Veterinary Parasitology 126 (2004) 91–120
www.elsevier.com/locate/vetpar
Review
Amebae and ciliated protozoa as causal agents of
waterborne zoonotic disease
Frederick L. Schustera,*, Govinda S. Visvesvarab
a
California Department of Health Services, Viral and Rickettsial Disease Laboratory,
850 Marina Bay Parkway, Richmond, CA 94804, USA
b
Centers for Disease Control and Prevention, Division of Parasitic Diseases,
4770 Buford Highway, NE, Atlanta, GA, USA
Abstract
The roles free-living amebae and the parasitic protozoa Entamoeba histolytica and Balantidium
coli play as agents of waterborne zoonotic diseases are examined. The free-living soil and water
amebae Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris are recognized
etiologic agents of mostly fatal amebic encephalitides in humans and other animals, with immunocompromised and immunocompetent hosts among the victims. Acanthamoeba spp. are also agents
of amebic keratitis. Infection is through the respiratory tract, breaks in the skin, or by uptake of water
into the nostrils, with spread to the central nervous system. E. histolytica and B. coli are parasitic
protozoa that cause amebic dysentery and balantidiasis, respectively. Both intestinal infections are
spread via a fecal–oral route, with cysts as the infective stage. Although the amebic encephalitides
can be acquired by contact with water, they are not, strictly speaking, waterborne diseases and are not
transmitted to humans from animals. Non-human primates and swine are reservoirs for E. histolytica
and B. coli, and the diseases they cause are acquired from cysts, usually in sewage-contaminated
water. Amebic dysentery and balantidiasis are examples of zoonotic waterborne infections, though
human-to-human transmission can occur. The epidemiology of the diseases is examined, as are
diagnostic procedures, anti-microbial interventions, and the influence of globalization, climate
change, and technological advances on their spread.
Published by Elsevier B.V.
Keywords: Free-living amebae; Amebic encephalitis; Amebic keratitis; Amebic dysentery; Balantidiasis;
Acanthamoeba; Balamuthia; Naegleria fowleri; Entamoeba histolytica; Balantidium coli
* Corresponding author. Tel.: +1 510 307 8651; fax: +1 510 307 8599.
E-mail address: [email protected] (F.L. Schuster).
0304-4017/$ – see front matter. Published by Elsevier B.V.
doi:10.1016/j.vetpar.2004.09.019
92
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. Current status of taxonomy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
92
93
2. Life cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1. Environmental persistence and infectivity . . . . . . . . . . . . . . . . . . . . . . . . . .
94
96
3. Infection and disease . . . . . . . . . . . . . . . . . . .
3.1. Central nervous system . . . . . . . . . . . . .
3.2. Corneal surface . . . . . . . . . . . . . . . . . . .
3.3. Nasopharyngeal and cutaneous infections .
3.4. Intestinal infections . . . . . . . . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
96
98
99
99
99
4. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Free-living amebae . . . . . . . . . . . . . . . . . . . .
4.2. Entamoeba histolytica and commensal amebae .
4.3. Balantidium coli . . . . . . . . . . . . . . . . . . . . . .
4.4. Developed and developing regions. . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
100
100
104
105
106
5. Diagnostic techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Recovery and identification of free-living amebae . . . . . . . . . . . . . . . . . . . .
5.2. Recovery and identification of Entamoeba histolytica and Balantidium coli . .
108
108
108
6. Control and management . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. The environment and free-living amebae . . . . . . . . . . . . . .
6.2. Transmission of Entamoeba histolytica and Balantidium coli
6.3. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
110
110
111
111
7. Future trends . . . . . . . . .
7.1. Globalization . . . .
7.2. Climate change . . .
7.3. Modern technology
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
112
112
114
115
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
116
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
116
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
117
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1. Introduction
The organisms dealt with in this section run the gamut from free-living to parasitic.
Among the free-living representatives are the amebae Acanthamoeba, Naegleria, and
Balamuthia. The parasitic forms include Entamoeba histolytica and Balantidium coli. The
free-living amebae have been described as facultative parasites, opportunistic pathogens,
and amphizoic amebae (Page, 1974), the latter term implying ability to live within and
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
93
outside of a host. Their role as pathogens was recognized since the 1960’s, while
Entamoeba (1870) and Balantidium (1860) have been known for more than a century
as etiologic agents of human disease. Because of their medical and economic impact on
human populations, E. histolytica, and to a lesser extent B. coli, have been the subjects of
intensive studies on transmission, possible zoonotic associations, and anti-microbial
therapy.
A parasite is an organism that lives at the expense of and causes damage to its host,
and which cannot survive for long in the absence of the host. Technically, the free-living
amebae are not parasites, although often described as such in the literature. Nevertheless,
they have made a niche for themselves in the field of parasitology, acquiring some
degree of notoriety while doing so. This has come about because the diseases caused
by these amebae—particularly the encephalitides—are mostly fatal, difficult to
diagnose premortem, and lack a well-defined optimal anti-microbial therapy. Several
factors over the past two decades have resulted in increased interest in these opportunistic
amebae: (1) the HIV/AIDS epidemic that led to increased numbers of cases of
Acanthamoeba and Balamuthia encephalitides; (2) the popularity of soft contact lenses that
became the vehicle for Acanthamoeba to reach the corneal surface to cause keratitis; and
(3) increased leisure time and affluence allowing vacationing in areas where there are warm
lakes and hot springs, exposing people to Naegleria fowleri, the cause of meningoencephalitis.
1.1. Current status of taxonomy
Taxonomy of the Protozoa has long been based upon morphologic criteria. In recent
years, morphology has taken a back seat to the use of molecular techniques as the moving
force in taxonomy, often with the resultant abandonment of comfortable assumptions about
relationships, and the recognition of new species and even genera based on sequencing the
16S and 18S rDNA genes. Once members of a single phylum, the major protozoan types
are now placed in their own different phyletic categories. The amebae dealt with in this
report represent a polyphyletic assemblage, encompassing a number of different
taxonomic Orders. We follow, in part, Page’s taxonomic scheme for the amebae (Page,
1988).
Phylum Rhizopoda
Class Heterolobosea
Order Schizopyrenida
Family Vahlkampfiidae
Naegleria fowleri
Class Lobosea
Order Acanthopodida
Family Acanthamoebidae
Acanthamoeba spp., Balamuthia mandrillaris
Of uncertain ordinal relationship
Family Entamoebidae
Entamoeba histolytica, E. dispar, E. coli, E. hartmanni, E. moshkovskii
94
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
(Note: B. mandrillaris, originally described as a leptomyxid ameba, is included in the
Family Acanthamoebidae, based on data from 16S rRNA gene sequencing (Amaral Zettler
et al., 2000; Booton et al., 2003)).
The one ciliate dealt with in this paper is the only one parasitizing humans. The
taxonomic scheme presented here is from Lynn and Small (2000).
Phylum Ciliophora
Class Litostomatea
Order Vestibuliferida
Family Balantidiidae
Balantidium coli
2. Life cycles
The general pattern of life cycles for the protozoa covered in this section is a trophic or
feeding stage, alternating with a resting, resistant cyst stage. There are no intermediate
hosts in the life cycles and, with the exception of B. coli, sexual reproduction is not known
to occur. None of these organisms requires vectors for transmission, an exception being E.
histolytica which can be carried by some insects as mechanical vectors.
Naegleria and Acanthamoeba, as free-living trophic amebae, present in soil or water,
feed primarily on bacteria. Balamuthia appears to feed on other Protozoa, probably other
amebae, found in the soil. Naegleria, in addition to the ameboid and cystic stages, has a
flagellated stage that arises from the trophic ameboid stage. The flagellate typically neither
feeds nor divides and is transitory, ultimately reverting to the ameboid stage. However,
species of Naegleria have been identified on the basis of sequencing data in which the
ability to flagellate has been lost, or in which the flagellate undergoes division (De
Jonckheere, 2002). The cysts of these amebae, which develop as growth conditions become
unfavorable (lack of food, waste product accumulation, desiccation), survive in nature until
growth conditions improve. The ameba cysts are enclosed by a wall that may be two or
three layers thick, and may or may not have pores through which the excysting ameba can
exit. They are all obligate aerobes but some can tolerate anaerobic conditions for short time
intervals. All have been grown in culture, either in the presence of bacteria as a food source
(xenically, from xeno-, G., stranger, or in this case, presence of another organism), or in the
absence of any other organisms (axenically) in a variety of enriched nutrient media
(Schuster, 2002). The trophic and cystic forms of Acanthamoeba are compared in Figs. 1
and 2, and the ameboid and flagellated stages of Naegleria are seen in Figs. 3 and 4.
The parasites E. histolytica and B. coli, similarly have trophic and cystic stages. The cyst
stage for both organisms is the transmissive stage in the life-cycle. In E. histolytica, a
precyst stage is recognized as intermediate between the trophozoite and the mature cyst.
The latter is quadrinucleate, with rod-like ribonucleoprotein elements called chromatoid
bodies and glycogen reserves, both of which disappear as the cyst ages. The cyst
germinates in the host’s small intestine to give rise to a quadrinucleate ameba that, by
nuclear and cytoplasmic divisions, produces eight small amebae that eventually localize in
the colon and caecum. Cysts are essential for transmission since the trophozoite cannot
survive passage through the stomach with its low pH. Cysts of E. histolytica are not as
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
95
Figs. 1–4. Photomicrographs of stained or unstained wet-mounts of amebae. (1) Acanthamoeba trophozoites seen
in an unstained wet-mount preparation with differential interference contrast optics. Note the characteristic
projecting pseudopods, called acanthapodia, over the ameba surface (arrow). Contractile vacuoles (V) and nuclei
(N) can be seen in two of the amebae. Bar represents 10 mm. (2) Unstained cysts of a large species of
Acanthamoeba (A. comandoni) are seen in a wet-mount preparation with differential interference contrast optics.
The cyst wall is made up of two layers, endocyst and ectocyst. The stellate form of the endocyst (I) is evident, as is
the outer wall or ectocyst (O) enclosing the entire structure. Pores (ostioles) are located at the junctures of the two
walls (arrow). Bar represents 10 mm. (3) Naegleria fowleri amebae stained with trichrome. The nucleus and its
dark staining nucleolus are clearly seen (N). Bar represents 10 mm. (4) The flagellate stage of N. fowleri is seen in
this stained wet-mount preparation. The shape of the organism is ovoid, with two (sometimes four) flagella seen at
the anterior end of the organism. The nucleus is at the anterior end of the flagellate, in close association with the
flagellar apparatus. Bar represents 5 mm.
highly resistant to harsh environmental stresses as those of free-living amebae, but
comparative studies of survival have not been performed. E. histolyctica is an anaerobe but
can tolerate trace amounts of oxygen for short time periods, perhaps through association
with intestinal bacteria that scavenge oxygen (Ravdin, 1986). As with other anaerobic
protozoa, mitochondria are lacking although the amebae retain a mitochondrial vestige, the
mitosome, as well as genes of mitochondrial origin (Bakatselou et al., 2003). The ameba is
cultured both xenically and axenically in a variety of enriched media (Diamond and Clark,
2002).
