Heterobilharzia americana Infection and Glomerulonephritis in a Dog

Introduction

Heterobilharzia (H.) americana is a trematode endemic to the South Atlantic and Gulf Coast regions of the United States.¹ Cases of canine schistosomiasis have not been commonly reported; only seven clinical case reports involving a total of 10 dogs with naturally occurring schistosomiasis have been described since 1961.^2–8 Only one of those reported cases described hypoalbuminemia and proteinuria as clinical findings.⁶ Another single case reported “chronic interstitial nephritis” without noting hypoalbuminemia and proteinuria.² Specific renal histopathological abnormalities have not been described.

Glomerulonephritis can trigger a series of histopathological abnormalities that result in persistent glomerular proteinuria and progressive renal failure in dogs.⁹ Accumulation of antigen-antibody complexes within the glomerular capillary wall stimulates an inflammatory cascade, leading to varying degrees of glomerular cell proliferation and thickening of the basement membrane.¹⁰ Ultimately, sclerosis and hyalinization occur, resulting in glomerular obsolescence and the eventual clinical signs of renal failure (polyuria, polydipsia, and azotemia) in chronically affected dogs.¹⁰

The pattern of inflammation allows division of glomerulonephritis into three categories: mesangioproliferative, membranous, and membranoproliferative. Mesangioproliferative glomerulonephritis is characterized by hypercellularity of the mesangial space, while membranous nephropathy is characterized by thickening of the glomerular basement membrane.^10–13 Membranoproliferative glomerulonephritis has characteristics of both membranous and mesangioproliferative forms. Membranoproliferative glomerulonephritis is thought to account for 10% to 60% of canine glomerulonephritis cases.^11–13

Glomerulonephritis has been associated with infectious (e.g., brucellosis, dirofilariasis, ehrlichiosis, leishmaniasis), inflammatory (e.g., pancreatitis), immune-mediated (e.g., systemic lupus erythematosus, immune-mediated polyarthritis), neoplastic, and endocrine diseases.⁹ Treatment goals include slowing or arresting the progression of proteinuria and glomerular pathology. As a result, affected dogs should be evaluated for an antigen source responsible for immune complex formation.⁹ One review article evaluated 137 cases of glomerular proteinuria.¹¹ Of the dogs in that review, 106 were diagnosed with glomerulonephritis, and of those 106, only 12 (11%) had an identifiable concomitant infectious disease.¹¹

The purpose of this report is to describe the diagnosis and treatment of a dog that developed membranoproliferative glomerulonephritis contemporaneous with H. americana infection.

Case Report

A 6-year-old, 22.7-kg, neutered male, mixed-breed dog was referred to Lexington Boulevard Animal Hospital for investigation of protein-losing nephropathy and weight loss. Ten months prior to the onset of clinical signs, the dog had been vaccinated with a killed rabies vaccine and modified live distemper, adenovirus, and parvovirus vaccine. The dog had a 4-month history of intermittent, mild gastrointestinal signs, including several episodes of anorexia and increased flatulence. No vomiting or diarrhea was reported by the owner. The diet consisted of an appropriate amount of a commercially available dry food. The dog went outdoors frequently, having access to a horse barn and low-lying fields in rural southeastern Texas.

Prior to referral, serial serum biochemical analyses revealed persistent hypoalbuminemia (1.9 to 2.0 g/dL, reference range 2.5 to 3.5 g/dL) and hyperglobulinemia (8.7 to 8.8 g/dL, reference range 3.5 to 4.5 g/dL). The dog initially had a normocytic, normochromic anemia (26% hematocrit, reference range 36% to 60%). A reticulocyte count was not performed at that time. Urinalysis revealed isosthenuria (1.011, reference range 1.015 to 1.050) and proteinuria (3+ on dipstick analysis). A urine protein:creatinine ratio was performed to confirm and quantify the proteinuria (6.8, reference range <0.5). Serum protein electrophoresis submitted to an outside laboratory showed decreased albumin and gamma fractions with polyclonal elevation in the beta fraction.^a Antibody titers for tickborne diseases (i.e., Ehrlichia canis, Anaplasma phagocytophilum, Neorickettsia risticii) and a Dirofilaria immitis antigen test were negative, as was fecal flotation for gastrointestinal parasites.

On presentation to the referral veterinarian, the dog was thin (body condition score 2/5). Over the preceding 15 months, the dog had lost 14% (3.6 kg) of its body weight, with the majority of weight loss subjectively occurring in the 2 months prior to presentation. The dog was normothermic; peripheral lymph nodes were normal in size; and no fundic abnormalities were detected on indirect ophthalmoscopic examination. The remainder of the physical examination was unremarkable. No parasite ova were seen on routine zinc sulfate fecal flotation or on direct saline preparation.

