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
a
Department of Internal Medicine, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur 3-5, Cluj-Napoca 400372, Cluj, Romania
b
Department of Parasitology and Parasitic Diseases, University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Calea Mănăştur 3-5, Cluj-Napoca 400372, Cluj, Romania
c
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
Keywords:
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-
tinuous expansion of their geographical distribution area, due to several
factors, including the climate change, the introduction of new compe-
tent vector species, traveling and relocation of infected dogs [1]. The
female nematodes release blood-circulating larvae (microfilariae)
which become infective after being ingested by mosquitoes (genera
Culex, Aedes, Anopheles), which act as intermediate host and vector [2].
The main veterinary concern is focused on Dirofilaria immitis, which
causes a severe and life-threatening cardio-pulmonary condition [3].
This species originates in Mediterranean basin, but during the last de-
cades, the infection has spread towards northern, central and eastern
countries [1,2]. A second species that has recently regained attention
is D. repens, due to its zoonotic importance in Europe [2]. In dogs, this
species causes subcutaneous dirofilariosis, which is generally asymp-
tomatic or associated with various dermatological conditions [4].
Despite the similarity of development requirements and favouring fac-
tors, this species seems to be spreading more rapidly than D. immitis,
currently having a larger distribution range in Europe [2].
⁎ Corresponding author.
E-mail address: ionica.angela@usamvcluj.ro (A.M. Ionică).
Until recently, Romania has been traditionally regarded as a non-
endemic country and only some reports on the occurrence of canine
dirofilarioses from the first half of the 20th Century were available.
The first comprehensive epidemiological studies were conducted few
years ago, highlighting the presence of both D. repens and D. immitis
and showing a relatively high frequency of co-infections [5].
While the pathological effects of the Dirofilaria spp. adults have been
intensively studied, the pathogenicity of microfilariae has not been
completely elucidated.
The present case report describes the clinical and pathological
features of a natural co-infection with D. repens and D. immitis in a dog
from Romania, revealing new possible erratic migration paths for
D. repens.
A 5 year-old male Boxer was referred to the Faculty of Veterinary
Medicine Cluj-Napoca, at the Small Animal Internal Medicine Clinic,
for haemodialysis treatment, after being diagnosed with chronic kidney
failure in a private medical practice in Satu-Mare town (north-western
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
body condition, poor coat condition (Fig. 1), two cutaneous swellings
on the right foreleg and one at the lateral surface of the thigh,

http://dx.doi.org/10.1016/j.parint.2017.02.003
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
of conjunctival mucous membranes and vital signs within the normal
ranges (rectal temperature = 38.4 °C, heart rate = 112/min, respiratory
rate = 32/min). The inspection of the conjunctival mucous membrane
revealed at the left eye, a small translucent mass. After surgical removal
of the conjunctival mass, a 13 cm long, slender, white nematode was re-
covered (Fig. 1) and morphologically identified as an adult female of D.
repens [6]. No treatments with antiparasitic drugs were specified in the
dog's health card record.
A thoracic radiograph and echocardiogram were performed, but
no alterations were observed. The ultrasound evaluation of the abdo-
men revealed a small accumulation of peritoneal liquid, a diffuse thick-
ening of the urinary bladder and an increased echogenicity of the renal
cortex.
Blood and urine samples were taken for a complete laboratory
evaluation. The haematological analysis revealed: non-regenerative
anaemia [RBC 4.67 × 106/μL, (reference interval 5.5–8.5 × 106/μL),
hemoglobin 10.7 g/dL (reference interval 12–18 g/dL), no reticulocytes
(reference interval b 70.000)], lymphopenia [210/μL, (reference interval
1000–4800/μL)], neutrophilia [14,200/μL, (reference interval 3000–
11,500/μL)] [7]. The serum biochemistry revealed increased values
of blood urea nitrogen (BUN) [300 mg/dL (reference interval 10–
28 mg/dL)], creatinine [10 mg/dL, (reference interval 0.5–1.5 mg/dL)],
aspartate aminotransferase (ASAT) [129 U/L, (reference value 23–
66 U/L)], alanine aminotransferase (ALAT) [334 U/L, (reference value
21–102 U/L)] and gamma-glutamyl transferase (GGT) [15.5 U/L, (refer-
ence value 1.2–6.4 U/L)]. A significant decrease in serum albumin was
also recorded [1.37 g/dL, (reference interval 2.6–3.3 g/dL)] [8]. Urine
analysis revealed proteinuria. Several microfilariae morphologically
identified as D. repens [6] were present in the urinary sediment.