Balantidium spp. are known only as commensals or parasites found in the intestinal tract
of various animal species; none is free-living. The trophic B. coli is covered with uniformly
arranged rows of cilia propelling it through the viscous mass of undigested food and
bacteria passing through the colon (Fig. 5). The name of the organism derives from its
pouch-like shape (balanti-, G., bag or sac). The organism has a cytostome through which
debris, bacteria and other particulate material are ingested and pass into food vacuoles. As
with other ciliates, there is a micro- and macronucleus, and two contractile vacuoles that
96
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
Figs. 5–6. Light microgrpahs of Balantidium coli from human stool. (5) Trophozoite with fringe of cilia evident
on its surface. Internal details are obscured by the density of the ciliate contents, although a contractile vacuole can
be seen at posterior end of the organism, and the outline of the macronucleus is also evident. Bar represents 10 mm.
(6) Cyst surrounded by a wall. The outline of the macronucleus can be seen in the cytoplasm. Bar represents
10 mm.
serve as osmoregulatory organelles. Conjugation has been observed in cultures (Zaman,
1978). The organism is the largest among protozoan parasites or commensals found in
humans, measuring up to 150 mm in length. Since the trophic ciliate cannot survive passage
through the stomach, the cyst with its protective wall is the infective stage (Fig. 6).
Encystment of trophic ciliates in the intestine is initiated during departure from the host’s
colon. Even though the ciliate is found in the same anaerobic environment as E. histolytica,
it is an aerobe. The organism can be grown in vitro in xenic cultures, but has not been grown
axenically (Diamond and Clark, 2002).
2.1. Environmental persistence and infectivity
The trophic protozoon, whether it is the ciliate, or ameba, is poorly equipped to survive
environmental conditions for prolonged periods of time. This is particularly true of
Entamoeba and Balantidium, in which the infective stage is the cyst. For the free-living
amebae, the infective stage can be either the trophic or cystic stages. Because of its wall and
the dormancy of the enclosed organism, the cyst allows survival during periods unfavorable
for growth. Under laboratory conditions, Acanthamoeba cysts have remained viable 20
years (Mazur et al., 1995). Accurate data on longevity of cysts in nature is lacking, but cyst
viability depends to a great extent on environmental conditions. Cysts of the free-living
amebae are better suited to survive prolonged desiccation and other environmental stresses,
while the cysts of Entamoeba and Balantidium are more fragile and temperature sensitive,
and remain viable only in a moist environment for limited time periods (see below).
3. Infection and disease
A summary of the disease-related properties of the organisms can be found in Table 1.
Parasite
Disease
Major site(s) in host
Portal of entry
Source of infection
Acanthamoeba spp.
Acanthamoeba encephalitis
(acanthamebiasis)
Acanthamoeba keratitis
Brain, skin, nasopharynx, lungs
Breaks in skin;
respiratory tract
Corneal surface
Soil, water
Breaks in skin;
respiratory tract
Nasal passages
Soil, watera
Balamuthia
mandrillaris
Naegleria fowleri
Entamoeba histolytica
Balamuthia encephalitis
(balamuthiasis)
Naegleria meningoencephalitis
(naegleriasis)
Amebic dysentery (amebiasis)
Balantidium coli
Dysentery (balantidiasis)
a
Potential for transmission but not reported.
Cornea
Brain, skin, nasopharynx, lungs
CNS
Colon, liver, lung, brain,
cutaneous lesions
Colon
Oral route
Oral route
Recreational waters (lakes,
pools, hot springs, etc.)
Feces-contaminated
water or food
Feces-contaminated
water or food
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
Table 1
Diseases caused by free-living amebae, Entamoeba histolytica, and Balantidium coli: sources of infection, portals of entry, and affected organs
97
98
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
3.1. Central nervous system
Prominent among the infections of the brain and spinal cord are the encephalitides
caused by Acanthamoeba spp. and N. fowleri. The disease caused by Naegleria is termed
primary amebic meningoencephalitis (PAM) and is acquired during swimming or other
water-related activities (Fig. 7). Naegleria as trophic amebae (or cysts) are carried into the
nostrils along with water and migrate from the nasal epithelial surface along the olfactory
nerves across the cribriform plate to the brain (Martinez et al., 1973). Once there, they
attack the olfactory and frontal lobes, as well as the base of the brain, the brainstem, and
cerebellum, and are found in a purulent exudate in the subarachnoid space and in the
cerebrospinal fluid (CSF). PAM is a fulminating infection. Onset occurs 1–2 days after
exposure to the amebas, and death occurs in 7–10 days following infection.
Acanthamoeba (Figs. 8 and 9) and Balamuthia (Fig. 10) are responsible for
granulomatous amebic encephalitis (GAE), usually without meningeal involvement
(Martinez, 1985; Martinez and Visvesvara, 1997, 2001). The name reflects the host’s
Figs. 7–10. Sections of human brain infected with amebae, as seen by light microscopy. (7) Concentrations of
Naegleria fowleri seen in a tangential section of the perivascular space. The wall of the blood vessel is not seen in
the section (cf. Fig. 8). Note the typical vesicular nucleus (N) of the trophic amebae (H&E stain). Bar represents
40 mm. (8) Acanthamoeba castellanii stained with H&E. Large numbers of trophic amebae are seen in the
perivascular region. The adjacent blood vessel (B) can be seen. The patient had a kidney transplant and was treated
with steroids. Bar represents 35 mm. (9) Trichrome stain of section of blood vessel wall showing large numbers of
encysted A. castellanii (C), identified by the enclosing cyst wall. Unlike Naegleria, Acanthamoeba encysts in host
tissue. Bar represents 35 mm. (10) Balamuthia mandrillaris trophic amebae (A) in an H&E-stained tissue section
from an experimentally infected immunocopromised mouse inoculated intranasally with about 1000 trophic
amebae and cysts. Note the large size of the amebae, as compared to Naegleria and Acanthamoeba. Bar represents
70 mm.
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
99
granulomatous response to the presence of the amebae. Portals of entry for both organisms
are either through breaks in the skin that become contaminated with soil, or as cysts taken
into the respiratory tract as dust or airborne soil particles. No association between
recreational water activity and infection has been reported, but this is a possible route of
infection. From the point of entry, the amebae spread hematogenously to the central
nervous system (CNS) where they cause GAE. Areas of the brain affected include the
cerebrum, cerebellum, and brainstem, but amebae are generally not found in the CSF.
Cutaneous or nasopharyngeal lesions are also produced, depending on the portal of entry
(see below). GAE is typically a chronic, insidious infection that can take weeks to years
after infection before becoming clinically apparent. E. histolytica is also found, though
rarely, in brain abscesses following hematogenous dissemination from other sites in the
body (Albach and Booden, 1978).
3.2. Corneal surface
Acanthamoeba spp. are also the causal agents of amebic keratitis, occurring as the result
of corneal trauma or, more often, improper care of contact lenses. Amebae present in water
can either directly infect the cornea, or can be carried to the cornea from the contact lens
storage case on the lens surface. In contact lens wearers, the source of amebae is typically
non-sterile tap or distilled water used in preparation of homemade saline solutions for lens
care. In corneal trauma, amebae are inoculated directly to the corneal surface by injury
with, to cite two actual cases, a stalk of hay or a cinder blown into the eye (Jones et al.,
1975; Ma et al., 1981). Usually only one eye is affected. The disease is characterized by a
ring-shaped stromal infiltrate, corneal ulceration, photophobia and pain, and is often
mistaken for viral keratitis, which delays effective treatment. Once established in the
corneal stroma, the amebae are difficult to eradicate. Apparent recovery from the disease
through anti-microbial treatment can be followed by recurrence when amebae encysted in
the stroma reactivate following therapy. Keratitis victims may require one or more corneal
transplants to repair damage or to reduce the parasite load in the eye. In a worst-case
scenario, enucleation has been necessary to provide relief, although the availability of
effective anti-microbials has made this unlikely.
3.3. Nasopharyngeal and cutaneous infections
These are due to Acanthamoeba and Balamuthia and develop when cysts or trophic
amebae are introduced into breaks in the skin or into the nasal passages. These infections
can remain localized, but usually amebae disseminate to other locations in the body,
particularly the CNS, and there is no sharp demarcation between these infections and
encephalitis.
3.4. Intestinal infections
Intestinal infections are generally spread by the classic fecal–oral route. Infection
results from ingestion of cysts in water or food; trophic organisms cannot survive passage
through the stomach, except in cases of low stomach acidity.
100
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
E. histolytica is responsible for amebic dysentery (Albach and Booden, 1978; Haque et
al., 2003; Ravdin, 1986). Once reaching the colon, the invasive amebae attack the intestinal
epithelial surface, causing flask-shaped ulcerations in the wall and producing a blood- and
mucus-flecked loose stool. In severe amebiasis cases, the intestinal wall can perforate
releasing amebae into the peritoneal cavity. Amebae can also disseminate from the
intestinal tract via the hepatic portal system to form discrete abscesses in the liver, lungs, or
brain. Cutaneous amebiasis, though rare, has been reported, often presenting as lesions in
the anogenital area, and related to passage of amebae from the intestinal tract (MagañaGarcı́a and Arista-Viveros, 1993). Primary cutaneous amebiasis has also been reported, in
which lesions form in regions not contiguous with the intestinal tract (Parshad et al., 2002).
A commensal variant of E. histolytica, now recognized as a distinct species E. dispar, is
found in the gut but is non-invasive (Diamond and Clark, 1993), and may be the basis for
large numbers of asymptomatic infections in humans.
In humans, E. histolytica is the sole parasitic ameba of the gut. Other amebae found
in the intestinal tract, which are present as commensals, are E. coli, E. hartmanni,
E. moshkovskii, Endolimax nana, Iodamoeba butschlii and, as already noted, E. dispar.
Dientamoeba fragilis, once regarded as an ameba, is now known to be a trichomonad
flagellate which, while retaining intracellular vestiges of a flagellar apparatus, lacks basal
bodies and flagella (Brugerolle and Lee, 2000).
