Diffuse Intralobular Liver Fibrosis in Dogs Naturally Infected with Leishmania(Leishmania) chagasi

Ferdinan Melo, Marina Amaral, Patricia Oliveira, Wanderson Lima, Marina Andrade, Marilene Michalick,Pedro Raso, Washington Tafuri, and Wagner Tafuri*

Departamento de Patologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Brazil; Departamento deAnatomie Patológica, Faculdade de Medicina, Universidade Federal de Minas Gerais, Brazil

Abstract. The aim of this study was to evaluate the diffuse intralobular fibrosis in dogs naturally infected withLeishmania (Leishmania) chagasi. One hundred five infected animals with positive serologic tests for Leishmania weredivided into two clinical groups: 69 symptomatic animals and 36 asymptomatic. Special staining with Gomori, Heiden-hain, Silver, and Picrosirius Red was applied to characterize fibrilopoesis. The tissue parasite load was measured byimmunohistochemistry and associated histomorphometric analyses. Intralobular fibrosis was observed in all dogs, andmore collagen deposition was confirmed in the infected animals than in the controls by these histomorphometric studies.There were significant differences among the distinct clinical groups. In fact, symptomatic dogs showed an increasedcollagen deposition in the liver compared with asymptomatic ones. A peculiar diffuse intralobular fibrosis, where thecollagen fibers encircled small groups of hepatocyte(s), was observed in two cases (1.9%).

INTRODUCTION

Canine visceral leishmaniasis (CVL) is caused by Leishma-nia infantum (syn. L. chagasi in America) and is transmittedby phlebotomine sand fly bites (Lutzomyia longipalpis spe-cies in America).1,2 Protozoa of the genus Leishmania aredimorphic obligate intracellular parasites that reside withinmononuclear phagocytes in the mammalian host. Classic his-topathologic lesions have been described mainly in organsrich in cells of the mononuclear-phagocytic system such asliver, spleen, lymph nodes, bone marrow, gastrointestinaltract, and skin. In general, an intense chronic inflammatoryreaction consisting of infiltration by mononuclear cells (mac-rophages, plasma cells, and lymphocytes) is observed in theliver and spleen,3–7 skin,6,8,9 bone marrow,9 lymph nodes,10,11

gastrointestinal tract,12,13 and kidneys.14,15

In many parts of the world, with some geographical varia-tion, liver disease and various associated histopathologicchanges have been described in CVL4–6,16 and human visceraldisease.17–23 These changes include hypertrophy and hyper-plasia of Kupffer cells, portal and intralobular granulomas,bilirubin deposition, and diffuse intralobular fibrosis. Fibrotictissue is formed in response to inflammation or direct toxicinsult to the liver.24–26 The liver seems to attract many para-sites, which may either inhabit the organ, pass through it dur-ing normal development, or be carried there and destroyed.The Kupffer cells are the target cells for Leishmania (Leish-mania donovani complex), causing visceral leishmaniasis orkala-azar.25

The protozoan, Leishmania, has been implicated in hepaticfibrosis in chronic cases of kala-azar in India,21,23,27,28 somecases in the Mediterranean,29 and one case in China.30 Fibro-sis has been reported in CVL in organs such as lung (chronicinterstitial pneumonitis)31,32 and liver (diffuse hepatic intra-lobular fibrosis).5

Hepatic fibrosis is characterized by increased deposition

and altered extracellular matrix composition in the portaltracts, around central veins, or in perisinusoidal spaces (intra-lobular fibrosis).24 The aim of this study was to verify theincidence of diffuse intralobular liver fibrosis in dogs natu-rally infected with Leishmania chagasi. Animals with definedparasitologic and clinical status were obtained from an en-demic area of Brazil, the municipality of Belo Horizonte, MG(Southern Brazil).

MATERIALS AND METHODS

Animals. One hundred five mongrel dogs of unknown agenaturally infected with L. chagasi were identified during anepidemiologic survey of CVL carried out by the municipalityof Belo Horizonte, MG (Southern Brazil). Enzyme-linked im-munosorbent assays (ELISA; optical density > 100 > 1:400dilutions) were positive for all infected animals. We also ana-lyzed serum samples using a commercial kit containing animmunochromatographic strip that uses recombinant leish-manial antigen k39 and a dominant amastigote antigen of L.chagasi (rK39), which is highly sensitive and specific forLeishmania donovani complex infection, as previously de-scribed.33–35 Sera from all infected dogs were also positive forthis test. Another 17 dogs with serologic examinations nega-tive for Leishmania were obtained as control animals.

