FREE-LIVING PROTOZOA & HUMAN DISEASE

Naegleria

Naegleria species are found in freshwater habitats and moist soil throughout the world. Primary amebic meningoencephalitis (PAM) was first recognized by Fowler in 1965 and N. fowleri is the only Naegleria species known to be pathogenic to humans. As of 1996, approximately 175 cases have been reported worldwide and 81 of these are in the U.S..

The life cycle consists of trophozoite and cyst stages and the trophozoite stage can be either ameboid or flagellated (Figure). The ameboid trophozoite feeds on bacteria and other organic matter and undergoes asexual replication. The ameboid form transforms into a pear-shaped flagellated form with two flagella at the broad end when placed in distilled water or deprived of nutrients. Flagellated trophozoites are non-feeding and do not replicate, but will convert back into the ameboid form when nutrients are restored. The ameboid forms can also encyst resulting in a stage resistant to desiccation. All stages are characterized by a single nucleus with a large karyosome and no peripheral chromatin. Only the ameboid form is found in tissue.

Naegleria produces a fulminant, and almosts always fatal, acute meningoencephalitis in children and young adults who were previously in excellent health (see PAM below). Almost all reported cases have been associated with swimming in warm or heated waters a few days prior to the onset of symptoms. The portal of entry appears to be the olfactory neuroepithelium in the nasal cavity. The trophozoites probably migrate along the olfactory nerves into the brain and CNS. The symptoms of PAM resemble bacterial meningoencephalitis and PAM is generally characterized by a sudden onset of headache and fever. Nausea, vomiting and other symptoms related to increase intracranial pressure may also be evident. There is a rapid progession from headache and fever to coma, with occasional seizures, and death. Diagnosis is almost always post-mortem and the prognosis is not good.

Acanthamoeba 

Acanthamoeba species are also ubiquitous in soil and water. Their pathogenic potential was first recognized in 1958 by Culbertson who produced an encephalitis in mice following inoculation with an Acanthamoeba-contaminated cell culture. The first definitive human cases were reported in the early 1970's. In contrast to PAM, Acanthamoeba infections are usually associated with chronically ill, immunocompromised, or other debilitated patients. Acanthamoeba, like Naegleria, is neurotropic and causes an encephalitis like disease. However the disease, or granulomatous amebic encephalitis (GAE), is more slowly progressing and chronic. Acanthamoeba infections are also associated with lesions in the cornea (amebic keratitis), lungs and skin.

The portal of entry for Acanthamoeba is not known but believed to the either the respiratory tract via inhalation of cysts or through wounds in the skin that become contaminated by soil. Presumably the trophozoites disseminate by a hematogenous route (i.e., via the circulatory system) to the central nervous system (CNS). However, Recavarren-Arce et al, (1999) have suggested a perivascular route of dessemination (see Box and discussion of Balamuthia). In other words, the ameba crawl along the outside of the blood vessels. The onset of symptoms is often insidious and the clinical manifestations include subtle headache, personality changes and slight fever. GAE has a prolonged clinical course and can take weeks to months to progress to coma and death. Diagnosis is difficult and usually done at autopsy.

Acanthamoeba exibits a typical protozoan life cycle consisting of an ameboid trophozoite stage and a cyst stage (see figure legend of Naegleria life cycle for explanation of trophozoites and cysts). In contrast to Naegleria, both cyst and trophozoite stages can be found in histological specimens. The cysts have a three-layered wall, a wrinkled appearance and are extremely resistant to desiccation. In histological preparations the trophozoites of Acanthamoeba are very similar to Naegleria trophozoites and cannot be distinguished on morphological criteria. However, in culture Acanthamoeba trophozoites can be distinguished by their spike-like pseudopodia (Figure).

Amebic keratitis [Keratitis due to Acanthamoeba] is a vision-threatening chronic inflammation of the cornea caused by Acanthamoeba. It was first reported in 1973 and until the mid-1980's was exceedingly rare and usually associated with ocular trama. The number of cases has increased since 1985 and it is estimated that there has been more than 700 cases as of 1996. This increase is attributed to the use of contact lenses and especially incomplete cleaning and disinfection.

Acanthamoeba probably gains access to the corneal stroma through small breaks in the corneal epithelium produced as a result of minor trauma or abrasion. The ameba can then further destroy the corneal stroma or permit secondary bacterial infections. A mild to moderate corneal inflammation is also associated with Acanthamoeba invasion. Clinical manifestations of amebic keratitis include severe ocular pain and corneal lesions refractory to antiviral, antibacterial, and antimycotic drugs. Diagnosis is confirmed by detecting the amebas in corneal scrapings or biopsies. Some success in treating amebic keratitis has been obtained with poly-hexa-methylene biguanide or propamidine isethionate (Brolene). Surgery is often need to correct the loss of vision.

Balamuthia

Balamuthia mandrilaris is another free-living ameba causing human disease. It was first reported in a mandrill baboon in 1990 and subsequently shown to be associated with human disease. The trophozoites and cysts of Balamuthia are morphologically similar to Acanthamoeba and it also causes GAE (see GAE Box). Phylogenetic analysis of rRNA sequences indicate that Balamuthia is a sister genus of Acanthamoeba and thus the two are closely related (Booton et al, 2003). But they are likely independent and monophyletic genera.

