Leishmania sp . Amastigotes Identification in Canine Transmissible Venereal Tumor

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


Introduction
Leishmaniasis is an anthropozoonotic disease with wide geographic distribution, affecting humans, dogs, and several wildlife species, and is caused by the obligate intracellular protozoa belonging to the genus Leishmania.Among Leishmania species, Leishmania chagasi and L. braziliensis are the documented species found in Arac ¸atuba, 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 mucocutaneous disease.Visceral leishmaniasis in dogs (CanVL) is associated with variable clinical manifestations ranging from unapparent subclinical infections to a systemic disease characterized 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 transmissible 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 been mostly reported in dogs (Canis familiaris) and foxes (Vulpes sp.).Clinically, CTVT lesions are red to tan, friable, verrucous to multilobulated masses, predominantly 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 extragenital 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 implantation 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 and CanVL 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 Leishmania-laden CTVT cells can successfully transmit the protozoan to another host.In veterinary medicine, CanVL has been previously identified concurrently with canine transmissible venereal tumor as well as Leishmania sp.amastigotes within CTVT neoplastic cells per se [10][11][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 impairment or, alternatively, neoplastic diseases could hamper the immune system, thus triggering the onset of clinical leishmaniasis 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 CTVT neoplastic cells [10].Furthermore, the fact that Leishmania amastigotes can be identified within CTVT neoplastic cells supports the hypothesis of a monocyte/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].

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 findings were within normal limits.At the time of CTVT diagnosis, Leishmania sp.serology as performed by Lima et al. [16] was negative.Therefore, standard chemotherapy protocol using intravenous vincristine sulphate 0.5 mg/m 2 was administered once weekly.After the fourth chemotherapy session, and with a reminiscent 3.0 × 2.0 cm mass, additional cytology sampling and biopsy were performed.Cytology smears consisted predominantly of mature keratinized epithelial cells admixed with neutrophils, lymphocytes, and plasma cells, as well as moderate numbers of mixed-morphology bacteria.Few 2-3 m 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 identified cytologically.Histopathology findings consisted of extensive 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).Immunohistochemistry 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 reminiscent CTVT lesion, but after this time it was lost to follow-up.

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 eosinstained 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 immunohistochemistry [18,19].CTVT diagnosis is routinely done based on its characteristic cytological findings.Tumors that receive chemotherapy and have concurrent regression response   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.Furthermore, the occurrence of Leishmania infection within the  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 Arac ¸atuba (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].

Conclusions
This report illustrates an asymptomatic Leishmania sp.infection 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 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 favoring 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 capabilities, is critically lacking.Thoroughly screening CTVTaffected 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 diseases.

Figure 3 :
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 lymphoplasmacytic infiltrate (H&E, 20x).

Figure 4 :
Figure 4: Dog.Photomicrograph of vaginal mucosa and canine venereal tumor cells (red arrow); accompanying inflammatory infiltrate 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).