Case Report
Leishmania sp. Amastigotes Identification in
Canine Transmissible Venereal Tumor

Vinicius Soares Carreira,1,2 Heitor Flávio Ferrari,1 Ingeborg Maria Langohr,3

Charles Mackenzie,4 Luiz Carlos Montezzo,5 Edson Taira,6

Lucile Maria Floeter-Winter,7 and Maria Cecília Rui Luvizotto1

1 Departamento de Cĺınica, Cirurgia e Reprodução Animal (DCCRA), Laboratório de Patologia Animal,
Universidade Estadual de São Paulo (UNESP), Campus Araçatuba, Rua Clóvis Pestana 793, 16050-680 Araçatuba, SP, Brazil

2 Department of Environmental Health, University of Cincinnati, 3223 Eden Avenue, Kettering Laboratory, Cincinnati,
OH 45267-0056, USA

3Department of Pathobiological Science, School of Veterinary Medicine, Louisiana State University, Skip Bertman Drive,
Baton Rouge, LA 70803, USA

4Department of Pathobiology and Diagnostic Investigation (PDI), Veterinary Medical Center F Wing, 784 Wilson Road,
Room F130, East Lansing, MI 48824-1314, USA

5Diagnostic Pathology Private Practice, Rua Treze de Maio 1671, 16901018 Andradina, SP, Brazil
6DCCRA, Hospital Veterinário, Universidade Estadual de São Paulo (UNESP), Campus Araçatuba, Rua Clovis Pestana 793,
6050-680 Araçatuba, SP, Brazil

7 Instituto de Biociências, Cidade Universitária/Universidade de São Paulo, Rua do Matão Travessa 14 No. 321,
05508-090 São Paulo, SP, Brazil

Correspondence should be addressed to Maria Cećılia Rui Luvizotto; analymendes@hotmail.com

Received 30 April 2014; Revised 9 July 2014; Accepted 16 July 2014; Published 24 August 2014

Academic Editor: Renato L. Santos

Copyright © 2014 Vinicius Soares Carreira et al.This is an open access article distributed under the Creative CommonsAttribution
License, which permits unrestricted use, distribution, and reproduction in anymedium, provided the originalwork is properly cited.

Leishmaniasis is a vector-borne disease with Leishmania chagasi being the etiological agent of canine visceral leishmaniasis in
South America. Canine venereal tumor is a transplantable round cell tumor of histiocytic origin which is mostly observed in
sexually active male and female intact dogs. It has been shown that Leishmania amastigotes have higher tropism for the canine
male genital tract tissues and venereal leishmaniasis transmission has been documented in dogs but, to date, a canine venereal
tumor-dependent transmission route has not been fully demonstrated. In this report, a 10-year-old, mixed breed, intact female
dog presented a vaginal venereal transmissible tumor but no other clinical abnormalities otherwise. Unexpectedly, tumor tissue
imprint smears examination revealed Leishmania sp. amastigotes within infiltrating macrophages. In addition to the cytological
direct identification, the protozoan was confirmedwithin the neoplastic tissue bymeans of immunohistochemistry and polymerase
chain reaction.This report illustrates an asymptomaticLeishmania sp. infection thatmay have started on or from the canine venereal
tumor tissue, the latter option further supporting previous evidence of such an alternative vector-independent route of transmission
for canine visceral leishmaniasis in areas where these diseases coexist.

1. Introduction

Leishmaniasis is an anthropozoonotic disease with wide
geographic distribution, affecting humans, dogs, and several
wildlife species, and is caused by the obligate intracellu-
lar protozoa belonging to the genus Leishmania. Among

Leishmania species, Leishmania chagasi and L. braziliensis
are the documented species found in Araçatuba, northwest
state of São Paulo, Brazil [1]. Depending on the infecting
Leishmania species and the immunocompetence of the host,
the infection can result in visceral, cutaneous, or mucocu-
taneous disease. Visceral leishmaniasis in dogs (CanVL) is