A hemogram, serum biochemical analysis, and urinalysis were repeated at the time of referral. Hemogram abnormalities included microcytic, normochromic, nonregenerative anemia (27% hematocrit, reference range 37% to 55%; red blood cell count 4.9 × 10⁶ cells/μL, reference range 5.5 to 8.5 × 10⁶ cells/μL; mean corpuscular volume 54 fL, reference range 60 to 77 fL; mean corpuscular hemoglobin 18 pg, reference range 19.5 to 24.5 pg; reticulocyte count 24,500 cells/μL, reference range >80,000 cells/μL) and mild relative eosinophilia (12%, reference range 2% to 10%). Hypoalbuminemia (1.9 g/dL, reference range 2.5 to 3.5 g/dL) and hyperglobulinemia (9.3 g/dL, reference range 3.5 to 4.5 g/dL) were confirmed on a serum biochemical panel. The dog was not azotemic, and the remainder of the serum biochemical analysis was considered unremarkable. Urinalysis of a sample obtained by cystocentesis revealed isosthenuria (1.010) with 4+ protein levels on commercial dipstick analysis.^b

Orthogonal radiographic views of the thorax and abdomen were considered normal. Fine-needle aspiration of the liver and spleen, followed by cytopathological examination, was unremarkable. Bone marrow aspiration and cytopathology were also performed to screen for occult neoplastic disease and to further characterize the nonregenerative anemia. The marrow was adequately cellular and revealed erythroid hyperplasia. All cell lines displayed orderly maturation, and moderately increased numbers of plasma cells were present. Despite only mild peripheral eosinophilia, eosinophil precursors in the bone marrow were quite numerous. No fungal elements or neoplastic cells were seen. Iron staining was not performed to evaluate iron stores. These findings suggested that the anemia did not result from intramarrow aplasia, neoplasia, or myelophthisis, but from extramarrow consumption or loss.

The documented persistent proteinuria was deemed significant enough to cause hypoalbuminemia; however, the dramatic weight loss and gastrointestinal signs suggested the possibility of concurrent enteric loss. In the absence of pyuria, bacteriuria, hematuria, and casts, differential diagnoses for the elevated urine protein:creatinine ratio included preglomerular (e.g., hemoglobinemia, myoglobinemia, myeloma) and glomerular (e.g., glomerulonephritis, amyloidosis) diseases. The elevated urine protein:creatinine ratio, combined with lack of evidence of preglomerular disease, supported a diagnosis of glomerular disease.

The nonregenerative microcytic anemia could have several etiologies. Occult enteric blood loss with subsequent iron deficiency is one consideration. Iron deficiency classically results in microcytic hypochromic anemia, and in this dog, the mean corpuscular hemoglobin concentration was normal. Serum iron concentration and total iron binding capacity were not measured in this case. Differential diagnoses for nonregenerative normochromic anemia, such as anemia of chronic inflammatory disease and anemia related to hepatic insufficiency, could not be excluded. The polyclonal gammopathy was felt to be the result of chronic infectious, autoimmune, or neoplastic disease.

Persistent hypoalbuminemia, renal proteinuria, polyclonal gammopathy, and unexplained nonregenerative anemia warranted further evaluation and biopsy of the kidneys, liver, and intestine. Differential diagnoses considered were infiltrative intestinal disease and occult neoplasia. Abdominal ultrasound was discussed with the owner and declined in favor of an exploratory celiotomy. Surgical biopsy was chosen, because it provided an opportunity to directly view and biopsy the kidneys, recognize and control hemorrhage, and acquire larger biopsy samples than could be gained through a percutaneous ultrasound-guided approach. A midline celiotomy was performed. Gross abnormalities affected numerous organs, including the liver, pancreas, small intestine, mesentery, and lymph nodes. All of the affected organs contained many focal nodules on serosal surfaces, measuring 2 to 5 mm in diameter [Figure 1]. The small intestine and pancreas were thickened and palpably nodular. Both kidneys were normal on gross examination. Liver, jejunum, pancreas, mesenteric lymph nodes, and the left kidney were biopsied in a routine manner. Samples were fixed in 10% formalin solution prior to submission to an outside laboratory for histopathology.^c Anesthetic recovery was uneventful. While waiting for histopathology results, the dog was given amoxicillin trihydrate/clavulanate potassium (11 mg/kg per os [PO] q 12 hours for 10 days) and enrofloxacin (3 mg/kg PO q 12 hours for 10 days).^d,e

Histopathological examination of the kidney demonstrated a marked interstitial, lymphoplasmacytic, inflammatory infiltrate. Moderate to marked periglomerular fibrosis was seen, and Bowman’s capsules were markedly thickened by connective tissue [Figure 2]. Glomerular tufts were interspersed with proliferative mesangial cells and fibrous connective tissue. Some glomeruli demonstrated fusion of the visceral and parietal layers of Bowman’s capsule. Focal regions of glomerulosclerosis and obsolete glomeruli were present. Tubular epithelial cells were hypertrophied and reactive, and the renal tubules were filled with dense, eosinophilic, proteinaceous material. Electron microscopy and immunofluorescence were not performed.

