Histological and immunohistochemical study of granuloma annulare and subcutaneous granuloma annulare in children

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Braz. J. vet. Res. anim. Sci., São Paulo, v. 44, n. 1, p. 5-11, 2007

Received: 03/02/2004Accepted: 06/02/2007

Histological and immunohistochemical study of thecentral nervous system of dogs naturally infected byLeishmania (Leishmania) chagasi

Fabiana Augusta IKEDA1

Márcia Dalastra LAURENTI2

Carlos Eduardo CORBETT2

Mary Marcondes FEITOSA3

Gisela Fabrino MACHADO3

Silvia Helena Venturoli PERRI3

Correspondece to:MARY MARCONDES FEITOSADepartamento de Clínica, Cirurgia eReprodução AnimalUniversidade Estadual PaulistaRua Clóvis Pestana, 793 – Bairro JardimDona Amélia16050-680 – Araçatuba – [email protected]

1 - Faculdade de Ciências Agrárias e Vaterinárias da Universidade EstadualPaulista, Jaboticabal - SP2 - Departamento de Patologia da Escola de Medicina da Universidade deSão Paulo, São Paulo - SP3 - Departamento de Clínica, Cirurgia e Reprodução Animal da UniversidadeEstadual Paulista, Araçatuba - SP

Abstract

The present study aimed to characterize the histopathologicalalterations and to detect, by immunohistochemistry, the presence ofamastigote forms of Leishmania in CNS tissue of dogs with andwithout neurological clinical signs of the disease. Two groups ofanimals were used: the first was composed of 18 dogs with visceralleishmaniasis without clinical evidence of neurological involvement,and the second, composed of 21 dogs with visceral leishmaniasis andneurological symptoms. The most frequent histopathologicalalterations found in the CNS of dogs of both groups were neuronaldegeneration with neuronophagia, gliosis, leptomeningitis, vascularcongestion, presence of perivascular lymphoplasmacytic infiltrate andareas of focal microhemorrhage. Antigen labeling for whole formsof Leishmania amastigotes was not observed in any fragment of theCNS of the dogs of either groups; however, most of them presentedlabeling of blood vessels walls, which suggests the presence ofcirculating parasite antigens.

Key-words:Leishmania chagasi.Dogs.Histopathology.Immunohistochemistry.Central nervous system.

Introduction

Visceral leishmaniasis, also known asKalazar, is an anthropozoonosis caused byprotozoa of the genus Leishmania. In Brazil,visceral leishmaniasis is caused by Leishmania(Leishmania) chagasi, which has already beenidentified in dogs, cats, wild canines,marsupials and rodents1,2,3,4. Transmissionbetween vertebrate hosts is carried out by thesting of the hematophagous phlebotomineLutzomyia longipalpis2,3,5,6 and, dogs, consideredthe main reservoir out of wildlife, are of greatimportance in maintaining the disease cycle.This importance comes from some facts:visceral leishmaniasis is more prevalent in thecanine population than in the humanpopulation, human cases are normallypreceded by canine cases, and dogs presenta greater amount of parasites in the skin than

humans, which favors the infestation ofvectors3,4,7.

Dog infection usually causes a chronicsystemic disease, which is characterized by longperiods of irregular fever, anemia, progressiveweight loss, and cachexia in the terminal stageof the disease1,2,3,4,7,8,9,10. Infected animals canalso present onychogryphosis, dermatological,ophtalmic, renal, and/or hepatic alterations,pneumonia, myocarditis, movement disorders,and hemorrhagic diathesis1,2,4,6,7,8,9,10,11,12,13,14,15,16.

In the infection by Leishmania, the cellsof the mononuclear phagocyte systeminfected by parasites act as antigen-presentingcells, which activate T-helper lymphocytes(CD4+) type 1 (TH1), or type 2 (TH2). Ifactivated, TH1 cells produce cytokines suchas interferon gamma (IFN-γ), tumor necrosisfactor (TNF), and interleukin 2 (IL-2), which,as they are considered proinflammatory,

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enhance the efficiency of phagocytic cellsand of cytotoxic lymphocytes. Activatedmacrophages, on their turn, produceoxygen-dependent free-radicals, which aretoxic to the parasites, and may lead toparasitism control with total elimination ofthe infection. On the opposite, when theinfection is associated with Type 2 T-helperlymphocyte (TH2), we have the productionof anti-inflammatory cytokines such asinterleukin 4 (IL-4), interleukin 5 (IL-5) andinterleukin 10 (IL-10), and the proliferation ofB cells with the production of antibodies. Theresulting response of the immunoglobulins isvery intense; however, it is deleterious and notprotective. Thus, the infection develops. Apotential danger of the impaired regulation ofT-lymphocytes and of the enhanced activityof B-lymphocytes is the generation of a greatamount of circulating immune complexes,which deposit on the walls of blood vessels,and may cause vasculitis, uveitis, dermatitis,polyarthritis, and glomerulonephritis. Thecirculating immune complexes may also bindthe complement to the blood cells, and thus,decrease their half life, and may be associatedto the development of pathologicalphenomena such as thrombocytopenia andimmune-mediated anemia4,10,13,17.

