Anatomomopathological and immunohistochemical analyses of the spleen and lymph node of dogs seropositives for leishmaniasis in serological tests

Aline Oliveira de Magalhães Lucilandia Maria Bezerra Diego Pereira Araújo Bruna Siqueira Gomes de Lima Leandro do Padro Assunção Eduardo de Paula Nascente Ana Paula Iglesias Santin Rodrigo Caldas Menezes Veridiana Maria Brianezi Dignani de Moura About the authors

Abstract

Canine leishmaniasis (CanL) is a zoonosis caused by the protozoan of the species Leishmania infantum. The spleen and lymph nodes undergo morphological changes during CanL. This research aimed to perform an anatomopathological and immunohistochemical study of these organs in dogs reactive to leishmaniasis in the Dual-path Platform chromatographic immunoassay (DPP®) and Enzyme Immunoabsorption Assay (ELISA). Twenty-seven dogs were evaluated for anatomopathological examination with 92.6% showing changes at gross evaluation, specially splenomegaly and lymphadenomegaly. All dogs showed changes in the spleen unrelated to the parasitic load, with granulomatous splenitis being the most severe change. Diffuse cortical and paracortical hyperplasia, and hyperplasia and hypertrophy of the medullary cords were observed in the lymph node. Amastigote forms of Leishmania spp. were found in the spleen and lymph node at histopathological and immunohistochemical evaluations, with good agreement between these evaluations (k = 0.55, p = 0.00124), but no difference was observed in the parasitic intensity of these organs at immunohistochemistry (p = 0.23). It was concluded that spleen and lymph node from dogs reactive to leishmaniasis on the DPP® and ELISA tests show histomorphological changes resulting from the disease, independent to the parasitic load, as well as these organs show similar parasitic load at immunohistochemical test.

Keywords:
amastigote; histopathology; immunostaining; Leishmania spp.; lymphoid tissue

Resumo

A leishmaniose canina (CanL) é uma zoonose causada pelo protozoário da espécie Leishmania infantum. O baço e os linfonodos sofrem alterações morfológicas durante o CanL. Esta pesquisa teve como objetivo realizar um estudo anatomopatológico e imuno-histoquímico desses órgãos em cães reativos para leishmaniose aos testes de Imunoensaio Cromatográfico “Dual Path Platform” (DPP®) e Ensaio de Imunoabsorção Enzimática (ELISA). Vinte e sete cães foram avaliados ao exame anatomopatológico, com 92,6% exibindo alterações à avaliação macroscópica, especialmente esplenomegalia e linfadenomegalia. Todos os cães apresentaram alterações no baço não relacionadas à carga parasitária, sendo a esplenite granulomatosa a alteração mais grave. Hiperplasia cortical e paracortical difusa e hiperplasia e hipertrofia dos cordões medulares foram observadas nos linfonodos. Formas amastigotas de Leishmania spp. foram encontradas no baço e linfonodo às avaliações histopatológica e imuno-histoquímica, com boa concordância entre os métodos (k = 0,55, p = 0,00124), mas não foi observada diferença na intensidade parasitária entre esses órgãos à imuno-histoquímica (p = 0,23). Conclui-se que baço e linfonodo de cães reativos para leishmaniose aos testes DPP® e ELISA apresentam alterações histomorfológicas decorrentes da doença, independente da carga parasitária, assim como esses órgãos apresentam carga parasitária semelhante ao método imuno-histoquímico.

Palavras-chave:
amastigota; histopatologia; imunocoloração; Leishmania spp.; tecido linfoide

Introduction

Canine leishmaniasis (CanL) is a systemic and zoonotic disease(11 Brasil, Ministério da Saúde. Manual de vigilância e controle da leishmaniose visceral. Ministério da Saúde, Brasília, Distrito Federal. 2014. 120p.,22 World Health Organization. Control of the leishmaniases. World Health Organization technical report series. 2010. 186p.). The parasite that causes the disease is transmitted specially by the bite of infected phlebotomines, which is represented in Brazil by the species Lutzomyia longipalpis(33 Coura-Vital W, Leal CGA, Marques LA, Pinheiro AC, Carneiro M, Reis AB. Effectiveness of deltamethrin-impregnated dog collars on the incidence of canine infection by Leishmania infantum: A large scale intervention study in an endemic area in Brazil. PLoS One [Internet]. 2018 [cited 2020 Dec 10]; 13(12): e0208613. Available from: https://doi.org/10.1371/journal.pone.0208613. English.
https://doi.org/10.1371/journal.pone.020...
) and Lutzomyia cruzi(11 Brasil, Ministério da Saúde. Manual de vigilância e controle da leishmaniose visceral. Ministério da Saúde, Brasília, Distrito Federal. 2014. 120p.) transmitting the protozoan of the species Leishmania infantum(44 Belo VS, Werneck GL, Barbosa DS, Simões TC, Nascimento BWL, Silva ES, et al. Factors Associated with Visceral Leishmaniasis in the Americas: A Systematic Review and Meta-Analysis. PLoS Negl Trop Dis. [Internet] 2013 [cited 2020 Dez 12]; 7(4):e2182. Available from: https://doi.org/10.1371/journal.pntd.0002182. Englisg.
https://doi.org/10.1371/journal.pntd.000...
). The dog (Canis familiaris) plays an important role in maintaining the disease, as it acts as a domestic reservoir for visceral leishmaniasis (VL) in urban areas (11 Brasil, Ministério da Saúde. Manual de vigilância e controle da leishmaniose visceral. Ministério da Saúde, Brasília, Distrito Federal. 2014. 120p.,44 Belo VS, Werneck GL, Barbosa DS, Simões TC, Nascimento BWL, Silva ES, et al. Factors Associated with Visceral Leishmaniasis in the Americas: A Systematic Review and Meta-Analysis. PLoS Negl Trop Dis. [Internet] 2013 [cited 2020 Dez 12]; 7(4):e2182. Available from: https://doi.org/10.1371/journal.pntd.0002182. Englisg.
https://doi.org/10.1371/journal.pntd.000...
,55 Oliveira VC, Boechat VC, Mendes Junior AAV, Madeira MF, Ferreira LC, Figueiredo FB, et al. Occurrence of Leishmania infantum in the central nervous system of naturally infected dogs: Parasite load, viability, co-infections and histological alterations. PLoS One [Internet]. 2017 [cited 2021 Jan 21]; 12(4):e0175588. Available from: https://doi.org/10.1371/journal.pone.0175588. Englisg.
https://doi.org/10.1371/journal.pone.017...
).

These protozoa are intracellular parasites that infect cells of the mononuclear phagocytic system in the vertebrate host(66 Tafuri WL, Michalick MS, Dias M, Genaro O, Leite VH, Barbosa AJ, et al. Optical and electron microscopic study of the kidney of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi. Rev Inst Med Trop Sao Paulo. 1989; 31(3):139-145.), being spread mainly to lymph nodes, spleen, bone marrow, and liver(77 Keenan CM, Hendricks LD, Lightner L, Johnson AJ. Visceral Leishmaniasis in the German Shepherd Dog. II. Pathology. Vet Pathol. 1984; 21:80-86.). The clinical signs of the disease result from the parasite interaction with the host’s immune system, and dogs susceptible to infection develop clinical signs due to a marked cellular and humoral immune response against the parasite(88 Almeida MAO, Jesus EEV, Sousa-Atta MLB, Alves LC, Berne MEA, Atta AM. Antileishmanial antibody profile in dogs naturally infected with Leishmania chagasi. Vet Immunol Immunopathol. [Internet]. 2005 [cited 2021 Jan 21]; 106:151-158. Available from: https://doi.org/10.1016/j.vetimm.2004.08.024. English.
https://doi.org/10.1016/j.vetimm.2004.08...
,99 Maia C, Campino L. Cytokine and phenotypic cell profiles of leishmania infantum infection in the dog. Journal Trop Med. [Internet]. 2012 [cited 2021 jan 23]. 2012: 541571. Available from: https://doi.org/10.1155/2012/541571. English.
https://doi.org/10.1155/2012/541571...
).