The liver contained multifocal granulomas, each with a central, collapsed fluke ovum (approximately 85 μm in contained dark hemosiderin pigment, consistent with waste produced from the blood-meal of the trematode parasite. A mesenteric lymph node, the pancreas, and the jejunum contained multiple fluke egg granulomas, similar to those in the liver [Figure 4]. All lesions were consistent with H. americana infection. Antigen-antibody complex deposition within the glomeruli and the resultant membranoproliferative glomerulonephritis were deemed responsible for the dog’s protein-losing nephropathy. After receipt of the histopathology report, direct fecal examination with a drop of physiological saline solution was performed on a fresh stool sample. Ova consistent with Heterobilharzia were readily demonstrated, and within minutes, several ciliated miracidia started to hatch.

Antitrematode therapy consisting of fenbendazole (24 mg/kg PO q 24 hours for 7 days) and praziquantel (10 mg/kg q 8 hours for 2 days) was administered.^f,g Twenty-four hours after initiation of therapy, the dog developed small-bowel diarrhea and vomiting that resolved within 2 days. Follow-up direct fecal examination and saline sedimentation failed to reveal the parasite at 2 and 3 months following treatment. Three months after treatment, the dog had gained 5.4 kg (19.2%) and had no signs of anorexia or flatulence. Serum biochemical analysis performed 16 months later confirmed restoration of normal serum albumin (3.6 mg/dL, reference range 2.5 to 3.5 mg/dL) and globulin (3.2 mg/dL, reference range 3.5 to 4.5 mg/dL) concentration, and a hemogram demonstrated resolution of the anemia (48%, reference range 36% to 60%). Urine dipstick analysis was negative for overt proteinuria, and a repeat urine protein:creatinine ratio was not performed. The referring veterinarian reported that the dog remained free of clinical signs 24 months after treatment.

Discussion

The life cycle of H. americana is dependent on both an intermediate and a definitive host. Wild mammals and domestic dogs serve as definitive hosts and reservoirs for the parasite.^1,2,7 Heterobilharzia ova are passed in the stool of the definitive host and hatch ciliated miracidia once in contact with water.^1,2 These miracidia penetrate the soft tissue of the freshwater snail (Lymnaea cubensis), where they reproduce and release free-swimming cercariae.^1,2 The cercariae can infiltrate the mucous membranes or intact skin of the definitive host, eventually gaining access to the lymphatic system or the bloodstream and migrating to the liver.^1,2

In the adult form, the fluke resides primarily in the liver and feeds on the erythrocytes of the definitive host.^1,2 The adults eventually migrate to the mesenteric veins, where male and female flukes conjoin for mating.^1,2 The female produces thousands of ova that lodge in the enteric venules, secrete proteolytic enzymes, and eventually traverse the intestinal wall to gain access to the lumen.^1,2 Eggs that bypass the enteric venules come to rest in other areas of the abdominal viscera (such as the hepatic parenchyma, lymph nodes, and pancreas), triggering a host cell-mediated immune response and formation of ova granulomas.² Histopathology of these lesions reveals fibroblasts, lymphocytes, and eosinophils surrounding an egg or fluke granuloma.¹ The life cycle is completed when sufficient eggs migrate through the bowel wall to be shed in the feces.²

Schistosomiasis can produce a variety of clinical signs in a heavily parasitized definitive host, presenting a diagnostic challenge. Multiple systems can be affected, because the dissemination and implantation of aberrant eggs occur in various tissues. The most consistent historical finding is weight loss, although hematochezia, melena, diarrhea, vomiting, and lethargy have been reported.^3–6 Anemia is present in many affected animals.^2–4,6 Anemia with low serum iron and iron-binding capacity has been reported in one dog infected with H. americana.⁶ In that case, a reticulocyte count was not reported, and enteric blood loss and anemia of chronic inflammatory disease were believed to be responsible for the anemia.⁶ Eosinophilia is not consistently noted in published reports of schistosomiasis.^3,6 The disseminated granulomatous host response to aberrant ova deposition can result in hypercalcemia, and elevated parathyroid hormone-related protein (PTHrP) has been reported in several cases.^4–6 When azotemia has been reported in the literature, it always has been encountered in the face of hypercalcemia.^4–6 In the current case, neither PTHrP nor ionized calcium analyses were performed.