Literature presents contradictionsconcerning the pathogenesis of visceralleishmaniasis in the central nervous system(CNS). While some authors reported theobservation of parasites in all parts of thebody, except in the CNS10, others observedintense deposition of parasite antigens andimmunoglobulins in the interstitial andintravascular spaces of the choroid plexus indogs with visceral leishmaniasis. This lead tochoroiditis characterized by hypercellularity,capillary congestion, inflammatory cellinfiltrate, thrombosis, epithelial metaplasia, andbuildup of amyloid substance in thecapillaries18,19. Concerning the encephalonand cerebellum, satellitosis, neuronophagia,intense mobilization of glial cells,degeneration of pyramidal neurons, andpresence of amyloid buildup could beobserved. Neuronal degeneration and

necrosis were also observed, besides edemain the Virchow-Robbin space, whichcontained amyloid intravascular buildup4,10,18,19.

Thus, the main goal of the presentstudy was to characterize the CNShistopathological alterations, as well as toassess, by immunohistochemistry, thepresence of Leishmania amastigotes in theCNS tissue of dogs with parasitologicaldiagnosis of leishmaniasis, with and withoutclinical neurological signs of the disease, inorder to clarify the mechanisms of theneurological lesion in canine visceralleishmaniasis.

Materials and Method

Two groups of animals were used.The first was composed of 18 dogs withvisceral leishmaniasis without clinical evidenceof neurological involvement, and the secondwas composed of 21 dogs with visceralleishmaniasis and neurological symptoms.The animals had been referred to theVeterinary Hospital of the VeterinaryMedicine Program of the Paulista StateUniversity “Júlio de Mesquita Filho”-UNESP – Campus Araçatuba, from June,2000 to May, 2002.

Visceral leishmaniasis was diagnosedby lymph node aspiration biopsy and/or byELISA serology test. All animals weresubjected to a complete physical andneurological examination, and the data wereregistered in individual charts. After clinicalevaluation, the animals were anesthetized withsodium pentobarbital (Hypnol 3%) andeuthanized with a 10 mL ampule of 19.1%potassium chloride.

Anatomical pathological examinations

The dogs were necropsied, theencephalon and the bone marrow werecollected and evaluated with regards to thepresence of macroscopic alterations. Afterthe separation in two brain hemispheres, oneof them was fixed in 10% phosphatebuffered formalin pH 7.2. After a fixationperiod of approximately 24 hours, fragments

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of the frontal cortex, lateral ventricle,piriform lobe, mesencephalon, cerebellarpeduncle, cerebellum, cervical medulla,thoracic medulla, and lumbar medulla werecollected. The fragments were embeddedin paraffin and subjected to the routinehistopathological procedure. Five-to six-micrometer-thick sections were stained bythe hematoxylin-eosin method.

Imunnohistochemistry

The immunohistochemical reaction wascarried out to search for Leishmania amastigotesand antigens in the CNS by the Streptavidin-Peroxidase method. The sections weredeparaffinized in a xylol wash at 60oC and re-hydrated in absolute alcohol washes of decreasingconcentrations, 95% and 70%, respectively.Antigen recovery was performed with thetreatment of the sections in citrate buffer (3M),using a pressure cooker. Then several blockageswere performed, such as of endogenousperoxidase, when hydrogen peroxide was usedin association with methanol, and of secondaryantibody, using normal serum of the same animalspecies from which the antibody was produced.Posteriorly, overnight incubation was carried outwith anti-Leishmania primary polyclonal antibody,adsorbed with normal dog CNS powder,produced in mouse by the Laboratory ofPathology of Infectious Diseases of theDepartment of Pathology of the Medical Schoolof the University of São Paulo. After incubationwith secondary biotinylated antibody, incubationwith Streptavidin-Peroxidase was carried out. Thefinal reaction was revealed with hydrogenperoxide (H

2O2) in the presence of

Diaminobenzidina (DAB). Tissue counterstainingwas performed with Harris hematoxylin. Aftersection drying, the slides were mounted withcoverslips using resin and were observed underan ordinary optical microscope to assess thepresence and localization of Leishmaniaamastigotes and antigens.