CanL is a progressive disease that causes a chronic inflammatory reaction consisting of macrophages, plasma cells, and lymphocytes(1010 Lima WG, Michalick MSM, Melo MN, Washington LT, Wagner LT. Canine visceral leishmaniasis: A histopathological study of lymph nodes. Acta Trop. [Internet]. 2004 [cited 2021 Jan 15]; 92:43-53. Available from: https://doi.org/10.1016/j.actatropica.2004.04.007. English.
https://doi.org/10.1016/j.actatropica.20...
,1111 Silva LC, Castro RS, Figueiredo MM, Michalick MSM, Washington LT, Wagner LT. Canine visceral leishmaniasis as a systemic fibrotic disease. International Jour Exp Pathol. [Internet]. 2013 [cited 2021 Jan 04]; 94:133-143. Available from: https://doi.org/10.1111/iep.12010. English.
https://doi.org/10.1111/iep.12010...
), with tissue remodeling and repair(1111 Silva LC, Castro RS, Figueiredo MM, Michalick MSM, Washington LT, Wagner LT. Canine visceral leishmaniasis as a systemic fibrotic disease. International Jour Exp Pathol. [Internet]. 2013 [cited 2021 Jan 04]; 94:133-143. Available from: https://doi.org/10.1111/iep.12010. English.
https://doi.org/10.1111/iep.12010...
). Affected dogs commonly develop lymphadenomegaly(1010 Lima WG, Michalick MSM, Melo MN, Washington LT, Wagner LT. Canine visceral leishmaniasis: A histopathological study of lymph nodes. Acta Trop. [Internet]. 2004 [cited 2021 Jan 15]; 92:43-53. Available from: https://doi.org/10.1016/j.actatropica.2004.04.007. English.
https://doi.org/10.1016/j.actatropica.20...
,1212 Cavalcanti AS, Ribeiro-Alves M, Pereira LOR, Mestre GL, Ferreira ABR, Morgado FN, et al. Parasite load induces progressive spleen architecture breakage and impairs cytokine mRNA expression in Leishmania infantum naturally infected dogs. PLoS One [Internet]. 2015 [cited 2021 Jan 20]; 10(4):e0123009. Available from: https://doi.org/10.1371/journal.pone.0123009. English.
https://doi.org/10.1371/journal.pone.012...
), splenomegaly, hepatomegaly, anemia, and cutaneous lesions(1212 Cavalcanti AS, Ribeiro-Alves M, Pereira LOR, Mestre GL, Ferreira ABR, Morgado FN, et al. Parasite load induces progressive spleen architecture breakage and impairs cytokine mRNA expression in Leishmania infantum naturally infected dogs. PLoS One [Internet]. 2015 [cited 2021 Jan 20]; 10(4):e0123009. Available from: https://doi.org/10.1371/journal.pone.0123009. English.
https://doi.org/10.1371/journal.pone.012...
). Spleen and lymph nodes are among the most affected organs in CanL, participating in specific immune responses(13,14) and where the presentation of antigens occurs(1414 Silva AV, Souza TL, Figueiredo FB, Mendes AAV, Ferreira LC, Filgueira COB, et al. Detection of amastigotes and histopathological alterations in the thymus of Leishmania infantum ‐infected dogs. Immun Inflamm Dis. [Internet]. 2020 [cited 2021 Jan 10]; 8:127-139. (https://doi.org/10.1002/iid3.285). English.
https://doi.org/10.1002/iid3.285...
,1515 Mebius RE, Kraal G. Structure and function of the spleen. Nat. Rev. Immunol. [Internet]. 2005 [cited 2021 Jan 10]; 5:606-616. Available from: https://doi.org/10.1038/nri1669. English.
https://doi.org/10.1038/nri1669...
).

The spleen is a lymphoid organ that directly influences the outcome of the infection(1616 Santana CC, Vassallo J, Freitas LAR, Oliveira GGS, Carvalho LCP, Santos WLS. Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: A study on naturally infected dogs. Parasite Immunol. [Internet]. 2008 [cited 2021 Jan 17]; 30:515-524. Available from: https://doi.org/10.1111/j.1365-3024.2008.01051.x. English.
https://doi.org/10.1111/j.1365-3024.2008...
). The white pulp is divided into a periarteriolar lymphatic sheath (PALS), nodular lymphoid follicles (NLF), and marginal zone (MZ). T lymphocytes predominate in PALS and B lymphocytes in NLF(1717 Lima IS, Silva JS, Almeida VA, Junior FGL, Souza PAN, Laranjeira DF, et al. Severe clinical presentation of visceral leishmaniasis in naturally infected dogs with disruption of the splenic white pulp. PLoS One [Internet]. 2014 [cited 2020 Jan 28]; 9(2)e87742. Available from: https://doi.org/10.1371/journal.pone.0087742. English.
https://doi.org/10.1371/journal.pone.008...
). The structure of the splenic lymphoid tissue allows the movement and differentiation of lymphocytes involved in immune responses and enables the ideal location of memory cells to respond to antigens. Follicular atrophy, reduction of lymphoid tissue boundaries(1818 Silva JS, Andrade AC, Santana CC, Santos LQ, Oliveira CI, Veras PST, et al. Low CXCL13 expression, splenic lymphoid tissue atrophy and germinal center disruption in severe canine visceral leishmaniasis. PLoS One [Internet]. 2012 [cited 2020 Jan 26]; 7(1): e29103. Available from: https://doi.org/10.1371/journal.pone.0029103. English.
https://doi.org/10.1371/journal.pone.002...
), cellular hyperplasia and hypertrophy, granulomatous inflammatory reaction, high parasitic load, and fibrosis(1414 Silva AV, Souza TL, Figueiredo FB, Mendes AAV, Ferreira LC, Filgueira COB, et al. Detection of amastigotes and histopathological alterations in the thymus of Leishmania infantum ‐infected dogs. Immun Inflamm Dis. [Internet]. 2020 [cited 2021 Jan 10]; 8:127-139. (https://doi.org/10.1002/iid3.285). English.
https://doi.org/10.1002/iid3.285...
) are among the splenic changes in CanL.

Lymph nodes are also affected by CanL(1414 Silva AV, Souza TL, Figueiredo FB, Mendes AAV, Ferreira LC, Filgueira COB, et al. Detection of amastigotes and histopathological alterations in the thymus of Leishmania infantum ‐infected dogs. Immun Inflamm Dis. [Internet]. 2020 [cited 2021 Jan 10]; 8:127-139. (https://doi.org/10.1002/iid3.285). English.
https://doi.org/10.1002/iid3.285...
) and lymphadenomegaly is a common clinical sign(1111 Silva LC, Castro RS, Figueiredo MM, Michalick MSM, Washington LT, Wagner LT. Canine visceral leishmaniasis as a systemic fibrotic disease. International Jour Exp Pathol. [Internet]. 2013 [cited 2021 Jan 04]; 94:133-143. Available from: https://doi.org/10.1111/iep.12010. English.
https://doi.org/10.1111/iep.12010...
). Among its three portions, the cortical region has lymphoid follicles with a predominance of B cells; the paracortical region has no follicles and is composed of T lymphocytes; and the medullary region contains medullary cords and B lymphocytes. Furthermore, all regions may have reticular cells, rare plasma cells, and macrophages(1919 Junqueira LC, Carneiro J. Histologia Básica. 13th ed. Guanabara Koogan; 2017. 568p.). Lymph nodes of dogs with CanL exhibit hyperplasia and hypertrophy of the cortical and medullary regions, granulomatous inflammation(77 Keenan CM, Hendricks LD, Lightner L, Johnson AJ. Visceral Leishmaniasis in the German Shepherd Dog. II. Pathology. Vet Pathol. 1984; 21:80-86.,2020 Reis AB, Martins-Filho OA, Carvalho AT, Giunchetti RC, Carneiro CM, Mayrink W, et al. Systemic and compartmentalized immune response in canine visceral leishmaniasis. Vet Immunol Immunopathol. [Internet]. 2009 [cited 2021 Jan 18]; 128:87-95. Available from: https://doi.org/10.1016/j.vetimm.2008.10.307. English.
https://doi.org/10.1016/j.vetimm.2008.10...
), and fibrosis(2121 Menezes RC, Madeira MF, Ferreira LC, Barbosa Filho CJL, Miranda LHM, Figueiredo FB. Cell-block immunohistochemistry of bone marrow aspirates: A novel tool to improve the diagnosis of leishmania infection in dogs. J. Comp. Pathol. [Internet]. 2016 [cited 2021 Jan 16]; 154:157-160. Available from: https://doi.org/10.1016/j.jcpa.2015.12.005. English.
https://doi.org/10.1016/j.jcpa.2015.12.0...
).

Considering the participation of the spleen and lymph nodes in the immune response of CanL, this study aimed to perform a histomorphological and immunohistochemical study of the spleen and lymph node of dogs reactive for leishmaniasis to the Dual Path Platform (DPP®) and enzyme-linked immunosorbent assay (ELISA) tests.

Material and methods

This research was approved by the Animal Ethics Committee of the Federal University of Goiás (CEUA/UFG), Goiânia, GO, Brazil, under protocol number 061/19. Were used 27 leishmaniasis reagent dogs to the rapid immunochromatographic tests Dual Path Platform (DDP® - Bio-Manguinhos, Rio de Janeiro, Brazil) and enzyme-linked immunosorbent assay (ELISA), from the routine of epidemiological surveillance for CanL performed by the Directorate of Zoonosis Surveillance of Goiânia (DVZ), Goiás, Brazil. As recommended by the Ministry of Health, the dogs were euthanized after positivity to DPP® and ELISA tests and the consent of their respective tutors, being sent to the Animal Pathology Service of the School of Veterinary and Animal Science of UFG (SPA/EVZ/UFG), Goiânia, GO, Brazil, for anatomopathological examination.