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Case Reports in Veterinary Medicine
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2 Case Reports in Veterinary Medicine

associated with variable clinical manifestations ranging from
unapparent subclinical infections to a systemic disease char-
acterized by progressive weight loss, hepatosplenomegaly,
lymphadenopathy, and a range of ocular and dermatological
manifestations [2]. Canine transmissible venereal tumor
(CTVT) is a unique neoplastic entity that is sexually trans-
missible and regarded as the oldest known mammalian
somatic cell neoplasm in continuous propagation [3]. CTVT
has worldwide distribution, with higher incidence in tropical
areas, and has beenmostly reported in dogs (Canis familiaris)
and foxes (Vulpes sp.). Clinically, CTVT lesions are red to
tan, friable, verrucous to multilobulated masses, predom-
inantly affecting genital organs, and are usually ulcerated
and inflamed [4]. Metastasis may occur, with lymphatic or
visceral dissemination generally associated with underlying
immunological impairment [5]. In addition, CTVT extragen-
ital lesions, such as cutaneous, are relatively common and
have been reported even in the absence of primary genital
lesions [5, 6]. CTVT transmission occurs by means of direct
neoplastic cell implantation, and successful CTVT implan-
tation has been linked to an ineffective immune response.
Conversely, an efficient postimplantation adaptive immune
response is believed to be associated with the mechanism of
natural regression that commonly occurs with this tumor [5].
CTVT andCanVL can overlap epidemiologically particularly
in regard to their geographical distribution, as in the case
presented herein. Leishmania sp. reportedly has tropism
for the canine male genital tract [7] although the same
was not observed in the female genital tract [8]. Venereal
transmission via semen has been demonstrated [9]. However,
it remains to be experimentally demonstrated whether Leish-
mania-laden CTVT cells can successfully transmit the proto-
zoan to another host. In veterinarymedicine, CanVLhas been
previously identified concurrently with canine transmissible
venereal tumor as well as Leishmania sp. amastigotes within
CTVT neoplastic cells per se [10–12]. Interestingly, in a
recent retrospective study, 5 out of 19 dogs affected by both
leishmaniasis and CTVT also had detectable amastigotes
within the CTVT neoplastic tissue [12].

In regard to the synergistic nature of systemic parasitism
and neoplasia, it has been proposed that concurrent diseases
may occur secondary to the CanVL-driven immune impair-
ment or, alternatively, neoplastic diseases could hamper the
immune system, thus triggering the onset of clinical leishma-
niasis in an already infected but asymptomatic dog [12, 13].
The coexistence in the same lesion of CTVT and Leishmania
has been previously attributed to the systemic dissemination
of the latter [10], where amastigote-laden macrophages get
recruited into the CTVT tissue. It has been suggested that
the histiocytic immunophenotype ofv CTVT [14] may play
an active role in the parasitic invasion of the CTVTneoplastic
cells [10]. Furthermore, the fact that Leishmania amastigotes
can be identified within CTVT neoplastic cells supports the
hypothesis of amonocyte/macrophage lineage histogenesis of
this tumor [6]. Finally, it has been suggested that Leishmania
amastigotes-laden neoplastic CTVT cells can represent an
alternative mode of transmission of canine leishmaniasis in
areas where these diseases coexist [11, 12, 15].

2. Case Report

A 10-year-old, mixed breed intact female dog was submitted
to the Veterinary Teaching Hospital with a 3-month history
of a 4.0 × 3.0 cm vaginal vestibule mass (Figure 1) without
any other significant systemic clinical signs. Examination of
tumor imprint smears revealed abundant typical CTVT cells
admixed with degenerate neutrophils. Hematological find-
ings were within normal limits. At the time of CTVT diag-
nosis, Leishmania sp. serology as performed by Lima et al.
[16]was negative.Therefore, standard chemotherapy protocol
using intravenous vincristine sulphate 0.5mg/m2 was admin-
istered once weekly. After the fourth chemotherapy session,
and with a reminiscent 3.0 × 2.0 cm mass, additional cytol-
ogy sampling and biopsy were performed. Cytology smears
consisted predominantly ofmature keratinized epithelial cells
admixed with neutrophils, lymphocytes, and plasma cells, as
well as moderate numbers of mixed-morphology bacteria.
Few 2-3 𝜇m oval Leishmania amastigotes with characteristic
perinuclear rod-shaped kinetoplast could be observed within
macrophages (Figure 2) as well as free in the smear. At this
point, no remaining CTVT neoplastic cells could be identi-
fied cytologically.Histopathology findings consisted of exten-
sive inflammatory infiltrate within the subepithelial vaginal
stroma composed of lymphocytes and plasma cells, moderate
amounts of neutrophils, and abundant macrophages, several
of which were loaded with amastigote organisms (Figures 3
and 4), as well as rare reminiscent CTVT neoplastic cells
within reactive newly formed collagen (fibrosis). Immuno-
histochemistry performed as described by Nogueira et al.
[17] labeled myriads of Leishmania sp. amastigotes within
the cytoplasm of histiocytoid cells (Figure 5), interpreted as
macrophages, and dispersed through the neoplastic tissue.
Furthermore, polymerase chain reaction (PCR) as performed
by Moreira et al. [18] confirmed the presence of Leishmania
sp. DNA in the tissue. Apart from the vaginal mass, the
dog was healthy, presenting no overt clinical signs of canine
leishmaniasis. This dog continued to have negative serology
for Leishmania and was devoid of clinical disease up to one
year following the last biopsy of the reminiscentCTVT lesion,
but after this time it was lost to follow-up.

