Echocardiographic quantification of Dirofilaria immitis in experimentally infected cats

The safety of heartworm preventives in heartworm-positive cats has traditionally been evaluated using adult Dirofilaria immitis removed from infected dogs and surgically implanted into the cats. An alternate study model uses infective larvae to establish adult infections in cats. Unfortunately, the number of adult worms resulting from the latter method varies widely from none to more than 30, both unacceptable for studies of natural heartworm infection and for studies evaluating product safety in heartworm-infected cats. We sought to determine infection severity in experimental infections via echocardiography to reduce the chances of enrolling uninfected and heavily infected cats into a study. Eighty adult cats were each inoculated with 60 infective D. immitis larvae and maintained for 8 months to allow for the development of adult worms. Antigen and antibody testing, as well as echocardiographic imaging, were performed to confirm and estimate adult worm burdens. Approximately 8 and 12 months post-infection, echocardiographic examination was performed to confirm and enumerate adult D. immitis populations in the cardiovascular system. Worm burdens were stratified as 0, 1-3, 4-11, and > 11 adults, with 0 being considered uninfected and more than 11 considered too heavily infected to be relevant for anthelmintic studies. Cats with clinically relevant infections (1-10 adults) subsequently received multiple treatments with the investigational drug, and worm burdens were confirmed by necropsy 30 days following the final treatment. Worm burden estimated with echocardiography correlated well, but not precisely, with post-mortem counts (p < 0.001, r2 = 0.67). Echocardiography under-, over-, and exactly estimated heartworm burden 53%, 27%, and 22% of the time, respectively. Although the correct category (0-4) was determined by echocardiography in only 54% of cats, positive cats were distinguished from negative cats 88% of the time and the heaviest infections (> 11) were correctly categorized 95% of the time. Both false negative and false positive results were observed. We conclude that echocardiography is useful for detecting mature experimental heartworm infections, identifying cats that have rejected mature infection, and detecting very heavy heartworm burdens, but it is only moderately accurate in classifying lesser burdens. While echocardiography cannot be relied upon to consistently determine the exact heartworm burden in experimentally infected cats, it is useful in stratifying worm burdens for anthelmintic safety studies.     

Correlation between loss of efficacy of macrocyclic lactone heartworm anthelmintics and P-glycoprotein genotype

Macrocyclic lactone (ML) molecules have been used for heartworm control for more than 25 years. However, in recent years, there have been reports of loss of efficacy of ML heartworm preventatives against Dirofilaria immitis in some locations in the United States. Macrocyclic lactone resistance is a common problem in nematode parasites of livestock, and more recently, evidence of ivermectin resistance has been reported in the human filarial nematode Onchocerca volvulus. In this study, four D. immitis sample groups from the United States with different treatment histories were investigated for evidence of ML-driven genetic selection. DNA from individual adult worms and microfilariae was amplified by polymerase chain reaction to investigate a gene encoding a P-glycoprotein, a protein class known to be involved in ML pharmacology. A significant correlation of a GG-GG genotype with ivermectin response phenotype was found. Moreover, a significant loss of heterozygosity was found in a low responder group; loss of heterozygosity is commonly seen in loci when a population has been under selection. Further studies are required to confirm ML resistance in heartworm populations. However, the genetic changes observed in this study may be useful as a marker to monitor for ML resistance in D. immitis.     

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.     

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.     

Current prevalence of Dirofilaria immitis in dogs, cats and humans from the island of Gran Canaria, Spain

The island of Gran Canaria is a hyperendemic area for canine dirofilariasis. The aim of the present study was to provide data on Dirofilaria immitis in dogs, cats, and humans on this island in 2010. The data confirms the prevalence in the overall canine population (19%), with a considerably higher prevalence (43%) in the autochthonous breed of Canarian Warren hound. The prevalence in the feline population (33%) is higher than that of the canine population, and the existence of specific D. immitis antibodies in the inhabitants of the island of Gran Canaria (12%) is confirmed. In both cats and humans, the prevalence, according to the different climate areas on the island, is related to the prevalence of D. immitis in dogs in the same area, which shows the key epidemiological role played by the canine host with regard to the transmission to other hosts of D. immitis.     

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.     

Pulmonary artery dissection causing haemothorax in a cat: potential role of Dirofilaria immitis infection and literature review

A 7-year-old male castrated domestic short-haired cat suddenly died. Gross examination revealed severe right-sided haemothorax with blood clots, four adult filarial nematodes in the blood clots and the caudal vena cava and haemorrhage dissecting into the tunica media of the right pulmonary artery. Histopathological investigation showed fibrosis of the tunica intima and disorganization/fragmentation of the elastic fibres accompanied by fibrous tissue deposition in the tunica media of both branches of pulmonary artery. Degenerative vasculopathy (intimal fibromuscular hyperplasia and medial hypertrophy/hyperplasia) involving pulmonary arteries was also observed. The polymerase chain reaction amplification and sequencing confirmed the identification of the parasite as Dirofilaria immitis. A diagnosis of pulmonary artery dissection with haemothorax and concomitant heartworm disease was formulated. Degenerative processes of the tunica media have been reported to cause pulmonary artery dissection in both humans and animals. Pulmonary artery remodelling induced by heartworms may be considered the underlying cause in the first case of feline pulmonary artery dissection, herein described.     

