Canine Hepatitis Associated with Intrahepatic Bacteria in Three Dogs
ABSTRACT
This case report describes the detection of intrahepatic bacteria in formalin-fixed paraffin-embedded histopathological sections from three dogs with neutrophilic, pyogranulomatous, or lymphoplasmacytic hepatitis and cholangiohepatitis. In each of these cases, eubacterial fluorescence in situ hybridization enabled colocalization of intrahepatic bacteria with neutrophilic and granulomatous inflammation in samples that were negative for bacteria when evaluated by routine hematoxylin and eosin histopathology augmented with histochemical stains. Positive responses to antimicrobial therapy were observed in of 2 out of 2 patients that were treated with antimicrobials. These findings suggest that eubacterial fluorescence in situ hybridization analysis of formalin-fixed paraffin-embedded histopathological sections is more sensitive than conventional histochemical stains for the diagnosis of bacteria-associated canine hepatitis.
Introduction
The term canine hepatitis encompasses a broad spectrum of conditions characterized by infiltration of the hepatic parenchyma with neutrophils, mononuclear cells, lymphocytes, or plasma cells with or without involvement of bile ducts.1,2 Bacteria are frequently postulated as a cause of canine hepatitis, but their presence within the liver is rarely documented.1–7 The application of culture-independent methods such as polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) to feline inflammatory liver disease (ILD) has enabled the detection of bacteria within the livers of cats with ILD, particularly those with neutrophilic and pyogranulomatous cholangitis.8–10 These findings in cats contrast with the absence of bacteria reported in dogs with canine granulomatous hepatitis evaluated with FISH in which inflammation was largely ascribed to copper-associated hepatopathy.7 This case report describes the presence of intrahepatic bacteria in three dogs with neutrophilic, pyogranulomatous, or lymphoplasmacytic hepatitis and cholangiohepatitis. In each of these cases, eubacterial FISH analysis enabled visualization of bacteria at the site of active inflammation in samples that were negative for bacteria by standard histopathological methods. The colocalization of intrahepatic bacteria with neutrophilic, granulomatous, and lymphoplasmacytic inflammation and clinical response to antimicrobial therapy support the concept that bacterial infections can be involved in the pathogenesis of canine hepatitis.
Case Report
Case 1
A 5 yr old 37.6 kg spayed female Labrador retriever was presented for lethargy, anorexia, and vomiting. Prior to referral, the dog had been treated with intravenous cefazolin (22 mg/kg q 12 hr), enrofloxacin (10 mg/kg q 24 hr), and meloxicam (0.1 mg/kg q 24 hr) by the primary care veterinarian for 3 days. The dog was quiet, alert, and responsive. Physical examination was normal except for pyrexia (104.9°F, reference interval [RI] 100–102.5°F) and a mildly tense abdomen. Hematological abnormalities included a normocytic, normochromic nonregenerative anemia (hematocrit 23%, RI 36–60%, reticulocyte count 23,100/mm3, RI 0–60,000/mm3), and thrombocytopenia (platelets 53,000/µL, RI 170,000–400,000/µL) accompanied by leukocytosis (22,000/µL, RI 4000–15,500/µL), neutrophilia (16,060/µL, RI 2060–10,600/µL), and a mild left shift (bands 440/µL, RI 0–300/µL). Serum biochemistry