Dirofilaria infections in animals and

humans

Claudio Genchi, Med Vet, PhD, EVPC Dipl

Dept of Veterinary Science and Public Health, Università

degli Studi of Milano

Bucharest, June 14th, 2013

Biology and epidemiology of Dirofilaria infections throughout

Europe and risk for public health

Pathogenesis and clinical presentation: Dirofilaria immitis

versus Dirofilaria repens

New insights in the prevention and treatment of canine and

feline dirofilarial infections

Guidelines for the diagnosis of Dirofilaria infection in dogs and

cats

Dirofilaria and D. immitis, the cruel worm of the dog: 400

years of history

Francesco Birago, a nobleman living in Lomellina (Lombardy, Italy), in his Trattato Cinegetico ouero della Caccia(1626) made the first observation of heartworms during necropsy of an hunting dog and he wrote:

The dog suffers of one more disease, which is not possible to be cured …. Two worms develop in the kidney. They are long as an half harm (Dioctophyma renale) wide as a finger and red as the fire … they move and migrate to the heart (Dirofilaria immitis)…

In 1850, Joseph Leidy, a physician and naturalist from Philadelphia, described 3 worms found in the heart of a dog as Filaria canis cordis

In 1856, the same author redescribing the parasite, used for the first time the name of Filaria immitis

The genus Dirofilaria, from the Latin, dirus (bad, cruel) and filum (thin, filiform), was erected in 1911 by Raillet and Henry in 1911, two parasitologists at the the School of Veterinary Medicine of Alfort, Paris.

In Europe, 5 filarial species has been reported in dogs and cats:

Dirofilaria immitis Leidy, 1856, Dirofilaria repens Raillet and Henry,

1911, Dipetalonema dracunculoides Cobbold, 1870,

Acanthocheilonema (syn. Dipetalonema) reconditum Grassi, 1890

(but a complete description of developmental stages in the fleas

was done by Calandruccio, 1892), Cercopithifilaria (syn.

Acanthocheilonema) grassii Noé, 1907.

Filarial parasite VectorsPrepatent

period

Length of adult

wormsLocation of

adult worms

Dirofilaria immitisMosquitoes

(Culicidae)120-180 days

M: 12-18 cm

F: 25-30

cm

Pulmonary arteries/right

heart

Dirofilaria repensMosquitoes

(Culicidae)189-259 days

M: 5-7 cm

F: 10-17

cm

Subcutaneous

tissue/muscular

fasciae

Acanthocheilonema

(formerly Dipetalonema)

reconditum

Flies and ticks 427-476 days

M: 9-17 mm

F: 21-25

mm

Subcutaneous

tissue/muscular

fasciae,

peritoneal cavity,

kidney

Acanthocheilonema

(formerly Dipetalonema)

dracunculoides

Fleas and ticks (R.

sanguineus)120 days

M: 15-31 mm

F: 33-55

mm

Peritoneal cavity

Cercopithifilaria spp.Ticks (R.

sanguineus)?

M: unknown

F: 23-24

mm

Subcutaneous

tissue/muscular

fasciae

www.ESCCAP.org Guideline 6 VBD

Dirofilaria diseases are vector-borne parasitic infections

mainly of dogs and cats (and wild carnivores) that in

Europe are caused by Dirofilaria immitis and D. repens

and D. immitis only in the Americas.

D. immitis, the cruel worm of dogs, is the causative agent

of canine and feline heartworm disease.

D. repens, is the causative agent of subcutaneous

infections and its interest is mainly due to the high

zoonotic potential.

Distribution of Dirofilaria infection

The life cycle of both parasites consists of 5 larval stages developing

both within an intermediate mosquito host [from embryo - microfilaria

- to infective L3), that also acts as vector, and in a definitive

vertebrate host [from L3 to the adult worms].

Adult D. immitis worms occur in the pulmonary arteries and right

heart chambers, causing a severe condition known as canine and

feline heartworm disease, while D. repens is found mainly in

subcutaneous tissues (subcutaneous dirofilariosis). Adult parasites

released microfilariae in the blood stream where they are picked up

by mosquitoes during their blood meal.

The development from L3 to adult stages lasts 6-8 months.

Mosquito becomes infected taking blood from a microfilaraemic

host. Mf remain in the mosquito midgut for approximately 24 h,

then larvae become shorter and stouter (‘sausage’ stage). Larvae

molt to the second stage 8 -10 days after infection and again to

the third stage (L3) 2 - 3 days later. L3 perforate the distal ends

of the Malpighian tubules and migrate to the head and

mouthparts where they become infective (1100–1300 µm long).

