,Vol. 141, No. 1, July 1992 AmericanJournal of Pathology Copyright X) American Association of Pathologists
Immunopathology of Glomerulonephritis Associated with Chronic Woodchuck Hepatitis Virus Infection in Woodchucks (Marmota monax) David N. Peters,* Howard Steinberg, * Wayne 1. Anderson,* William E. Hornbuckle,t Paul J. Cote, John L. Gerin, Robert M. Lewis,* and Bud C. Tennantt From the Departments of Pathology* and Clinical Sciences,t New York State College of Veterinary Medicine, Cornell University, Ithaca, Neuw York, and the Georgetown University Medical Center, Division of Molecular Virology, and
Immunology, Rockville, Maryland
Retrospective analysis of necropsy findings of 705 woodchucks was performed to determine the prevalence and morphology of immune-mediated glomerulonephritis, its relationship to woodchuck hepatitis virus (WHV) infection, and the presence of major WHV antigens. Twenty-six woodchucks had glomerular lesions. Renal tissue of the 26 animals was evaluated histologically and immunohistochemically for immune-mediated glomerulonephritis. Of these 26 animals, immune-mediated glomerulonephritis was diagnosed in six, all of which were chronic WHV carriers. Membranous glomerulonephritis was identified in three animals, two of which also had mesangial proliferation. Host immunoglobulin was present within the mesangium and along capillary loops in all three. Woodchuck hepatitis virus core antigen (WHcAg) was present along capillary loops of two of these animals, one membranous and one mixed4 and in the mesangium of all three. Woodchuck hepatitis virus surface antigen (WHsAg) deposition was similar to WHcAg deposition but was only present along capillaries in those animals with mixed nephritis. The remaining three animals had mesangial proliferation WHsAg and host immunoglobulin deposition were predominately mesangial; WHcAg was not detected. Transmission electron microscopy showed thickening of the capillary loop basement membranes and subepithelial electrondense deposits in animal one, and deposits in the
mesangium in animal six. (Am J Pathol 1992, 141:
143-152) An association between infection with hepatitis B virus (HBV) and glomerulonephritis has been well documented. The reported prevalence of chronic HBVassociated glomerulonephritis, among series of patients with glomerulonephritis, ranges from less than 1% in the United States to as high as 56% in some European countries,1 although the true prevalence of HBV-related glomerulonephritis is unknown. Several histologic forms of glomerulonephritis have been associated with HBV infection, including those characterized by minimal change and the membranous, mesangiocapillary (membranoproliferative), endocapillary proliferative, and endocapillary and extracapillary proliferative forms; and the mesangial proliferative form with IgA deposits.2 Membranous glomerulonephritis is the form most commonly associated with HBV infection. The association is particularly striking in children and teenagers, and the clinical course seems to be benign, although lengthy follow-up studies have not been done."19 The woodchuck (Marmota monax) has served as an excellent model for the study of HBV infection in humans because of similarities between HBV and the Woodchuck hepatitis virus (WHV) and between the hepatic manifestations associated with the two viruses.2>27 What is less clear is the prevalence and types of extrahepatic manifestations that are associated with hepadnaviral infection in these two species. This study determined the prevalence of immunemediated glomerulonephritis in a defined population of laboratory-maintained woodchucks. We also wanted to determine the relationship of immune-mediated glomerulonephritis to WHV infection, to define the major categories of immune-mediated glomerulonephritis associated Supported by NIH grants N01 Al 82698 and N01 Al 72623. Accepted for publication January 30, 1992. Address reprint requests to David N. Peters, James A. Baker Institute for Animal Health, Cornell University, Ithaca, New York 14853.
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with WHV infection, and to determine the presence or absence of the major WHV antigens.