B. coli is associated with infections in humans and in pigs, the reservoir for human
balantidiasis. In the colon, the organisms produce ulcerations resembling those produced
by Entamoeba, which can become secondarily infected by intestinal bacteria, or vice versa.
The ciliates are often found in clusters or ‘‘nests’’ in the ulcers or the mucosal surface
(Levine, 1961). In addition to the colon, spread can occur, though rarely, to the peritoneal
cavity (Zaman, 1978).
4. Epidemiology
4.1. Free-living amebae
The number of infections caused by free-living amebae is relatively small, given their
ubiquitous global distribution (Table 2). Reported cases number in the hundreds or, for
amebic keratitis, in the low thousands. The amebae are present in soil and water and, as
such, it is virtually impossible to avoid contacting them. Several studies have detected antiameba antibodies in surveyed human populations as evidence of contact with these amebae
(see below). Not all of the amebae encountered in the environment, however, have the
potential for causing disease.
Acanthamoeba spp. is the most common and widespread ameba found in the
environment and is almost invariably present in any soil sample plated for isolation of
amebae (Page, 1988). They have been isolated from fresh, brackish, and salt waters, soil
samples ranging from the Antarctic to arid desert-like areas, sewage dump sites, as well as
from the home environment in garden and flowerpot soils, water taps, humidifiers, home
aquaria, etc. The amebae have been recovered from laboratory eye wash stations, dental
irrigation systems, and from hospital plumbing and hydrotherapy pools, posing a threat to
Disease
Estimated number of human cases
Distribution and special habitats
Disease in non-human hosts
Acanthamoeba encephalitis
200 cases (1960’s to 2000)
World-wide; soil and water
Acanthamoeba keratitis
>3000 cases (1980’s to 2000)
Wild and domestic animals
(dogs, horse, Indian buffalo,
kangaroo, bull, monkeys, ovines)
Keratitis not reported
Balamuthia encephalitis
100 cases (1990 to 2000)
Apes, horse, sheep, dogs
Naegleria meningoencephalitis
Amebic dysentery
200 cases (1960’s to 2000)
World-wide; soil and
probably water
World-wide; naturally warm or
thermally polluted waters
Tropical and subtropical regions;
developing nations
Tropical and subtropical regions;
developing nations
Balantidium dysentery
500 million infected,
with 100,000 deaths/year
Incidence 1%, based on
surveys of stool samples
Cattle, tapir
Monkeys, apes, dogs
Pigs, monkeys, apes
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
Table 2
Epidemiological considerations of diseases caused by waterborne amebae and Balantidium coli
101
102
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
immunocompromised patients. Acanthamoeba spp. and Naegleria have been isolated from
nasal passages of humans, particularly during periods of high winds carrying dust, as
occurs during the harmattan in western Africa (Abraham and Lawande, 1982). They occur
as laboratory contaminants, appearing in tissue cultures, and have been misidentified as
‘‘transformed’’ tissue culture cells. They have been isolated from human and animal stool
samples, and their presence can be due to either their having passed through the host’s
intestinal tract as cysts which germinate in the stool, or as a result of secondary
(environmental) contamination of the stool sample. Acanthamoeba and other soil amebae
are regarded as coprophilic organisms (copros-, G., dung; philo-, G., liking), exploiting
environments rich in organic materials and bacteria, their natural food.
Approximately 17 species of Acanthamoeba have been described based upon phenotypic
characteristics such as size and cyst morphology (Page, 1988). Sequencing of 18S rDNA has
been the basis of more recent descriptions, with isolates falling into twelve (T1–T12) different
lineages containing either single species or complexes of species (Stothard et al., 1998).
Species that have been most often associated with human systemic infections are A. polyphaga
(T4), A. castellanii (T4), A. culbertsoni (T10), A. hatchetti (T11), and A. healyi (T12). Most
clinical isolates of Acanthamoeba are thermotolerant, growing at temperatures of 37 8C, but
there are also thermotolerant species that are non-pathogenic. Surprisingly, some
Acanthamoeba isolates from clinical cases do not grow well at 37 8C, but require a lower
temperature (30 8C) for optimal growth (Schuster and Visvesvara, 1998). In addition to human
cases, Acanthamoeba GAE has been reported from sheep, dogs, a kangaroo, a monkey, a horse,
and a bull (Greene, 1998; Kadlec, 1978; Martinez, 1985). Acanthamoeba spp. have also been
isolated from poikilotherms (fish and reptiles) (Dyková et al., 1999; Sesma and Ramos, 1989).
Individuals at risk for systemic Acanthamoeba infections include those who are
immunocompromised or immunodeficient. These infections paralleled to a great extent the
AIDS epidemic in the United States and elsewhere. With the development and use of more
effective therapies for treatment, GAE in HIV/AIDS patients has virtually disappeared, but
still remains a risk for organ transplant patients or those who are being administered antiinflammatory steroids (Steinberg et al., 2002).
Not all Acanthamoeba spp. are pathogenic. While it can be assumed that an ameba
isolated from brain or cutaneous lesions is a pathogen, the same assumption cannot be
made for those amebae isolated from the environment. The gold standard for assessing
pathogenicity is the ability to produce encephalitis in the mouse model, following
intranasal instillation of a suspension of amebae (Martinez et al., 1973, 1975). In addition
to infected brain tissue, amebae can also be found in the lungs of experimentally infected
mice (Fig. 11).
Acanthamoeba spp. isolated from air conditioning cooling towers and hospital
plumbing systems have been shown to harbor Legionella and Legionella-like bacteria.
Legionella spp. are fastidious in their growth requirements and it was something of a puzzle
that these bacteria should flourish in air conditioning cooling towers and plumbing pipes
where nutrients were limiting. The suggestion that Legionella could proliferate in amebae
found in the same water (Rowbotham, 1980) led to studies demonstrating the ability of
these bacteria to parasitize amebae, particularly Acanthamoeba, just as they invade and
destroy macrophages in the infected human host (Gao and Kwaik, 2000; Newsome et al.,
1998). Bacteria from the lysed amebae are released in aerosols, to be carried through the
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
103
Fig. 11. Transmission electron micrograph showing three trophic Acanthamoeba castellanii (A) in lung tissue of
an experimentally infected mouse inoculated intranasally with about 1000 amebae. Bar represents 10 mm.
ventilating system. The implication of this relationship is that the bacteria that cause
legionellosis can survive and proliferate in environments otherwise unsuitable for their
growth, causing disease in immunodepressed populations, such as hospital patients.
Acanthamoeba keratitis (AK) is caused by a much wider variety of Acanthamoeba spp.,
most of which are part of the T4 rDNA complex (Stothard et al., 1998). Thermotolerance is not
as critical for these organisms found at the corneal surface, which is slightly below normal
mammalian body temperature, as it is for those with systemic sites of infection. Corneal
trauma or improper care of contact lenses (daily wear or extended-wear soft lens types) are the
causes of AK, the former being more common among males and patients 50 years, and the
latter among females (Stehr-Green et al., 1989). Tap or distilled waters, used in preparation of
homemade saline solutions for lens care, are often the source of the amebae, which can then
bind to the lens, the lens case, and ultimately to the corneal surface. Data from 1989 indicated
that 64% of contact lens wearers with AK had prepared saline solutions from salt tablets using
distilled water (Stehr-Green et al., 1989). Typically, individuals developing AK are otherwise
in good health, with no evidence of immunodeficiency.
No naturally occurring AK cases have ever been described in animals, and attempts at
developing an animal model to study AK have met with mixed results. In an in vitro study
comparing attachment of amebae to corneas from a variety of animal eyes, attachment
occurred to human, pig, and hamster corneas but not to those of mice, rats, horses, dogs,
chickens, and guinea pigs (Niederkorn et al., 1992).
Balamuthia, another agent that causes GAE, is found in soil and perhaps water, but is
difficult to isolate from nature (Schuster et al., 2003). There have been about 100 cases of
104
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
GAE in humans reported in the literature since the first descriptions of this opportunistic
pathogen (Visvesvara et al., 1990, 1993). Given the difficulty in recognizing the pathogen,
it is likely that many cases go unreported. All clinical isolates of this organism are members
of a single species, as determined by DNA sequencing of clinical isolates (Booton et al.,
2003). Infection comes about as the result of inhalation of airborne soil particles, as well as
contamination of breaks in the skin with soil. Early descriptions of Balamuthia GAE were
in HIV/AIDS patients and others with impaired immune systems, such as the elderly, and
drug and alcohol abusers (Visvesvara et al., 1990). More recent cases have been from
immunocompetent children. The disease has also been reported in non-human primates
in zoos (Canfield et al., 1997; Rideout et al., 1997), dogs (Visvesvara, unpublished
observations), a sheep (Fuentealba et al., 1992), and a horse (Kinde et al., 1998). A lowland
gorilla at a zoo died of meningoencephalitis that was initially attributed to Acanthamoeba
(Anderson et al., 1986), but the ameba was subsequently identified as Balamuthia
(Visvesvara, unpublished observation).
N. fowleri is the causal agent of PAM. Although there are >30 species of Naegleria as
determined from sequencing data (De Jonckheere, 2002, 2004), N. fowleri is the only
species that has been recovered from clinical cases. Two other species, N. australiensis and
N. italica, are pathogenic in the mouse model of disease, but have never been associated
with any human cases. As for Acanthamoeba, thermotolerant species of Naegleria are
known (e.g. N. lovaniensis), but are not pathogenic for humans. Most victims of PAM are
children or young adults in good health with a history of swimming in naturally warm or
thermally polluted waters, where growth of the ameba is favored. In Baja California
(Mexico), irrigation canals were the source of infections in children who had been
swimming in the canal waters. In the United States, most PAM cases have been reported
from the southern tier of the country, with Florida, Virginia, Texas, and California
having relatively large percentages of victims (Martinez, 1985). Thermal effluents from
factories and power plants have also promoted growth of the pathogen, and cooling ponds
of nuclear power plants are monitored for the presence of N. fowleri (Reveiller et al., 2003).
Amebae have been isolated from sun-warmed domestic water supplies in both the United
States and Australia, but risk of infection from drinking water is minimal. More likely,
infection in these cases resulted from aspiration of water into the nostrils while washing.