The time required for the development of microfilariae to the

infective, third stage is temperature-dependent. At 27 °C and

80% relative humidity, development takes about 14 days.

Biology

Infective L3 are transmitted to the definitive host while the mosquito is

taking a blood meal: the tip of the labellum (fleshy part of the

mouthparts) ruptures and the L3 are deposited on the skin in a drop of

the mosquito’s haemolymph. Three days after infection, most of the

larvae are found in the subcutaneous tissues near their entry site. By

day 21, most of them have migrated to the abdomen of the dog,

The molt from L3 to L4 begins as early as day 3 days and as late as

day 9 - 12. L4 molt to the final stage [L5] at day 50 - 70; the first

worms entering the pulmonary artery on day 70 - 85 are 2 - 4 cm in

length. They become sexually mature (25-30 cm length) about day

120 post-infection. Dogs develop patent infections (circulating mf) as

early as 6 months, but usually by 7 - 9 months post-infection.

Biology

When juvenile heartworms first reach the lungs and the heart,

the pressure of venous blood forces them into the small

pulmonary arteries. As they increase in size, they migrate

upstream into larger arteries until the worms become fully

mature. The location of the adult worms depends on the size

of the dog and the worm burden.

A medium-sized dog with a low worm burden (i.e., 10) usually

has worms mainly in the lobar arteries and main pulmonary

artery. As the worm burden increases, worms are also located

in the right ventricle. Dogs with more than 40 worms are likely

to have caval syndrome, and most of worms migrate into the

right ventricle, right atrium and the caudal vena cava, thus

interfering with valvular function and/or blood flow.

Biology

Dog: aduld worms are able to survive until 7 years

mf: until 12-18 months

usually infected both by male and female worms

Cat: in most cases adult worms are able to survive 1½

year, but they can survive in asyptomatic cats until 4

years.

Most cats are amicrofilaraemic, when present, mf

can survive about 2 months

frequently infected by male or female worms only,

most infection abort (no adult worms in about 70% of

cats)

Biology

• Change in environmental ecology (global warming)

• Change in parasite ecology

• Decrease in natural resistance

• Economy

• Changed habit of human population

Factors affecting the spreading of parasitic

infections

The global average air temperature rose

0.74°± 0.18°C during the last 100 years

GLOBAL WARMING

Indeed, global climate change

will affect disease vector

behaviour, which in turn may

alter the current patterns of

vector-borne diseases

transmitted by the bite of

haematophagous arthropods

(Rogers and Randolph, 2006).

Arthropod vectors are cold-

blooded animals, meaning that

their internal temperature is

greatly affected by the

temperature of their

environment (Purse et al,

2005).

Factors for the spreading and establishment of infections

in pets

Furthermore, great concern has arisen following the introduction of less restrictions in the movement of pets throughout European countries, which can increase the risk of spreading of arthropod-borne diseases, such as filarial infections

In Practice 1999, 21: 482-491

Distibution of D. immitis in Italy: 1986-1988Genchi et al., 1988. Atti IV Seminario SCIVAC

Since the first

epidemiological studies,

north Italy, mainly the Po

River Valley, has been

shown the most endemic

area for canine HW

disease in Europe while

D. repens was distributed

mainly in central and

south regions of the

country

Starating and ending period of Dirofilaria transmisson risk in 74 Italian 1

provinces calculated for the decades 1980-1989, 1990-1999 and 2000-2012. 2

3

Decades

1980-1989

1990-1999

2000-2012

Transmission risk Number of weeks Number of days

strating-ending 16 Apr - 8 Oct

25

175

strating-ending 16 Apr - 29 Oct

28

196

strating-ending 2 Apr – 6 Nov

31

217

4

From south to north? From west to east?