revealed hypoalbuminemia (1.8 g/dL, RI 2.7–4.4 g/dL), hyperglobulinemia (3.8 g/dL, RI 1.6–3.6 g/dL), and elevated alkaline phosphatase (ALKP; 175 U/L, RI 5–131 U/L). Abdominal ultrasound revealed a mild peritoneal effusion, a hypoechoic pancreas with diffusely hyperechoic mesentery, mildly enlarged mesenteric lymph nodes, and a heterogeneous liver with undulating margins. Peritoneal fluid had a total protein of 4.3 g/dL and a nucleated cell count of 27,000/uL, composed of 90% mildly degenerative neutrophils, and low numbers of macrophages and mononuclear cells, without evidence of phagocytized organisms or neoplastic cells. A SNAP pancreatic health testa was normal, nonsupportive of pancreatitis. Cytological evaluation of a fine needle liver aspirate revealed pyogranulomatous inflammation with no visible organisms. Given the abnormal sonographic changes in the liver and cytological evidence of pyogranulomatous hepatitis and peritonitis, the decision was made to pursue laparoscopic abdominal exploratory surgery and hepatic biopsy. Laparoscopy revealed that the left lateral and caudate liver lobes were coated with a fibrinous membrane and were adhered to the body wall by granulation tissue. The pancreas appeared normal. Six wedge biopsies of the liver were obtained from affected left lateral and caudate lobes, and unaffected right middle lobe. Hepatic histopathology was characterized by moderate, chronic, centrilobular and portal, lymphohistiocytic hepatitis (Figure 1A). Periodic acid-Schiff staining and acid-fast staining were negative for fungal organisms and mycobacteria. The liver copper level was mildly increased (422 ppm, RI 120–400 ppm). There was no bacterial growth on aerobic and anaerobic cultures of the liver, and PCR for vector-borne diseasesb (including Anaplasma phagocytophilum, Anaplasma platys, Bartonella henselae, Bartonella vinsonii, Babesia canis, Babesia spp., Ehrlichia canis, Ehrlichia spp., M hemocanis/hematoparvum, Neorickettsia risticii, and Rickettsia rickettsii), Heterobilharzia (performed at Texas A&M University), and Leptospira species serologyc (including Leptospira pomona, Leptospira icterohaemorrhagiae, Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo, Leptospira autumnalis, and Leptospira bratislava [by microscopic agglutination test]) tested as a part of initial diagnostics returned as negative. Because the hepatic histopathology was consistent with bacterial infection, additional histological sections were evaluated by FISH at Cornell University with a eubacterial probe as previously described,11 revealing abundant slender semicurved bacteria dispersed throughout the liver, and a discrete nidus of rod-shaped bacteria (Figure 1B). The dog was treated with oral doxycyclined (5 mg/kg q 12 hr), enrofloxacine (10 mg/kg q 24 hr), clindamycinf (11 mg/kg q 12 hr), famotidineg (0.5 mg/kg q 12 hr), and maropitanth (2.2 mg/kg q 24 hr). At the 1-wk recheck examination, the owner reported a good appetite and activity level, and physical examination was normal. Convalescent Leptospira spp. serology (2 wk postdischarge) was negative. At 1 mo, the dog had made a complete recovery with normal hematologic and biochemical analyses. Doxycycline was discontinued and the dog was maintained on enrofloxacin and clindamycin for another mo. The dog remained in good health at re-evaluations at 2, 3, 4, and 5 mo with normal biochemistry and hematologic analyses.