Because of the short prodromal period, the association between infection and water-related
activities is readily apparent. The occurrence of the disease among young persons suggests
that diving, horseplay, prolonged immersion in water, and aspirating mud stirred up from
the bottom of lakes and ponds, are factors that promote infection and disease. In addition to
humans, PAM has been reported from cattle, and from a tapir (Daft et al., 1999; LozanoAlarcon et al., 1997). Willaertia sp., a free-living ameba closely related to N. fowleri,
was identified in the stomach wall of a dog with gastric ulcers and adenocarcinoma
(Steele et al., 1997)
4.2. Entamoeba histolytica and commensal amebae
E. histolytica is the only ameba parasitic in the human intestine. Once regarded as a
single species with pathogenic and non-pathogenic variants, the non-pathogenic form is
now recognized as a different species, E. dispar (Diamond and Clark, 1993). The latter
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
105
species is associated with individuals who are infected with Entamoeba, but are
asymptomatic for amebic dysentery. The picture is further complicated by another
intestinal ameba, E. hartmanni, which is morphologically similar to E. histolytica, but of
smaller size (3–10 mm versus 20–30 mm for E. histolytica) and non-pathogenic.
An estimated 500 million humans are infected with the parasite, but only 10–20%
exhibit dysenteric disease, the prevalence varying with different geographic locations
(Trager, 1986). Spread of E. histolytica is by the fecal–oral route, and water and food
contaminated with fecal waste containing cysts are the major vehicles of infection. Anal
intercourse practiced among gay males has also been recognized as a means of
transmission (Judson, 1984). Food handlers are another possible source of infection, as are
mechanical vectors such as flies and cockroaches. Dogs that have developed amebiasis by
ingestion of human feces, can be a potential source of human infections, though the reverse
is more likely (Barr, 1998; Botero, 1972). However, E. histolytica in the dog rarely encysts,
and trophic amebae in dog stools are not infective (Barr, 1998; Eyles et al., 1954). In the
mammalian host, ingested cysts germinate in the small intestine and are carried to the
colon, where the amebae attack the epithelial lining. Cysteine proteases are probably a
virulence determinant of the amebae (Bruchhaus et al., 2003). In cases of dysentery with
accompanying diarrhea, numerous trophic amebae, often containing ingested red blood
cells, are found in the unformed or loose stool. In asymptomatic cases, the formed stool
contains cysts whose development parallels the removal of water from the stool as it passes
to the rectal area. Trophic amebae in unformed stools do not encyst, nor are they infective
to others. The cyst, by virtue of its protective wall, is the prime infective stage in the ameba
life-cycle. The presence of non-pathogenic commensals in the gut seen upon examination
of stool samples can lead to erroneous laboratory diagnoses.
Cysts of Entamoeba can remain viable for 2–4 weeks outside of the host provided they
are in a moist or wet environment. Desiccation and extreme temperatures can render cysts
non-viable. Amebic dysentery is primarily a disease of tropical or sub-tropical areas of the
world, where climatic conditions favor cyst survival and sewage is apt to contaminate the
drinking water supply. But sporadic outbreaks of the disease have occurred throughout the
world, regardless of temperatures, and one such textbook example occurred in Chicago
during the 1933 World’s Fair with over 1000 cases (Albach and Booden, 1978). In addition
to humans, E. histolytica is found in a large number of non-human primates and, less
often, in dogs (Barr, 1998; Eyles et al., 1954). Another species, E. polecki, found in pigs
has been reported from humans but is not highly infective for humans (Levine, 1961).
E. moshkovskii, an ameba morphologically similar to E. histolytica but non-pathogenic,
has been isolated from sewage in several parts of the world and, recently, has been reported
in 21% of Bangladeshi children sampled as a non-invasive commensal (Ali et al., 2003).
Optimal temperature for growth of the organism is 25 8C, rather than 37 8C, and it is
hardier than E. histolytica. Other amebae that are found in the human gut as commensals
are also transmitted by the fecal–oral route.
4.3. Balantidium coli
This protozoon is the only ciliate known to cause infections in humans. Some fifty
species have been described, often on size differences, and the validity of these species is
106
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
unconfirmed. B. coli is spread by the fecal–oral route from pig-to-human and from
human-to-human, the latter mode of transmission occurring in institutionalized
populations (mental hospitals, orphanages, prisons). Balantidiasis is a disease of
tropical and sub-tropical regions, and the Philippines is cited as an endemic area (Zaman,
1978). Pigs, which harbor the ciliate, are typically the source of human infections,
although species-to-species transfer requires adaptation of the parasite. The ciliates in
pigs are non-invasive and non-pathogenic. Transmission occurs when swine are in close
contact with humans, and there is a lack of adequate sanitary facilities for sewage
treatment and disposal. The health of the host can be a factor, since individuals who are
malnourished, suffering from concurrent infections, or alcoholism are at greater risk of
developing balantidiasis. Nevertheless, the disease is uncommon in humans, and the
number of symptomatic cases of balantidiasis in the world is probably in the hundreds of
thousands.
Cysts can remain viable for weeks in pig feces, particularly if kept moist and away
from direct sunlight. Trophic ciliates can survive for as long as 10 days in the environment
(Zaman, 1978). Infection occurs when fecal material from swine contaminates
drinking water or food. Handling of pig intestine can also be a mode of transmission.
Once ingested, cysts give rise to active ciliates in the colon. These ciliates feed upon
bacteria and debris in the gut, but also release enzymes (hyaluronidase) that attack the
mucosal surface, producing flask-shaped ulcers in the wall of the colon and causing
diarrheic stools with blood and mucus. Secondary infection of the colonic lesions by
bacteria can worsen the clinical picture. Table 3 summarizes the epidemiological factors
involved in these protozoal diseases, along with their potential for zoonotic and waterborne
transmissions.
4.4. Developed and developing regions
Amebic dysentery and balantidiasis are a reflection of poor sanitation and inadequate
protection of the water supply from sewage contamination. Their prevalence, particularly
amebiasis, is high in developing countries. Although they occur in developed regions, their
appearance is sporadic and infection is most likely due to travel outside of developed areas
of North America, Australasia, Japan, and Western Europe.
Paradoxically, the situation is reversed in the case of diseases caused by free-living
amebae. They are reported mostly from developed regions with only sporadic reports from
the developing areas of the world. This anomaly may be due, however, not to the actual
incidence of the diseases but rather to the availability of diagnostic procedures and medical
care. Difficult enough to diagnose in developed nations, it is highly likely that amebic
encephlatides go undetected and undiagnosed in regions of Africa and Southeast Asia.
There are reports of encephalitis cases from South America (Recavarran-Arce et al., 1999)
and Thailand (Sangruchi et al., 1994), but these present only an incomplete picture of what
might actually exist. India has had a large number of keratitis cases associated with use of
contact lenses. Given the enormity of the HIV/AIDS epidemic and its large population of
immunodeficient victims, there are almost certainly opportunistic infections caused by
amebae that go unidentified in many developing nations.
Disease
Populations at risk
Zoonotic potential/animal source
Potential as waterborne infection
Acanthamoeba encephalitis
Immunocompromised hosts
(AIDS and transplant patients,
those with concurrent diseases)
Contact lens users
Not zoonotic
Minor
Not zoonotic
When unsterilized water is used
for preparation of ophthalmic
solutions; immersion in hot tubs
Minor
Acanthamoeba keratitis
Balamuthia encephalitis
Naegleria meningoencephalitis
Amebic dysentery
Balantidium dysentery
Immunocompromised and
immunocompetent hosts
Apparently healthy children,
young adults, active in water
Travelers to developing countries
Rural or agrarian populations;
institutionalized groups
Not zoonotic
Not zoonotic
Zoonotic/non-human
primates, dogs
Zoonotic/pigs,
non-human primates
Most infections contracted by
water-related activities
Sewage-polluted water and
vegetables
Sewage-polluted water; pig feces
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
Table 3
Opportunistic and parasitic diseases: populations at risk, and potential for zoonotic and waterborne transmission
107
108
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
5. Diagnostic techniques
5.1. Recovery and identification of free-living amebae
Infections caused by the free-living amebae are difficult to diagnose, and most are
recognized on autopsy or necropsy. Often, computed tomography and magnetic resonance
imaging of the brain of encephalitis patients indicate space-occupying lesions that lead to
biopsies and diagnoses. With infections caused by Acanthamoeba and Balamuthia, diagnoses
are made by finding the amebae in tissue sections stained with hematoxylin-eosin (H&E), or
by indirect immunofluorescence (IIF) staining using rabbit anti-ameba sera (Visvesvara et al.,
1993). The latter technique is preferred as a diagnostic tool because of its specificity in
identification of the etiologic agent. Few laboratories, however, are equipped to perform IIF
staining, the Centers for Disease Control and Prevention in Atlanta being one. Serological
screening of patients with suspect encephalitides can detect anti-ameba antibodies in
suspected Acanthamoeba and Balamuthia infections (Visvesvara et al., 1990, 1993). Because
these are chronic infections, there is time for an antibody response which can be detected by
indirect immunofluorescent staining. The polymerase chain reaction (PCR) has been used to
detect ameba DNA in tissue and CSF samples, but the technique is still in its early
developmental stages and requires more testing before its reliability can be certified.
Cutaneous infections by Acanthamoeba and Balamuthia are diagnosed using the same
techniques as for CNS infections. Because of their insidious nature and the difficulty in
antemortem recognition, the prognosis for recovery is poor and most cases are fatal.
In Acanthamoeba keratitis there is usually a strong association between contact lens
wear or corneal trauma and the occurrence of disease. Because of the pain associated with
the infection, victims are likely to seek medical help soon after symptoms develop. Cysts of
infecting amebae can be recognized in corneal scrapings that have been stained with
calcofluor white, a fluorescent stain that binds to polysaccharide polymers in the
Acanthamoeba cyst wall (Wilhelmus et al., 1986). The amebae can also be cultured onto
non-nutrient agar with bacteria for identification (Visvesvara, 1999; Schuster, 2002).
Naegleria meningoencephalitis, as mentioned above, is almost always associated
with swimming or other water activity. Initial diagnosis is made by finding trophic amebae
in the CSF following lumbar puncture. Naegleria amebae can be recognized under the
microscope by their characteristic limacine movement, with an anterior eruptive
pseudopod (Visvesvara, 1999). Because of its fulminant nature, early diagnosis is
essential, and recovery from the infection is rare, most cases being fatal. Here, too, staining
with H&E or IIF of brain tissue is helpful in confirming a diagnosis of PAM, but this is
usually done postmortem. Because Naegleria infections are often fatal within a 2-week
period, there is little or no antibody response to the presence of the amebae, and serological
procedures are of little avail. Microscopic observation of fresh CSF for active amebae is the
best opportunity for an on-the-spot identification.
