Infestación por "Dirofilaria immitis" en las Islas Canarias

TESIS DOCTORAL INFESTACIÓN POR DIROFILARIA IMMITIS EN LAS ISLAS CANARIAS José Alberto Montoya Alonso Las Palmas de Gran Canaria, 24 de septiembre de 2015 Anexo 1 DR. OCTAVIO LUIS PEREZ LUZARDO SECRETARIO DEL DEPARTAMENTO DE CIENCIAS CLINICAS DE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA, CERTIFICA, Que el Consejo de Doctores del Departamento en su sesi6n de fecha 22 de septiembre de 2015, tom6 el acuerdo de dar el consentimiento para su tramitaci6n, a la tesis doctoral titulada "INFESTACIÖN POR DIROFILARIA IMMITIS EN LAS ISLAS CANARIAS" presentada por el doctorando D. Jose Alberto Montoya Alonso y dirigida por el Dr. D. Fernando Lorenzo Simon Martin. Y para que asi conste, y a efectos de lo previsto en el Art0 6 del Reglamento para la elaboraci6n, defensa, tribunal y evaluaci6n de tesis doctorales de la Universidad de Las Palmas de Gran Canaria, firmo la presente en Las Palmas de Gran Canaria, a 22 de septiembre de dos mil quince. DEPARTAMENTO DE CIENCIAS CLÍNICAS PROGRAMA DE DOCTORADO EN MICROBIOLOGIA TESIS DOCTORAL INFESTACIÓN POR DIROFILARIA IMMITIS EN LAS ISLAS CANARIAS DOCTORANDO DIRECTOR Fernando Lorenzo Simón Martín Universidad de Salamanca Las Palmas de Gran Canaria, 24 de septiembre de 2015 José Alberto Montoya Alonso Fernando Simón Martín, Doctor en Biología y Catedrático de Parasitología del Departamento de Biología Animal, Parasitología, Ecología, Edafología y Química Agrícola de la Universidad de Salamanca, INFORMA: Que D. José Alberto Montoya-Alonso, Doctor en Veterinaria, ha realizado, bajo mi dirección y asesoramiento, la presente Tesis Doctoral titulada: “Infestación por Dirofilaria immitis en las islas Canarias”, que a mi juicio reúne las condiciones y calidad científica necesarias para su presentación y defensa, para optar al título de Doctor por la Universidad de Las Palmas de Gran Canaria. Lo que firmo, para que surta los efectos a que haya lugar, en Salamanca a quince de septiembre de 2015. Fdo.: Prof. Fernando Simón Martín. USAL Las ilustraciones de este trabajo han sido cedidas por mí hermano el Dr. Carlos Montoya Alonso Trabajo maquetado por Ernest Julio, Multimédica Ediciones Veterinarias A mi madre Pili (Q.E.P.D.) que siempre estuvo orgullosa de mí A Cande, Alberto y Nacho con todo mi cariño A toda mi familia por lo que me quiere y aguanta A Elena Carretón por ser como es Nuestra relación con las filarias comenzó hace muchos años. Siendo estudiante de 4º curso, en la Facultad de Veterinaria de Madrid, asistí a mi primera necropsia de una perra, de raza pastor alemán, que había muerto de un síndrome de vena cava, producido por los múltiples gusanos adultos de D. immitis, que invadían su corazón y sus vasos. Debo reconocer que la escena me produjo gran impresión. Al llegar a la isla de Gran Canaria, a principio de los 90, y gracias a muchos compañeros, pero principalmente a un magnífico veterinario canario como es Manolo Morales Doreste, empecé a trabajar con los perros parasitados de filaria y a reconocer sus síntomas, realizar diagnósticos y a aplicar las medidas terapéuticas disponibles en esa época. En 1998 publicamos el primer estudio epidemiológico de prevalencia de esta enfermedad en Gran Canaria, considerando las características isoclimáticas de la isla y sus particularidades geográficas. Este trabajo ha sido referente de muchos estudios epidemiológicos posteriores. Por aquella época, el querido y entrañable Prof. Zoilo González Lama (Q.E.P.D.) me propuso matricularme de doctorado en su Departamento de Ciencias Clínicas e iniciar esta Tesis Doctoral. Siempre me estuvo animando a seguir con el estudio de la dirofilariosis en Canarias, pero desgraciadamente no pudo ver la obra concluida. Más tarde conocí al Prof. Fernando Simón Martín, salmantino, amigo y catedrático de Parasitología de la Facultad de Farmacia de la Universidad de Salamanca y uno de los más importantes investigadores mundiales de dirofilariosis que, con mucho cariño, me invitó a investigar con su grupo y a dedicarme con más intensidad a trabajar con la enfermedad del gusano del corazón. Al grupo de medicina interna (cardio-respiratorio) de la Facultad de Veterinaria de Las Palmas de Gran Canaria, se incorporó hace unos años, con una beca de formación del personal investigador, Elena Carretón Gómez. La Dra. Carretón, Elena, es una bilbaína-canaria infatigable que nunca se desalienta, ni deja de trabajar con las filarias y sus pacientes, y una magnífica compañera. Lógicamente esto supuso un incremento exponencial de nuestra actividad investigadora. Necesariamente tuvimos que ampliar nuestros contactos con grupos multidisciplinares nacionales e internacionales, especialistas en esta zoonosis parasitaria (Profesores: Morchón, Genchi, Kramer, Guerrero, Mc.Call, Kartashev, Grandi, González-Miguel, Kozek, Siles-Lucas, Venco…) a los que agradecemos su apoyo y sin los que hubiera sido imposible avanzar en nuestro trabajo. Más becarios, estudiantes y proyectos han ido reforzando al equipo y nuestras investigaciones. Actualmente formamos parte activa de la European Dirofiliariasis Society (EDIS) y nuestro trabajo va adquiriendo reconocimiento internacional, con más de 30 artículos específicos de alto impacto, libros, monografías, proyectos, premios… Así, nuestro trabajo clínico e investigador, de varios años, sobre la enfermedad en Canarias y su importancia zoonósica ha quedado reflejando de manera sencilla, en esta tesis en formato de compendio de publicaciones. 2 OBJETIVOS 1. Determinar la prevalencia de Dirofilaria immitis en perros de la isla de Gran Canaria los años 1994,1995 y 1996. 2. Conocer la prevalencia de D. immitis en perros de la isla de Tenerife y sus variaciones en función de los diferentes isoclimas de la isla (años 2002-2003). 3. Valorar las variaciones de la incidencia de D. immitis en perros, en la isla de Gran Canaria, entre 2000 y 2008 y determinar si el podenco canario constituye un reservorio natural de la enfermedad. 4. Conocer la seroprevalencia de D. immitis en la población humana y su relación con la prevalencia en perros en la isla de Gran Canaria (año 2008). 5. Estudiar la incidencia de la zoonosis en perros, gatos y humanos en las diferentes zonas isoclimáticas de Gran Canaria, y evaluar la relación entre las prevalencias de las diferentes especies (año 2010). 6. Determinar la incidencia de la dirofilariosis cardiopulmonar en pequeños animales en todo el archipiélago Canario (años 2014-15). 3 REVISIÓN BIBLIOGRÁFICA Human and animal dirofilariasis: the emergence of a zoonotic mosaic Simón F, Siles-Lucas M, Morchón R, González-Miguel J, Mellado I, Carretón E, Montoya-Alonso JA Clinical Microbiology Reviews, 25 (3): 507-544, 2012 5 Human and Animal Dirofilariasis: the Emergence of a Zoonotic Mosaic Fernando Simón, a Mar Siles-Lucas, b Rodrigo Morchón, a Javier González-Miguel, a Isabel Mellado, a Elena Carretón, c and Jose Alberto Montoya-Alonso c Laboratory of Parasitology, Faculty of Pharmacy and IBSAL, University of Salamanca, Salamanca, Spain a ; Laboratory of Parasitology, IRNASA (CSIC), Salamanca, Spain b ; and Internal Medicine, Faculty of Veterinary Medicine, University of Las Palmas de Gran Canaria, Las Palmas, Spain c INTRODUCTION ............................................................................................................................................507 ESSENTIAL FEATURES OF THE BIOLOGY OF DIROFILARIA SPECIES .......................................................................................508 Life Cycle .................................................................................................................................................508 Development in the definitive host....................................................................................................................508 Development in mosquito vectors ....................................................................................................................509 Role of Wolbachia Symbiotic Bacteria in Development and Biology of Dirofilaria Species ...............................................................509 Classical and Proteomic Approaches to the Study of Proteins in Dirofilaria spp. .........................................................................510 Energy Generation and Molting in Dirofilaria Species ...................................................................................................510 Nutrient uptake and energy metabolism ..............................................................................................................510 Molting ................................................................................................................................................512 PREVALENCE AND DISTRIBUTION DYNAMICS.............................................................................................................513 Canine Dirofilariasis ......................................................................................................................................513 The Americas ..........................................................................................................................................513 Europe .................................................................................................................................................514 Africa...................................................................................................................................................514 Asia and Australia ......................................................................................................................................514 Feline Dirofilariasis........................................................................................................................................514 Dirofilariasis in Wild Carnivores ...........................................................................................................................515 Dirofilariasis in Human Hosts .............................................................................................................................515 Dirofilaria spp. in Vector Species..........................................................................................................................517 Relevant Research Approaches to Climatic Change and Other Factors That Influence Transmission and Distribution Dynamics of Dirofilaria spp.....517 Novel tools for the study and prediction of changes in the distribution of dirofilariasis ...............................................................518 CLINICAL FEATURES OF HUMAN AND ANIMAL DIROFILARIASIS .........................................................................................519 Heartworm Disease in Dogs, Cats, and Ferrets ...........................................................................................................519 Dogs ...................................................................................................................................................519 Cats ....................................................................................................................................................521 Ferrets .................................................................................................................................................521 Subcutaneous and Ocular Dirofilariasis in Animal Reservoirs.............................................................................................522 Human Dirofilariasis ......................................................................................................................................522 Pulmonary dirofilariasis ................................................................................................................................522 Subcutaneous/ocular dirofilariasis.....................................................................................................................523 Questionable paradigms related to human dirofilariasis...............................................................................................523 Diagnosis ................................................................................................................................................524 Laboratory diagnosis ..................................................................................................................................524 Clinical diagnosis ......................................................................................................................................526 Treatment and Prevention ...............................................................................................................................527 Wolbachia as a Therapeutic Target .......................................................................................................................528 THE HOST-PARASITE RELATIONSHIP IN DIROFILARIASIS .................................................................................................529 Immune Response: Basic Facts ...........................................................................................................................529 Both the Presence of Microfilariae and the Clinical Status of Dirofilaria-Infected Hosts Influence the Intensity of the Antibody Response ..............529 Immunopathogenic Mechanisms in Dirofilariasis ........................................................................................................530 Pathogenic Mechanisms Unrelated to the Immune Response...........................................................................................531 Parasite Survival Mechanisms ............................................................................................................................532 CONCLUSIONS AND FUTURE TRENDS .....................................................................................................................533 ACKNOWLEDGMENTS......................................................................................................................................534 REFERENCES ................................................................................................................................................534 INTRODUCTION ALombard noble named Francesco Birago made the first known reference to canine filariae in the 17th century by describing the presence of adult Dirofilaria immitis worms inside the hearts of his hunting dogs, although he erroneously identified them as larvae of another parasitic worm, possibly Dyoctophyma renale (49). Currently, dirofilariasis is understood as a group of parasitoses caused by species of the genus Dirofilaria transmitted by vectors. Among all Dirofilaria species, the most relevant are D. immitis and D. (Nochtiella)repens due to their severe pathological effects and their high prevalence and incidence. D. immitis proAddress correspondence to Fernando Simón, [email protected]. Supplemental material for this article may be found at http://cmr.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/CMR.00012-12 July 2012 Volume 25 Number 3 Clinical Microbiology Reviews p. 507–544 cmr.asm.org 507 duces both canine and feline cardiopulmonary dirofilariasis, whereas D. repens causes both canine and feline subcutaneous dirofilariasis. In addition, D. immitis and D. repens are responsible for human pulmonary and subcutaneous/ocular dirofilariasis, respectively, throughout the world (324,391). Therefore, these infections represent a zoonotic mosaic, which in practice includes two main filarial species that have adapted to canine, feline, and human hosts to various degrees. In each of these hosts, D. immitis and D. repens exhibit specific developmental patterns, each with distinct biological and clinical implications. At the same time, both D. immitis and D. repens are themselves hosts to symbiotic bacteria of the genus Wolbachia, the study of which has resulted in a profound shift in the understanding of filarial biology, the mechanisms of the pathologies that they produce in their hosts, and issues related to the treatment of this parasitosis. Additionally, the involvement of vectors in the parasite life cycle makes dirofilariasis transmission and distribution susceptible to global climate change, and rates have undergone rapid and significant changes in defined geographic regions in recent years. Despite advances in our knowledge of D. immitis and D. repens and the pathologies that they inflict on different hosts, dirofilariasis remains a priority subject of study in veterinary medicine nearly 400 years after its discovery. Therefore, there is greater interest in and attention to the increasing incidence and severity of human dirofilariasis cases. In consideration of all of these issues, we review human and animal dirofilariasis here, including the basic morphology, biology, protein composition, and metabolism of Dirofilaria species; the climate and human behavioral factors that influence distribution dynamics; disease pathology; the host-parasite relationship; the mechanisms involved in parasite survival; the immune response and pathogenesis; and the clinical management of human and animal infections. ESSENTIAL FEATURES OF THE BIOLOGY OF DIROFILARIA SPECIES Life Cycle The life cycle of Dirofilaria species comprises a definitive vertebrate host and a vector (Fig. 1). Both D. immitis and D. repens demonstrate poor vertebrate host specificity given that they can infect numerous mammalian species (34). Among mammalian hosts, they are best adapted to domesticated and wild dogs, which function as reservoirs. Humans and cats are less suitable hosts (261), in which parasite development is dramatically modified compared with the patterns in dogs. The vectors are females of various mosquito species of the Culicidae family (75). Development in the definitive host. During a blood meal, mosquitoes deposit a hemolymph on the wound, which carries infectious “larvae 3” (L3) stage larvae that penetrate the host’s skin on their own (435). The molt from L3 to L4 occurs soon after D. immitis infection, between 3 and 12 days postinfection (d.p.i.), and the subsequent molt, which produces preadult worms, takes place between 50 and 70 d.p.i. The first preadult worms arrive in the pulmonary artery and right ventricle of canine hearts at between 70 and 85 d.p.i. and reach sexual maturity at 120 d.p.i. Adult D. immitis worms have a filiform appearance, with females measuring 250 to 300 mm in length and 1 to 1.3 mm in diameter and males measuring 120 to 200 mm in length and 0.7 to 0.9 mm in diameter (252)(Fig. 2). Females start producing the first larval stage (microfilariae) between 6 and 9 months postinfection (m.p.i.) (261). Microfilariae that live in the bloodstream are 290 to 330 m in length and 5 to 7 m in diameter, with a straight tail and a spindle-shaped cephalic extremity. Adults can live over 7 years, and microfilariae live as long as 2 years (435). Some infected dogs do not harbor microfilariae in the blood, resulting in occult or amicrofilaremic infections, possibly due to factors such as the FIG 1 Biological life cycles of D. immitis and D. repens. mf, microfilaremia. Simón et al. 508 cmr.asm.org Clinical Microbiology Reviews aging of female worms, single-gender worm infections, and/or host immune responses (388). In cats, the maturation of adult D. immitis worms extends up to 8 m.p.i.; these worms are shorter than those found in dogs, with a shorter life expectancy (approximately 2 years maximum), and they generally do not produce microfilariae. When microfilaremia does occur in this host, it is transient and of a low intensity (252,261). Adult D. repens worms generally take residence in the subcutaneous tissues of definitive hosts, although they can also be found in the abdominal cavity and within connective muscular fasciae (148), where they achieve sexual maturity at 6 to 9 m.p.i. (252). D. repens worms are smaller than D. immitis worms: females are 100 to 170 mm in length and 4.6 to 6.3 mm in diameter, and males are 50 to 70 mm in length and 3.7 to 4.5 mm in diameter (252). Like those of D. immitis,D. repens microfilariae reside in the bloodstream, measuring between 350 and 385 m in length and 7 to 8 m in diameter, with a curved tail and rounded cephalic extremity (435). The finding that cats harbor D. repens microfilariae in their blood suggests that feline hosts may serve as reservoirs for this species (419). Humans are not suitable hosts for Dirofilaria species, as demonstrated by their deviation from the developmental patterns described above. Immature D. immitis worms can reach a branch of the human pulmonary artery, triggering an inflammatory response that destroys the worms, occasionally resulting in pulmonary nodules (391). D. repens worms cause subcutaneous nodules and can reach the ocular region in human patients (321). Both species can infect anatomical regions other than those described above but only incidentally (328,423). Development in mosquito vectors. D. immitis and D. repens microfilariae are ingested by mosquitoes during a blood meal on an infected host. Within approximately 24 h, the ingested microfilariae reach the Malpighian tubules from the digestive tract, where they molt to the L2 larval form within approximately 8 to 10 d.p.i. and from L2 to L3 approximately 3 days afterwards. The environmental temperature is the key factor that determines the length of the L3 developmental period in mosquitoes. The infective L3 larvae migrate to the mouthparts of the vector, where they reside until the next blood meal. L3 larvae are approximately 1 mm in length and grow to 1.5 mm in the subcutaneous tissues of definitive hosts upon inoculation (252). The