Signalment:  

Young male dog, Canis familiars.The dog was inoculated experimentally with Leptospira interrogans server icterohaemorrhagiae. The animal was euthanized for human reasons.

Histopathologic Description:

The liver is affected by a diffuse and severe lesion which is characterized by a dissociation of hepatocytes and hepatic plates (degeneration with loss of intercellular junction systems). Many hepatocytes have a hypereosinophilic cytoplasm and a karyorrhectic nucleus (individual hepatocyte necrosis). However, a number of hepatocytes are binucleated or show mitotic figures (hepatic proliferation). The sinusoids wall is not visible (endothelial cells lysis) and Kupffer cells show erythrophagocytosis (recent hemorrhages).

In portal tracts, bile ducts are distended by an amphophilic mucinous substance (moderate bile duct mucostasis). The wall of centrolobular veins is infiltrated by a few lymphocytes and neutrophils (discrete lymphocytic and neutrophilic transparietal vasculitis).

Etiology: Leptospira interrogans serovar icterohaemorrhagiae.

Lab Results:  

Special stain: A Warthin-Starry stain was negative.

Condition:  

Leptospirosis

Contributor Comment:  

Leptospirosis is a zoonosis of worldwide distribution, caused by infection with antigenically distinct serovars of the motile spirochete bacterium Leptospira interrogans sense alto.(2,3,4) Leptospirosis has been identified as one of the emerging infectious diseases.(4) Leptospira organisms are thin, flexible, filamentous bacteria made up of fine spirals with hook-� shaped ends. More than 200 different serovars have been identified in the Leptospira interrogans complex.(3) Canine leptospirosis was first described in 1899. Prior to 1960, serovars icterohemmorrhagiae and canicola were believed to be responsible for most clinical cases of canine leptospirosis. After a bivalent serovar-specific vaccine against canicola and icterohemmorrhagiae came into widespread use, the incidence of "classic" leptospirosis in dogs appeared to have decreased. However, these vaccines did not induce immunity against other serovars, leading to a relative increase in the incidence of disease attributed to other servers.(3) Leptospirosis can be transmitted directly between hosts in close contact through urine, venereal routes, placental transfer, bites, or ingestion of infected tissues. Recovered dogs can excrete organisms in urine intermittently for months to four years after infection. Indirect transmission happens more often, occurring through exposure of susceptible animals to a contaminated environment (e.g., soil, food or bedding). Water contact is the most common means of spread, and habitats with stagnant or slow moving warm water favor organism survival. Leptospires in contaminated water invade the host through skin wounds or through intact mucous membranes. Once in a susceptible host, leptospires begin to multiply as early as one day after entering the blood vascular space. They invade many organs, including the kidneys, liver, spleen, central nervous system, eyes and genital tract.(3)

Clinical signs associated with leptospirosis vary and depend on the serovar and the host. In maintenance hosts, leptospirosis generally is characterized by a low serological response, relatively mild acute clinical signs, and a prolonged renal carrier state which may be associated with chronic renal disease. In incidental hosts, leptospirosis can cause severe disease, associated with high titers of agglutinating antibody, and has a short or negligible renal carrier state. The clinical signs observed vary with the susceptibility of the host and with the infecting serovar. In general, young animals are more seriously affected than adults.(1,2) Clinical signs reported in dogs with leptospirosis include fever, inappetence, vomiting, abdominal pain, diarrhea, polyuria/polydipsia, myalgia, jaundice, epistaxis, hematuria and reproductive failure.(1,2) Signs of hepatic and renal dysfunction and of coagulation defects usually predominate in dogs with leptospirosis. The severity of clinical signs depends on the age and immunocompetence of the host, the environmental factors affecting the organisms, the serovar involved and its virulence, and the infecting dose. Younger dogs (less than six months) are more severely affected and develop more signs of hepatic dysfunction. A majority of leptospiral infections in dogs are subclinical. The peracute form is characterized by massive leptospiremia, causing shock and often death with few premonitory signs. Less severe infections are characterized by fever, anorexia, vomiting, dehydration, increased thirst and reluctance to move.(3)

