Etiology

Acanthamoeba is a genus of ubiquitous free-living amebas found in fresh and salt water, soil, dust, and sewage.1,22,22 The classification of the genus Acanthamoeba and separation from the closely related genus Hartmannella has had a confusing history that is becoming resolved only with the use of molecular techniques.^48,55 Hartmannella and Acanthamoeba are classified as distinct genera based on morphologic differences in the trophozoite shape and cyst wall structure, nutritional requirements, and serologic responses by infected hosts. Acanthamoeba has been further divided into three morphologic groups (I, II, and III) based on trophozoite and cyst size and structure. Although this morphologic classification is used, genetic comparisons have suggested the need for additional taxonomic reevaluation.⁴⁸ Fifteen genotypes (T1 to T15) of Acanthamoeba have been established based on differences in genetic sequences.⁵² Balamuthia mandrillaris is another free-living ameba that can cause fatal granulomatous amebic encephalitis. All the pathogenic ameba are known to harbor obligate intracellular prokaryotes including bacteria, chlamydiae, and viruses. The role of these endosymbionts in the production of disease or transport of these organisms is uncertain.

Epidemiology

Acanthamoeba organisms cause pneumonia and encephalitis in humans22,30,30 and animals^{2,12,22,23,38} and disseminated dermatitis and chronic keratitis in humans.^32,34 Although rare, rhinosinusitis, osteomyelitis, and disseminated infection have developed in human infections.

In dogs, epizootics of acanthamebiasis have been observed in greyhounds,22,23 whereas single cases have been described in two German shepherd dogs, one 6 months old and one 4 years old^2,7; in a 1-year-old Labrador-type dog¹⁴; and in an immunosuppressed Akita³⁸ and a 10-month-old boxer.²⁵ Young dogs appear to be most susceptible. Affected greyhounds ranged from 4 to 13 months of age. During outbreaks, organisms are thought to be acquired from a common environmental source rather than from other animals. The source of infection and the incubation period in dogs are unknown. Acanthamebiasis has not been described in cats.

Immunosuppression is likely involved in the development of infection. Although immunocompetency has not been evaluated in most infected dogs, T-cell function was determined to be abnormal based on lymphocyte blastogenesis in response to various mitogens, consistent with immunosuppression or T-cell mediated immunodeficiency, in one dog infected with A. castellanii.³⁸ However, most dogs with infection have received immunosuppressive therapy for suspected immune-mediated disease or neoplasia before the ultimate diagnosis of amebic infection.^15,25 Disseminated B. mandrillaris infection developed in a dog given immunosuppressive doses of glucocorticoids for suspected inflammatory bowel disease,¹⁶ and in another dog, infection may have disseminated after glucocorticoid and lomustine therapy for suspected CNS lymphoma.¹⁵ In these situations, initial resolution of signs has been accompanied by initial clinical improvement, with subsequent clinical deterioration as the infection becomes multisystemic. Concurrent acanthamebiasis and distemper have been confirmed by one of the authors of this chapter (EH) in the CNS of dogs from unpublished and previously published¹⁴ cases.²⁴ Distemper infection may result in immunosuppression facilitating Acanthamoeba proliferation, similar to what occurs in neosporosis (see Chapter 79). In any event, confusion may arise because the clinical signs of acanthamebiasis and distemper are similar (see Clinical Signs).

Immunosuppressed humans, such as those who have had organ transplants, have an acquired immunodeficiency syndrome, abuse alcohol, have diabetes, or have other debilitating conditions, are at high risk for infection and particularly susceptible to acanthamebiasis.30,57 In humans who are critically ill or debilitated, Acanthamoeba species typically cause a chronic granulomatous meningoencephalitis that may last for weeks or months before causing death.³⁰ Amebic keratitis has been seen in otherwise healthy humans who wear contact lenses or have minor corneal trauma.^32,34 Using tap water to make saline solutions for contact lenses has been suggested as a source of infection.²⁷ Acanthamoeba strains from humans with keratitis have a unique protein expressed on their cell surface that adheres to corneal epithelial surface glycoproteins.^10,17

Pathogenesis

The pathogenesis in dogs is unknown; however, in humans, routes of infection may include inhaling organisms from water while swimming or from contaminated air. The organisms can replicate in the upper or lower respiratory tract after inhalation or in skin or other tissues after penetrating injuries and then spread to the CNS by hematogenous means. Alternatively, infection of the cornea and nasal passage might lead to a spread to the nervous system via optic and olfactory nerves, respectively. In one dog with acanthamebiasis, distribution of the brain lesions suggested penetration of the cribriform plate.⁷ Experimentally, Balamuthia has been shown to infect the brain via the olfactory nerve in immunodeficient mice,²⁶ and this may be a likely route for natural infection.

Clinical Findings

Clinical manifestations of canine acanthamebiasis and canine distemper are remarkably similar.14,23,23 Initial signs include mild oculonasal discharge, anorexia, and lethargy. Rectal temperature varies from normal to as high as 40.5° C (105° F). Respiratory distress and neurologic signs then develop. Most dogs eventually develop neurologic dysfunction. Neurologic signs include incoordination, head tilt, stumbling, dysmetria, and seizures. Coughing and dyspnea may be observed after a few days. Severely affected dogs are tetraplegic and in lateral recumbency without the ability to right themselves. Less severely affected greyhounds have permanent disabilities that preclude performance racing. In one dog, progressive neurologic disease was caused by the meningoencephalitis; however, the brain damage also induced a syndrome of inappropriate antidiuretic hormone secretion.⁷ This syndrome was associated with impaired water excretion and normal sodium excretion leading to peripheral edema, serum hypoosmolality, and an inappropriately high serum sodium concentration.

