Heartworm (Dirofilaria Immitis) Disease in Dogs

Mappe Parassitologiche

Registered office Veterinary Parasitology and Parasitic Diseases Department of Pathology and Animal Health Faculty of Veterinary Medicine University of Naples Federico II Via della Veterinaria, 1 80137 Naples Italy

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First edition: February 2007

ISBN 88-89132-14-0 Dirofilaria M APPE P ARASSITOLOGICHE 8 Dirofilaria Claudio Genchi, Laura Rinaldi, Giuseppe Cringoli Giuseppe Rinaldi, Laura Genchi, Claudio D. repens D. Dirofilaria immitis Dirofilaria and human infections human and Editors in dog and cat and dog in and Dirofilaria L. Rinaldi, L. . . .15 Contributors ..13 Preface ..11 Preface Editor Series G. Grandi, T.Grandi, G. Z ..47 relationships host/parasite V Gabrielli S. Cancrini, G. ..39 cats and dogs parasitizing worms filarial of Biology Genchi M. Cerbo, M.T.Di A. Manfredi, . . .19 filariosis on experience applications: health in Systems Information Geographical Pathogenesis of Pathogenesis a role for role a cats: and dogs in infections filarial of Immunopathogenesis Kramer L.H. F. A possible role for role possible A Dirofilaria feline and canine of diagnosis laboratory the for Guideline Venco,Genchi L. M. Genchi, C. repens (Nochtiella) Dirofilaria VencoL. Human dirofilariasis due to due dirofilariasis Human F.Pampiglione, S. Rivasi ..73 infections Heartworm ( Heartworm VencoL. Heartworm ( Heartworm VencoL. ..81 2000 to 1995 from literature world of update an ectors of ectors

Simón, L.H. Kramer, R. Morchón, C. Genchi C. Kramer,Morchón, L.H. R. Simón, Wol Dirofilaria infections . . . .137 infections V. Dirofilaria immitis Dirofilaria Dirofilaria immitis Dirofilaria

bachia Musella, C. Genchi, G. Cringoli G. Genchi, C. Musella, ˇ Dirofilaria ivic ˇ Wol njak, R. Beck R. njak, Index noybot ..67 endosymbiont? nematodes: biology,nematodes: and behaviour bachia p.ifcin ..59 infections spp. Dirofilaria (Nochtiella) repens (Nochtiella) Dirofilaria in the diagnosis of diagnosis the in infection in dogs and cats ..133 cats and dogs in infection ) disease in cats ..127 cats in disease ) ies ndg ..117 dogs in disease ) Dirofilaria : 7 Dirofilaria 8 Hungary, ..181 present and past in humans and carnivores in dirofilariosis of importance The Fok É. ..175 Spain in update current Dirofilaria repens Dirofilaria V.Svobodova, Genchi Z. C. Svobodova, . .198 Republic Czech the in situation actual the - dogs in Dirofilariosis V.Svobodova, Z. Dobesova, Svobodova R. . . .197 Hungary Epidemiology,Parasitology,for of Center Budapest, National Department the at diagnosed dirofilariosis human of Review Danka J. Szénási, Z. Kucsera, I. ..195 Hungary in cats and dogs of dirofilariosis on survey epidemiological an of results Preliminary F. Dell’Omodarme, P.M. Magi, Gabrielli, M. S. Calderini, ( Heartworm Corbera J.A. Juste, M.C. Morales, M. Montoya, J.A. immitis with dogs in selamectin of efficacy and Safety R. McCall, S.D. Ard, M.B. P.Mansour,Supakorndej, A.E. N. Supakorndej, J.W.M.T.McCall, W.L. Dzimianski, Steffens, ..145 cats and dogs in of prevention and Epidemiology J.W.VencoGuerrero, L. J. McCall, Genchi, C. M. Gyurkovszky M. Farkas, R. Jacsó, O. Majoros, G. Kiss, G. Fok, É. ..193 Italy) (Central in parasites filarial on update An

Macchioni, M.C. Prati, G. Cancrini G. Prati, M.C. Macchioni, infection ..163 infection Free Communications presented at the at presented Communications Free Dirofilaria immitis Dirofilaria (Zagreb, Croatia, February 22-25, 2007) 22-25, February Croatia, (Zagreb, First European First neto ndg nteCehRpbi ..199 Republic Czech the in dogs in infection Index Hack, D. Domingo D. Hack, ) infection in dogs: in infection ) Dirofilaria V Dirofilaria ulpes vulpes ulpes infections Days Dirofilaria of Tuscanyof Dirofilaria morphological characteristics ..209 characteristics morphological W W . . .208 Turkey Province, Kayseri from dogs in of aspects epidemiological and Prevalence Inci A. Atalay,Duzlu, O. O. Ica, Yildirim, A. A. ..207 Turkey Province, Bursa of area Gemlik the in disease heartworm canine of Prevalence T. ..206 Turkey in Dirofilariasis T.Ulutas, B. Karagenc Savic´-Jevc´enic´,S. VidicB. ..201 Serbia in dogs in Filariosis Miladinovic´-TasicN. Dimitrijevic´,S. Tasic´,A. Tasic´,S. V. Adamovic i lxbebse ocp nadgwt aa ydoe..211 syndrome caval with dog a in forceps basket flexible via heartworms adult of removal surgical guided - Ultrasound Voyvoda,Atasoy H. Sarierler,A. Ulutas, M. B. T. T. Ba B. Coman, S. ..202 Vojvodina - Serbia in dirofilariosis of appearances The lvi ein ugra..205 Bulgaria region, Plovdiv the in practice animal small a in dogs among Dirofilariosis Kostadinov M. ..204 Bulgaria in carnivores wild and dogs in Dirofilariosis Ivanov,Georgieva A. D. Kirkova, Z. ..203 Romania in carnivores wild and dogs in Dirofilariosis

.J. Kozek, J.A. Gonzalez Jr.,Amigo Gonzalez L.A. J.A. Kozek, .J. olbachia Civelek, A. Yildirim, A. Ica, O. Duzlu O. Ica, Yildirim,A. A. Civelek, Petrut , , N. Bercaru, A. Amfim (Padure) Amfim A. Bercaru, N. , of Dirofilaria immitis Dirofilaria ˇ cescu, T. Coman, Gh. Pârvu, C. Dinu, C. T.Pârvu, cescu, Gh. Coman, ´ Index ´, Z ´, ˇ . Grgic´,. Zoran L. : an historical perspective and perspective historical an : Dirofilaria immitis Dirofilaria ´, T.´, Ilic´, 9 Series Editor Preface 11

The last years have seen an increasing use of cartographic representation of the distribution of parasitic diseases, particularly vector-borne (arthropod- and snail-borne) diseases. Geographical Information Systems (GIS) are finding increasing use in the study of geographical epidemiology and their recent application to medical and veterinary parasitology is rapidly advancing. MAPPE PARASSITOLOGICHE is a volume series aimed primarily to pub- lish Disease Maps and parasitological field researches dealing with applications of GIS. This Volume, the number 8th of the series, after a chapter dedicated to the use of GIS in health applications with particular regard to filariosis, contains the most important topics of canine, feline and human Dirofilaria infections and free communications presented at the First European Dirofilaria Days, held in Zagreb (Croatia), 22nd – 25th February 2007. The contents deal the different aspects concerning filarial worms, specifically biology, pathogenesis, diseases, treatment of Dirofilaria immitis and D. repens parasitizing dogs and cats; their vectors; the role of Wolbachia for immunopathogenesis and diagnosis; human dirofilariosis; and epidemiological updates of dirofilariosis in Italy, Spain, Hungary, Czech Republic, Bulgaria, Romania, Serbia, and Turkey. We thank the leading European experts and colleagues from the board of the American Heartworm Society for their contributions, and Pfizer Animal Health for having supported the costs of the Volume. We hope that the information presented in this Volume serves as inspiration for scientists and practitioners to join in the further development of activity and studies on filarial worms in veterinary and human field.

Giuseppe Cringoli Series Editor Preface 13

Although significant contributions to the understanding of canine and feline heartworm disease have come from many North American scientists, Dirofilaria immitis infection in the dog was first reported in southern Europe, in the Po River Valley of Italy. In 1626, Francesco Birago published a treatise on hunting (Trattato cinegetico, ouero della caccia. V. Sfondrato, Milano, pp. 77,1626) in which he refers to the presence of the worm in the right heart of a dog and of another worm (probably Dioctophyma renale) in the kidney of the same dog. Since then, the parasite has been recognized in many countries of the world and European researchers mainly in Italy and France have been active in the study of Dirofilaria spp. infections. Climatic changes, together with an increase in the movement of cats and dogs across Europe, have caused an increase in the geographical range of Dirofilaria worms and in the risk of infection for both animals and humans. At the same time, greater attention to animal welfare and improved social and economic conditions in many countries have contributed to an increase in pet owners’ awareness of potentially life- threatening conditions, such as canine and feline heartworm disease, or of less serious but equally distressing infections like subcutaneous dirofilariosis, both in animals and humans. This manual, which has been published on the occasion of the First European Dirofilaria Days, held in Zagabria (Croatia) in February 22nd-25th 2007, is intended as a contribution to and an update of current knowledge of Dirofilaria infection from leading European experts and colleagues from the board of the American Heartworm Society. The most important aspects of Dirofilaria infections are reviewed, from biology to prevention and treatment. Several chapters offer insights into new topics such as the application of the Geographic Information Systems (GIS) to Dirofilaria distribution in Europe and the role of Wolbachia endosymbionts in host immune response. The clinical aspects of the disease are described with the aim of giving practitioners suitable guidelines for diagnosis, management, prophylaxis and treatment of the disease. The last part includes a selection of short contributions presented during the meeting in Zagreb. Finally, we would like to thank Pfizer Animal Health for having supported the costs of this publication without any constraints to the opinions expressed by the contributors.

Claudio Genchi Albert Marinculic´

President International President Organizing Scientific Comitee Comitee Contributors 15

Mary B. Ard Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

Relja Beck Veterinary Faculty, University of Zagreb, Zagreb, Croatia

Gabriella Cancrini Dipartimento di Scienze di Sanità Pubblica, Sezione di Parassitologia, Università “La Sapienza”, Roma, Italy

J.A. Corbera Facultad de Veterinaria, Animal Medicine and Surgery, Universidad de Las Palmas de Gran Canaria, Canary Islands, Spain

Giuseppe Cringoli Dipartimento di Patologia e Sanità Animale, Facoltà di Medicina Veterinaria, Via della Veterinaria 1, 80137, Napoli, Italy

Annarita Di Cerbo Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Via Celoria 10, 20133 Milano, Italy

Dan Domingo Animal Health Group, Pfizer Inc., New York, New York, USA

Michael T. Dzimianski Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA

Éva Fok Department of Parasitology and Zoology, Faculty of Veterinary Science, Szent István University H-1400 Budapest, P.O.Box 2, Hungary

Simona Gabrielli Dipartimento di Scienze di Sanità Pubblica, Sezione di Parassitologia, Università “La Sapienza”, Roma, Italy

Claudio Genchi Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Via Celoria 10, 20133 Milano, Italy

Marco Genchi Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Via Celoria 10, 20133 Milano, Italy 16 Contributors

Giulio Grandi College of Veterinary Medicine, University of Parma, Parma, Italy

Jorge Guerrero Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

Richard Hack Animal Health Group, Pfizer Inc., Lee’s Summit, Missouri, USA

M.C. Juste Facultad de Veterinaria, Animal Medicine and Surgery, Universidad de Las Palmas de Gran Canaria, Canary Islands, Spain

Laura H. Kramer College of Veterinary Medicine, University of Parma, Parma, Italy

Maria Teresa Manfredi Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Via Celoria 10, 20133 Milano, Italy

Abdelmoneim E. Mansour TRS Labs, Inc., Athens, Georgia, USA

John W. McCall Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA

Scott D. McCall TRS Labs, Inc., Athens, Georgia, USA

J.A. Montoya Facultad de Veterinaria, Animal Medicine and Surgery, Universidad de Las Palmas de Gran Canaria, Canary Islands, Spain

M. Morales Facultad de Veterinaria, Animal Medicine and Surgery, Universidad de Las Palmas de Gran Canaria, Canary Islands, Spain

Rodrigo Morchón Laboratorio de Parasitología, Facultad de Farmacia, Universidad de Salamanca, Salamanca, Spain

Vincenzo Musella Dipartimento di Patologia e Sanità Animale, Facoltà di Medicina Veterinaria, Via della Veterinaria 1, 80137, Napoli, Italy Contributors 17

Silvio Pampiglione Dipartimento di Sanità Pubblica Veterinaria e Patologia Animale, Università di Bologna, Bologna, Italy

Laura Rinaldi Dipartimento di Patologia e Sanità Animale, Facoltà di Medicina Veterinaria, Via della Veterinaria 1, 80137, Napoli, Italy

F. Rivasi Dipartimento di Scienze Morfologiche e Medico Legali, Università di Modena e Reggio Emilia, Italy

Fernando Simón Laboratorio de Parasitología, Facultad de Farmacia, Universidad de Salamanca, Salamanca, Spain

W.L. Steffens Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

Nonglak Supakorndej TRS Labs, Inc., Athens, Georgia, USA

Prasit Supakorndej Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA

Luigi Venco Clinica Veterinaria Città di Pavia, Viale Cremona 179, 27100 Pavia, Italy

Tatjana Zˇ ivicˇ njak Veterinary Faculty, University of Zagreb, Zagreb, Croatia 1 Geographical Information Systems in health applications: experience on filariosis

Laura Rinaldi, Vincenzo Musella, Claudio Genchi, Giuseppe Cringoli Geographical Information Systems 21

Introduction journals, including the current launch of Geospatial Health (www.geospatial- The concept that geographic location health.unina.it) attest to the increased can influence health is a very old one in interest in these new technologies. The medicine. As far back as the time of establishment and maintenance of Hippocrates (460-377 BC), it was websites as a platform for sharing observed that certain diseases tend to data and exchanging opinions, expe- occur in some places and not in others. riences and expertise on GIS and RS, Disease mapping and environmental with an emphasis on animal and risk assessment using geographical public health, is also worth mentioning information systems (GIS) and remote (for example http://www.gnosisgis.org). sensing (RS) technologies are now In this paper, we first summarize gene- established as basic tools in the analy- ral aspects of GIS and RS and emphasis of both human and veterinary size the most important applications of health (for a review see Cringoli et al., these tools in veterinary parasitology, 2005b). However, epidemiologists have with particular regard to filarial needed time to become convinced that worms. Then, disease mapping, ecolo- the prospects offered by GIS and RS gical analyses and predicting parasite could be useful in their own discipline. occurrence/seasonality as useful tool The last 15-20 years have seen an ever for surveillance are summarized in the increasing reliance on cartographic next section. representation of infectious diseases, particularly parasitic infections and their vectors (Bergquist, 2006). In fact, Brief history of GIS and RS during the past two decades the publication of original research articles and Depending upon the application area, reviews in veterinary and human health a number of discordant definitions of with an emphasis on GIS and/or RS GIS have appeared in the literature has followed an exponential trend (Tim, 1995). One of the most widely (Hendrickx et al., 2004). In addition, used definitions of GIS is that propo- recent GIS/RS symposia organized at sed by Burrough (1986): “a powerful national and international conferences set of tools for collecting, retrieving at and several thematic issues on this will, transforming, and displaying spa- topic published in the peer-reviewed tial data from the real world”. Overall, international literature (e.g. special a GIS is a platform consisting of hard- theme issues in Advances in ware, software, data and people and Parasitology in 2000 and 2006; encompasses a fundamental and uni- Parassitologia in 2005) demonstrate versally applicable set of value-added the wide array of applications and tools for capturing, transforming, benefits of these tools (Cringoli et al., managing, analyzing, and presenting 2005b; Rinaldi et al., 2006). information that are geographically Furthermore, the publication of thema- referenced (geo-referenced). Digital tic books pertaining GIS and RS, as GIS data may be presented in map well as international peer-reviewed form using so called data layers repre- 22 Geographical Information Systems

senting the information collected. Two often called pixels, and attribute-values approaches can be used, namely: of the objects that the cells represent (i) a vector data model, and are assigned to corresponding pixels (ii) a raster data model. (Daniel et al., 2004). Raster data are The former model stores a table con- typically utilized to represent conti- taining coordinates of points together nuous phenomena, e.g. land cover with instructions on which points are maps, phenoclimatic maps, and digital alone and which points belong to a elevation models (Fig. 2). common set. In a vector data model, all The use of GIS in epidemiology can lines are represented by chains of vec- aid in answering some important que- tors, and all areas by polygons (Fig. 1). stions such as “what is?” and “where Attributes are coded in separate tables is?”. In addition, because also the time using alphanumeric characters as a domain - i.e. “when” - is important in label for a specific class or category of most environmental and epidemiologi- properties. The raster data model uses cal processes, it has also been suggested a net of adjacent polygons (termed that GIS should be replaced by STIS, “cells”) to provide a virtual cover of a which is an abbreviation for space-time given part of a territory. The cells are information systems (Kistemann et al., 2002; Hendrickx et al., 2004). There is currently a movement towards regarding GIS as a science (geographical information science) rather than a simple technology (Goodchild, 2000; Kistemann et al., 2002). One of the first major uses for GIS was in 1964 when the Canadian Geographical Information System (CGIS) was launched in an effort to assess the productivity of Canadian Fig. 1. Vector map of Europe and attribute farmland. Subsequently, GIS has beco- (spreadsheet), used in this example to extract Italy. me widely and effectively used in natural-resource management. Prominent examples are timber management, mine permitting, water quality assessment, wildlife and habitat management, land use planning, zoning, and transportation design; predictive modelling of earthquakes, forest fires, and flooding; utilities management, routing analysis; marketing and demographic analyses, etc. (Cox and Gifford, 1997). The capacity of these Fig. 2. Raster map of Europe, digital elevation systems justifies their wider application model. including veterinary and human medi- Geographical Information Systems 23

cal sciences. Several types of analyses causative agent of paramphistomosis in routinely used in GIS can be very use- ruminants (Biggeri et al., 2004), ful for spatial epidemiology. These Oncomelania hupensins, the interme- include: diate host snail of Schistosoma japoni- (i) neighbourhood analysis, i.e. all cum (Zhang et al., 2005), co-infection features which meet certain with S. mansoni and hookworm among criteria and are adjacent to a schoolchildren in Côte d’Ivoire (Raso et particular feature are found al., 2006), human filariosis (Brooker and listed; and Micheal, 2000) and canine filariosis (ii) buffer generation, i.e. genera- (Genchi et al., 2005; Vezzani and tion of buffer zones around or Carbajo, 2006). along certain features; The term “remote sensing” (RS) was (iii) overlay analysis, i.e. merging of used - for the first time - in the US two or more layers or maps to during the 1960s to designate the tech- identify areas of intersection; nique allowing the study of objects (iv) network analysis, i.e. modelling without any direct contact, through of networks and calculation of image capture. In 1970, in an article tit- parameters such as the shortest led “New eyes for epidemiologists: distance between two locations; aerial photography and other remote (v) surface area and distance calcu- sensing techniques” Cline recognised lations, and that RS could have applications in (vi) three-dimensional surface mo- detecting and monitoring disease out- delling (Ward and Carpenter, breaks (Cline, 1970, 2006). In the fol- 2000). lowing years, scientists of the National A very useful function of GIS is the Aeronautics and Space Administration kriging, i.e. a linear interpolation (NASA) in the US, used colour infrared method that predicts the values of a aerial photography to identify the habi- variable, at non-sampled locations, tats of Aedes sollecitans (Hay, 2000). based on observations at known loca- While aerial photographs were the tions, using a model of the covariance of first source of RS data, the subsequent a random function (Berke, 2004). development of satellite measuring Kriging is widely used in meteorology in instruments significantly improved order to interpolate values of climate both the spatial and temporal coverage data from observing stations, and have of the earth surface, generating a conti- also been used in epidemiology to model nuous and almost complete cover. the distribution of various parasites/dis- Satellites may be divided into two eases, e.g. Ixodes scapularis that trans- groups based on the orbit they follow, mits Lyme disease (Nicholson and namely: Mather, 1996), malaria (Kleinschmidt (i) geostationary satellites (e.g. et al., 2000), alveolar echinococcosis GOES, Meteosat, GMS), and (Conraths et al., 2003; Pleydell et al., (ii) near-polar-orbiting (or sun- 2004), tsetse flies that transmit human synchronous) satellites (e.g. African trypanosomosis (Sciarretta et NOAA, Landsat, MODIS, al., 2005), Calicophoron daubneyi, the SPOT, IKONOS, Quickbird). 24 Geographical Information Systems

The former group, which consists of mental health sciences for mapping satellites in orbits parallel with the and prediction, surveillance and moni- rotation of the earth, is dedicated to toring, particularly for vector-borne meteorological applications, while the diseases (Beck et al., 2000). Since the latter follows an elliptical orbit bet- disease vectors have specific require- ween 681 and 915 km, with a different ments regarding climate, vegetation, ground track after each rotation, soil and other edaphic factors, and are lasting about 100 minutes. The revisit sensitive to changes in these factors, period varies from 1 to 41 days but can RS can be used to determine their pre- be lowered. Most of the satellites are sent and future and predict distribu- sun-synchronous and offer different tion. Land cover classification refers to spatial resolutions (Herbreteau et al., the natural vegetative cover, function 2005). However, very few have found of the topography, soil or local climate, any application in epidemiology, the and can differentiate between different most important being the Landsat and covers up to the species communities. the NOAA series (Durr and Gatrell, Land use classification refers to the 2004). Satellites have several sensors description of human uses of the land, recording radiation in different wave- or immediate actions modifying the lengths that allow combination of spec- land cover, such as agriculture (e.g. tral signals (Cringoli et al., 2005a; de used for crop production, lying fallow, La Rocque et al., 2005). RS provides a irrigated, etc.), human settlements (e.g. unique source of data that can be urban, rural, isolated infrastructures, exploited to characterize climate and etc.), protected areas (e.g. national land surface variables at different spa- parks, forest reserves, etc.) (for recent tial resolutions. It permits the calcula- reviews see Daniel et al., 2004 and tion of vegetation indices, land surface Herbreteau et al., 2005). In Europe, temperatures, atmospheric and soil the corine (Coordination of moisture, rainfall indices, etc. Among Information on the Environment) land the vegetation indices obtained from cover (CLC, European Commission, RS, the most widely employed one is 2000) is widely used, which is a map of the normalized difference vegetation the European environmental landscape index (NDVI). It is defined as the dif- based on interpretation of satellite ima- ference between the visible (red) and ges. CLC provides comparable digital near-infrared (nir) bands of satellite maps of land cover for each country information over their sum: NDVI = for much of Europe. It is useful for (nir – red)/(nir + red). NDVI is a spe- environmental analysis and compari- cific measure of chlorophyll abundance sons over large scales (spatial resolu- and light absorption, but its use has tion = 1 km). When studies are focu- been extended to quantify herbaceous sed on small areas, aerial photographs vegetation biomass, vegetation primary can be used because of their capability productivity, vegetation coverage and to afford very high spatial resolution phenology. images (<1 m). The analysis of histori- The RS data are increasingly used for cal sequences of aerial photographs is investigations in the field of environ- an important method for determining Geographical Information Systems 25

the medium-term dynamics of land can manage without them. As we enter cover on a landscape scale (Acosta et the era of Global Earth Observation al., 2005). Most developed and deve- Systems (GEOS), the old adage that a loping countries have governmental picture is worth more than a 1000 programmes for aerial surveys but words rings truer than ever (Bergquist, campaigns are not reproduced on a 2006). regular time basis, and ground covera- The oldest examples date back more ge is often limited to areas of discreet than 200 years and consist of a world interest. map of diseases put forward by Finke However, aerial photography must be in 1792 (Barrett, 2000), and a map of considered as a supplementary source yellow fever occurrences in the har- of information (Herbreteau et al., bour of New York issued in 1798 2005). Classifications of landscape (Stevenson, 1965). One of the most from digital aerial photographs have prominent examples is the mapping of been scarcely used in veterinary parasi- cholera victims in relation to the loca- tology (Rinaldi et al., 2006), with the tion of water supplies in London’s majority of applications focusing on Soho district carried out by John Snow mosquito surveillance (see for exam- (1854). The street addresses of chole- ple, Kline and Wood, 1988; Brown and ra victims were recorded and close Sethi, 2002). proximity to putative pollution sources (i.e. water supply pumps) was identified as the key risk factor. With respect Disease mapping to parasites, in two papers published in 1903, Smith and Stiles indepen- One of the most useful functions of dently presented maps of Texas, which GIS in epidemiology continues to be its displayed the prevalence of hookworm utility in basic mapping. infection and demonstrated that infec- Usually, when data are collected tion was typically restricted to the either routinely or through purposely- eastern part of the state where the soils designed surveys, they are presented in contain the most sand (Brooker and tabular forms, which can be exploited Michael, 2000). for analytical usage. However, the rea- Development of methods for map- ding and interpretation of such data is ping diseases using GIS has progressed often a laborious and time-consuming considerably in recent years. task and does not permit easy decision- Disease maps can be drawn to a making (Paolino et al., 2005). On the demographic base or to a geographic other hand, representation of these base. In the first case, they are related data in the form of a map facilitates to the population and the epidemiolo- interpretation, synthesis and recogni- gical information they show are pre- tion of frequency and clusters of phe- sented in relation to population size. nomena. Today, GIS, RS and Global Geographically based maps are con- Positioning Systems (GPS) are well- structed according to the shape of a known tools of the trade and few scien- country or a region or any administra- tists working in the fields mentioned tive unit. They may be qualitative e.g. 26 Geographical Information Systems

point maps, distribution maps, point Brugia malayi) (Brooker and Michael, distribution maps (PDMs), indicating 2000). More recently, GIS and a data- location without specifying the amount base on the distribution of lymphatic of disease; or quantitative e.g. distribu- filariosis have been used to develop the tion maps with proportioned peaks, first maps at district-level in India proportional circle maps, choroplethic (Sabesan et al., 2000) and Egypt maps, choroplethic maps with propor- (Hassan, 2004). In addition, GIS and tioned peaks, PDMs with proportioned RS, combined with a landscape epide- peaks, PDMs with proportioned cir- miological approach, established the cles, isoplethic maps, displaying the geographical patterns of river blind- number of cases of disease, the popula- ness (caused by Onchocerca volvulus) tion at risk, infection prevalence or in southern Venezuela (Botto et al., intensity or incidence (Thrusfield, 2005) and Ethiopia (Gebre-Michael et 1995; Cringoli et al., 2005c; Rinaldi et al., 2005), and the relationship bet- al., 2006). With the appropriate data at ween environmental features and infec- hand, producing a map using GIS can tion prevalence was also assessed. be undertaken literally in a matter of GIS is also the tool used by the minutes; but therein lies one of the pro- African Programme for Onchocerciasis blems with GIS - one needs the spa- Control (APOC) in order to delineate tially explicit data - and collecting these zones of various levels of endemicity, may take months or even years (Durr, and this is considered a fundamental 2004). GIS is, however, not only a digi- step in the planning process for oncho- tal map representation but is indeed an cerciasis control (Noma et al., 2002; informational and analysing tool which Seketeli et al., 2002). permits the processing of space-related With respect to canine and feline fila- data. Certainly, maps themselves keep riosis, on the basis of a systematic on playing a prominent role and are, of review of the literature, in a recent course, the most frequently used out- paper, Trotz-Williams and Trees (2003) put of GIS (Kistemann et al., 2002). provided the first evidence-based GIS Disease maps drawn by GIS can be maps of the distribution of the major the results of both meta-analysis and vector-borne infections, including dedicated surveys. Dirofilaria immitis and Dirofilaria Regarding maps derived from meta- repens in dogs and cats in Europe by a analysis, examples on human filariosis thorough investigation of published are present in literature. For example, work on their distribution, prevalence the global geographical distribution of and incidence, which should be useful human filariosis prevalences, estimated both to practitioners and researchers in for 96 endemic countries in the 1993 endemic areas and to those in non- World Bank Global Burden of Disease endemic areas. study (Michael et al., 1996), showed Regarding maps derived from dedica- different patterns for the two lympha- ted surveys, GIS has become an impor- tic filariosis, namely Brancroftian fila- tant tool for designing a study and ter- riosis (caused by Wuchereria brancofti) ritorial sampling. The fundamental and Brugian filariosis (caused by steps which can be used to produce Geographical Information Systems 27

quality descriptive survey-based maps (Fig. 3), microfilariae were detected in within GIS are the following: 63 of the 351 dogs surveyed (prevalen- (i) selection of the study area; ce = 17.9%); in particular, 56 dogs (ii) selection of the study popula- (15.9%) showed only microfilariae of tion and calculation of the sam- Acanthocheilonema (Dipetalonema) ple size, using as parameters the reconditum, three dogs (0.8%) only study population, the expected microfilariae of D. repens, two dogs prevalence, the confidence (0.6%) microfilariae of both A. recon- level, and the standard error; ditum and D. repens and two dogs (iii) selection of the sample in the (0.6%) microfilariae of both D. immi- study area (e.g. random sam- tis and D. repens (Cringoli et al., pling, systematic sampling, pro- 2001). In the study conducted in the portional allocation, use of city of Naples (divided into 21 neigh- grids, transect sampling, etc.); bourhoods, Fig. 4), microfilariae were (iv) laboratory and/or field survey; detected in 8 of the 358 dogs surveyed (v) geo-referencing of the study (prevalence = 2.2%); in particular, 5 units (e.g. individuals, group of dogs (1.4%) showed only microfilariae animals, counties, municipali- of A. reconditum, three dogs (0.8%) ties, regions, or any other admi- only microfilariae of D. repens, and no nistrative unit); and finally D. immitis positive dogs were found (vi) drawing maps by GIS (Cringoli (Rinaldi et al., 2004). et al., 2005c). Figures 5-9 are distribution maps The sampling procedures in the study with proportioned peaks, which use area play obviously a key role in disea- the municipality (for the Mt. Vesuvius se mapping. As indicated at point 3 area) or the neighbourhood (for the above, several sampling methods can city of Naples) as the geographic unit be used. For example, in order to study of reference and display the following the distribution of filarial worms in information: dogs from a certain geographical area, (i) filarial species studied (D. proportional allocation can be used, immitis, D. repens and A. i.e. the number of dogs to be tested in reconditum); each administrative unit (e.g. neigh- (ii) total study area divided into bourhood, municipality, etc.) is propor- municipalities/neighbourhoods; tional to the dog population in that (iii) municipalities/neighbourhoods administrative unit, or, if the dog popu- with positive dogs; lation is unknown, is proportional to (iv) municipalities/neighbourhoods the administrative unit surface area. without positive dogs; and This latter approach has been used by (v) municipal/neighbourhood pre- us in order to study canine filariosis in valence (%) determined as fol- the Mt. Vesuvius area of southern Italy lows: (Cringoli et al., 2001) and in the city of (number of positive dogs in the Naples (Rinaldi et al., 2004). municipality/neighbourhood)/ Regarding the former study, conducted (number of dogs examined in in an area comprising 51 municipalities the total study area) x 100. 28 Geographical Information Systems

Fig. 3. The Mt. Vesuvius area of southern Italy divided into 51 municipalities (administrative boundaries on digital aerial photographs).

Fig. 4. The city of Naples divided into 21 neighbourhoods (administrative boundaries on digital aerial photographs). Geographical Information Systems 29

In both the studies, the general trends of the maps showed that D. immitis and D. repens were present only in a few municipalities/neighbourhoods, whereas A. reconditum was widely and homogeneously spread throughout the entire study areas, thus indicating that this filarial worm is the predominant filarial species in dogs in Fig. 6. Distribution map with proportioned peaks. Dirofilaria repens in dogs from the Mt. Vesuvius the Campania region of southern Italy. area of southern Italy (from Cringoli et al., 2001). The information derived from descriptive maps derived from dedicated surveys provides an operational tool for planning, monitoring and managing control programmes, and for deriving inferences about the relationship between the environment and diseases. In fact, without good descriptive maps of disease agent/vector species’ Fig. 7. Distribution map with proportioned peaks. distribution, based on ground observa- Dirofilaria immitis in dogs from the Mt. Vesuvius tion, we do not have the essential star- area of southern Italy (from Cringoli et al., 2001). ting point to generate predictive maps based on statistical pattern-matching (Randolph, 2000). The derivation of detailed epidemiological maps, at the relevant spatial resolution, is being increasingly recognized as vital to the effective design and implementation of successful for the control of parasites and their vectors (Sabesan et al., 2000). Fig. 8. Distribution map with proportioned peaks. Acanthocheilonema (Dipetalonema) reconditum in dogs from the city of Naples (from Rinaldi et al., 2004).

Fig. 5. Distribution map with proportioned peaks. Acanthocheilonema (Dipetalonema) reconditum Fig. 9. Distribution map with proportioned peaks. in dogs from the Mt. Vesuvius area of southern Dirofilaria repens in dogs from the city of Naples Italy (from Cringoli et al., 2001). (from Rinaldi et al., 2004). 30 Geographical Information Systems

Ecological analysis of lakes, rivers and other water bodies, temperatures, rainfall and humidity. Ecological analysis is targeted on the The extrapolation of environmental description of relations existing bet- variables can also be directly extracted ween the geographic distribution of from the study units instead of the buf- diseases and environmental risk factors fer zones. and their analysis by means of statisti- Ecological analysis has been pre- cal procedures (Kistemann et al., viously performed by us in order to 2002). A wide number of papers have study the climatic and environmental been published on the analysis of the factors that influence the distribution relationship between disease indicators of the rumen fluke C. daubneyi (e.g. positivity, incidence and prevalen- (Cringoli et al., 2004) and of the pro- ce) and the explanatory environmental tozoan Neospora caninum (Rinaldi et and climatic variables (Herbreteau et al., 2005) in southern Italy. al., 2005). In order to make ecological Environmental factors that may analysis, the following fundamental govern filarial worm distribution may steps can be utilized: be expected to affect the life history (i) GIS construction for the study parameters of both the mosquito vec- area utilizing data layers on tor and the filarial species. environmental and climatic fea- Traditionally, attention in this area has tures (Fig. 10); focussed primarily on temperature, (ii) geo-referencing the geographic humidity and elevation (Attenborough units of interest; et al., 1997; Brooker and Michael, (iii) creation of buffer zones of a 2000). For example, GIS functions given diameter centered on have been recently used by Sowilem et these geo-referenced points; al. (2006) to identify environmental (iv) extrapolation of values for each indicators of high-risk village for environmental feature within human filariosis in Egypt, as indicated each buffer zone; by mosquito density, human infection (v) databases with environmental rate, vector species composition, mean and parasitological data; life expectancy, as well as environmen- (vi) statistical analyses (univariate, tal variables (geology, hydrology, soil multivariate, etc.) and individua- types) and meteorological factors (tem- lize of environmental risk factors perature, rainfall and humidity); in this and/or development of forecast study, the most important landscape models (Cringoli et al., 2005c; Rinaldi et al., 2005, 2006). A part from the administrative boundaries, the data layers on environmental and climatic features most commonly used for ecological analysis in veterinary epidemiology are: NDVI, land cover and land use, elevation, slope, aspect, lithofacies map, presence Fig. 10. Data layers used for ecological analysis. Geographical Information Systems 31

elements associated with filarial prevalence were water and different vegetation types. In other recent study conducted in India, a range of geo-environmental variables were selected, and customized on GIS platform to develop a geo-environmental risk model for determining the areas of potential transmission of lymphatic filariosis (Sabesan et al., 2006). Studies on ecological analysis regarding canine filariosis are very scant in literature, probably due to the difficulties in obtaining geo-referenced data. We made an attempt to determine the environmental and climatic features that influence the distribution of D. repens in the Lazio region of central Italy (Scaramozzino et al., 2004). A GIS for the study area was constructed Fig. 11. NDVI map of the Lazio region obtained utilizing the following data layers: ele- from Landsat-5 TM (spatial resolution = 30 x 30 m) and 3 km diameter buffer zones centered on vation, slope, aspect, NDVI, land cover the dog sampling points. and minimum, maximum and mean temperature average covering a 30- Predicting parasite occurrence/seaso- years period. Data on each of the above nality: an useful tool for surveillance variables were then calculated for 3 km diameter buffer zones centered on the GIS and RS can be also used to pre- geo-referenced point of dog sampling dict disease seasonality based on the cli- (Fig. 11). However, statistical analysis matic and/or environmental characteri- did not show any significant association stics of a certain area and the informa- between the positivity to D. repens and tion about the climatic and/or environ- the environmental data. This was pro- mental requirement(s) of a certain spe- bably due to the non-uniform spatial cies. Climate-based forecast systems, distribution of the sample (the data employing the concept of growing used were obtained from a meta-analy- degree days (GDD), have been develo- sis, without a sampling design), and to ped for different diseases of parasitolo- the low number of positive dogs gical importance, such as fasciolosis, (1.9%). These latter results point out schistosomosis, onchocerciasis and once again that the use of GIS does by malaria (Malone, 2005; Gebre-Michael no means overcome the two major con- et al., 2005; Yang et al., 2006). cerns of any empirical research: data GIS have been also used in order to availability and data quality (Kistemann predict the occurrence and seasonality et al., 2002). This issue should always of D. immitis in Europe by us (Genchi be kept in mind by all GIS/RS users. et al., 2005) and in Argentina (Vezzani 32 Geographical Information Systems

and Carbajo, 2006). The heartworm wing the yearly average predicted num- (HW) disease models are based on the ber of HW generations (Fig. 15); 4) a fact that climate dictates the seasonal map showing the start and end of the occurrence of D. immitis transmission. HW transmission period in some The rate of maturation to infective third- European localities (Fig. 16). stage larvae (L3) in the mosquitoes The results of the model showed that depends mainly on the environmental HW transmission is markedly seasonal temperature, and there is a threshold of in Europe, with peaks in summer, from about 14°C below which development June to September. The study provided will not proceed (Fortin and Slocombe, HW risk assessment maps for Europe 1981). The total environment heat and suggests that if the actual trend of required for development may be temperature increase continues, filarial expressed in terms of degrees days in infection should spread into previously excess of this threshold (Heartworm infection-free areas (Genchi et al., Development Units – HDUs) (Fortin 2005). and Slocombe, 1981; Slocombe et al., The recent models performed by 1989). The seasonal HW transmission Vezzani and Carbajo (2006) in order to model formulated by Slocombe et al. assess spatial and seasonal D. immitis (1989) and re-evaluated by Lok and transmission risk throughout Argentina, Knight (1998) in Canada and US, showed that also in the Southern respectively, assumes a requirement of Hemisphere, HW transmission is mar- 130 HDUs for larvae to reach infectivity kedly seasonal, with peaks in January and a maximum life expectancy of 30 and February (summer). days for a vector mosquito. The model The D. immitis risk maps performed performed by us (Genchi et al., 2005) in Europe and Argentina could be used was applied using temperature records by practitioners and health authorities from 1846 European Meteorological sta- to efficiently direct surveillance and tions (Fig. 12), spanning a 14-year control efforts. From a general point of period, in order to assess the theoretic view, epidemiological surveillance transmission timing of HW in Europe. In employing the use of GIS is characteri- addition, a GIS based on thermal regime zed by an integrated set of planned epi- was constructed in order to produce pre- demiological activities whose aim is to dictive maps which assess the duration identify and prevent new cases of dis- of the HW transmission risk period, and ease. Such a system is justified provi- the efficient timing of HW chemo- ded the natural history of the disease prophylaxis and scheduling of diagnostic and its determinants are known and testing. The following maps were obtai- effective preventive strategies are avai- ned from the GIS: 1) monthly maps sho- lable (Rinaldi et al., 2006). wing the stations that reached the 130 HDUs at least once in the studied years (for example, see July in Fig. 13); 2) Conclusions monthly maps showing the average predicted number of generations (for exam- The trends over the past two decades ple, see July in Fig. 14); 3) a map sho- and their effects in the fields of hard- Geographical Information Systems 33

Fig. 12. European meteorological stations (n. 1846) used for predict HW transmission in Europe (from Genchi et al., 2005).

Fig. 13. HW predictive model based on growing degree days. Stations reaching the 130 HDUs at least once in the studied years in July (from Genchi et al., 2005). 34 Geographical Information Systems

Fig. 14. HW predictive model based on growing degree days. Average predicted number of D. immitis generations in July (from Genchi et al., 2005).

Fig. 15. HW predictive model based on growing degree days. Yearly average predicted number of D. immitis generation in Europe (from Genchi et al., 2005). Geographical Information Systems 35

Fig. 16. HW predictive model based on growing degree days. Starting and ending HW transmission period in some European localities (from Genchi et al., 2005). ware, software and network techno- ability to integrate different databases logy, in particular in the internet into a single environment; GIS may be domain, have created the prerequisites though of as a database of databases for a broad acceptance of GIS and RS (Cox and Gifford, 1997). In conclu- within the health sciences, including sion, the present review showed that, veterinary science. with the appropriate data at hand, GIS Recent studies showed that GIS – in can be very useful to study the epide- addition to their broad utility reviewed miological patterns of filarial infections here – holds promise in capturing data in veterinary and public health. on medicine usage (Ryan et al., 2005), as well as in organizing spatial information involving the distinction of ana- References tomic relationships (Ganai et al., Acosta A, Carranza ML, Giancola M, 2005. 2006). However, epidemiology remains Landscape change and ecosystem classification the main application field of GIS in in a municipal district of a small city (Isernia, Central Italy). Environ Monit Assess 108: 323- veterinary and public health and, since 335. epidemiology is inextricably bound to Attenborough RD, Burkot TR, Gardner DS, 1997. Altitude and the risk of bites from mos- “place”, it seems reasonable to expect quitoes infected with malaria and filariasis that GIS will further advance as scien- among the Mianmin people of Papua New ce (Jacquez, 2000). Perhaps one of the Guinea. Trans R Soc Trop Med Hyg 91: 8-10. Barrett FA, 2000. Finke’s 1792 map of human dis- most powerful benefits of GIS is its eases: the first world disease map? Soc Sci Med 36 Geographical Information Systems

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Maria Teresa Manfredi, Annarita Di Cerbo, Marco Genchi Biology of filarial worms 41

The nematodes of genus Dirofilaria Description of Dirofilaria immitis belong to family Onchocercidae and (Leidy, 1856) Railliet and Henry 1911 subfamily Dirofilariinae of the order Spirurida (Anderson, 2000). They are Dogs are the main hosts, however it slender nematodes and the larval sta- has been reported in wolves, dingoes, ges are generally found in the definiti- coyotes, foxes, various felines (inclu- ve host distant from their site of ori- ding the domestic cat), pinnipeds, gin. Embryos (microfilariae) develop mustelids (e.g. ferrets), bears, pandas, in the uteri of the female worm and beavers, coatis, rabbits, deer, horses, are surrounded by the egg membrane. non-human primates and humans. Females are ovoviparous and lay Most of these infections are sporadic microfilariae in the blood stream, and the microfilaremia could be absent. where they become available to hae- The distribution is worldwide and has matophagous vectors. been found in tropical, subtropical and Microfilariae are able of responding temperate regions (Lok, 1988). to physiological changes in the host and may flood into the peripheral cir- Morphology of adults culation at a certain time of the day or night and disappear from the periphe- D. immitis is a long, whitish thin fili- ral circulation at other time. form nematode. Mouth opening is ter- Microfilariae have structures that minal without lips and leads into the allow their passage through the fine oesophagus differentiated into muscu- blood capillaries and through the nar- lar and glandular regions without dis- row mouthparts and the gut wall of tinction. The oral aperture is surroun- the arthropod vectors. ded by 6 small median papillae and 2 The genus Dirofilaria is divided in lateral papillae (amphids). The adult two subgenera. The subgenus females measure 250 to 310 mm in Dirofilaria includes Dirofilaria immi- length and 1 to 1.3 mm in width and the tis and the subgenus Nochtiella inclu- males are 120-200 mm long and 0.7-0.9 des Dirofilaria repens. mm wide. The nematodes of Dirofilaria sub- Females have an obtuse caudal end; genus are large filarial worms the anus is subterminal and the vulvar without cuticular ridges except in the opening is located just posterior to the ventral surface of the caudal extre- junction of the oesophagus and intesti- mity of the male; the caudal papillae ne. The male caudal end is spirally of the males show only slight asym- coiled and has 2 lateral narrow alae. metry in number and distribution. The cloaca opens to 0.13 mm from the The nematodes of the subgenus tip of the tail. The ventral side bears Nochtiella are relatively small filarial pre-anal, per-anal and post-anal papil- worms with cuticular longitudinal lae. The spicules are unequal and with ridges; the caudal papillae of the different structure, enlarged at proximal males show a distinct asymmetry in end and acute at distal end. The left spi- number and distribution (Canestri cule is 300-375 µm and the right 175- Trotti et al., 1997). 299 µm; the gubernaculum is absent. 42 Biology of filarial worms

The cuticle is smooth and ridges and The larvae are deposited by the mos- striations are only present on the ventral quito throughout a drop of infected surface of the last coil of the male tail hemolymph in the proximity of bite (Uni and Takada, 1986; Lichtenfels et wound. L3 enter the bite wound and al., 1987). penetrate the connective tissues, The adults of D. immitis feed on pla- migrate from the subcutaneous or sub- sma and they can live several months or serosal tissues to the muscles and years in their hosts. undergo the 3rd and 4th moults. The This nematode is ovoviviparous, and fourth stage larva is 8.29-13.2 mm females release unsheathed microfilarie long (Lichtenfels et al., 1985). They into the bloodstream. They are 290- undertake extensive migration through 330 µm long and 5-7 µm wide; the the subcutis, which continues for some cephalic extremity is tapered and the 60-90 days until the final to L5 (imma- tail is straight. The tip of the tail is ture adult). Then, the juvenile worms pointed. The microfilarie can survive migrate from muscular tissues and tho- from 2 to 18 months in the blood- racic/abdominal cavities to pulmonary stream. arteries and right heart within a few days of their final moult, presumably Biology carried by the venous circulation (Kume and Itagaki, 1955; Webber and The microfilariae develop to L3 Hawking, 1955; Lichtenfels et al., (infective stage) in the intermediate 1985; Anderson, 2000). The immature host, a mosquito of the genera Culex, adults penetrate the jugular or other Aedes, Psorophora, Mansonia or veins leading them directly to hearth Anopheles. However, the most impor- and grow to a maximal length of 20 tant intermediate hosts are those spe- cm. Final maturation and mating occur cies without the bucco-paryngeal in the pulmonary arteries. After copu- armature, which is able to damage the lation, the females produce unsheathed microfilarial cuticle preventing its microfilariae approximately 6 and half development to the third larval stage. months after infection (Table 1). The microfilariae ingested by mos- Microfilariae are then released into the quitoes throughout the blood meal circulation. migrate to the Malpighian tubules where they moult into second larval stage and the third larval stage. Description of Dirofilaria repens Infective L3 migrate from the tubules to the lumen of the labial sheath in the D. repens Railliet and Henry, 1911 is vector mouthparts. Development in a parasite of the subcutaneous connec- the mosquito is temperature depen- tive tissues mainly of dogs. There are dent, requiring approximately two reports from domestic cats, genet cats weeks at temperature > 26°C. (Genetta tigrina), lions and foxes. Infection is transmitted to a new host Cases have been reported in humans. when an infected mosquito takes a fur- This species occurs in southern and ther blood meal in a competent host. Eastern Europe, Africa and Asia. Biology of filarial worms 43

Table 1. Development of Dirofilaria immitis in the mosquito and in dogs.

