Glycol methacrylate embedding for the histochemical study of the gastrointestinal tract of dogs naturally infected with Leishmania infantum

Abstract

In canine visceral leishmaniasis a diffuse chronic inflammatory exudate and an intense parasite load throughout the gastrointestinal tract (GIT) has been previously reported. However, these studies did not allow a properly description of canine cellular morphology details. The aim of our study was to better characterize these cells in carrying out a qualitative and quantitative histological study in the gastrointestinal tract of dogs naturally infected with Leishmania infantum by examining gut tissues embedded in glycol methacrylate. Twelve infected adult dogs were classified in asymptomatic and symptomatic. Five uninfected dogs were used as controls. After necropsy, three samples of each gut segment, including oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum were collected and fixed in Carnoy’s solution for glycol methacrylate protocols. Sections were stained with hematoxylin-eosin, toluidine blue borate, and periodic acid-Schiff stain. Leishmania amastigotes were detected by immunohistochemistry employed in both glycol methacrylate and paraffin embedded tissues. The quantitative histological analysis showed higher numbers of plasma cells, lymphocytes and macrophages in lamina propria of all segments of GIT of infected dogs compared with controls. The parasite load was more intense and cecum and colon, independently of the clinical status of these dogs. Importantly, glycol methacrylate embedded tissue stained with toluidine blue borate clearly revealed mast cell morphology, even after mast cell degranulation. Infected dogs showed lower numbers of mast cells in all gut segments than controls. Despite the glycol methacrylate (GMA) protocol requires more attention and care than the conventional paraffin processing, this embedding procedure proved to be especially suitable for the present histological study, where it allowed to preserve and observe cell morphology in fine detail.

The Roles of Mast Cells in Parasitic Protozoan Infections

“…Analysis of Th1, Th2, and Th17 cytokine responses by cultured peripheral blood mononuclear cells from patients who had developed kala-azar caused by Leishmania donovani , or who were protected against kala-azar, showed that IL-17 and IL-22 are the cytokines most strongly associated with protection against kala-azar (70). It has been reported that higher numbers of plasma cells, lymphocytes, and Mφ but lower number of MCs are present in the lamina propria of gastrointestinal tract of dogs naturally infected with L. infantum compared to non-infected controls, in all gastrointestinal tract segments (31). Thus, MCs may play different roles in visceral leishmaniasis and cutaneous leishmaniasis.…”