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Histopathological study in intestinal wall of dogs naturally infected with Leishmania in the intestines.

Wilma A. Starke-Buzetti1*, Diogo T. Silva1, Aline G. Lins1, Maria F. Neves1 and Nina Mari G. Queiroz1
  • 1 São Paulo State University-UNESP, Biologia e Zootecnia, Brazil

ABSTRACT
Canine Visceral Leishmaniasis (CVL) is caused by a protozoa parasite of the specie Leishmania (L.) infantum, endemic for humans and dogs in many regions of Brazil. For this study, the histopathological alterations in intestinal tissues from a group of naturally CVL infected dogs were evaluate by histo and immunohistochemical techniques. Leishmania amastigotes as well as the inflammatory cells detected in the intestinal tissues of dogs were quantified in the wall layers of small and large intestines, including mucosa, submucosa and muscular. A chronic infiltrate of mononuclear cells, particularly macrophages, lymphocytes and plasma cells was observed in the most intensely infected dogs. In contrast, mast cells, neutrophils and esosinophils were detected in dogs with lower parasitic burden, but with hemorrhagic enteritis. In conclusion, positive dogs by intestinal leishmaniasis with high parasitic load had a chronic mononuclear inflammation, while dogs with low intestinal parasitic burden had an acute inflammatory reaction characterized by hemorrhagic enteritis, mast cell and polymorph nuclear cell hyperplasia.
Key words: Leishmania (L.) infantum, Canine Visceral Leishmaniasis, dogs, intestines, mast cells, .polymorphonuclear cells.
INTRODUCTION
Canine Visceral Leishmaniasis (CVL) is a disease caused by protozoa of the genus Leishmania (Ross, 1903). The species Leishmania infantum (syn. L. chagasi) is the causative agent of visceral leishmaniasis in the New World with endemic regions extending from southern USA to northern Argentina, including Brazil (Kuhls et al., 2011). Inflammatory reactions in the gastrointestinal tract associated with the presence of intramacrophagic Leishmania amastigotes were reported in experimentally infected dogs (Keenan et al., 1984a, b) and in naturally infected dogs (Ferrer et al., 1991; Pinto et al., 2011).
The purpose of the present study was to evaluate the histopathological alterations in intestinal tissues from naturally infected dogs in a group of polisymptomatic CVL-animals.
MATERIALS AND METHODS
According to the Brazilian Ministry of Health (Interministerial Ordinance 1426 of July 11, 2008), animals infected with L. (L.) infantum must be submitted to euthanasia through the CVL Surveillance and Control Program. For the present study, intestinal tissue samples from euthanized polisymptomatic dogs for CVL were donated by the Zoonotic Disease Control Center (CCZ) from Ilha Solteira, São Paulo, Brazil.
The intestinal tissue samples were fixed in 0.01 M phosphate-buffered 10% formaldehyde solution for routine histological and immunohistochemical examination. For mast cell and eosinophil identification and quantification in the intestinal wall of the dogs the tissue samples were stained by Astra blue and Direct Red according to Duffy et al. (1993) and for neutrophils, hematoxylin and eosin (HE) was the appropriate staining. The presence of the parasite was evaluated under an optical microscope (objective lenses of 40x or 100x).
The immunohistochemical staining was done according to Tafuri et al. (2004 and Queiroz et al (2011), which the primary antibody was the hyperimmune serum from a naturally infected dog with L. (L.) infantum (IFAT; titer = 1: 2560). The secondary antibody was a biotinylated goat anti-rabbit reagent (canine cross-immune reaction) and the solution of avidin-biotin-complex peroxidase and with 3.3’ diaminobenzidine substrate were used for the reaction detection.
Positively immunostained macrophages containing amastigote profiles, mast cells, eosinophils and neutrophils were identified under the light microscope and these cells were counted in the lamina propria of the villi and the crypts (mucosa), submucosa and smooth muscle layer (tunica muscularis externa) of small and large intestines.
RESULTS AND DISCUSSION