Days in the Days in Stage Length Hosts Localization mosquito the dog cm Mf 0.030 dog blood 1 Mf 0.030 mosquito middle gut 5 sausage 0.015 mosquito Malpighian tubule cells 10 L2 0.05 mosquito Malpighian tubule lumen 15 L3 1 mosquito proboscis 1-15 L3 1.5 dog subcutaneous tissue 3-80 L4 3-7 dog subcutaneous/muscle tissues 70-150 L4-L5 4-13 dog muscle/subcutaneous tissues 100-160 L5 4-20 dog thoracic/abdominal cavities/ pulmonary arteries/right heart 150-270 gravid female/ 25-30 dog pulmonary arteries/right heart mf

Mf: Microfilariae Note: In cats the life cycle is about 30 days longer

Morphology of adults duce unsheathed microfilariae living in bloodstream. Microfilarie have the Adults of D. repens are smaller than anterior end obtuse, the posterior end D. immitis. The cuticle is white with tin and pointed ending curved in form distinct longitudinal and tender of an umbrella handle. The microfilarie transverse striations. Adult females are 300-360 µm long and 6-8 µm wide are 100 to 170 mm long and 4.6 to and should be differentiated by other 6.5 mm wide and males are 50-70 microfilariae which can be found in the mm long and 3.7-04.5 mm wide. The bloodstream such as D. immitis, male caudal end curves slightly Acanthocheilonema (syn. Dipetalonema) toward the ventral side. The tail is reconditum and Dipetalonema dracun- obtuse, with two lateral alae and culoides. oblong pedunculate papillae. The spicules are unequal; the larger spicule Biology is 0.43 mm long and has an acute tip; the smaller spicule is strongly chitini- D. repens life cycle requires a mos- zed, measures 0.175 mm in length quito of genera Aedes, Anopheles, and has an obtuse end. In the fema- Culex, Armigeres and Mansonoides as les, the vulva is situated 1.84 to 1.92 intermediate host. The duration of mm from cephalic end and it is encir- development depends on the species of cled by slightly projecting labia. The intermediate host (min 10 days max 21 tail has an obtuse tip and curve days at a temperature of 24 to 27°C). slightly toward ventral side. Under experimental conditions larvae D. repens is ovoviviparous and pro- developed also in horseflies and sand- 44 Biology of filarial worms flies (Coluzzi, 1964). taneous connective and reach the matu- Microfilariae of D. repens ingested by rity here (Table 2). The macrofilariae a blood meal reach the stomach of mos- are found between the connective quitoes and migrate to the body cavity layers in most part of the body. They within 1.5 days and settle in the were often found on the trunk and few Malpighian tubules where have 2 were found in the legs and head moults. After attaining the infective (Webber and Hawking, 1955). Usually, stage larvae migrate to the head and microfilarie do not show periodicity in mouthparts of the mosquito. the peripheral blood, but microfilare- In the definitive host the infective lar- mia has a tendency to increase seaso- vae were found in sections of the sub- nally (from August to September in cutaneous connective tissue up to 48 Italy) (Cancrini et al., 1975). The pre- hours after inoculation. The larvae do patent period is 27-34 weeks. Adults not have long migrations in the subcu- can live several years.

Table 2. Development of Dirofilaria repens in the mosquito host and in dogs.

Days in the Days in Stage Length Hosts Localization mosquito the dog cm Mf 0.035 dog blood 1 Mf 0.035 mosquito middle gut 2-7 sausage 0.015 mosquito Malpighian tubule cells 6-9 L2 0.05 mosquito Malpighian tubule lumen 10-20 L3 1.00 mosquito proboscis 1-15 L3 1.00 dog subcutaneous connective tissue 3-70 L4 n.d. dog n.d. 70-150 L5 n.d. dog n.d. 180-240 gravid female/ 10-17 dog subcutaneous tissue/ mf perimuscular connective fasciae

Mf: Microfilariae Note: The duration of the life cycle is unknown

Dirofilaria immitis Dirofilaria immitis Caudal end of male, ventral view Cephalic end of female, ventral view Biology of filarial worms 45

Dirofilaria immitis Dirofilaria immitis Cephalic end of female, ventral view Anterior end of female, ventral view

Dirofilaria immitis Dirofilaria immitis Posterior end of female, lateral view adult female (over) and male (under)

References Lichtenfelds JR, Pilitt PA, Kotani T, Powers KG, 1985. Morphogenesis of developmental Anderson RC, 2000. Nematode parasites of verte- stages of Dirofilaria immitis (Nematoda) in brates, their development and transmission. the dog. Proc Helmintol Soc Washington 52: 2nd Edition, CABI Publishing, Wallingford, 98-113. UK, 650 p. Lok JB, 1988. Dirofilaria sp: taxonomy and distri- Cancrini G, Coluzzi M, Balbo T, Gallo MG, 1975. bution. In: Dirofilariasis. Boreham P.F.L. and Variazioni stagionali della microfilariemia ed Atwell RB Eds, CRC Press, Boca Raton Florida, effetto della temperatura ambientale in cani 249 p. parassitati da Dirofilaria repens. Parassitologia Kume S, Itagaki S, 1955. On the life cycle of 17: 75-82. Dirofilaria immitis in the dog as the final host. Canestri Trotti G, Pampiglione S, Rivasi F, 1997. Brit Vet J 111: 16-24. The species of the genus Dirofilaria Railliet & Uni S, Takada S, 1986. The longitudinal cuticular Henry, 1911. Parassitologia 39: 369-374. markings of Dirofilaria immitis adult worm. Coluzzi M, 1964. Osservazioni sperimentali sul Japan J Parasitol 35 (3): 191-199. comportamento di Dirofilaria repens in diversi Webber WAF, Hawking F, 1955. Experimental gruppi di artropodi ematofagi. Parassitologia 6: maintenance of Dirofilaria repens and D. immi- 57-62. tis in dogs. Exp Parasitol 4: 143-164. 3 Vectors of Dirofilaria nematodes: biology, behaviour and host/parasite relationships

Gabriella Cancrini, Simona Gabrielli Vectors of Dirofilaria nematodes 49

In the last century many important are now strongly influencing the activi- aspects of the diseases due to ty patterns of mosquitoes in temperate Dirofilaria immitis and D. repens have areas. been studied and elucidated, including The male mosquito feeds mainly on pathogenesis and parasite transmission. fruit juices, whereas the female is also However, there are still many unan- hematophagous. This blood-sucking swered questions concerning the habit allows to the female participate to species that can act as vector of these the filariosis transmission. parasites in the world. Experimental studies have shown that dirofilarial nematodes can develop, Finding a host and host preference more or less completely, in numerous arthropods but, on the basis of current To find resting sites or environment knowledge, only the insects, namely the suitable for mating or ovoposition, or mosquitoes (order Diptera, suborder to find a source of blood host, mosqui- Nematocera, family Culicidae), act as toes move owing to phototropism and vector. in response to chemical stimuli. They The Culicidae have a cosmopolitan are attracted to carbon dioxide and distribution, with over 3500 species odour produced by the host, which is throughout the world. The high degree then localized by the warmth and mois- of adaptability of these insects has ture radiated from its body. Following assured their presence in every type of these stimuli, some mosquito species environmental habitat. In fact, mosqui- travel only a few hundred meters from toes are found in low-lying plains, hilly their larval habitats, whereas others areas, in mountainous zones and (several species of Aedes) can go as far marine habitats, where each species has as 50 km (Hocking, 1953). They may adapted its egg laying activity and lar- cover long distances carried mostly by val development to the types of water winds (passive movement) or by flying surfaces there available. Some species actively in successive stages. lay their eggs only in fresh rain water, The host-seeking behaviour of the others in stagnant pools or in any ves- mosquito follows different patterns, sel that contains water; others lay in depending on the species. Some are quiet pools at the edges of streams, and active only during the nocturnal hours some even in salt water. (Cx. pipiens and most of the In tropical areas, mosquitoes are Anopheles) whereas others predomi- active all year round, whereas in tem- nantly in the early morning (like An. perate climates adults are active during gambiae, An. balabacensis, An. mac- the late spring and summer, and have ulipennis), or on the daytime (Ae. different seasonal rhythms of activity. albopictus). Species such as Ae. aegyp- Environmental and climatic changes, in ti, Ae. caspius, Ae. vexans and Cx. particular the predicted rise of the modestus show two peaks of feeding mean temperature (+0.2°C in 10 years) activity, one at sundown and the other as a consequence of the “global warm- at dawn (Matting, 1969; Di Sacco et ing” (Romi, 2001; Genchi et al., 2005) al., 1992; Pollono et al., 1998). These 50 Vectors of Dirofilaria nematodes biological rhythms are not a fixed rule potential predators (therefore the life but the most common behaviour of the span of the mosquito). species, often the result of co-evolution As far the host preference, several with the parasite transmitted, and species are strictly zoophilic or “spe- therefore usually correlate to the possi- cialists” (limit feeding to specific types ble major success to fed on parasitized of hosts), several are less restrictive and animals. During this species-specific more largely zoophilic or “generalists” circadian window, other factors may (have the ability to identify and feed on modulate the expression of activity. For a wide variety of host types) and even example, the age of the female can anthropophilic. The last species are influence activity levels. Shortly after important for the dirofilariosis trans- the emergence, there may be a brief mission from animals to humans. Host period during which the insect is obliv- use patterns may vary seasonally ious to host stimuli, and holder individ- between geographic regions and, as the uals may respond much differently than relative abundance of different hosts, when they were younger (Nayar and changes in an area. Sauerman, 1973). In general, the older Mosquito feeding preferences may female is more epidemiologically depend on several factors, including the important by virtue of its increased behaviour and attractiveness of the host. opportunity to acquire and transmit Actually the mosquito never seeks a host pathogens. Nutritional state also affects as such, but simply responds to host the intensity of activity peaks; as the kairomones by orienting in their direc- period of blood deprivation lengthens, tion. These kairomones are released as activity patterns become more intense discontinuous filaments or packed of (Klowden, 1996). When all factors stimuli, broken up by the wind as they contribute to the increase in activity move downwind, like the dispersion levels, they make the female more like- pattern of a smoke plume from a chim- ly to encounter host stimuli, and it may ney. The host seeking steps may be then find itself in the vicinity of a host. bypassed in the laboratory experi- Based on their preference to feed out- ments, but are of critical importance in doors or inside dwellings, mosquito nature. In fact, those individuals that species can be divided into exophagic feed when placed directly on a host are and endophagic, respectively. As far not necessarily those that will seek it resting habits, species can be further from a distance. classified as endophilic (remaining Dog and cat baited traps, operated to after feeding within human habitations investigate on the species that can act for most of the gonotrophic cycle) or as vectors, showed that the dog attracts exophilic (spending most of their time a large number of Ae. caspius, Ae. outside). It is clear that the different scapularis, Ae. taeniorhynchus, Cx. behaviour patterns can influence sever- pipiens, Cx. quinquefasciatus, Cx. al epidemiological factors: the avail- declarator, Cx. nigripalpus, An. mac- ability of suitable hosts; the tempera- ulipennis and Cs. annulata and, under ture at which the filarial larvae develop the same field conditions, larger than within the mosquito; the exposure to does the cat (Di Sacco et al., 1992, Vectors of Dirofilaria nematodes 51

1994; Genchi et al., 1992; Iori et al., offer an ideal environment for the 1990; Pollono et al., 1994; Labarthe et insect (warmth, carbon dioxide). Cats, al., 1998) (Fig. 1). It is likely that the on the other hand, in natural condi- dog does not represent the preferred tions, are nocturnal hunters and move host of all individual mosquitoes of about quite a lot. It is possible that the these species (for example, Cx. pipiens mosquito, who needs sufficient contact and Cs. annulata were originally time with the host in order to feed, is ornithophilic and have adapted to bit- disturbed by the cat movement. This, ing man and other mammals; Ae. along with the cat smaller body mass, caspius, on the other hand, is highly may explain the insect feeding prefer- anthropophilic), but is however an ence for dogs and the higher prevalence important food source. Moreover it has of infection observed in dogs compared been shown that Culex species are not to cats, who likely play only a marginal particularly attracted to the cat, but role as reservoir for the disease. In fact, other studied species are even less so cat infections induced by simulated (Pollono et al., 1998; Labarthe et al., natural exposure to Ae. aegypti experi- 1998). The feeding activity of these mentally infected with D. repens or D. mosquito species begins at sundown immitis confirmed that mosquitoes do and lasts throughout the night. Dogs not feed willingly on the cat (Cancrini have a habit of sleeping during the noc- et al., 1979; Cancrini and Iori, 1981; turnal hours and their resting places Mansour et al., 1995).

Fig. 1. Activity patterns and feeding preferences of Cx. pipiens and Ae. caspius as regards the dog and the cat, taken from bait-capture studies in Pavia province (Italy). 52 Vectors of Dirofilaria nematodes

Development of Dirofilaria parasites distal end, migrate through the haemo- in the invertebrate host cele to reach the labium. During the mosquito probing and blood feeding on When the female mosquito fed on the vertebrate host infective L3 Dirofilaria infected animals, it takes up emerges from the folded labium and with the blood embryos (microfilariae) rests on the skin of the host immersed that can develop into infecting L3 only in a drop of haemolymph to avoid in the “competent” invertebrate host. dehydration. It then enters the host Moreover, the number of larvae that when the insect has pierced the skin. It can successfully complete their devel- is impossible to distinguish by mor- opment depends on the vector efficien- phology between the developing larvae cy of the mosquito individual. of D. immitis and D. repens (Nelson, During the blood meal the microfilar- 1959; Nelson et al., 1962). iae (270-365 µm in length and 6-8 µm The time required for larval develop- wide, depending on the species) pass ment in the mosquito is temperature- through the pharynx and reach the dependent: it takes 8-10 days at 28- mid-gut where they remain for approx- 30°C, 11-12 days at 24°C, and 16-20 imately 24 hours. They then migrate to days at 22°C. Larval development ceas- the Malpighian tubules and invade the es at temperatures below 18°C, but it cells of the distal end (Fig. 2), where can resume if the mosquitoes are they undergo transformation to the so- placed at higher temperatures called “sausage stage”(first stage larva (Cancrini et al., 1988) (Fig. 3). or L1, about 70 µm in length), mute to The initial invasion of the Malpighian the L2 stage and finally, into L3. The cells by L1 and the penetration of the L3 (1,100 µm in length) leaves the tubule walls by the exiting L3 are both Malpighian tubules by perforating their critical moments for the mosquito sur-

Fig. 2. Scansion electron microscopy at 2 days after experimental infection: a microfilaria of D. repens is penetrating in a primary cells of the Malpighian Fig. 3. Experimental infection of Cx. pipiens with tubules with the cefalic end (a), whereas its caudal D. repens: development times and location of the end is still free in the lumen of the tubules (b). larvae observed. Vectors of Dirofilaria nematodes 53 vival. When the parasite load is heavy, tubule function is compromised and the insect dies. The maximum number of larvae compatible with the insect survival depends on the species of mosquito and the species of Dirofilaria and it is lower for D. immitis than for D. repens (Le Coroller, 1957; Coluzzi, 1964; Christensen, 1978; Russell and Geary, 1996).

Host-parasite relationship and vector efficiency Fig. 4. Cibarial armature of Cx. pipiens, showing smooth teeth in a brush-like structure and pointed thorn-like projections. 1) Cross section of the phar- The percentage of microfilariae taken ingeal valve with teeth clearly evident (D). 2) up with a blood meal that complete Cibarial armature observed from the cibarial pump. their development to infective L3 can 3) Longitudinal section through the area zigrinata vary from 0 to 100%. In general, the (AZ) with thorn-like projections on top and cibarial armature, situated between the cibarial (PC) and efficient vector is the female that mod- pharingeal pumps (PF). 4) Thorn-like projections erates the parasite invasion and allows (S) in the area zigrinata (from Coluzzi et al., 1981). the development of the maximum number of infective larvae compatible with its survival. Therefore, different species or different individuals within a species can be more or less efficient vectors of the parasite, and it depends on various factors. Parasitic burden can affect the mosquito survival, so mosquitoes have different defence mechanisms they use to 1 2 block larval development and consequently to control infective larval load. The presence of the cibarial armature, for example, is an efficient mechanical tool for damaging microfilariae as they pass through the pharynx (Fig. 4). The rythmic opening-closing action of the 3 cibarial pump, armed with sharp teeth, can provoke serious lesions to the cuti- Fig. 5. D. repens microfilariae after ingestion by a cle of the microfilariae, leading to mosquito without (1), and with (2,3) cibarial armature. In the first case, the larva is intact, whereas in embryonic death and elimination the second, lesions to the cuticle are clearly evident (Coluzzi and Trabucchi, 1968) (Fig. 5). (2), even causing a complete break in the larva’s The coagulation time of the blood body (3) (from Coluzzi and Trabucchi, 1968). 54 Vectors of Dirofilaria nematodes

taken up during the meal is another factor that influences vector competence. In fact, if the blood coagulates quickly, the microfilariae remain trapped in the clot, and can not reach the tubules (Frizzi and Pedrotti, 1957; Grieve et al., 1983). Therefore, mosquitoes that produce anticoagulant substances (such as An. quadrimaculatus) are more receptive to infection because Fig. 6. Cross-section of D. immitis microfilaria they allow the microfilariae to more within a primary cell of the Malpighian tubules of easily reach the Malpighian tubules and Ae. aegypti. Transmission electron microscopy at go on their development. 6 days after experimental infection: the host cell Other defence mechanisms have been (on the left of each image) has produced electron- observed in different mosquito species. dense material that has begun to deposit on the cuticle wall of the embryo (mf, on the right of each For example, Ae. aegypti can secrete image), causing lysis. substances that induce epicuticle lysis (Fig. 6) and, like Ae. scapularis and Ae. taeniorhynchus, is able to encapsulate in a melanotic reaction microfilariae present in Malpighian tubules, causing microfilaria death (Kartman, 1953; Beernsten et al., 1994; Forton et al., 1985; Bradley and Nayar, 1985; Vegni- Talluri et al., 1993; Vegni-Talluri and Cancrini, 1994). On the contrary, haemocytes, are not able to cause any damage to the L3 that, having left the tubules (Fig. 7), migrate towards the head of the insect (Vegni-Talluri and Cancrini, 1991). Another defence mechanism, even if somewhat less efficient, is based upon the mosquito ability to control development times for the larvae, that may become incompatible with the insect life span (Cancrini and Iori, 1981; Cancrini et al., 1995). Fig. 7. Transmission electron microscopy of D. The receptiveness/refractoriness to repens L3 (cross-section) 10 days after experi- infection and the efficiency of the mos- mental infection of Ae. aegypti, situated in the quito as vector for both D. immitis haemocele. A cytoplasmic projection from a (Zielke, 1973; McGreevy et al., 1974) haemocyte (h), adhering to the L3 (l), has produced and deposited an electron-dense sub- and D. repens (Coluzzi and Cancrini, stance that has not, however, caused any damage 1974) is genetically determined and it to the cuticle. is controlled by a sex-linked recessive Vectors of Dirofilaria nematodes 55 allele. The genes that control the trait develop in numerous insects (several are different for D. repens and D. mosquito species and biting flies) and immitis. Variations in vector competen- that varying levels and modes of resist- cy have been identified in both natural ance to infection exist in other biting and laboratory mosquito populations. flies, simulides, phlebotomes, culicoids, The importance of a mosquito in the fleas, and in ticks (Coluzzi, 1964). epidemiology of dirofilarial infections Then, it has been demonstrated that not only depends upon the receptive- some mosquito species are more com- ness to infection and on the efficiency petent than others to transmit the para- in transmitting infective larvae, but site, and that within a single species also on the size of the vector popula- some individuals or strains are more tion, its feeding pattern, life span and efficient vectors than others (Coluzzi seasonality. In fact, in order to transmit and Trabucchi, 1968; McGreevy et al., the infection, the vector must take at 1974; Bemrick and Moorehouse, least two blood meals, and in this 1968). Capture studies carried out in respect autogenous species, which can endemic areas to identify natural vec- use protein reserves accumulated dur- tors have suggested a possible role of ing the larval stages to produce the first some species of Culicidae on the basis batch of eggs, are less important as vec- of their abundance. However, crucial tors than those species that require sev- progress in knowledge on the species eral blood meals for egg laying, thus actually involved in the parasite trans- increasing the chances for larval uptake mission has been made through the and transmission. The life span of the fieldwork based on animal-baited traps. insect must be long enough to guaran- This method allows to restrict the inter- tee larval development to the infective est only to species that are effectively stage and is thus influenced by the attracted to the host, and to evaluate exophagic and exophilic behaviour of the biological factors that affect the certain species. The seasonal activity is actual dirofilaria infection risk. These also important and those species that capture studies on host animals (dog, reproduce actively all year round or sev- cat, and/or man) have been carried out eral times during the summer are more in the United States of America, Italy efficient. Ambient temperature, humid- and Brasil, and have identified as pos- ity and predation are further factors sible vectors Cx. erraticus, Cx. modes- that influence the mosquito survival. tus, Cx. nigripalpis, Cx. pipiens, Cx. quinquefasciatus, Ae. canadensis, Ae. caspius, Ae. excrucians, Ae. scapularis, Arthropod vectors Ae. sierrensis, Ae. sollicitans, Ae. stim- ulans, Ae. taeniorhynchus, Ae. trivitta- Most of the data on the arthropod tus, Ae. vexans and An. maculipennis species that can act as vector derives s.l. (Sauerman, 1985; Favia et al., from laboratory experiments started in 1996). Further species, like Ae. can- the last century (Grassi and Noè, tans, Ae. cinereus, Ae. geniculatus, An. 1900). Experimental infections have claviger, Cq. richiardii, Cx. declarator, shown that dirofilarial nematodes can Cx. pipiens-restuans, Cx. sultanensis, 56 Vectors of Dirofilaria nematodes

Cx. territans and Cs. annulata feed less worm infection, in particular the sea- willingly on dogs and cats, and there- sonal transmission patterns in the dif- fore could be of minor interest in the ferent geographical areas and the mon- parasite transmission (Di Sacco et al., itoring of infection rates among differ- 1992; Genchi et al., 1992). ent vectors. Several studies have reported the These first results have been obtained mosquito species that, by microscopy, by studying several dirofilariosis areas have been found in nature infected in northern and central Italy. They are with dirofilarial larvae but, unfortu- of concern because the main natural nately, it is not possible to morphologi- vector (to date Ae. albopictus) is a cally distinguish developing larvae species proven very efficient in the par- belonging to the 27 recognized species asite transmission, “generalist” in the of Dirofilaria. First unambiguous host choice and highly anthropophilic. results have been achieved through the The presence now stable of this mos- analysis of the collected insects by quito in Italy suggests that the infection molecular techniques recently adjusted risk for animals and humans is (Favia et al., 1996), which allows to increased, at least because of the simul- reliably distinguish D. immitis and D. taneous presence of vectors showing repens larval stages developing in the diurnal and nocturnal activity patterns. invertebrate host (Cancrini and The same concern could be addressed Kramer, 2001). On this basis, entomo- to Croatia. In fact, although to the best logical investigations to date carried of our knowledge no data are available out in Italy and performed using PCR- for dirofilariosis natural vectors in the based technologies have shown that Ae. Country, the existence of a culicidofau- albopictus, Cx. pipiens and An. mac- na almost overlapping to that present ulipennis s.l. proved natural vectors for in Italy (Ramsdale et al., 2001; both D. immitis and D. repens, where- Ramsdale and Show, 2006; Merdic and as Cq. richiardii is almost certainty vec- Boca, 2004) suggests the possible tor for D. immitis. Moreover, importance of the same species. abdomens of Cx. modestus, Cx. torren- tium, Ae. punctor, Ae. cinereus, Ae. detritus, and Ae. geniculatus have been References found positive to D. immitis. Those species could act as vectors, but their Beerntsen BT, Severson DW, Christensen BM, role needs further confirmations, being 1994. Aedes aegypti: characterization of a the abdomen a location common to hemolymph polypeptide expressed during melanotic encapsulation of filarial worms. Exp either just ingested or infectious larvae Parasitol 79: 312-321. (Rossi et al., 2002; Cancrini et al., Bemrick WY, Moorehouse DE, 1968. Potential 2003a,b, 2004, 2006). A more exten- vectors of Dirofilaria immitis in the Brisbane sive application of the molecular tech- area of Queensland, Australia. J Med Entomol niques will allow a clearer and more 5: 269-272. Bradley TJ, Nayar JK, 1985. Intracellular melaniza- comprehensive understanding of the tion of the larvae of Dirofilaria immitis in the epidemiology of subcutaneous dirofi- malpighian tubules of the mosquito, Aedes sollic- lariosis and of feline and canine heart- itans. J Invertebr Pathol 45: 339-345. Vectors of Dirofilaria nematodes 57

Cancrini G, Iori A, 1981. Ulteriori osservazioni in relazione alle infezioni con Dirofilaria. sulla infestazione sperimentale del gatto con Parassitologia 10: 47-59. Dirofilaria repens. Parassitologia 23: 145-147. Di Sacco B, Cancrini G, Genchi C, 1992. Studio Cancrini G, Kramer LH, 2001. Insect vectors of del tropismo nei riguardi del cane e del gatto da Dirofilaria spp. In: Simon F and Genchi C (Eds), parte dei ditteri potenziali vettori delle filariosi Heartworm infection in humans and animals. in provincia di Pavia. Parassitologia 34: 11-12. Universidad Salamanca Ed, 63-82 pp. Di Sacco B, Del Zoppo B, Genchi C, 1994. Survey Cancrini G, Frangipane di Regalbono A, Ricci I, of dog Dirofilaria immitis mosquito vectors in Tessarin C, Gabrielli S, Pietrobelli M, 2003b. Milan and vicinity. Parassitologia 36 (suppl): 50. Aedes albopictus is a natural vector of Favia G, Lanfrancotti A, della Torre A, Cancrini Dirofilaria immitis in Italy. Vet Parasitol 118: G, Coluzzi M, 1996. A PCR- based method to 195-202. identify Dirofilaria repens and D. immitis. Cancrini G, Gabrielli S, Frangipane di Regalbono Parasitology 113: 567-571. A, Magi M, Scaramozzino P, Toma L, 2004. Forton KF, Christensen BM, Sutherland DR, Advances in the identification of natural vectors 1985. Ultrastructure of the melanization respon- of zoonotic filariae by PCR-based approaches. se of Aedes trivittatus against inoculated Proc IX EMOP, 400 p. Dirofilaria immitis microfilariae. J Parasitol 71: Cancrini G, Magi M, Gabrielli S, Arispici M, 331-341. Tolari F, Dell’Omodarme M, Prati MC, 2006. Frizzi G, Pedrotti D, 1957. Resistenza alla Natural Vectors of Dirofilariasis in Rural And Dirofilaria immitis (Leidy) in alcune specie di Urban Areas of the Tuscan region (Central Anopheles dei sottogeneri Nyssorhynchus e Italy). J Med Entomol 43: 574-579. Myzomyia. Simp Genet Pavia 2: 338-348. Cancrini G, Mantovani A, Coluzzi M, 1979. Genchi C, Di Sacco B, Cancrini G, 1992. Infestazione sperimentale del gatto con D. repens Epizoology of canine and feline heartworm di origine canina. Parassitologia 21: 89-90. infection in Northern Italy: possible mosquito Cancrini G, Pietrobelli M, Frangipane Di vectors. Proc Heartworm Symp, 39-46 pp. Regalbono A, Tampieri MP, della Torre A, Genchi C, Rinaldi L, Cascone C, Mortarino M, 1995. Development of Dirofilaria and Setaria Cringoli G, 2005. Is heartworm disease really nematodes in Aedes albopictus. Parassitologia spreading in Europe? Vet Parasitol 133: 37: 141-145. 137–141. Cancrini G, Romi R, Gabrielli S, Toma L, Di Paolo Grassi B, Noè G, 1900. The propagation of the M, Scaramozzino P, 2003a. First finding of filariae of the blood exclusively by means of the Dirofilaria repens in a natural population of puncture of peculiar mosquitoes. Brit Med J 3: Aedes albopictus. Med Vet Entomol 17: 448-451. 1306-1307. Cancrini G, Sun Yanchang, della Torre A, Coluzzi Grieve RB, Lok JB, Glickman LT, 1983. M, 1988. Influenza della temperatura sullo svi- Epidemiology of canine heartworm infection. luppo larvale di Dirofilaria repens in diverse Epidemiol Rev 5: 220-246. specie di zanzare. Parassitologia 30: 38. Iori A, Cancrini G, Vezzoni A, Del Ninno G, Tassi Christensen BM, 1978. Dirofilaria immitis: effect P, Genchi C, della Torre A, Coluzzi M, 1990. on the longevity of Aedes trivittatus. Exp Osservazioni sul ruolo di Culex pipiens nella tra- Parasitol 44: 116-123. smissione della filariasi del cane in Italia. Coluzzi M, 1964. Osservazioni sperimentali sul Parassitologia 32: 151-152. comportamento di Dirofilaria repens in diversi Hocking B, 1953. The intrinsic range and speed of gruppi di artropodi vettori. Parassitologia 4: fligth in insects. Trans R Ent Soc Lond 104: 223. 57-62. Kartman L, 1953. Factors influencing infection of Coluzzi M, Cancrini G, 1974. Genetica della the mosquito with Dirofilaria immitis Leidy, suscettibilità di Aedes aegypti a D. repens. 1856. Exp Parasitol 2: 27-78. Parassitologia 16: 239-256. Klowden MJ, 1996. Vector behavior. In: The Coluzzi M, Dallai R, Insom E, 1981. Morfologia e Biology of disease vectors. B.J. Beaty and W.C. funzione delle armature cibariche nelle zanzare. Marquard Es, University Press, Colorado. Parassitologia 23: 164-168. Labarthe N, Serrao ML, Melo YF, de Oliveira SJ, Coluzzi M, Trabucchi R, 1968. Importanza del- Lourenco-de-Oliveira R, 1998. Potential vectors l’armatura bucco-faringea in Anopheles e Culex of Dirofilaria immitis (Leidy 1856) in 58 Vectors of Dirofilaria nematodes

Itacoatiara, oceanic region of Niteroi municipal- (Diptera, Culicidae). Eur Mosq Bull 9. ity, state of Rio de Janeiro, Brazil. Mem Inst Ramsdale CD and Snow K, 2006. Distribution of Oswaldo Cruz 93: 425-432. the genus Anopheles in Europe. www.uel.ac.uk./ Le Corroler Y, 1957. Evolution de Dirofilaria mosquito/issue7/anopheles.htm immitis Leidy, 1856 chez Aedes (Stegomyia) Romi R, 2001. Malattie trasmesse da vettori e aegypti Lin. Souche “Orlando” (Floride). Bull cambiamenti climatici: analisi della situazione in Soc Pathol Exo 50: 542-555. Italia. Giorn Ital Med Trop 6: 131-140. Mansour AE, McCall JW, McTier TL, Ricketts R, Rossi L, Favia G, Ricci I, Cancrini G, 2002. 1995. Epidemiology of feline diroifilariasis. Proc Looking for the vectors of canine heartworm Heartworm Symp, 87-95 pp. infection in North of Italy. Parassitologia 36 Mattingly PF, 1969. The Biology of Mosquito- (suppl.): 127. Borne Disease. Carthy and Sutcliffe, London. Russell RC, Geary MJ, 1996. The influence of Merdic E, Boca I, 2004. Seasonal dynamics of the microfilarial density of dog heartworm Anopheles maculipennis complex in Osijek, Dirofilaria immitis on infection rate and survival Croatia. J Vector Ecol 29: 257-263. of Aedes notoscriptus and Culex annulirostris Mc Greevy PB, Mc Clelland GAH, Lavoipierre from Australia. Med Vet Entomol 10: 29-34. MMJ, 1974. Inheritance of susceptibility to Sauerman DM Jr, 1985. A survey for natural Dirofilaria immitis infection in Aedes aegypti. potential vectors of Dirofilaria immitis in Vero Ann Trop Med Parasitol 68: 97-109. beach. Mosq News 43: 222-225. Nayar JK, Sauerman DM, 1973. A comparative Vegni-Talluri M, Bigliardi E, Cancrini G, 1993. study of flight performance and fuel utilization Comparative ultrastructural study of Dirofilaria as a function of age of females of Florida repens (Nematoda, Filarioidea) development in mosquitoes. J Ins Physiol 19: 1977-1988. susceptible and refractory strains of Aedes Nelson GS, 1959. The identification of infective aegypti. Boll Zool 60: 377-383. filarial larvae mosquitoes: a note on the species Vegni-Talluri M, Cancrini G, 1991. Osservazioni found in wild mosquitoes on the Kenya Coast. J ultrastrutturali sullo sviluppo degli stadi di Helminthol 133: 233-256. Dirofilaria repens (Nematoda: Filarioidea) in Nelson GS, Heish RB, Furlong M, 1962. Studies in Aedes aegypti e Anopheles sinensis. Biologia filariasis in East Africa. II. Filarial infections in Oggi 5: 155-162. man, animals and mosquitoes on the Kenya Vegni-Talluri M, Cancrini G, 1994. An ultrastruc- Coast. Trans Roy Soc Trop Med Hyg 56: 202-217. tural study on the early cellular response to Pollono F, Cancrini G, Rossi L, 1994. Sampling of Dirofilaria immitis (Nematoda) in the dog attracted mosquitoes in Piedmont. Malpighian tubules of Aedes aegypti (refractory Parassitologia 36 (suppl.): 114. strains). Parasite 1: 343-348. Pollono F, Cancrini G, Rossi L, 1998. Indagine sui Zielke E, 1973. Untersuchungen zur Vererbung culicidi attratti da esca canina in Piemonte. der Empfänglichkeit gegenuber der Hundefilaie Parassitologia 40: 439-445. Dirofilaria immitis bei Culex pipiens fatigans Ramsdale CD, Alten B, Caglar SS, Ozer N, 2001. und Aedes aegypti. Z Tropenmed Parasitol 24: A revised, annotate checklist of the mosquitoes 36-44. 4 Pathogenesis of Dirofilaria spp. infections

Giulio Grandi, Tatjana Zˇ ivicˇnjak, Relja Beck Pathogenesis of Dirofilaria 61

Introduction pneumonitis and glomerulonephritis. Some individuals develop a hypersen- Dirofilaria spp., helminths belonging sitivity to microfilariae. Occasionally, to Dirofilariinae superfamily, are able to aberrant migration results in parasites cause a wide range of diseases. The best becoming trapped in ectopic locations known and studied is heartworm dis- like the anterior chamber of the eye ease, common in dogs and cats in areas (Weiner et al., 1980) or systemic arter- where Dirofilaria immitis is widespread. ies (Liu et al., 1966; Slonka et al., Other species of Dirofilaria, like D. 1977). Data regarding immunopatho- repens, are less important from a pathogenesis and in particular the role of genic point of view, but their differentia- cytokines, mediators as well as cellular tion from D. immitis is a fundamental components of the immunity in the diagnostic task. The zoonosic risks relat- development of heartworm-related ed to each species is also extremely lesions have been recently revised important. (Grandi et al., 2005). Although the spectrum of pathologies related to heartworm infection is broad, the most Pathogenesis of heartworm disease important clinical manifestation in (D. immitis) dogs is congestive heart failure (cor pulmonale). D. immitis infection is characterised by several different clinical pictures, caused both by adults and first stage Congestive heart failure (CHF, syn.: larvae (microfilariae). However, the cor pulmonale) pathophysiology of heartworm disease is mainly due to the presence of adult Heartworms are primary agents of vas- worms in the pulmonary arteries. The cular disease, rather than the cause of primary lesions occur in the pulmonary simple obstruction and/or blood flow arteries and lung parenchyma and are disturbances. Intimal proliferation mostly attributable to the intravascular occurs in arteries occupied by living adult parasites. They cause pulmonary worms and embolic worm fragments hypertension that, if not treated, pro- trigger thrombosis, both of which may gresses inevitably to congestive heart completely obstruct segments of the pul- failure. Other syndromes are related to monary arteries (Adcock, 1961). These the disturbance of blood flow due to effects, both leading to pulmonary the location of heartworms in the right hypertension, are strongly correlated to atrium at the level of tricuspidal valve. worm burden, in turn related to the This event causes massive haemolysis degree of their distribution through lung and related haemoglobinuria, responsi- parenchyma (Knight, 1980). ble for the vena caval syndrome (liver The juvenile adult heartworms are failure disease) (Ishihara et al., 1978; only about 2.5 cm long when they Kitagawa et al., 1986). Microfilariae reach the systemic venous circulation. play a relatively minor pathogenic role They passively embolize the pulmonary but may cause clinically significant arteries and are disbursed in propor- 62 Pathogenesis of Dirofilaria tion to the lobar blood flow. Generally, Furthermore, the disappearance of the larger right caudal lobar artery endothelial cells occurs without evi- accumulates more worms than the left dence of degeneration and is followed by (Atwell and Rezakhani, 1986). Contact an aggressive build-up of cells and struc- between the parasite and the intima of tural elements. This suggests that cells the pulmonary arteries is an important, have been dislodged rather than if not essential, initial step in the devel- destroyed in situ. Macrophages, granu- opment of the endovascular lesions. locytes, and platelets are attracted to the The earliest lesions are limited to the site of endothelial damage and adhere to small peripheral branches where the the exposed subendothelium. Shortly worms first come to rest. As the para- after their arrival, vascular smooth mus- site grows, lesions occur in more prox- cle cells migrate into the intima and a imal segments. Intimal thickening and very active process of myointimal prolif- narrowing of the vessel lumen in small eration produces rapid growth of the peripheral branches of the pulmonary lesions. The integrity of the endothelium arteries are the major cause of obstruct- covering the intraluminal villous-rugose ed blood flow and pulmonary hyperten- growths is restored and thrombosis is sion. The intimal proliferation is not a feature at this stage of the disease caused by migration of medial smooth (Munnel et al., 1980; Schaub et al., muscle cells through the internal elas- 1981). The prominence of platelets in tic laminae (Munnel et al., 1980; the acute lesions and their documented Patterson and Luginbuhl, 1963; ability to stimulate growth of the vascu- Schaub et al., 1981). lar smooth muscle, through the release The pathogenesis of the arteritis of platelet-derived growth factors caused by heartworms remains a matter (PDGFs) (Ross, 1986), are hypothe- of speculation. Disruption of endothelial sized to be a likely mechanism for trig- cell junction and denuding of the intimal gering and sustaining the growth of surface are characteristics of the first these lesions (Schaub and Rawlings, lesions that occur only after a few days 1980; Schaub et al., 1981). The circum- of presence of worms. Physical trauma, ferential lesions of the peripheral dis- metabolic and immune-mediated cyto- tributing arteries transcend into more toxicity induced by the parasite (Keith et discontinuous rugose ridges in the larg- al., 1983a), as well as other less likely er elastic branches (Keith et al., 1983b). mechanisms (Schaub and Rawlings, In cross section, these ridges have a 1980) have been considered. Several villous appearance and are considered facts seem to be more consistent with pathognomonic for heartworm infec- mechanical trauma as the initiating tion. Although the lesions thicken the event rather than cytotoxicity or induc- wall of these large elastic vessels and tion of host immune responses. The evi- produce a rough texture on the intimal dence suggests that injury to the surface, they do not obstruct blood flow endothelium occurs immediately upon by narrowing the lumen. On the con- arrival of the parasite, too soon for the trary, the large distributing arteries actu- components of an immune response to ally dilate as pulmonary hypertension fall in place without prior sensitisation. becomes increasingly severe. Pathogenesis of Dirofilaria 63

Pulmonary blood flow is impeded pri- the more severe the disease and active marily by the reduction in cross-sec- the patient, the more cardiac work tional area of the arterial vascular bed, must be performed. In advanced cases caused by obliterative endarteritis of of heartworm disease, low-output con- small peripheral branches. Recently, in gestive heart failure develops as a result heartworm infected dogs it has been of the right ventricle’s inability to gen- demonstrated that there are markedly erate and sustain the high perfusion increased plasma level of endothelin-1, pressures required to move blood a mediator that induces acute vasocon- through the lung. At a molecular level, striction and chronic vascular remodel- in the myocardium of heartworm ling. It is probable that both these infected dogs it has been observed a events contribute in turn to the devel- decrease of extracellular collagen opment of pulmonary hypertension matrix; this event can contribute to the (Uchide and Saida, 2005). Thrombosis dilatation of the ventricle, thereby and thromboembolism may further markedly affecting the systolic and compromise the pulmonary circulation. diastolic functions of the heart (Wang As worms accumulate, lesions also et al., 2005). Frequently, dogs at this develop in the large distributing arter- stage experience syncope when ies, which dilate and become stiffer as attempting to suddenly increase car- pulmonary blood pressure rises. The diac output. Right-sided congestive decreased distension of the large ves- heart failure (R-CHF) with ascites, sels significantly increases cardiac work hepatomegaly, and cachexia is a late by coupling the right ventricle directly sequela and may be precipitated by an to the high vascular resistance in the acute episode of pulmonary throm- obstructed peripheral vasculature. boembolism. Right ventricular hypertrophy is a compensatory response to the increased pressure load. As heartworm D. immitis infection in the cat infection impedes flow in an increasing number of branches, the pulmonary The general pulmonary pathology in vascular reserve diminishes. For a time, the cat is similar to the one observed in normal pulmonary blood pressure is dog. Muscular hypertrophy, villous preserved at rest and rises only modest- endarteritis, and cellular infiltrates of ly during exercise as patent arteries the adventitia are typically more severe reach full distension. Eventually, the in the caudal pulmonary arteries pulmonary arterial tree is restricted to (Dillon, 1998). A characteristic feature the point that it assumes the character- of the reaction in felines in response to istics of a system of rigid tubes and pul- heartworm infection is the development monary vascular resistance becomes of severe muscular hypertrophy in the fixed. This occurs about the time pul- smaller arteries (McCall et al., 1994). monary blood pressure becomes elevat- The host’s response to the parasite is ed at rest (Knight, 1977). At this stage, intense and the role of inflammation is pressure rises in direct proportion to very important, being platelet factors further increase in flow. Consequently, release less important in the cat rather 64 Pathogenesis of Dirofilaria than in the dog (Furlanello et al., 1998). tion with the presence of D. repens into The cause of the acute and often fatal two clinical syndromes (Scarzi, 1995). crisis in the cat is lung injury resulting The first one is characterized by a in respiratory distress. Frequently this nodular multifocal dermatitis, mainly event is associated with the death of as localized in facial region (Scott and little as one adult heartworm. The lung Vaughn, 1987), the second instead is can become acutely edematous and res- characterized by the presence of sever- piratory failure, instead of heart failure, al pruriginous papulae; this one is con- becomes the life threatening event. sidered similar to sarcoptic mange Being pulmonary hypertension an occa- (Halliwell and Gorman, 1989), sional event, right sided heart failure although the clinical experience of one and severe cor pulmonale is uncommon of us (Tatjana Zˇ ivicˇnjak) suggests that in feline heartworm infection (Dillon, pruritus rarely occurs in D. repens 1998; Genchi et al., 1995). microfilaremic dogs. The most frequent reports, however, are of dermatological signs (generalized dermatitis, localized D. repens infection alopecia, scratching and rubbing) associated with the presence of adults and Much less is known about the patho- microfilariae in the skin (Lee Gross et genesis of D. repens infection, which al., 1992; Kamalu, 1986; Mandelli and usually is characterized by the occur- Mantovani, 1966; Scarzi, 1995; rence of painless subcutaneous nodules Zˇ ivicˇnjak et al., 2006). in which adult parasites reside (Bredal It has been suggested that the pathol- et al., 1998). The localization of adult ogy may be significant in cases of mas- worms can vary, and recently it has sive infection (Kamalu, 1991; Mandelli been reported also the presence of a and Mantovani, 1966; Mantovani, female of D. repens in the conjunctiva 1965; Restani et al., 1962). In the few of a dog (Hermosilla et al., 2006). This cases found to be massively infected asymptomatic pattern of infection, with adult worms and with simultane- which seems to be the most common, ously high microfilaremia, gross and transforms the discovery of D. repens histopathological changes have been infection in an accidental finding. This reported in many organs, like spleen, occurs mainly in dogs (and cats) that liver, kidneys, lungs, heart and brain are showing other symptoms or dis- (Kamalu, 1991; Mandelli and eases, not related to the presence of Mantovani, 1966, Restani et al., 1962). these nematodes in the subcutaneous The nature of these lesions suggests tissues. Indeed, the majority of infected combined mechanical and immuno- animals do not present any clinical pathological effects elicited by both sign, notwithstanding the occurrence of micro- and macro-filariae (Mantovani, persistent microfilaremia, therefore 1965), also if it is not possible to deter- making it difficult to assess the patho- mine what underlying mechanisms may genicity of D. repens (Soulsby, 1982). be involved, also due to the lack of Some authors classified the rare clin- experimental infection with reproduc- ical manifestations observed in association of these clinical signs. Nothing is Pathogenesis of Dirofilaria 65 currently known about the potential Dirofilaria immitis: distribution within the pulmonary arteries. Am J Vet Res 47: 1044-1045. ability of microfilariae to cause hyper- Beaufils JP, Martin-Granel J, 1987. sensitivity and inflammatory reactions Polyparasitisme chez un chien, avec passage de at the cutaneous level, although this is leishmanies dans le sang circulant. Prat Méd Chir Anim Comp 22: 381-385. likely one of the major mechanisms Bonvicini A, 1910. Di un caso di filariosi in un involved. Furthermore, it has been sug- cane affetto da rogna demodettica. Med Zooiatr gested that D. repens-related dermatitis Torino 4: 46-55. is a conditioned pathology, that requires Bredal WP, Gjerde B, Eberhard ML, Aleksandersen M, Wilhelmsen DK, Mansfield the presence of other infectious agents LS, 1998. Adult Dirofilaria repens in a subcu- or stress (Bonvicini, 1910; Beaufils and taneous granuloma on the chest of a dog. J Martin-Granel, 1987; Cazelles and Small Anim Pract 39: 595-597. Cazelles C, Montagner C, 1995. Deux cas de fila- Montagner, 1995). Single case reports riose cutanee associee a une leishmaniose. Point of acute liver failure in the cat (Schwan Veterinaire 27(170): 71-73. et al., 2000) and cutaneous hyperpig- Dillon R, 1998. Clinical significance of feline heartworm disease. Vet Clin North Am: Small mentation in a dog have been attributed Anim Pract 28: 1547-1565. to D. repens infection (Kamalu, 1991). Furlanello T, Caldin M, Vezzoni A, Venco L, Kitagawa H, 1998. Patogenesi. In: La Filariosi cardiopolmonare del cane e del gatto (Genchi C, Venco L, Vezzoni A, Eds), Edizioni SCIVAC, Conclusions Cremona, 31-46 pp. Genchi C, Venco L, Vezzoni A, 1995. Aggiornamento sulla filariosi cardiopolmonare The pathogenesis of D. immitis is del gatto. Veterinaria-Cremona 9 (1): 53-58. well known, while that of D. repens is Grandi G, Kramer LH, Bazzocchi C, Mortarino M, still far from being understood, also if Venco L, Genchi C, 2005. Dirofilaria immitis and the endosymbiont Wolbachia: inflamma- in the last years this parasite has been tion and immune response in heartworm disea- suspected to be not as harmless as usu- se. Jpn J Vet Parasitol 4: 11-16. ally considered. However, both para- Halliwell REW, Gorman NT, 1989. Veterinary clinical immunology, Philadelphia. WB sites are important agents of zoonosis, Saunders, 239 p. in particular D. repens, and veterinari- Hermosilla C, Pantchev N, Dyachenko V, ans play an essential role in protecting Gutmann M, Bauer C, 2006. First autochthonous case of canine ocular Dirofilaria repens humans from infection. For this reason, infection in Germany. Vet Rec 158: 134-135. it is essential that clinical signs are rec- Ishihara K, Kitagawa H, Sasaki Y, Ojima M, Yagata ognized, correct diagnosis is made and Y, Suganuma Y, 1978. Clinicopathological studies on canine dirofilarial hemoglobinuria. Jpn J efficient treatment and prophylaxis are Vet Sci 40: 525-537. guaranteed not only to safeguard the Kamalu BP, 1986. Canine filariasis in southeastern health and well-being of our pets, but Nigeria. Bull Anim Health Prod Afr 34: 203-205. Kamalu BP, 1991. Canine filariasis caused by also that of our clients, neighbours and Dirofilaria repens in southeastern Nigeria. Vet ourselves. Parasitol 40: 335-338. Keith JC, Schaub RG, Rawlings CA, 1983a. Early arterial injury-induced myointimal proliferation in canine pulmonary arteries. Am J Vet Res 44: References 181-186. Keith JC, Rawlings CA, Schaub RG, 1983b. Adcock JL, 1961. Pulmonary arterial lesions in Pulmonary thromboembolism during therapy of canine dirofilariasis. Am J Vet Res 22: 655-662. dirofilariasis with thiacetarsamide: modification Atwell RB, Rezakhani A, 1986. Inoculation of with aspirin or prednisolone. Am J Vet Res 44: dogs with artificial larvae similar to those of 1278-1283. 66 Pathogenesis of Dirofilaria