The variables sex, age, and breed were considered for the epidemiological data tabulation. The animals were classified as young (up to two years old), adults (three to seven years old), or elderly (over eight years old) and divided into mongrel (MB) and purebred (PB) dogs, according to information provided by DVZ. Variables related to clinical signs of CanL were also considered at gross evaluation, including onychogryphosis, alopecia, desquamation, oral ulcer, nasal ulcer(2222 Boechat VC, Mendes Junior AAV, Madeira MF, Ferreira LC, Figueiredo FB, Rodrigues FC, et al. Occurrence of Leishmania infantum and associated histological alterations in the genital tract and mammary glands of naturally infected dogs. Parasitol. Res. [Internet]. 2016 [cited 2021 Jan 10]; 115:2371-2379. Available from: https://doi.org/10.1007/s00436-016-4987-4. English.
https://doi.org/10.1007/s00436-016-4987-...
,2323 Souza CC, Barreto TO, Silva SM, Pinto AWJ, Figueiredo MM, Rocha OGF, et al. A potential link among antioxidant enzymes, histopathology and trace elements in canine visceral leishmaniasis. Int J Exp Pathol. [Internet]. 2014 [[cited 2021 Jan 07]; 95:260-270. Available from: https://doi.org/10.1111/iep.12080. English.
https://doi.org/10.1111/iep.12080...
), and body score (scale from 1 to 5 points: 1- cachectic, visible ribs, without fat cover, showing palpable bony prominences, evident abdominal indentation with loss of muscle mass; 2 - low body score, lean animals, with easily palpable ribs showing minimal fat coverage, bony prominences easily palpable, abdominal indentation easily visible in the flank region and minimal abdominal fat; 3 - medium body, ideal score, palpable ribs with small fat cover, well-proportioned abdominal recess and minimal layer of abdominal fat; 4 - overweight, ribs difficult to be palpated with moderate fat coverage, minimal or absent abdominal indentation, rounded abdomen with moderate fat coverage, 5- obesity, difficult rib palpation, marked fat deposits, distended abdomen with exaggerated fat deposits, and fat deposits in the lumbar region, on the face and limbs(2424 Castro MCN, Vieira AB, Santos MCS, Gershony LC, Soares AMB, Ferreira AMR. Escore de condição corporal como indicador do prognóstico de gatos com doença renal crônica, Cienc Rural. [Internet]. 2010 [cited 2021 Jan 21]; 40:365-370. Available from: https://doi.org/10.1590/s0103-84782010005000010. Portuguese.
https://doi.org/10.1590/s0103-8478201000...
). According to the external and internal lesions at gross evaluation, the dogs were classified as asymptomatic or symptomatic(1717 Lima IS, Silva JS, Almeida VA, Junior FGL, Souza PAN, Laranjeira DF, et al. Severe clinical presentation of visceral leishmaniasis in naturally infected dogs with disruption of the splenic white pulp. PLoS One [Internet]. 2014 [cited 2020 Jan 28]; 9(2)e87742. Available from: https://doi.org/10.1371/journal.pone.0087742. English.
https://doi.org/10.1371/journal.pone.008...
).

Changes related to splenomegaly and lymphadenomegaly (regional or generalized) were also evaluated on necroscopic examination. Histomorphological and immunohistochemical evaluations were performed in the spleen and axillary lymph node samples fixed in 10% neutral buffered formalin for 48 hours(2222 Boechat VC, Mendes Junior AAV, Madeira MF, Ferreira LC, Figueiredo FB, Rodrigues FC, et al. Occurrence of Leishmania infantum and associated histological alterations in the genital tract and mammary glands of naturally infected dogs. Parasitol. Res. [Internet]. 2016 [cited 2021 Jan 10]; 115:2371-2379. Available from: https://doi.org/10.1007/s00436-016-4987-4. English.
https://doi.org/10.1007/s00436-016-4987-...
) and, sequentially, maintained in 70% alcohol until the histological processing and paraffin embedding.

Histopathological evaluation

From the paraffinized blocks containing spleen and lymph node samples were prepared 5 µm thick histological sections, which were stretched over histological slides and stained with hematoxylin and eosin (HE)(2222 Boechat VC, Mendes Junior AAV, Madeira MF, Ferreira LC, Figueiredo FB, Rodrigues FC, et al. Occurrence of Leishmania infantum and associated histological alterations in the genital tract and mammary glands of naturally infected dogs. Parasitol. Res. [Internet]. 2016 [cited 2021 Jan 10]; 115:2371-2379. Available from: https://doi.org/10.1007/s00436-016-4987-4. English.
https://doi.org/10.1007/s00436-016-4987-...
).

The structural organization of the lymphoid tissue and granuloma formation were considered in the spleen, as adapted from Silva et al.(2525 Silva AVA, Figueiredo FB, Menezes RC, Mendes-Junior AA, Miranda LHM, Cupolillo E, et al. Morphophysiological changes in the splenic extracellular matrix of Leishmania infantum-naturally infected dogs is associated with alterations in lymphoid niches and the CD4+ T cell frequency in spleens. PLoS Negl Trop Dis. [Internet]. 2018 [cited 2020 Jan 29]; 12(4):e0006445. Available from: https://doi.org/10.1371/journal.pntd.0006445. English.
https://doi.org/10.1371/journal.pntd.000...
). For this, the following scores were attributed: zero (0 - organized), lymphoid follicles showing all the structural regions developed and outlined, including the distinct periarteriolar lymphatic sheath, germinal center, mantle zone, and marginal zone; one (1 - slightly disorganized), lymphoid follicles with a reduction in a defined region or discrete disorganization of any structure of the lymph node, and discrete hyperplasia or rarefaction of the white pulp; two (2 - moderately disorganized), evident white pulp but with poorly defined or indistinct regions and moderate lymphoid hyperplasia; three (3 - extensively disorganized), indistinct or poorly differentiated lymphoid tissue of the red pulp, with fibroplasia and marked follicular hyperplasia; and four (4 - well-defined granulomas), samples with lymphoid tissue markedly disorganized, with boundaries established by the proliferation of fibrosis and epithelioid cells.

Splenic samples classified with scores three and four were subjected to Masson trichrome staining to identify and delimitate fibrosis formation in the splenic parenchyma. Also, splenic capsule thickness, perisplenitis according to the type of inflammatory infiltrate, hemosiderosis, extramedullary hematopoiesis, and macrophages parasitized by amastigote forms of Leishmania spp. were considered as spleen variables.

The criteria adapted from Toplu and Aydogan(2626 Toplu N, Aydogan A. An immunohistochemical study in cases with usual and unusual clinicopathological findings of canine visceral leishmaniosis. Parasitol Res. [Internet]. 2011 [cited 2021 Jan 19]; 109:1051-1057. Available from: https://doi.org/10.1007/s00436-011-2345-0. English.
https://doi.org/10.1007/s00436-011-2345-...
) were adopted in the lymph node evaluation, considering the variables diffuse cortical and paracortical hyperplasia; follicular hyperplasia with reactive germinal center in the cortical region; hyperplasia and hypertrophy of macrophages in subcapsular and medullary sinuses; hyperplasia and hypertrophy of medullary cords; capsular thickening; type of capsular inflammatory infiltrate; presence or absence of macrophages with amastigote forms of Leishmania spp.; and edema in cords and sinuses.

Immunohistochemical (IHC) evaluation

For immunohistochemical analysis, 4 µm histological sections were placed on silanized histological slides (StarFrost®) and subjected to deparaffinization in xylol and rehydration in decreasing ethanol concentrations. Antigenic recovery was performed in sodium citrate buffer (pH 6.0) at 95 °C for 30 minutes in a water bath. Endogenous peroxidase was blocked by immersing the sections in 30% hydrogen peroxide and 1:10 distilled water for 10 minutes. The sections were incubated in a background block reagent (Cell Marque, Rocklin, CA) for 12 minutes at room temperature in order to block nonspecific protein binding. Subsequently, the sections were incubated for 18 h at a temperature from 2-8 °C with polyclonal rabbit anti-Leishmania antibody diluted to a concentration of 1:1000 in antibody diluent (Diamond; Cell Marque, Rocklin, CA). The amplification of signals was done with the HiDef Detection HRP Polymer System kit in two stages, with sequential incubation of HiDef Detection™ Amplifier (Mouse and Rabbit), followed by HiDef Detection™ HRP Polymer Detector (HiDef Detection HRP Polymer System; Cell Marque, Rocklin, HERE). The amastigote forms of Leishmania spp. were visualized using DAB chromogen diluted in distilled water and urea, according to the manufacturer’s recommendations. The samples were counterstained in Harris’ hematoxylin, dehydrated in absolute ethanol, clarified in xylol, and covered with synthetic resin and coverslips. Two samples of canine tissue markedly parasitized with the amastigote forms of Leishmania spp. were used as a positive control of the reaction.

