24-year-old female chimpanzee (Pan troglodytes).Six days prior to death the chimpanzee was stiff, hesitant to move its neck and held its head down. The differential diagnosis included trauma or a neurologic disease. A complete blood count and clinical chemistry panel on the day following presentation revealed a mild leukocytosis and moderate hypokalemia. Radiographs of the cervical region were noncontributory, although physical examination of the area elicited a pain reaction even under anesthesia. Antibiotic and anti-inflammatory therapies were instituted with waxing and waning of results over the next six days and a relatively rapid decline culminating in death.

Gross Description:  

Numerous light brown foci (5-11 mm) were present on the surface (dorsal and ventral) and extending into the neuropil of the brain. Since this animal was housed in an outdoor corral, cross sections of the brainstem and a portion of the hippocampus were submitted for rabies diagnosis (negative).

Histopathologic Description:

The brain contains a large area of hemorrhage and necrosis with admixtures of neutrophils, mononuclear cells and multinucleated giant cells; cellular debris; fibrinoid necrosis of blood vessels; astrocytosis; and protozoal organisms consistent with Balamuthia mandrillaris. Trophozoites measure 30-60 μm. Also observed is a diffuse mild infiltration of mononuclear cells in the meninges and perivascular spaces.

Morphologic Diagnosis:  

Brain: Multifocal, marked, necrohemorrhagic and pyogranulomatous meningoencephalitis with fibrinoid vasculitis and protozoal organisms consistent with Balamuthia mandrillaris.

Lab Results:  

Day 1: Leukocytosis (16.3 k/μl); neutrophilia (13.3 k/μl); and hypokalemia (2.6 mEq/L).
Day 3: Leukocytosis (35.5 k/μl); neutrophilia (31.2 k/μl); and hypokalemia (2.5 mEq/L).
Day 4: Leukocytosis (28.7 k/μl); neutrophilia (22.4 k/μl); and hypokalemia (2.8 mEq/L).
Day 5: Leukocytosis (28.0 k/μl); neutrophilia (22.2 k/μl); and hypokalemia (2.7 mEq/L).


Balamuthia mandrillaris

Contributor Comment:  

Balamuthiasis is an emerging disease of humans and animals with fatal consequences. (4) Originally isolated and identified from a fatal case of meningoencephalitis in a pregnant mandrill (Papio sphinx), Balamuthia mandrillaris is a free-living amoeba; however, it was not recovered from the environment until 2003.(1,2,5) Encephalitis caused by free-living amoeba (e.g. Acanthamoeba and Balamuthia) is primarily a problem of immunocompromised patients, although immunocompetent patients are affected by both Balamuthia and Naegleria.(6) Granulomatous amoebic encephalitis associated with Acanthomeba and Balamuthia is typically slow in developing and insidious, and a hematogenous route of entry has been hypothesized but not proven for these disease agents.(6) By contrast, the rapidly fatal primary amoebic encephalitis associated with Naegleria is associated with exposure to freshwater lakes and swimming or water skiing with entry through the olfactory neuroepithelium.(6) The fact that this animal had a ruptured eardrum leads to somewhat of a quandary as to how the agent entered. Lesions in the brain appeared to represent a hematogenous spread; however, no parasitic lesions were observed outside of the central nervous system, and the presence of an open eardrum would appear to provide an open portal of entry.

JPC Diagnosis:  

Brain: Meningoencephalitis, necrohemorrhagic, histiocytic and neutrophilic, focally extensive, severe, with vasculitis, fibrin thrombi, and many amoebae.

Conference Comment:  

In a diagnostic setting, it is often important, but sometimes difficult, to determine whether vascular inflammatory lesions represent true vasculitis or are simply innocent bystander vessels caught within foci of inflammation. The conference moderator emphasized the importance of evaluating vessels distant from the most severely affected areas when making this histologic assessment. In this case, conference participants noted striking endothelial hypertrophy, inflammatory cells transmigrating and occasionally disrupting vessel walls, and perivascular accumulations of gitter cells both in severely and less affected areas of the section, and therefore agreed with the contributors diagnosis of vasculitis.

