impaginato spostato 2 - Parassitologia veterinaria · Romania, Serbia, and Turkey. We thank the leading European experts and colleagues from the board of the ... Dirofilaria infections

Mappe Parassitologiche

Series EditorGiuseppe CringoliCopyrigth© 2007 by Giuseppe Cringoli

Registered officeVeterinary Parasitology and Parasitic DiseasesDepartment of Pathology and Animal HealthFaculty of Veterinary MedicineUniversity of Naples Federico IIVia della Veterinaria, 180137 NaplesItaly

Tel +39 081 2536283 e-mail: [email protected] website: www.parassitologia.unina.it

All rights reserved – printed in Italy

No part of this publication may be reproduced in any form or by any means, electronically, mechani-cally, by photocopying, recording or otherwise, without the prior permission of the copyright owner.

Printed and bound by Litografia Vigilante srl, Rolando EditoreVia Nuova Poggioreale, 151 b/c80143 NaplesItaly

Tel/Fax +39 081 5846611e-mail: [email protected]

First edition: February 2007

ISBN 88-89132-14-0

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a MAPPE PARASSITOLOGICHE 8

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aDirofilaria immitis and

D. repens in dog and catand human infections

EditorsClaudio Genchi, Laura Rinaldi, Giuseppe Cringoli

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aSeries Editor Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

L. Rinaldi, V. Musella, C. Genchi, G. CringoliGeographical Information Systems in health applications: experience on filariosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

M.T. Manfredi, A. Di Cerbo, M. GenchiBiology of filarial worms parasitizing dogs and cats . . . . . . . . . .39

G. Cancrini, S. GabrielliVectors of Dirofilaria nematodes: biology, behaviour and host/parasite relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47

G. Grandi, T. Zivicnjak, R. BeckPathogenesis of Dirofilaria spp. infections . . . . . . . . . . . . . . . . .59

L.H. KramerImmunopathogenesis of filarial infections in dogs and cats: a role for Wolbachia endosymbiont? . . . . . . . . . . . . . . . . . . . . .67

F. Simón, L.H. Kramer, R. Morchón, C. GenchiA possible role for Wolbachia in the diagnosis of Dirofilariainfections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .73

S. Pampiglione, F. RivasiHuman dirofilariasis due to Dirofilaria (Nochtiella) repens: an update of world literature from 1995 to 2000 . . . . . . . . . . . .81

L. VencoHeartworm (Dirofilaria immitis) disease in dogs . . . . . . . . . . .117

L. VencoHeartworm (Dirofilaria immitis) disease in cats . . . . . . . . . . . .127

L. VencoDirofilaria (Nochtiella) repens infection in dogs and cats . . . . .133

C. Genchi, L. Venco, M. GenchiGuideline for the laboratory diagnosis of canine and felineDirofilaria infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .137

Index 7

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aC. Genchi, J. Guerrero, J.W. McCall, L. VencoEpidemiology and prevention of Dirofilaria infections in dogs and cats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .145

J.W. McCall, M.T. Dzimianski, W.L. Steffens, N. Supakorndej, P. Supakorndej, A.E. Mansour, M.B. Ard, S.D. McCall, R. Hack, D. DomingoSafety and efficacy of selamectin in dogs with Dirofilaria immitis infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .163

J.A. Montoya, M. Morales, M.C. Juste, J.A. CorberaHeartworm (Dirofilaria immitis) infection in dogs: current update in Spain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .175

É. FokThe importance of dirofilariosis in carnivores and humans inHungary, past and present . . . . . . . . . . . . . . . . . . . . . . . . . . . . .181

Free Communications presented at the First European Dirofilaria Days

(Zagreb, Croatia, February 22-25, 2007)

M. Magi, P. Calderini, S. Gabrielli, M. Dell’Omodarme, F. Macchioni, M.C. Prati, G. CancriniAn update on filarial parasites in Vulpes vulpes of Tuscany (Central Italy) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .193

É. Fok, G. Kiss, G. Majoros, O. Jacsó, R. Farkas, M. GyurkovszkyPreliminary results of an epidemiological survey on dirofilariosis of dogs and cats in Hungary . . . . . . . . . . . . . . . . . . . . . . . . . . . .195

I. Kucsera, Z. Szénási, J. DankaReview of human dirofilariosis diagnosed at the Department of Parasitology, National Center for Epidemiology, Budapest,Hungary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .197

R. Dobesova, Z. Svobodova, V. SvobodovaDirofilariosis in dogs - the actual situation in the Czech Republic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .198

Z. Svobodova, V. Svobodova, C. GenchiDirofilaria repens infection in dogs in the Czech Republic . . . .199

Index8

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aIndex 9

S. Dimitrijevic, A. Tasic, S. Tasic, V. Adamovic, T. Ilic, N. Miladinovic-TasicFilariosis in dogs in Serbia . . . . . . . . . . . . . . . . . . . . . . . . . . . .201

S. Savic-Jevcenic, B. Vidic, Z. Grgic, L. ZoranThe appearances of dirofilariosis in Serbia - Vojvodina . . . . . .202

S. Coman, B. Bacescu, T. Coman, Gh. Pârvu, C. Dinu, T. Petrut,, N. Bercaru, A. Amfim (Padure)Dirofilariosis in dogs and wild carnivores in Romania . . . . . . .203

Z. Kirkova, A. Ivanov, D. GeorgievaDirofilariosis in dogs and wild carnivores in Bulgaria . . . . . . . .204

M. KostadinovDirofilariosis among dogs in a small animal practice in the Plovdiv region, Bulgaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .205

B. Ulutas, T. KaragencDirofilariasis in Turkey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .206

T. Civelek, A. Yildirim, A. Ica, O. DuzluPrevalence of canine heartworm disease in the Gemlik area of Bursa Province, Turkey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .207

A. Yildirim, A. Ica, O. Atalay, O. Duzlu, A. InciPrevalence and epidemiological aspects of Dirofilaria immitisin dogs from Kayseri Province, Turkey . . . . . . . . . . . . . . . . . . .208

W.J. Kozek, J.A. Gonzalez Jr., L.A. AmigoWolbachia of Dirofilaria immitis: an historical perspective and morphological characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . .209

M. Sarierler, B. Ulutas, H. Voyvoda, A. AtasoyUltrasound - guided surgical removal of adult heartworms via flexible basket forceps in a dog with caval syndrome . . . . .211

Series Editor Preface

The last years have seen an increasing use of cartographic representation ofthe distribution of parasitic diseases, particularly vector-borne (arthropod-and snail-borne) diseases.

Geographical Information Systems (GIS) are finding increasing use in thestudy of geographical epidemiology and their recent application to medicaland veterinary parasitology is rapidly advancing.

MAPPE PARASSITOLOGICHE is a volume series aimed primarily to pub-lish Disease Maps and parasitological field researches dealing with applica-tions of GIS.

This Volume, the number 8th of the series, after a chapter dedicated to theuse of GIS in health applications with particular regard to filariosis, containsthe most important topics of canine, feline and human Dirofilaria infectionsand 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, specifical-ly biology, pathogenesis, diseases, treatment of Dirofilaria immitis andD. repens parasitizing dogs and cats; their vectors; the role of Wolbachia forimmunopathogenesis and diagnosis; human dirofilariosis; and epidemiologicalupdates 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 theAmerican Heartworm Society for their contributions, and Pfizer AnimalHealth for having supported the costs of the Volume.

We hope that the information presented in this Volume serves as inspirationfor scientists and practitioners to join in the further development of activityand studies on filarial worms in veterinary and human field.

Giuseppe CringoliSeries Editor

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Preface 13

Although significant contributions to the understanding of canine and felineheartworm 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 treatiseon 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 ofa dog and of another worm (probably Dioctophyma renale) in the kidney ofthe same dog. Since then, the parasite has been recognized in many countriesof the world and European researchers mainly in Italy and France have beenactive in the study of Dirofilaria spp. infections.

Climatic changes, together with an increase in the movement of cats anddogs across Europe, have caused an increase in the geographical range ofDirofilaria worms and in the risk of infection for both animals and humans.At the same time, greater attention to animal welfare and improved socialand economic conditions in many countries have contributed to an increasein pet owners’ awareness of potentially life- threatening conditions, such ascanine and feline heartworm disease, or of less serious but equally distressinginfections like subcutaneous dirofilariosis, both in animals and humans.

This manual, which has been published on the occasion of the FirstEuropean Dirofilaria Days, held in Zagabria (Croatia) in February 22nd-25th

2007, is intended as a contribution to and an update of current knowledge ofDirofilaria infection from leading European experts and colleagues from theboard of the American Heartworm Society. The most important aspects ofDirofilaria infections are reviewed, from biology to prevention and treatment.Several chapters offer insights into new topics such as the application of theGeographic Information Systems (GIS) to Dirofilaria distribution in Europeand the role of Wolbachia endosymbionts in host immune response. The clin-ical aspects of the disease are described with the aim of giving practitionerssuitable guidelines for diagnosis, management, prophylaxis and treatment ofthe disease. The last part includes a selection of short contributions present-ed during the meeting in Zagreb.

Finally, we would like to thank Pfizer Animal Health for having supportedthe costs of this publication without any constraints to the opinions expressedby the contributors.

Claudio Genchi Albert Marinculic

President International President OrganizingScientific Comitee Comitee

Contributors 15

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

Relja BeckVeterinary Faculty, University of Zagreb, Zagreb, Croatia

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

J.A. CorberaFacultad de Veterinaria, Animal Medicine and Surgery, Universidad de LasPalmas de Gran Canaria, Canary Islands, Spain

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

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

Dan DomingoAnimal Health Group, Pfizer Inc., New York, New York, USA

Michael T. DzimianskiDepartment of Infectious Diseases, College of Veterinary Medicine, Universityof Georgia, Athens, GA 30602, USA

Éva FokDepartment of Parasitology and Zoology, Faculty of Veterinary Science, SzentIstván University H-1400 Budapest, P.O.Box 2, Hungary

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

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

Marco GenchiDipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria,Università degli Studi di Milano, Via Celoria 10, 20133 Milano, Italy

Contributors

Giulio GrandiCollege of Veterinary Medicine, University of Parma, Parma, Italy

Jorge GuerreroDepartment of Pathobiology, School of Veterinary Medicine, University ofPennsylvania, Philadelphia, PA 19104, USA

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

M.C. JusteFacultad de Veterinaria, Animal Medicine and Surgery, Universidad de LasPalmas de Gran Canaria, Canary Islands, Spain

Laura H. KramerCollege of Veterinary Medicine, University of Parma, Parma, Italy

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

Abdelmoneim E. MansourTRS Labs, Inc., Athens, Georgia, USA

John W. McCallDepartment of Infectious Diseases, College of Veterinary Medicine, Universityof Georgia, Athens, GA 30602, USA

Scott D. McCallTRS Labs, Inc., Athens, Georgia, USA

J.A. MontoyaFacultad de Veterinaria, Animal Medicine and Surgery, Universidad de LasPalmas de Gran Canaria, Canary Islands, Spain

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

Rodrigo MorchónLaboratorio de Parasitología, Facultad de Farmacia, Universidad de Salamanca,Salamanca, Spain

Vincenzo MusellaDipartimento di Patologia e Sanità Animale, Facoltà di Medicina Veterinaria,Via della Veterinaria 1, 80137, Napoli, Italy

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Contributors

Silvio PampiglioneDipartimento di Sanità Pubblica Veterinaria e Patologia Animale, Università diBologna, Bologna, Italy

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

F. RivasiDipartimento di Scienze Morfologiche e Medico Legali, Università di Modena eReggio Emilia, Italy

Fernando SimónLaboratorio de Parasitología, Facultad de Farmacia, Universidad de Salamanca,Salamanca, Spain

W.L. SteffensDepartment of Pathology, College of Veterinary Medicine, University ofGeorgia, Athens, Georgia, USA

Nonglak SupakorndejTRS Labs, Inc., Athens, Georgia, USA

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

Luigi VencoClinica Veterinaria Città di Pavia, Viale Cremona 179, 27100 Pavia, Italy

Tatjana Z ivicnjakVeterinary Faculty, University of Zagreb, Zagreb, Croatia

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1 Geographical InformationSystems in health applications:experience on filariosis

Laura Rinaldi, Vincenzo Musella, Claudio Genchi,Giuseppe Cringoli

Geographical Information Systems

Introduction

The concept that geographic locationcan influence health is a very old one inmedicine. As far back as the time ofHippocrates (460-377 BC), it wasobserved that certain diseases tend tooccur in some places and not in others.Disease mapping and environmentalrisk assessment using geographicalinformation systems (GIS) and remotesensing (RS) technologies are nowestablished as basic tools in the analy-sis of both human and veterinaryhealth (for a review see Cringoli et al.,2005b). However, epidemiologists haveneeded time to become convinced thatthe prospects offered by GIS and RScould be useful in their own discipline.The last 15-20 years have seen an everincreasing reliance on cartographicrepresentation of infectious diseases,particularly parasitic infections andtheir vectors (Bergquist, 2006). In fact,during the past two decades the publi-cation of original research articles andreviews in veterinary and human healthwith an emphasis on GIS and/or RShas followed an exponential trend(Hendrickx et al., 2004). In addition,recent GIS/RS symposia organized atnational and international conferencesand several thematic issues on thistopic published in the peer-reviewedinternational literature (e.g. specialtheme issues in Advances inParasitology in 2000 and 2006;Parassitologia in 2005) demonstratethe wide array of applications andbenefits of these tools (Cringoli et al.,2005b; Rinaldi et al., 2006).Furthermore, the publication of thema-tic books pertaining GIS and RS, aswell as international peer-reviewed

journals, including the current launchof Geospatial Health (www.geospatial-health.unina.it) attest to the increasedinterest in these new technologies. Theestablishment and maintenance ofwebsites as a platform for sharingdata and exchanging opinions, expe-riences and expertise on GIS and RS,with an emphasis on animal andpublic health, is also worth mentioning(for example http://www.gnosisgis.org).In this paper, we first summarize gene-ral aspects of GIS and RS and empha-size the most important applications ofthese tools in veterinary parasitology,with particular regard to filarialworms. Then, disease mapping, ecolo-gical analyses and predicting parasiteoccurrence/seasonality as useful toolfor surveillance are summarized in thenext section.

Brief history of GIS and RS

Depending upon the application area,a number of discordant definitions ofGIS have appeared in the literature(Tim, 1995). One of the most widelyused definitions of GIS is that propo-sed by Burrough (1986): “a powerfulset of tools for collecting, retrieving atwill, transforming, and displaying spa-tial data from the real world”. Overall,a GIS is a platform consisting of hard-ware, software, data and people andencompasses a fundamental and uni-versally applicable set of value-addedtools for capturing, transforming,managing, analyzing, and presentinginformation that are geographicallyreferenced (geo-referenced). DigitalGIS data may be presented in mapform using so called data layers repre-

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senting the information collected. Twoapproaches can be used, namely:

(i) a vector data model, and (ii) a raster data model. The former model stores a table con-

taining coordinates of points togetherwith instructions on which points arealone and which points belong to acommon set. In a vector data model, alllines are represented by chains of vec-tors, and all areas by polygons (Fig. 1).Attributes are coded in separate tablesusing alphanumeric characters as alabel for a specific class or category ofproperties. The raster data model usesa net of adjacent polygons (termed“cells”) to provide a virtual cover of agiven part of a territory. The cells are

often called pixels, and attribute-valuesof the objects that the cells representare assigned to corresponding pixels(Daniel et al., 2004). Raster data aretypically utilized to represent conti-nuous phenomena, e.g. land covermaps, phenoclimatic maps, and digitalelevation models (Fig. 2).

The use of GIS in epidemiology canaid in answering some important que-stions such as “what is?” and “whereis?”. In addition, because also the timedomain - i.e. “when” - is important inmost environmental and epidemiologi-cal processes, it has also been suggestedthat GIS should be replaced by STIS,which is an abbreviation for space-timeinformation systems (Kistemann et al.,2002; Hendrickx et al., 2004). There iscurrently a movement towards regar-ding GIS as a science (geographicalinformation science) rather than a sim-ple technology (Goodchild, 2000;Kistemann et al., 2002).

One of the first major uses for GISwas in 1964 when the CanadianGeographical Information System(CGIS) was launched in an effort toassess the productivity of Canadianfarmland. Subsequently, GIS has beco-me widely and effectively used in natu-ral-resource management. Prominentexamples are timber management,mine permitting, water quality asses-sment, wildlife and habitat manage-ment, land use planning, zoning, andtransportation design; predictivemodelling of earthquakes, forest fires,and flooding; utilities management,routing analysis; marketing and demo-graphic analyses, etc. (Cox andGifford, 1997). The capacity of thesesystems justifies their wider applicationincluding veterinary and human medi-

Fig. 1. Vector map of Europe and attribute(spreadsheet), used in this example to extract Italy.

Fig. 2. Raster map of Europe, digital elevationmodel.

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cal sciences. Several types of analysesroutinely used in GIS can be very use-ful for spatial epidemiology. Theseinclude:

(i) neighbourhood analysis, i.e. allfeatures which meet certaincriteria and are adjacent to aparticular feature are foundand listed;

(ii) buffer generation, i.e. genera-tion of buffer zones around oralong certain features;

(iii) overlay analysis, i.e. merging oftwo or more layers or maps toidentify areas of intersection;

(iv) network analysis, i.e. modellingof networks and calculation ofparameters such as the shortestdistance between two locations;

(v) surface area and distance calcu-lations, and

(vi) three-dimensional surface mo-delling (Ward and Carpenter,2000).

A very useful function of GIS is thekriging, i.e. a linear interpolationmethod that predicts the values of avariable, at non-sampled locations,based on observations at known loca-tions, using a model of the covariance ofa random function (Berke, 2004).Kriging is widely used in meteorology inorder to interpolate values of climatedata from observing stations, and havealso been used in epidemiology to modelthe distribution of various parasites/dis-eases, e.g. Ixodes scapularis that trans-mits Lyme disease (Nicholson andMather, 1996), malaria (Kleinschmidtet al., 2000), alveolar echinococcosis(Conraths et al., 2003; Pleydell et al.,2004), tsetse flies that transmit humanAfrican trypanosomosis (Sciarretta etal., 2005), Calicophoron daubneyi, the

causative agent of paramphistomosis inruminants (Biggeri et al., 2004),Oncomelania hupensins, the interme-diate host snail of Schistosoma japoni-cum (Zhang et al., 2005), co-infectionwith S. mansoni and hookworm amongschoolchildren in Côte d’Ivoire (Raso etal., 2006), human filariosis (Brookerand Micheal, 2000) and canine filariosis(Genchi et al., 2005; Vezzani andCarbajo, 2006).

The term “remote sensing” (RS) wasused - for the first time - in the USduring the 1960s to designate the tech-nique allowing the study of objectswithout any direct contact, throughimage capture. In 1970, in an article tit-led “New eyes for epidemiologists:aerial photography and other remotesensing techniques” Cline recognisedthat RS could have applications indetecting and monitoring disease out-breaks (Cline, 1970, 2006). In the fol-lowing years, scientists of the NationalAeronautics and Space Administration(NASA) in the US, used colour infraredaerial photography to identify the habi-tats of Aedes sollecitans (Hay, 2000).

While aerial photographs were thefirst source of RS data, the subsequentdevelopment of satellite measuringinstruments significantly improvedboth the spatial and temporal coverageof the earth surface, generating a conti-nuous and almost complete cover.

Satellites may be divided into twogroups based on the orbit they follow,namely:

(i) geostationary satellites (e.g.GOES, Meteosat, GMS), and

(ii) near-polar-orbiting (or sun-synchronous) satellites (e.g.NOAA, Landsat, MODIS,SPOT, IKONOS, Quickbird).

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The former group, which consists ofsatellites in orbits parallel with therotation of the earth, is dedicated tometeorological applications, while thelatter follows an elliptical orbit bet-ween 681 and 915 km, with a differentground track after each rotation,lasting about 100 minutes. The revisitperiod varies from 1 to 41 days but canbe lowered. Most of the satellites aresun-synchronous and offer differentspatial resolutions (Herbreteau et al.,2005). However, very few have foundany application in epidemiology, themost important being the Landsat andthe NOAA series (Durr and Gatrell,2004). Satellites have several sensorsrecording radiation in different wave-lengths that allow combination of spec-tral signals (Cringoli et al., 2005a; deLa Rocque et al., 2005). RS provides aunique source of data that can beexploited to characterize climate andland surface variables at different spa-tial resolutions. It permits the calcula-tion of vegetation indices, land surfacetemperatures, atmospheric and soilmoisture, rainfall indices, etc. Amongthe vegetation indices obtained fromRS, the most widely employed one isthe normalized difference vegetationindex (NDVI). It is defined as the dif-ference between the visible (red) andnear-infrared (nir) bands of satelliteinformation over their sum: NDVI =(nir – red)/(nir + red). NDVI is a spe-cific measure of chlorophyll abundanceand light absorption, but its use hasbeen extended to quantify herbaceousvegetation biomass, vegetation primaryproductivity, vegetation coverage andphenology.

The RS data are increasingly used forinvestigations in the field of environ-

mental health sciences for mappingand prediction, surveillance and moni-toring, particularly for vector-bornediseases (Beck et al., 2000). Since thedisease vectors have specific require-ments regarding climate, vegetation,soil and other edaphic factors, and aresensitive to changes in these factors,RS can be used to determine their pre-sent and future and predict distribu-tion. Land cover classification refers tothe natural vegetative cover, functionof the topography, soil or local climate,and can differentiate between differentcovers up to the species communities.Land use classification refers to thedescription of human uses of the land,or immediate actions modifying theland cover, such as agriculture (e.g.used for crop production, lying fallow,irrigated, etc.), human settlements (e.g.urban, rural, isolated infrastructures,etc.), protected areas (e.g. nationalparks, forest reserves, etc.) (for recentreviews see Daniel et al., 2004 andHerbreteau et al., 2005). In Europe,the corine (Coordination ofInformation on the Environment) landcover (CLC, European Commission,2000) is widely used, which is a map ofthe European environmental landscapebased on interpretation of satellite ima-ges. CLC provides comparable digitalmaps of land cover for each countryfor much of Europe. It is useful forenvironmental analysis and compari-sons over large scales (spatial resolu-tion = 1 km). When studies are focu-sed on small areas, aerial photographscan be used because of their capabilityto afford very high spatial resolutionimages (<1 m). The analysis of histori-cal sequences of aerial photographs isan important method for determining

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the medium-term dynamics of landcover on a landscape scale (Acosta etal., 2005). Most developed and deve-loping countries have governmentalprogrammes for aerial surveys butcampaigns are not reproduced on aregular time basis, and ground covera-ge is often limited to areas of discreetinterest.

However, aerial photography must beconsidered as a supplementary sourceof information (Herbreteau et al.,2005). Classifications of landscapefrom digital aerial photographs havebeen scarcely used in veterinary parasi-tology (Rinaldi et al., 2006), with themajority of applications focusing onmosquito surveillance (see for exam-ple, Kline and Wood, 1988; Brown andSethi, 2002).

Disease mapping

One of the most useful functions ofGIS in epidemiology continues to be itsutility in basic mapping.

Usually, when data are collectedeither routinely or through purposely-designed surveys, they are presented intabular forms, which can be exploitedfor analytical usage. However, the rea-ding and interpretation of such data isoften a laborious and time-consumingtask and does not permit easy decision-making (Paolino et al., 2005). On theother hand, representation of thesedata in the form of a map facilitatesinterpretation, synthesis and recogni-tion of frequency and clusters of phe-nomena. Today, GIS, RS and GlobalPositioning Systems (GPS) are well-known tools of the trade and few scien-tists working in the fields mentioned

can manage without them. As we enterthe era of Global Earth ObservationSystems (GEOS), the old adage that apicture is worth more than a 1000words rings truer than ever (Bergquist,2006).

The oldest examples date back morethan 200 years and consist of a worldmap of diseases put forward by Finkein 1792 (Barrett, 2000), and a map ofyellow fever occurrences in the har-bour of New York issued in 1798(Stevenson, 1965). One of the mostprominent examples is the mapping ofcholera victims in relation to the loca-tion of water supplies in London’sSoho district carried out by John Snow(1854). The street addresses of chole-ra victims were recorded and closeproximity to putative pollution sources(i.e. water supply pumps) was identi-fied as the key risk factor. With respectto parasites, in two papers publishedin 1903, Smith and Stiles indepen-dently presented maps of Texas, whichdisplayed the prevalence of hookworminfection and demonstrated that infec-tion was typically restricted to theeastern part of the state where the soilscontain the most sand (Brooker andMichael, 2000).

Development of methods for map-ping diseases using GIS has progressedconsiderably in recent years.

Disease maps can be drawn to ademographic base or to a geographicbase. In the first case, they are relatedto the population and the epidemiolo-gical information they show are pre-sented in relation to population size.Geographically based maps are con-structed according to the shape of acountry or a region or any administra-tive unit. They may be qualitative e.g.

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point maps, distribution maps, pointdistribution maps (PDMs), indicatinglocation without specifying the amountof disease; or quantitative e.g. distribu-tion maps with proportioned peaks,proportional circle maps, choroplethicmaps, choroplethic maps with propor-tioned peaks, PDMs with proportionedpeaks, PDMs with proportioned cir-cles, isoplethic maps, displaying thenumber of cases of disease, the popula-tion at risk, infection prevalence orintensity or incidence (Thrusfield,1995; Cringoli et al., 2005c; Rinaldi etal., 2006). With the appropriate data athand, producing a map using GIS canbe undertaken literally in a matter ofminutes; but therein lies one of the pro-blems with GIS - one needs the spa-tially explicit data - and collecting thesemay take months or even years (Durr,2004). GIS is, however, not only a digi-tal map representation but is indeed aninformational and analysing tool whichpermits the processing of space-relateddata. Certainly, maps themselves keepon playing a prominent role and are, ofcourse, the most frequently used out-put of GIS (Kistemann et al., 2002).

Disease maps drawn by GIS can bethe results of both meta-analysis anddedicated surveys.

Regarding maps derived from meta-analysis, examples on human filariosisare present in literature. For example,the global geographical distribution ofhuman filariosis prevalences, estimatedfor 96 endemic countries in the 1993World Bank Global Burden of Diseasestudy (Michael et al., 1996), showeddifferent patterns for the two lympha-tic filariosis, namely Brancroftian fila-riosis (caused by Wuchereria brancofti)and Brugian filariosis (caused by

Brugia malayi) (Brooker and Michael,2000). More recently, GIS and a data-base on the distribution of lymphaticfilariosis have been used to develop thefirst maps at district-level in India(Sabesan et al., 2000) and Egypt(Hassan, 2004). In addition, GIS andRS, combined with a landscape epide-miological approach, established thegeographical patterns of river blind-ness (caused by Onchocerca volvulus)in southern Venezuela (Botto et al.,2005) and Ethiopia (Gebre-Michael etal., 2005), and the relationship bet-ween environmental features and infec-tion prevalence was also assessed.

GIS is also the tool used by theAfrican Programme for OnchocerciasisControl (APOC) in order to delineatezones of various levels of endemicity,and this is considered a fundamentalstep in the planning process for oncho-cerciasis control (Noma et al., 2002;Seketeli et al., 2002).

With respect to canine and feline fila-riosis, on the basis of a systematicreview of the literature, in a recentpaper, Trotz-Williams and Trees (2003)provided the first evidence-based GISmaps of the distribution of the majorvector-borne infections, includingDirofilaria immitis and Dirofilariarepens in dogs and cats in Europe by athorough investigation of publishedwork on their distribution, prevalenceand incidence, which should be usefulboth to practitioners and researchers inendemic areas and to those in non-endemic areas.

Regarding maps derived from dedica-ted surveys, GIS has become an impor-tant tool for designing a study and ter-ritorial sampling. The fundamentalsteps which can be used to produce

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quality descriptive survey-based mapswithin GIS are the following:

(i) selection of the study area;(ii) selection of the study popula-

tion and calculation of the sam-ple size, using as parameters thestudy population, the expectedprevalence, the confidencelevel, and the standard error;

(iii) selection of the sample in thestudy area (e.g. random sam-pling, systematic sampling, pro-portional allocation, use ofgrids, transect sampling, etc.);

(iv) laboratory and/or field survey;(v) geo-referencing of the study

units (e.g. individuals, group ofanimals, counties, municipali-ties, regions, or any other admi-nistrative unit); and finally

(vi) drawing maps by GIS (Cringoliet al., 2005c).

The sampling procedures in the studyarea play obviously a key role in disea-se mapping. As indicated at point 3above, several sampling methods canbe used. For example, in order to studythe distribution of filarial worms indogs from a certain geographical area,proportional allocation can be used,i.e. the number of dogs to be tested ineach administrative unit (e.g. neigh-bourhood, municipality, etc.) is propor-tional to the dog population in thatadministrative unit, or, if the dog popu-lation is unknown, is proportional tothe administrative unit surface area.

This latter approach has been used byus in order to study canine filariosis inthe Mt. Vesuvius area of southern Italy(Cringoli et al., 2001) and in the city ofNaples (Rinaldi et al., 2004).Regarding the former study, conductedin an area comprising 51 municipalities

(Fig. 3), microfilariae were detected in63 of the 351 dogs surveyed (prevalen-ce = 17.9%); in particular, 56 dogs(15.9%) showed only microfilariae ofAcanthocheilonema (Dipetalonema)reconditum, three dogs (0.8%) onlymicrofilariae of D. repens, two dogs(0.6%) microfilariae of both A. recon-ditum and D. repens and two dogs(0.6%) microfilariae of both D. immi-tis and D. repens (Cringoli et al.,2001). In the study conducted in thecity of Naples (divided into 21 neigh-bourhoods, Fig. 4), microfilariae weredetected in 8 of the 358 dogs surveyed(prevalence = 2.2%); in particular, 5dogs (1.4%) showed only microfilariaeof A. reconditum, three dogs (0.8%)only microfilariae of D. repens, and noD. immitis positive dogs were found(Rinaldi et al., 2004).

Figures 5-9 are distribution mapswith proportioned peaks, which usethe municipality (for the Mt. Vesuviusarea) or the neighbourhood (for thecity of Naples) as the geographic unitof reference and display the followinginformation:

(i) filarial species studied (D.immitis, D. repens and A.reconditum);

(ii) total study area divided intomunicipalities/neighbourhoods;

(iii) municipalities/neighbourhoodswith positive dogs;

(iv) municipalities/neighbourhoodswithout positive dogs; and

(v) municipal/neighbourhood pre-valence (%) determined as fol-lows:(number of positive dogs in themunicipality/neighbourhood)/(number of dogs examined inthe total study area) x 100.

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

Fig. 4. The city of Naples divided into 21 neighbourhoods (administrative boundaries on digital aerial pho-tographs).

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In both the studies, the generaltrends of the maps showed that D.immitis and D. repens were presentonly in a few municipalities/neighbour-hoods, whereas A. reconditum waswidely and homogeneously spreadthroughout the entire study areas, thusindicating that this filarial worm is thepredominant filarial species in dogs inthe Campania region of southern Italy.

The information derived fromdescriptive maps derived from dedica-ted surveys provides an operationaltool for planning, monitoring andmanaging control programmes, and forderiving inferences about the relations-hip between the environment and dis-eases. In fact, without good descriptivemaps of disease agent/vector species’distribution, based on ground observa-tion, we do not have the essential star-ting point to generate predictive mapsbased on statistical pattern-matching(Randolph, 2000). The derivation ofdetailed epidemiological maps, at therelevant spatial resolution, is beingincreasingly recognized as vital to theeffective design and implementation ofsuccessful for the control of parasitesand their vectors (Sabesan et al.,2000).

Fig. 5. Distribution map with proportioned peaks.Acanthocheilonema (Dipetalonema) reconditumin dogs from the Mt. Vesuvius area of southernItaly (from Cringoli et al., 2001).

Fig. 6. Distribution map with proportioned peaks.Dirofilaria repens in dogs from the Mt. Vesuviusarea of southern Italy (from Cringoli et al., 2001).

Fig. 7. Distribution map with proportioned peaks.Dirofilaria immitis in dogs from the Mt. Vesuviusarea of southern Italy (from Cringoli et al., 2001).

Fig. 8. Distribution map with proportioned peaks.Acanthocheilonema (Dipetalonema) reconditumin dogs from the city of Naples (from Rinaldi etal., 2004).

Fig. 9. Distribution map with proportioned peaks.Dirofilaria repens in dogs from the city of Naples(from Rinaldi et al., 2004).

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Ecological analysis

Ecological analysis is targeted on thedescription of relations existing bet-ween the geographic distribution ofdiseases and environmental risk factorsand their analysis by means of statisti-cal procedures (Kistemann et al.,2002). A wide number of papers havebeen published on the analysis of therelationship between disease indicators(e.g. positivity, incidence and prevalen-ce) and the explanatory environmentaland climatic variables (Herbreteau etal., 2005). In order to make ecologicalanalysis, the following fundamentalsteps can be utilized:

(i) GIS construction for the studyarea utilizing data layers onenvironmental and climatic fea-tures (Fig. 10);

(ii) geo-referencing the geographicunits of interest;

(iii) creation of buffer zones of agiven diameter centered onthese geo-referenced points;

(iv) extrapolation of values for eachenvironmental feature withineach buffer zone;

(v) databases with environmentaland parasitological data;

(vi) statistical analyses (univariate,multivariate, etc.) and individua-lize of environmental risk factorsand/or development of forecastmodels (Cringoli et al., 2005c;Rinaldi et al., 2005, 2006).

A part from the administrative boun-daries, the data layers on environmen-tal and climatic features most com-monly used for ecological analysis inveterinary epidemiology are: NDVI,land cover and land use, elevation,slope, aspect, lithofacies map, presence

of lakes, rivers and other water bodies,temperatures, rainfall and humidity.The extrapolation of environmentalvariables can also be directly extractedfrom the study units instead of the buf-fer zones.

Ecological analysis has been pre-viously performed by us in order tostudy the climatic and environmentalfactors that influence the distributionof the rumen fluke C. daubneyi(Cringoli et al., 2004) and of the pro-tozoan Neospora caninum (Rinaldi etal., 2005) in southern Italy.

Environmental factors that maygovern filarial worm distribution maybe expected to affect the life historyparameters of both the mosquito vec-tor and the filarial species.Traditionally, attention in this area hasfocussed primarily on temperature,humidity and elevation (Attenboroughet al., 1997; Brooker and Michael,2000). For example, GIS functionshave been recently used by Sowilem etal. (2006) to identify environmentalindicators of high-risk village forhuman filariosis in Egypt, as indicatedby mosquito density, human infectionrate, vector species composition, meanlife expectancy, as well as environmen-tal variables (geology, hydrology, soiltypes) and meteorological factors (tem-perature, rainfall and humidity); in thisstudy, the most important landscape

Fig. 10. Data layers used for ecological analysis.

30


elements associated with filarial preva-lence were water and different vegeta-tion types. In other recent study con-ducted in India, a range of geo-envi-ronmental variables were selected, andcustomized on GIS platform to developa geo-environmental risk model fordetermining the areas of potentialtransmission of lymphatic filariosis(Sabesan et al., 2006).

Studies on ecological analysis regar-ding canine filariosis are very scant inliterature, probably due to the difficul-ties in obtaining geo-referenced data.

We made an attempt to determine theenvironmental and climatic featuresthat influence the distribution of D.repens in the Lazio region of centralItaly (Scaramozzino et al., 2004). AGIS for the study area was constructedutilizing the following data layers: ele-vation, slope, aspect, NDVI, land coverand minimum, maximum and meantemperature average covering a 30-years period. Data on each of the abovevariables were then calculated for 3 kmdiameter buffer zones centered on thegeo-referenced point of dog sampling(Fig. 11). However, statistical analysisdid not show any significant associationbetween the positivity to D. repens andthe environmental data. This was pro-bably due to the non-uniform spatialdistribution of the sample (the dataused were obtained from a meta-analy-sis, without a sampling design), and tothe low number of positive dogs(1.9%). These latter results point outonce again that the use of GIS does byno means overcome the two major con-cerns of any empirical research: dataavailability and data quality (Kistemannet al., 2002). This issue should alwaysbe kept in mind by all GIS/RS users.

Predicting parasite occurrence/seaso-nality: an useful tool for surveillance

GIS and RS can be also used to pre-dict disease seasonality based on the cli-matic and/or environmental characteri-stics of a certain area and the informa-tion about the climatic and/or environ-mental requirement(s) of a certain spe-cies. Climate-based forecast systems,employing the concept of growingdegree days (GDD), have been develo-ped for different diseases of parasitolo-gical importance, such as fasciolosis,schistosomosis, onchocerciasis andmalaria (Malone, 2005; Gebre-Michaelet al., 2005; Yang et al., 2006).

GIS have been also used in order topredict the occurrence and seasonalityof D. immitis in Europe by us (Genchiet al., 2005) and in Argentina (Vezzani

Fig. 11. NDVI map of the Lazio region obtainedfrom Landsat-5 TM (spatial resolution = 30 x 30m) and 3 km diameter buffer zones centered onthe dog sampling points.

31

and Carbajo, 2006). The heartworm(HW) disease models are based on thefact that climate dictates the seasonaloccurrence of D. immitis transmission.The rate of maturation to infective third-stage larvae (L3) in the mosquitoesdepends mainly on the environmentaltemperature, and there is a threshold ofabout 14°C below which developmentwill not proceed (Fortin and Slocombe,1981). The total environment heatrequired for development may beexpressed in terms of degrees days inexcess of this threshold (HeartwormDevelopment Units – HDUs) (Fortinand Slocombe, 1981; Slocombe et al.,1989). The seasonal HW transmissionmodel formulated by Slocombe et al.(1989) and re-evaluated by Lok andKnight (1998) in Canada and US,respectively, assumes a requirement of130 HDUs for larvae to reach infectivityand a maximum life expectancy of 30days for a vector mosquito. The modelperformed by us (Genchi et al., 2005)was applied using temperature recordsfrom 1846 European Meteorological sta-tions (Fig. 12), spanning a 14-yearperiod, in order to assess the theoretictransmission timing of HW in Europe. Inaddition, a GIS based on thermal regimewas constructed in order to produce pre-dictive maps which assess the durationof the HW transmission risk period, andthe efficient timing of HW chemo-prophylaxis and scheduling of diagnostictesting. The following maps were obtai-ned from the GIS: 1) monthly maps sho-wing the stations that reached the 130HDUs at least once in the studied years(for example, see July in Fig. 13); 2)monthly maps showing the average pre-dicted number of generations (for exam-ple, see July in Fig. 14); 3) a map sho-

wing the yearly average predicted num-ber of HW generations (Fig. 15); 4) amap showing the start and end of theHW transmission period in someEuropean localities (Fig. 16).

The results of the model showed thatHW transmission is markedly seasonalin Europe, with peaks in summer, fromJune to September. The study providedHW risk assessment maps for Europeand suggests that if the actual trend oftemperature increase continues, filarialinfection should spread into previouslyinfection-free areas (Genchi et al.,2005).

The recent models performed byVezzani and Carbajo (2006) in order toassess spatial and seasonal D. immitistransmission risk throughout Argentina,showed that also in the SouthernHemisphere, HW transmission is mar-kedly seasonal, with peaks in Januaryand February (summer).

The D. immitis risk maps performedin Europe and Argentina could be usedby practitioners and health authoritiesto efficiently direct surveillance andcontrol efforts. From a general point ofview, epidemiological surveillanceemploying the use of GIS is characteri-zed by an integrated set of planned epi-demiological activities whose aim is toidentify and prevent new cases of dis-ease. Such a system is justified provi-ded the natural history of the diseaseand its determinants are known andeffective preventive strategies are avai-lable (Rinaldi et al., 2006).

Conclusions

The trends over the past two decadesand their effects in the fields of hard-

Geographical Information Systems32

Geographical Information Systems 33

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

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


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

Fig. 15. HW predictive model based on growing degree days. Yearly average predicted number ofD. immitis generation in Europe (from Genchi et al., 2005).

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ware, software and network techno-logy, in particular in the internetdomain, have created the prerequisitesfor a broad acceptance of GIS and RSwithin the health sciences, includingveterinary science.

Recent studies showed that GIS – inaddition to their broad utility reviewedhere – holds promise in capturing dataon medicine usage (Ryan et al., 2005),as well as in organizing spatial infor-mation involving the distinction of ana-tomic relationships (Ganai et al.,2006). However, epidemiology remainsthe main application field of GIS inveterinary and public health and, sinceepidemiology is inextricably bound to“place”, it seems reasonable to expectthat GIS will further advance as scien-ce (Jacquez, 2000). Perhaps one of themost powerful benefits of GIS is its

ability to integrate different databasesinto a single environment; GIS may bethough of as a database of databases(Cox and Gifford, 1997). In conclu-sion, the present review showed that,with the appropriate data at hand, GIScan be very useful to study the epide-miological patterns of filarial infectionsin veterinary and public health.

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Zhang ZY, Xu DZ, Zhou XN, Zhou Y, Liu SJ,2005. Remote sensing and spatial statisticalanalysis to predict the distribution ofOncomelania hupensis in the marshlands ofChina. Acta Trop 96: 205-212.

38

Biology of filarial worms parasitizing dogs and cats

Maria Teresa Manfredi, Annarita Di Cerbo, Marco Genchi

2

Biology of filarial worms

The nematodes of genus Dirofilariabelong to family Onchocercidae andsubfamily Dirofilariinae of the orderSpirurida (Anderson, 2000). They areslender nematodes and the larval sta-ges are generally found in the definiti-ve host distant from their site of ori-gin. Embryos (microfilariae) developin the uteri of the female worm andare surrounded by the egg membrane.Females are ovoviparous and laymicrofilariae in the blood stream,where they become available to hae-matophagous vectors.

Microfilariae are able of respondingto physiological changes in the hostand may flood into the peripheral cir-culation at a certain time of the day ornight and disappear from the periphe-ral circulation at other time.Microfilariae have structures thatallow their passage through the fineblood capillaries and through the nar-row mouthparts and the gut wall ofthe arthropod vectors.

The genus Dirofilaria is divided intwo subgenera. The subgenusDirofilaria includes Dirofilaria immi-tis and the subgenus Nochtiella inclu-des Dirofilaria repens.

The nematodes of Dirofilaria sub-genus are large filarial wormswithout cuticular ridges except in theventral surface of the caudal extre-mity of the male; the caudal papillaeof the males show only slight asym-metry in number and distribution.

The nematodes of the subgenusNochtiella are relatively small filarialworms with cuticular longitudinalridges; the caudal papillae of themales show a distinct asymmetry innumber and distribution (CanestriTrotti et al., 1997).

Description of Dirofilaria immitis(Leidy, 1856) Railliet and Henry 1911

Dogs are the main hosts, however ithas been reported in wolves, dingoes,coyotes, foxes, various felines (inclu-ding the domestic cat), pinnipeds,mustelids (e.g. ferrets), bears, pandas,beavers, coatis, rabbits, deer, horses,non-human primates and humans.

Most of these infections are sporadicand the microfilaremia could be absent.

The distribution is worldwide and hasbeen found in tropical, subtropical andtemperate regions (Lok, 1988).

Morphology of adults

D. immitis is a long, whitish thin fili-form nematode. Mouth opening is ter-minal without lips and leads into theoesophagus differentiated into muscu-lar and glandular regions without dis-tinction. The oral aperture is surroun-ded by 6 small median papillae and 2lateral papillae (amphids). The adultfemales measure 250 to 310 mm inlength and 1 to 1.3 mm in width and themales are 120-200 mm long and 0.7-0.9mm wide.

Females have an obtuse caudal end;the anus is subterminal and the vulvaropening is located just posterior to thejunction of the oesophagus and intesti-ne. The male caudal end is spirallycoiled and has 2 lateral narrow alae.The cloaca opens to 0.13 mm from thetip of the tail. The ventral side bearspre-anal, per-anal and post-anal papil-lae. The spicules are unequal and withdifferent structure, enlarged at proximalend and acute at distal end. The left spi-cule is 300-375 µm and the right 175-299 µm; the gubernaculum is absent.