Clinical evolution and radiographic findings of feline heartworm infection in asymptomatic cats

Clinical manifestations of heartworm disease in cats are variable; most cats seem to tolerate the infection well for extended periods. Heartworm-infected cats may undergo spontaneous self-cure due to the natural death of parasites without any symptomatology, or they may suddenly show dramatic and acute symptoms. Sudden death in apparently healthy cats is not a rare event. Thoracic radiographs are important tool for the diagnosis of cardiopulmonary disease. However, thoracic abnormalities are often absent or transient and highly variable in heartworm-infected cats. Findings, such as enlargement of the peripheral branches of the pulmonary arteries, with a varying degree of pulmonary parenchymal disease and hyperinflation, are the most typical features consistent with infection. A field study was performed for cats referred to the Veterinary Hospital Città di Pavia from January 1998 to December 2001 for routine health examinations and procedures to evaluate the clinical evolution and radiographic findings of feline heartworm infection. Thirty-four asymptomatic cats diagnosed with feline heartworm infection by antibody and antigen tests together with an echocardiogram that allowed worm visualization were included in the follow-up study. Cats were routinely examined every 3 months from the time of heartworm diagnosis until the outcome (self-cure or death). Self-cure was defined as no positive serology for heartworm antigens and no visualization of worms by echocardiography. A final examination for antibodies was carried after 12 months as a final confirmation of self-cure. Twenty-eight cats (82.4%) self-cured; including 21 that showed no clinical signs of infection throughout the study. Six cats died. The most common clinical features observed were acute respiratory symptoms and sudden death. Infection lasted over 3 years in the majority of the cats enrolled in the study. Thoracic radiograph appearance was variable, and the most commonly observed findings were focal and diffuse pulmonary parenchymal abnormalities.     

Dirofilaria immitis and Wolbachia-derived antigens: its effect on endothelial mammal cells

Antigens of both Dirofilaria immitis and Wolbachia symbiont bacteria are implicated in the inflammatory pathology of heartworm infection. The aim of the present study was to compare the stimulatory capacity of in vitro cultures of vascular endothelial cells by the adult somatic antigens of D. immitis (DiSA) and the recombinant form of the Wolbachia surface protein (rWSP), during the first 24h of stimulation. Our results indicate a different stimulatory activity of the two antigens. Both the DiSA and rWSP stimulate the production of the enzymes responsible of the arachidonic acid metabolism, cyclooxygenase-2, 5-lipoxygenase (5-LO), and leukotriene B4. Only DiSA stimulates the production of prostaglandin E2. Related to the adhesion molecules, the DiSA stimulates the expression of intercellular adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1), whereas rWSP stimulates ICAM-1, PECAM-1, and vascular cell adhesion molecule-1 (VCAM-1). Expression of E-cadherin and vascular endothelial growth factor also were stimulated by rWSP. Neither of the two antigens altered the basic physiological mechanisms of endothelial cells, such as cell proliferation, cell cycle, or apoptosis. The biological and pathological significance of these finding are discussed.     

Feline heartworm (Dirofilaria immitis) infection: a statistical elaboration of the duration of the infection and life expectancy in asymptomatic cats

A study was conducted to assess the duration and the outcome (self-cure or death) of feline heartworm infection and the life expectancy of infected cats. To be included in the study, cats had to be positive for heartworm antibody (Ab) and heartworm antigen (Ag) and had to demonstrate the presence of worms by echocardiography. Self-cure was defined as (1) negative for heartworm Ag and (2) no further visualization of worms by echocardiography. Of the 1962 eligible cats, 364 (18.5%) were positive for heartworm Ag and 131 were positive for heartworm Ag and for echocardiography diagnosis (prevalence 6.7%). None of the cats was microfilaremic. Of 43 asymptomatic cats included into the follow-up study with owners' consent, 34 (79%) self-cured and nine (21%) died. Eleven (26%) cats remained asymptomatic and self cured within 21-48 months, 23 (53%) showed symptoms but self-cured within 18-49 months, 6 (14%) died within 8-41 months of follow-up and 3 (7%) suddenly died after 38-40 months, which was related to heartworm infection. The probability for death or sudden death increased significantly with age at diagnosis, but no difference was detected by gender, survival time after diagnosis, or the presence or absence of symptoms. The presence/absence of symptoms showed significant interaction with the age at diagnosis (i.e., symptomatic cats showed increasing duration of heartworm infection along with age at diagnosis compared to that for asymptomatic cats. Heartworm-infected cats survived significantly longer than heartworm-negative cats affected by hypertrophic cardiomyopathy, chronic renal failure, or neoplastic diseases.