Renal colonization occurs in most infected animals because the organism replicates and persists in renal tubular epithelial cells, even in the presence of neutralizing antibodies. Acute impairment of renal function may result from decreased glomerular filtration caused by swelling that impairs renal perfusion. Renal function in some dogs that survive acute infections may return to normal within several weeks, or chronic compensated polyuric renal failure may develop.(3)

The liver is another major organ damaged during leptospirosis. The degree of icterus in both canine and human leptospirosis usually corresponds to the severity of hepatic necrosis. In contrast, the icterus, hemoglobinemia and hemoglobinuria which develop in cattle with leptospirosis result from a specific hemolytic toxin produced by serovar pomona. 

Tissue edema and disseminated intravascular coagulation may occur rapidly and result in acute endothelial injury and hemorrhagic manifestations. Leptospira lipopolysaccharides stimulate neutrophil adherence and platelet activation, which may be involved in inflammatory and coagulatory abnormalities.(3) Benign meningitis is produced when leptospires invade the CNS; however, it is not as common as in humans. Uveitis is occasionally present. Abortion and infertility resulting from transplacental transmission of leptospires associated with serovar bataviae infection have been described. Pulmonary manifestations include labored respiration and coughing. Interstitial pneumonia has been documented as the cause in humans, whereas lung changes in dogs with leptospirosis are associated with pulmonary hemorrhage most likely due to endothelial damage and vasculitis.(3)

Laboratory abnormalities usually include leukocytosis, thrombocytopenia, increased serum urea and creatinine, electrolyte disturbances, bilirubinemia and increased serum hepatic enzyme activities. Coagulation parameters may be altered in severely affected animals. Urinalysis abnormalities include bilirubinuria, sometimes glucosuria, proteinuria and increased numbers of granular casts, leukocytes and erythrocytes in the sediment.(3)

Establishment of a diagnosis is important because animals can serve as reservoirs and pose potential zoonotic risks.(1)

The diagnosis of leptospirosis can be accomplished by several techniques. Detection of antibodies using the microscopic agglutination test (MAT) is the most common diagnostic method; however, other methods to detect antibodies, such as immune-� fluorescent assays or enzyme-linked immunosorbent assay (ELISA), have been used.(2,3) Application of silver stains, or immunohistochemical stains to tissue sections are effective for detection of leptospires or leptospiral antigens in the renal tubules and interstitium of the kidney and liver. Low sensitivity is a disadvantage of this diagnostic technique. Leptospires are often present in small numbers in affected tissues, particularly in chronic leptospirosis or in dogs treated with antibiotics. Serological studies should also be conducted.(2) Bacteriologic culture of blood, urine, or tissue specimens is the definitive method for the diagnosis of leptospirosis. Leptospiremia occurs early in the clinical course of leptospirosis and is usually of short duration and low level. Therefore, blood is only useful for culture in the first few days of clinical illness and before antibiotic therapy. Leptospires are usually present in the urine of animals with leptospirosis four to ten days after the onset of clinical signs. Culture of leptospires is difficult, time-consuming and requires specialized culture medium. However, isolation of the organism allows definitive identification of the infecting server.(2)

The spectrum of human disease caused by leptospires is extremely wide, ranging from subclinical infection to a severe syndrome of multi-organ infection with high mortality.(4) Leptospirosis is presumed to be the most widespread zoonosis in the world. The source of infection in humans is usually either direct or indirect contact with the urine of an infected animal. The incidence is significantly higher in warm-climate countries than in temperate regions; this is due mainly to longer survival of leptospires in the environment in warm, humid conditions.(4)

The clinical presentation of leptospirosis is biphasic, with the acute or septicemic phase lasting about a week, followed by the immune phase, characterized by antibody production and excretion of leptospires in the urine. Most of the complications of leptospirosis are associated with localization of leptospires within the tissues during the immune phase and thus occur during the second week of the illness.(4) A comparison of the prevalence of anti-leptospiral antibodies with the prevalence of clinical signs indicates that subclinical infections are relatively common.(2)

JPC Diagnosis:  

Liver: Hepatitis, necrotizing, multifocal, random, with suppurative cholangitis, severe hepatocellular dissociation and single cell necrosis. 

