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ISRAEL JOURNAL OF


 
VETERINARY MEDICINE                      




Vol. 60 (1) 2005

KIDNEY PATHOLOGY IN CATTLE NATURALLY INFECTED BY FASCIOLA HEPATICA
Marques, S.T.1, Scroferneker, M.L.2, Edelweiss, M.I.3

  1. Veterinarian, Faculty of Veterinary, Universidade Federal do Rio Grande do Sul, Rua Aneron Corrêa de Oliveira 74/201, Porto Alegre, RS, Brasil. CEP 90410-070. Tel. + 55 51 3387 4141.

  2. Professor, Department of Microbiology, Institute of Basic Health Sciences, Universidade Federal do Rio Grande do Sul, Rua Vasco da Gama 176/604, Porto Alegre, RS, Brasil. CEP 90040-111. Tel.: +55 51 3311 0685.

  3. Professor, Department of Pathology, Medical School, Universidade Federal do Rio Grande do Sul , Rua Ramiro Barcelos, 1954/ 702, Porto Alegre, RS, Brasil. CEP: 90035-002 Tel-Fax: + 55 51 3330 7030

    Corresponding author: Dr. Maria Lúcia Scroferneker Rua Vasco da Gama 176/604 Porto Alegre - RS - Brazil. CEP 90420-111 Fax: +55 51 3316 3155 E-mail: scrofern@ufrgs.br






  Abstract

Kidney specimens of 51 animals, 27 with fascioliasis and 24 uninfected animals (controls), were examined by light microscopy, direct immunofluorescence (DIF), indirect immunofluorescence (IIF), and immunohistochemistry. In animals infected by Fasciola hepatica, the sections stained with hematoxylin and eosin (HE), periodic acid-Schiff (PAS) and Masson’s trichrome revealed mesangioproliferative glomerulonephritis (33.3%) and membranoproliferative glomerulonephritis (44.5%). On DIF, 100% of the animals with fascioliasis had granular and pseudolinear IgG deposits; while 70.4% (19 animals) had this lesion on IIF. Furthermore, Fasciola hepatica antigen was found in 20 infected animals by immunohistochemistry,. Our conclusion is that there is a glomerulopathy associated with fascioliasis, and that cattle can serve as an experimental model to detect immune-mediated renal injury caused by natural Fasciola hepatica infection.



Key words: fascioliasis, parasitic diseases, chronic renal failure, glomerulonephritis.


Introduction

    Liver fluke disease or fascioliasis is an important parasitic disease that affects cattle, sheep, goats, buffaloes, wild animals, and man worldwide. This disease causes significant economic losses, great expenses with antihelmintics, in addition to liver condemnation, production loss due to mortality, lower production of meat, milk and wool; reduced weight gain, and impaired fertility, also impeding the selection of animals (1-2). Renal involvement in parasitic diseases has several clinical manifestations, including hematuria, mild proteinuria, nephrotic syndrome, and chronic renal failure. Glomerular changes range from mild and transient mesangial proliferation to well-defined glomerulonephritis. In the acute phase of some parasitic diseases such as hydatid disease, trichinosis and leishmaniasis, mesangioproliferative glomerulonephritis is observed, but tends to disappear when the parasitic infection is under control. Deposits of IgG, IgM and C3 suggestive of circulating immune complexes can be observed in glomeruli (3-4). Glomerulonephritis in chronic parasitic diseases was reported in leishmaniasis, hydatid disease, schistosomiasis, malaria and dirofilariasis (5-10). The renal injuries caused by parasitic diseases involve nonspecific mechanisms, parasitic migration, and immunological mechanisms. The aim of our study was to assess glomerulopathy in cattle naturally infected by Fasciola hepatica in the State of Rio Grande do Sul, southern Brazil.

Materials and Methods

Animals

    Tissue specimens were collected from the kidneys of 51 animals, 27 with natural Fasciola hepatica infection, and 24 uninfected animals (controls). The cattle originated from different regions of the State of Rio Grande do Sul, Brazil, and were slaughtered at a meat packing house in compliance with regional laws.


Histopathology

    Kidney biopsies were fixed in Duboscq-Brasil for 10-12 hours, with post-fixation in 10% formalin, were dehydrated, clarified, embedded in paraffin, cut at a 4-µm thickness, stained with hematoxylin and eosin (HE), periodic acid-Schiff (PAS), and Masson’s trichrome (10).

 Direct immunofluorescence (DIF) and indirect immunofluorescence (IIF)

    For the DIF method, frozen kidney biopsies were sectioned in a cryostat at 4-µm thickness, fixed in acetone for five minutes, washed in phosphate-buffered saline (PBS, pH 7.2, 0.01 M, Laborclin), and placed in a humidity chamber for 30 minutes with anti-bovine IgG (whole molecule) FITC conjugate (Sigma- F7887), diluted 1:300 in PBS. The glass slides were washed with PBS three times, and the sections were then mounted onto gelatin-coated slides. For the IIF method, an FITC-conjugated antihuman IgG (Behring Institut), diluted 1:300 in PBS, was used as secondary antibody.



Immunohistochemical analysis

    Kidney biopsies, in paraffin-embedded blocks, were cut at 4-µm thickness. The labeled streptavidin-biotin (Dako LSAB + Kit Peroxidase, K 0690, CA, USA) method was used. The primary polyclonal rabbit antibody against Fasciola hepatica was prepared and diluted 1:100 in PBS (11,12). The primary antibody was replaced by PBS in the negative controls.


The rabbits were euthanized in compliance with the Rules for the Scientific Teaching of Animal Vivisection.
 