Kitagawa H, Sasaki Y, Ishihara K, 1986. Clinical Ross R, 1986. The pathogenesis of atherosclerosis: studies on canine dirofilarial hemoglobinuria: an update. N Engl J Med 314: 488-500. relationship between the heartworm mass at the Scarzi M, 1995. La dirofilariosi cutanea nel cane. tricuspid valve orifice and the plasma hemoglo- Obiettivi Documenti Vet 16 (6): 11-15. bin concentration. Jpn J Vet Sci 48: 99-103. Schaub RG, Rawlings CA, 1980. Pulmonary vas- Knight DH, 1977. Heartworm heart disease. Adv cular response during phases of canine heart- Vet Sci Comp Med 21: 107-149. worm disease: scanning electron microscopy Knight DH, 1980. Evolution of pulmonary artery study. Am J Vet Res 41: 1082-1089. disease in canine dirofilariasis: evaluation by Schaub RG, Rawlings CA, Keith JC, 1981. blood pressure measurements and angiography. Platelet adhesion and myointimal proliferation In Proceedings of the Heartworm Symposium in canine pulmonary arteries. Am J Pathol 104: ’80 (Otto GF Ed), Am Heart Soc, Bonner 13-22. Springs, KS, 55-62 pp. Schwan EV, Miller DB, de Kock D, van Heerden Knight DH, 1987. Heartworm infection. Vet. Clin. A, 2000. Dirofilaria repens in a cat with acute North Am: Small Anim Pract 17: 1463-1518. liver failure. J S Afr vet Assoc 71: 197-200. Lee Gross T, Ihrke PJ, Walder EJ, 1992. Veterinary Scott DW, Vaughn TC, 1987. Papulonodular der- dermatopathology. Mosby Year Book. matitis in a dog with occult filariasis. Comp Liu SK, Das KM, Tashjian RJ, 1966. Adult Anim Pract 1: 31. Dirofilaria immitis in the arterial system of a Slonka GF, Castleman W, Krum S, 1977. Adult dog. J Am Vet Med Assoc 148: 1501-1507. heartworms in arteries and veins of a dog. J Am Mandelli G, Mantovani A, 1966. Su di un caso di Vet Med Assoc 170: 717-719. infestazione massiva da Dirofilaria repens nel Soulsby EJL, 1982. Helminths. In Helminths, cane. Parassitologia 8: 21-28. Arthropods and Protozoa of Domesticated Mantovani A, 1965. Canine filariasis by Animals, 7th edition, Williams & Wilkins, Dirofilaria repens. Proceedings of the 32nd Baltimore, 311 p. Annual Meeting of the American Animal Uchide T, Saida K, 2005. Elevated endothelin-1 Hospital Association, 77-79 pp. expression in dogs with heartworm disease. J McCall JW, Calvert CA, Rawlings CA, 1994. Vet Med Sci 67: 1155-1161. Heartworm infection in cats: a life-threatening Wang JS, Tung KC, Huang CC, Lai CH, 2005. disease. Veterinary-Medicine 89: 639-647. Alteration of extracellular collagen matrix in the Munnel JF, Weldon JS, Lewis RE, Thrall DE, myocardium of canines infected with Dirofilaria McCall JW, 1980. Intimal lesions of the pul- immitis. Vet Parasitol 131: 261-265. monary artery in dogs with experimental dirofi- Weiner DJ, Aguirre G, Dubielzig R, 1980. lariasis. Am J Vet Res 41: 1108-1112. Ectopic-site filariid infection with immunologic Patterson DF, Luginbuhl HR, 1963. Clinico- follow-up of the host. In: Proceedings of the pathologic conference: case presentation. J Am Heartworm Symposium ’80 (Otto GF Ed), Am Vet Med Assoc 143: 619-628. Heart Soc, Bonner Springs, KS, 51-54. Restani R, Rossi G, Semproni G, 1962. Due inte- Zˇ ivicˇ njak T, Martinkovicˇ F, Beck R, 2006. ressanti reperti clinici in cani portatori di Dirofilariosis in Croatia: spread and public Dirofilaria repens. Atti della Società Italiana health impact. 5th Croatian congress on infec- delle Scienze Veterinarie 16: 406-412. tive diseases, Zadar, Croatia. Immunopathogenesis of filarial 5 infections in dogs and cats: a role for Wolbachia endosymbiont?

Laura H. Kramer Immonopathogenesis: a role for Wolbachia 69

Introduction (ii) the evolution of the bacteria match that of the filarial worms, Dirofilaria immitis is the causative and phylogenic studies have agent of canine and feline heartworm shown that the two organisms disease. Adult worms live in the pul- have been “walking hand-in- monary arteries, females produce first- hand” for millions of years; stage larvae (“microfilariae”) which are (iii) the bacteria are transmitted taken up by mosquitoes who then in from female to off-spring and in turn transmit the infection to other ani- this way Wolbachia increases its mals. In dogs, the infection is chronic own fitness by increasing the and leads to congestive heart failure. D. fitness of the host that is immitis, like most filarial worms stud- involved in its transmission; ied so far, harbour bacteria called (iv) removal of Wolbachia (antibi- Wolbachia which are thought to play otics/radiation) leads to sterility an essential role in the biology and of female worms and eventual reproductive functions of their filarial death of adults. hosts. Wolbachia pipientis, the only It is still unclear however exactly species thus far identified in the genus, what Wolbachia does to make it so are gram-negative bacteria belonging to important for its filarial host. It has the order Rickettsiales (just like been suggested that, while the filarial Ehrlichia spp and Anaplasma spp; worm likely supplies the bacteria with Bandi et al., 2001). In adult D. immitis, amino acids necessary for growth and Wolbachia is predominantly found replication, Wolbachia on the other throughout cells in the hypodermis, hand may produce several important which is directly under the worm’s molecules that are essential for heart- cuticle. In females, Wolbachia is also worms, like glutathione and haeme present in the ovaries, oocytes and (Foster et al., 2005). It is indeed a “one developing embryonic stages within the hand washing the other” situation that uteri. This suggests that the bacterium may, however, be the key to novel is vertically transmitted through the strategies for the control/treatment of cytoplasm of the egg. filarial infection, including canine and feline heartworm disease.

What does Wolbachia do in its filarial host? The role of Wolbachia in the inflammatory and immune response in D. There are several reasons why it is immitis-infected animals thought that the presence of Wolbachia is essential for a filarial worm’s survival: As a gram-negative bacteria, Wolb- (i) in those species of filarial achia has the potential to play an worms that have been identified important role in the pathogenesis and as harbouring Wolbachia, all of immune response to filarial infection. the individuals are infected: i.e. The possible consequences of the mas- 100% prevalence; sive release of Wolbachia in the filarial- 70 Immonopathogenesis: a role for Wolbachia

infected host has been evaluated. where microfilariae normally circulate Wolbachia are released both by living (Fig. 1). Furthermore, when we looked worms and following worm death at specific antibody responses to through natural attrition, microfilarial Wolbachia, we observed a stronger turnover and pharmacological inter- response in dogs with circulating vention (Taylor et al., 2001). In human microfilariae compared to dogs with and murine models of infection, the occult infection, supporting the release of bacteria has been shown to hypothesis that microfilarial turnover is be associated with the up-regulation of an important source of Wolbachia in pro-inflammatory cytokines, neu- dogs with heartworm disease. trophil recruitment and an increase in Furthermore, Wolbachia from D. specific immunoglobulins. Ongoing immitis has been shown to provoke studies in dogs with heartworm disease chemiokinesis and pro-inflammatory may shed light on what happens when cytokine production in canine neu- infected animals come into contact trophils (Bazzocchi et al., 2003, 2005). with the bacteria. For example, we Cats with heartworm disease also pro- recently tested the hypothesis that duce antibodies to Wolbachia. D. immitis-infected dogs come into Interestingly, it has recently been contact with Wolbachia either through reported that the development of a microfilarial turnover or natural death strong antibody response against D. of adult worms (Kramer et al., 2005). immitis occurs after one-two months of In our study, positive staining for infection and this may have important Wolbachia was observed in various tis- implications for early diagnosis sues from dogs who had died from nat- (Morchon et al., 2004). ural heartworm disease. Bacteria were Areas of future research should observed in the lungs and particularly include the possible diagnostic use of in organs like the kidney and liver, specific immune responses to

Fig. 1. Lung tissue from a D. immitis infected dog. A: The Wolbachia Surface Protein (WSP) is present within many neutrophils surrounding a pulmonary artery (ABC-HRP, X40). B: Free bacteria are also present in tissue (ABC-HRP, X100). Immonopathogenesis: a role for Wolbachia 71

Wolabchia, it’s potential immunomod- ment was able to block embryo- ulatory activity (prevention) and the genesis (Genchi et al., 1998; effects of antibiotic treatment in infect- Bandi et al., 1999); ed animals. (iii) adulticide effects: this a particularly intriguing aspect of antibiotic treatment of the filarial What are the effects of antibiotic treat- worm-infected host and one ment on filarial worms? that merits strict attention. Recently, clinical trials in human filari- Wolbachia can be eliminated from asis have reported extremely promising filarial worms through antibiotic thera- results: a recent placebo-controlled py of the infected host. Numerous stud- trial in humans infected with ies have shown that various treatment Wuchereria bancrofti has demonstrated protocols/dosages (tetracycline and a clear macrofilaricidal effect of doxy- synthetic derivatives appear to be the cycline (Taylor et al., 2005). When most effective), are able to drastically administered for 8 weeks at 200 reduce if not completely remove the mg/day, the treatment resulted in a endosymbiont from the worm host. complete amicrofilaremia in 28/32 Such depletion of Wolbachia is then patients assessed and a lack of scrotal followed by clear anti-filarial effects, worm nests (where adult worms reside) including: at 14 months post-treatment, as deter- (i) inhibition of larval develop- mined by ultrasonography in 21/27 ment: it has been shown that patients. In the other patients, the num- antibiotic treatment of filarial- ber of worm nests declined. This was infected hosts can inhibit moult- significantly different from placebo ing, an essential process in the patients, where lack of worm nests was maturation of worms from lar- only observed in 3/27. This is the first vae to adult; report of adulticide activity in a human (ii) female worm sterility: antibiotic filarial worm with antibiotics. Could treatment leads first to a reduc- antibiotic treatment have the same tion and then to the complete effect on D. immitis? Research is and sustained absence of micro- underway to answer this very impor- filariae. Researchers at the tant question. University of Milan, Italy have reported that D. immitis adults taken from naturally-infected What are the effects of antibiotic treat- dogs that had been treated with ment on the filarial worm-infected 20 mg kg-1 day-1 of doxycycline host? for 30 days showed morphlogi- cal alterations of uterine content It is very likely that antibiotic treat- with a dramatic decrease in the ment will have some beneficial effects number of pretzels and stretched on subjects with filariasis. First of all, microfilariae, indicating that those effects described above on the bacteriostatic antibiotic treat- worm will themselves lead to improved 72 Immonopathogenesis: a role for Wolbachia

clinical presentation: for example, a Wolbachia in filarial nematodes: evolutionary aspects and implications for the pathogenesis reduction in circulating microfilariae. and treatment of filarial diseases. Vet Parasitol However, if we consider Wolbachia as 98: 215-238. a potential cause of inflammation in the Bazzocchi C, Genchi C, Paltrinieri S, Lecchi., course of filarial disease, depletion of Mortarino M, Bandi C, 2003. Immunological role of the endosymbionts of Dirofilaria immi- the bacteria may be beneficial, inde- tis: the Wolbachia surface protein activates pendently of its effect on the worm. canine neutrophils with production of IL-8. Vet There is little data concerning the Parasitol 117: 73-83. Bazzocchi C, Mortarino M, Comazzi S, Bandi C, effects of antibiotic treatment in dogs Franceschi A, Genchi C, 2005. Expression and with natural heartworm disease. We function of toll-like receptor 2 in canine blood know however that such treatment phagocytes. Vet Immunol Immunopathol 104: 15-19. drastically reduces Wolbachia loads in Foster J, Ganatra M, Kamal I, Ware J, Makarova D. immitis. Preliminary trials in natu- K, Ivanova N, Bhattacharyya A, Kapatral V, rally infected dogs have shown that Kumar S, Posfai J, Vincze T, Ingram J, Moran L, Lapidus A, Omelchenko M, Kyrpides N, doxycycline treatment before adulticide Ghedin E, Wang S, Goltsman E, Joukov V, therapy with melarsomine may help Ostrovskaya O,Tsukerman K, Mazur M, reduce pro-inflammatory reactions due Comb D, Koonin E and Slatko B, 2005. The Wolbachia genome of Brugia malayi: to the death of adult worms (as seen by endosymbiont evolution within a human lower antibody levels against pathogenic nematode. PLoS Biology 3: 121- Wolbachia and lower levels of inter- 129. leukin 8, an inflammatory cytokine Genchi C, Sacchi L, Bandi C, Venco L, 1998. Preliminary results on the effect of tetacycline involved in nuetrophil recruitment). on the embryogenesis and symbiotic maceria These results have encouraged us to (Wolbachia) of Dirofilaria immitis. An continue evaluation of the clinical ben- update and discussion. Parassitologia 40: 247-249. efits of antibiotic treatment in naturally Kramer LH, Tamarozzi F, Morchon R, Lopez- infected dogs. Belmonte J, Marcos- Atxutegi C, Martin-Pacho Given the recent and very promising R, Simon F, 2005. Immune response to and tissue localization of the Wolbachia surface pro- developments in the use of tetracy- tein (WSP) in dogs with natural heartworm clines for micro-macrofilaricidal thera- (Dirofilaria immitis) infection. Vet Immunol py in human filariosis, it is hoped that Immunopathol 106: 303-308. Morchon R, Ferreira A C, Martin-Pacho J, similar attention will be given to canine Montoya A, Mortarino M, Genchi C, Simon F, and feline heartworm disease that 2004. Specific IgG antibody response against could greatly benefit from alternative antigens of Dirofilaria immitis and its Wolbachia endosymbiont bacterium in cats therapeutic strategies. with natural and experimental infections. Vet Parasitol 125: 313-321. Taylor MJ, Cross HF, Ford L, Makunde W H, References Prasad GBKS, Bilo K, 2001. Wolbachia bacteria in filarial immunity and disease. Parasite Bandi C, McCall JW, Genchi C, Corona S, Venco Immunol 23: 401-409. L and Sacchi L, 1999. Effects of tetracycline on Taylor MJ, Makunde WH, McGarry HF, Turner the filarial worms Brugia pahangi and JD, Mand S, and Hoerauf A, 2005. Dirofilaria immitis and their bacterial Macrofilaricidal activity after doxycycline treat- endosymbionts Wolbachia. Int J Parasitol 29: ment of Wuchereria bancrofti: a double-blind, 357-364. randomised placebo-controlled trial. Lancet Bandi C, Trees AJ and Brattig NW, 2001. 365: 2116-2121. 6 A possible role for Wolbachia in the diagnosis of Dirofilaria infections

Fernando Simón, Laura H. Kramer, Rodrigo Morchón, Claudio Genchi Diagnosis of Dirofilaria: a role for Wolbachia 75

Introduction tion between Wolbachia and the host immune system was obtained in mice Dirofilaria immitis is the aetiological immunized with crude D. immitis adult agent of cardiopulmonary dirofilariosis somatic and L3 antigens. These mice in dogs and cats, and pulmonary dirofi- develop specific antibodies against the lariosis in man, while Dirofilaria Wolbachia surface protein (WSP) (Nochtiella) repens causes subcuta- between 25 and 90 days post-inocula- neous dirofilariosis in animal reservoirs tion (Marcos-Atxutegi et al., 2003). In and subcutaneous/ocular dirofilariosis a different experiment, mice immu- in man. Like other filarial species, D. nized with WSP and/or a heat shock immitis and D. repens, harbour intra- protein (GroEl) from Wolbachia cellular bacteria of the genus showed a strong specific-antibody Wolbachia (Rickettsiales), whose par- response, demonstrating the contact of ticipation in the development of the Wolbachia molecules with murine inflammatory pathology of dirofilario- immune cells, and an intense expres- sis has been extensively investigated. sion of different cytokines and innate Nevertheless, the role of Wolbachia as inflammatory mediators a few days a possible diagnostic tool has been after inoculation (data not published). poorly studied until now. Dogs: Both microfilaremic and ami- Here we present our experience relat- crofilaremic naturally-infected dogs ed to Wolbachia in Dirofilaria spp. developed a high production of IgG canine, feline and human infections, as antibodies against WSP. Microfilaremic well as in a murine model of immu- dogs showed significantly higher IgG nization, and its possible importance antidody levels than amicrofilaremic for the diagnosis of Dirofilaria infec- dogs (Kramer et al., 2005a). Eighteen tion in humans. out of 128 dogs living in an endemic area were positive to a validated, commercial kit for circulating antigens. Wolbachia interacts with Dirofilaria- Among these, 15 were also positive for infected hosts IgG anti-WSP. Moreover, other 43 dogs that were negative for circulating anti- There is direct and indirect evidence gens showed elevated levels of IgG anti- that Wolbachia comes into contact WSP antibodies (data not published). with hosts infected with D. immitis and Evidence of the presence of D. repens. Direct evidence has been Wolbachia itself or its molecules in obtained in dogs and humans by Dirofilaria-infected hosts was obtained immunohistochemistry techniques, by an inmunohistochemistry using a while indirect evidence is based on the polyclonal antiserum against the presence of specific antibodies and Wolbachia surface protein (WSP). inflammatory mediators observed in Positive staining for WSP (Fig. 1a and dogs, cats and humans, as well as in 1b) was obtained in kidney tubules, mice immunized with Dirofilaria spp. glomerules, lung tissue (free bacteria) or Wolbachia-derived antigens. and alveolar macrophages and hepatic Mice: The first evidence of interac- monocytes of naturally infected dogs 76 Diagnosis of Dirofilaria: a role for Wolbachia

Fig.1. Immunohistochemistry (ABC-HRP method) for the localization of Wolbachia surface protein (WSP) in dogs and humans naturally infected with Dirofilaria immitis. A. Kidney tubule. B. Alveolar macrophage (dogs). C. Hypodermic chord. D. Morulae surrounded by immune cells (worms in subcutaneous nodules from humans).

(Kramer et al., 2005a,b). against WSP. Other 8 cats were positive Cats: Specific anti-WSP IgG antibod- to anti-WSP IgG but negative to the ies were detected in experimentally ELISA test for anti-WSP antibodies infected cats and in cats naturally (Morchón et al., 2004). exposed to a high risk of heartworm Humans: Specific IgG antibodies infection. Nine out of 49 cats living in against WSP were identified in patients an endemic area of the Canary Islands diagnosed as having clinical dirofilario- (Spain) were positive to a validated, sis (Fig. 2). The level of antibody commercial antibody kit and to an response was significantly higher in experimental ELISA for the detection of patients with pulmonary or subcuta- antibodies against synthetic peptides neous dirofilariosis than in clinically derived from molecules of 22 and 30 healthy individuals living in endemic kDa of the adult worms. Among these areas of canine dirofilariosis; among cats, 5 were positive to IgG-ELISA these only a part developed anti-WSP Diagnosis of Dirofilaria: a role for Wolbachia 77

Wolbachia stimulates a Th1-type response

Several studies have been conducted to identify the type of immune response stimulated by Wolbachia in different hosts of Dirofilaria spp. Mice immunized with WSP alone or in combination with GroEl developed a strong response of IgG2a antibodies (related to a Th1-type response in this species) immediately after inoculation. The highest levels were observed on Fig. 2. Specific IgG anti-WSP antibody response day 20 of the experiment (13 days in humans. A. Individuals diagnosed as having after the last inoculation) and similar pulmonary dirofilariosis. B. Humans diagnosed as levels were observed 10 days latter having subcutaneous dirofilariosis. C. Healthy donors residing in an endemic area. D. Healthy (data not published). In dogs with nat- donors living in a non endemic area. ural D. immitis infections, both microfilaremic and amicrofilaremic individ- antibodies (Simón et al., 2003 and data uals showed significantly higher IgG2 not published). antibody response against WSP than In humans diagnosed as having sub- negative controls. The response is sig- cutaneous dirofilariosis, positive stain- nificantly higher in microfilaremic ing for WSP was present in inflamma- than in amicrofilaremic dogs (Kramer tory cells (Fig. 1c and 1d), in very close et al., 2005b). Humans diagnosed as proximity to WSP+ oocytes and in one having pulmonary dirofilariosis also case WSP+ morulae were seen free develop significantly higher IgG1 anti- within inflammatory tissue. In those body levels (Th1-type response in nodules where worms were undergoing humans) than healthy individuals liv- degeneration, there were many cells ing in endemic areas. No antibodies within the inflammatory granuloma related to Th2-type response were that stained intensely positive for WSP detected in any human hosts (Marcos- (data not published). Atxutegi et al., 2004). An alternative to demonstrate that a worm removed from an infected person is a Dirofilaria, is the polymerase chain Specific anti-Wolbachia antibody lev- reaction (PCR). Amplification of els are modified by drug treatment Wolbachia DNA with specific primers can be achieved and may be applied Dogs with naturally acquired heart- directly on worms extracted from nod- worm disease and treated with melar- ules, or samples obtained by different somine showed a strong increase of histological techniques from nodules in specific IgG anti-WSP antibodies a few which the worms have been destroyed days after treatment (Fig. 3a). The by the granulomatous reaction. maximum antibody levels were detect- 78 Diagnosis of Dirofilaria: a role for Wolbachia

Fig. 3. Changes in specific IgG anti-WSP antibody levels in Dirofilaria immitis naturally infected dogs treated with melarsomine (A): a. Previous to treatment; b. One day after the treatment; c. 1 week (1-6) or 2 weeks (7) after the treatment; d. 2 weeks (1-6) or 6 weeks (7) after the treatment. Experimentally infected cats treated with ivermectin (B). ed at the end of the study (21 or 63 Nevertheless, the antibody response to days post-treatment depending the pro- both Dirofilaria and Wolbachia anti- tocol employed) (data not published). gens was measured during the follow- Change in the levels of anti- up of a subcunjuntival case due to D. Wolbachia and anti-Dirofilaria immitis repens, until 6 months after the antibodies following filaricide treat- removal of an intact, live female worm ment was observed in a group of exper- (Ruiz-Moreno et al., 1998). An ELISA imentally infected-, ivermectin treated- test for antibodies against D. repens cats (Morchón et al., 2004). A dramat- adult worms was positive until 3 ic increase of specific IgG antibodies months after the removal of the worm, against both WSP and Dirofilaria while the ELISA for anti-WSP antibod- immitis peptides (Dipp) was found 15 ies was positive before removal, but days after ivermectin treatment. At 3.5 was negative in the subsequent three months post-treatment the anti-Dipp blood samples (1, 3 and 6 months after antibodies decreased to values seen removal of the worm). before infection, while specific anti- WSP antibodies continued to increase till the end of the study 5.5 months Conclusions post-treatment. In the control group (infected-, untreated cats) the antibody The accurate diagnosis of canine and response against both WSP and Dipp feline D. immitis infections is the first increased continuously throughout the step towards the correct prevention of study (Fig. 3b). the spread of the disease in the animal Human pulmonary or subcutaneous reservoirs and human infections. The dirofilarioses do not require treatment. development of the parasite and the Diagnosis of Dirofilaria: a role for Wolbachia 79

signs it causes are different in dogs and interpreted as sign of drug effi- cats. Thus, the methods validated for cacy. Results of serological fol- the diagnosis in these two hosts are also low-up in the human subcon- different. Considering the excellent juntival case previously pre- performance of existing commercial sented confirms that massive kits for the detection of antigens and release of Wolbachia occurs antibodies for canine and feline dirofi- when worms are physically lariosis, respectively, the moderate/low destroyed, because in this case correspondence of Wolbachia antibody the removal of whole worm tests with these kits, and the existence causes an immediate and clear of other diagnostic techniques like fall of specific anti-Wolbachia detection of microfilariae and echocar- antibodies. diography, the application of serologic In the case of human pulmonary and diagnostic tests based on the detection subcutaneous/ocular dirofilariosis, the of anti-Wolbachia antibodies in animal suspicion of neoplasia caused by the dirofilariosis is not useful. presence of a nodule makes differential Nevertheless, the detection of anti- diagnosis of prime importance. This Wolbachia antibodies could be of inter- implies that once the dirofilarial origin est in: of a nodule is demonstrated, the spe- (i) epidemiological studies for cific identification is a secondary mat- evaluation of the real pressure ter (Simón et al., 2005). When worms of infection in an endemic area. are present inside the nodules, the spe- In this case, risk levels are rep- cific identification is possible by histol- resented not only by active ogy, but if they have been destroyed by infection (detected by commer- the inflammatory reaction or the physi- cial kits), but also by prepatent cian takes a conservative attitude, evi- infections, mainly in feline diro- dence for the presence of Wolbachia filariosis. In these cases, a test can contribute to the identification of for the detection of specific the filarial origin of the nodule. This antibodies against Wolbachia can be obtained by antibody tests, PCR could be an invaluable diagnos- or immunohistochemical techniques. tic tool, as shown by the results In the first case invasive techniques are discussed above; not necessary, while in the other two, (ii) evaluation of the efficacy of tissue samples must be obtained. macro/microfilaricidal treat- In conclusion, in spite of the exis- ment. The death of filarial tence of validated methods for the worms is the main factor for diagnosis of dirofilariosis in animal the release of Wolbachia, reservoirs based on the use or detec- increasing the stimulus of the tion of Dirofilaria molecules, the iden- host’s immune system. The tification of Wolbachia by both direct detection of a rise of anti- and indirect methods, seems an excel- Wolbachia antibody levels, lent complementary data and consti- together with a fall of the anti- tute an effective tool for epidemiologi- Dirofilaria antibodies can be cal studies and research related to 80 Diagnosis of Dirofilaria: a role for Wolbachia

inflammatory pathology. In humans role for Wolbachia? Vet Parasitol 112: 117-130. the detection of anti-Wolbachia anti- Marcos-Atxutegi C, Gabrielli S, Kramer LH, Cancrini G, Simón F, 2004. Antibody response bodies can be a fundamental tool in the against Dirofilaria immitis and Wolbachia differential diagnosis for the identifica- endosymbiont in naturally infected canine and tion of the genus Dirofilaria in cases in human hosts. Proc. IX European Multicolloquium which the worms have been previously of Parasitology 2: 297-302. destroyed by the granulomatous reac- Morchón R, Ferreira AC, Martín-Pacho R, Montoya A, Mortarino M, Genchi C, Simón F, 2004. tion of the host. Specific IgG antibody response against antigens of Dirofilaria immitis and Wolbachia endosym- References biont bacterium in cats with natural and experimental infections. Vet Parasitol 125: 313-321. Ruiz-Moreno JM, Bornay FJ, Prieto Maza G, Kramer LH, Tamarozzi F, Morchón R, López- Medrano M, Simón F Eberhard M, 1998. Belmonte J, Marcos-Atxutegi C, Martín-Pacho Subconjuntival infection with Dirofilaria R, Simón F, 2005a. Immune response to and tis- repens. Serological corfirmation of cure follo- sue localization of the Wolbachia surface pro- wing surgery. Arch Ophtalmol 116: 1370-1372. tein (WSP) in dogs with natural heartworm Simón F, Prieto G, Morchón R, Bazzocchi C, (Dirofilaria immitis) infection. Vet Immunol Bandi C, Genchi C, 2003. Immunoglobulin G Immunopathol 106: 303-308. antibodies against the endosymbionts of filarial Kramer LH, Simón F, Tamarozzi F, Genchi M, nematodes (Wolbachia) in patients with pulmo- Bazzocchi C, 2005b. Is Wolbachia complicating nary dirofilariosis. Clin Diagn Lab Immunol 10: the pathological effects of Dirofilaria immitis 180-181. infections? Vet Parasitol 133: 133-136. Simón F, López-Belmonte J, Marcos-Atxutegi C, Marcos-Atxutegi C, Kramer LH, Fernández I, Morchón R, Martín-Pacho R, 2005. What is hap- Simoncini L, Genchi M, Prieto G, Simón F, 2003. pening outside North America regarding human Th1 response in BALB/c mice immunized with dirofilariosis? Vet Parasitol 133: 181-189. Dirofilaria immitis soluble antigens: a possible Human dirofilariasis due to 7 Dirofilaria (Nochtiella) repens: an update of world literature from 1995 to 2000*

S. Pampiglione, F. Rivasi

* Paper from Parassitologia 42: 231-254, 2000 Human dirofilariasis 83

Introduction and have accepted them on trust. A few of other cases that occurred Following on from their 1995 review before 1995 but which escaped men- (Pampiglione et al.) of the world litera- tion in our previous enquiry have now ture on human dirofilariasis due to been included so as to offer as complete Dirofilaria (Nochtiella) repens the a general picture as possible of the authors have attempted to gather all the overall number and geographical distri- new cases published in the scientific lit- bution of subjects affected. erature from 1995 to 2000 for an In this context two recent reviews of update on the subject. Dirofilaria cases occurring in the ex-Soviet Union (Nochtiella) repens (Nematoda: (Avdiukhina et al., 1996, 1997) have Filarioidea, Onchocercidae) is a habitu- been extremely useful and have enabled al parasite of the subcutaneous tissue of us to include in our statistics many cases dogs and other carnivores. It is trans- published in Russian journals that are mitted by Culicidae and can also acci- unlikely to be available in European or dentally affect man. Of the species of American libraries. Similarly, as regards filariae found in the temperate regions Sri Lanka, the review recently published of the world it is the one most com- by Dissanaike et al. (1997), together monly observed. It is present only in the with their prompt personal communica- Old World, notably in Italy, where 181 tions of subsequent updates, have been human cases were reported up to 1995 of great value. The data missing for distributed practically over the whole of some of the cases reported in our 1995 the national territory. Endemic zones review have now been included (age, were also identified in France, Greece, sex, locality and location, together with Turkey, Sri Lanka, Ukraine, the Russian bibliographical reference, if applicable). Federation, Uzbekistan and other coun- They appear in the numerical listing tries for a total of 410 cases spread over with the number 0, since they were 30 different nations. already counted in the previous review.

Materials and methods Results

The cases published in the world liter- Geographical distribution ature during the last 5 years have been classified by nation and the essential There do not appear to be any signif- data for each one (year of publication, icant variations in the geographical dis- author’s name, patient’s sex, age, place tribution of the parasite in the dog and of residence, location of the parasite) other carnivores from that reported in set out in tabular form. In some cases in the previous review, exceptions being 5 which the authors did not provide the animals in Tenerife (Canary Isles) full anamnestic and morphological (Stenzenberger and Gothe, 1999), 3 in details necessary for a correct documen- Hungary (Fock et al., 1998; Szell et al., tation we have limited ourselves to 1999), several dogs (number not stat- recording what findings were reported ed) in the South African Republic 84 Human dirofilariasis

(Bredal et al., 1998; Schwan, personal (2000a) claim that dogs are more communication), all considered affected by D. repens than by D. immi- autochtonous infections by the respec- tis, in contrast to a previous study by tive observers. Rojo Vasquez et al. (1990), basing Imported cases from other endemic their results on 114 canine blood sam- countries are reported in Germany ples tested with Knott technique, PCR (Zahler et al., 1997; Glaser and Gothe, and ELISA for specific IgG detection. 1998) and also in the south of In Bulgaria, Kanev et al. (1996) report Switzerland (Bucklar et al., 1998), 2 dogs testing positive out of 192 even though in the latter instance it examined. may be suspected that the parasite’s life In Greece, prevalence is between 6.7 cycle completed itself in loco given the and 22% (Vakalis and Himonas, 1997). mildness of the climate in recent years. The geographical distribution of both In fact, Petruschke et al. (1998) report D. immitis and D. repens in the the presence of D. repens in the low- European Union in dogs is illustrated in lying Canton Ticino in 2 dogs that a map by Muro et al. (1999). apparently had never been outside the The possibility that the biological cycle area. The geographical distribution of of D. repens completes itself on Israeli canine dirofilariasis in France has been territory, and that therefore the human illustrated by Beuguet and Bourdasseau cases diagnosed there are autochtonous, (1996) and by Chauve (1997). The lat- is confirmed by the discovery of a dog ter claims that the parasitosis is present affected by the parasite but which had in 19 of the 62 districts investigated never left Israel (Harrus et al., 1998). and that it can affect as many as 22% In northern Italy, a study involving of the animals examined. Generally, in 2628 dogs (Rossi et al., 1996; Pollono France D. repens appears to be more et al., 1998) confirms the high inci- common than D. immitis, while neither dence of D. repens in Piedmont with a species has been reported there in cats. prevalence of up to 20.5%. In the Cazelles and Montagner (1995) report province of Varese (Lombardy), 2 cases of dogs simultaneously affected Petruschke et al. (1998) report a preva- by leishmaniasis and dirofilariasis due lence of 6.9% out of 216 dogs exam- to D. repens in Aveyron and Gard ined. In Sicily, it is confirmed that (southern France). autochtonous cases due to D. immitis In Spain, the presence of canine diro- are rare, maybe even non-existent, filariasis is reconfirmed in Alicante, whereas D. repens is widespread Cadiz and Salamanca, albeit with a (Pennisi and Furfaro, 1997; Giannetto prevalence of less than 2%, and, in the et al., 1999), particularly in the Ebro delta only, of 9.4% (Anguera province of Trapani, where Giannetto Galiana, 1995; Lucientes and Castello, et al. (1997) diagnosed a prevalence of 1996). Aranda et al. (1998) found no 22.8% for this latter species. In the evidence of the parasitosis in 188 dogs communes of Vesuvius (province of examined in the Barcelona area. In the Naples), microfilariae of D. repens Alicante and Elche areas (southeastern were found in 3% of 351 dogs exam- provinces of Spain) Cancrini et al. ined (Rinaldi et al., 2000). In Tuscany Human dirofilariasis 85

(central Italy), where canine dirofilaria- involving 25 countries. In Europe: sis due to D. repens affects 25-39% of Belgium, Bulgaria, France, Greece, dogs, Marconcini et al. (1996) found Hungary, Italy, Romania, Serbian microfilaraemia in 1% of 523 foxes Republic, Slovakia, Slovenia, Spain, examined, while autopsy revealed adult Ukraine; in Africa: Kenya and Tunisia; nematodes in 3 out of 28 foxes exam- in Asia: Georgia, India, Iran, Israel, ined. However, the authors in question Kazakhstan, Malaysia, Russia, Sri do not consider the fox to be a reservoir Lanka, Turkey, Turkmenistan and of parasitosis. Uzbekistan. The countries most affect- In South Africa, the presence of D. ed have been Italy (117 cases), Sri repens has been found for the first time Lanka (101), Russia (Siberia included, in a cat suffering from hepatic insuffi- 61), Ukraina (23) and France (23). In ciency (Schwan et al., 2000). Tarello the few cases found in Austria and (2000a,b) claims to have found micro- Germany the infection was picked up filariae of D. repens in the blood of 10 in other countries. In Belgium, where cats associated with pruritic dermatosis the parasite has never previously been and haemobartonellosis in Alessandria observed, a curious episode of dirofi- (northern Italy) and in another cat lariasis is reported (van den Ende et al., affected by ulceration on both flanks. 1995) involving three members of the In Romania, Olteanu, in a congres- same family; it may have been caused sional communication (1996) reports by a mosquito transported in luggage D. repens not only in the dog but also from an endemic zone or from a nearby in the fox, the wolf and the cat, as well international airport. Parasitological as in man, but without specifying the diagnosis (itching, cutaneous striae and number of cases. By way of confirma- a nodule containing a female of D. tion of the parasite on Romanian terri- repens) was possible in only one of the tory, said author quotes other three cases, but the presence of the researchers (Motas, 1903; Popovici, same type of cutaneous striae persisting 1916) who have, in the past, discov- for months in the other two family ered it in dogs. members would suggest a cryptic infec- In Sri Lanka the high prevalence of tion of the same nature. D. repens in dog, formerly signalled In a large part of Africa and in other (Dissanaike, 1961, and others) is con- developing regions of the world, as firmed to reach 60-70% in some Orihel and Eberhard (1998) point out, provinces by Dissanaike et al. (1997). it must be borne in mind that it is not Roncalli (1998), in a paper presented always possible to establish with any at the XX Congress of the Italian degree of certainty whether infections Parasitology Society, described the his- due to D. repens in man are rare or fre- tory of canine dirofilariasis from the quent because their diagnosis is deter- 17th century to the present with some mined by the level of development and observations on the geographic distri- the efficiency of the health services and bution for the past times. hence by the diagnostic resources avail- Cases of human dirofilariasis report- able. The only countries where the geo- ed since 1995 number 372 in all, graphical distribution of human dirofi- 86 Human dirofilariasis lariasis has been traced are France, Distribution by age and sex Greece, Italy, the Russian Federation and Sri Lanka. In Italy, cases of the The age-range of the subjects affected infection have been reported in 9 goes from 4 months to 100 years, most regions, prevalently in Piedmont. of them being in their 40s. In Sri Takings the cases recorded in the previ- Lanka, Dissanaike et al. (1997) report ous enquiry into account, the parasito- a considerable number of cases in chil- sis is found to be present in 19 regions dren less than 10 years old, a phenom- of Italy out of 20. In Greece enon not found in any other country. (Pampiglione et al., 1996d), when the There is a greater prevalence in females recent cases are added to those report- than in males (186 of the 322 cases in ed previously, human dirofilariasis is which the patient’s sex is specified, rep- also seen to affect virtually the whole of resenting 57.8%), but it is not statisti- the country. In France, Raccurt (1999, cally significant. 2000) claims that there are at least 14 departments affected by D. repens with Carriers the highest incidence in the Mediterranean coastal regions, Extensive research into the Culicidae although sporadic occurrences on the captured with canine bait in a zone Atlantic seaboard and above the 46° endemic for D. repens in Piedmont latitude North have been reported (north-western Italy) (Pollono et al., (Guillot et al., 1998; Weill et al., 1998) has shown that this natural reser- 1999). In Sri Lanka (Dissanaike, voir is very attractive to Culex molestus 1996), the zones most affected are and Aedes caspius, but less so to C. pipi- those of the western provinces. In the ens, Ae. vexans and Anopheles mac- ex-Soviet Union, today the Russian ulipennis. The Culicidae are at their Federation, although there is no dis- most active, at least in Piedmont, during tribution map properly covering this the month of July. Talbalaghi (1999) has vast area, it appears from the sketchy shown an increase in the density of Ae. literature and, in particular, from the caspius in the same area over the last 3 reviews of Avdiukhina et al. (1996 years and an increase in the spread of lar- and 1997), that the regions where the val foci. Ae. caspius is the species most parasitosis is most endemic are the aggressive towards man; it is capable of Caucasus (in the south of the Russian travelling 20 km without wind assistance Republic), which account for 62% of and bites all day long. Using both canine the cases cited, and Ukraine, account- and human bait in endemic zones of the ing for 21%. Kazakhstan, Uzbekistan Veneto and Friuli-Venezia Giulia (north- and Georgia each account for 4% eastern Italy), Pietrobelli et al. (2000) and Turkmenistan for 3%, while demonstrated a strong attraction for C. other small states account for the pipiens and Ae. caspius, much reduced remaining 2%. The northern limit to for Ae. vexans. In Nigeria, Anyanwu et the spread of D. repens seems to be al. (2000) between 6 different species of the 62nd parallel in the Russian Culicidae tested (Culex pipiens fatigans, Federation. C. p. pipiens, Anopheles gambiae, Human dirofilariasis 87

Mansonia africana, Aedes vittatus, Ae. ture, typical of the parasitosis and of aegypti) have found that this last is the which it may prove to be the sole clini- most suitable vector for a local strain of cal manifestation. It is noticed after a D. repens. In order to select strains of Ae. period of approximately 2-12 months aegypti refractory and otherwise to D. from the penetration of the parasite, repens, Favia et al. (1998) used molecu- although much longer periods cannot lar biology techniques with apparently be ruled out since it is often impossible reliable results. Pampiglione and Gupta to recall the moment when infection (1998) reported the presence of took place. At times the nodule appears immunocytes (plasmatocytes) of without any external phlogistic mani- Culicidae, probably Aedes sp., in a mam- festation, at others it is accompanied by mary nodule enclosing an immature local erythema and pruritus, localised specimen of D. repens. The authors or more widespread urticarial manifes- think it likely that, during the passage of tations lasting a few days but recurring the infecting larvae from the mosquito to for as much as a year and a half man, the carrier’s immunocytes are often (Thérizol-Ferly et al., 1996). The nod- borne along by the larvae themselves and ule may also suppurate and assume the so penetrate the subcutaneous tissue, appearance of an abscess. There have only to be regularly destroyed by the been very occasional reports of defensive reactions of the host. erysipeloid phlogistic reactions and satellite lymphoadenopathies as well as Clinical history and symptomatology of rises in temperature of modest entity or with hyperpyrexia due to infectious In the dog, the parasitosis is generally complications that are quickly brought asymptomatic even when microfilari- under control with antibiotics. In eye aemia is fairly developed. There may, cases, complications such as detached however, be cutaneous manifestations retina, crystalline opacity, glaucoma, such as erythematous patches, papulae, uveitis, episcleritis and a limited loss of localised alopecia, eczema accompa- vision have exceptionally been reported nied by pruritus and, rarely, nodule (Mizkievic and Leontieva, 1961; and/or ulceration (Bredal et al., 1998; Avdiukhina et al., 1996). In one case Tarello, 1999). affecting the orbit, Braun et al. (1996) In man, the carrier’s bite has some- report monolateral exophthalmos last- times been described as painful, fol- ing for a year. Patients have reported lowed by slight local acute phlogosis subcutaneous migrations of the para- (erythema, swelling, pruritus) lasting site sometimes over considerable dis- for 5-8 days. Generally speaking, how- tances, such as from the lower limbs to ever, no particular sensation attributa- the neck or head, or from one side of ble to the insect bite is recalled by the the body to the other, during periods of patient other than that of being bitten many months (Delage et al., 1995; by mosquitoes. The subsequent forma- Azarova et al., 1995; Artamonova and tion of the nodule in which the nema- Nagornyi, 1996). In one patient, migra- tode remains trapped by the defensive tions in the tissues of the head had reaction of the host is an essential fea- caused trigeminal neuralgia, which dis- 88 Human dirofilariasis appeared when the nematode, sudden- junctiva or eyelids, vigorous rubbing of ly appearing under the bulbar con- the affected eye by the patient caused junctiva, was promptly removed the spontaneous emergence of the nem- (Avdiukhina et al., 1997). atode (Avdiukhina et al., 1966, 1967). The speed of these migrations, when Peripheral hypereosinophilia is report- visible, has been estimated at 30 cm in ed only rarely, with values generally lit- two days. Jelinek et al. (1996) report 2 tle more than 10-15% of the leukocyte cases of patients whose insistently com- formula (Thérizol-Ferly et al., 1996; plaints of “a worm under the skin” had Pampiglione et al., 1996). However, led the doctors to diagnose delusory Petrocheilou et al. (1998) report a parasitosis and have them admitted to Greek case, diagnosed merely on psychiatric ward, until the appearance account of the presence of microfilariae of a nodule and its subsequent histo- in the bloodstream defined by the logical analysis cleared up the matter. A authors as Dirofilaria-like, in which the sensation of a nematode travelling eosinophil count amounted to 26% of under the skin had been reported by the leukocyte formula. A case of sub- 10% of the Russian patients included conjunctival dirofilariasis in an HIV- in the review. Artamonova and positive patient is reported in France Nagornyi (1996) report a case with (Basset et al., 1996) without there cutaneous manifestations as in a true being any apparent correlation between larva migrans. The application of hot the development of the parasitosis and steam compresses or ultrasound thera- the presence of HIV. py appears to stimulate the movements of the nematode under the skin. There Locations are rare cases of 2 nodules being present in the same patient, either appear- In the dog, reports of localised nodu- ing contemporaneously or months lar formations enclosing the nematode apart (Degardin and Simonart, 1996; are rare, since the parasite is almost Jelinek et al., 1996; Orihel et al., 1997). always more or less elongated and free The nodules had appeared 5 years ear- in the web of subcutaneous tissue. lier (Degardin and Simonart, 1996) in Localisation in the sternal region, the one case, 2 years earlier in others. The neck and the flanks has nevertheless maximum duration of the parasitosis is been reported by Bredal et al. (1998) reported as being 12 years (Avdiukhina and Tarello (1999). A dog with D. et al., 1996). The spontaneous emer- repens in the heart has been observed gence of the nematode from the suppu- by Isola et al. (2000), thus demonstrat- rating nodule is reported on at least 10 ing that the species can penetrate the occasions. In one case, localised near bloodstream. the junction of the lips, squeezing the In man, on the other hand, the para- nodule, which had been mistaken for a site nodule is always present, if one boil, caused the emergence of the nem- excludes localisation in the subcon- atode together with pus, after which junctiva where the nematode can be the lesion healed in the space of a few considered to be migratory and not yet days. In others involving the subcon- trapped by the host’s reaction. In the Human dirofilariasis 89 vast majority of cases, the nodule is quent cases affecting the male genitals located in the subcutaneous tissue, the (scrotum, epididymis, spermatic chord, deep dermis or the submucosa. There penis), particularly in children below 5 are rare cases of location in muscle tis- years of age; this is in contrast with sue (Pampiglione et al., 1996c; Gros et what occurs in other geographical al., 1996), in the lymph nodes regions, where the few cases reported (Alain,1997; Shekhar et al., 1996; are restricted to adults. One case affect- Auer, pers. comm., 1998; Cancrini et ing the subcutaneous tissue of the penis al., 1999) and in the deep viscera. is reported from Corsica (Pampiglione More frequent are reports of localisa- et al., 1999b), another from Israel tion in the upper half of the body (Stayermann et al., 1999) and two (74%), in particular the ocular region more from Sri Lanka (Dissanaike et al., (eyelid, subconjunctiva, orbit) 1997). Pulmonary cases are reported (35.3%) and also the upper limbs from Greece (Pampiglione et al., (11%). In the cases reported by 2000d) and from Italy (Pampiglione et Avdiukhina et al. (1997) localisation al., 1996e, 2000b). in the ocular region accounts for 45% Jelinek et al. (1996) and Fueter and of the total observed in the Russian Gebbers (1997) report 2 cases and one Federation. Localisation in the vitreous case, respectively, of pulmonary infec- body in 3 cases and in the crystalline tion, one from Corsica, one from Italy lens in one case is reported respective- and the third in a globetrotter possibly ly in the Slovakian Republic (Vasilkova affected in America – all treated in et al., 1992) and Uzbekistan Germany. These authors attribute the (Mizkievic and Leontieva, 1961). A infection to D. immitis; however, it is case of preretinal localisation is also possible that, by analogy with many reported in Kazakhstan (Glinciuk et other cases reported from Corsica and al., 1992), although the morphological Italy, these are due to D. repens, description may cast doubt on the although, given the advanced state of actual species. In Italy, two cases are decomposition of the nematodes, pre- reported of localisation in the orbital cise identification was not possible. cavity, operated on by means of anteri- Rare cases affecting the omentum and or orbitotomy (Strianese et al., 1998). the mesentery have been casually Another case of localisation in the observed during laparotomy in Italy orbital cavity was operated on and the Russian Federation transnasally (Braun et al., 1996; Groell (Avdiukhina et al., 1997; Dorofeiev et et al., 1999), the authors producing a al., 1997; Mastinu et al., 1998; remarkable videotape of the removal Pampiglione et al., 2000b). of the nematode. A subconjunctival There are single instances of localisa- localisation is reported by Bianchi tion in the parotid gland (Hoop, 1997; Rossi et al. (1991) where the parasite H. Braun, pers. comm.), in the submu- had previously passed through the cosa of the mouth, of the base of the retrobulbar area causing exophthal- tongue, of the isthmus of the fauces mos. In Sri Lanka, Dissanaike and and within a granuloma at the root of a coworkers (1997) report relatively fre- tooth (Avdiukhina et al., 1997). 90 Human dirofilariasis

Table 1. World distribution of human infections by Dirofilaria repens. The dates refer to publications. Abbreviations: n.s.=not specified; p.c.=personal communication; 0=cases already counted in the previous review (1995).

