Subcutaneous filariasis (Dirofilaria repens)

Dirofilaria repens (from the Latin dirus = nasty, filum = thread, repens = sudden) is a non-bursate nematode responsible for cutaneous filariasis in the dog (and in other definitive canid hosts of the parasite), a disease transmitted by haematophagous diptera: mosquitoes (there are more than 70 Culicidae, mostly of the genera Aedes, Culex, and Anopheles, recognised as the main biological vectors of both Dirofilaria immitis and D. repens). The adult parasites have a thread-like body of whitish colour with transverse and longitudinal cuticular striae. The cephalic extremity in both sexes is tapered, the buccal opening is small, circular and without lips and cephalic papillae are present. The male is 5-7 cm long while the female is 6-17 cm long (Figs. 1 and 2).

SPREAD AND BIOLOGICAL LIFE CYCLE

The spread of D. repensis increasing continuously in various areas of southern and central Europe, Africa and Asia. At present the USA is considered to be uninvolved.

The biological life-cycle of D. repens, like that of D. immitis, consists of five stages of development which involve a vertebrate host (domestic and wild canids) and an arthropod (the mosquito) as an intermediate host. The adult female lives subcutaneously and releases thousands of first stage larvae (L1), also called microfilariae, into the blood stream; the intermediate host ingests these larvae during a blood meal. The larvae reach the Malpighian tubules where they mutate into second stage larvae (L2) and then into third stage larvae (L3) before migrating through the thorax into the cephalic spaces and then to the labium of the insect.

During a subsequent meal, the infected mosquito deposits the third stage larvae on the skin in a drop of haemolymph; when the mosquito pierces the skin, the larvae migrate actively into the host through the breach and, within the vertebrate host, continue to develop until the adult stage. The prepatency period (the period between infestation by the L3 larvae from the mosquito and sexual maturity of the parasite) lasts from 6.5 months to 9 months.

The adults of D. repens live in the subcutaneous tissue of dogs and cats and do not normally cause any symptoms except in sporadic cases in which subcutaneous nodules, itchy skin and subcutaneous oedema occur. Most of the infestations are asymptomatic and diagnosed incidentally during post-mortem examinations or surgical operations.

DIAGNOSIS

The diagnosis is based on the finding of microfilariae in the peripheral blood. As for D. immitis, microscopic examination of thick films (Video 1) does not allow the species of parasite to be determined and, furthermore, is less sensitive than Knott’s test, which is the in vivo “gold standard” for the diagnosis of this parasitosis. Antigen tests for D. repens are currently not available and the tests that identify antigens of the adult parasites of D. immitis do not give cross reactions with the antigens of D. repens and are not, therefore, of use in the diagnosis of this latter.

The microfilariae of D. repens are easily distinguishable from other microfilariae that can be found in dogs in Italy (Table 1) (Figs. 3, 4 and 5). They are larger (length: 350-385 µm) with a rounded cephalic extremity whose transverse diameter is the same as that of the middle third of the body of the larva. The caudal extremity is typically hooked or shaped like an umbrella handle. This last feature is not, however, easily identified in all the larvae because the caudal extremity must be perpendicular and not parallel to the plane of vision and in most cases can only be seen by changing the focus of the microscope.

Table 1. Morphological characteristics of the first stage larvae (L1) of D. repens, D. immitis, and Dipetalonema reconditum.

In doubtful cases, the microfilariae can be concentrated and analysed after histochemical staining (acid phosphatase method, Table 2) or using molecular techniques (polymerase chain reaction).

Table 2. Characteristics of the microfilariae of D. repens, D. immitis and Dipetalonema reconditum after histochemical staining (acid phosphatase method).

The most common clinical manifestation of D. repens infestation in the dog is a nodule (Figs. 6 and 7) whereas the sporadically reported cases in cats have always been asymptomatic. The nodule varies in diameter from 1 to 3 cm and is not painful; however, following palpation or stimulation it may transiently increase in size with oedema (Darier’s sign). If this sign is present, cutaneous or subcutaneous neoformations, in particular mast cell tumour, must be included in the differential diagnosis.

Cytological examination of material collected by fine needle aspirate or biopsy and ultrasound examination of the nodule enable a definitive diagnosis to be made. Cytologically, there is always a mixed neutrophilic/eosinophilic inflammatory picture, often associated with the presence of microfilariae (Fig. 8).

Other findings that are almost always made when fragments of female parasite are sampled are oval structures of about 25-40 µm with a pale blue cytoplasm and 15-25 round nuclei: these structures are morulae, the pre-larval stage of the parasite (Fig. 9).

At ultrasonography (Figs. 10 and 11, Videos 2 and 3), carried out with a linear probe at high frequency (at least 10 mHz) the parasite within the nodule appears as two parallel, hyperechoic lines, resembling the adults of D. immitis within the pulmonary arterial circulation. However, in contrast to these latter, the parasites of D. repens may seem less distinct, because of the weaker echo-contrast resulting from the modest amount of fluid within the nodule.

TREATMENT

There are currently no registered adulticide treatments for D. repens and the off-label use of melarsomine has been reported in only a single case in which it was used, as an adulticide, in combination with doramectin as a microfilaricide: this combination treatment was successful in eliminating a D. repens infection in a dog.

Symptomatic treatment of dirofilariasis caused by D. repens is indicated only in those cases in which there are clinical signs of the infestation such as exudate, pruritus and subcutaneous nodules. In this case, the administration of corticosteroids and antibiotics may be useful as an alternative or in association with surgical removal of the parasite.

An easily performed mini-invasive procedure can be used to remove the parasite from within a nodule. The procedure, which does not require either sedation or local anaesthesia, involves a 5 ml syringe connected to a 19 gauge “butterfly” needle (Figs. 12 and 13, Video 4).