3. Discussion

Low cost and near 100% specificity make cytology the
most accessible method for diagnosing CTVT and canine
leishmaniasis (CanVL) provided that the examiner is familiar
with the unique cellular morphology of the CTVT cells and
the identification of Leishmania amastigotes. Identification
of Leishmania amastigotes within hematoxylin and eosin-
stained tissue sections is challenging and depends on the
affected tissue, stage of infection, severity of the secondary
inflammatory response, and number of organisms [18].
However, as in this case, such diagnostic challenges may be
circumvented by using ancillary techniques such as intissue
antibody-based identification of amastigotes via immunohis-
tochemistry [18, 19]. CTVT diagnosis is routinely done based
on its characteristic cytological findings. Tumors that receive
chemotherapy and have concurrent regression response



Case Reports in Veterinary Medicine 3

Figure 1: Dog. Vaginal vestibule, red verrucous, and lobulated mass
protruding from the vagina.

Figure 2: Dog. Photomicrograph of cytology imprint from the
vaginal mass with a histiocyte-like cell with three intracytoplasmic
protozoa amastigotes (arrow) (Diff-Quick, 100x).

Figure 3: Dog. Photomicrograph of vaginal mucosa with the
subepithelial stroma effaced by canine transmissible venereal tumor
cells, mixed with macrophages and with a mild diffuse lymphoplas-
macytic infiltrate (H&E, 20x).

are histologically characterized by decreased neoplastic cell
population, with predominantly degenerate remnant cells
amidst extensive stromal remodeling and lymphoplasmacytic
inflammatory infiltrate [20]. These changes are in agreement
with our findings of absent typical CTVT cells in the second
cytology sample as well as the predominantly inflammatory
and fibrosing morphology of the biopsy sample. Further-
more, the occurrence of Leishmania infection within the

Figure 4: Dog. Photomicrograph of vaginal mucosa and canine
venereal tumor cells (red arrow); accompanying inflammatory infil-
trate within the subepithelial stroma was composed of lymphocytes,
plasma cells, few neutrophils, and amastigote-laden macrophages
(black arrow) which are shown in higher magnification in the inset
(H&E, 40x and 63x).

Figure 5: Dog. Photomicrograph of vaginal mucosa and immuno-
histochemistry staining highlighting Leishmania sp. amastigotes
(DAB hematoxylin-counterstained, 40x and 63x).

tissue possibly resulted in added granulation tissue formation
that grossly resembled a neoplasm that was responding to
chemotherapy unsatisfactorily.

The coexistence of CTVT and leishmaniasis in this dog
is corroborated by the endemic nature of both diseases in
Araçatuba (northwest state of São Paulo, Brazil), particularly
among roaming dogs [1]. This dog had CTVT genital lesions
which could have provided a feeding site to the female
sand fly vector; the fact that Leishmania amastigotes could
have transplanted with CTVT cells to this new host, thereby
circumventing the vector in the classic transmission route of
canine leishmaniasis, is also a tempting possibility [12, 15].

4. Conclusions

This report illustrates an asymptomatic Leishmania sp. infec-
tion concurrent with a transmissible venereal tumor. We
speculate that the Leishmania sp. infection may have started
on or from the CTVT, the latter option further supporting
previous evidence of such an alternative vector-independent
route of transmission for CanVL in areas where these diseases
coexist. While it was not possible to determine whether this



4 Case Reports in Veterinary Medicine

dog had been previously infected with Leishmania prior to
CTVT, negative serology for Leishmania at the time of CTVT
diagnosis could indicate an early stage of infection favor-
ing the preexistence of the CTVT. Nonetheless, a negative
serology result due to a CanVL-driven impaired adaptive
immune system cannot be discarded. Experimental evidence
of amastigotes uptake by and viability within CTVT cells,
as well as amastigote-laden CTVT cells transmission capa-
bilities, is critically lacking. Thoroughly screening CTVT-
affected dogs for leishmaniasis in areas where these diseases
coexist is highly warranted. The present case underscores
the diagnostic challenges, complex nature, and the potential
epidemiological consequences of overlapping infectious dis-
eases.

Conflict of Interests

The authors declared no potential conflict of interests with
respect to the research, authorship, and/or publication of this
paper.

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