Citation: Journal of the American Animal Hospital Association 54, 1; 10.5326/JAAHA-MS-6492
Case 2
An 11 yr old 25 kg castrated male border collie was presented for lethargy, decreased appetite, and intermittent vomiting. The dog had not received any treatments prior to referral. Physical examination revealed a tense and painful abdomen and generalized icterus. Temperature, pulse, and respiratory rate were within normal limits. A complete blood count showed a normocytic, normochromic, nonregenerative anemia (hematocrit 22.4%, reticulocyte count 32,500/mm3), leukocytosis (27.74 10³/µL) with a mature neutrophilia (23,810/µL) and monocytosis (2480/µL, RI 0–840/µL), and moderate thrombocytopenia (platelets 59,000/µL). Serum biochemistry was characterized by increased alanine aminotransferase (ALT; 820 U/L, RI 12–118 U/L), ALKP (>2000 U/L), total bilirubin (21.1 mg/dL, RI 0.1–0.3 mg/dL), globulin (4.9 g/dL), and cholesterol (>520 mg/dL, RI 92–324 mg/dL), which were suggestive of biliary obstruction. Abdominal ultrasound showed a dilated common bile duct, multiple intrahepatic choleliths and choledocholiths, and hepatomegaly. Given the evidence of biliary obstruction, an abdominal exploratory surgery was performed. The liver appeared mottled and multiple intrahepatic choleliths and choledocholiths were found. A duodenostomy was performed. An 8-French red rubber catheter was introduced into the common bile duct, and choledocholiths were removed by flushing them through the distended major duodenal papilla. The intrahepatic choleliths upstream of the catheter were inaccessible and could not be removed. An 8-French red rubber stent was placed in the common bile duct to prevent reobstruction by the remaining choleliths and to improve bile flow into the duodenum. Multiple biopsies of the liver and samples of bile and choledocholiths were obtained. Unfortunately, the dog suffered a fatal cardiac arrest during the postoperative recovery period. Hepatic histopathology was characterized by severe, subacute to chronic, multifocal, supprative cholangiohepatitis with peribiliary fibrosis (Figure 1C). Aerobic and anaerobic microbial culture of the liver tissue was negative. However, culture of the choledocholiths and bile grew Escherichia coli and Enterococcus spp. The choleliths were determined to be 100% cholesterol. Eubacterial FISH revealed multifocal clusters of short rods to coccobacilli in areas of inflammation throughout the liver (Figure 1D), which were morphologically consistent with E coli and Enterococcus spp.
Case 3
A 5.5 yr old 17.9 kg spayed female German shorthaired pointer was presented for lethargy, anorexia, vomiting, polyuria, and polydipsia. Prior to referral, the dog had been treated with oral amoxicillin-clavulanic acidi (13.75 mg/kg q 12 hr) by the primary care veterinarian for 2 days. Pyrexia (105°F) at the primary care veterinarian had resolved at the time of referral. On physical examination, the dog was alert and responsive with cranial organomegaly. Hematology revealed leukocytosis (23,200/µL) with a mild left shift (bands 200/µL, mature neutrophils 19,000/µL), and thrombocytopenia (platelets 78,000/µL) with some platelet clumping. Serum biochemistry showed acidemia (pH 7.248, RI 7.35–7.45), hyperchloremia (128 mEq/L, RI 107–117 mEq/L), and low bicarbonate (12 mEq/L, RI 15–25 mEq/L) with normal anion gap (16 mEq/L, RI 13–25) suggestive of renal tubular acidosis. It also revealed hypoalbuminemia (1.7 g/dL), hyperglobulinemia (5.7 g/dL), hypoglycemia (48 mg/dL, RI 70–138 mg/dL), and increased ALT (231 U/L), aspartate aminotransferase (149 U/L), alkaline phosphatase (486 U/L), and total bilirubin (1.0 mg/dL). Urinalysis revealed isosthenuria (urine specific gravity 1.012, RI 1.015–1.050), alkaline urine (pH 8, RI 5.5–7.0), and no glucose. Microbial culture of the urine was negative. A SNAP vector-borne disease screening testj was negative for Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi, and Anaplasma phagocytophilum. Abdominal ultrasound revealed splenomegaly and a noncavitated splenic mass, a hypoechoic liver, mild hepatic lymphadenopathy, and mild peritoneal effusion. Because of the presence of multifocal undefined intra-abdominal abnormalities and the possibility of sepsis (hypoglycemia and hyperbilirubinemia accompanied by modest elevations in hepatic enzymes), an exploratory laparotomy was performed.12 The spleen was diffusely enlarged and lobulated, the liver was enlarged and mottled, the periportal, hepatic, and mesenteric lymph nodes were prominent, and there was mild peritoneal effusion and renal petechiation. A splenectomy was performed, the liver was biopsied, and a periportal lymph node was removed. A bone marrow aspirate, performed to detect multisystemic neoplasia or infectious agents, was characterized by myeloid hyperplasia. Histopathology of the liver revealed moderate, chronic neutrophilic