invasion of Malpighian tubules and migration to the mouthparts of the parasite are critical for mosquito survival. The vectors limit the number of larvae that can progress to L3 via antigenic recognition and humoral and cellular defense mechanisms (83). An antimicrobial polypeptide, defensin, has been identified in mosquito hemolymph inoculated with D. immitis L3 larvae. In addition, D. immitis vectors exhibit the capacity for the melanization and encapsulation of L3 larvae. Melanin is a pigment produced in hemocytes via the conversion of tyrosine to dopamine through hydroxylation by a phenoloxidase (181). Tyrosine, the limiting factor for the entire process, can be obtained exogenously or can be derived from phenylalanine by phenylalanine hydroxylase (PAH), which is an enzyme that is required for a complete melanization reaction in vectors (185). Melanization concludes with the formation of a membrane-like structure on the external zone of the cellular capsule (83). The melanization efficiency varies across species and among members of the same species due to differences in phenoloxidase activity, which is more robust during the initial 14 days of the life of the mosquito (91,232). Other mechanisms and structures contribute to the destruction of larvae, such as the buccopharyngeal armature (cibarial armature), which can damage microfilariae during a blood meal; the secretion of molecules that lyse the epicuticle of worms; and blood coagulation, which traps microfilariae in the digestive tract of mosquitoes, thereby impeding their passage to the Malpighian tubules (74). Mosquitoes’ susceptibility and resistance to infection are genetically determined and controlled by a gender-linked recessive allele, with separate genes coding for susceptibility and resistance to D. immitis and D. repens (74). Genetic differences that influence transmission may also exist within a single species: a study performed in a western region of Spain where D. immitis is endemic found D. immitis larvae only in the H1 haplotype of Culex pipiens but not in haplotypes H2 and H3 (286). Role of Wolbachia Symbiotic Bacteria in Development and Biology of Dirofilaria Species The first intracellular bacterium-like bodies in filariae were found in D. immitis (174). Later research demonstrated the bacterial nature of these bodies (266) and their presence in other species of filariae, such as Onchocerca volvulus (210). Two decades later, studies using electron microscopy and molecular techniques demonstrated that these bacteria belong to the order Rickettsiales (alpha-2-proteobacteria) and the genus Wolbachia (399). These bacteria have been also found in other organisms, including hexapods, crustaceans, and chelicerates. Wolbachia bacteria are intracellular, are observed in isolation or in clusters (81,133), and seem to have developed a symbiotic relationship with these and other organisms, including filariae of the family Onchocercidae, of which D. immitis and D. repens are members (134). Studies of the antibiotic treatment of filaria-infected hosts and genome sequencing of Wolbachia have provided information about the nature of the interactions between bacteria and filariae and the molecules involved (140,454). Those studies suggested that Wolbachia bacteria are involved in molting and embryogenesis of filariae (28), whereas filariae contribute amino acids for bacterial FIG 2 Male and female adult worms of D. immitis in the heart of a dog. Human and Animal Dirofilariasis July 2012 Volume 25 Number 3 cmr.asm.org 509 growth (140). Wolbachia are transmitted maternally, are found in all individuals at all filarial developmental stages, and are particularly abundant in larvae that develop in vertebrate hosts (L3 and L4), in the hypodermal cords of adults of both genders (Fig. 3), and in the genital organs of females. These findings suggest that symbiotic bacteria are essential for larval development in vertebrate hosts and for the long-term survival of adult worms (263). Wolbachia was recently found in new species of filariae from the family Onchocercidae in various organs, such as somatic gonads (epithelial layer) and the intestinal wall, suggesting that the bacterium-filaria relationship is far more complex and diverse than previously estimated (135). Classical and Proteomic Approaches to the Study of Proteins in Dirofilaria spp. Databases of protein sequences from the National Center for Biotechnology Information (NCBI) contain approximately 136 records for D. immitis, a very limited amount of genetic information compared with those for other nematodes (for example, 23,329 records for Brugia malayi). Several studies in the 1980s and 1990s identified single molecules in the different developmental stages of D. immitis and some of their characteristics. In adult worms, a significant proportion of the molecules identified were acidic polypeptides that ranged in size from 82 kDa to 200 kDa and were susceptible to various collagenases (381). Later, the antigenic repertoires of L2 and L3 larvae were found to be similar to but different from that of L4 larvae (382). Among the differences, a 35-kDa polypeptide was identified and characterized as an immunodominant surface antigen that is present in L3 larvae but not in larvae at subsequent developmental stages (340). A nonimmunogenic 6to 10-kDa glycolipid was also identified (381). Both the 35-kDa and 6-kDa molecules are shed from the surface of L3 larvae during the early days of development in vivo and in vitro, and the released material is not replenished (184). Later, several other proteins from D. immitis were identified, cloned, and characterized, among which the heat shock protein p27 is noteworthy due to its potential role in the host-parasite relationship. p27, to which repair functions during development have been attributed, is localized to the hypodermis of L3 and L4 larvae and adults (236). Additionally, various enzymes with redox potential have been described, including peroxiredoxins in somatic extracts and excretory-secretory (E/S) products from adults and microfilariae (88,463) and glutathione peroxidase in adult worms and L4 larvae (428), which is a precursor of a neutrophil chemotactic factor (320). Two nuclear receptors, Di-nh-7 and DiRXR-1, the latter being related to cell proliferation, differentiation, and apoptosis (99,385), have also been reported. A chitin synthase (175) and an ivermectin-sensitive glutamate-gated chloride channel subunit (458) have also been characterized. In addition to those single-molecule identification approaches, proteomics were later applied to the study of dirofilarial proteins. Together with mass spectrometry, proteomics allowed the simultaneous identification of numerous proteins, comprising 39 proteins from D. immitis and 15 from D. repens, many of which were represented by several isoforms (157,158,313). These proteins belong primarily to four functional groups, including metabolic enzymes, enzymes with redox or detoxification potential, and molecules involved in motility and the stress response (Table 1). The most represented enzymes are those involved in energy metabolism, eight of which participate in anaerobic glycolysis in D. immitis, among them enolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and lactate dehydrogenase. Five proteins with redox potential and four with roles in stress responses, including various heat shock proteins, were also identified. Many of these molecules have antioxidant and detoxification properties and have been linked to the parasites’ capacity to neutralize reactive oxygen species released by macrophages and neutrophils (158). Many of the proteins within these groups have also been found in D. repens albeit in a lower number than in D. immitis (Table 1). Antigenic extracts of D. immitis contain an abundance of glycosylated molecules, although glycosylated residues are not exposed on the surface of intact worms. Glycosylated residues contain primarily mannose, glucose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. N-Acetylgalactosamine has been found in an unusual terminal via a -linkage to the sequence GalNacbGlcNacbMan-R, which is a structure similar to N-union glycoproteins in mammals albeit with significant differences, such as the lack of both sialic acid and galactose (195,196,272). Energy Generation and Molting in Dirofilaria Species Nutrient uptake and energy metabolism. Although the molecules involved in Dirofilaria energy metabolism could provide excellent targets for future therapeutic or control measures, few studies have been conducted on this topic. Recently, studies of the relationship between filariae and Wolbachia and some proteomic analyses of worms have begun to yield valuable information about the molecules involved in the various metabolic processes that are key to the survival and development of D. immitis and D. repens. The routes for nutrient acquisition are different in the several developmental stages of Dirofilaria spp. Adult D. immitis worms have a functional digestive tract, whereas microfilariae do not, and the digestive tract of L3 larvae is nonfunctional in vectors but becomes functional in the vertebrate host (33,70). In adult worms, nutrient uptake is performed in the digestive tract, in which erythrocytes have been found (33), and the cuticle in vivo, whereas the transcuticular route is the main means for the uptake of low-molecular-weight molecules in vitro (207)(Fig. 4). Transcuticular uptake is a selective process, as demonstrated by the finding that worms acquire D-glucose and some precursors of nucleic acids but not L-glucose, sucrose, or thymidine via this route. FIG 3 Immunohistochemical positive reaction (red) against the Wolbachia surface protein (WSP) reveals the presence of symbiont bacteria in the hypodermal cords of a D. repens adult worm. (Courtesy of L. H. Kramer, University of Parma, Parma, Italy.) Simón et al. 510 cmr.asm.org Clinical Microbiology Reviews TABLE 1 Proteins of D. immitis and D. repens identified by mass spectrometry a GenBank accession no. Protein Species No. of isoforms Mascot score Biological process D. immitis proteins CAA34719 Actin Caenorhabditis elegans 4 202–450 Cell motility P30162 Actin-1 Onchocerca volvulus 1 321 Cell motility P30163 Actin-2 Onchocerca volvulus 2 245–367 Cell motility NP_508842 ACTin family member (act-4) Caenorhabditis elegans 3 146–180 Cell motility 1D4X_A Chain A, crystal structure of Mg-ATP actin Caenorhabditis elegans 1 355 Cell motility AAF32254 Heat shock protein 70 Wuchereria bancrofti 4 285–679 Stress response CAA61152 Small heat shock protein Brugia pahangi 4 86–116 Stress response AAB08736 Small heat shock protein p27 Dirofilaria immitis 1 203 Stress response CAA48632 OV25-1 protein Onchocerca volvulus 5 123–232 Stress response XP_001896281 Enolase Brugia malayi 6 115–427 Glycolysis AAB52600 Fructose-bisphosphate aldolase Onchocerca volvulus 7 112–248 Glycolysis XP_001899850 Glyceraldehyde-3-phosphate dehydrogenase Brugia malayi 4 100–349 Glycolysis AAV33247 Phosphoglycerate mutase Onchocerca volvulus 2 152–158 Glycolysis XP_001891892 Phosphoglycerate kinase Brugia malayi 3 104–149 Glycolysis EDP29666 Glucose phosphate isomerase Brugia malayi 2 128–133 Glycolysis XP_001897269 Triosephosphate isomerase Brugia malayi 4 163–448 Glycolysis XP_001900208 Lactate dehydrogenase Brugia malayi 1 113 Anaerobic glycolysis XP_001900957 Fumarase Brugia malayi 3 91–200 Aerobic metabolism A8NLA3 Hypothetical FAD-dependent oxidoreductase Brugia malayi 3 123–153 Electron transport XP_001901495 Nucleoside diphosphate kinase Brugia malayi 2 136–139 Nucleotide metabolism XP_001897743 Oxidoreductase, aldo/keto reductase family protein Brugia malayi 1 130 Redox process AAC24752 Transglutaminase precursor Dirofilaria immitis 3 109–115 Redox homeostasis CAE11787 Protein disulfide isomerase Brugia malayi 1 234 Redox homeostasis AAC38831 Thioredoxin peroxidase Dirofilaria immitis 2 118–124 Redox homeostasis P52033 Glutathione peroxidase, Di29 precursor Dirofilaria immitis 1 121 Oxidative stress response CAA73325 Glutathione transferase Brugia malayi 1 99 Detoxification AAC47233 Cyclophilin Ovcyp-2 Onchocerca volvulus 2 118–231 Protein folding AA799423 Peptidylprolyl isomerase Taenia solium 1 119 Protein folding XP_001902628 Bmcyp-2 Brugia malayi 1 144 Protein folding EDP37909 Rab GDP dissociation inhibitor alpha Brugia malayi 1 596 Protein transport BAA96354 Phosphatidyl-ethanolamine-binding protein Dirofilaria immitis 2 129–230 Signal transduction XP_001899662 OV-16 antigen precursor Brugia malayi 1 97 Signal transduction AAZ42332 G protein subunit Caenorhabditis remanei 1 134 Signal transduction AAF37720 Galectin Dirofilaria immitis 5 132–203 Immune response XP_001899521 Disorganized muscle protein 1 Brugia malayi 3 358–439 Cell adhesion Q27384 Pepsin inhibitor Dit33 precursor Dirofilaria immitis 4 178–379 XP_001670614 Hypothetical protein CBG05397 Caenorhabditis briggsae 4 85–140 AAD11968 P22U Dirofilaria immitis 2 510–582 BAA02004 Neutrophil chemotactic factor precursor Dirofilaria immitis 2 124 D. repens proteins ABH11671 Actin Chara contraria 3 128–145 Cell motility XP_001898433 Actin 2 Brugia malayi 2 485–541 Cell motility NP_508842 ACTin family member (act-4) Caenorhabditis elegans 8 132–274 Cell motility XP_001898461 Troponin family protein Brugia malayi 1 101 Cell motility XP_001895017 Heat shock 70-kDa protein C precursor Brugia malayi 2 124–138 Stress response XP_001896281 Enolase Brugia malayi 5 87–226 Glycolysis AAB52600 Fructose-bisphosphate aldolase Onchocerca volvulus 6 101–129 Glycolysis XP_001899850 Glyceraldehyde-3-phosphate dehydrogenase Brugia malayi 3 101–125 Glycolysis XP_001901359 Proteasome subunit alpha type 7-1 Brugia malayi 1 100 Protein catabolism AAC24752 Transglutaminase precursor Dirofilaria immitis 3 104–137 Redox homeostasis AAC47233 Cyclophilin Ovcyp-2 Onchocerca volvulus 1 112 Protein folding BAA96354 Phosphatidyl-ethanolamine-binding protein Dirofilaria immitis 1 110 Signal transduction XP_001898507 Immunoglobulin I-set domain-containing protein Brugia malayi 1 103 Signal transduction XP_001900812 Galectin Brugia malayi 3 98–191 Immune response XP_001899521 Disorganized muscle protein 1 Brugia malayi 3 124–328 Cell adhesion a The Mascot score is the score given as S10 log(P), where Pis the probability that the observed match would be a random event. Mascot score values above 80 are considered significant (P0.05). FAD, flavin adenine dinucleotide. Human and Animal Dirofilariasis July 2012 Volume 25 Number 3 cmr.asm.org 511 among them, and the prevalences of D. immitis among the canine populations of each zone are significantly different: 25.47% and 30.4% in the stepparic and mild climate zones, respectively; 13.57% in the dry desert climate zone; and 10% in the temperate cold climate zone (282). There is currently a scientific consensus regarding the phenomenon of climate change due to human activities (427). It is estimated that a total increase of 1.1°C to 6.4°C in temperature all over the world will be effected by the end of this century (186). Global warming affects host-parasite systems by influencing the amplification and emergence of parasite populations, inducing changes in the development and survival rates of both parasites and vectors, and altering seasonal transmission dynamics (68). With respect to dirofilariasis, climate change is lengthening annual periods of mosquito activity, shortening larval development periods, and increasing transmission in many geographical regions. Human activities such as the urbanization of wilderness areas at city peripheries and the construction of irrigation and artificial aqueduct systems also contribute to the spread of dirofilariasis by creating microhabitats that favor vector population growth in regions where such habitats do not naturally exist. The combination of those changes with climate change is driving mosquitoes into large areas of temperate regions and contributing to changes in the global distribution of dirofilariasis. One example is the Asian tiger mosquito, Ae. albopictus, which was introduced into the Americas and Europe by the commercial tire trade through tires harboring mosquito larvae and which is spreading rapidly in newly colonized areas (3,165,352) and becoming established in regions on both continents where dirofilariasis is endemic. Ae. albopictus feeds on a broad range of mammalian species, including humans, and has adapted its development to environmental conditions in temperate areas (120,361). Ae. albopictus has been shown to be a natural vector for both D. immitis and D. repens in Italy and for D. immitis in Taiwan and the United States (79,221,235). It is likely that Ae. albopictus vector activity for Dirofilaria will continue to spread; indeed, its introduction into other areas where dirofilariasis is endemic, such as the Mediterranean coasts of France and Spain, has already been observed (359,375). The stable presence of this vector increases the risk of infection for animals and humans because its diurnal activity adds to the nocturnal activity of indigenous mosquito species (74). The variety and abundance of vector species, their feeding predilections and activity patterns, the numbers of parasitic mosquitoes that survive infection, and the number of larvae that complete development to L3 are all factors that determine the efficiency of dirofilariasis transmission in a given area (75). For example, C. pipiens, which has been found to be infected by Dirofilaria spp., is highly abundant in various regions of endemicity in Europe and Asia, and its populations remain active throughout the summer; consequently, C. pipiens is considered a potential primary vector for dirofilariasis wherever it resides (10,30,286), although other species in the same areas can contribute to transmission. Therefore, the influences of all of these factors must be evaluated together. When mosquitoes are abundant, a reduction of the number of reservoirs is not sufficient to reduce the risk of infection. Otherwise, the spread of the disease across previously infectionfree regions permits parasite introduction into naïve canine populations, which are less resistant than those residing in regions of endemicity. Novel tools for the study and prediction of changes in the distribution of dirofilariasis. Because climate plays a fundamental role in the epidemiology of vector-borne diseases, including dirofilariasis, and considering the trend of increasing environmental temperatures, it is necessary to apply surveillance protocols to detect those changes and apply adequate preventive measures when needed (413). Geographic Information Systems (GIS) comprises tools for gathering, analyzing, and presenting spatial data, and remote sensing (RS) is a technique that allows the study of objects without direct contact via image capture. These systems have two important applications to health research: (i) disease mapping and (ii) the development of predictive models for disease transmission dynamics (356). Both of these applications are important as a basis for decision making regarding the control of parasitic diseases. As mentioned above, L3 development progresses exclusively at temperatures above 14°C until the accumulation of enough “degree-days” allows the maturation of infective L3 larvae. Degreedays are calculated in terms of heartworm development units (HDUs) (401). Complete L3 development requires 130 HDUs within the mosquito life span, which is estimated to be 30 days, and development will be completed independent of temperature fluctuations below the 14°C threshold during this period (245). Based on these calculations and on temperature and rainfall records from meteorological stations, together with information on localization and other temporal and geographical factors in the regions studied, a series of surveys using GIS and RS has been performed to create predictive models for dirofilariasis. Studies in Europe have allowed the determination of the number of annual D. immitis generations and the length of periods of high infection risk throughout several regions of the continent (Fig. 7)(148,149, 150). These studies have shown that summer temperatures are enough to foster the extrinsic incubation of Dirofilaria even in colder climates, if dirofilariasis were to be introduced to the reservoirs of these areas. Therefore, if the current trend of rising FIG 7 Approximate length of the transmission periods of Dirofilaria spp. per year in Europe (calculated with data from references 142 and 149). Simón et al. 518 cmr.asm.org Clinical Microbiology Reviews temperatures continues, it is estimated that within a few years, dirofilariasis could be introduced into areas in central and northern Europe where it is not currently endemic. These predictions seem to have been confirmed by epidemiological studies over the past decade that demonstrate both the emergence and the reemergence of dirofilariasis in humans and in animal reservoirs, as mentioned above. Related calculations regarding L3 development in vectors and the temperature requirements for it have been used by other authors to study the epidemiology of dirofilariasis in very specific areas. For example, Medlock et al. (270) demonstrated that in the United Kingdom, the temperatures during two summer seasons between 1995 and 2000 were warm enough to allow the complete development of Dirofilaria larvae in the southern regions, and in five of those summers, such conditions allowed L3 transmission in the areas surrounding London. Previously, Medlock et al. (269) studied the likelihood of the introduction of Ae. albopictus into England and concluded that this species could adapt to conditions in the south of the island with potential activity in 4 to 5 months per year, thereby