Macrophages with amastigote forms of Leishmania spp. were counted in five fields with a higher density of parasitized cells at 40x and using an optical grid of 1 mm2 and a manual cell counter for the evaluation of parasitic intensity. Subsequently, the average of parasitized macrophages in the five fields was obtained for each animal and the scores of parasite intensity were assigned as follows: absent (when amastigote forms were not observed), slightly to moderate (0.2 to 10 parasitized macrophages), and accentuated (more than 10 parasitized macrophages)(2222 Boechat VC, Mendes Junior AAV, Madeira MF, Ferreira LC, Figueiredo FB, Rodrigues FC, et al. Occurrence of Leishmania infantum and associated histological alterations in the genital tract and mammary glands of naturally infected dogs. Parasitol. Res. [Internet]. 2016 [cited 2021 Jan 10]; 115:2371-2379. Available from: https://doi.org/10.1007/s00436-016-4987-4. English.
https://doi.org/10.1007/s00436-016-4987-...
).

Statistical analyses

Descriptive statistics were used for the variables sex, age, breed, clinical classification, and histomorphological changes in the spleen and lymph node. The Kappa non-parametric test at the 5% significance level was used for the analysis of agreement regarding the number of positive cases in the histopathological and immunohistochemical examinations, followed by the application of the scores very good (0.8<k≤1), good (0.6<k≤0.8), moderate (0.4<k≤ 0.6), fair (0.2<k≤0.4) or poor (k≤0.2) to determine the degree of agreement between examinations. The Mann-Whitney test was applied to compare the parasite intensity between the spleen and lymph node, considering the 5% significance level. For this, Excel 2016 spreadsheets and the software R were used, including the libraries “irr” and “stats”.

Results

Among the 27 dogs reactive for leishmaniasis in the DPP® and ELISA tests, 51.9% (n=14) of the animals were young, 25.9% (n=7) adults, and 22.2% (n=6) elderly. Regarding sex, 66.7% (n=18) of the animals were females and 33.3% (n=9) males. Additionally, 51.9% (n=14) consisted of purebred and 48.1% (n=13) mongrel animals. At gross evaluation 77.8% (n=21) of the dogs presented skin desquamation, 55.5% (n=15) onychogryphosis, and 48.1% (n=13) alopecia. Moreover, one dog had nasal ulcer and another oral ulcer, which represents 3.7% for each of these changes. According to the variable body score, 14.8% (n=4) of the animals had a low body score, 51.8% (n=14) a mean body score, and 11.1% (n=3) a slight overweight. The body score was not established in six animals. According to the lesions at gross evaluation, 7.4% (n=2) of the dogs were asymptomatic and 92.6% (n=25) symptomatic.

At macroscopic evaluation 81.5% (n=22) of the animals had splenomegaly and 22.2% (n=6) some degree of splenic nodulation (Figure 1). Also, 63.0% (n=17) of the dogs exhibited generalized lymphadenomegaly, one dog had axillary lymphadenomegaly, and another one presented axillary and mandibular lymphadenomegaly.

Figure 1
Spleen of dogs reactive for CanL to DPP® and ELISA tests. A) Lymphoid tissue with a score 2 of disorganization. Slightly disorganized follicular regions with little evidence and delimitation of lymphoid regions. HE. B) Lymphoid tissue with a score 3 of disorganization. Splenic microarchitecture diffusely disorganized, with indistinct lymphoid follicle structures from the red pulp. HE. C) Macroscopic aspect of a spleen showing splenomegaly and a diffuse nodular surface. D) Lymphoid tissue with a score 4 of disorganization. Splenic parenchyma exhibits well-defined granulomas. HE. E) Higher magnification of splenic granuloma, showing an infiltration of lymphocytes, plasma cells, and epithelioid cells delimited by fibrosis (arrow). HE. F) Splenic granuloma with evidence of epithelioid cells (arrowhead) and collagen fibers (arrow). Masson trichrome.

Histopathological evaluation

Histomorphological evaluation of splenic samples showed 100% (n=27) of the dogs with some degree of disorganization of the spleen architecture (Table 1) and, among them, 18.5% (n=5) developed granulomas with evidence of fibrosis confirmed by Masson trichrome staining (Figure 1).

Table 1
Scores of disorganization of splenic lymphoid tissue and perisplenitis in the spleen of dogs reactive for leishmaniasis to DPP® and ELISA tests

Moreover, 55.6% (n=15) of the animals showed perisplenitis with varied cellularity (Table 1), 66.6% (n=18) splenic capsule thickening (Figure 2), and 88.8% (n=24) extramedullary hematopoiesis, characterized by the evidence of megakaryocytes in the splenic parenchyma. Also, 92.6% (n=25) of the dogs had some degree of hemosiderosis, and 14.8% (n=4) splenic macrophages were parasitized by Leishmania spp. (Figure 2). Two out of the five samples classified as score four exhibited amastigote forms of Leishmania spp. in the macrophages cytoplasm.

Figure 2
Photomicrographs of the spleen of dogs reactive for leishmaniasis to DPP® and ELISA tests. A) Perisplenitis. Splenic capsule thickened and interspersed with lymphoplasmocytic and macrophagic inflammatory infiltrate (asterisks). HE. B) Marked collagen deposition (fibrosis) in the splenic capsule (arrow). Masson trichrome. C) Amastigote forms of Leishmania spp. in the cytoplasm of macrophages of the splenic parenchyma (arrow). HE. D) Macrophages next to the capsule, showing amastigote forms of Leishmania spp. HE.

Histomorphological changes in the lymph node included diffuse cortical and paracortical hyperplasia (96.3%, n=26), follicular hyperplasia with a reactive germinal center in the cortical region (55.6%, n=15), hyperplasia and hypertrophy of macrophages in the subcapsular sinus (70.3%, n=19), hyperplasia and hypertrophy of spinal cord macrophages (88.9%, n=24), and hyperplasia and hypertrophy of medullar cord (100%, n=27). Also, 22.2% (n=6) of the lymph nodes presented capsular thickening due to fibrosis, and 85.2% (n=23) thickening due to fibrosis and capsular inflammatory infiltrate. Among those with capsular infiltrate, 59.3% (n=16) consisted of the lymphoplasmocytic type and 25.9% (n=7) of the lymphoplasmocytic and macrophage type. Moreover, 18.5% (n=5) of the lymph nodes presented macrophages with amastigote forms of Leishmania spp. and 40.7% (n=11) had edema of cords and sinuses. Figure 3 illustrates the main histomorphological changes in the lymph node of dogs reactive for leishmaniasis to DPP® and ELISA tests.

Figure 3
Photomicrograph of the lymph node of dogs reactive for CanL to DPP® and ELISA tests. A) Diffuse cortical and paracortical hyperplasia. HE. B) Capsular thickening due to fibrosis (arrow) and inflammatory infiltrate (asterisk). Masson trichrome. C) Capsular inflammatory infiltrate (arrow). HE. D) Amastigote forms of Leishmania spp. in the cytoplasm of macrophages (arrow) in the cortical region of interfolicular septum. HE.

Immunohistochemical evaluation

The IHC technique detected amastigotes of Leishmania spp. in the spleen and lymph node samples from dogs reactive for leishmaniasis to DPP® and ELISA tests (Figure 4), with 22.2% (n=6) of splenic immunostaining and 37.0% (n= 10) in the lymph node.

Figure 4
Photomicrographs of the spleen and lymph node of dogs reactive for leishmaniasis to DPP® and ELISA tests. Immunostaining of amastigote forms of Leishmania spp. in the cytoplasm of macrophages of the spleen (arrows) (A) and lymph node (arrows) (B). IHC, anti-Leishmania.

The analysis of agreement of the results regarding the number of positive cases on histopathological (n=5, higher number of positive samples between the spleen and lymph node) and immunohistochemical examinations (n=10, higher number of samples between the spleen and lymph node) showed a good agreement (k=0.55; p=0.00124). The evaluation of parasite intensity in the spleen and lymph node using the IHC technique showed animals with absent, slightly to moderate, and accentuated scores. Also, the comparison of the parasite intensity had no difference between the spleen and lymph node to the immunohistochemical evaluation (p= 0.23) (Table 2).

Table 2
Distribution of scores and comparison of means of the parasite intensity in the spleen and lymph node using the IHC technique

Data regarding the classification of lymphoid tissue disorganization scores and parasitic intensity in the immunohistochemistry technique are described in Table 3.

