Viral Diseases
Viral diseases associated with liver dysfunction include infectious canine hepatitis, canine herpesvirus, inadvertent parenteral injection of an intranasal Bordetella bronchiseptica vaccine in dogs, feline infectious peritonitis, and virulent systemic calicivirus infection in cats. Rarely, canine parvovirus can lead to hepatic injury as a result of portal systemic sepsis. A recently identified hepadnavirus in cats is associated with development of hepatocellular carcinoma.
Infectious canine hepatitis is caused by canine adenovirus 1. In addition to acute hepatic necrosis, chronic hepatitis and hepatic fibrosis can be sequelae if neutralizing antibody is inadequate to eliminate the infection during the active phase.
Canine herpesvirus affects neonatal puppies, causing hepatic necrosis as well as other systemic changes. It is usually fatal in puppies.
Accidental parenteral injection of intranasal B bronchiseptica vaccine in dogs can cause both a local inflammatory reaction at the injection site and acute, nonseptic hepatocellular degeneration and necrosis that evolves into chronic hepatitis. There is no known treatment other than supportive care for chronic inflammatory liver disease.
Feline infectious peritonitis virus is a coronavirus that causes diffuse pyogranulomatous inflammation and vasculitis. Icterus, abdominal effusion, vomiting, diarrhea, and fever are common clinical signs.
Virulent systemic calicivirus, a variant of feline calicivirus, can have mortality rates of 33%–60% in adult cats. Primarily identified in shelter or cattery populations, this virus causes profound fever, anorexia, marked subcutaneous edema (limbs and face especially), jaundice, alopecia, and crusting and ulceration of the nose, lips, ears, and feet. Adult cats are most severely affected. Individual hepatocyte necrosis ranging to centrilobular or more extensive necrosis is associated with neutrophilic inflammatory foci and intrasinusoidal fibrin deposits. Some cats develop inflammation invading portal structures. Subcapsular hemorrhage may develop secondary to vasculitis and regional necrosis.
An exploratory study of infectious viral disease in cats with FIV identified a novel member of the Hepadnaviridae in a single lymphoma sample in 2018, dubbed domestic cat hepadnavirus (DCH). A subsequent survey of 390 feline serum samples (by another research group) demonstrated hepadnavirus in 10.8% of samples. Using PCR assay and in situ hybridization (ISH) viral detection in formalin-fixed feline liver samples, virus was identified in 6 of 14 (43%) cats with chronic hepatitis, 8 of 29 (28%) with hepatocellular carcinoma (HCC), but not in cats with biliary carcinoma (n = 18), multilocular biliary cysts (n = 4), toxic hepatopathy (n = 1), cholangitis (n = 6), nodular hyperplasia (n = 8), or normal controls (n = 15). All PCR-positive HCC and 2 of 6 positive chronic hepatitis were also ISH positive.
Chronic hepatitis positive for DCH was characterized by lymphocytic periportal inflammation without ductal targeting (ie, no evidence of cholangitis). Inflammation at the portal-lobular interface was characterized as “piecemeal necrosis” with occasional single or aggregates of apoptotic/necrotic hepatocytes. Mixed lymphocytic and plasmacytic infiltrates were variably scattered in sinusoids, occasionally forming aggregates.
Rare neutrophils abutted necrotic hepatocytes. Occasional foci of dysplastic hepatocytes were present in all cats with DCH-associated chronic hepatitis but were not unique only to viral positive tissue. Both nuclear and cytoplasmic ISH signals were detected with patchy distribution in chronic hepatitis and HCC lesions.
Hepatocellular carcinomas displayed variable cellular atypia, were invasive, had loss of lobular architecture (either solid or stacked hepatic cord phenotypes), and most also displayed confluent foci of necrosis. Compared to canine HCC, these lesions were more similar to HCC in humans.
It remains to be proven whether DCH infection is apathogenic or is associated with subclinical or clinical signs of disease in cats. However, its association with HCC, which has histologic similarity and dysplastic features similar to HCC associated with hepatitis B in humans and hepadnavirus-associated HCC in woodchucks, is more than intriguing.
Bacterial Diseases
Leptospirosis in Hepatic Disease in Small Animals
Infections with Leptospira interrogans serovars Icterohaemorrhagiae and Pomona and chronic infections with L interrogansserotype Grippotyphosa and L kirschneri serotype Grippotyphosa have been associated with hepatic disease in dogs. Other serotypes may also involve the liver. Vacillating to markedly increased liver enzyme activity ± hyperbilirubinemia indicates hepatic involvement. However, these parameters may reflect hepatic response to a sepsis syndrome rather than specific organ invasion in acutely ill dogs. In some cases, clinical evidence of liver disease is weak.
Clinical and clinicopathologic features of liver involvement may worsen initially with treatment (fever, liver enzymes, hyperbilirubinemia) because of a Jarisch-Herxheimer reaction. Dogs with chronic Leptospira-associated hepatitis may present for clinical and clinicopathologic features charactering chronic liver injury with portal hypertension, APSSs, and ascites.
Microscopic Hepatic Findings
Leptospirosis infection causes vasculitis and endothelial damage leading to inflammatory infiltrates, localized ischemia, and microhemorrhage. Most often, the liver is but one of numerous organs damaged by this organism. However, some strains demonstrate a unique hepatic trophism causing isolated liver injury.
The classic vague microscopic leptospiral-associated liver lesion is dissociation of hepatocytes or disorganized hepatic cords. Rather than merely reflecting an organism invading the space of Disse, this lesion reflects disturbed structure of adherens junction (vascular endothelial cadherin and catenins) and actin filaments. Based on canine case reports and published case series, it is clear that numerous additional lesions are associated with leptospiral-induced liver injury.
Dogs with isolated hepatic injury may display mild to marked mixed inflammatory infiltrates with portal or centrilobular distribution (or both) as well as lobular aggregates. Inflammatory infiltrates are usually dominated by lymphocytes, with fewer macrophages and neutrophils, and occasional plasma cells. Portal infiltrates may breach the limiting plate and might display bile duct tropism (ie, cholangitis). However, in some dogs, a predominant pyogranulomatous hepatitis develops (portal to random distribution, of varying severity). Occasional necrotic hepatocytes may be evident.
More commonly, randomly scattered small coalesced aggregates of hemosiderin-laden foamy macrophages (iron-laden lipogranulomas) represent historic sites of hepatocyte loss. Concurrent glycogen-type hepatocyte vacuolation is common—an expected response in dogs with necroinflammatory liver injury. Depending on chronicity, mild periportal fibrosis, bridging portal fibrosis, regenerative nodules, and even cirrhosis may evolve. A ductular reaction (proliferation of cholangiocytes or ductal epithelium) and canalicular bile casts may also be apparent before fibrotic remodeling. Dogs with extensive fibrosis and remodeling develop portal hypertension and ascites.
Rare cases with fulminant hepatic failure display centrilobular hemorrhagic necrosis due to vascular inflammation and loss of sinusoidal endothelium. Widespread distribution of petechia in leptospiral infections has been long recognized, representing either direct capillary damage or indirect injury from some toxin produced by the organisms.
Diagnosis
Definitive diagnosis of leptospiral-associated hepatitis is not possible with routine staining (ie, hematoxylin and eosin); PCR assay of blood or urine is unreliable based on the timing of sample collection. Bacterial culture also is unreliable because organisms may require passage through another biologic host before they will grow in culture. Serologic titers are often difficult to interpret in light of vaccination history and do not prove leptospiral-associated lesions. Definitive identification of spirochetes in formalin-fixed biopsy sections has been possible but challenging with silver stains.
More sensitive and objective methods include direct tissue PCR assay, immunohistochemical methods, or in situ hybridization methods that directly confirm tissue- and lesion-associated organisms. Ultrastructural microscopy (transmission electron microscopy) has also been used to detect organism in liver tissue. Modified Steiner silver staining has demonstrated spirochetes within distended bile canaliculi. Immunohistochemical, in situ hybridization, and transmission electron microscopy methods also have confirmed intracanalicular organisms.
