Signalment:  

2.5-year-old, male American pit bull terrier (Canis familiaris).A young pit bull dog presented to a veterinary clinic with a 5-day history of lethargy and vomiting. Initial blood work revealed the following: BUN 76 mg/dl (7-25), creatinine 5.3 mg/dl (0.5-1.4), phosphorus 10 mg/dl (9-10.8), calcium 9 mg/dl (9-10.8), total protein 8.1 g/dl (5.4-7.1), albumin 2.4 g/dl (2.5-3.6), globulin 5.7 g/dl (2.4-3.6) and amylase 1,056 U/L (510-1,864); other values were within normal limits. The owner's initial concern was that the dog had been poisoned and the veterinarians presumptive diagnosis was ethylene glycol toxicity. Some treatment was attempted, but the dog was euthanized shortly after presentation.

Gross Description:  

The dog was in good body condition. Mucous membranes were pale with occasional random petechiae. The kidneys were bilaterally swollen and had prominent hyperemia of capsular vasculature. The subcapsular surface of the kidney and the corresponding cut surface of the cortex were pale tan and had numerous, disseminated, slightly raised, tan to white, pinpoint to 0.2 cm foci. A raised, fairly well-delineated, irregular, 0.2-0.5 cm band of similar coalescing nodular foci followed the corticomedullary junction; occasional sometimes discontinuous similar bands were in the adjacent outer medulla. A wider dark reddish purple medullary band that was up to 1.2 cm wide extended from the renal crest into the medulla. The renal pelvis was dilated. Some pale tan streaks radiated from the capsular surface into the medulla. The urinary bladder was distended with pale yellow urine. The lungs were red and some frothy fluid was present on the cut surface.

Histopathologic Description:

An intense predominantly mononuclear interstitial inflammatory infiltrate was within the cortex and outer medulla, although similar filtrates occurred in the inner medulla. Lymphocytes, plasma cells and macrophages predominated, although neutrophils were intermingled in multiple foci. These infiltrates often separated and effaced tubules within the cortex. Intermittent tubules had attenuation or irregularity of the tubular epithelial lining and luminal necrotic debris. The inner medulla along the renal crest had extensive necrosis and hemorrhage. Warthin Starry stain revealed clumps of black argyrophilic material within tubular lumina and in the apical cytoplasm of tubular epithelial cells. No discrete leptospires were identified. However, the argyrophilic material corresponded with discrete positive staining by immunohistochemistry (Purdue ADDL). The staining could be identified both within proximal convoluted tubules and collecting ducts.

Morphologic Diagnosis:  

Tubulointerstitial nephritis, lymphoplasmacytic, with necrosis, subacute, diffuse, severe. Etiology: Leptospira sp.

Lab Results:  

Serology (Texas VMDL) revealed the following titers:
- L. pomona	12,800
- L. grippotyphosa	12,800
- L. bratislava	12,800
- L. hardjo	3,200
- L. icterohaemorrhagiae	3,200
- L. canicola	3,200
PCR of kidney and urine for Leptospira sp. (Texas VMDL) was positive.

Condition:  

Leptospirosis

Contributor Comment:  

Leptospirosis is a ubiquitous and important bacterial infection resulting in significant disease in food animals, dogs and humans. Part of the enigma and mystery of leptospirosis is a consequence of cycling of infection in wildlife species resulting in sporadic spillover into domestic animals and humans. Although lately the presumed reemergence of leptospirosis has fallen into the opportunism of blaming global warming, a more likely explanation of the increased awareness is improved methods of detection. The organism has unique niche requirements and does not multiply outside of a host species, although it can survive quite well in wet soil, and, particularly, stagnant surface water. Transmission is typically by direct contact with infected urine or discharges subsequent to abortion; such contact may occur indirectly from water contaminated with the urine of wildlife carriers.

