Jorge Guerrero

Heartworm Disease in Animals and Humans

Heartworm disease due to Dirofilaria immitis continues to cause severe disease and even death in dogs and other animals in many parts of the world, even though safe, highly effective and convenient preventatives have been available for the past two decades. Moreover, the parasite and vector mosquitoes continue to spread into areas where they have not been reported previously. Heartworm societies have been established in the USA and Japan and the First European Dirofilaria Days (FEDD) Conference was held in Zagreb, Croatia, in February of 2007. These organizations promote awareness, encourage research and provide updated guidelines for the diagnosis, treatment and prevention of heartworm disease. The chapter begins with a review of the biology and life cycle of the parasite. It continues with the prevalence and distribution of the disease in domestic and wild animals, with emphasis on more recent data on the spreading of the disease and the use of molecular biology techniques in vector studies. The section on pathogenesis and immunology also includes a discussion of the current knowledge of the potential role of the Wolbachia endosymbiont in inflammatory and immune responses to D. immitis infection, diagnostic use of specific immune responses to the bacteria, immunomodulatory activity and antibiotic treatment of infected animals. Canine, feline and ferret heartworm disease are updated with regard to the clinical presentation, diagnosis, prevention, therapy and management of the disease, with special emphasis on the recently described Heartworm Associated Respiratory Disease (HARD) Syndrome in cats. The section devoted to heartworm infection in humans also includes notes on other epizootic filariae, particularly D. repens in humans in Europe. The chapter concludes with a discussion on emerging strategies in heartworm treatment and control, highlighting the potential role of tetracycline antibiotics in adulticidal therapy.

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.

Amphidial structure of ivermectin-resistant and susceptible laboratory and field strains of Haemonchus contortus

The development of anthelmintic resistance by nematode parasites is a growing problem for veterinarians, pet owners, and producers. The intensive use of the macrocyclic lactones for the treatment of a variety of parasitic diseases has hastened the development of resistance to this family of parasiticides. As a result, resistance to ivermectin, moxidectin, nemadectin, and doramectin by Haemonchus contortus has been documented throughout the world. Sensory neurons located in the cephalic end of nematodes are in close contact with the external environment. Through these neurons, important chemical and thermal cues are gathered by the parasite. Examination of serial electron micrographs of ivermectin-susceptible and ivermectin-resistant H. contortus allows for comparison of neuronal structure, arrangement of neurons within the amphidial channel, and distance of the tip of the dendritic processes to the amphidial pore. The latter of these characteristics provides a useful means by which to compare the association between the neurons and the external environment of the worm. Comparison of parental laboratory strains of ivermectin-susceptible strains of H. contortus with related selected, ivermectin-resistant strains and with a wild-type ivermectin-susceptible field strain of H. contortus from Louisiana reveal that the ivermectin-resistant worms examined have markedly shorter sensory cilia than their ivermectin-susceptible parental counterparts. Additionally, the amphidial neurons of ivermectin-resistant worms are characterized by generalized degeneration and loss of detail, whereas other neurons outside of the channels, such as the labial and cephalic neurons, are normal in structure. These findings raise a number of questions regarding the relationship between amphidial structure and ivermectin resistance as well as the role of amphids as a means of entry for ivermectin. While shortened amphidial sensilla are associated with ivermectin resistance, it remains unclear if such a structural modification facilitates survival of nematodes exposed to macrocyclic lactones.

Follow-up of the health conditions of an urban colony of free-roaming cats (Felis catus Linnaeus, 1758) in the city of Rio de Janeiro, Brazil

Abstract Similar to other urban areas where food and shelter are abundant, the zoological garden of Rio de Janeiro has dealt for years with a colony of feral or semi-feral domestic cats. A survey was conducted during 2002–2004 as a follow-up to a previous study in 2001 of the cat colony to identify pathogens circulating among the population and to annually follow the status of the cats to analyze morbidity coefficients and associations among infections and infestations identified in the colony. During the 3 years of the present study, 75 cats were sampled at least once, including 44 that were caught and examined only once, 14 that were examined twice, and 17 that were examined three times. For each cat that was caught, records were kept regarding sex, age, general health, and the presence of ectoparasites. Each year, a blood sample was taken for hematologic testing, platelet count, hemoparasite detection, antibodies to Toxoplasma gondii, and retrovirus detection. Blood counts were within normal range for the majority of cats tested. Feline immunodeficiency virus, fleas, and lice were detected in all years; however, incidence rates for each of these varied significantly throughout the years. Prevalence of Cytauxzoon spp., Mycoplasma spp., T. gondii infections were variable among the 3 years, although differences were not significant. Prevalence of feline leukemia virus increased significantly over the 3 years. Mycoplasma spp. and flea infestations were significantly associated, but no other associations among the pathogens were detected. Over the 3 years, the rate of new cat introductions decreased, and the pathogens showed a tendency to disseminate throughout the colony; however, there was virtually no evidence of clinically detectable disease. Therefore, it seems that stabilizing the population by a judicious control program facilitated the distribution of the pathogens throughout the colony, while the general well-being of the cats was not seriously affected.