No. Date Author(s) Sex Age Locality Location (yrs)

EUROPE Belgium

1 1995 van den Ende et al. F 33 n.s. upper eyelid*

* Two other suspected cases occorred in the same family. The locality where infections took place remained unknown.

Bulgaria

1 to 4 1996 Kaven et al. n.s. n.s. n.s. n.s.

France

1 1995 Nozais and Huerre M 39 Languedoc scrotum 2 1995 Delage et al. M 43 Ardèche eyelid 3 1996 Hautefort n.s. n.s. Arles thigh (cit. by Basset et al., 1996) 4 1995 Basset et al. M 37 Narbonne subconjunctival 5 1996 Masseron et al. M 61 Gironde subconjunctival 6 1996 Gros et al. M 19 Corsica arm 7 to 10 1996 Rabodonirina et al. n.s. n.s. Lyon? n.s. 11 1996 Thérizol-Ferly et al. F 35 Sologne? Côte méditerranéenne subconjunctival 12 1996 Jelinek et al. F 46 Corsica lung (D. immitis?) (surgery in Germany) 13 1997 Weill et al. F 70 Côte méditerranéenne subconjunctival 14 1997 Alain F 22 Loire and Cher groin lymphnode (cit. by Raccurt, 2000) 15 1998 Guillot et al. F 12 Gironde forehead 16 1998 Desruelles et al. F 47 Var subcutaneous n.s. 17 1999 Weill et al. M 66 Côte d’Azur or Corsica cheek 18 1999 Weill et al. F 39 Charante Maritime orbital region 19 1999 Pampiglione et al. M 64 Pinarello (Corsica) foot 20 1999 Pampiglione et al. M 23 Porto Vecchio (Corsica) penis 21 1999 Rouhette et al. M 65 Nice subconjunctival 0 1999 Morassin et al.* F 26 Tarn or Herault subclavicular region * This case was already recorded by Kramer 1993 (Raccurt, 2000).

22 1999 Calvet et al. M 47 Var arm 23 1999 Dei-Cas F 10 Nice or Corsica abdominal wall (cit. by Raccurt, 2000)

Greece

0 1996 Pampiglione et al. M 45 Athens abdominal wall 1 1996 Jelinek et al. n.s. adult n.s. hand, thigh (surgery in Germany) 2 1997 Arvanitis et al. M 68 n.s. subconjunctival 0 1998 Petrocheilov et al. M 70 Ionian islands worm not recovered* * Only microfilariae (210-230 µm long) were detected in peripheral blood with diagnosis of Dirofilaria-like.

continued Human dirofilariasis 91

No. Date Author(s) Sex Age Locality Location (yrs)

3 1998 Vakalis and Himonas n.s. n.s. n.s. nose region 4 1998 Vakalis and Himonas n.s. n.s. n.s. thoracic wall 0 1998 Vakalis and Himonas M 3 n.s. scrotum 0 1998 Vakalis and Himonas M 48 Athens or Epirus hand 5 1998 Vakalis et al. F 42 Messinia (Peloponnesos) breast 6 1999 Pampiglione et al. M 31 Aigios Peloponnesus lung

Hungary

1 2000 Parlagi et al. F 57 n.s. eyelid 2 2000 Szénasi, p.c. M 56 Szeged province subconjunctival 3 2000 Elek et al. F 52 Budapest province upper eyelid 4 2000 Elek et al. M 48 Hodmezovasarhely eye-brow 5 2000 Elek et al. F 64 Tisza river bank pelvis region 6 2000 Elek et al. F 76 Csobaj (Miscolc) arm

Italy 0 1986 Spina et al. M 60 Biancavilla (Catania) orbital region 0 1986 Spina et al. F 73 Ramacca (Catania) lower eyelid 0 1987 Toniolo et al. M 23 Cadore (Trento) jaw 1 1990 Stemberger H M adult Italy (locality n.s.) periocular region (cit. by Auer et al. 1997; surgery in Austria) 2 1991 Bianchi Rossi et al. F 79 Laiatico (Pisa) subconjunctival 0 1993 Bay et al. F 63 Torino lower eyelid 3 1995 Stemberger H. M adult Italy n.s. leg (cit. by Auer et al. 1997; surgery in Austria) 4 1996 Garaffini et al. F 72 Locality n.s. periocular region (surgery in France) 5 1996 Braun et al. F 61 Locality n.s. retrobulbar region (surgery in Austria) 6 1996 Cancrini et al. F 42 Augusta (Siracusa) leg 7 1996 Cancrini et al. F 26 Augusta (Siracusa) ankle 8 1996 Cancrini et al. F 48 Sortino (Siracusa) gluteal region 9 1996 Molet, p.c. F 35 Venice scalp 10 1996 Jelinek et al. F 30 Piedmont?Tuscany? lung* (surgery in Germany) * The diagnosis was D. immitis but probably it was D. repens.

11 1996 Jelinek et al. n.s. n.s. n.s. glabella (surgery in Germany) 12 1996 Jelinek et al. n.s. n.s. n.s. abdominal wall (surgery in Germany) 13 1996 Jelinek et al. n.s. n.s. n.s. shoulder (surgery in Germany) 0 1996c Pampiglione et al. F 43 Sciacca (Agrigento) temporal region 0 1996e Pampiglione et al. F 66 Pegognaga (Mantova) lung 0 1996e Pampiglione et al. M 69 Concordia (Modena) lung 0 1996a Pampiglione et al. M 55 Rivalta S (Alessandria) thigh 0 1996b Pampiglione et al. F 44 Asti iliac region 0 1996b Pampiglione et al. M 42 Gavorrano (Grosseto) leg 0 1996b Pampiglione et al. M 68 Surbo (Lecce) neck

continued 92 Human dirofilariasis

No. Date Author(s) Sex Age Locality Location (yrs)

0 1996a Pampiglione et al. M 38 Oristano zygomatic region 0 1996a Pampiglione et al. M 54 Oristano forehead 0 1996b Pampiglione et al. F 66 Staranzano (Gorizia) leg 0 1996b Pampiglione et al. M 40 Asti upper eyelid 14 1996 Pampiglione et al. M 58 Arbatax (Nuoro) subconjunctival 15 1996 Pampiglione et al. F 35 Olbia (Sassari) leg 16 1996 Vitullo, p.c. F 45 Isernia breast 17 1996 Vitullo, p.c. M 57 Venafro (Isernia) neck 0 1997 Pampiglione et al. M 52 Ravenna province spermatic chord 0 1997 Giannetto and Ubaldino F 38 Mazara del Vallo (Trapani) subconjunctival 18 1997 Auer et al. M 35 Udine province, or Corsica, epididymis (surgery in Austria) or Southern France 19 1997 Zardi et al. M 45 Gaeta thoracic wall 20 1997 Mastinu et al. F 42 Asti province arm 21 1997 Mastinu et al. M 36 Asti province subconjunctival 22 1997 Mastinu et al. F 45 Asti province periocular region 23 1997 Heep, p.c., Auer 1997 n.s. n.s. n.s. or Greece parotid gland (surgery in Austria) 24 1997 Garavelli, n.p. M 61 Bergamasco(Alessandria) scalp 25 1998 Mastinu et al. M 50 Asti province temporal region 26 1998 Mastinu et al. F 19 Asti province hand 27 1998 Mastinu et al. M 39 Asti province breast 28 1998 Mastinu et al. F 62 Asti province mesenteron 29 1998 Pampiglione et al. F 46 Milano breast 0 1998 Pampiglione et al. F 52 Garlasco (Pavia) breast 30 1998 Auer, p.c. M 43 Sardinia? Malta? (surgery in Austria) sacral region 31 1998 Cancrini et al. M 59 Grado (Gorizia) submandibular lymphnode 32 1998 Cancrini et al. M 45 Augusta (Siracusa) finger 33 1998 Cancrini et al. F 48 Augusta (Siracusa) leg 34 1998 Cancrini et al. F 60 Elba Island breast 35 1998 Cancrini et al. F 20 Mazara del Vallo (Trapani) eyelid 36 1998 Cancrini et al. M 74 Augusta (Siracusa) subconjunctival 37 1998 Cancrini et al. F 42 Siracusa subconjunctival 38 1998 Cancrini et al. F 56 Torino subconjunctival 39 1998 Cancrini et al. M 42 Siracusa eyelid 40 1998 Strianese et al. F adult Napoli province orbital region 41 1998 Strianese et al. F 47 Napoli province orbital region 42 1999 Pampiglione et al. M 37 Northern Italy or Hungary n.s (surgery in Hungary) spermatic chord 43 1999 Pampiglione et al. M 57 Odalengo (Alessandria) axilla 44 1999 Pampiglione et al. M 39 Novara province zigomatic region 45 1999 Pampiglione et al. F n.s. Novara province popliteal region 46 1999 Pampiglione et al. M 57 Novara province thoracic wall 47 1999 Pampiglione et al. F 83 Viarigi (Asti) abdominal wall 48 1999 Pampiglione et al. F 41 Castagneto Carducci (Livorno) abdominal wall 49 1999 Pampiglione et al. F 50 Piombino (Livorno) thigh 50 1999 Pampiglione et al. F 57 Crescentino (Vercelli) leg 51 1999 Pampiglione et al. F 50 Casale Monferrato (Alessandria) arm 52 1999 Pampiglione et al. M 25 Novara province thoracic wall 53 1999 Pampiglione et al. F 100 Casale Monferrato (Alessandria) thoracic wall 54 1999 Pampiglione et al. F 30 Novara province forearm 55 1999 Pampiglione et al. F 69 Novara province axilla

continued Human dirofilariasis 93

No. Date Author(s) Sex Age Locality Location (yrs)

56 1999 Pampiglione et al. M 59 Casorzo (Asti) scalp 57 1999 Pampiglione et al. F 34 Trino (Vercelli) thoracic wall 58 1999 Pampiglione et al. F 64 Fontanetto Po (Vercelli) knee 59 1999 Pampiglione et al. M 32 Casale Monferrato (Alessandria) scalp 60 1999 Pampiglione et al. F 69 Casale Monferrato (Alessandria) thoracic wall 61 1999 Pampiglione et al. F 40 Trino (Vercelli) neck 62 1999 Pampiglione et al. F 61 Moncalvo (Asti) scalp 63 1999 Pampiglione et al. F 41 Lecce upper eyelid 64 1999 Pampiglione et al. F 29 Asti hand back 65 1999 Pampiglione et al. M 39 Roatto (Asti) breast 66 1999 Pampiglione et al. F 27 Gela (Agrigento) cheek 67 1999 Pampiglione et al. M 50 Ravenna province subconjunctival 68 1999 Pampiglione et al. M 70 Novara province hand palm 69 1999 Pampiglione et al. F 52 Novara province axilla 70 1999 Pampiglione et al. F 56 Novara province abdominal wall 71 1999 Pampiglione et al. M 52 Saturnia (Grosseto) or Livorno epididymis 72 1999 Pampiglione et al. M 61 Alessandria scalp 73 1999 Pampiglione et al. F 69 Alfiano Natta (Alessandria) foot back 74 1999 Pampiglione et al. M adult Asti province n.s. 75 1999 Pampiglione et al. F 73 Asti province omentum 76 1999 Pampiglione et al. F 56 Asti province orbital region 77 1999 Pampiglione et al. F 56 Vercelli or Mazara del Vallo lung (Trapani) 78 1999 Pampiglione et al. M 42 Novara province arm 79 1999 Pampiglione et al. M 29 Asti province temporal region 80 1999 Pampiglione et al. M 49 Asti province lung 81 1999 Pampiglione et al. M 45 Asti province arm 82 1999 Pampiglione et al. F 66 Asti province breast 83 1999 Pampiglione et al. F 54 Baratili S.P. (Oristano) groin 84 1999 Pampiglione et al. M 37 Cantalupo (Alessandria) lower eyelid 85 1999 Pampiglione et al. M 64 Valenza Po (Alessandria) neck 86 1999 Pampiglione et al. F 68 Mirabello (Alessandria) nose region 87 1999 Pampiglione et al. M adult Ravenna province leg 88 1999 Pampiglione et al. M adult Ravenna province spermatic chord 89 1999 Pampiglione et al. F 68 Vercelli knee 90 1999 Pampiglione et al. M 4 Milano cheek 91 1999 Pampiglione et al. M 44 Novara province forehead 92 1999 Pampiglione et al. F 42 Novara forearm 93 1999 Pampiglione et al. M 76 Castellammare del Golfo abdominal wall (Trapani) 94 1999 Pampiglione et al. F 59 Castelleone (Cremona) shoulder or Ischia Island 95 1999 Pampiglione et al. M 36 Scano M. (Oristano) epididymis 96 1999 Pampiglione et al. F 34 Pontestura (Alessandria) abdominal wall 97 1999 Pampiglione et al. M 28 Novara province omentum 98 1999 Pampiglione et al. M 44 Crescentino (Vercelli) spermatic chord 99 1999 Pampiglione et al. F 40 Vignale M.(Alessandria) forearm 100 1999 Pampiglione et al. F 65 Villanova (Alessandria) forearm 101 1999 Pampiglione et al. F 63 Valenza Po (Alessandria) thigh 102 1999 Pampiglione et al. M 24 Reggio Emilia abdominal wall 103 2000 Giansanti, p.c. F 69 Petrelle (Perugia) breast 103 2000 Gobbo and Bisoffi, p.c. M 39 Venezia forearm 104 2000 Pastormerlo, p.c. F 49 Valenza Po (Alessandria) abdominal wall 105 2000 Pastormerlo, p.c. F 35 Motta di Conti (Vercelli) thigh

continued 94 Human dirofilariasis

No. Date Author(s) Sex Age Locality Location (yrs)

106 2000 Pastormerlo, p.c. F 74 Casale M.(Alessandria) knee 107 2000 Pastormerlo, p.c. F 62 Vignale M. (Alessandria) abdominal wall 108 2000 Speranza p.c M 35 Modena province groin 109 2000 Feyles, p.c F 74 Asti breast 110 2000 Feyles, p.c M 28 Asti province epididymis 111 2000 Feyles, p.c M 76 Asti province subcutaneous n.s. 112 2000 Pavesi, p.c. F 49 Valenza Po (Alessandria) abdominal wall 113 2000 Pavesi, p.c. F 47 Moncalvo (Asti) axillary region 114 2000 Pavesi, p.c. F 74 Casale Monferrato (Alessandria) knee 115 2000 Pavesi, p.c. F 36 Motta di Conti (Vercelli) thigh 116 2000 Pavesi, p.c. F 62 Vignale Monferrato abdominal wall (Alessandria) 117 2000 Elek et al. F 71 Rome province lower eyelid (surgery in Hungary)

Romania 1 1997 Olteanu n.s. n.s. n.s. n.s. 2 2000 Panaitescu et. al. n.s. n.s. n.s. n.s. 3 2000 Panaitescu et. al. n.s. n.s. n.s. n.s.

Serbian Republic

1 1996 Misic et al. F adult Smederevo head*

* Both female and male nematode were recovered in the same nodule. 2 1996 Misic et al. n.s. n.s. Smederevo head 3 1996 Misic et al. n.s. n.s. Beograd shoulder

Slovak Republic

1 1992 Vasilkova et al. M 18 Bardeiov vitreous body*

* From the description of the nematode it seems to be D. immitis.

Slovenia

1 1998 Auer (surgery in Austria) M 24 n.s. or Albania? groin lymphnode

Spain

1 1998 Ruiz-Moreno et al. M 43 Elche (Alicante) subconjunctival

Ukraine

1 1971 Melnicenko and Prosvetova F 55 Poltava subconjunctival 2 1971 Melnicenko and Prosvetova M 29 Poltava province subconjunctival 3 1977 Kondrazkyi and Parkomienko F 46 Kiev upper eyelid 4 to 11 1996 Davydov et al. n.s. n.s. Kiev province eye region (n.s) 12 1996 Pogolciuk M 60 Odessa province lower eyelid 13 to 16 1997 Dorofeev et al. n.s. n.s. n.s. n.s 17 1997 Dorofeiev et al. F 67 Crimea knee

continued Human dirofilariasis 95

No. Date Author(s) Sex Age Locality Location (yrs)

18 1997 Dorofeiev et al. n.s. n.s. n.s. mesenteron 19 1997 Avdiukhina et al. F 27 Simferopol lower eyelid 20 1997 Avdiukhina et al. M 60 Odessa province lower eyelid. 21 1997 Postnova et al. M 55 Soci neck 22 1997 Postnova et al. M 60 Belgorod Dniestrovski lower eyelid 23 1997 Postnova et al. F adult Belgorod Dniestrovski groin

AFRICA Kenya or Senegal

1 1997 Orihel et al. M 42 n.s. periocular region* (surgery in the States)

* Two worms were recovered at ten months apart between each other.

Tunisia

1 1990 Chaabouni et al. M 55 Kairouan subconjunctival 2 1995 Ben Said et al. F 39 Gabès axillary region 3 1995 Ben Said et al. F 48 Sousse forehead 4 1999 Mrad et al. F 32 n.s. breast ASIA Georgia

1 1967 Kamalov M n.s. Grusiya upper eyelid 2 1967 Kamalov F 36 Vostocnaya Grusyia upper eyelid

India

1 1999 Senthilvel & Pillai F 39 Ottapalan (Kerala) lip

Iran

1 1996 Degardin & Simonart M 26 n.s. leg* (surgery in Belgium)

* Long persistance (5 years) 3 worms in one nodule

Israel

1 1999 Stayerman et al. M 35 Safed? penis

Kazakhstan

1 1961 Mizkevi and Leontieva F 25 n.s. lower eyelid 2 1970 Lubova F 52 Ksil Orda lower eyelid 3 1970 Lubova F 57 Ksil Orda thoracic wall 4 1985 Tasberghenova and M 13 Alma Ata subconjunctival Anbakirova 5 1992 Glinciuk et. al. F 43 n.s. eye, preretinic*

* Long persistance (5 years) 3 worms in one nodule

continued 96 Human dirofilariasis

No. Date Author(s) Sex Age Locality Location (yrs)

Malaysia

1 1996 Shekhar et al. M 48 Penang inguinal lymphonode*

* Both male and female worms present in the nodule.

2 1996 Shekhar et al. M 39 Melaka cervical lymphonode

Russia (Siberia inclusive)

1 1954 Koroiev F 23 Northern Caucasus upper eyelid 2 1971 Prosvetova et al. n.s. n.s. n.s. n.s. 3 1977 Kondrazkii and F 46 Voronez province lower eyelid Parkomienko 4 1981 Merkusceva et al. M 46 Astrakhan province lower eyelid 5 1991 Maximova et al. F 41 Tula region zygomatic region 6 1993 Plotnikov et al. n.s. n.s. Tomsk (Siberia) subconjunctival 7 1993 Plotnikov et al. n.s. n.s. Saratov province subconjunctival 8 1993 Avdiukhina et al. M 35 Vladivostock (Siberia) submandibular 9 1995 Asarova et al. F 37 Barnaul (Siberia) upper eyelid 10 1995 Asarova et al. n.s. n.s. Barnaul (Siberia) eye region (n.s.) 11 1995 Asarova et al. n.s. n.s. Barnaul (Siberia) eye region (n.s.) 12 1995 Asarova et al. n.s. n.s. Barnaul (Siberia) eye region (n.s.) 13 1996 Artamonova and Nagornyi n.s. n.s. Northern Caucasus n.s. 14 1996 Avdiukhina et al. F 13 Astrakhan province upper eyelid 15 1996 Avdiukhina et al. F 36 Astrakhan province upper eyelid 16 1996 Avdiukhina et al. F 50 Astrakhan province subconjunctival 17 1996 Avdiukhina et al. F 42 Astrakhan province subconjunctival 18 1996 Avdiukhina et al. F 61 Astrakhan province subconjunctival 19 1996 Avdiukhina et al. F 49 Astrakhan province periocular region 20 1996 Avdiukhina et al. F 26 Astrakhan province lower eyelid 21 1996 Avdiukhina et al. F 33 Astrakhan province periocular region 22 1996 Avdiukhina et al. M 23 Krasnodar upper eyelid 23 1996 Avdiukhina et al. F 49 Astrakhan province periocular region 24 1996 Avdiukhina et al. F 26 Astrakhan province lower eyelid 25 1996 Avdiukhina et al. F 33 Astrakhan province periocular region 26 1996 Avdiukhina et al. F 37 Barnaul (Siberia) upper eyelid 27 1996 Avdiukhina et al. M 23 Krasnodar province upper eyelid 28 1997 Avdiukhina et al. F 55 Krasnodar province lower lip 29 1997 Avdiukhina et al. F 58 Krasnodar province subconjunctival 30 1997 Avdiukhina et al. F 57 Krasnodar province nape 31 1997 Avdiukhina et al. M 38 Krasnodar province thoracic wall 32 1997 Avdiukhina et al. M 23 Krasnodar upper eyelid 33 1997. Avdiukhina et al. M 50 Volgograd hip 34 1997 Avdiukhina et al. F adult Barnaul (Siberia) breast 35 1997 Avdiukhina et al. F adult Barnaul (Siberia) breast 36 1997 Avdiukhina et al. M 38 Volgograd thoracic wall 37 1997 Avdiukhina et al. M n.s. Barnaul (Siberia) shoulder 38 1997 Avdiukhina et al. M n.s. Barnaul (Siberia) hip 39 1997 Postnova et al. F 55 Soci neck 40 1997 Postnova et al. F 27 Moskow province lower eyelid 41 1997 Postnova et al. F 46 Astrakhan province elbow 42 1997 Postnova et al. F 41 Astrakhan province hand

continued Human dirofilariasis 97

No. Date Author(s) Sex Age Locality Location (yrs)

43 1997 Postnova et al. F 56 Astrakhan province shoulder 44 1997 Postnova et al. F 58 Astrakhan province knee 45 1997 Postnova et al. M 58 Astrakhan province hand 46 1997 Postnova et al. F 39 Astrakhan province forearm 47 1997 Postnova et al. M 31 Astrakhan province cheek 48 1997 Postnova et al. F 13 Astrakhan province upper eyelid 49 1997 Postnova et al. F 50 Astrakhan province subconjunctival 50 1997 Postnova et al. F 36 Krasnodar or Soci subconjunctival 51 1997 Postnova et al. F 34 Astrakhan province forearm 52 1997 Postnova et al. F 36 Astrakhan province shoulder 53 1997 Postnova et al. F 42 Astrakhan province upper eyelid 54 1997 Postnova et al. F 61 Astrakhan province subconjunctival 55 1997 Postnova et al. F 30 Astrakhan province thoracic wall 56 1997 Postnova et al. F 18 Astrakhan province cheek 57 1997 Postnova et al. M 23 Astrakhan province upper eyelid 58 1997 Postnova et al. F 49 Astrakhan province upper eyelid 59 1997 Postnova et al. F 26 Astrakhan province upper eyelid 60 1997 Postnova et al. F 35 Astrakhan province soft palate 61 1997 Postnova et al. F 33 Astrakhan province cheek

Sri Lanka [abbreviations: CP = Central Province; EP = Eastern Province; NP = Northern Province; NCP = North Central; Province; NWP = North Western; Province; SP = Southern Province; SabP = Sabaragamuwa Province; UP = Uva Province; WP = Western province]

0 1976 Wijesundera F 32 Moragolla (CP) forearm 0 1976 Wijesundera F n.s Kandy (CP) eye region (n.s.) 0 1980 Wijesundera M 19 n.s. scrotum 0 1980 Wijesundera F 63 Wattala (WP) breast 0 1988 Wijesundera M 1.6 Waharaka (SabP) scrotum 1 1997 Dissanaike et al. M 4 m Pannipityia (WP) scrotum 2 1997 Dissanaike et al. M 5 Angola (WP) subconjunctival 3 1997 Dissanaike et al. M 27 Karainagar (NP) forearm 4 1997 Dissanaike et al. M 38 Colombo (WP) forearm 5 1997 Dissanaike et al. M 43 Batticaloa (EP) subconjunctival 6 1997 Dissanaike et al. F 1 Colombo (WP) foot 7 1997 Dissanaike et al. M n.s. SP wrist 8 1997 Dissanaike et al. M 6 Waharaka (SabP) perianal region 9 1997 Dissanaike et al. F 22 Embilipitiya (Sab. P) lower eyelid 10 1997 Dissanaike et al. F 2 Moratuwa (WP) upper eyelid 11 1997 Dissanaike et al. F 72 Colombo (WP) subconjunctival 12 1997 Dissanaike et al. F 54 n.s. subconjunctival 13 1997 Dissanaike et al. M 10 Gandara (SP) abdominal wall 14 1997 Dissanaike et al. M 3 Lunuwila (NWP) cheek 15 1997 Dissanaike et al. M 60 Kolonnawa (WP) subconjunctival 16 1997 Dissanaike et al. M 4 Mirigama (WP) scrotum 17 1997 Dissanaike et al. F 10 m Kuliyapitiya (NWP) cheek 18 1997 Dissanaike et al. M 28 Tissamaharama (SP) cheek 19 1997 Dissanaike et al. M 9 m Ambalangoda (SP) scrotum 20 1997 Dissanaike et al. M 1.4 Galle (SP) scrotum 21 1997 Dissanaike et al. M 6 m Wanduramba (SP) penis 22 1997 Dissanaike et al. n.s. n.s. Hikkaduwa (SP) subconjunctival 23 1997 Dissanaike et al. M 40 Kandy (CP) thumb

continued 98 Human dirofilariasis

No. Date Author(s) Sex Age Locality Location (yrs)

24 1997 Dissanaike et al. M 5 m n.s. subconjunctival 25 1997 Dissanaike et al. M 5 m Kandy (CP) subconjunctival 26 1997 Dissanaike et al. F 6 CP eye region (n.s.) 27 1997 Dissanaike et al. n.s. n.s. CP eye region (n.s.) 28 1997 Dissanaike et al. F 52 Kandy (CP) breast 29 1997 Dissanaike et al. M 3 Ragama (WP) scrotum 30 1997 Dissanaike et al. M 40 CP lower eyelid 31 1997 Dissanaike et al. M 2.2 Kadugannawa (CP) scrotum 32 1997 Dissanaike et al. M 36 CP groin 33 1997 Dissanaike et al. M 40 Undugoda (Sab.P) eye (n.s.) 34 1997 Dissanaike et al. F 43 Gambola (CP) eye (n.s.) 35 1997 Dissanaike et al. M 1.6 Batapola (SP) scrotum 36 1997 Dissanaike et al. M 1.2 Panadura (WP). penis 37 1997 Dissanaike et al. M 11 SP face 38 1997 Dissanaike et al. M 11 Deraniyagala (Sab.P) neck 39 1997 Dissanaike et al. F 33 n.s. peritoneum 40 1998 Kumarasimghe et al. F 21 Jaffna (NP) forearm 41 1998 Dissanaike p.c. F 18 Rajagiriya (WP) subcutaneous n.s. 42 1998 Dissanaike p.c. F 7 m Apura (NCP) sclera 43 1998 Dissanaike p.c. M 11 Agalawatte (WP) neck 44 1998 Dissanaike p.c. F 65 Katunayake (WP) subconjunctival 45 1998 Dissanaike p.c. M 1.6 Negombe (WP) n.s. 46 1998 Dissanaike p.c. M 1 n.s. scrotum 47 1998 Dissanaike p.c. M 1.3 n.s. spermatic chord 48 1998 Dissanaike p.c. M 1.4 Ambalantota (SP) spermatic chord 49 1998 Dissanaike p.c. M 50 Negombo (WP) ankle 50 1998 Dissanaike p.c. F 30 Agalawatte (WP) forearm 51 1999 Dissanaike p.c. M 21 Kekirawa (NCP) scalp 52 1999 Dissanaike p.c. F 53 Kandy (CP) eyelid 53 1999 Dissanaike p.c. n.s. n.s. n.s. n.s. 54 1999 Dissanaike p.c. M 2.6 WP n.s. 55 1999 Dissanaike p.c. F 11 m Homagama (WP) scapular region 56 1999 Dissanaike p.c. F 1.7 Negombo (WP) scapular region 57 1999 Dissanaike p.c. n.s. n.s. n.s. n.s. 58 1999 Dissanaike p.c. F 9 n.s. hand 59 1999 Dissanaike p.c. F 41 Badulla (UP) neck 60 1999 Dissanaike p.c. M 1.1 WP scrotum 61 1999 Dissanaike p.c. F 1.7 Negombo (WP) scapular region 62 1999 Dissanaike p.c. M 2.3 n.s. scrotum 63 1999 Dissanaike p.c. M 48 Walasmulla (SP) knee 64 1999 Dissanaike p.c. M 7 m WP scrotum 65 1999 Dissanaike p.c. M 4.6 WP scrotum 66 1999 Dissanaike p.c. F 2.3 Kandy (CP) thumb 67 1999 Dissanaike p.c. F 55 Kandy (CP) subconjunctival 68 1999 Dissanaike p.c. F 30 Kandy (CP) subconjunctival 69 1999 Dissanaike p.c. F 30 Kandy (CP) hip 70 1999 Dissanaike p.c. M 35 Kandy (CP) forehead 71 1999 Dissanaike p.c. M 1,8 WP scrotum 72 1999 Dissanaike p.c. M 4.5 Matugama (WP) hip 73 1999 Ratnatunga and Wijesundera M 16 n.s. abdominal wall 74 1999 Ratnatunga and Wijesundera F 41 n.s. thigh 75 1999 Ratnatunga and Wijesundera F 65 n.s. cheek 76 1999 Ratnatunga and Wijesundera M 48 n.s. thoracic wall

continued Human dirofilariasis 99

No. Date Author(s) Sex Age Locality Location (yrs)

77 1999 Ratnatunga and Wijesundera F 45 n.s. breast 78 1999 Ratnatunga and Wijesundera M 5 n.s. subconjunctival 79 1999 Ratnatunga and Wijesundera M 16 n.s. forearm 80 1999 Ratnatunga and Wijesundera M 30 n.s. thoracic wall 81 1999 Ratnatunga and Wijesundera M 40 n.s. temporal region 82 1999 Ratnatunga and Wijesundera F 34 n.s. forehead 83 1999 Ratnatunga and Wijesundera F 22 n.s. thoracic wall 84 1999 Ratnatunga and Wijesundera M 1.6 n.s. scrotum 85 1999 Ratnatunga and Wijesundera M 45 n.s. cheek 86 1999 Ratnatunga and Wijesundera M 1.6 n.s. scrotum 87 1999 Ratnatunga and Wijesundera n.s. n.s. n.s. n.s. 88 1999 Ratnatunga and Wijesundera n.s. n.s. n.s. n.s. 89 1999 Fernando et al. M 3.4 Laxapana (CP) scrotum* * Two nodules with a male and female worms each.

90 1999 Pitakotuwage et al. F 80 Ampara (EP) cheek 91 1999 Dissanaike, p.c. F 35 Galle (SP) spermatic chord 92 2000 Dissanaike, p.c. M 7m n.s. n.s. 93 2000 Dissanaike, p.c. n.s. n.s. n.s. n.s. 94 2000 Dissanaike, p.c. n.s. n.s. n.s. n.s. 95 2000 Dissanaike, p.c. M 1 n.s. forearm 96 2000 Dissanaike, p.c. F n.s. Kandy (CP) n.s. 97 2000 Dissanaike, p.c. F 8 Ampara (EP) n.s. 98 2000 Dissanaike, p.c. M 1.4 Homagama (WP) eye region (n.s.) 99 2000 Dissanaike, p.c. M n.s. EP testis*

* As Professor Dissanaike claims, the location of this case is doubtful, being probably into the epidydimis.

100 2000 Dissanaike, p.c. F 50 Narahenpita (WP) subconjunctival 101 2000 Dissanaike, p.c. F 50 Maharagama (WP) subconjunctival

Turkey

1 1997 Otkun et al. F 44 Edirne Edirne

Turkmenistan

1 1970 Nurliyev n.s. n.s. n.s. n.s.

Uzbekistan

1 1961 Mizkievic & Leontieva F 35 Tashkent crystalline*

* The diagnosis of D. repens was only presumed. 100 Human dirofilariasis

Histopathology third, less numerous, group the nematode is in a state of more or less In an important work on parasites advanced decomposition and is sur- affecting human tissues, Orihel and rounded by scant mixed necrotic mat- Ash (1995) present a wide range of his- ter and occasional inflammatory cells. tological sections illustrating the vari- This area is delimited by reactive tissue ous Dirofilariae including D. repens, forming a dense fibrous ring of mainly that can infect man. Ratnatunga and fibroblastic tissue. As in the previous Mijesundera (1999) have described the cases, the surrounding soft tissues dis- histopathologic features observed in Sri play an aspecific acute and chronic Lanka in 14 subcutaneous nodules due phlogistic reaction. In the fourth group, to D. repens. Two fully illustrated comprising just a few cases, the histo- works on the histopathologic features logical pictures feature a dense inflam- of the lesions caused by the parasite in matory infiltrate consisting almost man have been recently published by entirely of lymphoid elements, some- Pampiglione et al. (1999e, 2000b): the times massing together to form germi- first is particularly concerned with the native centres. This reactive tissue dif- difficulties a histopathologist may fuses throughout the surrounding soft encounter in his attempt to arrive at a tissues. In the cases affecting the lungs, correct diagnosis of the species when the nematode is almost always in an the nematode is in a more or less advanced state of decomposition inside advanced stage of decomposition; the a thrombosed arteriole giving rise to a second takes 60 new cases and classi- small, roundish infarctual zone. The fies the histopathological features into adjacent pulmonary parenchyma dis- four basic groups, which appear to plays mainly acute inflammatory infil- depend on the length of time spent by trates with a redominance of the nematode in the host’s tissues and eosinophils. Flieder and Moran (1999), on the defence reaction triggered by it. in a documented article on human pul- In the first group, which includes the monary dirofilariasis, report on a clini- majority of cases, the phlogistic reac- copathological study of 41 infarctual tion is of an abscess-like type due to the nodules observed in the USA. Even presence of necrotic matter containing though the cases are all due to D. immi- neutrophil and eosinophil leukocytes tis, the study is very pertinent to D. and less numerous chronic inflammato- repens, for the clinical and histopatho- ry cells surrounding the nematode. logical pictures of the two species do A reactive granulation tissue is not differ greatly, apart from the mor- around, while acute and chronic phology of the parasites. Following on inflammatory cells infiltrate the sur- from the research of previous authors rounding soft tissues. In the second (Schaub and Rawlings, 1979; Kaiser et group, the central zone containing the al., 1992), it has recently been shown nematode is delimited by an actual val- that filarial parasites alter pulmonary lum consisting of epithelioid cells, his- artery endothelial cell relaxation. Thus, tiocytes and occasional plurinucleate vasoconstriction may play an important giant cells as of a foreign body. In the role in tissue necrosis in pulmonary Human dirofilariasis 101 human dirofilariasis (Mupanamunda et judging by the histological preparation. al., 1997). In 2 cases, a male and a female were The histological findings relating to found together in the same nodule nodules localised in the breast, epi- (Misic et al., 1996; Mrad et al., 1999), didymis, spermatic chord, omentum and in another case a male and a female and mesentery overlap: the nematode is were found in 2 separate nodules but immersed in fibrin-leukocyte necrotic close to each other in the skin of the material surrounded by demarcation scrotum (Fernando et al., 2000). The tissue consisting of fibroblast elements presence of 3 nematodes in the same together with lymphocytes, plasma nodule was reported by Degardin and cells and eosinophils. Simonart (1996) in a patient from Iran and by Avdiukhina et al. (1997) in Parasitology another patient in the Russian Federation. It has been known that, The samples comprised mostly imma- after being extracted from the nodule, ture females from a few centimetres to D. repens is capable of surviving for up 150 mm in length and with a maximum to 48 hours in physiological solution at thickness of µm 620. At times, the sam- 4°C (Thérizol-Ferly et al., 1996). In ples were already dead on surgical some rare cases it was noted that the removal; at others, they had been par- histopathological transverse sections of tially destroyed by the inflammatory females of D. repens inexplicably reaction of the host. Often, however, revealed an unusual number (up to ten they were still alive, as could be seen or more) of sex tubules (Figs. 1 and 2) from their lively movements; indeed, (Pampiglione et al., 1992, 1996). Dissanaike et al. (1997) found this to Orihel et al. (1997) have now provided be so in 42 out of 70 cases observed. an explanation. They were examining 2 In Italy, the ratio between living and females of D. repens extracted from the dead D. repens observed by the authors same site in the same patient, but at an was practically the same, although it interval of 10 months. was not always possible to be sure They deduced correctly that the two