increasing the risk of dirofilariasis transmission. Studies by Vezzani et al. (441,442) demonstrated that dirofilariasis in Argentina has a seasonal transmission pattern like that in Europe and established a potential transmission window of nearly 6 months for Buenos Aires. CLINICAL FEATURES OF HUMAN AND ANIMAL DIROFILARIASIS Heartworm Disease in Dogs, Cats, and Ferrets Dogs. Canine cardiopulmonary dirofilariasis (heartworm disease) is a serious and potentially fatal disease caused primarily by adult D. immitis worms (144,261,440,444) and their antigenic products, including Wolbachia symbiotic bacteria (216). Heartworm disease usually develops a chronic progression, first showing vascular and pulmonary effects and eventually affecting the right chambers of the heart (Fig. 8)(432). The first lesions occur on the walls of the pulmonary arteries and are key to the subsequent development of pulmonary and cardiac pathology. After the arrival of worms in the pulmonary arteries, an enlargement of endothelial cells in the vascular tunica intima occurs, resulting in the narrowing of vessels (endarteritis). Additionally, intercellular spaces increase, cells begin to deform (their longitudinal axes are modified due to mechanical trauma) (440), and the elasticity of the arterial walls is altered (193). The damaged arterial surface facilitates the passage of albumin, plasma, and blood cells into the perivascular space, stimulating the proliferation of smooth muscle cells in the vascular tunica media, which migrate to the lumen and induce intravascular villus formation (proliferative pulmonary endarteritis) (Fig. 9A), to which endothelial-like cells and collagen fibers also contribute (72,351). The severity of villus proliferation is related directly to the length of time of the infection, the parasite load, and the strength of the host’s immune response. The arterial wall becomes rough and velvety in appearance, with a concomitant reduction of both the lumen and the compliance of the pulmonary arteries (440,444). Pulmonary disease develops subsequent to these vascular changes. Fluids and proteins that diffuse across damaged vascular walls cause edema and inflammation of the lung parenchyma. Vascular lesions can lead to the rupture of lung vessels due to an abrupt increase in the cardiac load associated with exertion and due to hemoptysis or severe lung hemorrhage. Concurrent with the chronic progression of the disease, the death of worms, either spontaneously or by treatment with filaricides, causes acute adverse events characterized by thromboembolism (Fig. 9B) and severe inflammation, which threaten the survival of the affected animal. The alteration of the arterial wall and the reduction in the luminal diameter of pulmonary arteries, which may be occluded by villi, thrombi, and/or the presence of worms, in conjunction with inflammatory mediators, can lead to pulmonary hypertension. This generates an overload of the right side of the heart, inducing cor pulmonale, a congestive right heart insufficiency with corresponding hypertrophy and dilation (Fig. 10A) that is worsened by changes in the tricuspid valve. In congestive right heart failure, there is generalized venous congestion as a consequence of increased systemic venous pressure. D. immitis also causes severe renal dysfunction. Membranous glomerulonephritis has been described to be the result of alterations in the glomerular basal membrane in heartworm disease (2, 166,203,246). Glomerulonephritis is associated with the formation of immune complexes spurred by antigens from microfilariae, larvae, and adult worms, with the presence of microfilariae exacerbating the condition (321). Renal lesions can progress to severe nephrosis induced by proteinuria with renal insufficiency and azotemia. A serious condition that is most frequently observed in small dogs is vena cava syndrome (VCS). This occurs via the displacement of a mass of worms from the pulmonary arteries to the right ventricle, where they interfere with the kinetics and function of the tricuspid valve, resulting in increased pressure in the right ventriFIG 8 Progress of heartworm disease in dogs. The disease usually has a chronic progression. Initially, the damages affect the arteries, spreading later to the lung parenchyma and the right heart chambers. The simultaneous death of many worms contributes to the acute presentation. Human and Animal Dirofilariasis July 2012 Volume 25 Number 3 cmr.asm.org 519 cle and the obstruction of the valve lumen and circulating blood, thereby producing tricuspid insufficiency (144). These factors produce both volumetric and pressure overloads in the right atrium and the caudal vena cava, with a significant elevation in venous pressure and difficulty in return circulation. This situation often leads to the death of the animal due to hemolysis, hemoglobinuria, and disseminated intravascular coagulation (DIC). Some dogs with occult dirofilariasis exhibit eosinophilic pneumonia that produces severe respiratory distress. This respiratory syndrome is caused by an eosinophilic inflammatory reaction to microfilarial antigens, which leads to alveolar dysfunction and impaired gas exchange, resulting in hypoxemia, hypoxia, and severe respiratory insufficiency. Dirofilariae can produce lesions in other organs due to aberrant localizations, including the brain, liver, eyes, and peritoneal cavity. Many dogs do not manifest symptoms of these ailments for months or years, depending on parasite loads, individual reactivity, and animal exertion; arterial damage is greatest among animals that exercise vigorously. A frequent symptom in dogs is a persistent, chronic, unproductive cough, which increases with exercise, followed by moderate or severe dyspnea and/or stress tachypnea. Respiratory insufficiency increases with disease progression. Infected animals can present with epistaxis, hemoptysis, or pulmonary hemorrhage, which, if severe, can result in hypovolemic shock. Infected dogs exhibit intolerance to exercise and weakness that may be accompanied by syncope associated with excitement or increased physical activity. When congestive heart failure develops, ascites, peripheral edema, hydrothorax, and hydropericardium appear. Venous ingurgitation (jugular, cutaneous, episcleral, or retinal) and visceral congestion also occur, leading to varied symptomatologies. At the hepatic level, venous congestion causes hepatomegaly. This hepatopathy leads to liver failure, which may be accompanied by jaundice, increased levels of transferases, and coagulation disorders. Congestive splenomegaly also develops. Sudden death is rare but can occur as a consequence of cardiorespiratory insufficiency, cachexia, or severe thromboembolic phenomena. VCS produces orthopnea with severe respiratory insufficiency (hypoxemia/hypoxia), pansystolic FIG 9 Pathological alterations in pulmonary arteries associated with canine heartworm disease. (A) Surface of the vascular endothelium of a pulmonary artery from a dog with heartworm disease showing well-developed intravascular villi (yellow arrow). The black arrow indicates the presence of an adult worm. (B) Large thromboembolism (yellow arrow) in a pulmonary artery of a dog that died of cardiopulmonary dirofilariasis. (Courtesy of L. Venco, Clinica Veterinaria Citta `di Pavia, Pavia, Italy.) FIG 10 X-ray images of canine and feline heartworm disease. (A) Lateral thoracic radiograph of an 8-year-old dog heavily parasitized by D. immitis. Cardiomegaly on the right heart (white arrow), a mixed pulmonary pattern (red arrow), enlarged pulmonary vessels (blue arrow), perihilar edema (yellow arrow), and large areas of pulmonary densification are noted. (B) Lateral thoracic radiograph of a cat with heartworm-associated respiratory disease. Note the lung air trapping as evidenced by the flattened and caudally displaced diaphragm (white arrow) and the gas-filled stomach, caused by aerophagia (red arrow). Simón et al. 520 cmr.asm.org Clinical Microbiology Reviews heart murmur with tricuspid regurgitation, and hemoglobinuria by mechanical hemolysis due to turbulence caused by the interference of the accumulated worms with the blood flow (435). Extreme fatigue and complete exercise intolerance, as well as jugular pulse, DIC, decreased appetite, and, in the end, anorexia and death by cardiogenic shock, also occur. Finally, at the renal level, oliguria due to a decrease in filtration pressure and proteinuria following congestion-induced glomerular lesions may be present. Recently, IgG antibodies against Wolbachia were detected in the urine of dogs with heartworm disease, which is associated with the presence of microfilariae in renal capillaries (Fig. 11) and the release of Wolbachia when these microfilariae are destroyed (288). Cats. Heartworm infection in cats is primarily pulmonary in nature. Infected cats can be asymptomatic carriers of the parasite or can present nonspecific clinical signs, most frequently respiratory or digestive in origin, such as chronic coughing, labored breathing, and vomiting. Some infected cats die suddenly without any premonitory signs (17,239). When signs of infection are evident, they typically develop during the arrival of immature adult heartworms into the pulmonary vasculature and during the subsequent death of some of these worms, and signs may also develop during the death of adult heartworms. These signs are due to an acute vascular and parenchymal inflammatory response. Cats have specialized macrophages (pulmonary intravascular macrophages) in the capillary beds of the lung, and their activation is largely responsible for the exacerbated pulmonary reaction (119). The result is a nonfunctioning lung and an acute respiratory distress syndrome that is often misdiagnosed as asthma or allergic bronchitis but that is actually part of a syndrome known as heartworm-associated respiratory disease (Fig. 10B)(225). This term was coined to describe the lesions associated with the arrival and death of immature heartworms and can also be used for cats that develop the same symptoms due to the presence of adult heartworms (116). Heartworm-associated respiratory disease must be a part of the differential diagnosis when a cat presents with respiratory signs (119). In cats with either adult or immature heartworms, significant parenchymal and airway disease is manifested radiographically and histologically (17,117). In addition, arterial and airway disease was demonstrated by Dillon et al. (117), who showed that histopathology scores for pulmonary arterioles, capillaries, bronchioles, and alveoli in Dirofilaria-positive cats are significantly different from those of healthy cats (seronegative for D. immitis). Histologically, the lung responds with eosinophil infiltrates in the parenchyma, pulmonary vasculature, and air spaces, causing pneumonitis. The pulmonary vessels may leak plasma and produce pulmonary edema, and due to the death of immature or adult heartworms, there is an increased level of activity of pulmonary intravascular macrophages. In conjunction with endotoxins from dead worms, cytokines, and possibly other inflammatory mediators, injury to type I alveolar cells occurs, with subsequent hyperplasia of type II alveolar cells (118). The result is diminished pulmonary function, hypoxemia, dyspnea, and cough; in some cats, this can lead to chronic respiratory disease. These findings provide evidence that pulmonary disease occurs in heartworminfected cats, even when the infection does not progress to the adult worm stage, by inducing a strong vascular and parenchymal inflammatory response (53,69,114). This pronounced bronchoreactivity has been hypothesized to be due to the activity of pulmonary intravascular macrophages, a component of the reticuloendothelial system that cats, but not normal dogs, possess (118). The bronchial disease in cats with heartworm-associated respiratory disease is caused by a combination of epithelial disease, the proliferation of smooth muscle cells around the bronchioles, and an altered reactivity of bronchial smooth muscle cells to stimuli or an inhibited ability to respond to a bronchodilator; in fact, there is a loss of elasticity in the bronchioles, and the decreased lumen is more the result of epithelial infiltrates and smooth muscle cell proliferation than of bronchoconstriction (116). It has been hypothesized that the damaged cuticles of worms release large quantities of antigens and cause acute systemic shock. In an experimental model of acute systemic anaphylaxis in D. immitis antigen-sensitized cats, the intravenous inoculation of parasite antigens produced acute shock similar to that described above, which included dyspnea, hypoxia, and systemic hypotension (238) and for which a link between the severity of the response and the amount of inoculated antigen was demonstrated (237). Hyperacute dirofilariasis involves severe respiratory symptoms (including respiratory insufficiency) and gastrointestinal, cardiovascular, and neurological signs (431). The most frequent symptoms include dyspnea, cough, tachypnea, vomiting that is not associated with food intake, and diarrhea. Aberrant L4 localization is more frequent in cats than in dogs, and larvae have been found in body cavities and the nervous system (16). In cases with an ectopic localization of worms, ataxia, syncope, vestibular alterations, and blindness may occur. In nonfatal cases of acute infection, cats can transition to the chronic stage or become fully asymptomatic but later revert to the chronic form of the disease. Chronic dirofilariasis is generally associated with cases in which respiratory and/or gastrointestinal symptoms predominate, leading to severe organic degradation until the onset of cachexia (151,431). There are no reports of congestive heart failure. In contrast, arterial wall lesions, although similar to those in dogs, are better endured by cats, which is attributed to the presence of fewer worms and a shorter duration of infection (115). Ferrets. The ferret (Mustela putorius) has been an important hunting animal for centuries, and it has recently begun to be regarded as a pet. Ferrets are susceptible to dirofilariasis, and they have been used as an experimental host for heartworm studies (259). In ferrets, adult D. immitis worms are frequently located in FIG 11 Anti-Wolbachia surface protein (WSP)-positive immunohistochemical reaction in a microfilaria from the kidney of a dog with heartworm disease (arrow). Human and Animal Dirofilariasis July 2012 Volume 25 Number 3 cmr.asm.org 521 the cranial and cava veins as well as the pulmonary arteries and cardiac cavities (406). The presence of very few worms can cause serious symptoms and even the death of ferrets (198). Vena cava syndrome develops quite frequently, as indicated by a study by Supakorndej et al. (407) in which 1 out of 7 infected ferrets developed this syndrome. The symptoms are quite similar to those that occur in dogs, but they progress faster. These symptoms consist of lethargy, poor appetite, exercise intolerance, pleural effusion, cyanosis, and dyspnea. Hematological abnormalities such as anemia and monocytosis have also been shown. When sudden death occurs, this is caused by pulmonary embolisms (198). Subcutaneous and Ocular Dirofilariasis in Animal Reservoirs Subcutaneous dirofilariasis in dogs is commonly associated with the presence of adult D. repens worms in subcutaneous tissues (see Fig. S2 in the supplemental material) and/or subcutaneous nodules (Fig. 12)(164), although they can occupy other sites, including the ocular conjunctiva (178). The infection usually progresses asymptomatically. Clinical manifestations have been classified into two clinical syndromes (377): multifocal nodular dermatitis, which is generally localized to the face, and prurigo papularis dermatitis. However, diverse dermatological signs that recur seasonally for years are common, such as pruritus in 100% of animals, erythema (79%), papules (62%), focal or multifocal alopecia (55%), hyperkeratosis (18%), crusting (14%), nodules (12%), acanthosis (5%), eczema (3%), pyoderma (3%), and edema (1%). Extradermic symptoms include conjunctivitis (46%), anorexia (35%), vomiting (26%), fever (25%), lethargy (20%), and lymphadenomegaly (10%) (417,418). There are no experimental data regarding pathogenic mechanisms, although these alterations and lesions have been attributed to both mechanical and immunopathological processes (164). Some reports described alterations to internal organs such as the spleen, liver, kidneys, lungs, heart, and brain that are associated with massive infections with adult worms and microfilariae (164). Cases involving intravitreous infection in dogs are unusual and may be caused by D. repens (172,178) or, rarely, by D. immitis (102). The prognosis depends on the damage caused by the worms and the degree of success of surgical extraction (102). Human Dirofilariasis Pulmonary dirofilariasis. Human pulmonary dirofilariasis is characterized by the formation of pulmonary nodules (Fig. 13) around immature adult worms that have recently molted from L4 larvae. When L4 larvae reach a small or medium branch of the pulmonary artery, they block its passage, causing embolism and localized inflammation (301). The most significant event with farreaching consequences in human pulmonary dirofilariasis management is that the discovery of such nodules is frequently misdiagnosed as a malignant lesion (391). Gross histology reveals a central clot, which often traps a worm, surrounded by a yellowish or whitish fibrous wall 1 to 3 mm thick. In many cases, histopathology exposes worm structures at various stages of decomposition in the arterial lumen, surrounded by copious inflammatory infiltrates. In some cases, only a cellular reaction is observed because worms have already been destroyed by the time the nodule is found (391). Histological studies of lung nodules caused by D. immitis have shown that such cellular infiltration comprises eosinophils, lymphocytes, and plasma cells, accompanied by a histiocytic reaction and inflammatory changes in the tissues surrounding capillaries. These events are responsible for nodule forFIG12 Canine subcutaneous dirofilariasis caused by D. repens. (A) Subcutaneous nodule in the scrotum of a male dog. (B) Adult worm in an open subcutaneous nodule. (Courtesy of Sergey Kartashov, Rostov, Russia.) FIG 13 Human pulmonary dirofilariasis. Shown is a thoracic radiograph showing a pulmonary nodule attributed to D. immitis (arrow). Simón et al. 522 cmr.asm.org Clinical Microbiology Reviews mation rather than infarction stemming from embolism formation. Necrotic regions with pulmonary artery disruption due to exiting worms are also frequently observed (12). Although single nodules appear most frequently, multiple lesions have also been described, with a maximum of five occurring in the same individual (206). In general, radiological characteristics—spherical or ovoid nodules with well-defined borders and a homogeneous density—suggest a benign profile (301). Residual calcified lesions have also been described, which are consistent with the angiocentric lesions typical of dirofilariasis (97). In previous reports, times to nodule formation of 2, 3, and 8 months have been reported (191,206,304). To date, the longest-known residence of a single nodule is 13 years (42), during which the nodule underwent calcification, whereas a 2-year follow-up of a single nodule did not reveal any modifications to its radiological features (301). These lesions often disappear with time, suggesting that pulmonary dirofilariasis can present with transient lesions (98). There is evidence that nodules tend to be most frequently found in the right lung although with no differences in lobar distribution. Nodules are commonly found in peripheral locations, usually in subpleural regions. Pulmonary dirofilariasis is detected at the highest frequencies in male adults with a mean age of 53 years, although infected patients range in age from 10 to 79 years (301). Only a small number of patients present with symptoms associated with pulmonary dirofilariasis. When these symptoms do arise, they are nonspecific and include coughing with pleural or nonpleural thoracic pain (at comparable frequencies), purulent or hemoptoic sputum with hemoptysis and dyspnea in the minority of cases, fever, and other nonspecific signs such as malaise and myalgia. Only one case was initially diagnosed as a pulmonary embolism. Nevertheless, this diagnosis is considered a possible cause of the symptoms in most symptomatic cases that show nodules on an X ray. Auscultation is almost always normal, with crepitation, stertor, and wheezing being the most frequent signs among abnormal profiles. When present, pleural effusion is of a low magnitude (301). Subcutaneous/ocular dirofilariasis. Subcutaneous dirofilariasis, which is caused by adult and preadult D. repens worms in subcutaneous tissues, presents as a subcutaneous nodule (Fig. 14A and B) that grows gradually over a period of weeks or months. It has a firm, elastic consistency and is associated with erythema. Histology reveals four types of nodules, with diverse contents and characteristics (327). Although the highest incidence of subcutaneous cases occurs in individuals aged 40 to 49 years, infections in patients of all ages have been described, most notably in Sri Lanka, where 33.6% of the reported infections have occurred in children under 10 years of age. In contrast to pulmonary dirofilariasis, women seem to be more susceptible to subcutaneous dirofilariasis than men (55.4% versus 44.6%). The percentage of reported cases of ocular dirofilariasis has been increasing in recent years. Between 30% and 35% of D. repens-related infections occur in ocular regions (orbital zone, eyelids, and