A Mini-Invasive Procedure for Removal of Adult Dirofilaria Repens from Subcutane

A Mini-Invasive Procedure for Removal of Adult Dirofilaria Repens From Subcutane- ous Nodules in Dogs Venco Luigia Valenti Valentinaa Bertazzolo Waltera Genchi Marcob Grandi Giulioc a Veterinary Hospital “Città di Pavia” b Faculty of Veterinary Medicine , Università degli Studi di Milano c Faculty of Veterinary Medicine , Università degli Studi di Parma

KEY WORDS: Dog, Dirofilaria repens, symptoms. Therapy INTRODUCTION ABSTRACT Dirofilaria repens is the causative agent of Dirofilaria repens is the causative agent canine and feline subcutaneous dirofilariosis, of canine and feline subcutaneous a mosquito-borne disease that has become Dirofilariosis, a mosquito-borne disease that increasingly recognized in several countries has been increasingly reported in several in southern and central Europe, Africa and countries in southern and central Europe, Asia. The D. repens life cycle consists Africa and Asia. The natural hosts of D. of 5 developmental or larval stages in a repens are domestic and wild carnivores vertebrate host and an arthropod (mosquito) and the adults of these nematodes reside in intermediate host and vector. Adult female the subcutaneous tissues of dogs and cats. worms produce thousands of first-stage Infection occasionally causes mild clinical larvae or microfilariae that are ingested by signs such as subcutaneous nodules, itching, a blood-feeding insect. Some microfilariae and dermal swelling, but most affected have a unique circadian periodicity in animals are asymptomatic. Here, the authors the peripheral circulation over a 24-hour report a novel, mini-invasive procedure for period. Mosquitoes also have a circadian removal of adult worms in four naturally- rhythm in which they obtain blood meals. infected dogs presenting skin nodules. The highest concentration of microfilariae Ultrasonographic (US) and cytological usually occurs when the local vector is most diagnostic features are also presented. actively feeding. Microfilariae then undergo Worms were removed using a 19 Gauge 2 developmental moults in the insect. needle, connected to a vacuum syringe. No During subsequent feeding, the infected adulticide therapy is currently available for mosquito deposits third-stage larvae in a D. repens and therefore surgical removal drop of hemolymph in the proximity of of worms could represent a non-invasive the bite wound. From here, larvae actively treatment leading to definitive cure of migrate to the subcutaneous tissue. Larvae Intern J Appl Res Vet Med • Vol. 9, No. 2, 2011. 217 develop to the adult stage and prepatency microfilariae, ultrasonography and cytology lasts for six to nine months1,2,3,4,5. The adults may allow a diagnosis if nodules are of D. repens reside in the subcutaneous present9. tissues of dogs and cats and usually do To note that currently available tests to not cause any symptoms. Occasionally, detect adult heartworm (D immitis) antigens dermal swelling and subcutaneous nodules do not detect D. repens antigens and no may be observed3,6,7. Humans may act as cross reactivity has been described. Removal accidental and dead-end hosts of D. repens of the nodule and histo-pathological and there is currently rising concern over the identification of the parasite may be time- increase of reported zoonotic dirofilariosis consuming and costly. There is currently no in Europe. In most cases, the parasite is registered adulticide treatment for D. repens. not able to develop to the adult, sexually- The off-label use of the arsenic adulticide mature stage and infection is characterized melarsomine, combined with doramectin, a by the presence of pre-adult stages located macrocyclic lactone with microfilariacidal in subcutaneous nodules of different body activity, was recently described as effective areas, near the point of the mosquito vector in clearing D. repens infection in a dog7 bite. Ocular and pulmonary localization For those dogs suffering from clinical of the parasite has also been reported8. signs of this disease, such as dermal Subcutaneous or deep tissue localization swelling, sub-cutaneous nodules and of the parasitic nodule usually leads to a pruritus, symptomatic therapy with steroids clinical suspicion of neoplasia and requires and/or antibiotics may be indicated, biopsy or more invasive surgery for together with surgical removal of nodules3. differential diagnosis through histology and Several macrocyclic lactones (ivermectin, morphologic identification of the parasite, moxidectin, selamectin) have been reported thus causing emotional distress and costly as effective for the prevention of D. repens intervention8. In infected dogs and cats, infection in dogs10,11. However, while there diagnosis of infection is usually made by is no doubt that these drugs are able to clear detection of circulating microfilaria by microfilariemia in infected dogs (important the modified Knott’s test. Differentiation for breaking the transmission cycle and thus between D. immitis and D. repens larval for zoonotic implications), most field studies stages is made on the basis of morphological were not based on necropsy confirmation features, by histochemical methods or of infection status and concern still remains by PCR. In the absence of circulating about the ability of these compounds to completely prevent infection5. Here, the Figure 1.Subcutaneous nodules caused by Dirofilaria repens infection in the treated dogs Figure 2 Scrotal nodule and swelling in one (white circles) of the treated dogs