Austria Germany Switzerland Serbia Albania Bulgaria Ucraine Czech Republic Hungary

Hinaidy et al., J. Vet. Med., B 1987 34: 326-332 Reuther, Wschr Thierheilk 1888; 32: 429-430 Wenzel and Fallbericht, Kleintierpraxis 1898 34: 27-28 Leuterer and Gothe, Kleintierpraxis 1993 38: 633-646 Wohlsein and Brandes, Kleintierpraxis 1996 41: 367-374 Zahller et al., Tierärztl Prax 1997 25: 388-392. Arnold et al., Schweiz Arch Tierheilk 1994 136: 265-269 Deplazes et al., Schweiz Arch Tierheilk 1995 137:172-179 Bucklar et al., Schweiz Arch Tierheilk 1998 10 Tasić et al., Parasitol Res 2008 103: 1297-1302 Rapti and Rehbein, Parasitol Res 2010 107: 481-485 Georgieva et al., Bulgarian J Vet Med 1999 2: 121-124 Georgieva et al., Bulgarian J Vet Med 2001 4: 231-236 Mazurkevich et al., Vet Rec 2004 155: 638-639 Svobodova and Mišoňova, Vet Paasitol 2005 128: 137-140 Boros et al., Magyar Allatorvosok Lapja 1982 121 : 313-316 Zahler et al., Tierztlic. Praxis, 1997 25: 388-392 Szell et al., Magyar Allatorvosok Lapja 1999 121 : 100-104 Kleiter et al., Kleintierpraxis 2001 46: 283-288 Farkas, Helmithological Colloquium, 14th Nov, Vienna 2003

Country

Year

Prevalence (%)

No. of cases

Reference

Switzerland

1998

1.6% 8/479

Bucklar et al., Schweiz Arch Tierheilk 1998 140:255-260

Germany June 1993 - May 1996

72 Zahller et al., Tierärztl Prax 1997 25: 388-392

The Netherlands 1992-1993 1982-1992

7 9

Meyer et al., Vet. Quartely 1994 16: 169-174

Tourism-associated canine Dirofilaria infection in northern

Europe

Until recently, interest in Dirofilaria infections has been

focused primarily on D. immitis because of its

pathogenicity in companion animals.

However, the increasing spread of D. repens towards

eastern and northern Europe has renewed the interest for

this species. Furthermore, human infections caused by D.

repens are dramatically increasing in Europe.

To note …

Main factors allow the spread of Dirofilaria infection around

the world including socio/economic factors conditioning

the abundance and movement animal reservoirs able

to acts as donors of microfilariae to mosquitoes throughout

a blood meal and mosquito species and abundance,

however temperature plays a key role:

environmental temperature maintains/increases the

abundance of competent mosquitoes

temperature is critical for the development of mf and for

the duration of infection transmission season to new hosts

Worming and introduction

of exotic mosquito

species in Italy as a

consequence of trade

exchanges

1990: Aedes albopictus

1996: Aedes atroparvus

2011: Aedes koreicus

Distribution

D. immitis

D. repens

Anophelinae Anopheles claviger*

N, S, Si, Sa

-

-

Anopheles maculipennis N, S, Si mol/field mol/field

Culicinae Aedes albopictus

N, S, Sa

mol/field

mol/filed

Aedes cantans* N - - Aedes caspius* N, S, Si, Sa - - Aedes cinereus N mol/abd - Aedes geniculatus N, S, Si, Sa mol/abd - Aedes detritus N, S, Si, Sa mol/abd - Aedes punctor N, S mol/abd - Aedes vexans* N, S, Si, Sa - - Coquillettidia richiardii* N, S, Si, Sa mol/field - Culiseta annulata* N, S, Si, Sa - - Culex modestus N, S, Si, Sa mol/abd - Culex pipiens N, S, Si, Sa mol/field mol/field Culex torrentium N mol/abd - Culex territans* N, S - -

Proven or suspected Culicidae vectors of D. immitis and D. repens

in Italy

(North: N; South: S; Sicily: Si; Sardinia: Sa); studies by dissection or PCR of field collected samples

(mol/field). PCR positive abdomens (mol/abd) do not proving vector competence are also reported.

Central Italy: mosquitoes CO2 trapped and PCR for

Dirofilaria worms [2002-2003]

Tested

PCR positive

Mosquitoes

Individuals

Pools

head-torax

abdomen

%

Cx. pipiens Cx. hirritans Ae. albopictus Ae. caspius Ae. cinereus Cs. annulata

1,108

1 436 29 1 1

412

31 0 43 0 0 0

32 0 24

0 0 0

0.54

1.37

1 two D. repens, one D. immitis

2 one D. repens, two D. immitis

3 two D. repens, one D. immitis and D. repens

4 one D. repens and D. immitis

Cancrini et al, 2007

Aedes albopictus: introduced in Italy in 1991 and proved Dirofilaria vector in 2003

Cx. pipiens: 1 two D. repens and one D. immitis, 2 one D. repens and two D. immitis

Ae. albopictus: 3 two D. repens, one D. immitis; 4 one D. repens and one D. immitis

The rate of Dirofilaria maturation to infective third-

stage larvae (L3) in the mosquito vector(s)

depends mainly on the environmental

temperature, and there is a threshold of about

14°C below which development will not proceed.

