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Abstract
Bilateral chronic granulomatous nephritis and meningoencephalitis were diagnosed on necropsy of a 2-year-old male Great Dane dog. The causative agent was identified as Balamuthia mandrillaris, based on morphologic features, immunohistochemical staining, and deoxyribonucleic acid detection using the polymerase chain reaction with newly designed primer pairs. Trophozoite and cystic forms of the amoeba were evident within the kidneys and brain parenchyma. This is the first report on a B. mandrillaris infection in a dog.
Key words: Balamuthia mandrillaris; brain; canine; immunohistochemistry; kidney; polymerase chain reaction.
Balamuthia mandrillaris is a free-living amoeba of the order Leptomyxia capable of causing fatal granulomatous amoebic meningoencephalitis (GAE) in humans and animals. It was first isolated from a mandrill (Papio sphinx) at San Diego Zoo Wild Animal Park, then subsequently in gorillas (Gorilla gorilla gorilla),1 an orangutan (Pongo pygmaeus),5 and Old World primates, including a colobus monkey (Colobus guereza kikuyuensis) and a gibbon (Hylobates concolor leucogenys).12 In recent years, B. mandrillaris has also been increasingly identified as a cause of GAE in humans.9 So far, the only reports in a nonprimate species have involved a horse7 and a sheep.6 B. mandrillaris has been recently isolated from environmental samples, suggesting that it occupies similar habitats as other opportunistic amoeba such as Naegleria fowleri and Acanthamoeba spp.15 The route of invasion is still unknown; penetration of skin or respiratory tract and subsequently hematogenous spread has been postulated.8,9,10 Infections with B. mandrillaris are reported to be more common in immunosuppressed hosts such as patients with acquired immunodeficiency syndrome, although immunocompetent individuals also have been affected.9,10 Reports on canine disease caused by free-living amoebas are rare and include a report on Acanthamoeba culberstoni isolation from the lung of a naturally infected Greyhound puppy with granulomatous pneumonia and encephalitis.2 To the best of this author's knowledge, there is a single report on kidney lesions in a dog associated with dissemination of Acanthamoeba castellani.11 In this study, we report a systemic B. mandrillaris infection for the first time in an immunocompromised dog.
A 2-year-old, male, castrated Great Dane dog presented to the Veterinary Medical Teaching Hospital of the School of Veterinary Medicine at the University of California, Davis, California, with a several-day history of weight loss, vomiting, diarrhea, and urinary incontinence. The dog had been treated for 6 months with immunosuppressive doses of prednisone (1 mg/kg PO q 12 hours for 2 weeks) for chronic lymphocytic-plasmacytic enteritis (inflammatory bowel disease). Metronidazole was prescribed (10 mg/kg PO q 12 hours for 2 weeks), but weight loss and diarrhea continued, and 3 weeks later, profound lethargy and hematuria were noted. A hemolytic Escherichia coli was cultured from the urine. Treatment with amoxicillin-clavulanic acid (15 mg/kg PO q 8 hours) was initiated, and the owners were instructed to taper the prednisone dosage. Despite treatment, the clinical signs worsened, and 2 weeks later, the dog presented to the hospital recumbent, comatose, and with intermittent seizures and rotary nystagmus. Because of the poor prognosis, the owners elected the dog to be euthanatized.
A full necropsy was immediately performed and revealed numerous small, randomly distributed granulomas restricted to the kidneys and brain. In both kidneys, the granulomatous lesions were confined to the corticomedullary junction and renal cortex (Fig. 1). In the brain, lesions were found in the right occipital lobe (Fig. 2) and right caudal cerebellar peduncle. All tissues were immersion fixed in 10% buffered formalin, routinely processed by embedding in paraffin, sectioned (4 µm), and stained with hematoxylin and eosin (HE). In addition, selected sections of brain and kidney were stained with Giemsa, periodic acid–Schiff, and Gomori methanamine-silver nitrate.
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In the brain, within the grossly affected areas, there was fibrinoid necrosis of the blood vessels and a florid perivascular infiltration of neutrophils and macrophages admixed with lakes of fibrin (Fig. 7). Extensive areas of malacia, which was associated with astrogliosis, surrounded the affected vessels. Large numbers of trophozoites were concentrated primarily in the perivascular regions of the gray matter, the meninges, and the choroid plexus. The cystic forms were located further away from the blood vessels and were associated with less inflammation and necrosis.
