti, numerous species of fur mite (e.g. Cheyletiella spp. and Lynxacarus) and trombiculids ("chiggers") in cats, dogs and nontraditional or exotic pets.

Isometric responses of isolated intrapulmonary bronchioles from cats with and without adult heartworm infection

To determine the isometric responses of isolated intrapulmonary bronchioles from cats with and without adult heartworm infection. 13 purpose-bred adult cats. Cats were infected with 100 third-stage larvae or received a sham inoculation, and the left caudal lung lobe was collected 278 to 299 days after infection. Isometric responses of intrapulmonary bronchiolar rings were studied by use of a wire myograph. Three cycles of contractions induced by administration of 10 μM acetylcholine were followed by administration of the contractile agonists acetylcholine, histamine, and 5-hydroxy-tryptamine. To evaluate relaxation, intrapulmonary bronchiolar rings were constricted by administration of 10 μM 5-hydroxytryptamine, and concentration-response curves were generated from administration of sodium nitroprusside, isoproterenol, and substance P. Compared with tissues from control cats, contractile responses to acetylcholine and 5-hydroxytryptamine were reduced in tissues from heartworm-infected cats. Relaxation to isoproterenol was significantly reduced in tissues from heartworm-infected cats. Relaxation to substance P was increased in tissues from heartworm-infected cats, but relaxation to sodium nitroprusside was unchanged. Results suggested that despite increased bronchiolar wall thickness in heartworm-infected cats, a hyperreactive response of the bronchiolar smooth muscle is not the primary mechanism of respiratory tract clinical signs. Reduced response of the airway to isoproterenol may indicate refractoriness to bronchiolar relaxation in heartworm-infected cats.

Spontaneous pneumothorax in 35 cats (2001-2010).

Thirty-five cases of spontaneous pneumothorax were reviewed. In contrast to dogs, cats with an established etiology all had spontaneous pneumothorax associated with lung disease. Underlying diseases identified in affected cats included inflammatory airway disease, neoplasia, heartworm infection, pulmonary abscess and lungworm infection. Many cats were managed successfully with observation alone or needle thoracocentesis and specific therapy for their primary lung disease. Cats who present with spontaneous pneumothorax may be treated successfully with non-surgical therapies and appear to have a better prognosis than previously extrapolated from canine studies.

Treatment and prevention of natural heartworm (Dirofilaria immitis) infections in red pandas (Ailurus fulgens) with selamectin and ivermectin

Ten of the 48 red pandas in the Chengdu Research Base of Giant Panda Breeding, Sichuan province, China, died in 2006 after prolonged periods of depression, weight loss, and mucocutaneous membrane xanthochromia. During postmortem examination, live heartworms were found in the right cardiac ventricles and pulmonary arteries of all 10 animals. Selamectin and ivermectin were used for clinical prophylaxis in the remaining red pandas between December 2006 and November 2010. We observed a gradual decrease in morbidity and mortality during this period. As a consequence of our prophylaxis program, dirofilariosis did not occur in the remaining red pandas at Chengdu Research Base during 2010.

Serological screening of canine heartworm (Dirofilaria immitis) infections in Turkey.

This study was conducted to determine the seroprevalence of canine dirofilariosis in ten provinces of Turkey using antigen ELISA. A total of 317 blood samples were collected from 199 male and 118 female dogs between 2010 and 2011. 27 (8.5%) of 317 dog samples were positive for Dirofilaria immitis antigen by ELISA. No differences in seroprevalence were observed by sex (male: 9%; female: 8%; p > 0.05). Seroprevalence rates were the highest in Afyonkarahisar province (18%), followed by Eskişehir (15%), Nevşehir (14%), Aksaray (12%), Kars (10%), Malatya (9%), Niğde (4%), Mersin (3%) and Konya (2%) provinces. No positive results were obtained in Ankara province. The differences in seroprevalence of Afyonkarahisar, Eskişehir, Ankara, Konya and Mersin was statistically significant (p < 0.05). The seroprevalence of D. immitis infection varied with age. The highest seroprevalence was observed in dogs older than five years of age (the risk of infection increases with age).

