Signalment:  

Holstein heifer, unknown age, bovineBetween July 30 and September 28, 1998 and 1999, specimens from nine 10-20-month-old Holstein heifers with a history of acute CNS disease were submitted to our laboratory. All submissions were from one large heifer-raising operation located in southern California in an arid but highly productive agricultural area irrigated by Colorado River water supplied through a system of canals and ditches. The ranch relied on untreated canal water for livestock drinking water. These heifers had been diagnosed with fatal meningoencephalitis caused by Naegleria fowleri. Sporadic specimens from animals with similar clinical signs have been submitted in the successive years, including the formalin-fixed brain we are now examining.

Gross Description:  

No gross lesions.

Histopathologic Description:

Multifocal necrosuppurative hemorrhagic encephalitis, predominating within neuropil bordering the cerebral aqueduct, includes foci of malacia or necrosis, populated by degenerative neutrophils, hemorrhage, interspersed with neuropil populated by scattered lymphocytes and macrophages, and rare multinucleated giant cells. Numerous blood vessels are mildly (1-2 cell thickness) to markedly (9-10 cells thick) cuffed by lymphohistiocytic cells. Clustered and individual amoebic trophozoites (Fig. 4-2) are observed within and surrounding areas of necrosis/malacia and perivascularly. The amoebae are round to oval, approximately 5-10 μm in diameter, and have a pale eosinophilic, finely granular cytoplasm. The nuclei are small (approximately 2-3 μm in diameter), poorly delineated, mostly eccentric and weakly basophilic with a prominent basophilic karyosome.

Morphologic Diagnosis:  

Encephalitis, hemorrhagic, multifocal, necrosuppurative, severe, with presence of multifocal amoebic trophozoites.

Lab Results:  

Immunohistochemistry: Positive for Naegleria fowleri (Fig. 4-1). 

Condition:  

Naegleria fowleri

Contributor Comment:  

Primary amoebic meningoencephalitis is a fulminant infection of the human central nervous system caused by Naegleria fowleri, a free-living amoeba that thrives in artificially or naturally heated water. The infection usually is acquired while bathing or swimming in such waters. There are 4 genera of amoebae that cause CNS disease in mammals, namely Acanthamoeba (several species), Naegleria fowleri, Balamuthia mandrillaris, and the recently described Sappinia diploidea.⁹ Acanthamoeba and Naegleria are ubiquitous in soil and fresh water, including lakes, streams, and hot springs. These amoebae have also been isolated from various artificial water sources, such as swimming pools, tap water, heating and ventilation units, air conditioners, cooling water, sewage, contaminated cell cultures, and contact lens-storing fluid.^1,9 Their cysts have even been demonstrated in dust during dust storms.¹

Primary amoebic meningoencephalitis (PAM) is the term for the human disease caused by N. fowleri. It is an acute, usually fatal, necrotizing, and hemorrhagic meningoencephalitis.⁹ Although CNS infections due to Acanthamoeba and Balamuthia have been recorded in animals, such as dogs, sheep, cattle, primates, and horses, there has been only 1 report of naturally acquired PAM in animals, namely in a South American tapir at a zoo in Arizona.^1-3,7,8,10 Primary amoebic meningoencephalitis has been experimentally induced in mice, sheep, and monkeys.^1,10 Naegleria fowleri is thermophilic and tolerates temperatures of up to 45° C; hence, the frequent association of PAM with a history of contact with naturally warm or artificially heated waters.¹ The portal of entry is the olfactory mucosa. The parasite migrates to the brain via olfactory nerves. Incubation period is short, with onset of clinical signs several days following exposure. The disease rapidly progresses and usually culminates in death within 5-7 days.⁵

The life cycle of N. fowleri includes a trophozoite stage (amoebic form), a temporary flagellate stage, and, in unfavorable environments, a cyst stage. The cyst stage is susceptible to desiccation.⁹ Only the trophozoite stage of N. fowleri has been detected in CNS lesions. This is in contrast with other free-living opportunistic amoebae, where identification of cyst stages aids in their differentiation from Naegleria.⁵

Gross lesions vary and may or may not be present. Findings may include multifocal meningeal hemorrhages and tan-gray thickening of meninges, tan-gray deposits obscuring cerebellar cortical surface, and light brown and malacic brain parenchyma. Lesions may be bilateral and symmetrical.