Of dogs with pyogranulomatous Leptospira-associated hepatitis, clusters of organisms were shown in 8 of 10 dogs by in situ hybridization, potentially identifying reproducing organisms. Coinfecting bacteria were documented in 5 of 10 dogs with pyogranulomatous Leptospira-associated hepatitis, possibly implicating potential for compromised immune surveillance as a predisposing factor.
Treatment
Treatment includes supportive care and specific antimicrobial treatment. Penicillins are used initially for the acute phase (eg, ampicillin [22 mg/kg, IV, every 6 hours] or amoxicillin [22 mg/kg, PO, every 12 hours]). Aminoglycosides (dose depends on drug used; streptomycin was historically used to clear leptospirosis) or doxycycline (5 mg/kg, PO, every 12 hours for 4 weeks) is recommended to treat the carrier phase. Aminoglycosides are currently not recommended for treatment of leptospirosis owing to the high risk of nephrotoxicity.
Special precautions are recommended when handling animals suspected of having leptospirosis (and their urine specimens) because of the zoonotic potential.
Tyzzer Disease in Hepatic Disease in Small Animals
Tyzzer disease is a rare but fatal enterohepatic syndrome caused by the spore-forming, gram-variable, filamentous, obligate intracellular bacteriumClostridium piliforme. Infections in dogs or cats most commonly occur in immunocompromised hosts, either neonatal animals or adults affected with other conditions. Stresses associated with transport, weaning, overpopulation, unsanitary husbandry, or immunosuppression (ie, glucocorticoid administration, chemotherapy), or viral-induced infections are documented as antecedent conditions.
Because C piliforme is a commensal enteric organism in laboratory rodents, infection is usually acquired by fecal-oral transmission (ie, contact with or ingestion of rodent feces transporting bacterial spores) or acquired from another infected host. After ingestion, C piliforme enters and proliferates within intestinal epithelial cells, resulting in necrosis/enteritis. If conditions allow (ie, immunosuppression, severe enteric inflammation), organisms translocate into the portal circulation, colonize the liver, and may disseminate systemically. A characteristic multifocal necrotizing hepatitis ensues.
Filamentous bacteria within hepatocytes appear in a chaotic crisscross or more organized parallel orientation. Organisms are pale with routine hepatic encephalopathy (HE) staining, gram-variable (negative or variably positive) with Gram stain, purple with Giemsa, and distinctly outlined with silver stains (ie, Warthin-Starry method).
Clinical signs are peracute in onset and reflect hepatic and intestinal disease (ie, lethargy, anorexia, abdominal discomfort) with rapid progression to death within 24–48 hours. A marked increase in ALT activity immediately precedes death with occasional hyperbilirubinemia.
Special stains are needed to identify organisms because this bacterium does not grow in routine culture media. Immunohistochemistry (IHC) provides definitive diagnosis. There is no effective treatment other than prevention.
Mycobacterium Infections and Pyogranulomatous Hepatitis in Small Animals
Mycobacteria are rod-form, gram-positive, acid-fast bacteria, now classified into the groups mycobacterium tuberculosis complex (MTC; bacteria that form tubercle lesions) or nontuberculous mycobacterium (NTM; including Mycobacterium avium-intracellulare complex and environmental organisms [derived from soil, organic litter, tropical vegetation, aerosols, protozoa]) that are associated with hepatic disease. MTC occasionally associated with pyogranulomatous hepatitis are M tuberculosis, M microti, and M bovis. NTM occasionally associated with pyogranulomatous hepatitis are M avium and, rarely, unusual species that are slow-growing (eg, M kansasii, M marinum) or fast-growing (eg, M fortuitum, M chelonae).
Susceptibility to various Mycobacterium spp varies among mammals. Although M tuberculosis can induce progressive disease in humans, nonhuman primates, dogs, swine, and cats, infection in domestic pets is rare. More common among MTC organisms are infections with M bovis and M microti. Infections causing pyogranulomatous hepatitis often present as one component of disseminated disease.
M bovis (cattle-adapted mycobacterial species) infection in dogs has been linked with feeding of raw meat (large Foxhound kennel in England fed "dropped" stock animals) and to vector exposure (eg, dog-to-dog transmission). M bovis has been linked in multiple households to feline infections derived from consumption of unpasteurized milk or a commercially distributed raw meat diet (venison); each outbreak was documented in England. M marinum (rodent-adapted mycobacterial species) in cats is linked to consumption of raw meat via hunting behavior and to vector exposure.
Systemic and hepatic infections with M avium have been repeatedly reported in dogs and cats with suspected immune deficiencies. Case reports document M avium infections in numerous Bassett Hounds and Miniature Schnauzers. Specific predisposing immunodeficiencies remain unclarified.M avium infection is also documented in young Abyssinian and Somali cats where an unclarified innate immunodeficiency was suspected.
Clinical Signs
Animals with pyogranulomatous hepatitis may occasionally present with overt clinical signs of hepatic involvement. However, compared to acute forms of hepatic inflammation, copper-associated hepatopathy, or hepatotoxicities, clinical signs are usually vague. Early infection is subclinical, variably leading a protracted course and development of anorexia, cachexia, weakness, dyspnea, and a low-grade, fluctuating fever. Frequently the infection has been present for weeks to months before diagnosis.
Usually, animals with pyogranulomatous hepatitis present with modest vacillating increases in liver enzyme activity and occasionally are jaundiced. There may or may not be associated vomiting or diarrhea, and there often is no peripheral lymphadenopathy when pyogranulomatous hepatitis is the dominant lesion. Depending on the involved mycobacterial species, route of exposure, and extent of systemic dissemination, respiratory signs, marked diffuse interstitial pulmonary infiltrates, or discrete randomly distributed focal pulmonary densities may or may not be evident. Pulmonary involvement is usually accompanied by thoracic lymphadenopathy.
Pyogranulomatous hepatitis often is associated with hepatomegaly. There also may be palpable abdominal lymph nodes (especially in cats) or abdominal mass lesions representing pyogranulomatous lymphadenitis. Abdominal ultrasonography may substantiate hepatomegaly; the hepatic parenchyma may have a normal or vague nodular echotexture (even when gross nodules are obvious during biopsy or necropsy). Enlarged hepatic hilar or mesenteric lymph nodes may also be documented.
Diagnosis
Microscopic liver lesions: On routine H&E staining, histologic features diagnostic for pyogranulomatous hepatitis include multifocal large to small infiltrates dominated by macrophages and neutrophils with fewer lymphocytes and plasma cells. Depending on severity, pyogranulomatous inflammatory infiltrates may efface large areas of liver. There is typically no zonal tropism or evidence of cholangitis. With chronicity, expanding areas of extinct parenchyma may evolve (areas devoid of functional hepatic tissue).
Identification of large caseating granulomas is rare. Necrotic hepatocytes may be identified at the margin of pyogranulomatous foci. Canalicular bile casts are inconsistent and vary with the severity of infection. A diffuse or multifocal glycogen-type vacuolar change is common.
Additional histological stains and tissue tests: Once a pyogranulomatous process is identified, it is essential to use a rhodanine stain to identify copper to exclude mistaken copper granulomas as a pyogranulomatous process. Application of special stains to identify infectious agents is also mandatory. Gram stain, Ziehl-Neelsen (ZN), Fite-Faraco (FF), periodic acid-Schiff (PAS), Giemsa, and Gomori methenamine silver (GMS) are all employed to identify bacteria (gram-positive versus gram-negative), acid-fast bacteria (ZN, FF), protozoa (PAS, Giemsa), and fungal and spirochete (GMS) organisms.
If no obvious infectious agent is identified, after meticulous review of special stains (5 minutes per slide with abundant pyogranulomatous foci as investigated lesions), samples of formalin-fixed tissue should be submitted for PCR assay to detect mycobacterial or Leptospira organisms. PCR for mycobacteria should be submitted to a USDA-certified laboratory. Fluorescent in situ hybridization using a universal eubacterial primer should requested to investigate for lesional bacteria.