41


The cuticle is smooth and ridges andstriations are only present on the ventralsurface of the last coil of the male tail(Uni and Takada, 1986; Lichtenfels etal., 1987).

The adults of D. immitis feed on pla-sma and they can live several months oryears in their hosts.

This nematode is ovoviviparous, andfemales release unsheathed microfilarieinto the bloodstream. They are 290-330 µm long and 5-7 µm wide; thecephalic extremity is tapered and thetail is straight. The tip of the tail ispointed. The microfilarie can survivefrom 2 to 18 months in the blood-stream.

Biology

The microfilariae develop to L3(infective stage) in the intermediatehost, a mosquito of the genera Culex,Aedes, Psorophora, Mansonia orAnopheles. However, the most impor-tant intermediate hosts are those spe-cies without the bucco-paryngealarmature, which is able to damage themicrofilarial cuticle preventing itsdevelopment to the third larval stage.

The microfilariae ingested by mos-quitoes throughout the blood mealmigrate to the Malpighian tubuleswhere they moult into second larvalstage and the third larval stage.Infective L3 migrate from the tubulesto the lumen of the labial sheath in thevector mouthparts. Development inthe mosquito is temperature depen-dent, requiring approximately twoweeks at temperature > 26°C.

Infection is transmitted to a new hostwhen an infected mosquito takes a fur-ther blood meal in a competent host.

The larvae are deposited by the mos-quito throughout a drop of infectedhemolymph in the proximity of bitewound. L3 enter the bite wound andpenetrate the connective tissues,migrate from the subcutaneous or sub-serosal tissues to the muscles andundergo the 3rd and 4th moults. Thefourth stage larva is 8.29-13.2 mmlong (Lichtenfels et al., 1985). Theyundertake extensive migration throughthe subcutis, which continues for some60-90 days until the final to L5 (imma-ture adult). Then, the juvenile wormsmigrate from muscular tissues and tho-racic/abdominal cavities to pulmonaryarteries and right heart within a fewdays of their final moult, presumablycarried by the venous circulation(Kume and Itagaki, 1955; Webber andHawking, 1955; Lichtenfels et al.,1985; Anderson, 2000). The immatureadults penetrate the jugular or otherveins leading them directly to hearthand grow to a maximal length of 20cm. Final maturation and mating occurin the pulmonary arteries. After copu-lation, the females produce unsheathedmicrofilariae approximately 6 and halfmonths after infection (Table 1).Microfilariae are then released into thecirculation.

Description of Dirofilaria repens

D. repens Railliet and Henry, 1911 isa parasite of the subcutaneous connec-tive tissues mainly of dogs. There arereports from domestic cats, genet cats(Genetta tigrina), lions and foxes.Cases have been reported in humans.

This species occurs in southern andEastern Europe, Africa and Asia.

42


Morphology of adults

Adults of D. repens are smaller thanD. immitis. The cuticle is white withdistinct longitudinal and tendertransverse striations. Adult femalesare 100 to 170 mm long and 4.6 to6.5 mm wide and males are 50-70mm long and 3.7-04.5 mm wide. Themale caudal end curves slightlytoward the ventral side. The tail isobtuse, with two lateral alae andoblong pedunculate papillae. The spi-cules are unequal; the larger spiculeis 0.43 mm long and has an acute tip;the smaller spicule is strongly chitini-zed, measures 0.175 mm in lengthand has an obtuse end. In the fema-les, the vulva is situated 1.84 to 1.92mm from cephalic end and it is encir-cled by slightly projecting labia. Thetail has an obtuse tip and curveslightly toward ventral side.

D. repens is ovoviviparous and pro-

duce unsheathed microfilariae living inbloodstream. Microfilarie have theanterior end obtuse, the posterior endtin and pointed ending curved in formof an umbrella handle. The microfilarieare 300-360 µm long and 6-8 µm wideand should be differentiated by othermicrofilariae which can be found in thebloodstream such as D. immitis,Acanthocheilonema (syn. Dipetalonema)reconditum and Dipetalonema dracun-culoides.

Biology

D. repens life cycle requires a mos-quito of genera Aedes, Anopheles,Culex, Armigeres and Mansonoides asintermediate host. The duration ofdevelopment depends on the species ofintermediate host (min 10 days max 21days at a temperature of 24 to 27°C).

Under experimental conditions larvaedeveloped also in horseflies and sand-

Days in the

mosquito

Days in

the dog

Stage Length

cm

Hosts Localization

1

5

10

15

1-15

3-80

70-150

100-160

150-270

Mf

Mf

sausage

L2

L3

L3

L4

L4-L5

L5

gravid female/

mf

0.030

0.030

0.015

0.05

1

1.5

3-7

4-13

4-20

25-30

dog

mosquito

mosquito

mosquito

mosquito

dog

dog

dog

dog

dog

blood

middle gut

Malpighian tubule cells

Malpighian tubule lumen

proboscis

subcutaneous tissue

subcutaneous/muscle tissues

muscle/subcutaneous tissues

thoracic/abdominal cavities/

pulmonary arteries/right heart

pulmonary arteries/right heart

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

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

43


flies (Coluzzi, 1964). Microfilariae of D. repens ingested by

a blood meal reach the stomach of mos-quitoes and migrate to the body cavitywithin 1.5 days and settle in theMalpighian tubules where have 2moults. After attaining the infectivestage larvae migrate to the head andmouthparts of the mosquito.

In the definitive host the infective lar-vae were found in sections of the sub-cutaneous connective tissue up to 48hours after inoculation. The larvae donot have long migrations in the subcu-

taneous connective and reach the matu-rity here (Table 2). The macrofilariaeare found between the connectivelayers in most part of the body. Theywere often found on the trunk and fewwere found in the legs and head(Webber and Hawking, 1955). Usually,microfilarie do not show periodicity inthe peripheral blood, but microfilare-mia has a tendency to increase seaso-nally (from August to September inItaly) (Cancrini et al., 1975). The pre-patent period is 27-34 weeks. Adultscan live several years.

Mf: MicrofilariaeNote: The duration of the life cycle is unknown

Days in the

mosquito

Days in

the dog

Stage Length

cm

Hosts Localization

1

2-7

6-9

10-20

1-15

3-70

70-150

180-240

Mf

Mf

sausage

L2

L3

L3

L4

L5

gravid female/

mf

0.035

0.035

0.015

0.05

1.00

1.00

n.d.

n.d.

10-17

dog

mosquito

mosquito

mosquito

mosquito

dog

dog

dog

dog

blood

middle gut

Malpighian tubule cells

Malpighian tubule lumen

proboscis

subcutaneous connective tissue

n.d.

n.d.

subcutaneous tissue/

perimuscular connective fasciae

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

Dirofilaria immitisCaudal end of male, ventral view

Dirofilaria immitisCephalic end of female, ventral view

44


References

Anderson RC, 2000. Nematode parasites of verte-brates, their development and transmission.2nd Edition, CABI Publishing, Wallingford,UK, 650 p.

Cancrini G, Coluzzi M, Balbo T, Gallo MG, 1975.Variazioni stagionali della microfilariemia edeffetto della temperatura ambientale in caniparassitati da Dirofilaria repens. Parassitologia17: 75-82.

Canestri Trotti G, Pampiglione S, Rivasi F, 1997.The species of the genus Dirofilaria Railliet &Henry, 1911. Parassitologia 39: 369-374.

Coluzzi M, 1964. Osservazioni sperimentali sulcomportamento di Dirofilaria repens in diversigruppi di artropodi ematofagi. Parassitologia 6:57-62.

Lichtenfelds JR, Pilitt PA, Kotani T, PowersKG, 1985. Morphogenesis of developmentalstages of Dirofilaria immitis (Nematoda) inthe dog. Proc Helmintol Soc Washington 52:98-113.

Lok JB, 1988. Dirofilaria sp: taxonomy and distri-bution. In: Dirofilariasis. Boreham P.F.L. andAtwell RB Eds, CRC Press, Boca Raton Florida,249 p.

Kume S, Itagaki S, 1955. On the life cycle ofDirofilaria immitis in the dog as the final host.Brit Vet J 111: 16-24.

Uni S, Takada S, 1986. The longitudinal cuticularmarkings of Dirofilaria immitis adult worm.Japan J Parasitol 35 (3): 191-199.

Webber WAF, Hawking F, 1955. Experimentalmaintenance of Dirofilaria repens and D. immi-tis in dogs. Exp Parasitol 4: 143-164.

Dirofilaria immitisCephalic end of female, ventral view

Dirofilaria immitisAnterior end of female, ventral view

Dirofilaria immitisPosterior end of female, lateral view

Dirofilaria immitisadult female (over) and male (under)

45

Vectors of Dirofilaria nematodes:biology, behaviour and host/parasite relationships

Gabriella Cancrini, Simona Gabrielli

3

Vectors of Dirofilaria nematodes

In the last century many importantaspects of the diseases due toDirofilaria immitis and D. repens havebeen studied and elucidated, includingpathogenesis and parasite transmission.However, there are still many unan-swered questions concerning thespecies that can act as vector of theseparasites in the world.

Experimental studies have shown thatdirofilarial nematodes can develop,more or less completely, in numerousarthropods but, on the basis of currentknowledge, only the insects, namely themosquitoes (order Diptera, suborderNematocera, family Culicidae), act asvector.

The Culicidae have a cosmopolitandistribution, with over 3500 speciesthroughout the world. The high degreeof adaptability of these insects hasassured their presence in every type ofenvironmental habitat. In fact, mosqui-toes are found in low-lying plains, hillyareas, in mountainous zones andmarine habitats, where each species hasadapted its egg laying activity and lar-val development to the types of watersurfaces there available. Some specieslay their eggs only in fresh rain water,others in stagnant pools or in any ves-sel that contains water; others lay inquiet pools at the edges of streams, andsome even in salt water.

In tropical areas, mosquitoes areactive all year round, whereas in tem-perate climates adults are active duringthe late spring and summer, and havedifferent seasonal rhythms of activity.Environmental and climatic changes, inparticular the predicted rise of themean temperature (+0.2°C in 10 years)as a consequence of the “global warm-ing” (Romi, 2001; Genchi et al., 2005)

are now strongly influencing the activi-ty patterns of mosquitoes in temperateareas.

The male mosquito feeds mainly onfruit juices, whereas the female is alsohematophagous. This blood-suckinghabit allows to the female participate tothe filariosis transmission.

Finding a host and host preference

To find resting sites or environmentsuitable for mating or ovoposition, orto find a source of blood host, mosqui-toes move owing to phototropism andin response to chemical stimuli. Theyare attracted to carbon dioxide andodour produced by the host, which isthen localized by the warmth and mois-ture radiated from its body. Followingthese stimuli, some mosquito speciestravel only a few hundred meters fromtheir larval habitats, whereas others(several species of Aedes) can go as faras 50 km (Hocking, 1953). They maycover long distances carried mostly bywinds (passive movement) or by flyingactively in successive stages.

The host-seeking behaviour of themosquito follows different patterns,depending on the species. Some areactive only during the nocturnal hours(Cx. pipiens and most of theAnopheles) whereas others predomi-nantly in the early morning (like An.gambiae, An. balabacensis, An. mac-ulipennis), or on the daytime (Ae.albopictus). Species such as Ae. aegyp-ti, Ae. caspius, Ae. vexans and Cx.modestus show two peaks of feedingactivity, one at sundown and the otherat dawn (Matting, 1969; Di Sacco etal., 1992; Pollono et al., 1998). These

49


biological rhythms are not a fixed rulebut the most common behaviour of thespecies, often the result of co-evolutionwith the parasite transmitted, andtherefore usually correlate to the possi-ble major success to fed on parasitizedanimals. During this species-specificcircadian window, other factors maymodulate the expression of activity. Forexample, the age of the female caninfluence activity levels. Shortly afterthe emergence, there may be a briefperiod during which the insect is obliv-ious to host stimuli, and holder individ-uals may respond much differently thanwhen they were younger (Nayar andSauerman, 1973). In general, the olderfemale is more epidemiologicallyimportant by virtue of its increasedopportunity to acquire and transmitpathogens. Nutritional state also affectsthe intensity of activity peaks; as theperiod of blood deprivation lengthens,activity patterns become more intense(Klowden, 1996). When all factorscontribute to the increase in activitylevels, they make the female more like-ly to encounter host stimuli, and it maythen find itself in the vicinity of a host.

Based on their preference to feed out-doors or inside dwellings, mosquitospecies can be divided into exophagicand endophagic, respectively. As farresting habits, species can be furtherclassified as endophilic (remainingafter feeding within human habitationsfor most of the gonotrophic cycle) orexophilic (spending most of their timeoutside). It is clear that the differentbehaviour patterns can influence sever-al epidemiological factors: the avail-ability of suitable hosts; the tempera-ture at which the filarial larvae developwithin the mosquito; the exposure to

potential predators (therefore the lifespan of the mosquito).

As far the host preference, severalspecies are strictly zoophilic or “spe-cialists” (limit feeding to specific typesof hosts), several are less restrictive andmore largely zoophilic or “generalists”(have the ability to identify and feed ona wide variety of host types) and evenanthropophilic. The last species areimportant for the dirofilariosis trans-mission from animals to humans. Hostuse patterns may vary seasonallybetween geographic regions and, as therelative abundance of different hosts,changes in an area.

Mosquito feeding preferences maydepend on several factors, including thebehaviour and attractiveness of the host.Actually the mosquito never seeks a hostas such, but simply responds to hostkairomones by orienting in their direc-tion. These kairomones are released asdiscontinuous filaments or packed ofstimuli, broken up by the wind as theymove downwind, like the dispersionpattern of a smoke plume from a chim-ney. The host seeking steps may bebypassed in the laboratory experi-ments, but are of critical importance innature. In fact, those individuals thatfeed when placed directly on a host arenot necessarily those that will seek itfrom a distance.

Dog and cat baited traps, operated toinvestigate on the species that can actas vectors, showed that the dog attractsa large number of Ae. caspius, Ae.scapularis, Ae. taeniorhynchus, Cx.pipiens, Cx. quinquefasciatus, Cx.declarator, Cx. nigripalpus, An. mac-ulipennis and Cs. annulata and, underthe same field conditions, larger thandoes the cat (Di Sacco et al., 1992,

50


1994; Genchi et al., 1992; Iori et al.,1990; Pollono et al., 1994; Labarthe etal., 1998) (Fig. 1). It is likely that thedog does not represent the preferredhost of all individual mosquitoes ofthese species (for example, Cx. pipiensand Cs. annulata were originallyornithophilic and have adapted to bit-ing man and other mammals; Ae.caspius, on the other hand, is highlyanthropophilic), but is however animportant food source. Moreover it hasbeen shown that Culex species are notparticularly attracted to the cat, butother studied species are even less so(Pollono et al., 1998; Labarthe et al.,1998). The feeding activity of thesemosquito species begins at sundownand lasts throughout the night. Dogshave a habit of sleeping during the noc-turnal hours and their resting places

offer an ideal environment for theinsect (warmth, carbon dioxide). Cats,on the other hand, in natural condi-tions, are nocturnal hunters and moveabout quite a lot. It is possible that themosquito, who needs sufficient contacttime with the host in order to feed, isdisturbed by the cat movement. This,along with the cat smaller body mass,may explain the insect feeding prefer-ence for dogs and the higher prevalenceof infection observed in dogs comparedto cats, who likely play only a marginalrole as reservoir for the disease. In fact,cat infections induced by simulatednatural exposure to Ae. aegypti experi-mentally infected with D. repens or D.immitis confirmed that mosquitoes donot feed willingly on the cat (Cancriniet al., 1979; Cancrini and Iori, 1981;Mansour et al., 1995).

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

51


Development of Dirofilaria parasitesin the invertebrate host

When the female mosquito fed onDirofilaria infected animals, it takes upwith the blood embryos (microfilariae)that can develop into infecting L3 onlyin the “competent” invertebrate host.

Moreover, the number of larvae thatcan successfully complete their devel-opment depends on the vector efficien-cy of the mosquito individual.

During the blood meal the microfilar-iae (270-365 µm in length and 6-8 µmwide, depending on the species) passthrough the pharynx and reach themid-gut where they remain for approx-imately 24 hours. They then migrate tothe Malpighian tubules and invade thecells of the distal end (Fig. 2), wherethey undergo transformation to the so-called “sausage stage”(first stage larvaor L1, about 70 µm in length), mute tothe L2 stage and finally, into L3. TheL3 (1,100 µm in length) leaves theMalpighian tubules by perforating their

distal end, migrate through the haemo-cele to reach the labium. During themosquito probing and blood feeding onthe vertebrate host infective L3emerges from the folded labium andrests on the skin of the host immersedin a drop of haemolymph to avoiddehydration. It then enters the hostwhen the insect has pierced the skin. Itis impossible to distinguish by mor-phology between the developing larvaeof D. immitis and D. repens (Nelson,1959; Nelson et al., 1962).

The time required for larval develop-ment in the mosquito is temperature-dependent: it takes 8-10 days at 28-30°C, 11-12 days at 24°C, and 16-20days at 22°C. Larval development ceas-es at temperatures below 18°C, but itcan resume if the mosquitoes areplaced at higher temperatures(Cancrini et al., 1988) (Fig. 3).

The initial invasion of the Malpighiancells by L1 and the penetration of thetubule walls by the exiting L3 are bothcritical moments for the mosquito sur-

Fig. 2. Scansion electron microscopy at 2 days afterexperimental infection: a microfilaria of D. repensis penetrating in a primary cells of the Malpighiantubules with the cefalic end (a), whereas its caudalend is still free in the lumen of the tubules (b).

Fig. 3. Experimental infection of Cx. pipiens withD. repens: development times and location of thelarvae observed.

52

a

b


vival. When the parasite load is heavy,tubule function is compromised andthe insect dies. The maximum numberof larvae compatible with the insectsurvival depends on the species of mos-quito and the species of Dirofilaria andit is lower for D. immitis than for D.repens (Le Coroller, 1957; Coluzzi,1964; Christensen, 1978; Russell andGeary, 1996).

Host-parasite relationship and vectorefficiency

The percentage of microfilariae takenup with a blood meal that completetheir development to infective L3 canvary from 0 to 100%. In general, theefficient vector is the female that mod-erates the parasite invasion and allowsthe development of the maximum num-ber of infective larvae compatible withits survival. Therefore, different speciesor different individuals within a speciescan be more or less efficient vectors ofthe parasite, and it depends on variousfactors.

Parasitic burden can affect the mos-quito survival, so mosquitoes have dif-ferent defence mechanisms they use toblock larval development and conse-quently to control infective larval load.The presence of the cibarial armature,for example, is an efficient mechanicaltool for damaging microfilariae as theypass through the pharynx (Fig. 4). Therythmic opening-closing action of thecibarial pump, armed with sharp teeth,can provoke serious lesions to the cuti-cle of the microfilariae, leading toembryonic death and elimination(Coluzzi and Trabucchi, 1968) (Fig. 5).The coagulation time of the blood

Fig. 4. Cibarial armature of Cx. pipiens, showingsmooth teeth in a brush-like structure and pointedthorn-like projections. 1) Cross section of the phar-ingeal valve with teeth clearly evident (D). 2)Cibarial armature observed from the cibarial pump.3) Longitudinal section through the area zigrinata(AZ) with thorn-like projections on top and cibar-ial armature, situated between the cibarial (PC) andpharingeal pumps (PF). 4) Thorn-like projections(S) in the area zigrinata (from Coluzzi et al., 1981).

Fig. 5. D. repens microfilariae after ingestion by amosquito without (1), and with (2,3) cibarial arma-ture. In the first case, the larva is intact, whereas inthe second, lesions to the cuticle are clearly evident(2), even causing a complete break in the larva’sbody (3) (from Coluzzi and Trabucchi, 1968).

53

1

3

2


taken up during the meal is another fac-tor that influences vector competence.In fact, if the blood coagulates quickly,the microfilariae remain trapped in theclot, and can not reach the tubules(Frizzi and Pedrotti, 1957; Grieve etal., 1983). Therefore, mosquitoes thatproduce anticoagulant substances(such as An. quadrimaculatus) aremore receptive to infection becausethey allow the microfilariae to moreeasily reach the Malpighian tubules andgo on their development.

Other defence mechanisms have beenobserved in different mosquito species.For example, Ae. aegypti can secretesubstances that induce epicuticle lysis(Fig. 6) and, like Ae. scapularis and Ae.taeniorhynchus, is able to encapsulatein a melanotic reaction microfilariaepresent in Malpighian tubules, causingmicrofilaria death (Kartman, 1953;Beernsten et al., 1994; Forton et al.,1985; Bradley and Nayar, 1985; Vegni-Talluri et al., 1993; Vegni-Talluri andCancrini, 1994). On the contrary,haemocytes, are not able to cause anydamage to the L3 that, having left thetubules (Fig. 7), migrate towards thehead of the insect (Vegni-Talluri andCancrini, 1991). Another defencemechanism, even if somewhat less effi-cient, is based upon the mosquito abil-ity to control development times for thelarvae, that may become incompatiblewith the insect life span (Cancrini andIori, 1981; Cancrini et al., 1995).

The receptiveness/refractoriness toinfection and the efficiency of the mos-quito as vector for both D. immitis(Zielke, 1973; McGreevy et al., 1974)and D. repens (Coluzzi and Cancrini,1974) is genetically determined and itis controlled by a sex-linked recessive

Fig. 7. Transmission electron microscopy of D.repens L3 (cross-section) 10 days after experi-mental infection of Ae. aegypti, situated in thehaemocele. A cytoplasmic projection from ahaemocyte (h), adhering to the L3 (l), has pro-duced and deposited an electron-dense sub-stance that has not, however, caused any damageto the cuticle.

Fig. 6. Cross-section of D. immitis microfilariawithin a primary cell of the Malpighian tubules ofAe. aegypti. Transmission electron microscopy at6 days after experimental infection: the host cell(on the left of each image) has produced electron-dense material that has begun to deposit on thecuticle wall of the embryo (mf, on the right of eachimage), causing lysis.

54


allele. The genes that control the traitare different for D. repens and D.immitis. Variations in vector competen-cy have been identified in both naturaland laboratory mosquito populations.

The importance of a mosquito in theepidemiology of dirofilarial infectionsnot only depends upon the receptive-ness to infection and on the efficiencyin transmitting infective larvae, butalso on the size of the vector popula-tion, its feeding pattern, life span andseasonality. In fact, in order to transmitthe infection, the vector must take atleast two blood meals, and in thisrespect autogenous species, which canuse protein reserves accumulated dur-ing the larval stages to produce the firstbatch of eggs, are less important as vec-tors than those species that require sev-eral blood meals for egg laying, thusincreasing the chances for larval uptakeand transmission. The life span of theinsect must be long enough to guaran-tee larval development to the infectivestage and is thus influenced by theexophagic and exophilic behaviour ofcertain species. The seasonal activity isalso important and those species thatreproduce actively all year round or sev-eral times during the summer are moreefficient. Ambient temperature, humid-ity and predation are further factorsthat influence the mosquito survival.

Arthropod vectors

Most of the data on the arthropodspecies that can act as vector derivesfrom laboratory experiments started inthe last century (Grassi and Noè,1900). Experimental infections haveshown that dirofilarial nematodes can

develop in numerous insects (severalmosquito species and biting flies) andthat varying levels and modes of resist-ance to infection exist in other bitingflies, simulides, phlebotomes, culicoids,fleas, and in ticks (Coluzzi, 1964).

Then, it has been demonstrated thatsome mosquito species are more com-petent than others to transmit the para-site, and that within a single speciessome individuals or strains are moreefficient vectors than others (Coluzziand Trabucchi, 1968; McGreevy et al.,1974; Bemrick and Moorehouse,1968). Capture studies carried out inendemic areas to identify natural vec-tors have suggested a possible role ofsome species of Culicidae on the basisof their abundance. However, crucialprogress in knowledge on the speciesactually involved in the parasite trans-mission has been made through thefieldwork based on animal-baited traps.This method allows to restrict the inter-est only to species that are effectivelyattracted to the host, and to evaluatethe biological factors that affect theactual dirofilaria infection risk. Thesecapture studies on host animals (dog,cat, and/or man) have been carried outin the United States of America, Italyand Brasil, and have identified as pos-sible vectors Cx. erraticus, Cx. modes-tus, Cx. nigripalpis, Cx. pipiens, Cx.quinquefasciatus, Ae. canadensis, Ae.caspius, Ae. excrucians, Ae. scapularis,Ae. sierrensis, Ae. sollicitans, Ae. stim-ulans, Ae. taeniorhynchus, Ae. trivitta-tus, Ae. vexans and An. maculipenniss.l. (Sauerman, 1985; Favia et al.,1996). Further species, like Ae. can-tans, Ae. cinereus, Ae. geniculatus, An.claviger, Cq. richiardii, Cx. declarator,Cx. pipiens-restuans, Cx. sultanensis,

55


Cx. territans and Cs. annulata feed lesswillingly on dogs and cats, and there-fore could be of minor interest in theparasite transmission (Di Sacco et al.,1992; Genchi et al., 1992).

Several studies have reported themosquito species that, by microscopy,have been found in nature infectedwith dirofilarial larvae but, unfortu-nately, it is not possible to morphologi-cally distinguish developing larvaebelonging to the 27 recognized speciesof Dirofilaria. First unambiguousresults have been achieved through theanalysis of the collected insects bymolecular techniques recently adjusted(Favia et al., 1996), which allows toreliably distinguish D. immitis and D.repens larval stages developing in theinvertebrate host (Cancrini andKramer, 2001). On this basis, entomo-logical investigations to date carriedout in Italy and performed using PCR-based technologies have shown that Ae.albopictus, Cx. pipiens and An. mac-ulipennis s.l. proved natural vectors forboth D. immitis and D. repens, where-as Cq. richiardii is almost certainty vec-tor for D. immitis. Moreover,abdomens of Cx. modestus, Cx. torren-tium, Ae. punctor, Ae. cinereus, Ae.detritus, and Ae. geniculatus have beenfound positive to D. immitis. Thosespecies could act as vectors, but theirrole needs further confirmations, beingthe abdomen a location common toeither just ingested or infectious larvae(Rossi et al., 2002; Cancrini et al.,2003a,b, 2004, 2006). A more exten-sive application of the molecular tech-niques will allow a clearer and morecomprehensive understanding of theepidemiology of subcutaneous dirofi-lariosis and of feline and canine heart-

worm infection, in particular the sea-sonal transmission patterns in the dif-ferent geographical areas and the mon-itoring of infection rates among differ-ent vectors.

These first results have been obtainedby studying several dirofilariosis areasin northern and central Italy. They areof concern because the main naturalvector (to date Ae. albopictus) is aspecies proven very efficient in the par-asite transmission, “generalist” in thehost choice and highly anthropophilic.

The presence now stable of this mos-quito in Italy suggests that the infectionrisk for animals and humans isincreased, at least because of the simul-taneous presence of vectors showingdiurnal and nocturnal activity patterns.

The same concern could be addressedto Croatia. In fact, although to the bestof our knowledge no data are availablefor dirofilariosis natural vectors in theCountry, the existence of a culicidofau-na almost overlapping to that presentin Italy (Ramsdale et al., 2001;Ramsdale and Show, 2006; Merdic andBoca, 2004) suggests the possibleimportance of the same species.

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Bemrick WY, Moorehouse DE, 1968. Potentialvectors of Dirofilaria immitis in the Brisbanearea of Queensland, Australia. J Med Entomol5: 269-272.

Bradley TJ, Nayar JK, 1985. Intracellular melaniza-tion of the larvae of Dirofilaria immitis in themalpighian tubules of the mosquito, Aedes sollic-itans. J Invertebr Pathol 45: 339-345.

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Cancrini G, Iori A, 1981. Ulteriori osservazionisulla infestazione sperimentale del gatto conDirofilaria repens. Parassitologia 23: 145-147.

Cancrini G, Kramer LH, 2001. Insect vectors ofDirofilaria spp. In: Simon F and Genchi C (Eds),Heartworm infection in humans and animals.Universidad Salamanca Ed, 63-82 pp.

Cancrini G, Frangipane di Regalbono A, Ricci I,Tessarin C, Gabrielli S, Pietrobelli M, 2003b.Aedes albopictus is a natural vector ofDirofilaria immitis in Italy. Vet Parasitol 118:195-202.

Cancrini G, Gabrielli S, Frangipane di RegalbonoA, Magi M, Scaramozzino P, Toma L, 2004.Advances in the identification of natural vectorsof zoonotic filariae by PCR-based approaches.Proc IX EMOP, 400 p.

Cancrini G, Magi M, Gabrielli S, Arispici M,Tolari F, Dell’Omodarme M, Prati MC, 2006.Natural Vectors of Dirofilariasis in Rural AndUrban Areas of the Tuscan region (CentralItaly). J Med Entomol 43: 574-579.

Cancrini G, Mantovani A, Coluzzi M, 1979.Infestazione sperimentale del gatto con D. repensdi origine canina. Parassitologia 21: 89-90.

Cancrini G, Pietrobelli M, Frangipane DiRegalbono A, Tampieri MP, della Torre A,1995. Development of Dirofilaria and Setarianematodes in Aedes albopictus. Parassitologia37: 141-145.

Cancrini G, Romi R, Gabrielli S, Toma L, Di PaoloM, Scaramozzino P, 2003a. First finding ofDirofilaria repens in a natural population ofAedes albopictus. Med Vet Entomol 17: 448-451.

Cancrini G, Sun Yanchang, della Torre A, ColuzziM, 1988. Influenza della temperatura sullo svi-luppo larvale di Dirofilaria repens in diversespecie di zanzare. Parassitologia 30: 38.

Christensen BM, 1978. Dirofilaria immitis: effecton the longevity of Aedes trivittatus. ExpParasitol 44: 116-123.

Coluzzi M, 1964. Osservazioni sperimentali sulcomportamento di Dirofilaria repens in diversigruppi di artropodi vettori. Parassitologia 4:57-62.

Coluzzi M, Cancrini G, 1974. Genetica dellasuscettibilità di Aedes aegypti a D. repens.Parassitologia 16: 239-256.

Coluzzi M, Dallai R, Insom E, 1981. Morfologia efunzione delle armature cibariche nelle zanzare.Parassitologia 23: 164-168.

Coluzzi M, Trabucchi R, 1968. Importanza del-l’armatura bucco-faringea in Anopheles e Culex

in relazione alle infezioni con Dirofilaria.Parassitologia 10: 47-59.

Di Sacco B, Cancrini G, Genchi C, 1992. Studiodel tropismo nei riguardi del cane e del gatto daparte dei ditteri potenziali vettori delle filariosiin provincia di Pavia. Parassitologia 34: 11-12.

Di Sacco B, Del Zoppo B, Genchi C, 1994. Surveyof dog Dirofilaria immitis mosquito vectors inMilan and vicinity. Parassitologia 36 (suppl): 50.

Favia G, Lanfrancotti A, della Torre A, CancriniG, Coluzzi M, 1996. A PCR- based method toidentify Dirofilaria repens and D. immitis.Parasitology 113: 567-571.

Forton KF, Christensen BM, Sutherland DR,1985. Ultrastructure of the melanization respon-se of Aedes trivittatus against inoculatedDirofilaria immitis microfilariae. J Parasitol 71:331-341.

Frizzi G, Pedrotti D, 1957. Resistenza allaDirofilaria immitis (Leidy) in alcune specie diAnopheles dei sottogeneri Nyssorhynchus eMyzomyia. Simp Genet Pavia 2: 338-348.

Genchi C, Di Sacco B, Cancrini G, 1992.Epizoology of canine and feline heartworminfection in Northern Italy: possible mosquitovectors. Proc Heartworm Symp, 39-46 pp.

Genchi C, Rinaldi L, Cascone C, Mortarino M,Cringoli G, 2005. Is heartworm disease reallyspreading in Europe? Vet Parasitol 133:137–141.

Grassi B, Noè G, 1900. The propagation of thefilariae of the blood exclusively by means of thepuncture of peculiar mosquitoes. Brit Med J 3:1306-1307.

Grieve RB, Lok JB, Glickman LT, 1983.Epidemiology of canine heartworm infection.Epidemiol Rev 5: 220-246.

Iori A, Cancrini G, Vezzoni A, Del Ninno G, TassiP, Genchi C, della Torre A, Coluzzi M, 1990.Osservazioni sul ruolo di Culex pipiens nella tra-smissione della filariasi del cane in Italia.Parassitologia 32: 151-152.

Hocking B, 1953. The intrinsic range and speed offligth in insects. Trans R Ent Soc Lond 104: 223.

Kartman L, 1953. Factors influencing infection ofthe mosquito with Dirofilaria immitis Leidy,1856. Exp Parasitol 2: 27-78.

Klowden MJ, 1996. Vector behavior. In: TheBiology of disease vectors. B.J. Beaty and W.C.Marquard Es, University Press, Colorado.

Labarthe N, Serrao ML, Melo YF, de Oliveira SJ,Lourenco-de-Oliveira R, 1998. Potential vectorsof Dirofilaria immitis (Leidy 1856) in

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Itacoatiara, oceanic region of Niteroi municipal-ity, state of Rio de Janeiro, Brazil. Mem InstOswaldo Cruz 93: 425-432.

Le Corroler Y, 1957. Evolution de Dirofilariaimmitis Leidy, 1856 chez Aedes (Stegomyia)aegypti Lin. Souche “Orlando” (Floride). BullSoc Pathol Exo 50: 542-555.

Mansour AE, McCall JW, McTier TL, Ricketts R,1995. Epidemiology of feline diroifilariasis. ProcHeartworm Symp, 87-95 pp.

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Mc Greevy PB, Mc Clelland GAH, LavoipierreMMJ, 1974. Inheritance of susceptibility toDirofilaria immitis infection in Aedes aegypti.Ann Trop Med Parasitol 68: 97-109.

Nayar JK, Sauerman DM, 1973. A comparativestudy of flight performance and fuel utilizationas a function of age of females of Floridamosquitoes. J Ins Physiol 19: 1977-1988.

Nelson GS, 1959. The identification of infectivefilarial larvae mosquitoes: a note on the speciesfound in wild mosquitoes on the Kenya Coast. JHelminthol 133: 233-256.

Nelson GS, Heish RB, Furlong M, 1962. Studies infilariasis in East Africa. II. Filarial infections inman, animals and mosquitoes on the KenyaCoast. Trans Roy Soc Trop Med Hyg 56: 202-217.

Pollono F, Cancrini G, Rossi L, 1994. Sampling ofdog attracted mosquitoes in Piedmont.Parassitologia 36 (suppl.): 114.

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Ramsdale CD, Alten B, Caglar SS, Ozer N, 2001.A revised, annotate checklist of the mosquitoes

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Sauerman DM Jr, 1985. A survey for naturalpotential vectors of Dirofilaria immitis in Verobeach. Mosq News 43: 222-225.

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Pathogenesis of Dirofilaria spp.infections

Giulio Grandi, Tatjana Zivicnjak, Relja Beck

4

Pathogenesis of Dirofilaria

Introduction

Dirofilaria spp., helminths belongingto Dirofilariinae superfamily, are able tocause a wide range of diseases. The bestknown and studied is heartworm dis-ease, common in dogs and cats in areaswhere Dirofilaria immitis is widespread.Other species of Dirofilaria, like D.repens, are less important from a patho-genic point of view, but their differentia-tion from D. immitis is a fundamentaldiagnostic task. The zoonosic risks relat-ed to each species is also extremelyimportant.

Pathogenesis of heartworm disease(D. immitis)

D. immitis infection is characterisedby several different clinical pictures,caused both by adults and first stagelarvae (microfilariae). However, thepathophysiology of heartworm diseaseis mainly due to the presence of adultworms in the pulmonary arteries. Theprimary lesions occur in the pulmonaryarteries and lung parenchyma and aremostly attributable to the intravascularadult parasites. They cause pulmonaryhypertension that, if not treated, pro-gresses inevitably to congestive heartfailure. Other syndromes are related tothe disturbance of blood flow due tothe location of heartworms in the rightatrium at the level of tricuspidal valve.

This event causes massive haemolysisand related haemoglobinuria, responsi-ble for the vena caval syndrome (liverfailure disease) (Ishihara et al., 1978;Kitagawa et al., 1986). Microfilariaeplay a relatively minor pathogenic rolebut may cause clinically significant

pneumonitis and glomerulonephritis. Some individuals develop a hypersen-

sitivity to microfilariae. Occasionally,aberrant migration results in parasitesbecoming trapped in ectopic locationslike the anterior chamber of the eye(Weiner et al., 1980) or systemic arter-ies (Liu et al., 1966; Slonka et al.,1977). Data regarding immunopatho-genesis and in particular the role ofcytokines, mediators as well as cellularcomponents of the immunity in thedevelopment of heartworm-relatedlesions have been recently revised(Grandi et al., 2005). Although thespectrum of pathologies related toheartworm infection is broad, the mostimportant clinical manifestation indogs is congestive heart failure (corpulmonale).

Congestive heart failure (CHF, syn.:cor pulmonale)

Heartworms are primary agents of vas-cular disease, rather than the cause ofsimple obstruction and/or blood flowdisturbances. Intimal proliferationoccurs in arteries occupied by livingworms and embolic worm fragmentstrigger thrombosis, both of which maycompletely obstruct segments of the pul-monary arteries (Adcock, 1961). Theseeffects, both leading to pulmonaryhypertension, are strongly correlated toworm burden, in turn related to thedegree of their distribution through lungparenchyma (Knight, 1980).

The juvenile adult heartworms areonly about 2.5 cm long when theyreach the systemic venous circulation.They passively embolize the pulmonaryarteries and are disbursed in propor-

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tion to the lobar blood flow. Generally,the larger right caudal lobar arteryaccumulates more worms than the left(Atwell and Rezakhani, 1986). Contactbetween the parasite and the intima ofthe pulmonary arteries is an important,if not essential, initial step in the devel-opment of the endovascular lesions.

The earliest lesions are limited to thesmall peripheral branches where theworms first come to rest. As the para-site grows, lesions occur in more prox-imal segments. Intimal thickening andnarrowing of the vessel lumen in smallperipheral branches of the pulmonaryarteries are the major cause of obstruct-ed blood flow and pulmonary hyperten-sion. The intimal proliferation iscaused by migration of medial smoothmuscle cells through the internal elas-tic laminae (Munnel et al., 1980;Patterson and Luginbuhl, 1963;Schaub et al., 1981).

The pathogenesis of the arteritiscaused by heartworms remains a matterof speculation. Disruption of endothelialcell junction and denuding of the intimalsurface are characteristics of the firstlesions that occur only after a few daysof presence of worms. Physical trauma,metabolic and immune-mediated cyto-toxicity induced by the parasite (Keith etal., 1983a), as well as other less likelymechanisms (Schaub and Rawlings,1980) have been considered. Severalfacts seem to be more consistent withmechanical trauma as the initiatingevent rather than cytotoxicity or induc-tion of host immune responses. The evi-dence suggests that injury to theendothelium occurs immediately uponarrival of the parasite, too soon for thecomponents of an immune response tofall in place without prior sensitisation.

Furthermore, the disappearance ofendothelial cells occurs without evi-dence of degeneration and is followed byan aggressive build-up of cells and struc-tural elements. This suggests that cellshave been dislodged rather thandestroyed in situ. Macrophages, granu-locytes, and platelets are attracted to thesite of endothelial damage and adhere tothe exposed subendothelium. Shortlyafter their arrival, vascular smooth mus-cle cells migrate into the intima and avery active process of myointimal prolif-eration produces rapid growth of thelesions. The integrity of the endotheliumcovering the intraluminal villous-rugosegrowths is restored and thrombosis isnot a feature at this stage of the disease(Munnel et al., 1980; Schaub et al.,1981). The prominence of platelets inthe acute lesions and their documentedability to stimulate growth of the vascu-lar smooth muscle, through the releaseof platelet-derived growth factors(PDGFs) (Ross, 1986), are hypothe-sized to be a likely mechanism for trig-gering and sustaining the growth ofthese lesions (Schaub and Rawlings,1980; Schaub et al., 1981). The circum-ferential lesions of the peripheral dis-tributing arteries transcend into morediscontinuous rugose ridges in the larg-er elastic branches (Keith et al., 1983b).

In cross section, these ridges have avillous appearance and are consideredpathognomonic for heartworm infec-tion. Although the lesions thicken thewall of these large elastic vessels andproduce a rough texture on the intimalsurface, they do not obstruct blood flowby narrowing the lumen. On the con-trary, the large distributing arteries actu-ally dilate as pulmonary hypertensionbecomes increasingly severe.

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Pulmonary blood flow is impeded pri-marily by the reduction in cross-sec-tional area of the arterial vascular bed,caused by obliterative endarteritis ofsmall peripheral branches. Recently, inheartworm infected dogs it has beendemonstrated that there are markedlyincreased plasma level of endothelin-1,a mediator that induces acute vasocon-striction and chronic vascular remodel-ling. It is probable that both theseevents contribute in turn to the devel-opment of pulmonary hypertension(Uchide and Saida, 2005). Thrombosisand thromboembolism may furthercompromise the pulmonary circulation.

As worms accumulate, lesions alsodevelop in the large distributing arter-ies, which dilate and become stiffer aspulmonary blood pressure rises. Thedecreased distension of the large ves-sels significantly increases cardiac workby coupling the right ventricle directlyto the high vascular resistance in theobstructed peripheral vasculature.

Right ventricular hypertrophy is acompensatory response to theincreased pressure load. As heartworminfection impedes flow in an increasingnumber of branches, the pulmonaryvascular reserve diminishes. For a time,normal pulmonary blood pressure ispreserved at rest and rises only modest-ly during exercise as patent arteriesreach full distension. Eventually, thepulmonary arterial tree is restricted tothe point that it assumes the character-istics of a system of rigid tubes and pul-monary vascular resistance becomesfixed. This occurs about the time pul-monary blood pressure becomes elevat-ed at rest (Knight, 1977). At this stage,pressure rises in direct proportion tofurther increase in flow. Consequently,

the more severe the disease and activethe patient, the more cardiac workmust be performed. In advanced casesof heartworm disease, low-output con-gestive heart failure develops as a resultof the right ventricle’s inability to gen-erate and sustain the high perfusionpressures required to move bloodthrough the lung. At a molecular level,in the myocardium of heartworminfected dogs it has been observed adecrease of extracellular collagenmatrix; this event can contribute to thedilatation of the ventricle, therebymarkedly affecting the systolic anddiastolic functions of the heart (Wanget al., 2005). Frequently, dogs at thisstage experience syncope whenattempting to suddenly increase car-diac output. Right-sided congestiveheart failure (R-CHF) with ascites,hepatomegaly, and cachexia is a latesequela and may be precipitated by anacute episode of pulmonary throm-boembolism.

D. immitis infection in the cat

The general pulmonary pathology inthe cat is similar to the one observed indog. Muscular hypertrophy, villousendarteritis, and cellular infiltrates ofthe adventitia are typically more severein the caudal pulmonary arteries(Dillon, 1998). A characteristic featureof the reaction in felines in response toheartworm infection is the developmentof severe muscular hypertrophy in thesmaller arteries (McCall et al., 1994).The host’s response to the parasite isintense and the role of inflammation isvery important, being platelet factorsrelease less important in the cat rather

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than in the dog (Furlanello et al., 1998).The cause of the acute and often fatalcrisis in the cat is lung injury resultingin respiratory distress. Frequently thisevent is associated with the death of aslittle as one adult heartworm. The lungcan become acutely edematous and res-piratory failure, instead of heart failure,becomes the life threatening event.Being pulmonary hypertension an occa-sional event, right sided heart failureand severe cor pulmonale is uncommonin feline heartworm infection (Dillon,1998; Genchi et al., 1995).

D. repens infection

Much less is known about the patho-genesis of D. repens infection, whichusually is characterized by the occur-rence of painless subcutaneous nodulesin which adult parasites reside (Bredalet al., 1998). The localization of adultworms can vary, and recently it hasbeen reported also the presence of afemale of D. repens in the conjunctivaof a dog (Hermosilla et al., 2006). Thisasymptomatic pattern of infection,which seems to be the most common,transforms the discovery of D. repensinfection in an accidental finding. Thisoccurs mainly in dogs (and cats) thatare showing other symptoms or dis-eases, not related to the presence ofthese nematodes in the subcutaneoustissues. Indeed, the majority of infectedanimals do not present any clinicalsign, notwithstanding the occurrence ofpersistent microfilaremia, thereforemaking it difficult to assess the patho-genicity of D. repens (Soulsby, 1982).