Materials and Methods Animals Postmortem examinations were performed on 705 laboratory-maintained woodchucks during an 11-year period (January 1979-December 1989). These animals were from breeding and experimental colonies maintained at Cornell University for investigations of the pathogenesis of WHV infection. Of these 705 animals, 310 were colony born and the other 395 were trapped in central New York (Tompkins and adjacent counties), Maryland, or Delaware (Table 1). The age of trapped woodchucks was estimated on the basis of body size at the time they were added to the colony. Seronegative woodchucks were maintained individually in stainless-steel cages or in groups of two or three in animal runs with concrete floors. Woodchucks that were infected with WHV were housed in Horsfal isolation cages. Colony-born animals were infected with 105 infectious doses of a well-characterized virus pool (WHV 7) at 3 days of age.28 Infected wildcaught animals were infected as a result of natural infection. In some cases, woodchucks that had recovered from WHV infection (cleared WHV surface antigen and developed antibody to WHV surface antigen) were transferred to facilities similar to those used for seronegative woodchucks. Wooden nest boxes were provided for all woodchucks, and wood shavings were used as bedding. Tap water was supplied ad libitum, and animals were fed a commercial laboratory chow originally formulated for rabbits (Agway, Inc., Syracuse, NY).
Serologic Classification of Infection Serologic markers, measured by radioimmunoassay, included WHV surface antigen (WHsAg), antibody against WHV core antigen (antiWHc), and antibody against WHV surface antigen (antiWHs).'93 Serologic classification was based on the presence or absence of serum markers and included uninfected (WHsAg -, antiWHc -, antiWHs -) and chronic carriers (WHsAg +, antiWHc +, antiWHs -), recovered (WHsAg -, antiWHc +, antiWHs +), and window (antiWHc + only) animals.
Histologic Evaluation Kidney tissue was fixed in 10% neutral buffered formalin during necropsy. Sections were processed routinely and 2-,um-thick sections were stained with hematoxylin and eosin, periodic acid-Schiff (PAS), Jones methenamine silver, and Congo red for light microscopic evaluation.
Antisera Preparation Polyvalent rabbit anti-woodchuck antiserum was prepared in the following manner. Woodchuck serum was precipitated with 37.5% ammonium sulfate to isolate immunoglobulins and dialyzed against phosphate-buffered saline (PBS) at a pH of 7.2 and 40C for 2 days. Cellulose acetate serum protein electrophoresis and protein determination were performed before immunization of rabbits with 0.5 mg protein emulsified in 2.0 ml of complete Freund's adjuvant into six subcutaneous sites. The rabbits were given booster injections of antigen in incomplete Freund's adjuvant 10 days later. The rabbits were
Table 1. Ages and Serologic Status of Colont Born and Wild CaughtAnimals Infected
Age (yrs)
Immunopathology of Glomerulonephritis Associated with Chronic Woodchuck Hepatitis Virus Infection in Woodchucks (Marmota monax) David N. Peters,* Howard Steinberg, * Wayne 1. Anderson,* William E. Hornbuckle,t Paul J. Cote, John L. Gerin, Robert M. Lewis,* and Bud C. Tennantt From the Departments of Pathology* and Clinical Sciences,t New York State College of Veterinary Medicine, Cornell University, Ithaca, Neuw York, and the Georgetown University Medical Center, Division of Molecular Virology, and
Immunology, Rockville, Maryland
Retrospective analysis of necropsy findings of 705 woodchucks was performed to determine the prevalence and morphology of immune-mediated glomerulonephritis, its relationship to woodchuck hepatitis virus (WHV) infection, and the presence of major WHV antigens. Twenty-six woodchucks had glomerular lesions. Renal tissue of the 26 animals was evaluated histologically and immunohistochemically for immune-mediated glomerulonephritis. Of these 26 animals, immune-mediated glomerulonephritis was diagnosed in six, all of which were chronic WHV carriers. Membranous glomerulonephritis was identified in three animals, two of which also had mesangial proliferation. Host immunoglobulin was present within the mesangium and along capillary loops in all three. Woodchuck hepatitis virus core antigen (WHcAg) was present along capillary loops of two of these animals, one membranous and one mixed4 and in the mesangium of all three. Woodchuck hepatitis virus surface antigen (WHsAg) deposition was similar to WHcAg deposition but was only present along capillaries in those animals with mixed nephritis. The remaining three animals had mesangial proliferation WHsAg and host immunoglobulin deposition were predominately mesangial; WHcAg was not detected. Transmission electron microscopy showed thickening of the capillary loop basement membranes and subepithelial electrondense deposits in animal one, and deposits in the
mesangium in animal six. (Am J Pathol 1992, 141:
