Necrotizing suppurative nephritis in a Japanese black feedlot steer due to Proteus mirabilis infection.

A Japanese black feedlot steer suddenly died after exhibiting astasia and cramping of the extremities. Necropsy of the animal revealed that the right kidney was enlarged and pale with severe nephrolithiasis. The urinary bladder displayed mucosal hemorrhage. Upon bacteriological investigation, Proteus mirabilis was isolated from the liver, spleen, right kidney, lungs and urine. Histopathological examination revealed necrotizing suppurative nephritis with the presence of numerous gram-negative bacilli and fibrinous suppurative cystitis with no bacilli. Immunohistochemical analysis revealed that the bacteria and cytoplasm of the macrophages stained positively with P. mirabilis antiserum. Electron microscopy revealed the presence of numerous bacteria in the renal tubules. To our knowledge, this is the first report describing the histopathological aspects of nephritis caused by P. mirabilis in cattle.

The genus Proteus, which belongs to the Enterobacteriaceae family, comprises gram-negative, motile bacilli that are one of the main pathogens implicated in urinary tract infections [3, 13]. The genus Proteus comprises five species: Proteus hauseri, Proteus mirabilis, Proteus myxofaciens, Proteus penneri and Proteus vulgaris [10]. Although P. mirabilis is one of the most common bacteria infecting the urinary tract in humans and dogs [3, 4, 13], no reports to date have described histopathological findings. A recent report regarding urinary infections in cattle caused by Proteus did not describe histopathology [14]. Herein, we report a case of nephritis in a feedlot steer in which P. mirabilis infection was demonstrated by bacteriological, immunohistochemical and electron microscopic examinations.

A twenty-month-old Japanese black feedlot steer, belonging to a herd of 600 animals, died suddenly after exhibiting astasia and cramping of the extremities that had lasted for an hour on June 19, 2014. The steer had anorexia and claudication of the left hind limb; however, no medical treatment had been administered to the animal.

At necropsy, a large quantity of purple-red urine spouted out when we cut the urethra and there was a strong smell of urine upon cutting the adipose capsule of the right kidney. The right kidney was enlarged, pale with multiple red blotches, and had a rough outer surface. The cut surface displayed diffuse discoloration of the cortex, and many renal (up to 4 cm in diameter) and ureteral calculi (up to 1 cm in diameter) (Fig. 1). In contrast to the right kidney, the outer surface of the left kidney was smooth with some red blotches. The cut surface displayed a few radial lesions. The urinary bladder had severe mucosal hemorrhage and was covered by a thick false membrane (Fig. 2). The serosa adhered to the surrounding adipose tissue. Additionally, accumulation of a large quantity of ascites fluid in the peritoneal cavity and edema of the lungs were observed.

Fig. 1.

Gross findings of the right kidney. Diffuse discoloration of the cortex, roughened surface and renal calculi are seen. Bar=5 cm.

Fig. 2.

Gross findings of the urinary bladder. Severe mucosal hemorrhage and a thick false membrane are seen. Bar=5 cm.

The liver, spleen, right kidney, heart, lungs, brain and urine were cultured on 5% blood agar and deoxycholate hydrogen sulfide lactose agar under aerobic conditions at 37°C for 24 hr. Isolates were subjected to Gram staining and identification by API 20E (bioMérieux, Tokyo, Japan). Gram-negative bacteria with swarming motility on blood agar were isolated from the liver, spleen, right kidney, lungs and urine. The isolates were identified as P. mirabilis by API 20E (Profile No. 0736000, 99.9%).