The needle is fixed firmly within the nodule and moved slowly while exerting a negative pressure with the syringe until a white or pink fluid is seen within the butterfly needle. The negative pressure is maintained during extraction of the needle in order to enable complete removal of the undamaged parasite. A subsequent ultrasound control can be useful to confirm the complete removal of the parasites from the nodule.

PROPHYLAXIS

Some macrocyclic lactones seem to be effective at preventing infection by D. repens in the dog and, based on studies carried out in the field, in some countries (including Italy) they have been authorised for use for this purpose.

Although there are no doubts that these drugs are able to prevent microfilaraemia in dogs (which has important zoonotic implications), there are currently no post-mortem studies demonstrating that these agents are able to prevent the infection completely and not only the presence of microfilariae in the peripheral circulation.

ANTHROPOZOONOTIC ASPECTS

D. repens is considered to be a not uncommon, indirectly transmitted anthropozoonosis. The geographical distribution of the infestation in humans is the same as that in dogs, with a seroprevalence exceeding 30% in areas in which dirofilariasis is endemic in dogs. In Italy (a country in which a high number of cases have been reported) the mosquito species Culex pipiens and Aedes albopictus (the so-called tiger mosquito) are important vectors in urban and populated areas, while other species of the genus Aedes and Anopheles maculipennis are important vectors in suburban and rural areas.

In infested humans, the microfilariae and macrofilariae induce the formation of nodules, particularly in subcutaneous tissue and the lungs. The conjunctiva is another common localisation (Fig. 15). The lesions are completely benign but lack specific clinical features that differentiate them from tumours, pneumonia and sebaceous cysts and are, therefore, difficult to diagnose unless a histopathological examination is performed (Fig. 14).

Subcutaneous nodules are sometimes tender to palpation and associated with itching or burning; pulmonary nodules (the lower lobe of the right lung is classically affected) are often associated with fever and cough. Microfilariae are not found in the blood of humans infested with Dirofilaria repens and so, in the absence of a biopsy or asportation of a lesion, the diagnosis can only be made through serological examinations (ELISA, western blot and indirect immunofluorescence) to demonstrate the antibodies produced against the parasites or the endosymbiont bacteria of the genus Wolbachia. The species can be typed through molecular analyses (polymerase chain reaction) of adult specimens removed surgically.

Suggested readings

1. Anderson RC. Nematode Parasites of Vertebrates. Their Development and Transmission. 2nd ed. Wallingford, UK: CABI Publishing; 2000:1–650.
2. Soulsby EJL. Helminths. In: Helminths, Arthropods and Protozoa of Domesticated Animals 7th ed. Baltimore, MD: Williams & Wilkins; 1982:311.
3. Venco L  Dirofilaria (Nochtiella )repens  infection in dogs and cats Mappe Parassitologiche Dirofilaria immitis and D. repens in dog and cat and human infections  Genchi C, Rinaldi L, Cringoli G Eds 2007 :133:136
4. Webber WAF, Hawking F, 1955. Experimental maintenance of Dirofilaria repens and D. immitis in dogs. Exp Parasitol 4: 143-164.
5. Cancrini G, Tassi P, Coluzzi M 1989 Ivermectin against larval stages of Dirofilaria repens Parassitologia 31, 177-182
6. Živičnjak T, Martinković F, Beck R, 2006. Dirofilariosis in Croatia: spread and public health impact. 5th Croatian congress on infective diseases, Zadar, Croatia
7. Baneth G, Volansky Z, Anug Y, Favia G, Bain O, Goldstein RE, Harrus S2002 Dirofilaria repens infection in a dog: diagnosis and treatment with melarsomine and doramectin Veterinary parasitology 105, 173-178
8. Pampiglione S, Canestri Trotti G, Rivasi E. 1995 Human dirofilariosis due to Dirofilaria (Nochtiella) repens : a review of world lieterature. Parassitologia 37, 149-193
9. Giori L, Garbagnoli V, Venco L, Genchi M, Bazzocchi C, Bertazzolo W What is your diagnosis? Fine needle aspirate from a subcutaneous mass in a dog. Mixed neutrophilic-eosinophilic inflammation with Dirofilaria fragments. Vet Clin Pathol. 2010 Jun;39(2):255-6
10. Genchi C, Poglayen G, Kramer LH 2002 Efficacia di Selamectin nella profilassi delle infestazioni da Dirofilaria repens nel cane.Veterinaria 16, 69-71
11. Rossi L, Ferroglio E , Agostini A 2002 Use of moxidectin tablets in the control of canine subcutaneous dirofilariosis. Veterinary records 15 383
12. Venco L, Calzolari D, Mazzocchi D, Morini S. The Use of Echocardiography as a Diagnostic tool for Detection of Feline Heartworm (Dirofilaria immitis) Infections. Fel Pract 26 : 6-9 1998
13. Casiraghi M, Bazzocchi C, Mortarino M, Ottina E, Genchi C. A simple molecular method for discriminating common filarial nematodes of dogs (Canis familiaris). Vet Parasitol. 2006;141:368–372.
14. Gioia G, Lecová L, Genchi M, Ferri E, Genchi C, Mortarino M. Highly sensitive multiplex PCR for simultaneous detection and discrimination of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood. Vet Parasitol.2010 Aug 27;172(1-2):160-3.
15. Genchi C, Venco L, Genchi M Guidelines for the laboratory diagnosis of canine and feline Dirofilaria  infections Mappe Parassitologiche Dirofilaria immitis and D. repens in dog and cat and human infections  Genchi C, Rinaldi L, Cringoli G Eds 2007:137-144