Many cases of GAE originally ascribed to Acanthamoeba have retrospectively been assigned to Balamuthia. As of 2003 there have been approximately 100 cases of Balamuthia infections reported with about half of the cases being in the U.S.. Many of the reported cases involve children. In contrast to Acanthamoeba, Balamuthia can cause a subacute-to-chronic infection which is more rapidly progressing. In addition, Balamuthia appears more capable of infecting healthy individuals (see Box). In many of the infections a primary lesions in the respiratory tract or skin have been noted suggesting that the infections is acquired via inhalation or breaks in the skin. Thus far, only 3 survivors have been reported (Deetz et al, 2003; Jung et al, 2004). All were treated for an extensive time period with numerous drugs.

Only recently has Balamuthia been identified in the environment. The amebas were successfully grown from soil samples associated with a fatal case in a northern California child (Schuster et al, 2003) and have subsequently been cultivated from another soil source (Dunnebacke et al, 2004). The ameba are slow growing (generation time of 20-50 hours) and feed on other ameba (or cultivated mammalian cells) instead of bacteria. These features probably account for the difficulty in detecting Balamuthia in the environment. Interestingly, in two of the survivors acquisition of the infection was associated with gardening activities (Deetz et al, 2003; Jung et al, 2004).

Red Tides & Dinoflagellates

'Red tides', or algal blooms, are the result of large increases in the number of unicellular planktonic organisms. Dinoflagellates - claimed by protozoologists as protozoa and by phycologists as algae- are a major component of the microscopic zoo- and phytoplankton. Many of the dinoflagellates contain pigments and therefore will result in the water taking on a distinct colored appearance in the affected areas (usually coastal). Many dinoflagellates are colorless, but nonetheless, fall under the rubric of red tides. Recent increases in algal blooms are usually attributed to higher levels of nutrients in estuaries and coastal waters due to pollution and agricultural runoff. The shipping industry has also been blamed for increased worldwide distribution of organisms causing red tides.

An overabundance of metabolically-active microorganisms can cause 'dead zones' by depleting the water of oxygen. Fish and other macroscopic organisms will either die or avoid these dead zones, and therefore, can have an adverse economic impact on fishermen and the seafood industry. In some cases, though, the fish kills are the result of toxins produced by dinoflagellates.

These toxins can also accumulate in fatty tissues or organs (eg., liver) and pass up the food chain. Ciguatera and shellfish poisoning are examples of human disease caused indirectly by these toxic dinoflagellates (Table). Ciguatera - known in tropics for centuries and becoming more common elsewhere as tropical fish are more widely marketed - is an example of a fish poisoning associated with the accumulation of dinoflagellate toxins. Herbivorous fish may ingest dinoflagellates while feeding on seaweed and these fish are eaten by larger fish. These larger fish, such as barracuda, grouper, jack and snapper, can have toxin levels which will cause gastrointestinal, neurological, and/or cardiovascular symptoms in humans after a single meal. Initially the symptoms will resemble food poisoning and the neurological symptoms develop later. The neurological affects can last for years.

Similarly, shellfish, which are less sensitive to the toxins, will ingest the dinoflagellates and retain the toxins. The clinical outcome depends on the specific dinoflagellate species and are typically classified as diarrheic, paralytic, or neurotoxic. Temporary amnesia has been associated with eating contaminated shellfish containing neurotoxins. Developed countries typically monitor shellfish beds and other resources for dinoflagellates

Pfiesteria

Pfiestieria piscicida is a toxic dinoflagellate not associated with shellfish poisoning or ciguatera, but affects humans and fish through direct contact. Reports from fishermen and swimmers complaining of rashes, respiratory problems and neurological phenomenon and massive fish kills in North Carolina estuaries and the Chesapeake Bay during the late 1980's and early 1990's led to the identification of this previously undescribed dinoflagellate. Culture filtrates induce open ulcerative sores, hemorrhaging and death in finfish and shellfish. Human exposure to aerosols produces dramatic and rapid neurological effects (Box). Pfiesteria produces at least two toxins. One is a heat-stable, water-soluble neurotoxin which causes fish to become moribund within seconds and death within minutes. The other is a lipophilic compound which causes the epidermis of the fish to slough off.

These toxins likely play a role in the ambush-predator life style of Pfiesteria. Pfiesteria exhibits a complex life cycle with several morphologically distinct forms which undergo transformations depending on the types of food available and water conditions (eg., temperature, salinity, calmness). In the absence of fish, Pfiesteria can exist as a non-toxic zoospore (typical dinoflagellate morphology) which feeds on the planckton in the water column, or as an ameboid form which scavenges in the sediment (Figure). These trophic forms forms reproduce asexually and are capable of encysting.

Pfiesteria becomes toxic if fish linger in the area. One of the toxins causes the fish to become moribund and the other toxin damages the skin. The toxic zoospores feed on the fish tissues. Feeding on fish also induces a sexual cycle resulting in gametes and planozygotes which also feed on the fish. Planozygotes can convert back into zoospores, transform into ameba, or encyst. When the fish dies many of the zoospores will transform into ameba and continue feeding on the fish material. The toxic forms (both zoospores and ameba) will transform back into non-toxic forms or encyst when the fish disappear from the area.

(Tulane.edu/~wiser/protozoology)