Figs. 1 and 2. Paratransverse sections of D. repens where numerous sections of female sex tubules are visible (Haematoxylin/Eosin, 250). [Fig. 1: courtesy by Blackwell Ltd, Histopathology (in press)]. 102 Human dirofilariasis parasites must have been introduced by the carrier at the same time and that therefore one was older than the other. They explain that, as a female matures, her vagina lengthens, becomes coiled and looped and extends towards the head beyond the vulva and oesophagus. If the nematode is sectioned above that point, the sex tubules can thus appear to be very numerous. Orihel and Eberhard (1998) have helped to clarify Fig. 3. Transverse section of D. repens. The empty another important morphological space around the nematode is due to shrinkage of aspect for the recognition of D. repens its body diameter (Haematoxylin/Eosin, 250). in histological section: whereas Gutierrez (1990) stated that “key figures useful in identification of the species include longitudinal ridges sep- arated by a distance wider than the ridge itself, 95-105 ridges on the cir- cumference of the body”, Orihel and Eberhard point out that “the shape, height and interridge distances are actually quite variable at different levels of the body in the same worm or even within a single transverse section and hence do not constitute reliable criteria”. Fig. 4. Three transverse sections of D. repens. Another morphological feature whose Note the difference in diameter in the same speci- relative value needs to be reassessed is men sectioned at different points of the body (Haematoxylin/Eosin, 150). that of the diameter of the nematode’s body in transverse section. Apart from the possible variation in diameter that, of the 27 species considered valid, depending on the stage of development only 5 have been reported as having reached, on eventual regressive alter- infected man, apart from D. immitis ations and on shrinkage due to the and D. repens, namely: D. (N.) tenuis, worm being killed by the fixative while a parasite of the racoon in North still alive inside the nodule (as can often America; D. (N.) ursi, a parasite of the be seen from the empty space created brown bear in Canada, the northern around it) (Fig. 3), the diameter will USA, Siberia and Japan; D. (D.) normally vary by many microns in the spectans, a parasite of Mustelidae in same individual, depending on the point Brazil; D. (N.) magnilarvatum, a para- at which the section is taken (Fig. 4). site of catarrhine monkeys in Asia; D. From a review of the different species (N.) striata, a parasite of the puma and of Dirofilaria existing worldwide other American carnivores. The speci- (Canestri Trotti et al., 1997) it appears mens of D. ursi found in human cases Human dirofilariasis 103 have been termed D. ursi-like, since in the embryogenesis of filariae there is no absolute certainty that we are (Genchi et al., 1998), and so their sup- dealing with a single species, either for pression with tetracycline is supposed bears or for man, even though in histo- to inhibit the development of the logical section they appear to be practi- microfilariae. cally identical from a morphological Biomolecular techniques have recent- point of view. Since the zoonotic aspects ly been employed (Casiraghi et al., of dirofilariasis in Africa are little 2000; Favia et al., 2000) for filogenetic known, future research into the study of analysis. These authors have compared findings from that continent will bear in 10 different species of Onchocercidae, mind D. (N.) corynodes (a frequent par- among which D. repens, and confirm asite of African monkeys), given its great the clustering of the species of the similarity to D. repens (Orihel, 1969; genus Onchocerca with those of the Orihel and Eberhard, 1998). genus Dirofilaria. On examining the peripheral blood of a 64-year old patient living on a Greek Diagnosis Island in the Ionian sea (western Greece) Petrocheilou et al. (1998) dis- The clinical diagnosis of the parasito- covered microfilariae measuring 210- sis is almost always wrong, except for 230 µm in length (and therefore not some subconjunctival cases where the matching either D. repens or D. immi- oculist can see the nematode, given that tis), which they attribute to the the conjunctiva is transparent and so is Dirofilaria-like genus, but without able to diagnose a “helminth parasito- being able to find the adults. In Sri sis”, mistaken sometimes for onchocer- Lanka, rare cases of subcutaneous diro- ciasis. In cases of pulmonary infection, filariasis due to a different species but clinical diagnosis has been that of can- still belonging to the sub-genus cer or sarcoidosis (Jelinek et al., 1996) Nochtiella, D. linstowi (Dissanaike, and thoracotomy was always carried 1972), have been reported in man in out; a spermatic chord location, erro- jungle areas, home to the monkeys neously interpreted as a tumour or tes- Presbytis entellus and Macaca sinica ticular tuberculosis, was treated with which are its natural reservoir. Since it orchiectomy (Pampiglione et al., is difficult to differentiate between the 1999a); in breast infections, diagnosis two species, some cases due to this lat- of cancer or fibrous dysplasia of the ter species could have been interpreted breast was also frequently suspected. In as being due to D. repens a case of retroocular infection, the doc- (Abeyewickreme et al., 1997). tors diagnosed tumour of the orbit Studies of great interest have recently (Braun et al., 1996). In another case of shown the presence in D. repens of localisation in the vitreous body, the Wolbachia sp., intracellular bacteria clinical diagnosis was that of dissemi- already reported in insects and filariae, nated chorioretinitis of probable para- including D. immitis (McLaren et al., sitic origin (Vasilkova et al., 1992). In 1975), and transmitted transovarially. subcutaneous forms the most frequent They are held to play an important role diagnosis has been that of sebaceous 104 Human dirofilariasis cyst; other diagnoses have included tode revealed itself only because it lipoma, dermoid cyst, fibroadenoma, emerged spontaneously through the dental abscess, muscular haematoma, hole made by the needle (Kumara- trauma-induced detachment of the tem- singhe et al., 1997). But generally it is poralis muscle, collagen disease, angio- not recommended in dirofilariasis, as oedema, atypical Beliset syndrome, demonstrated by the American statis- periodical disease, larva migrans, tics for lung infections due to D. immi- recurrent thrombophlebitis, rheumatic tis, in which the outcome has invariably disease, herpetic keratitis, tendinous been negative (Flieder and Moran, cyst, neuroma, neurofibroma, inguinal 1999). lymphadenitis, cervical lymphadenitis, In the majority of cases, diagnosis is filariasis due to Wuchereria bancrofti, based on histological examination of the onchocerciasis, loiasis, tumour of the nodule with identification of the mor- parotid gland and, in two cases, acute phological features of the nematode. psychosis (Jelinek et al., 1996). The presence of the external longitu- With regard to the differential diagno- dinal cuticular ridges has proved essen- sis of the parasite, there is an article by tial for the diagnosis of the subgenus Orihel and Eberhard (1998) that Nochtiella and for its differentiation reports, with photographic illustra- from D. immitis, which is the most tions, the morphological data of the common of the other zoonotic various zoonotic filariae capable of Dirofilariae. The most useful stains, infecting man by targeting the subcu- apart from the common haema- taneous tissues and subconjunctiva, the toxylin/eosin, for the purpose of high- heart and pulmonary vessels, the lym- lighting the morphological details have phatic and nervous systems. proved to be PAS and Masson In this same context, there are two very Goldner’s trichrome. In cases in which recent reports of other zoonotic filari- the parasite had been dead for many ae: Brugia sp., probably ceylonensis, months or years, histological diagnosis localised under the conjunctiva of a 53- was more difficult; only a careful analy- year-old patient in Sri Lanka sis of the remains of the nematode’s (Dissanaike et al., 2000) and body and the existence at the same time Macacanema formosana, also localised of other recognisable sections of it under the conjunctiva in a 23-year-old enabled a diagnosis to be made woman in Taiwan (Lin-Ing Lau, per- (Pampiglione et al., 1999e). sonal communication), neither of There have been numerous studies car- which species has ever previously been ried out by various groups of researchers reported in that site in man and which aimed at perfecting a reliable and practi- can be borne in mind in the diagnostic cal diagnostic reaction on histological differentiation of D. repens. Fine sections both of the parasite and the car- Needle Aspiration Biopsy has proved riers (Chandrasekharan et al., 1994; successful in only 2 cases, one affecting Favia et al., 1996a,b, 1997, 1998, the breast (Bertoli et al., 1997), the 2000a,b,c; Cancrini et al., 1998a,b, other subcutaneous tissue. 2000; Favia, 1999; Ricci et al., 2000). In the latter case, however, the nema- Although they have succeeded in Human dirofilariasis 105 developing a PCR on fresh tissues or vention. In cases affecting the ocular those fixed in ethyl alcohol, they have region, however, Avdiukhina et al. not been able to reproduce the test on (1996) report a 10% incidence of com- tissues fixed in formalin. This is a seri- plications of a permanent nature, such ous drawback, for biopsies are normal- as detached retina, glaucoma, opacity ly sent to the histologist already fixed in of the vitreous body or the crystalline 10% formalin. And since the diagnosis lens, or other deterioration in visual of dirofilariasis is hardly ever suspected acuity. In cases affecting the visceral beforehand, it is virtually impossible to organs (lungs, mesentery, omentum) or get the surgeon doing the biopsy to fix the sexual organs (epididymis, spermat- the excised nodule in methyl alcohol or ic chord, scrotum, female breast), sur- send it fresh to the laboratory without gery (open-chest, laparotomy) can have any form of fixation. However, Vakalis relatively serious consequences or et al. (1999) have developed a PCR cause pointless mutilation, such as pul- technique which, in their hands, seems monary lobectomy (Jelinek et al., 1996; to have given good results on tissues Pampiglione et al., 2000d) or the fixed in formalin as well. removal of a breast, epididymus or As regards serological diagnosis, spermatic chord. progress has undoubtedly been made by research groups of various nationalities, Prophylaxis notably Spanish and Italian (Perera et al., 1994, 1998; Favia et al., 1996, 1997, Effective prevention of parasitosis had 2000; Simon et al., 1997; Cancrini et al., already been achieved in dogs exposed 1998, 1999), but they have not yet been to natural infection in endemic zones of able to perfect a reaction that is reliable, central Italy by treatment with ivermec- quick and simple to perform. tine per os (>6 mcg/kg) once a month However, Ruiz Moreno et al. (1998) for 7 consecutive months from May to did succeed in obtaining confirmation November (Marconcini et al., 1993). At of recovery in a case of human subcon- present, the use of ewable tablets of junctival dirofilariasis by measuring ivermectine/pirantel pamoate is yielding the reduction in the level of anti-D. good results in dogs exposed to natural repens serous antibodies by immu- infection (Pollono et al., 1998). noenzymatic means for 3-6 months In man, however, it would appear that from the time of surgery. no experiments in prevention against dirofilariasis due to D. repens or other Prognosis Dirofilariae have been undertaken. Since the presence of a large number of In the majority of subcutaneous cases mosquitoes, which are known carriers the prognosis can be considered of the parasite, and the high incidence benign, the lesion healing in a few days of the infection in dogs, which are the following the removal of the nodule. natural reservoir of the parasitosis, are Sometimes, healing occurs sponta- considered to be risk factors for man, it neously with the worm emerging from can be supposed that a reduction in the the nodule without any surgical inter- incidence of canine infection together 106 Human dirofilariasis with a drive to eliminate the carriers could cause permanent damage to the might considerably reduce the number visual organ. of human cases in endemic areas. This implies a preliminary study both of the prevalence and distribution of the para- Discussion sitosis in dogs and of the density and distribution of the carriers. In view of the number of publications that have appeared in the last 20 years, by comparison with previous decades, Therapy it is evident that the medical world has gradually become increasingly interest- The therapy of choice remains sur- ed in this zoonosis. This can be attrib- gery. Whereas both diethylcarbamazine uted not only to the clear increase in and ivermectine have often been used the incidence of the disease but also in in cases of canine dirofilariasis, they part to the reports of the parasite have only occasionally been used in infecting the viscera (lungs, mesentery) man (van den Ende et al., 1995; Jelinek as well as the female breast and the et al., 1996; Avdiukhina et al., 1997; male genitalia (scrotum, verga, sper- Petrocheilou et al., 1998) and not matic chord, epididymis). The involve- always to any apparent effect. A patient ment of these locations has almost with a nodule on one hand and who always led to diagnoses of forms of had been treated with the two drugs malignant tumour requiring drastic had a second nodule appear on his surgery and has thus highlighted the thigh 4 weeks after the end of treat- importance of the parasite in human ment (Jelinek et al., 1996). Other drugs pathology. It could be argued that the used have been levamisole (Dorofeiev increase in the number of cases, which et al., 1997), albendazole (van den until half a century ago were consid- Ende et al., 1995), thiabendazole and ered exceptional and have been report- cortisones (Delage et al., 1995), again ed with increasing frequency in the last with doubtful results. That drugs are few decades, is only an apparent not in current use is partly due to the increase resulting from a more careful fact that the clinical diagnosis of the examination of subjects affected and cases is generally wrong and appropri- from more refined diagnostic tech- ate therapy is not prescribed. In any niques. Yet the increase does seem to case, the use of filaricides with a certain be in part real, particularly in view of level of toxicity does not seem appro- the enhancement in the temperate priate in all those cases of subcuta- zones of the climatic conditions (tem- neous infection in which a minor surgi- perature, relative humidity, rainfall, cal operation performed in outpatients evaporation) that favour not only the can resolve the problem. The same growth of the carrier Culicidae (Martin holds for subconjunctival or periocular and Lefebvre, 1995) but also the devel- infections, where possible violent aller- opment of the larval phase of the nem- gic reactions induced by the drug atode inside the carrier. That this is so and/or by the death of the nematode is proved by the contemporaneous Human dirofilariasis 107 increase in the number of dogs infect- Lanka (Western province) and Russia ed, at least in endemic zones of (Caucasus) and which, being consid- Piedmont, as emerges from careful sur- ered by now an everyday complaint, are veys carried out in recent years (Rossi no longer reported and, finally, those et al., 1996). A similar increase in the occurring in the developing countries number of cases in recent decades has and which, given the lack of facilities been observed in some republics of the for histological diagnosis, are not even Russian Federation (Avdiukhina et al., screened by the histopathologist. 1997). Parasitosis due to D. repens can Taking into account the 1995 figures, therefore be considered an emergent Italy is still in number one position as zoonosis in many geographical areas of regards the number of cases reported the Old World. The number of cases (298) between 1885 and 2000, fol- reported worldwide since 1885 has lowed by Sri Lanka (132 cases), Russia reached 782 spread over 37 different (Siberia inclusive) (83 cases), France countries, all of which are in the Old (76 cases), Ukraine (51 cases), Greece World (Fig. 5). (27 cases), Turkey (18 cases), Hungary To this must be added a fair number (11 cases), and then the other countries of cases not diagnosed, and therefore with less than 10 cases each. However, not published, others that recovered if we relate these figures to the number spontaneously, others occurring in of inhabitants and territorial area, Sri highly endemic zones as in some Lanka, with a population of 18,300,000 provinces of Italy (Piedmont), Sri inhabitants and a surface area of 65,610

Fig. 5. World distribution of human dirofilariasis due to D. repens (cases recorded from 1885 to 2000). 108 Human dirofilariasis

km2 (FAO, 1997) is the country most body (Fig. 8) remains much the same as severely affected, while Italy, with a reported in 1995. The majority of cases population of 57,200,000 inhabitants (75.8%) affect the upper half of the and a surface area of 301,278 km2, body, particularly the ocular region, moves down to second place. which alone accounts for 30.5% of the The age group most commonly affect- total; the female breast (5.4%), the ed comprises adults, mainly between 40 male genital organs (6.5%), the lungs and 49 years of age (Fig. 6), as also (2.6%) and the abdominal viscera, reported in the 1995 review. The high comprising mesentery and omentum number of children affected is due (1.3%), also remain relatively impor- above all to those reported from Sri tant. The marked prevalence of cases Lanka, where 33.6% (44 out of 132) reported from Sri Lanka affecting the referred to children below the age of 10. genitalia, particularly of children Women are more commonly affected (Dissanaike et al., 1997), may be due to than men, even if not statistically sig- the clothing and sleeping habits of the nificant, as appeared in the previous local child population as well as to the review, accounting for 55.4% of all special tropisms of the carrier mosqui- cases reported between 1885 and the toes, that are perhaps attracted by present day (Fig. 7). Again with refer- ammonia smells and other odours in ence to overall figures published subjects with low levels of personal between 1995 and 2000, the distribu- hygiene. Of the various dirofilariases in tion of sites of infection in the human the world that can affect man, that due

160

140

120

100

60 Number of cases

0 0-9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 80-89 90-99 100-109 Age in years

Fig. 6. Distribution of human dirofilariasis due to D. repens according to age groups, when specified, from 1885 to 2000. Human dirofilariasis 109

from internal medicine to surgery, from immunology to molecular biology – they are all involved. But those most directly involved in the study of its aspects and as yet unresolved problems are, of course, the parasitologists and Fig. 7. Distribution of human dirofilariasis due to D. repens according to sex, when specified, from histopathologists. At present, the two 1885 to 2000. sectors in which research teams need to conduct a thoroughgoing investigative inquiry are diagnostics – with a view to developing practical techniques for histological and serological diagnosis of the parasitosis – and therapeutics, in particular to avoid drastic surgery in cases with involvement of lung, breast, male sexual organs or orbital cavity. Diagnostic research will also lead to improved analysis of the prevalence of current cases of human infection, apparent or not, in a given population and thus make for a clear understanding of the mechanisms underlying the relationship between parasites and man.

Acknowledgements

The Authors are indebted for valuable sugges- tions, advices, references and personal communi- Fig. 8. Locations of D. repens in the human body, cations of observed cases to Professors N.V. when specified, from 1885 to 2000 (the dashed Chandrasekharan, S.A. Dissanaike, J.O. Gebbers, lines refer to internal sites). Gh. Olteanu, C.P. Raccurt, N.C. Vakalis, I. Varga, M. de S. Wijesundera, and Doctors H. Auer, T. Avdiukhina, Z. Bisoffi, H. Braun, W.P. Bredal, A. to D. repens is the most common in Delage, E. Feyles, E. Fok, R. Fueter, P.L. Garavelli, terms of frequency, diffusion and vari- M. Giansanti, M. Gobbo, R. Groell, T. Jelinek, B. ety of localisation in the body; the oth- Molet, M. Pastormerlo, M. Pavesi, N. Ratnatunga, E. V. Schwan, K. C. Shekhar, G. Speranza, C. ers account overall for fewer than 300 Stayerman, R. Stenzenberger, D. Strianese, W. cases. The great variety of organs Tarello, M. Thérizol-Ferly, G. Vitullo, F.X. Weill. affected by dirofilariasis due to D. Thanks are also due to Mrs J. Petrova for translat- repens justifies the current interest ing texts from Russian, Mrs T.G. Afanasieva of the among medical practitioners in this State Russian Library, for providing published papers difficult to find, and to Dr M.L. Fioravanti zoonosis: specialists in various health and Mr Luca Fabiani for technical help. sectors, from dermatology to ophthal- From Sri Lanka, several colleagues provided mology, from urology to pneumology, professor Dissanaike with Cingalese cases referred 110 Human dirofilariasis

in our list as personal communications, namely of Barnaul, Altai territory. Medsk Parazitol 3: Professors J.S. Edirisinghe and M Wijesundera, 57-58 (in Russian). and Doctors W. Abeyewickreme, D. Ariyaratme, Bartoli C, Bono A, Di Palma S, Pilotti S, B.G. de Silva, P. Kumarasinghe, S. Samarasinghe, Pampiglione S, 1997. Unusual breast lumps. K. Vithanage, M. Weerasooriya, M.D. Weilgama, Tumori 83: 611-612. Basset D, Villain M, Bastien P, Taupin P, Jarry DM, and Mr R.L. Ihalamulla. Their contributions are Dedet JP, 1996. La dirofilariose en France: un highly appreciated. This study was carried out cas languedocien chez un sujet sérologiquement with the partial contribution of the Emilia positif pour le VIH. Presse Méd 25: 1372-1375. Romagna region. Ben Said M, Korbi S, Abdelhedi M, Sriha B, Mili A, Khochtali N, Aouini MT, 1995. La dirofilariose sous-cutanée humaine: à propos de deux nou- References veaux cas tunisiens. Méd Mal Infect 25:519-521. Beuguet F, Bourdosseau G, 1996. Les dirofilario- Abeyewickreme W, Wijesundera M de S, ses du chien en France metropolitaine. Act Vét Weerasooriya M, Ismail MM, Dissanaike AS, 1362: 31-38. 1997. Recent investigations on human dirofilar- Bianchi Rossi CA, Palamà C, Torre A, Lami O, iasis in Sri Lanka. Trans R Soc Trop Med Hyg Maggi C, 1991. Dirofilariosi orbitaria. 91: 245. Descrizione di un caso. Ann Oftalmol Clin Ocul Anguera Galiana M, 1995. La dirofilariosis canina 117: 157-160. en el Delta del Ebro. Med Veter 12: 242-245. Braun H, Eibel U, Stamberger H, Reinthaler FF, Anyanwu IN, Agbede RI, Ajanusi OJ, Umoh JU, 1996. Transnasal-endoskopische Entfernung Ibrahim ND, 2000. The incrimination of Aedes eines lebenden Wurmes der Gattung Dirofilaria (Stegomyia) aegypti as the vector of Dirofilaria aus der Orbita. Vortr 30 Tagung Österr Ges f repens in Nigeria. Vet Parasitol 92: 319-327. Tropenmed u Parasitol. Nov 21, Wien II. Aranda C, Panyella O, Eritja R, Castella J, 1998. Braun H, Eibel U, Stammberger H, Ranner G, Canine filariasis importance and transmission in Gröll R, 1999. Endoscopic removal of an intra- the Baix Llobregat area, Barcelona (Spain). Vet orbital tumor: a vital surprise. Am J Rhinol 13: Parasitol 77: 267-275. 469-472. Arnold P, Deplazes P, Ruckstuhl H, Fluckiger M, Bredal WP, Gjerde B, Eberhard ML, 1994. Fallbericht: Dirofilariose beim Hund. Aleksandersen M, Wilhelmsen DK, Mansfield Schweiz Arch Tierheilk 136: 265-269. LS, 1998. Adult Dirofilaria repens in a subcuta- Artamonova AA, Nagornyi SA, 1996. A case of neous granuloma on the chest of a dog. J Small Dirofilaria repens migrating in man. Medsk An Pract 39: 595-597. Parazitol 1: 44 (in Russian). Bucklar H, Scheu U, Mossi R, Deplazes P, 1998. Arvanitis PG, Vakalis NC, Damanakis AG, Breitet sich in der Südschweiz die Dirofilariose Theodossiadis GP, 1997. Ophthalmic dirofila- beim Hund aus? Schw Arch f Tierheilk 140: riasis. Am J Ophthalmol 123: 689-691. 255-259. Auer H, Weinkamer M, Bsteh A, Schnayder C, Calvet C, Terrier JP, Menard G, 1999. Dietze O, Kunit G, Aspöck H, 1997. Ein selte- Dirofilariose souscutanée. Méd Trop 59 (Suppl ner Fall einer Dirofilaria repensinfestation des 2): 65-68. Nebenhodens. Mitt Österr Ges Tropenmed Cancrini G, Allende E, Favia G, Bormy F, Antón F, Parasitol 19: 53-58. Simón F, 2000a. Canine dirofilariosis in two Avdiukhina TI, Lysenko A Ia, Supriaga VG, cities of southeastern Spain. Vet Parasitol 92: Postnova VF, 1996. Dirofilariasis of the vision 81-86. organ: registry and analysis of 50 cases in Cancrini G, Favia G, Giannetto S, Merulla R, Russian Federation and in the countries of the Russo R, Ubaldino V, Tringali R, Pietrobelli M, Commonwealth of Independent States. Vestn Del Nero L, 1998a. Nine more cases of human Ophthalmol Moskva 112: 35-39 (in Russian). infection by Dirofilaria repens diagnosed by Avdiukhina TI, Supriaga VG, Postnova VF, morphology and recombinant DNA technology. Kuimova RT, Mironova NI, Murashov NY, Parassitologia 40: 461-466. Putintseva YV, 1997. Dirofilariasis in the Cancrini G, Favia G, Merulla R, Russo R, Community of Independent States countries: Ubaldino R, Tringali R, Messina M, Pietrobelli analysis of cases from 1915 to 1996. Medsk M, Del Nero L, 1998b. Traditional and innova- Parazitol 4: 3-7 (in Russian). tive tools to diagnose 8 new cases of human Azarova AN, Zhgut Iu Iu, Kuimova RT, dirofilariosis occurred in Italy. Parassitologia 40 Mukhortova EM , 1995. A report of 4 cases of (Suppl 1): 24. human Dirofilaria repens infestation in the city Cancrini G, Prieto G, Favia G, Giannetto S, Human dirofilariasis 111

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Luigi Venco Heartworm disease in dogs 119

Introduction Lung disease occurs secondary to vascular changes. Fluid and protein leaking Despite the name “heartworm” sug- through the vessel wall of affected arter- gests a primitive cardiac involvement, ies produce oedema in the parenchyma. the main localization of the worms and Spontaneous death of some worms can the first damages are in pulmonary produce thromboembolism and severe arteries and heartworm disease should inflammatory reactions. be considered as a pulmonary disease The reduction of compliance and that in the last stage only involves the gauge of pulmonary arteries, that can be right cardiac chambers. also occluded by either thromboem- Few days after heartworms reach cau- bolism or severe villous proliferation dal pulmonary arteries, endothelial cells (Fig. 1), results in a hypertensive pul- become swollen with wide intercellular monary state and, as a consequence, in junctions and disoriented longitudinal an increased after load for the right ven- axes, as response to the trauma. tricle which can induce “cor pulmonare” Activates neutrophiles adhere to the and right cardiac congestive heart fail- endothelium surface and enter the ure. Protein and fluid leaking through space between endothelial cells. the vessel wall of affected arteries pro- Furthermore, as linear areas of sub duce further oedema and inflammation endothelium are exposed, platelet adhe- in the parenchyma (Dillon et al., 1995). sion and activation is greatly stimulated. Based on the pathogenesis the clinical Damaged arterial surface allows albu- evolution of heartworm disease in dogs min, plasma fluid and blood cells to is usually chronic. Most infected dogs reach the perivascular space. do not show any symptoms of the dis- After the endothelial changes, the ease for a long time, months or years, intima thickens with fluid, leukocytes depending on worm burden, individual invade the wall and smooth muscles reactivity and exercise, as arterial dam- cells multiply within tunica media and ages are more severe in dogs with migrate toward the endovascular surface as response to growth factor released by platelets. The multiplication and migration of smooth muscle cells cause the presence, on the internal arterial surface, of villi which are made by smooth muscle cells and collagen and covered by endothelial like-cells (Rawlings, 1986; Calvert and Rawlings, 1988). The gravity of villous proliferation is directly related to duration of infection and worm burden. The arterial surface of heavily infected dogs Fig. 1. Right ventricle outflow tract and pulmonary and cats appear rough and velvety, and artery of a dog with severe heartworm infections. both the lumen and the compliance of Note the large worm burden and the villous proli- the pulmonary arteries are reduced. feration of the pulmonary artery endothelium. 120 Heartworm disease in dogs intensive exercise than in dogs at rest Diagnosis (Dillon et al., 1995). Signs of the disease develop gradually and may begin Diagnosis of heartworm infection can with a chronic cough. be made in dogs by blood test detecting Coughing may be followed by dysp- circulating microfilariae or adult anti- noea, from moderate to severe, weak- gens but further diagnostic procedures ness, and sometimes lipothymias after are usually required to determine the exercise or excitement. At this time severity of disease and which is the best abnormal pulmonary sounds (crackles) treatment (Knight, 1995). over the caudal lung lobes and second heart sound splitting can be often heard. Blood test for microfilariae Later, when right cardiac congestive failure is developing, swelling of the Blood sample is examined after con- abdomen and sometimes legs from centration (Knott or Difill test) for fluid accumulation, anorexia, weight the presence of microfilariae. If micro- loss, dehydration, is usually noted. At filariae are seen and identified as this stage, cardiac murmur over the D. immitis, based on morphology that right side of the thorax due to tricuspid is considered a definitive proof of infec- valve insufficiency and abnormal car- tion (specificity 100 %). However up to diac rhythm due to atrial fibrillation are 30% of dogs do not have circulating common findings. Sudden death rarely microfilariae even though they harbour occurs and usually it happens following adult worms, due to the presence of respiratory distress or cachexia. only worms of the same sex (quite In the chronic pathway of the disease unusual in dogs), immune reactivity of sometimes acute symptomatology may the host to microfilariae or administra- occur. After severe spontaneous throm- tion of microfilaricidal drugs. The sensi- boembolism following the natural tivity of test for microfilariae is not death of many heartworms, dogs may therefore considered sufficient to rule show acute life threatening dyspnoea out the infection in case of negative test. and haemoptysis. In small sized dogs is furthermore a Blood test for adult female antigens common event the displacement of adult worms from pulmonary arteries Tests designed to detect heartworm to right cardiac chambers due to pul- adult antigens based on ELISA or col- monary hypertension and sudden fall loidal gold staining techniques are in right cardiac output. In this case considered highly specific as cross dogs affected shows the so called reactivity with other dogs parasites “caval syndrome”. Dyspnoea, tricuspid (i.e. D. repens, Dipetalonema sp.) cardiac murmur and emoglobinuria does not occur. (due to mechanical haemolysis in right These tests allow detection of adult cardiac chambers) are the most typical heartworm antigens produced only by signs and fatal outcome is usual female worms and may provide infor- (Kitagawa et al., 1987; Atwel and mation about worm burden (Knight, Buoro, 1988; Venco, 1993). 1995; Venco et al., 2003). The sensitiv- Heartworm disease in dogs 121 ity is actually very high, but false negative results may occur in prepatent or very light infections or when only male worms are present (McCall, 1992).

Thoracic radiographs

Thoracic radiographs may show, in the advanced stage, enlargement of the pulmonary arteries, abnormal pulmonary patterns and in the worst cases right-sided cardiomegaly. If congestive Fig. 2. Thoracic radiograph of a 12-year-old infected dog. With right sided cardiomegaly and severe right heart failure is present peritoneal pulmonary vascular disease. The level of circula- and pleural effusion can be noted ting antigens were very low. (Rawlings, 1986; Calvert and Rawlings, 1988). They are useful to assess the severity of the pulmonary lesions but not for evaluating worm burden (Venco et al., 2003). Since radiographic signs of advanced pulmonary vascular disease may persist long after an infection has run its course, some of the most severely diseased dogs may have dis- proportionately low worm burden (Fig. 2). On the contrary, some inactive dogs may have large worm burdens and be clinically asymptomatic with no or triv- Fig. 3. Thoracic radiograph of a 3 year old infec- ial radiographic lesions (Fig. 3). ted dog with just a trivial cranial pulmonary artery enlargement. From this dog 53 adult worms were surgically removed. Electrocardiography

As electrocardiogram displays the It also allows the visualization of para- electrical activity of the heart, abnor- sites in right cardiac chambers, caudal malities, (electrical axis right deviation, vena cava, main pulmonary artery and atrial fibrillation) are usually found proximal tract of both caudal pulmonary only in the last stage of the disease, arteries (Fig. 4). The heartworms are when right cardiac chambers present visualized as double, linear parallel severe damages. objects floating in the right cardiac chambers or into the lumen of vessels Echocardiography (Moise, 1988; Badertscher et al., 1988). It is performed mainly in cases where Echocardiography allows a direct clinical and radiographic findings sug- visualization of cardiac chambers and gest severe disease. Cardiac ultrasound connected vessels (Moise, 1988). can increase the accuracy in staging the 122 Heartworm disease in dogs

many worms are thought to be present based upon the ELISA tests performed and ultrasound examination (Venco et al., 2004), the size of the dog, the age of the dog (dogs ranging from 5 to 7 years are at high risk to harbour the largest worm burden; Venco et al., 2004), concurrent health factors, severity of the pulmonary disease, and the degree to which exercise can be restricted in the recovery period. Fig. 4. Echocardiogram of the same dog of Fig. 3. The categories into which patients are Heartworms are visualized as double, linear parallel objects (arrow) floating into the lumen of the grouped are as follows: right pulmonary artery. Low risk of thromboembolic complications (low worm burden and no disease and estimating the worm bur- parenchymal and/or pulmonary vascu- den, both of which affect the treatment lar lesions) program and the prognosis. Dogs included in this group must satisfy all this conditions: - No symptoms Therapy - Normal thoracic radiographs - Low level of circulating antigens or It has been said that the treatment of a negative antigen test with circulat- heartworm infection is difficult. There ing microfilariae are several strategies that can be used - No worms visualized by echocardio- including the option of not treating at graphy all. The important concept to realize is -No concurrent diseases that treating for heartworm infection is - Permission of exercise restriction neither simple nor safe in itself. Prior to therapy, the heartworm High risk of thromboembolic complica- patient is assessed and rated for risk of tions developing post-adulticide thromboem- In this group should be included all bolism. the dog that do not satisfy one or more Previously 4 classes were described in of these conditions: order to prevent the post-adulticide - Symptoms related to the disease thromboembolism and give a correct (coughing, lipotimias, swelling of prognosis (from class 1, low risk to the abdomen) class 4, very high risk) (Di Sacco and - Abnormal thoracic radiographs Vezzoni, 1992) but at present a more - High level of circulating antigens simple classification is usually pre- -Worms visualized by echocardiog- ferred and the patient is included into raphy one of two categories (low and high - Concurrent diseases risk). Important factors include: how -No permission of exercise restriction. Heartworm disease in dogs 123

Symptomatic therapy includes drugs 10% of female worms resulting there- and measures that can improve car- fore in 50% reduction of the worm diopulmonary circulation and lung burden (which is safer in terms of inflation in order to relief symptoms in embolism and shock). For this reasons, dogs that cannot undergo causal thera- the three-injection alternative protocol py or to prepare them for a adulticide is the treatment of choice of the or surgical therapy. American Heartworm Society and sev- Restriction of exercise and, in select- eral university teaching hospitals, ed cases, cage rest seems to be the most regardless of stage of disease. important measure to improve car- Pulmonary thromboembolism is an diopulmonary circulation and to reduce inevitable consequence of a successful pulmonary hypertension (Dillon et al., adulticide therapy. If several worms die 1995). widespread pulmonary thrombosis fre- Anti-inflammatory doses of glucocor- quently develops. Mild thromboem- ticosteroid (prednisolone 2 mg/kg s.i.d. bolism may be clinically unapparent, for four or five days) given at diminish- but in severe cases life threatening res- ing rate can control pulmonary inflam- piratory distress can occur. mation and thromboembolism. These complication can be reduced Diuretics (furosemide 1 mg/kg b.i.d.) by restriction of exercise (no walks, no are useful when right congestive heart running around; the dog must stay failure is present to reduce fluid effu- indoors or, in selected cases, a cage sions. Digoxin may be administered rest) during the 30-40 days following only to control atrial fibrillation. The the treatment and by administration of use of aspirin is debatable and as not calcium heparin and anti-inflammatory secure proofs of beneficial antithrom- doses of glucocorticosteroid to control botic effect have been reported, for this clinical signs of thromboembolism (Di reason the empiric use of aspirin is not Sacco and Vezzoni, 1992; Vezzoni et advised (Knight, 1995). al., 1992; Rawlings and McCall, 1996). The organical arsenical melarsomine It is now known that certain macro- dihydocloride is the only available com- cyclic lactones have adulticidal proper- pound to be used in the adulticide ties (McCall et al., 2001). heartworm therapy in dogs. Experimental studies have shown Two intramuscular injections of 2.5 ivermectin to have partial adulticidal mg/kg 24 hours apart is the standard properties when used continuously for regimen, but to reduce the risk of pul- at least 16 months at preventative monary thromboembolism a more doses (6-12 mcg/kg/month) and 100% gradual two step treatment is strongly adulticidal efficacy if administered con- advised by giving one injection and tinuously for over 30 months (McCall then administering the standard pair of et al., 2001). injections at least 50 days later (Keister While there may be a role for this et al., 1992; Rawlings and McCall, therapeutic strategy in few and selected 1996). In fact, one administration of cases in which patient age, or concur- melarsomine at the dose of 2.5 mg/kg rent medical problems prohibit melar- kills about 90% of male worms and somine therapy, the current recommen- 124 Heartworm disease in dogs dations are that ivermectin is not pulmonary arteries should be per- adapted as the primary adulticidal formed to determine if a sufficient approach, and that this kind of therapy number of worms are in accessible should be used carefully. In fact, the locations. adulticide effect of ivermectin generally Surgical removal of heartworm can requires long time span before heart- avoid pulmonary thromboembolism, as worms are eliminated completely. compared to pharmacologic adulti- Furthermore, the older are worms cides, such as melarsomine (Morini et when first exposed to ivermectin, the al., 1998). This procedure, however, slower they are to die. In the meantime, requires specialized training and instru- the infection persists and continues to mentation, including fluoroscopic cause disease. Clinical observations imaging capabilities. Nevertheless, it suggest that heartworm-positive active remains a very good and a safe alterna- dogs in prolonged ivermectin treatment tive for the management of high risk may worsen if ivermectin is given patients and the best choice in dogs monthly for 2 years (Venco et al., harbouring a large worm burden. 2004). Surgical therapy is advised when several worm displacement in the right References cardiac chambers produces the sudden Atwell RB, Buoro BJ, 1988. Caval syndrome. In: onset of severe symptoms (caval syn- Boreman PFL, Atwell RB Eds. Dirofilariasis drome). It can be accomplished under Boca Raton, CRC Press Inc, 191-203 pp. general anaesthesia with Flexible Badertscher RR , Losonsky JM , Paul AJ , Kneller SK, 1988. Two dimensional echocardiography Alligator Forceps introduced via jugu- for diagnosis of dirofilariasis in nine dogs. lar vein. JAVMA 193: 843-846. Flexible Alligator Forceps aided by Calvert CA, Rawlings CA, 1988. Canine fluoroscopic guidance can access not Heartworm Disease In: Canine and Feline Cardiology. Fox Pr, Ed. Churchill Livingstone only right cardiac chambers but also Inc, 519-549 pp. pulmonary arteries. The main pul- Dillon R, Brawner WR, Hanrahan L, 1995. monary artery and lobar branches can Influence of number of parasites and exercise on the severity of heartworm disease in dogs. In: be accessed with flexible alligator for- Soll MD and Knight DH Eds. Proceedings of the ceps, aided by fluoroscopic guidance ’95 Heartworm Symposium, American (Ishihara et al., 1990). Intra-operative Heartworm Society, Batavia, IL, 113-120 pp. Dillon R, Warner AE, Molina RM, 1995. mortality with this technique is very Pulmonary parenchymal changes in dogs and low. Overall, survival and rate of recov- cats after experimental transplantation of dead ery of dogs at high risk of pulmonary Dirofilaria immitis. In: Soll MD and Knight DH Eds. Proceedings of the heartworm Symposium thromboembolism is improved signifi- ’95, American Heartworm Society, Batavia, IL, cantly by physically removing as many 97-101 pp. worms as possible. When the facilities Di Sacco B, Vezzoni A, 1992. Clinical classification of heartworm disease for the purpose of are available, worm extraction is the adding objectivity to the assessment of thera- procedure of choice for the most heav- peutic efficacy of adulticidal drugs in the field. ily infected and high risk dogs. Before In: Soll M.D. Ed Proceedings of the Heartworm Symposium ’92, American Heartworm Society, electing this method of treatment, Batavia, 209-214 pp. echocardiographic visualization of the Ishihara K, Kitagawa H, Sasaky Y, 1990. Efficacy Heartworm disease in dogs 125

of heartworm removal in dogs with dirofilarial Moise NS, 1988. Echocardiography In: Canine hemoglobinuria using Flexible Alligator and Feline Cardiology, PR Fox Ed. New York, Forceps. Jpn J Vet Sci 53: 591-599. Churchill Livingstone Inc, 113-156 pp. Keister DM, Dzimianski MT, McTier TL, McCall Morini S, Venco L, Fagioli P, Genchi C, 1998. JW, Brown J, 1992. Dose selection and confir- Surgical removal of heartworms versus melar- mation of RM 340, a new filaricide for the treat- somine treatment of naturally-infected dogs ment of dogs with immature and mature with risk of thromboembolism. In Seward, L., Dirofilaria immitis. In: Soll MD, Ed Ed. Proceedings of the American Heartworm Proceedings of the Heartworm Symposium ’92: Symposium ‘98. American Heartworm Society, American Heartworm Society, Batavia, IL, 225- Batavia, IL, 235-240 pp. 240 pp. Rawlings CA, 1986. Heartworm disease in dogs Kitagawa H, Sasaky Y, Ishiara K, 1987. Canine and cats WB Saunders Co. Philadelphia. Dirofilaria hemoglobinuria: changes in right Rawlings CA, McCall JW, 1996. Melarsomine: A heart hemodinamics and heartworm migration new heartworm adulticide compound. from pulmonary artery toward right atrium fol- Continuing Education 18: 373-379. lowing beta - blocker administration. Jpn J Vet Venco L, 1993. Approccio diagnostico alla sin- Sci 49: 1081-1086. drome della vena cava. Veterinaria, SCIVAC Ed. Knight DH, 1995. Guidelines for diagnosis and (Cremona) 7: 11-18 [in Italian]. management of heartworm (Dirofilaria immitis) Venco L, Genchi C, Vigevani Colson P, Kramer L, infection. In: Bonagura JD Ed. Kirk’s Current 2003. Relative utility of echocardiography, radi- Veterinary Theraphy XII Small Animal Practice. ography, serologic testing and microfilariae WB Saunders Co. counts to predict adult worm burden in dogs McCall JW, 1992. A parallel between experimen- naturally infected with heartworms. In: Seward tally induced canine and feline heartworm dis- LE, Knight DH, Eds Recent Advances in ease. Proceedings of XVII WSAVA World Heartworm Disease. Symposium ’01. American Congress. Rome, Italy 24th-27th September. Heartworm Society. Batavia, IL, 111-124 pp. Delfino: 255-261. Venco L, McCall JW, Guerrero J, Genchi C, 2004. McCall JW, Guerrero J, Roberts RE, Supakorndej N, Efficacy of long-term monthly administration of Mansour AE, Dzimianski MT, McCall SD, 2001. ivermectin on the progress of naturally acquired Further evidence of clinical prophylactic (reach- heartworm infections in dogs. Vet Parasitol 124: back) and adulticidal activity of monthly adminis- 259-268. tration of ivermectin and pyrantel pamoate in Vezzoni A, Genchi C, Raynaud JP, 1992. Adulticide dogs experimentally infected with heartworms. In efficacy of RM 340 in dogs with mild and severe Seward, L., Ed. Proceedings of the American natural infections. In: Soll M.D., Ed. Proceedings Heartworm Symposium ‘01. American of the Heartworm Symposium ’92, American Heartworm Society, Batavia, IL, 189-200 pp. Heartworm Society, Batavia, IL, 231-240 pp. 9 Heartworm (Dirofilaria immitis) disease in cats

Luigi Venco Heartworm disease in cats 129

Cat is considered a susceptible but not ideal host for Dirofilaria immitis. Increased host resistance is reflected by the relatively low adult worm burden in natural infections (cats generally harbour 1 to 8 worms with 2 to 4 worms being the usual burden, Genchi et al., 1992), the low number of heartworms that develop after experimental inoculation with infective larvae, the prolonged pre-patent period (7-8 months), the low level and short duration of Fig. 1. Two adult female heartworms in a cat that microfilaremia, and the short life span experienced sudden death. of adult worms (2-3 years) (Dillon, 1984; Calvert, 1989; McCall et al., The onset of symptoms in most cases 1992; Atkins et al., 1995). seems to be related to the natural death In cats, changes in pulmonary arteries of parasites or to the first arriving of L5 and lungs after infection seem to be heartworms in the pulmonary arteries. similar to those found in dogs, but right cardiac chambers well bear pulmonary Diagnosis hypertension and right cardiac heart failure is an unusual finding (Dillon et Blood test for microfilariae al., 1995; Atkins et al., 1995). In cats, the clinical presentation is quite As microfilaremia in cats is unlikely, different than in the canine counterpart. sensitivity of test for detection of circu- Most cats seem to well bear the infection lating microfilariae is very low despite for long time. These cats may have a specificity is considered 100% as in spontaneous self-cure due to the natural dogs (Atkins et al., 1995). death of parasites without any kind of clinical signs or may suddenly show drama- Blood test for adult antigens tic acute symptoms. Respiratory signs as coughing, dyspnoea, haemoptysis are Test detecting adult female heart- usually seen but also vomiting frequently worm antigens can provide a definitive occurs. Sudden death in apparently proof of infections in cats because of healthy cats is furthermore not rarely the very high specificity. Nevertheless observed (McCall et al., 1994; Holmes, because the worm burden is usually 1995; Atkins et al., 1995) (Fig. 1). very light in cats, infections caused only Chronic symptoms including coughing, by male heartworms are not infrequent vomiting, diarrhoea, weight loss can be and symptomatology may be frequently less frequently observed. due to immature worms, these test On the contrary than in dogs, sympto- yield false negative result in a large ave- matology related to right ventricular rage. A negative test can not therefore heart failure is not considered consi- considered sufficient to rule out the stent with heartworm infection in cats. infection (Atkins et al., 1995). 130 Heartworm disease in cats

Blood test for antibodies to adult heart- monary artery cannot be observed worm because this tract of artery is obscured by cardiac silhouette. Right-sided car- Due to the low sensitivity of tests for diomegaly is not considered a typical circulating microfilariae and adult anti- finding in cat. gens in cats, test for detection of antibodies to adult heartworm can be useful Non selective angiocardiography used (Atkins et al., 1995; Prieto et al., 1997; Genchi et al., 1998). This kind of Non selective angiocardiography is test has high sensitivity but not comple- useful in visualizing the gross morpho- te specificity because of cross reactivity logy of the pulmonary arteries. Seldom with other parasites or antibodies to the heartworms can be seen as negative abortive infections. Consequently anti- filling defects within opacified arteries body tests should be interpreted care- (Atkins et al., 1995). fully, taking other relevant clinical information into consideration. Electrocardiography

Thoracic radiographs Heartworm infection does not involve right cardiac chambers. Consequently Thoracic radiography is an important electrocardiography cannot provide tool for the diagnosis of feline heart- useful information in infected cats. worm disease. Despite thoracic abnormalities in few cases are absent or Echocardiography transient (Selcer et al., 1996), typical findings as enlarged peripheral bran- Cardiac ultrasound allows the direct ches of the pulmonary arteries accom- visualization of the parasites in right panied by varying degrees of pulmo- atrium and ventricle, main pulmonary nary parenchymal disease are strongly artery and proximal tract of both its consistent with heartworm infection peripheral branches (Fig. 3). (Fig. 2). Enlargement of the main pul- Specificity is virtually 100% and sensi-

Fig. 2. Thoracic radiograph of a heartworm infect- Fig. 3. Echocardiogram of a heartworm infected cat. ed cat with of pulmonary parenchymal disease A worm is visualized as double, linear parallel lines (caudal lung lobes). (arrow) into the lumen of the right pulmonary artery. Heartworm disease in cats 131 tivity in cats seems to be very high debatable. Turner et al. (1989) reported (Venco et al., 1998, 1999b) because some toxicity in heartworm naive cats, the portion of caudal pulmonary arte- but later reports showed that thiacetar- ries that can not be thoroughly interro- samide delivered to normal cats produ- gate because of the acoustic impedance ced no respiratory distress or altered the of the air inflated lungs is very short body temperature (Dillon et al., 1992). when compared with the length of the However a large average heartworm adult parasite. Based on these conside- infected cats develop acute respiratory rations, cardiac ultrasonography distress or sudden death in the post- should be always performed when treatment period. These effects seem to heartworm infection is suspected. be due to embolization associated with worm death (Dillon et al., 1992). Transtracheal lavage The organical arsenical melarsomine dihydocloride is the only available com- The presence of eosinophiles in a tra- pound now on the market, but there is cheal wash, with or without eosinophi- insufficient experience about its use in lia, may be noted 4 to 7 months after cats until now. Furthermore, few data infection but this findings is not speci- suggests that melarsomine is toxic to fic and infection with other pulmonary cats at dosages >3.5 mg/kg. Ivermectin parasites (Paragonimus kellicotti, at 24 µg/kg monthly given for 2 years Aelurostrongylus abstrusus) and aller- has been reported to reduce worm bur- gic pneumonitis should be ruled out dens as compared to untreated cats. (Atkins et al., 1995). Since cats usually harbour low worm burdens and the main problem is the Therapy reaction that arise when the worms die (and not the worm mass by itself) this Diminishing doses of prednisone are reaction could probably occur even advised in cats in order to relief respira- when the ivermectin-treated worms tory distress. The dosage is 2 mg/kg daily die, but its intensity is unknown. initially, then declining to 0.5 mg/kg Anyway, there are no studies sugge- every other day for two weeks, and then sting that medical adulticidal therapy discontinuing treatment after an additio- increases the survival rate of heart- nal two weeks (Atkins et al., 1995). worm naturally infected cats (Knight et If crisis is due to embolization of dead al., 2002). Due to these reasons, worms high doses of prednisone (1-2 macrofilaricide treatment is not advi- mg/kg 3 times a day) are recommended sed in cats unless in selected cases. (Dillon, 1986). In case of caval syndrome or when a In previous studies, when thiacetarsa- heavy worm burden is visualized by mide was the only arsenical available echocardiography in right cardiac compound, some treatment regimen chambers, surgery may be attempted. was attempt against adult worms. The Worm can be extracted via jugular vein same dosage and regimen used in trea- using thin alligator forceps, horse hair ting dogs, 2.2 mg/kg twice daily for two brush or basket catheters (Glaus et al., days, was used for cats. The results were 1995; Borgarelli et al., 1997; Venco et 132 Heartworm disease in cats al., 1999b). Because of the small size of Symposium ‘98, American Heartworm Society, Batavia IL, 173-177 pp. the feline heart pulmonary, arteries can- Glaus TM, Jacobs GJ, Rawlings CA, Watson ED, not be accessed. Special care must be Calvert CA, 1995. Surgical removal of heart- taken during the heartworm removal worms from a cat with caval syndrome JAVMA 206: 663-666. because traumatic dissection of a worm Holmes RA, 1995. Feline heartworm disease: may result in circulatory collapse and Compend Cont Ed 15: 687-695. death (Venco et al., 1999b). Knight DH, Doiron DD, Longhofer S, Rubin SR, Downing R, Nelson CT, Mc Call JW Seward L, 2002. Guidelines for the diagnosis, prevention and management of heartworm (Dirofilaria References immitis) infection in cats In: Seward LE, Knight DH Eds Recent Advances in Heartworm Atkins CE , Atwell RB , Dillon AR , Genchi C , Disease: Symposium ’01 American Heartworm Hayasaky M , Holmes RA, Knight DH , Lukoff Society Batavia IL, 267-273 pp. DK , Mc Call JW, Slocombe JO, 1995. McCall JW, Calvert CA, Rawlings CA, 1994. Guidelines for the diagnosis, treatment, and pre- Heartworm infection in cats: a life threatening vention of heartworm (Dirofilaria immitis) disease. Vet Med 89: 639-647. Infection in cats. In: MD Soll and DH Knight McCall JW, Dzimiansky MT, McTier TL, Jernigan Eds. Proceedings of the heartworm symposium AD, Jun JJ, Mansour AE, Supakorndej P, Plue ’95. American Heartworm Society, Batavia, IL, RE, Clark JN, Wallace DH, Lewis RE, 1992. 309-312 pp. Biology of Experimental Heartworm Infections Borgarelli M, Venco L, Piga PM, Bonino F, Ryan in Cats in MD Soll Ed. Proceedings of the ’92 WG, 1997. Surgical removal of heartworms from Heartworm Symposium, American Heartworm the right atrium of a cat. JAVMA 211: 68-69. Society, Batavia IL, 71-79 pp. Calvert CA, 1989. Feline Heartworm disease. In: Prieto C, Venco L, Simon F, Genchi C, 1997. RG Sherding Ed. The Cat Diseases and Clinical Feline heartworm (Dirofilaria immitis) infec- Management Churchill Livingston Inc. New tion: detection of specific IgG for the diagnosis York, 95-510 pp. of occult infections. Vet Parasitol 70: 209-217. Dillon R, 1984. Feline Dirofilariasis. Vet Clin Selcer BA, Newell SM, Mansour AE, Mc Call JW, North Am 14: 1185. 1996. Radiographic and 2 D echocardiographic Dillon R, 1986. Feline heartworm disease. In: GF findings in eighteen cats experimentally exposed Otto Ed. Proceedings of the Heartworm to Dirofilaria immitis via mosquito bites. Vet Symposium ’86..: American Heartworm Society, Radiol Ultr 37: 137-144. Washington DC, 149-154 pp. Turner JL, Lees GE, Brown SA, 1989. Dillon R, Cox N, Brawner B, D’Andrea G, 1992. Thiacetarsamide in normal cats: pharmacokine- The effects of thiacetarsamide administration to tics, clinical, laboratory and pathologic features. normal cats. In: MD Soll Ed. Proceedings of the In GF Otto Ed. Proceedings of the Heartworm Heartworm Symposium ’92, American Symposium ’89. American Heartworm Society, Heartworm Society, Batavia IL, 133-137 pp. Washington DC, 135-141 pp. Dillon R, Warner AE, Molina RM, 1995. Pulmonary Venco L, Morini S, Ferrari E, Genchi C, 1999a. parenchymal changes in dogs and cats after expe- Technique for Identifying Heartworms in Cats rimental transplantation of dead Dirofilaria immi- by 2-D Echocardiography. In: Seward LE, tis. In MD Soll and DH Knight Eds. Proceedings Knight DH Eds Recent Advances in Heartworm of the Heartworm Symposium ’95. American Disease: Symposium ’98 American Heartworm Heartworm Society, Batavia IL, 97-101 pp. Society Batavia IL, 97-102 pp. Genchi C, Guerrero J, Di Sacco B, Formaggini L, Venco L, Borgarelli M, Ferrari E, Morini S, Genchi 1992. Prevalence of Dirofilaria immitis infec- C, 1999b. Surgical Removal of Heartworms tion in Italian cats. Proceedings of Heartworm from naturally-infected Cats. In: Seward LE, Symposium ‘92, American Heartworm Society, Knight DH Eds. Recent Advances in Batavia IL, 97-102 pp. Heartworm Disease: Symposium ’98 American Genchi C, Kramer LH, L Venco, Prieto G, Simon Heartworm Society Batavia IL, 241-246. F, 1998. Comparison of antibody and antigen Venco L, Calzolari D, Mazzocchi D, Morini S, testing with echocardiographic for the detection 1998. The Use of Echocardiography as a of heartworm (Dirofilaria immitis) in cats. In: Diagnostic tool for Detection of Feline Recent Advances in Heartworm Disease, in RL Heartworm (Dirofilaria immitis)Infections. Fel Seward Ed. Proceedings of Heartworm Pract 26: 6-9. 10 Dirofilaria (Nochtiella) repens infection in dogs and cats