If aerobic and anaerobic bacterial cultures were not already submitted, repeat sampling of the affected liver should be done. At that time, fresh tissue should be collected for PCR assay for mycobacteria and Leptospira because fresh tissue has better accuracy than formalin-fixed tissue for this procedure.
Interferon-gamma (IFN‐gamma) release assay (IGRA): The IGRA, originally developed for diagnosis of M bovis in cattle, has been adapted for dogs, cats, and humans. This test can be done using a heparinized whole blood test from which mononuclear cells are harvested. The test principle is a demonstration of IFN‐gamma production by sensitized peripherally circulating antigen‐specific effector memory T cells when exposed to mycobacterial antigens.
In cattle, this test has high sensitivity (81.8%–100%) and specificity (88%–99% ) for detection of M tuberculosiscomplex organisms. It has diagnostic utility equal to or exceeding the tuberculin skin test in humans and is easier to conduct. The IGRA was shown to have 100% sensitivity for the detection of mycobacterium tuberculosis complex infections in cats and was successfully applied to contain and control mycobacterium tuberculosis complex infections in a large Foxhound kennel and to identify infections in dogs with high risk of vector-spread M tuberculosis (exposed to sputum containing M tuberculosis in South Africa). The diagnostic utility of IGRA for diagnosis of nontuberculous mycobacteria is yet to be clearly demonstrated; the test can assist with confirming diagnosis of mycobacteria if acid-fast bacteria are identified on cytologic or histologic samples.
If thoracic radiography and ultrasonography have not yet been evaluated, these diagnostic assessments should be pursued. Finding pulmonary abnormalities, mass lesions, or large lymph nodes may provide additional tissues for sampling for histologic assessment, cytology, culture, and PCR assay. Cytologic aspirates showing a pyogranulomatous response should be meticulously reviewed for intracytosolic bacteria in macrophages with routine Wright-Giemsa, Gram stain, and acid-fast staining.
With Wright-Giemsa (a Romanowsky stain), bacteria appear as small unstained "white-clear" rods because of their impermeable waxy surface (acid-fast stain permeabilizes the waxy coat). A nonstained cytology smear can be used for PCR assay, although the inoculate is small and negative findings do not rule out infection.
Culture of mycobacterial organisms may require up to 3 months or longer to grow, and thus other testing methods should be used in the meantime. Even when mycobacterial organisms are confirmed with ZN staining, only 50% culture positive.
Treatment
Some animals with mycobacterial pyogranulomatous hepatitis present in the later stages of illness with disseminated disease warranting a grave prognosis. Those with less systemic illness are candidates for antimicrobial treatment. Recommended treatment protocols are based on expert opinion regarding management of human mycobacterial infections, refined by veterinary experience. Treatment protocols for cats are better scrutinized compared to those for dogs.
A first step in advising treatment is to inform the owner of the prolonged course of polymodal antimicrobial administration to achieve clinical remission; cure cannot be a promised outcome. Second, depending on the Mycobacterium species diagnosed, potential for zoonosis must be clarified.
When considering a treatment protocol, it is important to acknowledge that epidemiologic studies in humans show decreased emergence of drug-resistant mycobacterial strains with multimodal treatment. Studies also demonstrate unreliability of selecting therapeutic protocols based on culture sensitivity predictions. Human treatment protocols cannot be directly translated to veterinary patients because some of the more commonly advocated drugs may provoke renal, liver, or neurotoxic effects (eg, isoniazid and ethambutol) or optic neuritis (eg, ethambutol) or are ineffective against M bovis (pyrazinamide, the pyrazine analogue of nicotinamide, is also reported to be hepatotoxic to cats).
For cats, first-line drugs include:
rifampicin
pradofloxacin (better retinal safety profile over other fluoroquinolones and with proven efficacy against many mycobacteria compared to older fluoroquinolones
a macrolide, usually azithromycin due to its once-daily dosing pattern (alternatively clarithromycin or clindamycin)
All three drugs can be administered together once daily, increasing compliance. The combination protocol is administered over 3 months and then continued for 2 months beyond resolution of clinical signs.
Isoniazid, a commonly used drug for humans with mycobacterial infections, is administered only if rifampicin is not tolerated or there is evidence of organism resistance. Diverse drug-related toxicities are described in humans and animals treated with isoniazid. Notably, this drug can provoke acute renal failure and hepatotoxicity, although these are less common than neurologic effects.
Neurotoxicity is thought to reflect drug-induced depletion of tissue and circulating pyridoxine (vitamin B6) concentrations. Isoniazid binds directly with pyridoxine, forming isoniazid-pyridoxine hydrazones that are rapidly eliminated in urine. These products also competitively inhibit pyridoxine kinase, essential for conversion of pyridoxine to its physiologically active form (pyridoxal-5′-phosphate).
Pyridoxal 5'-phosphate is an essential cofactor for glutamic acid decarboxylase, the rate-limiting enzyme in synthesis of gamma aminobutyric acid (GABA), a major inhibitory neurotransmitter. Decreased CNS concentrations of GABA lead to neuroexcitation (ie, twitching, hypertonicity, seizures). Decreased pyridoxal-5' phosphate also provokes lactic acidemia, inhibiting conversion of lactate to pyruvate in the Krebs cycle. Pronounced seizure-induced muscle contracture can also escalate lactic acidemia. Finally, heightened risk for isoniazid hepatotoxicity in dogs is proposed to reflect their decreased ability to convert the parent drug to nontoxic metabolites by acetylation (ie, dogs lack N-acetyltransferase).
In animals with pulmonary involvement, treatment is extended to a minimum of 6 months, followed by 2 months after resolution of radiographic abnormalities. M avium and other NTMs have notoriously variable inherent drug resistance such that first-line protocol drugs are not always effective. Second-line drugs for these organisms include clarithromycin as an alternative to azithromycin and doxycycline as an alternative to the fluoroquinolone. Third-line drugs would include ethambutol, clofazimine, and isoniazid because of their risk for toxicity.
Zoonotic Risk
Because different mycobacteria cause overlapping clinical syndromes and have variable zoonotic risk, definitive discrimination of the involved species is important. There is zoonotic risk associated with the MTC group, and reporting these infections to state health officials is mandatory. Humans caring for or cohabiting with infected animals, especially those with decreased immune surveillance, should be advised to consult with their personal physician for appropriate monitoring and advice.
Hepatic Impact of Extrahepatic and Intrahepatic Bacterial Infections and Sepsis in Small Animals
Extrahepatic infection and sepsis can cause cholestasis and hyperbilirubinemia. Animals with chronic disorders causing stasis of bile flow or with chronic hepatic neoplasia are more likely to develop intrahepatic infections.
Bile formation depends on:
proper function of hepatic bile transporters
an intact cytoskeleton (orchestrates vesicle transport and canalicular contraction)
intact cell junctions barricading bile canaliculi from leakage; also essential for maintaining cell polarity and intercellular communications
intracellular signal cascades regulatory to membrane transporters (expression and localization)
Many functions essential to bile formation and choleresis are adversely impacted by sepsis, in the absence of redirect hepatic injury, leading to molecular cholestasis.
Sepsis denotes the presence of bacteria or other infectious organisms or their toxins spread systemically. Extrahepatic infections and the "sepsis syndrome" can cause cholestatic hyperbilirubinemia. Both gram-negative and gram-positive infections can provoke this phenomenon. Hyperbilirubinemia may be noted prior to suspected or documented infection. The liver plays a crucial role in sepsis, providing a first-line defense against bacterial dissemination. However, it also can be the recipient of injury due to host response (inflammation and cytokine elaboration).
Molecular cholestasis can be caused directly by bacterial products or secondary to host inflammatory response. Hyperbilirubinemia may range from moderate to marked, while increases in liver enzymes are typically modest.
