Detection of amastigotes and histopathological alterations in the thymus of Leishmania infantum-infected dogs

Abstract

Introduction: In canine visceral leishmaniasis (CVL), lymphopenia, and the disorganization of lymphoid organs such as spleen and lymph nodes have been demonstrated. However, the involvement of thymus in CVL has not been evaluated so far. Herein, we investigated whether the thymus can be colonized by Leishmania infantum in naturally infected dogs.

Methods: Thymus were obtained from 16 of 58 dogs and samples of this organ were submitted to immunohistochemistry for laminin and fibronectin detection, histopathology, in situ hybridization and polymerase chain reaction (PCR) targeting the gene ITS-1 for Leishmania and sequenced. Samples of spleen, skin and popliteal lymph nodes were collected and submitted to immunohistochemistry and parasitological culture followed by multilocus enzyme electrophoresis.

Results: L. infantum was identified in all dogs. DNA and amastigote forms of Leishmania were detected in the thymus from 16 dogs by PCR and in eight by immunohistochemistry. Besides thymus, parasites were detected in spleen, lymph nodes, and skin. A granulomatous or pyogranulomatous thymitis was observed in eight dogs associated to intact amastigotes forms of this parasite. Fibronectin deposition in thymus was higher in dogs with more clinical signs.

Conclusions: These results demonstrate that the thymus of dogs can be parasitized by L. infantum, which may generate inflammatory reactions leading to alterations in thymic microarchitecture.

Keywords: Leishmania infantum; canine visceral leishmaniasis; clinical signs; extracellular matrix; histopathology; parasite load; thymus.

Figure 1

Quantification of parasite load was performed counting the number of amastigotes/mm² of each animal. A Gaussian mixture (Normal distribution) was performed resulting in two groups: low and high parasite load

Figure 2

Histopathological evaluation of the thymus from Leishmania infantum‐naturally infected dogs. A, Brown‐stained amastigote forms of Leishmania spp. (arrow and inset) in the cytoplasm of macrophages (immunohistochemistry). B, Blue‐stained amastigote forms of Leishmania spp. (arrow and inset) in the cytoplasm of macrophages (in situ hybridization). C, Thymitis showing thickening of the interlobular trabeculae due to fibrosis and to an intense pyogranulomatous inflammatory infiltrate (HE). D, Detail of (C) showing an intense pyogranulomatous inflammatory infiltrate in the interlobular trabeculae. E, Detail of (D) showing an intense and poorly formed pyogranulomatous inflammatory infiltrate in the interlobular trabeculae composed mainly by macrophages and neutrophils with few lymphocytes and plasma cells. Several amastigote forms of Leishmania spp. (arrow and inset) are observed within the cytoplasm of macrophages (HE). F, Thymic cysts lined by ciliated epithelium in the parenchyma (HE). C, cortex; H , Hassall's corpuscle; HE, hematoxylin and eosin; M, medulla, T, interlobular trabecula; TC, thymic cyst

Figure 3

Analysis of the cortex:medulla ratio of the thymus from L. infantum‐naturally infected dogs. A,B, Representative images of the cortex:medulla ratio observed in two animals. C,D, Comparison of cortex:medulla ratio between groups according to clinical signs. E,F, Comparison of cortex:medulla ratio between groups according to parasite load

Figure 4

Analysis of laminin deposition in the thymus from L. infantum‐naturally infected dogs. A,B, Comparison of laminin deposition between groups according to clinical signs. C,D, Comparison of laminin deposition between groups according to parasite load

Figure 5

Analysis of fibronectin deposition in the thymus from L. infantum‐naturally infected dogs. A,B, Comparison of fibronectin deposition between groups according to clinical signs. C,D, Comparison of fibronectin deposition between groups according to parasite load

Figure 6

Correlations between (A) fibronectin deposition and clinical score and (B) laminin deposition and cortex:medulla ratio (Pearson correlation)