This case was reviewed in consultation with Dr. Christopher Gardiner, Consulting Parasitologist for the AFIPs Department of Veterinary Pathology; he noted that the large size of the Balamuthia mandrillaris trophozoites is helpful in distinguishing it from those of Naegleria fowleri and Acanthomoeba sp., the two other free-living amoebae most commonly implicated in human and animal disease. Only one case of amebic encephalitis has been attributed to a fourth free-living amoeba, Sappinia diploidea. During the conference, participants reviewed the freeliving amoebae, emphasizing the important distinguishing morphologic features. Acanthamoeba sp. and B. mandrillaris are closely related to one another phylogenetically, but are distant from N. fowleri and S. diploidea. While most B. mandrillaris trophozoites are uninucleate, binucleate forms may be seen, and the presence of multiple nucleoli may be useful in differentiating B. mandrillaris from Acanthamoeba sp. Although trophozoites are more numerous than cysts, the presence of cysts in brain tissue is useful in excluding N. fowleri, as only B. mandrillaris and Acanthamoeba sp. form cysts in the brain. However, the absence of cysts does not exclude a diagnosis of BAE, as cyst formation is not consistent, as the present case illustrates. When present, the cyst wall of B. mandrillaris is unique in that it is three-layered (i.e. composed of an outer ectocyst, inner endocyst, and intervening mesocyst) and lacks pores, whereas the cyst walls of Acanthamoeba sp. and N. fowleri are two-layered with pores.(3,4)

As mentioned by the contributor, differences in epidemiology and pathophysiology are also useful in differentiating the diseases caused by free-living amoebae. Acanthamoeba sp. generally only produces encephalitis in immunocompromised patients, whereas Balamuthia amebic encephalitis (BAE) is reported in both the immunocompetent and immunocompromised. By contrast, N. fowleri causes primary amebic meningoencephalitis (PAM) in immunocompetent children and young adults, classically within days of exposure to warm fresh waters. Primary amebic meningoencephalitis due to N. fowleri has also been reported naturally in bovids and a tapir (see WSC 2007-2008, Conference 22, case IV) and experimentally in a number of mammalian species. Of note, most humans with BAE present with characteristic skin lesions that predate central nervous system signs; such lesions are not characteristic of PAM produced by N. fowleri. Intriguingly, individuals of Hispanic origin are overrepresented among human BAE cases in the United States; unique environmental exposures and genetic predispositions have been proposed, but an explanation for this disparity has not been definitively proven. Among animals, BAE is most common in nonhuman primates, but has also been diagnosed in dogs, a sheep, and a horse.(3,4)

The major features that characterize and distinguish the pathogenic free-living amoebae are summarized in the following table, adapted from Schuster and Visvesvara:(3)
Pathogenic Free-living Amoebae
FeatureBalamuthia mandrillaris Acanthamoeba sp. Naegleria fowleri
DiseasesBalamuthia amebic encephalitis (BAE); cutaneous and sinus infectionsAmebic encephalitis; cutaneous and sinus infections*Primary amebic meningoencephalitis (PAM)
Risk FactorsImmunocompromised status (also occurs in immuno-competent); breaks in skin contaminated with soilImmunocompromised status Activity in warm fresh waters; diving; not associated with immuno-compromised status
Incubation periodWeeks to years Weeks to months Days
Trophozoite stage12-60 um 15-30 um 15-30 um
Flagellate stageNot found Not found Flagellate stage with 2 flagella
Cyst stage3-layered wall lacking pores; 10-30 um diameter; cysts form in brain tissue2-layered wall with pores; 10-15 um diameter; cysts form in brain tissue2-layered wall with pores; 7-15 um diameter; cysts do not form in brain tissue
*Acanthamoeba sp. also is associated with amebic keratitis in immunocompetent humans; risk factors include soft contact lens wear and wearing contact lenses while swimming; incubation period is days (versus weeks to months in systemic infections).


1. Rideout BA, Gardiner CH, Stalis IH, Zuba JR, Hadfield T, Visvesvara GS: Fatal Infections with Balamuthia mandrillaris (a free-living amoeba) in gorillas and other old world primates. Vet Pathol, 34:15-33, 1997
2. Schuster FL, Dunnebacke TH, Booton GC, Yagi S, Kohlmeier CK, Glaser C, Vugia D, Bakardjiev A, Azimi P, Maddux-Gonzalez M, Martinez AJ, Visvesvara GS: Environmental isolation of Balamuthia mandrillaris associated with a case of amebic encephalitis. Journal of Clin Microbiol 41:3175-3180, 2003
3. Schuster FL, Visvesvara GS: Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int J Parasitol 34:1001-1027, 2004
4. Siddiqui R, Khan NA: Balamuthia amoebic encephalitis: an emerging disease with fatal consequences. Microb Pathog 44:89-97, 2008
5. Visvesvara GS, Martinez AJ, Schuster FL, Leitch GJ, Wallace SV, Sawyer TK, Anderson M: Leptomyxid ameba, a new agent of amebic meningoencephalitis in humans and animals. J Clin Microbiol 28:2750-2756, 1990
6. Visvesvara GS, Moura H, Schuster FL: Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Microbiol 50:1-26, 2007

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