This type of jaundice has been documented in leptospirosis (dogs), septic pyometra, polyarthritis, endocarditis, and rhinosinusitis (after rhinoscopy and biopsy), and secondary to dental procedures, parvovirus enteritis, severe bacterial dermatitis, bite wound infection/abscesses, Cuterebra abscess, and ill-defined sepsis syndromes. Treatment targets the underlying disease process and organism causing infection.
Increasing liver enzyme activity in nonhepatic septicemia/sepsis may reflect bacterial dissemination to the liver (multifocal embolic sinusoidal dissemination causing inflammatory or necrotic foci), hepatocyte injury associated with cytokine release, hypoxemia impairing microvascular integrity, or hypotension declining hepatic perfusion. It can be difficult to ascertain whether there is direct hepatic injury or infection without a liver biopsy. Liver biopsy, however, is not recommended in such cases.
Molecular Cholestasis
Important mechanisms are briefly described. Endotoxins (lipopolysaccharide [LPS] and other bacterial toxins) provoke Kupffer cell release of pro-inflammatory cytokines (eg, tumor necrosis factor [TNF]-alpha, IL-6, IL-1-beta,). These lead to downregulation and impaired activity of or relocalization of bile transporters in hepatocyte, canalicular, and ductular membranes. These changes are coordinated by nuclear receptors and transcription factors.
Decreased expression of aquaporin-8 (a TNF-alpha-mediated post-transcriptional effect) limits canalicular water transport, important for normal bile flow. Increased production of nitric oxide (NO), driven by LPS induction of NO synthase in Kupffer and sinusoidal endothelial cells, also progresses molecular cholestasis:
NO induction decreases chloride and bicarbonate secretion into bile ductules, important for normal bile fluidity.
NO induction increases tight junction permeability (in canalicular membranes) that normally protects the osmotic gradient assisting bile production.
Canalicular contractions that normally help propel bile flow are decreased by local NO accumulation.
After the onset of molecular cholestasis, intrahepatocyte bile acid accumulation also augments molecular cholestasis. High cytosolic bile acid concentrations downregulate expression of certain bile acid transporters and also relocalize transporter expression, retracting them from surface to cytosolic orientation and thereby impairing their function. These changes, as well as others not described herein, are transient and reversible once infection is eradicated.
Molecular cholestasis may contribute to hyperbilirubinemia in animals with acute hepatic failure, chronic hepatobiliary disease, and hepatobiliary cholestasis. These patients are predisposed to systemic bacterial infection and endotoxemia because of compromised function of hepatic Kupffer cells. Because Kupffer cells represent the largest fixed macrophage population in the body, this substantially compromises hepatic clearance of bacteria circulating from the splanchnic circulation. Furthermore, mechanical cholestasis (ie, bile duct obstruction, severe cholangitis, developed ductopenia) also increases risk for enteric bacterial translocation.
This vulnerability reflects decreased biliary IgA at the enteric mucosal interface where it normally limits bacterial translocation. It also reflects enteric bacterial dysbiosis due to a disrupted bile acid milieu (decreased enterohepatic bile acid flux) that favors bacterial pathogens over commensals. In acute fulminant hepatic failure, the presence of sepsis and molecular cholestasis may be masked by fever, hypoglycemia, and leukocytosis that might also represent clinical signs of hepatic injury.
Animals with chronic liver disorders, mechanical cholestasis, or neoplastic mass lesions (ie, hepatocellular carcinomas) are more likely to develop intrahepatic infections. Risk factors associated with bileborne infection include advanced age, recent episodes of cholangitis, septic cholecystitis, cholelithiasis, and any cause of obstructive jaundice (mechanical cholestasis).
Hypoxic or Ischemic Hepatitis
Hypoxic or ischemic hepatitis may complicate metabolic cholestasis and sepsis, leading to increased liver enzyme activities. This syndrome is triggered by inadequate oxygen delivery (ie, hypoxemic hypoxia, lack of oxygen carriage [anemic hypoxia]) or decreased sinusoidal perfusion. Multifocal disruption of sinusoidal perfusion may reflect:
microthrombi or fibrin aggregates
clumped RBCs
neutrophil NETs (neutrophil extracellular traps that bind pathogens)
bacterial emboli
sinusoidal apertures narrowed by bulging activated Kupffer cells
altered function and vasoregulation of sinusoidal endothelium
These processes may also contribute to hyperbilirubinemia. A key diagnostic feature of hypoxic or ischemic hepatitis is its rapid reversibility once the underlying cause is corrected.
Ischemic or Sepsis-Associated Cholangiopathy
The exception to the above processes, in terms of reversibility, is when sepsis-associated hypotension is severe enough to compromise arterial perfusion of the peribiliary vascular plexus. In contrast to hepatic parenchyma that receives a dual blood supply (hepatic artery and portal vein), the intrahepatic biliary tree exclusively relies on small hepatic arterial branches. Consequently, persistent hypotension may cause small duct ischemic injury and irreversible loss of ductal elements (ductopenia).
This process is associated with rising alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) activities and persistent hyperbilirubinemia. Diagnosis requires hepatic ultrasonography to rule out obvious causes of mechanical cholestasis, and ultimately a liver biopsy to confirm ductular involution and loss.
Treatment of Suspected Metabolic Cholestasis and Hypoxic Hepatitis
Interventions should target the underlying process causing infection, hypotension, hypoxemia, or anemia. While awaiting culture and sensitivity results (from tissues, blood, effusions, or bile), antimicrobials protective against enteric opportunists should be administered, avoiding drugs extensively metabolized in the liver. The combination of a beta-lactamase–resistant penicillin, metronidazole (7.5 mg/kg, PO, every 12 hours, dose restriction because of cholestasis), and enrofloxacin (2.5–5 mg/kg, PO, IM, or IV, every 12 hours) provides broad-spectrum protection that may also cover the cause of sepsis.
General treatments aimed at reducing liver injury include administration of N-acetylcysteine, low-dose alpha-tocopherol, and bioavailable S-adenosylmethionine (SAMe). These strategies increase integrity of microvasculature, improve microvascular oxygen exchange in sepsis, help protect against enteric bacterial translocation and colonization, may augment innate immune response, and afford antioxidant protection against oxidant impact of sepsis-related inflammatory cytokines. Supplemental oxygen and vasopressors may be needed for persistent hypotension nonresponsive to volume expansion. Patients with clinical anemia should be supported with transfusion of packed RBCs.
Mycotic Diseases
Systemic Mycoses in Small Animals
Liver involvement can develop with systemic mycoses including blastomycosis, histoplasmosis, and coccidioidomycosis (dogs and cats), and cryptococcosis (cats), typically reflecting disseminated infection. Single hepatic involvement is extraordinarily rare. Clinical features of systemic fungal infections with liver involvement are dominated by respiratory, gastrointestinal, cutaneous, ocular, or CNS signs rather than clinical hepatopathy.
Consideration of geographic exposure helps inform diagnostic considerations. Systemic fungal infections are generally acquired from unique environmental niches recognized in North America. That said, there are numerous international locations where exposure is also recognized for each of these:
Blastomyces dermatitidis is endemic in the eastern US, especially around the Great Lakes, along the Ohio and Mississippi River valleys, and in the southeastern states, with small endemic foci in the northeast (the St. Lawrence River region extending into Canada and the Adirondacks in northern New York).
Histoplasmosis is endemic in North America, spanning from the central states to western Virginia to central Texas with sporadic cases encountered in California and western Canada.
Coccidioides immitis is endemic to lower Sonoran life zone regions (semiarid to arid soil, low sea level elevations) in the southwestern US, particularly in Arizona (Tucson and Phoenix) and in south-central California in the San Joaquin Valley.
Cryptococcus sppare endemic to British Columbia in Canada (especially Vancouver Island,C gatti) and the Pacific Northwest in the US. Additionally, cryptococcal infections have been acquired from intimate exposure to bird (pigeon) feces.