Evidence for Macrocyclic Lactone Anthelmintic Resistance in Dirofilaria immitis

Reports of loss-of-efficacy (LOE) events in dogs infected with Dirofilaria immitis despite adherence to accepted prophylaxis regimens with a macrocyclic lactone anthelmintic are attracting considerable attention. It is crucially important to distinguish among several possible causes for these LOE reports, one of which is the evolution of resistance to these drugs in heartworms. We review here recent evidence at the molecular level that supports the hypothesis that parasites derived from LOE cases have experienced a strong selection event and that these populations are characterized by very high frequencies of single-nucleotide polymorphisms in a D. immitis gene encoding a P-glycoprotein transporter, comprised of homozygous guanosine residues at 2 locations ("GG-GG" genotype). Furthermore, an infected dog adopted to Canada from the southern United States harbored a microfilarial population that was insensitive to very high doses of macrocyclic lactones and was characterized by a high frequency of the GG-GG genotype associated with LOE cases. We propose that this case be defined as a drug-resistant heartworm infection and suggest that a simple assay for the existence of resistant parasites is a 7-day microfilariae suppression test, which can be performed in a veterinary clinic as part of an effort to document the geographic distribution of this phenotype.

Anti-Wolbachia Surface Protein Antibodies Are Present in the Urine of Dogs Naturally Infected withDirofilaria immitiswith Circulating Microfilariae But Not in Dogs with Occult Infections

Heartworm infection (Dirofilaria immitis) can cause kidney damage due to the presence of circulating microfilariae (mf) that contribute to the production and deposit of immune complexes. It has been shown that mf are a major source of Wolbachia antigen during active infection. Here the authors compared urine samples from 19 naturally infected dogs with (mf+) and 12 without (mf-) microfilariae for the presence of proteinuria and anti-Wolbachia Surface Protein (-WSP) IgG in ELISA. Kidneys from 6 mf+ and 3 mf- dogs were also examined by anti-WSP immuno-histochemistry. All infected dogs showed proteinuria, but mf+ dogs had significantly higher values compared to mf-dogs. Mf+ dogs had optical density values for anti-WSP IgG consistently higher than established cut-off values and were significantly higher than values for mf- dogs. Kidneys from mf+ dogs showed Wolbachia+ mf in glomerular capillaries. Results strongly suggest that Wolbachia associated with circulating mf may contribute to immune-mediated kidney disease in dogs with heartworm infection.

Dirofilaria immitis infection in a Dachshund dog: Diagnosis and Treatment

Dirofilaria immitis is a filariid worm which typically lives as adult in the right ventricle of the heart and the pulmonary arteries of the dog, causing the canine heartworm disease.Therapy of canine dirofilariosis due to Dirofilaria immitis is indicated for dogs suffering from clinical signs of the disease (such as chronic cough). The present paper describes diagnostic and treatment features of a D. immitis affected dog in Ahvaz district, Southwest of Iran. The dog had coughing, tachypnea, labored breathing, panting, hind limb edema,weakness, and exercise intolerance, at the time of referral. Auscultation revealed grade III/VI systolic murmur over the left apex of the heart. Giemsa stained blood smears containing microfilaria was morphologically identified as D. immitis. CBC revealed an inflammatory leukogram and mild anemia. Combined therapy with ivermectin (440 μg/kg as single dose),levamisol (10 mg/kg q 24 h for 10 days) and aspirin (10 mg/kg q 12 h for 10 days), during 2 stages, was effective. The number of microfilaria dropped from 1250±50/ml blood pretreatment to 150±10/ml (following the first treatment stage, after 10 days) and reached 0 a day after the second treatment. The animal remained negative regarding D. immitis microfilaremia during a follow-up period of 180 days. This record confirms the efficacy of ivermectin with levamisol and aspirin in the treatment of adult heartworm infection in a dog.