 

Results



    Histopathological findings were classified according to the patterns of kidney diseases, which evaluated glomeruli, renal tubules, vessels, and interstitial spaces (13-14). Renal changes in response to bovine fascioliasis varied among the studied animals. Of the 27 infected animals, 21 (77.8%) had kidney disease, 12 (44.5%) presented with membranoproliferative glomerulonephritis, 9 (33.3%) had mesangioproliferative glomerulonephritis, and 6 (22.2%) showed normal renal biopsies.

    In addition to glomerular changes, renal tubules showed interstitial fibrosis and inflammatory infiltrations. The histopathological diagnosis of control animals revealed that 20 (83. 34%) did not present kidney disease. The renal biopsy of one animal (4.2%) of the control group revealed mesangioproliferative glomerulonephritis, and 3 (12.5%) had other alterations compatible with mild acute interstitial nephritis, and mild focal inflammatory cell infiltrate.

    DIF was positive in all infected animals. Granular and pseudolinear IgG deposits were found in the mesangial regions of the glomeruli. On IIF, the diagnosis was positive in 70.4% of infected animals (Figure 1). DIF and IIF were negative in the control group. The immunohistochemical analysis revealed a positive reaction for the antigen (the presence of diffuse brown antigens) of Fasciola hepatica in the glomerulus and vascular pole.
 

Figure 1 - Immunofluorescence microscopy showing granular deposits of IgG in mesangial areas and along some capillary walls X 40.

Discussion

    Fascioliasis in ruminants is an important parasitic disease in the State of Rio Grande do Sul, southern Brazil, with a prevalence rate of 13.2% (15) and with economic and health consequences. This study shows for the first time the relationship between fascioliasis and glomerulonephritis using naturally infected animals. There are several forms of glomerular disease in which, with or without complement, immunoglobulins and antigen complexes may be observed and considered to cause renal injury as the result of many etiological agents, including parasitic diseases (16). Nephritis caused by immune complex in kidneys of chickens infected by Plasmodium gallinaceum showed granular deposits in the glomerulus, associating glomerulonephritis with acute avian malaria (17). Membranoproliferative glomerulonephritis was found in cattle naturally infected by Tripanosoma congolensis (18). Our findings in cattle naturally infected by Fasciola hepatica in southern Brazil showed the same pattern of renal injury.

    In an experiment with rabbits infected by Schistosoma japonicum, 83% presented glomerulonephritis and IgG deposits in the subendothelial and mesangial regions of glomeruli, 78% had IgM deposits, and 28% presented deposits of C3 (19). Glomerulonephritis was observed in dogs infected by Leishmania infantum and immune complex deposits (IgM, IgG, C3) were detected in glomeruli, in the mesangial regions, and along the glomerular and tubular basement membrane (7,20). Tafuri et al. (8) studied dogs naturally infected by Leishmania chagasi to assess glomerular injury. Renal biopsies presented inflammatory cell infiltrate in glomeruli and interstitium, segmental glomerulosclerosis, diffuse membranoproliferative glomerulonephritis, diffuse mesangioproliferative glomerulonephritis, and crescentic glomerulonephritis, and the immunohistochemical analysis yielded positive results (8). Kidney biopsies of hamsters infected with Leishmania chagasi demonstrated the presence of IgG deposits, in addition to membranoproliferative and mesangioproliferative glomerulonephritis (9). The renal injury caused by leishmaniasis is similar to that of bovine fascioliasis. In addition, the immunohistochemical detection of Fasciola hepatica antigen in glomeruli, and the presence of IgG are suggestive of disease mediated by immune complexes. The antigen deposits shown by the streptavidin-peroxidase technique were not present in all infected animals. Similar results with experimental dirofilariasis showed glomerular injury in dogs, characterized by mesangial proliferation, neutrophil infiltrate, enhancement of the glomerular basement membrane, fibrin deposits, and presence of IgG, IgM and C3 deposits with a glomerular pattern, electrodense deposits in the mesangial, subendothelial and intramembrane regions of glomeruli (6). The positive results obtained through the immunofluorescent analysis of the renal tissue of cattle with Fasciola hepatica is clear evidence of antigen-antibody complexes with a granular and pseudolinear pattern in the mesangial and subendothelial regions.

    Glomerulonephritis in sheep naturally infected by Echinococcus granulosus in an endemic region in Southern Brazil was investigated. The results were membranoproliferative and mesangioproliferative glomerulonephritis, hyaline cylinders, and erythrocytes in the tubuli and antigens compatible with immunomediated disease (3). Renal involvement associated with parasitic disease was investigated in mice experimentally infected with Toxocara canis. Glomerulonephritis and granular deposits of IgG, IgM and C3 suggest the presence of immunomediated mechanisms in the pathogenesis of renal injury in toxocariasis (21). Kidneys of rhesus monkeys infected by Plasmodium inui showed IgG, C3, C4, albumin and fibrinogen, and glomerulopathy was associated with this parasitic infection (22).

    The absence of renal injury in 6 (22.2%) animals infected by Fasciola hepatica may be explained by low parasitic burden, or nutritional and health management. Focal mesangioproliferative glomerulonephritis, without any other renal alterations, was diagnosed in one control animal (4.2%). Biopsies conducted on 3 controls (12.5%) revealed diffuse inflammatory cell infiltrate and mild tubular atrophy, with no antigen-antibody complexes, which is possibly related to other pathologies or to sanitation and nutritional status. Our findings show for the first time, the occurrence of renal injury due to the formation of immune complexes causing glomerulonephritis associated with bovine fascioliasis.


 

 

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