Classically, hosts for leptospirosis have been subdivided into primary versus incidental or accidental. Therefore, dogs are regarded as the primary host for Leptospira canicola and accidental hosts for Leptospira icterohaemorrhagiae (primary host, rat). In the last 12 years or so, multiple outbreaks of leptospirosis have been attributed to non-canine serovars such as Leptospira grippotyphosa (primary host, vole), Leptospira pomona (primary host, cattle and pig) and Leptospira bratislava (primary reservoir, pig and horse). The dog in the case submitted had high serologic titers to all of the atypical canine serovars. Although it is fun to speculate that Leptospira grippotyphosa is the primary infection and that the other serovars are cross reactions, this is always arrogant speculation and not evidence-based science.

The Leptospira microscopic agglutination test (MAT) is ideally performed on paired sera 3-4 weeks apart, looking for a fourfold rise in antibody titer. This is likely more important with Leptospira canicola, particularly if a dog was previously vaccinated. The high levels seen in this case are more typical of an accidental host with a non-canine serovar. Antibodies are typically first detected 7-10 days after infection. Several other diagnostic techniques were pursued to confirm the diagnosis. PCR of kidney and urine was positive for Leptospira DNA (Texas VMDL). This is a genus-specific result only. Warthin-Starry silver staining was attempted to demonstrate leptospires in tubules and was unsuccessful. Immunohistochemistry was performed (Purdue ADDL) revealing convincing clumped Leptospira antigen in tubular epithelial cells and tubular lumina. This disparity was interpreted as a consequence of lack of viability of the leptospires due to the prolonged postmortem interval. Similarly, dark field microscopy of urine was inconclusive; this test also requires fresh urine to observe normal leptospires. Fluorescent anybody testing of centrifuged urine sediment also has the advantage of not requiring leptospires to be viable. Researchers at Mississippi State University have identified transcriptional regulator genes that are present in pathogenic Leptospira strains and absent in non-pathogenic strains. PCR primers have been developed that conceivably can easily separate pathogenic from nonpathogenic Leptospira.(4)

The stratified appearance of an intense white band in the inner cortex and zones of hemorrhage and necrosis in the medulla are a classic pattern of acute leptospirosis culminating in acute renal failure in dogs. A series of these cases was studied retrospectively and prospectively and determined to have a unique ultrasonographic pattern strongly suggestive of acute leptospirosis by Lisa Forrest and colleagues at the University of Wisconsin.(1) When dogs are presented in acute renal failure to veterinarians, initiation of appropriate antibiotic therapy requires timely decisions, and ultrasonography was determined to be a strongly suggestive finding for identifying leptospirosis due to atypical serovars. Otherwise, many of these dogs are euthanized with the presumption of chronic renal failure.

The classification of Leptospira has recently been changed.(3) Previously, one pathogenic species (Leptospira interrogans) and one nonpathogenic species (Leptospira biflexa) were recognized. These two species were subdivided into serovars-�-�200 in Leptospira interrogans and around 60 in Leptospira biflexa. Based on DNA hybridization Leptospira has been reclassified into 13 genomospecies. Since both pathogenic and nonpathogenic species may occur in a single genomospecies, the utility of this scheme for veterinarians, microbiologists, pathologists and others dealing with the disease has not been clarified. Therefore, serovars and serogroups will likely persist in diagnostic reports.

JPC Diagnosis:  

Kidney: Nephritis, tubulointerstitial, lymphoplasmacytic and neutrophilic, diffuse, severe, with tubular degeneration and necrosis and focally extensive medullary hemorrhage.

Conference Comment:  

The contributor provides an instructive example of this entity, the importance of which in both veterinary and human medicine is apparently ever-growing. In general, leptospirosis is characterized by any combination of septicemia, hepatitis, nephritis, and meningitis in humans and animals, and abortion and stillbirth in livestock. In maintenance hosts, which serve as the natural reservoir for the spirochete, the organism persists in the renal proximal convoluted tubules, and in some cases, the genital tract. As the contributor notes, recent taxonomic revisions have introduced tremendous complexity into the classification of Leptospira spp. Fortunately the serovar, which correlates most reasonably with epidemiology and disease, persists in the current nomenclature, and many circumvent the new taxonomy altogether by simply abbreviating to genus and serovar names, as in the contributors comments. The bulk of the discussion during conference was devoted to comparative pathology of leptospirosis, as outlined in the table that follows:(2,5-9)