Anaphylactoid reaction in a heartworm-infected dog undergoing lung lobectomy

A 7-year-old 23-kg (50.6-lb) spayed female Border Collie with a history of heartworm disease was evaluated for respiratory distress. Computed tomography of the thorax revealed possible pulmonary bullae or blebs, and on the basis of these findings, a tentative diagnosis of bullous emphysema was made. Exploratory median sternotomy revealed gross pathological lesions in the right caudal lung lobe; the most peripheral portion appeared discolored (pale pink), and a clear line of demarcation was observed. Upon excision of the affected lung lobe, a worm segment was noticed both on the remaining stump of the lung lobe in the dog and in the removed lobe. At this time, the dog had an anaphylactoid reaction. The reaction was successfully treated with fluid therapy, antihistamines, and corticosteroids. Lung lobectomy of the right caudal lobe resulted in clinical resolution of the bilateral pneumothorax. However, during the postoperative period, the dog developed a hemothorax and was returned to surgery. As no obvious cause for the hemothorax was seen at the second surgery, the dog was treated for a potential coagulopathy with fresh frozen plasma, which provided clinical resolution of the hemothorax. Parasitological examination of the worm segment confirmed a Dirofilaria immitis infection. The dog was discharged 5 days after surgery and treated with doxycycline for 30 days and monthly administration of heartworm preventative was prescribed. Heartworm infection should be a differential diagnosis in dogs with spontaneous pneumothorax. Anaphylactoid reactions associated with accidental dissection of adult heartworms should be managed with treatment aimed at cardiovascular stabilization.

Survey of Heartworm Prevention Practices Among Members of a National Hunting Dog Club

Surveillance data indicate that failures have been reported for virtually all heartworm prevention product categories. Resistance of third and fourth stage larvae of Dirofilaria immitis to macrocyclic lactones, lack of compliance, other unknown factors, or a combination of these reasons may be the cause of failure. A survey of members of a national hunting dog club was conducted to identify practices used to prevent canine heartworm infections. Questionnaires were completed by 708 dog owners. Year-round administration of heartworm preventive medication was reported by 208 (88%) respondents residing north of the 37th parallel. Dosing was based on the estimated weight of the dog by 54 (7%) respondents, 389 (55%) did not record the date prophylaxis was administered, and 89 (13%) observed the dogs spit out pills. Heartworm testing at least once per year was done by 556 (79%) respondents and test dates were spread throughout the year. Only 448 (64%) respondents tested newly acquired dogs for heartworm. These findings suggest that veterinarians should place a greater emphasis on the frequency and timing of heartworm diagnostic tests, the importance of weighing dogs, duration of administration, recording the date monthly heartworm prophylaxis is given, and observing dogs to ensure that oral medication is retained.

Assessment of the effectiveness of a combination product of spinosad and milbemycin oxime on the prophylaxis of canine heartworm infection

Three separate randomized, blinded, vehicle-controlled studies were conducted to determine the effectiveness of a single treatment and consecutive monthly treatments of a combination flavored tablet product containing spinosad and milbemycin oxime (MBO) in the prevention of the establishment of heartworm infections in dogs challenged with recent field isolates of the heartworm (HW), Dirofilaria immitis. For each study, dogs were allocated randomly based on pre-treatment body weights to treated or control groups of 10 animals each. Dogs were infected once with infective HW larvae, on Day-30, using either a Michigan isolate or a Georgia (MP3) isolate of D. immitis. Treatments of beef-flavored chewable tablets were administered in two studies one time either on Day 0 or Day 15, and in one study twice (Days 0 and 30, or Days 15 and 45) or 3 times (Days 0, 30 and 60). For the combination product groups, dosages were in the range of 30–45mg/kg (13.6–20.5mg/lb) for spinosad and 0.5–0.75mg/kg (0.2–0.34mg/lb) for MBO. Necropsies for heartworm counts were completed following euthanasia on Day 120 or Day 123.A single treatment with the combination product of spinosad and MBO 30 or 45 days post-inoculation with infective HW larvae was completely effective (100%) in preventing establishment of the Michigan D. immitis isolate, but efficacy against the Georgia MP3 isolate was incomplete, with geometric mean reductions in HW counts relative to vehicle treated controls of 99% reduction of the 30 day infection and a 98.9% reduction of the 45 day old infection. Against this same MP3 isolate, 3 consecutive monthly treatments provided complete prevention (100%) against establishment of D. immitis infections.The combination product of spinosad and MBO provides effective control of canine heartworms. A single treatment at 30 days post infection showed high but incomplete effectiveness against a heartworm isolate that had been shown to be partially refractory to treatment with marketed monthly heartworm preventives. Three consecutive monthly treatments provided complete control, providing support to the recommendation that heartworm prophylaxis should be maintained year round for optimal effectiveness.