Table 3
Disorganization scores of lymphoid tissue and frequency of parasitic intensity

Discussion

Twenty-four out of 27 dogs reactive for CanL to DDP® and ELISA tests were symptomatic, which showed mainly exfoliative desquamation, onychogryphosis, and alopecia. Studies have also shown that the common findings in CanL(2222 Boechat VC, Mendes Junior AAV, Madeira MF, Ferreira LC, Figueiredo FB, Rodrigues FC, et al. Occurrence of Leishmania infantum and associated histological alterations in the genital tract and mammary glands of naturally infected dogs. Parasitol. Res. [Internet]. 2016 [cited 2021 Jan 10]; 115:2371-2379. Available from: https://doi.org/10.1007/s00436-016-4987-4. English.
https://doi.org/10.1007/s00436-016-4987-...
,2323 Souza CC, Barreto TO, Silva SM, Pinto AWJ, Figueiredo MM, Rocha OGF, et al. A potential link among antioxidant enzymes, histopathology and trace elements in canine visceral leishmaniasis. Int J Exp Pathol. [Internet]. 2014 [[cited 2021 Jan 07]; 95:260-270. Available from: https://doi.org/10.1111/iep.12080. English.
https://doi.org/10.1111/iep.12080...
) include onychogryphosis(1212 Cavalcanti AS, Ribeiro-Alves M, Pereira LOR, Mestre GL, Ferreira ABR, Morgado FN, et al. Parasite load induces progressive spleen architecture breakage and impairs cytokine mRNA expression in Leishmania infantum naturally infected dogs. PLoS One [Internet]. 2015 [cited 2021 Jan 20]; 10(4):e0123009. Available from: https://doi.org/10.1371/journal.pone.0123009. English.
https://doi.org/10.1371/journal.pone.012...
) and skin lesions such as exfoliative desquamation, ulcers, and alopecia(2727 Tafuri WL, Santos RDL, Arantes RME, Gonçalves R, Melo MN, Michalick MSM, et al. An alternative immunohistochemical method for detecting Leishmania amastigotes in paraffin-embedded canine tissues. J Immunol Methods. [Internet]. 2004 [cited 2021 Jan 21]; 292:17-23. Available from: https://doi.org/10.1016/j.jim.2004.05.009. Portuguese.
https://doi.org/10.1016/j.jim.2004.05.00...
), besides a low body score(77 Keenan CM, Hendricks LD, Lightner L, Johnson AJ. Visceral Leishmaniasis in the German Shepherd Dog. II. Pathology. Vet Pathol. 1984; 21:80-86.,1010 Lima WG, Michalick MSM, Melo MN, Washington LT, Wagner LT. Canine visceral leishmaniasis: A histopathological study of lymph nodes. Acta Trop. [Internet]. 2004 [cited 2021 Jan 15]; 92:43-53. Available from: https://doi.org/10.1016/j.actatropica.2004.04.007. English.
https://doi.org/10.1016/j.actatropica.20...
), the latter not observed in most animals in this study. Thus, factors such as individual response to the disease and the degree of development of systemic lesions may directly mediate the progression of CanL and reflect the weight loss of affected animals. Splenomegaly(1212 Cavalcanti AS, Ribeiro-Alves M, Pereira LOR, Mestre GL, Ferreira ABR, Morgado FN, et al. Parasite load induces progressive spleen architecture breakage and impairs cytokine mRNA expression in Leishmania infantum naturally infected dogs. PLoS One [Internet]. 2015 [cited 2021 Jan 20]; 10(4):e0123009. Available from: https://doi.org/10.1371/journal.pone.0123009. English.
https://doi.org/10.1371/journal.pone.012...
) and lymphadenomegaly(77 Keenan CM, Hendricks LD, Lightner L, Johnson AJ. Visceral Leishmaniasis in the German Shepherd Dog. II. Pathology. Vet Pathol. 1984; 21:80-86.,1010 Lima WG, Michalick MSM, Melo MN, Washington LT, Wagner LT. Canine visceral leishmaniasis: A histopathological study of lymph nodes. Acta Trop. [Internet]. 2004 [cited 2021 Jan 15]; 92:43-53. Available from: https://doi.org/10.1016/j.actatropica.2004.04.007. English.
https://doi.org/10.1016/j.actatropica.20...
) are also described in most dogs of the present research.

Changes in lymphoid tissues are frequent in infections due to Leishmania, such as the spleen, which plays important role in CanL, as it has components of the immune response and acts in the interaction with the parasite, which reflects morphological changes(1616 Santana CC, Vassallo J, Freitas LAR, Oliveira GGS, Carvalho LCP, Santos WLS. Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: A study on naturally infected dogs. Parasite Immunol. [Internet]. 2008 [cited 2021 Jan 17]; 30:515-524. Available from: https://doi.org/10.1111/j.1365-3024.2008.01051.x. English.
https://doi.org/10.1111/j.1365-3024.2008...
), as observed in the spleen of animals in this research. These changes include mainly some degree of disorganization of the lymphoid tissue microstructure due to immunological reactions against the parasite(2525 Silva AVA, Figueiredo FB, Menezes RC, Mendes-Junior AA, Miranda LHM, Cupolillo E, et al. Morphophysiological changes in the splenic extracellular matrix of Leishmania infantum-naturally infected dogs is associated with alterations in lymphoid niches and the CD4+ T cell frequency in spleens. PLoS Negl Trop Dis. [Internet]. 2018 [cited 2020 Jan 29]; 12(4):e0006445. Available from: https://doi.org/10.1371/journal.pntd.0006445. English.
https://doi.org/10.1371/journal.pntd.000...
).

All animals in this study exhibited some degree of splenic structural disorganization, showing moderate disorganization of the lymphoid tissue in most cases, with little evident follicular regions or hyperplasia of lymphoid follicles. At the same time, cases with the formation of well-defined granulomas in association or not with amastigotes of Leishmania spp. were observed, with no relationship between these changes and the parasite presence or intensity. In this context, Santana et al.(1616 Santana CC, Vassallo J, Freitas LAR, Oliveira GGS, Carvalho LCP, Santos WLS. Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: A study on naturally infected dogs. Parasite Immunol. [Internet]. 2008 [cited 2021 Jan 17]; 30:515-524. Available from: https://doi.org/10.1111/j.1365-3024.2008.01051.x. English.
https://doi.org/10.1111/j.1365-3024.2008...
) and Silva et al.(2525 Silva AVA, Figueiredo FB, Menezes RC, Mendes-Junior AA, Miranda LHM, Cupolillo E, et al. Morphophysiological changes in the splenic extracellular matrix of Leishmania infantum-naturally infected dogs is associated with alterations in lymphoid niches and the CD4+ T cell frequency in spleens. PLoS Negl Trop Dis. [Internet]. 2018 [cited 2020 Jan 29]; 12(4):e0006445. Available from: https://doi.org/10.1371/journal.pntd.0006445. English.
https://doi.org/10.1371/journal.pntd.000...
) also described splenic changes in dogs with CanL, including atrophy or hyperplasia of the lymphoid follicle, resulting in loss of definition of follicular regions. On the other hand, some authors have described the formation of granulomas frequently associated with parasitic load(1212 Cavalcanti AS, Ribeiro-Alves M, Pereira LOR, Mestre GL, Ferreira ABR, Morgado FN, et al. Parasite load induces progressive spleen architecture breakage and impairs cytokine mRNA expression in Leishmania infantum naturally infected dogs. PLoS One [Internet]. 2015 [cited 2021 Jan 20]; 10(4):e0123009. Available from: https://doi.org/10.1371/journal.pone.0123009. English.
https://doi.org/10.1371/journal.pone.012...
,1616 Santana CC, Vassallo J, Freitas LAR, Oliveira GGS, Carvalho LCP, Santos WLS. Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: A study on naturally infected dogs. Parasite Immunol. [Internet]. 2008 [cited 2021 Jan 17]; 30:515-524. Available from: https://doi.org/10.1111/j.1365-3024.2008.01051.x. English.
https://doi.org/10.1111/j.1365-3024.2008...
).

The disorganized condition of the splenic architecture has been described in animals positive for serological tests and negative for parasitological tests for CanL, suggesting that the cause of this finding may be related to the prolonged inflammatory response in the spleen, with a consequent reduction in the parasite load(1111 Silva LC, Castro RS, Figueiredo MM, Michalick MSM, Washington LT, Wagner LT. Canine visceral leishmaniasis as a systemic fibrotic disease. International Jour Exp Pathol. [Internet]. 2013 [cited 2021 Jan 04]; 94:133-143. Available from: https://doi.org/10.1111/iep.12010. English.
https://doi.org/10.1111/iep.12010...
,2828 Vasconcelos TCB, Bruno SF, Miranda LHM, Silva FC, Belo VS, Figueiredo FB. Parasite load, iNOS and cytokine profiles, and histopathological aspects of Leishmania infantum infection in dogs with different clinical presentations. Cienc Rural. [Internet] 2019 [cited 2020 Jan 19]; 49(10): e20180984. Available from: https://doi.org/10.1590/0103-8478cr20180984. English.
https://doi.org/10.1590/0103-8478cr20180...
). Furthermore, a study that evaluated rats infected with Leishmania donovani showed splenic changes stimulated by high levels of tumor necrosis factor (TNF), without association with the parasite load(2929 Engwerda CR, Ato M, Cotterell SEJ, Mynott TL, Tschannerl A, Gorak-Stolinska PMA, et al. A role for tumor necrosis factor-α in remodeling the splenic marginal zone during Leishmania donovani infection. Am J Pathol. [Internet]. 2002 [cited 2021 Jan 18]; 161:429-437. (https://doi.org/10.1016/S0002-9440(10)64199-5. English.
https://doi.org/10.1016/S0002-9440(10)64...
). Although TNF was not evaluated in this research, this hypothesis may justify the fact that all dogs in this research showed splenic changes, regardless of the parasite presence or intensity.