Histopathology shows mainly a multifocal, necrosuppurative, and hemorrhagic meningoencephalitis. Most severely affected areas were the anterior cerebra, olfactory bulbs, and cerebella. Amoebae are not easily detected microscopically because they resemble degenerate macrophages and may be few in number. The tendency of the amoebae to accumulate in aggregates or in perivascular spaces within areas of necrosis is most helpful, as is the small, weakly basophilic karyosome within a poorly delineated, small nucleus. There are very few or no multinucleated giant cells or eosinophils to direct the pathologist toward parasitic etiology. Some amoebae form cysts, visualized by periodic acid-Schiff stain, but N. fowleri does not, possibly because death occurs during the acute stage of the disease. Routine special stains to identify bacteria, such as Giemsa, Brown-Brenn-modified Gram, and Steiner silver stain were not helpful in identifying amoeba. Immunohistochemistry of the brain is an important tool in the confirmation of the diagnosis and also the isolation of the organism from animal tissues and suspected sources can be attempted. Finally, it is advantageous to know that the disease is seasonal, occurs in areas with high ambient summer temperatures, and may be associated with the use of consumption of untreated surface water.¹

JPC Diagnosis:  

Brainstem: Encephalitis, necrotizing, subacute, multifocal, moderate, with hemorrhage and amoebic trophozoites, Holstein (Bos taurus), bovine.

Conference Comment:  

The contributor gives an excellent overview of Naegleria fowleri infection. The full pathogenesis and virulence factors of the organism are not completely understood. These infections are believed to be transmitted when water containing the organisms comes in contact with the host nasal mucosa. The amoebic trophozoites then migrate along the olfactory nerves and pass through the cribiform plate to enter the brain.^4,9 N. fowleri contains a surface protein, similar to the human integrin-like receptor, that facilitates adhesion to fibronectin, an extracellular matrix glycoprotein found at the basal lamina and surrounding cells.⁴ Two pore-forming proteins termed naegleriapores A and B, as well as proteases and phospholipases have all been implicated in host cell destruction.^4,9

Most animals infected with N. fowleri die before mounting a detectable adaptive immune response. The innate immune response, consisting of complement, neutrophils, and macrophages, appears to be a frequently inadequate defense mechanism. In vitro studies have shown that N. fowleri is able to evade complement damage by either expressing complement-regulatory proteins (CD59-like protein), shedding the MAC complex (C5b-C9) on surface membrane blebs, internalizing and degrading the MAC complex, or preventing the insertion of the MAC into the amoebic membrane.⁴ Although neutrophils play a major role in antiamoebic activity, their exact function is not clearly defined. TNF-_ does not have a direct effect on N. fowleri, but it appears that organism destruction by neutrophils cannot occur without TNF-_ being present.⁴ Production of high levels of proinflammatory cytokines such as IL-1_, IL-1β, IL-6, and TNF-_ do not appear to provide protection against infection.⁴ The roles of humoral and cell mediated immunity are also uncertain.

References:

1. Daft MD, Visvesvara GS, Read DH, Kinde H, Uzal FA, Manzer MD. Seasonal meningoencephalitis in Holstein cattle caused by Naegleria fowleri. J Vet Diag Invest 17:605-609, 2005
2. Fuentealba, IC, Wikse SE, Read WK, Edwards JF, Visvesvara GS: Amebic meningoencephalitis in a sheep. J Am Vet Med Assoc 200:363-365, 1992
3. Kinde H, Visvesvara GS, Barr BC, Nordhausen RW, Chiu PHW: Amebic meningoencephalitis caused by Balamuthia mandrillaris (leptomyxid ameba) in a horse. J Vet Diag Invest 10:378-381, 1998
4. Marciano-Cabral F, Cabral GA: The immune response to Naegleria fowleri amebae and pathogenesis of infection. FEMS Immunol Med Microbiol 51:243-259, 2007
5. Martinez JA, Visvesvara GS: Free-living, amphizoic and opportunistic amebas. Brain Pathol 7:583-598, 1997
6. Morales JA, Chaves AJ, Visvesvara GS, Dubey JP: Naegleria fowleri-associated encephalitis in a cow from Costa Rica. Vet Parasitol 139:221-223, 2006
7. Pearce JR, Powell HS, Chandler FW, Visvesvara GS: Amebic meningoencephalitis caused by Acanthamoeba castellani in a dog. J Am Vet Med Assoc 187:951-952, 1985
8. Rideout BA, Gardiner CH, Stalis IH, Zuba JR, Hadfield T, Visvesvara GS: Fatal infections with Balamuthia mandrillaris (a free-living amoeba) in forillas and other old world primates. Vet Path 34:15-22, 1997
9. 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
10. Wong MM, Karr SL, Balamuth WB: Experimental infections with pathogenic free-living amebae in laboratory primate hosts: I. (B) A study on susceptibility to Acanthamoeba culbertsoni. J Parasitol 61:682-690, 1995

A virtual slide is not available for this case.

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(Fig 4-1) Cerebrum, bovine

(Fig 4-2) Cerebrum

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