A rapid test (VetExpert Caniv-4) detecting antigens of adult female
of D. immitis was performed and a positive result was registered. Fol-
lowing DNA extraction from a 0.2 mL whole blood sample, a duplex
PCR amplifying a fragment of cytochrome c oxidase subunit 1 (cox 1) of
D. immitis (169 bp) and D. repens (479 bp) was performed according
to literature [9] and showed positivity for both species. The intensity
of microfilaremia was evaluated by means of modified Knott's test and
microfilariae were morphologically identified [6] and counted. Both D.
repens and D. immitis were present and the obtained values were 7780
microfilariae/mL and 427 microfilariae/mL respectively.
Based on the clinical and paraclinical exams, a diagnosis of kidney
failure, chronical cystitis and mixed infection with D. repens and D.
immitis was established. In order to determine the primary cause of
the kidney failure, an ultrasound guided kidney biopsy was performed.
The histopathological examination of two needle core biopsies showed
a severe and multifocal inflammatory process centered on the glomeruli
and interstitial space, associated with atrophy and fibrosis of the renal
parenchyma. Fragments of microfilariae were observed in the glomeru-
lar and peritubular capillaries. The histological findings were consistent
with chronic and severe membranoproliferative glomerulonephritis
and intralesional microfilariae. PCR evaluation of a third biopsy was pos-
itive only for D. repens DNA.
Taking into consideration the irreversible nature of the kidney le-
sions, the progressive degeneration of the animal's general status and
the palliative nature of treatment, 30 days after the diagnosis was
established, the owner requested euthanasia. During necropsy, the
general examination revealed an emaciated body and anaemia. In the
thoracic cavity, diffuse congestion of the lungs with multifocal prolifer-
ative pneumonia, mediastinal lymph node reactive hyperplasia and
fibrous epicarditis affecting the left atria were observed. Diffuse liver fi-
brosis (moderate), severe visceral and parietal lymph node hyperplasia,
multifocal ulcerative gastritis, splenomegaly (moderate) and follicular
cystitis were noted in the abdominopelvic cavity. The kidneys were
enlarged, diffusely pale, firm, with a striated medulla due to
mineralisation. The three clinically observed swellings were investigat-
ed during the necropsy, but no parasites were found. However, over 300
adult D. repens were recovered from the subcutaneous tissues (Fig. 1) of
the head, neck, thorax, limbs and testes. Two more adult nematodes
morphologically identified as D. repens were recovered from the pelvic
cavity (female) (Fig. 1) and the mesentery (male) (Fig. 1). The heart
and pulmonary arteries were dissected and two gravid female D. immitis
were recovered (Fig. 1).
As the localization of some specimens was quite unusual, in order to
exclude the possibility of the occurrence of other Dirofilaria species, ge-
nomic DNA was extracted from adult nematodes of both morphologi-
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
attained sequences were compared to those available in GenBank data-
base by using Basic Local Alignment Tool (BLAST) analysis. Sequence
analysis revealed a 100% similarity to other available sequences (acces-
sion numbers: LC107816, KR870344, EU159111 for D. immitis and
accession numbers: KX265049, KR998257 for D. repens).
For histological examination, tissue samples (kidney, liver, spleen,
lymph nodes, urinary bladder, lung) were fixed in 10% neutral buffered
formalin solution, (pH 7.0) for 24 h, routinely processed, embedded in
paraffin wax, cut into 3–4-μm sections, and stained with haematoxylin
and eosin (H & E). The histopathological examination of the kidney re-
vealed severe membranoproliferative glomerulonephritis affecting
most of the examined glomeruli, fibromuscular (and discreetly lipid)
atherosclerotic plaques, diffuse fibrous and lymphohistiocytic intersti-
tial nephritis, tubular mineralization (mainly in the medulla) and epi-
thelial desquamation. Microfilariae were present in a large number at
vascular, interstitial and intratubular levels (Fig. 2). In the liver, a chron-
ic congestion with diffuse moderate fibrosis was observed. Multiple
lipogranulomas and mild steatosis were also noted (Fig. 2). The splenic
lesions consisted in lymphoid depletion, presence of siderofibrotic
plaques and vascular fibrosis and mineralization. The presence of
microfilariae and several macrophages containing ceroids and hemosid-
erin were observed (Fig. 2). The lymph nodes presented severe diffuse
amyloidosis, lymphatic stasis, cortical lymphoid depletion and several
macrophages containing ceroids.