Groups and clinical aspects of infected dogs. All infecteddogs were clinically classified and divided into three groups asfollows: Group 1, symptomatic dogs—69 animals that exhib-ited the classic signs of the disease such as lymphadenopathy,cutaneous alterations (alopecia, dry exfoliative dermatitis, orulcers), onychogryphosis, keratoconjunctivitis, weight loss orcachexia, and anemia; Group 2, asymptomatic dogs—36 ap-parently healthy animals with no signs of the visceral disease;Group 3, control dogs—17 uninfected animals with serologicand parasitologic examinations negative for Leishmania.

Parasitological diagnosis of Leishmania infection. All dogswere anesthetized with 2.5% (1.0 mL/kg) intravenous thio-pental. The experimental protocol using dogs was approvedby CETEA-UFMG (Brazilian Animal Experimental College,number 106/2004). Touch aspirates of bone marrow were ob-tained for parasitologic diagnosis of infected and controlanimals. The smears were air-dried and stained with 10%Giemsa. Leishmania amastigotes were detected in all in-

* Address corresondence to Wagner Tafuri, Departamento de Pato-logia Geral, Instituto de Ciências Biológicas, Universidade Federalde Minas Gerais, Av Antonio Carlos, 6627, Campus Pampulha, BeloHorizonte, 31270-901, Brazil. E-mails: wagner@icb.ufmg.br andwltafuri@yahoo.com.br

Am. J. Trop. Med. Hyg., 79(2), 2008, pp. 198–204Copyright © 2008 by The American Society of Tropical Medicine and Hygiene

198

fected animals by light microscopy using oil immersion(×1,000 magnification). Control animals were parasitologi-cally negative.

Necropsy and histopathology. The animals were anesthe-tized with a dose of 0.5 mL/kg thiopental intravenously(2.5%) and killed with T-61 (0.3 mL/kg). During necropsy,the livers were weighed, and tissue touch preparations(smears) were obtained from small samples as described forthe bone marrow aspirates. Leishmania amastigotes were ob-served in slides stained with 10% Giemsa. Amastigote formsof Leishmania were observed in all smears of infected animallivers.

Other liver fragments were collected for histopathology.These samples were fixed in 10% neutral-buffered formalinand were dehydrated, cleared, embedded in paraffin, cut into4- to 5-�m-thick sections, and stained with hematoxylin andeosin (HE). For collagen studies, all liver fragments werestained with Gomori trichrome, Hedeihen blue, Gomori am-moniacal silver, and Picrosirius red. In Gomori trichrome,fibrillar collagen appears green and in Hedeihen, it appearsblue. In silver staining, the fibrillar collagen becomes black,and in Picrosirius, some fibers (probably type I) stain red oryellow, whereas reticular collagen (probably type III) appearsgreen under polarized light. For Picrosirius staining, fibrillarcollagen was estimated at ×110 resolution using polarizedlight.36,37

Histomorphometric analysis. Liver sections stained withGomori trichrome, Hedeihen blue, and Gomori ammoniacalsilver were analyzed morphometrically to characterize theintralobular collagen deposition, excluding perivascularcollagen. This analysis was carried out using an Axiolab lightmicroscope (Zeiss) with ×440 resolution. The images weretransferred to a computer video screen using software andrelayed to a computer-assisted image analysis system (Kon-tron Elektronic/Carl Zeiss, Oberkochen, Germany). Using adigital pad, the total area occupied by stained collagen fiberswas measured from real images and segmented to generatebinary images. The results are expressed in square microme-ters.32,38

Immunohistochemical method for labeling amastigotes ofLeishmania and morphometric analysis. The streptavidin–biotin immunohistochemical method for detecting Leishma-nia in formalin-fixed paraffin-embedded canine tissues de-scribed by Tafuri and others was used.39 Leishmania amasti-gotes were readily observed within macrophages in fragmentsof the livers from all naturally infected dogs.