The histopathogical analysis revealed that 5/11 (45.5%) of these polisymptomatic dogs had intramacrophagic L. (L.) infantum amastigotes in the wall of duodenum, jejunum, ileum and colon. Macrophages containing Leishmania amastigotes were found in the lamina propria of the mucosa in the majority of the cases (60-80%) in duodenum, jejunum, ileum and colon, most intensely infected in the mucosa. Few immunostained amastigotes were also noted in the circular smooth muscle layers of small intestines. Intramacrophagic Leishmania amastigotes have been reported in experimentally infected dogs (Keenan et al., 1984a; Keenan et al., 1984b) and in naturally infected dogs (Ferrer et al., 1991; Pinto et al., 2011) in agreement with our work.
Figure 1 shows numerous intramacrophagic amastigotes of L. infantum identified by immunohistochemistry in brown color, at the villus tips of the ileum, in a dog, which had an intense parasitic load.
Those dogs more intensely infected (60%) in the intestinal tissues showed a chronic inflammatory reaction evident in the submucosa represented by macrophages and numerous parasites; however, these animals had no evident diarrhea or any severe histopathological alterations in the mucosa. In contrast, 40% of dogs with hemorrhagic enteritis diarrhea had low intestinal parasitic loads. These animals showed a polymorphic inflammatory infiltration represented by intense eosinophilic and neutrophilic hyperplasia indicative of an acute rather than a chronic inflammation.
Mast cells are important effector cells in the immune response against parasite infections. Earlier studies have documented extensive mast cell degranulation at sites of L. major infection (Grilmadi et al., 1984). Here, mast cells were counted in all layers of the intestinal wall, including, mucosa, submucosa and muscular; with predominance of these cells in duodenum, followed by jejunum, ileum and colon, particularly in the mucosa and submucosa. But, the mast cells were not directly associated with the burden of Leishmania amastigotes in intestinal tissues of these infected dogs, probably because the most animals were suffering with a chronic inflammatory infection, which macrophages were the predominant cell.
Regarding about eosinophils, Grimaldi et al. (1984) showed accumulation of eosinophils and mast cells together with some mononuclear phagocytes in experimentally infected mice with Leishmania amastigotes in the early acute phase of the infection in the skin. Our study reported abundant eosinophils, particularly in the ileum, but not related with the presence of amastigote forms of the parasite. In contrast higher numbers of these cells were found in dogs with absence or low parasitic burden.
Neutrophils are also important effector cells related with leishmaniasis. In the present work, the quantification of neutrophils showed similar pattern of eosinophilic counting. Low numbers of eosinophils and neutrophils were observed in dogs with great burden of parasites suggesting a chronic phase of the disease with predominance of mononuclear cells. However, as compared to macrophages, the role of polymorphic nuclear cells (eosinophils and neutrophils) for immunity against Leishmania is less clear, particularly in intestinal tissues and needs further investigation.
This paper reports the intestinal inflammatory reaction in naturally infected dogs with amastigote forms of L. (L.) infantum. A chronic inflammatory reaction was correlated with a high parasitic burden represented by an infiltration of mononuclear cells, mainly by macrophages, while an acute inflammatory reaction was characterized by low parasitic burden, hemorrhagic enteritis, mast cell and polymorphic nuclear cell hyperplasia.

FIGURE CAPTION
Figure 1: Intestinal section showing the lamina propria at the tip of a villus from ileum tissue of a CVL-positive dog, showing numerous immunostained macrophages filled

Acknowledgements

To Brazilian Research Funding Agency FAPESP and the Zoonotic Disease Control Center (CCZ) of Ilha Solteira.

References

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Keywords: Canine Visceral Leishmaniasis, Dogs, intestines mast cells, polymorphonuclear leukocyte, Histopathological

Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.

Presentation Type: Abstract

Topic: Host-pathogen interactions

Citation: Starke-Buzetti WA, Silva DT, Lins AG, Neves MF and Queiroz NG (2013). Histopathological study in intestinal wall of dogs naturally infected with Leishmania in the intestines.. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01088

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Received: 28 Jun 2013; Published Online: 22 Aug 2013.

* Correspondence: Dr. Wilma A Starke-Buzetti, São Paulo State University-UNESP, Biologia e Zootecnia, Ilha Solteira, SP, 15385-000, Brazil, starke@bio.feis.unesp.br