subconjunctival and intravitreous tissues) (Fig. 14C) (148,328). Some of these cases have serious consequences, with symptoms including damaged vision, floaters, or loss of sight (148). Permanent complications, such as retinal detachment, glaucoma, opacity of the vitreous humor, crystalline lens, or other losses of visual acuity, will develop in 10% of patients (21). Additional risks and side effects are associated with the surgical extraction of worms from sensitive areas, such as the optic nerve (208). In cases with orbital localization, symptoms such as blepharedema, palpebral ptosis, and moderate ocular discomfort occur (404). Worms in the ocular conjunctiva can also cause inflammation in addition to hyperemic conjunctival tumefaction (364). Questionable paradigms related to human dirofilariasis. The traditional picture of human dirofilariasis includes three concepts: (i) D. immitis is associated with pulmonary nodules, and D. repens is associated with subcutaneous nodules and ocular locations; (ii) human infections are caused only by immature worms; and (iii) human infections are sporadic and accidental. These paradigms are currently changing as a consequence of the data obtained in the last 10 years. It is not infrequent to find worms of both species in anatomical locations distinct from those commonly associated with each species. D. immitis worms have been found in cranial, hepatic, intraocular, and mesenteric adipose tissues; testicular arteries; and conjunctival tissues (22,423), and D. repens worms have been found in the lungs, scrotum, penis, spermatic cord, epididymis, and female mammary glands (148,328). A questionable identification of the species causing dirofilariatic nodules (330) can be due to alterations in the parasite structures or to the decomposition of worms inside nodules. Under these conditions, the automatic attribution to a particular species due to the location of the nodule lesion and the use of diagnostic techniques with a reduced predictive value to confirm diagnosis, such as serological techFIG 14 Human subcutaneous and ocular dirofilariasis. (A) External appearance of a subcutaneous nodule in the ocular region. (B) Histological section of a nodule showing sections of adult D. repens worms. (C) Intravitreal location of an adult D. repens worm in a human patient. (Panels A and B courtesy of Vladimir Kartashev, University of Rostov Na Donu, Rostov, Russia; panel C reprinted from reference 208 with permission.) Human and Animal Dirofilariasis July 2012 Volume 25 Number 3 cmr.asm.org 523 niques, seem inadequate to ascertain the causative Dirofilaria species in each particular case. Regarding worm development in human patients, intact worms have been collected from subcutaneous nodules or ocular conjunctiva in some infections by D. repens (214,315,343,364). Some of the collected D. repens worms were mature females carrying intrauterine embryos or microfilariae (214,343). Moreover, at least three cases of subcutaneous human dirofilariasis showing circulating microfilariae have been reported (reviewed in reference 150). These data demonstrate that the full development and fertilization of D. repens worms in human hosts are feasible, contradicting the commonly accepted belief that Dirofilaria worms cannot fully develop in human patients. The reasons why these findings are restricted to D. repens must be carefully analyzed. It is likely that, as mentioned above, a subcutaneous/ocular localization facilitates a fast detection of the infection, being frequently referred to by the patients themselves, and as a result, many worms may still be intact upon diagnosis. In contrast, D. immitis pulmonary nodules are internal, and most are asymptomatic; thus, when they are diagnosed, they are more likely to be due to old infections, with the worms which caused the infection being unidentifiable due to the destruction and decomposition of parasite tissues. However, there are other factors that could influence the lack of reports of fully developed worms in patients with D. immitis to be considered. A recent analysis of D. immitis and D. repens proteomes and immunomes showed that D. immitis stimulates more vigorous antibody production against its energy metabolism and detoxification machinery than D. repens. If these specific antibodies can block the activity of parasitic enzymes, these data could account for the limited capacity for survival of infective D. immitis larvae over the infection time in human patients, due to worm metabolism blockage, and, consequently, could account for a lack of worm maturation in this host (158). Currently, human dirofilariasis is considered an emerging disease in some areas (214) because of the dramatic increase in the number of reported human cases (mostly subcutaneous/ocular cases) in the last 10 years. This contradicts the concept that human dirofilariasis is accidental and infrequent. Geographic expansion and the increased prevalence among canine populations are likely to run in parallel with the increase in numbers of human cases. The frequently asymptomatic character of D. repens in dogs and, thus, the lack of its clinical diagnosis could contribute to its silent spread through canine reservoirs, increasing the risk of infection in humans residing in the same areas (148). Some other factors that are not related directly with the epidemiology of the disease could have contributed to the detection of an increased number of human cases in the last years. In this respect, more than 80% of all human cases have been identified in a small set of countries (see “Dirofilariasis in Human Hosts”), countries in which research groups traditionally working with human dirofilariasis are found. Not surprisingly, other countries with frequent canine dirofilariasis infections show sporadic or no reports of human infections. This shows that the medical community’s knowledge of and vigilance for these parasites can decisively influence the successful detection of human cases, the numbers of which are likely to be underestimated in many areas of endemicity. A second point of discussion is that the increase in the number of human cases is due mostly to subcutaneous/ocular reports, with few pulmonary cases being reported in Europe. This has been attributed to the presence of two different variants of D. immitis in America and in Europe, with the Old World variant exhibiting no pathogenic role in humans. Furthermore, it has been pointed out that cases attributed to D. immitis in European patients may not be well characterized and instead represent D. repens infections (330), thereby rendering D. repens almost exclusively responsible for human infections in Europe. However, some data contradict this hypothesis. For example, it was demonstrated that D. immitis worms from diverse geographical origins, including Old World and New World locations, display genetic homogeneity (38,182). In addition, some of the pulmonary cases from Spain that were determined to be caused by D. immitis based on radiological and serological evidence (96,97,98) were those of patients who had resided exclusively in regions where only D. immitis has been detected in canine populations. Nevertheless, the difference in the numbers of human cases of D. repens and D. immitis infections are unquestionable. This difference cannot be attributed solely to the easier detection and diagnosis of D. repens nodules and the missed, asymptomatic D. immitis pulmonary cases. In this respect, more accurate and complete epidemiological and clinical information should be obtained to ascertain the fact(s) to which this disparity can be attributed. Diagnosis Laboratory diagnosis. Heartworm disease in dogs is diagnosed by the detection and specific identification of microfilariae and by using tests for the detection of circulating adult worm antigens, available only for D. immitis. Microfilariae in the blood are usually detected in concentrated blood specimens by microscopy using the Knott test or other tests to concentrate microfilariae (435). However, given the variety of canine filariae, the detection of microfilariae alone does not give an accurate diagnosis, because although filarial species can be identified by an evaluation of cephalic and caudal morphologies, these features are often difficult to differentiate. Species can also be defined by the histochemical staining of anatomical regions with phosphatase activity (84, 338) and by the amplification of microfilaria DNA by PCR (130, 131). D. immitis microfilariae harbor two phosphatase activity zones near the anal and excretory pores, whereas D. repens has only one near the anal pore. Other canine filariae exhibit different, distinguishable patterns of phosphatase activity. In PCR-based identification assays, primers based on the sequence of a D. immitis cuticle antigen-encoding gene with multiple tandem repeats (342) and also based on a second, highly repetitive sequence that constitutes 3% of the D. repens genome (86) have been used. Recently, a duplex real-time PCR able to detect D. immitis and differentiate it from D. repens in dogs and mosquitoes (223) and a multiplex PCR for the simultaneous detection of filarioids in dogs (224) have been described. Highly specific and sensitive enzymelinked immunosorbent assays (ELISAs) or immunochromatography-based assays that detect circulating antigens of adult D. immitis females are commercially available for the diagnosis of cardiopulmonary dirofilariasis (389,435). Serological tests allow the detection of amicrofilaremic infections, which are not detectable by using the techniques described above. In most cases, the use of a combination of the above-mentioned techniques allows the accurate detection of dirofilariasis. A positive microfilaria test followed by a positive antigen test conclusively confirms an infection with D. immitis. If microfilaria testing is positive but antigenic testing is negative, the infection is caused by a species other than D. immitis, which can be determined by histochemical phosphatase Simón et al. 524 cmr.asm.org Clinical Microbiology Reviews testing or by PCR. This potential diagnostic alternative is also useful in cases where the infection is caused by very few female worms or when adult parasites have died by natural causes or by treatment with adulticides, which leaves only circulating microfilariae in the peripheral blood. Moreover, both the transplacental and transfusion-based transmissions of microfilariae lead to a positive microfilaria test with a negative antigen test. A positive antigen test without the detection of circulating microfilariae indicates an amicrofilaremic infection with D. immitis. Feline cardiopulmonary dirofilariasis is seldom diagnosed due to its asymptomatic nature. When diagnosed, the erratic progression of many infections and the lack of microfilariae in most cases (261) make the use of combined diagnostic techniques to diagnose the disease necessary. Antigen detection, which is regarded as the gold standard for detection in dogs due to its sensitivity and specificity, does not yield the same benefits for cats (121). In feline dirofilariasis, infections by few adult and preadult worms are common, resulting in low concentrations of antigens that can occasionally be derived exclusively from male worms and are therefore undetectable by antigen testing. Consequently, antigen detection usually produces false-negative results and underestimates the number of feline infections (40). However, it is important to highlight the diagnostic utility of antigen detection when the parasite load consists of a single female worm. Another diagnostic procedure is antibody testing, which indicates the presence of specific anti-D. immitis antibodies in infected hosts. The immune response in feline cardiopulmonary dirofilariasis is generally strong after approximately 2 m.p.i., thereby enabling the early detection of single-worm infections (346,348). In contrast to antigen-detecting tests, circulating antibody tests can overestimate the number of infected animals by yielding false-positive results (40). Postmortem examinations of animals that previously yielded negative results by antibody tests have illustrated that false-negative results also occur. Moreover, cats with cardiopulmonary dirofilariasis without clinical symptoms may be more likely to produce negative antibody tests (307). Despite these limitations, serological testing is still a highly useful tool and can be employed in combination with other techniques to increase diagnostic precision in cats. In ferrets, due to transient microfilaremia, the detection of microfilariae is not a valid method of detection in most cases. Tests for the detection of circulating antigens become positive 1 month earlier than tests for detection in dogs, probably due to their higher concentrations in the small blood volume of this host (198, 406). Diagnosis is the key point in the management of human dirofilariasis. Human pulmonary and subcutaneous/ocular dirofilariasis pose different diagnostic challenges (Fig. 15). In the case of subcutaneous nodules or an ocular localization of worms, it is usually the patient who first discovers the infection and requests medical attention. In contrast, pulmonary nodules are located deep within the body and are asymptomatic in a high percentage of cases, and only a fraction of lung nodules are accidentally identified during chest X-ray procedures (Fig. 13), which are generally performed for reasons unrelated to dirofilariasis (98). However, in both cases, when nodules are detected, a malignancy is usually suspected, which makes human dirofilariasis an essential component of the differential detection of subcutaneous and pulmonary nodules (391). During the diagnostic process, two main issues must be addressed: adequate sample collection and the accurate identification of the causal agent. In the absence of microfilariae in the blood, detection is usually performed via biopsies that determine the presence of worms in nodules. This is an invasive procedure with a high iatrogenic potential, especially for pulmonary dirofiFIG 15 Management of human pulmonary and subcutaneous dirofilariasis. Human and Animal Dirofilariasis July 2012 Volume 25 Number 3 cmr.asm.org 525 lariasis. After the biopsy is performed, successful worm identification depends on several factors. One potential problem that is highly relevant to pulmonary dirofilariasis is the degree of worm decomposition in nodules, which makes their identification more difficult. An additional issue of concern is the similar morphologies of the cuticles of various species (262). In addition, some key features that are considered for worm species identification, including cuticular ridge size, ridge numbers, and ridge-to-ridge distance, are highly variable at different body areas of the same worm and even within a single transverse section, thereby rendering these criteria unreliable for diagnosis (315). According to the authors of that study, the localization of worms with smooth cuticles in subcutaneous tissues poses problems of identification because all species of the genus Dirofilaria, especially those described to infect humans, have cuticular ridges, except D. immitis and D. lutrae. In this respect, and because D. lutrae has not been found in humans to date and the initial stages of D. immitis develop within subcutaneous tissues, subcutaneous worms with smooth cuticles are usually identified as D. immitis. Molecular and immunological techniques are currently available as an alternative or complement to morphology-based diagnostic techniques. For cases in which parasites exhibit altered morphologies due to the host reaction, parasite detection by PCR is an invaluable technique due to its high sensitivity and specificity. Positive reactions are obtained with minimal quantities of parasite DNA, even from samples conserved in different fixatives (131). Immunohistochemical staining to confirm the existence of Wolbachia or its molecules in nodules can also be helpful, because a positive reaction indicates the prior presence of Dirofilaria (390). Nevertheless, none of these techniques preclude surgical intervention to obtain biological material from nodules. Despite the small number of worms that are required to cause infection, humans demonstrate a strong antibody response. Therefore, serology is used as a complementary technique to invasive methods. Different antigen complexes have been used to detect antibodies and diagnose human dirofilariasis (372,395). ELISAs have been refined with D. immitis somatic antigen (DiSA) and excretory antigen (DiE/S) from adult worms, which can be relatively easily obtained. Nevertheless, those crude antigens can cross-react among different Dirofilaria species and with other parasitic helminths of humans, primarily Toxocara canis (agent of visceral larva migrans) (395). This problem of specificity can be resolved by using epitopes from polypeptide sequences found in antigen complexes with previously demonstrated specificity for D. immitis or D. repens (336,397). The gene encoding Di35, a 35-kDa protein previously identified and characterized in its native state by Philipp and Davis (340), has already been cloned and produced as a recombinant antigen (405). When used in an ELISA, this recombinant protein demonstrated high sensitivity and specificity for human pulmonary dirofilariasis in studies of sera from patients with tropical filariasis. A group of molecules in the 22-kDa range (known as Di22) proved to be an excellent marker for pulmonary dirofilariasis (335) and has been used successfully in the diagnosis of clinical cases by Western blotting. In ELISAs, this protein exhibits 100% sensitivity and 90% specificity, with positive and negative predictive values of 75% and 100%, respectively (336). However, given the low pretest probability, positive results in serological studies need to be supplemented with other data, such as radiography, medical history, and region of residence, before any invasive diagnostic measures are initiated (302). With respect to D. repens, many specific polypeptides have been identified in the range of 26 to 40 kDa. These molecules allow discrimination not only between subcutaneous dirofilariasis and other parasitic or nonparasitic diseases but also between clinical cases and infections without subcutaneous or ocular changes (397). Finally,there isa goodcorrelation between PCR-basedand serologybased results, as evidenced by a study of eight cases with subcutaneous nodules or ocular localization that were analyzed in parallel by using molecular techniques, ELISA, and Western blotting (78). Clinical diagnosis. Visual assessments, such as chest X ray, echocardiography, and electrocardiography, provide insights into the clinical status of each individual patient. A chest X ray provides evidence of pulmonary artery enlargement (Fig. 10A), lung parenchymal changes, and right heart cardiomegaly in advanced stages of canine dirofilariasis (435,443). This technique also allows the confirmation of the presence of pleural effusions (351), although it cannot be used to assess the parasite load (436). Using echocardiography, clinicians can visualize parasites, which are seen as two parallel hyperechoic lines in the main pulmonary artery, left and right interlobe branches, or right heart atrium and ventricle (26, 276). Doppler echocardiography enables the precise determination of the presence and severity of pulmonary hypertension. Echocardiography must be considered in cases in which clinical and radiographic features suggest a severe infection, because they provide data on the stage of the disease and the parasite load, which are key factors in the choice of appropriate therapy (435). For diseased dogs in terminal stages that exhibit severe enlargement of the right atrium, electrocardiography can reveal alterations in both the electrical axis and rhythm (deviations to the right side of the axis and atrial fibrillation) (435). Several molecules released into the blood following cell damage in capillaries and myocardial tissue (311), inadequate perfusion (56), or lysis of thrombi (153) can serve as early markers for cardiovascular ailments and thereby assist in the treatment decision-making process (56,298). Levels of troponins T and I, myoglobin, and D-dimer in dogs with heartworm disease have recently been studied (80). The preliminary results obtained indicate the feasibility of the use of troponin I and myoglobin as markers of cardiac damage and of D-dimer as a confirmatory tool for the diagnosis of pulmonary thromboembolism in dogs (80). In feline cardiopulmonary dirofilariasis, a chest X ray allows a presumptive diagnosis and the evaluation of the severity of the infection based on characteristic changes of arteries and the lung parenchyma (Fig. 10B). Experimentally infected animals present with radiographic alterations 6 months after inoculation with infectious larvae (231). The presence of interstitial pulmonary patterning and radiodense zones in the lung parenchyma with enlargement, blunting, and tortuosity of one (usually the right) or both caudal pulmonary arteries must be regarded as suggestive of cardiopulmonary dirofilariasis in cats (18). Alterations in the cardiac silhouette are rare. It is also worth noting that radiographic profiles look normal in some cases despite the infection, and alterations in radiographic profiles may be transient (384), resulting in an erroneous diagnosis. Infections produced by a single parasite can also induce alterations that are not visible by radiography (437). Thus, it is essential that clinicians consider the advantages and limitations of each diagnostic technique and use all of the resources that they have at their disposal to reach an unequivocal