Notably, any of these systemic mycoses can be encountered in animals without known geographic exposure, with houseplant potting soil considered a plausible infection vector.
Clinicopathologic Features
Hematologic features are nonspecific and may include a mild nonregenerative anemia, mild neutrophilic leukocytosis, occasional left shift with toxic cells, and variable monocytosis. Serum biochemical findings often include a low-normal to subnormal albumin concentration and variable hyperglobulinemia (sometimes markedly increased). Hypercalcemia may develop secondary to macrophage 1 alpha-hydroxylase activity (this increases 1,25 OH calciferol concentrations). Alternatively, fungal bone invasion (ie, blastomycosis, coccidioidomycosis) may also contribute.
Increases in ALT and AST are often minor, if present, with more notable increases in ALP without coordinate increases in GGT. This likely reflects hepatic enzyme induction by inflammatory cytokines or osteolytic bone involvement. Hyperbilirubinemia develops in a small subset of dogs with disseminated disease; it is unclear if this implicates the liver directly or reflects systemic inflammation. Urinalysis is usually noncontributory with the exception that pathological proteinuria is often associated with coccidioidomycosis.
Imaging
Radiographic features are usually dominated by pulmonary pathology and bone involvement (with blastomycosis and coccidioidomycosis). Pyogranulomatous hepatitis may be associated with a normal to mildly large liver. Abdominal ultrasonography in disseminated disease may disclose mesenteric and/or hepatic hilar lymphadenopathy and occasional focal lesions in kidneys, spleen, or liver and an abdominal effusion. Dogs with pyogranulomatous hepatitis may have no discernible changes or may display ill-defined nodules. The gallbladder and extrahepatic biliary ductal structures are typically within normal limits.
Diagnosis
Diagnosis of disseminated systemic mycoses is often first suspected based on cytologic evaluation of tissue aspirates (lymph nodes, spleen, liver, or fluid collected from affected organs). With liver involvement, hepatic aspiration cytology may reveal the causal agent. However, fungal elements are erratically distributed such that a diagnosis can be overlooked. Consequently, testing for antibodies or antigens to the suspected agents, sample PCR assay, tissue special staining, or IHC is used to confirm a diagnosis.
Treatment
See Overview of Fungal Infections in Animals for a more detailed discussion of treatment of systemic mycosis. Pyogranulomatous hepatitis can complicate drug tolerance if severe lesions efface > 50% of functional hepatic mass or if the patient has cholestatic hepatopathy.
Opportunistic Uncommon Mycoses in Small Animals
Besides endemic systemic mycoses, hepatic involvement with uncommon opportunistic fungi may occur with disseminated infections. Such infections are most often associated with the following:
immunosuppressive predispositions (ie, chronic glucocorticoid administration, treatment with immunomodulation protocols, especially cyclosporine)
those being treated with cytotoxic drugs
diabetes mellitus
suspected breed‐associated immunodeficiency (ie, German Shepherd Dogs, congenital B12 insufficiency)
Infections may also occur subsequent to development of enteric dysbiosis (ie, chronic antibiotic administration) or gastrointestinal inoculation of unusual organisms (ie, NSAID ulceration, severe inflammatory bowel disease [IBD], surgical dehiscence of an enterotomy site, enteric lymphoma).
Hepatic infection causes a pyogranulomatous inflammation. In general, clinical signs reflect the inflammatory process and affected organs and do not directly implicate a fungal infection. Consequently, definitive diagnosis is delayed because supportive care is usually initially attempted. Young adult German Shepherd Dogs have a predilection for developing invasive infections with fungi and other saprophytic pathogens, suspected to reflect congenital immunodeficiency.
Clinical Signs
Clinical signs may include variable pyrexia, weight loss, hyporexia, lethargy, vomiting and diarrhea. In animals with CNS involvement or musculoskeletal involvement inoculations, gait abnormalities, ataxia, or other neurologic signs may be obvious (ie, bone infection, discospondylitis, osteomyelitis, or CNS invasion). Hematological and biochemical tests do not specify the nature of the infection. Demonstration of fungal elements in affected tissues and molecular classification of the invading fungus are necessary for definitive diagnosis.
Discovery of fungi in bile (fungibilia) without systemic fungal involvement is rare but has been associated with Candida spp and Cyniclomyces guttulatus in dogs with IBD treated with immunosuppressants. C guttulatus is part of the normal gastrointestinal flora of feral and domestic rabbits, guinea pigs, hares, and chinchillas. The fungus occupies a unique vegetative cell interface between gastric mucosa and ingesta without inciting host response. It has never been associated with pathological lesions in these host species.
A survey of dogs with diarrhea versus healthy dogs without diarrhea (studies in three countries) concluded there was no direct evidence of C guttulatus being a primary pathogen in dogs. One study confirmed lack of association between nystatin elimination of fecal C guttulatus and relapsing diarrhea (57 dogs with C guttulatus studied). Canine consumption of feces from host species is the suspected source of C guttulatus acquisition in dogs. The single report of C guttulatus in bile demonstrated concurrent polymicrobial bacterial infection consistent with translocation from the alimentary canal as the source of infection.
Candida spp in Small Animals
Candida spp are normal inhabitants of the alimentary, upper respiratory, and urogenital mucosa of mammals and may cause opportunistic local infections (in mouth, vagina, ears, perineum, skin folds). Systemic dissemination is rare and usually reflects an immunocompromised status. Systemic Candida infections also have followed chronic indwelling intravenous or urinary catheterization. Candida morphology in tissues is not always a simple yeast form; it also can transform into pseudohyphae (chains of elongated yeastlike structures and tubular hyphae).
In tissue sections, organisms are typically embedded in pyogranulomatous inflammation; organisms may be identified with H&E stained tissue sections but are more easily identified with periodic acid-Schiff (PAS), Gomori methenamine silver (GMS), and Gram staining.
Observation of numerous 2- to 6-mcm oval yeasts, chains of oval yeast forming pseudohyphae, and true hyphae on cytologic preps or histologic sections prioritizes consideration of Candida spp. Definitive diagnosis is confirmed using culture, IHC, or PCR assay. Invasive candidiasis is associated with a high mortality rate; C albicans is often involved.
Disseminated Aspergillosis in Small Animals
Systemic or disseminated aspergillosis (Aspergillus terreus and A deflectus are especially problematic causative species) and rare infections with Penicillium spp (P purpurogenum usually) are occasionally encountered. German Shepherd Dogs are predisposed, implicating a breed-related immunodeficiency. Widespread dissemination may involve the liver; however, these patients do not present for clinical signs or clinicopathologic features implicating a primary hepatopathy.
Clinical Signs
Clinical signs of disseminated aspergillosis are nonspecific and may include fever, anorexia, weight loss, weakness, lethargy, vomiting, cough and harsh lung sounds, and pain or neurologic disability associated with bone invasion (discospondylitis or osteomyelitis). When present, neurologic signs vary in severity (most to least common: ataxia, paresis, mental dullness, blindness [chorioretinitis, panophthalmitis], head tilt, circling, and seizures).
Clinicopathologic Features
Clinicopathologic features of disseminated aspergillosis also are variable and may include leukocytosis with a left shift, hyperglobulinemia, azotemia, hypercalcemia, hypoalbuminemia, increased ALT and ALP activities, and isosthenuria in azotemic dogs. Radiographic imaging may disclose discospondylitis or osteomyelitis as the predominant lesion leading to clinical presentation. Thoracic radiographs may disclose pleural effusion, pulmonary infiltrates, sternal lymphadenopathy, cranial mediastinal mass effect, or unusual cavitated nodules. Abdominal ultrasonography may disclose (most to least frequent): lymphadenomegaly, renal abnormalities, splenomegaly with hypoechoic nodules or masses, altered hepatic parenchymal echodensity (mottled hypoechogenicity), venous thrombi, and abdominal effusion.