Some authors classified the rare clin-ical manifestations observed in associa-

tion with the presence of D. repens intotwo clinical syndromes (Scarzi, 1995).The first one is characterized by anodular multifocal dermatitis, mainlylocalized in facial region (Scott andVaughn, 1987), the second instead ischaracterized by the presence of sever-al pruriginous papulae; this one is con-sidered similar to sarcoptic mange(Halliwell and Gorman, 1989),although the clinical experience of oneof us (Tatjana Zivicnjak) suggests thatpruritus rarely occurs in D. repensmicrofilaremic dogs. The most frequentreports, however, are of dermatologicalsigns (generalized dermatitis, localizedalopecia, scratching and rubbing) asso-ciated with the presence of adults andmicrofilariae in the skin (Lee Gross etal., 1992; Kamalu, 1986; Mandelli andMantovani, 1966; Scarzi, 1995;Zivicnjak et al., 2006).

It has been suggested that the pathol-ogy may be significant in cases of mas-sive infection (Kamalu, 1991; Mandelliand Mantovani, 1966; Mantovani,1965; Restani et al., 1962). In the fewcases found to be massively infectedwith adult worms and with simultane-ously high microfilaremia, gross andhistopathological changes have beenreported in many organs, like spleen,liver, kidneys, lungs, heart and brain(Kamalu, 1991; Mandelli andMantovani, 1966, Restani et al., 1962).

The nature of these lesions suggestscombined mechanical and immuno-pathological effects elicited by bothmicro- and macro-filariae (Mantovani,1965), also if it is not possible to deter-mine what underlying mechanisms maybe involved, also due to the lack ofexperimental infection with reproduc-tion of these clinical signs. Nothing is

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currently known about the potentialability of microfilariae to cause hyper-sensitivity and inflammatory reactionsat the cutaneous level, although this islikely one of the major mechanismsinvolved. Furthermore, it has been sug-gested that D. repens-related dermatitisis a conditioned pathology, that requiresthe presence of other infectious agentsor stress (Bonvicini, 1910; Beaufils andMartin-Granel, 1987; Cazelles andMontagner, 1995). Single case reportsof acute liver failure in the cat (Schwanet al., 2000) and cutaneous hyperpig-mentation in a dog have been attributedto D. repens infection (Kamalu, 1991).

Conclusions

The pathogenesis of D. immitis iswell known, while that of D. repens isstill far from being understood, also ifin the last years this parasite has beensuspected to be not as harmless as usu-ally considered. However, both para-sites are important agents of zoonosis,in particular D. repens, and veterinari-ans play an essential role in protectinghumans from infection. For this reason,it is essential that clinical signs are rec-ognized, correct diagnosis is made andefficient treatment and prophylaxis areguaranteed not only to safeguard thehealth and well-being of our pets, butalso that of our clients, neighbours andourselves.

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Keith JC, Rawlings CA, Schaub RG, 1983b.Pulmonary thromboembolism during therapy ofdirofilariasis with thiacetarsamide: modificationwith aspirin or prednisolone. Am J Vet Res 44:1278-1283.

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Kitagawa H, Sasaki Y, Ishihara K, 1986. Clinicalstudies on canine dirofilarial hemoglobinuria:relationship between the heartworm mass at thetricuspid valve orifice and the plasma hemoglo-bin concentration. Jpn J Vet Sci 48: 99-103.

Knight DH, 1977. Heartworm heart disease. AdvVet Sci Comp Med 21: 107-149.

Knight DH, 1980. Evolution of pulmonary arterydisease in canine dirofilariasis: evaluation byblood pressure measurements and angiography.In Proceedings of the Heartworm Symposium’80 (Otto GF Ed), Am Heart Soc, BonnerSprings, KS, 55-62 pp.

Knight DH, 1987. Heartworm infection. Vet. Clin.North Am: Small Anim Pract 17: 1463-1518.

Lee Gross T, Ihrke PJ, Walder EJ, 1992. Veterinarydermatopathology. Mosby Year Book.

Liu SK, Das KM, Tashjian RJ, 1966. AdultDirofilaria immitis in the arterial system of adog. J Am Vet Med Assoc 148: 1501-1507.

Mandelli G, Mantovani A, 1966. Su di un caso diinfestazione massiva da Dirofilaria repens nelcane. Parassitologia 8: 21-28.

Mantovani A, 1965. Canine filariasis byDirofilaria repens. Proceedings of the 32ndAnnual Meeting of the American AnimalHospital Association, 77-79 pp.

McCall JW, Calvert CA, Rawlings CA, 1994.Heartworm infection in cats: a life-threateningdisease. Veterinary-Medicine 89: 639-647.

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Patterson DF, Luginbuhl HR, 1963. Clinico-pathologic conference: case presentation. J AmVet Med Assoc 143: 619-628.

Restani R, Rossi G, Semproni G, 1962. Due inte-ressanti reperti clinici in cani portatori diDirofilaria repens. Atti della Società Italianadelle Scienze Veterinarie 16: 406-412.

Ross R, 1986. The pathogenesis of atherosclerosis:an update. N Engl J Med 314: 488-500.

Scarzi M, 1995. La dirofilariosi cutanea nel cane.Obiettivi Documenti Vet 16 (6): 11-15.

Schaub RG, Rawlings CA, 1980. Pulmonary vas-cular response during phases of canine heart-worm disease: scanning electron microscopystudy. Am J Vet Res 41: 1082-1089.

Schaub RG, Rawlings CA, Keith JC, 1981.Platelet adhesion and myointimal proliferationin canine pulmonary arteries. Am J Pathol 104:13-22.

Schwan EV, Miller DB, de Kock D, van HeerdenA, 2000. Dirofilaria repens in a cat with acuteliver failure. J S Afr vet Assoc 71: 197-200.

Scott DW, Vaughn TC, 1987. Papulonodular der-matitis in a dog with occult filariasis. CompAnim Pract 1: 31.

Slonka GF, Castleman W, Krum S, 1977. Adultheartworms in arteries and veins of a dog. J AmVet Med Assoc 170: 717-719.

Soulsby EJL, 1982. Helminths. In Helminths,Arthropods and Protozoa of DomesticatedAnimals, 7th edition, Williams & Wilkins,Baltimore, 311 p.

Uchide T, Saida K, 2005. Elevated endothelin-1expression in dogs with heartworm disease. JVet Med Sci 67: 1155-1161.

Wang JS, Tung KC, Huang CC, Lai CH, 2005.Alteration of extracellular collagen matrix in themyocardium of canines infected with Dirofilariaimmitis. Vet Parasitol 131: 261-265.

Weiner DJ, Aguirre G, Dubielzig R, 1980.Ectopic-site filariid infection with immunologicfollow-up of the host. In: Proceedings of theHeartworm Symposium ’80 (Otto GF Ed), AmHeart Soc, Bonner Springs, KS, 51-54.

Z ivicnjak T, Martinkovic F, Beck R, 2006.Dirofilariosis in Croatia: spread and publichealth impact. 5th Croatian congress on infec-tive diseases, Zadar, Croatia.

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Immunopathogenesis of filarialinfections in dogs and cats: a role for Wolbachiaendosymbiont?

Laura H. Kramer

5

Immonopathogenesis: a role for Wolbachia

Introduction

Dirofilaria immitis is the causativeagent of canine and feline heartwormdisease. Adult worms live in the pul-monary arteries, females produce first-stage larvae (“microfilariae”) which aretaken up by mosquitoes who then inturn transmit the infection to other ani-mals. In dogs, the infection is chronicand leads to congestive heart failure. D.immitis, like most filarial worms stud-ied so far, harbour bacteria calledWolbachia which are thought to playan essential role in the biology andreproductive functions of their filarialhosts. Wolbachia pipientis, the onlyspecies thus far identified in the genus,are gram-negative bacteria belonging tothe order Rickettsiales (just likeEhrlichia spp and Anaplasma spp;Bandi et al., 2001). In adult D. immitis,Wolbachia is predominantly foundthroughout cells in the hypodermis,which is directly under the worm’scuticle. In females, Wolbachia is alsopresent in the ovaries, oocytes anddeveloping embryonic stages within theuteri. This suggests that the bacteriumis vertically transmitted through thecytoplasm of the egg.

What does Wolbachia do in its filarialhost?

There are several reasons why it isthought that the presence of Wolbachiais essential for a filarial worm’s survival:

(i) in those species of filarialworms that have been identifiedas harbouring Wolbachia, all ofthe individuals are infected: i.e.100% prevalence;

(ii) the evolution of the bacteriamatch that of the filarial worms,and phylogenic studies haveshown that the two organismshave been “walking hand-in-hand” for millions of years;

(iii) the bacteria are transmittedfrom female to off-spring and inthis way Wolbachia increases itsown fitness by increasing thefitness of the host that isinvolved in its transmission;

(iv) removal of Wolbachia (antibi-otics/radiation) leads to sterilityof female worms and eventualdeath of adults.

It is still unclear however exactlywhat Wolbachia does to make it soimportant for its filarial host. It hasbeen suggested that, while the filarialworm likely supplies the bacteria withamino acids necessary for growth andreplication, Wolbachia on the otherhand may produce several importantmolecules that are essential for heart-worms, like glutathione and haeme(Foster et al., 2005). It is indeed a “onehand washing the other” situation thatmay, however, be the key to novelstrategies for the control/treatment offilarial infection, including canine andfeline heartworm disease.

The role of Wolbachia in the inflam-matory and immune response in D.immitis-infected animals

As a gram-negative bacteria, Wolb-achia has the potential to play animportant role in the pathogenesis andimmune response to filarial infection.The possible consequences of the mas-sive release of Wolbachia in the filarial-

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infected host has been evaluated. Wolbachia are released both by living

worms and following worm deaththrough natural attrition, microfilarialturnover and pharmacological inter-vention (Taylor et al., 2001). In humanand murine models of infection, therelease of bacteria has been shown tobe associated with the up-regulation ofpro-inflammatory cytokines, neu-trophil recruitment and an increase inspecific immunoglobulins. Ongoingstudies in dogs with heartworm diseasemay shed light on what happens wheninfected animals come into contactwith the bacteria. For example, werecently tested the hypothesis thatD. immitis-infected dogs come intocontact with Wolbachia either throughmicrofilarial turnover or natural deathof adult worms (Kramer et al., 2005).In our study, positive staining forWolbachia was observed in various tis-sues from dogs who had died from nat-ural heartworm disease. Bacteria wereobserved in the lungs and particularlyin organs like the kidney and liver,

where microfilariae normally circulate(Fig. 1). Furthermore, when we lookedat specific antibody responses toWolbachia, we observed a strongerresponse in dogs with circulatingmicrofilariae compared to dogs withoccult infection, supporting thehypothesis that microfilarial turnover isan important source of Wolbachia indogs with heartworm disease.Furthermore, Wolbachia from D.immitis has been shown to provokechemiokinesis and pro-inflammatorycytokine production in canine neu-trophils (Bazzocchi et al., 2003, 2005).Cats with heartworm disease also pro-duce antibodies to Wolbachia.Interestingly, it has recently beenreported that the development of astrong antibody response against D.immitis occurs after one-two months ofinfection and this may have importantimplications for early diagnosis(Morchon et al., 2004).

Areas of future research shouldinclude the possible diagnostic use ofspecific immune responses to

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

A B

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Wolabchia, it’s potential immunomod-ulatory activity (prevention) and theeffects of antibiotic treatment in infect-ed animals.

What are the effects of antibiotic treat-ment on filarial worms?

Wolbachia can be eliminated fromfilarial worms through antibiotic thera-py of the infected host. Numerous stud-ies have shown that various treatmentprotocols/dosages (tetracycline andsynthetic derivatives appear to be themost effective), are able to drasticallyreduce if not completely remove theendosymbiont from the worm host.Such depletion of Wolbachia is thenfollowed by clear anti-filarial effects,including:

(i) inhibition of larval develop-ment: it has been shown thatantibiotic treatment of filarial-infected hosts can inhibit moult-ing, an essential process in thematuration of worms from lar-vae to adult;

(ii) female worm sterility: antibiotictreatment leads first to a reduc-tion and then to the completeand sustained absence of micro-filariae. Researchers at theUniversity of Milan, Italy havereported that D. immitis adultstaken from naturally-infecteddogs that had been treated with20 mg kg-1 day-1 of doxycyclinefor 30 days showed morphlogi-cal alterations of uterine contentwith a dramatic decrease in thenumber of pretzels and stretchedmicrofilariae, indicating thatbacteriostatic antibiotic treat-

ment was able to block embryo-genesis (Genchi et al., 1998;Bandi et al., 1999);

(iii) adulticide effects: this a particu-larly intriguing aspect of antibi-otic treatment of the filarialworm-infected host and onethat merits strict attention.

Recently, clinical trials in human filari-asis have reported extremely promisingresults: a recent placebo-controlledtrial in humans infected withWuchereria bancrofti has demonstrateda clear macrofilaricidal effect of doxy-cycline (Taylor et al., 2005). Whenadministered for 8 weeks at 200mg/day, the treatment resulted in acomplete amicrofilaremia in 28/32patients assessed and a lack of scrotalworm nests (where adult worms reside)at 14 months post-treatment, as deter-mined by ultrasonography in 21/27patients. In the other patients, the num-ber of worm nests declined. This wassignificantly different from placebopatients, where lack of worm nests wasonly observed in 3/27. This is the firstreport of adulticide activity in a humanfilarial worm with antibiotics. Couldantibiotic treatment have the sameeffect on D. immitis? Research isunderway to answer this very impor-tant question.

What are the effects of antibiotic treat-ment on the filarial worm-infectedhost?

It is very likely that antibiotic treat-ment will have some beneficial effectson subjects with filariasis. First of all,those effects described above on theworm will themselves lead to improved

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clinical presentation: for example, areduction in circulating microfilariae.However, if we consider Wolbachia asa potential cause of inflammation in thecourse of filarial disease, depletion ofthe bacteria may be beneficial, inde-pendently of its effect on the worm.There is little data concerning theeffects of antibiotic treatment in dogswith natural heartworm disease. Weknow however that such treatmentdrastically reduces Wolbachia loads inD. immitis. Preliminary trials in natu-rally infected dogs have shown thatdoxycycline treatment before adulticidetherapy with melarsomine may helpreduce pro-inflammatory reactions dueto the death of adult worms (as seen bylower antibody levels againstWolbachia and lower levels of inter-leukin 8, an inflammatory cytokineinvolved in nuetrophil recruitment).These results have encouraged us tocontinue evaluation of the clinical ben-efits of antibiotic treatment in naturallyinfected dogs.

Given the recent and very promisingdevelopments in the use of tetracy-clines for micro-macrofilaricidal thera-py in human filariosis, it is hoped thatsimilar attention will be given to canineand feline heartworm disease thatcould greatly benefit from alternativetherapeutic strategies.

References

Bandi C, McCall JW, Genchi C, Corona S, VencoL and Sacchi L, 1999. Effects of tetracycline onthe filarial worms Brugia pahangi andDirofilaria immitis and their bacterialendosymbionts Wolbachia. Int J Parasitol 29:357-364.

Bandi C, Trees AJ and Brattig NW, 2001.

Wolbachia in filarial nematodes: evolutionaryaspects and implications for the pathogenesisand treatment of filarial diseases. Vet Parasitol98: 215-238.

Bazzocchi C, Genchi C, Paltrinieri S, Lecchi.,Mortarino M, Bandi C, 2003. Immunologicalrole of the endosymbionts of Dirofilaria immi-tis: the Wolbachia surface protein activatescanine neutrophils with production of IL-8. VetParasitol 117: 73-83.

Bazzocchi C, Mortarino M, Comazzi S, Bandi C,Franceschi A, Genchi C, 2005. Expression andfunction of toll-like receptor 2 in canine bloodphagocytes. Vet Immunol Immunopathol 104:15-19.

Foster J, Ganatra M, Kamal I, Ware J, MakarovaK, Ivanova N, Bhattacharyya A, Kapatral V,Kumar S, Posfai J, Vincze T, Ingram J, MoranL, Lapidus A, Omelchenko M, Kyrpides N,Ghedin E, Wang S, Goltsman E, Joukov V,Ostrovskaya O,Tsukerman K, Mazur M,Comb D, Koonin E and Slatko B, 2005. TheWolbachia genome of Brugia malayi:endosymbiont evolution within a humanpathogenic nematode. PLoS Biology 3: 121-129.

Genchi C, Sacchi L, Bandi C, Venco L, 1998.Preliminary results on the effect of tetacyclineon the embryogenesis and symbiotic maceria(Wolbachia) of Dirofilaria immitis. Anupdate and discussion. Parassitologia 40:247-249.

Kramer LH, Tamarozzi F, Morchon R, Lopez-Belmonte J, Marcos- Atxutegi C, Martin-PachoR, Simon F, 2005. Immune response to and tis-sue localization of the Wolbachia surface pro-tein (WSP) in dogs with natural heartworm(Dirofilaria immitis) infection. Vet ImmunolImmunopathol 106: 303-308.

Morchon R, Ferreira A C, Martin-Pacho J,Montoya A, Mortarino M, Genchi C, Simon F,2004. Specific IgG antibody response againstantigens of Dirofilaria immitis and itsWolbachia endosymbiont bacterium in catswith natural and experimental infections. VetParasitol 125: 313-321.

Taylor MJ, Cross HF, Ford L, Makunde W H,Prasad GBKS, Bilo K, 2001. Wolbachia bacte-ria in filarial immunity and disease. ParasiteImmunol 23: 401-409.

Taylor MJ, Makunde WH, McGarry HF, TurnerJD, Mand S, and Hoerauf A, 2005.Macrofilaricidal activity after doxycycline treat-ment of Wuchereria bancrofti: a double-blind,randomised placebo-controlled trial. Lancet365: 2116-2121.

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A possible role for Wolbachiain the diagnosis of Dirofilariainfections

Fernando Simón, Laura H. Kramer, Rodrigo Morchón, Claudio Genchi

6

Diagnosis of Dirofilaria: a role for Wolbachia

Introduction

Dirofilaria immitis is the aetiologicalagent of cardiopulmonary dirofilariosisin dogs and cats, and pulmonary dirofi-lariosis in man, while Dirofilaria(Nochtiella) repens causes subcuta-neous dirofilariosis in animal reservoirsand subcutaneous/ocular dirofilariosisin man. Like other filarial species, D.immitis and D. repens, harbour intra-cellular bacteria of the genusWolbachia (Rickettsiales), whose par-ticipation in the development of theinflammatory pathology of dirofilario-sis has been extensively investigated.Nevertheless, the role of Wolbachia asa possible diagnostic tool has beenpoorly studied until now.

Here we present our experience relat-ed to Wolbachia in Dirofilaria spp.canine, feline and human infections, aswell as in a murine model of immu-nization, and its possible importancefor the diagnosis of Dirofilaria infec-tion in humans.

Wolbachia interacts with Dirofilaria-infected hosts

There is direct and indirect evidencethat Wolbachia comes into contactwith hosts infected with D. immitis andD. repens. Direct evidence has beenobtained in dogs and humans byimmunohistochemistry techniques,while indirect evidence is based on thepresence of specific antibodies andinflammatory mediators observed indogs, cats and humans, as well as inmice immunized with Dirofilaria spp.or Wolbachia-derived antigens.

Mice: The first evidence of interac-

tion between Wolbachia and the hostimmune system was obtained in miceimmunized with crude D. immitis adultsomatic and L3 antigens. These micedevelop specific antibodies against theWolbachia surface protein (WSP)between 25 and 90 days post-inocula-tion (Marcos-Atxutegi et al., 2003). Ina different experiment, mice immu-nized with WSP and/or a heat shockprotein (GroEl) from Wolbachiashowed a strong specific-antibodyresponse, demonstrating the contact ofWolbachia molecules with murineimmune cells, and an intense expres-sion of different cytokines and innateinflammatory mediators a few daysafter inoculation (data not published).

Dogs: Both microfilaremic and ami-crofilaremic naturally-infected dogsdeveloped a high production of IgGantibodies against WSP. Microfilaremicdogs showed significantly higher IgGantidody levels than amicrofilaremicdogs (Kramer et al., 2005a). Eighteenout of 128 dogs living in an endemicarea were positive to a validated, com-mercial kit for circulating antigens.Among these, 15 were also positive forIgG anti-WSP. Moreover, other 43 dogsthat were negative for circulating anti-gens showed elevated levels of IgG anti-WSP antibodies (data not published).

Evidence of the presence ofWolbachia itself or its molecules inDirofilaria-infected hosts was obtainedby an inmunohistochemistry using apolyclonal antiserum against theWolbachia surface protein (WSP).Positive staining for WSP (Fig. 1a and1b) was obtained in kidney tubules,glomerules, lung tissue (free bacteria)and alveolar macrophages and hepaticmonocytes of naturally infected dogs

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(Kramer et al., 2005a,b).Cats: Specific anti-WSP IgG antibod-

ies were detected in experimentallyinfected cats and in cats naturallyexposed to a high risk of heartworminfection. Nine out of 49 cats living inan endemic area of the Canary Islands(Spain) were positive to a validated,commercial antibody kit and to anexperimental ELISA for the detection ofantibodies against synthetic peptidesderived from molecules of 22 and 30kDa of the adult worms. Among thesecats, 5 were positive to IgG-ELISA

against WSP. Other 8 cats were positiveto anti-WSP IgG but negative to theELISA test for anti-WSP antibodies(Morchón et al., 2004).

Humans: Specific IgG antibodiesagainst WSP were identified in patientsdiagnosed as having clinical dirofilario-sis (Fig. 2). The level of antibodyresponse was significantly higher inpatients with pulmonary or subcuta-neous dirofilariosis than in clinicallyhealthy individuals living in endemicareas of canine dirofilariosis; amongthese only a part developed anti-WSP

A B

C D

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. Alveolarmacrophage (dogs). C. Hypodermic chord. D. Morulae surrounded by immune cells (worms in subcu-taneous nodules from humans).

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antibodies (Simón et al., 2003 and datanot published).

In humans diagnosed as having sub-cutaneous dirofilariosis, positive stain-ing for WSP was present in inflamma-tory cells (Fig. 1c and 1d), in very closeproximity to WSP+ oocytes and in onecase WSP+ morulae were seen freewithin inflammatory tissue. In thosenodules where worms were undergoingdegeneration, there were many cellswithin the inflammatory granulomathat stained intensely positive for WSP(data not published).

An alternative to demonstrate that aworm removed from an infected personis a Dirofilaria, is the polymerase chainreaction (PCR). Amplification ofWolbachia DNA with specific primerscan be achieved and may be applieddirectly on worms extracted from nod-ules, or samples obtained by differenthistological techniques from nodules inwhich the worms have been destroyedby the granulomatous reaction.

Wolbachia stimulates a Th1-typeresponse

Several studies have been conductedto identify the type of immuneresponse stimulated by Wolbachia indifferent hosts of Dirofilaria spp. Miceimmunized with WSP alone or in com-bination with GroEl developed astrong response of IgG2a antibodies(related to a Th1-type response in thisspecies) immediately after inoculation.The highest levels were observed onday 20 of the experiment (13 daysafter the last inoculation) and similarlevels were observed 10 days latter(data not published). In dogs with nat-ural D. immitis infections, both micro-filaremic and amicrofilaremic individ-uals showed significantly higher IgG2antibody response against WSP thannegative controls. The response is sig-nificantly higher in microfilaremicthan in amicrofilaremic dogs (Krameret al., 2005b). Humans diagnosed ashaving pulmonary dirofilariosis alsodevelop significantly higher IgG1 anti-body levels (Th1-type response inhumans) than healthy individuals liv-ing in endemic areas. No antibodiesrelated to Th2-type response weredetected in any human hosts (Marcos-Atxutegi et al., 2004).

Specific anti-Wolbachia antibody lev-els are modified by drug treatment

Dogs with naturally acquired heart-worm disease and treated with melar-somine showed a strong increase ofspecific IgG anti-WSP antibodies a fewdays after treatment (Fig. 3a). Themaximum antibody levels were detect-

Fig. 2. Specific IgG anti-WSP antibody responsein humans. A. Individuals diagnosed as havingpulmonary dirofilariosis. B. Humans diagnosed ashaving subcutaneous dirofilariosis. C. Healthydonors residing in an endemic area. D. Healthydonors living in a non endemic area.

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ed at the end of the study (21 or 63days post-treatment depending the pro-tocol employed) (data not published).

Change in the levels of anti-Wolbachia and anti-Dirofilaria immitisantibodies following filaricide treat-ment was observed in a group of exper-imentally infected-, ivermectin treated-cats (Morchón et al., 2004). A dramat-ic increase of specific IgG antibodiesagainst both WSP and Dirofilariaimmitis peptides (Dipp) was found 15days after ivermectin treatment. At 3.5months post-treatment the anti-Dippantibodies decreased to values seenbefore infection, while specific anti-WSP antibodies continued to increasetill the end of the study 5.5 monthspost-treatment. In the control group(infected-, untreated cats) the antibodyresponse against both WSP and Dippincreased continuously throughout thestudy (Fig. 3b).

Human pulmonary or subcutaneousdirofilarioses do not require treatment.

Nevertheless, the antibody response toboth Dirofilaria and Wolbachia anti-gens was measured during the follow-up of a subcunjuntival case due to D.repens, until 6 months after theremoval of an intact, live female worm(Ruiz-Moreno et al., 1998). An ELISAtest for antibodies against D. repensadult worms was positive until 3months after the removal of the worm,while the ELISA for anti-WSP antibod-ies was positive before removal, butwas negative in the subsequent threeblood samples (1, 3 and 6 months afterremoval of the worm).

Conclusions

The accurate diagnosis of canine andfeline D. immitis infections is the firststep towards the correct prevention ofthe spread of the disease in the animalreservoirs and human infections. Thedevelopment of the parasite and the

Fig. 3. Changes in specific IgG anti-WSP antibody levels in Dirofilaria immitis naturally infected dogstreated 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. Experimentallyinfected cats treated with ivermectin (B).

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signs it causes are different in dogs andcats. Thus, the methods validated forthe diagnosis in these two hosts are alsodifferent. Considering the excellentperformance of existing commercialkits for the detection of antigens andantibodies for canine and feline dirofi-lariosis, respectively, the moderate/lowcorrespondence of Wolbachia antibodytests with these kits, and the existenceof other diagnostic techniques likedetection of microfilariae and echocar-diography, the application of serologicdiagnostic tests based on the detectionof anti-Wolbachia antibodies in animaldirofilariosis is not useful.

Nevertheless, the detection of anti-Wolbachia antibodies could be of inter-est in:

(i) epidemiological studies forevaluation of the real pressureof infection in an endemic area.In this case, risk levels are rep-resented not only by activeinfection (detected by commer-cial kits), but also by prepatentinfections, mainly in feline diro-filariosis. In these cases, a testfor the detection of specificantibodies against Wolbachiacould be an invaluable diagnos-tic tool, as shown by the resultsdiscussed above;

(ii) evaluation of the efficacy ofmacro/microfilaricidal treat-ment. The death of filarialworms is the main factor forthe release of Wolbachia,increasing the stimulus of thehost’s immune system. Thedetection of a rise of anti-Wolbachia antibody levels,together with a fall of the anti-Dirofilaria antibodies can be

interpreted as sign of drug effi-cacy. Results of serological fol-low-up in the human subcon-juntival case previously pre-sented confirms that massiverelease of Wolbachia occurswhen worms are physicallydestroyed, because in this casethe removal of whole wormcauses an immediate and clearfall of specific anti-Wolbachiaantibodies.

In the case of human pulmonary andsubcutaneous/ocular dirofilariosis, thesuspicion of neoplasia caused by thepresence of a nodule makes differentialdiagnosis of prime importance. Thisimplies that once the dirofilarial originof a nodule is demonstrated, the spe-cific identification is a secondary mat-ter (Simón et al., 2005). When wormsare present inside the nodules, the spe-cific identification is possible by histol-ogy, but if they have been destroyed bythe inflammatory reaction or the physi-cian takes a conservative attitude, evi-dence for the presence of Wolbachiacan contribute to the identification ofthe filarial origin of the nodule. Thiscan be obtained by antibody tests, PCRor immunohistochemical techniques.In the first case invasive techniques arenot necessary, while in the other two,tissue samples must be obtained.

In conclusion, in spite of the exis-tence of validated methods for thediagnosis of dirofilariosis in animalreservoirs based on the use or detec-tion of Dirofilaria molecules, the iden-tification of Wolbachia by both directand indirect methods, seems an excel-lent complementary data and consti-tute an effective tool for epidemiologi-cal studies and research related to

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inflammatory pathology. In humansthe detection of anti-Wolbachia anti-bodies can be a fundamental tool in thedifferential diagnosis for the identifica-tion of the genus Dirofilaria in cases inwhich the worms have been previouslydestroyed by the granulomatous reac-tion of the host.

References

Kramer LH, Tamarozzi F, Morchón R, López-Belmonte J, Marcos-Atxutegi C, Martín-PachoR, Simón F, 2005a. Immune response to and tis-sue localization of the Wolbachia surface pro-tein (WSP) in dogs with natural heartworm(Dirofilaria immitis) infection. Vet ImmunolImmunopathol 106: 303-308.

Kramer LH, Simón F, Tamarozzi F, Genchi M,Bazzocchi C, 2005b. Is Wolbachia complicatingthe pathological effects of Dirofilaria immitisinfections? Vet Parasitol 133: 133-136.

Marcos-Atxutegi C, Kramer LH, Fernández I,Simoncini L, Genchi M, Prieto G, Simón F, 2003.Th1 response in BALB/c mice immunized withDirofilaria immitis soluble antigens: a possible

role for Wolbachia? Vet Parasitol 112: 117-130.Marcos-Atxutegi C, Gabrielli S, Kramer LH,

Cancrini G, Simón F, 2004. Antibody responseagainst Dirofilaria immitis and Wolbachiaendosymbiont in naturally infected canine andhuman hosts. Proc. IX European Multicolloquiumof Parasitology 2: 297-302.

Morchón R, Ferreira AC, Martín-Pacho R, MontoyaA, Mortarino M, Genchi C, Simón F, 2004.Specific IgG antibody response against antigensof Dirofilaria immitis and Wolbachia endosym-biont bacterium in cats with natural and experi-mental infections. Vet Parasitol 125: 313-321.

Ruiz-Moreno JM, Bornay FJ, Prieto Maza G,Medrano M, Simón F Eberhard M, 1998.Subconjuntival infection with Dirofilariarepens. Serological corfirmation of cure follo-wing surgery. Arch Ophtalmol 116: 1370-1372.

Simón F, Prieto G, Morchón R, Bazzocchi C,Bandi C, Genchi C, 2003. Immunoglobulin Gantibodies against the endosymbionts of filarialnematodes (Wolbachia) in patients with pulmo-nary dirofilariosis. Clin Diagn Lab Immunol 10:180-181.

Simón F, López-Belmonte J, Marcos-Atxutegi C,Morchón R, Martín-Pacho R, 2005. What is hap-pening outside North America regarding humandirofilariosis? Vet Parasitol 133: 181-189.

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Human dirofilariasis due toDirofilaria (Nochtiella) repens:an update of world literaturefrom 1995 to 2000*

S. Pampiglione, F. Rivasi

7

* Paper from Parassitologia 42: 231-254, 2000

Human dirofilariasis

Introduction

Following on from their 1995 review(Pampiglione et al.) of the world litera-ture on human dirofilariasis due toDirofilaria (Nochtiella) repens theauthors have attempted to gather all thenew cases published in the scientific lit-erature from 1995 to 2000 for anupdate on the subject. Dirofilaria(Nochtiella) repens (Nematoda:Filarioidea, Onchocercidae) is a habitu-al parasite of the subcutaneous tissue ofdogs and other carnivores. It is trans-mitted by Culicidae and can also acci-dentally affect man. Of the species offilariae found in the temperate regionsof the world it is the one most com-monly observed. It is present only in theOld World, notably in Italy, where 181human cases were reported up to 1995distributed practically over the whole ofthe national territory. Endemic zoneswere also identified in France, Greece,Turkey, Sri Lanka, Ukraine, the RussianFederation, Uzbekistan and other coun-tries for a total of 410 cases spread over30 different nations.

Materials and methods

The cases published in the world liter-ature during the last 5 years have beenclassified by nation and the essentialdata for each one (year of publication,author’s name, patient’s sex, age, placeof residence, location of the parasite)set out in tabular form. In some cases inwhich the authors did not provide thefull anamnestic and morphologicaldetails necessary for a correct documen-tation we have limited ourselves torecording what findings were reported

and have accepted them on trust. A few of other cases that occurred

before 1995 but which escaped men-tion in our previous enquiry have nowbeen included so as to offer as completea general picture as possible of theoverall number and geographical distri-bution of subjects affected.

In this context two recent reviews ofcases occurring in the ex-Soviet Union(Avdiukhina et al., 1996, 1997) havebeen extremely useful and have enabledus to include in our statistics many casespublished in Russian journals that areunlikely to be available in European orAmerican libraries. Similarly, as regardsSri Lanka, the review recently publishedby Dissanaike et al. (1997), togetherwith their prompt personal communica-tions of subsequent updates, have beenof great value. The data missing forsome of the cases reported in our 1995review have now been included (age,sex, locality and location, together withbibliographical reference, if applicable).

They appear in the numerical listingwith the number 0, since they werealready counted in the previous review.

Results

Geographical distribution

There do not appear to be any signif-icant variations in the geographical dis-tribution of the parasite in the dog andother carnivores from that reported inthe previous review, exceptions being 5animals in Tenerife (Canary Isles)(Stenzenberger and Gothe, 1999), 3 inHungary (Fock et al., 1998; Szell et al.,1999), several dogs (number not stat-ed) in the South African Republic

83


(Bredal et al., 1998; Schwan, personalcommunication), all consideredautochtonous infections by the respec-tive observers.

Imported cases from other endemiccountries are reported in Germany(Zahler et al., 1997; Glaser and Gothe,1998) and also in the south ofSwitzerland (Bucklar et al., 1998),even though in the latter instance itmay be suspected that the parasite’s lifecycle completed itself in loco given themildness of the climate in recent years.In fact, Petruschke et al. (1998) reportthe presence of D. repens in the low-lying Canton Ticino in 2 dogs thatapparently had never been outside thearea. The geographical distribution ofcanine dirofilariasis in France has beenillustrated by Beuguet and Bourdasseau(1996) and by Chauve (1997). The lat-ter claims that the parasitosis is presentin 19 of the 62 districts investigatedand that it can affect as many as 22%of the animals examined. Generally, inFrance D. repens appears to be morecommon than D. immitis, while neitherspecies has been reported there in cats.Cazelles and Montagner (1995) report2 cases of dogs simultaneously affectedby leishmaniasis and dirofilariasis dueto D. repens in Aveyron and Gard(southern France).

In Spain, the presence of canine diro-filariasis is reconfirmed in Alicante,Cadiz and Salamanca, albeit with aprevalence of less than 2%, and, in theEbro delta only, of 9.4% (AngueraGaliana, 1995; Lucientes and Castello,1996). Aranda et al. (1998) found noevidence of the parasitosis in 188 dogsexamined in the Barcelona area. In theAlicante and Elche areas (southeasternprovinces of Spain) Cancrini et al.

(2000a) claim that dogs are moreaffected by D. repens than by D. immi-tis, in contrast to a previous study byRojo Vasquez et al. (1990), basingtheir results on 114 canine blood sam-ples tested with Knott technique, PCRand ELISA for specific IgG detection. In Bulgaria, Kanev et al. (1996) report2 dogs testing positive out of 192examined.

In Greece, prevalence is between 6.7and 22% (Vakalis and Himonas, 1997).

The geographical distribution of bothD. immitis and D. repens in theEuropean Union in dogs is illustrated ina map by Muro et al. (1999). The possibility that the biological cycleof D. repens completes itself on Israeliterritory, and that therefore the humancases diagnosed there are autochtonous,is confirmed by the discovery of a dogaffected by the parasite but which hadnever left Israel (Harrus et al., 1998).

In northern Italy, a study involving2628 dogs (Rossi et al., 1996; Pollonoet al., 1998) confirms the high inci-dence of D. repens in Piedmont with aprevalence of up to 20.5%. In theprovince of Varese (Lombardy),Petruschke et al. (1998) report a preva-lence of 6.9% out of 216 dogs exam-ined. In Sicily, it is confirmed thatautochtonous cases due to D. immitisare rare, maybe even non-existent,whereas D. repens is widespread(Pennisi and Furfaro, 1997; Giannettoet al., 1999), particularly in theprovince of Trapani, where Giannettoet al. (1997) diagnosed a prevalence of22.8% for this latter species. In thecommunes of Vesuvius (province ofNaples), microfilariae of D. repenswere found in 3% of 351 dogs exam-ined (Rinaldi et al., 2000). In Tuscany

84


(central Italy), where canine dirofilaria-sis due to D. repens affects 25-39% ofdogs, Marconcini et al. (1996) foundmicrofilaraemia in 1% of 523 foxesexamined, while autopsy revealed adultnematodes in 3 out of 28 foxes exam-ined. However, the authors in questiondo not consider the fox to be a reservoirof parasitosis.

In South Africa, the presence of D.repens has been found for the first timein a cat suffering from hepatic insuffi-ciency (Schwan et al., 2000). Tarello(2000a,b) claims to have found micro-filariae of D. repens in the blood of 10cats associated with pruritic dermatosisand haemobartonellosis in Alessandria(northern Italy) and in another cataffected by ulceration on both flanks.

In Romania, Olteanu, in a congres-sional communication (1996) reportsD. repens not only in the dog but alsoin the fox, the wolf and the cat, as wellas in man, but without specifying thenumber of cases. By way of confirma-tion of the parasite on Romanian terri-tory, said author quotes otherresearchers (Motas, 1903; Popovici,1916) who have, in the past, discov-ered it in dogs.

In Sri Lanka the high prevalence ofD. repens in dog, formerly signalled(Dissanaike, 1961, and others) is con-firmed to reach 60-70% in someprovinces by Dissanaike et al. (1997).

Roncalli (1998), in a paper presentedat the XX Congress of the ItalianParasitology Society, described the his-tory of canine dirofilariasis from the17th century to the present with someobservations on the geographic distri-bution for the past times.

Cases of human dirofilariasis report-ed since 1995 number 372 in all,

involving 25 countries. In Europe:Belgium, Bulgaria, France, Greece,Hungary, Italy, Romania, SerbianRepublic, Slovakia, Slovenia, Spain,Ukraine; in Africa: Kenya and Tunisia;in Asia: Georgia, India, Iran, Israel,Kazakhstan, Malaysia, Russia, SriLanka, Turkey, Turkmenistan andUzbekistan. The countries most affect-ed have been Italy (117 cases), SriLanka (101), Russia (Siberia included,61), Ukraina (23) and France (23). Inthe few cases found in Austria andGermany the infection was picked upin other countries. In Belgium, wherethe parasite has never previously beenobserved, a curious episode of dirofi-lariasis is reported (van den Ende et al.,1995) involving three members of thesame family; it may have been causedby a mosquito transported in luggagefrom an endemic zone or from a nearbyinternational airport. Parasitologicaldiagnosis (itching, cutaneous striae anda nodule containing a female of D.repens) was possible in only one of thethree cases, but the presence of thesame type of cutaneous striae persistingfor months in the other two familymembers would suggest a cryptic infec-tion of the same nature.

In a large part of Africa and in otherdeveloping regions of the world, asOrihel and Eberhard (1998) point out,it must be borne in mind that it is notalways possible to establish with anydegree of certainty whether infectionsdue to D. repens in man are rare or fre-quent because their diagnosis is deter-mined by the level of development andthe efficiency of the health services andhence by the diagnostic resources avail-able. The only countries where the geo-graphical distribution of human dirofi-

85


lariasis has been traced are France,Greece, Italy, the Russian Federationand Sri Lanka. In Italy, cases of theinfection have been reported in 9regions, prevalently in Piedmont.Takings the cases recorded in the previ-ous enquiry into account, the parasito-sis is found to be present in 19 regionsof Italy out of 20. In Greece(Pampiglione et al., 1996d), when therecent cases are added to those report-ed previously, human dirofilariasis isalso seen to affect virtually the whole ofthe country. In France, Raccurt (1999,2000) claims that there are at least 14departments affected by D. repens withthe highest incidence in theMediterranean coastal regions,although sporadic occurrences on theAtlantic seaboard and above the 46°latitude North have been reported(Guillot et al., 1998; Weill et al.,1999). In Sri Lanka (Dissanaike,1996), the zones most affected arethose of the western provinces. In theex-Soviet Union, today the RussianFederation, although there is no dis-tribution map properly covering thisvast area, it appears from the sketchyliterature and, in particular, from thereviews of Avdiukhina et al. (1996and 1997), that the regions where theparasitosis is most endemic are theCaucasus (in the south of the RussianRepublic), which account for 62% ofthe cases cited, and Ukraine, account-ing for 21%. Kazakhstan, Uzbekistanand Georgia each account for 4%and Turkmenistan for 3%, whileother small states account for theremaining 2%. The northern limit tothe spread of D. repens seems to bethe 62nd parallel in the RussianFederation.

Distribution by age and sex

The age-range of the subjects affectedgoes from 4 months to 100 years, mostof them being in their 40s. In SriLanka, Dissanaike et al. (1997) reporta considerable number of cases in chil-dren less than 10 years old, a phenom-enon not found in any other country.There is a greater prevalence in femalesthan in males (186 of the 322 cases inwhich the patient’s sex is specified, rep-resenting 57.8%), but it is not statisti-cally significant.

Carriers

Extensive research into the Culicidaecaptured with canine bait in a zoneendemic for D. repens in Piedmont(north-western Italy) (Pollono et al.,1998) has shown that this natural reser-voir is very attractive to Culex molestusand Aedes caspius, but less so to C. pipi-ens, Ae. vexans and Anopheles mac-ulipennis. The Culicidae are at theirmost active, at least in Piedmont, duringthe month of July. Talbalaghi (1999) hasshown an increase in the density of Ae.caspius in the same area over the last 3years and an increase in the spread of lar-val foci. Ae. caspius is the species mostaggressive towards man; it is capable oftravelling 20 km without wind assistanceand bites all day long. Using both canineand human bait in endemic zones of theVeneto and Friuli-Venezia Giulia (north-eastern Italy), Pietrobelli et al. (2000)demonstrated a strong attraction for C.pipiens and Ae. caspius, much reducedfor Ae. vexans. In Nigeria, Anyanwu etal. (2000) between 6 different species ofCulicidae tested (Culex pipiens fatigans,C. p. pipiens, Anopheles gambiae,

86


Mansonia africana, Aedes vittatus, Ae.aegypti) have found that this last is themost suitable vector for a local strain ofD. repens. In order to select strains of Ae.aegypti refractory and otherwise to D.repens, Favia et al. (1998) used molecu-lar biology techniques with apparentlyreliable results. Pampiglione and Gupta(1998) reported the presence ofimmunocytes (plasmatocytes) ofCulicidae, probably Aedes sp., in a mam-mary nodule enclosing an immaturespecimen of D. repens. The authorsthink it likely that, during the passage ofthe infecting larvae from the mosquito toman, the carrier’s immunocytes are oftenborne along by the larvae themselves andso penetrate the subcutaneous tissue,only to be regularly destroyed by thedefensive reactions of the host.

Clinical history and symptomatology

In the dog, the parasitosis is generallyasymptomatic even when microfilari-aemia is fairly developed. There may,however, be cutaneous manifestationssuch as erythematous patches, papulae,localised alopecia, eczema accompa-nied by pruritus and, rarely, noduleand/or ulceration (Bredal et al., 1998;Tarello, 1999).