For histomorphometric study, 40 randomly chosen imagesfrom histologic slides of liver tissue fragments were used toassess the number of immunolabeled amastigotes in a Kon-tron Elektronick/Carl Zeiss image analyzer (KS300 software)as described above, using an Axiolab light microscope (Zeiss)with a ×440 resolution.38

Statistical analysis. All collagen staining results were com-pared between asymptomatic and symptomatic dogs by a one-way analysis of variance (ANOVA) test. P < 0.05 was con-sidered significant.

RESULTS

Clinical aspects of the animals. The most frequent clinicalsigns were lymphadenopathy (67%), chronic dry desquama-

tion (61%) mainly restricted to the ears and limbs, followedby onychogryphosis (57%), weight loss (41%), alopecia(32%), anemia (27%), and skin ulcerations (19%).

Anatomic pathology. Macroscopically, the livers of thenaturally infected animals were generally enlarged. However,hepatomegaly was not necessarily found in symptomaticdogs; it varied markedly in association with a dark red color-ation indicating congestion. The relative liver weight (liverweight/body weight) was higher in symptomatic dogs (5.12%)than asymptomatic dogs (4.16%) or controls (4.08%; P <0.05).

We observed idiopathic macroscopic lesions in one symp-tomatic dog. In this case, the liver was pale with a slightlynodular surface, suggesting a prominent reticular pattern onthe surface and the cut section. The liver was of firm consis-tency.

Microscopically, histologic examination after HE stainingshowed a general chronic inflammatory reaction involving theentire architecture of the liver including the capsule, portaltracts, and central veins or perisinusoidal spaces. The portalspace showed no fibrosis, but there was mild infiltration oflymphocytes, plasma cells, and macrophages with or withoutamastigotes (Figure 1A and B). Kupffer cells showed hyper-plasia and hypertrophy. Additionally, intralobular granulo-mas comprising of plasma cells, epithelioid cells, and lympho-cytes were found in almost all the infected animals (Figure 1Cand D). The search for Leishmania parasites by HE and spe-cifically by immunohistochemistry showed amastigotesmainly in macrophage granulomas (Figure 1E) and Kupffercells (Figure 1F). Hepatocellular necrosis foci were found, butthey were not intensely or diffusely distributed throughoutthe liver parenchyma.

None of the hepatic lesions were specific to asymptomaticor symptomatic animals. All infected animals showed thesame qualitative histologic picture. The general histopatho-logic changes in the livers are summarized in Table 1.

Intralobular collagen deposition (intralobular fibrosis) wasalso observed in all infected animals, but the intensity washighly variable. Even thought it could be observed by HE,Gomori trichrome, and Heidenhain blue staining, it wasmost evident after Gomori ammoniacal silver staining (Figure2A–D).

Two cases of symptomatic animals merited attention. First,under HE analysis, we observed a peculiar and diffuse intra-lobular fibrosis compatible with the description of Rogers23

(so-called “Rogers’ cirrhosis”) in Indian Kala-azar. Here, thefibrosis had developed to link the portal areas and centralveins. Gomori trichrome and Heidenhain blue showed in-tense proliferation of intralobular collagen deposition, butthis histologic picture was again most evident after Gomoriammoniacal silver staining, as previously mentioned. In fact,silver staining showed conspicuous collagen (reticulin fibers)thickening in the space of Disse. In some areas, hepatic cellswere isolated from the sinusoidal blood by fibropoiesis (Fig-ure 2E and F). However, there was no subversion of thearchitecture of the organ with intersection of the classic lob-ules to produce pseudolobules. Swelling of liver cells was as-sociated with changes in fat content, but no intense necrosiswas observed. There was also hypertrophy and hyperplasia ofthe Kupffer cells, which were commonly parasitized. Portalinflammatory cellular exudate and intralobular granulomaswere also frequently found as described above. Regenerating