5.2. Recovery and identification of Entamoeba histolytica and Balantidium coli
E. histolytica is recovered from dysenteric stools as trophic amebae. During fulminant
disease, trophozoites do not encyst before being expelled with the unformed stool, and can
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
109
Fig. 12. Photomicrograph of Entamoeba histolytica, trophozoite (T) and cyst (C), seen in a trichrome-stained
fecal smear. Four nuclei can be seen in the cyst, as can a large dark-staining chromatoid body. A single nucleus is
evident in the trophic ameba. Bar represents 20 mm.
be seen by microscopic examination of stool samples (Fig. 12). In asymptomatic cases
or in carriers, the cyst is the stage that is found in the stool sample. The presence of cysts
of commensal amebae from the gut can be the cause of confusion or misdiagnosis, but
there are distinct differences between cysts of E. histolytica and those of E. coli,
Endolimax nana, and Iodamoeba butschlii that technicians in diagnostic laboratories
are trained to recognize (Leber and Novak, 1999). E. histolytica can be differentiated
from the non-pathogenic E. dispar and other commensal amebae in dysenteric stools
by the presence of ingested erythrocytes in the former (Tanyuksel and Petri, 2003).
Other manifestations of Entamoeba infections such as liver, lung, or brain abscesses
can be detected by sonographic or radiological imaging and, in cases where amebae have
invaded tissues, serology can be of use. PCR, ELISA, indirect hemagglutination assays are
used for laboratory detection of Entamoeba (Gonin and Trudel, 2003; Tanyuksel and Petri,
2003).
Balantidium infections are most often diagnosed on finding active ciliates or cysts in
stool samples of patients. History of contact with swine may also be indicative of infection.
Infection can occur among people directly involved in raising pigs (farmers), in treatment
of pigs for infections (veterinarians), and in handling of pig organs (slaughter house
personnel). Infection can occur if pork becomes contaminated with pig intestinal contents
or feces and is not adequately cooked before being consumed.
For all the above parasites, culturing is an additional and confirmatory means of
identification although, in many cases, culturing of organisms from clinical samples is apt
to be time- and labor-intensive and is not recommended for rapid diagnosis. Cultivation
techniques and appropriate media for Entamoeba and Balantidium are described in
Diamond and Clark (2002), and for free-living amebae, in Schuster (2002) and Visvesvara
(1999). Many of the organisms require complex media (with limited shelf lives) and growth
conditions that might be available only in reference or research laboratories.
110
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
6. Control and management
6.1. The environment and free-living amebae
Avoidance of the free-living amebae is virtually impossible since they are ubiquitous
in the environment, as evidenced by the presence of antibodies in screened human
populations (Cerva, 1989; Cursons et al., 1980; Marciano-Cabral et al., 1987; Huang et al.,
1999). Domestic and wild mammals, which might be expected to have closer contacts with
soil than humans, also exhibit antibodies against amebae (Cerva, 1981; Kollars and
Wilhelm, 1996). It is not clear, however, if these antibodies provide immunological
protection against infection. It is also not clear if victims of naegleriasis, which generally
occurs in immunocompetent humans, might have some inapparent immunological deficiency that renders them vulnerable to infections.
Some precautions can be taken to minimize contact with these opportunistic pathogens.
Immunodeficient patients are well advised to guard against exposure to airborne dust and
soil particles that might carry cysts of opportunistic amebae to the respiratory system, or
contamination of open wounds or sores with soil. N. fowleri, which has a clear association
with swimming and other activities that allow water to enter the nasal passages, is easier to
guard against. Natural or artificially warm waters, if they are not properly chlorinated,
could harbor Naegleria amebae. Maintaining effective levels of chlorine in recreational
waters is achievable in swimming pools and spas, but less so in the case of lakes and ponds.
However, adequate chlorination is not necessarily protective, as was seen in a retrospective
diagnosis of 16 PAM fatalities associated with a swimming pool in Czechoslovakia (Cerva
and Novak, 1968; Kadlec et al., 1980). In Florida, which has had and continues to have
sporadic cases of PAM associated with swimming in its warm lakes, the chance of infection
has been estimated to be about 1:2.6 million exposures (Wellings, 1977). Numerous
persons have been exposed to the amebae by swimming in the Florida waters, yet the
number of PAM cases remains negligible. In a survey covering a 1-year period in the
United States, four cases (6.8%) of Naegleria meningoencephalitis were found in 59
recreational waterborne disease outbreaks involving >2000 persons (Lee et al., 2002).
There is no evidence to indicate that infections by free-living amebae have any zoonotic
basis. Non-human animals also can develop these infections, but there is no likelihood of
their being able to pass the infection along to humans, either directly or indirectly through
pollution of drinking water or contamination of crop foods. Although free-living amebae
can be recovered from stool samples, their presence is not indicative of infection or disease,
and is more likely an indication of their being coprophiles.
In Acanthamoeba keratitis, the domestic water supply in the victim’s home, used in
preparation of homemade saline solutions for contact lens care, is the most likely source of
the amebae. Using molecular techniques for characterization of amebae, two different
studies identified the same ameba strain in the domestic water supply used for preparation
of wash solutions, the lens case, and the cornea of the patient (Kilvington et al., 1990;
Ledee et al., 1996). Compliance with procedures for contact lens care should protect
wearers against infection. Care should be taken to maintain a clean lens case in order
to prevent formation of bacterial films, a potential growth surface for the amebae. There
is evidence of protective IgA antibody levels at the corneal surface of normal individuals,
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
111
as compared to non-protective IgG serum levels in keratitis victims (Alizadeh et al.,
2001).
6.2. Transmission of Entamoeba histolytica and Balantidium coli
The situation with parasitic infections caused by E. histolytica and B. coli is somewhat
different in that animal species harbor these organisms, so the potential for zoonotic
transmission exists. Transmission is through fecal contamination of water or food by cysts
passed in stools. Humans appear to be the major source for infecting other humans through
fecal contamination of food or the water supply (Albach and Booden, 1978). Dogs, though
they can be infected with E. histolytica, are an unlikely source of human infection (Barr,
1998). Entamoeba is also found in a variety of non-human primates including baboons;
vervet, Sykes, and Debrazza’s monkeys; and in both black and grey mangabeys (Muriuki
et al., 1998). There is limited opportunity for primate feces to contaminate domestic water
supplies, even in parts of the world where there are large numbers of monkeys. Individuals
at risk include those involved in caring for monkeys (zoos or laboratory facilities,
veterinarians), butchering animals for food, or those keeping the animals as pets. Primates
in zoos and laboratory facilities can be monitored for the presence of Entamoeba and can
be treated to eliminate infection.
Domestic and wild swine represent a reservoir for human Balantidium infections and
several studies have found the incidence of infection to be 100% (Hindsbo et al., 2000;
Nakauchi, 1999). Although the organism is a commensal in the pig colon, it can be
pathogenic in humans. In a survey of 56 Japanese-bred mammalian species, primates and
pigs were positive for Balantidium (Nakauchi, 1999). A chimpanzee in this study was
found to be passing >1200 cysts and trophozoites per gram of feces. In rural or agrarian
areas where swine feces can contaminate the water supply, this would be a major source of
infection to the human population.
6.3. Treatment
There is no optimal anti-microbial therapy for the encephalitides caused by
Acanthamoeba and Balamuthia. Multiple drugs have been used in both these infections
with varying degrees of success, including amphotericin B, azithromycin, fluconazole,
flucytosine, pentamidine isethionate, and sulfa drugs (Schuster and Visvesvara, 2003).
Most Acanthamoeba encephalitis victims are immunodeficient, and succumb to multiple
opportunistic infections rather than to acanthamebiasis per se.
Amebic keratitis is treatable with one or more drugs (Kumar and Lloyd, 2002; Schuster
and Visvesvara, 2003). Chlorhexidine gluconate (a component of germicidal soaps) and
polyhexamethylene biguanide (a disinfectant and swimming pool cleaner) have become
the drugs of choice in treating keratitis cases. These drugs are well-tolerated in the eye,
although there are strains of Acanthamoeba that have shown resistance. Propamidine
isethionate, present in Brolene, an over-the-counter preparation available in Great Britain,
has also been used with some success, but is not as well-tolerated in the eye. In the presence
of these drugs, the amebae are likely to encyst in the corneal stroma and, with premature
cessation of treatment, excyst to give rise once again to trophozoites. Therefore, an
112
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
aggressive regimen of initial hourly drug applications should be followed, and
continued over several months at less frequent intervals (Schuster and Visvesvara,
2003).
Of the approximately 100 published cases of balamuthiasis, there are only two known
survivors, following intensive anti-microbial therapy (Deetz et al., 2003). In both cases,
following demonstration of brain lesions by magnetic resonance imaging, the diagnoses
were made by detection of Balamuthia amebae in biopsied brain tissue and Balamuthia
antibodies by immunofluorescence staining. A combination of anti-microbials was used in
treatment, including pentamidine isethionate, flucytosine, fluconazole, sulfadiazine, and a
macrolide antibiotic (azithromycin or clarithromycin). Recovery from GAE also depends
upon early diagnosis, the virulence of the infecting strain, the infectious dose of amebae,
and prompt initiation of treatment.
Few individuals infected with Naegleria meningoencephalitis have survived, in large
part because of the fulminant nature of the disease. Amphotericin B is the drug of choice in
treating victims, but early diagnosis is essential (Seidel et al., 1982).
Amebic dysentery is treatable with metronidazole and prospects for recovery are
excellent (Haque et al., 2003). Treatment, however, is often unavailable in developing
countries, and this is reflected in the disproportionate numbers of deaths from amebiasis in
these regions. Oxy- and chlorotetracycline and carbasone are used in the treatment of
balantidiasis but, for the same reasons as for amebiasis, people still suffer and die from the
disease. A summary of information about diagnostic techniques and anti-microbial therapy
can be found in Table 4.
7. Future trends
7.1. Globalization
With the increase in reciprocal travel between developed and developing nations of the
world, there is a greater likelihood of spread of infections caused by amebae and ciliated
protozoa. Globalization, however, probably would not affect the incidence of diseases
caused by free-living amebae because the potential for infection is already fairly uniform
throughout the world. The incidence of acanthamebiasis and balamuthiasis which occur in
immunocompromised hosts, will reflect the overall health of a population. In parts of the
globe where HIV/AIDS infections are rampant, opportunistic infections, including
the encephalitides, would be expected to have a higher occurrence. But since these
infections represent ‘‘dead ends’’ for the etiologic agent, human-to-human spread is most
unlikely.