Despite the disorganization of the splenic architecture is an important finding in this study, samples classified at scores three and four showed marked disorganization of the white pulp and formation of fibrosis associated or not with the parasite presence. In this sense, Santana et al.(1616 Santana CC, Vassallo J, Freitas LAR, Oliveira GGS, Carvalho LCP, Santos WLS. Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: A study on naturally infected dogs. Parasite Immunol. [Internet]. 2008 [cited 2021 Jan 17]; 30:515-524. Available from: https://doi.org/10.1111/j.1365-3024.2008.01051.x. English.
https://doi.org/10.1111/j.1365-3024.2008...
) described the rupture of the splenic architecture and fibrosis formation as a result of the production of a reticular web, seeking to prevent or slow the progression of the parasite to other tissues(1616 Santana CC, Vassallo J, Freitas LAR, Oliveira GGS, Carvalho LCP, Santos WLS. Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: A study on naturally infected dogs. Parasite Immunol. [Internet]. 2008 [cited 2021 Jan 17]; 30:515-524. Available from: https://doi.org/10.1111/j.1365-3024.2008.01051.x. English.
https://doi.org/10.1111/j.1365-3024.2008...
,3030 Alexandre-Pires G, Pais D, Correia M, Pina JAE. Leishmaniosis - A report about the microvascular and cellular architecture of the infected spleen in Canis familiaris. Microsc Res Tech. [Internet]. 2006 [cited 2021 Jan 21]; 69:227-235. Available from: https://doi.org/10.1002/jemt.20267. English.
https://doi.org/10.1002/jemt.20267...
). Moreover, many samples from this study presented perisplenitis, possibly due to the extension of the parenchymal splenic inflammatory reaction, associated or not with the parasite presence in these cases, which is also often described in cases of CanL, but normally related to the parasite load(1616 Santana CC, Vassallo J, Freitas LAR, Oliveira GGS, Carvalho LCP, Santos WLS. Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: A study on naturally infected dogs. Parasite Immunol. [Internet]. 2008 [cited 2021 Jan 17]; 30:515-524. Available from: https://doi.org/10.1111/j.1365-3024.2008.01051.x. English.
https://doi.org/10.1111/j.1365-3024.2008...
,3131 Tafuri WL, Tafuri WL, Barbosa AJA, Michalick MSM, Genaro O, França-Silva JC, et al. Histopathology and immunocytochemical study of type 3 and type 4 complement receptors in the liver and spleen of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi. Rev Inst Med Trop Sao Paulo. 1996; 38:81-89. (https://doi.org/10.1590/S0036-46651996000200001).
https://doi.org/10.1590/S0036-4665199600...
,3232 Tryphonas L, Zawidzka Z, Bernard MA, Janzen EA. Visceral leishmaniasis in a dog: clinical, hematological and pathological observations. Can J Comp Med. 1977. 41:1-12.).

A relevant number of animals with lymphadenomegaly was observed in this study, which is a frequent change in cases of CanL(77 Keenan CM, Hendricks LD, Lightner L, Johnson AJ. Visceral Leishmaniasis in the German Shepherd Dog. II. Pathology. Vet Pathol. 1984; 21:80-86.,1010 Lima WG, Michalick MSM, Melo MN, Washington LT, Wagner LT. Canine visceral leishmaniasis: A histopathological study of lymph nodes. Acta Trop. [Internet]. 2004 [cited 2021 Jan 15]; 92:43-53. Available from: https://doi.org/10.1016/j.actatropica.2004.04.007. English.
https://doi.org/10.1016/j.actatropica.20...
). The histopathological evaluation of the axillary lymph node showed hyperplasia and hypertrophy of the medullary and cortical regions, with no association with clinical signs, corroborating the findings of Lima et al.(1010 Lima WG, Michalick MSM, Melo MN, Washington LT, Wagner LT. Canine visceral leishmaniasis: A histopathological study of lymph nodes. Acta Trop. [Internet]. 2004 [cited 2021 Jan 15]; 92:43-53. Available from: https://doi.org/10.1016/j.actatropica.2004.04.007. English.
https://doi.org/10.1016/j.actatropica.20...
). Moreover, hypertrophy and hyperplasia of macrophages in capsular and/or medullary sinuses are among the most frequent changes in the lymph nodes of dogs infected with Leishmania spp.(2525 Silva AVA, Figueiredo FB, Menezes RC, Mendes-Junior AA, Miranda LHM, Cupolillo E, et al. Morphophysiological changes in the splenic extracellular matrix of Leishmania infantum-naturally infected dogs is associated with alterations in lymphoid niches and the CD4+ T cell frequency in spleens. PLoS Negl Trop Dis. [Internet]. 2018 [cited 2020 Jan 29]; 12(4):e0006445. Available from: https://doi.org/10.1371/journal.pntd.0006445. English.
https://doi.org/10.1371/journal.pntd.000...
), as also observed in this study. Also, Lima et al.(1010 Lima WG, Michalick MSM, Melo MN, Washington LT, Wagner LT. Canine visceral leishmaniasis: A histopathological study of lymph nodes. Acta Trop. [Internet]. 2004 [cited 2021 Jan 15]; 92:43-53. Available from: https://doi.org/10.1016/j.actatropica.2004.04.007. English.
https://doi.org/10.1016/j.actatropica.20...
) suggested that hyperplasia and hypertrophy in the cortical and medullary zones plus chronic and diffuse capsular inflammation are frequent and justify the occurrence of lymphadenomegaly in cases of CanL.

In addition, most lymph node samples from this study exhibited capsular thickening due to the lymphoplasmocytic and macrophagic inflammatory infiltrate aggregated to the proliferation of fibrosis, a frequent data in cases of CanL(1010 Lima WG, Michalick MSM, Melo MN, Washington LT, Wagner LT. Canine visceral leishmaniasis: A histopathological study of lymph nodes. Acta Trop. [Internet]. 2004 [cited 2021 Jan 15]; 92:43-53. Available from: https://doi.org/10.1016/j.actatropica.2004.04.007. English.
https://doi.org/10.1016/j.actatropica.20...
). In this context, Giunchetti et al.(3333 Giunchetti RC, Martins-Filho OA, Carneiro CM, Mayrink W, Marques MJ, Tafuri WL, et al. Histopathology, parasite density and cell phenotypes of the popliteal lymph node in canine visceral leishmaniasis, Vet Immunol Immunopathol. [Internet]. 2018 [cited 2021 Jan 06]; 121:23-33. Available from: https://doi.org/10.1016/j.vetimm.2007.07.009. English.
https://doi.org/10.1016/j.vetimm.2007.07...
) described that the capsular infiltrate is often composed of macrophages, including the presence of parasites, but these changes were not associated with the presence of amastigote forms of Leishmania spp. in this study.

Histopathological evaluation was used to observe morphological changes and identify amastigote forms of the parasite, and this identification was also carried out by IHC. The results of the identification for the two evaluations were compared and, in the qualitative analysis, the IHC of the spleen and lymph node expressed the best numerical result, but with no statistical difference. This data corroborates that described by authors who pointed out the IHC technique as a supplementary tool for CanL diagnosis, considering it has higher sensitivity and specificity(2121 Menezes RC, Madeira MF, Ferreira LC, Barbosa Filho CJL, Miranda LHM, Figueiredo FB. Cell-block immunohistochemistry of bone marrow aspirates: A novel tool to improve the diagnosis of leishmania infection in dogs. J. Comp. Pathol. [Internet]. 2016 [cited 2021 Jan 16]; 154:157-160. Available from: https://doi.org/10.1016/j.jcpa.2015.12.005. English.
https://doi.org/10.1016/j.jcpa.2015.12.0...
,2626 Toplu N, Aydogan A. An immunohistochemical study in cases with usual and unusual clinicopathological findings of canine visceral leishmaniosis. Parasitol Res. [Internet]. 2011 [cited 2021 Jan 19]; 109:1051-1057. Available from: https://doi.org/10.1007/s00436-011-2345-0. English.
https://doi.org/10.1007/s00436-011-2345-...
,2727 Tafuri WL, Santos RDL, Arantes RME, Gonçalves R, Melo MN, Michalick MSM, et al. An alternative immunohistochemical method for detecting Leishmania amastigotes in paraffin-embedded canine tissues. J Immunol Methods. [Internet]. 2004 [cited 2021 Jan 21]; 292:17-23. Available from: https://doi.org/10.1016/j.jim.2004.05.009. Portuguese.
https://doi.org/10.1016/j.jim.2004.05.00...
,3333 Giunchetti RC, Martins-Filho OA, Carneiro CM, Mayrink W, Marques MJ, Tafuri WL, et al. Histopathology, parasite density and cell phenotypes of the popliteal lymph node in canine visceral leishmaniasis, Vet Immunol Immunopathol. [Internet]. 2018 [cited 2021 Jan 06]; 121:23-33. Available from: https://doi.org/10.1016/j.vetimm.2007.07.009. English.
https://doi.org/10.1016/j.vetimm.2007.07...
). Tafuri et al.(2727 Tafuri WL, Santos RDL, Arantes RME, Gonçalves R, Melo MN, Michalick MSM, et al. An alternative immunohistochemical method for detecting Leishmania amastigotes in paraffin-embedded canine tissues. J Immunol Methods. [Internet]. 2004 [cited 2021 Jan 21]; 292:17-23. Available from: https://doi.org/10.1016/j.jim.2004.05.009. Portuguese.
https://doi.org/10.1016/j.jim.2004.05.00...
) also reported that IHC increases the effectiveness of diagnostic tests by 50% compared to histopathological evaluation using hematoxylin-eosin staining. On the other hand, the good agreement between the histopathological and immunohistochemical evaluations observed in this study, inferring similarity between the tests, may have occurred due to the small number of samples, a fact also mentioned by Xavier et al.(3434 Xavier SC, Andrade HM, Monte SJH, Chiarelli IM, Lima WG, Michalick MSM, et al. Comparison of paraffin-embedded skin biopsies from different anatomical regions as sampling methods for detection of Leishmania infection in dogs using histological, immunohistochemical and PCR methods. BMC Vet Res. [Internet]. 2006 [cited 2021 Jan 19]; 2:17. Available from: https://doi.org/10.1186/1746-6148-2-17. English.
https://doi.org/10.1186/1746-6148-2-17...
), who compared the same diagnostic methods for evaluation of parasite intensity in dogs with CanL.