diagnosis. Simón et al. 526 cmr.asm.org Clinical Microbiology Reviews Echocardiography allows detection when filariae are located in anatomical regions accessible to ultrasound, such as the right heart chambers, main pulmonary artery, proximal tracts of the right and left pulmonary arteries, and the distal portion of the caudal vena cava (277). The dimensions of adult filariae are significant compared to those of the pulmonary arteries of cats, facilitating their identification (26,71,439). Finally, necropsy is recommended for suspected cases in which death preceded diagnosis or for those cases in which cardiopulmonary dirofilariasis cannot be excluded despite a negative diagnosis. In addition to the predictable anatomical locations, other regions should be examined to exclude the presence of filariae in ectopic sites, including the systemic arterial circuit, body cavity, and brain and spinal cord, in cases with neurological symptoms. Radiographic changes in ferrets typically consist of cardiomegaly and severe pleural effusion (198,406). Ultrasound can detect adult worms 5 months postinfection, 1 1/2 months earlier than in cats (384). Treatment and Prevention Heartworm disease treatment in dogs is complex and frequently risky due to the side effects of the massive destruction of worms in the bloodstream. Therefore, it is necessary to choose an appropriate therapeutic strategy, and it may be best to withhold treatment in some cases. Before treatment, the individual situation of each animal must be evaluated, considering factors such as parasite load, age, and size. Other concomitant risk factors are the severity of pulmonary disease and the restrictions on physical activity that the dog can realistically endure (122,438). Based on all of these factors, the animal’s risk of thromboembolic complications and the presence of VCS can be determined. Dogs in the low-risk group exhibit a low parasite load, the absence of lung parenchyma and/or capillary lesions, normal clinical symptomatology and chest X ray, low levels of circulating antigens or negative microfilaremic antigen tests, negative parasite tests by echocardiography, the absence of concomitant ailments, and the ability to limit physical activity. Dogs with one or more of the following conditions must be included in the high-risk group for thromboembolic complications: symptoms related to the disease (coughing, exercise intolerance, and ascites), abnormal chest X ray, high levels of circulating antigens, visualization of parasites by echocardiography, concomitant ailments, or the impossibility of limiting physical activity (435). Supportive therapy is indicated for dogs with signs of cardiopulmonary dirofilariasis for which causal therapy is not recommended or for dogs before receiving adulticide or surgical therapy. This therapeutic approach includes the administration of drugs and/or the restriction of physical activity by crating (119). Corticosteroid use, such as prednisone at 1 mg/kg of body weight daily for 4 to 5 days, can control pulmonary inflammation and thromboembolic phenomena. Diuretics can be used to reduce pulmonary edema and pleural effusion when congestive heart failure is present, and digoxin is administered exclusively to control atrial fibrillation in severe cases. Oxygen therapy is advised when the animal exhibits respiratory difficulties. Treatment with adulticides must be performed exclusively with melarsomine hydrochloride. The standard protocol dictates the administration of two intramuscular injections every 24 h in doses of 2.5 mg/kg. This two-injection protocol kills only about 90% of the adult worms. The three-dose alternate protocol (a single dose followed by two injections, which are administered at least 30 days after the initial injection and 24 h apart) kills 98% of worms; besides, the first single dose of melarsomine eliminates 90% of male parasites and 10% of females, leading to a safe 50% reduction in the overall parasite load and thereby lessening the chance for embolic complications and allowing the recovery of the organism prior to the next 2-dose regimen, which will eliminate the rest of the adult worms (7). Adulticide therapy unavoidably leads to the development of pulmonary thromboembolism, especially in cases with high systemic levels of dead parasites. The risk of this condition can be reduced by restricting physical activity for 30 to 40 days following adulticide treatment and by administering heparin and glucocorticoids when necessary (433). It is known that some macrocyclic lactones have adulticide potential (259). Ivermectin has a partial adulticide effect if administered at a dose of 6 to 12 mg/kg of body weight monthly for 16 months, and the efficiency can reach 100% if treatment is extended to 30 months (259). Nevertheless, macrocyclic lactone administration is not recommended as the therapy of choice because the adulticide effect requires an extremely long treatment period. Throughout this period, the disease continues to progress, leading to damage to the dog’s health and, potentially, the development of thrombi, which can occur unpredictably. Indeed, some researchers have observed that the health of animals treated with ivermectin for 24 months can worsen (438). Surgical therapy is performed on dogs with VCS using flexible alligator forceps introduced via the jugular vein. This instrument, guided with the aid of a fluoroscope, reaches into the right cardiac cavity and the pulmonary artery down to its lobar branches to extract the infective worms (187). The intraoperative mortality risk is very low, and survival and recovery rates are positively correlated with the number of parasites removed. Unlike adulticide treatments, the surgical extraction of filariae can potentially avoid the risk of the formation of a pulmonary thromboembolism (297). Considering the severity of the disease and the difficulty and risks of therapy in infected dogs, prophylaxis is very important. The prophylactic treatment of choice in terms of safety and efficacy consists of the administration of macrocyclic lactones such as ivermectin, milbemycinoxime, moxidectin, or selamectin (205, 260,268). Prophylaxis must start 1 month prior to the transmission period and finish 1 month after this period ends. In zones of endemicity or in regions where the climate allows transmission throughout the year, the continuous annual administration of prophylaxis is recommended. The above-mentioned drugs do not prevent the inoculation of larvae, but they do impede larval development. Testing for microfilaremia and circulating antigens is necessary in dogs undergoing chemoprophylaxis for the first time, in dogs over 1 year old without prior medical history, and at the beginning and end of each season in animals receiving routinely scheduled chemoprophylaxis (146). In addition, the American Heartworm Society recommends that animals receiving ongoing continuous chemoprophylaxis be tested once per year. Currently, researchers are debating whether the possible loss of efficacy of macrocyclic lactones is due to the development of resistance (57) or the improper use of the drug during treatment. The prevention of patent infections by D. repens with macrocyclic lactones is questionable, and to date, only continuous-release moxidectin microspheres have demonstrated full efficacy in experimental studies (152). The characteristics of feline cardiopulmonary dirofilariasis Human and Animal Dirofilariasis July 2012 Volume 25 Number 3 cmr.asm.org 527 terest in this disease and heightened vigilance in recent years among physicians and researchers. GIS and RS techniques are contributing to the precise determination of how dirofilariasis is distributed and should be used for the reliable prediction of its changes and the potential emergence of new areas of endemicity, making these techniques invaluable for the design of parasite control measures. Knowledge of the genome, proteome, biochemistry, metabolism, molting, pathogenesis, and survival mechanisms of D. immitis and D. repens is limited to date. Further in-depth studies of these topics may provide information on novel therapeutic and infection control targets and explain the differences in the relationships between these parasites and their different hosts. New tools that allow the acquisition of objective parameters to evaluate and describe the damage in infected animals are being developed. These tools should contribute to improve prognosis and thus to define appropriate guidelines for the clinical management of dirofilariasis in different hosts. Knowledge of the role of Wolbachia in immune and immunopathogenic mechanisms of dirofilariasis and in treatment constitutes part of the most significant advancement in dirofilariasis research, e.g., by giving clues to improve the quality of life of treated animals by minimizing the inflammatory side effects of treatment. Research on additional antibiotics and routes of administration will most likely lead to improvements in treatment outcomes. Moreover, research that extends our knowledge of the symbiotic relationships between Wolbachia and filariae may yield further information on how to manipulate these relationships to avoid parasite development. This should not preclude further studies focused on Dirofilaria sp. antigens, since the available data suggest that they play important roles not only in parasite survival but also in pathogenic processes. Research on the development of vaccines for dirofilariasis has not progressed since the first tests were performed with irradiated larvae (393). The great molecular complexity and large number of isoforms of many antigens and the lack of immunogenicity of some proteins in natural infections are issues that require further investigation. This knowledge will enable researchers to perform protection studies with a more rational approach aimed toward interfering with multiple key processes in the life of the parasite simultaneously. ACKNOWLEDGMENTS We are grateful to Vladimir V. Kartashev, from the Rostov on Don University, Roston, Russia, for providing and translating publications on dirofilariasis from Russia. 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Heartworm infections in dogs in Northwestern United States and British Columbia, Canada, p 15–20. In Soll MD (ed), Proceedings of the Heartworm Symposium ‘92. American Heartworm Society, Batavia, IL. 463. Zipfel PF, Schrum S, Bialonski A, Büttner DW. 1998. The peroxidoxin 2 protein of the human parasite Onchocerca volvulus: recombinant expression, immunolocalization, and demonstration of homologous molecules in other species. Parasitol. Res. 84:623–631. Fernando Simón is Full Professor of Parasitology of the Department of Animal Biology and Parasitology, Faculty of Pharmacy and Institute of Biomedical Research of Salamanca (IBSAL), University of Salamanca, Salamanca, Spain. Mar Siles-Lucas is a Research Scientist in the Spanish National Research Council in Salamanca. She graduated from the Universidad Complutense (Madrid, Spain) and worked as a Postdoctoral and research fellow at the Institute of Parasitology in Berne, Switzerland, and the Rio Grande do Sul University (Porto Alegre, Brazil). She works with helminth parasites, focusing on the development of new diagnostic and control tools. Rodrigo Morchón is Assistant Professor Doctor (Ph.D.) of the Department of Animal Biology and Parasitology, Faculty of Pharmacy and Institute of Biomedical Research of Salamanca (IBSAL), University of Salamanca, Salamanca, Spain. Continued next page Human and Animal Dirofilariasis July 2012 Volume 25 Number 3 cmr.asm.org 543 () J.A. Montoya et al.rVeterinary Parasitology 75 1998 221–226222 this disease include right ventricle enlargement, enlargement of the main, lobar and peripheral pulmonary arteries, and perivascular parenchymal disease. Dirofilariosis is a disease of world wide distribution, but the most endemic areas are those with template, tropical and subtropical climates in the planet; where mosquito populations are high and stable. Other regions with cold weather, with hot summers and with rivers, lakes and wide irrigation lands are also suitable for the development of the disease. Frequency of infection is related to life style, with male dogs being more frequently than female dogs. In recent years, several epidemiological studies have been performed in different countries. Although heartworm disease is distributed widely in the world, the higher prevalences appear in South America, Central America and Caribe, North America, Mediterranean countries, Polynesia, Australia and Japan. Spain, belonging to the mediterranean countries, is one of the enzootic regions of D. immitis. The prevalence of the disease is high in the south of the Iberian Peninsula and Ž in the Canary Islands Valladares et al., 1987; Anguiano et al., 1985; Guerrero et al., 1989; Ortega-Mora et al., 1988, 1991; Castillo et al., 1989; Perez et al., 1989; ´ . Rojo-Vazquez et al., 1990; Gutierrez et al., 1995 . Canary Islands, situated in the 28th ´ parallel, on the African Atlantic coast has the highest prevalence in Spain, but only a Ž. few surveys has been performed in Canary Islands in the past Guerrero et al., 1989 . Most veterinarians practice in Canary Islands include heartworm disease in the differential diagnosis of patients with clinical signs referable to the lungs and heart. Our objective was to determine the prevalence of D. immitis in dogs in Gran Canaria Island, from 1994 to 1996. 2. Materials and methods Ž. We studied 2034 dogs 1143 males and 891 females for D. immitis infection over Ž .Ž . three consecutive years 1994 to 1996 , 567 dogs 306 males and 261 females were Ž .Ž analysed in 1994, 779 441 males and 338 females in 1995 and 688 396 males and 292 . females in 1996. Random subjects of the canine population brought to the veterinary clinics on Gran Canaria Island for routine health care examinations or procedures were included in the survey. All dogs were 6 months old or older, and many breeds were represented. None of the dogs in the study received heartworm chemoprophylaxis or chemotherapy. The dogs were randomly selected by the veterinarians, and the samples were obtained by cephalic venipuncture and kept refrigerated until assayed in our laboratory. A record form was completed for each dog, giving pertinent identification, history and demographic data. Blood was remitted to our laboratory where a haemagglutination test Žw. Vetred Dirofilaria , Rhone Merieux was used for serologic detection of D. immitis ˆ´ antigens. Gran Canaria Island is an approximately round island of 50 km in diameter, with four Ž. isoclimate zones: a zone with a dry and stepparic climate DS , where the average temperature is higher than 188C and with a dry summer; a zone with a dry and desertic Ž. climate DD where precipitation is lesser than 18 mm, temperatures higher than 188C () J.A. Montoya et al.rVeterinary Parasitology 75 1998 221–226 223 Table 1 Sex distribution of dogs with D. immitis infection Sex 1994 1995 1996 Mean No. of No. of Rate No. of No. of Rate No. of No. of Rate No. of No. of Rate Ž. Ž. Ž. Ž. animals positives % animals positives % animals positives % animals positives % Males 380 208 54.74 459 256 55.77 359 204 56.82 1198 668 55.76 Females 380 172 45.26 459 203 44.23 359 155 43.18 1198 530 44.24 )) ))) ))) ))) ))sp-0.01; ))) sp-0.001. and a very dry summer; a zone with a temperate climate but with a hot and dry summers Ž. and mild winters TM ; and a zone with a temperate climate but with cold winters and Ž. summers with a medium temperature below q228C TC . The results were computed using the SAS statistical package. For each variable and class, means and statistical signification were computed. The means were compared among classes by means of the Student–Newman–Keuls test. 3. Results Ž. Ž. Of the 2034 dogs, 1143 56.2% males and 891 43.8% females. The sex distribution Ž .Ž of dogs in each year was 53.96% males and 46.04% males in 1994; 56.61% males and .Ž . 43.39% females in 1995; and 57.55% males and 42.45% females in 1996. Of the 2034, 28.51% of dogs ranged in age between 6 months to 3 years old, 48.18% between 3 and 6, and 23.31% were older than 6 years. Dogs distribution within the different Ž. Ž. isoclimate included 707 dogs 34.8% in the DD zone, 617 30.3% in the TM zone, 442 Ž. Ž. 21.7% in the TC zone and 268 13.2% in the DS zone. The average seroprevalence of the disease in the three years studied was 58.89% Ž . Ž. 1198r2034 . The prevalences of the disease in each year were 67.02% 380r567 in Ž. Ž. 1994; 58.92% 459r779 in 1995; and 52.18% 359r688 in 1996. A significant difference was found between males and females affected dogs, more Ž .Ž . males than female dogs were affected p-0.001 Table 1 . Age distribution of the positive dogs is detailed on Table 2. Ž. The average prevalences in the different breeds were as follows; Bardino 69, 23% , Ž. Ž. Ž. Presa canario 64, 76% , Podenco canario 63, 84% , Large crossbreed 58, 25% , Table 2 Age distribution of dogs with D. immitis infection Age 1994 1995 1996 Mean groups No. of No. of Rate No. of No. of Rate No. of No. of Rate No. of No. of Rate Ž. Ž. Ž. Ž. animals positives % animals positives % animals positives % animals positives % 0.5–3 yr 380 73 19.21 459 92 20.04 359 60 16.71 1198 225 18.78 3–6 yr 380 198 52.10 459 252 54.90 359 195 54.31 1198 645 53.84 )6 yr 380 109 28.68 459 115 25.05 359 104 28.96 1198 328 27.37 () J.A. Montoya et al. r Veterinary Parasitology 75 1998 221–226224 Table 3 Isoclimate zone distribution of D. immitis in Gran Canaria Climate 1994 1995 1996 Mean No. of No. of Rate No. of No. of Rate No. of No. of Rate No. of No. of Rate Ž. Ž. Ž. Ž. animals positives % animals positives % animals positives % animals positives % DD 133 62 46.62 306 111 36.27 268 87 32.46 707 260 36.77 ))) ))) ))) NS DS 91 72 79.12 100 80 80 77 55 71.42 268 207 77.24 ))) ))) ))) ))) TM 196 133 67.86 212 146 68.87 209 130 62.20 617 409 66.28 ))) ))) TC 147 113 76.87 161 122 75.78 134 87 64.92 442 322 72.80 ))) ))) ))) ))) Ž. Ž.Ž Medium crossbreed 45, 08% and German Shepherd 41, 74% , only breeds represent- . ing more than 5% of total dogs are included . Table 3 shows the distribution of the different isoclimatic zones of the affected dogs Ž. in Gran Canaria. The Dry and Stepparic climate DS had the highest prevalence 77.24% Ž .Ž . Ž . 207r268 p-0.001 , and the Dry and Desertic climate DD had the lowest Ž. prevalence 36.77% 260r707 throughout the study. 4. Discussion Ž. Our present survey carried out in Gran Canaria Island Canary Islands reveals the highest prevalence of dirofilariosis ever reported in Spain, in contrast to Guerrero et al. Ž. 1989 who reported Huelva as the province of Spain with the highest prevalence Ž. 36.7% . In Guerrero et al. study the prevalence of Las Palmas de Gran Canaria was 36%. Our prevalence between 52.18% and 67.02% is similar to those described in Cuba Ž .Ž Dumenigo et al., 1988 and Japan Suenaga and Kitahara, 1978; Tanaka et al., 1985; . Hatsushika et al., 1992 ; although we have never found prevalence as high described in Ž. Ž . Papua New Guinea 86% by Hamir and Onaga 1986 . We observed some decrease in the prevalence of heartworm infection from 1994 Ž. Ž. Ž. 67.02% to 1996 52.18% . In the last year 1996 , no statistically significant difference was found between positive and negative dogs. This is probably due to education of pet owners and the increased use of chemoprophylaxis. Sex distribution of affected dogs coincide with the sex distribution in the population, but there were significant differences in male and female prevalences in the three years Ž. studied. This is similar to Selby et al. 1980 , who found that non-castrated male dogs had the highest relative risk. In Gran Canaria, more male dogs live in the open air, due to their use in defence of property. They are, therefore, more likely to be bitten by Ž. mosquitoes. For many authors Selby et al., 1980; Beugnet et al., 1994 , age is an important risk factor, determined by time of exposure in the endemic area. Therefore, () J.A. Montoya et al.rVeterinary Parasitology 75 1998 221–226 225 older dogs have a higher prevalence of dirofilariosis than younger dogs. We found the Ž. highest prevalence in animals between 3 and 6 years old 53.85% average , and the Ž. lowest in animals under 3 years of age 18.78% . Most hunting and farming breeds in our survey were affected. This is probably, because these dogs are maintained primarily outdoors in rural areas, and have low chemoprophylaxis, general and nutritional care. The most affected breeds were Canarian Ž. native dogs Presa canario, Bardino and Podenco canario , which represent more than 55% of the canine population studied. Presa canario and Bardino canario are custody and defence breeds, which live outdoors, in gardens, without protection against vectors. Ž For this reason, the prevalence of D. immitis in these breeds is high 64.73% and . 69.23%, respectively . German Shepherds, commonly live outdoors, but usually owners use chemoprophylaxis and the prevalence decreased to 41.74%. In all breeds, prevalence decrease gradually year after year. Weather is a critical factor in the prevalence of the disease. Transmission depends on Ž the intermediate host, which have certain climate requirements high relative humidity . and higher than 158C average temperature . Gran Canaria Island perfectly meets these Ž. climate requirements. DD dry and desertic zone, with very low rainfall and non-existence of water, lakes or rivers, has the lowest prevalence of Gran Canaria Island. In eastern Gran Canaria, Alisios winds blow continuously, making mosquito survival more difficult. Cities and villages are found mainly in the DD zone, and the majority of the canine population is under chemoprophylaxis. Ž. DS dry and stepparial zone had the highest prevalence. All the farming activity of the island is developed in this zone, with stagnant water, hot weather with suitable Ž temperatures for development of mosquito annual average temperature higher than . 