Parasitic infections such as amebiasis and balantidiasis can spread, as a result of
globalization, from tropical and subtropical areas to temperate zones of the globe. Persons
infected with the parasites and serving as carriers can bring their parasites to developed
areas of the world. But these diseases reflect the lack of adequate sanitary facilities and
clean drinking water, and are not transmissible where sewage disposal and water quality
control are rigorously practiced. In recent years, sewage-tainted produce (e.g. cantaloupes,
lettuce, and berries) and apple cider has caused outbreaks of bacterial, cryptosporidial, and
Disease
Available diagnostic procedures
Antimicrobial treatment
Prognosis for
recovery
Acanthamebiasis
H&Ea, immunofluorescence staining; PCRb;
CTc and MRId scans; cultivation from clinical samples
Corneal scrapings; cultivation
Amphotericin B, azithromycin, fluconazole, flucytosine,
pentamidine, sulfa drugs
Chlorhexidine, polyhexamethyl biguanide, propamidine
Poor
Excellent
H&E, immunofluorescence; PCR;
CT and MRI scans; cultivation
Trophic amebae in CSFe; H&E, immunofluorescence
staining; serology; cultivation
Trophs and/or cysts in stool; ELISAf
for stool antigen; PCR; sonography,
CT and MRI scans for extra-intestinal abscesses
(liver, lung, brain); cultivation
Trophs and/or cysts in stool; cultivation
Pentamidine, fluconazole, flucytosine, sulfadiazine
Poor
Amphotericin B
Poor
Metronidazole
Excellent
Oxy- and chlortetracycline, carbasone
Excellent
Acanthamoeba
keratitis
Balamuthiasis
Naegleriasis
Amebic dysentery
Balantidiasis
a
b
c
d
e
f
Hematoxylin and eosin.
Polymerase chain reaction.
Computerized tomography.
Magnetic resonance imaging.
Cerebrospinal fluid.
Enzyme-linked immunosorbent assay.
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
Table 4
Diagnostic techniques, treatment options, and prognoses for recovery from opportunistic and parasitic infections
113
114
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
cyclosporidial infections in North America and Europe (Ho et al., 2002; Millard et al.,
1994; Tauxe, 1997). But there have been no reported outbreaks of amebiasis and
balantidiasis as a result of food contamination. Cysts of these parasites are less likely than
bacteria to survive prolonged exposure to environmental stress. Surveys of pigs in Japan
and Denmark, neither of which is tropical or subtropical, have demonstrated a high
prevalence of B. coli. Yet Denmark has had no human cases of balantidiasis (Hindsbo et al.,
2000), emphasizing the effectiveness of sanitary precautions in prevention of human
infections.
7.2. Climate change
The generally accepted notion of global warming has caused concern regarding the
spread of infectious and parasitic diseases, particularly those transmitted by arthropod
vectors. Diseases caused by free-living amebae, amebic dysentery, and balantidiasis, are
only briefly mentioned, if referred to at all, in scenarios for disease spread due to global
warming (Cook, 1992; Patz et al., 2000).
For free-living soil amebae, increasing environmental temperatures might enhance
the growth of thermotolerant species which, in turn, might be better adapted to infect
humans and other mammals. But as noted before, thermotolerance is not necessarily
synonymous with pathogenicity and virulence, and growth at 37 8C, is no guarantee of
pathogencity in the mouse animal model of disease. Acanthamoeba spp., found in both
fresh and seawaters, may proliferate with rising environmental temperature and pose a
threat to swimmers. Since their primary food source is bacteria, the amebae would more
likely favor areas of pollution, with high bacterial counts. But those at risk for
acanthamebiasis are the immunocompromised or immunodeficient, and the disease is rare
in healthy individuals. Measures can be taken to insure that swimming pools and manmade lakes are adequately chlorinated, but this cannot be accomplished with large bodies
of water.
Increased environmental temperatures would lead to increased use of air conditioning
units in buildings. This, in turn, would provide opportunities for growth of Acanthamoeba
in the cooling towers. As noted previously, Acanthamoeba spp. can serve as hosts for
intracellular Legionella, which eventually cause lysis of the amebae and release of the
bacteria in aerosols. Thus, it is possible that these amebae have been vectors in outbreaks of
legionellosis and its less severe form, Pontiac fever. At particular risk would be patients
in hospitals and residents of homes for the elderly, both with concentrations of
immunoimpaired persons. If properly treated with biocides, proliferation of protozoa and
bacteria in cooling towers can be prevented. For extensive plumbing systems, periodic
flushing with hot water can minimize biofilm formation in the piping that serves as a
growth substrate for bacteria and amebae.
Acanthamoeba keratitis is caused by contamination of contact lenses or the lens
case with bacterial films that would serve as food for the amebae. The source of the
amebae is non-sterile water used in lens care, and it is unlikely that climate change
would affect their presence. More important is compliance with recommendations for
lens care, and not wearing lenses while swimming or relaxing in thermal pools or hot
tubs.
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
115
As for N. fowleri, the causal agent of PAM, the organism is already found in warm
waters and, with an increase in warmed lakes and such, they would be expected to extend
their distribution, leading to higher incidence of PAM in both humans and animals. As with
Acanthamoeba, growth of Naegleria can be checked by chlorination of pools and manmade swimming lakes, but that is not feasible for large bodies of water.
E. histolytica is endemic in many tropical and subtropical regions where climatic
conditions favor survival of cysts in the environment. Cyst viability, however, is not enough
to insure disease transmission and, in those areas of the globe that have adequate facilities
for treating sewage and protecting the water supply from contamination, there would likely
be no major change in the incidence of invasive amebiasis. There might be an increase in
sporadic cases of amebiasis, mostly among travelers returning from areas with substandard water quality, but no generalized explosion of infections. The same would be true
for B. coli, which shares a similar fecal–oral route of transmission.
7.3. Modern technology
Two aspects of human technological progress and their effect on the occurrence of some
of the diseases dealt with in this report are noteworthy. The development and extensive use
of soft and extended-wear contact lenses since the 1980’s have resulted in emergence of
Acanthamoeba keratitis as a significant ophthalmic disease. Prior to that period, the
occurrence of amebic keratitis was a rare event and limited to accidental injury to the
cornea, with subsequent infection by the ameba either from water or soil. With widespread
use of contact lenses and lack of clear guidelines for their care, amebic keratitis reached
epidemic proportions with cases being reported from all over the globe. The problem was
aggravated by the lack of effective anti-microbial agents for treatment. At present, medical
personnel are aware of amebic keratitis, effective therapy is available, and contact lens
users are better informed of the risks of non-compliance with recommendations for proper
lens care.
The extensive use of ventilation systems in otherwise sealed buildings has led to
sporadic outbreaks of legionellosis, associated with the cooling towers that are part of
the air conditioning systems. Acanthamoeba spp. have also been isolated from hospital
plumbing systems where they can serve as host to Legionella spp., releasing bacteria
from sink tap, shower head, and toilet aerosols into an environment having a
disproportionate number of patients with weakened immune systems (La Scola et al.,
2003). Hot tubs and physiotherapy pools, unless scrupulously maintained, can also favor
growth of thermotolerant amebae. Cases of amebic keratitis have been traced back to
contact lens users who wore their lenses while soaking in a hot tub or spa (Samples et al.,
1984). Among 108 patients with amebic keratitis surveyed from the literature, 3.7% had
spent time in a hot tub prior to developing the infection (Lang and von Heimburg-Elliger,
1991).
There was passing concern about possible release of Naegleria amebae in the
misting spray released over the fruit and vegetable displays in many supermarkets in the
United States. Since these displays release cool water, it is unlikely that growth of N.
fowleri and thermophilic amebae would be favored, but other species of Naegleria and
116
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
Acanthamoeba might be found. These amebae, however, are not transmitted by an oral
route.
8. Conclusions
Infections caused by opportunistic and pathogenic free-living amebae represent an
emerging group of diseases of humans and other animals recognized within the past few
decades. Generally, they are neither waterborne nor zoontic diseases. Escalating numbers
of immunocompromised patients, the result of the HIV/AIDS epidemic, has led to an
increase in some of these diseases, as has an increasing number of immunosuppressed
patients undergoing organ transplantation and other medical procedures. Because of
technical difficulties in diagnosis, these infections are reported mainly from the developed
world but are likely to go undiagnosed in the developing world.
In contrast, infections by the parasitic protozoa E. histolytica and B. coli were
recognized more than a century ago. They have the potential for zoonotic transmission but
their spread is, particularly for amebic dysentery, person-to-person. In the contemporary
world, amebiasis and balantidiasis remain public health problems chiefly in developing
countries where sewage-contaminated water and food, and lack of available therapy have
allowed them to persist.
Note added in proof
Addendum to veterinary parasitology paper (Schuster and Visvesvara)
Two reports concerning the free-living amebae update the literature on Acanthamoeba
and Balamuthia. The number of different sequence types recognized in Acanthamoeba has
increased to 15 (T15) with the report of Hewett et al. (2003).
A 2-year old Great Dane died of Balamuthia encephalitis (Foreman et al., 2004). The
animal was being treated for inflammatory bowel disease with immunosuppressive doses
of prednisone. During the 6-month course of therapy, the dog was taken to swim in a pond
containing stagnant water one to two times a week. Histopathology of brain and kidney
tissues from the dog showed amebic trophozoites and cysts, possibly Acanthamoeba. With
immunostaining, the polymerase chain reaction, and electron microscopy, the amebae were
identified as Balamuthia. This case is of importance because it is the first report in the
literature of balamuthiasis in a dog, and it also raises the possibility that the amebic
infection was acquired from water.
Acknowledgements
We gratefully acknowledge our late colleague and friend Dr. A. Julio Martinez as the
source of several micrographs of amebae used in this paper. Dr. Martinez, who died in
December 2002, was a neuropathologist who was expert in identification of amebae in
brain and other tissues and had done much in the way of increasing awareness of amebic
infections in humans through numerous publications and presentations at meetings. We
thank Henry Bishop (Centers for Disease Control and Prevention) for providing
photomicrographs of B. coli.
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
117
References
Abraham, S.N., Lawande, R.V., 1982. Incidence of free-living amoebae in the nasal passages of local population in
Zaria, Nigeria. J. Trop. Med. Hyg. 85, 217–222.
Albach, R.A., Booden, T., 1978. Amoebae. In: Kreier, J.P. (Ed.), Parasitic Protozoa, vol. 2. Academic Press, New
York, pp. 455–506.
Ali, I.K.M., Hossain, M.B., Roy, S., Ayeh-Kumi, P.F., Petri Jr., W.A., Haque, R., Clark, C.G., 2003. Entamoeba
moshkovskii infections in children in Bangladesh. Emerg. Infect. Dis. 9, 580–584.