Temperature dictates the development of

Dirofilaria larvae in the intermediate hosts

Temperature

Dirofilaria immitis

Dirofilaria repens

in Aedes spp. 28-30 °C 26 °C 22 °C 18 °C

8-9 days 10-14 days

17 days 29 days

8-10 days 10-11 days 16-20 days ~ 28 days

in Ae. albopictus 26 °C

14-18 days

16-18 days

Time requirements for Dirofilaria mf development to infective stage

at different temperatures

Temperature dictates the seasonal occurrence

of heartworm transmission in temperate

latitudes

The total environment heat required for

development may be expressed in terms of

degrees days in excess of this threshold

[Dirofilaria Development Units – DDUs]

The seasonal Dirofilaria transmission model

assumes a requirement of 130 Heartworm DUs for

larvae to reach infectivity and a maximum life

expectancy of 30 days for a vector mosquito (Slocombe et al., 1989; Lok and Knight, 1998)

Rationale

•A program was developed in order to calculate the

DDUs for 2556 European Meteorological Stations in

Europe

• Threshold value of 130 cumulative DDUs was

accepted only if it was reached in 30 consecutive days.

•Data interpolated utilizing the Linear Kriging of GIS

Model: Europe

1971-2000: 854 examined meteorological stations

Meteorological stations reaching the 130DDUs based on

the average temperature of the 15 studied years

May: 112 June: 356 July: 548

August: 550 September: 299 October: 65

Monthly average

predicted number of

HW generation

obtained by Linear

Kriging interpolation

May - November

ESCCAP Guideline 5, 2012

Control of vector-borne diseases

in dogs and cats

As for HW infection, D.

repens until recently was

mainly present in

northern Italy and

southern of France.

From 2000, the infection

was more and more

often diagnosed in

northern-eastern

countries.

Currently subcutaneous D. repens infection is

diagnosed in Italy, France, Spain, Switzerland, Austria,

Germany, The Netherlands, Poland, Czech Republic,

Hungary, Croatia, Slovakia, Serbia, Romania, Greece,

Russia

Interestingly, in several countries the spreading of

infection was firstly reported through human cases

Pantchev et al: Diagnosis of imported

canine filarial infections in Germany 2008-

2011. Pasitol Res 109 S61-S67 2011

Host

D. immitis

D. repens

A. reconditum

Reference

Dog [Vojvodina]

7.2%

49.2%

2.1%

Tasić et al 2008

Humans

>29 cases; most

ocular and periocular

Kranijčić-Zec et al 1998 Džamić et al 2004 Džamić et al 2009 Tasić et al 2011

Serbia

5.1%

38.3%

18.5%

2%

D. immitis

D. repens

Location

Reference

35% 23%

presence 3.5%

3.5% (1) 7%

0-31% (2)

presence

n.d. n.d. 81%

presence (1) 17% n.d.

whole country

Bucharest area whole country Timiş County whole country

south-western areas whole country

Olteanu et al, 1996 Coman et al, 2007 Fernoagă et al, 2010 Ciocan et al, 2012 Hammer et al, 2012 Ilie et al., 2012 Micean et al, 2012

(1) Dogs imported from Romania to Germany (2) Depending on the region

Prevalence of Dirofilaria infections in Romania

Geographic distribution of D. immitis in Romania [Ag Elisa]

Mircean et al, 2012 Vector-Borne Zoon Dis 12: 595-604

Humans are susceptible hosts for dirofilarial worms

[accidental hosts]

Usually the worm does not develop into adult stage and

humans are not microfilaremic, although at four cases of

microfilaraemic zoonotic infections have been reported

in Europe.

The severity of the infection depends on the localization

of the parasite

Though the first case of D. immitis human infection has

been found in Italy (histology and PCR), D. repens is the

most frequent cause of zoonotic infection in Europe and

Asia (more than 1500 cases vs 300-350 cases of HW

human infections in USA and Japan)

Human Dirofilaria repens infection in Europe: from

emergency to endemicity?