Luigi Venco D. repens infection in dogs and cats 135

Dirofilaria (Nochtiella) repens, is the zoonotic human cases is arising. Human causative agent of canine and feline infection manifests with either subcuta- subcutaneous dirofilariosis, a mosquito neous nodules, ocular or lung parenchy- borne disease that has become increas- mal nodules mostly asymptomatic. The ingly recognized in several countries in significance of infection in humans is southern and central Europe, Africa that pulmonary and some subcutaneous and Asia. lesions are commonly labelled as malig- D. repens life cycle, like that of nant tumours requiring invasive investi- Dirofilaria immitis, consists of 5 larval gation and surgery before a correct stages which develop both in a verte- diagnosis is made. The pathology of the brate host and an arthropod (mosqui- condition is associated with aberrant to), intermediate host and vector. Adult localization of immature worms that do female worms produce thousands of not reach adulthood; therefore, microfi- embryos (microfilariae) that are ingest- lariae are almost always absent ed by a blood-feeding insect. (Pampiglione et al., 1995). Microfilariae have a unique circadian periodicity in the peripheral circulation over a 24-hour period. The arthropod Diagnosis vectors also have a circadian rhythm in which they obtain blood meals. The Blood test for microfilariae highest concentration of microfilariae usually occurs when the local vector is Detection of circulating microfilariae most actively feeding. Microfilariae using the method developed by Knott is then undergo 2 developmental moults the best way for doing an in vivo diag- in the insect. During feeding, the infect- nosis but collection of hystopatological ed mosquito deposits third-stage larvae cutaneous specimens. throughout a drop of haemolymph in Larvae species determination is made the proximity of bite wound from where on the basis of morphological or hysto- larvae actively migrate to subcutis. chemical method or by using PCR. Larvae develop to the adult stage through moults in the vertebrate hosts. Blood test for adult antigens Prepatency lasts 6 1/2 to 9 months (Webber and Hawking, 1955; Cancrini Tests detecting adult heartworm (D. et al., 1989). The adults reside in the immitis) do not detect D. repens anti- subcutaneous tissues of dogs and cats gens and no cross reactivity is and may cause mild clinical signs such as described. pruritus, dermal swelling, subcutaneous nodules or no symptoms at all (Baneth et al., 2002; Zˇ ivicˇnjak et al., 2006). Therapy

Despite the usual hosts of D. repens No adulticide treatment for D. repens are domestic and wild carnivores, is registered and a off-label use of human beings may act as accidental and melarsomine has only recently been dead-end hosts and a big concern about described on the basis of a case report 136 D. repens infection in dogs and cats where combined therapy with the studies were not based on necropsy con- arsenic adulticide melarsomine and the firmation some concerns about the abil- avermectin microfilaricidal doramectin ity of completely preventing infection in was effective in clearing infection with dogs still remain (Cancrini et al., 1989). D. repens in a dog (Baneth et al., 2002), although the death of the patients does not allow conclusive evidences. References Symptomatic therapy of canine dirofi- Baneth G, Volansky Z, Anug Y, Favia G, Bain O, lariosis due to D. repens is indicated for Goldstein RE, Harrus S, 2002. Dirofilaria repens infection in a dog: diagnosis and treat- dogs suffering from clinical signs of ment with melarsomine and doramectin. Vet this disease, such as dermal swelling, Parasitol 105: 173-178. sub-cutaneous nodules and pruritus. Cancrini G, Tassi P, Coluzzi M, 1989. Ivermectin Steroids and/or antibiotics administra- against larval stages of Dirofilaria repens. Parassitologia 31: 177-182. tion and nodules surgical removal may Genchi C, Poglayen G, Kramer LH, 2002. be suggested in these cases for relieving Efficacia di Selamectin nella profilassi delle infe- symptoms. stazioni da Dirofilaria repens nel cane. Veterinaria 16: 69-71. Some macrocyclic lactones (iver- Pampiglione S, Canestri Trotti G, Rivasi E, 1995. mectin, moxidectin, selamectin) are Human dirofilariosis due to Dirofilaria claimed to be effective for the preven- (Nochtiella) repens: a review of world literature. Parassitologia 37: 149-193. tion of D. repens infection in dogs Rossi L, Ferroglio E, Agostini A, 2002. Use of (Genchi et al., 2002; Rossi et al., 2002) moxidectin tablets in the control of canine sub- and labelled for this use in some coun- cutaneous dirofilariosis. Vet Rec 15: 383. Webber WAF, Hawking F, 1955. Experimental try (Italy) on the basis of field study. maintenance of Dirofilaria repens and D. immi- While there is no doubt that they are tis in dogs. Exp Parasitol 4: 143-164. able to prevent mirofilaraemia in dogs Zˇ ivicˇ njak T, Martinkovicˇ F, Beck R, 2006. Dirofilariosis in Croatia: spread and public (and this is important for zoonotic health impact. 5th Croatian congress on infec- implications), as most of the performed tive diseases, Zadar, Croatia. 11 Guideline for the laboratory diagnosis of canine and feline Dirofilaria infections

Claudio Genchi, Luigi Venco, Marco Genchi Laboratory diagnosis: Guideline 139

Microfilariae To note that intensity of microfilaremia is not correlated to the adult worm bur- Fresh blood smear (not advised) den: in general, high microfilaremic dogs harbour few worms. A drop of fresh venous blood is placed on a clean microscopic slide and Modified Knott test covered with a coverslip and examined under low microscopic power. One ml of venous blood is mixed Microfilariae are seen throughout the with approximately 10 ml of 2% movement they cause to the blood red buffered formalin and the mixture cell layer. is centrifuged for 3-5 minutes at 1500 rpm. To note that the intensity of micro- The supernatant is decanted from the filaremia is not correlated to the centrifuge tube and the sediment is adult worm burden: in general, high mixed with equal parts of a 1:1000 microfilaremic dogs harbour few methylene blue stain. The stained sedi- worms. ment is placed on a slide, covered with When dogs are monthly treated with a coverslip and examined under a preventive drugs during heartworm microscope. transmission season, re-testing each year before starting again the preven- Advantage tive treatment is advisable. Sensitive in dogs and specific: microfilariae belonging to different species can be differ- Advantage entiate Rapid and inexpensive Disadvantages Time consuming, need of a skill operator with Disadvantages good knowledge of microfilarial morphology Very low sensitivity, frequent false negative, Specific but of low sensitivity in cats no species diagnosis (it is not possible to differentiate microfilariae) Not useful in cats Filter test One ml of venous blood with antico- Concentration methods agulant of either EDTA or heparin is added to approximately 10 ml of lysate Several methods can be used to con- solution. The mixture is injected centrate circulating microfilariae from through a filter (Millipore) chamber. the blood. These methods are sensitive The filter is removed from the chamber, and make possible to differentiate placed on a glass slide, stained, and microfilariae throughout morphologi- examined under a microscope. cal criteria (see Table 1 and Fig. 1). Microfilariae can be concentrated by To note that intensity of microfilaremia the modified Knott test or by a filter test. is not correlated to the adult worm bur- 140 Laboratory diagnosis: Guideline

Table 1. Morphological features of microfilariae1 from filarial worms of dogs and cats.

Species Length µm Width µm Features Dirofilaria immitis 290-330 5-7 No sheath, cephalic end pointed, tail straight with the end pointed

Dirofilaria repens 300-360 6-8 No sheath, cephalic end obtuse, tail sharp and filiform often ending as an umbrella handing

Acanthocheilonema reconditum 260-283 4 No sheath, cephalic end obtuse with a prominent cephalic hook, tail button hooked and curved

Acantocheilonema dracunculoides2 190-247 4-6.5 Sheath, cephalic end obtuse, caudal end sharp and extended

Cercopithifilaria grassii 567 12-25 Sheath, caudal end slightly curved

1 microfilariae measure by Knott test 2 microfilariae from the uterus den: in general, high microfilaremic described by Chalifoux and Hunt dogs harbour few worms. (1971), or frozen at -80°C in aliquot portions. After 2 hours at room tem- Advantage perature, the slide is air dried and Rapid and sensitive in dogs covered with a coverslip. No need for a centrifuge apparatus - D. immitis microfilariae show 2 acid Disadvantages phosphatase activity spots localized Expensive (tests are sold as kit, Difil Test around the anal and the excretory Evsco); the lysate solution shrinks the micro- pores, respectively. filariae and new measurement standards are - D. repens microfilariae show only 1 required to differentiate species acid phosphatase activity spot local- Low sensitivity in cats ized around the anal pore. - Acantocheilonema spp. microfilariae show acid phosphatase activity Histochemical stain throughout the body.

One ml of venous blood collected in Peribáñez et al. (2001) have EDTA is injected into 10 ml of deion- described an alternative method using a ized water and centrifuged at 1500 commercial kit with similar results. rpm for 15 minutes. The supernatant is discarded and the sediment placed Advantage on a slide and air-dried. The smear is Very specific then fixed with absolute acetone, air dried, and covered with acid phos- Disadvantages phatase substrate. The substrate Costly, time consuming, need for a skilled needs to be either made fresh, as laboratory technician Laboratory diagnosis: Guideline 141

Fig. 1. A and B: Dirofilaria immitis: cephalic(x400) and caudal end (x1000) C and D: Dirofilaria repens: cephalic (x400) and caudal end (x1000) E: cephalic end of Acantocheilonema reconditum (detail showing cephalic hook) (x1000)

ELISA and immunochromatographic the efficacy of an adulticide therapy. tests for adult female heartworm circu- However, the newest tests are quite sen- lating antigens sitive and for definitive diagnosis to confirm the success of the adulticide therapy Several ELISA and immunochro- dogs have to be retested 5 and 9 months matographic kits are commercially later. If the test at 5 months is negative, available to detect the presence of adult testing at 9 months can be avoided. female circulating antigens in serum, Because unisex infections consisting plasma and whole blood of dogs and of only male worms or symptomatic cats. Most are very specific, quite sensi- immature infections are not infrequent tive, rapid and easy to be performed. in cats, none of the presently available Most are in-clinic test kits for single antigen tests can be relied upon to rule diagnosis but ELISA plates for multi- out heartworm disease in cats. In cats test are also available. Manufacturers with heavy infections, detectable anti- claim for a positive result when 1 adult genemia develops at about 5.5 to 8 female worm is infecting the animal, months post infection. though many factors can affect the sensitivity of the tests such as age of To note that semi-quantitative and labo- worms, number of female worms ver- ratory ELISA tests (not immunochro- sus number of males and the dog size, matographic tests) have a direct, but and reliable and reproducible results imprecise, relationship to the adult can be obtained from 2-3 or more adult female worm burden. The utility of the female worms. Male worms are not ELISAs for assessing the degree of para- detectable by antigen tests. In dogs, sitism can be limited by the transient detectable antigenemia develops about increase in antigenemia as a conse- 5 to 6.5 months post infection. quence of recent worm death. Because the clearance of antigens is When animals are monthly treated quite rapid after the death of worms, with preventive drugs during heartworm these techniques can be used to assess transmission season, re-testing each year 142 Laboratory diagnosis: Guideline

before starting the preventive treatment Advantage in the following season is advisable. Very sensitive If the chemoprophylactic treatment is Able to detect the cat exposure to heartworm performed with a sustained release infection drug injection (only for dogs), periodic Suitable to asses the infection risk in cats testing (each 2 or 3 years) will ensure and for epidemiological survey there have been no efficacy breaks (Nelson et al., 2005b). Disadvantages Advantage Costly Very specific and sensitive for heartworm Not fully specific diagnosis [gold standard: when positive, the Difficult to be interpreted test is the definitive prove of heartworm infection in dogs and cats] PCR Disadvantages Costly, not available for other filarial infec- PCR (polymerase chain reaction) is a tions sensitive and accurate tool to discriminate microfilariae from the different Antibody tests filarial worms able to infect dogs and cats. Its use is advisable in case of Several antibody test kits are avail- morphological abnormalities of micro- able for the diagnosis of feline heart- filariae, not infrequent in dogs treated worm disease. These tests cannot be incorrectly with preventive drugs or used in dogs. when multiple infections with more Antibody tests have the advantage than one species of filarial worm to being able to detect the exposure makes difficult to differentiate microfi- of cats to the infection of both male lariae (Favia et al., 1996; Mar et al., and female adult worms and larvae, 2002; Casiraghi et al., 2006; Rishniw and the immune response in et al., 2006). detectable as early as 2 months post infection. However, their interpretation is complicate because positive results can be found both in aborted Advantage infections (developing larvae are Very sensitive, specific and destroyed by the host immune accurate response and will not be able to Able to discriminate all the develop to adult worms) and in filarial species patent infections. Furthermore, no proved data is available on whether the antibody level will decrease over Disadvantages the expected two-to-three year lifes- Costly, time consuming pan of an adult worm (Nelson et al., Need for specialized laboratory and skilled 2005a). technicians Laboratory diagnosis: Guideline 143

Guideline for the diagnosis of filarial infections in dogs.

Mf Knott Ag test Interpretation Comment Positive Positive Definitive diagnosis of HW ThR can help to manage the disease Clinical signs and the results of semiquantitative ELISA tests can help in discriminating between low and high risk of thromboembolism

Positive Negative Definitive diagnosis of HW: Normal ThR patterns very low HW burden if D. Low/very low risk of thromboembolic complications immitis Mf are present Histochemical stain or PCR can be used to differen- Filarial infection caused by tiate Mf other species than D. immitis

Negative Positive Definitive diagnosis of HW Dogs were previously treated incorrectly with pre- occult infection ventive drugs or with macrocyclic lactone injectable formulations ThR and ECHO can help to manage the disease Clinical signs and the results of semiquantitative ELISA tests can help in discriminating between low and high risk of thromboembolism Mf: microfilariae HW: heartworm TR: thoracic radiography ECHO: echocardiography

Guidelines for the diagnosis of filarial infections in cats.

Mf Knott Ag test Ab test Interpretation Comment

Positive Positive Positive Definitive diagnosis of HW ThR and ECHO can help to manage the disease Positive Negative Positive Definitive diagnosis of HW if ThR and ECHO can help to manage D. immitis Mf are present the disease Positive Negative Negative Filarial infection due to other Histochemical stain or PCR for differ- species than D. immitis entiate Mf and give specific diagnosis Negative Positive Positive Definitive diagnosis of HW ThR and ECHO can help to manage the disease Negative Negative Positive Low adult female worm burden ThR and ECHO are useful to confirm Immature worms the suspicion of HW infection Aborted infection Re-testing after 4-8 months can help Immune response to previous to confirm the suspicion patent infection, but worms are already died Mf: microfilariae HW: heartworm TR: thoracic radiography ECHO: echocardiography 144 Laboratory diagnosis: Guideline

References Buzhardt LF, Graham W, Longhofer SL, Guerrero J, Robertson-Plouch C, Paul A, 2005a. Casiraghi M, Mazzocchi C, Mortarino M, Ottina 2005 Guidelines for the diagnosis, prevention E, Genchi C, 2006. A simple molecular method and management of heartworm (Dirofilaria for discrimination common filarial nematodes immitis) infection in cats. Vet Parasitol 133: of dogs (Canis familiaris). Vet Parasitol 141: 255-266. 368-372. Nelson CT, McCall JW, Rubin SB, Buzhardt LF, Chalifoux L, Hunt RD, 1971. Histochemical dif- Doiron DW, Graham W, Longhofer SL, Guerrero ferentiation of Dirofilaria immitis and J, Robertson-Plouch C, Paul A, 2005b. 2005 Dipetalonema reconditum. J Am Vet Med Assoc Guidelines for the diagnosis, prevention and 5: 601-605. management of heartworm (Dirofilaria immitis) Favia G, Lanfrancott, A, Della Torre A, Cancrini infection in dogs. Vet Parasitol 133: 267-275. G, Coluzzi M, 1996. Polymerase chain reaction- Peribáñez MA, Lucientes J, Arce S, Morales M, identification of Dirofilaria repens and Castillo JA, Gracia MJ, 2001. Histochemical dif- Dirofilaria immitis. Parasitology 113 (Pt. 6): ferentiation of Dirofilaria immitis, Dirofilaria 567-571. repens and Acanthocheilonema dracunculoides Mar PH, Yang IC, Chang GN, Fei AC, 2002. microfilariae by staining with a commercial kit, Specific polymerase chain reaction for differen- Leucognost-SP®. Vet Parasitol 102: 173-175. tial diagnosis of Dirofilaria immitis and Rishniw M, Barr SC, Simpson KW, Frongillo MF, Dipetalonema reconditum using primers Franz M, Dominguez Alpizar JL, 2006. derived from internal transcribed spacer region Discrimination between six species of canine 2 (ITS2). Vet Parasitol 106: 243-252. microfilariae by a single polymerase chain reac- Nelson CT, Doiron DW, McCall JW, Rubin SB, tion. Vet Parasitol 135: 303-314. 12 Epidemiology and prevention of Dirofilaria infections in dogs and cats

Claudio Genchi, Jorge Guerrero, John W. McCall, Luigi Venco Epidemiology and prevention 147

Heartworm infection (Dirofilaria Epidemiology immitis) More than 70 species of mosquitoes Dirofilaria immitis is a parasite that have been shown to be capable of can be potentially fatal to a variety of developing microfilariae (first stage animal species. Dirofilariosis, however, larvae) to the infective, third-stage lar- is a fully preventable disease due to the vae (L3), but fewer than a dozen of availability of highly effective preven- these species are believed to be major tive drugs that are safe, effective, con- vectors (Otto et al., 1981). Although venient and easy to administer. the susceptibility of different geograp- According to the recently published hical strains may vary, the likelihood of American Heartworm Society guide- the presence of at least one susceptible lines, all animals that are at risk for vector species in a geographic area that contracting the disease should routine- is conducive to the propagation of ly receive heartworm preventive med- mosquitoes is high, and once the ubi- ications (Nelson et al., 2005a,b). quitous heartworm parasite is introdu- Chemoprophylatic drugs for heart- ced into an area, its transmission is vir- worm infection fall into two basic tually insured. classes, the macrocyclic lactones or Many countries are now endemic for macrolides (avermectins and milbe- heartworm infection. Furthermore, in mycins) and diethylcarbamazine spite of efforts aimed at prevention and (DEC). In heartworm-endemic areas, control, particularly in dogs, infection puppies and kittens that are born dur- appears to be spreading into areas pre- ing the transmission season should be viously considered to be free of the given their first dose of macrocyclic lac- disease (Genchi et al., 2005). The pre- tones between 2 to 8 weeks of age and valence and distribution is better 6 to 8 weeks of age, respectively. The known for dogs, but gradually more required daily administration of DEC is information on the frequency of diag- inconvenient, more than one missed nosis of infection in cats is becoming dose can result in a breakdown in pro- available. It is now generally accepted tection and the overall prevention pro- that heartworm disease may occur in gram is difficult to manage. The use of cats in any area where dogs are infected DEC is fairly limited in the United (Genchi et al., 1992b, 1998; Guerrero States and the drug is no longer avail- et al., 1992b; McCall et al., 1994; able in Europe. Kramer and Genchi, 2002) but the geo- The present chapter will briefly graphical distribution and level of review current information on the epi- infection are less predictable in cats demiology of D. immitis infections in than in dogs. In highly endemic areas, dogs and cats and the use of macro- with sufficient rainfall, essentially every cyclic lactones in the prevention of unprotected dog becomes infected heartworm infection, as well as their (McTier et al., 1992a). In contrast to effect on other important gastrointesti- dogs, about 75% of cats can be infec- nal nematode parasites. ted experimentally with D. immitis L3 148 Epidemiology and prevention

(McCall et al., 1992). However, the be partially protective (Dillon et al., prevalence rate of natural infections in 1996; Prieto et al., 2001); however, cats is between 5% and 20% of that for studies to determine levels of suscepti- dogs in the same geographical area bility have not been performed. For (Ryan and Newcomb, 1995). indoor cats, it seems that even one Cats with naturally acquired infec- encounter with an infective vector may tions usually harbor fewer adult worms lead to the development of a large pro- than dogs. Because of their small body portion of transmitted larvae to the size and exaggerated pulmonary vascu- adult stage, causing severe illness lar and parenchymal response to infec- (Genchi et al., 1992a). It has recently tion, cats with low worm burden can be been reported that between 9 and 27 considered to be relatively heavily percent of cats were seropositive for D. infected in terms of parasite biomass immitis in northern Italy, 19 percent of (Genchi et al., 1998). Microfilaremia, which were apartment-dwelling cats when present, is low and transient, (Kramer and Genchi, 2002). even in cats with experimentally indu- Chemoprophylactic treatment, there- ced infections. Mosquitoes fed on fore, is a viable option for cats residing heartworm microfilaremic cats develop in any area where heartworm is consid- L3, which are capable of producing an ered endemic in dogs, even cats living infection in dogs (Donhaoe, 1975), but more sheltered lives. As suggested by circulating microfilariae are seldom Atkins (1997), it seems rational to rec- found in cats. Thus, cats generally ommend chemoprophylaxis for feline become infected via mosquitoes that heartworm infection, given that the dis- have fed on microfilaremic dogs. ease has a higher incidence than both It is not currently possible to deter- Feline Leukemia Virus (FeLV) and mine which cats are resistant to heart- Feline Immunodeficiency Virus (FIV), worm infection and which are suscepti- infections for which vaccination proto- ble and will permit the infection to cols are increasingly advocated. develop to the adult stage. Such a dis- Heartworm transmission to dogs and tinction between resistant and suscepti- cats is influenced by several well- ble animals would require constant known epidemiological factors, among monitoring at very high costs. the most important of which is envi- Furthermore, environmental meas- ronmental heat. Development of D. ures taken to reduce the risk of infec- immitis to L3 in mosquitoes occurs at a tion seem to be of little consequence in rate that is dependent on ambient tem- the cat. In fact, keeping animals perature, and development may not indoors, one of the most important occur at a threshold temperature of measures in reducing infection rates in about 14 °C (Fortin and Slocombe, dogs, seems to be ineffective in protect- 1981). The effects of heat on larval ing cats from disease. Outdoor cats and development is assumed to be cumula- strays who are seemingly exposed to tive and may be calculated in terms of high numbers of bites from infective degree-days above the developmental mosquitoes may be able to mount an threshold, or heartworm development effective immune response that could units (HDU). One model of heartworm Epidemiology and prevention 149 seasonality assumes a requirement of along the Ohio and Missouri Rivers. 130 HDU (°C) for complete develop- New foci have been detected in nort- ment and a maximum life expectancy of hern California and Oregon, and 30 days for common vector mosquitoes autochthonous infections in dogs in the (Slocombe et al., 1989). Using this states of Wyoming, Utah, Idaho, and laboratory-derived model, which requi- Washington have been documented in res numerous inherent assumptions recent years (Zimmerman et al., 1992). and was designed to study only the There is a high probability that the influence of macroenvironmental tem- introduction of the tree-hole breeding perature on the heartworm develop- mosquito, Aedes sierrensis, in Salt ment period, investigators have predic- Lake City, Utah, during the past deca- ted the seasonal limits of transmission de or so is associated with the esta- in Canada (Slocombe et al., 1995), the blishment of enzootic heartworm USA (Knight and Lok, 1995) and transmission in the area (Scoles and Europe (Genchi et al., 2005) and for- Dickson, 1995). The tiger mosquito, mulated recommendations for timing A. albopictus, a known vector of heart- of heartworm chemoprophylaxis and worm in Japan, has spread rapidly scheduling of diagnostic testing. While throughout much of the USA and these model-based predictions are aca- Europe since it was introduced from demically appealing, they ignore seve- Asia in 1985. It breeds mainly in piles ral potentially important factors, such of discarded tires and is spread within as influence of microclimate and the the country by movement of tires from unique biological habits and adapta- place to place. This mosquito readily tions of the numerous mosquito vec- feeds on dogs and other mammals, and tors, on larval development. laboratory strains have shown it to be an excellent host for D. immitis. Two recent surveys conducted by Prevalence Merial in cooperation with the American Heartworm Society showed North America that the number of canine heartworm cases diagnosed in the US were close to Heartworm infection in dogs has now a quarter of a million. The first survey been diagnosed in all of the 50 states of conducted in 2002 requesting diagnos- the USA. Although transmission of tic data for 2001 had the participation infection has not been clearly docu- of 15,366 clinics which reported diag- mented for Alaska, the disease is consi- nosing heartworm infections in dered to be endemic in all of the remai- 244,291 dogs. The second survey con- ning 49 states. Heartworm is enzootic ducted in 2005 reviewing data for 2004 along the Atlantic Seaboard and Gulf had the participation of 12,173 clinics Coast areas, with the southeastern sta- (out of a total of 25,000), which report- tes generally showing the highest pre- ed 250,000 cases of canine heartworm valence values. Infection continues to infections diagnosed that year be diagnosed at a high frequency in the (Guerrero et al., 2006). Interestingly, Mississippi River basin and in states 8,800 of the responders in the second 150 Epidemiology and prevention survey had also responded in the first of the parasite and a resulting local poc- survey. Analyzing the data obtained in ket of infection (Zimmerman et al., the clinics that participated in both sur- 1992). Hunters, in previous years, had veys the following was seen: number of transported hunting dogs from Texas to canine heartworm cases decreased in this area, setting up new foci of infection. 17 states, in 3 states the numbers were the same and in 30 states plus South America Washington, DC the number of cases increased. Nationally, the reported pos- The prevalence and spread of heart- itive cases of heartworm infections in worm infection in South America has dogs increased slightly in those clinics been recently reviewed by Labarthe reporting in both 2002 and 2005. and Guerrero (2005). Surveys indicate In a parallel study, the national preva- that heartworm is endemic in several lence of heartworm infections in dogs countries of South America. In Brazil, was performed by evaluating medical the overall prevalence of canine heart- records of more than 500 Banfield Pet worm infection in the state of Rio de Hospitals that see approximately 80,000 Janeiro was 21.3% (Guerrero et al., pets on a weekly basis. Data collected 1992a), with the highest rate for dogs from January 1st 2002 to December 31st in the northern beaches (49%), fol- 2005 was evaluated. Results of this lowed by dogs from the mountain study show that 1.46% of the 871,839 towns near the cities of Rio de Janeiro dogs examined tested positive for circu- (27.4%) and Niteroi (26.4%). The rate lating antigen of D. immitis. Based on in the suburbs of Rio de Janeiro was this data, the estimates of pet dogs in the around 33% (Labarte et al., 1992). USA and the proportion of them proba- Labarthe and co-workers (1997 a,b) bly on heartworm prevention, the inves- confirmed and extended these findings, tigators (Apotheker et al., 2006) esti- and also reported heartworm infection mated that 509,932 dogs in the USA in random-source cats in the city. Alves had heartworm infections, a figure that and co-workers (1999) found a preva- is almost exactly double the number of lence rate, as determined by necropsy cases reported in the Merial-AHS sur- of dogs, in the city of Recife in north- veys of 2002 and 2005. Keep in mind eastern Brazil of 2.3%. In the same that in the 2005 Merial-AHS survey state (Pernambuco), prevalence in close to half of the total number of dogs on Itamaracá Island was found to Companion animal clinics in the USA be as high as 43%. Labarthe (1997) responded to the survey. reviewed the literature on prevalence in In Canada, the overall canine prevalen- Brazil and found reports of prevalence ce rate is 0.24% (Slocombe, 1992). rates as high as 45% in the state of São Prevalence is higher (8.4%) in endemic Paulo. In Argentina, endemic areas areas of southwestern Ontario. The most have been identified, with prevalence in significant reports of heartworm infec- dogs ranging from 5.0% in the greater tions in British Columbia are from the Buenos Aires area to 34.2% in the Okanagan Valley area, which represents northeastern province of Formosa a classic instance of recent introduction (Guerrero et al., 1992a). A rate of Epidemiology and prevention 151

10.9% was recorded for Corrientes. overall heartworm prevalence in dogs More recently, Peteta and co-workers was found to be 7.5%, with the highest (1998) reported a prevalence, deter- rates (20-42%) observed for dogs from mined by microfilarial and antigen test- the Gulf Coast cities of Tuxpan, ing, of 13.6% for dogs in Villa La Nàta, Tampico, and Ciudad Madero. which is located near the Parana Delta and surrounded by the Lujan River and Australia Villanueva and LaRioja Channels in the Tigre district of the province of Buenos Heartworm infection is enzootic Aires. In Argentina the prevalence along the northern coastal areas of ranges from 0% to 71% (Vezzani et al., Western Australia and the eastern sta- 2006). In Venezuela, examination of tes of Queensland, New South Wales, canine blood samples submitted to the and Victoria, where prevalence rates School of Veterinary Medicine, Central generally mimic those for the southeas- University of Venezuela in Aragua, tern states of the USA. The prevalence revealed that 2.3% were positive for of heartworm infection in dogs in microfilariae of D. immitis (Perez and Sidney was as high as 30% in the late Arlett, 1998). Recent studies performed 1980s, and cats were also found to be in Lima, Peru, reported 4.35% of the infected (Kendall et al., 1991). More blood samples from 140 randomly recently, the rate for dogs in this area selected dogs were positive for circulat- was reported to be only 11.4% ing antigen of D. immitis. These sam- (Bidgood and Collins, 1996). ples were examined by the ELISA Snap 3Dx test (Gonzales, 2002). Asia and the South Pacific

Central America and the Caribbean Heartworm disease is well established in most of the Islands of the Pacific and Kozek and co-workers (1995) revie- in many countries of Asia, but survey wed the prevalence of canine filariae in results are not readily available for the Caribbean islands and conducted a every country. A prevalence of 11.3% thorough epidemiological survey in for dogs from the Fars province of Iran Puerto Rico. Prevalence values for has been reported (Jafari et al., 1996). heartworm infection in Puerto Rico Heartworm is enzootic on the islands ranged from 3.1% to 20.4%, with the of Japan, where the disease is well highest rate recorded for the city of known and prevalence is well docu- Ponce, on the southern coast. In Cuba, mented. A survey conducted on stray prevalence for Havana ranged from 7% dogs and cats in the Kanto region of to 19% and from 37% to 63% on the Japan in 1985 revealed a prevalence Isla de Juventud. For Curacão, it ran- rate of 59% for dogs and 2% for cats ged from 9% to 11% and was 53% for (Tanaka et al., 1985). More recently, the Grand Bahamas and 18% for the Roncalli and co-workers (1998) repor- Dominican Republic. ted that prevalence rates for feline In a survey covering 15 cities in heartworm infection in Japan ranges Mexico (Guerrero et al., 1992a), the from 0.5% to 9.5% in stray cats and 152 Epidemiology and prevention from 3.0% to 5.2% in house cats. A drugs. The disease has recently spread survey of German shepherds in five northward into the provinces of Friuli- areas of South Korea revealed an ove- Venezia-Giulia. Furthermore, the spre- rall prevalence of 28.3%, by an antigen ading of A. albopictus in Italy and the test (Lee et al., 1996). Prevalence was evidence that this mosquito species can highest in Hoengsong-gun (84.4%), act as a natural vector for D. immitis while Yechon-gun and Chungwon-gun can enhance the risk of transmission areas had rates of 20.0% and 14.3%, from animals to humans, considering respectively. None of the dogs in the the aggressive anthropophylic behavior Kimhae-shi and Kwanju areas was of the species (30-48 bites/hour) positive. Kuo and co-workers (1995) (Cancrini et al., 2003). reported a 53.8% prevalence for dogs The highest rates for dogs in Spain are in the Taipei province of Taiwan, and reported for the southern provinces of Wu and Fan (2003) an overall preva- Huelva (37%), Cadiz (12%), and lence of 57% in stray dogs in Taiwan. Badajoz (8%) (Guerrero et al., 1992 a). During the past few years, D. immitis Europe infection appears to be spreading into other regions of Cataluña (Catalonia). A The prevalence and spread of heart- recent survey of Barcelona showed that worm infection in Europe has been 12.8% of the dogs were infected. The comprehensively reviewed by Genchi Canary Islands of Tenerife (20.0%) and and co-workers (2005). The disease is Las Palmas (36.0%) are highly endemic, diagnosed mainly in the southern and a recent report suggests that about European countries of Spain, Italy, 59% of the dogs on Gran Canaria Island Portugal, and France, with scattered are infected with heartworms. In reports from Greece, Turkey, and some Portugal, infection is diagnosed mainly in Eastern European countries. An increa- the southern regions, with prevalence sing number of cases are now being values ranging from 12% (Algarve) to diagnosed in northern countries such as 30% (Island of Madeira). Although limi- Austria, Germany, and The Netherlands ted survey data are available, prevalence in dogs that were either imported from values for dogs range from 2% to 17% the Mediterranean area or had accom- for Slovenia, Bulgaria, Greece, and panied their owners to the area. One Turkey and up to 65% for Romania, and possible exception is a heartworm-posi- some of these areas are considered to be tive dog from the Canton of Tessin endemic. In Croatia, canine heartworm (Switzerland), which appears to have infection has been reported from Istrian acquired an autochthonous infection. pensula (about 16% prevalence) and the For Europe, the area of highest pre- sutest regions (Dubrovnik; about 8%) valence values for dogs and cats is (Zˇ ivicˇnjak et al., 2006). along the Po River Valley in northern Italy. The prevalence rate for cats in Africa this area is high (up to 24%), and the rate for dogs ranges from 35% to 80% Heartworm is found in dogs from in animals not treated with preventive various regions of Africa, but no infor- Epidemiology and prevention 153 mation is available regarding infections tion is to treat year-round (Nelson et in cats. Infection in dogs appears to be al., 2005b). All three drugs have a wide common throughout western Africa range of efficacy, which allows them to and in eastern parts, extending from be administered every 30 days. This the Republic of South Africa and provides a safeguard in the case of Mozambique (Schwan and Durand, omission or delay of a monthly treat- 2002) to the Republic of Sudan. ment, or when the chemoprophylactic Matola (1991) reported a prevalence of history cannot be verified. Ivermectin 10.2% for dogs in Tanzania. and milbemycin oxime have both been found to provide a high degree of protection when administered on a regular Chemoprophylactic treatment of basis, beginning 3 months after infec- canine heartworm disease tion. In fact, monthly treatment with ivermectin over a one-year period has Since the discovery of ivermectin and been shown to be >95 percent effective the initial description of activity against in preventing development of D. immi- developmental stages of D. immitis a tis larvae that were 4 months old; how- large number of publications have ever, under the same conditions, milbe- appeared reporting on their attributes. mycin oxime was only 41.5 to 49.3 per- Monthly oral administrations of iver- cent effective as a clinical prophylactic mectin at 6-12 mcg/kg, milbemycin agent (McCall et al., 1992). This oxime at 500-999 mcg/kg, or oral mox- retroactive or “reachback” effect has idectin at 3 mcg/kg provide effective not been reported for moxidectin, protection against heartworm infec- although products with this compound tions in dogs. do have a label claim for efficacy of 2 An ivermectin/pyrantel chewable for- months duration. This so-called “safe mulation is also available in Europe net” or “reachback” effect of macro- and in the United States to expand the cyclic lactones is very useful to com- indications to include treatment and pensate for missed or delayed treat- control of certain gastrointestinal para- ments, but should not be considered as sites, including Toxocara canis, justification to modify the recommend- Toxascaris leonina, Ancylostoma can- ed monthly interval for treatment. inum and A. braziliense. Milbemycin A newly developed avermectin, oxime at the recommended dose for selamectin, has recently been approved heartworm prophylaxis is also effective in Europe and in the United States for against T. canis, T. leonina, A. caninum the prevention of heartworm infections and Trichuris vulpis. Treatment with in dogs and cats. The drug was also any one of these macrolide compounds 100% effective in preventing the devel- should begin within a month after the opment of heartworm infection in dogs beginning of the transmission season when administered as a single topical and the final dose should be adminis- dose of 3 or 6 mg/kg given at 30 or 45 tered within one month after the end of days after inoculation with L3 or a sin- mosquito activity although, at present gle topical dose of 6 mg/kg given 60 time the most accepted recommenda- days PI (Clemence et al., 2000; McTier 154 Epidemiology and prevention et al., 2000; Dzimianski et al., 2001; ally clear any microfilariae from the McCall et al., 2001). The drug was also blood, so that even if an infection has safe when given to dogs and cats with become patent during the therapy, it existing heartworm infections. There would not be detected by screening for are several important characteristics microfilariae. However, some cases of unique to selamectin: the drug is given microfilaraemic dogs can be observed, as a topical formulation, thereby avoid- mainly when some treatments are inad- ing problems associated with oral vertent omitted towards the end of the administration. At the dose recom- transmission season. Testing should be mended for heartworm prevention in performed a minimum of 6 months after dogs and cats (6 mg/kg) selamectin is the last administration of one of the also effective in preventing and control- macrolide compounds. It is also advis- ling flea infestations (Ctenocephalides able to repeat testing at the beginning of felis) and for treating and controlling each transmission season before the ear mites (Otodectes cynotis) and biting start of treatment in order to exclude the lice (Trichodectes canis and Felicola possibility of infection due to poor subrostratus) in dogs and cats; sarcoptic owner compliance during the preceding mange in dogs (Sarcoptes scabiei); and season, or to verify that there was no hookworm (Ancylostoma spp) and pre-existing infection. Annual retesting roundworm (Toxocara spp) in both should be advisable even if chemopro- dogs and cats. phylactic treatment has been utilized An injectable formulation of mox- continuously (year-round) due mainly to idectin to be solely utilized by veterinar- the overall low owner compliance ians in dogs for prevention of heart- (Nelson et al., 2005b). worm infections is sold in Italy and Australia. The commercial formulation (moxidectin sustained release injectable Chemoprophylactic treatment of feline for dogs) has been approved for use in heartworm disease dogs six months of age and older, but not growing dogs. It is able to protect Although the general guidelines and dogs for an entire heartworm transmis- criteria for the use of preventive drugs sion season (Genchi et al., 2002) and in dogs may be generally applied to also treats infections with A. caninum. heartworm infection in cats, several specific features of feline heartworm disease must first be considered when Control testing choosing the correct prevention regimen. D. immitis infection in cats can Retesting for D. immitis infection cause an unpredictable, and often fatal, should always be carried out after the disease. Cats are known to be suscepti- first season of preventive treatment and ble hosts for heartworm, but are must include testing for circulating adult extremely resistant to infection (Genchi antigens as well as for microfilariae. The et al., 1992a; McCall et al., 1992). reason for this is that regular chemopro- Preventive treatment in the cat fol- phylactic use of the macrolides will usu- lows the same regimen established for Epidemiology and prevention 155 the dog, i.e., monthly dosing should Using the same dosage as in the dog, begin within one month from the start 6 mg/kg by topical application, of the transmission season and the last selamectin is also active against T. cati, dose should be given within one month A. tubaeforme, C. felis, Felicola subros- from the end of the risk period. tratus and O. cynotis (Guerrero et al., Ivermectin is marketed for use as a 2002). prophylactic agent in cats given month- As with the dog, pre-treatment test- ly at the dose of 24 mcg/kg (McTier et ing is advisable in the cat, utilizing both al., 1992b). This oral dosage is also an antibody and an antigen test for highly effective for treatment and con- cats, to verify the absence of D. immi- trol of A. tubaeforme and A. tis infection; however, it is not manda- braziliense. The oral chewable formula- tory. Retesting of cats should be con- tion of ivermectin has been found to be sidered after the first season of preven- 100 percent effective in preventing tive treatment and is advisable at the development of D. immitis larvae when beginning of each new transmission administered 30 or 45 days after chal- season before preventive therapy is to lenge with infective larvae. be initiated, unless the clinician has Furthermore, McCall and co-workers wisely chosen to utilize chemoprophy- (2000, unpublished data) demonstrat- laxis year-round. The longer life cycle ed that the recommended prophylactic of the parasite in the cat, as well as the dosage of ivermectin administered difficulty in accurately diagnosing monthly for a maximum of 12 months infection, increase the risk of inadver- was 66.5 percent effective in clearing tently treating an infected animal; how- 7-month-old D. immitis that had been ever, based on data obtained by McCall transplanted from an infected dog. A et al. (2000, unpublished data), month- dramatic decrease in circulating anti- ly administration of preventive drugs to gen levels also was detected in iver- cats infected with adult worms did not mectin-treated cats. These findings are precipitate any negative reactions. highly noteworthy for the cat veterinarian since adulticide treatments are not considered a viable option for cats. Subcutaneous filarial infection Milbemycin oxime is also known to be (Dirofilaria repens) effective in cats for heartworm prophylaxis at a rate of 2 mg/kg (Genchi et al., As for heartworm infections, subcuta- 2004). In Europe, milbemycin oxime is neous filarisosis can be safely and effec- available in combination with prazi- tively prevented by chemoprophylactic quantel (5 mg/kg) and the product is treatment of both in dogs and cats. efficacious for preventing heartworm Although the disease is less severe infection and for the treatment and con- than heartworm infection and dogs can trol of A. tubeforme, T. cati, Dipylidium show cutaneous disorders of different caninum, Taenia spp and Echinococcus severity, such as pruritus, dermal swe- multilocularis. Selamectin is also avail- lling and subcutanous nodules contai- able for use in cats as a heartworm pre- ning the parasites (Baneth et al., 2002; ventative. Bredal et al., 1998), very severe infec- 156 Epidemiology and prevention tions have been reported (Restani et mentally induced infections of D. immi- al., 1962; Mandelli and Mantovani, tis (McCall, 1998). Laboratory studies 1966), with allergic reactions probably have shown the ferret to be highly sus- due to microfilariae. However, the main ceptible, with infection and recovery concerrn about this Dirofilaria species rates similar to those achieved in the is its ability to cause infections in dog and higher than those seen in cats humans in Europe. The number of zoo- (Supakorndej et al., 1994). The life notic infections has dramatically increa- span of adult heartworms in ferrets is sed in the last few decades thought to cover the entire life time of (Pampiglione et al., 1995) and the the ferret. Heartworms are somewhat infection now can be included in the list smaller in ferrets than in dogs, with of emerging zoonoses (Pampiglione et mean lengths of male and female worms al., 2001). The infection is spreading in recovered from the heart and associated many southern (Giannetto et al., 1997) vessels of 118 mm and 144 mm, res- and eastern European countries pectively, 140 days after infection (Mazurkevich et al., 2004; Fok, 2007), (Supakorndej et al., 1994). probably as a consequence of the move- Microfilaremia is characteristically of ment of infected dogs and global war- low concentration and transient in natu- ming that increase the transmission sea- re, similar to that seen in heartworm- son. Recently, Zˇ ivicˇnjak et al. (2006) infected cats. A definitive diagnosis can reported a prevalence ranging from 7- be made from ELISA-based antigen 18% in dogs from several regions of tests, echocardiography, and angio- Croatia. Cats, as well as dogs can be graphy, but suggestive radiographic fin- infected, but the prevalence seems quite dings require additional supportive low (0.2-0.5%; Genchi et al., 1993). information to confirm a tentative diag- Ivermectin, selamectin and moxidec- nosis (McCall, 1998; Sasai et al., 2000). tin (both tablets and subcutaneous Clinical signs upon presentation inclu- injectable) have been found to be effec- de lethargy, inappetence, exercise into- tive in preventing this subcutaneous lerance, pleura effusion, cyanosis and infection in dogs naturally exposed to dyspnea (Antinoff, 2001). infective mosquitoes, by treating Supakorndej and co-workers (2001) monthly (oral formulations) or once a found that adulticide treatment of year (moxidectin injectable formula- infected ferrets with melarsomine dihy- tion) and at the same doses that are drochloride enhanced cardiomegaly effective against D. immitis and alveolar infiltrates and increased (Marconcini et al., 1993; Genchi et al., the severity of interstitial disease. The 2002c; Rossi et al., 2002, 2004). drug at the dosage of 3.25 mg/kg was equally effective (80.6-83.3%) when given twice, 24 hours apart or one Domestic ferrets injection followed one month later by 2 injections, 24 hours apart, but several The domestic ferret (Mustela putorius animals in the treated and control furo) has been reported to be suscepti- groups died during the study. ble to naturally acquired and experi- Prevention has been shown to be Epidemiology and prevention 157 effective with currently used canine 6 months to exclude a possible pre- prophylactic compounds such as patent infection. monthly tretament with ivermectin at 6 mcg/kg (McCall, 1998) or cat chewa- 3. Retesting should always be carried ble ivermectin formulation (24 mcg/kg) out after the first season of preven- (Kemmerer, 1998), but effective treat- tive treatment and must include tes- ment of adult heartworms in ferrets has ting for circulating antigens as well not yet been confirmed by controlled as for microfilariae. It is also advi- studies. There is currently no approved sable to repeat testing at the begin- drug for prevention or treatment of D. ning of each transmission season, immitis in ferrets. before the start of treatment in order to exclude possible infections due to poor owner compliance or to Guideline for the chemoprophylactic verify that there was no pre-existing treatment of Dirofilaria infections in infection. In endemic areas where dogs year-round treatments are utilizad, yearly testing is recommended. 1. Test the dog for circulating microfi- lae (D. immitis and D. repens) and 4. Treatment must be given montly at adult female antigens (D. immitis) the recommended dosage, but for when the preventive treatment is moxidectin in the injectable formula- administered for the first time. tion, one injection is able to protect Testing cats is not necessary but it is dogs against Dirofilaria infection for advised; drugs are safe even in cats at least six months. The commercial with patent infection. formulations are sold at dosages that are able to cover different ranges of 2. When a 1 year-old dog with an body weight. The minimum effective unknown history, living in an ende- dosage of macrocyclic lactones for mic area (risk area) is prophylacti- prevention of Dirofilaria infection in cally treated for the first time, check dogs and cats and the indications for microfilariae and antigen before against other parasites are shown in starting treatment and recheck after the table below.