LIVER FIBROSIS IN DOGS INFECTED WITH LEISHMANIA (LEISHMANIA) CHAGASI 199

FIGURE 1. Liver sections of dogs naturally infected with L. (L.) chagasi. (A,B) Asymptomatic dog. (A) Portal space with a cellular infiltratedof plasma cells (arrows), lymphocytes and macrophages. Note hepatocytes swelling (hydropic degeneration) (arrowheads) HE (Bars � 16 �m).(B) Same field showing immunolabeled amastigotes forms of Leishmania in macrophages (large arrows). Note an intense swelling of thehepatocytes (“balloon cells”) (arrowheads). Streptoavidin-peroxidase method (Bars � 16 �m). BD � Bile duct; PV � portal vein; LV �lymphatic vessel; S � sinusoids. (C) Symptomatic dog. Observe three intralobular granulomas formation comprising macrophages (epithelioidcells) (large arrows), plasma cells and lymphocytes (small arrows). Hepatocytes with hydropic degeneration is also present (arrowheads) HE (Bars� 16 �m). (D,E) Asymptomatic dog. (D) Observe an intralobular granuloma formation comprising epithelioid cells with an elongated or ovalnucleus and a pink cytoplasm with indistinct cell boundaries (appearing to merge into one another) (large arrows), plasma cells (arrowhead) andlymphocytes (small arrow), HE (Bars � 16 �m). Fatty change (FC) of the hepatocytes (hepatic steatosis) is easily observed, HE (Bars � 16 �m).(E) Same field showing immunolabelled amastigotes into granulomas macrophages (arrows), Streptoavidin-peroxidase method (Bars � 16 �m).(F) Symptomatic dog. Note in the center of the figure Kupffer cells intensely parasitized (large arrows). On the right Kupffer cells with lessintracellular immunolabeled amastigotes (small arrows) (Bars � 16 �m). This figure appears in color at www.ajtmh.org.

MELO AND OTHERS200

liver cells plates were two cell layers thick, and acinar-likeregenerative processes were present. Hepatocellular necrosisfoci could be observed.

Picrosirius analysis showed that a large proportion of thetotal collagen was generally fibrillar collagen type I (yellow-red color). Fibrillar collagen with a reticular disposition(probably type III in green color) was less prominent.

Morphometric analysis of hepatic intralobular collagen de-position (Gomori trichrome, Heidenhain blue, Gomori am-moniacal silver-staining, and Picrosirius red methods). Thefocus of this study was to evaluate intralobular fibrosis. Thiswas apparent with all staining methods, but collagen fiberswere best visualized by silver staining. The morphometric re-sults obtained by all methods showed that infected animalshad more hepatic intralobular collagenopoiesis than controls.There were significant differences among the distinct clinicalgroups: symptomatic dogs showed more collagen depositionin the livers than asymptomatic ones (Table 2).

Parasitism burden. As depicted in Figure 1E and F, Leish-mania parasites were readily observed in Kupffer cells andmacrophage granulomas, and the tissue parasitism load wasevaluated. Our results showed a positive correlation betweenparasite load and collagenopoiesis. The difference was statis-tically significant (r2 � 0.0451; P � 0.0303, linear Pearsoncorrelation).The parasite tissue load was higher in symptom-atic dogs (20.1 ± 5.7) than asymptomatic (14.7 ± 3.7) dogs(P � 0.0310, unpaired t test). Although our results showedonly a weak correlation between parasite load and collage-nopoiesis (r2 � 0.0131), it was statistically significant (P �0.0303, linear Pearson correlation).

DISCUSSION

Diffuse intralobular fibrosis or a systemic form of fibrosis inthe sinusoids has been reported in human visceral leishma-niasis.17–23,40,41 Rogers23 described a peculiar diffuse hepaticintralobular fibrogenesis in a chronic case of kala-azar. AHindu male, ∼30 years of age, died with all the classic symp-toms of liver cirrhosis. The most striking histopathologic pic-ture of the liver was described by Rogers23: “. . . the universaldistribution of the cirrhotic process throughout the liver lob-ules, so that the hyperplastic connective tissue widely sepa-rates each column of epithelial liver cells. A careful study of

a number of sections showed that there is extremely littlealteration in the general arrangement of the liver lobules,which retain to a great extent their shape and size, althoughthe intralobular veins are somewhat less prominent thanusual.” Visentini,40 in southern Italy, described a case of hu-man visceral leishmaniasis with hepatic fibrosis similar toRogers’ description. However, as cited by Bogliolo,18 this au-thor did not know the previous work of Rogers.23 Thus, thispeculiar intralobular fibrosis was subsequently only cited byLaveran,41 but Nattan-Larrier,29 in a study of hepatic speci-mens from Indian adults, gave a detailed histologic descrip-tion. This author, who was familiar with the earlier work ofRogers,23 called the hepatic lesion “Rogers’ cirrhosis” butdescribed more details, showing that the collagen fibers iso-lated hepatocytes as single cells. He called the condition cir-rhoses dites monocelulaires. It was subsequently characterizedas “monocellular cirrhosis of Nattan-Larrier.”29