In man, the carrier’s bite has some-times been described as painful, fol-lowed by slight local acute phlogosis(erythema, swelling, pruritus) lastingfor 5-8 days. Generally speaking, how-ever, no particular sensation attributa-ble to the insect bite is recalled by thepatient other than that of being bittenby mosquitoes. The subsequent forma-tion of the nodule in which the nema-tode remains trapped by the defensivereaction of the host is an essential fea-

ture, typical of the parasitosis and ofwhich it may prove to be the sole clini-cal manifestation. It is noticed after aperiod of approximately 2-12 monthsfrom the penetration of the parasite,although much longer periods cannotbe ruled out since it is often impossibleto recall the moment when infectiontook place. At times the nodule appearswithout any external phlogistic mani-festation, at others it is accompanied bylocal erythema and pruritus, localisedor more widespread urticarial manifes-tations lasting a few days but recurringfor as much as a year and a half(Thérizol-Ferly et al., 1996). The nod-ule may also suppurate and assume theappearance of an abscess. There havebeen very occasional reports oferysipeloid phlogistic reactions andsatellite lymphoadenopathies as well asof rises in temperature of modest entityor with hyperpyrexia due to infectiouscomplications that are quickly broughtunder control with antibiotics. In eyecases, complications such as detachedretina, crystalline opacity, glaucoma,uveitis, episcleritis and a limited loss ofvision have exceptionally been reported(Mizkievic and Leontieva, 1961;Avdiukhina et al., 1996). In one caseaffecting the orbit, Braun et al. (1996)report monolateral exophthalmos last-ing for a year. Patients have reportedsubcutaneous migrations of the para-site sometimes over considerable dis-tances, such as from the lower limbs tothe neck or head, or from one side ofthe body to the other, during periods ofmany months (Delage et al., 1995;Azarova et al., 1995; Artamonova andNagornyi, 1996). In one patient, migra-tions in the tissues of the head hadcaused trigeminal neuralgia, which dis-

87


appeared when the nematode, sudden-ly appearing under the bulbar con-junctiva, was promptly removed(Avdiukhina et al., 1997).

The speed of these migrations, whenvisible, has been estimated at 30 cm intwo days. Jelinek et al. (1996) report 2cases of patients whose insistently com-plaints of “a worm under the skin” hadled the doctors to diagnose delusoryparasitosis and have them admitted topsychiatric ward, until the appearanceof a nodule and its subsequent histo-logical analysis cleared up the matter. Asensation of a nematode travellingunder the skin had been reported by10% of the Russian patients includedin the review. Artamonova andNagornyi (1996) report a case withcutaneous manifestations as in a truelarva migrans. The application of hotsteam compresses or ultrasound thera-py appears to stimulate the movementsof the nematode under the skin. Thereare rare cases of 2 nodules being pres-ent in the same patient, either appear-ing contemporaneously or monthsapart (Degardin and Simonart, 1996;Jelinek et al., 1996; Orihel et al., 1997).The nodules had appeared 5 years ear-lier (Degardin and Simonart, 1996) inone case, 2 years earlier in others. Themaximum duration of the parasitosis isreported as being 12 years (Avdiukhinaet al., 1996). The spontaneous emer-gence of the nematode from the suppu-rating nodule is reported on at least 10occasions. In one case, localised nearthe junction of the lips, squeezing thenodule, which had been mistaken for aboil, caused the emergence of the nem-atode together with pus, after whichthe lesion healed in the space of a fewdays. In others involving the subcon-

junctiva or eyelids, vigorous rubbing ofthe affected eye by the patient causedthe spontaneous emergence of the nem-atode (Avdiukhina et al., 1966, 1967).Peripheral hypereosinophilia is report-ed only rarely, with values generally lit-tle more than 10-15% of the leukocyteformula (Thérizol-Ferly et al., 1996;Pampiglione et al., 1996). However,Petrocheilou et al. (1998) report aGreek case, diagnosed merely onaccount of the presence of microfilariaein the bloodstream defined by theauthors as Dirofilaria-like, in which theeosinophil count amounted to 26% ofthe leukocyte formula. A case of sub-conjunctival dirofilariasis in an HIV-positive patient is reported in France(Basset et al., 1996) without therebeing any apparent correlation betweenthe development of the parasitosis andthe presence of HIV.

Locations

In the dog, reports of localised nodu-lar formations enclosing the nematodeare rare, since the parasite is almostalways more or less elongated and freein the web of subcutaneous tissue. Localisation in the sternal region, theneck and the flanks has neverthelessbeen reported by Bredal et al. (1998)and Tarello (1999). A dog with D.repens in the heart has been observedby Isola et al. (2000), thus demonstrat-ing that the species can penetrate thebloodstream.

In man, on the other hand, the para-site nodule is always present, if oneexcludes localisation in the subcon-junctiva where the nematode can beconsidered to be migratory and not yettrapped by the host’s reaction. In the

88


vast majority of cases, the nodule islocated in the subcutaneous tissue, thedeep dermis or the submucosa. Thereare rare cases of location in muscle tis-sue (Pampiglione et al., 1996c; Gros etal., 1996), in the lymph nodes(Alain,1997; Shekhar et al., 1996;Auer, pers. comm., 1998; Cancrini etal., 1999) and in the deep viscera.More frequent are reports of localisa-tion in the upper half of the body(74%), in particular the ocular region(eyelid, subconjunctiva, orbit)(35.3%) and also the upper limbs(11%). In the cases reported byAvdiukhina et al. (1997) localisationin the ocular region accounts for 45%of the total observed in the RussianFederation. Localisation in the vitreousbody in 3 cases and in the crystallinelens in one case is reported respective-ly in the Slovakian Republic (Vasilkovaet al., 1992) and Uzbekistan(Mizkievic and Leontieva, 1961). Acase of preretinal localisation is alsoreported in Kazakhstan (Glinciuk etal., 1992), although the morphologicaldescription may cast doubt on theactual species. In Italy, two cases arereported of localisation in the orbitalcavity, operated on by means of anteri-or orbitotomy (Strianese et al., 1998).Another case of localisation in theorbital cavity was operated ontransnasally (Braun et al., 1996; Groellet al., 1999), the authors producing aremarkable videotape of the removalof the nematode. A subconjunctivallocalisation is reported by BianchiRossi et al. (1991) where the parasitehad previously passed through theretrobulbar area causing exophthal-mos. In Sri Lanka, Dissanaike andcoworkers (1997) report relatively fre-

quent cases affecting the male genitals(scrotum, epididymis, spermatic chord,penis), particularly in children below 5years of age; this is in contrast withwhat occurs in other geographicalregions, where the few cases reportedare restricted to adults. One case affect-ing the subcutaneous tissue of the penisis reported from Corsica (Pampiglioneet al., 1999b), another from Israel(Stayermann et al., 1999) and twomore from Sri Lanka (Dissanaike et al.,1997). Pulmonary cases are reportedfrom Greece (Pampiglione et al.,2000d) and from Italy (Pampiglione etal., 1996e, 2000b).

Jelinek et al. (1996) and Fueter andGebbers (1997) report 2 cases and onecase, respectively, of pulmonary infec-tion, one from Corsica, one from Italyand the third in a globetrotter possiblyaffected in America – all treated inGermany. These authors attribute theinfection to D. immitis; however, it ispossible that, by analogy with manyother cases reported from Corsica andItaly, these are due to D. repens,although, given the advanced state ofdecomposition of the nematodes, pre-cise identification was not possible.Rare cases affecting the omentum andthe mesentery have been casuallyobserved during laparotomy in Italyand the Russian Federation(Avdiukhina et al., 1997; Dorofeiev etal., 1997; Mastinu et al., 1998;Pampiglione et al., 2000b).

There are single instances of localisa-tion in the parotid gland (Hoop, 1997;H. Braun, pers. comm.), in the submu-cosa of the mouth, of the base of thetongue, of the isthmus of the faucesand within a granuloma at the root of atooth (Avdiukhina et al., 1997).

89


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 pre-vious review (1995).

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

France

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

Locality Location

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

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

EUROPEBelgium

Bulgaria

01

20

19961996

19971998

Pampiglione et al.Jelinek et al. (surgery in Germany)Arvanitis et al.Petrocheilov et al.

Mn.s.

MM

45adult

6870

Athensn.s.

n.s.Ionian islands

abdominal wallhand, thigh

subconjunctivalworm not recovered*

Greece

* This case was already recorded by Kramer 1993 (Raccurt, 2000).

* Only microfilariae (210-230 µm long) were detected in peripheral blood with diagnosis of Dirofilaria-like.

123

456

7 to 101112

1314

151617181920210

199519951996

199519961996199619961996

19971997

19981998199919991999199919991999

Nozais and HuerreDelage et al.Hautefort (cit. by Basset et al., 1996)Basset et al.Masseron et al.Gros et al.Rabodonirina et al.Thérizol-Ferly et al.Jelinek et al. (surgery in Germany)Weill et al.Alain (cit. by Raccurt, 2000)Guillot et al.Desruelles et al.Weill et al.Weill et al.Pampiglione et al.Pampiglione et al.Rouhette et al.Morassin et al.*

MM

n.s.

MMM

n.s.FF

FF

FFMFMMMF

3943n.s.

376119n.s.3546

7022

1247663964236526

LanguedocArdècheArles

NarbonneGirondeCorsicaLyon?Sologne? Côte méditerranéenneCorsica

Côte méditerranéenneLoire and Cher

GirondeVarCôte d’Azur or CorsicaCharante MaritimePinarello (Corsica)Porto Vecchio (Corsica)NiceTarn or Herault

scrotumeyelidthigh

subconjunctivalsubconjunctivalarmn.s.subconjunctivallung (D. immitis?)

subconjunctivalgroin lymphnode

foreheadsubcutaneous n.s.cheekorbital regionfootpenissubconjunctivalsubclavicular region

2223

19991999

Calvet et al.Dei-Cas (cit. by Raccurt, 2000)

MF

4710

VarNice or Corsica

armabdominal wall

continued

90

Italy


Locality Location

340056

199819981998199819981999

Vakalis and HimonasVakalis and HimonasVakalis and HimonasVakalis and HimonasVakalis et al.Pampiglione et al.

n.s.n.s.MMFM

n.s.n.s.3484231

n.s.n.s.n.s.Athens or EpirusMessinia (Peloponnesos)Aigios Peloponnesus

nose regionthoracic wallscrotumhandbreastlung

123456

200020002000200020002000

Parlagi et al.Szénasi, p.c.Elek et al.Elek et al.Elek et al.Elek et al.

FMFMFF

575652486476

n.s.Szeged provinceBudapest provinceHodmezovasarhelyTisza river bankCsobaj (Miscolc)

eyelidsubconjunctivalupper eyelideye-browpelvis regionarm

Hungary

* The diagnosis was D. immitis but probably it was D. repens.

0001

203

4

5

6789

10

1986198619871990

199119931995

1996

1996

19961996199619961996

Spina et al.Spina et al.Toniolo et al.Stemberger H (cit. by Auer et al. 1997;surgery in Austria)Bianchi Rossi et al.Bay et al.Stemberger H. (cit. by Auer et al. 1997;surgery in Austria)Garaffini et al. (surgery in France)Braun et al. (surgery in Austria)Cancrini et al.Cancrini et al.Cancrini et al.Molet, p.c.Jelinek et al. (surgery in Germany)

MFMM

FFM

F

F

FFFFF

607323

adult

7963

adult

72

61

4226483530

Biancavilla (Catania)Ramacca (Catania)Cadore (Trento)Italy (locality n.s.)

Laiatico (Pisa)TorinoItaly n.s.

Locality n.s.

Locality n.s.

Augusta (Siracusa)Augusta (Siracusa)Sortino (Siracusa)VenicePiedmont?Tuscany?

orbital regionlower eyelidjawperiocular region

subconjunctivallower eyelidleg

periocular region

retrobulbar region

leganklegluteal regionscalplung*

11

12

13

0000000

1996

1996

1996

1996c1996e1996e1996a1996b1996b1996b

Jelinek et al. (surgery in Germany)Jelinek et al. (surgery in Germany)Jelinek et al. (surgery in Germany)Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.

n.s.

n.s.

n.s.

FFMMFMM

n.s.

n.s.

n.s.

43666955444268

n.s.

n.s.

n.s.

Sciacca (Agrigento)Pegognaga (Mantova)Concordia (Modena)Rivalta S (Alessandria)AstiGavorrano (Grosseto)Surbo (Lecce)

glabella

abdominal wall

shoulder

temporal regionlunglungthighiliac regionlegneck


continued

91



Locality Location

0000

1415161700

18

1920212223

2425262728290

30

31

3233343536373839404142

43444546474849505152535455

1996a1996a1996b1996b1996199619961996199719971997

19971997199719971997

19971998199819981998199819981998

1998

19981998199819981998199819981998199819981999

1999199919991999199919991999199919991999199919991999

Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Vitullo, p.c.Vitullo, p.c.Pampiglione et al.Giannetto and UbaldinoAuer et al. (surgery in Austria)Zardi et al.Mastinu et al.Mastinu et al.Mastinu et al.Heep, p.c., Auer 1997(surgery in Austria)Garavelli, n.p.Mastinu et al.Mastinu et al.Mastinu et al.Mastinu et al.Pampiglione et al.Pampiglione et al.Auer, p.c. (surgery in Austria)Cancrini et al.

Cancrini et al.Cancrini et al.Cancrini et al.Cancrini et al.Cancrini et al.Cancrini et al.Cancrini et al.Cancrini et al.Strianese et al.Strianese et al.Pampiglione et al. (surgery in Hungary)Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.

MMFMMFFMMFM

MFMF

n.s.

MMFMFFFM

M

MFFFMFFMFFM

MMFMFFFFFMFFF

3854664058354557523835

45423645n.s.

6150193962465243

59

4548602074425642

adult4737

5739n.s.578341505750251003069

OristanoOristanoStaranzano (Gorizia)AstiArbatax (Nuoro)Olbia (Sassari)IserniaVenafro (Isernia)Ravenna provinceMazara del Vallo (Trapani)Udine province, or Corsica, or Southern FranceGaetaAsti provinceAsti provinceAsti provincen.s. or Greece

Bergamasco(Alessandria)Asti provinceAsti provinceAsti provinceAsti provinceMilanoGarlasco (Pavia)Sardinia? Malta?

Grado (Gorizia)

Augusta (Siracusa)Augusta (Siracusa)Elba IslandMazara del Vallo (Trapani)Augusta (Siracusa)SiracusaTorinoSiracusaNapoli provinceNapoli provinceNorthern Italy or Hungary n.s

Odalengo (Alessandria)Novara provinceNovara provinceNovara provinceViarigi (Asti)Castagneto Carducci (Livorno)Piombino (Livorno)Crescentino (Vercelli)Casale Monferrato (Alessandria)Novara provinceCasale Monferrato (Alessandria)Novara provinceNovara province

zygomatic regionforeheadlegupper eyelidsubconjunctivallegbreastneckspermatic chordsubconjunctivalepididymis

thoracic wallarmsubconjunctivalperiocular regionparotid gland

scalptemporal regionhandbreastmesenteronbreastbreast

sacral regionsubmandibularlymphnodefingerlegbreasteyelidsubconjunctivalsubconjunctivalsubconjunctivaleyelidorbital regionorbital region

spermatic chordaxillazigomatic regionpopliteal regionthoracic wallabdominal wallabdominal wallthighlegarmthoracic wallthoracic wallforearmaxilla

continued

92



Locality Location

56575859606162636465666768697071727374757677

78798081828384858687888990919293

94

9596979899100101102103103104105

1999199919991999199919991999199919991999199919991999199919991999199919991999199919991999

1999199919991999199919991999199919991999199919991999199919991999

1999

199919991999199919991999199919992000200020002000

Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.

Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.

Pampiglione et al.

Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Pampiglione et al.Giansanti, p.c.Gobbo and Bisoffi, p.c.Pastormerlo, p.c.Pastormerlo, p.c.

MFFMFFFFFMFMMFFMMFMFFF

MMMMFFMMFMMFMMFM

F

MFMMFFFMFMFF

593464326940614129392750705256526169

adult735656

422949456654376468

adultadult684444276

59

363428444065632469394935

Casorzo (Asti)Trino (Vercelli)Fontanetto Po (Vercelli)Casale Monferrato (Alessandria)Casale Monferrato (Alessandria)Trino (Vercelli)Moncalvo (Asti)LecceAstiRoatto (Asti)Gela (Agrigento)Ravenna provinceNovara provinceNovara provinceNovara provinceSaturnia (Grosseto) or LivornoAlessandriaAlfiano Natta (Alessandria)Asti provinceAsti provinceAsti provinceVercelli or Mazara del Vallo(Trapani)Novara provinceAsti provinceAsti provinceAsti provinceAsti provinceBaratili S.P. (Oristano)Cantalupo (Alessandria)Valenza Po (Alessandria)Mirabello (Alessandria)Ravenna provinceRavenna provinceVercelliMilanoNovara provinceNovaraCastellammare del Golfo(Trapani)Castelleone (Cremona) or Ischia IslandScano M. (Oristano)Pontestura (Alessandria)Novara provinceCrescentino (Vercelli)Vignale M.(Alessandria)Villanova (Alessandria)Valenza Po (Alessandria)Reggio EmiliaPetrelle (Perugia)VeneziaValenza Po (Alessandria)Motta di Conti (Vercelli)

scalpthoracic wallkneescalpthoracic wallneckscalpupper eyelidhand backbreastcheeksubconjunctivalhand palmaxillaabdominal wallepididymisscalpfoot backn.s.omentumorbital regionlung

armtemporal regionlungarmbreastgroinlower eyelidnecknose regionlegspermatic chordkneecheekforeheadforearmabdominal wall

shoulder

epididymisabdominal wallomentumspermatic chordforearmforearmthighabdominal wallbreastforearmabdominal wallthigh

continued

93



Locality Location

106107108109110111112113114115116

117

20002000200020002000200020002000200020002000

2000

Pastormerlo, p.c.Pastormerlo, p.c.Speranza p.cFeyles, p.cFeyles, p.cFeyles, p.cPavesi, p.c.Pavesi, p.c.Pavesi, p.c.Pavesi, p.c.Pavesi, p.c.

Elek et al. (surgery in Hungary)

FFMFMMFFFFF

F

7462357428764947743662

71

Casale M.(Alessandria)Vignale M. (Alessandria)Modena provinceAstiAsti provinceAsti provinceValenza Po (Alessandria)Moncalvo (Asti)Casale Monferrato (Alessandria)Motta di Conti (Vercelli)Vignale Monferrato(Alessandria)Rome province

kneeabdominal wallgroinbreastepididymissubcutaneous n.s.abdominal wallaxillary regionkneethighabdominal wall

lower eyelid

Romania

1 1996 Misic et al. F adult Smederevo head*

23

19961996

Misic et al.Misic et al.

n.s.n.s.

n.s.n.s.

SmederevoBeograd

headshoulder

Serbian Republic

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

Slovak Republic

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

* Both female and male nematode were recovered in the same nodule.

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

123

199720002000

OlteanuPanaitescu et. al.Panaitescu et. al.

n.s.n.s.n.s.

n.s.n.s.n.s.

n.s.n.s.n.s.

n.s.n.s.n.s.

Slovenia

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

Spain

123

4 to 1112

13 to 1617

1971197119771996199619971997

Melnicenko and ProsvetovaMelnicenko and ProsvetovaKondrazkyi and ParkomienkoDavydov et al.PogolciukDorofeev et al.Dorofeiev et al.

FMF

n.s.M

n.s.F

552946n.s.60n.s.67

PoltavaPoltava provinceKievKiev provinceOdessa provincen.s.Crimea

subconjunctivalsubconjunctivalupper eyelideye region (n.s)lower eyelidn.sknee

Ukraine

continued

94


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

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

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

Tunisia

1234

1990199519951999

Chaabouni et al.Ben Said et al.Ben Said et al.Mrad et al.

MFFF

55394832

KairouanGabèsSoussen.s.

subconjunctivalaxillary regionforeheadbreast

181920212223

199719971997199719971997

Dorofeiev et al.Avdiukhina et al.Avdiukhina et al.Postnova et al.Postnova et al.Postnova et al.

n.s.FMMMF

n.s.27605560

adult

n.s.SimferopolOdessa provinceSociBelgorod DniestrovskiBelgorod Dniestrovski

mesenteronlower eyelidlower eyelid.necklower eyelidgroin

1 1997 Orihel et al. (surgery in the States)

M 42 n.s. periocular region*


Locality Location

AFRICAKenya or Senegal

12

19671967

KamalovKamalov

MF

n.s.36

GrusiyaVostocnaya Grusyia

upper eyelidupper eyelid

ASIAGeorgia

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

India

1 1996 Degardin & Simonart (surgery in Belgium)

M 26 n.s. leg*

Iran

1 1999 Stayerman et al. M 35 Safed? penis

Israel

1234

5

1961197019701985

1992

Mizkevi and LeontievaLubovaLubovaTasberghenova andAnbakirovaGlinciuk et. al.

FFFM

F

25525713

43

n.s.Ksil OrdaKsil OrdaAlma Ata

n.s.

lower eyelidlower eyelidthoracic wallsubconjunctival

eye, preretinic*

Kazakhstan

continued

95


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

2 1996 Shekhar et al. M 39 Melaka cervical lymphonode

Malaysia

123

456789

101112131415161718192021222324252627282930313233343536373839404142

195419711977

198119911993199319931995199519951995199619961996199619961996199619961996199619961996199619961996199719971997199719971997.199719971997199719971997199719971997

KoroievProsvetova et al.Kondrazkii andParkomienkoMerkusceva et al.Maximova et al.Plotnikov et al.Plotnikov et al.Avdiukhina et al.Asarova et al.Asarova et al.Asarova et al.Asarova et al.Artamonova and NagornyiAvdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Avdiukhina et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.

Fn.s.F

MF

n.s.n.s.MF

n.s.n.s.n.s.n.s.FFFFFFFFMFFFFMFFFMMMFFMMMFFFF

23n.s.46

4641n.s.n.s.3537n.s.n.s.n.s.n.s.1336504261492633234926333723555857382350

adultadult38n.s.n.s.55274641

Northern Caucasusn.s.Voronez province

Astrakhan provinceTula regionTomsk (Siberia)Saratov provinceVladivostock (Siberia)Barnaul (Siberia)Barnaul (Siberia)Barnaul (Siberia)Barnaul (Siberia)Northern CaucasusAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceKrasnodarAstrakhan provinceAstrakhan provinceAstrakhan provinceBarnaul (Siberia)Krasnodar provinceKrasnodar provinceKrasnodar provinceKrasnodar provinceKrasnodar provinceKrasnodarVolgogradBarnaul (Siberia)Barnaul (Siberia)VolgogradBarnaul (Siberia)Barnaul (Siberia)SociMoskow provinceAstrakhan provinceAstrakhan province

upper eyelidn.s.lower eyelid

lower eyelidzygomatic regionsubconjunctivalsubconjunctivalsubmandibularupper eyelideye region (n.s.)eye region (n.s.)eye region (n.s.)n.s.upper eyelidupper eyelidsubconjunctivalsubconjunctivalsubconjunctivalperiocular regionlower eyelidperiocular regionupper eyelidperiocular regionlower eyelidperiocular regionupper eyelidupper eyelidlower lipsubconjunctivalnapethoracic wallupper eyelidhipbreastbreastthoracic wallshoulderhipnecklower eyelidelbowhand

Russia (Siberia inclusive)


Locality Location

* Both male and female worms present in the nodule.

continued

96


000001234567891011121314151617181920212223

1976197619801980198819971997199719971997199719971997199719971997199719971997199719971997199719971997199719971997

WijesunderaWijesunderaWijesunderaWijesunderaWijesunderaDissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.

FFMFMMMMMMFMMFFFFMMMMFMMMM

n.s.M

32n.s19631.64 m

52738431

n.s.62227254103604

10 m28

9 m1.46 mn.s.40

Moragolla (CP)Kandy (CP)n.s.Wattala (WP)Waharaka (SabP)Pannipityia (WP)Angola (WP)Karainagar (NP)Colombo (WP)Batticaloa (EP)Colombo (WP)SPWaharaka (SabP)Embilipitiya (Sab. P)Moratuwa (WP)Colombo (WP)n.s.Gandara (SP)Lunuwila (NWP)Kolonnawa (WP)Mirigama (WP)Kuliyapitiya (NWP)Tissamaharama (SP)Ambalangoda (SP)Galle (SP)Wanduramba (SP)Hikkaduwa (SP)Kandy (CP)

forearmeye region (n.s.)scrotumbreastscrotumscrotumsubconjunctivalforearmforearmsubconjunctivalfootwristperianal regionlower eyelidupper eyelidsubconjunctivalsubconjunctivalabdominal wallcheeksubconjunctivalscrotumcheekcheekscrotumscrotumpenissubconjunctivalthumb


Locality Location

43 444546474849505152535455565758596061

1997199719971997199719971997199719971997199719971997199719971997199719971997

Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.Postnova et al.

FFMFMFFFFFFFFFMFFFF

56585839311350363436426130182349263533

Astrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceKrasnodar or SociAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan provinceAstrakhan province

shoulderkneehandforearmcheekupper eyelidsubconjunctivalsubconjunctivalforearmshoulderupper eyelidsubconjunctivalthoracic wallcheekupper eyelidupper eyelidupper eyelidsoft palatecheek

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 = UvaProvince; WP = Western province]

continued

97


2425262728293031323334353637383940414243444546474849505152535455565758596061626364656667686970717273747576

19971997199719971997199719971997199719971997199719971997199719971998199819981998199819981998199819981998199819991999199919991999199919991999199919991999199919991999199919991999199919991999199919991999199919991999

Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Dissanaike et al.Kumarasimghe et al.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Dissanaike p.c.Ratnatunga and WijesunderaRatnatunga and WijesunderaRatnatunga and WijesunderaRatnatunga and Wijesundera

MMF

n.s.FMMMMMFMMMMFFFFMFMMMMMFMF

n.s.MFF

n.s.FFMFMMMMFFFFMMMMFFM

5 m5 m

6n.s.523402.23640431.61.21111332118

7 m11651.61

1.31.450302153n.s.2.6

11 m1.7n.s.9411.11.72.348

7 m4.62.3553030351,84.516416548

n.s.Kandy (CP)CPCPKandy (CP)Ragama (WP)CPKadugannawa (CP)CPUndugoda (Sab.P)Gambola (CP)Batapola (SP)Panadura (WP).SPDeraniyagala (Sab.P)n.s.Jaffna (NP)Rajagiriya (WP)Apura (NCP)Agalawatte (WP)Katunayake (WP)Negombe (WP)n.s.n.s.Ambalantota (SP)Negombo (WP)Agalawatte (WP)Kekirawa (NCP)Kandy (CP)n.s.WPHomagama (WP)Negombo (WP)n.s.n.s.Badulla (UP)WPNegombo (WP)n.s.Walasmulla (SP)WPWPKandy (CP)Kandy (CP)Kandy (CP)Kandy (CP)Kandy (CP)WPMatugama (WP)n.s.n.s.n.s.n.s.

subconjunctivalsubconjunctivaleye region (n.s.)eye region (n.s.)breastscrotumlower eyelidscrotumgroineye (n.s.)eye (n.s.)scrotumpenisfaceneckperitoneumforearmsubcutaneous n.s.scleranecksubconjunctivaln.s.scrotumspermatic chordspermatic chordankleforearmscalpeyelidn.s.n.s.scapular regionscapular regionn.s.handneckscrotumscapular regionscrotumkneescrotumscrotumthumbsubconjunctivalsubconjunctivalhipforeheadscrotumhipabdominal wallthighcheekthoracic wall


Locality Location

continued

98


77787980818283848586878889

1999199919991999199919991999199919991999199919991999

Ratnatunga and WijesunderaRatnatunga and WijesunderaRatnatunga and WijesunderaRatnatunga and WijesunderaRatnatunga and WijesunderaRatnatunga and WijesunderaRatnatunga and WijesunderaRatnatunga and WijesunderaRatnatunga and WijesunderaRatnatunga and WijesunderaRatnatunga and WijesunderaRatnatunga and WijesunderaFernando et al.

FMMMMFFMMM

n.s.n.s.M

45516304034221.6451.6n.s.n.s.3.4

n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.n.s.Laxapana (CP)

breastsubconjunctivalforearmthoracic walltemporal regionforeheadthoracic wallscrotumcheekscrotumn.s.n.s.scrotum*

90919293949596979899

1999199920002000200020002000200020002000

Pitakotuwage et al.Dissanaike, p.c.Dissanaike, p.c.Dissanaike, p.c.Dissanaike, p.c.Dissanaike, p.c.Dissanaike, p.c.Dissanaike, p.c.Dissanaike, p.c.Dissanaike, p.c.

FFM

n.s.n.s.MFFMM

80357mn.s.n.s.1

n.s.8

1.4n.s.

Ampara (EP)Galle (SP)n.s.n.s.n.s.n.s.Kandy (CP)Ampara (EP)Homagama (WP)EP

cheekspermatic chordn.s.n.s.n.s.forearmn.s.n.s.eye region (n.s.)testis*

100101

20002000

Dissanaike, p.c.Dissanaike, p.c.

FF

5050

Narahenpita (WP)Maharagama (WP)

subconjunctivalsubconjunctival

1 1997 Otkun et al. F 44 Edirne Edirne


Locality Location

* Two nodules with a male and female worms each.

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

* The diagnosis of D. repens was only presumed.

Turkey

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

Turkmenistan

1 1961 Mizkievic & Leontieva F 35 Tashkent crystalline*

Uzbekistan

99


Histopathology

In an important work on parasitesaffecting human tissues, Orihel andAsh (1995) present a wide range of his-tological sections illustrating the vari-ous Dirofilariae including D. repens,that can infect man. Ratnatunga andMijesundera (1999) have described thehistopathologic features observed in SriLanka in 14 subcutaneous nodules dueto D. repens. Two fully illustratedworks on the histopathologic featuresof the lesions caused by the parasite inman have been recently published byPampiglione et al. (1999e, 2000b): thefirst is particularly concerned with thedifficulties a histopathologist mayencounter in his attempt to arrive at acorrect diagnosis of the species whenthe nematode is in a more or lessadvanced stage of decomposition; thesecond takes 60 new cases and classi-fies the histopathological features intofour basic groups, which appear todepend on the length of time spent bythe nematode in the host’s tissues andon the defence reaction triggered by it.In the first group, which includes themajority of cases, the phlogistic reac-tion is of an abscess-like type due to thepresence of necrotic matter containingneutrophil and eosinophil leukocytesand less numerous chronic inflammato-ry cells surrounding the nematode.

A reactive granulation tissue isaround, while acute and chronicinflammatory cells infiltrate the sur-rounding soft tissues. In the secondgroup, the central zone containing thenematode is delimited by an actual val-lum consisting of epithelioid cells, his-tiocytes and occasional plurinucleategiant cells as of a foreign body. In the

third, less numerous, group the nema-tode is in a state of more or lessadvanced decomposition and is sur-rounded by scant mixed necrotic mat-ter and occasional inflammatory cells.This area is delimited by reactive tissueforming a dense fibrous ring of mainlyfibroblastic tissue. As in the previouscases, the surrounding soft tissues dis-play an aspecific acute and chronicphlogistic reaction. In the fourth group,comprising just a few cases, the histo-logical pictures feature a dense inflam-matory infiltrate consisting almostentirely of lymphoid elements, some-times massing together to form germi-native centres. This reactive tissue dif-fuses throughout the surrounding softtissues. In the cases affecting the lungs,the nematode is almost always in anadvanced state of decomposition insidea thrombosed arteriole giving rise to asmall, roundish infarctual zone. Theadjacent pulmonary parenchyma dis-plays mainly acute inflammatory infil-trates with a redominance ofeosinophils. Flieder and Moran (1999),in a documented article on human pul-monary dirofilariasis, report on a clini-copathological study of 41 infarctualnodules observed in the USA. Eventhough the cases are all due to D. immi-tis, the study is very pertinent to D.repens, for the clinical and histopatho-logical pictures of the two species donot differ greatly, apart from the mor-phology of the parasites. Following onfrom the research of previous authors(Schaub and Rawlings, 1979; Kaiser etal., 1992), it has recently been shownthat filarial parasites alter pulmonaryartery endothelial cell relaxation. Thus,vasoconstriction may play an importantrole in tissue necrosis in pulmonary

100


human dirofilariasis (Mupanamunda etal., 1997).

The histological findings relating tonodules localised in the breast, epi-didymis, spermatic chord, omentumand mesentery overlap: the nematode isimmersed in fibrin-leukocyte necroticmaterial surrounded by demarcationtissue consisting of fibroblast elementstogether with lymphocytes, plasmacells and eosinophils.

Parasitology

The samples comprised mostly imma-ture females from a few centimetres to150 mm in length and with a maximumthickness of µm 620. At times, the sam-ples were already dead on surgicalremoval; at others, they had been par-tially destroyed by the inflammatoryreaction of the host. Often, however,they were still alive, as could be seenfrom their lively movements; indeed,Dissanaike et al. (1997) found this tobe so in 42 out of 70 cases observed.

In Italy, the ratio between living anddead D. repens observed by the authorswas practically the same, although itwas not always possible to be sure

judging by the histological preparation.In 2 cases, a male and a female werefound together in the same nodule(Misic et al., 1996; Mrad et al., 1999),and in another case a male and a femalewere found in 2 separate nodules butclose to each other in the skin of thescrotum (Fernando et al., 2000). Thepresence of 3 nematodes in the samenodule was reported by Degardin andSimonart (1996) in a patient from Iranand by Avdiukhina et al. (1997) inanother patient in the RussianFederation. It has been known that,after being extracted from the nodule,D. repens is capable of surviving for upto 48 hours in physiological solution at4°C (Thérizol-Ferly et al., 1996). Insome rare cases it was noted that thehistopathological transverse sections offemales of D. repens inexplicablyrevealed an unusual number (up to tenor more) of sex tubules (Figs. 1 and 2)(Pampiglione et al., 1992, 1996).Orihel et al. (1997) have now providedan explanation. They were examining 2females of D. repens extracted from thesame site in the same patient, but at aninterval of 10 months.

They deduced correctly that the two

Figs. 1 and 2. Paratransverse sections of D. repens where numerous sections of female sex tubules arevisible (Haematoxylin/Eosin, 250). [Fig. 1: courtesy by Blackwell Ltd, Histopathology (in press)].

101

1 2


parasites must have been introduced bythe carrier at the same time and thattherefore one was older than the other.They explain that, as a female matures,her vagina lengthens, becomes coiledand looped and extends towards thehead beyond the vulva and oesophagus.If the nematode is sectioned above thatpoint, the sex tubules can thus appearto be very numerous. Orihel andEberhard (1998) have helped to clarifyanother important morphologicalaspect for the recognition of D. repensin histological section: whereasGutierrez (1990) stated that “key fig-ures useful in identification of thespecies include longitudinal ridges sep-arated by a distance wider than theridge itself, 95-105 ridges on the cir-cumference of the body”, Orihel andEberhard point out that “the shape,height and interridge distances are actu-ally quite variable at different levels ofthe body in the same worm or even with-in a single transverse section and hencedo not constitute reliable criteria”.

Another morphological feature whoserelative value needs to be reassessed isthat of the diameter of the nematode’sbody in transverse section. Apart fromthe possible variation in diameterdepending on the stage of developmentreached, on eventual regressive alter-ations and on shrinkage due to theworm being killed by the fixative whilestill alive inside the nodule (as can oftenbe seen from the empty space createdaround it) (Fig. 3), the diameter willnormally vary by many microns in thesame individual, depending on the pointat which the section is taken (Fig. 4).

From a review of the different speciesof Dirofilaria existing worldwide(Canestri Trotti et al., 1997) it appears

that, of the 27 species considered valid,only 5 have been reported as havinginfected man, apart from D. immitisand D. repens, namely: D. (N.) tenuis,a parasite of the racoon in NorthAmerica; D. (N.) ursi, a parasite of thebrown bear in Canada, the northernUSA, Siberia and Japan; D. (D.)spectans, a parasite of Mustelidae inBrazil; D. (N.) magnilarvatum, a para-site of catarrhine monkeys in Asia; D.(N.) striata, a parasite of the puma andother American carnivores. The speci-mens of D. ursi found in human cases

Fig. 3. Transverse section of D. repens. The emptyspace around the nematode is due to shrinkage ofits body diameter (Haematoxylin/Eosin, 250).

Fig. 4. Three transverse sections of D. repens.Note the difference in diameter in the same speci-men sectioned at different points of the body(Haematoxylin/Eosin, 150).

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have been termed D. ursi-like, sincethere is no absolute certainty that we aredealing with a single species, either forbears or for man, even though in histo-logical section they appear to be practi-cally identical from a morphologicalpoint of view. Since the zoonotic aspectsof dirofilariasis in Africa are littleknown, future research into the study offindings from that continent will bear inmind D. (N.) corynodes (a frequent par-asite of African monkeys), given its greatsimilarity to D. repens (Orihel, 1969;Orihel and Eberhard, 1998).

On examining the peripheral blood ofa 64-year old patient living on a GreekIsland in the Ionian sea (westernGreece) Petrocheilou et al. (1998) dis-covered microfilariae measuring 210-230 µm in length (and therefore notmatching either D. repens or D. immi-tis), which they attribute to theDirofilaria-like genus, but withoutbeing able to find the adults. In SriLanka, rare cases of subcutaneous diro-filariasis due to a different species butstill belonging to the sub-genusNochtiella, D. linstowi (Dissanaike,1972), have been reported in man injungle areas, home to the monkeysPresbytis entellus and Macaca sinicawhich are its natural reservoir. Since itis difficult to differentiate between thetwo species, some cases due to this lat-ter species could have been interpretedas being due to D. repens(Abeyewickreme et al., 1997).

Studies of great interest have recentlyshown the presence in D. repens ofWolbachia sp., intracellular bacteriaalready reported in insects and filariae,including D. immitis (McLaren et al.,1975), and transmitted transovarially.They are held to play an important role

in the embryogenesis of filariae(Genchi et al., 1998), and so their sup-pression with tetracycline is supposedto inhibit the development of themicrofilariae.

Biomolecular techniques have recent-ly been employed (Casiraghi et al.,2000; Favia et al., 2000) for filogeneticanalysis. These authors have compared10 different species of Onchocercidae,among which D. repens, and confirmthe clustering of the species of thegenus Onchocerca with those of thegenus Dirofilaria.

Diagnosis

The clinical diagnosis of the parasito-sis is almost always wrong, except forsome subconjunctival cases where theoculist can see the nematode, given thatthe conjunctiva is transparent and so isable to diagnose a “helminth parasito-sis”, mistaken sometimes for onchocer-ciasis. In cases of pulmonary infection,clinical diagnosis has been that of can-cer or sarcoidosis (Jelinek et al., 1996)and thoracotomy was always carriedout; a spermatic chord location, erro-neously interpreted as a tumour or tes-ticular tuberculosis, was treated withorchiectomy (Pampiglione et al.,1999a); in breast infections, diagnosisof cancer or fibrous dysplasia of thebreast was also frequently suspected. Ina case of retroocular infection, the doc-tors diagnosed tumour of the orbit(Braun et al., 1996). In another case oflocalisation in the vitreous body, theclinical diagnosis was that of dissemi-nated chorioretinitis of probable para-sitic origin (Vasilkova et al., 1992). Insubcutaneous forms the most frequentdiagnosis has been that of sebaceous

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cyst; other diagnoses have includedlipoma, dermoid cyst, fibroadenoma,dental abscess, muscular haematoma,trauma-induced detachment of the tem-poralis muscle, collagen disease, angio-oedema, atypical Beliset syndrome,periodical disease, larva migrans,recurrent thrombophlebitis, rheumaticdisease, herpetic keratitis, tendinouscyst, neuroma, neurofibroma, inguinallymphadenitis, cervical lymphadenitis,filariasis due to Wuchereria bancrofti,onchocerciasis, loiasis, tumour of theparotid gland and, in two cases, acutepsychosis (Jelinek et al., 1996).With regard to the differential diagno-sis of the parasite, there is an article byOrihel and Eberhard (1998) thatreports, with photographic illustra-tions, the morphological data of thevarious zoonotic filariae capable ofinfecting man by targeting the subcu-taneous tissues and subconjunctiva, theheart and pulmonary vessels, the lym-phatic and nervous systems.In this same context, there are two veryrecent reports of other zoonotic filari-ae: Brugia sp., probably ceylonensis,localised under the conjunctiva of a 53-year-old patient in Sri Lanka(Dissanaike et al., 2000) andMacacanema formosana, also localisedunder the conjunctiva in a 23-year-oldwoman in Taiwan (Lin-Ing Lau, per-sonal communication), neither ofwhich species has ever previously beenreported in that site in man and whichcan be borne in mind in the diagnosticdifferentiation of D. repens. FineNeedle Aspiration Biopsy has provedsuccessful in only 2 cases, one affectingthe breast (Bertoli et al., 1997), theother subcutaneous tissue.In the latter case, however, the nema-

tode revealed itself only because itemerged spontaneously through thehole made by the needle (Kumara-singhe et al., 1997). But generally it isnot recommended in dirofilariasis, asdemonstrated by the American statis-tics for lung infections due to D. immi-tis, in which the outcome has invariablybeen negative (Flieder and Moran,1999).

In the majority of cases, diagnosis isbased on histological examination of thenodule with identification of the mor-phological features of the nematode.

The presence of the external longitu-dinal cuticular ridges has proved essen-tial for the diagnosis of the subgenusNochtiella and for its differentiationfrom D. immitis, which is the mostcommon of the other zoonoticDirofilariae. The most useful stains,apart from the common haema-toxylin/eosin, for the purpose of high-lighting the morphological details haveproved to be PAS and MassonGoldner’s trichrome. In cases in whichthe parasite had been dead for manymonths or years, histological diagnosiswas more difficult; only a careful analy-sis of the remains of the nematode’sbody and the existence at the same timeof other recognisable sections of itenabled a diagnosis to be made(Pampiglione et al., 1999e).

There have been numerous studies car-ried out by various groups of researchersaimed at perfecting a reliable and practi-cal diagnostic reaction on histologicalsections both of the parasite and the car-riers (Chandrasekharan et al., 1994;Favia et al., 1996a,b, 1997, 1998,2000a,b,c; Cancrini et al., 1998a,b,2000; Favia, 1999; Ricci et al., 2000).

Although they have succeeded in

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developing a PCR on fresh tissues orthose fixed in ethyl alcohol, they havenot been able to reproduce the test ontissues fixed in formalin. This is a seri-ous drawback, for biopsies are normal-ly sent to the histologist already fixed in10% formalin. And since the diagnosisof dirofilariasis is hardly ever suspectedbeforehand, it is virtually impossible toget the surgeon doing the biopsy to fixthe excised nodule in methyl alcohol orsend it fresh to the laboratory withoutany form of fixation. However, Vakaliset al. (1999) have developed a PCRtechnique which, in their hands, seemsto have given good results on tissuesfixed in formalin as well.

As regards serological diagnosis,progress has undoubtedly been made byresearch groups of various nationalities,notably Spanish and Italian (Perera etal., 1994, 1998; Favia et al., 1996, 1997,2000; Simon et al., 1997; Cancrini et al.,1998, 1999), but they have not yet beenable to perfect a reaction that is reliable,quick and simple to perform.

However, Ruiz Moreno et al. (1998)did succeed in obtaining confirmationof recovery in a case of human subcon-junctival dirofilariasis by measuringthe reduction in the level of anti-D.repens serous antibodies by immu-noenzymatic means for 3-6 monthsfrom the time of surgery.

Prognosis

In the majority of subcutaneous casesthe prognosis can be consideredbenign, the lesion healing in a few daysfollowing the removal of the nodule.Sometimes, healing occurs sponta-neously with the worm emerging fromthe nodule without any surgical inter-

vention. In cases affecting the ocularregion, however, Avdiukhina et al.(1996) report a 10% incidence of com-plications of a permanent nature, suchas detached retina, glaucoma, opacityof the vitreous body or the crystallinelens, or other deterioration in visualacuity. In cases affecting the visceralorgans (lungs, mesentery, omentum) orthe sexual organs (epididymis, spermat-ic chord, scrotum, female breast), sur-gery (open-chest, laparotomy) can haverelatively serious consequences orcause pointless mutilation, such as pul-monary lobectomy (Jelinek et al., 1996;Pampiglione et al., 2000d) or theremoval of a breast, epididymus orspermatic chord.