ML Presentation Species Dose Indications Minimum age of treatment

IVM Tablets/chewables Dog 6 mcg/kg Di; Dr 6 weeks Chewables Cat 24 mcg/kg Di, At, Ab 6 weeks IVM/PYR Chewables Dog 6 mcg/kg Dog: Di, Dr, Tc, 6 weeks Tl, Ac, Us

MBO Flavour tablets Dog 0.5 mcg/kg Di, Tc, Tl, Ac, Tv 2 weeks or 0.5 kg MBO/PZQ Tablets Dog 0.5 mcg/kg Di, Tc, Tl, Ac, Tv, 6 weeks Dc, Tae, Eg,Ms Cat 2 mg/kg Di, Tct, At, Dc, 6 weeks Tae, Em MBO/LFN Tablets Dog 0.5 mcg/kg Di, Tc, Ac, Tv, Cf 2 weeks

continued 158 Epidemiology and prevention

ML Presentation Species Dose Indications Minimum age of treatment

MOX Tablets Dog 3 mcg/kg Di, Dr 6 weeks Injectable 0.17 mg/kg Di, Dr, Ac adult

SLM Topical Dog 6 mg/kg Di, Dr, Tc, Cf, Ss, 6 weeks Oc, Trc Cat 6 mg/kg Di, Tct, At, Cf, 6 weeks Oc, Fs

VM: ivermectin Di: Dirofilaria immitis; Dr: D. repens; Ac: Ancylostoma caninum; PYR: pyrantel pamoate At: A. tubaeforme; Ab: A. braziliense, Tc: Toxocara canis; Tct: T. cati; MBO: milbemycine oxime Us: Uncinaria stenocephala; Tv: Trichuris vulpis; Dc: Dipylidium caninum; MOX: moxidectin Ms: Mesocestoides sp; Tae: Taenia spp, Eg: Echinococcus granulosus; SLM: selamectin Em: Echinococcus multilocularis; Cf: Ctenocephalides felis; Trc: Trichodectes canis; Fs: Felicola subrostrata; Oc: Otodectes cynotis; Ss: Sarcoptes scabiei Note: All of the compounds are intended for monthly administration except moxidectin injectable.

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13 Safety and efficacy of selamectin in dogs with Dirofilaria immitis infection

John W. McCall, Michael T. Dzimianski, W.L. Steffens, Nonglak Supakorndej, Prasit Supakorndej, Abdelmoneim E. Mansour, Mary B. Ard, Scott D. McCall, Richard Hack, Dan Domingo Safety and efficacy of selamectin 165

In both laboratory (McTier et al., monthly administration to dogs with 1998, 2000) and field studies patent D. immitis infections (study B); (Clemence et al., 2000; Boy et al., and the “reachback” or clinical prophy- 2000), selamectin has proven to be lactic effect in dogs inoculated with highly effective in the prevention of heartworm L3 3 months prior to initia- Dirofilaria immitis (heartworm) infection of long-term monthly prophylaxis tions in dogs and cats following (study C). monthly prophylactic administrations at a minimum dose rate of 6 mg/kg. In studies in which administration Materials and methods occurred 45 days (simulated delayed treatment) or 60 days (simulated Treatments delayed or missed treatment) after inoculation with infective third-stage Selamectin in the commercial formu- larvae (L3), a single treatment with lationa was administered as a unit dose selamectin was completely effective to provide a minimum dose rate of 6 (McTier et al., 2000). It has also been mg/kg of body weight. The placebo demonstrated that no adverse effects (vehicle only) was administered at the are likely if selamectin is inadvertently rate of 0.05 ml/kg of body weight to administered to animals with existing provide an equivalent volume. infections of adult D. immitis Treatments were applied to the skin (Novotny et al., 2000) In heartworm at a single site on each dog’s back at the endemic areas, there are several base of the neck and cranial to the potential scenarios for compliance scapulae. Treatments were adminis- failure in heartworm prevention, tered at 30-day intervals beginning on which a clinician may encounter. study day 0. These include dogs that have been exposed to infection for various peri- Animals ods prior to initiation of their heartworm prophylaxis, situations of Equal numbers of colony-bred male missed monthly dosing, and circum- and female Beagles were used. The ini- stances in which dogs with patent tial age and weight ranges for the dogs infections and various degrees of were 5 to 7 months and 6.45 to 15.4 microfilaremia are presented for treat- kg, respectively. Dogs were housed ment. The series of studies presented individually in mosquito-proof indoor here was designed to assess the per- pens inside purpose-built accommoda- formance of selamectin in 3 situations, tions, with controlled temperature and which hitherto had not been investi- ventilation systems. They were fed once gated. The studies included an assess- daily with an appropriate quantity of ment of the safety and efficacy of maintenance diet and water was sup- selamectin in heartworm-positive dogs plied ad libitum. Animals were main- with high microfilaremia (study A); the microfilaricidal and adulticidal a Stronghold®/Revolution®, Pfizer Inc., New York, efficacy of selamectin after long-term New York, USA. 166 Safety and efficacy of selamectin tained with due regard for their welfare heart and lungs (and liver in study C). and in accordance with applicable leg- The pre-cava, right atrium, right ventri- islation and guidelines. cle, and pulmonary arteries (and caudal vena cava as far as the liver in study C) were dissected and the worms removed, Parasite challenge recorded as male or female, recorded as alive or dead, and fixed in formalin. For dogs in studies A and B, mature adult D. immitis worms aged approxi- Design mately 10 to12 months were obtained from donor animals having experimentally induced infections. Twenty worms Study A. Safety and efficacy in dogs (10 males, 10 females) were intro- with high microfilarial counts duced surgically into the cranial vena cava via the external jugular vein while Eighteen dogs (9 males, 9 females) the dogs were under general anaesthe- that tested negative for heartworm anti- sia. For study C, D. immitis L3 were gen and microfilariae were inoculated obtained from experimentally infected with 20 adult D. immitis on day –222. Aedes agypti, and each dog was inocu- Sixteen dogs (8 males, 8 females) with lated with approximately 50 L3 by the highest D. immitis microfilarial subcutaneous injection into the counts (mean, >10,000 mf/ml on day inguinal region 3 months prior to initi- –5) were selected, weighed, and ran- ation of treatment. domly allocated to treatment with selamectin or a placebo. Treatments Dirofilaria immitis antigen testing were administered on days 0, 30, and and microfilarial counting 60. Clinical observations were made prior to each treatment, at 4 and 8 hours Dogs were tested for the presence of after treatment, and once daily for the D. immitis antigen by using a commer- next 7 days. General health observations cial test kitb. A modified Knott concen- were made daily on all other days tration technique was used to count throughout the study. Blood was collect- microfilariae. ed by venipuncture for microfilarial counting on the day of treatment (days Adult worm counts 0, 30, and 60) and on days 1, 7, and 14 after each of the 3 treatments. After euthanasia, dogs in studies B and C were necropsied for recovery of adult D. immitis. The pleural and peritoneal Study B. Efficacy of long-term treat- cavities were examined for adult worms, ment against adult D. immitis and and the cranial and caudal vena cavaee circulating microfilariae were clamped before removal of the Eighteen dogs (9 males, 9 females), b DiroChek® Canine Heartworm Antigen Test Kit, with negative results for blood microfi- Synbiotics Corporation, San Diego, California, USA. lariae and heartworm antigen on day Safety and efficacy of selamectin 167

32, were each inoculated with 20 adult each treatment, and then at 4, 8, and 24 D. immitis worms on day 30. On day hours after treatment. General health 3, 16 dogs (8 males, 8 females) with observations were made daily on all the highest levels of microfilariae and other days throughout the study. Blood antigen were selected, weighed, and samples for microfilarial counts and randomly allocated to treatment with antigen tests were collected at approxi- selamectin or a placebo. A total of 18 mately 30-day intervals and the dogs monthly treatments were administered were euthanized on day 363 (15 months on days 0, 31, 60, 90, 122, 152, 182, after inoculation) for necropsy and 213, 243, 273, 304, 334, 364, 395, recovery of adult D. immitis. 425, 455, 486, and 516. Clinical observations were made prior to each treat- Data analysis ment, and then within 10 minutes, and at 2, 4, 8, and 24 hours after treatment. Parasite counts were log transformed General health observations were made {ln(x+1)} prior to analysis by using a daily on all other days throughout the repeated- measures model. A priori study. Blood was collected by venipunc- contrasts among least squares means of ture for microfilarial counting and anti- the log transformed data were used to gen testing approximately every 30 assess the treatment differences on days. All dogs were euthanized on day each counting day at the 5% level of 546 for necropsy and recovery of adult significance (P 0.05). Geometric D. immitis. mean parasite counts for each treatment were calculated from least squares means of the log transformed Study C. Long-term monthly prophy- data, and these means were used to laxis beginning at 3 months after estimate percentage reductions in para- heartworm inoculation (“Reachback” site burden at each time point for those efficacy) animals treated with selamectin, compared with those receiving the placebo, Seventeen dogs that had tested nega- using the following formula: tive for microfilariae and D. immitis (Geometric mean count for placebo) – antigen on day –92 were each inoculat- (geometric mean count for ed with approximately 50 D. immitis L3 selamectin)/(Geometric mean count on day –90. On day –3, blood samples for placebo) x 100 = % reduction were obtained for microfilariae counting and antigen testing. On day –1, 16 dogs were selected and allocated to treatment Results with selamectin or a placebo (8 dogs/treatment). Monthly treatments were administered beginning on day 0 Study A. Safety and efficacy in dogs (90 days after infection), and continuing with high microfilarial counts thereafter for 11 months (total of 12 monthly treatments). Clinical observa- There were no adverse clinical obser- tions were made immediately prior to vations related either directly to treat- 168 Safety and efficacy of selamectin ment or to the effects of treatment on Study B. Efficacy of long-term treat- parasites within heartworm-positive ment against adult D. immitis and dogs during the study. Geometric mean circulating microfilariae microfilarial counts for placebo-treated dogs on days 0, 1, 30, 60, and 74 were At necropsy, the geometric mean 9,882, 7,117, 3,780, 6,322, and 2,523 adult D. immitis counts were 10 for mf/ml, respectively, compared with selamectin-treated dogs and 15 for selamectin-treated dogs, which had placebo-treated dogs (controls). There counts on the same days of 11,568, was no significant difference between 10,462, 459, 606, and 223 mf/ml, the treatments, even though the reduc- respectively (Table 1). Thus, the micro- tion in worm count for the selamectin- filarial count for the selamectin-treated treated dogs was 36.4%. All of the dogs gradually decreased during the live worms recovered from the month after the first treatment (87.5% selamectin-treated dogs and placebo- reduction on day 30), compared with treated dogs were normal in motility. the placebo-treated controls, and gener- Only 1 live worm recovered from the ally remained at approximately that control dogs was abnormal in appear- value for the remainder of the study. The ance (i.e., 1 female worm was opaque microfilarial counts were significantly (P white), whereas 6 of 8 selamectin- 0.05) lower for selamectin-treated treated dogs had abnormal appearing dogs, compared with placebo-treated worms. Twenty-one of the 51 worms dogs, on days 30, 31, 67, and 74. from selamectin-treated dogs were

Table 1. Geometric mean counts of Dirofilaria immitis microfilariae following monthly treatment of dogs with an initial high microfilaremia. Treatment began 222 days after experimentally acquired infection with adult worms.

Study A

Geometric mean D. immitis microfilarial counts (mf/ml)

Treatment* Day 0 Day 1 Day 7 Day 14 Day 30 Day 31 Day 37 Day 44 Day 60 Day 61 Day 67 Day 74

Placebo 9,882 7,117 6,776 917 3,780 3,653 4,401 4,938 6,322 5,228 3,878 2,523 (n=8)

Selamectin 11,568 10,462 2,620 432 459 496 654 796 606 458 336 223 (n=8)

% reduction** 0.0% 0.0% 61.3% 52.9% 87.9% 86.4% 85.1% 83.9% 90.4% 91.2% 91.3% 91.2%

P-value† NS NS NS NS 0.05 0.05 NS NS NS NS 0.05 0.05

* Dogs were treated on days 0, 30, and 60. ** For selamectin treatment compared with placebo. † Significance of difference between treatments. NS = Not significant. Safety and efficacy of selamectin 169 abnormal (i.e., opaque white with or no increase form day 3 to day 28 (348 without a darkened anterior end). All mf/ml), and then declined to reach 0 dogs in both treatments remained pos- at day 180. From day 180 through the itive for heartworm antigen through- end of the study (day 544, after 18 out the study, except for 1 selamectin- monthly treatments), the count treated dog that tested negative on 1 remained 0 except on days 271, 301, occasion (day 483). and 332 when there was a mean count On day –3 (prior to the first treat- of 1 mf/ml. From day 59 to the end of ment), the mean microfilarial counts the study, selamectin-treated dogs had were 382 and 315 mf/ml for the place- significantly (P 0.05) fewer microfi- bo- and selamectin-treatment groups, lariae than dogs treated with the respectively (Fig. 1). For dogs treated placebo. The reductions in geometric with the placebo, the count increased mean microfilarial count for dogs to >1,000 mf/ml on day 28 (1 month treated with selamectin, compared after the first dose) and to >3,000 with those treated with the placebo, mf/ml by day 59. The count was were 66.9% on day 28, 95.8% on day >10,000 mf/ml from day 119 through 59, 99.7% on day 90, 99.9% on day the end of the study, except on days 119, and approximately 100% from 362 (12 months) and 392 when counts day 150 to study completion. were 7,284 and 5,207 mf/ml respec- No adverse reactions attributed to tively. For dogs treated with selamectin, selamectin treatment were observed in the mean microfilarial count showed this study.

Study B Microfilaria counts (mf/ml)

Days

* Treatment differences were significant (P 0.05) from day 59 through completion (day 544).

Fig. 1. Geometric mean counts of Dirofilaria immitis microfilariae in dogs treated monthly, beginning 30 days after experimentally induced infection with adult worms. 170 Safety and efficacy of selamectin

Table 2. Geometric mean counts of adult D. immitis at necropsy following monthly treatment that began 90 days after inoculation with L3 in dogs.

Study C

Geometric mean counts of adult D. immitis worms recovered at necropsy on day 363

Treatment* Live worms Dead worms

Placebo 19.8 0.4 (n=8) Selamectin 0.3 0.0 (n=8) % reduction** 98.5% 100%

P-value† 0.05 NS * Dogs were treated on days 0, 30, 60, 90, 120,150, 180, 210, 240, 270, 300, and 330. ** For selamectin treatment compared with placebo. † Significance of difference between treatments. NS = Not significant.

Study C. Long-term monthly prophy- for selamectin-treated dogs, the percent- laxis beginning at 3 months after age never exceeded 38% and decreased heartworm inoculation (”Reachback” over time, with 12.5% of dogs antigen- efficacy) positive on day 361. Microfilarial counts were negative for all 16 dogs until day At necropsy (15 months after inocu- 118, and then only dogs treated with the lation), the geometric mean counts of placebo had positive counts. The live adult D. immitis from placebo- and selamectin-treated dogs remained nega- selamectin-treated dogs were 19.8 and tive throughout the study (Table 3). 0.3, respectively (Table 2). There was a significant (P 0.05) difference between the treatments, and the reduc- Discussion tion in the heartworm burden for selamectin-treated animals, compared Results of the first study presented with that for the control group, was here (study A) found that no adverse 98.5%. Dead heartworms were recov- effects were observed when selamectin ered at necropsy from the placebo-treat- was administered monthly for 3 ed dogs only, but there was no signifi- months to dogs with high microfi- cant difference between treatments for laremia (mean, >10,000 mf/ml) prior the number of dead heartworms. Adult to the first application. Furthermore, D. immitis antigen tests were negative just 3 treatments with selamectin at for all 16 dogs (both placebo- and monthly intervals was shown to have a selamectin-treated) from day –94 until substantial microfilaricidal effect, as day 57 (Fig. 2). From day 118 through demonstrated by reductions in microfi- day 361, the percentage of antigen-posi- laria count of >90%, compared with tive dogs for the placebo-treatment those for the control group (study A). group remained above 85%, whereas The microfilaricidal activity was con- Safety and efficacy of selamectin 171

Study C Dogs with positive antigen test (%)

Days

Fig. 2. Percentages of dogs positive for D. immitis antigen in dogs treated monthly, beginning 90 days after inoculation with (L3) larvae. firmed in dogs with lower initial levels administration (Knigth, 1998). Such of microfilariae (study B), where a reactions following the rapid death of reduction of 95% in microfilarial large numbers of microfilariae can counts was observed by the third occur with microfilaremias of at least month after commencing monthly 5,000 mf/ml, but are more usually treatment and 100% by the sixth associated with burdens greater than month. It is generally considered that if 10,000 mf/ml. Reactions include microfilaremic dogs are treated with an lethargy, inappetance, ptyalism, vomit- effective microfilaricide, there is an ing, tachycardia, pallor, and acute cir- increased risk of adverse effects occur- culatory collapse (Knigth, 1998). From ring during the first 4 to 8 hours after previous studies with other macrolides,

Table 3. Geometric mean counts of D. immitis microfilariae following monthly treatment that began 90 days after inoculation with L3 in dogs.

Study C Geometric mean D. immitis microfilarial counts (mf/ml)

Treatment* Day 28 Day 57 Day 88 Day 118 Day 148 Day 179 Day 209 Day 239 Day 270 Day 300 Day 330 Day 361 Placebo 0 0 0 2 80 986 1,205 3,325 3,353 13,131 2,554 2,219 (n=8) Selamectin 0 0 0 0 0 0 0 0 0 0 0 0 (n=8) % - - - 100% 100% 100% 100% 100% 100% 100% 100% 100% reduction** * Dogs were treated on days 0, 30, 60, 90, 120,150, 180, 210, 240, 270, 300, and 330. ** For selamectin treatment compared with placebo. 172 Safety and efficacy of selamectin notably ivermectin (20-50 mg/kg, ie, ment of a prophylactic regimen. higher than prophylactic dosages) and However, if for some reason this is milbemycin oxime, (prophylactic omitted, the majority of infected ami- dosage) treatment-induced reductions crofilaremic dogs should be identifiable in the levels of circulating microfilariae by using an adult antigen test. in dogs with patent infections have been It is not known at present whether demonstrated (Courtney et al., 1998). microfilarial counts will increase fol- Milbemycin oxime causes a more rapid lowing cessation of monthly adminis- reduction in microfilaremia than iver- tration of selamectin, as has been mectin (Courtney et al., 1998). Thus, it reported for milbemycin and iver- is possible to see adverse effects due to mectin (Courtney et al., 1998). The dead microfilariae, particularly follow- other macrolides have been shown to ing the use of milbemycin oxime (pro- effect embryogenesis in female heart- phylactic/microfilaricidal dosage), and worms (Lok and Knight, 1995); how- it is recommended that microfilaremic ever, at this time, the effects of dogs be carefully monitored for at least selamectin on adult worms, particular- 8 hours after treatment with microfila- ly female worms, have not been fully ricidal dosages of these drugs (Knigth, characterized. 1998). It has been shown previously Historically, as a class of com- that selamectin can be safely adminis- pounds, the macrolides have not been tered to dogs with mature adult heart- considered to have activity against worms and low levels of circulating mature adult heartworms. In study B, microfilariae (Novotny et al., 2000). when treatment was initiated against The efficacy of selamectin in gradual- mature adult heartworms (11 months ly reducing levels of microfilariae (90- old) and treatment was administered 100%) over time offers benefits in monthly for 18 consecutive months, terms of reducing transmission poten- there were 36.4% fewer adult heart- tial within a population. This effect worms present in selamectin-treated indicates the potential for selamectin dogs than in the controls, however, to be used following adulticidal thera- there was no statistical difference py to eliminate a residual microfi- between the worm counts in the 2 laremia in dogs with patent heartworm treatment groups. In addition, many infections. In common with other of the live selamectin-treated worms macrolides, such as ivermectin and were abnormal in appearance. It is milbemycin, when selamectin is possible that additional monthly treat- administered to dogs with patent infec- ments with selamectin would increase tions, the dogs may become amicrofi- the efficacy against adult heartworms. laremic resulting in “occult” infections. When ivermectin was administered Hence, in a dog with an unknown his- monthly for 16 consecutive months to tory or when it is possible that there dogs harbouring mature adult heart- has been exposure to infection, partic- worms, efficacy was 56.3% (McCall et ularly in young dogs or strays, it is al., 1998), and when treatment was important to establish the heartworm administered for 29 months, efficacy status of the dog prior to commence- improved to 94.9% (McCall et al., Safety and efficacy of selamectin 173

2001). It has also been suggested that Conclusions if an effective regimen could be devised, ivermectin might offer some Selamectin is microfilaricidal but kills clinical advantages in heartworm- microfilariae relatively slowly, even in infected dogs, because the slow killing dogs with high microfilaremia. This effect may reduce the risk of adverse gradual reduction of microfilariae vir- effects (particularly those associated tually eliminates, or at least greatly with pulmonary thromboembolism) minimizes potential adverse reactions from dead worms or worm fragments, due to large numbers of dead or dying compared with the risk of using adul- microfilariae. Selamectin appears to ticidal agents (melarsomine, thiac- have an adverse effect on mature adult etarsamide), which have a much more heartworms, but the effect is gradual rapid killing effect on adult heart- during long-term monthly prophylaxis. worms, which leads to the death of This slow killing effect on adult heart- some dogs (McCall et al., 1998). worms should minimize the potentially These studies demonstrated that severe adverse effects seen with a rapid selamectin, when administered as 12 kill of adult heartworms. In addition, monthly treatments beginning 3 selamectin has a 3-month reachback months after L3 infection (reachback), effect that is similar to the other is also highly effective in preventing the macrolide preventives. Thus, selamectin maturation of heartworms, with a has demonstrated exceptional safety in reduction of 98.5%. heartworm-positive dogs and, together Previously it has been shown that with robust reachback activity, increas- monthly prophylaxis for 13 months es the confidence with which this prod- with ivermectin or milbemycin, begin- uct can be used in the fight against ning 3 months after L3 infection, sig- heartworm disease in dogs. nificantly reduces the number of adult worms that develop by 97.7% and 96.8% for ivermectin and milbemycin, Acknowledgements respectively (McCall et al., 1996). It has also been found that monthly pro- The authors are most grateful to the laboratory staff and clinicians who were involved with these phylaxis for 12 months beginning 4 studies. Their expertise and commitment has months after L3–induced infection ensured the high quality of the data generated. leads to 95.1% and 41.4% reductions in maturation to adult worms for ivermectin and milbemycin, respectively, References indicating the potent reachback effects Boy MG, Six RH, Thomas CA, Novotny MJ, of ivermectin and the limits of reach- Smothers CD, Rowan TG, Jernigan AD, 2000. back activity for milbemycin. The Efficacy and safety of selamectin against fleas and heartworms in dogs and cats presented as effects of long-term monthly prophy- veterinary patients in North America. Vet lactic treatment with selamectin, com- Parasitol 91: 233-250. mencing 6 months after inoculation, is Clemence RG, Sarasola P, Genchi C, Smith DG, Shanks DJ, Jernigan AD, Rowan TG, 2000. currently under investigation, and Efficacy of selamectin in the prevention of adult results will be forthcoming. heartworm (Dirofilaria immitis) infection in 174 Safety and efficacy of selamectin

dogs in northern Italy. Vet Parasitol 91: 251-258. tions of ivermectin and pyrantel pamoate in Courtney CH, Zeng Q-Y, Maler MM, 1998. The dogs experimentally infected with heartworms. effect of chronic administration of milbemycin American Heartworm Society, 10thTriennial oxime and ivermectin on microfilaremias in Meeting, San Antonio, TX 20-22 April 2001. heartworm-infected dogs. In: Seward RL (ed). McCall JW, Ryan WG, Roberts RE, Dzimianski Recent Advances in Heartworm Disease: MT, 1998. Heartworm adulticidal activity of Symposium ’98. Am Heartworm Society, prophylactic doses of ivermectin (6 mg/kg) plus Batavia, IL, 193-199 pp. pyrantel administered monthly to dogs. In: Knight DH, 1998. 1999 Guidelines for the diag- Seward RL (ed). Recent Advances in nosis, prevention, and management of heart- Heartworm Disease: Symposium ’98. Am worm (Dirofilaria immitis) infection in dogs. In: Heartworm Society, Batavia, IL, 209-215 pp. Seward RL (ed). Recent Advances in McTier TL, McCall JW, Jernigan AD, Rowan TG, Heartworm Disease: Symposium ’98. Am Giles CJ, Bishop BF, Evans NA, Bruce CI, 1998. Heartworm Society, Batavia, IL, 257-264 pp. UK-124,114, a novel avermectin for the preven- Lok JB, Knight DH, 1995. Macrolide effects on tion of heartworms in dogs and cats. In: Seward reproductive function in male and female heart- RL (ed). Recent Advances in Heartworm worms. In: MD Soll and DH Knight (eds). Disease: Symposium ’98. Am Heartworm Proceeding of the American Heartworm Society, Batavia, IL, 187-192 pp. Symposium ‘95. American Heartworm Society, McTier TL, Shanks DJ, Watson P, McCall JW, Batavia, IL, 165-170 pp. Genchi C, Six RH, Thomas CA, Dickin SK, McCall JW, McTier TL, Ryan WG, Gross SJ, Soll Pengo G, Rowan TG, Jernigan AD, 2000. MD, 1996. Evaluation of ivermectin and milbe- Prevention of experimentally induced heart- mycin oxime efficacy against Dirofilaria immitis worm (Dirofilaria immitis) infections in dogs infections of three and four months duration in and cats with a single topical application of dogs. Am J Vet Res 57: 1189-1192. selamectin. Vet Parasitol 91: 259-268. McCall JW, Roberts RE, Supakorndej N, Mansour Novotny MJ, Krautmann MJ, Ehrhart JC, Godin A, Dzimianski MD, McCall SD, 2001. Further CS, Evans EI, McCall JW, Sun F, Rowan TG, evidence of clinical prophylactic (reachback) Jernigan AD, 2000. Safety of selamectin in dogs. and adulticidal activity of monthly administra- Vet Parasitol 91: 377-391. 14 Heartworm (Dirofilaria immitis) infection in dogs: current update in Spain

J.A. Montoya, M. Morales, M.C. Juste, J.A. Corbera Heartworm infection in dogs in Spain 177

Heartworm infection in dogs has heartworm endemic areas are at risk, been diagnosed around the globe. chemoprophylaxis is a priority. Relocation of infected, microfilaremic Furthermore, some evidence strongly dogs appears to be the most important suggests that by reducing the reservoir factor contributing to further dissemi- population through increasing the nation of the parasite. The ubiquitous number of dogs receiving chemopro- presence of one or more species of vec- phylaxis, a disproportionate decrease in tor competent mosquitoes makes trans- the prevalence of infection among mission possible wherever a reservoir unprotected dogs may occur relative to of infection and favourable climatic the percentage of additional dogs conditions co-exist. receiving chemoprophylaxis. This col- A climate that provides adequate lateral protection spreads the umbrella temperature and humidity to support a of chemoprophylaxis most effectively viable mosquito population, and also in communities where heartworm sustain sufficient heat to allow matura- prevalence and dog population density tion of ingested microfilariae to infec- are both relatively low. tive, third-stage larvae (L3) within this During the latest years several epi- intermediate host is a pivotal prerequi- demiological surveys on heartworm site for heartworm transmission to have been developed in many coun- occur. The length of the heartworm tries. The disease is distributed, mainly, transmission season in the temperate in temperate climates in the World. The latitudes is critically dependent on the more prevalent countries are in accumulation sufficient heat to incu- America, Africa, Polynesia, Australia bate larvae to the infective stage in the and Japan. In Europe the more preva- mosquito. The peak months for heart- lence (>10%) occurs in the worm transmission in the Northern Mediterranean countries. We must Hemisphere are July and August. point out that several studies in the Heartworm prevalence has increased north of Italy, south of France and dramatically during the latest years. Spain have elucidated prevalence over The disease has extended from tropical 20%. Therefore, Spain is an endemic or subtropical countries to others with area of Heartworm (Genchi et al., more temperate climate. The more fre- 2005). quent animal international travelling Inside Spain, the prevalence of the could contribute to dissemination of disease is higher in the southern coastal the parasite. Heartworm transmission areas. Also, Canary Islands are consid- is related to the dog’s life style, because ered an endemic area of the disease. outdoor permanence increases the pos- Specifically, in the Canaries, sited in sibility for mosquito contact. However, front of the north-western African breed or hair length has not been cor- coast, the available data has empha- related with the risk of heartworm sized the highest prevalence of the dis- infection. ease in Spain (Valladares et al., 1987; Heartworm infection is entirely pre- Guerrero et al., 1989). Veterinary ventable in dogs, despite their high sus- Surgeons in Canary Island include ceptibility. Since most dogs living in Heartworm Disease as the first differ- 178 Heartworm infection in dogs in Spain

Table 1. Prevalence of heartworm in Spain.

AUTONOMOUS COMMUNITY Prevalence References Province

ANDALUCIA 8.5% Guerrero et al., 1989 Cádiz 12% Guerrero et al., 1989; Ortega Mora et al., 1991 Cádiz-Málaga 5.5% Rojo-Vázquez et al., 1990 Córdoba 18.0% Anguiano et al., 1985 Córdoba 4% Guerrero et al., 1989 Huelva 36.7% Guerrero et al., 1989; Ortega Mora et al., 1991 Jaén 2.1% Guerrero et al., 1989 Málaga 2% Guerrero et al., 1989 Sevilla 1.5% Guerrero et al., 1989 ARAGON 4.3% Guerrero et al., 1989 Zaragoza 13.5% Castillo et al., 1989 Zaragoza 8 % Rodes, 2006 ASTURIAS 0 % Guerrero et al., 1989 CANTABRIA 0% Guerrero et al., 1989 CASTILLA-LA MANCHA 0% Guerrero et al., 1989 CASTILLA-LEON 0% Guerrero et al., 1989 Salamanca 12.0% Pérez-Sanchez et al., 1989 Salamanca (ribera Tormes) > 30% Pérez-Sanchez et al., 1989 CATALUÑA 2.17% Gutiérrez et al., 1995 CATALUÑA 0,6% Solano-Gallego et al., 2006 Barcelona 1.2% Rojo-Vázquez et al., 1990 Barcelona (bajo Llobregat) 12.8% Aranda et al., 1998 Barcelona 2% Solano-Gallego et al., 2006 Tarragona 0.85 Solano-Gallego et al., 2006 Tarragona (delta Ebro) 35.8 % Anguera, 1995 Tarragona (delta Ebro) 26 % Rodes, 2006 COMUNIDAD VALENCIANA Alicante 13% Guerrero et al., 1989 Alicante 1.6% Rojo-Vázquez et al., 1990 Alicante 18% Rodes, 2006 Elche 2.6% Cancrini et al., 2000 Valencia 4.1% Guerrero et al., 1989 EUSKADI 0% Guerrero et al., 1989 EXTREMADURA 6.7% Guerrero et al., 1989 Badajoz 8% Ortega Mora et al., 1991 GALICIA 0% Guerrero et al., 1989 ISLAS BALEARES 6.3% Guerrero et al., 1989 Mallorca 0.3% Solano-Gallego et al., 2006 Ibiza 39% Rodes, 2006 MADRID 2% Ortega Mora et al., 1988 Madrid 1.1% Guerrero et al., 1989 Madrid 1.9 Rojo-Vázquez et al., 1990 MURCIA 6.3% Guerrero et al., 1989 MURCIA 9% Rodes, 2006 NAVARRA 0% Guerrero et al., 1989 Heartworm infection in dogs in Spain 179

Table 2. Prevalence of heartworm disease in Canary Islands.

Canary Island Prevalence Reference

CANARIAS (total) 28% Guerrero et al., 1989 Gran Canaria 36% Guerrero et al., 1989 Gran Canaria 58.89% Montoya et al., 1998 Gran Canaria 23.87% Sosa et al., 2002 Tenerife 34.4% Valladares et al., 1987 Tenerife 20% Guerrero et al., 1989 Tenerife 23% Stenzenberger & Gothe, 1999 Tenerife 22.3% Morales et al., 2001 Tenerife 21% Montoya et al., 2006 ential diagnosis in dogs with cardiopul- of the disease to their clients. Also, it is monary signs. more frequent that clients demand the Previous studies in Spain (Guerrero use of the newest drugs for the treat- et al., 1989) showed that Huelva has ment of their infected pets; this increas- the highest prevalence in Spain ing demand is extending to rural areas (36.7%). This study determined that where population is not aware as far as the prevalence of Dirofilaria immitis in pet cares are concerned. Gran Canaria was 36%. Later studies revealed prevalence over 30% in References Salamanca (Muro et al., 1993) and the delta area of the river Ebro (Anguera, Anguera-Galiana M, 1995. La dirofilariosis canina en el Delta del Ebro. Medicina Veterinaria 12: 1995). More recently, the highest 242-246. prevalence (58%) of D. immitis in Anguiano A, Martínez-Cruz S, Gutiérrez PN, Spain has been described in Gran 1985. Epidemiología de la dirofilariasis canina en la provincia de Córdoba. IV Congreso Canaria (Montoya et al., 1998). The Nacional de Parasitología, Tenerife, Spain. prevalence in Gran Canaria is one of Aranda C, Panyella O, Eritja R, Castella J, 1998. the highest described in the World; Canine filariasis. Importance and transmission in the Baix Llobregat area, Barcelona (Spain). similar prevalence has been published Vet Parasitol 77(4): 267-275. in Cuba (Dumenigo et al., 1988) or Cancrini G, Allende E, Favia G, Bornay F, Antón Japan (Tanaka et al., 1985; Suenaga et F, Simón F, 2000. Canine dirofilariosis in two cities of southeastern Spain. Vet Parasitol 92: al., 1978; Hatsushika et al., 1992). 81-86. Otherwise, the highest prevalence ever Castillo JA, Lucientes J, Estévez C y Gortazar C, described (86%) is in Papua-New 1989. Epidemiología de la dirofilariosis en Zaragoza I. Estudio de la prevalencia en perro y Guinea (Hamir et al., 1986). zorro y su interrelación. VI Congreso Nacional y However, it is encouraging that the I Ibérico de Parasitología, Cáceres, Spain. prevalence of D. immitis in Gran Dumenigo BE, Espino AM, Bouza M, 1988. Prevalencia de filariasis caniana en la Isla de la Canaria is decreasing: more recent sur- Juventud. Revista de Salud Animal 10: 247- veys demonstrated prevalence under 250. 23% (Table 2). In our opinion, this Genchi C, Rinaldi L, Cascone C, Mortarino M, Cringoli G, 2005. Is heartworm disease really data could due to the increasing use of spreading in Europe? Vet Parasitol 133: 137- effectiveness drugs to prevent the dis- 148. ease, and mainly a very important vet- Guerrero J, Rojo F, Ródenas A, 1989. Estudio de la incidencia de la enfermedad del gusano del erinarian task in order to make aware corazón en la población canina española. 180 Heartworm infection in dogs in Spain

Medicina Veterinaria 6: 217-220. prevalence of canine filariasis in Spain. Prev Vet Gutiérrez J, Guerrero J, Ródenas A, Castella J, Med 11: 63-68. Muñoz E, Ferrer D, Florit F, 1995. Evolución de Pérez-Sanchez R, Goméz M, Encinas A, 1989. Dirofilaria immitis en Cataluña. Medicina Canine filariasis in Salamanca (northwest Veterinaria 12: 10. Spain). Ann Trop Med Parasitol 83: 143-150. Hamir AH and Onaga I, 1986. Canine spirocerco- Rodes D, 2006. Ultimos datos epidemiológicos sis and dirofilariasis infection in Papua New sobre filariosis canina. Argos: 52. Guinea. Aus Vet J 63: 98-99. Rojo-Vázquez FA, Valcárcel F, Guerrero J, Gómez Hatsushika R, Okino T, Shimizu M, Ohyama F, M, 1990. Prevalencia de la dirofilariosis canina 1992. The prevalence of dog heartworm en cuatro áreas geográficas de España. Medicina (Dirofilaria immitis) infection in stray dogs in Veterinaria 7: 297-305. Okayama, Japan. Kawasaki Medical Journal 18: Solano-Gallego L, Llull J, Osso M, Hegarty B, 75-83. Breitschwerdt E, 2006. A serological study of Montoya JA, Morales M, Ferrer O, Molina JM, exposure to arthropod-borne pathogens in dogs Corbera JA, 1998. The prevalence of Dirofilaria from northeastern Spain. Vet Res 37: 231-244. immitis in Gran Canaria, Canary Islands, Spain Sosa N, Montoya JA, Juste MC, 2002. Situación (1994-1996). Vet Parasitol 75: 221-226. epidemiológica actual de la dirofilariosis canina Montoya JA, Morales M, Juste MC, Bañares A, en la isla de Gran Canaria (Comunicación). I Simon F, Genchi C, 2006. Seroprevalence of Congreso Universitario de Ciencias Veterinarias canine heartworm disease (Dirofilaria immitis) y Afines, Madrid, Spain. in tenerife island: an epidemiological update. Stenzenberger R, Gothe R, 1999. Arthropod Parasitol Res 4. borne parasitic infections and tick infestations Morales M., Bañares A, Montoya JA, 2001. of dogs in Tenerife, Spain. Tierarztliche Praxis Prevalencia de la parasitación en perros por 27: 47-52. Dirofilaria immitis en la isla de Tenerife. 36 Suenaga O, Kitahara S, 1978. Studies on the Congreso Nacional de la Asociación de prevalence of Dirofilaria immitis among dogs Veterinarios Españoles Especialistas en and its vector mosquitoes in Sasebo city, Pequeños animales (A.V.E.P.A) Barcelona, Nagasaki Prefecture. Trop Med 20: 143-151. Spain. Tanaka H, Watanabe M, Ogawa Y, 1985. Parasites Ortega-Mora LM, Ferré I, Gómez M y Rojo- of stray dogs and cats in the Kanto region, Vázquez FA, 1988. Prevalencia de la infestación Honshu, Japan. J Vet Med 771: 657-661. por filarias en galgos en la zona centro de Valladares B, Gijón H, López-Román R, 1987. España. Medicina Veterinaria 5: 433-442. Dirofilaria immitis en la isla de Tenerife. Ortega-Mora LM, Gómez M, Rojo-Vázquez FA, Algunos aspectos de su fisiopatología. Revista Ródenas A, Guerrero J, 1991. A survey of the Ibérica Parasitología 47: 377-380. 15 The importance of dirofilariosis in carnivores and humans in Hungary, past and present

Éva Fok Dirofilariosis in Hungary 183

Introduction of the continent, D. repens is mostly widespread and nowadays dirofilariosis Several worm species of Superfamily is considered as an emerging zoonosis Filarioidea develop in human and ani- (Irwin, 2002). mal hosts and many may cause zoonotic infections. Two families are recognized: Filariidae and Onchocercidae Dirofilariosis in carnivores (Anderson, 2000). These filarioid nematodes are transmitted by Cardiovascular-dirofilariosis haematophagous arthropod vectors, including mosquitoes (Culicidae), The first recorded case of Dirofilaria- blackflies (Simulium spp.), biting infestation of carnivores in Hungary midges (Culicoides spp.), ticks was published more than 20 years ago (Rhipicephalus sp.), fleas and flies (Boros et al., 1982). In this report it (Kassai, 1999, 2003). Filariidae elicit was shown D. immitis infection in two skin lesions and release eggs and/or lar- female beagle dogs imported from the vae which attract arthropods, mainly United States. The adult worms were flies (Muscidae). The Onchocercidae, detected in the right cavity of the heart. on the other hand, have evolved blood These dogs, with the other beagles, or skin-inhabiting microfilariae and are were kept for experimental purpose in transmitted by arthropod vectors which closed recovers, so it was clear that the create lesions or pierce the skin and infection was imported. then suck blood. In the recent decade The next reported filarial infection in the importance of some zoonotic worms a dog was in 2000 (Vörös et al., 2000). of carnivores from Onchocercidae, such The dog lived in the USA for years with as Dirofilaria and Onchocerca spp. is the owner and they returned to increasing (Pampiglione and Rivasi, Hungary some months before the date 2000; Eberhard et al., 2000; of admission into clinic. This five-year- Pampiglione et al., 2001; Sréter et al., old male dog of mixed breed was 2002; Genchi, 2003; Salló et al., 2005). recovered with a two-days long history The climate changes and travelling of depression, exercise intolerance, dys- with pets may help both the spreading pnoea, anorexia, vomiting and voiding of vectors and the appearance of worm of reddish urine. The animal, in spite of infection at risk for human health in the intensive therapy died within some non-endemic or previously free areas hours after admission. Necropsy (Trotz-Williams and Trees, 2003). Two revealed several adult D. immitis in the species of genus Dirofilaria, D. immitis blood clots within the caudal vena cava and D. repens mostly are important and within the dilated right ventricle. agents of zoonotic infections in the Microfilariae were found within the Mediterranian area (Muro et al., 1999). capillaries of the myocardium, intersti- For both the parasites, the most tial pulmonary capillaries and in other important natural host is the dog, organs, including spleen and liver and which has a high prevalent and persist- in the peripheral blood smears. Vena ent microfilaremia. In the middle part cava syndrome due to the occlusion of 184 Dirofilariosis in Hungary

the caudal vena cava by adult worms between 1993-1996 in München. Out together with blood clots was diag- of five dogs found infected with D. nosed by necropsy and this complica- repens, two dogs were imported from tion was stated as the cause of the sud- Italy or Greece, and the third had trav- den crisis and the death of the patient. elled with its owner in Hungary and In spite of that, no newer imported case former Yugoslavia. The infection was of canine heartworm infection has been diagnosed by blood examinations with published in the scientific literature in Knott’s method. The origin of the infec- Hungary, we have got some information was not clear and it cannot be tion from personal communications excluded that the infection was really conducted with small animal practi- acquired in Hungary. tioners about some cases (e. g. import- The first three autochthonous D. ed Mastino Napoletano dogs from repens cases recorded in dogs were Canary Islands, Cane Corso puppies published in Hungary in 1998/1999 from Italy) where D. immitis infection (Fok et al., 1998; Széll et al., 1999). In was suspected. the very first case the worm was found An interesting case was detected in in a verruca-like nodule removed in 1999, when a 3-year-old mongrel dog autumn of 1995 from the subcutaneous living in Tisza river area was dissected tissue of the neck region of a 4-year-old after an accident and the veterinarian dog, which had spend the summer at found worms in the left ventricle of the river Tisza (Fok et al., 1998). In the heart. These worms were identified as other two cases, the worms were found D. immitis. This dog was living togeth- in a granuloma removed from the tho- er with his mother who was housed six rax region and from the subcutaneous years earlier by an owner from the tissue of the leg, respectively (Széll et street. The dog was living at the river al., 1999). One of them had spend shore, not far from a parking place of some days at Lake Balaton and the international trucks. However, it was other one was living in the Tolna not possible to clarify if the infection County, near the southern boarder of was autochthonous or acquired through the country. On the basis of the mor- the biting of an infected mosquito trans- phological features, the worms were ported by one of the trucks. identified as gravid females in all cases. Although proved autochthonous dog Furthermore, 101 dogs from Tolna heartworm infection has not so far County were examined for microfilari- recorded in Hungary, the described aemia by Knott’s method. The preva- cases demonstrate the risk of introduc- lence of D. repens microfilaremic dogs tion of this disease with infected animals was 9%. Considering that these dogs from endemic countries (Farkas, 2003). were never abroad, it was stated that there are endemic areas for D. repens Cutaneous-dirofilariosis in Hungary. In the recent years, according to the In 1997, Zahler et al. published the personal communications of colleagues results of an epidemiological survey from the main veterinary diagnostic (Zahler et al., 1997) carried out laboratories, more and more microfilar- Dirofilariosis in Hungary 185

ia positive blood samples of dogs as be useful for veterinarians working in accessory findings are detected in the the small animal praxis to help them in praxis. Most of these cases are suspect- the recognition of the clinical picture of ed D. repens infection, although the cutaneous dirofilariosis. Results con- detailed epidemiological background is cerning this zoonotic disease would be missing. Moreover, reports on human useful for the human doctors, as well, infection (see later) are frequently and can help in a more effective con- detected in Hungary. trol. Furthermore, the results of the Why is the number of cases increas- planned study could be important not ing in our country? What can be the only in Hungary, but also may con- epidemiological background? What is tribute to the European knowledge on the prevalence of infection in dogs and this mosquitoes transmitted disease. cats in Hungary? How it is possible (or weather it is necessary) to treat D. repens positive animals in Hungary? Dirofilariosis in humans Which mosquitoes species might be the vector of this worm in our coun- The first human filarioid infection in try? The cutaneous dirofilariosis can Hungary was detected by Babes in cause problems in the diagnostic dif- 1879 (cited by Kotlán, 1951). ferentiations, as well. What kind of However, an actual evaluation of the connections might be between the early human cases in Hungary was infection of the animals and the published in the middle of the last cen- increasing number of the human cases tury by Professor Academic Kotlán in our country? Because of such a (Kotlán, 1951). The author referred to questions and the lacking of D. repens Desportes (1939-1940), who in his prevalence data in domestic carni- paper presented a historical review of vores in Hungary, in 2005 we decided the cases of human filarioidosis know to start with a research work in our from literature, in which the parasites Department of Parasitology and playing part in the pathological process Zoology, including a PhD programme. could be identified with, or was found In parallel with the study, we plan to to be closely related to “Filaria con- monitor the human cases of junctivae” Addario, 1885 (cit. Kotlán, Dirofilaria infection to obtain more 1951). Having had the opportunity to information on the epidemiological examine a new case, Kotlán attempted background in our country. The aim to review all available data at that of this work is to survey the infection time, either verbal or published in of the most frequent final hosts, then Hungarian literature. It was stated in to study the role of the vectors in the his publication that at least 9 cases epidemiology and the possibilities of recorded from 1880 to 1950 in the spreading from carnivores to Hungary was probably identical with humans to achieve an effective control D. repens, however the infection with (see later the preliminary results of other filarioid species was not com- this work in a free presentation). pletely excluded. It was not possible to The baseline data of this work would clarify the epidemiological background 186 Dirofilariosis in Hungary

of these cases namely the sources of cat), and whether they live near water infection. It was supposed that some of shore where mosquitoes are abundant. these cases probably were autochtho- The answer may explain the spread of nous because the patients never had the worm in Hungary. left the country. It seems that in the recent six years The case published ten years later more and more new autochthonous (Németh and Kugler, 1968), was the human cases become known in our first ocular involvement in our country, country. However, we can have the and it was supposed that the removed information about most of them from worm pieces from a nodule on the scle- oral presentations in conferences or in ra of the 21 years old man were belong- personal communications, and only ing to a D. repens specimen. More than partly from the scientific publications. 30 years later, the next Hungarian In the new cases the worms were human case in which the worm was removed from the orbital tissues detected in the spermatic cord was (Parlagi et al., 2000), subconjuctival published by Pampiglione et al. (1999). space (Szénási et al., 2000; Hári et al., It was thought that these cases were 2002; Kucsera et al., 2002; Aczél et al., acquired abroad, too. 2005; Tornai et al., 2006), upper lid However, in a little while, six new (Salomváry et al., 2005), the fatty tis- cases were reported by pathologists sue of a subcutaneous nodule of the with the help of a parasitologist (Elek thigh (Lengyel et al., 2005), a cyst situ- et al., 2000). Two of these cases might ated in the cutaneous and subcuta- have been acquired in Italy during neous layers of the forearm (Péter et summer travels. Four patients have al., 2005) and the subcutaneous tissue never been abroad and these cases of the neck region (Csanády, 2005, per- must be considered as autochthonous sonal communication). All these cases, infections. According to the authors, after the detailed anamnesis and identi- the patients liked dogs and cats, and fication of the worms or examining of they were often living in sites along tissue sections were diagnosed as D. Danube and Tisza rivers, where mos- repens infection. quitoes are abundant. The thickness of Nowadays, the medical clinicians the multilayered cuticle of the worm, who suspect an human filarioid infec- diameter of the body and the size, tion in their patients can be supported form and number of the longitudinal in the etiological diagnosis by parasitol- ridges on its surface were used in the ogists sending removed worms for histological diagnosis of D. repens identification or dissected tissue from worms. In fact, the definitive diagno- the patient to specialized laboratories sis of human dirofilariosis is a patho- such as the Department of Parasitology logical and parasitological task. In this of ‘Johan Béla’ Human Health Institute report the authors called the attention (see later a free presentation about the of Hungarian human clinician to ask human cases by I. Kucsera) or the the patients whether they have been Department of Parasitology of the (and when and where) abroad, Veterinary Faculty of Szent István whether they have animals (dog or University, in Budapest. Dirofilariosis in Hungary 187