Diagnosis
Dogs with disseminated aspergillosis typically have advanced disease at diagnosis, usually > 1 month. Because these fungi are common environmental contaminants, immunosuppression or unusual inoculation exposures increase risk for infection. A terreus has greater potential for systemic dispersal because of the production of accessory spores growing directly on hyphae (shown in vitro and in infected tissue). These are considered to facilitate hematogenous dissemination and may interrupt microvascular (capillary) perfusion.
With these organisms, tissue aspirates or histologic sections disclose septate branching fungal hyphae. Diagnosis requires further detail, characterized by tissue, blood, or urine cultures; PCR testing; or serologic or urine antigen or antibody detection. While a grave prognosis is often warranted for disseminated aspergillosis, some dogs have achieved prolonged remissions (months to years) with chronic antifungal treatment. There are no large observational studies guiding treatment recommendations for systemic aspergillosis.
Drugs more often used include itraconazole, voriconazole, posaconazole, and/or amphotericin B. Fluconazole is not recommended because Aspergillus spp are intrinsically resistant to this drug. Disseminated aspergillosis infection carries a guarded to grave prognosis because this is typically a rapidly progressive disease refractory to treatment. There is one report of a young German Shepherd Dog with systemic Aspergillus surviving > 3 years without specific antifungal treatment.
Phaeohyphomycosis in Small Animals
Phaeohyphomycosis denotes infection with dematiaceous (pigmented) fungi belonging to over 60 genera (orders include Pleosporales, Ochroconiales, Chaetothyriales, Capnodiales, Dothideales, Botryosphaeriales, Microascales, Sordariales, Calosphaeriales, and Ophiostomatales). Species causing phaeohyphomycoses in veterinary patients have included Alternaria, Bipolaris, Cladophialophora, Curvularia, Exophiala, Fonsecaea, Moniliella, Phialophora, Ramichloridium, Scolecobasidium, and Ulocladium, among others.
Causal fungi are ubiquitous in nature with worldwide distribution and are typically nonpathogenic soil saprophytes. While more commonly causing cutaneous or subcutaneous infections, occasionally these organisms disseminate causing life-threatening opportunistic infections in immunocompromised hosts (ie, especially animals on cyclosporine) or hosts with extraordinary exposures.
Disseminated infection with hepatic involvement causes pyogranulomatous hepatitis and commonly is associated with abdominal effusion and peritonitis. The portal of entry in disseminated infection may remain unknown because of extended delay between infection and clinical illness and because of empirical antimicrobial treatments.
Clinical Signs
Liver biopsy demonstrates irregularly septate hyphae or yeastlike cells as solitary organisms or tangled aggregates. Fungi appear pigmented (brown/gray) on cytologic preps or histologic sections. However, melanic pigment may be inconspicuous in lightly pigmented hypha and may be identified on unstained smears with adjustment of the microscopic condenser to increase refractile contrast. The pigment also may be identified by use of a melanin stain (Fontana-Masson stain).
Diagnosis
Definitive diagnosis of phaeohyphomycosis is achieved by fungal culture of aspiration or biopsy samples collected from infected tissues or regional lymph nodes. Definitive identification is achieved based on colony and conidial morphology and ribosomal RNA gene sequencing. Results of culture implicating phaeohyphomycosis must be collaboratively interpreted considering morphological features in diagnostic samples; pigmented fungi are environmentally ubiquitous, and growth on culture might reflect contaminants (environment of patient’s skin or fur).
Treatment and Prognosis
Infection involving the liver is usually advanced at diagnosis and may not be responsive to antifungal treatment. Treatment of nonresectable lesions with itraconazole (10 mg/kg, PO, every 24 hours) may achieve remission. However, because recurrence is common, prolonged drug administration has been advised (ie, for 6–12 months or longer).
Voriconazole and posaconazole have been suggested as more effective than itraconazole but are more expensive. Furthermore, voriconazole has substantial adverse effects in cats and is not recommended in this species.
Longterm voriconazole at 7–10 mg/kg, PO, every 24 hours for 10–12 months was successful in controlling intracranial phaeohyphomycosis in one dog and mycotic peritonitis in another. Conditions provoking immunosuppression must be controlled or eliminated (ie, cyclosporine administration, hyperadrenocorticism).
A grave prognosis is warranted for patients with disseminated phaeohyphomycosis.
Protothecosis in Small Animals
Protothecosis is a rare opportunistic infection caused by environmental algae of the genus Prototheca, belonging to the Chlorellaceae family of unicellular saprophytic, aerobic, colorless algae. These organisms have worldwide distribution and are found abundantly in environmental niches with flowing or standing water contaminated by raw human or animal feces, soil, rotting food or vegetation, treated sewage, infected cow’s milk (Prototheca mastitis), and unsanitary fish tanks. Rare systemic infection in dogs can lead to a pyogranulomatous hepatitis. Immunosuppression increases risk, and Boxer and Collie breeds are seemingly predisposed.
Prototheca is considered an achlorophyllous mutant of Chlorella, a chlorophyll-containing green alga that very rarely causes infection in dogs. These organisms are similar in shape and size (round to oval, 5–30 mcm, with a thick cell wall) and reproduce asexually by endosporulation (sporangia release sporangiospores in tissue). Chlorella is green, due to a large single chloroplast that dominates the cell; it also has numerous starch granules (birefringent on polarized microscopy of unstained or hepatic encephalopathy (HE)-stained samples).
Starch granules and the cell walls in Chlorella are positive on PAS staining (negative after amylase [diastase] digestion). Prototheca has only small cytosolic protoplasts, no large chloroplast, no starch granules, but has a similar PAS-positive cell wall. Although the green color of Chlorella may be leached during tissue fixation/processing, wet mounts of Giemsa-stained samples still display its green hue. Discrete morphologic differences are best characterized ultrastructurally.
Clinical Signs
Disseminated protothecosis is a severe, relentlessly progressive illness. In dogs, a median duration of illness is ~4 months but is quite variable. Clinical signs include lethargy, vomiting, and episodic large bowel diarrhea with fresh blood, before overt dissemination. Systemic dissemination to eyes causes blindness (granulomatous chorioretinitis, uveitis, panophthalmitis, and retinal detachment); bone pain and lameness are caused by long bone osteomyelitis; and CNS signs are attributed to multifocal granulomatous meningoencephalitis or masslike effects (may progress to obtundation and seizures).
Liver disease does not present as a stand-alone illness. Diarrhea is often the first sign of infection and may persist for several months before progressive illness. The colon is considered the source of entry, implicating oral exposure.
Clinicopathologic Features
Clinicopathologic features vary widely, reflecting organ dissemination. The CBC may have no major abnormalities other than a nonregenerative anemia. Biochemical abnormalities include variable hyperglobulinemia, increased liver enzymes reflecting pyogranulomatous hepatitis, azotemia with renal invasion, and subnormal albumin and cholesterol, reflecting enteric losses. Urinalysis may disclose Prototheca organisms.
Radiographs may disclose osteomyelitis. Abdominal ultrasonography may disclose lymphadenomegaly or mural colonic thickening without notable change of hepatic parenchymal echogenicity. Colonoscopy reveals irregularly thickened hyperemic friable ulcerated mucosa.
Histologically, liver invasion causes a pyogranulomatous hepatitis, with variable hepatic necrosis, and free or phagocytized organisms. Organisms can be identified using H&E, GMS, and PAS stains. Colonic biopsies demonstrate marked mucosal and transmural inflammation and infiltration. Invasion into colonic microvasculature and intravascular protothecal thrombi have been documented. Large lymph nodes also display pyogranulomatous inflammation and organisms.
Diagnosis
Diagnosis of disseminated Prototheca sp has been achieved via rectal scrape cytology, endoscopic colonic biopsy, or culture of urine, feces, or tissue. The organism grows with 72 hours on blood agar or Sabouraud dextrose agar at 25°–37° C, producing smooth, white, creamy yeastlike colonies. Algal morphology (sporangiospores) are demonstrated using lactophenol cotton blue staining. Molecular speciation defines the infecting organism.