188C, in northern or north eastern coastal areas . Small villages and farm houses with Ž dogs living outdoor predominate in this zone. Prevalence in TM zone temperate . climate, with hot and dry summers and mild winters is also very high, probably due to available water and moderate temperatures. Chemoprophylaxis is not commonly used in many villages, although prevalence decreased in 1995 and 1996. The centre of Gran Canaria is located over 800 m above sea level, has a warm and regular climate, with high relative humidity. The prevalence of dirofilariosis in this area remains constant with Ž. inter annual variation below 7 points Table 3 . The importance and severity of dirofilariosis in Gran Canaria Island has been corroborated as the highest ever found in Spain. Hopefully, this data will encourage prophylaxis and result in a decreasing prevalence of D. immitis infection in Gran Canaria. New surveys must be done in coming years to verify the efficiency of this effort. Acknowledgements The authors wish to thank the Merck Sharp and Dohme AGVET for its financial assistance and the Fundacion Universitaria de Las Palmas for their aid in this study. Part ´ of the results of this study were reported in the IV Encontro dos Medicos Veterinarios ´´ das Regioes Autonomas da Madeira, Ac¸ores e Canarias in Faial, Ac¸ores, 1995. ˆ´ ´ () J.A. Montoya et al.rVeterinary Parasitology 75 1998 221–226226 References Anguiano, A., Martinez-Cruz, S., Gutierrez, P.N., 1985. Epidemiologıa de la dirofilariosis canina en la ´´ provincia de Cordoba. IV Congreso Nacional de Parasitologıa. Tenerife. ´´ Beugnet, F., Rous, V., Leurs, M., Chardonnet, L., 1994. Role du facteur age dans lXepidemiologie de la dirofilariose cardiopulmonaire du chien, choix de la date de mise en oeuvre de la chimioprevention. Rev. Ž. Med. Vet. 145 1 , 59–64. Castillo, J.A., Lucientes, J., Estevez, C., Gortazar, C., 1989. Epidemiologıa de la dirofilariosis en Zaragoza I. ´´ Estudio de la prevalencia en perro y zorro y su interrelacion. VI Congreso Nacional y I Iberico de ´´ Parasitologıa. Caceres, 128. ´´ Dumenigo, B.E., Espino, A.M., Bouza, M., 1988. Prevalencia de filariasis canina en la Isla de la Juventud. Ž. Rev. Salud Anim. 10 3 , 247–250. Guerrero, J., Rojo, F., Rodenas, A., 1989. Estudio de la incidencia de la enfermedad del gusano del corazon en ´ Ž. la poblacion canina espanola. Med. Vet. 6 4 , 217–220. ´˜ Gutierrez, J., Guerrero, J., Rodenas, A., Castella, J., Munoz, E., Ferrer, D., Florit, F., 1995. Evolucion de ´´ ˜ ´ Dirofilaria immitis en Cataluna. Med. Vet. 12, 10. ˜ Hamir, A.H., Onaga, I., 1986. Canine spirocercosis and dirofilariasis infection in Papua New Guinea. Aust. Ž. Vet. J. 63 3 , 98–99. Ž Hatsushika, R., Okino, T., Shimizu, M., Ohyama, F., 1992. The prevalence of dogs heartworm Dirofilaria . Ž. immitis infection in stray dogs in Okayama, Japan. Kawasaki Med. J. 18 3–4 , 75–83. Ortega-Mora, L.M., Ferre, I., Gomez, M., Rojo-Vazquez, F.A., 1988. Prevalencia de la infestacion por filarias ´´ ´ ´ en galgos en la zona centro de Espana. Med. Vet. 5, 433–442. ˜ Ortega-Mora, L.M., Gomez, M., Rojo-Vazquez, F., Rodenas, A., Guerrero, J., 1991. A survey of the ´ Ž. prevalence of canine filariasis in Spain. Prev. Vet. Med. 11 1 , 63–68. Ž. Perez, R., Gomez, M., Encinas, A., 1989. Canine filariasis in Salamanca northwest Spain . Ann. Trop. Med. ´´ Parasitol. 83, 143–150. Rojo-Vazquez, F.A., Valcarcel, F., Guerrero, J., Gomez, M., 1990. Prevalencia de la dirofilariosis canina en Ž. cuatro areas geograficas de Espana. Med. Vet. 7 5 , 297–305. ´˜ Selby, L.A., Corwin, R.M., Hayes, H.M. Jr, 1980. Risk factors associated with canine heartworm infection. J. Ž. Am. Vet. Med. Assoc. 176 1 , 33–35. Suenaga, O., Kitahara, S., 1978. Studies on the prevalence of Dirofilaria immitis among dogs and its vector mosquitos in Sasebo City, Nagasaki Prefecture. Trop. Med. 203, 143–151. Tanaka et al., 1985. Valladares, B., Gijon, H., Lopez-Roman, R., 1987. Presencia de Dirofilaria immitis en la isla de Tenerife. ´´ ´ Ž. Rev. Iberica Parasitol. 47 4 , 377–380. OBJETIVO 2 Conocer la prevalencia de D. immitis en perros de la isla de Tenerife y las variaciones en función de los diferentes isoclimas de la isla (años 2002-2003). ARTÍCULO Seroprevalence of canine heartworm disease (Dirofilaria immitis) in Tenerife Island: An epidemiological update. Parasitology Research, 100 (1): 103-105, 2006. 57 ORIGINAL PAPER Seroprevalence of canine heartworm disease (Dirofilaria immitis) on Tenerife Island: an epidemiological update J. A. Montoya &M. Morales &M. C. Juste &A. Bañares & F. Simon &C. Genchi Received: 1 March 2006 /Accepted: 2 June 2006 / Published online: 4 July 2006 #Springer-Verlag 2006 Abstract Blood samples from 823 dogs were tested for circulating Dirofilaria immitis antigen during a 1-year period (May 2002 to May 2003) on Tenerife Island, Canary Islands, Spain. Seroprevalence of heartworm infection was 21%. Heartworm infection was similar in males and females and was more common in dogs aged >6 years. Distribution of infection in varying climatic zones was not statistically different. Introduction Heartworm (HW) disease is a cosmopolitan parasitic infection of domestic and wild carnivores, caused by the filarial nematode Dirofilaria immitis. The adult parasite is located in the pulmonary arteries and right cardiac chambers, and infection causes severe to very severe disease in dogs and cats living in endemic areas. Areas with temperate, tropical, and subtropical climates, where mosquito populations are abundant and stable and where an active reservoir is present, are highly endemic. The frequency of infection is also related to the lifestyle of dogs and cats. In the same area, animals living outdoors show higher prevalence values than indoor dogs and cats (Glickman et al. 1984; Kramer and Genchi 2002). In Spain, high infection prevalence has been found mainly in southern regions (Pérez-Sánchez et al. 1989; Ortega-Mora et al. 1991; Guerrero et al. 1995). However, the highest HW prevalence was found in Canary Islands located at the 28th parallel on the African Atlantic coast (higher than 50%: Montoya et al. 1998). The aim of this survey was to update HW prevalence in dogs and to evaluate if the climatic factors that characterize Tenerife Island can influence the infection prevalence in dogs. Tenerife has three main isoclimatic zones: 1) a zone with a temperate climate, hot and dry summers and mild winters (TM zone), 2) a zone with temperate climate and cold winters (TC zone), and 3) a zone with a dry, desert climate and very dry summers (DD zone) (Table 1; data from Instituto de Astrofísica de Canarias). Materials and methods Eight hundred and twenty-three privately owned dogs presented to veterinary clinics from May 2002 and May 2003 for routine health examinations and procedures were included in the study. The criteria for inclusion were >6 months of age, no HW chemoprophylaxis, no previous history of heartworm infection and owner consensus to participate in the survey. Blood samples were obtained by cephalic venipuncture and were kept at +4°C until tested in Parasitol Res (2006) 100:103–105 DOI 10.1007/s00436-006-0257-1 J. A. Montoya :M. Morales :M. C. Juste :A. Bañares Department of Internal Medicine, Faculty of Veterinary Sciences, Las Palmas de Gran Canaria University, Las Palmas, Spain F. Simon Laboratorio de Parasitologia, Facultad de Farmacia, Universidad de Salamanca, Salamanca, Spain C. Genchi Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy C. Genchi (*) DIPAV, Università degli Studi di Milano, Via Caloria 10, 20133 Milano, Italy e-mail: [email protected] the laboratory. A complete record was kept for each dog, including identification (age, sex, breed), clinical history, and demographic data. Sera were examined using an ELISA test kit (Canine Heartworm Antigen, PetCheck PF IDEXX, Westbrook, Maine, USA) for detection of HW infection in dogs following manufacturer instructions. The results by age, sex, breed, and sampling zone were analyzed using the SAS statistical package (13.0). The prevalence values were compared by Chi-square test. Results Of 823 dogs, 406 (49.3%) were male and 417 (50.7%) were female. One hundred sixty-five dogs (20%) were between 6 months and 3 years of age, 387 (47%) were 4 to 6 years old, and 271 (33%) were older than 6 years of age. Three hundred sixty-three dogs (44%) were from the TM zone, 172 (21%) from the TC zone, and 288 (35%) from the DD zone. The overall seroprevalence was 21% (172 positive dogs of 823). Of the 172 seropositive dogs, 23 (13%) were 6 months to 3 years old, 70 (41%) were between 4 to 6 years old, and 79 (46%) were older than 6 years (Chisquare 17.848, p<0.001). No difference in seroprevalence was found between male dogs (84/406, 21%) and female dogs (88/417, 21%). One hundred and sixty-three dogs (95%) lived outdoors. Ninety dogs (53%) showed mild to severe symptoms of HW disease. Table 2shows the seroprevalence values within the different breeds: Podenco canario (45%), Dogo canario (previously named Presa canario) (34%), German shepherd (33%), large crossbreed (21%), medium crossbreed (18%), and Poodles (10%). Statistical analysis was performed only for those breeds accounting for more than 5% of the total sample. Seroprevalence values between large breeds (Padengo and Dogo canario, German shepherd and large crossbred), medium crossbreeds and small breeds differed significantly (Chi-square 4.684, p<0.05). Table 3reports HW seroprevalence in dogs from the different climate zones. Values were 22% in both TM and DD zones (80/363 and 64/288, respectively) and 16% in dogs from TC zone (28/172) (Chi-square 2.811, p=0.245). Discussion In the present survey, HW seroprevalence in dogs from Tenerife Island was 21%. Previous surveys have reported seroprevalences of 41.8% (Valladares et al. 1987) and 23% (Stenzernberger and Gothe 1999). Several factors can affect HW prevalence in endemic areas, not excluding owner Table 2 HW seroprevalence in dog breeds from Tenerife Island Breed 1 HW prevalence Indoor/outdoor positive dogs No. sampled No. positive dogs Within breed Within positive dogs Large breeds Podenco canario 106 48 45% 28% 0/48 Dogo canario 71 24 34% 14% 0/24 German Shepherd 64 21 33% 12% 3/18 Large crossbreed 193 41 21% 24% 1/40 Medium breed Crossbreed 142 26 18% 15% 2/24 Small breeds Poodles 43 4 10% 2% 2/2 Various breeds 204 8 4% 5% 1/9 Total 823 172 9/163 1 The statistic was performed for breeds representing more than 5% of total sample Table 1 Climate characteristics of Tenerife Island (Spain) Climatic zones Temperate/mild Temperate/cold Dry/desert Altitude m/sl North: 0–800 South: 500–1,100 North: >800 South: >1,000 0–600 Annual mean temperature °C min–max 20.1 16.6–23.5 16.1 11.7–20.6 19.9 17–22.9 Annual rainfall mm 500 464 280 Annual mean relative humidity % 70.8 68.4 64.2 104 Parasitol Res (2006) 100:103–105 compliance to chemoprophylactic treatment. In Tenerife, a commercial ivermectin formulation for canine HW prevention was first introduced in the late 1980s, but its use only became routine from the mid 1990s, mainly in companion dogs. HW preventives are usually not administered to hunting and guard dogs (Montoya and Morales, personal communication) Even though the routine use of preventives has resulted in a significant decrease of prevalence values compared to the 1987 survey (Valladares et al. 1987), no further decrease has been observed from 1999 to the present (Stenzernberger and Gothe 1999). This suggests that an active reservoir is present in the Island and it is able to maintain the HW prevalence high. The distribution of seroprevalence by sex, age (the risk of infection increases with age), exposure (inside vs outside), and breeds was in agreement with the epidemiological findings of HW infection (Glickman et al. 1984). Larger dogs are more attractive for mosquito intermediate hosts, and HW prevalence is usually higher in larger animals than in medium and small ones, as confirmed by our results. Furthermore, Padengo, Dogo, and German shepherds are frequently used as hunting or guard dogs; most live outdoors and are, therefore, exposed to a higher risk of mosquito bites. In this survey, only 9 of 172 HW seropositive dogs lived indoors. Climate is a critical factor in the prevalence of the disease. The rate of D. immitis maturation to infective thirdstage larvae (L3) in the mosquito vector(s) depends mainly on the environmental temperature, and there is a threshold of about 14°C below which development will not proceed (Fortin and Slocombe 1981; Genchi et al. 2005). Though the three main Tenerife zones studied here have different climatic characteristics, these differences are apparently not enough to influence infection prevalence. In conclusion, the data of this survey shows that canine HW disease is widely and homogeneously distributed in the canine population of Tenerife Island, and seroprevalence has remained quite high in spite of the introduction of preventive treatment more than 10 years ago. Nevertheless, because very few dogs are introduced onto the Island, and given the limited extension of the island (2,034 km 2 ), it could be of interest to involve practitioners in a control campaign to understand if and how many dogs should be treated to eradicate the parasite from the island. References Fortin JF, Slocombe JOD (1981) Temperature requirements for the development of Dirofilaria immitis in Aedes triseriatus and Ae. vexans. Mosq News 41:625–633 Genchi C, Rinaldi L, Cascone C, Mortarino M, Cringol G (2005) Is heartworm disease really spreading in Europe? Vet Parasitol 133:137–148 Glickman LT, Greve RB, Breitschwerdt EB, Mika-Grieve M, Patronek GJ, Domanski LM, Root RC, Malone JB (1984) Serologic pattern of canine heartworm (Dirofilaria immitis) infection. Am J Vet Res 45:1178–1183 Guerrero J, Rodenas A, Gutierrez Galindo J, Florit F (1995) The extension of the prevalence of Dirofilaria immitis in Cataluña, Spain. In: Soll MD, Knigh DH (eds) Proceedings of the Heartworm Symposium ‘95. American Heartworm Society, Batavia, IL, pp 73–77 Kramer L, Genchi C (2002) Feline heartworm infection: serological survey of asymptomatic cats living in northern Italy. Vet Parasitol 104:43–50 Montoya JA, Morales M, Ferrer O, Molina JM, Corbera JA (1998) The prevalence of Dirofilaria immitis in Gran Canaria, Canary Islands, Spain (1994–1996). Vet Parasitol 75:221–226 Ortega-Mora LM, Gómez-Bautista M, Rojo-Vazquez F, Rodenas A, Guerrero J (1991) A survey of the prevalence of canine filariasis in Spain. Prev Vet Med 11:63–68 Pérez-Sánchez R, Gómez-Bautista M, Encinas Grandes A (1989) Canine filariasis in Salamanca (northwest Spain). Ann Trop Med Parasitol 83:143–150 Stenzernberger R, Gothe R (1999) Arthropod borne parasitic infections and tick infestations of dogs in Tenerife, Spain. Tierarztl Praxis 27:47–52 Valladares B, Gijón H, López-Román R (1987) Presencia de Dirofilaria immitis en la isla de Tenerife. Rev Iber Parasitol 47:377–380 Table 3 HW seroprevalence in dogs from the different climate zones of Tenerife Island Climate zones No. sampled No. positive dogs HTW prevalence Indoor/outdoor positive dogs Within the area Within the positive dogs Temperate/mild (TM) 363 80 22% 47% 4/76 Temperate/cold (TC) 172 28 16% 16% 2/26 Dry/desert (DD) 288 64 22% 37% 3/61 Parasitol Res (2006) 100:103–105 105 168 J.A. Montoya-Alonso et al. / Veterinary Parasitology 173 (2010) 165–168 Cringoli, G. (Eds.), Dirofilaria immitis and D. repens in Dog and Cat and Human Infections. Rolando Editore, Italy, pp. 47–58. Genchi, C., Guerrero, J., McCall, J.W., Venco, L., 2007. Epidemiology and prevention of Dirofilaria infections in dogs and cats. In: Genchi, C., Rinaldi, L., Cringoli, G. (Eds.), Dirofilaria immitis and D. repens in Dog and Cat and Human Infections. Rolando Editore, Italy, pp. 145–162. Genchi, C., Kramer, L.H., Prieto, G., 2001. Epidemiology of canine and feline dirofilariasis: a global view. In: Simón, F., Genchi, C. (Eds.), Heartworm Infection in Humans and Animals. Ediciones Universidad de Salamanca, Spain, pp. 121–134. Genchi, C., Rinaldi, L., Cascone, C., Mortarino, M., Cringoli, G., 2005. Is heartworm disease really spreading in Europe? Vet. Parasitol. 133, 137–148. Genchi, C., Rinaldi, L., Mortarino, M., Genchi, M., Cringoli, G., 2009. Climate and Dirofilaria infection in Europe. Vet. Parasitol. 163, 286–292. Guerrero, J., Rojo, F., Rodenas, A., 1989. Estudio de la incidencia de la enfermedad del gusano del corazón en la población canina espa˜ nola. Med. Vet. 6, 217–220. Medlock, J.M., Barrass, I., Kerrod, E., Taylor, M.A., Leach, S., 2007. Analysis of climatic predictions for extrinsic incubation of Dirofilaria in the United Kingdom. Vector Borne Zoonotic Dis. 7, 4–14. Montoya, J.A., Morales, M., Ferrer, O., Molina, J.M., Corbera, J.A., 1998. The prevalence of Dirofilaria immitis in Gran Canaria, Canary Islands, Spain (1994–1996). Vet. Parasitol. 75, 221–226. Montoya, J.A., Morales, M., Juste, M.C., Corbera, J.A., 2007. Heartworm (Dirofilaria immitis) infection in dogs: current update in Spain. In: Genchi, C., Rinaldi, L., Cringoli, G. (Eds.), Dirofilaria immitis and D. repens in Dog and Cat and Human Infections. Rolando Editore, Italy, pp. 175–180. Morchón, R., Moya, I., González-Miguel, J., Montoya, M.N., Simón, F., 2010. Zoonotic Dirofilaria immitis infections in La Rioja, Northern Spain. Epidemiol. Infect. 138, 380–383. Otranto, D., Capelli, G., Genchi, C., 2009. Changing distribution patterns of canine vector borne diseases in Italy: leishmaniosis vs. dirofilariosis. Parasit. Vectors 2 (Suppl. 1), S2. Simón, F., Morchón, R., González-Miguel, J., Marcos-Atxutegi, C., SilesLucas, M., 2009. What is new about animal and human dirofilariosis? Trends Parasitol. 25, 404–409. Valladares, B., Gijón, H., López-Román, R., 1987. Dirofilaria immitis en la isla de Tenerife. Algunos datos de su fisiopatología. Rev. Ibér. Parasitol. 47, 377–380. Vezzani, D., Carbajo, A.E., 2006. Spatial and temporal transmission risk of Dirofilaria immitis in Argentina. Int. J. Parasitol. 36, 1463–1472. OBJETIVO 4 Conocer la seroprevalencia de D. immitis en la población humana y su relación con la prevalencia en perros en la isla de Gran Canaria (año 2008). ARTÍCULO Canine dirofilariosis caused by Dirofilaria immitis is a risk factor for the human population on the island of Gran Canaria, Canary Islands, Spain. Parasitology Research, 107: 1265-1269, 2010. 