One dog with disseminated balamuthiasis developed lethargy and hematuria followed by neurologic dysfunction, including intermittent seizures, rotary nystagmus, recumbency, and coma.¹⁶ A second dog had an 8-month history of generalized seizures that had been controlled with phenobarbital.¹⁵ Two months before referral, it developed hearing loss, head tilt, and circling that temporarily resolved with glucocorticoid and lomustine therapy before the dog was reevaluated with respiratory distress and hypotension. The owners requested euthanasia. Therapy had been instituted based on a clinical suspicion of lymphoma due to lymphocytic pleocytosis and atypical lymphocytes in the cerebrospinal fluid (CSF), but there was no evidence of lymphoma at necropsy.¹⁵ A third dog with CNS balamuthiasis had a sudden onset of central vestibular disease that was non responsive to glucocorticoid or antibacterial therapy and the dog was euthanized.^23a

Cats that harbor Hartmannella species on their ocular surface may be more likely to have clinical manifestations of keratitis or conjunctivitis such as photophobia, lacrimation, conjunctival hyperemia, and ocular discharge. However, a direct causal association between this ameba and ocular surface inflammation has not been proven as with Acanthamoeba. Neochlamydia co-infection may be important in the development of clinical illness (see Etiology, earlier).

Diagnosis

The clinical laboratory abnormalities in animals with systemic acanthamebiasis are nonspecific. Leukopenia in greyhounds is a result of marked lymphopenia²³ and in other breeds is caused by a reduction in all types of blood leukocytes.^2,38 The cause of the leukopenia is unknown but may be concurrent infectious disease, stress, or specific factors produced by the Acanthamoeba organisms. No laboratory findings are available for the two dogs that developed B. mandrillaris infection, except for CSF cytologic analysis in one dog.¹⁵ In that dog, a lymphocytic pleocytosis was found and initially misinterpreted as CNS lymphoma, which resulted in administration of glucocorticoids and lomustine. Lymphocytic CSF pleocytosis is the common cytologic finding in human amebic encephalitis.¹⁹ In another dog with acanthamebiasis, a mixed CSF pleocytosis consisting of approximately 30% neutrophils and 70% mononuclear cells was present.⁷ A dog with Balmuthia-induced meningoencephalitis, the CSF pleocytosis (234 cells/µL) consisted of 60% nondegenerate neutrophils and 40% mononuclear cells.^23a Using contrast enhancement, a computerized tomographic finding was a focal ring-enhancing lesion in the cerebellar parenchyma of this dog. In a 10-month-old boxer dog, neutrophilic CSF pleocytosis was present in CSF; the dog was treated with immunosuppressive doses of glucocorticoids and azathioprine for presumptive glucocorticoid-responsive meningitis, before the dog’s deterioration due to respiratory failure and final diagnosis of acanthamebic meningoencephalitis.²⁴

Identification of amebae in animals with keratitis is best accomplished using corneal scrapings or biopsy. Swabs are not suitable. The specimen should be inoculated as soon as possible onto specialized medium (Page’s agar, a recipe for which can be found at www.ccap.ac.uk/media/documents/NN.pdf). Specimens can be transported to the laboratory in a sterile vial containing a few drops of sterile saline. The organisms can be visualized in the specimen by microscopy, although trophozoites do not always stain predictably. Acanthamoeba and Hartmannella species have spikelike (acanth) projections, which help differentiate them from the genus Naegleria, Balamuthia, or other amebae.

Premortem diagnosis of amebic encephalitis in dogs or people has been rare. Prior or recent exposure or immersion in environmental water sources may increase the level of suspicion. Focal lesions are observed on neuroimaging. Culture or biopsy of affected tissues would be the most specific means of confirmation. Although lung and CNS tissues are not readily accessible, the possibility of finding organisms in CSF and specimens from tracheal or bronchoalveolar washings has not been evaluated. In one dog, a space-occupying cerebral granuloma was observed.^39a In humans, acanthamebic encephalitis is typically not associated with meningeal involvement, and consequently organisms are rarely identified in CSF.⁴¹

Amebas can be cultured from lesions by special methods that are not done in most laboratories⁵¹ because biocontainment facilities are needed.⁴ Organisms grow sparingly on potato dextrose agar, where amebas grow as individual colonies with many cysts, or on nonnutrient agar seeded with bacteria, where they grow more rapidly as a confluent expanding ringlike mass of trophozoites with cysts at the central portion. Organisms can also be isolated in cell culture, where they cause cytopathic effects. Mouse inoculation can be used to test the pathogenicity of isolated strains. Because these amebae are difficult to distinguish morphologically, genetic methods offer the most specific and reliable means of determining which species is involved in a particular infection.^29,39 Polymerase chain reaction (PCR) techniques have been used frequently on isolates in culture. PCR analysis of corneal epithelial and tear samples has been used in the premortem diagnosis of Acanthamoeba keratitis.²⁸ Similar genetic evaluation of blood samples, CSF, or brain biopsy specimens for premortem diagnosis of systemic infections has not been as commonly done in human or animal infections. Unfortunately, many of these infections have been fatal before the organisms can be successfully cultivated.