Prophylaxis

Effective prevention of parasitosis hadalready been achieved in dogs exposedto natural infection in endemic zones ofcentral Italy by treatment with ivermec-tine per os (>6 mcg/kg) once a monthfor 7 consecutive months from May toNovember (Marconcini et al., 1993). Atpresent, the use of ewable tablets ofivermectine/pirantel pamoate is yieldinggood results in dogs exposed to naturalinfection (Pollono et al., 1998).

In man, however, it would appear thatno experiments in prevention againstdirofilariasis due to D. repens or otherDirofilariae have been undertaken.Since the presence of a large number ofmosquitoes, which are known carriersof the parasite, and the high incidenceof the infection in dogs, which are thenatural reservoir of the parasitosis, areconsidered to be risk factors for man, itcan be supposed that a reduction in theincidence of canine infection together

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with a drive to eliminate the carriersmight considerably reduce the numberof human cases in endemic areas. Thisimplies a preliminary study both of theprevalence and distribution of the para-sitosis in dogs and of the density anddistribution of the carriers.

Therapy

The therapy of choice remains sur-gery. Whereas both diethylcarbamazineand ivermectine have often been usedin cases of canine dirofilariasis, theyhave only occasionally been used inman (van den Ende et al., 1995; Jelineket al., 1996; Avdiukhina et al., 1997;Petrocheilou et al., 1998) and notalways to any apparent effect. A patientwith a nodule on one hand and whohad been treated with the two drugshad a second nodule appear on histhigh 4 weeks after the end of treat-ment (Jelinek et al., 1996). Other drugsused have been levamisole (Dorofeievet al., 1997), albendazole (van denEnde et al., 1995), thiabendazole andcortisones (Delage et al., 1995), againwith doubtful results. That drugs arenot in current use is partly due to thefact that the clinical diagnosis of thecases is generally wrong and appropri-ate therapy is not prescribed. In anycase, the use of filaricides with a certainlevel of toxicity does not seem appro-priate in all those cases of subcuta-neous infection in which a minor surgi-cal operation performed in outpatientscan resolve the problem. The sameholds for subconjunctival or periocularinfections, where possible violent aller-gic reactions induced by the drugand/or by the death of the nematode

could cause permanent damage to thevisual organ.

Discussion

In view of the number of publicationsthat have appeared in the last 20 years,by comparison with previous decades,it is evident that the medical world hasgradually become increasingly interest-ed in this zoonosis. This can be attrib-uted not only to the clear increase inthe incidence of the disease but also inpart to the reports of the parasiteinfecting the viscera (lungs, mesentery)as well as the female breast and themale genitalia (scrotum, verga, sper-matic chord, epididymis). The involve-ment of these locations has almostalways led to diagnoses of forms ofmalignant tumour requiring drasticsurgery and has thus highlighted theimportance of the parasite in humanpathology. It could be argued that theincrease in the number of cases, whichuntil half a century ago were consid-ered exceptional and have been report-ed with increasing frequency in the lastfew decades, is only an apparentincrease resulting from a more carefulexamination of subjects affected andfrom more refined diagnostic tech-niques. Yet the increase does seem tobe in part real, particularly in view ofthe enhancement in the temperatezones of the climatic conditions (tem-perature, relative humidity, rainfall,evaporation) that favour not only thegrowth of the carrier Culicidae (Martinand Lefebvre, 1995) but also the devel-opment of the larval phase of the nem-atode inside the carrier. That this is sois proved by the contemporaneous

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increase in the number of dogs infect-ed, at least in endemic zones ofPiedmont, as emerges from careful sur-veys carried out in recent years (Rossiet al., 1996). A similar increase in thenumber of cases in recent decades hasbeen observed in some republics of theRussian Federation (Avdiukhina et al.,1997). Parasitosis due to D. repens cantherefore be considered an emergentzoonosis in many geographical areas ofthe Old World. The number of casesreported worldwide since 1885 hasreached 782 spread over 37 differentcountries, all of which are in the OldWorld (Fig. 5).

To this must be added a fair numberof cases not diagnosed, and thereforenot published, others that recoveredspontaneously, others occurring inhighly endemic zones as in someprovinces of Italy (Piedmont), Sri

Lanka (Western province) and Russia(Caucasus) and which, being consid-ered by now an everyday complaint, areno longer reported and, finally, thoseoccurring in the developing countriesand which, given the lack of facilitiesfor histological diagnosis, are not evenscreened by the histopathologist.Taking into account the 1995 figures,Italy is still in number one position asregards the number of cases reported(298) between 1885 and 2000, fol-lowed by Sri Lanka (132 cases), Russia(Siberia inclusive) (83 cases), France(76 cases), Ukraine (51 cases), Greece(27 cases), Turkey (18 cases), Hungary(11 cases), and then the other countrieswith less than 10 cases each. However,if we relate these figures to the numberof inhabitants and territorial area, SriLanka, with a population of 18,300,000inhabitants and a surface area of 65,610

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

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km2 (FAO, 1997) is the country mostseverely affected, while Italy, with apopulation of 57,200,000 inhabitantsand a surface area of 301,278 km2,moves down to second place.

The age group most commonly affect-ed comprises adults, mainly between 40and 49 years of age (Fig. 6), as alsoreported in the 1995 review. The highnumber of children affected is dueabove all to those reported from SriLanka, where 33.6% (44 out of 132)referred to children below the age of 10.

Women are more commonly affectedthan men, even if not statistically sig-nificant, as appeared in the previousreview, accounting for 55.4% of allcases reported between 1885 and thepresent day (Fig. 7). Again with refer-ence to overall figures publishedbetween 1995 and 2000, the distribu-tion of sites of infection in the human

body (Fig. 8) remains much the same asreported in 1995. The majority of cases(75.8%) affect the upper half of thebody, particularly the ocular region,which alone accounts for 30.5% of thetotal; the female breast (5.4%), themale genital organs (6.5%), the lungs(2.6%) and the abdominal viscera,comprising mesentery and omentum(1.3%), also remain relatively impor-tant. The marked prevalence of casesreported from Sri Lanka affecting thegenitalia, particularly of children(Dissanaike et al., 1997), may be due tothe clothing and sleeping habits of thelocal child population as well as to thespecial tropisms of the carrier mosqui-toes, that are perhaps attracted byammonia smells and other odours insubjects with low levels of personalhygiene. Of the various dirofilariases inthe world that can affect man, that due

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

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

160

140

120

100

80

60

40

20

0

Num

ber

of c

ases

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to D. repens is the most common interms of frequency, diffusion and vari-ety of localisation in the body; the oth-ers account overall for fewer than 300cases. The great variety of organsaffected by dirofilariasis due to D.repens justifies the current interestamong medical practitioners in thiszoonosis: specialists in various healthsectors, from dermatology to ophthal-mology, from urology to pneumology,

from internal medicine to surgery, fromimmunology to molecular biology –they are all involved. But those mostdirectly involved in the study of itsaspects and as yet unresolved problemsare, of course, the parasitologists andhistopathologists. At present, the twosectors in which research teams need toconduct a thoroughgoing investigativeinquiry are diagnostics – with a view todeveloping practical techniques for his-tological and serological diagnosis ofthe parasitosis – and therapeutics, inparticular to avoid drastic surgery incases with involvement of lung, breast,male sexual organs or orbital cavity.Diagnostic research will also lead toimproved analysis of the prevalence ofcurrent cases of human infection,apparent or not, in a given populationand thus make for a clear understandingof the mechanisms underlying the rela-tionship between parasites and man.

Acknowledgements

The Authors are indebted for valuable sugges-tions, advices, references and personal communi-cations of observed cases to Professors N.V.Chandrasekharan, S.A. Dissanaike, J.O. Gebbers,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.Delage, E. Feyles, E. Fok, R. Fueter, P.L. Garavelli,M. Giansanti, M. Gobbo, R. Groell, T. Jelinek, B.Molet, M. Pastormerlo, M. Pavesi, N. Ratnatunga,E. V. Schwan, K. C. Shekhar, G. Speranza, C.Stayerman, R. Stenzenberger, D. Strianese, W.Tarello, M. Thérizol-Ferly, G. Vitullo, F.X. Weill. Thanks are also due to Mrs J. Petrova for translat-ing texts from Russian, Mrs T.G. Afanasieva of theState Russian Library, for providing publishedpapers difficult to find, and to Dr M.L. Fioravantiand Mr Luca Fabiani for technical help.

From Sri Lanka, several colleagues providedprofessor Dissanaike with Cingalese cases referred

Fig. 7. Distribution of human dirofilariasis due toD. repens according to sex, when specified, from1885 to 2000.

Fig. 8. Locations of D. repens in the human body,when specified, from 1885 to 2000 (the dashedlines refer to internal sites).

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in our list as personal communications, namelyProfessors J.S. Edirisinghe and M Wijesundera,and Doctors W. Abeyewickreme, D. Ariyaratme,B.G. de Silva, P. Kumarasinghe, S. Samarasinghe,K. Vithanage, M. Weerasooriya, M.D. Weilgama,and Mr R.L. Ihalamulla. Their contributions arehighly appreciated. This study was carried outwith the partial contribution of the EmiliaRomagna region.

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Weill FX, Accoceberry I, Montané V, Le Moine JJ,Couprie B, Djossou F, Malvy D, Le Bras M,1999. Dirofilariose orbitaire à Dirofilaria repensen France. Un cas humain contracté sur le litoralatlantique. Méd Mal Infect 29: 642-645.

Weill FX, Bameul F, Tournaud M, Lanneluc C,Ferrari F, Tribouley J, 1997. Dirofilariosehumaine autochtone. Presse Méd 26: 1579.

Weill FX, Belleannée G, Accouberry I, Andres J,Revault-Gaye D, de Mascarel A, Couprie B,2000. Un curieux nodule sous-cutané d’ origineparasitaire. Ann Pathol 20: 163-164.

Zardi O, Zardi EM, Falagiani P, Zardi DM, ZardiMC, Barduagni F, Barduagni O, 1997.Dirofilariasi umana sottocutanea. Pathologica89: 31-35.

Zahler M, Glaser B, Gothe R, 1997. EingeschlepteParasiten bei Hunden: Dirofilaria repens undDipetalonema reconditum. Tierärztl Prax 25:388-392.

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Heartworm (Dirofilaria immitis)disease in dogs

Luigi Venco

8

Heartworm disease in dogs

Introduction

Despite the name “heartworm” sug-gests a primitive cardiac involvement,the main localization of the worms andthe first damages are in pulmonaryarteries and heartworm disease shouldbe considered as a pulmonary diseasethat in the last stage only involves theright cardiac chambers.

Few days after heartworms reach cau-dal pulmonary arteries, endothelial cellsbecome swollen with wide intercellularjunctions and disoriented longitudinalaxes, as response to the trauma.Activates neutrophiles adhere to theendothelium surface and enter thespace between endothelial cells.Furthermore, as linear areas of subendothelium are exposed, platelet adhe-sion and activation is greatly stimulated.Damaged arterial surface allows albu-min, plasma fluid and blood cells toreach the perivascular space.

After the endothelial changes, theintima thickens with fluid, leukocytesinvade the wall and smooth musclescells multiply within tunica media andmigrate toward the endovascular sur-face as response to growth factorreleased by platelets. The multiplica-tion and migration of smooth musclecells cause the presence, on the internalarterial surface, of villi which are madeby smooth muscle cells and collagenand covered by endothelial like-cells(Rawlings, 1986; Calvert andRawlings, 1988). The gravity of villousproliferation is directly related to dura-tion of infection and worm burden. Thearterial surface of heavily infected dogsand cats appear rough and velvety, andboth the lumen and the compliance ofthe pulmonary arteries are reduced.

Lung disease occurs secondary to vas-cular changes. Fluid and protein leakingthrough the vessel wall of affected arter-ies produce oedema in the parenchyma.Spontaneous death of some worms canproduce thromboembolism and severeinflammatory reactions.

The reduction of compliance andgauge of pulmonary arteries, that can bealso occluded by either thromboem-bolism or severe villous proliferation(Fig. 1), results in a hypertensive pul-monary state and, as a consequence, inan increased after load for the right ven-tricle which can induce “cor pulmonare”and right cardiac congestive heart fail-ure. Protein and fluid leaking throughthe vessel wall of affected arteries pro-duce further oedema and inflammationin the parenchyma (Dillon et al., 1995).

Based on the pathogenesis the clinicalevolution of heartworm disease in dogsis usually chronic. Most infected dogsdo not show any symptoms of the dis-ease for a long time, months or years,depending on worm burden, individualreactivity and exercise, as arterial dam-ages are more severe in dogs with

Fig. 1. Right ventricle outflow tract and pulmonaryartery of a dog with severe heartworm infections.Note the large worm burden and the villous proli-feration of the pulmonary artery endothelium.

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intensive exercise than in dogs at rest(Dillon et al., 1995). Signs of the dis-ease develop gradually and may beginwith a chronic cough.

Coughing may be followed by dysp-noea, from moderate to severe, weak-ness, and sometimes lipothymias afterexercise or excitement. At this timeabnormal pulmonary sounds (crackles)over the caudal lung lobes and secondheart sound splitting can be often heard.Later, when right cardiac congestivefailure is developing, swelling of theabdomen and sometimes legs fromfluid accumulation, anorexia, weightloss, dehydration, is usually noted. Atthis stage, cardiac murmur over theright side of the thorax due to tricuspidvalve insufficiency and abnormal car-diac rhythm due to atrial fibrillation arecommon findings. Sudden death rarelyoccurs and usually it happens followingrespiratory distress or cachexia.

In the chronic pathway of the diseasesometimes acute symptomatology mayoccur. After severe spontaneous throm-boembolism following the naturaldeath of many heartworms, dogs mayshow acute life threatening dyspnoeaand haemoptysis.

In small sized dogs is furthermore acommon event the displacement ofadult worms from pulmonary arteriesto right cardiac chambers due to pul-monary hypertension and sudden fallin right cardiac output. In this casedogs affected shows the so called“caval syndrome”. Dyspnoea, tricuspidcardiac murmur and emoglobinuria(due to mechanical haemolysis in rightcardiac chambers) are the most typicalsigns and fatal outcome is usual(Kitagawa et al., 1987; Atwel andBuoro, 1988; Venco, 1993).

Diagnosis

Diagnosis of heartworm infection canbe made in dogs by blood test detectingcirculating microfilariae or adult anti-gens but further diagnostic proceduresare usually required to determine theseverity of disease and which is the besttreatment (Knight, 1995).

Blood test for microfilariae

Blood sample is examined after con-centration (Knott or Difill test) forthe presence of microfilariae. If micro-filariae are seen and identified asD. immitis, based on morphology thatis considered a definitive proof of infec-tion (specificity 100 %). However up to30% of dogs do not have circulatingmicrofilariae even though they harbouradult worms, due to the presence ofonly worms of the same sex (quiteunusual in dogs), immune reactivity ofthe host to microfilariae or administra-tion of microfilaricidal drugs. The sensi-tivity of test for microfilariae is nottherefore considered sufficient to ruleout the infection in case of negative test.

Blood test for adult female antigens

Tests designed to detect heartwormadult antigens based on ELISA or col-loidal gold staining techniques areconsidered highly specific as crossreactivity with other dogs parasites(i.e. D. repens, Dipetalonema sp.)does not occur.

These tests allow detection of adultheartworm antigens produced only byfemale worms and may provide infor-mation about worm burden (Knight,1995; Venco et al., 2003). The sensitiv-

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ity is actually very high, but false nega-tive results may occur in prepatent orvery light infections or when only maleworms are present (McCall, 1992).

Thoracic radiographs

Thoracic radiographs may show, inthe advanced stage, enlargement of thepulmonary arteries, abnormal pul-monary patterns and in the worst casesright-sided cardiomegaly. If congestiveright heart failure is present peritonealand pleural effusion can be noted(Rawlings, 1986; Calvert and Rawlings,1988). They are useful to assess theseverity of the pulmonary lesions butnot for evaluating worm burden (Vencoet al., 2003). Since radiographic signsof advanced pulmonary vascular dis-ease may persist long after an infectionhas run its course, some of the mostseverely diseased dogs may have dis-proportionately low worm burden (Fig.2). On the contrary, some inactive dogsmay have large worm burdens and beclinically asymptomatic with no or triv-ial radiographic lesions (Fig. 3).

Electrocardiography

As electrocardiogram displays theelectrical activity of the heart, abnor-malities, (electrical axis right deviation,atrial fibrillation) are usually foundonly in the last stage of the disease,when right cardiac chambers presentsevere damages.

Echocardiography

Echocardiography allows a directvisualization of cardiac chambers andconnected vessels (Moise, 1988).

It also allows the visualization of para-sites in right cardiac chambers, caudalvena cava, main pulmonary artery andproximal tract of both caudal pulmonaryarteries (Fig. 4). The heartworms arevisualized as double, linear parallelobjects floating in the right cardiacchambers or into the lumen of vessels(Moise, 1988; Badertscher et al., 1988). It is performed mainly in cases whereclinical and radiographic findings sug-gest severe disease. Cardiac ultrasoundcan increase the accuracy in staging the

Fig. 2. Thoracic radiograph of a 12-year-old infec-ted dog. With right sided cardiomegaly and severepulmonary vascular disease. The level of circula-ting antigens were very low.

Fig. 3. Thoracic radiograph of a 3 year old infec-ted dog with just a trivial cranial pulmonary arteryenlargement. From this dog 53 adult worms weresurgically removed.

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disease and estimating the worm bur-den, both of which affect the treatmentprogram and the prognosis.

Therapy

It has been said that the treatment ofheartworm infection is difficult. Thereare several strategies that can be usedincluding the option of not treating atall. The important concept to realize isthat treating for heartworm infection isneither simple nor safe in itself.

Prior to therapy, the heartwormpatient is assessed and rated for risk ofdeveloping post-adulticide thromboem-bolism.

Previously 4 classes were described inorder to prevent the post-adulticidethromboembolism and give a correctprognosis (from class 1, low risk toclass 4, very high risk) (Di Sacco andVezzoni, 1992) but at present a moresimple classification is usually pre-ferred and the patient is included intoone of two categories (low and highrisk). Important factors include: how

many worms are thought to be presentbased upon the ELISA tests performedand ultrasound examination (Venco etal., 2004), the size of the dog, the ageof the dog (dogs ranging from 5 to 7years are at high risk to harbour thelargest worm burden; Venco et al.,2004), concurrent health factors, sever-ity of the pulmonary disease, and thedegree to which exercise can berestricted in the recovery period.

The categories into which patients aregrouped are as follows:

Low risk of thromboembolic complica-tions (low worm burden and noparenchymal and/or pulmonary vascu-lar lesions)

Dogs included in this group must sat-isfy all this conditions:

- No symptoms- Normal thoracic radiographs- Low level of circulating antigens or

a negative antigen test with circulat-ing microfilariae

- No worms visualized by echocardio-graphy

- No concurrent diseases- Permission of exercise restriction

High risk of thromboembolic complica-tions

In this group should be included allthe dog that do not satisfy one or moreof these conditions:

- Symptoms related to the disease(coughing, lipotimias, swelling ofthe abdomen)

- Abnormal thoracic radiographs- High level of circulating antigens- Worms visualized by echocardiog-

raphy- Concurrent diseases- No permission of exercise restriction.

Fig. 4. Echocardiogram of the same dog of Fig. 3.Heartworms are visualized as double, linear paral-lel objects (arrow) floating into the lumen of theright pulmonary artery.

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Symptomatic therapy includes drugsand measures that can improve car-diopulmonary circulation and lunginflation in order to relief symptoms indogs that cannot undergo causal thera-py or to prepare them for a adulticideor surgical therapy.

Restriction of exercise and, in select-ed cases, cage rest seems to be the mostimportant measure to improve car-diopulmonary circulation and to reducepulmonary hypertension (Dillon et al.,1995).

Anti-inflammatory doses of glucocor-ticosteroid (prednisolone 2 mg/kg s.i.d.for four or five days) given at diminish-ing rate can control pulmonary inflam-mation and thromboembolism.

Diuretics (furosemide 1 mg/kg b.i.d.)are useful when right congestive heartfailure is present to reduce fluid effu-sions. Digoxin may be administeredonly to control atrial fibrillation. Theuse of aspirin is debatable and as notsecure proofs of beneficial antithrom-botic effect have been reported, for thisreason the empiric use of aspirin is notadvised (Knight, 1995).

The organical arsenical melarsominedihydocloride is the only available com-pound to be used in the adulticideheartworm therapy in dogs.

Two intramuscular injections of 2.5mg/kg 24 hours apart is the standardregimen, but to reduce the risk of pul-monary thromboembolism a moregradual two step treatment is stronglyadvised by giving one injection andthen administering the standard pair ofinjections at least 50 days later (Keisteret al., 1992; Rawlings and McCall,1996). In fact, one administration ofmelarsomine at the dose of 2.5 mg/kgkills about 90% of male worms and

10% of female worms resulting there-fore in 50% reduction of the wormburden (which is safer in terms ofembolism and shock). For this reasons,the three-injection alternative protocolis the treatment of choice of theAmerican Heartworm Society and sev-eral university teaching hospitals,regardless of stage of disease.

Pulmonary thromboembolism is aninevitable consequence of a successfuladulticide therapy. If several worms diewidespread pulmonary thrombosis fre-quently develops. Mild thromboem-bolism may be clinically unapparent,but in severe cases life threatening res-piratory distress can occur.

These complication can be reducedby restriction of exercise (no walks, norunning around; the dog must stayindoors or, in selected cases, a cagerest) during the 30-40 days followingthe treatment and by administration ofcalcium heparin and anti-inflammatorydoses of glucocorticosteroid to controlclinical signs of thromboembolism (DiSacco and Vezzoni, 1992; Vezzoni etal., 1992; Rawlings and McCall, 1996).

It is now known that certain macro-cyclic lactones have adulticidal proper-ties (McCall et al., 2001).

Experimental studies have shownivermectin to have partial adulticidalproperties when used continuously forat least 16 months at preventativedoses (6-12 mcg/kg/month) and 100%adulticidal efficacy if administered con-tinuously for over 30 months (McCallet al., 2001).

While there may be a role for thistherapeutic strategy in few and selectedcases in which patient age, or concur-rent medical problems prohibit melar-somine therapy, the current recommen-

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dations are that ivermectin is notadapted as the primary adulticidalapproach, and that this kind of therapyshould be used carefully. In fact, theadulticide effect of ivermectin generallyrequires long time span before heart-worms are eliminated completely.Furthermore, the older are wormswhen first exposed to ivermectin, theslower they are to die. In the meantime,the infection persists and continues tocause disease. Clinical observationssuggest that heartworm-positive activedogs in prolonged ivermectin treatmentmay worsen if ivermectin is givenmonthly for 2 years (Venco et al.,2004).

Surgical therapy is advised when sev-eral worm displacement in the rightcardiac chambers produces the suddenonset of severe symptoms (caval syn-drome). It can be accomplished undergeneral anaesthesia with FlexibleAlligator Forceps introduced via jugu-lar vein.

Flexible Alligator Forceps aided byfluoroscopic guidance can access notonly right cardiac chambers but alsopulmonary arteries. The main pul-monary artery and lobar branches canbe accessed with flexible alligator for-ceps, aided by fluoroscopic guidance(Ishihara et al., 1990). Intra-operativemortality with this technique is verylow. Overall, survival and rate of recov-ery of dogs at high risk of pulmonarythromboembolism is improved signifi-cantly by physically removing as manyworms as possible. When the facilitiesare available, worm extraction is theprocedure of choice for the most heav-ily infected and high risk dogs. Beforeelecting this method of treatment,echocardiographic visualization of the

pulmonary arteries should be per-formed to determine if a sufficientnumber of worms are in accessiblelocations.

Surgical removal of heartworm canavoid pulmonary thromboembolism, ascompared to pharmacologic adulti-cides, such as melarsomine (Morini etal., 1998). This procedure, however,requires specialized training and instru-mentation, including fluoroscopicimaging capabilities. Nevertheless, itremains a very good and a safe alterna-tive for the management of high riskpatients and the best choice in dogsharbouring a large worm burden.

References

Atwell RB, Buoro BJ, 1988. Caval syndrome. In:Boreman PFL, Atwell RB Eds. DirofilariasisBoca Raton, CRC Press Inc, 191-203 pp.

Badertscher RR , Losonsky JM , Paul AJ , KnellerSK, 1988. Two dimensional echocardiographyfor diagnosis of dirofilariasis in nine dogs.JAVMA 193: 843-846.

Calvert CA, Rawlings CA, 1988. CanineHeartworm Disease In: Canine and FelineCardiology. Fox Pr, Ed. Churchill LivingstoneInc, 519-549 pp.

Dillon R, Brawner WR, Hanrahan L, 1995.Influence of number of parasites and exercise onthe severity of heartworm disease in dogs. In:Soll MD and Knight DH Eds. Proceedings of the’95 Heartworm Symposium, AmericanHeartworm Society, Batavia, IL, 113-120 pp.

Dillon R, Warner AE, Molina RM, 1995.Pulmonary parenchymal changes in dogs andcats after experimental transplantation of deadDirofilaria immitis. In: Soll MD and Knight DHEds. Proceedings of the heartworm Symposium’95, American Heartworm Society, Batavia, IL,97-101 pp.

Di Sacco B, Vezzoni A, 1992. Clinical classifica-tion of heartworm disease for the purpose ofadding objectivity to the assessment of thera-peutic efficacy of adulticidal drugs in the field.In: Soll M.D. Ed Proceedings of the HeartwormSymposium ’92, American Heartworm Society,Batavia, 209-214 pp.

Ishihara K, Kitagawa H, Sasaky Y, 1990. Efficacy

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of heartworm removal in dogs with dirofilarialhemoglobinuria using Flexible AlligatorForceps. Jpn J Vet Sci 53: 591-599.

Keister DM, Dzimianski MT, McTier TL, McCallJW, Brown J, 1992. Dose selection and confir-mation of RM 340, a new filaricide for the treat-ment of dogs with immature and matureDirofilaria immitis. In: Soll MD, EdProceedings of the Heartworm Symposium ’92:American Heartworm Society, Batavia, IL, 225-240 pp.

Kitagawa H, Sasaky Y, Ishiara K, 1987. CanineDirofilaria hemoglobinuria: changes in rightheart hemodinamics and heartworm migrationfrom pulmonary artery toward right atrium fol-lowing beta - blocker administration. Jpn J VetSci 49: 1081-1086.

Knight DH, 1995. Guidelines for diagnosis andmanagement of heartworm (Dirofilaria immitis)infection. In: Bonagura JD Ed. Kirk’s CurrentVeterinary Theraphy XII Small Animal Practice.WB Saunders Co.

McCall JW, 1992. A parallel between experimen-tally induced canine and feline heartworm dis-ease. Proceedings of XVII WSAVA WorldCongress. Rome, Italy 24th-27th September.Delfino: 255-261.

McCall JW, Guerrero J, Roberts RE, Supakorndej N,Mansour AE, Dzimianski MT, McCall SD, 2001.Further evidence of clinical prophylactic (reach-back) and adulticidal activity of monthly adminis-tration of ivermectin and pyrantel pamoate indogs experimentally infected with heartworms. InSeward, L., Ed. Proceedings of the AmericanHeartworm Symposium ‘01. AmericanHeartworm Society, Batavia, IL, 189-200 pp.

Moise NS, 1988. Echocardiography In: Canineand Feline Cardiology, PR Fox Ed. New York,Churchill Livingstone Inc, 113-156 pp.

Morini S, Venco L, Fagioli P, Genchi C, 1998.Surgical removal of heartworms versus melar-somine treatment of naturally-infected dogswith risk of thromboembolism. In Seward, L.,Ed. Proceedings of the American HeartwormSymposium ‘98. American Heartworm Society,Batavia, IL, 235-240 pp.

Rawlings CA, 1986. Heartworm disease in dogsand cats WB Saunders Co. Philadelphia.

Rawlings CA, McCall JW, 1996. Melarsomine: Anew heartworm adulticide compound.Continuing Education 18: 373-379.

Venco L, 1993. Approccio diagnostico alla sin-drome della vena cava. Veterinaria, SCIVAC Ed.(Cremona) 7: 11-18 [in Italian].

Venco L, Genchi C, Vigevani Colson P, Kramer L,2003. Relative utility of echocardiography, radi-ography, serologic testing and microfilariaecounts to predict adult worm burden in dogsnaturally infected with heartworms. In: SewardLE, Knight DH, Eds Recent Advances inHeartworm Disease. Symposium ’01. AmericanHeartworm Society. Batavia, IL, 111-124 pp.

Venco L, McCall JW, Guerrero J, Genchi C, 2004.Efficacy of long-term monthly administration ofivermectin on the progress of naturally acquiredheartworm infections in dogs. Vet Parasitol 124:259-268.

Vezzoni A, Genchi C, Raynaud JP, 1992. Adulticideefficacy of RM 340 in dogs with mild and severenatural infections. In: Soll M.D., Ed. Proceedingsof the Heartworm Symposium ’92, AmericanHeartworm Society, Batavia, IL, 231-240 pp.

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Heartworm (Dirofilaria immitis)disease in cats

Luigi Venco

9

Heartworm disease in cats

Cat is considered a susceptible butnot ideal host for Dirofilaria immitis.Increased host resistance is reflected bythe relatively low adult worm burden innatural infections (cats generally har-bour 1 to 8 worms with 2 to 4 wormsbeing the usual burden, Genchi et al.,1992), the low number of heartwormsthat develop after experimental inocu-lation with infective larvae, the prolon-ged pre-patent period (7-8 months),the low level and short duration ofmicrofilaremia, and the short life spanof adult worms (2-3 years) (Dillon,1984; Calvert, 1989; McCall et al.,1992; Atkins et al., 1995).

In cats, changes in pulmonary arteriesand lungs after infection seem to besimilar to those found in dogs, but rightcardiac chambers well bear pulmonaryhypertension and right cardiac heartfailure is an unusual finding (Dillon etal., 1995; Atkins et al., 1995).

In cats, the clinical presentation is quitedifferent than in the canine counterpart.Most cats seem to well bear the infectionfor long time. These cats may have aspontaneous self-cure due to the naturaldeath of parasites without any kind of cli-nical signs or may suddenly show drama-tic acute symptoms. Respiratory signs ascoughing, dyspnoea, haemoptysis areusually seen but also vomiting frequentlyoccurs. Sudden death in apparentlyhealthy cats is furthermore not rarelyobserved (McCall et al., 1994; Holmes,1995; Atkins et al., 1995) (Fig. 1).Chronic symptoms including coughing,vomiting, diarrhoea, weight loss can beless frequently observed.

On the contrary than in dogs, sympto-matology related to right ventricularheart failure is not considered consi-stent with heartworm infection in cats.

The onset of symptoms in most casesseems to be related to the natural deathof parasites or to the first arriving of L5heartworms in the pulmonary arteries.

Diagnosis


As microfilaremia in cats is unlikely,sensitivity of test for detection of circu-lating microfilariae is very low despitespecificity is considered 100% as indogs (Atkins et al., 1995).

Blood test for adult antigens

Test detecting adult female heart-worm antigens can provide a definitiveproof of infections in cats because ofthe very high specificity. Neverthelessbecause the worm burden is usuallyvery light in cats, infections caused onlyby male heartworms are not infrequentand symptomatology may be frequentlydue to immature worms, these testyield false negative result in a large ave-rage. A negative test can not thereforeconsidered sufficient to rule out theinfection (Atkins et al., 1995).

Fig. 1. Two adult female heartworms in a cat thatexperienced sudden death.

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Blood test for antibodies to adult heart-worm

Due to the low sensitivity of tests forcirculating microfilariae and adult anti-gens in cats, test for detection of anti-bodies to adult heartworm can be usefulused (Atkins et al., 1995; Prieto et al.,1997; Genchi et al., 1998). This kind oftest has high sensitivity but not comple-te specificity because of cross reactivitywith other parasites or antibodies toabortive infections. Consequently anti-body tests should be interpreted care-fully, taking other relevant clinical infor-mation into consideration.

Thoracic radiographs

Thoracic radiography is an importanttool for the diagnosis of feline heart-worm disease. Despite thoracic abnor-malities in few cases are absent ortransient (Selcer et al., 1996), typicalfindings as enlarged peripheral bran-ches of the pulmonary arteries accom-panied by varying degrees of pulmo-nary parenchymal disease are stronglyconsistent with heartworm infection(Fig. 2). Enlargement of the main pul-

monary artery cannot be observedbecause this tract of artery is obscuredby cardiac silhouette. Right-sided car-diomegaly is not considered a typicalfinding in cat.

Non selective angiocardiography

Non selective angiocardiography isuseful in visualizing the gross morpho-logy of the pulmonary arteries. Seldomthe heartworms can be seen as negativefilling defects within opacified arteries(Atkins et al., 1995).

Electrocardiography

Heartworm infection does not involveright cardiac chambers. Consequentlyelectrocardiography cannot provideuseful information in infected cats.

Echocardiography

Cardiac ultrasound allows the directvisualization of the parasites in rightatrium and ventricle, main pulmonaryartery and proximal tract of both itsperipheral branches (Fig. 3).Specificity is virtually 100% and sensi-

Fig. 2. Thoracic radiograph of a heartworm infect-ed cat with of pulmonary parenchymal disease(caudal lung lobes).

Fig. 3. Echocardiogram of a heartworm infected cat.A worm is visualized as double, linear parallel lines(arrow) into the lumen of the right pulmonary artery.

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tivity in cats seems to be very high(Venco et al., 1998, 1999b) becausethe portion of caudal pulmonary arte-ries that can not be thoroughly interro-gate because of the acoustic impedanceof the air inflated lungs is very shortwhen compared with the length of theadult parasite. Based on these conside-rations, cardiac ultrasonographyshould be always performed whenheartworm infection is suspected.

Transtracheal lavage

The presence of eosinophiles in a tra-cheal wash, with or without eosinophi-lia, may be noted 4 to 7 months afterinfection but this findings is not speci-fic and infection with other pulmonaryparasites (Paragonimus kellicotti,Aelurostrongylus abstrusus) and aller-gic pneumonitis should be ruled out(Atkins et al., 1995).

Therapy

Diminishing doses of prednisone areadvised in cats in order to relief respira-tory distress. The dosage is 2 mg/kg dailyinitially, then declining to 0.5 mg/kgevery other day for two weeks, and thendiscontinuing treatment after an additio-nal two weeks (Atkins et al., 1995).

If crisis is due to embolization of deadworms high doses of prednisone (1-2mg/kg 3 times a day) are recommended(Dillon, 1986).

In previous studies, when thiacetarsa-mide was the only arsenical availablecompound, some treatment regimenwas attempt against adult worms. Thesame dosage and regimen used in trea-ting dogs, 2.2 mg/kg twice daily for twodays, was used for cats. The results were

debatable. Turner et al. (1989) reportedsome toxicity in heartworm naive cats,but later reports showed that thiacetar-samide delivered to normal cats produ-ced no respiratory distress or altered thebody temperature (Dillon et al., 1992).However a large average heartworminfected cats develop acute respiratorydistress or sudden death in the post-treatment period. These effects seem tobe due to embolization associated withworm death (Dillon et al., 1992).

The organical arsenical melarsominedihydocloride is the only available com-pound now on the market, but there isinsufficient experience about its use incats until now. Furthermore, few datasuggests that melarsomine is toxic tocats at dosages >3.5 mg/kg. Ivermectinat 24 µg/kg monthly given for 2 yearshas been reported to reduce worm bur-dens as compared to untreated cats.Since cats usually harbour low wormburdens and the main problem is thereaction that arise when the worms die(and not the worm mass by itself) thisreaction could probably occur evenwhen the ivermectin-treated wormsdie, but its intensity is unknown.

Anyway, there are no studies sugge-sting that medical adulticidal therapyincreases the survival rate of heart-worm naturally infected cats (Knight etal., 2002). Due to these reasons,macrofilaricide treatment is not advi-sed in cats unless in selected cases.

In case of caval syndrome or when aheavy worm burden is visualized byechocardiography in right cardiacchambers, surgery may be attempted.Worm can be extracted via jugular veinusing thin alligator forceps, horse hairbrush or basket catheters (Glaus et al.,1995; Borgarelli et al., 1997; Venco et

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al., 1999b). Because of the small size ofthe feline heart pulmonary, arteries can-not be accessed. Special care must betaken during the heartworm removalbecause traumatic dissection of a wormmay result in circulatory collapse anddeath (Venco et al., 1999b).

References

Atkins CE , Atwell RB , Dillon AR , Genchi C ,Hayasaky M , Holmes RA, Knight DH , LukoffDK , Mc Call JW, Slocombe JO, 1995.Guidelines for the diagnosis, treatment, and pre-vention of heartworm (Dirofilaria immitis)Infection in cats. In: MD Soll and DH KnightEds. Proceedings of the heartworm symposium’95. American Heartworm Society, Batavia, IL,309-312 pp.

Borgarelli M, Venco L, Piga PM, Bonino F, RyanWG, 1997. Surgical removal of heartworms fromthe right atrium of a cat. JAVMA 211: 68-69.

Calvert CA, 1989. Feline Heartworm disease. In:RG Sherding Ed. The Cat Diseases and ClinicalManagement Churchill Livingston Inc. NewYork, 95-510 pp.

Dillon R, 1984. Feline Dirofilariasis. Vet ClinNorth Am 14: 1185.

Dillon R, 1986. Feline heartworm disease. In: GFOtto Ed. Proceedings of the HeartwormSymposium ’86..: American Heartworm Society,Washington DC, 149-154 pp.

Dillon R, Cox N, Brawner B, D’Andrea G, 1992.The effects of thiacetarsamide administration tonormal cats. In: MD Soll Ed. Proceedings of theHeartworm Symposium ’92, AmericanHeartworm Society, Batavia IL, 133-137 pp.

Dillon R, Warner AE, Molina RM, 1995. Pulmonaryparenchymal changes in dogs and cats after expe-rimental transplantation of dead Dirofilaria immi-tis. In MD Soll and DH Knight Eds. Proceedingsof the Heartworm Symposium ’95. AmericanHeartworm Society, Batavia IL, 97-101 pp.

Genchi C, Guerrero J, Di Sacco B, Formaggini L,1992. Prevalence of Dirofilaria immitis infec-tion in Italian cats. Proceedings of HeartwormSymposium ‘92, American Heartworm Society,Batavia IL, 97-102 pp.

Genchi C, Kramer LH, L Venco, Prieto G, SimonF, 1998. Comparison of antibody and antigentesting with echocardiographic for the detectionof heartworm (Dirofilaria immitis) in cats. In:Recent Advances in Heartworm Disease, in RLSeward Ed. Proceedings of Heartworm

Symposium ‘98, American Heartworm Society,Batavia IL, 173-177 pp.

Glaus TM, Jacobs GJ, Rawlings CA, Watson ED,Calvert CA, 1995. Surgical removal of heart-worms from a cat with caval syndrome JAVMA206: 663-666.

Holmes RA, 1995. Feline heartworm disease:Compend Cont Ed 15: 687-695.

Knight DH, Doiron DD, Longhofer S, Rubin SR,Downing R, Nelson CT, Mc Call JW Seward L,2002. Guidelines for the diagnosis, preventionand management of heartworm (Dirofilariaimmitis) infection in cats In: Seward LE, KnightDH Eds Recent Advances in HeartwormDisease: Symposium ’01 American HeartwormSociety Batavia IL, 267-273 pp.

McCall JW, Calvert CA, Rawlings CA, 1994.Heartworm infection in cats: a life threateningdisease. Vet Med 89: 639-647.

McCall JW, Dzimiansky MT, McTier TL, JerniganAD, Jun JJ, Mansour AE, Supakorndej P, PlueRE, Clark JN, Wallace DH, Lewis RE, 1992.Biology of Experimental Heartworm Infectionsin Cats in MD Soll Ed. Proceedings of the ’92Heartworm Symposium, American HeartwormSociety, Batavia IL, 71-79 pp.

Prieto C, Venco L, Simon F, Genchi C, 1997.Feline heartworm (Dirofilaria immitis) infec-tion: detection of specific IgG for the diagnosisof occult infections. Vet Parasitol 70: 209-217.

Selcer BA, Newell SM, Mansour AE, Mc Call JW,1996. Radiographic and 2 D echocardiographicfindings in eighteen cats experimentally exposedto Dirofilaria immitis via mosquito bites. VetRadiol Ultr 37: 137-144.

Turner JL, Lees GE, Brown SA, 1989.Thiacetarsamide in normal cats: pharmacokine-tics, clinical, laboratory and pathologic features.In GF Otto Ed. Proceedings of the HeartwormSymposium ’89. American Heartworm Society,Washington DC, 135-141 pp.

Venco L, Morini S, Ferrari E, Genchi C, 1999a.Technique for Identifying Heartworms in Catsby 2-D Echocardiography. In: Seward LE,Knight DH Eds Recent Advances in HeartwormDisease: Symposium ’98 American HeartwormSociety Batavia IL, 97-102 pp.

Venco L, Borgarelli M, Ferrari E, Morini S, GenchiC, 1999b. Surgical Removal of Heartwormsfrom naturally-infected Cats. In: Seward LE,Knight DH Eds. Recent Advances inHeartworm Disease: Symposium ’98 AmericanHeartworm Society Batavia IL, 241-246.

Venco L, Calzolari D, Mazzocchi D, Morini S,1998. The Use of Echocardiography as aDiagnostic tool for Detection of FelineHeartworm (Dirofilaria immitis) Infections. FelPract 26: 6-9.

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Dirofilaria (Nochtiella) repensinfection in dogs and cats

Luigi Venco

10

D. repens infection in dogs and cats

Dirofilaria (Nochtiella) repens, is thecausative agent of canine and felinesubcutaneous dirofilariosis, a mosquitoborne disease that has become increas-ingly recognized in several countries insouthern and central Europe, Africaand Asia.

D. repens life cycle, like that ofDirofilaria immitis, consists of 5 larvalstages which develop both in a verte-brate host and an arthropod (mosqui-to), intermediate host and vector. Adultfemale worms produce thousands ofembryos (microfilariae) that are ingest-ed by a blood-feeding insect.

Microfilariae have a unique circadianperiodicity in the peripheral circulationover a 24-hour period. The arthropodvectors also have a circadian rhythm inwhich they obtain blood meals. Thehighest concentration of microfilariaeusually occurs when the local vector ismost actively feeding. Microfilariaethen undergo 2 developmental moultsin the insect. During feeding, the infect-ed mosquito deposits third-stage larvaethroughout a drop of haemolymph inthe proximity of bite wound from wherelarvae actively migrate to subcutis.Larvae develop to the adult stagethrough moults in the vertebrate hosts.Prepatency lasts 6 1/2 to 9 months(Webber and Hawking, 1955; Cancriniet al., 1989). The adults reside in thesubcutaneous tissues of dogs and catsand may cause mild clinical signs such aspruritus, dermal swelling, subcutaneousnodules or no symptoms at all (Baneth etal., 2002; Zivicnjak et al., 2006).

Despite the usual hosts of D. repensare domestic and wild carnivores,human beings may act as accidental anddead-end hosts and a big concern about

zoonotic human cases is arising. Humaninfection manifests with either subcuta-neous nodules, ocular or lung parenchy-mal nodules mostly asymptomatic. Thesignificance of infection in humans isthat pulmonary and some subcutaneouslesions are commonly labelled as malig-nant tumours requiring invasive investi-gation and surgery before a correctdiagnosis is made. The pathology of thecondition is associated with aberrantlocalization of immature worms that donot reach adulthood; therefore, microfi-lariae are almost always absent(Pampiglione et al., 1995).

Diagnosis


Detection of circulating microfilariaeusing the method developed by Knott isthe best way for doing an in vivo diag-nosis but collection of hystopatologicalcutaneous specimens.

Larvae species determination is madeon the basis of morphological or hysto-chemical method or by using PCR.

Blood test for adult antigens

Tests detecting adult heartworm (D.immitis) do not detect D. repens anti-gens and no cross reactivity isdescribed.