69 SHORT COMMUNICATION Canine dirofilariosis caused by Dirofilaria immitis is a risk factor for the human population on the island of Gran Canaria, Canary Islands, Spain Jose Alberto Montoya-Alonso &Isabel Mellado & Elena Carretón &Elena Dolores Cabrera-Pedrero & Rodrigo Morchón &Fernando Simón Received: 7 June 2010 /Accepted: 13 July 2010 /Published online: 30 July 2010 #Springer-Verlag 2010 Abstract The aim of the present study was compare the prevalence of D. immitis in dogs and seroprevalence in humans of Gran Canaria (Canary Islands, Spain) taking into consideration the four isoclimatic areas of the island. A close relationship between the prevalence of Dirofilaria immitis in dogs and the seroprevalence in humans, in each isoclimatic area, was observed. The highest seroprevalence of infection in both canine and human hosts were found in a strip of mid-range altitude with 25.47% and 30.4% in dogs and 25.66% and 29.73% in humans, respectively. The coastal zone and the highest part of the island have prevalences significantly lower. These results demonstrate that the risk of infection by D. immitis in the human population in each area is tied to the prevalence in the canine population. Physicians should be alerted to the possibility of finding cases of human pulmonary dirofilariosis amongst the inhabitants of the island. Introduction Cardiopulmonary dirofilariosis (heartworm disease, HWD) caused by Dirofilaria immitis is a vector-borne disease, affecting primarily dogs and cats from temperate and tropical areas of the world. Different species of culicid mosquitoes of the genera Culex,Aedes, and Anopheles act as vectors for the dirofilariosis. Some of these species feed indistinctly on animal reservoirs and man. Thus, in endemic areas, zoonotic infections through D. immitis can occur (Simón et al. 2009a,b). In Europe, the highest prevalences have been reported in the canine populations of the Mediterranean countries. Moreover, heartworm disease is being detected with increasing frequency in central and Northern European countries as a consequence of the global warming and travels (Genchi et al. 2005). In Spain, the distribution of canine dirofilariosis is incompletely known because epidemiological studies have not been carried out in some provinces (Guerrero et al., 1989; Montoya et al., 2007). These studies do not reveal the existence of canine D. immitis infections in the Northern provinces of the Iberian Peninsula. Nevertheless, canine infections in two of these provinces have been recently reported (Morchón et al. 2009; Simón et al. 2009b), suggesting both that the lack of data is due more to the absence of studies than to the real lack of dirofilariosis and to a probable expansion of the infection from Southern endemic provinces. The climate of Canary Islands (Spain) is very different from those of the European continent because they are off the African Atlantic coast, only 95 km from the Western Sahara. The first epidemiological studies on canine dirofilariosis in the island of Gran Canaria showed a prevalence of 36% in dogs living in the capital of the island, Las Palmas de Gran Canaria (Guerrero et al. 1989). Prevalences of 67.02%, 58.92% and 52.18% were obtained in epidemiological surveys of the whole canine population of the island (Montoya et al. 1998), J. A. Montoya-Alonso :E. Carretón :E. D. Cabrera-Pedrero Internal Medicine, Faculty of Veterinary Medicine, University of Las Palmas de Gran Canaria, 35413 Arucas, Las Palmas, Spain I. Mellado :R. Morchón :F. Simón (*) Laboratory of Parasitology, Faculty of Pharmacy, University of Salamanca, Avda. Campo Charro s/n, 37007 Salamanca, Spain e-mail: [email protected] Parasitol Res (2010) 107:1265–1269 DOI 10.1007/s00436-010-1987-7 these being the highest prevalences observed in Spain. Data supporting the existence of the other important zoonotic species (D. repens) in the canine population of the island of Gran Canaria have not been published until now. Human dirofilariosis is habitually underdiagnosed (Simón et al. 2005). The reported clinical cases reveal only a part of the human infections (Simón et al. 2009a,b) because seroepidemiological studies carried out in exposed populations show that humans contact frequently with Dirofilaria species (Prieto et al. 2000). Moreover, the habitual asymptomatic course of human infections can contribute to the fact that some cases pass undetected (Simón et al. 2005). In the Canary Islands, a recent study showed a high seroprevalence of specific anti-D. immitis IgG and IgE antibodies in the human population of the island of Tenerife (Pou-Barreto et al. 2008). Nevertheless, data regarding human dirofilariosis on the island of Gran Canaria have not been published until now, in spite of the high prevalences observed in the canine population. In the present study, we analyze the current seroprevalence and distribution of human dirofilariosis and their relationship to the distribution of heartworm infection in the canine population of the island of Gran Canaria. Methods Physiographic and climate of the island of Gran Canaria The island is situated near the African coast of the Sahara at 28°N, 15°O. It is circular in shape with a diameter of approximately 80 km. It has a volcanic peak of 1,949 m over the sea at its centre. This structure, its geographical location, and the constant presence of the trade winds determine the existence of four concentric isoclimatic zones (Fig. 1) These are ascending in altitude, from the coast to the highest central peak of the island (Montoya et al. 1998): (1) in the dry, desert climate zone (DD), between 0 and 200 m, there is less than 18 mm/year rainfall, temperatures are higher than 18°C, and summers are very dry. Intensive agriculture is located in this zone, mainly bananas and tropical fruits. (2) In the dry, stepparic climate zone (DS), between 200 and 500 m, the average temperature is higher than 18°C, and the rainfall is 500 mm/year. The atmosphere is cool and pleasant, and there are vines, cereal, and fruits cultivated. (3) In the temperate, mild climate zone, there are dry summers and mild winters (TM), between 500 and 1,100 m. Average temperatures ranges are from 12°C to 16°C. In winter, the weather is cold and wet, with cloud formation that benefits the soil by the way of dew and fog. Precipitation reaches 500 to 1,000 mm/year. This area of the island is characterized by green foliage and cereals and potatoes are grown here. (4) The temperate, cold climate zone (TC) has cold winters and summers with temperatures below 22°C, it lies between 1,100 and ,2000 m, it has summers with hot days and winters with cold nights; it snows some years, and precipitation is about 400 mm. Temperatures may often fall below 0°C. Most of the human (60%) and canine (44%) populations are in the DD zone, followed by the DS zone with the 20% and 22%, respectively, the TM zone with the 15% and 21%, the TC zone being the least populated area with the 5% and 13%, respectively. Samples analyzed We analyzed 697 privately owned dogs (352 males and 345 females) which had been taken to veterinary clinics in 2008 for routine health examinations. The sample reflects the distribution of the canine population throughout the isoclimatic areas: 302 samples were from the DD zone, 157 from the DS, 148 from the TM, and 90 from the TC. The criteria for inclusion were >5 months of age, no HWD chemoprophylaxis, no previous history of heartworm infection, and the owner’s agreement to the participation in the survey. A complete record was kept for each dog, including identification (age, sex, and breed), clinical history, and demographic dates. Serum samples from 493 humans (208 men and 285 women) taken in 2008 in a local hospital were also analyzed. By ages, 40 samples were from individuals younger than 20 years, 256 from 20 to 40 years, 142 from 40 to 60, and 55 from individuals older than 60 years. The confidentiality of the information of the patients was always maintained. Two hundred and sixty serum samples were from the DD zone, 113 from the DS, 74 from the TM, and 46 from the TC. The study was performed in accordance Fig. 1 Isoclimate zones on the island of Gran Canaria. Dry and desert climate zone (DD), dry and stepparic climate zone (DS), temperate and mild climate zone (TM), and temperate and cold climate zone (TC) 1266 Parasitol Res (2010) 107:1265–1269 with deontological regulations and the present legislation on human and animal protection. Procedures The presence of circulating antigens and microfilariae of D. immitis were studied in the dog samples. To identify canine D. immitis infections, blood samples were analyzed by the Canine Heartworm Antigen, Petcheck PF IDEXX test (Westbrook, USA), for the detection of D. immitis circulating antigens, according to manufacturer instructions. The presence or absence of microfilariae was determined by a modified Knott (Acevedo et al. 1981). Human samples were analyzed by ELISAs to detect specific anti-D. immitis IgG antibodies using adult D. immitis somatic antigens as described previously (Simón et al. 1991). Briefly, 96-well microplates were coated with 0.8 μg of an extract of D. immitis adult worms. All serum samples were analyzed at a 1:100 dilution, and the secondary antibody (anti-human immunoglobulin G peroxidase-conjugated from Merck, Germany) was used at 1:4,000 dilution. Optical densities were measured at 492 nm in an Easy Reader (Bio-Rad Laboratories, Hercules, CA, USA). The cut-offs (OD=0.8) was established by calculating the mean value ±3 standard deviations of 20 serum samples from clinically healthy blood donors living in an area free of D. immitis. Statistical analysis The data were analyzed using the SPSS Base 17.0 software for Windows. The descriptive analysis of the variables considered was carried out studying the proportions in the qualitative variables. The chi-square test was performed to compare proportions. In all the cases, the significance level was established at p<0.05. Results One hundred and thirty-five dogs out of 697 dog samples analyzed were positive using the IDEXX test. Thus, the overall prevalence of D. immitis is 19.36% of which 83 (61.48%) were microfilaremic and 52 (38.52%) were amicrofilaremic infections. There are no significant differences between males (64/352, 18.18%) and females (71/345, 20.57%). Considering the isoclimatic areas, prevalences of 13.57% in DD (56.09% of microfilaremic infections), 25.47% in DS (62.50% of microfilaremic infections), 30.4% in TM (66.66% of microfilaremic infections), and 10% in TC (55.55% of microfilaremic infections) (Fig. 2) were observed. There are no significant differences between prevalences from TM and DS, nor are there among those of DD and TC, but there are significant differences (p<0.05) between prevalences observed in TM and DS and those observed in DD and TC. Ninety-two out of 493 human serum samples analyzed were positive in ELISA for D. immitis. Thus, total seroprevalence in humans is 18.66%. There are no significant differences among men (41/208, 19.71%) and women (51/ 285, 17.89%). When the isoclimatic areas were taken into consideration, seroprevalences of 13.85% in DD, 25.66% in DS, 29.73% in TM, and 10.86% in TC (Fig. 2) were observed. No significant differences were found between seroprevalences from TM and DS, nor were there seroprevalences between DD and TC areas. Nevertheless, as what occurs in dogs, there are significant differences (p<0.05) between seroprevalences of TM and DS and those observed in DD and TC. When age was considered, the highest seroprevalence was found in the youngest part of the population (<20 years), with 25%, followed by that observed in individuals between 20 and 40 years with 18.76%, and individuals between 40 and 60 years (18.7%). The lowest seroprevalence was observed in individuals older than 60 years (10.9%). There are significant differences between all the groups (p<0.05) except between groups of 20–40 and 40–60 years. Canine prevalences and human seroprevalences found in each isoclimatic area were very similar (Fig. 2). Statistical analysis showed no significant differences. Discussion The existence of canine and feline cardiopulmonary dirofilariosis in the island of Gran Canaria is well Fig. 2 Comparative canine and human D. immitis seroprevalences in different isoclimate areas on the island of Gran Canaria. Significant differences (p<0.05) were observed in the prevalences and seroprevalences of D. immitis infections in dogs and humans from TM and DS areas by one part and those observed in DD and TC areas by the other. No significant differences were observed between prevalences in dogs and seroprevalences in humans in each isoclimatic area Parasitol Res (2010) 107:1265–1269 1267 documented (Guerrero et al. 1989; Montoya et al. 1998; Morchón et al. 2004). Nevertheless, studies focusing on human dirofilariosis have not been carried out to date. In the present study, data demonstrating the existence of human D. immitis infections and their correlation with the canine prevalences are presented. The highest D. immitis prevalences and the highest microfilaremic infection rates are concentrated in a strip of mid-range altitude (DS and TM areas), the prevalences being lower in the coastal strip (DD) and in the highest part of the island (TC). These differences can be attributed to the fact that in the mid-range altitude zone, there are suitable conditions of humidity and temperature for the development of the mosquito vector populations. In fact, there exist irrigated cultures and many ponds and reservoirs of water in this area. In addition, many hunting dogs with a high prevalence of HWD resident in these areas are not included in a prophylactic regime. In the other two areas, the environmental conditions limit both the vector populations and their period of activity. Moreover, the main urban areas are located in the DD zone; thus in this area, the highest human and canine population appear, chemoprophylaxis being habitually performed. Probably, the habitual practice of chemoprophylaxis in this part of the canine population is the main factor affecting the decrease of the prevalence of cardiopulmonary dirofilariosis in the island of Gran Canaria, from 52.18% in 1996 (Montoya et al. 1998) to 19.36% in the present study. Although this decrease will be analyzed in depth in the future, the present study demonstrates that the situation is not the same throughout the whole insular territory. Therefore, research considering the climatic and social situation in the different zones of the island should be carried out to obtain an accurate picture of the epidemiological situation, as this picture does not appear when the canine population as a whole is analyzed. The seroprevalences observed in human populations living in each area show a high correlation with canine prevalences and with microfilaremic infection rates. This demonstrates, in the first place, that the existence of canine heartworm infections on the island of Gran Canaria is a potential risk for the resident human population, as has been observed in the nearby island of Tenerife (Pou-Barreto et al. 2008) and in other endemic European areas (Prieto et al. 2000; Simón et al. 1991). It is necessary to stress that the positive serology in humans only indicates a contact with the parasite (Pou-Barreto et al. 2008). Considering the high seroprevalence, the incidence of pulmonary nodules caused by D. immitis should be studied in the human population of the island of Gran Canaria to determine the real risk of the development of pulmonary dirofilariosis. This would enable physicians to be alerted the need for inclusion of the pulmonary dirofilariosis in the differential diagnosis of pulmonary nodules. An interesting fact is that the distribution of the human seroprevalences by age is different on the island of Gran Canaria than in other endemic areas previously analyzed. The highest seroprevalence of anti-D. immitis IgGs is usually detected in the oldest half of the exposed populations (Prieto et al. 2000); but in this study, the highest seroprevalence appears in the youngest part of the population. This seems to indicate that on the island of Gran Canaria, the exposure to the parasite is very early, probably due to the limited mobility of the resident individuals. Moreover, the species of vectors implicated in the transmission and their abundance and activity are other important factors. In fact, we have previously observed D. immitis DNA in Culex theileri,a strong anthropophilic species, captured in the island of Gran Canaria (own non-published data). In conclusion, the data presented here demonstrate the existence of a high correlation between the prevalences of D. immitis in canine and human populations in the different isoclimatic areas of the island of Gran Canaria, confirming the risk of contact for people living in areas where there is canine dirofilariosis and the importance of climatic characteristics. Periodic epidemiological studies are necessary to determine changes in the prevalence of canine dirofilariosis and to evaluate the effectiveness of the preventive measures in each specific area. The existence of a high seroprevalence in humans should alert physicians to the possibility of finding pulmonary nodules caused by D. immitis among the human population of the island of Gran Canaria. Acknowledgments This research is partly supported by Agencia Canaria de Investigación, Innovación y Sociedad de la Información, Gobierno de Canarias, España (cofinanced with FEDER funds) (grant C20080100093) and by Junta de Castilla y León (grant SA090/A09) References Acevedo RA, Theis JH, Kraus JF, Longhurst WM (1981) Combination of filtration and histochemical stain for detection and differentiation of Dirofilaria immitis and Dipetalonema reconditum in the dog. Am J Vet Res 42:537–540 Genchi C, Rinaldi L, Cascone C, Mortarino M, Cringoli G (2005) Is heartworm disease really spreading in Europe? Vet Parasitol 133:137–148 Guerrero J, Rojo F, Rodenas A (1989) Estudio de la incidencia de la enfermedad del gusano del corazón en la población canina española. Med Vet 6:217–220 Montoya JA, Morales M, Ferrer O, Molina JM, Corbera JA (1998) The prevalence of Dirofilaria immitis in Gran Canaria, Canary Islands, Spain (1994–1996). Vet Parasitol 75:221–226 Morchón R, Ferreira AC, Martín-Pacho JR, Montoya A, Mortarino M, Genchi C, Simón F (2004) Specific IgG antibody response against antigens of Dirofilaria immitis and its Wolbachia endosymbiont bacterium in cats with natural and experimental infections. Vet Parasitol 125:313–321 Morchón R, Moya I, González-Miguel J, Montoya MN, Simón F (2009) Zoonotic Dirofilaria immitis infections in La Rioja, Northern Spain. Epidemiol Infect 138:380–383 1268 Parasitol Res (2010) 107:1265–1269 Montoya JA, Morales M, Juste MC, Corbera JA (2007) Heartworm (Dirofilaria immitis) infection in dogs: current update in Spain, pp: 175-180. In: Dirofilaria immitis and D. repens in dog and cat and human infections (C. Genchi, L. Rinaldi, G. Cringoli, eds.) Rolando Editore, Naples, Italy, 211 pp. Pou-Barreto C, Quispe-Ricalpe MA, Morchón R, Vázquez C, Genchi M, Postigo I, Valladares B, Simón F (2008) Galectin and aldolase-like molecules are responsible for the specific IgE response in humans exposed to Dirofilaria immitis. Parasite Immunol 30:596–602 Prieto G, Cancrini G, Muro A, Genchi C, Simón F (2000) Seroepidemiology of Dirofilaria immitis and Dirofilaria repens in humans from three areas of souther Europe. Res Rev Parasitol 60:95–98 Simón F, Muro A, Cordero M, Martín JA (1991) Seroepidemiologic survey of human dorofilariosis in Western Spain. Trop Med Parasitol 42:106–108 Simón F, López-Belmonte J, Marcos-Atxutegi C, Morchón R, Martín-Pacho JR (2005) What is happening outside North America regarding human dirofilariasis? Vet Parasitol 133:181–189 Simón F, Morchón R, González-Miguel J, Marcos-Atxutegi C, SilesLucas M (2009a) What is new about animal and human dirofilariosis? Trends Parasitol 25:404–409 Simón F, Morchón R, González-Miguel J, Rodes-Moltó D (2009b) Dirofilariosis canina en La Coruña. Galicia. Argos 106:10–12 Parasitol Res (2010) 107:1265–1269 1269 OBJETIVO 5 Estudiar la incidencia de la zoonosis en perros, gatos y humanos en las diferentes zonas isoclimáticas de Gran Canaria, y evaluar la relación entre las prevalencias de las diferentes especies (año 2010). ARTÍCULO Current prevalence of Dirofilaria immitis in dogs, cats and humans from the island of Gran Canaria, Spain Veterinary Parasitology, 176: 291-294, 2011 77 CURRENT EPIDEMIOLOGICAL STATUS OF DIROFILARIA IMMITIS IN THE PET POPULATION OF THE CANARY ISLANDS J.A. Montoya-Alonsoa, E. Carretóna, R. Morchónb, L. Silveira-Vierac, Y. Falcóna, F. Simónb a Internal Medicine, Faculty of Veterinary Medicine, Research Institute of Biomedical and Health Sciences (IUIBS), University of Las Palmas de Gran Canaria, Las Palmas de Gran Canaria, Spain. b Laboratory of Parasitology, Faculty of Pharmacy, Institute of Biomedical Research of Salamanca (IBSAL) and University of Salamanca, Salamanca, Spain. c Laboratorios Taoro, Los Realejos, Santa Cruz de Tenerife, Spain ABSTRACT Cardiopulmonary dirofilariosis (heartworm) is a zoonotic vector borne disease caused by Dirofilaria immitis which affects domestic dogs and cats. Two of the seven Canary Islands are historically hyperendemic areas of dirofilariosis, although no epidemiological study has ever been done including the other islands. The aim of the study was to complete the epidemiological status of cardiopulmonary dirofilariosis in the canine and feline population including all the Canary Islands. 1643 client-owned dogs and 707 client-owned cats were tested for D.immitis antigens (dogs), and anti-D.immitis and antiWolbachia antibodies (cats). The prevalence of canine dirofilariosis on the Canary Islands was 15.7%, and the seroprevalence of feline dirofilariosis was 18.1%. A remarkable disparity was found evaluating the results by island separately, from 0% in Lanzarote and El Hierro and low prevalences and seroprevalences in Fuerteventura (1.8% and 2.5% in dogs and cats, respectively), to the other 4 islands which ranged from 15.7% (dogs) and 14.3% (cats) in La Palma to 22.5% (dogs) and 24.1% (cats) in Tenerife. In addition, prevalences and seroprevalences were very variable inside each island, being these differences associated to local climate conditions. The distribution and prevalence of dirofilariosis in the Canary Islands is heterogeneous and related to the climate, demographic factors and management of the pets in the studied areas. Dirofilariosis remains hyperendemic in 4 of the 7 islands. Since D.immitis is a zoonosis, veterinary and sanitary authorities should be aware of the current prevalence and seroprevalence of animal dirofilariosis. The results show the need of awareness campaigns for the implementation of prophylactic measures in pets, in order to achieve a decrease in the prevalence of animal dirofilariosis in the Canary Islands. KEYWORDS Animal heartworm, Dirofilaria immitis, prevalence, epidemiology, Canary Islands INTRODUCTION Cardiopulmonary dirofilariosis (heartworm) is caused by Dirofilaria immitis. It is a vector-borne disease which mainly affects domestic dogs and cats, although it has also been described in other pets, like ferrets, and in wild carnivores. It is transmitted by different species of culicid mosquitoes of the genera Culex, Aedes and Anopheles; therefore, the prevalence of the infection is influenced by the climatic conditions: those regions with high temperatures and humidity benefit the perpetuation and reproduction of the mosquito vectors and, hence, the transmission (Carretón et al., 2012; Simón et al., 2012). 