D. immitis is an important parasite of dogs, as infection invariably
leads to pulmonary hypertension and consecutive congestive heart fail-
ure [3]. The clinical presentation is generally chronic. Infected animals
may not show any clinical signs for months or even years. The signs of
the disease develop gradually and may begin with a chronic cough
that may be followed by dyspnoea, weakness, anorexia, weight loss
and sometimes lipothymia after exercise or excitement [3]. In contrast,
the majority of animals infected with D. repens shows no evident clinical
signs, or may develop a variety of dermatological conditions, including
erythema, alopecia, crusting and nodules [4]. The clinical presentation
of our patient was partly consistent with the manifestation of D. immitis
infection (progressive weight loss, irregular mild cough, poor appetite).
Most likely, the other clinical signs (lethargy, sporadic vomiting, poly-
dipsia, dysuria and pollakiuria) could be attributed to the injuries of
the kidneys and urinary bladder.
Occasionally, aberrant migration of D. immitis results in parasites be-
coming trapped in ectopic locations, such as the eyes, systemic arteries,
brain or testes [10,11]. Few data is available regarding the erratic migra-
tion of D. repens, with only such a report, describing an ocular localiza-
tion of the parasite [12]. In a study evaluating the therapeutical
efficacy of a spot-on product, Petry et al. [13] reported the presence of
D. repens adults in the abdominal cavity, on the serosa in experimentally
infected dogs. To the best of our knowledge, the present case is the first
documentation of aberrant localizations of D. repens within the pelvic
and abdominal cavities, in a naturally infected dog, and the second
one reporting the presence of the adult parasite in the eye.
Haematological and biochemical findings in dogs infected with D.
immitis may include anaemia, thrombocytopenia, leucocytosis,
neutrophilia, eosinophilia, monocytosis and increased values of alkaline
phosphatase (ALP), ALAT, ASAT, BUN, creatinine, direct and indirect bil-
irubin [14–16]. Urine exam can reveal proteinuria, a paraclinical finding
consistent with kidney injuries, even in cases when urine protein level is
low [15]. Although rarely evaluated, the occurrence of biochemical
modifications associated with D. repens infection have been suggested
in a dog from Poland, presenting increased values of ALAT and bilirubin,
as well as hypoalbuminemia [17]. These findings suggest that
microfilariae of D. repens could be responsible for liver and kidney dam-
age. Our laboratory results are similar to the ones described in literature.
However, as microfilariae of both Dirofilaria species seem to have the
same effect on internal organs, it is difficult to evaluate their individual
contribution.
Histopathological changes associated to D. immitis [14,18] or D.
repens [10,17] microfilariae, particularly in case of massive infection,
have been noted in several organs, such as kidney, lung, liver, heart,
spleen and brain, suggesting both immuno-pathological and mechani-
cal effects of microfilariae. In dogs experimentally infected with D.
immitis, fine or granular deposits of IgG were observed in the
mesangium and along the glomerular capillary wall, even in
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
immune-mediated [19]. Moreover, both Dirofilaria species are known to
harbour Wolbachia pipientis, a bacterium which is involved in the host's
inflammatory and immune response [20].
In the present case, the significant disproportion between the two
Dirofilaria species (both in number of adults and microfilariae) may in-
dicate a more important role of D. repens microfilariae in producing
histopathological lesions in different organs. Moreover, considering
that all microfilariae present in the urine samples were D. repens and
the molecular evaluation of kidney biopsy was positive exclusively for
this species, it can be assumed that renal damage is mainly a conse-
quence of D. repens microfilariae.
This is the first worldwide report of two simultaneous aberrant lo-
calizations of D. repens in a naturally infected dog and first report of
the ocular localisation of this parasite for Romania. The clinical and
paraclinical evaluation results suggest the role of D. repens microfilariae
in the aetiology of the histopathological lesions, stressing out the impor-
tance of further investigation of the actual pathogenic potential of this
frequently neglected parasite.
Acknowledgments
This study was supported by the CNCS-UEFISCDI Grant Agency
Romania, grant number TE 299/2015. The work of AMI and MOD was
performed under the frame of the EurNegVec COST Action TD1303.
All authors declare they have no conflict of interest.
All authors agree to the publication of the present manuscript, which
has not been published and is not under consideration for publication
elsewhere, and, if accepted, will not be published elsewhere in the
same form, in English or in any other language, including electronically,
without the written consent of the copyright-holder.
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