In American visceral leishmaniasis, a diffuse intralobularfibrosis such as that described by Rogers23 and Goswami21 inIndian Kala-azar (25–60%, respectively) is rarely found.20,42–44

In Brazil, Bogliolo18 gave a detailed description of an intenseand diffuse hepatic intralobular fibrosis in a 19-year-old malepatient diagnosed with visceral leishmaniasis. He describedthe formation of reticular collagen fibers on the sinusoidalwalls and around the hepatocytes, where they induced atro-phy of the hepatocytes. Since these studies, intralobular fi-brosis as depicted by Rogers23 has been shown by histopath-ologic studies in six isolated Brazilian cases of human visceralleishmaniasis.18,42–44 In this study, we found two morphologiccases (1.9%) of intralobular fibrosis of the kind described byRogers in Indian kala-azar.

Duarte and Corbett,42 in a study of 47 patients with visceralleishmaniasis, found a high incidence of intralobular fibrosis,with 74.4% of cases showing at least scattered small foci ofperisinusoidal fibrosis. In this study of 105 infected animals,we observed intralobular fibrilar collagen deposition in allcases, and this was confirmed by the histomorphometric data(Table 2).

According to Goswami21 and Duarte and Corbett,42 thisdiffuse intralobular fibrosis appears mainly in adults withlong-standing disease. Children often have a rapidly progress-ing disease, allowing no time for the fibrotic changes to occur.In parallel studies with beagles experimentally infected withL. chagasi, our group did not find intralobular fibrosis 1–2years after experimental infection (data not shown).

The “Rogers’ cirrhosis” as described is not nodular butpresents a marked diffuse fibrosis, isolating small groups ofliver cells with regenerative changes. In two cases describedherein, we observed no subversion of liver architecture withintersection of the classic lobules to produce pseudolobules.This means that “Rogers’ cirrhosis” does not represent realcirrhosis according to the 1997 definition of cirrhosis by aworking group sponsored by the World Health Organiza-tion.45 Fibrosis is usually part of the picture of chronic pro-gressive hepatic disease, and spreading changes in the sinu-soids may further compromise the liver cells. This very deli-cate and widely fenestrated structure is loosely associatedwith the hepatocyte cords and separated from them by theSpace of Disse, an arrangement that brings plasma into directcontact with the hepatic cells. As chronic fibrosis progresses,reticulin and subsequently collagen is deposited in the Spaceof Disse, and the hepatic cells become isolated from the si-

TABLE 1Histopathologic features of liver (hematoxilin–eosin staining) of

naturally infected dogs with Leishmania infantum from Belo Hori-zonte, MG, Brazil

Liver histologic analysis

Animals

Asymptomatic(N/%)

Symptomatic(N/%)

Infected(N/%)

Thickened capsule withchronic inflammation 30/83 42/61 72/69

Hypertrophy and hyperplasiaof Mø 36/100 62/90 98/93

Intralobular granulomas 33/92 63/91 63/90Congestion 32/89 65/94 97/92Portal tract inflammation 36/100 69/100 105/100Degenerative lesions

(swelling) 36/100 69/100 105/100Presence of leishmania 20/56 42/61 62/59Total 36 69 105

LIVER FIBROSIS IN DOGS INFECTED WITH LEISHMANIA (LEISHMANIA) CHAGASI 201

nusoidal blood. Eventually, the sinusoids begin to resemblecapillaries. In the two cases of “Rogers cirrhosis” describedherein, we observed areas of enlarged sinusoids with eryth-rocytes. This could be a result of splenic congestion. Indeed,the spleens of these animals were enlarged, tense, and cya-

notic, and on section, they freely exuded blood and the cap-sule became wrinkled. Cirrhosis may alternatively be definedas nodular regeneration of the liver combined with fibrosis.Thus, “pseudolobule formation,” a pathologic condition de-fined by derangement of the lobular and vascular architecture