Conclusion infection in two dogs in western United States. Vet Parasitol 90: 333-338. Elek G, Minik K, Pajor L, Parlagi Gy, Varga I, It seems to the parasitologists, that in Vetési F, Zombori J, 2000. New human dirofi- Hungary, the occurrence of D. repens is larioses in Hungary. Path Onc Res 6: 141-145. Farkas R, 2003. Dirofilariosis in Hungary. much more frequent than it was con- Helminthologische Fachgespräche [Helmintho- sidered earlier. The tourism with pets logical Colloquium]. November 14, 2003. and visiting of the different dog or cat Abstracts, 13 p. exhibitions abroad and in our country, Fok É, Szabó Z, Farkas R, 1998. Dirofilaria repens ferto´´zöttség elso´´hazai diagnosztizálása the repeated travelling of Italian kutyában sebészeti beavatkozás során [The first hunters with their dogs to Hungary, cli- autochthonous case of a dog infected with mate changing, frequent raining and Dirofilaria repens in Hungary]. Kisállatorvoslás 4: 218-219. flood may have increased the spreading Genchi C, 2003. Epidemiology and distribution of of dirofilariosis. A close cooperation Dirofilaria and dirofilariosis in Europe: state of not only with the parasitologists, but the art. Helminthologische Fachgespräche [Helminthological Colloquium]. November 14, also between human and veterinary cli- 2003. Abstracts, 6-11 pp. nicians in our country and with scien- Hári Kovács A, Szénási Zs, Tiszlavitz L, tists from surrounding countries are Kolozsvári L, Pampiglione S, Fioravanti ML, 2002. Ophthalmo-filarioidosis újabb esete needed to better understand the epi- Magyarországon [A new case of ocular dirofilar- demiology of dirofilariosis and to con- iosis]. Szemészet 139: 87-90. trol the risk of infection both for the Irwin PJ, 2002. Companion animal parasitology: a clinical perspective. Int J Parasitol 32: 581-593. public and animal health in Europe. Kassai T, 1999. Veterinary Helminthology. Butterworth & Heinemann. Oxford, 1999, 121- 124 pp. Kassai T, 2003. Helmintológia. Medicina Acknowledgements Könyvkiadó Rt., Budapest, 186-192 pp. Kotlán A, 1951. On a new case of human filari- I should like to thank to my PhD student, Olga idosis in Hungary. Acta Vet Acad Sci Hung 1: Jacsó who helped me to complete the list of the 69-79. references. I am also extremely grateful to Pfizer Kucsera I, Elek I, Danka J, Fok É, Varga EB, Ltd. Animal Health Hungary who supported my Birnbaum I, Szénási Zs, 2002. Ocularis filarioi- attendance to the First European Dirofilaria Days dosis-esetismertetés [A human case report]. in Zagreb. Magyar Mikrobiológiai Társaság és Alapítvány 2002. évi nagygyu´´lés, Összefoglalók [Abstracts], Balatonfüred, October 8-10, 2002, 80 p. Lengyel A, Kovács I, Nemes Z, Rábainé Pongrácz References J, Kovács J, Fok É, 2006. Humán dirofilariosis [A human case report]. 64. Pathologus Aczél K, Deák Gy, Farkas R, Majoros G, 1995. Kongresszus a Magyar Pathológusok társasága Subconjunctivalis fonálféreg-ferto´´zés Magyaro- és a Nemzetközi Pathologiai Akadémia (IAP) rszágon [Subconjunctival nematode infection in Magyar Divíziója rendezésében, Összefoglalók Hungary]. LAM-Tudomány 15: 465-467. [Abstracts], Pécs, September 22-24, 2005, 66 p. Anderson RC, 2000. Nematode parasites of verte- Muro A, Genchi C, Cordero M, Simón F, 1999. brates. Their development and transmission. Human dirofilariasis in the European Union. 2nd edition. C.A.B. International, Wallingford, Parasitol Today 15: 386-389. 467 p. Németh B, Kugler S, 1968. Ophthalmo-filariasis. Boros G, Janisch M, Sebestyén Gy, 1982. Orv Hetil 109: 195-197. Dirofilaria immitis ferto´´zöttség kutyában Pampiglione S, Elek G, Pálfi P, Vetési F, Varga I, [Dirofilaria immitis infestation in dogs]. Magyar 1999. Human Dirofilaria repens infection in Áo Lapja 37: 313-316. Hungary: A case in the spermatic cord and a Eberhard ML, Ortega Y, Dial Sh, Schiller CA, review of the literature. Acta Vet Acad Sci Hung Sears AW, Greiner A, 2000. Ocular Onchocerca 47: 77-83. 188 Dirofilariosis in Hungary

Pampiglione S, Rivasi F, 2000. Human dirofilaria- Széll Z, Sréter T, Csikós K, Kátai Z, Dobos- sis due to Dirofilaria (Nochtiella) repens: an Kovács M, Vetési F, Varga I, 1999. Autochton update of world literature from 1995 to 2000. Dirofilaria repens ferto´´zöttség kutyákban Parassitologia 42: 231-254. [Autochtonous Dirofilaria repens infection in Pampiglione S, Rivasi F, Angeli G, Boldorini R, dogs]. Magy Áo Lapja 121: 100-104. Incensati RM, Pastormerlo M, Pavesi M, Ramponi Szénási Zs, Hári Kovács A, Tiszlavicz L, A, 2001. Dirofilariasis due to Dirofilaria repens in Kolozsvári L, Nagy E, 2000. Humán ophthal- Italy, an emergent zoonosis: report of 60 new mo-filarioidosis. A Magyar Zoonózis Társaság cases. Histopathology 38: 344-354. Kiadványa [Publication of the Society of Parlagi Gy, Sumi Á, Elek G, Varga I, 2000. Hungarian Zoonosis]., Kiadó: Korzenszky E., Szemüregi dirofilariosis [Orbital dirofilariosis]. 124-130 pp. Szemészet 137: 105-107. Tornai I, Montovay E, Veress Cs, 2006. Humán Péter I, Czeyda-Pommersheim F, Kucsera I, ferto´´zés-szemészeti esetismertetés [A human Szénási Zs, Orosz Zs, 2005. Új humán dirofi- case report]. “A dirofilariosis, mint egyre növek- lariosis eset Magyarországon [A human case vo´´ állategészségügyi és közegészségügyi problé- report]. 64. Pathologus Kongresszus a Magyar ma hazánkban” c. rendezvény (conference) a Pathologusok Társasága és a Nemzetközi Magyar Parazitológusok Társasága, a Magyar Pathologiai Akadémia (IAP) Magyar Divíziója Zoonózis Társaság és a Pfizer Kft. Állate- rendezésében, Összefoglalók [Abstracts], Pécs, gészségügy rendezésében, March 24, 2006. September 22-24, 2005, 80 p. Trotz-Williams LA, Trees AJ, 2003. Systematic Salló F, Eberhard ML, Fok É, Baska F, Hatvani I, review of the distribution of the major vector- 2005. Zoonotic Intravitreal Onchocerca in borne parasitic infections in dogs and cats in Hungary. Ophthalmology 112: 502-504. Europe. Vet Rec 152: 97-105. Salomváry B, Korányi K, Kucsera I, Szénási Zs, Vörös K, Kiss G, Baska F, Bagdi N, Széll Z, 2000. Czirják S, 2005. Szemüregi dirofilariosis újabb Irodalmi áttekintés és esetismertetés esete Magyarországon. [A new case of orbital [Heartworm disease in dogs. Review article and dirofilariosis in Hungary]. Szemészet 142: 231- case report]. Magy Áo Lapja 122: 707-716. 235. Zahler M, Glaser B, Gothe R, 1997. Sréter T, Széll, Z, Egyed Zs, Varga I, 2002. Ocular Eingeschleppte Parasiten bei Hunden: onchocercosis in dogs: a review. Vet Rec 151: Dirofilaria repens und Dipetalonema recondi- 176-180. tum. Tierärztl Prax 25: 388-392. FACULTY OF VETERINARY MEDICINE UNIVERSITY OF ZAGREB PFIZER ANIMAL HEALTH CROATIAN VETERINARY SOCIETY CROATIAN VETERINARY CHAMBER

FIRST EUROPEAN DIROFILARIA DAYS

INTERNATIONAL SCIENTIFIC ORGANIZING COMITEE COMITEE Albert Marinculic´(president), Croatia Claudio Genchi (president), Italy Ljiljana Pinter (vice president), Croatia Horst Aspok, Austria Relja Beck (Croatia), Marijan Cergolj Peter Deplazes, Switzerland (Croatia), Claudio Genchi (Italy) Robert Farkas, Hungary Ivan Forgacˇ (Croatia), Drazˇen Gluvak Laura Kramer, Italy (Croatia), Jasminka Granic´ (Croatia), Albert Marinculic´, Croatia Boris Fabijanic´ (Croatia), Branko Juric´ Silvio Pampiglione, Italy (Croatia), Lidija Kozacˇinski (Croatia), Fernando Simon, Spain Milivoj Landeka (Croatia), Igor Natasa Tozon, Slovenia Markovic´ (Croatia), Drazˇen Maticˇic´ Alexander Trees, UK (Croatia), Mario Milesˇevic´ (Croatia), Luigi Venco, Italy Mirna Ladavac (Croatia), Darko Geresˇ (Croatia), Damir Zˇ ubcˇic´ (Croatia), Dagny Stojcˇevic´ (Croatia), Tatjana Zˇ ivicˇnjak (Croatia)

Zagreb, Croatia, February 22-25, 2007 Free Communications Free Communications 193

An update on filarial parasites in Vulpes vulpes of Tuscany (Central Italy)

M. Magi1, P. Calderini2, S. Gabrielli3, M. Dell’Omodarme4, F. Macchioni1, M.C. Prati4, G. Cancrini3 1Dip. di Patologia Animale, Profilassi ed Igiene degli Alimenti, Univ. di Pisa, V.le delle Piagge 2, 56124 Pisa, Italy; 2Ist. Zooprofilattico Sperimentale del Lazio e della Toscana, Dipartimento di Rieti, V. Tancia, 21, 02100 Rieti, Italy; 3Dip. di Scienze di Sanità Pubblica, Università “La Sapienza”, P.le Aldo Moro 2, 00185 Roma, Italy; 4Scuola Normale Superiore di Pisa, Italy, INFN Sezione di Pisa, Piazza dei Cavalieri 7, 56127 Pisa, Italy

Red foxes (Vulpes vulpes), like other people and for companion animals wild and domestic carnivores, can be prompted us to include filarioses affected by several species of filarial among the parasitic diseases that have nematodes, namely Dirofilaria immitis, to be checked on the foxes examined D. repens, Acanthocheilonema recon- during a survey on echinococcosis. ditum, and A. dracunculoides. The first With the aim of contribute to this species induces heartworm disease, subject of debate, and to compare the with severe/extremely severe pathologi- infection rates of filarial worms in wild cal changes in the animals, whereas the and domestic canidae of the region, other parasites are etiological agents of 112 foxes (42 males, and 72 females), subcutaneous/connective tissue filario- killed during the hunting season 2003- sis. Furthermore, at least dirofilarial 2004 in several areas of the Tuscany species proved zoonotic and, therefo- (Central Italy), were examined. The re, where the animal disease is ende- carcasses of the foxes, coming from the mic, the human population is at risk Monte Amiata (Grosseto; n=37), the for developing ocular lesions or pul- hills around Cecina (Livorno; n=26), monary/subcutaneous nodules that the zones around Cascina and Bientina always give rise to the suspicion of a (Pisa; n=33) and the hills around Siena malignant cause. (n=16), were sent to the laboratory of Literature data about the role of the Pisa. Data on sex, age, weight, and area fox as reservoir for filarial nematodes of origin were collected for each ani- are in disagreement. In fact, in spite of mal, and the carcasses were stored at adult worms found in this animal seem -20°C until necroscopy. Microfilariae often immature and microfilaraemia is were searched for in pulmonary and reported as sporadic (Meneguz et al, splenic blood smears, and species iden- Parassitologia 40 suppl: 105), it has tification was performed using both been suggested that the fox may play an morphologic and morphometric char- epizootiologic role in heartworm infec- acteristics, and Barka coloration tech- tion (Mañas et al, 2005, Vet J 169: 118- nique. As far the adults, it was only 120). The possible importance of this possible the searching for D. immitis in wild animal as reservoir of infection for the endocardium and in the pulmonary 194 Free Communications

artery, because organizational problems four areas no difference was evidenced hampered the examination of tissues in among positivity rates to adults of D. order to ascertain the presence of immitis, whereas there is a significant adults belonging to the other species. difference among those to microfilariae Finally, molecular diagnostics (DNA (P = 0.022). Molecular tools confirmed extraction, PCR using general “filarial” morphological identifications. primers S2-S16 and sequencing) were Our findings on A. reconditum, applied to adults and micriofilariae to D. immitis and/or D. repens circulating confirm all identifications. Data were microfilariae in about 10% and in less statistically analyzed with the Fisher than 2%, respectively, of the fox car- exact test, and results were considered casses examined fit in with data regard- statistically significant if P<0.05. ing Italy. As far Tuscany, after 25 years A total of 20 foxes (17.9%) turned the prevalence of microfilaraemic spec- out positive to filarial parasites. In 8 imens is about unchanged (19.4% vs specimens (7.1%) were found mature 17.9%) (Gradoni et al., 1980, Ann Ist adults of D. immitis (9 males and 14 Sup San 16: 251-256), and it is not so females), and two of them (1.8%), col- much different from that evidenced on lected around Cascina (Pisa), had also concentrated blood samples of alive microfilariae both at pulmonary and dogs (35.4%) (Marconcini & Magi, splenic level. One of these two foxes 1991, Ann Fac Med Vet Pisa, XLIV: was positive also to microfilariae of D. 153-156). Our findings, drawn by the repens. Amicriofilaraemic subjects har- examination of very few µl of blood boured at least an adult couple. A total obtained from perished carcasses, sup- of 12 foxes (10.7%) were positive to port the hypothesis that the fox can be microfilariae of A. reconditum, absent a source of infection for invertebrate only from specimens hunted on the hosts and, therefore, wild reservoir for hills around Livorno, where all foxes filarial nematodes that can be transmit- were found amicrofilaraemic. In the ted to companion animals and people. Free Communications 195

Preliminary results of an epidemiological survey on dirofilariosis of dogs and cats in Hungary

Éva Fok1, Gabriella Kiss2, Gábor Majoros1, Olga Jacsó1, Róbert Farkas1, Mónika Gyurkovszky1 1Department of Parasitology and Zoology, Faculty of Veterinary Science, Szent István University H-1400 Budapest, P.O. Box 2, Hungary; 2Pfizer Animal Health, 1123 Budapest, Alkotás u. 53., Hungary

Background (116/826; 14%) and in 2 samples from cats (2/29; 6.9%). Of the positive dogs, In the recent years more and more fre- 64 (55.2%) were living along Danube quently cases of mosquito-borne filari- river, 40 (34.5%) along Tisza river, 1 oid subcutaneous infection with (0.8%) on the shore of lake Balaton and Dirofilaria repens Railliet and Henry, 11 (9.5%) near other wet areas. The 1911 has been reported in animals positive cats were from the town of (mainly dogs) and in humans. However, Szeged, situated at both sides of Tisza limited information were available river. Of the positive dogs, 39.4% were about the frequency of D. repens infec- aged 3-6 years and 32.1% were 7-10 tion in carnivores in Hungary. year-old. The infection prevalence was For such a reason, epidemiological 53.2% in male dogs and 46.8% in surveys were carried out to assess the female dogs. Higher prevalence values status of this carnivore’s filarioid infec- were found among mongrel (20.7%) tion and to evaluate the risk for veteri- and German shepherds (19%). Other nary and medical public health. breeds such as various hunting breeds (21.6%) and 1 to 2 positives dogs from Materials and methods miscellaneous breeds (38.8%) were also found to be infected. From June 2005 to July 2006, 826 Skin lesions (such as erythema, dog and 29 cat blood samples collected papules, alopeca, nodules) were diag- from several areas of the country with nosed in 276 (33.4%) of the exam- the help of veterinary practitioners ined dogs and from these animals 37 were examined. The presence of micro- (13.4%) was microfilaria-positive. filariae in the blood samples was However, of the 116 positive dogs, 37 assessed by modified Knott’s tech- (31.9%) showed skin lesions. From nique. Larvae were identified by mor- the examined cats, 10 (34.5%) phological criteria. showed various skin disorders, but the microfilaria-positive cats were without Results symptoms. The prevalence of other parasitic D. repens microfilariae were found in infection was also evaluated according 116 blood samples from dogs to the other clinical records, animal 196 Free Communications

maintenance and control against the living in the regions where the positive ecto- and endoparasites. animals were found. Visiting or living at the coastal areas of rivers seems to Conclusion be a significant risk factor to acquire the infection. Our further aim is to con- It has been shown that D. repens tinue this survey to acquire more infor- infection in both dogs and cats is more mation about the occurrence of this important than it is currently consid- worm infection and to study the role of ered in Hungary. The survey highlights mosquitoes in spreading of the infec- the possible zoonotic risks for humans tion in our country. Free Communications 197

Review of human dirofilariosis diagnosed at the Department of Parasitology, National Center for Epidemiology, Budapest, Hungary

István Kucsera, Zsuzsanna Szénási, József Danka National Center for Epidemiology, Department of Parasitology, 1097 Budapest, Gyáli út 2-6, Hungary

In the temperate regions of Europe tion technique in 7 cases, with negative (mainly Italy, France and Greece, but results. over the last few years in Hungary as Based on the available epidemiologi- well) a specific filaroid worm, the cal data, it can be deduced that most of Dirofilaria (Nochtiella) repens, a para- these cases (8) are autochthon infec- site of dogs, cats and some other carni- tions. In 2 cases the data were ambigu- vores, transmitted by mosquitoes, has ous and in 4 cases there were no avail- occasionally infected people. In the able epidemiological data. The inci- world literature more than 800 cases dence of human dirofilariosis in have been recorded. Usually only one Hungary is sporadic, and does not worm can be detected in human tis- show seasonality. sues. In human cases the microfilar- It can be concluded that D. repens iemia and eosinophilia are usually not occurs in Hungary. The possibility of present. The diagnosis is ordinarily dirofilariosis has to be taken into con- based on morphology of the intact sideration in cases of predisposed worm and specific histopathological localized nodules. Increased interest findings. Surgical removal and identifi- of the clinicians and parasitologists cation of the worm are both diagnostic resulted in the newly detected cases and therapeutic. being completely diagnosed with both In the period from 2000 to August morphological and histopathological 2006 we diagnosed 14 cases of dirofi- examinations. The differential diagno- lariosis in 8 female and 6 male patients. sis from the other filaria species which Mean age was 57 years for female and occur in humans is not required in 62 for male (average 60 years). This these cases, because the patients’ his- higher prevalence in older-age corre- tories contain no data that refer to that sponds to literature data. Of the 14 possibility. Therefore, we consider cases, 7 had ocular localization, 6 sub- that most of our cases are auto- cutaneous and 1 was diagnosed in chthonal. Although the incidence of histopathological section of removed dirofilariosis is sporadic and the pub- lymph-node in a patient with lymphoid lic health importance is not high, the leukemia. Anamnestic eosinophilia was increasing number of cases suggests found in 2 cases. For detection of that direct attention must be paid to microfilariae we used Knott concentra- this zoonosis. 198 Free Communications

Dirofilariosis in dogs - the actual situation in the Czech Republic

R. Dobesova, Z. Svobodova, V. Svobodova Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic

Introduction Heartworm kit (Fuller Laboratories, USA) for the detection of specific anti- In the past, canine dirofilarial infection bodies against D. immitis. For compar- (Dirofilaria immitis and D. repens) in ison we inserted also 2 samples positive the Czech Republic was associated with for D. immitis antigen. import and was considered an imported infection. The present study shows at Results surveying the presence of autochthonous Dirofilaria infections in dogs in the Microfilariae were detected in 8 dogs. area of the Czech Republic where the The result of the acid phosphatase climatic conditions are favourable for staining for all eight samples agreed the life cycle of the parasite. with D. repens species. No microfilariae of D. immitis were detected. Material and Methods Serological testing detected D. immitis antigens in 7 dogs. D. repens positive We have investigated the occurrence dogs were negative on the ELISA for of dirofilariosis in dogs in the Czech D. immitis. Republic from Brˇeclav area known by Specific antibodies against D. immitis high abundance of mosquitoes. The were found in 6 out of 11 dogs includ- study was carried out from November ing 2 positive for D. immitis antigen. 2005 to November 2006. The group consisted of 104 dogs. The sampled Conclusions dogs had no history of travelling abroad and were free of clinical symp- Our results confirmed endemic diro- toms. Microfilariae were detected by filariosis in the Czech Republic. using the Knott test and determined Dirofilariosis in the Czech Republic has with the histochemical method based been slowly spreading as our previous on the activity of acid phosphatase. results show. Serum samples were examined with the PetChek kit (IDEXX Laboratories, References Portland, USA) for the detection of adult female D. immitis circulating Svobodova Z, Svobodova V, Genchi C, Forejte P, 2006. The first report of authochthonous dirofi- antigens. We have also examined 11 lariosis in dogs in the Czech Republic. Helmint- out of 104 animals with IFA hologia 43: 242-245. Free Communications 199

Dirofilaria repens infection in dogs in the Czech Republic

Z. Svobodova1, V. Svobodova2, C. Genchi3 1Small Animal Clinic, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic; 2Department of Parasitology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic; 3Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria, Università degli Studi di Milano, Milano, Italy

Introduction phology. Species identification was confirmed by histochemical staining of Dirofilaria repens is the agent of the acid phosphatase with naphthol AS- subcutaneous filariosis in dogs and TR-phosphate as a substrate, and cats. Its pathogenicity is often under- pararosaniline as a chromogen. estimated. Global warming, the PCR - DNA was extracted from 100 increasing abundance of mosquitoes, µl of canine blood samples positive for movement of dogs between endemic circulating mf using a commercial kit and non-endemic countries have (QIAamp DNA blood; QIAGEN increased the risk of spreading of filar- GmbH, Germany). PCR reactions for ial infections in formerly Dirofilaria- the amplification of the mitochondrial free areas. coxI gene were performed in a final volume of 10 µl using general filarial Material and Methods primers and thermal profile described in Casiraghi et al. (2004). PCR prod- The study was carried out from ucts were gel-purified and sequenced November 2005 to November 2006 on directly using ABI technology. The 80 dogs, 53 males and 27 females, obtained sequences were compared to aged 1-12 years. Most were hunting those deposited in gene banks. dogs that spend the majority of their time outdoors. The sampled dogs had Results no history of traveling abroad and were free of clinical symptoms. Seven of the 80 dogs tested were Samples of whole blood, blood serum microfilaremic (8.7 %). Five were male and 2 fresh blood smears were aging 6 - 11 years and 2 were female, obtained from each dog. both aged 2 years, different breeds - German Shorthaired Pointer (3), Microfilariae Jagterier (1), Labrador Retriever (1), Czech Pointer (1) and Tosa Inu (1). A modified Knott test was used to Acid phosphatase staining identified D. detect microfilariae (mf) that were then repens species in all the samples. PCR identified on the basis of their mor- definitely confirmed D. repens diagno- 200 Free Communications

sis in these samples. Numerous adult Svobodova V, Svobodova Z, Beladicova V, Valentova D, 2005. First cases of canine dirofi- D. repens were found in the subcuta- lariosis in Slovakia: a case report. Veterinarni neous tissue during necropsy of one D. Medicina 50: 510-511. repens microfilaremic dog euthanized Svobodova Z, Svobodova V, Genchi C, Forejtek P, 2006. The first report of autochthonous dirofi- following the owner’s request. lariosis in dogs in the Czech Republic. Helminthologia 43: 242-245. References Casiraghi M, Bain O, Guerriero R, Martin C, Pocacqua V, Gardner S, Franceschi A, Bandi C, Svobodova V, Misˇonˇva P, 2005. The potential risk 2004. Mapping the presence of Wolbachia pipi- of Dirofilaria immitis becoming established in entis on the phylogeny of filarial nematodes: the Czech Republic by imported dogs. Vet evidence for symbiont loss during evolution. Int Parasitol 128: 137-140. J Parasitol 34: 191-203. Free Communications 201

Filariosis in dogs in Serbia

S. Dimitrijevic´1, A. Tasic´2, S. Tasic´2, V. Adamovic´3, T. Ilic´1, N. Miladinovic´-Tasic´2 1Department of Parasitic Diseases, Faculty of Veterinary Medicine University of Belgrade, Serbia; 2Department of Microbiology and Parasitology, School of Medicine, University of Nisˇ, Serbia; 3Veterinary Clinic VEGE-VET, Pozˇarevac, Serbia

Systematic research on canine filar- mercial DIFIL test (EVSCO, BUENA, ioses caused by the species Dirofilaria NJ, USA) were used to detect microfi- repens, Dirofilaria immitis, and lariae in blood samples. Identification Acanthocheilonema (Dipetalonema) of species of microfilariae was per- reconditum in Serbia has not been formed according to their morphologi- reported. Aim of this study was to cal and morphometric characteristics. establish the prevalence of filarial All morphometric parameters were nematodes in dogs and to identify obtained using automatic image analy- species according to their morphologi- sis Lucia M (NIKON). Including mixed cal and morphometric characteristics. infections, microfilariae were found in A total of 208 blood samples were the peripheral blood of 101 examined drawn from dogs from different areas dogs. D. repens infection was found in of Vojvodina and Branicˇevo region. 97 dogs, D. immitis infection in 15 dogs, Modified Knott technique and com- and A. reconditum infection in 4 dogs. 202 Free Communications

The appearances of dirofilariosis in Serbia - Vojvodina

Sara Savic´-Jevc´enic´1, Branka Vidic´1, ˇZivoslav Grgic´1, Lolic´ Zoran2 1Scientific Veterinary Institute of “Novi Sad”, Novi Sad, Serbia; 2Private practice “Leo”, Novi Sad, Serbia

The first information on the existence samples were taken again from the ter- of dirofilariosis in Serbia was pub- itories where dirofilariosis was found lished in 1999 (Sanda Dimitrijevic´, previusely and analysed for dirofilario- 1999). The number of infected dogs is sis, again. The test was done on 50 getting higher every year. In the region blood samples from Srem region of Vojvodina, as the part of Serbia, the (Ledinci, Paragovo, Krusˇedol, Selisˇte, investigation was done and the cases of Venac, Bukovac and Petrovaradin). infection were found (Tasic´ et al., The samples were analysed by ELISA 2003). In the year of 2004, a group of method, again with the same test kits - 100 dogs was observed within the DiroCheck test kits, produced by region of Vojvodina (Bacˇka and Srem). Synobiotics, USA. The result was that The blood test was performed with a 5 of 50 dogs were positive to dirofilar- test kit diagnostic device for quick iosis, which makes 10% of the analysed diagnostic of dirofilariosis, named population. The positive samples were FASTest HW Antigen, produced by from the following places of the Srem MegaCor, Austria and ELISA test was region - Ledinci, Krusˇedol, Venac and performed in the same blood samples Bukovac. by a DiroCHECK test kit, produced by The number of positive dogs to diro- Synobiotics, USA. After the analysis filariosis has grown during the last two and the comparison of the results there years in the region where the disease were 7% of positive samples to dirofi- was primarly found and dirofilariosis is lariosis, gained by two different diag- still present at the same places of Srem nostic methods - fast test and ELISA. region with the tendency of spreading The positive samples were from the during the year 2006. Usually, dirofilar- dogs of Srem region, and the places iosis is a side finding during the dissec- were the following: Venas, Bukovac tion, and a dog is rarely suspected to and Ledinci. have dirofilariosis during the life time, During the year of 2006, the blood even it has clinical symptoms. Free Communications 203

Dirofilariosis in dogs and wild carnivores in Romania

Coman Sofia, B. Baˇcescu, T. Coman, Gh. Pârvu, Cristina Dinu, T. Petrut,, N. Bercaru, Adriana Amfim (Padure) The Veterinary Medicine Faculty, Spiru Haret University, 2 Jandarmeriei, Street, Bucharest, Romania

The increased number of dirofilaria We found positive 5 dogs, aged 3-5 cases and the impact on the human years old, 5 dogs aged 6-8, and 2 dogs health due to the zoonotical aspect of aged 10-14, from which 8 were females the helmintosis, it’s mainly the reason and 4 males. for the global scientifical research on The hematology examination revealed: the matter. microcytar cupripriv hypocrom ane- The research schedule was elaborated mia, neutrophily, lymphocytopeny, by the Parasitological laboratory of the eosinophily. Radiologicly was registerd Spiru Haret University, Faculty of cardiac hypertrophy with left ventricu- Veterinary Medicine, Bucharest, lar dilatation and densifications pul- Romania. The clinical findings were monary parenchim. studied on 52 dogs of different breed Electrocardiographic tests revealed and age. The clinical, parasitologic, and hipertrophic cardiomiopathy. pathologic features of this entity are Histopathological examination reve- discussed, during the 6 months trial. aled pulmonary edema, thromboses From the 52 studied dogs, 12 within arteryes, and expatiated alveolar (23.07%) were Dirofilaria positive. capillary. Among the perilobulary sep- Symptoms, commonly chest pain, tum and the conjunctive tissue, numer- cough, or hemoptysis, along with car- ous macrophage cells were found, with diac failure and ascites were present in the purpose of fagocitosing the erythro- 25% of 3 patients. cytes degradation product, hemosiderin. 204 Free Communications

Dirofilariosis in dogs and wild carnivores in Bulgaria

Zvezdelina Kirkova, Andrey Ivanov, Dimitrina Georgieva Department of Parasitology, Faculty of Veterinary Medicine, Trakia University, Stara Zagora, Bulgaria

A study of the prevalence of the diro- samples were antigen positive. filariosis in dogs and wild carnivores in The necropsy of 113 foxes, 56 jack- Bulgaria was conducted from 2001 to als, 22 wild cats and 21 martens 2006. Blood samples from 487 dogs revealed Dirofilaria immitis infection out of heartworm prophylaxis originat- in 4 (3%) of the foxes and 5 (8.9%) of ing from different parts of the country the jackals. The intensity of the infec- were tested. Microfilariemia was found tion was from 2 to 16 specimens. in 42 (8.62%) of the samples by the Our results show that wild canides modified Knott’s test. Infected animals are a natural reservoir and source of were found in all parts of the country. infection for dogs. The trend of increas- All microfilaria positive samples were ing prevalence of dirofilariosis in dogs, antigen positive by Pet Check when compared to previous studies, HTWMPT test (IDEXX). Three suggests that heartworm prophylaxis (6.6%) of 45 microfilaria negative should be considered in Bulgaria. Free Communications 205

Dirofilariosis among dogs in a small animal practice in the Plovdiv region, Bulgaria

Milen Kostadinov Private Veterinary Practitioner, Veterinary clinic “Novet”, Plovdiv, Bulgaria

Dirofilaria immitis is still considered ease for a first time when they present- a rare and imported disease for ed their sick animals. Bulgaria. It is studied by all veterinary During the period of investigation the students but it is not expected to be number of the diagnosed cases was found in the practice. Most practicing increasing every year. We believe this veterinarians regard the disease as observation doesn’t relate to the uncommon. As a result routine analy- dynamics of the disease in the region, ses are not performed, which leads to a but is rather due to technical factors. lack of up to date information about The increased cases identification is localization and numbers of dirofilario- more related to the fact that we sis cases. changed our approach from sporadic The data presented here are gathered tests (when the clinical condition indi- in a small animal practice for a period cates) to routine analysis of many dogs of 5 years (from 2001 until 2006), from coming to the practice. Another impor- the city of Plovdiv and its surroundings, tant factor is the expansion of our prac- southwestern Bulgaria (total population tice, thus the physical ability to check about 500,000 people). more dogs. Since 2001 we have registered a total The region of Plovdiv is serviced by of 87 cases, 56 percent of them in another 4 practices having the size of Plovdiv and 44 percent in the sur- ours and about 15 smaller practices. rounding towns and villages. We deduce that our results can be mul- We analyzed the cases by the follow- tiplied at least by a factor of 5 to derive ing categories: severity of disease at the total number of dirofilaria cases presentation; diagnostic method used; presented (but not necessarily diag- whether or not the dogs were treated; nosed) in Plovdiv. complications after treatment; dogs liv- Our results show that dirofilarosis is ing outdoors/indoors; use of dogs not a rare disease among dogs in (hunting dogs, guard dogs, pets); age; Plovdiv region. This imposes the need gender; location. to educate dog owners about preven- Practically no prevention was used on tion and to incorporate dirofilaria diag- any dog before presentation. The vast nostic methods into the routine checks majority of clients heard about the dis- of all dogs. 206 Free Communications

Dirofilariasis in Turkey

Bulent Ulutas1, Tulin Karagenc2 Department of Internal Medicine1 and Parasitology2, Veterinary Faculty, Adnan Menderes University, Aydın, Turkey

Dirofilariasis is a disease caused by prevalence ranged from 0.83 to filarial worms of the genus Dirofilaria 46.22% at local scale following years. transmitted by mosquitoes. Although The national prevalence of the disease dirofilariasis was originally considered has not been determined hitherto, yet. a disease of strict veterinary impor- Many cases of D. immitis have been tance, it has been recognized as an reported in dogs in Turkey. D. repens emerging zoonosis. Two Dirofilaria was first reported in one human in species, D. repens and D. immitis, are Turkey in 1944. It was only investigated of special interest to humans because of in dogs in the east part of Turkey, in their harmful effect on our company Elazıg province, seropositivity rate was pets (dogs and cats) and potential 7.07%. In Turkey, human dirofilariasis zoonotic role. Turkey is one of the were documented in 18 cases. Serologic world countries where the presence of methods for use in humans are needed D. immitis and D. repens is currently for clinical evaluations of patients with confirmed. The objective of this study pneumonitis living in highly enzootic was to provide information on dirofi- D. immitis regions. These results lariasis both on animals and humans in demonstrate that dirofilariasis is wide- Turkey. D. immitis was first reported in spread in dogs in Turkey and epidemio- one dog in Turkey in 1951 and was logical surveys are needed to determine found in different provinces and canine the real extent of this zoonotic infection. Free Communications 207

Prevalence of canine heartworm disease in the Gemlik area of Bursa Province, Turkey

T. Civelek1, A. Yildirim2, A. Ica2, O. Duzlu2 1Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of Internal Medicine, Afyon, Turkey; 2Erciyes University, Faculty of Veterinary Medicine, Department of Parasitology, Kayseri, Turkey

The purpose of this study was to of circulating microfilariae while sera determine the prevalence of heartworm were examined with commercial disease (Dirofilaria immitis) in dogs in ELISA test kit in order to detect the the Gemlik area of Bursa province, presence of circulating antigens. Turkey. The study was carried out No circulating microfilariae were between June and September 2004. A found in peripheral blood of examined total of 100 dogs with various ages and dogs by native and Modified Knott tech- sexes were included in the study. Of niques, whereas parasite antigens were these, 65 were between 0.5 and 2 years determined in the sera of two male dogs old, 25 were 3-6 years old and 10 were one of which was aged between 3-6 over 7 years old. years and the second was over 7 years. Heparinized blood samples obtained In conclusion, the prevalence of from dogs were separately examined D. immitis was determined as 2% with native and Modified Knott tech- among dogs (occult infection) in the niques in order to detect the presence Gemlik area of Bursa province, Turkey. 208 Free Communications

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

A. Yildirim1, A. Ica1, O. Atalay2, O. Duzlu1, A. Inci1 1Erciyes University, Faculty of Veterinary Medicine, Department of Parasitology, Kayseri, Turkey; 2Erciyes University, Faculty of Veterinary Medicine, Department of Internal Medicine, Kayseri, Turkey

This study was conducted to deter- mean number of microfilariae in infect- mine the prevalence of Dirofilaria ed dogs was 4730 ± 5479 per ml of immitis infection and to investigate the blood. The highest heartworm preva- risk factors related to heartworm dis- lence were observed in the 7-10 age ease in dogs from Kayseri, Turkey. group (28.6%), followed by the groups Blood samples were collected from 280 4-6 (17.1%) and 0.5-3 (4.8%) years dogs from May 2005 to March 2006 old. The differences between the 0.5-3 and were examined by membrane fil- age group and the other age groups tration-acid phosphatase histochemical were found significant, whereas no sta- staining and antigen Elisa techniques to tistically significant difference was detect circulating microfilariae and observed between the 4-6 and the 7-10 antigens of D. immitis, respectively. age groups. The infection was more Out of the total of 280 dogs, 27 were prevalent in males, larger breeds and in positive for D. immitis with a preva- the dogs not on prophylaxis. No statisti- lence value of 9.6%. In addition, cally significant difference was observed 29.6% of positive dogs determined to between stray and owned dogs. Our have occult D. immitis infections. results suggest that heartworm treat- D. immitis was the only canine filarial ment and prophylaxis should be consid- parasite present in the study area. The ered in Kayseri Province. Free Communications 209

Wolbachia of Dirofilaria immitis: an historical perspective and morphological characteristics

Wieslaw J. Kozek, Jose A. Gonzalez Jr., Lilia Ana Amigo Department of Microbiology and Medical Zoology, Medical Sciences Campus, University of Puerto Rico, San Juan, Puerto Rico

Collaborative studies between Starry. Its life cycle is complex and Marshall Hertig and Samuel Wolbach, may consist of two reproductive on the presence and identification of modes: by binary fission and by a microorganisms in arthropods, resulted Chlamydia-like cycle. The latter mech- in the discovery of Wolbachia in Culex anism offers a potential survival strat- pipientis in 1924 and description of the egy by producing more progeny than genus Wolbachia and the species pipi- multiplication by binary fission, and entis in 1936. Subsequent studies have appears to be more active during demonstrated that Wolbachia is wide- growth embryogenesis and develop- spread in arthropods, infecting about ment of the larvae. Wolbachia of 25-70% of species of insects, and is D. immits appears to be slightly larger now known as a remarkable genetic that Wolbachia of other filarids, often manipulator of the infected arthropod occurring in great numbers in oocytes. hosts. Application of electron micro- The Wolbachia are apparently mutu- scopy to elucidate the structure of alistic endosymbionts required for sur- nematodes revealed that many filariae vival of their hosts and embryogenesis (17 species reported to date, including of microfilariae, are present in all lar- Dirofilaria immitis and most of the val stages during the life cycle of filar- human-infecting species) harbor transo- iae, and contribute to some of the varially–transmitted bacterial endosym- inflammatory responses and patholog- bionts, determined by Sironi et al. as ical manifestations of filarial infec- belonging to the genus Wolbachia. We tions in the vertebrate hosts. Presence have examined Wolbachia of D. immi- of Wolbachia endosymbionts in tis in all stages of this parasite: in micro- D. immitis complicates our under- filariae, larvae developing in the mos- standing of dirofilariasis in dogs, but quito and in the vertebrate host, and also offers possible advantages in treat- male and female adult worms by histo- ment and control of this infection. logical techniques and by electron Susceptibility of Wolbachia of filari- microscopy, comparing it to the ae to certain antibiotics offers an Wolbachia of other filariae. Wolbachia attractive possibility of treatment and in D. immitis is polymorphic and can be control of dirofilariasis. Detection of demonstrated by standard histological wolbachial antigens provides the pos- stains such as Giemsa’s, Gimenez, sibility for early detection of D. immi- Pinkerton’s, Machiavello’s and Warthin- tis infections in animals and in man. 210 Free Communications

Recently sequenced genomes of additional characteristics that can be Wolbachia pipientis from mosquitoes applied towards treatment and control and Wolbachia from Brugia malayi of this filarial infection. have opened a new chapter in the studies on Wolbachia and could provide the means to fully characterize Acknowledgements the structure, composition and elucidate the nature of these organisms. Supported, in part, by University of Puerto Rico (Medical Sciences Campus) RCMI Award RR- Further studies on Wolbachia of 03051 from the NCRR, NIH, Bethesda, MD, D. immitis, will undoubtedly reveal U.S.A. Free Communications 211

Ultrasound - guided surgical removal of adult heartworms via flexible basket forceps in a dog with caval syndrome

Murat Sarierler1, Bulent Ulutas2, Huseyin Voyvoda2, Abidin Atasoy2 Departments of Surgery1, and Internal Medicine2,Veterinary Faculty, Adnan Menderes University, Aydın, Turkey

In this case study, ultrasonography- raphy, heartworm removal was per- guided heartworm removal was carried formed. Insertion of the forceps was out using flexible basket forceps in an repeated to remove heartworms as Anatolian Shepherd dog (Sivas Kangal much as possible. A total of 17 adult Dog). The dog was anaesthetized by heartworms could be removed. One day intramuscularly injection of 2mg/kg/BW later, the dog died because of poor clin- Xylazine HCL (Rompun®, Bayer) and ical condition. Immediately, necropsy 10 mg/kg/BW ketamine HCL was performed and 34 heartworms (Ketalar®, Eczacibasi) combination fol- remaining in the right heart and pul- lowing the subcutaneous administra- monary artery were collected. The ratio tion of 0.02 mg/kg/body weight of removed worms via flexible basket atropine sulphate (Atropin®, Vetas). forceps was 33.3%. The dog was positioned on the left The low rate of this ratio may be due lateral position. The region of right to the lack of our experience, because jugular vein was prepared for aseptic this is the first attempt for us and for surgery. And also the right 5th-7th Turkey. intercostal spaces were shaved and cou- It was concluded that it is possible to pling gel applied for ultrasonographic remove adult heartworms from right examination. Following the cut down of heart via flexible forceps with the guid- right jugular vein, the flexible basket ance of USG. However, the higher forceps was inserted in to the jugular experienced surgeons could give better vein. With the guidance of ultrasonog- results.