Histopathological and immunohistochemical study of the gastrointestinal tract from a dog naturally infected with Leishmania (Leishmania) chagasi: a case report

Histopatologia e imunoistoquímica do trato gastrintestinal em cão naturalmente infectado com Leishmania (Leishmania) chagasi: relato de caso

Histopathological and immunohistochemical study of the gastrointestinal tract from a dog naturally infected with Leishmania (Leishmania) chagasi: A case report

[Histopatologia e imunoistoquímica do trato gastrintestinal em cão naturalmente infectado com Leishmania (Leishmania) chagasi. Relato de caso]

F.L. Silva¹, W.L. Tafuri², M.R. Oliveira ³, Wg. L. Tafuri²

Departamento de Patologia Geral - Instituto de Ciências Biológicas

Universidade Federal de Minas Gerais

Av. Antônio Carlos, 6627

30270-901 - Belo Horizonte, MG

Recebido para publicação em 10 de agosto de 2001

Recebido para publicação, após modificações, em 17 de abril de 2002

E-mail: [e-mail]

ABSTRACT

Samples of stomach, duodenum, jejunun, ileum, cecum and colon were collected for Giemsa-smears ("imprints") from one asymptomatic mongrel dog, naturally infected with Leishmania (L) chagasi. Other fragments were obtained and fixed in formalin (10% and buffered) for histopathological and immunohistochemical studies. The immunohistochemistry was carried out by a streptavidin-peroxidase technique and it allowed to detect amastigote forms of Leishmania chagasi in the different paraffin gut sections. The principal lesion observed was a discrete to moderate chronic inflammatory reaction in the mucosa and submucosa in all fragments of the gastrointestinal tract (GIT). A chronic cellular exsudate was observed in all GIT tissues and it was composed by mononuclear cells (monocytes, plasmocytes and lymphocytes). A comparison between the two techniques showed that the immunohistochemistry study is the best method to detect amastigote forms of Leishmania.

Keywords: Dog, visceral leishmaniasis, histopathology, immunohistochemistry

RESUMO

Um cão assintomático e naturalmente infectado com Leishmania (L.) chagasi foi sacrificado e fragmentos do estômago, duodeno, jejuno, íleo, ceco e cólon foram coletados para confecção de esfregaços por aposição corados pelo Giemsa ("imprints"). Outros fragmentos foram obtidos e fixados em formol tamponado a 10% para estudos histopatológicos e imunoistoquímicos. Empregou-se a técnica imunoistoquímica de estreptavidina-peroxidase a qual possibilitou detecção de formas amastigotas de Leishmaniachagasi em todos os segmentos do trato gastrintestinal (TGI). A principal lesão observada foi a reação inflamatória crônica de intensidade variável, localizada principalmente na mucosa e submucosa de todos os segmentos do TGI. O exsudato celular era composto de monócitos, plasmócitos e linfócitos. O estudo imunoistoquímico mostrou a presença de amastigotas de Leishmania em todos os fragmentos do TGI. A pesquisa de parasitas não pôde ser observada de forma satisfatória pela técnica da HE. Concluiu-se que a técnica de imunoistoquímica como diagnóstico da doença no caso de envolvimento gastrintestinal é eficaz.

Palavras-chave:Cão, Leishmania chagasi, trato gastrintestinal, histopatologia, imunocitoquímica

INTRODUCTION

Visceral leishmaniasis is an endemic zoonotic disease, which affects men, dogs and several wild animals. The disease is endemic in tropical and subtropical areas, southern Europe, north Africa, sub-Saharan regions of east and west Africa, southwest Asia, and China (Tesh, 1995). In the New World the disease is caused by Leishmania (Leishmania) chagasi, which is transmitted bythe sandfly Lutzomyia (Lutzomyia) longipalpis. Dogs are the most important domestic reservoir and the life cycle of the parasite involves canids and phlebotomine sandflies. Leishmania is an intracellular protozoan parasite that is inoculated to the vertebrate host by the bite of an infected sandfly. Following injection into the skin, the extracellular promastigote form of the parasite rapidly enters its host cell, the macrophage. It is within macrophages that Leishmania survive and replicate as amastigotes, the intracellular form of the parasite. Amastigotes are responsible for the clinical manifestations of infection in the vertebrate host (Deane & Deane, 1962; Chang, 1979).

Canine visceral leishmaniasis has increased in suburban areas of major Brazilian cities such as Rio de Janeiro, Belo Horizonte, Fortaleza, Natal, Teresina, São Luís, Santarém and João Pessoa, where dogs seem to be the major reservoir of the parasite (Michalick et al., 1992; Marzochi et al., 1994). Classical canine leishmaniasis appears as a chronic disease with anaemia, generalised lymphadenomegaly, hepatosplenomegaly, onycogryphosis and cutaneous lesions, such as dry exfoliative dermatitis, ulcerations and alopecia (Slappendel & Greene, 1990; Ferrer et al., 1991; González et al., 1990; Ciaramella et al., 1997). Histopathological findings include hyperplasia and hypertrophy of mononuclear cells mainly observed in the spleen, liver, bone-marrow, lymph nodes and skin (Tryphonas et al., 1977; Keenan et al., 1984; Tafuri, 1995; Tafuri et al., 1996).