In addition, no difference was observed between the spleen and lymph node in terms of parasite intensity, indicating that both organs are useful for CanL diagnosis. However, no data were found in the literature to ratify or rectify the result relative to these organs. Nevertheless, Paparcone et al.(3535 Paparcone R, Fiorentino E, Cappiello S, Gizzarelli M, Gradoni L, Oliva G, et al. Sternal Aspiration of Bone Marrow in Dogs: A Practical Approach for Canine Leishmaniasis Diagnosis and Monitoring. J Vet Med. [Internet]. 2013 [cited 2021 Jan 27]; 2013:e217314. Available from: https://doi.org/10.1155/2013/217314. English.
https://doi.org/10.1155/2013/217314...
) compared different bone marrow collection sites for CanL diagnosis and also observed no difference regarding parasite intensity. Despite this, the authors mentioned that although the lymph node is equally rich in parasites, especially in symptomatic dogs, the access to the lymphoid tissue in this organ can be limited, particularly when there is no increase in volume, which commonly occurs in animals at early stage of the disease or in chronic asymptomatic animals.

Conclusions

Spleen and lymph node of dogs reactive for leishmaniasis to DPP® and ELISA tests develop histomorphological changes resulting from the immunological reactions caused by the disease, regardless of the presence and intensity of the parasite. Disorganization of lymphoid tissue in different degrees in the spleen comprises the main change, with granulomatous splenitis being the most serious lesion, as well as hyperplasia and hypertrophy of the medullary cords and hyperplasia of the cortical and paracortical regions prevail in the lymph node. In addition, the spleen and lymph node show similar parasitic load by immunohistochemical test.

Acknowledgments

To the Directorate of Zoonosis Surveillance of the municipality of Goiânia (DVZ), GO, Brazil, for providing the animals to the study and for all support during the development of the research. To the Laboratory of Clinical Research on Dermatozoonoses in Domestic Animals and the Pathological Anatomy Service, both belonging to the Evandro Chagas National Institute of Infectious Diseases, Oswaldo Cruz Foundation, Rio de Janeiro, Rio de Janeiro, Brazil, for the technical support in this research.