and lymphoplasmacytic cholangiohepatitis with sinusoidal neutrophilia (Figure 1E). Splenic histopathology was characterized by severe, coalescing, subacute, necrosuppurative splenitis. The portal lymph node showed marked lymphangiectasia and edema with sinusoidal histiocytosis. Giemsa, Gram, acid fast, and Gomori methenamine silver staining of liver and spleen samples were negative for bacterial and fungal organisms. Polymerase chain reaction for Bartonella spp. (performed at North Carolina State University; genus assayed included Bartonella henselae, Bartonella koehlerae, Bartonella vinsonii, Bartonella quintana, and Bartonella clarridgeiae) and serology for Toxoplasma gondii (performed at the University of Tennessee; Toxoplasma gondi IgG & IgM serology [by modified agglutination test]) and Leptospira spp. (performed at Cornell University; serogroup assayed included Leptospira pomona, Leptospira icterohaemorrhagiae, Leptospira canicola, Leptospira grippotyphosa, Leptospira hardjo, Leptospira autumnalis, and Leptospira Bratislava [by microscopic agglutination test]) were negative. Unfortunately, microbial culture of hepatic parenchyma or bile was not performed. Analysis of the liver with FISH revealed multifocal single pleiomorphic rods around the bile duct, hepatic vessels, and sinusoids (Figure 1F). Additionally, FISH analysis of the splenic sample yielded multiple single and clumped bacteria (Figure 1F). The patient was discharged and treated with oral cephalexink (22 mg/kg q 12 hr), famotidine (0.5 mg/kg q 12 hr), and tramadoll (2 mg/kg q 12 hr) for 2 wk. At the 2-wk recheck examination, the owner reported the patient having normal activity level and complete resolution of clinical signs. Hematology and biochemistry values were within the normal reference ranges and urine specific gravity was 1.037.
Discussion
This case report describes the presence of intrahepatic bacteria in a subset of dogs with hepatitis that were considered negative for bacteria by histopathological evaluation. The colocalization of intrahepatic bacteria with neutrophilic, granulomatous, and lymphoplasmacytic inflammation and clinical response to antimicrobial therapy of two out of two patients support the concept that bacterial infections can be involved in the pathogenesis of canine hepatitis. Our findings contrast with the uniformly negative results of a previous study employing FISH to detect intrahepatic bacteria in dogs with granulomatous hepatitis7 and underscore the need for comprehensive analytic methods to help direct appropriate therapy (antimicrobial versus immunosuppressive versus copper reduction) in dogs with hepatitis.
Canine hepatitis describes a broad spectrum of conditions characterized by infiltration of the hepatic parenchyma with neutrophils, mononuclear cells, lymphocytes, or plasma cells with or without involvement of bile ducts.1,2 According to the histological classification proposed by World Small Animal Veterinary Association guidelines, these conditions include (but are not restricted to) acute and chronic hepatitis, granulomatous hepatitis (GH), cholangiohepatitis, and lobular dissecting hepatitis.1 Recognized causes of canine hepatitis include infectious agents, toxins, adverse drug reactions, copper-associated hepatitis, and autoimmunity.1,2 Bacterial infection is considered a potential cause of most forms of canine hepatitis, and has been associated with acute and chronic hepatitis, hepatic abscessation, granulomatous and pyogranulomatous inflammation, and cholangiohepatitis.1–7 The most common enteric bacteria associated with canine hepatitis are E coli, Enterococcus spp., Bacteroides spp., Streptococcus spp., Clostridium spp., and Helicobacter canis.2,6,13,14 Nonenteric bacteria include Leptospira spp., Bartonella spp., Clostridium piliforme, and Mycobacterium spp.2,6,15–18
Definitive diagnosis of bacteria-associated canine hepatitis has until recently been based on the direct identification of bacteria by cytology, histopathology, histochemical stains, or microbial culture of hepatic parenchyma or bile.6,13–16 Advances in molecular microbiology have facilitated culture-independent DNA-based characterization of bacteria. Polymerase chain reaction is increasingly utilized as a diagnostic tool for detecting infectious organisms.4,7,8,17–19 Few studies to date have applied molecular techniques to canine and feline ILD. A PCR-based study of 98 dogs with hepatitis detected parvovirus in 2 out of 98 liver samples that were examined with a panel of primers directed against bacteria and viruses suspected of involvement in canine hepatitis.4 In one study of GH, there were 15 of 25 dogs with GH that were evaluated for infectious agents using special staining techniques, FISH, and Bartonella PCR assays, and there was no evidence of infectious agents found.7 In this study, 11 of the 15 dogs were evaluated by FISH.
