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Parasitology International 66 (2017) 331–334

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Parasitology International

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Clinical and pathological effects of Dirofilaria repens and Dirofilaria

immitis in a dog with a natural co-infection
Mircea Mircean a, Angela Monica Ionică b,⁎, Viorica Mircean b, Adriana Györke b, Andrei Răzvan Codea a,
Flaviu Alexandru Tăbăran c, Marian Taulescu c, Mirabela Oana Dumitrache b
Department of Internal Medicine, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur 3-5, Cluj-Napoca 400372, Cluj, Romania
Department of Parasitology and Parasitic Diseases, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur 3-5, Cluj-Napoca 400372, Cluj, Romania
Pathology Department, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur 3-5, Cluj-Napoca 400372, Cluj, Romania

a r t i c l e i n f o a b s t r a c t

Article history: Canine dirofilarioses are mosquito-borne zoonotic diseases with a continuous expansion of their geographical
Received 26 January 2017 distribution, as a consequence of different climatic and ecological factors. Dirofilaria immitis, the aetiological
Received in revised form 11 February 2017 agent of heartworm disease, has gained a major veterinary interest, mainly due to its severe clinical implication.
Accepted 12 February 2017
In the last decades, D. repens, despite of being regarded as a less pathogenic species, regained attention due to its
Available online 14 February 2017
recognized zoonotic potential. Romania has been traditionally regarded as a non-endemic country, but recent
epidemiological surveys are highlighting the presence of both D. repens and D. immitis. The present case report
Dirofilaria immitis describes the clinical and pathological features of a natural co-infection with D. repens and D. immitis in 5 year
Dirofilaria repens old male Boxer from north-western Romania. Based on clinical and laboratory investigations, a diagnosis of
Microfilariae kidney failure and chronical cystitis was established. The parasites were identified by morphology and confirmed
Co-infection by PCR and sequencing. To the best of our knowledge this is the first worldwide report of the ectopic presence of
Ectopic localization D. repens adults in the pelvic cavity and mesentery by natural infection and first report of the ocular localization of
this parasite for Romania. The clinical and paraclinical findings suggest the implication of D. repens microfilariae
in the aetiology of histopathological lesions. Further investigations are needed in order to establish the actual
pathogenic potential of D. repens, a frequently neglected parasite.
© 2017 Elsevier B.V. All rights reserved.

Canine dirofilarioses are mosquito-borne diseases undergoing a con- Until recently, Romania has been traditionally regarded as a non-
tinuous expansion of their geographical distribution area, due to several endemic country and only some reports on the occurrence of canine
factors, including the climate change, the introduction of new compe- dirofilarioses from the first half of the 20th Century were available.
tent vector species, traveling and relocation of infected dogs [1]. The The first comprehensive epidemiological studies were conducted few
female nematodes release blood-circulating larvae (microfilariae) years ago, highlighting the presence of both D. repens and D. immitis
which become infective after being ingested by mosquitoes (genera and showing a relatively high frequency of co-infections [5].
Culex, Aedes, Anopheles), which act as intermediate host and vector [2]. While the pathological effects of the Dirofilaria spp. adults have been
The main veterinary concern is focused on Dirofilaria immitis, which intensively studied, the pathogenicity of microfilariae has not been
causes a severe and life-threatening cardio-pulmonary condition [3]. completely elucidated.
This species originates in Mediterranean basin, but during the last de- The present case report describes the clinical and pathological
cades, the infection has spread towards northern, central and eastern features of a natural co-infection with D. repens and D. immitis in a dog
countries [1,2]. A second species that has recently regained attention from Romania, revealing new possible erratic migration paths for
is D. repens, due to its zoonotic importance in Europe [2]. In dogs, this D. repens.
species causes subcutaneous dirofilariosis, which is generally asymp- A 5 year-old male Boxer was referred to the Faculty of Veterinary
tomatic or associated with various dermatological conditions [4]. Medicine Cluj-Napoca, at the Small Animal Internal Medicine Clinic,
Despite the similarity of development requirements and favouring fac- for haemodialysis treatment, after being diagnosed with chronic kidney
tors, this species seems to be spreading more rapidly than D. immitis, failure in a private medical practice in Satu-Mare town (north-western
currently having a larger distribution range in Europe [2]. Romania). The dog had a history of progressive weight loss, irregular
mild cough, poor appetite, polydipsia, dysuria, pollakiuria, sporadic
vomiting and lethargy. The physical examination revealed: a score 2
⁎ Corresponding author. body condition, poor coat condition (Fig. 1), two cutaneous swellings
E-mail address: ionica.angela@usamvcluj.ro (A.M. Ionică). on the right foreleg and one at the lateral surface of the thigh,