Diagnostic, treatment, and prevention protocols for feline heartworm infection in animal sheltering agencies

Cats are at risk for heartworm infection ( Dirofilaria immitis) wherever the disease is endemic in dogs. Diagnosis is more difficult in cats, and little information is available regarding effective palliative and curative treatments for infected cats. In contrast to the challenges of diagnosis and treatment, chemoprophylaxis is highly effective, and current guidelines call for preventive medications to be administered to all cats in endemic areas. The purpose of this study was to survey feline heartworm management protocols used by 400 animal shelters and foster programs in the endemic states of Alabama, Florida, Georgia, and Mississippi. Only 23% of shelters performed feline heartworm testing. The most common reasons for not testing were expense (36%), lack of treatment options for infected cats (18%), and because the agency considers heartworm infections in cats to be less important than in dogs (12%). Most agencies (69%) did not provide preventive medication to cats. Reasons included because testing was not performed (36%), expense (35%), and the perception that local heartworm risk was low (10%). When preventive was provided, feline-labeled broad-spectrum products were used more commonly (81%) than livestock products (14%). The survey also indicated that many policy decisions were based on inaccurate knowledge of feline heartworm prevalence and pathogenesis. Issues of cost, feasibility, and education prevent most Southeastern sheltering agencies from adequately protecting cats against heartworm disease. Practical guidelines tailored to the needs of these agencies should be developed. Subsidized testing and preventive products may facilitate implementation of feline heartworm management protocols in sheltering agencies.

Renal effects of Dirofilaria immitis in experimentally and naturally infected cats

Canine heartworm infection has been associated with glomerular disease and proteinuria. We hypothesized that proteinuria, likely due to glomerular damage, would also be found in cats experimentally and naturally infected with Dirofilaria immitis. Two populations of cats were evaluated, including 80 that were each experimentally infected with 60 infective heartworm larvae as part of a drug safety study, and 31 that were naturally infected with D. immitis. Each had a control population with which to be compared. In the experimentally infected group, we evaluated urine from 64 cats. Ten of these cats were shown to have microalbuminuria 8 months post infection. No cat refractory to infection with larvae and no cats from the control group demonstrated microalbuminuria. All 10 microalbuminuric cats were shown to have significant proteinuria, as measured by the urine protein:creatinine ratio. There was a subtle, but significant, association between worm burden and proteinuria, and although the presence of adult heartworms was required for the development of proteinuria, both microfilaremic and amicrofilaremic cats were affected. Neither the presence of circulating heartworm antibodies and antigen nor the presence of antigenuria predicted the development of proteinuria. Both heavily infected cats (5–25 adult heartworms) and cats with worm burdens compatible with natural infections (1–4 adult heartworms) developed proteinuria, and the relative numbers of cats so affected were similar between heavily and more lightly infected cats. Naturally infected cats, for which only dipstick protein determinations were available, were shown to have a significantly greater incidence of proteinuria (90% vs 35%) than did those in an age- and gender-matched control population. Additionally, the proteinuria in heartworm-infected cats was 3- to 5-fold greater in severity. We conclude that cats infected with mature adult heartworms are at risk for developing proteinuria and that this is recognized relatively soon after infection. While heavier infections may predispose cats to developing proteinuria, this complication is seen in naturally infected cats and experimental cats with worm burdens similar to those seen in natural infections (i.e., “clinically appropriate” worm burdens). The clinical relevance of heartworm-associated proteinuria is yet to be determined.