Our findings contrast the uniformly negative results of FISH analysis in 11 dogs with granulomatous and pyogranulomatous hepatitis evaluated by Hutchins et al.7 The predominant clinical signs in our cases—lethargy, anorexia, vomiting (all cases), tense or painful abdomen (cases 1 and 2), and pyrexia (cases 1 and 3)—are similar to those reported in studies of canine GH.3,7 The neutrophilia, thrombocytopenia, hypoalbuminemia, hyperglobulinemia, and increased ALKP activity with lesser increases in transaminases observed in our case series also closely parallel the cases described by Hutchins et al.7 Proportionally higher increases in ALKP relative to ALT have also been observed in older reports of canine GH and suggest a predominance of cholestasis relative to hepatocellular necrosis.3 Given the similar clinical and clinicopathological findings, perhaps the discordance in FISH-based analysis reflects differences in the specific type of canine hepatitis. The 25 dogs evaluated by Hutchins et al. had predominantly granulomatous inflammation, although some dogs with pyogranulomatous lesions were included, and significant accumulation of copper, whereas the cases described herein were pyogranulomatous, had concurrent infiltration of the hepatic parenchyma and bile ducts, and copper levels did not support toxicosis.7
Although the cases in this series had broadly similar hepatic cellular infiltrates, they varied in the morphology of intrahepatic bacteria and concurrent disease processes. In case 1, the bacterial population was a mix of slender, semicurved morphology suggestive of Helicobacter spp. or Leptospira spp. and a focal cluster of short rods. Helicobacter canis has been associated with multifocal necrotizing hepatitis in a dog, and Leptospira spp. has been associated with chronic hepatitis in kenneled dogs.14,15 The negative convalescent Leptospira spp. titers do not support the Leptospira spp. infection in this dog. Because of the severity of its clinical condition, this dog was empirically treated with three antibiotics including a fluoroquinolone to cover a broad spectrum of microbes (gram positive, gram negative, Bartonella spp., and Mycobacterum spp.). Because of the positive clinical response to antimicrobial therapy in the absence of culture-based susceptibility testing, all three antibiotics were continued for 4–8 wk. It is notable that this dog had a classic ultrasonographic appearance of pancreatitis, but a negative SNAP cPL result. A pancreatic biopsy or canine pancreatic lipase immunoreactivity assay of peritoneal effusion was not performed to clarify this discordancy because the pancreas appeared grossly normal during laparoscopy. We suspect that the reported ultrasonographic changes reflect overestimation of the hyperechogenicity of the peripancreatic mesentery, secondary to the presence of peritoneal fluid. In case 2, positive bacterial cultures of bile and choledocholiths for E coli and Enterococcus spp. were consistent with the bacterial morphology observed on eubacterial FISH. The isolation of E coli and Enterococcus spp. from bile, but not liver, in this case echo the findings of Wagner et al., in which microbial culture performed on bile was more frequently positive than the liver. E coli and Enterococcus spp. are frequently associated with canine hepatitis2,6,13 and the multifocal clusters of intrahepatic bacteria observed with FISH in this case are similar to those reported in a cat with concurrent ILD and bile duct obstruction.10 Hepatobiliary production of bile, secretory immunoglobulin A, and physiologic choleresis are hepatic protective mechanisms limiting enteric and biliary bacterial colonization.6 The development of extrahepatic bile duct obstruction or other processes fostering cholestasis can disrupt these protective mechanisms, increasing the risk of biliary bacterial colonization.6 In case 3, the simultaneous presence of bacteria in the liver and spleen suggests bacteremia and hematogenous spread as the mode of bacterial dissemination, and could account for the hypoglycemia and hyperbilirubinemia accompanied by modest elevations in hepatic enzymes in this case.12