1383-5769/© 2017 Elsevier B.V. All rights reserved.
332 M. Mircean et al. / Parasitology International 66 (2017) 331–334

Fig. 1. Clinical and gross presentation of the Dirofilaria repens and D. immitis co-infection in a dog. A: Cachexia, with systemic muscle wasting and evident bony prominences; B: Intact D.
repens adult live worm recovered from the bulbar subconjunctival space of the left eye; C: Dorsal view of the lung and great vessels, the presence of D. immitis (arrow) in the pulmonary
artery; D: Serpentine adult D. repens in the subcutaneous tissue; E: adult D. repens in the retroperitoneal space of the pelvic cavity (arrow); F: Well demarked, coiled D. repens in the
mesentery (arrow).

enlargement of prescapular and popliteal lymph nodes, mild congestion repens and D. immitis were present and the obtained values were 7780
of conjunctival mucous membranes and vital signs within the normal microfilariae/mL and 427 microfilariae/mL respectively.
ranges (rectal temperature = 38.4 °C, heart rate = 112/min, respiratory Based on the clinical and paraclinical exams, a diagnosis of kidney
rate = 32/min). The inspection of the conjunctival mucous membrane failure, chronical cystitis and mixed infection with D. repens and D.
revealed at the left eye, a small translucent mass. After surgical removal immitis was established. In order to determine the primary cause of
of the conjunctival mass, a 13 cm long, slender, white nematode was re- the kidney failure, an ultrasound guided kidney biopsy was performed.
covered (Fig. 1) and morphologically identified as an adult female of D. The histopathological examination of two needle core biopsies showed
repens [6]. No treatments with antiparasitic drugs were specified in the a severe and multifocal inflammatory process centered on the glomeruli
dog's health card record. and interstitial space, associated with atrophy and fibrosis of the renal
A thoracic radiograph and echocardiogram were performed, but parenchyma. Fragments of microfilariae were observed in the glomeru-
no alterations were observed. The ultrasound evaluation of the abdo- lar and peritubular capillaries. The histological findings were consistent
men revealed a small accumulation of peritoneal liquid, a diffuse thick- with chronic and severe membranoproliferative glomerulonephritis
ening of the urinary bladder and an increased echogenicity of the renal and intralesional microfilariae. PCR evaluation of a third biopsy was pos-
cortex. itive only for D. repens DNA.
Blood and urine samples were taken for a complete laboratory Taking into consideration the irreversible nature of the kidney le-
evaluation. The haematological analysis revealed: non-regenerative sions, the progressive degeneration of the animal's general status and
anaemia [RBC 4.67 × 106/μL, (reference interval 5.5–8.5 × 106/μL), the palliative nature of treatment, 30 days after the diagnosis was