References

  • 1
    Brasil, Ministério da Saúde. Manual de vigilância e controle da leishmaniose visceral. Ministério da Saúde, Brasília, Distrito Federal. 2014. 120p.
  • 2
    World Health Organization. Control of the leishmaniases. World Health Organization technical report series. 2010. 186p.
  • 3
    Coura-Vital W, Leal CGA, Marques LA, Pinheiro AC, Carneiro M, Reis AB. Effectiveness of deltamethrin-impregnated dog collars on the incidence of canine infection by Leishmania infantum: A large scale intervention study in an endemic area in Brazil. PLoS One [Internet]. 2018 [cited 2020 Dec 10]; 13(12): e0208613. Available from: https://doi.org/10.1371/journal.pone.0208613 English.
    » https://doi.org/10.1371/journal.pone.0208613
  • 4
    Belo VS, Werneck GL, Barbosa DS, Simões TC, Nascimento BWL, Silva ES, et al. Factors Associated with Visceral Leishmaniasis in the Americas: A Systematic Review and Meta-Analysis. PLoS Negl Trop Dis. [Internet] 2013 [cited 2020 Dez 12]; 7(4):e2182. Available from: https://doi.org/10.1371/journal.pntd.0002182 Englisg.
    » https://doi.org/10.1371/journal.pntd.0002182
  • 5
    Oliveira VC, Boechat VC, Mendes Junior AAV, Madeira MF, Ferreira LC, Figueiredo FB, et al. Occurrence of Leishmania infantum in the central nervous system of naturally infected dogs: Parasite load, viability, co-infections and histological alterations. PLoS One [Internet]. 2017 [cited 2021 Jan 21]; 12(4):e0175588. Available from: https://doi.org/10.1371/journal.pone.0175588 Englisg.
    » https://doi.org/10.1371/journal.pone.0175588
  • 6
    Tafuri WL, Michalick MS, Dias M, Genaro O, Leite VH, Barbosa AJ, et al. Optical and electron microscopic study of the kidney of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi. Rev Inst Med Trop Sao Paulo. 1989; 31(3):139-145.
  • 7
    Keenan CM, Hendricks LD, Lightner L, Johnson AJ. Visceral Leishmaniasis in the German Shepherd Dog. II. Pathology. Vet Pathol. 1984; 21:80-86.
  • 8
    Almeida MAO, Jesus EEV, Sousa-Atta MLB, Alves LC, Berne MEA, Atta AM. Antileishmanial antibody profile in dogs naturally infected with Leishmania chagasi. Vet Immunol Immunopathol. [Internet]. 2005 [cited 2021 Jan 21]; 106:151-158. Available from: https://doi.org/10.1016/j.vetimm.2004.08.024 English.
    » https://doi.org/10.1016/j.vetimm.2004.08.024
  • 9
    Maia C, Campino L. Cytokine and phenotypic cell profiles of leishmania infantum infection in the dog. Journal Trop Med. [Internet]. 2012 [cited 2021 jan 23]. 2012: 541571. Available from: https://doi.org/10.1155/2012/541571 English.
    » https://doi.org/10.1155/2012/541571
  • 10
    Lima WG, Michalick MSM, Melo MN, Washington LT, Wagner LT. Canine visceral leishmaniasis: A histopathological study of lymph nodes. Acta Trop. [Internet]. 2004 [cited 2021 Jan 15]; 92:43-53. Available from: https://doi.org/10.1016/j.actatropica.2004.04.007 English.
    » https://doi.org/10.1016/j.actatropica.2004.04.007
  • 11
    Silva LC, Castro RS, Figueiredo MM, Michalick MSM, Washington LT, Wagner LT. Canine visceral leishmaniasis as a systemic fibrotic disease. International Jour Exp Pathol. [Internet]. 2013 [cited 2021 Jan 04]; 94:133-143. Available from: https://doi.org/10.1111/iep.12010 English.
    » https://doi.org/10.1111/iep.12010
  • 12
    Cavalcanti AS, Ribeiro-Alves M, Pereira LOR, Mestre GL, Ferreira ABR, Morgado FN, et al. Parasite load induces progressive spleen architecture breakage and impairs cytokine mRNA expression in Leishmania infantum naturally infected dogs. PLoS One [Internet]. 2015 [cited 2021 Jan 20]; 10(4):e0123009. Available from: https://doi.org/10.1371/journal.pone.0123009 English.
    » https://doi.org/10.1371/journal.pone.0123009
  • 13
    Haley PJ. The lymphoid system: A review of species differences, J. Toxicol. Pathol. [Internet]. 2017 [cited 2021 Jan 11]; 30:111-123. Available from: https://doi.org/10.1293/tox.2016-0075 English.
    » https://doi.org/10.1293/tox.2016-0075
  • 14
    Silva AV, Souza TL, Figueiredo FB, Mendes AAV, Ferreira LC, Filgueira COB, et al. Detection of amastigotes and histopathological alterations in the thymus of Leishmania infantum ‐infected dogs. Immun Inflamm Dis. [Internet]. 2020 [cited 2021 Jan 10]; 8:127-139. (https://doi.org/10.1002/iid3.285). English.
    » https://doi.org/10.1002/iid3.285
  • 15
    Mebius RE, Kraal G. Structure and function of the spleen. Nat. Rev. Immunol. [Internet]. 2005 [cited 2021 Jan 10]; 5:606-616. Available from: https://doi.org/10.1038/nri1669 English.
    » https://doi.org/10.1038/nri1669
  • 16
    Santana CC, Vassallo J, Freitas LAR, Oliveira GGS, Carvalho LCP, Santos WLS. Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: A study on naturally infected dogs. Parasite Immunol. [Internet]. 2008 [cited 2021 Jan 17]; 30:515-524. Available from: https://doi.org/10.1111/j.1365-3024.2008.01051.x English.
    » https://doi.org/10.1111/j.1365-3024.2008.01051.x
  • 17
    Lima IS, Silva JS, Almeida VA, Junior FGL, Souza PAN, Laranjeira DF, et al. Severe clinical presentation of visceral leishmaniasis in naturally infected dogs with disruption of the splenic white pulp. PLoS One [Internet]. 2014 [cited 2020 Jan 28]; 9(2)e87742. Available from: https://doi.org/10.1371/journal.pone.0087742 English.
    » https://doi.org/10.1371/journal.pone.0087742
  • 18
    Silva JS, Andrade AC, Santana CC, Santos LQ, Oliveira CI, Veras PST, et al. Low CXCL13 expression, splenic lymphoid tissue atrophy and germinal center disruption in severe canine visceral leishmaniasis. PLoS One [Internet]. 2012 [cited 2020 Jan 26]; 7(1): e29103. Available from: https://doi.org/10.1371/journal.pone.0029103 English.
    » https://doi.org/10.1371/journal.pone.0029103
  • 19
    Junqueira LC, Carneiro J. Histologia Básica. 13th ed. Guanabara Koogan; 2017. 568p.
  • 20
    Reis AB, Martins-Filho OA, Carvalho AT, Giunchetti RC, Carneiro CM, Mayrink W, et al. Systemic and compartmentalized immune response in canine visceral leishmaniasis. Vet Immunol Immunopathol. [Internet]. 2009 [cited 2021 Jan 18]; 128:87-95. Available from: https://doi.org/10.1016/j.vetimm.2008.10.307 English.
    » https://doi.org/10.1016/j.vetimm.2008.10.307
  • 21
    Menezes RC, Madeira MF, Ferreira LC, Barbosa Filho CJL, Miranda LHM, Figueiredo FB. Cell-block immunohistochemistry of bone marrow aspirates: A novel tool to improve the diagnosis of leishmania infection in dogs. J. Comp. Pathol. [Internet]. 2016 [cited 2021 Jan 16]; 154:157-160. Available from: https://doi.org/10.1016/j.jcpa.2015.12.005 English.
    » https://doi.org/10.1016/j.jcpa.2015.12.005
  • 22
    Boechat VC, Mendes Junior AAV, Madeira MF, Ferreira LC, Figueiredo FB, Rodrigues FC, et al. Occurrence of Leishmania infantum and associated histological alterations in the genital tract and mammary glands of naturally infected dogs. Parasitol. Res. [Internet]. 2016 [cited 2021 Jan 10]; 115:2371-2379. Available from: https://doi.org/10.1007/s00436-016-4987-4 English.
    » https://doi.org/10.1007/s00436-016-4987-4
  • 23
    Souza CC, Barreto TO, Silva SM, Pinto AWJ, Figueiredo MM, Rocha OGF, et al. A potential link among antioxidant enzymes, histopathology and trace elements in canine visceral leishmaniasis. Int J Exp Pathol. [Internet]. 2014 [[cited 2021 Jan 07]; 95:260-270. Available from: https://doi.org/10.1111/iep.12080 English.
    » https://doi.org/10.1111/iep.12080
  • 24
    Castro MCN, Vieira AB, Santos MCS, Gershony LC, Soares AMB, Ferreira AMR. Escore de condição corporal como indicador do prognóstico de gatos com doença renal crônica, Cienc Rural. [Internet]. 2010 [cited 2021 Jan 21]; 40:365-370. Available from: https://doi.org/10.1590/s0103-84782010005000010 Portuguese.
    » https://doi.org/10.1590/s0103-84782010005000010
  • 25
    Silva AVA, Figueiredo FB, Menezes RC, Mendes-Junior AA, Miranda LHM, Cupolillo E, et al. Morphophysiological changes in the splenic extracellular matrix of Leishmania infantum-naturally infected dogs is associated with alterations in lymphoid niches and the CD4+ T cell frequency in spleens. PLoS Negl Trop Dis. [Internet]. 2018 [cited 2020 Jan 29]; 12(4):e0006445. Available from: https://doi.org/10.1371/journal.pntd.0006445 English.
    » https://doi.org/10.1371/journal.pntd.0006445
  • 26
    Toplu N, Aydogan A. An immunohistochemical study in cases with usual and unusual clinicopathological findings of canine visceral leishmaniosis. Parasitol Res. [Internet]. 2011 [cited 2021 Jan 19]; 109:1051-1057. Available from: https://doi.org/10.1007/s00436-011-2345-0 English.
    » https://doi.org/10.1007/s00436-011-2345-0
  • 27
    Tafuri WL, Santos RDL, Arantes RME, Gonçalves R, Melo MN, Michalick MSM, et al. An alternative immunohistochemical method for detecting Leishmania amastigotes in paraffin-embedded canine tissues. J Immunol Methods. [Internet]. 2004 [cited 2021 Jan 21]; 292:17-23. Available from: https://doi.org/10.1016/j.jim.2004.05.009 Portuguese.
    » https://doi.org/10.1016/j.jim.2004.05.009
  • 28
    Vasconcelos TCB, Bruno SF, Miranda LHM, Silva FC, Belo VS, Figueiredo FB. Parasite load, iNOS and cytokine profiles, and histopathological aspects of Leishmania infantum infection in dogs with different clinical presentations. Cienc Rural. [Internet] 2019 [cited 2020 Jan 19]; 49(10): e20180984. Available from: https://doi.org/10.1590/0103-8478cr20180984 English.
    » https://doi.org/10.1590/0103-8478cr20180984
  • 29
    Engwerda CR, Ato M, Cotterell SEJ, Mynott TL, Tschannerl A, Gorak-Stolinska PMA, et al. A role for tumor necrosis factor-α in remodeling the splenic marginal zone during Leishmania donovani infection. Am J Pathol. [Internet]. 2002 [cited 2021 Jan 18]; 161:429-437. (https://doi.org/10.1016/S0002-9440(10)64199-5 English.
    » https://doi.org/10.1016/S0002-9440(10)64199-5
  • 30
    Alexandre-Pires G, Pais D, Correia M, Pina JAE. Leishmaniosis - A report about the microvascular and cellular architecture of the infected spleen in Canis familiaris. Microsc Res Tech. [Internet]. 2006 [cited 2021 Jan 21]; 69:227-235. Available from: https://doi.org/10.1002/jemt.20267 English.
    » https://doi.org/10.1002/jemt.20267
  • 31
    Tafuri WL, Tafuri WL, Barbosa AJA, Michalick MSM, Genaro O, França-Silva JC, et al. Histopathology and immunocytochemical study of type 3 and type 4 complement receptors in the liver and spleen of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi. Rev Inst Med Trop Sao Paulo. 1996; 38:81-89. (https://doi.org/10.1590/S0036-46651996000200001).
    » https://doi.org/10.1590/S0036-46651996000200001
  • 32
    Tryphonas L, Zawidzka Z, Bernard MA, Janzen EA. Visceral leishmaniasis in a dog: clinical, hematological and pathological observations. Can J Comp Med. 1977. 41:1-12.
  • 33
    Giunchetti RC, Martins-Filho OA, Carneiro CM, Mayrink W, Marques MJ, Tafuri WL, et al. Histopathology, parasite density and cell phenotypes of the popliteal lymph node in canine visceral leishmaniasis, Vet Immunol Immunopathol. [Internet]. 2018 [cited 2021 Jan 06]; 121:23-33. Available from: https://doi.org/10.1016/j.vetimm.2007.07.009 English.
    » https://doi.org/10.1016/j.vetimm.2007.07.009
  • 34
    Xavier SC, Andrade HM, Monte SJH, Chiarelli IM, Lima WG, Michalick MSM, et al. Comparison of paraffin-embedded skin biopsies from different anatomical regions as sampling methods for detection of Leishmania infection in dogs using histological, immunohistochemical and PCR methods. BMC Vet Res. [Internet]. 2006 [cited 2021 Jan 19]; 2:17. Available from: https://doi.org/10.1186/1746-6148-2-17 English.
    » https://doi.org/10.1186/1746-6148-2-17
  • 35
    Paparcone R, Fiorentino E, Cappiello S, Gizzarelli M, Gradoni L, Oliva G, et al. Sternal Aspiration of Bone Marrow in Dogs: A Practical Approach for Canine Leishmaniasis Diagnosis and Monitoring. J Vet Med. [Internet]. 2013 [cited 2021 Jan 27]; 2013:e217314. Available from: https://doi.org/10.1155/2013/217314 English.
    » https://doi.org/10.1155/2013/217314

Publication Dates

  • Publication in this collection
    27 Sept 2021
  • Date of issue
    2021

History

  • Received
    11 May 2021
  • Accepted
    04 Aug 2021
  • Published
    25 Aug 2021
Universidade Federal de Goiás Universidade Federal de Goiás, Escola de Veterinária e Zootecnia, Campus II, Caixa Postal 131, CEP: 74001-970, Tel.: (55 62) 3521-1568, Fax: (55 62) 3521-1566 - Goiânia - GO - Brazil
E-mail: revistacab@gmail.com