1900 - 1994: 267 cases 56/year

1995 - 1999: 264 cases 53/year

2000 - 2013: 1782 cases 137/year

Country

No.

cases

Ocular

Pulmonary

Other unusual and seriousness localization

France Greece Hungary Italy Ukraine Poland Russia (Siberia included) Turkey Other countries²

81 36 31

324 932 18

109¹

22 229

22 7

19 62 18

44

12 120

2 1

23 2

intradural tumor like scrotum abdominal cavity spermatic cord, spermatic duct, scrotum intravitreal, 7 intra-oral cases in Sri Lanka, 16 scrotal, 2 spermatic cord, 1 testis, 1 epididymis, 1 associated with meningoencephalitis (surgery in Germany)

Total

1782

305

27

¹ Sergiev et al [65] reported of worms from 140 individuals identified as D. repens; such a

figure has not been added to Russian cases because of incomplete description.

² Albania, Bulgaria, Croatia, Dubai, Former Yugoslavia, Georgia, Kazakhstan, Kenya, India,

Iran, Israel, Japan, Malaysia, Poland, Romania, Serbia and Montenegro, Slovenia, Spain, Sri

Lanka (more than 132 cases), Tunisia, Turkmenistan, Uzbekistan, USA

Human D. repens infections published from 1900-2013

Subcutaneous nodules

Deep localizations intraperitoneal in lungs mimicking

tumors

Ocular localization with impaired vision and floater-

like mobile shadow seem to be the most frequent

symptoms, but infection is seldom accompanied by

loss of vision or serious ocular complications;

intravitreal ocular infection is quite rare but possible

The severity of the infection depends on the

localization of the parasite

Human infection

Gherman 1967 (pers comm): hand dorsum

Olteanu 1997: many human cases, probably D. repens

Panaitescu et al, 2000: no data

Mănescu et al, 2009: a subcongiuntival nodule caused by D. repens

Popescu et al, 2012: unusual and severe clinical manifestations that

mimicked pathological conditions like cellulitis or deep venous

thrombosis. The diagnosis was performed by histology and PCR.

Romania

Typical features of D. repens are the external longitudinal ridges, the thick

cuticula, and the well-developed musculature. Around the parasite a

marked inflammatory reaction with numerous eosinophils, neutrophils,

lymphocytes, plasma cells, and macrophages. Many IgE-positive plasma

cells are present in the infiltration and near to the surface of the parasite

… and recently the first case of Dirofilaria autochtonous

infection in has been diagnosed in Poland

Most studies have reported D. repens in animals

mainly from north-eastern European countries (i.e.:

north eastern Austria, Germany, The Netherlands,

Hungary, Poland) and even when D. immitis is

present, D. repens shows higher prevalences (i.e.:

northern Serbia and Hungary).

Human D. repens infections showed the same trend:

the more recent and severe cases have been reported

from north eastern countries such as Hungary, Slovak

Republic, Poland, Russia and Romania.

Why D. repens is spreading more rapidly than D.

immitis?

Intermediate hosts competence? D. repens microfilariae are more

easily destroyed by the bucco-pharyngeal armature of some

mosquito species (e.g.: Culex pipiens). Very few mf can develop to

infective stage. In such a way, both the mosquito fitness and their

ability to transmit the infection are not affected.

The spreading throughout Europe of Ae. albopictus. This species

has shown to be infected by Dirofilaria larvae 2.5 times more than

C. pipens, which is a proven intermediate hosts for dirofilarial

worms.

D. repens has been found, when first established in the host, to be

able to stimulate a specific immune response able to reduce the

ability of D. immitis to develop into adult worms.

Some hypothesis

Most D. repens infections in dogs are asymptomatic while

HW infections usually cause severe clinical disease. It is

thus likely that dogs who travelled to endemic areas of

southern Europe become infected and when they return to

northern areas, having no apparent symptoms, act as

donors of microfilariae to local mosquito populations. On the

contrary, dogs with heartworm infection are usually referred

to veterinary clinics and cured. If such an hypothesis is

confirmed, considering that an increasing number of dogs

travel for holidays or relocation and that pet travel is now

facilitated by the new schemes in many European countries,

subcutaneous dirofilarial infection could continue its spread.

Some hypothesis

Dirofilaria immitis

Severe clinical signs

Mf in the blood by Knott test

Ag serology in dogs and Ab in

cats by Elisa

Very effective preventative drugs

Adulticide tharapy

Apparently uncommon

zoonotic infections in Europe

Dirofilaria repens

Mostly no clinical signs

Mf in the blood by Knott test

No serology

Only moxidectin has been confirmed as an effective preventative

No adulticide therapy

Most common agent of human

dirofilariosis in Europe