143-152) An association between infection with hepatitis B virus (HBV) and glomerulonephritis has been well documented. The reported prevalence of chronic HBVassociated glomerulonephritis, among series of patients with glomerulonephritis, ranges from less than 1% in the United States to as high as 56% in some European countries,1 although the true prevalence of HBV-related glomerulonephritis is unknown. Several histologic forms of glomerulonephritis have been associated with HBV infection, including those characterized by minimal change and the membranous, mesangiocapillary (membranoproliferative), endocapillary proliferative, and endocapillary and extracapillary proliferative forms; and the mesangial proliferative form with IgA deposits.2 Membranous glomerulonephritis is the form most commonly associated with HBV infection. The association is particularly striking in children and teenagers, and the clinical course seems to be benign, although lengthy follow-up studies have not been done."19 The woodchuck (Marmota monax) has served as an excellent model for the study of HBV infection in humans because of similarities between HBV and the Woodchuck hepatitis virus (WHV) and between the hepatic manifestations associated with the two viruses.2>27 What is less clear is the prevalence and types of extrahepatic manifestations that are associated with hepadnaviral infection in these two species. This study determined the prevalence of immunemediated glomerulonephritis in a defined population of laboratory-maintained woodchucks. We also wanted to determine the relationship of immune-mediated glomerulonephritis to WHV infection, to define the major categories of immune-mediated glomerulonephritis associated Supported by NIH grants N01 Al 82698 and N01 Al 72623. Accepted for publication January 30, 1992. Address reprint requests to David N. Peters, James A. Baker Institute for Animal Health, Cornell University, Ithaca, New York 14853.
143
144
Peters et al
AJPJu4l 1992, Vol. 141, No. 1
with WHV infection, and to determine the presence or absence of the major WHV antigens.
Materials and Methods Animals Postmortem examinations were performed on 705 laboratory-maintained woodchucks during an 11-year period (January 1979-December 1989). These animals were from breeding and experimental colonies maintained at Cornell University for investigations of the pathogenesis of WHV infection. Of these 705 animals, 310 were colony born and the other 395 were trapped in central New York (Tompkins and adjacent counties), Maryland, or Delaware (Table 1). The age of trapped woodchucks was estimated on the basis of body size at the time they were added to the colony. Seronegative woodchucks were maintained individually in stainless-steel cages or in groups of two or three in animal runs with concrete floors. Woodchucks that were infected with WHV were housed in Horsfal isolation cages. Colony-born animals were infected with 105 infectious doses of a well-characterized virus pool (WHV 7) at 3 days of age.28 Infected wildcaught animals were infected as a result of natural infection. In some cases, woodchucks that had recovered from WHV infection (cleared WHV surface antigen and developed antibody to WHV surface antigen) were transferred to facilities similar to those used for seronegative woodchucks. Wooden nest boxes were provided for all woodchucks, and wood shavings were used as bedding. Tap water was supplied ad libitum, and animals were fed a commercial laboratory chow originally formulated for rabbits (Agway, Inc., Syracuse, NY).
Serologic Classification of Infection Serologic markers, measured by radioimmunoassay, included WHV surface antigen (WHsAg), antibody against WHV core antigen (antiWHc), and antibody against WHV surface antigen (antiWHs).'93 Serologic classification was based on the presence or absence of serum markers and included uninfected (WHsAg -, antiWHc -, antiWHs -) and chronic carriers (WHsAg +, antiWHc +, antiWHs -), recovered (WHsAg -, antiWHc +, antiWHs +), and window (antiWHc + only) animals.
Histologic Evaluation Kidney tissue was fixed in 10% neutral buffered formalin during necropsy. Sections were processed routinely and 2-,um-thick sections were stained with hematoxylin and eosin, periodic acid-Schiff (PAS), Jones methenamine silver, and Congo red for light microscopic evaluation.
Antisera Preparation Polyvalent rabbit anti-woodchuck antiserum was prepared in the following manner. Woodchuck serum was precipitated with 37.5% ammonium sulfate to isolate immunoglobulins and dialyzed against phosphate-buffered saline (PBS) at a pH of 7.2 and 40C for 2 days. Cellulose acetate serum protein electrophoresis and protein determination were performed before immunization of rabbits with 0.5 mg protein emulsified in 2.0 ml of complete Freund's adjuvant into six subcutaneous sites. The rabbits were given booster injections of antigen in incomplete Freund's adjuvant 10 days later. The rabbits were
Table 1. Ages and Serologic Status of Colont Born and Wild CaughtAnimals Infected
Age (yrs)