Leptospira Serovars, Maintenance Hosts, and Disease
canicola	Dog	Dog: primarily renal disease; now rare due to vaccination
grippotyphosa	Raccoon, skunk, rodent	Dog: renal and hepatic disease; increasingly important
		Horse: abortion, premature foaling
icterohemorrhagiae	Rat	Dog: primarily hepatic disease; hyperacute disease in puppies; now rare due to vaccination
bratislava	Pig, horse, +/- dog	Dog: nephritis and abortion
		Pig: abortion, small litters of weak piglets
		Horse: abortion, premature foaling
pomona type kennewicki	Pig	Dog: renal and hepatic disease; increasingly important
		Pig: abortion, small litters of weak piglets
		Cattle: Acute form (calves): hemoglobinuria, hematuria, fever, anemia, icterus, dyspnea, +/- meningitis Acute form (adults): agalactia, abortion of decomposed fetus Chronic form (adults): abortion, stillbirth, or premature weak calves
		Horse: Chronic form: recurrent uveitis ("periodic ophthalmia"), abortion, premature foaling Acute form (rare): fever, icterus
		Pinnipeds: abortions, hemorrhagic syndromes in fetuses and neonates, interstitial nephritis and glomerulonephritis, hepatic necrosis
autumnalis	Rodents	Dog: renal and hepatic disease; increasingly important
		Pig: infertility syndrome ("repeat breeder")
hardjo type hardjobovis (North America)	Cattle, sheep	Cattle: chronic form; abortion, stillbirth, or premature weak calves
		Sheep: Lambs: acute form; similar to pomona in calves Ewes: late-term abortion, stillbirth, weak lambs, agalactia
hardjo type hardjoprajitno (Europe)	Cattle	Cattle: Subacute form: "milk drop syndrome," "flabby udder mastitis" Chronic form: abortion, stillbirth, or premature weak calves
		Sheep: Lambs: acute form; similar to pomona in calves Ewes: late-term abortion, stillbirth, weak lambs, agalactia
ballum	Mouse	Hamster: severe hemolysis, icterus, hemoglobinuria, nephritis, hepatitis

References:

1. Forrest LJ, OBrien RT, Tremelling MS, Steinberg HS, Cooley AJ, Kerlin RL: Sonographic renal findings in 20 dogs with leptospirosis. Vet Radiol Ultrasound 39:337-340, 1998
2. Greene CE, Sykes JE, Brown CA, Hartmann K: Leptospirosis. In: Infectious Diseases of the Dog and Cat, ed. Greene CE, 3rd ed., pp. 402-417. Saunders Elsevier, St. Louis, MO, 2006
3. Levett PN: Leptospirosis. Clin Microbiol Rev 14:296-326, 2001
4. Liu D, Lawrence ML, Austin FW, Ainsworth AJ, Pace LW. PCR detection of pathogenic Leptospira genomospecies targeting putative transcriptional regulator genes. Can J Microbiol 52:272-277, 2006
5. Newman SJ, Confer AW, Panciera RJ: Urinary system. In: Pathologic Basis of Veterinary Disease, ed. McGavin MD, Zachary JF, 4th ed., pp. 658-662. Mosby Elsevier, St. Louis, MO, 2007
6. Percy DH, Barthold SW: Pathology of Laboratory Rodents and Rabbits, 3rd ed., pp. 65, 152, 192, 211. Blackwell, Ames, IA, 2007
7. Prescot JF: Leptospirosis. In: Jubb, Kennedy, and Palmers Pathology of Domestic Animals, ed. Maxie MG, 5th ed., vol. 2, pp. 481-490. Elsevier Saunders, Philadelphia, PA, 2007
8. Radostits OM, Gay CC, Blood DC, Hinchcliff KW: Diseases caused by Leptospira spp. In: Veterinary Medicine: A textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses, eds. Radostits OM, Gay CC, Blood DC, Hinchcliff KW, 9th ed., pp. 971-982. W.B. Saunders, London, England, 2000
9. Szeredi L, Haake DA. Immunohistochemical identification and pathologic findings in natural cases of equine abortion caused by leptospiral infection. Vet Pathol 43:755-761, 2006

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4-1. Kidney

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