Therapy

No adulticide treatment for D. repensis registered and a off-label use ofmelarsomine has only recently beendescribed on the basis of a case report

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D. repens infection in dogs and cats

where combined therapy with thearsenic adulticide melarsomine and theavermectin microfilaricidal doramectinwas effective in clearing infection withD. repens in a dog (Baneth et al., 2002),although the death of the patients doesnot allow conclusive evidences.

Symptomatic therapy of canine dirofi-lariosis due to D. repens is indicated fordogs suffering from clinical signs ofthis disease, such as dermal swelling,sub-cutaneous nodules and pruritus.Steroids and/or antibiotics administra-tion and nodules surgical removal maybe suggested in these cases for relievingsymptoms.

Some macrocyclic lactones (iver-mectin, moxidectin, selamectin) areclaimed to be effective for the preven-tion of D. repens infection in dogs(Genchi et al., 2002; Rossi et al., 2002)and labelled for this use in some coun-try (Italy) on the basis of field study.

While there is no doubt that they areable to prevent mirofilaraemia in dogs(and this is important for zoonoticimplications), as most of the performed

studies were not based on necropsy con-firmation some concerns about the abil-ity of completely preventing infection indogs still remain (Cancrini et al., 1989).

ReferencesBaneth G, Volansky Z, Anug Y, Favia G, Bain O,

Goldstein RE, Harrus S, 2002. Dirofilariarepens infection in a dog: diagnosis and treat-ment with melarsomine and doramectin. VetParasitol 105: 173-178.

Cancrini G, Tassi P, Coluzzi M, 1989. Ivermectinagainst larval stages of Dirofilaria repens.Parassitologia 31: 177-182.

Genchi C, Poglayen G, Kramer LH, 2002.Efficacia di Selamectin nella profilassi delle infe-stazioni da Dirofilaria repens nel cane.Veterinaria 16: 69-71.

Pampiglione S, Canestri Trotti G, Rivasi E, 1995.Human dirofilariosis due to Dirofilaria(Nochtiella) repens: a review of world literature.Parassitologia 37: 149-193.

Rossi L, Ferroglio E, Agostini A, 2002. Use ofmoxidectin tablets in the control of canine sub-cutaneous dirofilariosis. Vet Rec 15: 383.

Webber WAF, Hawking F, 1955. Experimentalmaintenance of Dirofilaria repens and D. immi-tis in dogs. Exp Parasitol 4: 143-164.

Zivicnjak T, Martinkovic F, Beck R, 2006.Dirofilariosis in Croatia: spread and publichealth impact. 5th Croatian congress on infec-tive diseases, Zadar, Croatia.

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Guideline for the laboratorydiagnosis of canine and felineDirofilaria infections

Claudio Genchi, Luigi Venco, Marco Genchi

11

Laboratory diagnosis: Guideline

Microfilariae

Fresh blood smear (not advised)

A drop of fresh venous blood isplaced on a clean microscopic slide andcovered with a coverslip and examinedunder low microscopic power.

Microfilariae are seen throughout themovement they cause to the blood redcell layer.

To note that the intensity of micro-filaremia is not correlated to theadult worm burden: in general, highmicrofilaremic dogs harbour fewworms.

When dogs are monthly treated withpreventive drugs during heartwormtransmission season, re-testing eachyear before starting again the preven-tive treatment is advisable.

Concentration methods

Several methods can be used to con-centrate circulating microfilariae fromthe blood. These methods are sensitiveand make possible to differentiatemicrofilariae throughout morphologi-cal criteria (see Table 1 and Fig. 1).

Microfilariae can be concentrated bythe modified Knott test or by a filter test.

To note that intensity of microfilaremiais not correlated to the adult worm bur-den: in general, high microfilaremicdogs harbour few worms.

Modified Knott test

One ml of venous blood is mixedwith approximately 10 ml of 2%buffered formalin and the mixtureis centrifuged for 3-5 minutes at1500 rpm.

The supernatant is decanted from thecentrifuge tube and the sediment ismixed with equal parts of a 1:1000methylene blue stain. The stained sedi-ment is placed on a slide, covered witha coverslip and examined under amicroscope.

Filter test

One ml of venous blood with antico-agulant of either EDTA or heparin isadded to approximately 10 ml of lysatesolution. The mixture is injectedthrough a filter (Millipore) chamber.The filter is removed from the chamber,placed on a glass slide, stained, andexamined under a microscope.

To note that intensity of microfilaremiais not correlated to the adult worm bur-

AdvantageRapid and inexpensive

DisadvantagesVery low sensitivity, frequent false negative,no species diagnosis (it is not possible to dif-ferentiate microfilariae) Not useful in cats

AdvantageSensitive in dogs and specific: microfilariaebelonging to different species can be differ-entiateDisadvantagesTime consuming, need of a skill operator withgood knowledge of microfilarial morphologySpecific but of low sensitivity in cats

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den: in general, high microfilaremicdogs harbour few worms.

Histochemical stain

One ml of venous blood collected inEDTA is injected into 10 ml of deion-ized water and centrifuged at 1500rpm for 15 minutes. The supernatantis discarded and the sediment placedon a slide and air-dried. The smear isthen fixed with absolute acetone, airdried, and covered with acid phos-phatase substrate. The substrateneeds to be either made fresh, as

described by Chalifoux and Hunt(1971), or frozen at -80°C in aliquotportions. After 2 hours at room tem-perature, the slide is air dried andcovered with a coverslip.

- D. immitis microfilariae show 2 acidphosphatase activity spots localizedaround the anal and the excretorypores, respectively.

- D. repens microfilariae show only 1acid phosphatase activity spot local-ized around the anal pore.

- Acantocheilonema spp. microfilari-ae show acid phosphatase activitythroughout the body.

Peribáñez et al. (2001) havedescribed an alternative method using acommercial kit with similar results.

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

Species Length µm Width µm Features

Dirofilaria immitis

Dirofilaria repens

Acanthocheilonema reconditum

Acantocheilonema dracunculoides2

Cercopithifilaria grassii

290-330

300-360

260-283

190-247

567

5-7

6-8

4

4-6.5

12-25

No sheath, cephalic end pointed, tail straightwith the end pointed

No sheath, cephalic end obtuse, tail sharp andfiliform often ending as an umbrella handing

No sheath, cephalic end obtuse with a promi-nent cephalic hook, tail button hooked andcurved

Sheath, cephalic end obtuse, caudal end sharpand extended

Sheath, caudal end slightly curved

1 microfilariae measure by Knott test2 microfilariae from the uterus

AdvantageRapid and sensitive in dogsNo need for a centrifuge apparatus

DisadvantagesExpensive (tests are sold as kit, Difil TestEvsco); the lysate solution shrinks the micro-filariae and new measurement standards arerequired to differentiate speciesLow sensitivity in cats

AdvantageVery specific

DisadvantagesCostly, time consuming, need for a skilledlaboratory technician

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ELISA and immunochromatographictests for adult female heartworm circu-lating antigens

Several ELISA and immunochro-matographic kits are commerciallyavailable to detect the presence of adultfemale circulating antigens in serum,plasma and whole blood of dogs andcats. Most are very specific, quite sensi-tive, rapid and easy to be performed.Most are in-clinic test kits for singlediagnosis but ELISA plates for multi-test are also available. Manufacturersclaim for a positive result when 1 adultfemale worm is infecting the animal,though many factors can affect the sen-sitivity of the tests such as age ofworms, number of female worms ver-sus number of males and the dog size,and reliable and reproducible resultscan be obtained from 2-3 or more adultfemale worms. Male worms are notdetectable by antigen tests. In dogs,detectable antigenemia develops about5 to 6.5 months post infection.

Because the clearance of antigens isquite rapid after the death of worms,these techniques can be used to assess

the efficacy of an adulticide therapy.However, the newest tests are quite sen-sitive and for definitive diagnosis to con-firm the success of the adulticide therapydogs have to be retested 5 and 9 monthslater. If the test at 5 months is negative,testing at 9 months can be avoided.

Because unisex infections consistingof only male worms or symptomaticimmature infections are not infrequentin cats, none of the presently availableantigen tests can be relied upon to ruleout heartworm disease in cats. In catswith heavy infections, detectable anti-genemia develops at about 5.5 to 8months post infection.

To note that semi-quantitative and labo-ratory ELISA tests (not immunochro-matographic tests) have a direct, butimprecise, relationship to the adultfemale worm burden. The utility of theELISAs for assessing the degree of para-sitism can be limited by the transientincrease in antigenemia as a conse-quence of recent worm death.

When animals are monthly treatedwith preventive drugs during heartwormtransmission season, re-testing each year

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)

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before starting the preventive treatmentin the following season is advisable.

If the chemoprophylactic treatment isperformed with a sustained releasedrug injection (only for dogs), periodictesting (each 2 or 3 years) will ensurethere have been no efficacy breaks(Nelson et al., 2005b).

Antibody tests

Several antibody test kits are avail-able for the diagnosis of feline heart-worm disease. These tests cannot beused in dogs.

Antibody tests have the advantageto being able to detect the exposureof cats to the infection of both maleand female adult worms and larvae,and the immune response indetectable as early as 2 months postinfection. However, their interpreta-tion is complicate because positiveresults can be found both in abortedinfections (developing larvae aredestroyed by the host immuneresponse and will not be able todevelop to adult worms) and inpatent infections. Furthermore, noproved data is available on whetherthe antibody level will decrease overthe expected two-to-three year lifes-pan of an adult worm (Nelson et al.,2005a).

PCR

PCR (polymerase chain reaction) is asensitive and accurate tool to discrimi-nate microfilariae from the differentfilarial worms able to infect dogs andcats. Its use is advisable in case ofmorphological abnormalities of micro-filariae, not infrequent in dogs treatedincorrectly with preventive drugs orwhen multiple infections with morethan one species of filarial wormmakes difficult to differentiate microfi-lariae (Favia et al., 1996; Mar et al.,2002; Casiraghi et al., 2006; Rishniwet al., 2006).

AdvantageVery specific and sensitive for heartwormdiagnosis [gold standard: when positive, thetest is the definitive prove of heartworminfection in dogs and cats]

DisadvantagesCostly, not available for other filarial infec-tions

AdvantageVery sensitiveAble to detect the cat exposure to heartworminfectionSuitable to asses the infection risk in catsand for epidemiological survey

DisadvantagesCostlyNot fully specificDifficult to be interpreted

AdvantageVery sensitive, specific and accurateAble to discriminate all the filarial species

DisadvantagesCostly, time consumingNeed for specialized laboratory and skilledtechnicians

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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 diseaseClinical signs and the results of semiquantitative ELISA tests can help in discriminating between lowand high risk of thromboembolism

Positive Negative Definitive diagnosis of HW:very low HW burden if D.immitis Mf are present Filarial infection caused byother species than D. immitis

Normal ThR patterns Low/very low risk of thromboembolic complicationsHistochemical stain or PCR can be used to differen-tiate Mf

Negative Positive Definitive diagnosis of HWoccult infection

Dogs were previously treated incorrectly with pre-ventive drugs or with macrocyclic lactone injectableformulations ThR and ECHO can help to manage the disease Clinical signs and the results of semiquantitativeELISA tests can help in discriminating between lowand 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 managethe disease

Positive Negative Positive Definitive diagnosis of HW ifD. immitis Mf are present

ThR and ECHO can help to managethe disease

Positive Negative Negative Filarial infection due to otherspecies than D. immitis

Histochemical stain or PCR for differ-entiate Mf and give specific diagnosis

Negative Positive Positive Definitive diagnosis of HW ThR and ECHO can help to managethe disease

Negative Negative Positive Low adult female worm burdenImmature wormsAborted infectionImmune response to previouspatent infection, but worms arealready died

ThR and ECHO are useful to confirmthe suspicion of HW infectionRe-testing after 4-8 months can helpto confirm the suspicion

Mf: microfilariae HW: heartworm TR: thoracic radiography ECHO: echocardiography

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References

Casiraghi M, Mazzocchi C, Mortarino M, OttinaE, Genchi C, 2006. A simple molecular methodfor discrimination common filarial nematodesof dogs (Canis familiaris). Vet Parasitol 141:368-372.

Chalifoux L, Hunt RD, 1971. Histochemical dif-ferentiation of Dirofilaria immitis andDipetalonema reconditum. J Am Vet Med Assoc5: 601-605.

Favia G, Lanfrancott, A, Della Torre A, CancriniG, Coluzzi M, 1996. Polymerase chain reaction-identification of Dirofilaria repens andDirofilaria immitis. Parasitology 113 (Pt. 6):567-571.

Mar PH, Yang IC, Chang GN, Fei AC, 2002.Specific polymerase chain reaction for differen-tial diagnosis of Dirofilaria immitis andDipetalonema reconditum using primersderived from internal transcribed spacer region2 (ITS2). Vet Parasitol 106: 243-252.

Nelson CT, Doiron DW, McCall JW, Rubin SB,

Buzhardt LF, Graham W, Longhofer SL,Guerrero J, Robertson-Plouch C, Paul A, 2005a.2005 Guidelines for the diagnosis, preventionand management of heartworm (Dirofilariaimmitis) infection in cats. Vet Parasitol 133:255-266.

Nelson CT, McCall JW, Rubin SB, Buzhardt LF,Doiron DW, Graham W, Longhofer SL, GuerreroJ, Robertson-Plouch C, Paul A, 2005b. 2005Guidelines for the diagnosis, prevention andmanagement of heartworm (Dirofilaria immitis)infection in dogs. Vet Parasitol 133: 267-275.

Peribáñez MA, Lucientes J, Arce S, Morales M,Castillo JA, Gracia MJ, 2001. Histochemical dif-ferentiation of Dirofilaria immitis, Dirofilariarepens and Acanthocheilonema dracunculoidesmicrofilariae by staining with a commercial kit,Leucognost-SP®. Vet Parasitol 102: 173-175.

Rishniw M, Barr SC, Simpson KW, Frongillo MF,Franz M, Dominguez Alpizar JL, 2006.Discrimination between six species of caninemicrofilariae by a single polymerase chain reac-tion. Vet Parasitol 135: 303-314.

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Epidemiology and prevention ofDirofilaria infections in dogsand cats

Claudio Genchi, Jorge Guerrero, John W. McCall, Luigi Venco

12

Epidemiology and prevention

Heartworm infection (Dirofilariaimmitis)

Dirofilaria immitis is a parasite thatcan be potentially fatal to a variety ofanimal species. Dirofilariosis, however,is a fully preventable disease due to theavailability of highly effective preven-tive drugs that are safe, effective, con-venient and easy to administer.According to the recently publishedAmerican Heartworm Society guide-lines, all animals that are at risk forcontracting the disease should routine-ly receive heartworm preventive med-ications (Nelson et al., 2005a,b).

Chemoprophylatic drugs for heart-worm infection fall into two basicclasses, the macrocyclic lactones ormacrolides (avermectins and milbe-mycins) and diethylcarbamazine(DEC). In heartworm-endemic areas,puppies and kittens that are born dur-ing the transmission season should begiven their first dose of macrocyclic lac-tones between 2 to 8 weeks of age and6 to 8 weeks of age, respectively. Therequired daily administration of DEC isinconvenient, more than one misseddose can result in a breakdown in pro-tection and the overall prevention pro-gram is difficult to manage. The use ofDEC is fairly limited in the UnitedStates and the drug is no longer avail-able in Europe.

The present chapter will brieflyreview current information on the epi-demiology of D. immitis infections indogs and cats and the use of macro-cyclic lactones in the prevention ofheartworm infection, as well as theireffect on other important gastrointesti-nal nematode parasites.

Epidemiology

More than 70 species of mosquitoeshave been shown to be capable ofdeveloping microfilariae (first stagelarvae) to the infective, third-stage lar-vae (L3), but fewer than a dozen ofthese species are believed to be majorvectors (Otto et al., 1981). Althoughthe susceptibility of different geograp-hical strains may vary, the likelihood ofthe presence of at least one susceptiblevector species in a geographic area thatis conducive to the propagation ofmosquitoes is high, and once the ubi-quitous heartworm parasite is introdu-ced into an area, its transmission is vir-tually insured.

Many countries are now endemic forheartworm infection. Furthermore, inspite of efforts aimed at prevention andcontrol, particularly in dogs, infectionappears to be spreading into areas pre-viously considered to be free of thedisease (Genchi et al., 2005). The pre-valence and distribution is betterknown for dogs, but gradually moreinformation on the frequency of diag-nosis of infection in cats is becomingavailable. It is now generally acceptedthat heartworm disease may occur incats in any area where dogs are infected(Genchi et al., 1992b, 1998; Guerreroet al., 1992b; McCall et al., 1994;Kramer and Genchi, 2002) but the geo-graphical distribution and level ofinfection are less predictable in catsthan in dogs. In highly endemic areas,with sufficient rainfall, essentially everyunprotected dog becomes infected(McTier et al., 1992a). In contrast todogs, about 75% of cats can be infec-ted experimentally with D. immitis L3

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(McCall et al., 1992). However, theprevalence rate of natural infections incats is between 5% and 20% of that fordogs in the same geographical area(Ryan and Newcomb, 1995).

Cats with naturally acquired infec-tions usually harbor fewer adult wormsthan dogs. Because of their small bodysize and exaggerated pulmonary vascu-lar and parenchymal response to infec-tion, cats with low worm burden can beconsidered to be relatively heavilyinfected in terms of parasite biomass(Genchi et al., 1998). Microfilaremia,when present, is low and transient,even in cats with experimentally indu-ced infections. Mosquitoes fed onheartworm microfilaremic cats developL3, which are capable of producing aninfection in dogs (Donhaoe, 1975), butcirculating microfilariae are seldomfound in cats. Thus, cats generallybecome infected via mosquitoes thathave fed on microfilaremic dogs.

It is not currently possible to deter-mine which cats are resistant to heart-worm infection and which are suscepti-ble and will permit the infection todevelop to the adult stage. Such a dis-tinction between resistant and suscepti-ble animals would require constantmonitoring at very high costs.

Furthermore, environmental meas-ures taken to reduce the risk of infec-tion seem to be of little consequence inthe cat. In fact, keeping animalsindoors, one of the most importantmeasures in reducing infection rates indogs, seems to be ineffective in protect-ing cats from disease. Outdoor cats andstrays who are seemingly exposed tohigh numbers of bites from infectivemosquitoes may be able to mount aneffective immune response that could

be partially protective (Dillon et al.,1996; Prieto et al., 2001); however,studies to determine levels of suscepti-bility have not been performed. Forindoor cats, it seems that even oneencounter with an infective vector maylead to the development of a large pro-portion of transmitted larvae to theadult stage, causing severe illness(Genchi et al., 1992a). It has recentlybeen reported that between 9 and 27percent of cats were seropositive for D.immitis in northern Italy, 19 percent ofwhich were apartment-dwelling cats(Kramer and Genchi, 2002).

Chemoprophylactic treatment, there-fore, is a viable option for cats residingin any area where heartworm is consid-ered endemic in dogs, even cats livingmore sheltered lives. As suggested byAtkins (1997), it seems rational to rec-ommend chemoprophylaxis for felineheartworm infection, given that the dis-ease has a higher incidence than bothFeline Leukemia Virus (FeLV) andFeline Immunodeficiency Virus (FIV),infections for which vaccination proto-cols are increasingly advocated.

Heartworm transmission to dogs andcats is influenced by several well-known epidemiological factors, amongthe most important of which is envi-ronmental heat. Development of D.immitis to L3 in mosquitoes occurs at arate that is dependent on ambient tem-perature, and development may notoccur at a threshold temperature ofabout 14 °C (Fortin and Slocombe,1981). The effects of heat on larvaldevelopment is assumed to be cumula-tive and may be calculated in terms ofdegree-days above the developmentalthreshold, or heartworm developmentunits (HDU). One model of heartworm

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seasonality assumes a requirement of130 HDU (°C) for complete develop-ment and a maximum life expectancy of30 days for common vector mosquitoes(Slocombe et al., 1989). Using thislaboratory-derived model, which requi-res numerous inherent assumptionsand was designed to study only theinfluence of macroenvironmental tem-perature on the heartworm develop-ment period, investigators have predic-ted the seasonal limits of transmissionin Canada (Slocombe et al., 1995), theUSA (Knight and Lok, 1995) andEurope (Genchi et al., 2005) and for-mulated recommendations for timingof heartworm chemoprophylaxis andscheduling of diagnostic testing. Whilethese model-based predictions are aca-demically appealing, they ignore seve-ral potentially important factors, suchas influence of microclimate and theunique biological habits and adapta-tions of the numerous mosquito vec-tors, on larval development.

Prevalence

North America

Heartworm infection in dogs has nowbeen diagnosed in all of the 50 states ofthe USA. Although transmission ofinfection has not been clearly docu-mented for Alaska, the disease is consi-dered to be endemic in all of the remai-ning 49 states. Heartworm is enzooticalong the Atlantic Seaboard and GulfCoast areas, with the southeastern sta-tes generally showing the highest pre-valence values. Infection continues tobe diagnosed at a high frequency in theMississippi River basin and in states

along the Ohio and Missouri Rivers.New foci have been detected in nort-hern California and Oregon, andautochthonous infections in dogs in thestates of Wyoming, Utah, Idaho, andWashington have been documented inrecent years (Zimmerman et al., 1992).

There is a high probability that theintroduction of the tree-hole breedingmosquito, Aedes sierrensis, in SaltLake City, Utah, during the past deca-de or so is associated with the esta-blishment of enzootic heartwormtransmission in the area (Scoles andDickson, 1995). The tiger mosquito,A. albopictus, a known vector of heart-worm in Japan, has spread rapidlythroughout much of the USA andEurope since it was introduced fromAsia in 1985. It breeds mainly in pilesof discarded tires and is spread withinthe country by movement of tires fromplace to place. This mosquito readilyfeeds on dogs and other mammals, andlaboratory strains have shown it to bean excellent host for D. immitis.

Two recent surveys conducted byMerial in cooperation with theAmerican Heartworm Society showedthat the number of canine heartwormcases diagnosed in the US were close toa quarter of a million. The first surveyconducted in 2002 requesting diagnos-tic data for 2001 had the participationof 15,366 clinics which reported diag-nosing heartworm infections in244,291 dogs. The second survey con-ducted in 2005 reviewing data for 2004had the participation of 12,173 clinics(out of a total of 25,000), which report-ed 250,000 cases of canine heartworminfections diagnosed that year(Guerrero et al., 2006). Interestingly,8,800 of the responders in the second

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survey had also responded in the firstsurvey. Analyzing the data obtained inthe clinics that participated in both sur-veys the following was seen: number ofcanine heartworm cases decreased in17 states, in 3 states the numbers werethe same and in 30 states plusWashington, DC the number of casesincreased. Nationally, the reported pos-itive cases of heartworm infections indogs increased slightly in those clinicsreporting in both 2002 and 2005.

In a parallel study, the national preva-lence of heartworm infections in dogswas performed by evaluating medicalrecords of more than 500 Banfield PetHospitals that see approximately 80,000pets on a weekly basis. Data collectedfrom January 1st 2002 to December 31st

2005 was evaluated. Results of thisstudy show that 1.46% of the 871,839dogs examined tested positive for circu-lating antigen of D. immitis. Based onthis data, the estimates of pet dogs in theUSA and the proportion of them proba-bly on heartworm prevention, the inves-tigators (Apotheker et al., 2006) esti-mated that 509,932 dogs in the USAhad heartworm infections, a figure thatis almost exactly double the number ofcases reported in the Merial-AHS sur-veys of 2002 and 2005. Keep in mindthat in the 2005 Merial-AHS surveyclose to half of the total number ofCompanion animal clinics in the USAresponded to the survey.

In Canada, the overall canine prevalen-ce rate is 0.24% (Slocombe, 1992).Prevalence is higher (8.4%) in endemicareas of southwestern Ontario. The mostsignificant reports of heartworm infec-tions in British Columbia are from theOkanagan Valley area, which representsa classic instance of recent introduction

of the parasite and a resulting local poc-ket of infection (Zimmerman et al.,1992). Hunters, in previous years, hadtransported hunting dogs from Texas tothis area, setting up new foci of infection.

South America

The prevalence and spread of heart-worm infection in South America hasbeen recently reviewed by Labartheand Guerrero (2005). Surveys indicatethat heartworm is endemic in severalcountries of South America. In Brazil,the overall prevalence of canine heart-worm infection in the state of Rio deJaneiro was 21.3% (Guerrero et al.,1992a), with the highest rate for dogsin the northern beaches (49%), fol-lowed by dogs from the mountaintowns near the cities of Rio de Janeiro(27.4%) and Niteroi (26.4%). The ratein the suburbs of Rio de Janeiro wasaround 33% (Labarte et al., 1992).Labarthe and co-workers (1997 a,b)confirmed and extended these findings,and also reported heartworm infectionin random-source cats in the city. Alvesand co-workers (1999) found a preva-lence rate, as determined by necropsyof dogs, in the city of Recife in north-eastern Brazil of 2.3%. In the samestate (Pernambuco), prevalence indogs on Itamaracá Island was found tobe as high as 43%. Labarthe (1997)reviewed the literature on prevalence inBrazil and found reports of prevalencerates as high as 45% in the state of SãoPaulo. In Argentina, endemic areashave been identified, with prevalence indogs ranging from 5.0% in the greaterBuenos Aires area to 34.2% in thenortheastern province of Formosa(Guerrero et al., 1992a). A rate of

150


10.9% was recorded for Corrientes.More recently, Peteta and co-workers(1998) reported a prevalence, deter-mined by microfilarial and antigen test-ing, of 13.6% for dogs in Villa La Nàta,which is located near the Parana Deltaand surrounded by the Lujan River andVillanueva and LaRioja Channels in theTigre district of the province of BuenosAires. In Argentina the prevalenceranges from 0% to 71% (Vezzani et al.,2006). In Venezuela, examination ofcanine blood samples submitted to theSchool of Veterinary Medicine, CentralUniversity of Venezuela in Aragua,revealed that 2.3% were positive formicrofilariae of D. immitis (Perez andArlett, 1998). Recent studies performedin Lima, Peru, reported 4.35% of theblood samples from 140 randomlyselected dogs were positive for circulat-ing antigen of D. immitis. These sam-ples were examined by the ELISA Snap3Dx test (Gonzales, 2002).

Central America and the Caribbean

Kozek and co-workers (1995) revie-wed the prevalence of canine filariae inthe Caribbean islands and conducted athorough epidemiological survey inPuerto Rico. Prevalence values forheartworm infection in Puerto Ricoranged from 3.1% to 20.4%, with thehighest rate recorded for the city ofPonce, on the southern coast. In Cuba,prevalence for Havana ranged from 7%to 19% and from 37% to 63% on theIsla de Juventud. For Curacão, it ran-ged from 9% to 11% and was 53% forthe Grand Bahamas and 18% for theDominican Republic.

In a survey covering 15 cities inMexico (Guerrero et al., 1992a), the

overall heartworm prevalence in dogswas found to be 7.5%, with the highestrates (20-42%) observed for dogs fromthe Gulf Coast cities of Tuxpan,Tampico, and Ciudad Madero.

Australia

Heartworm infection is enzooticalong the northern coastal areas ofWestern Australia and the eastern sta-tes of Queensland, New South Wales,and Victoria, where prevalence ratesgenerally mimic those for the southeas-tern states of the USA. The prevalenceof heartworm infection in dogs inSidney was as high as 30% in the late1980s, and cats were also found to beinfected (Kendall et al., 1991). Morerecently, the rate for dogs in this areawas reported to be only 11.4%(Bidgood and Collins, 1996).

Asia and the South Pacific

Heartworm disease is well establishedin most of the Islands of the Pacific andin many countries of Asia, but surveyresults are not readily available forevery country. A prevalence of 11.3%for dogs from the Fars province of Iranhas been reported (Jafari et al., 1996).Heartworm is enzootic on the islandsof Japan, where the disease is wellknown and prevalence is well docu-mented. A survey conducted on straydogs and cats in the Kanto region ofJapan in 1985 revealed a prevalencerate of 59% for dogs and 2% for cats(Tanaka et al., 1985). More recently,Roncalli and co-workers (1998) repor-ted that prevalence rates for felineheartworm infection in Japan rangesfrom 0.5% to 9.5% in stray cats and

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from 3.0% to 5.2% in house cats. Asurvey of German shepherds in fiveareas of South Korea revealed an ove-rall prevalence of 28.3%, by an antigentest (Lee et al., 1996). Prevalence washighest in Hoengsong-gun (84.4%),while Yechon-gun and Chungwon-gunareas had rates of 20.0% and 14.3%,respectively. None of the dogs in theKimhae-shi and Kwanju areas waspositive. Kuo and co-workers (1995)reported a 53.8% prevalence for dogsin the Taipei province of Taiwan, andWu and Fan (2003) an overall preva-lence of 57% in stray dogs in Taiwan.

Europe

The prevalence and spread of heart-worm infection in Europe has beencomprehensively reviewed by Genchiand co-workers (2005). The disease isdiagnosed mainly in the southernEuropean countries of Spain, Italy,Portugal, and France, with scatteredreports from Greece, Turkey, and someEastern European countries. An increa-sing number of cases are now beingdiagnosed in northern countries such asAustria, Germany, and The Netherlandsin dogs that were either imported fromthe Mediterranean area or had accom-panied their owners to the area. Onepossible exception is a heartworm-posi-tive dog from the Canton of Tessin(Switzerland), which appears to haveacquired an autochthonous infection.

For Europe, the area of highest pre-valence values for dogs and cats isalong the Po River Valley in northernItaly. The prevalence rate for cats inthis area is high (up to 24%), and therate for dogs ranges from 35% to 80%in animals not treated with preventive

drugs. The disease has recently spreadnorthward into the provinces of Friuli-Venezia-Giulia. Furthermore, the spre-ading of A. albopictus in Italy and theevidence that this mosquito species canact as a natural vector for D. immitiscan enhance the risk of transmissionfrom animals to humans, consideringthe aggressive anthropophylic behaviorof the species (30-48 bites/hour)(Cancrini et al., 2003).

The highest rates for dogs in Spain arereported for the southern provinces ofHuelva (37%), Cadiz (12%), andBadajoz (8%) (Guerrero et al., 1992 a).During the past few years, D. immitisinfection appears to be spreading intoother regions of Cataluña (Catalonia). Arecent survey of Barcelona showed that12.8% of the dogs were infected. TheCanary Islands of Tenerife (20.0%) andLas Palmas (36.0%) are highly endemic,and a recent report suggests that about59% of the dogs on Gran Canaria Islandare infected with heartworms. InPortugal, infection is diagnosed mainly inthe southern regions, with prevalencevalues ranging from 12% (Algarve) to30% (Island of Madeira). Although limi-ted survey data are available, prevalencevalues for dogs range from 2% to 17%for Slovenia, Bulgaria, Greece, andTurkey and up to 65% for Romania, andsome of these areas are considered to beendemic. In Croatia, canine heartworminfection has been reported from Istrianpensula (about 16% prevalence) and thesutest regions (Dubrovnik; about 8%)(Zivicnjak et al., 2006).

Africa

Heartworm is found in dogs fromvarious regions of Africa, but no infor-

152


mation is available regarding infectionsin cats. Infection in dogs appears to becommon throughout western Africaand in eastern parts, extending fromthe Republic of South Africa andMozambique (Schwan and Durand,2002) to the Republic of Sudan.Matola (1991) reported a prevalence of10.2% for dogs in Tanzania.

Chemoprophylactic treatment ofcanine heartworm disease

Since the discovery of ivermectin andthe initial description of activity againstdevelopmental stages of D. immitis alarge number of publications haveappeared reporting on their attributes.

Monthly oral administrations of iver-mectin at 6-12 mcg/kg, milbemycinoxime at 500-999 mcg/kg, or oral mox-idectin at 3 mcg/kg provide effectiveprotection against heartworm infec-tions in dogs.

An ivermectin/pyrantel chewable for-mulation is also available in Europeand in the United States to expand theindications to include treatment andcontrol of certain gastrointestinal para-sites, including Toxocara canis,Toxascaris leonina, Ancylostoma can-inum and A. braziliense. Milbemycinoxime at the recommended dose forheartworm prophylaxis is also effectiveagainst T. canis, T. leonina, A. caninumand Trichuris vulpis. Treatment withany one of these macrolide compoundsshould begin within a month after thebeginning of the transmission seasonand the final dose should be adminis-tered within one month after the end ofmosquito activity although, at presenttime the most accepted recommenda-

tion is to treat year-round (Nelson etal., 2005b). All three drugs have a widerange of efficacy, which allows them tobe administered every 30 days. Thisprovides a safeguard in the case ofomission or delay of a monthly treat-ment, or when the chemoprophylactichistory cannot be verified. Ivermectinand milbemycin oxime have both beenfound to provide a high degree of pro-tection when administered on a regularbasis, beginning 3 months after infec-tion. In fact, monthly treatment withivermectin over a one-year period hasbeen shown to be >95 percent effectivein preventing development of D. immi-tis larvae that were 4 months old; how-ever, under the same conditions, milbe-mycin oxime was only 41.5 to 49.3 per-cent effective as a clinical prophylacticagent (McCall et al., 1992). Thisretroactive or “reachback” effect hasnot been reported for moxidectin,although products with this compounddo have a label claim for efficacy of 2months duration. This so-called “safenet” or “reachback” effect of macro-cyclic lactones is very useful to com-pensate for missed or delayed treat-ments, but should not be considered asjustification to modify the recommend-ed monthly interval for treatment.

A newly developed avermectin,selamectin, has recently been approvedin Europe and in the United States forthe prevention of heartworm infectionsin dogs and cats. The drug was also100% effective in preventing the devel-opment of heartworm infection in dogswhen administered as a single topicaldose of 3 or 6 mg/kg given at 30 or 45days after inoculation with L3 or a sin-gle topical dose of 6 mg/kg given 60days PI (Clemence et al., 2000; McTier

153


et al., 2000; Dzimianski et al., 2001;McCall et al., 2001). The drug was alsosafe when given to dogs and cats withexisting heartworm infections. Thereare several important characteristicsunique to selamectin: the drug is givenas a topical formulation, thereby avoid-ing problems associated with oraladministration. At the dose recom-mended for heartworm prevention indogs and cats (6 mg/kg) selamectin isalso effective in preventing and control-ling flea infestations (Ctenocephalidesfelis) and for treating and controllingear mites (Otodectes cynotis) and bitinglice (Trichodectes canis and Felicolasubrostratus) in dogs and cats; sarcopticmange in dogs (Sarcoptes scabiei); andhookworm (Ancylostoma spp) androundworm (Toxocara spp) in bothdogs and cats.

An injectable formulation of mox-idectin to be solely utilized by veterinar-ians in dogs for prevention of heart-worm infections is sold in Italy andAustralia. The commercial formulation(moxidectin sustained release injectablefor dogs) has been approved for use indogs six months of age and older, butnot growing dogs. It is able to protectdogs for an entire heartworm transmis-sion season (Genchi et al., 2002) andalso treats infections with A. caninum.

Control testing

Retesting for D. immitis infectionshould always be carried out after thefirst season of preventive treatment andmust include testing for circulating adultantigens as well as for microfilariae. Thereason for this is that regular chemopro-phylactic use of the macrolides will usu-

ally clear any microfilariae from theblood, so that even if an infection hasbecome patent during the therapy, itwould not be detected by screening formicrofilariae. However, some cases ofmicrofilaraemic dogs can be observed,mainly when some treatments are inad-vertent omitted towards the end of thetransmission season. Testing should beperformed a minimum of 6 months afterthe last administration of one of themacrolide compounds. It is also advis-able to repeat testing at the beginning ofeach transmission season before thestart of treatment in order to exclude thepossibility of infection due to poorowner compliance during the precedingseason, or to verify that there was nopre-existing infection. Annual retestingshould be advisable even if chemopro-phylactic treatment has been utilizedcontinuously (year-round) due mainly tothe overall low owner compliance(Nelson et al., 2005b).

Chemoprophylactic treatment of felineheartworm disease

Although the general guidelines andcriteria for the use of preventive drugsin dogs may be generally applied toheartworm infection in cats, severalspecific features of feline heartwormdisease must first be considered whenchoosing the correct prevention regi-men. D. immitis infection in cats cancause an unpredictable, and often fatal,disease. Cats are known to be suscepti-ble hosts for heartworm, but areextremely resistant to infection (Genchiet al., 1992a; McCall et al., 1992).

Preventive treatment in the cat fol-lows the same regimen established for

154


the dog, i.e., monthly dosing shouldbegin within one month from the startof the transmission season and the lastdose should be given within one monthfrom the end of the risk period.Ivermectin is marketed for use as aprophylactic agent in cats given month-ly at the dose of 24 mcg/kg (McTier etal., 1992b). This oral dosage is alsohighly effective for treatment and con-trol of A. tubaeforme and A.braziliense. The oral chewable formula-tion of ivermectin has been found to be100 percent effective in preventingdevelopment of D. immitis larvae whenadministered 30 or 45 days after chal-lenge with infective larvae.

Furthermore, McCall and co-workers(2000, unpublished data) demonstrat-ed that the recommended prophylacticdosage of ivermectin administeredmonthly for a maximum of 12 monthswas 66.5 percent effective in clearing7-month-old D. immitis that had beentransplanted from an infected dog. Adramatic decrease in circulating anti-gen levels also was detected in iver-mectin-treated cats. These findings arehighly noteworthy for the cat veterinar-ian since adulticide treatments are notconsidered a viable option for cats.Milbemycin oxime is also known to beeffective in cats for heartworm prophy-laxis at a rate of 2 mg/kg (Genchi et al.,2004). In Europe, milbemycin oxime isavailable in combination with prazi-quantel (5 mg/kg) and the product isefficacious for preventing heartworminfection and for the treatment and con-trol of A. tubeforme, T. cati, Dipylidiumcaninum, Taenia spp and Echinococcusmultilocularis. Selamectin is also avail-able for use in cats as a heartworm pre-ventative.

Using the same dosage as in the dog,6 mg/kg by topical application,selamectin is also active against T. cati,A. tubaeforme, C. felis, Felicola subros-tratus and O. cynotis (Guerrero et al.,2002).

As with the dog, pre-treatment test-ing is advisable in the cat, utilizing bothan antibody and an antigen test forcats, to verify the absence of D. immi-tis infection; however, it is not manda-tory. Retesting of cats should be con-sidered after the first season of preven-tive treatment and is advisable at thebeginning of each new transmissionseason before preventive therapy is tobe initiated, unless the clinician haswisely chosen to utilize chemoprophy-laxis year-round. The longer life cycleof the parasite in the cat, as well as thedifficulty in accurately diagnosinginfection, increase the risk of inadver-tently treating an infected animal; how-ever, based on data obtained by McCallet al. (2000, unpublished data), month-ly administration of preventive drugs tocats infected with adult worms did notprecipitate any negative reactions.

Subcutaneous filarial infection(Dirofilaria repens)

As for heartworm infections, subcuta-neous filarisosis can be safely and effec-tively prevented by chemoprophylactictreatment of both in dogs and cats.

Although the disease is less severethan heartworm infection and dogs canshow cutaneous disorders of differentseverity, such as pruritus, dermal swe-lling and subcutanous nodules contai-ning the parasites (Baneth et al., 2002;Bredal et al., 1998), very severe infec-

155


tions have been reported (Restani etal., 1962; Mandelli and Mantovani,1966), with allergic reactions probablydue to microfilariae. However, the mainconcerrn about this Dirofilaria speciesis its ability to cause infections inhumans in Europe. The number of zoo-notic infections has dramatically increa-sed in the last few decades(Pampiglione et al., 1995) and theinfection now can be included in the listof emerging zoonoses (Pampiglione etal., 2001). The infection is spreading inmany southern (Giannetto et al., 1997)and eastern European countries(Mazurkevich et al., 2004; Fok, 2007),probably as a consequence of the move-ment of infected dogs and global war-ming that increase the transmission sea-son. Recently, Zivicnjak et al. (2006)reported a prevalence ranging from 7-18% in dogs from several regions ofCroatia. Cats, as well as dogs can beinfected, but the prevalence seems quitelow (0.2-0.5%; Genchi et al., 1993).

Ivermectin, selamectin and moxidec-tin (both tablets and subcutaneousinjectable) have been found to be effec-tive in preventing this subcutaneousinfection in dogs naturally exposed toinfective mosquitoes, by treatingmonthly (oral formulations) or once ayear (moxidectin injectable formula-tion) and at the same doses that areeffective against D. immitis(Marconcini et al., 1993; Genchi et al.,2002c; Rossi et al., 2002, 2004).

Domestic ferrets

The domestic ferret (Mustela putoriusfuro) has been reported to be suscepti-ble to naturally acquired and experi-

mentally induced infections of D. immi-tis (McCall, 1998). Laboratory studieshave shown the ferret to be highly sus-ceptible, with infection and recoveryrates similar to those achieved in thedog and higher than those seen in cats(Supakorndej et al., 1994). The lifespan of adult heartworms in ferrets isthought to cover the entire life time ofthe ferret. Heartworms are somewhatsmaller in ferrets than in dogs, withmean lengths of male and female wormsrecovered from the heart and associatedvessels of 118 mm and 144 mm, res-pectively, 140 days after infection(Supakorndej et al., 1994).

Microfilaremia is characteristically oflow concentration and transient in natu-re, similar to that seen in heartworm-infected cats. A definitive diagnosis canbe made from ELISA-based antigentests, echocardiography, and angio-graphy, but suggestive radiographic fin-dings require additional supportiveinformation to confirm a tentative diag-nosis (McCall, 1998; Sasai et al., 2000). Clinical signs upon presentation inclu-de lethargy, inappetence, exercise into-lerance, pleura effusion, cyanosis anddyspnea (Antinoff, 2001).

Supakorndej and co-workers (2001)found that adulticide treatment ofinfected ferrets with melarsomine dihy-drochloride enhanced cardiomegalyand alveolar infiltrates and increasedthe severity of interstitial disease. Thedrug at the dosage of 3.25 mg/kg wasequally effective (80.6-83.3%) whengiven twice, 24 hours apart or oneinjection followed one month later by 2injections, 24 hours apart, but severalanimals in the treated and controlgroups died during the study.

Prevention has been shown to be

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effective with currently used canineprophylactic compounds such asmonthly tretament with ivermectin at6 mcg/kg (McCall, 1998) or cat chewa-ble ivermectin formulation (24 mcg/kg)(Kemmerer, 1998), but effective treat-ment of adult heartworms in ferrets hasnot yet been confirmed by controlledstudies. There is currently no approveddrug for prevention or treatment of D.immitis in ferrets.

Guideline for the chemoprophylactictreatment of Dirofilaria infections indogs

1. Test the dog for circulating microfi-lae (D. immitis and D. repens) andadult female antigens (D. immitis)when the preventive treatment isadministered for the first time.Testing cats is not necessary but it isadvised; drugs are safe even in catswith patent infection.

2. When a * 1 year-old dog with anunknown history, living in an ende-mic area (risk area) is prophylacti-cally treated for the first time, checkfor microfilariae and antigen beforestarting treatment and recheck after

6 months to exclude a possible pre-patent infection.

3. Retesting should always be carriedout after the first season of preven-tive treatment and must include tes-ting for circulating antigens as wellas for microfilariae. It is also advi-sable to repeat testing at the begin-ning of each transmission season,before the start of treatment inorder to exclude possible infectionsdue to poor owner compliance or toverify that there was no pre-existinginfection. In endemic areas whereyear-round treatments are utilizad,yearly testing is recommended.

4. Treatment must be given montly atthe recommended dosage, but formoxidectin in the injectable formula-tion, one injection is able to protectdogs against Dirofilaria infection forat least six months. The commercialformulations are sold at dosages thatare able to cover different ranges ofbody weight. The minimum effectivedosage of macrocyclic lactones forprevention of Dirofilaria infection indogs and cats and the indicationsagainst other parasites are shown inthe table below.