218 Vol. 9, No. 2, 2011 • Intern J Appl Res Vet Med. Table 1. Four dogs affected by subcutaneous dirofilariosis (D. repens) Breed Age Gender # Nodules Boxer 7 years Female 4 Italian Hunting Dog 5 years Male 3 Mixed breed 5 years Male 6 Petite Bleu da Gascogne 7 years Male 1

Table 2. Parasitological analyses of affected dogs.

Dog Worms Microfilaremia Boxer 7 D.repens + / D.immitis + (2 male, 5 female) Italian Hunting Dog 5 D.repens + / D.immitis + (1 male, 4 female) Mixed breed 6 D.repens - / D.immitis + (3 male, 3 female) Petite Bleu de Gascogne 1 D.repens - / D.immitis - (male) authors report a novel, mini-invasive parasites. A 5 ml syringe connected to a 19 procedure for removal of adult worms in Gauge Butterfly needle was used (Figure 2). four naturally-infected dogs presenting skin The needle was firmly inserted within the nodules. nodule and gently moved while negative MATERIALS AND METHODS pressure was applied to the syringe until a Four dogs from the province of Pavia (Po very small amount of white or pink fluid River valley, Northern Italy) presented with could be noted (Figure 3). The negative multiple subcutaneous nodules ranging in pressure was maintained during needle diameter from 1 to 2 cm. The number of skin withdrawal allowing removal of the intact nodules ranged from 1-6 (Table 1, Fig. 1a adult worm (Figure 4, 5, 6). Ultrasound and 1b). Only one on them was currently on control was performed in order to confirm chemoprophylaxis with ivermectin (dosage that all the worms in the nodule were 9 μg , oral route, once a month). removed. Fine needle aspirates were carried out Worms underwent identification based on all nodules in all dogs and smears were on morphological features (Figure 7a-7b), stained with a rapid, modified Romanowski which was subsequently confirmed by PCR 13,14,15. No (Diff-Quick®). Ultrasonographic analysis, as previously described examination of skin nodules was performed sedation or local anaesthesia treatment was by using a linear 12 mHz probe. performed. A modified Knotts’ test to detect RESULTS circulating microfilaria and discriminate Results of parasitological analyses are morphological features was performed in summarized in Table 2. In two of the dogs, each dog, as previously described15. a mixed infection with both D. immitis Following diagnosis of subcutaneous and D. repens microfilariae was found, dirofilariosis by D. repens, a mini-invasive only D. immitis larvae were present in the procedure was carried out to remove adult peripheral blood in one case while in the dog

Intern J Appl Res Vet Med • Vol. 9, No. 2, 2011. 219 Figure 4. The needle is firmly inserted within Figure 3. A 5 ml syringe connected to a 19 the nodule and gently moved while negative Gauge Butterfly needle used for adult Dirofi- pressure is applied to the syringe until a very laria repens removal small amount of white or pink fluid can be noted

Figure 5. The negative pressure is main- tained during needle withdrawal allowing to Figure 6. The adult worm is removed intact start the intact adult worm removal

Figure 7. Adult male worm of D.repens re- Figure 8. Female adult mature worm of Diro- moved from a scrotal nodule filaria repens after removal.

220 Vol. 9, No. 2, 2011 • Intern J Appl Res Vet Med. Figure 9 Male adult worm (magnification 40 x). Transverse striations are observed on the Figure 10 Multinucleated morulae (arrows) cuticles of D.repens together with longitu- , which represent the intrauterine pre-larval dinal ridges that are lacking on Dirofilaria stage of development of the parasite immitis adult worms

Figure 11. At ultrasound examination (12 Figure 12 Ultrasound examination (12 MHz linear probes) the adult parasite ap- MHz linear probes) of the scrotal nodule. pears as double, parallel and hyperechoic The white arrow shows the nodule with lines within the subcutaneous nodules (white inside double line filarid worm echoes. ( T : arrows) testicle)

Figure 13 Ultrasound examination (12 MHz linear probes) 7 days after worm removal (previous case). Resolution of the lesion.