Conference Comment:  

As the contributor states in this interesting review, leptospiruria may persist in an infected animal for months to years. It is believed that down-regulation or differential expression of Leptospira antigenic surface proteins and possibly the binding of complement regulatory proteins (i.e. plasma factor H) allow the leptospires to evade the host immune system. Although the virulence factors associated with leptospires have not been fully elucidated, it appears that their toxic components are associated with outer membrane proteins rather than the secretion of specific toxins. Attachment to the host cell is likely mediated by fibronectin-binding protein on the bacterial surface that binds to host extracellular matrix proteins. Infection of the host cell is via receptor-mediated endocytosis. Pathogenic species of Leptospira contain sphingomyelinases and other hemolysins, which may play roles in erythrocyte and endothelial cell membrane damage that results in hemolytic anemia, jaundice, hemoglobinuria and hemorrhage observed in acute leptospirosis. Once in the bloodstream, leptospires evade phagocytosis; the mechanism for this is thought to be induction of macrophage apoptosis. Leptospires are cytochemically gram negative bacteria; however, they differ from other gram negative organisms in several ways, including the following: The LPS of leptospires is not as endotoxic as other gram-negative bacteria; and leptospires, unlike other gram negative organisms, activate the host immune response through TLR-2 rather than TLR-4 pathway.

Conference participants discussed the serovars of Leptospira interrogans that commonly affect domestic animals and humans. The included chart summarizes these serovars and their hosts (both maintenance and incidental), as well as associated clinical conditions:(4)

Serovar	Maintenance host	Incidental host	Clinical conditions
Bratislava	Pigs, hedgehogs, horses	Dogs	Reproductive failure, abortions, stillbirths
Canicola	Dogs	Pigs, cattle	Acute nephritis in pups. Chronic renal disease in adult animals
Grippotyphosa	Rodents	Cattle, pigs, horses, dogs	Septicemic disease in young animals; abortion
Hardjo	Cattle, sheep, deer	Humans	Influenza-like illness; occasionally liver or kidney disease
Icterohaemorrhagiae	Rats	Domestic animals, humans	Acute septicemic disease in calves, piglets and lambs; abortions
Pomona	Pigs, cattle	Sheep, horses, dogs	Acute haemolytic disease in calves and lambs; abortions

* Adapted from Quinn PJ, Markey BK, Leonard FC, Hartigan P, Fanning S, FitzPatrick ES. Veterinary Microbiology and Microbial Disease. 2nd ed. 

References:

1. Birnbaum N, Barr SC, Center SA, Schermerhorn T, Randolph JF, Simpson KW. Naturally acquired leptospirosis in 36 dogs: serological and clinicopatholoical features. J. Small Anim. Pract. 1998;39:231- 236.
2. Bolin CA. Diagnosis of leptospirosis: a reemerging disease of companion animals. Semin. Vet. Med. Surg. (Small Anim.) 1996;11:166- 171.
3. Hartmann K, Creene GE. Diseases caused by systemic bacterial infection. In: Textbook of Veterinary Internal Medicine. 6th ed. St. Louis, MO: Elsevier Saunders; 2005;616- 619. 
4. Levett P. Leptospirosis. Clin. Microbiol. Rev. 2001;14: 296- 326.

*Quinn PJ, Markey BK, Leonard FC, Hartigan P, Fanning S, FitzPatrick ES. Veterinary Microbiology and Microbial Disease. Ames, IA: Wiley; 2011: Kindle Edition, location 13471 of 35051. 

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4-1. Liver

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