Immunohistochemistry (IHC) was performed as previously described7 using Acanthamoeba spp. (Visvesvara 1 : 800), N. fowleri (1 : 1,000), and B. mandrillaris (Visvesvara 1 : 1,500) antibodies. Balamuthia antibodies stained the cystic forms intensely and the cytoplasm of macrophages surrounding the cysts weakly (Fig. 5). The latter staining was interpreted to represent phagocytized antigen. IHC staining with Acanthamoeba spp. and N. fowleri antibodies was negative. Subsequently, formalin-fixed, paraffin-embedded sections of cerebrum and renal cortex were used for deoxyribonucleic acid (DNA) extraction. Separate scalpel blades were used to procure each piece of tissue. The DNA in each tissue section was extracted using the QIAmp blood and tissue kit (Qiagen Inc., Chatsworth, CA). Initially, polymerase chain reaction (PCR) primers were used for PCR amplification of B. mandrillaris and Acanthamoeba DNA as previously described (Table 1).4,14 These primers result in amplification of 1,075-bp and approximately 460- to 470-bp segments of DNA, respectively. Primers and positive-control B. mandrillaris and Acanthamoeba DNA were the kind gift of Dr. Gregory Booton. To ensure that the quality of the extracted DNA was adequate, a 214-bp segment of the canine histone 3.3 gene was amplified from the samples as previously described.13 Amplified products were subjected to agarose gel electrophoresis and were visualized using ultraviolet transillumination after staining with ethidium bromide. Unfortunately, although the positive-control amoeba DNA and the canine histone gene were successfully amplified, neither B. mandrillaris nor Acanthamoeba spp. DNA could be amplified from the tissue samples.
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The rapid progressive clinical signs, fatal outcome, and granulomatous lesions found in this dog are consistent with previous reports in humans and animals.5,6 So far, lesions associated with B. mandrillaris infection in animals and humans have been restricted to the central nervous system,6,7,10 cervical spinal cord,7 and lymph node.6 In this dog, however, granulomatous lesions and different life stages of the amoeba were also present in both kidneys. The renal involvement is most likely secondary to hematogenous spread, similar as described for the origin of the brain lesions in other species. The source of the amoebic infection in this dog is thought to be a pond of stagnant water, where the dog was taken periodically to swim, most recently 10 days before the onset of hematuria. Chronic administration of glucocorticoids at immunosuppressive doses most likely predisposed the dog to infection. The dog was housed with two other dogs that also were taken to the same pond. These dogs have remained asymptomatic.
Recently, a PCR assay was described for detection of B. mandrillaris.3,4 Unfortunately, our attempts to detect the organism using the primers described by these authors were unsuccessful, although the positive-control DNA was successfully amplified. This, together with a positive result using the canine histone gene primers, suggested the presence of formalin-induced cross-linking of DNA within the specimen. Ideally, PCR assays for use on formalin-fixed archival tissue specimens should be designed to amplify DNA segments approximately 200 bp in length. The primers designed by Booton et al. (2003) direct the amplification of a 1,075-bp product. We have described additional PCR primer pairs that may be more suitable for detection of Acanthamoeba spp. and B. mandrillaris in formalin-fixed tissues.
In summary, we have described a granulomatous nephritis and meningoencephalitis secondary to a B. mandrillaris infection in a dog. This dog was immunocompromised due to corticosteroid therapy, which is thought to have made this dog vulnerable to systemic amoebic disease. In contrast to other species, this dog had kidney lesions in addition to brain lesions. Staining with Giemsa stain was used successfully to highlight the organism in renal and brain tissues. IHC and PCR were necessary, however, to differentiate between amoeba species for a conclusive diagnosis. The PCR assays described in this report are the first to be used successfully to detect B. mandrillaris in archival tissue specimens and should be useful for other studies of this infection in the future. Additional studies will be required to determine routes of infection and the role of immune response in the pathophysiology of the disease.
References
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  A. Matin, R. Siddiqui, S. Jayasekera, and N. A. Khan Increasing Importance of Balamuthia mandrillaris Clin. Microbiol. Rev., July 1, 2008; 21(3): 435 - 448. [Abstract] [Full Text] [PDF]
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 S. Yagi, F. L. Schuster, and K. Bloch Demonstration of Presence of Acanthamoeba Mitochondrial DNA in Brain Tissue and Cerebrospinal Fluid by PCR in Samples from a Patient Who Died of Granulomatous Amebic Encephalitis J. Clin. Microbiol., June 1, 2007; 45(6): 2090 - 2091. [Full Text] [PDF]
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M. Tavares, J. M. Correia da Costa, S. S. Carpenter, L. A. Santos, C. Afonso, A. Aguiar, J. Pereira, A. I. Cardoso, F. L. Schuster, S. Yagi, et al. Diagnosis of first case of balamuthia amoebic encephalitis in portugal by immunofluorescence and PCR. J. Clin. Microbiol., July 1, 2006; 44(7): 2660 - 2663. [Abstract] [Full Text] [PDF]
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