Intern J Appl Res Vet Med • Vol. 9, No. 2, 2011. 221 on chemoprophylaxis no microfilariae were in one case) and that mixed infections by detected. Dirofilaria immitis and Dirofilaria repens Cytology was consistently characterized are not uncommon. To conclude, a mini- by a mixed, predominantly eosinophilic invasive surgical procedure for removing inflammatory infiltrate, the presence of adult Dirofilaria repens nodule in naturally several microfilariae and, in one case, by the infected dogs is a safe, easy and effective presence of multinucleated morulae, which procedure for relief of symptoms related to represent the intrauterine pre-larval stage of infection. development of the parasite (Figure 7). REFERENCES At US examination, the parasite 1) Anderson RC. Nematode Parasites of Vertebrates. appeared as double, parallel and hyperechoic Their Development and Transmission. 2nd ed. lines within the subcutaneous nodules (Fig. Wallingford, UK: CABI Publishing; 2000:1–650. 8a and 8 b), similar to that described for 2)Soulsby EJL. Helminths. In: Helminths, Arthropods 12 and Protozoa of Domesticated Animals 7th ed. Dirofilaria immitis in pulmonary arteries . Baltimore, MD: Williams & Wilkins; 1982:311. 9 Most nodules contained one intact 3) Venco L Dirofilaria (Nochtiella )repens infection in worm, while several others5 contained two dogs and cats Mappe Parassitologiche Dirofilaria intact worms. The procedure resulted in the immitis and D. repens in dog and cat and human infections Genchi C, Rinaldi L, Cringoli G Eds removal of a total of 12 female and 7 male 2007 :133:136 adult worms (Table 2). 4) Webber WAF, Hawking F, 1955. Experimental Morphological and PCR examination maintenance of Dirofilaria repens and D. immitis confirmed that the parasite wasD.repens in in dogs. Exp Parasitol 4: 143-164. 5) Cancrini G, Tassi P, Coluzzi M 1989 Ivermectin all of the cases. against larval stages of Dirofilaria repens All nodular lesions disappeared within Parassitologia 31, 177-182 a few days following the removal of the 6) Živičnjak T, Martinković F, Beck R, 2006. parasite with the mini-invasive technique, Dirofilariosis in Croatia: spread and public health impact. 5th Croatian congress on infective without any further treatment. diseases, Zadar, Croatia DISCUSSION 7) Baneth G, Volansky Z, Anug Y, Favia G, Bain O, Goldstein RE, Harrus S 2002 Dirofilaria repens The mini-invasive procedure described infection in a dog: diagnosis and treatment here is easy to perform and is followed by with melarsomine and doramectin Veterinary complete resolution of symptoms related to parasitology 105, 173-178 8) Pampiglione S, Canestri Trotti G, Rivasi E. infection by Dirofilaria repens when nodules 1995 Human dirofilariosis due to Dirofilaria are present. It requires no anaesthesia or (Nochtiella) repens : a review of world lieterature. sedation. However, it cannot be performed Parassitologia 37, 149-193 in asymptomatic dogs (no nodules) and 9) Giori L, Garbagnoli V, Venco L, Genchi M, Bazzocchi C, Bertazzolo W What is your therefore cannot be considered as curative, diagnosis? Fine needle aspirate from a from a parasitological point of view, for subcutaneous mass in a dog. Mixed neutrophilic- clearing the infection. eosinophilic inflammation with Dirofilaria fragments. Vet Clin Pathol. 2010 Jun;39(2):255-6 At this time, however, no adulticide drug 10) Genchi C, Poglayen G, Kramer LH 2002 Efficacia is currently registered for treatment against di Selamectin nella profilassi delle infestazioni da Dirofilaria repens and some concerns Dirofilaria repens nel cane.Veterinaria 16, 69-71 remain about its prevention, because off 11) Rossi L, Ferroglio E , Agostini A 2002 Use 5 of moxidectin tablets in the control of canine the results of experimental infections and subcutaneous dirofilariosis.Veterinary records 15 the findings of the dog of this study on 383 chemoprophilaxis. Therefore, in the authors 12) Venco L, Calzolari D, Mazzocchi D, Morini S. opinion, the described surgical method The Use of Echocardiography as a Diagnostic tool for Detection of Feline Heartworm (Dirofilaria appears to be the best option when nodules immitis) Infections. Fel Pract 26 : 6-9 1998 are present. Furthermore, as shown here, it is 13) Casiraghi M, Bazzocchi C, Mortarino M, Ottina important to remember that clinical infection E, Genchi C. A simple molecular method for may be present without microfilaremia (as discriminating common filarial nematodes of dogs (Canis familiaris). Vet Parasitol. 2006;141:368–

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