ML Presentation Species Dose Indications Minimum age of treatment

IVM

IVM/PYR

MBOMBO/PZQ

MBO/LFN

Tablets/chewablesChewablesChewables

Flavour tabletsTablets

Tablets

DogCatDog

DogDog

Cat

Dog

6 mcg/kg24 mcg/kg6 mcg/kg

0.5 mcg/kg0.5 mcg/kg

2 mg/kg

0.5 mcg/kg

Di; DrDi, At, Ab Dog: Di, Dr, Tc,Tl, Ac, Us

Di, Tc, Tl, Ac, TvDi, Tc, Tl, Ac, Tv,Dc, Tae, Eg,MsDi, Tct, At, Dc,Tae, EmDi, Tc, Ac, Tv, Cf

6 weeks6 weeks6 weeks

2 weeks or 0.5 kg6 weeks

6 weeks

2 weeks

continued

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MOX

SLM

TabletsInjectable

Topical

Dog

Dog

Cat

3 mcg/kg0.17 mg/kg

6 mg/kg

6 mg/kg

Di, DrDi, Dr, Ac

Di, Dr, Tc, Cf, Ss,Oc, TrcDi, Tct, At, Cf,Oc, Fs

6 weeksadult

6 weeks

6 weeks

VM: ivermectinPYR: pyrantel pamoateMBO: milbemycine oximeMOX: moxidectinSLM: selamectin

Di: Dirofilaria immitis; Dr: D. repens; Ac: Ancylostoma caninum; At: A. tubaeforme; Ab: A. braziliense, Tc: Toxocara canis; Tct: T. cati; Us: Uncinaria stenocephala; Tv: Trichuris vulpis; Dc: Dipylidium caninum;Ms: Mesocestoides sp; Tae: Taenia spp, Eg: Echinococcus granulosus; 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.

158


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Safety and efficacy ofselamectin in dogs withDirofilaria 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

13

Safety and efficacy of selamectin

In both laboratory (McTier et al.,1998, 2000) and field studies(Clemence et al., 2000; Boy et al.,2000), selamectin has proven to behighly effective in the prevention ofDirofilaria immitis (heartworm) infec-tions in dogs and cats followingmonthly prophylactic administrationsat a minimum dose rate of 6 mg/kg. Instudies in which administrationoccurred 45 days (simulated delayedtreatment) or 60 days (simulateddelayed or missed treatment) afterinoculation with infective third-stagelarvae (L3), a single treatment withselamectin was completely effective(McTier et al., 2000). It has also beendemonstrated that no adverse effectsare likely if selamectin is inadvertentlyadministered to animals with existinginfections of adult D. immitis(Novotny et al., 2000) In heartwormendemic areas, there are severalpotential scenarios for compliancefailure in heartworm prevention,which a clinician may encounter.

These include dogs that have beenexposed to infection for various peri-ods prior to initiation of their heart-worm prophylaxis, situations ofmissed monthly dosing, and circum-stances in which dogs with patentinfections and various degrees ofmicrofilaremia are presented for treat-ment. The series of studies presentedhere was designed to assess the per-formance of selamectin in 3 situations,which hitherto had not been investi-gated. The studies included an assess-ment of the safety and efficacy ofselamectin in heartworm-positive dogswith high microfilaremia (study A);the microfilaricidal and adulticidalefficacy of selamectin after long-term

monthly administration to dogs withpatent D. immitis infections (study B);and the “reachback” or clinical prophy-lactic effect in dogs inoculated withheartworm L3 3 months prior to initia-tion of long-term monthly prophylaxis(study C).


Treatments

Selamectin in the commercial formu-lationa was administered as a unit doseto provide a minimum dose rate of 6mg/kg of body weight. The placebo(vehicle only) was administered at therate of 0.05 ml/kg of body weight toprovide an equivalent volume.

Treatments were applied to the skinat a single site on each dog’s back at thebase of the neck and cranial to thescapulae. Treatments were adminis-tered at 30-day intervals beginning onstudy day 0.

Animals

Equal numbers of colony-bred maleand female Beagles were used. The ini-tial age and weight ranges for the dogswere 5 to 7 months and 6.45 to 15.4kg, respectively. Dogs were housedindividually in mosquito-proof indoorpens inside purpose-built accommoda-tions, with controlled temperature andventilation systems. They were fed oncedaily with an appropriate quantity ofmaintenance diet and water was sup-plied ad libitum. Animals were main-

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a Stronghold®/Revolution®, Pfizer Inc., New York,New York, USA.


tained with due regard for their welfareand in accordance with applicable leg-islation and guidelines.

Parasite challenge

For dogs in studies A and B, matureadult D. immitis worms aged approxi-mately 10 to12 months were obtainedfrom donor animals having experimen-tally induced infections. Twenty worms(10 males, 10 females) were intro-duced surgically into the cranial venacava via the external jugular vein whilethe dogs were under general anaesthe-sia. For study C, D. immitis L3 wereobtained from experimentally infectedAedes agypti, and each dog was inocu-lated with approximately 50 L3 bysubcutaneous injection into theinguinal region 3 months prior to initi-ation of treatment.

Dirofilaria immitis antigen testingand microfilarial counting

Dogs were tested for the presence ofD. immitis antigen by using a commer-cial test kitb. A modified Knott concen-tration technique was used to countmicrofilariae.

Adult worm counts

After euthanasia, dogs in studies B andC were necropsied for recovery of adultD. immitis. The pleural and peritonealcavities were examined for adult worms,and the cranial and caudal vena cavaeewere clamped before removal of the

heart and lungs (and liver in study C).The pre-cava, right atrium, right ventri-cle, and pulmonary arteries (and caudalvena cava as far as the liver in study C)were dissected and the worms removed,recorded as male or female, recorded asalive or dead, and fixed in formalin.

Design

Study A. Safety and efficacy in dogswith high microfilarial counts

Eighteen dogs (9 males, 9 females)that tested negative for heartworm anti-gen and microfilariae were inoculatedwith 20 adult D. immitis on day –222.Sixteen dogs (8 males, 8 females) withthe highest D. immitis microfilarialcounts (mean, >10,000 mf/ml on day–5) were selected, weighed, and ran-domly allocated to treatment withselamectin or a placebo. Treatmentswere administered on days 0, 30, and60. Clinical observations were madeprior to each treatment, at 4 and 8 hoursafter treatment, and once daily for thenext 7 days. General health observationswere made daily on all other daysthroughout the study. Blood was collect-ed by venipuncture for microfilarialcounting on the day of treatment (days0, 30, and 60) and on days 1, 7, and 14after each of the 3 treatments.

Study B. Efficacy of long-term treat-ment against adult D. immitis andcirculating microfilariae

Eighteen dogs (9 males, 9 females),with negative results for blood microfi-lariae and heartworm antigen on day

166

b DiroChek® Canine Heartworm Antigen Test Kit,Synbiotics Corporation, San Diego, California, USA.


32, were each inoculated with 20 adultD. immitis worms on day 30. On day 3, 16 dogs (8 males, 8 females) withthe highest levels of microfilariae andantigen were selected, weighed, andrandomly allocated to treatment withselamectin or a placebo. A total of 18monthly treatments were administeredon days 0, 31, 60, 90, 122, 152, 182,213, 243, 273, 304, 334, 364, 395,425, 455, 486, and 516. Clinical obser-vations were made prior to each treat-ment, and then within 10 minutes, andat 2, 4, 8, and 24 hours after treatment.General health observations were madedaily on all other days throughout thestudy. Blood was collected by venipunc-ture for microfilarial counting and anti-gen testing approximately every 30days. All dogs were euthanized on day546 for necropsy and recovery of adultD. immitis.

Study C. Long-term monthly prophy-laxis beginning at 3 months afterheartworm inoculation (“Reachback”efficacy)

Seventeen dogs that had tested nega-tive for microfilariae and D. immitisantigen on day –92 were each inoculat-ed with approximately 50 D. immitis L3on day –90. On day –3, blood sampleswere obtained for microfilariae countingand antigen testing. On day –1, 16 dogswere selected and allocated to treatmentwith selamectin or a placebo (8dogs/treatment). Monthly treatmentswere administered beginning on day 0(90 days after infection), and continuingthereafter for 11 months (total of 12monthly treatments). Clinical observa-tions were made immediately prior to

each treatment, and then at 4, 8, and 24hours after treatment. General healthobservations were made daily on allother days throughout the study. Bloodsamples for microfilarial counts andantigen tests were collected at approxi-mately 30-day intervals and the dogswere euthanized on day 363 (15 monthsafter inoculation) for necropsy andrecovery of adult D. immitis.

Data analysis

Parasite counts were log transformed{ln(x+1)} prior to analysis by using arepeated- measures model. A prioricontrasts among least squares means ofthe log transformed data were used toassess the treatment differences oneach counting day at the 5% level ofsignificance (P ) 0.05). Geometricmean parasite counts for each treat-ment were calculated from leastsquares means of the log transformeddata, and these means were used toestimate percentage reductions in para-site burden at each time point for thoseanimals treated with selamectin, com-pared with those receiving the placebo,using the following formula:(Geometric mean count for placebo) –(geometric mean count forselamectin)/(Geometric mean countfor placebo) x 100 = % reduction

Results

Study A. Safety and efficacy in dogswith high microfilarial counts

There were no adverse clinical obser-vations related either directly to treat-

167


ment or to the effects of treatment onparasites within heartworm-positivedogs during the study. Geometric meanmicrofilarial counts for placebo-treateddogs on days 0, 1, 30, 60, and 74 were9,882, 7,117, 3,780, 6,322, and 2,523mf/ml, respectively, compared withselamectin-treated dogs, which hadcounts on the same days of 11,568,10,462, 459, 606, and 223 mf/ml,respectively (Table 1). Thus, the micro-filarial count for the selamectin-treateddogs gradually decreased during themonth after the first treatment (87.5%reduction on day 30), compared withthe placebo-treated controls, and gener-ally remained at approximately thatvalue for the remainder of the study. Themicrofilarial counts were significantly (P) 0.05) lower for selamectin-treateddogs, compared with placebo-treateddogs, on days 30, 31, 67, and 74.

Study B. Efficacy of long-term treat-ment against adult D. immitis andcirculating microfilariae

At necropsy, the geometric meanadult D. immitis counts were 10 forselamectin-treated dogs and 15 forplacebo-treated dogs (controls). Therewas no significant difference betweenthe treatments, even though the reduc-tion in worm count for the selamectin-treated dogs was 36.4%. All of thelive worms recovered from theselamectin-treated dogs and placebo-treated dogs were normal in motility.Only 1 live worm recovered from thecontrol dogs was abnormal in appear-ance (i.e., 1 female worm was opaquewhite), whereas 6 of 8 selamectin-treated dogs had abnormal appearingworms. Twenty-one of the 51 wormsfrom selamectin-treated dogs were

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 (n=8)

9,882 7,117 6,776 917 3,780 3,653 4,401 4,938 6,322 5,228 3,878 2,523

Selamectin (n=8)

11,568 10,462 2,620 432 459 496 654 796 606 458 336 223

% 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

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

Study A

* Dogs were treated on days 0, 30, and 60. ** For selamectin treatment compared with placebo.† Significance of difference between treatments.NS = Not significant.

168


abnormal (i.e., opaque white with orwithout a darkened anterior end). Alldogs in both treatments remained pos-itive for heartworm antigen through-out the study, except for 1 selamectin-treated dog that tested negative on 1occasion (day 483).

On day –3 (prior to the first treat-ment), the mean microfilarial countswere 382 and 315 mf/ml for the place-bo- and selamectin-treatment groups,respectively (Fig. 1). For dogs treatedwith the placebo, the count increasedto >1,000 mf/ml on day 28 (1 monthafter the first dose) and to >3,000mf/ml by day 59. The count was>10,000 mf/ml from day 119 throughthe end of the study, except on days362 (12 months) and 392 when countswere 7,284 and 5,207 mf/ml respec-tively. For dogs treated with selamectin,the mean microfilarial count showed

no increase form day 3 to day 28 (348 mf/ml), and then declined to reach 0at day 180. From day 180 through theend of the study (day 544, after 18monthly treatments), the countremained 0 except on days 271, 301,and 332 when there was a mean countof 1 mf/ml. From day 59 to the end ofthe study, selamectin-treated dogs hadsignificantly (P ) 0.05) fewer microfi-lariae than dogs treated with theplacebo. The reductions in geometricmean microfilarial count for dogstreated with selamectin, comparedwith those treated with the placebo,were 66.9% on day 28, 95.8% on day59, 99.7% on day 90, 99.9% on day119, and approximately 100% fromday 150 to study completion. No adverse reactions attributed toselamectin treatment were observed inthis study.

Fig. 1. Geometric mean counts of Dirofilaria immitis microfilariae in dogs treated monthly, beginning30 days after experimentally induced infection with adult worms.

Study B

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

169M

icro

filar

ia c

ount

s (m

f/m

l)

Days


Study C. Long-term monthly prophy-laxis beginning at 3 months afterheartworm inoculation (”Reachback”efficacy)

At necropsy (15 months after inocu-lation), the geometric mean counts oflive adult D. immitis from placebo- andselamectin-treated dogs were 19.8 and0.3, respectively (Table 2). There was asignificant (P ) 0.05) differencebetween the treatments, and the reduc-tion in the heartworm burden forselamectin-treated animals, comparedwith that for the control group, was98.5%. Dead heartworms were recov-ered at necropsy from the placebo-treat-ed dogs only, but there was no signifi-cant difference between treatments forthe number of dead heartworms. AdultD. immitis antigen tests were negativefor all 16 dogs (both placebo- andselamectin-treated) from day –94 untilday 57 (Fig. 2). From day 118 throughday 361, the percentage of antigen-posi-tive dogs for the placebo-treatmentgroup remained above 85%, whereas

for selamectin-treated dogs, the percent-age never exceeded 38% and decreasedover time, with 12.5% of dogs antigen-positive on day 361. Microfilarial countswere negative for all 16 dogs until day118, and then only dogs treated with theplacebo had positive counts. Theselamectin-treated dogs remained nega-tive throughout the study (Table 3).

Discussion

Results of the first study presentedhere (study A) found that no adverseeffects were observed when selamectinwas administered monthly for 3months to dogs with high microfi-laremia (mean, >10,000 mf/ml) priorto the first application. Furthermore,just 3 treatments with selamectin atmonthly intervals was shown to have asubstantial microfilaricidal effect, asdemonstrated by reductions in microfi-laria count of >90%, compared withthose for the control group (study A).The microfilaricidal activity was con-

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

Treatment* Live worms Dead worms

Placebo (n=8)

19.8 0.4

Selamectin (n=8)

0.3 0.0

% reduction** 98.5% 100%

P-value† )0.05 NS

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

Study C

* 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.

170


firmed in dogs with lower initial levelsof microfilariae (study B), where areduction of 95% in microfilarialcounts was observed by the thirdmonth after commencing monthlytreatment and 100% by the sixthmonth. It is generally considered that ifmicrofilaremic dogs are treated with aneffective microfilaricide, there is anincreased risk of adverse effects occur-ring during the first 4 to 8 hours after

administration (Knigth, 1998). Suchreactions following the rapid death oflarge numbers of microfilariae canoccur with microfilaremias of at least5,000 mf/ml, but are more usuallyassociated with burdens greater than10,000 mf/ml. Reactions includelethargy, inappetance, ptyalism, vomit-ing, tachycardia, pallor, and acute cir-culatory collapse (Knigth, 1998). Fromprevious studies with other macrolides,

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 (n=8)

0 0 0 2 80 986 1,205 3,325 3,353 13,131 2,554 2,219

Selamectin (n=8)

0 0 0 0 0 0 0 0 0 0 0 0

% reduction**

- - - 100% 100% 100% 100% 100% 100% 100% 100% 100%

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

Study C

* Dogs were treated on days 0, 30, 60, 90, 120,150, 180, 210, 240, 270, 300, and 330.** For selamectin treatment compared with placebo.

Study C

Fig. 2. Percentages of dogs positive for D. immitis antigen in dogs treated monthly, beginning 90 daysafter inoculation with (L3) larvae.

171D

ogs

with

pos

itive

ant

igen

tes

t (%

)

Days


notably ivermectin (20-50 mg/kg, ie,higher than prophylactic dosages) andmilbemycin oxime, (prophylacticdosage) treatment-induced reductionsin the levels of circulating microfilariaein dogs with patent infections have beendemonstrated (Courtney et al., 1998).Milbemycin oxime causes a more rapidreduction in microfilaremia than iver-mectin (Courtney et al., 1998). Thus, itis possible to see adverse effects due todead microfilariae, particularly follow-ing the use of milbemycin oxime (pro-phylactic/microfilaricidal dosage), andit is recommended that microfilaremicdogs be carefully monitored for at least8 hours after treatment with microfila-ricidal dosages of these drugs (Knigth,1998). It has been shown previouslythat selamectin can be safely adminis-tered to dogs with mature adult heart-worms and low levels of circulatingmicrofilariae (Novotny et al., 2000).

The efficacy of selamectin in gradual-ly reducing levels of microfilariae (90-100%) over time offers benefits interms of reducing transmission poten-tial within a population. This effectindicates the potential for selamectinto be used following adulticidal thera-py to eliminate a residual microfi-laremia in dogs with patent heartworminfections. In common with othermacrolides, such as ivermectin andmilbemycin, when selamectin isadministered to dogs with patent infec-tions, the dogs may become amicrofi-laremic resulting in “occult” infections.Hence, in a dog with an unknown his-tory or when it is possible that therehas been exposure to infection, partic-ularly in young dogs or strays, it isimportant to establish the heartwormstatus of the dog prior to commence-

ment of a prophylactic regimen.However, if for some reason this isomitted, the majority of infected ami-crofilaremic dogs should be identifiableby using an adult antigen test.

It is not known at present whethermicrofilarial counts will increase fol-lowing cessation of monthly adminis-tration of selamectin, as has beenreported for milbemycin and iver-mectin (Courtney et al., 1998). Theother macrolides have been shown toeffect embryogenesis in female heart-worms (Lok and Knight, 1995); how-ever, at this time, the effects ofselamectin on adult worms, particular-ly female worms, have not been fullycharacterized.

Historically, as a class of com-pounds, the macrolides have not beenconsidered to have activity againstmature adult heartworms. In study B,when treatment was initiated againstmature adult heartworms (11 monthsold) and treatment was administeredmonthly for 18 consecutive months,there were 36.4% fewer adult heart-worms present in selamectin-treateddogs than in the controls, however,there was no statistical differencebetween the worm counts in the 2treatment groups. In addition, manyof the live selamectin-treated wormswere abnormal in appearance. It ispossible that additional monthly treat-ments with selamectin would increasethe efficacy against adult heartworms.When ivermectin was administeredmonthly for 16 consecutive months todogs harbouring mature adult heart-worms, efficacy was 56.3% (McCall etal., 1998), and when treatment wasadministered for 29 months, efficacyimproved to 94.9% (McCall et al.,

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2001). It has also been suggested thatif an effective regimen could bedevised, ivermectin might offer someclinical advantages in heartworm-infected dogs, because the slow killingeffect may reduce the risk of adverseeffects (particularly those associatedwith pulmonary thromboembolism)from dead worms or worm fragments,compared with the risk of using adul-ticidal agents (melarsomine, thiac-etarsamide), which have a much morerapid killing effect on adult heart-worms, which leads to the death ofsome dogs (McCall et al., 1998).

These studies demonstrated thatselamectin, when administered as 12monthly treatments beginning 3months after L3 infection (reachback),is also highly effective in preventing thematuration of heartworms, with areduction of 98.5%.

Previously it has been shown thatmonthly prophylaxis for 13 monthswith ivermectin or milbemycin, begin-ning 3 months after L3 infection, sig-nificantly reduces the number of adultworms that develop by 97.7% and96.8% for ivermectin and milbemycin,respectively (McCall et al., 1996). Ithas also been found that monthly pro-phylaxis for 12 months beginning 4months after L3–induced infectionleads to 95.1% and 41.4% reductionsin maturation to adult worms for iver-mectin and milbemycin, respectively,indicating the potent reachback effectsof ivermectin and the limits of reach-back activity for milbemycin. Theeffects of long-term monthly prophy-lactic treatment with selamectin, com-mencing 6 months after inoculation, iscurrently under investigation, andresults will be forthcoming.

Conclusions

Selamectin is microfilaricidal but killsmicrofilariae relatively slowly, even indogs with high microfilaremia. Thisgradual reduction of microfilariae vir-tually eliminates, or at least greatlyminimizes potential adverse reactionsdue to large numbers of dead or dyingmicrofilariae. Selamectin appears tohave an adverse effect on mature adultheartworms, but the effect is gradualduring long-term monthly prophylaxis.This slow killing effect on adult heart-worms should minimize the potentiallysevere adverse effects seen with a rapidkill of adult heartworms. In addition,selamectin has a 3-month reachbackeffect that is similar to the othermacrolide preventives. Thus, selamectinhas demonstrated exceptional safety inheartworm-positive dogs and, togetherwith robust reachback activity, increas-es the confidence with which this prod-uct can be used in the fight againstheartworm disease in dogs.

Acknowledgements

The authors are most grateful to the laboratorystaff and clinicians who were involved with thesestudies. Their expertise and commitment hasensured the high quality of the data generated.

References

Boy MG, Six RH, Thomas CA, Novotny MJ,Smothers CD, Rowan TG, Jernigan AD, 2000.Efficacy and safety of selamectin against fleasand heartworms in dogs and cats presented asveterinary patients in North America. VetParasitol 91: 233-250.

Clemence RG, Sarasola P, Genchi C, Smith DG,Shanks DJ, Jernigan AD, Rowan TG, 2000.Efficacy of selamectin in the prevention of adultheartworm (Dirofilaria immitis) infection in

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dogs in northern Italy. Vet Parasitol 91: 251-258.Courtney CH, Zeng Q-Y, Maler MM, 1998. The

effect of chronic administration of milbemycinoxime and ivermectin on microfilaremias inheartworm-infected dogs. In: Seward RL (ed).Recent Advances in Heartworm Disease:Symposium ’98. Am Heartworm Society,Batavia, IL, 193-199 pp.

Knight DH, 1998. 1999 Guidelines for the diag-nosis, prevention, and management of heart-worm (Dirofilaria immitis) infection in dogs. In:Seward RL (ed). Recent Advances inHeartworm Disease: Symposium ’98. AmHeartworm Society, Batavia, IL, 257-264 pp.

Lok JB, Knight DH, 1995. Macrolide effects onreproductive function in male and female heart-worms. In: MD Soll and DH Knight (eds).Proceeding of the American HeartwormSymposium ‘95. American Heartworm Society,Batavia, IL, 165-170 pp.

McCall JW, McTier TL, Ryan WG, Gross SJ, SollMD, 1996. Evaluation of ivermectin and milbe-mycin oxime efficacy against Dirofilaria immitisinfections of three and four months duration indogs. Am J Vet Res 57: 1189-1192.

McCall JW, Roberts RE, Supakorndej N, MansourA, Dzimianski MD, McCall SD, 2001. Furtherevidence of clinical prophylactic (reachback)and adulticidal activity of monthly administra-

tions of ivermectin and pyrantel pamoate indogs experimentally infected with heartworms.American Heartworm Society, 10thTriennialMeeting, San Antonio, TX 20-22 April 2001.

McCall JW, Ryan WG, Roberts RE, DzimianskiMT, 1998. Heartworm adulticidal activity ofprophylactic doses of ivermectin (6 mg/kg) pluspyrantel administered monthly to dogs. In:Seward RL (ed). Recent Advances inHeartworm Disease: Symposium ’98. AmHeartworm Society, Batavia, IL, 209-215 pp.

McTier TL, McCall JW, Jernigan AD, Rowan TG,Giles CJ, Bishop BF, Evans NA, Bruce CI, 1998.UK-124,114, a novel avermectin for the preven-tion of heartworms in dogs and cats. In: SewardRL (ed). Recent Advances in HeartwormDisease: Symposium ’98. Am HeartwormSociety, Batavia, IL, 187-192 pp.

McTier TL, Shanks DJ, Watson P, McCall JW,Genchi C, Six RH, Thomas CA, Dickin SK,Pengo G, Rowan TG, Jernigan AD, 2000.Prevention of experimentally induced heart-worm (Dirofilaria immitis) infections in dogsand cats with a single topical application ofselamectin. Vet Parasitol 91: 259-268.

Novotny MJ, Krautmann MJ, Ehrhart JC, GodinCS, Evans EI, McCall JW, Sun F, Rowan TG,Jernigan AD, 2000. Safety of selamectin in dogs.Vet Parasitol 91: 377-391.

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Heartworm (Dirofilaria immitis)infection in dogs: currentupdate in Spain

J.A. Montoya, M. Morales, M.C. Juste,J.A. Corbera

14

Heartworm infection in dogs in Spain

Heartworm infection in dogs hasbeen diagnosed around the globe.Relocation of infected, microfilaremicdogs appears to be the most importantfactor contributing to further dissemi-nation of the parasite. The ubiquitouspresence of one or more species of vec-tor competent mosquitoes makes trans-mission possible wherever a reservoirof infection and favourable climaticconditions co-exist.

A climate that provides adequatetemperature and humidity to support aviable mosquito population, and alsosustain sufficient heat to allow matura-tion of ingested microfilariae to infec-tive, third-stage larvae (L3) within thisintermediate host is a pivotal prerequi-site for heartworm transmission tooccur. The length of the heartwormtransmission season in the temperatelatitudes is critically dependent on theaccumulation sufficient heat to incu-bate larvae to the infective stage in themosquito. The peak months for heart-worm transmission in the NorthernHemisphere are July and August.Heartworm prevalence has increaseddramatically during the latest years.The disease has extended from tropicalor subtropical countries to others withmore temperate climate. The more fre-quent animal international travellingcould contribute to dissemination ofthe parasite. Heartworm transmissionis related to the dog’s life style, becauseoutdoor permanence increases the pos-sibility for mosquito contact. However,breed or hair length has not been cor-related with the risk of heartworminfection.

Heartworm infection is entirely pre-ventable in dogs, despite their high sus-ceptibility. Since most dogs living in

heartworm endemic areas are at risk,chemoprophylaxis is a priority.Furthermore, some evidence stronglysuggests that by reducing the reservoirpopulation through increasing thenumber of dogs receiving chemopro-phylaxis, a disproportionate decrease inthe prevalence of infection amongunprotected dogs may occur relative tothe percentage of additional dogsreceiving chemoprophylaxis. This col-lateral protection spreads the umbrellaof chemoprophylaxis most effectivelyin communities where heartwormprevalence and dog population densityare both relatively low.

During the latest years several epi-demiological surveys on heartwormhave been developed in many coun-tries. The disease is distributed, mainly,in temperate climates in the World. Themore prevalent countries are inAmerica, Africa, Polynesia, Australiaand Japan. In Europe the more preva-lence (>10%) occurs in theMediterranean countries. We mustpoint out that several studies in thenorth of Italy, south of France andSpain have elucidated prevalence over20%. Therefore, Spain is an endemicarea of Heartworm (Genchi et al.,2005).

Inside Spain, the prevalence of thedisease is higher in the southern coastalareas. Also, Canary Islands are consid-ered an endemic area of the disease.Specifically, in the Canaries, sited infront of the north-western Africancoast, the available data has empha-sized the highest prevalence of the dis-ease in Spain (Valladares et al., 1987;Guerrero et al., 1989). VeterinarySurgeons in Canary Island includeHeartworm Disease as the first differ-

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AUTONOMOUS COMMUNITYProvince

Prevalence References

ANDALUCIACádiz

Cádiz-MálagaCórdoba CórdobaHuelva

JaénMálagaSevillaARAGONZaragozaZaragozaASTURIASCANTABRIA CASTILLA-LA MANCHACASTILLA-LEONSalamancaSalamanca (ribera Tormes)CATALUÑACATALUÑABarcelona Barcelona (bajo Llobregat)BarcelonaTarragonaTarragona (delta Ebro)Tarragona (delta Ebro)COMUNIDAD VALENCIANAAlicanteAlicanteAlicanteElcheValenciaEUSKADIEXTREMADURABadajozGALICIAISLAS BALEARESMallorcaIbizaMADRIDMadridMadridMURCIAMURCIANAVARRA

8.5%12%

5.5%18.0%4%36.7%

2.1%2%1.5%4.3%13.5%8 %0 %0%0%0%12.0%> 30%2.17%0,6%1.2%12.8%2%0.8535.8 %26 %

13%1.6%18%2.6%4.1%0%6.7%8%0%6.3%0.3%39%2%1.1%1.96.3%9%0%

Guerrero et al., 1989Guerrero et al., 1989; Ortega Mora et al., 1991Rojo-Vázquez et al., 1990Anguiano et al., 1985Guerrero et al., 1989Guerrero et al., 1989; Ortega Mora et al., 1991Guerrero et al., 1989Guerrero et al., 1989Guerrero et al., 1989Guerrero et al., 1989Castillo et al., 1989Rodes, 2006Guerrero et al., 1989Guerrero et al., 1989Guerrero et al., 1989Guerrero et al., 1989Pérez-Sanchez et al., 1989Pérez-Sanchez et al., 1989Gutiérrez et al., 1995Solano-Gallego et al., 2006Rojo-Vázquez et al., 1990Aranda et al., 1998Solano-Gallego et al., 2006Solano-Gallego et al., 2006Anguera, 1995Rodes, 2006

Guerrero et al., 1989Rojo-Vázquez et al., 1990Rodes, 2006Cancrini et al., 2000Guerrero et al., 1989Guerrero et al., 1989Guerrero et al., 1989Ortega Mora et al., 1991Guerrero et al., 1989Guerrero et al., 1989Solano-Gallego et al., 2006Rodes, 2006Ortega Mora et al., 1988Guerrero et al., 1989Rojo-Vázquez et al., 1990Guerrero et al., 1989Rodes, 2006Guerrero et al., 1989

Table 1. Prevalence of heartworm in Spain.

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ential diagnosis in dogs with cardiopul-monary signs.

Previous studies in Spain (Guerreroet al., 1989) showed that Huelva hasthe highest prevalence in Spain(36.7%). This study determined thatthe prevalence of Dirofilaria immitis inGran Canaria was 36%. Later studiesrevealed prevalence over 30% inSalamanca (Muro et al., 1993) and thedelta area of the river Ebro (Anguera,1995). More recently, the highestprevalence (58%) of D. immitis inSpain has been described in GranCanaria (Montoya et al., 1998). Theprevalence in Gran Canaria is one ofthe highest described in the World;similar prevalence has been publishedin Cuba (Dumenigo et al., 1988) orJapan (Tanaka et al., 1985; Suenaga etal., 1978; Hatsushika et al., 1992).Otherwise, the highest prevalence everdescribed (86%) is in Papua-NewGuinea (Hamir et al., 1986).

However, it is encouraging that theprevalence of D. immitis in GranCanaria is decreasing: more recent sur-veys demonstrated prevalence under23% (Table 2). In our opinion, thisdata could due to the increasing use ofeffectiveness drugs to prevent the dis-ease, and mainly a very important vet-erinarian task in order to make aware

of the disease to their clients. Also, it ismore frequent that clients demand theuse of the newest drugs for the treat-ment of their infected pets; this increas-ing demand is extending to rural areaswhere population is not aware as far aspet cares are concerned.

References

Anguera-Galiana M, 1995. La dirofilariosis caninaen el Delta del Ebro. Medicina Veterinaria 12:242-246.

Anguiano A, Martínez-Cruz S, Gutiérrez PN,1985. Epidemiología de la dirofilariasis caninaen la provincia de Córdoba. IV CongresoNacional de Parasitología, Tenerife, Spain.

Aranda C, Panyella O, Eritja R, Castella J, 1998.Canine filariasis. Importance and transmissionin the Baix Llobregat area, Barcelona (Spain).Vet Parasitol 77(4): 267-275.

Cancrini G, Allende E, Favia G, Bornay F, AntónF, Simón F, 2000. Canine dirofilariosis in twocities of southeastern Spain. Vet Parasitol 92:81-86.

Castillo JA, Lucientes J, Estévez C y Gortazar C,1989. Epidemiología de la dirofilariosis enZaragoza I. Estudio de la prevalencia en perro yzorro y su interrelación. VI Congreso Nacional yI Ibérico de Parasitología, Cáceres, Spain.

Dumenigo BE, Espino AM, Bouza M, 1988.Prevalencia de filariasis caniana en la Isla de laJuventud. Revista de Salud Animal 10: 247-250.

Genchi C, Rinaldi L, Cascone C, Mortarino M,Cringoli G, 2005. Is heartworm disease reallyspreading in Europe? Vet Parasitol 133: 137-148.

Guerrero J, Rojo F, Ródenas A, 1989. Estudio dela incidencia de la enfermedad del gusano delcorazón en la población canina española.

Canary Island Prevalence Reference

CANARIAS (total)Gran CanariaGran CanariaGran CanariaTenerifeTenerifeTenerifeTenerifeTenerife

28%36%58.89%23.87%34.4%20%23%22.3%21%

Guerrero et al., 1989Guerrero et al., 1989Montoya et al., 1998Sosa et al., 2002Valladares et al., 1987Guerrero et al., 1989Stenzenberger & Gothe, 1999Morales et al., 2001Montoya et al., 2006

Table 2. Prevalence of heartworm disease in Canary Islands.

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Medicina Veterinaria 6: 217-220.Gutiérrez J, Guerrero J, Ródenas A, Castella J,

Muñoz E, Ferrer D, Florit F, 1995. Evolución deDirofilaria immitis en Cataluña. MedicinaVeterinaria 12: 10.

Hamir AH and Onaga I, 1986. Canine spirocerco-sis and dirofilariasis infection in Papua NewGuinea. Aus Vet J 63: 98-99.

Hatsushika R, Okino T, Shimizu M, Ohyama F,1992. The prevalence of dog heartworm(Dirofilaria immitis) infection in stray dogs inOkayama, Japan. Kawasaki Medical Journal 18:75-83.

Montoya JA, Morales M, Ferrer O, Molina JM,Corbera JA, 1998. The prevalence of Dirofilariaimmitis in Gran Canaria, Canary Islands, Spain(1994-1996). Vet Parasitol 75: 221-226.

Montoya JA, Morales M, Juste MC, Bañares A,Simon F, Genchi C, 2006. Seroprevalence ofcanine heartworm disease (Dirofilaria immitis)in tenerife island: an epidemiological update.Parasitol Res 4.

Morales M., Bañares A, Montoya JA, 2001.Prevalencia de la parasitación en perros porDirofilaria immitis en la isla de Tenerife. 36Congreso Nacional de la Asociación deVeterinarios Españoles Especialistas enPequeños animales (A.V.E.P.A) Barcelona,Spain.

Ortega-Mora LM, Ferré I, Gómez M y Rojo-Vázquez FA, 1988. Prevalencia de la infestaciónpor filarias en galgos en la zona centro deEspaña. Medicina Veterinaria 5: 433-442.

Ortega-Mora LM, Gómez M, Rojo-Vázquez FA,Ródenas A, Guerrero J, 1991. A survey of the

prevalence of canine filariasis in Spain. Prev VetMed 11: 63-68.

Pérez-Sanchez R, Goméz M, Encinas A, 1989.Canine filariasis in Salamanca (northwestSpain). Ann Trop Med Parasitol 83: 143-150.

Rodes D, 2006. Ultimos datos epidemiológicossobre filariosis canina. Argos: 52.

Rojo-Vázquez FA, Valcárcel F, Guerrero J, GómezM, 1990. Prevalencia de la dirofilariosis caninaen cuatro áreas geográficas de España. MedicinaVeterinaria 7: 297-305.

Solano-Gallego L, Llull J, Osso M, Hegarty B,Breitschwerdt E, 2006. A serological study ofexposure to arthropod-borne pathogens in dogsfrom northeastern Spain. Vet Res 37: 231-244.

Sosa N, Montoya JA, Juste MC, 2002. Situaciónepidemiológica actual de la dirofilariosis caninaen la isla de Gran Canaria (Comunicación). ICongreso Universitario de Ciencias Veterinariasy Afines, Madrid, Spain.

Stenzenberger R, Gothe R, 1999. Arthropodborne parasitic infections and tick infestationsof dogs in Tenerife, Spain. Tierarztliche Praxis27: 47-52.

Suenaga O, Kitahara S, 1978. Studies on theprevalence of Dirofilaria immitis among dogsand its vector mosquitoes in Sasebo city,Nagasaki Prefecture. Trop Med 20: 143-151.

Tanaka H, Watanabe M, Ogawa Y, 1985. Parasitesof stray dogs and cats in the Kanto region,Honshu, Japan. J Vet Med 771: 657-661.

Valladares B, Gijón H, López-Román R, 1987.Dirofilaria immitis en la isla de Tenerife.Algunos aspectos de su fisiopatología. RevistaIbérica Parasitología 47: 377-380.

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The importance of dirofilariosisin carnivores and humans inHungary, past and present

Éva Fok

15

Dirofilariosis in Hungary

Introduction

Several worm species of SuperfamilyFilarioidea develop in human and ani-mal hosts and many may cause zoonot-ic infections. Two families are recog-nized: Filariidae and Onchocercidae(Anderson, 2000). These filarioidnematodes are transmitted byhaematophagous arthropod vectors,including mosquitoes (Culicidae),blackflies (Simulium spp.), bitingmidges (Culicoides spp.), ticks(Rhipicephalus sp.), fleas and flies(Kassai, 1999, 2003). Filariidae elicitskin lesions and release eggs and/or lar-vae which attract arthropods, mainlyflies (Muscidae). The Onchocercidae,on the other hand, have evolved bloodor skin-inhabiting microfilariae and aretransmitted by arthropod vectors whichcreate lesions or pierce the skin andthen suck blood. In the recent decadethe importance of some zoonotic wormsof carnivores from Onchocercidae, suchas Dirofilaria and Onchocerca spp. isincreasing (Pampiglione and Rivasi,2000; Eberhard et al., 2000;Pampiglione et al., 2001; Sréter et al.,2002; Genchi, 2003; Salló et al., 2005).

The climate changes and travellingwith pets may help both the spreadingof vectors and the appearance of worminfection at risk for human health innon-endemic or previously free areas(Trotz-Williams and Trees, 2003). Twospecies of genus Dirofilaria, D. immitisand D. repens mostly are importantagents of zoonotic infections in theMediterranian area (Muro et al., 1999).

For both the parasites, the mostimportant natural host is the dog,which has a high prevalent and persist-ent microfilaremia. In the middle part

of the continent, D. repens is mostlywidespread and nowadays dirofilariosisis considered as an emerging zoonosis(Irwin, 2002).

Dirofilariosis in carnivores

Cardiovascular-dirofilariosis

The first recorded case of Dirofilaria-infestation of carnivores in Hungarywas published more than 20 years ago(Boros et al., 1982). In this report itwas shown D. immitis infection in twofemale beagle dogs imported from theUnited States. The adult worms weredetected in the right cavity of the heart.These dogs, with the other beagles,were kept for experimental purpose inclosed recovers, so it was clear that theinfection was imported.

The next reported filarial infection ina dog was in 2000 (Vörös et al., 2000).The dog lived in the USA for years withthe owner and they returned toHungary some months before the dateof admission into clinic. This five-year-old male dog of mixed breed wasrecovered with a two-days long historyof depression, exercise intolerance, dys-pnoea, anorexia, vomiting and voidingof reddish urine. The animal, in spite ofthe intensive therapy died within somehours after admission. Necropsyrevealed several adult D. immitis in theblood clots within the caudal vena cavaand within the dilated right ventricle.Microfilariae were found within thecapillaries of the myocardium, intersti-tial pulmonary capillaries and in otherorgans, including spleen and liver andin the peripheral blood smears. Venacava syndrome due to the occlusion of

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the caudal vena cava by adult wormstogether with blood clots was diag-nosed by necropsy and this complica-tion was stated as the cause of the sud-den crisis and the death of the patient. In spite of that, no newer imported caseof canine heartworm infection has beenpublished in the scientific literature inHungary, we have got some informa-tion from personal communicationsconducted with small animal practi-tioners about some cases (e. g. import-ed Mastino Napoletano dogs fromCanary Islands, Cane Corso puppiesfrom Italy) where D. immitis infectionwas suspected.

An interesting case was detected in1999, when a 3-year-old mongrel dogliving in Tisza river area was dissectedafter an accident and the veterinarianfound worms in the left ventricle of theheart. These worms were identified asD. immitis. This dog was living togeth-er with his mother who was housed sixyears earlier by an owner from thestreet. The dog was living at the rivershore, not far from a parking place ofinternational trucks. However, it wasnot possible to clarify if the infectionwas autochthonous or acquired throughthe biting of an infected mosquito trans-ported by one of the trucks. Although proved autochthonous dogheartworm infection has not so farrecorded in Hungary, the describedcases demonstrate the risk of introduc-tion of this disease with infected animalsfrom endemic countries (Farkas, 2003).

Cutaneous-dirofilariosis

In 1997, Zahler et al. published theresults of an epidemiological survey(Zahler et al., 1997) carried out

between 1993-1996 in München. Outof five dogs found infected with D.repens, two dogs were imported fromItaly or Greece, and the third had trav-elled with its owner in Hungary andformer Yugoslavia. The infection wasdiagnosed by blood examinations withKnott’s method. The origin of the infec-tion was not clear and it cannot beexcluded that the infection was reallyacquired in Hungary.

The first three autochthonous D.repens cases recorded in dogs werepublished in Hungary in 1998/1999(Fok et al., 1998; Széll et al., 1999). Inthe very first case the worm was foundin a verruca-like nodule removed inautumn of 1995 from the subcutaneoustissue of the neck region of a 4-year-olddog, which had spend the summer atriver Tisza (Fok et al., 1998). In theother two cases, the worms were foundin a granuloma removed from the tho-rax region and from the subcutaneoustissue of the leg, respectively (Széll etal., 1999). One of them had spendsome days at Lake Balaton and theother one was living in the TolnaCounty, near the southern boarder ofthe country. On the basis of the mor-phological features, the worms wereidentified as gravid females in all cases.Furthermore, 101 dogs from TolnaCounty were examined for microfilari-aemia by Knott’s method. The preva-lence of D. repens microfilaremic dogswas 9%. Considering that these dogswere never abroad, it was stated thatthere are endemic areas for D. repensin Hungary.

In the recent years, according to thepersonal communications of colleaguesfrom the main veterinary diagnosticlaboratories, more and more microfilar-

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ia positive blood samples of dogs asaccessory findings are detected in thepraxis. Most of these cases are suspect-ed D. repens infection, although thedetailed epidemiological background ismissing. Moreover, reports on humaninfection (see later) are frequentlydetected in Hungary.

Why is the number of cases increas-ing in our country? What can be theepidemiological background? What isthe prevalence of infection in dogs andcats in Hungary? How it is possible(or weather it is necessary) to treat D.repens positive animals in Hungary?Which mosquitoes species might bethe vector of this worm in our coun-try? The cutaneous dirofilariosis cancause problems in the diagnostic dif-ferentiations, as well. What kind ofconnections might be between theinfection of the animals and theincreasing number of the human casesin our country? Because of such aquestions and the lacking of D. repensprevalence data in domestic carni-vores in Hungary, in 2005 we decidedto start with a research work in ourDepartment of Parasitology andZoology, including a PhD programme.In parallel with the study, we plan tomonitor the human cases ofDirofilaria infection to obtain moreinformation on the epidemiologicalbackground in our country. The aimof this work is to survey the infectionof the most frequent final hosts, thento study the role of the vectors in theepidemiology and the possibilities ofthe spreading from carnivores tohumans to achieve an effective control(see later the preliminary results ofthis work in a free presentation).

The baseline data of this work would

be useful for veterinarians working inthe small animal praxis to help them inthe recognition of the clinical picture ofcutaneous dirofilariosis. Results con-cerning this zoonotic disease would beuseful for the human doctors, as well,and can help in a more effective con-trol. Furthermore, the results of theplanned study could be important notonly in Hungary, but also may con-tribute to the European knowledge onthis mosquitoes transmitted disease.