Treatment and Prognosis
Optimal treatment is not established because of the rarity of protothecal infections. Treatment with lipid-complexed amphotericin B is considered mandatory (weekly, biweekly treatments) combined with continuous administration of a tetracycline and itraconazole. This polymodal treatment has achieved remission for up to 1 year in a few canine cases. Solitary use of azole antifungals has limited effect; ketoconazole is not efficacious.
Most dogs with pyogranulomatous hepatitis and systemic dissemination are gravely ill at presentation, warranting euthanasia. Necropsy examination establishes the diagnosis. Rare dogs with cutaneous protothecosis have survived with treatment; in one case, organisms were also detected in urine, suggesting early dissemination before onset of typical clinical signs. Prototheca zopfii is incriminated as the most frequent cause of disseminated infection in dogs.
Protozoal Diseases
Toxoplasmosis in Small Animals
Toxoplasmosis can cause acute hepatic failure associated with hepatic necrosis. Toxoplasma gondii is more commonly encountered in cats positive for feline immunodeficiency virus and/or feline leukemia virus. In cats, fatal extraintestinal infection may follow acute infection due to overwhelming replication of tachyzoites (intracellular) in liver, lungs, pancreas, or CNS. Kittens with transplacental or transmammary infection develop severe illness that may present as severe liver disease.
Liver disease caused by toxoplasmosis is rare in dogs, but when encountered it is either in an immunocompromised host or in young dogs and also involves systemic infection. Young dogs may be concurrently infected with canine distemper virus; in these, illness has an acute onset and is rapidly fatal.
Clinical signs in animals with hepatic toxoplasmosis include fever, lethargy, vomiting, diarrhea, jaundice, and abdominal effusion, in addition to clinical signs reflecting pulmonary, ocular, or neuromuscular involvement.
Clinicopathologic features in animals with hepatic involvement include increased ALT, AST, and often CK (reflecting myonecrosis/inflammation in animals with disseminated infection); hyperbilirubinemia; and often hyperglobulinemia (polyclonal).
Definitive diagnosis is made when organisms are identified in histologic specimens, corroborated by PCR assay or IHC staining. Aspiration samples cannot definitively differentiate Neospora caninum from T gondii tachyzoites. Finding T gondii oocysts in feline feces cannot discern active infection. In dogs, this signifies coprophagia of feline stool.
Hepatic lesions are characterized by a necroinflammatory pyogranulomatous reaction associated with T gondii tachyzoites and/or bradyzoites. Finding this association supports diagnosis of clinical toxoplasmosis. Occasionally tissue inflammation obscures identification of organisms and requires IHC or PCR assay to achieve diagnosis.
Tissue diagnosis in dogs is complicated by the similar morphologic appearance of T gondii and N caninum. Immunohistochemistry and species-specific PCR assay can differentiate these organisms.
Diagnosis of toxoplasmosis can be difficult without a tissue-based diagnosis. A positive IgM titer indicates recent exposure or clinical signs of toxoplasmosis, whereas IgG titers may reflect chronic infections and animals lacking clinical signs of disease. See Toxoplasmosis in Animalsfor details on diagnostic testing options.
Treatment of T gondii is achieved with clindamycin (12.5 mg/kg, PO or IM, every 12 hours for 4 weeks), currently considered the drug of choice. Because clindamycin is metabolized in the liver, dosage decrease may be necessary in severe hepatic insufficiency. Oral clindamycin should be followed by a bolus of water or food to prevent esophageal irritation. In some cases, initial treatment is combined with anti-inflammatory glucocorticoids to protect against tissue injury due to host response to protozoal death. A trimethoprim-sulfa combination antimicrobial is considered second-line treatment because of potential for idiosyncratic sulfa-related drug reactions.
Patient prognosis depends on the extent of debilitation and stage of disease at initial diagnosis and predisposing immunosuppression. Despite improvement with treatment, animals should be considered chronically infected and thus must undergo surveillance for recrudescent disease. Reactivation of a latent infection can follow initiation of immunosuppressive treatment (ie, cyclosporine).
Leishmaniosis in Small Animals
Canine leishmaniosis is a multisystemic disease caused by protozoan parasites of the genus Leishmania, most commonly encountered in animals that have lived in Mediterranean countries, Portugal, the Middle East, and some parts of Africa, India, and Central and South America. A sandfly vector is required for host-to-host transfer.
Leishmania is occasionally encountered in dogs in the US (especially Foxhounds) where there is at present no identified sandfly or surrogate vector. Because leishmaniosis has a long incubation and prolonged disease course, travel-related leishmaniosis explains some sporadic cases. A serosurvey of > 12,000 dogs (Foxhounds, other breeds, including wild canids) and 185 humans in 35 states and 4 Canadian provinces identified Leishmania spp–infected Foxhounds in 18 states and 2 Canadian provinces but no evidence of infection in humans.
North America leishmaniosis appears widespread in Foxhounds and is limited to dog-to-dog transmission or by blood transfusion. The visceral form of leishmaniosis involves the liver, causing chronic progressive lymphoplasmacytic and macrophage-mediated inflammation and pyogranulomatous hepatitis.
Clinical signs of disease appear after chronic incubation of up to 7 years after inoculation. The outcome of Leishmania spp infection is widely variable; many dogs eliminate the infection, some remain persistently but subclinically infected, whereas a small percentage develop severe, life-threatening disease. It is this population that develops liver disease. Dogs resisting persistent infection manifest a strong T-cell immune response.
Clinical features associated with hepatic involvement include vacillating fevers with lethargy, insidious muscle wasting and weight loss, hyporexia, pallor, oral ulcerations, splenomegaly, prominent peripheral and abdominal lymphadenomegaly, and rare hepatomegaly. Immune-mediated hematologic complications may evolve thrombocytopenia and thrombopathia leading to spontaneous bleeding (ie, epistaxis or melena). Other immune complications include emergence of autoantibodies (antinuclear antibodies [ANA] in up to ~50%) and circulating immune complex-related disorders—ie, polyarthropathy, myositis, uveitis, vasculitis, and glomerulonephritis, often evolving a nephrotic syndrome. Stand-alone clinical liver disease does not occur.
Clinicopathologic features in dogs with liver involvement may include mild to moderate nonregenerative anemia, mild thrombocytopenia, and variable leukocyte counts and distributions. Occasionally, occurrence of lymphocytosis in light of peripheral and abdominal lymphadenomegaly warrants concern regarding lymphoma. Serum biochemical features may include variable hypoalbuminemia, mild to marked hyperglobulinemia, renal azotemia, and mild to moderate increases in liver enzymes (up to 5-fold reference limits, fold increase in ALP > fold increase of transaminases).
Increased ALP likely corresponds to an induction phenomenon attributable to inflammatory cytokines as concurrent glycogen-type vacuolation is common in these dogs. The ALP activity also may reflect periosteal proliferation associated with leishmanial bone invasion. Total bilirubin is usually within reference limits but may increase mildly. Abdominal radiography may disclose splenomegaly or modest hepatomegaly. Abdominal ultrasonography usually reveals lymphadenomegaly and splenomegaly, with or without hepatomegaly.
Hepatic histologic response is characterized by mixed lymphoplasmacytic and histiocytic infiltrates within and adjacent to portal tracts and surrounding central veins. With increasing severity, there are diffuse but randomly distributed granulomatous or pyogranulomatous lesions that may involve epithelioid macrophages and parasitized macrophages. Hepatic sinusoids display activated Kupffer cells (plump hyperplastic cells) and lymphocytes with fewer plasma cells. A diffuse or multifocal nondegenerative glycogen-type hepatocyte vacuolation is common as well as occasional hepatocytes undergoing ballooning degeneration.
Hepatocyte necrosis or apoptosis, dissecting sinusoidal fibrosis, cholangitis, and development of regenerative nodules are each uncommon.
