Immunoperoxidase Studies on Leptospiral Nephritis of Swine

Leptospiral infection in pigs results in localization of the organism in the kidney after a transient bacteremic phase. While the pathogenesis of acute leptospirosis in the pig has been explained, 1.3,5,8 little is known of renal reactive processes involved in eliminating leptospires during the later stages of chronic infection. Leptospirosis may be diagnosed by the presence of leptospires in tissue sections. A variety of silverstaining, immunofluorescence, immunoperoxidase, and immunogold silver techniques are used for this purpo~e . ~ . ~ This study investigates the relationship between Leptospira interrogans and renal lesions in spontaneously infected pigs. Kidneys of 78 9-month-old, 140-160-kg pigs from seven different groups were sampled at the slaughterhouse. Leptospiral infection in these groups was suspected on the basis of gross lesions on the kidneys and high antibody levels against Leptospira interrogans serovarpomona in the majority of the subjects. One to five fragments from each grossly changed or unchanged kidney were processed for light microscopy and stained with hematoxylin and eosin and the Warthin-Starry method. Immunohistochemical staining was done with the avidin-biotin complex pro~edure,~ using commercial immunoperoxidase kits (Vector Laboratories, Inc., Burlingame, CA). Rabbit antiserum to serovar pomona (courtesy of Dr. P. Mortarino, Istituto Zooprofilattico Sperimentale, Brescia, Italy) was used as the primary antibody. A high percentage of the kidneys showed numerous, macroscopic, grey-white focal lesions mainly located in the cortex and varying from 1 mm to 2 cm in diameter. The lesions were confluent or diffuse in the most severely affected kidneys. Microscopically subacute to chronic interstitial nephritis was present in most of the cases. Only six subjects showed no kidney lesions (grade 0, Fig. 1). In these cases, no leptospires were demonstrated in sections stained either by the Warthin-Starry method or immunohistochemically (Fig. 1). In mildly affected kidneys (grade I, Fig. l), vacuolar degeneration of the tubular epithelium and scattered interstitial foci consisting of lymphocytes and plasma cells were seen. Both methods revealed leptospires in nine out of 18 subjects. In silver-stained sections, leptospires were detected in the tubular lumen as black filiform organisms frequently grouped in masses adhering to the luminal surface of tubular cells (Fig. 2). In immunoperoxidase-stained sections, luminal leptospires were intensely brown (Fig. 3). Heavily infected tubules were never surrounded by inflammatory cells. In more involved kidneys (grade 11, Fig. 1) there were large foci of infiltrating lymphocytes, plasma cells, macrophages, and in some cases, neutrophils. The tubules surrounded by inflammatory cells were atrophic, deformed, had reduced lumen and, in some cases, were completely disrupted. In every kidney with severe interstitial nephritis, leptospires and/or leptospiral antigen could be demonstrated (Fig. 1). In seven of these cases, leptospiral organisms were detected only in the lumen of a few tubules. In addition to luminal organisms, other leptospiral structures were seen in the remaining cases. In areas of mononuclear infiltration, leptospires in the tubule lumens were lightly stained with silver and clumped into round masses lying free in the lumen. Moreover, minute, black, spherical bodies were seen in the cytoplasm of tubular cells. These black bodies were usually detected in tubules undergoing regressive changes (Fig. 4). When examined immunohistochemically, both the agglutinated leptospires and the intracytoplasmic bodies were positively stained. Immunoperoxidase staining revealed this intracellular positiv-