For histopathological examination, the liver, spleen, right kidney, heart, lungs, rumen, reticulum, omasum, abomasum, duodenum to rectum, gallbladder, adrenal glands, bladder, skeletal muscle, brain and jejunal, colic, superficial cervical, subiliac and superficial inguinal lymph nodes were fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. The left kidney was not examined. Additionally, the liver, spleen, right kidney, lungs and bladder sections were subjected to Gram staining. The right kidney was also subjected to Masson’s trichrome and periodic acid-methenamine-silver staining (PAM). Histologically, the right kidney showed multifocal, radial or wedge-shaped, necrotizing, suppurative, tubulointerstitial nephritis in the cortex and moderate fibrosis. The cortical structure was extensively collapsed with scattered, dense and cellular foci (Fig. 3). Masson’s trichrome-stained sections revealed dissociation of the tubulointerstitial space due to moderate fibrosis (Supplementary Figs. 1 and 2). Additionally, tubular remnants contained numerous gram-negative bacilli, neutrophils and macrophages, surrounded by accumulated neutrophils, macrophages and necrotizing debris (Figs. 4A, 5A and 5B). A few bacteria were seen in the phagocytes (Fig. 5A and 5B). Examination of PAM-stained sections revealed a basement membrane demarcating the tubular remnants (Fig. 4B). Outside the dense, cellular foci, necrotizing tissue was observed with less dense cellular infiltration with moderate fibrosis. Very mild atrophy was observed in glomeruli. Necrosis extended to the outer edge of the outer medulla. As we approached the inner portion of the outer medulla, the infiltrated cells in the tubules predominantly consisted of neutrophils. Structures of the inner medulla and renal pelvis had collapsed due to moderate edema and fibrosis. The structure of the mucous membrane had completely collapsed in the urinary bladder. In the innermost layer, approximately 900-µm thick false membrane consisted of the severe fibrin deposits and mild to moderate neutrophilic infiltration. In the submucosa under the false membrane, multiple hemorrhages, discrete vascular degenerations with thrombosis, moderate edema and laminated necrosis with severe neutrophilic infiltration were observed. The muscular layer was thickened with moderate edema, and mild fibroblast hyperplasia and the serous surface demonstrated diffuse fibrinous serositis. Additionally, erythrophagocytosis was often observed in the bleeding areas. Besides the urinary bladder, diffuse fibrinous serositis was also observed in the spleen, intestines and adrenal glands. In the liver, discrete foci of necrosis with mild infiltration of lymphocytes and macrophages, and local collapse of the hepatic tissue were observed. Diffuse severe edema was seen in the lungs. No significant lesions were observed in other tissues except those mentioned here, and no bacterium was observed in other tissues besides the kidney.

Fig. 3.

Histopathological findings of the right kidney. The cortical structure is extensively collapsed, with dense cellular foci including tubular remnants (asterisk). Hematoxylin and eosin. Bar=500 µm.

Fig. 4.

Histopathological findings of the right kidney. A: Tubular remnants (asterisks) in the lesion contain bacterial colonies, neutrophils and macrophages. The structures are surrounded by a large number of neutrophils and macrophages. Hematoxylin and eosin. B: Basement membranes are observed within each tubular remnant. PAM. Bar=50 µm.

Fig. 5.

Histopathological findings of the right kidney. A: Bacterial colonies and macrophages with engulfed bacilli are observed in the tubular remnant (asterisk). H&E. B: Bacteria are found to be gram-negative. C: Bacteria are positively stained by the anti-P. mirabilis antibody. Immunohistochemistry. Bar=10 µm.

Immunohistochemistry was performed using rabbit anti-P. mirabilis antiserum (Abcam, Tokyo, Japan) at a dilution of 1:1,024 together with Simple Stain Multi kit and Simple Stain AEC Solution (Nichirei, Tokyo, Japan). Antigen retrieval was performed by immersing sections in 0.01 M citrate buffer (pH 6.0), irradiating them in a microwave oven (preheated to 95°C at 500 W) for 3 cycles of 5 min each. A peace of liver taken from a healthy calf, in which a thick P. vulgaris or Escherichia coli suspension was injected, was fixed in 10% neutral buffered formalin and embedded in paraffin wax. Sections cut from the paraffin block served as controls. A positive reaction against anti-P. mirabilis antibody was found mainly within tubules in the dense cellular foci. Some phagocytes surrounding the tubules contained a lower number of bacteria and/or antigen (Fig. 5C). A relatively lower extent of positive reaction was observed in the necrotizing tissue with less dense cellular infiltration lying outside the dense cellular areas. Few positive reactions were observed in the false membrane or in the urinary bladder, and no positive reaction was observed in the liver, spleen and lungs. In control tissues, P. vulgaris was positively and E. coli was negatively stained with the antiserum.