Results

The main clinical neurologicalmanifestations observed in the animals were

generalized seizures; visual alterations such asblindness, anisocoria and bilateral mydriasis;signs of paralysis of cranial nerves (strabismus,facial ptosis, dysphagia); signs of vestibularand cerebellar involvement (head tilting,nystagmus, motor incoordination, falls,intention tremor); paraparesis, tetraparesis andtetraplegia; myoclonias; vocalization; walkingin circles, and episodes of tail chasing. Thesigns observed denote generalized CNSinvolvement, which reached the cerebralcortex, the encephalic trunk, the cerebellum,and the bone marrow.

The histopathological alterations mostfrequently found in the CNS of dogsnaturally infected by L. (L.) chagasi with andwithout neurological symptoms wereneuronal degeneration with neuronophagia,gliosis, which varied from focal to diffuse,leptomeningitis, vascular congestion andpresence of perivascular lymphoplasmacyticinfiltrate, and areas of focal microhemorrhages,among others. The number and percentage ofanimals in which the most frequenthistopathological alterations were observedare shown in table 1. Significant differenceswere found regarding the number of animalswith and without neurological symptoms thatpresented dark neurons, which characterizeneuronal degeneration, diffuse gliosis andperivascular lymphoplasmacytic infiltrate. Agreater number of animals with thosehistopathological alterations were found inthe group with neurological symptoms (p<0.05)

The immunohistochemistry reactionfor parasite detection was carried out insections of the same regions of the CNSpreviously stained with hematoxylin-eosin(HE): frontal cortex (FC), lateral ventricle(LV), piriform lobe (PL), mesencephalon(ME), cerebellar peduncle (CP), cerebellum(CE), cervical medulla (CM), thoracicmedulla (TM), and lumbar medulla (LM).Figures 1 and 2 show photomicrographs ofskin of dogs naturally infected by Leishmaniachagasi that were used as positive and negativecontrols of the immunohistochemicalreaction, respectively. Antigen labeling for

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Table 1 - Main histopathological alterations observed in the CNS fragments, stained with hematoxylin-eosin (HE), in dogs with visceral leishmaniasis with andwithout neurological symptoms, and descriptive level between control group and group with neurological symptoms (Araçatuba – SP, 2004)

(1) descriptive level of Fisher’s exact test (2) descriptive level of Qui-square test.

whole forms of Leishmania amastigotes wasnot observed in any fragment of the CNSof the dogs of either group; however, mostof them presented labeling of blood vesselswalls, which suggests the presence ofcirculating parasite antigens (Figure 3).

Discussion

Medical literature has scarceinformation concerning the description ofneurological symptoms associated to visceralleishmaniasis. Chunge, Gachihi and Muigai20

reported the occurrence of generalizedtremors in a patient with visceralleishmaniasis, and verified that the severityof the tremors was directly related to theparasitic load, for when parasite count wasnegative, indicating disease cure, the tremorsdisappeared. Despite these observations, thepathogenesis of the neurological involvementremained undetermined. Karak et al.21,described a case of a patient with visceralleishmaniasis with symptoms characteristic ofunilateral cerebellar disorder and unilateralfacial nerve paralysis, associated to a herpeszoster infection. In that case, the clinical signs

were justified by the presence of viralcerebellitis and peripheral neuropathy. In thedogs with meningoencephalitis associated toleishmaniasis, the signals observed denotegeneralized CNS involvement, which reachesthe cerebral cortex, encephalic trunk,cerebellum, and bone marrow.

Many histopathological alterationswere observed in the CNS of the animals inthe control group. This corroborates thefindings of Garcia-Alonso et al.18, whoverified a clear pathological reaction in theCNS of dogs with visceral leishmaniasiswithout neurological symptoms. However, thehistopathological alterations in the animals withneurological symptoms were more frequentand more intense. The histopathologicalalterations more frequently observed in theanimals in the control group were:leptomeningitis, satellitosis and neuronophagia,and dark neurons. On the other hand, thehistopathological alterations more frequentlyobserved in the animals with neurologicalsymptoms were leptomeningitis, satellitosis andneuronophagia, focal microgliosis, diffusegliosis, and dark neurons, which corroboratesthe reports of Garcia-Alonso et al.18 and

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Figure 1 - Photomicrograph of skin of a dog naturally infected by Leishmania chagasi, used as negative control of the immunohistochemicalreaction in which normal mouse serum was used as primary antibody. Note the absence of antigen labeling. (LSAB, AO 20x)