A potential limitation of this case report is the lack of a control group without hepatic disease. It could be argued that the presence of bacteria within the livers of the dogs in our case series reflects pre-existing colonization of the healthy liver, or nonspecific hepatic dysfunction with reduced Kupffer cell function and cholestasis.2,6,20 However, colocalization of bacteria in areas of active inflammation, the complete resolution of clinical signs and clinicopathological abnormalities in cases 1 and 3, and absence of bacteria in 11 dogs previously evaluated by FISH supports the involvement of bacteria in the disease process.7 The FISH methodology employed in this case series used a eubacterial probe in combination with a noneubacterial control probe to detect intrahepatic bacterial colonization. Further identification of intrahepatic bacteria using additional, best-guess probes directed against specific subgroups of bacteria (e.g., E coli and Streptococcus spp.) was not performed because of the limited availability of additional samples for the analysis and financial limitations of the owners.10 Additionally, 16S sequencing of DNA extracted from hepatic tissue sections can be used to identify colonizing bacterial species and guide the selection of FISH probes to evaluate the spatial distribution of specific bacteria. A further limitation of this case report is the small number of cases, restricted spectrum of disease, and lack of standardization in case management. These limitations should be addressed in prospective studies.
Conclusion
This case report serves to document the presence of intrahepatic bacteria in dogs with neutrophilic, pyogranulomatous, or lymphoplasmacytic hepatitis and cholangiohepatitis. Consistent clinical findings in these dogs were lethargy, anorexia, vomiting, abdominal pain, and pyrexia. Clinicopathological abnormalities included neutrophilia, thrombocytopenia, hypoalbuminemia, hyperglobulinemia, and increased ALKP activity with lesser magnitudes of increase in transaminases. In each of these cases, eubacterial FISH analysis enabled direct visualization of bacteria in samples that were negative for bacteria by routine histopathology, and guided therapy. These findings support the need for prospective studies to examine the relationship of bacteria to canine hepatitis.
Histopathology and eubacterial FISH on hepatic biopsies of the three dogs with hepatitis. In all images showing FISH, DAPI-stained nuclei are blue. Green background represents autofluorescence. Red blood cells autofluoresce yellow. Insets are arbitrary magnifications of fluorescent bacteria identified within the highlighted region(s) unless otherwise stated. (A) Case 1. Moderate, chronic, centrilobular and portal, lymphohistiocytic hepatitis. H&E stain. Bar = 250 μm. (B) Case 1. FISH with Eub338-cy3 probe showing slender semicurved bacteria (red, inset) on the left image, and focal clusters of rod shaped bacteria (red, inset) on the right image. Bar = 100 μm. (C) Case 2. Severe, subacute to chronic, multifocal, supprative cholangiohepatitis with peribiliary fibrosis. H&E stain. Bar = 248 μm. (D) Case 2. FISH with Eub338-cy3 probe showing multifocal clusters of short rods to cocco-bacilli (red, inset). Bar = 200 μm. (E) Case 3. Moderate, chronic neutrophilic and lymphoplasmacytic cholangiohepatitis with sinusoidal neutrophilia. H&E stain. Bar = 248 μm. (F) Case 3. FISH with Eub338-cy3 probe showing multifocal single pleiomorphic rods (red, inset). The inset in the lower left shows multiple single and clumps of bacteria (red) in the splenic parenchyma. Bar = 100 μm. DAPI, 4,6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; H&E, hematoxylin and eosin.
Contributor Notes