Characterization of ectoparasites in an urban cat (Felis catus Linnaeus, 1758) population of Rio de Janeiro, Brazil

Ectoparasites are capable of transmitting infectious diseases and, therefore, are of zoonotic concern. Cats submitted to a spay/neuter program in the city of Rio de Janeiro were examined to determine the distribution of ectoparasites in cats from a city with a tropical climate. Independent of gender, breed, or age, 292 cats were combed and subjected to otoscopic examination. Ectoparasites were collected, and blood samples were taken to determine packed cell volume. The majority of the 292 cats were female (71%), and most of them were categorized as domestic short hair cats (92%). Different species of ectoparasites that produced both single agent and multi-agent infestations were detected in these cats. Most cats were infested by Ctenocephalides felis (60%); however, other ectoparasites were found to a lesser degree in the sampled population (Felicola subrostratus (5.4%), Rhipicephalus sanguineus (1.4%), Lynxacarus radovskyi (1%), and Otodectes cynotis (6.2%)). Within the infested cats, 16.3% were also anemic, and there was a significant association between the occurrence of anemia and flea infestation. Of all sampled cats, 65% were infested by at least one species of ectoparasites, which suggests both a heavy infestation of the environment and owner negligence. Ectoparasites are not only nuisances to both cats and owners, but they are also significant carriers of disease. Therefore, the implementation of rigorous, safe preventive measures is of great importance.

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.

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.

ANTHELMINTIC ACTIVITY OF CLOSANTEL AGAINST ANCYLOSTOMA CANINUM IN DOGS

The efficacy and minimum therapeutic dose of closantel, an injectable salicylanilide anthelmintic, was evaluated on adult Ancylostoma caninum. Doses of 7.5 and 10 mg/kg had a marked anthelmintic effect on adult stages, removing 99 and 98%, respectively. A second experiment tested the efficacy of closantel against hypobiotic larval stages of A. caninum. A dose of 20 mg/kg did not affect the abundance of arrested hookworm larvae or prevent their subsequent development, but it may have inhibited their full maturation as adult worms so that patent, intestinal infections did not occur. Small numbers of adults were recovered from treated dogs at 29, 43, and 57 days post-treatment, but no eggs were passed in their feces. These studies bear on the practical problem of control of hookworms in dogs.

Effects of doxycycline on the endosymbiont Wolbachia in Dirofilaria immitis (Leidy, 1856)—Naturally infected dogs

Dirofilaria immitis carries intracellular endosymbiotic bacteria of the genus Wolbachia, known to be vital for the worms and sensitive to tetracycline antibiotics. With the purpose of studying the interaction between D. immitis and the endosymbiont Wolbachia sp., heartworm naturally infected microfilaremic or antigenemic dogs were treated with doxycycline (10mg/kg/day of the drug in three cycles of 21 days each, with 6-month intervals). Blood samples were collected on days 0, 7 and 21 of each treatment as well as on day 111 after the beginning of each cycle. A final sample was collected on day 723 from the beginning of the first treatment. The samples were examined for the presence and number of microfilariae and the presence of antigen as well as the presences of D. immitis and Wolbachia sp. DNA using PCR (polymerase chain reaction). With this approach, an evaluation of the effect of doxycycline on antigenemia and on the presence of Wolbachia sp. DNA in dogs with heartworm infection was possible. Doxycycline treatment did not alter the detection of adult parasite antigens with the exception of two animals, though the number of animals carrying Wolbachia sp. DNA decreased, despite the presence of the microfilariae. The effect of the antibiotic therapy on the worms may have interfered with the transmission of heartworm disease because the population of microfilariae and the number of microfilaremic dogs were reduced and the microfilariae positive samples that were found did not test positive for Wolbachia sp. in many cases. These findings suggest that in areas were doxycycline is extensively used D. immitis transmission may be impaired by the reduction on the number of microfilariae and on the endosymbiotic bacteria in the larvae turning them incapable of completing development once they infected a new host.

Heartworm disease: challenges in a changing world.

In the past three years since our State of Heartworm ymposium of 2007, much has been learned, and many pidemiological as well as client behavior challenges have een encountered by the veterinary practitioner. In spite of the magnificent tools available to the eterinarian for diagnosis, treatment, and prevention of eartworm disease, the prevalence either remains the ame or is increasing in the USA and other parts of the orld. New challenges to the lives of our pets, the health f their guardian families, and to the veterinary practiioner have been described, and we need to be prepared o face them. For that reason, in the 2010 Symposium of he American Heartworm Society, we have made a point o expand our traditional reach to include two nematode arasitic infections of growing importance in Europe and anada. The readers will find beneficial articles discussing mportant epidemiological as well as medical points about irofilaria repens and Angyostrongylus vasorum and the hreat they present to animals in the case of A. vasorum nd to the animals and their human families in the case of . repens. The relevance of climatic changes and the dissemination f heartworm as well as the use of geographic information