For transmission electron microscopy, renal tissues fixed in 10% neutral buffered formalin were washed with 0.1 M phosphate buffer and post-fixed in phosphate-buffered osmium tetroxide (1%) at 4°C overnight. After dehydrating them, the tissue sections were embedded in epoxy resin. Ultrathin sections were then cut, stained with uranyl acetate and lead citrate, and examined by using a transmission electron microscope (H-7500, Hitachi, Tokyo, Japan). A number of rod-shaped bacteria were observed in tubular epithelial cells and macrophages (Fig. 6A). The bacteria were approximately 0.5 µm in diameter and 2 µm in length, and had a thin cell wall that is characteristic of gram-negative bacteria. Surface of some bacteria had dense, stand of pili-like, 8 to 10 nm wide, filamentous structures. No flagellum was seen (Fig. 6B).

Fig. 6.

Transmission electron micrograph of a renal tubule. A: Several rod-shaped bacteria are seen inside the tubule basement membrane (arrow). Bar=2 µm. B: Rod-shaped bacteria show a thin cell wall and dense strands of pili-like, fine filamentous structures on their surface. Bar=200 nm.

For biochemical examination, we measured the quantity of blood urea nitrogen and creatinine in post mortem blood using Fuji DRY-CHEM system (7000Z, Fujifilm, Tokyo, Japan). Blood urea nitrogen and creatinine values were over 140 mg/dl and over 24 mg/dl, respectively.

In this study, we demonstrated the association between nephritis and P. mirabilis infection in a Japanese black feedlot steer using bacteriological, histopathological and electron microscopic examinations. To our knowledge, only one previous report has discussed renal histopathology caused by P. mirabilis in mice, wherein histologic changes were observed in the kidneys of mice, seven days post transurethral infection with P. mirabilis [1]. The most severe renal lesions in those mice were composed of large cortical necrosis with multiple foci of inflammation and necrosis. These lesions were similar to those in our case. Ultrastructural studies of P. mirabilis entry into cells and subsequent bacterial multiplication have been reported using cultured human renal epithelial cells [1, 2]. Bacterial invasion occurs within 30 min of exposure, while at 2 hr, P. mirabilis filamentous swarm cells begin to separate and divide into vegetative cells. Finally, within 3 hr, each epithelial cell contains numerous rods throughout the cytoplasm. Similar views were observed in our case by electron microscopy. We isolated P. mirabilis from multiple organs. However, as seen in previous reports, we believe that the infection may have ascended to the kidney based on necrotizing suppurative lesions in the nephrons and poor glomerular changes [1, 2].

Lesions of the right kidney are not attributable to P. mirabilis infection alone, because gross lesions of the right kidney also resemble chronic nephritis [8]. Additionally, moderate fibrosis was noted, which does not necessarily conform to necrotizing suppurative lesions observed by histopathological examination and the many large renal calculi observed by necropsy. Therefore, it was thought that necrotizing suppurative nephritis due to P. mirabilis happened after chronic changes due to renal calculi. Additionally, it was thought that existence of renal calculi induced the ascending infection of P. mirabilis [9]. However, the reason for severe cystitis remains unclear due to the low quantity of antigen and absence of bacteria within the lesions.

Proteus species are thought to enhance or induce formation of calculi [5, 6, 11, 12]. Purulent inflammation has been reported to be associated with renal calculi in mice experimentally infected with P. mirabilis [7]. In this case, association between P. mirabilis and enhancement of renal calculi remains unclear.

To our knowledge, this is the first report describing the histopathological aspects of nephritis caused by P. mirabilis in cattle. It is necessary to further examine the pathogenicity of P. mirabilis in cattle in future.