Alizadeh, H., Apte, S., El-Agha, M.S., Li, L., Hurt, M., Howard, K., Cavanagh, H.D., McCulley, J.P., Niederkorn,
J.Y., 2001. Tear IgA and serum IgG antibodies against Acanthamoeba in patients with Acanthamoeba keratitis.
Cornea 20, 622–627.
Amaral Zettler, L.A., Nerad, T.A., O’Kelly, C.J., Peglar, M.T., Gillevet, P.M., Silberman, J.D., Sogin, M.L., 2000.
A molecular reassessment of the leptomyxid amoebae. Protist 151, 275–282.
Anderson, M.P., Oosterhuis, J.E., Kennedy, S., Benirschke, K., 1986. Pneumonia and meningoencephalitis due to
amoeba in a lowland gorilla. J. Zoolog. Anim. Med. 17, 87–91.
Bakatselou, C., Beste, D., Kadri, A.O., Somanath, S., Clark, C.G., 2003. Analysis of genes of mitochondrial origin
in the genus Entamoeba. J. Eukaryot. Microbiol. 50, 210–214.
Barr, S.C., 1998. Enteric protozoal infections. In: Greene, C.E. (Ed.), Infectious Diseases of the Dog and Cat.
second ed. W.B. Saunders, Philadelphia, pp. 482–491.
Booton, G.C., Carmichael, J.R., Visvesvara, G.S., Byers, T.J., Fuerst, P.A., 2003. Genotyping of Balamuthia
mandrillaris based on nuclear 18S and mitochondrial 16S rRNA genes. Am. J. Trop. Med. Hyg. 68,
65–69.
Botero, D., 1972. Fatal case of acute amoebic dysentery in a naturally infected dog. Trans. R. Soc. Trop. Med. Hyg.
66, 517–518.
Bruchhaus, I., Loftus, B.J., Hall, N., Tannich, E., 2003. The intestinal protozoan parasite Entamoeba histolytica
contains 20 cysteine protease genes, of which only a small subset is expressed during in vitro cultivation.
Eukaryot. Cell 2, 501–509.
Brugerolle, G., Lee, J.J., 2000. Phylum Parabasalia. second ed. In: Lee, J.J., Leedale, G.F., Bradbury, P. (Eds.),
An Illustrated Guide to the Protozoa, vol. 2. Allen Press, Lawrence, Kansas, pp. 1196–1250.
Canfield, P.J., Vogelnest, L., Cunningham, M.L., Visvesvara, G.S., 1997. Amoebic meningoencephalitis caused by
Balamuthia mandrillaris in an orangutan. Aust. Vet. J. 75, 97–100.
Cerva, L., Novak, K., 1968. Amebic meningoencephalitis. 16 fatalities. Science 160, 92.
Cerva, L., 1981. Spontaneous occurrence of antibodies against pathogenic amoebae of the limax group in
domestic animals. Folia Parasitol. 28, 105–108.
Cerva, L., 1989. Acanthamoeba culbertsoni and Naegleria fowleri: occurrence of antibodies in man. J. Hyg.
Epidemiol. Microbiol. Immunol. 33, 99–103.
Cook, G.C., 1992. Effect of global warming on the distribution of parasitic and other infectious diseases: a review.
J. R. Soc. Med. 85, 688–691.
Cursons, R.T.M., Brown, T.J., Keys, E.A., Moriarty, K.M., Till, D., 1980. Immunity to pathogenic free-living
amoebae: role of humoral antibody. Infect. Immun. 29, 401–407.
Daft, B., Kinde, H., Read, D., Visvesvara, G.S., 1999. Naegleria fowleri meningoencephalitis in Holstein cattle
associated with drinking surface water. In: Proceedings of the 42nd Meeting of American Association of
Veterinary Laboratory Diagnosticians, San Diego, CA.
Deetz, T.R., Sawyer, M.H., Billman, G., Schuster, F.L., Visvesvara, G.S., 2003. Successful treatment of
Balamuthia amebic encephalitis: presentation of two cases. Clin. Infect. Dis. 37, 1304–1312.
De Jonckheere, J., 2002. A century of research on the amoeboflagellate genus Naegleria. Acta Protozool. 41, 309–
342.
De Jonckheere, J.F., 2004. Molecular definition and the ubiquity of species in the genus Naegleria. Protist 155, 89–
103.
Diamond, L.S., Clark, C.G., 1993. A redescription of Entamoeba histolytica Schaudinn, 1903 (Emended Walker,
1911) separating it from Entamoeba dispar Brumt, 1925. J. Eukaryot. Microbiol. 40, 340–344.
Diamond, L.S., Clark, C.G., 2002. Methods for cultivation of luminal parasitic protists of clinical importance.
Clin. Microbiol. Rev. 15, 329–341.
118
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
Dyková, I., Lom, J., Schroeder-Diedrich, J.M., Booton, G.C., Byers, T.J., 1999. Acanthamoeba strains isolated
from organs of freshwater fishes. J. Parasitol. 85, 1106–1113.
Eyles, D.E., Jones, F.E., Jumper, J.R., Drinnon, V.P., 1954. Amebic infections in dogs. J. Parasitol. 40, 163–166.
Foreman, O., Sykes, J., Ball, L., Yang, N., De Cock, H., 2004. Disseminated infection with Balamuthia
mandrillaris in a dog. Vet. Pathol. 41, 506–510.
Fuentealba, I.C., Wikse, S.E., Read, W.K., Edwards, J.F., Visvesvara, G.S., 1992. Amebic meningoencephallitis in
a sheep. J. Am. Vet. Med. Assoc. 200, 363–365.
Gao, L.-Y., Kwaik, Y.A., 2000. The mechanism of killing and exiting the protozoan host Acanthamoeba
polyphaga by Legionella pneumophila. Environ. Microbiol. 2, 79–90.
Gonin, P., Trudel, L., 2003. Detection and differentiation of Entamoeba histolytica and Entamoeba dispar isolates
in clinical samples by PCR and enzyme-linked immunosorbent assay. J. Clin. Microbiol. 41, 237–241.
Greene, C.E., 1998. Acanthamebiasis. In: Greene, C.E. (Ed.), Infectious Diseases of the Dog and Cat. second ed.
W.B. Saunders, Philadelphia, pp. 491–493.
Haque, R., Huston, C.D., Hughes, M., Houpt, E., Petri Jr., W.A., 2003. Amebiasis. N. Engl. J. Med. 348, 1565–
1573.
Hewett, M.K., Robinson, B.S., Monis, P.T., Saint, C.P., 2003. Identification of a new Acanthamoeba 18S rRNA
gene sequence type, corresponding to the species Acanthamoeba jacobsi Sawyer, Nerad and Visvesvara, 1992
(Lobosea: Acanthamoebidae). Acta Protozool. 42, 325–329.
Hindsbo, O., Nielsen, C.V., Andreassen, J., Willingham, A.L., Bendixen, M., Nielsen, M.A., Nielsen, N.O., 2000.
Age-dependent occurrence of the intestinal ciliate Balantidium coli in pigs at a Danish research farm. Acta Vet.
Scand. 41, 79–83.
Ho, A.Y., Lopez, A.S., Eberhart, M.G., Levenson, R., Finkel, B.S., da Silva, A.J., Roberts, J.M., Orlandi, P.A.,
Johnson, C.C., Herwaldt, B.L., 2002. Outbreak of cyclosporiasis associated with imported raspberries,
Philadelphia, Pennsylvania, 2000. Emerg. Infect. Dis. 8, 783–788.
Huang, Z.H., Ferrante, A., Carter, R.F., 1999. Serum antibodies to Balamuthia mandrillaris, a free-living amoeba
recently demonstrated to cause granulomatous amebic encephalitis. J. Infect. Dis. 179, 1305–1308.
Jones, D.B., Visvesvara, G.S., Robinson, N.M., 1975. Acanthamoeba polyphaga keratitis and Acanthamoeba
uveitis associated with fatal meningoencephalitis. Trans. Ophthal. Soc. UK 95, 221–232.
Judson, F.N., 1984. Sexually transmitted viral hepatitis and enteric pathogens. Urol. Clin. North Am. 11, 177–185.
Kadlec, V., 1978. The occurrence of amphizoic amebae in domestic animals. J. Protozool. 25, 235–237.
Kadlec, V., Skvarova, J., Cerva, L., Nebazniva, D., 1980. Virulent Naegleria fowleri in indoor swimming pool.
Folia Parasitol. 27, 11–17.
Kilvington, S., Larkin, D.F.P., White, D.G., Beeching, J.R., 1990. Laboratory investigation of Acanthamoeba
keratitis. J. Clin. Microbiol. 28, 2722–2725.
Kinde, H., Visvesvara, G.S., Barr, B.C., Nordhausen, R.W., Chiiui, P.H.W., 1998. Amebic meningoencephalitis
caused by Balamuthia mandrillaris (leptomyxid ameba) in a horse. J. Vet. Diagn. Invest. 10, 378–381.
Kollars Jr., T.M., Wilhelm, W.E., 1996. The occurrence of antibodies to Naegleria species in wild mammals. J.
Parasitol. 82, 73–77.
Kumar, R., Lloyd, D., 2002. Recent advances in the treatment of keratitis. Clin. Infect. Dis. 35, 434–441.
La Scola, B., Boyadjiev, I., Greub, G., Khamis, A., Martin, C., Raoult, D., 2003. Amoeba-resisting bacteria and
ventilator-associated pneumonia. Emerg. Infect. Dis. 9, 815–821.
Lang, G.E., von Heimburg-Elliger, A., 1991. Acanthamoeba keratitis in hard contact lens wearers. Case report and
review of the literature of 108 cases. Klin. Monatsbl. Augenheilkd. 198, 290–294 (in German).
Leber, A.L., Novak, S.M., 1999. Intestinal and urogenital amebae, flagellates, and ciliates. In: Murray, P.R.,
Baron, E.J., Pfaller, M.A., Tenover, F.C., Yolken, R.H. (Eds.), Manual of Clinical Microbiology. seventh ed.
ASM Press, Washington, DC, pp. 1391–1405.
Lee, S.H., Levy, D.A., Craun, G.F., Beach, M.J., Calderon, R.L., 2002. Surveillance for waterborne-disease
outbreaks: United States, 1999–2000. MMWR Surveill. Summ. 51, 1–47.
Ledee, D.R., Hay, J., Byers, T.J., Seal, D.V., Kirkness, C.M., 1996. Acanthamoeba griffini. Molecular characterization of a new corneal pathogen. Invest. Ophthalmol. Vis. Sci. 37, 544–550.
Levine, N., 1961. Protozoan Parasites of Domestic Animals and of Man. Burgess Publishing Co., Minneapolis,
MN, p. 412.