Dirofilariosis in humans

The first human filarioid infection inHungary was detected by Babes in1879 (cited by Kotlán, 1951).However, an actual evaluation of theearly human cases in Hungary waspublished in the middle of the last cen-tury by Professor Academic Kotlán(Kotlán, 1951). The author referred toDesportes (1939-1940), who in hispaper presented a historical review ofthe cases of human filarioidosis knowfrom literature, in which the parasitesplaying part in the pathological processcould be identified with, or was foundto be closely related to “Filaria con-junctivae” Addario, 1885 (cit. Kotlán,1951). Having had the opportunity toexamine a new case, Kotlán attemptedto review all available data at thattime, either verbal or published inHungarian literature. It was stated inhis publication that at least 9 casesrecorded from 1880 to 1950 inHungary was probably identical withD. repens, however the infection withother filarioid species was not com-pletely excluded. It was not possible toclarify the epidemiological background

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of these cases namely the sources ofinfection. It was supposed that some ofthese cases probably were autochtho-nous because the patients never hadleft the country. The case published ten years later(Németh and Kugler, 1968), was thefirst ocular involvement in our country,and it was supposed that the removedworm pieces from a nodule on the scle-ra of the 21 years old man were belong-ing to a D. repens specimen. More than30 years later, the next Hungarianhuman case in which the worm wasdetected in the spermatic cord waspublished by Pampiglione et al. (1999).It was thought that these cases wereacquired abroad, too.

However, in a little while, six newcases were reported by pathologistswith the help of a parasitologist (Eleket al., 2000). Two of these cases mighthave been acquired in Italy duringsummer travels. Four patients havenever been abroad and these casesmust be considered as autochthonousinfections. According to the authors,the patients liked dogs and cats, andthey were often living in sites alongDanube and Tisza rivers, where mos-quitoes are abundant. The thickness ofthe multilayered cuticle of the worm,diameter of the body and the size,form and number of the longitudinalridges on its surface were used in thehistological diagnosis of D. repensworms. In fact, the definitive diagno-sis of human dirofilariosis is a patho-logical and parasitological task. In thisreport the authors called the attentionof Hungarian human clinician to askthe patients whether they have been(and when and where) abroad,whether they have animals (dog or

cat), and whether they live near watershore where mosquitoes are abundant.The answer may explain the spread ofthe worm in Hungary.It seems that in the recent six yearsmore and more new autochthonoushuman cases become known in ourcountry. However, we can have theinformation about most of them fromoral presentations in conferences or inpersonal communications, and onlypartly from the scientific publications.In the new cases the worms wereremoved from the orbital tissues(Parlagi et al., 2000), subconjuctivalspace (Szénási et al., 2000; Hári et al.,2002; Kucsera et al., 2002; Aczél et al.,2005; Tornai et al., 2006), upper lid(Salomváry et al., 2005), the fatty tis-sue of a subcutaneous nodule of thethigh (Lengyel et al., 2005), a cyst situ-ated in the cutaneous and subcuta-neous layers of the forearm (Péter etal., 2005) and the subcutaneous tissueof the neck region (Csanády, 2005, per-sonal communication). All these cases,after the detailed anamnesis and identi-fication of the worms or examining oftissue sections were diagnosed as D.repens infection.

Nowadays, the medical clinicianswho suspect an human filarioid infec-tion in their patients can be supportedin the etiological diagnosis by parasitol-ogists sending removed worms foridentification or dissected tissue fromthe patient to specialized laboratoriessuch as the Department of Parasitologyof ‘Johan Béla’ Human Health Institute(see later a free presentation about thehuman cases by I. Kucsera) or theDepartment of Parasitology of theVeterinary Faculty of Szent IstvánUniversity, in Budapest.

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Conclusion

It seems to the parasitologists, that inHungary, the occurrence of D. repens ismuch more frequent than it was con-sidered earlier. The tourism with petsand visiting of the different dog or catexhibitions abroad and in our country,the repeated travelling of Italianhunters with their dogs to Hungary, cli-mate changing, frequent raining andflood may have increased the spreadingof dirofilariosis. A close cooperationnot only with the parasitologists, butalso between human and veterinary cli-nicians in our country and with scien-tists from surrounding countries areneeded to better understand the epi-demiology of dirofilariosis and to con-trol the risk of infection both for thepublic and animal health in Europe.

Acknowledgements

I should like to thank to my PhD student, OlgaJacsó who helped me to complete the list of thereferences. I am also extremely grateful to PfizerLtd. Animal Health Hungary who supported myattendance to the First European Dirofilaria Daysin Zagreb.

References

Aczél K, Deák Gy, Farkas R, Majoros G, 1995.Subconjunctivalis fonálféreg-fertozés Magyaro-rszágon [Subconjunctival nematode infection inHungary]. LAM-Tudomány 15: 465-467.

Anderson RC, 2000. Nematode parasites of verte-brates. Their development and transmission.2nd edition. C.A.B. International, Wallingford,467 p.

Boros G, Janisch M, Sebestyén Gy, 1982.Dirofilaria immitis fertozöttség kutyában[Dirofilaria immitis infestation in dogs]. MagyarÁo Lapja 37: 313-316.

Eberhard ML, Ortega Y, Dial Sh, Schiller CA,Sears AW, Greiner A, 2000. Ocular Onchocerca

infection in two dogs in western United States.Vet Parasitol 90: 333-338.

Elek G, Minik K, Pajor L, Parlagi Gy, Varga I,Vetési F, Zombori J, 2000. New human dirofi-larioses in Hungary. Path Onc Res 6: 141-145.

Farkas R, 2003. Dirofilariosis in Hungary.Helminthologische Fachgespräche [Helmintho-logical Colloquium]. November 14, 2003.Abstracts, 13 p.

Fok É, Szabó Z, Farkas R, 1998. Dirofilariarepens fertozöttség elso hazai diagnosztizálásakutyában sebészeti beavatkozás során [The firstautochthonous case of a dog infected withDirofilaria repens in Hungary]. Kisállatorvoslás4: 218-219.

Genchi C, 2003. Epidemiology and distribution ofDirofilaria and dirofilariosis in Europe: state ofthe art. Helminthologische Fachgespräche[Helminthological Colloquium]. November 14,2003. Abstracts, 6-11 pp.

Hári Kovács A, Szénási Zs, Tiszlavitz L,Kolozsvári L, Pampiglione S, Fioravanti ML,2002. Ophthalmo-filarioidosis újabb eseteMagyarországon [A new case of ocular dirofilar-iosis]. Szemészet 139: 87-90.

Irwin PJ, 2002. Companion animal parasitology: aclinical perspective. Int J Parasitol 32: 581-593.

Kassai T, 1999. Veterinary Helminthology.Butterworth & Heinemann. Oxford, 1999, 121-124 pp.

Kassai T, 2003. Helmintológia. MedicinaKönyvkiadó Rt., Budapest, 186-192 pp.

Kotlán A, 1951. On a new case of human filari-idosis in Hungary. Acta Vet Acad Sci Hung 1:69-79.

Kucsera I, Elek I, Danka J, Fok É, Varga EB,Birnbaum I, Szénási Zs, 2002. Ocularis filarioi-dosis-esetismertetés [A human case report].Magyar Mikrobiológiai Társaság és Alapítvány2002. évi nagygyulés, Összefoglalók [Abstracts],Balatonfüred, October 8-10, 2002, 80 p.

Lengyel A, Kovács I, Nemes Z, Rábainé PongráczJ, Kovács J, Fok É, 2006. Humán dirofilariosis[A human case report]. 64. PathologusKongresszus a Magyar Pathológusok társaságaés a Nemzetközi Pathologiai Akadémia (IAP)Magyar Divíziója rendezésében, Összefoglalók[Abstracts], Pécs, September 22-24, 2005, 66 p.

Muro A, Genchi C, Cordero M, Simón F, 1999.Human dirofilariasis in the European Union.Parasitol Today 15: 386-389.

Németh B, Kugler S, 1968. Ophthalmo-filariasis.Orv Hetil 109: 195-197.

Pampiglione S, Elek G, Pálfi P, Vetési F, Varga I,1999. Human Dirofilaria repens infection inHungary: A case in the spermatic cord and areview of the literature. Acta Vet Acad Sci Hung47: 77-83.

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Pampiglione S, Rivasi F, 2000. Human dirofilaria-sis due to Dirofilaria (Nochtiella) repens: anupdate of world literature from 1995 to 2000.Parassitologia 42: 231-254.

Pampiglione S, Rivasi F, Angeli G, Boldorini R,Incensati RM, Pastormerlo M, Pavesi M, RamponiA, 2001. Dirofilariasis due to Dirofilaria repens inItaly, an emergent zoonosis: report of 60 newcases. Histopathology 38: 344-354.

Parlagi Gy, Sumi Á, Elek G, Varga I, 2000.Szemüregi dirofilariosis [Orbital dirofilariosis].Szemészet 137: 105-107.

Péter I, Czeyda-Pommersheim F, Kucsera I,Szénási Zs, Orosz Zs, 2005. Új humán dirofi-lariosis eset Magyarországon [A human casereport]. 64. Pathologus Kongresszus a MagyarPathologusok Társasága és a NemzetköziPathologiai Akadémia (IAP) Magyar Divíziójarendezésében, Összefoglalók [Abstracts], Pécs,September 22-24, 2005, 80 p.

Salló F, Eberhard ML, Fok É, Baska F, Hatvani I,2005. Zoonotic Intravitreal Onchocerca inHungary. Ophthalmology 112: 502-504.

Salomváry B, Korányi K, Kucsera I, Szénási Zs,Czirják S, 2005. Szemüregi dirofilariosis újabbesete Magyarországon. [A new case of orbitaldirofilariosis in Hungary]. Szemészet 142: 231-235.

Sréter T, Széll, Z, Egyed Zs, Varga I, 2002. Ocularonchocercosis in dogs: a review. Vet Rec 151:176-180.

Széll Z, Sréter T, Csikós K, Kátai Z, Dobos-Kovács M, Vetési F, Varga I, 1999. AutochtonDirofilaria repens fertozöttség kutyákban[Autochtonous Dirofilaria repens infection indogs]. Magy Áo Lapja 121: 100-104.

Szénási Zs, Hári Kovács A, Tiszlavicz L,Kolozsvári L, Nagy E, 2000. Humán ophthal-mo-filarioidosis. A Magyar Zoonózis TársaságKiadványa [Publication of the Society ofHungarian Zoonosis]., Kiadó: Korzenszky E.,124-130 pp.

Tornai I, Montovay E, Veress Cs, 2006. Humánfertozés-szemészeti esetismertetés [A humancase report]. “A dirofilariosis, mint egyre növek-vo állategészségügyi és közegészségügyi problé-ma hazánkban” c. rendezvény (conference) aMagyar Parazitológusok Társasága, a MagyarZoonózis Társaság és a Pfizer Kft. Állate-gészségügy rendezésében, March 24, 2006.

Trotz-Williams LA, Trees AJ, 2003. Systematicreview of the distribution of the major vector-borne parasitic infections in dogs and cats inEurope. Vet Rec 152: 97-105.

Vörös K, Kiss G, Baska F, Bagdi N, Széll Z, 2000.Irodalmi áttekintés és esetismertetés[Heartworm disease in dogs. Review article andcase report]. Magy Áo Lapja 122: 707-716.

Zahler M, Glaser B, Gothe R, 1997.Eingeschleppte Parasiten bei Hunden:Dirofilaria repens und Dipetalonema recondi-tum. Tierärztl Prax 25: 388-392.

FACULTY OF VETERINARY MEDICINEUNIVERSITY OF ZAGREBPFIZER ANIMAL HEALTH

CROATIAN VETERINARY SOCIETYCROATIAN VETERINARY CHAMBER

FIRST EUROPEANDIROFILARIA

DAYS

INTERNATIONAL SCIENTIFICCOMITEE

Claudio Genchi (president), ItalyHorst Aspok, AustriaPeter Deplazes, SwitzerlandRobert Farkas, HungaryLaura Kramer, ItalyAlbert Marinculic, CroatiaSilvio Pampiglione, ItalyFernando Simon, SpainNatasa Tozon, SloveniaAlexander Trees, UKLuigi Venco, Italy

ORGANIZING COMITEE

Albert Marinculic (president), CroatiaLjiljana Pinter (vice president), CroatiaRelja Beck (Croatia), Marijan Cergolj(Croatia), Claudio Genchi (Italy) Ivan Forgac (Croatia), Drazen Gluvak(Croatia), Jasminka Granic (Croatia),Boris Fabijanic (Croatia), Branko Juric(Croatia), Lidija Kozacinski (Croatia),Milivoj Landeka (Croatia), IgorMarkovic (Croatia), Drazen Maticic(Croatia), Mario Milesevic (Croatia),Mirna Ladavac (Croatia), Darko Geres(Croatia), Damir Zubcic (Croatia), Dagny Stojcevic (Croatia), TatjanaZivicnjak (Croatia)

Zagreb, Croatia, February 22-25, 2007

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Red foxes (Vulpes vulpes), like otherwild and domestic carnivores, can beaffected by several species of filarialnematodes, namely Dirofilaria immitis,D. repens, Acanthocheilonema recon-ditum, and A. dracunculoides. The firstspecies induces heartworm disease,with severe/extremely severe pathologi-cal changes in the animals, whereas theother parasites are etiological agents ofsubcutaneous/connective tissue filario-sis. Furthermore, at least dirofilarialspecies proved zoonotic and, therefo-re, where the animal disease is ende-mic, the human population is at riskfor developing ocular lesions or pul-monary/subcutaneous nodules thatalways give rise to the suspicion of amalignant cause.

Literature data about the role of thefox as reservoir for filarial nematodesare in disagreement. In fact, in spite ofadult worms found in this animal seemoften immature and microfilaraemia isreported as sporadic (Meneguz et al,Parassitologia 40 suppl: 105), it hasbeen suggested that the fox may play anepizootiologic role in heartworm infec-tion (Mañas et al, 2005, Vet J 169: 118-120). The possible importance of thiswild animal as reservoir of infection for

people and for companion animalsprompted us to include filariosesamong the parasitic diseases that haveto be checked on the foxes examinedduring a survey on echinococcosis.

With the aim of contribute to thissubject of debate, and to compare theinfection rates of filarial worms in wildand domestic canidae of the region,112 foxes (42 males, and 72 females),killed during the hunting season 2003-2004 in several areas of the Tuscany(Central Italy), were examined. Thecarcasses of the foxes, coming from theMonte Amiata (Grosseto; n=37), thehills around Cecina (Livorno; n=26),the zones around Cascina and Bientina(Pisa; n=33) and the hills around Siena(n=16), were sent to the laboratory ofPisa. Data on sex, age, weight, and areaof origin were collected for each ani-mal, and the carcasses were stored at-20°C until necroscopy. Microfilariaewere searched for in pulmonary andsplenic blood smears, and species iden-tification was performed using bothmorphologic and morphometric char-acteristics, and Barka coloration tech-nique. As far the adults, it was onlypossible the searching for D. immitis inthe endocardium and in the pulmonary

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

M. Magi1, P. Calderini2, S. Gabrielli3, M. Dell’Omodarme4, F. Macchioni1,M.C. Prati4, G. Cancrini31Dip. 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, Dipartimentodi Rieti, V. Tancia, 21, 02100 Rieti, Italy; 3Dip. di Scienze di Sanità Pubblica, Università “LaSapienza”, 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

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artery, because organizational problemshampered the examination of tissues inorder to ascertain the presence ofadults belonging to the other species.Finally, molecular diagnostics (DNAextraction, PCR using general “filarial”primers S2-S16 and sequencing) wereapplied to adults and micriofilariae toconfirm all identifications. Data werestatistically analyzed with the Fisherexact test, and results were consideredstatistically significant if P<0.05.

A total of 20 foxes (17.9%) turnedout positive to filarial parasites. In 8specimens (7.1%) were found matureadults of D. immitis (9 males and 14females), and two of them (1.8%), col-lected around Cascina (Pisa), had alsomicrofilariae both at pulmonary andsplenic level. One of these two foxeswas positive also to microfilariae of D.repens. Amicriofilaraemic subjects har-boured at least an adult couple. A totalof 12 foxes (10.7%) were positive tomicrofilariae of A. reconditum, absentonly from specimens hunted on thehills around Livorno, where all foxeswere found amicrofilaraemic. In the

four areas no difference was evidencedamong positivity rates to adults of D.immitis, whereas there is a significantdifference among those to microfilariae(P = 0.022). Molecular tools confirmedmorphological identifications.

Our findings on A. reconditum,D. immitis and/or D. repens circulatingmicrofilariae in about 10% and in lessthan 2%, respectively, of the fox car-casses examined fit in with data regard-ing Italy. As far Tuscany, after 25 yearsthe prevalence of microfilaraemic spec-imens is about unchanged (19.4% vs17.9%) (Gradoni et al., 1980, Ann IstSup San 16: 251-256), and it is not somuch different from that evidenced onconcentrated blood samples of alivedogs (35.4%) (Marconcini & Magi,1991, Ann Fac Med Vet Pisa, XLIV:153-156). Our findings, drawn by theexamination of very few µl of bloodobtained from perished carcasses, sup-port the hypothesis that the fox can bea source of infection for invertebratehosts and, therefore, wild reservoir forfilarial nematodes that can be transmit-ted to companion animals and people.

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Background

In the recent years more and more fre-quently cases of mosquito-borne filari-oid subcutaneous infection withDirofilaria repens Railliet and Henry,1911 has been reported in animals(mainly dogs) and in humans. However,limited information were availableabout the frequency of D. repens infec-tion in carnivores in Hungary.

For such a reason, epidemiologicalsurveys were carried out to assess thestatus of this carnivore’s filarioid infec-tion and to evaluate the risk for veteri-nary and medical public health.


From June 2005 to July 2006, 826dog and 29 cat blood samples collectedfrom several areas of the country withthe help of veterinary practitionerswere examined. The presence of micro-filariae in the blood samples wasassessed by modified Knott’s tech-nique. Larvae were identified by mor-phological criteria.

Results

D. repens microfilariae were found in116 blood samples from dogs

(116/826; 14%) and in 2 samples fromcats (2/29; 6.9%). Of the positive dogs,64 (55.2%) were living along Danuberiver, 40 (34.5%) along Tisza river, 1(0.8%) on the shore of lake Balaton and11 (9.5%) near other wet areas. Thepositive cats were from the town ofSzeged, situated at both sides of Tiszariver. Of the positive dogs, 39.4% wereaged 3-6 years and 32.1% were 7-10year-old. The infection prevalence was53.2% in male dogs and 46.8% infemale dogs. Higher prevalence valueswere found among mongrel (20.7%)and German shepherds (19%). Otherbreeds such as various hunting breeds(21.6%) and 1 to 2 positives dogs frommiscellaneous breeds (38.8%) werealso found to be infected.

Skin lesions (such as erythema,papules, alopeca, nodules) were diag-nosed in 276 (33.4%) of the exam-ined dogs and from these animals 37(13.4%) was microfilaria-positive.However, of the 116 positive dogs, 37(31.9%) showed skin lesions. Fromthe examined cats, 10 (34.5%)showed various skin disorders, but themicrofilaria-positive cats were withoutsymptoms.

The prevalence of other parasiticinfection was also evaluated accordingto the other clinical records, animal

Preliminary results of an epidemiological survey ondirofilariosis 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 IstvánUniversity H-1400 Budapest, P.O. Box 2, Hungary; 2Pfizer Animal Health, 1123 Budapest,Alkotás u. 53., Hungary

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maintenance and control against theecto- and endoparasites.

Conclusion

It has been shown that D. repensinfection in both dogs and cats is moreimportant than it is currently consid-ered in Hungary. The survey highlightsthe possible zoonotic risks for humans

living in the regions where the positiveanimals were found. Visiting or livingat the coastal areas of rivers seems tobe a significant risk factor to acquirethe infection. Our further aim is to con-tinue this survey to acquire more infor-mation about the occurrence of thisworm infection and to study the role ofmosquitoes in spreading of the infec-tion in our country.

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In the temperate regions of Europe(mainly Italy, France and Greece, butover the last few years in Hungary aswell) a specific filaroid worm, theDirofilaria (Nochtiella) repens, a para-site of dogs, cats and some other carni-vores, transmitted by mosquitoes, hasoccasionally infected people. In theworld literature more than 800 caseshave been recorded. Usually only oneworm can be detected in human tis-sues. In human cases the microfilar-iemia and eosinophilia are usually notpresent. The diagnosis is ordinarilybased on morphology of the intactworm and specific histopathologicalfindings. Surgical removal and identifi-cation of the worm are both diagnosticand therapeutic.

In the period from 2000 to August2006 we diagnosed 14 cases of dirofi-lariosis in 8 female and 6 male patients.Mean age was 57 years for female and62 for male (average 60 years). Thishigher prevalence in older-age corre-sponds to literature data. Of the 14cases, 7 had ocular localization, 6 sub-cutaneous and 1 was diagnosed inhistopathological section of removedlymph-node in a patient with lymphoidleukemia. Anamnestic eosinophilia wasfound in 2 cases. For detection ofmicrofilariae we used Knott concentra-

tion technique in 7 cases, with negativeresults.

Based on the available epidemiologi-cal data, it can be deduced that most ofthese cases (8) are autochthon infec-tions. In 2 cases the data were ambigu-ous and in 4 cases there were no avail-able epidemiological data. The inci-dence of human dirofilariosis inHungary is sporadic, and does notshow seasonality.

It can be concluded that D. repensoccurs in Hungary. The possibility ofdirofilariosis has to be taken into con-sideration in cases of predisposedlocalized nodules. Increased interestof the clinicians and parasitologistsresulted in the newly detected casesbeing completely diagnosed with bothmorphological and histopathologicalexaminations. The differential diagno-sis from the other filaria species whichoccur in humans is not required inthese cases, because the patients’ his-tories contain no data that refer to thatpossibility. Therefore, we considerthat most of our cases are auto-chthonal. Although the incidence ofdirofilariosis is sporadic and the pub-lic health importance is not high, theincreasing number of cases suggeststhat direct attention must be paid tothis zoonosis.


Review of human dirofilariosis diagnosed at theDepartment of Parasitology, National Center forEpidemiology, Budapest, Hungary

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

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Introduction

In the past, canine dirofilarial infection(Dirofilaria immitis and D. repens) inthe Czech Republic was associated withimport and was considered an importedinfection. The present study shows atsurveying the presence of autochtho-nous Dirofilaria infections in dogs in thearea of the Czech Republic where theclimatic conditions are favourable forthe life cycle of the parasite.

Material and Methods

We have investigated the occurrenceof dirofilariosis in dogs in the CzechRepublic from Breclav area known byhigh abundance of mosquitoes. Thestudy was carried out from November2005 to November 2006. The groupconsisted of 104 dogs. The sampleddogs had no history of travellingabroad and were free of clinical symp-toms. Microfilariae were detected byusing the Knott test and determinedwith the histochemical method basedon the activity of acid phosphatase.

Serum samples were examined withthe PetChek kit (IDEXX Laboratories,Portland, USA) for the detection ofadult female D. immitis circulatingantigens. We have also examined 11out of 104 animals with IFA

Heartworm kit (Fuller Laboratories,USA) for the detection of specific anti-bodies against D. immitis. For compar-ison we inserted also 2 samples positivefor D. immitis antigen.

Results

Microfilariae were detected in 8 dogs.The result of the acid phosphatasestaining for all eight samples agreedwith D. repens species. No microfilari-ae of D. immitis were detected.Serological testing detected D. immitisantigens in 7 dogs. D. repens positivedogs were negative on the ELISA forD. immitis.

Specific antibodies against D. immitiswere found in 6 out of 11 dogs includ-ing 2 positive for D. immitis antigen.

Conclusions

Our results confirmed endemic diro-filariosis in the Czech Republic.Dirofilariosis in the Czech Republic hasbeen slowly spreading as our previousresults show.

References

Svobodova Z, Svobodova V, Genchi C, Forejte P,2006. The first report of authochthonous dirofi-lariosis in dogs in the Czech Republic. Helmint-hologia 43: 242-245.

Dirofilariosis in dogs - the actual situation in theCzech Republic

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

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Dirofilaria repens infection in dogs in the Czech Republic

Z. Svobodova1, V. Svobodova2, C. Genchi31Small Animal Clinic, Faculty of Veterinary Medicine, University of Veterinary andPharmaceutical Sciences Brno, Czech Republic; 2Department of Parasitology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences Brno, CzechRepublic; 3Dipartimento di Patologia Animale, Igiene e Sanità Pubblica Veterinaria,Università degli Studi di Milano, Milano, Italy

Introduction

Dirofilaria repens is the agent of thesubcutaneous filariosis in dogs andcats. Its pathogenicity is often under-estimated. Global warming, theincreasing abundance of mosquitoes,movement of dogs between endemicand non-endemic countries haveincreased the risk of spreading of filar-ial infections in formerly Dirofilaria-free areas.

Material and Methods

The study was carried out fromNovember 2005 to November 2006 on80 dogs, 53 males and 27 females,aged 1-12 years. Most were huntingdogs that spend the majority of theirtime outdoors. The sampled dogs hadno history of traveling abroad andwere free of clinical symptoms.Samples of whole blood, blood serumand 2 fresh blood smears wereobtained from each dog.

Microfilariae

A modified Knott test was used todetect microfilariae (mf) that were thenidentified on the basis of their mor-

phology. Species identification wasconfirmed by histochemical staining ofacid phosphatase with naphthol AS-TR-phosphate as a substrate, andpararosaniline as a chromogen.

PCR - DNA was extracted from 100µl of canine blood samples positive forcirculating mf using a commercial kit(QIAamp DNA blood; QIAGENGmbH, Germany). PCR reactions forthe amplification of the mitochondrialcoxI gene were performed in a final vol-ume of 10 µl using general filarialprimers and thermal profile describedin Casiraghi et al. (2004). PCR prod-ucts were gel-purified and sequenceddirectly using ABI technology. Theobtained sequences were compared tothose deposited in gene banks.

Results

Seven of the 80 dogs tested weremicrofilaremic (8.7 %). Five were maleaging 6 - 11 years and 2 were female,both aged 2 years, different breeds -German Shorthaired Pointer (3),Jagterier (1), Labrador Retriever (1),Czech Pointer (1) and Tosa Inu (1).Acid phosphatase staining identified D.repens species in all the samples. PCRdefinitely confirmed D. repens diagno-


sis in these samples. Numerous adultD. repens were found in the subcuta-neous tissue during necropsy of one D.repens microfilaremic dog euthanizedfollowing the owner’s request.

References

Svobodova V, Misonva P, 2005. The potential riskof Dirofilaria immitis becoming established inthe Czech Republic by imported dogs. VetParasitol 128: 137-140.

Svobodova V, Svobodova Z, Beladicova V,Valentova D, 2005. First cases of canine dirofi-lariosis in Slovakia: a case report. VeterinarniMedicina 50: 510-511.

Svobodova Z, Svobodova V, Genchi C, Forejtek P,2006. The first report of autochthonous dirofi-lariosis in dogs in the Czech Republic.Helminthologia 43: 242-245.

Casiraghi M, Bain O, Guerriero R, Martin C,Pocacqua V, Gardner S, Franceschi A, Bandi C,2004. Mapping the presence of Wolbachia pipi-entis on the phylogeny of filarial nematodes:evidence for symbiont loss during evolution. IntJ Parasitol 34: 191-203.

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Systematic research on canine filar-ioses caused by the species Dirofilariarepens, Dirofilaria immitis, andAcanthocheilonema (Dipetalonema)reconditum in Serbia has not beenreported. Aim of this study was toestablish the prevalence of filarialnematodes in dogs and to identifyspecies according to their morphologi-cal and morphometric characteristics.

A total of 208 blood samples weredrawn from dogs from different areasof Vojvodina and Branicevo region.Modified Knott technique and com-

mercial DIFIL test (EVSCO, BUENA,NJ, USA) were used to detect microfi-lariae in blood samples. Identificationof species of microfilariae was per-formed according to their morphologi-cal and morphometric characteristics.

All morphometric parameters wereobtained using automatic image analy-sis Lucia M (NIKON). Including mixedinfections, microfilariae were found inthe peripheral blood of 101 examineddogs. D. repens infection was found in97 dogs, D. immitis infection in 15 dogs,and A. reconditum infection in 4 dogs.

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Filariosis in dogs in Serbia

S. Dimitrijevic1, A. Tasic2, S. Tasic2, V. Adamovic3, T. Ilic1, N. Miladinovic-Tasic2

1Department of Parasitic Diseases, Faculty of Veterinary Medicine University of Belgrade,Serbia; 2Department of Microbiology and Parasitology, School of Medicine, University ofNis, Serbia; 3Veterinary Clinic VEGE-VET, Pozarevac, Serbia


The first information on the existenceof dirofilariosis in Serbia was pub-lished in 1999 (Sanda Dimitrijevic,1999). The number of infected dogs isgetting higher every year. In the regionof Vojvodina, as the part of Serbia, theinvestigation was done and the cases ofinfection were found (Tasic et al.,2003). In the year of 2004, a group of100 dogs was observed within theregion of Vojvodina (Backa and Srem).The blood test was performed with atest kit diagnostic device for quickdiagnostic of dirofilariosis, namedFASTest HW Antigen, produced byMegaCor, Austria and ELISA test wasperformed in the same blood samplesby a DiroCHECK test kit, produced bySynobiotics, USA. After the analysisand the comparison of the results therewere 7% of positive samples to dirofi-lariosis, gained by two different diag-nostic methods - fast test and ELISA.

The positive samples were from thedogs of Srem region, and the placeswere the following: Venas, Bukovacand Ledinci.

During the year of 2006, the blood

samples were taken again from the ter-itories where dirofilariosis was foundpreviusely and analysed for dirofilario-sis, again. The test was done on 50blood samples from Srem region(Ledinci, Paragovo, Krusedol, Seliste,Venac, Bukovac and Petrovaradin).The samples were analysed by ELISAmethod, again with the same test kits -DiroCheck test kits, produced bySynobiotics, USA. The result was that5 of 50 dogs were positive to dirofilar-iosis, which makes 10% of the analysedpopulation. The positive samples werefrom the following places of the Sremregion - Ledinci, Krusedol, Venac andBukovac.

The number of positive dogs to diro-filariosis has grown during the last twoyears in the region where the diseasewas primarly found and dirofilariosis isstill present at the same places of Sremregion with the tendency of spreadingduring the year 2006. Usually, dirofilar-iosis is a side finding during the dissec-tion, and a dog is rarely suspected tohave dirofilariosis during the life time,even it has clinical symptoms.

The appearances of dirofilariosis in Serbia - Vojvodina

Sara Savic-Jevcenic1, Branka Vidic1, Zivoslav Grgic1, Lolic Zoran2

1Scientific Veterinary Institute of “Novi Sad”, Novi Sad, Serbia; 2Private practice “Leo”, NoviSad, Serbia

Free Communications

The increased number of dirofilariacases and the impact on the humanhealth due to the zoonotical aspect ofthe helmintosis, it’s mainly the reasonfor the global scientifical research onthe matter.

The research schedule was elaboratedby the Parasitological laboratory of theSpiru Haret University, Faculty ofVeterinary Medicine, Bucharest,Romania. The clinical findings werestudied on 52 dogs of different breedand age. The clinical, parasitologic, andpathologic features of this entity arediscussed, during the 6 months trial.

From the 52 studied dogs, 12(23.07%) were Dirofilaria positive.Symptoms, commonly chest pain,cough, or hemoptysis, along with car-diac failure and ascites were present in25% of 3 patients.

We found positive 5 dogs, aged 3-5years old, 5 dogs aged 6-8, and 2 dogsaged 10-14, from which 8 were femalesand 4 males.

The hematology examination revealed:microcytar cupripriv hypocrom ane-mia, neutrophily, lymphocytopeny,eosinophily. Radiologicly was registerdcardiac hypertrophy with left ventricu-lar dilatation and densifications pul-monary parenchim.

Electrocardiographic tests revealedhipertrophic cardiomiopathy.

Histopathological examination reve-aled pulmonary edema, thromboseswithin arteryes, and expatiated alveolarcapillary. Among the perilobulary sep-tum and the conjunctive tissue, numer-ous macrophage cells were found, withthe purpose of fagocitosing the erythro-cytes degradation product, hemosiderin.

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Dirofilariosis in dogs and wild carnivores in Romania

Coman Sofia, B. Bacescu, 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


A study of the prevalence of the diro-filariosis in dogs and wild carnivores inBulgaria was conducted from 2001 to2006. Blood samples from 487 dogsout of heartworm prophylaxis originat-ing from different parts of the countrywere tested. Microfilariemia was foundin 42 (8.62%) of the samples by themodified Knott’s test. Infected animalswere found in all parts of the country.

All microfilaria positive samples wereantigen positive by Pet CheckHTWMPT test (IDEXX). Three(6.6%) of 45 microfilaria negative

samples were antigen positive. The necropsy of 113 foxes, 56 jack-

als, 22 wild cats and 21 martensrevealed Dirofilaria immitis infectionin 4 (3%) of the foxes and 5 (8.9%) ofthe jackals. The intensity of the infec-tion was from 2 to 16 specimens.

Our results show that wild canidesare a natural reservoir and source ofinfection for dogs. The trend of increas-ing prevalence of dirofilariosis in dogs,when compared to previous studies,suggests that heartworm prophylaxisshould be considered in Bulgaria.

Dirofilariosis in dogs and wild carnivores in Bulgaria

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

Free Communications

Dirofilaria immitis is still considereda rare and imported disease forBulgaria. It is studied by all veterinarystudents but it is not expected to befound in the practice. Most practicingveterinarians regard the disease asuncommon. As a result routine analy-ses are not performed, which leads to alack of up to date information aboutlocalization and numbers of dirofilario-sis cases.

The data presented here are gatheredin a small animal practice for a periodof 5 years (from 2001 until 2006), fromthe city of Plovdiv and its surroundings,southwestern Bulgaria (total populationabout 500,000 people).

Since 2001 we have registered a totalof 87 cases, 56 percent of them inPlovdiv and 44 percent in the sur-rounding towns and villages.

We analyzed the cases by the follow-ing categories: severity of disease atpresentation; diagnostic method used;whether or not the dogs were treated;complications after treatment; dogs liv-ing outdoors/indoors; use of dogs(hunting dogs, guard dogs, pets); age;gender; location.

Practically no prevention was used onany dog before presentation. The vastmajority of clients heard about the dis-

ease for a first time when they present-ed their sick animals.

During the period of investigation thenumber of the diagnosed cases wasincreasing every year. We believe thisobservation doesn’t relate to thedynamics of the disease in the region,but is rather due to technical factors.

The increased cases identification ismore related to the fact that wechanged our approach from sporadictests (when the clinical condition indi-cates) to routine analysis of many dogscoming to the practice. Another impor-tant factor is the expansion of our prac-tice, thus the physical ability to checkmore dogs.

The region of Plovdiv is serviced byanother 4 practices having the size ofours and about 15 smaller practices.We deduce that our results can be mul-tiplied at least by a factor of 5 to derivethe total number of dirofilaria casespresented (but not necessarily diag-nosed) in Plovdiv.

Our results show that dirofilarosis isnot a rare disease among dogs inPlovdiv region. This imposes the needto educate dog owners about preven-tion and to incorporate dirofilaria diag-nostic methods into the routine checksof all dogs.

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

Milen KostadinovPrivate Veterinary Practitioner, Veterinary clinic “Novet”, Plovdiv, Bulgaria


Dirofilariasis is a disease caused byfilarial worms of the genus Dirofilariatransmitted by mosquitoes. Althoughdirofilariasis was originally considereda disease of strict veterinary impor-tance, it has been recognized as anemerging zoonosis. Two Dirofilariaspecies, D. repens and D. immitis, areof special interest to humans because oftheir harmful effect on our companypets (dogs and cats) and potentialzoonotic role. Turkey is one of theworld countries where the presence ofD. immitis and D. repens is currentlyconfirmed. The objective of this studywas to provide information on dirofi-lariasis both on animals and humans inTurkey. D. immitis was first reported inone dog in Turkey in 1951 and wasfound in different provinces and canine

prevalence ranged from 0.83 to46.22% at local scale following years.The national prevalence of the diseasehas not been determined hitherto, yet.Many cases of D. immitis have beenreported in dogs in Turkey. D. repenswas first reported in one human inTurkey in 1944. It was only investigatedin dogs in the east part of Turkey, inElazıg province, seropositivity rate was7.07%. In Turkey, human dirofilariasiswere documented in 18 cases. Serologicmethods for use in humans are neededfor clinical evaluations of patients withpneumonitis living in highly enzooticD. immitis regions. These resultsdemonstrate that dirofilariasis is wide-spread in dogs in Turkey and epidemio-logical surveys are needed to determinethe real extent of this zoonotic infection.

Dirofilariasis in Turkey

Bulent Ulutas1, Tulin Karagenc2

Department of Internal Medicine1 and Parasitology2, Veterinary Faculty, Adnan MenderesUniversity, Aydın, Turkey

Free Communications

The purpose of this study was todetermine the prevalence of heartwormdisease (Dirofilaria immitis) in dogs inthe Gemlik area of Bursa province,Turkey. The study was carried outbetween June and September 2004. Atotal of 100 dogs with various ages andsexes were included in the study. Ofthese, 65 were between 0.5 and 2 yearsold, 25 were 3-6 years old and 10 wereover 7 years old. Heparinized blood samples obtainedfrom dogs were separately examinedwith native and Modified Knott tech-niques in order to detect the presence

of circulating microfilariae while serawere examined with commercialELISA test kit in order to detect thepresence of circulating antigens.

No circulating microfilariae werefound in peripheral blood of examineddogs by native and Modified Knott tech-niques, whereas parasite antigens weredetermined in the sera of two male dogsone of which was aged between 3-6years and the second was over 7 years.In conclusion, the prevalence ofD. immitis was determined as 2%among dogs (occult infection) in theGemlik area of Bursa province, Turkey.

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Prevalence of canine heartworm disease in theGemlik area of Bursa Province, Turkey

T. Civelek1, A. Yildirim2, A. Ica2, O. Duzlu2

1Afyon Kocatepe University, Faculty of Veterinary Medicine, Department of InternalMedicine, Afyon, Turkey; 2Erciyes University, Faculty of Veterinary Medicine, Department ofParasitology, Kayseri, Turkey


This study was conducted to deter-mine the prevalence of Dirofilariaimmitis infection and to investigate therisk factors related to heartworm dis-ease in dogs from Kayseri, Turkey.Blood samples were collected from 280dogs from May 2005 to March 2006and were examined by membrane fil-tration-acid phosphatase histochemicalstaining and antigen Elisa techniques todetect circulating microfilariae andantigens of D. immitis, respectively.

Out of the total of 280 dogs, 27 werepositive for D. immitis with a preva-lence value of 9.6%. In addition,29.6% of positive dogs determined tohave occult D. immitis infections.D. immitis was the only canine filarialparasite present in the study area. The

mean number of microfilariae in infect-ed dogs was 4730 ± 5479 per ml ofblood. The highest heartworm preva-lence were observed in the 7-10 agegroup (28.6%), followed by the groups4-6 (17.1%) and 0.5-3 (4.8%) yearsold. The differences between the 0.5-3age group and the other age groupswere found significant, whereas no sta-tistically significant difference wasobserved between the 4-6 and the 7-10age groups. The infection was moreprevalent in males, larger breeds and inthe dogs not on prophylaxis. No statisti-cally significant difference was observedbetween stray and owned dogs. Ourresults suggest that heartworm treat-ment and prophylaxis should be consid-ered in Kayseri Province.

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

A. Yildirim1, A. Ica1, O. Atalay2, O. Duzlu1, A. Inci11Erciyes University, Faculty of Veterinary Medicine, Department of Parasitology, Kayseri,Turkey; 2Erciyes University, Faculty of Veterinary Medicine, Department of Internal Medicine,Kayseri, Turkey

Free Communications

Collaborative studies betweenMarshall Hertig and Samuel Wolbach,on the presence and identification ofmicroorganisms in arthropods, resultedin the discovery of Wolbachia in Culexpipientis in 1924 and description of thegenus Wolbachia and the species pipi-entis in 1936. Subsequent studies havedemonstrated that Wolbachia is wide-spread in arthropods, infecting about25-70% of species of insects, and isnow known as a remarkable geneticmanipulator of the infected arthropodhosts. Application of electron micro-scopy to elucidate the structure ofnematodes revealed that many filariae(17 species reported to date, includingDirofilaria immitis and most of thehuman-infecting species) harbor transo-varially–transmitted bacterial endosym-bionts, determined by Sironi et al. asbelonging to the genus Wolbachia. Wehave examined Wolbachia of D. immi-tis in all stages of this parasite: in micro-filariae, larvae developing in the mos-quito and in the vertebrate host, andmale and female adult worms by histo-logical techniques and by electronmicroscopy, comparing it to theWolbachia of other filariae. Wolbachiain D. immitis is polymorphic and can bedemonstrated by standard histologicalstains such as Giemsa’s, Gimenez,Pinkerton’s, Machiavello’s and Warthin-

Starry. Its life cycle is complex andmay consist of two reproductivemodes: by binary fission and by aChlamydia-like cycle. The latter mech-anism offers a potential survival strat-egy by producing more progeny thanmultiplication by binary fission, andappears to be more active duringgrowth embryogenesis and develop-ment of the larvae. Wolbachia ofD. immits appears to be slightly largerthat Wolbachia of other filarids, oftenoccurring in great numbers in oocytes.

The Wolbachia are apparently mutu-alistic endosymbionts required for sur-vival of their hosts and embryogenesisof microfilariae, are present in all lar-val stages during the life cycle of filar-iae, and contribute to some of theinflammatory responses and patholog-ical manifestations of filarial infec-tions in the vertebrate hosts. Presenceof Wolbachia endosymbionts inD. immitis complicates our under-standing of dirofilariasis in dogs, butalso offers possible advantages in treat-ment and control of this infection.

Susceptibility of Wolbachia of filari-ae to certain antibiotics offers anattractive possibility of treatment andcontrol of dirofilariasis. Detection ofwolbachial antigens provides the pos-sibility for early detection of D. immi-tis infections in animals and in man.

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Wolbachia of Dirofilaria immitis: an historical perspective and morphological characteristics

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

Free Communications

Recently sequenced genomes ofWolbachia pipientis from mosquitoesand Wolbachia from Brugia malayihave opened a new chapter in thestudies on Wolbachia and could pro-vide the means to fully characterizethe structure, composition and eluci-date the nature of these organisms.

Further studies on Wolbachia ofD. immitis, will undoubtedly reveal

additional characteristics that can beapplied towards treatment and controlof this filarial infection.

Acknowledgements

Supported, in part, by University of Puerto Rico(Medical Sciences Campus) RCMI Award RR-03051 from the NCRR, NIH, Bethesda, MD,U.S.A.

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Free Communications

In this case study, ultrasonography-guided heartworm removal was carriedout using flexible basket forceps in anAnatolian Shepherd dog (Sivas KangalDog). The dog was anaesthetized byintramuscularly injection of 2mg/kg/BWXylazine HCL (Rompun®, Bayer) and10 mg/kg/BW ketamine HCL(Ketalar®, Eczacibasi) combination fol-lowing the subcutaneous administra-tion of 0.02 mg/kg/body weightatropine sulphate (Atropin®, Vetas).

The dog was positioned on the leftlateral position. The region of rightjugular vein was prepared for asepticsurgery. And also the right 5th-7thintercostal spaces were shaved and cou-pling gel applied for ultrasonographicexamination. Following the cut down ofright jugular vein, the flexible basketforceps was inserted in to the jugularvein. With the guidance of ultrasonog-

raphy, heartworm removal was per-formed. Insertion of the forceps wasrepeated to remove heartworms asmuch as possible. A total of 17 adultheartworms could be removed. One daylater, the dog died because of poor clin-ical condition. Immediately, necropsywas performed and 34 heartwormsremaining in the right heart and pul-monary artery were collected. The ratioof removed worms via flexible basketforceps was 33.3%.

The low rate of this ratio may be dueto the lack of our experience, becausethis is the first attempt for us and forTurkey.

It was concluded that it is possible toremove adult heartworms from rightheart via flexible forceps with the guid-ance of USG. However, the higherexperienced surgeons could give betterresults.

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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 MenderesUniversity, Aydın, Turkey

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impaginato spostato 2 - Parassitologia veterinaria · Romania, Serbia, and Turkey. We thank the leading European experts and colleagues from the board of the ... Dirofilaria infections