hemoglobin 10.7 g/dL (reference interval 12–18 g/dL), no reticulocytes established, the owner requested euthanasia. During necropsy, the
(reference interval b 70.000)], lymphopenia [210/μL, (reference interval general examination revealed an emaciated body and anaemia. In the
1000–4800/μL)], neutrophilia [14,200/μL, (reference interval 3000– thoracic cavity, diffuse congestion of the lungs with multifocal prolifer-
11,500/μL)] [7]. The serum biochemistry revealed increased values ative pneumonia, mediastinal lymph node reactive hyperplasia and
of blood urea nitrogen (BUN) [300 mg/dL (reference interval 10– fibrous epicarditis affecting the left atria were observed. Diffuse liver fi-
28 mg/dL)], creatinine [10 mg/dL, (reference interval 0.5–1.5 mg/dL)], brosis (moderate), severe visceral and parietal lymph node hyperplasia,
aspartate aminotransferase (ASAT) [129 U/L, (reference value 23– multifocal ulcerative gastritis, splenomegaly (moderate) and follicular
66 U/L)], alanine aminotransferase (ALAT) [334 U/L, (reference value cystitis were noted in the abdominopelvic cavity. The kidneys were
21–102 U/L)] and gamma-glutamyl transferase (GGT) [15.5 U/L, (refer- enlarged, diffusely pale, firm, with a striated medulla due to
ence value 1.2–6.4 U/L)]. A significant decrease in serum albumin was mineralisation. The three clinically observed swellings were investigat-
also recorded [1.37 g/dL, (reference interval 2.6–3.3 g/dL)] [8]. Urine ed during the necropsy, but no parasites were found. However, over 300
analysis revealed proteinuria. Several microfilariae morphologically adult D. repens were recovered from the subcutaneous tissues (Fig. 1) of
identified as D. repens [6] were present in the urinary sediment. the head, neck, thorax, limbs and testes. Two more adult nematodes
A rapid test (VetExpert Caniv-4) detecting antigens of adult female morphologically identified as D. repens were recovered from the pelvic
of D. immitis was performed and a positive result was registered. Fol- cavity (female) (Fig. 1) and the mesentery (male) (Fig. 1). The heart
lowing DNA extraction from a 0.2 mL whole blood sample, a duplex and pulmonary arteries were dissected and two gravid female D. immitis
PCR amplifying a fragment of cytochrome c oxidase subunit 1 (cox 1) of were recovered (Fig. 1).
D. immitis (169 bp) and D. repens (479 bp) was performed according As the localization of some specimens was quite unusual, in order to
to literature [9] and showed positivity for both species. The intensity exclude the possibility of the occurrence of other Dirofilaria species, ge-
of microfilaremia was evaluated by means of modified Knott's test and nomic DNA was extracted from adult nematodes of both morphologi-
microfilariae were morphologically identified [6] and counted. Both D. cally identified species, which were further confirmed by means of
M. Mircean et al. / Parasitology International 66 (2017) 331–334 333