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
119
Lozano-Alarcon, F., Bradley, G.A., Houser, B.S., Visvesvara, G.S., 1997. Primary amebic meningoencephalitis
due to Naegleria fowleri in a South American tapir. Vet. Pathol. 34, 239–243.
Lynn, D.H., Small, E.B., 2000. Phylum Ciliophora Doflein, 1901. second ed. In: Lee, J.J., Leedale, G.F., Bradbury, P. (Eds.), An Illustrated Guide to the Protozoa, 1. Allen Press, Lawrence, Kansas, pp. 371–656.
Ma, P., WIllaert, E., Jeuchter, K.B., Stevens, A.R., 1981. A case of keratitis due to Acanthamoeba in New York,
New York, and features of 10 cases. J. Infect. Dis. 143, 662–667.
Magaña-Garcı́a, M., Arista-Viveros, A., 1993. Cutaneous amebiasis in children. Pediatr. Dermatol. 10, 352–355.
Marciano-Cabral, F., Cline, M.L., Bradley, S.G., 1987. Specificity of antibodies from human sera for Naegleria
species. J. Clin. Microbiol. 25, 692–697.
Martinez, A.J., 1985. Free-Living Amoebas: Natural History, Prevention, Diagnosis, Pathology, and Treatment of
Disease. CRC Press, Boca Raton, FL, p. 156.
Martinez, A.J., Markowitz, S.M., Duma, R.J., 1975. Experimental acanthamoebic pneumonitis and encephalitis in
mice. J. Infect. Dis. 131, 692–699.
Martinez, A.J., Nelson, E.C., Duma, R.J., Moretta, F.L., 1973. Experimental Naegleria meningoencephalitis in
mice. Penetration of the olfactory mucosal epithelium by Naegleria and pathological changes produced. A
light and electron microscope study. Lab. Invest. 29, 121–133.
Martinez, A.J., Visvesvara, G.S., 1997. Free-living, amphizoic and opportunistic amebas. Brain Pathol. 7, 583–
598.
Martinez, A.J., Visvesvara, G.S., 2001. Balamuthia mandrillaris infection. J. Med. Microbiol. 50, 205–207
(editorial).
Mazur, T., Hadas, E., Iwanicka, I., 1995. The duration of the cyst stage and the viability and virulence of
Acanthamoeba isolates. Trop. Med. Parasitol. 46, 106–108.
Millard, P.S., Gensheimer, K.F., Addiss, D.G., Sosin, D.M., Beckett, G.A., Houck-Jankoski, A., Hudson, A., 1994.
An outbreak of cryptosporidiosis from fresh-pressed apple cider. JAMA 272, 1592–1596.
Muriuki, S.M.K., Murugu, R.K., Munene, E., Karere, G.M., Chai, D.C., 1998. Some gastro-intestinal parasites of
zoonotic (public health) importance commonly observed in old world non-human primates in Kenya. Acta
Trop. 71, 73–82.
Nakauchi, K., 1999. The prevalence of Balantidium coli infection in fifty-six mammalian species. J. Vet. Med. Sci.
61, 63–65.
Newsome, A.L., Scott, T.M., Benson, R.F., Fields, B.S., 1998. Isolation of an amoeba naturally harboring a
distinctive Legionella species. Appl. Environ. Microbiol. 64, 1688–1693.
Niederkorn, J.Y., Ubelaker, J.E., McCulley, J.P., Stewart, G.L., Meyer, D.R., Mellon, J.A., Silvany, R.E., He, Y.G.,
Pidherney, M., Martin, J.H., Alizadeh, H., 1992. Susceptibility of corneas from various animal species to in
vitro binding and invasion by Acanthamoeba castellanii. Invest. Ophthalmol. Vis. Sci. 33, 104–112.
Page, F.C., 1974. Rosculus ithacus Hawes, 1963 (Amoebida, Flabelluidae) and the amphizoic tendency in
amoebae. Acta Protozool. 13, 143–154.
Page, F.C., 1988. A New Key to Freshwater and Soil Gymnamoebae. Freshwater Biological Association,
Ambleside, Cumbria, UK, p. 122.
Parshad, S., Grover, P.S., Sharma, A., Verma, D.K., Sharma, A., 2002. Primary cutaneous amoebiasis: case report
with review of the literature. Int. J. Dermatol. 41, 676–680.
Patz, J.A., Graczyk, T.K., Geller, N., Vittor, A.Y., 2000. Effects of environmental change on emerging parasitic
diseases. Int. J. Parasitol. 30, 1395–1405.
Ravdin, J.I., 1986. Pathogenesis of disease caused by Entamoeba histolytica: studies of adherence, secreted toxins,
and contact-dependent cytolysis. Rev. Infect. Dis. 8, 247–258.
Recavarran-Arce, S., Velarde, C., Gotuzzo, E., Cabrera, J., 1999. Amoeba angeitic lesions of the central nervous
system in Balamuthia mandrillaris amoebiasis. Hum. Pathol. 30, 269–273.
Reveiller, F.L., Varenne, M.-P., Pougnard, C., Cabanes, P.-A., Pringuez, E., Pourima, B., Legastelois, S., Pernin, P.,
2003. An enzyme-linked immunosorbent assay (ELISA) for the identification of Naegleria fowleri in
environmental water samples. J. Eukaryot. Microbiol. 50, 109–113.
Rideout, B.A., Gardiner, C.H., Stalis, I.H., Zuba, J.R., Hadfield, T., Visvesvara, G.S., 1997. Fatal infections with
Balamuthia mandrillaris (a free-living amoeba) in gorillas and other old world primates. Vet. Pathol. 34, 15–22.
Rowbotham, T.J., 1980. Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil
amoeba. J. Clin. Pathol. 33, 1179–1183.
120
F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120
Samples, J.R., Binder, P.S., Luibel, F.J., Font, R.L., Visvesvara, G.S., Peter, C.R., 1984. Acanthamoeba keratitis
possibly acquired from a hot tub. Arch. Ophthalmol. 102, 707–710.
Sangruchi, T., Martinez, A.J., Visvesvara, G.S., 1994. Spontaneous granulomatous amebic encephalitis: report of
four cases from Thailand. Southeast Asian J. Trop. Med. Public Health 25, 309–313.
Schuster, F.L., 2002. Cultivation of pathogenic and opportunistic free-living amoebas. Clin. Microbiol. Rev. 15,
342–354.
Schuster, F.L., Dunnebacke, T.H., Booton, G.C., Yagi, S., Kohlmeier, C.K., Glaser, C., Vugia, D., Bakardjiev, A.,
Azimi, P., Maddux-Gonzalez, M., Martinez, A.J., Visvesvara, G.S., 2003. Environmental isolation of
Balamuthia mandrillaris associated with a case of amebic encephalitis. J. Clin. Microbiol. 41, 3175–3180.
Schuster, F.L., Visvesvara, G.S., 1998. Efficacy of novel anti-microbials against clinical isolates of opportunistic
amebas. J. Eukaryot. Microbiol. 45, 612–618.
Schuster, F.L., Visvesvara, G.S., 2003. Amebic encephalitides and amebic keratitis caused by pathogenic and
opportunistic free-living amebas. Curr. Treat. Options Infect. Dis. 5, 273–282.
Seidel, J.S., Harmatz, P., Visvesvara, G.S., Cohen, A., Edwards, J., Turner, J., 1982. Successful treatment of
primary amebic meningoencephalitis. N. Engl. J. Med. 306, 346–348.
Sesma, M.J.M., Ramos, L.Z., 1989. Isolation of free-living amoebas from the intestinal contents of reptiles. J.
Parasitol. 75, 322–324.
Stehr-Green, J.K., Bailey, T.M., Visvesvara, G.S., 1989. The epidemiology of Acanthamoeba keratitis in the
United States. Am. J. Ophthalmol. 107, 331–336.
Steele, K.E., Visvesvara, G.S., Bradley, G.A., Lipscomb, T.P., Gardiner, C.H., 1997. Amebiasis in a dog with
gastric ulcers and adenocarcinoma. J. Vet. Diagn. Invest. 9, 91–93.
Steinberg, J.P., Galindo, R.L., Kraus, E.S., Ghanem, K.G., 2002. Disseminated acanthamebiasis in a renal
transplant recipient with osteomyelitis and cutaneous lesions: case report and literature review. Clin. Infect.
Dis. 35, e43–e49.
Stothard, D.R., Schroeder-Diedrich, J.M., Awwad, M.H., Gast, R.J., Ledee, D.R., Rodriguez-Zaragoza, S., Dean,
C.L., Fuerst, P.A., Byers, T.J., 1998. The evolutionary history of the genus Acanthamoeba and the
identification of eight new 18S rRNA gene sequence types. J. Eukaryot. Microbiol. 45, 45–54.
Tanyuksel, M., Petri Jr., W.A., 2003. Laboratory diagnosis of amebiasis. Clin. Microbiol. Rev. 16, 713–729.
Tauxe, R.V., 1997. Emerging foodborne diseases: an evolving public health challenge. Emerg. Infect. Dis. 3, 425–
434.
Trager, W., 1986. Living Together. The Biology of Animal Parasitism. Plenum Press, New York, p. 467.
Visvesvara, G.S., 1999. Pathogenic and opportunistic free-living amebae. In: Murray, P.R., Baron, E.J., Pfaller,
M.A., Tenover, F.C., Yolken, R.H. (Eds.), Manual of Clinical Microbiology. seventh ed. ASM Press,
Washington, DC, pp. 1383–1390.
Visvesvara, G.S., Martinez, A.J., Schuster, F.L., Leitch, G.J., Wallace, S.V., Sawyer, T.K., Anderson, M., 1990.
Leptomyxid ameba, a new agent of amebic meningoencephalitis in humans and animals. J. Clin. Microbiol.
28, 2750–2756.
Visvesvara, G.S., Schuster, F.L., Martinez, A.J., 1993. Balamuthia mandrillaris, N. G., N. Sp. agent of amebic
meningoencephalitis in humans and other animals. J. Eukaryot. Microbiol. 40, 504–514.
Wellings, F.M., 1977. Amoebic meningoencephalitis. J. Florida Med. Assoc. 64, 327–328 (editorial).
Wilhelmus, K.R., Osato, M.S., Font, R.L., Robinson, N.M., Jones, D.B., 1986. Rapid diagnosis of Acanthamoeba
keratitis using calcofluor white. Arch. Ophthalmol. 104, 1309–1312.
Zaman, V., 1978. Balantidium coli. In: Kreier, J.P. (Ed.), Parasitic Protozoa, vol. 2. Academic Press, New York,
pp. 633–653.