Definitive diagnosis of Leishmania as a cause of liver disease requires demonstration of organisms in liver tissue by IHC. Testing using molecular methods using peripheral blood can be misleading because clinically normal animals in endemic regions may harbour chronic evidence of infection.
Treatment is rarely curative, and prognosis for debilitated animals with active hepatic disease is poor. Owners should be informed that control rather than cure is the therapeutic goal and that relapses may require repeated or lifelong treatment. In the absence of renal insufficiency, a high-protein diet is recommended. Current first-line treatment involves the combined administration of meglumine antimoniate (100 mg/kg, SC, every 24 hours for 4 weeks) and allopurinol (10 mg/kg, PO, every 12 hours until clinical signs resolve and quantitative serology converts to negative).
Complementary effects thought to augment treatment efficacy involve pentavalent antimonials' inhibition of protozoal enzymes and damage of protozoal DNA, along with allopurinol's interference with protozoal protein synthesis. This protocol has achieved complete or near-complete clinical remission ranging from ~65% to 100% of cases. In patients without clinically overt renal disease, clinical improvement should be evident within several months.
Specific treatment recommended in the US is allopurinol (7–20 mg/kg, PO, every 8–24 hours) given for 3–24 months or indefinitely; other first-line treatments include meglumine antimoniate (100 mg/kg, IV or SC, every 24 hours), sodium stibogluconate (30–50 mg/kg, IV or SC, every 24 hours), or liposomal amphotericin B (0.25–0.5 mg/kg, IV, every 48 hours until a total dose of 5–10 mg/kg is achieved). Numerous other second-line drugs have also helped control infections. The reader is referred to a more comprehensive therapeutic discussion elsewhere in this text.
Pyogranulomatous Hepatitis in Small Animals
Granulomatous or pyogranulomatous hepatitis can reflect a wide spectrum of causes including a diversity of infectious diseases, drug- or toxin-initiated liver injury (DILI), autoimmune or idiopathic immune-mediated inflammation, or an idiopathic process. Differentiation of the probable cause requires thoughtful scrutiny of the patient’s medical history and husbandry practices, clinical illness, physical and clinicopathologic findings, and details disclosed on examination of the biopsy sample, with meticulous inspection for infectious agents.
Often a pyogranulomatous hepatitis is not a stand-alone disease entity. A first step is to consider the patient’s systemic clinical signs and reevaluate physical findings, inspecting for features implicating a disseminated disease process. Specific considerations should also be given to:
environmental exposures (travel, farm, industrial, boarding)
animal exposures (domesticated and feral)
husbandry practices, including diet fed (raw meat in homemade or commercially prepared rations), nutritional supplements including holistic, nutraceutical, and traditional Chinese medicine/tinctures
feeding of special treats (ie, jerky treats, rawhides), especially those manufactured in countries with loosely enforced manufacturing standards
onset of newly introduced medications, including prophylactic anthelmintics; flea, tick, and heartworm preventatives; and any recently administered vaccinations
The timeline between illness onset and changes in any of these variables should be scrutinized for coincidental associations. Plausible or endemic infectious disorders should be considered in light of historical, clinical, and clinicopathologic findings. Reliable serologic or molecular methods of testing for plausible infectious diseases should be undertaken, including rickettsial agents.
Biopsy sections must be meticulously inspected for infectious agents. However, confusion with development of copper granulomas (sometimes characterized as pyogranulomatous foci) must be excluded. This is done by application of a rhodanine stain on tissue sections; rhodanine stains copper a vivid red-orange. This process illustrates association between pathological copper-initiated cell necrosis, hepatocyte copper accumulation, and multifocal granulomas.
Use of rubeanic acid stain for copper detection is strongly discouraged, because these are difficult to evaluate in the midst of an inflammatory process. Thereafter, special stains should be requested that might elucidate the presence of infective organisms, otherwise inconspicuous on routine H&E staining.
If aerobic and anaerobic bacterial and fungal cultures have not yet been submitted, these should be retroactively enacted with a second sampling procedure. If this is done, portions of this biopsy should be frozen for future testing strategies (ie, PCR procedures done on frozen tissue are more accurate than formalin-fixed samples).
If special stains fail to disclose infectious causes, bacterial and fungal cultures are negative, and serology for possible infectious disorders do not implicate a plausible cause, then sections of liver biopsy should be submitted for fluorescent in situ hybridization (FISH) for general detection of eubacterial agents and specifically for Leptospira. A PCR assay for mycobacterial pathogens also should be submitted (using either formalin-fixed or frozen biopsy sections) to a certified testing laboratory.
A variety of bacterial agents can initiate this pathological response, including systemic and unusual opportunistic fungal agents; numerous bacteria, including mycobacteria; and protozoal and algal agents. While infection with Bartonella rarely evolves this injury pattern in humans, this has not been documented in feline or canine patients to the author’s knowledge. Importantly, there are dogs with pyogranulomatous hepatitis that are negative on all tests for infectious causes but still respond to broad-spectrum antimicrobial treatment. Single-agent treatment with doxycycline, enrofloxacin, azithromycin, and amoxicillin-clavulanic acid has resolved pyogranulomatous hepatopathy in select cases.
After thorough attention to diagnostic considerations as described above, submission of an antinuclear antibody (ANA) test is recommended. If results of an ANA test are positive, a lupus erythematosus (LE) cell prep should then be requested. This consideration is pursued because pyogranulomatous inflammation also can have an autoimmune or immune-mediated pathogenesis.
What is considered a high positive ANA varies with the laboratory conducting the test; the on-site clinical pathologist should be consulted. Positive results of an ANA test with or without positive results of an LE cell prep suggests immune dysregulation. Such patients fit into a category of immune-mediated granulomatous/pyogranulomatous hepatitis, an exclusion diagnosis that requires immunomodulation.
Any discovered environmental or husbandry-related issues should be immediately rectified. Any recent new supplements or medications should be suspended (replaced using alternatives from a different drug class). Unfortunately, with DILI, a pyogranulomatous process may not resolve with supplementation or drug discontinuation, as shown with ketoconazole and diclofenac in dogs. Some of these patients need immunomodulation.
Cases of pyogranulomatous hepatitis with no demonstrable cause remain enigmatic. These are treated with immunomodulation after conscientious evaluation, as described herein. Immunomodulation is usually done with a combined low-dose intermittent (every 48 hours) glucocorticoid with azathioprine or mycophenolate rather than starting with cyclosporine. There is general belief that more complicating infections evolve after starting cyclosporine treatment.
Patients treated with azathioprine should have baseline and then sequential liver enzyme evaluations (0, 1, 2, 4, 8 weeks after starting treatment) to detect possible serious hepatotoxicosis (ALT rising to > 5-fold coincident with initiated treatment). If mycophenolate is used, the total daily dose should be divided into an every-8-hours dosing strategy to avoid gastrointestinal effects and to achieve efficacious plasma drug concentrations. Efficacy of this drug can be assessed by evaluation for IL-2 suppression. Any sign suggesting emergence of an infectious syndrome warrants immediate suspension of immunomodulation.
As the pathogenesis of pyogranulomatous inflammation involves inflammatory cytokines associated with T cells, dendritic cells, and macrophages, oxidative injury is imposed on regional cells. Hepatoprotectants and antioxidants including bioavailable SAMe, low-dose vitamin E, and phosphatidylcholine should be administered as suggested for chronic hepatitis.
Expected response to control of an infectious etiology is a cure of the pyogranulomatous hepatitis. In dogs in which this has become an immune-mediated process, immunomodulation will curtail lesions but not entirely eliminate them. Lesion regression is difficult to ascertain based only on sequential liver enzyme profiles because pyogranulomatous hepatitis usually does not coordinate with extreme increases in ALT or AST activities. A liver biopsy is needed to document resolution. If this is done, it is essential to sample multiple liver lobes because of erratic or spotty distribution of lesions within and between liver lobes in some patients.