The antemortem diagnosis of schistosomiasis can be problematic, because the eggs are intermittently shed and, once shed, quickly hatch upon contact with water. Routine fecal centrifugation and flotation methods usually fail to sufficiently concentrate the sinking ova.² Saline sedimentation provides a more sensitive tool for demonstration of the organism.² Alternatively, a miracidia hatching technique has been described.¹⁴ The hatching technique immerses a washed fecal sample in distilled water to encourage the motile miracidia to hatch, thereby improving detection.¹⁴

An enzyme-linked immunosorbent assay intended for human use has been successful in identifying schistosome-related antigens in at least one dog.³ In that dog, schistosome antigen levels were used, in addition to fecal sedimentation, to gauge the response to therapy.³ An indirect hemagglutination test has been developed specifically for the dog, but it is not likely to be commercially available or practical for use in dogs.¹⁵ More recently, a polymerase chain reaction (PCR) assay has become commercially available for the detection of minute quantities of schistosome ova deoxyribonucleic acid in fresh-frozen feces.^h The PCR test was not available at the time of presentation of this dog.

Standard radiographs have failed to reveal specific findings in previously reported cases of schistosomiasis. Sternal lymphadenopathy, splenomegaly, and decreased serosal detail have been described.^3,5 Upper gastrointestinal contrast radiography has been used to demonstrate diffuse, small intestinal infiltration in one dog with Heterobilharziasis.⁶ Ultrasound findings reported in the veterinary literature include peritoneal effusion, hyperechoic kidneys, small intestinal wall thickening, lymphadenomegaly, and splenomegaly.^3,5 Humans infected by a similar digenic trematode, Schistosoma mansoni, have comparable ultrasound findings.¹⁶ Advanced imaging, such as computed tomography, has also proven useful in human medicine,¹⁷ but it has not yet been described in cases of canine schistosomiasis. Dogs may undergo biopsy and histopathology of lesions in the mesenteric arteries, small intestine, and liver as part of the diagnostic process. Histopathology is a definitive test, demonstrating the adult flukes and eggs encased within granulomatous lesions in various tissues.²

Treatment of schistosomiasis is usually successful. The use of fenbendazole at a dose of 40 mg/kg q 24 hours for 10 days has been described.¹⁸ Praziquantel has also been proposed at a dose of 25 mg/kg q 8 hours for 2 days, which is five to 10 times higher than that used for treatment of cestode infections.² Concurrent administration of both fenbendazole and praziquantel, as done in this dog, was successful in another reported case.⁶ Treated dogs may have inadequate responses when administered lower doses of praziquantel, with eggs being shed for weeks after the initiation of treatment.³

While glomerulonephritis can have significant long-term detrimental effects, clinicopathological abnormalities and clinical signs may improve with correction of the underlying cause. Therefore, it is important to search for antigens that can be eliminated and to verify that proteinuria has subsided after treatment. Clinicopathological abnormalities common in cases of glomerulonephritis include renal proteinuria, hypoalbuminemia, azotemia, and anemia.¹⁰ In the early stages of the disease, proteinuria (with or without hyaline casts) may be the only clinical abnormality.⁹ Proteinuria detected on dipstick analysis should be further investigated through evaluation of urine sediment and urine culture to rule out infection. In addition, quantification of proteinuria using a urine protein:creatinine ratio is superior to dipstick analysis.⁹ In a retrospective study of 41 dogs with glomerulonephritis, 44% were anemic, and (as in this case) none of those were regenerative.¹⁰ A portion of the dogs (13 of 41) in that study were also azotemic, and it is possible that the anemia was the result of reduced erythropoietin levels in some of those dogs.¹⁰ Confirmation of glomerulonephritis is achieved through renal biopsy and histopathology.¹⁰ Electron microscopy and immunofluorescence can provide additional information about the type of glomerular pathology.¹⁰

Other undetected causes of glomerulonephritis or occult neoplasia could explain the signs seen in the dog of this report. Although trematode eggs were observed in numerous vessels on histopathology, serology documenting the presence of circulating trematode antigen would have further supported the link between Heterobilharzia infection and glomerulonephritis. Electron microscopy and immunofluorescence would have provided supplementary information regarding the deposition of antigen-antibody complexes and the pattern of injury to the glomeruli. All of the renal biopsy specimens were fixed in 10% formalin solution, and no additional tissue was available for electron microscopy or immunofluorescence evaluation.

Conclusion

Glomerulonephritis and H. americana infection were diagnosed in a dog. Treatment for the parasite resulted in clinical recovery and resolution of proteinuria. This case suggests that Heterobilharziasis should be considered as a cause of protein-losing nephropathy in dogs living in the Gulf Coast regions of the United States.