Figure 2 - Photomicrograph of skin of a dog naturally infected by Leishmania chagasi, used as positive control of the immunohistochemicalreaction. Note the dark brown antigen labeling with no interference of nonspecific labeling. (LSAB, AO 20x)

Figure 3 - Photomicrograph of frontal cortex of a dog naturally infected by Leishmania chagasi with neurological symptoms. Note antigen labelinginside blood vessels. (LSAB, AO 40x)

of Viñuelas et al.22. All the neurologicalsymptoms observed in the dogs werecompatible with the lesions observed in thehistopathological examination, althoughthere was no correlation between theintensity of the lesions and the severity ofthe clinical picture.

The immunohistochemical methoddid not evidence the presence of amastigoteforms of the parasite in the CNS of theanimals naturally infected by L. (L.) chagasiwith and without neurological symptoms;however, it showed antigen labeling in theblood vessel walls. Our findings corroboratethose described by Garcia-Alonso et al.18,who reported the presence of intenseLeishmania antigen deposits in the choroidplexus of two dogs with visceral

leishmaniasis without neurological symptoms.Those findings suggest that the neurologicalpicture associated to visceral leishmaniasis inthe animals studied is due to the buildup ofimmune complexes, as occurs in other partsof the organism. Most CNS sections of thedogs in this study, when subjected to theimmunohistochemical method, showedlabeling inside the blood vessels, which mayhave occurred due to the presence ofcirculating Leishmania antigens.

Prasad and Sen23 described chronicmeningitis in a 10-year-old boy with visceralleishmaniasis. According to the authors, theneurological involvement started one yearafter the diagnosis of the disease. The specifictreatment was not successful and there wereseveral recurrence episodes within a period

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of 20 months. In that period, several samplesof CSF were collected from the patient, andin none of them there was evidence of thepresence of amastigote forms of Leishmaniain the CNS; only after splenectomy parasiticforms were observed in the CSF samples.The authors argue that, probably, aftersplenectomy the parasites migrated to the CSF,a safe site for proliferation, as it was notreached by most anti-Leishmania drugs that donot cross the hematoencephalic barrier. Thiscould explain why repeated treatmentprotocols did not cure the patient. Accordingto the authors, this was the first report inliterature about the presence of Leishmania inthe CSF of a patient with visceral leishmaniasis.

Despite some literature descriptions ofthe presence of amastigote forms of

Leishmania in the CNS of dogs with visceralleishmaniasis18,22, we did not detect the parasitein those tissues. Those findings were reportedmainly in Europe - Spain and Italy - wherethe dogs are usually treated of visceralleishmaniasis. However, none of the reportsmention the treatment the dogs underwentbefore euthanasia. If we follow the reasoningof Prasad and Sen23, a possible explanationfor the appearance of parasites in the CNSof those dogs would be the attempt of theparasite to find a safer site in the organismwhere it could be less vulnerable to the attacksof the drugs used. This would also explainwhy the parasite was absent in our animals, asnone of them were treated of the disease,having been euthanized as soon as the diseasewas diagnosed.

Resumo

O objetivo do presente estudo foi caracterizar as alteraçõeshistopatológicas e detectar por imunoistoquímica, a presença deformas amastigotas de Leishmania no sistema nervoso central de cãescom e sem sintomatologia neurológica da doença. Foram utilizadosdois grupos de animais; o primeiro foi composto de 18 cães comleishmaniose visceral sem evidência clínica de envolvimentoneurológico, e o segundo, composto de 21 cães com leishmaniosevisceral e sintomas neurológicos. As mais freqüentes alteraçõeshistopatológicas encontradas no sistema nervoso central de cães deambos os grupos foram degeneração neuronal com neuronofagia,gliose, leptomeningite, congestão vascular, presença de infiltradolinfoplasmocitário perivascular e áreas de microhemorragia. Não foramobservadas marcações antigênicas para formas amastigotas deLeishmania em nenhum fragmento do sistema nervoso central de cãesde ambos os grupos; entretanto, a maioria deles apresentava marcaçãoem parede de vasos sangüíneos, o que sugere a presença de antígenosparasitários circulantes.

Estudo histológico e imunoistoquímico do sistema nervoso central decães naturalmente infectados por Leishmania (Leishmania) chagasi

Palavras-chave:Leishmania chagasi.Cães.Histopatologia.Imunoistoquímica.Sistema Nervoso Central.

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Histological and immunohistochemical study of granuloma annulare and subcutaneous granuloma annulare in children