Geographical information systems as a tool in the control of heartworm infections in dogs and cats

Geospatial tools (e.g., geographical information systems, remote sensing, global positioning systems, and virtual globes) are very useful for the simultaneous visualization of health data with environmental data, which holds promise to understand environmental-health linkages and to generate new hypotheses to be tested in future research. Current epidemiological studies clearly show that the distribution patterns of vector-borne infections are changing; for example, in Europe, heartworm infection and subcutaneous dirofilariosis are spreading throughout areas that previously had little to no incidence of heartworm. In view of the changes of the distribution patterns of Dirofilaria immitis and Dirofilaria repens, geospatial tools are now more useful for mapping (including territorial sampling), monitoring, ecological analysis, risk assessment, forecasting (including the choose of the timing of treatment), early warning, and surveillance of both heartworm and subcutaneous dirofilariosis. All these issues have control of these infections as the ultimate goal.

Dirofilaria immitis infection in dogs: Cardiopulmonary biomarker levels

Cardiopulmonary biomarkers are biological parameters that can be objectively measured and quantified as indicators of pathogenic processes (heartworm disease) or as indicators of response to therapeutic intervention. To determine levels of cardiopulmonary biomarkers in canine dirofilariasis, measurements of cardiac troponin T, cardiac troponin I, myoglobin, and D-dimer concentrations were performed for dogs with and without evidence of adult heartworm infection. The results showed that levels of cardiac troponin T were undetectable in all dogs studied while levels of cardiac troponin I were higher in dogs infected with Dirofilaria immitis. In healthy dogs, levels of myoglobin and D-dimer were below detection limits of the instrument and were significantly higher in heartworm-infected dogs, notably in microfilaremic dogs. The results suggest the possibility of using troponin I and myoglobin as markers for cardiac damage and the D-dimer as a supportive tool for a diagnosis of pulmonary thromboembolism in dogs with cardiopulmonary dirofilariasis.

Diagnostic, treatment, and prevention protocols for canine heartworm infection in animal sheltering agencies

The high prevalence of heartworm infection in shelter dogs creates a dilemma for shelter managers, who frequently operate with insufficient funding, staffing, and expertise to comply with heartworm guidelines developed for owned pet dogs. The purpose of this study was to survey canine heartworm management protocols used by 504 animal sheltering agencies in the endemic states of Alabama, Florida, Georgia, and Mississippi. Open-admission shelters, which tended to be larger and more likely to perform animal control functions, were less likely (41%) to test all adult dogs than were limited-admission shelters (80%), which tended to be smaller non-profit humane agencies, and foster programs (98%) based out of private residences. Open-admission shelters were more likely to euthanize infected dogs (27%) or to release them without treatment (39%), whereas limited-admission shelters and foster programs were more likely to provide adulticide therapy (82% and 89%, respectively). Of the 319 agencies that treated infections, 44% primarily used a standard two-dose melarsomine protocol, and 35% primarily used a three-dose split-treatment melarsomine protocol. Long-term low-dose ivermectin was the most common treatment used in 22% of agencies. Open-admission shelters were less likely (35%) to provide preventive medications for all dogs than were limited-admission shelters (82%) and foster programs (97%). More agencies used preventives labeled for monthly use in dogs (60%) than ivermectin products labeled for livestock (38%). The most common reason diagnostic testing and preventive medication was not provided was cost. These results indicate a lack of protocol uniformity among agencies and insufficient resources to identify, treat, and prevent infection. Sheltering agencies and companion animal health industries should develop guidelines that are feasible for use in sheltering agencies and provide improved access to preventive and treatment strategies for management of Dirofilaria immitis.

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.

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.