85 Canine dirofilariosis constitutes a risk for the human population since D.immitis can also be transmitted to humans, in which produce benign pulmonary nodules; these are often confused with lung cancer in X-ray and thoracic CT scans (Simón et al., 2009). Europe currently presents high prevalences of canine dirofilariosis in the southern Mediterranean countries; in addition, it has been reported a spreading of the disease towards non-endemic areas and countries previously considered free of dirofilariosis (Genchi et al., 2009; Morchón et al., 2012). Although the Canary Islands belong politically to the European Union, they are located near the Western Sahara coast. Dirofilariosis is hyperendemic in the two most populated islands, showing prevalences from 41.8% in 1987 to 21% in 2003 in dogs from Tenerife (Valladares et al., 1987; Guerrero et al., 1989; Stenzernberger and Gothe, 1999; Montoya et al., 2006) and a decrease from 67% in 1994 to 19% in 2011 in dogs from Gran Canaria (Montoya et al., 1998; Montoya-Alonso et al., 2010; 2011). With regard to the cats, a seroprevalence of 33% was found in Gran Canaria in 2011, being the first time that a higher seroprevalence in cats over prevalence in dogs has been described in the same endemic area. Since the study detected antibodies and, therefore, exposure to D.immitis, demonstrated that the feline population was at high risk of infection (Montoya-Alonso et al., 2011). A geo-environmental model for the prediction of potential transmission risk of Dirofilaria in Spain predicted the highest number of generations in the SouthWestern quadrant of the Iberian Peninsula, Mediterranean regions and the Canary and Balearic islands; furthermore, the longest period of transmission of Dirofilaria appeared in the Canary Islands where it occurs throughout the year (Simón, 2014). Despite this worrying evidence, no epidemiological study including all the Canary Islands has been ever done. For this reason, the aim of the present study was to complete the epidemiological status of cardiopulmonary dirofilariosis in the canine and feline population of the Canary Islands. METHODS 1. Location and climate of the Canary Islands The Canary Islands are situated 97 kilometres (60 miles) from the African coast of the Sahara, between the meridians 13° and 19° W and the parallels 27° and 30° N. The Archipelago consists of seven islands, all of which are volcanic in origin. The islands are from largest to smallest: Tenerife, Fuerteventura, Gran Canaria, Lanzarote, La Palma, La Gomera and El Hierro. Further demographic and geographic data are included in table 1. Overall, the climate is subtropical and desert, moderated by the sea and in summer by the trade winds. According to the position of the islands with respect to the north-east trade winds and to the altitude of each island, the climate varies from mild and wet to very dry. Indeed, different climates can be found by travelling only a few kilometres in each island, by ascending in altitude from the coast to the central peak. According to the Köppen Climate Classification (Production Department of the State Meteorological Agency of Spain, 2012), the Canary Islands present hot desert (BWh) and cold desert (BWk) climates, characterized because the evaporation exceeds precipitation on average but is less than half potential evaporation, and average temperature is >18°C (BWh) and <18ºC (BWk). The hot steppe (BSh) and cold steppe (BSk) climates are characterized because the evaporation exceeds precipitation on average but is less than potential evaporation and the difference between these two measures is less than what is found in a BW climate. Average temperature is >18°C (BSh) and <18ºC (BSk). The temperate with hot and dry summer (Csa) climate is characterized by having hot summers with the average temperature in the warmest month above 22ºC. The temperate with dry and warm summers (Csb) climate is characterized by having warm summers with the average tem86 perature in the hottest month below or equal to 22ºC, and with 4 months or more with the average temperatures above 10ºC. The areas in which the described climates are present in each island can be observed in figure 1. 2. Sample collection 1643 client-owned dogs, presented to veterinary clinics for routine health examination between May 2014 and February 2015, were included in the study. Of these, 884 were female and 759 were male; 1209 were pure-bred dogs and 434 were mixed-bred dogs. 707 client-owned cats, presented to veterinary clinics for routine health examination in the same interval of date, were included. Of these, 368 were female and 339 were male; 159 were pure-bred cats and 548 were mixed-bred cats. The criteria for inclusion of dogs and cats were being over 6 months of age, never having received treatment for heartworm disease, no previous history of heartworm infection, and owner consensus to participate in the survey. A complete record was kept for each animal, including identification by age, sex and breed; clinical history, and demographic data. The distribution by islands of dogs and cats included in the study are enumerated in table 1. The study was approved by the ethical committee of Veterinary Medicine Service of Las Palmas de Gran Canaria University and was carried out in accordance with the current European legislation on animal protection. 3. Laboratory analysis Blood samples were collected from the cephalic or jugular vein, placed in 3 ml serum tubes and centrifuged. Serum was kept at –20°C until tests were performed. All canine serum samples were analysed for circulating D. immitis antigens using a commercial immunochromatographic test kit (Urano test Dirofilaria®, Urano Vet SL, Barcelona, Spain) according to manufacturer’s instructions. Feline D. immitis infection was identified using serological techniques for anti-D. immitis and antiWolbachia antibody detection (Morchón et al., 2004) with some modifications. In brief, the plates were coated with 0.8 mg of D. immitis somatic antigen and Wolbachia surface protein (WSP). Serum samples were prepared at 1/100 for anti-D. immitis serum antibodies and 1/40 for anti-WSP antibody detection. Horseradish peroxidase-labelled anti-feline IgG antibody (Kirkegaard and Perry Laboratories, Gaithersburg, Maryland, USA), was applied at 1/4000 dilution. The optical densities were measured in an Easy-Reader (Bio-Rad Laboratories, Hercules, California, USA) at 492 nm. Cut-off points of enzyme-linked immunosorbent assay (ELISA) D. immitis 0.8 and ELISA WSP 0.6 were obtained as arithmetic mean optical density±3 standard deviations of sera of clinically healthy cats. Seropositivity was considered when animals were positive to both tests, as registered by previous epidemiological studies (Montoya-Alonso et al., 2011; 2015; Vieira et al., 2015). 4. Statistical analysis Data were analysed using SPSS Base 20.0 software for Windows (SPSS Inc./IBM, Chicago, Illinois, USA). Descriptive analysis of the considered variables was carried out considering the proportions of the qualitative variables. We performed chi-square and Fischer exact tests to compare proportions. In all cases, the significance level was established at P<0.05. 87 RESULTS The prevalence of canine dirofilariosis on the Canary Islands was 15.7%, with a greater prevalence of 17.1% in females compared with 14.1% in males (p<0.5). By age, prevalence was 16.4% for dogs <3 years, 17.1% for dogs aged 3–6 years and 12.3% for those >6 years. There were no statistically significant differences among the three groups. The results obtained by islands are illustrated in figure 2. The mean age of the infected dogs was 4.6 years, being found infected dogs from 9 months to 16 years old. By breeds, 18.5% of pure-bred dogs were infected vs 7.8% of mixed-bred dogs (p<0.05). However, when the Canary Hound was analysed separately from the rest of the breeds, a prevalence of 30.7% was obtained compared to 9.3% in the rest of the studied dogs. There were statistically significant differences between the prevalences found in the Canary Hound and the total canine prevalence (p<0.05) and between the Canary Hound and the rest of the studied breeds (p<0.05). Prevalences of Canary Hound compared with the other dogs by islands are shown in figure 3. When cats were evaluated, the seroprevalence in the Canary Islands was 18.1%. There were no statistically significant differences in the prevalence between males (18.3%) and females (17.9%). By age, seroprevalence was 20.1% for cats <3 years, 16.8% for cats aged 3–6 years and 14.6% for those >6 years There were significant differences among the three groups (p<0.05). The mean age of the seropositive cats was 3.6 years, being found seropositive cats from 6 months to 19 years old. By breeds, 20.2% of the seropositive cats were mixed-bred compared to 7.4% of pure-bred cats seropositive to D.immitis (p<0.05). The results obtained by islands are illustrated in figure 2. When climates were evaluated separately, the highest prevalences were found in the Csb climate (23.8% and 25.5% in dogs and cats, respectively). In dogs, prevalence was followed by the BS climate (21%) and the Csa climate (19.5%). In cats, the Csa climate presented the next highest prevalence (22.1%) followed by the BS climate (16.7%). There were no statistically significant differences between the canine and feline prevalences in the BS, Csa and Csb climate zones. In both dogs and cats, the lowest prevalences were found in the BW climate (2% and 13.8%, respectively). There were statistically significant differences between the BW climate and all the other described climates (p<0.05). The results found in each climate by islands are described in table 2. DISCUSSION Epidemiological studies on dirofilariosis in the Canary Islands have been conducted to the present exclusively in two islands, Gran Canaria and Tenerife (Valladares et al., 1987; Guerrero et al., 1989; Montoya et al., 1998; Stenzernberger and Gothe, 1999; Montoya et al., 2006; Montoya-Alonso et al., 2010; 2011). The present epidemiological study includes the 7 islands of the Archipelago, which reveals a remarkable disparity in the prevalences between islands and also between different areas within a same island. These differences can be attributed to the climatic singularities present in each island and to demographic factors (Montoya-Alonso et al., 2011; Production Department of the State Meteorological Agency of Spain, 2012). According to the data obtained in the present study, canine and feline dirofilariosis are absent or present with very low prevalences where the desert climate predominates, like in the easternmost Canary Islands Fuerteventura and Lanzarote. 88 Dirofilariosis was absent in El Hierro, despite its climatic conditions which are favourable for the transmission. This situation is attributed to the low population density of the island and the unusual movement of animals from other islands, which prevents from the introduction of reservoir dogs. The rest of the islands presented moderate to high prevalences of D. immitis, both in dogs and cats, predominantly in areas with steparic and temperate climates, where moisture is provided by rainfall, influence of humid trade winds and/or existence of open ponds for water storage (Simón et al., 2014). Moreover, the coastal areas present optimal climatic conditions for heartworm transmission, in addition to higher concentrations of inhabitants, since the biggest cities and towns of the islands are located there (Montoya et al., 2006; Montoya-Alonso et al., 2011). In the islands were canine dirofilariosis was present, the Canary Hound presented prevalences significantly greater, compared to the other breeds. Similar results were described in previous studies in Gran Canaria and Tenerife, being the Canary Hound considered the most important natural reservoir for D. immitis, mostly because is kept outdoors in rural areas in unhygienic conditions exposed to mosquito bites and generally do not receive preventative therapy (Montoya et al., 2006; MontoyaAlonso et al., 2010; 2011). By age, the highest prevalence was found in dogs from 3 to 6 years, similar to the results from other studies (Montoya-Alonso et al., 2011). It can be considered that the current canine epidemiological situation is more or less stable with a slightly growing trend in Gran Canaria and Tenerife: previous studies showed prevalences of 20% in 1989, 22.6% in 1999 and 21% in 2003 in Tenerife, which not differ much from the current prevalence of 22.5%(Guerrero et al., 1989; Stenzernberger and Gothe, 1999; Montoya et al., 2006). Regarding Gran Canaria, since 1994 canine dirofilariosis has declined from 67.02%, to 30.19% in 2000, 19.36% in 2008 and 19.2% in 2011 (Montoya et al., 1998; Montoya-Alonso et al., 2010; 2011) while the current result of 20.7% breaks the downward trend. Furthermore, compared to a previous study (Montoya-Alonso et al., 2011), there is a fall of the prevalence from 12% to 3.42% in the urban areas, while a high increase from 32.1% to 40% is observed in rural communities and crop areas of Gran Canaria. The most important factor influencing these changes could be the current economic crisis and the differences in the economic level of each population, since many families are terminating the veterinary services and interrupting the administration of chemoprophylaxis or giving concentrated ivermectin formulas not approved for heartworm prevention (Montoya-Alonso and Carretón, personal communication). Because the lack of previous data in La Palma and La Gomera, we cannot discuss possible changes in the distribution and prevalence of canine dirofilariosis in those islands. As in dogs, this is the first complete epidemiological study of D. immitis in cats in the Canary Islands. Only previous studies have demonstrated the existence of feline seropositivity in Gran Canaria using similar serological techniques as described in this study (Morchón et al., 2004; Montoya-Alonso et al., 2011). Feline heartworm disease has spread and increased during the past few years throughout the world (Simón et al., 2012). However, unlike in dogs, the diagnosis of feline dirofilariosis presents a challenge, mainly because cats usually show a low parasite burden, frequently leading to a negative antigen test. Since the present study was designed to detect antibodies of anti-D.immitis in feline serum samples, the results demonstrate exposure to the parasite rather than necessarily active infection and further research is required to confirm the infection with adult parasites (Litster and Atwell, 2008). Cats living in the Canary Islands showed an important rate of exposure to D. immitis in those islands where canine dirofilariosis is present and, consequently, a high risk of developing heartworm disease. For the first time, a complete epidemiological study of canine and feline dirofilariosis in the Canary Archipelago is conducted, revealing that the distribution and prevalence of the infection is related 89 to the different climates present in the islands, demographic factors and human factors related to the management of the pets. Consequently, 4 of the 7 islands are hyperendemic while dirofilariosis is absent or present a low prevalence in the other 3 islands; therefore, it is not appropriate to refer to the Canary Islands as a single hyperendemic area. The Canary Hound presents significantly higher prevalences than the other breeds, being a dangerous reservoir of the disease for both other pets and humans, considering the zoonotic aspect of the infection (Montoya-Alonso et al., 2010b). Consequently, veterinarians, physicians and owners of pets should be alerted about the need for an adequate management of dirofilariosis, including the widespread use of diagnostic tools and prophylactic measures, in order to achieve a decrease in the prevalence of animal dirofilariosis in those Canary Islands where heartworm infection is present. ACKNOWLEDGEMENTS The authors are grateful to all owners and veterinarians who kindly collaborated in this research; without them this study could not have been realized. This study has been partially supported by Urano Vet S.L. REFERENCES Carretón, E., Morchón, R., Montoya-Alonso, J.A., 2012. Dirofilariosis car-diopulmonar canina. In: Montoya-Alonso, J.A., Carretón, E. (Eds.), Dirofilariosis: Pautas de manejo clínico. Multimédica Ediciones Veterinarias, Barcelona, Spain, pp. 1–130. Genchi, C., Rinaldi, L., Mortarino, M., Genchi, M., Cringoli, G., 2009. Climate and Dirofilaria infection in Europe. Vet. Parasitol. 163, 286-292. Guerrero, J., Rojo, F., Ródenas, A., 1989. Estudio de la incidencia de la enfermedad del gusano del corazón en la población canina española. Med. Vet. 6, 217-220. Litster, A.L., Atwell, R.B., 2008. Feline heartworm disease: a clinical review. J. Feline Med. Surg. 10, 137-144. Montoya, J.A., Morales, M., Juste, M.C., Bañares, A., Simón, F., Genchi, C., 2006. Seroprevalence of canine heartworm disease (Dirofilaria immitis) in Tenerife island: an epidemiological update. Parasitol. Res. 100, 103-105. Montoya, J.A., Morales, M., Ferrer, O., Molina, J.M., Corbera, J.A., 1998. The prevalence of Dirofilaria immitis in Gran Canaria, Canary Islands, Spain (1994-1996). Vet. Parasitol. 75, 221-226. Montoya-Alonso, J.A., Carretón, E., Corbera, J.A., Juste, M.C., Mellado, I., Morchón, R., Simón, F., 2011. Current prevalence of Dirofilaria immitis in dogs, cats and humans from the island of Gran Canaria, Spain. Vet. Parasitol. 176, 291-294. Montoya-Alonso, J.A., Carretón, E., García-Guasch, L., Expósito, J., Armario, B., Morchón, R., Simón, F., 2014. First epidemiological report of feline heartworm infection in the Barcelona metropolitan area (Spain). Parasit. Vectors 7, 506. Montoya-Alonso, J.A., Carretón, E., Juste, M.C., Mellado, I., Morchón, R., Simón, F., 2010a. Epidemiological survey of canine heartworm disease on the island of Gran Canaria (Canary Islands - Spain) between 2000 and 2008. Vet. Parasitol. 173, 165-168. Montoya-Alonso, J.A., Mellado, I., Carretón, E., Cabrera-Pedrero, E.D., Morchón, R., Simón, F. , 2010b. Canine dirofilariosis caused by Dirofilaria immitis is a risk factor for the human population on the island of Gran Canaria, Canary Islands, Spain. Parasitol. Res. 107, 1265-1269. 90 Morchón, R., Carretón, E., González-Miguel, J., Mellado-Hernández, I., 2012. Heartworm Disease (Dirofilaria immitis) and Their Vectors in Europe - New Distribution Trends. Front. Physiol. 3, 196. Morchón, R., Ferreira, A.C., Martín-Pacho, J.R., Montoya-Alonso, J.A., Mortarino, M., Genchi, C., Simón, F., 2004. Specific IgG antibody response against antigens of Dirofilaria immitis and its Wolbachia endosymbiont bacterium in cats with natural and experimental infections. Vet. Parasitol. 125, 313–321. Production Department of the State Meteorological Agency of Spain, Department of Meteorology and Climatology of the Institute of Meteorology of Portugal. (Eds.), 2012. Climate Atlas of the Archipelagos of the Canary Islands, Madeira and the Azores. Agencia Estatal de Meteorología, Ministerio de Agricultura, Alimentación y Medio Ambiente, Spain, 80 pp. Available at: http://www. aemet.es/documentos/es/conocermas/publicaciones/2Atlas_climatologico/Atlas_Clima_Macaronesia___Baja.pdf Simón, F., Morchón, R., González-Miguel, J., Marcos-Atxutegi, C., Siles-Lucas, M., 2009. What is new about animal and human dirofilariosis? Trends Parasitol. 25, 404-499. Simón, F., Siles-Lucas, M., Morchón, R., González-Miguel, J., Mellado, I., Carretón, E., Montoya-Alonso, J.A., 2012. Human and animal dirofilariasis: the emergence of a zoonotic mosaic. Clin. Microbiol. Rev. 25, 507-544. Simón, L., Afonin, A., López-Díez, L.I., González-Miguel, J., Morchón, R., Carretón, E., Montoya-Alonso, J.A., Kartashev, V., Simón, F., 2014. Geo-environmental model for the prediction of potential transmission risk of Dirofilaria in an area with dry climate and extensive irrigated crops. The case of Spain. Vet. Parasitol. 200, 257-264. Stenzernberger, R., Gothe, R., 1999. Arthropod borne parasitic infections and tick infestations of dogs in Tenerife, Spain. Tierarztl. Praxis 27, 47–52. Valladares, B., Gijón, H., López-Román, R., 1987. Dirofilaria immitis en la isla de Tenerife. Algunos aspectos de su fisiopatología. Rev. Iber. Parasitol. 47, 377-380. Vieira, L., Silvestre-Ferreira, A.C., Fontes-Sousa, A.P., Balreira, A.C., Morchón, R., Carretón, E., Vilhena, H., Simón, F., Montoya-Alonso, J.A., 2015. Seroprevalence of heartworm (Dirofilaria immitis) in feline and canine hosts from central and northern Portugal. J. Helminthol. 89, 625-629. Island Area (Km2) * Inhabitants * * Density** (inhabitants/ km2) Studied Dogs Studied Cats Tenerife 2.034 889.936 437 409 166 Gran Canaria 1.560 851.157 546 478 338 La Gomera 370 20.721 56 98 36 La Palma 708 83.456 118 305 56 El Hierro 269 10.675 40 74 17 Fuerteventura 1.660 106.930 64 162 40 Lanzarote 846 141.940 168 117 54 Total 7.447 2.104.815 283 1643 707 TABLES Table 1. Distribution of the studied animals by islands and additional demographic data of the Canary Islands. Source: Instituto Canario de Estadística (ISTAC) www.gobiernodecanarias.org/istac, data from (*) Instituto Geográfico Nacional (**) Instituto Nacional de Estadística (INE). 91 DOGS Tenerife Gran Canaria La Gomera La Palma El Hierro Fuerteventura Lanzarote BW 8.3% (1/12) 3.4% (6/175) 0% (0/23) -- 0% (0/6) 1.8% (3/162) 0% (0/117) BS 26.5% (39/147) 25.6% (43/168) 20.9% (9/43) 15.2% (7/46) 0% (0/20) -- -- CSa 16% (12/75) 40% (26/65) 43.7% (7/16) 10.6% (16/151) 0% (0/6) -- -- CSb 28% (49/175) 34.3% (24/70) 0% (0/16) 23.1% (25/108) 0% (0/41) -- -- CATS Tenerife Gran Canaria La Gomera La Palma El Hierro Fuerteventura Lanzarote BW 33.3% (2/6) 19.8% (38/192) 0% (0/0) -- 0% (0/4) 2.5% (1/40) 0% (0/54) BS 8.1% (5/62) 25.9% (15/58) 22.2% (2/9) 11.1% (1/9) 0% (0/0) -- -- CSa 39.1% (9/23) 22.8% (16/70) 23.8% (5/21) 14.9% (7/47) 0% (0/6) -- -- CSb 32% (24/75) 16.7% (3/18) 0% (0/6) 0% (0/0) 0% (0/7) -- -- Table 2. Canine prevalence (%) and feline seroprevalence (%) by climates in the Canary Islands according to the Köppen Climate Classification. (positive animals/total animals studied) (--): No climate present in the island or presence of the climate not significant (BW: desert climate, BS: steppe climate, Csa: temperate with hot and dry summer, Csb: temperate with dry and warm summers). FIGURES Figure 1. (Next page up) Köppen-Geiger climate classification of the Canary Islands. Legend: BWh (hot desert), BWk (cold desert), BSh (hot steppe), BSk (cold steppe), Csa (temperate with hot and dry summer), Csb (temperate with dry and warm summers), CSc (temperate with dry and cool summers), DSc (cold without a dry season and a fresh summer). Map extracted from the Climate Atlas of the Archipelagos of the Canary Islands, Madeira and the Azores (Production Department of the State Meteorological Agency of Spain, 2012). Figure 2. (Next page down) Geographical location of the Canary Islands and prevalences obtained in each islands (D: dogs, C: cats). 92 93