FIGURE 2. Liver sections of dogs naturally infected with L. (L.) chagasi. Intralobular Fibrosis. (A–D) Symptomatic dog. (A) Lower magni-fication showing a diffuse intralobular fibrosis. Note collagen fibers extend through sinusoids from the portal tract (PT), HE (Bars � 62 �m); (B)Higher mgnification showing an intense proliferation of collagen and reticulin fibers detected by Gomori ammoniacal silver-staining (Bars � 16�m). (C,D) Higher magnifications showing intralobular proliferation of collagen fibers (arrows) detected by Gomori’s trichrome and Heidenhainblue (arrows), respectively (Bars � 16 �m). (E,F) Symptomatic dog “Rogers’ Cirrhosis.” (E) Observe dense and coiled fibers extend throughsinusoids from the portal tract (white arrows). (PV) Portal vein. Gomori ammoniacal silver-staining (Bars � 16 �m). (F) Note conspicuouscollagen thickening in the space of Disse. Hepatic cells had become isolated from the sinusoidal blood by the fibropoiesis (white arrowheads).Gomori ammoniacal silver-staining (Bars � 16 �m). This figure appears in color at www.ajtmh.org.

MELO AND OTHERS202

of the liver, may be the final common pathway through whichalmost all chronic liver diseases produce morbidity and mor-tality.26

In this work, we identified an intralobular fibrosis patternin dogs naturally infected with L. chagasi. Also, we founddifferences in fibrilopoesis among the clinically definedgroups, and we observed a positive correlation between thetissue parasite load and collagen deposition. Moreover, thetwo cases with “Rogers’ cirrhosis” (two symptomatic dogs)suggest that this intralobular fibrosis pattern may representchronic progression of long-standing disease.

Received November 23, 2007. Accepted for publication May 5, 2008.

Acknowledgments: The authors thank Dr. Hélio Chiarini-Garcia(Associate Professor), Departamento de Morfologia, Instituto deCiências Biológicas da UFMG, Belo Horizonte, Brazil, for the pho-tographic support.

Financial support: This work was supported by Conselho Nacional deDesenvolvimento da Pesquisa Tecnológica e Científica (CNPq)(Grants 477427/2006-5; 301301/2006-9 CNPq), Brazil.

Authors’ addresses: Ferdinan Melo, Marina Amaral, Patricia Oliveira,Wanderson Lima, Marina Andrade, Marilene Michalick, and WagnerTafuri, Departamento de Patologia Geral, Instituto de Ciências Biológi-cas, Universidade Federal de Minas Gerais, Av Antonio Carlos, 6627,Campus Pampulha, Belo Horizonte–MG, 31270-901, Brazil, E-mails:ferdinanmelo@yahoo.com.br, marinaelamaral@yahoo.com.br,pattybio2@yahoo.com.br, limaw_g@yahoo.com.br, debrot@gmail.com,michalik@icb.ufmg.br, wagner@icb.ufmg.br, and wltafuri@yahoo.com.br.Pedro Raso and Washington Tafuri, Departamento de Anatomia Pa-tológica, Faculdade de Medicina, Universidade Federal de Minas Gerais,Av. Prof. Alfredo Balena, 190, Belo Horizonte–MG, Brasil Cep 30130-100,Brazil, E-mails: altafuri@terra.com.br and washington.tafuri@cnpq.br.

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TABLE 2Morphometrical analysis of collagen (average of collagen deposition

in �m2) stained by Gomori trichrome, Hedeihen blue, and Gomoriammoniacal silver methods in liver of infected dogs with Leishma-nia infantum

Groups

Average (in �m2)

Gomoritrichrome

Hedeihenblue

Gomori ammoniacalsilver

Control 130 198 2211*Infected animals 291† 348† 3692*†Asymptomatic 332‡ 363‡ 3173*Symptomatic group 287§ 364§ 3955*§

* The averages from infected dogs and asymptomatic and symptomatic dogs vs. controlwere extremely significant under Gomori ammoniacal silver collagen staining analysis(ANOVA test with P < 0.0001).

† The averages from infected dogs were statistically different vs. controls for all collagenstaining (ANOVA test with P < 0.001).

‡ The averages from asymptomatic dogs were statistically different vs. controls for allcollagen staining (ANOVA test with P < 0.01).

§ The averages from symptomatic dogs were statistically different vs. controls for allcollagen staining (ANOVA test with P < 0.001).

LIVER FIBROSIS IN DOGS INFECTED WITH LEISHMANIA (LEISHMANIA) CHAGASI 203

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