Intestinal disorders in dogs naturally (Tryphonas et al., 1977; Anderson et al., 1980; Slappendel, 1988; González et al., 1990; Ferrer et al., 1991) and experimentally (Keenan, 1984) infected have been reported to occur during visceral leishmaniasis. However, these reports have been limited to some clinical aspects and the nature of the inflammatory response of dogs infected mainly with Leishmania infantum.

The present study describes the pathological aspects of the gastrointestinal tract of one dog naturally infected with Leishmania (Leishmania) chagasi from João Pessoa, PB, Brazil.

CASUISTIC

An asymptomatic mongrel dog was selected by an epidemiological survey of canine visceral leishmaniasis from the City Hall (Zoonosis Department) of João Pessoa- Paraíba, based on indirect fluorescence antibody test (IFAT) and complement fixation reaction (CFR) test (Pellegrino & Brener, 1958).

The animal was considered asymptomatic, since no classical symptoms of the disease, such as a weight loss, anaemia, generalized lymphadenomegaly, onycogryphosis and cutaneous lesions were seen.

The parasites had been previously classified as Leishmania chagasi by PCR using conserved regions of kinetoplastidae and hybridization with kDNA probes to complex (data not shown) (Tafuri et al., 2001).

The dog was sacrificed with an overdose of thiopenthal sodic (ThionembutalÒ) (33%, 5ml/kg dose, IV). During necropsy, small samples of stomach, duodenum, jejune, ileum, cecum and colon were obtained for slides "imprint smears". Smears were stained with Giemsa and examined in an optical microscope (1000´ magnification) for detection of amastigotes of Leishmania. Other fragments from the same organs were fixed in 10% buffered formalin solution, dehydrated, cleared, embedded in paraffin, cut (3-4mm thick) and stained with hematoxilin and eosin for the histopathological characterization.

An immunocytochemical method was carried out for demonstration of Leishmania in paraffin tissue samples. Samples were stained using a biotin-streptoavidin peroxidase immunostainning technique. Heterologous serum collected from a dog naturally infected with Leishmania chagasi (IFAT > 1:40) from João Pessoa, PB, Brazil, diluted 1:100 was used as primary antibody. Deparaffinized tissue sections were rehydrated and incubated in 3% H₂O₂ in PBS (0,01M, pH 7.2) to block endogenous peroxidase activity, and then in normal goat serum to reduce non-specific binding by the secundary antibody. The primary antibody was applied (overnight at 4ºC, in a dry chamber) and the fragments were washed with PBS. Anti-mouse/rabbitt biotin labeled (Dako) antibody was used as a secondary antibody for 20 minutes at room temperature. The slides were washed again and incubated with the streptavidin peroxidase complex for 20 minutes at room temperature. The reaction was revealed with diaminobenzidine-hydrogen peroxidase reaction. The sections were counter stained with Mayer's Hematoxylin and mounted in Canadian balsam. As negative control infected serum was replaced by PBS, normal mouse serum and serum from dogs serologically negative to Leishmania. Fragments of spleen and liver from dogs naturally infected with Leishmania (Leishmania) chagasi were used as positive controls.

Macroscopical examination did not show any important lesions on the gastrointestinal tract. However, the microscopic study showed a chronic inflammatory reaction with parasites along gastrointestinal tract. The cellular exsudate included macrophages, lymphocytes and plasmocytes with rare neutrophils and eosinophils. Giant cell formations could be noted associated to the mononuclear exsudate. Many macrophages in the mucosa (lamina propria), muscularis mucosae and submucosa layers showed a peculiar morphology. They had a pale and abundant cytoplasm, with the nucleous less dense than other cells and intracellular amastigotes into vacuoles ("parasitophorus vacuole") (Fig. 1).

In the stomach, the inflammatory reaction was discrete foccused in the mucosa (lamina propria) in the proximity of the submucosa layer. The other intestinal segments showed an intense inflammatory reaction mainly in the duodenum mucosa and the jejune, ileum, cecum and colon mucosa and submucosa. Polipoides formation with intestinal villous enlargement was observed in the small intestine a result of the cellular exsudate presented on the mucosa layer.

Imunolabelled amastigote forms of Leishmania were observed along all gastrointestinal tract and parasites were seen inside macrophages of the mucosa, muscalaris mucosae and submucosa.

DISCUSSION

In this work, the main histopathological lesion observed along the gastrointestinal tract was a chronic inflammatory reaction associated to the presence of tissue amastigotes of Leishmania in all segments, as reported by other authors. González et al. (1990) and Ferrer et al. (1991), working with naturally infected dogs with Leishmania infantum, observed an inflammatory reaction in the colon mucosa and submucosa, but they did not indicate giant cell formation. Hervás et. al. (1996) also observed the same pathological picture in the mucosa and submucosa of the small and large intestines from a wild reservoir (jackal).

In the present study, the inflammatory lesions observed were very similar to those described in the literature regarding animals naturally infected with Leishmania infantum. This study also shows that the immunocytochemical reaction was more appropriate to detected parasites in the tissue than the hematoxilin-eosin stained smears. Immunolabelled parasites were easily detected in all segments and layers of the gastrointestinal tract, whereas, the microscopic analysis by hematoxilin-eosin staining was inappropriate to detect parasites in the gastrointestinal tissues.

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Publication Dates

• Publication in this collection
  16 Dec 2002
• Date of issue
  Aug 2002

History

• Received
  10 Aug 2001
• Accepted
  17 Apr 2002