PCR and sequencing (performed at Macrogen Europe, Amsterdam). The Most likely, the other clinical signs (lethargy, sporadic vomiting, poly-
attained sequences were compared to those available in GenBank data- dipsia, dysuria and pollakiuria) could be attributed to the injuries of
base by using Basic Local Alignment Tool (BLAST) analysis. Sequence the kidneys and urinary bladder.
analysis revealed a 100% similarity to other available sequences (acces- Occasionally, aberrant migration of D. immitis results in parasites be-
sion numbers: LC107816, KR870344, EU159111 for D. immitis and coming trapped in ectopic locations, such as the eyes, systemic arteries,
accession numbers: KX265049, KR998257 for D. repens). brain or testes [10,11]. Few data is available regarding the erratic migra-
For histological examination, tissue samples (kidney, liver, spleen, tion of D. repens, with only such a report, describing an ocular localiza-
lymph nodes, urinary bladder, lung) were fixed in 10% neutral buffered tion of the parasite [12]. In a study evaluating the therapeutical
formalin solution, (pH 7.0) for 24 h, routinely processed, embedded in efficacy of a spot-on product, Petry et al. [13] reported the presence of
paraffin wax, cut into 3–4-μm sections, and stained with haematoxylin D. repens adults in the abdominal cavity, on the serosa in experimentally
and eosin (H & E). The histopathological examination of the kidney re- infected dogs. To the best of our knowledge, the present case is the first
vealed severe membranoproliferative glomerulonephritis affecting documentation of aberrant localizations of D. repens within the pelvic
most of the examined glomeruli, fibromuscular (and discreetly lipid) and abdominal cavities, in a naturally infected dog, and the second
atherosclerotic plaques, diffuse fibrous and lymphohistiocytic intersti- one reporting the presence of the adult parasite in the eye.
tial nephritis, tubular mineralization (mainly in the medulla) and epi- Haematological and biochemical findings in dogs infected with D.
thelial desquamation. Microfilariae were present in a large number at immitis may include anaemia, thrombocytopenia, leucocytosis,
vascular, interstitial and intratubular levels (Fig. 2). In the liver, a chron- neutrophilia, eosinophilia, monocytosis and increased values of alkaline
ic congestion with diffuse moderate fibrosis was observed. Multiple phosphatase (ALP), ALAT, ASAT, BUN, creatinine, direct and indirect bil-
lipogranulomas and mild steatosis were also noted (Fig. 2). The splenic irubin [14–16]. Urine exam can reveal proteinuria, a paraclinical finding
lesions consisted in lymphoid depletion, presence of siderofibrotic consistent with kidney injuries, even in cases when urine protein level is
plaques and vascular fibrosis and mineralization. The presence of low [15]. Although rarely evaluated, the occurrence of biochemical
microfilariae and several macrophages containing ceroids and hemosid- modifications associated with D. repens infection have been suggested
erin were observed (Fig. 2). The lymph nodes presented severe diffuse in a dog from Poland, presenting increased values of ALAT and bilirubin,
amyloidosis, lymphatic stasis, cortical lymphoid depletion and several as well as hypoalbuminemia [17]. These findings suggest that
macrophages containing ceroids. microfilariae of D. repens could be responsible for liver and kidney dam-
D. immitis is an important parasite of dogs, as infection invariably age. Our laboratory results are similar to the ones described in literature.
leads to pulmonary hypertension and consecutive congestive heart fail- However, as microfilariae of both Dirofilaria species seem to have the
ure [3]. The clinical presentation is generally chronic. Infected animals same effect on internal organs, it is difficult to evaluate their individual
may not show any clinical signs for months or even years. The signs of contribution.
the disease develop gradually and may begin with a chronic cough Histopathological changes associated to D. immitis [14,18] or D.
that may be followed by dyspnoea, weakness, anorexia, weight loss repens [10,17] microfilariae, particularly in case of massive infection,
and sometimes lipothymia after exercise or excitement [3]. In contrast, have been noted in several organs, such as kidney, lung, liver, heart,
the majority of animals infected with D. repens shows no evident clinical spleen and brain, suggesting both immuno-pathological and mechani-
signs, or may develop a variety of dermatological conditions, including cal effects of microfilariae. In dogs experimentally infected with D.
erythema, alopecia, crusting and nodules [4]. The clinical presentation immitis, fine or granular deposits of IgG were observed in the
of our patient was partly consistent with the manifestation of D. immitis mesangium and along the glomerular capillary wall, even in
infection (progressive weight loss, irregular mild cough, poor appetite). amicrofilaremic dogs, suggesting that glomerular lesions are in fact

Fig. 2. Histopathological changes in the kidney (A, D), liver (B, E) and spleen (C, F) following Dirofilaria repens and D. immitis co-infection. A: kidney, severe interstitial fibrosis and mild
lymphohistiocytic infiltrate, associated with cystic dilatation of the renal tubules and intravascular presence of many microfilariae, H&E stain, 20×; B: liver, fibrosis and focal
hepatocyte vacuolar degeneration (blue arrow), with intralesional presence of a microfilaria, H&E stain, 40×; C: spleen, focal vascular fibrosis and mineralization (arrow), H&E stain,
20×; D: many intravascular curvilinear microfilariae with visible nuclear columns (detailed aspect of the area demarked by the square from image A), H&E stain, 100×; E: longitudinal
sections of microfilaria between hepatic cords, H&E stain, 100×; F: oblique section of a microfilaria surrounded by many hemosiderin and lipofuscin laden macrophages, lymphocytes
and plasma cells from the splenic red pulp, H&E stain, 100×.
334 M. Mircean et al. / Parasitology International 66 (2017) 331–334

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